EXPRESSIONAL REGULATION OF THE LEPTIN AND IT’S RECEPTOR BY N-3 PUFAs IS NOT RELATED TO THE DNA METHYLATION OF THEIR PROMOTERS IN DIET INDUCED OBESE MICE

C. Fan (1), X. Liu (1), W. Shen (1), K. Qi (1)

1 Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University

In this study, we investigated the effects of dietary n-3 PUFAs on methylation of CpG islands in the promoter regions of leptin, leptin receptor (leptin-R) and pro-opiomelanocortin (POMC) genes, as well as the effects of the n-3 PUFA status in early life on the DNA modification of the three genes’ promoters. Male C57 BL/6J mice were fed with four high-fat diets with different fat types – sunflower oil (n-3 PUFA deficient), soy oil, fish oil, and mixture of soy and fish oil (soy;fish oil =1:1), with two low-fat diets -- sunflower oil and soy oil as control. Female mice were fed with two breeding diets -- sunflower oil and mixture of soy and fish oil (soy;fish oil =1:1) during pregnancy and lactation to breed new pups. Compared to the mice fed with the control diets, the expressions of leptin in fats and leptin-R and POMC in hypothalamus were increased in the DIO mice, and n-3 PUFAs in the DIO diets reversed their increased expressions. The mean methylation level of CpG sites in the promoter regions of leptin and POMC genes showed no differences between the DIO and the control groups as well as between the n-3 PUFA containing and deficient diet groups. For the CpG sites in the promoter regions of leptin-R, no methylation was found both in all the DIO and the control groups. Still feeding the mice with the n-3 PUFA diet during maternal pregnancy and lactation did not affect CpG methylation in the leptin and POMC promoters. Our findings indicate that the promoter DNA methylation may not relate to the expressions of leptin, leptin-R and its related hypothalamic neuropeptides POMC.

SINGLE NUCLEOTIDE POLYMORPHISMS OF PROTEIN TYROSINE PHOSPHATASE 1B AND RISK OF HYPERTENSION

P. Gu (1), H. Du (1), B. Lu (1), J. Shao (1), D. Zou (2)

1 Department of Endocrinology, Nanjing General Hospital of Nanjing Military Command, Nanjing, China

2 Department of Endocrinology, Changhai Hospital, Second Military Medical University, Shanghai, China

Aim: To investigate whether Protein tyrosine phosphatase (PTP1B) Single nucleotide polymorphisms (SNPs) are associated with hypertension and hypertension related metabolic traits in Chinese subjects.

Methods A total of 239 Chinese patients with hypertension and 141 non-hypertensive subjects were screened. The genotypes of PTP-1B gene polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods.

Results: The case-control study showed associations between the frequencies of T allelic g54281 T>A and A allele of I5/37C>A and hypertension. (p<0.0001, P=0.0371 respectively). In addition, significant associations were observed between the IVS6 + G82A polymorphism and waist circumference, total cholesterol and LDL levels in hypertensive patients (P=0.0005~0.0260). And g54281 T allele was associated with higher plasma triglyceride (p=0.0390) and LDL- cholesterol concentration (P =0.0141). While g58585 T>C was associated with BMI (P =0.0308), waist circumference (P =0.0216) and HOMA-IR (p=0.047). 0.047). Multivariate logistic regression analysis showed that T allele carriers of g54281T>A and A allele carriers of I5/37C>A had higher risks of hypertension independent of age, gender, BMI, glucose levels and lipids profiles (OR: 1.79, 95% CI: 1.09– 2.96, P =0.02; OR: 1.66, 95% CI: 1.13– 2.44, P =0.01, respectively).

Conclusions: PTP1B polymorphisms contribute to pathogenesis of hypertension in Chinese subjects, and PTP1B SNPs may be involved in the development of several features including dyslipidemia and obesity in hypertension subjects.

GENETIC POLYMORPHISMS OF THE VISFATIN GENE AND ITS CORRELATION WITH DIFFERENT METABOLIC PARAMETERS IN OBESE CHILDREN

S.Q. Ooi (1), R.M.E. Chan (2), K.S. Poh (2), S.U. Gan (3), K.O. Lee (1), Y.S. Lee (2)

1 Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore

2 Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore

3 Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore

Visfatin is a novel adipocytokine which was reported to be predominantly secreted by visceral adipose tissues and had insulin-mimetic properties. Serum level of visfatin was reported to be associated with insulin resistance and obesity. Some studies revealed associations between variants of the visfatin gene and different metabolic parameters such as glucose and lipids. In this study, we aim to examine the role of visfatin gene variants in the pathophysiology of obesity in children. We genotyped the upstream promoter region, exon-intron boundaries and 11 exons of the visfatin gene in 24 DNA samples of local obese children through direct DNA sequencing. Two upstream variants -3187G>A and -1537C>T in perfect linkage disequilibrium were detected and the sequencing of the exons region of the visfatin gene also identified synonymous variants +7849T>C at exon 2 and +21426G>A at exon 7 respectively. Direct sequencing of specific regions of interest with these variants was then performed in 222 other obese paediatric subjects of the same study cohort. The upstream variants, -3187A/A and -1537T/T of minor allele frequency (MAF) of 0.451 were shown to correlate significantly with higher level of triglycerides. In addition, the variants at exon 2, +7849T>C (MAF=0.033) and at exon 7, +21426G>A (MAF=0.12) were also found to be associated with fasting glucose level and birth-weight respectively. The associations between the variants and metabolic parameters remained significant (p<0.05) after adjusting for ethnicity. These findings indicate a possible contribution of visfatin gene variants in the development of co-morbidities and cardio-metabolic risk in childhood obesity.

ELEVATED EXPRESSION AND THE FUNCTION OF FFAR2 IN PBMCs FROM TYPE 1 DIABETIC PATIENTS

G. Shi (1,2,3), W. Gu (1,2,3), X. Zhang (1,2,3), G. Ning (1,2,3)

1 Shanghai Institute of Endocrine and Metabolic Diseases

2 Department of Endocrine and Metabolic Diseases

3 Shanghai Clinical Center for Endocrine and Metabolic Diseases, Rui Jin Hospital, Shanghai Jiaotong University School of Medicine

Objective: FTo study the expression and the regulatory mechanism of FFAR2 in PBMCs from type 1 diabetic patients, and further study the function of FFAR2 in diabetic mouse model.

Methods: F‡@PBMCs from recent onset type 1 diabetic patients and healthy controls were collected, and the mRNA expression profiles were analyzed by microarray; ‡AHuman FFAR2 promoter region was cloned and the transcription activity were analyzed in macrophage cell line; ‡BThe regulatory effect of FFAR2 on LPS or PMA induced inflammation response and cell proliferation were analyzed; ‡CMulti low dose streptozocin induced diabetic mice were treated with FFAR2 agonist, Sodium acetate, and diabetic indexes were analyzed to investigate the protective effect of FFAR2 in this model.

Results:F‡@ The microarray result of PBMCs from 10 patients and 10 matched controls showed that FFAR2 mRNA increased by 4.9 fold; ‡AThe promoter region of FFAR2 (-2266 to +48) was cloned into pGL-luc plasmid; Our results showed that the promoter was activated by LPS and PMA stimulation in Raw264.7 macrophage cell line; ‡BActivation of FFAR2 by sodium acetate inhibits IL-6 mRNA expression induced by LPS in Raw264.7 3T3-L1 adipocyte; Further, Activation of FFAR2 by sodium acetate differently regulated cell proliferation in different cell lines, such as Raw264.7, THP-1 and MCF-7; ‡CSodium acetate treatment in MLDS diabetic mice significantly attenuated blood glucose and increased serum insulin compared with untreated group under feeding state; Blood glucose was also significantly lower after introperitoneal glucose challenge or insulin injection.

THE ROLE OF SIK1 IN DIABETES WITH HYPERTENSION

X. Wen (1), Q. Liao (1), W. Xu (1), J. Yu (1), K. Lu (1)

1 Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology

Objectives: Diabetes usually accompany with hypertension, which is relevant with the enhanced sodium reabsorption in renal tubular. Newly discovered salt-induced kinase 1 (SIK1) not only involved in glucose and lipid metabolism, but also activated  Na+,K+-ATPase activity through signaling pathways, leading to high blood pressure. SIK1 activity was significantly elevated in renal proximal tubule cells from Milan hypertensive rats. However, whether SIK1 is expressed in the kidney of diabetic rats has not evaluated yet. This study investigates the abundance of the renal SIK1 and Na+,K+-ATPase α1 mRNA and protein, as well as the activity of Na+,K+-ATPase in experimental diabetic rats, and also analyzes the role of SIK1 in diabetes with hypertension.

Methods: Male Wistar rats were randomly divided into three groups: normal group, type 1 dibetic(DM1) group,and type 2 diabetic(DM2) group(n = 12 in each group). Rats in normal and DM1 groups were fed regular chow, and rats in DM2 group fed high-fat diet. After 4 weeks, rats received STZ of 60mg/kg and 30mg/kg body weight intraperitoneally in DM1 and DM2 group respectively. Metabolic parameters and renal function were measured. Tail arterial blood pressure was checked. Kidney stained with HE and PAS were examined by light microscope. The level of mRNA and protein expression of SIK1 and Na+,K+-ATPase α1 in  renal tissue were detected by RT-PCR and Western blot. Meanwhile Na+,K+-ATPase activity were measured.

