Endothelin rapidly stimulates tyrosine phosphorylation in osteoblast-like cells.

The mitogenic activity of endothelin (ET) was studied in osteoblast-like cells, MC3T3-E1. [3H] Thymidine incorporation induced by ET was markedly lower than that of platelet-derived growth factor (PDGF). ET synergistically stimulated [3H] thymidine incorporation induced by PDGF with an apparent ED50 value of 2.5 nM. Treatment of MC3T3-E1 cells with ET and subsequent immunoblotting of the cell extracts with antiphosphotyrosine antibodies followed by labeling with [125I] protein A resulted in the identification of several phosphotyrosine-containing proteins. The intensity of these labeled phosphoproteins significantly increased when the cells were treated with a combination of ET and PDGF. Genistein, an inhibitor of tyrosine kinases, blocked [3H] thymidine incorporation as well as protein tyrosine phosphorylation stimulated by either ET, PDGF or the combination of ET and PDGF. These findings suggest that tyrosine phosphorylation could play a role in the comitogenic activity of ET in osteoblast-like cells.     

Insulin and insulin-like growth factor I (IGF I) stimulate phosphorylation of a Mr 175,000 cytoskeleton-associated protein in intact FRTL5 cells

FRTL5 rat thyroid cells possess separate high affinity receptors for insulin and insulin-like growth factor I (IGF I) that undergo beta-subunit phosphorylation upon interaction with the specific ligand. Phosphorylation is rapid and dose-dependent and occurs primarily on tyrosine residues. Within 2 min, both insulin and IGF I also give rise to a Mr 175,000 phosphoprotein (pp175) that can be immunoprecipitated by anti-phosphotyrosine antibody (alpha-Tyr(P]. Phosphorylation of pp175 occurs on serine and threonine as well as tyrosine residues. When FRTL5 cells are solubilized with 1% Triton X-100, alpha-Tyr(P) immunoprecipitates phosphorylated insulin and IGF I receptors but little pp175 from the Triton-soluble fraction. After treatment of the Triton-insoluble portion with 1% sodium dodecyl sulfate at 100 degrees C, pp175 can be identified by immunoprecipitation with alpha-Tyr(P). The fraction of FRTL5 cells that remains after extraction of an attached monolayer with 1% Triton for 5 min at 22 degrees C contains most of the cytoskeleton and also nuclei. Extraction of this 32P-labeled cytoskeleton preparation with sodium dodecyl sulfate followed by alpha-Tyr(P) immunoprecipitation results in almost complete recovery of the pp175 content of the cells. When a nuclear fraction was prepared from FRTL5 cells by differential centrifugation, pp175 was not found in the nuclear pellet from labeled cells, but greater than 80% of pp175 was recovered in the supernatant. We conclude that pp175 is a common substrate for insulin and IGF I receptor kinases which, in FRTL5 cells, is associated with the cytoskeleton. It is suggested that phosphorylation of proteins associated with cytoskeletal elements could be involved in insulin and IGF I action in cells.     

PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells

A phosphatidylinositol (PI) kinase activity associated with certain protein tyrosine kinases important in cell proliferation phosphorylates the 3' hydroxyl position of PI to produce phosphatidylinositol-3-phosphate (PI-3-P). Here we report that, in addition to PI-3' kinase activity, anti-phosphotyrosine (alpha-P-tyr) immunoprecipitates from platelet-derived growth factor (PDGF)-stimulated smooth muscle cells (SMC) contain lipid kinase activities that utilize the substrates phosphatidylinositol-4-phosphate (PI-4-P) and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). These activities are absent in alpha-P-tyr immunoprecipitates from quiescent SMC. The product of PI-4-P phosphorylation appears to be phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), a lipid not previously reported. The product of PI-4,5-P2 phosphorylation is phosphatidylinositol-trisphosphate (PIP3). PI-3-P was detected in quiescent SMC and increased only slightly in response to PDGF. PIP3 and the putative PI-3,4-P2 appeared only after the addition of mitogen. Both the temporal production of these novel phospholipids after PDGF stimulation and the observation of the enzymatic activities that produce them in alpha-P-tyr immunoprecipitates suggest that these phospholipids are excellent candidates for mediators of the PDGF mitogenic response.     

Epidermal growth factor stimulates tyrosine phosphorylation of human glucocorticoid receptor in cultured cells

Human breast epithelial HBL100 cells, which bind both epidermal growth factor (EGF) and glucocorticoids, were labelled to steady state specific activity with 32Pi and the glucocorticoid receptor was immunoprecipitated from cell lysates with polyclonal antiserum GR884. Immunoprecipitated receptor was resolved by NaDodSO4-polyacrylamide gel electrophoresis and identified by autoradiography. Immunoprecipitated receptor also was characterized by western blot analysis and affinity labelling with [3H]dexamethasone-21-mesylate. Phosphoamino acid analysis of 32P-glucocorticoid receptor revealed 89% phosphoserine and 11% phosphotyrosine. Treatment of steady state 32Pi-labelled cells with EGF stimulated total and alkali-stable phosphorylation in the 97 kDa receptor band by about 35%. Prior incubation with dexamethasone inhibited EGF stimulated, alkali-stable phosphorylation of the 97 kDa glucocorticoid receptor band.     

Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.

Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.     

Insulin-like growth factor I stimulates elastin synthesis by bovine pulmonary arterial smooth muscle cells

Insulin-like growth factor I stimulates mitogenesis in smooth muscle cells, and upregulates elastin synthesis in embryonic aortic tissue. Increased smooth muscle elastin synthesis may play an important role in vascular remodeling in chronic pulmonary hypertension. Therefore, we studied the effect of IGF-I on elastin and total protein synthesis by pulmonary arterial smooth muscle cells in vitro. Tropoelastin synthesis was measured by enzyme immunoassay, and total protein synthesis was measured by [3H]-leucine incorporation. In addition, the steady-state levels of tropoelastin mRNA were determined by slot blot hybridization. Incubation of confluent cultures with various concentrations of IGF-I resulted in a dose-dependent stimulation of elastin synthesis, with a 2.4-fold increase over control levels at 1000 ng/ml of IGF. The increase in elastin synthesis was reflected by a stimulation of the steady-state levels of tropoelastin mRNA. We conclude that IGF-I has potent elastogenic effects on vascular smooth muscle cells, and speculate that it may contribute to vascular wall remodeling in chronic hypertension.     

Bovine skeletal growth factor stimulates protein phosphorylation of chicken bone cells in vitro

1. Bovine skeletal growth factor (SGF), a potent bone cell mitogen, stimulated protein phosphorylation in cultured chicken calvarial cells. 2. SDS-PAGE followed by autoradiographic analysis of the cellular proteins indicated that [32P] incorporation was enhanced in several proteins in response to 10 ng/ml of SGF (the maximum stimulatory mitogenic dose for these cells). 3. Under conditions favoring tyrosine kinases, SGF stimulated phosphorylation of at least 6 proteins in crude calvarial cell membrane fraction, and caused a time-dependent stimulation of phosphorylation of angiotensin II by crude calvarial cell membrane fractions. 4. Thus, our data demonstrate that SGF stimulates protein phosphorylation in bone cells, and suggest that at least some of the protein phosphorylation involves tyrosine residues.     

Nerve growth factor stimulates the tyrosine phosphorylation of MAP2 kinase in PC12 cells.

NGF treatment of PC12 cells results in the rapid activation of MAP2 kinase. We report here that the induction of enzyme activity was correlated with the phosphorylation of MAP2 kinase, detected by metabolic labeling of the enzyme and with anti-phosphotyrosine antibodies. NGF stimulated the phosphorylation of MAP2 kinase on tyrosine, as well as serine and threonine residues. Western blot analysis using a polyclonal anti-phosphotyrosine antibody demonstrated that the tyrosine phosphorylation of MAP2 kinase was maximal within 2 min following NGF exposure and preceded the induction of MAP2 kinase activity. The NGF-stimulated tyrosine phosphorylation of an identified substrate provides direct evidence for the participation of a tyrosine kinase in the mechanism of action of NGF.     

Hydrogen peroxide stimulates tyrosine phosphorylation of the insulin receptor and its tyrosine kinase activity in intact cells.

