Xiaomei Zhang,1,2 Ran Yang,1,2 Yan Jia,1,2 Demin Cai,2 Bo Zhou,2 Xiaoli Qu,2 Huihua Han,2 Liang Xu,1,2 Linfeng Wang,2

Yanan Yao,1 and Guoqing Yang1,2

Hypermethylation of Sp1 BindingSite Suppresses HypothalamicPOMC in Neonates and MayContribute to MetabolicDisorders in Adults: Impact ofMaternal Dietary CLAs

Epigenetic regulation of neuropeptide genesassociated with central appetite control plays animportant part in the development of nutritionalprogramming. While proopiomelanocortin (POMC) iscritical in appetite control, the molecular mechanismof methylation-related regulation of POMC remainsunclear. Based on the report that the proximalspecificity protein 1 (Sp1) binding site in POMCpromoter is crucial for the leptin-mediated activationof POMC, the methylation of this site wasinvestigated in this study in both cultured cells andpostnatal mice reared by the dams with dietarysupplementation of conjugated linoleic acids (CLAs).The change of milk composition made the offspringundergo the increase of food intake, suppression ofPOMC, attenuation of Sp1–promoter interaction, andthe hypermethylation of cytosine guanine (CpG)dinucleotides at 2100 and 2103 within the Sp1binding site of POMC promoter, which may beassociated with the decrease of hypothalamic Sp1and/or plasma S-adenosylhomocystein. In culturedcells, the methylation of the 2100 CpG dinucleotides

of the POMC promoter blocked both the formationof Sp1–promoter complex and the leptin-inducedactivation of POMC. In addition, a catch-up growthand adult metabolic changes like adulthyperglycemia and insulin resistance were observedin these postnatal pups, suggesting that this CLA-mediated hypermethylation may contribute, at leastin part, to the metabolic disorders.Diabetes 2014;63:1475–1487 | DOI: 10.2337/db13-1221

Chronic metabolic diseases, such as obesity, type 2 di-abetes, hypertension, and heart diseases, result fromcomplicated gene and environmental abnormalities (1).The poor eating habits and availability of food containingor not containing various additives in modern societymake it susceptive for humans to suffer from metabolicillness (2). Moreover, the programming in response tosome abnormal insult in early life can affect metabolicbehavior in adulthood (3–5). While the major inves-tigations are performed with the hypothesis of fetalorigins of adult-onset diseases, little attention has been

1Laboratory of Animal Gene Engineering, College of Life Sciences, HenanAgricultural University, Zhengzhou, People’s Republic of China2Key Laboratory of Animal Biochemistry and Nutrition, Ministry ofAgriculture, Henan Agricultural University, Zhengzhou, People’s Republicof China

Corresponding author: Guoqing Yang, gqyang@yeah.net.

Received 9 August 2013 and accepted 23 December 2013.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1221/-/DC1.

© 2014 by the American Diabetes Association. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

Diabetes Volume 63, May 2014 1475

METABOLISM

paid to the postnatal programming. However, like a fe-tus, suckling pups with developmental plasticity can alsogrow up into adults with metabolic disorders, which re-sult from early malnutrition.

Hypothalamic control of appetite is considered asa focus of the study on perinatal nutritional pro-gramming (6). Hypothalamus contains not onlyorexigenic neurons expressing neuropeptide Y (NPY) andagouti-related protein (AgRP), but also anorexigenicneurons expressing cocaine- and amphetamine-regulatedtranscript (CART) and proopiomelanocortin (POMC),which exerts the key role in appetite control (7). Evidenceis accumulating that DNA methylation contributes to thedevelopment of metabolic diseases by changing the ex-pression of genes controlling whole-body energy homeo-stasis (8,9); however, the studies on the epigeneticregulation of POMC expression in postnatal animals are few.

The expression of POMC in hypothalamus is facili-tated by leptin, the fat-derived satiety signal, throughOBRb (the long-form leptin receptor) signaling pathwayto inhibit appetite of animals (10). DNA deletion, DNA–protein, and protein–protein interaction assays indicatethat the specificity protein 1 (Sp1) binding site around2100 of mouse POMC promoter is crucial for thetransduction of OBRb signaling from signal transducerand activator of transcription 3 (STAT3) to POMC (11).Nevertheless, the methylation of this site and its con-sequences have not been reported yet.

One of the nutritional stimuli that causes postnatalprogramming is maternal dietary supplementation ofconjugated linoleic acids (CLAs) (12), which is a group ofpositional and geometric isomers of fatty acids widely usedas food additives for humans. Studies show that adultrodents fed CLAs suffer from metabolic disorders (13–15).

Generally, this study was designed to investigate themethylation status of the Sp1 binding site in the POMCpromoter, the methylation-related regulation of hypo-thalamic POMC, and metabolic changes in postnatal micereared by the lactating dams with dietary supplementa-tion of CLAs.

