Hypothalamic SIRT1 prevents age-associated weight gain by improving leptin sensitivity in mice

Aims/hypothesis

Obesity is associated with ageing and increased energy intake, while restriction of energy intake improves health and longevity in multiple organisms; the NAD⁺-dependent deacetylase sirtuin 1 (SIRT1) is implicated in this process. Pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons in the arcuate nucleus (ARC) of the hypothalamus are critical for energy balance regulation, and the level of SIRT1 protein decreases with age in the ARC. In the current study we tested whether conditional Sirt1 overexpression in mouse POMC or AgRP neurons prevents age-associated weight gain and diet-induced obesity.

Methods

We targeted Sirt1 cDNA sequence into the Rosa26 locus and generated conditional Sirt1 knock-in mice. These mice were crossed with mice harbouring either Pomc-Cre or Agrp-Cre and the metabolic variables, food intake, energy expenditure and sympathetic activity in adipose tissue of the resultant mice were analysed. We also used a hypothalamic cell line to investigate the molecular mechanism by which Sirt1 overexpression modulates leptin signalling.

Results

Conditional Sirt1 overexpression in mouse POMC or AgRP neurons prevented age-associated weight gain; overexpression in POMC neurons stimulated energy expenditure via increased sympathetic activity in adipose tissue, whereas overexpression in AgRP neurons suppressed food intake. SIRT1 improved leptin sensitivity in hypothalamic neurons in vitro and in vivo by downregulating protein-tyrosine phosphatase 1B, T cell protein-tyrosine phosphatase and suppressor of cytokine signalling 3. However, these phenotypes were absent in mice consuming a high-fat, high-sucrose diet due to decreases in ARC SIRT1 protein and hypothalamic NAD⁺ levels.

Conclusions/interpretation

ARC SIRT1 is a negative regulator of energy balance, and decline in ARC SIRT1 function contributes to disruption of energy homeostasis by ageing and diet-induced obesity.     

Epigenetic silencing of WIF-1 in hepatocellular carcinomas

Purpose

To examine the expression profile and promoter methylation status of WIF-1 in hepatocellular carcinoma (HCC) and identify the possible relationship between the WIF-1 expression pattern and promoter methylation status.

Methods

Quantitative real-time PCR was performed to detect mRNA level of WIF-1 in 4 HCC cell lines, 15 paired HCC clinical samples and 3 normal liver tissues. Methylation-specific PCR and bisulfite DNA sequencing were used in methylation analysis. In vitro assays for HCC cells, colony formation and cell proliferation assay were carried out to observe the effect of WIF-1 on cell growth; TOP-flash luciferase analysis was employed to determine its role in the Wnt pathway.

Results

Quantitative real-time PCR analysis showed the extensive low expression of WIF-1 mRNA in HCC, and this down-regulation was generally dependent on the degree of methylation at its promoter region. In vitro assays indicated WIF-1 can inhibit cell growth by blocking Wnt signaling in HCC cells.

Conclusions

WIF-1 silencing as a result of its promoter hypermethylation may be a frequent event in HCC.     

Expression of MUC5AC, an early marker of pancreatobiliary cancer, is regulated by DNA methylation in the distal promoter region in cancer cells

Background and purpose

High de novo expression of MUC5AC (a gastric-type secreted mucin) is observed in many types of pancreatobiliary neoplasms, including precursor lesions. In this study, we show that the DNA methylation pattern is intimately correlated with MUC5AC expression in ten cancer cell lines (breast, lung, pancreas, and colon).

Methods

The CpG methylation status of the MUC5AC promoter from ?3855 to +321 was mapped using MassARRAY analysis, which utilizes base-specific cleavage of nucleic acids. ChIP assays and micro-RNA (miRNA) microarray expression profiling were also carried out in both MUC5AC-positive cells and in those with no or low MUC5AC expression.

Results

In the distal region from ?3718 to ?3670 of the promoter, MUC5AC-negative cancer cells (e.g., MDA-MB-453) were highly methylated, whereas MUC5AC-positive cells (e.g., MCF-7) had low methylation levels. The modification status of histone H3 lysine 9 (H3-K9) was also closely related to MUC5AC expression. Expression levels of miRNAs in the cancer cells were not correlated with MUC5AC expression.

Conclusion

Our results indicate that MUC5AC is regulated by CpG methylation and histone H3-K9 modification of the MUC5AC promoter distal region, but not by miRNAs. An understanding of the epigenetic regulation of MUC5AC may be of importance for the diagnosis of carcinogenic risk in the pancreatobiliary system.     

