摘要
目的：烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase, Nampt)能够调节胰岛β细胞分泌胰岛素的过程,在糖尿病的发生发展中可能发挥重要的直接或间接作用。本课题旨在分析Nampt基因-948G>T和-3186C>T突变在中国湖南地区汉族T2DM人群中的分布频率及其与T2DM发生的关系；探讨这些突变对瑞格列奈治疗T2DM的疗效的影响；在细胞水平研究Nampt对胰岛素分泌的调节作用及其对与胰岛素分泌相关的重要转录因子PDX-1、FoxO1基因表达的影响。
方法：采用聚合酶链式反应-限制性片断长度多态性(PCR-RFLP)对154名2型糖尿病患者和142名健康对照者进行Nampt-948G>T基因型分析,对170名2型糖尿病患者和129名健康对照者进行Nampt-3186C>T基因型分析,随机选择部分样品应用直接测序法验证PCR-RFLP结果；选取-3186C>T不同基因型的40名糖尿病患者进行OATPIB1 521T>C基因分型；选择35名OATPIB1 521TT基因型相同的个体,每天口服3mg瑞格列奈,持续8周,服药前和服药后第8周末收集血标本5ml；采用酶氧化法测定血糖、放射免疫法测定胰岛素、高效液相色谱法测定HbAlc等反映疗效的指标。用大鼠胰岛素ELISA试剂盒检测RIN-m5f细胞胰岛素分泌水平。用Real-time PCR的方法检测RIN-m5f细胞PDX-1和FoxO1的mRNA表达水平。用western-blotting的方法检测RIN-m5f细胞PDX-1蛋白表达水平。
结果：Nampt基因-948位点在糖尿病患者和健康对照中均未检测到突变。Nampt-3186C＞T各基因型及等位基因频率在中国汉族T2DM患者和健康对照人群中的分布差异无统计学意义(P=0.763,P=0.987)。瑞格列奈治疗后CT基因型患者PINS的升高值明显低于CC基因型与TT基因型(P=0.022),CT基因型患者CHO的改变与CC基因型及TT基因型比较差异也有统计学意义(P=0.033)。用瑞格列奈10nM+NMN100μM处理RIN-m5f细胞48h,与空白对照及DMSO对照组相比,胰岛素分泌量均显著增高(P=0.023,P=0.029),与NMN50μM组比较胰岛素分泌量的增高也有统计学意义(P=0.049)。10μM,50μM和100μM的NMN作用RIN-m5f细胞36h,PDX-1的mRNA表达量均上调(P值分别为0.049,0.006,0.000),100μM剂量组与10μM和50μM剂量组比较差异也有统计学意义(P值均为0.000)。50μM,100μM和200μM的NMN作用RIN-m5f细胞36h或48h,PDX-1的蛋白表达量与对照组比较差异无统计学意义。
结论：Nampt基因-948G>T和-3186C>T的基因多态性与中国2型糖尿病发病无明显相关性,但-3186C>T对瑞格列奈的疗效存在一定的影响。NMN可以调控RIN-m5f细胞中胰岛素的分泌及PDX-1的mRNA表达水平。
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.
引文
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