Glucose effects on beta-cell growth and survival require activation of insulin receptors and insulin receptor substrate 2

Abstract

Insulin and insulin-like growth factor I (IGF-I) are ubiquitous hormones that regulate growth and metabolism of most mammalian cells, including pancreatic beta-cells. In addition to being an insulin secretagogue, glucose regulates proliferation and survival of beta-cells. However, it is unclear whether the latter effects of glucose occur secondary to autocrine activation of insulin signaling proteins by secreted insulin. To examine this possibility we studied the effects of exogenous glucose or insulin in beta-cell lines completely lacking either insulin receptors (betaIRKO) or insulin receptor substrate 2 (betaIRS2KO). Exogenous addition of either insulin or glucose activated proteins in the insulin signaling pathway in control beta-cell lines with the effects of insulin peaking earlier than glucose. Insulin stimulation of betaIRKO and betaIRS2KO cells led to blunted activation of phosphatidylinositol 3-kinase and Akt kinase, while surprisingly, glucose failed to activate either kinase but phosphorylated extracellular signal-regulated kinase. Control beta-cells exhibited low expression of IGF-1 receptors compared to compensatory upregulation in betaIRKO cells. The signaling data support the slow growth and reduced DNA and protein synthesis in betaIRKO and betaIRS2KO cells in response to glucose stimulation. Together, these studies provide compelling evidence that the growth and survival effects of glucose on beta-cells require activation of proteins in the insulin signaling pathway.

FIG. 1.

Insulin activates proteins in the insulin/IGF-1 signaling pathway in control β-cells, but not in βIRKO cells. Cells were stimulated with 100 nM insulin for 0, 1, 5, or 15 min. (a) Tyrosine phosphorylation of the insulin receptor and IGF-1 receptor. (b) Tyrosine phosphorylation of IRS-1 and IRS-2. The immunoprecipitates (IP) with anti-IR, anti-IRS-1, or anti-IRS-2 from the indicated cell lysates with or without insulin stimulation were immunoblotted (IB) with anti-PY (top panels) or the p85 antibody (bottom panels). (c) PI3K activity associated with the phosphotyrosine complex. PI3K activity was measured after immunoprecipitation with anti-PY from the indicated cell lysates. (d) Phosphorylation of Akt and ERK. The cell lysates were immunoblotted with the indicated antibodies. Representative blots are shown from three independent experiments.

FIG. 2.

Glucose activates proteins in the insulin/IGF-1 signaling pathway in control β-cells, but not in βIRKO cells. Cells were treated with glucose (25 mM) for 0, 1, 5, or 15 min. (a) Tyrosine phosphorylation of the insulin receptor and IGF-1 receptor. (b) Tyrosine phosphorylation of IRS-1 and IRS-2. The immunoprecipitates (IP) with anti-IR, anti-IRS-1, or anti-IRS-2 from the indicated cell lysates with or without glucose stimulation were immunoblotted (IB) with anti-PY (top panels) or the p85 antibody (bottom panels). (c) PI3K activity associated with the phosphotyrosine complex. PI3K activity was measured in the immunoprecipitation with anti-PY from the indicated cell lysates. (d) Phosphorylation of Akt and ERK. The cell lysates were immunoblotted with the indicated antibodies. Representative blots are shown from three independent experiments.

FIG. 3.

βIRKO cells exhibit a compensatory increase in expression of IGF-1 receptors. (a) To determine the presence and cross-reactivity of insulin and IGF-1 receptors we treated control β-cells with IGF-I (100 nM) and performed immunoprecipitation and blotting for either the insulin receptor or the IGF-1 receptor. For experiments in panels b to d, cells were treated with IGF-I (100 nM) for 0, 1, 5 or 15 min. (b) Tyrosine phosphorylation of insulin receptor (right panel) and IGF-1 receptor (left panel). (c) Tyrosine phosphorylation of IRS-1 and IRS-2. The immunoprecipitates (IP) with anti-IRS-1 or anti-IRS-2 from the indicated cell lysates with or without IGF-1 stimulation were immunoblotted (IB) with anti-PY (top panels) or the p85 antibody (bottom panels). (d) PI3K activity associated with the phosphotyrosine complex. PI3K activity was measured in the immunoprecipitation reaction with anti-PY from the indicated cell lysates. (e) Phosphorylation of Akt and ERK. The cell lysates were immunoblotted with the indicated antibodies. Representative blots are shown from three independent experiments.

FIG. 4.

