ppar , inflamació i resistència a la insulina en...

PPAR��, inflamació i resistència a la insulina en adipòcits

Lucía Serrano Marco

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UNIVERSITAT�DE�BARCELONA��

FACULTAT�DE�FARMÀCIA��

DEPARTAMENT�DE�FARMACOLOGIA�I�QUÍMICA�TERAPÈUTICA�UNITAT�DE�FARMACOLOGIA�I�FARMACOGNÒSIA�

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PPAR��,�INFLAMACIÓ�I�RESISTÈNCIA�A�LA�INSULINA�EN�ADIPÒCITS�

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LUCIA�SERRANO�MARCO�2011�

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UNIVERSITAT�DE�BARCELONA��

FACULTAT�DE�FARMÀCIA��

Departament��de�Farmacologia�i�Química�Terapèutica�Unitat�de�Farmacologia�i�Farmacognòsia�

�Programa�de�Doctorat�:�Biologia�Cel�lular�i�Molecular�

�2006�2008�

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PPAR��,�INFLAMACIÓ�I�RESISTÈNCIA�A�LA�INSULINA�EN�ADIPÒCITS�

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Memòria�presentada�per��

Lucía�Serrano�Marco��per�optar�al�títol�de�Doctor�per�la�Universitat�de�Barcelona�

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�director,�� La�doctoranda,��

Dr.�Manuel�Vàzquez�Carrera� � � � � � �Lucía�Serrano�Marco��

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�LUCÍA�SERRANO�MARCO�2011�

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Ministerio�de�Ciencia�e�Innovación�(PROJECTE�SAF�2006�01475�i�SAF�2009�06939)�(Beca�de�Formación�de�Personal�Investigador)�

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Centro�de�Investigación�Biomédica�en�Red�Diabetes�i�Enfermedades�Metabólicas�Asociadas�

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AGRAÏMENTS�

Per�fi!�Per�fi�després�de�sis�anys�i�mig�veig�el�final�d’aquest�camí.�Ha�costat�però�ja�he�arribat�a�

la�meta.�Com�es�diu�habitualment,�m’ha�costat� sang,� suor� i� llàgrimes,� i�m’atreviria�a�dir�que�

literalment.� Però� una� altra� dita� popular� diu� que� l’ésser� humà� és� l’únic� animal� que� ensopega�

dues� vegades� amb� la� mateixa� pedra,� i� sembla� que� aquest� camí� que� vaig� iniciar� fa� un� temps�

tindrà� la� seva�continuïtat.�Durant� tot�aquest� temps�m’ha�acompanyat�molta�gent,�a�qui�avui�

vull�donar�les�gràcies�de�tot�cor.��

En�primer�lloc�agraeixo�al�Dr.�Manel�Vázquez�i�al�Dr.�Joan�Carles�Laguna�per�haver�me�donat�la�

oportunitat�de�formar�part�del�seu�grup�de�recerca�en�els�meus�inicis�com�a�“masteranda”�.�I�li�

torno� a� donar� les� gràcies� al� Dr.� Manel� Vázquez� per� donar�me� una� nova� oportunitat� per�

realitzar�aquesta�tesi�doctoral�sota�la�seva�direcció.�No�hagués�pogut�desitjar�un�millor�director�

de�tesi,�de�vegades�has�confiat�en�mi�més�del�que�jo�ho�feia,�però�al�final�ho�hem�aconseguit!�

Gràcies�Manel.�

Agraeixo� també� a� tots� els� professors� del� departament� i� membres� de� PAS,� tots� han� sigut� un�

exemple�i�de�tots�he�aprés�alguna�cosa.�No�puc�deixar�d’esmentar�a�alguns.�Gràcies�Mar,�per�

tota� la� paperassa� amb� la� que� m’has� ajudat� sempre� i� per� estar� tan� guapa� cada� matí.� Gràcies�

Sílvia,� una� tècnic� vinguda� del� cel.� Dra.� Núria� Roglans,� que� t’he� de� dir,� gràcies� pel� teu� bon�

humor.�Dr.�Xavier�Palomer,�gràcies�per�tots�els�consells,�pel�teu�punt�de�vista,�pel�teu�humor�i�

pels�teus�mimos.�I�al�grup�de�Farmacognòsia,�per�compartir�tants�bons�moments�i�pica�picas.�

Gràcies�a�tots.�

I� no� puc� deixar� d’agrair� a� tots� el� companys� que� han� passat� pel� laboratori,� sense� ells� si� que�

hauria� llençat� la� tovallola.�En�primer� lloc�el�Dr.�Ricardo�Rodríguez,�que�va�ser�un�exemple�de�

passió�per� la� feina.�Algo� se�me�acabó� pegando,espero�que� te�guste� la� tesis.� A� la�Dra.�Emma�

Barraso,� companya,� amiga,� confident,� un� pilar� insubstituïble� en� aquests� anys,� ¿de� qué� no�

hemos�hablado�en�el�laboratorio�6?�Muchas�gracias�Emma!!�Gràcies�també�a�les�Dres.�Teresa�

Coll� i� Laia� Vilà,� indispensables� en� els� inicis� i� amigues� per� molts� anys.� Gràcies� al� Dr.� David�

Alvàrez� per� aportar� sempre� un� punt� de� vista� diferent� i� pel� seu� bon� humor.� A� la� resta� dels�

naranjitos,� el� Gerard,� la� Eva� i� la� Laia� que� m’han� acompanyat� aquests� últims� temps� fent�me�

sentir� la� veterana,� que� m’han� donat� tants� bons� moments� i� fins� i� tot� una� companya� de� pis!�

Moltes�gràcies.�I�que�no�se�m’oblidin�les�internacionals,�des�de�Mèxic,�la�Saray�i�des�de�Portugal�

la�Anna,�muchas�gracias�chicas�por�acompañarme�y�darme�vuestra�amistad�en�tan�poco�tiempo�

i�por�hacer�el�laboratorio�6�un�lugar�mejor.�

Gràcies�al�grup�dels�azulitos�o�pitufos,�encara�falta�determinar�el�nom.�Alba,�qué�grande�eres,�

gracias�por�tus�consejos�antes�de�un�EMSA�y�en�la�vida�en�general,�gracias�por�ser�mi�amiga�y�

por�tus�mimos.�Al�Dr.�Jordi�Pou,�gràcies�pel�teu�bon�humor,�la�teva�amistat�i�per�ser�com�ets.�Al�

nou,�Miguel,�el�optimismo�hecho�oscense�i�a�l’Anna�Padrosa,�una�noia�que�deixa�empremta�allà�

on�va.�

Continuo�amb�la�Sara,� la�Loli,�el�Carlos,� la�Sònia,�el�Jose� i�el�Raúl,�qué�bé�ens�ho�hem�passat.�

Davant�seu�i�de�tant�en�tant�treballant�colze�amb�colze�el�grup�de�neuro,�segur�que�me’n�deixo�

algun.�Dani,�Marc�Yeste,�Javi�G,�Javi�P.�(el�mac�dels�somriures),�Abrisqueta�(amic�de�tants�anys�i�

més�que�seran),�Laura�Altimira,�Paolo�i�Katherina,�tots�m’heu�fet�somriure,��gràcies.�Agraeixo�a�

la�Dra.�Natàlia�Crespo,�per�la�seva�amistat�aquests�anys.�Gràcies�Dra.�Marta�Tajes,�no�sé�com�

t’ho�fas�per�estar�sempre,�ets�un�àngel.�Agraeixo�al�Sergi,�que�té�un�cor�més�fort�que�les�seves�

cames,� al� David� pel� seu� bon� humor� i� les� estones� dinant,� a� la� Caro,� la� dulzura� mejicana,� a� la�

Luisa�un�altre�àngel�però�aquest�cop�portuguès�i�a�l’Aureli,�mi�impu,�no�podria�dir�tot�en�lo�que�

m’has�ajudat,�gràcies�per�la�teva�amistat.�

La�gent�de�fora�del�laboratori�també�ha�col�laborat�en�aquesta�feina,�donant�me�suport�i�fent�

me�la�vida�una�mica�més�feliç.�Eva,�Marc,�Vane,�Vicenç,�Mireya,�Sandra,�Fulgen,�Joan,�Núria�i�

Laura.�Gràcies�per�fer�me�una�mica�més�feliç�cada�vegada�que�us�veig,�gràcies�pels�viatges,�pels�

sopars,�per� les�festes,� i�per� les�xerrades�friquis�de�cèl�lules� i�ciència,�per� les�visites�gratuïtes� i�

guiades�a�l’aquari,�concerts,�etct.�En�definitiva�gràcies�per�ser�com�sou,�per�estar�amb�mi�i�per�

la�vostra�amistat.�La�vida�tindria�menys�colors�sense�vosaltres.�Laia�Miret,�gràcies�per� la�teva�

companyia�i�amistat,�el�pis�està�més�alegre�i�divertit�des�de�que�hi�has�entrat.�Jaume�i�Gerard,�

gràcies�per�continuar�la�nostra�amistat�tants�anys.�

Ah!� Y� tengo� dos� pueblos,� Monterde� de� Albarracín� i� Alacón,� que� van� dónde� yo� voy.� Gracias�

familia�y�peña�Trokimoche.�Los�veranos�de�mi�vida�hubieran�sido�muy�aburridos�sin�vosotros.�

Els�més�importants�de�tots,�la�meva�família.�Gràcies�a�ells�soc�qui�soc�i�estic�orgullosa�de�ser�

ho.�Gracias�Mama,�por�ti�hoy�soy�cómo�soy,�tu�tienes�la�culpa�de�todo,�gracias�por�tu�paciencia�

y� tu�apoyo� incondicional,�estoy� tan�orgullosa�de� ti.�Gracias�Papa,�por�acompañarme�en�cada�

paso�que�doy�en�mi�vida,�espero�que�estés�orgulloso.�Gracias�Tío,�por�estar�tan�orgulloso�de�tu�

sobrina,�y�por�querernos�tanto.�Gracias�Marta,�eres�mi�espejo�y�creo�que�tienes�la�mitad�de�mi�

alma,�gracias�por�apoyarme�siempre�y�por�cantarme� las� cuarenta�cuando�hace� falta.�Gracias�

Fran,�por�ayudarme�y�por�hacer�feliz�a�mi�otra�mitad.��

Gracias�Raúl,�por�llegar�a�mi�vida�para�apoyarme,�por�intentar�entenderme�a�mi�y�a�mi�trabajo,�

que�ni�si�quiera�yo�entiendo.�Gracias�por�estar�ahí.�Gracias�Amor.�

GRÀCIES�A�TOTS!�

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No�sé�qué�té�la�ciència�que�enganxa.�He�aprés�a�voler�aprendre�més,�he�aprés�que�encara�no�sé�

res,�i�que�mai�ho�sabré�tot.�

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ABREVIATURES.......................................................................................................................1�

INTRODUCCIÓ........................................................................................................................9�

1. TEIXIT�ADIPÓS..........................................................................................................15�

1.1. TIPUS�DE�TEIXIT�ADIPÓS...........................................................................................15�

1.1.1. TEIXIT�ADIPÓS�MARRÓ................................................................................16�

1.1.2. TEIXIT�ADIPÓS�BLANC..................................................................................16�

1.2. EL�EL�TAB�COM�A�ÒRGAN�ENDOCRÍ.........................................................................18�

1.3. VIA�DE�SENYALITZACIÓ�DE�LA�INSULINA�AL�TEIXIT�ADIPÓS.....................................21�

1.3.1. VIA�DE�SENYALITZACIÓ�PI3K/AKT................................................................22�

1.3.2. RESISTÈNCIA�A�LA�INSULINA.......................................................................26�

2. RECEPTORS�ACTIVATS�PER�PROLIFERADORS�PEROXISÒMICS......................................28�

2.1. ESTRUCTURA�DELS�PPARs........................................................................................29�

2.2. MECANISMES�D’ACCIÓ�DELS�PPARs.........................................................................30�

2.2.1. TRANS�ACTIVACIÓ.......................................................................................30�

2.2.2. TRANS�REPRESSIÓ.......................................................................................31�

2.3. PPAR�......................................................................................................................33�

2.3.1. DISTRIBUCIÓ�TISSULAR...............................................................................33�

2.3.2. LLIGANDS�DE�PPAR�...................................................................................33�

2.3.3. FUNCIONS�DE�PPAR�..................................................................................34�

2.4. PPAR�.......................................................................................................................35�

2.4.1. DISTRIBUCIÓ�TISSULAR...............................................................................35�

2.4.2. LLIGANDS�DE�PPAR�....................................................................................35�

2.4.3. FUNCIONS�DE�PPAR�...................................................................................36�

2.5. PPAR��...................................................................................................................37�

2.5.1. DISTRIBUCIÓ�TISSULAR�..............................................................................37�

2.5.2. LLIGANDS�DE�PPAR��................................................................................37�

2.5.3. FUNCIONS�DE�PPAR��...............................................................................38�

3. FACTOR�NUCLEAR��B...............................................................................................40�

3.1. FAMÍLIA�I�ESTRUCTURA�DE�NF��B...........................................................................41��

3.2. REGULACIÓ�DE�NF��B..............................................................................................42�

3.2.1. COMPLEX�IKK..............................................................................................42�

3.2.2. UBIQUITINITZACIÓ�AL�PROTEASOMA.........................................................44�

3.2.3. ACETILACIÓ�I�DESACETILACIÓ.....................................................................45�

4. INTERLEUCINA�6......................................................................................................48�

4.1. IL�6�I�LA�RI................................................................................................................49�

4.2. �VIES�DE�SENYALITZACIÓ�DE�LA�IL�6.........................................................................50�

4.2.1. RECEPTORS�DE�LA�IL�6:�IL�6R��I�GP130.......................................................50�

4.2.2. VIA�DE�SENYALITZACIÓ�JAK/STAT3.............................................................52�

4.2.3. INHIBICIÓ�DE�LA�SENYALITZACIÓ�DE�LA�IL�6...............................................56�

5. FACTOR�DE�NECROSIS�TUMORAL��..........................................................................58�

5.1. TNF��I�RI..................................................................................................................59�

5.1.1. TNF��ACTIVA�NF��B....................................................................................60�

5.1.2. TNF��INHIBEIX�LA�VIA�DE�SENYALITZACIÓ�DE�LA�INSULINA.......................60�

OBJECTIUS............................................................................................................................63�

RESULTATS...........................................................................................................................67�

1. Activation� of� Peroxisome� Proliferator�Activated� Receptor� �� (PPAR�� ameliorates�

insulin� signaling� and� reduces� SOCS3� levels� by� inhibiting� STAT3� in� interleukin�6�

adipocytes......................................................................................................................69 �

2. TNF�� inhibits� PPAR�� activity� and� SIRT1� expression� through� NF��B� in� human�

adipocytes......................................................................................................................83 �

DISCUSSIÓ�GLOBAL.............................................................................................................119�

CONCLUSIONS....................................................................................................................129�

BIBLIOGRAFIA....................................................................................................................133�

ANNEX...............................................................................................................................159�

� The�Peroxisome�Proliferator�Activated�Receptor��(PPAR��)�agonist�GW501516�

inhibits� IL�6�induced� STAT3� activation� and� insulin� resistance� in� human� liver�

cells........................................................................................................................163 �

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ABREVIATURES�

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8(S)�HETE�� àcid�8(S)�hidroxieicosatetraenoic�

AACT�� 1�antiquimotripsina�

ACC�� acetyl�CoA�Carboxilase�

ACO� � acil�CoA�oxidasa�

AF�1�� activation�function�1�

AF�2�� activation�function�2�

AGP� � �1�acid�glicoproteïna��

Akt�ó�PKB�� proteïna�cinasa�B�

AP�1�� activator�protein�1�

aP2�� fatty�acid�binding�protein�

CBM�� módul�d’unió�a�citocines�

CBP/p300�� (cyclic�AMP�response�element)�CREB�binding�protein�

cIAP� � inhibitor�of�cellular�apoptosis�proteins�

CIS�� cytokine�inducible�SH2�proteins�

CNTF�� ciliary�neurotrophic�factor�

CPT�1� � carnitin�palmitoil�transferasa�1��

CRP�� proteïna�C�reactiva�

CT�1�� cardiotrophin�like�citokine�

DBD� �� DNA�binding�domain�

Domini�PTB� domini�d’unió�a�fosfotirosines�

dominis�SH2�� domini�Src�homology�2�

ABREVIATURES

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DR�1� � Direct�Repeat�1�

EGF�� epidermal�growth�factor�

ERK1/2�� extracellular�signal�related�kinase�

FAS�� fatty�acid�sinthase�

FAS�� fatty�acid�synthase�

FAT�� fatty�acid�translocase�

FATP�� fatty�acid�transport�protein�

FNIII�� fibronectin�type�III�like�

G�6�PDH�� glucose�6�phosphat�dehydrogenase�

Gab1�� GRB2�associated�binding�protein�1�

GLUT�4�� transportador�de�glucosa�4�

GPAT�� glycerol�3�phosphate�acyltransferase�

Grb2�� growth�factor�receptor�bound�protein�2�

HAT� � histone�acetyl�transferase�

HDAC� � histone�deacetylase�

HDL�� high�density�lipoprotein�

HLH�� hèlix�loop�hèlix�

hsp90�� heatshock�protein�90�

IKK� � I�B�cinasa�

IKKK�� cinasa�de�IKKs�

IL�6� � interleucina�6��

INS� � insulina�

IRF�1�� interferon�regulatory�factor�1�

ABREVIATURES

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IRS�� substrats�del�receptor�de�la�insulina��

IRS�1�� receptor�de�la�insulina�1�

JAK�� janus�kinase�

JH1�� Jak�homology1�

JNK�� c�Jun�N�terminal�kinase�

LBD�� ligand�binding�domain�

LDL�� low�density�lipoprotein�

LIF�� leukaemia�inhibitor�factor�

LPL�� lipoprotein�lipase�

LZ� � leucine�zipper�

MAPK�� mitogen�activated�protein�kinase�

MCAD�� acil�CoA�deshidrogenasa�de�cadena�mitja�

MEKK�3�� MAP/ERK�cinasa�3�

NFAT�� Nuclear�Factor�of�Activated�T�cells�

NIK�� NF��B�inducing�kinase�

NLS�� senyals�de�localització�nuclear�

OSM�� oncostatin�M�

PCAF�� CBP/p300�associated�factor�

PDGF�� platelet��derived�growth�factor�

PDK�1� � phosphoinositide�dependent�kinase�1�

PDK4�� pyruvate�dehydrogenase�4�

PEPCK�� phosphoenolpyruvate�carboxykinase�

PGD�� phophogluconate�dehydrogenase�

ABREVIATURES

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PH�� pleckstrin�homology�

PI3K�� fosfatidilinositol�3’�cinasa��

PIP2� � fosfoinositol�difosfat��

PIP3� � fosfoinositol�trifosfat��

PKB�� proteïna�cinasa�B�

PKC�/�� proteïna�cinasa�C�/��

PKC�� protein�kinase�C��

PLTP�� phospholipid�transfer�protein�

PPARs�� receptors�activats�per�proliferadors�peroxisòmics��

Ppp1r3C�� protein�phosphatase�1�regulatory�subunit�

PPRE�� elements�de�resposta�a�proliferadors�peroxisòmics��

PTP1B�� protein�tyrosin�phospatase�1B�

RHD� � domini�d’homologia�Rel�

RIP�1�� receptor�interacting�protein�1�

RXR�ó�NR2B�� receptor�de�l’àcid�9�cis�retinoic�

SCD�1�� steaoryl�CoA�desaturase�1�

SH2�� Src�homology�2dominis��

SH3�like�� Src�homology�3�like�

SIRT�1�� silent�information�regulator�T1��

SOCS� � supressor�of�citokine�signaling�

SODD�� silencer�of�death�domains�

STAT3�� Signal�transducer�and�activator�of�transcription�3�

TAK1�� (transforming�growth�factor�beta�TGF��)�activated�kinase�

ABREVIATURES

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TBP�� TATA�binding�protein�

TIMP�3�� metalloproteinase�inhibitor�3�

TNF�� receptor�associated�death�domain�

TNF�� factor�de�necrosis�tumoral��

TRAF2�� TNFR�associated�factor�2�

TZD� � tiazolidindiona�

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ABREVIATURES

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INTRODUCCIÓ�

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9

La� incidència�de� l’obesitat�arreu�del�món�ha�augmentat�dràsticament�durant�els�últims�anys.�

L’Organització� Mundial� de� la� Salut� estima� que� més� de� mil� milions� d’adults� a� tot� el� món�

pateixen�de�sobrepès� i,�d’aquests,�300�milions�són�clínicament�obesos.�Es�considera�obesitat�

quan�l’índex�de�massa�corporal�(BMI,�Body�Mass�Index),��cocient�que�s’obté�dividint�el�pes�en�

Kg�per�l’alçada�en�metres�quadrats,�és�igual�o�superior�a�30�Kg/m2.�La�importància�de�l’obesitat�

deriva� en� gran� part� per� la� seva� associació� amb� l’aparició� d’altres� problemes� de� salut� com� el�

increment� del� risc� de� patir� resistència� a� la� insulina� (RI),� diabetis� mellitus� de� tipus� 2� (DM2)� i�

aterosclerosi,�entre�d’altres�patologies�(Hotamisligil,�2006).��

La� DM2,� és� la� forma� més� freqüent� de� la� diabetis� mellitus.� Es� caracteritza� per� presentar� alts�

nivells�de�glucosa�en�sang�degut�a�la�presència�de�resistència�a�la�insulina�(RI),�combinada�amb�

una� secreció� insuficient� d’insulina� pel� pàncrees.� La� RI� es� defineix� com� la� disminució� de� la�

resposta� dels� teixits� perifèrics� a� l’acció� de� la� insulina.� Aquest� factor� precedeix� i� prediu� el�

desenvolupament� de� la� DM2� i� és� clau� per� a� la� progressió� de� la� malaltia� i� de� les� seves�

complicacions.� La� manca� de� les� funcions� de� la� insulina� porta� a� un� metabolisme� cel�lular�

defectuós� que� acaba� provocant� un� increment� dels� àcids� grassos� i� dels� nivells� circulants� de�

triglicèrids,�a�més�d’una�disminució�de�la�concentració�de�la�lipoproteïna�d’alta�densitat�(HDL).��

Alguns� factors� de� risc� que� predisposen� a� un� individu� a� desenvolupar� DM2� inclouen�

antecedents� familiars,� el� sedentarime,� una� mala� alimentació� i� l’obesitat� abdominal,� entre�

d’altres.� De� fet,� s’ha� demostrat� que� existeix� una� forta� correlació� entre� obesitat� i� RI� � tant� en�

pacients�diabètics�com�en�no�diabètics�(Ludvik�i�col.,�1995).�Així,�hi�ha�estudis�que�demostren�

que�el�risc�de�patir�diabetis�augmenta�onze�vegades�quan�el�BMI�s’incrementa�de�20�a�30Kg/m2�

(Carey� i�col.,�1997).�Tanmateix,� la� falta�de�sensibilitat�a� la� insulina�produïda�en� la�RI�ha�estat�

reconeguda�com� la�característica�essencial�de� l’anomenada�síndrome�metabòlica,�que� inclou�

intolerància� a� la� glucosa,� RI,� obesitat,� hipertrigliceridèmia,� colesterol� d’HDL� baix� (cHDL),�

hipertensió�i�aterosclerosis�(Xu�i�col.,�2003).�La�insulina�és�la�hormona�anabòlica�més�potent�de�

l’organisme�i�té�un�paper�significatiu�en�el�metabolisme�de�la�glucosa�i�dels�àcids�grassos,�però�

també�afecta�al�creixement�i�a�la�diferenciació�cel�lular�(Duvnjak�i�Duvnjak,�2009).��

Amb� les�dades�anteriors�es�pot�concloure�que� l’obesitat,� la�RI� i� la�DM2�tenen�algun�tipus�de�

relació�causa�efecte.�En�aquest�sentit�cal�destacar�que�en�els�últims�anys�ha�estat�descrit�que�

un� estat� inflamatori� crònic� de� baixa� intensitat� podria� ser� el� punt� d’encreuament� d’aquestes�

tres�patologies.�Aquest�estat�inflamatori�es�caracteritza�per�la�producció�anòmala�de�citocines�

com� la� interleucina� 6� (IL�6),� el� factor� de� necrosis� tumoral��TNF�� o� la� proteïna� C� reactiva�

(CRP),�així�com�per�la�producció�de�mediadors�de�la�fase�aguda�de�la�inflamació�i�l’activació�de�

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11

vies�de�senyalització�inflamatòries�(Wellen�i�Hotamisligil,�2005).��Aquestes�i�altres�citocines�pro�

inflamatòries�semblen�participar�en�la�inducció�i�el�manteniment�d’aquest�estat�inflamatori.�

La� resposta� inflamatòria� que� sorgeix� en� presència� d’obesitat� podria� desencadenar�se�

predominantment� al� teixit� adipós,� encara� que� altres� teixits� com� el� fetge� també� poden� estar�

involucrats�en�el�seu�manteniment�al�llarg�del�transcurs�de�la�malaltia�(Shoelson�i�col.,�2006).�El�

teixit�adipós�juga�un�paper�important�en�el�metabolisme�dels�àcids�grassos,�encarregant�se�del�

seu�emmagatzematge�a�l’interior�dels�adipòcits.�D’altra�banda,�estudis�recents�(Laustsen�i�col.,�

2002;�Moitra�i�col.,�1998;�Shimomura�i�col.,�1998;�Sovik�i�col.,�1996)�han�situat�el�teixit�adipós�

com�un�òrgan�crucial�en� la�producció�de�citocines� i�mediadors�pro�inflamatoris.�Curiosament�

s’ha�observat�que�la�manca�de�teixit�adipós�produeix�un�increment�dels�triglicèrids�i�dels�àcids�

grassos� circulants� que� desencadenen� l’aparició� de� RI� en� ratolins� i� humans.� Tanmateix,� la�

presència� de� teixit� adipós� és� necessària� per� a� la� secreció� normal� d’adipocines� com� leptina� i�

adiponectina,� que� incrementen� la� sensibilitat� a� la� insulina.� Per� tant,� totes� aquestes�

observacions�suggereixen�que�la�sensibilitat�a�la�insulina�i�l’homeòstasi�de�la�glucosa�necessiten�

d’un� teixit� adipós� funcional� i� en� proporció� adequada� a� la� mida� corporal.� Així,� en� casos�

d’obesitat� en� que� hi� ha� un� augment� de� la� ingesta� calòrica� que� incrementa� la� mida� dels�

adipòcits�per� l’acumulació�de�triglicèrids�al�seu�citosol,�es�veu�alterada� la�capacitat�d’aquests�

per�actuar� com�a�cèl�lules�endocrines� (Kershaw� i� Flier,�2004;�Qatanani� i� Lazar,�2007;�Rajala� i�

Scherer,�2003).�

La� relació� de� la� inflamació� � i� la� diabetis� no� és� nova.� Fa� més� d’un� segle� es� va� demostrar� que�

l’administració� d’elevades� dosis� de� salicilat� de� sodi� (5�75g/d)� disminuïa� la� glucosúria� en�

pacients� diabètics� que� patien� la� forma� aleshores� coneguda� com� � “la� forma� més� lleu� de� la�

malaltia”,�probablement,�DM2� (Williamson,�1902).� L’efecte�va�ser� redescobert�el�1957,�quan�

un�pacient�diabètic�tractat�amb�insulina�que�prenia�elevades�dosis�d’aspirina�per�tal�de�tractar�

l’artritis� reumatoide� no� va� necessitar� més� injeccions� d’insulina.� Quan� es� va� analitzar� la�

concentració�de�glucosa�d’aquest�individu�es�va�veure�que�era�propera�als�valors�normals.�Però�

quan� es� va� interrompre� el� tractament� amb� aspirina� el� pacient� va� tornar� a� presentar�

intolerància�a�la�glucosa�(REID�i�col.,�1957).�No�va�ser�fins�molt�més�endavant�que�estudis�que�

buscaven� quin� paper� jugava� la� inflamació� en� la� patogènesi� de� la� RI� van� analitzar� les� accions�

hipoglicèmiques� dels� salicilats,� trobant� que� la� diana� molecular� sobre� la� qual� actuen� aquests�

salicilats�és� la�via� I�B�cinasa��I�K�� factor�nuclear��B�(NF��B)� (Yuan� i�col.,�2001;�Shoelson� i�

col.,� 2003;� Hundal� i� col.,� 2002).� NF��B� és� el� principal� factor� de� transcripció� encarregat� de� la�

transcripció� de� nombrosos� marcadors� i� mediadors� d’inflamació,� com� ara� la� IL�6� i� el� TNF��

(Jellema� i� col.,� 2004).� Aquest� factor� de� transcripció� també� activa� diverses� cascades� de�

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12

transducció� de� senyals� que� inclouen� vies� importants� per� a� la� senyalització� de� la� insulina�

(Hotamisligil� i� col.,� 1993).� Per� exemple,� en� ratolins� obesos,� la� falta� de� les� funcions� de�

TNF��resulta� en� la� millora� de� la� sensibilitat� a� la� insulina� i� de� l’homeòstasi� de� la� glucosa,�

confirmant�que�aquesta�resposta�inflamatòria�juga�un�paper�crític�en�la�regulació�de�l’acció�de�

la�insulina�en�l’obesitat�(Uysal�i�col.,�1997;�Ventre�i�col.,�1997).�

A� mitjans� dels� anys� 90,� Hotamisligil� G.� S.� i� col.� (1993),� van� observar� que� el� TNF��es� trobava�

sobreexpressat� en� teixit� adipós� de� ratolins� obesos.� � En� teixit� adipós� humà,� l’expressió� de�

TNF��es� correlaciona� amb� l’increment� del� BMI,� el� percentatge� de� greix� corporal� i� la�

hiperinsulinèmia,� mentre� que� els� nivells� d’aquesta� citocina� disminueixen� amb� la� pèrdua� de�

pes.� Posteriorment,� es� va� observar� que� a� més� del� TNF��el� teixit� adipós� de� models� animals�

obesos� genètics� i� induïts� amb� la� dieta� també� presentaven� major� expressió� de� citocines� pro�

inflamatòries�com�la�IL�1�i�la�IL�6.�D’aquestes�citocines,�la�IL�6�és�la�que�presenta�una�associació�

més�forta�amb�la�RI� i� la�DM2�(Kern�i�col.,�2001;�Pickup�i�col.,�1997;�Pradhan�i�col.,�2001)� i�els�

seus�nivells�plasmàtics�augmenten�de�2�3�vegades�en�pacients�amb�obesitat�i�DM2�comparats�

amb� individus� control� (Pickup� i� col.,� 1997).� Tenint� en� compte� això� es� podria� suggerir� que� la�

deficiència�d’IL�6� tindria�un�efecte�protector� sobre� l’obesitat� i� la�RI,�però�diversos�estudis�en�

ratolins� deficients� en� IL�6� han� donat� resultats� controvertits� (Kristiansen� i� Mandrup�Poulsen,�

2005).�Per�una�banda,�aquests�ratolins�no�presenten�ni�obesitat�ni�hiperglucèmia�anormals�(Di�

Gregorio� i� col.,� 2004).� Per� l’altra� banda,� sembla� que� la� deficiència� d’IL�6� provoca� més�

susceptibilitat�a�desenvolupar�obesitat�i�RI�(Wallenius�i�col.,�2002). �

Tant�els�processos�metabòlics�com�inflamatoris�poden�ser�coordinadament�regulats�per�lípids�

(Yu�i�col.,�2002).�Diversos�factors�de�transcripció,�particularment�la�família�del�Receptor�Activat�

per� Proliferadors� Peroxisòmics� (PPAR)� sembla� ser� crucial� per� la� regulació� conjunta� d’aquests�

processos,�ja�que�l’activació�d’aquests�factors�de�transcripció�inhibeix�l’expressió�de�diferents�

gens� involucrats� en� la� resposta� inflamatòria� i� regulen� l’expressió� de� gens� implicats� en� el�

metabolisme�(Chawla� i�col.,�2001;�Glass� i�Ogawa,�2006).�La�família�dels�PPARs�consta�de�tres�

tipus,� PPAR�,� PPAR�� i� PPAR��amb� diferent� distribució� tissular� i� funcions� sobre� el�

metabolisme� lipídic� i� de� la� glucosa� (Boden� i� Laakso,� 2004).� Dels� tres� subtipus,� potser� el� més�

desconegut� és� PPAR�� La� manca� de� lligands� específics� d’aquest� subtipus� de� PPAR,� ha� fet�

difícil�fins�fa�poc�estudiar�els�efectes�produïts�per�la�seva�activació.�GW501516�va�ser�el�primer�

lligand�d’alta�afinitat�disponible,�amb�una�selectivitat�per�PPAR��1000�vegades�superior�que�

per� la�resta�dels�PPARs�(Takahashi� i�col.,�2006).�Recentment,�s’ha�observat�que� l’activació�de�

PPAR��interfereix�amb�la�reacció�inflamatòria�de�fase�aguda�mediada�per�IL�6�en�hepatòcits�

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13

mitjançant�la�inhibició�de�l’activitat�transcripcional�de�STAT3�(Kino�i�col.,�2007),�encara�que�els�

mecanismes� implicats�són�encara�desconeguts.�Per�altra�banda,�en�el�nostre�grup�de�recerca�

s’havia� dut� a� terme� un� estudi� en� que� s’havia� descrit� que� GW501516� inhibia� l’activitat�

transcripcional�de�NF��B�a�través�de�la�inhibició�de�l’activitat�de�l’ERK1/2�(Extracellular�signal�

related�kinase�1/2)�en�adipócits�(Rodriguez�Calvo�i�col.,�2008),�disminuint�d’aquesta�manera�la�

producció� de� citocines� pro�inflamatòries� implicades� en� el� desenvolupament� de� RI.� Malgrat�

aquests�resultats,�encara�es�desconeixia�si�l’activació�de�PPAR��en�adipòcits�podia�prevenir�la�

RI�a�través�d’altres�mecanismes.�Tanmateix�es�desconeix�quins�són�els�efectes�de�la�presència�

d’obesitat�sobre�l’activitat�i�nivells�de�PPAR��al�teixit�adipós�humà.��

En�definitiva,�totes�aquestes�dades�semblen�implicar�als�PPARs�,�i�especialment�al�PPAR��en�

la� relació� entre� inflamació� i� RI.� Per� això� és� d’interès� científic� l’estudi� i� descripció� dels�

mecanismes�moleculars�mitjançant�els�quals�els�agonistes�de�PPAR��com�ara�el�GW501516,�

regulen�el�procés�inflamatori��i�la�RI�en�adipòcits�per�tal�d’establir�noves�dianes�terapèutiques�

que� millorin� la� sensibilitat� a� la� insulina.� Tanmateix,� resulta� interessant� estudiar� com� la�

presència� d’obesitat� i� els� mediadors� pro�inflamatoris� regulen� l’activitat� i� l’expressió� de�

PPAR��en�adipòcits�humans.�

�

�

�

�

�

�

�

�

�

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14

�

�

�

�

�

�

�

�

�

�

1. TEIXIT�ADIPÓS�

Durant� molts� anys,� les� funcions� del� teixit� adipós� van� estar� associades� a� l’aïllament� tèrmic,� a�

l’emmagatzematge� de� l’excés� dels� àcids� grassos� lliures� per� a� ser� alliberats� quan� es�

necessitessin,�i�a�la�protecció�mecànica�dels�òrgans.�Però�des�del�descobriment�i�la�identificació�

de� les� anomenades� adipocines,� el� teixit� adipós� ha� passat� a� ser� considerat� com� un� òrgan�

important� en� el� desenvolupament� de� la� fisiopatologia� de� la� RI� i� de� la� síndrome� metabòlica�

(Gustafson,� 2010).� A� més,� el� teixit� adipós� té� nombrosos� receptors� que� li� permeten� donar�

resposta�a�diferents�estímuls�hormonals�i�al�sistema�nerviós�central�(Kershaw�i�Flier,�2004).�

�

1.1. TIPUS�DE�TEIXIT�ADIPÓS�

En�mamífers�existeixen�dos�tipus�principals�de�teixit�adipós,�el�teixit�adipós�marró�(TAM)� i�el�

teixit�adipós�blanc�(TAB).��

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15

1.1.1. TEIXIT�ADIPÓS�MARRÓ�(TAM)�

El� teixit� adipós� marró� existeix� en� gran� quantitat� de� mamífers,� però� només� és� especialment�

important�en�aquells�que�duen�a� terme� la�hibernació.�Per�a�aquests�animals� la�producció�de�

calor� és� essencial,� i� aquesta� funció� calorigènica� la� realitza� el� TAM.� Per� tal� d’optimitzar�

l’escalfament�del�cos,�el�TAM�es�localitza�estratègicament�en�regions�superficials�com�són�les�

àrees� interescapular,�cervical� i�axil�lar,�o�en�regions�profundes�que�corresponen�a� les�regions�

perirrenals,�periaòrtica,�inguinal�i,�especialment,�al�voltant�del�cor�i�dels�vasos�sanguinis.�Això�

permet�escalfar�els�òrgans�vitals�mitjançant�la�sang�que�els�irriga.�En�humans,�el�TAM�envolta�el�

cor�i�els�grans�vasos�sanguinis�però�desapareix�en�els�primers�anys�de�vida�essent�substituït�pel�

teixit� adipós� blanc.� Tanmateix,� estudis� recents� suggereixen� que� en� adults� encara� romanen�

dipòsits� de� TAM� metabòlicament� actius� que� poden� ser� induïts� per� fred� i� donar� resposta� a�

l’activació� del� sistema� nerviós� simpàtic.� El� TAM� també� emmagatzema� i� sintetitza� triglicèrids,�

controla�activament�el�metabolisme�de�la�glucosa�i�és�un�dipòsit�de�glicogen.��

�

1.1.2. TEIXIT�ADIPÓS�BLANC�(TAB)�

El�TAB�representa�el�teixit�adipós�majoritari�en�adults�humans�i�comprèn�entre�un�20�25%�del�

pes�corporal,�però�en�casos�d’obesitat�la�quantitat�de�TAB�pot�arribar�a�ser�el�50%�de�la�seva�

massa�corporal.�De�fet,�és� l’únic�teixit�amb�un�potencial�de�creixement� il�limitat�en�qualsevol�

etapa�de�la�nostra�vida,�i�es�localitza�àmpliament�per�tot�l’organisme.�

El�TAB�està�altament�irrigat� i�és�format�majoritàriament�per�adipòcits�en�una�matriu�de�teixit�

connectiu� (formada� per� col�lagen� i� fibres� reticulars),� fibres� nervioses,� cèl�lules�

estromavasculars,� cèl�lules� del� sistema� immune� (macròfags),� fibroblasts� i� preadipòcits�

(adipòcits� no� diferenciats).� Les� seves� funcions� estan� relacionades� amb� el� control� del�

metabolisme� energètic,� tant� de� l’homeòstasi� lipídica,� com� de� la� glucosa,� i� fa� un� parell� de�

dècades� se� li� ha� assignat� a� aquest� teixit� un� paper� important� en� la� regulació� de� processos�

inflamatoris� i�hormonals.�Això�és�possible�gràcies�a�que�els�adipòcits�contenen�diversos�tipus�

de�receptors�de�membrana�que�donaran�resposta�a�diferents�estímuls�externs.�Per�exemple,�

són� rics� en� el� receptor� de� la� insulina� (IR)� i� receptors� adrenèrgics,� també� contenen� receptors�

pels� glucocorticoides,� receptors� per� la� hormona� del� creixement� i� hormones� tiroidees� o�

receptors�per�les��hormones�sexuals.�

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Dependent�de�la�localització�corporal�d’aquest�teixit�adipós,�es�poden�trobar�diferències�en�les�

seves�funcions�secretores.�

�

� Teixit�adipós�perivascular�

Es� troba� envoltant� la� majoria� de� vasos� sanguinis� del� cos,� i� primerament� se� li� va� atribuir�

funcions�mecàniques�de�suport.�Ara�es�coneix�que�aquest�teixit�adipós�expressa�i�secreta�gran�

varietat�de�citocines�i�quimiocines�que�podrien�afectar�la�morfologia�vascular�i�contribuir�així�al�

desenvolupament�de�malalties�vasculars�com� l’arteriosclerosi� (Henrichot� i� col.,�2005).�Així,� la�

hipertròfia�d’aquest�teixit�observada�en�estats�d’obesitat�pot�afectar� la�secreció�d’adipocines�

com�TNF��i�IL�6�i�interferir�amb�la�senyalització�de�la�insulina�en�els�teixits�perifèrics�(Torres�

Leal�i�col.,�2010).�Aquesta�hipertròfia�del�teixit�perivascular�en�obesitat�també�pot�contribuir�a�

la�RI�a�través�d’efectes�vasculars,�promovent�la�infiltració�de�monòcits�i�la�seva�diferenciació�a�

macròfags� que� contribuirien� a� la� producció� de� citocines� en� aquest� teixit.� Aquestes� citocines�

poden�inhibir�la�via�de�senyalització�de�la�insulina�via�fosfatidilinositol�3�cinasa�(PI3�K)�i�l’Akt�en�

les� cèl�lules� de� l’endoteli� causant� una� disminució� de� la� producció� d’òxid� nítric.� Tot� això� pot�

acabar� afectant� la� vasodilatació� de� l’endoteli� i� el� flux� circulatori� d’insulina� cap� al� múscul,�

resultant� en� una� reducció� de� la� captació� de� glucosa� induïda� per� la� insulina� en� aquest� teixit�

(Yudkin�i�col.,�2005).�

�

� Teixit�adipós�subcutani�

Aquest� teixit� es� localitza� a� les� regions� perifèriques� del� cos� com� la� regió� mamària� o�

gluteofemoral.�Metabòlicament�no�és�molt�actiu,�però�s’ha�descrit�que�secreta�leptina�i�àcids�

grassos�lliures�(Wajchenberg,�2000),�raó�per�la�qual�aquest�teixit�s’ha�relacionat�amb�l’aparició�

de�hiperleptinèmia.��

�

� Teixit�adipós�visceral�

Localitzat� més� internament� a� les� regions� mesentèrica� i� epicardial.� Comprèn� el� 20%� del� greix�

corporal�en�homes�i�només�un�6%�en�dones�pre�menopàusiques.�Hi�ha�nombroses�evidències�

que� suggereixen� que� l’acumulació� de� greix� en� el� compartiment� visceral� és� la� que� comporta�

més�risc�metabòlic� (Weyer� i�col.,�2000;�Smith� i�col.,�2001).�La�RI,�precursora�de� la�DM2,�està�

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17

estretament� relacionada� a� la� hipertròfia� en� aquest� compartiment.� Els� dipòsits� viscerals�

secreten� més� citocines� pro�inflamatòries� com� la� IL�6,� la� visfatina� i� la� MCP�1,� que� han� estat�

relacionades�amb�el�desenvolupament�de�la�RI�(Greenberg�i�Obin,�2006;�Madani�i�col.,�2009).��

El�teixit�visceral�epicardial�es�troba�al�llarg�de�les�artèries�coronàries�i�sobre�la�superfície�dels�

ventricles�i�de�l’àpex�del�cor.�S’ha�descrit�que�el�gruix�del�teixit�epicardial�reflexa�la�quantitat�de�

greix� visceral� intra�abdominal� (Iacobellis� i� col.,� 2003).� Així,� en� humans� amb� RI� i� DM2� s’ha�

observat�un�gruix�major�d’aquest�teixit�que�en�individus�sense�aquestes�patologies�(Iacobellis�i�

col.,�2003;�Iacobellis�i�col.,�2008;�Wang�i�col.,�2009).�

�

� Acumulació�ectòpica�de�greix�

Es�defineix�com�el�dipòsit�de�triglicèrids�dintre�de�cèl�lules�de�teixit�no�adipós,�que�normalment�

conté�poca�quantitat�de�greix.�Un�excés�energètic�produeix�l’acumulació�de�l’excés�de�lípids�al�

fetge,�al�múscul�esquelètic�i�al�pàncrees,�indicant�que�el�teixit�adipós�és�incapaç�de�captar�els�

lípids�nutricionals�(Heilbronn�i�col.,�2004).�L’acumulació�de�greix�al�múscul�esquelètic�i�al�fetge�

s’ha� associat� amb� RI,� malaltia� cardiovascular� i� DM2.� Un� cor� carregat� de� lípids� es� desrregula�

metabòlicament�apareixent�RI� i� resultant�en�una�deficient�oxidació�de� la�glucosa�que�genera�

insuficiència�cardíaca�(Sharma�i�col.,�2004).�Malgrat�aquestes�evidències,�encara�no�es�coneix�si�

l’acumulació�ectòpica�de�greix�precedeix�o�succeeix�a�la�RI.�

�

1.2. �EL�TAB�COM�A�ÒRGAN�ENDOCRÍ�

Com�s’ha�esmentat�anteriorment,�uns�vint�anys�enrere�se�li�va�sumar�al�TAB,�a�més�de�la�seva�

capacitat�de�regular�l’homeòstasi�i�el�metabolisme�energètic,�la�capacitat�de�secretar�un�gran�

nombre� de� substàncies� amb� activitat� biològica.� Aquestes� proteïnes� es� van� anomenar�

adipocines,� i� poden� actuar� de� manera� autocrina� i� paracrina� per� regular� el� metabolisme� dels�

propis� adipòcits,� o� de� forma� endocrina� quan� són� secretades� a� la� circulació� sanguínia� i�

produeixen�efectes�sistèmics.�

A�la�Taula�1�apareixen�algunes�de�les�adipocines�i�altres�molècules�produïdes�al�teixit�adipós.�

Totes� aquestes� proteïnes� permeten� al� teixit� adipós� comunicar�se� amb� altres� teixits� i� òrgans.�

Així,�a� través�d’aquesta�xarxa�de�comunicació,�el�TAB�modula� importants�processos�biològics�

com� són� la� ingesta,� el� metabolisme� energètic,� funcions� neuroendocrines� i� immunitàries,�

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angiogènesi,�pressió�sanguínia�i�inflamació�(Kershaw�i�Flier,�2004).�Per�això,�tots�aquells�factors�

que�afectin�als�adipòcits,�ja�siguin�la�hiperplàsia�o�la�hipertròfia�que�apareixen�en�l’obesitat�o�

un� increment� d’àcids� grassos� lliures� o� canvis� positius� o� negatius� en� el� balanç� energètic�

afectaran�també�a�la�secreció�d’adipocines�i,�per�tant,�tindran�efectes�a�nivell�sistèmic�(Bourlier�

i�col.,�2008;�Lago�i�col.,�2007).�

Adipocines� Efectes�sobre Referència�

Leptina� Ingesta,�greix�corporal Zhang�i�col.�1994�

Adiponectina� RI�i�inflamació Cook�i�col.�1985�

Resistina� RI�i�inflamació Steppan�i�col.�2001�

Cholesterol�Ester�Transfer�Protein�(CETP) Metabolisme�de�lípids Dusserre�i�col.�2000�

Lipoproteina�Lipasa�(LPL)� Metabolisme�de�lípids Fried�i�DiGirolamo�1986�

Adipocyte�Fatty�Acid�Binding�Protein�4 (FABP�4) Metabolisme�de�lípids Xu�i�col.�2006�

Retinol�Binding�Protein�4�(RBP�4)� Metabolisme�de�lípids Zovich�i�col.�1992�

Plasminogen�Activator�Inhibitor�1�(PAI�1) Fibrinòlisi Shimomura�i�col.�1996�

Angiotensinogen�(AGT)� Pressió�sanguínia Cassis�i�col.�1988�

TNF�� Inflamació Hotamisligil�i�col.�1993�

IL�6� Inflamació Fried�i�col.�1998�

Proteïna�C�Reactiva�(CRP)� Inflamació Ouchi�i�col.�2003�

Monocyte�Chemoattractant�Protein�1 (MCP�1) Atracció�de�m nòcits Gerhardt�i�col.�2001��

Taula�1.��Exemples�d’algunes�adipocines�secretades�pel�teixit�adipós�amb�efectes�endocrins.�

�

� Obesitat,�inflamació�i�RI�en�TAB�

Com�s’ha�exposat�anteriorment,�la�incidència�de�l’obesitat�ha�augmentat�en�els�darrers�30�anys�

degut�principalment�a�canvis�en�la�dieta�i�a�un�increment�del�sedentarisme.�Particularment,�un�

excés�d’acumulació�de�greix�visceral�comporta�un�increment�del�risc�per�desenvolupar�DM2�i�

RI,�entre�d’altres�patologies�(Trayhurn�i�col.,�2006;�Fonseca�Alaniz�i�col.,�2007).�L’acumulació�de�

greixos�durant�el�desenvolupament�d’obesitat�es�caracteritza�per�la�hiperplàsia�(increment�del�

nombre� d’adipòcits� causat� per� la� diferenciació� dels� preadipòcits� a� adipòcits� madurs)� i� per� la�

hipertròfia� dels� adipòcits� (increment� en� la� mida� dels� adipòcits� causat� per� un� excessiu�

emmagatzematge� de� lípids).� Aquests� canvis� en� el� teixit� adipós� s’associen� a� l’augment� de�

l’angiogènesi,�a�l’increment�de�la�infiltració�de�macròfags,�a�la�producció�de�components�de�la�

matriu� extracel�lular,� a� l’activació� de� cèl�lules� endotelials� i� a� la� producció� i� alliberament� de�

diversos� mediadors� inflamatoris� (Lago� i� col.,� 2007;� Bourlier� i� col.,� 2008).� Les� citocines� pro�

inflamatòries� secretades� pel� teixit� adipós� provenen� dels� adipòcits� i� també� dels� macròfags�

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infiltrats�al�TAB.�La�desregulació�en�la�funció�i�producció�d’aquestes�citocines,�com�el�TNF��i�la�

IL�6,�en�individus�obesos�serien�en�gran�part�causants�de�la�RI�i�la�DM2�associades�a�obesitat.�

�

� TNF��

La�primera�vegada�que�es�va�descriure�el�TNF��com�un�mediador�que�relacionava�obesitat,�la�

inflamació�i�la�RI�va�ser�en�un�estudi�desenvolupat�per�Hotamisligil�i�col.�(1993),�en�el�qual�van�

observar�que�l’expressió�de�TNF��era�més�alta�en�diferents�models�de�ratolins�obesos.�A�més,�

si� es� bloquejava� l’efecte� del� TNF�� la� sensibilitat� a� la� insulina� millorava� (Hotamisligil� i� col.,�

1993).� Després� s’han� realitzat� nombrosos� experiments� que� confirmen� la� correlació� entre�

l’increment� del� TNF��i� l’aparició� de� RI� en� individus� obesos,� i� també� s’ha� descrit� que� una�

pèrdua�de�pes�s’acompanya�de�la�disminució�d’aquesta�adipocina�(Dandona�i�col.,�1998;�Maury�

i� Brichard,� 2010).� Tots� aquests� resultats� suggereixen� que� l’efecte� negatiu� del� TNF��sobre�

l’acció� de� la� insulina.� Un� dels� mecanismes� pels� quals� es� creu� que� el� TNF��podria� causar� RI�

podria�ser�la�fosforilació�en�serina�del�substrat�del�receptor�de�la�insulina�1�(IRS1)�a�través�de�

JNK� (stress�activated� protein� kinase/Jun�amino�terminal� kinase� SAPK/JNK),� la� qual� podria�

inhibir� la� fosforilació� normal� en� el� residu� tirosina� d’IRS1,� causant� així� una� reducció� de�

l’activació�de�la�cascada�de�senyalització�de�la�insulina�(Rui�i�col.,�2001).�

Al� TAB,� el� TNF�� redueix� l’expressió� de� PPAR�,� de� la� lipoproteïna� lipasa� (LPL)� i� de�GLUT�4,�

resultant�en�la�disminució�de�la�captació�de�glucosa�(Guilherme�i�col.,�2008).�En�cèl�lules�3T3�L1�

diferenciades�s’ha�observat�que�la�incubació�amb�TNF��provoca�un�increment�de�l’expressió�

gènica�de�la�IL�6�de�manera�dosi�i�temps�dependent,�raó�per�la�qual�part�dels�efectes�del�TNF�

��han� estat� atribuïts,� almenys� en� part,� a� una� alliberació� simultània� de� la� IL�6� (Rotter� i� col.,�

2003).�

�

� IL�6�

Aquesta� citocina� és� secretada� per� una� gran� varietat� de� cèl�lules:� endotelials,� queratinòcits,�

osteoblasts,� miòcits,� adipòcits,� cèl�lules�� pancreàtiques,� monòcits,� macròfags,� i� nombrosos�

teixits� i� fins� i� tot� tumors.� La� IL�6� és� fonamental� en� la� reducció� del� procés� inflamatori� agut�

perquè� promou� la� síntesis� de� citocines� anti�inflamatòries� i� regula� negativament� dianes�

inflamatòries� (Steensberg� i� col.,� 2003;� Xing� i� col.,� 1998).� Per� això,� aquesta� citocina� s’ha�

classificat�com��anti�inflamatòria�en�processos�aguts�i�pro�inflamatòria�en�processos�crònics.�

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El� TAB�produeix�el� 10�35%� de� la� IL�6� circulant� basal� en� individus� sans�en�estat� de� repòs.� Les�

concentracions� plasmàtiques� d’aquesta� adipocina� augmenten� amb� l’obesitat� i,� tal� i� com�

succeeix�amb�el�TNF��,�els�nivells�de� la� IL�6�disminueixen�amb� la�pèrdua�de�pes.�Nombrosos�

estudis� revelen�una�clara� relació�entre�nivells�elevats� d’IL�6� i� la� presència� de� RI� (Guilherme� i�

col.,�2008;�Iacobellis�i�col.,�2003;�Pickup�i�col.,�1997;�Fernandez�Real�i�col.,�2001;�Pradhan�i�col.,�

2001).�També�ha�estat�descrit�que�les�concentracions�elevades�d’IL�6�són�predictores�de�patir�

DM2�(Pedersen�i�Febbraio,�2007).�

Durant� la� última� dècada� s’han� demostrat� efectes� inhibidors� de� la� IL�6� sobre� l’acció� de� la�

insulina�al�fetge�i�al�TAB.�Aquesta�adipocina�redueix�la�síntesi�de�glicogen�hepàtica�dependent�

d’insulina�(Klover�i�col.,�2003;�Senn�i�col.,�2002)�i�la�captació�de�glucosa�en�adipòcits�(Coppack,�

2001;� Rotter� i� col.,� 2003).� Aquests� efectes� podrien� produir�se� a� través� de� l’increment� de� la�

proteïna� supressora� de� la� senyalització� de� citocines� (SOCS3),� que� s’uneix� al� receptor� de� la�

insulina�i�promou�la�degradació�proteasomal�d’IRS1�(Sabio�i�col.,�2008;�Rotter�i�col.,�2003).�

Més�endavant,�en�altres�apartats,�s’ampliarà� la� informació�sobre�aquestes�dues�citocines� i�el�

seu�paper�en�relació�a�l’obesitat�i�l’aparició�de�RI.�

�

1.3. �VIA�DE�SENYALITZACIÓ�DE�LA�INSULINA�AL�TEIXIT�ADIPÓS�

La� insulina�és� la�hormona�més�important�en�la�regulació�de�les�concentracions�de�glucosa�en�

sang,�i�és�essencial�en�l’estat�post�prandial,�moment�en�que�les�concentracions�sanguínies�de�

glucosa� augmenten� i� la� insulina� és� secretada� per� les� cèl�lules� ��pancreàtiques.� La� insulina�

estimula�la�captació�de�glucosa�al�teixit�adipós�i�al�múscul,�i�promou�el�seu�emmagatzematge�

en�forma�de�triglicèrids�i�de�glicogen,�respectivament.�A�més,�la�insulina�inhibeix�la�producció�

de�glucosa�hepàtica�(gluconeogènesi�i�glicogenòlisi).�D’aquesta�manera�es�mantenen�els�nivells�

de� glucosa� en� un� rang� fisiològic� bastant� estret,� entre� 90�110� mg/dL,� en� persones� sanes�

(Shulman,�2000).�Però�aviat�en� la�DM2,� les�concentracions�de�glucosa�plasmàtica�augmenten�

malgrat� la�presència�d’elevats�nivells�d’insulina,�aquest�fet�és�degut�a�que�el�múscul,�el� teixit�

adipós�i�el�fetge�han�desenvolupat�RI�(Khan�i�Pessin,�2002).�

La�reducció�dels�nivells�de�glucosa�circulant�depenen�de�la�translocació,�estimulada�per�insulina�

(Abel� i� col.,� 2001)� del� GLUT�4� a� la� membrana� cel�lular.� Encara� que� el� teixit� adipós� és� el�

responsable�només�de�la�captació�d’una�petita�porció�de�glucosa�dependent�de�la�insulina,�els�

ratolins� knockout� selectius� en� greix� per� al� gen� Glut�4� presenten� un� empitjorament� de� la�

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tolerància� a� la� glucosa,� fet� que� suggereix� que� el� bon� funcionament� d’aquest� teixit� és�

fonamental�en�el�control�de�la�glucèmia�(Abel�i�col.,�2001).�La�distribució�d’aquest�GLUT�4�a�la�

membrana� plasmàtica� està� sota� el� control� de� la� via� de� senyalització� de� la� fosfatidilinositol� 3�

cinasa�(PI3K)�i�l’Akt�(Smith�i�col.,�1991;�Saltiel,�2001)�(Figura�1).�

INS

IRS1P

p85 p110

PI3K

PIP2 PIP3 PDK

Akt

VesículaGLUT�4

GLUT�4

Glucosa

�

Figura� 1.� Esquema� de� la� via� de� senyalització� de� la� insulina.� La� unió� de� la� insulina� al� seu� receptor� provoca� una�cascada�de�fosforilacions�que�acabaran�amb�la�translocació�de�GLUT�4�a�la�membrana,�permetent�així�l’entrada�de�glucosa�a�la�cèl�lula.�INS:�insulina;�IRS1:�substrat�del�receptor�de�la�insulina�1;�PI3K:�fosfatidilinositol�3’�cinasa;�PIP2:�fosfoinositol� difosfat;� PIP3:� fosfoinositol� trifosfat;� PDK:�phosphoinositide�dependent� kinase�1;� Akt:� (PKB),� proteïna�cinasa�B;�GLUT�4:�transportador�de�glucosa�4.�

�

1.2.1. VIA�DE�SENYALITZACIÓ�PI3K/AKT�

� Receptor�de�la�inuslina�(IR)�

La�senyalització�de�la�insulina�comença�amb�la�unió�d’aquesta�al�seu�receptor�de�membrana,�IR�

(insulin�receptor).�Aquest�receptor�és�una�glicoproteïna�heterotetramèrica��que�pertany�a�

la�família�dels�receptors�amb�activitat� intrínseca�tirosin�cinasa,�RTK�(Hubbard�i�Till,�2000).�Les�

dos�subunitats��estan�situades�a� la�part�extracel�lular� i�contenen�el� lloc�d’unió�a� la� insulina,�

mentre�que�les�dos�subunitats��es�troben�a�la�porció�intracel�lular�i�contenen�l’activitat�tirosin�

cinasa� (DeFronzo� i� col.,� 1992).� Quan� la� insulina� s’uneix� a� les� subunitats� ��té� lloc�

l’autofosforilació� de� la� subunitat� ��a� diferents� residus� de� tirosina� (Lee� i� col.,� 1993;� De� i�

Whittaker,� 2002).� Un� cop� fosforilat� el� IR� recluta� diferents� substrats� intracel�lulars� entre� els�

quals�es�troben�els�substrats�del�receptor�de�la�insulina�IRS1�i�IRS2.�

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� IRS�

La� família� de� substrats� del� receptor� de� la� insulina� (IRS),� dels� quals� IRS1� i� IRS2� són� els� més�

implicats�en�el�metabolisme�de�la�glucosa�(Cai�i�col.,�2003),�també�regula�processos�de�síntesis�

proteica,�diferenciació,�creixement�i�supervivència�cel�lular.�

IRS1� i� IRS2� interaccionen� amb�el� IR� fosforilat�a� través�del� seu� domini�d’unió�a� fosfotirosines,�

PTB,�a�l’extrem�amino�terminal.�A�més,�a�l’extrem�carboxi�terminal,�també�contenen�diversos�

residus�de�tirosina�susceptibles�de�ser�fosforilats�que�seran�els�responsables�de�la�transmissió�

de�la�senyalitació�intracel�lular�de�la�insulina�(Thirone�i�col.,�2006;�White,�2002)�(Figura�2).�En�

unir�se�al�IR�la�proteïna�IRS1�és�fosforilada�per�aquest�mateix�receptor�en�diversos�residus�de�

tirosines,�que�seran�llocs�d’anclatge�per�a�proteïnes�amb�dominis�SH2�(Src�homology�2),�entre�

les�quals�s’inclou�la�subunitat�p85�de�la�PI3K.�

�

Figura� 2.� Estructura� en�dominis� de� IRS1.� S’indiquen� cinases� i� fosfatases� que� poden� actuar� sobre� la� regulació� de�l’activitat�d’aquest�proteïna�i�per�tant�que�controlen�la�via�de�senyalització�de�la� insulina.�Cal�destactar�els�factors�que�fosforilen�en�la�posició�serina�307,�els�quals�poden�jugar�un�paper�important�en�l’aparició�de�la�RI.�

�

IRS1� té� un� pes� molecular� de� 132� KDa,� i� la� seva� activitat� es� regula� a� través� de� serin�treonin�

cinases,� defosforilacions� mitjançant� fosfatases� i� la� seva� pròpica� degradació,� la� qual� sembla�

promoure� la�RI�a� llarg�termini.�També�s’ha�observat�que� les� fosforilacion�en� la�serina�307�en�

ratolí� (serina� 312� en� humans)� impedeixen� la� unió� de� la� IRS1� al� IR� i,� per� tant,� eviten� la� seva�

activació� i,� conseqüentment,� inhibeixen� la� via� de� senyalització� de� la� insulina� (Aguirre� i� col.,�

2002).�La�citocina�proinflamatòria�TNF��,�a�través�de�JNK,�és�un�dels�factors�que�promouen�la�

fosforilació�en�aquest�residu�i�és�per�això�que�s’ha�establert�una�relació�entre�l’aparició�de�RI�i�

la�inflamació�(Hotamisligil,�2006).�

�

� PI3K�

La� PI3K� es� troba� al� citosol� en� forma� d’heterodímer� amb� la� subunitat� reguladora� p85� i� la�

subunitat� catalítica� p110� (Fruman� i� col.,� 1998).� La� subunitat� p85� conté� dominis� SH2� capaços�

d’unir�se�a�la�IRS1�fosforilada�(Sun�i�col.,�1993)�i�d’aquesta�manera�apropar�a�la�subunitat�p110�

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a� la� membrana� plasmàtica� on� serà� capaç� de� fosforilar� el� seu� substrat,� PIP2� (fosfatidilinositol�

4,5�bifosfat)� i� convertir�lo� en� PIP3� (fosfatidilinositol� 3,4,5�trifosfat)� que� té� efectes� sobre� la�

supervivència� i� el� creixement� cel�lular� i� el� metabolisme� de� la� glucosa� (Cengel� i� col.,� 1998).�

Aquests� fosfoinositols� fosforilats� recluten� i� activen� altres� proteïnes� com� la� PDK�1�

(phosphoinositide�dependent�kinase�1)�i�l’Akt,�també�coneguda�com�proteïna�cinasa�B�(PKB).�Al�

seu�torn�la�PDK�1�fosforila�i�activa�altres�proteïnes�incloent�l’Akt�i�la�proteïna�cinasa�C�/��(PKC�

�/�)�(Sampson�i�Cooper,�2006).�

�

� Akt�

La�família�de�l’Akt�està�formada�per�tres�isoformes�Akt�1�(PKB��),�Akt�2�(PKB��)�i�Akt�3�(PKB��).�

D’aquestes,�l’Akt�2�és�la�que�s’expressa�a�tots�els�teixits�sensibles�a�la�insulina�(Kitamura�i�col.,�

1998).�Aquestes�serin�treonin�cinases�tenen�un�domini�PH�(pleckstrin�homology)�d’alta�afinitat�

pel� PIP3� que� provoca� que� transloquin� a� la� membrana� (Bellacosa� i� col.,� 1998).� Allà� són�

fosforilades�per�PDK�1�i�PDK�2�a�la�treonina�308�i�a�la�serina�473,�respectivament�(Welsh�i�col.,�

2005).� La� fosforilació� en� treonina� 308� és� necessària� per� l’activació� de� l’Akt� mentre� que� la�

fosforilació�en�serina�473�li�dóna�la�màxima�activació�(Downward,�1998).�Un��cop�activada�l’Akt�

pot�regular�la�captació�de�glucosa�promovent�la�translocació�de�GLUT�4�a�la�membrana,�regular�

la�supervivència�cel�lular,�la�síntesis�de�glicogen,�de�lípids�i�de�proteïnes�així�com�la�transcripció�

de�gens�(Zdychova�i�Komers,�2005).��

�

� PKC�

La�família�de�les�PKCs�està�composta�per�12�membres�d’aquestes�serin�treonin�cinases�que�es�

divideixen�en�tres�subgrups�segons�la�seva�afinitat�pel�calci�i�els�lípids.�Les�PKCs�clàssiques�(PKC�

�,��I,��II� i� �)� són� activades� per� calci� i� DAG.� Les� PKCs� noves� (PKC��)� que� són�

independents�de�calci�però�necessiten�DAG�per�a�ser�activades.�I�finalment,�les�PKCs�atípiques�

(PKC��)�que�no�necessiten�ni� calci�ni�DAG�per� ser�activades.�En�estat� inactiu� les�PKCs�es�

troben� al� citoplasma� i� un� cop� actives� migren� a� la� membrana� plasmàtica,� al� nucli� o� a� altres�

orgànuls� cel�lulars� on� duran� a� terme� la� seva� acció� (Sampson� i� Cooper,� 2006).� La� localització�

cel�lular�d’aquestes�proteïnes�ve�determinada�per�la�fosforilació�de�tres�llocs�molt�conservats�

del�domini�catalític�(Cenni�i�col.,�2002).�

INTRODUCCIÓ

24

Als�principals�teixits�diana�de�l’acció�de�la�insulina�com�són�el�teixit�adipós,�el�múscul�esquelètic�

i�el�fetge�s’expressen�les�isoformes��,��I,��II�de�les�PKCs�clàssiques�i�les�isoformes�� i��de�les�

atípiques�i�és�per�això�que�són�aquestes�isoformes�les�que�s’han�relacionat�amb�la�RI�(Sampson�

i�Cooper,�2006).�

�

� Transportadors�de�la�glucosa�

Una�de�les�funcions�més�importants�de�la�insulina�és�controlar�l’homeòstasi�de�la�glucosa,�que�

ha�de�mantenir�les�seves�concentracions�plasmàtiques�dintre�d’uns�marges�molt�estrets.�Per�tal�

d’assolir�aquest�objectiu�existeixen�tres�processos�com�són� la�captació�de�glucosa�pels�teixits�

perifèrics,� la� producció� de� glucosa� al� fetge� i� la� secreció� d’insulina� per� part� de� les� cèl�lules��

pancreàtiques.�Per�a�que�la�glucosa�sigui�captada�pels�teixits�perifèrics�existeixen�dos�tipus�de�

transportadors�que�afavoreixen�el�pas�d’aquesta�molècula�a�l’interior�cel�lular.�Per�una�banda,�

es�pot�parlar�dels�transportadors�dependents�de�sodi,�SGLT,�localitzats�a�l’intestí�i�al�ronyó�que�

transporten� sodi� i� glucosa� en� contra� del� gradient� de� concentracions.� D’altra� banda,� els�

transportadors� de� la� família� GLUT� transporten� la� glucosa� a� favor� del� gradient� de�

concentracions�(Shepherd�i�Kahn,�1999).�

Dels�transportadors�de�la�família�GLUT�s’han�descrit�13�membres�agrupats�en�tres�classes.�La�

classe� I� (GLUT1�4)� transporta� glucosa,� la� classe� II� (GLUT5,� 7,� 9� i� 11)� transporta� fructosa� i� la�

classe� III� (GLUT6,� 8,� 10,� 12� i� HMIT1)� és� poc� coneguda� fins� al� moment� (Bryant� i� col.,� 2002).�

D’aquests,� el� GLUT4� és� el� tranportador� de� glucosa� dependent� d’insulina� més� important� i�

majoritari�al�teixit�adipós�(Mueckler,�2001).�

En� absència� d’estímul,� GLUT4� es� troba� a� l’interior� cel�lular� en� diferents� compartiments� com�

l’aparell�de� Golgi,� vesícules� recobertes�de� clatrina� i� endosomes� (Rea� i� James,�1997),� però�on�

s’emmagatzema� majoritàriament� és� a� les� vesícules� d’emmagatzematge� de� GLUT4� les� GSV�

(GLUT4� storage� vesicles)� situades� al� citoplasma� molt� properes� a� la� membrana� plasmàtica�

(Watson� i� Pessin,� 2001).� L’estímul� de� la� insulina� provoca� l’exocitosi� d’aquestes� vesícules�

provocant�un�increment�dels�nivells�de�GLUT4�a�la�superfície�de�la�membrana�plasmàtica�i,�per�

tant,�un� increment�de� la�captació�de� la�glucosa�(Watson� i�Pessin,�2001).�Per�a�que�tingui� lloc�

aquesta�exocitosi�és� fonamental� l’activació�de� la�PI3K� i� la�generació�de�PIP3� (Shepherd� i� col.,�

1998)� que� desencadena� una� cascada� de� fosforilacions� que� acaben� en� l’activació� de� l’Akt,� la�

qual� juga� un� paper� important� en� la� translocació� de� GLUT4� a� la� membrana� (Toker� i� Newton,�

2000;�Balendran�i�col.,�1999;�Kotani�i�col.,�1998).�

INTRODUCCIÓ

25

La� translocació� de� GLUT4� a� la� membrana� és� el� primer� pas� limitant� per� a� la� utilització� de� la�

glucosa� al� teixit� adipós� (Fink� i� col.,� 1992).� Per� tant,� les� alteracions� en� aquest� sistema� de�

transport�degudes�a�processos�que�impliquen�RI�són�claus�per�al�desenvolupament�de�la�DM2.�

�

1.3.2. RESISTÈNCIA�A�LA�INSULINA�

La�RI�apareix�quan�el�múscul�i�el�teixit�adipós�són�incapaços�de�captar�la�glucosa�plasmàtica,�i�el�

fetge�és�incapaç�de�suprimir� la�gluconeogènesi� i� la�glicogenòlisi�produïnt�un�increment�de�les�

concentracions�plasmàtiques�de�glucosa�malgrat�els�nivells�normals�o�elevats�d’insulina�(Usui�i�

Tobe,�2011).�

El� teixit� adipós� és� un� dels� majors� responsables� del� control� de� l’homeòstasi� de� la� glucosa�

(Kershaw�i�Flier,�2004)�i,�a�més,�com�s’ha�vist�anteriorment,�és�un�òrgan�secretor�de�diverses�

adipocines�pro�inflamatòries�que�promouen� l’aparició�de� la�RI.�Aquestes�adipocines�actuen�a�

diferents�nivells�sobre�la�regulació�de�la�via�de�senyalització�de�la�insulina.�Per�exemple,�la�IL�6�

provoca� la� disminució� de� la� captació� de� glucosa� en� adipòcits� (Rotter� i� col.,� 2003),� i� estimula�

l’expressió�de�SOCS3�que�al�seu�torn�evita�la�la�fosforilació�d’IRS1�i�promou�la�seva�degradació�

proteasomal� (Emanuelli� i� col.,� 2000;� Kamura� i� col.,� 1998;� Rui� i� col.,� 2002).� El� TNF��fosforila�

IRS1�en�la�Ser307,�inhibint�la�seva�unió�amb�IR�(Rui�i�col.,�2001)�(Figura�3).�

INS

IRS1p85 p110

PI3K

PIP2 PIP3 PDK

Akt

VesículaGLUT�4

GLUT�4

Glucosa

IL�6

P

TNF�SOCS3

PROTEASOMA

�

Figura�3.�Els� factors�pro�inflamatoris�col�laboren�en� l’aparició�de�RI.�La�via�de�senyalització� de� la� insulina�es�pot�veure�afectada�per�l’efecte�de�diversos�factors�pro�inflamatoris�a�diversos�nivells�de�la�cascada�de�senyalització.�INS:�insulina;�IRS1:�substrat�del�receptor�de�la�insulina�1;�PI3K:�fosfatidilinositol�3’�cinasa;�PIP2:�fosfatidilinositol�difosfat;�PIP3:� fosfatidilinositol� trifosfat;� PDK:�phosphoinositide�dependent� kinase�1;� Akt:� (PKB),� proteïna� cinasa� B;� GLUT�4:�transportador�de�glucosa�4;�IL�6:�interleucina�6;�TNF��factor�de�necrosis�tumoral��;�SOCS3:�proteïna�supressora�de�la�senyalització�de�citocines.�

INTRODUCCIÓ

26

Tots�aquests�aspectes�que�involucren�factors�pro�inflamatoris�en�l’aparició�de�la�RI�es�tractaran�

detalladament� al� llarg� d’aquesta� memòria.� Però� en� general,� es� podria� dir� que� el� control� de�

l’estat� inflamatori�crònic�que�vincula� l’obesitat�amb�l’aparició�de�RI� i�DM2,�s’ha�postulat�com�

una�diana�farmacològica�crucial�en�la�prevenció�de�l’aparició�de�RI.�

�

�

�

�

�

�

�

�

�

�

�

�

�

�

�

�

�

�

INTRODUCCIÓ

27

��

�

�

�

�

�

�

�

�

�

�

2. RECEPTORS� ACTIVATS� PER� PROLIFERADORS�PEROXISÒMICS�

�

Els� receptors� activats� per� proliferadors� peroxisòmics� (PPARs)� es� van� descriure� als� anys� 90� i�

pertanyen� a� la� superfamília� dels� receptors� nuclears� hormonals.� Els� PPARs� són� factors� de�

transcripció�que�tenen�com�a�lligands�compostos�que�indueixen�la�proliferació�del�peroxisoma,�

com�els�àcids�grassos�lliures�provinents�de�la�dieta�i�els�seus�derivats,�els�quals�serviran�com�a�

sensors�de� lípids� i�podran�control�lar�el�metabolisme� (Evans� i� col.,�2004).�També�s’ha�descrit�

que� alguns� eicosanoids,� i� alguns� fàrmacs� hipolipemiants� i� antidiabètics� s’uneixen� a� aquests�

receptors�(Willson�i�Wahli,�1997).�

D’aquesta�subfamília�s’ha�descrit�tres�subtipus,�el�PPAR��(segons�la�nomenclatura�unificada�de�

receptors�nuclears,�NR1C1)�(Issemann�i�Green,�1990),�el�PPAR��(NR1C2)�i�el�PPAR�� (NR1C3)�

(Dreyer� i� col.,� 1992).� Tots� els� subtipus� tenen� una� estructura� molecular� comuna,� però� una�

INTRODUCCIÓ

28

distribució�tissular�específica�de�cadascun.�Estan�codificats�per�gens�independents�i�situats�en�

cromosomes� diferents� (Desvergne� i� Wahli,� 1999).� Els� PPARs� participen� en� la� regulació� de� la�

transcripció�dels�seus�gens�diana,�els�quals�poden�estar�implicats�en�gran�varietat�de�processos�

metabòlics� com� el� metabolisme� dels� lípids,� el� balanç� energètic,� la� sensibilitat� a� la� insulina� o�

l’homeòstasi� de� la� glucosa,� així� com� també� intervenen� en� la� regulació� de� processos�

inflamatoris�(Chawla�i�col.,�2001;�Glass�i�Ogawa,�2006;�Willson�i�Wahli,�1997).��

�

2.1. ESTRUCTURA�DELS�PPARs�

Els� PPARs� presenten� una� estructura� compartida� amb� la� superfamília� dels� receptors� nuclears�

hormonals�a� la�qual� pertanyen� (Blanquart� i� col.,�2003).�Aquesta� estructura�consta�de�quatre�

dominis� funcionals� independents,� però� que� interactuen� entre� ells� (Figura� 4).� A� la� regió� NH2�

terminal�es�troba�el�domini�A/B,�que�inclou�la�regió�AF�1�(activation�function�1),�responsable�

de�l’activitat�de�transactivació�independent�de�lligand�del�receptor.�El�domini�C�o�domini�DBD�

(DNA�binding�domain)�és�el�més�conservat�i�està�format�per�dos�dits�de�zinc�que�permeten�la�

unió�dels�PPARs�als�elements�de�resposta�a�proliferadors�peroxisòmics�(PPRE).�El�domini�D�està�

implicat� en� la� interacció� dels� receptors� amb� cofactors� que� regulen� la� seva� activitat�

transcripcional.�Finalment,�el�domini�E�o�domini�LBD�(ligand�binding�domain)�es� localitza�a� la�

regió�C�terminal�i�conté�la�regió�AF�2�(activation�function�2),�que�requereix�la�unió�del�lligand�

per� induir� l’activació� transcripcional.� La� diferència� en� la� seqüència� d’aminoàcids� d’aquest�

domini�és�la�responsable�de�la�selectivitat�pels�diferents�lligands�(Gehin�i�col.,�1999).�

�

Figura�4.�Estructura�en�dominis�dels�PPARs.�

�

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2.2. MECANISMES�D’ACCIÓ�DELS�PPARs�

Els� PPARs� són� receptors� nuclears� que� poden� regular� la� transcripció� gènica� mitjançant� dos�

mecanismes�principals,�la�trans�activació�i�la�trans�repressió.�

�

2.2.1. TRANS�ACTIVACIÓ�

A�través�d’aquest�mecanisme,�els�PPARs�funcionen�com�a�factors�de�transcripció�unint�se�als�

seus� elements� de� resposta� situats� als� promotors� dels� gens� diana,� els� PPRE,� que� són� una�

repetició� directa� de� la� seqüència� AGGTCA� separada� per� un� nucleòtid� (DR�1,�Direct�Repeat�1)�

(Kliewer� i�col.,�1992).�Per�a�poder�unir�se�als�PPRE,�els�PPARs�han�de�formar�un�heterodímer�

amb�un�altre�receptor�nuclear,�el�receptor�de�l’àcid�9�cis�retinoic�(RXR�ó�NR2B)�donant�lloc�al�

complex� PPAR:RXR.� De� fet,� els� PPARs� només� poden� unir�se� a� l’ADN� en� aquesta� forma�

heterodimèrica,� ja�que�no�actuen�mai�com�a�homodímers�o�monòmers.�La�unió�amb�el�PPRE�

sempre� té� lloc� de� la� mateixa� manera,� el� PPAR� interactua� amb� la� seqüència� hexanucleotídica�

upstream�i�l’element�RXR�ho�fa�amb�la�seqüència�downstream�[Desvergne�i�Wahli,�1999b].�

