Hypermethylation of Sp1 Binding Site Suppresses Hypothalamic POMC in Neonates and May Contribute to Metabolic Disorders in Adults: Impact of Maternal Dietary CLAs

  1. Guoqing Yang1,2
  1. 1Laboratory of Animal Gene Engineering, College of Life Sciences, Henan Agricultural University, Zhengzhou, People’s Republic of China
  2. 2Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture, Henan Agricultural University, Zhengzhou, People’s Republic of China
  1. Corresponding author: Guoqing Yang, gqyang{at}yeah.net.


Epigenetic regulation of neuropeptide genes associated with central appetite control plays an important part in the development of nutritional programming. While proopiomelanocortin (POMC) is critical in appetite control, the molecular mechanism of methylation-related regulation of POMC remains unclear. Based on the report that the proximal specificity protein 1 (Sp1) binding site in POMC promoter is crucial for the leptin-mediated activation of POMC, the methylation of this site was investigated in this study in both cultured cells and postnatal mice reared by the dams with dietary supplementation of conjugated linoleic acids (CLAs). The change of milk composition made the offspring undergo the increase of food intake, suppression of POMC, attenuation of Sp1–promoter interaction, and the hypermethylation of cytosine guanine (CpG) dinucleotides at −100 and −103 within the Sp1 binding site of POMC promoter, which may be associated with the decrease of hypothalamic Sp1 and/or plasma S-adenosylhomocystein. In cultured cells, the methylation of the −100 CpG dinucleotides of the POMC promoter blocked both the formation of Sp1–promoter complex and the leptin-induced activation of POMC. In addition, a catch-up growth and adult metabolic changes like adult hyperglycemia and insulin resistance were observed in these postnatal pups, suggesting that this CLA-mediated hypermethylation may contribute, at least in part, to the metabolic disorders.


Chronic metabolic diseases, such as obesity, type 2 diabetes, hypertension, and heart diseases, result from complicated gene and environmental abnormalities (1). The poor eating habits and availability of food containing or not containing various additives in modern society make it susceptive for humans to suffer from metabolic illness (2). Moreover, the programming in response to some abnormal insult in early life can affect metabolic behavior in adulthood (35). While the major investigations are performed with the hypothesis of fetal origins of adult-onset diseases, little attention has been paid to the postnatal programming. However, like a fetus, suckling pups with developmental plasticity can also grow up into adults with metabolic disorders, which result from early malnutrition.

Hypothalamic control of appetite is considered as a focus of the study on perinatal nutritional programming (6). Hypothalamus contains not only orexigenic neurons expressing neuropeptide Y (NPY) and agouti-related protein (AgRP), but also anorexigenic neurons expressing cocaine- and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC), which exerts the key role in appetite control (7). Evidence is accumulating that DNA methylation contributes to the development of metabolic diseases by changing the expression of genes controlling whole-body energy homeostasis (8,9); however, the studies on the epigenetic regulation of POMC expression in postnatal animals are few.

The expression of POMC in hypothalamus is facilitated by leptin, the fat-derived satiety signal, through OBRb (the long-form leptin receptor) signaling pathway to inhibit appetite of animals (10). DNA deletion, DNA–protein, and protein–protein interaction assays indicate that the specificity protein 1 (Sp1) binding site around −100 of mouse POMC promoter is crucial for the transduction of OBRb signaling from signal transducer and activator of transcription 3 (STAT3) to POMC (11). Nevertheless, the methylation of this site and its consequences have not been reported yet.

One of the nutritional stimuli that causes postnatal programming is maternal dietary supplementation of conjugated linoleic acids (CLAs) (12), which is a group of positional and geometric isomers of fatty acids widely used as food additives for humans. Studies show that adult rodents fed CLAs suffer from metabolic disorders (1315).

Generally, this study was designed to investigate the methylation status of the Sp1 binding site in the POMC promoter, the methylation-related regulation of hypothalamic POMC, and metabolic changes in postnatal mice reared by the lactating dams with dietary supplementation of CLAs.