RESEARCH DESIGN AND METHODS

Animals and Experiment Design

All animals in the experiments were approved by theInstitutional Animal Care and Use Committee of HenanProvince. Wild-type Kunming mice were purchased fromHenan Laboratory Animal Center (Zhengzhou, China).Animals were kept under standard conditions with12/12-h light/dark cycles at 22°C and had free access towater and diet.

The main purpose of the animal experiments was tostudy whether only 10 days’ exposure to the modifiedmilk from the lactating dams with dietary supplemen-tation of CLAs would make the pups undergo: 1) thealternation of methylation status and methylation-associated items, which would be found even when theywere fed chow diet for a week after weaning; and 2)

metabolic behaviors afterward. The 3-month-old femalemice were crossed with normal adult males. Once pupswere born (d0; Fig. 1), the litter size was adjusted to12 pups per dam. All dams before or during conceptionwere fed chow diet until day 3 postpartum (d3, Fig. 1A),when dams with pups were divided into linoleic acids(LA) group and CLA group randomly. From days 3–13,the dams of the CLA group (CLA-dams) were fed chowdiet supplemented with CLAs (1.5% weight for weight)(CLA-diet), while those of LA group (LA-dams), as thecontrol, were fed chow diet supplemented with LA (1.5%weight for weight) (LA-diet). The pups suckling the milkof CLA-dams (CLA-milk) or LA-dams (LA-milk) werenamed LA-pups or CLA-pups, respectively. From day 14to the end of the lactating period, the supplements wereremoved from the diet of dams in both groups. At day 9(i.e., the seventh day of the supplement feeding), themilk was collected for composition analysis; at day 13(i.e., the last day of the supplement feeding), the bloodparameters were measured.

Correspondingly, the pups of both groups were suck-ling normal milk from day 0 (d0, Fig. 1B) to day 2 afterbirth and LA- or CLA-milk from days 3–13. But the pupswere suckling the milk with unknown components fromdays 14 to 21 (weaning) since the milk analysis was notperformed during this period. From day 21, the pups ofboth groups were fed chow diet without supplement untilthe end of experiment. The body weight of the offspringwas traced in the whole experiment period from days0–140, in which the measurements of plasma glucosefrom days 49–119 and insulin tolerance test (ITT) at day119 were performed. The food intake was measured fromdays 21–35. Further measurements were performedmainly at two time points (days 13 and 28). At day 13,hypothalamic Sp1 protein, blood leptin, and insulin wereassessed. At day 28, the hypothalamic gene expression, DNAmethylation, DNA–protein interaction, epididymal fat mass,as well as blood leptin, insulin, S-adenosylmethionine(SAM), and S-adenosylhomocystein (SAH) were assessed.Different batches of pups were used on the sameschedule of milk/diet feeding and various operations.

DNA Constructs

The 5.5 kb of POMC promoter–luciferase construct(pGL3-POMC) was a generous gift from Dr. DomenicoAccili (Department of Medicine, Columbia UniversityMedical Center, New York, New York), and the pN3-Sp1FL-complete was from Dr. Guntram Suske (Institut fürMolekularbiologie und Tumorforschung, Marburg,Germany). pXJ40-FLAG-STAT3 was described previously(16). pRL-CMV was included in the Dual-Luciferase Re-porter Assay System (Promega, Madison, WI). pMD19-Twas purchased from TaKaRa (Dalian, China).

Analysis of Fatty Acids in Milk

Milk samples were collected as reported (17). Themethodology of fatty acid analysis was as described (18).

1476 DNA Methylation and Metabolic Disorders Diabetes Volume 63, May 2014

Analysis of Energy Substances in Milk

The concentration of triglycerides in milk was determinedusing commercial kit (Beijing BHKT Clinical Reagent Co.,Ltd., Beijing, China) following the instructions in themanual, protein concentration was measured by Bio-Radprotein assay (Bio-Rad, Hercules, CA), and lactose concen-tration was assessed by a modified Teles method (19).

Measurement of Metabolic Parametersin Blood and Milk

With EDTA as anticoagulant,;1 mL of blood sample wasdrawn from the abdominal vein of mice anesthetized bythe intraperitoneal injection of pelltobarbitalumnatricum (80 mg/kg body weight). Blood glucosewas measured using OneTouch Ultra Easy (LifeScan,Shanghai, China). Milk glucose was measured by glucoseoxidase method using a Glucose Assay Kit (Applygen,Beijing, China) according to the instructions. Plasma ormilk insulin was measured using an [125I] insulin radio-immunoassay kit (Beijing North Institute of BiologicalTechnology, Beijing, China) following the manual. Plasma

or milk leptin levels were detected using a Mouse LeptinImmunoassay kit (R&D Systems, Minneapolis, MN)according to the instructions. Plasma SAM and SAH weredetermined by a commercial service of Beijing AminoMedical Research Co., Ltd. (Beijing, China). The protocolused was as described.

