Epigenetics of Adipose Tissue and its related disorders
Abstract
Obesity and metabolic syndrome are increasing epidemic worldwide, especially in developed countries. Obesity and metabolic disease increase the risk of suffering certain diseases such as diabetes, cardiovascular disease, NASH, neurodegenerative disorders or some type of cancers like colorectal cancer. Adipose tissue is nowadays considered as an important organ that can regulate body homeostasis and modulate other tissues function through the delivery of a wide range of molecules. It has been shown that in obesity and metabolic disease context an adipose tissue dysfunction exists, producing changes in the adipokine secretion profile that in turn can prone to the disorders usually associated to these conditions. Besides, metabolic disease and obesity development are closely related to environmental factors such as food, smoking, sedentary habits, etc. However, obesity and metabolic syndrome also have a genetic and an epigenetic component, being regarded the epigenetic landscape as a mediator between environmental and genetic factors. In this sense, epigenetics mechanisms can be shaped by nutrition, smoking or exercise among other lifestyle factors. There are two main epigenetics mechanism: 1) DNA methylation, which occurs in the C5 of a cytosine pyrimidine near to a guanine (position called CpG); and 2) histone modifications, which mainly occurs in the N terminal of histone proteins and that have a more diverse nature (acetylation, methylation, phosphorylation, sumoylation, ubiquitination, glycosylation,…). Both mechanism have been shown to be deregulated in metabolic syndrome and obesity, and could be implied in the etiology and associated risks observed in these conditions.
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AUTOR: Daniel Cas ellano Cas illo
h p://o cid.o g/0000-0001-8041-8244!
EDITA: Publicaciones y Di ulgación Cien í ica. Uni e sidad de Málaga
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Es a Tesis Doc o al es á deposi ada en el Reposi o io Ins i ucional de la Uni e sidad de Málaga
(RIUMA): iuma.uma.es
Facul ad de Ciencias
P og ama de Doc o ado:
Biología Celula y Molecula
Thesis by compendium o publica ions
EPIGENETICS OF ADIPOSE TISSUE AND
ITS RELATED DISORDERS
Daniel Cas ellano Cas illo
Di ec o es:
F ancisco Tinahones
Ma ía Isabel Queipo O uño
Isabel Mo eno Indias
D. F ancisco José Tinahones Madueño, Doc o en Medicina y Ci ugía, Di ec o de
la Unidad de Endoc inología y Nu ición del Hospi al Vi gen de la Vic o ia de
Málaga y P o eso Ti ula del Depa amen o de Medicina y De ma ología de la
Facul ad de Medicina de la Uni e sidad de Málaga
CERTIFICA: Que el abajo expues o en la memo ia de la Tesis Doc o al desa ollada
po Daniel Cas ellano Cas illo con el í ulo “EPIGENETICS OF ADIPOSE TISSUE
AND ITS RELATED DISORDERS” co esponde ielmen e a los esul ados
ob enidos.
La p esen e memo ia ha sido ealizada bajo mi di ección, conside ando que iene el
con enido y igo cien í ico necesa io pa a se some ida a juicio po el ibunal
nomb ado po la Uni e sidad de Málaga pa a op a al g ado de Doc o .
Y pa a que cons e, en cumplimien o de las disposiciones legalmen e igen es a los
e ec os opo unos, i mo el p esen e ce i icado, con lo que au o izo la lec u a de la
misma.
Di ec o de la esis:
D . F ancisco José Tinahones Madueño
Málaga, No iemb e de 2018
Dña. Ma ía Isabel Queipo O uño, Doc o a en Biología po la Uni e sidad de
Málaga
CERTIFICA: Que el abajo expues o en la memo ia de la Tesis Doc o al desa ollada
po Daniel Cas ellano Cas illo con el í ulo “EPIGENETICS OF ADIPOSE TISSUE
AND ITS RELATED DISORDERS” co esponde ielmen e a los esul ados
ob enidos.
La p esen e memo ia ha sido ealizada bajo mi di ección, conside ando que iene el
con enido y igo cien í ico necesa io pa a se some ida a juicio po el ibunal
nomb ado po la Uni e sidad de Málaga pa a op a al g ado de Doc o .
Y pa a que cons e, en cumplimien o de las disposiciones legalmen e igen es a los
e ec os opo unos, i mo el p esen e ce i icado, con lo que au o izo la lec u a de la
misma.
Di ec o a de la esis:
D a. Ma ía Isabel Queipo O uño
Málaga, No iemb e de 2018
Dña. Isabel Mo eno Indias, Doc o a po la Uni e sidad de las Palmas de G an
Cana ia
CERTIFICA: Que el abajo expues o en la memo ia de la Tesis Doc o al desa ollada
po Daniel Cas ellano Cas illo con el í ulo “EPIGENETICS OF ADIPOSE TISSUE
AND ITS RELATED DISORDERS” co esponde ielmen e a los esul ados
ob enidos.
La p esen e memo ia ha sido ealizada bajo mi di ección, conside ando que iene el
con enido y igo cien í ico necesa io pa a se some ida a juicio po el ibunal
nomb ado po la Uni e sidad de Málaga pa a op a al g ado de Doc o .
Y pa a que cons e, en cumplimien o de las disposiciones legalmen e igen es a los
e ec os opo unos, i mo el p esen e ce i icado, con lo que au o izo la lec u a de la
misma.
Di ec o a de la esis:
D a. Isabel Mo eno Indias
Málaga, No iemb e de 2018
Index
ABBREVIATIONS!19!
INTRODUCTION!33!
1. OBESITY 35!
2. ADIPOSE TISSUE 36!
2.1. Adipose Tissue cellula i y: Adipocy e p ecu so s and adipogenesis 38!
2.2. Adipose Tissue cellula i y: Immune cells 41!
3. ADIPOSE TISSUE METABOLISM: LIPID AND GLUCOSE METABOLISM 43
3.1. Lipid me abolism 43!
3.2. Glucose me abolism 47!
4. ROLE OF ADIPOSE TISSUE IN METABOLIC DISORDERS 48!
4.1. Me abolic ac o s ela ed o me abolic diso de s 50!
4.1.1. Lipop o ein Lipase (LPL) 50!
4.1.2. Low-densi y lipop o ein ecep o - ela ed p o ein 1 (LRP1) 53!
4.1.3. Glucose anspo e ype 4 (GLUT4) 53!
4.1.4. Pe oxisome p oli e a o -ac i a ed ecep o s (PPARs) 54!
4.1.5. S e ol egula o y elemen binding ac o s (SREBFs) 55!
4.1.6. S ea oyl-CoA-desa u ase (SCD) 56!
4.1.7. Li e X ecep o be a (LXRb) 56!
4.1.8. Lep in (LEP) 58!
4.2. In lamma o y ac o s in me abolic diso de s 60!
5. ADIPOSE TISSUE AND COLORECTAL CANCER 64!
6. EPIGENETICS 67!
6.1. DNA me hyla ion 67!
6.2. His one modi ica ions 71!
6.3. In e play be ween DNA me hyla ion and his one modi ica ions 73!
6.4. In e play be ween me abolic s a us and epigene ics 77!
Index
6.4.1. Role o li es yle and nu i ional condi ions in he epigene ics o obesi y and me abolic
disease 80!
6.4.2. Epigene ics al e a ions in colo ec al cance 83!
HYPOTHESIS!85!
OBJECTIVES!89!
RESULTS!93!
MANUSCRIPT 1. Adipose Tissue LPL Me hyla ion is Associa ed wi h T iglyce ide
Concen a ions in he Me abolic Synd ome 95!
MANUSCRIPT 2. Adipose Tissue DNA Me hyla ion o Adipogenic, Lipid Me abolism and
In lamma o y Genes in Me abolic Synd ome 107!
MANUSCRIPT 3. Complemen Fac o C3 Me hyla ion and mRNA Exp ession Is Associa ed
o BMI and Insulin Resis ance in Obesi y 121!
MANUSCRITP 4. Ch oma in Immunop ecipi a ion Imp o emen s o he P ocessing o Small
F ozen Pieces o Adipose Tissue 129!
MANUSCRIPT 5. Human Adipose Tissue H3K4me3 in Adipogenic, Lipid and In lamma o y
genes a e Posi i ely Associa ed o BMI and HOMA-IR 139!
MANUSCRIPT 6. Adipose Tissue In lamma ion and VDR Exp ession and Me hyla ion in
Colo ec al Cance 151!
GENERAL DISCUSSION!161!
CONCLUSIONS!171!
LITERATURE!175!
SUPPLEMENTAL DATA!207!
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ABBREVIATIONS
Abb e ia ions
21
Body mass index: BMI
Tumo nec osis ac o : TNF/ TNFα
In e leukin 6: IL6
Insulin esis ance: IR
Ca dio ascula disease: CVD
Me abolic Synd ome: Me S
T iglyce ides: TG/Tg
High-densi y lipop o ein choles e ol: HDL-cho/HDL-C
Adipose issue: AT
Whi e adipose issue: WAT
B own adipose issue: BAT
Uncoupling p o ein: UCP
Subcu aneous adipose issue: SAT
Visce al adipose issue: VAT
Weigh /hip a ion: WHR
S omal ascula ac ion: SVF
Adipose issue-de i ed mesenchymal s em cells: ASCs
Sca enge ecep o class A membe 5: SCARA5
Bone mo phogene ic p o ein 2: BMP2
Bone mo phogene ic p o ein 4: BMP4
T ans o ming g ow h ac o be a: TGFβ
Pla ele -de i ed g ow h ac o ecep o A: PDGFRα
Pla ele -de i ed g ow h ac o ecep o B: PDGFRβ
Pla ele -de i ed g ow h ac o subuni A: PDGFA
SMAD amily membe 1: SMAD1
Abb e ia ions
22
SMAD amily membe 4: SMAD4
SMAD amily membe 5: SMAD5
SMAD amily membe 8: SMAD8
Zinc inge p o ein 423: Z p423
Lysyl oxidase: Lox
Fib oblas g ow h ac o 2: FGF2
Pe oxisome p oli e a o ac i a ed ecep o gamma: PPARγ
Pe oxisome p oli e a o ac i a ed ecep o gamma 2: PPARγ2
Pe oxisome p oli e a o ac i a ed ecep o alpha: PPAR
CCAAT enhance binding p o ein be a: C/EBPβ
CCAAT enhance binding p o ein del a: C/EBPδ
CCAAT enhance binding p o ein alpha: C/EBPα
K üppel-like ac o 5: KLF5
K üppel-like ac o 15: KLF15
K üppel-like ac o 2: KLF2
S e ol egula o y elemen binding ansc ip ion ac o 1: SREBP1
S e ol egula o y elemen binding ansc ip ion ac o 1 iso o m c: SREBP1c
S e ol egula o y elemen binding ansc ip ion ac o 1 iso o m a: SREBP1a
S e ol egula o y elemen binding ansc ip ion ac o 2: SREBP2
Lipopolysaccha ides: LPS
In e e on-γ: IFN-γ
In e leukin 12: IL12
In e leukin 4: IL4
In e leukin 14: IL14
In e leukin 10: IL10
Abb e ia ions
23
Chi inase-like 3: Ym1
A ginase 1: ARG1
Chylomic ons: CM
Apolipop o ein B48: ApoB48
Apolipop o ein CII: ApoCII
Apolipop o ein CIII: ApoCIII
Lipop o ein lipase: LPL
Apolipop o ein E: ApoE
Low-densi y lipop o ein choles e ol: LDL-cho/LDL-C
Low densi y lipop o ein ecep o : LDLR
Low-densi y lipop o ein (LDL)- ela ed p o ein 1: LRP1
Ve y low-densi y lipop o ein: VLDL
In e media e densi y lipop o eins: IDL
Hepa ic lipop o ein lipase: HLPL
Insulin ecep o : INSR
Lep in: LEP
Lep in ecep o Ob-Rb: Ob-Rb
F ee a y acids: FFA
Clus e o di e en ia ion 36: CD36
Fa y acid binding p o ein 4: FABP4
Acyl-CoA syn hase: ACS
Glyce ol-3 phospha e: glyce ol-3P
Ace yl-CoA ca boxylase: ACC
Fa y acid syn hase: FAS
Adipose TG lipase: ATGL
Abb e ia ions
24
Ho mone sensi i e lipase: HSL
Pe ilipin 1: PLIN1
Ca eolin 1: CAV-1
Janus kinase: JAK
Signal ansduce and ac i a o o ansc ip ion: STAT
Low-molecula -weigh lep in ime : LMW
Medium-molecula -weigh lep in hexame : MMW
High-molecula -weigh lep in complex: HMW
Phosphoenolpy u a e ca boxykinase: PEPCK
Glucose-6-phospha ase: G6P
AMP-ac i a ed p o ein kinase: AMPK
Be a cell: β-cell
Wais ci cum e ence: WC
Blood p essu e: BP
Sys olic blood p essu e: SBP
Dias olic blood p essu e: DBP
Non-alcoholic s ea ohepa i is: NASH
Neu ological diso de s: ND
Endoplasmic e iculum: ER
Lipase ma u a ion ac o 1: LMF1
Sel-1 supp esso o lin-12-like: Sel1L
Hepa an sul a e p o eoglycans: HSPG
Glycosylphospha idylinosi ol (GPI)-ancho ed glycop o ein 1: GPIHBP1
Apolipop o ein A5: ApoA5
Angiopoie in-like p o ein 3: Angp l3
Abb e ia ions
31
Glycop o ein ( ansmemb ane) nmb: Gpnmb
Ch oma in inmunop ecipi a ion: ChIP
Homeos a ic model assessmen o insulin esis ance: HOMA-IR
Pos p andial iglyce ides: Pos TG
Glu ama e-Oxaloace a e T ansaminase: GOT
Glu ama e-Py u a e T ansaminase: GPT
Gamma Glu amyl T anspep idase: GGT
C-Reac i e P o ein: CRP
P o einase K: PK
E2F ansc ip ion ac o 1: E2F1
T ansc ip ion S a Si e: TSS
Lean NG: Lean no moglycemic
MO NG: Mo bid obese no moglycemic
MO PD: Mo bid obese p ediabe ic
Pa a hy oid ho mone: PT
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!INTRODUCTION
In odu ion
35
1. OBESITY
Nowadays, obesi y has become one o he g ea es causes o heal h diso de s in
de eloped coun ies. Mo e han a hi d o he wo ld popula ion su e s o o e weigh o
obesi y, being expec ed o ise up o 38% o o e weigh and 20% o obesi y by 2030,
eaching he 85% o o e weigh /obese people in he USA (1).
Obesi y is de ined as an excess o body weigh , which unde lines wi h an accumula ion
o adiposi y (adipose issue). Obesi y is usually in e p e ed by he Body mass index
(BMI), measu ed as a a io weigh /heigh 2 (2). Though, subjec s a e mos ly classi ied
acco ding o his index as lean o no mal-weigh subjec s (when BMI<25), o e weigh
subjec s (when BMI≥25 and <30), obese subjec s (BMI≥30 and <40) and mo bid obese
subjec s (BMI≥40) (3,4).
The excess o adiposi y is usually accompanied by a se ies o me abolic dis u bances
and a highe isk o su e ing ce ain diseases (1). Obesi y inc eases he isk o being
diabe ic be ween 4.9% and 9%, and in e sely 60% o 90% o all diabe ics a e o ha e
been obese (5). In ac , al hough ba ia ic su ge y has as i s consequence he weigh
loss, is also one o he mos e ec i e ea men o imp o e diabe es and me abolic
disease, demons a ing he close ela ionship be ween BMI and diabe es (6). Al hough
he connec ion be ween bo h, obesi y and diabe es, is no o ally clea , mechanisms in
which a e in ol ed p o-in lamma o y cy okines ( umo nec osis ac o (TNF),
in e leukin 6 (IL6)), insulin esis ance (IR), de anged a y acid me abolism and cellula
p ocesses such as e iculum s ess and mi ochond ial dys unc ion ha e been shown o
be in ol ed (7).
