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Sample records for insulin receptor activator

  1. 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.

  2. Activation of insulin signal transduction pathway and anti-diabetic activity of small molecule insulin receptor activators.

    PubMed

    Qureshi, S A; Ding, V; Li, Z; Szalkowski, D; Biazzo-Ashnault, D E; Xie, D; Saperstein, R; Brady, E; Huskey, S; Shen, X; Liu, K; Xu, L; Salituro, G M; Heck, J V; Moller, D E; Jones, A B; Zhang, B B

    2000-11-24

    We recently described the identification of a non-peptidyl fungal metabolite (l-783,281, compound 1), which induced activation of human insulin receptor (IR) tyrosine kinase and mediated insulin-like effects in cells, as well as decreased blood glucose levels in murine models of Type 2 diabetes (Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M. T. , Pelaez, F., Ruby, C., Kendall, R. L., Mao, X., Griffin, P., Calaycay, J., Zierath, J. R., Heck, J. V., Smith, R. G. & Moller, D. E. (1999) Science 284, 974-977). Here we report the characterization of an active analog (compound 2) with enhanced IR kinase activation potency and selectivity over related receptors (insulin-like growth factor I receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor). The IR activators stimulated tyrosine kinase activity of partially purified native IR and recombinant IR tyrosine kinase domain. Administration of the IR activators to mice was associated with increased IR tyrosine kinase activity in liver. In vivo oral treatment with compound 2 resulted in significant glucose lowering in several rodent models of diabetes. In db/db mice, oral administration of compound 2 elicited significant correction of hyperglycemia. In a streptozotocin-induced diabetic mouse model, compound 2 potentiated the glucose-lowering effect of insulin. In normal rats, compound 2 improved oral glucose tolerance with significant reduction in insulin release following glucose challenge. A structurally related inactive analog (compound 3) was not effective on insulin receptor activation or glucose lowering in db/db mice. Thus, small molecule IR activators exert insulin mimetic and sensitizing effects in cells and in animal models of diabetes. These results have implications for the future development of new therapies for diabetes mellitus.

  3. 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.

  4. 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.

  5. Corticosterone-induced insulin resistance is not associated with alterations of insulin receptor number and kinase activity in chicken kidney.

    PubMed

    Bisbis, S; Taouis, M; Derouet, M; Chevalier, B; Simon, J

    1994-12-01

    Chicken renal insulin receptors have been recently characterized; their number and kinase activities vary in response to altered nutritional status. In the present study, the effect of chronic corticosterone treatment was examined in 5-week-old chickens. The development of an insulin resistance following corticosterone was suggested after 1 and 2 weeks of treatment by a significant increases in plasma insulin levels (1.63 +/- 0.13 vs 0.56 +/- 0.14 ng insulin/ml in controls) and in renal cytosolic phosphoenolpyruvate carboxykinase activity (17.2 +/- 0.8 vs 13.7 +/- 0.7 nm/mn/mg tissue in controls). No significant changes were present at the level of insulin receptor number and kinase activity. Therefore, in kidney and, as previously observed, in muscles, corticosterone can induce insulin resistance at postreceptor steps in the cascade of events leading to insulin action.

  6. Solubilized placental membrane protein inhibits insulin receptor tyrosine kinase activity

    SciTech Connect

    Strout, H.V. Jr.; Slater, E.E.

    1987-05-01

    Regulation of insulin receptor (IR) tyrosine kinase (TK) activity may be important in modulating insulin action. Utilizing an assay which measures IR phosphorylation of angiotensin II (AII), the authors investigated whether fractions of TX-100 solubilized human placental membranes inhibited IR dependent AII phosphorylation. Autophosphorylated IR was incubated with membrane fractions before the addition of AII, and kinase inhibition measured by the loss of TSP incorporated in AII. An inhibitory activity was detected which was dose, time, and temperature dependent. The inhibitor was purified 200-fold by sequential chromatography on wheat germ agglutinin, DEAE, and hydroxyapatite. This inhibitory activity was found to correlate with an 80 KD protein which was electroeluted from preparative slab gels and rabbit antiserum raised. Incubation of membrane fractions with antiserum before the IRTK assay immunoprecipitated the inhibitor. Protein immunoblots of crude or purified fractions revealed only the 80 KD protein. Since IR autophosphorylation is crucial to IRTK activity, the authors investigated the state of IR autophosphorylation after treatment with inhibitor; no change was detected by phosphoamino acid analysis.

  7. Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

    PubMed

    Takayama, S; White, M F; Kahn, C R

    1988-03-05

    The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

  8. Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects.

    PubMed Central

    Goodyear, L J; Giorgino, F; Sherman, L A; Carey, J; Smith, R J; Dohm, G L

    1995-01-01

    To determine whether the impaired insulin-stimulated glucose uptake in obese individuals is associated with altered insulin receptor signaling, we measured both glucose uptake and early steps in the insulin action pathway in intact strips of human skeletal muscle. Biopsies of rectus abdominus muscle were taken from eight obese and eight control subjects undergoing elective surgery (body mass index 52.9 +/- 3.6 vs 25.7 +/- 0.9). Insulin-stimulated 2-deoxyglucose uptake was 53% lower in muscle strips from obese subjects. Additional muscle strips were incubated in the basal state or with 10(-7) M insulin for 2, 15, or 30 min. In the lean subjects, tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), measured by immunoblotting with anti-phosphotyrosine antibodies, was significantly increased by insulin at all time points. In the skeletal muscle from the obese subjects, insulin was less effective in stimulating tyrosine phosphorylation (maximum receptor and IRS-1 phosphorylation decreased by 35 and 38%, respectively). Insulin stimulation of IRS-1 immunoprecipitable phosphatidylinositol 3-kinase (PI 3-kinase) activity also was markedly lower in obese subjects compared with controls (10- vs 35-fold above basal, respectively). In addition, the obese subjects had a lower abundance of the insulin receptor, IRS-1, and the p85 subunit of PI 3-kinase (55, 54, and 64% of nonobese, respectively). We conclude that impaired insulin-stimulated glucose uptake in skeletal muscle from severely obese subjects is accompanied by a deficiency in insulin receptor signaling, which may contribute to decreased insulin action. Images PMID:7537758

  9. 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...