Body Weight and Food Intake of Postnatal Pups

From days 0–21, litter weight was recorded every 3 daysand divided by pup number to get the body weight ofeach pup. From day 21, when pups were housed in-dividually, to day 140, daily body weight of each pup wasrecorded at 5:00 P.M. From days 23–35, the food intakewas measured every other day.

Real-Time PCR

The protocol of real-time PCR was the same as previouslyreported (11). Briefly, total RNA was isolated from hy-pothalamus and reversely transcribed to cDNA. Real-timePCR in triplicates was performed with SYBR Green Uni-versal PCR Master Mix in Realplex2 (Eppendorf,

Figure 1—Flow chart of operation schedule on mice. The days after delivery (dam) or birth (pup) are represented by “d.” Various itemswere assessed at different time points (arrow) or in different durations (brackets). A: Dams were fed different diets (italics) in differentperiods of lactation that are divided by dotted lines. Milk compositions and plasma parameters were analyzed at days 9 and 13, re-spectively. B: Before weaning (day 21), the pups were exposed to different milk (italics) in different periods divided by dotted lines; afterweaning, the pups were fed chow diet (italics). WAT, white adipose tissue.

diabetes.diabetesjournals.org Zhang and Associates 1477

Hamburg, Germany). The mRNA level of target genes wasnormalized by that of mouse glyceraldehyde-3-phosphatedehydrogenase (GAPDH). Primer sequences for GAPDH,NPY, AGRP, POMC, CART, DNA methyltransferase 1(DNMT1), DNMT3a, and DNMT3b are listed in Supple-mentary Table 1.

Bisulfite Modification of Genomic DNA

Genomic DNA of hypothalamus was isolated by phenol/chloroform method and treated for bisulfate modifica-tion using the EZ DNA Methylation Kit (Zymo Research,Irvine, CA) according to the manufacturer’s instructions.The bisulfite-modified DNA was used immediately orstored at 280°C.

Bisulfite Sequencing PCR

A DNA fragment between 2163 and 221 base pairs (bp)in the POMC promoter including eight cytosines of cy-tosine guanine (CpG) dinucleotide was amplified by PCR.The primers are listed in Supplementary Table 1. Theprotocol of bisulfite sequencing PCR (BSP) was previouslyreported (20), with the modification of reaction condi-tion as follows: one cycle of 94°C for 15 min, 45 cycles of94°C for 20 s, 58°C for 30 s, and 72°C for 1 min, anda final incubation at 72°C for 3 min. The PCR products werepurified and cloned into the pMD19-T vector (TaKaRa,Dalian, China) followed by sequencing analysis via a com-mercial service (Sangon Biotech, Shanghai, China).

Generation of Construct with Site-Specific Methylation

Site-specific methylation of the POMC promoter wasgenerated using modified primers (Supplementary Table1) from Sangon Biotech (Shanghai, China), which con-tained either a methylated or an unmethylated CpGdinucleotide at the site of interest and spanned 27 bpon either strain of DNA. Using the primers above andpGL3-POMC as a template (see schema in Fig. 4A),PCR was carried out according to the protocol in theQuickChange site-directed mutagenesis kit (Stratagene,La Jolla, CA). After a digestion with DpnI for 2 h, thePCR products were purified using the SanPrep DNAPurification Kit (Sangon Biotech), assessed with agarosegel electrophoresis, and then used for transfectioninto the cells.

Cell Culture and Luciferase Assay

The methodology of cell culture and luciferase assay wasthe same as reported (11). Briefly, OBRb 293 cells (293-OBRb) were cultured in Dulbecco’s minimal essentialmedium (11995; Invitrogen, Carlsbad, CA) containing10% FBS and transfected with relevant DNA constructsusing FuGENE 6 (Roche Applied Sciences, Indianapolis,IN). After the treatment of recombinant leptin (Invi-trogen) at a dose of 1 mg/well or vehicle for 20 h, the cellswere lysed in 200 mL of 13 passive lysis buffer includedin a Dual Luciferase Rporter Assay System (Promega).Luciferase activity was measured from cell extracts ona luminometer (Molecular Devices, Sunnyvale, CA). The

firefly luciferase activity was normalized against Renillaluciferase activity.

