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Biomedical subjects

C R Kahn

Publications and source records attributed to C R Kahn.

At least 73 records · Page 4Linked to original sources
PubMed1999

Dilated cardiomyopathy and atrioventricular conduction blocks induced by heart-specific inactivation of mitochondrial DNA gene expression.

Mutations of mitochondrial DNA (mtDNA) cause several well-recognized human genetic syndromes with deficient oxidative phosphorylation and may also have a role in ageing and acquired diseases of old age. We report here that hallmarks of mtDNA mutation disorders can be reproduced in the mouse using a conditional mutation strategy to manipulate the expression of the gene encoding mitochondrial transcription factor A (Tfam, previously named mtTFA), which regulates transcription and replication of mtDNA. Using a loxP-flanked Tfam allele (TfamloxP) in combination with a cre-recombinase transgene under control of the muscle creatinine kinase promoter, we have disrupted Tfam in heart and muscle. Mutant animals develop a mosaic cardiac-specific progressive respiratory chain deficiency, dilated cardiomyopathy, atrioventricular heart conduction blocks and die at 2-4 weeks of age. This animal model reproduces biochemical, morphological and physiological features of the dilated cardiomyopathy of Kearns-Sayre syndrome. Furthermore, our findings provide genetic evidence that the respiratory chain is critical for normal heart function.

Animals↗
PubMed1999

Crosstalk between insulin and angiotensin II signalling systems.

Insulin resistance and hypertension commonly occur together. Pharmacological inhibition of the renin-angiotensin system has been found to reduce not only hypertension, but also insulin resistance. This raises the possibility that the renin-angiotensin system may interact with insulin signalling. We have investigated the relationship between insulin and angiotensin II (AII) intracellular signalling in vivo using an intact rat heart model, and in vitro using rat aorta smooth muscle cells (RASMC). Results generated in the in vivo studies indicate that, like insulin, AII stimulates tyrosine phosphorylation of the insulin receptor substrates IRS-1 and IRS-2. This leads to binding of IRS-1 and IRS-2 to PI3-kinase. However, in contrast to the effect of insulin. IRS-1- and IRS-2-associated PI3-kinase activity is inhibited by AII in a dose-dependent manner. Moreover, AII inhibits insulin-stimulated IRS-1/IRS-2-associated PI3-kinase activity. The in vivo effects of AII are mediated via the AT1 receptor. The results of the in vitro studies indicate that AII inhibits insulin-stimulated, IRS-1-associated PI3-kinase activity by interfering with the docking of IRS-1 with the p85 regulatory subunit of PI3-kinase. It appears that AII achieves this effect by stimulating serine phosphorylation of the insulin receptor beta-subunit IRS-1, and the p85 regulatory subunit of PI3-kinase. These actions result in the inhibition of normal interactions between the insulin signalling pathway components. Thus, we believe that AII negatively modulates insulin signalling by stimulating multiple serine phosphorylation events in the early components of the insulin signalling cascade. Overactivity of the renin-angiotensin system is likely to impair insulin signalling and contribute to insulin resistance observed in essential hypertension.

Angiotensin II↗
PubMed1999

Exercise modulates postreceptor insulin signaling and glucose transport in muscle-specific insulin receptor knockout mice.

Physical exercise promotes glucose uptake into skeletal muscle and makes the working muscles more sensitive to insulin. To understand the role of insulin receptor (IR) signaling in these responses, we studied the effects of exercise and insulin on skeletal muscle glucose metabolism and insulin signaling in mice lacking insulin receptors specifically in muscle. Muscle-specific insulin receptor knockout (MIRKO) mice had normal resting 2-deoxy-glucose (2DG) uptake in soleus muscles but had no significant response to insulin. Despite this, MIRKO mice displayed normal exercise-stimulated 2DG uptake and a normal synergistic activation of muscle 2DG uptake with the combination of exercise plus insulin. Glycogen content and glycogen synthase activity in resting muscle were normal in MIRKO mice, and exercise, but not insulin, increased glycogen synthase activity. Insulin, exercise, and the combination of exercise plus insulin did not increase IR tyrosine phosphorylation or phosphatidylinositol 3-kinase activity in MIRKO muscle. In contrast, insulin alone produced a small activation of Akt and glycogen synthase kinase-3 in MIRKO mice, and prior exercise markedly enhanced this insulin effect. In conclusion, normal expression of muscle insulin receptors is not needed for the exercise-mediated increase in glucose uptake and glycogen synthase activity in vivo. The synergistic activation of glucose transport with exercise plus insulin is retained in MIRKO mice, suggesting a phenomenon mediated by nonmuscle cells or by downstream signaling events.

Animals↗
PubMed1999

Altered function of insulin receptor substrate-1-deficient mouse islets and cultured beta-cell lines.

Insulin receptor substrate-1 (IRS-1) is pivotal in mediating the actions of insulin and growth factors in most tissues of the body, but its role in insulin-producing beta islet cells is unclear. Freshly isolated islets from IRS-1 knockout mice and SV40-transformed IRS-1-deficient beta-cell lines exhibit marked insulin secretory defects in response to glucose and arginine. Furthermore, insulin expression is reduced by about 2-fold in the IRS-1-null islets and beta-cell lines, and this defect can be partially restored by transfecting the cells with IRS-1. These data provide evidence for an important role of IRS-1 in islet function and provide a novel functional link between the insulin signaling and insulin secretion pathways. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Animals↗
PubMed1999

Postnatal growth responses to insulin-like growth factor I in insulin receptor substrate-1-deficient mice.

Organ weight was compared in adult mice with deletion of one (IRS-1-/+) or both (IRS-1-/-) copies of the insulin receptor substrate-1 (IRS-1) gene and IRS-1+/+ littermates. IRS-1-/+ mice showed modest reductions in weight of most organs in proportion to a decrease in body weight. IRS-1-/- mice showed major reductions in weight of heart, liver, and spleen that were directly proportional to a decrease in body weight. In IRS-1-/- mice, kidney and particularly small intestine and brain exhibited proportionately smaller weight reductions, and gastrocnemius muscle showed a proportionately greater weight reduction than the decrease in body weight. Growth deficits in IRS-1-/- mice could reflect impaired actions of multiple hormones or cytokines that activate IRS-1. To assess the requirement for IRS-1 in insulin-like growth factor I (IGF-I)-dependent postnatal growth, IRS-1-/+ mice were cross-bred with mice that widely overexpress a human IGF-I transgene (IGF+) to generate IGF+ and wild-type mice on an IRS-1+/+, IRS-1-/+, and IRS-1-/- background. IGF-I overexpression increased body weight and weight of brain, small intestine, kidney, spleen, heart, and gastrocnemius muscle in IRS-1+/+ mice. IGF-I overexpression could not completely reverse the body growth retardation in IRS-1-/- mice. Absolute or partial IRS-1 deficiency impaired IGF-I-induced body overgrowth more in females than in males. In males and females, IGF-I stimulated similar overgrowth of brain regardless of IRS-1 status, and intestine and spleen showed dose dependence on IRS-1 for IGF-I-induced growth. IGF-I-induced growth of gastrocnemius muscle had an absolute requirement for IRS-1. IGF-I-induced growth of kidney and heart was impaired by IRS-1 deficiency only in females. In vivo, therefore, most organs do not require IRS-1 for IGF-I-induced growth and can use alternate signaling molecules to mediate IGF-I action. Other organs, such as gastrocnemius muscle, require IRS-1 for IGF-I-induced growth in vivo.

Animals↗
PubMed1999

Calorie restriction increases insulin-stimulated glucose transport in skeletal muscle from IRS-1 knockout mice.

Calorie restriction (CR), even for brief periods (4-20 days), results in increased whole-body insulin sensitivity, in large part due to enhanced insulin-stimulated glucose transport by skeletal muscle. Evidence suggests that the cellular alterations leading to this effect are postreceptor steps in insulin signaling. To determine whether insulin receptor substrate (IRS)-1 is essential for the insulin-sensitizing effect of CR, we measured in vitro 2-deoxyglucose (2DG) uptake in the presence and absence of insulin by skeletal muscle isolated from wild-type (WT) mice and transgenic mice lacking IRS-1 (knockout [KO]) after either ad libitum (AL) feeding or 20 days of CR (60% of ad libitum intake). Three muscles (soleus, extensor digitorum longus [EDL], and epitrochlearis) from male and female mice (4.5-6 months old) were studied. In each muscle, insulin-stimulated 2DG uptake was not different between genotypes. For EDL and epitrochlearis, insulin-stimulated 2DG uptake was greater in CR compared to AL groups, regardless of sex. Soleus insulin-stimulated 2DG uptake was greater in CR compared with AL in males but not females. The diet effect on 2DG uptake was not different for WT and KO animals. Genotype also did not alter the CR-induced decrease in plasma constituents (glucose, insulin, and leptin) or body composition (body weight, fat pad/body weight ratio). Consistent with previous studies in rats, IRS-1 protein expression in muscle was reduced in WT-CR compared with WT-AL mice, and muscle IRS-2 abundance was unchanged by diet. Skeletal muscle IRS-2 protein expression was significantly lower in WT compared with KO mice. These data demonstrate that IRS-1 is not essential for the CR-induced increase in insulin-stimulated glucose transport in skeletal muscle, and the absence of IRS-1 does not modify any of the characteristic adaptations of CR that were evaluated.

Adipose Tissue↗
PubMed1999

Insulin signaling in insulin receptor substrate (IRS)-1-deficient brown adipocytes: requirement of IRS-1 for lipid synthesis.

Immortalized fetal brown adipocyte cell lines have been generated from homozygous (-/-) and heterozygous (+/-) insulin receptor substrate (IRS)-1-deficient mice, as well as from wild-type mice (+/+). Under growing conditions, these cell lines maintained the expression of the adipogenic marker fatty acid synthase and uncoupling protein-1, a tissue-specific thermogenic marker. The IRS-1 (-/-) brown adipocytes lacked IRS-1 protein expression and had a significant increase in IRS-2 protein expression. Insulin-induced tyrosine phosphorylation of IRS-1 was reduced by 50% in heterozygous IRS-1-deficient cells and was totally absent in homozygous cells, while tyrosine phosphorylation of IRS-2 showed a gradual increase. Insulin receptor alpha-subunit protein content and beta-subunit tyrosine kinase activity remained unchanged upon insulin stimulation, regardless of the lack of IRS-1. Brown adipocytes from homozygous IRS-1-deficient mice showed no IRS-1-associated p85alpha subunit of phosphatidylinositol 3-kinase (PI 3-kinase) or IRS-1-associated PI 3-kinase activity in response to insulin, but exhibited enhanced IRS-2-associated p85alpha subunit and IRS-2-associated PI 3-kinase activity. Overall insulin-induced PI 3-kinase activity associated to antiphosphotyrosine immune complexes was decreased by 30% in the homozygous IRS-1-deficient brown adipocytes. Downstream PI 3-kinase, activated Akt (protein kinase B) was decreased by 92% in an insulin-stimulated homozygous IRS-1-deficient brown adipocyte cell line, whereas the expression of Akt was similar in the three cell lines. However, activated p70 S6 kinase (p70s6k) remained unchanged. Although brown adipocyte cell lines showed similar cytosolic lipid content in the presence of 10% fetal calf serum, cytosolic lipid content was reduced in both serum-deprived heterozygous and homozygous IRS-1-deficient cells. Insulin treatment for 24 h doubled the cytosolic lipid content in wild-type and heterozygous IRS-1-deficient brown adipocyte cell lines but failed to increase the cytosolic lipid content in homozygous IRS-1-deficient cells. Our results strongly suggest that IRS-1/PI 3-kinase/Akt activation is an essential requirement for insulin stimulation of lipid synthesis in brown adipocytes.

Adipocytes↗
PubMed1999

Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance of early postreceptor insulin signaling events in skeletal muscle.

To explore potential cellular mechanisms by which activation of the hexosamine pathway induces insulin resistance, we have evaluated insulin signaling in conscious fasted rats infused for 2-6 h with saline, insulin (18 mU x kg(-1) x min(-1)), or insulin and glucosamine (30 micromol x kg(-1) x min(-1)) under euglycemic conditions. Glucosamine infusion increased muscle UDP-N-acetylglucosamine concentrations 3.9- and 4.3-fold over saline- or insulin-infused animals, respectively (P < 0.001). Glucosamine induced significant insulin resistance to glucose uptake both at the level of the whole body and in rectus abdominis muscle, and it blunted the insulin-induced increase in muscle glycogen content. At a cellular level, these metabolic effects were paralleled by inhibition of postreceptor insulin signaling critical for glucose transport and glycogen storage, including a 45% reduction in insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation (P = 0.02), a 44% decrease in IRS-1 association with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase (P = 0.03), a 34% reduction in IRS-1-associated PI 3-kinase activity (P = 0.03), and a 51% reduction in insulin-stimulated glycogen synthase activity (P = 0.03). These alterations in postreceptor insulin signaling were time-dependent and paralleled closely the progressive inhibition of systemic glucose disposal from 2 to 6 h of glucosamine infusion. We also demonstrated that glucosamine infusion results in O-linked N-acetylglucosamine modification of IRS-1 and IRS-2. These data indicate that activation of the hexosamine pathway may directly modulate early postreceptor insulin signal transduction, perhaps via posttranslation modification of IRS proteins, and thus contribute to the insulin resistance induced by chronic hyperglycemia.

Animals↗
PubMed1998

Obesity associated with a mutation in a genetic regulator of adipocyte differentiation.

BACKGROUND: There is increasing evidence of genetic factors leading to obesity, but the exact genes involved have not been defined. Peroxisome-proliferator-activated receptor gamma2 (PPARgamma2) is a transcription factor that has a key role in adipocyte differentiation, and therefore mutations of the gene for this factor might predispose people to obesity. METHODS: We studied 358 unrelated German subjects, including 121 obese subjects (defined as those with a body-mass index [the weight in kilograms divided by the square of the height in meters] of more than 29). We evaluated these subjects for mutations in the gene for PPARgamma2 at or near a site of serine phosphorylation at position 114 that negatively regulates the transcriptional activity of the protein, using a polymerase-chain-reaction-based assay coupled with specific endonuclease digestion. The activity of the mutation identified was analyzed by retroviral transfection and overexpression in murine fibroblasts. RESULTS: Four of the 121 obese subjects had a missense mutation in the gene for PPARgamma2 that resulted in the conversion of proline to glutamine at position 115, as compared with none of the 237 subjects of normal weight (P=0.01). All the subjects with the mutant allele were markedly obese, with body-mass-index values ranging from 37.9 to 47.3, as compared with a mean of 33.6 in the other obese subjects. Overexpression of the mutant gene in murine fibroblasts led to the production of a protein in which the phosphorylation of serine at position 114 was defective, as well as to accelerated differentiation of the cells into adipocytes and greater cellular accumulation of triglyceride than with the wild-type PPARgamma2. These effects were similar to those of an in vitro mutation created directly at the Ser114 phosphorylation site. CONCLUSIONS: A Pro115Gln mutation in PPARgamma2 accelerates the differentiation of adipocytes and may cause obesity.

Adipocytes↗
PubMed1998

The ras-related protein rad associates with the cytoskeleton in a non-lipid-dependent manner.

Rad is the prototypic member of a new family of Ras-related proteins (Rad, Gem, and Kir) which lack typical C-terminal amino acid motifs for isoprenylation. In mouse C2C12 muscle cell lines about 50% of Rad protein resides in the cytosol and behaves as a hydrophilic protein partitioning away from TX-114. The remainder of Rad is associated with plasma and internal membranes. The association of Rad with the membrane does not occur through the lipid bilayer, but instead depends on the interaction of Rad with the cytoskeleton or membrane skeleton. In contrast to Ras, biosynthetic labeling of cellular proteins in C2Cl2 cells with [3H]palmitic acid demonstrates that Rad is not modified with this fatty acid, and inhibition of isoprenylation with lovastatin treatment has no effect on Rad subcellular distribution. Furthermore, removal of the C-terminal 11 amino acids that are precisely conserved in all three Rad family members has no effect on Rad subcellular distribution. Addition of the 9 amino acids from the C-terminus of H-Ras to the truncated Rad protein results in a redistribution of Rad from the cytosol to the membrane skeleton without the presence of any detectable lipid modification of the chimeric protein. These data suggest that Rad possesses unique cellular localization signals which, in contrast to other Ras-related family members, do not depend on the lipid modification of the C-terminus.

Amino Acid Sequence↗
PubMed1998

Effects of phosphorylation on function of the Rad GTPase.

Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278-297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometries in vitro of 0.2-1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290 and Ser299. Incubation of Rad with PKA decreases GTP binding by 60-70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273-->Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases.

Binding Sites↗
PubMed1998

Dynamics of insulin signaling in 3T3-L1 adipocytes. Differential compartmentalization and trafficking of insulin receptor substrate (IRS)-1 and IRS-2.

The ability of the insulin receptor to phosphorylate multiple substrates and their subcellular localization are two of the determinants that contribute to diversity of signaling. We find that insulin receptor substrate (IRS)-1 is 2-fold more concentrated in the intracellular membrane (IM) compartment than in cytosol, whereas IRS-2 is 2-fold more concentrated in cytosol than in IM. Insulin stimulation induces rapid tyrosine phosphorylation of both IRS-1 and IRS-2. This occurs mainly in the IM compartment, even though IRS-2 is located predominantly in cytosol. Furthermore, after insulin stimulation, both IRS-1 and IRS-2 translocate from IM to cytosol with a t1/2 of 3.5 min. Using an in vitro reconstitution assay, we have demonstrated an association between IRS-1 and internal membranes and have shown that the dissociation of IRS-1 from IM is dependent on serine/threonine phosphorylation of IM. By comparison, within 1 min after insulin stimulation, 40% of the total pool of the 85-kDa subunit of phosphatidylinositol 3-kinase (p85) is recruited from cytosol to IM, the greater part of which can be accounted for by binding to IRS-1 present in the IM. The p85 binding and phosphatidylinositol 3-kinase activity associated with IRS-2 rapidly decrease in both IM and cytosol, whereas those associated with IRS-1 stay at a relatively high level in IM and increase with time in cytosol despite a return of p85 to the cytosol and decreasing tyrosine phosphorylation of cytosolic IRS-1. These data indicate that IRS-1 and IRS-2 are differentially distributed in the cell and move from IM to cytosol following insulin stimulation. Insulin-stimulated IRS-1 and IRS-2 signaling occurs mainly in the IM and shows different kinetics; IRS-1-mediated signaling is more stable, whereas IRS-2-mediated signaling is more transient. These differences in substrate utilization and compartmentalization may contribute to the complexity and diversity of the insulin signaling network.

3T3 Cells↗
PubMed1998

Bidirectional modulation of insulin action by amino acids.

Amino acids have been shown to stimulate protein synthesis, inhibit proteolysis, and decrease whole-body and forearm glucose disposal. Using cultured hepatoma and myotube cells, we demonstrate that amino acids act as novel signaling elements in insulin target tissues. Exposure of cells to high physiologic concentrations of amino acids activates intermediates important in the initiation of protein synthesis, including p70 S6 kinase and PHAS-I, in synergy with insulin. This stimulatory effect is largely due to branched chain amino acids, particularly leucine, and can be reproduced by its transamination product, ketoisocaproic acid. Concurrently, amino acids inhibit early steps in insulin action critical for glucose transport and inhibition of gluconeogenesis, including decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2, decreased binding of grb 2 and the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 and IRS-2, and a marked inhibition of insulin-stimulated phosphatidylinositol 3-kinase. Taken together, these data support the hypothesis that amino acids act as specific positive signals for maintenance of protein stores, while inhibiting other actions of insulin at multiple levels. This bidirectional modulation of insulin action indicates crosstalk between hormonal and nutritional signals and demonstrates a novel mechanism by which nutritional factors contribute to insulin resistance.

Adaptor Proteins, Signal Transducing↗
PubMed1998

A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance.

Skeletal muscle insulin resistance is among the earliest detectable defects in humans with type 2 diabetes mellitus. To determine the contribution of muscle insulin resistance to the metabolic phenotype of diabetes, we used the Cre-loxP system to disrupt the insulin receptor gene in mouse skeletal muscle. The muscle-specific insulin receptor knockout mice exhibit a muscle-specific > 95% reduction in receptor content and early signaling events. These mice display elevated fat mass, serum triglycerides, and free fatty acids, but blood glucose, serum insulin, and glucose tolerance are normal. Thus, insulin resistance in muscle contributes to the altered fat metabolism associated with type 2 diabetes, but tissues other than muscle appear to be more involved in insulin-regulated glucose disposal than previously recognized.

Animals↗
PubMed1998

The insulin receptor--a critical link in glucose homeostasis and insulin action.

We have achieved significant progress in understanding the central role of the insulin receptor in an increasingly complicated web of intracellular signal transduction leading to the ultimate biological actions of insulin on glucose, lipid, and other metabolic pathways. The excitement for the future lies not only in clarifying these pathways but also returning to whole-body physiology to readdress basic mechanisms of insulin action in known and novel insulin-sensitive tissues. Hopefully, these new techniques and new perspectives will bring us closer to understanding the pathophysiology of type 2 diabetes mellitus.

Animals↗
PubMed1998

An association between NIDDM and a GAA trinucleotide repeat polymorphism in the X25/frataxin (Friedreich's ataxia) gene.

Friedreich's ataxia is the most common hereditary ataxia and is frequently associated with disturbances of glucose metabolism. This autosomal recessive disease is caused by the decreased expression of a mitochondrial protein, frataxin, encoded by the X25 gene. Homozygous expansion of a GAA repeat in the first intron of X25 inhibits frataxin expression and is associated with clinical disease. We evaluated whether heterozygous expansions of the triplet repeat in the frataxin gene X25 may be associated with NIDDM in two genetically distinct populations--one in Germany (n = 358) and the other in the U.S. (n = 292)--using a polymerase chain reaction-based assay. Intermediate expansions (10-36 repeats), which are longer than normal but not sufficient for the appearance of the ataxia phenotype, were found in 24.7 and 27.3% of these two NIDDM cohorts compared with 7.6 and 6.3% of the matched control subjects (both P < 0.001). The odds ratios were 3.36 (95% CI 1.72-6.55) for the German group and 4.01 (2.08-7.74) for the U.S. group. Therefore, we conclude that the X25/frataxin GAA repeat polymorphism is associated with NIDDM in a frequency higher than any other mutation heretofore described. Further studies are needed to elucidate the possible role of frataxin in the pathogenesis of NIDDM.

Aged↗