Dataset Information

Addition of exogenous sodium palmitate increases the IAPP/insulin mRNA ratio via GPR40 in human EndoC-?H1 cells.

ABSTRACT:

Background

Enhanced IAPP production may contribute to islet amyloid formation in type 2 diabetes. The objective of this study was to determine the effects of the saturated fatty acid palmitate on IAPP levels in human ?-cells.

Methods

EndoC-?H1 cells and human islets were cultured in the presence of sodium palmitate. Effects on IAPP/insulin mRNA expression and secretion were determined using real-time qPCR/ELISA. Pharmacological activators and/or inhibitors and RNAi were used to determine the underlying mechanisms.

Results

We observed that EndoC-?H1 cells exposed to palmitate for 72?h displayed decreased expression of Pdx-1 and MafA and increased expression of thioredoxin-interacting protein (TXNIP), reduced insulin mRNA expression and glucose-induced insulin secretion, as well as increased IAPP mRNA expression and secretion. Further, these effects were independent of fatty acid oxidation, but abolished in response to GPR40 inhibition/downregulation. In human islets both a high glucose concentration and palmitate promoted increased IAPP mRNA levels, resulting in an augmented IAPP/insulin mRNA ratio. This was paralleled by elevated IAPP/insulin protein secretion and content ratios.

Conclusions

Addition of exogenous palmitate to human ?-cells increased the IAPP/insulin expression ratio, an effect contributed to by activation of GPR40. These findings may be pertinent to our understanding of the islet amyloid formation process.

SUBMITTER: Krizhanovskii C

PROVIDER: S-EPMC5649320 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Publications

Addition of exogenous sodium palmitate increases the IAPP/insulin mRNA ratio via GPR40 in human EndoC-βH1 cells.

Krizhanovskii Camilla C Fred Rikard G RG Oskarsson Marie E ME Westermark Gunilla T GT Welsh Nils N

Upsala journal of medical sciences 20170801 3

<h4>Background</h4>Enhanced IAPP production may contribute to islet amyloid formation in type 2 diabetes. The objective of this study was to determine the effects of the saturated fatty acid palmitate on IAPP levels in human β-cells.<h4>Methods</h4>EndoC-βH1 cells and human islets were cultured in the presence of sodium palmitate. Effects on IAPP/insulin mRNA expression and secretion were determined using real-time qPCR/ELISA. Pharmacological activators and/or inhibitors and RNAi were used to de ...[more]

PMID: 28980863

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Project description:OBJECTIVE:To characterize the EndoC-?H1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. METHODS:EndoC-?H1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. RESULTS:Transplantation of EndoC-?H1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-?H1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. CONCLUSIONS:Overall, the EndoC-?H1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-?H1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates.