Insulin Receptor Substrate 1-induced Inhibition of Endoplasmic Reticulum Ca2+ Uptake in β-Cells

Abstract

To understand the role of the insulin receptor pathway in ␤-cell function, we have generated stable ␤-cells (␤IRS1-A) that overexpress by 2-fold the insulin receptor substrate-1 (IRS-1) and compared them to vector-expressing controls. IRS-1 overexpression dramatically increased basal cytosolic Ca 2؉ levels from 81 to 278 nM, but it did not affect Ca 2؉ response to glucose. Overexpression of the insulin receptor also caused an increase in cytosolic Ca 2؉ . Increased cytosolic Ca 2؉ was due to inhibition of Ca 2؉ uptake by the endoplasmic reticulum, because endoplasmic reticulum Ca 2؉ uptake and content were reduced in ␤IRS1-A cells. Fractional insulin secretion was significantly increased 2-fold, and there was a decrease in ␤IRS1-A insulin content and insulin biosynthesis. Steady-state insulin mRNA levels and glucose-stimulated ATP were unchanged. High IRS-1 levels also reduced ␤-cell proliferation. These data demonstrate a direct link between the insulin receptor signaling pathway and the Ca 2؉ -dependent pathways regulating insulin secretion of ␤-cells. We postulate that during regulated insulin secretion, released insulin binds the ␤-cell insulin receptor and activates IRS-1, thus further increasing cytosolic Ca 2؉ by reducing Ca 2؉ uptake. We suggest the existence of a novel pathway of autocrine regulation of intracellular Ca 2؉ homeostasis and insulin secretion in the ␤-cell of the endocrine pancreas.

Roles of Insulin Receptor Substrate-1, Phosphatidylinositol 3-Kinase, and Release of Intracellular Ca2+ Stores in Insulin-stimulated Insulin Secretion in β-Cells

“…Our data have shown that insulin-stimulated insulin secretion is mediated by functional insulin receptors (18), IRS-1, and PI3-K activation. Activation of these substrates by insulin in ␤-cells has previously been reported (16,19), and our results illustrate that these effects are directly linked to insulin secretion and increases in [Ca 2ϩ ] i . The increase in [Ca 2ϩ ] i evoked by insulin appears to be mediated by release of Ca 2ϩ from intracellular Ca 2ϩ stores based on the localization of the increase, the effects of thapsigargin, and the occurrence of [Ca 2ϩ ] i changes in the absence of extracellular Ca 2ϩ .…”

“…This report also showed that insulin could evoke an increase in intracellular [Ca 2ϩ ] ([Ca 2ϩ ] i ). A recent study with clonal ␤-cells demonstrated that overexpression of IRS-1 and insulin receptor elevated [Ca 2ϩ ] i levels and enhanced fractional insulin secretion (19), in good agree-ment with the studies on application of exogenous insulin.…”

“…One possible explanation of the increases in [Ca 2ϩ ] i is due to the inhibition of SERCA pumps on the ER. IRS-1 has been shown to interact with SERCA proteins (37), and it has recently been demonstrated that overexpression of IRS-1 in the clonal ␤-cell line ␤TC6-F7 inhibits the uptake of Ca 2ϩ into ER, possibly by inhibition of the SERCA pump (19 We have shown that inhibition of PI3-K blocks both the [Ca 2ϩ ] i increase and exocytosis evoked by insulin. PI3-K may be involved in releasing intracellular Ca 2ϩ through an interaction with the ER, and the resulting rise in [Ca 2ϩ ] i may be sufficient for activating secretion; however, we cannot rule out that PI3-K has other roles in activating exocytosis.…”

Biogenesis of endoplasmic reticulum proteins involved in Ca2+ signalling during megakaryocytic differentiation: an in vitro study

“…This might imply a reconstruction of the ER from pre-existing SERCA2b-and InsP $ -RI-associated intracellular Ca# + stores towards additional SERCA3-and InsP $ -RII-and III-associated ER subcompartments in connection with plasma membrane. Again, this new ER network may be needed to create links between agonists and their fine spatial tuning of Ca# + -dependent signalling processes, as shown recently for SERCA and insulin-receptor substrate, IRS-1 [53].…”

IRS proteins and beta-cell function.

“…Experiments performed with a ␤-cell tumor line derived from IRS1 -/-mice have revealed that insulin stimulation fails to elevate cytosolic Ca 2+ in these IRS-1-deficient cells; insulin evokes release of intracellular cell stores of Ca 2+ in wild-type transformed ␤-cells (39). Interestingly, studies in the INS-1 ␤-cell line have demonstrated that overexpression of IRS-1 increases cytosolic Ca 2+ levels due to inhibition of uptake by the endoplasmic reticulum (40). Owing perhaps to this dysregulation of intracellular Ca 2+ , insulin secretion was increased two-fold in INS cells.…”