Insulin decreases blood glucose concentration. It increases cell permeability to monosaccharides, amino acids and fatty acids. It accelerates glycolysis, the pentose phosphate cycle, and glycogen synthesis in liver.
The action characteristic of a hormone, any substance formed in very small amounts in one specialized organ or group of cells and carried (sometimes in the bloodstream) to another organ or group of cells in the same organism, upon which it has a specific regulatory action. The term was originally applied to agents with a stimulatory physiological action in vertebrate animals (as opposed to a chalone, which has a depressant action). Usage is now extended to regulatory compounds in lower animals and plants, and to synthetic substances having comparable effects; all bind receptors and trigger some biological process.
Insulin isolated from the pancreas of a diabetic patient with fasting hyperinsulinaemia showed decreased activity in binding to cell membrane insulin receptors and in stimulating cellular 2-deoxyglucose transport and glucose oxidation. Chemical studies suggest that the isolated hormone is a mixture of normal insulin and an abnormal variant which contains a leucine for phenylalanine substitution at position 24 or 25 of the insulin B-chain.
PURPOSE: Nitric oxide, a messenger molecule has been reported as having various antineoplastic properties. The activation of insulin-activated nitric oxide synthase (IANOS) was found to be related to the production of NO as a result of the binding of insulin to its receptor through the activation of tyrosine kinase in erythrocyte membrane. As nitric oxide is reported to be a systemic anticancer agent, studies were carried out to determine the role of insulin receptor binding that lead to the activation of tyrosine kinase and IANOS in erythrocytes in breast cancer. METHODS: Blood samples were collected from female breast cancer patients who, at the time of participation in the study, had not undergone any therapeutic intervention but had opted for surgery. The binding of insulin to its receptor in erythrocyte membrane and the activation of both receptor tyrosine kinase and IANOS due to the hormone binding were determined and compared with the appropriate control. RESULTS: It was found that the impaired NO synthesis in erythrocyte membrane in breast cancer was related to the marked decrease of insulin binding sites of the high-affinity hormone receptor population. This impaired insulin binding to high-affinity receptors resulted in the impairment of both reaction velocity (Vmax) of the IANOS and receptor tyrosine kinase activation. CONCLUSION: These results indicated that the impairment of interaction between insulin and its high-affinity receptors in erythrocyte membrane might be a critical pathophysiological event in the development of breast cancer.
Eur. J. Pediatr. 157, 456-460 (1998)[PubMed:9667398]
Familial hyperproinsulinaemia is a rare genetic disorder characterized by point mutations in the insulin gene which impair the conversion of proinsulin to insulin. We report here three members of a two-generation Caucasian family in whom this syndrome was identified by unexplained hyperinsulinism associated with normal glucose tolerance and normal insulin sensitivity. Plasma insulin immunoreactivity showed a reduced affinity for the insulin receptor and eluted mainly, on Biogel chromatography, at the position of proinsulin. Analysis of the PCR-amplified insulin gene by restriction enzyme mapping revealed a new recognition site for the enzyme Nla III, indicating a Arg65 to His mutation. Sequence analysis of exon 3 confirmed this mutation in one allele of the gene. CONCLUSION: This study reports a two-generation European-Caucasian family with hyperproinsulinaemia due to a substitution of His for Arg at position 65 in proinsulin, the seventh now identified worldwide and the second from Europe. The mutation generated a new restriction site on the insulin gene suggesting the usefulness of restriction enzyme mapping as a screening procedure.
Previous studies have suggested that human and porcine insulin exert identical effects on blood glucose and counter-regulatory hormones but elicit different neurophysiological reactions. A major goal of the present study was to investigate whether this could be caused by different relative affinities of the insulins from different species to insulin receptors from the brain compared to other tissues. Insulin receptors isolated from human brain, muscle or adipocytes as well as from cultured cells over-expressing either of the human insulin receptor isoforms (exon 11- or exon 11 +) were immobilized to microwells coated with monoclonal anti-insulin receptor antibody. Subsequently the binding of human, porcine and bovine insulin was measured. While the receptors derived from the different tissues had different affinities for insulin, there were no tissue-specific differences in the relative binding of the insulins of the three species. The insulins of the three species were also not different with regard to their binding to the receptor isoforms. Finally, in human brain homogenates no differences in the degradation rates for human, porcine and bovine insulin were detected. Thus, our data do not support the hypothesis that different neurophysiological reactions during hypoglycaemia due to human or porcine insulin are caused by differences of the binding of the insulins to human brain insulin receptors or their degradation in the human brain.
Evidence
2:
Inferred from Physical InteractionBHF-UCL
Biochem. J. 290 ( Pt 2), 419-426 (1993)[PubMed:8452530]
Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.
Eur. J. Pediatr. 157, 456-460 (1998)[PubMed:9667398]
Familial hyperproinsulinaemia is a rare genetic disorder characterized by point mutations in the insulin gene which impair the conversion of proinsulin to insulin. We report here three members of a two-generation Caucasian family in whom this syndrome was identified by unexplained hyperinsulinism associated with normal glucose tolerance and normal insulin sensitivity. Plasma insulin immunoreactivity showed a reduced affinity for the insulin receptor and eluted mainly, on Biogel chromatography, at the position of proinsulin. Analysis of the PCR-amplified insulin gene by restriction enzyme mapping revealed a new recognition site for the enzyme Nla III, indicating a Arg65 to His mutation. Sequence analysis of exon 3 confirmed this mutation in one allele of the gene. CONCLUSION: This study reports a two-generation European-Caucasian family with hyperproinsulinaemia due to a substitution of His for Arg at position 65 in proinsulin, the seventh now identified worldwide and the second from Europe. The mutation generated a new restriction site on the insulin gene suggesting the usefulness of restriction enzyme mapping as a screening procedure.
Biochem. J. 290 ( Pt 2), 419-426 (1993)[PubMed:8452530]
Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.
Insulin-like peptide 3 (INSL3) is an insulin superfamily peptide hormone, primarily expressed in the testes and playing a key role in the fetus testes descent and suppression of male germ cell apoptosis. Insulin-degrading enzyme (IDE) is a zinc-metalloprotease, responsible for in vivo degradation of insulin, Abeta, and other peptide hormones. IDE has high expression level in the testes, implying it might be involved in INSL3 turnover in vivo. In present work, we studied in vitro degradation of INSL3 by IDE. Recombinant human IDE degraded human INSL3, but its degradation rate for INSL3 is more than a magnitude lower than that for insulin. However, IDE bound INSL3 and insulin with almost same affinity. IDE cleaved the peptide bond between B26R and B27W of INSL3, and released a pentapeptide, WSTEA, from the C-terminal of B-chain. Our present work suggested that IDE might play a role in INSL3 degradation in vivo.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
J. Am. Soc. Nephrol. 9, 1759-1766 (1998)[PubMed:9773776]
Renal clearance is a major pathway for regulating the levels of insulin and other low molecular weight polypeptide hormones in the systemic circulation. Previous studies have shown that the reabsorption of insulin from the glomerular filtrate occurs by binding to as yet unidentified sites on the luminal surface of proximal tubule cells followed by endocytosis and degradation in lysosomes. In this study, an insulin binding site was identified in renal microvillar membranes by chemical cross-linking procedures. By immunoprecipitation it was demonstrated that this binding site is megalin, the large multiligand binding endocytic receptor that is abundantly expressed in clathrin-coated pits on the apical surface of proximal tubule cells. Moreover, using cytochemical procedures, it was also shown that megalin is able to internalize insulin into endocytic vesicles. In ligand blotting assays, megalin also bound several other low molecular weight polypeptides, including beta2-microglobulin, epidermal growth factor, prolactin, lysozyme, and cytochrome c. These data suggest that megalin may play a significant role as a renal reabsorption receptor for the uptake of insulin and other low molecular weight polypeptides from the glomerular filtrate.
Evidence
2:
Inferred from Physical InteractionBHF-UCL
J. Biol. Chem. 272, 30729-30734 (1997)[PubMed:9388210]
The insulin-like growth factors (IGFs) are transported by a family of high-affinity binding proteins (IGFBPs) that protect IGFs from degradation, limit their binding to IGF receptors, and modulate IGF actions. The six classical IGFBPs have been believed to have no affinity for insulin. We now demonstrate that IGFBP-7/mac25, a newly identified member of the IGFBP superfamily that binds IGFs specifically with low affinity is a high-affinity insulin binding protein. IGFBP-7 blocks insulin binding to the insulin receptor and thereby inhibiting the earliest steps in insulin action, such as autophosphorylation of the insulin receptor beta subunit and phosphorylation of IRS-1, indicating that IGFBP-7 is a functional insulin-binding protein. The affinity of other IGFBPs for insulin can be enhanced by modifications that disrupt disulfide bonds or remove the conserved COOH terminus. Like IGFBP-7, an NH2-terminal fragment of IGFBP-3 (IGFBP-3((1-87))), also binds insulin with high affinity and blocks insulin action. IGFBPs with enhanced affinity for insulin might contribute to the insulin resistance of pregnancy, type II diabetes mellitus, and other pathological conditions.
J. Biol. Chem. 271, 21920-21926 (1996)[PubMed:8702995]
Akt is a serine/threonine kinase that is stimulated by receptor tyrosine kinases and contains a pleckstrin homology domain. One model proposed to explain this activation suggests that receptor tyrosine kinases stimulate a phosphatidylinositol 3-kinase whose lipid products directly activate Akt kinase by interacting with its pleckstrin homology domain. In the present study, we show, in three cell types, that Akt does not require its pleckstrin homology domain to respond to either insulin or platelet-derived growth factor. Moreover, attachment of the src myristoylation signal to target Akt, without its pleckstrin homology domain, to the membrane constitutively activates Akt by causing an increase in its basal level of phosphorylation. This constitutively active form of Akt can also activate p70(S6K), indicating that the pleckstrin homology domain is not necessary for downstream interactions. Fusion of the inter src homology 2 domain from the p85 regulatory subunit of the phosphatidylinositol 3-kinase to Akt also constitutively activated Akt and induced an association with the lipid kinase. Phosphorylation of this fusion protein still critically contributes toward its increased activity. The sum of these results indicates that the primary mechanism of Akt activation is via protein phosphorylation.
An acute inflammatory response that involves non-antibody proteins whose concentrations in the plasma increase in response to infection or injury of homeothermic animals.
BACKGROUND: Severe burn induces the hepatic acute phase response. In this study, we wondered whether continuous insulin treatment decreases acute phase protein levels in the severely burned. METHODS: Eighteen children aged 2 to 17 years with burns >40% of total body surface area were randomized to receive either insulin (n=9) or no treatment (n=9) within 72 hours after injury until the wounds were 95% healed. Insulin was given at a continuous rate of > or =1.5 microU/kg/min to maintain euglycemia (serum glucose 100-140 microg/dL). Plasma was examined at days 7, 14, 21, and 28 for acute phase protein levels including C-reactive protein, C3 complement, alpha1-acid glycoprotein, haptoglobin, alpha2-macroglobulin, prealbumin, transferrin, and retinol-binding protein. Statistical analysis was by ANOVA and t test. RESULTS: With insulin treatment, alpha1-acid glycoprotein, C3 complement, alpha2-macroglobulin, and haptoglobin levels decreased (P<.05) after a severe burn compared with control, especially at days 21 and 28. Additionally, the hepatic constitutive proteins (prealbumin, transferrin, and retinol-binding protein) were lower in the insulin-treatment group than those of the control group at day 21 (P<.05). CONCLUSIONS: Continuous insulin treatment decreases acute phase protein levels after a severe burn. The results suggest insulin downregulation of the hepatic acute phase response to injury.
The change in morphology and behavior of an alpha-beta T cell resulting from exposure to a mitogen, cytokine, chemokine, cellular ligand, or an antigen for which it is specific.
CD8+ T cells down-regulate a variety of immune responses. For example, porcine and human insulin do not stimulate Abs in C57BL/6 mice because CD8+ T cells inhibit CD4+ helper T cells. By contrast, bovine insulin induces Ab in C57BL/6 mice, and removal of CD8+ T cells does not alter this response. This raises the question of whether porcine, but not bovine, insulin activates CD8+ T cells or whether both insulins activate CD8+ T cells but CD4+ helper T cells are differentially inhibited by them. In this study, we show that insulin-specific CD8+ CTL can be cultured from C57BL/6 mice primed with either bovine or human insulin in CFA. Thus, exogenous Ags, besides OVA, induce CD8+ CTL when administered in an adjuvant, suggesting this is a typical response. These CTL are H-2Kb restricted and produce IL-5, IL-10, IFN-gamma, and small amounts of IL-4, which is distinct from IFN-gamma and TNF-alpha that are typically secreted by virus-specific CTL. Moreover, the CTL primed with either bovine or human insulin recognize an A-chain peptide that is identical to the mouse insulin sequence. That foreign proteins, which are closely related to self-proteins, activated autoreactive, CD8+ T cells in vivo is a novel finding. It raises the possibility that self-reactive CTL may be activated by cross-reacting Ags and once activated they might participate in autoimmunity. These results also suggest that down-regulation of insulin-specific responses by autoreactive CD8+ T cells is most likely due to the differential sensitivity of bovine and human insulin-specific CD4+ T cells.
Previous studies have suggested that human and porcine insulin exert identical effects on blood glucose and counter-regulatory hormones but elicit different neurophysiological reactions. A major goal of the present study was to investigate whether this could be caused by different relative affinities of the insulins from different species to insulin receptors from the brain compared to other tissues. Insulin receptors isolated from human brain, muscle or adipocytes as well as from cultured cells over-expressing either of the human insulin receptor isoforms (exon 11- or exon 11 +) were immobilized to microwells coated with monoclonal anti-insulin receptor antibody. Subsequently the binding of human, porcine and bovine insulin was measured. While the receptors derived from the different tissues had different affinities for insulin, there were no tissue-specific differences in the relative binding of the insulins of the three species. The insulins of the three species were also not different with regard to their binding to the receptor isoforms. Finally, in human brain homogenates no differences in the degradation rates for human, porcine and bovine insulin were detected. Thus, our data do not support the hypothesis that different neurophysiological reactions during hypoglycaemia due to human or porcine insulin are caused by differences of the binding of the insulins to human brain insulin receptors or their degradation in the human brain.
The present study was designed to test the hypothesis that physiological concentrations of glucagon may increase plasma ketone body concentration when sufficient free fatty acid substrate is available to support hepatic ketogenesis. Physiological elevations of plasma glucagon concentration were produced by a constant infusion of hormone, and increased plasma-free fatty acid availability was produced by simultaneous heparin injection to induce intravascular lipolysis. In the five insulin-dependent subjects studied, when plasma glucagon concentration remained at the normal basal level of 72+/-14 pg/ml during control saline infusion, the heparin-induced increase in free fatty acid availability resulted in approximately a 20% increase in plasma ketone body concentration. In contrast, when plasma glucagon concentration was elevated by hormone infusion to the physiological level of 215+/-35 pg/ml, the heparin-induced increases in free fatty acid availability resulted in approximately an 80% increase in plasma ketone body concentration. These results suggest that physiological elevations in plasma glucagon concentration may augment ketonemia in diabetic man when simultaneous elevations in plasma-free fatty acid arepresent.
A series of molecular signals that proceeds with an activated receptor promoting the exchange of GDP for GTP on the alpha-subunit of an associated heterotrimeric G-protein complex. The GTP-bound activated alpha-G-protein then dissociates from the beta- and gamma-subunits to further transmit the signal within the cell. The pathway begins with receptor-ligand interaction, or for basal GPCR signaling the pathway begins with the receptor activating its G protein in the absence of an agonist, and ends with regulation of a downstream cellular process, e.g. transcription.
J. Biol. Chem. 272, 10135-10143 (1997)[PubMed:9092559]
Human fat cells possess a multireceptor-linked H2O2-generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H2O2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H2O2 generation by Mn2+ and insulin was blocked by GDPbetaS (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-Galphai1-2 or the corresponding peptide. Consistently, manganese could be replaced by micromolar concentrations of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTPgammaS to the left (>10-fold) and increased the maximal response. In the presence of 10 microM GTPgammaS, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. The insulin receptor and Gi were co-adsorbed on anti-Galphai and anti-insulin receptor beta-subunit (anti-IRbeta) affinity columns. Partially purified insulin receptor preparations contained Galphas, Galphai2, and Gbetagamma (but no Galphai1 or Galphai3). The functional nature of the insulin receptor-Gi2 complex was made evident by insulin's ability to modulate labeling of Gi by bacterial toxins. Insulin action was mimicked by activated Galphai, but not by Galphao or Gbetagamma, indicating that insulin's signal was transduced via Galphai2. Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.
Insulin isolated from the pancreas of a diabetic patient with fasting hyperinsulinaemia showed decreased activity in binding to cell membrane insulin receptors and in stimulating cellular 2-deoxyglucose transport and glucose oxidation. Chemical studies suggest that the isolated hormone is a mixture of normal insulin and an abnormal variant which contains a leucine for phenylalanine substitution at position 24 or 25 of the insulin B-chain.
The chemical reactions and pathways involving glucose, the aldohexose gluco-hexose. D-glucose is dextrorotatory and is sometimes known as dextrose; it is an important source of energy for living organisms and is found free as well as combined in homo- and hetero-oligosaccharides and polysaccharides.
The directed movement of the hexose monosaccharide glucose into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore.
The present work examines whether insulin and NO can act as regulators of glucose transport in placenta. Glucose uptake (2-deoxy D-[(3)H]glucose) was measured in the absence (control or basal values) and in the presence of insulin (1200 microU/ml) or SNP (20 microM) in isolated perfused cotyledons and tissue slices preparations of human placenta. Both insulin and NO significantly increased glucose uptake by 20 and 27 per cent, respectively. Insulin decreased the Km of glucose transport from 42.5 +/- 2.69 to 35.1 +/- 2.58 mM. The stimulatory effect of SNP was mimicked by 8-CPT-cGMP and was completely blocked by the guanylate cyclase inhibitor, ODQ (10 microM). ODQ and the NOS inhibitor, L-NAME (100 microM), decreased basal glucose uptake but did not affect insulin-stimulated glucose transport. Taken together, these findings indicate that insulin and NO stimulate glucose uptake in human placenta and suggest that both potential regulators of glucose transport use different signaling pathways.
OBJECTIVE: In cultured human vascular smooth muscle cells, insulin increases cyclic GMP production by inducing nitric oxide (NO) synthesis. The aim of the present study was to determine whether in these cells the insulin-stimulated NO/cyclic GMP pathway plays a role in the regulation of glucose uptake. METHODS AND RESULTS: Glucose transport in human vascular smooth muscle cells was measured as uptake of 2-deoxy-d-[3H]glucose, cyclic GMP synthesis was checked by radioimmunoassay, and GLUT4 recruitment into the plasma membrane was determined by immunofluorescence. Insulin-stimulated glucose transport and GLUT4 recruitment were blocked by an inhibitor of NO synthesis and mimicked by NO-releasing drugs. Insulin- and NO-elicited glucose uptake were blocked by inhibitors of soluble guanylate cyclase and cyclic GMP-dependent protein kinase; furthermore, glucose transport was stimulated by an analog of cyclic GMP. CONCLUSIONS: Our results suggest that insulin-elicited glucose transport (and the corresponding GLUT4 recruitment into the plasma membrane) in human vascular smooth muscle cells is mediated by an increased synthesis of NO, which stimulates the production of cyclic GMP and the subsequent activation of a cyclic GMP-dependent protein kinase.
An intracellular protein kinase cascade containing at least a MAPK, a MAPKK and a MAP3K. The cascade can also contain two additional tiers: the upstream MAP4K and the downstream MAP Kinase-activated kinase (MAPKAPK). The kinases in each tier phosphorylate and activate the kinases in the downstream tier to transmit a signal within a cell.
Incubation of rat hepatoma Fao cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins. This is followed by elevation in their P-Ser/Thr content, and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the increase in the P-Ser/Thr content of IRS-1, its dissociation from the IR, and the decrease in its P-Tyr content following 60 min of insulin treatment, indicating that the Ser kinases that negatively regulate IRS-1 function are downstream effectors of PI3K. PKCzeta fulfills this criterion, being an insulin-activated downstream effector of PI3K. Overexpression of PKCzeta in Fao cells, by infection of the cells with adenovirus-based PKCzeta construct, had no effect on its own, but it accelerated the rate of insulin-stimulated dissociation of IR.IRS-1 complexes and the rate of Tyr dephosphorylation of IRS-1. The insulin-stimulated negative regulatory role of PKCzeta was specific and could not be mimic by infecting Fao cells with adenoviral constructs encoding for PKC alpha, delta, or eta. Because the reduction in P-Tyr content of IRS-1 was accompanied by a reduced association of IRS-1 with p85, the regulatory subunit of PI3K, it suggests that this negative regulatory process induced by PKCzeta, has a built-in attenuation signal. Hence, insulin triggers a sequential cascade in which PI3K-mediated activation of PKCzeta inhibits IRS-1 functions, reduces complex formation between IRS-1 and PI3K, and inhibits further activation of PKCzeta itself. These findings implicate PKCzeta as a key element in a multistep negative feedback control mechanism of IRS-1 functions.
J. Clin. Endocrinol. Metab. 86, 3257-3265 (2001)[PubMed:11443198]
In view of the fact that insulin resistance is associated with atherogenesis and that troglitazone, an insulin sensitizer, has anti-inflammatory effects, which may be potentially antiatherogenic in the long term, we have now investigated whether insulin has potential anti-inflammatory effects. We infused 2.0 to 2.5 IU/h in 5% dextrose (100 mL/h) iv into 10 obese subjects for 4 h followed by 5% dextrose alone for 2 h. The rate of insulin infusion was varied to maintain glucose concentrations as close to the baseline as possible. Blood samples were obtained before and at 2, 4, and 6 h. Subjects were also infused with 5% dextrose without insulin and with saline on separate occasions. Intranuclear nuclear factor kappaB (NFkappaB) in mononuclear cells fell at 2 and further at 4 h, reverting toward the baseline at 6 h (P < 0.05). IkappaB increased significantly at 2 h, increasing further at 4 h and remaining elevated at 6 h (P < 0.001). Reactive oxygen species (ROS) generation by mononuclear cells fell significantly at 2 h and fell further at 4 h; it partially reverted to baseline at 6 h (P < 0.005). p47(phox) subunit, the key protein of nicotinamide adenine dinucleotide phosphate oxidase also fell at 2 h and 4 h, reverting toward the baseline at 6 h (P < 0.05). In addition, soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) fell significantly following insulin infusion. Glucose or saline infusions without insulin caused no alteration in NFkappaB, IkappaB, ROS generation, p47(phox) subunit, sICAM-1, MCP-1, or PAI-1. We conclude that insulin has a potent acute anti-inflammatory effect including a reduction in intranuclear NFkappaB, an increase in IkappaB, and decreases in ROS generation, p47(phox) subunit, plasma soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1. This acute anti-inflammatory effect, if demonstrated in the long term, may have implications for atherosclerosis and its complications.
Acta Biochim. Biophys. Sin. (Shanghai) 38, 241-248 (2006)[PubMed:16604263]
In order to investigate the neuroprotection of insulin in retinal neurons, we used retinal neuronal culture as a model system to study the protective effects of insulin against H2O2-induced cytotoxicity and apoptotic death. Primary retinal neuronal cultures were grown from retinas of 0-2-day old Sprague-Dawley rats. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling assay, and by DNA laddering analysis. Phosphoinositide 3-kinase (PI3K) activity was measured using phosphoinositide 4,5-bisphophate and [gamma-32P]ATP as substrate. Western blot analysis with anti-phospho-Akt (pS473) antibody was performed to examine the level of phosphorylated Akt. We observed that treatment with 100 microM H2O2 for 24 h significantly decreased cell viability and induced apoptotic death of retinal neurons, and that pretreatment with 10 nM insulin significantly inhibited or attenuated H2O2-induced cytotoxicity and apoptosis. Pretreatment with LY294002, a specific PI3K inhibitor, abolished the cytoprotective effect of insulin. Insulin also strongly activated both PI3K and the downstream effector Akt. These results suggest that insulin protects retinal neurons from oxidative stress-induced apoptosis and that the PI3K/Akt signal pathway is involved in insulin-mediated retinal neuroprotection.
The present study was designed to test the hypothesis that physiological concentrations of glucagon may increase plasma ketone body concentration when sufficient free fatty acid substrate is available to support hepatic ketogenesis. Physiological elevations of plasma glucagon concentration were produced by a constant infusion of hormone, and increased plasma-free fatty acid availability was produced by simultaneous heparin injection to induce intravascular lipolysis. In the five insulin-dependent subjects studied, when plasma glucagon concentration remained at the normal basal level of 72+/-14 pg/ml during control saline infusion, the heparin-induced increase in free fatty acid availability resulted in approximately a 20% increase in plasma ketone body concentration. In contrast, when plasma glucagon concentration was elevated by hormone infusion to the physiological level of 215+/-35 pg/ml, the heparin-induced increases in free fatty acid availability resulted in approximately an 80% increase in plasma ketone body concentration. These results suggest that physiological elevations in plasma glucagon concentration may augment ketonemia in diabetic man when simultaneous elevations in plasma-free fatty acid arepresent.
Our aim was to study the feeding behavior of healthy term infants in the first week of life and determine whether this was related to cord blood leptin, ghrelin, and insulin. A total of 100 healthy bottle-fed infants were studied by weighing bottles of milk before and after feeds. Leptin, total ghrelin, and insulin concentrations were measured in cord blood. Mean (SD) birth weight was 3.46 (0.43) kg. Mean milk intake increased from 196.7 (83.0) g on d 1 to 585.0 (128.4) g on d 7. Milk intake over the first 6 d was significantly associated with weight gain to d 7. There was no relationship between cord ghrelin or leptin and milk intake or feed frequency. Cord blood insulin was inversely related to the mean daily number of feeds over the first 6 d (r = -0.21, p < 0.05). Birth weight and milk intake are the major determinants of weight gain in the first week of life in healthy bottle-fed infants. Total cord ghrelin and leptin are not directly related to milk intake or feed frequency in the first week of life. Circulating insulin concentrations may have a role in the initiation of feeding behavior.
The epidemic of type 2 diabetes and impaired glucose tolerance is one of the main causes of morbidity and mortality worldwide. In both disorders, tissues such as muscle, fat and liver become less responsive or resistant to insulin. This state is also linked to other common health problems, such as obesity, polycystic ovarian disease, hyperlipidaemia, hypertension and atherosclerosis. The pathophysiology of insulin resistance involves a complex network of signalling pathways, activated by the insulin receptor, which regulates intermediary metabolism and its organization in cells. But recent studies have shown that numerous other hormones and signalling events attenuate insulin action, and are important in type 2 diabetes.
Any process that stops, prevents, or reduces the frequency, rate or extent of the chemical reactions and pathways resulting in the breakdown of glycogen.
Insulin isolated from the pancreas of a diabetic patient with fasting hyperinsulinaemia showed decreased activity in binding to cell membrane insulin receptors and in stimulating cellular 2-deoxyglucose transport and glucose oxidation. Chemical studies suggest that the isolated hormone is a mixture of normal insulin and an abnormal variant which contains a leucine for phenylalanine substitution at position 24 or 25 of the insulin B-chain.
Any process that stops, prevents, or reduces the frequency, rate or extent of the chemical reactions and pathways resulting in the breakdown of lipids.
The epidemic of type 2 diabetes and impaired glucose tolerance is one of the main causes of morbidity and mortality worldwide. In both disorders, tissues such as muscle, fat and liver become less responsive or resistant to insulin. This state is also linked to other common health problems, such as obesity, polycystic ovarian disease, hyperlipidaemia, hypertension and atherosclerosis. The pathophysiology of insulin resistance involves a complex network of signalling pathways, activated by the insulin receptor, which regulates intermediary metabolism and its organization in cells. But recent studies have shown that numerous other hormones and signalling events attenuate insulin action, and are important in type 2 diabetes.
J. Clin. Endocrinol. Metab. 86, 3257-3265 (2001)[PubMed:11443198]
In view of the fact that insulin resistance is associated with atherogenesis and that troglitazone, an insulin sensitizer, has anti-inflammatory effects, which may be potentially antiatherogenic in the long term, we have now investigated whether insulin has potential anti-inflammatory effects. We infused 2.0 to 2.5 IU/h in 5% dextrose (100 mL/h) iv into 10 obese subjects for 4 h followed by 5% dextrose alone for 2 h. The rate of insulin infusion was varied to maintain glucose concentrations as close to the baseline as possible. Blood samples were obtained before and at 2, 4, and 6 h. Subjects were also infused with 5% dextrose without insulin and with saline on separate occasions. Intranuclear nuclear factor kappaB (NFkappaB) in mononuclear cells fell at 2 and further at 4 h, reverting toward the baseline at 6 h (P < 0.05). IkappaB increased significantly at 2 h, increasing further at 4 h and remaining elevated at 6 h (P < 0.001). Reactive oxygen species (ROS) generation by mononuclear cells fell significantly at 2 h and fell further at 4 h; it partially reverted to baseline at 6 h (P < 0.005). p47(phox) subunit, the key protein of nicotinamide adenine dinucleotide phosphate oxidase also fell at 2 h and 4 h, reverting toward the baseline at 6 h (P < 0.05). In addition, soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) fell significantly following insulin infusion. Glucose or saline infusions without insulin caused no alteration in NFkappaB, IkappaB, ROS generation, p47(phox) subunit, sICAM-1, MCP-1, or PAI-1. We conclude that insulin has a potent acute anti-inflammatory effect including a reduction in intranuclear NFkappaB, an increase in IkappaB, and decreases in ROS generation, p47(phox) subunit, plasma soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1. This acute anti-inflammatory effect, if demonstrated in the long term, may have implications for atherosclerosis and its complications.
Any process that stops, prevents, or reduces the frequency, rate or extent of the chemical reactions and pathways resulting in the breakdown of a protein by the destruction of the native, active configuration, with or without the hydrolysis of peptide bonds.
J. Pediatr. Surg. 39, 839-844 (2004)[PubMed:15185208]
BACKGROUND/PURPOSE: Infants requiring extracorporeal membrane oxygenation (ECMO) have the highest rates of protein catabolism ever reported. Recent investigations have found that such extreme protein breakdown is refractory to conventional nutritional management. In this pilot study, the authors sought to use the anabolic hormone insulin to reduce the profound protein degradation in this cohort. METHODS: Four parenterally fed infants on ECMO were enrolled in a prospective, randomized, crossover trial. Subjects were administered an insulin infusion using a 4-hour hyperinsulinemic euglycemic clamp followed by a control saline infusion on consecutive days in random order. Whole-body protein flux and breakdown were quantified using a primed continuous infusion of the stable isotope L-[1-13C]leucine. Statistical analyses were performed using paired t tests. RESULTS: Serum insulin levels were increased 15-fold during the insulin clamp compared with the saline control (407 +/- 103 v 26 +/- 12 microU/mL; P <.05). During the insulin infusion, infants had decreased rates of total leucine flux (214 +/- 25 v 298 +/- 38 micromol/kg/h; P <.05) and leucine flux derived from protein breakdown (156 +/- 40 v 227 +/- 54 micromol/kg/h; P <.05) when compared with saline control. Overall, insulin administration produced a 32% reduction in protein breakdown (P <.05). CONCLUSIONS: In this pilot study, the anabolic hormone insulin markedly reduced protein breakdown in critically ill infants on ECMO. Because elevated protein breakdown correlates with mortality and morbidity, the administration of intravenous insulin may ultimately have broad applicability to the metabolic management of critically ill infants.
