Chronic hyperglycemia and hyperlipidemia cause deleterious effects on β-cell function. Interestingly, increased circulating amino acid (AA) levels are also a characteristic of the prediabetic and diabetic state. The chronic effects of AAs on β-cell function remain to be determined. Isolated mouse islets and INS-1E cells were incubated with or without excess leucine. After 72 h, leucine increased basal insulin secretion and impaired glucose-stimulated insulin secretion in both mouse islets and INS-1E cells, corroborating the existence of aminoacidotoxicity-induced β-cell dysfunction. This took place concomitantly with alterations in proteins and genes involved in insulin granule transport, trafficking (e.g. collapsin response mediator protein 2 and GTP-binding nuclear protein Ran), insulin signal transduction (proteasome subunit α type 6), and the oxidative phosphorylation pathway (cytochrome c oxidase). Leucine downregulated insulin 1 gene expression but upregulated pancreas duodenum homeobox 1 and insulin 2 mRNA expressions. Importantly, cholesterol (CH) accumulated in INS-1E cells concomitantly with upregulation of enzymes involved in CH biosynthesis (e.g. 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate (diphospho) decarboxylase, and squalene epoxidase) and LDL receptor, whereas triglyceride content was decreased. Our findings indicate that chronic exposure to elevated levels of leucine may have detrimental effects on both β-cell function and insulin sensitivity. Aminoacidotoxicity may play a pathogenic role in the development of type 2 diabetes.
Zhenping Liu, Per Bendix Jeppesen, Søren Gregersen, Lotte Bach Larsen and Kjeld Hermansen
Lorraine O’Driscoll, Patrick Gammell, Eadaoin McKiernan, Eoin Ryan, Per Bendix Jeppesen, Sweta Rani and Martin Clynes
The long-term potential to routinely use replacement β cells/islets as cell therapy for type 1 diabetes relies on our ability to culture such cells/islets, in vitro, while maintaining their functional status. Previous β cell studies, by ourselves and other researchers, have indicated that the glucose-stimulated insulin secretion (GSIS) phenotype is relatively unstable, in long-term culture. This study aimed to investigate phenotypic and gene expression changes associated with this loss of GSIS, using the MIN-6 cell line as model. Phenotypic differences between MIN-6(L, low passage) and MIN-6(H, high passage) were determined by ELISA (assessing GSIS and cellular (pro)insulin content), proliferation assays, phase contrast light microscopy and analysis of alkaline phosphatase expression. Differential mRNA expression was investigated using microarray, bioinformatics and real-time PCR technologies. Long-term culture was found to be associated with many phenotypic changes, including changes in growth rate and cellular morphology, as well as loss of GSIS. Microarray analyses indicate expression of many mRNAs, including many involved in regulated secretion, adhesion and proliferation, to be significantly affected by passaging/ long-term culture. Loss/reduced levels, in high passage cells, of certain transcripts associated with the mature β cell, together with increased levels of neuron/glia-associated mRNAs, suggest that, with time in culture, MIN-6 cells may revert to an early (possibly multi-potential), poorly differentiated, ‘precursor-like’ cell type. This observation is supported by increased expression of the stem cell marker, alkaline phosphatase.