| Objective:Nicotinamide phosphoribosyltransferase (Nampt) could regulate insulin secretion process of pancreaticβ-cell and it may play an important role in the occurrence and development of diabetes. This study aimed to analyze the frequency distribution of Nampt gene-948G>T and-3186C>T polymorphisms in Chinese Han type 2 diabetes mellitus (T2DM) in Hunan province and their action to development of T2DM; and to explore the effect of these polymorphisms on the therapeutic efficacy to repaglinide in the treatment of T2DM. Moreover, we were to investigate the effect of Nampt on insulin secretion and gene expressions of PDX-1 and FoxO1 which were important transcription factors associated with insulin secretion.Methods:One hundred and fifty-four T2DM patients and 142 healthy controls were genotyped for Nampt-948G>T locus by PCR-RFLP assay.170 T2DM patients and 129 healthy controls were genotyped for Nampt-3186C>T locus. Some samples were randomly selected to directly sequence to verify the reliability of PCR-RFLP assay.40 T2DM patients with different-3186C>T genotypes were selected for OATP1B1 521T>C genotyping.35 OATP1B1 521TT individuals were chose to undergo daily oral administration of 3mg repaglinide for 8 consecutive weeks. Blood samples were collected preadministration and postadministration for 8 weeks. Blood glucose was determined using enzymatic oxidation kit and insulin level was measured by radioimmunoassay. The hemoglobin A1c (HbA1c) level was measured by high performance liquid chromatography. Insulin secretion level in RIN-m5f cells was detected by rat insulin ELISA detection kit. The mRNA expression levels of pancreatic and duodenal homeobox 1 (PDX-1) and Forkhead box-containing protein O-1 (FoxO1) in RIN-m5f cells were detected by Real-time PCR method. The protein expression of PDX-1 was detected by western-blotting.Results:Nampt gene-948G>T mutation was not found in T2DM patients and healthy controls. There is no significant difference in distributive frequencies of Nampt-3186C>T genotypes and allele frequencies between Chinese Han T2DM patients and healthy controls (P=0.763, P=0.987). The elevated value of postprandial insulin (PINS) in patients with CT genotypes was significantly lower than CC genotype and TT genotype (P=0.022) after repaglinide treatment. There are significant differences in total cholesterol (CHO) between patients with CT genotype and patients with CC genotype or TT genotype (P=0.033). Insulin secretion level of RIN-m5f cells treated with repaglinide 10 nM added 100μM nicotinamide mononucleotide (NMN) were significantly higher than blank control and DMSO control group (P=0.023, P =0.029), and also significant difference compared with 50μM NMN (P=0.049). The mRNA expression level of PDX-1 of RIN-m5f cells treated with 10μM,50μM and 100μM NMN for 36h were significantly higher than control group (P=0.049, P=0.006, P=0.000), and significant differences in mRNA expression level of PDX-1 were also found between 100μM dose group and 10μMor 50μM dose group (P=0.000). The protein expression level of PDX-1 in RIN-m5f cells treated by 50μM,100μM, and 200μM NMN for 36h or 48h were no significant difference compared with control group.Conclusion:The genetic polymorphisms of-948G>T and-3186C>T in Nampt gene were not associated with development of type 2 diabetes. However,-3186C>T polymorphism affected therapetic efficacy of repaglinide in Chinese patients with T2DM. NMN stimulated the secretion of insulin and upregulated mRNA expression level of PDX-1 in RIN-m5f cells.
|
PDF Full Text Request