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Facultad!de!Farmacia!

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Ana!Elsa!Huerta!Hernández!

PAMPLONA,!2016!

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EFFECTS!OF!ORAL!SUPPLEMENTATION!WITH!α

EFECTOS!DE!LA!SUPLEMENTACIÓN!ORAL!CON!ÁCIDO!α

Memoria!presentada!por!Dña.!Ana!Elsa!Huerta!Hernández!para!aspirar!al!grado!de!Doctor!por! la!Universidad!de!Navarra.!!

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Dña.!Ana!Elsa!Huerta!Hernández!

El!presente!trabajo!ha!sido!realizado!bajo!nuestra!dirección!en!el!Departamento!de!Ciencias!de! la!Alimentación!y!Fisiología!y!autorizamos!su!presentación!ante!el!tribunal!que!lo!ha!de!juzgar.!!

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Pamplona,!2016!!

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Dra.!María!Jesús!Moreno!Aliaga!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Dr.!J.!Alfredo!Martínez!Hernández!

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Este! trabajo! ha! sido! posible! gracias! a! la! financiación! de! diversas! entidades:! Ministerio! de! Economía! y! Competitividad! del! Gobierno! de! España! (ref.! AGL! 2009T10873/ALI! and! BFU2012T 36089),!Departamento!de!Salud!del!Gobierno!de!Navarra!(!ref.!67/2015),!Asociación!de!amigos!de! la! Universidad! de! Navarra! (beca! predoctoral! 2012T2016);!Línea!Especial!“Nutrición,!Obesidad!y! Salud”! Universidad! de! Navarra;! Centro! de! Investigación! en! Nutrición! de! la! Universidad! de! Navarra;! Centro! de! Investigación! Biomédica! en! Red,! Fisiopatología! de! la! Obesidad! y! Nutrición! (CIBERobn).!

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AGRADECIMIENTOS!

Después!de!unos!años!de!trabajo,!por!fin!llega!el!momento!de!escribir!los!agradecimientos.!Me! gustaría! aprovechar! este! espacio! para! agradecer! a! todas! las! instituciones! y! personas! que! han! hecho!posible!que!este!proyecto!se!llevara!a!cabo,!así!como!también!a!aquellos!y!aquellas!que!me! han!apoyado!durante!este!camino!y!han!estado!ahí.!!

Antes! que! nada,! me! gustaría! agradecer! al! Departamento! de! Ciencias! de! la! Alimentación! y! Fisiología! de! la! Universidad! de! Navarra,! a! la! Asociación! de! Amigos! y! a! todas! las! instituciones! financiadoras,!incluyendo!el!Ministerio!de!Economía!y!Competitividad,!Gobierno!de!Navarra!y!el! Centro!de!Nutrición,!que!han!hecho!posible!el!desarrollo!de!esta!investigación.!!

Me!gustaría!agradecer!especialmente!a!mis!directores!de!tesis,!la!Prof.!María!Jesús!Moreno! Aliaga! y! el! Prof.! Alfredo! Martínez! por! su! apoyo! durante! estos! años! de! tesis.! De! ambos! he! aprendido! cosas! muy! valiosas! que! estoy! segura! que! me! han! hecho! mejor! profesional! y! me! ayudarán! en! años! futuros.! Gracias! María! Jesús! por! la! confianza! depositada! en! mí! desde! un! principio,! por! tu! estar! hasta! el! final! y! por! fijarte! en! los! pequeños! detalles! que! siempre! se! nos! escapaban.!Gracias!Alfredo!por!tu!disponibilidad!incondicional!para!cualquier!duda!o!problema! que!surgiera.!!

Gracias! al! Dr.! Santiago! Navas,! la! Dra.! Marta! Cuervo,! la! Dra.! Blanca! Martínez! de! Morentín,! María!Hernández,!Salomé!Pérez!y!todo!el!personal!de!la!Unidad!Metabólica,!por!su!apoyo!en!la! realización! de! este! estudio,! pues! sin! él! no! hubiera! sido! posible.! Especialmente! me! gustaría! agradecer!a!María!Zabala!y!a!Pedro!Prieto!por!su!soporte!en!mis!primeros!años!de!tesis.!Mery,! gracias!por!tu!paciencia,!por!enseñarme!el!cómo!trabajar!en!un!laboratorio!y!por!estar!siempre! transmitiendo!buena!vibra.!!

También!agradezco!a!todas!aquellas!personas!que!en!estos!últimos!años!o!meses!de!tesis!me! han!ayudado!de!alguna!u!otra!forma!y!que!son!parte!del!esfuerzo!que!ha!sido!depositado!en!este! proyecto.!Como!las!técnicos!de!laboratorio,!Vero!y!Ana,!que!siempre!están!ahí!para!ayudarnos!y! resolver!nuestras!dudas.!También!a!Neira!y!a!Asun,!que!a!pesar!de!todo!el!trabajo!diario,!siempre! con!una!sonrisa!y!buena!gana!estuvieron!ahí.!A!José!Ignacio,!que!ha!sido!parte!esencial!en!este! último!capítulo!de!la!tesis,!no!solo!por!el!apoyo!científico!sino!también!moral.!A!Xavi,!igual!por!la! ayuda!técnica!y!la!proTactividad!que!contagia.!Me!gustaría!mencionar!igual!a!Ana!Romo,!Fermín,! Pedro! González,! Jaione,! Marian! Zulet! y! a! todos! aquellos! investigadores! e! investigadoras! que! trabajan!en!el!departamento.!!

Agradezco!a!todos!mis!compis!de!trabajo!del!área!de!ordenadores,!muchas!gracias!por!generar! tan!buen!ambiente!de!trabajo!y!hacer!que!de!gusto!llegar!a!la!Universidad.!Gracias!a!vosotros!y! vosotras!porque!creo!que!he!recibido!mucho!apoyo!y!ayuda!de!su!parte,!no!solo!en!la!cuestión!

! AGRADECIMIENTOS! técnica! o! de! investigación! sino! también! moral.! Especialmente! al! Granada! team,! todas! súper! trabajadoras,!perseverantes!y!una!inspiración!para!mí,!sé!que!vuestro!trabajo!no!será!en!vano!y! para!mí!sois!unas!campeonas.!En!este!apartado!incluyo!al!equipo!Brasileño!que!estuvo!de!paso,! José,!Júlia!y!Carol,!fue!un!placer!haber!convivido!con!ustedes.!A!cada!uno!de!vosotras!y!vosotros! los!llevaré!en!el!corazón!y!pensamiento!siempre.!!

Gracias!a!todas!aquellas!personas!que!han!hecho!que!para!mí!Pamplona!sea!como!mi!segundo! hogar.!Gracias!por!todos!aquellos!momentos!compartidos,!gracias!por!los!paseos!en!familia,!por! las!comidas,!escaladas!y!acampadas.!Estando!tan!lejos!de!casa!se!agradece!mucho!conocer!gente! así.!Especialmente!a!las!Moon!girls,!Oihane!y!Perla,!por!hacerme!sentir!acompañada!y!apoyada,! por!estar!en!momentos!difíciles!y!de!crisis,!por!su!filosofía!de!vida,!su!sabiduría!y!buenos!consejos.! A! mi! laguna! Koldo,! por! querer! enseñarme! todo! lo! que! puede! de! su! tierra,! por! transmitir! esas! ganas!de!vivir,!de!conocer!y!de!saber,!por!estar!ahí,!por!hacerme!sentir!en!casa!fuera!de!casa.!!

Gracias!esposo!por!estar!ahí!siempre,!por!tu!apoyo!incondicional!en!momentos!difíciles,!por! todos! los! buenos! momentos! que! hemos! pasado! y! por! creer! en! mí.! Gracias! por! ser! mi! familia.! Gracias!por!ser!cómo!eres,!por!tu!perseverancia,!por!siempre!estar!buscando!y!querer!ir!más!allá.!!

Por!último!quisiera!agradecer!a!mis!padres!y!a!mi!abuela,!aunque!ya!no!esté,!pues!sin!ustedes! ni!siquiera!existiría,!les!agradezco!por!la!confianza!depositada!en!mí,!por!quererme!tanto!así!como! soy,!les!agradezco!su!amor!incondicional.!Gracias!a!ustedes!soy!lo!que!soy.!Siempre!han!sido!mi! soporte!y!los!amo.!

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“Nuestra(recompensa(se(encuentra(en(el(esfuerzo(y(no(en(el(resultado.(Un(esfuerzo(total(es( una(victoria(completa”!

Mahatma!Gandhi!

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ABSTRACT!

Abstract!

Obesity!is!a!multiTfactorial!chronic!condition!that!increases!the!risk!of!some!chronic!diseases! such!as!type!2!diabetes,!hypertension!and!dyslipidemia,!among!others.!There!is!strong!scientific! evidence! that! the! proTinflammatory! state! induced! by! enhanced! adiposity! levels! plays! an! important! role! in! the! occurrence! of! obesityTrelated! metabolic! impairments.! Actually,! adipose! tissue! is! an! important! endocrine! organ! with! a! key! role! in! the! inflammatory! response! during! obesity! through! secretion! of! adipokines,! and! other! mediators! either! proinflammatory! or! antiT inflammatory.! In! this! context,! it! has! been! suggested! that! the! supplementation! with! antiT inflammatory! or! antiToxidant! compounds! could! contribute! not! only! to! counteract! the! obesityT related!clinical!complications!but!also!to!induce!weight!loss.!!!

The!general!aim!of!the!current!work!was!to!analyze!the!efficacy!of!a!dietary!supplementation! with!eicosapentaenoic!acid!(EPA)!and/or!αTlipoic!acid!in!combination!with!a!moderately!energyT restricted!diet!on!body!weight!loss!and!on!some!metabolic!and!inflammatory!status!biomarkers!in! metabolically!healthy!overweight/obese!women,!as!well!as!to!characterize!the!transcriptomic!and! epigenetic!mechanisms!that!could!be!involved.!

In!order!to!achieve!these!goals,!Caucasian!healthy!overweight/obese!women!(n=!77)!aged!20T 50!years!followed!a!10Tweeks!nutritional!intervention!with!a!hypocaloric!diet!(T30%!from!the!total! energy! expenditure)! after! being! randomly! assigned! to! one! of! the! four! parallel! experimental! groups:!1)!Control!group!(placebo);!2)!EPA!group!(1.3!g/day!of!EPA);!3)!αTlipoic!acid!group!(0.3! g/day!of!αTlipoic!acid)!and!4)!EPA!+!αTlipoic!acid!(1.3!g/day!of!EPA!+!0.3!g/day!of!αTlipoic!acid).!At! the! end! of! the! study,! blood! samples! from! 73! volunteers! were! available! and! subcutaneous! abdominal!adipose!tissue!(SAAT)!biopsies!from!57!women!were!obtained.!Moreover,!within!an!in# vitro!approach,!human!cultured!adipocytes!were!treated!with!EPA!(100T200!µM)!or!αTlipoic!acid! (100T250!µM)!during!24!h.!!

The!findings!reported!in!Chapter!1,!evidenced!that!αTlipoic!acid!supplementation!promoted!a! significant! greater! decrease! in! body! weight! loss.! Moreover,! although! EPA! did! not! exert! any! additional!effect!on!anthropometric!measurements,!it!was!able!to!significantly!attenuate!the!drop! in! leptin! levels! that! normally! occurs! during! weight! loss.! Body! weight! loss! improved! lipid! and! glucose! metabolism! indicators,! however,! no! statistically! significant! differences! were! observed! between!the!four!experimental!groups.!!

In! Chapter! 2,! αTlipoic! acid! supplementation! induced! a! greater! reduction! of! systemic! inflammatory! markers,! such! as! C! reactive! protein! and! leukocytes,! while! prevented! the! subsequent!decrease!of!apelin!that!occurs!after!weightTloss,!which!has!been!suggested!to!be!a!

! ABSTRACT! beneficial!adipokine/myokine!with!antiTobesity!and!cardioprotective!properties.!Although!EPA!did! not!modify!the!circulating!levels!of!the!inflammatory!and!cardiovascular!risk!markers!measured,! in!SAAT!biopsies,!the!supplementation!with!this!nT3!polyunsaturated!fatty!acid!downregulated!the! expression!of!the!macrophage!marker,!ADGRE1,!and!upregulated!the!expression!of!IL10,!an!antiT inflammatory!adipokine.!!

In!addition,!in!Chapter! 3,!the!effects!of!EPA!and/or!αTlipoic!acid!on!irisin,!an!adipomyokine! with! a! potential! role! in! obesity! and! related! metabolic! disorders,! were! evaluated.! Our! data! revealed! that! irisin! circulating! levels! were! positively! associated! with! fasting! glucose! levels.! However,! the! data! from! the! oral! glucose! tolerance! test! suggested! that! glucose! is! not! a! direct! contributing!factor!of!irisin!release.!Studies!in!cultured!human!adipocytes!showed!that! αTlipoic! acid!treatment!(250!µM),!but!not!EPA,!upregulated!FNDC5!mRNA!levels!and!irisin!secretion.!In! overweight/obese!women,!circulating!levels!of!irisin!decreased!significantly!after!weight!loss!in!all! groups,! but! the! supplementation! with! EPA,! αTlipoic! acid! or! both! did! not! modify! plasma! irisin! concentrations.!

Chapter!4!reports!a!microarray!analysis!of!SAAT!in!a!subsample!of!6!subjects!per!group.!The! bioinformatic!study!revealed!that!supplementation!with!EPA!promoted!changes!in!the!expression! of!ECM!remodeling!genes,!besides!to!a!general!increase!of!genes!considered!either!chemotactic! factors! or! macrophage! markers,! specially! those! associated! with! wound! repair.! αTLipoic! acid! decreased! the! expression! of! genes! related! with! chemotactic! factos! and! inflammatory! signals.! Also,! αTlipoic! acid,! particularly! in! combination! with! EPA,! upregulated! the! expression! of! genes! associated!with!lipid!catabolism,!while!downregulated!genes!involved!in!lipid!storage.!!

Finally,! the! methylation! analyses! described! in! Chapter! 5,!performed!in!a!subsample!of!6T7! subjects!per!group,!identified!some!CpG!sites!differentially!methylated!by!EPA!and/or!αTlipoic!acid! treatment! from! control! group.! Thus,! the! changes! in! the! cg10320884! (TRRAP)! methylation! site! were!associated!with!changes!in!the!Framingham!coronary!heart!disease!risk!score.!Interestingly,! αTlipoic!acid!upregulated!the!expression!of! NCK2,!TRRAP! and!RPTOR! genes!in!peripheral!blood! mononuclear!cells.!In!addition,!the!expression!of!these!genes!at!the!end!of!the!intervention!was! associated!with!the!changes!in!body!weight!loss!and!fat!mass!reduction.!!

Overall!the!current!investigation!suggests!that!αTlipoic!acid!and/or!EPA!supplementation!could! be!beneficial!in!body!weight!regulation/management!and!in!preventing!inflammation!onset!and! other! associated! metabolic! complications! commonly! found! in! overweight/obese! women.! The! current! data! also! revealed! the! ability! of! αTlipoic! acid! and! EPA! to! regulate! the! transcriptomic!

! ABSTRACT! profile! and! the! inflammation! in! adipose! tissue.! Finally,! the! available! data! also! support! the! involvement!of!some!epigenetic!mechanisms!in!the!metabolic!actions!of!αTlipoic!acid!and!EPA.!

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RESUMEN!

Resumen!

La!obesidad!es!una!enfermedad!crónica!multifactorial! que! incrementa! el! riesgo! de! padecer! otras!condiciones!crónicas,!tales!como!diabetes!tipo!2,!hipertensión!y!dislipidemias,!entre!otras.!El! estado!proTinflamatorio!inducido!por!el!incremento!de!la!adiposidad!juega!un!papel!importante! en!la!aparición!de!complicaciones!metabólicas!asociadas!a!la!obesidad.!El!tejido!adiposo!es!un! importante! órgano! endocrino! con! un! papel! clave! en! la! respuesta! inflamatoria! durante! la! obesidad,!a!través!de!un!desequilibrio!en!la!secreción!de!adipoquinas!y!otros!mediadores,!ya!sean! proTinflamatorios! o! antiTinflamatorios.! En! este! contexto,! se! ha! sugerido! que! la! suplementación! con! compuestos! antiTinflamatorios! o! antiToxidantes! podría! ayudar! no! solo! a! contrarrestar! las! patologías!relacionadas!con!la!obesidad,!sino!también!a!inducir!pérdida!de!peso.!!

El!objetivo!general!del!presente!trabajo!fue!analizar!la!eficacia!de!la!suplementación!dietética! con!ácido!eicosapentaenoico!(EPA)!y/o!ácido!αTlipoico!en!combinación!con!una!dieta!hipocalórica! moderada!sobre!la!pérdida!de!peso!y!sobre!biomarcadores!del!estado!metabólico!e!inflamatorio! en! mujeres! metabólicamente! sanas! con! sobrepeso/obesidad,! así! como! caracterizar! los! mecanismos!transcriptómicos!y!epigenéticos!que!pudieran!estar!involucrados.!!

Con!el!fin!de!conseguir!los!objetivos!propuestos,!se!seleccionó!un!grupo!de!mujeres!Caucásicas! sanas! con! sobrepeso/obesidad! (n=! 77)! con!una!edad!entre!20T50!años.! Todas! las! participantes! siguieron! una! intervención! nutricional! de! 10! semanas! con! una! restricción! energética! (T30%! del! total! del! gasto! energético)! después! de! ser! asignadas! aleatoriamente! a! uno! de! los! 4! grupos! experimentales!paralelos:!1)!Grupo!control!(placebo);!2)!Grupo!EPA!(1.3!g/día!de!EPA);!3)!Grupo! ácido!αTlipoico!(0.3!g/día!de!ácido!αTlipoico)!y!4)!grupo!EPA!+!ácido!αTlipoico!(1.3!g/día!de!EPA!+! 0.3!g/día!de!ácido!αTlipoico).!Al!final!del!estudio,!estuvieron!disponibles!muestras!de!sangre!de!73! voluntarias! y! se! obtuvieron! biopsias! de! tejido! adiposo! abdominal! subcutáneo! (TAAS)! de! 57! voluntarias.!Además,!desde!un!enfoque!in#vitro,!se!trataron!cultivos!de!adipocitos!humanos!con! EPA!(100T200!µM)!o!ácido!αTlipoico!(100T250!µM)!durante!24!h.!!

Los!principales!hallazgos!reportados!en!el!Capítulo!1,!evidenciaron!que!la!suplementación!con! ácido! αTlipoico! promovió! una! mayor! reducción! en! la! pérdida! de! peso.! Asimismo,! aunque! la! suplementación! con! EPA! no! tuvo! un! efecto! adicional! sobre! las! medidas! antropométricas,! fue! capaz!de!atenuar!significativamente!la!caída!en!los!niveles!de!leptina!que!normalmente!ocurren! durante! la! pérdida! de! peso.! El! peso! perdido! mejoró! marcadores! circulantes! del! metabolismo! glucídico! y! lipídico,! sin! embargo,! no! se! observaron! diferencias! estadísticamente! significativas! entre!los!cuatro!grupos!experimentales.!!

! RESUMEN!

En! el! Capítulo! 2,! la! suplementación! con! ácido! αTlipoico! indujo! una! mayor! reducción! de! marcadores! sistémicos! de! inflamación,! como! la! proteína! C! reactiva! y! los! leucocitos,! mientras! previno!la!disminución!de!apelina,!una!adipomioquina!con!propiedades!antiTobesidad!y!cardioT protectoras,!que!se!observa!tras!la!pérdida!de!peso.!A!pesar!de!que!el!EPA!no!modificó!los!niveles! en!sangre!de!los!marcadores!de!inflamación!y!riesgo!cardiovascular,!en!las!biopsias!de!TAAS,!la! suplementación! con! este! ácido! graso! poliTinsaturado! omegaT3,! disminuyó! la! expresión! del! gen! marcador!de!macrófagos,!ADGRE1,!y!aumentó!la!expresión!génica!de!IL10,!una!adipoquina!antiT inflamatoria.!

En! el! Capítulo! 3,! se! evaluaron! los! efectos! del! EPA! y/o! ácido! αTlipoico! sobre! irisina,! una! adipomioquina! con! un! papel! potencial! en! obesidad! y! en! las! complicaciones! metabólicas! asociadas.! Nuestros! datos,! evidenciaron! que! los! niveles! circulantes! de! irisina! estuvieron! positivamente! asociados! con! los! niveles! de! glucosa! en! ayunas.! Sin! embargo,! los!resultados! del! test! de! tolerancia! oral! a! la! glucosa! sugirieron! que! la! glucosa! no! es! un! factor! que! contribuya! directamente! a! la! liberación! de! irisina.! Estudios! en! cultivos! de! adipocitos! humanos! mostraron,! que!el!tratamiento!con!ácido!αTlipoico!(250!µM),!pero!no!con!EPA,!aumentó!el!ARNm!de!FNDC5!y! la! secreción! de! irisina.! En! mujeres! con! sobrepeso/obesidad,! los! niveles! circulantes! de! irisina! disminuyeron! significativamente! después! de! perder! peso! en! todos! los! grupos,! pero! ni! la! suplementación! con! EPA! o! ácido! αTlipoico,! ni! la! combinación! de! ambos,! modificó! las! concentraciones!de!irisina!en!plasma.!!

El!Capítulo! 4!describe!el!análisis!transcriptómico!del!TAAS!mediante!microarray!en!una!subT muestra!de!6! sujetos!por!grupo.!El!estudio!bioTinformático,!mostró! que!la!suplementación!con! EPA! promovió! cambios! en! la! expresión! de! genes! implicados! en! la! remodelación! de! la! matriz! extracelular,! e! incrementó! la! expresión! de! genes! considerados! factores! quimiotácticos! y! marcadores!de!macrófagos,!especialmente!aquellos!relacionados!con!la!reparación!de!lesiones.!El! ácido! αTlipoico! disminuyó! la! expresión! de! genes! relacionados! con! factores! quimiotácticos! y! señales! inflamatorias.! También,! el! ácido! αTlipoico,! especialmente! en! combinación! con! EPA,! promovió! la! expresión! de! genes! asociados! al! catabolismo! lipídico! mientras! que! reprimió! la! de! genes!involucrados!con!el!almacenamiento!de!lípidos.!!

Finalmente,! el! análisis! de! metilación! descrito! en! el! Capítulo! 5,!llevado!a!cabo!en!una!subT muestra!de!6T7!voluntarias!por!grupo,!identificó!algunos!sitios!CpG!diferencialmente!metilados! por! el! tratamiento! con! EPA! y/o! ácido! αTlipoico! en! comparación! con! el! grupo! control.! Así,! los! cambios!de!metilación!en!el!sitio!cg10320884!(TRRAP)!estuvieron!asociados!con!los!cambios!en!el! score!de!riesgo!de!enfermedad!coronaria!de!Framingham.!El!grupo!suplementado!con!ácido!αT

! RESUMEN! lipoico!mostró!una!sobreTexpresión!de!los!genes!NCK2,!TRRAP!y!RPTOR!en!células!mononucleares! periféricas!de!la!sangre.!Además,!la!expresión!de!estos!genes!al!final!de!la!intervención!estuvo! correlacionada!con!los!cambios!en!el!peso!corporal!y!la!masa!grasa.!!

En!general,!el!presente!proyecto!sugiere!que!la!suplementación!con!ácido!αTlipoico!y/o!EPA! podría!ser!beneficiosa!en!la!regulación!del!peso!corporal!y!en!la!prevención!de!la!inflamación!y!la! aparición! de! futuras! complicaciones! metabólicas,! comúnmente! encontradas! en! mujeres! con! sobrepeso/obesidad.!Los!datos!presentados,!también!revelaron!la!habilidad!del!ácido!αTlipoico!y! del! EPA! de! regular! el! perfil! transcriptómico! y! la! inflamación! del! tejido! adiposo.! Finalmente,! nuestros! datos,! apoyan! la! participación! de! ciertos! mecanismos! epigenéticos! en! las! acciones! metabólicas!del!ácido!αTlipoico!y!del!EPA.!!

!

!

TABLE!OF!CONTENTS!

TABLE!OF!CONTENTS!

I.! INTRODUCTION!...... !1!

1.!Health!implications!of!obesity!...... !3! 1.1.!Obesity:!definition,!causes!and!prevalence!...... !3! 1.2.!Obesity!as!a!lowTgrade!chronic!inflammatory!condition!...... !5! 1.2.1!Adipose!tissue!and!inflammation!...... !5! 1.2.2.!Triggers!of!the!inflammatory!response!...... !7! 2.!Obesity:!role!of!the!genome!and!the!epigenome!...... !9! 3.!Omega<3!polyunsaturated!fatty!acids!(n<3!PUFAs)!...... !12! 3.1.!NT3!PUFAs!and!weight!loss!...... !13! 3.1.1.!Effects!of!marine!nT3!PUFAs!in!the!context!of!an!energy!restricted!diet!...... !13! 3.1.2.!Effects!of!marine!nT3!PUFAs!in!the!context!of!an!isocaloric!diet!...... !15! 3.2.!Effects!of!nT3!PUFAs!on!lipid!and!glucose!metabolism!...... !19! 3.2.1.!Lipid!metabolism!...... !19! 3.2.2.!Glucose!metabolism!...... !20! 3.3.!NT3!PUFAs:!role!in!inflammation!...... !31! 3.3.1!Inflammation!and!nT3!PUFAs:!evidence!from!human!studies!...... !32! 3.4.!NT3!PUFAs!and!nutrigenomics!in!obesity!...... !35! 3.5.!Summary!...... !36! 4.!Alpha

4.1.!αTLipoic!acid!and!weight!loss!in!humans!...... !38! 4.2.!αTLipoic!acid:!effects!on!glucose!and!lipid!metabolism!...... !43! 4.2.1.!Glucose!metabolism!...... !43! 4.2.2.!Lipid!metabolism!...... !43! 4.3.!αTLipoic!acid!and!inflammation!...... !44! 4.4.!Summary!...... !45! 5.!References!...... !46!

II.! HYPOTHESIS!AND!AIMS!...... !59!

1.!Justification!for!the!study!...... !61! 2.!Hypothesis!...... !62! 3.!Objectives!...... !63! 4.!References!...... !64!

III.! SUBJECTS!AND!METHODS!...... !67!

1.!Study!design!and!intervention!...... !69! 2.!Study!population!...... !69! 3.!Data!collection!...... !72! 3.1.!Dietary!assessment!...... !72! 3.2.!Energy!expenditure!...... !73!

! TABLE!OF!CONTENTS!

3.3.!Anthropometric!measurements!and!body!composition!...... !73! 3.4.!Blood!pressure!measurements!...... !73! 3.5.!Blood!samples!...... !74! 3.6.!Oral!glucose!tolerance!test!(OGTT)!...... !74! 3.7.!Insulin!sensitivity!indexes!...... !74! 3.8.!Adipose!tissue!biopsies!...... !74! 4.!Biochemical!measurements!...... !74! 5.!Studies!in!cultured!human!subcutaneous!adipocytes!...... !75! 6.!Gene!expression!analyses!(real

IV.! RESULTS!...... !85!

CHAPTER!1!...... !87! CHAPTER!2!...... !117! CHAPTER!3!...... !141! CHAPTER!4!...... !163! CHAPTER!5!...... !197!

V.! GENERAL!DISCUSSION!...... !221!

VI.! CONCLUSIONS!...... !241!

VII.!!ANNEXES!...... !249!

!

!

! LIST!OF!ABBREVIATIONS!

LIST!OF!ABBREVIATIONS!!

AA:! ! Arachidonic!acid! AADAC:! Arylacetamide!deacetylase!

ACTG2:! Actin,!gamma!2! ADGRE1:! Adhesion!G!proteinTcoupled!receptor!E1! ADIPOQ:! Adiponectin! ADMA:! Asymmetric!dimethylarginine! ALA:!! AlphaTlinolenic!acid!

ALCAM:! Activated!leukocyte!cell!adhesion!molecule! AHA:! American!Heart!Association! ANOVA:! Analysis!of!Variance!

ANCOVA:! Analysis!of!Covariance! APLNR:! Apelin!receptor! APOH:! Apolipoprotein!H! BAT:!! Brown!adipose!tissue! BMI:! Body!mass!index! ch:! ! Cholesterol! CRP:!! C!reactive!protein! CVD:! Cardiovascular!disease! CCL:!! Chemokine!(cTc!motif)!ligand! CCR2:! Chemokine!(cTc!motif)!receptor!2! CHIT1:! Chitinase!1! CREBBP:!! CREB!binding!protein!1! COX:! Cyclooxygenase! DHA:! Docosahexaenoic!acid! DNA:! Deoxyribonucleic!acid! DNMT:! DNA!methyltransferase! DXA:! Dual!XTray!absorptiometry! ECM:! Extracellular!matrix! EE:! ! Ethyl!ester! EFSA:! European!Food!Safety!Authority! ELISA:! EnzymeTlinked!immunosorbent!assay!

! LIST!OF!ABBREVIATIONS!

EPA:!! Eicosapentaenoic!acid!

ER:! ! Endoplasmic!reticulum! FBG:! Fasting!blood!glucose! FBI:! ! Fasting!blood!insulin!

FDA:! Food!Drug!Administration! FFA:!! Free!fatty!acids! FITM2:! Fat!storage!inducing!transmembrane!protein!2!

GAPDH:! Glyceraldehyde!3Tphosphate!dehydrogenase! GWAS:! Genome!wide!association!studies!

HbA1c:! Glycosylated!hemoglobin! HDL:! High!density!lipoprotein! HIF1A:! HypoxiaTinducible!factor!1Talpha! HOMATIR:! Homeostatic!model!assessment!of!insulin!resistance! iAUC:! Incremental!Area!under!curve! IL:! ! Interleukin!

IFNTγ:! InterferonTgamma! IPA:!! Ingenuity!pathway!analysis! ISI:! ! Insulin!sensitivity!index! i.v.:! ! Intravenous! JNK:!! cTJun!NTterminal!kinase! LASY:! Lipoic!acid!synthase! LDL:!! LowTdensity!lipoprotein! LPS:!! Lypopolyssacharide! MCPT1:! Monocyte!chemoattractant!protein!1! MetS:! Metabolic!syndrome! MU:!! Metabolic!unit! MMLV:! Moloney!murine!leukaemia!virus! MMPs:! Matrix!metalloproteinases! MSR1:! Macrophage!scavenger!receptor!1! MYH11:! Myosin,!heavy!chain!11! nT3!PUFAs:! OmegaT3!Polyunsaturated!fatty!acids!! nT6!PUFAs:! OmegaT6!Polyunsaturated!fatty!acids!

NCK2:! NCK!adaptor!protein!2!

! LIST!OF!ABBREVIATIONS!

OECD:! Organization!for!Economics!Cooperation!and!Development!

OGTT:! Oral!glucose!tolerance!test! PAIT1:! Plasminogen!activator!inhibitorT1! PBMC:! Peripheral!blood!mononuclear!cells!

PCR:!! Polymerase!chain!reaction! PLA2G7:! Phospholipase!A2,!group!VII!

PGCT1α:! ProliferatorTactivated!receptor!gamma,!coactivator!1α!

PPARγ:! Peroxisome!proliferatorTactivated!receptor!γ! ROS:! Reactive!oxygen!species! RMR:! Resting!metabolic!rate!

RNA:!! Ribonucleic!acid! RPTOR:! Regulatory!associated!protein!of!MTOR!complex!1! SAA:! Serum!amyloid!A! SAAT:! Subcutaneous!abdominal!adipose!tissue! SAM:! STadenosyl!methionine! sICAMT1:! Soluble!Intracellular!adhesion!molecule!1! SNP:! Single!nucleotide!polymorphism! SPMs:! Specialized!proTresolving!lipid!mediators! sVCAMT1:! Soluble!Vascular!cell!adhesion!molecule!1! T2DM:! Type!2!Diabetes!Mellitus! TEE:!! Total!energy!expenditure! TG:! ! Triglycerides! TGFB1:! Transforming!growth!factor!beta!1! TIMPs:! Tissue!inhibitor!of!metalloproteinases! TLR:!! Toll!like!receptor! TRRAP:! Transcription!domain!associated!protein! TSS:!! Total!symptoms!score! TyG:!! TriglycerideTglucose!index! VLCD:! VeryTlow!calorie!diet! VAT:! Visceral!adipose!tissue! VEGF:! Vascular!endothelial!growth!factor! VLDL:! VeryTlow!densitiy!lipoprotein!

! LIST!OF!ABBREVIATIONS!

WAT:! White!adipose!tissue!

WHO:! World!Health!Organization! WHR:! Waist!to!hip!ratio! WHtR:! Waist!to!height!ratio!

!

! !

I. INTRODUCTION!

!

!

I.!INTRODUCTION!

1.!Health!implications!of!obesity!

1.1.#Obesity:#definition,#causes#and#prevalence#

Obesity!is!a!multifactorial!chronic!disease,!whose!etiology!involved!genetic!and!environmental! factors! (Abete!et#al.,! 2012).!Thus,! twin!studies!have!suggested!that!the!estimate!heritability!of! body!mass!index!(BMI)!is!about!40T70%!in!children!and!adults!(Herrera!et#al.,!2011),!while!the! environmental! factors! are! involved! in! the! 95%! of! the! cases! and! are! linked! to! sedentary! and! dietary! habits! that! promote! a! positive! energy! balance! between! energy! intake! and! energy! expenditure!(Aranceta!et#al.,!2007).!The!positive!energy!balance!maintained!across!the!time,!leads! to!an!excessive!fat!accumulation!with!the!subsequent!increase!in!body!weight!(WHO,!2014).!!

Beyond!the!aesthetic!issue!and!social!stigma,!obesity!has!a!negative!effect!in!the!quality!of!life! of! population,! because! the! associated! comorbidities.! Actually,! obesity! increases! the! risk! of! cardiovascular! diseases! (CVD),! hypertension,! dyslipidemia,! type! 2! Diabetes! Mellitus! (T2DM),! insulin! resistance,! certain! types! of! cancer! and! gallbladder! disease! (WHO,! 2014).! Additionally,! other! health! problems! associated! with! obesity! include:! respiratory! difficulties,! chronic! musculoskeletal! problems,! skin! alterations! and! infertility! (WHO,! 2014).! The! adverse! effects! of! obesity! are! influenced! for! different! factors! such! as! the! amount! of! excessive! weight,! fat! mass! accumulation!and!distribution,!the!magnitude!of!weight!gain!during!the!adulthood!and!sedentary! lifestyle!(WHO,!2014).!!

In!this!context,!according!with!the!Organization!for!Economics!CoToperation!and!Development! (OECD)!report,!people!severely!obese!die!eight!to!ten!years!before!than!lean!people;!each!fifteen! extra!Kg,!increases!the!likelihood!of!untimely!death!about!30%!(OECD,!2010).!Moreover,!it!has! been! estimated! that! around! both! 3.4! million! of! deaths! per! year! and! 93.6! million! of! disabilityT adjusted!lifeTyear,!were!associated!to!overweight!or!obesity!during!2010!(Lim!et#al.,!2012).!The! last!World!Health!Organization!(WHO)!report!(WHO,!2015)!showed!a!worldwide!increasing!trend! in! the! prevalence! of! obesity! and! overweight! and! an! increment! in! the! prevalence! of! chronic! diseases!as!T2DM.!In!2008,!the!prevalence!of!obesity!had!almost!doubled!since!1980,!reaching!in! 2014!to!a!currency!of!11!%!for!men!and!15!%!for!women!in!persons!aged!18!years!or!older!(WHO,! 2014).!

BMI!is!defined!as!weight!in!kilograms!divided!by!the!square!of!the!height!in!meters!(kg/m2),! which!has!been!used!to!estimate!the!prevalence!of!obesity!within!a!population!and!to!classify! underweight,! overweight! and! obesity! in! adults! (Table! 1).! In! fact,! the! WHO! considers! that! for! achieving!optimal!health,!the!BMI!for!individuals!should!be!maintained!in!the!range!18.5!to!24.9!

3! I.!INTRODUCTION! kg/m2!and!the!risk!of!obesity!comorbidities!increases!from!a!BMI!of!25.0!kg/m2,!considering!as!a! moderate!to!severe!risk!of!comorbidities!a!BMI!greater!than!30!kg/m2!(WHO,!2014).!However,!it!is! important!to!take!into!account!that!the!BMI!may!not!correspond!in!all!cases!with!the!same!degree! of!fatness!or!associated!health!risk!in!different!individuals!and!etnic!populations!(Report!of!a!WHO! Consultation,!2004).!!

Besides!these!considerations,!it!has!been!observed!that!the!distribution!of!the!excessive!fat! mass! affects! the! risk! to! develop! pathologies! associated! with! obesity! (Report! of! a! WHO! Consultation,!2004).!In!fact,!obesity!can!be!classified!according!with!the!fat!mass!distribution!as! android! (observed! generally! in! men)! or! gynoid! (more! common! in! women).! The! android! fat! distribution! is! defined! by! the! accumulation! of! visceral! and! subcutaneous! adipose! tissue! in! the! central! area! affecting! visceral! and! abdominal! vital! organs! such! as! pancreas,! liver,! stomach,! intestine!or!colon,!moreover!it!has!been!correlated!with!increasing!risk!of!cardiovascular!health! diseases! (Lee! et# al.,! 2013).! Otherwise,! in! the! gynoid! distribution,! the! fat! is! accumulated! in! the! gluteofemoral! area! and! leg;! some! authors! have! suggested! that! it! could! play! a! protective! role,! decreasing!the!risk!of!cardiovascular!diseases!(Karpe!and!Pinnick,!2015).!!!

Indeed,!based!on!its!relation!with!the!central!fat!accumulation,!it!has!been!accepted!that!a! higher! waist! to! hip! ratio! (WHR)! is! linked! with! a! higher! risk! of! cardiovascular! disease! or! other! obesity! comorbidities! (WHR! >! 1.0! in! men! and! >! 0.85! in! women).! Therefore,! some! evidence! suggested! that! measurement! of! waist! circumference! alone! provides! a! more! simple! practical! method!of!identifying!the!risk!of!cardiovascular!disease!(Lee!et#al.,!2013).!

! Table!1.!Classification!of!obesity!according!with!the!BMI! Classification! BMI! Risk!comorbidities! Underweight! <18.50! Low!(but!risk!of!other!clinical!problems! increased)! Normal!range! 18.50!–!24.99! Average! Overweight! ≥!25.00! ! Preobese! 25.00!–!29.99! Increased! Obese!class!I! 30.00!–!34.99! Moderate! Obese!class!II! 35.00!–!39.99! Severe! Obese!class!III! ≥!40.00! Very!severe! Source:!WHO.!Obesity.!Preventing!and!Managing!the!Global!Epidemic.!Technical!Report!Series! 894.!Geneva:!WHO,!2000.!! !

4! I.!INTRODUCTION!

1.2.#Obesity#as#a#lowIgrade#chronic#inflammatory#condition#

1.2.1!Adipose!tissue!and!inflammation!

In! general! terms,! inflammation! is! a! normal! localized! protective! reaction! of! a! living! tissue! to! injury!or!infection,!which!is!characterized!by!heat,!redness,!swelling!and!pain.!The!inflammatory! mechanism! involves! the! activation! not! only! of! proTinflammatory! mediators! to! the! site! of! inflammation!but!also!of!antiTinflammatory!molecules!that!help!to!maintain!the!homeostasis!and! restore!the!injured!tissue!(Calder!et#al.,!2011;!Hotamisligil,!2006).!Moreover,!a!lowTgrade!chronic! inflammation! is! referred! as! an! inflammatory! response! prolonged! during! time,! in! which! the! resolution! mechanisms! of! inflammation! are! impaired! and! accompanied! by! a! moderate! but! persistent!increase!of!systemic!indicators!of!inflammation!(Suganami!and!Ogawa,!2010).!

Several!epidemiological!studies!have!pointed!out!the!strong!association!between!acuteTphase! proinflammatory! proteins! and! markers,! such! as! leukocytes,! fibrinolytic! factors,! plasminogen! activator!inhibitor!1!(PAIT1)!and!C!reactive!protein!(CRP)!among!others,!with!either!CVD!or!T2DM! (Crook!et#al.,!1993;!Dandona!et#al.,!2004;!Gregor!and!Hotamisligil,!2011;!Koenig!et#al.,!1999).!Also,! numerous!human!studies!have!reported!that!obesity!is!closely!correlated!with!the!increment!of! proinflammatory!systemic!proteins,!which!increases!the!likelihood!of!chronic!metabolic!diseases! (Calder!et#al.,!2011;!Das,!2001;!Gregor!and!Hotamisligil,!2011).!In!this!context,!the!inflammation! pathway! has! been! proposed! as! the! link! between! obesity! and! the! development! of! obesity! comorbidities!including!arterial!disease!and!T2DM!(Calder!et#al.,!2011;!Gregor!and!Hotamisligil,! 2011).!Since!adipose!tissue!is!able!to!secrete!an!array!of!inflammatory!mediators,!it!is!considered! a!key!metabolic!organ!that!participates!in!the!systemic!inflammatory!response!observed!in!obese! subjects!(Choe!et#al.,!2016).!!

Generally!in!humans,!adipose!tissue!can!be!divided!in!white!adipose!tissue!(WAT)!and!brown! adipose! tissue! (BAT),! being! WAT! the! most! abundant! (Tilg! and! Moschen,! 2006).! In! this! sense,! broadly! the! main! physiological! role! of! WAT! is! to! be! the! site! of! lipid! storage! whereas! the! BAT! function,!which!is!found!mainly!in!human!neonates!and!rodents,!is!to!be!involved!in!nonTshivering! thermogenesis!through!lipid!oxidation!(Choe!et#al.,!2016).!Recently,!it!was!discovered!other!third! type!of!adipocytes!called!“brite!or!beige”!adipocytes,!which!are!brownTlike!adipocytes!occurring! within!WAT,!and!when!stimulated!by!cold!exposure!or!betaTadrenergic!factors,!are!able!to!induce! UCP1!and!a!thermogenic!response,!similar!to!BAT!(SanchezTGurmaches!and!Guertin,!2014).!!

Just!few!years!ago,!it!was!thought!that!the!principal!role!of!WAT!was!being!a!reservoir!of!fat! (Tilg!and!Moschen,!2006).!Nowadays,!it!is!largely!accepted!that!WAT!beyond!to!be!an!important! energy! reservoir! organ,! is! also! an! important! secretory! organ! with! autocrine,! paracrine! and!

5! I.!INTRODUCTION! endocrine! functions! (Gregor! and! Hotamisligil,! 2011;! MorenoTAliaga# et# al.,! 2010;! Tilg! and! Moschen,!2006).!!WAT!secretes!a!large!number!of!biologically!active!molecules!that!have!been! defined! as! adipocytokines! or! adipokines! (eg.! tumor! necrosis! factorTα! (TNFTα),! PAIT1,! leptin,! adiponectin,! monocyte! chemoattractant! protein! 1! (MCPT1),! serum! amyloid! A! (SAA),! resistin,! among! others),! which! are! capable! to! modulate! inflammatory! responses! not! only! in! the! own! tissue,!but!also!in!other!important!organs!associated!with!energy!metabolism!such!as!the!liver,! pancreas! or! skeletal! muscle! (Calder! et# al.,! 2011;! Furukawa! et# al.,! 2004;! Suganami! and! Ogawa,! 2010).! Also,! WAT,! through! leptin! production,! influences! food! intake! and! energy! balance! by! directly!acting!in!the!hypothalamus!(Calder!et#al.,!2011).!Moreover,!several!adipokines!have!been! related! with! the! regulation! of! glucose! and! lipid! metabolism! (e.g.! adiponectin,! apelin,! among! others)!(Bertrand!et#al.,!2015;!Wang!and!Scherer,!2016).!Additionally,!WAT!is!able!to!respond!to! afferent!signals!from!other!tissues!by!the!fact!that!it!expresses!several!related!receptors!(Kershaw! and!Flier,!2004;!Scherer,!2006).!!

Although!mature!adipocytes!constitute!the!major!part!of!WAT,!this!tissue!also!has!a!stromal! vascular!fraction,!which!contains!not!only!preadipocytes!and!endothelial!cells!but!also!fibroblast! and! immune! cells! (Calder! et# al.,! 2011;! Suganami! and! Ogawa,! 2010;! Tilg! and! Moschen,! 2006).! Furthermore,! WAT! has! been! recognized! as! the! principal! site! of! production! of! inflammatory! mediators! (Calder! et# al.,! 2011).! In! a! normal! condition,! there! is! a! homeostasis! between! the! production!of!inflammatory!and!proTinflammatory!compounds!(Suganami!and!Ogawa,!2010).!In! obesity,! the! increment! in! the! adiposity! levels! disrupts! the! organism! equilibrium! in! the! inflammatory!pathways,!promoting!the!activation!and!infiltration!of!WAT!with!macrophages!and! other!immune!cells,!which!stimulates!the!inflammatory!processes!in!this!tissue!(Flock!et#al.,!2013;! Suganami!and!Ogawa,!2010),!creating!a!vicious!cycle!with!an!unresolved!inflammation!(Serhan!et! al.,! 2015a).! At! the! same! time,! the! increment! in! the! production! of! proTinflammatory! molecules! could!trigger!metabolic!impairments!that!promote!inflammatory!pathways!and!affect!the!normal! signaling!cascade!of!insulin!and!glucose!(Flock!et#al.,!2013).!!

Macrophages,!which!are!part!of!the!innate!immune!system,!have!been!recognized!for!playing! a! principal! role! in! adipocyte! homeostasis! through! the! secretion! of! antiTinflammatory! and! proT inflammatory!mediators!(Calder!et#al.,!2011;!Schipper!et#al.,!2012).!Normally,!in!order!to!prevent! adipose!tissue!damage,!the!macrophage!function!is!to!remove!the!remnants!from!the!apoptosis! of!the!adipocyte!and!prevent!the!ectopic!accumulation!of!fat!by!removing!free!fatty!acids!(Dalmas! et#al.,!2011).!In!obese!subjects,!it!has!been!suggested!that!the!disruption!of!the!normal!function!of! adipose! tissue! macrophages! could! be! promoted! by! the! increment! of! macrophage! levels! in! the!

6! I.!INTRODUCTION! adipose!tissue!(from!4%!in!visceral!adipose!tissue!(VAT)!of!lean!subjects!to!approximately!12%!in! obese!patients),!as!described!elsewhere!(Schipper!et#al.,!2012).!

In!this!context,!Lumeng!et#al.!(2007)!found!different!hallmarks!between!the!adipose!tissue!of! lean!mice!and!of!obese!mice,!which!promote!the!polarization!to!different!types!of!macrophages.! The!hypertrophy!of!the!adipocytes!and!the!increment!in!the!adiposity!levels!induced!by!nutrient! excess,! activate! a! phenotypic! switch! from! the! antiTinflammatory! M2! state! towards! the! proT inflammatory! M1! state.! Thus,! M2! state! macrophages,! which! are! produced! in! response! to! interleukin! 4! (ILT4)! and! ILT13,! are! known! to! favour! tissue! repair,! angiogenesis,! production! of! insulin! sensitizing! chemokines! (ILT10)! and! antioxidant! enzymes! (arginase);! whereas! the! M1! macrophages,!which!are!produced!in!response!to!interferon!(IFN)!γ!and!lipopolysaccharide!(LPS),! promote!the!secretion!of!MCPT1!(also!known!as!chemokine!(CTC!Motif)!ligand!2!/!CCL2),!ILT12,! TNFTα,! ILT6! and! other! proTinflammatory! cytokines! (Lumeng! et# al.,! 2007;! Suganami! and! Ogawa,! 2010).!Additionally,!it!has!been!hypothesized!that!in!lean!animals,!the!resident!macrophages!are! derived!principally!from!chemokine!(CTC!Motif)!receptor!2!negative!(CCR2T)!monocytes,!which!are! associated! with! the! M2! phenotype! and! that! during! adipose! tissue! expansion,! the! CCR2+! monocytes,!which!are!typically!differentiated!to!the!M1!phenotype,!are!attracted!into!the!adipose! tissue! by! monocyte! chemoattractant! proteins! (Lumeng! et# al.,! 2007).! Other! hypothesis! is! that! according!with!the!environmental!stimuli,!the!resident!monocytes!are!able!to!be!polarized!into! both,!M1!and!M2!phenotypes!(Lumeng!et#al.,!2007).!!

Besides! macrophage! infiltration,! other! immune! cells! are! increased! in! the! hypertrophied! adipocyte,!such!as!neutrophils,!natural!killer!cells!and!mast!cells!(Gregor!and!Hotamisligil,!2011;! Suganami! and! Ogawa,! 2010).! Studies! in! murine! models! of! obesity! have! suggested! that! the! infiltration! of! adipose! tissue! by! macrophages! is! preceded! by! an! influx! of! TTlymphocytes,! suggesting! that! these! cells! may! initiate! the! proTinflammatory! response! in! the! adipose! tissue! (Calder!et#al.,!2011).!!

1.2.2.!Triggers!of!the!inflammatory!response!

Several! studies! have! suggested! that! the! extracellular! matrix! (ECM)! remodeling! or! the! endoplasmic! reticulum! (ER)! stress,! as! well! as! impairments! in! adipose! tissue! function! and! the! foods'! nutrients! for! themselves,! could! be! important! players! in! triggering! the! inflammatory! response! (Gregor! and! Hotamisligil,! 2011).! In! addition,! it! is! known! that! the! proTinflammatory! response!is!activated!either!by!the!NFTκB!transcription!factor!or!through!cTJun!NTterminal!kinase! (JNK)!protein!family!(Calder!et#al.,!2011;!Gregor!and!Hotamisligil,!2007;!Keophiphath!et#al.,!2009).!

7! I.!INTRODUCTION!

The! ECM! allows! the! cell! to! communicate! with! the! exterior! and! it! is! involved! in! both! the! constant! communication! and! the! response! between! the! cell! and! the! extracellular! components! (Williams! et#al.,! 2015).! The! ECM! plays! a! key! role! in! important!biological!processes!such!as!cell! migration!and!adhesion,!as!well!as!cell!repair!and!death!(Lu!et#al.,!2011;!Williams!et#al.,!2015).! Strong!scientific!evidence!suggests! that! during! obesity! there! is! an! imbalance! between! proteins! that! promote! ECM! degradation! (e.g.! metalloproteinases/MMPs)! and! proteins! that! act! as! inhibitors!of!these!signals!(e.g.!tissue!inhibitor!of!metalloproteinases/TIMPs).!The!rise!of!the!ECM! components,! triggers! the! production! of! both,! angiogenic! growth! factors! and! chemoattractant! signals,! which! in! turn! favors! the! infiltration! of! immune! cells! into! adipose! tissue! and! a! proT inflammatory!environment!(Keophiphath!et#al.,!2009;!Liu!et#al.,!2015).!!

Additionally,! the! ER! together! with! the! Golgi! apparatus! are! sites! of! protein! synthesis! and! triglyceride!(TG)!droplet!formation!(Gregor!and!Hotamisligil,!2007).!The!uncontrolled!demand!of! protein!synthesis!started!by!nutrient!excess!and!adipose!tissue!expansion,!lead!to!a!prolonged! activation!of!unfolded!protein!response,!which!in!turn!activates!inflammatory!signaling!pathways! (JNK! and! NFTκB)! and! favors! the! production! of! reactive! oxygen! species! (ROS)! (Gregor! and! Hotamisligil,!2007).!It!has!been!also!suggested!that!in!obesity!the!increment!in!oxygen!demand!of! the! growing! adipose! tissue! mass! and! the! limited! vascularization! of! adipose! tissue! favors! the! development! of! hypoxia! (Trayhurn,! 2014),! which! in! turn! could! drive! to! ER! stress! and! to! the! activation!of!proteins!with!either!chemoattractant!or!inflammatory!properties!(Calder!et#al.,!2011;! Hotamisligil,!2006;!Suganami!and!Ogawa,!2010).!

Also,! different! researchers! have! suggested! that! the! increase! of! free! fatty! acids! (FFA)! bloodstream! levels! observed! in! not! only! obese,! but! also! in! insulinTresistant! individuals! are! associated! with! impairments! in! the! adipose! tissue! storage! function,! which! favors! lipolysis! and! reduces! fat! storage,! leading! to! both! an! increased! flux! of! FFA! and! its! ectopic! accumulation! in! important!metabolic!organs!causing!lipotoxicity!(Christodoulides!et#al.,!2009;!Dalmas!et#al.,!2011).! The!FFA!are!toll!like!receptor!(TLR)!4!ligands,!which!belongs!to!a!transmembrane!receptors!that!by! activating!NFTκB!participate!in!the!immune!response.!Moreover,!it!was!proposed!that!the!TLR4! signaling! is! one! of! the! links! between! obesity,! inflammation! and! insulin! resistance! (Song! et# al.,! 2006).!!!

Interestingly,!the!nutrients!by!themselves!are!able!to!induce!different!inflammatory!responses.! In!this!sense,!different!epidemiological!or!clinical!trials!have!identified!that!the!dietary!patterns! and/or!food!components!can!promote!or!retard!the!inflammatory!response!(Calder!et#al.,!2011;! Wu!and!Schauss,!2012).! Overall,! the! consumption! of! fresh! vegetables! and! fruits,! whole! grains,! dietary! fiber,! legumes,! dried! fruits,! omegaT3! rich! foods! and! low! glycemic! index! carbohydrates!

8! I.!INTRODUCTION! have!been!associated!with!a!low!inflammatory!response!and!low!CVD!risk,!whereas!the!intake!of! saturated! highTfat! products,! refined! grains,! carbohydrate! beverages,! high! glycemic! index! carbohydrates,!high!intake!of!red!meals!and!sugary!products!has!been!linked!with!an!increment!of! either!proTinflammatory!mediators!or!CVD!risk!(Calder!et#al.,!2011;!Kolb!and!MandrupTPoulsen,! 2009;!LopezTLegarrea!et#al.,!2014;!Wu!and!Schauss,!2012;!Zulet!et#al.,!2012).!In!this!context,!the! study!of!Esposito!et#al.!(2004)!reported!that!after!2Tyear!period!of!following!the!Mediterranean! dietary!pattern,!whose!one!of!the!features!is!the!high!intake!of!omegaT3!polyunsaturated!fatty! acids! (nT3! PUFAs),! the! inflammatory! biomarkers! and! insulin! sensitivity! were! improved! whereas! the!prevalence!of!metabolic!syndrome!(MetS)!features!was!decreased.!!

2.!Obesity:!role!of!the!genome!and!the!epigenome!

Although! the! environmental! factors! and! dietary! habits! are! involved! in! the! major! part! of! obesity!prevalence!and!it!is!overall!accepted!that!obesity!increases!the!risk!of!chronic!metabolic! diseases,! the! complex! interaction! between! diet! and! genes! cannot! be! underestimated,! if! the! objective! is! to! move! towards! a! more! personalized! therapy.! In! this! sense,! in! the! recent! years,! different!questions!have!arisen!as:!1)!Why!people!do!not!respond!equal!to!the!same!treatment?;! 2)!Why!not!all!obese!people!develop!metabolic!diseases?;!3)!Why!are!there!some!people!with!a! certain!either!resistance!or!susceptibility!to!the!dietTinduced!obesity?!

The!discovery!of!the!entire!human!genome!by!the!International!Human!Genome!Sequencing! Consortium! (IHGS! Consortium,! 2004)! and! the! development! of! "omics"! technologies! as! transcriptomics! are! considered! a! watershed! in! the! study! of! the! role! of! geneTenvironmental! interactions! in! some! chronic! and! metabolic! diseases,! such! as! obesity! (Goni! et# al.,! 2016).! It! is! important! to! note! that! the! single! nucleotide! polymorphism! (SNP)! is! a! fixed! throughoutTlife! variation!of!a!single!nucleotide!that!occurs!in!a!specific!position!in!the!genome!that!is!present!in! approximately!more!than!1%!of!the!population,!though!its!prevalence!could!vary!depending!of! the! geographical! zone! or! ethnic! group! (Gibbs! et# al.,! 2003).! In! this! line,! despite! more! that! 130! polymorphisms!related!to!obesity!have!been!identified!through!different!approaches,!including! the!Genome!Wide!Association!Studies!(GWAS)!(Goni!et#al.,!2016),!the!total!influences!of!these! variants!on!BMI!have!been!described!as!modest!(Cordero!et#al.,!2015;!Goni!et#al.,!2016).!!

Moreover,! epigenetic! modifications! are! stable! and! tissue! specific! heritable! marks! of! the! genome! that! may! alter! gene! expression,! influencing! the! phenotype! without! changes! in! DNA! coding!sequence!(Goni!et#al.,!2016;!Remely!et#al.,!2015a).!The!most!extensively!studied!epigenetic! changes!include!not!only!DNA!methylation!and!covalent!reactions!in!histone!amino!acid!chains,! which!could!interact!at!transcriptional!level,!but!also!RNATinterference!(such!as!small!interfering!

9! I.!INTRODUCTION!

RNAs! and! microRNAs),! which! could! modulate! gene! expression! at! postTtranscriptional! level! (Cordero!et#al.,!2015;!McKay!and!Mathers,!2011;!Milagro!et#al.,!2013;!Remely!et#al.,!2015a).!The! total! of! epigenetic! marks! on! the! genome! are! known! as! the! epigenome! (McKay! and! Mathers,! 2011)!(Figure!1).!!

DNA!methylation,!which!is!the!most!widely!studied!epigenetic!mark,!occurs!principally!in!the! fifth!carbon!in!the!cytosines!that!are!followed!by!a!guanine!(CpG)!(Milagro!et#al.,!2013;!Portela! and!Esteller,!2010).!Though!the!methylation!process!normally!takes!place!in!CpG!islands!that!are! zones!with!a!high!density!of!CpG!dinucleotides,!it!also!happens!either!in!regions!with!lower!CpG! density!that!lie!in!close!proximity!with!CpG!islands!called!CpG!island!shores!or!in!the!gene!bodies! (Kim!et#al.,!2011;!Portela!and!Esteller,!2010).!Generally!the!methylation!in!the!promoter!region! has!been!associated!with!the!inhibition!of!gene!transcription!(Portela!and!Esteller,!2010;!Remely! et#al.,!2015a).!Furthermore,!DNA!methylation!is!mediated!by!the!DNA!methyltransferase!(DNMT)! family!of!enzymes!that!catalyze!the!transfer!of!methyl!group!from!STadenosyl!methionine!(SAM)! to!DNA!(Portela!and!Esteller,!2010).!!

In! addition,! histones! are! proteins! responsible! for! DNA! packaging! that! through! their! postT transcriptional! modifications! such! as! acetylation,! methylation,! phosphorylation,! ubiquitination,! SUMOylation!and!ADPTribosylation!in!their!NTterminal!tails,!are!modulating!important!processes! as!the!translation!of!DNA!information!(Cordero!et#al.,!2015;!Portela!and!Esteller,!2010).!!!

!

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!

!

!

!

!

!

! Figure! 1.! Scheme! for! the! main! ! mechanisms!of! epigenetic!regulation.! Figure!from!Kim!et#al.!(2011).! !

10! I.!INTRODUCTION!

From!a!nutritional!point!of!view,!besides!to!the!influence!of!the!genome!to!the!response!of!the! treatment! or! dietary! habits,! the! studies! derived! from! the! Dutch! Famine! (1944T1945)! (Kyle! and! Pichard,! 2006;! Painter! et# al.,! 2008),! have! suggested! that! through! epigenetic! mechanisms,! maternal! nutrition! could! modulate,! from! birth,! susceptibility! either! to! some! chronic! diseases,! including!CVD,!or!to!certain!metabolic!conditions!such!as!obesity!and!glucose!intolerance.!In!this! context,! growing! scientific! evidence! has! highlighted! that! the! gestation! period! is! a! critical! time! window!in!which!the!epigenome!is!particularly!plastic!and!susceptible!to!modifications!by!dietary! patterns!or!circumstances!and!other!environmental!factors!(McKay!and!Mathers,!2011).!

Because!the!epigenome!is!considered!a!moldable!system!throughout!life,!external!factors!such! as! smoking! habits,! physical! activity! or! diet! are! constantly! interacting! with! the! epigenome,! promoting!a!change!or!maintenance!of!the!different!epigenetic!marks!(Feil,!2006).!In!this!context,! Fraga!et#al.!(2005)!showed!that!although!monozygotic!twins!are!genetically!identical,!they!have! different!epigenetic!marks,!which!even!though!being!comparable!in!early!life,!they!may!affect!the! disease!susceptibility.!Moreover,!they!observed!that!the!epigenetic!marks!become!more!distinct! in! older! twins! or! in! twins! that! had! spent! less! time! together,! had! different! lifestyles! or! with! a! dissimilar! clinical! history,! pointing! out! the! significant! role! of! environmental! factors! in! the! modulation!of!both!genotype!and!phenotype.!

In!this!sense,!studies!in!humans!have!reported!both,!that!the!initial!methylation!marks!may! help! to! predict! the! response! to! a! weight! loss! treatment! (Campión! et# al.,! 2009;! Cordero! et# al.,! 2011;!Milagro!et#al.,!2011,!2012;!Moleres!et#al.,!2013)!and!that!the!beneficial!effects!of!a!healthy! dietary!pattern!(Abete!et#al.,!2015),!a!high!physical!activity!(Rönn!et#al.,!2013)!or!weight!loss!after! bariatric!surgery!(Barres!et#al.,!2013;!Benton!et#al.,!2015;!Nicoletti!et#al.,!2016),!are!mediated,!at! least!in!some!manner,!by!epigenetic!changes.!

Also,! some! nutrients! and! food! components! such! as! folate,! vitamin! B6,! vitamin! B12,! zinc,! choline,! biotin,! methionine,! among! others,! are! known! to! induce! epigenetic! changes! through! increasing! methyl! availability! and! by! promoting! changes! in! the! activity! of! enzymes! involved! in! oneTcarbon!metabolism!(McKay!and!Mathers,!2011;!Milagro!et#al.,!2013;!Remely!et#al.,!2015a).! Additionally,!in!the!recent!years,!in!order!to!improve!the!treatment!of!chronic!diseases!such!as! cancer! or! MetS,! the! epigenetic! mechanisms! whereby! some! bioactive! compounds! as! PUFAs,! vitamins!(e.g.!vitamin!C,!vitamin!E),!polyphenols!(e.g.!resveratrol)!and!other!dietary!components! (e.g.! lipoic! acid,! carotenoid,! curcumin)! promote! beneficial! effects! on! health! have! been! under! study!(Milagro!et#al.,!2013;!Remely!et#al.,!2015b).! ! !

11! I.!INTRODUCTION!

3.!Omega<3!polyunsaturated!fatty!acids!(n<3!PUFAs)!!

According!with!the!previous!information,!obesity!could!be!considered!both!a!metabolic!and!an! inflammatory! disease,! which! increases! the! risk! of! CVD! and! insulin! resistance! (Gregor! and! Hotamisligil,! 2011).! In! this! sense,! the! supplementation! with! antiTinflammatory! food! bioactive! components,!as!nT3!PUFAs,!has!been!proposed!as!potential!adjuvant!in!the!obesity!treatment.!!

Generally,! nT3! PUFAs! can! be! categorized! in! either! vegetable! derived! (αTlinolenic! acid! (ALA),! C18:3)!or!marine!derived!(eicosapentaenoic!acid!(EPA),!C20:5!and!docosahexaenoic!acid!(DHA),! C22:6,!mainly).!Vegetable!oil!from!flaxseeds,!canola!and!soybean!are!the!principal!sources!of!ALA,! while! EPA! and! DHA! are! principally! found! in! marine! sources! such! as! fatty! fishes! (e.g.! salmon,! herring,!mackerel!and!sardines)!and!krill!(Ghasemifard!et#al.,!2014;!LorenteTCebrián!et#al.,!2013).! Moreover,!though!EPA!and!DHA!are!mainly!found!in!fatty!fishes,!they!also!could!be!obtained!from! the! microalgae! (Crypthecodinium# cohnii! and! Schizochytrium! sp.)! sources,! predominantly! DHA! (Cottin! et# al.,! 2011).! The! beneficial! effects! of! nT3! PUFAs! on! cardiovascular! health! have! been! principally! attributed! to! those! from! marine! origin! (EPA! and! DHA),! and! although! they! can! be! derived! from! ALA! in! the! organism,! the! conversion! rate! of! ALA! to! either! EPA! or! DHA! is! controversial!and!apparently!modest,!making!necessary!the!direct!intake/supplementation!with! the!marine!derived!nT3!PUFAs!to!assure!optimal!levels!(Burdge!and!Wootton,!2002;!Burdge!et#al.,! 2002;!Emken!et#al.,!1994).!!

Since! the! first! observations! from! Bang! et# al.! (1980),! where! they! proposed! that! the! low! prevalence!of!CVD!in!Eskimos!could!be!attributable!to!the!high!intake!of!fatty!fish!in!their!habitual! diet,! several! studies! evaluating! the! effects! of! nT3! PUFAs! on! cardiovascular! health! and! MetS! features!have!been!performed!(LorenteTCebrián!et#al.,!2013).!!

Nowadays,! different! international! organizations! have! recognized! the! beneficial! effect! of! the! marine! nT3! PUFAs! in! the! reduction! of! cardiovascular! risks! (EFSA! Panel! on! Dietetic! Products,! Nutrition!and!Allergies,!2009)!and!in!hypertriglyceridemia!treatment!(KrisTEtherton!et#al.,!2003).! The! Food! Drug! Administration! (FDA)! has! stated! that! fatty! fish! oil! foods! decrease! the! risk! of! coronary!disease,!being!considered!an!intake!up!to!3!g/day!of!marine!nT3!PUFAs!as!safe!(EFSA! Panel! on! Dietetic! Products,! Nutrition! and! Allergies,! 2012).! Moreover,! the! American! Heart! Association! (AHA)! recommends! consuming! at! least! 2! meals! of! fish! per! week! for! primary! prevention! of! CVD! and! the! use! of! EPA/DHA! supplements! in! a! dose! of! 2T4! g/day! for! hypertriglyceridemia! treatment! (KrisTEtherton! et# al.,! 2003).! ! Accordingly,! the! European! Food! Safety!Authority!(EFSA)!considers!that!250!mg/day!of!EPA/DHA!could!help!to!maintain!the!normal! cardiac!function!(EFSA!Panel!on!Dietetic!Products,!Nutrition!and!Allergies!(NDA),!2009),!while!the!

12! I.!INTRODUCTION!

WHO!also!establishes!that!EPA/DHA!at!doses!of!200T500!mg/day!could!protect!against!coronary! heart!disease!(WHO!Nutrition!topics,!revised:!July!2016).!!

Furthermore,!in!murine!models!of!obesity,!strong!evidence!suggests!that!the!supplementation! with!nT3!PUFAs!could!protect!from!dietTinduced!obesity,!by!decreasing!body!weight!and!reducing! body!fat!accumulation!(Buckley!and!Howe,!2009;!MartínezTFernández!et#al.,!2015).!In!this!context,! although!some!studies!in!humans!have!reported!a!negative!significant!correlation!between!the! percentage! of! nT3! PUFAs! in! plasma! phospholipids! with! measures! of! adiposity! (Karlsson! et# al.,! 2006;!KleinTPlatat!et#al.,!2005;!Micallef!and!Garg,!2009),!until!now,!there!is!not!a!consensus!about! if!supplementation!could!induce!changes!in!weight!and!body!composition.!!

3.1.#NI3#PUFAs#and#weight#loss#

RandomizedTplacebo! controlled! trials! evaluating! the! effects! of! marine! nT3! PUFAs! supplementation! on! weight! loss! have! reached! contradictory! outcomes! (Table! 2! and! Table! 3).! Most!of!the!studies!have!been!performed!using!a!combination!of!EPA!and!DHA,!with!or!without! an!energyTrestricted!regime,!and!with!or!without!exercise.!!!

3.1.1.!Effects!of!marine!n<3!PUFAs!in!the!context!of!an!energy!restricted!diet!

Studies!evaluating!the!effects!of!marine!nT3!PUFAs!supplementation!on!body!weight!loss!in!a! context!of!a!hypocaloric!diet!have!arrived!to!conflicting!outcomes.!Thus,!the!study!of!Tapsell!et#al.! (2013),! observed! no! additional! effects! of! either! fatty! fish! (180! g/week)! or! marine! nT3! PUFAs! supplementation! (420! mg! EPA! +! 210! mg! DHA)! on! body! weight! loss! and! fat! mass! in! healthy! overweight! or! obese! subjects! following! a! moderate! energyTrestricted! diet! during! 12! months.! Similarly,! DeFina! et# al.! (2011)! showed!that!supplementation!during!24!weeks!with!marine!nT3! PUFAs!at!a!dose!of!3!g/day!(EPA!+!DHA!at!a!5:1!ratio!in!a!60%!concentration)!had!no!additional! effects! on! body! weight! loss! and! adiposityTrelated! anthropometric! variables! to! the! energyT controlled!diet,!which!was!accompanied!by!exercise!and!dietary!counseling.!In!the!same!way,!a! study!of!Munro!and!Garg!(2013a)!observed!that!supplementation!during!12!weeks!with!tuna!fish! oil!capsules!(6!x!1!g!capsules/day!containing!70!mg!EPA!+!270!mg!DHA)!did!not!promote!additional! benefits!to!a!moderate!energyTrestricted!diet!in!anthropometric!variables!and!body!composition.!!

Moreover,!some!trials!have!evaluated!whether!the!effects!of!marine!nT3!PUFAs!could!have!any! effect!during!the!maintenance!period!after!weightTloss!treatment.!In!this!context,!another!study! of! Munro! and! Garg! (2012)! reported! that! supplementation! with! marine! nT3! PUFAs! (6! x! 1! g! capsules/day!containing!70!mg!EPA!plus!270!mg!DHA)!during!14!weeks!in!healthy!subjects!had!no! additional!effects!on!anthropometric!and!body!composition!variables!than!placebo!during!either!

13! I.!INTRODUCTION! the! weight! loss! phase! (4! weeks)! with! a! veryTlow! energy! restricted! diet! or! the! 10Tweeks! maintenance!period.!Similarly,!a!study!from!Krebs!et#al.!(2006),!performed!in!hyperinsulinaemic! overweight/obese!females,!showed!no!additional!effects!of!supplementation!(1.3!g/day!EPA!+!2.9! g/day!DHA)!during!24!weeks!to!an!energy!restricted!diet!on!body!weight!loss!neither!during!the! weightTloss!phase!(12!weeks)!nor!in!the!maintenance!period!(12!weeks).!!

