Glucose Regulation Of Gene Gmrp-1 Expression And Preliminary Functional And Ncex

OBJECTIVE: To clone and express human glucose metabolism related protein-1 (GMRP-1) gene, and its function was preliminary investigated. METHODS: Full length of GMRP-1 gene was cloned into prokaryotic expression vector pET-32a and eukaryotic expression vector pEGFP—C3 by Using subclone technique. GMRP-1 protein was highly effectively expressed in E.col ROSSET (DE3) strain and the fused protein was purified to immunize the Newzealand rabbit to generate polyclonal antibody against GMRP-1 protein .Laser confocal microscopy was employed to reveal the localization of GMRP-1 in HEK293 cell line. Using Northern Blot and Western Blot, GMRP-1mRNA and protein expression profile was demonstrated in various tissues. Using double staining immunofluorescence immunochemistry and laser cofocal scan microscopy, localization of GMRP-1 in pancreas was also demonstrated. GMRP-1mRNA and protein expression inP-TC3 cell line was analyzed by Northern Blot and Western Blot when treated with various concentrations of glucose for different time periods. Through transfection and RNA interference technologies, relationship between GMRP-1 and insulin secretion fromβ-TC3 was investigated. RESULTS: Polyclonal antibody with higher specificity and higher potency was prepared successfully. The antibody was tested by Western Blot, and the results showed a specific 56kd band when the antibody was diluted with 1:2000. GMRP-1was located in cytoplasm of HEK293 cell under laser confocalmicroscope. GMRP-1 was widely expressed in tissues and particularly high in brain, skeletal muscle, lipid tissue, liver, aorta, β -TC3 cell. Double staining immunofluorescence immunochemistry and laser cofocalmicroscope showed that GMRP-1 was located in pancreas islet, no expression was seen in excrine gland, furthermore GMRP-1 and insulin were colocalized partly. GMRP-1 expression responding to glucose was dose-and time-dependent manner in β-TC3 cells. As concentration increased, the gene and protein expression levels of GMRP-1 increased. At concentration of 20mmol/L of glucose, the highest expression level was achieved.And GMRP-1 expression increased as treatment time was prolonged and reached peak at 72h. Overexpression of GMRP-1 and gene silence of GMRP-1 using RNAi technology did not result in significant variations in insulin seretion in β-TC3 cell. CONCLUSIONS: GMRP-1 is specifically located in pancreas islet in rats, which expression in βcells in pancreas islet is regulated by glucose. And it plays a important role in functional changes of βcells resulted from high glucose. There is no direct association between GMRP-1 and insulin secretion.OBJECTIVE This study is to clone and express the gene of NCEx and to investigate primarily the relationship between NCEx and diabetic macrovascular complications. METHODS The full-length cDNA sequence was subcloned into green fluorescence protein vector pEGFP-C3 to study the location of NCEx in the HEK293 cells and mouse insulinoma β-TC3 cells by confocal microscope. The calcium concentration was measured by fluorescence spectrophotometer. The protein expression of NCEx was analyzed in the normal and diabetic monkeys and humans macrovascular tissues by Western Blotting. And the gene expression of NCEX was detected in the human aortal endothelial cell(HAEC)and human aortal smooth muscle cell (HAMSC)and human macrophage cell(THP-l) using RT-PCR. The effect of high glucose on the expression NCEX mRNA in the HAEC was further confirmed by Northern Blot. RESULTS The location of NCEX in the HEK293 cells and mouse insulinoma β-TC3 cell was membrane of the cell . Significant calcium increasing after over-expression of NCEx in the HEK293 was seen when compared that in normal control group( P<0.01). when extracellular Na at concentration of 145 mmol/L was completely exchanged by Li at concentration of 145 mmol/L, calcium concentration increased significantly in NCEx-overexpressed HEK293 cells. Addition of KC1 at end concentration of 5mmol/L during the assay did not result in calcium changes. NCEx protein was up-regulated in the aoeta of the monkey with high plasma glucose and patients with diabetes, but no changes were seen in other tissues. NCEx expression was found mainly in HAEC, but low in HASMC and THP-1. Data from RT-PCR and Northern Blotting demonstrated that NCEx expression was positively regulated by glucose, as D-glucose increased, its expression was gradually upregulated after 72h . CONCLUSIONS Reverse transportation mode of NCEx during bidirectional transportation is the main pattern which is responsible for influx of Ca2+.NCEx plays a important role in diabetic macrovascular complications.