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C R Kahn

Publications and source records attributed to C R Kahn.

At least 109 records · Page 6Linked to original sources
PubMed1996

Cross-talk between the insulin and angiotensin signaling systems.

Angiotensin II (AII), acting via its G-protein linked receptor, is an important regulator of cardiac, vascular, and renal function. Following injection of AII into rats, we find that there is also a rapid tyrosine phosphorylation of the major insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) in the heart. This phenomenon appears to involve JAK2 tyrosine kinase, which associates with the AT1 receptor and IRS-1/IRS-2 after AII stimulation. AII-induced phosphorylation leads to binding of phosphatidylinositol 3-kinase (PI 3-kinase) to IRS-1 and IRS-2; however, in contrast to other ligands, AII injection results in an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity. The latter occurs without any reduction in insulin receptor or IRS phosphorylation or in the interaction of the p85 and p110 subunits of PI 3-kinase with each other or with IRS-1/IRS-2. These effects of AII are inhibited by AT1 receptor antagonists. Thus, there is direct cross-talk between insulin and AII signaling pathways at the level of both tyrosine phosphorylation and PI 3-kinase activation. These interactions may play an important role in the association of insulin resistance, hypertension, and cardiovascular disease.

Angiotensin II↗
PubMed1996

Overexpression of Rad inhibits glucose uptake in cultured muscle and fat cells.

Rad is a Ras-like GTPase that was isolated by subtraction cloning of human muscle and shown to have increased expression in some individuals with Type II diabetes. To ascertain the potential role of Rad in insulin-mediated signaling, we have overexpressed Rad in myocyte and adipocyte cell lines. Expression of Rad resulted in a 50-90% reduction in insulin-stimulated 2-deoxyglucose glucose uptake in C2C12 murine myotubes, L6 rat myotubes, and 3T3-L1 adipocytes and a 25% reduction in 3-O-methylglucose uptake in 3T3-L1 adipocytes. This occurred despite unaltered levels of glucose transporter expression, with no detectable change in Glut4 translocation and with no alteration in insulin receptor or substrate phosphorylation or phosphatidylinositol 3-kinase activity. These data indicate that Rad is a negative regulator of glucose uptake and that this effect may be due to a decrease in the intrinsic activity of the transporter molecules, rather than an effect on the translocation of Glut4.

Adipocytes↗
PubMed1996

A common amino acid polymorphism in insulin receptor substrate-1 causes impaired insulin signaling. Evidence from transfection studies.

Insulin receptor substrates-1 (IRS-1) is the major cytoplasmic substrate of the insulin and IGF-1 receptors. Recent studies have identified multiple sequence variants of IRS-1, especially in patients with non-insulin-dependent diabetes mellitus. In the present study, we have examined insulin-stimulated processes in 32D(IR) cells, a myeloid progenitor cell stably overexpressing the insulin receptor, transfected with wild-type human-IRS-1 or the most common human variant of IRS-1 in which glycine 972 is replaced by arginine. As compared to wild-type IRS-1, insulin stimulation of cells transfected with mutant IRS-1 exhibited a 32% decrease in incorporation of [3H]thymidine into DNA (P = 0.002), a 36% decrease in IRS-1 associated phosphatidylinositol (PI) 3-kinase activity (P = 0.004) and a 25% decrease in binding of the p85 regulatory subunit of PI 3-kinase to IRS-1 (P = 0.002). There was also a tendency for a decrease in Grb2 binding to IRS-1 and insulin-stimulated mitogen-activated protein kinase activity, however, these were not statistically significant. The changes occurred with no change in insulin receptor or IRS-1 tyrosine phosphorylation. These data indicate that the mutation in codon 972 in IRS-1 impairs insulin-stimulated signaling, especially along the PI 3-kinase pathway, and may contribute to insulin resistance in normal and diabetic populations.

Arginine↗
PubMed1996

Rad, a novel Ras-related GTPase, interacts with skeletal muscle beta-tropomyosin.

