Judith A. Smith

Insulin-Induced Protein Tyrosine Phosphorylation Cascade and Signalling Molecules Are Localized in a Caveolin-Enriched Cell Membrane Domain

The cellular localisation of time- and temperature-dependent 125I-insulin binding, insulin-sensitive signalling proteins and the insulin-induced protein tyrosine phosphorylation cascade were assessed in subcellular fractions isolated on Iodixanol gradients from control and insulin-treated H35 hepatoma cells. Western blot analysis demonstrated that the concentrations of IRS-1, Shc, GRB-2, SOS, Syp, PI 3-kinase, MAP kinase and Gi alpha were at least 10-fold higher in cell surface-derived, caveolin-enriched fraction than in a cell surface-derived, caveolin-poor fraction (i.e., the plasma membranes). Insulin treatment caused a 15-fold increase in tyrosine phosphorylation of IRS-1 in the caveolin-enriched fraction in 5 min at 37 degrees C compared with a 3-fold increase in plasma membranes and a 6-fold increases in the cytosol and endosomes. Insulin also increased tyrosine phosphorylation of both a 72-kDa protein and the 46-kDa Shc isoform only in the caveolin-enriched fraction. Insulin treatment did not change the concentrations of insulin receptors or Shc but increased IRS-1 in the caveolin-enriched fraction, possibly recruited from the cytosolic pool. Insulin also increased the concentrations of insulin receptors, IRS-1 and Shc in endosomes, suggesting insulin-induced internalization of the insulin receptors and proteins activated with them. Electron microscopic analysis, with the use of a combination of colloidal gold-labelled insulin to label the insulin receptor and immunolabelling to detect caveolin or IRS-1, demonstrated the co-localisation of insulin receptors in caveolin- and IRS-1 containing vesicular structures. Differences in the insulin-induced protein tyrosine phosphorylation and concentrations of these proximal signalling proteins in the caveolin-enriched fraction, plasma membranes, and cytosol suggest that insulin receptors in the caveolae play a major role in initiating insulins signal transduction processes.

Insulin-induced egr-1 and c-fos Expression in 32D Cells Requires Insulin Receptor, Shc, and Mitogen-activated Protein Kinase, but Not Insulin Receptor Substrate-1 and Phosphatidylinositol 3-Kinase Activation*

Many studies suggest that insulin utilizes multiple signal transduction pathways. Insulins effects are initiated by insulin binding to the insulin receptor, resulting in tyrosine phosphorylation of insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1), IRS-2, or Shc. We recently demonstrated that immediate-early gene egr-1 transcription was fully induced without phosphorylation of IRS-1 in Chinese hamster ovary cells (Harada, S., Smith, R. M., Smith, J. A., Shah, N., Hu, D.-Q. & Jarett, L. (1995) J. Biol. Chem. 270, 26632-26638). In the present study, we examined the effects of insulin on immediate-early gene egr-1 and c-fos expression in 32D cells overexpressing the insulin receptor (32D/IR), IRS-1 (32D/IRS), or both (32D/IR+IRS) and compared these effects with insulin-induced tyrosine phosphorylation. Insulin (17 nM) increased egr-1 and c-fos expression in 32D/IR and 32D/IR+IRS cells, but not in parental cells or 32D/IRS cells, as determined by Northern blot analysis. Insulin treatment (5 min at 37°C) markedly increased tyrosine phosphorylation of several proteins, including the insulin receptor, IRS-1, and Shc, in 32D/IR+IRS cells as determined by immunoprecipitation and Western blot analysis with anti-phosphotyrosine antibody. In contrast, only two tyrosine-phosphorylated proteins, i.e. insulin receptor and Shc, were detected in 32D/IR cells. These data suggest that insulin receptor and Shc phosphorylation is necessary for insulin-induced egr-1 and c-fos expression, but IRS-1 phosphorylation is not necessary or sufficient for the expression of these genes. Furthermore, the effect of specific inhibitors on insulin-induced egr-1 expression was examined. Wortmannin (25 nM), a phosphatidylinositol 3-kinase inhibitor, had no effect on insulin-induced egr-1 expression. In contrast, PD 98059 (30 μM), a mitogen-activated protein kinase kinase inhibitor, totally blocked egr-1 expression induced by insulin. These data indicate that mitogen-activated protein kinase activation, but not phosphatidylinositol 3-kinase activation, is involved in insulin-induced egr-1 expression. Taken together, insulin receptor tyrosine phosphorylation, Shc tyrosine phosphorylation, and mitogen-activated protein kinase activation appear to be the signal transduction pathway responsible for insulin-induced egr-1 expression in 32D cells. These data demonstrate that insulin has multiple signal transduction pathways that vary from cell to cell.