H-35 rat hepatoma cells were labelled with [32P]orthophosphate and their insulin receptors isolated on wheat germ agglutinin (WGA)-agarose and anti-(insulin receptor) serum. The incubation of these cells with 10 mM-H2O2 for 10 min increased the phosphorylation of both the serine and tyrosine residues of the beta subunit of the insulin receptor. Next, insulin receptors were purified on WGA-agarose from control and H2O2-treated H-35 cells and the purified fractions incubated with [gamma-32P]ATP and Mn2+. Phosphorylation of the beta subunit of insulin receptors obtained from H2O2-treated cells was 150% of that of control cells. The kinase activity of the WGA-purified receptor preparation obtained from H2O2-treated cells, as measured by phosphorylation of src-related synthetic peptide, was increased about 4-fold over control cells. These data suggest that in intact cell systems, H2O2 may increase the insulin receptor kinase activity by inducing phosphorylation of the beta subunit of insulin receptor.     

Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-dalton substrate in intact A-431 cells

We have previously reported the isolation of a 35-kDa protein from A-431 cells that, in the presence of Ca2+, is an excellent in vitro substrate for the epidermal growth factor (EGF) receptor/kinase present in membrane preparations (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). In this communication we demonstrate that the phosphorylation of the 35-kDa protein is markedly enhanced in intact, 32P-labeled, A-431 cells following exposure of the cells to EGF. The 35-kDa protein immunoprecipitated from cells treated with EGF is phosphorylated to a 20-120-fold greater extent than comparable preparations from control cells. Both phosphotyrosine and phosphoserine residues are detected in the protein after treatment of the cells with EGF. EGF-dependent phosphorylation of the 35-kDa protein is barely detected unless the intact cells are exposed to EGF for periods greater than 5 min. We suggest that endosomes containing internalized EGF X receptor/kinase complexes are primarily responsible for the observed phosphorylation of the 35-kDa protein in intact cells.     

Epidermal growth factor induces tyrosine phosphorylation of epidermal growth factor receptors not occupied with ligand in intact A431 cells.

The mechanism of epidermal growth factor receptor (EGF-R) autophosphorylation in intact A431 cells was studied. We detected epidermal growth factor (EGF) induced tyrosine phosphorylation of EGF-R not occupied with ligand. Cell monolayers were subjected to irradiation after incubation with photoreactive derivative of EGF and uncoupled EGF was extracted by acidic treatment. Subsequent immunoprecipitation with antiphosphotyrosine antibodies resulted in precipitation of both EGF-R complexes with EGF and EGF-R with unoccupied ligand-binding site. The fact of precipitation of EGF-R with unoccupied ligand-binding site in conjunction with our finding of rapid dephosphorylation of EGF-R after EGF extraction by acidic treatment, strongly supports the interpretation that cross-phosphorylation of EGF-R may take place in intact cells.     

Epidermal growth factor stimulates tyrosine phosphorylation of specific proteins in permeabilized human fibroblasts

We have investigated the epidermal growth factor (EGF)-stimulated tyrosine-specific protein kinase activity in quiescent cultures of diploid human fibroblasts that have a well characterized mitogenic response to EGF. We developed a method of permeabilizing cells with digitonin or other agents that permitted the rapid labeling of cellular proteins with exogenously added [gamma-32P]ATP while allowing only about 25% of marker cytosolic enzymes to escape from the cells. When phosphatases were inhibited with zinc and vanadate, EGF induced up to 8-fold stimulation of the incorporation of radioactivity from [gamma-32P]ATP into a 35-kDa band on sodium dodecyl sulfate gels. Alkali treatment of gels showed that EGF stimulated the phosphorylation of bands with apparent molecular masses of 170, 45, 35, 26, 22, and 21 kDa. Phosphoamino acid analysis was performed on the 170- and 35-kDa bands and revealed that the EGF-stimulated phosphorylation was on tyrosyl residues. The 35-kDa band was resolved into four spots by two-dimensional gel electrophoresis. The most acidic form was the most prominent and it was precipitated by an antiserum against a 35-kDa protein from A-431 cells; heretofore, this protein has only been reported to be phosphorylated in an EGF-dependent manner by A-431 membranes in vitro (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). This antiserum also precipitated a 35-kDa phospho-protein from extracts of intact [32P]orthophosphate-labeled fibroblasts which was phosphorylated on tyrosine in an EGF-dependent manner. None of the forms of the 35-kDa phosphoproteins labeled in permeabilized cells were immunologically related to the 34-kDa protein that is a substrate for the tyrosyl kinase encoded by Rous sarcoma virus. Other mitogens (serum, insulin, platelet-derived growth factor, and thrombin) did not detectably stimulate phosphorylation in permeabilized cells.     

