Biomedical subjects
Publications and source records attributed to C R Kahn.
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Phosphotyrosine phosphatase (PTPase) activity in rat liver was measured using a phosphopeptide substrate containing sequence identity to the major site of insulin receptor autophosphorylation. PTPase activity was detected in both cytosolic and particulate fractions of rat liver and produced linear dephosphorylation over a 15-min time course. In rats made insulin-deficient diabetic by streptozotocin treatment (STZ), cytosolic PTPase activity increased to 180% of the control values after 2 d of diabetes and remained elevated at 30 d (P less than 0.02). Gel filtration on Sephadex-75 revealed a single peak of activity in the cytosol in both control and diabetic animals and confirmed the increased levels. In BB diabetic rats, another model of insulin deficiency, the PTPase activity in the cytosolic fraction was increased to approximately 230% of control values. PTPase activity in the particulate fraction of liver was also increased by 30 and 80% after 2 and 8 d of STZ diabetes, respectively. However, this increase was not sustained and after 30 d of STZ diabetes, PTPase activity associated with the particulate fraction in the BB diabetic rat was reduced to approximately 70% of the control levels. Treatment of STZ diabetic rats with subcutaneous insulin or vanadate in their drinking water for 3 d reduced tyrosine PTPase activity in the particulate, but not in the cytosolic fraction. This was associated with a change in blood glucose toward normal. These data indicate insulin deficient diabetes is accompanied by significant changes in hepatic PTPase activity. Since tyrosine phosphorylation plays a central role in the cellular action of insulin receptor, an increase in PTPase activity may be an important factor in the altered insulin action associated with these diabetic states.
Leprechaunism is a rare genetic disorder characterized by severe growth retardation and insulin resistance. Maximal epidermal growth factor (EGF) binding was reduced in fibroblasts from three unrelated patients with leprechaunism (Ark-1, Can-1, and Minn-1) compared with control (0.8-2.2%/mg protein vs. 5.5%/mg protein). This was due to a decrease in receptor affinity in Ark-1 and Can-1 and a decrease in receptor number in Minn-1. In all cell lines, EGF-stimulated receptor autophosphorylation was also decreased to 18-60% of control, whereas EGF internalization and degradation was normal. Sphingosine (40 microM), a protein kinase C inhibitor, increased EGF receptor affinity twofold in control cells and six- to nine-fold in cells of leprechaunism. However, sphingosine did not enhance EGF-stimulated receptor autophosphorylation in either the controls or the patients' cells. By contrast, only one of the three cell lines of patients with the type A syndrome demonstrated a decrease in EGF binding and all demonstrated normal or near normal EGF-stimulated receptor autophosphorylation. These data indicate that in patients with leprechaunism, there are functional abnormalities of the EGF receptor, as well as of the insulin receptor, that may contribute to the severity of the syndrome. These data also suggest a role for the insulin receptor in maintaining normal EGF receptor function in these cells.
Insulin and insulin-like growth factor-I (IGF-I) effects on protein phosphorylation were investigated in intact skeletal muscle cells at different stages of differentiation. In undifferentiated L6 myoblasts, stimulation by either insulin or IGF-I, but not IGF-II, led to a 3- to 5-fold increase in phosphorylation of insulin and IGF receptor beta-subunits and the appearance of a 175,000 mol wt (Mr) phosphoprotein (pp175). These effects reached a maximum within 3 min, were maintained for 12 min, and then declined. Dose-response curves for pp175 phosphorylation in response to insulin (ED50 = 2 nM) and IGF-I (ED50 = 0.2 nM) were consistent with occupancy and stimulation of each receptor kinase by its specific hormone. The 175,000 Mr phosphoprotein was not precipitated by antireceptor antibodies, and the phosphoamino acid composition differed markedly from that of insulin and IGF-I receptors, with a 10-fold lower phosphotyrosine/phosphoserine ratio after insulin stimulation. In contrast to insulin and IGF-I receptors, pp175 was not extracted by the nonionic detergent Triton X-100, but required sodium dodecyl sulfate for solubilization. When experiments were carried out with L6 cells after differentiation into skeletal muscle myotubes, hormone-induced phosphorylation of pp175 was almost undetectable. We conclude that pp175 is a phosphoprotein distinct from insulin and IGF-I receptors that is involved in the early phosphorylation events that follow the activation of the insulin and IGF-I receptor kinases. Its disappearance after terminal differentiation of the L6 cells is consistent with a role in hormonal stimulation of cell proliferation.
