Showing papers by "Jerrold M. Olefsky published in 1993"

Relative expression of insulin receptor isoforms does not differ in lean, obese, and noninsulin-dependent diabetes mellitus subjects.

Abstract: The insulin receptor is expressed as two isoforms that differ by a 12-amino acid region at the carboxy-terminus of the alpha-subunit encoded by exon 11. These isoforms are produced by tissue-specific alternate splicing of the insulin receptor mRNA. To determine whether the relative expression of the isoforms is altered in skeletal muscle in two insulin-resistant states, NIDDM and obesity, relative mRNA levels were measured using a polymerase chain reaction technique. There were no differences in the relative amounts of skeletal muscle mRNA encoding the exon 11-containing form compared to the exon 11-lacking form of the insulin receptor among lean normal (30 +/- 2% Ex11-), obese nondiabetic (32 +/- 2%), and NIDDM (31 +/- 1%) subjects. We conclude that altered expression of insulin receptor isoform mRNAs does not account for skeletal muscle insulin resistance in NIDDM and obesity.

Insulin Resistance and the Pathogenesis of Non-Insulin Dependent Diabetes Mellitus: Cellular and Molecular Mechanisms

Abstract: Non-insulin dependent diabetes mellitus (NIDDM) is a complex metabolic disorder of heterogeneous etiology (1–4). There is clearly a strong hereditary component to the disease, but the exact genetic abnormalities are likely to differ among different population groups (1,5). In addition, NIDDM is likely to be multigenic, in that more than one discrete gene defect needs to complement before the NIDDM phenotype manifests. Thus, from a genetic point of view, NIDDM is heterogeneous and polygenic making identification of “diabetes” genes particularly difficult. Numerous biochemical abnormalities have been identified in NIDDM and the relative contribution of different physiologic or cellular defects differs among different patient groups (6,7). Regardless of the exact pathophysiologic sequence in a particular patient, once full blown fasting hyperglycemia develops, a characteristic set of metabolic derangements can be identified in the great majority of NIDDM patients (Fig. 1). This consists of abnormalities at the level of the pancreatic islets, the liver, and peripheral insulin target tissues which, taken together, represent a final common metabolic pathway for the pathogenesis of hyperglycemia (1,8).

Effect of a major histocompatibility complex class I peptide on insulin-like growth factor-I receptor internalization and biological signaling

Abstract: Previous studies have shown that a peptide derived from the murine major histocompatibility complex class 1 (MHC-1) antigen inhibits insulin receptor internalization and enhances insulin action. Consequently, we have studied the effect of this peptide [Dk-(62-85)] derived from the MHC-1 antigen on insulin-like growth factor-I (IGF-I) internalization and action. When Chinese hamster ovary cells that overexpress human IGF-I receptors were incubated in the presence or absence of 30 microM Dk-(62-85), IGF-I internalization was inhibited by 43.9 +/- 1.7% (P < 0.005). This inhibitory effect was dose responsive, with a half-maximal effect at 8 microM and a maximal effect at 30 microM. The peptide did not affect IGF-I receptor autophosphorylation or ligand-induced phosphorylation of an endogenous substrate, pp185, in vivo. When added alone, the peptide increased glucose transport into Chinese hamster ovary cells by 66.3 +/- 16.0%, whereas when the peptide was added together with IGF-I, ligand-stimulated glucose transport was inhibited. In conclusion, 1) the MHC-1-derived peptide Dk-(62-85) inhibits IGF-I internalization in a dose-responsive fashion; 2) Dk-(62-85) does not affect receptor kinase activity, as IGF-I receptor autophosphorylation and endogenous substrate phosphorylation are unaltered; and 3) Dk-(62-85) stimulates glucose transport by itself, but inhibits IGF-I stimulated transport.