Obesi y has also been widely associa ed o ca dio ascula disease (CVD) (8). I has
been shown ha an inc ease in a uni o BMI is associa ed o a 4% inc ease in he isk
o ischemic s oke and 6% inc ease in hemo hagic s oke (9). Inc ease in BMI was also
In odu ion
36
associa ed o an inc ease in blood p essu e, which was aduced o a 12% inc ease in
co ona y hea disease and 24% inc ease isk o s oke (9). O he s udy showed an
inc ease in subclinical ca dio ascula disease associa ed o a ise in BMI compa ed o
lean subjec s (10). Obesi y was posi i ely associa ed wi h a 1.17-1.28 ela i e isk
inc ease in co ona y a e y calcium, 1.45 ela i e isk inc ease o common ca o id
a e y in imal medial hickness, 1.32 ela i e isk inc ease o in e nal ca o id a e y
in imal medial hickness and 2.69 ela i e isk inc ease in le en icula mass (10).
A he oscle osis has also been associa ed o obesi y in a p ocess in which adipose issue,
low-g ade in lamma ion, oxida i e s ess, impai ed au ophagy and gu mic obio a
among o he ac o s could be ela ed (11,12).
I is clea ha cance is one o he g ea es heal h conce ns in mode n socie ies. Obesi y
has been s ongly associa ed o se e al cance s, and wha is mo e, has been p oposed as
he second bigges cause o cance in he wo ld only su passed by smoking (13). Thus,
he es ima ion is ha o e 20% o all cance s a e caused o excess body weigh , ising
o 50% in pos menopausal women (14). Mo eo e , obesi y also a ec s dea h a es in
cance , up o 20% o dea h in women and 14% o dea h in men ha can be a ibu able
o obesi y (14).
Finally, obesi y has adi ionally been associa ed o wha is known as me abolic
synd ome (Me S). Me S is de ined by a clus e o me abolic al e a ions which al oge he
inc ease he isk o su e ing diabe es, ca dio ascula disease and cance (15,16).
2. ADIPOSE TISSUE
The e a e di e en adipose issue (AT) depo s wi h di e en me abolic oles (17).
Whi e AT (WAT) is composed by unilocula cells wi h a high capaci y o lipid s o age
in he o m o iglyce ides (TG), since is he issue in cha ge o accumula ing he
In odu ion
37
su plus o ene gy in he o ganism. On he o he hand, B own AT (BAT) is made ou o
mul ilocula adipocy es wi h a high mi ochond ial con en ha can consume high le els
o ene gy when a e s imula ed by ad ene gic signals o cold; he ene gy is used in hea
p oduc ion, a p ocess ha is ca ied ou by he mi ochond ial uncoupling p o ein (UCP)
(17).
The e a e wo ypes o WAT, which a e mainly de ined by hei depo loca ion: he
subcu aneous AT (SAT) and he isce al AT (VAT) (18) (Figu e 1).
Figu e 1. Pa e ns o a dis ibu ion and i s ela ionship wi h BMI, weigh /hip
ci cum e ence and isk o su e ing disease. Pea shape (2) is associa ed o SAT and is
usually e e s as woman a dis ibu ion. Apple shape (3) is associa ed o VAT and is mos
common o men a dis ibu ion. Risk o associa ed ch onic disease such as ca dio ascula
disease o diabe es is display by he ed line in ensi y om he lowes isk o lean subjec s (1)
o he g ea es isk cha ac e is ic o apple a dis ibu ion (3). Abb e ia ions: Subcu aneous
In odu ion
38
adipose issue (SAT); isce al adipose issue (VAT); Body mass index (BMI); weigh /hip a io
(WHR). Adap ed om (19).
Bo h SAT and VAT no only di e by hei loca ion, bu also by hei molecula p o ile,
hei me abolism and hei implica ion in he e iology o he me abolic disease (18,20–
22). Also he emb yonic o igin o bo h a depo s seems o be di e en . While VAT
adipocy es a e hough o ha e a mesode mal o igin, SAT adipocy es come om
di e en emb yonic o igins depending on he exac depo s (mesode m, neu oec ode m),
and in some cases s ill unde e mined wha hei o igin a e (17). Besides, while
expansion o SAT has been long conside ed as a p o ec i e ac o agains me abolic
diso de s, a accumula ion in VAT (cha ac e is ic o cen al obesi y o high wai /hip
a io) has been deemed o p omp me abolic diso de s (19,23). Howe e , he e is an
ongoing discussion abou which issue is i s damaged and which a e hei oles in he
gene a ion o hese diseases (20–22,24). Wha is undoub edly is ha AT has eme ged as
an impo an media o o he whole body homeos asis, and i s ole as an endoc ine
o gan has ocused g ea in e es s.
2.1. Adipose Tissue cellula i y: Adipocy e p ecu so s and adipogenesis
AT is composed by a my iad o cell ypes in which adipocy es, he p edominan cell,
a e in cha ge o lipid accumula ion. Mo eo e , in i s s omal ascula ac ion (SVF)
he e a e mac ophages (which modula e adipose issue unc ion), lymphocy es, ascula
cells, pe icy es, adipose issue-de i ed mesenchymal s em cells (ASCs) (wi h au o-
enew p ope ies and can di e en ia e o p e-adipocy es) all o hem known as s omal
ascula cells (SVC). The adipocy e cell u no e is ca ied ou o ASCs, al hough he
con ibu ion o pe icy es, endo helial cells and non- esiden p ogeni o s wi h
In odu ion
39
hema opoie ic o igin has been p oposed o con ibu e a di e en deg ee depending on
he speci ic a depo and he me abolic condi ion o he subjec (17). Thus, a p ope
balance among all hese ype o cells is needed o a co ec unc ion o AT.
Adipogenesis, he mechanism in which new adipose issue is gene a ed, assu es he AT
u no e and hype plasia (AT expansion by cell numbe inc ease). ASCs ha e been
shown o be able o di e en ia e o a wide ange o cell ypes, including adipocy es,
chond ocy es, os eocy es and miocy es. Al hough p e-adipocy es di e en ia ion o
adipocy es is a well-known p ocess much less is known abou ASCs commi men o
p e-adipocy es. Basically, he di e en ia ion om ASC o ull di e en ia ed adipocy e
equi es o ou s ages: 1) Commi men o ASCs o p e-adipocy es; 2) clonal expansion
o p e-adipocy es; 3) g ow h a es ; 4) e minal di e en ia ion o adipocy es (25). The
commi men o ASC o p e-adipocy e lineage implies se e al ac o s as Sca enge
ecep o class A membe 5 (SCARA5), Bone mo phogene ic p o ein 2 (BMP2), Bone
mo phogene ic p o ein 4 (BMP4), T ans o ming g ow h ac o be a (TGFβ) signaling,
Pla ele -de i ed g ow h ac o ecep o A (PDGFRα), Pla ele -de i ed g ow h ac o
ecep o B (PDGFRβ), Pla ele -de i ed g ow h ac o subuni A (PDGFA), SMAD
amily membe 1 (SMAD1), SMAD amily membe 4 (SMAD4), SMAD amily
membe 5 (SMAD5), SMAD amily membe 8 (SMAD8), Zinc inge p o ein 423
(Z p423), Lysyl oxidase (Lox) o Fib oblas g ow h ac o 2 (FGF2) among o he s (25–
27). TGFβ signaling inhibi s p e-adipocy e commi men , BMP ( om he TGFβ p o ein
amily) exe s gene ally a posi i e e ec o e ASC o p e-adipocy e commi men , and
he PDGF has been p oposed o ha e bo h, adipogenic and an i-adipogenic e ec s.
BMP2 and BMP4 ha e been demons a ed o s imula e he he e odime Smad1/Smad4
(p ocess ha Z p423 is hough o egula e) and he exp ession o Pe oxisome
p oli e a o ac i a ed ecep o gamma 2 (PPARγ2) and Lox ha a e necessa y o p e-
In odu ion
40
adipocy e commi men . Ano he p o-adipogenic e ec di e en om he BMP
signaling is he p o-adipogenic e ec ha FGFs p o okes o e ASCs. Thus, FGF2 has
been shown o s imula e PPARγ2 o e exp ession and exogenous addi ion o FGF2 in
SCV s imula es WAT o ma ion (26,27).
Much mo e is known abou p e-adipocy e di e en ia ion o ma u e adipocy e. The
adipogenesis pa hway is con olled by se e al ansc ip ion ac o s, wi h PPARG as
cen al ac o in his p ocess. Besides, se e al CCAAT enhance binding p o ein
(C/EBP) ac o s a e in ol ed a di e en s ages o adipogenesis. Fi s , an
o e exp ession o C/EBPβ and C/EBPδ akes place, s imula ing he exp ession o
C/EBPα and PPARγ. In u n, bo h PPARγ and C/EBPα can s imula e each o he in a
egula o y loop. E en ually, PPARγ and C/EBPα ac i a e he exp ession o genes
ela ed o adipocy e me abolism, p oducing inal adipocy e ma u a ion (Figu e 2). O he
ac o s ha e been also shown o be impo an in adipocy e di e en ia ion. Fo ins ance,
se e al genes om he K üppel-like ac o s (KLFs) amily a e in ol ed in he
adipogenic pa hway. KLF5 and KLF15 ha e been obse ed o s imula e PPARγ
exp ession in ea ly and e minal di e en ia ion s ages, espec i ely. Con a y, KFL2 has
been shown o ha e an an i-adipogenic e ec by inhibi ing PPARγ exp ession (28,29).
O he ac o ha has been p oposed o play a ole in adipocy e di e en ia ion is he
S e ol egula o y elemen binding ansc ip ion ac o 1 (SREBP1), especially he S e ol
egula o y elemen binding ansc ip ion ac o 1 iso o m c (SREBP1c), which is able o
ac i a e adipogenesis ia PPARγ s imula ion, al hough mice wi h o e esp ession o
nuclea SREBP1c has been shown o su e o lipodys ophy (28).
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3.2. Glucose me abolism
AT no only plays a ole in lipid me abolism bu he closed in e play be ween bo h, lipid
and glucose egula ion, gi es AT an impo an weigh in glucose homeos asis. This is
e iden since AT-speci ic KO-mouse o GLUT4 has been shown o p o oke a ailu e in
sys emic glucose homeos asis leading o insulin esis ance. I has been shown ha
GLUT4 is down- egula ed du ing as ing s a e and up- egula ed du ing pos p andial
s a e. In AT, glucose and speci ically he in e media y me aboli e in glycolysis glyce ol-
3P, is necessa y o he e-es e i ica ion o FFA o TG. When glucose is limi ed (as in
as ing s a es) adipocy es ely on glyce oneogenesis in o de o ob ain he necessa y
sou ce o glyce ol-3P (48).
Mo eo e , some adipokines sec e ed by AT can exe a egula o y e ec o e glucose
homeos asis (48). Thus, LEP has been shown o di ec ly inhibi insulin gene (ISN)
exp ession in β-cells ia he ac i a ion o JAK/STAT signaling. LEP has also been
demons a ed o inhibi insulin sec e ion and o egula e β-cell mass in panc eas (49)
(Figu e 5).
Figu e 5. Schema ic ep esen a ion o he di ec e ec s exe ed by LEP o e glucose
me abolism. LEP inhibi s insulin by down- egula ion o p o-insulin exp ession, inhibi ion o
insulin sec e ion in β-cell and by con olling β-cell mass.
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Adiponec in is ano he molecule sec e ed by AT, wi h an ac i e ole in glucose
homeos asis. This molecule is sec e ed in h ee iso o ms: low-molecula -weigh (LMW)
ime s, medium-molecula -weigh (MMW) hexame s and high-molecula -weigh
(HMW) complexes. Ou o he h ee iso o ms, he HMW has been desc ibed as he mos
ac i e o m. Adiponec in enhances hepa ic insulin sensi i i y and a oid
gluconeogenesis by down- egula ing bo h phosphoenolpy u a e ca boxykinase (PEPCK)
and glucose-6-phospha ase (G6P). The inc eased hepa ic sensi i i y o adiponec in is
hough o occu ia ei he ecep o -media ed ac i a ion o AMPK pa hway o lowe ing
hepa ic ce amide le els (50,51). Apa om he e ec on pe iphe al issues, adiponec in
a ec s β-cells. Thus, adiponec in can p e en β-cells om apop osis, has a p o ec i e
ole agains lipid cy o oxici y and i is hough o s imula e insulin sec e ion du ing
challenged s a es (50).
4. ROLE OF ADIPOSE TISSUE IN METABOLIC DISORDERS
Since obesi y is cha ac e ized by an inc ease o AT mass, he p emise ha adipose
issue may pa icipa e in he e iology and gene a ion o me abolic diso de s usually
associa ed o obesi y came ou long ime ago.
I is known ha AT can deli e a wide ange o molecules, as cy okines, p os aglandins,
adipokines o ee a y acids, which can exe hei ac i i y in o he issues, which in
cases can lead o disease (52–55). The e o e, AT has gained a cen al ole in explaining
me abolic dis u bances and has been ela ed o he appea ance and de elopmen o
diseases like dyslipidemia, hype ension, p oin lamma o y s a es, insulin esis ance,
Me S, diabe es, CVD, s oke, o cance (37,56–59).
Al hough AT abno mali ies has been usually associa ed o obesi y, me abolic
dis u bances also accoun in no mal-weigh subjec s (37), which ag ees wi h he new
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49
ha moniza ion c i e ia o de ine Me S, in which cen al obesi y is no longe a manda o y
ac o (15).
Me S is de ined by a clus e o me abolic al e a ions, which al oge he inc eases he isk
o su e ing diabe es, ca dio ascula disease and cance (15,16) (Figu e 6). Gene ic and
li es yle ac o s ha e been shown o be impo an o he de elopmen and e iology o
Me S (60). Me S ep esen s a se ious p oblem in de eloped coun ies, wi h a high
p e alence (30-40% by he age o 65 yea s) and bo h, i s p e alence and incidence a e
inc easing (61). The appea ance o Me S has been mainly associa ed wi h li es yle
ea u es like physical inac i i y, smoking, alcohol in ake and die . Howe e , gene ic and
epigene ic ac o s a e now eme ging as ac o s o pa amoun impo ance in i s
pa hophysiology (62,63).
Figu e 6. Me abolic synd ome a iables and Me S associa ed diso de s. Me S is composed
by a leas he al e a ion in 3 o he ollowing pa ame e s: HDL, TG, glucose, blood p essu e
and WC. Some o he pa ame e s a e sex dependen (as HDL o WC) and aze dependen (as
WC, he showed alues a e o wes coun ies). The Me S pa ame e s a e ep esen ed acco ding
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50
o he es ablished in (15). Abb e ia ions: Me abolic synd ome (Me S); wais ci cum e ence
(WC); blood p essu e (BP); sys olic blood p essu e (SBP); dias olic blood p essu e (DBP);
high-densi y lipop o ein (HDL); iglyce ides (TG); Ca dio ascula diseases (CVD); Non-
alcoholic s ea ohepa i is (NASH); Neu ological diso de s (ND).