Although! some! studies! have! suggested! a! possible! sexTbased! interaction! of! the! marine! nT3! PUFAs,!this!interaction!is!not!clear.!Thus,!a!study!by!Thorsdottir!et#al.!(2007)!suggested!that!either! the!consumption!of!fatty!fish!(150!g!of!cod!or!salmon!3!times/week)!or!the!supplementation!with! marine! nT3! PUFAs! capsules! (1.3! g/day! of! EPA+DHA)! promoted! a! greater! weight! loss! and! waist! circumference!only!in!men.!However,!other!studies!in!females!have!observed!that!nT3!PUFAs!are! able!to!modulate!changes!in!either!anthropometric!variables!or!body!composition!(Kunesova!et# al.,!2006;!Munro!and!Garg,!2013b;!Tardivo!et#al.,!2014).!In!this!sense,!a!study!of!Tardivo!et#al.! (2014)!found!that!supplementation!with!marine!nT3!PUFAs!(3!capsules!of!1!g!containing!180!mg! EPA!+!120!mg!DHA!per!day)!during!6!months!induced!a!moderate!decrease!in!fat!mass!without! changes!in!body!weight,!muscle!mass!and!waist!circumference!in!postmenopausal!women!with! MetS.!Other!study!in!severely!obese!women!following!a!veryTlow!calorie!diet!in!conjunction!with! exercise!during!3!weeks,!observed!in!the!supplemented!groups!(2.98!g/day!of!EPA+DHA!at!a!2:1! ratio)!a!greater!decrease!in!body!weight,!BMI!and!hip!circumference!besides!a!greater!increase!in! βThydroxybutyrate!levels!(Kunesova!et#al.,!2006).!Contrary,!the!study!of!Su!et#al.!(2014),!using!a! supplementation! of! 2.13! g/day! of! marine! nT3! PUFAs! (1280! mg! EPA! +! 850! mg! DHA)! during! 12! weeks!showed!no!significant!differences!both!in!the!decrease!of!body!weight!and!in!the!changes! of! body! composition! in! the! treated! groups! as! compared! with! placebo! group.! Interestingly,! another!study!of!Munro!and!Garg!(2013b)!suggested!that!4Tweeks!of!supplementation!with!nT3! PUFAs!prior!to!4!weeks!with!a!veryTlowTenergy!restricted!diet!could!help!to!promote!a!greater! reduction!of!body!weight!as!compared!with!non!supplemented!group.!

Though!several!studies!in!adult!population!have!evaluated!the!potential!body!weight!lowering! effects!of!marine!nT3!PUFAs,!the!differential!effects!between!EPA!and!DHA!have!not!been!widely! assessed.!In!this!context,!the!previously!reported!study!by!Kunesova!et#al.!(2006)!hypothesized! that!docosahexaenoate!(22:6!nT3)!could!be!the!active!compound!that!induced!the!changes!in!BMI.! In! addition,! the! Japan! Obesity! and! Metabolic! Syndrome! Study,!using!a!supplementation!of!1.8! g/day!of!highly!purified!EPA!during!3!months!in!combination!with!a!hypocaloric!diet!(25!kcal/kg!of! ideal!body!weight),!reported!no!significant!changes!in!both!BMI!and!waist!circumference!(Itoh!et# al.,!2007;!Yamada!et#al.,!2008).!!

14! I.!INTRODUCTION!

3.1.2.!Effects!of!marine!n<3!PUFAs!in!the!context!of!an!isocaloric!diet!

The!study!of!Ebrahimi!et#al.!(2009)!in!Iranian!males!and!females,!giving!a!general!dietary!advice! to!both!study!groups!and!using!a!supplementation!of!1!g/day!of!fish!oils!(containing!180!mg!EPA! and!120!mg!DHA)!for!6!months,!observed!that!marine!nT3!PUFAs!promoted!weight!loss.!Similarly,! the!study!from!Kabir!et#al.!(2007),!which!also!gave!a!dietary!counseling,!using!a!supplementation! with!1.8!g/day!of!marine!nT3!PUFAs!(1080!mg!EPA!+!720!mg!DHA)!during!2!months,!showed!that! although!the!treatment!had!no!effects!in!body!weight!loss,!it!promoted!a!reduction!in!both!fat! mass! and! adipocyte! diameter.! ! Also,! Hill! et# al.! (2007)! observed! a! greater! decrease! in! fat! mass! independently!of!the!exercise!effect!in!those!subjects!taking!6!g/day!of!tuna!fish!oil!(60!mg!EPA!+! 260!mg!DHA!per!g)!during!12!weeks!as!compared!with!control!group.!!

Contrary!to!these!observations,!studies!from!Simão!et#al.!(2012)!found!that!supplementation! of!3!g/day!of!marine!nT3!PUFAs!(180!mg!EPA!+!120!mg!DHA!per!capsule)!during!90!days!with!the! habitual!diet,!did!not!have!any!effect!in!body!weight!and!waist!circumference.!In!the!same!way,!a! study! of! Tousoulis! et# al.! (2014)! showed!that!the!supplementation!with!2! g/day! of! marine! nT3! PUFAs! (46%! EPA,! 38%! DHA)! during! 12! weeks! with! a! washout! period! of! four! weeks! between! treatments!did!not!promote!a!reduction!in!body!weight.!

!

15! I.#INTRODUCTION#

Table# 2.# Clinical' trials' evaluating' the' effects' of' marine' n33' PUFAs' supplementation' on' body' weight' loss' and' anthropometric' parameters' in' overweight/obese'subjects,'following'a'hypocaloric'diet# References# Study#design# n<3#PUFAs#treatment# Outcomes# Randomized'placebo3controlled'trial'with'parallel' 5' capsules/d' (3' g/d' of' EPA' +' DHA' at' a' design' in' overweight' and' obese' females' and' 5:1'ratio'in'a'60%'concentration)'for'243 males,' 30%' with' MetS' (n=' 81;' age:' 45.6' ±' 8.3' ↔'Body'weight,'body'fat' (DeFina'et#al.,'2011)' weeks' (with' exercise' and' dietary' years'in'n33'PUFAs'group'and'47.0'±'7.8'years'in' mass,'waist'circumference# counseling'and'a'calorie3controlled'diet)# placebo'group)# Randomized'single3blind'trial'with'parallel'design' 1.8'g'of'EPA'rich3capsules'(>98%'as'EE)' in' obese' females' and' males' with' MetS' (n=' 52' (Itoh'et#al.,'2007;'Yamada'et# for'33months'(energy3restricted'diet'25' ↔'BMI'and'waist' (Itoh' et# al.,' 2007);' n=' 60' (Yamada' et# al.,' 2008);' al.,'2008)' kcal/kg'of'ideal'body'weight)' circumference' age:'51.9'±'2.5'years)' 1.3'g/d'EPA'+'2.9'g/d'DHA'(EE'capsules)' Randomized'placebo3controlled'trial'with'parallel' for' 243weeks' (123first' weeks' with' an' design'in'hyperinsulinaemic'overweight'or'obese' (Krebs'et#al.,'2006)' energy3restricted' diet' followed' by' ↔'Body'weight' females'(n='93;'age:'21369'years)' maintenance'period)' Randomized,' placebo3controlled' trial' in' severely' 2.98'g/d'of'n33'PUFAs'(EPA'+'DHA'at'a' ↓'Body'weight,'BMI,'hip' obese'women'(n='20;'age:'54.3'±'5.4'years'in'n33' 2:1' ratio)' for' 33weeks' (with' a' VLCD' of' circumferences' (Kunesova'et#al.,'2006)' PUFAs' group' and' 49.8' ±' 12.3' years' in' placebo' 2200' kJ/d' and' light' to' moderate' ↑β3hydroxybutyrate'' group)' physical'activity'60'min/d)' ↔'Waist'circumference' 6x1'g'capsules/d'(70'mg'EPA'+'270'mg' Randomized'double3blind'placebo3controlled'trial' DHA' per' fish' tuna' oil' capsule)' for' 43 ↔'Body'weight,'fat'mass,' with'parallel'design'in'healthy'obese'females'and' weeks' (43first' weeks' with' a' VLCD' of' (Munro'and'Garg,'2012)' fat'free'mass,'waist'and'hip' males'(n='32;'age:'18360'years)' 3000'kJ/d'with'a'meal'replacement'food' circumferences,'WHR.' followed'by'a'maintenance'period)' ' '

16# I.#INTRODUCTION#

Table#2.#Continuation' References# Study#design# n<3#PUFAs#treatment# Outcomes# 6x1'g'tuna'fish'oil'capsules/d'(70'mg'EPA' Randomized'double3blind'placebo3controlled'trial' ↔'Body'weight,'fat'mass,' +' 270' mg' DHA' per' gram)' for' 12' weeks' (Munro'and'Garg,'2013a)' with'parallel'design'in'obese'healthy'females'and' fat'free'mass,'waist'and' (with'an'energy3restricted'diet'of'5000'kJ' males'(n='33;'age:'18360'years)' hip'circumferences' for'females'and'6000'kJ'for'males)' 6x1'g'tuna'fish'oil'capsules/d'(70'mg'EPA' ↓'Body'weight'and'BMI' Randomized'double3blind'placebo3controlled'trial' +' 270' mg' DHA' per' gram)' for' 8' weeks' (only'in'females)' (Munro'and'Garg,'2013b)' with'parallel'design'in'obese'healthy'females'and' (with' prior' 4' weeks' n33' PUFAs' ↔'Fat'mass,'fat'free' males'(n='36;'age:'18360'years)' supplementation' followed' by' a' VLCD' of' mass,'waist'and'hip' 3000'kJ/d'with'a'meal'replacement'food)' circumferences' 2.13' g/d' of' n33' PUFAs' (1280' mg' EPA' +' Randomized' controlled' trial' with' parallel' design' ↔'Body'weight,'BMI,' 850' mg' DHA)' for' 12' weeks' (calorie' (Su'et#al.,'2014)' in' overweight' and' obese' females' with' MetS' (n=' waist'circumference,'fat' restriction' of' 5003800' kcal/d' with' or' 136,'age:'30365'years)' mass,'fat'free'mass' without'meal'replacement'diet)' 180'g/week'of'fatty'fish'or'1'g/d'fish'oil' Randomized' controlled' trial' with' parallel' design' capsule'(420'mg'EPA'+'210'mg'DHA)'for' (Tapsell'et#al.,'2013)' in' overweight' and' obese' healthy' males' and' ↔'Body'weight,'fat'mass' 123months'(with'an'energy'restriction'of' females'(n='64;'age:'18360'years)' 2'MJ'and'dietary'education)' ↓'Fat'mass'(moderately)' Randomized' prospective' controlled' trial' in' 3'capsules/d'(180'mg'EPA'+'120'mg'DHA' ↔'Body'weight,'muscle' (Tardivo'et#al.,'2014)' overweight' and' obese' postmenopausal' women' per' capsule)' for' 63months' (with' energy3 mass,'waist' with'MetS'(n='63;'age:'45370'years)' restricted'diet)' circumference' 2' groups' with' 150' g' of' fish' three' Randomized' controlled' trial' with' parallel' design' times/week' (either' cod' or' salmon)' and' ↓'Body'weight'and'waist' (Thorsdottir'et#al.,'2007)' in' overweight' and' obese' healthy' females' and' other' with' 6x0.5' g' fish' oil' capsules/d' circumference'(only'in' males'(n='324;'age:'20340'years)' (ratio' DHA:EPA' ns)' for' 8' weeks' (with' males)' energy3restricted'diet'<30%'Kcal)' Abbreviations:'↔:'no'change;'↑:'increase;'↓:'decrease;'BMI:'body'mass'index;'d:'day;'DHA:'docosahexaenoic'acid;'EE:'ethyl'ester;'EPA:'eicosapentaenoic'acid;'MetS:' metabolic'syndrome;'n33'PUFAs:'omega33'polyunsaturated'fatty'acids;'ns:'non3specified;'VLCD:'very3low'calorie'diet;'WHR:'waist'to'hip'ratio.'

17# I.#INTRODUCTION#

Table# 3.# Clinical' trials' evaluating' the' effects' of' marine' n33' PUFAs' supplementation' on' body' weight' loss' and' anthropometric' parameters' in' overweight/obese'subjects,'using'an'isocaloric'diet# References# Study#design# n<3#PUFAs#treatment# Outcomes#

Randomized' controlled3trial' with' parallel' design' 1'g'capsule/d'of'n33'PUFAs'(180'mg'EPA' (Ebrahimi'et#al.,'2009)# in'overweight'and'obese'females'and'males'with' and'120'mg' DHA)'for'6'months'(general' ↓'Body'weight# MetS'(n='89;'age:'40370'years)# dietary'advice)#

Randomized'placebo3controlled'trial'with'parallel' 6'g/d'of'tuna'fish'oil'(60'mg'EPA'+'260'mg' design' in' overweight' and' obese' females' and' DHA' per' gram)' for' 12' weeks' (usual' diet' ↓'Fat'mass' males' with' at' list' one' risk' factor:' mild' (Hill'et#al.,'2007)' with'or'without'3'times/week'of'45'min' ↔'Body'weight' hypertension,' elevated' TG' or' elevated' total3 exercise)' cholesterol'(n='65,'67%'males;'age:'25365'years)'

Randomized,' double3blind,' placebo3controlled' 1.8'g/d'of'n33'PUFAs'(1.08'g'EPA'+'0.72'g' ↓'Fat'mass' trial'with'parallel'design'in'overweight'and'obese' DHA)' for' 2' months' of' run3in' period' ↓'Adipocyte'diameter' (Kabir'et#al.,'2007)' postmenopausal' women' with' T2DM,' 50%' with' followed'by'2'months'treatment'(dietary' ↔'Body'weight' hypoglycemic'treatment'(n='26;'age:'40360'years)' counseling)'

3'g/d'of'n33'PUFAs'(180'mg'EPA'plus'120' Randomized' controlled3trial' with' parallel' design' mg'DHA'per'capsule)'for'90'days'alone'or' ↔'Body'weight'and'waist' in' obese' females' with' MetS' (n=65;' age:' 47.9' ±' (Simão'et#al.,'2012)' in' combination' with' kinako' (29' g/d' of' circumference' 9.9'years)' toasted'ground'soya'bean)'(usual'diet)' Randomized,' double3blind,' placebo' controlled,' 2'g/d'of'n33'PUFAs'(46%'EPA,'38%'DHA)' cross3over'trial'in'normal'weight,'overweight'and' for' 12' weeks' with' 4' weeks' of' washout' ↔'Body'weight' (Tousoulis'et#al.,'2014)' obese'females'and'males'with'MetS'(n='29;'age:' period'between'treatments'(usual'diet)' 26370'years)' Abbreviations:'↔:'no'change;'↑:'increase;'↓:'decrease;'d:'day;'DHA:'docosahexaenoic'acid;'EPA:'eicosapentaenoic'acid;'MetS:'metabolic'syndrome;'n33'PUFAs:'omega33' polyunsaturated'fatty'acids;'T2DM:'type'2'diabetes'mellitus;'TG:'triglycerides.'

18# I.#INTRODUCTION#

3.2.$Effects$of$n-3$PUFAs$on$lipid$and$glucose$metabolism$

Studies( evaluating( the( effects( of( marine( n43( PUFAs( supplementation( not( only( in( overweight/obese(subjects(but(also(with(some(features(of(MetS(on(lipid(and(glucose(metabolism( are(described(in(Table#4.((

3.2.1.#Lipid#metabolism#

Strong( evidence( suggests( that( n43( PUFAs( supplementation( is( able( to( decrease( TG( concentrations.(In(this(sense,(the(systematic(review(from(Balk(et$al.((2006),(taking(into(account( not(only(studies(in(healthy(individuals(but(also(with(T2DM,(dyslipidemia(or(CVD(published(through( 2003,(found(an(overall(net(effect(of(fish(oils(to(decrease(TG(blood(levels.(Moreover,(they(observed( that(each(increase(in(fish(oil(dose(of(1(g/day(was(associated(with(a(decrease(of(approximately(8( mg/dL(in(the(TG(concentrations.(Also,(those(subjects(with(higher(TG(levels(had(more(benefits,( decreasing(more(the(TG(after(fish(oil(treatment.(Similarly,(the(meta4analysis(from(Hartweg(et$al.( (2007),( described( that( n43( PUFAs( supplementation( promoted( a( decrease( in( TG( levels( in( type( 2( diabetic(subjects.(Other(trials(carried(out(in(subjects(with(MetS(characteristics(have(highlited(the( ability(of(n43(PUFAs(to(decrease(not(only(fasting(TG(levels((Bragt(and(Mensink,(2012;(Browning(et$ al.,(2007;(Dewell(et$al.,(2011;(Hill(et$al.,(2007;(Itariu(et$al.,(2012;(Kelley(et$al.,(2007;(Krebs(et$al.,( 2006;(Lovegrove(et$al.,(2004;(Munro(and(Garg,(2013a,(2013b;(Nestel(et$al.,(2002;(Satoh4Asahara( et$al.,(2012;(Simão(et$al.,(2014;(Spencer(et$al.,(2013;(Tousoulis(et$al.,(2014;(Wong(et$al.,(2013;( Yamada(et$al.,(2008),(but(also(postprandial(TG(response((Kelley(et$al.,(2007;(Wong(et$al.,(2014)( (Table# 4).((Although(no(differential(effects(on(TG(reduction(between(EPA(and(DHA(have(been( reported((Balk(et$al.,(2006;(Egert(et$al.,(2009;(Nestel(et$al.,(2002),(Itariu(et$al.((2012)(suggested( that(EPA(rather(than(DHA(appears(to(be(associated(with(the(hypotriglyceridemic(effects(of(n43( PUFAs,( based( on( the( significantly( negative( correlation( between( EPA( plasma( phospholipids( concentration( with( serum( TG.( In( this( sense,( the( 54years( duration( JELIS( study( (Yokoyama( et$ al.,( 2007),(observed(that(supplementation(with(1.8(g/day(of(EPA(decreased(both(the(hazard(ratio(of( coronary(events(and(the(frequency(of(non4fatal(coronary(events.((

According( with( the( available( scientific( information,( although( total( cholesterol( appears( to( be( not(affected(by(n43(PUFAs(supplementation((Balk(et$al.,(2006;(Cottin(et$al.,(2011;(Hartweg(et$al.,( 2007;(Marqués(et$al.,(2008),(the(information(referred(to(low(density(lipoprotein((LDL)(and(high( density(lipoprotein((HDL)(cholesterol((ch)(fractions(is(not(consistent.(Some(studies(suggested(that( DHA(could(have(a(differential(effect(as(compared(with(EPA,(by(promoting(a(slight(increment(in( LDL4ch(and(HDL4ch,(however(it(has(been(suggested(that(the(increase(is(clinically(negligible((Cottin(

19# I.#INTRODUCTION# et$ al.,( 2011).( Furthermore,( the( data( suggest( that( although( supplementation( with( marine( n43( PUFAs(has(not(a(clear(effect(in(total(LDL4ch,(these(could(promote(a(fall(of(the(pro4atherogenic( small( dense( LDL4ch,( favoring( the( formation( of( a( less( atherogenic( large( buoyant( LDL4ch( particle( (Cottin(et(al.,(2011;(Kelley(et$al.,(2012;(Mori(et$al.,(2000).(Likewise,(studies(have(suggested(that( supplementation(with(marine(n43( PUFAs( could( increase( HDL24ch,( which( is( capable( to( transport( greater(cholesterol(amounts(than(the(HDL34ch,(without(necessarily(increasing(total(HDL4ch((Cottin( et$al.,(2011;(Mori(et$al.,(2000).(Also,(the(studies(that(have(tested(supplementation(of(either(EPA(or( DHA,(have(observed(that(DHA(but(not(EPA(promoted(an(increment(of(both(large(buoyant(LDL4ch( and(HDL24ch(sub4fractions((Kelley(et$al.,(2007,(2012;(Mori(et$al.,(2000).(

3.2.2.#Glucose#metabolism#

Although( animal( studies( have( evidenced( that( marine( n43( PUFAs( can( regulate( glucose( metabolism( (Martínez4Fernández( et$ al.,( 2015),( in( humans,( the( effects( of( n43( PUFAs( supplementation( on( glucose( metabolism( and( insulin( sensitivity( either( in( healthy( overweight/obese(subjects(or(persons(with(T2DM(as(well(as(in(individuals(with(some(feature(of( MetS,(are(not(conclusive((Balk(et$al.,(2006;(Hartweg(et$al.,(2008).(

The(systematic(review((Balk(et$al.,(2006),(previously(mentioned,(observed(that(neither(fish(nor( fish( oil( had( a( significant( effect( on( fasting( blood( glucose( and( glycosylated( hemoglobin( (HbA1c).( Furthermore,(higher(doses(of(fish(oil(were(associated(with(greater(fasting(glucose(levels,(being( this( effect( of( fish( oils( on( fasting( glucose( more( marked( in( those( subjects( with( greater( fasting( glucose(levels(at(baseline.(Moreover,(a(recent(study(from(Veleba(et$al.((2015),(observed(in(type(2( diabetic( individuals( that( the( treatment( with( a( n43( PUFAs( concentrate( (providing( 2.8( g/day( of( EPA+DHA)(promoted(an(increase(not(only(in(postprandial(glucose,(but(also(in(both(fasting(glucose( and(HbA1c(without(any(effect(in(insulin(sensitivity.(Other(meta4analysis(conducted(by(Hartweg(et$ al.( (2008),(which(considered(those(trials( published( up( to( 2006( and( performed( in( subjects( with(

T2DM(diagnosis,(concluded(that(neither(EPA(nor(DHA(had(a(significant(effect(in(HbA1c(and(both( fasting( glucose( and( insulin.( ( Additionally,( Kabir( et$ al.( (2007),(reported(that(in(postmenopausal( type(2(diabetic(women,(supplementation(with(1.8(g/day(of(marine(n43(PUFAs(had(no(a(differential( effect( in( any( parameter( of( glucose( metabolism( (glucose,( insulin,( insulin( sensitivity( and( HbA1c).( Although(some(studies(have(suggested(a(slight(increase(in(fasting(glucose(with(fish(oil(treatment,( some(authors(have(proposed(that(it(could(be(related(with(the(increment(of(gluconeogenesis(as(a( result( of( either( the( decrease( of( de( novo( lipogenesis( or( the( drop( in( TG( release( as( an( energy( substrate(by(the(liver,(rather(than(owing(to(any(adverse(effects(of(fish(oils(in(insulin(sensitivity( (Wu(et$al.,(2013).(

20# I.#INTRODUCTION#

Most( of( the( trials( carried( out( in( subjects( with( some( characteristic( of( the( MetS( and( using( different(ratios(of(EPA:DHA(at(different(doses((144(g/day)(reported(no(significant(effect(of(marine( n43( PUFAs( supplementation( on( glucose( metabolism( or( insulin( sensitivity( (Bragt( and( Mensink,( 2012;(Browning(et$al.,(2007;(DeFina(et$al.,(2011;(Dewell(et$al.,(2011;(Itariu(et$al.,(2012;(Kelley(et( al.,(2012;(Krebs(et$al.,(2006;(Lovegrove(et$al.,(2004;(Munro(and(Garg,(2013a,(2013b;(Skulas4Ray(et( al.,(2011;(Spencer(et$al.,(2013;(Su(et$al.,(2014;(Tapsell(et$al.,(2013;(Tousoulis(et$al.,(2014;(Wong(et( al.,(2013,(2014).((

Though(the(majority(of(the(studies(have(used(a(supplementation(of(EPA+DHA,(some(trials(have( evaluated(the(effects(of(these(marine(n43(PUFAs(separately,(in(the(context(of(an(isocaloric(diet( and( in( subjects( with( MetS( features.( Supplementation( with( 243( g/day(of(DHA(did(not(exert(any( effect( in( glucose( parameters( in( subjects( with( some( characteristics( of( the( MetS( (Kelley( et$ al.,( 2012).(Moreover,(the(study(of(Mori(et$al.((2000),(which(evaluated(the(differential(effect(of(DHA( and(EPA((4(g/day)(in(mildly(hypercholesterolemic(men(following(their(usual(diet(found(that(both( EPA( and( DHA( promoted( an( increment( in( fasting( insulin( levels( without( significant( changes( in( fasting(glucose.((

On(the(other(hand,(trials(evaluating(the(potential(body4weight(lowering(effects(of(n43(PUFAs( (EPA( +( DHA)( in( healthy( overweight/obese( individuals,( have( observed( no( additional( effects( of( supplementation( (146( g/day)( to( the( weight4loss( regime( in( neither( glucose( nor( insulin( fasting( plasma(levels((Krebs(et$al.,(2006;(Munro(and(Garg,(2013a,(2013b;(Su(et$al.,(2014;(Tapsell(et$al.,( 2013;(Wong(et$al.,(2013).(Contrary(with(these(results,(the(study(of(Ramel(et$al.((2008),(performed( in( overweight/obese( young( adults( following( an( energy4restricted( diet,( detected( that( the( supplementation(with(fish(oil((1.3(g/day(of(EPA(+(DHA)(improved(insulin(sensitivity(measured(by( the(Homeostatic(Model(Assessment(of(Insulin(Resistance((HOMA4IR),(independently(of(changes(in( body(weight.((

Despite(that(the(major(part(of(the(trials(have(found(no(therapeutic(effects(of(n43(PUFAs(on( insulin(sensitivity,(as(suggested(by(Flock(et$al.((2013),(it(cannot(be(discard(that(consumption(of(n43( PUFAs(could(be(important(at(primary(prevention(of(insulin(resistance.((

(

21# I.#INTRODUCTION#

Table#4.#Clinical'trials'evaluating'the'effects'of'marine'n33'PUFAs'supplementation'on'lipid'and'glucose'metabolism,'as'well'as'in'inflammatory'markers# References# Study#design# n<3#PUFAs#treatment# Outcomes# Lipid%metabolism% ↓'TG' 8'fish'oil'capsules/d'providing'a'total'of' ↓'VLDL'particles' 3.7' g' of' n33' PUFAs' (1.7' g' EPA' +' 1.2' g' ↑'LDL3ch,'sdLDL3ch,'large'LDL3ch' Randomized,' double3blind,' placebo3 DHA).' Three' consecutive' periods' of' 6' controlled'trial'with'crossover'design'in' ↔'HDL3ch' weeks'intervention'(fish'oil;'fenofibrate' dyslipidemic' females' and' males,' some' ↔'FFA' (Bragt'and'Mensink,'2012)# (200' mg/d)' and' placebo)' with' 2' weeks' with' moderate' hypertension' or' with' Glucose%metabolism% of'washout'period'between'treatments' MetS'(n='20;'age:'52'±'12'years)' (usual'diet)' ↔'FBG' # ' Inflammation% # ↔''TNFR1,'TNFR2,'CRP,'TNF3α,'IL36,' sICAM31,'sVCAM31,'MCP31,'sE3 selectin# Lipid%metabolism# ↓'TG' Randomized' crossover' trial' in' 5'fish'oil'capsules/day'(1.3'g'EPA'+'2.9'g' ↔'Total3ch,'HDL3ch,'LDL3ch' overweight' or' obese' women' DHA).' Two' consecutive' periods' of' 12' categorized' into' 2' groups:' high' Glucose%metabolism% (Browning'et#al.,'2007)' weeks' intervention' (fish' oil' and' inflammatory' status/low' inflammatory' ↔'FBG,'FBI# placebo)' with' 4' weeks' of' washout' status'based'on'serum'sialic'acid'(n='30;' period'between'treatments'(usual'diet)' Inflammation% age:'ns)' ↓'IL36,'CRP' ↔'PAI31,'SAA# Randomized' placebo3controlled' trial' Lipid%metabolism% with' parallel' design' in' overweight' and' 5' capsules/d' (3' g/d' of' EPA' +' DHA' at' a' ↑'LDL3ch' obese' females' and' males,' 30%' with' 5:1'ratio'in'a'60%'concentration)'for'243 (DeFina'et#al.,'2011)' ↔'HDL3ch,'TG' MetS'(n='81;'age:'45.6'±'8.3'years'in'n33' weeks' (with' exercise' and' dietary' PUFA' group' and' 47.0' ±' 7.8' years' in' counseling)' Glucose%metabolism% placebo'group)' ↔'FBG,'FBI# '

22" I.#INTRODUCTION#

Table#4.#Continuation# References# Study#design# n<3#PUFAs#treatment# Outcomes# Lipid%metabolism% ↓'TG' Randomized,' single' blind' placebo3 Low'and'high'dose'of'ALA'(2.2'g/d'and' controlled' trial' with' parallel' design' in' 6.6'g/d'respectively);'low'and'high'dose' ↑'LDL3ch' overweight' and' obese' females' and' of' fish' oil' (1.2' g/d' (700' mg' EPA' +' 500' ↔'Total3ch,'HDL3ch' (Dewell'et#al.,'2011)' males' with' MetS,' without' anti3 mg' DHA' as' TG' or' 3.6' g/d' (2.1' g' EPA' +' Glucose%metabolism% hypertensive'drugs'(n='98;'age:'50'±'10' 1.5' g' DHA)' as' TG,' respectively)' for' 83 ↔'FBG,'FBI' years)' weeks'(usual'diet)' Inflammation% ↔'MCP31,'IL36,'sICAM31' Randomized' placebo3controlled' trial' with' parallel' design' in' overweight' and' 6'g/d'of'tuna'fish'oil'(60'mg'EPA'+'260' Lipid%metabolism% obese' females' and' males' with' at' list' mg'DHA'per'gram)'for'123wk'(usual'diet' (Hill'et#al.,'2007)' ↓'TG' one' risk' factor:' mild' hypertension,' with'or'without'3'times/week'of'45'min' ↑'HDL3ch' elevated'TG'or'elevated'total3ch'(n='65,' exercise)' 67%'males;'age:'25365'years)' Lipid%metabolism% ↓'TG' ↔'Total3ch,'LDL3ch,'HDL3ch,'apo'B' Randomized,'open3label,'controlled'trial' Glucose%metabolism%

with' parallel' design' in' severely' obese' ↔'FBG,'FBI,'HbA1c,'AUC'glucose,' (BMI'≥40)'and'non3diabetic'females'and' 3.36'g/day'(460'mg'EPA'+'380'mg'DHA' AUC'insulin,'HOMA3IR,'fasting'C3 (Itariu'et#al.,'2012)' males' that' undergo' elective' bariatric' per' gram)' for' 8' weeks' (isocaloric' peptide,'clamp3like'index' surgery'(n='55;'age'20365'years).'Both,' balanced3diet)' Inflammation% SAT' and' VAT' were' used' for' gene' ↓'IL36' expression'analyses' ↔'Adiponectin,'leptin,'CRP,' leukocytes' ↓'CCL2,'CCL3,'HIF1A,'TGFB1,'CD40# gene'expression'(only'in'SAT)'

23" I.#INTRODUCTION#

Table#4.#Continuation# References# Study#design# n<3#PUFAs#treatment# Outcomes# ↔#IL6,'ADIPOQ,'CD68,'CD163,'MRC1# gene'expression'(only'in'SAT)' ↑'173HDHA,'PD1'(only'in'VAT)' (Itariu'et#al.,'2012)' Continuation' Continuation' ↑'RvE1,'RvD1'(only'in'VAT,'in'the' control'group'were'no'detected)' ↔'183HEPE,'PGE2,'153HETE,'123HETE' Lipid%metabolism% ↓TG,'ratio'TG:HDL3ch' ↓'apo'CIII' ↓'Diameter'of'VLDL3ch'particles' ↓'sdLDL,'LDL'particle'size' ↓Post3prandial'TG' ↓'AUC'for'small'LDL,'small'HDL'and' large'VLDL'particles' Randomized,' double3blind' placebo3 ↑'AUC'for'large'LDL,'large'HDL,'small' 7.5'g/d'of'DHA'oil/d'(containing'3'g'of' controlled' trial' with' parallel' design' in' VLDL'particles' (Kelley'et#al.,'2007,'2009,' DHA)' for' 903d' (usual' diet).' Test' normal' weight,' overweight' and' obese' ↔'Total3ch,'HDL3ch,'apo'A1,'apo'B,' 2012)' breakfast'with'total'intake'of'850'kcal' hypertriglyceridemic' males' with' MetS' apo'E' ' features'(n='34;'age'39366'years)' ↓'FFA' Glucose%metabolism% ↔'FBG,'FBI,'HOMA3IR,'Matsuda' index,'glucose'or'insulin'AUC' Inflammation% ↓'CRP,'IL36' ↑'MMP32' ↔'ICAM31,'VCAM31,'E3selectin' ↔'NO,'IL31β,'IL32,'IL310,'TNF3α,'SAA' '

24" I.#INTRODUCTION#

Table#4.#Continuation# References# Study#design# n<3#PUFAs#treatment# Outcomes# Lipid%metabolism% ↓'TG' ↔'Total3ch,'LDL3ch,'HDL3ch' Randomized' placebo3controlled' trial' 1.3'g/d'EPA'+'2.9'g/d'DHA'(EE'capsules)' Glucose%metabolism% with' parallel' design' in' for' 24' weeks' (12' first' weeks' with' an' (Krebs'et#al.,'2006)' ↔'FBG,'FBI,'HbA ,'HOMA3IR' hyperinsulinaemic'overweight'or'obese' energy3restricted' diet' followed' by' 1c ↔'Glucose'and'insulin'AUC' females'(n='93;'age:'21369'years)' maintenance'period)' Inflammation% ↑'Adiponectin' ↔'TNF3α,'IL36,'CRP,'leptin' Lipid%metabolism% Randomized,' double3blind,' placebo3 4' g/d' of' fish' oil' capsules' (containing' ↓'TG,'apo'B348' controlled' trial' with' parallel' design' in' 60%' of' n33' PUFAs' with' 367' mg' EPA' +' ↑'HDL3ch' (Lovegrove'et#al.,'2004)' normal'weight'and'overweight'females' 255'mg'DHA'per'capsule)'for'12'weeks' ↔'Total3ch,'LDL3h,'FFA,'apo'B## and'males'without'hypertension'(n='84;' (usual'diet)' age:'25370'years)' Glucose%metabolism% ↔'FBI,'HOMA3IR,'HbA1c# Lipid%metabolism% ↓'TG' ↑'LDL3ch'(only'DHA)' Randomized,' double3blind,' placebo3 ↑'LDL3ch'particle'size'(only'DHA)' controlled' trial' with' parallel' design' in' 2'groups'supplemented'with'4'g/d'of'n3 ↓'HDL33ch'(only'EPA)' mildly' hyperlipidemic,' normotensive' (Mori'et#al.,'2000)' 3'PUFAs:'either'EPA'(96%'as'EE)'or'DHA' ↑'HDL23ch'(only'DHA)' men' without' anti3hypertensive' and' (92%'as'EE)'for'6'weeks'(usual'diet)' ↔'Total3ch,'HDL3ch' lipid3lowering' drugs' (n=' 56;' age:' 20365' years)' Glucose%metabolism% ↑'FBI' ↓'Ratio'FBG:FBI'(only'DHA)' ↔'FBG' '

25" I.#INTRODUCTION#

Table#4.#Continuation# References# Study#design# n<3#PUFAs#treatment# Outcomes# Lipid%metabolism% ↓'TG' 6x1' g' tuna' fish' oil' capsules/d' (70' mg' Randomized' double3blind' placebo3 ↔'Total3ch,'LDL,'HDL3ch' EPA' +' 270' mg' DHA' per' gram)' for' 12' controlled' trial' with' parallel' design' in' Glucose%metabolism% (Munro'and'Garg,'2013a)' weeks'(with'an'energy3restricted'diet'of' obese' healthy' females' and' males' (n=' ↔'FBG' 5000' kJ' for' females' and' 6000' kJ' for' 33;'age:'18360'years)' males)' Inflammation% ↔'Leptin,'adiponectin,'TNF3α,'IL36,' CRP' Lipid%metabolism% 'TG' 6x1' g' tuna' fish' oil' capsules/d' (70' mg' ↓ Randomized' double3blind' placebo3 EPA' +' 270' mg' DHA' per' gram)' for' 8' ↔'Total3ch,'LDL,'HDL3ch' controlled' trial' with' parallel' design' in' weeks' (with' prior' 4' weeks' n33' PUFAs' Glucose%metabolism% (Munro'and'Garg,'2013b)' obese' healthy' females' and' males' (n=' supplementation'followed'by'a'VLCD'of' ↔'FBG' 36;'age:'18360'years)' 3000' kJ/d' with' a' meal' replacement' Inflammation% food)' ↔'Leptin,'adiponectin,'TNF3α,'IL36,' CRP' 2'groups'supplemented'with'4'g/d'of'n3 Lipid%metabolism% Randomized,' double3blind,' placebo3 3'PUFAs:'either'EPA'(85%'EPA'as'EE)'or' ↓'TG'and'VLDL3TG' controlled' trial' with' parallel' design' in' (Nestel'et#al.,'2002)' DHA'(70.7%'DHA'as'EE)'for'2'weeks'of' ↔'Total3ch,'LDL3ch,'HDL3ch' overweight'hyperlipidemic'females'and' familiarization' period' followed' by' 7' males'(n='38;'age:'40369'years)' No'differences'between'EPA'and' weeks'of'treatment'(usual'diet)' DHA'treatments# Randomized,' double' blind,' placebo3 Inflammation% 2x1' g' capsules/d' of' either' EPA3rich' oil' controlled' trial' with' parallel' design' in' ↓'Platelet'aggregation'(EPA'more'in' supplement' (500:100' mg' EPA:DHA)' or' (Phang'et#al.,'2013)'' normal' weight' and' overweight' healthy' males'while'DHA'more'in'women)' DHA3rich' oil' supplement' (100:500' mg' females'and'males'(n='94;'age:' 39.6' ±' ↓PAI31'(only'EPA'in'males)' EPA:DHA)'' 1.7'years)' ↔'TXB2,'P3sel'and'PAI31' '

26" I.#INTRODUCTION#

Table#4.#Continuation# References# Study#design# n<3#PUFAs#treatment# Outcomes# 2' groups' with' 150' g' of' fish' three' Randomized' controlled' trial' with' Lipid%metabolism% times/week'(either'cod'or'salmon)'and' (Marqués'et#al.,'2008;'Ramel' parallel'design'in'overweight'and'obese' ↔'TG,'total3ch,'HDL3ch,'LDL3ch' other' with' 6x0.5' g' fish' oil' capsules/d' et#al.,'2008)# healthy'females'and'males'(n='324;'age:' (ratio' DHA:EPA' ns)' for' 8' weeks' (with' Glucose%metabolism% 20340'years)# energy3restricted'diet'<30%'Kcal)# ↑'Insulin'sensitivity# Lipid%metabolism% ↓'TG' ↔Total3ch,'HDL3ch,'LDL3ch' Glucose%metabolism% Randomized' single3blind' trial' with' ↓'HbA ' (Itoh'et#al.,'2007;'Satoh3 parallel' design' in' obese' females' and' 1.8'g'of'EPA'rich3capsules'(>98%'as'EE)' 1c ↔'FBG' Asahara'et#al.,'2012;'Yamada' males'with'MetS'(n='82'(Satoh3Asahara' for'33months'(energy3restricted'diet'25' et#al.,'2008)' et' al.,' 2012),' age:' 53.1' ±' 13;' n=' 60' Kcal/kg'of'ideal'body'weight)' Inflammation% (Yamada'et'al.,'2008),'age:'51.9'±'2.5;)' ↓'sICAM31,'sVCAM31' ↑'Adiponectin,'IL310'and'IL10#gene' expression'(PBMC)' ↔'CRP;'and'TNF,'IL6,'CD163/CD14# gene'expression'(PBMC)' Lipid%metabolism% ↓'TG' Randomized,'placebo3controlled'trial'in' ↔'Total3ch,'LDL3ch,'HDL3ch' overweight' and' obese' non3diabetic' Glucose%metabolism% males'and'females'with'either'impaired' 4'g/d'of'fish'oils'capsules'(1'g'containing' ↔'FBG,'23h'glucose,'insulin' (Spencer'et#al.,'2013)' glucose' tolerance,' impaired' fasting' at' least' 465' mg' EPA' +' 375' mg' DHA' as' sensitivity'and'secretion' glucose' or' MetS' (n=' 34;' age:' 48' ±' 2.3' EE)'for'123months'(diet:'no'specified)' years'in'n33'PUFA'group'and'53.3'±'2.2' Inflammation% years'in'placebo'group)' ↓'MCP31'and'MCP1'and'CD68'gene' expression'(SAT)' ↓'Decreased'CLSs'(SAT)'

27" I.#INTRODUCTION#

' Table#4.#Continuation# References# Study#design# n<3#PUFAs#treatment# Outcomes# ↔'Leptin,'IL36,'IL310,'IL312,'TNF3α',' (Spencer'et#al.,'2013)' Continuation' Continuation' resistin,'PAI31,'leptin;'and'TNF,'IL1,' IL12'and'IL6#gene'expression'(SAT)' Lipid%metabolism% ↓'TG' 3'g/d'of'n33'PUFAs'in'10'capsules'(180' ↑'Total3ch,'LDL3ch' Randomized' controlled3trial' with' mg'EPA'plus'120'mg'DHA'per'capsule)' ↔'HDL3ch' parallel' design' in' obese' females' and' (Simão'et#al.,'2012,'2014)' for' 90' days' alone' or' in' combination' Glucose%metabolism% males' with' MetS' (n=' 65;' age:' 47.9' ±' with' kinako' (29' g/d' of' toasted' ground' ↑'FBG,'FBI,'HOMA3IR' 10.0'years)' soya'bean)'(usual'diet)' Inflammation% ↑'Adiponectin,'NO' ↔'CRP,'IL36,'TNF3α# n33' PUFAs' (≈' 465' mg' EPA' +' ≈' 375' mg' Lipid%metabolism% DHA'at'ratio'1.2:1'per'gram'as'EE)'at'2' Randomized,' double3blind' placebo3 ↓'TG'(only'high'dose)' different' doses:' low' (0.85' g/d' controlled'trial'with'crossover'design'in' ↔'Total3ch,'LDL3ch,'HDL3ch' EPA+DHA)'and'high'(3.4'g/d)'EPA+DHA.' (Skulas3Ray'et#al.,'2011,'2012)' normal' weight,' overweight' and' obese' Glucose%metabolism% Three' consecutive' periods' of' 8' weeks' hypertriglyceridemic'males'(88.5%)'and' ↔'FBG,'FBI' intervention'(placebo;'low'and'high'n33' females'(n='26;'age:'21365'years)' PUFAs)'with'6'weeks'of'wash3out'period' Inflammation% (usual'diet)' ↔'IL31β,'IL36,'TNF3α# Lipid%metabolism% Randomized' controlled' trial' with' 2.13'g/d'of'n33'PUFAs'(1280'mg'EPA'+' ↔'TG' parallel'design'in'overweight'and'obese' 850' mg' DHA)' for' 12' weeks' (calorie' (Su'et#al.,'2014)' ↔'Total3ch,'HDL3ch' females'with'MetS'(n=' 136;'age:'30365' restriction' of' 5003800' kcal/d' with' or' ↓'LDL3ch'(with'the'meal'replacement' years)' without'meal'replacement'diet)' diet)' '

28" I.#INTRODUCTION#

' Table#4.#Continuation# References# Study#design# n<3#PUFAs#treatment# Outcomes# Glucose%metabolism% ↔'FBG,'FBI,'HOMA3IR' (Su'et#al.,'2014)' Continuation' Continuation' ↓'MetS'severity'(with'the'meal' replacement'diet)' Lipid%metabolism% 180'g/week'of'fatty'fish'or'1'g/d'of'fish' ↔'TG,'total3ch,'LDL3ch,'HDL3ch' Randomized' controlled' trial' with' oil'capsule'(420'mg'EPA'+'210'mg'DHA)' parallel'design'in'overweight'and'obese' Glucose%metabolism% (Tapsell'et#al.,'2013)' for' 12' months' (with' an' energy' healthy'males'and'females'(n='64;'age:' ↔'FBG,'FBI,'HOMA3IR' restriction' of' 2' MJ' and' dietary' 18360'years)' education)' Inflammation% ↔'Leptin' Lipid%metabolism% ↓'TG,'total3ch,'LDL3ch' Randomized,' double3blind' placebo' 2' g/d' of' n33' PUFAs' (46%' EPA' +' 38%' ↔'HDL3ch' controlled,' cross3over' trial' in' normal' DHA).' Two' consecutive' periods' of' 12' weight,' overweight' and' obese' females' weeks' intervention' (placebo' and' n33' Glucose%metabolism% (Tousoulis#et#al.,'2014)' and' males' with' MetS' without' statin' PUFAs)'with'4'weeks'of'washout'period' ↔'FBG' treatment'(n='29;'age:'44'±'12'years)' (usual'diet)' Inflammation% ' ' ↓'IL36' ↑'PAI31' ' ' '

29" I.#INTRODUCTION#

Table#4.#Continuation# References# Study#design# n<3#PUFAs#treatment# Outcomes# Lipid%metabolism% ↓'TG,'VLDL3apo'B100,'apo'B48' ↓Postprandial'TG'and'apo'B48' Randomized,' single' blind' with' parallel' 4' g/d' of' n33' PUFAs' (46%' EPA' and' 38%' response' design'in'normotensive,'centrally'obese,' DHA' as' EE)' for' 163weeks' (12' weeks' of' 'LDL3ch' (Wong'et#al.,'2013,'2014)' dyslipidemic' and' insulin3resistant' energy3restricted' diet' followed' by' 43 ↔ females' and' males' (n=' 99,' age:' 18375' weeks' of' maintenance' period).' Test' Glucose%metabolism% years)' meal'with'a'high'fat'liquid'formula' ↔'FBG,'FBI,'HOMA3IR' Inflammation% ↔'Adiponectin' Abbreviations:'↔:'no'change;'↑:'increase;'↓:'decrease;'ADIPOQ:'adiponectin;'ALA:'alpha3linoleic'acid;'AUC:'area'under'curve;'ch:'cholesterol;'CCL:'chemokine'(C3C'Motif)' ligand;'CLS:'crown3like'structures;'CRP:'C'reactive'protein;'d:'day;'DHA:'docosahexaenoic'acid;'EE:'ethyl'ester;'EPA:'eicosapentaenoic'acid;'FBG:'fasting'blood'glucose;'FBI:' fasting' blood' insulin;' FFA:' free' fatty' acids;' HbA1c:' glycosylated' hemoglobin;' HIF1A:' hypoxia3inducible' factor' 13alpha;' HDHA:' hydroxydocosahexaenoic' acid;' HDL:' high' density' lipoprotein;' HEPE:' hydroxyeicosapentaenoic' acid;' HETE:' hydroxyeicosatetraenoic' acid;' HOMA3IR:' homeostatic' model' assessment' of' insulin' resistance;' ICAM:' intercellular'adhesion'molecule;'IL:'interleukin;'LDL:'low'density'lipoprotein;'MCP31'(also'CCL2):'monocyte'chemoattractant'protein'1;'MetS:'metabolic'syndrome;'MMP:' matrix'metalloproteinases;'MRC1:'mannose'receptor'C'type'1;'NO:'nitric'oxide;'n33'PUFAs:'omega33'polyunsaturated'fatty'acids;'PAI31:'plasminogen'activator'inhibitor'1;' PD1:'protectin'D1;'P3sel:'p3selectin;'Rv:'resolvin;'SAA:'serum'amyloid'A;'SAT:'subcutaneous'adipose'tissue;'sdLDL:'small'dense'LDL;'TG:'triglycerides;'TNF:'tumor'necrosis' factor;' TNFR:' tumor' necrosis' factor' receptor;' TXB2:' thromboxane' B2;' VAT:' visceral' adipose' tissue;' VCAM:' vascular' cell' adhesion' molecule;' VLDL:' very' low' density' lipoprotein.' '

'

'

30" I.#INTRODUCTION#

3.3.#N%3#PUFAs:#role#in#inflammation#

N"3$ PUFAs$ are$ considered$ as$ anti"inflammatory$ compounds.$ According$ with$ the$ available$ evidence,$they$could$exert$its$anti"inflammatory$properties$through$different$mechanism:$$

First,$by$binding$to$peroxisome$proliferator"activated$receptor$gamma$(PPARγ),$EPA$and$DHA$ decrease$ activation$ of$ TLR4,$ which$ is$ known$ to$ participate$ in$ pathogen$ recognition$ and$ innate$ immune$system,$blocking$IκB$phosphorylation$and$leading$to$inhibition$of$NF"κB$activity.$Other$ proposed$mechanism$by$which$marine$n"3$PUFAs$inhibit$NF"κB$activation$is$through$decreasing$ the$cell$membrane$saturated$fatty$acid$content,$driving$to$TLR4$inhibition$and$thereby$preventing$ NF"κB$activation$(Flock$et#al.,$2013).$$

Second,$it$is$known$that$the$n"3$PUFAs$anti"inflammatory$properties$are$in$part$mediated$by$ their$ incorporation$ in$ phospholipids$ cell$ membrane.$ Generally,$ cell$ membrane$ contains$ higher$ amounts$of$omega"6$(n"6)$PUFAs$(linoleic$acid$and$arachidonic$acid$/AA)$than$n"3$PUFAs$(Flock$et# al.,$2013).$Moreover$both$the$content$of$n"3$PUFAs$and$the$ratio$n"6:n"3$could$be$modulated$by$ the$diet.$In$this$sense,$the$consumption$ratio$n"6:n"3$in$a$typical$Western$diet$is$about$10:1$to$ 20:1,$while$in$populations$with$high$intake$of$fish$this$ratio$could$be$around$4:1$(Kalupahana$et# al.,$ 2011).$ Several$ studies$ have$ confirmed$ that$ supplementation$ with$ n"3$ PUFAs$ could$ be$ accumulated$into$phospholipid$cell$membrane,$favoring$a$decrease$in$the$ratio$n"6:n"3$(Itariu$et# al.,$ 2012;$ Phang$ et# al.,$ 2013;$ Satoh"Asahara$ et# al.,$ 2012).$ Both$ n"3$ PUFAs$ and$ n"6$ PUFAs$ are$ sources$for$the$eicosanoid$synthesis,$which$are$compounds$involved$in$the$inflammatory$process$ (Serhan$et#al.,$2015b).$The$AA"derived$eicosanoids$are$generally$considered$as$pro"inflammatory,$ while$ the$ n"3$ PUFAs$ (EPA$ and$ DHA)"derived$ eicosanoids$ are$ recognized$ as$ anti"inflammatory$ (Lorente"Cebrián$et#al.,$2013).$In$this$sense$the$n"3$PUFAs,$by$competing$for$the$same$enzymes$ (cyclooxygenase$(COX)$and$lipoxygenase$(LOX))$with$n"6$PUFAs,$could$decrease$the$formation$of$ pro"inflammatory$eicosanoids$(Cottin$et#al.,$2011;$Lorente"Cebrián$et#al.,$2013;$Titos$and$Claria,$ 2013)$(Figure#2).$$

Third,$through$the$production$of$the$commonly$called$specialized$pro"resolving$lipid$mediators$ (SPMs),$maresins,$protectins$and$resolvins,$the$n"3$PUFAs$actively$participate$in$the$resolution$of$ inflammation,$which$in$turn$could$decrease$the$low"grade$chronic$pro"inflammatory$environment$ characterizing$obesity$(Lorente"Cebrián$et#al.,$2013;$Martínez"Fernández$et#al.,$2015;$Spite$et#al.,$ 2014).$Additionally,$it$has$been$suggested$that$the$supplementation$with$n"3$PUFAs$enhance$the$ polarization$of$macrophages$to$the$anti"inflammatory$M2$macrophages$phenotype$(Wang$et#al.,$ 2015)$ by$ different$ pathways,$ including$ both$ the$ production$ of$ SPMs,$ as$ resolvins$ (Spite$ et# al.,$

31# I.#INTRODUCTION#

2014);$and$by$stimulation$of$adiponectin$production,$a$molecule$with$insulin$sensitizing$actions,$ through$PPARγ$activation$(Flock$et#al.,$2013).$$$

$

$

#

#

#

#

#

$

$ Figure#2.#Endogenous$biosynthesis$of$omega"6$and$omega"3$derived$eicosanoids.$Figure$from$Titos$and$ Claria$(2013).#

3.3.1#Inflammation#and#n<3#PUFAs:#evidence#from#human#studies#

Since$the$finding$that$marine$n"3$PUFAs$exert$anti"inflammatory$properties,$a$large$number$of$ studies$have$assessed$their$effects$on$several$inflammatory$markers$(Table#4).$Some$of$the$more$ relevant$ inflammatory$ proteins$ analyzed$ in$ the$ different$ studies$ are$ shown$ in$ Table# 5.$ It$ is$ important$to$note$that$the$effects$of$EPA$or$DHA$separately$have$not$been$widely$studied,$by$the$ fact$ that$ the$ majority$ of$ reported$ trials$ have$ used$ supplements$ including$ both$ EPA$ and$ DHA$ (Myhrstad$et#al.,$2011;$Robinson$and$Mazurak,$2013).$

In$ order$ to$ evaluate$ the$ mechanisms$ by$ which$ the$ n"3$ PUFAs$ could$ modulate$ the$ inflammatory$ status,$ some$ studies$ have$ also$ evaluated$ the$ effects$ of$ marine$ n"3$ PUFAs$ supplementation$on$the$expression$of$inflammatory$markers$(including$gene$expression$profile$by$ microarray$ approach)$ in$ peripheral$ blood$ mononuclear$ cells$ (PBMC)$ and/or$ adipose$ tissue$ (Bouwens$et#al.,$2009;$Itariu$et#al.,$2012;$Rudkowska$et#al.,$2013a,$2013b;$Satoh"Asahara$et#al.,$ 2012;$Tsunoda$et#al.,$2015).$$ $ $

32# I.#INTRODUCTION#

Table#5.#Inflammatory$markers$commonly$evaluated$in$n"3$PUFAs$studies# Protein# Symbol# Function# Acute&phase&reactants& Acute$phase$protein,$which$increase$with$ C$reactive$protein$ CRP$ inflammatory$processes$ Adhesion&Molecules& Soluble$intercellular$adhesion$ sICAM"1$ molecule"1$ Cell$adhesion$molecules,$which$bind$ Soluble$vascular$cell$adhesion$ monocytes$and$lymphocytes$to$endothelial$ sVCAM"1$ molecule"1$ cells.$Play$a$role$in$the$development$of$ sE"$selectin$ sE"sel$ arterial$plaque.$ sP"selectin$ sP"sel$ Cytokines& Major$mediators$of$inflammatory$response$ and$considered$as$pro"inflammatory$ Interleukin"1α,$interleukin"1β$ IL"1α,$IL"1β$ cytokines.$Involved$in$various$immune$ responses$and$inflammatory$processes$ It$is$a$potent$inducer$of$the$acute$phase$ Interleukin"6$ IL"6$ response.$Involved$in$lymphocyte$and$ monocyte$differentiation.$$ Pro"inflammatory$cytokine.$Stimulates$ Interleukin"18$ IL"18$ interferon$gamma$production$in$T"helper$ type$I$cells.$ Anti"inflammatory$cytokine,$which$is$mainly$ produced$by$monocytes$and$inhibits$the$ Interleukin"10$ IL"10$ synthesis$of$a$number$cytokines,$including$ IFN"gamma,$IL"2,$IL"3,$TNF"α$and$GM"CSF.$

Granulocyte$macrophage$ Controls$the$production,$differentiation$and$ GM"CSF$ colony$stimulating$factor$ function$of$granulocytes$and$macrophages.$ Pro"inflammatory$cytokine,$which$is$involved$ Tumor$necrosis$factor"α$ TNF"α$ inflammatory$and$immune$responses$and$is$ mainly$secreted$by$macrophages$ Chemokines# Attracts$neutrophils,$basophils$and$T"cells.$It$ Interleukin"8$ IL"8$ is$released$from$several$cell$types$in$ response$to$an$inflammatory$stimulus.$ Attracts$monocytes$and$basophils.$May$be$ Monocyte$chemoattractant$ MCP"1$ involved$in$the$recruitment$of$monocytes$ protein"1$ into$arterial$wall$during$atherosclerosis.$ Adipokines& Anti"atherogenic$and$anti"inflammatory$ Adiponectin$ Adiponectin$ activities.$It$is$involved$in$the$control$of$fat$ metabolism$and$insulin$sensitivity.$ Plays$a$role$in$the$regulation$of$body$weight,$ Leptin$ Leptin$ inhibiting$food$intake$and$regulating$energy$ expenditure.$ Source:$Catalog$from$Universal$Protein$Resource$(UniProt).$Available$online$(www.uniprot.org)$

33# I.#INTRODUCTION#

Though$observational$trials$have$spotted$an$inverse$relationship$between$both$plasma$levels$ of$ n"3$ PUFAs$ and$ high$ intake$ of$ n"3$ PUFAs,$ with$ plasma$ levels$ of$ circulating$ inflammatory$ markers,$the$evidence$derived$from$the$randomized$controlled$trials$is$not$consistent$(Robinson$ and$Mazurak,$2013).$According$with$the$systematic$reviews$from$Robinson$and$Mazurak$(2013),$ which$evaluated$studies$until$2012,$and$Myhrstad$et#al.$(2011),$which$followed$studies$published$ up$ to$ 2009,$ there$ are$ not$ a$ clear$ effect$ of$ n"3$ PUFAs$ in$ the$ different$ cytokines,$ acute$ phase$ proteins,$ adhesion$ molecules$ and$ chemokines$ measured.$ Also,$ these$ authors$ mentioned$ the$ difficulty$to$compare$the$different$studies$by$the$fact$of$the$multiple$ratios$used$for$EPA:DHA,$the$ characteristics$of$the$population$included,$the$duration,$the$doses,$health$status,$etc.$$

Additionally,$although$Kelley$and$Adkins$(2012),$described$the$same$inconsistencies$about$the$ effects$of$marine$n"3$PUFAs$in$circulating$blood$inflammatory$markers,$they$also$highlight$that$ contrary$to$the$findings$in#vitro#in$human$endothelial$cells,$monocytes$and$lymphocytes,$in$human$ intervention$studies,$EPA$may$be$more$effective$than$DHA$in$decreasing$the$circulating$levels$of$ adhesion$molecules.$Accordingly,$the$study$from$Yamada$et#al.$(2008),$which$is$part$of$the$Japan$ Obesity$and$Metabolic$Syndrome$Study,$revealed$that$EPA$supplementation$(1.8$g/day$during$3$ months)$promoted$a$decrease$of$soluble$intracellular$adhesion$molecule$1$(sICAM"1)$and$soluble$ vascular$cell$adhesion$molecule$1$(sVCAM"1)$in$subjects$with$MetS.$Furthermore,$the$study$from$ Phang$ et# al.$ (2013),$ using$ a$ supplementation$ of$ EPA$ and$ DHA$ in$ healthy$ normal/overweight$ participants,$ separately$ in$ a$ dose$ of$ 2$ g/day$ during$ 4$ weeks$ showed$ a$ gender*treatment$ interaction$ in$ the$ platelet$ aggregation$ outcome,$ concluding$ that$ men$ could$ benefit$ more$ with$ EPA$treatment$whereas$women$could$have$more$benefits$with$DHA$supplementation.$

Moreover,$it$has$been$suggested$that$the$beneficial$effects$of$n"3$PUFAs,$at$least$in$part,$could$ be$mediated$by$the$increment$of$either$anti"inflammatory$cytokines,$as$IL"10$and$adiponectin,$or$ SPMs,$ which$ could$ favor$ the$ resolution$ of$ the$ low"grade$ chronic$ inflammation$ associated$ with$ obesity.$IL"10$is$an$anti"inflammatory$cytokine$expressed$mainly$by$adipose$tissue$macrophages$ (González"Périz$ and$ Clària,$ 2010),$ whose$ decreased$ production$ has$ been$ associated$ with$ increased$odds$ratio$for$T2DM$and$major$predisposition$to$MetS$(van$Exel$et#al.,$2002).$In$this$ context,$Satoh"Asahara$et#al.$(2012),$observed$in$patients$with$dyslipidemia$that$supplementation$ with$1.8$g/day$of$highly$purified$EPA$promoted$an$increase$in$serum$levels$of$adiponectin$and$IL" 10,$as$well$as$IL10$mRNA$levels$in$PBMC,$without$appreciable$changes$in$the$expression$of$other$ pro"inflammatory$genes$(TNFα,$IL6$and$CD163).$$

Another$molecule$of$interest$that$could$help$to$elucidate$the$mechanism$by$which$n"3$PUFAs$ improve$ cardiovascular$ health$ and$ insulin$ sensitivity$ is$ adiponectin$ (Gray$ et# al.,$ 2013).$ Adiponectin$is$an$adipokine$with$important$insulin"sensitizing$and$anti"inflammatory$properties$

34# I.#INTRODUCTION#

(Stern$et#al.,$2016).$Prospective$cohort$studies$have$found$that$higher$blood$concentrations$of$ adiponectin$are$associated$with$a$lower$risk$of$T2DM$and$coronary$heart$disease$(Davis$et#al.,$ 2015;$ Koenig$ et# al.,$ 2006;$ Li$ et# al.,$ 2009;$ Schulze$ et# al.,$ 2005).$ In$ this$ context,$ two$ published$ systematic$ reviews$ have$ observed$ that$ n"3$ PUFAs$ supplementation$ promotes$ a$ moderate$ but$ statistically$ significant$ increase$ in$ blood$ adiponectin$ circulating$ levels$ (von$ Frankenberg$ et# al.,$ 2014;$Wu$et#al.,$2013).$In$this$sense,$whereas$Wu$et#al.$(2013)$reported$that$the$effects$of$n"3$ PUFAs$ on$ adiponectin$ could$ be$ stronger$ in$ younger$ or$ obese$ persons,$ the$ study$ from$ von$ Frankenberg$et#al.$(2014)$mentioned$that$the$type$of$placebo$oil$could$affect$the$increment$of$ adiponectin$by$n"3$PUFAs,$finding$that$it$was$greater$when$paraffin$oil$was$used$as$placebo$as$ compared$with$other$vegetable$oils$(e.g.$sunflower$oil,$soy$oil,$olive$oil).$Neither$of$the$systematic$ reviews$mentioned$differential$effects$between$EPA$and$DHA$in$circulating$levels$of$adiponectin.$$$

Though,$ adipose$ tissue$ is$ considered$ as$ a$ target$ organ$ for$ n"3$ PUFA$ and$ their$ effects$ on$ adipose$tissue$gene$expression$have$been$widely$studied$in$animal$models$(Martínez"Fernández$ et#al.,$2015;$Moreno"Aliaga$et#al.,$2010;$Pérez"Echarri$et#al.,$2008;$Pérez"Matute$et#al.,$2007),$in$ humans$ the$ information$ available$ is$ limited,$ perhaps$ because$ the$ problems$ arising$ to$ obtain$ adipose$tissue$samples.$In$this$sense,$Itariu$et#al.$(2012),$reported$in$severely$obese$non"diabetic$ subjects$that$supplementation$with$marine$n"3$PUFAs$(4$g/day$providing$460$mg$EPA$+$380$mg$ DHA$ per$ gram)$ during$ 8$ weeks$ was$ able$ not$ only$ to$ decrease$ expression$ of$ pro"inflammatory$ (CCL2,$CCL3,$IL6,$hypoxia%inducible#factor#1α$(HIF1A),$transforming#growth#factor#beta#1$(TGFB1))$ and$ macrophage$ marker$ (CD40)$ genes,$ but$ also$ to$ increase$ adiponectin$ gene$ expression$ (ADIPOQ)$in$subcutaneous$adipose$tissue,$this$without$significantly$modify$some$of$the$circulating$ inflammatory$ markers$ measured$ (TNF"α,$ CRP,$ leukocytes,$ adiponectin$ and$ leptin).$ Moreover,$ they$observed$greater$amounts$of$some$of$the$SPMs$measured$(resolvin$E1$and$D1,$17"hydroxy$ docosahexaenoic$acid$and$protectin$D1)$in$VAT$from$treated$subjects$(Table#4).$$

3.4.#N%3#PUFAs#and#nutrigenomics#in#obesity#

In$ general,$ there$ are$ not$ many$ studies$ evaluating$ the$ effects$ of$ n"3$ PUFAs$ in$ either$ obese$ subjects$or$individuals$with$MetS$features$using$microarray$approaches.$$

The$ study$ from$ Rudowska$ et# al.$ (2013a),$ performed$ in$ overweight/obese$ but$ otherwise$ healthy$ subjects,$ using$ a$ supplementation$ of$ 3$ g/day$ (1.9$ g$ EPA$ +$ 1.1$ g$ DHA)$ during$ 6$ weeks,$ observed$ that$ n"3$ PUFAs$ supplementation$ promoted$ differential$ metabolic$ effects$ between$ responders$ and$ non"responders,$ categorized$ according$ with$ the$ $ reduction$ in$ plasma$ TG$ concentrations,$finding$that$those$subjects$with$a$more$deteriorated$biochemical$parameters$at$ the$beginning$of$the$trial$had$more$favorable$changes$with$the$treatment$by$decreasing$TG$and$

35# I.#INTRODUCTION# increasing$ HDL"ch.$Also,$these$improvements$were$accompanied$by$modifications$ in$ the$ PBMC$ gene$expression$pattern$as$compared$with$the$non"responders.$Thus,$the$modified$pathways$in$ the$ responders$ group$ included$ those$ related$ with$ fatty$ acid$ oxidation$ and$ with$ hydrolysis$ and$ storage$of$TG.$In$this$sense$Schmidt$et#al.$(2012a,$2012b,$2012c),$who$studied$the$effects$on$gene$ expression$from$total$white$blood$cells$RNA$of$supplementation$with$n"3$PUFAs$(1.14$g$DHA$+$ 1.56$g$EPA$per$day)$during$12$weeks$in$normo$and$dyslipidemic$men,$found$that$the$n"3$PUFAs$ treatment$upregulated$antioxidative$genes$while$downregulated$stress"related$genes,$as$well$as$ modulated$expression$of$genes$related$to$TG$synthesis$and$clearance$and$promoted$changes$in$ immune$ response$ genes$ (including$ those$ anti"inflammatory$ and$ pro"inflammatory)$ in$ dyslipidemic$subjects.$

Moreover,$ either$ the$ gender$ or$ the$ dose$ appears$ to$ affect$ the$ n"3$ PUFAs$ effects$ on$ gene$ expression$pattern.$Thus,$other$analyses$derived$from$the$same$study$of$Rudowska$et#al.$(2013b),$ described$ above,$ revealed$ that$ although$ the$ n"3$ PUFAs$ did$ not$ induce$ changes$ in$ plasma$ inflammatory$ markers,$ it$ promoted$ in$ PBMC$ a$ down"regulation$ of$ the$ NF"κB$ inflammatory$ pathway$related"genes$only$in$men;$moreover,$the$effects$of$supplementation$in$others$pathways$ related$with$oxidative$stress$protection$(Nrf2)$and$hypoxia$inducible$factor$(HIF)$were$significantly$ and$ differentially$ affected$ by$ the$ gender.$ Furthermore,$ Bouwens$ et# al.$ (2009),$ evaluating$ the$ effects$of$n"3$PUFAs$at$two$different$doses$by$a$microarray$approach,$observed$that$the$highest$ dose$of$EPA$+$DHA$$(1.8$g/day)$during$26$weeks$decreased$the$expression$of$genes$involved$in$ inflammatory$pathways,$atherosclerotic$plaque$formation$and$angiogenesis$in$PBMC$from$healthy$ elderly$(>65$years$old)$subjects.$

Although$it$has$been$proposed$that$EPA$and$DHA$may$have$different$metabolic$effects,$studies$ evaluating$either$the$differences$or$using$these$supplements$separately$are$not$sufficient.$In$this$ context,$ a$ recent$ study$ from$ Tsunoda$ et# al.$ (2015),$ assessed$ the$ differential$ effects$ of$ supplementation$with$EPA$and$DHA$at$a$dose$of$1.8$g/day$during$6$weeks$in$subjects$with$mild$ elevation$ in$ plasma$ lipoprotein"associated$ phospholipase$ A2$ (an$ enzyme$ involved$ in$ the$ development$of$atherosclerosis),$finding$in$PBMC$that$both$n"3$PUFAs$promoted$positive$changes$ in$ inflammatory$ pathways,$ but$ the$ genes$ significantly$ modified$ were$ not$ the$ same.$ Moreover,$ their$data$also$suggest$that$EPA$could$have$greater$anti"inflammatory$effects$than$DHA$based$on$ the$genes$and$pathways$significantly$affected.$$

3.5.#Summary#

Most$ of$ the$ studies$ evaluating$ the$ effects$ of$ n"3$ PUFAs$ in$ body$ weight,$ glucose$ and$ lipid$ metabolism$as$well$as$in$inflammation,$have$been$performed$using$a$combination$of$EPA$+$DHA.$

36# I.#INTRODUCTION#

Moreover,$the$studies$suggest$that$the$metabolic$status$at$the$beginning$of$the$supplementation$ with$n"3$PUFAs$are$influencing$significantly$the$outcomes$observed.$The$main$effect$of$n"3$PUFAs$ appears$ to$ be$ related$ with$ the$ improvements$ in$ triglyceride$ metabolism.$ Regarding$ glucose$ metabolism,$the$outcomes$are$not$consistent$and$though$some$studies$have$suggested$a$slight$ increase$in$fasting$glucose,$this$increment$may$have$not$negative$effects$in$persons$with$glucose$ metabolic$impairments.$Finally,$some$of$the$benefits$of$n"3$PUFAs$have$been$associated$with$the$ anti"inflammatory$properties$of$these$compounds,$however$the$evidence$in$human$trials$is$not$ consistent$and$perhaps$the$study$either$in$specific$blood$fractions$or$in$adipose$tissue$expression$ could$help$to$elucidate$if$the$supplementation$with$n"3$PUFAs$could$exert$some$benefits$in$the$ inflammatory$profile$of$obese$subjects.$$

4.#Alpha

Alpha"lipoic$ acid$ (thioctic$ acid,$ 5"(1,2"dithiolan"3"yl)$ pentanoic$ acid)$ is$ an$ organosulfur$ compound,$which$contains$a$single$chiral$center$and$asymmetric$carbon,$resulting$in$two$possible$ optical$isomers:$R"lipoic$acid$and$S"lipoic$acid$(Fernández"Galilea$et#al.,$2013).$$Although$in$small$ quantities,$α"lipoic$acid$can$be$found$in$different$food$sources$such$as$vegetables$(e.g.$spinach,$ tomato,$broccoli,$brussel$sprout$and$rice$bran),$red$meat$and$entrails$(e.g.$liver$and$kidney)$in$ form$ of$ lipoyllysine$ (lipoic$ acid$ with$ binding$ lysine$ residues),$ being$ 2.6$ mg/g$ dry$ weight,$ the$ highest$ concentration$ of$ lipoyllysine$ detected$ in$ beef$ kidney$ (Fernández"Galilea$ et# al.,$ 2013;$ Ghibu$et#al.,$2009).$$