Insulin-responsive human adipocytes express two glucose transporter isoforms and target them to different vesicles

Abstract: We have characterized the insulin-dependent increase in glucose transport in human adipocytes using subcellular fractionation and antibodies specific for the two isoforms of the glucose transporter that are expressed in these cells. Plasma membranes isolated from untreated human fat cells contain the erythroid/GLUT1 isoform of the glucose transporter almost exclusively whereas the muscle-fat/GLUT4 transporter isoform is most abundant in intracellular microsomal membranes in resting cells. After exposure of adipocytes to insulin, the muscle-fat isoform is dramatically increased in the plasma membrane whereas the erythroid isoform barely changes in response to insulin. Thus, the total insulin-mediated increase in plasma membrane glucose transporters, confirmed by affinity labeling of both transporter isoforms, must be due to the increase in the muscle-fat/GLUT4 transporter. The two isoforms exist in different vesicle populations as shown by immunoadsorption of the muscle fat isoform-containing vesicles which are essentially devoid of the erythroid transporter. These data indicate that the insulin-mediated increases in glucose transport in human fat cells is a result of the translocation of vesicles uniquely containing the muscle-fat glucose transporter isoform.

The insulin receptor C-terminus is involved in regulation of the receptor kinase activity.

Abstract: During the insulin receptor activation process, ligand binding and autophosphorylation induce two distinct conformational changes in the C-terminal domain of the receptor beta-subunit. To analyze the role of this domain and the involvement of the C-terminal autophosphorylation sites (Tyr1316 and Tyr1322) in receptor activation, we used (i) antipeptide antibodies against three different C-terminal sequences (1270-1281, 1294-1317, and 1309-1326) and (ii) an insulin receptor mutant (Y/F2) where Tyr1316 and Tyr1322 have been replaced by Phe. We show that the autophosphorylation-induced C-terminal conformational change is preserved in the Y/F2 receptor, indicating that this change is not induced by phosphorylation of the C-terminal sites but most likely by phosphorylation of the major sites in the kinase domain (Tyr1146, Tyr1150, and Tyr1151). Binding of antipeptide antibodies to the C-terminal domain modulated (activated or inhibited) both mutant and wild-type receptor-mediated phosphorylation of poly(Glu/Tyr). In contrast to the wild-type receptor, Y/F2 exhibited the same C-terminal configuration before and after insulin binding, evidencing that mutation of Tyr1316 and Tyr1322 introduced conformational changes in the C-terminus. Finally, the mutant receptor was 2-fold more active than the wild-type receptor for poly(Glu/Tyr) phosphorylation. In conclusion, the whole C-terminal region of the insulin receptor beta-subunit is likely to exert a regulatory influence on the receptor kinase activity. Perturbations of the C-terminal region, such as binding of antipeptides or mutation of Tyr1316 and Tyr1322, provoke alterations at the receptor kinase level, leading to activation or inhibition of the enzymic activity.

Potential role for insulin and cycloheximide in regulating the intrinsic activity of glucose transporters in isolated rat adipocytes.

Abstract: We examined the hypothesis that insulin stimulation of cellular glucose transport may involve a protein synthesis-dependent regulation of glucose transporter (GTer) activity independent of GTer translocation to the cell surface. Rat adipocytes were isolated, incubated with or without 10 micrograms/ml (36 microM) cycloheximide (CHX) for 60 min, and then with or without 7 nM insulin for 30 min. Glucose transport rates were assessed in intact cells, and both glucose transport rates and GTer levels were assessed in subcellular fractions of membrane vesicles before and after reconstitution into artificial liposomes. GTer functional and intrinsic activities were calculated as the ratio between these transport rates and GTer levels in native and reconstituted membranes, respectively. Insulin increased functional activity by 340% in native plasma membrane (PM) vesicles and intrinsic activity by 60% in reconstituted membranes (from 54 +/- 4 to 86 +/- 4 molecules transported per GTer/sec, P < 0.02). CHX preincubati...

Deletion of exon 21 of the insulin receptor eliminates tyrosine kinase activity but preserves mitogenic signaling.

