Adipose tissue hypoxia induces inflammatory M1 polarity of macrophages in an HIF-1α-dependent and HIF-1α-independent manner in obese mice.

AIMS/HYPOTHESIS: As obesity progresses, adipose tissue exhibits a hypoxic and inflammatory phenotype characterised by the infiltration of adipose tissue macrophages (ATMs). In this study, we examined how adipose tissue hypoxia is involved in the induction of the inflammatory M1 and anti-inflammatory M2 polarities of ATMs. METHODS: The hypoxic characteristics of ATMs were evaluated using flow cytometry after the injection of pimonidazole, a hypoxia probe, in normal-chow-fed or high-fat-fed mice. The expression of hypoxia-related and inflammation-related genes was then examined in M1/M2 ATMs and cultured macrophages. RESULTS: Pimonidazole uptake was greater in M1 ATMs than in M2 ATMs. This uptake was paralleled by the levels of inflammatory cytokines, such as TNF-α, IL-6 and IL-1ß. The expression level of hypoxia-related genes, as well as inflammation-related genes, was also higher in M1 ATMs than in M2 ATMs. The expression of Il6, Il1ß and Nos2 in cultured macrophages was increased by exposure to hypoxia in vitro but was markedly decreased by the gene deletion of Hif1a. In contrast, the expression of Tnf, another inflammatory cytokine gene, was neither increased by exposure to hypoxia nor affected by Hif1a deficiency. These results suggest that hypoxia induces the inflammatory phenotypes of macrophages via Hif1a-dependent and -independent mechanisms. On the other hand, the expression of inflammatory genes in cultured M2 macrophages treated with IL-4 responded poorly to hypoxia. CONCLUSIONS/INTERPRETATION: Adipose tissue hypoxia induces an inflammatory phenotype via Hif1a-dependent and Hif1a-independent mechanisms in M1 ATMs but not in M2 ATMs.

Deletion of platelet-derived growth factor receptor-ß improves diabetic nephropathy in Ca²âº/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice.

AIMS/HYPOTHESIS: The activation of platelet-derived growth factor receptor-ß (PDGFR-ß) signalling is increased in the glomeruli and tubules of diabetic animals. In this study, we examined the role of PDGFR-ß signalling during the development of diabetic nephropathy. METHODS: We recently generated pancreatic beta cell-specific Ca(2+)/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice (CaMKIIα mice), which show very high plasma glucose levels up to 55.5 mmol/l and exhibit the features of diabetic nephropathy. These mice were crossed with conditional knockout mice in which Pdgfr-ß (also known as Pdgfrb) was deleted postnatally. The effect of the deletion of the Pdgfr-ß gene on diabetic nephropathy in CaMKIIα mice was evaluated at 10 and 16 weeks of age. RESULTS: The plasma glucose concentrations and HbA(1c) levels were elevated in the CaMKIIα mice from 4 weeks of age. Variables indicative of diabetic nephropathy, such as an increased urinary albumin/creatinine ratio, kidney weight/body weight ratio and mesangial area/glomerular area ratio, were observed at 16 weeks of age. The postnatal deletion of the Pdgfr-ß gene significantly decreased the urinary albumin/creatinine ratio and mesangial area/glomerular area ratio without affecting the plasma glucose concentration. Furthermore, the increased oxidative stress in the kidneys of the CaMKIIα mice as shown by the increased urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion and the increased expression of NAD(P)H oxidase 4 (NOX4), glutathione peroxidase 1 (GPX1) and manganese superoxide dismutase (MnSOD) was decreased by Pdgfr-ß gene deletion. CONCLUSIONS/INTERPRETATION: The activation of PDGFR-ß signalling contributes to the progress of diabetic nephropathy, with an increase in oxidative stress and mesangial expansion in CaMKIIα mice.

Mammalian target of rapamycin pathway regulates insulin signaling via subcellular redistribution of insulin receptor substrate 1 and integrates nutritional signals and metabolic signals of insulin.