4.1. Me abolic ac o s ela ed o me abolic diso de s
4.1.1. Lipop o ein Lipase (LPL)
LPL plays a key ole in lipid me abolism (64) by hyd olyzing iglyce ide- ich
lipop o ein (CM o VLDL) o ee a y acids, ha can be hen inco po a ed in o he AT
o s o age o ene gy u iliza ion in o he issues (64,65) (Figu e 7). LPL is syn he ized
and sec e ed o he endoplasmic e iculum (ER) whe e is olded wi h he help o Lipase
ma u a ion ac o 1 (LMF1). LPL/LMF1 o m a complex wi h Sel-1 supp esso o lin-
12-like (Sel1L) and his LPL/LMF1/Sel1L complex helps o s abilize LPL homo-dime s,
he ac i e o m o LPL, and allows LPL exi s he ER (65).
A e being sec e ed, LPL is bound o Hepa an sul a e p o eoglycans (HSPG) a he
su ace o he cell. HSPG no only se es as an ancho age molecule bu also con ibu e
o LPL ansloca ion o he endo helial su ace and ac s as co ac o o LPL ac i i y.
Ano he ac o , he GPIHBP1 (which is a Glycosylphospha idylinosi ol (GPI)-ancho ed
glycop o ein belonging o he lymphocy e an igen 6 amily) has been shown o play an
impo an ole in LPL anspo a ion om he in e s i ial space o he luminal space a
he endo helial su ace. Lack o his ac o has been obse ed o impai ed ansloca ion
o LPL o he endo helial su ace p o oking hype iglyce idemia. Besides, i has been
shown ha GPIHBP1 is able o ac o e LPL ac i i y by keeping he ca aly ic domain o
he enzyme un olded (65).
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Figu e 7. Schema ic o e iew o LPL sec e ion and ancho age o EC su aces. LPL o ms a
dime in he endoplasmic e iculum and is hen sec e ed. LPL is bound o HSPG in he su ace
o he adipocy e and hen ansloca ed o GPIHBP1. LPL hyd olyze TG om T iglyce ide- ich
lipop o eins as CM and VLDL p oducing FFA ha a e anspo ed o he adipocy e whe e a e
e-es e i ied gi ing TG o be s o ed in LD. Abb e ia ions: Endo helial cells (EC); hepa an
sul a e p o eoglycans (HSPG); iglyce ides (TG); chylomic ons (CM); e y-low densi y
lipop o eins (VLDL); ee a y acid (FFA); lipid d ople s (LD).
Gi en he impo ance o LPL in lipid homeos asis he enzyme unde goes a igh
egula ion a all le els. Thus, some ac o s ha e been pos ula ed o con ol LPL ac i i y
like ApoCI/II/III, ApoA5, angiopoie ins and ho mones. ApoCII, which is ca ied in CM,
has been shown o be a s imula o o LPL ac i i y, while ApoCI/III a e inhibi o y
ac o s. ApoA5 ha is p esen in VLDL, HDL and CM o a less ex end has been
desc ibed o s imula e LPL ac i i y, al hough he e ec seems o be weake han he
posi i e ac ion ca ies ou by ApoCII. Angiopoie in-like p o ein 3 (Angp l3) has been
desc ibed o inhibi LPL ac i i y as well as by enhancing i s clea age by p o-p o ein
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52
con e ases. Angp l4 is p oduced in he li e and AT and has been shown o inc ease
wi h as ing, causing an inhibi o y e ec o e LPL. I is hough ha inhibi s LPL
dime iza ion and educes LPL a ini y o GPIHBP1. Ano he membe o angiopoie ins,
Angp l8 has also been desc ibed o inhibi LPL ac i i y (65). I has been obse ed ha
insulin and glucose can egula e LPL. Thus, insulin leads o a aise o LPL mRNA
le els, while glucose can ac i a e LPL ac i i y by glycosyla ion in i s ca aly ic si e (66).
O he ho mones o de i a i e ho mones like P os aglandin E2 (PGE2), 7-β-
hyd oxycholes e ol and 25-hyd oxycholes e ol has been shown o exe an inhibi o y
e ec o e LPL p oduc ion in mac ophage cul u es. Mo eo e , LPL gene exp ession
has been shown o be egula ed by miRNAs such miR-27 and miR-29 in AT (65). De-
egula ion o any o hese ac o s can lead o de- egula ion o LPL exp ession o i s
ac i i y, which in u n migh p omo e lipid me abolic ailu e. By con as , he
unde s anding o he egula ion o LPL can also gi e clues o new ea men app oaches
based on he inhibi ion o s imula ion o hese ac o s. Fo ins ance, some molecules
such as s a ins, ib a es, nico inic acid, Eze imibe o O lis a ha e been used o ea
hype iglyce idemia by mean o hei s imula o y e ec o e ApoCII, while o he
ea men as ib a es ha e been desc ibed o inhibi ApoCIII, he e o e p o oking LPL
ac i i y enhancemen (65).
Indeed, AT is he main TG s o age issue. LPL mRNA inc eases du ing he adipocy e
di e en ia ion, being one o he ea lies ma ke s de ined in he p eadipocy e lineage
(67). On he o he hand, AT LPL mRNA exp ession has been nega i ely associa ed o
he BMI (58). As we s a ed be o e, LPL ac i i y in AT has been ela ed wi h
hype iglyce idemia (68). Thus, lowe ac i i y o LPL can esul in g ea e le els o
plasma TG, which in u n can be accumula ed in o he issues causing insulin- esis ance
(69).
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4.1.2. Low-densi y lipop o ein ecep o - ela ed p o ein 1 (LRP1)
LRP1 is a membe o he LDL ecep o amily ha egula es lipid and glucose
me abolism in he li e and AT (70). LRP1 is in ol ed in CM- emnan s, insulin
ecep o a icking and egula ion, and glucose me abolism, he e o e being ela ed o
a he oscle osis (i is an a he op o ec i e ac o ) and diabe es (71). LRP1 ansloca ion o
he cell su ace a e glucose and insulin s imula ion has been desc ibed in adipocy es.
Mo eo e , his LRP1 induc ion by insulin and glucose is accompanied by an inc ease in
he up ake o CM- emnan s by adipocy es (71). In ac , i has been demons a ed ha
LRP1 a e con ained a GLUT4 ich esicles and is deli e ed a e insulin s imula ion
al oge he . I has been shown ha adipocy e-speci ic LRP1-knockou mice p o okes a
dec ease o GLUT4, Insulin- egula ed aminopep idase (IRAP) and so ilin exp essions,
and ha his h ee ac o s oge he wi h LRP1 o m a complex ha is in ol ed in
GLUT4 a icking. Thus, LRP1-deple ed 3T3-L1 adipocy es p esen s up o 50%
dec ease in glucose up ake, demons a ing he impo ance o LRP1 in he e iology o
insulin esis ance (71). Adipose-speci ic LRP1-knockou in mice has also been ela ed
o AT dys unc ion (wi h he s imula ion o CD68, MCP1/CCL2, IL6 and TNF)
p omo ing a he oscle osis (72). VAT LRP1 mRNA has been desc ibed o be
o e exp essed bo h, in mice ed wi h high a die (HDF) compa ed o no mal die and
in obese people compa ed o no mal-weigh subjec s (73).
4.1.3. Glucose anspo e ype 4 (GLUT4)
GLUT4 in AT is he a e-con olling s ep in insulin-media ed glucose disposal, being i s
le els diminished in insulin esis ance s a e and obesi y. Glucose is necessa y as a
eliable sou ce o glyce ol-3P (a glycoly ic in e media y) ha is used o e-es e i ied
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F ee a y acid (FFA) aken om he blood s eam o o m TG (48). A down- egula ion
o GLUT4 could impai his sou ce o glyce ol-3P o wha glyce oneogenesis eme ges
as an impo an pa hway o TG o ma ion. In his pa hway, lac a e o py u a e en e s
he T ica boxylic acid cycle (TCA cycle) ha p oduces oxaloace a e, which is hen
ans o med o phosphoenolpy u a e by he cy osolic enzyme Phosphoenolpy u a e
ca boxykinase (PEPCK). Phosphoenolpy u a e is hen con e ed o glyce ol-3P by
e e se glycolysis. A huge in ake o FFA in subjec s wi h insulin- esis ance could
o e whelmed his pa hway, hinde ing FFA es e i ica ion and leading o FFA
accumula ion in pe iphe al issues con ibu ing o insulin esis ance wo sening.
Thiazolidinediones (TZDs) is a po en insulin sensi izing used o ea diabe es. This
enzyme can up- egula e glyce ol kinase and PEPCK ac i i y, igge ing in an
imp o emen in FFA emo al by inc easing FFA es e i ica ion in TG and esul ing in
an inc ease o insulin sensi i i y (48).
4.1.4. Pe oxisome p oli e a o -ac i a ed ecep o s (PPARs)
PPARγ is a key ac o o adipocy e di e en ia ion. Adipose-speci ic PPARγ knockou
mice, has been ca ied ou . In his model, he e was an impai ed AT expansion,
p esen ed adipocy e hype ophy, and highe le els o plasma ic FFA. Besides, hese
mice we e mo e sensi i e o insulin esis ance and li e s ea osis induced by HFD.
When hese mice we e ea ed wi h TZDs (an i-diabe ic d ug ha has PPARγ as a a ge )
li e insulin esis ance emi ed al hough no o he e ec s we e obse ed (74). O he
membe o PPAR ecep o amily, PPARα, is a a ge o he ib a es, molecules ha a e
used o he ea men o hype iglyce idemia. PPARα is a ansc ip ion ac o ha is
in ol ed in a y acid oxida ion in issues wi h a high le el o a oxida ion and
pe oxisomal me abolism (li e , hea muscle o b own adipose issue). In AT, PPARα
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has been shown o a enua e adiposi y and o s imula e he sec e ion o adiponec in,
which is an insulin-sensi izing ho mone (75). Thus, 3T3L1 p e-adipocy e line and mice
ea ed wi h PPARα agonis s ha e been shown o induce adipogenesis, and o inc ease
a oxida ion by di ec induc ion o PPARα o e adipogenic and a oxida ion genes (75).
Bo h membe s o he PPAR amily, PPARγ and PPARα ca y ou hei unc ion as
he e odime wi h he Re inoid X ecep o alpha (RXRα) (76). Thus, i has been
desc ibed ha a adipocy e-speci ic RXRα-knockou mice can cause adipogenesis ailu e,
and canno inc emen AT e en a e HFD adminis a ion (77,78).
4.1.5. S e ol egula o y elemen binding ac o s (SREBFs)
SREBFs a e ansc ip ion ac o s known o con ol choles e ol and a y acid
biosyn hesis, as well as adipogenesis in AT. SREBF1 has wo iso o ms, he SREBF1c
( he mos exp essed in AT wi h a key ole in adipogenesis) and SREBF1a. These
iso o ms a e de e mined by he ansc ip ion s a ing si e (79). Al hough he di e en
iso o ms o SREBF p esen some unc ional o e lap, SREBF1c has a mo e p e alen
ac ion in lipid biosyn hesis while SREBF2 has a p e alen ole in choles e ol
biosyn hesis. By con as , SREBF1a has a ole in bo h, a y acid and choles e ol
biosyn hesis (80–82). SREBF1c and SREBF2 ha e lowe gene exp ession le els in
VAT o obese people espec o lean people. I has al eady been shown ha his
exp ession is ees ablished o lean-like alues a e ba ia ic su ge y in obese people
(83). Fu he mo e, SREB1c exp ession in AT has been demons a ed o espond o ood
and calo ic in ake. Thus, ed can p omo e SREBF1c down- egula ion while as ing and
calo ic es ic ion ha e been shown o s imula e AT SREBF1 exp ession (79). SREBF1
o e exp ession is esponsible o he oxida i e s ess imp o emen obse ed in calo ic
es ic ion die s (79). In he o he way a ound, oxida i e s ess is capable o inhibi ing
In odu ion
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heal hy AT expansion h ough he supp ession o he SREBF1c-media ed lipogenic
pa hway (84). In animal models, i has been demons a ed ha SREBF2 p o ein in
adipocy e hype ophy is ac i a ed, which leads o an inc ease in he p oduc ion o
cheme in, and adipokine posi i ely ela ed o Me S (85).
4.1.6. S ea oyl-CoA-desa u ase (SCD)
SCD is a key enzyme ha ca alyzes he a e-limi ing s ep o monounsa u a ed a y
acids (MUFFAs) om sa u a ed a y acids, specially om s ea oyl-CoA and palmi oyl-
CoA, gi ing as a esul olea e and palmi olea e (86,87). I is long known ha
polyunsa u a ed ee a y acids (PUFFAs) exe s an inhibi o y e ec o e SCD gene
exp ession (87). I has been p o en in mice, ha a lack o SCD lowe s adiposi y by he
inc ease in he me abolic a e, he he mogenesis and he β-oxida ion, and a dec ease in
he lipogenesis pa hway (87).
4.1.7. Li e X ecep o be a (LXRb)
LXRb is a ansc ip ion ac o ac i a ed by ligand, which belongs o he nuclea ecep o
amily and ha is in ol ed in gluconeogenesis, choles e ol and in lamma ion (88).
LXRb is ac i a ed upon choles e ol b eakdown p oduc s oxys e ols and hen dime ize
wi h i s pa ne RXRα o ca y ou i s unc ion (89,90). This biological unc ion is
mainly ela ed o he lipogenesis pa hway, being o ins ance Fa y acid syn hase (FAS)
and Ace yl-CoA ca boxylase (ACC) some o he a ge genes implied. O he a ge
genes o LXR in ol e he choles e ol anspo e s ATP binding casse e anspo e s A
(ABCA) and G (ABCG), he SREBF1c ansc ip ion ac o , and he key a e-limi ing
enzyme o he biosyn hesis o monounsa u a ed a y acids, he SCD (89). Thus, LXR
ac i a ion has been shown o ha e bene icial e ec s in me abolism by: 1) inc easing
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in lamma ion indi ec ly ia lipo oxici y, bu a di ec modula ion o insulin pa hway in
he adipocy e has been desc ibed. This e ec o e insulin signaling implies he
inhibi ion o he kinase ac i i y by he insulin ecep o (IR), ia he inac i a ion o he
Insulin ecep o subs a e 1 (IRS1) and by he des abiliza ion o he in e ac ion
IR/ca eolin 1 (CAV1) (109) (Figu e 11).
Figu e 11. TNF signaling on AT. TNF in e ac s wi h i s ecep o TNFR1 which can induce
apop osis, ce amide p oduc ion, lypolisis, ER oxida i e s ess, mi ochond ial dys unc ion and
al e ed adipokine p o ile, ac o s ha can e en ually lead o inhibi ion o insulin signaling and
he e o e o insulin esis ance. TNF migh also ac h ough TNFR2 which could also p o oke an
induc ion o apop osis and a dec ease o insulin sensi i i y by down- egula ion o GLUT4 and
IRS-2 (47).
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Since he low-g ade in lamma ion has been associa ed o me abolic disease, ea men s
agains TNF and/o i s pa hway could be sui able o ea diso de s associa ed wi h his
ch onic in lamma ion, impai ed glucose ole ance and dyslipidemia (47). I has also
shown ha TNF co ela es posi i ely wi h he size o he adipocy es, which in u n has
been ela ed o he gene a ion o me abolic diso de s (109,110). Gi en he di ec and
indi ec ac ion o TNF o e he biology o AT and he lipid and glucose me abolism, i
is no su p ising he ele an ole his ac o could ha e in he e iology o he Me S and
i s complica ions (111).
5. ADIPOSE TISSUE AND COLORECTAL CANCER
AT can deli e molecules and p omo e me abolic s a es, which in u n can a ec o he
issues and p omo e disease. In his sense, he possible ole o AT in he de elopmen o
some kind o cance s has gained in e es . As i is desc ibed p e iously, obesi y and AT
dys unc ion has been ela ed o a ch onic low-g ade in lamma ion (112). In connec ion
wi h his, ch onic in lamma ion is well known o be a isk ac o o de eloping cance
including colo ec al cance (CRC) (113,114). In his manne , se e al s udies ha e no ed
he ela ionship be ween CRC and low g ade in lamma ion (115). CRC subjec s p esen
a dys unc ional AT, which migh be a key con ibu o o he in lamma o y s a e h ough
he sec e ion o se e al p oin lamma o y ac o s such as TNF, IL-6, and NFκB pa hway
(116).