  10. The association of phosphoinositide 3-kinase enhancer A with hepatic insulin receptor enhances its kinase activity.

    PubMed

    Chan, Chi Bun; Liu, Xia; He, Kunyan; Qi, Qi; Jung, Dae Y; Kim, Jason K; Ye, Keqiang

    2011-07-01

    Dysfunction of hepatic insulin receptor tyrosine kinase (IRTK) causes the development of type 2 diabetes. However, the molecular mechanism regulating IRTK activity in the liver remains poorly understood. Here, we show that phosphoinositide 3-kinase enhancer A (PIKE-A) is a new insulin-dependent enhancer of hepatic IRTK. Liver-specific Pike-knockout (LPKO) mice display glucose intolerance with impaired hepatic insulin sensitivity. Specifically, insulin-provoked phosphoinositide 3-kinase/Akt signalling is diminished in the liver of LPKO mice, leading to the failure of insulin-suppressed gluconeogenesis and hyperglycaemia. Thus, hepatic PIKE-A has a key role in mediating insulin signal transduction and regulating glucose homeostasis in the liver.

  11. 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

  12. 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.

  13. Ligand-dependent intersubunit association with the insulin receptor complex activates its intrinsic kinase activity

    SciTech Connect

    Boeni-Schnetzler, M.; Kaligian, A.; DelVecchio, R.; Pilch, P.F.

    1988-05-15

    Insulin receptor halves (..cap alpha beta..) were obtained upon selective reduction of the holoreceptor (..cap alpha../sub 2/..beta../sub 2/) and were isolated in concentrated form. Autophosphorylation of concentrated ..cap alpha beta.. receptor halves can be stimulated by insulin an average of 4.0-fold, whereas nonreduced holoreceptor can be stimulated 5.4-fold. If ..cap alpha beta.. half-receptors are immobilized on wheat germ agglutinin-agarose, no insulin-stimulated autophosphorylation is observed, whereas immobilized holoreceptor retains insulin responsiveness. Treatment of ..cap alpha beta.. half-receptors with glutathione in the presence of insulin results in reoxidation to the holoreceptor form (..cap alpha../sub 2/..beta../sub 2/) with an efficiency of 60-70% as visualized by immunoblotting, thus providing evidence that two ..cap alpha beta.. halves are in close physical proximity. This reoxidation reaction, which is evident prior to autophosphorylation, is rapid and strictly dependent on the presence of insulin, consistent with the hypothesis that insulin promotes the association of two ..cap alpha beta.. halves. Furthermore, the insulin-induced reoxidation reaction and the insulin-induced autophosphorylation show the same dose dependence suggesting that the noncovalent association of ..cap alpha beta.. half-receptors upon insulin binding is a prerequisite for insulin-stimulated autophosphorylation in concentrated ..gamma beta.. half-receptor preparations. If the ..cap alpha beta.. half-receptor forms are phosphorylated in the presence of an anti-phosphotyrosine antibody and separated from nonphosphorylated ..cap alpha beta.. receptors, we observe that the phosphorylated ..cap alpha beta.. receptor halves contain bound insulin.

  14. Contraction inhibits insulin-stimulated insulin receptor substrate-1/2-associated phosphoinositide 3-kinase activity, but not protein kinase B activation or glucose uptake, in rat muscle.

    PubMed Central

    Whitehead, J P; Soos, M A; Aslesen, R; O'rahilly, S; Jensen, J

    2000-01-01

    The initial stages of insulin-stimulated glucose uptake are thought to involve tyrosine phosphorylation of insulin receptor substrates (IRSs), which recruit and activate phosphoinositide 3-kinase (PI 3-kinase), leading to the activation of protein kinase B (PKB) and other downstream effectors. In contrast, contraction stimulates glucose uptake via a PI 3-kinase-independent mechanism. The combined effects of insulin and contraction on glucose uptake are additive. However, it has been reported that contraction causes a decrease in insulin-stimulated IRS-1-associated PI 3-kinase activity. To investigate this paradox, we have examined the effects of contraction on insulin-stimulated glucose uptake and proximal insulin-signalling events in isolated rat epitrochlearis muscle. Stimulation by insulin or contraction produced a 3-fold increase in glucose uptake, with the effects of simultaneous treatment by insulin and contraction being additive. Wortmannin completely blocked the additive effect of insulin in contracting skeletal muscle, indicating that this is a PI 3-kinase-dependent effect. Insulin-stimulated recruitment of PI 3-kinase to IRS-1 was unaffected by contraction; however, insulin produced no discernible increase in PI 3-kinase activity in IRS-1 or IRS-2 immunocomplexes in contracting skeletal muscle. Consistent with this, contraction inhibited insulin-stimulated p70(S6K) activation. In contrast, insulin-stimulated activation of PKB was unaffected by contraction. Thus, in contracting skeletal muscle, insulin stimulates glucose uptake and activates PKB, but not p70(S6K), by a PI 3-kinase-dependent mechanism that is independent of changes in IRS-1- and IRS-2-associated PI 3-kinase activity. PMID:10903138

  15. 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

  16. 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.

  17. 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.

  18. Internalization and activation of the rat liver insulin receptor kinase in vivo.

    PubMed

    Khan, M N; Baquiran, G; Brule, C; Burgess, J; Foster, B; Bergeron, J J; Posner, B I

    1989-08-05

    The preparation of clearly delineated plasmalemma (PM) and endosomal subcellular fractions from rat liver has allowed us to compare insulin receptor (IR) kinase activity at the cell surface and in hepatic endosomes (ENs) as a function of dose and time after injected insulin. Tyrosine kinase activity in PM and ENs was measured, after solubilization and partial purification by wheat germ agglutinin chromatography (lectin-purified), using poly(Glu:Tyr) as substrate. Following the injection of a subsaturating dose of insulin (1.5 micrograms/100 g body weight), lectin-purified receptor showed peak activation at 30 s in PM and at 2 min in ENs. As observed previously (Khan, M. N., Savoie, S., Bergeron, J. J. M., and Posner, B. I. (1986) J. Biol. Chem. 261, 8462-8472) autophosphorylation activity was also augmented following insulin injection. In a pattern virtually identical to that of exogenous kinase activity, autophosphorylation attained peak activity at 30 s in PM and at 2 min in ENs. The time course of IR autophosphorylation in intact membranes was very similar to that observed for lectin purified receptors and was seen with an injected insulin dose as low as 150 ng/100 g body weight. Phosphatase treatment of the solubilized endosomal receptor abolished its enhanced activity. Hence, insulin treatment led to in vivo receptor phosphorylation which was reflected in the enhancement of both tyrosine kinase and autophosphorylation activities. Significant differences in the phosphorylation activities of PM and ENs were observed. Phosphoamino acid analyses revealed that the activated IR of intact PM was autophosphorylated in vitro, at both serine (55%) and tyrosine (45%) residues; whereas the activated IR of intact ENs was phosphorylated in vitro exclusively on tyrosine autophosphorylation specific activity for the activated IR of ENs was 3- to 4-fold that of the IR of PM. This was observed for the lectin purified IRs as well as for IRs of intact cell fractions. The reduced

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Insulin receptor substrate-2 phosphorylation is necessary for protein kinase C zeta activation by insulin in L6hIR cells.