Obesity is associated with insulin resistance and type II diabetes mellitus. In the present study, we have characterized hepatic insulin receptor function in two animal models of obesity: the Zucker fatty rat (ZFR), a model of genetic obesity with severe hyperinsulinemia, and the Sprague-Dawley rat with dietary obesity, a model of acquired obesity. Zucker fatty rats were also treated with streptozotocin (STZ) in an effort to examine the effects of relative insulin deficiency and hyperglycemia in the setting of obesity. Using wheat germ agglutinin-purified insulin receptor extracted from liver, no significant difference in insulin binding was identified in either model of obesity. beta-Subunit autophosphorylation was significantly decreased in both obese models relative to that in controls (72% in the obese ZFR and 49% in the overfed Sprague-Dawley model). Kinase activity, as measured by phosphorylation of the 1142-1153 synthetic peptide, was also decreased in both models of obesity by 22% and 64%, respectively. In the Zucker rat, STZ treatment led to an 80% increase in receptor concentration and a further 70% increase in beta-subunit autophosphorylation per receptor, whereas tyrosine kinase activity toward substrate was not altered. Since kinase activity is closely linked to autophosphorylation, we determined the fraction of autophosphorylated (activated) receptors vs. non-phosphorylated (inactive) receptors by using antiphosphotyrosine antibody to precipitate receptors bound with [125I]insulin. There was no significant difference in the percentage of activated insulin receptors in the dietary obese, ZFR, or STZ-treated Zucker rat vs. that in the controls. In all models, the percentage of activated receptors ranged from 32-46% of the total receptor pool. These data suggest that in genetic and acquired obesity, autophosphorylation of the beta-subunit is reduced and is a limiting factor in insulin receptor activation. A similar fraction of all receptors appears to undergo some level of autophosphorylation; however, full autophosphorylation and, thus, activation of the receptor do not occur, and this results in a decrease in kinase activity. This block in autophosphorylation may account for significant reductions in insulin receptor kinase function in obesity.
We used a ribonuclease cleavage assay to screen for insulin receptor mRNA sequence alterations in 12 patients with syndromes of severe insulin resistance. Uniformly labeled [32P]antisense RNA probes complementary to insulin receptor mRNA were prepared by an SP6 or T7 RNA polymerase transcription reaction. Four probes ranging in size from 670-1470 bases were used to examine the entire 4.2-kilobase receptor protein-coding region. Patient RNA samples were hybridized to individual probes in solution, and mismatched sequences were detected by susceptibility to cleavage by a mixture of RNAses A and T1. The method was validated with insulin receptor mRNAs from cells transfected with cDNA constructs bearing known point and deletion mutations. Alterations in the insulin receptor mRNA sequence of two patients were detected. A patient with the type A syndrome of severe insulin resistance (A2-Boston) had a mutation in the insulin receptor beta-subunit mRNA sequence that localized to the region coding for amino acid residues 1174-1211 near the tyrosine kinase domain. The second alteration was a sequence polymorphism in the insulin receptor alpha-subunit mRNA in a patient with lipoatropic diabetes (LA-2) that localized to a region within amino acids 268-272. Direct sequence analysis revealed that the ribonuclease cleavage sites in patients A2-Boston and LA-2 were due to distinct single base changes in the insulin receptor gene and mRNA. Additional insulin receptor mRNA sequence polymorphisms were also identified as mismatches between the labeled RNA probes used and mRNA from several cultured human cell types. This study demonstrates that ribonuclease cleavage can rapidly detect and localize insulin receptor mRNA sequence mutations and polymorphic variations as small as single base changes. Further analysis of insulin receptor mRNA sequence alterations identified in this way may elucidate a possible genetic basis for functional insulin receptor defects in patients with severe insulin resistance and can also reveal some insulin receptor sequence polymorphisms that occur in the population at large.