Results: Compared with normal group, fasting blood glucose, insulin, total cholesterol and triglycerides were increased markedly (P＜0.01).Serum creatinine, urea nitrogen, and urine albumin were significantly increased in the rats of two model groups (P＜0.01). Insulin sensitivity index decreased significantly in the diabetic rats(P＜0.01). Blood pressure in two model groups was significantly higher than normal (P＜0.01). Light microscopic examination showed markedly increased glomerular area and mesangial region, thickened glomerular basement membrane, swollen renal tubular epithelial cells, narrowed lumina in two model groups. The expression levels of SIK1 and Na+,K+-ATPase α1 mRNA  and  protein were significantly increased in diabetic rats(P <0.01). Na+,K+-ATPase activity was significantly increased in two model groups compared with normal group (P＜0.01).All of the above results between the two model groups was not significant different.

Conclusion: SIK1 mRNA expression and protein level increased in DM1 and DM2 rats. At the same time, mRNA and protein of Na+,K+-ATPase α1 were up-regulated in both DM1 and DM2 rats. Na+,K+-ATPase was over-activated. Rats in both model groups showed a characteristic pathological changes of diabetic nephropathy and hypertension. SIK1 itself or via regulating Na+,K+-ATPase involved in the process of diabetic nephropathy accompanied with hypertension in DM1 and DM2 rat. This work was supported by National Natural Science Foundation of China (No.30672730).

ASSOCIATION OF PARAOXONASE 1 GENE POLYMORPHISM WITH INTIMA-MEDIA THICKNESS (IMT) OF THE CAROTID ARTERIES IN JAPANESE TYPE 2 DIABETIC PATIENTS

I. Yamamoto (1), T. Honda (1), K. Takada (1), M. Maeda (2), S. Nonen (2), T. Motomura (3), K. Maeda (3), Y. Fujio (4), J. Azuma (2)

1 Azabu University, Kanagawa, Japan

2 Hyogo University of Health Science, Hyogo, Japan

3 NTT West Osaka Hospital, Osaka, Japan

4 Graduate School of Pharmaceutical Science, Osaka University, Osaka, Japan

Aim: We investigated the association between paraoxonase 1 (PON1)-192 genotypes and IMT values of carotid arteries that correlate with the progression of systemic atherosclerosis in diabetic Japanese patients.

Methods: One hundred and fifty-five Japanese type 2 diabetic patients aged from 40 to 79 years without major cardiovascular events and severe nephropathy were enrolled in this study.  Genotypes of the patients were determined by the PCR-RFLP method. IMT of carotid arteries of the subjects was recorded by B-mode ultrasound imaging. 

Results:  The genotypes QQ, QR and RR were found in 18(0.116), 70(0.452) and 67(0.432) patients respectively. IMT values of the RR group were significantly greater (1.08±0.41mm, n=67) than those of the Q group, consisting of patients carrying one or two Q alleles (0.95±0.27mm, n=88, P=0.023). There was no significant difference in clinical characteristics between the two groups.

Conclusion: The results of this study indicate that PON1-192RR genotype is associated with development of atherosclerosis.

ASSOCIATION OF CIDEA AND CIDEB GENE WITH OBESITY AMONG HAN CHINESE

L. Zhang (1), J. Wu (1), J. Zhang (1), Y. Dai (1), L. Bian (1), W. Wang (1)

1 Department of Epidemiology and Biostatistics, School of Public Health and Family Medicine, Capital Medical University, Beijing, China

Objective: To assess the association of the SNPs of CIDEA and CIDEB gene and the haplotype structure of CIDEA gene with obesity.

Material and Methods: A sample of 347 subjects was included in a case-control study for genotyping. Blood samples of subjects and anthropometric measurements were collected. SPSS 13.0, Haploview 4.0 and Multifactor Dimensionality Reduction (MDR) software were used for statistical analysis.

Results: The rs4796955 (G/T) and rs8092502 (C/T) polymorphisms in CIDEA gene were associated with obesity [odds ratio (OR), 1.640; 95% confidence interval (95%CI), 1.206-2.232.829; and OR, 0.971; 95%CI, 0.666-1.415, respectively]. There was no significant difference in the allelic and genotypic frequencies of the rs34740317, rs1053648 and rs3742509 polymorphisms in CIDEB gene between the two groups. Haplotype GTTGC and TTTGC in CIDEA gene were significant different between obesity and normal groups [OR, 1.612; 95%CI, 1.017-2.555.829; and OR, 0.478; 95%CI, 0.241-0.946, respectively]. By MDR analysis, the best model was a four-locus site model composed of SNPs rs45619832, rs4796955, rs8092502, and rs12962340 in the CIDEA gene.

Conclusion: No significant result of CIDEB gene was obtained in our study. The CIDEA genotypes were associated with obesity in Chinese Han Population. G allele of rs4796955 and T allele of rs8092502 were a risk facto for obesity. The strong interaction between rs45619832, rs4796955, rs8092502, and rs12962340 could increase the risk of obesity significantly. The multiplicative effect of the CIDEA genotype on obesity should be taken more attention in further researches.