A pathway sensitive to rapamycin, a selective inhibitor of mammalian target of rapamycin (mTOR), down-regulates effects of insulin such as activation of Akt (protein kinase B) via proteasomal degradation of insulin receptor substrate 1 (IRS-1). We report here that the pathway also plays an important role in insulin-induced subcellular redistribution of IRS-1 from the low-density microsomes (LDM) to the cytosol. After prolonged insulin stimulation, inhibition of the redistribution of IRS-1 by rapamycin resulted in increased levels of IRS-1 and the associated phosphatidylinositol (PI) 3-kinase in both the LDM and cytosol, whereas the proteasome inhibitor lactacystin increased the levels only in the cytosol. Since rapamycin but not lactacystin enhances insulin-stimulated 2-deoxyglucose (2-DOG) uptake, IRS-1-associated PI 3-kinase localized at the LDM was suggested to be important in the regulation of glucose transport. The amino acid deprivation attenuated and the amino acid excess enhanced insulin-induced Ser/Thr phosphorylation and subcellular redistribution and degradation of IRS-1 in parallel with the effects on phosphorylation of p70 S6 kinase and 4E-BP1. Accordingly, the amino acid deprivation increased and the amino acid excess decreased insulin-stimulated activation of Akt and 2-DOG uptake. Furthermore, 2-DOG uptake was affected by amino acid availability even when the degradation of IRS-1 was inhibited by lactacystin. We propose that subcellular redistribution of IRS-1, regulated by the mTOR-dependent pathway, facilitates proteasomal degradation of IRS-1, thereby down-regulating Akt, and that the pathway also negatively regulates insulin-stimulated glucose transport, probably through the redistribution of IRS-1. This work identifies a novel function of mTOR that integrates nutritional signals and metabolic signals of insulin.

Growth hormone induces cellular insulin resistance by uncoupling phosphatidylinositol 3-kinase and its downstream signals in 3T3-L1 adipocytes.

Growth hormone (GH) is well known to induce in vivo insulin resistance. However, the molecular mechanism of GH-induced cellular insulin resistance is largely unknown. In this study, we demonstrated that chronic GH treatment of differentiated 3T3-L1 adipocytes reduces insulin-stimulated 2-deoxyglucose (DOG) uptake and activation of Akt (also known as protein kinase B), both of which are downstream effects of phosphatidylinositol (PI) 3-kinase, despite enhanced tyrosine phosphorylation of insulin receptor substrate (IRS)-1, association of IRS-1 with the p85 subunit of PI 3-kinase, and IRS-1-associated PI 3-kinase activity. In contrast, chronic GH treatment did not affect 2-DOG uptake and Akt activation induced by overexpression of a membrane-targeted form of the p110 subunit of PI 3-kinase (p110(CAAX)) or Akt activation stimulated by platelet-derived growth factor. Fractionation studies indicated that chronic GH treatment reduces insulin-stimulated translocation of Akt from the cytosol to the plasma membrane. Interestingly, chronic GH treatment increased insulin-stimulated association of IRS-1 with p85 and IRS-1-associated PI 3-kinase activity preferentially in the cytosol. These results indicate that cellular insulin resistance induced by chronic GH treatment in 3T3-L1 adipocytes is caused by uncoupling between activation of PI 3-kinase and its downstream signals, which is specific to the insulin-stimulated PI 3-kinase pathway. This effect of GH might result from the altered subcellular distribution of IRS-1-associated PI 3-kinase.

Pioglitazone ameliorates tumor necrosis factor-alpha-induced insulin resistance by a mechanism independent of adipogenic activity of peroxisome proliferator--activated receptor-gamma.

Tumor necrosis factor (TNF)-alpha is one of the candidate mediators of insulin resistance associated with obesity, a major risk factor for the development of type 2 diabetes. The insulin resistance induced by TNF-alpha is antagonized by thiazolidinediones (TZDs), a new class of insulin-sensitizing drugs. The aim of the current study was to dissect the mechanism whereby pioglitazone, one of the TZDs, ameliorates TNF-alpha-induced insulin resistance in 3T3-L1 adipocytes. Pioglitazone restored insulin-stimulated 2-deoxyglucose (DOG) uptake, which was reduced by TNF-alpha, with concomitant restorations in tyrosine phosphorylation and protein levels of insulin receptor (IR) and insulin receptor substrate (IRS)-1, as well as association of the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase with IRS-1 and PI 3-kinase activity. Adenovirus-mediated gene transfer of either wild-type human peroxisome proliferator-activated receptor (PPAR)-gamma2 or a mutant carrying a replacement at the consensus mitogen-activated protein kinase phosphorylation site (hPPAR-gamma2-S112A) promoted adipogenesis of 3T3-L1 fibroblasts and restored TNF-alpha-induced decrease of triglyceride in adipocytes as effectively as pioglitazone. Overexpression of the PPAR-gamma proteins in TNF-alpha-treated adipocytes restored protein levels of IR/IRS-1, but did not improve insulin-stimulated tyrosine phosphorylation of IR/IRS-1 or insulin-stimulated 2-DOG uptake. These results indicate that the ability of pioglitazone to restore insulin-stimulated tyrosine phosphorylation of IR/IRS-1, which is necessary for amelioration of TNF-alpha-induced insulin resistance, may be independent of the adipogenic activity of PPAR-gamma that regulates protein levels of IR/IRS-1.

Evidence for functional roles of Crk-II in insulin and epidermal growth factor signaling in Rat-1 fibroblasts overexpressing insulin receptors.

We examined the potential role of Crk-II in insulin and epidermal growth factor (EGF) signaling in Rat-1 fibroblasts overexpressing insulin receptors. Crk is an SH2 and SH3 domain-containing adaptor protein that has been reported to associate with p130cas, paxillin, c-cbl, c-abl, Sos, and C3G in vitro. Insulin- and EGF-induced association of Crk-II with these molecules was assessed by immunoblotting of anti-Crk-II precipitates in Rat-1 fibroblasts overexpressing insulin receptors. Neither insulin nor EGF treatment induced Crk-II association with either Sos or C3G. Basal tyrosine phosphorylation of c-abl and its constitutive association with Crk-II were not further increased by insulin or EGF. p130cas and paxillin were heavily tyrosine phosphorylated in the basal state. Both insulin and EGF stimulated their dephosphorylation, followed by p130cas-Crk-II dissociation and paxillin-Crk-II association, although the magnitude of these effects was greater with insulin than with EGF. Interestingly, EGF, but not insulin, stimulated tyrosine phosphorylation of c-cbl and its association with Crk-II. To investigate the functional roles of Crk-II in mitogenesis and cytoskeletal rearrangement, we performed microinjection analysis. Cellular microinjection of anti-Crk-II antibody inhibited EGF-induced, but not insulin-induced, DNA synthesis. Insulin, but not EGF, stimulated cytoskeletal rearrangement in the cells, and microinjection of anti-Crk-II antibody effectively inhibited insulin-induced membrane ruffling, suggesting that Crk-II is involved in insulin-induced cytoskeletal rearrangement. These results indicate that Crk-II functions as a multifunctional adaptor molecule linking insulin and EGF receptors to their downstream signals. The presence of c-cbl-Crk-II association may partly determine the signal specificities initiated by insulin and EGF.

Synergistic role of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase cascade in the regulation of insulin receptor trafficking.

To examine the molecular mechanism of insulin receptor trafficking, we investigated the intracellular signaling molecules that regulate this process in Rat1 fibroblasts overexpressing insulin receptors. Cellular localization of insulin receptors was assessed by confocal laser microscopy with indirect immunofluorescence staining. Insulin receptors were visualized diffusely in the basal state. Insulin treatment induced the change of insulin receptor localization to perinuclear compartment. This insulin-induced insulin receptor trafficking was not affected by treatment of the cells with PI3-kinase inhibitor (wortmannin), whereas treatment with MEK [mitogen-activated protein (MAP) kinase-Erk kinase] inhibitor (PD98059) partly inhibited the process in a dose-dependent manner. Interestingly, treatment with both wortmannin and PD98059 almost completely inhibited insulin receptor trafficking. The functional importance of PI3-kinase and MAP kinase in the trafficking process was directly assessed by using single cell microinjection analysis. Microinjection of p85-SH2 and/or catalytically inactive MAP kinase ([K71A]Erk1) GST fusion protein gave the same results as treatment with wortmannin and PD98059. Furthermore, to determine the crucial step for the requirement of PI3-kinase and MAP kinase pathways, the effect of wortmannin and PD98059 on insulin receptor endocytosis was studied. Insulin internalization from the plasma membrane and subsequent insulin degradation were not affected by treatment with wortmannin and PD98059. In contrast, insulin receptor down-regulation from the cell surface and insulin receptor degradation, after prolonged incubation with insulin, were markedly impaired by the treatment. These results suggest that PI3-kinase and MAP kinase pathways synergistically regulate insulin receptor trafficking at a step subsequent to the receptor internalization.