Rad, a prototypic member of a subfamily of Ras-related GTPases, is overexpressed in skeletal muscle of type II diabetic humans. By expression screening of mouse embryo and human skeletal muscle cDNA libraries, we found that Rad interacted with skeletal muscle beta-tropomyosin. In the mouse skeletal muscle cell line C2C12, this interaction was significantly increased by the calcium ionophore A23187. A23187 also caused a time- and concentration-dependent decrease in total cellular Rad with increased interaction between tropomyosin and Rad in the detergent-soluble fraction and the appearance of Rad in the cytoskeleton. In C2C12 cells stably overexpressing a putative dominant negative mutant of Rad (S105N), there was an increase in the amount of tropomyosin in Rad immunoprecipitates. In cells overexpressing wild type Rad, much of Rad was associated with the cytoskeleton and was no longer responsive to A23187. In far-Western blotting and guanine nucleotide saturation studies, GDP-Rad bound to tropomyosin far better than GTP-Rad. We conclude that Rad interacts with skeletal muscle beta-tropomyosin and the cytoskeleton in a guanine nucleotide-dependent manner. These data suggest that Rad may be involved in skeletal muscle motor function and cytoskeletal organization.

Animals↗
PubMed1996

Molecular scanning of the insulin receptor substrate-1 (IRS-1) gene in Japanese patients with NIDDM: identification of five novel polymorphisms.

Since the insulin receptor substrate-1 (IRS-1) is the major substrate of the insulin receptor tyrosine kinase and has been shown to activate phosphatidylinositol (PI) 3-kinase and promote GLUT4 translocation, the IRS-1 gene is a potential candidate for development of non-insulin-dependent diabetes mellitus (NIDDM). In this study, we have identified IRS-1 gene polymorphisms, evaluated their frequencies in Japanese subjects, and analysed the contribution of these polymorphisms to the development of NIDDM. The entire coding region of the IRS-1 gene of 94 subjects (47 NIDDM and 47 control subjects) was screened by polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) analysis. Seven SSCP polymorphisms were identified. These corresponded to two previously identified polymorphisms [Gly971 --> Arg (GGG --> AGG) and Ala804 (GCA --> GCG)] as well as five novel polymorphisms [Pro190 --> Arg (CCC --> CGC), Met209 --> Thr (ATG --> ACG), Ser809 --> Phe (TCT --> TTT), Leu142 (CTT --> CTC), and Gly625 (GGC --> GGT)]. Although the prevalence of each of these polymorphisms was not statistically different between NIDDM and control subjects, the prevalence of the four IRS-1 polymorphisms with an amino acid substitution together was significantly higher in NIDDM than in control subjects (23.4 vs 8.5%, p < 0.05), and two substitutions (Met 209 --> Thr and Ser809 --> Phe) were found only in NIDDM patients. Equilibrium glucose infusion rates during a euglycaemic clamp in NIDDM and control subjects with the IRS-1 polymorphisms decreased by 29.5 and 22.0%, respectively on the average when compared to those in comparable groups without polymorphisms, although they were not statistically significant. Thus, IRS-1 polymorphisms may contribute in part to the insulin resistance and development of NIDDM in Japanese subjects; however, they do not account for the major part of the decrease in insulin-stimulated glucose uptake which is observed in subjects with clinically apparent NIDDM.

Amino Acid Sequence↗
PubMed1996

Expression of the gene encoding glycogen phosphorylase is elevated in diabetic rat skeletal muscle and is regulated by insulin and cyclic AMP.

Glycogen phosphorylase regulates the breakdown of glycogen into glucose, but as previous studies have demonstrated, the control of glycogen metabolism becomes deregulated in diabetes mellitus. Messenger RNA levels encoding several different proteins are altered in skeletal muscle biopsies of patients with insulin-dependent and non-insulin-dependent diabetes. The possible alteration of expression of the gene encoding the skeletal muscle isoform of glycogen phosphorylase during diabetes has not previously been investigated. We examined the effect of streptozotocin-induced diabetes and insulin treatment on glycogen phosphorylase mRNA in rat skeletal muscle; glycogen phosphorylase mRNA levels were elevated in diabetic rat muscle tissue, but were partially suppressed in diabetic rat muscle following insulin treatment. To distinguish between the effects of insulin and counter-regulatory hormones on glycogen phosphorylase mRNA levels, we employed differentiating rat L6 myoblasts in culture. Insulin stimulated the accumulation of glycogen phosphorylase mRNA as determined by Northern blot analysis. Moreover, insulin and dibutyryl cAMP stimulated expression of a transiently transfected chloramphenicol acetyl transferase reporter gene under the control of the muscle glycogen phosphorylase promoter in differentiating myotubes in culture, suggesting that the effects of insulin and counter-regulatory hormones on glycogen phosphorylase mRNA are at the level of transcription. These results suggest that insulin and epinephrine may participate in the induction of the glycogen phosphorylase gene during myogenesis; moreover, activation of this gene in muscle tissue may be a contributing factor in impaired glycogen storage during uncontrolled diabetes.

