Original Data

Chronic Exposure to Proline Causes Aminoacidotoxicity and Impaired Beta-Cell Function: Studies In Vitro

Zhenping Liu^1,2, Per B. Jeppesen¹, Søren Gregersen¹, Lotte Bach Larsen³, Kjeld Hermansen¹

Manuscript submitted August 31, 2015; resubmitted October 7, 2015; accepted December 12, 2015.

Keywords: type 2 diabetes, amino acid, pancreatic β-cell, cytochrome c oxidase, cholesterol biosynthesis, INS-1E, insulin

Abstract

BACKGROUND: Pancreatic islet-cell dysfunction is a hallmark in the development of diabetes, but the reasons for the primary β-cell defect are still elusive. Elevated circulating proline levels have been found in subjects with insulin resistance, obesity, and type 2 diabetes. Therefore, we assessed β-cell function, gene expressions, and cell death after long-term exposure of pancreatic β-cells to excess proline in vitro. METHODS: Isolated mouse islets and INS-1E cells were incubated with and without excess proline. After 72 h, we examined: (1) β-cell function, including basal insulin secretion (BIS) and glucose-stimulated insulin secretion (GSIS), (2) transcription factors related to insulin gene expression and enzymes involved in the tricarboxylic acid cycle and cholesterol biogenesis, (3) cellular triglycerides (TG) and cholesterol content, (4) the death of INS-1E cells and 3H thymidine incorporation, and (5) protein expression of INS-1E cells in response to proline by proteomics. RESULTS: We found that high doses of proline increased BIS and decreased GSIS in both isolated mouse islets and INS-1E cells. MafA, insulin 1, and the cytochrome c oxidase subunit VIa polypeptide 2 mRNA expressions were all downregulated, indicating that proline impaired insulin gene transcription and mitochondrial oxidative phosphorylation. In contrast, mevalonate decarboxylase gene expression was upregulated, and simultaneously, cholesterol content in INS-1E cells was enhanced. Protein profiling of INS-1E cells revealed that cytosolic non-specific dipeptidase and α enolase were differentially expressed. CONCLUSIONS: Our results indicate that proline-induced insulin transcription and mitochondrial oxidative phosphorylation impairment may contribute to the β-cell dysfunction observed in type 2 diabetes. Caution should be applied in interpreting the pathophysiological role of proline since very high proline concentrations were used in the experiments.

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