Abstract: To study the function of exon 21 of the insulin receptor, a mutant human insulin receptor lacking this domain was constructed. The mutant HIR delta E21 cDNA was transfected into Rat-1 fibroblasts and stable cell lines were selected. The HIR delta E21 receptors were expressed on the cell surface, and they bound insulin with the same affinity as did the wild-type-expressing cell line, hIRcB. The HIR delta E21 receptors did not display detectable autophosphorylation or kinase activity, and as expected, internalization was impaired and metabolic signaling properties were absent. Unexpectedly, insulin's ability to stimulate DNA synthesis in cells expressing HIR delta E21 receptors was far greater than that in the parental Rat-1 cells and equal to that measured in the hIRcB cell line. The enhanced mitogenic signaling properties of the HIR delta E21 receptors was confirmed by showing that treatment of HIR delta E21 cells with a human-specific insulin-mimetic anti-insulin receptor antibody also led to enhanced DNA synthesis. Thus, although no insulin receptor autophosphorylation or kinase activity was detectable in HIR delta E21 cells, these cells displayed enhanced insulin-induced mitogenic signaling. These results suggest that an alternative non-kinase-dependent stimulus-response pathway exists for the long-term biological effects of insulin.

Transmembrane signaling by an insulin receptor lacking a cytoplasmic beta-subunit domain.

Abstract: To assess the function of the cytoplasmic domain of the insulin receptor (IR) beta subunit, we have studied a mutant IR truncated by 365 aa (HIR delta 978), thereby deleting > 90% of the cytoplasmic domain. HIR delta 978 receptors were processed normally to homodimers that were expressed at the cell surface where they bind insulin with normal affinity. Although these truncated IRs were inactive with respect to ligand-induced internalization and autophosphorylation, insulin stimulated endogenous substrate (pp185) phosphorylation significantly more in HIR delta 978 cells than in untransfected Rat1 cells. Importantly, despite absence of the beta-subunit cytoplasmic domain, fibroblasts expressing HIR delta 978 receptors displayed enhanced sensitivity to insulin for stimulation of glucose incorporation into glycogen, alpha-aminoisobutyric acid uptake, thymidine incorporation, and S6 kinase activity compared with parental fibroblasts. Insulin also induced the expression of the protooncogene c-fos and the early growth response gene Egr-1 in HIR delta 978 cells far greater than in parental Rat1 fibroblasts. Furthermore, an agonistic monoclonal antibody specific for the human IR stimulated insulin action in fibroblasts expressing wild-type human IR but had no effect on HIR delta 978 cells. In conclusion, the HIR delta 978 truncated IRs appear to confer enhanced insulin sensitivity by augmenting the signaling properties of the endogenous rodent IRs.

Deletion of the insulin receptor beta-subunit acidic domain results in enhanced metabolic signaling.

Abstract: The contribution of the insulin receptor beta-subunit acidic domain, amino acids 1262-1291, to receptor function was analyzed. A mutant insulin receptor complementary DNA lacking this domain was created. Rat-1 fibroblasts were stably transfected with plasmids containing the mutant insulin receptor complementary DNA and clonal cell lines derived (hIR1262). Compared with cells overexpressing the wild type insulin receptor, metabolic signaling was enhanced in hIR1262 cells whereas the mitogenic response to insulin was unchanged. hIR1262 had normal kinase activity and insulin-stimulated receptor internalization in spite of substantially reduced autophosphorylation (70% decreased in vitro). Additionally, polylysine, a polycation postulated to interact with the insulin receptor beta-subunit acidic domain, increased autophosphorylation and facilitated insulin-induced phosphorylation of calmodulin in the wild type as well as the hIR1262 receptors. We conclude: 1) The acidic domain is not the site of interaction b...

A truncated human insulin receptor missing the COOH-terminal 365 amino acid residues does not undergo insulin-mediated receptor migration or aggregation

Abstract: A previous study of tyrosine kinase-defective insulin receptors demonstrated that receptor autophosphorylation or tyrosine kinase activity was required for concentrating insulin receptors in coated pits, but not for their migration or aggregation on the cell surface. Furthermore, receptor migration and aggregation on the cell surface were not sufficient to cause internalization of the occupied receptors in coated pits. In the present study, biochemical and ultrastructural techniques were used to compare insulin receptor mobility and internalization in Rat 1 fibroblasts expressing wild-type human insulin receptors (HIRc) with those in cells expressing receptors truncated at residues 978 (HIR delta 978) or 1301 of the carboxyl-terminus (HIR delta CT). There were no significant differences in the mobility or internalization of insulin receptors on HIR delta CT cells compared to those of insulin receptors on HIRc cells. Ultrastructural analysis revealed that truncated insulin receptors on HIR delta 978 cells ...