Research Design and Methods

Animals and Experiment Design

All animals in the experiments were approved by the Institutional Animal Care and Use Committee of Henan Province. Wild-type Kunming mice were purchased from Henan Laboratory Animal Center (Zhengzhou, China). Animals were kept under standard conditions with 12/12-h light/dark cycles at 22°C and had free access to water and diet.

The main purpose of the animal experiments was to study whether only 10 days’ exposure to the modified milk from the lactating dams with dietary supplementation of CLAs would make the pups undergo: 1) the alternation of methylation status and methylation-associated items, which would be found even when they were fed chow diet for a week after weaning; and 2) metabolic behaviors afterward. The 3-month-old female mice were crossed with normal adult males. Once pups were born (d0; Fig. 1), the litter size was adjusted to 12 pups per dam. All dams before or during conception were 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 chow diet supplemented with CLAs (1.5% weight for weight) (CLA-diet), while those of LA group (LA-dams), as the control, were fed chow diet supplemented with LA (1.5% weight for weight) (LA-diet). The pups suckling the milk of CLA-dams (CLA-milk) or LA-dams (LA-milk) were named LA-pups or CLA-pups, respectively. From day 14 to the end of the lactating period, the supplements were removed from the diet of dams in both groups. At day 9 (i.e., the seventh day of the supplement feeding), the milk was collected for composition analysis; at day 13 (i.e., the last day of the supplement feeding), the blood parameters were measured.

Figure 1

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

Correspondingly, the pups of both groups were suckling normal milk from day 0 (d0, Fig. 1B) to day 2 after birth and LA- or CLA-milk from days 3–13. But the pups were suckling the milk with unknown components from days 14 to 21 (weaning) since the milk analysis was not performed during this period. From day 21, the pups of both groups were fed chow diet without supplement until the end of experiment. The body weight of the offspring was traced in the whole experiment period from days 0–140, in which the measurements of plasma glucose from days 49–119 and insulin tolerance test (ITT) at day 119 were performed. The food intake was measured from days 21–35. Further measurements were performed mainly at two time points (days 13 and 28). At day 13, hypothalamic Sp1 protein, blood leptin, and insulin were assessed. At day 28, the hypothalamic gene expression, DNA methylation, 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 same schedule 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. Domenico Accili (Department of Medicine, Columbia University Medical Center, New York, New York), and the pN3-Sp1 FL-complete was from Dr. Guntram Suske (Institut für Molekularbiologie und Tumorforschung, Marburg, Germany). pXJ40-FLAG-STAT3 was described previously (16). pRL-CMV was included in the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). pMD19-T was purchased from TaKaRa (Dalian, China).

Analysis of Fatty Acids in Milk

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

Analysis of Energy Substances in Milk

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

Measurement of Metabolic Parameters in Blood and Milk

With EDTA as anticoagulant, ∼1 mL of blood sample was drawn from the abdominal vein of mice anesthetized by the intraperitoneal injection of pelltobarbitalum natricum (80 mg/kg body weight). Blood glucose was measured using OneTouch Ultra Easy (LifeScan, Shanghai, China). Milk glucose was measured by glucose oxidase method using a Glucose Assay Kit (Applygen, Beijing, China) according to the instructions. Plasma or milk insulin was measured using an [125I] insulin radioimmunoassay kit (Beijing North Institute of Biological Technology, Beijing, China) following the manual. Plasma or milk leptin levels were detected using a Mouse Leptin Immunoassay kit (R&D Systems, Minneapolis, MN) according to the instructions. Plasma SAM and SAH were determined by a commercial service of Beijing Amino Medical Research Co., Ltd. (Beijing, China). The protocol used was as described.

Body Weight and Food Intake of Postnatal Pups

From days 0–21, litter weight was recorded every 3 days and divided by pup number to get the body weight of each pup. From day 21, when pups were housed individually, to day 140, daily body weight of each pup was recorded at 5:00 p.m. From days 23–35, the food intake was measured every other day.