    PubMed

    Oriente, F; Formisano, P; Miele, C; Fiory, F; Maitan, M A; Vigliotta, G; Trencia, A; Santopietro, S; Caruso, M; Van Obberghen, E; Beguinot, F

    2001-10-05

    We have investigated glycogen synthase (GS) activation in L6hIR cells expressing a peptide corresponding to the kinase regulatory loop binding domain of insulin receptor substrate-2 (IRS-2) (KRLB). In several clones of these cells (B2, F4), insulin-dependent binding of the KRLB to insulin receptors was accompanied by a block of IRS-2, but not IRS-1, phosphorylation, and insulin receptor binding. GS activation by insulin was also inhibited by >70% in these cells (p < 0.001). The impairment of GS activation was paralleled by a similarly sized inhibition of glycogen synthase kinase 3 alpha (GSK3 alpha) and GSK3 beta inactivation by insulin with no change in protein phosphatase 1 activity. PDK1 (a phosphatidylinositol trisphosphate-dependent kinase) and Akt/protein kinase B (PKB) activation by insulin showed no difference in B2, F4, and in control L6hIR cells. At variance, insulin did not activate PKC zeta in B2 and F4 cells. In L6hIR, inhibition of PKC zeta activity by either a PKC zeta antisense or a dominant negative mutant also reduced by 75% insulin inactivation of GSK3 alpha and -beta (p < 0.001) and insulin stimulation of GS (p < 0.002), similar to Akt/PKB inhibition. In L6hIR, insulin induced protein kinase C zeta (PKC zeta) co-precipitation with GSK3 alpha and beta. PKC zeta also phosphorylated GSK3 alpha and -beta. Alone, these events did not significantly affect GSK3 alpha and -beta activities. Inhibition of PKC zeta activity, however, reduced Akt/PKB phosphorylation of the key serine sites on GSK3 alpha and -beta by >80% (p < 0.001) and prevented full GSK3 inactivation by insulin. Thus, IRS-2, not IRS-1, signals insulin activation of GS in the L6hIR skeletal muscle cells. In these cells, insulin inhibition of GSK3 alpha and -beta requires dual phosphorylation by both Akt/PKB and PKC zeta.

  4. 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.

  5. The antidiabetic agent sodium tungstate activates glycogen synthesis through an insulin receptor-independent pathway.

    PubMed

    Domínguez, Jorge E; Muñoz, M Carmen; Zafra, Delia; Sanchez-Perez, Isabel; Baqué, Susanna; Caron, Martine; Mercurio, Ciro; Barberà, Albert; Perona, Rosario; Gomis, Ramon; Guinovart, Joan J

    2003-10-31

    Sodium tungstate is a powerful antidiabetic agent when administered orally. In primary cultured hepatocytes, tungstate showed insulin-like actions, which led to an increase in glycogen synthesis and accumulation. However, this compound did not significantly alter the insulin receptor activation state or dephosphorylation rate in cultured cells (CHO-R) or in primary hepatocytes, in either short or long term treatments. In contrast, at low concentrations, tungstate induced a transient strong activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) after 5-10 min of treatment, in a similar way to insulin. Moreover, this compound did not significantly delay or inhibit the dephosphorylation of ERK1/2. ERK1/2 activation triggered a cascade of downstream events, which included the phosphorylation of p90rsk and glycogen synthase-kinase 3beta. Experiments with a specific inhibitor of ERK1/2 activation and kinase assays indicate that these proteins were directly involved in the stimulation of glycogen synthase and glycogen synthesis induced by tungstate without a direct involvement of protein kinase B (PKB/Akt). These results show a direct involvement of ERK1/2 in the mechanism of action of tungstate at the hepatic level.

  6. Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses.

    PubMed

    Zhang, Manchao; Deng, Youping; Tandon, Ruchi; Bai, Cheng; Riedel, Heimo

    2008-01-01

    The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.

  7. [Peroxisome proliferator-activated receptors (PPARs) in obesity and insulin resistance development].

    PubMed

    Alemán, Gabriela; Torres, Nimbe; Tovar, Armando R

    2004-01-01

    The peroxisome proliferator-activated receptors (PPARs) are a family of nuclear transcription factors that belong to the steroid receptor superfamily. PPARs family includes PPARalpha, PPARbeta/delta, PPARgamma1 and PPARgamma2. PPARs form an heterodimer with the 9-cis retinoic acid receptor (RXR) and bind to response elements present in target genes activated by these transcription factors. PPARs control the expression of genes involved in fatty acid synthesis, oxidation and storage. PPARs are present in most tissues, where PPARalpha is most abundant in liver and skeletal muscle, whereas PPARgamma is found mainly in adipose tissue. Natural ligands for PPARs are polyunsaturated fatty acids (PUFAs) and some eicosanoids, however they are also activated by compounds such as fibrates and thiazolidinediones (TZDs). In this review is shown the different PPARs isoforms, identification, and regulation of their expression and activity. Also shows which are the natural ligands, and the chemical compounds that activate PPARs. Finally, it shows the target genes activated by the different isoforms of PPARs, the metabolic integration between the different PPAR isoforms to maintain a balance between fatty acid synthesis and oxidation and the association with the development of obesity and insulin resistance. Also shows information about the nutritional requirements of PUFAs that are the main natural ligands of PPARs.

  8. Role of insulin and insulin receptor in learning and memory.

    PubMed

    Zhao, W Q; Alkon, D L

    2001-05-25

    As one of the most extensively studied protein hormones, insulin and its receptor have been known to play key roles in a variety of important biological functions. Until recent years, the functions of insulin and insulin receptor (IR) in the central nervous system (CNS) have largely remained unclear. IR is abundantly expressed in several specific brain regions that govern fundamental behaviors such as food intake, reproduction and high cognition. The IR from the periphery and CNS exhibit differences in both structure and function. In addition to that from the peripheral system, locally synthesized insulin in the brain has also been identified. Accumulated evidence has demonstrated that insulin/IR plays important roles in associative learning, as suggested by results from both interventive and correlative studies. Interruption of insulin production and IR activity causes deficits in learning and memory formation. Abnormal insulin/IR levels and activities are seen in Alzheimer's dementia, whereas administration of insulin significantly improves the cognitive performance of these patients. The synaptic bases for the action of insulin/IR include modifying neurotransmitter release processes at various types of presynaptic terminals and modulating the activities of both excitatory and inhibitory postsynaptic receptors such as NMDA and GABA receptors, respectively. At the molecular level, insulin/IR participates in regulation of learning and memory via activation of specific signaling pathways, one of which is shown to be associated with the formation of long-term memory and is composed of intracellular molecules including the shc, Grb-r/SOS, Ras/Raf, and MEK/MAP kinases. Cross-talk with another IR pathway involving IRS1, PI3 kinase, and protein kinase C, as well as with the non-receptor tyrosine kinase pp60c-src, may also be associated with memory processing.

  9. 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.

  10. Molecular docking studies of banana flower flavonoids as insulin receptor tyrosine kinase activators as a cure for diabetes mellitus.

    PubMed

    Ganugapati, Jayasree; Baldwa, Aashish; Lalani, Sarfaraz

    2012-01-01

    Diabetes mellitus is a metabolic disorder caused due to insulin deficiency. Banana flower is a rich source of flavonoids that exhibit anti diabetic activity. Insulin receptor is a tetramer that belongs to a family of receptor tyrosine kinases. It contains two alpha subunits that form the extracellular domain and two beta subunits that constitute the intracellular tyrosine kinase domain. Insulin binds to the extracellular region of the receptor and causes conformational changes that lead to the activation of the tyrosine kinase. This leads to autophosphorylation, a step that is crucial in insulin signaling pathway. Hence, compounds that augment insulin receptor tyrosine kinase activity would be useful in the treatment of diabetes mellitus. The 3D structure of IR tyrosine kinase was obtained from PDB database. The list of flavonoids found in banana flower was obtained from USDA database. The structures of the flavonoids were obtained from NCBI Pubchem. Docking analysis of the flavonoids was performed using Autodock 4.0 and Autodock Vina. The results indicate that few of the flavonoids may be potential activators of IR tyrosine kinase.

  11. Role of endosomal trafficking dynamics on the regulation of hepatic insulin receptor activity: models for Fao cells.

    PubMed

    Hori, Sharon S; Kurland, Irwin J; DiStefano, Joseph J

    2006-05-01

    Evidence indicates that endosomal insulin receptor (IR) trafficking plays a role in regulating insulin signal transduction. To evaluate its importance, we developed a series of biokinetic models for quantifying activated surface and endosomal IR dynamics from published experimental data. Starting with a published two-compartment Fao hepatoma model, a four-pool model was formulated that depicts IR autophosphorylation after receptor binding, IR endosomal internalization/trafficking, insulin dissociation from and dephosphorylation of internalized IR, and recycling of unliganded, dephosphorylated IR to the plasma membrane. Quantification required three additional data sets, two measured, but unmodeled by the same group. A five-pool model created to include endosomal trafficking of the nonphosphorylated insulin-IR complex was fitted using the same data sets, augmented with another published data set. Creation of a six-pool model added the physiologically relevant dissociation of insulin ligand from the activated endosomal IR. More importantly, all three models, validated against additional data not used in model fitting, predict that, mechanistically, internalization of activated IR is a rate-limiting step, at least under the receptor saturating conditions of the fitting data. This rate includes the transit time to a site where insulin dissociation from and/or dephosphorylation of the IR occurs by docking with protein-tyrosine phosphatases (PTPases), or where a sufficient conformational change occurs in the IR, perhaps due to insulin-IR dissociation, where associated PTPases may complete IR dephosphorylation. Our new models indicate that key events in endosomal IR trafficking have significance in mediating IR activity, possibly serving to regulate insulin signal transduction.

  12. Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic β cells

    PubMed Central

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

    2012-01-01

    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 β cells. In the present study, we have identified the expression of TGR5 in pancreatic β cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated Gαs and caused an increase in intracellular cAMP and Ca2+. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective Gαs 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 Gs/cAMP/Ca2+ pathway. 8-pCPT-2′-O-Me-cAMP, a cAMP analogue, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic β cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis. PMID:23022524

  13. Diabetes reduces basal retinal insulin receptor signaling: reversal with systemic and local insulin.

    PubMed

    Reiter, Chad E N; Wu, Xiaohua; Sandirasegarane, Lakshman; Nakamura, Makoto; Gilbert, Kirk A; Singh, Ravi S J; Fort, Patrice E; Antonetti, David A; Gardner, Thomas W

    2006-04-01

    Diabetic retinopathy is characterized by early onset of neuronal cell death. We previously showed that insulin mediates a prosurvival pathway in retinal neurons and that normal retina expresses a highly active basal insulin receptor/Akt signaling pathway that is stable throughout feeding and fasting. Using the streptozotocin-induced diabetic rat model, we tested the hypothesis that diabetes diminishes basal retinal insulin receptor signaling concomitantly with increased diabetes-induced retinal apoptosis. The expression, phosphorylation status, and/or kinase activity of the insulin receptor and downstream signaling proteins were investigated in retinas of age-matched control, diabetic, and insulin-treated diabetic rats. Four weeks of diabetes reduced basal insulin receptor kinase, insulin receptor substrate (IRS)-1/2-associated phosphatidylinositol 3-kinase, and Akt kinase activity without altering insulin receptor or IRS-1/2 expression or tyrosine phosphorylation. After 12 weeks of diabetes, constitutive insulin receptor autophosphorylation and IRS-2 expression were reduced, without changes in p42/p44 mitogen-activated protein kinase or IRS-1. Sustained systemic insulin treatment of diabetic rats prevented loss of insulin receptor and Akt kinase activity, and acute intravitreal insulin administration restored insulin receptor kinase activity. Insulin treatment restored insulin receptor-beta autophosphorylation in rat retinas maintained ex vivo, demonstrating functional receptors and suggesting loss of ligand as a cause for reduced retinal insulin receptor/Akt pathway activity. These results demonstrate that diabetes progressively impairs the constitutive retinal insulin receptor signaling pathway through Akt and suggests that loss of this survival pathway may contribute to the initial stages of diabetic retinopathy.

  14. Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

    PubMed

    Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

    1995-03-01

    Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context

  15. Protein kinase C activators selectively inhibit insulin-stimulated system A transport activity in skeletal muscle at a post-receptor level.

