Paul Rothenberg

Regulation of insulin receptor substrate-1 in liver and muscle of animal models of insulin resistance.

Insulin rapidly stimulates tyrosine phosphorylation of a protein of approximately 185 kD in most cell types. This protein, termed insulin receptor substrate-1 (IRS-1), has been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the present study we have examined the levels of IRS-1 and the phosphorylation state of insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo in two rat models of insulin resistance, i.e., insulinopenic diabetes and fasting, and a mouse model of non-insulin-dependent diabetes mellitus (ob/ob) by immunoblotting with anti-peptide antibodies to IRS-1 and anti-phosphotyrosine antibodies. As previously described, there was an increase in insulin binding and a parallel increase in insulin-stimulated receptor phosphorylation in muscle of fasting and streptozotocin-induced (STZ) diabetic rats. There was also a modest increase in overall receptor phosphorylation in liver in these two models, but when normalized for the increase in binding, receptor phosphorylation was decreased, in liver and muscle of STZ diabetes and in liver of 72 h fasted rats. In the hyperinsulinemic ob/ob mouse there was a decrease in insulin binding and receptor phosphorylation in both liver and muscle. The tyrosyl phosphorylation of IRS-1 after insulin stimulation reflected an amplification of the receptor phosphorylation in liver and muscle of hypoinsulinemic animals (fasting and STZ diabetes) with a twofold increase, and showed a significant reduction (approximately 50%) in liver and muscle of ob/ob mouse. By contrast, the levels of IRS-1 protein showed a tissue specific regulation with a decreased level in muscle and an increased level in liver in hypoinsulinemic states of insulin resistance, and decreased levels in liver in the hyperinsulinemic ob/ob mouse. These data indicate that: (a) IRS-1 protein levels are differentially regulated in liver and muscle; (b) insulin levels may play a role in this differential regulation of IRS-1; (c) IRS-1 phosphorylation depends more on insulin receptor kinase activity than IRS-1 protein levels; and (d) reduced IRS-1 phosphorylation in liver and muscle may play a role in insulin-resistant states, especially of the ob/ob mice.

Insulin Receptor Signaling in the β-Cell Influences Insulin Gene Expression and Insulin Content: Evidence for Autocrine β-Cell Regulation

The insulin receptor (IR) is expressed by insulin-secreting β-Cells, but its cellular function is unknown. We transfected βTC6-F7 β-Cells with cDNAs encoding either wild-type or mutant kinase-inactive (A/K1018) IRs, and by fluorescence-activated cell sorting generated polyclonal β-cell lines that overexpressed each receptor type at levels two- to fourfold higher than the parental cells. β-Cells overexpressing wild-type IRs had a proportional increase in insulin-stimulated tyrosine kinase activity; no change occurred in β-Cells expressing the mutant IR. We observed a threefold increase in cellular insulin content in β-Cells that overexpressed the wildtype IR, as determined by radioimmunoassay. This increase occurred despite a fivefold elevated rate of both basal and 10 mmol/l glucose-induced insulin secretion. Fractional insulin secretion (percentage of total cell insulin releasable at 10 mmol/l glucose) was unchanged in β-Cells overexpressing the wild-type IR compared with the parental β-cell line. Insulin content and insulin secretion were unaffected by overexpression of kinase-inactive IRs. Steady-state insulin mRNA levels were elevated twofold in the β-Cells overexpressing the wild-type IR and unchanged in the β-Cells expressing the kinase-inactive receptor, as determined by Northern blot analysis. The rate of insulin mRNA degradation measured in the presence of 5 μg/ml actinomycin D was not significantly affected in either cell line. In the absence of glucose, the basal level of (pro)insulin biosynthesis in the β-Cells overexpressing the wild-type IR increased significantly (61%) compared with the pcells transfected with the kinase-inactive IR. These data indicate that IR signaling can regulate insulin gene transcription and can modulate the steady-state insulin content of β-Cells.

Expression of Insulin Receptor mRNA and Insulin Receptor Substrate 1 in Pancreatic Islet β-Cells

The expression of insulin receptor mRNA was examined in rat pancreatic islet cells by single-cell reverse transcriptase (RT)–polymerase chain reaction (PCR). Single cells from disaggregated islets were individually isolated in a microcapillary pipet, and the β-cells were identified by amplification of the mRNA for insulin. We found that in single β-cells, the mRNA for the insulin receptor was also expressed. The fraction of single islet cells expressing both insulin receptor and insulin mRNAs corresponds closely to the fraction of β-cells in the disaggregated islet cell preparation. These results indicate that normal β-cells have the potential to express authentic insulin receptors. Immunohistochemical analysis was insufficiently sensitive for assaying insulin receptor protein; however, insulin receptor substrate 1 (IRS-1) was readily immunolocalized in islet β-cells. Since IRS-1 links several cell surface receptors, including those for insulin and IGF-I, to distal signal transduction pathways, our observations indicate that hormonal regulation of islet β-cells potentially involves the same signal transduction pathway that mediates insulin and growth factor signaling in peripheral insulin target tissue cell types.

