Proteomic analysis of the INS-1E secretome identify novel vitamin D-regulated proteins.

BACKGROUND: Experimental evidence indicates that vitamin D may have a beneficial role in pancreatic ß-cell function. METHODS: In the present study, stable isotope labelling by amino acids in cell culture (SILAC) in combination with liquid chromatography-tandem mass spectrometry was used to quantitatively assess the impact of the active vitamin D metabolite, 1,25-(OH)2 D3 , on global protein expression in INS-1E cell secretome. RESULTS: Twenty-one proteins were found up-regulated (≥1.5 fold changes) and three down-regulated (≤0.67) after treatment of INS-1E cells with 1,25-(OH)2 D3 . Up-regulation of proteins implicated in ß-cell growth and proliferation, such as IGF2, IGFBP7 and gelsolin, suggest that 1,25-(OH)2 D3 has a positive effect on ß-cell growth and proliferation. Moreover, modulations of several proteins implicated in prohormone processing and insulin exocytosis (IGF2, IGFBP7, Scg5, ProSAAS, Fabp5, Ptprn2 and gelsolin) appear to support the hypothesis that 1,25-(OH)2 D3 plays positive modulatory role in insulin processing and secretion. CONCLUSIONS: Together, we reveal a number of novel vitamin D-regulated proteins that may contribute to a better understanding of the reported beneficial effects of vitamin D on pancreatic ß-cells. Copyright © 2016 John Wiley & Sons, Ltd.

Discovery of novel vitamin D-regulated proteins in INS-1 cells: a proteomic approach.

BACKGROUND: Experimental evidence indicates that vitamin D may have a beneficial role in pancreatic ß-cell function. Global gene expression studies have shown that the active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3 ] modulates genes involved in ion transport, lipid metabolism and insulin secretion. METHODS: We employed stable isotope labelling by amino acids in cell culture in combination with liquid chromatography-tandem mass spectrometry to quantitatively assess the impact of two vitamin D metabolites, 1,25-(OH)2 D3 and 25-hydroxyvitamin D3 [25-(OH)D3 ], on global protein expression on a model rat ß-cell line, insulinoma-derived INS-1 cells. RESULTS: Although treatment with 1,25-(OH)2 D3 resulted in 31 differentially expressed proteins, 25-(OH)D3 had no impact on protein expression. Of these 31 proteins, 29 were upregulated, whereas two showed a decrease in abundance. Proteins whose expression levels markedly increased in the presence of 1,25-(OH)2 D3 included Crat, Hmgn2, Protein Tmsbl1 and Gdap1. One of the most important findings in this study is upregulation of proteins implicated in insulin granule motility and insulin exocytosis, suggesting a positive effect on insulin secretion. Moreover, modulation of several membrane transport proteins suggests that 1,25-(OH)2 D3 has an impact on the homeostatic regulation of ions, which is critical for most functions in the ß-cell. CONCLUSIONS: In this study, we discovered a number of novel 1,25-(OH)2 D3 -regulated proteins, which may contribute to a better understanding of the reported beneficial effects of vitamin D on pancreatic ß-cells. All in all, our findings should pave the way for future studies providing insights into molecular mechanisms by which 1,25-(OH)2 D3 regulates protein expression in pancreatic ß-cells.

Vitamin D metabolites influence expression of genes concerning cellular viability and function in insulin producing ß-cells (INS1E).

BACKGROUND: Studies have shown that vitamin D can enhance glucose-stimulated insulin secretion (GSIS) and change the expression of genes in pancreatic ß-cells. Still the mechanisms linking vitamin D and GSIS are unknown. MATERIAL AND METHODS: We used an established ß-cell line, INS1E. INS1E cells were pre-treated with 10 nM 1,25(OH)2vitamin D or 10 nM 25(OH)vitamin D for 72 h and stimulated with 22 mM glucose for 60 min. RNA was extracted for gene expression analysis. RESULTS: Expression of genes affecting viability, apoptosis and GSIS changed after pre-treatment with both 1,25(OH)2vitamin D and 25(OH)vitamin D in INS1E cells. Stimulation with glucose after pre-treatment of INS1E cells with 1,25(OH)2vitamin D resulted in 181 differentially expressed genes, whereas 526 genes were differentially expressed after pre-treatment with 25(OH)vitamin D. CONCLUSION: Vitamin D metabolites may affect pancreatic ß-cells and GSIS through changed gene expression for genes involved in ß-cell function and viability.

Vitamin D Increases Glucose Stimulated Insulin Secretion from Insulin Producing Beta Cells (INS1E).

BACKGROUND: Vitamin D affects the pancreatic beta cell function and in vitro studies have shown that vitamin D may influence insulin secretion, apoptosis, and gene regulation. However, the outcomes have differed and there has been uncertainty regarding the effect of different vitamin D metabolites on insulin secretion. OBJECTIVES: We hypothesized that vitamin D could increase insulin secretion in insulin producing beta cells and investigated the effect of 25(OH) vitamin D and 1,25(OH)2 vitamin D on insulin secretion. METHODS: The study was conducted in INS1E cells, an established insulinoma cell line from rat. The cells were divided into three groups; a control group, a group with 1,25(OH)2 vitamin D enriched medium (10 nM), and a group with 25(OH) vitamin D (10 nM) supplemented medium. After 72 hours of treatment, the cells underwent glucose stimulation at different concentrations (0, 5, 11, and 22 mM) for 60 minutes. RESULTS: INS1E cells treated with 1,25(OH)2 vitamin D showed a trend towards increased insulin secretion at all glucose concentrations compared to control cells and at 22 mM glucose, the difference was significant (18.40 +/- 1.97 vs 12.90 +/- 2.22 nmol/L, P < 0.05). However, pretreatment with 25(OH) vitamin D did not show any significant increase in insulin secretion compared to cells without vitamin D treatment. There was no difference in insulin secretion in cells not stimulated with glucose. CONCLUSIONS: Treatment with 1,25(OH)2 vitamin D combined with high levels of glucose increased insulin secretion in INS1E cells, whereas 25(OH) vitamin D had no effect. This suggests that glucose stimulated insulin secretion in INS1E beta cells appears to be related to the type of vitamin D metabolite treatment.

Pharmacology of neurotransmitter transport into secretory vesicles.

Many neuropsychiatric disorders appear to involve a disturbance of chemical neurotransmission, and the mechanism of available therapeutic agents supports this impression. Postsynaptic receptors have received considerable attention as drug targets, but some of the most successful agents influence presynaptic processes, in particular neurotransmitter reuptake. The pharmacological potential of many other presynaptic elements, and in particular the machinery responsible for loading transmitter into vesicles, has received only limited attention. The similarity of vesicular transporters to bacterial drug resistance proteins and the increasing evidence for regulation of vesicle filling and recycling suggest that the pharmacological potential of vesicular transporters has been underestimated. In this review, we discuss the pharmacological effects of psychostimulants and therapeutic agents on transmitter release.