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Sample records for insulin receptor activator

  1. Monoclonal Antibodies to the Human Insulin Receptor that Activate Glucose Transport but not Insulin Receptor Kinase Activity

    NASA Astrophysics Data System (ADS)

    Forsayeth, John R.; Caro, Jose F.; Sinha, Madhur K.; Maddux, Betty A.; Goldfine, Ira D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the α subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  2. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor.

    PubMed

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick; Negishi, Masahiko

    2016-05-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)-forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally. PMID:26994072

  3. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor

    PubMed Central

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick

    2016-01-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)–forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally. PMID:26994072

  4. Transgenic silkworms expressing human insulin receptors for evaluation of therapeutically active insulin receptor agonists.

    PubMed

    Matsumoto, Yasuhiko; Ishii, Masaki; Ishii, Kenichi; Miyaguchi, Wataru; Horie, Ryo; Inagaki, Yoshinori; Hamamoto, Hiroshi; Tatematsu, Ken-ichiro; Uchino, Keiro; Tamura, Toshiki; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2014-12-12

    We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptor agonists. PMID:25449269

  5. Differential Activation of Insulin Receptor Substrates 1 and 2 by Insulin-Like Growth Factor-Activated Insulin Receptors▿

    PubMed Central

    Denley, Adam; Carroll, Julie M.; Brierley, Gemma V.; Cosgrove, Leah; Wallace, John; Forbes, Briony; Roberts, Charles T.

    2007-01-01

    The insulin-like growth factors (insulin-like growth factor I [IGF-I] and IGF-II) exert important effects on growth, development, and differentiation through the IGF-I receptor (IGF-IR) transmembrane tyrosine kinase. The insulin receptor (IR) is structurally related to the IGF-IR, and at high concentrations, the IGFs can also activate the IR, in spite of their generally low affinity for the latter. Two mechanisms that facilitate cross talk between the IGF ligands and the IR at physiological concentrations have been described. The first of these is the existence of an alternatively spliced IR variant that exhibits high affinity for IGF-II as well as for insulin. A second phenomenon is the ability of hybrid receptors comprised of IGF-IR and IR hemireceptors to bind IGFs, but not insulin. To date, however, direct activation of an IR holoreceptor by IGF-I at physiological levels has not been demonstrated. We have now found that IGF-I can function through both splice variants of the IR, in spite of low affinity, to specifically activate IRS-2 to levels similar to those seen with equivalent concentrations of insulin or IGF-II. The specific activation of IRS-2 by IGF-I through the IR does not result in activation of the extracellular signal-regulated kinase pathway but does induce delayed low-level activation of the phosphatidylinositol 3-kinase pathway and biological effects such as enhanced cell viability and protection from apoptosis. These findings suggest that IGF-I can function directly through the IR and that the observed effects of IGF-I on insulin sensitivity may be the result of direct facilitation of insulin action by IGF-I costimulation of the IR in insulin target tissues. PMID:17325037

  6. The insulin receptor activation process involves localized conformational changes.

    PubMed

    Baron, V; Kaliman, P; Gautier, N; Van Obberghen, E

    1992-11-15

    The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation. PMID:1331080

  7. Thrombin stimulates insulin secretion via protease-activated receptor-3

    PubMed Central

    Hänzelmann, Sonja; Wang, Jinling; Güney, Emre; Tang, Yunzhao; Zhang, Enming; Axelsson, Annika S; Nenonen, Hannah; Salehi, Albert S; Wollheim, Claes B; Zetterberg, Eva; Berntorp, Erik; Costa, Ivan G; Castelo, Robert; Rosengren, Anders H

    2015-01-01

    The disease mechanisms underlying type 2 diabetes (T2D) remain poorly defined. Here we aimed to explore the pathophysiology of T2D by analyzing gene co-expression networks in human islets. Using partial correlation networks we identified a group of co-expressed genes (‘module’) including F2RL2 that was associated with glycated hemoglobin. F2Rl2 is a G-protein-coupled receptor (GPCR) that encodes protease-activated receptor-3 (PAR3). PAR3 is cleaved by thrombin, which exposes a 6-amino acid sequence that acts as a ‘tethered ligand’ to regulate cellular signaling. We have characterized the effect of PAR3 activation on insulin secretion by static insulin secretion measurements, capacitance measurements, studies of diabetic animal models and patient samples. We demonstrate that thrombin stimulates insulin secretion, an effect that was prevented by an antibody that blocks the thrombin cleavage site of PAR3. Treatment with a peptide corresponding to the PAR3 tethered ligand stimulated islet insulin secretion and single β-cell exocytosis by a mechanism that involves activation of phospholipase C and Ca2+ release from intracellular stores. Moreover, we observed that the expression of tissue factor, which regulates thrombin generation, was increased in human islets from T2D donors and associated with enhanced β-cell exocytosis. Finally, we demonstrate that thrombin generation potential in patients with T2D was associated with increased fasting insulin and insulinogenic index. The findings provide a previously unrecognized link between hypercoagulability and hyperinsulinemia and suggest that reducing thrombin activity or blocking PAR3 cleavage could potentially counteract the exaggerated insulin secretion that drives insulin resistance and β-cell exhaustion in T2D. PMID:26742564

  8. Adipocyte insulin receptor activity maintains adipose tissue mass and lifespan.

    PubMed

    Friesen, Max; Hudak, Carolyn S; Warren, Curtis R; Xia, Fang; Cowan, Chad A

    2016-08-01

    Type 2 diabetes follows a well-defined progressive pathogenesis, beginning with insulin resistance in metabolic tissues such as the adipose. Intracellular signaling downstream of insulin receptor activation regulates critical metabolic functions of adipose tissue, including glucose uptake, lipogenesis, lipolysis and adipokine secretion. Previous studies have used the aP2 promoter to drive Cre recombinase expression in adipose tissue. Insulin receptor (IR) knockout mice created using this aP2-Cre strategy (FIRKO mice) were protected from obesity and glucose intolerance. Later studies demonstrated the promiscuity of the aP2 promoter, casting doubts upon the tissue specificity of aP2-Cre models. It is our goal to use the increased precision of the Adipoq promoter to investigate adipocyte-specific IR function. Towards this end we generated an adipocyte-specific IR knockout (AIRKO) mouse using an Adipoq-driven Cre recombinase. Here we report AIRKO mice are less insulin sensitive throughout life, and less glucose tolerant than wild-type (WT) littermates at the age of 16 weeks. In contrast to WT littermates, the insulin sensitivity of AIRKO mice is unaffected by age or dietary regimen. At any age, AIRKO mice are comparably insulin resistant to old or obese WT mice and have a significantly reduced lifespan. Similar results were obtained when these phenotypes were re-examined in FIRKO mice. We also found that the AIRKO mouse is protected from high-fat diet-induced weight gain, corresponding with a 90% reduction in tissue weight of major adipose depots compared to WT littermates. Adipose tissue mass reduction is accompanied by hepatomegaly and increased hepatic steatosis. These data indicate that adipocyte IR function is crucial to systemic energy metabolism and has profound effects on adiposity, hepatic homeostasis and lifespan. PMID:27246738

  9. Defect in tyrosine kinase activity of the insulin receptor from a patient with insulin resistance and acanthosis nigricans.

    PubMed

    Yamamoto, R; Shiba, T; Tobe, K; Shibasaki, Y; Koshio, O; Izumi, T; Odawara, M; Mikami, Y; Matsuura, N; Akanuma, Y

    1990-04-01

    We report here a defect in tyrosine kinase activity of the insulin receptor from an insulin-resistant patient with acanthosis nigricans using cultured Ebstein-Barr virus (EBV)-transformed B-lymphocytes. As judged by affinity labeling and immunoblotting, the alpha- and beta-subunits of insulin receptors from the patient's lymphocytes exhibited the same mol wt as those from control subjects. Lectin-purified extracts from lymphocytes of the patient and the control subjects containing the same insulin-binding capacity were assayed for autophosphorylation and the ability to phosphorylate histone H2B. The degree of insulin-dependent autophosphorylation and the tyrosine kinase activity of the insulin receptor from the patient's lymphocytes were decreased to 15% and 13%, respectively, in a cell-free system. The insulin-dependent autophosphorylation of the insulin receptor was also impaired in intact EBV lymphocytes from the patient. Consistent with these results, we found that one of this patient's alleles had a mutation in which valine is substituted for Gly996, the third glycine in the conserved Gly-X-Gly-X-X-Gly motif in the kinase domain. Thus, it seems likely that the defect in tyrosine kinase activity of the insulin receptor cause the insulin resistance in this patient. The EBV lymphocyte can be a good system to detect genetically determined abnormalities in the insulin receptor. PMID:2180980

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

    PubMed

    Kaliman, P; Baron, V; Alengrin, F; Takata, Y; Webster, N J; Olefsky, J M; Van Obberghen, E

    1993-09-21

    During the insulin receptor activation process, ligand binding and autophosphorylation induce two distinct conformational changes in the C-terminal domain of the receptor beta-subunit. To analyze the role of this domain and the involvement of the C-terminal autophosphorylation sites (Tyr1316 and Tyr1322) in receptor activation, we used (i) antipeptide antibodies against three different C-terminal sequences (1270-1281, 1294-1317, and 1309-1326) and (ii) an insulin receptor mutant (Y/F2) where Tyr1316 and Tyr1322 have been replaced by Phe. We show that the autophosphorylation-induced C-terminal conformational change is preserved in the Y/F2 receptor, indicating that this change is not induced by phosphorylation of the C-terminal sites but most likely by phosphorylation of the major sites in the kinase domain (Tyr1146, Tyr1150, and Tyr1151). Binding of antipeptide antibodies to the C-terminal domain modulated (activated or inhibited) both mutant and wild-type receptor-mediated phosphorylation of poly(Glu/Tyr). In contrast to the wild-type receptor, Y/F2 exhibited the same C-terminal configuration before and after insulin binding, evidencing that mutation of Tyr1316 and Tyr1322 introduced conformational changes in the C-terminus. Finally, the mutant receptor was 2-fold more active than the wild-type receptor for poly(Glu/Tyr) phosphorylation. In conclusion, the whole C-terminal region of the insulin receptor beta-subunit is likely to exert a regulatory influence on the receptor kinase activity. Perturbations of the C-terminal region, such as binding of antipeptides or mutation of Tyr1316 and Tyr1322, provoke alterations at the receptor kinase level, leading to activation or inhibition of the enzymic activity. PMID:7690586

  11. Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

    SciTech Connect

    Niessen, Markus . E-mail: markus.niessen@usz.ch; Jaschinski, Frank; Item, Flurin; McNamara, Morgan P.; Spinas, Giatgen A.; Trueb, Thomas

    2007-02-15

    Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the {beta}-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH and PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission.

  12. Insulin receptor binding and protein kinase activity in muscles of trained rats

    SciTech Connect

    Dohm, G.L.; Sinha, M.K.; Caro, J.F.

    1987-02-01

    Exercise has been shown to increase insulin sensitivity, and muscle is quantitatively the most important tissue of insulin action. Since the first step in insulin action is the binding to a membrane receptor, the authors postulated that exercise training would change insulin receptors in muscle and in this study they have investigated this hypothesis. Female rats initially weighing approx. 100 g were trained by treadmill running for 2 h/day, 6 days/wk for 4 wk at 25 m/min (0 grade). Insulin receptors from vastus intermedius muscles were solubilized by homogenizing in a buffer containing 1% Triton X-100 and then partially purified by passing the soluble extract over a wheat germ agglutinin column. The 4 wk training regimen resulted in a 65% increase in citrate synthase activity in red vastus lateralis muscle, indicating an adaptation to exercise ( SVI). Insulin binding by the partially purified receptor preparations was approximately doubled in muscle of trained rats at all insulin concentrations, suggesting an increase in the number of receptors. Training did not alter insulin receptor structure as evidenced by electrophoretic mobility under reducing and nonreducing conditions. Basal insulin receptor protein kinase activity was higher in trained than untrained animals and this was likely due to the greater number of receptors. However, insulin stimulation of the protein kinase activity was depressed by training. These results demonstrate that endurance training does alter receptor number and function in muscle and these changes may be important in increasing insulin sensitivity after exercise training.

  13. Insulin receptor activation in the nucleus accumbens reflects nutritive value of a recently ingested meal.

    PubMed

    Woods, C A; Guttman, Z R; Huang, D; Kolaric, R A; Rabinowitsch, A I; Jones, K T; Cabeza de Vaca, S; Sclafani, A; Carr, K D

    2016-05-15

    With respect to feeding, insulin is typically thought of as a satiety hormone, acting in the hypothalamus to limit ingestive behavior. However, accumulating evidence suggests that insulin also has the ability to alter dopamine release in the striatum and influence food preferences. With increased access to high calorie foods, Western societies have a high prevalence of obesity, accompanied by insulin insensitivity. Little is known about how insulin is trafficked into the brain following food consumption and whether insulin insensitivity in the periphery is mirrored in the central nervous system. We investigated insulin receptor activation in the ventral striatum of rats receiving water or 16% glucose either orally or intragastrically. We also investigated whether glucose-induced insulin receptor activation was altered in food-restricted (FR) or diet-induced obesity (OB) rat models. Lastly, we examined whether insulin plays a significant role in flavor-nutrient preference learning. Glucose intake stimulated a rapid increase in insulin receptor activity in the ventral striatum of FR and ad libitum (AL) fed rats, but not OB rats. Similarly, both AL and FR, but not OB rats demonstrated significant flavor-nutrient preferences. However AL rats receiving brief inhibition of insulin activity during conditioning failed to acquire a significant flavor-nutrient preference. These findings suggest that impaired insulin receptor activation in the ventral striatum may result in inaccurate valuation of nutritive foods, which could lead to overconsumption of food or the selection of foods that don't accurately meet the body's current physiological needs. PMID:26988281

  14. Metabolic, anabolic, and mitogenic insulin responses: A tissue-specific perspective for insulin receptor activators

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insulin acts as the major regulator of the fasting-to-fed metabolic transition by altering substrate metabolism, promoting energy storage, and helping activate protein synthesis. In addition to its glucoregulatory and other metabolic properties, insulin can also act as a growth factor. The metabolic...

  15. Interaction of the C-terminal acidic domain of the insulin receptor with histone modulates the receptor kinase activity.

    PubMed

    Baron, V; Kaliman, P; Alengrin, F; Van Obberghen, E

    1995-04-01

    In this study, we investigated the role of the insulin receptor domain 1270-1280, an acid-rich sequence located in the receptor C-terminus. Antipeptide IgG raised against this sequence were obtained and used to analyze their effect on receptor function. Antipeptide IgG inhibited receptor autophosphorylation at Tyr1146, Tyr1150 and Tyr1151. These sites are known to be key modulators of the receptor activity. Autophosphorylation at other sites may also have been inhibited. The antipeptide antibody decreased the receptor kinase activity measured with poly(Glu80Tyr20) and a synthetic peptide corresponding to the proreceptor sequence 1142-1158. We provide evidence that the effect of the antibody on substrate phosphorylation may result from the control of the phosphorylation level of the receptor. Concerning the action of the antipeptide IgG on the receptor kinase activity, histone did not behave similarly to poly(Glu80Tyr20). The antibody recognizing sequence 1270-1280 competed with histone for an overlapping binding site. Histone also modulated insulin receptor autophosphorylation, supporting the idea that interference with domain 1270-1280 alters the receptor kinase. Our data suggest that the acidic region including residues 1270-1280 of the insulin receptor C-terminus is involved in the following events: (a) receptor binding with histone, an exogenous substrate of the receptor kinase, and (b) the regulation of receptor autophosphorylation and kinase activity. Based on these observations, we would like to propose that this insulin receptor domain could interact with cellular proteins modulating the receptor kinase. PMID:7744039

  16. Structural insights into ligand-induced activation of the insulin receptor

    SciTech Connect

    Ward, C.; Lawrence, M.; Streltsov, V.; Garrett, T.; McKern, N.; Lou, M.-Z.; Lovrecz, G.; Adams, T.

    2008-04-29

    The current model for insulin binding to the insulin receptor proposes that there are two binding sites, referred to as sites 1 and 2, on each monomer in the receptor homodimer and two binding surfaces on insulin, one involving residues predominantly from the dimerization face of insulin (the classical binding surface) and the other residues from the hexamerization face. High-affinity binding involves one insulin molecule using its two surfaces to make bridging contacts with site 1 from one receptor monomer and site 2 from the other. Whilst the receptor dimer has two identical site 1-site 2 pairs, insulin molecules cannot bridge both pairs simultaneously. Our structures of the insulin receptor (IR) ectodomain dimer and the L1-CR-L2 fragments of IR and insulin-like growth factor receptor (IGF-1R) explain many of the features of ligand-receptor binding and allow the two binding sites on the receptor to be described. The IR dimer has an unexpected folded-over conformation which places the C-terminal surface of the first fibronectin-III domain in close juxtaposition to the known L1 domain ligand-binding surface suggesting that the C-terminal surface of FnIII-1 is the second binding site involved in high-affinity binding. This is very different from previous models based on three-dimensional reconstruction from scanning transmission electron micrographs. Our single-molecule images indicate that IGF-1R has a morphology similar to that of IR. In addition, the structures of the first three domains (L1-CR-L2) of the IR and IGF-1R show that there are major differences in the two regions governing ligand specificity. The implications of these findings for ligand-induced receptor activation will be discussed. This review summarizes the key findings regarding the discovery and characterization of the insulin receptor, the identification and arrangement of its structural domains in the sequence and the key features associated with ligand binding. The remainder of the review

  17. Novel method demonstrates differential ligand activation and phosphatase-mediated deactivation of insulin receptor tyrosine-specific phosphorylation.

    PubMed

    Cieniewicz, Anne M; Cooper, Philip R; McGehee, Jennifer; Lingham, Russell B; Kihm, Anthony J

    2016-08-01

    Insulin receptor signaling is a complex cascade leading to a multitude of intracellular functional responses. Three natural ligands, insulin, IGF1 and IGF2, are each capable of binding with different affinities to the insulin receptor, and result in variable biological responses. However, it is likely these affinity differences alone cannot completely explain the myriad of diverse cellular outcomes. Ligand binding initiates activation of a signaling cascade resulting in phosphorylation of the IR itself and other intracellular proteins. The direct catalytic activity along with the temporally coordinated assembly of signaling proteins is critical for insulin receptor signaling. We hypothesized that determining differential phosphorylation among individual tyrosine sites activated by ligand binding or dephosphorylation by phosphatases could provide valuable insight into insulin receptor signaling. Here, we present a sensitive, novel immunoassay adapted from Meso Scale Discovery technology to quantitatively measure changes in site-specific phosphorylation levels on endogenous insulin receptors from HuH7 cells. We identified insulin receptor phosphorylation patterns generated upon differential ligand activation and phosphatase-mediated deactivation. The data demonstrate that insulin, IGF1 and IGF2 elicit different insulin receptor phosphorylation kinetics and potencies that translate to downstream signaling. Furthermore, we show that insulin receptor deactivation, regulated by tyrosine phosphatases, occurs distinctively across specific tyrosine residues. In summary, we present a novel, quantitative and high-throughput assay that has uncovered differential ligand activation and site-specific deactivation of the insulin receptor. These results may help elucidate some of the insulin signaling mechanisms, discriminate ligand activity and contribute to a better understanding of insulin receptor signaling. We propose this methodology as a powerful approach to characterize

  18. Insulin binding and receptor tyrosine kinase activity in skeletal muscle of carnivorous and omnivorous fish.

    PubMed

    Párrizas, M; Planas, J; Plisetskaya, E M; Gutiérrez, J

    1994-06-01

    We characterized the insulin receptors in skeletal muscle from several fish species with different nutritional preferences: brown trout (Salmo trutta fario), gilthead sea bream (Sparus aurata), tilapia (Tilapia mossambica), and carp (Cyprinus carpio), semipurified by affinity chromatography (wheat germ agglutinin-agarose). Total specific binding and number of receptors per unit weight of piscine white skeletal muscle were lower than those values found in mammalian skeletal muscle. The same parameters in carp muscle receptor preparations were severalfold higher than in trout muscle (binding capacity 440 +/- 47 fmol/mg glycoprotein in carp and 82 +/- 23 fmol/mg glycoprotein in trout). Piscine insulin receptors phosphorylated exogenous substrate poly(Glu,Tyr) but less so than mammalian receptors. Tyrosine kinase activity of receptors, calculated as percent of 32P incorporated into substrate in the presence of insulin compared with basal incorporation, was also highest in carp (210 +/- 4%) and lowest in trout (150 +/- 2%). In both trout and carp deprived of food for 15 days, specific binding of insulin decreased. Nevertheless, differences between the two species were retained. Our results demonstrate that particular properties of insulin receptors in fish skeletal muscle may be related to nutritional preferences. This finding coincides with the phenomenon of differential glucose tolerance in fish: carnivorous fish, such as trout, are less tolerant, whereas omnivorous fish, such as carp, readily utilize a carbohydrate-rich diet. PMID:8024051

  19. Insulin Excites Anorexigenic Proopiomelanocortin Neurons via Activation of Canonical Transient Receptor Potential Channels

    PubMed Central

    Qiu, Jian; Zhang, Chunguang; Borgquist, Amanda; Nestor, Casey C; Smith, Arik W.; Bosch, Martha A.; Ku, Stephen; Wagner, Edward J.; Rønnekleiv, Oline K.; Kelly, Martin J.

    2014-01-01

    SUMMARY Proopiomelanocortin (POMC) neurons within the hypothalamic arcuate nucleus are vital anorexigenic neurons. Although both the leptin receptor and insulin receptor are coupled to activation of phosphatidylinositide3-kinase (PI3K) in POMC neurons, they are thought to have disparate actions on POMC excitability. Using whole-cell recording and selective pharmacological tools, we have found that similar to leptin, purified insulin depolarized POMC, and adjacent kisspeptin neurons via activation of TRPC5 channels, which are highly expressed in these neurons. In contrast, insulin hyperpolarized and inhibited NPY/AgRP neurons via activation of KATP channels. Moreover, Zn2+, which is found in insulin formulations at nanomolar concentrations, inhibited POMC neurons via activation of KATP channels. Finally as predicted, insulin given intracerebroventrically robustly inhibited food intake and activated c-fos expression in arcuate POMC neurons. Our results show that purified insulin excites POMC neurons in the arcuate nucleus, which we propose is a major mechanism by which insulin regulates energy homeostasis. PMID:24703699

  20. Phosphorylation of insulin receptor substrate-1 serine 307 correlates with JNK activity in atrophic skeletal muscle

    NASA Technical Reports Server (NTRS)

    Hilder, Thomas L.; Tou, Janet C L.; Grindeland, Richard E.; Wade, Charles E.; Graves, Lee M.

    2003-01-01

    c-Jun NH(2)-terminal kinase (JNK) has been shown to negatively regulate insulin signaling through serine phosphorylation of residue 307 within the insulin receptor substrate-1 (IRS-1) in adipose and liver tissue. Using a rat hindlimb suspension model for muscle disuse atrophy, we found that JNK activity was significantly elevated in atrophic soleus muscle and that IRS-1 was phosphorylated on Ser(307) prior to the degradation of the IRS-1 protein. Moreover, we observed a corresponding reduction in Akt activity, providing biochemical evidence for the development of insulin resistance in atrophic skeletal muscle.

  1. Insulin glulisine: insulin receptor signaling characteristics in vivo.

    PubMed

    Hennige, Anita M; Lehmann, Rainer; Weigert, Cora; Moeschel, Klaus; Schäuble, Myriam; Metzinger, Elisabeth; Lammers, Reiner; Häring, Hans-Ulrich

    2005-02-01

    In recent years, recombinant DNA technology has been used to design insulin molecules that overcome the limitations of regular insulin in mealtime supplementation. However, safety issues have been raised with these alternatives, as the alteration of the three-dimensional structure may alter the interaction with the insulin and/or IGF-I receptors and therefore lead to the activation of alternate metabolic as well as mitogenic signaling pathways. It is therefore essential to carefully study acute and long-term effects in a preclinical state, as insulin therapy is meant to be a lifelong treatment. In this study, we determined in vivo the insulin receptor signaling characteristics activated by insulin glulisine (Lys(B3), Glu(B29)) at the level of insulin receptor phosphorylation, insulin receptor substrate phosphorylation, and downstream signaling elements such as phosphatidylinositol (PI) 3-kinase, AKT, and mitogen-activated protein kinase. C57BL/6 mice were injected with insulin glulisine or regular insulin and Western blot analysis was performed for liver and muscle tissue. The extent and time course of insulin receptor phosphorylation and activation of downstream signaling elements after insulin glulisine treatment was similar to that of human regular insulin in vivo. Moreover, insulin signaling in hypothalamic tissue determined by PI 3-kinase activity was comparable. Therefore, insulin glulisine may be a useful tool for diabetes treatment. PMID:15677493

  2. In situ autoradiography and ligand-dependent tyrosine kinase activity reveal insulin receptors and insulin-like growth factor I receptors in prepancreatic chicken embryos.

    PubMed Central

    Girbau, M; Bassas, L; Alemany, J; de Pablo, F

    1989-01-01

    We previously reported specific cross-linking of 125I-labeled insulin and 125I-labeled insulin-like growth factor I (IGF-I) to the alpha subunit of their respective receptors in chicken embryos of 20 somites and older. To achieve adequate sensitivity and localize spatially the receptors in younger embryos, we adapted an autoradiographic technique using whole-mounted chicken blastoderms. Insulin receptors and IGF-I receptors were expressed and could be localized as early as gastrulation, before the first somite is formed. Relative density was analyzed by a computer-assisted image system, revealing overall slightly higher binding of IGF-I than of insulin. Structures rich in both types of receptors were predominantly of ectodermal origin: Hensen's node in gastrulating embryos and neural folds, neural tube and optic vesicles during neurulation. The signal transduction capability of the receptors in early organogenesis was assessed by their ability to phosphorylate the exogenous substrate poly(Glu80Tyr20). Ligand-dependent tyrosine phosphorylation was demonstrable with both insulin and IGF-I in glycoprotein-enriched preparations from embryos at days 2 through 6 of embryogenesis. There was a developmentally regulated change in ligand-dependent tyrosine kinase activity, with a sharp increase from day 2 to day 4, in contrast with a small increase in the ligand binding. Binding of 125I-labeled IGF-I was, with the solubilized receptors, severalfold higher than binding of 125I-labeled insulin. However, the insulin-dependent phosphorylation was as high as the IGF-I-dependent phosphorylation at each developmental stage. Images PMID:2548191

  3. The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

    NASA Astrophysics Data System (ADS)

    Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

    2015-03-01

    The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

  4. Insulin receptor in Drosophila melanogaster

    SciTech Connect

    Petruzzelli, L.; Herrera, R.; Rosen, O.

    1986-05-01

    A specific, high affinity insulin receptor is present in both adult Drosophila and in Drosophila embryos. Wheat germ lectin-enriched extracts of detergent-solubilized membranes from embryos and adults bind insulin with a K/sub d/ of 15 nM. Binding is specific for insulin; micromolar concentrations of proinsulin, IGFI, and IGFII are required to displace bound /sup 125/I-insulin. Insulin-dependent protein tyrosine kinase activity appears during embryogenesis. It is evident between 6 and 12 hours of development, peaks between 12 and 18 hours and falls in the adult. During 0-6 hours of embryogenesis, and in the adult, a specific protein band (Mr = 135,000) is crosslinked to /sup 125/I-insulin. During 6-12 and 12-18 hours of embryogenesis stages in which insulin-dependent protein tyrosine kinase is high, an additional band (Mr = 100,000) becomes crosslinked to /sup 125/I-insulin. Isolation and DNA sequence analysis of genomic clones encoding the Drosophila insulin receptor will be presented as will the characterization of insulin receptor mRNA's during development.

  5. Tyrosine kinase activity of a chimeric insulin-like-growth-factor-1 receptor containing the insulin receptor C-terminal domain. Comparison with the tyrosine kinase activities of the insulin and insulin-like-growth-factor-1 receptors using a cell-free system.

    PubMed

    Mothe, I; Tartare, S; Kowalski-Chauvel, A; Kaliman, P; Van Obberghen, E; Ballotti, R

    1995-03-15

    In a previous study, we showed that a chimeric insulin-like-growth-factor-1 (IGF-1) receptor, with the beta subunit C-terminal part of the insulin receptor was more efficient in stimulating glycogen synthesis and p44mapk activity compared to the wild-type IFG-1 receptor [Tartare, S., Mothe, I., Kowalski-Chauvel, A., Breittmayer, J.-P., Ballotti, R. & Van Obberghen, E. (1994) J. Biol. Chem. 269, 11449-11455]. These data indicate that the receptor C-terminal domain plays an important role in the transmission of biological effects. To understand the molecular basis of the differences in receptor specificity, we studied the characteristics of insulin, IGF-1 and chimeric receptor tyrosine kinase activities in a cell-free system. We found that, compared to wild-type insulin and IGF-1 receptors, the chimeric receptor showed a decrease in (a) autophosphorylation, (b) tyrosine kinase activity towards insulin receptor substrate-1 and the insulin receptor-(1142-1158)-peptide, and (c) the ability to activate phosphatidylinositol 3-kinase. However, for all the effects measured in a cell-free system, the chimeric receptor displayed an increased response to IGF-1 compared to the native IGF-1 receptor. Concerning the cation dependence of the tyrosine kinase activity, we showed that, at 10 mM Mg2+, the ligand-stimulated phosphorylation of poly(Glu80Tyr20) by both insulin receptor and chimeric receptor was increased by Mn2+. Conversely at 50 mM Mg2+, the chimeric receptor behaved like the IGF-1 receptor, since the presence of Mn2+ decreased the stimulatory effect of IGF-1 on their kinase activity. Furthermore, the Km of the chimeric receptor for ATP was increased compared to the wild-type receptors. These data demonstrate that the replacement of the C-terminal tail of the IGF-1 receptor by that of the insulin receptor has changed the receptor characteristics studied in a cell-free system. Our findings indicate that the C-terminal domain of the insulin receptor beta subunit plays a

  6. GLUT 4 and insulin receptor binding and kinase activity in trained human muscle.

    PubMed Central

    Dela, F; Handberg, A; Mikines, K J; Vinten, J; Galbo, H

    1993-01-01

    1. Physical training enhances sensitivity and responsiveness of insulin-mediated glucose uptake in human muscle. This study examines if this effect of physical training is due to increased insulin receptor function or increased total concentration of insulin-recruitable glucose transporter protein (GLUT 4). 2. Seven healthy young subjects carried out single leg bicycle training for 10 weeks at 70% of one leg maximal oxygen uptake (VO2,max). Subsequently biopsies were taken from the vastus lateralis muscle of both legs. 3. Single leg VO2,max increased for the trained leg (46 +/- 3 to 52 +/- 2 ml min-1 kg-1 (means +/- S.E.M., P < 0.05), and cytochrome c oxidase activity was higher in this compared to the untrained leg (2.0 +/- 0.1 vs. 1.4 +/- 0.1 nmol s-1 (mg muscle)-1, P < 0.05). Insulin binding as well as basal- and insulin-stimulated receptor kinase activity did not differ between trained and untrained muscle. The concentration of GLUT 4 protein was higher in the former (14.9 +/- 1.9 vs. 11.6 +/- 1.0 arbitrary units (micrograms protein)-1 in crude membranes, P < 0.05). The training-induced increase in GLUT 4 (26 +/- 11%) matched a previously reported increase in maximum insulin-stimulated leg glucose uptake (25 +/- 7%) in the same subjects, and individual values of the two variables correlated (correlation coefficient (r) = 0.84, P < 0.05). 4. In conclusion, in human muscle training induces a local contraction-dependent increase in GLUT 4 protein, which enhances the effect of insulin on glucose uptake. On the other hand, insulin receptor function in muscle is unlikely to be affected by training. PMID:8271219

  7. Insulin receptor substrate-3, interacting with Bcl-3, enhances p50 NF-{kappa}B activity

    SciTech Connect

    Kabuta, Tomohiro; Hakuno, Fumihiko; Cho, Yoshitake; Yamanaka, Daisuke; Chida, Kazuhiro; Asano, Tomoichiro; Wada, Keiji; Takahashi, Shin-Ichiro

    2010-04-09

    The insulin receptor substrate (IRS) proteins are major substrates of both insulin receptor and insulin-like growth factor (IGF)-I receptor tyrosine kinases. Previously, we reported that IRS-3 is localized to both cytosol and nucleus, and possesses transcriptional activity. In the present study, we identified Bcl-3 as a novel binding protein to IRS-3. Bcl-3 is a nuclear protein, which forms a complex with the homodimer of p50 NF-{kappa}B, leading to enhancement of transcription through p50 NF-{kappa}B. We found that Bcl-3 interacts with the pleckstrin homology domain and the phosphotyrosine binding domain of IRS-3, and that IRS-3 interacts with the ankyrin repeat domain of Bcl-3. In addition, IRS-3 augmented the binding activity of p50 to the NF-{kappa}B DNA binding site, as well as the tumor necrosis factor (TNF)-{alpha}-induced transcriptional activity of NF-{kappa}B. Lastly, IRS-3 enhanced NF-{kappa}B-dependent anti-apoptotic gene induction and consequently inhibited TNF-{alpha}-induced cell death. This series of results proposes a novel function for IRS-3 as a transcriptional regulator in TNF-{alpha} signaling, distinct from its function as a substrate of insulin/IGF receptor kinases.

  8. Characterization of the chicken muscle insulin receptor

    SciTech Connect

    Adamo, M.; Simon, J.; Rosebrough, R.W.; McMurtry, J.P.; Steele, N.C.; LeRoith, D.

    1987-12-01

    Insulin receptors are present in chicken skeletal muscle. Crude membrane preparations demonstrated specific /sup 125/I-insulin binding. The nonspecific binding was high (36-55% of total binding) and slightly lower affinity receptors were found than are typically observed for crude membrane insulin binding in other chicken tissues. Affinity crosslinking of /sup 125/I-insulin to crude membranes revealed insulin receptor alpha-subunits of Mr 128K, intermediate between those of liver (134K) and brain (124K). When solubilized and partially purified on wheat germ agglutinin (WGA) affinity columns, chicken muscle insulin receptors exhibited typical high affinity binding, with approximately 10(-10) M unlabeled insulin producing 50% inhibition of the specific /sup 125/I-insulin binding. WGA purified chicken muscle insulin receptors also exhibited insulin-stimulated autophosphorylation of the beta-subunit, which appeared as phosphorylated bands of 92- and 81K. Both bands were immunoprecipitated by anti-receptor antiserum (B10). WGA purified membranes also demonstrated dose-dependent insulin-stimulated phosphorylation of the exogenous substrate poly(Glu,Tyr)4:1. However, unlike chicken liver, chicken muscle insulin receptor number and tyrosine kinase activity were unaltered by 48 hr of fasting or 48 hr of fasting and 24 hr of refeeding. Thus, despite the presence of insulin receptors in chicken muscle showing normal coupling to receptor tyrosine kinase activity, nutritional alterations modulate these parameters in a tissue-specific manner in chickens.

  9. Peroxisome Proliferator-Activated Receptor Agonist Treatment of Alcohol-Induced Hepatic Insulin Resistance

    PubMed Central

    de la Monte, Suzanne M.; Pang, Maoyin; Chaudhry, Rajeeve; Duan, Kevin; Longato, Lisa; Carter, Jade; Ouh, Jiyun; Wands, Jack R.

    2011-01-01

    Chronic ethanol exposure impairs insulin signaling in the liver. Peroxisome-proliferator activated receptor (PPAR) agonists function as insulin sensitizers and are used to treat type 2 diabetes mellitus. We examined the therapeutic effectiveness of PPAR agonists in reducing alcoholic hepatitis and hepatic insulin resistance in a model of chronic ethanol feeding. Adult male Long Evans rats were pair fed with isocaloric liquid diets containing 0% (control) or 37% ethanol (caloric content; 9.2% v/v) for 8 weeks. After 3 weeks on the diets, the rats were treated with vehicle, or a PPAR-α, PPAR-δ, or PPAR-γ agonist twice weekly by i.p. injection. Livers were harvested for histopathological, gene expression (RT-PCR), protein (Western and ELISA), and receptor binding studies. Ethanol-fed rats developed steatohepatitis with disordered hepatic chord architecture, increased hepatocellular apoptosis, reduced binding to the insulin, IGF-1, and IGF-2 receptors, and decreased expression of glyceraldehyde-3-phosphate dehydrogenase and aspartyl-(asparaginyl)-β-hydroxylase (mediates remodeling), which are regulated by insulin/IGF signaling. PPAR-α, PPAR-δ, or PPAR-γ agonist treatments reduced the severity of ethanol-mediated liver injury, including hepatic architectural disarray and steatosis. In addition, PPAR-δ and PPAR-γ agonists reduced insulin/IGF resistance and increased insulin/IGF-responsive gene expression. In conclusion, PPAR agonists may help reduce the severity of chronic ethanol-induced liver injury and insulin/IGF resistance, even in the context of continued high-level ethanol consumption. PMID:21426453

  10. mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR.

    PubMed

    Yin, Yancun; Hua, Hui; Li, Minjing; Liu, Shu; Kong, Qingbin; Shao, Ting; Wang, Jiao; Luo, Yuanming; Wang, Qian; Luo, Ting; Jiang, Yangfu

    2016-01-01

    Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor(+/+) MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor(-/-) MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation. PMID:26584640

  11. Defective insulin secretion by chronic glucagon receptor activation in glucose intolerant mice.

    PubMed

    Ahlkvist, Linda; Omar, Bilal; Valeur, Anders; Fosgerau, Keld; Ahrén, Bo

    2016-03-01

    Stimulation of insulin secretion by short-term glucagon receptor (GCGR) activation is well characterized; however, the effect of long-term GCGR activation on β-cell function is not known, but of interest, since hyperglucagonemia occurs early during development of type 2 diabetes. Therefore, we examined whether chronic GCGR activation affects insulin secretion in glucose intolerant mice. To induce chronic GCGR activation, high-fat diet fed mice were continuously (2 weeks) infused with the stable glucagon analog ZP-GA-1 and challenged with oral glucose and intravenous glucose±glucagon-like peptide 1 (GLP1). Islets were isolated to evaluate the insulin secretory response to glucose±GLP1 and their pancreas were collected for immunohistochemical analysis. Two weeks of ZP-GA-1 infusion reduced insulin secretion both after oral and intravenous glucose challenges in vivo and in isolated islets. These inhibitory effects were corrected for by GLP1. Also, we observed increased β-cell area and islet size. We conclude that induction of chronic ZP-GA-1 levels in glucose intolerant mice markedly reduces insulin secretion, and thus, we suggest that chronic activation of the GCGR may contribute to the failure of β-cell function during development of type 2 diabetes. PMID:26698567

  12. Separate domains of the insulin receptor contain sites of autophosphorylation and tyrosine kinase activity

    SciTech Connect

    Goren, H.J.; White, M.F.; Khan, C.R.

    1987-04-21

    The authors have studied the structure and function of the solubilized insulin receptor before and after partial proteolytic digestion to define domains in the ..beta..-subunit that undergo autophosphorylation and contain the tyrosine kinase activity. Wheat germ agglutinin purified insulin receptor from Fao cells was digested briefly at 22/sup 0/C with low concentrations of trypsin, staphylococcal V8 protease, or elastase. Autophosphorylation of the ..beta..-subunit was carried out before and after digestion, and the (/sup 32/P)phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, detected by autoradiography, and analyzed by tryptic peptide mapping by use of reverse-phase high-performance liquid chromatography. The 85-kDa fragment was not immunoprecipitated by an antibody directed against the C-terminal domain of the ..beta..-subunit (..cap alpha..Pep-1), indicating that this region of the receptor was lost. The 85-kDa fragment contained about half of the (/sup 32/P)phosphate originally found in the ..beta..-subunit, and tryptic peptide mapping showed that two major tryptic phosphopeptides (previously called pY2 and pY3) were removed. Three other tryptic phosphopeptides (pY1, pY1a, and pY4) were found in the 85- and 70-kDa fragments. To determined the structural requirements for kinase activity, the insulin receptor was subjected to tryptic digestion for 30 s-30 min, such that the receptor was composed exclusively of 85- and 70-kDa fragments of the ..beta..-subunit. The 85-kDa fragment exhibited autophosphorylation at pY1, pY1a, and pY4. Both the 85- and 70-kDa fragments phosphorylated tyrosine residues in a synthetic decapeptide that has the sequence of the C-terminal domain of the ..beta..-subunit of human insulin rare in the receptor.

  13. Insulin Receptor-Overexpressing β-Cells Ameliorate Hyperglycemia in Diabetic Rats through Wnt Signaling Activation

    PubMed Central

    Lee, Moon-Kyu

    2013-01-01

    To investigate the therapeutic efficacy and mechanism of β-cells with insulin receptor (IR) overexpression on diabetes mellitus (DM), rat insulinoma (INS-1) cells were engineered to stably express human insulin receptor (INS-IR cells), and subsequently transplanted into streptozotocin- induced diabetic rats. Compared with INS-1 cells, INS-IR cells showed improved β-cell function, including the increase in glucose utilization, calcium mobilization, and insulin secretion, and exhibited a higher rate of cell proliferation, and maintained lower levels of blood glucose in diabetic rats. These results were attributed to the increase of β-catenin/PPARγ complex bindings to peroxisome proliferator response elements in rat glucokinase (GK) promoter and the prolongation of S-phase of cell cycle by cyclin D1. These events resulted from more rapid and higher phosphorylation levels of insulin-signaling intermediates, including insulin receptor substrate (IRS)-1/IRS-2/phosphotylinositol 3 kinase/v-akt murine thymoma viral oncogene homolog (AKT) 1, and the consequent enhancement of β-catenin nuclear translocation and Wnt responsive genes including GK and cyclin D1. Indeed, the higher functionality and proliferation shown in INS-IR cells were offset by β-catenin, cyclin D1, GK, AKT1, and IRS-2 gene depletion. In addition, the promotion of cell proliferation and insulin secretion by Wnt signaling activation was shown by 100 nM insulin treatment, and to a similar degree, was shown in INS-IR cells. In this regard, this study suggests that transferring INS-IR cells into diabetic animals is an effective and feasible DM treatment. Accordingly, the method might be a promising alternative strategy for treatment of DM given the adverse effects of insulin among patients, including the increased risk of modest weight gain and hypoglycemia. Additionally, this study demonstrates that the novel mechanism of cross-talk between insulin and Wnt signaling plays a primary role in the higher

  14. Autoantibodies to Insulin Receptor Spontaneously Develop as Anti-Idiotypes in Mice Immunized with Insulin

    NASA Astrophysics Data System (ADS)

    Shechter, Yoram; Maron, Ruth; Elias, Dana; Cohen, Irun R.

    1982-04-01

    Mice immunized with insulin developed antibodies to both insulin and the insulin receptor. The antibodies to insulin receptor displaced labeled insulin from insulin receptors and mimicked the actions of insulin in stimulating the oxidation of glucose and its incorporation into lipids, and in inhibiting lipolysis. The antibodies to insulin receptor could be blocked by or bound to the antibodies to insulin, and therefore were identified as anti-idiotypes. Thus, immunization against a hormone may activate spontaneously an idiotype-anti-idiotype network resulting in antibodies to the hormone receptor.

  15. Activated central galanin type 1 receptor alleviated insulin resistance in diabetic rat muscle.

    PubMed

    Bu, Le; Chang, Xusheng; Cheng, Xiaoyun; Yao, Qian; Su, Bin; Sheng, Chunjun; Qu, Shen

    2016-10-01

    Evidence indicates that central galanin is involved in regulation of insulin resistance in animals. This study investigates whether type 1 galanin receptor (GAL1) in the brain mediates the ameliorative effect of galanin on insulin resistance in skeletal muscles of type 2 diabetic rats. Rats were intracerebroventricularly (i.c.v.) injected with galanin(1-13)-bradykinin(2-9) amide (M617), a GAL1 agonist, and/or Akti-1/2, an Akt inhibitor, via caudal veins once per day for 10 days. Insulin resistance in muscle tissues was evaluated by glucose tolerance and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) tests, peroxisome proliferator-activated receptor-γ (PPARγ), glucose transporter 4 (GLUT4) mRNA expression levels, Akt phosphorylation, and GLUT4 and vesicle-associated membrane protein 2 (VAMP2) concentration at plasma membranes in muscle cells. The results show that i.c.v. treatment with M617 increased glucose tolerance, 2-NBDG uptake, PPARγ levels, Akt phosphorylation, GLUT4 protein, and GLUT4 mRNA expression levels as well as GLUT4 and VAMP2 concentration at plasma membranes. All increases may be blocked by pretreatment with Akti-1/2. These results suggest that activated central GAL1 may trigger the Akt signaling pathway to alleviate insulin resistance in muscle cells. Therefore, the impact of galanin on insulin resistance is mediated mainly by GAL1 in the brain, and the GAL1 agonist may be taken as a potential antidiabetic agent for treatment of type 2 diabetes mellitus. © 2016 Wiley Periodicals, Inc. PMID:27410235

  16. Insulin receptor binding motif tagged with IgG4 Fc (Yiminsu) works as an insulin sensitizer to activate Akt signaling in hepatocytes.

    PubMed

    Wang, J; Zou, T; Yang, H X; Gong, Y Z; Xie, X J; Liu, H Y; Liao, D F

    2015-01-01

    Insulin resistance is a key feature of obesity and type 2 diabetes mellitus (T2DM). Interaction of insulin with the insulin receptor (IR) leads to both its auto-phosphorylation and phosphorylation of tyrosine residues on the IR substrate (IRS) proteins, initiating the activation of intracellular signaling cascades. The metabolic effects of IRS are known to be mediated through pathways involving phosphatidyl-inositol 3-kinase (PI-3K), which result in the activation of Akt signaling. The C-terminal region of the IR ectodomain is required to facilitate the conformational changes that are required for high-affinity binding to insulin. Furthermore, the CH2 and CH3 domains in the Fc fragments of immunoglobulins are responsible for their binding to the Fc receptor, which triggers transcytosis. In this study, we created a fusion peptide of the C-terminal end of the human IR ectodomain with the IgG4 Fc fragment, including an intervening polyG fragment to ensure enough space for insulin binding. We named this new peptide "Yiminsu", meaning an insulin sensitizer. The results of our analyses show that Yiminsu significantly facilitates insulin signaling via the activation of Akt in hepatocytes in a dose- and time-dependent manner. Further studies are required to determine whether Yiminsu can act as an insulin sensitizer. PMID:26345813

  17. Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor.

    PubMed

    Kimura, Kumi; Tanida, Mamoru; Nagata, Naoto; Inaba, Yuka; Watanabe, Hitoshi; Nagashimada, Mayumi; Ota, Tsuguhito; Asahara, Shun-ichiro; Kido, Yoshiaki; Matsumoto, Michihiro; Toshinai, Koji; Nakazato, Masamitsu; Shibamoto, Toshishige; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

    2016-03-15

    Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR) on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity. PMID:26947072

  18. Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation.

    PubMed

    Law, Nathan C; Hunzicker-Dunn, Mary E

    2016-02-26

    The ubiquitous phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)-dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, granulosa cells secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser(789). Knockdown of PP1β blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through protein kinase B (AKT) and FOXO1 (forkhead box protein O1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT. PMID:26702053

  19. Canavanine induces insulin release via activation of imidazoline I3 receptors.

    PubMed

    Yang, Ting-Ting; Niu, Ho-Shan; Chen, Li-Jen; Ku, Po-Ming; Lin, Kao-Chang; Cheng, Juei-Tang

    2015-03-01

    The aim of the present study was to identify the effect of canavanine on the imidazoline receptor because canavanine is a guanidinium derivative that has a similar structure to imidazoline receptor ligands. Transfected Chinese hamster ovary-K1 cells expressing imidazoline receptors (nischarin (NISCH)-CHO-K1 cells) were used to elucidate the direct effects of canavanine on imidazoline receptors. In addition, the imidazoline I3 receptor has been implicated in stimulation of insulin secretion from pancreatic β-cells. Wistar rats were used to investigate the effects of canavanine (0.1, 1 and 2.5 mg/kg, i.v.) on insulin secretion. In addition the a specific I3 receptor antagonist KU14R (4 or 8 mg/kg, i.v.) was used to block I3 receptors. Canavanine decreased blood glucose by increasing plasma insulin in rats. In addition, canavanine increased calcium influx into NISCH-CHO-K1 cells in a manner similar to agmatine, the endogenous ligand of imidazoline receptors. Moreover, KU12R dose-dependently attenuated canavanine-induced insulin secretion in HIT-T15 pancreatic β-cells and in the plasma of rats. The data suggest that canavanine is an agonist of I3 receptors both in vivo and in vitro. Thus, canavanine would be a useful tool in imidazoline receptor research. PMID:25482045

  20. Peroxisome proliferator-activated receptor gamma agonists as insulin sensitizers: from the discovery to recent progress.

    PubMed

    Cho, Nobuo; Momose, Yu

    2008-01-01

    An epidemic of metabolic diseases including type 2 diabetes and obesity is undermining the health of people living in industrialized societies. There is an urgent need to develop innovative therapeutics. The peroxisome proliferator-activated receptor gamma (PPARgamma) is one of the ligand-activated transcription factors in the nuclear hormone receptor superfamily and a pivotal regulator of glucose and lipid homeostasis. The discovery of PPARgamma as a target of multimodal insulin sensitizers, represented by thiazolidinediones (TZDs), has attracted remarkable scientific interest and had a great impact on the pharmaceutical industry. With the clinical success of the PPARgamma agonists, pioglitazone (Actos) and rosiglitazone (Avandia), development of novel and potent insulin-sensitizing agents with diverse clinical profiles has been accelerated. Currently, a number of PPARgamma agonists from different chemical classes and with varying pharmacological profiles are being developed. Despite quite a few obstacles to the development of PPAR-related drugs, PPARgamma-targeted agents still hold promise. There are new concepts and encouraging evidence emerging that suggest this class can yield improved anti-diabetic agents. This review covers the discovery of TZDs, provides an overview of PPARgamma including the significance of PPARgamma as a drug target, describes the current status of a wide variety of novel PPARgamma ligands including PPAR dual and pan agonists and selective PPARgamma modulators (SPPARgammaMs), and highlights new approaches for identifying agents targeting PPARgamma in the treatment of type 2 diabetes. PMID:19075761

  1. Human biliverdin reductase: a member of the insulin receptor substrate family with serine/threonine/tyrosine kinase activity.

    PubMed

    Lerner-Marmarosh, Nicole; Shen, Jenny; Torno, Michael D; Kravets, Anatoliy; Hu, Zhenbo; Maines, Mahin D

    2005-05-17

    We describe here the tyrosine kinase activity of human biliverdin reductase (BVR) and its potential role in the insulin-signaling pathway. BVR is both a substrate for insulin receptor (IR) tyrosine kinase (IRK) activity and a kinase for serine phosphorylation of IR substrate 1 (IRS-1). Our previous studies have revealed serine/threonine kinase activity of BVR. Y198, in the YMKM motif found in the C-terminal domain of BVR, is shown to be a substrate for insulin-activated IRK. This motif in IRS proteins provides a docking site for proteins that contain a Src homology 2 domain. Additionally, Y228 in the YLSF sequence and Y291 are IRK substrates; the former sequence provides optimum recognition motif in the tyrosine phosphatase, SHP-1, and for SHC (Src homology 2 domain containing transfroming protein 1). BVR autophosphorylates N-terminal tyrosines Y72 and Y83. Serine residues in IRS-1 are targets for BVR phosphorylation, and point mutation of serine residues in the kinase domain of the reductase inhibits phosphotransferase activity. Because tyrosine phosphorylation of IRS-1 activates the insulin signaling pathway and serine phosphorylation of IRS-1 blocks insulin action, our findings that insulin increases BVR tyrosine phosphorylation and that there is an increase in glucose uptake in response to insulin when expression of BVR is "knocked down" by small interfering RNA suggest a potential role for BVR in the insulin signaling pathway. PMID:15870194

  2. Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic {beta} cells

    SciTech Connect

    Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila; Mirshahi, Faridoddin; Grider, John R.; Murthy, Karnam S.; Sanyal, Arun J.

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer G protein coupled receptor TGR5 is expressed in mouse and human islets. Black-Right-Pointing-Pointer TGR5 is coupled to activation of Gs and Ca{sup 2+} release via cAMP/Epac/PLC-{epsilon} pathway. Black-Right-Pointing-Pointer Activation of TGR5 by bile salts and selective ligands causes insulin secretion. Black-Right-Pointing-Pointer TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic {beta} cells. In the present study, we have identified the expression of TGR5 in pancreatic {beta} cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated G{alpha}{sub s} and caused an increase in intracellular cAMP and Ca{sup 2+}. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective G{alpha}{sub s} inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G{sub s}/cAMP/Ca{sup 2+} pathway. 8-pCPT-2 Prime -O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic {beta} cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

  3. Activation of Aryl Hydrocarbon Receptor Dissociates Fatty Liver from Insulin Resistance by Inducing FGF21

    PubMed Central

    Lu, Peipei; Yan, Jiong; Liu, Ke; Garbacz, Wojciech G.; Wang, Pengcheng; Xu, Meishu; Ma, Xiaochao; Xie, Wen

    2015-01-01

    The aryl hydrocarbon receptor (AHR), also known as the dioxin receptor, was originally characterized as a xenobiotic receptor that senses xenotoxicants. Here we investigated the endobiotic and hepatic role of AHR in fatty liver and energy metabolism, and identified the endocrine factor that mediates the metabolic function of AHR. Wild type and liver-specific constitutively activated human AHR transgenic (TG) mice were used to investigate the role of AHR in fatty liver and energy homeostasis. Adenovirus expressing short hairpin RNA targeting the fibroblast growth factor 21 (FGF21) were used to determine the involvement of FGF21 in the metabolic effect of AHR. We showed that despite their severe fatty liver, the TG mice were protected from diet-induced obesity and type 2 diabetes. We identified the endocrine hormone FGF21 as a mediator for the metabolic benefit of AHR and established FGF21 as a direct transcriptional target of AHR. Interestingly, the transactivation of FGF21 by AHR contributed to both hepatic steatosis and systemic insulin hypersensitivity, both of which were largely abolished upon FGF21 knockdown. Conclusions The AHR-FGF21 endocrine signaling pathway establishes AHR as a pivotal environmental modifier that integrates signals from chemical exposure in the regulation of lipid and energy metabolism. PMID:25614121

  4. Acute treatment with XMetA activates hepatic insulin receptors and lowers blood glucose in normal mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It has been proposed that monoclonal antibodies may become therapeutics for metabolic diseases such as diabetes mellitus. We have previously characterized an allosteric monoclonal antibody to the human insulin receptor (IR), XMetA, that activated metabolic signaling leading to enhanced glucose tran...

  5. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by xmeta, an allosteric partial agonist antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

  6. The Novel Functions of High-Molecular-Mass Complexes Containing Insulin Receptor Substrates in Mediation and Modulation of Insulin-Like Activities: Emerging Concept of Diverse Functions by IRS-Associated Proteins

    PubMed Central

    Hakuno, Fumihiko; Fukushima, Toshiaki; Yoneyama, Yosuke; Kamei, Hiroyasu; Ozoe, Atsufumi; Yoshihara, Hidehito; Yamanaka, Daisuke; Shibano, Takashi; Sone-Yonezawa, Meri; Yu, Bu-Chin; Chida, Kazuhiro; Takahashi, Shin-Ichiro

    2015-01-01

    Insulin-like peptides, such as insulin-like growth factors (IGFs) and insulin, induce a variety of bioactivities, such as growth, differentiation, survival, increased anabolism, and decreased catabolism in many cell types and in vivo. In general, IGFs or insulin bind to IGF-I receptor (IGF-IR) or insulin receptor (IR), activating the receptor tyrosine kinase. Insulin receptor substrates (IRSs) are known to be major substrates of receptor kinases, mediating IGF/insulin signals to direct bioactivities. Recently, we discovered that IRSs form high-molecular-mass complexes (referred to here as IRSomes) even without IGF/insulin stimulation. These complexes contain proteins (referred to here as IRSAPs; IRS-associated proteins), which modulate tyrosine phosphorylation of IRSs by receptor kinases, control IRS stability, and determine intracellular localization of IRSs. In addition, in these complexes, we found not only proteins that are involved in RNA metabolism but also RNAs themselves. Thus, IRSAPs possibly contribute to modulation of IGF/insulin bioactivities. Since it is established that disorder of modulation of insulin-like activities causes various age-related diseases including cancer, we could propose that the IRSome is an important target for treatment of these diseases. PMID:26074875

  7. Involvement of PRMT1 in hnRNPQ activation and internalization of insulin receptor

    SciTech Connect

    Iwasaki, Hiroaki

    2008-07-25

    Insulin signaling in skeletal L6 myotubes is known to be affected by arginine methylation catalyzed by protein N-arginine methyltransferase 1 (PRMT1), however, the mechanism by which this occurs has not yet been defined. This study aimed to determine the exact substrate involved in the methylation and regulating insulin signaling in cells. Insulin enhanced arginine methylation of a 66-kDa protein (p66) concomitant with translocation of PRMT1 to the membrane fraction. Peptide mass fingerprinting identified p66 as a heterogeneous nuclear ribonucleoprotein, hnRNPQ that was bound to and methylated by PRMT1. Pharmacological inhibition of methylation (MTA) and small interfering RNA against PRMT1 (PRMT1-siRNA) attenuated insulin-stimulated tyrosine phosphorylation of hnRNPQ and insulin receptor (IR), and the interaction between hnRNPQ and IR. MTA, PRMT1-siRNA, and hnRNPQ-siRNA inhibited internalization of IR in the same manner. These data suggest that the PRMT1-mediated methylation of hnRNPQ is implicated in IR trafficking and insulin signaling in skeletal L6 myotubes.

  8. Inhibition of human insulin gene transcription by peroxisome proliferator-activated receptor γ and thiazolidinedione oral antidiabetic drugs

    PubMed Central

    Schinner, S; Krätzner, R; Baun, D; Dickel, C; Blume, R; Oetjen, E

    2009-01-01

    Background and purpose: The transcription factor peroxisome proliferator-activated receptor γ (PPARγ) is essential for glucose homeostasis. PPARγ ligands reducing insulin levels in vivo are used as drugs to treat type 2 diabetes mellitus. Genes regulated by PPARγ have been found in several tissues including insulin-producing pancreatic islet β-cells. However, the role of PPARγ at the insulin gene was unknown. Therefore, the effect of PPARγ and PPARγ ligands like rosiglitazone on insulin gene transcription was investigated. Experimental approach: Reporter gene assays were used in the β-cell line HIT and in primary mature pancreatic islets of transgenic mice. Mapping studies and internal mutations were carried out to locate PPARγ-responsive promoter regions. Key results: Rosiglitazone caused a PPARγ-dependent inhibition of insulin gene transcription in a β-cell line. This inhibition was concentration-dependent and had an EC50 similar to that for the activation of a reporter gene under the control of multimerized PPAR binding sites. Also in normal primary pancreatic islets of transgenic mice, known to express high levels of PPARγ, rosiglitazone inhibited glucose-stimulated insulin gene transcription. Transactivation and mapping experiments suggest that, in contrast to the rat glucagon gene, the inhibition of the human insulin gene promoter by PPARγ/rosiglitazone does not depend on promoter-bound Pax6 and is attributable to the proximal insulin gene promoter region around the transcription start site from −56 to +18. Conclusions and implications: The human insulin gene represents a novel PPARγ target that may contribute to the action of thiazolidinediones in type 2 diabetes mellitus. PMID:19338578

  9. n-3 Fatty acids preserve insulin sensitivity in vivo in a peroxisome proliferator-activated receptor-alpha-dependent manner.

    PubMed

    Neschen, Susanne; Morino, Katsutaro; Dong, Jianying; Wang-Fischer, Yanlin; Cline, Gary W; Romanelli, Anthony J; Rossbacher, Jörg C; Moore, Irene K; Regittnig, Werner; Munoz, David S; Kim, Jung H; Shulman, Gerald I

    2007-04-01

    Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fat diet-induced insulin resistance through peroxisome proliferator-activated receptor (PPAR)-alpha activation and a subsequent decrease in intracellular lipid abundance. To directly test this hypothesis, we fed PPAR-alpha null and wild-type mice for 2 weeks with isocaloric high-fat diets containing 27% fat from either safflower oil or safflower oil with an 8% fish oil replacement (fish oil diet). In both genotypes the safflower oil diet blunted insulin-mediated suppression of hepatic glucose production (P < 0.02 vs. genotype control) and PEPCK gene expression. Feeding wild-type mice a fish oil diet restored hepatic insulin sensitivity (hepatic glucose production [HGP], P < 0.002 vs. wild-type mice fed safflower oil), whereas in contrast, in PPAR-alpha null mice failed to counteract hepatic insulin resistance (HGP, P = NS vs. PPAR-alpha null safflower oil-fed mice). In PPAR-alpha null mice fed the fish oil diet, safflower oil plus fish oil, hepatic insulin resistance was dissociated from increases in hepatic triacylglycerol and acyl-CoA but accompanied by a more than threefold increase in hepatic diacylglycerol concentration (P < 0.0001 vs. genotype control). These data support the hypothesis that n-3 fatty acids protect from high-fat diet-induced hepatic insulin resistance in a PPAR-alpha-and diacylglycerol-dependent manner. PMID:17251275

  10. The adaptor protein insulin receptor substrate 2 inhibits alternative macrophage activation and allergic lung inflammation.

    PubMed

    Dasgupta, Preeta; Dorsey, Nicolas J; Li, Jiaqi; Qi, Xiulan; Smith, Elizabeth P; Yamaji-Kegan, Kazuyo; Keegan, Achsah D

    2016-01-01

    Insulin receptor substrate 2 (IRS2) is an adaptor protein that becomes tyrosine-phosphorylated in response to the cytokines interleukin-4 (IL-4) and IL-13, which results in activation of the phosphoinositide 3-kinase (PI3K)-Akt pathway. IL-4 and IL-13 contribute to allergic lung inflammation. To examine the role of IRS2 in allergic disease, we evaluated the responses of IRS2-deficient (IRS2(-/-)) mice. Unexpectedly, loss of IRS2 resulted in a substantial increase in the expression of a subset of genes associated with the generation of alternatively activated macrophages (AAMs) in response to IL-4 or IL-13 in vitro. AAMs secrete factors that enhance allergic responses and promote airway remodeling. Moreover, compared to IRS2(+/+) mice, IRS2(+/-) and IRS2(-/-) mice developed enhanced pulmonary inflammation, accumulated eosinophils and AAMs, and exhibited airway and vascular remodeling upon allergen stimulation, responses that partially depended on macrophage-intrinsic IRS2 signaling. Both in unstimulated and IL-4-stimulated macrophages, lack of IRS2 enhanced phosphorylation of Akt and ribosomal S6 protein. Thus, we identified a critical inhibitory loop downstream of IRS2, demonstrating an unanticipated and previously unrecognized role for IRS2 in suppressing allergic lung inflammation and remodeling. PMID:27330190

  11. Biologically active insulin-derived peptides.

    PubMed

    Fawcett, Janet

    2014-06-01

    Insulin has many actions within cells many of which are dependent on the cell type. For example, insulin stimulates glucose uptake in adipose tissue and skeletal muscle but not in liver. In liver glucose influx will increase as insulin stimulates the phosphorylation of glucose and eventual storage in the form of glycogen. Insulin also increases glucose oxidation, decreases glucose production, decreases lipolysis, increases protein synthesis and inhibits protein degradation in addition to others. Many actions have been related to insulin binding to its receptor and subsequent phosphorylation cascades, but insulin action on protein degradation has been shown to be linked to insulin degradation, specifically insulin degradation by the insulin-degrading enzyme (IDE). This activity has been shown to be due to an interaction of IDE with the proteasome, which is responsible for degradation of ubiquitin-tagged proteins. Smaller fragments of insulin that are produced by the action of IDE that do not bind to the insulin receptor show a small effect on protein degradation and a modest effect on mitogenesis. These small fragments do however inhibit lipolysis in a similar manner to insulin. If fragments are larger and can bind to the receptor they have been shown to increase glucose oxidation. Studies show that fragments of the insulin molecule have cellular activity, and that the varied actions of insulin are not completely controlled by insulin binding to the insulin receptor, even though the mechanisms may not be mutually exclusive. PMID:24559166

  12. Peroxisome Proliferator-activated Receptor γ Regulates Genes Involved in Insulin/Insulin-like Growth Factor Signaling and Lipid Metabolism during Adipogenesis through Functionally Distinct Enhancer Classes*

    PubMed Central

    Oger, Frédérik; Dubois-Chevalier, Julie; Gheeraert, Céline; Avner, Stéphane; Durand, Emmanuelle; Froguel, Philippe; Salbert, Gilles; Staels, Bart; Lefebvre, Philippe; Eeckhoute, Jérôme

    2014-01-01

    The nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ is a transcription factor whose expression is induced during adipogenesis and that is required for the acquisition and control of mature adipocyte functions. Indeed, PPARγ induces the expression of genes involved in lipid synthesis and storage through enhancers activated during adipocyte differentiation. Here, we show that PPARγ also binds to enhancers already active in preadipocytes as evidenced by an active chromatin state including lower DNA methylation levels despite higher CpG content. These constitutive enhancers are linked to genes involved in the insulin/insulin-like growth factor signaling pathway that are transcriptionally induced during adipogenesis but to a lower extent than lipid metabolism genes, because of stronger basal expression levels in preadipocytes. This is consistent with the sequential involvement of hormonal sensitivity and lipid handling during adipocyte maturation and correlates with the chromatin structure dynamics at constitutive and activated enhancers. Interestingly, constitutive enhancers are evolutionary conserved and can be activated in other tissues, in contrast to enhancers controlling lipid handling genes whose activation is more restricted to adipocytes. Thus, PPARγ utilizes both broadly active and cell type-specific enhancers to modulate the dynamic range of activation of genes involved in the adipogenic process. PMID:24288131

  13. Isohumulones, bitter acids derived from hops, activate both peroxisome proliferator-activated receptor alpha and gamma and reduce insulin resistance.

    PubMed

    Yajima, Hiroaki; Ikeshima, Emiko; Shiraki, Maho; Kanaya, Tomoka; Fujiwara, Daisuke; Odai, Hideharu; Tsuboyama-Kasaoka, Nobuyo; Ezaki, Osamu; Oikawa, Shinichi; Kondo, Keiji

    2004-08-01

    The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of fibrate drugs and the therapeutic benefits of the thiazolidinedione drugs are due to their activation of PPARalpha and -gamma, respectively. In this study, isohumulones, the bitter compounds derived from hops that are present in beer, were found to activate PPARalpha and -gamma in transient co-transfection studies. Among the three major isohumulone homologs, isohumulone and isocohumulone were found to activate PPARalpha and -gamma. Diabetic KK-Ay mice that were treated with isohumulones (isohumulone and isocohumulone) showed reduced plasma glucose, triglyceride, and free fatty acid levels (65.3, 62.6, and 73.1%, respectively, for isohumulone); similar reductions were found following treatment with the thiazolidinedione drug, pioglitazone. Isohumulone treatment did not result in significant body weight gain, although pioglitazone treatment did increase body weight (10.6% increase versus control group). C57BL/6N mice fed a high fat diet that were treated with isohumulones showed improved glucose tolerance and reduced insulin resistance. Furthermore, these animals showed increased liver fatty acid oxidation and a decrease in size and an increase in apoptosis of their hypertrophic adipocytes. A double-blind, placebo-controlled pilot study for studying the effect of isohumulones on diabetes suggested that isohumulones significantly decreased blood glucose and hemoglobin A1c levels after 8 weeks (by 10.1 and 6.4%, respectively, versus week 0). These results suggest that isohumulones can improve insulin sensitivity in high fat diet-fed mice with insulin resistance and in patients with type 2 diabetes. PMID:15178687

  14. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    SciTech Connect

    Wang, Feng; Yang, Yong

    2014-10-03

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers.

  15. Down-regulation of insulin receptors is related to insulin internalization

    SciTech Connect

    Geiger, D.; Carpentier, J.L.; Gorden, P.; Orci, L. )

    1989-11-01

    In the present study, we have tested the influence of inhibition of endocytosis by hypertonic medium on the regulation of cell surface insulin receptors. We show that active internalization of {sup 125}I-insulin is markedly inhibited by hypertonic media and that, in parallel, cell surface invaginations are significantly diminished. These two events are accompanied by a marked inhibition of cell surface insulin receptor down-regulation. These data provide further strong evidence that receptor-mediated endocytosis is the major mechanism by which insulin receptors are regulated at the surface of target cells.

  16. Insulin and insulin like growth factor II endocytosis and signaling via insulin receptor B

    PubMed Central

    2013-01-01

    Background Insulin and insulin-like growth factors (IGFs) act on tetrameric tyrosine kinase receptors controlling essential functions including growth, metabolism, reproduction and longevity. The insulin receptor (IR) binds insulin and IGFs with different affinities triggering different cell responses. Results We showed that IGF-II induces cell proliferation and gene transcription when IR-B is over-expressed. We combined biotinylated ligands with streptavidin conjugated quantum dots and visible fluorescent proteins to visualize the binding of IGF-II and insulin to IR-B and their ensuing internalization. By confocal microscopy and flow cytometry in living cells, we studied the internalization kinetic through the IR-B of both IGF-II, known to elicit proliferative responses, and insulin, a regulator of metabolism. Conclusions IGF-II promotes a faster internalization of IR-B than insulin. We propose that IGF-II differentially activates mitogenic responses through endosomes, while insulin-activated IR-B remains at the plasma membrane. This fact could facilitate the interaction with key effector molecules involved in metabolism regulation. PMID:23497114

  17. A Novel Allosteric Insulin Receptor-Activating Antibody Reduces Hyperglycemia without Hypoglycemia in Diabetic Cynomolgus Monkeys.

    PubMed

    Bezwada, Padma; Zhao, Jingsong; Der, Ken; Shimizu, Bob; Cao, Liching; Ahene, Ago; Rubin, Paul; Johnson, Kirk

    2016-02-01

    XMetA is a fully human, allosteric monoclonal antibody that binds the insulin receptor with high affinity and mimics the glucoregulatory, but not the mitogenic, actions of insulin. Here we evaluated the efficacy of both single and repeat s.c. administrations of XMetA in reducing hyperglycemia in obese cynomolgus monkeys with naturally developed type 2 diabetes, a model that shares many features of human diabetes. The data show that a single s.c. administration of XMetA at dose levels ranging from 1.5 to 10 mg/kg markedly reduced fasting hyperglycemia, with a peak effect occurring 1 to 2 days after administration, and sustained for up to 1 week. XMetA's effect on hyperglycemia was observed without elevations in serum insulin and was concomitant with reduced serum C-peptide levels, even at the lowest dose. Subchronic effects were evaluated via once weekly s.c. administration of XMetA, 10 mg/kg, for 6 weeks. XMetA treatment resulted in robust weekly decreases in fasting glucose levels averaging approximately 30% throughout the study, along with a significant absolute reduction from the vehicle control baseline of 1.2% in hemoglobin A1c, a marker of long-term glycemic status. XMetA treatment was well tolerated with no injection-site reactions, no body weight gain, and no episodes of clinical hypoglycemia. Thus, XMetA shows acute and subchronic improvements in glycemic control in spontaneously diabetic cynomolgus monkeys with a broad safety margin. This profile supports the development of XMetA as a novel glucose-lowering therapeutic agent for the management of type 2 diabetes. PMID:26578267

  18. Implications of Insulin-like Growth Factor 1 Receptor Activation in Lung Cancer

    PubMed Central

    Nurwidya, Fariz; Andarini, Sita; Takahashi, Fumiyuki; Syahruddin, Elisna; Takahashi, Kazuhisa

    2016-01-01

    Insulin-like growth factor 1 receptor (IGF1R) has been intensively investigated in many preclinical studies using cell lines and animal models, and the results have provided important knowledge to help improve the understanding of cancer biology. IGF1R is highly expressed in patients with lung cancer, and high levels of circulating insulin-like growth factor 1 (IGF1), the main ligand for IGF1R, increases the risk of developing lung malignancy in the future. Several phase I clinical trials have supported the potential use of an IGF1R-targeted strategy for cancer, including lung cancer. However, the negative results from phase III studies need further attention, especially in selecting patients with specific molecular signatures, who will gain benefits from IGF1R inhibitors with minimal side effects. This review will discuss the basic concept of IGF1R in lung cancer biology, such as epithelial-mesenchymal transition (EMT) induction and cancer stem cell (CSC) maintenance, and also the clinical implications of IGF1R for lung cancer patients, such as prognostic value and cancer therapy resistance. PMID:27418865

  19. Candesartan cilexetil prevents diet-induced insulin resistance via peroxisome proliferator-activated receptoractivation in an obese rat model

    PubMed Central

    YAN, WEN-HUA; PAN, CHANG-YU; DOU, JING-TAO; MENG, JUN-HUA; WANG, BAO-AN; MU, YI-MING

    2016-01-01

    Angiotensin II type 1 receptor (AT1R) blockers (ARBs) have been shown to reduce the incidence of type 2 diabetes mellitus; however, the underlying molecular mechanism is unknown. Peroxisome proliferator-activated receptor γ (PPARγ) is the central regulator of insulin and glucose metabolism, which improves insulin sensitivity. Whether candesartan cilexetil, as a prodrug of the AT1R blocker candesartan, has PPARγ-activating properties remains to be elucidated. The aim of the present study was to investigate the effects of oral administration of candesartan cilexetil on glucose tolerance and the actions of PPARγ on liver and adipose tissue in the insulin-resistant obese rat induced by high-fat diet. Animals treated with candesartan cilexetil showed an improved glucose tolerance after oral glucose challenge. Whole-body insulin sensitivity was evaluated using the hyperinsulinemic-euglycemic clamp technique. During high-fat feeding in high-fat diet (HF) rats, the glucose infusion rate (GIR) was 52.3% lower than that in normal chow (NC) rats. However, the GIR was significantly enhanced following candesartan cilexetil treatment. Angiotensin II receptor antagonism also resulted in significant increases in PPARγ protein expression in adipose and liver tissue. These results indicate that PPARγ activation by candesartan cilexetil may provide novel therapeutic options in the treatment of patients with metabolic syndrome. PMID:27347049

  20. Activation of phosphatidylinositol 3-kinase by insulin.

    PubMed Central

    Ruderman, N B; Kapeller, R; White, M F; Cantley, L C

    1990-01-01

    Insulin action appears to require the protein-tyrosine kinase domain of the beta subunit of the insulin receptor. Despite this, the identities and biochemical functions of the cellular targets of this tyrosine kinase are unknown. A phosphatidylinositol 3-kinase (PI 3-kinase) that phosphorylates the D-3 position of the inositol ring associates with several protein-tyrosine kinases. Here we report that PI 3-kinase activity is immunoprecipitated from insulin-stimulated CHO cells by antiphosphotyrosine and anti-insulin receptor antibodies. Insulin as low as 0.3 nM increased immunoprecipitable PI 3-kinase activity within 1 min. Increases in activity were much greater in CHO cells expressing the human insulin receptor (100,000 receptors per cell) than in control CHO cells (2000 receptors per cell). During insulin stimulation, various lipid products of the PI 3-kinase either appeared or increased in quantity in intact cells, suggesting that the appearance of immunoprecipitable PI 3-kinase reflects an increase in its activity in vivo. These results indicate that insulin at physiological concentrations regulates the PI 3-kinase and suggest that this regulation involves a physical association between the insulin receptor and the PI 3-kinase and tyrosyl phosphorylation. Images PMID:2154747

  1. Effect of Insulin Analogues on Insulin/IGF1 Hybrid Receptors: Increased Activation by Glargine but Not by Its Metabolites M1 and M2

    PubMed Central

    Pierre-Eugene, Cécile; Pagesy, Patrick; Nguyen, Tuyet Thu; Neuillé, Marion; Tschank, Georg; Tennagels, Norbert; Hampe, Cornelia; Issad, Tarik

    2012-01-01

    Background In diabetic patients, the pharmacokinetics of injected human insulin does not permit optimal control of glycemia. Fast and slow acting insulin analogues have been developed, but they may have adverse properties, such as increased mitogenic or anti-apoptotic signaling. Insulin/IGF1 hybrid receptors (IR/IGF1R), present in most tissues, have been proposed to transmit biological effects close to those of IGF1R. However, the study of hybrid receptors is difficult because of the presence of IR and IGF1R homodimers. Our objective was to perform the first study on the pharmacological properties of the five marketed insulin analogues towards IR/IGF1R hybrids. Methodology To study the effect of insulin analogues on IR/IGF1R hybrids, we used our previously developed Bioluminescence Resonance Energy Transfer (BRET) assay that permits specific analysis of the pharmacological properties of hybrid receptors. Moreover, we have developed a new, highly sensitive BRET-based assay to monitor phophatidylinositol-3 phosphate (PIP3) production in living cells. Using this assay, we performed a detailed pharmacological analysis of PIP3 production induced by IGF1, insulin and insulin analogues in living breast cancer-derived MCF-7 and MDA-MB231 cells. Results Among the five insulin analogues tested, only glargine stimulated IR/IGF1R hybrids with an EC50 that was significantly lower than insulin and close to that of IGF1. Glargine more efficiently stimulated PIP3 production in MCF-7 cells but not in MDA-MB231 cells as compared to insulin. In contrast, glargine metabolites M1 and M2 showed lower potency for hybrid receptors stimulation, PIP3 production, Akt and Erk1/2 phosphorylation and DNA synthesis in MCF-7 cells, compared to insulin. Conclusion Glargine, possibly acting through IR/IGF1R hybrids, displays higher potency, whereas its metabolites M1 and M2 display lower potency than insulin for the stimulation of proliferative/anti-apoptotic pathways in MCF-7 cells. PMID:22848683

  2. Elevated insulin receptor content in human breast cancer.

    PubMed Central

    Papa, V; Pezzino, V; Costantino, A; Belfiore, A; Giuffrida, D; Frittitta, L; Vannelli, G B; Brand, R; Goldfine, I D; Vigneri, R

    1990-01-01

    The growth of breast cancer cells is under the regulation of hormones, growth factors, and their receptors. In the present study, we have employed a new, sensitive, and specific radioimmunoassay for the direct measurement of insulin receptors in surgical specimens of breast cancers. In 159 specimens the insulin receptor content was 6.15 +/- 3.69 ng/0.1 mg protein. This value was more than sixfold higher than the mean value found in both 27 normal breast tissues obtained at total mastectomy (0.95 + 0.68, P less than 0.001) and in six normal specimens obtained from reduction mammoplasty (0.84 +/- 0.78, P less than 0.001). The insulin receptor content in breast cancer tissues was also higher than in any normal tissue investigated including liver (Pezzino, V., V. Papa, V. Trischitta, A. Brunetti, P.A. Goodman, M.K. Treutelaar, J.A. Williams, B.A. Maddux, R. Vigneri, and I.D. Goldfine, 1989. Am. J. Physiol. 257:E451-457). The insulin receptor in breast cancer retained its ability to both bind insulin and undergo insulin-induced tyrosine kinase activation. Immunostaining of the specimens revealed that the insulin receptor was present in malignant epithelial cells, but was not detected in stromal and inflammatory cells. Univariant analysis revealed that the insulin receptor content of the tumors correlated positively with tumor size (P = 0.014), histological grading (P = 0.030), and the estrogen receptor content (P = 0.035). There were no significant correlations between insulin receptor content and the age, body weight, menopausal status, and nodal involvement of the patients. These studies indicate, therefore, that the insulin receptor content is increased in breast cancers and raise the possibility that the insulin receptor may have a role in the biology of these tumors. Images PMID:2243127

  3. GQ-16, a Novel Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand, Promotes Insulin Sensitization without Weight Gain

    PubMed Central

    Amato, Angélica A.; Rajagopalan, Senapathy; Lin, Jean Z.; Carvalho, Bruno M.; Figueira, Ana C. M.; Lu, Jenny; Ayers, Stephen D.; Mottin, Melina; Silveira, Rodrigo L.; Souza, Paulo C. T.; Mourão, Rosa H. V.; Saad, Mário J. A.; Togashi, Marie; Simeoni, Luiz A.; Abdalla, Dulcinéia S. P.; Skaf, Munir S.; Polikparpov, Igor; Lima, Maria C. A.; Galdino, Suely L.; Brennan, Richard G.; Baxter, John D.; Pitta, Ivan R.; Webb, Paul; Phillips, Kevin J.; Neves, Francisco A. R.

    2012-01-01

    The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the β-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby “ideal” PPARγ-based therapeutics stabilize the β-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects. PMID:22584573

  4. Agonism and Antagonism at the Insulin Receptor

    PubMed Central

    Knudsen, Louise; Hansen, Bo Falck; Jensen, Pia; Pedersen, Thomas Åskov; Vestergaard, Kirsten; Schäffer, Lauge; Blagoev, Blagoy; Oleksiewicz, Martin B.; Kiselyov, Vladislav V.; De Meyts, Pierre

    2012-01-01

    Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B’29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1–10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints (3H-thymidine incorporation), and not on metabolic endpoints (14C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity (3H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B’29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed. PMID:23300584

  5. CMHX008, a Novel Peroxisome Proliferator-Activated Receptor γ Partial Agonist, Enhances Insulin Sensitivity In Vitro and In Vivo

    PubMed Central

    Song, Ying; Liu, Zhiguo; Li, Jibin; Gao, Rufei; Zhang, Yuyao; Mei, Hu; Guo, Tingwang; Xiao, Ling; Wang, Bochu; Wu, Chaodong; Xiao, Xiaoqiu

    2014-01-01

    The peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in adipocyte differentiation and insulin sensitivity. Its ligand rosiglitazone has anti-diabetic effect but is frequently accompanied with some severe unwanted effects. The aim of the current study was to compare the anti-diabetic effect of CMHX008, a novel thiazolidinedione-derivative, with rosiglitazone. A luciferase assay was used to evaluate in vitro PPARγ activation. 3T3-L1 cells were used to examine adipocyte differentiation. High fat diet (HFD) mice were used to examine in vivo insulin sensitivity. The mRNA levels were evaluated by real-time RT-PCR. Serum biochemical and hormonal variables were assessed using a clinical chemistry analyser. CMHX008 displayed a moderate PPARγ agonist activity, and promoted 3T3-L1 preadipocyte differentiation with lower activity than rosiglitazone. CMHX008 regulated the expression of PPARγ target genes in a different manner from rosiglitazone. CMHX008 increased the expression and secretion of adiponectin with the similar efficacy as rosiglitazone, but only 25% as potent as rosiglitazone for the induction of adipocyte fatty acid binding protein. Treatment of CMHX008 and rosiglitazone protected mice from high fat diet (HFD)-induced glucose intolerance, hyperinsulinemia and inflammation. CMHX008 reduced the mRNA expression of M1 macrophage markers, and significantly increased the expressions of M2 markers. In conclusion, CMHX008 shared the comparable insulin-sensitizing effects as rosiglitazone with lower adipogenic capacity and might potentially be developed into an effective agent for the treatment of diabetes and metabolic disorders. PMID:25004107

  6. Insulin Receptor Substrate 2 Is a Negative Regulator of Memory Formation

    ERIC Educational Resources Information Center

    Irvine, Elaine E.; Drinkwater, Laura; Radwanska, Kasia; Al-Qassab, Hind; Smith, Mark A.; O'Brien, Melissa; Kielar, Catherine; Choudhury, Agharul I.; Krauss, Stefan; Cooper, Jonathan D.; Withers, Dominic J.; Giese, Karl Peter

    2011-01-01

    Insulin has been shown to impact on learning and memory in both humans and animals, but the downstream signaling mechanisms involved are poorly characterized. Insulin receptor substrate-2 (Irs2) is an adaptor protein that couples activation of insulin- and insulin-like growth factor-1 receptors to downstream signaling pathways. Here, we have…

  7. Insulin receptors in the mammary gland

    SciTech Connect

    Smith, D.H.

    1986-01-01

    Insulin binding studies were conducted using mammary membrane preparations to further the authors understanding of insulin's role in regulating mammary metabolism, particularly ruminant mammary metabolism. Specific objectives were to: (1) characterize insulin binding to bovine mammary microsomes and determine if the specificity and kinetics of binding indicate the presence of insulin receptors in bovine mammary gland; (2) examine and compare insulin binding by liver and mammary microsomes of the pig and dairy cow; (3) examine insulin binding to bovine milk fat globule membranes (MFGM) and evaluate this model's usefulness in assessing insulin receptor regulation in the mammary gland of the cow; (4) examine the effect of dietary fat in insulin binding by rat mammary and liver microsomes. The specificity and kinetics of /sup 125/I-insulin binding of bovine mammary microsomes indicated the presence of insulin receptors in bovine mammary gland. Bovine liver and mammary microsomes specifically bound less /sup 125/I-insulin than did the corresponding porcine microsomes, and mammary microsomes, regardless of species, specifically bound less /sup 125/I-insulin than did liver microsomes. These differences in binding suggest differences in insulin responsiveness between pigs and cattle, as well as between the liver and mammary glands.

  8. Ligand-independent activation of peroxisome proliferator-activated receptor-gamma by insulin and C-peptide in kidney proximal tubular cells: dependent on phosphatidylinositol 3-kinase activity.

    PubMed

    Al-Rasheed, Nawal M; Chana, Ravinder S; Baines, Richard J; Willars, Gary B; Brunskill, Nigel J

    2004-11-26

    Peroxisome proliferator-activated receptor gamma (PPARgamma) has key roles in the regulation of adipogenesis, inflammation, and lipid and glucose metabolism. C-peptide is believed to be inert and without appreciable biological functions. Recent studies suggest that C-peptide possesses multiple functions. The present study investigated the effects of insulin and C-peptide on PPARgamma transcriptional activity in opossum kidney proximal tubular cells. Both insulin and C-peptide induced a concentration-dependent stimulation of PPARgamma transcriptional activity. Both agents substantially augmented thiazolidinedione-stimulated PPARgamma transcriptional activity. Neither insulin nor C-peptide had any effect on the expression levels of PPARgamma. GW9662, a PPARgamma antagonist, blocked PPARgamma activation by thiazolidinediones but had no effect on either insulin- or C-peptide-stimulated PPARgamma transcriptional activity. Co-transfection of opossum kidney cells with dominant negative mitogen-activated protein kinase kinase significantly depressed basal PPARgamma transcriptional activity but had no effect on that induced by either insulin or C-peptide. Both insulin- and C-peptide-stimulated PPARgamma transcriptional activity were attenuated by wortmannin and by expression of a dominant negative phosphatidylinositol (PI) 3-kinase p85 regulatory subunit. In addition PI 3-kinase-dependent phosphorylation of PPARgamma was observed after stimulation by C-peptide or insulin. C-peptide effects but not insulin on PPARgamma transcriptional activity were abolished by pertussis toxin pretreatment. Finally both C-peptide and insulin positively control the expression of the PPARgamma-regulated CD36 scavenger receptor in human THP-1 monocytes. We concluded that insulin and C-peptide can stimulate PPARgamma activity in a ligand-independent fashion and that this effect is mediated by PI 3-kinase. These results support a new and potentially important physiological role for C-peptide in

  9. Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor.

    PubMed

    Lawrence, Callum F; Margetts, Mai B; Menting, John G; Smith, Nicholas A; Smith, Brian J; Ward, Colin W; Lawrence, Michael C

    2016-07-22

    Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucine-rich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor α-chain (termed αCT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective αCT residues Phe(701) and Phe(705) The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor. PMID:27281820

  10. Toll-like receptors 2 and 4 impair insulin-mediated brain activity by interleukin-6 and osteopontin and alter sleep architecture.

    PubMed

    Sartorius, Tina; Lutz, Stefan Z; Hoene, Miriam; Waak, Jens; Peter, Andreas; Weigert, Cora; Rammensee, Hans-Georg; Kahle, Philipp J; Häring, Hans-Ulrich; Hennige, Anita M

    2012-05-01

    Impaired insulin action in the brain represents an early step in the progression toward type 2 diabetes, and elevated levels of saturated free fatty acids are known to impair insulin action in prediabetic subjects. One potential mediator that links fatty acids to inflammation and insulin resistance is the Toll-like receptor (TLR) family. Therefore, C3H/HeJ/TLR2-KO (TLR2/4-deficient) mice were fed a high-fat diet (HFD), and insulin action in the brain as well as cortical and locomotor activity was analyzed by using telemetric implants. TLR2/4-deficient mice were protected from HFD-induced glucose intolerance and insulin resistance in the brain and displayed an improvement in cortical and locomotor activity that was not observed in C3H/HeJ mice. Sleep recordings revealed a 42% increase in rapid eye movement sleep in the deficient mice during daytime, and these mice spent 41% more time awake during the night period. Treatment of control mice with a neutralizing IL-6 antibody improved insulin action in the brain as well as cortical activity and diminished osteopontin protein to levels of the TLR2/4-deficient mice. Together, our data suggest that the lack of functional TLR2/4 protects mice from a fat-mediated impairment in insulin action, brain activity, locomotion, and sleep architecture by an IL-6/osteopontin-dependent mechanism. PMID:22278939

  11. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by XMetA, an allosteric partial agonist antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

  12. Activation of Distinct P2Y Receptor Subtypes Stimulates Insulin Secretion in MIN6 Mouse Pancreatic β Cells

    PubMed Central

    Balasubramanian, Ramachandran; de Azua, Inigo Ruiz; Wess, Jürgen; Jacobson, Kenneth A.

    2010-01-01

    Extracellular nucleotides and their receptor antagonists have therapeutic potential in disorders such as inflammation, brain disorders, and cardiovascular diseases. Pancreatic β cells express several purinergic receptors, and reported nucleotide effects on insulin secretion are contradictory. We studied the effect of P2Y receptors on insulin secretion and cell death in MIN6, mouse pancreatic β cells. Expression of P2Y1 and P2Y6 receptors was revealed by total mRNA analysis using RT-PCR. MIN6 cells were stimulated in the presence of 16.7 mM glucose with or without P2Y1 and P2Y6 agonists, 2-MeSADP and Up3U, respectively. Both the agonists increased insulin secretion with EC50 values of 44.6±7.0 nM and 30.7±12.7 nM respectively. The insulin secretion by P2Y1 and P2Y6 agonists was blocked by their selective antagonists MRS2179 and MRS2578, respectively. Binding of the selective P2Y1 receptor antagonist radioligand [125I]MRS2500 in MIN6 cell membranes was saturable (KD 4.74±0.47 nM), and known P2Y1 ligands competed with high affinities. Inflammation and glucose toxicity leads to pancreatic β cell death in diabetes. Flow cytometric analysis revealed that Up3U but not 2-MeSADP protected MIN6 cells against TNF-α induced apoptosis. Overall, the results demonstrate that selective stimulation of P2Y1 and P2Y6 receptors increases insulin secretion that accompanies intracellular calcium release, suggesting potential application of P2Y receptor ligands in the treatment of diabetes. PMID:20067775

  13. Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

    SciTech Connect

    Levy, J.R.; Olefsky, J.M.

    1988-05-05

    The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4/sup 0/C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37/sup 0/C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation.

  14. Insulin receptors in normal and disease states.

    PubMed

    Grunberger, G; Taylor, S I; Dons, R F; Gorden, P

    1983-03-01

    The binding of insulin to its receptor has been studied under various physiological and pathological conditions. Quantitative studies have involved human circulating cells such as monocytes and erythrocytes, adipocytes, placental cells, and cultured cells such as fibroblasts and transformed lymphocytes. In animals, other target tissues such as liver and muscle have been studied and correlated with the human studies. Various physiological conditions such as diurnal rhythm, diet, age, exercise and the menstrual cycle affect insulin binding; in addition, many drugs perturb the receptor interaction. Disease affecting the insulin receptor can be divided into five general categories: (1) Receptor regulation--this involves diseases characterized by hyper- or hypoinsulinaemia. Hyperinsulinaemia in the basal state usually leads to receptor 'down' regulation as seen in obesity, type II diabetes, acromegaly and islet cell tumours. Hypoinsulinaemia such as seen in anorexia nervosa or type I diabetes may lead to elevated binding. (2) Antireceptor antibodies--these immunoglobulins bind to the receptor and competitively inhibit insulin binding. They may act as agonists, antagonists or partial agonists. (3) Genetic diseases which produce fixed alterations in both freshly isolated and cultured cells. (4) Diseases of receptor specificity where insulin may bind with different affinity to its own receptor or related receptors such as receptors for insulin-like growth factors. (5) Disease of affinity modulation where physical factors such as pH, temperature, ions, etc. may modify binding. In this review, we have considered primarily abnormality in insulin receptor binding. There are numerous other functions of the receptor such as coupling and transmission of the biological signal. These mechanisms are frequently referred to as postreceptor events, but more properly should be referred to as postbinding events since the receptor subserves other functions in addition to recognition and

  15. Insulin Action is Blocked by a Monoclonal Antibody That Inhibits the Insulin Receptor Kinase

    NASA Astrophysics Data System (ADS)

    Morgan, David O.; Ho, Lisa; Korn, Laurence J.; Roth, Richard A.

    1986-01-01

    Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor β subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens, and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor β subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.

  16. Brain Insulin Receptor Causes Activity-Dependent Current Suppression in the Olfactory Bulb Through Multiple Phosphorylation of Kv1.3

    PubMed Central

    FADOOL, D. A.; TUCKER, K.; PHILLIPS, J. J.; SIMMEN, J. A.

    2015-01-01

    Insulin and insulin receptor (IR) kinase are found in abundance in discrete brain regions yet insulin signaling in the CNS is not understood. Because it is known that the highest brain insulin-binding affinities, insulin-receptor density, and IR kinase activity are localized to the olfactory bulb, we sought to explore the downstream substrates for IR kinase in this region of the brain to better elucidate the function of insulin signaling in the CNS. First, we demonstrate that IR is postnatally and developmentally expressed in specific lamina of the highly plastic olfactory bulb (OB). ELISA testing confirms that insulin is present in the developing and adult OB. Plasma insulin levels are elevated above that found in the OB, which perhaps suggests a differential insulin pool. Olfactory bulb insulin levels appear not to be static, however, but are elevated as much as 15-fold after a 72-h fasting period. Bath application of insulin to cultured OB neurons acutely induces outward current suppression as studied by the use of traditional whole-cell and single-channel patchclamp recording techniques. Modulation of OB neurons is restricted to current magnitude; IR kinase activation does not modulate current kinetics of inactivation or deactivation. Transient transfection of human embryonic kidney cells with cloned Kv1.3 ion channel, which carries a large proportion of the outward current in these neurons, revealed that current suppression was the result of multiple tyrosine phosphorylation of Kv1.3 channel. Y to F single-point mutations in the channel or deletion of the kinase domain in IR blocks insulininduced modulation and phosphorylation of Kv1.3. Neuromodulation of Kv1.3 current in OB neurons is activity dependent and is eliminated after 20 days of odor/sensory deprivation induced by unilateral naris occlusion at postnatal day 1. IR kinase but not Kv1.3 expression is downregulated in the OB ipsilateral to the occlusion, as demonstrated in cryosections of right (control

  17. [Severe type A insulin resistance syndrome due to a mutation in the insulin receptor gene].

    PubMed

    Ros, P; Colino-Alcol, E; Grasso, V; Barbetti, F; Argente, J

    2015-01-01

    Insulin resistance syndromes without lipodystrophy are an infrequent and heterogeneous group of disorders with variable clinical phenotypes, associated with hyperglycemia and hyperinsulinemia. The three conditions related to mutations in the insulin receptor gene are leprechaunism or Donohue syndrome, Rabson-Mendenhall syndrome, and Type A syndrome. A case is presented on a patient diagnosed with type A insulin resistance, defined by the triad of extreme insulin resistance, acanthosis nigricans, and hyperandrogenism, carrying a heterozygous mutation in exon 19 of the insulin receptor gene coding for its tyrosine kinase domain that is crucial for the catalytic activity of the receptor. The molecular basis of the syndrome is reviewed, focusing on the structure-function relationships of the insulin receptor, knowing that the criteria for survival are linked to residual insulin receptor function. It is also pointed out that, although type A insulin resistance appears to represent a somewhat less severe condition, these patients have a high morbidity and their treatment is still unsatisfactory. PMID:25027621

  18. Activation of the PI3K/Akt signal transduction pathway and increased levels of insulin receptor in protein repair-deficient mice.

    PubMed

    Farrar, Christine; Houser, Carolyn R; Clarke, Steven

    2005-02-01

    Protein L-isoaspartate (D-aspartate) O-methyltransferase is an enzyme that catalyses the repair of isoaspartyl damage in proteins. Mice lacking this enzyme (Pcmt1-/- mice) have a progressive increase in brain size compared with wild-type mice (Pcmt1+/+ mice), a phenotype that can be associated with alterations in the PI3K/Akt signal transduction pathway. Here we show that components of this pathway, including Akt, GSK3beta and PDK-1, are more highly phosphorylated in the brains of Pcmt1-/- mice, particularly in cells of the hippocampus, in comparison with Pcmt1+/+ mice. Examination of upstream elements of this pathway in the hippocampus revealed that Pcmt1-/- mice have increased activation of insulin-like growth factor-I (IGF-I) receptor and/or insulin receptor. Western blot analysis revealed an approximate 200% increase in insulin receptor protein levels and an approximate 50% increase in IGF-I receptor protein levels in the hippocampus of Pcmt1-/- mice. Higher levels of the insulin receptor protein were also found in other regions of the adult brain and in whole tissue extracts of brain, liver, heart and testes of both juvenile and adult Pcmt1-/- mice. There were no significant differences in plasma insulin levels for adult Pcmt1-/- mice during glucose tolerance tests. However, they did show higher peak levels of blood glucose, suggesting a mild impairment in glucose tolerance. We propose that Pcmt1-/- mice have altered regulation of the insulin pathway, possibly as a compensatory response to altered glucose uptake or metabolism or as an adaptive response to a general accumulation of isoaspartyl protein damage in the brain and other tissues. PMID:15659208

  19. Insulin receptor-related receptor as an extracellular alkali sensor

    PubMed Central

    Deyev, Igor E.; Sohet, Fabien; Vassilenko, Konstantin P.; Serova, Oxana V.; Popova, Nadezhda V.; Zozulya, Sergey A.; Burova, Elena B.; Houillier, Pascal; Rzhevsky, Dmitry I.; Berchatova, Anastasiya A.; Murashev, Arkady N.; Chugunov, Anton O.; Efremov, Roman G.; Nikol’sky, Nikolai N.; Bertelli, Eugenio; Eladari, Dominique; Petrenko, Alexander G.

    2011-01-01

    SUMMARY The insulin receptor-related receptor (IRR), an orphan receptor tyrosine kinase of the insulin receptor family, can be activated by alkaline media both in vitro and in vivo at pH>7.9. The alkali-sensing property of IRR is conserved in frog, mouse and human. IRR activation is specific, dose-dependent, quickly reversible and demonstrates positive cooperativity. It also triggers receptor conformational changes and elicits intracellular signaling. The pH sensitivity of IRR is primarily defined by its L1F extracellular domains. IRR is predominantly expressed in organs that come in contact with mildly alkaline media. In particular, IRR is expressed in the cell subsets of the kidney that secrete bicarbonate into urine. Disruption of IRR in mice impairs the renal response to alkali loading attested by development of metabolic alkalosis and decreased urinary bicarbonate excretion in response to this challenge. We therefore postulate that IRR is an alkali sensor that functions in the kidney to manage metabolic bicarbonate excess. PMID:21641549

  20. Insulin resistance and muscle insulin receptor substrate‐1 serine hyperphosphorylation

    PubMed Central

    Stuart, Charles A.; Howell, Mary E. A.; Cartwright, Brian M.; McCurry, Melanie P.; Lee, Michelle L.; Ramsey, Michael W.; Stone, Michael H.

    2014-01-01

    Abstract Insulin resistance in metabolic syndrome subjects is profound in spite of muscle insulin receptor and insulin‐responsive glucose transporter (GLUT4) expression being nearly normal. Insulin receptor tyrosine kinase phosphorylation of insulin receptor substrate‐1 (IRS‐1) at Tyr896 is a necessary step in insulin stimulation of translocation of GLUT4 to the cell surface. Serine phosphorylation of IRS‐1 by some kinases diminishes insulin action in mice. We evaluated the phosphorylation status of muscle IRS‐1 in 33 subjects with the metabolic syndrome and seventeen lean controls. Each underwent euglycemic insulin clamps and a thigh muscle biopsy before and after 8 weeks of either strength or endurance training. Muscle IRS‐1 phosphorylation at six sites was quantified by immunoblots. Metabolic syndrome muscle IRS‐1 had excess phosphorylation at Ser337 and Ser636 but not at Ser307, Ser789, or Ser1101. Ser337 is a target for phosphorylation by glycogen synthase kinase 3 (GSK3) and Ser636 is phosphorylated by c‐Jun N‐terminal kinase 1 (JNK1). Exercise training without weight loss did not change the IRS‐1 serine phosphorylation. These data suggest that baseline hyperphosphorylation of at least two key serines within muscle IRS‐1 diminishes the transmission of the insulin signal and thereby decreases the insulin‐stimulated translocation of GLUT4. Excess fasting phosphorylation of muscle IRS‐1 at Ser636 may be a major cause of the insulin resistance seen in obesity and might prevent improvement in insulin responsiveness when exercise training is not accompanied by weight loss. PMID:25472611

  1. Development of a Quantitative PCR Assay for Detection of Human Insulin-Like Growth Factor Receptor and Insulin Receptor Isoforms.

    PubMed

    Flannery, Clare A; Rowzee, Anne M; Choe, Gina H; Saleh, Farrah L; Radford, Caitlin C; Taylor, Hugh S; Wood, Teresa L

    2016-04-01

    The biological activity of insulin and the insulin-like growth factor (IGF) ligands, IGF-I and IGF-II, is based in part on the relative abundance and distribution of their target receptors: the insulin receptor (IR) splice variants A (IR-A) and B (IR-B) and IGF 1 receptor (IGF-1R). However, the relative quantity of all three receptors in human tissues has never been measured together on the same scale. Due to the high homology between insulin receptor (IR)-A and IR-B proteins and lack of antibodies that discern the two IR splice variants, their mRNA sequence is the most reliable means of distinguishing between the receptors. Hence, highly specific primers for IR-A, IR-B, and IGF-1R mRNA were designed to accurately detect all three receptors by quantitative RT-PCR and enable direct quantification of relative receptor expression levels. A standard concentration curve of cDNA from each receptor was performed. Assay specificity was tested using competition assays and postamplification analysis by gel electrophoresis and cloning. Forward and reverse primer concentrations were optimized to ensure equal efficiencies across primer pairs. This assay enables a specific molecular signature of IGF/insulin signaling receptors to be assayed in different tissues, cell types, or cancers. PMID:26862994

  2. Sweet Taste Receptor Expressed in Pancreatic β-Cells Activates the Calcium and Cyclic AMP Signaling Systems and Stimulates Insulin Secretion

    PubMed Central

    Nakagawa, Yuko; Nagasawa, Masahiro; Yamada, Satoko; Hara, Akemi; Mogami, Hideo; Nikolaev, Viacheslav O.; Lohse, Martin J.; Shigemura, Noriatsu; Ninomiya, Yuzo; Kojima, Itaru

    2009-01-01

    Background Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. Methodology/Principal Findings The expression of the sweet taste receptor was determined by RT–PCR and immunohistochemistry. Changes in cytoplasmic Ca2+ ([Ca2+]c) and cAMP ([cAMP]c) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca2+]c. The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5)-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca2+]c response. The effect of sucralose on [Ca2+]c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a Gq inhibitor. Sucralose also induced sustained elevation of [cAMP]c, which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. Conclusions Sweet taste receptor is expressed in β-cells, and activation of this receptor induces insulin secretion by Ca2+ and cAMP-dependent mechanisms. PMID:19352508

  3. Specific activation of insulin-like growth factor-1 receptor by ginsenoside Rg5 promotes angiogenesis and vasorelaxation.

    PubMed

    Cho, Young-Lai; Hur, Sung-Mo; Kim, Ji-Yoon; Kim, Ji-Hee; Lee, Dong-Keon; Choe, Jongeon; Won, Moo-Ho; Ha, Kwon-Soo; Jeoung, Dooil; Han, Sanghwa; Ryoo, Sungwoo; Lee, Hansoo; Min, Jeong-Ki; Kwon, Young-Guen; Kim, Dong-Hyun; Kim, Young-Myeong

    2015-01-01

    Ginsenoside Rg5 is a compound newly synthesized during the steaming process of ginseng; however, its biological activity has not been elucidated with regard to endothelial function. We found that Rg5 stimulated in vitro angiogenesis of human endothelial cells, consistent with increased neovascularization and blood perfusion in a mouse hind limb ischemia model. Rg5 also evoked vasorelaxation in aortic rings isolated from wild type and high cholesterol-fed ApoE(-/-) mice but not from endothelial nitric-oxide synthase (eNOS) knock-out mice. Angiogenic activity of Rg5 was highly associated with a specific increase in insulin-like growth factor-1 receptor (IGF-1R) phosphorylation and subsequent activation of multiple angiogenic signals, including ERK, FAK, Akt/eNOS/NO, and Gi-mediated phospholipase C/Ca(2+)/eNOS dimerization pathways. The vasodilative activity of Rg5 was mediated by the eNOS/NO/cGMP axis. IGF-1R knockdown suppressed Rg5-induced angiogenesis and vasorelaxation by inhibiting key angiogenic signaling and NO/cGMP pathways. In silico docking analysis showed that Rg5 bound with high affinity to IGF-1R at the same binding site of IGF. Rg5 blocked binding of IGF-1 to its receptor with an IC50 of ∼90 nmol/liter. However, Rg5 did not induce vascular inflammation and permeability. These data suggest that Rg5 plays a novel role as an IGF-1R agonist, promoting therapeutic angiogenesis and improving hypertension without adverse effects in the vasculature. PMID:25391655

  4. Specific Activation of Insulin-like Growth Factor-1 Receptor by Ginsenoside Rg5 Promotes Angiogenesis and Vasorelaxation*

    PubMed Central

    Cho, Young-Lai; Hur, Sung-Mo; Kim, Ji-Yoon; Kim, Ji-Hee; Lee, Dong-Keon; Choe, Jongeon; Won, Moo-Ho; Ha, Kwon-Soo; Jeoung, Dooil; Han, Sanghwa; Ryoo, Sungwoo; Lee, Hansoo; Min, Jeong-Ki; Kwon, Young-Guen; Kim, Dong-Hyun; Kim, Young-Myeong

    2015-01-01

    Ginsenoside Rg5 is a compound newly synthesized during the steaming process of ginseng; however, its biological activity has not been elucidated with regard to endothelial function. We found that Rg5 stimulated in vitro angiogenesis of human endothelial cells, consistent with increased neovascularization and blood perfusion in a mouse hind limb ischemia model. Rg5 also evoked vasorelaxation in aortic rings isolated from wild type and high cholesterol-fed ApoE−/− mice but not from endothelial nitric-oxide synthase (eNOS) knock-out mice. Angiogenic activity of Rg5 was highly associated with a specific increase in insulin-like growth factor-1 receptor (IGF-1R) phosphorylation and subsequent activation of multiple angiogenic signals, including ERK, FAK, Akt/eNOS/NO, and Gi-mediated phospholipase C/Ca2+/eNOS dimerization pathways. The vasodilative activity of Rg5 was mediated by the eNOS/NO/cGMP axis. IGF-1R knockdown suppressed Rg5-induced angiogenesis and vasorelaxation by inhibiting key angiogenic signaling and NO/cGMP pathways. In silico docking analysis showed that Rg5 bound with high affinity to IGF-1R at the same binding site of IGF. Rg5 blocked binding of IGF-1 to its receptor with an IC50 of ∼90 nmol/liter. However, Rg5 did not induce vascular inflammation and permeability. These data suggest that Rg5 plays a novel role as an IGF-1R agonist, promoting therapeutic angiogenesis and improving hypertension without adverse effects in the vasculature. PMID:25391655

  5. Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system.

    PubMed Central

    O'Neill, T J; Craparo, A; Gustafson, T A

    1994-01-01

    Insulin receptor substrate 1 (IRS-1) is a major substrate of the insulin receptor and has been implicated in insulin signaling. Although IRS-1 is thought to interact with the insulin receptor, the nature of the interaction has not been defined. In this study, we used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to study the interaction between human IRS-1 and the insulin receptor. We demonstrate that IRS-1 forms a specific complex with the cytoplasmic domain of the insulin receptor when both are expressed as hybrid proteins in yeast cells. We show that the interaction is strictly dependent upon receptor tyrosine kinase activity, since IRS-1 shows no interaction with a kinase-inactive receptor hybrid containing a mutated ATP-binding site. Furthermore, mutation of receptor tyrosine 960 to phenylalanine eliminates IRS-1 interaction in the two-hybrid assay. These data suggest that the interaction between IRS-1 and the receptor is direct and provide evidence that the juxtamembrane domain of the receptor is involved. Furthermore, we show that a 356-amino-acid region encompassed by amino acids 160 through 516 of IRS-1 is sufficient for interaction with the receptor in the two-hybrid assay. Lastly, in agreement with our findings for yeast cells, we show that the insulin receptor is unable to phosphorylate an IRS-1 protein containing a deletion of amino acids 45 to 516 when expressed in COS cells. The two-hybrid assay should provide a facile means by which to pursue a detailed understanding of this interaction. Images PMID:7935368

  6. Activation of insulin-like growth factor 1 receptor in patients with non-small cell lung cancer.

    PubMed

    Kim, Jin-Soo; Kim, Edward S; Liu, Diane; Lee, J Jack; Behrens, Carmen; Lippman, Scott M; Hong, Waun Ki; Wistuba, Ignacio I; Lee, Euni; Lee, Ho-Young

    2015-06-30

    According to previous reports demonstrating the implication of insulin-like growth factor receptor (IGF-1R) signaling in non-small cell lung cancer (NSCLC), in this study, the potential prognostic values of IGF-1R expression/activation were analyzed. The expression and activation of IGF-1R were evaluated in two tissue microarray (TMA) sets from NSCLC patients (N = 352 for TMA I, and N = 353 for TMA II). Alterations in IGF-1R protein or mRNA expression in NSCLC patients were evaluated using publicly available data from The Cancer Genome Atlas (TCGA). We found that membranous and cytoplasmic IGF-1R expressions were significantly associated with squamous cell carcinoma (SCC) in both of the TMAs. Analysis of the TCGA data revealed increased mRNA levels in NSCLC patients, which was significantly associated with reductions in overall survival (OS) (median survival 26.51 vs. 47.77 months, P = 0.017) and disease-free survival (median survival 17.44 vs. 37.65 months, P = 0.045) only in NSCLC patients with adenocarcinoma (ADC). These data suggest that IGF-1R is activated in patients with NSCLC, particularly those with SCC. IGF-1R mRNA expression is a potential prognostic factor in patients with NSCLC, especially those with ADC. Further studies are warranted to investigate the prognostic value of IGF-1R in NSCLC patients. PMID:25944691

  7. Insulin receptor alternative splicing is regulated by insulin signaling and modulates beta cell survival

    PubMed Central

    Malakar, Pushkar; Chartarifsky, Lital; Hija, Ayat; Leibowitz, Gil; Glaser, Benjamin; Dor, Yuval; Karni, Rotem

    2016-01-01

    Type 2 Diabetes (T2DM) affects more than 300 million people worldwide. One of the hallmarks of T2DM is peripheral insulin resistance, in part due to unproductive insulin signaling through the insulin receptor. The insulin receptor (INSR) exists as two isoforms, INSR-A and INSR-B, which results from skipping or inclusion of exon 11 respectively. What determines the relative abundance of the different insulin receptor splice variants is unknown. Moreover, it is not yet clear what the physiological roles of each of the isoforms are in normal and diseased beta cells. In this study, we show that insulin induces INSR exon 11 inclusion in pancreatic beta cells in both human and mouse. This occurs through activation of the Ras-MAPK/ERK signaling pathway and up-regulation of the splicing factor SRSF1. Induction of exon 11 skipping by a splice-site competitive antisense oligonucleotide inhibited the MAPK-ERK signaling pathway downstream of the insulin receptor, sensitizing the pancreatic β-cell line MIN6 to stress-induced apoptosis and lipotoxicity. These results assign to insulin a regulatory role in INSR alternative splicing through the Ras-MAPK/ERK signaling pathway. We suggest that in beta cells, INSR-B has a protective role, while INSR-A expression sensitizes beta cells to programmed cell death. PMID:27526875

  8. Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons

    PubMed Central

    Storozhevykh, Tatiana P; Senilova, Yana E; Persiyantseva, Nadezhda A; Pinelis, Vsevolod G; Pomytkin, Igor A

    2007-01-01

    Background Accumulated evidence suggests that hydrogen peroxide (H2O2) generated in cells during insulin stimulation plays an integral role in insulin receptor signal transduction. The role of insulin-induced H2O2 in neuronal insulin receptor activation and the origin of insulin-induced H2O2 in neurons remain unclear. The aim of the present study is to test the following hypotheses (1) whether insulin-induced H2O2 is required for insulin receptor autophosphorylation in neurons, and (2) whether mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in insulin receptor autophosphorylation in neurons. Results Insulin stimulation elicited rapid insulin receptor autophosphorylation accompanied by an increase in H2O2 release from cultured cerebellar granule neurons (CGN). N-acetylcysteine (NAC), a H2O2 scavenger, inhibited both insulin-stimulated H2O2 release and insulin-stimulated autophosphorylation of insulin receptor. Inhibitors of respiratory chain-mediated H2O2 production, malonate and carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), inhibited both insulin-stimulated H2O2 release from neurons and insulin-stimulated autophosphorylation of insulin receptor. Dicholine salt of succinic acid, a respiratory substrate, significantly enhanced the effect of suboptimal insulin concentration on the insulin receptor autophosphorylation in CGN. Conclusion Results of the present study suggest that insulin-induced H2O2 is required for the enhancement of insulin receptor autophosphorylation in neurons. The mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in the insulin receptor autophosphorylation in neurons. PMID:17919343

  9. Mechanical stretch augments insulin-induced vascular smooth muscle cell proliferation by insulin-like growth factor-1 receptor

    SciTech Connect

    Liu, Gang; Hitomi, Hirofumi; Hosomi, Naohisa; Lei, Bai; Nakano, Daisuke; Deguchi, Kazushi; Mori, Hirohito; Masaki, Tsutomu; Ma, Hong; Griendling, Kathy K.; Nishiyama, Akira

    2011-10-15

    Insulin resistance and hypertension have been implicated in the pathogenesis of cardiovascular disease; however, little is known about the roles of insulin and mechanical force in vascular smooth muscle cell (VSMC) remodeling. We investigated the contribution of mechanical stretch to insulin-induced VSMC proliferation. Thymidine incorporation was stimulated by insulin in stretched VSMCs, but not in un-stretched VSMCs. Insulin increased 2-deoxy-glucose incorporation in both stretched and un-stretched VSMCs. Mechanical stretch augmented insulin-induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation. Inhibitors of epidermal growth factor (EGF) receptor tyrosine kinase and Src attenuated insulin-induced ERK and Akt phosphorylation, as well as thymidine incorporation, whereas 2-deoxy-glucose incorporation was not affected by these inhibitors. Moreover, stretch augmented insulin-like growth factor (IGF)-1 receptor expression, although it did not alter the expression of insulin receptor and insulin receptor substrate-1. Insulin-induced ERK and Akt activation, and thymidine incorporation were inhibited by siRNA for the IGF-1 receptor. Mechanical stretch augments insulin-induced VSMC proliferation via upregulation of IGF-1 receptor, and downstream Src/EGF receptor-mediated ERK and Akt activation. Similar to in vitro experiment, IGF-1 receptor expression was also augmented in hypertensive rats. These results provide a basis for clarifying the molecular mechanisms of vascular remodeling in hypertensive patients with hyperinsulinemia. -- Highlights: {yields} Mechanical stretch augments insulin-induced VSMC proliferation via IGF-1 receptor. {yields} Src/EGFR-mediated ERK and Akt phosphorylation are augmented in stretched VSMCs. {yields} Similar to in vitro experiment, IGF-1 receptor is increased in hypertensive rats. {yields} Results provide possible mechanisms of vascular remodeling in hypertension with DM.

  10. Peroxisome proliferator-activated receptorβ/δ activation is essential for modulating p-Foxo1/Foxo1 status in functional insulin-positive cell differentiation

    PubMed Central

    Li, L; Li, T; Zhang, Y; Pan, Z; Wu, B; Huang, X; Zhang, Y; Mei, Y; Ge, L; Shen, G; Ge, R-s; Zhu, D; Lou, Y

    2015-01-01

    Peroxisome proliferator-activated receptors (PPARs) participate in energy homeostasis and play essential roles in diabetes therapy through their effects on non-pancreas tissues. Pathological microenvironment may influence the metabolic requirements for the maintenance of stem cell differentiation. Accordingly, understanding the mechanisms of PPARs on pancreatic β-cell differentiation may be helpful to find the underlying targets of disrupted energy homeostasis under the pancreatic disease condition. PPARs are involved in stem cell differentiation via mitochondrial oxidative phosphorylation, but the subtype member activation and the downstream regulation in functional insulin-positive (INS+) cell differentiation remain unclear. Here, we show a novel role of PPARβ/δ activation in determining INS+ cell differentiation and functional maturation. We found PPARβ/δ expression selectively upregulated in mouse embryonic pancreases or stem cells-derived INS+ cells at the pancreatic mature stage in vivo and in vitro. Strikingly, given the inefficiency of generating INS+ cells in vitro, PPARβ/δ activation displayed increasing mouse and human ES cell-derived INS+ cell numbers and insulin secretion. This phenomenon was closely associated with the forkhead box protein O1 (Foxo1) nuclear shuttling, which was dependent on PPARβ/δ downstream PI3K/Akt signaling transduction. The present study reveals the essential role of PPARβ/δ activation on p-Foxo1/Foxo1 status, and in turn, determining INS+ cell generation and insulin secretion via affecting pancreatic and duodenal homeobox-1 expression. The results demonstrate the underlying mechanism by which PPARβ/δ activation promotes functional INS+ cell differentiation. It also provides potential targets for anti-diabetes drug discovery and hopeful clinical applications in human cell therapy. PMID:25855963

  11. Peroxisome proliferator-activated receptorβ/δ activation is essential for modulating p-Foxo1/Foxo1 status in functional insulin-positive cell differentiation.

    PubMed

    Li, L; Li, T; Zhang, Y; Pan, Z; Wu, B; Huang, X; Zhang, Y; Mei, Y; Ge, L; Shen, G; Ge, R-s; Zhu, D; Lou, Y

    2015-01-01

    Peroxisome proliferator-activated receptors (PPARs) participate in energy homeostasis and play essential roles in diabetes therapy through their effects on non-pancreas tissues. Pathological microenvironment may influence the metabolic requirements for the maintenance of stem cell differentiation. Accordingly, understanding the mechanisms of PPARs on pancreatic β-cell differentiation may be helpful to find the underlying targets of disrupted energy homeostasis under the pancreatic disease condition. PPARs are involved in stem cell differentiation via mitochondrial oxidative phosphorylation, but the subtype member activation and the downstream regulation in functional insulin-positive (INS+) cell differentiation remain unclear. Here, we show a novel role of PPARβ/δ activation in determining INS+ cell differentiation and functional maturation. We found PPARβ/δ expression selectively upregulated in mouse embryonic pancreases or stem cells-derived INS+ cells at the pancreatic mature stage in vivo and in vitro. Strikingly, given the inefficiency of generating INS+ cells in vitro, PPARβ/δ activation displayed increasing mouse and human ES cell-derived INS+ cell numbers and insulin secretion. This phenomenon was closely associated with the forkhead box protein O1 (Foxo1) nuclear shuttling, which was dependent on PPARβ/δ downstream PI3K/Akt signaling transduction. The present study reveals the essential role of PPARβ/δ activation on p-Foxo1/Foxo1 status, and in turn, determining INS+ cell generation and insulin secretion via affecting pancreatic and duodenal homeobox-1 expression. The results demonstrate the underlying mechanism by which PPARβ/δ activation promotes functional INS+ cell differentiation. It also provides potential targets for anti-diabetes drug discovery and hopeful clinical applications in human cell therapy. PMID:25855963

  12. Rapid internalization of the insulin receptor in rat hepatoma cells

    SciTech Connect

    Backer, J.M.; White, M.F.; Kahn, C.R.

    1987-05-01

    The authors have studied the internalization of the insulin receptor (IR) in rat hepatoma cells (Fao). The cells were surface-iodinated at 4C, stimulated with insulin at 37C, and then cooled rapidly, trypsinized at 4C and solubilized. The IR was immunoprecipitated with a specific antibody, and internalization of the IR was assessed by the appearance of trypsin-resistant bands on SDS-PAGE. Insulin induced the internalization of surface receptors with a t 1/2 of 9-10 mins; cells not exposed to insulin internalized less than 20% of the IR during 1 h at 37C. Further experiments demonstrated that the accumulation of trypsin-resistant IR paralleled a loss of receptor from the cell surface. Insulin-stimulated cells were chilled and iodinated at 4C, followed by solubilization, immunoprecipitation and SDS-PAGE; alternatively, insulin-stimulated cells were chilled, surface-bound ligand removed by washing the cells at pH 4.2, and specific ( SVI)insulin binding measured at 4C. Both techniques confirmed the disappearance of IR from the cell surface at rates comparable to the insulin-stimulated internalization described above. The total amount of phosphotyrosine-containing IR, as assessed by immunoprecipitation with an anti-phosphotyrosine antibody, remained constant during this time interval, suggesting that active kinase is translocated into the cell. In summary, the authors data indicate that insulin binding increases the rate of IR internalization of Fao cells. This relocation may facilitate the interaction of the activated tyrosine kinase in the IR with intracellular substrates, thus transmitting the insulin signal to metabolic pathways.

  13. Direct method for detection and characterization of cell surface receptors for insulin by means of 125I-labeled autoantibodies against the insulin receptor.

    PubMed Central

    Jarrett, D B; Roth, J; Kahn, C R; Flier, J S

    1976-01-01

    Autoantibodies directed against the cell surface receptors for insulin are found in some patients with extreme insulin resistance. These antibodies specifically inhibit the binding of insulin to its receptor. A purified IgG fraction from one patient's plasma was labeled with 125I. The 125I-labeled antireceptor antibody, which initially represented about 0.3% of the total 125I-IgG, was enriched by selective adsorption and subsequent elution from cells rich in insulin receptors. The 125I-antireceptor antibody bound to cells and the binding was inhibited by whole plasma and purified IgG from this patient, as well as whole plasma from another patient with autoantibodies to the insulin receptor. Insulins that differed 300-fold in biological potency and affinity inhibited binding of 125I-antireceptor antibody in direct proportion to their ability to bind to the insulin receptor. The binding of 125I-antireceptor antibody was closely correlated with the binding of 125I-insulin over a wide range of receptor concentrations on different cell types. Experimentally induced reduction of the insulin receptor concentration was associated with parallel decreases in the binding of 125I-antireceptor antibody and 125I-insulin. The preparation of 125I-antireceptor antibody with a high specific activity by cytoadsorption and elution has provided a sensitive method for the detection of receptors and autoantibodies to cell surface components. PMID:1069300

  14. Autophagy downregulation contributes to insulin resistance mediated injury in insulin receptor knockout podocytes in vitro

    PubMed Central

    Xin, Wei; Li, Zhaoping; Chen, Liyong

    2016-01-01

    It is unknown whether autophagy activity is altered in insulin resistant podocytes and whether autophagy could be a therapeutic target for diabetic nephropathy (DN). Here we used shRNA transfection to knockdown the insulin receptor (IR) gene in cultured human immortalized podocytes as an in vitro insulin resistant model. Autophagy related proteins LC3, Beclin, and p62 as well as nephrin, a podocyte injury marker, were assessed using western blot and immunofluorescence staining. Our results show that autophagy is suppressed when podocytes lose insulin sensitivity and that treatment of rapamycin, an mTOR specific inhibitor, could attenuate insulin resistance induced podocytes injury via autophagy activation. The present study deepens our understanding of the role of autophagy in the pathogenesis of DN. PMID:27077005

  15. Anticancer activity of the type I insulin-like growth factor receptor antagonist, ganitumab, in combination with the death receptor 5 agonist, conatumumab.

    PubMed

    Tabernero, Josep; Chawla, Sant P; Kindler, Hedy; Reckamp, Karen; Chiorean, E Gabriela; Azad, Nilofer S; Lockhart, A Craig; Hsu, Cheng-Pang; Baker, Nigel F; Galimi, Francesco; Beltran, Pedro; Baselga, José

    2015-03-01

    Agents targeting the insulin-like growth factor receptor type 1 (IGF1R) have shown antitumor activity. Based on the evidence for interaction between the IGF-1 and TRAIL pathways, we hypothesized that the combination of ganitumab (monoclonal antibody to IGF1R) with the pro-apoptotic death receptor 5 agonist, conatumumab, might increase antitumor response. Ganitumab and conatumumab were tested in combination in a Colo-205 xenograft model. Part 1 of the clinical study was a phase Ib program of three doses of conatumumab (1, 3, 15 mg/kg) in combination with 18 mg/kg ganitumab to determine the maximum tolerated dose (MTD) in patients with advanced solid tumors. Part 2 was conducted in six cohorts with advanced non-small cell lung cancer (squamous or non-squamous histology), colorectal cancer, sarcoma, pancreatic cancer, or ovarian cancer, treated at the recommended doses of the combination. The combination was significantly more active in the Colo-205 xenograft model than either single agent alone (p < 0.0015). In part 1 of the clinical study, no dose-limiting toxicities were observed and the MTD of conatumumab was 15 mg/kg in combination with 18 mg/kg ganitumab. In part 2, 78 patients were treated and there were no objective responses but 28 patients (36 %) had stable disease (median 46 days, range 0-261). The combination was well-tolerated with no new toxicities. In conclusion, the combination of ganitumab and conatumumab was well-tolerated but had no objective responses in the population tested. The successful future application of this combination of antitumor mechanisms may rely on the identification of predictive biomarkers. PMID:24816908

  16. H2O2 Signalling Pathway: A Possible Bridge between Insulin Receptor and Mitochondria

    PubMed Central

    Pomytkin, Igor A

    2012-01-01

    This review is focused on the mechanistic aspects of the insulin-induced H2O2 signalling pathway in neurons and the molecules affecting it, which act as risk factors for developing central insulin resistance. Insulin-induced H2O2 promotes insulin receptor activation and the mitochondria act as the insulin-sensitive H2O2 source, providing a direct molecular link between mitochondrial dysfunction and irregular insulin receptor activation. In this view, the accumulation of dysfunctional mitochondria during chronological ageing and Alzheimer’s disease (AD) is a risk factor that may contribute to the development of dysfunctional cerebral insulin receptor signalling and insulin resistance. Due to the high significance of insulin-induced H2O2 for insulin receptor activation, oxidative stress-induced upregulation of antioxidant enzymes, e.g., in AD brains, may represent another risk factor contributing to the development of insulin resistance. As insulin-induced H2O2 signalling requires fully functional mitochondria, pharmacological strategies based on activating mitochondria biogenesis in the brain are central to the treatment of diseases associated with dysfunctional insulin receptor signalling in this organ. PMID:23730255

  17. C333H ameliorated insulin resistance through selectively modulating peroxisome proliferator-activated receptor γ in brown adipose tissue of db/db mice.

    PubMed

    Zhang, Ning; Chen, Wei; Zhou, Xinbo; Zhou, Xiaolin; Xie, Xinni; Meng, Aimin; Li, Song; Wang, Lili

    2013-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) is a unique target for insulin sensitizer agents. These drugs have been used for the clinical treatment of type 2 diabetes for almost twenty years. However, serious safety issues are associated with the PPARγ agonist thiazolidinediones (TZDs). Selective PPARγ modulators (SPPARMs) which retain insulin sensitization without TZDs-like side effects are emerging as a promising new generation of insulin sensitizers. C333H is a novel structure compound synthesized by our laboratory. In diabetic rodent models, C333H has insulin-sensitizing and glucose-lowering activity comparable to that of TZDs, and causes no significant increase in body weight or adipose tissue weight in db/db mice. In diabetic db/db mice, C333H elevated circulating high molecular weight adiponectin isoforms, decreased PPARγ 273 serine phosphorylation in brown adipose tissue and selectively modulated the expression of a subset of PPARγ target genes in adipose tissue. In vitro, C333H weakly recruited coactivator and weakly dissociated corepressor activity. These findings suggest that C333H has similar properties to SPPARMs and may be a potential therapeutic agent for the treatment of type 2 diabetes. PMID:23563593

  18. Contribution of TyrB26 to the Function and Stability of Insulin: STRUCTURE-ACTIVITY RELATIONSHIPS AT A CONSERVED HORMONE-RECEPTOR INTERFACE.

    PubMed

    Pandyarajan, Vijay; Phillips, Nelson B; Rege, Nischay; Lawrence, Michael C; Whittaker, Jonathan; Weiss, Michael A

    2016-06-17

    Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. We investigated the role of Tyr(B26), a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, non-aromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakest-binding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the Glu(B26) analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although Tyr(B26) is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stability and self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences. PMID:27129279

  19. Therapeutic potential of the dual peroxisome proliferator activated receptor (PPAR)α/γ agonist aleglitazar in attenuating TNF-α-mediated inflammation and insulin resistance in human adipocytes.

    PubMed

    Massaro, Marika; Scoditti, Egeria; Pellegrino, Mariangela; Carluccio, Maria Annunziata; Calabriso, Nadia; Wabitsch, Martin; Storelli, Carlo; Wright, Matthew; De Caterina, Raffaele

    2016-05-01

    Adipose tissue inflammation is a mechanistic link between obesity and its related sequelae, including insulin resistance and type 2 diabetes. Dual ligands of peroxisome proliferator activated receptor (PPAR)α and γ, combining in a single molecule the metabolic and inflammatory-regulatory properties of α and γ agonists, have been proposed as a promising therapeutic strategy to antagonize adipose tissue inflammation. Here we investigated the effects of the dual PPARα/γ agonist aleglitazar on human adipocytes challenged with inflammatory stimuli. Human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes were treated with aleglitazar or - for comparison - the selective agonists for PPARα or γ fenofibrate or rosiglitazone, respectively, for 24h before stimulation with TNF-α. Aleglitazar, at concentrations as low as 10nmol/L, providing the half-maximal transcriptional activation of both PPARα and PPARγ, reduced the stimulated expression of several pro-inflammatory mediators including interleukin (IL)-6, the chemokine CXC-L10, and monocyte chemoattractant protein (MCP)-1. Correspondingly, media from adipocytes treated with aleglitazar reduced monocyte migration, consistent with suppression of MCP-1 secretion. Under the same conditions, aleglitazar also reversed the TNF-α-mediated suppression of insulin-stimulated ser473 Akt phosphorylation and decreased the TNF-α-induced ser312 IRS1 phosphorylation, two major switches in insulin-mediated metabolic activities, restoring glucose uptake in insulin-resistant adipocytes. Such effects were similar to those obtainable with a combination of single PPARα and γ agonists. In conclusion, aleglitazar reduces inflammatory activation and dysfunction in insulin signaling in activated adipocytes, properties that may benefit diabetic and obese patients. The effect of aleglitazar was consistent with dual PPARα and γ agonism, but with no evidence of synergism. PMID:26976796

  20. A novel insulin receptor-signaling platform and its link to insulin resistance and type 2 diabetes.

    PubMed

    Alghamdi, Farah; Guo, Merry; Abdulkhalek, Samar; Crawford, Nicola; Amith, Schammim Ray; Szewczuk, Myron R

    2014-06-01

    Insulin-induced insulin receptor (IR) tyrosine kinase activation and insulin cell survival responses have been reported to be under the regulation of a membrane associated mammalian neuraminidase-1 (Neu1). The molecular mechanism(s) behind this process is unknown. Here, we uncover a novel Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with neuromedin B G-protein coupled receptor (GPCR), which is essential for insulin-induced IR activation and cellular signaling. Neu1, MMP-9 and neuromedin B GPCR form a complex with IRβ subunit on the cell surface. Oseltamivir phosphate (Tamiflu®), anti-Neu1 antibodies, broad range MMP inhibitors piperazine and galardin (GM6001), MMP-9 specific inhibitor (MMP-9i), and GPCR neuromedin B specific antagonist BIM-23127 dose-dependently inhibited Neu1 activity associated with insulin stimulated rat hepatoma cells (HTCs) that overly express human IRs (HTC-IR). Tamiflu, anti-Neu1 antibodies and MMP-9i attenuated phosphorylation of IRβ and insulin receptor substrate-1 (IRS1) associated with insulin-stimulated cells. Olanzapine, an antipsychotic agent associated with insulin resistance, induced Neu3 sialidase activity in WG544 or 1140F01 human sialidosis fibroblast cells genetically defective in Neu1. Neu3 antagonist 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) and anti-Neu3 antibodies inhibited sialidase activity associated with olanzapine treated murine Neu4 knockout macrophage cells. Olanzapine attenuated phosphorylation of IGF-R and IRS1 associated with insulin-stimulated human wild-type fibroblast cells. Our findings identify a novel insulin receptor-signaling platform that is critically essential for insulin-induced IRβ tyrosine kinase activation and cellular signaling. Olanzapine-induced Neu3 sialidase activity attenuated insulin-induced IGF-R and IRS1 phosphorylation contributing to insulin resistance. PMID:24583283

  1. Activation of α7 Nicotinic Acetylcholine Receptor Decreases On-site Mortality in Crush Syndrome through Insulin Signaling-Na/K-ATPase Pathway

    PubMed Central

    Fan, Bo-Shi; Zhang, En-Hui; Wu, Miao; Guo, Jin-Min; Su, Ding-Feng; Liu, Xia; Yu, Jian-Guang

    2016-01-01

    On-site mortality in crush syndrome remains high due to lack of effective drugs based on definite diagnosis. Anisodamine (Ani) is widely used in China for treatment of shock, and activation of α7 nicotinic acetylcholine receptor (α7nAChR) mediates such antishock effect. The present work was designed to test whether activation of α7nAChR with Ani decreased mortality in crush syndrome shortly after decompression. Sprague-Dawley rats and C57BL/6 mice with crush syndrome were injected with Ani (20 mg/kg and 28 mg/kg respectively, i.p.) 30 min before decompression. Survival time, serum potassium, insulin, and glucose levels were observed shortly after decompression. Involvement of α7nAChR was verified with methyllycaconitine (selective α7nAChR antagonist) and PNU282987 (selective α7nAChR agonist), or in α7nAChR knockout mice. Effect of Ani was also appraised in C2C12 myotubes. Ani reduced mortality and serum potassium and enhanced insulin sensitivity shortly after decompression in animals with crush syndrome, and PNU282987 exerted similar effects. Such effects were counteracted by methyllycaconitine or in α7nAChR knockout mice. Mortality and serum potassium in rats with hyperkalemia were also reduced by Ani. Phosphorylation of Na/K-ATPase was enhanced by Ani in C2C12 myotubes. Inhibition of tyrosine kinase on insulin receptor, phosphoinositide 3-kinase, mammalian target of rapamycin, signal transducer and activator of transcription 3, and Na/K-ATPase counteracted the effect of Ani on extracellular potassium. These findings demonstrated that activation of α7nAChR could decrease on-site mortality in crush syndrome, at least in part based on the decline of serum potassium through insulin signaling-Na/K-ATPase pathway. PMID:27065867

  2. Regulation of insulin resistance and adiponectin signaling in adipose tissue by liver X receptor activation highlights a cross-talk with PPARγ.

    PubMed

    Zheng, Fenping; Zhang, Saifei; Lu, Weina; Wu, Fang; Yin, Xueyao; Yu, Dan; Pan, Qianqian; Li, Hong

    2014-01-01

    Liver X receptors (LXRs) have been recognized as a promising therapeutic target for atherosclerosis; however, their role in insulin sensitivity is controversial. Adiponectin plays a unique role in maintaining insulin sensitivity. Currently, no systematic experiments elucidating the role of LXR activation in insulin function based on adiponectin signaling have been reported. Here, we investigated the role of LXR activation in insulin resistance based on adiponectin signaling, and possible mechanisms. C57BL/6 mice maintained on a regular chow received the LXR agonist, T0901317 (30 mg/kg.d) for 3 weeks by intraperitoneal injection, and differentiated 3T3-L1 adipocytes were treated with T0901317 or GW3965. T0901317 treatment induced significant insulin resistance in C57BL/6 mice. It decreased adiponectin gene transcription in epididymal fat, as well as serum adiponectin levels. Activity of AMPK, a key mediator of adiponectin signaling, was also decreased, resulting in decreased Glut-4 membrane translocation in epididymal fat. In contrast, adiponectin activity was not changed in the liver of T0901317 treated mice. In vitro, both T0901317 and GW3965 decreased adiponectin expression in adipocytes in a dose-dependent manner, an effect which was diminished by LXRα silencing. ChIP-qPCR studies demonstrated that T0901317 decreased the binding of PPARγ to the PPAR-responsive element (PPRE) of the adiponectin promoter in a dose-dependent manner. Furthermore, T0901317 exerted an antagonistic effect on the expression of adiponectin in adipocytes co-treated with 3 µM Pioglitazone. In luciferase reporter gene assays, T0901317 dose-dependently inhibited PPRE-Luc activity in HEK293 cells co-transfected with LXRα and PPARγ. These results suggest that LXR activation induces insulin resistance with decreased adiponectin signaling in epididymal fat, probably due to negative regulation of PPARγ signaling. These findings indicate that the potential of LXR activation as a therapeutic

  3. Insulin decreases atherosclerosis by inducing endothelin receptor B expression

    PubMed Central

    Park, Kyoungmin; Mima, Akira; Li, Qian; Rask-Madsen, Christian; He, Pingnian; Mizutani, Koji; Katagiri, Sayaka; Maeda, Yasutaka; Wu, I-Hsien; Khamaisi, Mogher; Preil, Simone Rordam; Maddaloni, Ernesto; Sørensen, Ditte; Rasmussen, Lars Melholt; Huang, Paul L.; King, George L.

    2016-01-01

    Endothelial cell (EC) insulin resistance and dysfunction, caused by diabetes, accelerates atherosclerosis. It is unknown whether specifically enhancing EC-targeted insulin action can decrease atherosclerosis in diabetes. Accordingly, overexpressing insulin receptor substrate-1 (IRS1) in the endothelia of Apoe−/− mice (Irs1/Apoe−/−) increased insulin signaling and function in the aorta. Atherosclerosis was significantly reduced in Irs1/ApoE−/− mice on diet-induced hyperinsulinemia and hyperglycemia. The mechanism of insulin’s enhanced antiatherogenic actions in EC was related to remarkable induction of NO action, which increases endothelin receptor B (EDNRB) expression and intracellular [Ca2+]. Using the mice with knockin mutation of eNOS, which had Ser1176 mutated to alanine (AKI), deleting the only known mechanism for insulin to activate eNOS/NO pathway, we observed that IRS1 overexpression in the endothelia of Aki/ApoE−/− mice significantly decreased atherosclerosis. Interestingly, endothelial EDNRB expression was selectively reduced in intima of arteries from diabetic patients and rodents. However, endothelial EDNRB expression was upregulated by insulin via P13K/Akt pathway. Finally EDNRB deletion in EC of Ldlr−/− and Irs1/Ldlr−/− mice decreased NO production and accelerated atherosclerosis, compared with Ldlr−/− mice. Accelerated atherosclerosis in diabetes may be reduced by improving insulin signaling selectively via IRS1/Akt in the EC by inducing EDNRB expression and NO production. PMID:27200419

  4. Inhibition of clathrin-mediated endocytosis selectively attenuates specific insulin receptor signal transduction pathways.

    PubMed

    Ceresa, B P; Kao, A W; Santeler, S R; Pessin, J E

    1998-07-01

    To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor. PMID:9632770

  5. Inhibition of Clathrin-Mediated Endocytosis Selectively Attenuates Specific Insulin Receptor Signal Transduction Pathways

    PubMed Central

    Ceresa, Brian P.; Kao, Aimee W.; Santeler, Scott R.; Pessin, Jeffrey E.

    1998-01-01

    To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor. PMID:9632770

  6. Acute up-regulation of the rat brain somatostatin receptor-effector system by leptin is related to activation of insulin signaling and may counteract central leptin actions.

    PubMed

    Perianes-Cachero, A; Burgos-Ramos, E; Puebla-Jiménez, L; Canelles, S; Frago, L M; Hervás-Aguilar, A; de Frutos, S; Toledo-Lobo, M V; Mela, V; Viveros, M P; Argente, J; Chowen, J A; Arilla-Ferreiro, E; Barrios, V

    2013-11-12

    Leptin and somatostatin (SRIF) have opposite effects on food seeking and ingestive behaviors, functions partially regulated by the frontoparietal cortex and hippocampus. Although it is known that the acute suppression of food intake mediated by leptin decreases with time, the counter-regulatory mechanisms remain unclear. Our aims were to analyze the effect of acute central leptin infusion on the SRIF receptor-effector system in these areas and the implication of related intracellular signaling mechanisms in this response. We studied 20 adult male Wister rats including controls and those treated intracerebroventricularly with a single dose of 5 μg of leptin and sacrificed 1 or 6h later. Density of SRIF receptors was unchanged at 1h, whereas leptin increased the density of SRIF receptors at 6h, which was correlated with an elevated capacity of SRIF to inhibit forskolin-stimulated adenylyl cyclase activity in both areas. The functional capacity of SRIF receptors was unaltered as cell membrane levels of αi1 and αi2 subunits of G inhibitory proteins were unaffected in both brain areas. The increased density of SRIF receptors was due to enhanced SRIF receptor subtype 2 (sst2) protein levels that correlated with higher mRNA levels for this receptor. These changes in sst2 mRNA levels were concomitant with increased activation of the insulin signaling, c-Jun and cyclic AMP response element-binding protein (CREB); however, activation of signal transducer and activator of transcription 3 was reduced in the cortex and unchanged in the hippocampus and suppressor of cytokine signaling 3 remained unchanged in these areas. In addition, the leptin antagonist L39A/D40A/F41A blocked the leptin-induced changes in SRIF receptors, leptin signaling and CREB activation. In conclusion, increased activation of insulin signaling after leptin infusion is related to acute up-regulation of the SRIF receptor-effector system that may antagonize short-term leptin actions in the rat brain. PMID

  7. Bile acids acutely stimulate insulin secretion of mouse β-cells via farnesoid X receptor activation and K(ATP) channel inhibition.

    PubMed

    Düfer, Martina; Hörth, Katrin; Wagner, Rebecca; Schittenhelm, Björn; Prowald, Susanne; Wagner, Thomas F J; Oberwinkler, Johannes; Lukowski, Robert; Gonzalez, Frank J; Krippeit-Drews, Peter; Drews, Gisela

    2012-06-01

    Type 2 diabetes mellitus is associated with alterations in bile acid (BA) signaling. The aim of our study was to test whether pancreatic β-cells contribute to BA-dependent regulation of glucose homeostasis. Experiments were performed with islets from wild-type, farnesoid X receptor (FXR) knockout (KO), and β-cell ATP-dependent K(+) (K(ATP)) channel gene SUR1 (ABCC8) KO mice, respectively. Sodium taurochenodeoxycholate (TCDC) increased glucose-induced insulin secretion. This effect was mimicked by the FXR agonist GW4064 and suppressed by the FXR antagonist guggulsterone. TCDC and GW4064 stimulated the electrical activity of β-cells and enhanced cytosolic Ca(2+) concentration ([Ca(2+)](c)). These effects were blunted by guggulsterone. Sodium ursodeoxycholate, which has a much lower affinity to FXR than TCDC, had no effect on [Ca(2+)](c) and insulin secretion. FXR activation by TCDC is suggested to inhibit K(ATP) current. The decline in K(ATP) channel activity by TCDC was only observed in β-cells with intact metabolism and was reversed by guggulsterone. TCDC did not alter insulin secretion in islets of SUR1-KO or FXR-KO mice. TCDC did not change islet cell apoptosis. This is the first study showing an acute action of BA on β-cell function. The effect is mediated by FXR by nongenomic elements, suggesting a novel link between FXR activation and K(ATP) channel inhibition. PMID:22492528

  8. Impact of peroxisome proliferator-activated receptor γ on angiotensin II type 1 receptor-mediated insulin sensitivity, vascular inflammation and atherogenesis in hypercholesterolemic mice

    PubMed Central

    Becher, Ulrich M.; Camara, Bakary; Yildirimtürk, Cihan; Aksoy, Adem; Kebschull, Moritz; Werner, Nikos; Nickenig, Georg; Müller, Cornelius

    2015-01-01

    Introduction The angiotensin II type 1 receptor (AT1R) and the peroxisome proliferator-activated receptor γ (PPARγ) have been implicated in the pathogenesis of atherosclerosis. A number of studies have reported that AT1R inhibition or genetic AT1R disruption and PPARγ activation inhibit vascular inflammation and improve glucose and lipid metabolism, underscoring a molecular interaction of AT1R and PPARγ. We here analyzed the hypothesis that vasculoprotective anti-inflammatory and metabolic effects of AT1R inhibition are mediated by PPARγ. Material and methods Female ApoE–/–/AT1R–/– mice were fedwith a high-fat and cholesterol-rich diet and received continuous treatment with the selective PPARγ antagonist GW9662 or vehicle at a rate of 700 ng/kg/min for 4 weeks using subcutaneously implanted osmotic mini-pumps. Additionally, one group of female ApoE–/– mice served as a control group. After treatment for 4 weeks mice were sacrificed and read-outs (plaque development, vascular inflammation and insulinsensitivity) were performed. Results Using AT1R deficient ApoE–/– mice (ApoE–/–/AT1R–/– mice) we found decreased cholesterol-induced endothelial dysfunction and atherogenesis compared to ApoE–/– mice. Inhibition of PPARγ by application of the specific PPARγ antagonist GW9662 significantly abolished the anti-atherogenic effects of AT1R deficiency in ApoE–/–/AT1R–/– mice (plaque area as % of control: ApoE–/–: 39 ±5%; ApoE–/–/AT1R–/–: 17 ±7%, p = 0.044 vs. ApoE–/–; ApoE–/–/AT1R–/– + GW9662: 31 ±8%, p = 0.047 vs. ApoE–/–/AT1R–/–). Focusing on IL6 as a pro-inflammatory humoral marker we detected significantly increased IL-6 levels in GW9662-treated animals (IL-6 in pg/ml: ApoE–/–: 230 ±16; ApoE–/–/AT1R–/–: 117 ±20, p = 0.01 vs. ApoE–/–; ApoE–/–/AT1R–/– + GW9662: 199 ±20, p = 0.01 vs. ApoE–/–/AT1R–/–), while the anti-inflammatory marker IL-10 was significantly

  9. Pancreatic Insulin Content Regulation by the Estrogen Receptor ERα

    PubMed Central

    Alonso-Magdalena, Paloma; Ropero, Ana B.; Carrera, M. Pilar; Cederroth, Christopher R.; Baquié, Mathurin; Gauthier, Benoit R.; Nef, Serge; Stefani, Enrico; Nadal, Angel

    2008-01-01

    The function of pancreatic β-cells is the synthesis and release of insulin, the main hormone involved in blood glucose homeostasis. Estrogen receptors, ERα and ERβ, are important molecules involved in glucose metabolism, yet their role in pancreatic β-cell physiology is still greatly unknown. In this report we show that both ERα and ERβ are present in pancreatic β-cells. Long term exposure to physiological concentrations of 17β-estradiol (E2) increased β-cell insulin content, insulin gene expression and insulin release, yet pancreatic β-cell mass was unaltered. The up-regulation of pancreatic β-cell insulin content was imitated by environmentally relevant doses of the widespread endocrine disruptor Bisphenol-A (BPA). The use of ERα and ERβ agonists as well as ERαKO and ERβKO mice suggests that the estrogen receptor involved is ERα. The up-regulation of pancreatic insulin content by ERα activation involves ERK1/2. These data may be important to explain the actions of E2 and environmental estrogens in endocrine pancreatic function and blood glucose homeostasis. PMID:18446233

  10. Insulin and insulin-like growth factor-I (IGF-I) receptor phosphorylation in µ-calpain knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Numerous cellular processes are controlled by insulin and IGF-I signaling pathways. Due to previous work in our laboratories, we hypothesized that insulin (IR) and type 1 IGF-I (IGF-IR) receptor signaling is decreased due to increased protein tyrosine phosphatase 1B (PTP1B) activity. C57BL/6J mice...

  11. Interaction of insulin with the rat diaphragm. Subcellular distribution of insulin and its receptor

    SciTech Connect

    Brush, J.S.; Guzman-Diaz, A.; Celis, J.

    1987-05-01

    In studying the uptake and processing of A-14( SVI)monoiodoinsulin by isolated rat hemidiaphragms it was found that major amounts of the hormone are associated with the debris fraction. A method was developed for separating debris components by discontinuous sucrose density gradient centrifugation. Major amounts of radioactivity were associated with its myofibril component with much lower amounts in its sarcolemmal elements. Using a modified method of Marshall, et. al. insulin receptor was measurable in these fractions with greatest amounts in myofibril and microsomal fractions. Receptor was also detectable in the latter after gel electrophoresis and immunoblotting with receptor antiserum. Sarcolemmal marker enzyme (K -stimulated, ouabain-suppressible p-nitrophenylphosphate phosphatase) activity was insignificant in sarcolemmal and myofibril fractions, but was significant in the microsomal fraction. Sarcolemmal activity becomes significant after hemidiaphragm incubation with 1 M insulin for 90 sec. but does not change in the microsomal fraction. It is concluded that 1) a component bound to the myofibrils in muscle is important in insulin processing, and 2) the largest part of sarcolemmal insulin receptors are incorporated in the microsomal fraction of homogenized tissue.

  12. Complementation analysis demonstrates that insulin cross-links both alpha subunits in a truncated insulin receptor dimer.

    PubMed

    Chan, Shu Jin; Nakagawa, Satoe; Steiner, Donald F

    2007-05-01

    The insulin receptor is a homodimer composed of two alphabeta half receptors. Scanning mutagenesis studies have identified key residues important for insulin binding in the L1 domain (amino acids 1-150) and C-terminal region (amino acids 704-719) of the alpha subunit. However, it has not been shown whether insulin interacts with these two sites within the same alpha chain or whether it cross-links a site from each alpha subunit in the dimer to achieve high affinity binding. Here we have tested the contralateral binding mechanism by analyzing truncated insulin receptor dimers (midi-hIRs) that contain complementary mutations in each alpha subunit. Midi-hIRs containing Ala(14), Ala(64), or Gly(714) mutations were fused with Myc or FLAG epitopes at the C terminus and were expressed separately by transient transfection. Immunoblots showed that R14A+FLAG, F64A+FLAG, and F714G+Myc mutant midi-hIRs were expressed in the medium but insulin binding activity was not detected. However, after co-transfection with R14A+FLAG/F714G+Myc or F64A+FLAG/F714G+Myc, hybrid dimers were obtained with a marked increase in insulin binding activity. Competitive displacement assays revealed that the hybrid mutant receptors bound insulin with the same affinity as wild type and also displayed curvilinear Scatchard plots. In addition, when hybrid mutant midi-hIR was covalently cross-linked with (125)I(A14)-insulin and reduced, radiolabeled monomer was immunoprecipitated only with anti-FLAG, demonstrating that insulin was bound asymmetrically. These results demonstrate that a single insulin molecule can contact both alpha subunits in the insulin receptor dimer during high affinity binding and this property may be an important feature for receptor signaling. PMID:17339314

  13. Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes.

    PubMed

    Hiriart, Marcia; Sanchez-Soto, Carmen; Diaz-Garcia, Carlos Manlio; Castanares, Diana T; Avitia, Morena; Velasco, Myrian; Mas-Oliva, Jaime; Macias-Silva, Marina; González-Villalpando, Clicerio; Delgado-Coello, Blanca; Sosa-Garrocho, Marcela; Vidaltamayo, Román; Fuentes-Silva, Deyanira

    2014-01-01

    It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l(-1) insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance. PMID:24995000

  14. Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes

    PubMed Central

    Hiriart, Marcia; Sanchez-Soto, Carmen; Diaz-Garcia, Carlos Manlio; Castanares, Diana T.; Avitia, Morena; Velasco, Myrian; Mas-Oliva, Jaime; Macias-Silva, Marina; González-Villalpando, Clicerio; Delgado-Coello, Blanca; Sosa-Garrocho, Marcela; Vidaltamayo, Román; Fuentes-Silva, Deyanira

    2014-01-01

    It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l−1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance. PMID:24995000

  15. Protective hinge in insulin opens to enable its receptor engagement

    PubMed Central

    Menting, John G.; Yang, Yanwu; Chan, Shu Jin; Phillips, Nelson B.; Smith, Brian J.; Whittaker, Jonathan; Wickramasinghe, Nalinda P.; Whittaker, Linda J.; Pandyarajan, Vijay; Wan, Zhu-li; Yadav, Satya P.; Carroll, Julie M.; Strokes, Natalie; Roberts, Charles T.; Ismail-Beigi, Faramarz; Milewski, Wieslawa; Steiner, Donald F.; Chauhan, Virander S.; Ward, Colin W.; Weiss, Michael A.; Lawrence, Michael C.

    2014-01-01

    Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated “microreceptors” that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of PheB24 to a 60° rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor's L1–β2 sheet. Opening of this hinge enables conserved nonpolar side chains (IleA2, ValA3, ValB12, PheB24, and PheB25) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function. PMID:25092300

  16. Central insulin signaling is attenuated by long-term insulin exposure via insulin receptor substrate-1 serine phosphorylation, proteasomal degradation, and lysosomal insulin receptor degradation.

    PubMed

    Mayer, Christopher M; Belsham, Denise D

    2010-01-01

    Central insulin signaling is critical for the prevention of insulin resistance. Hyperinsulinemia contributes to insulin resistance, but it is not yet clear whether neurons are subject to cellular insulin resistance. We used an immortalized, hypothalamic, clonal cell line, mHypoE-46, which exemplifies neuronal function and expresses the components of the insulin signaling pathway, to determine how hyperinsulinemia modifies neuronal function. Western blot analysis indicated that prolonged insulin treatment of mHypoE-46 cells attenuated insulin signaling through phospho-Akt. To understand the mechanisms involved, time-course analysis was performed. Insulin exposure for 4 and 8 h phosphorylated Akt and p70-S6 kinase (S6K1), whereas 8 and 24 h treatment decreased insulin receptor (IR) and IR substrate 1 (IRS-1) protein levels. Insulin phosphorylation of S6K1 correlated with IRS-1 ser1101 phosphorylation and the mTOR-S6K1 pathway inhibitor rapamycin prevented IRS-1 serine phosphorylation. The proteasomal inhibitor epoxomicin and the lysosomal pathway inhibitor 3-methyladenine prevented the degradation of IRS-1 and IR by insulin, respectively, and pretreatment with rapamycin, epoxomicin, or 3-methyladenine prevented attenuation of insulin signaling by long-term insulin exposure. Thus, a sustained elevation of insulin levels diminishes neuronal insulin signaling through mTOR-S6K1-mediated IRS-1 serine phosphorylation, proteasomal degradation of IRS-1 and lysosomal degradation of the IR. PMID:19887566

  17. Dexamethasone effects on creatine kinase activity and insulin-like growth factor receptors in cultured muscle cells

    NASA Technical Reports Server (NTRS)

    Whitson, Peggy A.; Stuart, Charles A.; Huls, M. H.; Sams, Clarence F.; Cintron, Nitza M.

    1989-01-01

    The effect of dexamethasone on the activity of creatine kinase (CK) and the insulin-like growth factor I (IGF-I) binding were investigated using skeletal- and cardiac-muscle-derived cultured cell lines (mouse, C2C12; rat, L6 and H9c2). It was found that, in skeletal muscle cells, dexamethasone treatment during differentiation of skeletal-muscle cells caused dose-dependent increases in CK activity and increases in the degree of myotube formation, whereas cardiac cells (H9c2) exhibited very low CK activity during culture or dexamethasone treatment. Results for IGF-I binding were similar in all three cell lines. The IGF-I binding to dexamethasone-treated cells (50 nM for 24 hr on the day prior to confluence) resulted in an increased number of available binding sites, with no effect on the binding affinities.

  18. Insulin-like Growth Factor 1-mediated Hyperthermia Involves Anterior Hypothalamic Insulin Receptors*

    PubMed Central

    Sanchez-Alavez, Manuel; Osborn, Olivia; Tabarean, Iustin V.; Holmberg, Kristina H.; Eberwine, James; Kahn, C. Ronald; Bartfai, Tamas

    2011-01-01

    The objective is to investigate the role of insulin-like growth factor 1 (IGF-1) in the regulation of core body temperature. Sequencing cDNA libraries from individual warm-sensitive neurons from the preoptic area (POA) of the hypothalamus, a region involved in the central control of thermoregulation, identified neurons that express both IGF-1 receptor (IGF-1R) and insulin receptor transcripts. The effects of administration of IGF-1 into the POA was measured by radiotelemetry monitoring of core temperature, brown adipose tissue (BAT) temperature, metabolic assessment, and imaging of BAT by positron emission tomography of 2-[18F]fluoro-2-deoxyglucose uptake combined with computed tomography. IGF-1 injection into the POA caused dose-dependent hyperthermia that could be blocked by pretreatment with the IGF-1R tyrosine kinase inhibitor, PQ401. The IGF-1-evoked hyperthermia involved activation of brown adipose tissue and was accompanied by a switch from glycolysis to fatty acid oxidation as a source of energy as shown by lowered respiratory exchange ratio. Transgenic mice that lack neuronal insulin receptor expression in the brain (NIRKO mice) were unable to mount the full hyperthermic response to IGF-1, suggesting that the IGF-1 mediated hyperthermia is partly dependent on expression of functional neuronal insulin receptors. These data indicate a novel thermoregulatory role for both IGF-1R and neuronal insulin receptors in IGF-1 activation of BAT and hyperthermia. These central effects of IGF-1 signaling may play a role in regulation of metabolic rate, aging, and the risk of developing type 2 diabetes. PMID:21330367

  19. Insulin-like growth factor 1-mediated hyperthermia involves anterior hypothalamic insulin receptors.

    PubMed

    Sanchez-Alavez, Manuel; Osborn, Olivia; Tabarean, Iustin V; Holmberg, Kristina H; Eberwine, James; Kahn, C Ronald; Bartfai, Tamas

    2011-04-29

    The objective is to investigate the role of insulin-like growth factor 1 (IGF-1) in the regulation of core body temperature. Sequencing cDNA libraries from individual warm-sensitive neurons from the preoptic area (POA) of the hypothalamus, a region involved in the central control of thermoregulation, identified neurons that express both IGF-1 receptor (IGF-1R) and insulin receptor transcripts. The effects of administration of IGF-1 into the POA was measured by radiotelemetry monitoring of core temperature, brown adipose tissue (BAT) temperature, metabolic assessment, and imaging of BAT by positron emission tomography of 2-[(18)F]fluoro-2-deoxyglucose uptake combined with computed tomography. IGF-1 injection into the POA caused dose-dependent hyperthermia that could be blocked by pretreatment with the IGF-1R tyrosine kinase inhibitor, PQ401. The IGF-1-evoked hyperthermia involved activation of brown adipose tissue and was accompanied by a switch from glycolysis to fatty acid oxidation as a source of energy as shown by lowered respiratory exchange ratio. Transgenic mice that lack neuronal insulin receptor expression in the brain (NIRKO mice) were unable to mount the full hyperthermic response to IGF-1, suggesting that the IGF-1 mediated hyperthermia is partly dependent on expression of functional neuronal insulin receptors. These data indicate a novel thermoregulatory role for both IGF-1R and neuronal insulin receptors in IGF-1 activation of BAT and hyperthermia. These central effects of IGF-1 signaling may play a role in regulation of metabolic rate, aging, and the risk of developing type 2 diabetes. PMID:21330367

  20. S961, an insulin receptor antagonist causes hyperinsulinemia, insulin-resistance and depletion of energy stores in rats

    SciTech Connect

    Vikram, Ajit; Jena, Gopabandhu

    2010-07-23

    Research highlights: {yields}Insulin receptor antagonist S961 causes hyperglycemia, hyperinsulinemia and insulin resistance in rats. {yields}Peroxysome-proliferator-activated-receptor-gamma agonist pioglitazone improves S961 induced hyperglycemia and glucose intolerance. {yields}Long term treatment with insulin receptor antagonist S961 results in the decreased adiposity and hepatic glycogen content. {yields}Improvement in the hyperglycemia and glucose intolerance by pioglitazone clearly demonstrates that S961 treated rats can be successfully used to screen the novel therapeutic interventions having potential to improve glucose disposal through receptor independent mechanisms. -- Abstract: Impairment in the insulin receptor signaling and insulin mediated effects are the key features of type 2 diabetes. Here we report that S961, a peptide insulin receptor antagonist induces hyperglycemia, hyperinsulinemia ({approx}18-fold), glucose intolerance and impairment in the insulin mediated glucose disposal in the Sprague-Dawley rats. Further, long-term S961 treatment (15 day, 10 nM/kg/day) depletes energy storage as evident from decrease in the adiposity and hepatic glycogen content. However, peroxysome-proliferator-activated-receptor-gamma (PPAR{gamma}) agonist pioglitazone significantly (P < 0.001) restored S961 induced hyperglycemia (196.73 {+-} 16.32 vs. 126.37 {+-} 27.07 mg/dl) and glucose intolerance ({approx}78%). Improvement in the hyperglycemia and glucose intolerance by pioglitazone clearly demonstrates that S961 treated rats can be successfully used to screen the novel therapeutic interventions having potential to improve glucose disposal through receptor independent mechanisms. Further, results of the present study reconfirms and provide direct evidence to the crucial role of insulin receptor signaling in the glucose homeostasis and fuel metabolism.

  1. Inter-domain tagging implicates caveolin-1 in insulin receptor trafficking and Erk signaling bias in pancreatic beta-cells

    PubMed Central

    Boothe, Tobias; Lim, Gareth E.; Cen, Haoning; Skovsø, Søs; Piske, Micah; Li, Shu Nan; Nabi, Ivan R.; Gilon, Patrick; Johnson, James D.

    2016-01-01

    Objective The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells. Methods We generated functional insulin receptors tagged with pH-resistant fluorescent proteins between domains. Confocal, TIRF and STED imaging revealed a trafficking pattern of inter-domain tagged insulin receptors and endogenous insulin receptors detected with antibodies. Results Surprisingly, interdomain-tagged and endogenous insulin receptors in beta-cells bypassed classical Rab5a- or Rab7-mediated endocytic routes. Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes. Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact. Conclusions We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation. PMID:27110488

  2. Peroxisome proliferator-activated receptor gamma (PPARγ) in yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA expression and transcriptional regulation by insulin in vivo and in vitro.

    PubMed

    Zheng, Jia-Lang; Zhuo, Mei-Qin; Luo, Zhi; Pan, Ya-Xiong; Song, Yu-Feng; Huang, Chao; Zhu, Qing-Ling; Hu, Wei; Chen, Qi-Liang

    2015-02-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is ligand-inducible transcription factor and has important roles in lipid metabolism, cell proliferation and inflammation. In the present study, yellow catfish Pelteobagrus fulvidraco PPARγ cDNA was isolated from liver by RT-PCR and RACE, and its molecular characterization and transcriptional regulation by insulin in vivo and in vitro were determined. The generation of PPARγ1 and PPARγ2 was due to alternative promoter of PPARγ gene. PPARγ1 and PPARγ2 mRNA covered 2426 bp and 2537 bp, respectively, with an open reading frame (ORF) of 1584 bp encoding 527 amino acid residues. Yellow catfish PPARγ gene was organized in a manner similar to that of their mammalian homologs, implying a modular organization of the protein's domains. A comparison between the yellow catfish PPARγ amino acid sequence and the correspondent sequences of several other species revealed the identity of 55-76.2%. Two PPARγ transcripts (PPARγ1 and PPARγ2) mRNAs were expressed in a wide range of tissues, but the abundance of each PPARγ mRNA showed the tissue- and developmental stage-dependent expression patterns. Intraperitoneal injection of insulin in vivo significantly stimulated the mRNA expression of total PPARγ and PPARγ1, but not PPARγ2 in the liver of yellow catfish. In contrast, incubation of hepatocytes with insulin in vitro increased the mRNA levels of PPARγ1, PPARγ2 and total PPARγ. To our knowledge, for the first time, the present study provides evidence that PPARγ1 and PPARγ2 are differentially expressed with and among tissues during different developmental stages and also regulated by insulin both in vivo and in vitro, which serves to increase our understanding on PPARγ physiological function in fish. PMID:25637673

  3. Dissecting Mannose 6-Phosphate-Insulin-like Growth Factor 2 Receptor Complexes That Control Activation and Uptake of Plasminogen in Cells*

    PubMed Central

    Leksa, Vladimir; Pfisterer, Karin; Ondrovičová, Gabriela; Binder, Brigitte; Lakatošová, Silvia; Donner, Clemens; Schiller, Herbert B.; Zwirzitz, Alexander; Mrvová, Katarína; Pevala, Vladimir; Kutejová, Eva; Stockinger, Hannes

    2012-01-01

    The plasminogen (Plg) activation cascade on the cell surface plays a central role in cell migration and is involved in a plethora of physiological and pathological processes. Its regulation is coordinated by many receptors, in particular the urokinase-type plasminogen activator receptor (uPAR, CD87), receptors that physically interact and functionally cooperate with uPAR, and Plg binding molecules. Here we studied the impact of one of the Plg binding molecules, the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P-IGF2R, CD222), on cellular Plg activation. By developing both in vitro and in vivo Plg activation assays on size-fractionated lysates of M6P-IGF2R-silenced cells, we identified Plg-associated complexes with M6P-IGF2R as the regulatory factor. Using lipid raft preserving versus dissolving detergents, we found lipid dependence of the Plg regulatory function of these complexes. Furthermore, M6P-IGF2R-silencing in uPAR-positive human cell lines reduced internalization of Plg, resulting in elevated Plg activation. In contrast, the expression of human M6P-IGF2R in mouse embryonic fibroblasts derived from M6P-IGF2R knock-out mice enhanced Plg internalization. Finally, peptide 18–36 derived from the Plg-binding site within M6P-IGF2R enhanced Plg uptake. Thus, by targeting Plg to endocytic pathways, M6P-IGF2R appears to control Plg activation within cells that might be important to restrict plasmin activity to specific sites and substrates. PMID:22613725

  4. Dissociation of insulin receptor phosphorylation and stimulation of glucose transport in BC3H-1 myocytes

    SciTech Connect

    Mojsilovic, L.P.; Standaert, M.L.; Rosic, N.K.; Pollet, R.J.

    1986-05-01

    The authors have investigated insulin receptor phosphorylation in differentiated cultured BC3H-1 myocytes. As for other insulin-responsive cell systems in partially purified wheat germ agglutinin receptor preparations, insulin stimulates the phosphorylation of its own receptor (95K ..beta..-subunits) in a dose dependent manner (0-400 nM), as identified by immunoprecipitation with antiinsulin receptor antibodies and SDS-PAGE. In the same preparations they show that 12-0-tetradecanyl phorbol acetate (TPA), which in many respect ..beta..-subunits in the same dose dependent manner (0-5 ..mu..M). In addition, antiinsulin receptor antibodies (B-10) also induced phosphorylation of mimics insulin action, also induced phosphorylation of the insulin receptor and HPLC tryptic maps of the /sup 32/P-labeled ..beta..-subunit were identical to those for insulin-induced receptor phosphorylation. However, while insulin and TPA are potent stimulators of glucose transport in these muscle cells, the antireceptor antibodies alone failed to provoke glucose transport at any concentration. The specificity and activity of these antibodies were confirmed in their system by their ability to inhibit insulin binding and insulin-stimulated glucose transport in a concentration-dependent manner. Their results indicate that phosphorylation of insulin receptor is not a crucial event in mediating insulin action, at least with respect to glucose transport. While the effects of the B-10 antibody in the BC3H-1 myocyte differ from those in the adipocyte, their results provide independent confirmation of their essential conclusion that phosphorylation of the insulin receptor may not be necessary nor sufficient for its acute action in promoting glucose transport.

  5. Involvement of Sp1 and SREBP-1a in transcriptional activation of the LDL receptor gene by insulin and LH in cultured porcine granulosa-luteal cells.

    PubMed

    Sekar, Natesampillai; Veldhuis, Johannes D

    2004-07-01

    Luteinizing hormone (LH) and insulin stimulate transcriptional activity of the porcine low-density lipoprotein (LDL) receptor (LDLR) promoter supra-additively in primary cultures of granulosa-luteal cells. The mechanistic basis of this bihormonal interaction is unknown. The pig LDLR gene promoter includes three putative Sp1/Sp3-binding sites and one sterol response element (SRE) site 5' upstream to the transcriptional start site. To assess the role of SRE-binding protein (SREBP) in LDLR gene regulation, swine granulosa-luteal cells were cotransfected with CMV/SREBP-1a or SREBP-2 and the pLDLR1076/luc promoter. SREBP-1a and SREBP-2 stimulated LDLR gene transcription eight- and fourfold, respectively. LH alone augmented stimulation by SREBP-1 twofold. Conversely, cotransfection of a dominant-negative mutant form of SREBP-1a repressed basal and hormonally stimulated LDLR promoter activity by >80% (P < 0.01). Mutation of the SRE -167 ATCACCCCATG -157 to -167 ATCACCgCATG -157 bp decreased basal expression by 50% and LH + insulin- and LH + IGF-I-stimulated transcriptional activity by 80% and >90%, respectively (both P < 0.01). Mutations within each of the three flanking putative Sp1/Sp3 sites at -216/-211, -201/-196, and -151/-146 bp in the LDLR gene promoter also reduced basal activity (by >85%) and hormonal responsiveness (>95%, P < 0.05). EMSA confirmed that presumptive SRE-1 and Sp1/Sp3 elements bind respective peptides. Mithramycin, an inhibitor of Sp1/Sp3 protein(s) binding, blocked hormonally induced LDLR promoter expression by 80%. Basal transcription and supra-additive stimulation of porcine LDLR gene transcription by LH and insulin in granulosa-luteal cells require SREBP-1a and Sp1/Sp3-binding elements. PMID:14998783

  6. Thyrotropin via cyclic AMP induces insulin receptor expression and insulin Co-stimulation of growth and amplifies insulin and insulin-like growth factor signaling pathways in dog thyroid epithelial cells.

    PubMed

    Burikhanov, R; Coulonval, K; Pirson, I; Lamy, F; Dumont, J E; Roger, P P

    1996-11-15

    Despite the similarity of their receptors and signal transduction pathways, insulin is regarded as a regulator of glucose, protein, and lipid metabolism, whereas insulin-like growth factors (IGF-I and IGF-II) mainly act as mitogenic hormones. In the dog thyroid primary culture model, the triggering of DNA synthesis by thyrotropin (TSH) through cAMP, or by cAMP-independent factors including epidermal growth factor, hepatocyte growth factor and phorbol esters, requires insulin or IGFs as comitogenic factors. In the present study, in TSH-treated cells, IGF-I receptors and insulin receptors were paradoxically equivalent in their capacity to elicit the comitogenic pathway, which, however, was mediated only by IGF-I receptors in dog thyroid cells stimulated by cAMP-independent mitogens. Moreover, prior cell exposure to TSH or forskolin increased their responsiveness to insulin, IGF-I, and IGF-II, as seen on DNA synthesis and activation of a common insulin/IGF signaling pathway. To understand these observations, binding characteristics and expression of insulin and IGF-I receptors were examined. To analyze IGF-I receptor characteristics, the unexpected interference of a huge presence of IGF-binding proteins at the cell membrane was avoided using labeled Long R3 IGF-I instead of IGF-I. Strikingly, TSH, through cAMP, time-dependently induced insulin binding and insulin receptor mRNA and protein accumulation without any effect on IGF-I receptors. These findings constitute a first example of an induction of insulin receptor gene expression by a cAMP-mediated hormone. In dog thyroid cells, this allows low physiological insulin concentrations to act as a comitogenic factor and might explain in part the enhanced responsiveness to IGFs in response to TSH. This raises the possibility that TSH-insulin interactions may play a role in the regulation of thyroid growth and function in vivo. PMID:8910605

  7. Insulin growth factor-1 (IGF-1) enhances hippocampal excitatory and seizure activity through IGF-1 receptor-mediated mechanisms in the epileptic brain.

    PubMed

    Jiang, Guohui; Wang, Wei; Cao, Qingqing; Gu, Juan; Mi, Xiujuan; Wang, Kewei; Chen, Guojun; Wang, Xuefeng

    2015-12-01

    Insulin-like growth factor-1 (IGF-1) is known to promote neurogenesis and survival. However, recent studies have suggested that IGF-1 regulates neuronal firing and excitatory neurotransmission. In the present study, focusing on temporal lobe epilepsy, we found that IGF-1 levels and IGF-1 receptor activation are increased in human epileptogenic tissues, and pilocarpine- and pentylenetetrazole-treated rat models. Using an acute model of seizures, we showed that lateral cerebroventricular infusion of IGF-1 elevates IGF-1 receptor (IGF-1R) signalling before pilocarpine application had proconvulsant effects. In vivo electroencephalogram recordings and power spectrogram analysis of local field potential revealed that IGF-1 promotes epileptiform activities. This effect is diminished by co-application of an IGF-1R inhibitor. In an in vitro electrophysiological study, we demonstrated that IGF-1 enhancement of excitatory neurotransmission and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor- and N-methyl-D-aspartate receptor-mediated currents is inhibited by IGF-1R inhibitor. Finally, activation of extracellular signal-related kinase (ERK)-1/2 and protein kinase B (Akt) in seizures in rats is increased by exogenous IGF-1 and diminished by picropodophyllin. A behavioural study reveals that the ERK1/2 or Akt inhibitor attenuates seizure activity. These results indicate that increased IGF-1 levels after recurrent hippocampal neuronal firings might, in turn, promote seizure activity via IGF-1R-dependent mechanisms. The present study presents a previously unappreciated role of IGF-1R in the development of seizure activity. PMID:26286172

  8. Immunoreactive insulin and insulin-degrading activity in rat fecal extracts

    SciTech Connect

    Pillion, D.J.

    1986-05-01

    The insulin-degrading activity and insulin content of aqueous extracts of rat fecal material isolated from the proximal colon were measured by radioimmunoassay in the presence and absence of various inhibitors of proteolysis. In the absence of protease inhibitors, up to 40% of the (/sup 125/I)-insulin present in radioimmunoassay tubes was degraded to a form that was not precipitated by trichloroacetic acid. In the presence of bacitracin, EDTA or a mixture of protease inhibitors, insulin degradation was diminished to < 10%. The concentration of insulin in aqueous extracts of rat proximal colon fecal material was found to be small but significant, ranging from 10-120 ..mu..U/g wet weight when measured in the presence of protease inhibitors. The efficacy of various protease inhibitors differed when added to the aqueous fecal extract than when added to rat serum suggesting that a distinct insulinase activity was present in rat feces. Degradation of exogenously added (/sup 125/I)-insulin by the aqueous fecal extract was time- and temperature-dependent. Fecal samples obtained from streptozotocin-diabetic rats contained reduced amounts of insulin, suggesting that the fecal insulin was produced partially, but not exclusively, in the pancreas. These results indicate that fecal material in the rat proximal colon contains small amounts of insulin, as well as a rich supply of insulinase activity, and that insulin receptors located on the mucosal surface of proximal colon epithelial cells may be exposed to insulin in vivo.

  9. Insertion of isolated insulin receptors into placental membrane vesicles.

    PubMed Central

    Christiansen, K; Carlsen, J

    1992-01-01

    Purified human insulin receptors were inserted into placental plasma-membrane vesicles by fusion of membranes with receptor-lysophosphatidylcholine micelles. Scatchard analysis of insulin binding showed that about 10-15% of the added receptors became inserted into the membrane. The receptor number could be increased about 3-fold, corresponding to approx. 5 pmol of receptor/mg of membrane protein. The receptors became firmly bound to the membrane, as they could not be removed by extensive wash. The insertion of exogenous receptors could be demonstrated by immunoblotting. The inserted insulin receptor had the same insulin-binding affinity as the isolated receptor and the endogenous receptor of the membrane. Insulin binding in the presence or absence of Triton X-100 revealed that more than 80% of the exogenous receptors had a right-side-out orientation. Function of the inserted receptors, as observed by insulin-stimulated autophosphorylation, could be demonstrated. About 80% of the added lysophospholipid, corresponding to approx. 160 nmol of lysophospholipid/mg of membrane protein, became integrated into the membrane and was partly metabolized to phospholipid and to non-esterified fatty acid. The method of insertion of isolated insulin receptors using the natural detergent, lysophospholipid, may be a method for insertion of receptors into intact cells, where the lysophospholipid, as in the plasma-membrane vesicles, will be acylated to phospholipid. Images Fig. 3. Fig. 4. PMID:1736892

  10. Insulin-like factor regulates neural induction through an IGF1 receptor-independent mechanism

    PubMed Central

    Haramoto, Yoshikazu; Takahashi, Shuji; Oshima, Tomomi; Onuma, Yasuko; Ito, Yuzuru; Asashima, Makoto

    2015-01-01

    Insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) signalling is required for normal embryonic growth and development. Previous reports indicated that the IGF/IGF1R/MAPK pathway contributes to neural induction and the IGF/IGF1R/PI3K/Akt pathway to eye development. Here, we report the isolation of insulin3 encoding a novel insulin-like ligand involved in neural induction. Insulin3 has a similar structure to pro-insulin and mature IGF ligands, but cannot activate the IGF1 receptor. However, similar to IGFs, Insulin3 induced the gene expression of an anterior neural marker, otx2, and enlarged anterior head structures by inhibiting Wnt signalling. Insulin3 are predominantly localised to the endoplasmic reticulum when otx2 is induced by insulin3. Insulin3 reduced extracellular Wnts and cell surface localised Lrp6. These results suggest that Insulin3 is a novel cell-autonomous inhibitor of Wnt signalling. This study provides the first evidence that an insulin-like factor regulates neural induction through an IGF1R-independent mechanism. PMID:26112133

  11. Linking Functional Domains of the Human Insulin Receptor with the Bacterial Aspartate Receptor

    NASA Astrophysics Data System (ADS)

    Ellis, Leland; Morgan, David O.; Koshland, Daniel E.; Clauser, Eric; Moe, Gregory R.; Bollag, Gideon; Roth, Richard A.; Rutter, William J.

    1986-11-01

    A hybrid receptor has been constructed that is composed of the extracellular domain of the human insulin receptor fused to the transmembrane and cytoplasmic domains of the bacterial aspartate chemoreceptor. This hybrid protein can be expressed in rodent (CHO) cells and displays several functional features comparable to wild-type insulin receptor. It is localized to the cell surface, binds insulin with high affinity, forms oligomers, and is recognized by conformation-specific monoclonal antibodies. Although most of the expressed protein accumulates as a 180-kDa proreceptor, some processed 135-kDa receptor can be detected on the cell surface by covalent cross-linking. Expression of the hybrid receptor inhibits the insulin-activated uptake of 2-deoxyglucose by CHO cells. Thus, this hybrid is partially functional and can be processed; however, it is incapable of native transmembrane signaling. The results indicate that the intact domains of different types of receptors can retain some of the native features in a hybrid molecule but specific requirements will need to be satisfied for transmembrane signaling.

  12. Insulin Receptor Substrate-1 Activation Mediated p53 Downregulation Protects Against Hypoxic-Ischemia in the Neonatal Brain.

    PubMed

    Tu, Yi-Fang; Jiang, Si-Tse; Chow, Yen-Hung; Huang, Chao-Ching; Ho, Chien-Jung; Chou, Ya-Ping

    2016-08-01

    This study determined if dietary restriction (DR) protects against hypoxic-ischemia (HI) in the neonatal brain via insulin receptor substrate-1 (IRS-1)/Akt pathway-mediated downregulation of p53 in the neurovascular unit. On postnatal (P) day 7, HI was induced in rat pups grouped from P1 into normal litter size (NL, 12 pups/dam) and increased litter size (DR, 18 pups/dam). In vivo IRS-1 anti-sense oligonucleotide and IRS-1 overexpressed recombinant adenovirus were given, and neurovascular damage was assessed. In vitro models of oxygen-glucose deprivation (OGD) examined the inhibition and overexpression of IRS-1 on p53 and cell death in neurons and endothelial cells. Compared to NL pups, DR pups had significantly higher IRS-1, p-IRS-1, and pAkt levels, decreased p53, more tight junction proteins, reduced blood-brain barrier (BBB) damage after HI, and less infarct volumes at P21. Immunofluorescence revealed that IRS-1 was upregulated in the endothelial cells and neurons of DR pups. IRS-1 downregulation in DR pups reduced p-Akt, increased p53, worsened BBB damage, and increased brain injury, whereas IRS-1 overexpression in NL pups upregulated p-Akt, decreased p53, attenuated BBB damage, and decreased brain injury. In vitro, IRS-1 downregulation aggravated cell death in neurons and endothelial cells and is associated with decreased p-Akt and increased p53. In contrast, IRS-1 overexpression reduced cell death in endothelial cells with increased p-Akt and decreased p53. In conclusion, DR reduces neurovascular damage after HI in the neonatal brain through an IRS-1/Akt-mediated p53 downregulation, suggesting that IRS-1 signaling is a therapeutic target for hypoxic brain injury in neonates. PMID:26111627

  13. [Differences in dynamics of insulin and insulin-like growth I (IGF-I) receptors internalization in isolated rat hepatocytes].

    PubMed

    2013-01-01

    Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is the most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones, the internalization dynamics of 125I-insulin and 125I-IGF-I was traced in isolated rat hepatocytes at 37 and 12 degrees C. There were established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37 degrees C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. However, essential differences in the internalization course of these two related peptide were obvious at the temperature of 12 degrees C. The internalization level of insulin receptors at 12 degrees C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocytes plasma membrane. At 12 degrees C a slight decrease of the proportion of intracellular 125I-IGF-I correlated with a decrease in the 125I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12 degrees C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar "inhibition mechanism" of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates. PMID:25509050

  14. [Differences in dynamics of insulin and insulin-like growth I (IGF-I) receptors internalization in isolated rat hepatocytes].

    PubMed

    Kolychev, A P; Ternovskaya, E E; Arsenieva, A V; Shapkina, E V

    2013-01-01

    Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is the most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones, the internalization dynamics of 125I-insulin and 125I-IGF-I was traced in isolated rat hepatocytes at 37 and 12 degrees C. There were established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37 degrees C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. However, essential differences in the internalization course of these two related peptide were obvious at the temperature of 12 degrees C. The internalization level of insulin receptors at 12 degrees C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocytes plasma membrane. At 12 degrees C a slight decrease of the proportion of intracellular 125I-IGF-I correlated with a decrease in the 125I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12 degrees C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar "inhibition mechanism" of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates. PMID:25490849

  15. Acute physical exercise reverses S-nitrosation of the insulin receptor, insulin receptor substrate 1 and protein kinase B/Akt in diet-induced obese Wistar rats

    PubMed Central

    Pauli, José R; Ropelle, Eduardo R; Cintra, Dennys E; Carvalho-Filho, Marco A; Moraes, Juliana C; De Souza, Cláudio T; Velloso, Lício A; Carvalheira, José B C; Saad, Mario J A

    2008-01-01

    Early evidence demonstrates that exogenous nitric oxide (NO) and the NO produced by inducible nitric oxide synthase (iNOS) can induce insulin resistance. Here, we investigated whether this insulin resistance, mediated by S-nitrosation of proteins involved in early steps of the insulin signal transduction pathway, could be reversed by acute physical exercise. Rats on a high-fat diet were subjected to swimming for two 3 h-long bouts, separated by a 45 min rest period. Two or 16 h after the exercise protocol the rats were killed and proteins from the insulin signalling pathway were analysed by immunoprecipitation and immunoblotting. We demonstrated that a high-fat diet led to an increase in the iNOS protein level and S-nitrosation of insulin receptor β (IRβ), insulin receptor substrate 1 (IRS1) and Akt. Interestingly, an acute bout of exercise reduced iNOS expression and S-nitrosation of proteins involved in the early steps of insulin action, and improved insulin sensitivity in diet-induced obesity rats. Furthermore, administration of GSNO (NO donor) prevents this improvement in insulin action and the use of an inhibitor of iNOS (l-N6-(1-iminoethyl)lysine; l-NIL) simulates the effects of exercise on insulin action, insulin signalling and S-nitrosation of IRβ, IRS1 and Akt. In summary, a single bout of exercise reverses insulin sensitivity in diet-induced obese rats by improving the insulin signalling pathway, in parallel with a decrease in iNOS expression and in the S-nitrosation of IR/IRS1/Akt. The decrease in iNOS protein expression in the muscle of diet-induced obese rats after an acute bout of exercise was accompanied by an increase in AMP-activated protein kinase (AMPK) activity. These results provide new insights into the mechanism by which exercise restores insulin sensitivity. PMID:17974582

  16. A novel domain mediates insulin-induced proteasomal degradation of insulin receptor substrate 1 (IRS-1).

    PubMed

    Boura-Halfon, Sigalit; Shuster-Meiseles, Timor; Beck, Avital; Petrovich, Katia; Gurevitch, Diana; Ronen, Denise; Zick, Yehiel

    2010-11-01

    Insulin receptor substrate-1 (IRS-1) plays a pivotal role in insulin signaling, therefore its degradation is exquisitely regulated. Here, we show that insulin-stimulated degradation of IRS-1 requires the presence of a highly conserved Ser/Thr-rich domain that we named domain involved in degradation of IRS-1 (DIDI). DIDI (amino acids 386-430 of IRS-1) was identified by comparing the intracellular degradation rate of several truncated forms of IRS-1 transfected into CHO cells. The isolated DIDI domain underwent insulin-stimulated Ser/Thr phosphorylation, suggesting that it serves as a target for IRS-1 kinases. The effects of deletion of DIDI were studied in Fao rat hepatoma and in CHO cells expressing Myc-IRS-1(WT) or Myc-IRS-1(Δ386-430). Deletion of DIDI maintained the ability of IRS-1(Δ386-434) to undergo ubiquitination while rendering it insensitive to insulin-induced proteasomal degradation, which affected IRS-1(WT) (80% at 8 h). Consequently, IRS-1(Δ386-434) mediated insulin signaling (activation of Akt and glycogen synthesis) better than IRS-1(WT). IRS-1(Δ386-434) exhibited a significant greater preference for nuclear localization, compared with IRS-1(WT). Higher nuclear localization was also observed when cells expressing IRS-1(WT) were incubated with the proteasome inhibitor MG-132. The sequence of DIDI is conserved more than 93% across species, from fish to mammals, as opposed to approximately 40% homology of the entire IRS-1. These findings implicate DIDI as a novel, highly conserved domain of IRS-1, which mediates its cellular localization, rate of degradation, and biological activity, with a direct impact on insulin signal transduction. PMID:20843941

  17. Dietary modulation of erythrocyte insulin receptor interaction and the regulation of adipose tissue pyruvate dehydrogenase enzyme activity in growing rats; a mechanism of action of dietary fiber in metabolism

    SciTech Connect

    Ogunwole, J.O.A.

    1984-01-01

    The metabolic effects of graded cellulose (a dietary fiber) intake were studied at minimal (10%) and maximal (20%) protein levels in male weanling Sprague Dawley rats. The hypothesis was tested that the hypoglycemic effect of high fiber diets is partly mediated through increased tissue sensitivity to insulin at the cell receptor level. Erythrocyte insulin receptor interaction (IRI) and percent insulin stimulation of adipose tissue pyruvate dehydrogenase (PDH) activity (PDS) were used as indices of tissue sensitivity to insulin. IRI was determined by a standardized radioceptor assay PDS by the rate of oxidation of 1-/sup 14/C-pyruvate to /sup 14/CO/sub 2/ in epidymal fat pads and serum insulin levels by radioimmunoassay. In both protein groups, the addition of fiber in the diet resulted in a significant (P < 0.05) increase in food intake (FI) for calorie compensation. Fiber and protein intake had a significant (P < 0.01) effect on IRI and both basal (PDB) and PDS activities of PDH. At all fiber levels, specific percent /sup 125/I-insulin binding (SIB) was higher in the 20% protein groups while in the fiber-free group, a higher SIB was observed in the 10% protein group.

  18. Oncogenic transformation by the signaling adaptor proteins insulin receptor substrate (IRS)-1 and IRS-2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insulin receptor substrates (IRSs) are adaptor proteins that link signaling from upstream activators to multiple downstream effectors to modulate normal growth, metabolism, survival, and differentiation. Recent cell culture studies have shown that IRSs can interact with, and are functionally require...

  19. Molecular Basis of Signaling Specificity of Insulin and IGF Receptors: Neglected Corners and Recent Advances

    PubMed Central

    Siddle, Kenneth

    2011-01-01

    Insulin and insulin-like growth factor (IGF) receptors utilize common phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways to mediate a broad spectrum of “metabolic” and “mitogenic” responses. Specificity of insulin and IGF action in vivo must in part reflect expression of receptors and responsive pathways in different tissues but it is widely assumed that it is also determined by the ligand binding and signaling mechanisms of the receptors. This review focuses on receptor-proximal events in insulin/IGF signaling and examines their contribution to specificity of downstream responses. Insulin and IGF receptors may differ subtly in the efficiency with which they recruit their major substrates (IRS-1 and IRS-2 and Shc) and this could influence effectiveness of signaling to “metabolic” and “mitogenic” responses. Other substrates (Grb2-associated binder, downstream of kinases, SH2Bs, Crk), scaffolds (RACK1, β-arrestins, cytohesins), and pathways (non-receptor tyrosine kinases, phosphoinositide kinases, reactive oxygen species) have been less widely studied. Some of these components appear to be specifically involved in “metabolic” or “mitogenic” signaling but it has not been shown that this reflects receptor-preferential interaction. Very few receptor-specific interactions have been characterized, and their roles in signaling are unclear. Signaling specificity might also be imparted by differences in intracellular trafficking or feedback regulation of receptors, but few studies have directly addressed this possibility. Although published data are not wholly conclusive, no evidence has yet emerged for signaling mechanisms that are specifically engaged by insulin receptors but not IGF receptors or vice versa, and there is only limited evidence for differential activation of signaling mechanisms that are common to both receptors. Cellular context, rather than intrinsic receptor activity, therefore appears

  20. Cytoplasmic domains determine signal specificity, cellular routing characteristics and influence ligand binding of epidermal growth factor and insulin receptors.

    PubMed Central

    Riedel, H; Dull, T J; Honegger, A M; Schlessinger, J; Ullrich, A

    1989-01-01

    The cell surface receptors for insulin and epidermal growth factor (EGF) both employ a tyrosine-specific protein kinase activity to fulfil their distinct biological roles. To identify the structural domains responsible for various receptor activities, we have generated chimeric receptor polypeptides consisting of major EGF and insulin receptor structural domains and examined their biochemical properties and cellular signalling activities. The EGF-insulin receptor hybrids are properly synthesized and transported to the cell surface, where they form binding competent structures that are defined by the origin of their extracellular domains. While their ligand binding affinities are altered, we find that these chimeric receptors are fully functional in transmitting signals across the plasma membrane and into the cell. Thus, EGF receptor and insulin receptor cytoplasmic domain signalling capabilities are independent of their new heterotetrameric or monomeric environments respectively. Furthermore, the cytoplasmic domains carry the structural determinants that define kinase specificity, mitogenic and transforming potential, and receptor routing. Images PMID:2583088

  1. EGCG inhibits activation of the insulin-like growth factor-1 receptor in human colon cancer cells

    SciTech Connect

    Shimizu, Masahito; Deguchi, Atsuko; Hara, Yukihiko; Moriwaki, Hisataka; Weinstein, I. Bernard . E-mail: ibw1@columbia.edu

    2005-09-02

    The IGF/IGF-1R system, which includes the IGF, IGF-1R, and IGFBPs proteins, plays an important role in the development and growth of colorectal cancer. We previously reported that in the HT29 human colon cancer cell line EGCG, the major biologically active component of green tea, inhibits activation of the RTKs EGFR, HER2, and HER3, and that this is associated with inhibition of multiple downstream signaling pathways. Since IGF-1R is also a RTK, in this study we examined the effects of EGCG on the activity of IGF/IGF-1R system in human colon cancer cells. We found that the colon cancer cell lines Caco2, HT29, SW837, and SW480 express high levels of the IGF-1R receptor, and that both SW837 and SW480 cells display constitutive activation of this receptor. Treatment of SW837 cells with 20 {mu}g/ml of EGCG (the IC{sub 50} concentration for growth inhibition) caused within 6 h a decrease in the phosphorylated (i.e., activated) form of the IGF-1R protein. At 12 h, there was a decrease in the levels of both IGF-1 protein and mRNA and within 3-6 h there was an increase in the levels of both IGFBP-3 protein and mRNA. The increased expression of the latter protein was sustained for at least 48 h. When SW837 cells were treated with EGCG for a longer time, i.e., 96 h, a very low concentration (1.0 {mu}g/ml) of EGCG also caused inhibition of activation of IGF-1R, a decrease in the IGF-1 protein, and an increase in the IGFBP-3 protein. EGCG also caused a decrease in the levels of mRNAs that encode MMPs-7 and -9, proteins that proteolyze IGFBP-3. In addition, treatment with EGCG caused a transient increase in the expression of TGF-{beta}2, an inducer of IGFBP-3 expression. These findings expand the roles of EGCG as an inhibitor of critical RTKs involved in cell proliferation, providing further evidence that EGCG and related compounds may be useful in the chemoprevention or treatment of colorectal cancer.

  2. Lnk is an important modulator of insulin-like growth factor-1/Akt/peroxisome proliferator-activated receptor-gamma axis during adipogenesis of mesenchymal stem cells

    PubMed Central

    Lee, Jun Hee; Lee, Sang Hun; Lee, Hyang Seon; Ji, Seung Taek; Jung, Seok Yun; Kim, Jae Ho; Bae, Sun Sik

    2016-01-01

    Adipogenic differentiation of mesenchymal stem cells (MSCs) is critical for metabolic homeostasis and nutrient signaling during development. However, limited information is available on the pivotal modulators of adipogenic differentiation of MSCs. Adaptor protein Lnk (Src homology 2B3 [SH2B3]), which belongs to a family of SH2-containing proteins, modulates the bioactivities of different stem cells, including hematopoietic stem cells and endothelial progenitor cells. In this study, we investigated whether an interaction between insulin-like growth factor-1 receptor (IGF-1R) and Lnk regulated IGF-1-induced adipogenic differentiation of MSCs. We found that wild-type MSCs showed greater adipogenic differentiation potential than Lnk–/– MSCs. An ex vivo adipogenic differentiation assay showed that Lnk–/– MSCs had decreased adipogenic differentiation potential compared with wild-type MSCs. Interestingly, we found that Lnk formed a complex with IGF-1R and that IGF-1 induced the dissociation of this complex. In addition, we observed that IGF-1-induced increase in the phosphorylation of Akt and mammalian target of rapamycin was triggered by the dissociation of the IGF-1R–Lnk complex. Expression levels of a pivotal transcription factor peroxisome proliferator-activated receptor gamma (PPAR-γ) and its adipogenic target genes (LPL and FABP4) significantly decreased in Lnk–/– MSCs. These results suggested that Lnk adaptor protein regulated the adipogenesis of MSCs through the IGF-1/Akt/PPAR-γ pathway. PMID:27610032

  3. Lnk is an important modulator of insulin-like growth factor-1/Akt/peroxisome proliferator-activated receptor-gamma axis during adipogenesis of mesenchymal stem cells.

    PubMed

    Lee, Jun Hee; Lee, Sang Hun; Lee, Hyang Seon; Ji, Seung Taek; Jung, Seok Yun; Kim, Jae Ho; Bae, Sun Sik; Kwon, Sang-Mo

    2016-09-01

    Adipogenic differentiation of mesenchymal stem cells (MSCs) is critical for metabolic homeostasis and nutrient signaling during development. However, limited information is available on the pivotal modulators of adipogenic differentiation of MSCs. Adaptor protein Lnk (Src homology 2B3 [SH2B3]), which belongs to a family of SH2-containing proteins, modulates the bioactivities of different stem cells, including hematopoietic stem cells and endothelial progenitor cells. In this study, we investigated whether an interaction between insulin-like growth factor-1 receptor (IGF-1R) and Lnk regulated IGF-1-induced adipogenic differentiation of MSCs. We found that wild-type MSCs showed greater adipogenic differentiation potential than Lnk (-/-) MSCs. An ex vivo adipogenic differentiation assay showed that Lnk (-/-) MSCs had decreased adipogenic differentiation potential compared with wild-type MSCs. Interestingly, we found that Lnk formed a complex with IGF-1R and that IGF-1 induced the dissociation of this complex. In addition, we observed that IGF-1-induced increase in the phosphorylation of Akt and mammalian target of rapamycin was triggered by the dissociation of the IGF-1R-Lnk complex. Expression levels of a pivotal transcription factor peroxisome proliferator-activated receptor gamma (PPAR-γ) and its adipogenic target genes (LPL and FABP4) significantly decreased in Lnk (-/-) MSCs. These results suggested that Lnk adaptor protein regulated the adipogenesis of MSCs through the IGF-1/Akt/PPAR-γ pathway. PMID:27610032

  4. Growth Hormone-Releaser Diet Attenuates Cognitive Dysfunction in Klotho Mutant Mice via Insulin-Like Growth Factor-1 Receptor Activation in a Genetic Aging Model

    PubMed Central

    Park, Seok Joo; Chung, Yoon Hee; Lee, Jeong Hyun; Dang, Duy-Khanh; Nam, Yunsung; Jeong, Ji Hoon; Kim, Yong Sun; Nabeshima, Toshitaka

    2014-01-01

    Background It has been recognized that a defect in klotho gene expression accelerates the degeneration of multiple age-sensitive traits. Accumulating evidence indicates that aging is associated with declines in cognitive function and the activity of growth hormone (GH)/insulin-like growth factor-1 (IGF-1). Methods In this study, we examined whether a GH-releaser diet could be effective in protecting against cognitive impairment in klotho mutant mice. Results The GH-releaser diet significantly induced the expression of IGF-1 and IGF-1 receptors in the hippocampus of klotho mutant mice. Klotho mutant mice showed significant memory impairments as compared with wild-type mice. In addition, the klotho mutation significantly decreased the expression of cell survival/antiapoptotic factors, including phospho-Akt (p-Akt)/phospho-glycogen synthase kinase3β (p-GSK3β), phospho-extracellular signal-related kinase (p-ERK), and Bcl-2, but significantly increased those of cell death/proapoptotic factors, such as phospho-c-jun N-terminal kinase (p-JNK), Bax, and cleaved caspase-3 in the hippocampus. Treatment with GH-releaser diet significantly attenuated both decreases in the expression of cell survival/antiapoptotic factors and increases in the expression of cell death/proapoptotic factors in the hippocampus of klotho mutant mice. In addition, klotho mutation-induced oxidative stress was significantly attenuated by the GH-releaser diet. Consequently, a GH-releaser diet significantly improved memory function in the klotho mutant mice. GH-releaser diet-mediated actions were significantly reversed by JB-1, an IGF-1 receptor antagonist. Conclusion The results suggest that a GH-releaser diet attenuates oxidative stress, proapoptotic changes and consequent dysfunction in klotho mutant mice by promoting IGF-1 expression and IGF-1 receptor activation. PMID:25309793

  5. Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway

    PubMed Central

    Bernis, María E.; Oksdath, Mariana; Dupraz, Sebastián; Nieto Guil, Alvaro; Fernández, Marisa M.; Malchiodi, Emilio L.; Rosso, Silvana B.; Quiroga, Santiago

    2013-01-01

    Initial axonal elongation is essential for neuronal polarization and requires polarized activation of IGF-1 receptors (IGF-1r) and the phosphatidylinositol 3 kinase (PI3k) pathway. Wingless-type family growth factors (Wnts) have also been implied in the regulation of axonal development. It is not known, however, if Wnts have any participation in the regulation of initial axonal outgrowth and the establishment of neuronal polarity. We used cultured hippocampal neurons and growth cone particles (GCPs) isolated from fetal rat brain to show that stimulation with the wingless family factor 3A (Wnt3a) was sufficient to promote neuronal polarization in the absence of IGF-1 or high insulin. We also show that Wnt3a triggered a strong activation of IGF-1r, PI3k, and Akt in developmental Stage 2 neurons and that the presence of activatable IGF-1r and PI3k activation were necessary for Wnt3a polarizing effects. Surface plasmon resonance (SPR) experiments show that Wnt3a did not bind specifically to the IGF-1r. Using crosslinking and immuno-precipitation experiments, we show that stimulation with Wnt3a triggered the formation of a complex including IGF-1r-Wnt3a-Frizzled-7. We conclude that Wnt3a triggers polarization of neurons via cross-activation of the IGF-1r/PI3k pathway upon binding to Fz7. PMID:24298236

  6. Estradiol Binds to Insulin and Insulin Receptor Decreasing Insulin Binding in vitro

    PubMed Central

    Root-Bernstein, Robert; Podufaly, Abigail; Dillon, Patrick F.

    2014-01-01

    Rationale: Insulin (INS) resistance associated with hyperestrogenemias occurs in gestational diabetes mellitus, polycystic ovary syndrome, ovarian hyperstimulation syndrome, estrogen therapies, metabolic syndrome, and obesity. The mechanism by which INS and estrogen interact is unknown. We hypothesize that estrogen binds directly to INS and the insulin receptor (IR) producing INS resistance. Objectives: To determine the binding constants of steroid hormones to INS, the IR, and INS-like peptides derived from the IR; and to investigate the effect of estrogens on the binding of INS to its receptor. Methods: Ultraviolet spectroscopy, capillary electrophoresis, and NMR demonstrated estrogen binding to INS and its receptor. Horse-radish peroxidase-linked INS was used in an ELISA-like procedure to measure the effect of estradiol on binding of INS to its receptor. Measurements: Binding constants for estrogens to INS and the IR were determined by concentration-dependent spectral shifts. The effect of estradiol on INS binding to its receptor was determined by shifts in the INS binding curve. Main Results: Estradiol bound to INS with a Kd of 12 × 10−9 M and to the IR with a Kd of 24 × 10−9 M, while other hormones had significantly less affinity. Twenty-two nanomolars of estradiol shifted the binding curve of INS to its receptor 0.8 log units to the right. Conclusion: Estradiol concentrations in hyperestrogenemic syndromes may interfere with INS binding to its receptor producing significant INS resistance. PMID:25101056

  7. Two truncated forms of rat insulin receptor-related receptor.

    PubMed

    Itoh, N; Jobo, K; Tsujimoto, K; Ohta, M; Kawasaki, T

    1993-08-25

    The insulin receptor-related receptor (IRR) (1271 amino acids) is expected to have unique functions as a novel member of the insulin receptor family. In this paper, we report two alternatively spliced variants of rat IRR mRNA, which are predicted to encode two truncated forms of IRR, sIRR-1 (410 amino acids) and sIRR-2 (469 amino acids). The amino acid sequence of sIRR-1 is identical to the N-terminal 410-amino acid sequence of IRR. sIRR-2 has an additional 59-amino acid insertion in the C-terminal region. Both truncated forms retain the N-terminal and cysteine-rich domains but lack the transmembrane and intracellular tyrosine kinase domains, indicating that the truncated forms are the secreted forms. The translation products of the truncated form mRNAs were detected in the stomach and kidney by Western analysis. However, the physiological significance of the secreted forms remains to be elucidated. PMID:7688734

  8. Inhibition of net HepG2 cell apolipoprotein B secretion by the citrus flavonoid naringenin involves activation of phosphatidylinositol 3-kinase, independent of insulin receptor substrate-1 phosphorylation.

    PubMed

    Borradaile, Nica M; de Dreu, Linda E; Huff, Murray W

    2003-10-01

    The flavonoid naringenin improves hyperlipidemia and hyperglycemia in streptozotocin-treated rats. In HepG2 human hepatoma cells, naringenin inhibits apolipoprotein B (apoB) secretion primarily by inhibiting microsomal triglyceride transfer protein and enhances LDL receptor (LDLr)-mediated apoB-containing lipoprotein uptake. Phosphatidylinositol 3-kinase (PI3K) activation by insulin increases sterol regulatory element-binding protein (SREBP)-1 and LDLr expression and inhibits apoB secretion in hepatocytes. Thus, we determined whether naringenin activates this pathway. Insulin and naringenin induced PI3K-dependent increases in cytosolic and nuclear SREBP-1 and LDLr expression. Similar PI3K-mediated increases in SREBP-1 were observed in McA-RH7777 rat hepatoma cells, which express predominantly SREBP-1c. Reductions in HepG2 cell media apoB with naringenin were partially attenuated by wortmannin, whereas the effect of insulin was completely blocked. Both treatments reduced apoB100 secretion in wild-type and LDLr(-/-) mouse hepatocytes to the same extent. Insulin and naringenin increased HepG2 cell PI3K activity and decreased insulin receptor substrate (IRS)-2 levels. In sharp contrast to insulin, naringenin did not induce tyrosine phosphorylation of IRS-1. We conclude that naringenin increases LDLr expression in HepG2 cells via PI3K-mediated upregulation of SREBP-1, independent of IRS-1 phosphorylation. Although this pathway may not regulate apoB secretion in primary hepatocytes, PI3K activation by this novel mechanism may explain the insulin-like effects of naringenin in vivo. PMID:14514640

  9. The C-terminus of the B-chain of human insulin-like peptide 5 is critical for cognate RXFP4 receptor activity.

    PubMed

    Patil, Nitin A; Bathgate, Ross A D; Kocan, Martina; Ang, Sheng Yu; Tailhades, Julien; Separovic, Frances; Summers, Roger; Grosse, Johannes; Hughes, Richard A; Wade, John D; Hossain, Mohammed Akhter

    2016-04-01

    Insulin-like peptide 5 (INSL5) is an orexigenic peptide hormone belonging to the relaxin family of peptides. It is expressed primarily in the L-cells of the colon and has a postulated key role in regulating food intake. Its G protein-coupled receptor, RXFP4, is a potential drug target for treating obesity and anorexia. We studied the effect of modification of the C-terminus of the A and B-chains of human INSL5 on RXFP4 binding and activation. Three variants of human INSL5 were prepared using solid phase peptide synthesis and subsequent sequential regioselective disulfide bond formation. The peptides were synthesized as C-terminal acids (both A- and B-chains with free C-termini, i.e., the native form), amides (both chains as the C-terminal amide) and one analog with the C-terminus of its A-chain as the amide and the C-terminus of the B-chain as the acid. The results showed that C-terminus of the B-chain is more important than that of the A-chain for RXFP4 binding and activity. Amidation of the A-chain C-terminus does not have any effect on the INSL5 activity. The difference in RXFP4 binding and activation between the three peptides is believed to be due to electrostatic interaction of the free carboxylate of INSL5 with a positively charged residue (s), either situated within the INSL5 molecule itself or in the receptor extracellular loops. PMID:26661035

  10. Mammary tumorigenesis and metastasis caused by overexpression of insulin receptor substrate 1 (IRS-1) or IRS-2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insulin receptor substrates (IRSs) are signaling adaptors that play a major role in the metabolic and mitogenic actions of insulin and insulin-like growth factors. Reports have recently noted increased levels, or activity, of IRSs in many human cancers, and some have linked this to poor patient prog...

  11. Effect of a thermogenic agent, BRL 26830A, on insulin receptors in obese mice.

    PubMed

    Rochet, N; Tanti, J F; Grémeaux, T; Van Obberghen, E; Le Marchand-Brustel, Y

    1988-08-01

    The effect of a new type of antidiabetic agent, BRL 26830A, has been tested in obese mice. Since this drug increases thermogenesis, insulin receptor binding and kinase activity were studied in brown adipose tissue and skeletal muscle of mice made obese by gold thioglucose. At 1 mg.kg-1.day-1, a 3-wk treatment normalized the glycemia and increased the uncoupling protein content of brown adipose tissue. The insulin receptor number and its associated kinase activity increased only in brown adipose tissue. At 2 mg.kg-1.day-1, additional effects, i.e., a 20% reduction in body weight and a normalization of insulin receptor number both in brown adipose tissue and in skeletal muscle, were observed. All those results were obtained even though hyperinsulinemia was not corrected. At the higher drug dosage, insulin receptor kinase activity evolved in direct proportion to the receptor number in brown adipose tissue. By contrast, in skeletal muscle, the receptor kinase activity toward exogenous substrates increased more than the receptor number, suggesting that the alteration of insulin receptor kinase activity previously reported in skeletal muscle of obese mice was partly reversed by BRL 26830A. None of these parameters was modified by the drug in lean mice. These results show that, even without affecting obesity, BRL 26830A improves insulin resistance in obese mice, probably through its effect on insulin receptors. This action prevails in brown adipose tissue, supporting the idea that this tissue plays an important role in glucose homeostasis. Thermogenic drugs could thus be powerful agents for the treatment of noninsulin-dependent diabetics. PMID:2841863

  12. β3-Adrenoceptor activation relieves oxidative inhibition of the cardiac Na+-K+ pump in hyperglycemia induced by insulin receptor blockade.

    PubMed

    Karimi Galougahi, Keyvan; Liu, Chia-Chi; Garcia, Alvaro; Fry, Natasha A; Hamilton, Elisha J; Figtree, Gemma A; Rasmussen, Helge H

    2015-09-01

    Dysregulated nitric oxide (NO)- and superoxide (O2 (·-))-dependent signaling contributes to the pathobiology of diabetes-induced cardiovascular complications. We examined if stimulation of β3-adrenergic receptors (β3-ARs), coupled to endothelial NO synthase (eNOS) activation, relieves oxidative inhibition of eNOS and the Na(+)-K(+) pump induced by hyperglycemia. Hyperglycemia was established in male New Zealand White rabbits by infusion of the insulin receptor antagonist S961 for 7 days. Hyperglycemia increased tissue and blood indexes of oxidative stress. It induced glutathionylation of the Na(+)-K(+) pump β1-subunit in cardiac myocytes, an oxidative modification causing pump inhibition, and reduced the electrogenic pump current in voltage-clamped myocytes. Hyperglycemia also increased glutathionylation of eNOS, which causes its uncoupling, and increased coimmunoprecipitation of cytosolic p47(phox) and membranous p22(phox) NADPH oxidase subunits, consistent with NADPH oxidase activation. Blocking translocation of p47(phox) to p22(phox) with the gp91ds-tat peptide in cardiac myocytes ex vivo abolished the hyperglycemia-induced increase in glutathionylation of the Na(+)-K(+) pump β1-subunit and decrease in pump current. In vivo treatment with the β3-AR agonist CL316243 for 3 days eliminated the increase in indexes of oxidative stress, decreased coimmunoprecipitation of p22(phox) with p47(phox), abolished the hyperglycemia-induced increase in glutathionylation of eNOS and the Na(+)-K(+) pump β1-subunit, and abolished the decrease in pump current. CL316243 also increased coimmunoprecipitation of glutaredoxin-1 with the Na(+)-K(+) pump β1-subunit, which may reflect facilitation of deglutathionylation. In vivo β3-AR activation relieves oxidative inhibition of key cardiac myocyte proteins in hyperglycemia and may be effective in targeting the deleterious cardiac effects of diabetes. PMID:26063704

  13. β3-Adrenoceptor activation relieves oxidative inhibition of the cardiac Na+-K+ pump in hyperglycemia induced by insulin receptor blockade

    PubMed Central

    Karimi Galougahi, Keyvan; Liu, Chia-Chi; Garcia, Alvaro; Fry, Natasha A.; Hamilton, Elisha J.; Figtree, Gemma A.

    2015-01-01

    Dysregulated nitric oxide (NO)- and superoxide (O2·−)-dependent signaling contributes to the pathobiology of diabetes-induced cardiovascular complications. We examined if stimulation of β3-adrenergic receptors (β3-ARs), coupled to endothelial NO synthase (eNOS) activation, relieves oxidative inhibition of eNOS and the Na+-K+ pump induced by hyperglycemia. Hyperglycemia was established in male New Zealand White rabbits by infusion of the insulin receptor antagonist S961 for 7 days. Hyperglycemia increased tissue and blood indexes of oxidative stress. It induced glutathionylation of the Na+-K+ pump β1-subunit in cardiac myocytes, an oxidative modification causing pump inhibition, and reduced the electrogenic pump current in voltage-clamped myocytes. Hyperglycemia also increased glutathionylation of eNOS, which causes its uncoupling, and increased coimmunoprecipitation of cytosolic p47phox and membranous p22phox NADPH oxidase subunits, consistent with NADPH oxidase activation. Blocking translocation of p47phox to p22phox with the gp91ds-tat peptide in cardiac myocytes ex vivo abolished the hyperglycemia-induced increase in glutathionylation of the Na+-K+ pump β1-subunit and decrease in pump current. In vivo treatment with the β3-AR agonist CL316243 for 3 days eliminated the increase in indexes of oxidative stress, decreased coimmunoprecipitation of p22phox with p47phox, abolished the hyperglycemia-induced increase in glutathionylation of eNOS and the Na+-K+ pump β1-subunit, and abolished the decrease in pump current. CL316243 also increased coimmunoprecipitation of glutaredoxin-1 with the Na+-K+ pump β1-subunit, which may reflect facilitation of deglutathionylation. In vivo β3-AR activation relieves oxidative inhibition of key cardiac myocyte proteins in hyperglycemia and may be effective in targeting the deleterious cardiac effects of diabetes. PMID:26063704

  14. Differential Effects of Camel Milk on Insulin Receptor Signaling – Toward Understanding the Insulin-Like Properties of Camel Milk

    PubMed Central

    Abdulrahman, Abdulrasheed O.; Ismael, Mohammad A.; Al-Hosaini, Khaled; Rame, Christelle; Al-Senaidy, Abdulrahman M.; Dupont, Joëlle; Ayoub, Mohammed Akli

    2016-01-01

    Previous studies on the Arabian camel (Camelus dromedarius) showed beneficial effects of its milk reported in diverse models of human diseases, including a substantial hypoglycemic activity. However, the cellular and molecular mechanisms involved in such effects remain completely unknown. In this study, we hypothesized that camel milk may act at the level of human insulin receptor (hIR) and its related intracellular signaling pathways. Therefore, we examined the effect of camel milk on the activation of hIR transiently expressed in human embryonic kidney 293 (HEK293) cells using bioluminescence resonance energy transfer (BRET) technology. BRET was used to assess, in live cells and real-time, the physical interaction between hIR and insulin receptor signaling proteins (IRS1) and the growth factor receptor-bound protein 2 (Grb2). Our data showed that camel milk did not promote any increase in the BRET signal between hIR and IRS1 or Grb2 in the absence of insulin stimulation. However, it significantly potentiated the maximal insulin-promoted BRET signal between hIR and Grb2 but not IRS1. Interestingly, camel milk appears to differentially impact the downstream signaling since it significantly activated ERK1/2 and potentiated the insulin-induced ERK1/2 but not Akt activation. These observations are to some extent consistent with the BRET data since ERK1/2 and Akt activation are known to reflect the engagement of Grb2 and IRS1 pathways, respectively. The preliminary fractionation of camel milk suggests the peptide/protein nature of the active component in camel milk. Together, our study demonstrates for the first time an allosteric effect of camel milk on insulin receptor conformation and activation with differential effects on its intracellular signaling. These findings should help to shed more light on the hypoglycemic activity of camel milk with potential therapeutic applications. PMID:26858689

  15. Differential Effects of Camel Milk on Insulin Receptor Signaling - Toward Understanding the Insulin-Like Properties of Camel Milk.

    PubMed

    Abdulrahman, Abdulrasheed O; Ismael, Mohammad A; Al-Hosaini, Khaled; Rame, Christelle; Al-Senaidy, Abdulrahman M; Dupont, Joëlle; Ayoub, Mohammed Akli

    2016-01-01

    Previous studies on the Arabian camel (Camelus dromedarius) showed beneficial effects of its milk reported in diverse models of human diseases, including a substantial hypoglycemic activity. However, the cellular and molecular mechanisms involved in such effects remain completely unknown. In this study, we hypothesized that camel milk may act at the level of human insulin receptor (hIR) and its related intracellular signaling pathways. Therefore, we examined the effect of camel milk on the activation of hIR transiently expressed in human embryonic kidney 293 (HEK293) cells using bioluminescence resonance energy transfer (BRET) technology. BRET was used to assess, in live cells and real-time, the physical interaction between hIR and insulin receptor signaling proteins (IRS1) and the growth factor receptor-bound protein 2 (Grb2). Our data showed that camel milk did not promote any increase in the BRET signal between hIR and IRS1 or Grb2 in the absence of insulin stimulation. However, it significantly potentiated the maximal insulin-promoted BRET signal between hIR and Grb2 but not IRS1. Interestingly, camel milk appears to differentially impact the downstream signaling since it significantly activated ERK1/2 and potentiated the insulin-induced ERK1/2 but not Akt activation. These observations are to some extent consistent with the BRET data since ERK1/2 and Akt activation are known to reflect the engagement of Grb2 and IRS1 pathways, respectively. The preliminary fractionation of camel milk suggests the peptide/protein nature of the active component in camel milk. Together, our study demonstrates for the first time an allosteric effect of camel milk on insulin receptor conformation and activation with differential effects on its intracellular signaling. These findings should help to shed more light on the hypoglycemic activity of camel milk with potential therapeutic applications. PMID:26858689

  16. 14-3-3 proteins interact with the insulin-like growth factor receptor but not the insulin receptor.

    PubMed Central

    Furlanetto, R W; Dey, B R; Lopaczynski, W; Nissley, S P

    1997-01-01

    We have used a yeast two-hybrid system to identify proteins which bind to the cytosolic portion of the type 1 insulin-like growth factor (IGF) receptor (IGFIR) but not the insulin receptor (IR). This analysis identified 14-3-3beta and zeta proteins. 14-3-3beta also binds to the IGFIR but not the IR in vitro and 14-3-3-IGFIR complexes are present in insect cells overexpressing the IGFIR cytoplasmic domain. 14-3-3 proteins are substrates of the IGFIR in the yeast system and in vitro. The interaction of 14-3-3 with the IGFIR requires receptor-kinase activity and maps to the C-terminus of the receptor, but does not depend on tyrosine residues in this or the juxtamembrane regions. Instead, the binding maps to serine residue 1283 and requires phosphorylation of this residue. 14-3-3 proteins are phosphoserine-binding proteins which have been shown to interact directly with components of the mitogenic and apoptotic signalling pathways, suggesting that they participate in growth regulation. Our findings suggest that 14-3-3 proteins may play a role in IGFIR signal transduction and may contribute to the differences in IGF and IR signalling capabilities. PMID:9581554

  17. Amelioration of Diabetic Mouse Nephropathy by Catalpol Correlates with Down-Regulation of Grb10 Expression and Activation of Insulin-Like Growth Factor 1 / Insulin-Like Growth Factor 1 Receptor Signaling

    PubMed Central

    Yang, Shasha; Deng, Huacong; Zhang, Qunzhou; Xie, Jing; Zeng, Hui; Jin, Xiaolong; Ling, Zixi; Shan, Qiaoyun; Liu, Momo; Ma, Yuefei; Tang, Juan; Wei, Qianping

    2016-01-01

    Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that can negatively regulate the insulin-like growth factor 1 receptor (IGF-1R). The IGF1-1R pathway is critical for cell growth and apoptosis and has been implicated in kidney diseases; however, it is still unknown whether Grb10 expression is up-regulated and plays a role in diabetic nephropathy. Catalpol, a major active ingredient of a traditional Chinese medicine, Rehmannia, has been reported to possess anti-inflammatory and anti-aging activities and then used to treat diabetes. Herein, we aimed to assess the therapeutic effect of catalpol on a mouse model diabetic nephropathy and the potential role of Grb10 in the pathogenesis of this diabetes-associated complication. Our results showed that catalpol treatment improved diabetes-associated impaired renal functions and ameliorated pathological changes in kidneys of diabetic mice. We also found that Grb10 expression was significantly elevated in kidneys of diabetic mice as compared with that in non-diabetic mice, while treatment with catalpol significantly abrogated the elevated Grb10 expression in diabetic kidneys. On the contrary, IGF-1 mRNA levels and IGF-1R phosphorylation were significantly higher in kidneys of catalpol-treated diabetic mice than those in non-treated diabetic mice. Our results suggest that elevated Grb10 expression may play an important role in the pathogenesis of diabetic nephropathy through suppressing IGF-1/IGF-1R signaling pathway, which might be a potential molecular target of catalpol for the treatment of this diabetic complication. PMID:26986757

  18. A novel regulation of IRS1 (insulin receptor substrate-1) expression following short term insulin administration

    PubMed Central

    2005-01-01

    Reduced insulin-mediated glucose transport in skeletal muscle is a hallmark of the pathophysiology of T2DM (Type II diabetes mellitus). Impaired intracellular insulin signalling is implicated as a key underlying mechanism. Attention has focused on early signalling events such as defective tyrosine phosphorylation of IRS1 (insulin receptor substrate-1), a major target for the insulin receptor tyrosine kinase. This is required for normal induction of signalling pathways key to many of the metabolic actions of insulin. Conversely, increased serine/threonine phosphorylation of IRS1 following prolonged insulin exposure (or in obesity) reduces signalling capacity, partly by stimulating IRS1 degradation. We now show that IRS1 levels in human muscle are actually increased 3-fold following 1 h of hyperinsulinaemic euglycaemia. Similarly, transient induction of IRS1 (3-fold) in the liver or muscle of rodents occurs following feeding or insulin injection respectively. The induction by insulin is also observed in cell culture systems, although to a lesser degree, and is not due to reduced proteasomal targeting, increased protein synthesis or gene transcription. Elucidation of the mechanism by which insulin promotes IRS1 stability will permit characterization of the importance of this novel signalling event in insulin regulation of liver and muscle function. Impairment of this process would reduce IRS1 signalling capacity, thereby contributing to the development of hyperinsulinaemia/insulin resistance prior to the appearance of T2DM. PMID:16128672

  19. Role of insulin receptor substrate-1 serine 307 phosphorylation and adiponectin in adipose tissue insulin resistance in late pregnancy.

    PubMed

    Sevillano, Julio; de Castro, Javier; Bocos, Carlos; Herrera, Emilio; Ramos, M Pilar

    2007-12-01

    Insulin resistance is a hallmark of late pregnancy both in human and rat. Adipose tissue is one of the tissues that most actively contributes to this reduced insulin sensitivity. The aim of the present study was to characterize the molecular mechanisms of insulin resistance in adipose tissue at late pregnancy. To this end, we analyzed the insulin signaling cascade in lumbar adipose tissue of nonpregnant and pregnant (d 20) rats both under basal and insulin-stimulated conditions. We found that the levels of relevant signaling proteins, such as insulin receptor (IR), IR substrate-1 (IRS-1), phosphatidylinositol 3-kinase, 3-phosphoinositide-dependent kinase-1, ERK1/2, and phosphatase and tensin homolog (PTEN) did not change at late pregnancy. However, insulin-stimulated tyrosine phosphorylation of both IR and IRS-1 were significantly decreased, coincident with decreased IRS-1/p85 association and impaired phosphorylation of AKR mouse thymoma viral protooncogene (Akt) and ERK1/2. This impaired activation of IRS-1 occurred together with an increase of IRS-1 phosphorylation at serine 307 and a decrease in adiponectin levels. To corroborate the role of IRS-1 in adipose tissue insulin resistance during pregnancy, we treated pregnant rats with the antidiabetic drug englitazone. Englitazone improved glucose tolerance, and this pharmacological reversal of insulin resistance was paralleled by an increase of adiponectin levels in adipose tissue as well as by a reduction of IRS-1 serine phosphorylation. Furthermore, the impaired insulin-stimulated tyrosine phosphorylation of IRS-1 in adipose tissue of pregnant animals could be restored ex vivo by treating isolated adipocytes with adiponectin. Together, our findings support a role for adiponectin and serine phosphorylation of IRS-1 in the modulation of insulin resistance in adipose tissue at late pregnancy. PMID:17823255

  20. Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex

    SciTech Connect

    Žáková, Lenka; Kletvíková, Emília; Lepšík, Martin; Collinsová, Michaela; Watson, Christopher J.; Turkenburg, Johan P.; Jiráček, Jiří; Brzozowski, Andrzej M.

    2014-10-01

    [AsnB26]- and [GlyB26]-insulin mutants attain a B26-turn like fold without assistance of chemical modifications. Their structures match the insulin receptor interface and expand the spectrum of insulin conformations. The structural characterization of the insulin–insulin receptor (IR) interaction still lacks the conformation of the crucial B21–B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms.

  1. Insulin and insulin-like growth factor I exert different effects on plasminogen activator production or cell growth in the ovine thyroid cell line OVNIS.

    PubMed

    Degryse, B; Maisonobe, F; Hovsépian, S; Fayet, G

    1991-11-01

    Insulin and Insulin-like Growth Factor I (IGF-I) are evaluated for their capacity to affect cell proliferation and plasminogen activator (PA) activity production in an ovine thyroid cell line OVNIS. Insulin at physiological and supraphysiological doses induces cell proliferation and increases PA activity. IGF-I, which is also clearly mitogenic for these cells, surprisingly does not modulate PA activity. The results indicate that the growth promoting effect is mediated through the insulin and IGF-I receptors whereas PA activity is solely regulated via the insulin receptors. PMID:1802921

  2. Insulin Receptor Signaling in Normal and Insulin-Resistant States

    PubMed Central

    Boucher, Jérémie; Kleinridders, André; Kahn, C. Ronald

    2014-01-01

    In the wake of the worldwide increase in type-2 diabetes, a major focus of research is understanding the signaling pathways impacting this disease. Insulin signaling regulates glucose, lipid, and energy homeostasis, predominantly via action on liver, skeletal muscle, and adipose tissue. Precise modulation of this pathway is vital for adaption as the individual moves from the fed to the fasted state. The positive and negative modulators acting on different steps of the signaling pathway, as well as the diversity of protein isoform interaction, ensure a proper and coordinated biological response to insulin in different tissues. Whereas genetic mutations are causes of rare and severe insulin resistance, obesity can lead to insulin resistance through a variety of mechanisms. Understanding these pathways is essential for development of new drugs to treat diabetes, metabolic syndrome, and their complications. PMID:24384568

  3. Statin induces apoptosis of human colon cancer cells and downregulation of insulin-like growth factor 1 receptor via proapoptotic ERK activation

    PubMed Central

    JANG, HYUN JOO; HONG, EUN MI; PARK, SE WOO; BYUN, HYUN WOO; KOH, DONG HEE; CHOI, MIN HO; KAE, SEA HYUB; LEE, JIN

    2016-01-01

    Insulin-like growth factor 1 (IGF-1) and IGF-1 receptor (IGF-1R) signaling plays an important role in tumor progression in patients with certain gastrointestinal tract cancers. In addition to lowering cholesterol in serum, statins have pleiotropic effects, including anti-oxidative, anti-inflammatory or anti-neoplastic effects. Therefore, the present study investigated whether statins could induce the apoptosis of colon cancer cells and regulate the expression of IGF-1R and its associated signaling pathways in the present study. It was demonstrated that simvastatin and pravastatin suppressed cell proliferation and induced cell death in human HT-29 cells, but simvastatin was more potent than pravastatin. Simvastatin induced apoptosis in a concentration-dependent manner. In addition, simvastatin suppressed the expression of IGF-1R and inhibited the activity of phosphorylated-extracellular signal-regulated kinase (ERK)1/2 and phosphorylated-Akt activated by IGF-1. Simvastatin and IGF-1 each stimulated the activity of phosphorylated ERK1/2. However, simvastatin inhibited cell proliferation and IGF-1 stimulated cell proliferation. Mevalonic acid and PD98059 reversed the ERK activation and apoptosis induced by treatment with simvastatin. It was concluded that simvastatin induces the apoptosis of human colon cancer cells and inhibits IGF-1-induced ERK and Akt expression via the downregulation of IGF-1R expression and proapoptotic ERK activation. Simvastatin may be beneficial for the treatment of colon cancer. The present study suggested that statin may possess therapeutic potential for the treatment of colon cancer. PMID:27347133

  4. Methanolic leaf extract of Gymnema sylvestre augments glucose uptake and ameliorates insulin resistance by upregulating glucose transporter-4, peroxisome proliferator-activated receptor-gamma, adiponectin, and leptin levels in vitro

    PubMed Central

    Kumar, Puttanarasaiah Mahesh; Venkataranganna, Marikunte V.; Manjunath, Kirangadur; Viswanatha, Gollapalle L.; Ashok, Godavarthi

    2016-01-01

    Aims: The present study was undertaken to evaluate the effect of methanolic leaf extract of Gymnema sylvestre (MLGS) on glucose transport (GLUT) and insulin resistance in vitro. Materials and Methods: Peroxisome proliferator-activated receptor-gamma (PPAR-γ) and GLUT-4 expression were assessed in L6 myotubes for concluding the GLUT activity, and adiponectin and leptin expression was studied in 3T3 L1 murine adipocyte cell line to determine the effect of MLGS (250-750 μg/ml) on insulin resistance. Results: The findings of the experiments have demonstrated a significant and dose-dependent increase in glucose uptake in all the tested concentrations of MLGS, further the glucose uptake activity of MLGS (750 μg/ml) was at par with rosiglitazone (50 μg/ml). Concomitantly, MLGS has shown enhanced GLUT-4 and PPAR-γ gene expressions in L6 myotubes. Furthermore, cycloheximide (CHX) had completely abolished the glucose uptake activity of MLGS when co-incubated, which further confirmed that glucose uptake activity of MLGS was linked to enhanced expression of GLUT-4 and PPAR-γ. In addition, in another experimental set, MLGS showed enhanced expression of adiponectin and leptin, thus confirms the ameliorative effect of MLGS on insulin resistance. Conclusion: These findings suggest that MLGS has an enhanced glucose uptake activity in L6 myotubes, and ameliorate the insulin resistance in 3T3 L1 murine adipocyte cell line in vitro. PMID:27104035

  5. An insulin with the native sequence but virtually no activity.

    PubMed

    Wollmer, A; Gilge, G; Brandenburg, D; Gattner, H G

    1994-03-01

    The B24-B25 peptide bond of insulin was replaced by an ester bond. To our knowledge this is the first replacement of a main chain atom reported for the hormone. It is meant to eliminate a structurally important H-bond between the imino group of B25 and the carbonyl oxygen of A19, and consequently to enhance detachment of the C-terminal B chain from the underlying A chain. On the basis of independent experimental evidence this very conformational change is believed to be a prerequisite for receptor binding. It was thus anticipated that increased flexibility would increase receptor binding and activity. Intriguingly, porcine [B24-B25 CO-O]insulin (depsi-insulin) and likewise [B24-B25 CO-O]des-(B26-B30)insulin-B25-amide (depsi-DPI-amide) were found to be only 3-4% potent. PMID:8011179

  6. Determination of Alkali-Sensing Parts of the Insulin Receptor-Related Receptor Using the Bioinformatic Approach

    PubMed Central

    Deyev, I. E.; Popova, N. V.; Petrenko, A. G.

    2015-01-01

    IRR (insulin receptor-related receptor) is a receptor tyrosine kinase belonging to the insulin receptor family, which also includes insulin receptor and IGF-IR receptor. We have previously shown that IRR is activated by extracellular fluid with pH > 7.9 and regulates excess alkali excretion in the body. We performed a bioinformatic analysis of the pH-sensitive potential of all three members of the insulin receptor family of various animal species (from frog to man) and their chimeras with swapping of different domains in the extracellular region. An analysis using the AcalPred program showed that insulin receptor family proteins are divided into two classes: one class with the optimal working pH in the acidic medium (virtually all insulin receptor and insulin-like growth factor receptor orthologs, except for the IGF-IR ortholog from Xenopus laevis) and the second class with the optimal working pH in the alkaline medium (all IRR orthologs). The program had predicted that the most noticeable effect on the pH-sensitive property of IRR would be caused by the replacement of the L1 and C domains in its extracellular region, as well as the replacement of the second and third fibronectin repeats. It had also been assumed that replacement of the L2 domain would have the least significant effect on the alkaline sensitivity of IRR. To test the in silico predictions, we obtained three constructs with swapping of the L1C domains, the third L2 domain, and all three domains L1CL2 of IRR with similar domains of the insulin-like growth factor receptor. We found that replacement of the L1C and L1CL2 domains reduces the receptor’s ability to be activated with alkaline pH, thus increasing the half-maximal effective concentration by about 100%. Replacement of the L2 domain increased the half-maximal effective concentration by 40%. Thus, our results indicate the high predictive potential of the AcalPred algorithm, not only for the pH-sensitive enzymes, but also for p

  7. A study of brain insulin receptors, AChE activity and oxidative stress in rat model of ICV STZ induced dementia.

    PubMed

    Agrawal, Rahul; Tyagi, Ethika; Shukla, Rakesh; Nath, Chandishwar

    2009-03-01

    In the present study, role of brain insulin receptors (IRs) in memory functions and its correlation with acetylcholinesterase (AChE) activity and oxidative stress in different brain regions were investigated in intracerebroventricular (ICV) streptozotocin (STZ) induced dementia model. Rats were treated with STZ (3 mg/kg, ICV) on day 1 and 3. Donepezil (5 mg/kg po) and melatonin (20 mg/kg ip) were administered in pre- and post-treatment schedules. Morris water maze test was done on day 14 and animals were sacrificed on day 21 from 1st STZ injection. Memory deficit was found in STZ group as indicated by no significant decrease in latency time antagonized by donepezil and melatonin. IR protein level was found significantly increased in trained group as compared to control, whereas STZ decreased IR level significantly as compared to trained rats in hippocampus which indicates that IR is associated with memory functions. STZ induced decrease in IR was reversed by melatonin but not by donepezil. Melatonin per se did not show any significant change in IR level as compared to control. AChE activity (DS and SS fraction) was found to be increased in hippocampus in STZ group as compared to trained which was inhibited by donepezil and melatonin. Increase in MDA level and decrease in GSH level were obtained in STZ group indicating oxidative stress, which was attenuated by donepezil and melatonin. Effectiveness of antioxidant, melatonin but not of anti-cholinesterase, donepezil against STZ induced changes in IR indicates that IR is more affected with oxidative stress than cholinergic changes. PMID:19705549

  8. Oxidative enhancement of insulin receptor signaling: experimental findings and clinical implications.

    PubMed

    Dröge, Wulf

    2005-01-01

    Signaling through the insulin receptor and several other receptor tyrosine kinases is subject to redox regulation. Prolonged exposure to hydrogen peroxide impairs the action of insulin, and may account to some extent for the decreased insulin responsiveness in hyperglycemic diabetic patients. However, insulin receptor kinase (IRK) autophosphorylation and/or kinase activity were found to be markedly enhanced by a more limited exposure to hydrogen peroxide or by an oxidative shift in the thiol/disulfide redox status. Oxidative enhancement of IRK function may be mediated by two different mechanisms with similar effects, i.e., by direct oxidative activation of IRK activity or by oxidative inactivation of a protein tyrosine phosphatase, which otherwise down-regulates IRK-mediated signaling. As both mechanisms enhance IRK activity in the absence of insulin, there is a strong possibility that the background IRK activity in the postabsorptive period may be abnormally increased in certain oxidative conditions and thereby disturb the metabolism of glucose and other energy substrates. This remains to be tested. In line with the oxidative enhancement of IRK activity, clinical studies have shown that treatment with a thiol-containing antioxidant increases the postabsorptive glucose and/or insulin concentrations (i.e., the HOMA-R index) at least under certain conditions. This effect may have therapeutic implications. PMID:15998262

  9. A Comparative Structural Bioinformatics Analysis of the Insulin Receptor Family Ectodomain Based on Phylogenetic Information

    PubMed Central

    Rentería, Miguel E.; Gandhi, Neha S.; Vinuesa, Pablo; Helmerhorst, Erik; Mancera, Ricardo L.

    2008-01-01

    The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight ‘twist’ rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals. PMID:18989367

  10. Insulin Restores Gestational Diabetes Mellitus–Reduced Adenosine Transport Involving Differential Expression of Insulin Receptor Isoforms in Human Umbilical Vein Endothelium

    PubMed Central

    Westermeier, Francisco; Salomón, Carlos; González, Marcelo; Puebla, Carlos; Guzmán-Gutiérrez, Enrique; Cifuentes, Fredi; Leiva, Andrea; Casanello, Paola; Sobrevia, Luis

    2011-01-01

    OBJECTIVE To determine whether insulin reverses gestational diabetes mellitus (GDM)–reduced expression and activity of human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelium cells (HUVECs). RESEARCH DESIGN AND METHODS Primary cultured HUVECs from full-term normal (n = 44) and diet-treated GDM (n = 44) pregnancies were used. Insulin effect was assayed on hENT1 expression (protein, mRNA, SLC29A1 promoter activity) and activity (initial rates of adenosine transport) as well as endothelial nitric oxide (NO) synthase activity (serine1177 phosphorylation, l-citrulline formation). Adenosine concentration in culture medium and umbilical vein blood (high-performance liquid chromatography) as well as insulin receptor A and B expression (quantitative PCR) were determined. Reactivity of umbilical vein rings to adenosine and insulin was assayed by wire myography. Experiments were in the absence or presence of l-NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) or ZM-241385 (an A2A-adenosine receptor antagonist). RESULTS Umbilical vein blood adenosine concentration was higher, and the adenosine- and insulin-induced NO/endothelium-dependent umbilical vein relaxation was lower in GDM. Cells from GDM exhibited increased insulin receptor A isoform expression in addition to the reported NO–dependent inhibition of hENT1-adenosine transport and SLC29A1 reporter repression, and increased extracellular concentration of adenosine and NO synthase activity. Insulin reversed all these parameters to values in normal pregnancies, an effect blocked by ZM-241385 and l-NAME. CONCLUSIONS GDM and normal pregnancy HUVEC phenotypes are differentially responsive to insulin, a phenomenon where insulin acts as protecting factor for endothelial dysfunction characteristic of this syndrome. Abnormal adenosine plasma levels, and potentially A2A-adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM. PMID:21515851

  11. Effects of a combination of puerarin, baicalin and berberine on the expression of proliferator-activated receptor-γ and insulin receptor in a rat model of nonalcoholic fatty liver disease

    PubMed Central

    ZHAO, WEIHAN; LIU, LIJUAN; WANG, YUNLIANG; MAO, TANGYOU; LI, JUNXIANG

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) is a prevalent disease, with a clinical spectrum ranging from simple fatty liver disease to nonalcoholic steatohepatitis and cirrhosis. Puerarin, baicalin and berberine are herbal products widely used in Asia, which are believed to possess therapeutic benefits for alleviating the symptoms of NAFLD. In the present study, a rat model of NAFLD, induced by a high-fat diet, was established and orthographical experimentation was used to investigate the effects of various combinations of puerarin, baicalin and berberine on the hepatic expression of proliferator-activated receptor (PPAR)-γ and insulin receptor (IR). The present study demonstrated that serum levels of total cholesterol, alanine transaminase and low-density lipoproteins were significantly decreased in the puerarin-dominated groups (P<0.05 vs. the model group), whereas the concentrations of tumor necrosis factor-α and interleukin-6 were significantly improved in the baicalin- and berberine-dominated groups (P<0.05 vs. the model group). Furthermore, as compared with the control group, the levels of PPAR-γ/IR mRNA and protein expression were significantly decreased in the model group (P<0.01), and significantly increased in the rosiglitazone group and some of the orthogonal experiment groups (P<0.01). In conclusion, a combination of puerarin, baicalin and berberine induced favorable effects on NAFLD by upregulating hepatic PPAR-γ and IR expression levels, and different proportions of monomer compositions exerted variable positive effects on various stages of NAFLD. PMID:26889237

  12. Metabolic effect and receptor signalling profile of a non-metabolisable insulin glargine analogue

    PubMed Central

    Korn, Marcus; Schmidt, Ronald; Wendrich, Thomas M.; Tennagels, Norbert

    2014-01-01

    Context Insulin glargine (GLA) is rapidly metabolized in vivo to metabolite M1, which has in vitro metabolic and mitogenic profiles comparable with human insulin (HI). Objective To investigate the pharmacologic and signalling profiles of a non-metabolizable analogue (A21Gly,DiD-Arg) insulin (D-GLA). Methods Rats were injected s.c. with 1, 12.5 or 200 U/kg of GLA or D-GLA; blood glucose and phosphorylation status of the insulin receptor (IR), Akt and IGF-1 receptor (IGF1R) in tissue samples were investigated after 1 h. Plasma samples were analysed for insulin by LC-MS/MS. Results Blood glucose lowering was prolonged with D-GLA. D-GLA comprised ≥98% of insulin after D-GLA injection; M1 comprised 76–92% after GLA injection. IR and Akt phosphorylation were comparable with GLA and D-GLA. Neither analogue stimulated IGF1R phosphorylation. Conclusions Suprapharmacological doses of D-GLA did not activate IGF1R in vivo. Mitogenic effects of insulin and insulin analogues might be solely based on IR growth-promoting activity. PMID:25144413

  13. Insulin-degrading enzyme as a downstream target of insulin receptor signaling cascade: implications for Alzheimer's disease intervention.

    PubMed

    Zhao, Lixia; Teter, Bruce; Morihara, Takashi; Lim, Giselle P; Ambegaokar, Surendra S; Ubeda, Oliver J; Frautschy, Sally A; Cole, Greg M

    2004-12-01

    Insulin-degrading enzyme (IDE) is one of the proteins that has been demonstrated to play a key role in degrading beta-amyloid (Abeta) monomer in vitro and in vivo, raising the possibility of upregulating IDE as an approach to reduce Abeta. Little is known, however, about the cellular and molecular regulation of IDE protein. Because one of the main functions of IDE is to degrade insulin, we hypothesized that there is a negative feedback mechanism whereby stimulation of insulin receptor-mediated signaling upregulates IDE to prevent chronic activation of the pathway. We show that treatment of primary hippocampal neurons with insulin increased IDE protein levels by approximately 25%. Insulin treatment also led to phosphatidylinositol-3 (PI3) kinase activation evidenced by Akt phosphorylation, which was blocked by PI3 kinase inhibitors, wortmannin and LY 294002. Inhibition of PI3 kinase abolished the IDE upregulation by insulin, indicating a cause-effect relationship between insulin signaling and IDE upregulation. Further support for this link was provided by the findings that deficient insulin signaling (decreased PI3 kinase subunit P85) was correlated with reduced IDE in Alzheimer's disease (AD) brains and in Tg2576 Swedish amyloid precursor protein transgenic mice fed a safflower oil-enriched ("Bad") diet used to accelerate pathogenesis. Consistent with IDE function in the degradation of Abeta monomer, the IDE decrease in the Bad diet-fed Tg2576 mice was associated with increased Abeta monomer levels. These in vitro and in vivo analyses validate the use of enhanced CNS insulin signaling as a potential strategy for AD intervention to correct the IDE defects occurring in AD. PMID:15590928

  14. The Insulin Receptor: A New Target for Cancer Therapy

    PubMed Central

    Malaguarnera, Roberta; Belfiore, Antonino

    2011-01-01

    A large body of evidences have shown that both the IGF-I receptor (IGF-IR) and the insulin receptor (IR) play a role in cancer development and progression. In particular, IR overactivation by IGF-II is common in cancer cells, especially in dedifferentiated/stem-like cells. In spite of these findings, until very recently, only IGF-IR but not IR has been considered a target in cancer therapy. Although several preclinical studies have showed a good anti-cancer activity of selective anti-IGF-IR drugs, the results of the clinical first trials have been disappointing. In fact, only a small subset of malignant tumors has shown an objective response to these therapies. Development of resistance to anti-IGF-IR drugs may include upregulation of IR isoform A (IR-A) in cancer cells and its overactivation by increased secretion of autocrine IGF-II. These findings have led to the concept that co-targeting IR together with IGF-IR may increase therapy efficacy and prevent adaptive resistance to selective anti-IGF-IR drugs. IR blockade should be especially considered in tumors with high IR-A:IGF-IR ratio and high levels of autocrine IGF-II. Conversely, insulin sensitizers, which ameliorate insulin resistance associated with metabolic disorders and cancer treatments, may have important implications for cancer prevention and management. Only few drugs co-targeting the IR and IGF-IR are currently available. Ideally, future IR targeting strategies should be able to selectively inhibit the tumor promoting effects of IR without impairing its metabolic effects. PMID:22654833

  15. Insulin receptors and downstream substrates associate with membrane microdomains after treatment with insulin or chromium(III) picolinate.

    PubMed

    Al-Qatati, Abeer; Winter, Peter W; Wolf-Ringwall, Amber L; Chatterjee, Pabitra B; Van Orden, Alan K; Crans, Debbie C; Roess, Deborah A; Barisas, B George

    2012-04-01

    We have examined the association of insulin receptors (IR) and downstream signaling molecules with membrane microdomains in rat basophilic leukemia (RBL-2H3) cells following treatment with insulin or tris(2-pyridinecarbxylato)chromium(III) (Cr(pic)(3)). Single-particle tracking demonstrated that individual IR on these cells exhibited reduced lateral diffusion and increased confinement within 100 nm-scale membrane compartments after treatment with either 200 nM insulin or 10 μM Cr(pic)(3). These treatments also increased the association of native IR, phosphorylated insulin receptor substrate 1 and phosphorylated AKT with detergent-resistant membrane microdomains of characteristically high buoyancy. Confocal fluorescence microscopic imaging of Di-4-ANEPPDHQ labeled RBL-2H3 cells also showed that plasma membrane lipid order decreased following treatment with Cr(pic)(3) but was not altered by insulin treatment. Fluorescence correlation spectroscopy demonstrated that Cr(pic)(3) did not affect IR cell-surface density or compete with insulin for available binding sites. Finally, Fourier transform infrared spectroscopy indicated that Cr(pic)(3) likely associates with the lipid interface in reverse-micelle model membranes. Taken together, these results suggest that activation of IR signaling in a cellular model system by both insulin and Cr(pic)(3) involves retention of IR in specialized nanometer-scale membrane microdomains but that the insulin-like effects of Cr(pic)(3) are due to changes in membrane lipid order rather than to direct interactions with IR. PMID:22101510

  16. Cross-talk between insulin and IGF-1 receptors in the cortical collecting duct principal cells: implication for ENaC-mediated Na+ reabsorption

    PubMed Central

    Ilatovskaya, Daria V.; Levchenko, Vladislav; Brands, Michael W.; Pavlov, Tengis S.

    2015-01-01

    Insulin and IGF-1 are recognized as powerful regulators of the epithelial Na+ channel (ENaC) in the aldosterone-sensitive distal nephron. As previously described, these hormones both acutely increase ENaC activity in freshly isolated split open tubules and cultured principal cortical collecting duct cells. The present study was aimed at differentiating the effects of insulin and IGF-1 on Na+ transport in immortalized mpkCCDcl4 cells and defining their interrelations. We have shown that both insulin and IGF-1 applied basolaterally, but not apically, enhanced transepithelial Na+ transport in the mpkCCDcl4 cell line with EC50 values of 8.8 and 14.5 nM, respectively. Insulin treatment evoked phosphorylation of both insulin and IGF-1 receptors, whereas the effects of IGF-1 were more profound on its own receptor rather than the insulin receptor. AG-1024 and PPP, inhibitors of IGF-1 and insulin receptor tyrosine kinase activity, diminished insulin- and IGF-1-stimulated Na+ transport in mpkCCDcl4 cells. The effects of insulin and IGF-1 on ENaC-mediated currents were found to be additive, with insulin likely stimulating both IGF-1 and insulin receptors. We hypothesize that insulin activates IGF-1 receptors in addition to its own receptors, making the effects of these hormones interconnected. PMID:25651558

  17. Insulin-Like Growth Factor 1 Receptor and p38 Mitogen-Activated Protein Kinase Signals Inversely Regulate Signal Transducer and Activator of Transcription 3 Activity to Control Human Dental Pulp Stem Cell Quiescence, Propagation, and Differentiation

    PubMed Central

    Vandomme, Jerome; Touil, Yasmine; Ostyn, Pauline; Olejnik, Cecile; Flamenco, Pilar; El Machhour, Raja; Segard, Pascaline; Masselot, Bernadette; Bailliez, Yves; Formstecher, Pierre

    2014-01-01

    Dental pulp stem cells (DPSCs) remain quiescent until activated in response to severe dental pulp damage. Once activated, they exit quiescence and enter regenerative odontogenesis, producing reparative dentin. The factors and signaling molecules that control the quiescence/activation and commitment to differentiation of human DPSCs are not known. In this study, we determined that the inhibition of insulin-like growth factor 1 receptor (IGF-1R) and p38 mitogen-activated protein kinase (p38 MAPK) signaling commonly activates DPSCs and promotes their exit from the G0 phase of the cell cycle as well as from the pyronin Ylow stem cell compartment. The inhibition of these two pathways, however, inversely determines DPSC fate. In contrast to p38 MAPK inhibitors, IGF-1R inhibitors enhance dental pulp cell sphere-forming capacity and reduce the cells' colony-forming capacity without inducing cell death. The inverse cellular changes initiated by IGF-1R and p38 MAPK inhibitors were accompanied by inverse changes in the levels of active signal transducer and activator of transcription 3 (STAT3) factor, inactive glycogen synthase kinase 3, and matrix extracellular phosphoglycoprotein, a marker of early odontoblast differentiation. Our data suggest that there is cross talk between the IGF-1R and p38 MAPK signaling pathways in DPSCs and that the signals provided by these pathways converge at STAT3 and inversely regulate its activity to maintain quiescence or to promote self-renewal and differentiation of the cells. We propose a working model that explains the possible interactions between IGF-1R and p38 MAPK at the molecular level and describes the cellular consequences of these interactions. This model may inspire further fundamental study and stimulate research on the clinical applications of DPSC in cellular therapy and tissue regeneration. PMID:24266654

  18. Insulin-like growth factor 1 receptor and p38 mitogen-activated protein kinase signals inversely regulate signal transducer and activator of transcription 3 activity to control human dental pulp stem cell quiescence, propagation, and differentiation.

    PubMed

    Vandomme, Jerome; Touil, Yasmine; Ostyn, Pauline; Olejnik, Cecile; Flamenco, Pilar; El Machhour, Raja; Segard, Pascaline; Masselot, Bernadette; Bailliez, Yves; Formstecher, Pierre; Polakowska, Renata

    2014-04-15

    Dental pulp stem cells (DPSCs) remain quiescent until activated in response to severe dental pulp damage. Once activated, they exit quiescence and enter regenerative odontogenesis, producing reparative dentin. The factors and signaling molecules that control the quiescence/activation and commitment to differentiation of human DPSCs are not known. In this study, we determined that the inhibition of insulin-like growth factor 1 receptor (IGF-1R) and p38 mitogen-activated protein kinase (p38 MAPK) signaling commonly activates DPSCs and promotes their exit from the G0 phase of the cell cycle as well as from the pyronin Y(low) stem cell compartment. The inhibition of these two pathways, however, inversely determines DPSC fate. In contrast to p38 MAPK inhibitors, IGF-1R inhibitors enhance dental pulp cell sphere-forming capacity and reduce the cells' colony-forming capacity without inducing cell death. The inverse cellular changes initiated by IGF-1R and p38 MAPK inhibitors were accompanied by inverse changes in the levels of active signal transducer and activator of transcription 3 (STAT3) factor, inactive glycogen synthase kinase 3, and matrix extracellular phosphoglycoprotein, a marker of early odontoblast differentiation. Our data suggest that there is cross talk between the IGF-1R and p38 MAPK signaling pathways in DPSCs and that the signals provided by these pathways converge at STAT3 and inversely regulate its activity to maintain quiescence or to promote self-renewal and differentiation of the cells. We propose a working model that explains the possible interactions between IGF-1R and p38 MAPK at the molecular level and describes the cellular consequences of these interactions. This model may inspire further fundamental study and stimulate research on the clinical applications of DPSC in cellular therapy and tissue regeneration. PMID:24266654

  19. The role of thermosensitive TRP (transient receptor potential) channels in insulin secretion.

    PubMed

    Uchida, Kunitoshi; Tominaga, Makoto

    2011-01-01

    Insulin secretion from pancreatic β-cells is the only efficient means to decrease blood glucose concentrations. Glucose is the principal stimulator of insulin secretion with the ATP-sensitive K+ channel-voltage-gated Ca2+ channel-mediated pathway being the primary one involved in glucose-stimulated insulin secretion. Recently, several reports demonstrated that some transient receptor potential (TRP) channels are expressed in pancreatic β-cells and contribute to pancreatic β-cell functions. Interestingly, six of them (TRPM2, TRPM4, TRPM5, TRPV1, TRPV2 and TRPV4) are thermosensitive TRP channels. Thermosensitive TRP channels in pancreatic β-cells can function as multimodal receptors and cause Ca2+ influx and membrane depolarization at physiological body temperature. TRPM channels (TRPM2, TRPM4 and TRPM5) control insulin secretion levels by sensing intracellular Ca2+ increase, NAD metabolites, or hormone receptor activation. TRPV2 is involved not only in insulin secretion but also cell proliferation, and is regulated by the autocrine effects of insulin. TRPV1 expressed in sensory neurons is involved in β-cell stress and islet inflammation by controlling neuropeptide release levels. It is thus clear that thermosensitive TRP channels play important roles in pancreatic β-cell functions, and future analyses of TRP channel function will lead to better understanding of the complicated mechanisms involved in insulin secretion and diabetes pathogenesis. PMID:21785227

  20. Identification of Host Insulin Binding Sites on Schistosoma japonicum Insulin Receptors

    PubMed Central

    Stephenson, Rachel J.; Toth, Istvan; Liang, Jiening; Mangat, Amanjot; McManus, Donald P.; You, Hong

    2016-01-01

    Schistosoma japonicum insulin receptors (SjIRs) have been identified as encouraging vaccine candidates. Interrupting or blocking the binding between host insulin and the schistosome insulin receptors (IRs) may result in reduced glucose uptake leading to starvation and stunting of worms with a reduction in egg output. To further understand how schistosomes are able to exploit host insulin for development and growth, and whether these parasites and their mammalian hosts compete for the same insulin source, we identified insulin binding sites on the SjIRs. Based on sequence analysis and the predicted antigenic structure of the primary sequences of the SjIRs, we designed nine and eleven peptide analogues from SjIR-1 and SjIR-2, respectively. Using the Octet RED system, we identified analogues derived from SjIR-1 (10) and SjIR-2 (20, 21 and 22) with insulin-binding sequences specific for S. japonicum. Nevertheless, the human insulin receptor (HIR) may compete with the SjIRs in binding human insulin in other positions which are important for HIR binding to insulin. However, no binding occurred between insulin and parasite analogues derived from SjIR-1 (2, 7 and 8) and SjIR-2 (14, 16 and 18) at the same locations as HIR sequences which have been shown to have strong insulin binding affinities. Importantly, we found two analogues (1 and 3), derived from SjIR-1, and two analogues (13 and 15) derived from SjIR-2, were responsible for the major insulin binding affinity in S. japonicum. These peptide analogues were shown to have more than 10 times (in KD value) stronger binding capacity for human insulin compared with peptides derived from the HIR in the same sequence positions. Paradoxically, analogues 1, 3, 13 and 15 do not appear to contain major antigenic determinants which resulted in poor antibody responses to native S. japonicum protein. This argues against their future development as peptide-vaccine candidates. PMID:27441998

  1. Ultrastructural evidence for the accumulation of insulin in nuclei of intact 3T3-L1 adipocytes by an insulin-receptor mediated process

    SciTech Connect

    Smith, R.M.; Jarett, L.

    1987-01-01

    Monomeric ferritin-labeled insulin (F/sub m/-Ins), a biologically active, electron-dense marker of occupied insulin receptors, was used to characterize the internalization of insulin in 3T3-L1 adipocytes. F/sub m/-Ins bound specifically to insulin receptors and was internalized in a time- and temperature-dependent manner. In the nucleus, several F/sub m/-Ins particles usually were found in the same general location-near nuclear pores, associated with the periphery of the condensed chromatin. Addition of a 250-fold excess of unlabeled insulin or incubation at 15/sup 0/C reduced the number of F/sub m/-Ins particles found in nuclei after 90 min by 99% or 92%, respectively. Nuclear accumulation of unlabeled ferritin was only 2% of that found with F/sub m/-Ins after 90 min at 37/sup 0/C. Biochemical experiments utilizing /sup 125/I-labeled insulin and subcellular fractionation indicated that intact 3T3-L1 adipocytes internalized insulin rapidly and that approx. = 3% of the internalized ligand accumulated in nuclei after 1 hr. These data provide biochemical and high-resolution ultrastructural evidence that 3T3-L1 adipocytes accumulate potentially significant amounts of insulin in nuclei by an insulin receptor-mediated process. The transport of insulin or the insulin-receptor complex to nuclei in this cell or in others may be directly involved in the long-term biological effects of insulin - in particular, in the control of DNA and RNA synthesis.

  2. Structural and Biochemical Characterization of the KRLB Region in Insulin Receptor Substrate-2

    SciTech Connect

    Wu,J.; Tseng, Y.; Xu, C.; Neubert, T.; White, M.; Hubbard, S.

    2008-01-01

    Insulin receptor substrates 1 and 2 (IRS1 and -2) are crucial adaptor proteins in mediating the metabolic and mitogenic effects of insulin and insulin-like growth factor 1. These proteins consist of a pleckstrin homology domain, a phosphotyrosine binding domain and a C-terminal region containing numerous sites of tyrosine, serine and threonine phosphorylation. Previous yeast two-hybrid studies identified a region unique to IRS2, termed the kinase regulatory-loop binding (KRLB) region, which interacts with the tyrosine kinase domain of the insulin receptor. Here we present the crystal structure of the insulin receptor kinase in complex with a 15-residue peptide from the KRLB region. In the structure, this segment of IRS2 is bound in the kinase active site with Tyr628 positioned for phosphorylation. Although Tyr628 was phosphorylated by the insulin receptor, its catalytic turnover was poor, resulting in kinase inhibition. Our studies indicate that the KRLB region functions to limit tyrosine phosphorylation of IRS2.

  3. PTPRD silencing by DNA hypermethylation decreases insulin receptor signaling and leads to type 2 diabetes.

    PubMed

    Chen, Yng-Tay; Lin, Wei-D; Liao, Wen-Lin; Lin, Ying-Ju; Chang, Jan-Gowth; Tsai, Fuu-Jen

    2015-05-30

    Genome-wide association study (GWAS) data showed that the protein tyrosine phosphatase receptor type delta (PTPRD) is associated with increased susceptibility to type 2 diabetes (T2D) in Han Chinese. A replication study indicated that PTPRD is involved in the insulin signaling pathway; however, the underlying mechanism remains unclear. We evaluated PTPRD expression in patients with T2D and controls. PTPRD expression levels were lower in patients and were correlated with the duration of the disease. Overexpression of the human insulin receptor PPARγ2 in HepG2 cells induced overexpression of PTPRD and the insulin receptor. PTPRD knockdown, using a shRNA, resulted in down-regulation of the insulin receptor. These results indicate that PTPRD activates PPARγ2 in the insulin signaling pathway. Similar results for PTPRD expression were found using a T2D mouse model. Silencing of PTPRD was caused by DNA methylation in T2D mice and patients, and correlated with DNMT1 expression. Furthermore, we showed that a DNMT1 SNP (rs78789647) was correlated with susceptibility to T2D. This study shows for the first time that DNMT1 caused PTPRD DNA hypermethylation and induced insulin signaling silencing in T2D patients. Our findings contribute to a better understanding of the crucial roles of these regulatory elements in human T2D. PMID:26079428

  4. PTPRD silencing by DNA hypermethylation decreases insulin receptor signaling and leads to type 2 diabetes

    PubMed Central

    Chen, Yng-Tay; Lin, Wei-De; Liao, Wen-Lin; Lin, Ying-Ju; Chang, Jan-Gowth; Tsai, Fuu-Jen

    2015-01-01

    Genome-wide association study (GWAS) data showed that the protein tyrosine phosphatase receptor type delta (PTPRD) is associated with increased susceptibility to type 2 diabetes (T2D) in Han Chinese. A replication study indicated that PTPRD is involved in the insulin signaling pathway; however, the underlying mechanism remains unclear. We evaluated PTPRD expression in patients with T2D and controls. PTPRD expression levels were lower in patients and were correlated with the duration of the disease. Overexpression of the human insulin receptor PPARγ2 in HepG2 cells induced overexpression of PTPRD and the insulin receptor. PTPRD knockdown, using a shRNA, resulted in down-regulation of the insulin receptor. These results indicate that PTPRD activates PPARγ2 in the insulin signaling pathway. Similar results for PTPRD expression were found using a T2D mouse model. Silencing of PTPRD was caused by DNA methylation in T2D mice and patients, and correlated with DNMT1 expression. Furthermore, we showed that a DNMT1 SNP (rs78789647) was correlated with susceptibility to T2D. This study shows for the first time that DNMT1 caused PTPRD DNA hypermethylation and induced insulin signaling silencing in T2D patients. Our findings contribute to a better understanding of the crucial roles of these regulatory elements in human T2D. PMID:26079428

  5. Nymphaea nouchali Burm. f. hydroalcoholic seed extract increases glucose consumption in 3T3-L1 adipocytes through activation of peroxisome proliferator-activated receptor gamma and insulin sensitization.

    PubMed

    Parimala, Mabel; Debjani, M; Vasanthi, Hannah Rachel; Shoba, Francis Gricilda

    2015-01-01

    Nymphaea nouchali Burm. f. (Family - Nymphaeaceae) is a well-known medicinal plant used in the Indian ayurvedic system of medicine for treating diabetes. The seeds especially have been prescribed for diabetes. The hydroalcoholic extract of N. nouchali seeds has been demonstrated to possess anti-hyperglycemic effects in diabetic rats, but the functional mechanism remains unknown. The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ) is noted to play an important role in glucose and lipid homeostasis. This study was hence focused in evaluating the effect of the extract on PPARγ activation, adipocyte differentiation, and glucose consumption in 3T3-L1 cells. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), followed by adipogenesis assay using Oil Red O technique. Glucose consumption of preadipocytes and adipocytes in the presence of the extract was also determined. Real-time polymerase chain reaction was performed to identify the expression of genes involved in glucose consumption in the adipocytes. MTT assay confirmed the extract to be nontoxic, and Oil Red O staining confirmed enhanced adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner. The extract also increased the expression of PPARγ target gene, which in turn enhanced the expression of GLUT-4. The data, therefore, suggests that N. nouchali seed extract promotes adipocyte differentiation and glucose consumption by inducing PPARγ activation, which in turn increases mRNA GLUT-4 expression and subsequently enhances insulin-responsiveness in insulin target tissues. PMID:26605160

  6. Nymphaea nouchali Burm. f. hydroalcoholic seed extract increases glucose consumption in 3T3-L1 adipocytes through activation of peroxisome proliferator-activated receptor gamma and insulin sensitization

    PubMed Central

    Parimala, Mabel; Debjani, M.; Vasanthi, Hannah Rachel; Shoba, Francis Gricilda

    2015-01-01

    Nymphaea nouchali Burm. f. (Family – Nymphaeaceae) is a well-known medicinal plant used in the Indian ayurvedic system of medicine for treating diabetes. The seeds especially have been prescribed for diabetes. The hydroalcoholic extract of N. nouchali seeds has been demonstrated to possess anti-hyperglycemic effects in diabetic rats, but the functional mechanism remains unknown. The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ) is noted to play an important role in glucose and lipid homeostasis. This study was hence focused in evaluating the effect of the extract on PPARγ activation, adipocyte differentiation, and glucose consumption in 3T3-L1 cells. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), followed by adipogenesis assay using Oil Red O technique. Glucose consumption of preadipocytes and adipocytes in the presence of the extract was also determined. Real-time polymerase chain reaction was performed to identify the expression of genes involved in glucose consumption in the adipocytes. MTT assay confirmed the extract to be nontoxic, and Oil Red O staining confirmed enhanced adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner. The extract also increased the expression of PPARγ target gene, which in turn enhanced the expression of GLUT-4. The data, therefore, suggests that N. nouchali seed extract promotes adipocyte differentiation and glucose consumption by inducing PPARγ activation, which in turn increases mRNA GLUT-4 expression and subsequently enhances insulin-responsiveness in insulin target tissues. PMID:26605160

  7. Antibodies to the extracellular receptor domain restore the hormone-insensitive kinase and conformation of the mutant insulin receptor valine 382.

    PubMed

    Lebrun, C; Baron, V; Kaliman, P; Gautier, N; Dolais-Kitabgi, J; Taylor, S; Accili, D; Van Obberghen, E

    1993-05-25

    A mutation substituting a valine for phenylalanine at residue 382 in the insulin receptor alpha-subunit has been found in two sisters with a genetic form of extreme insulin resistance. This receptor mutation impairs the ability of the hormone to activate autophosphorylation of solubilized receptors and phosphorylation of substrates (Accili, D., Mosthaf, L., Ullrich, A., and Taylor, S. I. (1991) J. Biol. Chem. 266, 434-439). We have previously demonstrated that in native receptors insulin induces a conformational change in the receptor beta-subunit, which is thought to be necessary for receptor activation (Baron, V., Gautier, N., Komoriya, A., Hainaut, P., Scimeca, J. C., Mervic, M., Lavielle, S., Dolais-Kitabgi, J., and Van Obberghen, E. (1990) Biochemistry 29, 4634-4641). Hence, it was thought that a defect in this conformational change might explain the functional defect of the mutant receptor. This appears to be the case, as we demonstrate here that the mutant receptor is locked in its inactive configuration. However, we found two monoclonal antibodies, directed to the extracellular domain, which are capable of restoring the mutant receptor kinase activity. The activation of the mutant receptor was accompanied by restoration of conformational changes in the beta-subunit C terminus. From these data, we draw the two following conclusions. (i) A causal link exists between receptor kinase activation and the occurrence of conformational changes. (ii) Ligands other than insulin, such as antibodies, which perturb the extracellular domain, can function as alternative ways to restore the mutant receptor kinase. PMID:8388389

  8. Insulin Directly Regulates Steroidogenesis via Induction of the Orphan Nuclear Receptor DAX-1 in Testicular Leydig Cells*

    PubMed Central

    Ahn, Seung Won; Gang, Gil-Tae; Kim, Yong Deuk; Ahn, Ryun-Sup; Harris, Robert A.; Lee, Chul-Ho; Choi, Hueng-Sik

    2013-01-01

    Testosterone level is low in insulin-resistant type 2 diabetes. Whether this is due to negative effects of high level of insulin on the testes caused by insulin resistance has not been studied in detail. In this study, we found that insulin directly binds to insulin receptors in Leydig cell membranes and activates phospho-insulin receptor-β (phospho-IR-β), phospho-IRS1, and phospho-AKT, leading to up-regulation of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) gene expression in the MA-10 mouse Leydig cell line. Insulin also inhibits cAMP-induced and liver receptor homolog-1 (LRH-1)-induced steroidogenic enzyme gene expression and steroidogenesis. In contrast, knockdown of DAX-1 reversed insulin-mediated inhibition of steroidogenesis. Whether insulin directly represses steroidogenesis through regulation of steroidogenic enzyme gene expression was assessed in insulin-injected mouse models and high fat diet-induced obesity. In insulin-injected mouse models, insulin receptor signal pathway was activated and subsequently inhibited steroidogenesis via induction of DAX-1 without significant change of luteinizing hormone or FSH levels. Likewise, the levels of steroidogenic enzyme gene expression and steroidogenesis were low, but interestingly, the level of DAX-1 was high in the testes of high fat diet-fed mice. These results represent a novel regulatory mechanism of steroidogenesis in Leydig cells. Insulin-mediated induction of DAX-1 in Leydig cells of testis may be a key regulatory step of serum sex hormone level in insulin-resistant states. PMID:23589295

  9. Insulin regulation of Na/K pump activity in rat hepatoma cells

    SciTech Connect

    Gelehrter, T.D.; Shreve, P.D.; Dilworth, V.M.

    1984-05-01

    Insulin rapidly increases Na/K pump activity in HTC rat hepatoma cells in tissue culture, as measured by the ouabain-sensitive influx of the potassium analogue 86Rb+. Increased influx is observed within minutes and is maximal (70% above control) within 1-2 h. The effect appears to be mediated by the insulin receptors, as: the concentration dependence on insulin is identical to that for insulin induction of tyrosine aminotransferase and stimulation of 2-aminoisobutyric acid transport, proinsulin is 6% as potent as insulin, and the effect is blocked by anti-receptor antibodies. The early stimulation of potassium influx is not blocked by cycloheximide and is not associated with an increased number of pump sites as measured by /sup 3/H-ouabain binding. The insulin effect is blocked by amiloride, which blocks sodium influx, and is mimicked by the sodium ionophore monensin, which increases sodium influx and intracellular accumulation. Insulin also rapidly increases the initial rate of /sup 22/Na+ influx, suggesting that insulin may enhance Na/K pump activity, in part, by increasing intracellular sodium concentration. Incubation of HTC cells with insulin for 24 h causes complete unresponsiveness to the insulin induction of transaminase and stimulation of amino acid transport, a phenomenon mediated by postbinding mechanisms. In contrast, similar incubation with insulin does not cause unresponsiveness to the insulin stimulation of Na/K pump activity. Therefore, the site of regulation of responsiveness to insulin must be distal to, or separate from, those events causing stimulation of ion fluxes.