Preparation of Nuclear Extract and ElectrophoreticMobility Shift Assay

The protocol of nuclear extraction and electrophoreticmobility shift assay (EMSA) was the same as reported(11). Briefly, the 293Rb cells were treated with hypotonicbuffer. After a spin, the pellet was suspended in high-saltbuffer followed by a centrifugation at 13,000 rpm for10 min at 4°C. The supernatant was collected as the nu-clear extract. Pairs of oligonucleotides unmethylated ormethylated (Supplementary Table 1) were annealed, re-spectively. The purified double strains were labeled with50 mCi of [32P] deoxycytidine triphosphate by klenowexo-

(New England Biolabs, Ipswich, MA). The nuclear proteinwas incubated with the probes and resolved by 4% PAGEgel in 0.53 Tris/borate/EDTA. After the gel was dried at80°C, supersensitive X-ray film (Kodak, Rochester, NY)was exposed for 48 h at 280°C and then developed.

Western Blotting

The protocol of Western blotting was the same asreported (11). Briefly, hypothalamus was homogenized inRIPA lysis buffer (Sigma-Aldrich, St. Louis, MO) con-taining 1 mmol/L phenylmethylsulfonyl fluoride. Thelysate was rocked on ice for 20 min and centrifuged at14,000g for 10 min at 4°C. The protein concentration ofthe supernatant was adjusted and analyzed by SDS-PAGE, and then immunoblotting was performed usingthe antibody against Sp1 (Santa Cruz Biotechnology,Santa Cruz, CA).

Chromatin Immunoprecipitation Assays

The protocol of chromatin immunoprecipitation (ChIP)assay was the same as reported previously (21). Briefly,hypothalamuses were fixed in minimal medium con-taining 1% formaldehyde followed by homogenization.The homogenates of two hypothalamuses were pooledtogether and sonicated for DNA fragmentation. ChIPassay was conducted using 2 mg of Sp1 antibody (Abcam,Cambridge, MA) at 4°C overnight. DNA was extractedand resuspended in 25 mL of distilled water, and 2 mL ofDNA solution was used for real-time PCR with primers(Supplementary Table 1). The conditions of real-timePCR were as follows: 30 s at 95°C followed by 40 cycles of5 s at 95°C and 5 s at 60°C.

ITT

Prior to the test, mice had been fasted for 6 h. Themeasurement of blood glucose with OneTouch Ultra Easy(LifeScan) was conducted with tail bleeding at 0, 15, 30,60, 90, and 120 min posterior to the intraperitonealinjection of insulin (Novolin R; Novo Nordisk, Bagsvaerd,Denmark) at a dose of 1 unit/kg body weight.

Statistics Analysis

Data are shown as the means 6 SEM. Comparisons ofdata were made using two-tailed Student t tests for

1478 DNA Methylation and Metabolic Disorders Diabetes Volume 63, May 2014

independent data. The significance limit was set at P ,0.05.

RESULTS

CLA-Diet Feeding Changed the Milk Components ofLactating Dams

At day 9, the milk components were analyzed. The con-centration of milk glucose and milk insulin from CLA-dams increased about twofold (P , 0.05) and sevenfold(P , 0.001), respectively, compared with that of thecontrol group (Table 1). Concomitantly, these parametersalso elevated in blood of dams (Supplementary Fig. 1),agreeing with the point that the increased glucose andinsulin in milk was attributed to the diffusion frommaternal circulation (22,23). Interestingly, while the milkleptin did not differ significantly between the twogroups of dams (Table 1), the blood leptin level of CLA-dams increased nearly twofold compared with that incontrol dams (P , 0.05) (Supplementary Fig. 1). Themilk of CLA-dams grossly appeared more watery thanthat of the control group (data not shown). The con-centrations of both lactose (P , 0.05) and triglyceride(P , 0.001) in milk of CLA-dams were significantlylower than those of LA-dams (Table 1). CLAs in the milkof CLA-dams remarkably increased compared with thatof LA-dams, mirroring the differences of dietary sup-plementation between the two groups. Besides thesechanges, the pattern of some other fatty acids alsochanged (Table 2).

CLA-Milk Caused Metabolic Responses of thePostnatal Offspring

At days 13 and 28, there were no differences in bloodglucose between the two groups of pups (data notshown). At day 13, plasma insulin reduced 54.2% (P ,0.05) (Fig. 2A), and plasma leptin strikingly reduced96.7% (P , 0.01) in CLA-pups (Fig. 2C) compared withthose of the controls; but at day 28, the levels of bothplasma insulin (Fig. 2B) and leptin (Fig. 2D) of CLA-pupsdid not differ from that of LA-pups. Interestingly, at day28, the content of epididymal fat of CLA-pups reduced71.1% compared with that of the control group (P, 0.01)

(Fig. 2E), nonproportional to plasma leptin. The bodyweight reduction of suckling CLA-pups began at day 6,when exposed to CLA milk for 3 days, and lasted to day 21(Fig. 2F). As expected, the retarded growth of CLA-pupscontinued after day 21 (weaning) (Fig. 2G), even whenboth groups of pups consumed the same chow diet, con-sistent with the previous report (24). However, the foodintake normalized by body weight of CLA-pups was sig-nificantly increased compared with that of the controlgroup (Fig. 2H), suggesting that in CLA-pups, some eventsaffecting appetite happened during their suckling periodand lasted at least to day 33.