The insulin receptor and its substrate: molecular determinants of early events in insulin action.

Publisher Summary This chapter describes insulin receptor and its substrate. It discusses the two earliest molecular events, binding and activation of the insulin receptor kinase and phosphorylation and subsequent signal transduction by the insulin receptor substrate IRS-1. Defining these events has helped add a whole new dimension to the understanding of insulin action. At the same time, studies defining the molecular events at the end of the insulin action cascade, such as activation of glucose transport and regulation of gene expression, have also begun to clarify the specific components required for these signaling events. Although a black box remains between the early and late events in insulin action, it is becoming smaller.

Wortmannin Inhibits Insulin Secretion in Pancreatic Islets and β-TC3 Cells Independent of Its Inhibition of Phosphatidylinositol 3-Kinase

Glucose is the primary stimulus for insulin secretion by pancreatic β-cells, and it triggers membrane depolarization and influx of extracellular Ca2+. Cholinergic agonists amplify insulin release by several pathways, including activation of phospholipase C, which hydrolyzes membrane polyphosphoinositides. A novel phospholipid, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3[, a product of phosphatidylinositol 3-kinase (PI 3-kinase), has recently been found in various cell types. We demonstrate by immunoblotting that PI 3-kinase is present in both cytosolic and membrane fractions of insulin-secreting β-TC3 cells and in rat islets. The catalytic activity of PI 3-kinase in immunoprecipitates of islets and β-TC3 cells was measured by the production of radioactive phosphatidylinositol 3-monophosphate from phosphatidylinositol (Ptdlns) in the presence of [γ-32P[ATP. Wortmannin, a fungal metabolite, dose dependency inhibited PI 3-kinase activity of both islets and P-TC3 cells, with an IC50 of 1 nmol/l and a maximally effective concentration of 100 nmol/l, when it was added directly to the kinase assay. However, if intact islets were incubated with wortmannin and PI 3-kinase subsequently was determined in islet immunoprecipitates, ∼50% inhibition of PI 3-kinase activity (but no inhibition of glucose- and carbachol-stimulated insulin secretion) from intact islets was obtained at wortmannin concentrations of 100 µmol/l. Wortmannin, at higher concentrations (1 and 10 µmol/l), inhibited glucose- and carbachol-induced insulin secretion of intact rat islets by 58 and 92%, respectively. Wortmannin had no effect on the basal insulin release from rat islets. A similar dose curve of inhibition of glucose- and carbachol-induced insulin secretion by wortmannin was obtained when β-TC3 cells were used. Cellu-lar metabolism was not changed by any wortmannin concentrations tested (0.01–10 µmol/l). Both basal cytosolic [Ca2+]1 and carbamyl choline-induced increases of [Ca2+]1 were unaffected by wortmannin in the presence of 2.5 mmol/l Ca2+, while Ca2+ mobilization from intracellular stores was partially decreased by wortmannin. Together, these data suggest that wortmannin at concentrations that inhibit PI 3-kinase does not affect insulin secretion. PI 3-kinase is unlikely to have a major role in insulin secretion induced by glucose and carbachol.Diabetes 45:854–862, 1996

Quantifying monocyte infiltration in response to intradermal tetanus toxoid injection

AIMS To characterize monocyte response in a delayed-type hypersensitivity reaction to intradermal tetanus toxoid (TT) injection. MATERIALS & METHODS Men with positive serum anti-tetanus titers were stratified by last TT vaccination. Subjects were administered three intradermal injections of TT and one saline control on the same side of the back. Skin biopsies were taken post-injection. After 2 weeks, the procedure was repeated on the contralateral side. RESULTS Men who received TT booster vaccination 1 month before the study showed greater reproducibility and lower variability in monocyte responses than those who were not revaccinated. Monocyte concentration in subjects re-vaccinated within 1 month of study start appeared maximal at 48 h post-injection. CONCLUSION This assay represents a novel approach that allows for quantification of dermal monocyte/macrophage influx. This clinical methodology has potential utility in the pharmacodynamic evaluation of therapies targeting inflammatory disorders, which involve monocyte tissue recruitment, like the delayed-type hypersensitivity response.