J. Biol. Chem. 273, 32730-32738 (1998)[PubMed:9830016]
Excessive cerebral accumulation of the 42-residue amyloid beta-protein (Abeta) is an early and invariant step in the pathogenesis of Alzheimer's disease. Many studies have examined the cellular production of Abeta from its membrane-bound precursor, including the role of the presenilin proteins therein, but almost nothing is known about how Abeta is degraded and cleared following its secretion. We previously screened neuronal and nonneuronal cell lines for the production of proteases capable of degrading naturally secreted Abeta under biologically relevant conditions and concentrations. The major such protease identified was a metalloprotease released particularly by a microglial cell line, BV-2. We have now purified and characterized the protease and find that it is indistinguishable from insulin-degrading enzyme (IDE), a thiol metalloendopeptidase that degrades small peptides such as insulin, glucagon, and atrial natriuretic peptide. Degradation of both endogenous and synthetic Abeta at picomolar to nanomolar concentrations was completely inhibited by the competitive IDE substrate, insulin, and by two other IDE inhibitors. Immunodepletion of conditioned medium with an IDE antibody removed its Abeta-degrading activity. IDE was present in BV-2 cytosol, as expected, but was also released into the medium by intact, healthy cells. To confirm the extracellular occurrence of IDE in vivo, we identified intact IDE in human cerebrospinal fluid of both normal and Alzheimer subjects. In addition to its ability to degrade Abeta, IDE activity was unexpectedly found be associated with a time-dependent oligomerization of synthetic Abeta at physiological levels in the conditioned media of cultured cells; this process, which may be initiated by IDE-generated proteolytic fragments of Abeta, was prevented by three different IDE inhibitors. We conclude that a principal protease capable of down-regulating the levels of secreted Abeta extracellularly is IDE.
BACKGROUND: Severe burn induces the hepatic acute phase response. In this study, we wondered whether continuous insulin treatment decreases acute phase protein levels in the severely burned. METHODS: Eighteen children aged 2 to 17 years with burns >40% of total body surface area were randomized to receive either insulin (n=9) or no treatment (n=9) within 72 hours after injury until the wounds were 95% healed. Insulin was given at a continuous rate of > or =1.5 microU/kg/min to maintain euglycemia (serum glucose 100-140 microg/dL). Plasma was examined at days 7, 14, 21, and 28 for acute phase protein levels including C-reactive protein, C3 complement, alpha1-acid glycoprotein, haptoglobin, alpha2-macroglobulin, prealbumin, transferrin, and retinol-binding protein. Statistical analysis was by ANOVA and t test. RESULTS: With insulin treatment, alpha1-acid glycoprotein, C3 complement, alpha2-macroglobulin, and haptoglobin levels decreased (P<.05) after a severe burn compared with control, especially at days 21 and 28. Additionally, the hepatic constitutive proteins (prealbumin, transferrin, and retinol-binding protein) were lower in the insulin-treatment group than those of the control group at day 21 (P<.05). CONCLUSIONS: Continuous insulin treatment decreases acute phase protein levels after a severe burn. The results suggest insulin downregulation of the hepatic acute phase response to injury.
In vivo leucine metabolism was studied after an overnight fast in nine type I diabetic patients and nine healthy control subjects using L-[1-13C] leucine as a tracer. In the insulin-deprived state, leucine flux (reflecting proteolysis), leucine oxidation, and plasma leucine concentrations were higher in the diabetic patients than in the control subjects (P less than .001). In 4 of the 9 insulin-deprived diabetic patients, a four-hour intravenous insulin treatment decreased plasma glucose and leucine concentrations and leucine flux, but failed to decrease leucine oxidation. In the remaining 5 of the 9 diabetic patients, uninterrupted insulin treatment prior to the study and a seven-hour intravenous insulin treatment during the study period decreased not only the concentrations of plasma glucose and leucine and leucine flux, but also leucine oxidation (P less than .01). In all 9 diabetic patients the nonoxidative portion of leucine flux (reflecting protein synthesis) decreased during insulin treatment (P less than .01), but this decrease was lower than that of leucine flux (reflecting proteolysis), and therefore protein was conserved during insulin treatment. We conclude that the effect of insulin on proteolysis (reflected by leucine flux) is more rapid than its effect on leucine oxidation, but on aggressive insulin treatment accelerated leucine oxidation also was decreased in type I diabetic patients.
Negative regulation of respiratory burst involved in inflammatory responsedefinition[GO:0060266]
Any process that decreases the rate, frequency or extent of a phase of elevated metabolic activity, during which oxygen consumption increases made as a defense response ; this leads to the production, by an NADH dependent system, of hydrogen peroxide (H2O2), superoxide anions and hydroxyl radicals.
J. Clin. Endocrinol. Metab. 86, 3257-3265 (2001)[PubMed:11443198]
In view of the fact that insulin resistance is associated with atherogenesis and that troglitazone, an insulin sensitizer, has anti-inflammatory effects, which may be potentially antiatherogenic in the long term, we have now investigated whether insulin has potential anti-inflammatory effects. We infused 2.0 to 2.5 IU/h in 5% dextrose (100 mL/h) iv into 10 obese subjects for 4 h followed by 5% dextrose alone for 2 h. The rate of insulin infusion was varied to maintain glucose concentrations as close to the baseline as possible. Blood samples were obtained before and at 2, 4, and 6 h. Subjects were also infused with 5% dextrose without insulin and with saline on separate occasions. Intranuclear nuclear factor kappaB (NFkappaB) in mononuclear cells fell at 2 and further at 4 h, reverting toward the baseline at 6 h (P < 0.05). IkappaB increased significantly at 2 h, increasing further at 4 h and remaining elevated at 6 h (P < 0.001). Reactive oxygen species (ROS) generation by mononuclear cells fell significantly at 2 h and fell further at 4 h; it partially reverted to baseline at 6 h (P < 0.005). p47(phox) subunit, the key protein of nicotinamide adenine dinucleotide phosphate oxidase also fell at 2 h and 4 h, reverting toward the baseline at 6 h (P < 0.05). In addition, soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) fell significantly following insulin infusion. Glucose or saline infusions without insulin caused no alteration in NFkappaB, IkappaB, ROS generation, p47(phox) subunit, sICAM-1, MCP-1, or PAI-1. We conclude that insulin has a potent acute anti-inflammatory effect including a reduction in intranuclear NFkappaB, an increase in IkappaB, and decreases in ROS generation, p47(phox) subunit, plasma soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1. This acute anti-inflammatory effect, if demonstrated in the long term, may have implications for atherosclerosis and its complications.
Insulin resistance is a risk factor for atherosclerosis and is associated with hyperinsulinemia, abnormal lipid profile, and hypertension. Whether hyperinsulinemia affects vascular function independent of insulin resistance or other metabolic risk factors is unknown. This investigation aimed to assess the effects of hyperinsulinemia on endothelial function in subjects with a spectrum of insulin sensitivity and lipid profile. Endothelium-dependent (flow-mediated dilation, FMD) and -independent (nitroglycerin) responses of the brachial artery were studied by high-resolution ultrasound before and during hyperinsulinemia (euglycemic clamp) in 25 normoglycemic, normotensive subjects. Participants were divided into an insulin-sensitive and an insulin-resistant subgroup based on their sensitivity index values, with a cutoff of 8, and into a normal-cholesterol and a high-cholesterol subgroup based on their total cholesterol levels, with a cutoff of 5.2 mmol/l (200 mg/dl). In the whole population, FMD was lower during hyperinsulinemia compared with baseline (2.3 +/- 0.6% vs. 6 +/- 0.6%; P < 0.001). Resting FMD was lower in the insulin-resistant subgroup compared with the insulin-sensitive subgroup (4.2 +/- 0.9% vs. 7.4 +/- 0.8%; P = 0.014) and in the high-cholesterol subjects compared with the normal-cholesterol subjects (4.4 +/- 0.7% vs. 8 +/- 0.7%; P = 0.002). Hyperinsulinemia decreased FMD in both the insulin-sensitive (from 7.4 +/- 0.8% to 3.6 +/- 0.4%; P < 0.001) and insulin-resistant (from 4.2% to 1.22%; P = 0.012) subgroups and in both the normal-cholesterol (from 8 +/- 0.7% to 3.9 +/- 0.4%; P < 0.001) and high-cholesterol (from 4.4 +/- 0.7% to 1.1 +/- 0.8%; P = 0.01) participants. Acute hyperinsulinemia impairs conduit vessel endothelial function independent of insulin sensitivity and lipid profile. Insulin may trigger endothelial dysfunction and promote atherosclerosis.
Any process that increases the rate, frequency, or extent of brown fat cell differentiation. Brown fat cell differentiation is the process in which a relatively unspecialized cell acquires specialized features of a brown adipocyte, an animal connective tissue cell involved in adaptive thermogenesis. Brown adipocytes contain multiple small droplets of triglycerides and a high number of mitochondria.
Leptin, the ob gene product, is produced by adipose tissue and is submitted to a complex hormonal and metabolic regulation. Leptin plays a critical role in the balance of body weight. Here we report on secretion and hormonal regulation of leptin by brown adipocytes. Using the recently established T37i cell line, we show that leptin expression and secretion occurred as a function of cell differentiation. In differentiated T37i cells, insulin induced leptin release ( approximately 0.25 ng/10(6) cells/h) in a concentration-dependent manner (EC50=0.1 nM), and this was totally suppressed by beta3-adrenergic ligand, thiazolidinedione, cycloheximide, or actinomycin D. Insulin induced a strong, rapid (within 2 h) but transient fivefold increase in leptin mRNA levels. This transcriptional control of ob gene expression by insulin involved both phosphatidylinositol 3-kinase- and MAP kinase-dependent pathways. Glucocorticoids inhibited both insulin-stimulated leptin secretion and ob gene expression without affecting leptin mRNA stability (t(1/2)=3h05). Altogether, our results demonstrate that brown adipocytes express and secrete leptin, whose hormonal regulation clearly differs from that described in white adipose tissue. These findings point to tissue-specific molecular mechanisms and suggest that leptin might exert direct effects on energy homeostasis through an autocrine mechanism.
The epidemic of type 2 diabetes and impaired glucose tolerance is one of the main causes of morbidity and mortality worldwide. In both disorders, tissues such as muscle, fat and liver become less responsive or resistant to insulin. This state is also linked to other common health problems, such as obesity, polycystic ovarian disease, hyperlipidaemia, hypertension and atherosclerosis. The pathophysiology of insulin resistance involves a complex network of signalling pathways, activated by the insulin receptor, which regulates intermediary metabolism and its organization in cells. But recent studies have shown that numerous other hormones and signalling events attenuate insulin action, and are important in type 2 diabetes.
The epidemic of type 2 diabetes and impaired glucose tolerance is one of the main causes of morbidity and mortality worldwide. In both disorders, tissues such as muscle, fat and liver become less responsive or resistant to insulin. This state is also linked to other common health problems, such as obesity, polycystic ovarian disease, hyperlipidaemia, hypertension and atherosclerosis. The pathophysiology of insulin resistance involves a complex network of signalling pathways, activated by the insulin receptor, which regulates intermediary metabolism and its organization in cells. But recent studies have shown that numerous other hormones and signalling events attenuate insulin action, and are important in type 2 diabetes.
The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R(-) cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-Rs(A)) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs(A), insulin activated the IGF-I half-receptor beta-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-Rs(B) bound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-R(A)-containing cells than in Hybrid-R(B)-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.
Recently, single chain peptides have been designed that target the insulin receptor and mimic insulin action. The aim of this study is to explore if activation of the insulin receptor with such an optimized peptide (S597) leads to the same activation of signaling pathways and biological endpoints i.e. stimulation of glycogen synthesis and cell proliferation as stimulation with insulin. We find that surface activation of the insulin receptor A-isoform with S597 leads to activation of protein kinase B (PKB) and glycogen synthesis comparable to activation by insulin, even though the level of insulin receptor phosphorylation is lower. In contrast, both Src homology 2/alpha collagen-related (Shc) and extracellular signal-regulated kinase (ERK) 2 activation are virtually absent upon stimulation with S597. Cell proliferation is only stimulated slightly by S597, suggesting that it depends on signals from Shc and ERK. The differences in signaling response could explain both the earlier reported differences in gene expression, and the reported differences in cell proliferation and glycogen synthesis induced by insulin and S597. In conclusion, despite binding equipotency, insulin, and S597 initiate different signaling and biological responses through the same insulin receptor isoform. We show for the first time that it is possible to design insulin receptor ligand mimetics with metabolic equipotency but low mitogenicity.
Any process that activates or increases the frequency, rate or extent of the chemical reactions and pathways involving a protein, occurring at the level of an individual cell.
In vivo leucine metabolism was studied after an overnight fast in nine type I diabetic patients and nine healthy control subjects using L-[1-13C] leucine as a tracer. In the insulin-deprived state, leucine flux (reflecting proteolysis), leucine oxidation, and plasma leucine concentrations were higher in the diabetic patients than in the control subjects (P less than .001). In 4 of the 9 insulin-deprived diabetic patients, a four-hour intravenous insulin treatment decreased plasma glucose and leucine concentrations and leucine flux, but failed to decrease leucine oxidation. In the remaining 5 of the 9 diabetic patients, uninterrupted insulin treatment prior to the study and a seven-hour intravenous insulin treatment during the study period decreased not only the concentrations of plasma glucose and leucine and leucine flux, but also leucine oxidation (P less than .01). In all 9 diabetic patients the nonoxidative portion of leucine flux (reflecting protein synthesis) decreased during insulin treatment (P less than .01), but this decrease was lower than that of leucine flux (reflecting proteolysis), and therefore protein was conserved during insulin treatment. We conclude that the effect of insulin on proteolysis (reflected by leucine flux) is more rapid than its effect on leucine oxidation, but on aggressive insulin treatment accelerated leucine oxidation also was decreased in type I diabetic patients.
BACKGROUND: We aimed to evaluate whether insulin influences vascular endothelial growth factor (VEGF) synthesis and secretion in cultured vascular smooth muscle cells (VSMCs) via nitric oxide (NO) and whether these putative effects are lost in insulin-resistant states. MATERIALS AND METHODS: In VSMC derived from human arterioles and from aortas of insulin-sensitive Zucker fa/+rats and insulin-resistant Zucker fa/fa rats incubated with different concentrations of human regular insulin with or without inhibitors of phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3-K), mitogen-activated protein kinase (MAPK), nitric oxide synthase (NOS) and guanosine 3',5'cyclic monophosphate(cGMP)-dependent protein kinase (PKG), we measured protein expression (Western blot) and secretion (ELISA) of VEGF. RESULTS: We found that in VSMCs from humans and from insulin-sensitive Zucker fa/+rats, insulin increases VEGF protein expression and secretion, with mechanisms blunted by wortmannin and LY294002 (PI3-K inhibitors), PD98059 (MAPK inhibitor), L-NMMA (NOS inhibitor) and Rp-8pCT-cGMPs (PKG inhibitor). Also the NO donor sodium nitroprusside (SNP) and the cGMP analogue 8-Bromo-cGMP increase VEGF protein expression and secretion, with mechanisms inhibited by wortmannin and PD98059. The insulin effects on VEGF are impaired in VSMCs from Zucker fa/fa rats, which also present a reduced insulin ability to increase NO. CONCLUSIONS: In VSMCs from humans and insulin-sensitive Zucker fa/+rats: (i) insulin increases VEGF protein expression and secretion via both PI3-K and MAPK; (ii) the insulin effects on VEGF are mediated by nitric oxide. The insulin action on both nitric oxide and VEGF is impaired in VSMCs from Zucker fa/fa rats, an animal model of metabolic and vascular insulin-resistance.
Insulin receptors and insulin-like growth factor-1 (IGF-1) receptors are present in circulating human B lymphocytes (B cells) and certain B cell malignancies, but no function has been attributed to either receptor. We report a human myeloma cell line, RPMI 8226, that exhibits insulin and IGF-1-dependent receptor and substrate tyrosine phosphorylation as well as hormone-responsive cellular metabolism. Competitive hormone-binding analysis revealed that the cell line expressed approximately 4 x 10(3) high affinity insulin binding sites and 1.1 x 10(4) high affinity IGF-1 binding sites per cell. The Kd of the insulin-binding sites for insulin was 0.32 nM. The Kd of high affinity IGF-1 binding sites for IGF-1 was 0.89 nM. Insulin receptor autophosphorylation was maximum at 200 nM as was tyrosine phosphorylation of the 180-kDa cytosolic receptor substrate. Insulin-dependent activation of phosphatidylinositol 3-kinase paralleled receptor phosphorylation. In contrast, IGF-1 produced its maximum effects at 200 nM for receptor phosphorylation and 20 nM for substrate phosphorylation and PI 3-kinase activation. In growth synchronized cells, IGF-1 and insulin at 200 nM increased DNA synthesis by 122 +/- 18% and 101 +/- 27%, respectively. IGF-1 increased DNA synthesis 88 +/- 21% at 2 nM and the effect of insulin at 2 nM was 34 +/- 12%. Flux through the glycolytic pathway was also increased by insulin and IGF-1. At 200 and 2 nM, insulin increased production of lactate by 33 +/- 9% and 19 +/- 11%, respectively. IGF-1 increased lactate production 47 +/- 3% and 23 +/- 3% at identical hormone concentrations. Finally, in two additional myeloma cell lines, U266 (human) and Ag8.653 (mouse), insulin and IGF-1 increased tyrosine phosphorylation of receptor beta-subunit (95 kDa), the prominent 180-kDa substrate (pp185), and several other substrates. Thus, functional insulin and IGF-1 receptors are present in myeloma cell lines. Through these receptors, insulin and IGF-1 regulate mitogenesis and glucose metabolism, and may be important in potentiating plasma cell malignancy.
OBJECTIVE: In cultured human vascular smooth muscle cells, insulin increases cyclic GMP production by inducing nitric oxide (NO) synthesis. The aim of the present study was to determine whether in these cells the insulin-stimulated NO/cyclic GMP pathway plays a role in the regulation of glucose uptake. METHODS AND RESULTS: Glucose transport in human vascular smooth muscle cells was measured as uptake of 2-deoxy-d-[3H]glucose, cyclic GMP synthesis was checked by radioimmunoassay, and GLUT4 recruitment into the plasma membrane was determined by immunofluorescence. Insulin-stimulated glucose transport and GLUT4 recruitment were blocked by an inhibitor of NO synthesis and mimicked by NO-releasing drugs. Insulin- and NO-elicited glucose uptake were blocked by inhibitors of soluble guanylate cyclase and cyclic GMP-dependent protein kinase; furthermore, glucose transport was stimulated by an analog of cyclic GMP. CONCLUSIONS: Our results suggest that insulin-elicited glucose transport (and the corresponding GLUT4 recruitment into the plasma membrane) in human vascular smooth muscle cells is mediated by an increased synthesis of NO, which stimulates the production of cyclic GMP and the subsequent activation of a cyclic GMP-dependent protein kinase.
Recently, single chain peptides have been designed that target the insulin receptor and mimic insulin action. The aim of this study is to explore if activation of the insulin receptor with such an optimized peptide (S597) leads to the same activation of signaling pathways and biological endpoints i.e. stimulation of glycogen synthesis and cell proliferation as stimulation with insulin. We find that surface activation of the insulin receptor A-isoform with S597 leads to activation of protein kinase B (PKB) and glycogen synthesis comparable to activation by insulin, even though the level of insulin receptor phosphorylation is lower. In contrast, both Src homology 2/alpha collagen-related (Shc) and extracellular signal-regulated kinase (ERK) 2 activation are virtually absent upon stimulation with S597. Cell proliferation is only stimulated slightly by S597, suggesting that it depends on signals from Shc and ERK. The differences in signaling response could explain both the earlier reported differences in gene expression, and the reported differences in cell proliferation and glycogen synthesis induced by insulin and S597. In conclusion, despite binding equipotency, insulin, and S597 initiate different signaling and biological responses through the same insulin receptor isoform. We show for the first time that it is possible to design insulin receptor ligand mimetics with metabolic equipotency but low mitogenicity.
Insulin receptors and insulin-like growth factor-1 (IGF-1) receptors are present in circulating human B lymphocytes (B cells) and certain B cell malignancies, but no function has been attributed to either receptor. We report a human myeloma cell line, RPMI 8226, that exhibits insulin and IGF-1-dependent receptor and substrate tyrosine phosphorylation as well as hormone-responsive cellular metabolism. Competitive hormone-binding analysis revealed that the cell line expressed approximately 4 x 10(3) high affinity insulin binding sites and 1.1 x 10(4) high affinity IGF-1 binding sites per cell. The Kd of the insulin-binding sites for insulin was 0.32 nM. The Kd of high affinity IGF-1 binding sites for IGF-1 was 0.89 nM. Insulin receptor autophosphorylation was maximum at 200 nM as was tyrosine phosphorylation of the 180-kDa cytosolic receptor substrate. Insulin-dependent activation of phosphatidylinositol 3-kinase paralleled receptor phosphorylation. In contrast, IGF-1 produced its maximum effects at 200 nM for receptor phosphorylation and 20 nM for substrate phosphorylation and PI 3-kinase activation. In growth synchronized cells, IGF-1 and insulin at 200 nM increased DNA synthesis by 122 +/- 18% and 101 +/- 27%, respectively. IGF-1 increased DNA synthesis 88 +/- 21% at 2 nM and the effect of insulin at 2 nM was 34 +/- 12%. Flux through the glycolytic pathway was also increased by insulin and IGF-1. At 200 and 2 nM, insulin increased production of lactate by 33 +/- 9% and 19 +/- 11%, respectively. IGF-1 increased lactate production 47 +/- 3% and 23 +/- 3% at identical hormone concentrations. Finally, in two additional myeloma cell lines, U266 (human) and Ag8.653 (mouse), insulin and IGF-1 increased tyrosine phosphorylation of receptor beta-subunit (95 kDa), the prominent 180-kDa substrate (pp185), and several other substrates. Thus, functional insulin and IGF-1 receptors are present in myeloma cell lines. Through these receptors, insulin and IGF-1 regulate mitogenesis and glucose metabolism, and may be important in potentiating plasma cell malignancy.
Insulin isolated from the pancreas of a diabetic patient with fasting hyperinsulinaemia showed decreased activity in binding to cell membrane insulin receptors and in stimulating cellular 2-deoxyglucose transport and glucose oxidation. Chemical studies suggest that the isolated hormone is a mixture of normal insulin and an abnormal variant which contains a leucine for phenylalanine substitution at position 24 or 25 of the insulin B-chain.
Insulin receptors and insulin-like growth factor-1 (IGF-1) receptors are present in circulating human B lymphocytes (B cells) and certain B cell malignancies, but no function has been attributed to either receptor. We report a human myeloma cell line, RPMI 8226, that exhibits insulin and IGF-1-dependent receptor and substrate tyrosine phosphorylation as well as hormone-responsive cellular metabolism. Competitive hormone-binding analysis revealed that the cell line expressed approximately 4 x 10(3) high affinity insulin binding sites and 1.1 x 10(4) high affinity IGF-1 binding sites per cell. The Kd of the insulin-binding sites for insulin was 0.32 nM. The Kd of high affinity IGF-1 binding sites for IGF-1 was 0.89 nM. Insulin receptor autophosphorylation was maximum at 200 nM as was tyrosine phosphorylation of the 180-kDa cytosolic receptor substrate. Insulin-dependent activation of phosphatidylinositol 3-kinase paralleled receptor phosphorylation. In contrast, IGF-1 produced its maximum effects at 200 nM for receptor phosphorylation and 20 nM for substrate phosphorylation and PI 3-kinase activation. In growth synchronized cells, IGF-1 and insulin at 200 nM increased DNA synthesis by 122 +/- 18% and 101 +/- 27%, respectively. IGF-1 increased DNA synthesis 88 +/- 21% at 2 nM and the effect of insulin at 2 nM was 34 +/- 12%. Flux through the glycolytic pathway was also increased by insulin and IGF-1. At 200 and 2 nM, insulin increased production of lactate by 33 +/- 9% and 19 +/- 11%, respectively. IGF-1 increased lactate production 47 +/- 3% and 23 +/- 3% at identical hormone concentrations. Finally, in two additional myeloma cell lines, U266 (human) and Ag8.653 (mouse), insulin and IGF-1 increased tyrosine phosphorylation of receptor beta-subunit (95 kDa), the prominent 180-kDa substrate (pp185), and several other substrates. Thus, functional insulin and IGF-1 receptors are present in myeloma cell lines. Through these receptors, insulin and IGF-1 regulate mitogenesis and glucose metabolism, and may be important in potentiating plasma cell malignancy.
The epidemic of type 2 diabetes and impaired glucose tolerance is one of the main causes of morbidity and mortality worldwide. In both disorders, tissues such as muscle, fat and liver become less responsive or resistant to insulin. This state is also linked to other common health problems, such as obesity, polycystic ovarian disease, hyperlipidaemia, hypertension and atherosclerosis. The pathophysiology of insulin resistance involves a complex network of signalling pathways, activated by the insulin receptor, which regulates intermediary metabolism and its organization in cells. But recent studies have shown that numerous other hormones and signalling events attenuate insulin action, and are important in type 2 diabetes.
J. Cell. Biochem. 82, 610-618 (2001)[PubMed:11500939]
Insulin-like growth factor II (IGF-II) plays a key role in mitogenesis during development and tumorigenesis and is believed to exert its mitogenic functions mainly through the IGF-I receptor. Recently, we identified the insulin receptor isoform A (IR(A)) as an additional high affinity receptor for IGF-II in both fetal and cancer cells. Here we investigated the mitogenic signaling of IGF-II via the Akt/Glycogen synthase kinase 3 (Gsk3) axis employing R-IR(A) cells that are IGF-I receptor null mouse embryonic fibroblasts expressing the human IR(A). IGF-II induced activation of the proto-oncogenic serine kinase Akt, reaching maximal at 5-10 min. IGF-II also caused the rapid and sustained deactivation of glycogen synthase kinase 3-beta (Gsk3beta), reaching maximal at 1-3 min, shortly preceding, therefore, maximal activation of Akt. Under our conditions, IGF-II and insulin induced 70-80% inhibition of Gsk3betaactivity. In these cells IGF-II also deactivated Gsk3alpha although less effectively than Gsk3beta. In parallel experiments, we found that IGF-II induced transient activation of extracellular-signal-regulated kinases (Erk) reaching maximal at 5-10 min and decreasing thereafter. Time courses and potencies of regulation of both mitogenic pathways (Akt/Gsk3beta and Erk) by IGF-II via IR(A) were similar to those of insulin. Furthermore, IGF-II like insulin effectively stimulated cell cycle progression from the G0/G1 to the S and G2/M phases. Interestingly, AP-1-mediated gene expression, that was reported to be negatively regulated by Gsk3beta was only weakly increased after IGF-II stimulation. Our present data suggest that the coordinated activation or deactivation of Akt, Gsk3beta, and Erk may account for IGF-II mitogenic effects and support an active role for IR(A) in IGF-II action.
PSM/SH2-B has been described as a cellular partner of the FcepsilonRI receptor, insulin receptor (IR), insulin-like growth factor-I (IGF-I) receptor (IGF-IR), and nerve growth factor receptor (TrkA). A function has been proposed in neuronal differentiation and development but its role in other signaling pathways is still unclear. To further elucidate the physiologic role of PSM we have identified additional mitogenic receptor tyrosine kinases as putative PSM partners including platelet-derived growth factor (PDGF) receptor (PDGFR) beta, hepatocyte growth factor receptor (Met), and fibroblast growth factor receptor. We have mapped Y740 as a site of PDGFR beta that is involved in the association with PSM. We have further investigated the putative role of PSM in mitogenesis with three independent experimental strategies and found that all consistently suggested a role as a positive, stimulatory signaling adapter in normal NIH3T3 and baby hamster kidney fibroblasts. (1) PSM expression from cDNA using an ecdysone-regulated transient expression system stimulated PDGF-BB-, IGF-I-, and insulin- but not EGF-induced DNA synthesis in an ecdysone dose-responsive fashion; (2) Microinjection of the (dominant negative) PSM SH2 domain interfered with PDGF-BB- and insulin-induced DNA synthesis; and (3) A peptide mimetic of the PSM Pro-rich putative SH3 domain-binding region interfered with PDGF-BB-, IGF-I-, and insulin- but not with EGF-induced DNA synthesis in NIH3T3 fibroblasts. This experiment was based on cell-permeable fusion peptides with the Drosophila antennapedia homeodomain which effectively traverse the plasma membrane of cultured cells. These experimental strategies independently suggest that PSM functions as a positive, stimulatory, mitogenic signaling mediator in PDGF-BB, IGF-I, and insulin but not in EGF action. This function appears to involve the PSM SH2 domain as well as the Pro-rich putative SH3 domain binding region. Our findings support the model that PSM participates as an adapter in various mitogenic signaling mechanisms by linking an activated (receptor) phospho-tyrosine to the SH3 domain of an unknown cellular partner.
J. Cell. Biochem. 82, 610-618 (2001)[PubMed:11500939]
Insulin-like growth factor II (IGF-II) plays a key role in mitogenesis during development and tumorigenesis and is believed to exert its mitogenic functions mainly through the IGF-I receptor. Recently, we identified the insulin receptor isoform A (IR(A)) as an additional high affinity receptor for IGF-II in both fetal and cancer cells. Here we investigated the mitogenic signaling of IGF-II via the Akt/Glycogen synthase kinase 3 (Gsk3) axis employing R-IR(A) cells that are IGF-I receptor null mouse embryonic fibroblasts expressing the human IR(A). IGF-II induced activation of the proto-oncogenic serine kinase Akt, reaching maximal at 5-10 min. IGF-II also caused the rapid and sustained deactivation of glycogen synthase kinase 3-beta (Gsk3beta), reaching maximal at 1-3 min, shortly preceding, therefore, maximal activation of Akt. Under our conditions, IGF-II and insulin induced 70-80% inhibition of Gsk3betaactivity. In these cells IGF-II also deactivated Gsk3alpha although less effectively than Gsk3beta. In parallel experiments, we found that IGF-II induced transient activation of extracellular-signal-regulated kinases (Erk) reaching maximal at 5-10 min and decreasing thereafter. Time courses and potencies of regulation of both mitogenic pathways (Akt/Gsk3beta and Erk) by IGF-II via IR(A) were similar to those of insulin. Furthermore, IGF-II like insulin effectively stimulated cell cycle progression from the G0/G1 to the S and G2/M phases. Interestingly, AP-1-mediated gene expression, that was reported to be negatively regulated by Gsk3beta was only weakly increased after IGF-II stimulation. Our present data suggest that the coordinated activation or deactivation of Akt, Gsk3beta, and Erk may account for IGF-II mitogenic effects and support an active role for IR(A) in IGF-II action.