Though$ the$ R"α"lipoic$ acid$ isomer$ is$ the$ naturally$ found$ in$ food,$ when$ synthesized$ in$ a$ laboratory$ α"lipoic$ acid$ is$ normally$ a$ racemic$ mixture$ of$ the$ R"$ and$ S"$ forms.$ Thereby$ for$ supplementation$therapy$the$racemic$α"lipoic$acid$is$usually$used$(Fernández"Galilea$et#al.,$2013).$ Moreover,$the$safety$and$toxicity$of$α–lipoic$acid$have$been$widely$tested,$being$in$humans$the$ greatest$dose$administered$2.4$g/day$during$a$2$years$period$(Shay$et#al.,$2009)$with$no$reported$ adverse$effects.$$

α"Lipoic$ acid$ can$ be$ also$ synthesized$ endogenously$ by$ the$ lipoic$ acid$ synthase$ (LASY)$ (Morikawa$ et# al.,$ 2001).$ Interestingly,$ it$ has$ been$ observed$ in$ murine$ models$ of$ obesity$ and$ T2DM$that$LASY$gene$expression$is$decreased$in$both$muscle$and$adipose$tissues,$which$results$in$ a$ reduction$ of$ α"lipoic$ acid$ content$ (Padmalayam$ et# al.,$ 2009).$ Moreover,$in# vitro,$ it$ has$ been$ observed$that$LASY$inhibition$is$able$to$induce$not$only$a$reduction$of$enzymes$involved$in$the$ antioxidant$defense,$such$as$glutathione$or$superoxide$dismutase,$but$also$a$decrease$in$glucose$ uptake$ and$ mitochondrial$ function$ in$ a$ human$ skeletal$ muscle$ cell$ line,$ and$ to$ enhance$ pro" inflammatory$ signals$ in$ human$ microvascular$ endothelial$ cells$ (Padmalayam$ et# al.,$ 2009).$

37# I.#INTRODUCTION#

Additionally,$several$studies$have$demonstrated$anti"oxidant$properties$for$α"lipoic$acid,$which$is$ able$ to$ counteract$ the$ production$ of$ ROS$ through$ either$ stimulating$ anti"oxidant$ defenses$ (including$ glutathione,$ superoxide$ dismutase,$ catalase)$ or$ enhancing$ the$ function$ of$ other$ antioxidants,$such$vitamins$as$C$and$E$(Fernández"Galilea$et#al.,$2013;$Padmalayam$et#al.,$2009).$ In$ this$ sense,$ since$ the$ first$ observations$ that$ patients$ with$ liver$ cirrhosis,$ T2DM$ and$ polyneuropathy$had$lower$levels$of$lipoic$acid$(Biewenga$et#al.,$1997),$supplementation$with$this$ compound$ has$ been$ proposed$ to$ improve$ pathological$ states$ defined$ by$ the$ increment$ of$ oxidative$ stress$ and$ inflammation,$ such$ as$ T2DM,$ rheumatoid$ arthritis,$ vascular$ disease$ or$ asthma$(Fernández"Galilea$et#al.,$2013;$Moura$et#al.,$2015).$$

Moreover,$ several$ studies$ conducted$ in$ different$ experimental$ models$ of$ obesity$ have$ suggested$that$α"lipoic$acid$is$able$to$promote$body$weight$loss$through$different$mechanisms$ including$not$only$the$decrease$of$food$intake$(Kim$et#al.,$2004)$and$the$inhibition$of$intestinal$ sugar$absorption$(Prieto"Hontoria$et#al.,$2009),$but$also$the$diminution$of$food$efficiency$(Prieto" Hontoria$et#al.,$2009)$and$the$stimulation$of$energy$expenditure$(Kim$et#al.,$2004;$Wang$et#al.,$ 2010).$$

In$addition,$recent$studies$from$our$group$have$reported$that$treatment$with$α"lipoic$acid$in$ adipocytes$ from$ overweight/obese$ subjects$ inhibited$ de$ novo$ lipogenesis$ and$ decreased$ triglyceride$ accumulation$ through$ the$ down"regulation$ of$ key$ lipogenic$ enzymes$ (Fernández" Galilea$ et# al.,$ 2014),$ and$ increased$ fatty$ acid$ oxidation$ enzymes,$ mitochondrial$ biogenesis$ and$ brown"like$adipocyte$markers$(Fernández"Galilea$et#al.,$2015).$$

Furthermore,$different$trials$have$demonstrated$the$ability$of$α"lipoic$ acid$ to$ modulate$ the$ secretion$of$some$adipokines$involved$in$inflammation$and$metabolism.$In$this$regard,$it$has$been$ observed$that$the$α"lipoic$acid$treatment$could$stimulate$not$only$adiponectin$in$a$murine$model$ of$obesity$(Prieto"Hontoria$et#al.,$2013)$but$also$apelin,$which$is$another$adipokine$involved$in$ fatty$acid$oxidation$and$mitochondrial$function$(Fernández"Galilea$et#al.,$2011),$while$inhibits$the$ production$of$leptin$and$chemerin$(Prieto"Hontoria$et#al.,$2011,$2016)$in$3T3"L1$adipocytes.$

4.1.#α%Lipoic#acid#and#weight#loss#in#humans#

In$humans,$most$of$the$trials$evaluating$the$potential$anti"obesity$effects$of$α"lipoic$acid$have$ been$ performed$ in$ subjects$ with$ either$ T2DM$ or$ with$ impaired$ glucose$ tolerance$ (Table# 6).$ Moreover,$because$in$most$of$them$the$diet$followed$is$not$specified,$it$is$assumed$that$they$have$ been$conducted$with$the$usual$diet.$To$our$knowledge$there$is$only$one$study$that$has$evaluated$ the$ additional$effects$of$α"lipoic$acid$to$a$hypocaloric$diet$on$anthropometric$variables.$ In$ this$ sense,$ the$ study$ from$ Koh$ et# al.$ (2011),$ performed$ in$ subjects$ with$ some$ features$ of$ MetS$

38# I.#INTRODUCTION# following$an$energy"restricted$diet,$observed$that$supplementation$with$α"lipoic$acid$(1.2$and$1.8$ g/day)$during$20$weeks$promoted$a$reduction$in$BMI,$body$weight$and$waist$circumference,$but$ only$was$significantly$different$as$compared$with$the$placebo$group$at$the$highest$dose$tested.$$ Similarly,$the$trial$of$Carbonelli$et#al.$(2010),$which$evaluated$the$effects$of$0.8$g/day$α"lipoic$acid$ orally$ during$ a$ 4"months$ period$ in$ a$ large$ population,$ found$ that$ supplementation$ decreased$ body$ weight,$ fat$ mass$ and$ waist$ circumference$ in$ overweight$ and$ obese$ subjects,$ but$ not$ in$ normal$ weight$ individuals.$ Interestingly,$ other$ recent$ study$ performed$ in$ subjects$ with$ T2DM$ using$a$lower$oral$dose$of$α"lipoic$acid$(0.6$g/day)$during$20$weeks$reported$a$decreased$body$ weight$in$the$supplemented$group$(Okanović$et#al.,$2015).$

Opposite$to$these$findings,$other$studies$with$a$range$duration$between$8$weeks$to$1$year,$ performed$ in$ overweight/obese$ subjects$ with$ T2DM$ or$ impaired$ glucose$ tolerance$ following$ a$ usual$diet$have$observed$no$statistically$significant$effects$of$α"lipoic$acid$oral$supplementation$ (0.3$–$1$g/day)$in$body$weight$(Ansar$et#al.,$2011;$Manning$et#al.,$2012;$McNeilly$et#al.,$2011)$and$ fat$mass,$as$well$as$in$hip$and$waist$circumferences$(McNeilly$et#al.,$2011).$$Analogous$outcomes$ in$ anthropometric$ variables$ have$ been$ found$ with$ 0.6$ g/day$ of$ α"lipoic"acid$ administered$ i.v.$ during$2$weeks$(Zhang$et#al.,$2011).$$

$

39# I.#INTRODUCTION#

Table#6.#Clinical'trials'evaluating'the'effects'of'α2lipoic'acid'supplementation'on'body'weight'loss'and'anthropometric'parameters,'as'well'as'on'glucose'and' lipid'metabolism'and'inflammatory'and'oxidative'stress'markers# References# Study#design# α

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'

40# I.#INTRODUCTION#

Table#6.#Continuation# References# Study#design# α

41# I.#INTRODUCTION#

Table#6.#Continuation# References# Study#design# α

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42# I.#INTRODUCTION#

4.2.$α%Lipoic$acid:$effects$on$glucose$and$lipid$metabolism$

4.2.1.#Glucose#metabolism#

The$effects$of$α*lipoic$acid$in$the$treatment$for$diabetic$polyneuropathy$have$been$extensively$ studied.$In$this$context,$the$meta*analysis$conducted$by$Ziegler$et$al.$(2004)$taking$into$account$ those$studies$published$until$2003,$revealed$that$α*lipoic$acid$at$a$dose$of$0.6$g/day$intravenously$ (i.v.)$ for$ 3$ weeks$ significantly$ improved$ the$ symptoms$ of$ diabetic$ neuropathy$ to$ a$ clinically$ meaningful$ degree.$ Similarly,$ the$ meta*analysis$ from$ Han$ et$ al.$ (2012),$ which$ included$ studies$ published$until$2009,$concluded$that$the$treatment$with$α*lipoic$acid$(0.3*0.6$g/day$i.v.$for$2*4$ weeks)$is$capable$to$ameliorate$neuropathic$symptoms$without$any$adverse$effect.$In$this$sense,$ Ziegler$et$al.$(2006),$that$evaluated$the$effects$of$α*lipoic$acid$at$different$oral$doses$ranged$0.6$* 1.8$ g/$ day$ during$ 5$ weeks,$ observed$ that$ similarly$ to$ the$ effects$ observed$ intravenously,$ supplementation$with$α*lipoic$acid$was$able$to$decrease$total$symptoms$score$as$well$as$stabbing$ pain$and$burning$pain,$being$0.6$g/day$the$most$appropriate$dose$because$a$greater$dose$caused$ in$some$patients$gastrointestinal$side$effects.$$

It$ is$important$to$remark$that$although$appears$to$be$a$clear$effect$of$ α*lipoic$ acid$ in$ the$ amelioration$ of$ diabetic$ polyneuropathy$ symptoms,$ it$ is$ not$ necessarily$ accompanied$ by$ significant$ improvements$ in$ other$ glucose$ metabolic$ parameters$ such$ as$ fasting$ glucose$ and$ insulin,$as$well$as$HOMA*IR$and$HbA1c.$In$this$sense,$according$with$the$information$available,$all$ in$subjects$with$impairments$in$glucose$metabolism,$the$major$part$of$the$trials$have$shown$no* effects$of$α*lipoic$acid$administered$either$orally$or$intravenously$in$glucose$fasting$parameters$ (Kamenova,$2006;$Manning$et$al.,$2012;$McNeilly$et$al.,$2011;$Zhang$et$al.,$2011),$only$the$study$ of$Ansar$et$al.$(2011),$reported$that$supplementation$with$α*lipoic$acid$promoted$a$reduction$in$ fasting$blood$glucose$and$HOMA*IR.$Importantly,$the$studies$from$Kamenova$(2006)$and$Zhang$et$ al.$ (2011),$ which$ evaluated$ insulin$ sensitivity$ by$ the$ hyperinsulinaemic$ euglycaemic$ clamp$ technique,$found$that$supplementation$with$α*lipoic$acid$is$able$to$improve$both$glucose$disposal$ rate$and$insulin$sensitivity$index$(ISI)$in$type$2$diabetic$subjects.$However,$whether$α*lipoic$acid$ could$ improve$ glucose$ metabolism$ in$ overweight/obese$ healthy$ individuals$ is$ still$ an$ open$ question.$$

4.2.2.#Lipid#metabolism#

There$is$limited$information$about$the$effects$of$α*lipoic$acid$on$lipid$metabolism.$While$some$ studies$ have$ reported$ that$ α*lipoic$ acid$ could$ decrease$ not$ only$ FFA$ and$ TG$ (Okanović$ et$ al.,$ 2015;$Zhang$et$al.,$2011)$levels,$but$also$the$total*ch$and$LDL*ch$(Zhang$et$al.,$2011),$other$studies$

43# I.#INTRODUCTION# have$reported$no$effect$of$this$antioxidant$in$TG$and$cholesterol$levels$(Koh$et$al.,$2011;$Manning$ et$al.,$2012;$McNeilly$et$al.,$2011).$Contrary$to$the$expectation,$McNeilly$et$al.$(2011),$observed$a$ greater$LDL$oxidation$rate$in$subjects$with$impaired$glucose$tolerance$supplemented$with$α*lipoic$ acid$alone,$suggesting$that$it$could$favor$the$development$of$CVD$(Table#6).$$

4.3.$α%Lipoic$acid$and$inflammation$

Although$the$alleviation$of$inflammation$and$oxidative$stress$are$the$proposed$mechanisms$by$ which$ α*lipoic$ acid$ appears$ to$ alleviate$ symptoms$ of$ diabetic$ polyneuropathy$ and$ other$ inflammatory$conditions$such$as$multiple$sclerosis$(Khalili$et$al.,$2014;$Rochette$et$al.,$2013),$in$ human$trials$the$modulation$by$α*lipoic$acid$of$both$the$inflammatory$status$and$oxidative$stress$ in$obesity$have$not$been$widely$described$ (Table# 6).$All$these$studies$have$been$conducted$in$ subjects$following$their$usual$diet.$

In$this$context$the$study$of$Carbonelli$et$al.$(2010)$showed$that$supplementation$with$α*lipoic$ acid$decreased$the$erythrocyte$sedimentation$rate,$TNF*α$and$IL*6$circulating$levels$in$overweight$ and$obese$subjects$while$promoted$a$reduction$of$CRP$only$in$the$obese$group.$Moreover,$other$ study$ performed$ in$ overweight/obese$ healthy$ individuals$ with$ a$ randomized,$ controlled$ cross* over$design$with$a$4*week$wash*out$period$between$the$8*week$intervention,$observed$that$oral$ supplementation$with$1.2$g/day$of$α*lipoic$acid$did$not$promote$significant$changes$in$oxidized* LDL$and$8*iso*prostaglandin$F2α$blood$circulating$levels$(Yan$et$al.,$2013).$$

Among$ the$ studies$ performed$ in$ overweight/obese$ type$ 2$ diabetic$ individuals$ that$ have$ analyzed$either$inflammation$or$oxidative$stress,$only$the$trial$of$Zhang$et$al.$(2011),$which$used$ supplementation$with$0.6$g/day$intravenously$of$α*lipoic$acid$during$2$weeks,$observed$a$drop$in$ TNF*α,$IL*6$and$oxidative$markers$(8*iso*protaglandin$and$malondialdehyde)$in$parallel$with$an$ increase$ in$ adiponectin$ levels$ in$ the$ treated$ group.$ Contrary$ to$ these$ findings,$ the$ study$ of$ Manning$et$al.$(2012)$showed$that$oral$supplementation$with$α*lipoic$acid$(0.6$g/day)$during$one$ year$did$not$promote$any$change$on$any$of$the$inflammatory$markers$measured$(CRP,$IL*6,$TNF*α$ and$ adiponectin).$ Additionally,$ the$ study$ from$ McNeilly$ et$ al.$ (2011),$ observed$ that$ though$ supplementation$with$α*lipoic$acid$(1$g/day$during$12$weeks)$did$not$induce$changes$in$CRP,$it$ increased$ the$ total$ antioxidant$ capacity.$ Finally,$ the$ study$ from$ Ansar$ et$ al.$ (2011),$ which$ was$ performed$in$normal$weight/overweight$subjects,$observed$that$0.3$g/day$of$α*lipoic$acid$during$ 2$ months$ did$ not$ induce$ significant$ changes$ in$ glutathione$ peroxidase,$ which$ is$ an$ enzyme$ involved$in$the$antioxidant$defense.$$

44# I.#INTRODUCTION#

In$ summary,$ there$ is$ not$ clear$ evidence$ that$ α*lipoic$ acid$ could$ modulate$ circulating$ inflammatory$and$oxidative$stress$markers.$Also,$it$is$important$to$note$that$the$effects$of$the$ treatment$are$possibly$affected$not$only$by$the$baseline$metabolic$characteristics$of$the$subjects,$ but$also$by$the$route$of$administration$and$the$diet$followed$during$treatment.$In$this$sense$it$ could$be$interesting$to$analyze$the$effects$of$α*lipoic$acid$in$the$context$of$a$controlled$energy* restricted$diet.$$

4.4.$Summary$

Most$ of$ the$ trials$ evaluating$ the$ effects$ of$ α*lipoic$ acid$ in$ body$ weight,$ glucose$ and$ lipid$ metabolism$ as$ well$ as$ in$ inflammation,$ have$ been$ performed$ in$ subjects$ with$ T2DM$ following$ their$habitual$diet.$Although$initially$α*lipoic$acid$supplementation$was$proposed$as$a$treatment$ to$improve$the$symptoms$of$diabetic$polyneuropathy,$in$the$recent$years$the$anti*obesity$effects$ of$this$supplement$have$gained$attention;$however$there$are$not$enough$trials$that$have$assessed$ its$effects$in$overweight/obese$subjects$as$an$adjuvant$to$the$hypocaloric$diet.$Moreover,$though$ it$is$generally$accepted$that$the$α*lipoic$acid$is$an$anti*oxidant$compound,$the$evidence$about$its$ effects$in$inflammation$and$oxidative$stress$in$human$subjects$is$limited.$ # #

45# I.#INTRODUCTION#

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53# I.#INTRODUCTION#

long*chain$n*3$fatty$acids$included$in$an$energy*restricted$diet$on$insulin$resistance$in$overweight$and$ obese$European$young$adults.$Diabetologia$51,$1261–1268.$

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Ziegler,$ D.,$ Ametov,$ A.,$ Barinov,$ A.,$ Dyck,$ P.J.,$ Gurieva,$ I.,$ Low,$ P.A.,$ Munzel,$ U.,$ Yakhno,$ N.,$ Raz,$ I.,$ Novosadova,$ M.,$ et$ al.$ (2006).$ Oral$ treatment$ with$ alpha*lipoic$ acid$ improves$ symptomatic$ diabetic$ polyneuropathy:$the$SYDNEY$2$trial.$Diabetes$Care$29,$2365–2370.$

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57#

$

II. HYPOTHESIS#AND#AIMS#

$

#

II.#HYPOTHESIS#AND#AIMS#

1.#Justification#for#the#study#

Obesity$is$associated$with$a$wide$range$of$chronic$clinical$manifestations$such$as$hypertension,$ dyslipidemia$ and$ insulin$ resistance$ among$ other$ pathologies$ related$ with$ MetS$ (Report$ from$ WHO$ Consultation,$ 2004).$ Nowadays,$ it$ is$ accepted$ that$ apart$ of$ the$ excessive$ fat$ mass$ accumulation,$ obesity$ is$ characterized$ by$ impairments$ in$ adipocytokine$ production,$ which$ promotes$increased$secretion$of$acute$phase$reactants$and$activate$inflammatory$pathways$(Tilg$ and$ Moschen,$ 2006).$ Moreover,$ both$ the$ inflammation$ and$ the$ oxidative$ stress$ induced$ by$ obesity$ are$ linked$ phenomena$ that$ are$ affecting$ the$ normal$ insulin$ pathway$ function$ and$ are$ promoting$ an$ oxidative$ stress$ damage,$ which$ in$ turn$ could$ trigger$ CVD$ and$ insulin$ resistance$ onset$(Furukawa$et$al.,$2004;$Tinahones$et$al.,$2009).$$

For$ these$ reasons,$ the$ supplementation$ with$ either$ antioxidant$ compounds$ as$ α*lipoic$ acid$ (Fernández*Galilea$et$al.,$2013;$Prieto*Hontoria$et$al.,$2013)$or$anti*inflammatory$complexes$as$n* 3$PUFAs$(Lorente*Cebrián$et$al.,$2013;$Pérez*Echarri$et$al.,$2008)$has$been$proposed$not$only$to$ counteract$the$negative$health$effects$of$obesity$but$also$to$increase$the$amount$of$body$weight$ loss.$$

In$this$sense,$in$murine$models$of$obesity,$the$supplementation$with$α*lipoic$acid$has$been$ shown$to$effectively$reduce$weight$and$fat$mass$by$decreasing$food$intake$(Kim$et$al.,$2004)$and$ intestinal$ sugar$ transport$ (Prieto*Hontoria$ et$ al.,$ 2009),$ as$ well$ as$ by$ stimulating$ energy$ expenditure$ (Wang$ et$ al.,$ 2010).$ However,$ though$ recent$ studies$ in$ human$ adipocytes$ from$ overweight/obese$subjects$showed$that$α*lipoic$acid$treatment$was$able$both$to$decrease$the$TG$ content$ by$ inhibiting$ de$ novo$ lipogenesis$ (Fernández*Galilea$ et$ al.,$ 2014)$ and$ to$ increase$ mitochondrial$ biogenesis$ and$ fatty$ acid$ oxidation$ (Fernández*Galilea$ et$ al.,$ 2015),$ the$ trials$ evaluating$the$body*weight$lowering$effects$of$this$antioxidant$in$humans$are$insufficient,$being$ difficult$to$conclude$about$either$the$most$appropriate$dose$or$the$real$efficacy$of$α*lipoic$acid$to$ promote$body$weight$loss$in$association$to$a$hypocaloric$diet.$$

On$ the$ other$ hand,$ most$ studies$that$have$evaluated$the$anti*obesity$ effects$ of$ n*3$ PUFAs$ have$been$performed$using$a$combination$of$the$marine$derived$EPA$and$DHA$(Lorente*Cebrián$ et$al.,$2013).$Additionally$some$studies$have$pointed$out$differential$effects$of$these$n*3$PUFAs$in$ body$ weight$loss$and$biochemical$variables$(Kunesova$et$al.,$ 2006;$ Mori$ and$ Woodman,$ 2006;$ Mori$ et$ al.,$2000).$In$this$sense,$the$evaluation$of$the$effects$of$either$EPA$or$DHA$could$be$ interesting$ to$ elucidate$ their$ specific$ actions.$ In$ addition,$ the$ assessment$ about$ whether$ the$ combination$of$α*lipoic$acid$with$the$n*3$PUFA,$EPA,$could$exert$synergistic$effects$in$obesity$has$ not$been$explored$until$now.$$

# 61$ II.#HYPOTHESIS#AND#AIMS#

Furthermore,$some$studies$in$both$animal$models$of$obesity$(Deiuliis$et$al.,$2011;$Fernández* Galilea$et$al.,$2011;$Prieto*Hontoria$et$al.,$2013)$and$cultured$adipocytes$from$mice$(Fernández* Galilea$et$al.,$2011,$2012;$Prieto*Hontoria$et$al.,$2011,$2016)$and$humans$(Fernández*Galilea$et$ al.,$2014,$2015)$have$suggested$that$adipose$tissue$is$a$target$organ$for$α*lipoic$acid$anti*obesity$ actions.$However,$there$is$not$evidence$about$α*lipoic$acid$effects$in$human$adipose$tissue.$$

Moreover,$ strong$ scientific$ evidence$ has$ pointed$ out$ that$ n*3$ PUFAs$ could$ exert$ their$ anti* inflammatory$ effects$ through$ being$ accumulating$ in$ adipose$ tissue;$ thus$ modulating$ the$ production$of$both$anti*inflammatory$adipokines$and$pro*resolving$lipid$mediators$(Lopategi$et$ al.,$2016;$Martínez*Fernández$et$al.,$2015).$In$this$sense,$although$their$effects$in$adipose$tissue$ from$animal$models$have$been$widely$studied$(Martínez*Fernández$et$al.,$2015;$Moreno*Aliaga$et$ al.,$2010),$in$humans$the$information$available$is$limited$(Itariu$et$al.,$2012;$Spencer$et$al.,$2013).$ Then,$ further$ studies$ are$ needed$ to$ better$ elucidate$ the$ actions$ of$ the$ anti*inflammatory$ and$ metabolic$effects$of$n*3$PUFAs$in$adipose$tissue.$$

Finally,$some$studies$have$suggested$that$the$beneficial$metabolic$effects$of$both,$n*3$PUFAs$ and$α*lipoic$acid,$could$be$driven$by$epigenetic$mechanisms$(do$Amaral$et$al.,$2014;$Dashwood$ and$Ho,$2007;$Khaire$et$al.,$ 2015;$ Milagro$ et$al.,$ 2013).$ In$ this$ sense,$ though$ some$ trials$ have$ observed$ that$ supplementation$ with$ n*3$ PUFAs$ is$ able$ to$ modify$ methylation$ pattern$ during$ pregnancy$ and$ lactation$ (Hernando$ Boigues$ and$ Mach,$ 2015;$ Kulkarni$ et$ al.,$ 2011;$ Lee$ et$ al.,$ 2013),$ only$ few$ studies$ have$ evaluated$ whether$ the$ dietary$ supplementation$ with$ n*3$ PUFAS$ could$modulate$methylation$marks$(do$Amaral$et$al.,$2014).$Regarding$α*lipoic$acid,$though$it$has$ been$proposed$that$this$compound$could$inhibit$histone$deacetylase$activity,$making$chromatin$ more$accessible$to$DNA$repair$or$to$epigenetic$modifications$(Dashwood$and$Ho,$2007;$Suh$et$al.,$ 2004),$to$our$knowledge,$there$is$not$evidence$in$humans$about$the$effects$of$this$compound$on$ methylation$arrangement$in$obesity.$$

2.#Hypothesis#

Based$on$the$available$evidence,$we$hypothesized$that$oral$supplementation$with$α*lipoic$acid$ and/or$ EPA,$ in$ conjunction$ with$ a$ moderate$ energy*restricted$ diet,$ will$ improve$ outcomes$ in$ weight$ management,$ increasing$ not$ only$ the$ amount$ of$ weight$ loss$ but$ also$ improving$ the$ inflammatory$status$associated$with$obesity.$$

Also,$we$envisage$that$the$improvements$in$metabolic$parameters$could$be$related$in$part$to$ the$ transcriptional/epigenetic$ modulation$ of$ gene$ expression$ in$ adipose$ tissue$ and$ peripheral$ white$blood$cells.$

62# # II.#HYPOTHESIS#AND#AIMS#

3.#Objectives#

The$general#objective$of$this$research$was$to$analyze$the$efficacy$of$a$dietary$supplementation$ with$ EPA$ and/or$ α*lipoic$ acid$ in$ combination$ with$ a$ moderate$ energy*restricted$ diet$ on$ body$ weight$loss$and$on$some$metabolic$and$inflammatory$status$biomarkers$in$metabolically$healthy$ overweight/obese$ women,$ as$ well$ as$ to$ characterize$ the$ transcriptomic$ and$ epigenetic$ mechanisms$that$could$be$involved.$$

$

The$specific#objectives$were:$$

$ 1. To$ evaluate$ the$ potential$ body$ weight*lowering$ effects$ of$ dietary$ supplementation$ with$ α* lipoic$acid$and$EPA$separately$or$in$combination$in$healthy$overweight/obese$women$during$ an$energy*restricted$nutritional$intervention$(Chapter$1).$$ $ 2. To$ assess$ the$ influence$ of$ the$ supplementation$ with$ α*lipoic$ acid$ and/or$ EPA$ in$ promoting$ additional$effects$to$a$weight*loss$treatment$on$biochemical$parameters$of$glucose$and$lipid$ metabolism$(Chapter$1).$$

$ 3. To$investigate$the$role$of$the$dietary$supplementation$with$α*lipoic$acid$and/or$EPA$on$some$ systemic$and$adipose$tissue$inflammatory$biomarkers$(Chapter$2).$$ $ 4. To$analyze$the$influence$of$α*lipoic$acid$and/or$EPA$treatment$in$the$production$of$irisin$not$ only$ in$ vivo$ but$ also$ in$ vitro$ using$ cultured$ human$ adipocytes,$ as$ well$ as$ to$ explore$ the$ relationship$between$irisin$and$glucose$metabolism$parameters$(Chapter$3).$$

$ 5. To$characterize$the$transcriptomic$changes$promoted$by$supplementation$with$α*lipoic$acid$ and/or$ EPA$ into$ adipose$ tissue$ after$ weight*loss$treatment$using$a$microarray$approach,$as$ well$as$to$evaluate$possible$associations$between$gene$expression$with$some$metabolic$and$ inflammatory$biomarkers$(Chapter$4).$$ $ 6. To$study$the$epigenetic$modifications$induced$by$supplementation$with$α*lipoic$acid$and/or$ EPA,$ as$ well$ as$ to$ explore$ possible$ associations$ between$ methylation$ changes$ in$ peripheral$ white$blood$cells$with$some$metabolic$biomarkers$(Chapter$5).$$

# 63$ II.#HYPOTHESIS#AND#AIMS#

4.#References# do$Amaral,$C.L.,$Milagro,$F.I.,$Curi,$R.,$and$Martínez,$J.A.$(2014).$DNA$methylation$pattern$in$overweight$ women$under$an$energy*restricted$diet$supplemented$with$fish$oil.$BioMed$Res.$Int.$2014,$No.$675021.$

Dashwood,$R.H.,$and$Ho,$E.$(2007).$Dietary$histone$deacetylase$inhibitors:$from$cells$to$mice$to$man.$Semin.$ Cancer$Biol.$17,$363–369.$

Deiuliis,$J.A.,$Kampfrath,$T.,$Ying,$Z.,$Maiseyeu,$A.,$and$Rajagopalan,$S.$(2011).$Lipoic$acid$attenuates$innate$ immune$infiltration$and$activation$in$the$visceral$adipose$tissue$of$obese$insulin$resistant$mice.$Lipids$ 46,$1021–1032.$

Fernández*Galilea,$M.,$Pérez*Matute,$P.,$Prieto*Hontoria,$P.,$Martínez,$J.A.,$and$Moreno*Aliaga,$M.J.$(2011).$ Effects$of$lipoic$acid$on$apelin$in$3T3*L1$adipocytes$and$in$high*fat$fed$rats.$J.$Physiol.$Biochem.$67,$479– 486.$

Fernández*Galilea,$ M.,$ Pérez*Matute,$ P.,$ Prieto*Hontoria,$ P.L.,$ Martínez,$ J.A.,$ and$ Moreno*Aliaga,$ M.J.$ (2012).$Effects$of$lipoic$acid$on$lipolysis$in$3T3*L1$adipocytes.$J.$Lipid$Res.$53,$2296–2306.$

Fernández*Galilea,$ M.,$ Prieto*Hontoria,$ P.L.,$ Martínez,$ J.A.,$ and$ Moreno*Aliaga,$ M.J.$ (2013).$ Antiobesity$ effects$of$α*lipoic$acid$supplementation.$Clin.$Lipidol.$8,$371–383.$

Fernández*Galilea,$ M.,$ Pérez*Matute,$ P.,$ Prieto*Hontoria,$ P.L.,$ Sáinz,$ N.,$ López*Yoldi,$ M.,$ Houssier,$ M.,$ Martínez,$J.A.,$Langin,$D.,$and$Moreno*Aliaga,$M.J.$(2014).$α*Lipoic$acid$reduces$fatty$acid$esterification$ and$lipogenesis$in$adipocytes$from$overweight/obese$subjects.$Obesity$(Silver$Spring)$22,$2210–2215.$

Fernández*Galilea,$ M.,$ Pérez*Matute,$ P.,$ Prieto*Hontoria,$ P.L.,$ Houssier,$ M.,$ Burrell,$ M.A.,$ Langin,$ D.,$ Martínez,$ J.A.,$ and$ Moreno*Aliaga,$ M.J.$ (2015).$ α*Lipoic$ acid$ treatment$ increases$ mitochondrial$ biogenesis$ and$ promotes$ beige$ adipose$ features$ in$ subcutaneous$ adipocytes$ from$ overweight/obese$ subjects.$Biochim.$Biophys.$Acta$BBA$*$Mol.$Cell$Biol.$Lipids$1851,$273–281.$

Furukawa,$S.,$Fujita,$T.,$Shimabukuro,$M.,$Iwaki,$M.,$Yamada,$Y.,$Nakajima,$Y.,$Nakayama,$O.,$Makishima,$ M.,$ Matsuda,$ M.,$ and$ Shimomura,$ I.$ (2004).$ Increased$ oxidative$ stress$ in$ obesity$ and$ its$ impact$ on$ metabolic$syndrome.$J.$Clin.$Invest.$114,$1752–1761.$

Hernando$Boigues,$J.F.,$and$Mach,$N.$(2015).$The$effect$of$polyunsaturated$fatty$acids$on$obesity$through$ epigenetic$modifications.$Endocrinol.$Nutr.$Órgano$Soc.$Esp.$Endocrinol.$Nutr.$62,$338–349.$

Itariu,$B.K.,$Zeyda,$M.,$Hochbrugger,$E.E.,$Neuhofer,$A.,$Prager,$G.,$Schindler,$K.,$Bohdjalian,$A.,$Mascher,$D.,$ Vangala,$ S.,$ Schranz,$ M.,$ et$ al.$ (2012).$ Long*chain$ n−3$ PUFAs$ reduce$ adipose$ tissue$ and$ systemic$ inflammation$in$severely$obese$nondiabetic$patients:$a$randomized$controlled$trial.$Am.$J.$Clin.$Nutr.$96,$ 1137–1149.$

Khaire,$A.A.,$Kale,$A.A.,$and$Joshi,$S.R.$(2015).$Maternal$omega*3$fatty$acids$and$micronutrients$modulate$ fetal$lipid$metabolism:$A$review.$Prostaglandins$Leukot.$Essent.$Fatty$Acids$98,$49–55.$

Kim,$M.S.,$Park,$J.Y.,$Namkoong,$C.,$Jang,$P.G.,$Ryu,$J.W.,$Song,$H.S.,$Yun,$J.Y.,$Namgoong,$I.S.,$Ha,$J.,$Park,$ I.S.,$ et$ al.$ (2004).$ Anti*obesity$ effects$ of$ alpha*lipoic$ acid$ mediated$ by$ suppression$ of$ hypothalamic$ AMP*activated$protein$kinase.$Nat.$Med.$10,$727–733.$

Kulkarni,$ A.,$ Dangat,$ K.,$ Kale,$ A.,$ Sable,$ P.,$ Chavan*Gautam,$ P.,$ and$ Joshi,$ S.$ (2011).$ Effects$ of$ altered$ maternal$ folic$ acid,$ vitamin$ B12$ and$ docosahexaenoic$ acid$ on$ placental$ global$ DNA$ methylation$ patterns$in$Wistar$rats.$PloS$One$6,$e17706.$

Kunesova,$ M.,$ Braunerova,$ R.,$ Hlavaty,$ P.,$ Tvrzicka,$ E.,$ Stankova,$ B.,$ Skrha,$ J.,$ Hilgertova,$ J.,$ Hill,$ M.,$ Kopecky,$J.,$Wagenknecht,$M.,$et$al.$(2006).$The$influence$of$n*3$polyunsaturated$fatty$acids$and$very$

64# # II.#HYPOTHESIS#AND#AIMS#

low$ calorie$ diet$ during$ a$ short*term$ weight$ reducing$ regimen$ on$ weight$ loss$ and$ serum$ fatty$ acid$ composition$in$severely$obese$women.$Physiol.$Res.$Acad.$Sci.$Bohemoslov.$55,$63–72.$

Lee,$H.*S.,$Barraza*Villarreal,$A.,$Hernandez*Vargas,$H.,$Sly,$P.D.,$Biessy,$C.,$Ramakrishnan,$U.,$Romieu,$I.,$ and$ Herceg,$ Z.$ (2013).$ Modulation$ of$ DNA$ methylation$ states$ and$ infant$ immune$ system$ by$ dietary$ supplementation$with$ω*3$PUFA$during$pregnancy$in$an$intervention$study.$Am.$J.$Clin.$Nutr.$98,$480– 487.$

Lopategi,$A.,$López*Vicario,$C.,$Alcaraz*Quiles,$J.,$García*Alonso,$V.,$Rius,$B.,$Titos,$E.,$and$Clària,$J.$(2016).$ Role$of$bioactive$lipid$mediators$in$obese$adipose$tissue$inflammation$and$endocrine$dysfunction.$Mol.$ Cell.$Endocrinol.$419,$44–59.$

Lorente*Cebrián,$ S.,$ Costa,$ A.G.,$ Navas*Carretero,$ S.,$ Zabala,$ M.,$ Martínez,$ J.A.,$ and$ Moreno*Aliaga,$ M.J.$ (2013).$ Role$ of$ omega*3$ fatty$ acids$ in$ obesity,$ metabolic$ syndrome,$ and$ cardiovascular$ diseases:$ a$ review$of$the$evidence.$J.$Physiol.$Biochem.$69,$633*651.$

Martínez*Fernández,$L.,$Laiglesia,$L.M.,$Huerta,$A.E.,$Martínez,$J.A.,$and$Moreno*Aliaga,$M.J.$(2015).$Omega* 3$fatty$acids$and$adipose$tissue$function$in$obesity$and$metabolic$syndrome.$Prostaglandins$Other$Lipid$ Mediat.$121,$24*41.$

Milagro,$ F.I.,$ Mansego,$ M.L.,$ De$ Miguel,$ C.,$ and$ Martínez,$ J.A.$ (2013).$ Dietary$ factors,$ epigenetic$ modifications$and$obesity$outcomes:$progresses$and$perspectives.$Mol.$Aspects$Med.$34,$782–812.$

Moreno*Aliaga,$M.J.,$Lorente*Cebrián,$S.,$and$Martínez,$J.A.$(2010).$Regulation$of$adipokine$secretion$by$n* 3$fatty$acids.$Proc.$Nutr.$Soc.$69,$324–332.$

Mori,$ T.A.,$ and$ Woodman,$ R.J.$ (2006).$ The$ independent$ effects$ of$ eicosapentaenoic$ acid$ and$ docosahexaenoic$acid$on$cardiovascular$risk$factors$in$humans.$Curr.$Opin.$Clin.$Nutr.$Metab.$Care$9,$ 95–104.$

Mori,$ T.A.,$ Burke,$ V.,$ Puddey,$ I.B.,$ Watts,$ G.F.,$ O’Neal,$ D.N.,$ Best,$ J.D.,$ and$ Beilin,$ L.J.$ (2000).$ Purified$ eicosapentaenoic$and$docosahexaenoic$acids$have$differential$effects$on$serum$lipids$and$lipoproteins,$ LDL$particle$size,$glucose,$and$insulin$in$mildly$hyperlipidemic$men.$Am.$J.$Clin.$Nutr.$71,$1085–1094.$

Pérez*Echarri,$N.,$Pérez*Matute,$P.,$Marcos*Gómez,$B.,$Baena,$M.J.,$Marti,$A.,$Martínez,$J.A.,$and$Moreno* Aliaga,$ M.J.$ (2008).$ Differential$ inflammatory$ status$ in$ rats$ susceptible$ or$ resistant$ to$ diet*induced$ obesity:$effects$of$EPA$ethyl$ester$treatment.$Eur.$J.$Nutr.$47,$380–386.$

Prieto*Hontoria,$ P.L.,$ Pérez*Matute,$ P.,$ Fernández*Galilea,$ M.,$ Barber,$ A.,$ Martínez,$ J.A.,$ and$ Moreno* Aliaga,$M.J.$(2009).$Lipoic$acid$prevents$body$weight$gain$induced$by$a$high$fat$diet$in$rats:$effects$on$ intestinal$sugar$transport.$J.$Physiol.$Biochem.$65,$43–50.$

Prieto*Hontoria,$ P.L.,$ Pérez*Matute,$ P.,$ Fernández*Galilea,$ M.,$ Martínez,$ J.A.,$ and$ Moreno*Aliaga,$ M.J.$ (2011).$Lipoic$acid$inhibits$leptin$secretion$and$Sp1$activity$in$adipocytes.$Mol.$Nutr.$Food$Res.$55,$1059– 1069.$

Prieto*Hontoria,$ P.L.,$ Pérez*Matute,$ P.,$ Fernández*Galilea,$ M.,$ Martínez,$ J.A.,$ and$ Moreno*Aliaga,$ M.J.$ (2013).$Effects$of$lipoic$acid$on$AMPK$and$adiponectin$in$adipose$tissue$of$low*$and$high*fat*fed$rats.$ Eur.$J.$Nutr.$52,$779–787.$

Prieto*Hontoria,$ P.L.,$ Pérez*Matute,$ P.,$ Fernández*Galilea,$ M.,$ López*Yoldi,$ M.,$ Sinal,$ C.J.,$ Martínez,$ J.A.,$ and$Moreno*Aliaga,$M.J.$(2016).$Effects$of$alpha*lipoic$acid$on$chemerin$secretion$in$3T3*L1$and$human$ adipocytes.$Biochim.$Biophys.$Acta$BBA$*$Mol.$Cell$Biol.$Lipids$1861,$260–268.$

Report$ of$ a$ WHO$ Consultation$ (2004).$ Obesity:$ preventing$ and$ managing$ the$ global$ epidemic.$ WHO$ Technical$Report$Series$894.$

# 65$ II.#HYPOTHESIS#AND#AIMS#

Spencer,$M.,$Finlin,$B.S.,$Unal,$R.,$Zhu,$B.,$Morris,$A.J.,$Shipp,$L.R.,$Lee,$J.,$Walton,$R.G.,$Adu,$A.,$Erfani,$R.,$et$ al.$(2013).$Omega*3$Fatty$Acids$Reduce$Adipose$Tissue$Macrophages$in$Human$Subjects$With$Insulin$ Resistance.$Diabetes$62,$1709–1717.$

Suh,$ J.H.,$ Shenvi,$ S.V.,$ Dixon,$ B.M.,$ Liu,$ H.,$ Jaiswal,$ A.K.,$ Liu,$ R.*M.,$ and$ Hagen,$ T.M.$ (2004).$ Decline$ in$ transcriptional$activity$of$Nrf2$causes$age*related$loss$of$glutathione$synthesis,$which$is$reversible$with$ lipoic$acid.$Proc.$Natl.$Acad.$Sci.$U.$S.$A.$101,$3381–3386.$

Tilg,$ H.,$ and$ Moschen,$ A.R.$ (2006).$ Adipocytokines:$ mediators$ linking$ adipose$ tissue,$ inflammation$ and$ immunity.$Nat.$Rev.$6,$772–783.$

Tinahones,$F.J.,$Murri*Pierri,$M.,$Garrido*Sanchez,$L.,$Garcia*Almeida,$J.M.,$Garcia*Serrano,$S.,$Garcia*Arnes,$ J.,$ and$ Garcia*Fuentes,$ E.$ (2009).$ Oxidative$ stress$ in$ severely$ obese$ persons$ is$ greater$ in$ those$ with$ insulin$resistance.$Obesity$(Silver$Spring)$17,$240–246.$

Wang,$Y.,$Li,$X.,$Guo,$Y.,$Chan,$L.,$and$Guan,$X.$(2010).$alpha*Lipoic$acid$increases$energy$expenditure$by$ enhancing$ adenosine$ monophosphate*activated$ protein$ kinase*peroxisome$ proliferator*activated$ receptor*gamma$coactivator*1alpha$signaling$in$the$skeletal$muscle$of$aged$mice.$Metabolism.$59,$967– 976.$ $

$

66# # $

III. SUBJECTS#AND#METHODS#

#

III.#SUBJECTS#AND#METHODS#

1.#Study#design#and#intervention#

The$study$about$the$effects$of$α*lipoic$acid$and$EPA$in$human$obesity$(OBEPALIP$study)$was$a$ short*term$randomized$double$blind$placebo*controlled$weight$loss$trial$with$4$parallel$nutritional$ intervention$groups$(Figure#1a):$1)$Control$group:$3$placebo*I$capsules$(containing$sunflower$oil)$ and$3$placebo*II$capsules$(containing$same$excipients$as$the$α*lipoic$acid$capsules),$2)$EPA$group:$ 1300$mg/day$of$EPA$distributed$in$3$capsules$of$EPA$80$(provided$by$Solutex®,$Madrid,$Spain)$ containing$433.3$mg$of$EPA$and$13.8$mg$of$DHA$as$ethyl*ester$and$3$placebo*II$capsules,$3)$α* lipoic$acid$group:$300$mg/day$of$α*lipoic$acid$from$3$capsules$containing$100$mg$of$α*lipoic$acid$ (Nature’s$ Bounty®,$ NY,$ USA)$ and$ 3$ placebo*I$ capsules;$ and$ 4)$ EPA$ +$ α*lipoic$ acid$ group:$ 1300$ mg/day$ of$ EPA$ (distributed$ in$ 3$ capsules$ of$ EPA$ 80)$ and$ 300$ mg/day$ of$ α*lipoic$ acid$ (from$ 3$ capsules$containing$100$mg$of$α*lipoic$acid).$Therefore,$each$daily$dose$was$administered$into$ three$equal$doses$to$minimize$the$impact$of$decline$in$plasma$levels$after$oral$supplementation,$ and$each$participant$consumed$a$total$of$6$capsules$per$day$(2$at$breakfast,$2$at$lunch$and$2$at$ dinner).$$

Both$placebo*I$(sunflower$oil)$and$EPA$capsules$were$provided$by$Solutex,$and$were$similar$in$ shape$ and$ size.$ Placebo*II$ capsules$ were$ similar$ in$ appearance$ to$ the$ α*lipoic$ acid$ capsules.$ Analysis$of$the$EPA$capsules$verified$that$they$contained$81.3%$EPA,$2.7%$DHA,$3.9%$arachidonic,$ 3.3%$ stearidonic,$ 0.25%$ linoleic,$ 0.10%$ oleic$ and$ small$ amounts$ of$ other$ fatty$ acids.$ Oxidation$ levels$ of$ the$ EPA$ supplements,$ based$ on$ peroxide$ and$ anisidine$ values,$ were$ below$ maximum$ permitted$levels$(Codex$Alimentarius$Commission,$2015).$Moreover,$our$independent$analysis$of$ the$ α*lipoic$ acid$ capsules$ (using$ an$ Agilent$ Technologies$ 1200$ liquid$ chromatographic$ system$ equipped$ with$ a$ 6220$ Accurate*Mass$ TOF$ LC/MS)$ verified$ that$ the$ content$ was$ approximately$ 100$(105.6$±$4.1)$mg$of$α*lipoic$acid$per$capsule.$

All$intervention$groups$followed$a$weight*reduction$program$consisting$of$a$calorie*restricted$ balanced$diet$(55%$carbohydrates;$30%$lipids,$15%$proteins)$in$accordance$with$American$Heart$ Association$guidelines,$and$prescribed$individually$by$a$dietitian.$During$the$baseline$visit,$each$ subject$was$instructed$to$follow$an$energy*restricted$diet$accounting$for$30%$less$than$her$total$ energy$ expenditure$ (TEE),$ and$ to$ not$ change$ the$ physical$ activity$ pattern$ during$ the$ 10*week$ intervention$period.$The$main$outcome$of$the$study$was$the$amount$of$weight$loss.$$

2.#Study#population#

Enrollment$ started$ in$ May$ 2010,$ and$ follow*up$ ended$ in$ December$ 2011.$ Pre*menopausal$ women$ aged$ 20*50$ years,$ with$ a$ BMI$ between$ 27.5$ and$ 40$ Kg/m2,$ were$ recruited$ by$

# 69$ III.#SUBJECTS#AND#METHODS# advertisement$in$local$newspapers$and$by$calls$for$volunteers$from$the$database$of$the$MU$of$the$ University$of$Navarra.$The$inclusion$and$exclusion$criteria$are$detailed$in$Table#7.$Medical$history$ and$physical$examination$were$conducted$by$the$physician$of$the$Metabolic$Unit$(MU).$

$ Table#7.#Inclusion$and$exclusion$criteria# Inclusion#criteria# • Female$ • BMI$between$27.5$and$40$Kg/m2$ • Age$between$20*50$years$old$ • Regular$menstrual$cycles$ • Stable$weight$(±$3$kg)$for$the$last$3$months$ • Overall$healthy$and$physiological$condition$ Exclusion#criteria# • Use$of$prescription$medications$ • Having$a$pre*existent$obesity*related$disease:$hypertension,$severe$dyslipidemia,$diabetes,$ thyroid$disorders,$renal$dysfunction,$cirrhosis,$fatty$liver$etc.$ • Pre*existing$digestive$diseases$such$as$Crohn’s$disease,$irritable$bowel$syndrome$etc.$ • Allergies$or$intolerance$to$food$ • Following$special$diets$(ketogenic,$vegetarian,$etc.)$3$months$prior$to$the$beginning$of$the$study$ • Eating$disorders$ • Having$previously$undergone$obesity*related$surgery$ • Pregnancy$or$lactation$ • Alcohol$or$drug$abuse$ $

Based$on$previous$investigations$(Abete$et$al.,$2009)$and$considering$as$primary$outcome$the$ weight$loss,$the$number$of$subjects$per$arm$of$intervention$was$estimated$at$sixteen$subjects,$ which$would$allow$a$detection$of$approximately$a$difference$of$5$kg$with$a$dispersion$of$5$kg$(5$±$ 5$kg)$in$weight$loss$between$experimental$groups,$at$a$0.05$level$of$significance,$with$a$statistical$ power$of$80%.$Since$the$expected$dropout$rate$was$around$25%,$the$final$sample$size$estimation$ was$established$in$at$least$20$participants$per$group$to$be$recruited.$

A$ total$ of$ 122$ potential$ participants$ were$ asked$ to$ attend$ a$ screening$ session.$ Finally,$ 103$ subjects$ that$ fulfilled$ the$ inclusion$ criteria$ were$ randomly$ assigned$ to$ one$ of$ the$ four$ experimental$groups$and$97$volunteers$received$the$allocated$treatment$(Figure#1b).$$ $ $

70# # III.#SUBJECTS#AND#METHODS#

$$

a$#

$ ! Personalized'energy@restricted'diet'(<30'%'of'TEE)' ! 55'%'carbohydrates,'30'%'fats,'15'%''proteins' $ Fas4ng:'10@12'h' $

$

$

$ Start( Follow%up(visits( Final( $ !Weight'and'other'anthropometric'measurements' !Body'composi4on'(DXA'and'bioimpedance)' $ !Res4ng'metabolic'rate'(indirect'calorimetry)' !Blood'samples' $ Subcutaneous'abdominal' !Blood'pressure'' adipose'biopsies'were'extracted' !Oral'glucose'tolerance'test' !72@h'recall'ques4onnaire' at'the'end'of'the'nutri4onal' $ interven4on'period.'' $ b$# $ Assessed%for%eligibility% (n=122)% Excluded%(n=19)% $ ?Not%meeAng%inclusion%criteria% (n=17)% # ?Declined%to%parAcipate%(n=2)% Randomly%assigned% # (%n=103)%

#

#

# Alloca&on( Allocated(to(EPA+α0 Allocated(to(α0lipoic( # lipoic(acid(group( acid(group!(n=26)% (n=26)% Allocated(to(Control( Allocated(to(EPA( ?Received%allocated% # ?Received%allocated% group((n=31)% group((n=20)% intervenAon%(n=23)% intervenAon%(n=23)% ?Received%allocated% ?Received%allocated% ?Did%not%receive% # ?Did%not%receive% intervenAon%(n=31)% intervenAon%(n=20)% allocated%intervenAon% allocated%intervenAon% (did%not%come%to%first% # (did%not%come%to%first% visit%n=%3)% visit%n%=%3)% #

Figure#1.$Study$design$from$the$OBEPALIP$study$(a)$and$flowchart$of$participants$(b).$EPA:$eicosapentaenoic$ acid;$DXA:$dual$X*ray$absorptiometry;$TEE:$total$energy$expenditure.$$ $ $ $

# 71$ III.#SUBJECTS#AND#METHODS#

Throughout$ the$ study,$ volunteers$ were$ advised$ to$ avoid$ any$ marketed$ omega*3$ enriched$ products:$milk,$eggs,$biscuits,$butter,$margarine,$juices,$soy$beverages$and$olives$or$others$oily$ products.$ The$ subjects$ and$ researchers$ directly$ assessing$ the$ outcomes$ were$ blinded$ to$ the$ intervention.$

Follow*up$ visits$ were$ scheduled$ with$ each$ volunteer$ every$ two$ weeks$ to$ monitor$ weight,$ assess$the$compliance$with$the$assigned$diet,$motivate$participants$and$ensure$that$all$capsules$ were$consumed$accordingly.$$

The$ trial$ was$ approved$ by$ the$ Research$ Ethics$ Committee$ of$ the$ University$ of$ Navarra$ No.# 007/2009$ in$ compliance$ with$ Helsinki$ Declaration$ and$ registered$ at$ clinicaltrials.gov$ as$ NCT01138774.$ All$ recruited$ volunteers$ received$ an$ information$ sheet$ and$ additional$ verbal$ explanation$of$the$protocol.$Once$the$participants$gave$their$written$consent,$they$were$allocated$ by$ a$ scientist$ of$ the$ Metabolic$ Unit$ (MU)$ of$ the$ University$ of$ Navarra$ to$ one$ of$ the$ four$ experimental$ groups$ by$ simple$ randomization$ using$ the$ Microsoft$ Excel$ Office$ 2003$ Software$ (Microsoft$Inc.,$USA)$that$generates$random$numbers.$

3.#Data#collection##

At$ baseline$ and$ at$ end$ point$ the$ volunteers$ underwent$ 10–12$ h$ fasting$ and$ met$ with$ the$ physician,$ the$ dietitian$ and$ the$ nurse$ at$ the$ MU.$ Anthropometric$ measurements,$ body$ composition$ analysis,$ physical$ activity,$ food$ intake,$ respiratory$ exchange$ measurements$ by$ indirect$calorimetry$and$blood$pressure$were$evaluated.$A$catheter$was$then$inserted$into$the$ antecubital$vein$for$a$fasting$blood$sample$extraction.$In$order$to$attenuate$bias,$either$data$or$ measurements$were$taken$by$the$same$person$using$standard$protocols.$$

3.1.$Dietary$assessment$

In$order$to$evaluate$the$approximate$energy$intake,$at$the$screening$session$a$food$scale$was$ provided$to$facilitate$the$precise$weighting$registration$of$the$72*hour$recall$questionnaire.$The$ three*day$estimated$food$record$was$delivered$to$the$volunteers$in$the$screening$session$and$at$ the$8th$week.$This$procedure$had$to$be$handed$over$at$beginning$and$endpoint$of$the$study$and$ the$ information$ was$ processed$ using$ the$ program$ DIAL$ version$ 2.16$ (Alce$ Ingenieria$ Madrid,$ Spain),$ for$ the$ evaluation$ of$ diets$ and$ management$ of$ nutrition$ data,$ as$ previously$ described$ (Ibero*Baraibar$et$al.,$2014).$

72# # III.#SUBJECTS#AND#METHODS#

3.2.$Energy$expenditure$

Total$ energy$ expenditure$ was$ estimated$ utilizing$ the$ Harris*Benedict$ formula,$ using$ a$ correction$ factor$ to$ take$ into$ account$ the$ overweight$ status$ of$ the$ subjects$ and$ the$ resting$ metabolic$rate$(RMR)$measured$by$indirect$calorimetry$(Deltatrac$II®$metabolic$monitor$MBM* 200$ Datex*Ohmeda,$ Finland)$ at$ the$ beginning$ of$ the$ morning$ after$ 10*12$ h$ of$ fasting,$ as$ previously$ described$ (Thorsdottir$ et$ al.,$2007).$$For$calculating$the$RMR,$the$abbreviated$Weir$ equation,$which$gives$a$similar$product$to$the$full$version$and$does$not$have$the$disadvantage$of$ 24$h$urine$collection,$was$used$(Matarese,$1997).$Additionally,$the$adjustment$for$physical$activity$ level$was$added.$At$baseline,$information$about$physical$activity$patterns$was$collected$using$a$ questionnaire$ in$ order$ to$ evaluate$ the$ physical$ activity$ factor$ (National$ Research$ Council$ (US),$ 1989).$

3.3.$Anthropometric$measurements$and$body$composition$

Anthropometric$measurements$of$weight,$height,$waist$circumference$and$hip$circumference$ were$performed$according$to$established$protocols$(Pérez$et$al.,$2005).$The$assessment$material$ was$calibrated$and$standardized$before$starting$the$measurements;$height$was$evaluated$using$a$ stadiometer$(Seca$220,$Vogel$&$Halke,$Germany)$and$body$weight$using$a$digital$scale$accurate$to$ 0.1$Kg$(TBF*410GS,$TANITA,$Tokyo,$Japan).$The$waist$and$hip$circumferences$were$measured$with$ a$flexible$tape$with$a$precision$of$1$mm$and$were$used$for$calculating$the$waist$to$hip$ratio.$Also,$ the$waist$to$height$ratio$(WHtR)$was$estimated$as$waist$circumference$divided$by$height,$both$ measured$in$centimeters.$

Body$composition$was$determined$by$two$methods:$bioimpedance$using$a$Tanita$(TBF*410GS,$ TANITA,$Tokyo,$Japan)$and$Dual$X*ray$Absorptiometry$(DXA)$using$the$Lunar,$Prodigy,$software$ version$6.0$(Madison,$WI,$USA)$following$standardized$methods$(Bolanowski$and$Nilsson,$2001).$$

The$android$and$gynoid$fat$were$defined$by$DXA$as$the$fat$deposition$in$the$upper$(central)$ body$region$and$the$fat$deposition$in$the$gluteofemoral$area,$respectively$(Wiklund$et$al.,$2008).$$

3.4.$Blood$pressure$measurements$

Systolic$and$diastolic$blood$pressures$were$measured$with$a$sphygmomanometer$taken$on$the$ right$arm$and$in$resting$sitting$position$as$described$by$the$WHO$Expert$Committee$guidelines$ (WHO$Expert$Committee,$1996).$

# 73$ III.#SUBJECTS#AND#METHODS#

3.5.$Blood$samples$

Fasting$blood$samples$were$drawn$on$weeks$0$and$10$into$Serum$Clot$Activator$tubes$(4$mL$ Vacuette$®)$and$into$tubes$containing$tripotassium$EDTA$(4$mL$Vacuette$®)$between$8:00$–$9:00$ am.$Plasma$and$buffy$coat$were$extracted$from$EDTA$tubes$after$centrifugation$at$1500$g$during$ 15$ min$ at$ 4$ oC.$ The$ buffy$ coat$ was$ either$ kept$ for$ DNA$ extraction$ or$ was$ processed$ by$ standardized$ methods$ according$ with$ the$ PolymorphprepTM$ protocol$ to$ obtain$ PBMC$ for$ RNA$ extraction.$All$samples$were$separated$appropriately$and$stored$at$*80$oC$for$further$analysis.$

3.6.$Oral$glucose$tolerance$test$(OGTT)$

All$ participants$ underwent$ a$ 2*h$ oral$ OGTT$ with$ 75$ g$ of$ glucose$ according$ with$ the$ WHO$ protocol$(Alberti$and$Zimmet,$1998)$at$beginning$and$at$the$end$of$the$nutritional$intervention.$ Additionally,$during$the$OGTT,$blood$samples$were$extracted$at$the$different$times$(30’,$60’$and$ 120’)$in$either$EDTA$or$serum$tubes.$

3.7.$Insulin$sensitivity$indexes$

The$incremental$area$under$the$curve$(iAUC)$were$calculated$by$first$estimating$the$total$area$ under$curve$in$accordance$with$Tai's$Model$(Tai,$1994)$and$then$subtracting$the$area$from$the$ baseline$concentration$over$the$2*h$period.$The$HOMA*IR$was$defined$ as$fasting$serum$insulin$ (mU/L)$x$fasting$plasma$glucose$(mmol/L)$/$22.5,$as$described$elsewhere$(Matthews$et$al.,$1985).$ The$triglyceride*glucose$(TyG)$index$(Guerrero*Romero$et$al.,$2010)$was$estimated$as$ln[fasting$ triglycerides$(mg/dL)$x$fasting$glucose$(mg/dL)/2].$

3.8.$Adipose$tissue$biopsies$

At$the$end$of$the$intervention,$biopsies$from$subcutaneous$abdominal$periumbilical$area$(1*2$ g)$ were$ obtained$ in$ the$ Clínica$ Universidad$ de$ Navarra$ by$ liposuction$ under$ local$ anesthesia$ following$an$overnight$fast.$The$samples$were$washed$and$stored$at$−80$°C$until$their$utilization.$ Adipose$tissue$biopsies$from$57$subjects$were$available,$15$from$each$Control,$EPA$and$EPA+α* lipoic$acid$groups$and$12$from$the$α*lipoic$acid$group.$

4.#Biochemical#measurements#

Serum$ levels$ of$ glucose,$ total*cholesterol,$ HDL*cholesterol,$ TG,$ FFA$ and$ β*hydroxybutyrate$ were$ measured$ using$ the$ auto$ analyzer$ PENTRA$ C200$ (HORIBA$ medical,$ Madrid,$ Spain).$ The$ values$ of$ LDL*cholesterol$ were$ calculated$ using$ the$ Friedewald$ equation$ defined$ as$ LDL* cholesterol$ =$ Total*cholesterol$ –$ HDL*cholesterol$ –$ TG/5$ (Friedewald$ et$ al.,$ 1972).$ Non*HDL*

74# # III.#SUBJECTS#AND#METHODS# cholesterol$was$estimated$as$the$difference$between$total$cholesterol$and$LDL*cholesterol.$The$ white$ blood$ cells$ (WBC)$ recounts,$ extracted$ from$ tubes$ containing$ tripotassium$ EDTA,$ were$ analyzed$using$an$automated$cell$counter$(ABX$®$Pentra$120,$HORIBA).$$

Plasma$ samples$ were$ used$ for$ assessing$ insulin$ and$ circulating$ inflammatory$ markers$ by$ enzyme*linked$ immunosorbent$ assay$ (ELISA)$ using$ the$ appropriate$ kit$ and$ following$ the$ manufacturer’s$instructions$(Table#8).$Insulin$was$determined$using$a$Human$Sensitive$ELISA$Kit$ from$ Mercodia$ (Uppsala,$ Sweden).$ Leptin,$ total$ adiponectin,$ chemerin$ and$ PAI*1$ were$ determined$using$ELISA$kits$from$BioVendor$(Brno,$Czech$Republic).$IL*6$and$vascular$endothelial$ growth$ factor$ (VEGF)$ were$ assessed$ with$ ELISA$ kits$ from$ R&D$ Systems$ (MN,$ USA).$ SAA$ and$ haptoglobin$were$evaluated$with$ELISA$kits$from$SunRed$Biological$Technology$(Shanghai,$China).$ Other$ ELISA$kits$were$used$for$the$determination$of$human$CRP$(Immundiagnostik,$Bensheim,$ Germany),$apelin$C*terminus$(RayBiotech,$GA,$USA),$asymmetric$dimethylarginine$(ADMA)$(DLD$ Diagnostika$ GmbH,$ Hamburg,$ Germany)$ and$ high$ molecular$ weight$ adiponectin$ (ALPCO$ Diagnostics,$Salem,$NH,$USA).$$

The$ plasma$ concentrations$ of$ ghrelin,$ an$ orexigenic$ hormone$ synthesized$ mainly$ by$ the$ stomach$ (Kojima$ et$ al.,$ 1999),$ were$ measured$ with$ the$ Human$ ELISA$ kit$ from$ RayBiotech$ (Norcross,$ GA,$ USA),$ but$ also$ the$ levels$ of$ irisin$ with$ the$ human$ ELISA$ kit$ of$ Phoenix$ Pharmaceuticals$(Burlingame,$CA,$USA)$(Table#8).$

5.#Studies#in#cultured#human#subcutaneous#adipocytes#

Commercially$available$cryopreserved$human$subcutaneous$preadipocytes$from$non*diabetic$ overweight/obese$ female$ donors$ (BMI:$ 26.85–33.37$ kg/m2)$ were$ purchased$ from$ Zen*Bio$ Inc.$ (Research$ Triangle$ Park,$ NC)$ and$ differentiated$ with$ the$ DM*2$ Zen*Bio$ commercial$ medium,$ according$ to$ manufacturer’s$ instructions.$ Fourteen$ days$ after$ the$ induction$ of$ differentiation,$ cells$contained$large$lipid$droplets$and$were$considered$mature$adipocytes.$α*Lipoic$acid$(Sigma;$ St.$ Louis,$ MO)$ and$ EPA$ (Cayman$ Chemical,$ Ann$ Arbor,$ MI)$ were$ dissolved$ in$ ethanol.$ Stocks$ (1000×)$were$prepared,$and$1$μL/mL$of$media$was$added.$Adipocytes$were$treated$with$α*lipoic$ acid$(100–250$μM)$or$EPA$(100–200$μM)$during$24$h,$as$previously$described$(Fernández*Galilea$ et$al.,$2014;$Pérez*Matute$et$al.,$2005).$

$

$

$

$$

# 75$ III.#SUBJECTS#AND#METHODS#

$ Table#8.$List$of$commercial$ELISA$kits$used$ Antibody# Manufacturer# Reference# Sensitivity# HMW$adiponectin$ ALPCO$Diagnostics$ 47*ADPHU*E01$ 0.019$ng/mL$ Total$adiponectin$ BioVendor$ RD191023100$ 0.47$ng/mL$ Chemerin$ BioVendor$ RD191136200R$ 0.1$ng/mL$ Leptin$ BioVendor$ RD191001100$ 0.2$ng/mL$ PAI*1$ BioVendor$ RBMS2033R$ 29.0$pg/mL$ ADMA$ DLD$Diagnostika$GmbH$ EA201/96$ 0.05$µmol/L$ CRP$ Immundiagnostik$ K9710s$ 0.921$ng/mL$ Insulin$ Mercodia$ 10*1113*10$ 1.0$mU/L$ Irisin$ Phoenix$Pharmaceuticals$ EK*067*52$ 7.0$ng/mL$ Interleukin*6$ R&D$Systems$ HS600B$ 0.039$pg/mL$ VEGF$ R&D$Systems$ DVE00$ 9.0$pg/mL$ Apelin$C*Terminus$ RayBiotech$ EIA*APC*1$ 29.1$pg/mL$ Ghrelin$ RayBiotech$ EIA*GHR*1$ 161$pg/mL$ Haptoglobin$ SunRed$Biological$Technology$ 201*12*1118$ 0.05$µmol/L$ SAA$ SunRed$Biological$Technology$ 201*12*1226$ 1.05$µg/mL$ ADMA:$asymmetric$dimethylarginine;$CRP:$C$reactive$protein;$HMW:$high$molecular$weight;$PAI*1:$ plasminogen$activator$inhibitor*1;$SAA:$serum$amyloid$A;$VEGF:$vascular$endothelial$growth$factor.$

6.#Gene#expression#analyses#(realZtime#polymerase#chain#reaction#and#microarray)##

Gene$ expression$ analyses$ in$ adipose$ tissue$ biopsies,$ obtained$ at$ the$ end$ of$ the$ trial,$ were$ conducted$ by$ two$ different$ approaches.$ First,$ by$ using$ a$ microarray$ technology$ in$ 6$ selected$ subjects$per$group.$Second$by$using$the$quantitative$real*time$Polymerase$Chain$Reaction$(RT* PCR)$for$validation$of$microarray$data$and$for$measuring$the$expression$of$some$interesting$genes$ related$with$inflammation$in$all$adipose$tissue$available$samples.$Also,$gene$expression$of$some$ genes$was$measured$in$human$cultured$adipocytes.$

Moreover,$ expression$ genes$ associated$ with$ inflammation$ and$ lipid$ metabolism$ were$ also$ measured$in$PBMC$at$baseline$and$at$the$end$of$the$nutritional$intervention$using$RT*PCR.$

6.1.$Quantitative$real%time$PCR$

Total$RNA$from$adipose$tissue$biopsies,$human$cultured$adipocytes$and$PBMC$was$extracted$ using$ TRIzol®$ reagent$ (Invitrogen,$ Carlsbad,$ CA,$ USA)$ according$ to$ the$ manufacturer’s$ instructions.$ RNA$ concentrations$ and$ quality$ were$ measured$ with$ the$ Nanodrop$ ND*1000$

76# # III.#SUBJECTS#AND#METHODS#

Spectrophotometer$ (Thermo$ Scientific,$ Waltham,$ MA,$ USA).$ Then,$ RNA,$ either$ from$ adipose$ tissue$(1$μg)$or$PBMC$(1*4$μg),$was$incubated$with$the$DNA*free$kit$DNAse$(Ambion,$TX,$USA)$for$ 30$min$at$37$oC.$RNA$was$reverse*transcribed$to$cDNA$using$the$Moloney$Murine$Leukaemia$Virus$ (MMLV)$reverse$transcriptase$(Invitrogen).$$

The$ expression$ of$ several$ genes$ was$ determined$ by$ RT*PCR$ using$ predesigned$ TaqMan®$ Assays*on*Demand$primers$and$TaqMan$Universal$Master$Mix$(Applied$Biosystems,$CA,$USA)$with$ the$ABI$PRISM$7900HT$Fast$System$Sequence$Detection$System$(Applied$Biosystems)$(Table# 9).$ Finally,$the$relative$expression$level$of$each$gene$was$calculated$as$2*ΔΔCt$(Livak$and$Schmittgen,$ 2001),$using$18S$as$housekeeping$gene$in$both$adipose$tissue$and$human$cultured$adipocytes,$as$ well$with$glyceraldehyde*3*phosphate$dehydrogenase$(GAPDH)$as$housekeeping$gene$in$PBMC.$

6.2.$Microarray$analysis$$

Both,$the$RNA$extraction$and$the$microarray$study$were$performed$at$Progenika$Biopharma$ SA$(Grifols$Company,$Bizkaia,$Spain).$Total$RNA$was$extracted$using$RNeasy$lipid$tissue$mini$kit$ (QIAGEN,$ Hilden,$ Germany)$ following$ the$ manufacturer’s$ instructions.$ RNA$ concentration$ and$ purity$were$measured$with$a$Nanodrop$ND*1000$Spectrophotometer$(Thermo$Fisher$Scientific).$ High*quality$RNA$was$confirmed$using$an$Agilent$2100$Bioanalyzer$(Agilent$Technologies,$Bizkaia,$ Spain).$From$300$ng$of$RNA,$the$Ambion®$WT$Expression$kit$in$conjunction$with$the$Affymetrix$ GeneChip®$ WT$ Terminal$ Labeling$ kit$ were$ used$ for$ the$ preparation$ of$ labeled$ cDNA$ according$ with$common$protocols.$Labeled$samples$were$hybridized$on$Affymetrix$GeneChip$HumanGene$ 1.1$ST$arrays,$provided$in$plate$format.$Hybridization,$washing$and$scanning$of$the$array$plates$ was$ performed$ on$ an$ Affymetrix$ GeneTitanTM$ Instrument,$ according$ to$ the$ manufacturer’s$ recommendations.$