Hepatic DNA methylation modifications in early development of rats resulting from perinatal BPA exposure contribute to insulin resistance in adulthood

Aims/hypothesis

Perinatal exposure to bisphenol A (BPA), a widely distributed environmental endocrine disruptor, is associated with insulin resistance and diabetes in offspring. The underlying molecular mechanisms could involve epigenetics, as adverse effects induced by environmental exposure in early life are suggested through DNA methylation. In this study we sought to elucidate the relationship between perinatal BPA exposure and alteration of hepatic DNA methylation.

Methods

Pregnant Wistar rats were administered BPA (50 μg/kg/day) or corn oil by oral gavage throughout gestation and lactation. Variables associated with insulin resistance and hepatic DNA methylation were examined at postnatal week 3 and week 21 in male offspring.

Results

In BPA-treated offspring, serum insulin and HOMA-insulin resistance were increased, and the insulin sensitivity index and hepatic glycogen storage were decreased compared with controls at week 21. At week 3, none of these variables were significantly changed. However, hepatic global DNA methylation was decreased, accompanied by overexpression of DNA methyltransferase 3B mRNA at week 3. Meanwhile, perinatal exposure to BPA induced promoter hypermethylation and a reduction in gene expression of hepatic glucokinase. Moreover, increased promoter hypermethylation of Gck became more pronounced in BPA-treated offspring at week 21.

Conclusions/interpretation

Abnormal DNA methylation in hepatic tissue precedes development of insulin resistance induced by perinatal BPA exposure. These findings support the potential role of epigenetics in fetal reprogramming by BPA-induced metabolic disorders.     

Young men with low birthweight exhibit decreased plasticity of genome-wide muscle DNA methylation by high-fat overfeeding

Aims/hypothesis

The association between low birthweight (LBW) and risk of developing type 2 diabetes may involve epigenetic mechanisms, with skeletal muscle being a prime target tissue. Differential DNA methylation patterns have been observed in single genes in muscle tissue from type 2 diabetic and LBW individuals, and we recently showed multiple DNA methylation changes during short-term high-fat overfeeding in muscle of healthy people. In a randomised crossover study, we analysed genome-wide DNA promoter methylation in skeletal muscle of 17 young LBW men and 23 matched normal birthweight (NBW) men after a control and a 5 day high-fat overfeeding diet.

Methods

DNA methylation was measured using Illumina’s Infinium BeadArray covering 27,578 CpG sites representing 14,475 different genes.

Results

After correction for multiple comparisons, DNA methylation levels were found to be similar in the LBW and NBW groups during the control diet. Whereas widespread DNA methylation changes were observed in the NBW group in response to high-fat overfeeding, only a few methylation changes were seen in the LBW group (χ², p?<?0.001).

Conclusions/interpretation

Our results indicate lower DNA methylation plasticity in skeletal muscle from LBW vs NBW men, potentially contributing to understanding the link between LBW and increased risk of type 2 diabetes.     

DNA Methylation of NDRG2 in Gastric Cancer and Its Clinical Significance

Background

Gastric cancer is one of the most common digestive malignancies worldwide. N-myc downstream-regulated gene 2 (NDRG2) is a differentiation-related gene that is considered to be a metastasis suppressor gene. In this study, we examined the expression and DNA methylation of NDRG2 in gastric cancer cell lines and tissues, as well as its clinical significance.

Methods

Six gastric cancer cell lines and 42 paired normal and gastric cancer tissue samples were used to assess NDRG2 mRNA expression using RT-PCR. NDRG2 DNA methylation status was evaluated by methylation-specific PCR (MSP) in gastric cancer cell lines and tissues. The suppression of NDRG2 in BGC823 cells by siRNA transfection was utilized to detect the role of NDRG2 in gastric cancer progression.

Results

NDRG2 mRNA was down-regulated in gastric cancer cell lines and tissues, and its expression was just related to lymph node metastasis (p = 0.032). MSP showed methylation of NDRG2 in 54.0 % (47/87) of primary gastric cancer specimens and in 20.0 % (16/80) of corresponding nonmalignant gastric tissues. NDRG2 methylation was related to depth of tumor invasion, Borrmann classification and TNM stage (p < 0.05). Upon treatment with 5-aza-2′-deoxycytidine and trichostatin A, NDRG2 expression was upregulated in HGC27 cells, and demethylation of the highly metastatic cell line, MKN45, inhibited cell invasion. Furthermore, the suppression of NDRG2 by siRNA transfection enhanced BGC823 cells invasion.