POMC Expression Was Impaired in the Hypothalamusof CLA-Pups

Based on the prediction that the alternation of hypo-thalamic neuropeptides would be responsible for the in-crease of food intake, we detected the expression of NPY,POMC, AgRP, and CART in the hypothalamus of the pupsat day 28, when the difference of food intake was sig-nificant between the two groups. The data indicated thatPOMCmRNA of CLA-pups reduced 66.9% compared withthat of LA-pups (P , 0.05) (Fig. 3A). The mRNA levels ofthe other three neuropeptides did not differ significantlybetween the two groups. It was noteworthy that at day28, the concentration of circulating leptin in CLA-pupswas normal and similar to that in LA-pups (Fig. 2D),excluding the possibility that the suppression of POMCin CLA-pups was attributed to the insufficiency of bloodleptin. This phenomenon was possibly associated withthe blockage of the OBRb signaling pathway.

The Methylation of Sp1 Binding Site in POMC PromoterWas Enhanced in the Hypothalamus of CLA-Pups

Leptin-mediated activation of POMC requires the for-mation of the STAT3–Sp1–promoter complex in whichthe Sp1 binding site is essential (11). It is reported thatthe methylation of this site abrogates the binding of Sp1(25) with DNA. These facts led us to test the hypothesisthat the suppression of POMC may result from thehypermethylation of POMC promoter. Therefore, at day28, genomic DNA was extracted from the hypothalamusof the pups for methylation analysis by BSP. The PCRproducts covering the 143 bp of POMC promoter regionincluding eight CpG dinucleotides (2140, 2126, 2110,2103,2100,290,279, and262) (Fig. 3B) were cloned.The sequencing results of individual clones from at leastthree independent experiments showed that the Sp1binding sites at 2100 and 2103 were hypermethylatedin CLA-pups compared with those of LA-pups (Fig. 3C).This finding indicated that DNA methylation patterns inpostnatal hypothalamus can undergo dynamic changes inresponse to early nutritional alternation and thata higher level of DNA methylation correlates with a lowerlevel of POMC expression in the neurons. To understandhow the hypermethylation of Sp1 binding site happened,Sp1 protein level in hypothalamus of CLA-pups wasdetected. Given that Sp1 occupies its binding site and

Table 1—Milk composition of lactating dams

Milk composition LA (n = 5) CLA (n = 6) P values

Glucose (mmol/L) 3.47 6 0.45 9.55 6 2.11 0.046

Insulin (ng/mL) 5.26 6 0.56 37.6 6 3.88 ,0.001

Leptin (ng/mL) 1.89 6 0.29 1.62 6 0.21 0.53

Triglycerides (g/L) 40.84 6 5.91 14.33 6 1.87 ,0.001

Lactose (g/L) 18.76 6 0.7 14.6 6 1.02 0.02

Protein (g/L) 115.21 6 6.36 106.22 6 5.24 0.35

Data are least square means. Mouse milk was collected at day9 postpartum. n = animal numbers.

diabetes.diabetesjournals.org Zhang and Associates 1479

prevents it from methylation (26), we hypothesized thatshortage of this protein would facilitate the access of themethyltransferases to the site and consequently pro-mote the site-specific methylation (27). The data fromWestern blotting indicated that Sp1 protein decreasedin hypothalamus of CLA-pups at day 13 compared withthat of LA-pups (Fig. 3D and E). In contrast, the hy-pothalamic expression of methyltransferases DNMT1,DNMT3a, and DNMT3b at either day 13 (Fig. 3G) or 28(Fig. 3H) did not differ between the two groups. Therewere no significant differences in plasma SAM (Fig. 3I)and SAM/SAH ratio (Fig. 3K) between the two groups,but the plasma SAH decreased obviously in CLA-pupscompared with that in the control group (P , 0.05)(Fig. 3J).

The Methylation of Sp1 Binding Site ImpairedSp1–Promoter Interaction and the Leptin-MediatedActivation of POMC In Vivo and In Vitro

To determine if the hypothalamic POMC suppressionresulted from the failure of Sp1–promoter formation,

ChIP assays were conducted. The result indicated thatthe formation of Sp1–promoter complex in the hypo-thalamus of CLA-pups at day 28 was greatly compro-mised compared with that in LA-pups (Fig. 3F).Collectively, these observations suggested a correlationamong the hypermethylation of Sp1 binding site, theimpairment of Sp1–promoter interaction, the suppres-sion of POMC, and the increase of food intake. Based onthe established 293Rb cell line (11), we performed an invitro experiment to clarify if the methylation of the Sp1binding site could affect the leptin-mediated action ofPOMC. With primers (Fig. 4A and Supplementary Table1) containing either a methylated or an unmethylatedCpG and pGL3-POMC as template, an around-the-circlePCR (28) was performed; the methylation was in-corporated into the promoter, illustrated in Fig. 4A. The5.5-kb plasmid pGL3-POMC included 440 bp of themouse POMC promoter. Fig. 4B showed molecular weightof the PCR products was 5.5 kb, identical to that ofthe plasmid template (data not shown). The leptin-stimulated luciferase activity in the cells with methylated

