Abstract
BackgroundProlonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB) is required for stabilization of insulin mRNA and the PTB mRNA 3′-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates.Methodology/Principal FindingsHuman islets were cultured for 24 hours in the presence of low (5.6 mM) or high glucose (20 mM). Islets were also exposed to sodium palmitate or the proinflammatory cytokines IL-1β and IFN-γ, since saturated free fatty acids and cytokines also cause islet dysfunction. RNA was then isolated for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB was analyzed by immunoblotting following culture at low or high glucose. Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146. Cytokines increased the contents of miR-146. The insulin and PTB mRNA contents were unaffected by high glucose. However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose. The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose.ConclusionProlonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism contributes to hyperglycemia-induced beta-cell dysfunction.
Highlights
Type 2 diabetes is characterized by a decrease in b-cell mass and function either alone or in combination with insulin resistance, which results in an insufficient insulin production and subsequent hyperglycemia [1,2,3]
Prolonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism contributes to hyperglycemia-induced beta-cell dysfunction
Metabolic factors, and proinflammatory cytokines promote islet dysfunction leading to loss of glucose-stimulated insulin secretion, lowered insulin contents [16,17], and b-cell death through apoptosis [18,19]
Summary
Type 2 diabetes is characterized by a decrease in b-cell mass and function either alone or in combination with insulin resistance, which results in an insufficient insulin production and subsequent hyperglycemia [1,2,3]. It has long been known that prolonged exposure to high glucose concentrations results in human islet dysfunction and death [10], a phenomenon known as glucotoxicity [11]. Culture of human islets for 4 to 9 days resulted in lowered insulin mRNA contents, insulin biosynthesis rates, insulin/proinsulin ratios, glucose-stimulated insulin release and insulin contents [10,12]. Metabolic factors, and proinflammatory cytokines promote islet dysfunction leading to loss of glucose-stimulated insulin secretion, lowered insulin contents [16,17], and b-cell death through apoptosis [18,19]. Prolonged periods of high glucose exposure results in human islet dysfunction in vitro. The aim of this study was to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates
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