    PubMed Central

    Gumà, A; Camps, M; Palacín, M; Testar, X; Zorzano, A

    1990-01-01

    We have investigated the role of phorbol esters on different biological effects induced by insulin in muscle, such as activation of system A transport activity, glucose utilization and insulin receptor function. System A transport activity was measured by monitoring the uptake of the system A-specific analogue alpha-(methyl)aminoisobutyric acid (MeAIB), by intact rat extensor digitorum longus muscle. The addition of 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.5 microM) for 60 or 180 min did not modify basal MeAIB uptake by muscle, suggesting that insulin signalling required to stimulate MeAIB transport does not involve protein kinase C activation. However, TPA added 30 min before insulin (100 nM) markedly inhibited insulin-stimulated MeAIB uptake. The addition of polymyxin B (0.1 mM) or H-7 (1 mM), protein kinase C inhibitors, alone or in combination with TPA leads to impairment of insulin-stimulated MeAIB uptake. This paradoxical pattern is incompatible with a unique action of Polymyxin B or H-7 on protein kinase C activity. Therefore these agents are not suitable tools with which to investigate whether a certain insulin effect is mediated by protein kinase C. TPA did not cause a generalized inhibition of insulin action. Thus both TPA and insulin increased 3-O-methylglucose uptake by muscle, and their effects were not additive. Furthermore, TPA did not modify insulin-stimulated lactate production by muscle. In keeping with this selective modification of insulin action, treatment of muscles with TPA did not modify insulin receptor binding or kinase activities. In conclusion, phorbol esters do not mimic insulin action on system A transport activity; however, they markedly inhibit insulin-stimulated amino acid transport, with no modification of insulin receptor function in rat skeletal muscle. It is suggested that protein kinase C activation causes a selective post-receptor modification on the biochemical pathway by which insulin activates system A amino acid

  16. 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...

  17. 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...

  18. Activation of oncogenic tyrosine kinase signaling promotes insulin receptor-mediated cone photoreceptor survival

    PubMed Central

    Rajala, Ammaji; Wang, Yuhong; Rajala, Raju V.S.

    2016-01-01

    In humans, daylight vision is primarily mediated by cone photoreceptors. These cells die in age-related retinal degenerations. Prolonging the life of cones for even one decade would have an enormous beneficial effect on usable vision in an aging population. Photoreceptors are postmitotic, but shed 10% of their outer segments daily, and must synthesize the membrane and protein equivalent of a proliferating cell each day. Although activation of oncogenic tyrosine kinase and inhibition of tyrosine phosphatase signaling is known to be essential for tumor progression, the cellular regulation of this signaling in postmitotic photoreceptor cells has not been studied. In the present study, we report that a novel G-protein coupled receptor–mediated insulin receptor (IR) signaling pathway is regulated by non-receptor tyrosine kinase Src through the inhibition of protein tyrosine phosphatase IB (PTP1B). We demonstrated the functional significance of this pathway through conditional deletion of IR and PTP1B in cones, in addition to delaying the death of cones in a mouse model of cone degeneration by activating the Src. This is the first study demonstrating the molecular mechanism of a novel signaling pathway in photoreceptor cells, which provides a window of opportunity to save the dying cones in retinal degenerative diseases. PMID:27391439

  19. Acute Treatment With XMetA Activates Hepatic Insulin Receptors and Lowers Blood Glucose in Normal Mice.

    PubMed

    Bedinger, Daniel H; Kieffer, Dorothy A; Goldfine, Ira D; Roell, Marina K; Adams, Sean H

    2015-09-01

    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 transport in cultured cells, and chronically reduced fasting blood glucose levels in mouse models of diabetes mellitus. Under acute dosing conditions, the large size of an IR-binding antibody like XMetA (∼ 150 kDa) could lead to a more rapid access into liver, an insulin sensitive tissue with well-fenestrated capillaries, when compared to other insulin sensitive tissues with non-fenestrated capillaries, such as muscle and adipose. Thus, in the present study we administered XMetA (10 mg/kg) and insulin (0.5 U/kg) via IV injection, and for 90 min compared their effects on blood glucose lowering and IR activation in three of the major insulin-sensitive tissues of the normal fasted mouse: liver, adipose, and muscle. Like insulin, XMetA lowered blood glucose levels, although the effect was less rapid. Insulin activated IR autophosphorylation and Akt phosphorylation in liver, fat, and muscle. In contrast, IR activation by XMetA was primarily observed in the liver. Both insulin and XMetA lowered β-hydroxybutyrate levels in plasma; however, only insulin reduced both non-esterified fatty acids (NEFA) and glycerol concentrations. These data indicate that, in normal mice, acute glucose regulation by XMetA is largely mediated by its action on the liver.

  20. 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

  1. Upregulated insulin secretion in insulin-resistant mice: evidence of increased islet GLP1 receptor levels and GPR119-activated GLP1 secretion.

    PubMed

    Ahlkvist, L; Brown, K; Ahrén, B

    2013-06-01

    We previously demonstrated that the overall incretin effect and the β-cell responsiveness to glucagon-like peptide-1 (GLP1) are increased in insulin-resistant mice and may contribute to the upregulated β-cell function. Now we examined whether this could, first, be explained by increased islet GLP1 receptor (GLP1R) protein levels and, secondly, be leveraged by G-protein-coupled receptor 119 (GPR119) activation, which stimulates GLP1 secretion. Female C57BL/6J mice, fed a control (CD, 10% fat) or high-fat (HFD, 60% fat) diet for 8 weeks, were anesthetized and orally given a GPR119 receptor agonist (GSK706A; 10 mg/kg) or vehicle, followed after 10 min with gavage with a liquid mixed meal (0.285 kcal). Blood was sampled for determination of glucose, insulin, intact GLP1, and glucagon, and islets were isolated for studies on insulin and glucagon secretion and GLP1R protein levels. In HFD vs CD mice, GPR119 activation augmented the meal-induced increase in the release of both GLP1 (AUCGLP1 81±9.6 vs 37±6.9 pM×min, P=0.002) and insulin (AUCINS 253±29 vs 112±19 nM×min, P<0.001). GPR119 activation also significantly increased glucagon levels in both groups (P<0.01) with, however, no difference between the groups. By contrast, GPR119 activation did not affect islet hormone secretion from isolated islets. Glucose elimination after meal ingestion was significantly increased by GPR119 activation in HFD mice (0.57±0.04 vs 0.43±0.03% per min, P=0.014) but not in control mice. Islet GLP1R protein levels was higher in HFD vs CD mice (0.8±0.1 vs 0.5±0.1, P=0.035). In conclusion, insulin-resistant mice display increased islet GLP1R protein levels and augmented meal-induced GLP1 and insulin responses to GPR119 activation, which results in increased glucose elimination. We suggest that the increased islet GLP1R protein levels together with the increased GLP1 release may contribute to the upregulated β-cell function in insulin resistance.

  2. 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.

  3. Simulation model of defective insulin receptors as byproducts of receptor recycling.

    PubMed

    Kurbel, B; Kurbel, S; Kristek, Z; Jakić, M; Jurić, M; Sulava, D

    1997-08-01

    Our simulation model assumes that the defective insulin-binding receptors in non-insulin-dependent diabetes (NIDDM) patients result from functional receptor recycling. The model is a short program written in MS DOS 5.0 Qbasic. MODEL DESIGN: Receptors with intracellular portions damaged in the process of recycling are considered defective since they bind insulin but do mediate insulin effects, or recycle. Their occurrence depends on the average activation rate of functional receptors. The insulin-binding receptors (defective and functional) are objects of slow and time-dependent turnover defined by the turnover rate. Recycled receptors rejoin functional receptors or enter the pool of defective receptors. The waste in the functional receptors' pool is covered by a limited amount of newly synthesized receptors. The defective receptors often accumulate in cases of increased activation of functional receptors. SIMULATION RESULTS: The insulin-binding receptor quantity is determined, in the model, only by the number of newly synthesized receptors, reflecting the intensity of insulin stimulation. Synthesis is increased following variable insulin stimulations and decreased after sustained, intensive insulin stimulation. The number of functional receptors inversely reflects the average activation rate of functional receptors compared with the insulin-binding receptors turnover rate. High activation rates can diminish the proportion of functional receptors to less than 5% of that of all insulin-binding receptors. The model predicts that cells bearing only functional receptors show progressively shortened half-lives of receptors, reflecting the receptor activation intensity. On the other hand, cells bearing both defective and functional receptors show stable receptors' half-lives (20-36% of the defective receptors' half-life). Simulation results suggest that reduced functional receptor proportions in NIDDM patients might reflect the imbalance between the activation of

  4. 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

  5. [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.

  6. The neuronal insulin receptor in its environment.

    PubMed

    Gralle, Matthias

    2017-02-01

    Insulin is known mainly for its effects in peripheral tissues, such as the liver, skeletal muscles and adipose tissue, where the activation of the insulin receptor (IR) has both short-term and long-term effects. Insulin and the IR are also present in the brain, and since there is evidence that neuronal insulin signaling regulates synaptic plasticity and that it is impaired in disease, this pathway might be the key to protection or reversal of symptoms, especially in Alzheimer's disease. However, there are controversies about the importance of the neuronal IR, partly because biophysical data on its activation and signaling are much less complete than for the peripheral IR. This review briefly summarizes the neuronal IR signaling in health and disease, and then focuses on known differences between the neuronal and peripheral IR with regard to alternative splicing and glycosylation, and lack of data with respect to phosphorylation and membrane subdomain localization. Particularities in the neuronal IR itself and its environment may have consequences for downstream signaling and impact synaptic plasticity. Furthermore, establishing the relative importance of insulin signaling through IR or through hybrids with its homolog, the insulin-like growth factor 1 receptor, is crucial for evaluating the consequences of brain IR activation. An improved biophysical understanding of the neuronal IR may help predict the consequences of insulin-targeted interventions.

  7. 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.

  8. Immunological demonstration of the accumulation of insulin, but not insulin receptors, in nuclei of insulin-treated cells

    SciTech Connect

    Soler, A.P.; Thompson, K.A.; Smith, R.M.; Jarett, L. )

    1989-09-01

    Although insulin is known to regulate nuclear-related processes, such as cell growth and gene transcription, the mechanisms involved are poorly understood. Previous studies suggested that translocation of insulin or its receptor to cell nuclei might be involved in some of these processes. The present investigation demonstrated that intact insulin, but not the insulin receptor, accumulated in nuclei of insulin-treated cells. Cell fractionation studies demonstrated that the nuclear accumulation of {sup 125}I-labeled insulin was time-, temperature-, and insulin-concentration-dependent. Electron microscopic immunocytochemistry demonstrated that the insulin that accumulated in the nucleus was immunologically intact and associated with the heterochromatin. Only 1% of the {sup 125}I-labeled insulin extracted from isolated nuclei was eluted from a Sephadex G-50 column as {sup 125}I-labeled tyrosine. Plasma membrane insulin receptors were not detected in the nucleus by immuno electron microscopy or when wheat germ agglutinin-purified extracts of the nuclei were subjected to PAGE, electrotransfer, and immunoblotting with anti-insulin receptor antibodies. These results suggested that internalized insulin dissociated from its receptor and accumulated in the nucleus without its membrane receptor. The authors propose that some of insulin's effects on nuclear function may be caused by the translocation of the intact and biologically active hormone to the nucleus and its binding to nuclear components in the heterochromatin.

  9. Insulin-receptor phosphotyrosyl-protein phosphatases.

    PubMed Central

    King, M J; Sale, G J

    1988-01-01

    Calmodulin-dependent protein phosphatase has been proposed to be an important phosphotyrosyl-protein phosphatase. The ability of the enzyme to attack autophosphorylated insulin receptor was examined and compared with the known ability of the enzyme to act on autophosphorylated epidermal-growth-factor (EGF) receptor. Purified calmodulin-dependent protein phosphatase was shown to catalyse the complete dephosphorylation of phosphotyrosyl-(insulin receptor). When compared at similar concentrations, 32P-labelled EGF receptor was dephosphorylated at greater than 3 times the rate of 32P-labelled insulin receptor; both dephosphorylations exhibited similar dependence on metal ions and calmodulin. Native phosphotyrosyl-protein phosphatases in cell extracts were also characterized. With rat liver, heart or brain, most (75%) of the native phosphatase activity against both 32P-labelled insulin and EGF receptors was recovered in the particulate fraction of the cell, with only 25% in the soluble fraction. This subcellular distribution contrasts with results of previous studies using artificial substrates, which found most of the phosphotyrosyl-protein phosphatase activity in the soluble fraction of the cell. Properties of particulate and soluble phosphatase activity against 32P-labelled insulin and EGF receptors are reported. The contribution of calmodulin-dependent protein phosphatase activity to phosphotyrosyl-protein phosphatase activity in cell fractions was determined by utilizing the unique metal-ion dependence of calmodulin-dependent protein phosphatase. Whereas Ni2+ (1 mM) markedly activated the calmodulin-dependent protein phosphatase, it was found to inhibit potently both particulate and soluble phosphotyrosyl-protein phosphatase activity. In fractions from rat liver, brain and heart, total phosphotyrosyl-protein phosphatase activity against both 32P-labelled receptors was inhibited by 99.5 +/- 6% (mean +/- S.E.M., 30 observations) by Ni2+. Results of Ni2+ inhibition

  10. Insulin/receptor binding: the last piece of the puzzle? What recent progress on the structure of the insulin/receptor complex tells us (or not) about negative cooperativity and activation.

    PubMed

    De Meyts, Pierre

    2015-04-01

    Progress in solving the structure of insulin bound to its receptor has been slow and stepwise, but a milestone has now been reached with a refined structure of a complex of insulin with a "microreceptor" that contains the primary binding site. The insulin receptor is a dimeric allosteric enzyme that belongs to the family of receptor tyrosine kinases. The insulin binding process is complex and exhibits negative cooperativity. Biochemical evidence suggested that insulin, through two distinct binding sites, crosslinks two receptor sites located on each α subunit. The structure of the unliganded receptor ectodomain showed a symmetrical folded-over conformation with an antiparallel disposition. Further work resolved the detailed structure of receptor site 1, both without and with insulin. Recently, a missing piece in the puzzle was added: the C-terminal portion of insulin's B-chain known to be critical for binding and negative cooperativity. Here I discuss these findings and their implications.

  11. Insulin-Independent GABAA Receptor-Mediated Response in the Barrel Cortex of Mice with Impaired Met Activity

    PubMed Central

    Lo, Fu-Sun; Erzurumlu, Reha S.

    2016-01-01

    Autism spectrum disorder (ASD) is a neurodevelopmental disorder caused by genetic variants, susceptibility alleles, and environmental perturbations. The autism associated gene MET tyrosine kinase has been implicated in many behavioral domains and endophenotypes of autism, including abnormal neural signaling in human sensory cortex. We investigated somatosensory thalamocortical synaptic communication in mice deficient in Met activity in cortical excitatory neurons to gain insights into aberrant somatosensation characteristic of ASD. The ratio of excitation to inhibition is dramatically increased due to decreased postsynaptic GABAA receptor-mediated inhibition in the trigeminal thalamocortical pathway of mice lacking active Met in the cerebral cortex. Furthermore, in contrast to wild-type mice, insulin failed to increase GABAA receptor-mediated response in the barrel cortex of mice with compromised Met signaling. Thus, lacking insulin effects may be a risk factor in ASD pathogenesis. SIGNIFICANCE STATEMENT A proposed common cause of neurodevelopmental disorders is an imbalance in excitatory neural transmission, provided by the glutamatergic neurons, and the inhibitory signals from the GABAergic interneurons. Many genes associated with autism spectrum disorders impair synaptic transmission in the expected cell type. Previously, inactivation of the autism-associated Met tyrosine kinase receptor in GABAergic interneurons led to decreased inhibition. In thus report, decreased Met signaling in glutamatergic neurons had no effect on excitation, but decimated inhibition. Further experiments indicate that loss of Met activity downregulates GABAA receptors on glutamatergic neurons in an insulin independent manner. These data provide a new mechanism for the loss of inhibition and subsequent abnormal excitation/inhibition balance and potential molecular candidates for treatment or prevention. PMID:27030755

  12. Deletion of 3 basepairs resulting in the loss of lysine-121 in the insulin receptor alpha-subunit in a patient with leprechaunism: binding, phosphorylation, and biological activity.

    PubMed

    Jospe, N; Zhu, J; Liu, R; Livingston, J N; Furlanetto, R W

    1994-11-01

    We have identified a novel mutation of the human insulin receptor gene in a previously unreported patient with leprechaunism, leprechaun Rochester. This mutation consists of deletion of three nucleotides (GAA) in exon 2 and results in loss of the lysine-121 in the putative ligand-binding domain of the alpha-subunit. To analyze this mutation, we prepared a corresponding mutant insulin receptor by site-directed mutagenesis and expressed the receptor in Chinese hamster ovary cells. Although the mutant receptor displayed normal insulin binding, abnormalities were found in autophosphorylation and in phosphorylation of endogenous and exogenous protein substrates. These abnormalities consisted of increased basal kinase activity, but blunted insulin-stimulated responsiveness. Importantly, cells that expressed the mutant receptor showed markedly decreased insulin- and serum-stimulated DNA synthesis compared to untransfected control cells and cells transfected with the wild-type insulin receptor. These findings suggest that deletion of lysine-121 in conjunction with a presumed, but thus far unidentified, second mutant allele contributed significantly to the lethal insulin-resistant state in this patient with leprechaunism.

  13. Skeletal muscle peroxisome proliferator- activated receptor-gamma expression in obesity and non- insulin-dependent diabetes mellitus.

    PubMed Central

    Kruszynska, Y T; Mukherjee, R; Jow, L; Dana, S; Paterniti, J R; Olefsky, J M

    1998-01-01

    The two isoforms of peroxisome proliferator-activated receptor-gamma (PPARgamma1 and PPARgamma2), are ligand-activated transcription factors that are the intracellular targets of a new class of insulin sensitizing agents, the thiazolidinediones. The observation that thiazolidinediones enhance skeletal muscle insulin sensitivity in obesity and in patients with non-insulin-dependent diabetes mellitus (NIDDM), by activating PPARgamma, and possibly by inducing its expression, suggests that PPARgamma expression in skeletal muscle plays a key role in determining tissue sensitivity to insulin, and that PPARgamma expression may be decreased in insulin resistant subjects. We used a sensitive ribonuclease protection assay, that permits simultaneous measurement of the two isoforms, to examine the effects of obesity and NIDDM, and the effects of insulin, on skeletal muscle levels of PPARgamma1 and PPARgamma2 mRNA. We studied seven patients with NIDDM (body mass index, 32+/-1 kg/m2), seven lean (24+/-1 kg/m2), and six obese (36+/-1 kg/m2) normal subjects. Biopsies from the vastus lateralis muscle were taken before and after a 5-h hyperinsulinemic (80 mU/m2 per minute) euglycemic clamp. The obese controls and NIDDM patients were insulin resistant with glucose disposal rates during the last 30 min of the clamp that were 67 and 31%, respectively, of those found in the lean controls. PPARgamma1, but not PPARgamma2 mRNA was detected in skeletal muscle at 10-15% of the level found in adipose tissue. No difference was found in PPARgamma1 levels between the three groups, and there was no change in PPARgamma1 levels after 5 h of hyperinsulinemia. In obese subjects, PPARgamma1 correlated with clamp glucose disposal rates (r = 0.92, P < 0.01). In the lean and NIDDM patients, muscle PPARgamma1 levels correlated with percentage body fat (r = 0.76 and r = 0.82, respectively, both P < 0.05) but not with body mass index. In conclusion: (a) skeletal muscle PPARgamma1 expression does not differ