Insulin-induced egr-1 Expression in Chinese Hamster Ovary Cells Is Insulin Receptor and Insulin Receptor Substrate-1 Phosphorylation-independent EVIDENCE OF AN ALTERNATIVE SIGNAL TRANSDUCTION PATHWAY

Insulins effects primarily are initiated by insulin binding to its plasma membrane receptor and the sequential tyrosine phosphorylation of the insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1). However, studies suggest some insulin effects, including those at the nucleus, may not be regulated by this pathway. The present study compared the levels of insulin binding, insulin receptor and IRS-1 tyrosine phosphorylation, and phosphatidylinositol 3′-kinase activity to immediate early gene c-fos and egr-1 mRNA expression in Chinese hamster ovary (CHO) cells expressing only neomycin-resistant plasmid (CHONEO), overexpressing wild type human insulin receptor (CHOHIRc) or ATP binding site-mutated insulin receptors (CHOA1018K). Insulin binding in CHONEO cells was markedly lower than that in other cell types. 10 nM insulin significantly increased tyrosine phosphorylation of insulin receptor and IRS-1 in CHOHIRc cells. Phosphorylation of insulin receptor and IRS-1 in CHONEO and CHOA1018K cells was not detected in the presence or absence of insulin. Similarly, insulin increased phosphatidylinositol 3-kinase activity only in CHOHIRc cells. As determined by Northern blot, nuclear run-on analysis, and in situ hybridization, insulin induced c-fos mRNA expression, through transcription, in CHOHIRc cells but not in CHONEO and CHOA1018K cells, consistent with previous reports. In contrast, all three cell types showed a similar insulin dose-dependent increase of egr-1 mRNA expression through transcription. These data indicated that insulin-induced egr-1 mRNA expression did not correlate with the levels of insulin binding to insulin receptor or phosphorylation of insulin receptor and IRS-1. These results suggest that different mechanisms are involved in induction of c-fos and egr-1 mRNA expression by insulin, the former by the more classic insulin receptor tyrosine kinase pathway and the latter by a yet to be determined alternative signal transduction pathway.

Effect of adenosine on insulin activation of rat adipocyte pyruvate dehydrogenase

Adenosine and its analogue N 6‐phenylisopropyladenosine stimulated pyruvate dehydrogenase activity of isolated rat adipocytes. Maximal stimulation was obtained with concentrations between 50 and 100 μM, with the effect decreasing at higher concentrations. The effects of insulin on this enzyme was modified by adenosine. The concentration of insulin (10 ) that produced almost half‐maximal stimulation, had little or no effect, when adenosine deaminase was present. Adenosine also enhanced the effect of suboptimal but not optimal concentrations of insulin. Thus, the mechanism of adenosine action on adipocyte pyruvate dehydrogenase could in some way be similar or related to that of insulin.

Activation and inhibition of insulin receptor autophosphorylation by trypsin treatment of intact H35 cells.

1. Treatment of intact cultured H35 cells with trypsin (1 mg/ml) for 15 min at low temperature (4 degrees C) or for 30 sec at 37 degrees C causes activation of the insulin receptor subsequently isolated from the cells. 2. Receptor activation was assessed by increased phosphotyrosine content of the beta-subunit of the receptor, and increased autophosphorylation using [32P]-ATP. 3. Treatment of the cells for 15 min at 37 degrees C however completely abolished insulin binding and all insulin receptor kinase activity. 4. These data demonstrate that proteolytic damage of the extracellular domain of the insulin receptor can render the receptor kinase inactive and lead to a cell which is unresponsive to insulin.

Adenosine and oxytocin reverse antagonism of cyclic AMP elevating agents to insulin activation of adipocyte pyruvate dehydrogenase

ACTH, isoprenaline, forskolin, and dibutyryl cyclic AMP prevented insulin from stimulating adipocyte pyruvate dehydrogenase in the presence of adenosine deaminase. Antagonism was reversed by N 6‐phenyliso‐propyladenosine as well as oxytocin. The stimulatory effects of insulin, adenosine and oxytocin on adipocyte pyruvate dehydrogenase appear to be through (a) mechanism(s) which is (are) similar or related.