In lamma o y p ocesses could no be he only ela ionship be ween AT and CRC.
Me abolic de e io a ion has been also desc ibed o inc ease he cance incidence,
including CRC (117–120). Thus, he ela ionship be ween in lamma ion and me abolic
de egula ion has been desc ibed. Fo ins ance, N κB p o-in lamma o y pa hway has
been desc ibed o be a p omo e o in lamma ion in AT, leading o me abolic diso de s
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(121). In his line, s a egies based on he dis up ion o N κB ac ion has been p oposed
o amelio a e diabe es, hype glycemia and insulin esis ance (121).
Gi en his ela ionship be ween in lamma ion and CRC, and he capaci y o AT o
gene a e low-g ade in lamma ion, i is o in e es he s udy o he possible mechanisms
ha lead o his in lamma ion in AT. Besides, i would be o g ea in e es he sea ch o
he apeu ic app oxima ions o a oid his p o-in lamma o y s a e in AT. In his sense,
me o min, one o he ea lies d ugs used o ea diabe es, has been desc ibed o inhibi
in lamma ion in a p ocess in which N κB is in ol ed. Me o min has been shown o
amelio a e adipose issue in lamma ion, and o p omo e mac ophage pola iza ion o he
an i-in lamma o y M2 pheno ype (122). In addi ion, a lowe isk o su e ing cance
disease has been desc ibed in subjec s who unde go me o min ea men (123,124).
A po en an i-in lamma o y molecule is i amin D (VD) (125–127). VD was i s
iden i ied by i s ole in s imula ing in es inal calcium in ake and bone mine aliza ion,
being low le els o his i amin ela ed o dec eased bone mine al densi y and
os eopo osis (128). VD is syn he ized om 7-dehyd ocholes e ol in he skin in a
eac ion ca ied ou by UVB ligh , gi en as a esul he p e- i amin D, which is u he
con e ed by hea o VD. This o m is no ac i e ye , and VD is u ned in o 25-
hyd oxy i amin D (25(OH)D) by CYP27A1, hyd oxyla ion ha occu s mainly in he
li e . 25(OH)D is he majo o m o se um i amin D and i s le els ha e been obse ed
o be a good indica o o VD s a us. This o m is hen u he con e e in he kidney o
he ac i e o m, 1,25-dihyd oxy i amin D (1,25(OH)2D) by CYP27B1. Kidney
CYP27B1 exp ession is s imula ed by pa a hy oid ho mone (PTH) and down- egula ed
by FGF23 and i s own p oduc 1,25(OH)2D (129). 1,25(OH)2D s imula es i s own
deg ada ion by he 24-hyd oxylase CYP24A1, which ca alyzes he con e sion o
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25(OH)D and 1,25(OH)2D o calci oic acid and o he inac i e me aboli es (129,130)
(Figu e 12).
Figu e 12. Vi amin D me abolism om p e- i amin D o he ac i e o m 1,25-dihyd oxy
i amin D.
VD ecep o (VDR) has been desc ibed o be exp essed in a wide ange o issues and
cell ypes, and i is hough ha he non-classical ac ions o VD (such as inhibi ion o
p oli e a ion, mac ophage modula ion, e minal di e en ia ion s imula ion o insulin
p oduc ion s imula ion) a e exe ed ia i s in e ac ion wi h i (128). Lack o VD,
speci ically he majo plasma o m o VD, he 25(OH)D has been ela ed o an inc ease
in he de elopmen o CRC (131). AT can exp ess p o eins ela ed o VD me abolism
(132), and i has been p oposed ha i can ac as VD s o age issue (133). I has been
shown ha he ac i e o m o VD, 1,25(OH)2D3, is able o modi y adipocy e and AT
physiology ia VDR ac ion (134,135), dec easing he exp ession o p o-in lamma o y
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cy okines in AT (136). The e o e, VD could be o he ac o ha could be in ol ed in
he egula ion o AT in lamma ion and could con ibu e o he biology o CRC.
6. EPIGENETICS
Epigene ics conce ns he in o ma ion con eyed h ough cell di ision and ha no
implies changes a he DNA sequence. Epigene ics shape di e en ia ion p ocesses, and
i is he oo o he di e ences obse ed be ween cellula ypes o o gans (137). The e
a e basically wo epigene ic egula ion landscapes: DNA me hyla ion, which occu s a
cy osines adjacen o guanines (CpG); and his one modi ica ions, which is mo e
a iable and di e se han DNA me hyla ion a CpG (137).
6.1. DNA me hyla ion
DNA me hyla ion is a p ocess in which a co alen me hyl-g oup is added o he ca bon
5 o a cy osine-py imidine ing (5mC) in a CpG nucleo ide. Mos o he DNA
me hyla ions a e sp ead in ansposons and mobile DNA sequences (SINE, LINE, e c),
while DNA me hyla ion a he p omo e o i s exon o genes ep esen s a small
pe cen age o whole DNA me hyla ion in he genome (138). I is hough ha DNA
me hyla ion was i s selec ed e olu iona y as a mechanism o s op he eplica ion o
mobile sequences in he genome (138). Un-me hyla ed CpG can accumula e in he
p omo e o genes, elemen s ha a e called CpG islands. Un-me hyla ed CpG island
would assu e ha ansc ip ion ac o s and he ansc ip ion machine y a e bound o he
igh place in he p omo e (138). A ound 75% o all genes ha e CpG island in hei
p omo e , being suscep ible o DNA me hyla ion con ol (138). While DNA
me hyla ion a CpG islands in he p omo e o genes a e ela ed o gene ep ession,
DNA me hyla ion in he body o he gene (which a e usually hype -me hyla ed) has
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been epo ed o ac i a e gene exp ession, maybe by inc easing he e iciency o he
ansc ip ion (139–141).
DNA me hyla ion is ca alyzed by DNA me hyl- ans e ases (DNMTs). These enzymes
ca alyze he con e sion o un-me hyla ed o me hyla ed CpG in a p ocess in which S-
adenosylme hionine (SAM) ac s as me hyl-dono . In humans, he e a e se e al
DNMTs: DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L. F om hese,
DNMT1, DNMT3A and DNMT3B ha e DNA-me hyl ans e ase ac i i y while
DNMT3L and DNMT2 do no conse e his capaci y e en hough can ei he ac as
co ac o s o he o he DNMTs (in he case o DNMT3L) o me hyla e RNA ( o
DNMT2) (142). DNMT3A and DNMT3B ha e been desc ibed as me hyl ans e ases
“de no o”, which a e in cha ge o es ablishing new DNA me hyla ed ma ks
ubiqui ously and in an un-speci ic way. Once he ma k is es ablished, DNMT1 has been
shown o main ain he DNA pa e n ough he eplica i e cell cycles by ecognizing
hemi-me hyla ed DNA (142,143). Con a y o DNMT1 and DNMT3A/B, DNMT2 has
been demons a ed o me hyla e RNA ins ead o DNA, modi ica ion ha has been
p oposed o a oid RNA agmen a ion and egula ed p o ein ansduc ion. E en hough
is a RNA modi ie , he enzyme e ol ed om DNMTs ha wi h sub le changes inally
acqui ed his new unc ion (142) (Figu e 13).
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Figu e 13. DNA me hyla ion eac ion. DNMTs use as subs a es a cy osine nea a guanine and
S-adenosylme hionine (SAM). In he eac ion, he me hyl g oup is ans e ed o he ca bon 5 o
he cy osine ing, p oducing 5-me hyl-cy osine. As a esul S-adenosylhomocys eine (SAH) is
also p oduced ha can be ecycle o o m SAM. Abb e ia ions: Cy osine (C); DNA-
me hyl ans e ases (DNMTs); 5-me hyl-cy osine (5mC).
DNA me hyla ion is a dynamic p ocess and can be modi ied acco ding o en i onmen al,
gene ics and s ochas ic ac o s. The e o e, a ce ain CpG can be me hyla ed and un-
me hyla ed (144). DNA de-me hyla ion can occu s bo h, by enzyma ic ac ion o by
passi e de-me hyla ion. Passi e de-me hyla ion would ake place in successi e
eplica ions wi h a low ac i i y o he me hyla ion machine y, hough p oducing a
dilu ion o 5-me hyl-cy osine (5mC) ( o med by he ans e s o he me hyl g oup o he
ca bon 5 o he cy osine ing) gi ing as a esul un-me hyla ed DNA (145,146). The
ac i e de-me hyla ion p ocess implies he ac ion o Ten-ele en ansloca ion (TET)
membe amily. These enzymes ha e been epo ed o ca alyze he oxida ion o 5mC o
5-hyd oxime hylcy osine (5hmC) in a p ocess in which molecula oxygen and α-
ke oglu a a e ( ha is con e ed o succina e) a e necessa y. In u n, 5hmC can be u he
oxidized by TET o 5- o mylci osine (5 C), and 5 C o 5-ca boxylci osine (5caC), again
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wi h he pa icipa ion o oxygen and α-ke oglu a a e (145,146). 5hmC has been ela ed
o ac i e genes and is hough o con ibu e o he cell-speci ic se o exp essed genes
obse ed among cellula ypes, al hough he exac mechanism by which exe s his
egula ion is no ully unde s ood (147–149). The es o a ion o hese oxidized o ms o
cy osine can be ca ied ou in a passi e way as well, wi h dilu ion du ing ounds o cell
eplica ion, o in an ac i e way. In he ac i e way, Thymine DNA glycosylase (TDG)
has been shown o excise 5caC and 5 C, a e wha he cy osine is es o ed by he base
excision epai (BER) pa hway (145,146) (Figu e 14). The e o e, unlike he me hyla ion
p ocess, he DNA de-me hyla ion mechanism is a complex pa hway whe e mo e s udies
would be necessa y o expand he knowledge and weigh he impo ance o he di e en
ac o s in ol ed.
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Figu e 14. DNA de-me hyla ion p ocess. A e me hyla ion o cy osine by DNMTs, TET
successi ely oxidizes 5mC o 5hmC, 5 C and 5caC. 5hC, 5 C and 5caC can be dilu ed o
cy osine du ing he ollowing ounds o eplica ion (b oken a ows) in wha is known as passi e
de-me hyla ion. Addi ionally, 5 C and 5caC can be excised by TDG a e wha he cy osine can
be es o ed by he BER pa hway. Abb e ia ions: Cy osine (C); 5-me hylcy osine (5mC); 5-
hyd oxyme hylcy osine (5hmC); 5- o mylcy osine (5 C); 5-ca boxylcy osine (5caC); DNA-
me hyl ans e ases (DNMTs); S-adenosylme hionine (SAM); S-adenosylhomocys eine (SAH);
en-ele en ansloca ion enzymes (TET); α-ke oglu a a e (α-KG); hymine DNA glycosylase
(TDG); base excision epai pa hway (BER).
6.2. His one modi ica ions
DNA in he nucleus is associa ed o his ones in wha is called he nucleosome. A pai o
each H2A, H2B, H3 and H4 his one ypes o ms he nucleosome (Figu e 15). These
his ones a e suscep ible o pos - ansla ional modi ica ions ha can modi ied he
s eng h o he DNA union wi h he complex, a ac o he egula o y ac o s and in u n
egula e ch oma in s uc u e, gene ac i i y and DNA epai . Fo ins ance, his one
ace yla ion o lysine esidues p oduces de blockage o posi i e cha ges, esul ing in a
weake in e ac ion wi h he DNA and making he DNA mo e accessible o o he
egula o y ac o s (polyme ase, ansc ip ion ac o s, e c.) (150–152).
While DNA modi ica ions a e basically educed o 5mC, his ones can be modi ied a
he same deg ee as any o he p o eins, modi ica ions ha mainly ake place in he N-
e minal ail. Tha means, he e is a wide ange o possible modi ica ions such as
me hyla ion, phospho yla ion, sumoyla ion, ubiqui ina ion, glycosyla ion, ace yla ion,
p opionyla ion, bu y yla ion, c o onyla ion o ci ullina ion. Lysines a e he esidue
mo e o en modi ied, al hough modi ica ions in o he amino acids like a ginine, se ine
o h eonine has been epo ed. These modi ica ions ha e been desc ibed o be ca ied
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ou o a se ies o his one modi ie such as his one ace yl ans e ases (HATs), his one
meh yl ans e ases (HMTs) o phospha ases among o he s. In u n, hese ma ks ha e
been shown o be emo ed by p o eins called e ase s, o ins ance his one deace ylases
(HDACs) ( ha emo e ace yl g oups) o his one deme hylases (HDMs) (which emo e
me hyl g oups). Di e en om DNA me hyla ion ha is usually linked o gene
ep ession (al hough as i is discussed he e a e some excep ions), his one modi ica ions
can be in ol ed in gene ep ession, gene ac i a ion, DNA epai , ch oma in s uc u e e c.
Besides, he ole o some modi ica ions a e di icul o unde s and since many o hese
modi ica ions occu s a he same his one and my iads o possible combina ions makes
his one egula o y landscape a complex ield o s udy (150,151).
Figu e 15. Nucleosome and his one modi ica ions. Nucleosome is composed by a pai o each
H2A, H2B, H3 and H4 his one ypes (1), whe e DNA is w apped a ound he s uc u e.
Addi ionally, his ones can su e pos - ansla ional modi ica ions in hei aminoacidic chain. (2)
Summa iza ion o he mos common N- e minal modi ica ions a H2A, H2B, H3 and H4 his one
ypes. Adap a ion om (153).
E en hough i s complexi y, some his one modi ica ions ha e been la gely s udied.
Thus, di-me hyla ion a lysine (K) 4 and i-me hyla ion a K4, K36 and K79 o H3 ha e
been associa ed o ansc ip ion ac i a ion. On he con a y, T i-me hyla ion a K9 and
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pe missi e ch oma in, which gi es o a aise o gene exp ession o genes ela ed o
g ow h o p oli e a ion (152,176). By con as , si uins (class III HDAC) has been
shown o pe o m his one de-ace yla ion in a p ocess in which oxidized nico inamide
adenine dinucleo ide (NAD+) is equi ed. NAD+ is a senso o he me abolic s a us and
i is implied in se e al oxida i e pa hways such us glycolysis, β-oxida ion and he TCA
cycle. NAD+ de iciency in diabe es, aging o in mice ed wi h HFD has been shown o
impai ed si uin ac ion (152). I has been desc ied ha si uin 6 (SIRT6) can be
s imula ed by FFA. Lack o SIRT6 has been shown o up- egula e he glycoly ic
pa hway, leading o a se e e hypoglycemic s a e. Thus, i is hough ha SIRT6
s imula ion h ough FFA ( o example du ing β-oxida ion) could p oduce he
inac i a ion o glycoly ic genes (152) (Figu e 13).
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Figu e 13. In e play be ween me abolism and epigene ic modi ica ions. E ec s o me abolic
in e media ies on his one ace yla ion (A), his one de-ace yla ion (B), and DNA and his one
me hyla ion and de-me hyla ion (C). Adap a ion om (152).