En�la�trans�activació�els�PPARs�actuen�com�a�factors�de�transcripció�dependents�de�lligands.�La�

interacció�amb�el�lligand�del�complex�PPAR:RXR�indueix�canvis�conformacionals�que�provoquen�

l’alliberament�d’aquest�heterodímer�del�seu�co�repressor�de�manera�que�pot�unir�se�al�PPRE.�

Degut� a� aquests� canvis� conformacionals� apareixe� noves� superfícies� de� contacte� proteïna�

proteïna,� que� permetren� la� unió� específica� amb� co�activadors� [Desvergne� i� Wahli,� 1999c].�

L’estimulació� del� promotor� és� màxima� quan� ambdós� membres� de� l’heterodímer� s’uneixen�

simultàniament�amb�els�seus�lligands�(Keller�i�col.,�1993;�Kersten�i�col.,�2000)�(Figura�5).�

PPRE (DR-1)

5’

3’

AACT AGGTCA n AGGTCA

TTGA AGGTCA n AGGTCA 5’

3’gen diana

ÀC.�GRASSOS ÀC.�9�CIS�RETINOIC

NUCLI

PPAR RXR

PPAR RXR TRANSCRIPCIÓ

�

Figura�5.�Mecanisme�de�trans�activació.�Els�PPARs�s’uneixen�a� l’ADN�en�forma�d’heterodímers.�La�unió�dels�seus�lligands�provoca�l’alliberament�del�complex�PPAR:RXR�del�seu�co�repressor�i�l’activació�de�l’activitat�transcripcional.�

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30

2.2.2. TRANS�REPRESSIÓ�

Els�PPARs�poden� interferir�amb�altres� factors�de� transcripció�d’una� forma� independent�de� la�

unió�a� l’ADN,�mitjançant�un�mecanisme�anomenat�trans�repressió.�Aquest�mecanisme�és,�en�

molts�casos,�el�responsable�dels�efectes�antiinflamatoris�dels�PPARs�(Blanquart�i�col.,�2003).�A�

través�d’aquest�mecanisme�es�suprimeix�l’activitat�de�diversos�factors�de�transcripció�com�NF�

�B,� STAT� (Signal� Transducer� and� Activator� of� Transcription)� i� AP�1� (Activator� Protein�1)�

(Delerive�i�col.,�1999;�Zhou�i�Waxman,�1999).�S’ha�demostrat�que�la�inhibició�de�l’activitat�de�

NF��B� és� un� mecanisme� a� través� del� qual� els� agonistes� PPAR�� milloren� la� sensibilitat� a� la�

insulina�in�vivo�i�que�l’NF��B�adipocitari�és�una�diana�terapèutica�potencial�per�a�l’obesitat�i�la�

DM2�(Ruan� i� col.,�2003).�Actualment�es�coneixen� tres�mecanismes�de� trans�repressió� (Figura�

6).�

�

� Segrest�de�co�activadors�

Aquest�mecanisme�suposa�una�competició�per��quantitats�limitades�de�co�activadors�essencials�

que� són� compartits� pels� complexes� PPAR:RXR� i� altres� factors� de� transcripció.� Així,� l’activació�

del�complex�PPAR:RXR�utilitzarà�aquests�co�activadors�que� llavors�no�estaran�disponibles�per�

altres�factors�de�transcripció�que�els�necessitin,�suprimint�la�seva�activació�(Li�i�col.,�2000).�

�

� Antagonisme�mutu�de�receptors�

Els� heterodímers� PPAR:RXR� tenen� la� capacitat� d’interactuar� físicament� amb� altres� factors� de�

transcripció�com�NF��B,�STAT,�AP�1�o�NFAT�(Nuclear�Factor�of�Activated�T�cells).�Aquesta�unió�

evita�que�aquests�altres�factors�de�transcripció�puguin�unir�se�als�seus�elements�de�resposta�i�

no�puguin�dur�a�terme�la�transcripció�dels�seus�gens�diana�(Delerive�i�col.,�1999).��

�

� Inhibició�de�la�cascada�de�la�MAPK�

Els� PPARs� poden� inhibir� la� fosforilació� i� l’activació� de� certs� membres� de� la� cascada� de�

senyalització� de� les� MAPK� (mitogen�activated� protein� kinase),� evitant� així� l’activació� d’altres�

factors�de�transcripció�(Desreumaux�i�col.,�2001;�Daynes�i�Jones,�2002).�

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CBPSRC1

1.�SEGREST�DE�CO�ACTIVADORS

FT:�AP1,�NFAT,�STAT,�NF��BCBP:�cAMP REB�Binding protein

SRC1:�Steroid receptor�coactivatorMAPK:�Mitogen�activated protein kinase

Gen diana MCP1 Gen diana

2.�ANTAGONISME�MUTU�DE�RECEPTORS

3.�INHIBICIÓ DE�LA�MAPK

MAPK

PPAR RXR PPAR RXR

NF��Bp65

NF��Bp65

CBPSRC1

PPAR RXR

FT FT

�

Figura� 6.� Mecanisme� de� trans�repressió.� Els� PPARs� poden� regular� l’activitat� d’altres� factors� de� transcripció�independentment�de�la�unió�a�l’ADN�a�través�de�tres�mecanismes�de�tran�repressió.�

�

L’activitat� dels� PPARs� no� tan� sols� es� veu� afectada� pels� mecanismes� vistos� fins� ara� de� trans�

activació�i�trans�repressió�si�no�també�per�processos�de�fosforilació�i�ubiquitinització.�

�

� Fosforilació�

Determinats� factors� extracel�lulars� poden� modificar� l’estat� de� fosforilació� de� les� proteïnes�

cel�lulars.� La� fosforilació� dels�PPARs�és� un� dels�mecanismes�que� determinen� la� seva�activitat�

transcripcional.� Per� exemple,� la� fosforilació� de� PPAR�� augmenta� en� resposta� a� la� insulina� i�

aquest�procés�es�correspon�amb�un�increment�de�la�seva�activitat�transcripcional�[Shalev�i�col.,�

1996;� Juge�Aubry� i� col.,� 1999].� Aquesta� fosforilació� es� produeix� per� la� via� de� les� MAPK� i�

incrementa�l’activitat�de�la�regió�AF�1�de�PPAR� �

En� el� cas� de� PPAR��les� fosforilacions� que� rep� per� part� de� la� PKA,� de� les� MAPK,� de� l’EGF�

(epidermal� growth� factor)� o� el� PDGF� (platelet�derived� growth� factor)� poden� tenir� efectes�

positius� o� negatius� sobre� la� seva� activitat� transcripcional� [Zhang� i� col.,� 1996a;� Adams� i� col.,�

1997;�Camp�i�Tafuri,�1997]�

�

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� Ubiquitinització�

El� sistema� de� degradació� proteasomal� per� ubiquitinització� està� implicat� en� la� regulació� de�

diverses�proteïnes�de�curta�vida� involucrades�en� funcions�cel�lulars�essencials�com�el� control��

del�cicle�cel�lular,� la�regulació�de�la�transcripció�o�la�transducció�de�senyals�(Mimnaugh�i�col.,�

1999).�A�les�proteïnes�degradades�per�aquest�sistema�se’ls�uneix�covalentment�la�ubiquitina�en�

residus� de� lisina.� És� llavors� quan� les� proteïnes� multi�ubiquitinitzades� són� ràpidament�

degradades�per�la�subunitat�26S�del�proteasoma.�Aquest�procés�de�degradació�el�pateixen�els�

PPARs�com�a��mecanisme�de�regulació�de�la�seva�activitat�transcripcional,�i�sembla�ser�que�en�

el� cas� de� PPAR�� la� unió� del� lligand� desencadenaria� aquest� procés,� mentre� que� en� el� cas� de�

PPAR��la� unió� del� lligand� seria� protectora� en� front� de� la� degradació� per� ubiquitinització�

(Hodges�i�col.,�1998;�Hauser�i�col.,�2000;�Blanquart�i�col.,�2002).�

�

2.3. PPAR��

2.3.1. DISTRIBUCIÓ�TISSULAR�

El� subtipus� PPAR��va� ser� el� que� primer� que� es� va� descriure� (Issemann� i� Green,� 1990).� La�

distribució� tissular� d’aquest� subtipus� de� PPAR� es� correlaciona� amb� teixits� amb� una� elevada�

taxa�mitocondrial,�és�a�dir,�amb�elevada�activitat�catabòlica,�tal� i�com�ho�demostren�els�seus�

nivells�d’expressió�gènica�elevats�en�cardiomiòcits,�cèl�lules�dels�túbuls�proximals�del�ronyó,�i�al�

fetge.� També� es� troben� nivells� elevats� de� PPAR�� en� la� mucosa� de� l’estòmac,� el� duodè,� la�

retina,� glàndules� adrenals,� múscul� esquelètic� i� illots� pancreàtics� (Braissant� i� col.,� 1996;�

Lemberger�i�col.,�1996).��

�

2.3.2. LLIGANDS�DE�PPAR��

Des�del�moment�en�què�PPAR��va�ser�descrit�es�va�observar�que�era�el�receptor�de�diversos�

proliferadors�peroxisòmics�i,�de�fet,�per�aquesta�raó�va�rebre�aquest�nom�(Issemann�i�Green,�

1990).� En� estudis� amb� ratolins� knockout� per� a� aquest� receptor� es� va� observar� que� el�

tractament� amb� clofibrat� i� Wy14,643� no� tenia� cap� efecte� sobre� aquests� ratolins� (Lee� i� col.,�

1995).�A�més,�altres�estudis�posteriors�van�demostrar�que�aquests�ratolins�sense�PPAR��tenien�

nivells� de� colesterol� basals� més� elevats� i� esdevenien� obesos� amb� l’edat� (Peters� i� col.,� 1997;�

INTRODUCCIÓ

33

Costet�i�col.,�1998).�Aquestes�dades�suggerien�que�aquest�receptor�nuclear�mediava�els�efectes�

hipolipemiants�dels�fibrats�i�altres�proliferadors�peroxisòmics.��

Els� fibrats,�com�el� fenofibrat�o�el�bezafibrat,� són� fàrmacs�utilitzats�per� reduïr�els� triglicèrids� i�

disminuir�el�risc�de�malaltia�cardiovascular.�També�són�capaços�d’augmentar�el�colesterol�HDL�

(high�density� lipoprotein)� i�de� reduir�el�colesterol�LDL� (low�density� lipoprotein).�El� fet�que�els�

fibrats�fossin�poc�selectius�per�PPAR��respecte�els�altres�dos�subtipus�de�PPARs�(Brown�i�col.,�

1999),�va�fer�que�es�sintetitzessin�compostos�anàlegs�com�els�ureido�fibrats,�que�presentaven�

major�especificitat �Aquests�ureido�fibrats�van�mostrar�que�també�eren�capaços�de�prevenir�la�

hiperinsulinèmia�en�models�murins�de�RI�(Guerre�Millo�i�col.,�2000).�

Els� lligands� naturals� d’aquest� subtipus� de� PPAR� són� els� àcids� grassos� insaturats� com� l’àcid�

araquidònic,�linoleic�i�oleic,�i�àcids�grassos�saturats�com�l’àcid�palmític�(Berger�i�Moller,�2002)�o�

l’àcid�8(S)�hidroxieicosatetraenoic�(8(S)�HETE)�(Kliewer�i�col.,�1997).�

�

2.3.3. FUNCIONS�DEL�PPAR��

� Efectes�sobre�el�metabolisme�lipídic�

L’activació�de�PPAR��té�efectes�sobre�el�metabolisme�dels�àcids�grassos,�tant�regulant�la�seva�

captació�com�la�seva��oxidació�als�peroxisomes�i�al�mitocondri.�De�fet,�els�primers�gens�diana�

descrits�per�a�aquest�PPAR�codificaven�per�enzims�perixosomals�involucrats�en�la�via�de�la��

oxidació,� com� la� acil�CoA� oxidasa� (ACO).� Aquesta� ��oxidació� mitocondrial� contribueix� a� la�

producció� d’energia� a� través� de� fosforilacions� oxidatives� que� generen� ATP.� PPAR��indueix� la�

transcripció� del� gen� codificant� per� la� carnitina�palmitoil� transferasa�1� (CPT�1),� el� qual� és� un�

dels�components�crítics�dels�sistema�de�transport�dependent�de�carnitina.�Aquest�sistema�de�

transport�és�considerat�el�primer�pas�limitant�de�la��oxidació�mitocondrial,�controlant�el�flux�

dels� àcids� grassos� a� l’interior� del� mitocondri� (Mascaro� i� col.,� 1998;� Yu� i� col.,� 1998).� A� més,�

PPAR��regula�aquest�procés�metabòlic�mitjançant�el�control�de�l’expressió�del�gen�de�la�acil�

CoA�deshidrogenasa�de�cadena�mitja�(MCAD)�(Gulick�i�col.,�1994).�

També� són� regulats� per� aquest� factor� de� transcripció� els� gens� codificants� per� les� proteïnes�

FATP� (fatty� acid� transport� protein)� i� FAT� (fatty� acid� translocase),� responsables� del� transport�

dels�àcids�grassos�a�través�de� la�membrana,�o�gens� implicats�en� la�síntesi�de�novo�dels�àcids�

grassos�com�l’ACC�(acetyl�CoA�Carboxilase),�la�FAS�(fatty�acid�sinthase)�i�la�SCD�1�(steaoryl�CoA�

desaturase�1)�(Jump,�2011).�

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� Efectes�sobre�el�metabolisme�de�la�glucosa�i�la�RI�

Tal�com�s’ha�esmentat�anteriorment,�en�un�model�de�ratolí�amb�RI�l’activació�del�PPAR��amb�

compostos� sintètics,� com� els� ureido� fibrats,� va� demostrar� � que� prevenia� la� hiperinsulinèmia�

(Guerre�Millo�i�col.,�2000).�En�aquests�estudis,�l’activació�de�PPAR��disminuia�els�elevats�nivells�

de�glucosa�plasmàtica�i�les�concentracions�d’insulina,�incrementant�l’acció�de�la�insulina�sobre�

la�captació�de�glucosa.�

En�altres�experiments�amb�ratolins�knockout�per�a�aquest�factor�de�transcripció�es�va�observar�

que�aquests�presentaven�hipoglucèmia�en�dejú�deguda�a�una�disminució�de�la�gluconeogènesi�

hepàtica�i�de�la��oxidació�hepàtica�(Im�i�col.,�2011;�Kersten�i�col.,�2000).��

D’altra� banda� ha� estat� descrit� que� l’acumulació� de� triglicèrids� a� múscul� s’associa� amb� la� RI� i�

l’obesitat.� L’activació� de� PPAR��pels� seus� lligands� podria� disminuir� el� contingut� plasmàtic� de�

triglicèrids,� raó� per� la� qual� disminuirien� els� triglicèrids� acumulats� al� múscul� i� s’atenuaria�

d’aquesta�manera� la�RI.�També�ha�estat�descrit�que�l’activació�d’aquesta� isoforma�inhibeix� la�

senyalització�de�NF��B,�disminuint�la�producció�de�citocines�com�la�IL�6�i�el�TNF��(Staels�i�col.,�

1998;�Madej�i�col.,�1998).�

�

2.4. PPAR��


�En�humans�s’expressen�tres�isoformes�de�PPAR�:�PPAR�1,�PPAR�2�i�PPAR�3.�(Fajas�i�col.,�1998;�

Houseknecht� i� col.,�2002).�El�PPAR��és�el� subtipus� majoritari� al� teixit� adipós� (Tontonoz� i� col.,�

1994a),�i�és�menys�abundant�al�ronyó,�al�fetge,�a�l’intestí,�a�la�retina,�al�múscul�esquelètic�i�al�

cor��(Saez�i�col.,�1998;�Braissant�i�col.,�1996;�Braissant�i�Wahli,�1998).�

�


El� subtipus� PPAR��a� l’igual� que� els� altres� dos� subtipus,� té� com� a� lligands� naturals� els� àcids�

grassos� poliinsaturats� com� l’àcid� linoleic,� araquidònic� i� eicosapentanoic� (Xu� i� col.,� 2001),� un�

derivat�de�la�prostaglandia�D2�(15�deoxi��12,14�PGJ2)�(Bell�Parikh�i�col.,�2003)�i�els�metabolits�

oxidats�derivats�de�l’àcid�linoleic�presents�en�les�LDL,�l’àcid�9�hidroxioctadecadienoic�(9�HODE)�

i�el�13�HODE�(Nagy�i�col.,�1998;�Waku�i�col.,�2009).�

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35

Per�una�altra�banda,�dels�lligands�sintètics�cal�destacar�les�tiazolidindiones�(TZD)�o�glitazones,�

que�són�fàrmacs�antidiabètics�(Sohda�i�col.,�1982).�Aquestes�molècules�disminueixen�els�nivells�

de�glucosa�en�models�de�ratolins�resistents�a�la�insulina,�sense�afectar�a�la�secreció�d’insulina.�

Per� aquesta� raó� se’ls� anomena� sensibilitzadors� a� la� insulina.� D’aquests� compostos,� la�

pioglitazona�s’utilitza�actualment�per�al�tractament�de�la�DM2.�

�


� Diferenciació�dels�adipòcits�

La� seva� implicació� en� el� procés� de� diferenciació� dels� adipòcits� ha� estat� recolzada� per�

nombroses�evidències.�Per�exemple,�en�cèl�lules�3T3�L1� i�3T3�F442A� l’expressió�de�PPAR��en�

l’estat�de�preadipòcits�gairebé�és�nul�la,�mentre�que�augmenta�en�el�procés�de�diferenciació.�A�

més,� l’activació� d’aquest� PPAR� promou� la� conversió� de� preadipòcits� a� adipòcits� (Tontonoz� i�

col.,� 1994a;� Tontonoz� i� col.,� 1994b).� PPAR��seria� l’efector� final� de� la� cascada� transcripcional�

que�inclou�membres�de�la�família�del�factor�transcripcional�C/EBP�(Wu�i�col.,�1999;�Mandrup�i�

Lane,�1997).�

�

� Metabolisme�lipídic�

A�més�de�controlar� la�diferenciació�dels�adipòcits,�PPAR�� també�s’encarrega�de�mantenir� les�

seves� funcions� bàsiques� com� l’emmagatzematge� de� lípids� al� TAB� i� la� dissipació� d’energia� al�

TAM.� D’aquesta� manera,� els� gens� que� controla� aquest� factor� de� transcripció� codifiquen� per�

enzims�implicats�en�l’alliberament�d’àcids�grassos,�com�la�LPL�(lipoprotein�lipase)�(Schoonjans�i�

col.,�1996;�Frohnert�i�col.,�1999),�i�per�proteïnes�implicades�en�el�transport�d’aquests�greixos�a�

l’interior�dels�adipòcits,�com�la�FAT�i�la�FATP�(Frohnert�i�col.,�1999).�L’activació�de�PPAR��també�

incrementa�els�nivells�de�la�FABP�o�de�l’acil�CoA�sintasa.�

La�síntesi�d’àcids�grassos�i�triglicèrids�també�és�regulada�per�l’activació�induïda�per�PPAR��del�

gen� de� l’enzim� màlic� (Castelein� i� col.,� 1994),� i� del� gen� de� la� PEPCK� (phosphoenolpyruvate�

carboxykinase),�involucrat��en�la�producció�de�glicerol�per�emmagatzemar�els�àcids�grassos�en�

forma�de�triglicèrids�(Devine�i�col.,�1999).�

�

INTRODUCCIÓ

36


Les�TZD�són�lligands�de�PPAR��amb�efectes�antidiabètics,�que�es�comporten�com�a�compostos�

sensibilitzadors�a�la�insulina.�Fins�ara�s’han�considerat�dos�mecanismes�pels�quals�aquest�factor�

de� transcripció� pot� dur� a� terme� els� seus� efectes� antidiabètics.Per� una� banda,� com� s’ha� vist,�

l’activació� de� PPAR��al� teixit� adipós� promou� la� captació� i� l’emmagatzematge� de� lípids.�

D’aquesta� manera� es� redueix� la� seva� lipotoxicitat� a� nivell� muscular� i� hepàtic� i� es� millora� la�

sensibilitat�a� la� insulina� (Yamauchi� i�col.,�2001a).�Per�una�altra�banda,� l’activació�de�PPAR��al�

teixit�adipós�disminueix�la�secreció�d’adipocines�que�produeixen�RI�com�el�TNF��i�la�resistina,�i�

incrementa�la�producció�d’adiponectina,�que�promou�l’oxidació�dels�àcids�grassos�i�afavoreix�la�

sensibilitat�a�la�insulina�al�múscul�i�al�fetge�(Peraldi�i�col.,�1997;�Yamauchi�i�col.,�2001b).�

�

2.5. PPAR��

La�doble�nomenclatura�de�PPAR��es�deu�a�que�va�ser�descrit�primer�en�Xenopus�per�Dreyer�i�

col.�(1992)�i�el�van�anomenar�PPAR�.�Posteriorment,�va�ser�identificat�en�humans,�en�ratolins�i�

rates,� rebent� el� nom� de� PPAR�� ja� que� la� seqüència� proteica� presentava� algunes� diferències�

amb�la�de�Xenopus�(Schmidt�i�col.,�1992;�Kliewer�i�col.,�1994;�Mukherjee�i�col.,�1994).�Més�tard,�

es� va� observar� que� ambdós� gens� tenien� el� mateix� origen� evolutiu� i� les� mateixes� funcions�

biològiques,� per� això� ara� aquest� receptor� se’l� coneix� habitualment� amb� al� nom� de�

PPAR��(Takada�i�col.,�2000).�

�


Aquest�subtipus�de�PPAR�és�el�que�presenta�una�localització�més�ubíqua�per�tot�l’organisme.�La�

seva�expressió�és�més�elevada�a�cor,�múscul�esquelètic,�teixit�adipós,�cèl�lules� inflamatòries� i�

pell�(Wagner�i�Wagner,�2010).�

�


S’han�descrit�molts�lligands�nuclears�sintètics�per�al�receptor�nuclear�PPAR��.�Aquest�fet�pot�

ser�degut�a�que� la�cavitat�d’unió�al�domini�LBD�presenta�unes�dimensions�més�grans�que�els�

altres�dos�subtipus�de�PPAR�(Takada�i�col.,�2000;�Xu�i�col.,�2001).�

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37

Com� a� lligands� endògens� del� PPAR��s’han� descrit� els� àcids� grassos,� els� eicosanoides�

(especialment�la�prostaciclina)�i�l’àcid�retinoic.�Alguns�dels�compostos�sintètics�que�actuen�com�

a� lligands� d’aquest� PPAR� són� el� GW0742,� GW2433,� GW9578,� L�782483,� L�165041� o� el�

GW501516.� També� s’han� desenvolupat� compostos� que� actuen� com� antagonistes� del�

PPAR��com� el� fàrmac� antiinflamatori� no� esteroideu� sulindac� o� el� GSK0660� (Bishop�Bailey� i�

Wray,�2003;�Wagner�i�Wagner,�2010).�

�


� Metabolisme�lipídic�

PPAR��juga� un� paper� important� en� la� regulació� de� l’homeòstasi� energètica� mitjançant� la�

inducció� de� gens� implicats� en� el� catabolisme� dels� àcids� grassos� i� la� termogènesis� adaptativa�

(Peters�i�col.,�2000;�Barak�i�col.,�2002).�

En� ratolins�db/db,� un� model� ben� establert� per� l’estudi� de� la� diabetis,� es� va� observar� que� el�

tractament�amb�el� lligand�de�PPAR��GW501516�produïa�un� increment�de� l’HDL�colesterol� i�

una�disminució�dels�nivells�de�triglicèrids�(Lee�i�col.,�2006).�En�aquest�cas�el�teixit�que�més�va�

respondre�al�tractament�va�ser�el�fetge,�on�es�va�trobar�incrementada�l’expressió�de�gens�que�

codificaven�per�proteïnes�involucrades�en�la�síntesi�d’àcids�grassos�i�en�la�seva�elongació,�com�

l’ACC��(acetyl�CoA�carboxylase),� la� FAS� (fatty�acid� synthase)� o� la� GPAT� (glycerol�3�phosphate�

acyltransferase),�així�com�gens�implicats�en�el�transport�d’àcids�grassos�com�el�CD36�o�el�que�

codificava�per�la�PLTP�(phospholipid�transfer�protein).�En�aquest�estudi�també�es�va�examinar�

el�catabolisme�lipídic�al�múscul�després�del�tractament�amb�el�GW501516,�observant�se�que�la�

��oxidació�estava� incrementada,� fet�que�concordava�amb� l’augment�de� l’expressió�de�CPT�1.�

Altres� estudis� realitzats� en� micos� obesos� i� en� humans� tractats� amb� GW501516� han� mostrat�

resultats�similars�als�obtinguts�en�ratolins�diabètics�(Oliver,�Jr.�i�col.,�2001;�Riserus�i�col.,�2008).�

�


En� el� mateix� estudi� de� Lee� i� col.� (2006),� el� tractament� de� ratolins�db/db� amb� GW501516� va�

afectar� l’expressió� de� gens� implicats� en� el� metabolisme� de� la� glucosa� com� la� PEPCK,� que�

codifica�per�un�enzim�limitant�per�la�gluconeogènesi,�que�va�veure�disminuïda�la�seva�expressió�

al�fetge.�A�més,�el�tractament�també�va�suprimir�l’acció�de�la�Ppp1r3C�(protein�phosphatase�1�

INTRODUCCIÓ

38

regulatory� subunit),� inhibint� la� glicogenòlisi.� Per� altra� banda,� al� fetge,� la� via� de� les� pentoses�

fosfat� es� va� afavorir� per� què� el� tractament� incrementava� la� PGD� (phophogluconate�

dehydrogenase)�i�la�G�6�PDH�(glucose�6�phosphat�dehydrogenase).�Tots�aquests�canvis�induïts�

per�l’activació�de�PPAR��van�produir�una�disminució�de�la�producció�de�glucosa�hepàtica�i�un�

increment�del�seu�catabolisme�(Lee�i�col.,�2006).��

Aquests�resultats�suggereixen�que�PPAR��podria�exercir�efectes�sensibilitzadors�a�la�insulina�

a�través�de�l’augment�de�la�lipogènesi� i�de�la�glicòlisi�al�fetge.�I�per�altra�banda,� induiria�la��

oxidació�al�múscul�per�tal�de�reduir�l’excés�de�càrrega�d’àcids�grassos.�

Altres� resultats� que� recolzaven� una� acció� sensibilitzadora� dels� efectes� de� la� insulina� per�

activació� de� PPAR�� es� van� obtenir� al� nostre� grup� de� recerca,� que� va� demostrar� que� el�

GW501516� evitava� la� inflamació� i� la� RI� induïdes� per� palmitat� en� cèl�lules� musculars�

esquelètiques.�El� tractament�amb� l’agonista�de�PPAR��revertia� la� reducció�de� la��oxidació�

causada�per�palmitat�a�través�del�increment�de�PDK4�(pyruvate�dehydrogenase�4)�i�de�CPT�1.�A�

més,�l’activació�de�PPAR��en�aquestes�cèl�lules�també�disminuïa�l’activitat�d’unió�a�l’ADN�de�

NF��B�induïda�per�palmitat�així�com�l’expressió�d’IL�6,�gen�diana�de�NF��B�(Coll�i�col.,�2010),�i�

reconeguda�citocina�implicada�en�l’aparició�de�RI.��

Altres�experiments�en�adipòcits�3T3�L1,�també�realitzats�en�el�nostre�grup�d’investigació,�han�

demostrat� que� l’activació� de� PPAR��amb� GW501516� evita� la� secreció� i� l’expressió� d’IL�6�

induïda� per� un� estímul� pro�inflamatori� com� el� lipopolisacàrid� (LPS).� Aquests� efectes� eren�

conseqüència�de�la�inhibició�per�part�del�GW501516�de�l’activació�de�NF��B�(Rodriguez�Calvo�i�

col.,�2008),�la�qual�requeria�la�inducció�de�la�via�ERK1/2�(extracellular�signal�related�kinase).�

Una�altra�citocina�pro�inflamatòria�implicada�en�l’aparició�de�RI,�el�TNF��,�també�es�afectat�per�

l’activació� de� PPAR��.� S’ha� demostrat� que� PPAR��,� a� través� de� TIMP�3� (metalloproteinase�

inhibitor�3),� �és�capaç�de�bloquejar�la�migració�dels�macròfags�a�la�placa�d’ateroma�i�d’inhibir�

TNF��post�transcripcionalment�(Rosenson�i�col.,�2002).�

En� resum,� totes� aquestes� dades� suggereixen� que� PPAR��presenta� efectes� que� milloren� la�

sensibilitat�a�la�insulina�a�través�del�control�de�la�producció�i�de�l’eliminació�dels�àcids�grassos�i�

de�la�glucosa�al�fetge.�A�més,�al�múscul�també�regula,�en�part,� la�producció�de�citocines�pro�

inflamatòries�com�la�IL�6�i�el�TNF��

INTRODUCCIÓ

39

�

�

�

�

�

��

�

�

�

��

3. FACTOR�NUCLEAR��B�

�

En� casos� d’obesitat� s’ha� observat� una� alteració� de� la� regulació� de� la� producció� d’adipocines�

pro��i�antiinflamatòries�al�teixit�adipós�que�juga�un�paper�important�en�l’aparició�de�RI,�DM2�i�

altres� complicacions� associades� a� l’obesitat.� En� aquest� sentit� s’ha� descrit� un� increment� dels�

nivells� plasmàtics� de� TNF�� i� IL�6� procedents� en� gran� part� del� teixit� adipòs� en� situacions�

d’obesitat�(Hotamisligil�i�col.,�1993).�

El�factor�de�transcripció�NF��B�és�un�regulador�primordial�de�la�resposta�inflamatòria,�i�juga�un�

paper� crític� en� gran� diversitat� de� processos� patològics� (Barnes� i� Karin,� 1997).� En� diferents�

models�murins�d’obesitat� s’ha�demostrat�que�NF��B�s’activa�en� teixits� sensibles�a� la� insulina�

com�el�múscul�esquelètic�i�el�fetge�(Cai�i�col.,�2005;�Arkan�i�col.,�2005),�i�podria�estar�implicat�

en�l’aparició�de�RI�en�aquests�teixits.��

Un� dels� efectes� antiinflamatoris� de� PPAR��es� produeix� per� la� inhibició� de� NF��B,� però� els�

mecanismes� implicats� són� encara� poc� coneguts.� En� cèl�lules� musculars� esquelètiques�

estimulades� amb� palmitat,� el� tractament� amb� l’agonista� de� PPAR��GW501516� disminuïa�

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40

l’activitat�d’unió�a� l’ADN�de�NF��B�induïda�per�palmitat�així�com�l’expressió�d’IL�6�(Coll� i�col.,�

2010).� En� teixit� adipós� humà� existeixen� estudis� que� demostren� que� la� inhibició� de� NF��B�

suprimeix� la�producció�de�citocines�pro�inflamatòries�(Lappas� i�col.,�2005).�Posteriorment,�en�

adipòcits� 3T3�L1� ha� estat� descrit� que� l’activació� de� PPAR�� evita� l’activació� de� NF��B�

mitjançant�la�inhibició�de�la�ERK1/2�(Rodriguez�Calvo�i�col.,�2008).�

Malgrat� aquestes� dades,� els� mecanismes� que� connecten� la� inflamació� crònica� de� baixa�

intensitat�amb�el�desenvolupament�de�RI�induïda�per�obesitat�només�es�coneixen�parcialment.�

Per� això� és� important� trobar� els� mecanismes� pels� quals� noves� dianes� farmacològiques,� com�

PPAR�/��puguin�explicar�la�capacitat�d’aquest�receptor�per�a�prevenir�l’aparició�de�RI�induïda�

per�obesitat.�

�

3.1. FAMÍLIA�I�ESTRUCTURA�DE�NF��B�

La� família� de� NF��B� s’expressa� ubíquament� per� tot� l’organisme� de� la� qual� s’han� descrit� cinc�

membres:� c�Rel,� NF��B1� (p50/p105),� NF��B2� (p52/p100),� RelA� (p65)� i� RelB.� Cadascuna�

d’aquestes� proteïnes� conté� un�domini� d’homologia� Rel� (RHD)� que� s’encarrega� de� la� unió� a�

l’ADN,� de� la�dimerització� i� de� la� interacció� amb� les� proteïnes� I�B� (Siebenlist� i� col.,�1994).� Els�

membres�NF��B1�(p105)�i�NF��B2�(p100)�contenen�a�la�regió�C�terminal�múltiples�còpies�de�la�

repetició�anquirina,� formada� per� la� repetició�de� 33� aminoàcids� en� tàndem� que� permeten� la�

interacció�proteïna�proteïna�(Figura�7).�

�

Figura�7.�Estructura�de�la�família�NF��B/Rel.�Es�caracteritza�per�que�contenen�un�domini�RHD�i�els�membres�NF��B1�i�NF��B2�contenen�múltiples�còpies�de�la�repetició�d’anquirina.��

�

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D’acord� amb� la� seva� activitat� transcripcional,� la� família� NF��B/Rel� conté� també� dominis�

d’activació� transcripcional� (Blair� i� col.,� 1994;� Schmitz� i� col.,� 1994).� A� més,� p65� i� c�Rel�

interaccionen� amb� la� TATA�binding� protein� (TBP).� Estudis� in� vitro� i� in� vivo� indiquen� que�

diferents� dímers� de� NF��B� tenen� diferents� propietats� transcripcionals� (Lin� i� col.,� 1995).�

L’habilitat�de�diferents�dímers�per�reconèixer�dianes�d’ADN�lleugerament�diferents�augmenta��

la�capacitat� de� les� subunitats�de� NF��B�per� regular�diferencialment� l’expressió�gènica.�Altres�

diferències� entre� els� dímers� de� NF��B� són� l’especificitat� pel� tipus� cel�lular,� una� localització�

subcel�lular� diferencial,� interaccions� específiques� amb� diferents� tipus� de� proteines� I�B�

(inhibidors� de� �B)� i� diferents� formes� d’activació� (Baeuerle� i� Henkel,� 1994;� Siebenlist� i� col.,�

1994).��

�

3.2. REGULACIÓ�DE�NF��B�

3.2.1. COMPLEX�IKK�

NF��B�en�la�seva�forma�inactiva�es�localitza�al�citoplasma�interaccionant�amb�molècules�de�la�

família� I�B� que� emmascaren� les� senyals� de� localització� nuclear� (NLS)� d’aquest� factor� de�

transcripció.� En� resposta� a� múltiples� estímuls� com� citocines� inflamatòries,� productes� virals� o�

bacterians,� o� altres� estímuls� d’estrès,�aquestes� proteïnes� I�B� es� fosforilen� en� dos� residus� de�

serina.� Aquesta� modificació� permet� la� seva� poliubiquitinització� i� la� seva� destrucció� al�

proteasoma.�Com�a�conseqüència,�els�heterodímers�de�NF��B,�com�p50/p65,�queden�lliures�i�

poden� translocar� al� nucli� per� activar� la� transcripció� dels� seus� gens� diana� implicats� en� la�

resposta� inflamatòria� i� immune,� en� l’adhesió� cel�lular,� en� el� control� del� creixement� i� en� la�

protecció�en�front�de�l’apoptosi�(Hoffmann�i�Baltimore,�2006).�

Les� cinases� responsables� de� la� fosforilació� dels� inhibidors� I�B� són� la� IKK�,� IKK��(Chen� i� col.,�

1996;�DiDonato�i�col.,�1997)�i�la�IKK�/NEMO�(Yamaoka�i�col.,�1998).�IKK��i��IKK��presenten�una�

estructura� similar� que� inclou� un� domini� cinasa� amino�terminal,� un� domini� hèlix�loop�hèlix�

(HLH)�que�controla�l’activitat�cinasa�d’IKK,�una�cremallera�de�leucines�(LZ,� leucine�zipper)�que�

permet� la� homo�� o� heterodimerització� de� les� cinases� i,� finalment,� a� l’extrem� C�terminal� una�

regió�d’uns�40�aminoàcids�necessària�per�la�interacció�amb�NEMO�(Figura�8).�

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�

Figura� 8.� Subunitats� IKK�� i� IKK� �A� la� figura� s’indiquen� els� dominis� de� les� subunitats� IKK�� i� IKK� � Es� mostra� un�domini�ubiquitin�like,�que�sembla�estar�involucrat�en�l’activitat�catalítica�d’IKK� �

�

Per�a�ser�actives,�IKK�� i�IKK��necessiten�ser�fosforilades�a�dos�residus�de�serina�(Ser177�i�181�

per� la� IKK��i� Ser176� i� 180� per� la� IKK�)� localitzats� al� loop� d’activació.� Aquesta� fosforilació�

probablement� produeix� un� canvi� conformacional� que� comporta� l’activació� de� les� cinases.� Es�

creu�que�TAK1�podria�comportar�se�com�una�IKKK�(cinasa�d’IKKs)�en�resposta�a�alguns�estímuls�

(Ninomiya�Tsuji� i� col.,� 1999),� fet� que� li� permetria� fosforilar� IKK��al� loop� d’activació,� així� com�

participar�com�a�cinasa�de�la�via�JNK.�

Una�altra�via�d’activació�d’aquestes� IKK�és� l’activació�de�NF��B�per�certes�proteïnes�virals,� la�

qual�no�requeriria�l’activació�d’una�via�de�cinases�upstream�sinó�que�actuaria�a�través�de�canvis�

conformacionals�o�oligomeritzacions�dependents�d’estímuls�i�de�la�presència�de�NEMO�(Israel,�

2010).�

A�més,� l’activació�de� les� IKKs�no�depèn�sempre�de� la�seva� fosforilació.� �El�2001� (Senftleben� i�

col.,�2001)�van�descriure� una�via�d’activació�que� sembla� estar� involucrada�en� la� resposta� als�

lligands� BAFF,� CD40� i� LT�� i� associada� a� la� organogènesi� limfoide.� Aquesta� via� no� requereix�

IKK��ni� NEMO,� només� depèn� d’IKK�� i� de� NIK� (NF��B� inducing� kinase),� una� cinasa�upstream�

d’aquesta� IKK� (Park� i� col.,� 2005).� L’activació� d’aquesta� via� produeix� � la� degradació� parcial� al�

proteasoma�de�la�subunitat�p100�de�NF��B�que�resulta�en�p52,�que�llavors�s’associa�amb�relB.��

Altres�substrats�del�complex�IKK�són�els�components�de�la�cascada�de�senyalització�de�NF��B�

tals�com�NEMO,�p65,�c�rel�o�Bcl10,�o�bé�molècules�que�no�tenen�relació�directa� �amb�NF��B�

com�IRS1�(Nakamori�i�col.,�2006),�implicada�en�l’aparició�de�RI�induïda�per�l’activació�per�TNF�

��del�complex�IKK.�

�

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3.2.2. UBIQUITINITZACIÓ�AL�PROTEASOMA�

El� complex� IKK� és� l’encarregat� de� fosforilar� les� proteïnes� I�B� per� a� ser� posteriorment�

poliubiquitinitzades� i�degradades�al�proteasoma.�Aquesta� fosforilació�permet�que�el�complex�

ligasa�d’ubiquitines�SCF�TrCP�(Skp�1/Cul/Fbox)�reconegui�les�I�B�com�a�dianes�i�les�marqui�per�a�

ser�degradades.�El�mateix�procés�té�lloc�quan�la�subunitat�p100�és�fosforilada�per�NIK.�Aquesta�

fosforilació�fa�que�el�complex�ligasa�d’ubiquitines�SCF�TrCP�la�reconegui�i�la�ubiquitinitzi�per�a�ser�

processada�al�proteasoma,�donant�com�a�resultat�la�subunitat�p52�que�és�transcripcionalment�

competent� juntament� amb� RelB� (Vallabhapurapu� i� Karin,� 2009).� La� ubiquitinització� és� un�

mecanisme� crític� per� regular� l’activitat� de� NF��B� dintre� de� les� diferents� cascades� d’activació�

d’aquest.�

La�ubiquitina�és�una�proteïna�de�76�aminoàcids�amb�set�residus�de�lisina,�i�qualsevol�d’ells�pot�

participar� en� la� formació� de� la� cadena� de� poliubiquitines.� Aquesta� proteïna� quan� s’uneix�

covalentment� amb� les� seves� proteïnes� diana� promou� canvis� en� la� seva� vida� mitja,� la� seva�

localització�o�la�seva�funció.�Una�cascada�enzimàtica�composta�per�tres�proteïnes�s’encarrega�

de�la�unió�de�la�ubiquitina�a�les�seves�proteïnes�substrat�(Figura�9).�La�proteïna�E1,�és�l’enzim�

activador� d’ubiquitina,� l’E2,� l’enzim� que� conjuga� la� ubiquitina,� i� la� proteïna� E3,� la� ligasa�

d’ubiquitines.� Múltiples� seqüències� d’ubiquitinització� generen� cadenes� de� poliubiquitina.� Es�

poden� formar� diferents� tipus� de� cadenes� d’ubiquitina� dependent� de� quin� residu� de� lisina�

s’utilitzi�per� la�poliubiquitinització.�Segons�quina�sigui� la�cadena�d’ubiquitines�podrà�dirigir�se�

cap� a� la� degradació� proteasomal� o� facilitar� la� unió� de� complexes� de� senyalització� (Pickart� i�

Fushman,�2004;�Ikeda�i�Dikic,�2008).�

També� existeixen� cicles� de� desubiquitinització� de� NF��B� duts� a� terme� per� les� proteïnes� DUB�

(deubiquitinases)� (Figura� 9),� considerades� també� reguladores� importants� de� les� vies� de�

senyalització�de�NF��B�(Lee�i�col.,�2000).�Aquests�cicles�d’ubiquitinització�i�desubiquitinització,�

cadascun�regulat�per�enzims�diferents,�controlen�processos�essencials�per�al�manteniment�de�

l’homeòstasi�cel�lular.�

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�

Figura�9.�Processos�d’ubiquitinització�i�desubiquitinització.�La�ubiquitinització�pot�dirigir�cap�a�la�degradació�al�proteasoma�o�pot�facilitar�la�unió�de�complexes�proteics�importants�per�a�la�senyalització�cel�lular.�

�

3.2.3. ACETILACIÓ�I�DESACETILACIÓ�

L’acetilació� de� proteïnes� afecta� molts� processos� cel�lulars� incloent� diversos� aspectes� de� la�

regulació� transcripcional� a� través� del� reclutament� d’enzims� desacetilases� (HDACs,� histone�

deacetylases)� i� acetiltransferases� (HATs,� histone� acetyl� transferase).� En� cèl�lules� eucariotes,�

l’empaquetament�de�l’ADN�en�forma�de�cromatina�interfereix�amb�l’accessibilitat�dels�factors�

de� transcripció.� L’acetilació� de� residus�específics� de� lisines� a� les� cues� amino�terminals� de� les�

histones� nucleosomals� provoca� la� disrupció� de� la� cromatina� i� l’activació� transcripcional� de�

gens.� De� fet,� diversos� co�activadors� transcripcionals� com� CBP/p300� ((cyclic� AMP� response�

element)� CREB�binding� protein),� PCAF� (CBP/p300�associated� factor)� i� SRC�1,� tenen� activitat�

acetiltransferasa�i�alguns�co�repressors�tenen�activitat�desacetilasa�(Roth�i�col.,�2001;�Khochbin�

i�col.,�2001).�A�més,�aquesta�acetilació�reversible�s’ha�identificat�en�proteïnes�no�histones�que�

inclouen� diferents� factors� de� transcripció.� Dependent� del� domini� modificat,� l’acetilació� pot�

regular�diferents� funcions�d’aquests� factors�de� transcripció,� com�el� reconeixement� de� l’ADN,�

l’estabilitat�de�la�proteïna,�interaccions�proteïna�proteïna�i�la�localització�subcel�lular�(Sterner�i�

Berger,�2000;�Bannister�i�Miska,�2000).�

�

� CBP/p300�

L’activitat� transcripcional� de� NF��B� requereix� co�activadors� que� posseeixen� activitat� HAT�

(CBP/p300,� P/CAF� i� SRC�1/NcoA�1)� i� HDAC� (Roth� i� col.,� 2001;� Ashburner� i� col.,� 2001).�

L’heterodímer�de�NF��B�més�estudiat�és�el�p50/p65,� i�ambdues�subunitats�poden�acetilar�se�

en�diferents�residus�de�lisina.�L’acetilació�en�un�residu�o�un�altre�regula�diferents�funcions�de�

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NF��B,� com� l’activació� transcripcional,� l’afinitat� per� la� unió� a� l’ADN� i� la� unió� amb� I�B�.� El�

reclutament� de� p300� i� PCAF� a� la� regió� promotora� dels� gens� diana� de� NF��B� resulta� en�

l’activació�de�la�transcripció�a�través�de�la�remodelació�de�la�cromatina�i�en�l’acetilació�de�p65�

en� les� lisines� 221� i� 310,� permetent�li� la� unió� als� elements� �B� i� iniciant� la� transcripció.� La�

posterior�acetilació�de�p65�per�p300�o�PCAF�als�residus�de�lisina�122�i�123�disminueix�la�seva�

afinitat� per� l’ADN� facilitant� l’alliberació� dels� promotors� i,� per� tant,� inhibeixen� NF��B.� És�

interessant�destacar�que�la�proteïna�I�B��és�un�dels�gens�diana�de�NF��B,�i�que�en�el�moment�

en�què�p65�és�acetilada�i�perd�afinitat�per�l’ADN,�la�nova�I�B��s’uneix�a�NF��B�i� la�retorna�al�

citosol,� on� romandrà� de� forma� latent� unit� a� la� proteïna� I�B,� o� servirà� per� altres� cicles�

d’activació,�acetilació�i�desacetilació�(Quivy�i�Van,�2004)�(Figura�10).�

�

� SIRT1�

SIRT�1� (silent� information�regulator�T1)� �és�una�desacetilasa�dependent�de�NAD+�que� juga�un�

paper�important�en�la�regulació�de�NF��B,�desacetilant�la�subunitat�p65�en�el�residu�lisina�310�i�

inactivant�aquest�factor�de�transcripció�(Yeung�i�col.,�2004)�(Figura�10).�

�

Figura�10.�Acetilació�i�desacetilació�de�NF��B.�NF��B�en�la�seva�forma�inactiva�es�troba�al�citosol�unit�a�les�proteïnes�I�B.�Quan�arriba�un�estímul�extern�aquestes�proteïnes�són�fosforilades�i�ubiquitinitzades,�de�manera�que�NF��B�pot�entrar�al�nucli,�on�necessita�l’acció�de�co�activadors�com�p300�amb�activitat�acetiltransferasa�per�poder�transcriure�els�seus�gens�diana.�L’acció�de�desacetilases�com�el�SIRT1�inactiva�aquest�factor�de�transcripció.�A�més,�p300�també�pot�acetilar�NF��B�en�les�Lys�122�i�123�fent�li�perdre�afinitat�per�la�unió�a�l’ADN.�Les�proteïnes�I�B��transcrites�per�NF��B�podran�segrestar�NF��B,�que�quedarà�lliure�al�nucli,� i�retornar�lo�al�citosol,�on�romandrà�fins�a�tornar�a�ser�activat.�

�

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Donat�que�NF��B�és�un�factor�de�transcripció�pro�inflamatori�que�té�com�a�gens�diana�la�IL�6,�el�

TNF�� o� la� MCP�1,� la� seva� inactivació� podria� atenuar� el� procés� inflamatori� crònic� de� baixa�

intensitat� que� causa� RI� en� estats� d’obesitat.� De� fet,� estudis� recents� demostren� que� els�

adipòcits� 3T3�L1� knockdown� per� SIRT1� mostren� un� augment� de� l’expressió� de� gens� pro�

inflamtoris� com� la� IL�6,� el�TNF�� o� la�MCP�1,� acompanyada� per� una� disminució� de� la� via� de�

senyalització�de�la�insulina�i�de�la�captació�de�glucosa,�portant�a�un�estat�de�RI.�A�més,�quan�es�

van� tractar� les� cèl�lules� amb� un� activador� de� SIRT1,� s’observaven� efectes� antiinflamatoris,�

millorava�la�sensibilitat�a�la�insulina�i�es�protegia�contra�la�RI�induïda�pel�TNF��(Yoshizaki�i�col.,�

2009).�

Resulta�d’especial�interès�que�PPAR�/��també�és�capaç�de�regular�l’activitat�de�SIRT1�a�través�

Sp1�(Okazaki�i�col.,�2010).�Així�doncs,�la�regulació�de�NF��B�és�un�factor�important�sobre�el�qual�

es�podria�actuar�a�diferents�nivells�per�tal�de�millorar�la�sensibilitat�a�la�insulina.��

�

�

�

�

�

�

�

�

�

�

�

�

�

�

INTRODUCCIÓ

47

�

�

�

�

�

�

�

�

�

�

4. INTERLEUCINA�6�

La�IL�6�pertany�a�la�família�de�les�citocines�tipus�IL�6,�que�està�composta�per�la�pròpia�IL�6,�IL�

11,�LIF�(leukaemia�inhibitor�factor),�OSM�(oncostatin�M),�CNTF�(ciliary�neurotrophic�factor)�i�CT�

1� (cardiotrophin�like� citokine).� Totes� elles� tenen� una� estructura� comuna� de� quatre� ��hèlix,�

designades� amb� les� lletres� A� a� D,� i� una� mida� d’uns� 20� kDa.� Activen� gens� involucrats� en� la�

diferenciació,� l’apoptosi� i� la� proliferació,� tenen� propietats� anti�� i� pro�inflamatòries� i� són�

fonamentals�en�l’hematopoiesi,�en�la�resposta�de�fase�aguda�i�immune�de�l’organisme.�

La� interleucina�6� (IL�6)� és� una� citocina� produïda� per� una� gran� varietat� de� tipus� cel�lulars,� és�

essencial� en� la� regulació� de� la� inflamació,� amb� efectes� anti�� i� pro�inflamatoris,� en�

l’hematopoiesi,�en�la�resposta�immune�i�en�els�mecanismes�de�defensa�de�l’hoste�(Akira�i�col.,�

1993).�És�una�proteïna� formada�per�una�cadena�de�polipèptids�de�185�aminoàcids� (Somers� i�

col.,�1997).�El�seu�pes�molecular�oscila�de�21�a�28�kDa,�dependent�del�seu�estat�de�glicosilació�i�

de� fosforilació� (Gudmundsson� i� col.,� 1997;� May� i� col.,� 1988).� En� humans� sans� les� seves�

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48

concentracions� plasmàtiques� són� molt� baixes,� i� poden� variar� de� 1�9� pg/mL� segons� siguin�

persones� primes� o� obeses� (Gudmundsson� i� col.,� 1997).� No� obstant,� durant� processos�

inflamatoris�aquesta�citocina�assoleix�concentracions�més�altes,�podent�augmentar�fins�a�10�o�

1000�vegades�els�nivells�normals�en�casos�de�septicèmia,�estrés�greu�o�cirurgia�(Martin�i�col.,�

1997).��

�

4.1. IL�6�I�RI�

Fa�més�d’una�dècada�es�va�observar�que�una�de�les�principals�fonts�de�mediadors�inflamatoris�

era�el�teixit�adipós�(Hotamisligil�i�col.,�1993).�De�fet,�ha�estat�descrit�que�entre�el�15�35%�dels�

nivells� circulants� d’IL�6� són� produïts� pel� teixit� adipós� (Mohamed�Ali� i� col.,� 1997),� i� que� els�

adipòcits�són�capaços�de�secretar�IL�6�com�també�ho�són�altres�cèl�lules�de�la�matriu�cel�lular�

d’aquest�teixit�(Fain�i�col.,�2004;�Rodriguez�Calvo�i�col.,�2008).�

Com� s’ha� esmentat� en� aquest� treball,� l’obesitat� i� la� DM2� es� relacionen� amb� un� estat�

inflamatori�crònic�de�baixa�intensitat�caracteritzat�per�la�presència�de�nivells�plasmàtics�elevats�

de� citocines� pro�inflamatòries� com� el� TNF�� i� la� IL�6.� Tanmateix,� els� marcadors� inflamatoris�

detectats�en�l’obesitat�és�consideren�predictors�de�l’aparició�de�RI�i�de�DM2�(Festa�i�col.,�2002).�

S’ha� observat� que� els� nivells� d’IL�6� circulants� són� dos� o� tres� vegades� més� elevats� en� obesos�

amb�DM2�comparats�amb�obesos�sans�(Mohamed�Ali�i�col.,�1997;�Kern�i�col.,�2001).�Un�estudi�

clínic�amb�20�voluntaris�sans�va�demostrar�que�la�injecció�de�LPS�incrementava�el�TNF��i�la�IL�

6� tant� a� nivell� sistèmic� com� a� nivell� del� teixit� adipós,� i� va� causar� RI� (Anderson� i� col.,� 2007).�

Altres� estudis� han� demostrat� una� correlació� significativa� entre� nivells� elevats� d’IL�6� i� d’àcids�

grassos� lliures� i� la�presència�de�RI�en� teixit�adipós�humà�(Bastard� i� col.,�2002).�La� IL�6� també�

redueix�la�síntesi�hepàtica�de�glicogen�dependent�d’insulina�(Klover�i�col.,�2003)�i�la�captació�de�

glucosa� en� adipòcits� (Rotter� i� col.,� 2003).� També� ha� estat� demostrat� que� l’administració�

perifèrica� d’IL�6,� imitant� les� concentracions� observades� en� obesitat,� indueix� hiperlipidèmia,�

hiperglucèmia�i�RI�en�ratolins�i�humans�(Tsigos�i�col.,�1997)�(Figura�11).�

Experiments� realitzats� amb� GW501516,� un� agonista� de� PPAR��,� han� demostrat� que� aquest�

agonista�és�capaç�de�suprimir�l’expressió�gènica�de�molècules�de�la�fase�aguda�induïdes�per�IL�

6� com� el� ��fibrinogen,� l’�1�acid� glicoproteïna� (AGP),�l’�1�antiquimotripsina� (AACT)� i� l’�2�

macroglobulina� en� hepatòcis.� A� més,� el� mateix� estudi� demostra� que� l’activació� de�

PPAR��suprimeix� l’expressió� d’AACT� induïda� per� la� IL�6� a� través� del� bloqueig� de� l’activitat�

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49

transcripcional�d’STAT3�(Signal�transducer�and�activator�of�transcription�3)�(Kino�i�col.,�2007),�

tot�i�que�el�mecanisme�implicat�no�va�ser�descrit.�

Malgrat�totes�aquestes�dades�encara�es�desconeixen�els�mecanismes�a�través�dels�quals�la�IL�6�

produeix�els�seus�efectes�sobre�la�sensibilitat�a�la�insulina,�així�com�PPAR��pot�evitar�aquests�

efectes�en�teixit�adipós.�

�Figura�11.�Efectes�de�la�IL�6�implicats�en�el�desenvolupament�de�RI.�

�

4.2. VIES�DE�SENYALITZACIÓ�DE�LA�IL�6�

La�família�de�citocines�tipus�IL�6�s’uneix�a�complexes�de�receptors�de�membrana�plasmàtics�els�

quals� contenen� tots� la� glicoproteïna� 130� (gp130)� com� a� receptor� transductor� de� senyals.� La�

transducció�de�senyals�activa�les�proteïnes�JAK�(janus�kinase)�portant�a�l’activació�de�la�família�

de�factors�de�transcripció�STAT.�Una�altra�via�de�senyalització�per�a�aquesta�família�de�citocines�

inclou�la�cascada�de�les�MAPK�(Heinrich�i�col.,�2003).�

�

4.2.1. RECEPTORS�DE�LA�IL�6:�IL�6R��i�GP130�

Els� receptors� que� reconeixen� a� aquesta� família� de� citocines� tipus� IL�6� es� poden� dividir� en�

receptors��no�senyalitzadors,�on�s’inclou�el�receptor� IL�6R�,� i�els�receptors�transductors�de�

senyals�que�inclou�la�gp130�entre�d’altres.�

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50

Tots�els�receptor�d’IL�6�contenen�una�sèrie�de�dominis�FNIII� (fibronectin�type� III�like)� i� Ig�like.�

Cada� receptor� conté� almenys� un� mòdul� d’unió� a� citocines� (CBM)� que� comprèn� dos� dominis�

FNIII.�A�més,�els�receptors�encarregats�de�la�transducció�de�senyals,�com�la�gp130,�tenen�tres�

dominis� FNIII� proximals� a� la� membrana� i� una� llarga�cua� citoplasmàtica� que� reflecteix� la� seva�

funció�com�a�transductors�de�senyals�(Heinrich�i�col.,�1998)�(Figura�12).�

Les�citocines�d’aquesta�família�s’uneixen�específicament�a�un�receptor�de�membrana,�i�aquesta�

especificitat� ve�donada�per�àrees�de� la� seva�superfície�anomenades�sites.�El�site� I�determina�

l’especificitat�pel� receptor��,�el�site� II�és�comú�en�totes� les�citocines� i� reconeix�el�CBM�de� la�

gp130.�El�site�III�varia�i�determina�si�es�formaran�homodímers�amb�la�gp130,�com�és�el�cas�de�la�

IL�6,� o� si� s’uniran� altres� receptors� de� membrana� formant� heterodímers� (Hammacher� i� col.,�

1998;�Kurth�i�col.,�1999).�

�

Figura�12.�Estructura�de�la�IL�6�i�dels�seus�receptors�de�membrana.�El�receptor�IL�6R��només�s’encarrega�de�la�unió�de�la�IL�6,�mentre�que�la�gp130�és�la�responsable�de�la�internalització�de�la�IL�6�i�de�la�transducció�de�senyals.�

�

� IL�6R��

La�IL�6�primer�s’uneix�al�seu�receptor��mitjançant�els�dominis�CBM�i�Ig�like.�Aquest�receptor�

dóna� sensibilitat� a� la� cèl�lula� per� la� citocina.� Aquesta� funció� de� sensibilització� també� pot� ser�

duta� a� terme� mitjançant� les� formes� solubles� (sIL�6R�)� que� no� posseeixen� les� parts�

transmembrana�ni�citoplasmàtiques�(Taga�i�col.,�1989).��

�

� gp130�

La� IL�6� no� indueix� la� dimerització� del� receptor,� si� no� que� estabilitza� el� complex� del� receptor�

gp130� i� el� receptor�� preformat� prèviament� i� inicia� l’activació� del� receptor� induïnt� un� canvi�

conformacional�(Livnah�i�col.,�1999;�Chan�i�col.,�2000).�Un�cop�activat�el�receptor�és�capaç�de�

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51

reclutar� els� components� necessaris� per� a� la� transducció� de� senyals.� Els� dominis� FNIII�

addicionals�que�posseeix�la�gp130�són�necessaris�per�l’acoblament�dels�lligands�implicats�en�la�

senyalització� (Timmermann� i� col.,� 2002).� Les� cadenes� transductores� de� senyals�

citoplasmàtiques� d’aquest� receptor� s’uneixen� amb� les� proteïnes� JAK.� Aquestes� proteïnes�

s’uneixen�a�la�regió�proximal�de�la�membrana�de�gp130�que�conté�els�motius�box1� i�box2.�La�

primera�és�essencial�per�a�la�unió�de�les�proteïnes�JAK�i� la�segona�contribueix�a�aquesta�unió�

(Haan� i� col.,� 2002).� A� més,� la� regió� interbox1�2� és� essencial� per� a� aquesta� unió� (Haan� i� col.,�

2000).�El�receptor�gp130�no�només�és�un�lloc�d’unió�per�als�factors�de�senyalització�sinó�que�

també�és�essencial�per�a�l’activació�de�JAK�(Haan�i�col.,�2002).�

Diverses� publicacions� descriuen� altres� cinases� que� s’associen� a� la� transducció� de� senyals�

induïda� per� la� IL�6.� Per� exemple� la� deleció� de� la� regió� acídica� de� la� gp130,� que� inclou� els�

aminoàcids�711�811,�redueix�l’activitat�cinasa�de�la�Hck�i�l’activació�de�l’ERK.�Per�altra�banda,�la�

cinasa�PKC�� (protein�kinase�C�),� implicada�en� la� fosforilació�en�serina�d’STAT3,�s’ha�observat�

que� s’associa� amb� el� receptor� gp130� i� incrementa� la� unió� al� receptor� d’STAT3� (Novotny�

Diermayr�i�col.,�2002).�

�

4.2.2. VIA�DE�SENYALITZACIÓ�JAK/STAT3�

El�1994�es�va�descobrir�que�les�citocines�tipus�IL�6�utilitzaven�tirosin�cinases�de�la�família�Jak�i�

factors� de� transcripció� de� la� família� STAT� com� a� principals� mediadors� de� la� transducció� de�

senyals�(Lutticken�i�col.,�1994;�Stahl�i�col.,�1995).��

Al� receptor� gp130� s’associen� les� cinases� Jak1,� Jak2� i� Tyk2� que� després� de� l’estimulació� del�

receptor� s’activen� i� fosforilen� en� tirosina� la� cua� citoplasmàtica� de� gp130.� Aquestes�

fosfotirosines�serviran�com�a�punts�d’unió�pels�dominis�SH2�(Src�homology�2)�dels� factors�de�

transcripció�STAT,�particularment,�STAT1�i�STAT3�(Stahl�i�col.,�1995).�Aquestes�proteïnes�STAT�

es� fosforilen,� formen� dímers� i� transloquen� al� nucli,� on� regulen� la� transcripció� dels� seus� gens�

diana.��

La�tirosin�fosfatasa�SHP2�també�s’uneix�a�la�gp130�fosforilada,�i�possiblement�ès�la�responsable�

de�l’activació�de�la�via�de�les�MAPK,�que�també�s’activa�després�de�l’estimulació�per�citocines�

tipus�IL�6�(Stahl�i�col.,�1995)�(Figura�13).�

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52

�

Figura�13.�La�IL�6�activa�la�via�JAK/STAT�i�la�via�de�les�MAPK.�La�unió�de�la�IL�6�a�gp130�provoca�l’activació�de�les�cinases� Jak� que� hi� estan� associades� i� d’aquesta� manera� s’inicia� una� cascada� de� fosforilacions� que� resultarà� amb�l’activació�de�les�proteïnes�STAT�i�en�la�transcripció�dels�seus�gens�diana.�

�

�

� JAK�

Les�proteïnes�JAK�són�tirosin�cinases�amb�masses�moleculars�entre�120�140�kDa.�Se’n�coneixen�

quatre�membres�en�mamífers,�Jak1,�Jak2�i�Tyk2�que�s’expressen�a�tot�l’organisme,�i�Jak3,�que�

es�troba�principalment�a�cèl�lules�d’origen�hematopoiètic.�

Les�proteïnes�JAK�s’organitzen�en�un�domini�cinasa�anomenat�JH1�(Jak�homology1)�a�l’extrem�

C�terminal�que�conté�un� loop�d’activació�que�regula�l’activitat�cinasa.�Un�domini�tipus�cinasa,�

JH2,� i� a� l’extrem� N�terminal�algunes� Jaks,� contenen�cinc� regions� responsables�de� l’associació�

amb�el�receptor�(Ihle,�1995)�(Figura�14).�

�

Figura�14.�Estructura�de�les�proteïnes�Jak,�STAT�i�SHP2.�

�

Ha�estat�demostrat�que�la�senyalització�de�la�IL�6�en�presència�del�seu�receptor�soluble�depèn�

de�la�presència�de�Jak1,�i�que�Jak2�i�Tyk2�formen�part�d’un�complex�proteic�sense�el�qual�no�es�

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53

podria�activar�Jak1,�que�és�la�única�capaç�de�continuar�els�següents�passos�de�la�senyalització�

intracel�lular�(Guschin�i�col.,�1995).�L’associació�amb�el�receptor�gp130�només�té�lloc�a�la�regió�

proximal�de�membrana�del�domini�citoplasmàtic�que�conté�els�motius�box1�i�box2.�

�

� STATs�

La� família� STAT� de� factors� de� transcripció� comprèn� set� membres� descrits� en� mamífers�

anomenats� STAT1,� �2,� �3,� �4,� �5a,� �5b,� 6.� La� seva� activitat� està� regulada� principalment� per�

modificacions� postraduccionals� com� les� fosforilacions� en� serina� i� en� tirosina.� Són� activades�

després�de�l’associació�amb�els�receptors�de�citocines.�Totes�les�citocines�de�la�família�tipus�IL�

6�activen�STAT1�i�STAT3�a�través�del�receptor�comú�gp130�(Heinrich� i�col.,�1998).�Tanmateix,�

existeixen�estudis�que�demostren�que�també�poden�ser�activats�per�receptors�tirosin�cinases�

(EGF�receptor,�FGF�receptor,�PDGF�receptor,�etc.)�(Briscoe�i�col.,�1994;�Yamamoto�i�col.,�1996;�

Park�i�col.,�1996).�

L’organització�estructural�és�molt�conservada�dintre�de�la�família�STAT�(Figura�13).�Tenen�uns�

750�850� aminoàcids� (STAT3,� 770� aminoàcids)� que� inclouen� diferents� dominis.� A� l’extrem� N�

terminal�tenen�un�domini�de�tetramerització,�també�anomenat�feix�de�4�hèlix,�i�una�cremallera�

de�leucines.�Al�domini�intermig�conté�una�regió�d’unió�a�l’ADN�o��barrel�i�un�domini�SH3�like�

(Src�homology�3�like).� I�a� l’extrem�C�terminal�es� troben�un�domini�SH2� i�un�domini�de� trans�

activació.� El� domini� SH2� és� el� reponsable� de� la� unió� de� les� STAT� als� motius� del� receptor�

fosforilats�en�tirosina� i� també�de� la�homo��o�heterodimerització�amb�altres�STAT�fosforilades�

en� tirosina� (Heim� i� col.,� 1995;� Shuai� i� col.,� 1994).� Totes� les� STATs� són� fosforilades� a� prop� de�

l’extrem�C�terminal�quan�s’activa�el�receptor.�En�el�cas�d’STAT3�aquesta�fosforilació�té� lloc�al�

residu�de�tirosina�705.�

Els�elements�d’unió�a�les�proteïnes�STAT�de�l’ADN�són�molt�similars�ja�que�el�domini�d’unió�a�

l’ADN�d’aquests�factors�de�transcripció�és�molt�conservat.�L’afinitat�d’unió�a�unes�seqüències�o�

a�unes�altres�en�funció�de�la�composició�dels�dímers�de�les�proteïnes�STAT.�

L’activitat�del�domini�de�trans�activació�està�parcialment�regulada�per�la�fosforilació�en�serina,�

en�el�cas�d’STAT3�al�residu�serina�727.�La�cinasa�responsable�d’aquesta�fosforilació�depèn�de�la�

via� de� senyalització� i� del� context� cel�lular.� Algunes� de� les� cinases� descrites� responsables�

d’aquesta�fosforilació�són�PKC�,�p38�MAPK�i�ERK1/2�i�JNK�(Schuringa�i�col.,�2000;�Uddin�i�col.,�

2002;� Abe� i� col.,� 2001).� Sembla� que� aquesta� fosforilació� en� serina� incrementaria� l’activitat�

transcripcional�d’STAT3�(Abe�i�col.,�2001;�Uddin�i�col.,�2002).�

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L’activació� de� les� proteïnes� STAT� té� lloc� al� citosol� però� per� a� realitzar� la� seva� funció� han� de�

translocar�al�nucli�(Figura�13).�Després�de�l’estimulació�amb�IL�6,�STAT3,�que�es�troba�per�tot�el�

citoplasma,�dimeritza�en� resposta�a� la� fosforilació�en� tirosina� i�es�concentra�al�nucli� (Zhang� i�

col.,� 1995).� Degut� a� la� seva� mida� de� 90� kDa,� necessita� ser� activament� translocada� al� nucli,� i�

quan� és� defosforilada� és� exportada� al� citoplasma� (Haspel� i� Darnell,� Jr.,� 1999),� però� es�

desconeixen�els�mecanismes�implicats.��

Un�cop�al� nucli� les�STAT� s’uneixen� a� seqüències�específiques�de� l’ADN� i� activen,�o�en�alguns�

casos�reprimeixen,� la� transcripció�dels�seus�gens�diana.�S’ha�descrit�un�gran�nombre�de�gens�

diana� de� les� STAT� després� de� l’activació� per� citocines� tipus�IL�6,� com� aquells� que� codifiquen�

per� la� CRP� (proteïna� C�reactiva),� l’�2�macroglobulina� (Wegenka� i� col.,� 1993;� Zhang� i� col.,�

1996b),�la�proteïna�d’unió�al�lipopolisacàrid�(Schumann�i�col.,�1996),�o�gens�que�codifiquen�per�

factors� de� transcripció� com� Jun� B� (Coffer� i� col.,� 1995),� c�Fos� (Hill� i� Treisman,� 1995),� IRF�1�

(interferon� regulatory� factor�1)� (Harroch� i� col.,� 1994)� o� altres� gens� com� l’hsp90� (heat� shock�

protein�90)�(Stephanou�i�col.,�1998),�la�gp130�i�SOCS�(O'Brien�i�Manolagas,�1997;�Auernhammer�

i�col.,�1999).�Cal�destacar�que�els� llocs�d’unió�a� l’ADN�dels� factors�de�transcripció�STAT�solen�

estar�pròxims�a�llocs�d’unió�d’altres�factors�de�transcripció,�fet�que�suggereix�una�cooperació�

d’aquests�factors�per�la�regulació�dels�gens,�seria�el�cas�de�NF��B�(Brown�i�col.,�1995)�o�d’AP�1�

(Korzus� i� col.,� 1997).� A� més,� aquests� llocs� d’unió� solen� estar� en� tàndem,� suggerint� que� les�

proteïnes�STAT�poden�formar�multímers�(Xu�i�col.,�1996).�En�conjunt,�tot��fa�pensar�que�existeix�

un�procés�integrador�que�regularia�l’expressió�dels�gens�diana�de�les�proteïnes�STAT.�

�

� SHP2�i�la�via�MAPK�

SHP2�és�una�fosfatasa�que�conté�dos�dominis�N�terminals�SH2�i�un�domini�fosfatasa�catalític�a�

la�regió�C�terminal�(Figura�14).�La�unió�dels�dominis�SH2�a�les�fosfotirosines�de�gp130�desplega�

la�proteïna� i� l’activa�(Pluskey� i�col.,�1995).�SHP2�també�s’activa�per� la�fosforilació�de�tirosines�

de�la�regió�fosfatasa�(Lu�i�col.,�2001).�

El� receptor� gp130� conté� un� lloc� d’unió� per� SHP2� a� la� tirosina� 759.� Quan� aquesta� tirosin�

fosfatasa� és� reclutada� es� fosforila� de� manera� dependent� de� Jak1� (Schaper� i� col.,� 1998),�

seguidament�SHP2�interacciona�amb�Grb2�(growth�factor�receptor�bound�protein�2)�(Fukada�i�

col.,�1996),��fet�que�permet�l’activació�de�la�via�Ras�Raf�MAPK�(Hermanns�i�col.,�2000)�(Figura�

13).�

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A� més,� Gab1� (GRB2�associated�binding� protein� 1)� també� està� implicada� en� l’activació� de� la�

cascada� Ras�Raf�MAPK.� Aquesta� proteïna� conté� llocs� d’unió� per� SHP2,� entre� d’altres,� i� en�

resposta� a� la� IL�6� es� fosforila� en� tirosina� i� interactúa� amb� SHP2� i� PI3K,� portant� a� l’activació�

d’ERK1/2�(Takahashi�Tezuka�i�col.,�1998).�

�

4.2.3. INHIBICIÓ�DE�LA�SENYALITZACIÓ�DE�LA�IL�6�

� SHP2�

Malgrat�que�SHP2�afavoreix� l’activació� de� la� cascada�de� les� MAPK,� té�efectes�negatius� sobre�

l’activació�de�la�via�JAK/STAT�(Lehmann�i�col.,�2003),�ja�que�la�mutació�del�residu�tirosina�759�

de�gp130�evita�la�unió�de�SHP2�i�afavoreix�la�senyalització�via�JAK/STAT�(Symes�i�col.,�1997).�A�

més,�la�presència�d’una�SHP2�mutada�fa�que�la�fosforilació�de�JAK�i�STAT�augmenti�i�que�també��

augmenti�l’expressió�dels�gens�diana�d’STAT3�(Symes�i�col.,�1997).�

�

� SOCS�

La� família� de� les� proteïnes� SOCS� està� formada� per� vuit� membres� anomenats� CIS� (cytokine�

inducible�SH2�proteins)�i�SOCS1�7.�Tots�contenen�un�domini�central�SH2�i�a�l’extrem�C�terminal�

un�domini�anomenat�SOCS�box�(Starr�i�col.,�1997;�Naka�i�col.,�1997)�(Figura�15).�

�

Figura�15.�Estructura�de�SOCS.�El�domini�SH2�pot�unir�se�al� IR� impedint� la�fosforilació�d’IRS1.�A�través�del�domini�SOCS�box�les�proteïnes�SOCS�poden�portar�a�la�degradació�proteasomal.�

�

Tots� els� membres� de� la� família� són� induïts� per� la� senyalització� de� les� citocines� tipus� IL�6.�

Concretament,� la� IL�6� incrementa� els� nivells� de� CIS,� SOCS1,� SOCS2� i� SOCS3� (Blanchard� i� col.,�

2001).� SOCS1� i� SOCS3� estan� més� relacionats� amb� la� inhibició� de� la� senyalització� d’aquesta�

citocina,� actuant� com� un� feedback�negatiu� clàssic.� Ambdós� actuen� sobre� les� proteïnes� JAK� i�

conseqüentment� inhibeixen� la� fosforilació� i� l’activació� de� gp130,� d’STAT� i� de� les� pròpies� JAK�

(Naka�i�col.,�1997;�Starr�i�col.,�1997;�Krebs�i�Hilton,�2001).�A�més�de�la�interacció�directa�amb�la�

via� JAK/STAT,� les� proteïnes� SOCS� a� través� del� domini� SOCS�box� són� capaces� d’interaccionar�

INTRODUCCIÓ

56

amb�les�proteïnes�E2�i�E3�i�ubiquitinitzar�proteïnes�de�senyalització�portant�les�a�la�degradació�

proteasomal.�

Existeixen�estudis�que�han�demostrat�que�els�nivells�d’expressió�de�SOCS3�estan�més�elevats�

en�teixit�adipós�de�ratolins�obesos�(Emanuelli� i�col.,�2001)� i� també�es�sobreexpressa�en�teixit�

adipós� d’humans� obesos� i� diabétics� (Shi� i� col.,� 2004).� A� més,� els� nivells� de� RNAm� d’IL�6� es�

correlacionen�amb�els�nivells�de�RNAm�de�SOCS3�en�teixit�adipós�d’humans�obesos,�suggerint�

que�la�IL�6�podria�regular�l’expressió�de�SOCS3�a�través�de�mecanismes�autocrins�i�paracrins�en�

teixit�adipós�humà�(Rieusset�i�col.,�2004).�

De�fet,�les�proteïnes�SOCS,�a�més�de�ser�inhibidores�de�la�senyalització�de�la�IL�6,�també�ho�són�

de�la�via�de�senyalització�de�la�insulina�(Emanuelli� i�col.,�2000).�S’ha�descrit�que�la�deficiència�

de�SOCS3�incrementa�la�fosforilació�d’IRS1�i�IRS2�induïda�per�insulina,�incrementa�l’activitat�de�

la�PI3K� i,� finalment,� incrementa� la� captació�de� glucosa� induïda� per� insulina� (Shi� i� col.,� 2004).�

Mentre� que� la� sobreexpressió� d’aquesta� proteïna� indueix� una� reducció� de� la� fosforilació� en�

tirosina� d’IRS1� induïda� per� la� insulina� i� una� disminució� de� l’associació� de� la� PI3K� amb� IRS1�

(Emanuelli�i�col.,�2001;�Senn�i�col.,�2003).�Diversos�estudis�han�descrit�diferents�mecanismes�a�

través� dels� quals� SOCS3� podria� interferir� amb� la� via� de� senyalització� de� la� insulina.� D’una�

banda,�en�hepatòcits,�SOCS3�és�capaç�d’induir� la�degradació�proteasomal�d’IRS1� i� IRS2� (Rui� i�

col.,� 2002;� Kamura� i� col.,� 1998).� Per� altra� banda,� també� s’ha� demostrat� que� SOCS3� pot�

interactuar�directament�amb�el� residu�de� tirosina�960�del� IR�a� través�del� seu�domini�SH2� fet�

que� impediria� l’associació� d’IRS1� amb� el� receptor� i� inhibiria� la� fosforilació� en� tirosina� d’IRS1�

(Emanuelli�i�col.,�2000;�Mooney�i�col.,�2001;�White�i�col.,�1988).�L’estudi�de�Rieusset�i�col�(2004)�

dóna� suport� a� aquest� mecanisme.� En� aquest� estudi� observen� que� l’estimulació� de� miotubs�

humans� amb� IL�6� inhibeix� la� fosforilació� d’IRS1� mentre� que� la� fosforilació� del� IR� no� es� veu�

afectada� (Rieusset� i� col.,� 2004).� Ambdós� mecanismes� exposats,� és� a� dir,� la� degradació�

proteasomal�i�la�interacció�amb�el�IR,�són�complementaris.��

En� conjunt,� totes� aquestes� dades� suggereixen� que� SOCS3� juga� un� paper� important� com� a�

mediador�de�la�RI�induïda�per�la�IL�6.�Per�tant,�els�factors�que�controlen�l’expressió�i�la�funció�

de� SOCS3� poden� ser� determinants� pel� desenvolupament� d’obesitat� i� diabetis� i� es� poden�

considerar�potencials�dianes�terapèutiques�pel�tractament�d’aquestes�patologies.�

�

�

�

INTRODUCCIÓ

57

�

�

�

�

�

�

�

�

�

�

5. FACTOR�DE�NECROSI�TUMORAL��

�

El� factor� de� necrosi� tumoral�� (TNF��)� va� ser� descrit� la� primera� vegada� com� un� factor� del�

sèrum�induït�per�endotoxines�que�causava�necrosi�dels�tumors�(Carswell�i�col.,�1975).�Més�tard�

es�va�demostrar�que�era� igual�que� la�molècula�anomenada�caquectina,�present�en�cultius�de�

macròfags� exposats� a� endotoxina� i� que� induïa� caquèxia� en� animals� (Tracey� i� col.,� 1988).�

S’expressa� com� un� monòmer� de� 26� kDa� unit� a� la� membrana� cel�lular,� que� després� de� ser�

proteolitzat�queda�en�forma�de�trímer�soluble�de�17�kDa.�Es�creu�que�la�part�que�queda�lligada�

a�la�membrana�(mTNF��)�podria�tenir�diverses�funcions�a�través�del�contacte�cel�lular,�mediant�

principalment�efectes�locals�del�TNF��(Grell,�1995).�

TNF��és� una� citocina� produïda� per� diversos� tipus� cel�lulars,� però� els� macròfags� o� altres�

cèl�lules� de� llinatge� monocític� són� les� cèl�lules� que� més� en� produeixen� (Flynn� i� col.,� 1995;�

INTRODUCCIÓ

58

Pfeffer�i�col.,�1993).�Està�involucrada�en�processos�inflamatoris�i�en�la�inducció�de�la�resposta�

de� fase� aguda,� és� capaç� d’induir� apoptosi,� inflamació� i� tumorigènesis.� La� desregulació�

d’aquesta�citocina�s’ha�observat�en�un�gran�nombre�de�malalties�humanes�com�l’Alzheimer,�el�

càncer,�la�depressió�o�la�RI,�entre�d’altres�(Parameswaran�i�Patial,�2010).�

El�TNF��excerceix�els�seus�efectes�a�través�de�dos�receptors�transmembrana,�TNFR1�i�TNFR2�

(receptor�de�TNF��de�tipus�1� i�de� tipus�2,� respectivament).�En� funció�de�quin�receptor�sigui�

activat�es�poden�disparar�vies�intracel�lulars�totalment�oposades,�per�exemple�pot�tenir�efectes�

pro�� o� anti�apoptòtics� (Aggarwal,� 2003;� Dirks� i� Leeuwenburgh,� 2006).� Ambdós� receptors�

contenen� quatre� repeticions� riques� en� cisteïna� en� els� seus� dominis� extracel�lulars� que�

interaccionen� amb�el� trímer� TNF��(Banner� i� col.,�1993).�TNF�R1� s’expressa� constitutivament�

en�la�majoria�dels�teixits�dels�mamífers,�mentre�que�l’expressió�de�TNF�R2�és�més�específica�en�

cèl�lules�del�sistema�immune.��

�

5.1. TNF��I�RI�

TNF��,�de�la�mateixa�manera�que�la�IL�6,�ha�estat�proposat�com�un�dels�mediadors�inflamatoris�

que� vinculen� l’obesitat� amb� el� desenvolpament� de� RI� i� DM2� (Hotamisligil,� 2003).� Aquest�

citocina�pro�inflamatòria�és�secretada�per�macròfags�i�per�adipòcits��(Hotamisligil�i�col.,�1993)�i�

sembla� actuar� per� mitjà� de� mecanismes� autocrins� i� paracrins� tenint� efectes� sobre� la� RI� i� la�

inducció�d’IL�6�(Arner,�2003;�Rotter�i�col.,�2003).�També�s’ha�observat�que�l’expressió�de�TNF��

és� més� elevada� en� teixit� adipós� d’homes� insulino�resistents� i� obesos� (Kern� i� col.,� 2001).� La�

deficiència�de�TNF��en�ratolins�obesos�millora�la�sensibilitat�a�la� insulina�(Uysal� i�col.,�1997),�

mentres�que�en�múscul�esquelètic�el�TNF��augmenta� la� lipòlisi�provocant�un�increment�dels�

nivells� d’àcids� grassos� lliures� que� afavoreix� l’aparició� de� RI� (Arner,� 2003).� A� més,� TNF��

interacciona�directament�amb�la�via�de�senyalització�de�la�insulina.�Per�exemple,�en�adipòcits,�

l’exposició�crónica�a�baixes�dosis�de�TNF��causa�la�disminució�de�l’autofosforilació�induïda�per�

insulina�del� IR� i�de� la� fosforilació�en�tirosina�d’IRS1� (Hotamisligil� i� col.,�1994),� i� incrementa� la�

fosforilació�en�serina�307�d’IRS1,�impedint�d’aquesta�manera�que�es�pugui�unir�al�IR�(Rui�i�col.,�

2001).�

�

�

INTRODUCCIÓ

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5.1.1. TNF��ACTIVA�NF��B�

TNF��s’uneix�al�domini�extracel�lular�de�TNFR1�alliberant�la�proteïna�SODD�(silencer�of�death�

domains)� del� domini� intracel�lular� del� receptor,� que� pot� unir�se� a� proteïnes� adaptadores�

TRADD� (TNF�� receptor�associated� death� domain)� (Hsu� i� col.,� 1995).� Al� seu� torn� el� complex�

SODD�TRADD�recluta�les�proteïnes�adaptadores,�RIP�1�(receptor�interacting�protein�1),�que�són�

unes� serin/treonin� cinases� (Hsu� i� col.,� 1996),� i� TRAF2� (TNF�R�associated� factor� 2),� una� ligasa�

d’ubiquitines�E3�(Takeuchi�i�col.,�1996).�Aquest�complex�és�internalitzat�i�el�complexe�TRADD�

RIP�1�TRAF2�s’allibera�de�TNFR1.�Llavors,�aquestes�proteïnes�adaptadores�activen�altres�vies�de�

senyalització.�

RIP�1� recluta� MEKK�3� (MAP/ERK�cinasa� 3)� i� la� cinasa� TAK1� (transforming� growth� factor�beta�

(TGF��)�activated�kinase)�i�activa�el�complex�IKK.�Aquesta�cinasa��fosforila�les�proteïnes�I�B�que�

es�degraden�al�proteasoma,�tot�alliberant��NF��B�que�llavors�transloca�al�nucli�i�activa�els�seus�

gens�diana�(Chen,�2005;�Hayden�i�Ghosh,�2004).�També�ha�estat�demostrat�que�TRAF2�activa�

NF��B�unint�se�amb�el�complex�IKK�(Devin�i�col.,�2001)�i�reclutant�cIAP�1�i�cIAP�2�(inhibitor�of�

cellular�apoptosis�proteins),�inhibidors�de�caspases�amb�activitat�ubiquitin�ligasa�mitjançant�la�

qual�degraden�I�B�(Chen,�2005).�Dependent�del�tipus�cel�lular�hi�ha�moltes�variacions�respecte�

a� l’activació� de� NF��B� per� TNF��.� Per� exemple,� en� macròfags� aquesta� activació� pot� ser�

regulada�per�la�tirosin�cinasa�c�Src�(bu�Amer�i�col.,�1998).��

Finalment,� cal� tornar� a� esmentar� que� la� regulació� d’aquest� factor� de� transcripció�

proinflamatori�(explicada�detalladament�a�l’apartat�3�d’aquesta�memòria)�ha�estat�proposada�

com�una�diana�farmacològica�per��a�prevenir�o�tractar�la�RI.�Per�tant,�almenys�part�dels�efectes�

sobre�la�RI�produïts�per�TNF��podrien�ser�mediats�per�l’activació�de�NF��B.�

�

5.1.2. TNF��INHIBEIX�LA�VIA�DE�SENYALITZACIÓ�DE�LA�INSULINA�

El�TNF��a�més�de�ser�un�factor�important�que�col�labora�en�l’aparició�de�RI�per�l’activació�del�

factor� de� transcripció� pro�inflamatori� NF��B,� també� inhibeix� la� via� de� senyalització� de� la�

insulina�a�través�dels�seus�receptors�de�membrana.��

La�unió�de� la� insulina�al� seu�receptor� IR,�que�té�activitat� tirosin�cinasa,� inicia�una�cascada�de�

fosforilacions�que�comença�amb�l’autofosforilació�d’ell�mateix�en�diversos�residus�de�tirosina�i�

continua�amb�els�seus�substrats�com�IRS1�(Garvey�i�col.,�1991;�Kasuga�i�col.,�1982).�Aquest�és�

capaç�d’interaccionar�amb�proteïnes�que�contenen�dominis�SH2�com�la�PI3K�o�com�la�fosfatasa�

INTRODUCCIÓ

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SHP2.� A� través� de� la� senyalització� de� la� PI3K� s’inicia� la� fosforilació� i� senyalització� de� la� via�

PKB/Akt�que�porta�a�la�captació�de�glucosa.�Per�una�altra�banda,�la�cascada�de�senyalització�de�

la� insulina� es� regula� negativament� per� fosfatases� tant� de� tirosines� com� de� serines� com� per�

exemple�la�PTP1B��(protein�tyrosin�phospatase�1B)�que�defosforila�residus�dels�IR�i�de�la�IRS1.��

El�TNF��pot�interferir�en�la�via�de�senyalització�de�la�insulina�mitjançant�diversos�mecanismes.�

Per�una�banda,�pot�induïr�l’expressió�de�SOCS3�que�pot�marcar�IRS1�per�a�la�seva�degradació�

proteasomal�en�adipòcits�3T3�L1�(Shi�i�col.,�2004)�fet�que�resulta�en�la�disminució�de�l’activitat�

de�la�PI3K�i�del�transport�de�glucosa�(Liu�i�col.,�1998;�Hotamisligil� i�col.,�1994).�A�més,�SOCS3,�

com� s’ha� exposat� prèviament,� pot� unir�se� al� IR� i� impedir� la� fosforilació� d’IRS1.� De� fet,� la�

supressió�de�l’expressió�de�SOCS3�en�adipòcits�atenua�parcialment�la�inhibició�de�la�fosforilació�

en� tirosina� d’IRS1� induïda� per� TNF��(Ueki� i� col.,� 2004).� Aquests� resultats� es� repeteixen� en�

experiments� realitzats� en� fetge� de� ratolins� diabètics� en� que� el� silenciament� de� SOCS3�

normalitza� la� fosforilació� d’IRS1� (Uysal� i� col.,� 1997).� Per� altra� banda,� TNF��pot� activar� JNK�

provocant� la� fosforilació� en� la� serina� 307� d’IRS1� en� adipòcits� (White,� 1997;� Hirosumi� i� col.,�

2002)�fet�que�disminuiria� la�capacitat�d’IRS1�per�unir�se�amb�el� IR� i,�per�tant,�no�s’iniciaria� la�

cascada� de� senyalització� de� la� insulina� (Rui� i� col.,� 2001).� De� fet,� s’ha� observat� que� ratolins�

deficients�per�JNK1�presenten�nivells�de�fosforilació�més�baixos�en�el�residu�serina�307�d’IRS1�i�

major�sensibilitat�a�la�insulina�(Hirosumi�i�col.,�2002).�

�

�

�

�

�

�

�

�

�

�

INTRODUCCIÓ

61

�

�

�

�

�

�

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�

�

OBJECTIUS�

�

�

�

�

63

L’obesitat� i� la� DM2� s’han� associat� a� la� presència� d’un� estat� inflamatori� crònic� de� baixa�

intensitat�que�s’ha�postulat�com�un�dels�principals�causants�de�l’aparició�de�RI.�De�fet,�aquesta�

RI� es� correlaciona� amb� nivells� alts� de� citocines� pro�inflamatòries� com� el� TNF�� o� la� IL�6�

(Hotamisligil,�2003).��

El�receptor�nuclear�PPAR��presenta��efectes�antiinflamatoris�que�podrien�evitar�l’aparició�de�

la� RI,� però� els� mecanismes� implicats� encara� no� són� prou� coneguts.� D’una� banda,� ha� estat�

descrit� que� PPAR�� reprimeix� la� via� de� senyalització� JAK/STAT3� induïda� per� la� IL�6� en�

hepatòcits�(Kino�i�col.,�2007),�encara�que�els�mecanismes�implicats�es�desconeixen.�Un�efecte�

similar�en�adipòcits�podria�contribuir�a�reduir�la�RI.�D’altra�banda,�el�manteniment�de�l’activitat�

PPAR�/��pot�ser�fonamental�per�a�evitar�el�desenvolupament�del�procés�inflamatori�i�la�RI�en�

el�teixit�adipós,�donat�que�ha�estat�demostrat�que�evita�el�procés�inflamatori�(Rodriguez�Calvo�

i�col.,�2008),�però�encara�es�desconeix�quins�efectes�tenen�les�citocines�pro�inflamatòries,�com�

la� IL�6� i� el� TNF�,� sobre� l’expressió� i� l’activitat� d’aquests� receptors� nuclears� en� adipòcits� de�

pacients�i�en�cultius�d’adipòcits�humans.�Per�tot�això,�l’objectiu�general�d’aquesta�Tesi�Doctoral�

ha�estat�trobar�els�mecanismes�pels�quals�PPAR��pot�contribuir�a�prevenir�la�inflamació�i�la�RI�

en�adipòcits.�Més�concretament,�els�objectius�específics�d’aquesta�Tesi�Doctoral�han�estat:�

�

I. Determinar� si� l’activador� de� PPAR�� GW501516� preveu� l’activació� de� la� via� IL�

6/STAT3/SOCS3� i� l’aparició� de� RI� en� adipòcits� i� descriure� els� mecanismes� moleculars�

implicats�responsables�d’aquests�efectes.�

�

II. Avaluar� l’efecte� de� les� citocines� pro�inflamatòries� sobre� l’expressió� i� l’activitat� de�

PPAR�/��al�teixit�adipós�de�pacients�amb�obesitat�mòrbida�i�en�adipòcits�humans�en�cultiu.�

�

�

�

�

�

�

OBJECTIUS

65

�

�

�

�

�

�

�

�

�

�

�

�

�

RESULTATS�

�

�

�

�

67

�PUBLICACIÓ�1��

��

Activation�of�Peroxisome�Proliferator–Activated�Receptor�b/�d�(PPAR�b/�d)�Ameliorates�Insulin�

Signaling�and�Reduces�SOCS3�Levels�by�Inhibiting�STAT3�in�Interleukin�6–Stimulated�Adipocytes�

Lucía�Serrano�Marco,�Ricardo�Rodríguez�Calvo,�Ilhem�El�Kochairi,�Xavier�

Palomer,�Liliane�Michalik,�Walter�Wahli�and�Manuel�Vázquez�Carrera�

��

Diabetes�60:1990–1999,�2011�

RESULTATS

69

�La� DM2� s’ha� associat� amb� un� estat� inflamatori� crònic� de� baixa� intensitat.� A� més,� la� RI,� que�

precedeix� i� prediu� l’aparició� de� DM2,� es� correlaciona� amb� nivells� alts� de� marcadors�

inflamatoris� com� TNF�� i� IL�6�(Hotamisligil� i� col.,� 1993;� Bastard� i� col.,� 2002).� D’aquests�

marcadors� pro�inflamatoris,� la� IL�6� és� la� que� mostra� una� major� associació� amb� l’obesitat�

(Vozarova�i�col.,�2001).�

La� IL�6� a� través� del� seu� receptor� de� membrana� gp130� activa� la� via� JAK/STAT� que� resulta� en�

l’activació�de�la�transcripció�de�SOCS3,�una�proteïna�inhibidora�de�la�senyalització�per�citocines�

que�pot� inhibir� la�via�de�senyalització�de� la� insulina� (Emanuelli� i�col.,�2000;�Starr� i�col.,�1997)�

mitjançant�la�interacció�directa�amb�IRS�1�(Rui�i�col.,�2002).�

Ha� estat� descrit� que� els� PPARs� tenen� funcions� antiinflamatòries� mitjançant� la� reducció� de�

l’alliberament� de� factors� inflamatoris� o� per� estabilització� de� complexes� repressors� als�

promotors�dels�gens�inflamatoris�(Lee�i�col.,�2003;�Daynes�i�Jones,�2002).�Dels�tres�subtipus�de�

PPARs,� PPAR�� incrementa� el� catabolisme� dels� àcids� grassos� en� teixit� adipós� i� en� múscul�

esquelètic� i� redueix� el� procés� inflamatori,� raons� per� les� quals� ha� estat� proposat� com� una�

possible�diana�terapèutica�per�la�prevenció�de�la�RI�(Barish�i�col.,�2006).�Recentment�ha�estat�

publicat�que�l’activació�de�PPAR��per�l’agonista�GW501516�interfereix�amb�la�reacció�de�fase�

aguda�al� fetge�mitjançant� la� inhibició�de� l’activitat�transcripcional�d’STAT3�(Kino� i�col.,�2007),�

malgrat�que�els�mecanismes�implicats�es�desconeixen.�

Donada� la� importància� de� la� via� de� senyalització� STAT3�SOCS3� en� la� RI� mediada� per� IL�6� en�

adipòcits,�vam�decidir�avaluar��si� l’activació�de�PPAR��per�GW501516�evitava�la�RI�mediada�

per�IL�6�en�adipòcits�i�els�mecanismes�implicats.�Per�tal�d’assolir�aquest�objectiu�es�van�utilitzar�

en�aquest�estudi�ratolins�deficients�en�PPAR��adipòcits�diferenciats�3T3�L1�i�ratolins�tractats�

amb�IL�6.�

En� primer� lloc,� vam� observar� que� el� tractament� amb� IL�6� reduïa� la� captació� de� glucosa� i� la�

fosforilació� d’Akt� induïdes� per� insulina,� indicant� que� la� IL�6� induïa� RI,� però� la� incubació� amb�

l’agonista� de� PPAR��evitava� aquest� efecte.� El� GW501516� també� evitava� la� inducció� dels�

nivells�d’ARNm�de�SOCS3�i�dels�nivells�de�fosforilació�de�la�proteïna�STAT3�causats�per�la�IL�6.��

A�més,�l’acció�inhibidora�de�GW501516�sobre�l’activació�de�la�via�STAT3�induïda�per�la�IL�6��es�

va�confirmar�tant�en�teixit�adipós�blanc�de�ratolins�deficients�en�PPAR��com�en�teixit�adipós�

de�ratolins�tractats�amb�IL�6.�Els�primers�presentaven�un�increment�de�l’activitat�d’unió�a�l’ADN�

d’STAT3�i�del�seu�estat�de�fosforilació�comparats�amb�ratolins�salvatges.�En�els�ratolins�tractats�

amb� IL�6�es�va�observar�que�el�GW501516�revertia�els�efectes�de� la� IL�6� sobre�els�nivells�de�

RESULTATS

71

proteics� de� SOCS3� i� sobre� els� nivells� de� fosforilació� d’STAT3.� Per� altra� banda,� l’activació� de�

PPAR��promovia�la�dissociació�d’STAT3�de�Hsp90�en�adipòcits�mentre�que�aquesta�associació�

era�molt�més�elevada�en�ratolins�deficients�en�PPAR��en�comparació�als�ratolins�salvatges.�A�

més,� s’ha� demostrat� que� la� fosforilació� d’STAT3� en� el� residu� serina� 727� és� necessària� per� a�

adquirir� la�seva�màxima�activitat�transcripcional� i�una�de�les�cinases�responsables�de�dur�la�a�

terme� és� l’ERK1/2� (Decker� i� Kovarik,� 2000).� El� GW501516� va� evitar� la� inducció� de� l’ERK1/2�

induïda�per�la�IL�6.�

En�resum,�els�resultats�presentats�en�aquest�estudi�suggereixen�que�l’activació�de�PPAR��pot�

atenuar�la�RI�en�teixit�adipós�evitant�l’activació��de�la�via�STAT3�SOCS3�per�la�IL�6.�

�

�

�

�

�

�

�

�

�

�

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RESULTATS

72

Activation of Peroxisome Proliferator–ActivatedReceptor-b/-d (PPAR-b/-d) Ameliorates InsulinSignaling and Reduces SOCS3 Levels by InhibitingSTAT3 in Interleukin-6–Stimulated AdipocytesLucía Serrano-Marco,

1Ricardo Rodríguez-Calvo,

1Ilhem El Kochairi,

2Xavier Palomer,

1

Liliane Michalik,2Walter Wahli,

2and Manuel Vázquez-Carrera

1

OBJECTIVE—It has been suggested that interleukin (IL)-6 is oneof the mediators linking obesity-derived chronic inflammation withinsulin resistance through activation of STAT3, with subsequentupregulation of suppressor of cytokine signaling 3 (SOCS3). Weevaluated whether peroxisome proliferator–activated receptor(PPAR)-b/-d prevented activation of the IL-6-STAT3-SOCS3 path-way and insulin resistance in adipocytes.

RESEARCH DESIGN AND METHODS—Adipocytes and whiteadipose tissue from wild-type and PPAR-b/-d-null mice were usedto evaluate the effect of PPAR-b/-d on the IL-6-STAT3-SOCS3pathway.

RESULTS—First, we observed that the PPAR-b/-d agonistGW501516 prevented both IL-6–dependent reduction in insulin-stimulated Akt phosphorylation and glucose uptake in adipocytes.In addition, this drug treatment abolished IL-6–induced SOCS3expression in differentiated 3T3-L1 adipocytes. This effect wasassociated with the capacity of the drug to prevent IL-6–inducedSTAT3 phosphorylation on Tyr705 and Ser727 residues in vitro andin vivo. Moreover, GW501516 prevented IL-6–dependent inductionof extracellular signal–related kinase (ERK)1/2, a serine-threonine-protein kinase involved in serine STAT3 phosphorylation. Further-more, in white adipose tissue from PPAR-b/-d–null mice, STAT3phosphorylation (Tyr705 and Ser727), STAT3 DNA-binding activity,and SOCS3 protein levels were higher than in wild-type mice. Sev-eral steps in STAT3 activation require its association with heatshock protein 90 (Hsp90), which was prevented by GW501516as revealed in immunoprecipitation studies. Consistent with thisfinding, the STAT3-Hsp90 association was enhanced in whiteadipose tissue from PPAR-b/-d–null mice compared with wild-type mice.

CONCLUSIONS—Collectively, our findings indicate that PPAR-b/-d activation prevents IL-6–induced STAT3 activation by inhibit-ing ERK1/2 and preventing the STAT3-Hsp90 association, an effectthat may contribute to the prevention of cytokine-induced insulinresistance in adipocytes. Diabetes 60:1990–1999, 2011

Accumulating evidence suggests that type 2 di-abetes is associated with a cytokine-relatedacute-phase reaction, as part of an overall infla-mmatory state. Indeed, insulin resistance cor-

relates with increased acute-phase response marker levels,including tumor necrosis factor-a (TNF-a) (1), interleukin(IL)-1b (2), and IL-6 (3–5). Of these cytokines, IL-6 showsa strong association with obesity in both human and ro-dent models. Thus elevated levels of IL-6 in humans posi-tively correlate with obesity and insulin resistance andpredict the development of type 2 diabetes (5–7), whereasdepletion of IL-6 ameliorates insulin signaling in obesemice (8).IL-6 signals through a transmembrane receptor complex

containing the common signal transducing receptor gly-coprotein gp130, which activates Janus tyrosine kinases(Jak1, Jak2, Tyk2), with subsequent Tyr705 phosphoryla-tion of STAT3 (9–11). Phosphorylated STAT3 dimerizesand translocates to the nucleus, where it regulates thetranscription of target genes through binding to specificDNA-responsive elements (12). In addition to activation byTyr705 phosphorylation, STAT3 also requires phosphory-lation on Ser727 to achieve maximal transcriptional activity(13,14). Protein kinases involved in STAT3 serine phos-phorylation include protein kinase C, Jun NH2-terminalkinase, extracellular signal-regulated kinase (ERK), themitogen-activated protein kinase p38, and mammaliantarget of rapamycin (mTOR) (15). Interestingly, interactionof STAT3 with the chaperone heat shock protein 90 (Hsp90)contributes to many steps in STAT3 activation (16).Suppressor of cytokine signaling (SOCS) is a family of

target genes that are upregulated through IL-6–mediatedactivation of STAT3. These SOCS proteins were originallydescribed as cytokine-induced molecules involved in anegative feedback loop of cytokine (17) and insulin sig-naling (18). Several studies have reported that SOCS3 caninhibit insulin signaling (18–20) by direct interaction withthe insulin receptor and by preventing the coupling of in-sulin receptor substrate (IRS)-1 with the insulin receptor,thereby inhibiting IRS-1 tyrosine phosphorylation anddownstream insulin signaling (18,19). In addition, SOCS3inhibits insulin signaling by proteasomal-mediated degra-dation of IRS-1 (20). Thus overexpression of SOCS3 inadipocytes inhibits insulin signal transduction (19,21),whereas SOCS3 deficiency in adipocytes increases insulin-stimulated IRS-1 phosphorylation and glucose uptake (22).

From the 1Pharmacology Unit, Department of Pharmacology and TherapeuticChemistry, University of Barcelona, Institut de Biomedicina de la UB(IBUB), and CIBER de Diabetes y Enfermedades Metabólicas (CIBERDEM),Instituto de Salud Carlos III, Barcelona, Spain; and the 2Center for Integra-tive Genomics, National Research Center Frontiers in Genetics, Universityof Lausanne, Lausanne, Switzerland.

Corresponding author: Manuel Vázquez-Carrera, [email protected] 17 May 2010 and accepted 16 April 2011.DOI: 10.2337/db10-0704� 2011 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit,and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

1990 DIABETES, VOL. 60, JULY 2011 diabetes.diabetesjournals.org

ORIGINAL ARTICLE

Peroxisome proliferator–activated receptors (PPARs)are members of the nuclear receptor superfamily of ligand-inducible transcription factors that form heterodimerswith retinoid X receptors (RXRs) and bind to consensusDNA sites (23). In addition, PPARs may suppress infla-mmation through diverse mechanisms, such as reducedrelease of inflammatory factors or stabilization of repres-sive complexes at inflammatory gene promoters (24–27).Of the three PPAR isotypes found in mammals, PPAR-a(NR1C1) (28) and PPAR-g (NR1C3) are the targets forhypolipidemic (fibrates) and antidiabetic (thiazolidine-diones) drugs, respectively. Finally, activation of the thirdisotype, PPAR-b/-d (NR1C2, called PPAR-d below), en-hances fatty acid catabolism in adipose tissue and skeletalmuscle; therefore, it has been proposed as a potentialtreatment for insulin resistance (29). Recently, it was re-ported that agonist-activated PPAR-d interferes with IL-6–mediated acute phase reaction in the liver by inhibitingthe transcriptional activity of STAT3 (30), although theexact molecular mechanism involved remains unknown.Given the prominent role of the STAT3-SOCS3 pathway inIL-6–mediated insulin resistance in adipocytes, we ex-plored whether PPAR-d activation by GW501516 preventedIL-6–mediated insulin resistance in adipocytes and themechanisms involved. PPAR-d activation by GW501516prevented the reduction in insulin-stimulated Akt phos-phorylation and glucose uptake, indicating that this drugprevents IL-6–induced insulin resistance. In addition, wefound that this drug prevented IL-6–mediated induction ofSOCS3 mRNA levels and STAT3 phosphorylation in 3T3-L1adipocytes. Consistent with the role of PPAR-d in blockingIL-6–induced STAT3 activity, STAT3-DNA binding activityand STAT3 phosphorylation was higher in white adiposetissue from PPAR-d–null mice than in wild-type mice. Ourfindings also show that PPAR-d activation elicited STAT3dissociation from Hsp90 in adipocytes, whereas the asso-ciation of these two proteins was greatly enhanced inwhite adipose tissue in PPAR-d–null mice compared withwild-type mice. Overall, on the basis of our findings, wesuggest that PPAR-d activation can ameliorate insulin re-sistance in adipose tissue by preventing activation of theSTAT3-SOCS3 pathway by cytokines.

RESEARCH DESIGN AND METHODS

Materials. The PPAR-d ligand GW501516 was obtained from Biomol ResearchLaboratories (Plymouth Meeting, PA). Other chemicals were from Sigma(St. Louis, MO).Cell culture. 3T3-L1 preadipocytes [American Type Culture Collection(ATCC)] were grown to confluence in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 10% bovine calf serum. Two days after conflu-ence (day 0), differentiation of the 3T3-L1 cells was induced in DMEM con-taining 10% FBS, methylisobutylxanthine (500 mmol/L), dexamethasone (0.25mmol/L), and insulin (5 mg/mL) for 48 h. The cells were then incubated in 10%FBS/DMEM with insulin for 3 days and this was then replaced with FBS/DMEM. Medium was changed every 2 days. Fat droplets were observed inmore than 90% of cells after day 10. Adipocytes were then incubated with 10mmol/L GW501516 and IL-6 (10 or 100 ng/mL) for the times indicated. Afterincubation, RNA and total and nuclear protein extracts were extracted fromadipocytes as described below. Inhibitors were added 30 min before in-cubation with IL-6.Animals. Obese male ZDF rats (ZDF/Gmi, fa/fa) and their lean littermates (fa/+or +/+) were used. Both strains were maintained under standard light-dark cycle(12-h light/dark cycle) and temperature (21 6 1°C) conditions and fed withPurina 5008 chow. Male ZDF and lean rats were killed at 12 weeks of age. CD-1male mice (12 weeks old) were treated for 48 h with vehicle (100 mL PBS-0.1%BSA and 0.5% w/v carboxymethylcellullose medium viscosity), IL-6 (0.8 mg/gbody wt i.p.), or IL-6 plus GW501516 (one daily oral gavage of 3 mg/kg/dayGW501516 dissolved in carboxymethylcellullose). Epididymal white adipose

tissue of rats and mice was rapidly removed, frozen in liquid nitrogen, andstored at 280°C. All procedures were conducted in accordance with the prin-ciples and guidelines established by the University of Barcelona BioethicsCommittee, as stated in Law 5/1995, 21 July, passed by the Generalitat deCatalunya.

The generation of PPAR-d–null mice was described previously (31). Eightmale PPAR-d–null mice and eight of their control male PPAR-d wild-typemice were used (5 to 6 months of age). In agreement with the guidelinesspecified by the veterinary office of Lausanne (Switzerland), the mice werehoused under standard light-dark cycle (12-h light/dark cycle) and temper-ature (21 6 1°C) conditions and fed with Provimi Kliba 3436 chow. Epidid-ymal white adipose tissue was rapidly removed, frozen in liquid nitrogen, andstored at 280°C.2-Deoxy-D-[

14C]glucose uptake experiment. Determination of 2-Deoxy-D-

[14C]glucose (2-DG) uptake was performed as reported elsewhere (32).Measurements of mRNA. Levels of mRNA were assessed by RT-PCR aspreviously described (33). Total RNA was isolated using the Ultraspec reagent(Biotecx, Houston, TX). The total RNA isolated by this method is nondegradedand free of protein and DNA contamination. The sequences of the sense andantisense primers used for amplification were: Socs3 (suppressor of cytokinesignaling 3) 59-TTTTCGCTGCAGAGTGACCCC-39 and 59-TGGAGGAGAGAGGTCGGCTCA-39; early growth response (Egr-1), 59-CTTCCTCTGCCTCCCA-GAGCC-39 and 59-TGGGAACCTGGAAACCACCCT-39, and Aprt (adenosylphosphoribosyl transferase), 59-GCCTCTTGGCCAGTCACCTGA-39 and 59-CCA-GGCTCACACACTCCACCA-39. Amplification of each gene yielded a single bandof the expected size (Socs3: 250 bp, Egr-1: 210 bp, and Aprt: 329 bp). Preliminaryexperiments were carried out with various amounts of cDNA to determinenonsaturating conditions of PCR amplification for all the genes studied. Underthese conditions, relative quantification of mRNA was then assessed by the RT-PCR method used in this study (34). Radioactive bands were quantified by video-densitometric scanning (Vilbert Lourmat Imaging). The results for the expressionof specific mRNAs are always presented relative to the expression of the controlgene (Aprt).Isolation of nuclear extracts. Nuclear extracts were isolated as previouslydescribed (35). Cells were scraped into 1.5 mL of cold PBS, pelleted for 10 s,and resuspended in 400 mL cold Buffer A (10 mM HEPES [pH 7.9 at 4°C],1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, and 5 mg/mL apro-tinin) by flicking the tube. Cells were allowed to swell on ice for 10 min andthen vortexed for 10 s. Samples were then centrifuged for 10 s, and the su-pernatant fraction was discarded. Pellets were resuspended in 50 mL of coldBuffer C (20 mM HEPES-KOH [pH 7.9 at 4°C], 25% glycerol, 420 mM NaCl,1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 5 mg/mL apro-tinin, and 2 mg/mL leupeptin) and incubated on ice for 20 min for high-saltextraction. Cellular debris was removed by centrifugation for 2 min at 4°C,and the supernatant fraction (containing DNA-binding proteins) was storedat 280°C. Nuclear extract concentration was determined by the Bradfordmethod.Electrophoretic mobility shift assay. Electrophoretic mobility shift assay(EMSA) was performed using double-stranded oligonucleotides (Santa Cruz) forthe consensus binding site of the STAT3 nucleotide (59-GATCCTTCTGGGAA-TTCCTAGATC-39). Oligonucleotides were labeled in the following reaction: 2mL of oligonucleotide (1.75 pmol/mL), 2 mL 53 kinase buffer, 1 mL T4 poly-nucleotide kinase (10 units/mL), and 2.5 mL [g-32P]ATP (3,000 Ci/mmol at10 mCi/mL) incubated at 37°C for 2 h. The reaction was stopped by adding 90 mLof TE buffer (10 mmol/L Tris-HCl [pH 7.4] and 1 mmol/L EDTA). To separate thelabeled probe from the unbound ATP, the reaction mixture was eluted in a Nickcolumn (Amersham) following the manufacturer’s instructions. Crude nuclearprotein (mg) was incubated for 10 min on ice in binding buffer (10 mmol/L Tris-HCl [pH 8.0], 25 mmol/L KCl, 0.5 mmol/L DTT, 0.1 mmol/L EDTA [pH 8.0], 5%glycerol, 5 mg/mL BSA, and 50 mg/mL poly[dI-dC]), in a final volume of 15 mL.Labeled probe (~75,000 cpm) was added, and the reaction was incubated for30 min at 4°C. Where indicated, specific competitor oligonucleotide was addedbefore the labeled probe and incubated for 20 min on ice. STAT3 antibody wasadded 15 min before incubation with the labeled probe at 4°C. Protein-DNAcomplexes were resolved by electrophoresis at 4°C on a 5% acrylamide gel andsubjected to autoradiography.Antibodies, immunoprecipitation, and immunoblotting. Antibodies againsttotal and phospho-ERK1/2 and phospho-STAT3 (Tyr705 and Ser727) were pur-chased from Cell Signaling. Antibodies against total STAT3 and Hsp90 werepurchased from Santa Cruz.

To obtain total protein, cells and adipose tissue were homogenized inradioimmunoprecipitation assay (RIPA) buffer (Sigma) with phosphataseinhibitors (0.2 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodiumorthovanadate, 5.4 mg/mL aprotinin). The homogenate was centrifuged at16,700g for 30 min at 4°C. Protein concentration was measured by theBradford method.

L. SERRANO-MARCO AND ASSOCIATES

diabetes.diabetesjournals.org DIABETES, VOL. 60, JULY 2011 1991

Whole-cell lysates and nuclear extracts were mixed with various antibodies(as specified under RESULTS) and protein A coupled to agarose beads. Proteinsfrom whole-cell lysates, nuclear extracts, and immunoprecipitates were sepa-rated by SDS-PAGE and then transferred to immobilon polyvinylidene diflouridemembranes (Millipore, Bedford, MA) and blotted with various antibodies (asspecified in RESULTS). Detection was achieved using the EZ-ECL chemilumi-nescence kit (Amersham). Size of detected proteins was estimated using proteinmolecular-mass standards (Invitrogen, Barcelona, Spain).Statistical analyses. Data are presented as mean 6 SD of five separateexperiments. Significant differences were established by one-way ANOVA,using the GraphPad InStat program (GraphPad Software V2.03, GraphPadSoftware, San Diego, CA). When significant variations were found, the Tukey-Kramer multiple comparisons test was applied. Differences were consideredsignificant at P , 0.05.

RESULTS

PPAR-d activation restores IL-6 defects in Akt acti-vation and glucose uptake in response to insulin. Wefirst examined the effects of the PPAR-d agonist GW501516on IL-6–induced insulin resistance, which was assessed asthe inhibition of insulin-stimulated Akt phosphorylation andglucose uptake. Differentiated 3T3-L1 adipocytes werestimulated with IL-6 in the absence or in the presence ofGW501516, a selective ligand for PPAR-d with a 1,000-foldhigher affinity toward PPAR-d than PPAR-a and PPAR-g(36). Exposure of adipocytes to IL-6 for 24 h caused

a reduction in insulin-stimulated Akt phosphorylation (Fig.1A). In contrast, when cells preincubated with 10 mmol/LGW501516 were exposed to IL-6 the inhibitory effect of thiscytokine on insulin-stimulated Akt phosphorylation wasprevented. The effect of GW501516 on insulin-stimulatedAkt phosphorylation was also observed at lower concen-trations and the effect attained at 10 mmol/L was dependenton PPAR-d since it was abolished by coincubation with thePPAR-d antagonist GSK0660 (Fig. 1B). Similarly, GW501516significantly reversed the reduction observed in glucoseuptake in IL-6–stimulated cells (Fig. 1C). Drug treatment inthe absence of insulin did not affect the phosphorylationstatus of Akt (data not shown). Thus GW501516 treatmentoffered protection against a reduction in insulin re-sponsiveness by IL-6.PPAR-d activation inhibits IL-6–induced SOCS3expression in 3T3-L1 adipocytes by preventing STAT3activation. Because IL-6–induced insulin resistance in3T3-L1 adipocytes has been attributed to SOCS3 (22), wethen examined the effect of PPAR-d activation on themRNA levels of the STAT3-target gene SOCS3. Differen-tiated 3T3-L1 adipocytes were stimulated with 10 ng/mLof IL-6 for 1 h in the absence or in the presence ofGW501516. Under these conditions, the increase in SOCS3

FIG. 1. PPAR-d activation antagonizes IL-6 action by restoring insulin responsiveness. Differentiated adipocytes were stimulated with 100 nmol/L

insulin for 30 min, with or without pretreatment with either 10 mmol/L GW501516 or 100 ng/mL IL-6 for 24 h. A: Cell lysates were subjected to

Western blot analysis for phospho-Akt(Ser473

) and total Akt and b-actin. B: Different concentrations of GW501516 were assayed on insulin-stimulated Akt-phosphorylation in cells exposed to IL-6. Indicated cells were pretreated for 30 min with 10 mmol/L GSK0660 before treatment with

GW501516. ***P < 0.001 vs. control cells stimulated with insulin; ###P < 0.001 vs. IL-6-stimulated cells in the presence of insulin. C: 2-Deoxyglucoseuptake was assessed without or with insulin. Values are means 6 SD of six independent experiments. ***P < 0.001 vs. control cells without insulin

stimulation; ###P < 0.001 vs. control cells stimulated with insulin; @P < 0.05 vs. IL-6–exposed cells.

PPAR-d INHIBITS STAT3 IN ADIPOCYTES


Aprt

Socs3

A

CTIL-6

IL-6+GW50

1516

IL-6+GELD GW

0

1

2

3

4 ***

###

@

Socs

3m

RNA

leve

ls (A

.U.)

CTIL-6

IL-6+GW50

1516

GW5015

160.0

0.5

1.0

1.5 *

#

phos

pho-

STAT

3-Ty

r705

prot

ein

leve

ls (A

.U.)

CTIL-6

IL-6+GW50

1516

GW5015

160.00

0.25

0.50

0.75

1.00 **

###

phos

pho-

STAT

3-Se

r727

prot

ein

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ls (A

.U.)