For$ the$ analyses,$ microarray$ raw$ files$ were$ imported$ into$ Partek$ Genomics$ Suite$ V6.11$ program$(Partek$Inc.,$Chesterfield,$MO,$USA).$Normalized$intensity$values$were$obtained$either$by$ using$the$Robust$Multichip$Average$method$or$by$removing$those$sequences$near$to$background$ values.$ In$ addition,$ probe$ sets$ that$ remained$ unchanged$ across$ the$ experiment$ were$ also$ removed,$which$left$a$total$of$8562$genes$for$further$analyses.$

Differentially$ expressed$ genes$ from$ control$ group$ were$ detected$ using$ a$ linear$ regression$ model,$using$the$equation$Yi=$condition$+$ε,$where$Yi$is$the$intensity$“Y”$for$each$sequence$“i”,$ “condition”$ is$ referred$ to$ the$ condition$ effect$ in$ the$ intensity$ observed$ and$ $ “ε”$ is$ the$ non* measurable$error$that$gathered$the$non*predicted$part$of$the$rest$of$variables.$$ $ $

# 77$ III.#SUBJECTS#AND#METHODS#

$ Table# 9.$List$of$TaqMan$probes$for$real*time$PCR$used$in$adipose$tissue$biopsies$and/or$ PBMC,$as$well$as$in$human$subcutaneous$cultured$adipocytes$ Symbol# Name# Reference# 18S1$ 18S$ribosomal$ Hs00917508_m1$ AADAC1$ Arylacetamide$deacetylase$ Hs00153677_m1$ ACTG21$ Actin,$gamma$2$ Hs01123712_m1$ ADGRE11,$2$ Adhesion$G$protein*coupled$receptor$E1$ Hs00892591_m1$ ADIPOQ1,$2$ Adiponectin$ Hs00605917_m1$ ALCAM1$ Activated$leukocyte$cell$adhesion$molecule$ Hs00977641_m1$ APLNR1$ Apelin$receptor$ Hs00766613_m1$ CCL21,$2$ Chemokine$(C*C$motif)$ligand$2$ Hs00234140_m1$ CHIT11$ Chitinase$1$ Hs00185753_m1$ CREBBP2$ CREB$binding$protein$ Hs00231733_m1$ FITM22$ Fat$storage$inducing$transmembrane$protein$2$ Hs00380930_m1$ FNDC53$ Fibronectin$type$III$domain*containing$protein$5$ Hs00401006_m1$ FRZB1$ Fizzled*related$protein$ Hs00173503_m1$ GAPDH2$ Glyceraldehyde*3*phosphate$dehydrogenase$ Hs02758991_g1$ IL61,$2$ Interleukin$6$ Hs00985639_m1$ IL101,$2$ Interleukin$10$ Hs00961622_m1$ MSR11$ Macrophage$scavenger$receptor$1$ Hs00234007_m1$ MYH111$ Myosin,$heavy$chain$11$ Hs00224610_m1$ NCK21,$2$ NCK$adaptor$protein$2$ Hs02561903_s1$ PLA2G71$ Phospholipase$A2,$group$VII$ Hs00965837_m1$ RPTOR2$ Regulatory$associated$protein$of$MTOR$complex$1$ Hs00375332_m1$ TRRAP2$ Transcription$domain$associated$protein$ Hs00268883_m1$ 1Probes$used$in$adipose$tissue.$ 2Probes$used$in$peripheral$blood$mononuclear$cells$(PBMC).$ 3Probes$used$in$human$subcutaneous$adipocytes.$ $ $ $

78# # III.#SUBJECTS#AND#METHODS#

The$ determination$ of$ enriched*pathways$ was$ conducted$ by$ using$ the$ Ingenuity$ Pathway$ Analysis$(IPA)$(Ingenuity$Systems$Inc.,$Redwood$City,$CA,$USA),$whose$database$includes$manually$ curated$and$fully$traceable$data$derived$from$literature$sources,$at$the$Centro$de$Investigación$ Médica$Aplicada$(CIMA,$University$of$Navarra,$Spain).$The$input$was$all$differentially$regulated$ genes$ respecting$ to$ control$ group$ in$ each$ group$ (nominal$ P*value<$ 0.05),$ considering$ as$ a$ significant$ enriched$ pathway$ those$ with$ a$ z*score$≥$1.6$and$≤$1.6.$The$z*score,$ whose$ primary$ purpose$is$to$infer$the$activation$state$of$a$specific$pathway,$is$calculated$as$described$elsewhere$ (Ingenuity$Systems$website:$revised$on$July$2016).$$

Furthermore,$some$differentially$expressed$genes$regarding$to$control$group$in$the$microarray$ analysis$were$selected$for$validation,$using$RT*PCR$based$on$following$criteria:$$

1.$ Genes$ implicated$ in$ enriched$ pathways,$ preferably$ in$ more$ than$ one$ and$ with$ a$ fold$ change$of$more$than$±$1.5$$

2.$ Genes$ with$ biological$ significance$ involved$ in$ either$ inflammatory$ or$ lipid$ metabolic$ pathways.$$

Both,$RNA$preparation$for$RT*PCR$and$gene$expression$analyses,$were$conducted$according$ with$ standard$ protocols$ (see# point# 6.1),$ using$ 18S$ gene$ as$ housekeeping$ and$ calculating$ gene$ expression$by$the$2*ΔΔCt$method$(Livak$and$Schmittgen,$2001).$

7.#Genomic#DNA#isolation#and#methylation#analyses##

Genomic$ DNA$ from$ buffy$ coat$ obtained$ at$ baseline$ and$ at$ the$ end$ of$ the$ nutritional$ intervention$was$extracted$using$the$MaterPureTM$DNA$purification$Kit$(Epicentre,$Madison,$WI,$ USA)$according$with$manufacturer’s$instructions.$The$microarray$preparation,$hybridization$and$ scanning$ was$ performed$ at$ INCLIVA$ Health$ Research$ Institute$ (Valencia,$ Spain)$ while$ the$ preprocessing$ and$ normalization$ of$ methylation$ data$ were$ performed$ at$ CIMA$ (Univesity$ of$ Navarra).$As$previously$described$by$Mansego$et$al.$(2015),$DNA$was$quantified$using$PicoGreen$ double*stranded$ DNA$ (dsDNA)$ Quantification$ Reagent®$ (Invitrogen).$ EZ$ DNA$ methylation$ kit$ (Zymo$Research,$Irvine,$CA,$USA)$was$used$to$bisulfite$modification$of$500$ng$of$genomic$DNA$ according$ with$ manufacturer’s$ protocol.$ $ Bisulfite*treated$ genomic$ DNA$ was$ amplified$ and$ hybridized$using$the$Infinitum$Human$Methylation$450K$BeadChips$(Illumina,$San$Diego,$CA,$USA)$ and$scanned$using$the$Illumina$HiScanSQ$platform.$$DNA$methylation$signals$from$scanned$arrays$ were$preprocessed$and$normalized$using$the$R$scripts$(Gentleman$R$et$al.,$2005)$of$Touleimat$ and$Tost$(2012).$After$normalization$with$the$DASEN$method$(Pidsley$et$al.,$2013),$methylation$ difference$ between$ the$ beginning$ and$ the$ end$ of$ the$ nutritional$ intervention$ was$ calculated.$

# 79$ III.#SUBJECTS#AND#METHODS#

LIMMA$(Linear$Models$for$Microarray$Data)$(Smyth$et$al.,$2005)$was$then$used$to$identify$the$ probes$ with$ significant$ differential$ methylation$ between$ experimental$ conditions.$ The$ linear$ model$was$adjusted$for$body$weight$loss.$$

Moreover,$in$order$to$explore$possible$associations$between$methylation$changes$with$some$ biochemical$and$metabolic$biomarkers$related$to$the$phenotype,$some$CpG$sites$were$selected$ based$on$the$following$criteria:$

1.$ Genes$differentially$methylated$(P<$0.001)$as$compared$with$control$group$in$not$only$the$ three$supplemented$groups$(EPA,$α*lipoic$acid$and$EPA+α*lipoic$acid)$but$also$in$both$groups$with$ EPA$supplementation$or$in$both$groups$with$α*lipoic$acid$supplementation.$$

2.$ Genes$implicated$in$either$inflammation$or$lipid$metabolism.$$

Both,$RNA$preparation$for$RT*PCR$and$gene$expression$analyses,$were$conducted$according$ with$standard$protocols$(see#point#6.1),$using$GADPH$gene$as$housekeeping$and$calculating$gene$ expression$by$the$2*ΔΔCt$method$(Livak$and$Schmittgen,$2001).$

8.#Statistical#analyses#

Statistical$ analyses$ were$ performed$ using$ Stata$ Statistical$ Software$ (Release$ 12.$ College$ Station,$ TX:$ StataCorp$ LP).$ Differences$ were$ considered$ significant$ at$ two*sided$ P*value<$ 0.05.$ Moreover,$ the$ multiple$ testing$ correction$ (Benjamini–Hochberg)$ method$ was$ performed$ when$ appropriate$(Benjamini$and$Hochberg,$1995).$

To$select$the$adequate$test,$normal$distribution$was$assessed$not$only$by$the$Shapiro*Wilk$test$ and$normal$probability$plots,$but$also$using$the$heteroskedasticity$by$the$Breusch*Pagan/Cook* Weisberg$test.$Comparisons$between$groups$at$baseline$were$evaluated$by$one*way$Analysis$of$ Variance$(ANOVA)$test$or$by$the$Kruskal*Wallis$test.$Both,$the$contrasts$between$baseline$and$ endpoint,$ as$ well$ as$ the$ differences$ between$ two$ means$ or$ medians,$ were$ assessed$ by$ paired$ Student’s$t$test$or$the$Wilcoxon$signed*rank$test$as$applicable.$$

In$ order$ to$ evaluate$ the$ differential$ effects$ between$ treatments,$ the$ changes$ within$ each$ group$(after*before)$were$compared$between$groups$by$two*way$ANOVA$analysis,$adjusting$in$ some$ cases$ by$ potential$ confounders$ as$ body$ weight$ and$ the$ respective$ value$ at$ baseline$ as$ pertinent.$Moreover$when$a$statistically$significant$interaction$was$found$(EPA$x$α*lipoic$acid),$a$ contrast$ analysis$ was$ applied$ to$ identify$ which$ conditions$ were$ different$ from$ each$ other.$ Depending$of$the$normality$of$the$sample,$the$relationships$between$variables$were$assessed$by$ using$either$Pearson$or$Spearman$correlations.$$

80# # III.#SUBJECTS#AND#METHODS#

Additionally,$ to$ evaluate$ whether$ the$ changes$ in$ leukocytes$ were$ related$ with$ the$ changes$ in$ Framingham$score,$in$chapter$2,$the$change$in$the$leukocyte$count$was$categorized$according$to$ the$ change$ in$ the$ Framingham$ score$ stratified$ into$ tertiles,$ and$ was$ compared$ by$ Analysis$ of$ Covariance$(ANCOVA)$test,$adjusting$for$body$weight$loss$and$the$respective$value$at$baseline.$ Furthermore,$in$chapter$4,$the$non*normally$distributed$variables$were$log*transformed$before$an$ appropriate$application$of$parametric$test.$ $ $

# 81$ III.#SUBJECTS#AND#METHODS#

9.#References#

Abete,$ I.,$ Parra,$ D.,$ and$ Martínez,$ J.A.$ (2009).$ Legume*,$ fish*,$ or$ high*protein*based$ hypocaloric$ diets:$ effects$on$weight$loss$and$mitochondrial$oxidation$in$obese$men.$J.$Med.$Food$12,$100–108.$

Alberti,$K.G.,$and$Zimmet,$P.Z.$(1998).$Definition,$diagnosis$and$classification$of$diabetes$mellitus$and$its$ complications.$ Part$ 1:$ diagnosis$ and$ classification$ of$ diabetes$ mellitus$ provisional$ report$ of$ a$ WHO$ consultation.$Diabet.$Med.$J.$Br.$Diabet.$Assoc.$15,$539–553.$

Benjamini,$ Y.,$ and$ Hochberg,$ Y.$ (1995).$ Controlling$ the$ False$ Discovery$ Rate:$ A$ Practical$ and$ Powerful$ Approach$to$Multiple$Testing.$J.$R.$Stat.$Soc.$B$Methodol.$57,$289–300.$

Bolanowski,$M.,$and$Nilsson,$B.E.$(2001).$Assessment$of$human$body$composition$using$dual*energy$x*ray$ absorptiometry$ and$ bioelectrical$ impedance$ analysis.$ Med.$ Sci.$ Monit.$ Int.$ Med.$ J.$ Exp.$ Clin.$ Res.$ 7,$ 1029–1033.$

Codex$Alimentarius$Comimission$(2015).$Report$of$the$twenty$forth$session$of$the$codex$committee$on$fats$ and$oils,$Malaysia.$

Fernández*Galilea,$ M.,$ Pérez*Matute,$ P.,$ Prieto*Hontoria,$ P.L.,$ Sáinz,$ N.,$ López*Yoldi,$ M.,$ Houssier,$ M.,$ Martínez,$J.A.,$Langin,$D.,$and$Moreno*Aliaga,$M.J.$(2014).$α*Lipoic$acid$reduces$fatty$acid$esterification$ and$lipogenesis$in$adipocytes$from$overweight/obese$subjects.$Obesity$(Silver$Spring)$22,$2210–2215.$

Friedewald,$W.T.,$Levy,$R.I.,$and$Fredrickson,$D.S.$(1972).$Estimation$of$the$concentration$of$ low*density$ lipoprotein$cholesterol$in$plasma,$without$use$of$the$preparative$ultracentrifuge.$Clin.$Chem.$18,$499– 502.$

Gentleman$ R,$ Carey$ VJ,$ Huber$ W,$ Irizarry$ RA,$ Dudoit$ S$ (Eds)$ (2005).$ Bioinformatics$ and$ Computational$ Biology$Solutions$Using$R$and$Bioconductor,$New$York,$NY:$Springer.$

Guerrero*Romero,$F.,$Simental*Mendía,$L.E.,$González*Ortiz,$M.,$Martínez*Abundis,$E.,$Ramos*Zavala,$M.G.,$ Hernández*González,$ S.O.,$ Jacques*Camarena,$ O.,$ and$ Rodríguez*Morán,$ M.$ (2010).$ The$ product$ of$ triglycerides$ and$ glucose,$ a$ simple$ measure$ of$ insulin$ sensitivity.$ Comparison$ with$ the$ euglycemic* hyperinsulinemic$clamp.$J.$Clin.$Endocrinol.$Metab.$95,$3347–3351.$

Ibero*Baraibar,$ I.,$ Abete,$ I.,$ Navas*Carretero,$ S.,$ Massis*Zaid,$ A.,$ Martínez,$ J.A.,$ and$ Zulet,$ M.A.$ (2014).$ Oxidised$LDL$levels$decreases$after$the$consumption$of$ready*to*eat$meals$supplemented$with$cocoa$ extract$within$a$hypocaloric$diet.$Nutr.$Metab.$Cardiovasc.$Dis.$NMCD$24,$416–422.$

Ingenuity$Systems$Upstream$Regulator$Analysis.$Available$online:$http://www.ingenuity.com/products/ipa/$ [revised:$July$2016].$

Kojima,$M.,$Hosoda,$H.,$Date,$Y.,$Nakazato,$M.,$Matsuo,$H.,$and$Kangawa,$K.$(1999).$Ghrelin$is$a$growth* hormone*releasing$acylated$peptide$from$stomach.$Nature$402,$656–660.$

Livak,$ K.J.,$ and$ Schmittgen,$ T.D.$ (2001).$ Analysis$ of$ relative$ gene$ expression$ data$ using$ real*time$ quantitative$PCR$and$the$2(*Delta$Delta$C(T))$Method.$Methods$San$Diego$Calif$25,$402–408.$

Mansego,$M.L.,$Milagro,$F.I.,$Zulet,$M.Á.,$Moreno*Aliaga,$M.J.,$and$Martínez,$J.A.$(2015).$Differential$DNA$ Methylation$in$Relation$to$Age$and$Health$Risks$of$Obesity.$Int.$J.$Mol.$Sci.$16,$16816–16832.$

Matarese,$L.E.$(1997).$Indirect$calorimetry:$technical$aspects.$J.$Am.$Diet.$Assoc.$97,$S154–S160.$

Matthews,$ D.R.,$ Hosker,$ J.P.,$ Rudenski,$ A.S.,$ Naylor,$ B.A.,$ Treacher,$ D.F.,$ and$ Turner,$ R.C.$ (1985).$ Homeostasis$model$assessment:$insulin$resistance$and$beta*cell$function$from$fasting$plasma$glucose$ and$insulin$concentrations$in$man.$Diabetologia$28,$412–419.$

National$ Research$ Council$ (US)$ (1989).$ Recommended$ Dietary$ Allowances.$ Washington$ D.C:$ National$ Academy$Press.$

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Pérez,$ S.,$ Parra,$ M.D.,$ Moretín,$ B.M.$ de,$ Rodríguez,$ M.C.,$ and$ Martínez,$ J.A.$ (2005).$ Evaluación$ de$ la$ variabilidad$ intraindividual$ de$ la$ medida$ de$ composición$ corporal$ mediante$ bioimpedancia$ en$ voluntarias$sanas$y$su$relación$con$el$índice$de$masa$corporal$y$el$pliegue$tricipital.$Enferm.$Clínica$15,$ 307–314.$

Pérez*Matute,$P.,$Martí,$A.,$Martínez,$J.A.,$Fernández*Otero,$M.P.,$Stanhope,$K.L.,$Havel,$P.J.,$and$Moreno* Aliaga,$ M.J.$ (2005).$ Eicosapentaenoic$ fatty$ acid$ increases$ leptin$ secretion$ from$ primary$ cultured$ rat$ adipocytes:$role$of$glucose$metabolism.$Am.$J.$Physiol.$Integr.$Comp.$Physiol.$288,$R1682–R1688.$

Pidsley,$R.,$Y$Wong,$C.C.,$Volta,$M.,$Lunnon,$K.,$Mill,$J.,$and$Schalkwyk,$L.C.$(2013).$A$data*driven$approach$ to$preprocessing$Illumina$450K$methylation$array$data.$BMC$Genomics$14,$293.$

WHO$Expert$Committee$(1996).$Hypertension$control.$World$Health$Organization$Technical$Report$Series$ 862.$

Smith,$M.L.,$Baggerly,$K.A.,$Bengtsson,$H.,$Ritchie,$M.E.,$and$Hansen,$K.D.$(2013).$illuminaio:$An$open$source$ IDAT$parsing$tool$for$Illumina$microarrays.$F1000Research$2,$264.$

Smyth,$G.K.,$Ritchie,$M.,$Thorne,$N.,$and$Wettenhall,$J.$(2005).$LIMMA:$linear$models$for$microarray$data.$In$ Bioinformatics$and$Computational$Biology$Solutions$Using$R$and$Bioconductor.$Statistics$for$Biology$and$ Health.$

Tai,$M.M.$(1994).$A$mathematical$model$for$the$determination$of$total$area$under$glucose$tolerance$and$ other$metabolic$curves.$Diabetes$Care$17,$152–154.$

Thorsdottir,$ I.,$ Tomasson,$ H.,$ Gunnarsdottir,$ I.,$ Gisladottir,$ E.,$ Kiely,$ M.,$ Parra,$ M.D.,$ Bandarra,$ N.M.,$ Schaafsma,$G.,$and$Martínez,$J.A.$(2007).$Randomized$trial$of$weight*loss*diets$for$young$adults$varying$ in$fish$and$fish$oil$content.$Int.$J.$Obes.$31,$1560–1566.$

Touleimat,$ N.,$ and$ Tost,$ J.$ (2012).$ Complete$ pipeline$ for$ Infinium(®)$ Human$ Methylation$ 450K$ BeadChip$ data$ processing$ using$ subset$ quantile$ normalization$ for$ accurate$ DNA$ methylation$ estimation.$ Epigenomics$4,$325–341.$

Wiklund,$P.,$Toss,$F.,$Weinehall,$L.,$Hallmans,$G.,$Franks,$P.W.,$Nordström,$A.,$and$Nordström,$P.$(2008).$ Abdominal$and$gynoid$fat$mass$are$associated$with$cardiovascular$risk$factors$in$men$and$women.$J.$ Clin.$Endocrinol.$Metab.$93,$4360–4366.$

# 83$

$

IV. RESULTS#

#

$

$

CHAPTER$1$

$ $ Post%print$version$of$the$article:$$ $ $ $ $ Effects# of# αZlipoic# acid# and# eicosapentaenoic# acid# in# overweight# and# obese# women# during# weight#loss## # Ana$ E.$ Huerta1,2,$ Santiago$ Navas*Carretero1,2,3,$ Pedro$ L.$ Prieto*Hontoria1,3,4,$ J.$ Alfredo$ Martínez1,2,3,$María$J.$Moreno*Aliaga1,2,3$ $ 1Department$of$Nutrition,$Food$Science$and$Physiology.$University$of$Navarra,$Pamplona,$Spain.$ 2Centre$for$Nutrition$Research.$Faculty$of$Pharmacy.$University$of$Navarra,$Pamplona,$Spain.$ 3Biomedical$ Research$ Centre$ Network$ in$ Physiopathology$ of$ Obesity$ and$ Nutrtition$ (CIBERobn).$ National$Institute$of$Health$Carlos$III$(ISCIII),$Madrid,$Spain.$ 4Current$affiliation:$Faculty$of$Health$and$Physical$Activity$Science.$University$SEK,$Santiago$de$Chile$ (Chile).$ $ $ Obesity$(Silver$Spring),$2015,$23:$313*321$$ doi:$10.1002/oby.20966$ $ $ $ $ $ $ Impact$factor$(2015):$3.614$ 18/78$in$Nutrition$&$Dietetics$$ 41/131$in$Endocrinology$&$Metabolism$

#

IV.#RESULTS#(CHAPTER(1)#

Abstract#

Objective:$ To$ evaluate$ the$ potential$ body$ weight*lowering$ effects$ of$ dietary$ supplementation$ with$ eicosapentaenoic$ acid$ (EPA)$ and$ α*lipoic$ acid$ separately$ or$ combined$ in$ healthy$ overweight/obese$women$following$a$hypocaloric$diet.$

Methods:$ This$ is$ a$ short*term$ double*blind$ placebo*controlled$ study$ with$ parallel$ design$ that$ lasted$10$weeks.$Of$the$randomized$participants,$97$women$received$the$allocated$treatment$ [Control,$EPA$(1.3$g/d),$α*lipoic$acid$(0.3$g/d),$and$EPA$+$α*lipoic$acid$(1.3$g/d$+$0.3$g/d)],$and$77$ volunteers$completed$the$study.$All$groups$followed$an$energy*restricted$diet$of$30%$less$than$ total$energy$expenditure.$Body$weight,$anthropometric$measurements,$body$composition,$resting$ energy$expenditure,$blood$pressure,$serum$glucose,$and$insulin$and$lipid$profile,$as$well$as$leptin$ and$ghrelin$levels,$were$assessed$at$baseline$and$after$nutritional$intervention.$

Results:$Body$weight$loss$was$significantly$higher$(P<$0.05)$in$those$groups$supplemented$with$α* lipoic$acid.$EPA$supplementation$significantly$attenuated$(P<$0.001)$the$decrease$in$leptin$levels$ that$ occurs$ during$ weight$ loss.$ Body$ weight$ loss$ improved$ lipid$ and$ glucose$ metabolism$ parameters$but$without$significant$differences$between$groups.$

Conclusions:$ The$ intervention$ suggests$ that$ α*lipoic$ acid$ supplementation$ alone$ or$ in$ combination$ with$ EPA$ may$ help$ to$ promote$ body$ weight$ loss$ in$ healthy$ overweight/obese$ women$following$energy*restricted$diets.$

Keywords:$omega*3,$nutritional$intervention,$lipoic$acid,$nutritional$supplementation$and$obesity$ $ $

# 89$ IV.#RESULTS#(CHAPTER(1)#

Introduction#

Obesity$is$considered$as$an$important$public$health$disease$that$predisposes$people$to$a$wide$ range$ of$ other$ chronic$ pathologies$ such$ as$ type*2$ diabetes,$ coronary$ heart$ disease,$ stroke,$ obstructive*sleep$apnea$and$certain$forms$of$cancer$(World$Health$Organization,$2011).$$

α*Lipoic$ acid$ (thioctic$ acid,$ 5*(1,2*dithiolan*3*yl)$ pentanoic$ acid)$ is$ a$ naturally$ occurring$ antioxidant$ and$ co*factor$ for$ mitochondrial$ enzymes$ (Fernández*Galilea$ et$ al.,$ 2013).$ Several$ studies$ have$ suggested$ important$ anti*obesity$ properties$ for$ α*lipoic$ acid.$ Thus,$ in$ rodents,$ α* lipoic$acid$supplementation$promotes$the$reduction$of$body$weight$and$fat$mass$(Prieto*Hontoria$ et$al.,$2009,$2011,$2012)$by$decreasing$not$only$food$intake,$but$also$by$reducing$feed$efficiency$ (Prieto*Hontoria$ et$ al.,$ 2009)$ and$ stimulating$ energy$ expenditure$ (Wang$ et$ al.,$ 2010).$ Furthermore,$some$trials$in$overweight,$obese$and/or$diabetic$subjects$have$also$suggested$that$ supplementation$with$α*lipoic$acid$could$in$some$cases$promote$weight$loss,$reduce$fat$mass$and$ increase$ satiety$ (Carbonelli$ et$al.,$ 2010;$ Koh$ et$al.,$ 2011).$ However,$ studies$ in$ humans$ with$ α* lipoic$ acid$ supplementation$ are$ limited$ and$ with$ controversial$ outcomes,$ and$ it$ is$ difficult$ to$ reach$ firm$ conclusions$ regarding$ the$ proper$ dose$ and$ its$ potential$ role$ in$ the$ treatment$ of$ obesity.$

Although$several$studies$have$demonstrated$that$n*3$polyunsaturated$fatty$acids$(n*3$PUFAs)$ could$improve$cardiovascular$health,$the$use$of$these$fatty$acids$on$obesity$treatment$remains$ unclear$(Lorente*Cebrián$et$al.,$2013).$Thus,$some$studies$described$that$n*3$PUFAs$could$have$ positive$ effects$ on$ body$ weight$ reduction$ (Kunesova$ et$ al.,$ 2006;$ Thorsdottir$ et$ al.,$ 2007),$ whereas$others$have$reported$no$effects$on$adiposity$(DeFina$et$al.,$ 2011;$ Krebs$ et$al.,$ 2006).$ Most$ of$ the$ studies$ in$ humans$ have$ been$ performed$ using$ a$ combination$ of$ docosahexaenoic$ acid$ (DHA)$ and$ eicosapentaenoic$ acid$ (EPA),$ but$ only$ few$ trials$ have$ evaluated$ the$ effects$ of$ these$ n*3$ PUFAs$ separately.$ Several$ investigations$ have$ suggested$ that$ EPA$ and$ DHA$ have$ different$ hemodynamic$ properties$ and$ actions$ on$ cell$ function$ (Mori$ and$ Woodman,$ 2006;$ Tishinsky$ et$ al.,$ 2011).$ Furthermore,$ the$ effects$ of$ EPA$ supplementation$ in$ healthy$ overweight/obese$subjects$on$body$weight$and$fat$mass$loss$have$been$scarcely$investigated$until$ now.$

Hence,$ the$ aim$ of$ this$ trial$ was$ to$ evaluate$ the$ potential$ body$ weight$ lowering$ effects$ of$ dietary$ supplementation$ with$ EPA$ and$ α*lipoic$ acid$ separately$ or$ in$ combination,$ in$ healthy$ overweight/obese$women$during$a$hypocaloric$diet.$$

#

90# # IV.#RESULTS#(CHAPTER(1)#

Design#and#Methods#

Participants$

Women$aged$20*50$years$were$recruited$by$advertisement$in$local$newspapers$and$by$calls$for$ volunteers$ from$ the$ database$ of$ the$ Metabolic$ Unit$ (MU)$ of$ the$ University$ of$ Navarra.$ The$ inclusion$criteria$were:$1)$female,$2)$regular$menstrual$cycles,$3)$BMI$between$27.5$and$40$Kg/m2,$ 3)$ unchanged$ weight$ (±$ 3$ Kg)$ for$ the$ last$ 3$ months$ and$ 4)$ all$ subjects$ should$ have$ an$ overall$ healthy$physical$and$psychological$condition.$$

Study$design$and$intervention$

This$ study$ was$ a$ parallel,$ short*term$ randomized$ double$ blind$ placebo*controlled$ trial$ performed$in$compliance$with$the$Helsinki$Declaration.$The$trial$was$approved$by$the$Research$ Ethics$Committee$of$the$University$of$Navarra$and$registered$at$clinicaltrials.gov$as$NCT01138774.$

Out$of$the$103$participants,$who$gave$their$signed$consent$and$were$assigned$by$a$scientist$of$ the$MU$to$one$of$the$four$experimental$groups$by$simple$randomization$using$the$Microsoft$Excel$ Office$ 2003$ Software$ (Microsoft$ Inc.,$ USA)$ that$ generates$ random$ numbers,$ finally$ 97$ women$ received$the$allocated$treatment,$with$77$finishing$the$trial$$(Figure#1).$The$intervention$groups$ varied$ in$ the$ type$ of$ daily$ supplement:$ 1)$ Control$ group:$ 3$ placebo*I$ capsules$ (containing$ sunflower$ oil)$ and$ 3$ placebo*II$ capsules$ (containing$ same$ excipients$ than$ the$ α*lipoic$ acid$ capsules),$ 2)$ EPA$ group:$ 1300$ mg/d$ of$ EPA$ distributed$ in$ 3$ capsules$ of$ EPA$ 80$ (provided$ by$ Solutex®,$Madrid,$Spain),$containing$433.3$mg$of$EPA$and$13.8$mg$of$DHA$as$ethyl*ester;$and$3$ placebo*II$capsules,$3)$α*lipoic$acid$group:$300$mg/d$of$α*lipoic$acid$from$3$capsules$containing$ 100$mg$of$α*lipoic$ acid$ (Nature’s$ Bounty®,$ NY,$ USA),$ and$ 3$ placebo*I$ capsules;$ and$ 4)$ EPA+$ α* lipoic$acid$group:$1300$mg/d$of$EPA$(distributed$in$3$capsules$of$EPA$80)$and$300$mg/d$of$α*lipoic$ acid$$(from$3$capsules$containing$100$mg$of$α*lipoic$acid),$respectively.$Therefore,$each$daily$dose$ was$divided$into$three$equal$doses$to$minimize$the$impact$of$decline$in$plasma$levels$after$oral$ supplementation,$and$each$participant$consumed$a$total$of$6$capsules$per$day$(2$at$breakfast,$2$ at$ lunch$ and$ 2$ at$ dinner).$ Both$ placebo*I$ (sunflower$ oil)$ and$ EPA$ capsules$ were$ provided$ by$ Solutex,$and$were$similar$in$shape$and$size.$Placebo*II$capsules$were$similar$in$appearance$to$the$ α*lipoic$acid$capsules.$ $ $

# 91$ IV.#RESULTS#(CHAPTER(1)#

$ Assessed%for%eligibility% $ (n=122)% Excluded%(n=19)% $ ?Not%meeAng%inclusion%criteria%(n=17)% ?Declined%to%parAcipate%(n=2)%

Randomly%assigned% $ (%n=103)%

$

$ Alloca&on(

Allocated(to(EPA+α0 $ Allocated(to(α0lipoic( lipoic(acid(treatment( acid(treatment!(n=26)% (n=26)% Allocated(to(Control( Allocated(to(EPA( ?Received%allocated% $ ?Received%allocated% treatment((n=31)% treatment((n=20)% intervenAon%(n=23)% intervenAon%(n=23)% ?Received%allocated% ?Received%allocated% ?Did%not%receive% ?Did%not%receive% intervenAon%(n=31)% intervenAon%(n=20)% allocated%intervenAon% $ allocated%intervenAon% (did%not%come%to%first% (did%not%come%to%first% visit%n=%3)% $ visit%n%=%3)%

$ Follow0up(

?Lost%to%follow?up% ?Lost%to%follow?up% ?Lost%to%follow?up% ?Lost%to%follow?up% (n=4)% $ (n=7)% (n=1)% (n=2)% ?DisconAnued% ?DisconAnued% ?DisconAnued% ?DisconAnued% intervenAon%(viral% intervenAon%(health% $ intervenAon%(health% intervenAon%(health% infecAon%n=1,%did%not% problem%n=1,%Ame% problem%n=1)% problem%n=1)% follow%assigned% incompaAbiliAes%n=1)% treatment%n=1)% $

$ Analysis(

$ Analyzed0primary( Analyzed0primary( Analyzed0primary( outcome((n=22)% outcome((n=18)% outcome((n=20)% Analyzed0primary( Analyzed0biochemical( Analyzed0biochemical( Analyzed0biochemical( outcome((n=17)% $ variables((n=21)% variables((n=16)% variables((n=19)% Analyzed0biochemical( ?Excluded%from%analysis% ?Excluded%from%analysis% ?Excluded%from%analysis% variables((n=17)% (problems%with%blood% (problems%with%blood% (problems%with%blood% Analyzed0OGTT((n=16)% $ collecAon%n=1)% collecAon%n=2)% collecAon%n=1)% ?Excluded%from%analysis% Analyzed0OGTT((n=19)% Analyzed0OGTT((n=15)% Analyzed0OGTT((n=18)% (incomplete%data%n=1)% # ?Excluded%from%analysis% ?Excluded%from%analysis% ?Excluded%from%analysis% % (incomplete%data%n=3)% (incomplete%data%n=3)% (incomplete%data%n=2)% #

Figure# 1.# Flowchart$ of$ participants.$ Of$ the$ 103$ randomized$ women$ who$ met$ the$ inclusion$ criteria,$ 97$ started$ the$ allocated$ intervention,$ of$ these,$ 20$ participants$ (21%)$ did$ not$ completed$ the$ study$ as$ they$ either$discontinued$the$follow*up$because$of$unexpected$health$problems$(n=4),$withdrew$from$the$study$ and$did$not$came$to$all$visits$(n=15)$or$were$noncompliant$with$the$assigned$treatment$(n=1).$The$dropout$ rates$were$29%$(n=9),$10%$(n=2),$13%$(n=3)$and$26%$(n=6)$for$the$control,$EPA,$α*lipoic$acid$and$EPA$+$α* lipoic$ acid$ groups,$ respectively$ and$ no$ significant$ statistical$ differences$ were$ found$ in$ the$ dropout$ rates$ after$the$chi*square$statistic$test.$For$the$analysis$of$biochemical$variables,$4$volunteers$(1$control$group,$2$ EPA$group,$and$1$α*lipoic$acid$group)$were$excluded$due$to$problems$with$blood$collection.$Similarly,$in$the$ analysis$of$the$OGTT,$9$volunteers$(3$control$group,$3$EPA,$2$α*lipoic$acid$group$and$1$EPA$+$α*lipoic$acid$ group)$were$excluded$because$of$incomplete$data$as$a$result$of$complications$during$the$intravenous$blood$ collection.$# $ $

92# # IV.#RESULTS#(CHAPTER(1)#

All$intervention$groups$followed$a$weight*reduction$program$consisting$of$a$calorie*restricted$ balanced$ diet$ (55%$ carbohydrates;$ 30%$ lipids,$ 15%$ proteins)$ in$ accordance$ with$ the$ American$ Heart$Association$guidelines,$and$prescribed$individually$by$a$dietitian.$During$the$baseline$visit,$ each$subject$was$instructed$to$follow$an$energy*restricted$diet$accounting$to$30%$less$than$her$ total$ energy$ expenditure,$ and$ to$ not$ change$ the$ physical$ activity$ pattern$ during$ the$ 10*weeks$ intervention$ period.$ Additionally,$ volunteers$ were$ advised$ to$ avoid$ any$ marketed$ omega*3$ supplement$and$enriched$products.$

The$ main$ outcome$ of$ the$ study$ was$ the$ amount$ of$ weight$ loss.$ Follow*up$ visits$ were$ scheduled$with$each$volunteer$every$two$weeks$to$monitor$weight,$assess$the$compliance$with$ the$assigned$diet,$motivate$participants$and$ensure$that$all$capsules$were$consumed.$

At$baseline$and$at$endpoint$the$volunteers$went$under$10*12$h$fasting$conditions$to$the$MU$ and$ met$ with$ the$ physician,$ the$ dietitian$ and$ the$ nurse.$ Anthropometric$ measurements,$ body$ composition$ analysis,$ physical$ activity,$ food$ intake,$ respiratory$ exchange$ measurements$ by$ indirect$calorimetry$and$blood$pressure$were$evaluated$(See#Supporting#information).$A$catheter$ was$then$inserted$into$the$antecubital$vein$for$a$fasting$blood$sample$extraction.$Thereafter,$all$ participants$underwent$an$oral$glucose$tolerance$test$(OGTT).$In$order$to$evaluate$the$effect$of$ supplementation,$biochemical$determinations$of$glucose$and$lipid$metabolism$as$well$as$of$leptin$ and$ghrelin$were$assessed$(See#Supporting#Information).$$

Statistics$

The$ number$ of$ subjects$ per$ arm$ of$ intervention$ was$ estimated$ at$ sixteen$ subjects,$ which$ would$allow$a$detection$of$approximately$a$difference$of$5$kg$with$a$dispersion$of$5$kg$(5$±$5$kg)$ in$ weight$ loss$ between$ experimental$ groups,$ at$ a$ 0.05$ level$ of$ significance,$ with$ a$ statistical$ power$of$80%.$Since$a$dropout$rate$of$25%$was$expected,$the$final$sample$size$estimation$was$ established$in$at$least$20$participants$per$group$to$be$recruited.$

A$per*protocol$analysis$was$performed$including$all$volunteers$for$which$end*of*intervention$ values$ were$ available,$ however,$ intention*to*treat$ analysis$ using$ the$ last$ measured$ reported$ during$at$least$one$follow*up$visit$was$also$conducted$without$relevant$differences$between$both$ analyses$(Supplemental# Table# I).$For$categorical$variables,$differences$were$examined$by$using$ the$ chi*square$ test.$ Normality$ was$ evaluated$ using$ the$ Shapiro*Wilk$ test.$ Differences$ were$ considered$ significant$ if$ P*value<$ 0.05.$ Additionally$ for$ secondary$ outcomes$ the$ Benjamini* Hochberg$ approach$ (Benjamini$ and$ Hochberg,$ 1995)$ was$ used$ to$ correct$ for$ multiple$ comparisons$in$order$to$counteract$the$false$discovery$rate.$The$differences$after$the$nutritional$

# 93$ IV.#RESULTS#(CHAPTER(1)# intervention$ trial$ within$ each$ group$ were$ analyzed$ by$ Student's$ t$ test$ or$ by$ Wilcoxon$ test,$ depending$of$the$sample$distributions.$Comparisons$at$baseline$and$in$the$effects$between$the$ four$experimental$groups$in$subjects$who$completed$the$study$were$evaluated$using$the$two*way$ ANOVA.$When$the$differences$were$statistically$significant$at$the$interaction$level$(EPA$x$α*lipoic$ acid$ supplementation),$ a$ Student's$ t$ test$ was$ performed$ to$ compare$ the$ effects$ of$ each$ treatment.$ Also,$ some$ values$ affected$ by$ potential$ confounders$ were$ adjusted$ accordingly.$ Statistical$analysis$was$performed$using$Stata$Statistical$Software$(Release$12.$College$Station,$TX:$ StataCorp$LP).$$

Supporting#Information:#Design#and#Methods#

Oxidation$ levels$ of$ EPA$ supplements,$ based$ on$ peroxide$ and$ anisidine$ values,$ were$ below$ maximum$permitted$levels$(EFSA$Panel$on$Biological$Hazards,$2010).$The$subjects$and$researchers$ directly$assessing$the$outcomes$were$blinded$to$the$intervention.$$

Dietary$Intervention$

The$ total$ energy$ expenditure$ was$ estimated$ using$ the$ Harris*Benedict$ formula,$ using$ a$ correction$ factor$ to$ take$ into$ account$ the$ overweight$ status$ of$ the$ subjects$ and$ the$ resting$ metabolic$rate$(RMR)$measured$by$indirect$calorimetry$(Deltatrac$II®$metabolic$monitor$MBM* 200$Datex*Ohmeda,$Finland)$as$previously$described$(Thorsdottir$et$al.,$2007).$Additionally,$the$ adjustment$for$physical$activity$level$was$added.$At$baseline,$information$about$physical$activity$ patterns$ was$ collected$ using$ a$ questionnaire$ in$ order$ to$ evaluate$ the$ physical$ activity$ factor$ (National$Research$Council$(US),$1989).$

Energy$expenditure$

Indirect$calorimetry$was$performed$at$the$beginning$of$the$morning$after$10*12$h$of$fasting.$ For$calculating$the$RMR,$the$abbreviated$Weir$equation,$which$gives$a$similar$product$to$the$full$ version$and$does$not$have$the$disadvantage$of$24$h$urine$collection,$was$used$(Matarese,$1997).$$$

Anthropometric$measurements$and$body$composition$

Anthropometric$measurements$of$weight,$height,$waist$circumference$and$hip$circumference$ were$performed$according$to$established$protocols$(Pérez$et$al.,$2005).$The$assessment$material$ was$calibrated$and$standardized$before$starting$the$measurements;$height$was$evaluated$using$a$ stadiometer$(Seca$220,$Vogel$&$Halke,$Germany)$and$body$weight$using$a$digital$scale$accurate$to$

94# # IV.#RESULTS#(CHAPTER(1)#

0.1$Kg$(TBF*410GS,$TANITA,$Tokyo,$Japan).$The$waist$and$hip$circumferences$were$measured$with$ a$flexible$tape$with$a$precision$of$1$mm$and$were$used$for$calculating$the$waist$to$hip$ratio.$$

Body$composition$was$determined$by$two$methods:$bioimpedance$using$a$Tanita$(TBF*410GS,$ TANITA,$Tokyo,$Japan)$and$Dual$X*ray$Absorptiometry$(DXA)$using$the$Lunar,$Prodigy,$software$ version$6.0$(Madison,$WI,$USA)$following$standardized$methods.$$

The$android$and$gynoid$fat$were$defined$by$DXA$as$the$fat$deposition$in$the$upper$(central)$ body$region$and$the$fat$deposition$in$the$gluteofemoral$area$respectively$(Wiklund$et$al.,$2008).$$

Blood$pressure$

Systolic$and$diastolic$blood$pressures$were$measured$with$a$sphygmomanometer$taken$on$the$ right$arm$and$in$resting$sitting$position$as$described$by$the$WHO$Expert$Committee$(WHO$Expert$ Committee,$1996).$

Oral$glucose$tolerance$test$(OGTT)$

In$ accordance$ with$ the$ WHO$ standard$ protocol,$ study$ participants$ underwent$ a$ 2*h$ 75*g$ OGTT.$The$incremental$area$under$the$curve$(iAUC)$were$calculated$by$first$estimating$the$total$ area$under$curve$in$accordance$with$Tai's$Model$(Tai,$1994)$and$then$subtracting$the$area$from$ the$ baseline$ concentration$ over$ the$ 2*h$ period.$ Insulin$ resistance$ was$ estimated$ using$ the$ homeostasis$ model$ assessment$ (HOMA*IR)$ with$ the$ formula:$ fasting$ serum$ insulin$ (mU/L)$ x$ fasting$plasma$glucose$(mmol/L)/22.5$(Matthews$et$al.$1985).$$

Dietary$assessment$

In$order$to$evaluate$the$approximate$energy$intake,$at$the$screening$session$a$food$scale$was$ provided$to$facilitate$the$precise$weighting$registration$of$the$72*hour$recall$questionnaire.$The$ three*day$estimated$food$record$was$delivered$to$the$volunteers$in$the$screening$session$and$at$ the$8th$week.$This$procedure$had$to$be$handed$over$at$beginning$and$endpoint$of$the$study$and$ the$information$was$processed$using$the$program$DIAL$version$2.16,$for$the$evaluation$of$diets$ and$management$of$nutrition$data$(Alce$Ingenieria$Madrid,$Spain).$

Biochemical$determinations$

Serum$levels$of$glucose,$total$cholesterol,$HDL$cholesterol,$triglycerides$(TG),$free$fatty$acids$ (FFA)$ and$ β*hydroxybutyrate$ were$ measured$ using$ the$ auto$ analyzer$ PENTRA$ C200$ (HORIBA$

# 95$ IV.#RESULTS#(CHAPTER(1)# medical,$ Madrid,$ Spain).$ The$ values$ of$ LDL$ cholesterol$ were$ calculated$ using$ the$ Friedewald$ equation$(Friedewald$et$al.,$1972).$$

Plasma$ samples$ were$ used$ for$ assessing$ insulin,$ leptin$ and$ ghrelin$ following$ the$ manufacturer's$protocols$of$each$Enzyme$Immunoassay$(ELISA)$kit.$Insulin$was$determined$using$ a$Human$Sensitive$ELISA$Kit$(Mercodia$AB,$Sylveniusgatan,$Sweden)$with$a$sensitivity$of$1$mU/L.$ Leptin$was$evaluated$using$a$Human$ELISA$Kit$for$clinical$range$(BioVendor,$Brno,$Czech$Republic)$ with$a$sensitivity$of$0.2$ng/ml.$Ghrelin$was$measured$using$the$Human$ELISA$Kit$(RayBiotech$Inc.,$ Norcross,$GA,$USA)$with$a$sensitivity$of$161$pg/ml.$$

Results#

Effects$on$body$weight$and$body$composition$

There$ were$ no$ significant$ differences$ between$ the$ four$ experimental$ groups$ in$ all$ subjects$ whom$came$to$the$first$visit$(Supplemental#Table#I).$On$average,$the$physical$activity$factor$was$ 1.40$±$0.08,$which$corresponds$to$sedentary$activity$and$was$maintained$during$the$study$period;$ the$rate$of$capsules$intake$was$99%$in$all$the$groups.$In$general$no$adverse$effects$were$reported$ during$the$trial.$

After$ the$ 10$ weeks$ of$ nutritional$ intervention,$ all$ groups$ showed$ significant$ differences$ (P<$ 0.05)$in$the$decrease$in$body$weight,$as$well$as$in$waist$and$hip$circumference$and$the$waist$to$ hip$ratio$(WHR)$as$compared$to$baseline$(Table# 1).$Additionally$the$changes$in$weight$loss$and$ anthropometric$ measurements$ were$ accompanied$ by$ a$ significant$ reduction$ of$ total$ fat$ mass,$ lean$ mass,$ android$ fat,$ gynoid$ fat$ and$ the$ android/gynoid$ ratio$ in$ all$ the$ intervention$ groups$ (Table#1).$$Weight$loss$was$also$accompanied$by$a$statistically$significant$(P<$0.001)$decrease$in$ resting$metabolic$rate$(RMR)$in$all$intervention$groups.$

$

96# # IV.$RESULTS$(CHAPTER(1)$

Table$1.$Effects#of#10-wk#intervention#with#EPA#and#α-lipoic#acid#on#anthropometry,#body#composition,#RMR,#energy#intake#and#energy#balance1# EPA$+$$ Parameters$ Control$ EPA$ α

! 97# IV.$RESULTS$(CHAPTER(1)$

Table$1.$Continuation# Lean%mass%(Kg)% # # # # # # # Before# 42.6#±#6.3# 45.2#±#5.2# 42.1#±#3.9# 42.5#±#5.1# ns# ns# ns# Change## -1.0#±#1.12# -0.6#±#1.4# -1.4#±#1.42# -0.8#±#1.34# ns# ns# ns# Android%fat%(%)% # # # # # # # Before# 57.1#±#5.3# 57.1#±#5.1# 56.1#±#6.2# 57.3#±#6.1# ns# ns# ns# Change## -3.2#±#2.72# -3.7#±#2.42# -4.7#±#3.12# -4.7#±#3.52# ns# ns# ns# Gynoid%fat%(%)% # # # # # # # Before# 57.0#±#4.7# 57.6#±#3.8# 58.0#±#4.2# 56.6#±#4.0# ns# ns# ns# Change## -2.1#±#1.82# -2.3#±#1.42# -3.0#±#1.92# -3.2#±#2.32# ns# 0.039# ns# Adjusted#change5# -2.3#(0.4)# -2.5#(0.4)# -2.7#(0.4)# -3.1#(0.4)# ns# ns# ns# Android/Gynoid%ratio# # # # # # # Before# 1.00#±#0.11# 0.99#±#0.09# 0.97#±#0.09# 1.01#±#0.09# ns# ns# ns# Change## -0.02#±#0.044# -0.03#±#0.043# -0.03#±#0.043# -0.03#±#0.054# ns# ns# ns# RMR%(KJ/d)6# # # # # # # # Before# 6576#(139)# 6866#(164)# 6479#(150)# 6501#(154)# ns# ns# ns# Change# -399.2#(37.6)2# -305.0#(44.5)#2# -448.8#(40.6)#2# -370.3#(41.8)#2# 0.039# ns# ns# Adjusted#change5# -416.2#(34.6)# -327.5#(41.0)# -421.2#(#37.8)# -358.7#(38.3)# 0.049# ns# ns# Energy%intake%(KJ/d)# # # # # # # # Before# 7650#±#1178# 9072#±#1865# 8485#±#2617# 8346#±#1704# ns# ns# ns# -2731.6#±# -3045.1#±# Change# -2543.6#±#1713.22# -3693.2#±-1887.32# ns# ns# ns# 2594.02# 2056.82# Adjusted#change7# -3201.0#(260.3)# -3029.0#(282.8)# -2612.9#(269.8)# -3055.5#(287.0)# ns# ns# ns# 1Means#±#SDs#(all#unadjusted#such#values).#EPA,#eicosapentaenoic#acid;#RMR,#resting#metabolic#rate.#For#all#secondary#outcomes#the#P-values#were#adjusted#by#the#Benjamini- Hochberg#multiple-testing#correction#(Benjamini#and#Hochberg,#1995).#Data#from#all#subjects#for#whom#baseline#and#follow-up#measurements#were#available#were#included.# 2,3,4Significantly#different#from#baseline#(paired#samples%t#test):#2P<#0.001#3P<#0.01#4P<#0.05.## 5,6,7Means#(SEMs):#5adjusted#for#the#changes#in#body#weight;#6adjusted#by#the#age#and#lean#mass;#7adjusted#by#energy#intake#at#baseline.# 8Differences#between#groups#at#baseline#and#in#changes#(10#wk#–#before)#were#evaluated#by#2-way#ANOVA#(P<#0.05;#ns,#non-significant).#No#significant#differences#between#groups# were#found#in#secondary#outcomes#after#the#adjustment#by#Benjamini-Hochberg.#

98! ! IV.$RESULTS$(CHAPTER(1)$

# Supplemental$Table$I.$Intention#to#treat#analysis#of#the#effects#of#10-wk#intervention#with#EPA#and#α-lipoic#acid#on#anthropometry#and#body#composition#of# the#97#subjects#that#received#the#assigned#treatment1$ EPA$+$$ Parameters# Control$ EPA$ α

! 99# IV.$RESULTS$(CHAPTER(1)$

Supplemental$Table$I.$Continuation$ Fat%mass%(Kg)% # # # # # # # Before# 39.4#±#9.7# 41.1#±#5.5# 40.0#±#10.3# 38.8#±#8.3# ns# ns# ns# Change## -3.5#±#2.32# -4.1#±#1.72# -5.6#±#2.42# -4.2#±#3.12# ns# 0.02# ns# Adjusted#change5# -4.2#(0.2)# -4.3#(0.2)# -4.4#(0.2)# -4.2#(0.2)# ns# ns# ns# Lean%mass%(Kg)% # # # # # # # Before# 42.2#±#7.9# 45.9#±#5.4# 43.4#±#5.8# 45.1#±#6.2# ns# ns# ns# Change## -0.8#±#1.24# -0.6#±#1.3# -1.2#±#1.43# -0.8#±#1.24# ns# ns# ns# 1Means#±#SDs#(all#unadjusted#such#values).#EPA,#eicosapentaenoic#acid;#For#all#secondary#outcomes#the#P-values#were#adjusted#by#the#Benjamini-Hochberg#multiple-testing# correction.#Data#from#all#volunteers#that#received#the#allocated#treatment#and#which#data#were#available#in#one#of#the#four#follow-up#visits.### 2,3,4Significantly#different#from#baseline#(paired#samples%t#test):#2P<#0.0001#3P<#0.001#4P<#0.01.## 5Means#(SEMs)#and#adjusted#for#the#changes#in#body#weight.# 6Differences#between#groups#at#baseline#and#in#changes#(10#wk#–#before)#were#evaluated#by#2-way#ANOVA#(ns,#non-significant).#No#significant#differences#between#groups# were#found#in#secondary#outcomes#after#the#adjustment#by#Benjamini-Hochberg.# #

100!! IV.$RESULTS$(CHAPTER(1)$

When#the#differences#between#groups#were#evaluated,#it#was#observed#that#those#receiving#α: lipoic#acid#supplementation#showed#a#higher#statistically#significant#reduction#(P<#0.05)#in#body# weight,#hip#circumference#and#fat#mass#comparing#to#the#others#at#the#end#of#the#trial#(Table$1).# This# pattern# was# observed# in# the# α:lipoic# acid# supplemented# groups# from# the# first# weeks# of# treatment# and# became# more# prominent# during# the# trial# (Supplemental$ Figure$ I).# Supplementation# with# EPA# did# not# significantly# modify# body# weight,# but# caused# a# moderate# decrease# in# the# WHR# (P<# 0.05),# even# though# when# the# change# in# WHR# was# adjusted# for# body# weight#loss.#Interestingly,#EPA#supplementation#was#able#to#partly#prevent#(P<#0.05)#the#drop#in# RMR#secondary#to#weight#loss.##In#the#α:lipoic#acid#supplemented#groups,#the#fall#in#RMR#was#not# significantly#different#from#the#RMR#changes#observed#in#the#control#group,#besides#the#higher# weight# loss# observed# in# α:lipoic# acid# groups# (Table$ 1).# Moreover,# there# were# not# differences# between#groups#either#in#changes#(Table$1)#or#endpoint#values#of#energy#intake#(Supplemental$ Figure$II)#and#the#decrease#in#weight#loss#were#not#significantly#affected#by#the#changes#in#energy# intake#(R2=#0.007#P=#0.48).## # # # Control EPA

# -lipoic acid EPA+-lipoic acid # # a b 100 Week: P< 0.001 100 Week: P < 0.001 # Week*Group : P = 0.033 Week*Group: P = 0.009 # 95 # * 95 90 * # * # * * * 85 * * * % Decreased fat mass % Decreased ** # weight body % Decreased **

# 90 80 0 2 4 6 8 10 0 2 4 6 8 10 # Weeks Weeks # # c 110 Supplemental* Figure* I.! Changes( in( body( # Week: P < 0.001 Week*Group: P = 0.393 weight' (a),# fat# mass# (b)" and" lean" mass" (c)" # 105 during' the' nutritional' intervention.! Data$ # are$expressed$as$mean$±!SEM."Differences) 100 # were$ evaluated$ by$ repeated$ measures$ # 95 ANOVA% followed% by% multiple% comparison% # % Decreased lean mass lean % Decreased tests$ for$ evaluated$ significant$ differences% # 90 (*P

! 101# IV.$RESULTS$(CHAPTER(1)$

#

# 6000

# 5500

# $ 5000

4500 $ Energy intake at endpoint (KJ/d) at endpoint intake Energy 4000 $ Control EPA α-lipoic acid EPA+α-lipoic acid Intervention Group

Supplemental$ Figure$ II.$ Mean# (±# SEM)# of# energy# intake# at# endpoint.# The# values# of# energy# intake# were# adjusted#by#the#energy#intake#at#baseline.#The#comparisons#between#groups#were#evaluated#by#two:way# ANOVA#and#no#differences#were#found#(P>#0.05).#

Effects(on(lipid(and(glucose(metabolism(profile(

After#the#10#weeks#of#nutritional#intervention,#the#groups#supplemented#with#EPA#showed#a# significant#increase#(P<#0.05)#in#the#β:hydroxybutyrate#levels,#even#after#adjusting#by#body#weight# loss# (Table$ 2).# Serum# levels# of# total# cholesterol# were# significantly# (P<# 0.05)# reduced# in# all# experimental#groups,#while#the#LDL:cholesterol#only#decrease#significantly#(P<#0.05)#in#the#groups# with#EPA.#Furthermore,#there#was#a#statistically#significant#drop,#compared#with#baseline#levels,#in# triglycerides# (P<# 0.01)# and# diastolic# blood# pressure# (DBP)# (P<# 0.05)# in# the# group# supplemented# with#EPA+#α:lipoic#acid.#Besides,#no#significant#differences#were#found#between#groups#for#any#of# these#variables#(Supplemental$Table$II).##

Levels#of#fasting#glucose#were#reduced#in#the#four#experimental#groups#at#the#end#of#the#trial# as# compared# with# baseline,# although# statistically# significant# decreases# (P<# 0.01)# were# only# displayed#in#the#Control#and#EPA+#α:lipoic#acid#groups.#Furthermore,#insulin#levels#as#well#as#the# HOMA:IR# were# also# significantly# reduced# at# the# endpoint# in# Control# and# in# both# groups# supplemented#with#α:lipoic#acid#(Table$2).#However,#when#the#differences#between#groups#were# evaluated,# no# significant# changes# were# obtained,# although# a# significant# interaction# between# supplements#(P<#0.01)#was#observed#(Table$2).#

#

102!! IV.$RESULTS$(CHAPTER(1)$

Table$2.$Effects$of$10,wk$intervention$with$EPA$and$α,lipoic$acid$on$glucose$metabolism,$β,hydroxybutyrate,$leptin$and$ghrelin1$ EPA$+$$ Parameters$ Control$ EPA$ α

! 103$ IV.$RESULTS$(CHAPTER(1)$

Table$2.$Continuation$ Leptin((ng/mL)$ $ $ $ $ $ $ $ Before$ 23.6$±$7.6$ 21.7$±$8.0$ 22.5$±$10.0$ 20.4$±$7.0$ ns$ ns$ ns$ Change$ ,6.4$±$6.42$ ,0.9$±$5.2$ ,8.7$±$8.32$ ,3.5$±$5.44$ 0.0009*$ ns$ ns$ Adjusted$change8$ ,7.2$(1.2)$ ,1.8$(1.4)$ ,7.6$(1.3)$ ,2.9$(1.3)$ 0.0003*$ ns$ ns$ Ghrelin((ng/mL)( $ $ $ $ $ $ $ 240.8$±$ Before$ 230.1$±$103.3$ 251.2$±$98.0$ 170.1$±$91.3$ ns$ ns$ ns$ 136.3$ Change$ 15.2$±$117.5$ 11.4$±$79.9$ 8.8$±$76.7$ 56.7$±$56.43$ ns$ ns$ ns$ Adjusted$change8,10$ 18.3$(17.9)$ 21.6$(21.4)$ 13.0$(19.0)$ 38.2$(21.1)$ ns$ ns$ ns$ 1Means$±$SDs$(all$unadjusted$such$values).$EPA,$eicosapentaenoic$acid.$For$all$secondary$outcomes$the$P,values$were$adjusted$by$the$Benjamini,Hochberg$multiple,testing$ correction$(Benjamini$and$Hochberg,$1995).$$ 2,3,4Significantly$different$from$baseline$in$normally$distributed$samples$(paired$samples(t$test):$2P<$0.001$3P<$0.01$4P<.$0.05.$ 5,6,7Significantly$different$from$baseline$in$non,normally$distributed$variables$(Wilcoxon’s$test):$5P<$0.001$6P<$0.01$7P<.$0.05.$ 8,$9,$10Means$(SEMs):$8adjusted$for$the$changes$in$body$weight;$9adjusted$changes$for$the$insulin$levels$at$baseline;$10adjusted$by$ghrelin$levels$at$baseline.$ 11Differences$ between$ groups$ at$ baseline$ and$ in$ changes$ (10$ wk$ –$ before)$ were$ evaluated$ by$ 2,way$ ANOVA$ (P<$ 0.05;$ ns,$ non,significant).$ *Statistically$ significant$ differences$ between$ groups$ after$ the$ adjustment$ by$ Benjamini,Hochberg.$ When$ a$ significant$ interaction$ between$ groups$ was$ found$ (P<$ 0.05)$ it$ was$ performed$ an$ unpaired$samples$t$test,$means$that$do$not$share$a$common$superscript$letter$in$a$horizontal$line$were$significantly$different$(P<$0.05).$ $ $ $

104!! IV.$RESULTS$(CHAPTER(1)$

Supplemental$Table$II.$Effects$of$10,wk$intervention$with$EPA$and$α,lipoic$acid$on$lipid$metabolism$profile$and$blood$pressure1$ EPA$+$$$ $ Control$ EPA$ α

$

$

$

! 105$ IV.$RESULTS$(CHAPTER(1)$

Supplemental$Table$II.$Continuation$ SBP((mmHg)( $ $ $ $ $ $ $ Before$ 111.6$±$12.7$ 108.7$±$8.1$ 107.6$±$11.5$ 110.6$±$11.8$ ns$ ns$ ns$ Change$$ ,2.1$±$11.5$ 0.3$±$14.5$ ,0.3$±$10.2$ ,4.4$±$9.5$ ns$ ns$ ns$ DBP((mmHg)( $ $ $ $ $ $ $ Before$ 71.6$±$7.8$ 70.3$±$7.6$ 70.6$±$6.6$ 71.2$±$10.2$ ns$ ns$ ns$ Change$ ,3.2$±$8.7$ 0.0$±$12.9$ ,1.8$±$6.4$ ,5.0$±$7.34$ ns$ ns$ ns$ 1Means$±$SDs$(all$unadjusted$such$values).$DBP,$diastolic$blood$pressure;$EPA,$eicosapentaenoic$acid;$SBP,$systolic$blood$pressure.$For$all$secondary$outcomes$ the$P,values$were$adjusted$by$the$Benjamini,Hochberg$multiple,testing$correction.$$Data$from$all$subjects$for$whom$baseline$and$follow,up$measurements$were$ available$are$included.$ 2,3,4Significantly$different$from$baseline$in$normally$distributed$samples$(paired$samples(t$test):$2P<$0.001$3P<$0.01$4P<.$0.05.$ 5Significantly$different$from$baseline$in$non,normally$distributed$variables$(Wilcoxon’s$test):$P$<$0.01.$ 6Differences$ between$ groups$ at$ baseline$ and$ in$ changes$ (10$ wk$ –$ before)$ were$ evaluated$ by$ 2,way$ ANOVA$ (ns,$ non,significant).$ No$ significant$ differences$ between$groups$were$found$in$secondary$outcomes$after$the$adjustment$by$Benjamini,Hochberg.$ 7Differences$ between$ groups$ at$ baseline$ and$ in$ changes$ (10$ wk$ –$ before)$ were$ evaluated$ by$ 2,way$ ANOVA$ (P<$ 0.05;$ ns,$ non,significant).$ No$ significant$ differences$between$groups$were$found$in$secondary$outcomes$after$the$adjustment$by$Benjamini,Hochberg.$ $

$

106!! IV.$RESULTS$(CHAPTER(1)$

The$ data$ of$ the$ OGTT$ revealed$ that$ the$ EPA+$ α7lipoic$ acid$ group$ had$ the$ major$ changes$ in$ glucose$ during$ the$ OGTT$ (Figure$ 2a),$ but$ without$ significant$ modifications$ at$ the$ end$ of$ the$ nutritional$intervention$in$the$glucose$incremental$area$under$curve$(iAUC)$(Figure$2b).$Although$ all$ groups,$ excepting,$ the$ group$ supplemented$ with$ EPA,$ appears$ to$ improve$ the$ changes$ in$ insulin$during$the$OGTT$(Figure$2c),$no$significant$differences$were$found$between$groups$in$the$ insulin$iAUC$(Figure$2d).$

$

$

!

!

!

!

!

!

!

!

!

Figure$2.$Mean$(±SEM)$plasma$glucose$(a,$b)$and$insulin$(c,$d)$during$the$27hour$757g$OGTT.$Data$from$all$ subjects$for$whom$baseline$and$follow7up$measurements$were$available$are$included$(in$the$glucose$and$ insulin$incremental$area$under$curve$(iAUC):$control,$n=$19;$EPA,$n=$15;$α7lipoic$acid,$n=$18;$EPA$+$α7lipoic$ acid,$ n=$ 16).$ (a,$ c)$ Solid$ lines$ represent$ pre7study$ values;$ dotted$ lines$ represent$ post7study$ values.$ The$ comparisons$in$each$group$before$and$after$the$nutritional$intervention$were$assessed$by$Wilcoxon’s$test$ (**P<$0.01,$*P<$0.05).$(b,$d)$Differences$between$groups$in$the$changes$of$the$iAUC$for$glucose$and$insulin$ were$evaluated$by$two7way$ANOVA,$and$no$significant$differences$were$observed$(P>$0.05).$$ 1Adjusting$by$body$weight$loss$and$baseline$values.$$

!

! !

! 107$ IV.$RESULTS$(CHAPTER(1)$

Effects!on!leptin!and!ghrelin!levels!!

After$ the$ nutritional$ intervention,$ a$ significant$ (P<$ 0.01)$ reduction$ in$ leptin$ levels$ was$ demonstrated$in$almost$all$groups,$except$in$the$EPA$supplemented$group$(Table$ 2).$Thus,$the$ groups$supplemented$with$α7lipoic$acid$showed$an$important$drop$in$leptin$in$parallel$with$the$ reduction$of$fat$mass.$Interestingly,$the$groups$with$EPA$supplementation$showed$a$significantly$ lower$decline$(P<$0.001)$in$leptin$levels$with$respect$to$the$others$(Table$2);$also$it$was$observed$ that$the$changes$in$leptin$had$a$positive$significant$association$with$the$RMR$modifications$(r=$ 0.42,$P<$0.001).$$The$EPA$+$α7lipoic$acid$group$showed$a$statistically$significant$(P<$0.05)$increase$ in$ghrelin$at$the$end$of$the$trial,$but$without$differences$between$groups$(Table$2).$$

Adjustment!for!multiple!comparisons!