Epidermal growth factor stimulates serine and tyrosine phosphorylation in a 59-kD protein in purified plasma membranes from rat liver

Preincubation of purified plasma membranes from rat liver with EGF stimulates the level of phosphorylation on serine and tyrosine residues in a 59-kD protein. Such an increased phosphoserine and phosphotyrosine content of the 59-kD protein occurs at the expense of the phosphorylation on threonine residues. The effect is observed under conditions where the plasma membranes have been extracted at pH 10. It is not observed when the membranes are simply washed at pH 7.5 before further purification. A number of experiments, including TBR-IgG phosphorylation in immunoprecipitates and partial hydrolysis with varying concentrations of the V8 protease, suggest that the 59-kD protein modified upon EGF treatment could be a representative of the c-src gene product from hepatocytes.     

Heparin rapidly and selectively regulates protein tyrosine phosphorylation in vascular smooth muscle cells

Aberrant vascular smooth muscle cell (VSMC) hyperplasia is the hallmark of atherosclerosis and restenosis seen after vascular surgery. Heparin inhibits VSMC proliferation in animal models and in cell culture. To test our hypothesis that heparin mediates its antiproliferative effect by altering phosphorylation of key mitogenic signaling proteins in VSMC, we examined tyrosine phosphorylation of cellular proteins in quiescent VSMC stimulated with serum in the presence or absence of heparin. Western blot analysis with anti-phosphotyrosine antibodies shows that heparin specifically alters the tyrosine phosphorylation of only two proteins (42 kDa and 200 kDa). The 200 kDa protein (p200) is dephosphorylated within 2.5 min after heparin treatment with an IC50 that closely parallels the IC50 for growth inhibition. Studies using the tyrosine phosphatase inhibitor, sodium orthovanadate, indicate that heparin blocks p200 phosphorylation by inhibiting a kinase. Phosphorylation of p200 is not altered in heparin-resistant cells, supporting a role for p200 in mediating the antiproliferative effect of heparin. Purification and sequence analysis indicate that p200 exhibits very high homology to the heavy chain of nonmuscle myosin IIA. The 42 kDa protein, identified as mitogen activated protein kinase (MAPK), undergoes dephosphorylation within 15 min after heparin treatment, and this effect is also not seen in heparin-resistant cells. The identification of only two heparin-regulated tyrosine phosphoproteins suggests that they may be key mediators of the antiproliferative effect of heparin.     

Epidermal growth factor, but not insulin, stimulates tyrosine phosphorylation of an endogenous protein of Mr 95,000 in triton extracts of human placental syncytiotrophoblast membranes.

1. Triton extracts of syncytiotrophoblast membranes were incubated with [gamma-32P]ATP, MgCl2 and MnCl2. Addition of epidermal growth factor (EGF) resulted in increased phosphorylation not only of the EGF receptor and a Mr-35,000 protein as previously described, but also a protein of Mr 95,000 on both tyrosine and serine residues. In addition, a small increase in the phosphorylation of a protein of Mr 105,000 was observed. Spermine had a similar effect on the phosphorylation of the Mr-95,000 protein, without affecting the phosphorylation of the other proteins. In the absence of MnCl2, the effect of spermine on the phosphorylation of Mr-95,000 protein was still evident, whereas that of EGF was greatly diminished. 2. The Mr-95,000 protein bound poorly to wheat-germ-lectin-Sepharose and was not precipitated by antisera specific for insulin and EGF receptors. The protein continued to exhibit serine and tyrosine phosphorylation on addition of [gamma-32P]ATP, MgCl2 and MnCl2 to a glycoprotein-depleted fraction prepared by chromatography on wheat-germ-lectin-Sepharose. The extent of phosphorylation was no longer increased by spermine or EGF, but was inhibited by heparin. 3. It is suggested that the Mr-95,000 protein not only is a possible direct substrate for the EGF-receptor (but not the insulin receptor) tyrosine kinase but is a substrate for other endogenous kinases, including a protein tyrosine kinase which is probably not a glycoprotein, and a protein serine kinase with properties similar to those of casein kinase II.     

Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.

We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.     

Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease

Insulin-like growth factor (IGF)-I and its binding protein IGF binding protein 5 (IGFBP-5) were highly expressed in inflamed and fibrotic intestine in experimental Crohn's disease. IGF-I induced proliferation and increased collagen synthesis by smooth muscle cells and fibroblasts/myofibroblasts in vitro. Here we studied IGF-I and IGFBP-5 in Crohn's disease tissue. Tissue was collected from patients undergoing intestinal resection for Crohn's disease. IGF-I and IGFBP-5 mRNAs were quantitated by RNase protection assay and Northern blot analysis, respectively. In situ hybridization was performed to localize mRNA expression, and Western immunoblot was performed to quantitate protein expression. IGF-I and IGFBP-5 mRNAs were increased in inflamed/fibrotic intestine compared with normal-appearing intestine. IGF-I mRNA was expressed in multiple cell types in the lamina propria and fibroblast-like cells of the submucosa and muscularis externa. IGFBP-5 mRNA was highly expressed in smooth muscle of the muscularis mucosae and muscularis externa as well as fibroblast-like cells throughout the bowel wall. Tissue IGFBP-5 protein correlated with collagen type I (r = 0.82). These findings are consistent with a mechanism whereby IGF-I acts on smooth muscle and fibroblasts/myofibroblasts to increase collagen synthesis and cellular proliferation; its effects may be modulated by locally expressed IGFBP-5.     

Platelet-derived growth factor stimulates the phosphorylation of ribosomal protein S6

The human platelet derived-growth factor (PDGF) is both a potent mitogen and a strong chemoattractant protein for cells involved in inflammation and repair. In seeking mechanisms by which PDGF might initiate specific activities in target cells, it was found that highly purified PDGF stimulates the phosphorylation of an Mr approximately 33000 protein in confluent Swiss mouse 3T3 cells [Biochem. Biophys. Res. Commun. (1981) 103, 355-361]. The Mr approximately 33000 protein has now been recovered in polysomes by differential centrifugation and identified as ribosomal protein S6 by two-dimensional polyacrylamide gel electrophoresis.     

Insulin-stimulated protein tyrosine phosphorylation in intact cells evaluated by giant two-dimensional gel electrophoresis

We have studied the insulin-stimulated phosphorylation of proteins in NIH 3T3 cells expressing high numbers of human insulin receptors (HIR 3.5 cells) using the technique of giant two-dimensional gel electrophoresis. In serum-deprived cells, insulin stimulated the phosphorylation of more than 25 proteins; all but two of these were also phosphorylated in response to 15% (v/v) fetal bovine serum, which also stimulated the phosphorylation of additional proteins thought to be direct substrates for protein kinase C. In cells pretreated insulin specifically stimulated the phosphorylation insulin specifically stimulated the phosphorylation of at least 26 predominantly cytosolic proteins, only one of which was observed in insulin-treated cells not exposed to phenylarsine oxide. Serum was without effect in cells pretreated with phenylarsine oxide. In phenylarsine oxide-pretreated cells, phosphoamino acid analysis of 10 of the most highly labeled insulin-stimulated phosphoproteins showed that all 10 were labeled predominantly or exclusively on tyrosine residues. The phosphorylation of several of these could be stimulated in vitro by the addition of insulin to a detergent extract of cells in the presence of Mn2+ and ATP. In general, the insulin-stimulated phosphorylations observed in the presence of phenylarsine oxide were more rapid than those observed in its absence. Finally, a variety of other growth factors and mitogens did not stimulate any of the insulin-stimulated phosphorylations in the presence of phenylarsine oxide. Thus, the use of this inhibitor apparently unmasked a number of novel insulin-specific protein phosphorylations that were ordinarily undetectable. We suggest that at least some of these proteins may be direct substrates for the insulin receptor protein tyrosine kinase and may play significant roles in insulin action.