Real-Time PCR

The protocol of real-time PCR was the same as previously reported (11). Briefly, total RNA was isolated from hypothalamus and reversely transcribed to cDNA. Real-time PCR in triplicates was performed with SYBR Green Universal PCR Master Mix in Realplex2 (Eppendorf, Hamburg, Germany). The mRNA level of target genes was normalized by that of mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences for GAPDH, NPY, AGRP, POMC, CART,DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b are listed in Supplementary Table 1.

Bisulfite Modification of Genomic DNA

Genomic DNA of hypothalamus was isolated by phenol/chloroform method and treated for bisulfate modification using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA) according to the manufacturer's instructions. The bisulfite-modified DNA was used immediately or stored at −80°C.

Bisulfite Sequencing PCR

A DNA fragment between −163 and −21 base pairs (bp) in the POMC promoter including eight cytosines of cytosine guanine (CpG) dinucleotide was amplified by PCR. The primers are listed in Supplementary Table 1. The protocol of bisulfite sequencing PCR (BSP) was previously reported (20), with the modification of reaction condition as follows: one cycle of 94°C for 15 min, 45 cycles of 94°C for 20 s, 58°C for 30 s, and 72°C for 1 min, and a final incubation at 72°C for 3 min. The PCR products were purified and cloned into the pMD19-T vector (TaKaRa, Dalian, China) followed by sequencing analysis via a commercial service (Sangon Biotech, Shanghai, China).

Generation of Construct with Site-Specific Methylation

Site-specific methylation of the POMC promoter was generated using modified primers (Supplementary Table 1) from Sangon Biotech (Shanghai, China), which contained either a methylated or an unmethylated CpG dinucleotide at the site of interest and spanned 27 bp on either strain of DNA. Using the primers above and pGL3-POMC as a template (see schema in Fig. 4A), PCR was carried out according to the protocol in the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). After a digestion with DpnI for 2 h, the PCR products were purified using the SanPrep DNA Purification Kit (Sangon Biotech), assessed with agarose gel electrophoresis, and then used for transfection into the cells.

Cell Culture and Luciferase Assay

The methodology of cell culture and luciferase assay was the same as reported (11). Briefly, OBRb 293 cells (293-OBRb) were cultured in Dulbecco’s minimal essential medium (11995; Invitrogen, Carlsbad, CA) containing 10% FBS and transfected with relevant DNA constructs using FuGENE 6 (Roche Applied Sciences, Indianapolis, IN). After the treatment of recombinant leptin (Invitrogen) at a dose of 1 μg/well or vehicle for 20 h, the cells were lysed in 200 μL of 1× passive lysis buffer included in a Dual Luciferase Rporter Assay System (Promega). Luciferase activity was measured from cell extracts on a luminometer (Molecular Devices, Sunnyvale, CA). The firefly luciferase activity was normalized against Renilla luciferase activity.

Preparation of Nuclear Extract and Electrophoretic Mobility Shift Assay

The protocol of nuclear extraction and electrophoretic mobility shift assay (EMSA) was the same as reported (11). Briefly, the 293Rb cells were treated with hypotonic buffer. After a spin, the pellet was suspended in high-salt buffer followed by a centrifugation at 13,000 rpm for 10 min at 4°C. The supernatant was collected as the nuclear extract. Pairs of oligonucleotides unmethylated or methylated (Supplementary Table 1) were annealed, respectively. The purified double strains were labeled with 50 μCi of [32P] deoxycytidine triphosphate by klenowexo- (New England Biolabs, Ipswich, MA). The nuclear protein was incubated with the probes and resolved by 4% PAGE gel in 0.5× Tris/borate/EDTA. After the gel was dried at 80°C, supersensitive X-ray film (Kodak, Rochester, NY) was exposed for 48 h at −80°C and then developed.

Western Blotting

The protocol of Western blotting was the same as reported (11). Briefly, hypothalamus was homogenized in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO) containing 1 mmol/L phenylmethylsulfonyl fluoride. The lysate was rocked on ice for 20 min and centrifuged at 14,000g for 10 min at 4°C. The protein concentration of the supernatant was adjusted and analyzed by SDS-PAGE, and then immunoblotting was performed using the 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 containing 1% formaldehyde followed by homogenization. The homogenates of two hypothalamuses were pooled together and sonicated for DNA fragmentation. ChIP assay was conducted using 2 μg of Sp1 antibody (Abcam, Cambridge, MA) at 4°C overnight. DNA was extracted and resuspended in 25 μL of distilled water, and 2 μL of DNA solution was used for real-time PCR with primers (Supplementary Table 1). The conditions of real-time PCR were as follows: 30 s at 95°C followed by 40 cycles of 5 s at 95°C and 5 s at 60°C.


Prior to the test, mice had been fasted for 6 h. The measurement 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 intraperitoneal injection 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 ± SEM. Comparisons of data were made using two-tailed Student t tests for independent data. The significance limit was set at P < 0.05.


CLA-Diet Feeding Changed the Milk Components of Lactating Dams

At day 9, the milk components were analyzed. The concentration 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 the control group (Table 1). Concomitantly, these parameters also elevated in blood of dams (Supplementary Fig. 1), agreeing with the point that the increased glucose and insulin in milk was attributed to the diffusion from maternal circulation (22,23). Interestingly, while the milk leptin did not differ significantly between the two groups of dams (Table 1), the blood leptin level of CLA-dams increased nearly twofold compared with that in control dams (P < 0.05) (Supplementary Fig. 1). The milk of CLA-dams grossly appeared more watery than that of the control group (data not shown). The concentrations of both lactose (P < 0.05) and triglyceride (P < 0.001) in milk of CLA-dams were significantly lower than those of LA-dams (Table 1). CLAs in the milk of CLA-dams remarkably increased compared with that of LA-dams, mirroring the differences of dietary supplementation between the two groups. Besides these changes, the pattern of some other fatty acids also changed (Table 2).

Table 1

Milk composition of lactating dams

Table 2

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

CLA-Milk Caused Metabolic Responses of the Postnatal Offspring

At days 13 and 28, there were no differences in blood glucose between the two groups of pups (data not shown). At day 13, plasma insulin reduced 54.2% (P < 0.05) (Fig. 2A), and plasma leptin strikingly reduced 96.7% (P < 0.01) in CLA-pups (Fig. 2C) compared with those of the controls; but at day 28, the levels of both plasma insulin (Fig. 2B) and leptin (Fig. 2D) of CLA-pups did not differ from that of LA-pups. Interestingly, at day 28, the content of epididymal fat of CLA-pups reduced 71.1% compared with that of the control group (P < 0.01) (Fig. 2E), nonproportional to plasma leptin. The body weight 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-pups continued after day 21 (weaning) (Fig. 2G), even when both groups of pups consumed the same chow diet, consistent with the previous report (24). However, the food intake normalized by body weight of CLA-pups was significantly increased compared with that of the control group (Fig. 2H), suggesting that in CLA-pups, some events affecting appetite happened during their suckling period and lasted at least to day 33.

Figure 2

Physiological parameters of the pups. The concentration of plasma insulin (A and B) and leptin (C and D) in CLA-pups at days 13 (A and C) and 28 (B and D) was detected using the [125I] Insulin Radioimmunoassay Kit and Mouse Leptin Immunoassay Kit (R&D Systems), respectively. At day 28, the male pups were killed by cervical dislocation; the weight of epididymal fat pad normalized by their body weight was measured (E). The body weight of suckling pups (F) was recorded every 3 days. After weaning, the pups had been fed chow diet for 2 weeks; during this period, the body weight was measured every other day (G), coupled with the measurement of food intake. H: Data are the food intake normalized by body weight. Data represented the means ± SEM. *P < 0.05; **P < 0.01. WAT, white adipose tissue.

