The effect of statin exposure on clonal pancreatic beta cells (INS-1) metabolism and insulin secretion
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OBJECTIVE: To investigate the effects of statin exposure on insulin secretion and metabolism in pancreatic beta cells in order to determine possible mechanisms that may contribute to the increased risk of developing Type 2 Diabetes that has been associated with taking statins. METHODS: Simvastatin and Pitavastatin were prepared to resemble the intracellular active form of the compound by converting the lactone, when present to a carboxyl group. These statins were then incubated at varying concentrations (0-200 nM) with clonal pancreatic β-cells (INS-1) for variable amounts of time (24-72 hours). Glucose-stimulated insulin secretion was measured from INS-1 cells cultured at low (4 mM) and high (11 mM) glucose with and without statin incubation. Results were measured using a homogenous time-resolved fluorescence (HTRF) insulin assay kit (Cisbio). The effect of statins on INS-1 cellular metabolism was determined by measuring oxygen consumption rate using a Seahorse XFe96 analyzer (Agilent Technologies). Statin effects on intracellular Ca2+ ([Ca2+]i) was examined in INS-1 cells cultured on glass bottom dishes loaded with the calcium indicator Fura-2 AM (Invitrogen) and mounted on an Olympus fluorescence microscope. RESULTS: Simvastatin significantly inhibited GSIS and depleted insulin content in a dose-dependent manner (25-200 nM) after 72-hour exposure. However, when normalized for insulin content, inhibition was not observed. In contrast, pitavastatin did not affect GSIS, but did decrease insulin content in a dose-dependent manner (25-200 nM). At both high and low glucose, simvastatin (200 nM) increased the frequency and amplitude of intracellular calcium ([Ca2+]) oscillations at 1, 3, and 12 mM glucose. Furthermore, simvastatin increased mitochondrial respiration at low glucose, in cells exposed to the highest dose of simvastatin (200 nM). CONCLUSION: Inhibition of GSIS by simvastatin and not pitavastatin, confirmed previous results from our lab that indicate that statins may have differential mechanistic effects on β-cells. Furthermore, intracellular calcium ([Ca2+])i imaging revealed that cells exposed to simvastatin (200 nM) had increased oscillation frequency and amplitude at basal (1, 3 mM) and high (12 mM) glucose conditions, indicating a potential difference in basal insulin secretion that may contribute to hyperinsulinemia. Lastly, mitochondrial respiration at low glucose increased in cells exposed to the highest dose of simvastatin (200 nM), suggesting that statins may play a role in decreasing energy efficiency.