Insulin signaling protects β-cells from apoptosis and promotes growth. (a) Glucose protects β-cells from serum deprivation-induced apoptosis. Cells were cultured in the indicated glucose concentrations without serum for 24 h. The levels of apoptosis were assessed by determining the levels of cleaved caspase-3 by Western blotting (IB). Shown is a representative result from three independent experiments. Data (lower graph) for the changes are expressed as the ratio of cleaved caspase-3 to total caspase. *, P < 0.05 versus the respective control (FCS plus glucose). (b) Insulin protects β-cells from serum deprivation-induced apoptosis. Cells were cultured in the indicated concentration of insulin without serum for 24 h. The levels of apoptosis were assessed using an enzyme-linked immunosorbent assay for nucleosomal DNA as described in Materials and Methods. Data are expressed as the percentage of cells rescued from apoptosis. Data are from three independent experiments. (c) Glucose promotes activation of cleaved caspase-3 in βIRKO but not in control β-cells. Data are representative of three independent experiments. (d) Blocking the insulin receptor with anti-insulin receptor antibody enhances serum depletion-induced cleaved caspase-3 in control β-cells. Data are representative of three independent experiments. (e) Insulin or glucose stimulates p4E-BP1 in control but not βIRKO β-cells. Cells were stimulated with insulin (100 nM) or glucose (20 mM) for 30 min, and the cell lysates were subjected to immunoblotting (IB) with anti-p-4E-BP1. Data are representative of three independent experiments. (f) βIRKO β-cells exhibit significantly reduced [³H]thymidine incorporation compared to control β-cells over a 96-h incubation period. *, P < 0.05 versus the respective control (day 0); #, P < 0.05 for βIRKO versus control.

FIG. 5.

Glucose and insulin promote expression of GLUT2. (a) Insulin and glucose enhance expression of GLUT2. Cells were starved overnight and stimulated with insulin (100 nM), IGF-I (100 nM), or glucose (20 mM) for 12 h. The right panel shows the the data normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. *, P < 0.05 for untreated versus insulin or IGF-I or glucose. Data are representative of three independent experiments. (b) Inhibition of PI3K blocks the effect of glucose on the expression of GLUT2. Cells were preincubated with 10 μM LY294002 for 30 min and stimulated with glucose (20 mM) in the presence of the inhibitor for 12 h. The right panel shows data normalized to GAPDH levels. *, P < 0.05 for untreated versus glucose; #, P < 0.05 for glucose versus glucose plus LY. Data are representative of three independent experiments. (c) Expression of the dominant negative p85 in control β-cells downregulates the expression of GLUT2. Cells were infected with the indicated adenovirus for 48 h and stimulated with glucose (20 mM) for 12 h. Total RNA was isolated and subjected to semiquantitative RT-PCR for detecting GLUT2. The lower panel shows quantification of the data. Comparisons with controls: *, P < 0.05 for LacZ glucose treated versus LacZ glucose untreated or for p85 glucose untreated versus LacZ glucose untreated; #, P < 0.05 for p85 glucose treated versus LacZ glucose treated. Comparisons with βIRKOs: #, P < 0.05 for p85 glucose untreated or glucose treated versus LacZ glucose untreated versus glucose treated. For comparisons of LacZ controls versus LacZ βIRKOs: *, P < 0.05 for glucose treated or untreated. Representative data from three independent experiments are shown.

FIG. 6.

Insulin activates proteins in the insulin/IGF-I signaling pathway in control but not in βIRS2KO β-cells. Cells were stimulated with 100 nM insulin for 0, 1, 5, or 15 min. (a) Tyrosine phosphorylation of insulin receptor and IGF-1 receptor. (b) Tyrosine phosphorylation of IRS-1 and IRS-2. The immunoprecipitates (IP) with anti-IR, anti-IRS-1, or anti-IRS-2 antibodies from the indicated cell lysates with or without insulin stimulation were immunoblotted (IB) with anti-PY (top panels) or the p85 antibody (bottom panels). (c) PI3K activity associated with the phosphotyrosine complex. PI3K activity was measured in the immunoprecipitate with anti-PY from the indicated cell lysates. (d) Phosphorylation of Akt kinase and ERK. The cell lysates were immunoblotted with the indicated antibodies. Representative blots are shown from three independent experiments.

FIG. 7.

Glucose activates proteins in the insulin/IGF-I signaling pathway in control but not in βIRS2KO β-cells. Cells were treated with glucose (25 mM) for 0, 1, 5, or 15 min. (a) Tyrosine phosphorylation of insulin receptor and IGF-1 receptor. (b) Tyrosine phosphorylation of IRS-1 and IRS-2. The immunoprecipitates (IP) with anti-IR, anti-IRS-1, or anti-IRS-2 from the indicated cell lysates with or without glucose stimulation were immunoblotted (IB) with anti-PY (top panels) or the p85 antibody (bottom panels). (c) PI3K activity associated with the phosphotyrosine complex. PI3K activity was measured in the immunoprecipite with anti-PY from the indicated cell lysates. (d) Phosphorylation of Akt kinase and ERK. The cell lysates were immunoblotted with the indicated antibodies. Representative blots are shown from three independent experiments.

FIG. 8.

Glucose effects on ERK occur independently of insulin signaling. Cells were exposed to glucose (25 mM) (a) or 2-DOG (25 mM) (b) for 0, 1, 5, or 15 min. The cell lysates were immunoblotted (IB) with the indicated antibody. Representative blots from three experiments are shown.

FIG. 9.

Schematic of a link between glucose and insulin signaling in β-cells. (A) Potential direct effects of glucose and/or its metabolites on proteins in the insulin/IGF-1 signaling pathway. (B) Potential indirect effects of glucose and direct effects of insulin following exocytosis of insulin. Akt, v-akt murine thymoma viral oncogene homolog; FoxO-1, forkhead box O1; GRB2, growth factor receptor-bound protein 2; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; mTOR, mammalian target of rapamycin; 4EBP1, translation initiation factor 4e binding protein 1.