To$control$the$false$discovery$rate$and$give$more$forcefulness$to$the$results,$the$Benjamini7 Hochberg$adjustment$for$multiple$comparisons$was$used,$finding$that$in$the$secondary$outcomes,$ only$the$change$in$leptin$remains$significantly$different$between$groups$(Tables$1$and$2).$$

Discussion$

In$this$study$we$found$that$the$supplementation$with$α7lipoic$acid$(alone$or$in$combination$ with$EPA)$at$lower$doses$(300$mg/day)$could$help$to$promote$weight$loss$and$fat$mass$reduction$ in$healthy$overweight/obese$women$following$an$energy7restricted$balanced$diet.$In$this$context,$ Koh$et!al.$(2011)$found$that$α7lipoic$acid$supplementation$(120071800$mg/day)$together$with$a$ calorie$restricted$diet$in$Asian$overweight$and$obese$subjects$promotes$weight$loss$as$well$as$BMI$ and$ waist$ circumference$ reduction$ in$ a$ dose7dependent$ manner,$ but$ this$ effect$ was$ only$ significant$ at$ the$ highest$ dose$ tested.$ Carbonelli$ et! al.$ (2010)$ described$ how$ supplementation$ with$ α7lipoic$ acid$ (800$ mg/day)$ promotes$ the$ reduction$ of$ weight,$ fat$ mass$ and$ waist$ circumference$ only$ in$ overweight$ and$ obese$ Caucasian$ subjects,$ but$ not$ in$ normal7weight$ subjects,$suggesting$that$these$effects$could$be$due$to$the$increased$satiety$induced$by$α7lipoic$ acid.$ Besides$ the$ positive$ effects$ on$ weight$ loss$ observed$ in$ some$ trials,$ the$ anti7obesity$ properties$of$α7lipoic$acid$in$humans$remain$controversial.$Thus,$other$studies$in$overweight$and$ obese$subjects$with$impaired$glucose$tolerance$or$type72$diabetes,$following$a$regular$diet,$have$ observed$ no$ effect$ on$ anthropometric$ measurements$ after$ supplementation$ with$ α7lipoic$ acid$ (Ansar$et!al.,$2011;$McNeilly$et!al.,$2011).$Thereby,$the$discrepancies$between$the$different$trials$ could$be$related$to$the$specific$features$of$the$subjects,$the$diet$assigned$and$the$duration$of$the$ trials.$

108!! IV.$RESULTS$(CHAPTER(1)$

Studies$ in$ animal$ models$ have$ suggested$ that$ α7lipoic$ acid$ could$ promote$ weight$ loss$ by$ reducing$food$intake$and$stimulating$energy$expenditure$(Prieto7Hontoria$et!al.,$2009;$Kim$et!al.,$ 2004).$However,$our$current$data$do$not$support$a$role$of$increased$satiety$for$α7lipoic$acid$at$the$ dose$used$in$this$trial,$since$differences$in$energy$intake$were$not$detected.$Moreover,$our$data$ suggest$that$the$RMR$changes$are$not$differentially$affected$by$α7lipoic$ acid$ supplementation,$ since$ after$ adjusting$ for$ body$ weight$ loss,$ the$ RMR$ decrease$ in$ the$ α7lipoic$ acid$ groups$ was$ similar$ to$ control$ group.$ Adipose$ tissue$ is$ a$ target$ organ$ whereby$ α7lipoic$ acid$ exerts$ its$ anti7 adiposity$ effects$ by$ inhibiting$ lipogenesis$ (Fernández7Galilea$ et! al.,$ 2014),$ increasing$ lipolysis$ (Fernández7Galilea$ et! al.,$ 2012)$ and$ inhibiting$ adipogenesis$ (Fernández7Galilea$ et! al.,$ 2013).$ Therefore$these$mechanisms$could$be$also$contributing$to$the$body$and$fat$mass$lowering$actions$ of$α7lipoic$acid,$without$necessarily$involving$an$increment$of$energy$expenditure.$

Studies$ evaluating$ the$ effects$ of$ n73$ PUFAs$ on$ weight$ loss$ in$ obese$ humans$ have$ reported$ contradictory$outcomes$(Kunesova$et!al.,$2006;$Itoh$et!al.,$2007;$Munro$and$Garg,$2012),$which$ largely$depend$on$the$different$amounts$and$ratios$of$n73$PUFAs$type$(EPA$or$DHA),$as$well$as$the$ phenotypical$characteristics$of$the$recruited$subjects.$Most$of$the$studies$analyzing$the$effects$of$ n73$PUFAs$have$used$supplements$that$include$both$EPA$and$DHA.$In$fact,$several$studies$have$ suggested$that$EPA$and$DHA$have$different$effects$on$adipocyte$function$(Oster$et!al.,$2010).$In$ agreement$ with$ the$ present$ study,$ Itoh$ et! al.$ (2007),$ who$ used$ highly$ purified$ (>98%)$ EPA$ in$ obese$subjects,$showed$that$supplementation$with$this$n73$PUFA$(1.8$g/day)$for$3$months$had$no$ effects$on$waist$circumference$reduction$and$BMI$loss.$$

A$remarkable$finding$of$the$present$trial$was$the$fact$that$the$reduction$of$leptin$secondary$to$ fat$mass$loss$was$significantly$lower$in$the$group$supplemented$with$EPA.$In$accordance$with$this,$ in! vitro$ and$ in! vivo$ studies$have$evidenced$the$ability$of$EPA$in$stimulating$the$production$of$ leptin$ in$ rodents$ (Pérez7Matute$ et! al.,$ 2005,$ 2007).$ In$ this$ context,$ Hinkle$ et! al.$ (2013)$ have$ suggested$that$the$decrease$of$leptin$during$weight$loss$could$contribute$to$hunger,$a$lowered$ metabolic$rate,$and$further$weight$regain;$therefore,$leptin$replacement$therapy$could$prevent$ subsequent$ RMR$ fall$ after$ weight$ loss$ and$ the$ weight$ regain$ in$ weight7reduced$ subjects.$ Moreover,$the$positive$and$significant$association$between$RMR$and$leptin$could$suggest$that$the$ less$drop$in$the$RMR$found$in$the$EPA$treated$groups$can$be$related$with$the$effect$of$EPA$on$ leptin.$In$this$regard,$Doucet$et!al.$(2000)$have$proposed$that$after$weight$loss,$the$decrease$in$ RMR$adjusted$by$fat$mass$and$fat$free$mass,$is$explained$mainly$by$changes$in$circulating$plasma$ leptin.$Although$EPA$supplementation$did$not$promote$weight$loss,$its$actions$on$leptin$seem$to$ suggest$ that$ supplementation$ with$ this$ fatty$ acid$ could$ become$ important$ for$ weight$ loss$ maintenance.$ Few$ studies$ have$ addressed$ the$ beneficial$ effects$ of$ n37PUFAs$ on$ weight$

! 109$ IV.$RESULTS$(CHAPTER(1)$ maintenance$after$a$weight$loss$program,$and$no$significant$effects$have$been$described$(Krebs$et! al.,$2006,$Munro$and$Garg,$2012).$However,$it$has$been$suggested$that$the$effects$of$n37PUFAs$ could$ be$ evident$ after$ a$ long$ time$ period$ and$ that$ their$ accumulation$ in$ the$ body$ could$ contribute$to$weight$management$(Munro$and$Garg,$2012,$2013).$$

The$ changes$ noted$ in$ serum$ metabolic$ profile$ in$ this$ study$ clearly$ confirm$ the$ benefits$ of$ weight$loss$and$fat$mass$reduction$by$an$energy7restricted$diet$on$several$metabolic$alterations$ associated$with$the$harmful$effects$of$obesity,$as$previously$demonstrated$(Chang$et!al.,$2010).$ The$effects$of$the$n73$PUFAs$on$glucose$and$lipid$profile$are$not$consistent,$depending$mainly$on$ the$baseline$metabolic$characteristics.$The$main$outcome$observed$in$the$studies$with$n73$PUFAs$ in$ overweight/obese$ subjects$ is$ the$ reduction$ in$ triglyceride$ levels$ (Munro$ and$ Garg,$ 2012;$ Yamada$et!al.,$2008),$however,$the$doses$used$in$these$trials$range$between$1.6$and$4$g$per$day,$ and$are$therefore$higher$than$the$dose$used$in$this$study$(1.3$g/day).$Furthermore,$the$effects$of$ n73$PUFAs$on$the$cholesterol$profile$are$not$clear$(Egert$et!al.,$2009;$Hill$et!al.,$2007;$Kurabayashi$ et!al.,$2000;$Mori$et!al.,$1999).$Similar$to$our$observations,$other$trials$have$described$that$the$ levels$ of$ LDL7cholesterol,$ HDL7cholesterol$ and$ total7cholesterol$ remain$ unchanged$ after$ n73$ PUFAs$treatment$(DeFina$et!al.,$2011;$Munro$and$Garg,$2012;$Yamada$et!al.,$2008).$Although$the$ increase$ in$ β7hydroxybutyrate$ levels$ was$ not$ significantly$ different$ between$ groups$ after$ the$ Benjamini7Hochberg$correction,$it$cannot$be$discarded$some$effect$of$EPA$in$fatty$acid$oxidation,$ as$ has$ been$ previously$ reported$ in$ other$ studies$ in$ both$ humans$ (Kunesova$ et! al.,$ 2006)$ and$ rodents$(Flachs$et!al.,$2005).$

Several$studies$in$type$2$diabetic$and$impaired$glucose$tolerance$subjects$have$exhibited$the$ properties$ of$ α7lipoic$ acid$ to$ ameliorate$ glucose$ metabolic$ complications$ (Ansar$ et! al.,$ 2011;$ Ziegler$ et! al.,$ 2006).$ Furthermore,$ studies$ analyzing$ the$ effects$ of$ n73$ PUFAs$ in$ glucose$ metabolism$have$reached$contradictory$outcomes$(DeFina$et!al.,$2011;$Ramel$et!al.,$2008;$Griffin$ et!al.,$2006).$In$the$current$study,$a$significant$interaction$between$both$supplements$in$glucose$ concentration$ changes$ was$ found;$ however,$ it$ is$ important$ to$ note,$ that$ the$ major$ decreases$ found$ in$ glucose$ and$ HOMA7IR$ in$ the$ control$ and$ EPA+α7lipoic$ acid$ group,$ may$ have$ been$ conditioned$by$the$higher$levels$at$baseline$in$these$groups$on$insulin$levels.$Though,$the$effects$ of$ supplementation$ are$ influenced$ possibly$ by$ the$ metabolic$ status$ at$ baseline,$ it$ could$ be$ interesting$to$further$evaluate$the$possible$coadjuvant$properties$of$n73$PUFAs$and$α7lipoic$acid$ on$glucose$metabolism.$

The$safety$and$toxicity$of$the$α–lipoic$acid$have$been$tested$by$several$authors$at$different$ doses$and$times,$being$the$greatest$dose$administered$2400$mg/day$during$a$2$years$period$(Shay$

110!! IV.$RESULTS$(CHAPTER(1)$ et!al.,$2009)$with$no$reported$adverse$effects$versus$placebo.$The$dose$used$in$our$study$is$much$ lower$than$the$dose$considered$safe.$$

On$the$other$hand,$it$is$important$to$take$into$account$the$limitations$of$this$study,$including$ the$ sample$ size,$ the$ short$ duration$ of$ the$ nutritional$ intervention,$ the$ lack$ of$ a$ maintenance$ weight$loss$period,$the$absence$of$blood$samples$of$some$volunteers$and$especially$the$fact$that$ the$outcomes$could$have$been$conditioned$by$the$degree$of$adherence$to$the$hypocaloric$diet.$ Thus,$the$findings$of$our$trial$suggest$the$importance$of$performing$longer$randomized$controlled$ trials$followed$by$sustained$weight$maintenance$periods.$$

In$summary,$our$data$suggest$that$α–lipoic$ acid$ supplementation$ at$ a$ dose$ of$ 300$ mg/day$ combined$ with$ an$ energy7restricted$ diet$ might$ help$ to$ promote$ weight$ loss$ and$ fat$ mass$ reduction$in$healthy$obese$women.$Although$EPA$supplementation$did$not$have$any$additional$ effect$on$the$reduction$of$body$and$fat$mass,$it$prevents$the$fall$of$leptin$during$weight$loss.$It$is$ essential$ that$ these$ observations$ be$ further$ explored$ and$ the$ underlying$ mechanisms$ better$ elucidated.$

Competing!interest!

The$authors$have$no$competing$interests.$

Acknowledgments!

Designed$ research,$ raised$ grants,$ and$ coordinated$ the$ complete$ project:$ MJM7A$ and$ JAM;$ Conducted$ laboratory$research:$AEH,$and$PLPH;$Performed$the$statistical$analyses$and$wrote$the$ manuscript:$ AEH;$ Help$ in$ the$ implementation$ of$ the$ clinical$ research$ plan:$ SNC;$ Critically$ reviewed$ the$ manuscript:$ SNC,$ PLPH,$JAM$and$MJMA.$All$authors$have$approved$the$final$version$of$the$manuscript.$$

AE$ Huerta$ was$ supported$ by$ a$ predoctoral$ grant$ from$ “Asociación$ de$ Amigos$ de$ la$ Universidad$ de$ Navarra”.$ This$ work$ was$ supported$ by$ a$ grant$ (AGL$ 2009710873/ALI)$ from$ Ministerio$ de$ Economía$ y$ Competitividad,$ Spain$ $ (principal$ investigator$ MJM7A)$ and$ by$ Línea$ Especial$ de$ Investigación$ “Nutrición,$ Obesidad$y$Salud”,$University$of$Navarra7Spain$(principal$investigator$JAM).$EPA$and$sunflower$oil$capsules$ were$ generously$ provided$ by$ Solutex$ (Madrid,$ Spain).$ $ Solutex$ had$ no$ role$ in$ the$ study$ design,$ data$ collection,$analysis$and$interpretation,$or$writing$of$the$manuscript.$$ We$thank$all$participants$of$the$study$and$the$clinical$investigators$of$the$Metabolic$Unit$Blanca$Martínez$ de$Morentin$(M.D.),$María$Hernández$(Dietitian)$and$Salomé$Pérez$(Nurse)$for$the$execution$of$the$study$ throughout$and$for$taking$care$of$study$participants.$We$also$thank$María$Zabala$for$her$excellent$technical$ assistance$and$Marta$Cuervo$for$her$support$in$diet$design.$ $ $

! 111$ IV.$RESULTS$(CHAPTER(1)$

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114!! IV.$RESULTS$(CHAPTER(1)$

WHO$Expert$Committee$(1996).$Hypertension$control.$World$Health$Organization$Technical$Report$Series$ 862.$

Yamada,$H.,$Yoshida,$M.,$Nakano,$Y.,$Suganami,$T.,$Satoh,$N.,$Mita,$T.,$Azuma,$K.,$Itoh,$M.,$Yamamoto,$Y.,$ Kamei,$ Y.,$ et$ al.$ (2008).$ In$ vivo$ and$ in$ vitro$ inhibition$ of$ monocyte$ adhesion$ to$ endothelial$ cells$ and$ endothelial$adhesion$molecules$by$eicosapentaenoic$acid.$Arterioscler.$Thromb.$Vasc.$Biol.$28,$2173– 2179.$

Ziegler,$ D.,$ Ametov,$ A.,$ Barinov,$ A.,$ Dyck,$ P.J.,$ Gurieva,$ I.,$ Low,$ P.A.,$ Munzel,$ U.,$ Yakhno,$ N.,$ Raz,$ I.,$ Novosadova,$ M.,$ et$ al.$ (2006).$ Oral$ treatment$ with$ alpha7lipoic$ acid$ improves$ symptomatic$ diabetic$ polyneuropathy:$the$SYDNEY$2$trial.$Diabetes$Care$29,$2365–2370.$ $

! 115$

$

! CHAPTER!2! $ $ Post?print!version!of!the!article:!! ! $ Supplementation$with$αFlipoic$acid$alone$or$in$combination$with$EPA$modulates$inflammatory$ status$in$healthy$overweight/obese$women$following$an$energyFrestricted$diet$$ $ Ana$ E.$ Huerta1,2,$ Pedro$ L.$ Prieto7Hontoria$ 1,5,$ Neira$ Sáinz1,2,$ J.$ Alfredo$ Martínez1,2,3,4,$ María$ J.$ Moreno7Aliaga1,2,3,4$ $ 1Department$of$Nutrition,$Food$Science$and$Physiology,$University$of$Navarra,$Pamplona,$Spain$$ 2Centre$for$Nutrition$Research,$University$of$Navarra,$Pamplona,$Spain$ 3Biomedical$ Research$ Centre$ Network$ in$ Physiopathology$ of$ Obesity$ and$ Nutrition$ (CIBERobn),$ National$Institute$of$Health$Carlos$III$(ISCIII),$Madrid,$Spain$ 4Navarra$Institute$for$Health$Research$(IdiSNA),$Pamplona,$Spain$ 5Current$affiliation:$Faculty$of$Health$and$Physical$Activity$Science,$University$SEK,$Santiago$de$Chile,$ Chile$ $ $ $ $ J$Nutr,$2016,$146:$889S7896S$$ doi:$10.3945/jn.115.224105$ $ $ $ $ $ $ Impact$factor$(2015):$3.740$ 17/78$in$Nutrition$&$Dietetics$

! Huerta AE, Prieto-Hontoria P, Sáinz N, Martínez JA, Moreno-Aliaga MJ. Supplementation with α-Lipoic Acid Alone or in Combination with Eicosapentaenoic Acid Modulates the Inflammatory Status of Healthy Overweight or Obese Women Consuming an Energy-Restricted Diet. The Journal of Nutrition. Abril 2016, 146(4):8895–8965. &

'

CHAPTER'3'

& & Post,print'version'of'the'article:'' & Circulating$irisin$and$glucose$metabolism$in$overweight/obese$women:$effects$of$αJlipoic$acid$ and$eicosapentaenoic$acid$$ $ Ana&E.&Huerta1,2,&Pedro&L.&Prieto:Hontoria1,4,&M.&Fernández:Galilea1,5,&N.&Sáinz2,&M.&Cuervo1,2,3,&J.& Alfredo&Martínez1,2,3,&María&J.&Moreno:Aliaga1,2,3& & 1Department&of&Nutrition,&Food&Science&and&Physiology.&University&of&Navarra.&Pamplona,&Spain.& 2Centre& for& Nutrition& Research.& School& of& Pharmacy.& University& of& Navarra.& Irunlarrea& St.& E:31008& Pamplona,&Spain.& 3Biomedical& Research& Centre& Network& in& Physiopathology& of& Obesity& and& Nutrition& (CIBERobn).& National&Institute&of&Health&Carlos&III&(ISCIII),&Madrid,&Spain.& 4Current&affiliation:&Faculty&of&Health&and&Physical&Activity&Science.&University&SEK.&Santiago&de&Chile,& Chile.& 5Current&affiliation:&Department&of&Nutrition,&Diabetes&and&Metabolism.&School&of&Medicine.&Pontificia& Universidad&Católica&de&Chile.&Santiago&de&Chile,&Chile.& & & & & J&Physiol&Biochem,&2015,&71:&547:558& doi:&10.1007/s13105:015:0400:5& & & & & Impact&factor&(2015):&2.054& 45/83&in&Physiology& 201/289&in&Biochemistry&&&Molecular&Biology&&

!

IV.$RESULTS$(CHAPTER(3)$

Abstract$

Irisin& is& a& myokine/adipokine& with& potential& role& in& obesity& and& diabetes.& The& objectives& of&the& present&study&were&to&analyze&the&relationship&between&irisin&and&glucose&metabolism&at&baseline& and& during& an& oral& glucose& tolerance& test& (OGTT)& and& to& determine& the& effects& of& EPA& (eicosapentaenoic& acid)& and/or& α:lipoic& acid& treatment& on& irisin& production& in& cultured& human& adipocytes& and& in' vivo& in& healthy& overweight/obese& women& following& a& weight:loss& program.& Seventy:three& overweight/obese& women& followed& a& 30%& energy:restricted& diet& supplemented& without& (Control)& or& with& EPA& (1.3& g/d),& α:lipoic& acid& (0.3& g/d)& or& both& EPA+α:lipoic& acid& (1.3& g/d+0.3&g/d)&during&10&weeks.&An&OGTT&was&performed&at&baseline.&Moreover,&human&adipocytes& were& treated& with& EPA& (100:200& μM)& or& α:lipoic& acid& (100:250& μM)& during& 24& h.& At& baseline& plasma&irisin&circulating&levels&were&positively&associated&with&glucose&levels;&however&serum&irisin& concentrations&were¬&affected&by&the&increment&in&blood&glucose&or&insulin&during&the&OGTT.& Treatment&with&α:lipoic&acid&(250&μM)&upregulated&FNDC5&mRNA&and&irisin&secretion&in&cultured& adipocytes.& In& overweight/obese& women,& irisin& circulating& levels& decreased& significantly& after& weight:loss& in& all& groups,& while& no& additional& differences& were& induced& by& EPA& or& α:lipoic& acid& supplementation.& Moreover,& plasma& irisin& levels& were& positively& associated& with& higher& glucose& concentrations&at&beginning&and&at&endpoint&of&the&study.&The&data&from&the&OGTT&suggest&that& glucose& is& not& a& direct& contributing& factor& of& irisin& release.& The& higher& irisin& levels& observed& in& overweight/obese& conditions& could& be& a& protective& response& of& organism& to& early& glucose& impairments.&

Keywords:&obesity;&myokine/adipokine;&weight&loss;&eicosapentaenoic&acid;&α:lipoic&acid;&Fndc5& & & &

! 143& IV.$RESULTS$(CHAPTER(3)$

Introduction$

Irisin&is&a&myokine&identified&in&mice&and&humans,&which&has&been&proposed&to&mediate&some& of&the&beneficial&effects&of&exercise&on¯onutrient&metabolism&(Bostrom&et'al.,&2012;&Huh&et' al.,&2012).&Thus,&the&increment&in&the&expression&of&the&transcriptional&co:activator&factor&PGC:1α,& activates& the& membrane& protein,& fibronection& type& III& domain& containing& 5& (FNDC5),& which& is& proteolytically&cleaved&and&secreted&into&circulation&as&irisin&(Bostrom&et'al.,&2012).&Fndc5&is&also& expressed&in&kidney,&liver,&heart,&lung&and&adipose&tissues&(Huh&et'al.,&2012),&but&the&expression&in& muscle&samples&has&been&found&to&be&200:fold&greater&than&in&adipose&tissue&(Moreno:Navarrete& et'al.,&2013).&&

The&role&of&irisin&in&obesity&and&glucose&metabolism&has¬&been&fully&established&yet.&While& some&researchers&observed&that&plasma&irisin&levels&are&decreased&in&obesity&(Gutierrez:Repiso&et' al.,&2014;&Moreno:Navarrete&et'al.,&2013),&others&found&the&opposite&outcome&(Huh&et'al.,&2012;& Stengel&et'al.,&2013).&Additionally,&it&has&been&suggested&that&Fndc5&expression&is&decreased&in&the& adipose&tissue&of&obese&and&type&2&diabetic&subjects&and&positively&correlated&with&some&insulin: pathway&related&genes&(Moreno:Navarrete&et'al.,&2013).&Furthermore,&despite&that&some&studies& in& type& 2& diabetic& patients& have& observed& that& blood& irisin& concentration& negatively& correlated& with&glucose¶meters&(Choi&et'al.,&2013;&Liu&et'al.,&2013),&other&trials&in&overweight&or&obese& subjects&have&found&that&blood&irisin&levels&positively&correlated&with&insulin&(Stengel&et'al.,&2013),& fasting&blood&glucose&(Zhang&et'al.,&2013)&and&insulin&resistance&(Sesti&et'al.,&2014).&Therefore,&the& first&aim&of&the&present&study&was&to&analyze&the&relationship&between&basal&levels&of&irisin&with& glucose& metabolism& in& healthy& overweight/obese& women.& Moreover,& previous& studies& have& reported& that& the& acute& increment& in& blood& glucose& levels& can& promote& changes& in& circulating& levels&of&some&adipo/myokines&such&as&IL:6&and&FGF21&(Esposito&et'al.,&2002;&Manning&et'al.,&2008)& and&there&were&no&data&available&about&whether&serum&irisin&is&responsive&to´&changes&in& glucose&and&insulin.&Therefore,&we&also&aimed&to&assess&the&possible&changes&on&irisin&circulating& levels&during&an&oral&glucose&tolerance&test&(OGTT)&in&overweight/obese&subjects.&

α:Lipoic& acid& is& a& short& chain& fatty& acid,& acting& as& an& important& co:factor& for& mitochondrial& enzymes& function,& which& can& be& found& in& little& amounts& in& some& vegetables& and& entrails& (Fernández:Galilea& et' al.,& 2013).& Moreover,& the& eicosapentaenoic& acid& (EPA)& is& an& omega:3& polyunsaturated&fatty&acid&(n:3&PUFAs),&which&occurs&predominantly&in&oily&fishes&and&may&have& important& anti:inflammatory& properties& (Lorente:Cebrián& et' al.,& 2013;& Moreno:Aliaga& et' al.,& 2010).&Studies&in&rodents&and&humans&have&suggested&that&the&supplementation&with&either&α: lipoic& acid& or& EPA& could& exert& beneficial& effects& in& obesity,& promoting& body& weight& loss& and/or&

144!! IV.$RESULTS$(CHAPTER(3)$ improving& glucose& disturbances& (Carbonelli& et' al.,& 2010;& Huerta& et' al.,& 2014;& Koh& et' al.,& 2011).& These&effects&have&been&partly&related&with&their&ability&to&modulate&some&adipokines&involved&in& energy&metabolism&such&as&leptin,&adiponectin,&visfatin&and&apelin&(Fernández:Galilea&et'al.,&2011,& 2013,& 2014;& Moreno:Aliaga& et' al.,& 2010;& Pérez:Echarri& et' al.,& 2009;& Pérez:Matute& et' al.,& 2005,& 2007;&Prieto:Hontoria&et'al.,&2009,&2011).&

There& is& not& information& available& about& the& effects& of& α:lipoic& acid& or& EPA& on& irisin& levels.& Therefore,& in& the& present& study,& we& also& aimed& to& analyze& their& effects& on& irisin& production& in& cultured& human& adipocytes& and& to& evaluate& the& influence& of& EPA& and/or& α:lipoic& acid& dietary& supplementation&on&irisin&levels&in&healthy&overweight/obese&women&who&underwent&a&weight& loss&treatment.&

Material$and$methods$

Subjects'and'Study'design'

Ninety:seven& overweight/obese& women& (BMI:& 27.5:39.9& kg/m2;& Age:& 20:45& years:old)& were& enrolled&in&the&trial,&after&signing&the&informed&consent.&The&study&was&approved&by&the&Research& Ethics& Committee& of& the& University& of& Navarra& and& was& performed& in& compliance& with& the& Declaration&of&Helsinki&guidelines,&and®istered&at&Clinical&Trials&(NCT01138774).&&

This&study&is&an&ancillary&analysis&of&the&OBEPALIP&study,&which&primary&outcomes&have&been& recently&reported&(Huerta&et'al.,&2014).&Briefly,&overall&healthy&women&with&a&BMI&between&27.5: 40&kg/m2&and&with&unchanged&weight&(±&3&kg)&during&the&last&three&months&participated&into&this& randomized,&short:term&(10&weeks)&double&blind&placebo:controlled&trial&with¶llel&design&that& consisted& in& a& weight:loss& program& with& a& moderate& energy:restriction& of& 30%& from& the& total& energy&expenditure,&and&with&a¯onutrient&distribution&(55%&carbohydrates;&30%&lipids,&15%& proteins)&according&with&the&American&Heart&Association&(AHA)&guidelines.&The&four&intervention& groups&varied&in&the&type&of&the&administered&daily&supplement&as&follows:&(1)&control&group:&3& placebo:I& capsules& (containing& sunflower& oil)& and& 3& placebo:II& capsules& (containing& same& excipients&than&the&α:lipoic&acid&capsules),&(2)&EPA&group:&1300&mg/day&of&EPA&distributed&in&3& capsules&of&EPA80&(provided&by&Solutex®,&Madrid,&Spain),&containing&433.3&mg&of&EPA&and&13.8&mg& of&DHA&asðyl:ester;&and&3&placebo:II&capsules,&(3)&α:lipoic&acid&group:&300&mg/day&of&α:lipoic& acid&from&3&capsules&containing&100&mg&of&α:lipoic&acid&(Nature’s&Bounty®,&NY),&and&3&placebo:I& capsules;& and& (4)& EPA+& α:lipoic& acid& group:& 1300& mg/day& of& EPA& (distributed& in& 3& capsules& of& EPA80)& and& 300& mg/day& of& α:lipoic& acid& & (from& 3& capsules& containing& 100& mg& of& α:lipoic& acid),& respectively.&At&the&beginning&of&the&study,&information&about&the&physical&activity&patterns&were&

! 145& IV.$RESULTS$(CHAPTER(3)$ collected&using&a&questionnaire&in&order&to&evaluate&the&physical&activity&factor;&then&the&women& were& instructed& to& not& change& the& physical& activity& pattern& during& the& intervention& period.& In& accordance&with&World&Health&Organization&(WHO)&criteria&and&as&previously&described&(Huerta&et' al.,&2014),&the&physical&activity&factor&was&1.40&±&0.08,&which&corresponds&to&sedentary&activity&and& was&maintained&during&the&study&period.&

Out&of&the&77&healthy&overweight/obese&women&who&completed&the&intervention&period&of&10& weeks& only& the& blood& samples& of& 73& were& available.& At& baseline& and& endpoint,& the& volunteers& arrived&under&fasting&conditions&to&the&Metabolic&Unit&(MU)&of&the&University&of&Navarra&and&met& with&the&physician,&the&dietician&and&the&nurse.&Anthropometric&measurements&of&weight,&height,& waist& circumference& and& hip& circumference& were& performed& following& validated& protocols.& Additionally,& body& composition& by& Dual& X:ray& Absorptiometry& (DXA)& and& respiratory& exchange& measurements& by& indirect& calorimetry& were& evaluated& as& previously& described& (Huerta& et' al.,& 2014).& A& catheter& was& then& inserted& into& the& antecubital& vein& for& a& fasting& blood& sample& extraction.&

Oral'Glucose'Tolerance'test'

Before&nutritional&supplementation,&a&75:g&oral&glucose&tolerance&test&(OGTT)&was&performed& in& 15& subjects& (4& from& each& supplemented& group& and& 3& from& control& group).& The& baseline& characteristics&of&this&sub:sample&did¬&differ&from&the&total.&In&particular&their&age&was&38.9&±& 6.7&years&and&BMI&was&32.67&±&3.39&kg/m2.&Blood&samples&were&extracted&at&baseline&and&each&30& minutes&during&2&hours&for&determining&glucose,&insulin&and&irisin&levels.&&

Laboratory'measurements'

Plasma&and&serum&were&conventionally&separated&from&blood&cells&and&stored&at&:80&°C&until& further& analyses.& Serum& levels& of& glucose,& triglycerides,& free& fatty& acids& (FFA)& and& β: hydroxybutyrate&were&measured&using&the&auto:analyzer&PENTRA&C200&(HORIBA&medical,&Japan).&&

Other& specific& biochemical& determinations& in& plasma& and& serum& were& measured& with& the& appropriate& enzyme& immunoassay& (ELISA)& kits& following& the& manufacturer's& protocols.& Thus,& insulin&and&irisin&were&assessed&in&plasma&samples&at&the&beginning&and&at&the&endpoint&of&the& nutritional&intervention&and&also&in&serum&samples&during&the&OGTT.&Insulin&was&determined&using& a&Human&Sensitive&ELISA&Kit&(Mercodia&AB,&Sylveniusgatan,&Sweden).&Irisin&was&measured&using& the&Human&ELISA&Kit&(Phoenix&Pharmaceuticals,&Inc,&CA.).&&

146!! IV.$RESULTS$(CHAPTER(3)$

HOMA:IR&was&defined&as&fasting&serum&insulin&(mU/L)&x&fasting&plasma&glucose&(mmol/L)/22.5.& The&TyG&index&(Simental:Mendía&et'al.,&2008)&was&estimated&as&ln[fasting&triglycerides&(mg/dL)&x& fasting&glucose&(mg/dL)&/&2].&

Studies'in'cultured'human'subcutaneous'adipocytes''

Commercially&available&cryopreserved&human&subcutaneous&preadipocytes&from&non:diabetic& overweight:obese& female& donors& (BMI:& 26.85:33.37& kg/m2)& were& purchased& from& Zen:Bio& Inc.& (Research& Triangle& Park,& NC)& and& differentiated& with& the& DM:2& Zen:Bio& commercial& medium,& according& to& manufacturer’s& instructions.& Fourteen& days& after& the& induction& of& differentiation,& cells&contained&large&lipid&droplets&and&were&considered&mature&adipocytes.&α:Lipoic&acid&(Sigma;& St.&Louis,&MO)&and&EPA&(Cayman&Chemical,&Ann&Arbor,&MI)&were&dissolved&inðanol.&1000x&stocks& were&prepared&and&1&µl/ml&of&media&was&added.&Adipocytes&were&treated&with&α:lipoic&acid&(100: 250& μM)& or& EPA& (100:200& μM)& during& 24& h.& The& total& amount& of& irisin& secreted& into& culture& medium&was&determined&through&the&use&of&the&Human&ELISA&Kit&(Phoenix&Pharmaceuticals,&Inc,& CA.).&Total&RNA&was&extracted&using&TRIzol®&reagent&(Invitrogen;&Carlsbad,&CA)&and&Fndc5&mRNA& levels& were& determined& using& predesigned& TaqMan®& Assays:on:Demand& primers& and& TaqMan& Universal& Master& Mix& (Applied& Biosystems,& Foster& City,& CA)& using& the& ABI& PRISM& 7900HT& Fast& System&Sequence&Detection&System&(Applied&Biosystems).&Fndc5&mRNA&levels&were&normalized&by& the& housekeeping& gene& 18S.& Ct& values& (the& cycle& where& the& emitted& fluorescence& signal& is& significantly&above&background&levels&and&is&inversely&proportional&to&the&initial&template©& number)&were&generated&by&the&ABI&software.&Finally,&the&relative&expression&level&of&each&gene& was&calculated&as&2:ΔΔCt.&

Statistical'analyses'

The&power&calculation&estimated&that&sixteen&participants&per&group&would&allow&a&detection& of&approximately&5&±&5&Kg&difference&in&weight&loss&between&supplemented&and&control&groups&at& a& 0.05& level& of& significance,& with& a& statistical& power& of& 80%.& Since& a& dropout& rate& of& 25%& was& expected,&it&was&estimated&that&at&least&twenty&participants&per&group&should&be&recruited.&&

A& per:protocol& (PP)& analysis& was& performed& including& all& volunteers& for& which& end:of: intervention&values&and&blood&samples&were&available.&The&relationship&between&paired&data&was& analyzed& by& the& bivariate& Pearson’s& correlation.& Changes& during& the& OGTT& were& assessed& with& repeated& measures& ANOVA& followed& by& a& Dunnett’s& post& hoc& test.& The& effects& of& the& supplementation&and&the&weight&loss&in&each&intervention&group&were&evaluated&by&Student’s&t& test&or&by&Wilcoxon&test&depending&of&the&distribution&normality&of&data,&which&was&assessed&with&

! 147& IV.$RESULTS$(CHAPTER(3)$ the&Shapiro:Wilk&test.&Values&are&represented&as&mean&±&SD&or&median&(interquartile&range).&The& differential& effects& between& the& intervention& groups& were& estimated& by& a& two:way& factorial& ANOVA.&Volunteers&were÷d&by&the&median&levels&of&irisin&at&baseline,&considering&a&cut:off& where&low&irisin&levels&were&those&values&<&171.72&ng/mL&and&high&irisin&levels&were&those&values&≥& 171.72&ng/mL;&because&n>&30,&normality&of&the&sample&was&assumed,&then&the&differences&were& evaluated& by& Student’s& t& test;& the& Breusch:Pagan/Cook:Weisberg&test&was&performed&to&detect& the&heteroskedasticity,&and&when&it&was&found,&the&Student’s&t&test&with&Welch&approximation&was& carried& out.& Differences& were& considered& significant& if& P<& 0.05.& Additionally,& the& Benjamini: Hochberg&(B:H)&approach&(Benjamini&and&Hochberg,&1995)&was&used&as&a&correction&for&multiple& comparisons.&&

In&the&adipocyte&culture&study,&the&statistical&analyses&were&performed&by&repeated&measures& one:way& ANOVA& followed& by& Tukey’s& post& hoc& test& or& by& a& paired& Student’s& t& test& in& order& to& determine&differences&between&groups.&Statistical&analyses&were&performed&using&Stata&Statistical& Software& (Release& 12.& College& Station,& TX:& StataCorp& LP)& or& GraphPad& Prism& software& 6.0& (GraphPad&Software,&San&Diego,&CA).&

Results$

Relationship'between'irisin'and'other'parameters'at'baseline'

The&baseline&anthropometric&and&clinical&features&of&all&participants&are&detailed&in&Table$ 1.& Correlation&analyses&of&plasma&irisin&levels&at&the&beginning&of&the&study&revealed&that&circulating& irisin&had&positive&statistically&significant&correlation&(P<&0.05)&with&glucose,&insulin,&HOMA:IR,&β: hydroxybutyrate& and& FFA& (Table$ 1),& while& no& significant& associations& were& found& with& body& weight,&BMI,&and&other&anthropometric&variables.&Furthermore,&after&the&correction&by&multiple& comparisons&only&the&correlation&between&irisin&and&glucose&remained&significant&(Table$1).&& & &

148!! IV.$RESULTS$(CHAPTER(3)$

Table$1.$Baseline&characteristics&of&the&volunteers&and&correlation&with&irisin& plasma&levels$ Variable$ $ r( PJvalue$ Number&of&subjects& 73& ' & Age&(years)& 38.5&±&7.1& 0.07' 0.55& Body&weight&(Kg)& 85.76&±&12.72& 0.16& 0.18& BMI&(Kg/m2)& 32.80&±&4.37& 0.21' 0.07& Waist&circumference&(cm)& 98.61&±&10.68& 0.19' 0.11& Waist&to&hip&ratio& 0.85&±&0.07& 0.15' 0.20& Fat&mass&(Kg)& 47.58&±&3.82& 0.14' 0.25& Fat&free&mass&(Kg)& 45.82&±&5.41& 0.09' 0.47& RMR&(Kcal/day)& 1572&±&187& 0.23' 0.05& Triglycerides&(mg/dL)& 88.45&±&40.99& 0.13& 0.26& β:hydroxybutyrate&(mmol/L)& 0.33&±&0.22& 0.25& 0.03& FFA&(mmol/L)& 0.52&±&0.18& 0.23& 0.04& Glucose&(mg/dL)& 91.07&±&7.57& 0.38' <0.001*& Insulin&(mU/L)& 8.37&±&6.62& 0.27' 0.02& HOMA:IR& 1.83&±&1.51& 0.31& <0.01& & TyG&index& 8.20&±&0.46& 0.24& 0.04& Irisin&(ng/mL)& 176.2&±&59.5& :::& :::& Data& are& represented& as& mean& ±& SD.& FFA:& free& fatty& acids;& RMR:& resting& metabolic& rate;&TyG&index:&triglyceride:glucose&index.& *Statistically&different&after&Benjamini:Hochberg&correction.&& &

Irisin'changes'during'the'OGTT'

In& order& to& evaluate& if& acute& changes& in& blood& glucose& or& insulin& are& determining& the& irisin& levels,&a&2:h&OGTT&was&performed&in&15&subjects&at&the&baseline&period,&prior&to&the&nutritional& intervention.& As& expected,& the& glucose& overload& promoted& a& significant& increase& in& insulin& and& glucose& plasma& levels& during& the& OGTT& (P<& 0.05),& but& not& relevant& variations& were& detected& in& irisin&concentrations&along&the&120&min&of&the&test&(P=&0.73)&(Figure$1aJc).& & &

! 149& IV.$RESULTS$(CHAPTER(3)$

a 180 Time P<0.0001 b 100 Time P<0.0001 ! *** 160 80 *** *** *** ! *** *** 140 *** ! 60

120 *** 40

100 (mU/L) Insulin 20 Glucose (mg/dL) Glucose

80 0 0 30 60 90 120 0 30 60 90 120 Time in minutes Time in minutes

c 125 Time P=0.73 Figure' 1.' Changes(in(serum((a)(glucose,((b)(insulin( and( (c)( irisin( during( the( 26h( OGTT.' Data( (mean( ±(

100 SEM)(from(15(subjects(with(normal(isolate(glucose( tolerance( during( the( OGTT.( The( effect( of( glucose(

75 overload( (756g)( was( evaluated( by( repeated(

Irisin (ng/mL) Irisin measures( ANOVA( followed( by( Dunnett( post6hoc( test( for( comparing( the( differences( with( the( 50 0 30 60 90 120 baseline(concentration((***P<(0.001).(( Time in minutes

&&

In'vitro'and'in'vivo'effects'of'α,lipoic'acid'and'EPA'on'irisin'

As&shown&in&Figure$2,&α:lipoic&acid,&but¬&EPA,&stimulated&both&Fndc5&gene&expression&and& the& amount& of& irisin& released& to& media& (P<& 0.01& for& α:lipoic& acid& 250& µM)& in& subcutaneous& adipocytes&from&overweight/obese&subjects.&However,&this&stimulatory&effect&of&α:lipoic&acid&on& Fndc5'was¬&observed&in&C2C12&murine&myotubes&(data¬&shown).&&

& a" b"

Figure$2.$Effects&of&α:lipoic&acid&(100&and&250&µM)&and&EPA&(100&and&200&µM)&treatment&for&24&h&on&irisin& secretion&(a)&and'Fndc5&mRNA&levels&(b)&in&subcutaneous&adipocytes&from&overweight/obese&subjects.&Data& are&means&±&SEM&of&at&least&four&independent&experiments.&**P<&0.01&vs.&control&(vehicle:treated&cells).& &

&

150!! IV.$RESULTS$(CHAPTER(3)$

The& in' vivo& effects& of& α:lipoic& acid& and& EPA& supplementation& were& also& analyzed& in& overweight/obese& women& following& a& hypocaloric& diet.& After& the& 10& weeks& of& nutritional& intervention,& all& groups& exhibited& a& significant& decrease& (P<& 0.01)& in& body& weight,& BMI& and& fat& mass&(Table$ 2).&Regarding&glucose&metabolism&markers,&the&levels&of&glucose&had&a&statistically& significant& decrease& only& in& the& control& group& and& the& EPA+α:lipoic& acid& group,& while& insulin,& HOMA:IR&and&TyG&index&fell&significantly&in&roughly&all&groups&except&in&the&EPA&supplemented& group.&&

Interestingly,& irisin& levels& decreased& after& weight& loss& in& all& groups,& but& the& drop& was& not& significant&in&the&group&supplemented&with&EPA&(Table$ 2).& However,& not& statistically& significant& differences&were&found&in&changes&of&irisin&between&groups,&even&adjusting&by&body&weight&loss& (Figure$3).&Moreover,&although&the&fall&in&irisin&levels&after&intervention&was¬&associated&with& the&changes&in&body&weight&(r=&:0.049&P=&0.68),&fat&mass&(r=&:0.087&P=&0.47)&or&fat&free&mass&(r=&: 0.009& P=& 0.93),& changes& in& irisin& were& positively& correlated& (P<& 0.05)& with& changes& in& insulin.& Additionally,&a&marginally&significant&association&(P=&0.06)&was&observed&between&the&changes&in& irisin&levels&and&the&changes&in&HOMA:IR&(Figure$4).&

&

&

&

&

&

&

&

&

Figure$3.$Irisin&changes&after&10&weeks&of&nutritional&intervention.&Data&are&means&±&SEM&and&were&analyzed& by& two:way& ANOVA.& No& significant& differences& were& observed& between& groups,& even& adjusting& by& body& weight&loss&(P>&0.05).& &

! 151& IV.$RESULTS$(CHAPTER(3)$

Table&2.&Changes!after&10&weeks&of&nutritional&intervention&in&anthropometric&and&biochemical¶meters$ Variable$ Control$ EPA$ α-lipoic&acid$ EPA+α-lipoic&acid$ No.$of$subjects! 21! 16! 19! 17! $ Baseline$ Endpoint$ Baseline$ Endpoint$ Baseline$ Endpoint$ Baseline$ Endpoint$ Body%weight%(Kg)! 85.5$±$14.2! 80.2%±%14.0**! 89.4%±%11.2! 84.1%±%11.9**! 83.8$±$11.7! 76.8%±%10.9**! 84.9%±%13.6! 78.4%±%12.2**! BMI$(Kg/m2)! 33.2$±$6.0! 31.1$±$5.7**! 33.0$±$3.0! 31.0%±%3.3**! 32.2$±$4.0! 29.5%±%3.8**! 32.7%±%3.7! 30.3$±$3.7**! Fat$mass$(Kg)! 39.5%±%9.3! 35.1%±%9.0**! 42.0%±%5.6! 37.7$±$5.9**! 38.7%±%9.0! 33.2$±$8.6**! 39.3$±$8.7! 33.9$±$8.1**$! Fat$free$mass$(Kg)! 45.6%±%6.3! 44.6$±$6.3**! 48.1%±%5.3! 47.5%±%5.6! 44.9$±$4.1! 43.4$±$3.9**! 45.0%±%5.3! 44.2$±$5.2*!

Glucose((mg/dL)! 90.4%±%6.8! 86.2%±%7.2**! 89.6%±"5.9! 88.9$±$8.4! 89.9$±$7.8! 89.2%±%7.8! 94.6%±%9.0! 90.1%±%7.7**! Insulin'(mU/L)a! 6.5$(5.3,$10.0)! 4.4#(3.5,#6.7)**! 6.1$(3.7,$8.2)! 4.7$(4.0,$8.7)! 5.7$(5.3,$9.2)! 4.6$(3.1,$6.4)*! 7.6$(5.5,$12.7)! 4.6$(3.4,$9.1)**! HOMA"IRa! 1.4$(1.0,$2.1)! 0.9$(0.7,$1.3)**! 1.2$(0.7,&1.7)! 1.0$(0.8,$1.7)! 1.4$(1.0,$2.1)! 1.0$(0.8,$1.4)**! 1.7$(1.2,$3.1)! 1.0$(0.7,$1.9)**! TyG$index! 8.1$±$0.4! 8.0$±$0.4*! 8.1$±$0.4! 8.0$±$0.5! 8.2$±$0.4! 8.1$±$0.3*! 8.2$±$0.6! 7.9$±$0.5**!

Irisin&(ng/mL)a! 187.7%%%%%! 165.4&! 168.2&! 152.5%! 161.8%! 151.8%! 188.4%! 162.7&! (142.9,(226.0)! (133.2,'186.4)*! (127.6,(208.7)! (111.6,&179.8)! (114.0,'200.7)! (132.7,(185.3)*! (148.1,'217.0)! (146.7,(174.1)*! Significantly+different+from+baseline+(**P<"0.01;"*P<"0.05)."The"P!value&was&obtained&by&paired!t!test$for$normal$distributed$variables$and$represented$as$mean$±$SD.$The$P! value&was&corrected&by&Benjamini"Hochberg)method.)! EPA:%eicosapentaenoic%acid;%TyG!index:"triglyceride"glucose(index.(! aThe$P"value&was&obtained&by&Wilcoxon´s&test&for&non"normally(distributed)variables)and)represented)as)median)(interquartile)range).!

!

!

!

152!! IV.$RESULTS$(CHAPTER(3)$

a 100 r= 0.23 P= 0.049 b 100 r= 0.21 P= 0.068

50 50

0 0

-50 -50

-100 -100

-150 -150 Plasma irisin changes (ng/mL) changes irisin Plasma Plasma irisin changes (ng/mL) changes irisin Plasma -20 -10 0 10 -6 -4 -2 0 2 Insulin changes (mU/L) HOMA-IR Figure$4.$Pearson’s$correlation$between$changes$in$irisin$with$changes$in$insulin$(a)$and$HOMA=IR$(b)$after$ 10$weeks$of$nutritional$intervention.$ $

In$ order$ to$ further$ explore$ the$ potential$ relationship$ between$ irisin$ and$ glucose$ levels$ and$ because$no$differences$between$groups$were$detected,$the$volunteers$were$categorized$by$the$ median,$as$cut$off,$of$the$baseline$irisin$level.$Then,$it$was$observed$that$although$the$subjects$ with$higher$circulating$levels$of$irisin$at$beginning$had$the$greatest$significant$decrease$of$irisin,$at$ the$endpoint$the$levels$of$this$hormone$in$this$group$remained$higher$than$in$the$other$group.$ Interestingly,$ those$ subjects$ who$ started$ the$ intervention$ with$ higher$ levels$ of$ irisin$ had$ also$ higher$ HOMA=IR,$ insulin$ and$ glucose$ levels$ both$ at$ the$ beginning$ and$ at$ the$ end$ of$ the$ intervention$ (P<$ 0.05),$ but$ after$ the$ correction$ by$ B=H,$ only$ the$ differences$ between$ glucose$ endpoint$ values$ remained$ significant.$ Changes$ on$ glucose$ metabolism=related$ markers$ were$ similar$(P>$0.05)$in$both$groups$(Table$3).$$

! 153$ IV.$RESULTS$(CHAPTER(3)$

Table$3.$Comparisons*between*low*and*high*irisin*levels*at*baseline*in*anthropometric*and*biochemical*parameters$ Low$Irisin$ High$Irisin$ P>value$ P>value$ P>value$ Variable$ n(=36$ n(=37$ baseline$ endpoint$ changes$ $ Baseline$ Endpoint$ Δ$ Baseline$ Endpoint$ Δ$ $ $ $ Irisin*(ng/mL)* 128.4*±*31.0* 130.6*±*41.4* 2.1*±*32.9* 222.6*±*40.7* 185.1*±*37.5* F37.6*±*39.7* <0.0001** <0.0001** <0.0001** Body*weight*(Kg)* 84.64*±*10.64* 78.42*±*10.80* F6.22*±*2.82* 86.85*±*14.53* 81.07*±*13.80* F5.77*±*3.14* 0.46* 0.36* 0.52* BMI*(Kg/m2)* 32.47*±*3.77* 30.0*±*3.71* F2.41*±*1.12* 33.12*±*4.91* 30.91*±*4.78* F2.20*±*1.13* 0.53* 0.40* 0.43* Fat*mass*(Kg)* 39.0*±*7.24* 33.75*±*1.26* F5.0*±*2.40* 40.5*±*9.26* 35.68*±*8.70* F4.81*±*2.36* 0.45* 0.31* 0.73* Fat* free* mass* 45.85*±*4.82* 44.71*±*4.80* F1.24*±*1.43* 45.80*±*5.97* 45.01*±*6.05* F0.79*±*1.08* 0.97* 0.82* 0.13* (Kg)* Glucose*(mg/dL)* 88.72*±*7.08* 85.97*±*6.55* F2.75*±*5.43* 93.36*±*7.43* 90.94*±*8.09* F2.41*±*5.69* 0.01* <0.01** 0.79* Insulin*(mU/L)* 6.56*±*2.99* 4.91*±*2.12* F1.64*±*2.90* 10.13*±*8.52* 7.00*±5.59* F3.13*±*5.84* 0.02* 0.04* 0.17* HOMAFIR* 1.30*±*0.66* 1.01*±*0.45* F0.38*±*0.63* 2.25*±*1.94* 1.54*±*1.28* F0.72*±*1.35* 0.01* 0.02* 0.17* TyG*index* 8.17*±*0.47* 7.91*±*0.38* F0.25*±*0.36* 8.22*±*0.46* 8.09*±0.48* F0.14*±*0.31* 0.62* 0.09* 0.14* Data*are*mean*±*SD.*The*data*were*divided*by*the*median*levels*of*plasma*irisin*at*baseline,*considering*as*low*irisin*levels*those*values*<171.72*ng/mL*and*as*high*irisin* levels*those*values*≥171.72*ng/mL.*The*differences*between*groups*were*evaluated*by*twoFtail!t*Student*test*with*Welch*approximation*when*heteroskedasticity*was* observed.*EPA:*eicosapentaenoic*acid;*TyG*index:*triglycerideFglucose*index.* *Statistically*significant*after*BenjaminiFHochberg*correction.* *

154!! IV.$RESULTS$(CHAPTER(3)$

Discussion$

The#present#study#revealed#that#in#overweight/obese#non8diabetic#subjects,#fasting#irisin#levels# are#not#significantly#related#to#body#weight,#BMI,#fat#mass#or#fat#free#mass#content.#Similarly#to# our#study,#Sesti#et#al.#(2014)#in#a#cohort#of#healthy#obese#individuals#did#not#find#any#significant# association# between# irisin# with# BMI.# However,# the# associations# between# irisin# with# anthropometric#variables#in#different#populations#are#controversial.#Thus,#the#study#of#Stengel#et# al.# (2013)# in# a# small# sample# of# patients# with# normal8weight,# obesity# and# anorexia# nervosa# described# that# irisin# was# positively# correlated# with# BMI,# adiposity# levels# and# fat# free# mass.# Similarly,#Pardo#et#al.#(2014)#also#analyzed#irisin#levels#in#conditions#of#extreme#BMI,#and#found# that#irisin#correlated#positively#with#body#weight,#BMI,#and#fat#mass,#and#suggested#that#fat#mass# contributed#to#most#of#the#variability#of#the#irisin#plasma#levels.#Moreover,#the#study#of#Huh#et#al.# (2012)#in#middle8aged#normal#weight#and#obese#women#found#that#irisin#is#positively#associated# with# BMI# and# fat# free# mass.# However,# Moreno8Navarrete# et# al.# (2013)# have# observed# that# circulating#irisin#was#negatively#associated#with#BMI#and#fat#mass#in#a#men#cohort#of#non8diabetic# subjects.#In#accordance#with#this,#the#study#of#Gutierrez8Repiso#et#al.#(2014)#revealed#higher#irisin# concentrations#in#non8obese#subjects#than#in#morbidly#obese#subjects,#being#the#waist#to#hip#ratio# the#main#predictor#of#serum#irisin#levels.#

Our# current# data# also# revealed# that# after# weight# loss# irisin# levels# decreased,# although# this# change#was#independent#of#the#amount#of#weight#and#fat#mass#loss.#These#data#are#in#agreement# with#previous#trials#that#also#reported#a#drop#in#irisin#levels#after#weight#loss#(Huh#et#al.,#2012;#de# la#Iglesia#et#al.,#2013;#Lopez8Legarrea#et#al.,#2014).##Huh#et#al.#(2012)#suggested#that#muscle#mass# could#be#the#main#predictor#of#irisin#concentration,#while#Pardo#et#al.#(2014)#proposed#that#fat# mass# is# the# main# contributing# factor# in# irisin# levels# in# obesity.# However,# neither# this# nor# other# trials#have#reported#that#the#changes#of#irisin#occurring#during#weight#loss#are#in#parallel#with#the# changes#in#the#adiposity#or#muscle#mass#levels.##

The#present#trial#also#evidenced#that#fasting#irisin#levels#are#highly#correlated#with#circulating# glucose#concentrations.#Similarly,#the#study#of#Huh#et#al.#(2012)#in#middle8aged#normal#weight#and# obese#women#found#that#irisin#is#positively#associated#with#serum#glucose#levels.#Some#studies# have#suggested#that#irisin#levels#are#associated#with#insulin#resistance#and#with#signs#of#vascular# atherosclerosis# (Sesti# et# al.,# 2014).# However,# other# trials# reported# that# the# blood# irisin# concentrations# are# decreased# in# type# 2# diabetic# individuals# (Choi# et# al.,# 2013;# Liu# et# al.,# 2013;# Moreno8Navarrete#et#al.,#2013)#and#in#those#with#metabolic#syndrome#(Yan#et#al.,#2014).#In#this# context,#Liu#et#al.#(2013)#found#decreased#irisin#levels#in#type#2#diabetic#subjects,#and#a#positive#

! 155# IV.$RESULTS$(CHAPTER(3)$ association# between# blood# fasting# levels# of# irisin# with# glucose,# triglycerides# and# BMI# in# non8 diabetic#controls#with#normal#weight#or#moderately#overweight.#According#to#this,#the#study#of# Choi#et#al.#(2013)#showed#that#normal#glucose#tolerance#volunteers#exhibited#greater#irisin#levels# than# their# new8onset#type#2#diabetics#counterparts.#Moreover,#Moreno8Navarrete#et#al.# (2013)# have#observed#reduce#blood#irisin#levels#in#type#2#diabetic#individuals,#and#that#in#non8diabetic# subjects,#circulating#irisin#was#positively#correlated#with#insulin#sensitivity.#Also,#Yan#et#al.#(2014)# described#in#a#large#population#of#obese#individuals#with#central#obesity,#decreased#levels#of#irisin# in#those#subjects#with#metabolic#syndrome#and#higher#levels#of#fasting#glucose;#however,#there# was#no#association#between#irisin#and#BMI,#body#fat#and#muscle#mass.#All#these#findings#seem#to# suggest#that,#in#overweight/obese#individuals,#the#body#weight#and#body#composition#are#not#the# main#predictors#of#circulating#irisin#concentrations,#and#that#there#are#other#factors,#such#as#the# metabolic# status# that# regulate# the# levels# of# this# hormone.# Furthermore,# the# discrepancies# between# studies# could# be# attributed# to# presence# of# metabolic# disturbances# and# the# degree# of# obesity.##

Additionally,# our# trial# found# that# when# the# study# population# was# divided# into# low# and# high# irisin#levels,#higher#concentrations#of#the#hormone#were#associated#with#higher#levels#of#glucose,# insulin#and#HOMA#index.#Previous#studies#with#metabolic#syndrome#or#pre8diabetic#subjects#(de#la# Iglesia#et#al.,#2013;#Lopez8Legarrea#et#al.,#2014;#Norheim#et#al.,#2014)#revealed#that#those#subjects# whom#started#a#weight#loss#program#with#higher#levels#of#irisin#have#greater#reductions#in#lipid# and#glucose8related#parameters#(de#la#Iglesia#et#al.,#2013;#Lopez8Legarrea#et#al.,#2014).#Our#study# in# healthy# overweight/obese# subjects# showed# that# those# individuals# with# higher# levels# of# irisin# exhibited#a#slightly,#but#non8significant,#higher#drop#in#insulin#and#HOMA8IR#in#response#to#the# weight#loss,#but#this#group#still#remained#with#higher#levels#in#glucose#metabolism#parameters.# The#differences#between#healthy#and#diseased#situations#may#explain#the#apparent#discrepancies# in# this# outcome# between# the# current# and# the# previous# mentioned# studies.# Additionally,# it# has# been#suggested#that#similar#to#leptin,#it#could#exist#a#degree#of#resistance#to#irisin#in#overweight#or# obese# subjects# that# would# promote# an# increased# production# of# irisin# to# face# metabolic# complications#(Roca8Rivada#et#al.,#2013;#Stengel#et#al.,#2013).#In#this#context,#other#authors#have# suggested#(Sesti#et#al.,#2014;#Stengel#et#al.,#2013)#that#the#increment#in#circulating#irisin#levels#in# obesity#could#be#an#early#protective#response#of#organism#to#restore#glucose#metabolism#or#to# counteract#metabolic#disturbances.#

Although#the#outcomes#of#several#studies#could#suggest#that#increased#chronic#glucose#could# be#a#key#regulator#of#irisin#levels,#a#recent#study#has#described#that#glucose#treatment#decreased# the#expression#of#Fndc5#gene#in#human#primary#muscle#cells#from#lean#and#diabetic#individuals#

156!! IV.$RESULTS$(CHAPTER(3)$

(Kurdiova#et#al.,#2014).##Our#current#data#from#the#OGTT#evidenced#that#the#production#of#irisin#is# apparently#independent#of#the#acute#glucose#and#insulin#changes.#In#support#of#this,#Kurdiova#et# al.# (2014)# have# recently# shown# that# irisin# circulating# levels# remained# unaffected# during# the# euglycaemic8hyperinsulinaemic# clamp# in# humans.# These# facts# argue# against# acute# changes# in# glucose# and# insulin# as# direct# key# regulators# of# irisin# production# in# healthy# overweight/obese# subjects,# however;# considering# the# duration# of# the# OGTT# (2# hours),# we# cannot# discard# later# changes#on#irisin#levels.#Therefore,#the#relationship#between#irisin#levels#and#chronic#(long8term)# changes#in#glucose#and#insulin#deserve#to#be#further#characterized.#

Another#important#outcome#of#this#study#is#the#finding#that#in#vitro# treatment#with#α8lipoic# acid# increases# both# Fndc5# mRNA# levels# and# irisin# release# in# cultured# human# adipocytes# from# overweight/obese#subjects.#This#rise#in#irisin#release#coincides#with#the#reduction#of#triglyceride# accumulation# and# leptin# production# induced# by# α8lipoic# acid# in# these# adipocytes,# that# we# previously# reported# (Fernández8Galilea# et# al.,# 2014).# We# have# performed# our# study# in# cultured# adipocytes#of#non8diabetic#overweight/obese#subjects,#but#it#would#be#interesting#to#determine#if# the#stimulatory#effects#of#α8lipoic#acid#on#irisin#could#be#also#observed#in#adipocytes#from#obese8 diabetic# subjects# or# if# could# be# affected# by# the# levels# of# glucose.# Moreover,# it# is# important# to# mention# that# the# stimulatory# actions# of# α8lipoic# acid# on# Fndc5# in# human# adipocytes# were# not# observed# in# murine# C2C12# myocytes,# suggesting# a# possible# cell8type# and/or# species8dependent# effects# of# α8lipoic# acid# on# this# gene.# In# this# context,# a# previous# study# of# our# group# has# also# observed# a# tissue# specific# response# to# α8lipoic# acid# supplementation# on# AMPK# between# white# adipose#tissue#and#muscle#in#rats#(Prieto8Hontoria#et#al.,#2012).##

Regarding# the# factors# involved# in# the# molecular# control# of# irisin# production,# it# has# been# proposed#that#the#peroxisome#proliferator8activated#receptor#gamma,#coactivator#1#alpha##(PGC8 1α)#is#a#key#regulator#of#irisin#production#in#muscle#(Bostrom#et#al.,#2012).#Thus,#the#increment#in# the# expression# of# PGC81α# in# skeletal# muscle,# increases# the# expression# of# the# transmembrane# protein#FNDC5,#which#after#cleaving#is#released#as#irisin#into#circulation#(Bostrom#et# al.,# 2012).# Moreover,# increased# expression# of# AMPK# and# its# phosphorylation# has# been# shown# to# activate# PGC81α#and#Fndc5#in#muscle#(Shan#et#al.,#2013).#However,#promotion#of#irisin#release#from#muscle# independently#of#AMPK#pathway#activation#has#been#observed#after#Metformin#treatment#(Li#et# al.,#2014).#Regarding#the#regulation#of#irisin#in#adipose#tissue,#it#has#been#shown#that#Fndc5#gene# expression# was# negatively# associated# with# leptin# and# positively# associated# with# PGC81α# mRNA# levels#in#human#subcutaneous#adipose#tissue#(Moreno8Navarrete#et#al.,#2013).#Previous#studies# from# our# group# (Fernández8Galilea# et# al.,# 2014)# and# from# others# (Wang# et# al.,# 2010)# have# described# the# ability# of# α8lipoic# acid# to# activate# AMPK# and# PGC81α# in# adipocytes# and# muscle,#

! 157# IV.$RESULTS$(CHAPTER(3)$ suggesting# their# potential# involvement# in# the# stimulatory# action# of# α8lipoic# acid# on# irisin.# However,#the#supplementation#with#α8lipoic#acid#in#obese#humans#following#an#energy#restricted# diet,#apparently#did#not#have#any#additional#effects#on#irisin#circulating#levels#than#those#observed# for# weight# loss.# These# contradictory# outcomes# may# be# explained# by# the# fact# that# plasma# irisin# concentrations# are# not# representative# of# the# adipose# tissue# irisin# secretion# since# other# tissues# could#contribute#to#blood#irisin#levels.#Moreover,#we#only#tested#one#dose#of#α8lipoic#acid#(300# mg/day)#and#it#cannot#be#ruled#out#that#other#doses#of#α8lipoic#acid#could#be#necessary#to#observe# differences#on#circulating#irisin#levels.#In#this#context,#it#has#been#reported#that#the#plasma#levels# of#α8lipoic#acid#reached#after#supplementation#with#a#higher#dose#than#the#used#in#our#trial#(600# mg)# in# humans# ranged# around# 508250# μM# (Carlson# et# al.,# 2007),# which# coincide# with# the# concentrations# tested# in# our# in# vitro# study.# We# also# found# that# supplementation# with# the# n83# PUFA#EPA#did#not#have#any#significant#effect#on#irisin#either#in#vivo#or#in#vitro.#In#this#context,#the# study#of#Sánchez#et#al.#(2013)#revealed#that#oleic#and#linoleic#acid#did#not#modify#Fndc5#mRNA# levels#in#myotubes.#According#with#Murata#et#al.#(2000),#the#concentrations#of#EPA#used#in#our# studies#in#adipocytes#are#comparable#with#its#plasma#levels#after#EPA#dietary#ingestion.#To#our# knowledge,#the#effects#of#supplementation#with#EPA#and/or#α8lipoic#acid#on#irisin#have#not#been# evaluated#to#date,#but#our#data#suggest#that#the#beneficial#effects#of#these#supplements#in#obese# subjects#at#the#doses#tested#are#not#mediated#by#irisin.###

In#summary,#the#current#data#suggest#that#in#healthy#overweight/obese#women,#fasting#irisin# levels#are#highly#correlated#with#fasting#glucose.#However,#acute#changes#in#glucose#and#insulin# are#not#able#to#increase#irisin8circulating#levels,#suggesting#that#they#are#not#direct#regulators#of# the#secretion#of#this#hormone.#Our#results#also#revealed#that#α8lipoic#acid#is#able#to#stimulate#irisin# production# by# cultured# adipocytes;# however# supplementation# with# this# acid# (alone# or# in# combination#with#EPA)#did#not#have#any#additional#effect#on#the#reduction#of#irisin#induced#by#a# hypocaloric#diet#in#overweight/obese#women.#

Conflict#of#interest#

No#conflict#of#interest#is#disclosed#for#any#of#the#authors.#

Acknowledgments#

This# work# was# supported# by# grants# from# Ministerio# de# Economía# y# Competitividad,# Spain# # (AGL# 20098 10873/ALI# and# BFU2012836089)# and# by# Línea# Especial# de# Investigación# “Nutrición,# Obesidad# y# Salud”,# University#of#Navarra8Spain.#AE#Huerta#was#supported#by#a#predoctoral#grant#from#“Asociación#de#Amigos# de#la#Universidad#de#Navarra”.#We#thank#María#Zabala#for#her#excellent#technical#assistance#and#Dr.#Ana# Pérez8Ruiz#from#CIMA#(University#of#Navarra)#for#kindly#providing#the#C2C12#myotubes.# # #

158!! IV.$RESULTS$(CHAPTER(3)$

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160!! IV.$RESULTS$(CHAPTER(3)$

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CHAPTER#4#

# # Post=print#version#of#the#article:## # # Effects$ of$ dietary$ supplementation$ with$ EPA$ and/or$ αElipoic$ acid$ on$ adipose$ tissue$ transcriptomic$profile$of$healthy$overweight/obese$women$following$a$hypocaloric$diet$ $ Ana# E.# Huerta1,2,# Pedro# L.# Prieto8Hontoria1,5,# M.# Fernández8Galilea1,6,# X.# Escoté1,2,# J.# Alfredo# Martínez1,2,3,4,#María#J.#Moreno8Aliaga1,2,3,4# # 1Department#of#Nutrition,#Food#Science#and#Physiology,#University#of#Navarra,#Pamplona,#Spain## 2Centre#for#Nutrition#Research,#University#of#Navarra,#Pamplona,#Spain# 3Biomedical# Research# Centre# Network# in# Physiopathology# of# Obesity# and# Nutrition# (CIBERobn),# National#Institute#of#Health#Carlos#III#(ISCIII),#Madrid,#Spain# 4Navarra#Institute#for#Health#Research#(IdiSNA),#Pamplona,#Spain# 5Current#affiliation:#Faculty#of#Health#and#Physical#Activity#Science.#University#SEK.#Santiago#de#Chile,# Chile.# 6Current#affiliation:#Department#of#Nutrition,#Diabetes#and#Metabolism.#School#of#Medicine.#Pontificia# Universidad#Católica#de#Chile.#Santiago#de#Chile,#Chile.# # # # Biofactors,#2016#[Epub#ahead#of#print]# doi:#10.1002/biof.1317# # # # # Impact#factor#(2015):#4.504# 26/131#in#Endocrinology#&#Metabolism# 63/289#in#Biochemistry#&#Molecular#Biology##

!