Conclusions

Our results suggest that the aberrant methylation of NDRG2 may be mainly responsible for its downregulation in gastric cancer, and may play an important role in the metastasis of gastric cancer.     

Detection of TFPI2 Methylation in the Serum of Hepatocellular Carcinoma Patients

Background

DNA methylation plays a key role in hepatocellular carcinogenesis and progression. Analysis of aberrant methylation in serum DNA might provide a strategy for noninvasive detection of hepatocellular carcinoma (HCC).

Methods

To explore the feasibility of this approach, we compared TFPI2 methylation status in serum samples of HCC, chronic hepatitis B (CHB) patients and normal control groups using methylation-specific polymerase chain reaction.

Results

Our results showed that the percentage of serum TFPI2 promoter methylation was significantly higher in the HCC group (46.5 %, 20/43) compared with the CHB group (16.7 %, 4/24; p = 0.015) and the normal control group (19.2 %, 5/26; p = 0.022), respectively, indicating that TFPI2 methylation frequently existed in the serum of HCC patients. In our study, the detection rate of HCC using serum TFPI2 methylation was 46.5 % (20/43), which was quite close to the reported detection rate of α-fetoprotein (54 %). In cases where we combined both markers, the detection rate was 61.0 %, suggesting that serum TFPI2 methylation could be used as a potential marker for noninvasive detection of HCC. Then, we evaluated the correlation between the serum TFPI2 methylation status of HCC patients and their clinicopathological parameters. Patients with advanced TNM stage (III–IV) showed a significantly elevated serum methylation percentage of TFPI2 in comparison with those with early TNM stage (I–II) (p = 0.025). Moreover, TFPI2 methylation was observed more frequently according to the progression of TNM stage.

Conclusions

Our present study suggested that TFPI2 methylation in serum tended to be detected more easily in patients with advanced HCC and might be used as a predictor of HCC progression.     

Identification of Disease-Associated DNA Methylation in B Cells from Crohn’s Disease and Ulcerative Colitis Patients

Background

Changes in the methylation status of inflammatory bowel disease (IBD)-associated genes could significantly alter levels of gene expression, thereby contributing to disease onset and progression. We previously identified seven disease-associated DNA methylation loci from intestinal tissues of IBD patients using the Illumina GoldenGate BeadArray assay.

Aims

In this study, we extended this approach to identify IBD-associated changes in DNA methylation in B cells from 18 IBD patients [9 Crohn??s disease (CD) and 9 ulcerative colitis (UC)]. B cell DNA methylation markers are particularly favorable for diagnosis due to the convenient access to peripheral blood.

Methods

We examined DNA methylation profiles of B cell lines using the Illumina GoldenGate BeadArray assay. Disease-associated CpGs/genes with changes in DNA methylation were identified by comparison of methylation profiles between B cell lines from IBD patients and their siblings without IBD. BeadArray data were validated using a bisulfite polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) method. To verify that observed changes in DNA methylation were not due to virus transformation, we compared specific CpG DNA methylation levels of GADD45A and POMC between B cell lines and matching peripheral blood B lymphocytes from five individuals.

Results

Using this approach with strict statistical analysis, we identified 11 IBD-associated CpG sites, 14 CD-specific CpG sites, and 24 UC-specific CpG sites with methylation changes in B cells.

Conclusions

IBD- and subtype-specific changes in DNA methylation were identified in B cells from IBD patients. Many of these genes have important immune and inflammatory response functions including several loci within the interleukin (IL)-12/IL-23 pathway.     

Hypothalamic pro-opiomelanocortin gene delivery ameliorates obesity and glucose intolerance in aged rats