Table 2—Effect of CLA on fatty acid composition of milk in lactating dams (day 9 postpartum) (g/100 g)

Fatty acid LA (n = 6) CLA (n = 7) P value

C8 0.0557 6 0.0104 0.0215 6 0.0061 0.0151

C10 1.0935 6 0.1342 0.3801 6 0.079 0.0007

C12 1.6956 6 0.1638 0.4584 6 0.0968 ,0.0001

C14 2.0523 6 0.2174 0.4124 6 0.0814 ,0.0001

C14:1 0.0118 6 0.0073 0 6 0 0.1218

C16 2.9584 6 0.3192 1.6473 6 0.0795 0.0063

C16:1 0.2254 6 0.0427 0.2521 6 0.0111 0.6276

C17 0.012 6 0.0005 0.0131 6 0.0005 0.1586

C18 0.2962 6 0.0294 0.2511 6 0.0199 0.2333

T9C18:1 0.0015 6 0 0.0079 6 0.0007 0.0020

T11C18:1 0.0033 6 0.0004 0.0052 6 0.0008 0.1306

C9C18:1 1.522 6 0.1067 2.1742 6 0.0953 0.0026

C9C12C18:2 2.4993 6 0.1409 1.3771 6 0.0365 0.0003

C20:0 0.0323 6 0.0046 0.0216 6 0.0039 0.1376

C18:3 0.1164 6 0.0083 0.1394 6 0.0531 0.7224

C9T11CLA 0.0648 6 0.0066 0.4211 6 0.0312 ,0.0001

T10C12CLA 0.0089 6 0.0021 0.2044 6 0.0348 0.0006

C20:2 0.1165 6 0.0125 0.0484 6 0.0051 0.0012

C22 0 6 0 0.0047 6 0.0035 0.2841

C20:3 (8,11,14) 0.0997 6 0.0101 0.0217 6 0.0035 0.0005

C20:3 (11,14,17) 0 6 0 0.0198 6 0.0184 0.3774

C23 0.1102 6 0.0108 0.0785 6 0.013 0.1101

C20:5 0.0421 6 0.0065 0.0467 6 0.0036 0.5798

C22:4 0.0858 6 0.0044 0.0391 6 0.0038 ,0.0001

C22:6 0.0938 6 0.0104 0.1144 6 0.0065 0.1401

Data are least square means. n = animal numbers.

1480 DNA Methylation and Metabolic Disorders Diabetes Volume 63, May 2014

Figure 2—Physiological parameters of the pups. The concentration of plasma insulin (A and B) and leptin (C and D) in CLA-pups at days13 (A and C ) and 28 (B and D) was detected using the [125I] Insulin Radioimmunoassay Kit and Mouse Leptin Immunoassay Kit (R&DSystems), respectively. At day 28, the male pups were killed by cervical dislocation; the weight of epididymal fat pad normalized by theirbody weight was measured (E). The body weight of suckling pups (F ) was recorded every 3 days. After weaning, the pups had been fedchow diet for 2 weeks; during this period, the body weight was measured every other day (G), coupled with the measurement of foodintake. H: Data are the food intake normalized by body weight. Data represented the means 6 SEM. *P < 0.05; **P < 0.01. WAT, whiteadipose tissue.

diabetes.diabetesjournals.org Zhang and Associates 1481

promoter was significantly lower than that withunmethylated promoter (Fig. 4C). In order to clarify ifthe methylation of CpG would prevent the site frombinding with Sp1, an EMSA was conducted posterior tothe preparation of DNA probe containing Sp1 binding

site and nuclear extract from 293 cells expressing Sp1.The band representing the DNA–protein complex wasobserved when the probe was unmethylated and wassupershifted in the addition of Sp1 antibody, butwas not seen when the probe was methylated (Fig. 4D),