POMC Expression Was Impaired in the Hypothalamus of CLA-Pups

Based on the prediction that the alternation of hypothalamic neuropeptides would be responsible for the increase of food intake, we detected the expression of NPY, POMC, AgRP, and CART in the hypothalamus of the pups at day 28, when the difference of food intake was significant between the two groups. The data indicated that POMC mRNA of CLA-pups reduced 66.9% compared with that of LA-pups (P < 0.05) (Fig. 3A). The mRNA levels of the other three neuropeptides did not differ significantly between the two groups. It was noteworthy that at day 28, the concentration of circulating leptin in CLA-pups was normal and similar to that in LA-pups (Fig. 2D), excluding the possibility that the suppression of POMC in CLA-pups was attributed to the insufficiency of blood leptin. This phenomenon was possibly associated with the blockage of the OBRb signaling pathway.

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 of POMC promoter containing eight cytosines (boldface Cs) of CpG dinucleotides between −163 and −21 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 DNA Methylation Kit (Zymo Research). The region between −163 and −21 bp in POMC promoter was amplified using BSP system. The PCR products were cloned into the pMD19-T vector followed by DNA sequencing. Results of sequencing analysis (C) represented more than three separate experiments. Open circles represent unmethylated cytosines and filled circles the methylated cytosines. D: Sp1 was detected from the homogenates of hypothalamus of the pups at day 13 using Western blotting. E: The bands of Sp1 were scanned using CanoScan 9900F for intensity analysis. Hypothalamuses of the pups at day 28 were fixed and sonicated for DNA fragmentation. ChIP was conducted 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 using real-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 by that of SAH equals SAM/SAH ratio (K). For A and EK, the data represent means ± SEM. *P < 0.05.

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

Leptin-mediated activation of POMC requires the formation of the STAT3–Sp1–promoter complex in which the Sp1 binding site is essential (11). It is reported that the methylation of this site abrogates the binding of Sp1 (25) with DNA. These facts led us to test the hypothesis that the suppression of POMC may result from the hypermethylation of POMC promoter. Therefore, at day 28, genomic DNA was extracted from the hypothalamus of the pups for methylation analysis by BSP. The PCR products covering the 143 bp of POMC promoter region including eight CpG dinucleotides (−140, −126, −110, −103, −100, −90, −79, and −62) (Fig. 3B) were cloned. The sequencing results of individual clones from at least three independent experiments showed that the Sp1 binding sites at −100 and −103 were hypermethylated in CLA-pups compared with those of LA-pups (Fig. 3C). This finding indicated that DNA methylation patterns in postnatal hypothalamus can undergo dynamic changes in response to early nutritional alternation and that a higher level of DNA methylation correlates with a lower level of POMC expression in the neurons. To understand how the hypermethylation of Sp1 binding site happened, Sp1 protein level in hypothalamus of CLA-pups was detected. Given that Sp1 occupies its binding site and prevents it from methylation (26), we hypothesized that shortage of this protein would facilitate the access of the methyltransferases to the site and consequently promote the site-specific methylation (27). The data from Western blotting indicated that Sp1 protein decreased in hypothalamus of CLA-pups at day 13 compared with that of LA-pups (Fig. 3D and E). In contrast, the hypothalamic expression of methyltransferases DNMT1, DNMT3a, and DNMT3b at either day 13 (Fig. 3G) or 28 (Fig. 3H) did not differ between the two groups. There were 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-pups compared with that in the control group (P < 0.05) (Fig. 3J).