IV.$RESULTS$(CHAPTER(4)$

Abstract$

In#obesity,#the#increment#of#adiposity#levels#disrupts#the#whole#body#homeostasis,#promoting#an# over#production#of#oxidants#and#inflammatory#mediators.#The#current#study#aimed#to#characterize# the#transcriptomic#changes#promoted#by#supplementation#with#eicosapentaenoic#acid#(EPA,#1.3# g/day),#α8lipoic#acid#(0.3#g/day)#or#both#(EPA+α8lipoic#acid,#1.3#g/day+0.3#g/day)#in#subcutaneous# abdominal# adipose# tissue# from# overweight/obese# healthy# women,# who# followed# a# hypocaloric# diet#(30%#of#total#energy#expenditure)#during#ten#weeks,#by#using#a#microarray#approach.#At#the# end#of#the#intervention,#a#total#of#33,297#genes#were#analyzed#using#Affymetrix#GeneChip#arrays.# EPA#promoted#changes#in#extracellular#matrix#remodeling#gene#expression,#besides#a#rise#of#genes# associated#with#either#chemotaxis#or#wound#repair.#α8Lipoic#acid#decreased#expression#of#genes# related#with#cell#adhesion#and#inflammation.#Furthermore,#α8lipoic#acid,#especially#in#combination# with# EPA,# upregulated# the# expression# of# genes# associated# with# lipid# catabolism# while# downregulated#genes#involved#in#lipids#storage.#Together,#all#these#data#suggest#that#some#of#the# metabolic#effects#of#EPA#and#α8lipoic#acid#could#be#related#to#their#regulatory#actions#on#adipose# tissue#metabolism.#

#

Keywords:#microarray;#n38PUFAs;#gene#expression;#adipose#tissue;#obesity# # #

! 165# IV.$RESULTS$(CHAPTER(4)$

Introduction$

The#important#role#of#white#adipose#tissue#in#obesity8related#clinical#complications#has#been# widely# described# (Calder# et# al.,# 2011;# Camargo# et# al.,# 2013;# Moreno8Aliaga# et# al.,# 2010).# The# increment#in#the#adiposity#levels#disrupts#the#homeostasis#of#the#organism,#promoting#both#an# overproduction#of#oxidants#as#well#as#the#activation#and#infiltration#of#macrophages#and#other# immune# cells,# thereby# stimulating# inflammatory# processes# within# the# adipose# tissue# and# other# organs#(Calder#et#al.,#2011;#Furukawa#et#al.,#2004)#and#creating#a#vicious#cycle#accompanied#by#an# unresolved#chronic#inflammation#(Spite#et#al.,#2014).##

Adipose# tissue# extracellular# matrix# (ECM),# which# is# composed# of# a# miscellaneous# of# multifunctional# proteins# and# molecules,# modulates# a# broad# of# biological# processes# including# differentiation,#cell#migration#and#adhesion,#as#well#as#cell#repair,#development#and#cell#death#(Lu# et# al.,# 2011).# Moreover# it# has# been# considered# that# during# obesity,# either# the# hyperplasia# of# adipocytes#or#the#overload#of#nutrients,#promote#a#disruption#in#the#ECM#remodeling,#favoring# the#production#of#growth#factors#and#chemotactic#signals#besides#the#excessive#accumulation#of# ECM#components#(Keophiphath#et#al.,#2009).#These#factors#increase#macrophage#recruitment#into# the#adipose#tissue#and#promote#the#overproduction#of#pro8inflammatory#mediators#(Sun#et#al.,# 2013).##

Eicosapentaenoic# acid# (EPA)# and# docosahexaenoic# acid# (DHA)# are# the# principal# omega83# polyunsaturated# fatty# acids# (n83# PUFAs)# from# marine# origin,# which# are# considered# essential# nutrients# with# anti8inflammatory# properties# (Calder# and# Yaqoob,# 2009;# Lorente8Cebrián# et# al.,# 2013).#The#beneficial#actions#attributed#to#these#compounds#are#mediated,#in#some#manner,#by# their#accumulation#into#the#adipose#tissue#(Lorente8Cebrián#et#al.,#2013).#Although#some#studies# in# humans# have# observed# the# ability# of# these# n83# PUFAS# to# regulate# the# expression# of# energy# metabolism#and#inflammation8related#genes#into#adipose#tissue#(Itariu#et#al.,#2012;#Kabir#et#al.,# 2007),#most#studies#have#been#performed#using#a#combination#of#both#n83#PUFAs#(EPA#and#DHA).#

Additionally,# α8lipoic# acid,# also# known# as# thioctic# acid,# is# an# essential# cofactor# for# mitochondrial#enzymes#with#antioxidant#properties#(Bast#and#Haenen,#2003;#Fernández8Galilea#et# al.,#2013).#Studies#in#animal#models#of#obesity#(Deiuliis#et#al.,#2011;#Fernández8Galilea#et#al.,#2011;# Prieto8Hontoria# et# al.,# 2013)# and# also# in# cultured# human# adipocytes# (Fernández8Galilea# et# al.,# 2014,#2015)#have#pointed#out#that#adipose#tissue#is#a#target#organ#for#α8lipoic#acid#anti8obesity# actions,# as# well# as# for# its# regulation# of# glucose# and# lipid# metabolism.#In# humans,# some# studies# have#proposed#that#the#supplementation#with#this#compound#could#be#effective#as#an#adjuvant# for#the#weight8loss#treatment#(Carbonelli#et#al.,#2010;#Huerta#et#al.,#2015;#Koh#et#al.,#2011)#and#for#

166!! IV.$RESULTS$(CHAPTER(4)$ the# management# of# the# obesity8associated# metabolic# disturbances# (Manning# et# al.,# 2012;# McNeilly#et#al.,#2011).#However,#there#is#scarce#information#about#whether#the#beneficial#effects# observed# after# the# dietary# supplementation# with# α8lipoic# acid# in# humans# are# related# to# the# modulation#of#adipose#tissue#gene#expression.#

Thus,# the# current# research# aimed# to# characterize# the# transcriptomic# profiles# promoted# by# supplementation# with# EPA# and/or# α8lipoic# acid# into# subcutaneous# abdominal# adipose# tissue# (SAAT)# of# overweight/obese# healthy# women# following# an# energy8restricted# diet,# using# a# microarray#approach,#as#well#as#to#evaluate#possible#associations#between#gene#expression#with# some#specific#biochemical#and#metabolic#biomarkers.#

Methods$

SAAT#biopsies##

SAAT# biopsies# were# obtained# from# the# groups# of# healthy# overweight/obese# women# that# participated# in# the# OBEPALIP# study# (Huerta# et# al.,# 2016).# Inclusion# and# exclusion# criteria# to# participate# in# the# study# have# been# previously# reported# (Huerta# et# al.,# 2015).# Before# biopsy,# volunteers#underwent#an#energy8restricted#dietary#intervention#of#30%#calorie#reduction#based# on# the# total# energy# expenditure# with# a# macronutrient# distribution# as# defined# by# the# American# Heart#Association#(55%#carbohydrates;#30%#lipids,#15%#proteins)#during#108weeks.#At#beginning,# the#subjects#were#assigned#to#one#of#the#four#parallel#intervention#groups,#which#varied#only#in# the# product# supplementation:# 1)# Control# group:# three# placebo8I# capsules# (containing# sunflower# oil)#and#three#placebo8II#capsules#(containing#same#excipients#than#the#α8lipoic#acid#capsules);#2)# EPA#group:#1300#mg/day#of#EPA#distributed#in#three#capsules#of#EPA#80#(provided#by#Solutex®,# Madrid,#Spain,#containing#433.3#mg#of#EPA#and#13.8#mg#of#DHA#as#ethyl8ester);#and#three#placebo8 II#capsules;#3)#α8lipoic#acid#group:#300#mg/day#of#α8lipoic#acid#from#three#capsules#containing#100# mg#of#α8lipoic#acid#(Nature’s#Bounty®,#NY),#and#three#placebo8I#capsules;#and#4)#EPA+α8lipoic#acid# group:#1300#mg/day#of#EPA#(distributed#in#three#capsules#of#EPA#80)#and#300#mg/day#of#α8lipoic# acid#(from#3#capsules#containing#100#mg#of#α8lipoic#acid),#respectively.#The#trial#was#approved#by# the# Research# Ethics# Committee# of# the# University# of# Navarra# No.$ 007/2009# and# registered# at# clinicaltrials.gov# as# NCT01138774.# All# volunteers# signed# the# informed# consent# before# being# enrolled#in#the#trial.#Moreover,#additional#information#about#the#supplementation#protocol#and# anthropometric#measurements,#as#well#as#the#biochemical#and#inflammatory#markers#assessment# have#been#previously#detailed#(Huerta#et#al.,#2015,#2016).#Briefly,#serum#levels#of#glucose,#free# fatty# acids# (FFA)# and# β8hydroxybutyrate# were# measured# using# the# auto# analyzer# PENTRA# C200# (HORIBA# medical,# Madrid,# Spain),# while# plasma# levels# of# insulin# and# leptin# were# assessed#

! 167# IV.$RESULTS$(CHAPTER(4)$ according# with# the# manufacturer’s# protocol# by# commercial# Enzyme# Immunoassay# (ELISA)# kits# from#Mercodia#AB#(Uppsala,#Sweden)#and#BioVendor#(Brno,#Czech#Republic),#respectively.#

Furthermore,# at# beginning# of# the# study,# volunteers# were# advised# to# avoid# foods# with# high# amounts#of#n83#PUFAs,#fish8oil#supplements#or#related8enriched#products.#The#energy#intake#was# evaluated# at# the# beginning# and# at# the# end# of# the# intervention,# by# a# three8day# estimated# food# recall#questionnaire,#as#previously#detailed#(Huerta#et#al.,#2015).##

At#the#end#of#the#intervention,#because#some#participants#declined#to#be#adipose#tissue#biopsy# donors,#out#of#the#73#volunteers#that#finished#the#intervention#and#whose#blood#samples#were# available,# enough# amount# of# 40# adipose# tissue# biopsies# from# subcutaneous# abdominal# periumbilical#area#(182#g)#were#obtained#from#some#women#at#the#Clínica#Universidad#de#Navarra# by#liposuction#under#local#anesthesia#following#an#overnight#fast#and#used#for#the#gene#expression# studies.#Finally,#based#on#the#sample#amount,#24#samples#(6#from#each#group#intervention#group)# were#selected#for#the#microarray#analysis#(Figure$1).##

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Figure$ 1.$ Flowchart# describing# the# available# biopsy# samples# from# the# OBEPALIP# study# and# the# samples# selected#for#the#microarray#analysis.$

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168!! IV.$RESULTS$(CHAPTER(4)$

RNA#extraction#and#microarray#analysis#

Once#obtained,#SAAT#biopsies#were#rapidly#washed,#snap#frozen#in#liquid#nitrogen#and#stored# at# 880# oC# until# their# utilization# for# analysis.# Both,# the# RNA# extraction# and# the#microarray# study# were# performed# at# Progenika# Biopharma# SA.# (Grifols# Company,# Bizkaia,# Spain).# Total# RNA# was# extracted# using# RNeasy# lipid# tissue# mini# kit# (QIAGEN,# Hilden,# Germany)# following# the# manufacturer’s#instructions.#RNA#concentration#and#purity#were#measured#with#a#Nanodrop#ND8 1000# Spectrophotometer# (Thermo# Fisher# Scientific,# Waltham,# MA).# High8quality# RNA# was# confirmed#by#using#an#Agilent#2100#Bioanalyzer#(Agilent#Technologies,#Bizkaia,#Spain).#From#300# ng# of# RNA,# the# Ambion®# WT# Expression# kit# in# conjunction# with# the# Affymetrix# GeneChip®# WT# Terminal# Labeling# kit# were# used# for# the# preparation# of# labeled# cDNA# according# with# common# protocols.#Labeled#samples#were#hybridized#on#Affymetrix#GeneChip#HumanGene#1.1#ST#arrays,# provided#in#plate#format.#Hybridization,#washing#and#scanning#of#the#array#plates#was#performed# on#an#Affymetrix#GeneTitanTM#Instrument,#according#to#the#supplier’s#recommendations.#

For# the# analyses,# microarray# raw# files# were# imported# into# Partek# Genomics# Suite# V6.11# program#(Partek#Inc,#Chesterfield,#MO).#Normalized#intensity#values#were#obtained#either#by#using# the# Robust# Multichip# Average# method# or# by# removing# those# sequences# near# to# background# values.#In#addition,#according#with#the#standard#deviation#of#normalized#intensity#values,#those# probe#sets#that#did#not#present#gene#expression#change#across#all#samples#were#removed,#which# left#a#total#of#8,562#genes#for#further#analyses.#

The# determination# of# enriched8pathways# was# conducted# by# using# the# Ingenuity# Pathway# Analysis#(IPA)#(Ingenuity#Systems,#Redwood#City,#CA),#whose#database#includes#manually#curated# and#fully#traceable#data#derived#from#literature#sources,#at#the#Centro#de#Investigación#Médica# Aplicada# (CIMA,# University# of# Navarra,# Spain).# The# input# was# all# differentially# regulated# genes# compared#to#control#group#in#each#group#(nominal# P8value<# 0.05),# considering# as# a# significant# enriched#pathway#those#with#a#z8score#≥#1.6#and#≤#1.6.#The#z8score,#which#primary#purpose#is#to# infer#the#activation#state#of#an#specific#pathway,#is#calculated#as#described#elsewhere#(Ingenuity# Systems#website:#revised#on#July#2016).#Moreover,#for#the#purpose#of#this#study,#the#analysis#of# pathways#was#focused#in#those#biological#functions#implicated#in#both#the#inflammatory#response# and#metabolism.##

Microarray#validation#by#quantitative#real=time#polymerase#chain#reaction#analysis##

Total#RNA#from#40#volunteers#(10#volunteers#of#each#group),#including#the#6#samples#where# the# microarray# analysis# was# performed# (Figure$ 1),# were# extracted# using# TRIzol®# reagent#

! 169# IV.$RESULTS$(CHAPTER(4)$

(Invitrogen,#Carlsbad,#CA)#according#to#the#manufacturer’s#guidelines.#Then,#as#described#before,# RNA#concentrations#and#quality#were#measured.#RNA#was#then#incubated#with#the#DNA8free#kit# DNAse#(Ambion,#Austin,#TX)#for#30#min#at#37#oC.#RNA#(1#µg)#was#reverse8transcribed#to#cDNA#using# the# MMLV# reverse# transcriptase.# The# expression# of# the# selected# genes# were# determined# using# predesigned# Taqman®# Assays8on8Demand# primers# and# Taqman# Universal# Master# Mix# (Applied# Biosystems,#Foster#City,#CA)#using#the#ABI#PRISM#7900HT#Fast#System#Sequence#Detection#System# (Applied# Biosystems).# Nine# genes# were# selected# for# validation# of# the# microarray# data# by# using# real8time#polymerase#chain#reaction#(PCR):#(AADAC:#Hs00153677_m1;#ACTG2:#Hs01123712_m1;# CHIT1:# Hs00185753_m1;# FRZB:# Hs00173503_m1;# MYH11:# Hs00224610_m1;# ALCAM:# Hs00977641_m1;# MSR1:# Hs00234007_m1;# PLA2G7:# Hs00965837_m1# and# APLNR:# Hs00766613_m1)# on# the# basis# of# the# following# two# criteria:# 1)# genes# implicated# in# enriched# pathways,#preferably#in#more#than#one#and#with#a#fold#change#of#more#than#±#1.5,#and#2)#genes# with#biological#significance,#involved#in#either#the#inflammatory#or#the#lipid#metabolism#pathways.# The# mRNA# levels# were# normalized# by# the# housekeeping# gene# 18S# (Hs00917508_m1),# which# is# stably#expressed#in#adipose#tissue#(Catalán#et#al.,#2007).#Finally,#the#relative#expression#level#of# each#gene#was#calculated#as#28ΔΔCt.#

Statistical#analyses#

Preprocessing# and# normalization# of# gene# expression# data# were# performed# at# Progenika# Biopharma# SA.# (Grifols# Company).# Differentially# expressed# genes# from# control# group# were# detected#using#a#linear#regression#model,#using#the#equation#Yi=# condition#+#ε,#where#Yi#is#the# intensity#“Y”#for#each#sequence#“i”,#“condition”#is#referred#to#the#condition#effect#in#the#intensity# observed#and#“ε”#is#the#non8measurable#error#that#gathered#the#non8predicted#part#of#the#rest#of# variables.# Additionally,# a# one8way# Analysis# of# Variance# (ANOVA)# test# was# also# performed# to# compare#the#four#experimental#groups.#The#significance#threshold#was#set#at# P<#0.05#and#false# discovery#rate#(FDR)#approach#was#used#to#control#multiple#testing.#

Statistical# analyses# for# evaluating# characteristics# of# participants# and# validation# data# were# performed#using#Stata#Statistical#Software#(Release#12.#College#Station,#TX:#StataCorp#LP).#For#all# tests,# the# significance# level# was# set# at# α<# 0.05.# The# Shapiro8Wilk#analysis#was#used#to#test# the# normality.# Depending# on# the# data# distribution# (parametric# or# non8parametric),# differences# between# 108weeks# and# baseline# values# were# compared# using# either# the# Student’s# t# test# (for# paired# samples)# or# Wilcoxon’s# test,# respectively,# taking# into# account# the# two8sided# P8value.# Anthropometric# and# biochemical# variables# were# compared# at# the# beginning# of# the# nutritional# intervention#by#one8way#ANOVA.#Moreover#either#the#endpoint#value#or#the#changes#(10#weeks#–#

170!! IV.$RESULTS$(CHAPTER(4)$ baseline)#were#compared#between#groups#by#a#two8way#ANOVA.#Additionally,#the#data#from#the# validation# of# microarray8selected# genes# by# real8time# PCR# were# evaluated# using# unpaired# t8test# (with# Welch’s# correction).# In# order# to# evaluate# the# potential# interaction# between# both# supplements#(EPA#and#α8lipoic#acid),#a#two8way#ANOVA#was#performed.#Non8normally#distributed# variables#(CHIT1,#ALCAM,#MSR1,#PLA2G7,#ACTG2,#FRZB,#MYH11)#were#log8transformed#before#the# application# of# a# parametric# test.# The# Spearman# correlation# coefficient# was# used# to# identify# associations#among#variables.#

Results$

Anthropometric#and#biochemical#characteristics#of#the#biopsy#donors#are#described#in#Table$1.# At# beginning# of# the# trial# no# significant# differences# were# found# between# groups# in# any# of# the# variables# assessed.# All# groups# decreased# significantly# body# weight# and# fat# mass# (Table$ 1).# Differences# between# groups# in# changes# were# evaluated,# observing# that# those# groups# supplemented#with#α8lipoic#acid#presented#a#greater#decrease#of#fat#mass#than#the#others,#while# having# the# lower# circulating# levels# of# leptin.# Furthermore,# EPA# supplementation# promoted# a# significant#inhibition#in#the#drop#of#systemic#leptin#levels#that#usually#accompanies#fat#mass#loss.# Similar#observations#were#found#taking#into#account#only#those#volunteers#where#the#microarray# analyses# were# performed# (Supplementary$ Table$ I).# Moreover# no# differences# between# groups# were#observed#in#fatty#acids#intake#at#the#beginning#and#at#the#end#of#nutritional#intervention# (Supplementary$Table$II).#

! 171# IV.$RESULTS$(CHAPTER(4)$

Table$1.$Biochemical*and*anthropometric*characteristics*of*the*biopsy*donors* EPA$+$$ Control$ EPA$ α>lipoic$acid$ Parameters$ α>lipoic$acid$ Two>way$ANOVAc$ (n$=$10)$ (n$=$10)$ (n$=$10)$ (n$=$10)$ $α>lipoic$ EPA$x$$ * $ $ $ $ EPA$ acid$ α>lipoic$acid$ Age*(years)* 38.7*±*8.4* 36.0*±*9.6* 38.5*±*4.5* 39.8*±*5.5* * * * Body%weight%(Kg)* * * * * * * Baseline* 84.5*±*10.4* 89.4*±*9.4* 85.3*±*11.5* 81.0*±*10.2* * * * 10*weeksa* 79.4*±*8.7*** 84.0*±*10.4**** 78.0*±*11.7**** 74.2*±*9.1**** NS* NS* NS* Change** J5.1*±*3.7* J5.4*±*2.2* J7.3*±*3.7* J6.8*±*2.8* NS* NS* NS* Fat%mass%(%)% * * * * * * * Baseline* 40.8*±*3.9* 42.4*±*2.8* 42.2*±*4.4* 41.6*±*3.8* * * * 10*weeksa* 39.0±*4.0** 40.6*±*3.6** 38.4*±*5.4*** 37.7*±*3.4**** NS* NS* NS* Change* J1.8*±*2.5* J1.8*±*2.5* J3.8*±*2.6* J3.9*±*1.7* NS* 0.01* NS* Insulin%(mU/L)* * * * * * * Baseline* 7.1*±*3.9* 6.8*±*4.0* 6.8*±*3.2* 11.6*±*11.9* * * * 10*weeksb* 4.2*±*1.6** 6.7*±*4.6* 4.5*±*2.7** 7.3*±*7.5*** NS* NS* NS* Change** J2.9*±*3.6* J0.0*±*4.9* J2.4*±*3.6* J4.3*±*4.7* NS* NS* NS* Glucose%(mg/dL)* * * * * * * Baseline* 88.2*±*7.2* 90.9*±*6.9* 85.9*±*5.0* 93.9*±*8.6* * * * 10*weeksb* 85.8*±*7.1* 90.9*±*9.7* 85.7*±*5.6* 91.2*±*7.0* 0.03* NS* NS* Change** J2.4*±*4.8* J0.0*±*5.8* J0.2*±*6.5* J2.7*±*4.1* NS* NS* NS* *

* * *

172!! IV.$RESULTS$(CHAPTER(4)$

Table$1.$Continuation* * HOMAAIR* * * * * * * Baseline* 1.5*±*0.8* 1.5*±*0.9* 1.4*±*0.7* 2.6*±*2.7* * * * * 10*weeksb* 0.9*±*0.4** 1.5*±*1.2* 0.9*±*0.5** 1.6*±*1.7*** NS* NS* NS* Change** J0.6*±*0.7* 0.0*±*1.2* J0.5*±*0.8* J1.0*±*1.1* NS* NS* NS* * FFA%(mmol/L)% * * * * * * * * Baseline* 0.47*±*0.16* 0.51*±*0.12* 0.60*±*0.25* 0.59*±*0.13* * * * b 10*weeks * 0.50*±*0.21* 0.56*±*0.15* 0.55*±*0.14* 0.61*±*0.16* NS* NS* NS* * Change** 0.03*±*0.20* 0.05*±*0.13* J0.06*±*0.21* 0.02*±*0.20* NS* NS* NS* βAhydroxybutyrate%(mmol/L)* * * * * * * * Baseline* 0.34*±*0.17* 0.24*±*0.16* 0.36*±*0.15* 0.34*±*0.11* * * * * 10*weeksb* 0.34*±*0.29* 0.39*±*0.45* 0.37*±*0.34* 0.44*±0.37* NS* NS* NS* Change** 0.00*±*0.25* 0.15*±*0.38* 0.01*±*0.35* 0.10*±*0.40* NS* NS* NS* * Leptin%(ng/mL)* * * * * * * Baseline* 23.7*±*5.7* 20.2*±*5.7* 23.6*±*8.6* 18.4*±*3.0* * * * * 10*weeksa* 17.3*±*7.3** 20.9*±*5.6* 14.7*±*8.2*** 14.3*±*4.8* NS* 0.03* NS* * Change** J6.4*±*6.4* 0.7*±*4.4* J8.9*±*8.4* J4.1*±*5.5* 0.006* NS* NS* Values*are*presented*as*mean*±*SD,*no*significant*differences*between*groups*were*found*at*baseline.*EPA:*eicosapentaenoic*acid,*FFA:*free* * fatty*acids.* a,*b a b Differences*between*baseline*and*10*weeks*(*P<*0.05,***P<*0.01,****P<*0.001):* Student’s%t*test*or* Wilcoxon’s*test.** * cDifferences*between*groups*at*10*weeks*and*in*changes*(10*weeks*–*before)*were*evaluated*by*2Jway*ANOVA*(P<*0.05;*NS,*nonJsignificant).* * * *

! 173* IV.$RESULTS$(CHAPTER(4)$

Supplemental$Table$I.$Biochemical*and*anthropometric*characteristics*of*the*biopsy*donors,*in*which*the*microarray*analysis*was*performed* EPA$+$$ Control$ EPA$ α>lipoic$acid$ Parameters$ α>lipoic$acid$ Two>way$ANOVAc$ (n$=$6)$ (n$=$6)$ (n$=$6)$ (n$=$6)$ $α>lipoic$ EPA$x$$ * $ $ $ $ EPA$ acid$ α>lipoic$acid$ Age*(years)* 41.8*±*6.6* 38.3*±*7.8* 35.3*±*8.4* 38.7*±*5.7* * * * Body%weight%(Kg)% * * * * * * * Baseline* 87.4*±*10.2* 92.1*±*11.5* 88.0*±*13.3* 82.0*±*11.3* * * * 10*weeksa* 80.9*±*8.9** 86.3*±*11.8** 78.6*±*13.4** 74.0*±*10.7** NS* NS* NS* Change** J6.5*±*3.2* J5.8*±*0.8* J9.4*±*2.4* J8.1*±*2.1* NS* 0.01* NS* Fat%mass%(%)% * * * * * * * Baseline* 42.0*±*3.6* 43.8*±*3.1* 43.2*±*5.2* 42.2*±*4.2* * * * 10*weeksa* 40.3*±*3.7* 41.0*±*3.6** 38.4*±*6.2** 37.6*±*4.2** NS* NS* NS* Change* J1.7*±*2.5* J2.8*±*1.5* J4.7*±*2.4* J4.6*±*1.5* NS* 0.008* NS* Insulin%(mU/L)% * * * * * * * Baseline* 8.2*±*4.8* 7.0*±*4.9* 7.4*±*2.9* 8.8*±*6.3* * * * 10*weeksb* 4.8*±*1.6* 6.0*±*3.8* 4.7*±*3.5* 5.3*±*3.8** NS* NS* NS* Change** J3.5*±*4.6* J1.0*±*5.3* J2.7*±*3.9* J3.5*±*3.0* NS* NS* NS* Glucose%(mg/dL)% * * * * * * * Baseline* 88.2*±*8.2* 91.9*±*6.7* 87.8*±*4.0* 89.7*±*6.6* * * * 10*weeksb* 85.3*±*6.6* 90.7*±*9.4* 87.8*±*7.9* 88.5*±*7.2* NS* NS* NS* Change** J3.0*±*3.8* J1.2*±*6.0* 0.0*±*9.1* J1.3*±*3.1* NS* NS* NS* HOMAAIR% * * * * * * * Baseline* 1.7*±*1.0* 1.5*±*1.1* 1.6*±*0.7* 1.9*±*1.5* * * * 10*weeksb* 1.0*±*0.4* 1.3*±*0.8* 1.0*±*0.7* 1.1*±*0.8** NS* NS* NS* Change** J0.7*±*0.9* J0.3*±*1.2* J0.6*±*0.8* J0.8*±*0.7* NS* NS* NS* *

174!! IV.$RESULTS$(CHAPTER(4)$

Supplemental$Table$I.$Continuation* FFA%(mmol/L)% * * * * * * * Baseline* 0.43*±*0.18* 0.53*±*0.13* 0.53*±*0.14* 0.63*±*0.15* * * * 10*weeksb* 0.44*±*0.21* 0.59*±*0.14* 0.56*±*0.12* 0.60*±*0.14* NS* NS* NS* Change** 0.01*±*0.26* 0.06*±*0.15* 0.03*±*0.18* J0.03*±*0.17* NS* NS* NS* βAhydroxybutyrate% * * * * * * * (mmol/L)% Baseline* 0.38*±*0.18* 0.32*±*0.24* 0.34*±*0.16* 0.36*±*0.12* * * * 10*weeksb* 0.31*±*0.38* 0.50*±*0.52* 0.41*±*0.39* 0.34*±*0.27* NS* NS* NS* Change** J0.07*±*0.24* 0.18*±*0.54* 0.07*±*0.43* J0.02*±*0.22* NS* NS* NS* Leptin%(ng/mL)% * * * * * * * Baseline* 23.0*±*7.6* 21.9*±*6.8* 24.2*±*9.0* 19.3*±*3.4* * * * 10*weeksa* 13.9*±*6.2** 20.7*±*5.6* 12.2*±*7.4** 14.5*±*4.7** NS* NS* NS* Change** J9.1*±*5.5* J1.2*±*3.4* J12.0*±*7.5* J4.5*±*5.9* 0.004* NS* NS* Data*are*means*±*SDs,*no*significant*differences*between*groups*were*found*at*baseline.*EPA:*eicosapentaenoic*acid;*FFA:*free*fatty*acids.* a,*bDifferences*between*baseline*and*10*weeks*(*P<*0.05):*aWilcoxon’s*test*or*bStudent’s*t*test.** cDifferences*between*groups*at*10*weeks*and*in*changes*(10*weeks*–*before)*were*evaluated*by*2Jway*ANOVA*(NS,*nonJsignificant).** *

*

! 175* IV.$RESULTS$(CHAPTER(4)$

Supplemental$ Table$ II.$ Dietary( fatty( acids( intake( in( subjects( in( where( the( validation( from( the( microarray(analysis(was(performed( EPA$+$ Parameter$ Control$ EPA$ αAlipoic$acid$ αAlipoic$acid$ TwoAway$ANOVAa$ (g$/100$g)$ (n$=$10)$ (n$=$10)$ (n$=$10)$ (n$=$10)$ EPA$x$$ $αAlipoic$ ( $ $ $ $ EPA$ αAlipoic$ acid$ acid$ SFAs% ( ( ( ( ( ( ( Baseline( 29.9(±(7.4( 31.1(±(10.8( 24.3(±(10.3( 31.6(±(12.1( ( ( ( Final( 15.7(±(9.4( 13.7(±(6.1( 12.6(±(3.3( 15.8(±(7.3( NS( NS( NS( Change(( J14.2(±(9.1( J17.4(±(9.6( J11.7(±(11.5( J15.8(±(12.4( NS( NS( NS( MUFAs% ( ( ( ( ( ( ( Baseline( 43.0(±(11.0( 43.2(±(12.9( 39.0(±(13.1( 44.5(±(15.0( ( ( ( Final( 26.6(±(11.2( 25.3(±(6.3( 24.3(±(6.1( 25.4(±(8.6( NS( NS( NS( Change( J16.4(±(17.1( J18.0(±(13.5( J14.7(±(11.5( J19.2(±(19.0( NS( NS( NS( PUFAs% ( ( ( ( ( ( ( Baseline( 10.5(±(3.1( 11.7(±(2.8( 11.3(±(3.1( 11.2(±(2.9( ( ( ( Final( 6.8(±(2.8( 6.2(±(1.3( 6.0(±(1.3( 6.8(±(3.4( NS( NS( NS( Change(( J3.7(±(4.9( J5.5(±(2.3( J5.4(±(3.4( J4.4(±(3.1( NS( NS( NS( N3+PUFAs% ( ( ( ( ( ( ( Baseline( 0.30(±(0.20( 0.26(±(0.11( 0.25(±(0.04( 0.23(±(0.07( ( ( ( Final( 0.17(±(0.09( 0.17(±(0.05( 0.16(±(0.06( 0.16(±(0.06( NS( NS( NS( Change(( J0.13(±(0.21( J0.09(±(0.10( J0.09(±(0.06( J0.07(±(0.10( NS( NS( NS( N6+PUFAs% ( ( ( ( ( ( ( Baseline( 3.4(±(2.8( 3.2(±(1.4( 3.0(±(0.9( 2.6(±(0.7( ( ( ( Final( 1.8(±(1.0( 1.9(±(0.5( 1.8(±(0.6( 1.9(±(0.8( NS( NS( NS( Change(( J1.6(±(2.6( J1.3(±(1.2( 1.2(±(1.1( J0.7(±(1.2( NS( NS( NS( Values( are( presented( as( mean( ±( SD.( Differences( between( groups( at( baseline( were( evaluated( by( oneJway( ANOVA(and(no(significant(differences(were(observed.(MUFAs:(monounsaturated(fatty(acids,(N3:(omegaJ3,(N6:( omegaJ6,(PUFAs:(polyunsaturated(fatty(acids,(SFAs:(saturated(fatty(acids.( aDifferences(between(groups(at(final(of(the(nutritional(intervention(were(evaluated(by(twoJway(ANOVA(and( no(significant(differences(were(observed.(

Microarray%analysis%

Upon(comparing(the(gene(expression(profile(of(SAAT(biopsies,(of(the(8,562(probe(sets(present( on(the(genome(array,(580(genes(were(differentially(expressed((ANOVA(P<(0.05)(between(the(four( groups.(As(shown(in(the(Venn(diagram((Figure$2),(72(genes,(27(of(which(were(downregulated(and( 45(of(which(were(upregulated,(were(commonly(differentiated(in(the(three(supplemented(groups.( Furthermore,(a(total(of(120(genes(were(typically(either(downregulated((48(genes)(or(upregulated( (72( genes)( in( both( groups( supplemented( with( αJlipoic( acid.( A( total( of( 83( genes( were( either(

176!! IV.$RESULTS$(CHAPTER(4)$ downregulated((26(genes)(or(upregulated((57(genes)(in(all(groups(supplemented(with(EPA.(Also,( within( the( 563( genes( differentially( expressed( in( the( group( supplemented( with( EPA( alone( as( compared(with(control(group,(262(genes(were(downregulated(and(301(genes(were(upregulated,( whereas(between(the(617(genes(differentially(expressed(in(the(group(supplemented(with(αJlipoic( acid( alone( regarding( control( group,( 248( genes( were( downregulated( and( 369( genes( were( upregulated((Figure$2).(

(

(

(

(

(

(

(

(

Figure$2.(Venn(diagram(illustrating(the(genes(differentially(expressed(regarding(Control(group((nominal(PJ value<(0.05).(Genes(that(were(commonly(downregulated(or(upJregulated(by(the(treatments(were(specified( in(each(compartment.$

Tables$2(and(3(report(the(highest(differentially(upregulated(and(downregulated(genes(with(a( fold(change(>(1.5(or(<(J1.5(respectively,(in(at(least(one(of(the(three(supplemented(groups(and(with( a( nominal( PJvalue<( 0.05.( In( this( sense,( it( was( noted( that( in( the( group( supplemented( with( EPA( alone,(most(of(the(top(38(differentially(regulated(genes(were(upregulated((27(genes)(while(in(the( group(supplemented(with(αJlipoic(acid(alone,(most(of(the(top(35(differentially(regulated(genes( were(downregulated((25(genes).(

! 177( IV.$RESULTS$(CHAPTER(4)$

Table$2.$The!highest!upregulated!nominally3significant!(P<#0.05)!genes!in!supplemented!groups!compared!with!control!group$ ! Biological$process$ Gene$name$ Gene$symbol$ Fold$change$ ! EPA+αClipoic$ ! ! ! EPA$ αClipoic$acid$ acid$ ! Angiogenesis! ! Zinc!finger!protein!7! ZNF7# NS! 1.68! 1.28! ! ! Tenomodulin! TNMD# 1.58! 1.47! NS! Adipogenesis! ! # ! ! ! ! ! Glypican!4! GPC4# 1.74! NS! 1.73! ! ! Stathmin!1! STMN1# 1.51! NS! 1.37! Cell#adhesion#and#migration# ! ! Activated!leukocyte!cell!adhesion!molecule! ALCAM# 2.01! NS! 1.96! ! Complement!component!6! C6# 1.20! NS! 1.84! ! ! Chemokine!(C3C!Motif)!Ligand!14315! CCL15DCCL14# 1.52! NS! 1.83! ! ! Leukocyte!Antigen!CD84! CD84# 1.61! NS! NS! ! Calsyntenin!2! CLSTN2# NS! 1.61! 1.55! ! ! Phospholipase!A2,!group!VII! PLA2G7# 2.52! NS! 2.89! ! Retinoic!acid!receptor!responder!1! RARRES1# NS! NS! 2.18! ! ! Secreted!phosphoprotein!1! SPP1# 3.24! NS! 3.50! ! Extracellular#matrix#remodelling#and#cytoskeleton# ! Capping!protein,!gelsolin3like! CAPG# 1.40! NS! 1.57! ! ! Cathepsin!H! CTSH# 1.55! NS! NS! ! Matrix!metallopeptidase!7!! MMP7# 3.08! NS! NS! ! ! Suppression!of!tumorigenicity!14! ST14# 1.79! NS! 2.02! ! Glucose#metabolism! ! # ! ! ! ! ATPase,!Na+/K+!transporting,!beta!1!polypetide! ATP1B1# 1.77! NS! 1.98! ! ! Fructose31,!63bisphosphatase!1! FBP1# NS! NS! 1.99! !

178!! IV.$RESULTS$(CHAPTER(4)$

Table$2.!Continuation$ ! Inflammatory#and#immune#response! ! ! Acid!phosphatase!5,!tartrate!resistant$ ACP5( 2.06$ NS$ 2.24$ ! CD52!molecule! CD52# NS! NS! 1.58! ! ! Chitinase!1!(chitotriosidase)! CHIT1# 5.87! 4.06! 5.39! ! Chitinase!33like!1!! CHI3L1# 2.92! NS! NS! ! ! Dendrocyte!expressed!seven!transmembrane!protein! DCSTAMP# 2.98! NS! 3.09! ! ! Hematopoietic!cell!signal!transducer! HCST# 1.79! NS! 1.68! ! Hematopoietic!prostaglandin!D!synthase! HPGDS# 1.99! NS! 2.00! ! ! MORC!family!CW3type!zing!finger!4! MORC4# NS! 1.50! 1.70! ! Macrophage!scavenger!receptor!1! MSR1# 1.95! NS! 2.01! ! Membrane3spanning!43domains,!subfamily!A,!member! ! MS4A6E# 4.21! NS! 4.60! 6E! ! ! Transferrin!receptor! TFRC# 1.53! NS! 1.62! ! Lipid#metabolism# ! Alcohol!dehydrogenase!1A!! ADH1A# NS! 1.54! NS! ! ! Apolipoprotein!C31! APOC1# NS! NS! 2.16! ! Apolipoprotein!L,!6! APOL6# 1.25! 1.67! 1.36! ! ! Arylacetamide!deacetylase! AADAC# 3.26! 7.96! 7.45! ! ! Cell!death3inducing!DFFA3like!effector!a! CIDEA# NS! 2.00! 1.91! ! Cytochrome!P450,!family!4,!subfamily!B,!polypeptide!1! CYP4B1# NS! NS! 1.78! ! ! Cytochrome!P450,!family!27,!subfamily!A,!polypeptide!1! CYP27A1# NS! NS! 1.66! ! Glycerol333phosphate!dehydrogenase!13like! GPD1L# NS! 1.61! 1.61! ! ! Lipase!A,!lysosomal!acid,!cholesterol!esterase! LIPA# NS! NS! 1.80! ! ! Neutral!cholesterol!ester!hydrolase!1! NCEH1# 2.18! NS! 2.24! Lysosome#transport# ! ! Major!facilitator!superfamily!domain!containing!12! MFSD12# 1.67! NS! 1.53! ! Solute!Carrier!Family!15,!Member!3! SLC15A3# 1.52! NS! NS! !

! 179! IV.$RESULTS$(CHAPTER(4)$

!

Table$2.$Continuation! ! Oxidative#metabolism! ! ! ! ! ! ! Glutathione!S3transferase!theta!2! GSTT2# NS! 1.51! NS! ! Transcriptional#regulation# ! ! Histone!Cluster!2,!H3a! HIST2H3A# 1.56! NS! NS! Transport! ! # ! ! ! ! ! Solute!carrier!family!31!(copper!transporter),!member!2! SLC31A2# 1.41! 1.39! 1.50! Others# ! ! Transmembrane!protein!100! TMEM100# NS! NS! 1.55! ! Transmembrane!4!L!six!family!member!19! TM4SF19# 3.54! NS! 3.49! ! Fold!change!values!with!50%!of!variation!respecting!control!group!in!at!least!one!supplemented!group.!EPA:!eicosapentaenoic!acid,!NS:!non3significant.!! ! ! !

180!! IV.$RESULTS$(CHAPTER(4)$

Table$3.$The!highest!downregulated!nominally3significant!(P<#0.05)!genes!in!supplemented!groups!compared!with!control!group$ Biological$process$ Gene$name$ Gene$symbol$ Fold$change$ ! EPA+αClipoic$ ! ! ! EPA$ αClipoic$acid$ acid$ ! Angiogenesis! ! Apelin!receptor! APLNR# NS! 31.67! 32.12! ! ! C3fos!induced!growth!factor! FIGF# 31.50! NS! NS! ! EGF3like!repeats!and!discoidin!I3like!domains!3! EDIL3# 31.60! 31.43! NS! ! Adipogenesis# ! # ! ! ! ! ! Fizzled3related!protein! FRZB# 31.68! 31.85! 32.01! ! MicroRNA!21! MiRD21# NS! 31.83! NS! ! Apoptosis! ! # ! ! ! ! Family!with!sequence!similarity!105,!member!A! FAM105A# NS! 31.52! NS! ! ! Family!with!sequence!similarity!96,!member!A! FAM96A# 31.52! 31.35! NS! ! Hypermethylated!in!cancer!1! HIC1# NS! NS! 31.50! ! ! Immediate!early!response!3! IER3# NS! 31.51! NS! ! Interleukin!7!receptor! IL7R# NS! 31.85! NS! ! Cell#adhesion#and#migration# ! β31,43galactosyltransferase$ B4GALT6( NS! 31.62$ NS$ ! ! Chromosome!4!Open!Reading!Frame!31! C4orf31# 31.71! NS! NS! ! ! FBJ!murine!osteosarcoma!viral!oncogene!homolog! FOS# NS! 32.38! NS! ! Fibronectin!type!III!domain!containing!1! FNDC1# NS! 31.86! NS! ! ! Kallmann!syndrome!1!sequence! KAL1# NS! 31.55! 31.50! ! Latrophilin!3! LPHN3# 31.49! NS! 31.58! ! ! Myelin!protein!zero3like!2! MPZL2# 31.52! NS! 31.68! ! Purinergic!receptor!P2Y,!G3protein!coupled,!12! P2RY12# NS! 31.53! 31.58! ! ! Plasminogen!activator,!tissue! PLAT# 31.65! NS! NS! ! Small!Cell!Adhesion!Glycoprotein! SMAGP# NS! NS! 31.75! ! ! Thromboxane!A2!receptor! TBXA2R# NS! 31.37! 31.65! ! Thrombospondin! THBS4# NS! NS! 32.24! ! ! Tetraspanin!7! TSPAN7# NS! NS! 31.60! ! Tetraspanin!18! TSPAN18# NS! NS! 31.59! !

! 181! IV.$RESULTS$(CHAPTER(4)$

Table$3.$Continuation$ ! Extracellular#matrix#remodelling#and#cytoskeleton! ! ! Actin,!gamma!2! ACTG2# 32.42! 32.43! 32.04! ! Matrix3remodelling!associated!5! MXRA5# NS! 31.88! NS! ! ! Myosin,!heavy!chain!11! MYH11# 31.92! 32.10! 31.89! ! Synaptopodin!2! SYNPO2# NS! 31.70! NS! ! Glucose#metabolism! ! # ! ! ! ! Double!C23like!domains,!beta! DOC2B# NS! NS! 31.53! ! Inflammatory#and#immune#response! ! Butyrophilin,!subfamily!3,!member!A2! BTN3A2# NS! NS! 31.53! ! ! C1q!and!tumor!necrosis!factor!related!protein!3! C1QTNF3# NS! 32.31! NS! ! Complement!component!5a!receptor!1! C5AR1# NS! 31.87! NS! ! ! Cystatin!F!(leukocystatin)! CST7# NS! 31.97! NS! ! Endoplasmic!reticulum!aminopeptidase!2!! ERAP2# NS! NS! 33.21! ! ! High!mobility!group!nucleosomal!binding!domain!2! HMGN2# NS! 32.26! 32.33! ! ! Killer!cell!lectin3like!receptor!subfamily!K,!member!1! KLRK1# NS! 31.76! NS! ! Toll3like!receptor!1! TLR1# NS! 31.53! NS! ! ! Toll3like!receptor!2! TLR2# NS! 31.83! NS! Lipid#metabolism# ! ! Diacylglycerol!O3acyltransferase!2! DGAT2# NS! 31.50! NS! Transcriptional#regulation# ! ! Histone!cluster!1,!H4F! HIST1H4F# 31.71! 31.66! 31.81! ! Indolethylamine!N3methyltransferase! INMT# NS! 31.45! 31.91! ! ! RNA,!U11!small!nuclear! RNU11# 31.47! NS! 31.52! ! Small!nuclear!RNA!activating!complex,!polypeptide!1! SNAPC1# 31.53! NS! NS! ! Transport# ! ! Plasmolipin! PLLP# NS! NS! 31.56! ! Solute!carrier!family!38,!member!1! SLC38A1# NS! 31.53! NS! ! Fold!change!values!with!50%!of!variation!respecting!control!group!in!at!least!one!supplemented!group.!EPA:!eicosapentaenoic!acid,!NS:!non3significant.!! !

182!! IV.$RESULTS$(CHAPTER(4)$

Microarray$analysis$revealed$that$both$CHIT1&(which$is$either$preferentially$expressed$by$M2$ anti;inflammatory$ macrophages$ or$ could$ mediate$ M2$ macrophage$ polarization,$ and$ AADAC$ (which$ shares$ sequence$ homology$ with$ the$ hormone$ sensitive$ lipase$ and$ participates$ in$ triglyceride$ hydrolysis)$ were$ genes$ with$ the$ higher$ expression$ in$ all$ supplemented$ groups$ as$ compared$ with$ control$ group.$ Moreover,$ the$ APOL6$ gene,$ which$ encodes$ an$ apolipoprotein$ involved$ in$ autophagy$ and$ apoptosis,$ was$ upregulated$ in$ all$ supplemented$ groups.$ Among$ the$ other$genes$downregulated$in$the$three$supplemented$groups,$it$was$found$an$inhibitor$of$Wnt$ signaling$pathway$(FRZB)$and$two$genes$that$encode$for$cytoskeletal$organization$proteins$that$ participate$in$the$ECM$integrin$signaling$(ACTG2$and$MYH11).$

In$groups$supplemented$with$EPA$(EPA$and$EPA+α;lipoic$acid),$there$were$an$upregulation$of$ genes$with$chemotactic$activity,$involved$in$cell$adhesion$and$migration$(ALCAM,$C6,$CCL14;CCL15$ and$ SPP1).$ Moreover,$ either$ genes$ considered$ as$ a$ M2$ alternatively$ activated$ macrophage$ markers$ (ACP5,$ MSR1,$ TFRC),$ and$ a$ gene$ involved$ in$ the$ degradation$ of$ the$ platelet$ activated$ factor$(PLA2G7)$were$also$upregulated$by$EPA$treatment.$$

The$commonly$downregulated$genes$in$both$groups$supplemented$with$α;lipoic$acid$included$ APLNR$(a$gene$that$participates$in$the$positive$regulation$of$angiogenesis),$HMGN2$gene$(whose$ inhibition$attenuates$the$response$to$LPS$induced$NF;κB$pathway)$and$the$CIDEA$gene$(which$in$ humans$ is$ expressed$ mainly$ in$ white$ adipose$ tissue$ and$ increases$ the$ lipid$ storage$ capacity$ in$ adipocytes).$

Analysis&of&biological&pathways&and&functions&&

In$ order$ to$ get$ a$ better$ characterization$ of$ the$ biological$ pathways$ and$ functions$ that$ are$ regulated$ by$ supplementation$ with$ EPA$ and/or$ α;lipoic$ acid,$ a$ determination$ of$ enriched$ pathways$was$performed$using$the$IPA$software.$$

EPA$treatment$activated$pathways$associated$with$ECM$function$as$“proliferation$of$fibroblast$ cell$ lines”,$ “adhesion$ of$ fibroblast”,$ “formation$ of$ focal$ adhesions”,$ and$ “cell$ movement$ of$ endothelial$cell$lines”$(Table$4).$$

The$ group$ supplemented$ with$ α;lipoic$ acid,$ showed$ an$ activation$ of$ “lipolysis$ of$ adipose$ tissue”.$Additionally,$the$“quantity$of$lymphatic$system$cells”$biological$function$was$activated,$ which$ suggest$ a$ modulation$ of$ immune$ system.$ In$ contrast,$ functions$ related$ with$ both,$ “adhesion$of$endothelial$cells”$and$“cell$spreading”$were$negatively$regulated.$$

Interestingly,$ the$ group$ supplemented$ with$ EPA+α;lipoic$ acid$ had$ a$ greater$ number$ of$ enriched$pathways$than$the$others.$Genes$related$with$lipid$metabolism,$were$clearly$affected,$

! 183$ IV.$RESULTS$(CHAPTER(4)$ observing$an$increment$of$not$only$the$“catabolism$of$lipids”$but$also$an$activation$of$“hydrolysis$ of$ triacylglycerol”,$ whereas$ the$ “deposition$ of$ lipids”$ was$ decreased.$ In$ addition,$ biological$ pathways$ related$ with$ both,$ cell$ migration$ and$ adhesion$ were$ downregulated$ in$ this$ group$ (“transmigration$of$blood$cells”,$“attachment$of$cells”,$“transmigration$of$leukocytes”,$“migration$ of$ myeloid$ cells”,$ “transmigration$ of$ cells”),$ besides$ a$ negative$ regulation$ of$ the$ necrosis$ biological$function,$which$is$associated$with$adipocyte$death$and$infiltration$of$pro;inflammatory$ macrophages.$The$“vasculogenesis”,$which$shares$genes$associated$with$angiogenesis,$was$also$ significantly$inhibited$(Table$4).$$ $ $

184!! IV.$RESULTS$(CHAPTER(4)$

Table$4.$Top$biological$functions$identified$by$Ingenuity$global$function$analysis$ Activation$ No.$ Biological$function$ P>value$ z>score$ Molecules$ $ $ $ EPA(group(versus(control(group( $ $$$Proliferation$of$fibroblast$cell$lines$ 3.055$ 4.58E;03$ 23$ $$$Formation$of$focal$adhesions$ 2.126$ 5.64E;03$ 10$ $$$Adhesion$of$fibroblasts$ 1.951$ 6.52E;03$ 5$ $$$Cell$movement$of$endothelial$cell$lines$ 1.709$ 6.16E;03$ 8$ $$$Size$of$lesion$ ;1.720$ 1.54E;02$ 14$ $ α6lipoic(acid(group(versus(control(group( $ $ $ $$$Lipolysis$of$adipose$tissue$ 1.951$ 1.52E;02$ 4$ $$$Quantity$of$lymphatic$system$cells$ 1.843$ 6.62E;03$ 17$ $$$Cell$spreading$ ;2.304$ 6.54E;04$ 20$ $$$Adhesion$of$endothelial$cells$ ;1.842$ 1.46E;02$ 10$ $ EPA+α6lipoic(acid(group(versus(control(group$ $ $ $ $$$Catabolism$of$lipid$ 2.787$ 3.73E;06$ 15$ $$$Hydrolysis$of$triacylglycerol$ 1.960$ 1.04E;02$ 4$ $$$Transmigration$of$blood$cells$ ;2.606$ 4.45E;04$ 12$ $$$Attachment$of$cells$ ;2.592$ 1.32E;03$ 12$ $$$Transmigration$of$leukocytes$ ;2.439$ 1.46E;03$ 11$ $$$Necrosis$ ;2.223$ 2.20E;03$ 122$ $$$Migration$of$myeloid$cells$ ;2.401$ 1.19E;03$ 14$ $$$Deposition$of$lipid$ ;1.969$ 3.74E;03$ 5$ $$$Transmigration$of$cells$$ ;1.929$ 7.86E;05$ 15$ $$$Vasculogenesis$ ;1.707$ 1.96E;05$ 50$ The$Ingenuity$Pathway$Analysis$(IPA)$software$was$used$for$the$assessment$of$biological$functions$ affected$by$the$treatment$with$EPA$and/or$α;lipoic$acid$as$compared$with$Control$group.$EPA:$ eicosapentaenoic$acid.$$ $ $

Gene& validation& by& realOtime& PCR& and& correlation& analyses& with& anthropometric& and& metabolic&outcomes&

Nine$ genes$ more$ differentially$ expressed$ were$ selected$ for$ validation$ by$ real;time$ PCR.$ Accordingly$ to$ the$ microarray$ data$ analysis,$ the$ PCR$ data$ were$ analyzed$ by$ comparing$ each$ supplemented$ group$ versus$ the$ control$ group,$ using$ the$ unpaired$ Student$ t;test$ with$ Welch’s$ correction,$ to$ confirm$ the$ microarray$ data.$ Additionally,$ with$ the$ aim$ of$ detecting$ potential$ interactions$between$the$two$supplements$used,$a$two;way$ANOVA$analysis$of$the$PCR$data$was$ also$ performed.$ Although$ the$ data$ obtained$ by$ PCR$ had$ a$ similar$ fold;change$ pattern$ to$ the$

! 185$ IV.$RESULTS$(CHAPTER(4)$ microarray,$ some$ results$ were$ partially$ confirmed$ because$ not$ all$ changes$ were$ statistically$ significant$(Figure$3).$

In$ this$ sense,$ similarly$ to$ microarray$ data,$ AADAC$ gene$ expression$ was$ upregulated$ in$ all$ treatments,$ but$ statistically$ significant$ differences$ were$ only$ observed$ (P<$ 0.01)$ in$ the$ EPA+α; lipoic$ acid$ supplemented$ group$ as$ compared$ to$ control$ group.$ The$ two;way$ ANOVA$ analysis$ revealed$ the$ ability$ of$ both$ treatments$ to$ upregulate$ AADAC$ expression$ (P<$ 0.05),$ without$ interactions$ between$ both$ supplements.$ In$ accordance$ with$ the$ microarray$ data$ CHIT1$ mRNA$ levels$ were$ higher$ in$ all$ supplemented$ groups,$ but$ being$ only$ significant$ (P<$ 0.05)$ in$ the$ EPA$ supplemented$group.$In$addition,$the$expression$of$FRZB$was$significantly$downregulated$in$all$ supplemented$groups,$without$significant$interactions$between$EPA$and$α;lipoic$acid.$Moreover,$ though$the$genes$ACTG2$and$MYH11&were$decreased$in$all$supplemented$groups,$only$the$change$ was$significantly$different$in$the$groups$supplemented$with$EPA$(Figure$3).$$

The$validation$data$confirmed$that$gene$expression$of&ALCAM$was$commonly$and$significantly$ upregulated$ (P<$ 0.05)$ in$ both$ groups$ supplemented$ with$ EPA$ (EPA$ and$ EPA+α;lipoic$ acid)$ as$ compared$ with$ control$ group.$ Moreover,$ although$ MSR1$ and$ PLA2G7& mRNA$ levels$ were$ increased$ in$ groups$ supplemented$ with$ EPA,$ only$ the$ group$ with$ EPA$ supplementation$ alone,$ showed$ statistically$ significant$ (P<$ 0.05)$ differences$ regarding$ control$ group.$ In$ this$ sense,$ the$ two;way$ ANOVA$ analysis$ further$ supported$ the$ ability$ of$ EPA$ to$ upregulate$ (P<$ 0.05)$ ALCAM,$ MSR1$ and$ PLA2G7$ mRNA$ levels,$ and$ no$ interactions$ with$ α;lipoic$ acid$ supplementation$ were$ observed$(Figure$3).$$

When$ comparing$ with$ the$ control$ group,$ APLNR$ gene$ expression$ was$ significantly$ downregulated$ only$ in$ the$ group$ supplemented$ with$ EPA+α;lipoic$ acid.$ The$ two;way$ ANOVA$ analysis$ confirmed$ that$ this$ effect$ on$ APLNR$ expression$ was$ caused$ (P<$ 0.01)$ by$ α;lipoic$ acid$ treatment,$and$no$effect$or$interaction$was$observed$by$EPA$supplementation$(Figure$3).$$ $ $

186!! IV.$RESULTS$(CHAPTER(4)$

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Figure$ 3.$ Validation$ by$ RT;PCR$ of$ selected$ differentially$ expressed$ genes$ identified$ by$ microarray.$ Top$ panel)$Relative$gene$expression$of$validated$genes.$Values$are$means$of$the$fold;change$from$control$group$ ±$SEM$(n=$10$per$group)#.$Comparisons$versus$control$group$were$performed$using$unpaired$t;test$(with$ Welch’s$correction)$(**P<$0.01,$*P<$0.05,$+P<$0.1).$Bottom$panel)$P;values$from$the$two;way$ANOVA$test$ (NS:$ non;significant).$ Non;normally$ distributed$ variables$ (CHIT1,$ ALCAM,$ MSR1,$ PLA2G7,$ ACTG2,$ FRZB,$ MYH11)$ were$ log;transform$ before$ the$ test.$ AADAC:$ arylacetamide$ deacetylase;$ ACTG2:$ actin$ gamma$ 2;$ ALCAM:$ activated$ leukocyte$ cell$ adhesion$ molecule;$ APLNR:$ apelin$ receptor;$ CHIT1:$ chitinase$ 1;$ FRZB:$ fizzled;related$protein;$MSR1:$macrophage$scavenger$receptor$1;$MYH11:$myosin$heavy$chain$11;$PLA2G7:$ purinergic$receptor$P2Y$G;protein$coupled$12.$ #Similar$observations$were$found$taking$into$account$only$ those$volunteers$where$the$microarray$analyses$were$performed$(Supplemental$Figure$I).$

The$ correlation$ analysis$ revealed$ that$ the$ expression$ of$ the$ APLNR$ gene$ was$ not$ only$ positively$associated$with$the$changes$(after;before$nutritional$intervention)$in$body$weight$(rho=$ 0.43,$P=$0.007)$and$fat$mass$(rho=$0.42,$P=$0.009),$but$also$negatively$related$with$the$final$levels$ and$ with$ the$ changes$ of$ circulating$ FFA$ (rho=$ ;0.69$ and$ P<$ 0.0001;$ rho=$ ;0.32$ and$ P=$ 0.04,$

! 187$ IV.$RESULTS$(CHAPTER(4)$ respectively)$ and$ β;hydroxybutyrate$ (rho=$ ;0.47$ and$ P=$ 0.002;$ rho=$ ;0.45$ and$ P=$ 0.004,$ respectively).$$

On$the$other$hand,$changes$in$plasma$leptin$levels$were$positively$associated$with$MSR1&(rho=$ 0.49,$P=$0.002)$and$PLA2G7$(rho=0.44,$P=$0.005)$genes$and$negatively$with$ACTG2$(rho=$;0.33,$P=$ 0.04)$ gene.$ The$ FFA$ levels$ at$ the$ end$ of$ the$ intervention$ were$ negatively$ associated$ with$ the$ expression$ of$ AADAC$ (rho=$ ;0.40,$ P=$ 0.02)$ and$ positively$ correlated$ with$ CHIT1$ (rho=$ 0.54,$ P<$ 0.001)$gene$expression.$

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Supplemental$Figure$I.$Gene$expression$of$the$genes$in$the$volunteers$in$where$the$microarray$analyses$ were$ performed.$ Values$ are$ means$ of$ the$ fold;change$ from$ control$ group$ ±$ SEM$ (n=$ 6).$ Comparisons$ versus$ control$ group$ were$ performed$ using$ unpaired$ t;test$ (with$ Welch’s$ correction)$ (***P<$ 0.001,$ *P<$ 0.05,$+P<$0.1).$ $

Discussion$

The$ current$ study$ reports$ differences$ in$ gene$ expression$ levels$ and$ metabolic$ pathways$ in$ SAAT$from$healthy$overweight/obese$women,$who$followed$an$energy;restricted$diet$during$10$ weeks$ with/without$ a$ dietary$ supplementation$ with$ EPA$ and/or$ α;lipoic$ acid.$ As$ previously$ reported$(Huerta$et&al.,$2015),$at$the$end$of$the$intervention,$the$hypocaloric$diet$promoted$not$ only$a$decrease$in$body$weight,$waist$circumference$and$waist$to$hip$ratio$in$all$groups,$but$also$ improved$ some$ biochemical$ parameters$ such$ as$ fasting$ insulin$ and$ insulin$ sensitivity.$ Interestingly,$ the$ groups$ supplemented$ with$ α;lipoic$ acid$ presented$ a$ greater$ decrease$ of$ fat$ mass$and$reduced$expression$of$some$inflammatory$markers$than$the$others$(Huerta$et&al.,$2015,$ 2016).$

188!! IV.$RESULTS$(CHAPTER(4)$

In$ obesity,$ adipose$ tissue$ expansion$ leads$ to$ an$ increment$ of$ angiogenesis$ accompanied$ by$ increased$production$of$chemotactic$factors,$inflammatory$cells$and$upregulation$of$extracellular$ matrix$ remodeling$ factors$ (Cao,$ 2007).$ In$ addition,$ in$ order$ to$ inhibit$ fat$ mass$ expansion$ and$ improve$ insulin$ sensitivity,$ therapy$ with$ anti;angiogenic$ agents$ in$ obesity$ has$ been$ proposed$ (Cao,$2007).$In$this$sense,$our$study$revealed$a$downregulation$of$genes$encoding$proteins$that$ positively$ regulate$ angiogenesis$ (APLNR,$ FIGF,$ EDIL3)$ (O’Carroll$ et& al.,$ 2013),$ which$ was$ accompanied$by$the$upregulation$of$genes$considered$as$angiogenesis$inhibitors$(ZNF7,$TNMD)$ (Shukunami$and$Hiraki,$2007;$Song$et&al.,$2006)$not$only$in$those$groups$supplemented$with$α; lipoic$acid,$but$also$in$those$with$EPA.$Accordingly,$Larghero$et&al.$(2007)$demonstrated$that$α; lipoic$acid$treatment$inhibits$endothelial$cell$migration$and$has$anti;angiogenic$properties$both,$ in$ vitro$ and$ in& vivo.$ Interestingly,$ APLNR$ presented$ a$ negative$ association$ with$ the$ circulating$ levels$of$FFA$and$the$catabolism$of$lipids.$In$this$sense,$Yue$et&al.$(2011),$described$an$increased$ abdominal$ adiposity$ and$ serum$ FFA$ levels$ in$ apelin$ null;mice,$ which$ was$ counteracted$ by$ an$ infusion$ of$ apelin.$ Previous$ studies$ of$ our$ group$ also$ support$ the$ ability$ of$ α;lipoic$ acid$ to$ modulate$the$apelin$system$in$human,$rodents$and$cultured$adipocytes,$which$could$contribute$ to$the$cardiometabolic$protective$effects$of$this$antioxidant$molecule$(Fernández;Galilea$et&al.,$ 2011;$Huerta$et&al.,$2016)$

In$ α;lipoic$ acid$ supplemented$ groups,$ both$ biological$ functions$ and$ genes$ related$ with$ attachment$ and$ adhesion$ of$ cells$ were$ inhibited$ as$ compared$ with$ control$ group.$ Moreover,$ although$ the$ migration$ of$ immune$ cells$ as$ leukocytes$ was$ only$ inhibited$ in$ the$ group$ supplemented$ with$ EPA+α;lipoic$ acid,$ genes$ related$ with$ inflammation$ and$ immune$ response$ were$not$only$downregulated$in$this$group,$but$also$in$the$group$supplemented$with$α;lipoic$acid$ alone$ (as$ TLR1,$ TLR2$ and$ KLRK1).$ In$ this$ sense,$ it$ could$ be$ suggested$ that$ α;lipoic$ acid$ supplementation$is$modulating$inflammation$into$adipose$tissue,$which$in$turn$could$affect$the$ expression$ of$ chemotactic$ factors$ or$ perhaps$ this$ could$ happen$ in$ the$ opposite$ way.$ Though$ there$ is$ not$ available$ information$ about$ the$ in& vivo$ effects$ of$ α;lipoic$ acid$ on$ human$ SAAT$ transcriptome,$ Deiullis$ et& al.$ (2011),$ using$ a$ murine$ model$ of$ obesity,$ reported$ that$ treatment$ with$α;lipoic$acid$inhibited$the$expression$of$TLR4$and$the$pro;inflammatory$cytokines$TNF;α$and$ MCP;1$ in$ adipose$ tissue$ through$ the$ inhibition$ of$ NF;κB$ activation.$ Additionally,$ in$ a$ previous$ study,$ we$ reported$ that$ the$ supplementation$ with$ α;lipoic$ acid$ was$ able$ to$ decrease$ some$ systemic$inflammatory$markers$(CRP$and$leukocyte$count)$in$overweight/obese$women$following$ an$energy$moderate$restricted$diet$(Huerta$et&al.,$2016).$

Through$ different$ signals,$ the$ macrophages$ are$ called$ to$ the$ site$ of$ wound$ for$ healing$ the$ affected$tissue.$Moreover$in$the$obese$subjects,$the$inflammatory$response$is$disturbed,$finding$

! 189$ IV.$RESULTS$(CHAPTER(4)$ an$unresolved$inflammation$(Hummasti$and$Hotamisligil,$2010).$Depending$of$the$environmental$ stimulus,$the$macrophages$could$be$differentiated$to$either$M1$pro;inflammatory$phenotype$or$ M2;anti;inflammatory$phenotype$(Dalmas$et&al.,$2011).$In$this$regard,$besides$the$upregulation$of$ chemotactic$factors$in$the$EPA$supplemented$group,$we$observed$an$increment$of$macrophage$ marker$ genes$ either$ M1$ markers$ (CHI3L1)$ (Di$ Rosa$ et& al.,$2013)$or$M2$markers$(ACP5,$ CHIT1,$ MSR1$ and$ TFRC)$ (Ahlin$ et& al.,$ 2013;$ Bune$ et& al.,$ 2001;$ Di$ Rosa$ et& al.,$ 2013;$ Kratz$ et& al.,$ 2014;$ Mališová$et&al.,$2014;$Orr$et&al.,$2014).$In$this$context,$in$either$a$murine$model$of$obesity$(Kosteli$ et&al.,$2010)$or$humans$(Capel$et&al.,$2009;$Mališová$et&al.,$2014),$an$increase$in$the$adipose$tissue$ macrophages$during$the$early$phase$of$weight$loss$has$been$described,$which$was$not$necessarily$ associated$ with$ the$ upregulation$ of$ inflammatory$ genes.$ Additionally,$ Kitamoto$ et& al.$ (2013)$ observed$ that$ the$ inhibition$ of$ CHIT1$ promoted$ an$ increment$ of$ pro;inflammatory$ signals$ and$ decreased$ M2$ macrophage$ polarization$ in$ a$ macrophage$ cell$ line,$ and$ in$ rodents.$ Taking$ into$ account$the$information$available,$we$suggest$that$the$increment$of$chemotactic$factors$observed$ in$ the$ EPA$ supplemented$ group,$ are$ promoting$ a$ migration$ of$ macrophages$ into$ the$ adipose$ tissue,$especially$M2$macrophages.$In$the$same$line,$a$previous$study$in$the$same$subjects,$found$ that$the$supplementation$with$EPA$was$able$to$increase$the$expression$of$IL10$in$SAAT,$which$is$ an$anti;inflammatory$cytokine$(Huerta$et&al.,$2016).$$

Integrins$binds$to$ECM$and$transduce$signals$through$the$plasma$membrane$to$activate$some$ intracellular$signaling$pathways$(Williams$et&al.,$2015).$Also,$either$during$overfeeding$or$weight$ loss,$important$changes$in$the$ECM$remodeling$expression$genes$have$been$reported,$suggesting$ an$adaptive$and$physiological$response$of$the$adipose$tissue$(Alligier$et&al.,$2011;$Dahlman$et&al.,$ 2010;$ Liu$ et& al.,$2015).$In$this$sense,$it$is$known$that$fibroblast$are$primary$producers$of$ECM$ components$(Song$et&al.,$2000);$and$through$these$ECM$components,$the$cell$is$able$to$respond$ to$peripheral$signals$and$to$communicate$with$the$exterior,$being$the$focal$adhesions$part$of$the$ machinery$that$allows$the$migration$and$proliferation$of$a$variety$of$compounds$(Mason$et&al.,$ 2012).$Although$the$supplementation$with$EPA$and$α;lipoic$acid$did$not$promote$similar$changes$ in$ genes$ related$ with$ ECM$ remodeling,$ genes$ involved$ in$ the$ integrin$ signaling$ pathway$ (GeneCards$ Database,$ revised:$ June$ 2016),$ were$ downregulated$ in$ all$ supplemented$ groups$ (ACTG2$and$MYH11).$

Also,$ the$ analysis$ of$ biological$ pathways$ suggests$ that$ α;lipoic$ acid$ could$ modulate$ lipid$ metabolism.$In$this$sense,$in$the$group$supplemented$with$EPA+α;lipoic$acid,$the$catabolism$of$ lipids$was$increased$while$the$deposition$of$lipids$was$decreased.$This$finding$was$supported$by$ the$ upregulation$ of$ genes$ encoding$ enzymes$ involved$ in$ the$ oxidation$ of$ triglyceride$ and$ cholesterol$(CYP4B1$and$CYP27A1)$or$a$cholesteryl$ester$hydrolase$LIPA.$Moreover,$in$the$group$

190!! IV.$RESULTS$(CHAPTER(4)$ supplemented$with$α;lipoic$acid$the$biological$function$of$lipolysis$of$adipose$tissue$was$positively$ regulated$ and$ it$ was$ observed$ that$ one$ of$ the$ main$ acyltransferases$ responsible$ for$ the$ esterification$ of$ triacylglycerol$ in$ adipose$ tissue,$ DGAT2,$was$between$the$top$ downregulated$ genes.$ Furthermore,$ AADAC,$ which$ encodes$ an$ intracellular$ triglyceride$ lipase$ and$ shares$ sequence$ homology$ with$ the$ HSL$ (Lo$ et& al.,$2010),$was$one$of$the$top$commonly$upregulated$ genes$in$the$three$supplemented$groups.$In$this$sense,$also$the$gene$encoding$for$HSL,$one$of$the$ major$ lipolytic$ enzymes$ responsible$ for$ triglyceride$ hydrolysis$ into$ the$ adipose$ tissue$ (Ghosh,$ 2012),$ was$ commonly,$ although$ marginally,$ upregulated$ in$ all$ supplemented$ groups$ (data$ not$ shown).$ Though$ the$ effects$ in$ SAAT$ gene$ expression$ of$ α;lipoic$ acid$ has$ not$ widely$ studied$ in$ humans,$in$accordance$with$our$findings,$several$trials$have$suggested$that$the$supplementation$ with$this$compound$could$be$an$adjuvant$for$weight$and$fat$mass$loss$treatment$(Carbonelli$et& al.,$ 2010;$ Huerta$ et& al.,$ 2015;$ Koh$ et& al.,$2011).$In$this$context,$previous$studies$of$our$group$ carried$out$either$in$3T3;L1$cell$line$or$in$human$adipocytes,$have$evidenced$the$ability$of$α;lipoic$ acid$to$induce$lipolysis$and$to$inhibit$triglyceride$accumulation$by$modulating$the$activity$of$key$ lipolytic$proteins$as$HSL$(Fernández;Galilea$et&al.,$2012)$and$by$reducing$enzymes$involved$in&de& novo$lipogenesis$as$DGAT1,$FAS$and$SCD1$(Fernández;Galilea$et&al.,$2014).$

The$FRZB$gene$encodes$one$of$the$fizzled;related$proteins,$which$through$binding$Wingless; type$mouse$mammary$tumor$virus$integration$site$(WNTs),$inhibits$their$signal.$The$Wnt$signaling$ is$ known$ to$ play$ a$ role$ in$ the$ regulation$ of$ fat$ distribution$ by$ the$ inhibition$ of$ adipogenesis$ (Christodoulides$ et& al.,$2009).$FRZB$was$found$to$be$increase$in$SAAT$of$obese$subjects$and$is$ primarily$ expressed$ in$ the$ stromal;vascular$ fraction$ (Ehrlund$ et& al.,$ 2013)$ (where$ there$ are$ different$cell$types,$including$preadipocytes).$The$lower$expression$of$FRZB$in$the$supplemented$ groups,$ suggests$ an$ inhibition$ of$ adipogenesis$ through$ the$ regulation$ of$ Wnt$ signal.$ Previous$ studies$have$also$suggested$anti;adipogenic$properties$for$both$EPA$and$α;lipoic$acid$(Fernández; Galilea$et&al.,$2013;$Manickam$et&al.,$2010;$Pérez;Matute$et&al.,$2007).$

One$of$the$strengths$of$this$trial$is$the$use$of$microarray$technology$that$allows$to$measure$ thousands$ of$ genes$ at$ the$ same$ time,$ facilitating$ the$ study$ of$ gene$ expression$ patterns$ and$ pathways$(Moreno;Aliaga$et&al.,$2001).$Furthermore,$it$is$important$to$consider$that$few$studies$ have$ evaluated$ the$ transcriptomic$ changes$ in$ human$ adipose$ tissue$ biopsies$ after$ supplementation$with$either$n;3$PUFAs$and/or$α;lipoic$acid$(Itariu$et&al.,$2012;$Kabir$et&al.,$2007;$ Spencer$et&al.,$2013).$

Moreover,$because$the$EPA$supplements$contained$only$a$very$small$percentage$of$other$fatty$ acids$ (Huerta$ et& al.,$ 2016),$ it$ can$ be$ assumed$ that$ the$ observed$ effects$ in$ both$ groups$ supplemented$with$EPA$capsules$were$mainly$promoted$by$this$n;3$PUFA.$

! 191$ IV.$RESULTS$(CHAPTER(4)$

However,$it$should$be$taken$into$account$some$limitations$of$the$present$investigation,$as$the$ absence$of$SAAT$biopsies$at$baseline$of$the$study.$Moreover,$in$the$present$study,$whole$adipose$ tissue$ (which$ is$ composed$ not$ only$ of$ adipocytes$ but$ also$ by$ other$ cell;types$ as$ fibroblast,$ immune$cells$and$preadipocytes)$was$used$for$gene$expression$profile$analysis.$This$fact$raises$ the$ question$ about$ which$ of$ these$ cell$ fractions$ are$ mainly$ contributing$ to$ the$ observed$ gene$ expression$changes$in$adipose$tissue.$Also,$it$is$important$to$note$that$this$study$was$performed$ in$healthy$women,$and$that$it$would$be$of$interest$to$evaluate$if$the$effects$are$similar$in$men$or$ in$subjects$with$metabolic$disturbances.$Further$studies$in$larger$samples$could$be$of$relevance$to$ confirm$the$current$findings.$

As$ general$ conclusion,$ our$ data$ suggest$ that:$ 1)$ EPA$ is$ promoting$ changes$ in$ the$ ECM$ remodeling$genes$besides$an$increment$of$chemotactic$factors$and$macrophages$associated$with$ wound$ repair;$ 2)$ α;lipoic$ acid$ is$ mediating$ a$ general$ decrease$ of$ chemotactic$ factors$ and$ pro; inflammatory$signals;$and$3)$supplementation$with$α;lipoic$acid,$especially$in$combination$with$ EPA,$is$able$to$upregulate$genes$involved$in$lipid$catabolism$while$downregulates$lipid$storage$ genes.$In$summary,$these$data$suggest$that$acting$through$different$pathways$both$EPA$and$α; lipoic$ acid$ could$ modulate$ adipose$ tissue$ inflammation$ and$ remodeling.$ Furthermore,$ these$ observations$point$out$that$the$metabolic$effects$of$EPA$and$α;lipoic$supplementation$could$be$ related$to$their$regulatory$actions$on$adipose$tissue$genomics$and$metabolism.$

Acknowledgments&&

We$thank$to$the$Ministry$of$Economy$and$Competitiveness$from$the$Government$of$Spain$(ref.$AGL$2009; 10873/ALI$ and$ BFU2012;36089),$ to$ Navarra$ Government$ (Department$ of$ Health$ ref.$ 67/2015),$ to$ the$ Special$ Research$ Line$ of$ “Nutrition,$ Obesity$ and$ Health”$ from$ the$ University$ of$ Navarra;Spain$ and$ to$ CIBERobn$for$the$grants$received.$AE$Huerta$was$supported$by$a$predoctoral$grant$from$“Asociación$de$ Amigos$de$la$Universidad$de$Navarra”.$EPA$and$sunflower$oil$capsules$were$provided$by$Solutex®$(Madrid,$ Spain).$Solutex$had$no$role$in$the$study$design,$data$collection,$analysis$and$interpretation,$or$writing$of$the$ manuscript.$The$authors$have$declared$no$conflict$of$interest.$

Authors&Contribution&

M.J.M;A$and$J.A.M.$conceived$and$designed$the$study;$M.J.M;A,$P.L.P;H,$M.F;G.$and$A.E.H.$supervised$or$ conducted$research;$A.E.H.$performed$the$statistical$analysis;$A.E.H.,$P.L.P;H.$and$X.E.$carried$out$the$gene$ expression$ analyses;$ M.J.M;A,$ J.A.M.$ and$ A.E.H.$ wrote$ the$ paper;$ M.J.M;A$ and$ J.A.M.$ had$ the$ primary$ responsibility$for$the$final$content.$All$authors$have$read$and$approved$the$final$manuscript.$

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192!! IV.$RESULTS$(CHAPTER(4)$

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194!! IV.$RESULTS$(CHAPTER(4)$

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$ $

196!! $

CHAPTER&5&

$ $ Version&in&preparation:&& & $ Differential$ peripheral$ blood$ methylation$ by$ α>lipoic$ acid$ and$ EPA$ supplementation$ in$ overweight/obese$women$during$a$weight$loss$program$

Ana$E.$Huerta1,2,$José$I.$Riezu;Boj1,2,4,$Fermín$I.$Milagro1,2,3,4,$E.$Guruceaga4,5,$María$J.$Moreno; Aliaga1,2,3,4,*,$J.$Alfredo$Martínez1,2,3,4,*$$

$ 1Department$of$Nutrition,$Food$Science$and$Physiology,$University$of$Navarra,$Pamplona,$Spain$$ 2Centre$for$Nutrition$Research,$University$of$Navarra,$Pamplona,$Spain$ 3Biomedical$ Research$ Centre$ in$ Physiopathology$ of$ Obesity$ and$ Nutrition$ (CIBERobn),$ Institute$ of$ Health$Carlos$III$(ISCIII),$Madrid,$Spain$ 4Navarra$Institute$for$Health$Research$(IdiSNA),$Pamplona,$Spain$ 5Proteomics,$Genomics$&$Bioinformatics$Unit,$Center$for$Applied$Medical$Research$(CIMA)$ *Both,$María$J.$Moreno;Aliaga$and$J.$Alfredo$Martínez$shared$equal$senior$authorship.$ $ $ $ $ $ $ $ $ $ $ $ $ $

!

