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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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:Aims/hypothesisStudies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-?H1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.MethodsCells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.ResultsGlucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-?H1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-?H1 cells, similar to those observed in human islets. Respiration in EndoC-?H1 cells was more similar to that in human islets than in INS-1 832/13 cells.Conclusions/interpretationFunctions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-?H1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-?H1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.

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