B

phospho-STAT3-Tyr705

phospho-STAT3-Ser727

STAT3

C

D

CTIL-6

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1516

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160

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2

3

4

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Egr-

1m

RNA

leve

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phospho-ERK1/2

ERK1/2

Aprt

Egr-1

CT GW501516

FIG. 2. The PPAR-d agonist GW501516 prevents IL-6–induced SOCS3 expression and STAT3 phosphorylation in 3T3-L1 adipocytes. A: Analysis of

the mRNA levels of Socs3 in serum-starved differentiated adipocytes untreated or treated with 10 mmol/L GW501516 for 24 h or 2 mmol/Lgeldanamycine for 30 min before stimulation with 10 ng/mL IL-6 for 1 h. Total RNA was isolated and analyzed by RT-PCR. A representative au-

toradiogram and the quantification normalized to the AprtmRNA levels are shown. Data are the means6 SD of five independent experiments. 3T3-L1 adipocytes were treated with 10 mmol/L GW501516 for 24 h before stimulation with 10 ng/mL IL-6 for 24 h. Nuclear (B) or total cell extracts (C)

were subjected to Western blot analysis with phospho-STAT3 (Tyr705

and Ser727

) or STAT3 antibodies (B) or phospho-ERK1/2 and ERK1/2 (C)antibodies. D: Analysis of the mRNA levels of Egr-1 in 3T3-L1 serum-starved differentiated adipocytes untreated or treated with 10 mmol/L

GW501516 for 24 h before stimulation with 10 ng/mL IL-6 for 1 h. Total RNA was isolated and analyzed by RT-PCR. Bars are the means 6 SD of five

independent experiments. *P< 0.05; **P< 0.01; ***P< 0.001 vs. control; @P< 0.05 vs. IL-6+GW501516-exposed cells; #P< 0.05 and ###P< 0.001vs. IL-6–stimulated cells. Dividing lines indicate grouping of images from different parts of the same gel.



mRNA levels caused by IL-6 exposure (23-fold induction;P , 0.001) was reduced in cells coincubated with IL-6plus GW501516 (sevenfold induction; P , 0.001 vs. IL-6–stimulated cells) (Fig. 2A). Furthermore, because of re-cent reports showing suppression of IL-6 signaling throughinhibition of STAT3-Hsp90 interaction by the selectiveHsp90 inhibitor geldanamycin (16,37,38), we also evalu-ated the effects of the latter. Geldanamycin treatmentsignificantly reduced the increase in SOCS3 mRNA levelscaused by IL-6 (P , 0.05 vs. IL-6–stimulated cells). Overall,these findings suggest that PPAR-d activation inhibitsSTAT3. Dimerization, nuclear translocation, and increasein transcriptional activity of STAT3 require its phos-phorylation on tyrosine residue 705. In addition, STAT3phosphorylation on Ser727 is required for its maximalactivation (13,14). When we analyzed the phosphoryla-tion status of STAT3 we observed that IL-6 exposure in-creased both Tyr705 and Ser727 phosphorylation, whereasin the presence of GW501516 these changes were pre-vented (Fig. 2B). Because IL-6 activates ERK1/2 (10), whichhas been reported to be a kinase for STAT3 phosphory-lation on Ser727 (15), and we have previously reportedthat GW501516 prevents LPS-induced ERK1/2 activationin adipocytes (36), we evaluated the effect of the PPAR-dagonist on the activation of this kinase. IL-6 exposurecaused a slight increase in ERK1/2 phosphorylation,whereas GW501516 strongly suppressed ERK1/2 proteinphosphorylation (Fig. 2C). Consistent with these changes,

the increase in Egr-1 mRNA levels, which has been at-tributed to IL-6–mediated activation of ERK1/2 (10), wasabolished by GW501516 (Fig. 2D). To demonstrate theinvolvement of the ERK1/2 activation in IL-6–inducedinsulin resistance in adipocytes, we took advantage ofU0126, a potent and specific ERK1/2 inhibitor, which bindsto mitogen-activated protein kinase (MAPK)–ERK1/2(MEK1/2), thereby inhibiting its catalytic activity as wellas phosphorylation of ERK1/2. Similarly to GW501516,U0126 prevented the reduction in insulin-stimulated Aktphosphorylation (Fig. 3A) and glucose-uptake (Fig. 3B) andprevented the increase in STAT3 phosphorylation on Ser727

(Fig. 3C) caused by IL-6. U0126 treatment alone did notaffect the phosphorylation status of Akt (data not shown).GW501516 prevents the increase in SOCS3 proteinlevels and STAT3 activity induced by IL-6 in vivo. Wehave previously reported that PPAR-d expression andactivity is reduced, whereas ERK1/2 phosphorylation isincreased in white adipose tissue from an animal modelof obesity and insulin resistance, the ZDF (fa/fa) rat(36). In the current study we found that STAT3 phos-phorylation at Ser727 and SOCS3 protein levels were in-creased in white adipose tissue of ZDF rats comparedwith lean animals (Fig. 4A and B). Hence, we hypothe-sized that reduced PPAR-d activity and enhanced ERK1/2activation may contribute to the increase in STAT3 ac-tivity in the white adipose tissue of the ZDF rat. We thenevaluated whether GW501516 might prevent the increase

FIG. 3. ERK1/2 inhibition prevents IL-6–induced insulin resistance and STAT3 phosphorylation on Ser727

. Differentiated adipocytes were stimu-

lated with 100 nmol/L insulin for 30 min, with or without pretreatment with either 10 mmol/L U0126, 10 mmol/L GW501516, or 100 ng/mL IL-6 for

24 h. A: Cell lysates were subjected to Western blot analysis for phospho-Akt(Ser473

) and total Akt and b-actin. B: 2-Deoxyglucose uptakewas assessed without or with insulin. Values are means 6 SD of six independent experiments. C: Nuclear cell extracts were subjected to Western

blot analysis with phospho-STAT3 (Ser727

), STAT3, or Lamin B antibodies. ***P< 0.001 vs. control cells without insulin stimulation; ##P< 0.01 vs.control cells stimulated with insulin; @@P < 0.01 vs. IL-6–exposed cells. Dividing lines indicate grouping of images from different parts of the

same gel.



FIG. 4. The PPAR-d agonist GW501516 prevents IL-6–induced SOCS3 expression and STAT3 phosphorylation in white adipose tissue. Phospho-

STAT3 (Ser727

) and SOCS3 protein levels are increased in white adipose tissue of ZDF rats. A: Analysis of phospho-STAT3 (Ser727

) and totalSTAT3 by immunoblotting of nuclear or total protein extracts from white adipose tissue of lean and ZDF rats. B: Total cell extracts from white

adipose tissue of lean and ZDF rats were subjected to Western blot analysis with SOCS3 and b-actin antibodies. Mice were treated for 48 h withvehicle, IL-6, or IL-6 plus GW501516. SOCS3 (C), phospho-STAT3 (Tyr

705and Ser

727) (D), and phospho-ERK1/2 (E) protein levels. Nuclear

(phospho-STAT3-Ser727

) or total cell extracts were subjected to Western blot analysis with phospho-STAT3 (Tyr705

) or STAT3 antibodies or phospho-

ERK1/2 and ERK1/2 antibodies. Bars are the means6 SD of four independent experiments. ***P< 0.001; **P< 0.01 vs. control; ###P< 0.001 vs.IL-6–treated mice. Dividing lines indicate grouping of images from different parts of the same gel.



in STAT3-SOCS3 pathway in white adipose tissue afterIL-6 stimulation in vivo in a similar fashion as observedin vitro. When we examined the SOCS3 protein levels inmice exposed to IL-6 (Fig. 4C) we observed that the in-crease caused by IL-6 treatment was prevented in thosemice treated with the PPAR-d agonist. Similarly, drugtreatment prevented the increase in the phosphorylationstatus of STAT3 in both Tyr705 and Ser727 (Fig. 4D). In agree-ment with data obtained in vitro, GW501516 inhibited theincrease in phospho-ERK1/2 levels induced by IL-6 treatment(Fig. 4E).Increased STAT3 activity in the white adipose tissueof the PPAR-d–null mouse. To clearly demonstrate theinvolvement of PPAR-d in the regulation of STAT3 activity inwhite adipose tissue we took advantage of the PPAR-d–nullmouse. In the absence of PPAR-d, Tyr705 and Ser727 phos-phorylation of STAT3 and SOCS3 protein levels were in-creased compared with wild-type mice (Fig. 5A). Consistentwith these changes, the DNA-binding activity of STAT3 wasstrongly increased in nuclear extracts of white adipose tis-sue of PPAR-d–null mice compared with wild-type animals(Fig. 5B).PPAR-d elicits STAT3 dissociation from Hsp90. Hsp90is thought to contribute to many steps in STAT3 activation,such as binding to its docking sites on gp130 and sub-sequent phosphorylation by associated JAKs, as well asenhanced trafficking of the activated cytosolic STAT3 to the

nucleus (16,37). Thus we examined whether PPAR-d sup-pressed IL-6 signaling through inhibition of STAT3-Hsp90interaction. This interaction was studied by using nuclearextracts of 3T3-L1 adipocytes stimulated with IL-6 in thepresence or in the absence of GW501516 and the Hsp90inhibitor geldanamycin, which were immunoprecipitatedwith anti-Hsp90, and analyzed by Western blot (Fig. 6A). IL-6stimulation caused an increase in the interaction of Hsp90with STAT3, whereas in the presence of GW501516 orgeldanamycin the IL-6–induced recruitment of Hsp90 toSTAT3 was blocked. Consistent with this, the associationof STAT3 with Hsp90 was very faint in white adipose tissueof wild-type mice but strongly increased in PPAR-d–nullmice (Fig. 6B). Overall, these findings indicate that PPAR-dmay inhibit IL-6–induced STAT3 activation by promotingSTAT3 dissociation from Hsp90.

DISCUSSION

Insulin resistance and type 2 diabetes are closely associ-ated with low-grade chronic inflammation characterizedby abnormal proinflammatory cytokine production (39),such as TNF-a (1) and IL-6 (4,5,21). Of these cytokines,IL-6 plasma levels correlate more strongly with the sever-ity of insulin resistance in insulin-resistant patients thanwith TNF-a (3,6). Adipocytes play an important role inIL-6–induced insulin resistance since adipose tissue is an

FIG. 5. The PPAR-d–null mouse shows STAT3 activation and enhanced SOCS3 protein levels in white adipose tissue. A: Cellular extracts from wild-type (WT) or PPAR-d–null (knockout [KO]) mouse white adipose tissue were analyzed by Western blot with phospho-STAT3 (Tyr

705and Ser

727),

STAT3, SOCS3, and b-actin antibodies as indicated. B: Autoradiograph of EMSA performed with a32P-labeled STAT3 nucleotide and nuclear

protein extracts (NE). One specific complex (I), based on competition with a molar excess of unlabeled probe, is shown. An analysis performed byincubating NE with an antibody directed against STAT3 is also shown.



important source (40) and a major target of this cytokine.IL-6 acts primarily by activating STAT3 and upregulatingthe transcription of its target gene SOCS3, which causesinsulin resistance by interfering with insulin receptors and/or IRS-1 (19,41–43). Our findings demonstrate that PPAR-dactivation by GW501516 prevents IL-6–induced expressionof SOCS3 in adipocytes, showing a similar effect to thatreported for the PPAR-g activators rosiglitazone (21) andpioglitazone (44). These data suggest that PPAR-d preventsSTAT3 activation in adipocytes, which is in agreementwith a previous report showing that GW501516 preventedIL-6–induced STAT3 activation in hepatocytes (30), althoughin this latter study the molecular mechanism involved wasnot elucidated. The activity of STAT3 is dependent onits phosphorylation status. Thus STAT3 phosphorylation onTyr705 leads to dimerization and translocation to the nucleus,where it regulates the transcription of its target genes (12),whereas phosphorylation of Ser727 is required to achievemaximal activity (13,14). Our findings show that the PPAR-dactivator GW501516 inhibits IL-6–induced STAT3 phosphor-ylation on both residues. The increase in STAT3 phosphor-ylation, STAT3 DNA-binding activity, and SOCS3 proteinlevels in white adipose tissue from PPAR-d2null mice com-pared with wild-type animals clearly indicates that PPAR-dinhibits STAT3 activation in adipocytes. Interestingly, whiteadipose tissue from ZDF rats showed increased STAT3serine phosphorylation and SOCS3 protein levels. Thesechanges could be related to the reduction in PPAR-d ex-pression observed in white adipose tissue of ZDF rats (36).In addition, because overexpression of SOCS3 in adiposetissue causes local but not systemic insulin resistance (45),these findings suggest that the increase in SOCS3 levels inwhite adipose tissue from ZDF rats might, at least, exacer-bate insulin resistance in this tissue, thereby contributing tothe metabolic alterations observed in this animal model oftype 2 diabetes.The prevention of IL-6–induced STAT3 activation after

PPAR-d activation might be the result of several mecha-nisms of action. First, protein kinases responsible for STAT3serine phosphorylation include, among others, ERK1/2 (46),and we have previously reported that GW501516 inhibitsERK1/2 phosphorylation in adipocytes (36). GW501516abolished both ERK1/2 phosphorylation and the expres-sion of Egr-1, which is upregulated after ERK1/2 activation(10), suggesting that inhibition of this protein kinase byPPAR-d activation might be involved in the suppression ofSTAT3 Ser727 phosphorylation in IL-6–exposed cells. These

data are consistent with previous studies reporting that theMEK/ERK inhibitor PD98059 suppresses STAT3 serinephosphorylation, whereas STAT3 tyrosine phosphoryla-tion was blunted by the JAK2 inhibitor AG490 (47). How-ever, our findings also showed a reduction of STAT3 Tyr705

phosphorylation after GW501516 treatment, suggesting thatadditional mechanisms were involved. This second mecha-nism might involve a reduction in the interaction of STAT3with Hsp90. In fact, activation of STAT3 requires its asso-ciation with Hsp90 in many steps, including binding to itsdocking sites on gp130 and subsequent phosphorylation byassociated JAKs and its translocation to the nucleus (16).Thus it has been reported that both geldanamycin, a selec-tive Hsp90 inhibitor, and pyrrolidine dithiocarbamate(PDTC) inhibit STAT3 tyrosine and serine phosphorylationand its translocation to the nucleus by reducing the asso-ciation of STAT3 with Hsp90 (16). Our immunoprecipita-tion studies in adipocytes exposed to IL-6 indicate thatPPAR-d activation by GW501516 dissociates Hsp90 fromSTAT3, a mechanism that may prevent STAT3 activationby phosphorylation. In agreement with this, white adiposetissue from PPAR-d knockout mice showed enhancedSTAT3 phosphorylation and DNA-binding activity. Ourdata do not explain how PPAR-d activation reduces thephysical interaction between STAT3 and Hsp90. However,because it has been reported that PPAR-d interacts withHsp90 (48), it is likely that competition between PPAR-dand STAT3 for binding to Hsp90 may reduce the availabilityof this heat shock protein to interact with STAT3.The mechanisms of action responsible for STAT3 in-

hibition by PPAR-d agonists reported here are different tothat previously reported for PPAR-g activators (49,50).Thus ligand binding to PPAR-g promotes its dissociationfrom the corepressor silencing mediator for retinoid andthyroid hormone receptors (SMRT), which, in turn, inter-acts with STAT3 and inhibits its transcriptional activity. Itremains a matter of further study whether PPAR-g ligandsinhibit STAT3 by promoting its dissociation from Hsp90.However, this possibility seems unlikely since it has beenreported that, in contrast with PPAR-d and PPAR-a, PPAR-gdoes not interact with Hsp90 (48). Further studies arenecessary to evaluate whether PPAR-d–mediated inhibitionof STAT3 involves enhanced interaction between SMRT andSTAT3.It has been suggested that the proinflammatory cytokine

IL-6 is one of the mediators linking obesity-derived chronicinflammation with insulin resistance. In addition, it haspreviously been reported that STAT3 activation is requiredfor IL-6 inhibition of insulin signaling in hepatocytes (51)and that the negative effect of IL-6 on insulin signaling inadipocytes is linked to the upregulation of SOCS3 (21).Thus we wanted to explore whether PPAR-d activationprevented IL-6–induced insulin resistance in adipocytes. Inagreement with the reported inhibition of the STAT3-SOCS3 by GW501516, PPAR-d activation by this drugprevented the reduction in insulin-stimulated Akt phos-phorylation and glucose uptake caused by IL-6 exposure.These findings suggest that the inhibition of STAT3 and thesubsequent reduction in SOCS3 levels after PPAR-d acti-vation in IL-6–stimulated adipocytes might contribute to-ward preventing IL-6–induced insulin resistance. Giventhat impairment of insulin resistance in adipocytes by ex-posure to TNF-a (52) and IL-1a (53) has been largely as-sociated with IL-6 production and SOCS3 induction, it islikely that the effects of PPAR-d on the improvement ininsulin sensitivity can also be extended to these cytokines.

FIG. 6. PPAR-d activation dissociates the complex formed between

Hsp90 and STAT3. A: Differentiated adipocytes untreated or treatedwith 10 mmol/L GW501516 for 24 h or 2 mmol/L geldanamycine for

30 min before stimulation with 10 ng/mL IL-6 for 24 h. Nuclear extracts

were immunoprecipitated (IP) with anti-Hsp90 followed by Westernblot analysis using antibodies directed against STAT3.B: Cellular extractsfrom wild-type (WT) or PPAR-d–null (KO) mouse white adipose tissuewere immunoprecipitated (IP) with anti-STAT3 followed by Western blot

analysis using antibodies directed against Hsp90 or STAT3.



In summary, on the basis of our findings in adipocytes,we suggest that PPAR-d activation prevents IL-6–inducedSTAT3 activation and SOCS3 upregulation, thereby con-tributing toward preventing the cytokine-mediated devel-opment of insulin resistance.

ACKNOWLEDGMENTS

This study was partly supported by funds from the SwissNational Science Foundation, the Spanish Ministerio deCiencia e Innovación (SAF2006-01475 and SAF2009-06939),and European Union ERDF funds. CIBER de Diabetes yEnfermedades Metabólicas (CIBERDEM) is an Instituto deSalud Carlos III project. L.S.-M. was supported by an FPIgrant from the Spanish Ministerio de Ciencia e Innovación.R.R.-C. was supported by a grant from the FundaciónRamón Areces.No potential conflicts of interest relevant to this article

were reported.L.S.-M., R.R.-C., and I.E.K. performed experiments. X.P.

performed experiments and contributed to discussion.L.M. and W.W. developed the PPAR-b/-d–null mice andcontributed to discussion. M.V.-C. wrote the manuscriptand performed experiments.The authors thank the University of Barcelona’s Lan-

guage Advisory Service for its help.

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�PUBLICACIÓ�2��

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TNF-� inhibits PPAR�/� activity and SIRT1 expression through NF-�B in human adipocytes

Lucía�Serrano�Marco,�Matilde�R.�Chacón,�Elsa�Maymó�Masip,�Lourdes�Garrido�Sánchez,�

Francisco�J.�Tinahones,�Xavier�Palomer,�Joan�Vendrel,�Manuel�Vázquez�Carrera�

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(En avaluació)

RESULTATS

83

L’obesitat,� la�RI� i� la�DM2� i� s’associen�a�un�estat� inflamatori� crònic�de�baixa� intensitat�que� es�

caracteritza� per� la� producció� anormal� de� citocines� (Hotamisligil,� 2006).� Els� adipòcits� són�

productors� d’adipocines� com�el� TNF��,� la� IL�6�o� la�MCP�1.� La� secreció�d’aquestes�adipocines�

produeix�l’activació�de�diverses�cascades�de�senyalització�involucrades�en�la�RI�induïdes�per�la�

presència�d’obesitat�(Tataranni�i�Ortega,�2005).�Diversos�estudis�han�suggerit�que�el�factor�de�

transcripció�NF��B�forma�part�d’aquestes�vies�inflamatòries�que�relacionen�l’obesitat�amb�la�RI�

(Barbarroja� i� col.,� 2010).� En� la� seva� forma� inactiva,� aquest� factor� de� transcripció� es� troba� al�

citoplasma�unit�a�proteïnes�inhibidores�I�B.�En�resposta�a�estímuls�com�el�TNF��les�proteïnes�

inhibidores�són�degradades�al�proteasoma,�alliberant�se�NF��B�que� llavors�transloca� �al�nucli�

on�activa�la�transcripció�dels�seus�gens�diana�com�la�IL�6�i�el�TNF��(Baldwin,�2004).�

Els� mecanismes� que� vinculen� la� RI� induïda� per� obesitat� amb� un� estat� inflamatori� crònic� de�

baixa�intensitat�es�coneixen�parcialment.�Per�això,�el�descobriment�dels�factors�claus�implicats�

en� aquesta� associació� pot�permetre�arribar�a�establir�noves�dianes� farmacològiques� per�a� la�

prevenció� d’aquesta� patologia.� Entre� aquests� nous� factors� ha� estat� proposat� que� PPAR��

podria�arribar�a�convertir�se�en�una�diana�potencial�pel�tractament�de�la�RI�(Barish�i�col.,�2006;�

Barroso�i�col.,�2011;�Rodriguez�Calvo�i�col.,�2008;�Coll�i�col.,�2010).�De�fet,�ha�estat�demostrat�

que� l’activació� d’aquest� receptor� en� adipòcits� evita� el� procés� inflamatori� induït� per� LPS�

(Rodriguez�Calvo�i�col.,�2008),�fet�que�podria�contribuir�a�evitar�l’aparició�de�RI.�Un�altre�factor�

important�que�vincula�la� inflamació�amb�la�RI� induïda�per�obesitat�es�SIRT1,�una�desacetilasa�

que� regula� l’activitat�de� NF��B� i�millora� la� sensibilitat�a� la� insulina� (Yoshizaki� i� col.,�2009).�És�

interessant� comentar� que� s’ha� descrit� que� l’activació� de� PPAR��incrementa� l’expressió� de�

SIRT1�(Okazaki�i�col.,�2010).�

En�aquest�estudi�es�van�avaluar�els�nivells�de�PPAR��,�de�SIRT1�i�la�translocació�al�nucli��de�p65�

en� teixit� adipós� visceral� (TAV)� de� pacients� insulino�resistents� obesos� respecte� d’individus�

control.�Els�nostres�resultats�demostren�que�els�pacients�amb�RI� i�obesos�presentaven�nivells�

més�alts�de�citocines�pro�inflamatòries�al�TAV�a�més�d’una�disminució�dels�nivells�proteics�de�

SIRT1� i� un� increment� dels� nivells� d’ARNm� de� PPAR�� El� tractament� in� vitro� d’adipòcits�

humans�SGBS�amb�TNF��reproduïa�aquestes�condicions,�malgrat�que�l’augment�observat�en�

l’expressió�de�PPAR�/��s’acompanyava�de�la�reducció�de�l’expressió�dels�seus�gens�diana�i�de�la�

seva� activitat� d’unió� a� l’ADN.� Aquest� fet� podria� indicar� una� possible� reducció� de� l’activitat�

PPAR,�que�es�va�confirmar�amb�la�reducció�de�l’activitat�d’unió�a�l’ADN�de�PPAR�en�presència�

de�TNF��.�El�tractament�amb�l’agonista�de�PPAR�/�,�GW501516,�o�amb�un�inhibidor�de�NF��B�

RESULTATS

85

evitava� els� canvis� produïts� per� TNF�� Aquests� resultats� indicaven� que� TNF��modula�

l’expressió�i�l’activitat�PPAR�/��mitjançant�l’activació�de�NF��B.�

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1

TNF-α inhibits PPARβ/δ activity and SIRT1 expression through NF-κB in human adipocytes 1

2

Running title: TNF-α inhibits PPARβ/δ activity and SIRT1 via NF-κB 3

Lucía Serrano-Marco1,2, Matilde R. Chacón2,3*, Elsa Maymó-Masip2,3, Lourdes Garrido-4

Sánchez2,3, Francisco J. Tinahones4, Xavier Palomer1,2, Joan Vendrell2,3, Manuel Vázquez-5

Carrera1,2* 6

1Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry and Institut de 7

Biomedicina de la UB (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain, 8

2CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)-Instituto de Salud 9

Carlos III, Barcelona, Spain, 3Endocrinology and Diabetes Unit, Research Department, University 10

Hospital of Tarragona Joan XXIII, Pere Virgili Institute, and Rovira i Virgili University, Tarragona, 11

Spain, 4Endocrinology and Nutrition Unit, Virgen de la Victoria Hospital (Fundación IMABIS) and 12

CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN)-Instituto de Salud Carlos III, 13

Málaga. 14

*Corresponding authors: 15

Manuel Vázquez-Carrera. Unitat de Farmacologia. Facultat de Farmàcia. Diagonal 643. E-08028 16

Barcelona. Spain. Phone 93 4024531. Fax 93 4035982. E-mail: [email protected]. 17

Matilde R. Chacón. Research Unit. University Hospital of Tarragona Joan XXIII C/ Dr. Mallafré 18

Guasch, 4. 43007 Tarragona. Spain. Phone and FAX: +34 977295823. E-mail: 19

[email protected] 20

21

Grant support: This study was partly supported by funds from the Spanish Ministerio de Ciencia 22

e Innovación (SAF2009-06939), Fondo de Investigación Sanitaria (FIS) (PI08/0733 and 23

PS09/00997), Junta de Andalucía (P08-CTS-04369), StemOb project from CIBERDEM and the 24

European Union ERDF funds. L. S.-M. is supported by a FPI grant from the Spanish Ministerio de 25

Ciencia e Innovación. M.R.C. is supported by a fellowship from the Fondo de Investigación 26

Sanitaria (FIS) “Miguel Servet” CP 06/00119. 27

28

2

Abstract 1

Introduction: The mechanisms linking low-grade chronic inflammation with obesity-induced 2

insulin resistance have only been partially elucidated. PPARβ/δ and SIRT1 might play a role in 3

this association. 4

Methods and results: In visceral adipose tissue (VAT) from obese insulin-resistant patients we 5

observed enhanced p65 nuclear translocation and elevated expression of the pro-inflammatory 6

cytokines TNF-α and IL-6 compared to control subjects. Inflammation was accompanied by a 7

reduction in the levels of SIRT1 protein and an increase in PPARβ/δ mRNA levels. Stimulation of 8

human mature SGBS adipocytes with TNF-α caused similar changes in PPARβ/δ and SIRT1 to 9

those reported in obese patients. Unexpectedly, PPAR DNA-binding activity and the expression 10

of PPARβ/δ-target genes was reduced following TNF-α stimulation, suggesting that the activity of 11

this transcription factor was inhibited by cytokine treatment. Interestingly, the PPARβ/δ ligand 12

GW501516 prevented the expression of inflammatory markers and the reduction in the 13

expression of PPARβ/δ-target genes in adipocytes stimulated with TNF-α. Consistent with a role 14

for NF-κB in the changes caused by TNF-α, treatment with the NF-κB inhibitor parthenolide 15

restored PPAR DNA-binding activity, the expression of PPARβ/δ-target genes and the expression 16

of SIRT1 and PPARβ/δ. 17

Conclusion: These findings suggest that the reduction in PPARβ/δ activity and SIRT1 expression 18

caused by TNF-α stimulation through NF-κB helps perpetuate the inflammatory process in human 19

adipocytes. 20

21

Key words: PPARβ/δ, TNF-α, NF-κB, SIRT1. 22

23

24

25

26

27

3

Introduction 1

Obesity, insulin resistance and type 2 diabetes mellitus are closely associated with low-grade 2

chronic inflammation characterized by abnormal cytokine production (1). The adipocyte plays a 3

crucial role in this process, since this cell is a source of cytokines (TNF-α, IL-6, MCP-1), which 4

are secreted as a result of the activation of several signaling cascades involved in obesity-5

induced insulin resistance (2). A number of studies have implicated chronic activation of the pro-6

inflammatory transcription factor NF-κB as part of one of these signaling pathways that link 7

inflammation with obesity and insulin resistance (3;4). For instance, overexpression of the NF-κB 8

activator IκB kinase (IKK)β in mice results in increased inflammatory cytokine production and the 9

onset of diabetes (5). Furthermore, in human adipose tissue, inhibition of NF-κB suppresses the 10

release of pro-inflammatory cytokines (6). This transcription factor can be activated by a wide 11

array of exogenous and endogenous stimuli. In mammals the NF-κB/Rel family includes five 12

known members: p50 (NF-κB1), p52 (NF-κB2), p65 (RelA), c-Rel, and RelB. The most abundant 13

form of NF-κB is a heterodimer consisting of p50 and p65. In unstimulated cells, NF-κB is 14

sequestered in the cytoplasm in an inactive form through the interaction with the IκB inhibitory 15

proteins. In the canonical activation pathway, activation of cells by specific stimuli, such as the 16

pro-inflammatory cytokine TNF-α, results in phosphorylation of IκB by the IKK complex, leading to 17

its degradation by the 26S proteasome. This releases NF-κB, which then translocates to the 18

nucleus, where it activates the transcription of a wide variety of genes, such as those of TNF-α 19

and IL-6 (7). 20

21

Since the specific mechanisms linking the presence of low-grade chronic inflammation and the 22

development of obesity-induced insulin resistance have only been partially elucidated, the 23

discovery of new key factors involved in this association might provide new pharmacological 24

targets for preventing obesity-induced insulin resistance. Among these new factors, Peroxisome 25

Proliferator-Activated Receptors (PPARs) could play an important role. PPARs are members of 26

the nuclear receptor superfamily of ligand-inducible transcription factors that regulate the 27

4

expression of genes involved in many important biological processes (8). They form heterodimers 1

with retinoid X receptors (RXRs) and bind to consensus DNA sites composed of direct repeats 2

(DRs) of hexameric DNA sequences usually separated by 1 bp (DR1) (9). In addition, PPARs 3

suppress inflammation through diverse mechanisms, for example by reducing the release of 4

inflammatory factors or stabilizing repressive complexes at inflammatory gene promoters (10-13). 5

The PPAR family consists of three members: PPARα (NR1C1 according to the unified 6

nomenclature system for the nuclear receptor superfamily), PPARβ/δ (NR1C2) and PPARγ 7

(NR1C3) (9). PPARα and PPARγ are the targets for hypolipidemic (fibrates) and anti-diabetic 8

(thiazolidinediones) drugs, respectively. Finally, activation of the third isotype, PPARβ/δ, by high-9

affinity ligands (including GW501516) has been proposed as a potential treatment for insulin 10

resistance (14-17). 11

Another potential player in the relationship between inflammation and obesity-induced insulin 12

resistance is sirtuin 1 (SIRT1). This is a prominent member of the family of NAD+-dependent 13

enzymes that deacetylate lysine residues on various proteins. It has recently been proposed that 14

SIRT1 could play a role in the protection against proinflammatory responses in adipose tissue 15

(18). In fact, SIRT1 activation represses proinflammatory gene expression through NF-κB 16

deacetylation at lysine 310 and improves insulin signaling, whereas exposure to a high-fat diet 17

downregulates SIRT1 in white adipose tissue (18). Interestingly, it has recently been reported that 18

PPARβ/δ activation can increase the expression of SIRT1 (19). 19

20

In this study we assessed the levels of PPARβ/δ and SIRT1 and nuclear p65 translocation in 21

visceral adipose tissue (VAT) from severely obese insulin-resistant patients and from control 22

subjects. Our findings demonstrate that severely obese insulin-resistant patients show enhanced 23

inflammation in VAT that is accompanied by a reduction in SIRT1 protein levels and an increase 24

in PPARβ/δ mRNA levels. By treating human adipocytes with TNF-α we reproduced in vitro the 25

increase in PPARβ/δ expression and the reduction in SIRT1 levels found in obese insulin-26

resistant patients. However, the increase in PPARβ/δ expression was linked to reduced 27

5

expression of its target genes and PPAR-DNA binding activity. Interestingly, treatment with either 1

the PPARβ/δ agonist GW501516 or a NF-κB inhibitor prevented the changes caused by TNF-α. 2

These findings indicate that TNF-α reduces PPARβ/δ activity and SIRT1 expression through NF-3

κB activation. Given the role of SIRT1 and PPARβ/δ in inflammation and insulin signaling, the 4

changes induced by TNF-α in these genes may help to perpetuate the inflammatory process in 5

adipocytes. 6

7

6

Materials and Methods 1

Materials 2

GW501516 was provided by Alexis Biochemicals (Lausen, Switzerland). [γ-32P]dATP (3000 3

Ci/mmol) was purchased from Perkin Elmer (Waltham, MA). All other chemicals, except where 4

specified, were from Sigma-Aldrich (St. Louis, MO). 5

6

Subjects 7

The study included a cohort of 23 severely obese subjects (Body mass index, BMI 57.4 ± 7.3 8

kg/m2) recruited at the Malaga Clinic Hospital (Malaga, Spain) (Table 1). For inter-group 9

comparisons (control group), we selected an age and gender-matched population of 35 10

overweight subjects (BMI 26.2 ± 3.6 kg/m2) recruited at the University Hospital Joan XXIII 11

(Tarragona, Spain) as a control group. 12

Patients were excluded if they had cardiovascular disease, arthritis, acute inflammatory disease, 13

infectious disease, or were receiving drugs that could alter their lipid profile or metabolic 14

parameters at the time of inclusion in the study. None of the morbidly obese patients was being 15

treated with insulin therapy, oral antidiabetic agents, or diet. The weight of all persons had been 16

stable for at least 1 month, and none had renal involvement. The Hospitals’ Ethics Committees 17

approved the study, and informed consent was obtained from all participants. 18

VAT and subcutaneous adipose tissue (SAT) were obtained during bariatric surgery in the 19

severely obese patients or during elective abdominal surgery procedures in control subjects. The 20

biopsy samples were washed in physiological saline and immediately frozen in liquid nitrogen. 21

Biopsy samples were maintained at -80ºC until analysis. 22

23

Laboratory measurements 24

Blood samples were collected after a 12-h fast. The serum was separated and immediately frozen 25

at -80ºC. Serum biological parameters were measured in duplicate. Serum glucose, cholesterol, 26

high-density lipoprotein (HDL) cholesterol, triglycerides (Randox Laboratories Ltd., Antrium, UK) 27

and FFA (Wako Chemicals, Richmond, VA) were measured by using standard enzymatic 28

7

methods. Low-density lipoprotein cholesterol was calculated according to the Friedewald formula. 1

Insulin was analyzed via an immunoradiometric assay (Biosource International, Camarillo, CA). 2

Leptin (DSL, Webster, TX) and adiponectin (DRG Diagnostics GmbH, Germany) were analyzed 3

via enzyme immunoassay (ELISA) kits. HOMA-IR was calculated from fasting insulin and glucose 4

according to the following equation: HOMA-IR=fasting insulin (μIU/mL) x fasting glucose 5

(mmol/L)/22.5. 6

7

Cell culture 8

The human Simpson-Golabi-Behmel Syndrome (SGBS) cell line of preadipocytes was induced to 9

differentiate to mature adipocytes as described previously (20). Before applying the different 10

stimuli, cells were seeded in duplicate in 6- or 12-well tissue culture plates and differentiated into 11

mature adipocytes. At day 14 of differentiation, adipocytes were cultured for 6 h in DMEM/F-12 12

(Lonza, Barcelona, Spain) without serum and in the presence of 0.2% BSA, preincubated with or 13

without 10 μM GW501516 for 30 min and then stimulated with either 100 ng/ml human 14

recombinant TNF-α or 100 ng/ml human recombinant IL-6 (BioNova, Barcelona, Spain) for 8 h. 15

GSK0660 and parthenolide were added 30 min before incubation with GW501516 and TNF-α, 16

respectively. After incubation, RNA, whole cell lysates, and cytosolic and nuclear protein extracts 17

were extracted from cells as described below. 18

19

Measurements of mRNA 20

RNA extraction and assessment of the levels of mRNA via the reverse transcription-polymerase 21

chain reaction (RT-PCR) in VAT were performed as described previously (21). The sequences of 22

the sense and antisense primers used for amplification were: TNF-α, 5’-23

AAGCTGAGGGGCAGCTCCAGT-3’ and 5’-TCTGGTAGGAGACGGCGATGC-3’; IL-6, 5’-24

AAGATGTAGCCGCCCCACACA-3’ and 5’-TCTGCCAGTGCCTCTTTGCTG-3’; PPARβ/δ, 5’-25

CATCGGCCTTCCACTACGGTG-3’ and 5’-TCTGGAAGCGGCAGTACTGGC-3’; and 18S, 5’-26

CCAAAGTCTTTGGGTTCCGGG-3’ and 5’-GCTCAATCTCGGGTGGCTGAA-3’. Amplification of 27

8

each gene yielded a single band of the expected size (TNF-α: 204 bp, IL-6: 151 bp, PPARβ/δ: 1

153 bp, 18S: 333 bp). Preliminary experiments were carried out with various amounts of cDNA to 2

determine the non-saturating conditions of PCR amplification for all genes studied. Under these 3

conditions, relative quantification of mRNA was assessed via the RT-PCR method used in this 4

study (22). Radioactive bands were quantified by video-densitometric scanning (Vilber Lourmat 5

Imaging, France). The results for the expression of specific mRNAs are presented relative to the 6

expression of the control gene (18S). 7

RNA extraction and measurement of mRNA from mature SGBS adipocytes was performed by 8

qRT-PCR as previously described (23). Approximately 20 ng of cDNA per gene was used in real-9

time PCR quantification analysis, which was performed on a 7900HT Fast Real-Time PCR 10

System using TaqMan® Fast Universal PCR MasterMix (Applied Biosystems, Spain). The results 11

are expressed relative to the expression levels of the housekeeping gene PPIA expression levels 12

and were analyzed using the comparative Ct method (2-��Ct). The following TaqMan gene 13

expression assays were used: SIRT1 (Hs01009006_m1), CPT1b (Hs00992664_m1), PDK4 14

(Hs01037704_m1), PGC1�. (Hs01016721_m1), PPARβ/� (Hs00606407_m1) and PPIA 15

(Hs99999904_m1). 16

17

Isolation of nuclear extracts 18

Nuclear extracts were isolated as described previously (24). Cells were scraped into 1.5 ml of 19

cold phosphate-buffered saline, pelleted for 10 s and resuspended in 400 μl of cold Buffer A (10 20

mM HEPES pH 7.9 at 4 ºC, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, and 5 μg/ml 21

aprotinin) by flicking the tube. Cells were allowed to swell on ice for 10 min and were then 22

vortexed for 10 s. Samples were subsequently centrifuged for 10 s and the supernatant fraction 23

was discarded. Pellets were resuspended in 50 μl of cold Buffer C (20 mM HEPES-KOH pH 7.9 24

at 4 ºC, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 25

5 μg/ml aprotinin, and 2 μg/ml leupeptin) and incubated on ice for 20 min for high-salt extraction. 26

Cellular debris were removed by centrifugation for 2 min at 4 ºC and the supernatant fraction 27

9

(containing DNA-binding proteins) was stored at –80 ºC. The concentration of the nuclear extract 1

was determined by the Bradford method. 2

3

Electrophoretic mobility shift assay (EMSA) 4

EMSA was performed using double-stranded oligonucleotides for the consensus binding site of 5

the NF-κB (5'-AGTTGAGGGGACTTTCCCAGGC-3', Promega, Madison, WI) and PPAR 6

(Peroxisome Proliferator Response Element, PPRE probe; 5’-CAAAACTAGGTCAAAGGTCA-3’, 7

Santa Cruz Biotechnology, Santa Cruz, CA). Oligonucleotides were labeled in the following 8

reaction: 2 μl of oligonucleotide (1.75 pmol/μl), 2 μl of 5x kinase buffer, 1 μl of T4 polynucleotide 9

kinase (10 U./μl) (Gibco Invitrogen, Barcelona, Spain), and 2.5 μl of [γ-32P] ATP (3000 Ci/mmol at 10

10 mCi/ml), incubated at 37 ºC for 1 h. The reaction was stopped by adding 90 μl of TE buffer (10 11

mM Tris-HCl pH 7.4 and 1 mM EDTA). To separate the labeled probe from the unbound ATP, the 12

reaction mixture was eluted in a Nick column (GE Healthcare, CA) according to the 13

manufacturer’s instructions. Eight micrograms of crude nuclear protein were incubated for 10 min 14

on ice in binding buffer (10 mM Tris-HCl pH 8.0, 25 mM KCl, 0.5 mM DTT, 0.1 mM EDTA pH 8.0, 15

5% glycerol, 5 mg/ml BSA, and 50 μg/ml poly(dI-dC)), in a final volume of 15 μl. Labeled probe 16

(approximately 60,000 cpm) was added and the reaction was incubated for 15 min at 4 ºC. Where 17

indicated, specific competitor oligonucleotide was added before the labeled probe and incubated 18

for 10 min on ice. Protein-DNA complexes were resolved by electrophoresis at 4 ºC on a 5% 19

acrylamide gel and subjected to autoradiography. 20

21

Immunoblotting 22

Antibodies against p65 and SIRT1 (Santa Cruz Biotechnology) were used. Cytosolic and nuclear 23

protein extracts of visceral adipose tissue were prepared as described previously (25). 24

To obtain whole-cell lysates, cells were homogenized in RIPA buffer (Sigma Aldrich) with 25

phosphatase and protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 1 mM sodium 26

10

orthovanadate, and 5.4 μg/ml aprotinin). The homogenate was centrifuged at 17,000x g for 30 1

min at 4 ºC. Protein concentration was measured by the Bradford method. 2

Proteins from cytosolic and nuclear extracts were separated by SDS-PAGE and transferred to 3

immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA) and blotted with various 4

antibodies (as specified in the “Results”). Detection was achieved using the ECL plus 5

chemiluminescence kit (GE Healthcare, CA). The size of detected proteins was estimated using 6

protein molecular-mass standards (Invitrogen, Barcelona, Spain). 7

8

Statistical analyses 9

The results of the controls and morbidly obese patients were compared using the non-parametric 10

Mann–Whitney test. The results for in vitro studies are expressed as the means ± S.D. of six 11

separate experiments. Significant differences were established by Student’s t-test or one-way 12

ANOVA, according to the number of groups compared, using the GraphPad Instat program 13

(GraphPad Software V2.03) (GraphPad Softwware Inc., San Diego, CA). In the latter case, when 14

significant differences were found, the Tukey-Kramer multiple comparisons post-tes was applied. 15

Differences were considered significant at P<0.05. 16

17

18

19

20

11

Results 1

VAT from morbidly obese-insulin resistant patients shows inflammation, reduced SIRT1 protein 2

levels and increased PPARβ/δ expression 3

Table 1 summarizes the characteristics of the control subjects and the morbidly obese insulin-4

resistant patients. As expected, weight and anthropometric data were significantly greater in the 5

morbidly obese insulin-resistant patients. In addition, diastolic blood pressure, plasma free fatty 6

acids, insulin, HOMA-IR and plasma IL-6 levels were higher in patients with severe obesity 7

compared to control subjects. VAT samples were obtained from a representative subgroup of six 8

control and six obese patients for subsequent analysis. First we evaluated whether the presence 9

of severe obesity and insulin resistance resulted in increased expression of pro-inflammatory 10

cytokines in VAT. In obese patients the mRNA levels of TNF-α (1.6-fold induction, p<0.01) and IL-11

6 (1.8-fold induction, p<0.001) (Figures 1A-B) were higher than in control subjects, showing the 12

presence of inflammation. Since the expression of these cytokines is under the control of the pro-13

inflammatory transcription factor NF-κB (7), we then examined the nuclear translocation of the 14

p65 subunit of NF-κB. In agreement with the increase in the expression of TNF-α and IL-6, the 15

protein levels of p65 were higher in the nuclear fraction of obese patients compared to non-obese 16

subjects, suggesting that NF-κB activity was increased in the adipose tissue of these patients. 17

Under these conditions of increased inflammation in VAT from obese patients, SIRT1 protein 18

levels were lower in nuclear VAT fractions from obese patients compared to non-obese subjects 19

(Figure 1D). Likewise, the mRNA levels of the nuclear transcription factor PPARβ/δ were higher 20

(p<0.01) in obese patients than in control subjects (Figure 1E). In order to establish whether the 21

increase in PPARβ/δ expression was specific to VAT, we assessed the mRNA levels of this 22

transcription factor in SAT. Regardless of the localization of the fat tissue, the PPARβ/δ mRNA 23

levels were higher in obese insulin-resistant patients than in control subjects (Figure 1F). 24

25

TNF-α increases the expression of PPARβ/δ in human SGBS adipocytes but reduces the 26

expression of its target genes 27

12

To study the mechanisms responsible for the increase in PPARβ/δ expression and the reduction 1

of SIRT1 levels in adipocytes we took advantage of the human SGBS adipocytes. These cells 2

have a high capacity for adipose differentiation and, therefore, represent a unique tool for 3

studying human fat cell development and metabolism (20). In order to induce inflammation in 4

these adipocytes they were exposed to TNF-α for 8 h. Incubation with TNF-α led to an increase in 5

the mRNA levels of PPARβ/δ (1.4-fold induction, p<0.05) and a reduction in SIRT1 mRNA levels 6

(25% reduction, p<0.05) (Figure 2A-B). These changes were similar to those found in VAT from 7

obese patients. Unexpectedly, the increase in PPARβ/δ expression was accompanied by a 8

reduction in the expression of its target genes (26;27) CPT-1b (17% reduction, p<0.05), PDK4 9

(80% reduction, p<0.05) and PGC-1α (79% reduction, p<0.05) (Figure 2C-E). Similar changes, 10

but of lower intensity, were observed when adipocytes were incubated with IL-6 (Figure 3). Given 11

that it has been reported that TNF-α activates the transcription factor NF-κB (7), we then 12

performed EMSA to confirm this possibility. The NF-κB probe formed three complexes (I to III) 13

with adipocyte nuclear proteins (Figure 4). The specificity of these DNA-binding complexes was 14

assessed in competition experiments by adding an excess of unlabeled NF-κB oligonucleotide. 15

NF-κB DNA-binding activity was higher in cells exposed to TNF-α compared to control cells. 16

Addition of antibody against p65 supershifted complexes I and II, thereby indicating that these 17

complexes contained this subunit of NF-κB. 18

19

The PPARβ/δ agonist GW501516 prevents the reduction in PPARβ/δ-target genes and the 20

increase in pro-inflammatory cytokines 21

Since TNF-α induces NF-κB, activation of this pro-inflammatory transcription factor could be 22

responsible for the changes in PPARβ/δ activity. To confirm this possibility we evaluated the effect 23

of the NF-κB inhibitor parthenolide on PPAR DNA-binding activity. This compound specifically 24

inhibits activation of NF-κB by preventing IκB degradation (28). The PPRE probe formed two 25

main complexes when incubated with nuclear proteins from control adipocytes (Figure 5A). 26

Interestingly, cells exposed to TNF-α showed a strong reduction in PPAR DNA-binding activity, 27

13

whereas this reduction was prevented in cells co-incubated with TNF-α and parthenolide. Addition 1

of antibody against PPARβ/δ reduced complexes I and II, whereas an unrelated antibody against 2

Oct-1 did not (data not shown), indicating the presence of this nuclear receptor. These findings 3

confirm that the PPARβ/δ DNA-binding activity is reduced by TNF-α-induced NF-κB activation. 4

5

As we have previously reported that the PPARβ/δ agonist GW501516 inhibits NF-κB activation by 6

LPS (15), we next explored whether this drug can prevent the increase in the expression of the 7

NF-κB-target genes TNF-α and IL-6 caused by TNF-α exposure. As expected, drug treatment 8

prevented the induction of these pro-inflammatory genes in response to TNF-α, confirming that 9

this drug prevents NF-κB activation by this cytokine. The effect of GW501516 was blocked in 10

cells co-incubated with the PPARβ/δ antagonist GSK0660, indicating that the effect of GW501516 11

was dependent on this nuclear receptor (Figure 5B-C). Consistent with a role for NF-κB in the 12

reduction of PPARβ/δ activity, co-incubation of adipocytes with TNF-α and GW501516 restored 13

the reduction in the expression of the PPARβ/δ-target genes PGC-1α and PDK4 caused by TNF-14

α (Figure 5 D-E). 15

16

NF-κB inhibition prevents the reduction in PPARβ/δ-target genes and SIRT1 and the increase in 17

PPARβ/δ expression 18

Finally, to clearly demonstrate that TNF-α-induced NF-κB activation was responsible for the 19

reduction in PPARβ/δ activity we evaluated the effects of parthenolide on the expression of its 20

target genes PGC-1α and PDK4, as well as on SIRT1 and PPARβ/δ expression. Co-incubation of 21

adipocytes with TNF-α plus parthenolide prevented the reduction in the expression levels of PGC-22

1α and PDK4 caused by TNF-α (Figure 6 A-B). In addition, parthenolide prevented the reduction 23

in SIRT1 and the increase in PPARβ/δ expression, indicating that these changes were also 24

dependent on TNF-α-induced NF-κB activation (Figure 6 C-D). 25

Discussion 26

14

At the cellular level, insulin resistance and enhanced expression of pro-inflammatory cytokines by 1

adipose tissue during obesity, and also under a high-fat diet, have been linked to activation of the 2

transcription factor NF-κB (29). In agreement with a previous study (4), we report here that VAT 3

from obese insulin-resistant patients shows enhanced levels of expression of IL-6 and TNF-α and 4

increased NF-κB activity. Interestingly, we show that the presence of inflammation in this adipose 5

tissue was accompanied by enhanced PPARβ/δ expression and reduced SIRT1 protein levels. 6

The findings of this study suggest that cytokines could be responsible for the increase in PPARβ/δ 7

expression and the reduction in SIRT1 levels, since treatment with both TNF-α and IL-6 led to 8

similar changes in human SGBS adipocytes. The increase in the expression of PPARβ/δ in 9

human VAT from obese insulin-resistant patients conflicts with a previous study in which we 10

found a reduction in PPARβ/δ expression in white adipose tissue from ZDF rats (15). In 11

agreement with the reduction in the expression of PPARβ/δ in the adipose tissue of ZDF rats, the 12

expression of its target gene Pdk4 and the PPAR DNA-binding activity were reduced (15), 13

whereas an increase in IL-6 expression was observed, suggesting that the association between 14

the presence of low-grade chronic systemic inflammation and the development of obesity and 15

insulin resistance involves a decrease in PPARβ/δ activity and the concomitant activation of NF-16

κB. In fact, the white adipose tissue of PPARβ/δ-null mice shows higher NF-κB binding activity 17

and IL-6 expression levels when compared to wild-type mice (15), confirming the anti-18

inflammatory effect of PPARβ/δ in this tissue. We do not know the reasons for these species 19

differences regarding the changes in PPARβ/δ mRNA levels, but it is worth noting that regardless 20

of the changes in PPARβ/δ expression, obesity-induced insulin resistance and inflammation in 21

humans and animal models lead to reduced PPARβ/δ activity, as demonstrated by the reduction 22

in PPAR DNA-binding activity and in the expression of its target genes. The consequences of the 23

reduction in PPARβ/δ activity might affect both the metabolic and the anti-inflammatory capacity 24

of the adipocyte. Thus, several PPARβ/δ target genes, including PDK4, CPT-1b and PGC-1α, are 25

involved in the utilization of fatty acids, suggesting that a reduction in the expression of these 26

genes as a result of the lower activity of PPARβ/δ in obesity and insulin resistance might lead in 27

15

the long-term to an increase in the accumulation of fatty acids in adipocytes in the form of 1

triglycerides. In addition, the reduction in PPARβ/δ activity might compromise the anti-2

inflammatory defense, perpetuating the inflammatory process of the adipocyte. 3

4

We observed a reduction in the levels of SIRT1 in VAT from obese insulin-resistant patients and 5

in SGBS adipocytes exposed to TNF-α. This protein deacetylates NF-κB and inhibits NF-κB 6

binding to the promoters of its target genes in adipocytes (18). Therefore, it is tempting to 7

speculate that the reduction in SIRT1 observed in obese-insulin resistant patients and in SGBS 8

adipocytes exposed to TNF-α contributes to the increase in NF-κB-mediated inflammation 9

observed in these cells. In fact, SIRT1 represses pro-inflammatory gene expression, including 10

TNF-α and IL-6, in adipocytes (18). In addition, obese and insulin-resistant mice fed a high fat diet 11

show a dramatic reduction in SIRT1 protein levels in adipose tissue (18). In humans, it has been 12

reported that SIRT1 transcription is decreased in VAT of obese insulin-resistant patients (30) and 13

in morbidly obese patients with severe hepatic steatosis (31). Overall, these findings suggest that 14

SIRT1 downregulation may be involved in the development of NF-κB-mediated inflammation in 15

adipose tissue. 16

17

A causal link between obesity, inflammation and insulin resistance was first reported by 18

Hotamisligil et al. (32) and Feinstein et al. (33). Both studies demonstrated that overproduction of 19

TNF-α by the adipose tissue of obese subjects can cause insulin resistance. Recently, Barbarroja 20

et al. (4) demonstrated that morbidly obese insulin-sensitive individuals show less inflammation in 21

their VAT than BMI-matched individuals who are insulin-resistant. These insulin-resistant subjects 22

show increased NF-κB activation in VAT. Our in vitro findings indicate that both the increase in 23

PPARβ/δ expression and the reduction in SIRT1 levels seem to be dependent on NF-κB 24

activation, since the NF-κB inhibitor parthenolide prevented these changes. Therefore, the 25

reduction in SIRT1 and PPARβ/δ activity associated with NF-κB activation may also contribute to 26

16

the development of insulin resistance in obese patients. Further studies are necessary to confirm 1

this hypothesis. 2

3

Interestingly, PPARβ/δ activation by GW501516 reversed the reduction in the expression of 4

PPARβ/δ-target genes and prevented the increase in the expression of NF-κB-target genes, such 5

as TNF-α and IL-6, caused by TNF-α exposure. The latter is consistent with a previous study in 6

which we reported that GW501516 inhibits LPS-induced NF-κB activation (15). These findings 7

support the fact that some of the antidiabetic effects of PPARβ/δ activators might result from their 8

ability to inhibit NF-κB activity in adipocytes, and demonstrate that NF-κB inhibition is a 9

pharmacological target for preventing obesity-induced metabolic dysregulation. 10

11

In summary, our findings indicate that human adipocytes exposed to TNF-α show reduced 12

PPARβ/δ activity and reduced expression of SIRT1. These changes were dependent on NF-κB 13

activation and were prevented by PPARβ/δ agonists, suggesting that inhibition of NF-κB by these 14

drugs can prevent metabolic dysregulation in obese subjects. 15

16

17 Conflict of interest: The authors declare no conflict of interest 18

19

20

Acknowledgements- We would like to thank Dr. M. Wabistch for kindly providing the SGBS 21

cell line. This study was partly supported by funds from the Spanish Ministerio de Ciencia e 22

Innovación (SAF2009-06939), Fondo de Investigación Sanitaria (FIS) (PI08/0733 and 23

PS09/00997), Junta de Andalucía (P08-CTS-04369), StemOb project from CIBERDEM and the 24

European Union ERDF funds. L. S.-M. is supported by a FPI grant from the Spanish Ministerio de 25

Ciencia e Innovación. M.R.C. is supported by a fellowship from the Fondo de Investigación 26

Sanitaria (FIS) “Miguel Servet” CP 06/00119. 27

17

We would like to thank the University of Barcelona’s Language Advisory Service for its help. 1

CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) is an initiative of ISCIII 2

(Ministerio de Ciencia e Innovación). 3

18

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24

Table 1. Clinical and anthropometric characteristics of the patients.

Control (n=35)

Obese-IR (n=23)

p

Age (years)

44.5±8.3

40±0.4

ns

Gender (male/female)

23/12

9/14

ns

BMI (kg/m2)

26.2±3.6

57.4±7.3

<0.001

Waist circumference (cm)

90.8±13

146.2±23.5

<0.001

SBP (mm Hg)

122.4±13.3

139±23.6

ns

DBP (mm Hg)

70.6±9.4

84.5±14.2

0.004

Cholesterol (mmol/L)

4.8±1

5.1±1.1

ns

HDL-Cholesterol (mmol/L)

1.3±0.3

0.9±0.6

ns

LDL-Cholesterol (mmol/L)

2.9±0.9

3.5±1.1

ns

Triglycerides (mmol/L)

1.2±0.7

1.4±0.8

ns

Glucose (mmol/L)

5.3±0.7

5.6±1

ns

Insulin (pmol/L)

5.9±4.5

31±18.8

<0.001

HOMA-IR

1.4±1.1

8.1±5.9

<0.001

sIL-6 (pg/mL)

1.9±1.4

6.1±4.5

<0.001

The results are given as the mean ± SD. DBP: Diastolic blood pressure. SBP: Systolic blood pressure. ns: not significant

25

Figure legends

FIG. 1. VAT from obese insulin-resistant patients shows inflammation, reduced SIRT1 protein

levels and increased PPARβ/δ expression. mRNA levels of TNF-α (A) and IL-6 (B) in VAT from

control and obese insulin-resistant patients. Total RNA was isolated and analyzed by RT-PCR. A

representative autoradiogram and the quantification normalized to the 18S mRNA levels are

shown. Data are expressed as means ± S.D. (n=6 per group). Analysis of the protein levels of the

p65 subunit of NF-κB (C) and SIRT1 (D) by immunoblotting in protein extracts from VAT.

Autoradiograph data are representative of three separate experiments. mRNA levels of PPARβ/δ

in VAT (E) and SAT (F) from obese insulin-resistant patients. Total RNA was isolated from VAT

and SAT and analyzed by qRT-PCR. Data are expressed as means ± S.D. (15 control subjects

and 23 severe obese insulin resistant patients). Adipose tissue expression levels were normalized

using β-actin. *p<0.05, **p<0.01 and ***p<0.001 vs. control subjects. C: Cytosolic; N, nuclear

protein extracts.

FIG. 2. TNF-α treatment increases PPARβ/δ expression, but reduces the mRNA levels of its

target genes in human SGBS adipocytes. Analysis of the mRNA levels of PPARβ/δ (A), SIRT1

(B), CPT-1b� (C), PDK4 (D) and PGC-1α (E) in SGBS cells untreated or treated with 100 ng/ml

TNF-α for 8 h. Total RNA was isolated and analyzed by qRT-PCR. *p<0.05 vs. control cells

FIG. 3. IL-6 treatment reduces the mRNA levels of PPARβ/δ−target genes in human SGBS

adipocytes. Analysis of the mRNA levels of PPARβ/δ (A), SIRT1 (B), CPT-1b � (C), PDK4 (D) and

PGC-1α (E) in SGBS cells untreated or treated with 100 ng/ml IL-6 for 8 h. Total RNA was

isolated and analyzed by qRT-PCR. *p<0.05 vs. control cells

FIG. 4. TNF-α increases NF-κB DNA binding activity in SGBS human adipocytes. Cells were

stimulated with 100 ng/ml TNF-α for 8 h. Autoradiograph of EMSA performed with a 32P-labeled

26

NF-κB nucleotide and nuclear protein extracts (NE). Three specific complexes (I to III), based on

competition with a molar excess of unlabeled probe, are formed. A supershift analysis performed

by incubating NE with an antibody directed against the p65 subunit of NF-κB is also shown.

Autoradiograph is representative of three separate experiments. IC: Immunocomplex.

FIG. 5. Parthenolide prevents the decrease in PPAR DNA-binding activity caused by TNF-α in

SGBS human adipocytes. Cells were stimulated with 100 ng/ml TNF-α for 8 h. A, Autoradiograph

of EMSA performed with a 32P-labeled PPRE nucleotide and nuclear protein extracts (NE). Two

specific complex, based on competition with a molar excess of unlabeled probe, is shown.

Analysis of the mRNA levels of PGC-1α (B), PDK4 (C), TNF-α (D) and IL-6 (E) in SGBS cells

untreated or treated with 10 μM GW501516 in the absence or presence of 1 μM GSK0660 for 30

min before stimulation with 100 ng/ml TNF-α for 8 h. ***p<0.001 and *p<0.05 and vs. control cells.

###p<0.001, ##p<0.01, #p<0.05 vs. TNF-α-stimulated cells. @p<0.05 vs. cells co-incubated with

TNF-α plus GW501516. Parth: Parthenolide.

FIG. 6. Parthenolide prevents the changes in PPARβ/δ and SIRT1 expression caused by TNF-α

stimulation in SGBS human adipocytes. Analysis of the mRNA levels of PGC-1α (A), PDK4 (B),

SIRT1 (C) and PPARβ/δ (D) in SGBS cells untreated or treated with 10 μM parthenolide for 30

min before stimulation with 100 ng/ml TNF-α for 8 h. *p<0.05 and vs. control cells. ###p<0.001,

##p<0.01, #p<0.05 vs. TNF-α-stimulated cells. CT: Control. Parth: Parthenolide.

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DISCUSSIÓ�GLOBAL�

�

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�

�

119

L’obesitat,� la� RI� i� la� DM2� s’associen� a� un� estat� inflamatori� crònic� de� baixa� intensitat�

caracteritzat�per�una�producció�més�alta�de�citocines�pro�inflamatòries�com�el�TNF��i� la� IL�6�

(Fernandez�Real�i�col.,�2001;�Hotamisligil�i�col.,�1993;�Kern�i�col.,�2001).��

El�teixit�adipós�en�el�seu�estat�normal�actua�com�a�òrgan�endocrí�secretant�adipocines,�entre�

elles� la� IL�6,� i� d’aquesta� manera� modulant� molts� processos� biològics� a� nivell� local� i� sistèmic�

(Path� i� col.,� 2001;� Kershaw� i� Flier,� 2004).� A� més,� el� teixit� adipós� és� un� dels� teixits� més�

importants� que� donen� resposta� a� la� insulina� i� que� regulen� l’homeòstasi� de� la� glucosa� i� dels�

lípids�(Guilherme�i�col.,�2008;�Trayhurn�i�col.,�2006).�En�estat�d’obesitat�es�produeixen�canvis�

en�aquest�teixit�com�l’augment�de� la�reserva�de�greixos�que�va�acompanyat�de� la�hipertròfia�

del� teixit� adipós,� especialment� del� teixit� adipós� visceral.� Tots� aquells� factors� que� afectin� a�

aquest� teixit� afectaran�a� la� secreció�d’adipocines� i,�per� tant,� tindran�efectes�a�nivell� sistèmic�

(Frayn� i� col.,� 2003),� com� per� exemple� contribuiran� al� desenvolupament� de� RI� associada� a�

obesitat.�S’hi�sembla�molt�a�la�introducció�de�la�tesi.�

En�els�últims�anys�s’ha�proposat�que�PPAR�/��podria�ser�una�diana�farmacològica�per�a�inhibir�

molts�processos� implicats� en�el� desenvolupament� del� procés� inflamatori�associat�a� l’aparició�

de� RI.� Així,� per� exemple,� ha� estat� descrit� que� l’activació� en� cèl�lules� epitelials� l’activació� de�

PPAR�� evitava� l’activació� de� NF��B� (Barroso� i� col.,� 2011)� i� també� disminuïa� l’activitat�

transcripcional�de�NF��B,�així�com�la�secreció�d’IL�6�induïdes�per�LPS�en�adipòcits�(Rodriguez�

Calvo�i�col.,�2008;�Barroso�i�col.,�2011).�Tanmateix,�ha�estat�descrit�que�l’activació�de�PPAR�/��

en� hepatòcits� inhibia� la� via� IL�6/JAK/STAT3,� tot� i� que� el� mecanisme� responsable� d’aquesta�

inhibició�no�havia�estat�descrit�(Kino�i�col.,�2007),�(Barroso�i�col.,�2011;�Rodriguez�Calvo�i�col.,�

2008).�Donat�que�la�via� IL�6/STAT3�juga�un�paper�fonamental�en� l’aparició�de�resistència�a� la�

insulina� en� adipòcits,� en� aquesta� tesi� doctoral� vam� avaluar� els� efectes� de� l’activació� de�

PPAR�/��sobre�aquesta�via�podrien�contribuir�a�explicar�els�seus�efectes�per�a�millorar�la�RI�en�

adipòcits.��

�

I. L’agonista� de� PPAR�� GW501516� evita� l’activació� de� la� via� STAT3/SOCS3�

induïda�per�IL�6�i�així�preveu�l’aparició�de�RI�en�adipòcits.�

�

Els�nivells�de�IL�6�circulants�en�individus�obesos�amb�DM2�són�dos�o�tres�vegades�més�elevats�

respecte� als� nivells� observats� en� individus� sans,� i� aquests� nivells� es� correlacionen� amb� més�

àcids�grassos�lliures�circulants�i�menys�sensibilitat�a�la�insulina�(Bastard�i�col.,�2002;�Kern�i�col.,�

DISCUSSIÓ GLOBAL

121

2001).�Aquesta�citocina�pro�inflamatòria�porta�a� terme�els� seus�efectes�a� través�de� la�via�de�

senyalització� JAK/STAT3� que� incrementa� la� transcripció� de� SOCS3.� Llavors,� SOCS3� és� capaç�

d’interferir�amb�el�IR�i�la�IRS�1�causant�RI�(Emanuelli�i�col.,�2000;�Krebs�i�Hilton,�2001;�Shi�i�col.,�

2006).�

Els�nostres�resultats�demostren�que,�a� l’igual�que�ja�havia�estat�descrit�en�hepatòcits� � (Kino�i�

col.,�2007),� l’activació�de�PPAR��per�GW501516�en�adipòcits�evita� l’augment�de� l’expressió�

de� SOCS3� induït� per� IL�6� a� través� de� la� inhibició� de� l’activitat� transcripcional� d’STAT3.�

L’agonista�GW501516�va�inhibir�la�fosforilació�d’STAT3�en�els�residus�de�tirosina�705�i�de�serina�

727� induïda� per� la� IL�6.� L’activitat� transcripcional� d’aquest� factor� de� transcripció� depèn,�

justament,�de�les�fosforilacions�en�aquests�dos�residus.�La�fosforilació�en�el�residu�tirosina�705�

és�necessària�per�la�dimerització�i�la�translocació�al�nucli�d’STAT3,�mentre�que�la�fosforilació�en�

serina�727�es�requereix�per�a�que�aquest�adquireixi�la�màxima�activitat�transcripcional�(Wen�i�

col.,�1995;�Decker�i�Kovarik,�2000).�Aquests�resultats�concorden�amb�els�prèviament�obtinguts�

en� teixit� adipós� de� rates� obeses� ZDF,� en� les� quals� els� nivells� de� fosforilació� en� la� serina� 727�

d’STAT3�i�els�nivells�proteics�de�SOCS3�eren�més�elevats�en�rates�obeses�que�en�rates�control,�

fet�que� coincidia�amb�una� reducció�de� l’expressió�de�PPAR�� (Rodriguez�Calvo� i� col.,�2008).�

Aquests�canvis�observats�en�aquestes�rates�obeses�ZDF�podrien�indicar�que�la�sobreexpressió�

de� SOCS3� al� teixit� adipós� podria� � contribuir� a� les� alteracions� metabòliques� en� aquest� model�

animal�de�DM2.�

Segons�els�nostres�resultats,� la� inhibició�de� l’activació�d’STAT3� induïda�per� la� IL�6�després�de�

l’activació� de� PPAR��podria� haver� tingut� lloc� a� través� de� dos� mecanismes.� Per� una� banda,�

s’ha�descrit�que� la� fosforilació�en�serina�d’STAT3�es�duu�a�terme�per�diferents�cinases� (Abe� i�

col.,�2001;�Schuringa�i�col.,�2000;�Uddin�i�col.,�2002),�entre�elles�l’ERK1/2�(Chung�i�col.,�1997).�

Estudis�previs�realitzats�al�nostre�grup�de�recerca�van�demostrar�que�el�GW501516�inhibia� la�

fosforilació� d’ERK1/2� en� adipòcits� (Rodriguez�Calvo� i� col.,� 2008).� En� el� present� estudi,�

GW501516�no�només�inhibia�la�fosforilació�d’ERK1/2�si�no�que�també�va�disminuir�l’expressió�

d’Egr�1� (early�growth�response�1),�gen�que�es�troba�sota�el�control�d’aquesta�cinasa,� fet�que�

confirmava� la� inhibició� sobre� ERK1/2� (Kamimura� i� col.,� 2003).� La� inhibició� de� l’ERK1/2� per�

GW501516� suggereix� que� la� inhibició� d’aquesta� cinasa� podria� estar� involucrada� en� la�

disminució� de� la� fosforilació� en� la� serina� 727� d’STAT3� induïda� en� cèl�lules� exposades� a� IL�6.�

D’altra�banda,�l’activació�d’STAT3�requereix�la�seva�associació�amb�altres�proteïnes,�entre�elles�

Hsp90,� per� tal� de� poder� unir�se� al� receptor� gp130� o� per� poder� translocar� al� nucli� (He� i� col.,�

2006).De� fet,� estudis� realitzats� en� hepatòcits� exposats� a� IL�6� i� tractats� amb� pirrolidin�

DISCUSSIÓ GLOBAL

122

ditiocarbamat�(PDTC),�un�inhibidor�de�NF��B,�i�geldanamicina,�un�inhibidor�específic�de�Hsp90,�

van�demostrar�que�les�fosforilacions�en�serina�i�tirosina�d’STAT3�induïdes�per�la�IL�6,�així�com�la�

seva�translocació�al�nucli�disminuïen�en�presència�dels�dos�inhibidors�mitjançant�la�reducció�de�

l’associació� d’STAT3� amb� Hsp90� (He� i� col.,� 2006).� Els� nostres� resultats� obtinguts� per�

immunoprecipitació� mostren� que� l’activació� de� PPAR��provoca� la� dissociació� de� Hsp90� i�

STAT3�que�havia�estat�induïda�amb�l’estimulació�dels�adipòcits�amb�la�IL�6.�Aquests�resultats�es�

van� confirmar� en� utilitzar� el� teixit� adipós� blanc� de� ratolins� deficients� per� PPAR��on� vam�

observar�una�associació�entre�Hsp90�i�STAT3�molt�més�elevada�en�els�ratolins�deficients�per�a�

aquest�receptor�nuclear,�i�a�més,�nivells�de�fosforilació�més�alts�d’STAT3,�així�com�més�activitat�

d’unió� a� l’ADN� en� aquests� ratolins� deficients� en� PPAR��respecte� els� ratolins� salvatges.�

Malgrat�que�els�nostres�resultats�no�expliquen�com�PPAR��disminueix�aquesta�associació,�ha�

estat�descrit�que�aquest�receptor�nuclear�pot� interaccionar�amb�Hsp90�(Sumanasekera� i�col.,�

2003),� suggerint� que� podria� existir� una� competició� entre� STAT3� i� PPAR��per� la� unió� amb�

Hsp90�i�aquesta�podria�ser�la�causa�de�la�disminució�de�la�interacció�d’aquesta�proteïna�amb�

STAT3.�

Finalment,� l’activació� per� la� IL�6� de� la� via� STAT3/SOCS3� redueix� tant� la� fosforilació� de� l’Akt�

induïda� per� la� insulina� com� la� captació� de� glucosa.� En� canvi,� l’addició� a� les� cèl�lules� de�

GW501516� va� revertir� aquests� efectes.� Donat� que� la� via� STAT3/SOCS3� juga� un� paper� molt�

important�en�la�resistència�a�la�insulina�induïda�per�IL�6�en�adipòcits,�la�inhibició�d’aquesta�via�

per� part� del� GW501516� podria� ser� responsable� de� l’increment� de� la� sensibilitat� a� la� insulina�

causat� per� aquest� fàrmac.� Tanmateix,� donat� que� la� inhibició� de� l’ERK1/2� amb� U0126� també�

reverteix�la�fosforilació�de�l’Akt�induïda�per�insulina�en�presència�d’IL�6�és�molt�probable�que�

part�dels�efectes�de�GW501516�siguin�deguts�a�la�seva�capacitat�per�a�inhibir�aquesta�cinasa.��

Tots� aquests� resultats,� juntament� amb� altres� publicats� prèviament� pel� nostre� grup� on� es� va�

demostrar�que�GW501516�reduïa�el�procés�inflamatori�en�adipòcits�per�inhibició�del�factor�de�

transcripció�NF��B�(Rodriguez�Calvo� i� col.,�2008),�confirmen�que�PPAR�/�� juga�un�paper�clau�

en� la� regulació� del� procés� inflamatori� i� la� RI� en� adipòcits.� En� conseqüència,� la� regulació� dels�

nivells�d’expressió�de�PPAR�/��i�de�la�seva�activitat�en�adipòcits�pot�resultar�clau�en�l’aparició�

d’aquests�processos.�Per�aquesta�raó,�en�el�segon�treball�d’aquesta�tesi�doctoral�vam�estudiar�

com� la� presència� d’obesitat� mòrbida� i� les� citocines� pro�inflamatòries� afectaven� l’expressió� i�

l’activitat�de�PPAR�/��en�adipòcits.�

�

DISCUSSIÓ GLOBAL

123

II. L’exposició�a�TNF��d’adipòcits�humans�provoca�la�disminució�de�l’activitat�de�

PPAR��i�de�l’expressió�de�Sirt1�a�través�de�NF��B.�Aquests�canvis�es�poden�

revertir�amb�l’activació�de�PPAR��

�

Quan�una�cèl�lula�és�estimulada�per�la�citocina�pro�inflamatòria�TNF��resulta�en�la�fosforilació�

de� les� proteïnes� I�B� que� mantenen� NF��B� inactiu� al� citoplasma� i� són� degradades� al�

proteasoma.�D’aquesta�manera�NF��B,�un�factor�de�transcripció�pro�inflamatori,�queda�lliure�i�

pot�translocar�al�nucli�on�inicia�la�transcripció��dels�seus�gens�diana�(Baldwin,�Jr.,�2001)�com�la�

IL�6,�el�TNF��o�la�MCP1.��

El�TNF��és�una�de�les�citocines�que�es�troben�elevades�en�estats�d’obesitat,�RI�i�DM2.�A�més�

s’ha�observat�que�amb�la�pèrdua�de�pes�disminueixen�els�nivells�plasmàtics�de�TNF��(Maury�i�

Brichard,�2010).�De�fet,�ha�estat�considerat�un�dels�responsables�de�l’aparició�de�RI�a�través�de�

la�fosforilació�en�la�serina�307�de�IRS�1,�fet�que�impediria�la�senyalització�de�la�insulina�(Rui� i�

col.,�2001;�White,�2002;�Hotamisligil�i�col.,�1993;�Feinstein�i�col.,�1993).�D’acord�amb�aquestes�

dades�en�el�nostre�estudi�vam�trobar�nivells�d’expressió�elevats�de�TNF��i�d’IL�6�i�l’activitat�de�

NF��B�més�alta�en�pacients�obesos�amb�RI�respecte�a�pacients�no�obesos.�En�el� teixit�adipós�

visceral�d’aquests� individus�obesos� i� insulino�resistents�també�es�va�observar�un�augment�de�

l’expressió� de� PPAR��i� una� reducció� dels� nivells� de� SIRT1.� Quan� van� estimular� adipòcits�

humans� SGBS� amb� TNF��i� IL�6� també� vam� observar� un� increment� en� els� nivells� d’ARNm� de�

PPAR�/��i�una�reducció�de�SIRT1,�fet�que�suggeria�que�les�citocines�pro�inflamatòries�podrien�

ser� les� responsables� dels� canvis� observats� en� els� pacients� obesos.� Aquests� resultats� no�

concorden�amb�els�descrits�prèviament�en�rates�obeses�ZDF,�en�els�quals�l’expressió�i�l’activitat�

de� PPAR�� estaven� reduïdes� (Rodriguez�Calvo� i� col.,� 2008).� Malgrat� aquestes� diferències,�

l’increment� de� l’expressió� de� PPAR�/�� en� adipòcits� humans,� l’activitat� d’aquest� receptor�

nuclear�semblava�estar�reduïda,�ja�que�tant�la�seva�activitat�d’unió�a�l’ADN�com�la�reducció�de�

l’expressió� dels� seus� gens� diana,�PDK4,�CPT�1b� i�PGC�1�,� així� ho� confirmaven.� Aquests� gens�

diana�de�PPAR��desenvolupen�papers�essencials�en�el�metabolisme�de� la�glucosa� i�en� la��

oxidació�dels�àcids�grassos.� La� seva�disminució�contribuiria�al�manteniment�de� l’estat�de�RI� i�

d’obesitat.� A� més,� la� disminució� de� l’activitat� de� PPAR��podria� reduir� la� seva� activitat�

antiinflamatòria�en�aquest�teixit�contribuint�així�al�manteniment�del�procés�inflamatori.�

Per� altra� banda,� els� adipòcits� SGBS� exposats� a� TNF��i� el� teixit� adipós� dels� pacients� obesos�

resistents�a�insulina�van�mostrar�una�disminució�dels�nivells�de�la�desacetilasa�SIRT1.�L’activitat�

DISCUSSIÓ GLOBAL

124

transcripcional�de�NF��B�es�regula�mitjançant�acetilacions�(Quivy�i�Van,�2004).�Aquesta�sirtuïna�

desacetila�NF��B�i�inhibeix�la�unió�d’aquest�als�promotors�dels�seus�gens�diana�(Yoshizaki�i�col.,�

2009).�En�aquest�estudi�de�Yoshizaki�i�col.�(2009)�van�demostrar�que�SIRT1�presentava�nivells�

baixos�en� ratolins�obesos� i� resistents�a� la� insulina� i�que� reprimia� la� transcripció�de�gens�pro�

inflamatoris�com�el�TNF��i�la�IL�6�en�adipòcits.�En�conjunt,�s’ha�suggerit�que�la�disminució�dels�

nivells�de�SIRT1�podrien�contribuir�a�l’augment�del�procés�inflamatori�mediat�per�NF��B.�

Els�resultats�obtinguts�in�vitro,�l’augment�en�l’expressió�de�PPAR��i�la�disminució�dels�nivells�

de� SIRT1,� semblen� ser� dependents� de� NF��B� ja� que� l’ús� d’un� inhibidor� d’aquest� factor� de�

transcripció,� el� parthenolide,� evitava� aquests� canvis.� A� més,� l’activació� de� PPAR�� per�

GW501516�va�revertir�la�reducció�en�els�gens�diana�de�PPAR��i�va�evitar�l’augment�dels�gens�

diana� de� NF��B,� el� TNF��i� la� IL�6.� Aquests� resultats� concorden� amb� els� obtinguts� per�

Rodriguez�Calvo�i�col.�(2008)�en�els�quals�es�va�demostrar�que�el�GW501516�era�capaç�d’inhibir�

l’activació�de�NF��B�induïda�per�LPS�(Rodriguez�Calvo�i�col.,�2008).�

En�conclusió,�els�resultats�obtinguts�en�aquest�estudi�suggereixen�que�els�canvis�provocats�pel�

TNF��en�adipòcits�humans,�és�a�dir,� la�disminució�de� l’activitat� de�PPAR�� i� la� reducció�de�

SIRT1,�són�dependents�de�NF��B�i�que�l’activació�de�PPAR��podria�revertir�aquests�efectes,�

possiblement�disminuint�l’activitat�de�NF��B.�

�

�

En� conjunt,� ambdós� estudis� presentats� en� aquesta� Tesi� Doctoral� confirmen� el� paper� de� les�

citocines�pro�inflamatòries�IL�6�i�TNF��com�a�mediadors�fonamentals�en�el�desenvolupament�

de�RI�a�nivell�del�teixit�adipós.�Ambdues�citocines�interfereixen�en�diferents�punts�del�procés�

de� senyalització� de� la� insulina.� Per� una� banda,� en� adipòcits� 3T3�L1,� la� IL�6� activa� la� via� de�

senyalització� JAK/STAT3/SOCS3.� L’augment� dels� nivells� de� SOCS3� resultants� provoquen�

l’ubiquitinització� de� la� IRS�1� per� a� ser� degradada� al� proteasoma� (Rui� i� col.,� 2001).� Per� altra�

banda,�el�TNF��és�el�responsable�de�la�fosforilació�de�IRS�1�en�el�residu�de�serina�307,�fet�que�

provoca� una� reducció� de� l’activitat� d’aquesta� via� (Rui� i� col.,� 2001).� Les� dues� citocines�

convergeixen�en�la� inhibició�de�la�senyalització�de�la� insulina�sobre�el�mateix�punt,�d’aquesta�

manera� s’interromp� la� cascada� de� fosforilacions� induïda� per� la� unió� de� la� insulina� al� seu�

receptor� i� s’impedeix� la� captació� de� glucosa.� Val� la� pena� esmentar� l’estudi� de� Rotter� i� col.�

(2003)� en� el� qual� atribueixen� part� dels� efectes� del� TNF�� a� l’augment� de� l’expressió� de� IL�6.�

DISCUSSIÓ GLOBAL

125

receptor� i� s’impedeix� la� captació� de� glucosa.� Val� la� pena� esmentar� l’estudi� de� Rotter� i� col.�

(2003)� en� el� qual� atribueixen� part� dels� efectes� del� TNF�� a� l’augment� de� l’expressió� de� IL�6.�

Amb� aquestes� dades� es� podria� suggerir� un� mecanisme� de� retroalimentació� del� procés�

inflamatori�amb�la�finalitat�de�mantenir�lo�actiu�(Figura�16).�

A�més,�ambdues�citocines�són�gens�diana�del�factor�de�transcripció�NF��B,�és�més,�s’ha�descrit�

que� TNF��activa� aquest� factor� de� transcripció� (Baldwin,� Jr.,� 2001),� donant� força� a� aquesta�

teoria� de� retroalimentació� positiva� del� procés� inflamatori.� Tots� aquells� factors� que� regulin�

l’activació� de� NF��B� seran� potencials� dianes� farmacològiques� per� a� la� regulació� del� procés�

inflamatori�associat�a�la�RI,�a�l’obesitat�i�a�la�DM2.�En�aquesta�línia�es�troben�les�proteïnes�I�B,�

que� el� mantenen� inactiu� al� nucli,� o� el� complex� IKK,� que� provoca� la� degradació� d’aquestes�

proteïnes� inhibidores� (Baldwin,� Jr.,� 2001).� També� es� pot� actuar� sobre� aquells� enzims� que�

regulen� la�capacitat� transcripcional�de�NF��B,� ja�sigui�afavorint�la�com�CBP/p300�(Roth� i�col.,�

2001)�o�inhibint�la�com�SIRT1�(Yoshizaki�i�col.,�2009).�

INS

IRS1p85 p110

PI3K

PIP2 PIP3 PDK

Akt

VesículaGLUT�4

GLUT�4

Glucosa

PROTEASOMA

JAK JAK

STAT3SOCS3

ERK1/2

IL�6 TNF�

PROTEASOMA

NUCLI

STAT3SOCS3

SIRT1

TNF�IL�6

�

Figura� 16.� Les� citocines� pro�inflamatòries,� IL�6� i� TNF��,�inhibeixen� la� via� de� senyalització� de� la� insulina.�D’una�banda,� la�IL�6�interfereix�amb�la�senyalització�de�la� insulina�a�través�de�l’activació�de�la�via�JAK/STAT3/SOCS3.�Per�altra�banda,�el�TNF��activa�NF��B�que�transcriu�citocines�pro�inflamatòries.�Ambdós�processos�interfereixen�amb�la�cascada�de�fosforilacions�que�té�lloc�quan�la�insulina�(INS)�s´uneix�al�seu�receptor.�(Els�efectes�negatius�de�la�IL�6�i�del�TNF��sobre�la�via�de�senyalització�de�la�insulina�estan�assenyalats�a�la�figura�amb�les�fletxes�vermelles).�

�

Arribats�a�aquest�punt,�el�receptor�nuclear�PPAR��s’ha�postulat�com�un�ferm�candidat�per�al�

control�d’aquest�procés� inflamatori�que� involucra�a� la� IL�6,�al�TNF�� i�al�NF��B.�En� la�present�

Tesi� Doctoral� s’ha� demostrat� que� PPAR��podria� un� factor� clau� en� el� control� del� procés�

inflamatori� tant� en� models� in� vivo� d’obesitat� i� DM2� com� són� les� rates� ZDF� o� els� ratolins�

PPAR��,� però� també� en� estudis� in� vitro� en� adipòcits� murins� i� humans.� Els� resultats�

DISCUSSIÓ GLOBAL

126

presentats�demostren�que� l’activació�de�PPAR��és�capaç�d’inhibir� la�senyalització�de� la� IL�6�

millorant�la�captació�de�glucosa�que�apareixia�reduïda�degut�a�l’estímul�d’aquesta�citocina.�És�a�

dir,�l’activació�de�PPAR��seria�capaç�d’atenuar�la�RI�en�adipòcits�en�cultiu�induïda�per�la�IL�6.�

A�més,� i�en�concordança�amb�altres�estudis�realitzats�en�el�nostre�grup�d’investigació�en�que�

PPAR��evita�l’activació�de�NF��B�en�cèl�lules�epitelials,�en�cèl�lules�musculars�esquelètiques�i�

en�adipòcits�estimulats�amb�LPS�(Barroso�i�col.,�2011;�Coll� i�col.,�2010;�Rodriguez�Calvo� i�col.,�

2008),�s’ha�demostrat�en�que�els�agonistes�d’aquest�subtipus�de�PPAR�també�eviten�l’activació�

de�NF��B�induïda�per�TNF��en�adipòcits�humans�(Figura�17).�

INS

IRS1p85 p110

PI3K

PIP2 PIP3 PDK

Akt

VesículaGLUT�4

GLUT�4

Glucosa

PROTEASOMA

JAK JAK

STAT3SOCS3

ERK1/2

IL�6 TNF�

PROTEASOMA

NUCLI

STAT3SOCS3

SIRT1

TNF�IL�6

PPAR��

Figura�17.�L’activació�de�PPAR��és�capaç�d’inhibir�el�procés�inflamatori�que�causa�RI.�PPAR��pot�inhibir�la�via�de� senyalització� de� la� IL�6,� de� manera� que� la� via� de�senyalització� de� la� insulina� continua� activa� i� afavoreix� així� el�transport�de�glucosa�al�nucli.�Per�altra�banda,�l’activació�de�PPAR��inhibeix�l’activació�de�NF��B�induïda�per�TNF��i�per�tant,�disminueix�la�transcripció�dels�seus�gens�diana.�(Els�efectes�de�PPAR��inhibint�la�senyalització�de�les�citocines�pro�inflamatòries�s’assenyalen�a�la�figura�amb�creus�negres).�

DISCUSSIÓ GLOBAL

127

�

�

�

�

�

�

�

�

�

�

�

�

�

CONCLUSIONS�

�

�

�

�

129

�

�

Les�principals�conclusions�del�treball�experimental�d’aquesta�Tesi�Doctoral�són�les�següents:�

�

I. L’activació�de�PPAR��amb�GW501516�evita�l’activació�de�la�via�STAT3/SOCS3�

a�través�de�dos�mecanismes�diferents:�

a. La�inhibició�de�la�fosforilació�de�l’ERK1/2.�

b. La�inhibició�de�la�interacció�proteïna�proteïna�entre�STAT3�i�Hsp90.�

� Aquests� dos� mecanismes� podrien� explicar� la� millora� en� la� sensibilitat� a� la�

� insulina�produïda�per�GW501516�en�adipòcits.�

�

II. Al� teixit� adipós� de� pacients� obesos� mòrbids� l’expressió� de� PPAR��es� troba�

incrementada�respecte�a�pacients�no�obesos.�

III. Adipòcits�humans�exposats�a�TNF��presenten�un� increment�de� l’expressió�de�

PPAR�/�,�però�la�seva�activitat�d’unió�a�l’ADN�i�l’expressió�dels�seus�gens�diana�

està� reduïda,� fet� que� suggereix� una� reducció� de� l’activitat� d’aquest� receptor�

nuclear.��

IV. L’exposició�a�TNF��d’adipòcits�humans�provoca�la�disminució�de�l’expressió�de�

SIRT1.��

V. Els�canvis� induïts�per�TNF��en� l’activitat�de�PPAR�/�� i�en� l’expressió�de�SIRT1�

en� adipòcits� humans� es� deuen� a� l’activació� de� NF��B,� ja� que� l’addició� d’un�

inhibidor�específic�d’aquest�factor�de�transcripció�els�reverteix.��

VI. Els�activadors�de�PPAR��reverteixen�els�efectes�deguts�a�l’activació�de�NF��B�

induïda�per�TNF��en�adipòcits�humans.�

�

CONCLUSIONS

131

�

�

�

�

�

�

�

�

�

�

�

�

�

BIBLIOGRAFIA�

133

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Lucía�Serrano�Marco,�Emma�Barroso1,�Ilhem�El�Kochairi2,�Xavier�Palomer1,�Liliane�Michalik2,Walter�Wahli2�and�Manuel�Vázquez�Carrera1.�

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caracteritzat�per�la�producció�anormal�de�citocines�pro�inflamatòries�com�el�TNF��,�la�IL�1��o�

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consegüent� inducció� de� SOCS3� el� qual� inhibeix� la� senyalització� de� la� insulina� a� través� de�

diversos�mecanismes�que�involucren�l’IR�i�IRS�1.�

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induïda�per�la�IL�6�al�fetge�mitjançant�la�inhibició�de�l’activitat�transcripcional�de�STAT3,�però�el�

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senyalització� STAT3/SOCS3� en� l’aparició� de� RI� induïda� per� IL�6� en� hepatòcits� és� interessant�

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THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR�/� (PPAR�/�) AGONIST

GW501516 INHIBITS IL-6-INDUCED STAT3 ACTIVATION AND INSULIN RESISTANCE IN

HUMAN LIVER CELLS

Lucía Serrano-Marco1, Emma Barroso1, Ilhem El Kochairi2, Xavier Palomer1, Liliane Michalik2,

Walter Wahli2 and Manuel Vázquez-Carrera1.

1Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, Faculty of Pharmacy,

University of Barcelona, Institut de Biomedicina de la UB (IBUB), and CIBER de Diabetes y

Enfermedades Metabólicas - CIBERDEM-Instituto de Salud Carlos III, Diagonal 643, E-08028

Barcelona, Spain and 2Center for Integrative Genomics, National Research Center Frontiers in Genetics,

University of Lausanne, CH-1015 Lausanne, Switzerland.

Running title: PPAR� Inhibits STAT3 in HepG2 cells

Key words: PPAR�, IL-6, STAT3, SOCS3, AMPK, ERK1/2.

Corresponding author: Manuel Vázquez-Carrera

Unitat de Farmacologia. Facultat de Farmàcia.

Diagonal 643. E-08028 Barcelona. Spain

Phone 93 4024531

Fax 93 4035982

E-mail: [email protected]

Abbreviations: AMPK, AMP-activated protein kinase; ERK1/2, extracellular-related kinase 1/2; IL-6,

Interleukin 6; IRS, insulin receptor substrate, PPAR, Peroxisome Proliferator-Activated Receptor; SOCS3,

suppressor of cytokine signaling 3; STAT3, transducer and activator of transcription 3.

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Abstract

Aim/hypothesis IL-6 is one of the mediators linking obesity-derived chronic inflammation with insulin

resistance through activation of signal transducer and activator of transcription 3 (STAT3), with subsequent

up-regulation of suppressor of cytokine signaling 3 (SOCS3). Here we evaluated whether the Peroxisome

Proliferator-Activated Receptor (PPAR)�/� activator GW501516 prevented the activation of the IL-6-

STAT3-SOCS3 pathway and insulin resistance in human hepatic HepG2 cells.

Methods Studies were conducted with human HepG2 cells and livers form PPAR�/�-null mice and wild-

type mice.

Results GW501516 prevented IL-6-dependent reductions in insulin-stimulated Akt phosphorylation and in

IRS-1 and IRS-2 protein levels. In addition, this drug treatment abolished IL-6-induced STAT3

phosphorylation on Tyr705 and Ser727 and prevented the increase in SOCS3 caused by this cytokine.

Moreover, GW501516 prevented IL-6-dependent induction of ERK1/2, a serine-threonine-protein kinase

involved in serine STAT3 phosphorylation, and the livers of PPAR�/�-null mice showed increased Tyr705-

and Ser727-STAT3 as well as phospho-ERK1/2 levels. Furthermore, drug treatment prevented the IL-6-

dependent reduction in phospho-AMPK, a kinase reported to inhibit STAT3 phosphorylation on Tyr705. In

agreement with the recovery in phospho-AMPK levels observed following GW501516 treatment, this drug

increased the AMP to ATP ratio and decreased the ATP to ADP ratio.

Conclusions Collectively, our findings indicate that the PPAR�/��activator GW501516 prevents IL-6-

induced STAT3 activation by inhibiting ERK1/2 phosphorylation and by preventing the reduction in

phospho-AMPK levels. These effects of GW501516 may contribute to the prevention of cytokine-induced

insulin resistance in hepatic cells.

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Introduction

Insulin resistance and type 2 diabetes mellitus are closely associated with low-grade chronic inflammation

characterized by abnormal production of pro-inflammatory cytokines, such as tumor necrosis factor alpha

(TNF-�) (1), interleukin 1-beta (2) and interleukin 6 (IL-6) (3-5). Of these cytokines, IL-6 shows a strong

association with obesity in both rodent and human models. Thus, depletion of IL-6 improves insulin action

in a mouse model of obesity (6), whereas in humans, elevated plasma IL-6 levels correlate positively with

obesity and insulin resistance and predict the development of type 2 diabetes mellitus (5;7;8). In addition,

administration of IL-6 to healthy subjects induces blood glucose increases (9). In vitro, IL-6 has been

shown to induce insulin resistance in hepatic cells (10;11). Although the contribution of IL-6 to the

development of insulin resistance in adipose tissue and skeletal muscle is still being debated, it is generally

accepted that, at least in liver, IL-6 causes insulin resistance (12;13)

IL-6 signals through a transmembrane receptor complex containing the common signal transducing receptor

glycoprotein gp130, which activates Janus tyrosine kinases (Jak1, Jak2, Tyk2), with subsequent Tyr705

phosphorylation of signal transducer and activator of transcription 3 (STAT3) (14-16). Phosphorylated

STAT3 dimerizes and translocates to the nucleus, where it regulates the transcription of target genes

through binding to specific DNA-responsive elements (17). In addition to activation by Tyr705

phosphorylation, STAT3 also requires phosphorylation on Ser727 to achieve maximal transcriptional activity

(18;19). Protein kinases involved in STAT3 serine phosphorylation include protein kinase C, Jun N-

terminal kinase, extracellular signal-related kinase (ERK), the mitogen-activated protein kinase p38 and

mammalian target of rapamycin (mTOR) (20).

The mechanism by which IL-6 induces insulin resistance in liver involves the activation of STAT3 and

subsequent induction of suppressor of cytokine signaling 3 (SOCS3) (6;21;22), a negative regulator of

cytokine signaling (23). Several cytokines and hormones associated with insulin resistance induce the

expression of SOCS proteins, which inhibit insulin signaling through several distinct mechanism, including

directly interfering with insulin receptor activation, blocking insulin receptor substrate (IRS) activation, and

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inducing IRS degradation (24). In liver, overexpression of SOCS3 causes insulin resistance, whereas

antisense suppression of SOCS3 in obese diabetic mice (db/db) ameliorates insulin resistance (25).

Peroxisome Proliferator-Activated Receptors (PPARs) are members of the nuclear receptor superfamily of

ligand-inducible transcription factors that form heterodimers with retinoid X receptors (RXRs) and bind to

consensus DNA sites (26). In addition, PPARs may suppress inflammation through diverse mechanisms,

such as reduced release of inflammatory factors or stabilization of repressive complexes at inflammatory

gene promoters (27-30). Of the three PPAR isotypes found in mammals, PPAR��(NR1C1) and PPAR�

(NR1C3) (31) are the targets for hypolipidemic (fibrates) and anti-diabetic (thiazolidinediones) drugs,

respectively. Finally, activation of the third isotype, PPAR�/� (NR1C2, called PPAR��below), enhances

fatty acid catabolism in adipose tissue and skeletal muscle and, therefore, it has been proposed as a potential

treatment for insulin resistance (32). Recently, it was reported that agonist-activated PPAR� interferes with

IL-6-mediated acute phase reaction in the liver by inhibiting the transcriptional activity of STAT3 (33),

although the exact molecular mechanism involved remains unknown. It is worth noting that a recent study

demonstrated that AMP-activated protein kinase (AMPK) regulates IL-6 signaling in HepG2 cells by

inhibiting STAT3 (34) and that the PPAR� activator GW501516 can increase the activity of AMPK (35).

Given the prominent role of the STAT3-SOCS3 pathway in IL-6-mediated insulin resistance in

hepatocytes, we explored whether PPAR� activation by GW501516 prevented IL-6-mediated insulin

resistance in human hepatic cells and the mechanisms involved. PPAR� activation by GW501516

prevented IL-6-mediated induction of SOCS3 mRNA levels and STAT3 phosphorylation on Tyr705 and

Ser727 in HepG2 cells. Consistent with the role of PPAR� in blocking IL-6-induced STAT3 activity, STAT3

phosphorylation on Tyr705 and Ser727 was higher in liver from PPAR�-null mice than in wild-type mice. In

agreement with the inhibition of the STAT3-SOCS3 pathway caused by GW501516, this drug prevented

the reduction in insulin-stimulated Akt phosphorylation and in IRS-1 and IRS-2 protein levels. GW501516

prevented the increase in ERK-1/2 phosphorylation caused by IL-6 exposure, suggesting that this

mechanism contributed to its effects on STAT3 phosphorylation on Ser727. Our findings also show that

GW501516 prevented the reduction in phospho-AMPK levels observed in IL-6-exposed cells by increasing

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the AMP/ATP ratio. This mechanism can explain the reduction in STAT3 phosphorylation on Tyr705

observed following GW501516 treatment. Overall, on the basis of our findings, we suggest that PPAR�

activation can ameliorate insulin resistance in hepatic cells by preventing IL-6-induced activation of the

STAT3-SOCS3 pathway through ERK1/2 inhibition and by restoring phospho-AMPK levels.

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Methods

Materials

The PPAR� ligand GW501516 was obtained from Biomol Research Labs Inc. (Plymouth Meeting, PA).

Other chemicals were from Sigma (St. Louis, MO).

Cell culture

The HepG2 cells (hepatocellular carcinoma, American Type Culture Collection, Manassas, VA, USA) were

maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Lonza, Barcelona, Spain) including 4.5 g/l

glucose and L-glutamine, supplemented with 10% (v/v) FBS (Invitrogen, San Diego, CA, USA), penicillin-

streptomycin (Invitrogen) and non-essential amino acids. Cell density was adjusted to 2 x 105 cells/ml and 1

ml of the cell suspension was added per well to 12-well cell culture plates (NUNC, Roskilde, Denmark).

HepG2 cells were then incubated with 10 µM GW501516 and IL-6 (20 ng/ml) for the times indicated. After

incubation, RNA, total and nuclear proteins extracts were extracted as described below. Inhibitors were

added 30 min prior to incubation with IL-6.

Animals

The generation of PPAR� null mice was as described previously (36). Six male PPAR� null mice and 6 of

their control male PPAR� wild type mice were used (5 to 6 months old). In agreement with the guidelines

specified by the veterinary office of Lausanne (Switzerland), the mice were housed under standard light-

dark cycle (12-h light/dark cycle) and temperature (21 � 1ºC) conditions, and fed with Provimi Kliba 3436

chow. Liver tissue was rapidly removed, frozen in liquid nitrogen and stored at -80ºC.

Measurements of mRNA

Levels of mRNA were assessed by the reverse transcription-polymerase chain reaction (RT-PCR) as

previously described (37). Total RNA was isolated using the Ultraspec reagent (Biotecx, Houston). The total

RNA isolated by this method is non-degraded and free of protein and DNA contamination. The sequences of

the sense and antisense primers used for amplification were: SOCS3 (Suppressor of cytokine signaling 3) 5’-

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TTTTCGCTGCAGAGTGACCCC-3’ and 5’-TGGAGGAGAGAGGTCGGCTCA-3’; and 18S, 5’-

ATGACTTCCAAGCTGGCCGTG-3’ and 5’-GCGCAGTGTGGTCCACTCTCA-3’. Amplification of each

gene yielded a single band of the expected size (SOCS3: 250 bp and 18S: 333 bp). Preliminary experiments

were carried out with various amounts of cDNA to determine non-saturating conditions of PCR amplification

for all the genes studied. Then, under these conditions, relative quantification of mRNA was assessed by the

RT-PCR method used in this study (38). Radioactive bands were quantified by video-densitometric scanning

(Vilbert Lourmat Imaging). The results for the expression of specific mRNAs are always presented relative to

the expression of the control gene (18S).

Isolation of nuclear extracts

Nuclear extracts were isolated as previously described (39). Cells were scraped into 1.5 ml of cold phosphate-

buffered saline, pelleted for 10 seconds and re-suspended in 400�l of cold Buffer A (10mM HEPES pH 7.9 at

4ºC, 1.5mM MgCl2, 10mM KCl, 0.5mM DTT, 0.2mM PMSF, and 5�g/ml aprotinin) by flicking the tube.

Cells were allowed to swell on ice for 10 min, and then vortexed for 10 sec. Samples were then centrifuged for

10 sec and the supernatant fraction was discarded. Pellets were re-suspended in 50�l of cold Buffer C (20mM

HEPES-KOH pH 7.9 at 4ºC, 25% glycerol, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.5mM DTT,

0.2mM PMSF, 5�g/ml aprotinin and 2�g/ml leupeptin) and incubated on ice for 20 min for high-salt

extraction. Cellular debris was removed by centrifugation for 2 min at 4ºC and the supernatant fraction

(containing DNA-binding proteins) was stored at –80ºC. Nuclear extract concentration was determined by the

Bradford method.

Antibodies and immunoblotting

Antibodies against total and phospho-AMPK(Thr172), total and phospho-Akt (Ser473), phospho-ERK1/2

(Thr202/Tyr204) and phospho-STAT3 (Tyr705 and Ser727) were purchased from Cell Signaling. Antibody

against total STAT3 was purchased from Santa Cruz.

To obtain total protein, cells and livers were homogenized in RIPA buffer (Sigma) with phosphatase

inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 5.4 �g/ml aprotinin). The

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homogenate was centrifuged at 16,700 x g for 30 min at 4ºC. Protein concentration was measured by the

Bradford method.

Proteins from whole-cell lysates and nuclear extracts were separated by SDS-PAGE, then transferred to

immobilon polyvinylidene diflouride membranes (Millipore, Bedford, MA) and blotted with various

antibodies (as specified in “Results”). Detection was achieved using the EZ-ECL chemiluminescence kit

(Amersham). Size of detected proteins was estimated using protein molecular-mass standards (Invitrogen,

Barcelona, Spain).

High Performance Liquid Chromatography Measurement of ATP, ADP, and AMP

Adenine nucleotides were separated by high performance liquid chromatography using an X-Bridge column

with a 3.5 �m outer diameter (100 x 4.6 cm). Elution was performed with 0.1 mM potassium dihydrogen

phosphate, pH 6, containing 4 mM tetrabutylammonium hydrogen sulfate and 15% (v/v) methanol. The

conditions were as follows: 20 �l sample injection, column at room temperature, flow rate of 0.6 ml min-1

and UV monitoring at 260 nm.

Statistical Analyses

Data are presented as mean � S.D. of 5 separate experiments. Significant differences were established by

one-way ANOVA, using the GraphPad InStat program (GraphPad Software V2.03) (GraphPad Softwware

Inc., San Diego, CA). When significant variations were found, the Tukey-Kramer multiple comparisons test

was applied. Differences were considered significant at P<0.05.

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Results

PPAR� activation prevents the reduction in insulin-stimulated Akt phosphorylation and IRS degradation

caused by IL-6 It has been previously reported that IL-6 induces insulin resistance in human

hepatocarcinoma HepG2 cells (10;11), a frequently used in vitro system for studying insulin’s effects on

hepatic cells. Cells exposed to IL-6 stimulation significantly dampened their response to insulin, as

measured by Akt phosphorylation (Figure 1A). Interestingly, when cells were preincubated with IL-6 in the

presence of 10 �M GW501516, a selective ligand for PPAR� with a 1000-fold higher affinity toward

PPAR� than PPAR� and PPAR� (40), the inhibitory effect of this cytokine on insulin-stimulated Akt

phosphorylation was prevented. Drug treatment in the absence of insulin did not affect the phosphorylation

status of Akt (data not shown).

In addition, since IL-6-induced insulin resistance in hepatic cells has been attributed to SOCS3 (41) and this

protein inhibits insulin signaling by proteasomal-mediated degradation of IRS-1 and IRS-2 (42), we also

examined their protein levels. As shown in Figure 1B, IRS-1 and IRS-2 protein levels were reduced

following IL-6 exposure, but these effects were abolished in the presence of GW501516. Thus, GW501516

treatment offered protection against the effects of IL-6 on insulin signaling.

PPAR� activation inhibits IL-6-induced SOCS3 expression in HepG2 cells We then examined the effect of

PPAR� activation on the mRNA levels of the STAT3-target gene SOCS3. HepG2 cells exposed to IL-6

showed increased SOCS3 mRNA levels (2.7-fold induction, p<0.01), whereas in cells co-incubated with

IL-6 plus GW501516 (p<0.001 vs. IL-6-stimulated cells) this induction was abolished (Figure 2A).

Dimerization, nuclear translocation and increase in transcriptional activity of STAT3 require its

phosphorylation on tyrosine residue 705. In agreement with this, IL-6 exposure increased STAT3

phosphorylation on Tyr705, and GW501516 treatment reduced STAT3 phosphorylation on Tyr705 (Figure

2B). In addition, STAT3 phosphorylation on Ser727 is required for its maximal activation (43;44). As

expected, IL-6 stimulation enhanced STAT3 phosphorylation on Ser727, whereas it was prevented in the

presence of GW501516 (Figure 2B). Since IL-6 activates ERK1/2 (45), which has been reported to be a

kinase for STAT3 phosphorylation on Ser727 (46), and we have previously reported that GW501516

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prevents IL-6-induced ERK1/2 activation in adipocytes (47), we evaluated the effect of this PPAR� agonist

on the activation of this kinase. IL-6 exposure increased ERK1/2 phosphorylation, whereas in the presence

of GW501516, phospho-ERK1/2 levels were strongly suppressed (Figure 3A). To confirm that in our

conditions IL-6-induced ERK1/2 phosphorylation was involved in STAT3 phosphorylation on Ser727, we

took advantage of U0126, a potent and specific ERK1/2 inhibitor, which binds to MEK1/2 (mitogen-

activated protein kinase (MAPK)–ERK 1/2), thereby inhibiting its catalytic activity as well as

phosphorylation of ERK1/2. Similarly to GW501516, U0126 prevented IL-6-induced STAT3

phosphorylation on Ser727 (Figure 3B). In addition, U0126 prevented the increase in SOCS3 mRNA levels

caused by IL-6 (Figure 3C). These findings confirm that IL-6-induced ERK1/2 phosphorylation contributes

to STAT3 phosphorylation on Ser727, leading to increased expression of its target gene SOCS3.

Increased levels of phospho-STAT3 (Ser727) and phospho-ERK1/2 in the liver of the PPAR�-null mouse To

clearly demonstrate the involvement of PPAR� in the regulation of STAT3 phosphorylation we used the

PPAR�-null mouse. Livers of these mice showed a significant increase in STAT3 Ser727 and Tyr705

phosphorylation compared to wild-type mice (Figure 4A). In agreement with this, the phosphorylation

status of ERK1/2 was increased in PPAR�-null mice (Figure 4B). These findings demonstrate that PPAR�

regulates ERK1/2 and STAT3 phosphorylation in vivo.

PPAR� activation inhibits IL-6-induced AMPK down-regulation in HepG2 cells The involvement of

ERK1/2 inhibition in the effects of GW501516 does not provide an explanation for the reduction of Tyr705-

STAT3 phosphorylation following treatment with this PPAR� activator. Therefore, we explored the effects

of IL-6 and GW501516 on AMPK, since activation of this kinase prevents IL-6-induced STAT3 activation

in HepG2 cells by inhibiting STAT3 phosphorylation on Tyr705 (34), suggesting that this kinase is a

potential pharmacological target to inhibit the deleterious effects of IL-6 in liver cells. AMPK can be

activated by several kinases and by allosteric binding of AMP to the regulatory ��subunit (48). Interestingly,

it has been reported that GW501516 increases the AMP to ATP ratio both in vitro (35) and in vivo (49).

Thus, we first examined the effects of IL-6 and its co-incubation with GW501516 on AMPK-

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phosphorylation. IL-6 stimulation reduced phospho-AMPK levels compared to control cells, but this

reduction was blocked by the presence of GW501516 (Figure 5A). Next we measured adenine nucleotide

concentrations by high performance liquid chromatography in HepG2 cells to determine the cellular

ATP:ADP and AMP:ATP ratios. Cells exposed to IL-6 did not show significant changes. In contrast,

GW501516 significantly increased the AMP to ATP ratio (Figure 5B) and decreased the ATP to ADP ratio

(Figure 5C).

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Discussion

Chronic production of pro-inflammatory cytokines, which is associated with obesity in both human and

rodent models, is considered a major link between obesity and insulin resistance (50). In contrast to

adipose-derived TNF-�, which may act locally in an autocrine and paracrine manner, adipose-derived IL-6

can enter circulation and play a systemic role in modulating insulin actions (51). IL-6 acts primarily by

activating STAT3 and up-regulating the transcription of its target gene SOCS3, which causes insulin

resistance by interfering with insulin receptors and/or IRS-1 (52-55). Our findings demonstrate that

GW501516 confers protection against the effects of IL-6 on insulin signaling in hepatic cells, as

demonstrated by its effects on insulin-stimulated Akt phosphorylation and on IRS-1 and IRS-2 protein

levels. These effects of GW501516 are consistent with the capacity of this drug to prevent IL-6-induced

SOCS3 expression in HepG2 cells, suggesting that drug treatment prevented IL-6-induced STAT3

activation. Activation of STAT3 is dependent on its phosphorylation status, and, in fact, GW501516

prevented the increase induced by IL-6 in STAT3 phosphorylation on Tyr705 and on Ser727 phosphorylation

The effect of GW501516 on Tyr705 and on Ser727 phosphorylation seems to be dependent on PPAR� since in

the livers of PPAR�-null mice we observed an increase in the levels of Tyr705- and Ser727-STAT3. Several

kinases can phosphorylate STAT3 on Ser727, including ERK1/2 (56). In agreement with a role for ERK1/2

on STAT3-Ser727 phosphorylation following IL-6 stimulation, we report that this cytokine increased

phospho-ERK1/2 levels and that the ERK1/2 inhibitor U0126 reduced the levels of Ser727-STAT3 in IL-6

exposed cells. In addition, this inhibitor prevented the increase in SOCS3 mRNA levels caused by IL-6,

suggesting that ERK1/2 inhibition is sufficient to prevent the activation of the STAT3-SOCS3 pathway.

Interestingly, GW501516 completely abolished the increase in phospho-ERK1/2 levels caused by IL-6,

suggesting that inhibition of this kinase was responsible for the reduction in Ser727-STAT3 in cells co-

incubated with IL-6 plus GW501516. In agreement with the inhibition of STAT3 phosphorylation on

Ser727, the reduction in ERK1/2 phosphorylation caused by GW501516 treatment also seems to be

PPAR��dependent, since the livers of PPAR�-null mice showed increased phospho-protein levels of this

kinase.

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The findings of this study provide an additional mechanism by which GW501516 can inhibit IL-6-mediated

activation of STAT3 in human liver cells. This mechanism involves AMPK, a kinase reported to regulate

IL-6 signaling in HepG2 cells by inhibiting STAT3 (34). The authors of this study showed that AMPK

agonists reduce the IL-6-stimulated expression of inflammatory markers and SOCS3 in HepG2 cells by

preventing STAT3 phosphorylation on Tyr705. These data are consistent with the reported activation of

AMPK as an attractive strategy for the treatment of insulin resistance and type 2 diabetes (57) and suggest

that down-regulation of AMPK would promote the STAT3-SOCS3 pathway contributing to insulin

resistance. However, when they studied the effects of IL-6 on phospho-AMPK, no changes were observed.

In contrast to this study, here we report that cells exposed to IL-6 showed a reduction in AMPK

phosphorylation. The discrepancy between these studies can be attributed to differences in the

concentration of IL-6 used. In our study we exposed cells to 20 ng/ml IL-6 compared to the 10 ng/ml used

by Nerstedt et al. (34). In agreement with our findings, a previous study reported a reduction in AMPK and

IRS-1 protein levels in the heart of mice treated with IL-6 (58). The authors of this study also reported that

the potential mechanism by which IL-6 can reduce AMPK levels might involve increased protein-protein

interaction between SOCS3 and AMPK, leading to ubiquitin-mediated degradation of AMPK, as reported

for IRS-1 (58). Of note, GW501516 treatment prevented the reduction in phospho-AMPK levels caused by

IL-6 stimulation. As we observed an increase in the AMP:ATP ratio in cells incubated with GW501516, the

recovery in phospho-AMPK levels induced by GW501516 could be the result of a modification of the

cellular energy status. This effect of GW501516 on the AMP:ATP ratio has previously been reported in

human skeletal muscle cells (35) and in liver (49), and it has been considered the result of a specific

inhibition of one or more complexes of the respiratory chain, an effect of the ATP synthase system, or

mitochondrial uncoupling (35). These changes would reduce the yield of ATP synthesis by the

mitochondria, leading to AMPK activation.

In summary, on the basis of our findings, we suggest that PPAR� activation prevents IL-6-induced STAT3

activation and SOCS3 up-regulation, thereby contributing to the prevention of the cytokine-mediated

development of insulin resistance in hepatic cells.

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Acknowledgements This study was partly supported by funds from the Swiss National Science

Foundation, the Spanish Ministerio de Ciencia e Innovación (SAF2009-06939) and European Union ERDF

funds. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) is an Instituto de Salud

Carlos III project. L. S.-M. was supported by a FPI grant from the Spanish Ministerio de Ciencia e

Innovación. We would like to thank the University of Barcelona’s Language Advisory Service for help.

Duality of interest The authors declare that they have no duality of interest associated with this

manuscript.

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FIGURE LEGENDS

FIG. 1. PPAR� activation antagonizes IL-6 action and protects against its effects on insulin signaling.

HepG2 cells were stimulated with 100 nM insulin for 3 min, with or without pretreatment with either 10

µM GW501516 for 18 h or 20 ng/ml IL-6 for 10 min. Cell lysates were subjected to Western blot analysis

for phospho-Akt(Ser473) and total Akt (A), IRS-1 and IRS-2 (C). Values are means � S.D. of five

independent experiments. ***p<0.001 vs. control cells without insulin stimulation. ###p<0.001 vs. control

cells stimulated with insulin.

FIG. 2. The PPAR� agonist GW501516 prevents IL-6-induced SOCS3 expression and STAT3

phosphorylation in HepG2 cells. A, Analysis of the mRNA levels of SOCS3 in human hepatic cells

untreated or treated with 10 µM GW501516 for 18 h prior to stimulation with 20 ng/ml IL-6 for 24 h. Total

RNA was isolated and analyzed by RT-PCR. A representative autoradiogram and the quantification

normalized to 18S mRNA levels are shown. Data are the means � S.D. of five independent experiments. B,

Total cell extracts (Tyr705-STAT3) or nuclear (Ser727-STAT3) were subjected to Western blot analysis with

phospho-STAT3 (Tyr705 and Ser727) or total STAT3 antibodies. HepG2 cells untreated or treated with 10

µM GW501516 for 18 h prior to stimulation with 20 ng/ml IL-6 for either 10 min (Ser727-STAT3) or 2.5 h

(Tyr705-STAT3). Bars are the means � S.D. of five independent experiments. ***P<0.001 vs. control,

#P<0.05 and ###P<0.001 vs. IL-6-stimulated cells. A.U.: Arbitrary Units.

FIG. 3. PPAR� activation inhibits IL-6-induced ERK1/2 phosphorylation. HepG2 cells were pretreated

with or without 10 µM U0126 or 10 µM GW501516 prior stimulation with 20 ng/ml IL-6 for 2.5 h. Cell

lysates were subjected to Western blot analysis for phospho-ERK1/2 (Thr202/Tyr204) (A) or phospho-STAT3

(Ser727) (B). C, Analysis of the mRNA levels of SOCS3 in HepG2 cells untreated or treated with 10 µM

GW501516 for 18 h or with 10 µM GW501516 prior to stimulation with 20 ng/ml IL-6 for 24 h. Total

RNA was isolated and analyzed by RT-PCR. A representative autoradiogram and the quantification

normalized to 18S mRNA levels are shown. Data are the means � S.D. of five independent experiments.

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***P<0.001, **P<0.01, *P<0.05 vs. control, ###P<0.001, ##p<0.01 #p<0.05 vs. IL-6-stimulated cells. A.U.:

Arbitrary Units.

FIG. 4. The PPAR�-null mouse shows enhanced STAT3 and ERK1/2 phosphorylation in liver. Cellular

extracts from wild-type (WT) or PPAR�-null (KO) mouse liver were analyzed by Western blot with

phospho-STAT3 (Tyr705 and Ser727) (A) and phospho-ERK1/2 (Thr202/Tyr204) (B) antibodies as indicated.

Bars are the means � S.D. of five independent experiments. ***P<0.001, *P<0.05 vs. wild-type animals.

FIG. 5. The PPAR� agonist GW501516 prevents the reduction in phospho-AMPK protein levels caused by

IL-6. A, Analysis of phospho-AMPK(Thr172) and total AMPK by immunoblotting of total protein extracts

from HepG2 cells pretreated with or without 10 µM GW501516 prior stimulation with 20 ng/ml IL-6 for

2.5 h. AMP/ATP (B) and ATP/ADP (C) ratio in HepG2 cells pretreated with or without 10 µM GW501516

prior stimulation with 20 ng/ml IL-6 for 2.5 h. Bars are the means � S.D. of five independent experiments.

***P<0.001, **P<0.01 vs. control, ###P<0.001, ##P<0.01 vs. IL-6-stimulated cells. @ P<0.05 vs. IL-6 plus

GW501516-stimulated cells.

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