The Methylation of Sp1 Binding Site Impaired Sp1–Promoter Interaction and the Leptin-Mediated Activation of POMC In Vivo and In Vitro

To determine if the hypothalamic POMC suppression resulted from the failure of Sp1–promoter formation, ChIP assays were conducted. The result indicated that the formation of Sp1–promoter complex in the hypothalamus of CLA-pups at day 28 was greatly compromised compared with that in LA-pups (Fig. 3F). Collectively, these observations suggested a correlation among the hypermethylation of Sp1 binding site, the impairment of Sp1–promoter interaction, the suppression of POMC, and the increase of food intake. Based on the established 293Rb cell line (11), we performed an in vitro experiment to clarify if the methylation of the Sp1 binding site could affect the leptin-mediated action of POMC. With primers (Fig. 4A and Supplementary Table 1) containing either a methylated or an unmethylated CpG and pGL3-POMC as template, an around-the-circle PCR (28) was performed; the methylation was incorporated into the promoter, illustrated in Fig. 4A. The 5.5-kb plasmid pGL3-POMC included 440 bp of the mouse POMC promoter. Fig. 4B showed molecular weight of the PCR products was 5.5 kb, identical to that of the plasmid template (data not shown). The leptin-stimulated luciferase activity in the cells with methylated promoter was significantly lower than that with unmethylated promoter (Fig. 4C). In order to clarify if the methylation of CpG would prevent the site from binding with Sp1, an EMSA was conducted posterior to the preparation of DNA probe containing Sp1 binding site and nuclear extract from 293 cells expressing Sp1. The band representing the DNA–protein complex was observed when the probe was unmethylated and was supershifted in the addition of Sp1 antibody, but was not seen when the probe was methylated (Fig. 4D), indicating that Sp1 only bound to unmethylated DNA sequence.

Figure 4

Manipulation of site-directed methylation in vitro. A: Using primers containing either a methylated or an unmethylated CpG dinucleotide at the site of interest, PCR was carried out. The lollipop represents the methyl group in cytosine of CpG. The PCR product was assessed on agarose gel (B), digested with Dpn I, and purified for transfection assay in 293Rb cells. M, 1 kb DNA ladder (New England Biolabs). C: Luciferase activity in the cell lysate was assessed in a Dual Luciferase Reporter Assay System (Promega). 293Rb cells were transfected with relevant DNA constructs followed by the treatment of recombinant leptin or vehicle. pGL3-POMC was the original plasmid amplified in Escherichia coli. pGL3-POMC-un (unmethylated) and pGL3-POMC-me (methylated) were the products of around-the-circle PCR. Data are means ± SEM and represented three independent experiments. Nuclear protein was extracted from 293Rb cells transfected 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 the absence (Un) or presence of Sp1 antibody (Ab). The mixture was resolved by 4% PAGE gel, which was then dried and set to expose X-ray film. *P < 0.05; **P < 0.01. ctrl, control.

The Catch-up Growth and Insulin Resistance of CLA-Pups in Adulthood

The pups were exposed to CLA-milk at suckling period when their development of neural system was incomplete. To find out whether under this nutrient stimulation these pups undergo postnatal programming, we traced their body weight for 140 days and blood glucose for 70 days. The body weight of female CLA-pups caught up around day 48, and significantly exceeded around day 78, to that of female LA-pups (Fig. 5A). The body weight of male CLA-pups caught up around day 54 and kept similar afterward to that of male LA-pups (Fig. 5B). The blood glucose of CLA-pups was significantly higher than that of LA-pups from days 49–119 (Fig. 5C). The result of ITT indicated that the insulin sensitivity of CLA-pups was lower than that of LA-pups (Fig. 5D). Collectively, the CLA-pups may suffer from adult metabolic disorders associated with programming.

Figure 5

Growth curve and metabolic indicators. From weaning (day 21), the body weight of female (A) and male (B) pups was measured at 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 on mice fasted overnight. ITT (D) was performed at day 119 on mice fasted beforehand for 6 h, which included the measurement of blood glucose 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 results represented the means ± SEM. *P < 0.05; **P < 0.01.


We have reported that the leptin-mediated activation of POMC relies on a Sp1–DNA complex (11), so we hypothesized that the abrogation of the complex by DNA methylation might block this action. In this study, we demonstrated, like the previous report (25), that the methylation of Sp1 binding site blocked the formation of Sp1–promoter complex and diminished the leptin-induced activation of POMC in cultured cells. Importantly, the hypermethylation-associated POMC suppression was observed in animals accompanied with increased appetite. The hypermethylation happened at a time when circulating leptin level was normal, making it possible that the suppression of POMC resulted from the blockage of leptin signaling rather than the deficiency of blood leptin. In this study, we raised a novel hypothesis that besides the forkhead box O1–involved inhibition of leptin action (11), the methylation of Sp1 binding site abrogated the formation of Sp1–promoter complex, thus attenuated the leptin-mediated activation of POMC (Fig. 6).

Figure 6

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

Although little is known, regulation of DNA methylation in mammals can be associated with the alternation of some nutritional and developmental factors. The status of one-carbon metabolism and the activity of DNA methyltransferases are closely related to the machinery of genome-wide DNA methylation. The level of SAM, the direct donor of methyl group for the methylation of carbon 5′ position of cytosine within the CpG dinucleotide, and SAH, the product formed when the methyl group of SAM was transferred to the acceptor, is affected by dietary changes (29). As a biomarker, plasma SAM and SAH represent the ability of global methylation. It is reported that the concentration of plasma SAH negatively correlates with global methylation status in apolipoprotein E–deficient mice (30). Another work addresses that the variation of seasonal nutrition in periconceptional rural African women results in the decline of plasma SAH, which is associated with the global hypermethylation in the offspring (31). Thus, the hypermethylation identified in this study may be attributed to the low plasma SAH (31) in CLA-pups exposed to CLA-milk. Moreover, given that the Sp1 protein decreased in the hypothalamus of CLA-pups, we also supposed that the methylation of Sp1 binding site may result from the lack of Sp1, a protector of Sp1 binding site, preventing methyltransferases from access to the DNA sequence (26). This kind of sequence-specific methylation plays an important role in gene regulation in time and space in mammals (27). How the Sp1 was downregulated in the hypothalamus of CLA-pups needs to be investigated in the future.

The transient exposure to the modified milk during the suckling period made CLA-pups exhibit an abnormal phenotype. The lactating dams were fed CLA-diet only from days 3–13, because earlier onset or longer feeding caused high mortality of the pups, which would interrupt the experiment (data not shown). The retarded growth of CLA-pups, consistent with the previous report (24), was followed by a catch-up growth, especially in the females. Moreover, higher blood glucose and insulin resistance was observed in these mice in adulthood, as reported previously (12,32,33). These disorders were closely associated with the postnatal programming. It should be noted that this study has examined only postnatal anorexigenic pathways via the regulation of POMC. The growth retardation of the objectives would be also relevant to other factors such as growth hormone and glucocorticoid, which need to be addressed in a future study.

Finally, CLA is a widely used food additive for human beings. The observations in this study may provide us insights on human beings regarding the diet supplements, like CLAs, of lactating mothers that result in the change of their milk compositions, which would cause the long-term metabolic dysregulation in progeny. Therefore, special attention should be paid to the diet composition of mothers in lactation.

Article Information

Acknowledgments. The authors thank Dr. Chunxiang Zhang (Deborah R. 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 technical support, Jiazi Ren for critical comments in manuscript proofreading, and Yijing Fu 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 this article were reported.

Author Contributions. X.Z. carried out the experiments and analyzed the 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. assisted in animal experiments and participated in manuscript discussion. X.Q. and H.H. contributed to cell culture and luciferase assay. L.X. contributed to the measurement of plasma SAM and SAH. L.W. and Y.Y. contributed to the analysis of milk 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 the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

  • Received August 9, 2013.
  • Accepted December 23, 2013.

Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.


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  1. Diabetes vol. 63 no. 5 1475-1487
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