Animals↗
PubMed1996

The early intracellular signaling pathway for the insulin/insulin-like growth factor receptor family in the mammalian central nervous system.

Several studies support the idea that the polypeptides belonging to the family of insulin and insulin-like growth factors (IGFs) play an important role in brain development and continue to be produced in discrete areas of the adult brain. In numerous neuronal populations within the olfactory bulb, the cerebral and cerebellar cortex, the hippocampus, some diencephalic and brainstem nuclei, the spinal cord and the retina, specific insulin and IGF receptors, as well as crucial components of the intracellular receptor signaling pathway have been demonstrated. Thus, mature neurons are endowed with the cellular machinery to respond to insulin and IGF stimulation. Studies in vitro and in vivo, using normal and transgenic animals, have led to the hypothesis that, in the adult brain, IGF-I not only acts as a trophic factor, but also as a neuromodulator of some higher brain functions, such as long-term potentiation and depression. Furthermore, a trophic effect on certain neuronal populations becomes clearly evident in the ischemic brain or neurodegenerative disorders. Thus, the analysis of the early intracellular signaling pathway for the insulin/IGF receptor family in the brain is providing us with new intriguing findings on the way the mammalian brain is sculpted and operates.

Adult↗
PubMed1996

Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.

We have identified two novel alternatively spliced forms of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus- produced human insulin receptor substrate 1. One form is identical to p85alpha throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85alpha on the 5' side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of approximately 53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85alpha, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a approximately 7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85alpha itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85alpha regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action.

Alternative Splicing↗
PubMed1996

Genetics of non-insulin-dependent (type-II) diabetes mellitus.

Both genetic and environmental factors contribute to the etiology of non-insulin-dependent diabetes. The genetic component is heterogeneous and in some patients is probably complex, involving multiple genes. Specific genetic defects have been identified for rate monogenic forms of NIDDM: maturity-onset diabetes of the young, or MODY (which is due to glucokinase mutations in about 40% of families), syndromes of extreme insulin resistance (which often involve the insulin receptor), and diabetes-deafness syndromes (with defects in mitochondrial genes). In contrast, the genes involved in common forms of NIDDM are still uncertain. Mutations have been extensively searched in genes regulating insulin signaling and secretion. Some evidence of involvement has been produced for insulin-receptor substrate-1, glycogen synthase, the glucagon receptor, a ras-related protein (Rad), histocompatibility antigens, PC-1, and fatty acid binding protein, but the contributions of these genes to NIDDM is probably small. Other candidate genes (e.g. insulin, insulin receptor, glucose transporters) have been excluded as major diabetogenes. New insights are expected in the near future from the systematic scanning of the genome for linkage with NIDDM.

DNA, Mitochondrial↗
PubMed1995

4PS/insulin receptor substrate (IRS)-2 is the alternative substrate of the insulin receptor in IRS-1-deficient mice.

Insulin receptor substrate-1 (IRS-1) is the major cytoplasmic substrate of the insulin and insulin-like growth factor (IGF)-1 receptors. Transgenic mice lacking IRS-1 are resistant to insulin and IGF-1, but exhibit significant residual insulin action which corresponds to the presence of an alternative high molecular weight substrate in liver and muscle. Recently, Sun et al. (Sun, X.-J., Wang, L.-M., Zhang, Y., Yenush, L. P., Myers, M. G., Jr., Glasheen, E., Lane, W.S., Pierce, J. H., and White, M. F. (1995) Nature 377, 173-177) purified and cloned 4PS, the major substrate of the IL-4 receptor-associated tyrosine kinase in myeloid cells, which has significant structural similarity to IRS-1. To determine if 4PS is the alternative substrate of the insulin receptor in IRS-1-deficient mice, we performed immunoprecipitation, immunoblotting, and phosphatidylinositol (PI) 3-kinase assays using specific antibodies to 4PS. Following insulin stimulation, 4PS is rapidly phosphorylated in liver and muscle, binds to the p85 subunit of PI 3-kinase, and activates the enzyme. Insulin stimulation also results in the association of 4PS with Grb 2 in both liver and muscle. In IRS-1-deficient mice, both the phosphorylation of 4PS and associated PI 3-kinase activity are enhanced, without an increase in protein expression. Immunodepletion of 4PS from liver and muscle homogenates removes most of the phosphotyrosine-associated PI 3-kinase activity in IRS-1-deficient mice. Thus, 4PS is the primary alternative substrate, i.e. IRS-2, which plays a major role in physiologic insulin signal transduction via both PI 3-kinase activation and Grb 2/Sos association. In IRS-1-deficient mice, 4PS/IRS-2 provides signal transduction to these two major pathways of insulin signaling.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Common and distinct elements in insulin and PDGF signaling.