6.4.1. Role o li es yle and nu i ional condi ions in he epigene ics o obesi y and
me abolic disease
Gi en he in e play obse ed be ween epigene ics and me abolism, i is no su p ising
he e o s ha esea che s a e pu ing in ying o unde s and wha could be he ole o
epigene ics in he e iology o he me abolic diseases. In his sense, Du ch amine
( amine ha ook place in Ne he land du ing he 1944-1945 win e a he end o he Wa
Wo ld II) has shed ligh on he nu i ional e ec s du ing in-u e us condi ion o e DNA
me hyla ion and adul hood me abolic disease. Thus, unde -nu i ion condi ion has been
ela ed o BMI and me abolic disease h ough speci ic DNA me hyla ion ma ks a genes
ha egula ed lipid o glucose homeos asis as well as adipogenesis (177). Fo ins ance,
i has been shown in his Du ch amine popula ion ha DNA me hyla ion a
se ine/ h eonine-p o ein kinase pim-3 (PIM3) ( ac o in ol ed in glucose me abolism)
could explain BMI in adul hood. DNA me hyla ion a hio edoxin in e ac ing p o ein
(TXNIP), gene ha egula es β-cell unc ion, and ATP binding casse e sub amily G
Membe 1 (ABCG1), which is in ol ed in lipid me abolism, was able o explain
( oge he wi h o he CpG posi ions) up o 80% o he associa ion obse ed be ween he
amine and he TG le els. Du ing ea ly s age ges a ional amine CpG ma ks nea 6-
phospho uc o-2-kinase/F uc ose-2,6-biphospha ase 3 (PFKFB3) (in ol ed in
glycolysis) and Me hyl ans e ase like 8 (METTL8) (adipogenesis) we e shown o
in luence TG le els as well (177).
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As i is desc ibed abo e, DNA me hyla ion elies on SAM as he me hyl-g oup dono .
SAM le els depend on me hyl-g oup dono nu ien s such as choline, me hionine, ola e,
e c. I has been desc ibed ha ma e nal consump ion le els o hese me hyl-dono s jus
be o e p egnancy and du ing p egnancy can de e mined he DNA me hyla ion le els in
he newbo n o genes ela ed o me abolism and adipogenesis like RXRα, insulin like
g ow h ac o 2 (IGF2) and LEP, as well as o he DNA me hyl- ans e ase DNMT1
(178).
DNA me hyla ion pa e n canno only be es ablished du ing he de elopmen al pe iod,
bu can also be modi ied in adul hood unde se e al condi ions. Fo ins ance, in a
andomized con ol ial whe e subjec s we e exposed o Sa u a ed o Polyunsa u a ed
a y acids (SFA and PUFA, espec i ely) o e eeding du ing 7 weeks, DNA
me hyla ion changes speci ic o SFA and PUFA eme ged. The e we e changes a genes
in ol ed in me abolism and in lamma ion in AT like FTO, INSR, Neu onal g ow h
egula o 1 (NEGR1), Fa y acid binding p o ein 1 (FABP1), Fa y acid binding p o ein
2 (FABP2), PPARG coac i a o 1 alpha (PPARGC1α), Melanoco in 2 ecep o
(MC2R), melanoco in 3 ecep o (MC3R), TNF o IL-6, among o he s. Mo eo e ,
DNA me hyla ion a se e al loci a baseline we e associa ed wi h he pe cen age o
body weigh inc ease a e he ial (179). As well as ood, exe cise has also been ela ed
o DNA changes in adipose issue. In a six-mon h in e en ional s udy, i has been
desc ibed changes in DNA me hyla ion in AT a se e al CpGs o genes which ha e
been associa ed o obesi y, diabe es and adipocy e me abolism such as T ansc ip ion
ac o 7 like 2 (TCF7L2), Po assium ol age-ga ed channel sub amily Q membe 1
(KCNQ1), His one deace ylase 4 (HDAC4) o Nuclea ecep o co ep esso (NCOR)
(180). Li es yle habi s as smoking has been ela ed o changes a DNA me hyla ion in
AT. These changes we e a speci ic loci ha we e in u n associa ed o u u e weigh
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gain and me abolic disease isk a e smoking cessa ion. Fu he mo e, i has been shown
ha a e smoking cessa ion, he DNA me hyla ion smoking-signa u e pa e n had a
longe las ing in luence on DNA me hyla ion han he mRNA pa e n (181).
His one de egula ion has also been ela ed o obesi y, me abolic disease and ela ed
diso de s. Fo example, Plan homeodomain inge wo (PHF2) (which is an his one
deme hylase) has been shown o egula e CEBPα and PPARγ exp ession. Thus, i has
been epo ed ha speci ic Plan homeodomain inge wo (PHF2) knockou mice
display abno mal adipogenesis and a subsequen dec ease in AT mass. Mo eo e , i is
hough ha PHF2 is impo an in he egula ion o se e al me abolic issues being
in ol ed in he me abolism o glucose and lipids (182). A o al epigene ic emodeling a
he AT LEP p omo e has been desc ibed in Die induced obese (DIO) mice eed wi h
n-3 PUFAs. These changes comp ised he p omo e binding inc ease o Me hyl-CpG-
binding domain p o ein 2 (MBD2), DNMTs and se e al HDACs, wi h a subsequen
inc ease o DNA me hyla ion and a dec ease in he ace yla ion o H3 and H4.
Fu he mo e, a dec ease o H3K4me3 was obse ed. All o hese changes would be
in e p e ed as a compensa o y mechanism ying o deal wi h he ex a-ene ge ic
consump ion expe imen ed (183). This s udy poin s ou how he epigene ic s a e o a
ce ain ac o in ol ed in he con ol o ene gy balance can deeply change in adul hood
igge ed o changes in he nu i ional s a us.
As p e iously desc ibed, his one modi ica ions a e s ongly associa ed o me abolism,
and some o he e ase s and w i e s in ol ed ha e been associa ed o me abolic
diso de s (152). Howe e , he e a e no s udies abou he his one ma ks p o ile in human
AT, which could gi e us a be e knowledge abou he ac ual epigene ic s a us and abou
i s possible ole in he e iology o he AT ela ed dis u bances. Conce ning AT, his one
modi ica ions s udies ha e been mainly ca ied ou in cul u es (3T3L1 o p ima y p e-
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adipocy e cul u es) (184,185), and o a less ex end in mice. Thus, i has been epo ed
an inc ease o H3K4me2 en ichmen in db/db mice compa ed wi h db/m a he p omo e
o ATPase, H+ anspo ing, Lysosomal V0 subuni D2 (A p6 0d2), Ma ix
me allopep idase 12 (Mmp12), T igge ing ecep o exp essed on myeloid cells 2
(T em2) and C- ype lec in domain amily 4, membe d (Clec4d) genes, while his ma k
was lowe in Glycop o ein ( ansmemb ane) nmb (Gpnmb) (186).
6.4.2. Epigene ics al e a ions in colo ec al cance
Epigene ic abe a ions in he con ex o CRC ha e been s udied, which could lead o
new sub ypes classi ica ion based on pha macological esponse, and he e o e leading
o new ea men s a egies (187). Indeed, AT has been ela ed o CRC appea ance and
p og ession (188), al hough he ole o AT epigene ic egula ion and i s ela ionship o
CRC has been poo ly s udied. Epigene ic modi ica ions in pe i- umo al AT ha e been
shown o b eas and p os a e cance s. Thus, an al e ed DNA me hyla ion pa e n has
been desc ibed in he su ounding b eas malignan cells, al e a ions ha a e ela ed o
ch omosomal o ganiza ion and wi h ad e se clinical ou come (189). AT DNA
me hyla ion pa e n has also been implica ed no only in he appea ance o me abolic
diseases bu also o he de elopmen o cance by al e ing he me abolism and
inc easing he in lamma o y en i onmen (190). Di e en ia ed DNA me hyla ion has
been epo ed o genes ela ed o lipid me abolism and immune sys em (as Acyl-CoA
dehyd ogenase medium chain (ACADM), Ca ni ine palmi oyl ans e ase 1B (CPT1B),
Ca ni ine palmi oyl ans e ase 1C CPT1C, Fa y acid desa u ase 1 (FADS1),
Monoacylglyce ol O-acyl ans e ase 1 (MOGAT1), Monoacylglyce ol O-
acyl ans e ase 2 (MOGAT2), Solu e ca ie amily 44 membe 2 (CTL2) o TAP
binding p o ein (TAPBP)) in pe i-p os a ic AT o obese and o e weigh e sus lean
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subjec s wi h p os a e cance , which could be ul ima ely con ibu ing o he wo sening
and di e en cance p og ession obse ed in subjec s wi h highe adiposi y (190).
Since epigene ic can be he esul o gene ic, age, issue speci ici y and a gi en
en i onmen al condi ions, i s s udy can poin ou unc ional ac o s o pa hways ha
could be a ec ing o he AT unc ioning and con ibu ing o me abolic disease and o he
diso de s associa ed (as CRC), being a mo e accu a e ool han gene ic s udies (which
gi es us a ixed scena io) o in e his ela ionships (137).
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!HYPOTHESIS
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87
1- Adipose issue DNA me hyla ion a LPL p omo e could be egula ing LPL
mRNA and be ela ed o se um iglyce ide le els, hus pa icipa ing in he
e iology o me abolic synd ome. Besides, hese le els o LPL me hyla ion could
be ela ed o he esponse in iglyce ide clea ance a e a a o e load.
2- O e all DNA me hyla ion s a e o he adipose issue ia LINE-1 as well as he
DNA me hyla ion p omo e egions o genes ela ed o adipogenesis, lipid
me abolism and in lamma ion could be al e ed in me abolic synd ome playing a
ole in he e iology o he me abolic disease.
3- Adipose issue DNA me hyla ion a C3 p omo e could be egula ing C3 mRNA
and be ela ed o se um ASP le els in obesi y, being e ol ed in he highe p o-
in lamma o y s a us and impai ed lipid s o age p esen in ex eme obesi y.
4- Adipose issue H3K4me3 en ichmen a he p omo e o se e al genes ela ed o
adipogenesis, adipose issue me abolism and in lamma ion could be modi ied in
acco dance wi h he obesi y deg ee and me abolic s a us, which could explain in
pa he de elopmen o obesi y and me abolic disease.
5- Low le els o i amin D could be ela ed o adipose issue in lamma ion and
adipose issue DNA me hyla ion in subjec s wi h colo ec al cance , which in
u n could be con ibu ing o colo ec al cance de elopmen .
Resul s
95
MANUSCRIPT 1. Adipose Tissue LPL Me hyla ion is Associa ed wi h
T iglyce ide Concen a ions in he Me abolic Synd ome
Cas ellano-Cas illo D. e al. Clinical Chemis y. 2018;64(1):210-218.
Resul s
96
In o de o ca y ou he objec i e 1, he le els o DNA me hyla ion o se e al LPL-
p omo e -CpG dinucleo ides in a CpG island egion we e analyzed and ela ed o he
gene and p o ein exp ession le els isce al adipose issue (VAT) in indi iduals wi h
(Me S) and wi hou (non-Me S) me abolic synd ome. To pe o m his, VAT samples
we e collec ed om lapa oscopic su gical pa ien s, and le els o LPL mRNA, LPL
p o ein and LPL DNA me hyla ion we e measu ed by qPCR, wes e n blo and
py osequencing. Biochemical and an h opome ic a iables we e analyzed. Mo eo e , a
subse o indi iduals unde wen a die a y a challenge es and pos p andial
iglyce ides we e de e mined.
An h opome ic and biochemical cha ac e is ics o he pa ien s
Table 1A shows he an h opome ic and biochemical pa ame e s o he non-Me S and
Me S pa ien s. As expec ed, he Me S pa ien s had signi ican ly inc eased glucose,
iglyce ides, wais ci cum e ence, sys olic and dias olic blood p essu es, BMI, insulin,
HOMA-IR, o al choles e ol, LDL choles e ol, ApoA1, ApoB and se um lep in le els in
compa ison o he non-Me S subjec s, whe eas HDL choles e ol, ApoA1 and
adiponec in alues we e signi ican ly lowe compa ed wi h he non-Me S subjec s.
Table 1. Biochemical and an h opome ic pa ame e s in non-me abolic synd ome subjec s
(Non-Me S) and me abolic synd ome subjec s (Me S) in he desc ip i e s udy (A) and he
subpopula ion who unde wen he a o e load es (B).
(A) S udy popula ion
(B) Fa o e load
subpopula ion
Non-Me S
(N=70)
Me S
(N=64)
Non-Me S
(n=11)
Me S (n=26)
Age (yea s)
48.0±13.38
49.79±15.0
44.0±7.9
42.6±6.9
Male/Female (%)
51/49
40/60
27/73
48/52
BMI (Kg/m2)
32.2±10.8**
41.5±12.5
51.6±7.3
52.3±6.9
Wais ci cum e ence (cm)
101.5±21.2**
118.7±23.5
137.0±18.2
139.5±17.7
SBP (mm Hg)
124.7±17.6**
139.7±19.7
128.8±19.6
137.6±19.9
Resul s
97
DBP (mm Hg)
76.2±11.6**
82.6±11.1
83.6±11.9
82.6±12.1
Glucose (mg/dL)
92.7±11.9**
116.4±30.7
93.0±7.9
104.4±18.0
Insulin (pmol/L)
12.0±13.6*
17.8±11.4
26.0±27.4
23.2±13.8
HOMA-IR
2.5±2.0**
5.1±3.4
4.3±3.2
6.1±4.1
U ic acid (mg/dL)
4.7±1.3**
5.7±1.3
5.4±1.0
6.1±1.4
TG (mg/dL)
102.7±43.0**
160.1±64.6
105.1±62.0
142.3±53.5
Pos TG (mg/dL)
-
-
177.7±69.6
193.9±72.2
Choles e ol (mg/dL)
194.1±34.9**
212.3±42.1
174.7±48.5
201.5±38.6
HDL choles e ol (mg/dL)
54.5±12.8**
47.1±12.8
50.4±13.8
44.6±10.0
LDL choles e ol (F iedwald)
118.8±30.7*
133.2±32.5
95.6±37.0
124.9±33.6*
ApoA1 (mg/dL)
169.8±25.4*
155.6±26.6
158.3±30.9
148.0±21.6
ApoB (mg/dL)
93.8±22.9*
108.2±22.5
89.5±33.3
104.1±22.9
GOT (mg/dL)
21.0±12.3
20.0±11.5
22.3±8.8
24.8±13.7
GPT (mg/dL)
41.2±22.2
46.3±21.0
46.6±13.9
55.3±20.9
GGT (mg/dL)
56.6±186.5
40.4±27.7
28.6±10.2
34.8±22.9
CRP (mg/dL)
7.4±15.5
5.6±3.4
6.7±6.3
5.2±3.4
Lep in (ng/ml)**
22.5±26.3
47.1±31.3
58.3±22.0
65.8±26.1
Adiponec in (ug/ml)**
12.3±7.2
8.6±4.5
8.7±4.4
7.8±3.3
De ini ions: Body Mass Index (BMI), Homeos a ic Model Assessmen o Insulin Resis ance
(HOMA-IR), Baseline T iglyce ides (TG), Pos p andial T iglyce ides (Pos TG), High Densi y
Lipop o ein (HDL), Low Densi y Lipop o ein (LDL), Apolipop o ein A1 (ApoA1),
Apolipop o ein B (ApoB), Glu ama e-Oxaloace a e T ansaminase (GOT), Glu ama e-Py u a e
T ansaminase (GPT), Gamma Glu amyl T anspep idase (GGT), Sys olic Blood P essu e (SBP),
Dias olic Blood P essu e (DBP), C-Reac i e P o ein (CRP). * P<0.05 and ** P<0.01 conside ed
s a is ically signi ican be ween Non-Me S and Me S in each popula ion (A) and (B).
On he o he hand, Table 1B shows he an h opome ic and biochemical pa ame e s o
he subse o people who unde wen a o e load. Only mean LDL choles e ol was
ound signi ican ly highe in Me S compa ed o non-Me S pa ien s.