Figure 3—Methylation-associated detections in vivo. From the hypothalamus of pups at day 28 (Fig. 1), total RNA was isolated for real-time PCR analysis. A: Relative mRNA levels of NPY, POMC, AGRP, and CART normalized by that of mouse GAPDH. B: DNA sequence ofPOMC promoter containing eight cytosines (boldface Cs) of CpG dinucleotides between 2163 and 221 bp, Sp1 binding site (rectangle),and transcription start site (arrow). From pups at day 28, hypothalamic genomic DNA was isolated and modified using the EZ DNAMethylation Kit (Zymo Research). The region between 2163 and 221 bp in POMC promoter was amplified using BSP system. The PCRproducts were cloned into the pMD19-T vector followed by DNA sequencing. Results of sequencing analysis (C) represented more thanthree separate experiments. Open circles represent unmethylated cytosines and filled circles the methylated cytosines. D: Sp1 wasdetected from the homogenates of hypothalamus of the pups at day 13 using Western blotting. E: The bands of Sp1 were scanned usingCanoScan 9900F for intensity analysis. Hypothalamuses of the pups at day 28 were fixed and sonicated for DNA fragmentation. ChIP wasconducted using Sp1 antibody. The DNA fragments were extracted and used as template for real-time PCR. F shows the result of real-time PCR. Relative mRNA levels of DNMT1, DNMT3a, and DNMT3b in hypothalamus of pups at day 13 (G) or 28 (H) were detected usingreal-time PCR. The plasma SAM (I) and SAH (J) in pups at day 28 was measured by a commercial service. The value of SAM divided bythat of SAH equals SAM/SAH ratio (K). For A and E–K, the data represent means 6 SEM. *P < 0.05.

1482 DNA Methylation and Metabolic Disorders Diabetes Volume 63, May 2014

indicating that Sp1 only bound to unmethylatedDNA sequence.

The Catch-up Growth and Insulin Resistance ofCLA-Pups in Adulthood

The pups were exposed to CLA-milk at suckling periodwhen their development of neural system was in-complete. To find out whether under this nutrientstimulation these pups undergo postnatal programming,we traced their body weight for 140 days and bloodglucose for 70 days. The body weight of female CLA-pupscaught up around day 48, and significantly exceededaround day 78, to that of female LA-pups (Fig. 5A). Thebody weight of male CLA-pups caught up around day 54and kept similar afterward to that of male LA-pups

(Fig. 5B). The blood glucose of CLA-pups was significantlyhigher than that of LA-pups from days 49–119 (Fig. 5C).The result of ITT indicated that the insulin sensitivity ofCLA-pups was lower than that of LA-pups (Fig. 5D).Collectively, the CLA-pups may suffer from adult meta-bolic disorders associated with programming.

DISCUSSION

We have reported that the leptin-mediated activation ofPOMC relies on a Sp1–DNA complex (11), so we hy-pothesized that the abrogation of the complex by DNAmethylation might block this action. In this study, wedemonstrated, like the previous report (25), that themethylation of Sp1 binding site blocked the formation ofSp1–promoter complex and diminished the leptin-induced

Figure 4—Manipulation of site-directed methylation in vitro. A: Using primers containing either a methylated or an unmethylated CpGdinucleotide at the site of interest, PCR was carried out. The lollipop represents the methyl group in cytosine of CpG. The PCR productwas assessed on agarose gel (B), digested with Dpn I, and purified for transfection assay in 293Rb cells. M, 1 kb DNA ladder (New EnglandBiolabs). C: Luciferase activity in the cell lysate was assessed in a Dual Luciferase Reporter Assay System (Promega). 293Rb cells weretransfected with relevant DNA constructs followed by the treatment of recombinant leptin or vehicle. pGL3-POMC was the originalplasmid amplified in Escherichia coli. pGL3-POMC-un (unmethylated) and pGL3-POMC-me (methylated) were the products of around-the-circle PCR. Data are means 6 SEM and represented three independent experiments. Nuclear protein was extracted from 293Rb cellstransfected with pN3-Sp1 FL-complete; pairs of primers annealed and labeled with [32P]deoxycytidine triphosphate were used as probes.For EMSA (D), nuclear protein was incubated with the labeled probes including methylated (Me) or unmethylated Sp1 binding site in theabsence (Un) or presence of Sp1 antibody (Ab). The mixture was resolved by 4% PAGE gel, which was then dried and set to expose X-rayfilm. *P < 0.05; **P < 0.01. ctrl, control.

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Figure 5—Growth curve and metabolic indicators. From weaning (day 21), the body weight of female (A) and male (B) pups was measuredat 5:00 P.M. every 3 days until day 140 (Fig. 1). From days 49–119, the measurement of plasma glucose level (C ) was performed weekly onmice fasted overnight. ITT (D) was performed at day 119 on mice fasted beforehand for 6 h, which included the measurement of bloodglucose at 0, 15, 30, 60, 90, and 120 min posterior to the intraperitoneal injection of insulin at a dose of 1 unit/kg body weight. The resultsrepresented the means 6 SEM. *P < 0.05; **P < 0.01.