The receptors for insulin and PDGF are tyrosine kinases that mediate distinct effects in identical cellular backgrounds. Each receptor must therefore engage a unique subset of the available signaling elements--at least partly through the selection of proteins with src-homology 2 domains (SH2 proteins). Autophosphorylation sites in the PDGFr directly bind SH2 proteins, whereas activation of the insulin receptor leads to phosphorylation of IRS-1, which in turn binds SH2 proteins. In HIR 3.5 cells, which contain similar numbers of PDGF and insulin receptors, insulin, but not PDGF, stimulated tyrosyl phosphorylation of IRS-1. Similarly, insulin, but not PDGF, treatment of HIR 3.5 stimulated the association of IRS-1 with PtdIns 3'-kinase, although PDGF stimulated the association of PtdIns 3'-kinase with the tyrosine-phosphorylated PDGFr. Association with IRS-1 activated PtdIns 3'-kinase more effectively than association with the PDGFr. Whereas the PDGFr associated with PtdIns 3'-kinase, ras-GAP, GRB-2, and phospholipase C gamma, only GRB-2 and PtdIns 3'-kinase associated with IRS-1. Moreover, PDGF, but not insulin, caused tyrosine phosphorylation of phospholipase C gamma in HIR 3.5 cells. Thus, the insulin signal differs from that of PDGF by the insertion of a cytosolic, nonreceptor SH2 domain docking protein (IRS-1). Furthermore, IRS-1 binds a different subset of SH2 domain-containing proteins than does the PDGFr and regulates at least one common element (PtdIns 3'-kinase) differently than the PDGFr. These results support the hypothesis that IRS-1 differentiates the signals generated by the insulin receptor and PDGFr tyrosine kinases by binding and regulating a specific subset of SH2 domain-containing signaling molecules.

3T3 Cells↗
PubMed1995

Insulin-dependent phosphatidylinositol 3'-kinase activity co-precipitates with insulin receptor in human circulating mononuclear cells.

In order to establish a working cellular model for the study of post-receptor signaling events, insulin-dependent phosphatidylinositol 3' (PtdIns 3')-kinase activity was investigated in circulating mononuclear cells from normal subjects. The p85 alpha regulatory subunit of PtdIns 3'-kinase co-precipitated with activated insulin receptor as revealed by immunoblotting. Whereas insulin receptor substrate-1 was weakly detected, insulin increased 5.5 +/- 1.5-fold (mean +/- SD) PtdIns 3'-kinase activity in receptor immunoprecipitates. We conclude that insulin-stimulated PtdIns 3'-kinase activity is measurable in circulating mononuclear cells which may constitute an easily available cellular model for the detection of post-receptor defects in insulin-resistant states.

Adult↗
PubMed1995

Characterization of Rad, a new member of Ras/GTPase superfamily, and its regulation by a unique GTPase-activating protein (GAP)-like activity.

We have recently identified a new member of the Ras/GTPase superfamily termed Rad which has unique sequence features and is overexpressed in the skeletal muscle of humans with type II diabetes (Reynet, C., and Kahn, C. R. (1993) Science, 262, 1441-1444). When expressed in bacteria as a glutathione S-transferase fusion protein, Rad bound [alpha-32P]GTP quickly and saturably. Binding was specific for guanine nucleotides and displayed unique magnesium dependence such that both GTP and GDP binding were optimal at relatively high Mg2+ concentrations (1-10 mM). Rad had low intrinsic GTPase activity which was greatly enhanced by a GTPase-activating protein (GAP) activity present in various tissues and cell lines. Several known GAPs had no stimulatory effect toward Rad. Conversion of Ser to Asn at position 66 in Rad (equivalent to position 12 in Ras) resulted in a total loss of GTP binding. Mutation of Pro61 (equivalent to Gly12 in Ras) or Gln109 (equivalent to Gln61 in Ras) had no effect on Rad GTPase activity, whereas creation of a double mutation at these positions resulted in exceptionally high intrinsic GTPase activity. In vitro, Rad was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (PK). Phosphopeptide mapping indicated two PKA phosphorylation sites near the COOH terminus. Rad also co-precipitated a serine/threonine kinase activity from extracts of various tissues and cell lines which catalyzed phosphorylation on Rad but was not inhibited by PKA inhibitor. Thus, Rad is a GTP-binding protein and a GTPase which has some structure/function similarities to Ras, but displays unique features. Rad may also be phosphorylated on serine/threonine residues by PKA and other kinases, as well as regulated by its own GAP which is present in many tissues and cell types.

Amino Acid Sequence↗
PubMed1995

Role of the transmembrane domain and flanking amino acids in internalization and down-regulation of the insulin receptor.

We have characterized the internalization and down-regulation of the insulin receptor and nine receptors with mutations in the transmembrane (TM) domain and/or flanking charged amino acids to define the role of this domain in receptor cycling. When expressed in Chinese hamster ovary cells, all had normal tetrameric structure and normal insulin-stimulated autophosphorylation/kinase activity. Replacement of the TM domain with that of the platelet-derived growth factor receptor, insertion of 3 amino acids, and substitution of Asp for Val938 or of Ala for either Gly933 or Pro934 had no effect on internalization. Replacement of the TM domain with that of c-neu or conversion of the charged amino acids on the cytoplasmic flank to uncharged amino acids, on the other hand, resulted in a 40-60% decrease in insulin-dependent internalization rate constants. By contrast, substitution of Ala for both Gly933 and Pro934 increases lateral diffusion mobility and accelerates internalization rate. These changes in internalization were due to decreased or increased rates of redistribution of receptors from microvilli to the nonvillous cell surface. In all cases, receptor down-regulation and receptor-mediated insulin degradation paralleled the changes in internalization. Thus, the structure of the transmembrane domain of the insulin receptor and flanking amino acids are major determinants of receptor internalization, insulin degradation, and receptor down-regulation.

Amino Acid Sequence↗
PubMed1995

Decreased muscle glucose transport/phosphorylation is an early defect in the pathogenesis of non-insulin-dependent diabetes mellitus.

Recent studies have demonstrated that reduced insulin-stimulated muscle glycogen synthesis is the major cause of insulin resistance in patients with non-insulin-dependent diabetes mellitus (NIDDM). This reduced rate has been assigned to a defect in either glucose transport or hexokinase activity. However it is unknown whether this is a primary or acquired defect in the pathogenesis of NIDDM. To examine this question, we measured the rate of muscle glycogen synthesis and the muscle glucose 6-phosphate (G6P) concentration using 13C and 31P NMR spectroscopy as well as oxidative and nonoxidative glucose metabolism in six lean, normoglycemic offspring of parents with NIDDM and seven age/weight-matched control subjects under hyperglycemic (approximately 11 mM)-hyperinsulinemic (approximately 480 pM) clamp conditions. The offspring of parents with NIDDM had a 50% reduction in total glucose metabolism, primarily due to a decrease in the nonoxidative component. The rate of muscle glycogen synthesis was reduced by 70% (P < 0.005) and muscle G6P concentration was reduced by 40% (P < 0.003), which suggests impaired muscle glucose transport/hexokinase activity. These changes were similar to those previously observed in subjects with fully developed NIDDM. When the control subjects were studied at similar insulin levels (approximately 440 pM) but euglycemic plasma glucose concentration (approximately 5 mM), both the rate of glycogen synthesis and the G6P concentration were reduced to values similar to the offspring of parents with NIDDM. We conclude that insulin-resistant offspring of parents with NIDDM have reduced nonoxidative glucose metabolism and muscle glycogen synthesis secondary to a defect in muscle glucose transport/hexokinase activity prior to the onset of overt hyperglycemia. The presence of this defect in these subjects suggests that it may be the primary factor in the pathogenesis of NIDDM.

Adenosine Diphosphate↗