AIMS/HYPOTHESIS: Extracellular pre-B cell colony-enhancing factor/nicotinamide phosphoribosyltransferase/visfatin (ePBEF/NAMPT/visfatin) is an adipocytokine, whose circulating levels are enhanced in metabolic disorders, such as diabetes mellitus and obesity. Here, we explored the ability of ePBEF/NAMPT/visfatin to promote vascular inflammation, as a condition closely related to atherothrombotic diseases. We specifically studied the ability of PBEF/NAMPT/visfatin to directly activate pathways leading to inducible nitric oxide synthase (iNOS) induction in cultured human aortic smooth muscle cells, as well as the mechanisms involved. METHODS: iNOS levels and extracellular signal-regulated kinase (ERK) 1/2 activity were determined by western blotting. Nuclear factor (NF)-kappaB activity was assessed by electrophoretic mobility shift assay. RESULTS: ePBEF/NAMPT/visfatin (10-250 ng/ml) induced iNOS in a concentration-dependent manner. At a submaximal concentration (100 ng/ml), ePBEF/NAMPT/visfatin time-dependently enhanced iNOS levels up to 18 h after stimulation. Over this time period, ePBEF/NAMPT/visfatin elicited a sustained activation of NF-kappaB and triggered a biphasic ERK 1/2 activation. By using the respective ERK 1/2 and NF-kappaB inhibitors, PD98059 and pyrrolidine dithiocarbamate, we established that iNOS induction by ePBEF/NAMPT/visfatin required the consecutive upstream activation of ERK 1/2 and NF-kappaB. The pro-inflammatory action of ePBEF/NAMPT/visfatin was not prevented by insulin receptor blockade. However, exogenous nicotinamide mononucleotide, the product of NAMPT activity, mimicked NF-kappaB activation and iNOS induction by ePBEF/NAMPT/visfatin, while the NAMPT inhibitor APO866 prevented the effects of ePBEF/NAMPT/visfatin on iNOS and NF-kappaB. CONCLUSIONS/INTERPRETATION: Through its intrinsic NAMPT activity, ePBEF/NAMPT/visfatin appears to be a direct contributor to vascular inflammation, a key feature of atherothrombotic diseases linked to metabolic disorders.
Any process that activates or increases the frequency, rate or extent of the chemical reactions and pathways resulting in the formation of nitric oxide.
OBJECTIVE: In cultured human vascular smooth muscle cells, insulin increases cyclic GMP production by inducing nitric oxide (NO) synthesis. The aim of the present study was to determine whether in these cells the insulin-stimulated NO/cyclic GMP pathway plays a role in the regulation of glucose uptake. METHODS AND RESULTS: Glucose transport in human vascular smooth muscle cells was measured as uptake of 2-deoxy-d-[3H]glucose, cyclic GMP synthesis was checked by radioimmunoassay, and GLUT4 recruitment into the plasma membrane was determined by immunofluorescence. Insulin-stimulated glucose transport and GLUT4 recruitment were blocked by an inhibitor of NO synthesis and mimicked by NO-releasing drugs. Insulin- and NO-elicited glucose uptake were blocked by inhibitors of soluble guanylate cyclase and cyclic GMP-dependent protein kinase; furthermore, glucose transport was stimulated by an analog of cyclic GMP. CONCLUSIONS: Our results suggest that insulin-elicited glucose transport (and the corresponding GLUT4 recruitment into the plasma membrane) in human vascular smooth muscle cells is mediated by an increased synthesis of NO, which stimulates the production of cyclic GMP and the subsequent activation of a cyclic GMP-dependent protein kinase.
BACKGROUND: Hyperglycemia impairs functional properties of cytosolic and nuclear proteins via O-linked glycosylation modification (O-GlcNAcylation). We studied the effects of O-GlcNAcylation on insulin signaling in human coronary artery endothelial cells. METHODS AND RESULTS: O-GlcNAcylation impaired the metabolic branch of insulin signaling, ie, insulin receptor (IR) activation of the IR substrate (IRS)/phosphatidylinositol 3-kinase (PI3-K)/Akt, whereas it enhanced the mitogenic branch, ie, ERK-1/2 and p38 (mitogen-activated protein kinase). Both in vivo and in vitro phosphorylation of endothelial nitric oxide synthase (eNOS) by Akt were reduced by hyperglycemia and hexosamine activation. Insulin-induced eNOS activity in vivo was reduced by hyperglycemia and hexosamine activation, which was coupled to increased activation and expression of matrix metalloproteinase-2 and -9; these phenomena were reversed by inhibition of the hexosamine pathway. Finally, carotid plaques from type 2 diabetic patients showed increased endothelial O-GlcNAcylation with respect to nondiabetics. CONCLUSIONS: Our data show that hyperglycemia, through the hexosamine pathway, impairs activation of the IR/IRS/PI3-K/Akt pathway, resulting in deregulation of eNOS activity.
Leptin, the ob gene product, is produced by adipose tissue and is submitted to a complex hormonal and metabolic regulation. Leptin plays a critical role in the balance of body weight. Here we report on secretion and hormonal regulation of leptin by brown adipocytes. Using the recently established T37i cell line, we show that leptin expression and secretion occurred as a function of cell differentiation. In differentiated T37i cells, insulin induced leptin release ( approximately 0.25 ng/10(6) cells/h) in a concentration-dependent manner (EC50=0.1 nM), and this was totally suppressed by beta3-adrenergic ligand, thiazolidinedione, cycloheximide, or actinomycin D. Insulin induced a strong, rapid (within 2 h) but transient fivefold increase in leptin mRNA levels. This transcriptional control of ob gene expression by insulin involved both phosphatidylinositol 3-kinase- and MAP kinase-dependent pathways. Glucocorticoids inhibited both insulin-stimulated leptin secretion and ob gene expression without affecting leptin mRNA stability (t(1/2)=3h05). Altogether, our results demonstrate that brown adipocytes express and secrete leptin, whose hormonal regulation clearly differs from that described in white adipose tissue. These findings point to tissue-specific molecular mechanisms and suggest that leptin might exert direct effects on energy homeostasis through an autocrine mechanism.
Incubation of rat hepatoma Fao cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins. This is followed by elevation in their P-Ser/Thr content, and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the increase in the P-Ser/Thr content of IRS-1, its dissociation from the IR, and the decrease in its P-Tyr content following 60 min of insulin treatment, indicating that the Ser kinases that negatively regulate IRS-1 function are downstream effectors of PI3K. PKCzeta fulfills this criterion, being an insulin-activated downstream effector of PI3K. Overexpression of PKCzeta in Fao cells, by infection of the cells with adenovirus-based PKCzeta construct, had no effect on its own, but it accelerated the rate of insulin-stimulated dissociation of IR.IRS-1 complexes and the rate of Tyr dephosphorylation of IRS-1. The insulin-stimulated negative regulatory role of PKCzeta was specific and could not be mimic by infecting Fao cells with adenoviral constructs encoding for PKC alpha, delta, or eta. Because the reduction in P-Tyr content of IRS-1 was accompanied by a reduced association of IRS-1 with p85, the regulatory subunit of PI3K, it suggests that this negative regulatory process induced by PKCzeta, has a built-in attenuation signal. Hence, insulin triggers a sequential cascade in which PI3K-mediated activation of PKCzeta inhibits IRS-1 functions, reduces complex formation between IRS-1 and PI3K, and inhibits further activation of PKCzeta itself. These findings implicate PKCzeta as a key element in a multistep negative feedback control mechanism of IRS-1 functions.
Insulin receptors and insulin-like growth factor-1 (IGF-1) receptors are present in circulating human B lymphocytes (B cells) and certain B cell malignancies, but no function has been attributed to either receptor. We report a human myeloma cell line, RPMI 8226, that exhibits insulin and IGF-1-dependent receptor and substrate tyrosine phosphorylation as well as hormone-responsive cellular metabolism. Competitive hormone-binding analysis revealed that the cell line expressed approximately 4 x 10(3) high affinity insulin binding sites and 1.1 x 10(4) high affinity IGF-1 binding sites per cell. The Kd of the insulin-binding sites for insulin was 0.32 nM. The Kd of high affinity IGF-1 binding sites for IGF-1 was 0.89 nM. Insulin receptor autophosphorylation was maximum at 200 nM as was tyrosine phosphorylation of the 180-kDa cytosolic receptor substrate. Insulin-dependent activation of phosphatidylinositol 3-kinase paralleled receptor phosphorylation. In contrast, IGF-1 produced its maximum effects at 200 nM for receptor phosphorylation and 20 nM for substrate phosphorylation and PI 3-kinase activation. In growth synchronized cells, IGF-1 and insulin at 200 nM increased DNA synthesis by 122 +/- 18% and 101 +/- 27%, respectively. IGF-1 increased DNA synthesis 88 +/- 21% at 2 nM and the effect of insulin at 2 nM was 34 +/- 12%. Flux through the glycolytic pathway was also increased by insulin and IGF-1. At 200 and 2 nM, insulin increased production of lactate by 33 +/- 9% and 19 +/- 11%, respectively. IGF-1 increased lactate production 47 +/- 3% and 23 +/- 3% at identical hormone concentrations. Finally, in two additional myeloma cell lines, U266 (human) and Ag8.653 (mouse), insulin and IGF-1 increased tyrosine phosphorylation of receptor beta-subunit (95 kDa), the prominent 180-kDa substrate (pp185), and several other substrates. Thus, functional insulin and IGF-1 receptors are present in myeloma cell lines. Through these receptors, insulin and IGF-1 regulate mitogenesis and glucose metabolism, and may be important in potentiating plasma cell malignancy.
Insulin stimulates the autophosphorylation of tyrosine residues of the beta subunit of the insulin receptor (IR); this modified insulin-independent kinase has increased activity toward exogenous substrates in vitro. We show here that replacement of one or both of the twin tyrosines (residues 1162 and 1163) with phenylalanine results in a dramatic reduction in or loss of insulin-activated autophosphorylation and kinase activity in vitro. In vivo, these mutations not only result in a substantial decrease in insulin-stimulated IR autophosphorylation but also in a parallel decrease in the insulin-activated uptake of 2-deoxyglucose. Furthermore, a truncated IR protein (lacking the last 112 amino acids) has an unstable beta subunit; this mutant has no kinase activity in vitro or in vivo and does not mediate insulin-stimulated uptake of 2-deoxyglucose. IR autophosphorylation is thus implicated in the regulation of IR activities, with tyrosines 1162 and 1163 as major sites of this regulation.
Any process that activates or increases the frequency, rate or extent of the protein kinase B signaling cascade, a series of reactions mediated by the intracellular serine/threonine kinase protein kinase B.