IV.$RESULTS$(CHAPTER(5)$

Abstract$

Scope:$To$assess$methylation$modifications$in$blood$cell$genes$induced$by$eicosapentaenoic$acid$

(EPA)$ and/or$ α;lipoic$ acid$ supplementation,$ and$ their$ possible$ relationship$ with$ metabolic$ risk$ biomarkers.$$

Methods$ and$ results:$ This$ is$ an$ ancillary$ analysis$ of$ the$ OBEPALIP$ study,$ where$ healthy$ overweight/obese$women$followed$a$hypocaloric$diet$(;30%)$during$10$wk$after$being$randomly$ assigned$to$1$of$4$parallel$experimental$groups:$a$control$group$or$groups$supplemented$with$1.3$ g$EPA/d,$0.3$g$α;lipoic$acid/d,$or$both.$DNA$from$white$blood$cells$was$hybridized$in$a$Human$

450K$ methylation$ microarray.$ Differentially$ methylated$ changes$ (after;before)$ were$ identified$ concerning$EPA$(228$CpGs),$α;lipoic$acid$(125$CpGs)$and$EPA+α;lipoic$acid$(279$CpGs)$groups$as$ compared$ with$ control$ group.$ A$ total$ of$ 5$ CpG$ regions$ from$ NCK2,$ FITM2,$ TRRAP,$ RPTOR$ and$

CREBBP$ were$ selected.$ Expression$ of$ selected$ genes$ was$ measured$ in$ peripheral$ blood$ mononuclear$cells$(PBMC).$α;Lipoic$acid$significantly$upregulated$NCK2,$TRRAP&and$RPTOR$genes,$ which$ were$ significantly$ associated$ with$ changes$ in$ body$ weight$ and$ fat$ mass.$ Changes$ in$ cg10320884$ (TRRAP)$ methylation$ site$ significantly$ correlated$ with$ changes$ in$ TRRAP$ gene$ expression$in$PBMC$and$with$changes$in$the$Framingham$score.$$

Conclusion:$These$results$suggest$the$involvement$of$epigenetic$mechanisms$in$some$metabolic$ actions$of$α;lipoic$acid$and$EPA.$

Keywords:$$

Epigenetics$/$Lipoic$acid$/$Methylation$/$Omega;3$/$Weight$loss$$

$ $ $ $ $ $

! 199$ IV.$RESULTS$(CHAPTER(5)$

Introduction$

Obesity$is$a$multifactorial$chronic$disease,$whose$etiology$involves$genetic$and$environmental$ interactions$ (WHO,$ 2014).$ Although$ unhealthy$ diets$ and$ sedentary$ lifestyles$ are$ known$ to$ be$ important$contributors$to$the$obesity$prevalence$by$promoting$a$positive$energy$balance$(WHO,$ 2015),$studies$in$twins$have$suggested$that$the$estimated$heritability$of$body$mass$index$(BMI)$is$ about$40;70%$in$children$and$adults$(Herrera$et&al.,$2011).$$

In$this$context,$epigenetic$modifications$are$stable$and$tissue$specific$heritable$marks$of$the$ genome$ that$ may$ alter$ gene$ expression,$ influencing$ the$ phenotype$ without$ changes$ in$ DNA$ coding$ sequence$ (Goni$ et& al.,$ 2016;$ Remely$ et& al.,$ 2015).$ DNA$ methylation,$ which$ is$ the$ most$ widely$ studied$ epigenetic$ signature,$ occurs$ mainly$ in$ the$ fifth$ carbon$ in$ the$ cytosines$ that$ are$ followed$ by$ a$ guanine$ (CpG)$ as$ reported$ elsewhere$ (Portela$ and$ Esteller,$ 2010).$ Moreover,$ because$the$epigenome$is$considered$a$moldable$system,$throughout$life,$external$factors$such$as$ smoking$habits,$physical$activity$or$dietary$intake$are$constantly$interacting$with$the$epigenome,$ promoting$a$change$or$maintenance$of$different$epigenetic$marks$(Glier$et&al.,$2014;$Remely$et& al.,$2015).$

In$ this$ sense,$ the$ epigenetic$ mechanisms$ whereby$ some$ bioactive$ compounds,$ such$ as$ polyunsaturated$ fatty$ acids$ (PUFAs),$ vitamins$ (e.g.$ vitamin$ C,$ vitamin$ E),$ polyphenols$ (e.g.$ resveratrol)$ and$ other$ dietary$ food$ components$ (e.g.$ α;lipoic$ acid,$ carotenoid,$ curcumin)$ that$ promote$beneficial$effects$on$health$have$been$investigated$(Milagro&et&al.,$2013).$

According$ to$ their$ natural$ source,$ the$ n;3$ PUFAs$ could$ be$ categorized$ into$ marine;derived,$ including$eicosapentaenoic$acid$(EPA)$and$docosahexaenoic$acid$(DHA),$and$vegetable$derived$(α; linolenic$ acid),$ as$ reviewed$ elsewhere$ (Martínez;Fernández$ et& al.,$ 2015).$ Strong$ scientific$ evidence$ has$ demonstrated$ the$ ability$ of$ marine$ n;3$ PUFAs$ to$ improve$ the$ inflammatory$ response$ through$ the$ modulation$ of$ genes$ expression$ in$ inflammation$ in$ different$ cells$ and$ tissues,$including$peripheral$blood$mononuclear$(PBMC)$cells$(Huerta$et&al.,$2016a,$2016b;$Kabir$ et&al.,$2007;$Satoh;Asahara$et&al.,$2012).$In$addition,$some$studies$have$suggested$that$n;3$PUFAs$ supplementation$ could$ modify$ not$ only$ global$ methylation$ levels$ when$ administered$ during$ pregnancy$ (Lee$ et& al.,$ 2013),$ but$ also$ the$ methylation$ pattern$ of$ some$ genes$ related$ to$ inflammation$in$overweight$young$adults$(do$Amaral$et&al.,$2014).$

Additionally,$ α;lipoic$ acid,$ also$ known$ as$ thioctic$ acid,$ is$ an$ essential$ cofactor$ for$ mitochondrial$enzymes$with$antioxidant$properties$(Fernández;Galilea$et&al.,$2013;$Rochette$et& al.,$ 2013;$ Ying$ et& al.,$ 2011).$ Studies$ not$ only$ in& vitro$ (Fernández;Galilea$ et& al.,$ 2011;$ Prieto; Hontoria$ et& al.,$ 2016),$ but$ also$ in$ murine$ models$ (Bitar$ et& al.,$ 2010;$ Deiuliis$ et& al.,$ 2011;$

200!! IV.$RESULTS$(CHAPTER(5)$

Fernández;Galilea$et&al.,$2011;$Prieto;Hontoria$et&al.,$2013;$Yang$et&al.,$2008)$have$found$that$α; lipoic$acid$could$improve$obesity$related;diseases$by$modulating$the$expression$of$genes$related$ to$ oxidative$ stress$ and$ inflammation.$ In$ addition,$ though$ it$ has$ been$ suggested$ that$ the$ supplementation$with$α;lipoic$acid$could$act$across$epigenetic$mechanisms$(Dashwood$and$Ho,$ 2007),$the$evidence$in$humans,$is$scarce.$

Hence,$the$aim$of$the$present$study$was$to$assess$the$methylation$modifications$induced$by$ supplementation$with$EPA$and/or$α;lipoic$acid$in$white$blood$cells$after$a$weight$loss$nutritional$ intervention,$ as$ well$ as$ to$ explore$ possible$ associations$ between$ both$ methylation$ and$ transcriptomic$ changes$ with$ some$ biochemical$ and$ metabolic$ biomarkers$ related$ to$ the$ phenotype.$

Material$and$Methods$

Participants&and&study&design&

The$current$study$is$an$ancillary$analysis$conducted$in$the$framework$of$the$OBEPALIP$study,$a$ double$blind$randomized$placebo;controlled$trial$with$a$parallel$nutritional$intervention,$where$a$ subsample$of$26$healthy$overweight/obese$sedentary$women$(37.3$±$7.6$years$old$and$31.6$±$3.1$ BMI)$were$selected.$All$participants$followed$a$hypocaloric$diet$during$10$weeks$with$an$energy$ restriction$of$30%$according$with$the$total$subject’s$energy$expenditure$as$measured$by$indirect$ calorimetry$(Huerta$et&al.,$2015a).$Macronutrient$distribution$was$prescribed$according$with$the$ American$Heart$Association$dietary$guidelines$(de$la$Iglesia&et&al.,$2014).$During$the$baseline$visit,$ the$dietary$prescription$was$individually$instructed$by$a$trained$dietitian$and$the$volunteers$were$ advised$ about$ not$ change$ their$ physical$ activity$ patterns$ through$ the$ duration$ of$ the$ trial.$ Moreover,$ in$ this$ first$visit,$women$were$assigned$to$one$of$the$four$experimental$groups:$1)$ Control$ group$ (n=$ 6):$ 3$ placebo;I$ capsules$ (containing$ sunflower$ oil)$ and$ 3$ placebo;II$ capsules$ (containing$the$same$excipients$as$the$α;lipoic$acid$capsules),$2)$EPA$group$(n=$7):$1300$mg/d$of$ EPA$distributed$in$3$capsules$of$EPA$80$(provided$by$Solutex®,$Madrid,$Spain),$containing$433.3$ mg$of$EPA$and$13.8$mg$of$DHA$as$ethyl;ester;$and$3$placebo;II$capsules,$3)$α;lipoic$acid$group$(n=$ 7):$ 300$ mg/d$ of$ α;lipoic$ acid$ from$ 3$ capsules$ containing$ 100$ mg$ of$ α;lipoic$ acid$ (Nature’s$ Bounty®,$ NY),$ and$ 3$ placebo;I$ capsules;$ and$ 4)$ EPA+α;lipoic$ acid$ (n=$ 6):$ 1300$ mg/d$ of$ EPA$ (distributed$in$3$capsules$of$EPA80)$and$300$mg/d$of$α;lipoic$acid$(from$3$capsules$containing$100$ mg$of$α;lipoic$acid),$respectively$(Huerta$et&al.,$2015a,$2016b).$The$study$was$approved$by$the$ Research$Committee$of$the$University$of$Navarra$No.$007/2009$and$registered$at$clinicaltrials.gov$

! 201$ IV.$RESULTS$(CHAPTER(5)$ as$NCT01138774.$All$volunteers$signed$the$informed$consent$before$being$enrolled$in$the$trial.$In$ addition,$the$trial$was$conducted$in$compliance$with$the$Helsinki$Declaration$guidelines.$$

In$the$present$analyses,$the$volunteers$were$selected$according$with$their$initial$values$based$ on$ the$ following$ parameters:$ 1)$ BMI$ <40$ kg/m2;$ 2)$ LDL$ <130$ mg/dL$ and$ HDL$ >40$ mg/dL;$ 3)$ Triglycerides$<150$mg/dL;$4)$Glucose$<100$mg/dL.$All$groups$were$balanced$and$included$a$similar$ number$of$overweight$or$obese$subjects.$$

At$ baseline$ and$ at$ endpoint$ the$ volunteers,$ under$ 10;12$ h$ fasting$ conditions,$ visited$ the$ Metabolic$Unit$of$the$University$of$Navarra$to$meet$the$physician,$the$dietitian$and$the$nurse.$ Anthropometric$ measurements$ were$ performed$ according$ with$ standardized$ methods,$ as$ previously$detailed$(Huerta$et&al.,$2015a,$2015b,$2016b).$

Blood&samples&and&biochemical&analyses&

Fasting$blood$samples$were$drawn$on$weeks$0$and$10$into$Serum$Clot$Activator$tubes$(4$mL$ Vacuette®)$and$into$tubes$containing$tripotassium$EDTA$(4$mL$Vacuette®).$Plasma$and$buffy$coat$ were$extracted$from$EDTA$tubes$after$centrifugation$at$1500$g$during$15$min$at$4$oC.$The$buffy$ coat$ was$ either$ kept$ for$ DNA$ extraction$ or$ was$ processed$ by$ standardized$ methods$ according$ with$ the$ PolymorphprepTM$ protocol$ to$ obtain$ PBMC$ for$ RNA$ extraction.$ All$ samples$ were$ appropriately$separated$and$stored$at$;80$oC$for$further$analysis.$$

Serum$ levels$ of$ glucose$ and$ free$ fatty$ acids$ (FFA)$ were$ measured$ using$ the$ Pentra$ C200$ (HORIBA$medical,$Madrid,$Spain)$auto;analyzer,$whereas$white$blood$cells$were$analyzed$with$the$ use$of$an$automated$cell$counter$ABX$Pentra$120$(HORIBA$medical,$Madrid,$Spain).$In$addition,$ plasma$concentrations$of$insulin,$asymmetric$dimethylarginine,$apelin$and$leptin$were$assessed$ according$ with$ the$ manufacturer’s$ protocols$ by$ commercial$ ELISA$ kits$ from$ Mercodia$ AB$ (Uppsala,$ Sweden),$ DLD$ Diagnostika$ GmbH$ (Hamburg,$ Germany),$ RayBiotech$ (GA,$ USA)$ and$ BioVendor$ (Brno,$ Czech$ Republic),$ respectively$ (Huerta$ et& al.,$ 2016b).$ The$ homeostasis$ model$ assessment$was$defined$as$fasting$serum$insulin$(mU/L)$x$fasting$plasma$glucose$(mmol/L)/22.5.$ Additionally,$the$Framingham$score,$which$is$used$to$estimate$cardiovascular$risk$(Expert$Panel$on$ Detection,$Evaluation,$and$Treatment$of$High$Blood$Cholesterol$in$Adults,$2001),$was$evaluated$at$ the$beginning$and$at$the$end$of$the$study,$as$detailed$elsewhere$(Huerta$et&al.,$2016b).$

Genomic&DNA&and&methylation&study&

Genomic$ DNA$ from$ buffy$ coat$ obtained$ at$ baseline$ and$ at$ the$ end$ of$ the$ nutritional$ intervention$was$extracted$using$the$MasterPureTM$DNA$purification$Kit$(Epicentre,$Madison,$WI,$

202!! IV.$RESULTS$(CHAPTER(5)$

USA)$according$with$manufacturer’s$instructions.$The$microarray$preparation,$hybridization$and$ scanning$ was$ performed$ at$ INCLIVA$ Health$ Research$ Institute$ (Valencia,$ Spain),$ while$ the$ preprocessing$ and$ normalization$ of$ methylation$ data$ were$ performed$ at$ CIMA$ (University$ of$ Navarra).$As$previously$described$by$Mansego$et&al.$(2015),$DNA$was$quantified$using$PicoGreen$ double;stranded$ DNA$ Quantification$ Reagent®$ (Invitrogen,$ Carlsbad,$ CA,$ USA).$ The$ EZ$ DNA$ methylation$kit$(Zymo$Research,$Irvine,$CA,$USA)$was$used$for$bisulfite$modification$of$500$ng$of$ genomic$ DNA$ according$ with$ manufacturer’s$ protocols.$ Bisulfite;treated$ genomic$ DNA$ was$ amplified$and$hybridized$using$the$Infinitum$Human$Methylation$450K$BeadChips$(Illumina,$San$ Diego,$CA,$USA)$and$scanned$using$the$IIlumina$HiScanSQ$platform.$DNA$methylation$signals$from$ scanned$arrays$were$preprocessed$and$normalized$using$the$R$scripts$(Gentleman$et&al.,$2005)$of$ Touleimat$and$Tost$(2012).$After$data$normalization$with$the$DASEN$method$(Pidsley$et&al.,$2013),$ methylation$difference$between$the$beginning$and$the$end$of$the$nutritional$intervention$was$ calculated.$ LIMMA$ (Linear$ Models$ for$ Microarray$ Data)$ (Smyth$ et& al.,$ 2005)$ was$ then$ used$ to$ identify$ the$ probes$ with$ significant$ differential$ methylation$ between$ experimental$ conditions.$ The$linear$model$was$adjusted$for$body$weight$loss$and$CpG$sites$were$selected$as$significant$ using$a$P;value$threshold$of$<0.001.$$

RNA&extraction&and&gene&expression&analyses&

Total$RNA$from$PBMC$was$extracted$using$TRIzol®$reagent$(Invitrogen;$Carlsbad,$CA)$according$ to$ the$ manufacturer’s$ instructions.$ RNA$ concentrations$ and$ quality$ were$ assessed$ with$ the$ Nanodrop$ND;1000$Spectrophotometer$(Thermo$Scientific,$Wilminton,$DE).$Then,$RNA$(2$μg)$was$ incubated$with$the$DNA;free$kit$DNAse$(Ambion,$Austin,$TX)$for$30$min$at$37$oC.$RNA$was$reverse; transcribed$ to$ cDNA$ using$ the$ Moloney$ Murine$ Leukaemia$ Virus$ (MMLV)$ reverse$ transcriptase$ (Invitrogen).$Gene$expression$of$5$selected$genes$was$determined$by$quantitative$real$time$PCR$ using$ predesigned$ Taqman®$ Assays;on;Demand$ primers$ (NCK$ adaptor$ protein$ 2$ (NCK2):$ Hs02561903_s1;$ fat$ storage$ inducing$ transmembrane$ protein$ 2$ (FITM2):$ $ Hs00380930_m1;$ transcription$domain;associated$protein$(TRRAP):$Hs00268883_m1;$regulatory$associated$protein$ of$ mTOR$ (RPTOR):$ Hs00375332_m1;$ CREB$ binding$ protein$ (CREBBP):$ $ Hs00231733_m1)$ and$ Taqman$Universal$Master$Mix$(Applied$Biosystems,$Foster$City,$CA)$with$the$ABI$PRISM$7900HT$ Fast$ System$ Sequence$ Detection$ System$ (Applied$ Biosystems).$ Finally,$ the$ relative$ expression$ level$of$each$gene$was$calculated$as$2;ΔΔCt$(Livak$and$Schmittgen,$2001),$using$glyceraldehyde;3; phosphate$dehydrogenase$(GAPDH:$Hs02758991_g1)$as$the$housekeeping$gene.$

&

! 203$ IV.$RESULTS$(CHAPTER(5)$

Statistical&analyses&&

Statistical$analyses$for$evaluating$characteristics$of$participants$and$gene$expression$data$were$ performed$using$the$Stata$Statistical$Software$(Release$12.$College$Station,$TX:$StataCorp$LP).$For$ all$ tests,$ the$ significance$ level$ was$ set$ at$ α<$ 0.05$ and$ all$ comparisons$ were$ two;sided.$ The$ Shapiro;Wilk$ analysis$ was$ used$ to$ test$ the$ normality.$ Depending$ on$ the$ parametric$ or$ non; parametric$ data$ distribution,$ comparisons$ between$ 10;weeks$ and$ baseline$ values$ were$ performed$using$either$the$Student’s$t$test$(for$paired$samples)$or$Wilcoxon’s$test,$respectively.$ Both,$ anthropometric$ and$ biochemical$ variables$ at$ beginning$ of$ the$ nutritional$ intervention$ as$ well$ as$ the$ changes$ in$ gene$ expression$ analyses$ were$ compared$ between$ groups$ by$ one;way$ analysis$ of$ variance$ (ANOVA)$ or$ Kruskal;Wallis$ tests,$ as$appropriate.$ Additionally,$ the$ post$ hoc$ Dunnett’s$test$was$used$to$evaluate$differences$in$the$changes$in$gene$expression$as$compared$ with$ the$ control$ group.$ The$ Pearson’s$ correlation$ coefficient$ was$ used$ to$ identify$ associations$ among$variables.$Additionally,$the$Benjamini;Hochberg$false$discovery$rate$(FDR)$approach$was$ used$as$a$correction$for$multiple$comparisons$(Benjamini$and$Hochberg,$1995).$

Results$

Our$ previous$ data$ in$ the$ OBEPALIP$ study$ showed$ that$ supplementation$ with$ α;lipoic$ acid$ promotes$body$weight$and$fat$mass$loss$(Huerta$et&al.,$2015a)$conjointly$with$a$hypocaloric$diet.$ A$similar$trend$was$observed$in$the$selected$individuals$per$group$included$in$the$methylation$ study,$ although$ no$ significant$ differences$ were$ observed,$ probably$ as$ a$ consequence$ of$ the$ reduced$number$of$subjects$per$group.$Interestingly,$in$these$selected$subgroups,$α;lipoic$acid$ promoted$a$greater$and$significant$decrease$in$the$Framingham$score$and$prevented$the$drop$in$ apelin$that$normally$accompanied$body$weight$loss$(Supplemental$Table$I).$In$addition,$a$lower$ decline$ of$ leptin$ circulating$ levels$ that$ usually$ parallel$ weight$ loss$ was$ observed$ in$ the$ EPA$ supplemented$groups.$No$other$significant$differences$were$observed$among$groups$concerning$ phenotypic$features$(Supplemental$Table$I).$$

204!! IV.$RESULTS$(CHAPTER(5)$

Supplemental$Table$I.$Biochemical$and$anthropometric$characteristics$of$selected$volunteers$at$baseline$and$the$changes$(after:before)$at$the$end$of$the$ 10:week$energy:restricted$diet$in$the$4$parallel$experimental$groups$

Control$ EPA$ α?Lipoic$acid$ EPA+α?lipoic$acid$ e Parameters$ Two?way$ANOVA $ (n$=$6)$ (n$=$7)$ (n$=$7)$ (n$=$6)$ EPA$x$α?lipoic$ $ $ $ $ $ EPA$ $LA$ acid$ Age$(years)$ 39.7$±$8.9$ 35.6$±$9.8$ 35.6$±$6.9$ 39.2$±$4.4$ ns$ ns$ ns$ Body%weight%(Kg)% $ $ $ $ $ $ $ Baselinea$ 82.1$±$4.5$ 82.3$±$8.0$ 84.8$±$12.4$ 82.8$±$10.8$ $ $ $ Changec$$ :6.5$±$1.2*$ :5.9$±$1.6*$ :6.7$±$3.0*$ :5.7$±$2.0*$ ns$ ns$ ns$ Fat%mass%(Kg)$ $ $ $ $ $ $ $ Baselinea$ 33.5$±$4.1$ 33.9$±$4.5$ 35.7$±$9.7$ 34.4$±$6.7$ $ $ $ Changec$ :4.9$±$1.3***$ :4.6$±$1.9***$ :5.4$±$2.8***$ :4.6$±$1.2***$ ns$ ns$ ns$ WHtR% $ $ $ $ $ $ $ Baselinea$ 0.59$±$0.02$ 0.60$±$0.04$ 0.59$±$0.07$ 0.59$±$0.03$ $ $ $ Changed$$ :0.03$±$0.02***$ :0.04$±$0.02***$ :0.04$±$0.02***$ :0.04$±$0.02***$ ns$ ns$ ns$ Glucose%(mg/dL)$ $ $ $ $ $ $ $ Baselineb$ 87.7$±$4.7$ 89.3$±$5.7$ 87.5$±$4.3$ 92.8$±$8.1$ $ $ $ Changed$$ :3.7$±$4.7$ :0.5$±$5.2$ 0.8$±$5.7$ :2.4$±$4.9$ ns$ ns$ ns$ Insulin%(mU/L)$ $ $ $ $ $ $ $ Baselineb$ 5.4$±$2.3$ 4.6$±$1.9$ 7.8$±$3.5$ 7.5$±$4.4$ $ $ $ Changed$$ :1.2$±$2.8$ :0.9$±$2.5$ :2.8$±$3.4*$ :2.3$±$1.8*$ ns$ ns$ ns$ HOMABIR% $ $ $ $ $ $ $ Baselineb$ 1.13$±$0.48$ 0.97$±$0.44$ 1.62$±$0.76$ 1.62$±$0.88$ $ $ $ Changec$$ :0.29$±$0.56$$ 0.21$±$0.60$ :0.57$±$0.69*$ :0.51$±$0.37*$ ns$ ns$ ns$

! 205$ IV.$RESULTS$(CHAPTER(5)$

Supplemental$Table$I.$Continuation$ FFA%(mmol/L)$ $ $ $ $ $ $ $ Baselinea$ 0.44$±$0.12$ 0.49$±$0.13$ 0.52$±$0.12$ 0.59$±$0.17$ $ $ $ Changed$$ 0.04$±$0.23$ 0.07$±$0.14$ :0.08$±$0.09$ 0.01$±$0.14$ ns$ ns$ ns$ Framingham%Score$ $ $ $ $ $ $ Baselinea$ 2.2$±$3.1$ 1.9$±$5.6$ 3.4$±$3.8$ 4.7$±$4.1$ $ $ $ Changed$$ :0.2$±$1.6$ 0.7$±$1.1$ :1.7$±$1.8*$ :1.3$±$1.9$ ns$ 0.009$ ns$ Leukocytes%(103/mm3)% $ $ $ $ $ $ $ Baselineb$ 5.0$±$0.7$ 4.8$±$0.9$ 6.2$±$1.5$ 6.1$±$1.5$ $ $ $ Changec$$ :0.4$±$0.9$ 0.3$±$1.0$ :0.9±$1.1$ :0.7$±$1.2$ ns$ ns$ ns$ ADMA%(µmol/L)$ $ $ $ $ $ $ $ Baselineb$ 0.46$±$0.18$ 0.86$±$0.56$ 0.53$±$0.31$ 0.60$±$0.29$ $ $ $ Changec$$ 0.02$±$0.08$ :0.12$±$0.29$ :0.13$±$0.29$ :0.24$±$0.34$ ns$ ns$ ns$ Apelin%(pg/mL)$ $ $ $ $ $ $ $ Baselineb$ 95.8$±$61.3$ 83.0$±$32.3$ 63.7$±$24.0$ 63.9$±$38.1$ $ $ $ Changec$$ :22.7$±$26.3$ :11.7$±$49.9$ :0.2$±$19.7$ 17.2$±$13.0*$ ns$ 0.04$ ns$ Leptin%(ng/mL)$ $ $ $ $ $ $ $ Baselinea$ 26.7$±$7.8$ 19.6$±$8.4$ 23.7$±$10.2$ 20.6$±$6.2$ $ $ $ Changed$$ :10.4$±$5.0$ :1.2$±$4.7$ :8.2$±$8.3$ :3.1$±$5.8$ 0.007$ ns$ ns$ Values$ are$ presented$ as$ mean$ ±$ SD.$ ADMA:$ asymmetric$ dimethylarginine;$ EPA:$ eicosapentaenoic$ acid;$ FFA:$ free$ fatty$ acids;$ HOMA:IR;$ homeostatic$ model$ assessment$of$insulin$resistance;$ns:$non:significant;$WHtR:$waist$to$height$ratio.$ a,$bDifferences$between$groups$at$baseline$were$evaluated$by:$aone:way$anova$or$bKruskall:wallis$test.$No$significant$differences$were$observed$between$groups.$ c,$dDifferences$between$baseline$and$10$weeks$(*P<$0.05,$**P<$0.01,$***P<$0.001):$cWilcoxon’s$test$or$dStudent’s$t$test.$$ eDifferences$between$groups$in$changes$(10$weeks:baseline)$were$assessed$by$two:way$ANOVA.$ $

$

206!! IV.$RESULTS$(CHAPTER(5)$

Methylation+microarray+analyses+

As$ illustrated$ in$ the$ Venn$ diagram$ (Figure$ 1),$ the$ microarray$ revealed$ that$ the$ methylation$ changes$(after

$

$ $ $ $ $ Figure$ 1.$ Venn$ diagram$ illustrating$ the$ CpG$ sites$ with$ a$ differential$ (P

! 207$ IV.$RESULTS$(CHAPTER(5)$

Table$1.$CpG$sites$and$their$respective$genes$with$a$differentially$(P<$0.001)$methylated$change$(after=before)$at$the$end$of$the$10=week$energy$restricted$diet$ as$compared$with$control$group$ EPA$ LA$ EPA+LA$ Ilumina$ID$ Gene$ Gene$name$ CpG$region$ Δ a$ P@value$ Δ a$ P@value$ Δ a$ P@value$ CpG(sites(shared(between(all(treated8groups((EPA,(α8lipoic(acid(and(EPA+α8lipoic(acid)( cg18709306$ NCK2% NCK$Adaptor$Protein$2$ 5UTR$ 4.5$±$0.7$ 1.40E=05$ 3.9$±$0.7$ 7.02E=05$ 4.9$±$0.9$ 9.41E=06$ cg02962499$ FITM2% Fat$Storage=Inducing$Transmembrane$Protein$2$ TSS1500$ 5.7$±$1.5$ 4.89E=04$ 5.1$±$1.4$ 7.09E=04$ 6.8$±$1.2$ 1.15E=04$ cg12236761$ APLP2% Amyloid$Beta$(A4)$Precursor=Like$Protein$2$ Body;3UTR$ =4.0$±$0.9$ 2.18E=04$ =4.9$±$1.2$ 3.48E=05$ =3.7$±$1.0$ 6.09E=04$ cg16334795$ BACE2% Beta=Site$APP=Cleaving$Enzyme$2$ TSS1500$ =8.5$±$3.1$ 9.39E=04$ =9.1$±$3.3$ 9.16E=04$ =8.7$±$2.9$ 9.48E=04$ CpG(sites(shared(between(the(groups(supplemented(with(α8lipoic(acid((α8lipoic(acid(and(EPA+α8lipoic(acid)( cg04006839$ APOH% Apolipoprotein$H$ 1stExon$ 1.4$±$0.6$ ns$ 5.3$±$1.0$ 1.75E=05$ 5.2$±$1.0$ 5.21E=05$ cg17968037$ ZCWPW1% Zinc$Finger,$CW$Type$With$PWWP$Domain$1$ 5UTR$ 4.7$±$3.7$ ns$ 12.3$±$2.7$ 7.94E=04$ 16.5$±$3.5$ 7.52E=05$ cg23666299$ GRIK2% Glutamate$Receptor,$Ionotropic,$Kainate$2$ Body$ 3.0$±$1.2$ ns$ 5.5$±$1.5$ 1.72E=04$ 5.8$±$1.1$ 8.35E=05$ cg10320884$ TRRAP% Transcription$Domain=Associated$Protein$ Body$ =2.3$±$1.5$ ns$ =6.8$±$1.6$ 1.77E=05$ =5.7$±$1.9$ 1.22E=04$ cg21804148$ LLPH% LLP$homolog,$long=term$synaptic$facilitation$ TSS1500$ =1.0$±$0.8$ ns$ =3.3$±$0.9$ 2.41E=04$ =3.2$±$0.7$ 2.44E=04$ cg14736210$ KCNB1% Potassium$voltage=gated$channel$subfamily$B$1$ 1stExon$ =2.4$±$0.8$ ns$ =3.9$±$1.2$ 9.58E=04$ =4.3$±$1.1$ 2.48E=04$ cg07344315$ RAP1B% RAP1B,$member$of$RAS$oncogene$family$ 3UTR$ =4.4$±$1.9$ ns$ =7.4$±$1.8$ 4.72E=04$ =8.3$±$1.7$ 3.36E=04$ cg03043296$ AP3B2% Adaptor$related$protein$complex$3$beta$2$subunit$ Body$ 2.7$±$1.2$ ns$ 4.2$±$1.2$ 8.55E=04$ 5.1$±$1.1$ 3.97E=04$ cg23287547$ MYO5A% Myosin$VA$ Body$ 2.7$±$0.8$ ns$ 5.0$±$1.4$ 1.96E=04$ 4.5$±$1.4$ 4.04E=04$ TSS200;1st$ cg08466051$ TTC21B% Tetratricopeptide$repeat$domain$21B$ 4.5$±$1.8$ ns$ 6.8$±$2.3$ 9.10E=04$ 7.7$±$1.8$ 4.57E=04$ Exon$ cg03402775$ DZIP3% DAZ$Interacting$Zinc$Finger$Protein$3$ TSS200$ 2.0$±$0.9$ ns$ 3.5$±$1.0$ 2.86E=04$ 3.1$±$1.0$ 4.57E=04$ cg22321985$ CREBBP% CREB$binding$protein$ 3UTR$ =2.9$±$1.4$ ns$ =5.1$±$1.4$ 2.29E=04$ =4.6$±$1.3$ 5.69E=04$ cg17346650$ B3GNTL1% Beta1,3=N=Acetylglucosaminyltransferase=Like$Prot$1$ Body$ =3.4$±$2.0$ ns$ =7.2$±$1.7$ 2.43E=04$ =6.6$±$1.9$ 6.43E=04$ cg16278512$ VWDE% Von$Willebrand$Factor$D$And$EGF$Domains$ 5UTR;1stExon$ =2.0$±$0.9$ ns$ =3.9$±$1.1$ 2.69E=04$ =3.3$±$1.2$ 7.30E=04$ $ $ $ $

208!! IV.$RESULTS$(CHAPTER(5)$

Table$1.$Continuation$ CpG(sites(shared(between(the(groups(supplemented(with(EPA((EPA(and(EPA+α8lipoic(acid)% cg08855288$ NKAIN3% Na+/K+$Transporting$ATPase$Interacting$3$ 1stExon;5UTR$ =8.4$±$2.6$ 3.64E=04$ =7.1$±$2.2$ ns$ =11.4$±$2.5$ 1.39E=05$ cg10938221$ VWA8% Von$Willebrand$Factor$A$Domain$Containing$8$ Body$ =4.4$±$1.6$ 7.83E=04$ =2.8$±$1.7$ ns$ =5.9$±$1.6$ 7.01E=05$ cg07540386$ CCDC135% Coiled=Coil$Domain$Containing$135$ TSS1500$ 3.7$±$1.0$ 9.71E=04$ 0.7$±$1.1$ ns$ 4.2$±$1.3$ 3.57E=04$ cg18194426$ RGS13% Regulator$Of$G=Protein$Signaling$13$ TSS1500$ 3.8$±$1.0$ 9.14E=04$ 3.3$±$1.0$ ns$ 4.3$±$1.2$ 3.96E=04$ cg17480554$ ITGA2% Integrin,$Alpha$2$(CD49B)$ Body$ =5.3$±$1.9$ 3.20E=04$ =4.0$±$2.0$ ns$ =5.0$±$2.0$ 6.14E=04$ cg17094065$ TNS2% Tensin$2$ TSS1500;5UTR;Body$ =5.5$±$9.9$ 6.12E=04$ =2.0$±$1.8$ ns$ =5.5$±$2.1$ 7.59E=04$ cg20653986$ C6orf203% Chromosome$6$Open$Reading$Frame$203$ 5UTR;1stExon$ 4.6$±$1.0$ 8.62E=04$ 2.6$±$1.3$ ns$ 4.8$±$1.3$ 7.69E=04$ cg22366698$ ZNF195% Zinc$Finger$Protein$195$ TSS200$ =5.7$±$1.5$ 8.43E=04$ =3.8$±$1.8$ ns$ =5.8$±$1.6$ 7.98E=04$ cg08150315$ RPTOR% Regulatory$Associated$Protein$of$mTOR$ Body$ 4.8$±$1.3$ 9.19E=04$ 4.0$±$1.3$ ns$ 5.0$±$1.3$ 9.82E=04$ Δ:$change;$C:$control;$EPA:$eicosapentaenoic$acid.$ aMean$change$of$the$difference$from$the$control$group.$Data$are$mean$±$SE.$ $ $

! 209$ IV.$RESULTS$(CHAPTER(5)$

In# order# to# further# explore# the# relationship# between# methylation# changes# with# some# anthropometric# and# metabolic# parameters,# from# the# CpG# sites# described# in# Table$ 1,# 5# CpG# regions# with# a# differential# change# in# methylation# as# compared# with# control# group# (NCK2# (cg18709306),# FITM2# (cg02962499),# TRRAP# (cg10320884),# CREBBP# (cg22321985)# and# RPTOR# (cg08150315)# were# selected# for# further# analyses# (Figure$ 2),# based# on# their# involvement# in# pathways#related#with#insulin#resistance,#inflammation,#lipid#metabolism#and/or#adipogenesis.# # # # # # # # # # # # Figure$2.#Selected#CpG#sites#with#differential#change#(#P<#0.001)#in#methylation#in#supplemented#groups#as# compared# with# C# group.# Δ:# change# (afterNbefore);# C:# control;# CREBBP:# CREB# binding# protein;# EPA:# eicosapentaenoic#acid;#FITM2:#Fat#storageNinducing#transmembrane#protein#2;#LA:#αNlipoic#acid;/NCK2:#NCK# adaptor#protein#2;#RPTOR:#regulatory#associated#protein#of#mTOR;#TRRAP:#transcription#domainNassociated# protein.#

In# addition,# the# correlation# analyses# between# the# changes# of# those# CpG# regions# selected,# revealed# that# the# cg18709306# (NCK2)# CpG# region# was# significantly# (P<# 0.01)# associated# with# almost#all,#except#for#the#cg22321985#(CREBBP)#site#(P=#0.06).#Similarly,#the#cg08150315#(RPTOR)# CpG#region#was#significantly#correlated#(P<#0.01)#with#most#of#the#CpG#regions#assessed,#except# with# the# cg10320884# (TRRAP)# CpG# site.# Finally,# the# CpG# region# cg10320884# (TRRAP)# was# significantly#associated#(P<#0.01)#with#the#CpG#region#from#CREBBP/gene#(Supplemental$Table$II).#/

#

# # # # #

210!! IV.$RESULTS$(CHAPTER(5)$

# # Supplemental$ Table$ II.#Pearson’s#correlation#between#the#methylation#changes#(beforeNafter)#of#the# selected#CpG#regions#according#with#the#association#with#inflammation#and#lipid#metabolism#in#the#4# parallel#experimental#groups# Δ$Cg18709306$ Δ$Cg02962499$ Δ$Cg10320884$ Δ$Cg22321985$ Δ$Cg08150315$ CpG$regions$ NCK2/ FITM2/ TRRAP/ CREBBP/ RPTOR/ r$$ P( r$$ P$ r$$ P$ r$$ P$ r$$ P$ Δ#Cg18709306$ 1# NN# NNN# NNN# NNN# NNN# NNN# NNN# NNN# NNN# NCK2$ Δ$Cg02962499$ 0.61$ 0.001$ 1# NNN# NNN# NNN# NNN# NNN# NNN# NNN# FITM2/ Δ$Cg10320884$ H0.50$ 0.009$ N0.31# ns# 1# NNN# NNN# NNN# NNN# NNN# TRRAP/ Δ$Cg22321985$ N0.38# ns# N0.26# ns# 0.54$ 0.005$ 1# NNN# NNN# NNN# CREBBP/ Δ$Cg08150315$ H0.58$ 0.002$ H0.59$ 0.001$ N0.30# ns# H0.55$ 0.004$ 1# NNN# RPTOR/ Δ:#change#(afterNbefore);#CREBBP:#CREB#binding#protein;#FITM2:#Fat#storageNinducing#transmembrane#protein#2;# NCK2:# NCK# adaptor# protein# 2;# ns:# nonNsignificant;# RPTOR:# regulatory# associated# protein# of# mTOR;# TRRAP:# transcription#domainNassociated#protein.#

Gene/expression/analyses/in/PBMC/

#Also,#gene#expressions#of#NCK2,#FITM2,#TRRAP,#CREBBP#and#RPTOR#genes#were#measured#in# PBMC,# where# only# the# αNlipoic# acid# group# showed# a# significant# increase# (P<# 0.05)# in# the# expression# of# genes# NCK2,# TRRAP# and# RPTOR# as# compared# with# control# group# and# a# marginal# increase#in#the#CREBBP#gene#expression#(Figure$3).##

#

##

# # # # # # Figure$3.#Gene#expression#change#(afterNbefore)#at#the#end#of#the#10Nweeks#nutritional#intervention.#Data# are# mean# ±# SE.# Genes# were# normalized# by# the# GAPDH/ housekeeping# gene# and# analyzed# by# the# 2NΔΔCt# method.#Comparisons#between#groups#were#performed#by#oneNway#ANOVA#test#followed#by#the#Dunnett’s# post# hoc# analysis# (*P<# 0.05# as# compared# with# C# group).# C:# control;# CREBBP:# CREB# binding# protein;# EPA:# eicosapentaenoic#acid;#FITM2:#Fat#storageNinducing#transmembrane#protein#2;#LA:#αNlipoic#acid;/NCK2:#NCK# adaptor#protein#2;#RPTOR:#regulatory#associated#protein#of#mTOR;#TRRAP:#transcription#domainNassociated# protein.#

! 211# IV.$RESULTS$(CHAPTER(5)$

Concerning#NCK2,#TRRAP,#CREBBP#and#RPTOR/gene#expression,#a#high#significant#correlation# (P<# 0.0001)# between# them# was# observed# not# only# at# the# start# and# end# of# the# nutritional# intervention#(data#not#shown),#but#also#in#the#changes#(Supplemental$Table$III).#

# Supplemental$Table$III.#Pearson’s#correlation#between#changes#(beforeNafter)#in#gene#expression#of# the#selected#genes#in#the#4#experimental#groups# Δ$NCK2( Δ$FITM2( Δ$TRRAP( Δ$CREBBP( Δ$RPTOR( Parameters$ r$ P( r$ P$ r$ P$ r$ P$ r$ P$ Δ$NCK2$ 1# NNN# NNN# NNN# NNN# NNN# NNN# NNN# # # Δ$FITM2$ 0.21# ns# 1# NNN# NNN# NNN# NNN# NNN# # # Δ$TRRAP$ 0.91$ <0.0001$ 0.40$ 0.047$ 1# NNN# NNN# NNN# # # Δ$CREBBP$ 0.86$ <0.0001$ 0.19# ns# 0.91$ <0.0001$ 1# NNN# # # Δ$RPTOR$ 0.92$ <0.0001$ 0.21# ns# 0.92$ <0.0001$ 0.89$ <0.0001$ 1# NNN# Δ:#change#(afterNbefore);#CREBBP:#CREB#binding#protein;#FITM2:#Fat#storageNinducing#transmembrane#protein#2;# NCK2:# NCK# adaptor# protein# 2;# ns:# nonNsignificant;# RPTOR:# regulatory# associated# protein# of# MTOR;# TRRAP:# transcription#domainNassociated#protein.# #

Furthermore,#it#was#evidenced#a#negative#significant#(r=# N0.44#P=#0.03)#association#between# the# changes# in# the# expression# of# TRRAP# gene# with# the# changes# in# the# methylation# of# the# cg10320884#CpG#region#(Figure$4).#No#other#significant#associations#were#found#between#the#DNA# methylation#and#mRNA#expression#in#the#other#analyzed#genes.##

/

/

/

/

/

Figure$4.#Pearson’s#correlation#analyses,#between/TRRAP#gene#expression#with#the#methylation#changes#in# the# CpG# position# cg10320884# (TRRAP)# Δ:# change# (afterNbefore);# LA:# αNlipoic# acid;# TRRAP:# transcription# domainNassociated#protein.#

Correlation/ analyses/ between/ changes/ in/ methylation/ and/ gene/ expression/ with/ anthropometric/and/metabolic/outcomes/

Regarding# associations# between# changes# in# methylation# and# metabolic# outcomes,# the# Pearson’s# correlation# revealed# that# the# methylation# change# in# the# CpG# position# cg10320884#

212!! IV.$RESULTS$(CHAPTER(5)$

(TRRAP)#had#a#highly#significant#positive#correlation#with#the#changes#in#the#Framingham#score,# which#remained#significant#after#the#FDR#correction#(Figure$5a).#Furthermore,#a#negative#marginal# association#(r=#N0.36#P=#0.08)#was#observed#between#TRRAP#gene#expression#at#the#end#of#the#10N week#nutritional#intervention#with#the#changes#in#the#Framingham#score#(Figure$5b).##

#

#

#

#

#

# $ Figure$5.#Pearson’s#correlation#analyses,#between#the#methylation#changes#in#the#CpG#position#cg10320884# (TRRAP)#with#the#changes#in#the#Framingham#score#(a)#and#between#the#final#TRRAP#gene#expression#with# the#changes#in#the#Framingham#score#(b).#Δ:#change#(afterNbefore);#LA:#αNlipoic#acid;#TRRAP:#transcription# domainNassociated#protein.#

Moreover,# the# gene# expressions# at# the# end# of# the# nutritional# intervention# of# NCK2,# TRRAP,# CREBBP#and#RPTOR#were#negatively#and#significantly#associated#(P<#0.05)#with#the#changes#(afterN before)#in#body#weight#and#fat#mass#(Table$2).##

#

Table$2.#Pearson’s#correlations#between#gene#expression#at#the# end#of#the#10Nweeks#nutritional#intervention#with#changes#(afterN before)#in#body#weight#and#fat#mass#in#the#4#experimental#groups# Δ$Body$weight$(kg)$ Δ$Fat$mass$(kg)$ Gene$(AU)$ r$ P( r$ P( NCK2/ N0.50# 0.01# N0.44# 0.03# FITM2/ N0.29# ns# N0.23# ns# TRRAP/ N0.58# 0.002# N0.51# 0.01# CREBBP/ N0.49# 0.01# N0.46# 0.02# RPTOR/ N0.49# 0.01# N0.42# 0.03# Genes# were# normalized# by# the# GAPDH# housekeeping# gene# and# analyzed# by# the# 2NΔΔCt# method,# as# described# elsewhere# (Livak# and# Schmittgen,#2001).#CREBBP:#CREB#binding#protein;#FITM2:#Fat#storageN inducing#transmembrane#protein#2;#NCK2:#NCK#adaptor#protein#2;#ns:# nonNsignificant;#RPTOR:#regulatory#associated#protein#of#MTOR;#TRRAP:# transcription#domainNassociated#protein.# #

Interestingly,#similar#negative#associations#were#found#between#the#final#circulating#fasting#FFA# levels# at# the# end# of# the# study# with# the# changes# in# the# expression# of# NCK2# (r=# N0.49,# P=0.01),#

! 213# IV.$RESULTS$(CHAPTER(5)$

TRRAP# (r=# N0.38,# P=# 0.06),# CREBBP# (r=# N0.42,# P=# 0.04)# and# RPTOR# (r=# N0.45# P=# 0.02)# genes,# suggesting# that# volunteers# with# higher# increases# in# the# expression# of# these# genes,# had# lower# serum#circulating#FFA#levels#at#final#of#the#nutritional#intervention.#Although#no#association#was# observed#between#the#CpG#sites#selected#and#the#changes#in#FFA,#the#final#methylation#of#the# cg18709306#(NCK2)#was#positively#associated#with#the#final#FFA#levels#(r=#0.60#P=#0.001).#$

Discussion$

In# the# current# study,# we#sought#to#evaluate#whether#the#dietary#supplementation#with#EPA# and/or# αNlipoic# acid# was# able# to# induce# methylation# changes# as# well# as# to# explore# possible# associations#between#the#methylation#in#the#selected#CpG#sites#in#white#blood#cells#with#some# transcriptomic,#biochemical#and#metabolic#biomarkers.#In#this#sense,#5#CpG#regions#of#interest#in# genes#(NCK2,#FITM2,#TRRAP,#CREBBP#and#RPTOR)#that#have#been#related#with#insulin#resistance,# inflammation#and#lipid#metabolism#and/or#adipogenesis#were#identified#(Dusseault#et/al.,#2016;# GeneCards# Database,# revised:# August# 2016;# Herzig# et/ al.,# 2003;# Murr# et/ al.,# 2006;# Unno# et/ al.,# 2005),#which#were#differentially#methylated#as#compared#with#control#group#by#at#least#one#of#the# treatments.#

A#previous#study#of#our#group#involving#a#larger#sample#of#this#trial#found#that#αNlipoic#acid# supplementation#was#able#to#improve#inflammatory#and#cardiovascular#risk#markers#(Huerta/et/ al.,#2016b).#Several#studies#in#cell#lines,#murine#models#(Cheng#et/al.,#2011;#FernándezNGalilea#et/ al.,#2014,#2015;#Park#et/al.,#2008)#and#also#in#humans##(Huerta#et/al.,#2016a)#have#related#these# actions#of#αNlipoic#acid#with#the#expression/regulation#of#genes#involved#in#these#processes.#To# our# knowledge,# the# current# trial# shows# for# the# first# time# that# αNlipoic# acid# supplementation# in# humans#induces#methylation#changes,#which#could#be#also#contributing#to#the#metabolic#effects# described#for#αNlipoic#acid#in#overweight/obesity.#In#this#context,#the#present#results#suggest#that# the#changes#in#the#methylation#of#CpG#site#cg10320884#located#in#the#TRRAP#gene#region#could#be# related# with# the# global# improvement# of# some# cardiovascular# risk# markers# (Framingham# score)# observed#after#αNlipoic#acid#supplementation.#In#this#sense,#Unno#et/al.#(2005)#have#suggested#the# involvement#of#TRRAP#in#the#regulation#of#lipid#metabolism#and#cholesterol#efflux#through#the#coN activation#of#nuclear#receptors#(liver#X#receptor#and#farnesoid#X#receptor),#which#modulate#gene# expression#of#enzymes#that#control#lipid#metabolism.#

Interestingly,#the#methylation#changes#in#the#CpG#sites#related#with#the#genes#NCK2,#TRRAP,# CREBBP#and#RPTOR#were#significantly#associated,#as#well#as#the#changes#in#the#expression#of#these# genes#in#PBMC,#suggesting#that#they#could#be#involved#in#a#common#pathway.#In#this#sense,#the# histone#acyltransferases#(HAT)#complexes#are#implicated#in#DNA#repair#through#making#chromatin#

214!! IV.$RESULTS$(CHAPTER(5)$ more#accessible#to#DNA#repair#proteins#(Murr#et/al.,#2006).#In#addition,#TRRAP#is#known#to#be#part# of#many#HAT#complexes#including#p300/CREBBPNassociated#factor#complex#(Loizou#et/al.,#2009).# Indeed,#either#TRRAP/or#CREBBP#genes,#are#considered#to#be#involved#in#important#processes#as# DNA#repair,#cell#cycle#and#transcriptional#regulation#of#genes#participating#not#only#in#metabolism,# but#also#in#inflammatory#signals#(Leduc#et/al.,#2014;#Vo#and#Goodman,#2001).#Furthermore,#it#has# been#suggested#that#αNlipoic#acid#could#inhibit#histone#deacetylase#activity,#which#in#turn#allows# HAT#complexes#to#recruit#acetyl#groups#to#histone#tails,#making#chromatin#more#accessible#to#DNA# repair#or#to#epigenetic#modifications#(Dashwood#and#Ho,#2007;#Suh#et/al.,#2004).##

On# the# other# hand,# the# mTORC1# complex,# which# includes# mTOR# and# RPTOR# proteins,# participates# in# glucose# and# lipid# metabolism# as# a# nutrient# sensor,# which# regulates# biosynthetic# processes# and# energy# storage# in# the# organism# (Zoncu# et/ al.,# 2011).# In# these# analyses,# EPA# treatment# promoted# changes# in# methylation# of# a# CpG# site# related# with# RPTOR,# but# without# affecting#expression#of#RPTOR#gene#in#PBMC.#Contrariwise,#although#αNlipoic#acid#did#not#induce# significant#methylation#changes#in#RPTOR,#it#was#able#to#regulate#RPTOR#expression,#suggesting# that#others#putative#mechanism#different#to#the#methylation#in#the#regulation#of#this#gene#by#αN lipoic# acid# could# be# involved.# Additionally,# both# mTOR# (TORC1)# and# TRRAP,# belong# to# the# phosphatidylinositol# 3Nkinase# related# kinases# family,# which# respond# to# a# large# number# of# cell# stimulus,#such#as#stress#and#nutrient#availability,#as#well#as#DNANrepair,#cell#cycle#progression#and# apoptosis#(De#Cicco#et/al.,#2015).##

In#this#sense,#the#close#relationship#observed#between#the#expression#of#genes#RPTOR,#TRRAP# and#CRREBP#with#not#only#weight#loss#and#fat#mass#decrease,#but#also#with#the#fasting#FFA#levels# at#the#end,#suggest#that#the#previously#described#changes#(Huerta#et/al.,#2015a,#2016a,#2016b)# promoted#by#αNlipoic#acid#in#body#weight#and#lipid#metabolism,#could#be#mediated#by#a#common# mechanism#that#pass#through#the#regulation#of#these#genes.##

A#recent#study#(Dusseault#et/al.,#2016),#has#reported#that#NCK2#is#a#regulator#of#the#glucose# and#lipid#metabolism#balance,#since#NCK2#deficient#mice#display#progressive#increased#adiposity# and#adipocyte#hypertrophy.#In#fact,#NCK2#deficiency#not#only#promoted#the#adipogenic#program# and#increased#the#droplet#formation,#but#also#a#dysfunctional#increase#in#lipogenesis#and#lipolysis# activities.#Also,#NCK2#deficient#mice#developed#glucose#intolerance,#insulin#resistance#and#hepatic# steatosis.#The#current#data#also#suggest#that#both#changes#in#methylation#and#gene#expression#of# NCK2#could#be#relevant#in#humans#and#could#account#for#the#metabolic#actions#of#αNlipoic#acid# supplementation.#Thus,#we#observed#a#negative#relationship#between#NCK2#gene#expression#and# changes#in#body#weight#and#fat#mass.#Moreover,#NCK2#changes#were#also#negatively#associated# with#the#levels#of#FFA#at#the#end#of#the#nutritional#intervention.##

! 215# IV.$RESULTS$(CHAPTER(5)$

However,#it#is#important#to#consider#the#limitations#of#this#study,#including#the#sample#size,#the# relatively# short# duration# of# the# nutritional# intervention,# and# the# fact# that# the# gene# expression# changes#were#assessed#in#PBMCs#and#the#methylation#changes#in#buffy#coat#(which#contains#most# of#the#white#blood#cells,#including#the#PBMCs,#and#may#also#have#some#platelets).#Our#trial#was# performed# in# healthy# overweight/obese# subjects,# given# the# interest# to# assess# the# effects# on# methylation#in#a#larger#sample#with#and#without#metabolic#disturbances.##

In#summary,#our#current#data#suggest#that#the#regulation#of#some#CpG#methylation#in#white# blood#cells#and/or#gene#expression#of#RPTOR,#TRRAP,#CREBBP#and#NCK2#could#contribute#to#the# beneficial#actions#of#αNlipoic#acid#on#body#weight#and#fat#mass#as#well#as#on#cardiovascular#risk# markers# in# healthy# overweight/obese# women.# Overall,# the# outcomes# of# our# study# support# the# role#of#epigenetic#mechanisms#in#the#actions#of#αNlipoic#acid#and#EPA#supplementation#in#humans,# and# highlight# the# importance# of# performing# longer# randomized# controlled# trials# to# assess# the# relevance#of#these#mechanisms#in#longNtime#period#or#in#subjects#with#metabolic#disturbances.###

Acknowledgments#

We#thank#to#the#Ministry#of#Economy#and#Competitiveness#from#the#Government#of#Spain#(ref.#AGL#2009N 10873/ALI,# BFU2012N36089# and# AGL2013N45554NR),# to# Navarra# Government# (Department# of# Health# ref.# 67/2015),#to#the#Special#Research#Line#of#“Nutrition,#Obesity#and#Health”#from#the#University#of#NavarraN Spain# and# to# CIBERobn# for# the# grants# received.# AE# Huerta# was# supported# by# a# predoctoral# grant# from# “Asociación#de#Amigos#de#la#Universidad#de#Navarra”.#EPA#and#sunflower#oil#capsules#were#provided#by# Solutex®# (Madrid,# Spain).# Solutex# had# no# role# in# the# study# design,# data# collection,# analysis# and# interpretation,#or#writing#of#the#manuscript.##

The#authors#have#declared#no#conflict#of#interest#

Authors/Contribution/

M.J.MNA# and# J.A.M.# conceived# and# designed# the# study;# M.J.MNA,# F.I.M,# J.I.RNB# and# A.E.H.# supervised# or# conducted#research;#E.G.#performed#the#bioinformatic#analysis#of#methylation#data;#A.E.H.#performed#the# statistical#analysis#and#carried#out#the#gene#expression#analyses;#M.J.MNA,#J.A.M.,#A.E.H.#wrote#the#paper;# M.J.MNA#and#J.A.M.#had#the#primary#responsibility#for#the#final#content#and#provided#the#financial#support.# All#authors#have#read#and#approved#the#final#manuscript.# # # # #

216!! IV.$RESULTS$(CHAPTER(5)$

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220!! #

V. GENERAL$DISCUSSION$

!

V.$GENERAL$DISCUSSION$

The#general#objective#of#the#present#trial#was#to#assess#the#additional#effects#to#an#energyN restricted#diet#(<30%#of#TEE)#of#a#supplementation#with#EPA#and/or#αNlipoic#acid#on#body#weight# loss#and#body#composition,#as#well#as#on#some#metabolic#and#inflammatory#markers.##

In#this#study,#we#found#that#supplementation#with#αNlipoic#acid#at#a#dose#of#300#mg/day,#alone# or#in#combination#with#EPA#could#help#to#promote#weight#loss#and#fat#mass#reduction#in#healthy# overweight/obese# premenopausal# women,# but# also# might# benefit# health# by# improving# inflammatory# conditions# and# decreasing# cardiovascular# risk.# Moreover,# supplementation# either# with# αNlipoic# acid# or# EPA,# was# able# to# induce# changes# in# SAAT# gene# expression# and# in# the# methylation#pattern#in#peripheral#white#blood#cells#in#a#subNsample#of#volunteers#participating#in# this#study.###

The#primary#aim#of#the#study#was#to#characterize#the#effects#of#αNlipoic#acid#and/or#EPA#on# body#weight#reduction#within#a#hypocaloric#approach.#In#this#sense,#the#supplementation#with#αN lipoic# acid# was# able# to# induce# a# greater# weight# loss# and# fat# mass# reduction# than# the# observed# alone# with# the# hypocaloric# diet.# In# this# context,# Koh# et/ al.# (2011)# found# that# αNlipoic# acid# supplementation#(1200N1800#mg/day)#together#with#an#energy#restricted#diet#in#Asian#overweight# and# obese# subjects# promoted# weight# loss,# BMI# and# waist# circumference# reduction,# being# the# effect#only#significant#at#the#highest#dose#tested.#Similarly,#Carbonelli#et/al.#(2010)#described#how# supplementation#with#αNlipoic#acid#(800#mg/day)#promoted#a#reduction#of#weight,#fat#mass#and# waist# circumference# only# in# obese# and# overweight# Caucasian# subjects,# but# not# in# those# with# normal#weight.#Thereby,#it#is#important#to#note#that#in#the#present#trial#the#body#weightNlowering# effects# of# αNlipoic# acid# were# observed# at# a# lower# dose# (300# mg/day).# On# the# contrary,# other# studies#that#have#evaluated#the#effects#of#αNlipoic#acid#(300N1000#mg/day)#in#subjects#with#MetS# or#with#T2D,#following#a#regular#diet,#did#not#observe#body#weight#lowering#properties#for#this# acid#(Ansar#et/al.,#2011;#Manning#et/al.,#2012;#McNeilly#et/al.,#2011).#The#apparent#discrepancies# between#the#different#trials#regarding#the#effectiveness#of#αNlipoic#acid#to#promote#weight#loss# and#the#lack#of#consistency#regarding#the#doses#required#to#observe#such#effect#could#be#related# with#the#specific#features#of#the#subjects#participating#in#each#trial#(ethnicity,#age,#presence#or# absence#of#pathologies#associated#to#obesity),#the#type#of#diet#assigned#(hypocaloric#vs#isocaloric)# and#the#duration#of#the#trials.##

Also,#the#current#study#showed#that#EPA#treatment#did#not#induce#a#greater#weight#loss#than# the#observed#in#the#nonNsupplemented#groups.#Several#studies#have#evaluated#the#potential#body# weight# lowering# effects# of# nN3# PUFAs# using# different# doses# and# ratios# of# DHA:EPA,# arriving# to# contradictory# outcomes# (DeFina# et/ al.,# 2011;# Kunesova# et/ al.,# 2006;# Munro# and# Garg,# 2013a;#

! 223# V.$GENERAL$DISCUSSION$

Tapsell#et/al.,#2013;#Tardivo#et/al.,#2014;#Thorsdottir#et/al.,#2007).#However#the#differential#effects# between#EPA#and#DHA#have#not#been#widely#explored.#In#this#sense,#some#authors#(Kunesova#et/ al.,#2006)#have#hypothesized#that#the#docosahexaenoate#could#be#the#bioactive#compound#that# induces#changes#in#BMI.#In#agreement#with#our#data,#the#results#derived#from#the#Japan#Obesity# and#Metabolic#Syndrome#Study,#observed#that#supplementation#with#highly#purified#EPA#(>98%)# at#a#dose#of#1800#mg/day#during#3#months#did#not#promote#significant#changes#in#both#BMI#and# waist#circumference#(Itoh#et/al.,#2007;#Yamada#et/al.,#2008).#

Interestingly,# in# the# current# trial# EPA# prevented# the# drop# of# leptin# levels# that# normally# accompanies#the#decrease#in#fat#mass.#In#this#context,#it#has#been#reported#that#after#weight#loss# there# is# a# metabolic# adaptation# in# where# the# RMR# decreases# more# than# the# predicted# for# the# changes#in#body#composition,#which#could#be#related#with#the#weight#loss#rebound#effect#(Galgani# and# Santos,# 2016;# Hall,# 2012).# Interestingly,# it# has# been# proposed# that# the# leptin# fall# could# contribute#not#only#to#hunger,#but#also#to#a#lower#RMR#and#further#weight#regain,#suggesting#that# leptin#replacement#therapy#could#be#useful#to#prevent#both#the#decrease#of#RMR#and#the#weight# regain#in#weightNreduced#subjects#(Doucet#et/al.,#2000;#Hinkle#et/al.,#2013).#In#this#sense,#the#fact# that#EPA#is#capable#of#preventing#the#drop#of#leptin#associated#to#weight#loss,#and#the#significant# association#observed#between#the#change#of#RMR#and#leptin,#suggest#that#the#effects#of#EPA#on# leptin#could#become#important#for#weight#loss#maintenance.#However,#it#is#important#to#take#into# account# that# in# this# trial# we# did# not# follow# the# participants# during# a# maintenance# weight# loss# period.# Nevertheless,# studies# that# have# evaluated# the# effects# of# nN3# PUFAs# supplementation# during# a# maintenance# weight# period# have# described# no# significant# actions# of# these# fatty# acids# (Krebs#et/al.,#2006;#Munro#and#Garg,#2012).#Other#researchers#have#suggested#that#the#effects#of# nN3#PUFAs#could#be#evident#after#a#long#time#period#and#that#their#accumulation#in#the#body#could# contribute#to#weight#management#(Munro#and#Garg,#2012,#2013b).#

The# overall# changes# observed# in# biochemical# parameters# from# carbohydrate# and# lipid# metabolism#markers,#including#not#only#glucose,#insulin#and#HOMANIR,#but#also#totalNcholesterol,# LDLNcholesterol#and#TG,#clearly#confirm#the#beneficial#effects#of#a#weightNloss#treatment#with#a# hypocaloric# diet# in# overweight/or# obese# subjects,# as# reported# elsewhere# (IberoNBaraibar# et/ al.,# 2015;#de#la#Iglesia#et/al.,#2014).##The#present#research#showed#that#supplementation#with#neither# EPA# nor# αNlipoic# acid# promoted# additional# effects# to# the# hypocaloric# diet# on# lipid# and# glucose# blood#metabolic#parameters#in#healthy#overweight/obese#individuals.#The#effects#of#nN3#PUFAs#on# glucose# and# lipid# profile# are# not# consistent,# and# depend# mainly# on# the# baseline# metabolic# characteristics#of#subjects#(Balk#et/al.,#2006;#Cottin#et/al.,#2011;#Hartweg#et/al.,#2007).#The#most# common#outcome#reported#with#nN3#PUFAs#supplementation#is#the#reduction#of#TG#levels#(Dewell#

224!! V.$GENERAL$DISCUSSION$ et/al.,#2011;#Hill#et/al.,#2007;#Kelley#et/al.,#2007;#Spencer#et/al.,#2013;#Tousoulis#et/al.,#2014;#Wong# et/al.,#2013).#However,#the#doses#administered#in#these#trials#ranged#between#1600#and#4000#mg# per#day,#and#are#therefore#higher#than#the#dose#used#in#this#study#(1300#mg/#day).#Thus,#the#trial# of#Yamada#et/al.#(2008),#performed#in#subjects#with#MetS,#observed#that#supplementation#with# highly# purified# EPA# (1800# mg/day)# decreased# TG# levels.# The# apparent# discrepancy# with# our# current# data# may# rely# on# the# fact# that# the# participants# of# our# trial# can# be# considered# healthy# overweight/obese,#as#they#did#not#suffer#MetS#or#any#other#relevant#metabolic#or#cardiovascular# disorder.#In#this#context,#in#the#present#study#it#was#observed#that#TG#decreased#more#in#those# subjects#with#higher#levels#at#the#beginning#of#the#intervention,#but#no#significant#effects#were# promoted# by# supplementations# (See$ Annex$ 5).# Furthermore,# the# effects# of# nN3# PUFAs# on# the# cholesterol#profile#are#not#clear#(Balk#et/al.,#2006;#Cottin#et/al.,#2011;#Hartweg#et/al.,#2007).#Similar# to#our#findings,#other#trials#have#described#that#the#levels#of#LDLNcholesterol,#HDLNcholesterol#and# totalNcholesterol#remain#unchanged#after#nN3#PUFAs#treatment#(Itariu#et/al.,#2012;#Krebs# et/al.,# 2006;#Munro#and#Garg,#2013a,#2013b;#SatohNAsahara#et/al.,#2012;#Spencer#et/al.,#2013;#Yamada#et/ al.,# 2008).# Although# the# increase# in# βNhydroxybutyrate# levels# was# not# significant# after# the# adjustment# of# BenjaminiNHochberg# correction# for# multiple# comparisons,# some# effect# of# EPA# in# fatty# acid# oxidation# cannot# be# discarded,# as# it# has# been# previously# described# in# both# humans# (Kunesova#et/al.,#2006)#and#rodents#(Flachs#et/al.,#2005).##

Moreover,#in#the#present#study,#αNlipoic#acid#did#not#exert#significant#effects#in#glucoseNrelated# biochemical#parameters#(fasting#glucose#or#insulin,#HOMANIR#and#glucose#tolerance#test).#In#this# sense,#although#the#evidence#suggests#that#αNlipoic#acid#is#able#to#improve#insulin#sensitivity#in# type#2#diabetic#individuals#(Kamenova,#2006;#Zhang#et/al.,#2011),#its#actions#are#not#necessarily# accompanied#by#significant#changes#in#fasting#glucose#concentrations#(Kamenova,#2006;#Manning# et/al.,#2012;#McNeilly#et/al.,#2011;#Zhang#et/al.,#2011).#Additionally,#to#our#knowledge,#the#effects# of#this#antioxidant#in#overweight/obese#healthy#individuals#have#not#been#widely#described,#and# the#outcomes#might#be#conditioned#by#the#metabolic#status#of#subjects#at#baseline.###

A# remarkable# finding# of# this# study# was# that# the# supplementation# with# αNlipoic# acid# in# conjunction# with# an# energyNrestricted# diet,# promoted# a# decrease# on# CRP# levels# and# leukocyte# count,#which#are#considered#important#acute#inflammatory#factors#implicated#in#insulin#resistance# and#cardiovascular#disease,#which#are#often#increased#in#obese#subjects#(Coffman#and#RichmondN Bryant,#2015;#Farhangi#et/al.,#2013;#Kao#et/al.,#2006;#Oda,#2013;#Tamakoshi#et/al.,#2007).#Only#few# studies# have# evaluated# the# antiNinflammatory# effects# of# αNlipoic# acid# in# overweight# and# obese# individuals#(Carbonelli#et/al.,#2010;#Manning#et/al.,#2012;#McNeilly#et/al.,#2011).#In#this#context,#the# study#from#Carbonelli#et/al.#(2010)#showed#that#supplementation#with#αNlipoic#acid#(800#mg/day)#