Measu es o DNA me hyla ion, LPL mRNA, and p o ein le els in adipose issue
Figu e 1 shows he LPL DNA me hyla ion and mRNA le els. The Me S pa ien s had
signi ican ly highe le els o DNA me hyla ion (P<0.001) (Figu e 1A) and lowe le els
o mRNA gene exp ession (P=0.012) (Figu e 1B) compa ed o he non-Me S subjec s.
Resul s
98
In e es ingly, his associa ion was con i med wi h a co ela ion analysis, which showed
a nega i e co ela ion ( =-0.306, P=0.004) be ween he DNA me hyla ion le els and
mRNA le els o LPL (Figu e 1C).
Figu e 1.- Mean and SE o LPL DNA me hyla ion le els (n=41 Non-Me S and n=46 Me S) (A)
and LPL ela i e mRNA (n=70 Non-Me S and n=64 Me S) (B) in non-me abolic (Non-Me S)
and me abolic synd ome (Me S) subjec s. Figu e 1C shows he co ela ion be ween bo h LPL
me hyla ion le els and LPL mRNA le els.
Finally, we used wes e n blo s o examine he p o ein le el o LPL o assess whe he he
exp ession le els we e ansla ed in o he inal p o ein p oduc s, con i ming a
signi ican ly lowe LPL p o ein exp ession in he Me S pa ien s in compa ison wi h he
non-Me S subjec s (Figu e 2A and Figu e 2B).
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99
Figu e 2.- A e age and SE o LPL p o ein le els quan i ied by wes e n blo in bo h non-
me abolic synd ome (Non-Me S) and me abolic synd ome (Me S) subjec s (n=10).
Associa ions be ween LPL le els and me abolic synd ome
We pe o med a co ela ion analysis o analyze he ela ionships be ween LPL DNA
me hyla ion and he LPL mRNA exp ession le els and he me abolic and
an h opome ic a iables p esen in he s udy subjec s (Table 2). We ound ha he
numbe o Me S componen s, BMI, wais ci cun e ence, glucose, as ing iglyce ide
concen a ions and se um lep in had signi ican and posi i e co ela ions wi h he LPL
DNA me hyla ion le els. Likewise, we ound signi ican nega i e co ela ions be ween
he LPL mRNA exp ession le els and he numbe o Me S componen s, BMI, wais
ci cum e ence, HOMA-IR, glucose concen a ions, baseline iglyce ide concen a ions,
and ApoB (Table 2). Finally, we ound signi ican posi i e co ela ions be ween LPL
mRNA exp ession and he se um concen a ions o HDL choles e ol and adiponec in
le els (Table 2). These esul s a e consis en wi h he ole o he LPL gene in
me abolism.
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100
Table 2. Co ela ions be ween LPL DNA me hyla ion (LPLme ) and LPL ela i e mRNA (LPL
mRNA) and se e al an h opome ic and biochemical pa ame e s.
De ini ions: Numbe o a iables o Me S p esen (Me S a iables), Body Mass Index (BMI),
Homeos a ic Model Assessmen o Insulin Resis ance (HOMA-IR), High Densi y Lipop o ein
(HDL), Low Densi y Lipop o ein (LDL), Log10 o Fas ing iglyce ides (Log10(TG)), Log 10
o pos p andial iglyce ides (Log10(Pos TG)), Sys olic Blood P essu e (SBP), Dias olic Blood
P essu e (DBP), Apolipop o ein A1 (ApoA1), Apolipop o ein B (ApoB). * P<0.05 and ** P
<0.01 conside ed s a is ically signi ican .
LPL me hyla ion
LPL mRNA
Me S a iables (0-5)
0.421**
-0.244**
BMI (kg/m2)
0.344*
-0.239**
Wais ci cum e ence (cm)
0.297**
-0.222*
HOMA-IR
0.175
-0.183*
Insulin (pmol/L)
0.155
-0.165
Glucose (mg/dL)
0.269*
-0.220*
Choles e ol (mg/dL)
0.182
0.035
HDL choles e ol (mg/dL)
-0.068
0.210*
LDL choles e ol (mg/dL)
0.098
0.039
Log10(TG) (mg/dL)
0.246*
-0.259**
Log10(Pos TG)
0.467**
-0.360*
SBP (mm Hg)
0.135
0.057
DBP (mm Hg)
0.111
0.026
ApoA1 (mg/dL)
-0.126
0.130
ApoB (mg/dL)
0.147
-0.301*
Lep in (ng/ml)
0.402**
-0.139
Adiponec in (ug/ml)
-0.039
0.378**
Resul s
101
Epigene ic ac o s associa ed wi h me abolic synd ome
In o de o s udy he ela ionship be ween LPL DNA me hyla ion and he pa ame e s
associa ed wi h Me S, and be ween he me hyla ion and mRNA le els o LPL we
pe o med eg ession analyses. Reg ession model 1 (Table 3 model 1) a e adjus men
o age, gende , BMI and HOMA-IR showed ha he s a is ically signi ican a iables
in p edic ing he inc ease in LPL p omo e me hyla ion le els we e he numbe o
componen s o Me S and he BMI. When we conside ed LPL mRNA as dependen
a iable and adjus ed by age, gende , BMI and HOMA-IR we ound ha LPL
me hyla ion was he only a iable signi ican ly associa ed wi h LPL mRNA a iabili y
(Table 3 model 2).
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102
Table 3. Reg ession analysis wi h LPL DNA me hyla ion (model 1) and LPL mRNA (model 2) le els as dependen a iable.
Model 1: LPL DNA Me hyla ion
(R=0.472; R2=0.223)
Model 2; LPL mRNA
(R=0.382; R2=0.147)
P
95% CI
P
95% CI
Age
0.066
0.584
-0.023-0.041
-0.029
0.814
-0.012-0.010
BMI
0.298
0.037
0.003-0.080
-0.104
0.492
-0.018-0.009
Gende
-0.115
0.302
-1.121-0.383
0.113
0.331
-0.140-0.410
Me S a iables (0-5)
0.349
0.009
0.126-0.837
-
-
-
HOMA-IR
-0.118
0.351
-0.216-0.078
-0.128
0.337
-0.077-0.027
LPL me hyla ion
-
-
-
-0.266
0.025
-0.166-(-0.011)
Independen a iables o model 1 a e Age, BMI, Gende , MS a iables and HOMA-IR; and age, BMI, gende , HOMA-IR and LPL
me hyla ion o model 2. Abb e ia ions: Body Mass Index (BMI), Numbe o a iables o Me S p esen (Me S a iables), Homeos a ic
Model Assessmen o Insulin Resis ance (HOMA-IR), Lipop o ein Lipase (LPL).
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103
Fu he mo e, we used logis ic eg ession analysis o assess he isk o su e ing Me S
isk acco ding o he me hyla ion le els in LPL DNA p omo e . LPL me hyla ion le el
was ound o be he only a iable signi ican ly p edic i e o he Me S s a e (dependen
a iable). In his eg ession, adjus ed by age, gende and LPL mRNA le els, an inc ease
in one uni in LPL me hyla ion esul ed in a mo e han wo- old highe likelihood o
ha ing Me S (Table 4).
Table 4. Logis ic eg ession wi h membe ship o no membe ship o Me S g oup as dependen
a iable, and age, gende , LPL me hyla ion and LPL mRNA as independen a iables. Men a e
used as e e ence gende (0).
NonMe S/Me S
OR (95% CI)
P
Age
1.003 (0.96-1.04)
0.865
Gende
Male
Female
1 ( e e ence)
1.927 (0.73-6.41)
0.158
LPL me hyla ion
2.092 (1.38-3.06)
0.000
LPL mRNA
0.464 (0.23-1.53)
0.282
Abb e ia ions: Lipop o ein Lipase (LPL).
Die a y Fa challenge
We designed a die a y a challenge es in humans o e alua e he ela ionship be ween
pos p andial iglyce ide and LPL me hyla ion le els. Posi i e co ela ions we e ound
be ween he baseline and pos p andial iglyce ide concen a ions and he LPL DNA
me hyla ion le els (Table 2, Figu e 3A and Figu e 3B). Acco dingly, we ound a
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nega i e co ela ion be ween LPL mRNA and bo h, he baseline and pos p andial
iglyce ide concen a ions (Table 2, Figu e 3C and Figu e 3D).
Figu e 3. The igu e shows he co ela ion o LPL DNA me hyla ion wi h he Log o as ing
iglyce ides le els (A) and Log o pos p and ial iglyce ides le els (B). (C) shows he
co ela ion be ween LPL gene exp ession and Log o as ing iglyce ides le els and (D) he
associa ion be ween LPL gene exp ession and Log o pos p and ial iglyce ides le els.
In a linea eg ession model adjus ed o age, gende , BMI and HOMA-IR we ound
ha LPL me hyla ion was he only a iable which explained he pos p andial
iglyce ide concen a ions (Table 5).
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111
Table 3. Pea son’s co ela ion be ween LINE-1 CpG posi ions (P1, P2, P3, P4, P5, P6) and he an h opome ic and biochemical a iables ela ed
o Me S. * p<0.05 and ** p<0.01 we e conside ed s a is ically signi ican .
Me S index
BMI
Wais
Glucose
Tg
HDL-cho
LDL-cho
SBP
DBP
HOMA-IR
LINE-1 P1
-0.167
0.057
-0.031
-0.246*
-0.088
0.113
0.082
0.162
0.02
-0.114
LINE-1 P2
-0.233*
0.025
-0.068
-0.334**
-0.208
0.074
0.028
0.171
0.010
-0.199
LINE-1 P3
-0.136
0.018
-0.011
-0.168
-0.072
-0.115
0.093
0.220
0.155
-0.101
LINE-1 P4
-0.068
0.042
0.012
-0.158
0.039
-0.112
0.077
0.168
0.010
-0.041
LINE-1 P5
-0.137
0.093
-0.037
-0.238*
0.016
-0.139
0.05
0.136
0.100
-0.088
LINE-1 P6
-0.19
-0.055
-0.05
-0.137
-0.166
0.028
0.052
0.066
0.016
-0.126
Abb e ia ions: Numbe o me abolic synd ome a iables p esen in he subjec o s udy (Me S index); Body mass index (BMI); T iglyce ides (TG); High-
densi y lipop o ein choles e ol (HDL-cho); Low-densi y lipop o ein choles e ol (LDL-cho); Sys olic blood p essu e (SBP); Dias olic blood p essu e (DBP);
Homeos a ic model assessmen o insulin esis ance (HOMA-IR); Long in e spe sed elemen 1 DNA me hyla ion a posi ions 1 o 6 (LINE-1 P1-P6). * and **
mean p<0.05 and p<0.01 espec i ely acco ding o Pea son’s co ela ion.
Resul s
112
Gene speci ic DNA me hyla ion in Me S e sus Non Me S
Adipogenic and lipid me abolism ac o s
We s udied genes ela ed o adipose issue de elopmen , as PPARA, PPARG and hei
he e odime pa ne RXRA. The e we e no di e ences a any o he CpG si es included
o PPARA, PPARG and RXRA (Figu e 1). Ne e heless, a endency o highe le els
o DNA me hyla ion in PPARA o Me S subjec s han in Non Me S was obse ed.
Resul s o he associa ion analyses showed a posi i e co ela ion be ween PPARA P2
wi h Me S index, TG le els and HOMA-IR. PPARG P1 co ela ed posi i ely wi h BMI,
while PPARG P1 and P3 we e nega i ely associa ed o DBP. In he case o he PPAR’s
pa ne RXRA, we ound a nega i e co ela ion be ween RXRA P1 wi h BMI and wais
ci cum e ence.
Resul s
113
Figu e 1. Adipogenic ac o s DNA me hyla ion le els. DNA me hyla ion p o ile ac oss he
CpG analyzed a he p omo e s o he adipogenic ac o s PPARA (A), PPARG (B) and he
PPARs pa ne RXRA (C) in bo h, Non Me S and Me S g oups. Values a e gi en as he
mean±SE. Pe oxisome p oli e a o -ac i a ed ecep o alpha (PPARA); Pe oxisome p oli e a o -
ac i a ed ecep o gamma (PPARG); Re inoid X ecep o alpha (RXRA).
Fu he mo e, a se o CpG si es inside genes ela ed only o lipid me abolism was also
py osequenced. No di e ences we e ound a any o he CpGs analyzed o SREBF1
and SREBF2 egula o s (Figu e 2A and Figu e 2B, espec i ely). The e we e no
signi ican DNA me hyla ion di e ences a any o he LRP1 CpG si es s udied ei he
(Figu e 2C). In he case o LPL, we ound an inc ease o DNA me hyla ion o he CpG
si ua ed a he posi ion 2 (LPL P2) (Figu e 2D). We did no ind di e en le els o
DNA me hyla ion a any o he CpG s udied o SCD and LXRB genes (Figu es 2E and
2F).
Fo hese genes, we obse ed ha Me S index co ela ed nega i ely wi h SCD P6, while
SCD P3 was nega i ely associa ed o BMI. Posi i e associa ions exis ed be ween TG
le els and LPL P3, and be ween HDL-cho and LRP1 P2. Fu he mo e, he e was a
nega i e associa ion be ween he choles e ol egula o SREBF2 and DBP, speci ically
wi h SREBF2 P2.
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Figu e 2. Lipid me abolism DNA me hyla ion. The igu e shows he DNA me hyla ion in Non
Me S and Me S g oups a each CpG o se e al ac o s ela ed o lipid me abolism as SREBF1
(A), SREBF2 (B), LRP1 (C), LPL (D), SCD (E) and LXRB (F). Values a e gi en as he
mean±SE. S e ol egula o y elemen binding ansc ip ion ac o 1 (SREBF1); S e ol egula o y
elemen binding ansc ip ion ac o 2 (SREBF2); Low densi y lipop o ein ecep o - ela ed
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115
p o ein 1 (LRP1); Lipop o ein lipase (LPL); S ea oyl-CoA desa u ase (SCD); Li e X ecep o
be a (LXRB). * means p<0.05 acco ding o a S uden ’s T- es .
In lamma ion ac o s
Due o he ela ionship be ween adipose issue and in lamma ion, we analyzed some
ac o s in ol ed in his p ocess. We analyzed 7 CpG si es inside he C3 gene p omo e ,
and we did no ind di e en DNA me hyla ion le els be ween bo h, Non Me S and
Me S subjec s (Figu e 3A). We s udied 5 CpG si es o he umo nec osis ac o (TNF)
as well. In his case, Me S subjec s p esen ed a lowe DNA me hyla ion le els a 3 ou
o he 5 CpG si es ha we e analyzed, conc e ely a posi ion 1, 2 and 3 (TNF P1-P3)
(Figu e 3B). The hi d ac o we s udied was lep in (LEP), in which we analyzed 4 CpG
si es a lep in sequence, which did no p esen signi ican di e ences be ween Non
Me S and Me S subjec s (Figu e 3C).
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116
Figu e 3. In lamma o y p omo e s DNA me hyla ion. Compa isons be ween he Non Me S and
he Me S g oup o DNA me hyla ion a di e en CpG om genes implied in in lamma o y
p ocesses as C3 (A), TNF (B) and LEP (C). Values a e gi en as he mean±SE. Complemen
ac o 3 (C3); Tumo nec osis ac o (TNF); Lep in (LEP). * means p<0.05 acco ding o a
S uden ’s T- es .
On he o he hand, a nega i e ela ionship was ound be ween Me S index and he DNA
me hyla ion le els o TNF P2, TNF P3, TNF P4 and TNF P5 (Table 4). Glucose
co ela ed in a nega i e way wi h he DNA me hyla ion o TNF P4 (Table 4).
Acco ding o iglyce ide le els, he e we e a nega i e co ela ions we e ound wi h
TNF P2 and P5. In e sely o TG, HDL-cho co ela ed posi i ely wi h TNF P1, P2, P5
(Table 4).