J. Cell. Biochem. 82, 610-618 (2001)[PubMed:11500939]
Insulin-like growth factor II (IGF-II) plays a key role in mitogenesis during development and tumorigenesis and is believed to exert its mitogenic functions mainly through the IGF-I receptor. Recently, we identified the insulin receptor isoform A (IR(A)) as an additional high affinity receptor for IGF-II in both fetal and cancer cells. Here we investigated the mitogenic signaling of IGF-II via the Akt/Glycogen synthase kinase 3 (Gsk3) axis employing R-IR(A) cells that are IGF-I receptor null mouse embryonic fibroblasts expressing the human IR(A). IGF-II induced activation of the proto-oncogenic serine kinase Akt, reaching maximal at 5-10 min. IGF-II also caused the rapid and sustained deactivation of glycogen synthase kinase 3-beta (Gsk3beta), reaching maximal at 1-3 min, shortly preceding, therefore, maximal activation of Akt. Under our conditions, IGF-II and insulin induced 70-80% inhibition of Gsk3betaactivity. In these cells IGF-II also deactivated Gsk3alpha although less effectively than Gsk3beta. In parallel experiments, we found that IGF-II induced transient activation of extracellular-signal-regulated kinases (Erk) reaching maximal at 5-10 min and decreasing thereafter. Time courses and potencies of regulation of both mitogenic pathways (Akt/Gsk3beta and Erk) by IGF-II via IR(A) were similar to those of insulin. Furthermore, IGF-II like insulin effectively stimulated cell cycle progression from the G0/G1 to the S and G2/M phases. Interestingly, AP-1-mediated gene expression, that was reported to be negatively regulated by Gsk3beta was only weakly increased after IGF-II stimulation. Our present data suggest that the coordinated activation or deactivation of Akt, Gsk3beta, and Erk may account for IGF-II mitogenic effects and support an active role for IR(A) in IGF-II action.
Any process that increases the rate frequency or extent of a phase of elevated metabolic activity, during which oxygen consumption increases; this leads to the production, by an NADH dependent system, of hydrogen peroxide (H2O2), superoxide anions and hydroxyl radicals.
J. Biol. Chem. 272, 10135-10143 (1997)[PubMed:9092559]
Human fat cells possess a multireceptor-linked H2O2-generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H2O2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H2O2 generation by Mn2+ and insulin was blocked by GDPbetaS (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-Galphai1-2 or the corresponding peptide. Consistently, manganese could be replaced by micromolar concentrations of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTPgammaS to the left (>10-fold) and increased the maximal response. In the presence of 10 microM GTPgammaS, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. The insulin receptor and Gi were co-adsorbed on anti-Galphai and anti-insulin receptor beta-subunit (anti-IRbeta) affinity columns. Partially purified insulin receptor preparations contained Galphas, Galphai2, and Gbetagamma (but no Galphai1 or Galphai3). The functional nature of the insulin receptor-Gi2 complex was made evident by insulin's ability to modulate labeling of Gi by bacterial toxins. Insulin action was mimicked by activated Galphai, but not by Galphao or Gbetagamma, indicating that insulin's signal was transduced via Galphai2. Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.
Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process. Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS-/- mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of alphaIIbbeta3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO-G kinase-dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.
In vivo leucine metabolism was studied after an overnight fast in nine type I diabetic patients and nine healthy control subjects using L-[1-13C] leucine as a tracer. In the insulin-deprived state, leucine flux (reflecting proteolysis), leucine oxidation, and plasma leucine concentrations were higher in the diabetic patients than in the control subjects (P less than .001). In 4 of the 9 insulin-deprived diabetic patients, a four-hour intravenous insulin treatment decreased plasma glucose and leucine concentrations and leucine flux, but failed to decrease leucine oxidation. In the remaining 5 of the 9 diabetic patients, uninterrupted insulin treatment prior to the study and a seven-hour intravenous insulin treatment during the study period decreased not only the concentrations of plasma glucose and leucine and leucine flux, but also leucine oxidation (P less than .01). In all 9 diabetic patients the nonoxidative portion of leucine flux (reflecting protein synthesis) decreased during insulin treatment (P less than .01), but this decrease was lower than that of leucine flux (reflecting proteolysis), and therefore protein was conserved during insulin treatment. We conclude that the effect of insulin on proteolysis (reflected by leucine flux) is more rapid than its effect on leucine oxidation, but on aggressive insulin treatment accelerated leucine oxidation also was decreased in type I diabetic patients.
OBJECTIVE: In cultured human vascular smooth muscle cells, insulin increases cyclic GMP production by inducing nitric oxide (NO) synthesis. The aim of the present study was to determine whether in these cells the insulin-stimulated NO/cyclic GMP pathway plays a role in the regulation of glucose uptake. METHODS AND RESULTS: Glucose transport in human vascular smooth muscle cells was measured as uptake of 2-deoxy-d-[3H]glucose, cyclic GMP synthesis was checked by radioimmunoassay, and GLUT4 recruitment into the plasma membrane was determined by immunofluorescence. Insulin-stimulated glucose transport and GLUT4 recruitment were blocked by an inhibitor of NO synthesis and mimicked by NO-releasing drugs. Insulin- and NO-elicited glucose uptake were blocked by inhibitors of soluble guanylate cyclase and cyclic GMP-dependent protein kinase; furthermore, glucose transport was stimulated by an analog of cyclic GMP. CONCLUSIONS: Our results suggest that insulin-elicited glucose transport (and the corresponding GLUT4 recruitment into the plasma membrane) in human vascular smooth muscle cells is mediated by an increased synthesis of NO, which stimulates the production of cyclic GMP and the subsequent activation of a cyclic GMP-dependent protein kinase.
We compared the acute effect of insulin on the human colonic intestinal epithelial cell line CaCo-2 and the transformed human hepatic cell line HepG2. Over 24 h, 100 nM and 10 microM insulin significantly inhibited the secretion of apolipoprotein (apo) B-100 from HepG2 cells to 63 and 49% of control, respectively. Insulin had no effect on the secretion of apoB-48 from CaCo-2 cells. There was no effect of insulin on the cholesterol ester or free cholesterol concentrations in HepG2 or CaCo-2 cells. HepG2 and CaCo-2 cells bound insulin with high affinity, leading to similar stimulation of insulin receptor protein tyrosine kinase activation. Protein kinase C or mitogen-activated protein kinase activity in the presence or absence of insulin was not correlated with apoB-48 production in CaCo-2 cells. Therefore, insulin acutely decreases the secretion of apoB-100 in hepatic HepG2 cells, but does not acutely modulate the production or secretion of apoB-48 from CaCo-2 intestinal cells.
The human insulin receptor (IR) exists in two isoforms (IR-A and IR-B). IR-A is a short isoform, generated by the skipping of exon 11, a small exon encoding for 12 amino acid residues at the carboxyl terminus of the IR alpha-subunit. Recently, we found that IR-A is the predominant isoform in fetal tissues and malignant cells and binds with a high affinity not only insulin but also insulin-like growth factor-II (IGF-II). To investigate whether the activation of IR-A by the two ligands differentially activate post-receptor molecular mechanisms, we studied gene expression in response to IR-A activation by either insulin or IGF-II, using microarray technology. To avoid the interfering effect of the IGF-IR, IGF-II binding to the IR-A was studied in IGF-IR-deficient murine fibroblasts (R- cells) transfected with the human IR-A cDNA (R-/IR-A cells). Gene expression was studied at 0.5, 3, and 8 h. We found that 214 transcripts were similarly regulated by insulin and IGF-II, whereas 45 genes were differentially transcribed. Eighteen of these differentially regulated genes were responsive to only one of the two ligands (12 to insulin and 6 to IGF-II). Twenty-seven transcripts were regulated by both insulin and IGF-II, but a significant difference between the two ligands was present at least in one time point. Interestingly, IGF-II was a more potent and/or persistent regulator than insulin for these genes. Results were validated by measuring the expression of 12 genes by quantitative real-time reverse transcriptase-PCR. In conclusion, we show that insulin and IGF-II, acting via the same receptor, may differentially affect gene expression in cells. These studies provide a molecular basis for understanding some of the biological differences between the two ligands and may help to clarify the biological role of IR-A in embryonic/fetal growth and the selective biological advantage that malignant cells producing IGF-II may acquire via IR-A overexpression.
OBJECTIVE: In cultured human vascular smooth muscle cells, insulin increases cyclic GMP production by inducing nitric oxide (NO) synthesis. The aim of the present study was to determine whether in these cells the insulin-stimulated NO/cyclic GMP pathway plays a role in the regulation of glucose uptake. METHODS AND RESULTS: Glucose transport in human vascular smooth muscle cells was measured as uptake of 2-deoxy-d-[3H]glucose, cyclic GMP synthesis was checked by radioimmunoassay, and GLUT4 recruitment into the plasma membrane was determined by immunofluorescence. Insulin-stimulated glucose transport and GLUT4 recruitment were blocked by an inhibitor of NO synthesis and mimicked by NO-releasing drugs. Insulin- and NO-elicited glucose uptake were blocked by inhibitors of soluble guanylate cyclase and cyclic GMP-dependent protein kinase; furthermore, glucose transport was stimulated by an analog of cyclic GMP. CONCLUSIONS: Our results suggest that insulin-elicited glucose transport (and the corresponding GLUT4 recruitment into the plasma membrane) in human vascular smooth muscle cells is mediated by an increased synthesis of NO, which stimulates the production of cyclic GMP and the subsequent activation of a cyclic GMP-dependent protein kinase.
BACKGROUND: Insulin plus glucose, given for 7 days to hypermetabolic burn patients, has been shown to stimulate limb protein anabolism. We hypothesized that insulin plus glucose given to burn patients would also stimulate wound healing. METHODS: Six patients with burns >40% total body surface area were randomized to receive insulin or placebo in a crossover study during the healing of their first and second donor sites. Insulin treatment was titrated at 25 to 49 U/h to achieve a plasma insulin level of 400 to 900 microU/mL for 7 days. Patients receiving insulin received dextrose 50 at 20 to 50 mL/h, titrated to maintain euglycemia. Donor-site biopsies were taken at 7 days and evaluated by three observers blinded to the treatment. RESULTS: The mean (+/-SD) donor-site healing time was reduced from 6.5 +/- 1.0 days with placebo to 4.7 +/- 1.2 days during insulin infusion (p < 0.05). Laminin showed intense staining along the basal lamina and blood vessels. Collagen type IV staining also increased after insulin therapy compared with placebo. CONCLUSION: Data indicate that high doses of insulin and glucose can be safely administered to massively burned patients to improve wound matrix formation.
Protein participating in biochemical reactions in which carbohydrates are involved. Carbohydrate is a general term for sugars and related compounds with the general formula Cn(H2O)n. The smallest are monosaccharides (e.g. glucose); polysaccharides (e.g. starch, cellulose, glycogen) can be large and vary in length.
Protein which functions as a hormone, a biochemical substance secreted by specialized cells that affects the metabolism or behavior of other cells which possess functional receptors for the hormone. Hormones may be hydrophilic, like insulin, in which case the receptors are on the cell surface, or lipophilic, like the steroids, where the receptor can be intracellular.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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