The insulin receptor signaling pathway is present in beta-cells and is believed to be important in beta-cell function. We show here that insulin directly regulates beta-cell function in isolated rodent islets. Long-term insulin treatment caused a sustained increase in [Ca(2+)](i) and enhanced glucose-stimulated insulin secretion in rat islets, but failed to increase insulin content. Chronic activation of insulin receptor signaling by IRS-1 overexpression in the beta-cell inhibited gene expression of SERCA3, an endoplasmic reticulum Ca(2+)-ATPase. Insulin gene transcription was stimulated by insulin receptor signaling and insulin mimetic compound (L-783 281) in a glucose- and Grb2-dependent manner. Thus, beta-cell SERCA3 is a target for insulin regulation, which implies that beta-cell Ca(2+) homeostasis is regulated in an autocrine feedback loop by insulin. This study identifies a novel regulatory pathway of insulin secretion at the molecular level with two main components: (1) regulation of intracellular Ca(2+) homeostasis via SERCA3 and (2) regulation of insulin gene expression.

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.

We have previously characterized an insulin receptor substrate 1 (IRS-1)-overexpressing ␤-cell line. These ␤-cells demonstrated elevated fractional insulin secretion and elevated cytosolic Ca 2؉ levels compared with wildtype and vector controls. This effect of IRS-1 may be mediated via an interaction with the sarco-endoplasmic reticulum calcium ATPase (SERCA). Here we demonstrate that IRS-1 and IRS-2 localize to an endoplasmic reticulum (ER)-enriched fraction in ␤-cells using subcellular fractionation. We also observe co-localization of both IRS-1 and IRS-2 with ER marker proteins using immunofluorescent confocal microscopy. Furthermore, immuno-electron microscopy studies confirm that IRS-1 and SERCA3b localize to vesicles derived from the ER. In Chinese ham- Taken together, our data suggest that interaction between IRS proteins and SERCA is an important regulatory step in insulin secretion.The pancreatic ␤-cell plays a key role in glucose homeostasis by secretion of the hormone insulin. The first step in insulin secretion is the metabolism of glucose in ␤-cells (1-3). Glucose enters the ␤-cell through GLUT2 transporters on the plasma membrane. Glucose metabolism begins with glucokinase, the ␤-cell glucose sensor (4), and results in an increase in the intracellular ATP/ADP ratio. This causes closure of ATP-dependent K ϩ channels and ␤-cell plasma membrane depolarization. The depolarization event leads to Ca 2ϩ influx through voltage-gated L-type Ca 2ϩ channels (5, 6). Increased cytosolic Ca 2ϩ stimulates insulin exocytosis by the ␤-cell through Ca 2ϩ -dependent protein kinase pathways.Insulin acts on a variety of tissues by binding to the insulin receptor (IR).1 Insulin binds to the dimerized insulin receptor and causes activation of the catalytic tyrosine kinase in the IR ␤-subunit. The IR tyrosine kinase then autophosphorylates intracellular tyrosine residues on the insulin receptor itself, which act as docking sites for insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) and Shc. These proteins are then phosphorylated by the insulin receptor and interact with several downstream effectors including phosphoinositide 3-kinase and Grb2. These effectors mediate glucose transport, cell growth, and various other important cellular functions.A growing body of evidence has confirmed that the insulin receptor-signaling pathway is active in the pancreatic ␤-cell and is involved in regulating key cellular processes (7-16). Insulin stimulation of the ␤-cell IR results in tyrosine phosphorylation of the catalytic IR ␤-subunit and IRS proteins (8). Mice with a pancreatic ␤-cell-specific knockout of IR exhibit hyperinsulinemia and impaired glucose tolerance that develops after 6 months (11). In addition, these mice lose their acute firstphase glucose-stimulated insulin secretion response. The loss of IRS-1 leads to mild insulin resistance, hyperinsulinemia, and ␤-cell hyperplasia but no overt diabetic phenotype (17)(18)(19)(20). Islets and ␤-cells derived from these IRS-1 knockout mice show decreased insulin content of 51 ...