Aims/hypothesis Age-related obesity is associated with impaired hypothalamic pro-opiomelanocortin (Pomc) gene expression. We assessed whether overproduction of POMC in the hypothalamus ameliorates age-related obesity in rats.Methods Recombinant adeno-associated virus (rAAV) encoding Pomc (rAAV-Pomc) or control vector was delivered bilaterally into the basomedial hypothalamus of aged obese rats with coordinates targeting the arcuate nucleus. Energy balance, glucose metabolism, brown adipose tissue thermogenesis and mRNA levels of hypothalamic neuropeptides and melanocortin receptors were assessed.Results Forty-two days after Pomc gene delivery, hypothalamic Pomc expression increased 12-fold while agouti-related protein and neuropeptide Y mRNA levels remained unchanged. Using a punch technique, we detected the highest Pomc RNA level in the arcuate nucleus. Pomc overexpression reduced food consumption from day 10 after vector injection, but this anorexic effect abated by day 30. In contrast, there was a steady decrease in body weight without apparent attenuation. Pomc gene delivery decreased visceral adiposity and induced uncoupling protein 1 in brown adipose tissue in aged rats. Serum NEFA and triglyceride levels were also diminished by rAAV-Pomc treatment. Improved glucose metabolism and insulin sensitivity were observed on day 36 but not day 20 after Pomc gene delivery. The expression of hypothalamic melanocortin 3 and 4 receptor decreased by 17% and 25%, respectively in rAAV-Pomc rats.Conclusions/interpretation This study demonstrates that targeted Pomc gene therapy in the hypothalamus reduces body weight and visceral adiposity, and improves glucose and fat metabolism in aged obese rats. Thus long-term activation of the central melanocortin system may be a viable strategy to combat age-related obesity and diabetes.     

Identification of CpG-SNPs associated with type 2 diabetes and differential DNA methylation in human pancreatic islets

Aims/hypothesis

To date, the molecular function of most of the reported type 2 diabetes-associated loci remains unknown. The introduction or removal of cytosine–phosphate–guanine (CpG) dinucleotides, which are possible sites of DNA methylation, has been suggested as a potential mechanism through which single-nucleotide polymorphisms (SNPs) can affect gene function via epigenetics. The aim of this study was to examine if any of 40 SNPs previously associated with type 2 diabetes introduce or remove a CpG site and if these CpG-SNPs are associated with differential DNA methylation in pancreatic islets of 84 human donors.

Methods

DNA methylation was analysed using pyrosequencing.

Results

We found that 19 of 40 (48%) type 2 diabetes-associated SNPs introduce or remove a CpG site. Successful DNA methylation data were generated for 16 of these 19 CpG-SNP loci, representing the candidate genes TCF7L2, KCNQ1, PPARG, HHEX, CDKN2A, SLC30A8, DUSP9, CDKAL1, ADCY5, SRR, WFS1, IRS1, DUSP8, HMGA2, TSPAN8 and CHCHD9. All analysed CpG-SNPs were associated with differential DNA methylation of the CpG-SNP site in human islets. Moreover, six CpG-SNPs, representing TCF7L2, KCNQ1, CDKN2A, ADCY5, WFS1 and HMGA2, were also associated with DNA methylation of surrounding CpG sites. Some of the type 2 diabetes CpG-SNP sites that exhibit differential DNA methylation were further associated with gene expression, alternative splicing events determined by splice index, and hormone secretion in the human islets. The 19 type 2 diabetes-associated CpG-SNPs are in strong linkage disequilibrium (r ²?>?0.8) with a total of 295 SNPs, including 91 CpG-SNPs.

Conclusions/interpretation

Our results suggest that the introduction or removal of a CpG site may be a molecular mechanism through which some of the type 2 diabetes SNPs affect gene function via differential DNA methylation and consequently contributes to the phenotype of the disease.     

Estrogen receptor promoter methylation predicts survival in low-grade ovarian carcinoma patients

Purpose

Ovarian carcinoma is the third most common gynecological cancer and only short recurrence-free survival and overall survival times are archived. The role of the estrogen receptor expression is well studied in breast cancer and breast cancer cell lines. Patients with positive estrogen receptor expression have a lower risk for recurrence and a better overall survival. Previous studies have shown that ESR1 methylation influences ovarian cancer development and might thus play a role regarding prognosis of ovarian carcinoma.

Methods

A total of 75 patients were identified that were treated for ovarian carcinoma by debulking surgery and adjuvant standard chemotherapy. Isolation and bisulfite treatment of genomic DNA from serial sections of surgically resected ovarian carcinoma tissue was performed using commercially available kits. For the detection of methylated ESR1 promoter sequences, real-time methylation-specific PCR was used.

Results

Promoter methylation did not show a correlation between clinical–pathological data for all patients. However, within the subgroup of low-grade ovarian carcinoma patients and patients with an ovarian tumor of low malignant potential methylation of the ESR1 promoter inversely correlated with survival (p = 0.031).

Conclusions

Although small numbers of ovarian carcinoma patients were analyzed, methylation status might be useful as a prognostic marker within the subgroup of low-grade ovarian carcinoma patients. Further studies should investigate a larger cohort and also address the use of demethylation agents with respect to improve patient’s prognosis in this subgroup of ovarian carcinoma patients.