IRS -1 inhibition of SERCA: A potential molecular interaction mechanism for regulation of insulin secretion and calcium homeostasis in pancreatic β-cells

Although many components of the insulin-signaling pathway are present in pancreatic β-cells, the exact role of insulin feedback on β-cell function remains controversial. In a previously characterized Insulin Receptor Substrate 1 (IRS-1) overexpressing β-cell line, fractional insulin secretion and cytosolic calcium levels were increased compared to wild type and vector controls. This effect of IRS-1 may be mediated by inhibition of the sarco-endoplasmic reticulum calcium ATPase (SERCA). In this work, we investigate the potential mechanisms by which IRS-1 inhibits SERCA function. Our hypothesis is that IRS-1 interacts directly with SERCA to modulate calcium homeostasis and insulin secretion in pancreatic β-cells. Overexpression of IRS-1 resulted in a decrease in SERCA3 gene expression compared to wild-type cells. Calcium uptake in ER-enriched fractions prepared from wild-type and IRS-1 overexpressing cell lines however, shows no significant difference, indicating that neither a reduction of SERCA3b expression nor the expression of defective SERCA protein can account for the previously observed decrease in calcium uptake by IRS-1 overexpressing cells. Treatment of wild-type β-cells with thapsigargin (Tg), an inhibitor of SERCA that binds directly to the protein, resulted in an increase in cytosolic calcium and glucose-stimulated fractional insulin secretion similar to that observed in IRS-1 overexpressing cells. Given the apparent similarity in inhibition mechanism, IRS-1 most likely acts by directly binding to SERCA. To further illustrate the probability of IRS-1/SERCA interaction, we demonstrate using subcellular fractionation, immunofluorescence, and immuno-electron microscopy that IRS proteins localize to endoplasmic reticulum derived intracellular membranes where SERCA is also present, increasing the likelihood that these proteins can interact with one another. In CHO-T cells transiently transfected with SERCA3b alone or together with IRS-1, SERCA3b co-immunoprecipitates with IRS-1. This interaction is enhanced with insulin treatment. SERCA3b also co-immunoprecipitates with IRS-1 in wild type and IRS-1 overexpressing β-cell lines. Co-immunoprecipitation of IRS-1 and SERCA in CHO-T cells and β-cells confirms that these proteins do indeed interact directly. Taken together, our data indicate the existence of a novel positive feedback loop of insulin on insulin secretion in β-cells that appears to be mediated by a direct interaction between IRS proteins and SERCA