  10. Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex.

    PubMed

    Záková, Lenka; Kletvíková, Emília; Lepšík, Martin; Collinsová, Michaela; Watson, Christopher J; Turkenburg, Johan P; Jiráček, Jiří; Brzozowski, Andrzej M

    2014-10-01

    The structural characterization of the insulin-insulin receptor (IR) interaction still lacks the conformation of the crucial B21-B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms. PMID:25286859

  11. Aleglitazar, a dual peroxisome proliferator-activated receptor-α/γ agonist, improves insulin sensitivity, glucose control and lipid levels in people with type 2 diabetes: findings from a randomized, double-blind trial.

    PubMed

    Stirban, A O; Andjelkovic, M; Heise, T; Nosek, L; Fischer, A; Gastaldelli, A; Herz, M

    2016-07-01

    The present single-centre, randomized, double-blind, placebo-controlled phase II study investigated the effect of the balanced dual peroxisome proliferator-activated receptor-α/γ agonist aleglitazar on whole-body and liver insulin sensitivity, β-cell function and other components of cardiometabolic syndrome after 16 weeks of treatment in patients with type 2 diabetes inadequately controlled with metformin monotherapy who received once-daily 150 µg aleglitazar or matching placebo as add-on therapy to metformin. Baseline and 16-week assessments included a two-step hyperinsulinaemic-euglycaemic clamp, followed by a hyperglycaemic clamp, as well as evaluation of glycated haemoglobin (HbA1c), lipids and safety variables. The primary endpoint was change in whole-body insulin sensitivity (M-value) from baseline compared with placebo, derived from the second clamp step. M-value improved significantly from baseline with aleglitazar (n = 16) compared with placebo (n = 24; p = 0.05 for difference between arms). We found statistically significant treatment differences with aleglitazar versus placebo in fasting hepatic insulin resistance index (p = 0.01), and in total glucose disposal (p = 0.03) at the second insulin infusion step. Aleglitazar treatment resulted in significant improvements in HbA1c and lipids and was well tolerated. PMID:26663152

  12. [Insulin receptor defect as a cause of Rabson-Mendenhall syndrome and other rare genetic insulin resistance syndromes].

    PubMed

    Rojek, Aleksandra; Niedziela, Marek

    2010-01-01

    Insulin plays a very important role in maintaining homeostasis of the whole organism. It regulates glucose metabolism, glycogen synthesis, lipid and protein metabolism. Insulin receptors are present in virtually all cells, which is reflected by the diversity of regulatory processes in which this hormone is involved. Any dysfunction of insulin signalling pathway as a result of insulin receptor gene mutations is linked with various forms of insulin resistance, including insulin resistance type A, Donohue or Rabson-Mendenhall syndrome, which differ in the level of severity. Molecular analysis of insulin receptor gene may lead to a better understanding of molecular mechanisms underlying various types of insulin resistance and help to develop a more efficient treatment. They may also be used as a powerful tool in prenatal diagnostics as well as in pregnancy planning. PMID:21092701

  13. Insulin receptor mutation results in insulin resistance and hyperinsulinemia but does not exacerbate Alzheimer's-like phenotypes in mice.

    PubMed

    Murakami, Kazuma; Yokoyama, Shin-ichi; Murata, Nakaba; Ozawa, Yusuke; Irie, Kazuhiro; Shirasawa, Takuji; Shimizu, Takahiko

    2011-05-27

    Obesity is a risk factor for Alzheimer's disease (AD), which is characterized by amyloid β depositions and cognitive dysfunction. Although insulin resistance is one of the phenotypes of obesity, its deleterious effects on AD progression remain to be fully elucidated. We previously reported that the suppression of insulin signaling in a mouse with a heterozygous mutation (P1195L) in the gene for the insulin receptor showed insulin resistance and hyperinsulinemia but did not develop diabetes mellitus [15]. Here, we generated a novel AD mouse model carrying the same insulin receptor mutation and showed that the combination of insulin resistance and hyperinsulinemia did not accelerate plaque formation or memory abnormalities in these mice. Interestingly, the insulin receptor mutation reduced oxidative damage in the brains of the AD mice. These findings suggest that insulin resistance is not always involved in the pathogenesis of AD. PMID:21549686

  14. Theoretical and Computational Studies of Peptides and Receptors of the Insulin Family

    PubMed Central

    Vashisth, Harish

    2015-01-01

    Synergistic interactions among peptides and receptors of the insulin family are required for glucose homeostasis, normal cellular growth and development, proliferation, differentiation and other metabolic processes. The peptides of the insulin family are disulfide-linked single or dual-chain proteins, while receptors are ligand-activated transmembrane glycoproteins of the receptor tyrosine kinase (RTK) superfamily. Binding of ligands to the extracellular domains of receptors is known to initiate signaling via activation of intracellular kinase domains. While the structure of insulin has been known since 1969, recent decades have seen remarkable progress on the structural biology of apo and liganded receptor fragments. Here, we review how this useful structural information (on ligands and receptors) has enabled large-scale atomically-resolved simulations to elucidate the conformational dynamics of these biomolecules. Particularly, applications of molecular dynamics (MD) and Monte Carlo (MC) simulation methods are discussed in various contexts, including studies of isolated ligands, apo-receptors, ligand/receptor complexes and intracellular kinase domains. The review concludes with a brief overview and future outlook for modeling and computational studies in this family of proteins. PMID:25680077

  15. All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors

    PubMed Central

    Mohammadiarani, Hossein; Vashisth, Harish

    2016-01-01

    The receptor tyrosine kinase superfamily comprises many cell-surface receptors including the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) that are constitutively homodimeric transmembrane glycoproteins. Therefore, these receptors require ligand-triggered domain rearrangements rather than receptor dimerization for activation. Specifically, binding of peptide ligands to receptor ectodomains transduces signals across the transmembrane domains for trans-autophosphorylation in cytoplasmic kinase domains. The molecular details of these processes are poorly understood in part due to the absence of structures of full-length receptors. Using MD simulations and enhanced conformational sampling algorithms, we present all-atom structural models of peptides containing 51 residues from the transmembrane and juxtamembrane regions of IR and IGF1R. In our models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxtamembrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. We also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane–solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane–solvent interfaces. Our metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. Our all-atom structural models suggest potentially unique structural organization of kinase domains in each receptor. PMID

  16. Insulin-like activity in the retina

    SciTech Connect

    Das, A.

    1986-01-01

    A number of studies have recently demonstrated that insulin or a homologous peptide may be synthesized outside the pancreas also. The present study was designed to investigate whether insulin-like activity exists in the retina, and if it exists, whether it is due to local synthesis of insulin or a similar peptide in the retina. To determine whether the insulin-like immunoreactivity in retinal glial cells is due to binding and uptake or local synthesis of insulin, a combined approach of immunocytochemistry and in situ DNA-RNA hybridization techniques was used on cultured rat retinal glial cells. Insulin-like immunoreactivity was demonstrated in the cytoplasma of these cells. In situ hybridization studies using labeled rat insulin cDNA indicated that these cells contain the mRNA necessary for de novo synthesis of insulin or a closely homologous peptide. Since human retinal cells have, as yet, not been conveniently grown in culture, an ocular tumor cell line, human Y79 retinoblastoma was used as a model to extend these investigations. The presence of insulin-like immunoreactivity as well as insulin-specific mRNA was demonstrated in this cell line. Light microscopic autoradiography following incubation of isolated rat retinal cells with /sup 125/I-insulin showed the presence of insulin binding sites on the photoreceptors and amarcine cells. On the basis of these observations that rat retina glial cells, including Muller cells are sites of synthesis of insulin or a similar peptide, a model for the pathogenesis of dabetic retinopathy is proposed.

  17. Interacting with the Human Insulin Receptor.

    PubMed

    Kidmose, Rune T; Andersen, Gregers R

    2016-03-01

    Insulin is an essential regulator of glucose homeostasis. In this issue of Structure, Croll et al. (2016) reports a significantly improved model of the Fab-complexed IR ectodomain refined against a dataset extending to 3.3 Å. PMID:26933970

  18. Assessing the range of kinase autoinhibition mechanisms in the insulin receptor family

    PubMed Central

    Artim, Stephen C.; Mendrola, Jeannine M.; Lemmon, Mark A.

    2012-01-01

    To investigate the range of autoinhibitory mechanisms used by TKDs (tyrosine kinase domains) from the insulin receptor family of RTKs (receptor tyrosine kinases), we determined crystal structures of TKDs from TrkA (tropomyosin receptor kinase A, a nerve growth factor receptor) and Ror2 (receptor tyrosine kinase-like orphan receptor 2, an unconventional Wnt receptor). TrkA autoinhibition closely resembles that seen for the insulin receptor, relying on projection of an activation loop tyrosine residue into the substrate-binding site and occlusion of the ATP-binding site by the activation loop. Ror2 employs similar mechanisms, but the unusual replacement of the phenylalanine residue in its Asp-Phe-Gly motif with leucine necessitates occlusion of the ATP-binding site by other means. The unusual Asp-Leu-Gly motif in Ror2 is displaced compared with other inactive kinases, allowing the activation loop to interact directly with the TKD's αC helix, in another mode of autoinhibition that is characteristic of the other extreme of this receptor family: ALK (anaplastic lymphoma kinase) and Met. These findings provide insight into the expected range of activating mutations in these TKDs in cancer. We also describe symmetrical dimers of the inactive TrkA TKD resembling those found in other RTKs, possibly reflecting an arrangement of kinase domains in a pre-formed TrkA dimer. PMID:22992069

  19. MARCH1 regulates insulin sensitivity by controlling cell surface insulin receptor levels.

    PubMed

    Nagarajan, Arvindhan; Petersen, Max C; Nasiri, Ali R; Butrico, Gina; Fung, Annie; Ruan, Hai-Bin; Kursawe, Romy; Caprio, Sonia; Thibodeau, Jacques; Bourgeois-Daigneault, Marie-Claude; Sun, Lisha; Gao, Guangping; Bhanot, Sanjay; Jurczak, Michael J; Green, Michael R; Shulman, Gerald I; Wajapeyee, Narendra

    2016-01-01

    Insulin resistance is a key driver of type 2 diabetes (T2D) and is characterized by defective insulin receptor (INSR) signalling. Although surface INSR downregulation is a well-established contributor to insulin resistance, the underlying molecular mechanisms remain obscure. Here we show that the E3 ubiquitin ligase MARCH1 impairs cellular insulin action by degrading cell surface INSR. Using a large-scale RNA interference screen, we identify MARCH1 as a negative regulator of INSR signalling. March1 loss-of-function enhances, and March1 overexpression impairs, hepatic insulin sensitivity in mice. MARCH1 ubiquitinates INSR to decrease cell surface INSR levels, but unlike other INSR ubiquitin ligases, MARCH1 acts in the basal state rather than after insulin stimulation. Thus, MARCH1 may help set the basal gain of insulin signalling. MARCH1 expression is increased in white adipose tissue of obese humans, suggesting that MARCH1 contributes to the pathophysiology of T2D and could be a new therapeutic target. PMID:27577745

  20. SORLA facilitates insulin receptor signaling in adipocytes and exacerbates obesity.

    PubMed

    Schmidt, Vanessa; Schulz, Nadja; Yan, Xin; Schürmann, Annette; Kempa, Stefan; Kern, Matthias; Blüher, Matthias; Poy, Matthew N; Olivecrona, Gunilla; Willnow, Thomas E

    2016-07-01

    In humans, genetic variation of sortilin-related receptor, L(DLR class) A repeats containing (SORL1), which encodes the intracellular sorting receptor SORLA, is a major genetic risk factor for familial and sporadic forms of Alzheimer's disease. Recent GWAS analysis has also associated SORL1 with obesity in humans and in mouse models, suggesting that this receptor may play a role in regulating metabolism. Here, using mouse models with genetic loss or tissue-specific overexpression of SORLA as well as data from obese human subjects, we observed a gene-dosage effect that links SORLA expression to obesity and glucose tolerance. Overexpression of human SORLA in murine adipose tissue blocked hydrolysis of triacylglycerides and caused excessive adiposity. In contrast, Sorl1 gene inactivation in mice accelerated breakdown of triacylglycerides in adipocytes and protected animals from diet-induced obesity. We then identified the underlying molecular mechanism whereby SORLA promotes insulin-induced suppression of lipolysis in adipocytes. Specifically, we determined that SORLA acts as a sorting factor for the insulin receptor (IR) that redirects internalized receptor molecules from endosomes to the plasma membrane, thereby enhancing IR surface expression and strengthening insulin signal reception in target cells. Our findings provide a molecular mechanism for the association of SORL1 with human obesity and confirm a genetic link between neurodegeneration and metabolism that converges on the receptor SORLA. PMID:27322061

  1. Autoreactive T-cell receptor (Vbeta/D/Jbeta) sequences in diabetes are homologous to insulin, glucagon, the insulin receptor, and the glucagon receptor.

    PubMed

    Root-Bernstein, Robert

    2009-01-01

    The hypervariable (Vbeta/D/Jbeta) regions of T-cell receptors (TCR) have been sequenced in a variety of autoimmune diseases by various investigators. An analysis of some of these sequences shows that TCR from both human diabetics and NOD mice mimic insulin, glucagon, the insulin receptor, and the glucagon receptor. Such similarities are not found in the TCR produced in other human autoimmune diseases. These data may explain how insulin, glucagon, and their receptors are targets of autoimmunity in diabetes and also suggest that TCR mimicking insulin and its receptor may be targets of anti-insulin autoantibodies. Such intra-systemic mimicry of self-proteins also raises complex questions about how "self" and "nonself" are regulated during TCR production, especially in light of the complementarity of insulin for its receptor and glucagon for its receptor. The data presented here suggest that some TCR may be complementary to other TCR in autoimmune diseases, a possibility that is experimentally testable. Such complementarity, if it exists, could either serve to down-regulate the clones bearing such TCR or, alternatively, trigger an intra-immune system civil war between them. PMID:19051206

  2. Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex

    PubMed Central

    Žáková, Lenka; Kletvíková, Emília; Lepšík, Martin; Collinsová, Michaela; Watson, Christopher J.; Turkenburg, Johan P.; Jiráček, Jiří; Brzozowski, Andrzej M.

    2014-01-01

    The structural characterization of the insulin–insulin receptor (IR) interaction still lacks the conformation of the crucial B21–B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms. PMID:25286859

  3. [PPAR receptors and insulin sensitivity: new agonists in development].

    PubMed

    Pégorier, J-P

    2005-04-01

    Thiazolidinediones (or glitazones) are synthetic PPARgamma (Peroxisome Proliferator-Activated Receptors gamma) ligands with well recognized effects on glucose and lipid metabolism. The clinical use of these PPARgamma agonists in type 2 diabetic patients leads to an improved glycemic control and an inhanced insulin sensitivity, and at least in animal models, to a protective effect on pancreatic beta-cell function. However, they can produce adverse effects, generally mild or moderate, but some of them (mainly peripheral edema and weight gain) may conduct to treatment cessation. Several pharmacological classes are currently in pre-clinical or clinical development, with the objective to retain the beneficial metabolic properties of PPARgamma agonists, either alone or in association with the PPARalpha agonists (fibrates) benefit on lipid profile, but devoid of the side-effects on weight gain and fluid retention. These new pharmacological classes: partial PPARgamma agonists, PPARgamma antagonists, dual PPARalpha/PPARgamma agonists, pan PPARalpha/beta(delta)/gamma agonists, RXR receptor agonists (rexinoids), are presented in this review. Main results from in vitro cell experiments and animal model studies are discussed, as well as the few published short-term studies in type 2 diabetic patients. PMID:15959400

  4. Low-Density Lipoprotein Receptor-Related Protein-1 Protects Against Hepatic Insulin Resistance and Hepatic Steatosis.

    PubMed

    Ding, Yinyuan; Xian, Xunde; Holland, William L; Tsai, Shirling; Herz, Joachim

    2016-05-01

    Low-density lipoprotein receptor-related protein-1 (LRP1) is a multifunctional uptake receptor for chylomicron remnants in the liver. In vascular smooth muscle cells LRP1 controls reverse cholesterol transport through platelet-derived growth factor receptor β (PDGFR-β) trafficking and tyrosine kinase activity. Here we show that LRP1 regulates hepatic energy homeostasis by integrating insulin signaling with lipid uptake and secretion. Somatic inactivation of LRP1 in the liver (hLRP1KO) predisposes to diet-induced insulin resistance with dyslipidemia and non-alcoholic hepatic steatosis. On a high-fat diet, hLRP1KO mice develop a severe Metabolic Syndrome secondary to hepatic insulin resistance, reduced expression of insulin receptors on the hepatocyte surface and decreased glucose transporter 2 (GLUT2) translocation. While LRP1 is also required for efficient cell surface insulin receptor expression in the absence of exogenous lipids, this latent state of insulin resistance is unmasked by exposure to fatty acids. This further impairs insulin receptor trafficking and results in increased hepatic lipogenesis, impaired fatty acid oxidation and reduced very low density lipoprotein (VLDL) triglyceride secretion. PMID:27322467

  5. Acetylation of insulin receptor substrate-1 is permissive for tyrosine phosphorylation

    PubMed Central

    Kaiser, Christina; James, Stephen R

    2004-01-01

    Background Insulin receptor substrate (IRS) proteins are key moderators of insulin action. Their specific regulation determines downstream protein-protein interactions and confers specificity on growth factor signalling. Regulatory mechanisms that have been identified include phosphorylation of IRS proteins on tyrosine and serine residues and ubiquitination of lysine residues. This study investigated other potential molecular mechanisms of IRS-1 regulation. Results Using the sos recruitment yeast two-hybrid system we found that IRS-1 and histone deacetylase 2 (HDAC2) interact in the cytoplasmic compartment of yeast cells. The interaction mapped to the C-terminus of IRS-1 and was confirmed through co-immunoprecipitation in vitro of recombinant IRS-1 and HDAC2. HDAC2 bound to IRS-1 in mammalian cells treated with phorbol ester or after prolonged treatment with insulin/IGF-1 and also in the livers of ob/ob mice but not PTP1B knockout mice. Thus, the association occurs under conditions of compromised insulin signalling. We found that IRS-1 is an acetylated protein, of which the acetylation is increased by treatment of cells with Trichostatin A (TSA), an inhibitor of HDAC activity. TSA-induced increases in acetylation of IRS-1 were concomitant with increases in tyrosine phosphorylation in response to insulin. These effects were confirmed using RNA interference against HDAC2, indicating that HDAC2 specifically prevents phosphorylation of IRS-1 by the insulin receptor. Conclusions Our results show that IRS-1 is an acetylated protein, a post-translational modification that has not been previously described. Acetylation of IRS-1 is permissive for tyrosine phosphorylation and facilitates insulin-stimulated signal transduction. Specific inhibition of HDAC2 may increase insulin sensitivity in otherwise insulin resistant conditions. PMID:15522123

  6. Insulin enhances striatal dopamine release by activating cholinergic interneurons and thereby signals reward

    PubMed Central

    Stouffer, Melissa A.; Woods, Catherine A.; Patel, Jyoti C.; Lee, Christian R.; Witkovsky, Paul; Bao, Li; Machold, Robert P.; Jones, Kymry T.; de Vaca, Soledad Cabeza; Reith, Maarten E. A.; Carr, Kenneth D.; Rice, Margaret E.

    2015-01-01

    Insulin activates insulin receptors (InsRs) in the hypothalamus to signal satiety after a meal. However, the rising incidence of obesity, which results in chronically elevated insulin levels, implies that insulin may also act in brain centres that regulate motivation and reward. We report here that insulin can amplify action potential-dependent dopamine (DA) release in the nucleus accumbens (NAc) and caudate–putamen through an indirect mechanism that involves striatal cholinergic interneurons that express InsRs. Furthermore, two different chronic diet manipulations in rats, food restriction (FR) and an obesogenic (OB) diet, oppositely alter the sensitivity of striatal DA release to insulin, with enhanced responsiveness in FR, but loss of responsiveness in OB. Behavioural studies show that intact insulin levels in the NAc shell are necessary for acquisition of preference for the flavour of a paired glucose solution. Together, these data imply that striatal insulin signalling enhances DA release to influence food choices. PMID:26503322

  7. Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation.

    PubMed

    Yunn, Na-Oh; Koh, Ara; Han, Seungmin; Lim, Jong Hun; Park, Sehoon; Lee, Jiyoun; Kim, Eui; Jang, Sung Key; Berggren, Per-Olof; Ryu, Sung Ho

    2015-09-18

    Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors. PMID:26245346

  8. Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation

    PubMed Central

    Yunn, Na-Oh; Koh, Ara; Han, Seungmin; Lim, Jong Hun; Park, Sehoon; Lee, Jiyoun; Kim, Eui; Jang, Sung Key; Berggren, Per-Olof; Ryu, Sung Ho

    2015-01-01

    Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors. PMID:26245346

  9. Interaction of myocardial insulin receptor and IGF receptor signaling in exercise-induced cardiac hypertrophy

    PubMed Central

    Ikeda, Hiroyuki; Shiojima, Ichiro; Ozasa, Yukako; Yoshida, Masashi; Holzenberger, Martin; Kahn, C Ronald; Walsh, Kenneth; Igarashi, Takashi; Abel, E Dale; Komuro, Issei

    2016-01-01

    Insulin-like growth factor-1 (IGF-1) signaling has recently been implicated in the development of cardiac hypertrophy after long-term endurance training, via mechanisms that may involve energetic stress. Given the potential overlap of insulin and IGF-1 signaling we sought to determine if both signaling pathways could contribute to exercise-induced cardiac hypertrophy following shorter-term exercise training. Studies were performed in mice with cardiac-specific IGF-1 receptor (IGF1R) knockout (CIGFRKO), mice with cardiac-specific insulin receptor (IR) knockout (CIRKO), CIGFRKO mice that lacked one IR allele in cardiomyocytes (IGFR−/−IR+/−), and CIRKO mice that lacked one IGF1R allele in cardiomyocytes (IGFR+/−IR−/−). Intravenous administration of IGF-1 or 75 hours of swimming over 4 weeks increased IGF1R tyrosine phosphorylation in the heart in control and CIRKO mice but not in CIGFRKO mice. Intriguingly, IR tyrosine phosphorylation in the heart was also increased following IGF-1 administration or exercise training in control and CIGFRKO mice but not in CIRKO mice. The extent of cardiac hypertrophy following exercise training in CIGFRKO and CIRKO mice was comparable to that in control mice. In contrast, exercise-induced cardiac hypertrophy was significantly attenuated in IGFR−/−IR+/− and IGFR+/−IR−/− mice. Thus, IGF-1 and exercise activates both IGF1R and IR in the heart, and IGF1R- and IR-mediated signals may serve redundant roles in the hypertrophic responses of the heart to exercise training. PMID:19744489

  10. Metformin Disrupts Crosstalk Between G protein-Coupled Receptor and Insulin Receptor Signaling Systems and Inhibits Pancreatic Cancer Growth

    PubMed Central

    Kisfalvi, Krisztina; Eibl, Guido; Sinnett-Smith, James; Rozengurt, Enrique

    2009-01-01

    Recently we identified a novel crosstalk between insulin and G-protein-coupled receptor (GPCR) signaling pathways in human pancreatic cancer cells. Insulin enhanced GPCR signaling through a rapamycin-sensitive mTOR-dependent pathway. Metformin, the most widely used drug in the treatment of type-2 diabetes, activates AMP kinase (AMPK), which negatively regulates mTOR. Here, we determined whether metformin disrupts crosstalk between insulin receptor and GPCR signaling in pancreatic cancer cells. Treatment of human pancreatic cancer cells (PANC-1, MIAPaCa-2, BxPC-3) with insulin (10ng/ml) for 5 min markedly enhanced the increase in intracellular [Ca2+] induced by GPCR agonists (e.g. neurotensin, bradykinin, angiotensin II). Metformin pretreatment completely abrogated insulin-induced potentiation of Ca2+ signaling but did not interfere with the effect of GPCR agonists alone. Insulin also enhanced GPCR agonist-induced growth, measured by DNA synthesis, and numbers of cells cultured in adherent or non-adherent conditions. Low doses of metformin (0.1-0.5 mM) blocked stimulation of DNA synthesis, anchorage-dependent and independent growth induced by insulin and GPCR agonists. Treatment with metformin induced striking and sustained increase in the phosphorylation of AMPK at Thr172 and a selective AMPK inhibitor (compound C, at 5μM) reversed the effects of metformin on [Ca2+]i, and DNA synthesis, indicating that metformin acts through AMPK activation. In view of these results we tested whether metformin inhibits pancreatic cancer growth. Administration of metformin significantly decreased the growth of MIAPaCa-2 and PANC-1 cells xenografted on the flank of nude mice. The results raise the possibility that metformin could be a potential candidate in novel treatment strategies for human pancreatic cancer. PMID:19679549

  11. Characterization of an endogenous substrate of the insulin receptor in cultured cells

    SciTech Connect

    White, M.F.; Stegmann, E.W.; Dull, T.J.; Ullrich, A.; Kahn, C.R.

    1987-07-15

    Using antiphosphotyrosine antibodies, we have characterized the tyrosine phosphorylation of an endogenous substrate of the insulin receptor in Fao hepatoma cells and in Chinese hamster ovary cells transfected with a eukaryotic expression vector containing the human insulin receptor cDNA. In Fao cells, besides the beta-subunit of the insulin receptor, a protein with a molecular mass between 170 and 210 kDa designated pp185, undergoes tyrosine phosphorylation immediately after insulin stimulation reaching a maximum level within 30 s. After 4 h of continuous insulin stimulation, the labeling of pp185 decreased to less than half of its original intensity, whereas the insulin receptor was unchanged. After 24 h of insulin stimulation, the phosphotyrosine-containing insulin receptor decreased by 75% owing to down-regulation, whereas the pp185 was completely undetectable. By several biochemical and physiological criteria, the pp185 is distinct from the insulin receptor. The pp185 and the beta-subunit of the insulin receptor were strongly labeled with (/sup 32/P)orthophosphate, but in contrast to the insulin receptor, the pp185 was not labeled by cross-linking with /sup 125/I-insulin or surface 125I iodination. Unlike the insulin receptor, the pp185 was extracted from Fao cells without detergent, and tryptic phosphopeptide mapping of the pp185 and the insulin receptor yielded distinct patterns. Thus, the pp185 is not located at the external face of the plasma membrane and does not bind insulin. Treatment of Fao cells with the phorbol ester, phorbol 12-myristate 13-acetate, stimulated the phosphorylation of two proteins with molecular weights of 170 and 210 kDa which were immunoprecipitated with the anti-phosphotyrosine antibody. Subsequent insulin stimulation increased the phosphorylation of the 210 kDa protein, but the pp185 was not detected.

  12. MicroRNA-1225-5p inhibits proliferation and metastasis of gastric carcinoma through repressing insulin receptor substrate-1 and activation of β-catenin signaling.

    PubMed

    Zheng, Haiyin; Zhang, Fuxing; Lin, Xinjian; Huang, Changming; Zhang, Yiqin; Li, Yun; Lin, Jianyin; Chen, Wannan; Lin, Xu

    2016-01-26

    Emerging evidence has linked aberrantly expressed microRNAs (miRNAs) with oncogenesis and malignant development in various human cancers. However, their specific roles and functions in gastric carcinoma (GC) remain largely undefined. In this study we identify and report a novel miRNA, miR-1225-5p, as tumor suppressor in GC development and progression. Microarray analysis revealed that there were fifty-six differentially expressed miRNAs (thirty-two upregulated and twenty-four downregulated) in GC tumor samples compared to their corresponding nontumorous tissues. Downregulation of miR-1225-5p was frequently detected in GC and strongly correlated with more aggressive phenotypes and poor prognosis. Functional assays demonstrated that ectopic overexpression of miR-1225-5p could inhibit cell proliferation, colony formation, migration and invasion in vitro, as well as suppress tumor growth and metastasis in nude mice. Further integrative and functional studies suggested insulin receptor substrate 1 (IRS1) as a downstream effector of miR-1225-5p which acted through β-catenin signaling pathway. These results demonstrate that miR-1225-5p serves to constrain GC growth and metastatic potential via inhibition of IRS1 and β-catenin signaling. Therefore, downregulation of miR-1225-5p is likely to be one of major molecular mechanisms accounting for the development and progression of GC. PMID:26684358

  13. MicroRNA-1225-5p inhibits proliferation and metastasis of gastric carcinoma through repressing insulin receptor substrate-1 and activation of β-catenin signaling

    PubMed Central

    Lin, Xinjian; Huang, Changming; Zhang, Yiqin; Li, Yun; Lin, Jianyin; Chen, Wannan; Lin, Xu

    2016-01-01

    Emerging evidence has linked aberrantly expressed microRNAs (miRNAs) with oncogenesis and malignant development in various human cancers. However, their specific roles and functions in gastric carcinoma (GC) remain largely undefined. In this study we identify and report a novel miRNA, miR-1225-5p, as tumor suppressor in GC development and progression. Microarray analysis revealed that there were fifty-six differentially expressed miRNAs (thirty-two upregulated and twenty-four downregulated) in GC tumor samples compared to their corresponding nontumorous tissues. Downregulation of miR-1225-5p was frequently detected in GC and strongly correlated with more aggressive phenotypes and poor prognosis. Functional assays demonstrated that ectopic overexpression of miR-1225-5p could inhibit cell proliferation, colony formation, migration and invasion in vitro, as well as suppress tumor growth and metastasis in nude mice. Further integrative and functional studies suggested insulin receptor substrate 1 (IRS1) as a downstream effector of miR-1225-5p which acted through β-catenin signaling pathway. These results demonstrate that miR-1225-5p serves to constrain GC growth and metastatic potential via inhibition of IRS1 and β-catenin signaling. Therefore, downregulation of miR-1225-5p is likely to be one of major molecular mechanisms accounting for the development and progression of GC. PMID:26684358

  14. In vivo stimulation of oestrogen receptor α increases insulin-stimulated skeletal muscle glucose uptake

    PubMed Central

    Gorres, Brittany K; Bomhoff, Gregory L; Morris, Jill K; Geiger, Paige C

    2011-01-01

    Abstract Previous studies suggest oestrogen receptor α (ERα) is involved in oestrogen-mediated regulation of glucose metabolism and is critical for maintenance of whole body insulin action. Despite this, the effect of direct ERα modulation in insulin-responsive tissues is unknown. The purpose of the current study was to determine the impact of ERα activation, using the ER subtype-selective ligand propylpyrazoletriyl (PPT), on skeletal muscle glucose uptake. Two-month-old female Sprague–Dawley rats, ovariectomized for 1 week, were given subcutaneous injections of PPT (10 mg kg−1), oestradiol benzoate (EB; 20 μg kg−1), the ERβ agonist diarylpropionitrile (DPN, 10 mg kg−1) or vehicle every 24 h for 3 days. On the fourth day, insulin-stimulated skeletal muscle glucose uptake was measured in vitro and insulin signalling intermediates were assessed via Western blotting. Activation of ERα with PPT resulted in increased insulin-stimulated glucose uptake into the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles, activation of insulin signalling intermediates (as measured by phospho-Akt (pAkt) and pAkt substrate (PAS)) and phosphorylation of AMP-activated protein kinase (AMPK). GLUT4 protein was increased only in the EDL muscle. Rats treated with EB or DPN for 3 days did not show an increase in insulin-stimulated skeletal muscle glucose uptake compared to vehicle-treated animals. These new findings reveal that direct activation of ERα positively mediates glucose uptake and insulin action in skeletal muscle. Evidence that oestrogens and ERα stimulate glucose uptake has important implications for understanding mechanisms of glucose homeostasis, particularly in postmenopausal women. PMID:21486807

  15. Dephosphorylation of human insulin-like growth factor I (IGF-I) receptors by membrane-associated tyrosine phosphatases.

    PubMed Central

    Peraldi, P; Hauguel-de Mouzon, S; Alengrin, F; Van Obberghen, E

    1992-01-01

    The insulin-like growth factor-I (IGF-I) receptor exhibits structural and functional similarities to the insulin receptor. Although the regulation of the insulin-receptor tyrosine kinase has been extensively investigated, the mechanisms involved in phosphorylation/dephosphorylation of the IGF-I receptor have received only little attention. To obtain a better understanding of the mode of IGF-I action, we have investigated the effects of protein phosphotyrosine phosphatases (PTPases) on the phosphorylation status of the IGF-I receptor. The dephosphorylation of the human IGF-I receptor by membrane-associated tyrosine phosphatases was studied by an immuno-enzymic assay based on the recognition of phosphotyrosine residues by anti-phosphotyrosine antibodies. Using intact IGF-I receptors as substrates, we show that they could be completely dephosphorylated by different cellular PTPases. Three pieces of evidence indicate that receptor dephosphorylation takes place on phosphotyrosine, i.e. the inhibition profile of phosphatase activity by zinc and vanadate, its absolute requirement for thiol compounds and the diminution of [32P]phosphotyrosine labelling of the beta subunit assessed by SDS/PAGE and phosphoamino acid analysis. Tyrosine kinase activity and autophosphorylation of the IGF-I receptor were decreased in a dose-dependent manner by PTPases, indicating that partial dephosphorylation of the receptor was associated with a decrease in its intrinsic activity. The sensitivity of the activated human IGF-I receptor to dephosphorylation on tyrosine leads to the speculation that IGF-I receptor activity might be regulated by mechanisms such as those described for the insulin receptor. Further investigation of the pathways of IGF-I receptor dephosphorylation will contribute to define the role(s) of PTPases in the overall mechanism of IGF-I signalling. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1322128

  16. Molecular Recognition of Insulin by a Synthetic Receptor

    SciTech Connect

    Chinai, Jordan M.; Taylor, Alexander B.; Ryno, Lisa M.; Hargreaves, Nicholas D.; Morris, Christopher A.; Hart, P. John; Urbach, Adam R.

    2011-08-29

    The discovery of molecules that bind tightly and selectively to desired proteins continues to drive innovation at the interface of chemistry and biology. This paper describes the binding of human insulin by the synthetic receptor cucurbit[7]uril (Q7) in vitro. Isothermal titration calorimetry and fluorescence spectroscopy experiments show that Q7 binds to insulin with an equilibrium association constant of 1.5 x 10{sup 6} M{sup -1} and with 50-100-fold selectivity versus proteins that are much larger but lack an N-terminal aromatic residue, and with >1000-fold selectivity versus an insulin variant lacking the N-terminal phenylalanine (Phe) residue. The crystal structure of the Q7{center_dot}insulin complex shows that binding occurs at the N-terminal Phe residue and that the N-terminus unfolds to enable binding. These findings suggest that site-selective recognition is based on the properties inherent to a protein terminus, including the unique chemical epitope presented by the terminal residue and the greater freedom of the terminus to unfold, like the end of a ball of string, to accommodate binding. Insulin recognition was predicted accurately from studies on short peptides and exemplifies an approach to protein recognition by targeting the terminus.

  17. Regulator of insulin receptor affinity in rat skeletal muscle sarcolemmal vesicles

    SciTech Connect

    Whitson, R.H.; Barnard, K.J.; Kaplan, S.A.; Itakura, K.

    1986-05-01

    Wheat germ agglutinin (WGA) affinity purification of detergent solubilized insulin receptors (IR) from rat skeletal muscle sarcolemmal vesicles resulted in an apparent increase in total insulin binding activity of greater than 5-fold, suggesting that an inhibitory component had been removed. This was verified when the flow-through fraction from the WGA column was dialyzed and added back to the partially purified receptor. The addition of a 100-fold dilution of the inhibitor preparation caused a 50% reduction in binding to trace amounts of /sup 125/I-insulin. Scatchard analysis revealed that the effect of the inhibitor was to decrease the affinity of the muscle IR. The inhibitor appeared to be tissue specific, inasmuch as the I/sub 50/'s for WGA-purified IR from rat fat and liver were 10 times the I/sub 50/ for muscle IR. The I/sub 50/ for insulin binding to intact IM-9 cells was 30 times the value for muscle IR. The inhibitor eluted in the void volume of Sephadex G-50 columns. Its activity was not destroyed by heating at 90/sup 0/C for 10 minutes, or by prolonged incubation with trypsin or dithiothreitol. The inhibitor described here may have a role in modulating insulin sensitivity in skeletal muscle.

  18. Non-small cell lung cancer cell survival crucially depends on functional insulin receptors.

    PubMed

    Frisch, Carolin Maria; Zimmermann, Katrin; Zilleßen, Pia; Pfeifer, Alexander; Racké, Kurt; Mayer, Peter

    2015-08-01

    Insulin plays an important role as a growth factor and its contribution to tumor proliferation is intensely discussed. It acts via the cognate insulin receptor (IR) but can also activate the IGF1 receptor (IGF1R). Apart from increasing proliferation, insulin might have additional effects in lung cancer. Therefore, we investigated insulin action and effects of IR knockdown (KD) in three (NCI-H292, NCI-H226 and NCI-H460) independent non-small cell lung cancer (NSCLC) cell lines. All lung cancer lines studied were found to express IR, albeit with marked differences in the ratio of the two variants IR-A and IR-B. Insulin activated the classical signaling pathway with IR autophosphorylation and Akt phosphorylation. Moreover, activation of MAPK was observed in H292 cells, accompanied by enhanced proliferation. Lentiviral shRNA IR KD caused strong decrease in survival of all three lines, indicating that the effects of insulin in lung cancer go beyond enhancing proliferation. Unspecific effects were ruled out by employing further shRNAs and different insulin-responsive cells (human pre-adipocytes) for comparison. Caspase assays demonstrated that IR KD strongly induced apoptosis in these lung cancer cells, providing the physiological basis of the rapid cell loss. In search for the underlying mechanism, we analyzed alterations in the gene expression profile in response to IR KD. A strong induction of certain cytokines (e.g. IL20 and tumour necrosis factor) became obvious and it turned out that these cytokines trigger apoptosis in the NSCLC cells tested. This indicates a novel role of IR in tumor cell survival via suppression of pro-apoptotic cytokines. PMID:26113601

  19. Production of recombinant insulin-like androgenic gland hormones from three decapod species: In vitro testicular phosphorylation and activation of a newly identified tyrosine kinase receptor from the Eastern spiny lobster, Sagmariasus verreauxi.

    PubMed

    Aizen, Joseph; Chandler, Jennifer C; Fitzgibbon, Quinn P; Sagi, Amir; Battaglene, Stephen C; Elizur, Abigail; Ventura, Tomer

    2016-04-01

    In crustaceans the insulin-like androgenic gland hormone (IAG) is responsible for male sexual differentiation. To date, the biochemical pathways through which IAG exerts its effects are poorly understood and could be elucidated through the production of a functional recombinant IAG (rIAG). We have successfully expressed glycosylated, biologically active IAG using the Pichia pastoris yeast expression system. We co-expressed recombinant single-chain precursor molecules consisting of the B and A chains (the mature hormone) tethered by a flexible linker, producing rIAGs of the following commercially important species: Eastern spiny lobster Sagmariasus verreauxi (Sv), redclaw crayfish Cherax quadricarinatus (Cq) and giant freshwater prawn Macrobrachium rosenbergii (Mr). We then tested the biological activity of each, through the ability to increase phosphorylation in the testis; both Sv and Cq rIAGs significantly elevated phosphorylation specific to their species, and in a dose-dependent manner. Mr rIAG was tested on Macrobrachium australiense (Ma), eliciting a similar response. Moreover, using bioinformatics analyses of the de novo assembled spiny lobster transcriptome, we identified a spiny lobster tyrosine kinase insulin receptor (Sv-TKIR). We validated this discovery with a receptor activation assay in COS-7 cells expressing Sv-TKIR, using a reporter SRE-LUC system designed for RTKs, with each of the rIAG proteins acting as the activation ligand. Using recombinant proteins, we aim to develop specific tools to control sexual development through the administration of IAG within the critical sexual differentiation time window. The biologically active rIAGs generated might facilitate commercially feasible solutions for the long sought techniques for sex-change induction and monosex population culture in crustaceans and shed new light on the physiological mode of action of IAG in crustaceans. PMID:26883686

  20. Flexibility in the insulin receptor ectodomain enables docking of insulin in crystallographic conformation observed in a hormone-bound microreceptor.

    PubMed

    Vashisth, Harish

    2014-01-01

    Insulin binding to the insulin receptor (IR) is the first key step in initiating downstream signaling cascades for glucose homeostasis in higher organisms. The molecular details of insulin recognition by IR are not yet completely understood, but a picture of hormone/receptor interactions at one of the epitopes (Site 1) is beginning to emerge from recent structural evidence. However, insulin-bound structures of truncated IR suggest that crystallographic conformation of insulin cannot be accommodated in the full IR ectodomain due to steric overlap of insulin with the first two type III fibronectin domains (F1 and F2), which are contributed to the insulin binding-pocket by the second subunit in the IR homodimer. A conformational change in the F1-F2 pair has thus been suggested. In this work, we present an all-atom structural model of complex of insulin and the IR ectodomain, where no structural overlap of insulin with the receptor domains (F1 and F2) is observed. This structural model was arrived at by flexibly fitting parts of our earlier insulin/IR all-atom model into the simulated density maps of crystallized constructs combined with conformational sampling from apo-IR solution conformations. Importantly, our experimentally-consistent model helps rationalize yet unresolved Site. PMID:25309993

  1. INSULIN INDUCED EPIDERMAL GROWTH FACTOR ACTIVATION IN VASCULAR SMOOTH MUSCLE CELLS IS ADAM-DEPENDENT

    PubMed Central

    Roztocil, Elisa; Nicholl, Suzanne M.; Davies, Mark G.

    2008-01-01

    Background With the rise in metabolic syndrome, understanding the role of insulin signaling within the cells of vasculature has become more important but yet remains poorly defined. The study examines the role of insulin actions on a pivotal cross-talk receptor, Epidermal Growth Factor Receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor linked tyrosine kinases and is key to many of their responses. Objective To determine the pathway of EGFR transactivation by insulin in human coronary smooth muscle cells (VSMC) Methods VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to insulin in the presence and absence of the plasmin inhibitors (e-aminocaproic acid and aprotinin) matrix metalloprotease (MMP) inhibitor GM6001, the ADAM (A Disintegrin And Metalloproteinase Domain) inhibitors TAPI-0 and TAPI-1, Heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies and the EGFR inhibitor AG1478. Results Insulin induced time-dependent EGFR phosphorylation, which was inhibited by AG1478 in a concentration dependent manner. Application of the plasmin inhibitors did not block the response. EGFR phosphorylation by insulin was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs TAPIs, or HB-EGF. Direct blockade of the EGFR prevented activation by both insulin and EGF. Conclusion Insulin can induce transactivation of EGFR by an ADAM-mediated, HB-EGF dependent process. This is the first description of crosstalk via ADAM between insulin and EGFR in vascular SMC. Targeting a pivotal cross-talk receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases is an attractive molecular target. PMID:18656632

  2. Phosphorylation of the insulin receptor in cultured hepatoma cells and a solubilized system

    SciTech Connect

    Kasuga, M.; White, M.F.; Kahn, C.R.

    1985-01-01

    Methods are described which have been used successfully to study insulin receptor autophosphorylation in cultured cells (hepatoma cell line Fao) and detergent solubilized receptor systems. Intact cultured cells were labelled with /sup 32/PO/sub 4//sup 3 -/. Details are given for the solubilization and purification of the insulin receptor and insulin dose-response curves for phosphorylation of the solubilized insulin receptor. Trypsin digestion of a phosphorylated subunit suggests that at least peptides containing sites of /sup 32/P incorporation exist in the receptor molecule.