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activation of POMC in cultured cells. Importantly, thehypermethylation-associated POMC suppression wasobserved in animals accompanied with increased ap-petite. The hypermethylation happened at a time whencirculating leptin level was normal, making it possiblethat the suppression of POMC resulted from theblockage of leptin signaling rather than the deficiencyof blood leptin. In this study, we raised a novel hy-pothesis that besides the forkhead box O1–involvedinhibition of leptin action (11), the methylation of Sp1binding site abrogated the formation of Sp1–promotercomplex, thus attenuated the leptin-mediated activationof POMC (Fig. 6).

Although little is known, regulation of DNA methyl-ation in mammals can be associated with the alternationof some nutritional and developmental factors. The sta-tus of one-carbon metabolism and the activity of DNAmethyltransferases are closely related to the machineryof genome-wide DNA methylation. The level of SAM, thedirect donor of methyl group for the methylation ofcarbon 59 position of cytosine within the CpG di-nucleotide, and SAH, the product formed when themethyl group of SAM was transferred to the acceptor, isaffected by dietary changes (29). As a biomarker, plasmaSAM and SAH represent the ability of global methylation.It is reported that the concentration of plasma SAHnegatively correlates with global methylation status inapolipoprotein E–deficient mice (30). Another workaddresses that the variation of seasonal nutrition inpericonceptional rural African women results in the de-cline of plasma SAH, which is associated with the globalhypermethylation in the offspring (31). Thus, thehypermethylation identified in this study may be at-tributed to the low plasma SAH (31) in CLA-pups

exposed to CLA-milk. Moreover, given that the Sp1protein decreased in the hypothalamus of CLA-pups, wealso supposed that the methylation of Sp1 binding sitemay result from the lack of Sp1, a protector of Sp1binding site, preventing methyltransferases from accessto the DNA sequence (26). This kind of sequence-specificmethylation plays an important role in gene regulation intime and space in mammals (27). How the Sp1 wasdownregulated in the hypothalamus of CLA-pups needsto be investigated in the future.

The transient exposure to the modified milk duringthe suckling period made CLA-pups exhibit an abnormalphenotype. The lactating dams were fed CLA-diet onlyfrom days 3–13, because earlier onset or longer feedingcaused high mortality of the pups, which would interruptthe experiment (data not shown). The retarded growth ofCLA-pups, consistent with the previous report (24), wasfollowed by a catch-up growth, especially in the females.Moreover, higher blood glucose and insulin resistancewas observed in these mice in adulthood, as reportedpreviously (12,32,33). These disorders were closely as-sociated with the postnatal programming. It should benoted that this study has examined only postnatalanorexigenic pathways via the regulation of POMC. Thegrowth retardation of the objectives would be alsorelevant to other factors such as growth hormoneand glucocorticoid, which need to be addressed ina future study.

Finally, CLA is a widely used food additive for humanbeings. The observations in this study may provide usinsights on human beings regarding the diet supple-ments, like CLAs, of lactating mothers that result in thechange of their milk compositions, which would causethe long-term metabolic dysregulation in progeny.

Figure 6—Diagram of potential mechanism of methylation-related regulation of POMC. A: In normal conditions, the binding of leptin toOBRb activates STAT3, which in turn translocates into the nucleus and activates POMC through its interaction with the Sp1–POMCpromoter complex. B: When leptin resistance exists, forkhead box O1 (FOXO1) binds to the activated STAT3 in nucleus, prevents the latterfrom interacting with the Sp1–POMC promoter complex, and inhibits STAT3-mediated leptin activation of POMC (11). C: In this study, themethylation of Sp1 binding site of POMC promoter blocked the formation of the promoter–protein complex and consequently impaired thetranscription initiation of POMC. CM, cell membrane; NE, nuclear envelope.

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Therefore, special attention should be paid to the dietcomposition of mothers in lactation.

Acknowledgments. The authors thank Dr. Chunxiang Zhang (DeborahR. and Edgar D. Jannotta Presidential Professor, Department of Pharmacology,Rush Medical College, Rush University) for editorial assistance, Dr. Qun He(professor of Microbiology, China Agricultural University, China) for technicalsupport, Jiazi Ren for critical comments in manuscript proofreading, and YijingFu for animal care.

Funding. This work was supported by the Ministry of Agriculture, China(2012-Z29 to G.Y.).

Duality of Interest. No potential conflicts of interest relevant to thisarticle were reported.

Author Contributions. X.Z. carried out the experiments and analyzedthe data. R.Y. contributed to the animal experiments and ChIP assay. Y.J.contributed to Western blotting and manuscript editing. D.C. and B.Z. assistedin animal experiments and participated in manuscript discussion. X.Q. and H.H.contributed to cell culture and luciferase assay. L.X. contributed to the mea-surement of plasma SAM and SAH. L.W. and Y.Y. contributed to the analysis ofmilk fatty acids. G.Y. designed the experiments and wrote the manuscript. G.Y.is the guarantor of this work and, as such, had full access to all the data in thestudy and takes responsibility for the integrity of the data and the accuracy ofthe data analysis.

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