Comparison of the insulin and insulin-like growth factor 1 mitogenic intracellular signaling pathways.

We compared the intracellular insulin-like growth factor-1 (IGF-1) and insulin signaling pathways in Rat1 fibroblasts expressing the equivalent number of insulin receptors and endogenous IGF-1 receptors. Insulin and IGF-1 stimulated tyrosine phosphorylation of IRS-1 and Shc in a similar dose- and time-dependent manner. The time course of Shc phosphorylation by both IGF-1 and insulin was slower than that of IRS-1. Both phosphorylated IRS-1 and Shc associated with Grb2.Sos complexes, leading to p21ras activation. To compare the functional importance of p21ras for IGF-1-and insulin-induced DNA synthesis, single cell microinjection studies were performed. BrdU incorporation into newly synthesized DNA was measured by immunofluorescence microscopy to assess the functional importance of p21ras. Both IGF-1 and insulin stimulated BrdU incorporation, but the effect of IGF-1 was greater. Microinjection of anti-p21ras antibody completely inhibited both IGF-1-and insulin-induced DNA synthesis, indicating the central role of p21ras in signaling by both hormones. Signal transduction from these receptors to Grb2.Sos complexes can occur through IRS-1 and/or Shc. To assess these two possible pathways, we performed Western blots for Grb2 in anti-Shc and anti-IRS-1 immunoprecipitates and found that 5-fold more Grb2 was associated with Shc than with IRS-1 after either IGF-1 or insulin stimulation. Microinjection of anti-Shc antibody inhibited IGF-1 and insulin stimulation of DNA synthesis by 78% and 74%, respectively. By microinjecting Shc subdomains of GST fusion proteins, we found that Shc N-terminus, but not the Shc SH2, was the functionally important domain through which Shc interacts with IGF-1 and insulin receptors. Insulin stimulation caused hyperphosphorylation and decreased electrophoretic mobility of Sos, and a similar effect was seen with IGF-1, although the time course was delayed compared with insulin. Finally, IGF-1 activated mitogen-activated proten kinase activity more effectively than insulin. These data indicate that Shc, rather than IRS-1, appears to be the predominant functional link to Grb2.Sos complexes from the IGF-1 receptor, as it is from the insulin receptor. Although IGF-1 and insulin stimulate cell cycle progression with similar coupling mechanisms from the receptor to Shc, to Grb2.Sos, to p21ras, the delayed IGF-1 induced mobility shift of Sos could lead to, at least in part, more efficient coupling to mitogen-activated protein kinase. These findings might explain the greater mitogenic activity of IGF-1 compared with insulin.

Glucosamine enhances platelet-derived growth factor-induced DNA synthesis via phosphatidylinositol 3-kinase pathway in rat aortic smooth muscle cells.

Vascular smooth muscle cells play a key role in the development of atherosclerosis. Culture of vascular smooth muscle A10 cells with high glucose for 4 weeks enhanced platelet-derived growth factor (PDGF)-induced BrdU incorporation. Since a long period of high glucose incubation was required for the effect, and it was inhibited by co-incubation with azaserine, the role of hexosamine biosynthesis in the development of atherosclerosis in diabetes was studied in A10 cells. Addition of glucosamine to the culture media enhanced PDGF-stimulated BrdU incorporation, and PDGF-induced tyrosine phosphorylation of the PDGF beta-receptor was increased by glucosamine treatment. Of the subsequent intracellular signaling pathways, PDGF-induced PDGF beta-receptor association with PLC gamma was not affected, whereas tyrosine phosphorylation of Shc, subsequent association of Shc with Grb2, and MAP kinase activation were relatively decreased. In contrast, PDGF-induced PDGF beta-receptor association with the p85 regulatory subunit of PI3-kinase and PI3-kinase activation were increased by 20% (P<0.01) and 36% (P<0.01), respectively. The intracellular signaling molecules responsible for the glucosamine effect were further examined using pharmacological inhibitors. Pretreatment with PLC inhibitor (U73122) had negligible effects, and MEK1 inhibitor (PD98059) showed only a slight inhibitory effect on the PDGF-induced BrdU incorporation. In contrast, pretreatment with PI3-kinase inhibitor (LY294002) significantly inhibited glucosamine enhancement of PDGF-induced BrdU incorporation. These findings suggest that glucosamine is involved in the development of atherosclerosis by enhancing PDGF-induced mitogenesis specifically via the PI3-kinase pathway.

Homozygous serum amyloid P component-deficiency does not enhance regression of AA amyloid deposits.

Serum amyloid P component (SAP) is a common protein constituent of all types of amyloid deposits. Using SAP-deficient mice generated through gene targeting, we and others have shown that SAP significantly promotes amyloid deposition. It has been speculated that SAP protects amyloid fibrils from degradation by coating their exterior surface. To assess potential ways of treating individuals with amyloidosis, we examined the persistence of splenic AA amyloid fibrils in SAP-deficient and wild-type mice. No enhancement in the rate of regression of splenic AA amyloid was observed in the SAP-deficient mice relative to wild-type mice. These results present, for the first time, evidence that lack of SAP in AA amyloid deposits does not enhance regression of the deposits in vivo and suggest that dissociation of bound SAP from AA amyloid deposits would not significantly accelerate regression of the deposits in vivo.

The dominant negative effect of a kinase-defective insulin receptor on insulin-like growth factor-I-stimulated signaling in Rat-1 fibroblasts.

To study the interaction between insulin receptor (IR) and insulin-like growth factor-I (IGF-I) receptor (IGF-IR) tyrosine kinases, we examined IGF-I action in Rat-1 cells expressing a naturally occurring tyrosine kinase-deficient mutant IR (Asp 1048 IR). IGF-I normally stimulated receptor autophosphorylation, IRS-I phosphorylation, and glycogen synthesis in cells expressing Asp 1048 IR. However, the Asp 1048 IR inhibited IGF-I-stimulated thymidine uptake by 45% to 52% and amino acid uptake (aminoisobutyric acid [AIB]) by 58% in Asp 1048 IR cells. Furthermore, IGF-I-stimulated tyrosine kinase activity toward synthetic polymers, Shc phosphorylation, and mitogen-activated protein (MAP) kinase activity was inhibited. The inhibition of mitogenesis and AIB uptake was restored with the amelioration of the impaired tyrosine kinase activity and Shc phosphorylation by the introduction of abundant wild-type IGF-IR in Asp 1048 IR cells. These results suggest that the Asp 1048 IR causes a dominant negative effect on IGF-IR in transmitting signals to Shc and MAP kinase activation, which leads to decreased IGF-I-stimulated DNA synthesis, and that the kinase-defective insulin receptor does not affect IGF-I-stimulated IRS-I phosphorylation, which leads to the normal IGF-I-stimulated glycogen synthesis.

Effect of glimepiride (HOE 490) on insulin receptors of skeletal muscles from genetically diabetic KK-Ay mouse.

A new sulfonylurea, glimepiride (HOE 490), has been developed for the glycemic control in non-insulin-dependent diabetes mellitus. We examined the effect of glimepiride on glucose and insulin levels in KK-Ay mice, an animal model of non-insulin-dependent diabetes mellitus, which is characterized by hyperglycemia and hyperinsulinemia. Administration of glimepiride (0.5 mg/kg/day) for 8 weeks to KK-Ay mice resulted in decrease in glucose (297 +/- 36 to 250 +/- 51 mg/dl) and insulin (76 +/- 14 to 41 +/- 14 microU/ml) levels. To clarify the mechanism of the agent, we examined the effect of this new drug on insulin receptors in the skeletal muscles. There was no difference in insulin binding to the receptors from both glimepiride-treated and -untreated KK-Ay mice muscles. The insulin-stimulated autophosphorylation of insulin receptors from KK-Ay mice was decreased compared to that from normal mice (5 +/- 1 vs. 39 +/- 13% over basal). Glimepiride did not ameliorate impaired insulin-stimulated insulin receptor autophosphorylation. To determine the effect of glimepiride on post-insulin receptor signaling pathway, we measured 2-[3H]glycerol incorporation into diacylglycerol in the cultured rat fibroblast cell line overexpressing human insulin receptors. Glimepiride (100 microM) as well as insulin (10 nM) significantly stimulated diacylglycerol production. These results suggest that glimepiride has a potent extrapancreatic effect on glucose metabolism and may directly stimulate glucose transport activity through phospholipid signaling pathway, but not through insulin receptor kinase signaling pathway.

[CT of the pelvic kidney].