  3. Disruption of insulin receptor function inhibits proliferation in endocrine-resistant breast cancer cells.

    PubMed

    Chan, J Y; LaPara, K; Yee, D

    2016-08-11

    The insulin-like growth factor (IGF) system is a well-studied growth regulatory pathway implicated in breast cancer biology. Clinical trials testing monoclonal antibodies directed against the type I IGF receptor (IGF1R) in combination with estrogen receptor-α (ER) targeting have been completed, but failed to show benefits in patients with endocrine-resistant tumors compared to ER targeting alone. We have previously shown that the closely related insulin receptor (InsR) is expressed in tamoxifen-resistant (TamR) breast cancer cells. Here we examined if inhibition of InsR affected TamR breast cancer cells. InsR function was inhibited by three different mechanisms: InsR short hairpin RNA, a small InsR-blocking peptide, S961 and an InsR monoclonal antibody (mAb). Suppression of InsR function by these methods in TamR cells successfully blocked insulin-mediated signaling, monolayer proliferation, cell cycle progression and anchorage-independent growth. This strategy was not effective in parental cells likely because of the presence of IGFR /InsR hybrid receptors. Downregulation of IGF1R in conjunction with InsR inhibition was more effective in blocking IGF- and insulin-mediated signaling and growth in parental cells compared with single-receptor targeting alone. Our findings show TamR cells were stimulated by InsR and were not sensitive to IGF1R inhibition, whereas in tamoxifen-sensitive parental cancer cells, the presence of both receptors, especially hybrid receptors, allowed cross-reactivity of ligand-mediated activation and growth. To suppress the IGF system, targeting of both IGF1R and InsR is optimal in endocrine-sensitive and -resistant breast cancer. PMID:26876199

  4. Disruption of insulin receptor function inhibits proliferation in endocrine resistant breast cancer cells

    PubMed Central

    Chan, Jie Ying; LaPara, Kelly; Yee, Douglas

    2015-01-01

    The insulin-like growth factor (IGF) system is a well-studied growth regulatory pathway implicated in breast cancer biology. Clinical trials testing monoclonal antibodies directed against the type I IGF receptor (IGF1R) in combination with estrogen receptor-α (ER) targeting have been completed, but failed to show benefits in patients with endocrine resistant tumors compared to ER targeting alone. We have previously shown that the closely related insulin receptor (InsR) is expressed in tamoxifen resistant breast cancer cells. Here we examined if inhibition of InsR affected tamoxifen-resistant (TamR) breast cancer cells. InsR function was inhibited by three different mechanisms: InsR shRNA, a small InsR blocking peptide, S961 and an InsR monoclonal antibody (mAb). Suppression of InsR function by these methods in TamR cells successfully blocked insulin-mediated signaling, monolayer proliferation, cell cycle progression and anchorage-independent growth. This strategy was not effective in parental cells likely due to the presence of IGFR/InsR hybrid receptors. Down-regulation of IGF1R in conjunction with InsR inhibition was more effective in blocking IGF- and insulin-mediated signaling and growth in parental cells compared to single receptor targeting alone. Our findings show TamR cells were stimulated by InsR and were not sensitive to IGF1R inhibition, whereas in tamoxifen-sensitive parental cancer cells, the presence of both receptors, especially hybrid receptors, allowed cross-reactivity of ligand-mediated activation and growth. To suppress the IGF system, targeting of both IGF1R and InsR is optimal in endocrine sensitive and resistant breast cancer. PMID:26876199

  5. Preclinical and first-in-human phase I studies of KW-2450, an oral tyrosine kinase inhibitor with insulin-like growth factor receptor-1/insulin receptor selectivity.

    PubMed

    Schwartz, Gary K; Dickson, Mark A; LoRusso, Patricia M; Sausville, Edward A; Maekawa, Yoshimi; Watanabe, Yasuo; Kashima, Naomi; Nakashima, Daisuke; Akinaga, Shiro

    2016-04-01

    Numerous solid tumors overexpress or have excessively activated insulin-like growth factor receptor-1 (IGF-1R). We summarize preclinical studies and the first-in-human study of KW-2450, an oral tyrosine kinase inhibitor with IGF-1R and insulin receptor (IR) inhibitory activity. Preclinical activity of KW-2450 was evaluated in various in vitro and in vivo models. It was then evaluated in a phase I clinical trial in 13 patients with advanced solid tumors (NCT00921336). In vitro, KW-2450 inhibited human IGF-1R and IR kinases (IC50 7.39 and 5.64 nmol/L, respectively) and the growth of various human malignant cell lines. KW-2450 40 mg/kg showed modest growth inhibitory activity and inhibited IGF-1-induced signal transduction in the murine HT-29/GFP colon carcinoma xenograft model. The maximum tolerated dose of KW-2450 was 37.5 mg once daily continuously; dose-limiting toxicity occurred in two of six patients at 50 mg/day (both grade 3 hyperglycemia) and in one of seven patients at 37.5 mg/day (grade 3 rash). Four of 10 evaluable patients showed stable disease. Single-agent KW-2450 was associated with modest antitumor activity in heavily pretreated patients with solid tumors and is being further investigated in combination therapy with lapatinib/letrozole in patients with human epidermal growth factor receptor 2-postive metastatic breast cancer. PMID:26850678

  6. Central Administration of Galanin Receptor 1 Agonist Boosted Insulin Sensitivity in Adipose Cells of Diabetic Rats

    PubMed Central

    Zhang, Zhenwen; Fang, Penghua; He, Biao; Guo, Lili; Runesson, Johan; Langel, Ülo; Shi, Mingyi; Zhu, Yan; Bo, Ping

    2016-01-01

    Our previous studies testified the beneficial effect of central galanin on insulin sensitivity of type 2 diabetic rats. The aim of the study was further to investigate whether central M617, a galanin receptor 1 agonist, can benefit insulin sensitivity. The effects of intracerebroventricular administration of M617 on insulin sensitivity and insulin signaling were evaluated in adipose tissues of type 2 diabetic rats. The results showed that central injection of M617 significantly increased plasma adiponectin contents, glucose infusion rates in hyperinsulinemic-euglycemic clamp tests, GLUT4 mRNA expression levels, GLUT4 contents in plasma membranes, and total cell membranes of the adipose cells but reduced the plasma C-reactive protein concentration in nondiabetic and diabetic rats. The ratios of GLUT4 contents were higher in plasma membranes to total cell membranes in both nondiabetic and diabetic M617 groups than each control. In addition, the central administration of M617 enhanced the ratios of pAkt/Akt and pAS160/AS160, but not phosphorylative cAMP response element-binding protein (pCREB)/CREB in the adipose cells of nondiabetic and diabetic rats. These results suggest that excitation of central galanin receptor 1 facilitates insulin sensitivity via activation of the Akt/AS160 signaling pathway in the fat cells of type 2 diabetic rats. PMID:27127795

  7. H2O2-Activated Mitochondrial Phospholipase iPLA2γ Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein–Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic β-Cells

    PubMed Central

    Ježek, Jan; Dlasková, Andrea; Zelenka, Jaroslav; Jabůrek, Martin

    2015-01-01

    Abstract Aims: Pancreatic β-cell chronic lipotoxicity evolves from acute free fatty acid (FA)–mediated oxidative stress, unprotected by antioxidant mechanisms. Since mitochondrial uncoupling protein-2 (UCP2) plays antioxidant and insulin-regulating roles in pancreatic β-cells, we tested our hypothesis, that UCP2-mediated uncoupling attenuating mitochondrial superoxide production is initiated by FA release due to a direct H2O2-induced activation of mitochondrial phospholipase iPLA2γ. Results: Pro-oxidant tert-butylhydroperoxide increased respiration, decreased membrane potential and mitochondrial matrix superoxide release rates of control but not UCP2- or iPLA2γ-silenced INS-1E cells. iPLA2γ/UCP2-mediated uncoupling was alternatively activated by an H2O2 burst, resulting from palmitic acid (PA) β-oxidation, and it was prevented by antioxidants or catalase overexpression. Exclusively, nascent FAs that cleaved off phospholipids by iPLA2γ were capable of activating UCP2, indicating that the previously reported direct redox UCP2 activation is actually indirect. Glucose-stimulated insulin release was not affected by UCP2 or iPLA2γ silencing, unless pro-oxidant activation had taken place. PA augmented insulin secretion via G-protein–coupled receptor 40 (GPR40), stimulated by iPLA2γ-cleaved FAs (absent after GPR40 silencing). Innovation and Conclusion: The iPLA2γ/UCP2 synergy provides a feedback antioxidant mechanism preventing oxidative stress by physiological FA intake in pancreatic β-cells, regulating glucose-, FA-, and redox-stimulated insulin secretion. iPLA2γ is regulated by exogenous FA via β-oxidation causing H2O2 signaling, while FAs are cleaved off phospholipids, subsequently acting as amplifying messengers for GPR40. Hence, iPLA2γ acts in eminent physiological redox signaling, the impairment of which results in the lack of antilipotoxic defense and contributes to chronic lipotoxicity. Antioxid. Redox Signal. 23, 958–972. PMID:25925080

  8. Nimesulide, a cyclooxygenase-2 selective inhibitor, suppresses obesity-related non-alcoholic fatty liver disease and hepatic insulin resistance through the regulation of peroxisome proliferator-activated receptor γ

    PubMed Central

    Tsujimoto, Shunsuke; Kishina, Manabu; Koda, Masahiko; Yamamoto, Yasutaka; Tanaka, Kohei; Harada, Yusuke; Yoshida, Akio; Hisatome, Ichiro

    2016-01-01

    Cyclooxygenase (COX)-2 selective inhibitors suppress non-alcoholic fatty liver disease (NAFLD); however, the precise mechanism of action remains unknown. The aim of this study was to examine how the COX-2 selective inhibitor nimesulide suppresses NAFLD in a murine model of high-fat diet (HFD)-induced obesity. Mice were fed either a normal chow diet (NC), an HFD, or HFD plus nimesulide (HFD-nime) for 12 weeks. Body weight, hepatic COX-2 mRNA expression and triglyceride accumulation were significantly increased in the HFD group. Triglyceride accumulation was suppressed in the HFD-nime group. The mRNA expression of hepatic peroxisome proliferator-activated receptor γ (PPARγ) and the natural PPARγ agonist 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) were significantly increased in the HFD group and significantly suppressed in the HFD-nime group. Glucose metabolism was impaired in the HFD group compared with the NC group, and it was significantly improved in the HFD-nime group. In addition, the plasma insulin levels in the HFD group were increased compared with those in the NC group, and were decreased in the HFD-nime group. These results indicate that HFD-induced NAFLD is mediated by the increased hepatic expression of COX-2. We suggest that the production of 15d-PGJ2, which is mediated by COX-2, induces NAFLD and hepatic insulin resistance by activating PPARγ. Furthermore, the mRNA expression of tissue inhibitor of metalloproteinases-1 (TIMP-1), procollagen-1 and monocyte chemoattractant protein-1 (MCP-1), as well as the number of F4/80-positive hepatic (Kupffer) cells, were significantly increased in the HFD group compared with the NC group, and they were reduced by nimesulide. In conclusion, COX-2 may emerge as a molecular target for preventing the development of NAFLD and insulin resistance in diet-related obesity. PMID:27431935

  9. Peroxisome proliferator-activated receptor alpha1 in yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA tissue expression and transcriptional regulation by insulin in vivo and in vitro.

    PubMed

    Zheng, Jia-Lang; Zhuo, Mei-Qin; Luo, Zhi; Song, Yu-Feng; Pan, Ya-Xiong; Huang, Chao; Hu, Wei; Chen, Qi-Liang

    2015-05-01

    Peroxisome proliferator-activated receptor alpha1 (PPARα1) cDNA was isolated from liver of yellow catfish Pelteobagrus fulvidraco by RT-PCR and RACE. Its molecular characterization, tissue expression and transcriptional regulation by insulin in vitro and in vivo were determined. PPARα1 mRNA covered 1879 bp, with an open reading frame (ORF) of 1410 bp encoding 469 amino acid residues, a 5'-untranslated region (UTR) of 49 bp, and a 3'-UTR of 421 bp. PPARα1 consisted of 4 domains, the A/B domain, DNA-binding domain (DBD), D domain, and ligand-binding domain (LBD). The predicted tertiary structure of yellow catfish PPARα1 showed an increased size of the main cavity that was made up of side chains from helices 3, 5, 10, 11, and 12. Changes of PPARα1 structure might affect binding of mammalian PPARα1-specific ligand and cofactor in yellow catfish and may endow yellow catfish PPARα1 with new ligand-independent or -dependent transactivation activity. PPARα1 was differentially expressed in various tissues during development. Furthermore, intraperitoneal injection in vivo and incubation in vitro of insulin reduced the mRNA expression of PPARα1 in the liver and hepatocytes of yellow catfish. Based on the observation above, the present study provides evidence that PPARα1 is differentially expressed within and among tissues during three developmental stages and also regulated by insulin both in vivo and in vitro, which warrants further investigation of PPARα1 physiological function in fish. PMID:25645400

  10. A novel antitumor activity of deguelin targeting the insulin-like growth factor (IGF) receptor pathway via up-regulation of IGF-binding protein-3 expression in breast cancer

    PubMed Central

    Suh, Young-Ah; Kim, Jai-Hyun; Sung, Myung A; Boo, Hye-Jin; Yun, Hye Jeong; Lee, Sun-Hye; Lee, Hyo-Jong; Suh, Young-Ger; Kim, Kyu-Won; Lee, Ho-Young

    2013-01-01

    In this study, we investigated the antitumor effects of deguelin in several human breast cancer cells in vitro and in vivo. Deguelin inhibited cell viability and the anchorage-dependent and anchorage-independent colony formation of triple-negative (MDA-MB-231 and MDA-MB-468) and triple-positive (MCF-7) breast cancer cells, and it significantly reduced the growth of MCF-7 cell xenograft tumors. The induction of apoptosis, inhibition of insulin-like growth factor-1 receptor (IGF-1R) signaling activation, and up-regulation of IGF-binding protein-3 (IGFBP-3) expression may be associated with deguelin-mediated antitumor effects. Our findings suggest a potential therapeutic use for deguelin in patients with triple-negative breast cancer and for those with breast cancers who are sensitive to endocrine- and HER2-targeted therapies. PMID:23348700

  11. Does adrenergic activity suppress insulin secretion during surgery? A clinical experiment with halothane anesthesia.

    PubMed Central

    Aärimaa, M; Syvälahti, E; Ovaska, J

    1978-01-01

    Peroperative inhibition of insulin release is widely attributed to increased alpha-adrenergic activity. To test this hypothesis serum insulin and glucose concentrations were measured at short intervals in 11 patients who underwent major surgery. Five patients were anesthetized with halothane and six with general anesthesia without halothane. The results were similar in both patient groups; halothane had no effect on insulin. This suggests that suppression of insulin under operations is probably not due to activation of the alpha-adrenergic receptors of the pancreatic beta-cells. The authors propose that suppression of insulin secretion during surgery may be caused by adrenaline, which, in competing for the glucose receptors, insensitizes the pancreatic beta-cells. PMID:202205

  12. Transition from passive to active targeting of oral insulin nanomedicines: enhancement in bioavailability and glycemic control in diabetes.

    PubMed

    Kaklotar, Dhansukh; Agrawal, Poornima; Abdulla, Allabakshi; Singh, Rahul P; Mehata, Abhishesh K; Singh, Sanjay; Mishra, Brahmeshwar; Pandey, Bajarangprasad L; Trigunayat, Anshuman; Muthu, Madaswamy S

    2016-06-01

    Oral insulin nanomedicines are effective tools for therapy and management of both Type I and Type II diabetes. This review summarizes the various nanocarriers developed so far in the literature for oral delivery of insulin. It includes lipid-based (i.e., solid lipid nanoparticles and liposomes) and polymeric-based insulin nanomedicines (i.e., chitosan nanoparticles, alginate nanoparticles, dextran nanoparticles and nanoparticles of synthetic polymers) for sustained, controlled and targeted oral delivery of insulin. Mainly, goblet cell-targeting, vitamin B12 receptor-targeting, folate receptor-targeting and transferrin receptor-targeting aspects were focused. Currently, passive and active targeting approaches of oral insulin nanomedicines have improved the oral absorption of insulin and its bioavailability (up to 14%) that produced effective glycaemic control in in vivo models. These results indicate a promising future of oral insulin nanomedicines for the treatment of diabetes. PMID:27171572

  13. Momordica charantia and its novel polypeptide regulate glucose homeostasis in mice via binding to insulin receptor.

    PubMed

    Lo, Hsin-Yi; Ho, Tin-Yun; Lin, Chingju; Li, Chia-Cheng; Hsiang, Chien-Yun

    2013-03-13

    Momordica charantia (MC) has been used as an alternative therapy for diabetes mellitus. This study analyzed and elucidated therapeutic targets contributing to the hypoglycemic effect of aqueous extract of MC seeds (MCSE) by transcriptomic analysis. Protein ingredients aimed at the hypoglycemic target were further identified by proteomic, docking, and receptor-binding assays. The data showed that MSCE (1 g/kg) significantly lowered the blood glucose level in normal and diabetic mice. Moreover, MCSE primarily regulated the insulin signaling pathway in muscles and adipose tissues, suggesting that MCSE might target insulin receptor (IR), stimulate the IR-downstream pathway, and subsequently display hypoglycemic activity in mice. It was further revealed that inhibitor against trypsin (TI) of MC directly docked into IR and activated the kinase activity of IR in a dose-dependent manner. In conclusion, the findings suggested that MCSE regulated glucose metabolism mainly via the insulin signaling pathway. Moreover, TI was newly identified as a novel IR-binding protein of MC that triggered the insulin signaling pathway via binding to IR. PMID:23414136

  14. Concentrations of Insulin Glargine and Its Metabolites During Long-Term Insulin Therapy in Type 2 Diabetic Patients and Comparison of Effects of Insulin Glargine, Its Metabolites, IGF-I, and Human Insulin on Insulin and IGF-I Receptor Signaling

    PubMed Central

    Varewijck, Aimee J.; Yki-Järvinen, Hannele; Schmidt, Ronald; Tennagels, Norbert; Janssen, Joseph A.M.J.L.

    2013-01-01

    We investigated 1) the ability of purified glargine (GLA), metabolites 1 (M1) and 2 (M2), IGF-I, and NPH insulin to activate the insulin receptor (IR)-A and IR-B and IGF-I receptor (IGF-IR) in vitro; 2) plasma concentrations of GLA, M1, and M2 during long-term insulin therapy in type 2 diabetic patients; and 3) IR-A and IR-B activation in vitro induced by serum from patients treated with GLA or NPH insulin. A total of 104 patients (age 56.3 ± 0.8 years, BMI 31.4 ± 0.5 kg/m2, and A1C 9.1 ± 0.1% [mean ± SE]) were randomized to GLA or NPH insulin therapy for 36 weeks. Plasma concentrations of GLA, M1, and M2 were determined by liquid chromatography–tandem mass spectrometry assay. IR-A, IR-B, and IGF-IR autophosphorylation was induced by purified hormones or serum by kinase receptor activation assays. In vitro, M1 induced comparable IR-A, IR-B, and IGF-IR autophosphorylation (activation) as NPH insulin. After 36 weeks, M1 increased from undetectable (<0.2 ng/mL) to 1.5 ng/mL (0.9–2.1), while GLA and M2 remained undetectable. GLA dose correlated with M1 (r = 0.84; P < 0.001). Serum from patients treated with GLA or NPH insulin induced similar IR-A and IR-B activation. These data suggest that M1 rather than GLA mediates GLA effects and that compared with NPH insulin, GLA does not increase IGF-IR signaling during long-term insulin therapy in type 2 diabetes. PMID:23569175

  15. The Drosophila Insulin Receptor Independently Modulates Lifespan and Locomotor Senescence

    PubMed Central

    Boylan, Michael; Achall, Rajesh; Shirras, Alan; Broughton, Susan J.

    2015-01-01

    The Insulin/IGF-like signalling (IIS) pathway plays an evolutionarily conserved role in ageing. In model organisms reduced IIS extends lifespan and ameliorates some forms of functional senescence. However, little is known about IIS in nervous system ageing and behavioural senescence. To investigate this role in Drosophila melanogaster, we measured the effect of reduced IIS on senescence of two locomotor behaviours, negative geotaxis and exploratory walking. Two long-lived fly models with systemic IIS reductions (daGAL4/UAS-InRDN (ubiquitous expression of a dominant negative insulin receptor) and d2GAL/UAS-rpr (ablation of insulin-like peptide producing cells)) showed an amelioration of negative geotaxis senescence similar to that previously reported for the long-lived IIS mutant chico. In contrast, exploratory walking in daGAL4/UAS-InRDN and d2GAL/UAS-rpr flies declined with age similarly to controls. To determine the contribution of IIS in the nervous system to these altered senescence patterns and lifespan, the InRDN was targeted to neurons (elavGAL4/UAS-InRDN), which resulted in extension of lifespan in females, normal negative geotaxis senescence in males and females, and detrimental effects on age-specific exploratory walking behaviour in males and females. These data indicate that the Drosophila insulin receptor independently modulates lifespan and age-specific function of different types of locomotor behaviour. The data suggest that ameliorated negative geotaxis senescence of long-lived flies with systemic IIS reductions is due to ageing related effects of reduced IIS outside the nervous system. The lifespan extension and coincident detrimental or neutral effects on locomotor function with a neuron specific reduction (elavGAL4/UAS-InRDN) indicates that reduced IIS is not beneficial to the neural circuitry underlying the behaviours despite increasing lifespan. PMID:26020640

  16. The Drosophila insulin receptor independently modulates lifespan and locomotor senescence.

    PubMed

    Ismail, Mohd Zamri Bin Haji; Hodges, Matt D; Boylan, Michael; Achall, Rajesh; Shirras, Alan; Broughton, Susan J

    2015-01-01

    The Insulin/IGF-like signalling (IIS) pathway plays an evolutionarily conserved role in ageing. In model organisms reduced IIS extends lifespan and ameliorates some forms of functional senescence. However, little is known about IIS in nervous system ageing and behavioural senescence. To investigate this role in Drosophila melanogaster, we measured the effect of reduced IIS on senescence of two locomotor behaviours, negative geotaxis and exploratory walking. Two long-lived fly models with systemic IIS reductions (daGAL4/UAS-InRDN (ubiquitous expression of a dominant negative insulin receptor) and d2GAL/UAS-rpr (ablation of insulin-like peptide producing cells)) showed an amelioration of negative geotaxis senescence similar to that previously reported for the long-lived IIS mutant chico. In contrast, exploratory walking in daGAL4/UAS-InRDN and d2GAL/UAS-rpr flies declined with age similarly to controls. To determine the contribution of IIS in the nervous system to these altered senescence patterns and lifespan, the InRDN was targeted to neurons (elavGAL4/UAS-InRDN), which resulted in extension of lifespan in females, normal negative geotaxis senescence in males and females, and detrimental effects on age-specific exploratory walking behaviour in males and females. These data indicate that the Drosophila insulin receptor independently modulates lifespan and age-specific function of different types of locomotor behaviour. The data suggest that ameliorated negative geotaxis senescence of long-lived flies with systemic IIS reductions is due to ageing related effects of reduced IIS outside the nervous system. The lifespan extension and coincident detrimental or neutral effects on locomotor function with a neuron specific reduction (elavGAL4/UAS-InRDN) indicates that reduced IIS is not beneficial to the neural circuitry underlying the behaviours despite increasing lifespan. PMID:26020640

  17. Coupling between insulin binding and activation of glucose transport in rat adipocytes

    SciTech Connect

    Ahn, N.G.; Lipkin, E.W.; Teller, D.C.; de Haeen, C.

    1986-05-01

    Previous studies have shown that the kinetics of binding of insulin (I) to its receptor (R) in isolated rat adipocytes at 15/sup 0/C, where insulin degradation was observed to be negligible, could best be described by the model: R+I in equilibrium RI in equilibrium R'I. According to this model, bound insulin is distributed between two kinetically distinct states of the occupied receptor, RI and R'I. The quantities of RI and R'I contributing to the observed total binding of insulin to cells can be obtained from the four rate constants describing the model. In order to examine the possible roles of RI and R'I in mediating hormone action, insulin stimulation of carrier-mediated 3-0-methyl-(U-/sup 14/C) glucose transport at 15/sup 0/C was studied. The results show that insulin activation of the rate of glucose transport was sigmoidal with time, and this was qualitatively similar to the formation of R'I with time. In contrast, formation of RI was described by an exponential approach to a plateau. This finding raises the possibility that R'I is the form of the insulin receptor directly mediating insulin activation of glucose transport.

  18. Synchronization in G0/G1 enhances the mitogenic response of cells overexpressing the human insulin receptor A isoform to insulin

    PubMed Central

    Nelander, Gitte-Mai; Hansen, Bo Falck; Jensen, Pia; Krabbe, Jonas S.; Jensen, Marianne B.; Hegelund, Anne Charlotte; Svendsen, Jette E.; Oleksiewicz, Martin B.

    2009-01-01

    Evaluating mitogenic signaling specifically through the human insulin receptor (IR) is relevant for the preclinical safety assessment of developmental insulin analogs. It is known that overexpression of IR sensitizes cells to the mitogenic effects of insulin, but it is essentially unknown how mitogenic responses can be optimized to allow practical use of such recombinant cell lines for preclinical safety testing. We constitutively overexpressed the short isoform of the human insulin receptor (hIR-A, exon 11-negative) in L6 rat skeletal myoblasts. Because the mitogenic effect of growth factors such as insulin is expected to act in G0/G1, promoting S-phase entry, we developed a combined topoinhibition + serum deprivation strategy to explore the effect of G0/G1 synchronization as an independent parameter in the context of serum deprivation, the latter being routinely used to reduce background in mitogenicity assays. G0/G1 synchronization significantly improved the mitogenic responses of L6-hIR cells to insulin, measured by 3H-thymidine incorporation. Comparison with the parental L6 cells using phospho-mitogen-activated protein kinase, phospho-AKT, as well as 3H-thymidine incorporation end points supported that the majority of the mitogenic effect of insulin in L6-hIR cells was mediated by the overexpressed hIR-A. Using the optimized L6-hIR assay, we found that the X-10 insulin analog was more mitogenic than native human insulin, supporting that X-10 exhibits increased mitogenic signaling through the hIR-A. In summary, this study provides the first demonstration that serum deprivation may not be sufficient, and G0/G1 synchronization may be required to obtain optimal responsiveness of hIR-overexpressing cell lines for preclinical safety testing. PMID:19898946

  19. Electrophysiological and behavioral phenotype of insulin receptor defective mice

    PubMed Central

    Das, P.; Parsons, A.D.; Scarborough, J.; Hoffman, J.; Wilson, J.; Thompson, R.N.; Overton, J.M.; Fadool, D.A.

    2009-01-01

    The olfactory bulb expresses one of the highest levels of insulin found in the brain. A high level of expression of the concomitant insulin receptor (IR) kinase is also retained in this brain region, even in the adult. We have previously demonstrated in a heterologous system that insulin modulates the voltage-dependent potassium channel, Kv1.3, through tyrosine phosphorylation of three key residues in the amino and carboxyl terminus of the channel protein. Phosphorylation also induces current suppression of the Kv1.3-contributed current in cultured olfactory bulb neurons (OBNs) of rodents. In order to explore the behavioral importance of this kinase-induced modulation of the channel for the olfactory ability of the animal, mice with a targeted-gene deletion of the insulin receptor were electrophysiologically and behaviorally characterized. Mice heterozygous for the insulin receptor kinase (IR+/−) gene performed the same as wild-type (+/+) mice when challenged with a traditional, non-learning-based task to test gross anosmia. There was also no significant difference across the two genotypes in tests designed to measure exploratory behavior or in a battery of systems physiology experiments designed to assess metabolic energy usage (locomotion, ingestive behaviors, weight, oxygen consumption, and respiratory quotient). Object memory recognition tests suggest that IR+/− mice have an impairment in recognition of familiarized objects; IR+/− mice demonstrate poor performance for both short-term (1 h) and long-term (24 h) memory tests in comparison to that of wild-type mice. Electrophysiological experiments indicate that mitral cell neurons cultured from both heterozygous and homozygous-null mice (IR+/− and IR−/−) have an decreased peak current amplitude compared with that recorded for wild-type (+/+) animals matched for days in vitro (DIV). These data indicate that the loss of one allele of the IR kinase gene modifies the electrical phenotype of the mitral

  20. Coevolution of insulin-like growth factors, insulin and their receptors and binding proteins in New World Monkeys.

    PubMed

    Wallis, Michael

    2015-08-01

    Previous work has shown that the evolution of both insulin-like growth factor 1 (IGF1) and insulin shows an episode of accelerated change on the branch leading to New World Monkeys (NWM). Here the possibility that this is accompanied by a corresponding episode of accelerated evolution of IGF1 receptor (IGF1R), insulin receptor (IR) and/or IGF binding proteins (IGFBPs) was investigated. Analysis of receptor sequences from a range of primates and some non-primate mammals showed that accelerated evolution did indeed occur on this branch in the case of IGF1R and IR, but not for the similar insulin receptor-related receptor (IRRR) which does not bind insulin or IGF1. Marked accelerated evolution on this branch was also seen for some IGFBPs, but not the mannose 6-phosphate/IGF2 receptor or epidermal growth factor receptor. The rate of evolution slowed before divergence of the lineages leading to the NWM for which sequences are available (Callithrix and Saimiri). For the IGF1R and IR, the accelerated evolution was most marked for the extracellular domains (ectodomains). Application of the branch-site method showed dN/dS ratios significantly greater than 1.0 for both receptor ectodomains and for IGFBP1, and allowed identification of residues likely to have been subject to selection. These residues were concentrated in the N-terminal half of the IGF1R ectodomain but the C-terminal half of the IR ectodomain, which could have implications for the formation of hybrid receptors. Overall the results suggest that adaptive coevolution of IGF1, insulin and their receptors and some IGFBPs occurred during the evolution of NWM. For the most part, the residues that change on this branch could not be associated with specific functional aspects (ligand binding, receptor dimerization, glycosylation) and the physiological significance of this coevolution remains to be established. PMID:26072449

  1. Surface-expressed insulin receptors as well as IGF-I receptors both contribute to the mitogenic effects of human insulin and its analogues.

    PubMed

    Lundby, Anders; Bolvig, Pernille; Hegelund, Anne Charlotte; Hansen, Bo F; Worm, Jesper; Lützen, Anne; Billestrup, Nils; Bonnesen, Christine; Oleksiewicz, Martin B

    2015-07-01

    There is a medical need for new insulin analogues. Yet, molecular alterations to the insulin molecule can theoretically result in analogues with carcinogenic effects. Preclinical carcinogenicity risk assessment for insulin analogues rests to a large extent on mitogenicity assays in cell lines. We therefore optimized mitogenicity assay conditions for a panel of five cell lines. All cell lines expressed insulin receptors (IR), IGF-I receptors (IGF-IR) and hybrid receptors, and in all cell lines, insulin as well as the comparator compounds X10 and IGF-I caused phosphorylation of the IR as well as IGF-IR. Insulin exhibited mitogenicity EC(50) values in the single-digit nanomolar to picomolar range. We observed correlations across cell types between (i) mitogenic potency of insulin and IGF-IR/IR ratio, (ii) Akt phosphorylation and mitogenic potency and (iii) Akt phosphorylation and IR phosphorylation. Using siRNA-mediated knockdown of IR and IGF-IR, we observed that in HCT 116 cells the IR appeared dominant in driving the mitogenic response to insulin, whereas in MCF7 cells the IGF-IR appeared dominant in driving the mitogenic response to insulin. Together, our results show that the IR as well as IGF-IR may contribute to the mitogenic potency of insulin. While insulin was a more potent mitogen than IGF-I in cells expressing more IR than IGF-IR, the hyper-mitogenic insulin analogue X10 was a more potent mitogen than insulin across all cell types, supporting that the hyper-mitogenic effect of X10 involves the IR as well as the IGF-IR. These results are relevant for preclinical safety assessment of developmental insulin analogues. PMID:25413577

  2. Evidence that the angiotensin IV (AT(4)) receptor is the enzyme insulin-regulated aminopeptidase.

    PubMed

    Albiston, A L; McDowall, S G; Matsacos, D; Sim, P; Clune, E; Mustafa, T; Lee, J; Mendelsohn, F A; Simpson, R J; Connolly, L M; Chai, S Y

    2001-12-28

    Central infusion of angiotensin IV or its more stable analogues facilitates memory retention and retrieval in normal animals and reverses amnesia induced by scopolamine or by bilateral perforant pathway lesions. These peptides bind with high affinity and specificity to a novel binding site designated the angiotensin AT(4) receptor. Until now, the AT(4) receptor has eluded molecular characterization. Here we identify the AT(4) receptor, by protein purification and peptide sequencing, to be insulin-regulated aminopeptidase (IRAP). HEK 293T cells transfected with IRAP exhibit typical AT(4) receptor binding characteristics; the AT(4) receptor ligands, angiotensin IV and LVV-hemorphin 7, compete for the binding of [(125)I]Nle(1)-angiotensin IV with IC(50) values of 32 and 140 nm, respectively. The distribution of IRAP and its mRNA in the brain, determined by immunohistochemistry and hybridization histochemistry, parallels that of the AT(4) receptor determined by radioligand binding. We also show that AT(4) receptor ligands dose-dependently inhibit the catalytic activity of IRAP. We have therefore demonstrated that the AT(4) receptor is IRAP and propose that AT(4) receptor ligands may exert their effects by inhibiting the catalytic activity of IRAP thereby extending the half-life of its neuropeptide substrates. PMID:11707427

  3. A novel insulin receptor-binding protein from Momordica charantia enhances glucose uptake and glucose clearance in vitro and in vivo through triggering insulin receptor signaling pathway.

    PubMed

    Lo, Hsin-Yi; Ho, Tin-Yun; Li, Chia-Cheng; Chen, Jaw-Chyun; Liu, Jau-Jin; Hsiang, Chien-Yun

    2014-09-10

    Diabetes, a common metabolic disorder, is characterized by hyperglycemia. Insulin is the principal mediator of glucose homeostasis. In a previous study, we identified a trypsin inhibitor, named Momordica charantia insulin receptor (IR)-binding protein (mcIRBP) in this study, that might interact with IR. The physical and functional interactions between mcIRBP and IR were clearly analyzed in the present study. Photo-cross-linking coupled with mass spectrometry showed that three regions (17-21, 34-40, and 59-66 residues) located on mcIRBP physically interacted with leucine-rich repeat domain and cysteine-rich region of IR. IR-binding assay showed that the binding behavior of mcIRBP and insulin displayed a cooperative manner. After binding to IR, mcIRBP activated the kinase activity of IR by (5.87 ± 0.45)-fold, increased the amount of phospho-IR protein by (1.31 ± 0.03)-fold, affected phosphoinositide-3-kinase/Akt pathways, and consequently stimulated the uptake of glucose in 3T3-L1 cells by (1.36 ± 0.12)-fold. Intraperitoneal injection of 2.5 nmol/kg mcIRBP significantly decreased the blood glucose levels by 20.9 ± 3.2% and 10.8 ± 3.6% in normal and diabetic mice, respectively. Microarray analysis showed that mcIRBP affected genes involved in insulin signaling transduction pathway in mice. In conclusion, our findings suggest that mcIRBP is a novel IRBP that binds to sites different from the insulin-binding sites on IR and stimulates both the glucose uptake in cells and the glucose clearance in mice. PMID:25144709

  4. Insulin Receptor Signaling in Long-Term Memory Consolidation Following Spatial Learning

    ERIC Educational Resources Information Center

    Dou, Jing-Tao; Chen, Min; Dufour, Franck; Alkon, Daniel L.; Zhao, Wei-Qin

    2005-01-01

    Evidence has shown that the insulin and insulin receptor (IR) play a role in cognitive function. However, the detailed mechanisms underlying insulin's action on learning and memory are not yet understood. Here we investigated changes in long-term memory-associated expression of the IR and downstream molecules in the rat hippocampus. After…

  5. Insulin/glucose induces natriuretic peptide clearance receptor in human adipocytes: a metabolic link with the cardiac natriuretic pathway.

    PubMed

    Bordicchia, M; Ceresiani, M; Pavani, M; Minardi, D; Polito, M; Wabitsch, M; Cannone, V; Burnett, J C; Dessì-Fulgheri, P; Sarzani, R

    2016-07-01

    Cardiac natriuretic peptides (NP) are involved in cardiorenal regulation and in lipolysis. The NP activity is largely dependent on the ratio between the signaling receptor NPRA and the clearance receptor NPRC. Lipolysis increases when NPRC is reduced by starving or very-low-calorie diet. On the contrary, insulin is an antilipolytic hormone that increases sodium retention, suggesting a possible functional link with NP. We examined the insulin-mediated regulation of NP receptors in differentiated human adipocytes and tested the association of NP receptor expression in visceral adipose tissue (VAT) with metabolic profiles of patients undergoing renal surgery. Differentiated human adipocytes from VAT and Simpson-Golabi-Behmel Syndrome (SGBS) adipocyte cell line were treated with insulin in the presence of high-glucose or low-glucose media to study NP receptors and insulin/glucose-regulated pathways. Fasting blood samples and VAT samples were taken from patients on the day of renal surgery. We observed a potent insulin-mediated and glucose-dependent upregulation of NPRC, through the phosphatidylinositol 3-kinase pathway, associated with lower lipolysis in differentiated adipocytes. No effect was observed on NPRA. Low-glucose medium, used to simulate in vivo starving conditions, hampered the insulin effect on NPRC through modulation of insulin/glucose-regulated pathways, allowing atrial natriuretic peptide to induce lipolysis and thermogenic genes. An expression ratio in favor of NPRC in adipose tissue was associated with higher fasting insulinemia, HOMA-IR, and atherogenic lipid levels. Insulin/glucose-dependent NPRC induction in adipocytes might be a key factor linking hyperinsulinemia, metabolic syndrome, and higher blood pressure by reducing NP effects on adipocytes. PMID:27101299

  6. Glucose enhances insulin promoter activity in MIN6 beta-cells independently of changes in intracellular Ca2+ concentration and insulin secretion.

    PubMed Central

    Kennedy, H J; Rafiq, I; Pouli, A E; Rutter, G A

    1999-01-01

    Recent studies have suggested that glucose may activate insulin gene transcription through increases in intracellular Ca(2+) concentration, possibly acting via the release of stored insulin. We have investigated this question by dynamic photon-counting imaging of insulin- and c-fos-promoter-firefly luciferase reporter construct activity. Normalized to constitutive viral promoter activity, insulin promoter activity in MIN6 beta-cells was increased 1.6-fold after incubation at 30 mM compared with 3 mM glucose, but was unaltered at either glucose concentration by the presence of insulin (100 nM) or the Ca(2+) channel inhibitor, verapamil (100 microM). Increases in intracellular [Ca(2+)] achieved by plasma membrane depolarization with KCl failed to enhance either insulin or c-fos promoter activity in MIN6 cells, but increased c-fos promoter activity 5-fold in AtT20 cells. Together, these results demonstrate that glucose can exert a direct effect on insulin promoter activity in islet beta-cells, via a signalling pathway which does not require increases in intracellular [Ca(2+)] nor insulin release and insulin receptor activation. PMID:10455011

  7. Growth-stimulatory monoclonal antibodies against human insulin-like growth factor I receptor.

    PubMed

    Xiong, L; Kasuya, J; Li, S L; Kato, J; Fujita-Yamaguchi, Y

    1992-06-15

    Monoclonal antibodies (mAbs) against purified human placental insulin-like growth factor I (IGF-I) receptors were prepared and characterized. Three IgG mAbs were specific for the human IGF-I receptor and displayed negligible crossreactivity with the human insulin receptor. They stimulated 125I-labeled IGF-I (125I-IGF-I) or 125I-IGF-II binding to purified human placental IGF-I receptors and to IGF-I receptors expressed in NIH 3T3 cells in contrast to the well-studied mAb alpha IR-3, which inhibits 125I-IGF-I or 125I-IGF-II binding to both forms of IGF-I receptors. The mAbs introduced in this study stimulated DNA synthesis in NIH 3T3 cells expressing human IGF-I receptors approximately 1.5-fold above the basal level and the IGF-I- or IGF-II-stimulated level. In contrast, alpha IR-3 inhibited both basal and IGF-I or IGF-II-stimulated DNA synthesis by approximately 30%. Inhibition of IGF-II-stimulated DNA synthesis by alpha IR-3 was as potent as its inhibition of IGF-I-stimulated DNA synthesis, although IGF-II binding to the IGF-I receptors was not inhibited by IGF-II as potently as was IGF-I. With the purified IGF-I receptors, both inhibitory and stimulatory mAbs were shown to activate autophosphorylation of the IGF-I receptor beta subunit and to induce microaggregation of the receptors. These results suggest that conformational changes resulting from receptor dimerization in the presence of either type of mAb may affect the signal-transducing function of the IGF-I receptor differently. These additional mAbs and alpha IR-3 immunoprecipitated nearly 90% of IGF-I binding activity from Triton X-100-solubilized human placental membranes, indicating that IGF-I receptor reactive with these mAbs is the major form of the IGF-I receptor in human placenta. PMID:1319060

  8. Diabetic state, high plasma insulin and angiotensin II combine to augment endothelin-1-induced vasoconstriction via ETA receptors and ERK

    PubMed Central

    Kobayashi, T; Nogami, T; Taguchi, K; Matsumoto, T; Kamata, K

    2008-01-01

    Background and purpose: Mechanisms associated with the enhanced contractile response to endothelin-1 in hyperinsulinaemic diabetes have been examined using the rat aorta. Functions for angiotensin II, endothelin-1 receptor expression and extracellular signal-regulated kinase (ERK) have been investigated. Experimental approach: Streptozotocin-induced diabetic rats were infused with angiotensin II or, following insulin treatment, were treated with losartan, an angiotensin II receptor antagonist. Contractions of aortic strips with or without endothelium, in response to endothelin-1 and angiotensin II, were examined in vitro. Aortic ETA receptors and ERK/MEK expression were measured by western blotting. Key results: Insulin-treated diabetic rats exhibited increases in plasma insulin, angiotensin II and endothelin-1. The systolic blood pressure and endothelin-1-induced contractile responses in aortae in vitro were enhanced in insulin-treated diabetic rats and blunted by chronic losartan administration. LY294002 (phosphatidylinositol 3-kinase inhibitor) and/or PD98059 (MEK inhibitor) diminished the enhanced contractile response to endothelin-1 in aortae from insulin-treated diabetic rats. ETA and ETB receptors, ERK-1/2 and MEK-1/2 protein expression and endothelin-1-stimulated ERK phosphorylation were all increased in aortae from insulin-treated diabetic rats. Such increases were blunted by chronic losartan administration. Endothelin-1-induced contraction was significantly higher in aortae from angiotensin II-infused diabetic rats. angiotensin II-infusion increased ERK phosphorylation, but the expression of endothelin receptors and ERK/MEK proteins remained unchanged. Conclusions and implications: These results suggest that the combination of high plasma angiotensin II and insulin with a diabetic state induced enhancement of endothelin-1-induced vasoconstriction, ETA receptor expression and ERK expression/activity in the aorta. Losartan improved both the diabetes

  9. In vitro and in vivo antitumor effects of the dual insulin-like growth factor-I/insulin receptor inhibitor, BMS-554417.

    PubMed

    Haluska, Paul; Carboni, Joan M; Loegering, David A; Lee, Francis Y; Wittman, Mark; Saulnier, Mark G; Frennesson, David B; Kalli, Kimberly R; Conover, Cheryl A; Attar, Ricardo M; Kaufmann, Scott H; Gottardis, Marco; Erlichman, Charles

    2006-01-01

    The insulin-like growth factor receptor (IGF-IR) and insulin receptor are either overactivated and/or overexpressed in a wide range of tumor types and contribute to tumorigenicity, proliferation, metastasis, and drug resistance. Here, we show that BMS-554417, a novel small molecule developed as an inhibitor of IGF-IR, inhibits IGF-IR and insulin receptor kinase activity and proliferation in vitro, and reduces tumor xenograft size in vivo. In a series of carcinoma cell lines, the IC50 for proliferation ranged from 120 nmol/L (Colo205) to >8.5 micromol/L (OV202). The addition of stimulatory ligands was unnecessary for the antiproliferative effect in MCF-7 and OV202 cells. BMS-554417 treatment inhibited IGF-IR and insulin receptor signaling through extracellular signal-related kinase as well as the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased Akt phosphorylation at Ser473. At doses that inhibited proliferation, the compound also caused a G0-G1 arrest and prevented nuclear accumulation of cyclin D1 in response to LR3 IGF-I. In Jurkat T-cell leukemia cells, this agent triggered apoptotic cell death via the mitochondrial pathway. BMS-554417 was orally bioavailable and significantly inhibited the growth of IGF1R-Sal tumor xenografts in vivo. BMS-554417 is a member of a novel class of IGF-IR/insulin receptor inhibitors that have potential clinical applications because of their antiproliferative and proapoptotic activity in vitro and in vivo. PMID:16397250

  10. Genomic organization and expression of insulin receptors in grass carp, Ctenopharyngodon idellus.

    PubMed

    Cai, Wenjing; Liang, Xu-Fang; Yuan, Xiaochen; Li, Aixuan; He, Yuhui; He, Shan

    2016-01-01

    Insulin receptors have been demonstrated to be involved in embryogenesis, food intake regulation and glucose metabolism in several fish, while more researchis needed for further understanding. In this study, the complete coding sequence (CDS) of insulin receptor a (insra) gene and insulin receptor b (insrb) gene in grass carp were obtained, the CDS were 4068 bp and 4514 bp in length, encoding 1355 aa protein and 1351 aa protein. Both of insra and insrb in grass carp showed high amino acid identities with other fish. Insra and insrb genes were widely expressed in all tested tissues with an overlapping but distinct expressions. The high levels of insra mRNA were distributed in hindgut and heart tissues. The insrb gene showed the highest expression levels in liver and hindgut. We also proved that two forms of grass carp insulin receptors participate in the regulation of blood glucose and might act differently. Phylogenetic analysis confirmed that different isoforms of fish insulin receptors are derived from two distinct genes, which was inconsistent with the generation of mammalian insulin receptors. Synteny analyses of insulin receptor genes showed that genes surrounding the insulin receptor genes were conserved in fish. Arhgef18, PEX11G, humanC19orf45 genes were highly conserved among mammal species. However, no conserved synteny was observed among fish, mammals, avians and amphibians. PMID:26772721