Using the well differentiated rat hepatoma Fao we have studied the regulation of phosphoenolpyruvate carboxykinase (PEPCK) mRNA by insulin and glucose and compared these results to glucose production as estimated by glucose release into the medium. Fao cells possess an active gluconeogenic pathway and, when grown in glucose-free medium, release glucose for over 8 h. Addition of the cAMP analog, 8-(4-chlorophenyl-thio) cAMP (8-CTP-cAMP) or increasing the concentration of dihydroxyacetone and oxaloacetate results in an increase in glucose release which can be suppressed by insulin at concentrations between 1 and 100 nM. These effect of cAMP and insulin are associated with parallel changes in the level of mRNAPEPCK. Insulin treatment reduces mRNAPEPCK levels in these cells by 80%; this effect is transient reaching a maximum at 2-4 h. Addition of glucose to cells grown in glucose-free (G-) medium produces a decrease in mRNAPEPCK which is similar in magnitude and kinetics to that produced by insulin. Conversely, when cells grown in normal medium are placed in G- medium mRNAPEPCK levels triple over a period of 8 h, then return toward the basal value. Cells grown in G- medium or in G- medium plus 10nM insulin for 1 yr exhibit only slightly increased levels of mRNAPEPCK and respond to both 8-CTP-cAMP, and insulin, although the response to 8-CTP-cAMP is slightly blunted. These data indicate that glucose and insulin can play independent roles in regulation of PEPCK gene expression, and that these regulatory effects are usually transient.
We studied the structure of the insulin-receptor gene in normal individuals and in four unrelated patients with leprechaunism (Minn-1, Ark-1, Ark-2, Can-1) and four unrelated patients with the type A syndrome of insulin resistance, both disorders associated with genetic alterations in affinity, binding capacity, and kinase activity of the insulin receptor. Genomic cloning and Southern blot analysis indicate that the normal human insulin-receptor gene is greater than or equal to 150 kilobases long and consists of a minimum of 17 exons, 6 in the genomic region of the alpha-subunit and 11 in the region of the beta-subunit. Three of the patients, one with leprechaunism and two with type A syndrome, have decreases in insulin-receptor mRNA but on genomic blot analysis have no obvious abnormalities in the insulin-receptor gene. No distinctive pattern of restriction-fragment-length polymorphisms or evidence for major insertion or deletion mutations of the insulin-receptor gene was found in any of the patients. These data indicate that the insulin-receptor gene is greater than 35 times larger than coding regions and has a complex structure. Although leprechaunism and type A syndrome are most likely due to defects in the structure and expression of the insulin-receptor gene, they are likely to be associated with specific point mutations rather than major changes in gene structure.
Insulin-producing beta-cells were selectively absent from the islets of Langerhans in postmortem specimens from two patients with Wolfram's syndrome. In families with multiple cases of this syndrome, we found a very high concordance rate (r = .910, P less than .001) among siblings for age at onset of diabetes mellitus. Taken together with the lack of markers for an autoimmune process, these findings suggest that diabetes mellitus in this syndrome results from genetically programmed selective beta-cell death.
Leprechaunism is a genetic form of insulin resistance characterized by severe growth retardation and early death. To clarify the molecular basis of the insulin resistance, we investigated the insulin binding and kinase properties of the insulin receptor and the receptor gene in cultured skin fibroblasts of two patients (Ark-1 and Ark-2) with leprechaunism and in those of three of their parents. Specific insulin binding to fibroblasts was markedly reduced (less than 25% of control) in both patients with leprechaunism but was essentially normal in the parents. In contrast, insulin receptor autophosphorylation in 1% Triton X-100 cell lysates was reduced in both patients and parents. In Ark-1, the 70% reduction in autophosphorylation correlated with the decrease in binding, whereas in Ark-2 and in the three parents included in the study, autophosphorylation of the insulin receptor was reduced below the level accounted for by a change in receptor content. Analysis of the insulin receptor gene by hybridization with the receptor cDNA probes revealed no gross defect in either Ark-1 or Ark-2. Both parents of Ark-2 were heterozygous for a restriction fragment length polymorphism in the beta-subunit detected with Bam HI digestion (observed in 15% of controls). Ark-2 was homozygous for the more common allele of this polymorphism (observed in 84% of controls). Thus, we have biochemically characterized a new family of leprechaunism (Ark-2) and have found insulin receptor phosphorylation defects in their phenotypically normal parents.
Lipocortin 1 is a steroid-induced, calcium-regulated membrane binding protein (Mr = 39,000) which is a substrate for the epidermal growth factor receptor kinase in intact cells. Using a polyclonal antibody to human recombinant lipocortin 1, we have identified a 170-kDa phosphoprotein in freshly isolated rat hepatocytes which shares antigenic determinants with lipocortin 1. The protein was recognized by four different anti-lipocortin 1 antisera, and antibody binding was inhibited by a 100-fold molar excess of human recombinant lipocortin 1 over antibody. Based on Coomassie Blue staining, the 170-kDa lipocortin-related protein is abundant (approximately 100 ng/10(6) cells) in rat liver, while lipocortin 1 itself is found in very low amounts. Epidermal growth factor and insulin stimulated phosphorylation of this 170-kDa protein in intact rat hepatocytes. The increase in phosphorylation was more pronounced in hepatocytes from dexamethasone-treated animals. The phosphorylation occurred exclusively on serine residues and was maximal 30-60 min after hormone addition. The 170-kDa protein was localized in the cytoplasm in the absence of calcium, while increasing calcium concentration led to partial association with the membrane compartment in rat liver. This 170-kDa protein represents a new member of the class of proteins whose serine phosphorylation is regulated by insulin and EGF and may belong to the family of lipocortin-related molecules.
Insulin modifies cellular responsiveness to some hormones which operate via guanine nucleotide binding proteins (G-proteins); also, G-proteins have been implicated in some actions of insulin. Using pertussis toxin-catalyzed [32P]ADP-ribosylation of Gi as an index of G-protein conformation, we evaluated interaction of insulin receptors with G-proteins. In isolated rat liver plasma membranes, insulin treatment for 10 min inhibited [32P]ADP-ribosylation of Gi by 50%. This effect was half-maximal at 2 x 10(-8) M. A similar effect was observed with rat adipocyte plasma membranes with half-maximal effect at 1 x 10(-8) M. Pertussis toxin activity itself was uninfluenced by insulin, as ribosylation of tubulin or heat-treated bovine serum albumin was unaltered. Elevated Mg2+ diminished basal ADP-ribosylation, but insulin inhibition occurred at all Mg2+ levels between 0 and 1 mM. Insulin inhibition was independent of ATP (20 microM to 10 mM), and GTP (0-100 microM) concentrations. Because both protein kinase C and purified insulin receptor phosphorylate purified Gi in vitro, we examined Gi as a substrate for the insulin receptor tyrosine kinase in vivo. Triton-extracts of isolated rat hepatocytes which had been 32Pi labeled and treated with insulin were immunoprecipitated with a polyclonal anti-Gi antiserum. The dominant labeled phosphoprotein had a molecular weight of 42 kDa, consistent with the alpha-subunit of Gi, contained only phosphoserine, and was unaffected in its phosphorylation by insulin. These results indicate the existence of a novel pathway for physiological "cross-talk" between insulin and other hormones and further suggests that the insulin receptor may interact with regulatory G-proteins via biochemical mechanisms not directly involving the tyrosine kinase activity of the insulin receptor.
To evaluate the synthesis and initial processing of the insulin receptor precursor, we compared cell-free translation of rat liver poly(A)+ RNA in a reticulocyte lysate system with metabolically labeled rat hepatoma (Fao) cells. In in vitro translation assays, the primary L-[35S]cysteine-labeled products of rat liver mRNA specifically immunoprecipitable with insulin receptor antiserum were two closely migrating polypeptides with a Mr range of 160,000-164,000 (n = 7). This is similar to the size predicted by the insulin receptor cDNA sequence. When heterologous microsomal membranes were included in the cell-free system to process newly synthesized proteins co-translationally, the receptor precursors migrated as larger species of 180 +/- 2 kDa (n = 3). For comparison, when Fao cells were treated with tunicamycin to block core N-glycosylation and pulse-labeled with L-[35S]methionine, two closely migrating precursors were labeled that co-migrated with the unprocessed in vitro translation products (approximately 160 kDa). Pulse labeling of Fao cells in the absence of tunicamycin revealed receptor precursor species of 188 and 198 kDa that rapidly disappeared (t1/2 = 54 min) as the receptor subunits were observed. Thus, the initial products of insulin receptor mRNA translation are two approximately 162-kDa polypeptides that are rapidly processed in intact cells and can only be observed by in vitro studies or by using inhibitors of core glycosylation. Insulin proreceptor species can also be partially glycosylated during cell-free translation by added microsomal membranes. This is the first description of cell-free translation of the insulin proreceptor in a system that will allow detailed characterization of the earliest steps in insulin receptor biogenesis.
Tyrosyl phosphorylation is implicated in the mechanism of insulin action. Mutation of the beta-subunit of the insulin receptor by substitution of tyrosyl residue 960 with phenylalanine had no effect on insulin-stimulated autophosphorylation or phosphotransferase activity of the purified receptor. However, unlike the normal receptor, this mutant was not biologically active in Chinese hamster ovary cells. Furthermore, insulin-stimulated tyrosyl phosphorylation of at least one endogenous substrate (pp185) was increased significantly in cells expressing the normal receptor but was barely detected in cells expressing the mutant. Therefore, beta-subunit autophosphorylation was not sufficient for the insulin response, and a region of the insulin receptor around Tyr-960 may facilitate phosphorylation of cellular substrates required for transmission of the insulin signal.
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Lipocortins 1 and 2 are major substrates for the epidermal growth factor receptor and the pp60v-src tyrosine kinases in transformed cells. In the present study, we have characterized the phosphorylation of lipocortins 1 and 2 by the insulin receptor tyrosine kinase in vitro and in vivo. In vitro, the solubilized insulin receptor, partially purified from rat liver, catalyzed phosphorylation of human recombinant lipocortin 1 and purified bovine lipocortin 2. Phosphorylation of lipocortin 1 was increased 15-fold upon stimulation with 10(-7) M insulin. The apparent Km of the reaction was 3.3 microM and was not affected by insulin stimulation. Insulin stimulated phosphate incorporation into lipocortin 2 by 20-fold (apparent Km greater than 20 microM). Both lipocortins were phosphorylated exclusively on tyrosine residues as judged by phosphoamino acid analysis. Based upon peptide mapping, lipocortin 1 was phosphorylated on Tyr-21, a site phosphorylated by other tyrosine kinases. Polyclonal anti-phosphotyrosine antibodies recognized the tyrosine-phosphorylated lipocortin 2, but not lipocortin 1 in its phosphorylated form. In hepatocytes from normal and dexamethasone-treated rats, lipocortin 1 content was less than 50 ng/10(6) cells. Insulin-induced phosphorylation of lipocortin 1 was detected in intact hepatocytes from corticosteroid-treated animals but not in cells from normal rats. No phosphorylation of lipocortin 2 was found, although its content was approximately 100 ng/10(6) cells from normal animals and increased to approximately 1 microgram/10(6) cells following treatment of rats with dexamethasone for 4 days. Thus, although lipocortins 1 and 2 are in vitro substrates of the insulin receptor kinase, only lipocortin 1 is phosphorylated in an insulin-dependent manner in intact hepatocytes, and this is only observed after dexamethasone treatment of the rats.
The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.
Trypsin exerts insulin-like effects in intact cells and on partially purified preparations of insulin receptors. To elucidate the mechanism of these insulinomimetic effects, we compared the structures of insulin- and trypsin-activated receptor species with their functions, including insulin binding, autophosphorylation, and tyrosine kinase activity. In vitro treatment of wheat germ agglutinin-purified receptor preparations with trypsin resulted in proteolysis of both alpha- and beta-subunits. The activated form of the receptor had an apparent molecular mass of 110 kDa under nonreducing conditions, compared to the 400-kDa intact receptor, and was separated following reduction into an 85-kDa beta-subunit related fragment and a 25-kDa alpha-subunit related fragment. Treatment of whole cells with trypsin prior to isolation of the insulin receptor resulted in proteolytic modification of the alpha-subunit only. In this case, the total molecular mass of the activated species was 116 kDa, comprised of an intact 92-kDa beta-subunit and again a 25-kDa alpha-subunit related fragment. Values of Km for peptide substrate phosphorylation and Ki for inhibition of receptor autophosphorylation, and sites of autophosphorylation within the beta-subunits were similar for receptors activated either by insulin or trypsin. Insulin had no additional effect on the rate of autophosphorylation of the truncated receptor, and no binding of insulin by the truncated receptor was detected either by direct assay or cross-linking with bifunctional reagents. Based on the deduced amino acid sequence of the insulin receptor and the structural studies presented here we concluded that this activated form of the receptor resulted from tryptic cleavage at the dibasic site Arg576-Arg577. This was accompanied by loss of the insulin binding site and separation of alpha-beta heterodimers. As truncation of the alpha-subunit results in beta-subunit activation, it appears that the beta-subunit is a constitutively activated kinase and that the function of the alpha-subunit in the intact receptor is to inhibit the beta-subunit.