! 225# V.$GENERAL$DISCUSSION$ for# 4# months# promoted# a# decrease# in# TNFNα# and# ILN6# in# overweight# and# obese# subjects,# while# induced#a#reduction#of#CRP#only#in#the#obese#group.#Other#study,# performed# in# patients# with# either#T2DM#or#with#impaired#glucose#metabolism,#all#following#the#habitual#diet,#observed#that# intravenous# supplementation# of# αNlipoic# acid# (600# mg/day)# for# 2# weeks# could# improve# inflammatory#markers#(decreases#TNFNα#and#ILN6#while#increases#adiponectin)#(Zhang#et/al.,#2011).# However,# other# trial# in# overweight/obese# subjects# with# MetS# showed# that# oral# αNlipoic# acid# supplementation#(600N1000#mg/day)#during#1Nyear#period#did#not#promote#such#effects#on#TNFNα,# ILN6,# CRP# and# adiponectin# (Manning# et/ al.,#2012).#It#is#important#to#highlight#that#the# route# of# administration#and#the#type#of#dietary#intervention#used#in#the#different#trials#may#be#affecting# the# different# outcomes.# In# this# context,# our# current# data# suggest# that# αNlipoic# acid# action# on# inflammatory# markers# might# be# more# effective# when# administered# as# a# coadyuvant# of# a# hypocaloric#diet.##

Irisin#is#a#recently#discovered#adipoNmyokine#identified#in#mice#and#humans,#which#has#been# proposed# to# mediate# some# of# the# beneficial# effects# of# exercise# on# macronutrient# metabolism# (Boström# et/ al.,# 2012;# Huh# et/ al.,# 2012).# Some# studies# have# suggested# a# relationship# between# irisin# levels# and# glucose# metabolism# parameters,# but# the# role# of# irisin# in# obesity# and# glucose# metabolism#has#not#been#fully#established#yet.#In#this#context,#our#data#showed#that#in#healthy# overweight/obese# women,# fasting# irisin# levels# are# highly# correlated# with# fasting# glucose.# Accordingly,#the#study#of#Huh#et/al.#(2012)#in#middle#aged#normal#weight#and#obese#women#found# that# irisin# is# positively# associated# with# serum# glucose# levels.# In# addition,# some# authors# have# proposed# that# irisin# levels# are# associated# with# insulin# resistance# and# signs# of# vascular# atherosclerosis# in# a# cohort# of# nondiabetic# adult# subjects# (Sesti# et/ al.,# 2014).# However,# other# studies#described#that#irisin#circulating#levels#are#reduced#in#type#2#diabetic#individuals#(Choi#et/al.,# 2013;#Liu#et/al.,#2013;#MorenoNNavarrete#et/al.,#2013)#and#in#subjects#with#MetS#features#(Yan#et/ al.,#2014).#In#summary,#while#glucose#metabolism#is#related#in#some#manner#with#irisin#release,# our#current#data#revealed#that#acute#changes#in#the#concentrations#of#glucose#and#insulin,#at#least# through# the# 2# hours# of# the# OGTT,# were# not# affecting# blood# concentrations# of# this# protein.# In# support#of#these#findings,#a#recent#study#(Kurdiova#et/al.,#2014)#has#shown#that#irisin#circulating# levels#remained#unaffected#during#the#euglycaemicNhyperinsulinaemic#clamp#in#humans.##

On#the#other#hand,#similar#to#leptin,#it#could#exist#a#degree#of#resistance#to#irisin#in#overweight# or# obese# subjects# that# could# promote# an# increased# production# of# irisin,# in# order# to# face# and# counteract# metabolic# complications# (RocaNRivada# et/ al.,# 2013;# Stengel# et/ al.,# 2013).# Moreover,# some#authors#(Sesti#et/al.,#2014;#Stengel#et/al.,#2013)#have#hypothesized#that#the#increment#in#the# circulating#irisin#levels#in#obesity#could#be#an#early#protective#response#of#the#organism#to#restore#

226!! V.$GENERAL$DISCUSSION$ glucose# metabolism# or# to# counteract# metabolic# disturbances.# Our# study# in# cultured# human# adipocytes# of# overweight/obese# subjects# revealed# that# αNlipoic# acid# was# able# to# increase# both# FNDC5# mRNA# levels# and# irisin# release.# Hence,# this# led# us# to# evaluate# whether# the# beneficial# actions#of#αNlipoic#acid#supplementation#alone#or#in#combination#with#EPA#were#mediated,#at#least# in#part,#by#changes#in#irisin.#Nevertheless,#the#supplementation#with#αNlipoic#acid#and/or#EPA#in# overweight/obese#humans#following#an#energyNrestricted#diet#did#not#have#any#additional#effects# on#irisin#circulating#levels#than#those#induced#by#the#calorieNrestricted#diet,#suggesting#that#the# beneficial#effects#of#these#supplements#in#overweight/obese#subjects#at#the#doses#tested#in#this# study#are#not#mediated#by#irisin.#

In#the#current#trial#we#have#not#found#any#significant#effect#of#EPA#supplementation#in#any#of# the# systemic# inflammatory# and# cardiovascular# risk# markers# measured.# In# this# context,# different# systematic#reviews#(Myhrstad#et/al.,#2011;#Robinson#and#Mazurak,#2013)#have#pointed#out#that# the# effects# of# nN3# PUFAs# on# the# different# blood# acute# phase# proteins,# adhesion# molecules# and# chemokines# are# not# clear# and# that# the# difficulty# lies# in# both,# the# different# features# of# the# population#involved#and#the#different#study#designs,#including#the#administered#doses.#Oppositely# to#our#results,#the#trials#from#the#Japan#Obesity#and#Metabolic#Syndrome#Study#(Itoh#et/al.,#2007;# SatohNAshara# et/ al.,# 2012),# observed# that# supplementation# with# highly# purified# EPA# (1800# mg/day)#increased#adiponectin#levels,#but#no#significant#effects#were#observed#in#CRP#and#TNFNα.# Hence,#the#different#outcomes#for#EPA#effects#are#probably#modulated#by#the#baseline#metabolic# status#of#the#participating#subjects.##

Though#EPA#did#not#promote#significant#changes#in#inflammatory#plasma#circulating#levels,#the# EPA#supplemented#groups#exhibited#lower#expression#of#the#macrophage#marker#ADGRE1#gene# and#a#greater#expression#of#the#antiNinflammatory#IL10#in#SAAT,#which#lead#us#to#suggest#that#EPA# could#decrease#macrophage#infiltration#and#attenuate#inflammation#in#human#adipose#tissue.#In# this#sense,#several#studies#in#obese#rodents#have#insinuated#the#ability#of#EPA#to#combat#adipose# tissue#inflammation,#in#part#by#decreasing#macrophage#infiltration#and#inflammation#(LeMieux#et/ al.,# 2015;# PérezNEcharri# et/ al.,# 2008;# PérezNMatute# et/ al.,# 2007).# However,# in# humans,# the# information#available#is#limited,#probably#in#part#for#the#problems#arising#to#obtain#adipose#tissue# sample# biopsies.# The# study# from# Itariu# et/ al./ (2012),# performed# in# severely# obese# nonNdiabetic# individuals,#observed#that#supplementation#with#3360#mg/day#of#nN3#PUFAs#promoted#a#decrease# in#the#expression#of#proNinflammatory#(IL6,#HIF1A,#TGFB1)#and#macrophage#(CCL2,#CCL3)#genes,# while#increased#the#gene#expression#of#the#antiNinflammatory#adipokine#ADIPOQ/in#SAAT#and#the# production# of# antiNinflammatory# and# proresolving# eicosanoids# in# VAT.# # Similarly,# Spencer# et/ al.# (2013)#found#in#subjects#with#impaired#fasting#glucose#or#MetS,#that#the#supplementation#with#

! 227# V.$GENERAL$DISCUSSION$

4000# mg/day# of# nN3# PUFAs# (including# both# EPA# and# DHA)# promoted# a# decrease# in# crownNlike# structures# and# in# the# expression# of# macrophage# markers# (CCL2# and# CD68)# in# SAAT.# Thus,# our# results#reinforce#the#hypothesis#that#adipose#tissue#is#a#target#organ#for#EPA#actions.#This#move#us# to# better# characterize# the# actions# of# EPA# and/or# lipoic# acid# supplementation# on# adipose# tissue# transcriptomic# profile.# For# this# purpose# a# microarray# analysis# was# performed# in# SAAT# biopsies# from#a#selected#subsample#of#volunteers.#

The# microarray# analysis# revealed# that# EPA# upregulated# genes# related# with# increments# of# chemotactic# factors# together# with# some# macrophage# marker# genes# associated# with# either# M1# (CHI3L1)#(Di#Rosa#et/al.,#2013)#or#M2#phenotypes#(ACP5,#CHIT1,#MSR1,#TFRC)#(Ahlin#et/al.,#2013;# Bune#et/al.,#2001;#Di#Rosa#et/al.,#2013;#Kratz#et/al.,#2014;#Mališová#et/al.,#2014;#Orr#et/al.,#2014)#as# compared#with#the#control#group.#In#this#context,#human#studies#have#described#an#increase#in# adipose#tissue#macrophages#during#the#early#phase#of#weight#loss#without#necessarily#involving#an# increment#of#inflammatory#genes#(Capel#et/al.,#2009;#Mališová#et/al.,#2014).#In#addition,#Kitamoto# et/al.#(2013)#observed#that#the#inhibition#of#CHIT1#promoted#an#increment#of#proNinflammatory# signals# and# decreased# M2# macrophage# polarization# in# a# macrophage# cell# line# and# in# rodents.# Taking# into# account# the# available# information,# our# results# suggest# that# the# increment# of# chemotactic# factors# observed# in# the# EPA# supplemented# group,# promotes# a# migration# of# macrophages#into#adipose#tissue#specially#M2#macrophages.###

In# SAAT,# αNlipoic# acid# supplementation# also# promoted# an# inhibition# of# some# biological# functions# related# to# the# attachment# and# adhesion# of# cells# as# compared# with# control# group.# Moreover,#genes#related#with#inflammation#and#immune#responses# (as# TLR1,# TLR2# and# KLRK1)# were# downregulated# in# both# groups# supplemented# with# αNlipoic# acid,# suggesting# a# possible# modulation# by# this# antioxidant# in# adipose# tissue# inflammation.# Even# if# there# are# not# enough# information#about#the#effects#of#αNlipoic#acid#treatment#on#human#SAAT#transcriptome,#Deiuliis#et/ al.#(2011),#using#a#murine#model#of#obesity,#found#that#the#treatment#with#αNlipoic#acid#inhibited# the#expression#of#TLR4#besides#the#production#of#proNinflammatory#cytokines#in#adipose#tissue,# this#through#the#inhibition#of#NFNκβ#activation.#It#is#important#to#mention#that#this#ability#of#αN lipoic# acid# to# downregulate# inflammatory# pathways# in# adipose# tissue# paralleled# with# the# reduction#of#proinflammatory#factors#at#systemic#level,#as#we#have#previously#described.###

Also,#the#analysis#of#biological#pathways#arising#from#the#microarray,#suggests#that#αNlipoic#acid# modulates#lipid#metabolism#in#SAAT,#especially#in#the#group#supplemented#with#EPA+αNlipoic#acid,# in#which#the#pathways#of#catabolism#of#lipids#were#increased#while#the#deposition#of#lipids#was# decreased.# This# observation# was# supported# by# the# upregulation# of# genes# encoding# enzymes# involved# in# the# oxidation# of# TG# and# cholesterol# (CYP4B1# and# CYP27A1)# or# a# cholesteryl# ester#

228!! V.$GENERAL$DISCUSSION$ hydrolase,#LIPA.#Moreover#in#the#group#supplemented#with#αNlipoic#acid#the#biological#function#of# lipolysis# of# adipose# tissue# was# positively# regulated# and# it# was# observed# that# one# of# the# main# acyltransferases#responsible#for#the#esterification#of#triacylglycerol#in#adipose#tissue#(DGTA2),#was# among# the# top# downNregulated# genes.# Additionally,# AADAC,#which#encodes#an#intracellular#TG# lipase#and#shares#sequence#homology#with#HSL#(Lo#et/al.,#2010)#was#one#of#the#top#upregulated# common#genes#in#the#three#supplemented#groups.#Though#the#effects#of#αNlipoic#acid#on#adipose# tissue#gene#expression#have#not#been#widely#studied#in#humans,#previous# assays# of# our# group# carried#out#either#in#3T3NL1#cell#line#or#in#human#adipocytes,#have#evidenced#the#ability#of#αNlipoic# acid#to#induce#lipolysis#and#to#inhibit#TG#accumulation#by#modulating#the#activity#of#key#lipolytic# proteins# as# HSL# (FernándezNGalilea# et/ al.,#2012)#and#by#reducing#enzymes#involved#in# de/ novo# lipogenesis#as#DGAT1,#FAS#and#SCD1#(FernándezNGalilea# et/ al.,#2014).#In#this#sense,#these#data# suggest#that#the#body#weightNlowering#actions#observed#for#αNlipoic#acid#could#be#related#with#its# properties# to# modulate# lipid# metabolism# in# adipose# tissue.# In# summary,# altogether# these# data# reinforce# the# idea# that# supplementation# with# lower# doses# of# αNlipoic# acid# (300# mg/day)# in# the# context#of#a#hypocaloric#diet#not#only#might#help#to#loss#more#weight,#in#part#by#stimulating#lipid# catabolic#pathways#in#adipose#tissue,#but#also#to#attenuate#both#local#and#systemic#inflammation# associated#to#obesity.#

The# bioinformatic# analysis# of# the# microarray# assay# in# SAAT# also# identified# some# genes# commonly#downregulated#in#the#three#supplemented#groups#(EPA,#αNlipoic#acid#or#EPA+#αNlipoic# acid)#including#genes#encoding#proteins#that#positively#regulate#angiogenesis#processes#such#as# the# apelin# receptor# (APLNR),# whereas# genes# considered# angiogenesis# inhibitors# (ZNF7,# TNMD)# (Shukunami# and# Hiraki,# 2007;# Song# et/ al.,# 2006)# were# upregulated# as# compared# with# control# group.# Accordingly,# Larghero# et/ al.# (2007)# demonstrated# that# αNlipoic# acid# treatment# inhibited# endothelial#cell#migration#and#had#antiNangiogenic#properties#both,#in/vitro#and#in/vivo.#In#obesity,# adipose# tissue# enlargement# leads# to# an# increment# of# angiogenesis# accompanied# by# increased# production# of# chemotactic# factors,# inflammatory# cells# and# upregulation# of# extracellular# matrix# remodeling#factors#(Cao,#2007).#In#addition,#in#order#to#inhibit#fat#mass#expansion#and#improve# insulin#sensitivity,#therapy#with#antiNangiogenic#agents#has#been#proposed#in#obesity#(Cao,#2007).# The#apelin/APJ#signaling#system#has#been#proposed#as#a#potential#link#between#adipose#tissue#and# endothelial# angiogenic# processes# (Kunduzova# et/ al.,# 2008).# Apelin# is# an# adipoNmyokine,# which# levels#are#increased#in#obesity#and#decrease#after#weight#loss#(Boucher#et/al.,#2005;#Japp#et/al.,# 2010).#However,#it#has#been#suggested#that#the#overNproduction#of#apelin#in#obesity#could#be#a# protective#mechanism#to#counteract#the#emergence#of#obesityNrelated#diseases#including#T2DM# and# cardiovascular# disorders# (CastanNLaurell# et/ al.,#2005).#In#the#current#trial,#supplementation#

! 229# V.$GENERAL$DISCUSSION$ with# αNlipoic# acid# prevented# the# decline# in# apelin# concentrations# induced# by# weight# loss.# In# a# same#way,#it#has#been#found#that#treatments#with#insulinNsensitizing#drugs#such#as#metformin#and# rosiglitazone#promoted#an#increase#in#apelin#concentrations#and#a#decrease#in#CRP#(Kadoglou#et/ al.,#2010).#On#the#contrary,#Yu#et/al./(2012)#found#that#intravenous#administration#of#αNlipoic#acid# (600#mg/day)#for#2#weeks#in#combination#with#hypoglycemic#drugs#(metformin#or#pioglitazone)# induced#a#reduction#in#the#levels#of#this#adipokine#in#patients#with#T2DM,#however#it#is#important# to#note#that#this#decrease#was#compared#only#to#baseline#values#and#in#the#absence#of#a#control# group#to#analyze#these#findings.#Our#observations#regarding#both,#changes#in#plasma#circulating# levels#of#apelin#and#changes#in#apelin#receptor#(APLNR)#gene#expression#in#adipose#tissue,#suggest# that#αNlipoic#acid#appears#to#modulate#the#apelin#system#in#humans.#This#finding#is#in#accordance# with#previous#observations#of#our#group#analyzing#the#effects#of#αNlipoic#acid#on#apelin#in#rodents# and# cultured# adipocytes# (FernándezNGalilea# et/ al.,#2011).#Further#studies#are#needed# to# better# understand#the#crosstalk#between#αNlipoic#acid#and#the#apelin#system#and#the#potential#relevance# of#the#apelin#system#in#the#cardiometabolic#protective#effects#of#αNlipoic#acid.##

Finally,#we#evaluated#whether#the#dietary#supplementation#with#EPA#and/or#αNlipoic#acid#was# able# to# induce# methylation# changes# in# white# blood# cells# from# a# subsample# of# volunteers# that# participated#in#this#study.#The#possible#associations#between#the#methylation#in#the#selected#CpG# sites#with#some#biochemical#and#metabolic#biomarkers#were#also#explored.#In#this#context,#5#CpG# regions# of# interest# related# with# inflammation# and# lipid# metabolism# that# were# differentially# methylated#as#compared#with#the#control#group#by#at#least#one#of#the#treatments#were#identified.#

To# our# knowledge,# the# current# project# showed# for# the# first# time# that# αNlipoic# acid# supplementation#in#humans#promotes#methylation#changes,#which#could#be#also#contributing#to# the#metabolic#effects#described#for#αNlipoic#acid#in#overweight/obese#subjects#under#a#hypocaloric# diet.# Our# current# results# suggest# that# the# changes# in# the# methylation# of# CpG# site# cg10320884# located#in#the#TRRAP#gene#region#could#be#related#with#the#improvement#of#some#cardiovascular# risk#markers#(Framingham#score)#observed#after#αNlipoic#acid#supplementation.#

Interestingly,#the#methylation#changes#in#the#CpG#sites#related#with#the#genes#NCK2,#TRRAP,# CREBBP#and#RPTOR#were#significantly#associated,#as#well#as#the#changes#in#the#expression#of#these# genes# in# PBMC,# suggesting# that# they# could# be# involved# in# a# common# pathway.# In# this# sense,# TRRAP# is# known# to# be# part# of# many# histone# acyltransferases# (HAT)# complexes# including# p300/CREBBPNassociated#factor#complex#(Loizou#et/al.,#2009).#Indeed,#αNlipoic#acid#could#inhibit# histone# deacetylase# activity,# which# in# turn# allows# HAT# complexes# to# recruit# acetyl# groups# to# histone# tails,# making# chromatin# more# accessible# to# DNA# repair# or# to# epigenetic# modifications# (Dashwood#and#Ho,#2007;#Suh#et/al.,#2004).#Additionally,#both#mTOR#(TORC1)#and#TRRAP,#belong#

230!! V.$GENERAL$DISCUSSION$ to# the# phosphatidylinositol# 3Nkinase# related# kinases# (PIKKs)# family,# which# respond# to# a# large# number#of#cellular#stimuli,#such#as#stress#and#nutrient#availability#as#well#as#DNANrepair,#cell#cycle# progression#and#apoptosis#(De#Cicco#et/al.,#2015).#

Moreover,# the# close# relationship# observed# between# the# expression# of# genes# RPTOR,# TRRAP# and#CRREBP#with#not#only#weight#loss#and#fat#mass#decrease,#but#also#with#the#fasting#FFA#levels# at# the# end,# suggests# that# the# changes# promoted# by# αNlipoic# acid# in# body# weight# and# lipid# metabolism#could#be#mediated#by#a#common#mechanism#that#occurs#through#the#regulation#of# these#genes.##

Also,#either#EPA#or#αNlipoic#acid#decreased#the#methylation#in#the#cg18709306#CpG#position# from#NCK2/gene#in#peripheral#white#blood#cells,#however#only#the#αNlipoic#acid#supplementation# induced#an#increment#in#the#expression#of#this#gene#in#PBMC.#To#date,#the#function#of#NCK2#gene# in#lipid#or#glucose#metabolism#was#mostly#unknown,#but#a#recent#study#(Dusseault#et/al.,#2016)# has#reported#that#NCK2#may#be#an#important#regulator#of#glucose#and#lipid#metabolism#balance,# since#NCK2#deficient#mice#display#progressive#increased#adiposity#and#adipocyte#hypertrophy.#In# fact#NCK2#deficiency#promoted#the#adipogenic#program#and#increased#the#droplet#formation,#and# also# caused# a# dysfunctional# increase# in# lipogenesis# and# lipolysis# activities.# Also,# NCK2# deficient# mice#develop#glucose#intolerance,#insulin#resistance#and#hepatic#steatosis.#Our#current#data#also# suggest# that# both,# changes# in# methylation# and# gene# expression# of# NCK2,/ could# be# relevant# in# humans#and#could#account#for#the#metabolic#actions#of#αNlipoic#acid#supplementation.#Thus,#we# observed#a#negative#relationship#between#NCK2/gene#expression#and#changes#in#body#weight#and# fat#mass.#Moreover,#NCK2#changes#were#also#negatively#associated#with#the#levels#of#FFA#at#the# end#of#the#nutritional#intervention.##

Strengths$and$limitations$$

Some#limitations#of#the#present#study#should#be#stated.#The#principal#drawback#of#this#study#is# the# fact# that# the# outcomes# could# have# been# conditioned# by# the# degree# of# adherence# to# the# hypocaloric# diet# and# treatment.# In# this# sense,# it# should# be# interesting# to# find# an# objective# metabolite#that#confirm#the#adherence#to#the#αNlipoic#acid#treatment,#as#well#as#to#measure#the# content# of# nN3# PUFAs# in# erythrocytes# or# blood# in# order# to# assess# the# adherence# to# each# treatment.# Other# limitations# that# are# important# to# take# into# account# are# the# sample# size,# the# relatively# short# duration# of# the# nutritional# intervention# (10# weeks),# the# lack# of# a# maintenance# weight# loss# period,# the# absence# of# samples# from# some# volunteers# and# the# interNindividual# variability#that#probably#is#affecting#the#supplementation#effects#and#could#contribute#to#bias.#In# addition,#another#limitation#was#the#absence#of#SAAT#biopsies#at#baseline#of#the#study.#Moreover,#

! 231# V.$GENERAL$DISCUSSION$ whole#adipose#tissue#(which#is#composed#not#only#of#adipocytes#but#also#of#other#cellNtypes#like# fibroblasts,#immune#cells#and#preNadipocytes)#was#used#for#gene#expression#profile#analysis.#This# fact# raises# the# question# about# which# of# these# cell# fractions# were# mainly# contributing# to# the# observed#gene#expression#changes#in#adipose#tissue.##

The#changes#in#methylation#were#studied#in#buffy#coat,#which#contains#most#of#the#white#blood# cells#including#the#PBMC#and#might#also#have#some#platelets.#However,#because#of#the#absence#of# enough# buffy# coat,# the# gene# expression# changes# were# evaluated# in# PBMC,# which# could# be# considered#as#a#proxy.##

In# addition,# it# is# important# to# consider# that# it# could# exist# a# possible# treatment*gender# interaction.#In#the#present#trial#the#effects#of#supplementation#with#EPA#and/or#αNlipoic#acid#were# only#tested#in#women,#placing#the#issue#whether#similar#effects#could#be#observed#in#men.##

On#the#other#hand,#one#of#the#main#strengths#of#this#trial#is#the#study#design.#The#randomized# placeboNcontrolled# trial# is# considered# the# gold# standard# of# intervention# studies,# because# it# provides# not# only# the# strongest# possible# evidence# of# causation# but# also,# the# use# of# a# placebo,# permits#to#investigators#of#study#and#volunteers#to#be#blinded.#Furthermore,#the#randomization# prevents#the#selection#bias#that#could#occur#if#either#the#research#staff#or#the#volunteers#decide# the#treatment#(Misra,#2012).#Moreover,#the#fact#that#all#the#volunteers#participating#in#the#study# were#healthy#women,#allowed#us#to#avoid#gender#bias#and#to#really#evaluate#the#effect#of#the# treatment#without#the#bias#that#could#generate#the#use#of#some#medications#(e.g.#hypoglycemic,# antihypertensive#or#hypolipidemic#medications).##

In# addition,# in# this# study# the# effects# of# the# treatments# were# assessed# in# different# types# of# samples#and#from#different#perspectives.#The#use#of#SAAT#biopsies#allowed#us#to#study#whether# the#supplementation#either#with#αNlipoic#acid#or#EPA#were#exerting#their#effects#by#modulating# gene# expression# in# this# tissue.# Also,# the# use# of# microarray# technology# that# permits# to# evaluate# thousands#of#genes#and#CpG#methylation#sites#at#the#same#time#facilitated#not#only#the#study#of# gene# expression# patterns# and# pathways,# but# also# to# assess# if# treatments# were# able# to# alter# methylation#pattern.#Furthermore,#the#adjustment#for#multiple#comparisons#allowed#us#to#control# the#false#discovery#rate,#given#more#robustness#to#our#results.#

Finally,#it#is#important#to#mention#that#our#trial#was#performed#in#healthy#overweight/obese# subjects,# and# it# would# be# of# interest# to# assess# the# effects# of# αNlipoic# acid# and# EPA# supplementation# in# a# larger# sample# of# overweight/obese# subjects# with# and# without# metabolic# disturbances.#

232!! V.$GENERAL$DISCUSSION$

Altogether,#the#present#data#provide#novel#findings#about#the#effects#of#EPA#and/or#αNlipoic# acid#not#only#in#body#weight#but#also#in#inflammation#and#lipid#metabolism.#The#most#remarkable# findings# included:# 1)# the# ability# of# αNlipoic# acid# to# promote# a# greater# weight# loss# and# fat# mass# decrease#than#the#hypocaloric#diet#alone;#2)#the#inhibition#of#the#subsequent#decrease#of#leptin# parallel# to# fat# mass# loss# by# EPA,# which# could# become# important# in# a# weight# loss# maintenance# phase;# 3)# the# capability# of# αNlipoic# acid# to# decrease# systemic# inflammatory# biomarkers# such# as# CRP# and# leukocytes,# independently# of# weight# loss,# which# was# accompanied# by# a# general# downregulation#of#genes#encoding#chemotactic#factors#and#proNinflammatory#signals#in#adipose# tissue;#4)#the#actions#of#EPA#to#modulate#adipose#tissue#inflammation,#by#increasing#chemotactic# factors# and# favoring# expression# of# genes# associated# with# antiNinflammatory# signals# and# wound# repair;#5)#αNlipoic#acid,#especially#in#combination#with#EPA,#was#able#to#upregulate#genes#involved# in#lipid#catabolism#while#downregulated#lipid#storage#genes#in#adipose#tissue;#6)#both,#EPA#and#αN lipoic#acid#modulated#methylation#changes#in#white#blood#cells;#7)#the#regulation#of#methylation# and/or#gene#expression#of#RPTOR,# TRRAP,# CREBBP# and#NCK2/by#αNlipoic# acid# supplementation,# could# contribute# to# its# beneficial# actions# on# body# weight# and# fat# mass,# as# well# as# on# cardiovascular#risk#markers#in#healthy#overweight/obese#women.#

# # #

! 233# V.$GENERAL$DISCUSSION$

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236!! V.$GENERAL$DISCUSSION$

Larghero,#P.,#Venè,#R.,#Minghelli,#S.,#Travaini,#G.,#Morini,#M.,#Ferrari,#N.,#Pfeffer,#U.,#Noonan,#D.M.,#Albini,#A.,# and# Benelli,# R.# (2007).# Biological# assays# and# genomic# analysis# reveal# lipoic# acid# modulation# of# endothelial#cell#behavior#and#gene#expression.#Carcinogenesis#28,#1008–1020.#

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Munro,#I.A.,#and#Garg,#M.L.#(2012).#Dietary#supplementation#with#nN3#PUFA#does#not#promote#weight#loss# when#combined#with#a#veryNlowNenergy#diet.#Br.#J.#Nutr.#108,#1466N1474.#

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Munro,#I.A.,#and#Garg,#M.L.#(2013b).#Prior#supplementation#with#long#chain#omegaN3#polyunsaturated#fatty# acids#promotes#weight#loss#in#obese#adults:#a#doubleNblinded#randomised#controlled#trial.#Food#Funct.#4,# 650–658.#

Myhrstad,#M.C.W.,#Retterstøl,#K.,#TelleNHansen,# V.H.,# Ottestad,# I.,# Halvorsen,# B.,# Holven,# K.B.,# and# Ulven,# S.M.#(2011).#Effect#of#marine#nN3#fatty#acids#on#circulating#inflammatory#markers#in#healthy#subjects#and# subjects#with#cardiovascular#risk#factors.#Inflamm.#Res.#Off.#J.#Eur.#Histamine#Res.#Soc.#Al#60,#309–319.#

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! 237# V.$GENERAL$DISCUSSION$

Orr,#J.S.,#Kennedy,#A.,#AndersonNBaucum,#E.K.,#Webb,#C.D.,#Fordahl,#S.C.,#Erikson,#K.M.,#Zhang,#Y.,#Etzerodt,# A.,#Moestrup,#S.K.,#and#Hasty,#A.H.#(2014).#Obesity#Alters#Adipose#Tissue#Macrophage#Iron#Content#and# Tissue#Iron#Distribution.#Diabetes#63,#421–432.#

PérezNEcharri,#N.,#PérezNMatute,#P.,#MarcosNGómez,#B.,#Baena,#M.J.,#Marti,#A.,#Martínez,#J.A.,#and#MorenoN Aliaga,# M.J.# (2008).# Differential# inflammatory# status# in# rats# susceptible# or# resistant# to# dietNinduced# obesity:#effects#of#EPA#ethyl#ester#treatment.#Eur.#J.#Nutr.#47,#380–386.#

PérezNMatute,# P.,# PérezNEcharri,# N.,# Martínez,# J.A.,# Martí,# A.,# and# MorenoNAliaga,# M.J.# (2007).# Eicosapentaenoic#acid#actions#on#adiposity#and#insulin#resistance#in#control#and#highNfatNfed#rats:#role#of# apoptosis,#adiponectin#and#tumour#necrosis#factorNalpha.#Br.#J.#Nutr.#97,#389–398.#

Robinson,#L.E.,#and#Mazurak,#V.C.#(2013).#NN3#Polyunsaturated#Fatty#Acids:#Relationship#to#Inflammation#in# Healthy#Adults#and#Adults#Exhibiting#Features#of#Metabolic#Syndrome.#Lipids#48,#319–332.#

RocaNRivada,#A.,#Castelao,#C.,#Senin,#L.L.,#Landrove,#M.O.,#Baltar,#J.,#Crujeiras,#A.B.,#Seoane,#L.M.,#Casanueva,# F.F.,#and#Pardo,#M.#(2013).#FNDC5/irisin#is#not#only#a#myokine#but#also#an#adipokine.#PloS#One#8,#e60563.#

SatohNAsahara,#N.,#Shimatsu,#A.,#Sasaki,#Y.,#Nakaoka,#H.,#Himeno,#A.,#Tochiya,#M.,#Kono,#S.,#Takaya,#T.,#Ono,# K.,# Wada,# H.,# et# al.# (2012).# Highly# purified# eicosapentaenoic# acid# increases# interleukinN10# levels# of# peripheral#blood#monocytes#in#obese#patients#with#dyslipidemia.#Diabetes#Care#35,#2631–2639.#

Sesti,#G.,#Andreozzi,#F.,#Fiorentino,#T.V.,#Mannino,#G.C.,#Sciacqua,#A.,#Marini,#M.A.,#and#Perticone,#F.#(2014).# High# circulating# irisin# levels# are# associated# with# insulin# resistance# and# vascular# atherosclerosis# in# a# cohort#of#nondiabetic#adult#subjects.#Acta#Diabetol.#51,#705N13.#

Shukunami,# C.,# and# Hiraki,# Y.# (2007).# ChondromodulinNI# and# tenomodulin:# the# negative# control# of# angiogenesis#in#connective#tissue.#Curr.#Pharm.#Des.#13,#2101–2112.#

Song,#J.,#Jie,#C.,#Polk,#P.,#Shridhar,#R.,#Clair,#T.,#Zhang,#J.,#Yin,#L.,#and#Keppler,#D.#(2006).#The#candidate#tumor# suppressor#CST6#alters#the#gene#expression#profile#of#human#breast#carcinoma#cells:#downNregulation#of# the# potent# mitogenic,# motogenic,# and# angiogenic# factor# autotaxin.# Biochem.# Biophys.# Res.# Commun.# 340,#175–182.#

Spencer,#M.,#Finlin,#B.S.,#Unal,#R.,#Zhu,#B.,#Morris,#A.J.,#Shipp,#L.R.,#Lee,#J.,#Walton,#R.G.,#Adu,#A.,#Erfani,#R.,#et# al.#(2013).#OmegaN3#Fatty#Acids#Reduce#Adipose#Tissue#Macrophages#in#Human#Subjects#With#Insulin# Resistance.#Diabetes#62,#1709–1717.#

Stengel,#A.,#Hofmann,#T.,#GoebelNStengel,#M.,#Elbelt,#U.,#Kobelt,#P.,#and#Klapp,#B.F.#(2013).#Circulating#levels# of#irisin#in#patients#with#anorexia#nervosa#and#different#stages#of#obesity–correlation#with#body#mass# index.#Peptides#39,#125–130.#

Suh,# J.H.,# Shenvi,# S.V.,# Dixon,# B.M.,# Liu,# H.,# Jaiswal,# A.K.,# Liu,# R.NM.,# and# Hagen,# T.M.# (2004).# Decline# in# transcriptional#activity#of#Nrf2#causes#ageNrelated#loss#of#glutathione#synthesis,#which#is#reversible#with# lipoic#acid.#Proc.#Natl.#Acad.#Sci.#U.#S.#A.#101,#3381–3386.#

Tamakoshi,# K.,# Toyoshima,# H.,# Yatsuya,# H.,# Matsushita,# K.,# Okamura,# T.,# Hayakawa,# T.,# Okayama,# A.,# Ueshima,# H.,# and# Group,# T.N.D.R.# (2007).# White# Blood# Cell# Count# and# Risk# of# AllNCause# and# Cardiovascular#Mortality#in#Nationwide#Sample#of#Japanese.#Circ.#J.#71,#479–485.#

Tapsell,#L.C.,#Batterham,#M.J.,#Charlton,#K.E.,#Neale,#E.P.,#Probst,#Y.C.,#O’Shea,#J.E.,#Thorne,#R.L.,#Zhang,#Q.,# and# Louie,# J.C.Y.# (2013).# Foods,# nutrients# or# whole# diets:# effects# of# targeting# fish# and# LCn3PUFA# consumption#in#a#12mo#weight#loss#trial.#BMC#Public#Health#13,#1231.#

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238!! V.$GENERAL$DISCUSSION$

Thorsdottir,# I.,# Tomasson,# H.,# Gunnarsdottir,# I.,# Gisladottir,# E.,# Kiely,# M.,# Parra,# M.D.,# Bandarra,# N.M.,# Schaafsma,#G.,#and#Martínez,#J.A.#(2007).#Randomized#trial#of#weightNlossNdiets#for#young#adults#varying# in#fish#and#fish#oil#content.#Int.#J.#Obes.#31,#1560–1566.#

Tousoulis,#D.,#Plastiras,#A.,#Siasos,#G.,#Oikonomou,#E.,#Verveniotis,#A.,#Kokkou,#E.,#Maniatis,#K.,#Gouliopoulos,# N.,# Miliou,# A.,# Paraskevopoulos,# T.,# et# al.# (2014).# OmegaN3# PUFAs# improved# endothelial# function# and# arterial# stiffness# with# a# parallel# antiinflammatory# effect# in# adults# with# metabolic# syndrome.# Atherosclerosis#232,#10–16.#

Wong,#A.T.,#Chan,#D.C.,#Barrett,#P.H.,#Adams,#L.A.,#and#Watts,#G.F.#(2013).#Supplementation#with#n3#fatty# acid#ethyl#esters#increases#large#and#small#artery#elasticity#in#obese#adults#on#a#weight#loss#diet.#J.#Nutr.# 143,#437–441.#

Yamada,#H.,#Yoshida,#M.,#Nakano,#Y.,#Suganami,#T.,#Satoh,#N.,#Mita,#T.,#Azuma,#K.,#Itoh,#M.,#Yamamoto,#Y.,# Kamei,# Y.,# et# al.# (2008).# In# vivo# and# in# vitro# inhibition# of# monocyte# adhesion# to# endothelial# cells# and# endothelial#adhesion#molecules#by#eicosapentaenoic#acid.#Arterioscler.#Thromb.#Vasc.#Biol.#28,#2173– 2179.#

Yan,#B.,#Shi,#X.,#Zhang,#H.,#Pan,#L.,#Ma,#Z.,#Liu,#S.,#Liu,#Y.,#Li,#X.,#Yang,#S.,#and#Li,#Z.#(2014).#Association#of#Serum# Irisin#with#Metabolic#Syndrome#in#Obese#Chinese#Adults.#PLoS#ONE#9,#e94235.#

Yu,#S.,#Zhang,#Y.,#Li,#M.NZ.,#Xu,#H.,#Wang,#Q.,#Song,#J.,#Lin,#P.,#Zhang,#L.,#Liu,#Q.,#Huang,#Q.NX.,#et#al.#(2012).# Chemerin#and#apelin#are#positively#correlated#with#inflammation#in#obese#type#2#diabetic#patients.#Chin.# Med.#J.#(Engl.)#125,#3440–3444.#

Zhang,#Y.,#Han,#P.,#Wu,#N.,#He,#B.,#Lu,#Y.,#Li,#S.,#Liu,#Y.,#Zhao,#S.,#Liu,#L.,#and#Li,#Y.#(2011).#Amelioration#of#lipid# abnormalities#by#alphaNlipoic#acid#through#antioxidative#and#antiNinflammatory#effects.#Obesity#(Silver# Spring)#19,#1647–1653.# #

! 239#

#

VI. CONCLUSIONS$

!

VI.$CONCLUSIONS$

1. Supplementation# with# αNlipoic# acid# at# a# dose# of# 300# mg/day# combined# with# an# energyN restricted# diet# promoted# additional# weight# loss# and# fat# mass# reduction# in# healthy# overweight/obese#women.## # 2. Supplementation#with#EPA#did#not#have#remarkable#effects#on#weight#loss,#but#prevented#the# decrease#in#leptin#levels#that#occurs#during#weight#loss,#that#has#been#related#with#increased# hunger,#lowered#metabolic#rate,#and#further#weight#regain.#No#synergistic#effects#were#found# between#treatments.## # 3. The#energyNrestricted#diet#improved#the#biochemical#profile#for#glucose#(glucose,#insulin#and# HOMANIR)#and#lipid#(totalNcholesterol,#LDLNcholesterol#and#TG)#metabolism.#However,#EPA#and# αNlipoic#acid#had#no#additional#effects#to#weight#loss#reduction#on#these#metabolic#markers.## # 4. Fasting#levels#of#the#adipoNmyokine#irisin#were#highly#correlated#with#fasting#glucose,#but#irisin# levels#were#not#modified#in#response#to#a#glucose#tolerance#challenge,#suggesting#that#glucose# is#not#a#direct#regulator#of#the#secretion#of#this#hormone.#αNLipoic#acid,#but#not#EPA,#was#able# to# induce# the# production# of# irisin# and# the# expression# of# FNDC5# gene# in# cultured# human# adipocytes;# however# supplementation# with# either# EPA# or# αNlipoic# acid# did# not# have# any# additional#effect#on#the#reduction#of#irisin#induced#by#a#hypocaloric#diet#in#overweight/obese# women.## # 5. Dietary# supplementation# with# αNlipoic# acid# promoted# a# greater# decrease# in# CRP# circulating# levels#and#leukocyte#count,#and#tended#to#reduce#the#Framingham#score#at#the#end#of#the#10N weeks# nutritional# intervention.# Additionally,# αNlipoic# acid# prevented# the# drop# in# apelin# that# normally#accompanies#weight#loss.## # 6. EPA#did#not#modify#the#levels#of#systemic#inflammatory#markers#after#the#10Nweeks#nutritional# intervention,#but#in#subcutaneous#abdominal#adipose#tissue#induced#a#decrease#in#the#gene# expression# of# ADRGE1,# a# macrophage# marker,# and# an# increase# in# the# antiNinflammatory# cytokine# IL10/ mRNA# levels,# suggesting# the# ability# of# EPA# to# attenuate# adipose# tissue# inflammation.## # 7. The# transcriptomic# study# of# adipose# tissue# obtained# at# the# end# of# the# 10Nweeks# nutritional# intervention# revealed# that# αNlipoic# acid# supplementation# downregulated# genes# related# with# chemotactic# factors# and# inflammatory# signals.# Moreover,# αNlipoic# acid,# especially# in#

! 243# VI.$CONCLUSIONS$

combination# with# EPA,# was# able# to# upregulate# genes# involved# in# lipid# catabolism,# while# downregulated# those# associated# with# lipid# storage.# In# addition,# EPA# supplementation# promoted#a#general#increase#of#chemotactic#factors#and#macrophage#gene#markers,#especially# those# known# as# antiNinflammatory,# as# well# as# changes# in# ECM# remodeling# genes.# These# observations# point# out# that# the# metabolic# effects# of# EPA# and# αNlipoic# acid# supplementation# could#be#related#to#their#regulatory#actions#on#adipose#tissue#genomics#and#metabolism.# # 8. Both#EPA#and#αNlipoic#acid#induced#in#peripheral#blood#white#cells,#changes#in#the#methylation# and#in#the#expression#of#genes#related#with#inflammation#and#lipid#metabolism.#Interestingly,#a# negative#relationship#between#the#methylation#changes#in#the#cg10320884#CpG#region#located# in# TRRAP# gene# with# the# changes# in# the# Framingham# score# was# observed,# suggesting# its# involvement#in#the#improvement#of#cardiovascular#risk.## # 9. αNLipoic# acid# induced# an# increment# in# the# expression# of# NCK2,# TRRAP,# CRREBP# and# RPTOR# genes#in#PBMC.#In#addition#the#expression#of#these#genes#at#the#end#of#the#intervention#was# associated# with# the# changes# in# body# weight# loss# and# fat# mass# reduction.# Moreover,# the# changes#in#the#expression#of#these#genes#were#related#with#the#FFA#levels#at#the#end#of#the# study.#Overall#these#results#suggest#a#potential#role#of#these#genes#in#the#effects#of#αNlipoic# acid#in#body#weight#regulation.##

General$conclusion$

The#current#research#suggests#that#αNlipoic#acid#could#be#useful#in#promoting#body#weight#and# fat#mass#loss#in#overweight/obese#healthy#women#in#the#context#of#a#hypocaloric#diet.#In#addition,# dietary#supplementation#with#αNlipoic#acid#may#help#to#improve#some#systemic#inflammatory#and# cardiovascular# disease–related# risk# markers# in# healthy# overweight# or# obese# women# independently#of#weight#loss.#Although#EPA#supplementation#did#not#have#any#additional#effect# on# the# reduction# of# body# and# fat# mass,# it# may# be# helpful# for# attenuating# adipose# tissue# inflammation# and# preventing# the# fall# of# leptin# during# weight# loss,# which# could# be# relevant# for# weight# loss# maintenance.# These# metabolic# effects# of# αNlipoic# acid# and# EPA# supplementation# appear#to#be#mediated,#at#least#in#part,#by#the#modulation#of#the#transcriptome#profile#of#adipose# tissue.#Moreover,#our#study#suggests#a#role#of#epigenetic#mechanisms#in#the#actions#of#αNlipoic# acid#and#EPA#supplementation#in#humans.#Overall,#the#current#project#suggests#that#αNlipoic#acid# and#or#/EPA#supplementation#could#be#beneficial#in#body#weight#regulation#and#in#preventing#the# appearance#of#future#metabolic#complications#in#healthy#overweigh/obese#women.##

244!! VI.$CONCLUSIONES$

1. La#suplementación#con#ácido# αNlipoico# a# una# dosis# de# 300# mg/día,# en# combinación# con# una# dieta#de#restricción#energética,#promovió#la#pérdida#de#peso#y#la#reducción#en#la#masa#grasa#en# mujeres#sanas#con#sobrepeso/obesidad.## # 2. La# suplementación# con# ácido# eicosapentaenoico# (EPA)# no# mostró# ningún# efecto# remarcable# sobre#la#pérdida#de#peso,#pero#previno#la#disminución#de#los#niveles#de#leptina#que#tienen#lugar# durante# la# pérdida# de# peso,# la# cual# se# ha# relacionado# con# el# incremento# en# el# apetito,# la# disminución# de# la# tasa# metabólica# y# la# recuperación# del# peso# perdido.# No# se# encontraron# efectos#sinérgicos#entre#tratamientos.## # 3. La# dieta# hipocalórica# mejoró# el# perfil# bioquímico# de# marcadores# del# metabolismo# glucídico# (glucosa,#insulina#e#índice#HOMA)#y#lipídico#(colesterol#total,#colesterol#LDL,#y#triglicéridos).#Sin# embargo,#el#EPA#y#el#ácido#αNlipoico#no#tuvieron#efectos#adicionales#a#la#pérdida#de#peso#en# estos#marcadores#metabólicos.## # 4. Los#niveles#en#ayunas#de#la#adipomioquina#irisina#se#correlacionaron#con#la#glucosa#en#ayunas;# sin#embargo,#los#niveles#de#irisina#no#se#vieron#afectados#en#respuesta#al#test#de#tolerancia#oral# a# la# glucosa,# sugiriendo# que# la# glucosa# no# es# un# regulador# directo# de# la# secreción# de# esta# hormona.#El#ácido#αNlipoico#pero#no#el#EPA,#fue#capaz#de#inducir#la#producción#de#irisina#y#la# expresión#del#gen#FNDC5#en#cultivos#de#adipocitos#humanos.#Sin#embargo,#ni#la#suplementación# con# EPA# ni# con# ácido# αNlipoico# tuvieron# algún# efecto# adicional# sobre# la# reducción# de# irisina# inducida#por#la#dieta#hipocalórica#en#mujeres#con#sobrepeso/obesidad.## # 5. Con# respecto# a# los# biomarcadores# de# inflamación# y# riesgo# cardiovascular,# la# suplementación# dietética# con# ácido# αNlipoico# promovió# una# mayor# disminución# de# los# niveles# circulantes# de# proteína# C# reactiva# y# del# recuento# de# leucocitos.# Además# tendió# a# disminuir# el# score# de# Framingham#al#final#de#la#intervención#nutricional#de#10#semanas.#Adicionalmente,#el#ácido#αN lipoico#previno#la#caída#en#apelina#que#normalmente#acompaña#a#la#pérdida#de#peso.## # 6. El#EPA#no#modificó#los#niveles#sistémicos#de#los#marcadores#de#inflamación#estudiados#tras#la# intervención#nutricional#de#10#semanas,#pero#en#tejido#adiposo#subcutáneo#abdominal#indujo# una# menor# expresión# del# gen# ADRE1,# considerado# un# marcador# de# macrófagos,# y# un# incremento# de# los# niveles# de# ARNm# de# la# citoquina# antiNinflamatoria# IL10,# sugiriendo# la# habilidad#del#EPA#para#atenuar#la#inflamación#en#el#tejido#adiposo.##

! 245# VI.$CONCLUSIONES$

# 7. El# estudio# transcriptómico# en# el# tejido# adiposo,# reveló# que# la# suplementación# con# ácido# αN lipoico,# disminuyó# la# expresión# de# genes# relacionados# con# factores# quimiotácticos# y# señales# inflamatorias.#Además,#el#ácido#αNlipoico,#especialmente#en#combinación#con#EPA,#fue#capaz#de# regular# positivamente# genes# involucrados# con# el# catabolismo# lipídico# mientras# reprimió# aquellos#genes#asociados#con#el#almacenamiento#de#lípidos.#Adicionalmente,#la#suplementación# con#EPA,#promovió#un#incremento#general#de#factores#quimiotácticos#y#de#genes#marcadores# de# macrófagos,# especialmente# de# aquellos# conocidos# como# antiNinflamatorios,# así# como# un# incremento# de# genes# involucrados# en# la# remodelación# de# la# matriz# extracelular.# Estas# observaciones#señalan#que#los#efectos#metabólicos#de#la#suplementación#con#EPA#y#con#ácido# αNlipoico# podrían# estar# relacionados# con# sus# acciones# reguladoras# de# la# genómica# y# el# metabolismo#del#tejido#adiposo.### # 8. Tanto# el# EPA# como# el# ácido# αNlipoico,# indujeron# en# células# blancas# periféricas# de# la# sangre# cambios# en# la# metilación# y# en# la# expresión# de# genes# relacionados# con# la# inflamación# y# el# metabolismo#de#los#lípidos.#Es#de#destacar,#la#asociación#negativa#observada#entre#los#cambios# de#metilación#en#la#región#CpG#cg10320884#localizada#en#el#gen#TRRAP#con#los#cambios#en#el# score#de#Framingham,#lo#que#sugiere#su#implicación#en#la#mejora#del#riesgo#cardiovascular.# # 9. El#ácido#αNlipoico#indujo#un#incremento#en#la#expresión#de#los#genes#NCK2,#TRRAP,#CREBBP#y## RPTOR#en#células#mononucleares#periféricas#de#la#sangre.#Además,#la#expresión#de#estos#genes# al#final#de#la#intervención,#estuvo#asociada#con#la#pérdida#de#peso#corporal#y#con#la#reducción# de#masa#grasa.#Por#otra#parte,#los#cambios#en#la#expresión#de#estos#genes#estuvieron#también# relacionados# con# los# niveles# de# ácido# grasos# libres# al# final# del# estudio.# En# conjunto,# estos# resultados#sugieren#una#implicación#potencial#de#estos#genes#en#los#efectos#del#ácido#αNlipoico# en#la#regulación#del#peso#corporal.##

Conclusión$general$

La# evidencia# presentada# en# este# trabajo# sugiere# que# el# ácido# αNlipoico# puede# ser# útil# para# promover# tanto# la# pérdida# peso# como# la# pérdida# de# masa# grasa# en# mujeres# sanas# con# sobrepeso/obesidad# en# el# contexto# de# una# dieta# hipocalórica.# Asimismo,# la# suplementación# dietética# con# ácido# αNlipoico# podría# mejorar# algunos# marcadores# sistémicos# de# inflamación# y# relacionados# con# el# riesgo# cardiovascular# en# mujeres# con# sobrepeso/obesidad,# independientemente#de#la#pérdida#de#peso.#Aunque#la#suplementación#con#EPA#no#tuvo#efectos#

246!! VI.$CONCLUSIONES$ adicionales#en#la#reducción#del#peso#y#la#masa#grasa,#éste#podría#ayudar#a#atenuar#la#inflamación# del#tejido#adiposo#y#a#prevenir#la#caída#de#leptina#observada#durante#la#pérdida#de#peso,#lo#que# podría# ser# relevante# para# mantener# el# peso# perdido.# Los# efectos# metabólicos# de# la# suplementación# con# ácido# αNlipoico# y# EPA# parecen# estar# mediados,# al# menos# en# parte,# por# la# modulación#del#perfil#transcriptómico#del#tejido#adiposo.#Adicionalmente,#nuestro#estudio#sugiere# una#implicación#de#mecanismos#epigéneticos#en#las#acciones#metabólicas#del#ácido#αNlipoico#y#el# EPA#en#humanos.#En#general,#el#presente#proyecto#sugiere#que#la#suplementación#con#ácido#αN lipoico#y#EPA#podría#ser#beneficiosa#en#la#regulación#del#peso#y#en#la#prevención#de#la#aparición#de# futuras#complicaciones#metabólicas#en#mujeres#sanas#con#sobrepeso/obesidad.##

$

! 247#

#

VII. ANNEXES$

!

VII.$ANNEX$1:$INFORMED$CONSENT$

HOJA$INFORMATIVA$PARA$EL$PARTICIPANTE$

Esta#hoja#informativa#le#invita#a#participar#en#el#proyecto#“Efectos$celulares$y$moleculares$ del$ácido$lipoico$y$del$eicosapentaenoico$(EPA)$en$tejido$adiposo:$aplicación$potencial$ en$ obesidad$ humana”,# en# concreto# en# el# ESTUDIO# DEL# EFECTO# DEL# CONSUMO# DE# SUPLEMENTOS#DE#ÁCIDO#LIPOICO#(LA)#Y#ÁCIDO#EICOSAPENTAENOICO#(EPA)#SOBRE# EL#PESO#CORPORAL,#LA#COMPOSICIÓN#CORPORAL#Y#MARCADORES#DE#INSULINON RESISTENCIA#Y#RIESGO#CARDIOVASCULAR.##

El# objetivo# concreto# del# estudio# es# establecer# si# una# dieta# hipocalórica# y# equilibrada,# junto#con#el#consumo#habitual#de#suplementos#de#EPA#y/o#LA#tiene#efectos#beneficiosos# sobre:##

N#El#peso#y#la#composición#corporal#(porcentaje#de#grasa#y#de#músculo#de#su#organismo).#

N#El#metabolismo#lipoproteico#(colesterol#y#triglicéridos),#niveles#de#glucosa#y#de#insulina.##

N#La#función#de#los#adipocitos#(células#de#almacenamietno#de#grasa)#mediate#estudios#de# expresión# génica,# que# estudiarán# si# la# capacidad# de# funcionamiento# de# las# células# ha# variado#y#si#esta#variación#resulta#positiva#o#negativa,#después#de#seguir#un#determinado# tratamiento#dietético.##

Para# participar# en# este# proyecto# usted# tiene# que# estar# de# acuerdo# en# acudir# al# Departamento#de#Ciencias#de#la#Alimentación,#Fisiología#y#Toxicología#de#la#Universidad# de#Navarra#en#varias#ocasiones:##

En#esta#primera#cita#se#le#hace#entrega#de#esta#hoja#informativa#para#que#usted#la#lea#y# pregunte# dudas# sobre# el# proyecto.# A# continuación# se# le# hace# entrega# de# la# hoja# de# consentimiento#informado,#por#duplicado#y#aprobado#por#el#Comité#de#Ética,#para#que# muestre#su#conformidad.#El#estudio#comezará#con#una#breve#anamnesis#e#historia#clínica# con#exploración#llevada#a#cabo#por#una#Licenciada#en#Medicina.#La#enfermera#procederá# haciéndole#entrega#de#un#cuestionario#de#consumo#de#alimentos#y#hábitos#de#vida#(SUN)# y# un# registro# de# pesada# de# 72# horas# con# las# aclaraciones# correspondientes# acerca# de# cómo# se# deben# cumplimentar# y# errores# frecuentes# que# se# cometen# a# la# hora# de#

! 251# VII.$ANNEX$1:$INFORMED$CONSENT$ rellenarlos,#que#completará#en#su#casa#y#se#le#recogerá#en#la#segunda#cita.#El#tiepo#que# deberá#emplear#en#cumplimentar#los#cuestionarios#es#de#2#horas.##

En#este#estudio#se#va#a#ensayar#una#dieta#hipocalórica,#con#una#restricción#moderada#de# energía,#y#además#tendrá#que#tomar#uno#de#estos#suplementos:#Control#→#Cápsulas#sin# contenido# nutricional,# llamadas# placebo;# EPA# →# Cápsulas# que# contienen# EPA;# LA# →# Cápsulas#que#contienen#LA;#EPA+LA#→#Cápsulas#que#contienen#EPA#y#LA.##

Su#inclusión#en#un#grupo#u#toro#se#realizará#de#modo#aleatorio,#y#no#sabrá#a#qué#grupo# pertenece,#para#que#no#exista#ningún#tipo#de#condicionamiento#en#su#evolución.##

En# caso# de# que# usted# cumpla# los# criterios# de# inclusión,# se# le# seleccionará# para# una# segunda#cita#en#la#que#se#le#volverá#a#recordar#en#qué#consiste#su#participación#y#se#le# tomarán#las#medidas#de#peso,#talla,#pliegues#cutáneos#y#de#composición#corporal#(Día#0).# A#continuación#se#le#tomará#la#presión#arterial#y#la#enfermera#extraerá#una#muestra#de# sangre#para#llevar#a#cabo#análisis#bioquímicos#de#rutina#relacionados#con#el#metabolismo# lipídico,# glucídico# y# proteico.# También# se# le# realizará# un# test# de# tolerancia# oral# a# la# glucosa.# La# dietista# le# dará# las# directrices# necesarias# para# el# comienzo# del# estudio# de# intervención# nutricional# basado# en# la# dieta# a# la# que# se# le# habrá# asignado# y# se# le# proporcionarán#las#correspondientes#cápsulas.##

Deberá$ajustarse$a$la$ingesta$de$alimentos$y$de$las$cápsulas$del$modo$que$se$le$indica.##

Durante#el#estudio#acudirá#al#final#de#la#semana#2,#4,#6#y#8#para#comprobar#seguimiento#y# adherencia#a#la#dieta,#lo#cual#se#llevará#a#cabo#mediante#entrevistas#dietéticas,#control#de# peso#y#registros#de#pesados#de#72#horas.##

El# estudio# concluirá# con# la# valoración# de# la# composición# corporal,# historia# dietética,# presión#arterial#y#extracción#de#una#muestra#de#sangre#tras#10#semanas#de#participación.# También#se#le#extraerá#una#biopsia#de#tejido#adiposo#subcutáneo#abdominal#(1N2#gramos,# en#ayunas).#Una#vez#desinfectada#la#zona#(2,6#mm#de#diámetro#de#la#región#periumbilical)# se#procederá#a#la#anestecia#con#500#µL#de#liocaína#al#1%#intradermal#y#posterior#succión# en# una# jeringuilla# de# 10# mL# con# 2# mL# de# solución# salina.# Este# procedimiento# le# será# realizado#por#personal#cualificado#y#experimentado.##

252!! VII.$ANNEX$1:$INFORMED$CONSENT$

En#total#deberá#acudir#a#nuestras#instalaciones#7#ocasiones,#3#para#realizarse#una#exámen# completo,# en# ayunas# y# 4# veces# para# pesarse# y# realizar# una# breve# entrevista# de# seguimiento#con#la#dietista.##

Los#posibles#riesgos#de#esta#intervención#nutricional#están#relacionados#con#la#extracción# de#sangre#por#punción#venosa#y#por# la# biopsia# de# tejido# adiposo.# Estos# procedimietos# pueden# conllevar# algunas# molestias# para# usted# como# ligera# molestia# en# la# zona# de# punción#o#presencia#posterior#de#hematoma#en#esta#misma#zona#y#en#casos#excepcionales# lipotimias# o# infección.# En# cuanto# a# los# suplementos# que# se# les# va# a# proporcionar,# son# productos#comerciales,#y#no#se#espera#la#aparición#de#efectos#adversos.#En#cualquier#caso,# si#usted#está#embarazada#o#planea#estarlo#durante#la#intervención,#no#podrá#participar#en# el#estudio.##

Toda# la# información# que# nos# proporcione,# así# como# los# resultados# de# los# análisis# de# sangre#se#tratarán#según#la#Ley#Orgánica#15/1999,#de#13#de#diciembre,#de#Protección#de# Datos# de# Carácter# Personal,# utilizando# códigos# para# asegurar# la# confidencialidad# y# garantizar#el#anonimato.#Sólo#dos#miembros#del#equipo#investigador#conocerán#sus#datos# personales,# ya# que# serán# los# encargados# de# contactar# con# usted# para# cualquier# evento# relacionado#con#el#estudio.##

El#resto#de#miembros#del#equipo#trabajarán#con#códigos,#ignorando#a#qué#voluntario#le# corresponde#cada#código.##

Usted#puede#abandonar#el#estudio#en#cualquier#momento#,#sin#dar#explicaciones.#Tras#el# procedimiento#de#los#datos,#se#le#informará#de#los#resultados#de#las#pruebas#realizadas#y# se#mantendrá#la#confidencialidad#propia#de#todo#procedimiento#médico.##

SU#PARTICIPACIÓN#EN#EL#ESTUDIO#NO#ESTÁ#REMUNERADA.##

# #

! 253# VII.$ANNEX$1:$INFORMED$CONSENT$

Formulario$de$consentimieto$(COPIA$1)$

Proyecto:$ “Efectos$ celulares$ y$ moleculares$ del$ ácido$ lipoico$ y$ del$ eicosapentaenoico$ (EPA)$en$tejido$adiposo:$aplicación$potencial$en$obesidad$humana”$

Objetivo:#Estudio#del#efecto#del#consumo#de#suplementos#del#ácido#lipoico#(LA)#y#ácido# eicosapentaenoico#(EPA)#sobre#el#peso#corporal,#la#composición#corporal#y#marcadores#de# insulinoNresistencia#y#riesgo#cardiovascular,#así#como#sobre#la#expresión#génica#del#tejido# adiposo.#

Yo#(nombre#y#apellidos)#______# o He#leído#la#hoja#de#información#que#se#me#ha#entregado.# o He#podido#hacer#preguntas#sobre#el#estudio.## o He#hablado#con:##(nombre#del#investigador):#______#

Entiendo#que#mi#participación#es#voluntaria.##

Entiendo#que#puedo#retirarme#del#studio:##

1. Cuando#quiera.# 2. Sin#tener#que#dar#explicaciones.## 3. Sin#que#esto#repercuta#en#mis#cuidados#médicos.##

Accedo$ a$ que$ las$ muestras$ de$ sangre$ y$ tejido$ adipose$ obtenidas$ en$ el$ estudio$ sean$almacenadas$para$futuras$determinaciones$en$estudios$de$la$misma$línea$ de$investigación$y$que$serán$aprobados$por$el$Comité$de$Ética$de$Investigación$ Clínica$de$la$Universidad$de$Navarra$(escriba$Sí$o$No)$______.$

Presto$libremente$mi$conformidad$para$participar$en$el$estudio.$$

#

Fecha################################################################################Firma#del#participante#

#

#

Fecha###############################################################################Firma#del#investigador# # #

254!! VII.$ANNEX$1:$INFORMED$CONSENT$

Formulario$de$consentimieto$(COPIA$2)$

Proyecto:$ “Efectos$ celulares$ y$ moleculares$ del$ ácido$ lipoico$ y$ del$ eicosapentaenoico$ (EPA)$en$tejido$adiposo:$aplicación$potencial$en$obesidad$humana”$

Objetivo:#Estudio#del#efecto#del#consumo#de#suplementos#del#ácido#lipoico#(LA)#y#ácido# eicosapentaenoico#(EPA)#sobre#el#peso#corporal,#la#composición#corporal#y#marcadores#de# insulinoNresistencia#y#riesgo#cardiovascular,#así#como#sobre#la#expresión#génica#del#tejido# adiposo.#

Yo#(nombre#y#apellidos)#______# o He#leído#la#hoja#de#información#que#se#me#ha#entregado.# o He#podido#hacer#preguntas#sobre#el#estudio.## o He#hablado#con:##(nombre#del#investigador):#______#

Entiendo#que#mi#participación#es#voluntaria.##

Entiendo#que#puedo#retirarme#del#studio:##

4. Cuando#quiera.# 5. Sin#tener#que#dar#explicaciones.## 6. Sin#que#esto#repercuta#en#mis#cuidados#médicos.##

Accedo$ a$ que$ las$ muestras$ de$ sangre$ y$ tejido$ adipose$ obtenidas$ en$ el$ estudio$ sean$almacenadas$para$futuras$determinaciones$en$estudios$de$la$misma$línea$ de$investigación$y$que$serán$aprobados$por$el$Comité$de$Ética$de$Investigación$ Clínica$de$la$Universidad$de$Navarra$(escriba$Sí$o$No)$______.$

Presto$libremente$mi$conformidad$para$participar$en$el$estudio.$$

#

Fecha################################################################################Firma#del#participante#

#

#

Fecha###############################################################################Firma#del#investigador#

! 255#

VII.$ANNEX$2:$ADDITIONAL$NOTES$TO$THE$VOLUNTEER$

! # # # Facultad#de#Farmacia## Departamento#de#Ciencias#de#la#Alimentación,# Fisiología#y#Toxicología# # # Notas$importantes$ # PESCADOS$ PERMITIDOS:#abadejo,#bacalao,#bacaladilla,#cazón,#gallo,#lenguado,#merluza,# palometa,#pescadilla,#rape,#rosada.# # EXCLUIR$DE$LA$DIETA#PESCADO#GRASO,#SEMIGRASOS,#CRUSTÁCEOS#Y#MOLUSCOS.# # EXCLUIR$ DE$ LA$ DIETA# TODOS# LOS# ALIMENTOS# ENRIQUECIDOS# EN# OMEGA# 3:# Leches# y# derivados,#huevos,#galletas,#mantequillas,#margarinas,#zumos,#bebidas#de#soja,#aceitunas# o#preparados#grasos#a#base#de#mezclas#de#aceites.# # OTROS$ALIMENTOS$A$EXCLUIR#SON:#frutos#secos,#aceite#de#algas,#semillas#de#lino,#pan#de# cereales.#

CONSEJOS:#

- Medir#el#aceite#de#todo#el#día#en#un#vasito#de#chupito#para#controlarlo#mejor.# - Pesar#los#alimentos#en#crudo#y#en#limpio,#siempre#que#sea#posible.#Ej:#acelga,# después#de#limpiar#pero#antes#de#cocinar# - Todos#los#pesos#de#la#dieta#son#en#crudo#y#en#limpio,#si#no#se#especifica#lo# contrario.#Ej:#175g#de#naranja,#una#vez#pelada.# - Carne#magra:#retirar#la#grasa#visible#antes#de#cocinar,#ya#que#es#mucho#más#fácil#e# higiénico.#En#el#caso#del#jamón#serrano,#antes#de#comer.# - Tanto#los#postres#de#comida#y#cena,#como#las#medias#mañanas#y#meriendas,#son# intercambiables,#dentro#del#mismo#día.# - No#dejar#las#cápsulas#para#el#final#de#la#comida#

! 257#

VII.$ANNEX$3:$72HHOUR$RECALL$QUESTIONNAIRE$

INSTRUCCIONES$PARA$LA$CUPLIMENTACIÓN$DEL$REGISTRO$DE$PESADA$PRECISA$DE$ ALIMENTOS$DE$72$HORAS$

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Este#cuestionario#consta#de#un#total#de#4#páginas,#aunque#usted#puede#añadir#más#si#lo# considera#necesario.##

Las#tres#primeras#páginas#corresponden#cada#una#a#un#día,#recuerde#anotar#el#día#de#la# semana#y#la#fecha#correspondiente.##

Usted#deberá#pesar#todos#los#alimentos#que#consuma#cada#día#que#rellene#el#cuestionario# intentando#no#olvidar#el#aceite#que#usa#para#cocinar#y#aliñar,#el#azúcar#(en#café#y#postres)# así#como#todas#la#bebidas#que#ingiera,#incluido#el#agua#de#grifo#y#alimentos#consumidos# entre#horas.##

Para#evitar#que#se#le#olvide#algún#alimento#o#plato,#intente#anotarlos#nada#más#comerlos# siempre#que#sea#posible.##

Recuerde# también,# en# el# caso# de# alimentos# envasados,# anotar# la# marca# y# si# es# posible# adjuntarnos#la#etiqueta#del#producto.##

Si#alguna#de#las#comidas#le#es#imposible#pesar#(por#comer#fuera#de#casa,#por#ejemplo),# intente#anotar#con#medidas#caseras,#es#decir,#si#el#plato#era#llano#u#hondo,#si#estaba#lleno,# a# la# mitad,# etc.,# tamaño# de# la# fruta# (pequeña,# mediana# o# grande),# usar# medidas# como# cucharadas,#tazas#o#puñados,#etc..#

En#el#caso#de#que#el#plato#que#tomado#se#un#plato#elaborado#con#varios#ingredients#(por# ejemplo,# paella),# utilice# la# hoja# adjuntada# al# final# para# describir# con# toda# la# precisión# posible# dicho# plato.# Si# lo# necesita# puede# utilizer# más# hojas# como# la# que# le# hemos# proporcionado.##

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! 261$ VII.$ANNEX$3:$72-HOUR$RECALL$QUESTIONNAIRE$

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262! ! VII.$ANNEX$3:$72-HOUR$RECALL$QUESTIONNAIRE$

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! 263$ VII.$ANNEX$3:$72-HOUR$RECALL$QUESTIONNAIRE$

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264! ! VII.$ANNEX$4:$EXAMPLE$OF$DIET$

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! 265$ VII.$ANNEX$4:$EXAMPLE$OF$DIET$

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266!! VII.$ANNEX$5:$COMPARISONS$ACCORDING$WITH$BASELINE$TRIGLYCERIDE$LEVELS$

$$$ a Low TG levels at baseline b High TG baseline levels 4 0 $ -8 0 -16 $ -4 -24 -32

TG (mg/mL) -8 TG (mg/mL) $ Δ Δ -40 -12 -48 $ Control EPA LA EPA+LA Control EPA LA EPA+LA Intervention group Intervention group

Figure$ 1.$Comparisons$of$the$changes$(after6before)$in$triglyceride$(TG)$concentrations$after$10$ weeks$nutritional$intervention$between$groups$according$with$the$triglyceride$levels$at$baseline.$ Data$are$adjusted$means$by$body$weight$loss$±$SE.$The$data$were$divided$by$the$median$levels$of$ serum$TG$at$baseline,$considering$as$low$TG$levels$(a)$those$values$≤83$mg/dL$(Control=$13;$EPA=$ 8;$LA=$6;$EPA+LA$=$9)$and$as$high$TG$levels$(b)$those$values$>83$mg/dL$(Control=$8;$EPA=$7;$LA=$ 12;$ EPA+LA=$ 8).$ Comparisons$ between$ groups$ were$ performed$ by$ two6way$ ANOVA$ and$ no$ significant$differences$were$observed$between$groups$(P>$0.05).$EPA:$eicosapentaenoic$acid;$LA:$ α6lipoic$acid.$

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$ 6 $ 0 -6 $ -12 $ -18

TG (mg/dL) -24

$ Δ -30 $ -36 *** Low TG High TG $ Baseline TG levels

Figure$2.$$Comparison$of$the$changes$(after6before)$in$triglyceride$(TG)$concentration$according$ with$the$TG$levels$at$baseline.$Data$are$means$±$SE.$The$data$were$divided$by$the$median$levels$of$ serum$TG$at$baseline,$considering$as$low$TG$levels$those$values$≤83$mg/dL$(n=$36)$and$as$high$TG$ levels$those$values$>83$mg/dL$(n=$35).$Comparisons$between$groups$were$performed$by$unpaired$ t$test$(***P<$0.001).$$

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