Six cases of unilateral pelvic kidney are presented. CT features are summarized as follows: (1) an absence of a kidney in the renal fossa with normal sized opposite kidney: (2) a pelvic kidney whose hilus faces in various directions; and (3) homogeneous parenchymal enhancement and visualization of the pyelocalyceal system on post-contrast CT. In the study of suspected pelvic kidney, CT is a useful diagnostic tool.

Long-term interleukin-1alpha treatment inhibits insulin signaling via IL-6 production and SOCS3 expression in 3T3-L1 adipocytes.

Proinflammatory cytokines are well-known to inhibit insulin signaling to result in insulin resistance. IL-1alpha is also one of the proinflammatory cytokines, but the mechanism of how IL-1alpha induces insulin resistance remains unclear. We have now examined the effects of IL-1alpha on insulin signaling in 3T3-L1 adipocytes. Prolonged IL-1alpha treatment for 12 to 24 hours partially decreased the protein levels as well as the insulin-stimulated tyrosine phosphorylation of IRS-1 and Akt phosphorylation. mRNA for SOCS3, an endogenous inhibitor of insulin signaling, was dramatically augmented 4 hours after IL-1alpha treatment. Concomitantly, the level of IL-6 in the medium and STAT3 phosphorylation were increased by the prolonged IL-1alpha treatment. Addition of anti-IL-6 neutralizing antibody to the medium or overexpression of dominant-negative STAT3 decreased the IL-1alpha-stimulated STAT3 activation and SOCS3 induction, and ameliorated insulin signaling. These results suggest that the IL-1alpha-mediated deterioration of insulin signaling is largely due to the IL-6 production and SOCS3 induction in 3T3-L1 adipocytes.

The wear of a polyethylene socket articulating with a zirconia ceramic femoral head in canine total hip arthroplasty.

Wear of yttria-zirconia (zirconia) in the femoral head was investigated in mature mongrel dogs weighing 10 to 13 kg. Two dogs, which were used as a control group, were sacrificed 18 months after implantation of the uncemented modular hip system with an alumina ceramic (alumina) femoral head. A zirconia femoral head was implanted in five dogs: one was sacrificed 12 months after implantation, two at 18 months, and two at 24 months. In each femoral head and polyethylene (PE) socket, the surface was observed by means of scanning electron microscopy (SEM); the mean articulation surface roughness on the femoral head and PE socket and the thickness of the PE socket were measured. Wear was not seen on the surface of either the zirconia or the alumina heads. In both groups, minute white spots on the smooth surface of the PE socket were visible by SEM. In the alumina and zirconia groups the mean roughness was 0.1 microm. The mean thickness of the PE socket was reduced by 0.2 mm in the alumina group. In the zirconia group it was reduced by 0.2 to 0.3 mm. However, the mechanical strength of zirconia is known to be greater than that of alumina and it may be possible to reduce the diameter of the femoral head. The smaller zirconia head may contribute to the reduction of the wear of the PE socket in an uncemented modular total hip system.

Retinoblastoma protein phosphorylation via PI 3-kinase and mTOR pathway regulates adipocyte differentiation.

In the early phase of adipocyte differentiation, transient increase of DNA synthesis, called clonal expansion, and transient hyperphosphorylation of retinoblastoma protein (Rb) are observed. We investigated the role of these phenomena in insulin-induced adipocyte differentiation of 3T3-L1 cells. Insulin-induced clonal expansion, Rb phosphorylation and adipocyte differentiation were all inhibited by the PI 3-kinase inhibitors and rapamycin, but not the MEK inhibitor, whereas the MEK inhibitor, but not PI 3-kinase inhibitors or rapamycin, decreased c-fos induction. We conclude that insulin induces hyperphosphorylation of Rb via PI 3-kinase and mTOR dependent pathway, which promotes clonal expansion and adipocyte differentiation of 3T3-L1 cells.

Functional importance of heat shock protein 90 associated with insulin receptor on insulin-stimulated mitogenesis.