The e was also a nega i e co ela ion be ween TNF P4 wi h LDL-cho and DBP.
Fu he mo e, he e we e posi i e and signi ican co ela ions be ween LEP P1 wi h
LDL-cho, SBP and DBP (Table 4).
Resul s
117
Table 4. Co ela ion analyses be ween an h opome ic and biochemical a iables associa ed o Me S wi h some o he DNA me hyla ion a he CpG analyzed.
Only CpG ha p esen ed any signi ican associa ion a e ep esen ed.
Me S V
BMI
Wais
Glucose
Tg
HDL-cho
LDL-cho
SBP
DBP
HOMA-IR
PPARA P2
0.276*
0.076
0.165
0.166
0.392**
0.061
0.08
0.066
-0.025
0.229*
PPARG P1
-0.072
0.306*
0.169
-0.224
-0.194
0.015
-0.197
-0.2
-0.293*
-0.058
PPARG P3
-0.078
0.138
0.174
0.03
-0.139
0.021
-0.218
0.037
-0.283*
0.112
RXRA P1
-0.102
-0.298**
-0.229*
-0.052
0.025
-0.095
0.127
-0.066
-0.225
-0.032
SREBF2 P2
0.056
0.006
0.144
0.112
0.136
-0.032
0.189
-0.224
-0.262*
0.121
LRP1 P2
0.09
-0.065
-0.048
0.114
-0.215
0.373*
-0.055
0.192
0.180
0.251
LPL P3
0.135
0.029
0.089
0.128
0.245*
-0.102
0.085
0.126
-0.111
0.149
SCD P3
-0.056
-0.340*
-0.283
-0.096
-0.018
0.108
0.22
0.087
-0.117
-0.03
SCD P6
-0.325*
-0.116
-0.17
-0.141
-0.134
0.121
0.102
-0.275
-0.232
-0.172
TNF P1
-0.212
0.132
0.046
-0.034
-0.188
0.283*
-0.02
-0.010
-0.115
0.029
TNF P2
-0.420**
0.054
-0.061
-0.192
-0.273*
0.304*
-0.195
-0.188
-0.217
-0.196
TNF P3
-0.320*
0.151
-0.021
-0.094
-0.155
0.222
-0.109
-0.237
-0.242
-0.03
TNF P4
-0.330*
-0.006
-0.096
-0.278*
-0.203
0.098
-0.295*
-0.245
-0.305*
-0.133
TNF P5
-0.281*
0.132
-0.100
-0.153
-0.281*
0.380**
-0.132
-0.097
-0.008
-0.074
LEP P1
0.088
0.081
-0.159
0.061
-0.071
0.015
0.229*
0.264*
0.230*
0.028
Resul s
118
Abb e ia ions: Numbe o me abolic synd ome a iables p esen in he subjec o s udy (Me S
V); Body mass index (BMI); T iglyce ides (TG); High-densi y lipop o ein choles e ol (HDL-
cho); Low-densi y lipop o ein choles e ol (LDL-cho); Sys olic blood p essu e (SBP); Dias olic
blood p essu e (DBP); Homeos a ic model assessmen o insulin esis ance (HOMA-IR);
Pe oxisome p oli e a o -ac i a ed ecep o alpha DNA me hyla ion a posi ion 2 (PPARA P2);
Re inoid X ecep o alpha me hyla ion a posi ion 1 (RXRA P1); Lep in DNA me hyla ion a
posi ion 1 (LEP P1); S e ol egula o y elemen binding ansc ip ion ac o DNA me hyla ion a
posi ion 2 (SREBF2 P2); S ea oyl-CoA desa u ase DNA me hyla ion a posi ions 3 and 6 (SCD
P3 and P6); Tumo nec osis ac o DNA me hyla ion a posi ions P1 o P5 (TNF P1-P5);
Pe oxisome p oli e a o -ac i a ed ecep o gamma DNA me hyla ion a posi ions 1and 2
(PPARG P1 and P2); Lipop o ein lipase DNA me hyla ion a posi ion 3 (LPL P3); Low densi y
lipop o ein ecep o - ela ed p o ein 1 DNA me hyla ion a posi ion 2 (LRP1 P2). * and **
means p<0.05 and p<0.01 espec i ely
Reg ession analyses
To s udy he s eng h o he associa ion obse ed in he co ela ion analyses we
pe o med lineal eg ession analyses co ec ed by age, gende and BMI. We obse ed
ha he DNA me hyla ion le els o PPARA P2 and LPL P3 could explain TG le els
(Table 5).
Table 5. Lineal eg ession analysis wi h as ing iglyce ides as dependen a iable and PPARA
P2, LPL P3 and TNF P2 as independen a iables and co ec ed by age, gende and BMI.
Fas ing iglyce ides (R=0.566; R2=0.320)
β
P
CI 95 %
Age
0.111
0.425
-0.582-1.358
Gende
-0.268
0.047
-48.377-(-.312)
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119
BMI
-0.101
0.446
-1.825-0.816
PPARA P2
0.332
0.012
1.32-10.012
LPL P3
0.264
0.046
0.099-10.72
TNF P2
-0.117
0.347
-1.867-0.669
Body mass index (BMI); Pe oxisome p oli e a o -ac i a ed ecep o alpha DNA me hyla ion a
posi ion 2 (PPARA P2); Lipop o ein lipase DNA me hyla ion a posi ion 3 (LPL P3); Tumo
nec osis ac o DNA me hyla ion a posi ion 2 (TNF P2).
Fu he mo e, we pe o med a logis ic eg ession analyses (ha monized by s ep me hod)
o de e mine wha ac o s could p edic he isk o ha ing Me S. We obse ed ha TNF
P2 emained as a p o ec i e a iable; wi h a educ ion o 23% o p obabili y o being
Me S pe uni o DNA me hyla ion inc eased (Table 6).
Table 6. Logis ic eg ession analysis: isk o Me S. Va iables ha showed a signi ican
associa ion wi h Me S V a he co ela ion analyses such as age, gende , PPARA P2, SCD P6,
TNF P2 and P5 we e in oduced as independen a iables. A ha monized model in which
gende , PPARA P2 and TNF P2 was main ained was gene a ed.
Non Me s/Me S (R2=0.506-0.686)
β
P
CI 95%
Gende
5.813
0.094
0.739-45.699
PPARA P2
1.630
0.246
0.714-3.719
TNF P2
0.791
0.008
0.664-0.942
Non me abolic synd ome g oup (Non Me S); Me abolic synd ome g oup (Me S); Pe oxisome
p oli e a o -ac i a ed ecep o alpha DNA me hyla ion a posi ion 2 (PPARA P2); Tumo
nec osis ac o DNA me hyla ion a posi ion 2 (TNF P2).
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127
B
HOMA-IR (R=0.59, R2=0.35)
p
CI (95%)
Age
-0.00
0.96
-0.06-0.05
Gende
-0.71
0.32
-2.14-0.72
BMI
0.13
0.00
0.04-0.21
C3 mRNA
0.74
0.00
0.26-1.22
C3 me hyla ion
0.07
0.44
-0.12-0.28
Abb e ia ions: HOMA-IR: Homeos asis Model Assessmen o Insulin Resis ance; CI:
Con idence In e al.
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129
MANUSCRITP 4. Ch oma in Immunop ecipi a ion Imp o emen s o
he P ocessing o Small F ozen Pieces o Adipose Tissue
Cas ellano-Cas illo D. e al. PloS One. 2018;13(2):e0192314.
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130
In his sec ion we aimed o accomplish he objec i e 4. In his objec i e, we op imized
he s anda d p o ocol o ch oma in immunop ecipi a ion (ChIP) o small pieces o
ozen human adipose issue. In addi ion, we es ChIP o he his one ma k H3K4m3,
which is ela ed o ac i e p omo e s, and alida e he pe o mance o he ChIP by
analyzing gene p omo e s o ac o s usually s udied in adipose issue using qPCR.
We ha e in oduced c ucial changes o he s anda d ChIP p o ocol, imp o ing he
homogeniza ion, ixa ion and de-c osslinking s eps, allowing enough immunop ecipi ed
ma e ial o be ob ained o pe o m u he s eps, as we demons a ed by es ing
H3K4me3 modi ica ions. Thus, we ha e shown ha he use o only 100 mg o ozen
AT is enough o ChIP es s, which will help o ad ance knowledge abou epigene ic
ma ks o AT and hei signi icance o me abolic homeos asis.
The high lipid con en o he AT makes he ixa ion and subsequen s eps di icul o
wo k wi h. Adipocy es loa in he uppe laye due o hei lipid con en (Figu e 1.1),
which leads o a high loss o issue in he p ocessing. Thus, s anda d homogeniza ion
me hods (mo a and pes le) we e no able o ex ac a p ope quan i y o DNA, showing
a e y low pe o mance. In his me hod, a high quan i y o issue emained s uck o he
su aces o he pes le and mo a ha esul ed in a high issue loss, a e y low nuclei
eco e y and no ch oma in ha es . We he e o e pe o med wo o he al e na i e
me hods whe e he ixa ion and washing o he issue we e p io o he homogeniza ion
s ep. This allowed be e issue manipula ion, indeed a oiding loss o issue. We
compa ed he dounce homogenize (Figu e 1.2) wi h he ul a u ax homogenize
(Figu e 1.3).
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131
Figu e 1. Figu e shows he h ee di e en wo k lows pe o med, using he pes le and mo a
(1.1), he Dounce homogenize (1.2) o he Ul a u ax homogenize (1.3). In (1.1), he
homogeniza ion s ep was pe o med using liquid ni ogen, a e which i was ixed. A e his,
he nuclei we e pelle ed and nucleus lysis bu e was added. Once incuba ed, he sample was
shea ed and he ch oma in agmen a ion and eco e y we e checked. In he o he wo
al e na i e me hods p oposed (1.2 and 1.3), he issue was cu in small pieces (3 mm) and he
ixa ion s ep was pe o med p io o he homogeniza ion. Once homogenized, he nuclei we e
eco e ed by cen i uga ion, nucleus lysis bu e was added and he sample was shea ed and he
ch oma in agmen a ion and eco e y we e checked.
The use o he ul a u ax homogenize esul s in a highe o al DNA eco e y a e he
pu i ica ion s ep (Figu e 2).
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132
Figu e 2. The igu e shows he di e en e iciency in o al DNA eco e y be ween he Dounce
and he Ul a u ax homogenize . 100 mg o ozen adipose issue was ixed in 1%
pa a o maldehyde, homogenized ei he , using he Dounce o Ul a u ax homogenize and
shea ed o 40 cycles (30 seconds ON and 30 seconds OFF). A sample o 50 l o he
homogenized ma e ial was hen aken, and he ch oma in was de-c osslinked using he as
Chelex-100 me hod. To al DNA was ex ac ed and quan i ied by nanod op. Da a a e gi en as
means wi h e o ba s. Abb e ia ions: Dounce, Dounce homogeniza ion me hod; Ul a u ax,
ul a u ax homogeniza ion me hod. (n=6).
Once he op imal homogeniza ion me hod was es ablished, a p ope ixa ion me hod o
AT was ound. Concen a ion o o maldehyde o c osslinking DNA/p o ein is an
impo an s ep, which can a ec he shea ing o he DNA and, consequen ly, he
pe o mance o he immunop ecipi a ion (IP) and he eco e y o DNA. Fo his eason,
we ied wo di e en o maldehyde concen a ions in o de o imp o e DNA eco e y:
he classical concen a ion o 1% o maldehyde was compa ed o a lowe concen a ion
o 0.5% o maldehyde. In addi ion, we also ied di e en incuba ion imes (10, 8, 5
minu es) and empe a u es (RT o 37ºC) o ix he sample, al hough no good esul s
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133
we e ob ained o incuba ion imes longe han 5 minu es and empe a u es highe han
RT (da a no shown). 5 ml o each ixa ion solu ion we e used o ca y ou he ixa ion
s ep o 5 minu es a RT and shaking. Fu he mo e, he sonica ion s ep is highly a iable
depending on he sonica o and he e is e en mode a e a ia ion be ween di e en
de ices o he same echnology. Indeed, i is ecommendable o se up he p ope
shea ing me hod no only o each kind o issue bu also o each de ice. A he same
ime, we also de e mined he sonica ion ime o p ope ly shea he DNA using a
Bio up o sonica o a e issue ixa ion. We es ed 20, 30 and 40 cycles o 30 seconds
ON / 30 seconds OFF a high powe . We ob ained be e esul s using a low
concen a ion o 0.5% o maldehyde oge he wi h a numbe o 40 cycles o DNA
eco e y (Figu e 3A and Figu e 3B). Howe e , 1% o maldehyde hinde ed shea ing o
he ch oma in, independen ly o he numbe o sonica ion cycles.
Figu e 3. 100 mg o ozen adipose issue was ixed in ei he 1% o 0.5% o pa a o maldehyde
and hen homogenized using he Ul a u ax me hod. I was hen shea ed o 40 cycles (30
seconds ON and 30 seconds OFF), a e which a sample o 50 µl o he homogenized ma e ial
was aken, and he ch oma in was de-c osslinked using he as Chelex-100 me hod. To al DNA
was ex ac ed and quan i ied by nanod op. Fixa ion a 0.5% p esen s highe le els o DNA
eco e y (A) a e DNA pu i ica ion and a be e ch oma in shea es ed by elec opho esis in
2% aga ose gel. (B) Compa ison o he use o PBS+1% o PBS+0.5% o maldehyde in he
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134
pe o mance o DNA eco e y a e de-c osslinking and pu i ying he DNA. (n=6). Da a a e
gi en as means wi h e o ba s.
Finally, due o he small pieces and na u e o AT i sel , added o he ac ha i was
ozen, he de-c osslinking and DNA eco e y s eps may be de e minan o he success
o he IP and downs eam p ocedu es. Two me hods o ch oma in de-c osslinking we e
es ed: he s anda d me hod, which consis s o incuba ing he ch oma in a 65ºC o 5
hou s ollowed by p o einase K (PK) ea men a 55º o 1 hou ; and a as e me hod in
which ch oma in is hea ed o 100ºC du ing a sho e pe iod o ime o 10 minu es wi h
10% chelex-100 o p o ec he DNA. Once he DNA was pu i ied, he da a e ealed a
highe pe o mance o he s anda d me hod, in which he ch oma in is submi ed o a
mode a e empe a u e o a long ime (Fig 4).
Figu e 4. 100 mg o ozen adipose issue was ixed in 0.5% pa a o maldehyde and hen
homogenized using he Ul a u ax me hod. I was hen shea ed o 40 cycles (30 seconds ON
and 30 seconds OFF), a e which a sample o 50 µl o he homogenized ma e ial was aken,
and he ch oma in was de-c osslinked using ei he he as Chelex-100 me hod o a mode a e
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135
empe a u e o 5 hou s plus PK ea men . Figu e shows he de-c osslinking s ep a a mode a e
empe a u e o 5 hou s and hen a PK s ep imp o es he quan i y o DNA wi h espec o he
me hod based on he use o Chelex-100. (n=6). To al DNA was ex ac ed and quan i ied by
nanod op. Da a a e gi en as means wi h e o ba s.
The e o e, a e es ing di e en s eps du ing he egula ChIP p o ocol, se e al changes
ha e been in oduced in o de o ma ch he me hod o small ozen AT samples. The
high lipid con en in AT hinde s issue manipula ion, DNA ex ac ion and e en nucleus
elease and nucleus b eakdown. This has led o he de elopmen o specialized
ex ac ion ki s o AT, o example o RNA ex ac ion. Thus, based on ou da a in AT
manipula ion (da a no shown), we decided o inc ease he p opo ion o bu e wi h
espec o he sample quan i y compa ed o egula p ocedu es o he ollowing s eps:
ixa ion, washes a e ixa ion, cell lysis and elease o nuclei. This allowed us o deal
wi h he high lipid con en , a oiding a e y hick cell lysa e, which could hinde nucleus
elease. Fu he mo e, his allowed eco e y o a cleane nucleus pelle , imp o ing he
sonica ion and ch oma in elease. In hese s eps, we ecommend he use o glass
pipe es o emo e he liquid disca ded in each s ep since he high lipìd con en o AT
can become s uck o plas ic su aces, hinde ing manipula ion and leading o issue loss.
Mo eo e , we de e mined use o he ul a u ax homogenize as he bes me hod o
homogeniza ion, a ixing solu ion o PBS+0.5% o maldehyde a RT and he s anda d
de-c osslinking me hod as he mos sui able p ocedu es o small pieces o ozen AT.
Up o now, he use o ChIP o AT has been limi ed o big amoun s o issue, and
especially o mouse AT whe e he condi ions a e less limi ing. Thus, he imp o emen s
shown in his wo k could help esea che s s udy he p o eome-DNA in e ac ion in
human AT, which is s o ed ozen in la ge issue banks.
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136
Once he bes p ocedu e was es ablished, we applied he me hod o 100 mg o ozen
samples o human AT. The yield o he me hod a e he IP esul ed in an a e age o
almos 100 ng o DNA, enough o pe o m a pos e io high h oughpu sequencing
hanks o he high esolu ion o he la es nex gene a ion sequencing me hods. On he
o he hand, in o de o imp o e he pe o mance and DNA eco e y, we encou age
o he s o pe o m ChIP expe imen s in small ounds o samples
Al hough we ha e p o ided an imp o ed me hod o wo k wi h small ozen pieces o
AT, we needed o con i m he co ec assessmen o he IP. We alida ed ou ChIP
p o ocol in mouse and human AT by es ing H3K4me3 modi ica ions, a ma k o ac i e
p omo e egions. By qPCR we iden i ied H3K4me3 en ichmen on se e al p omo e s
o genes usually exp essed in AT, such as PPARG, SCD, LPL, LEP, SREBF2, as well
as a sequence 30 kb be o e PPARG TSS (T ansc ip ion S a Si e) in mice (PPARG Ou )
and a sequence 35 kb be o e SCD TSS (SCD Ou ) o humans, bo h as con ol egions.
We ob ained a high pe cen age o en ichmen in bo h mice and humans (Figu e 5A and
Figu e 5B espec i ely) o genes usually exp essed in AT, like SCD, PPARG, LPL o
SREBF2, while con ol egions p esen ed esidual exp essions. The p esence o
H3K4m3 a hese p omo e s has al eady been demons a ed in se e al issues and cell
lines (ENCODE p ojec ), bu o he bes o ou knowledge, no esul s a e a ailable in
whi e AT. Ne e heless, hese gene exp essions a e usually assessed in AT, which could
ag ee wi h he high pe cen age o DNA immunop ecipi a ion obse ed in hese
p omo e genes o H3K4m3, a his one associa ed wi h ac i e genes.
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143
(E2F1); Lipop o ein Lipase (LPL); S e ol egula o y elemen -binding ac o 2 (SREBF2);
S ea oyl-CoA desa u ase 1 (SCD1); Pe oxisome p oli e a o -ac i a ed ecep o gamma
(PPARG); In e leukin 6 (IL6); Tumo nec osis ac o (TNF).
Acco dingly, associa ion analysis o he H3K4me3 ma k in he s udied genes wi h he
measu ed clinical a iables showed us a posi i e co ela ion o he H3K4me3 ma k a
E2F1, LEP, LPL, SREBF2, SCD1, PPARG, IL6 and TNF p omo e s wi h he BMI,
HOMA-IR and insulin le els (Table 2). Mo eo e , he e was a posi i e co ela ion
be ween glucose and H3K4me3 ma k en ichmen a he p omo e o SCD1, PPARG,
E2F1 and IL6 (Table 2). We also obse ed a posi i e co ela ion be ween he
H3K4me3 ma k a E2F1, SREBF2 and SCD p omo e s wi h he numbe o me abolic
synd ome (Me S) componen s (Me S Va ) p esen s in he subjec .
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144
Table 2. Spea man co ela ion analysis be ween H3K4me3 ma k en ichmen a he s udy gene p omo e s and he an h opome ic and biochemical a iables. *
and ** mean p<0.05 and p<0.01 espec i ely.
H3K4me3
en ichmen
Age
BMI
Glucose
Insulin
HOMA-IR
Tg
Chol
HDL-C
LDL-C
SBP
DBP
Me S Va
E2F1
-0.214
0.530**
0.552**
0.573**
0.594**
-0.01
-0.256
-0.163
-0.257
0.123
0.072
0.448*
LEP
-0.148
0.364
0.361
0.348
0.367
0.044
-0.133
-0.12
-0.143
0.075
0.006
0.279
LPL
-0.168
0.430*
0.395*
0.437*
0.463*
-0.008
-0.208
-0.141
-0.198
0.154
0.064
0.333
SREBF2
-0.236
0.467*
0.442*
0.403*
0.441*
-0.048
-0.178
-0.232
-0.113
0.088
0.062
0.463*
SCD
-0.242
0.528**
0.513**
0.529**
0.548**
-0.034
-0.214
-0.186
-0.213
0.157
0.071
0.420*
PPARG
-0.243
0.488**
0.399*
0.460*
0.479**
-0.051
-0.263
-0.212
-0.246
0.073
0.038
0.369
IL6
-0.131
0.430*
0.497**
0.472*
0.501**
0.062
-0.094
-0.15
-0.065
0.191
0.047
0.327
TNF
0.009
0.23
0.379*
0.295
0.305
-0.051
-0.093
-0.008
-0.105
0.085
0.056
0.163
Abb e ia ions: E2F ansc ip ion ac o 1 (E2F1); Lep in (LEP); Lipop o ein Lipase (LPL); S e ol egula o y elemen -binding ac o 2 (SREBF2); S ea oyl-
CoA desa u ase 1 (SCD1); Pe oxisome p oli e a o -ac i a ed ecep o gamma (PPARG); In e leukin 6 (IL6); Tumo nec osis ac o (TNF); Body mass index
(BMI); Homeos a ic model assessmen o insulin esis ance (HOMA-IR); T iglyce ides (Tg); To al choles e ol (Chol); High-densi y lipop o ein choles e ol
(HDL-C); Low-densi y lipop o ein choles e ol (LDL-C); Sys olic blood p essu e (SBP); Dias olic blood p essu e (DBP); Numbe o Me S a iables (Me S
Va ).
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145
Gene exp ession le els
Le els o gene exp ession in he s udied genes a e despic ed in Figu e 2. Wi h espec
o he Lean NG g oup, LPL, SCD and PPARG mRNA le els we e lowe in he MO PD
g oup, whils highe mRNA le els we e desc ibed o IL6 and TNF genes.
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146
Figu e 2. G oup compa isons o he ela i e mRNA le els o he s udy genes. Di e en le e s
mean signi ican di e ences a p<0.05 acco ding o K uskall-Wallis and Mann-Wi hney U-Tes .
Abb e ia ions: Lean No moglycemic (Lean NG); Mo bid obese no moglycemic (MO NG);
Mo bid obese p ediabe ic (MO PD); E2F ansc ip ion ac o 1 (E2F1); Lipop o ein Lipase
(LPL); S e ol egula o y elemen -binding ac o 2 (SREBF2); S ea oyl-CoA desa u ase 1
(SCD1); Pe oxisome p oli e a o -ac i a ed ecep o gamma (PPARG); In e leukin 6 (IL6);
Tumo nec osis ac o (TNF).
Co ela ion analyses we e in line wi h he exp ession esul s. Thus, LEP, IL6 and TNF
mRNA le els we e posi i ely associa ed o BMI, while LPL, SCD and PPARG mRNA
le els dec eased in line wi h BMI (Table 3). On he o he hand, HOMA-IR co ela ed
wi h LEP, IL6 and TNF mRNA in a posi i e way, and nega i ely wi h SREBF2 and
SCD (Table 3).
Rega ding he es o measu ed a iables, posi i e associa ions we e desc ibed be ween
HDL-C and LPL, SCD and PPARG gene exp essions. In e es ingly, E2F1 mRNA le els
we e nega i ely associa ed wi h o al choles e ol (Chol), HDL-C and LDL-C (Table 3).
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Table 3. Spea man co ela ion analysis be ween he ela i e mRNA le els a he s udy genes and he an h opome ic and biochemical a iables. * and **
mean p<0.05 and p<0.01 espec i ely.
Rela i e
mRNA
Age
BMI
Glucose
Insulin
HOMA-IR
Tg
Chol
HDL-C
LDL-C
SBP
DBP
Me S Va
E2F1
-0.256
0.359
0.116
0.194
0.19
-0.151
-0.559**
-0.407*
-0.534**
-0.144
0.097
0.284
LEP
-0.321
0.522**
0.334
0.673**
0.683**
0.179
-0.213
-0.308
-0.093
0.04
-0.095
0.533**
LPL
0.103
-0.500**
-0.295
-0.33
-0.327
-0.189
0.31
0.516**
0.203
0.008
-0.102
-0.454*
SREBF2
0.088
-0.258
-0.178
-0.391*
-0.409*
-0.238
-0.082
-0.024
-0.069
-0.209
-0.238
-0.373
SCD
0.375*
-0.681**
-0.319
-0.526**
-0.525**
-0.124
0.381*
0.555**
0.277
0.054
-0.165
-0.597**
PPARG
0.061
-0.408*
-0.214
-0.216
-0.204
-0.112
0.221
0.371*
0.182
0.097
0.161
-0.28
IL6
-0.291
0.729**
0.486*
0.571**
0.590**
0.036
-0.285
-0.223
-0.322
-0.025
0.144
0.533**
TNF
-0.298
0.679**
0.322
0.594**
0.612**
0.024
-0.263
-0.175
-0.335
0.048
-0.108
0.444*
E2F ansc ip ion ac o 1 (E2F1); Lep in (LEP); Lipop o ein Lipase (LPL); S e ol egula o y elemen -binding ac o 2 (SREBF2); S ea oyl-CoA desa u ase 1
(SCD1); Pe oxisome p oli e a o -ac i a ed ecep o gamma (PPARG); In e leukin 6 (IL6); Tumo nec osis ac o (TNF); Body mass index (BMI);
Homeos a ic model assessmen o insulin esis ance (HOMA-IR); T iglyce ides (Tg); To al choles e ol (Chol); High-densi y lipop o ein choles e ol (HDL-C);
Low-densi y lipop o ein choles e ol (LDL-C); Sys olic blood p essu e (SBP); Dias olic blood p essu e (DBP); Numbe o Me S a iables (Me S Va ).
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148
Rela ionship be ween H3K4me3 ma k le els and gene exp ession le els
In o de o analyze whe he he p omo e H3K4me3 le els could be ela ed o gene
exp ession we pe o med spea man’s co ela ion analysis in he whole popula ion
be ween he mRNA le els and H3K4me3 en ichmen a each gene. We did no obse e
any signi ican associa ion be ween he p omo e H3K4me3 le els and he mRNA
le els o any gene excep o E2F1, in which a posi i e co ela ion was obse ed
( =0.422, p=0.04).
Mul i a ia e models
In addi ion, a ha monized lineal eg ession analyses showed ha BMI was hea ily
explained by H3K4me3 en ichmen le els a he p omo e o E2F1 and LPL, and by he
mRNA le els o LEP and SCD (Table 4). In his model, hese ou a iables could
explain up o 83% o he BMI a iabili y p esen in ou s udied popula ion.
Table 4. Ha monized lineal eg ession analysis wi h BMI as dependen a iable, which was
co ec ed by age and sex. H3K4me3 en ichmen a gene p omo e s and gene exp ession o
genes ha showed signi ican associa ion in he spea man co ela ion analysis we e in oduced
in he model,.
BMI (R=0.91, R2=0.83)
Be a
p
95% CI
E2F1 H3K4me3
0.979
0.001
0.844 o 2.922
LPL H3K4me3
-0.813
0.003
-3.640 o -0.840
LEP mRNA
0.344
0.002
38.32 o 152.97
SCD mRNA
-0.516
0.000
-3.650 o -1.459
Resul s
149
Abb e ia ions: Body mass index (BMI); E2F ansc ip ion ac o 1 (E2F1); Lipop o ein Lipase
(LPL); Lep in (LEP); S ea oyl-CoA desa u ase 1 (SCD1).
On he o he hand, when he s udied a iable is HOMA-IR, a ha monized lineal
eg ession showed ha he H3K4me3 en ichmen a he p omo e o SCD and IL6,
oge he wi h he mRNA le els o LEP and SCD could explain a 79% o he a ia ion
obse ed in he HOMA-IR (Table 5).
Table 5. Ha monized lineal eg ession analysis wi h HOMA-IR as dependen a iable, which
was co ec ed by age and sex. H3K4me3 en ichmen a gene p omo e s and gene exp ession o
genes ha showed signi ican associa ion in he spea man co ela ion analysis we e in oduced
in he model,.
HOMA-IR (R=0.89, R2=0.79)
Be a
p
95% CI
Gende
-0.259
0.047
-3.598 o -0.027
SCD H3K4me3
0.792
0.016
0.049 o 0.417
IL6 H3K4me3
-0.666
0.030
-1.769 o -0.105
LEP mRNA
0.564
0.000
16.60 o 44.82
SCD mRNA
-0.261
0.065
-0.541 o 0.018
Homeos a ic model assessmen o insulin esis ance (HOMA-IR); S ea oyl-CoA desa u ase 1
(SCD1); In e leukin 6 (IL6); Lep in (LEP).
Resul s
151
MANUSCRIPT 6. Adipose Tissue In lamma ion and VDR Exp ession
and Me hyla ion in Colo ec al Cance
Cas ellano-Cas illo D. e al. Clinical Epigene ics. 2018;10:60.
Resul s
152
The objec i e 6 was accomplished in his sec ion. The aim o his s udy was o explo e
he ela ionship be ween se um 25-hyd oxy i amin D (25(OH)D), adipose issue gene
exp ession o VD ecep o (VDR), p o-in lamma o y ma ke s and he epigene ic ac o
DNA me hyl ans e ase 3a (DNMT3A) as well as VDR and NFκB1 p omo e s
me hyla ion in subjec s wi h colo ec al cance (CRC) and wi ou h CRC (Con ol).
Blood and isce al adipose issue om 57 CRC and 50 heal hy con ol subjec s we e
collec ed. mRNA was measu ed by qPCR using Taqman echnology while bisul i e
ea ed DNA was py osequensed using he Py oma kQ96 echnology in o de o analyze
DNA me hyla ion. P o ein le els we e measu ed by Wes e n-blo .
An h opome ic and biochemical a iables
Table 1 shows he biochemical and an h opome ic cha ac e is ics o he s udy g oups.
The e we e no di e ences in age, BMI o gende be ween he con ol and CRC g oups.
The CRC g oup had lowe le els o insulin, o al choles e ol, HDL-C and LDL-C han
he con ol g oup. In con as , he CRC g oup p esen ed highe le els o plasma
iglyce ides when compa ed wi h he con ol g oup.
Table 1. An h opome ic and biochemical a iables o he s udy g oups
Con ol
(n=57)
CRC
(n=50)
Age (yea s)
64.94±8.84
68.035±8.43
Male/Female (%)*
68/32
45/55
BMI (kg/m2)
28.51±4.21
27.61±3.91
Wais (cm)
96.55±11.64
97±12.74
Glucose (mg/dl)
111.72±28.77
125.035±46.87