The role of stress proteins on the function of insulin receptor is not well understood. In the rat-1 fibroblasts overexpressing human insulin receptors, heat shock protein (Hsp) 90 was co-immunoprecipitated with insulin receptors and the association was not affected by insulin stimulation. A GST-fusion protein containing the intracellular insulin receptor beta subunit was associated with Hsp 90 in vitro, suggesting the direct interaction of this protein with insulin receptor beta-subunit. Furthermore, microinjection of anti-Hsp 90 antibody into these cells completely inhibited insulin-stimulated mitogenesis. However, neither epidermal growth factor-stimulated nor serum-stimulated mitogenic signal in the cells was affected by the antibody microinjection. These results suggest that Hsp 90 constitutively binds to insulin receptor beta-subunit, which may be necessary for insulin signaling in mitogenesis.

Fatty acid induced insulin resistance in rat-1 fibroblasts overexpressing human insulin receptors: impaired insulin-stimulated mitogen-activated protein kinase activity.

Saturated fatty acids cause insulin resistance but the underlying molecular mechanism is still unknown. We examined the effect of saturated nonesterified fatty acids on insulin binding and action in transfected Rat-1 fibroblasts, which over-expressed human insulin receptors. Incubation with 1.0 mmol/l palmitate for 1-4 h did not affect insulin binding, insulin receptor autophosphorylation, insulin-stimulated tyrosine kinase activity toward poly(Glu4:Tyr1), pp185 and Shc phosphorylation and PI3-kinase activity in these cells. However, the dose response curve of insulin-stimulated glucose transport was right-shifted. Palmitate inhibited the maximally insulin-stimulated mitogen activated protein (MAP) kinase activity toward synthetic peptide to 7% that of control. The palmitate treatment influenced neither cytosolic protein kinase A activity nor cAMP levels. These results suggested that 1) palmitate did not inhibit the early steps of insulin action from insulin binding to pp185 or Shc phosphorylation but inhibited insulin-stimulated MAP kinase, and that 2) palmitate decreased insulin sensitivity as manifested by inhibited insulin-stimulated glucose uptake. In conclusion, the mechanism of saturated non-esterified fatty acid induced insulin resistance in glucose uptake may reside at post PI3-kinase or Shc steps, including the level of MAP kinase activation.

Functional importance of amino-terminal domain of Shc for interaction with insulin and epidermal growth factor receptors in phosphorylation-independent manner.

Shc has two distinct domains, amino-terminal and SH2 domain, which can interact with activated growth factor receptors. Shc interacts with insulin receptor via Shc-amino-terminal (N) domain, whereas Shc associates with epidermal growth factor (EGF) receptor through both Shc-N and -SH2 domains. In accordance with the different functional roles between insulin and EGF receptors, EGF stimulated tyrosine phosphorylation of Shc faster than insulin. To clarify the functional importance of three distinct Shc domains on insulin and EGF signaling, we microinjected glutathione S-transferase (GST) fusion proteins containing the amino terminus plus collagen homology domain (NCH), collagen homology domain (CH), and Src homology 2 domain (SH2) into Rat1 fibroblasts expressing insulin receptors (HIRc). Bromodeoxyuridine (BrdUrd) incorporation into newly synthesized DNA was subsequently studied to assess the importance of the three distinct domains of Shc. Microinjection of the NCH-GST fusion protein inhibited BrdUrd incorporation induced by both EGF and insulin, whereas microinjection of the SH2-GST fusion protein inhibited EGF, but not insulin stimulation of DNA synthesis. Neither EGF- nor insulin-induced BrdUrd incorporation was inhibited by the CH-GST fusion protein. Following EGF or insulin stimulation, Shc is phosphorylated on single Tyr-317 residue serving as a docking site for Grb2. Microinjection of Shc-N+CH GST fusion protein with Tyr-317 --> Phe replacement (Y317F) also inhibited insulin stimulation of DNA synthesis. Next, we stably overexpressed wild-type Shc or Y317F mutant Shc into HIRc cells. Insulin-induced tyrosine phosphorylation of IRS-1 was compared among the transfected cell lines, since IRS-1 and Shc could competitively interact with insulin receptor. Insulin-stimulated tyrosine phosphorylation of IRS-1 was decreased in both WT-Shc and Y317F-Shc cells compared with that in HIRc cells. Furthermore, overexpression of the Shc-SH2 domain or Shc-N+CH domain with Y317F mutation interfered with EGF-stimulated endogenous Shc phosphorylation. These results suggest that the amino terminus domain of Shc is functionally important in insulin- and EGF-induced cell cycle progression and that the phosphorylation of Shc Tyr-317 residue is independent of Shc interaction with these receptors.