  14. Impact of Oxidative Stress and Peroxisome Proliferator–Activated Receptor γ Coactivator-1α in Hepatic Insulin Resistance

    PubMed Central

    Kumashiro, Naoki; Tamura, Yoshifumi; Uchida, Toyoyoshi; Ogihara, Takeshi; Fujitani, Yoshio; Hirose, Takahisa; Mochizuki, Hideki; Kawamori, Ryuzo; Watada, Hirotaka

    2008-01-01

    OBJECTIVE—Recent studies identified accumulation of reactive oxygen species (ROS) as a common pathway causing insulin resistance. However, whether and how the reduction of ROS levels improves insulin resistance remains to be elucidated. The present study was designed to define this mechanism. RESEARCH DESIGN AND METHODS—We investigated the effect of overexpression of superoxide dismutase (SOD)1 in liver of obese diabetic model (db/db) mice by adenoviral injection. RESULTS—db/db mice had high ROS levels in liver. Overexpression of SOD1 in liver of db/db mice reduced hepatic ROS and blood glucose level. These changes were accompanied by improvement in insulin resistance and reduction of hepatic gene expression of phosphoenol-pyruvate carboxykinase and peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α), which is the main regulator of gluconeogenic genes. The inhibition of hepatic insulin resistance was accompanied by attenuation of phosphorylation of cAMP-responsive element-binding protein (CREB), which is a main regulator of PGC-1α expression, and attenuation of Jun NH2-terminal kinase (JNK) phosphorylation. Simultaneously, overexpression of SOD1 in db/db mice enhanced the inactivation of forkhead box class O1, another regulator of PGC-1α expression, without the changes of insulin-induced Akt phosphorylation in liver. In hepatocyte cell lines, ROS induced phosphorylation of JNK and CREB, and the latter, together with PGC-1α expression, was inhibited by a JNK inhibitor. CONCLUSIONS—Our results indicate that the reduction of ROS is a potential therapeutic target of liver insulin resistance, at least partly by the reduced expression of PGC-1α. PMID:18487450

  15. 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

  16. GQ-16, a novel peroxisome proliferator-activated receptor γ (PPARγ) ligand, promotes insulin sensitization without weight gain.

    PubMed

    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

    2012-08-10

    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.

  17. 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

  18. Insulin receptors mediate growth effects in cultured fetal neurons. II. Activation of a protein kinase that phosphorylates ribosomal protein S6

    SciTech Connect

    Heidenreich, K.A.; Toledo, S.P. )

    1989-09-01

    As an initial attempt to identify early steps in insulin action that may be involved in the growth responses of neurons to insulin, we investigated whether insulin receptor activation increases the phosphorylation of ribosomal protein S6 in cultured fetal neurons and whether activation of a protein kinase is involved in this process. When neurons were incubated for 2 h with 32Pi, the addition of insulin (100 ng/ml) for the final 30 min increased the incorporation of 32Pi into a 32K microsomal protein. The incorporation of 32Pi into the majority of other neuronal proteins was unaltered by the 30-min exposure to insulin. Cytosolic extracts from insulin-treated neurons incubated in the presence of exogenous rat liver 40S ribosomes and (gamma-32P)ATP displayed a 3- to 8-fold increase in the phosphorylation of ribosomal protein S6 compared to extracts from untreated cells. Inclusion of cycloheximide during exposure of the neurons to insulin did not inhibit the increased cytosolic kinase activity. Activation of S6 kinase activity by insulin was dose dependent (seen at insulin concentration as low as 0.1 ng/ml) and reached a maximum after 20 min of incubation. Addition of phosphatidylserine, diolein, and Ca2+ to the in vitro kinase reaction had no effect on the phosphorylation of ribosomal protein S6. Likewise, treatment of neurons with (Bu)2cAMP did not alter the phosphorylation of ribosomal protein S6 by neuronal cytosolic extracts. We conclude that insulin activates a cytosolic protein kinase that phosphorylates ribosomal S6 in neurons and is distinct from protein kinase-C and cAMP-dependent protein kinase. Stimulation of this kinase may play a role in insulin signal transduction in neurons.

  19. 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

  20. Knockdown of the gene encoding Drosophila tribbles homologue 3 (Trib3) improves insulin sensitivity through peroxisome proliferator-activated receptor-γ (PPAR)-γ activation in a rat model of insulin resistance

    PubMed Central

    Weismann, D.; Erion, D. M.; Ignatova-Todorava, I.; Nagai, Y.; Stark, R.; Hsiao, J. J.; Flannery, C.; Birkenfeld, A. L.; May, T.; Kahn, M.; Zhang, D.; Yu, X. X.; Murray, S. F.; Bhanot, S.; Monia, B. P.; Cline, G. W.; Shulman, G. I.; Samuel, V. T.

    2014-01-01

    Aims/hypothesis Insulin action is purportedly modulated by Drosophila tribbles homologue 3 (TRIB3), which in vitro prevents thymoma viral proto-oncogene (AKT) and peroxisome proliferator-activated receptor (PPAR)-γ activation. However, the physiological impact of TRIB3 action in vivo remains controversial. Methods We investigated the role of TRIB3 in rats treated with either a control or Trib3 antisense oligonucleotide (ASO). Tissue-specific insulin sensitivity was assessed in vivo using a euglycaemic–hyperinsulinaemic clamp. A separate group was treated with the PPAR-γ antagonist, bisphenol-A-diglycidyl ether (BADGE) to assess the role of PPAR-γ in mediating the response to Trib3 ASO. Results Trib3 ASO treatment specifically reduced Trib3 expression by 70 to 80% in liver and white adipose tissue. Fasting plasma glucose, insulin concentrations and basal rate of endogenous glucose production were unchanged. However, Trib3 ASO increased insulin-stimulated whole-body glucose uptake by ~50% during the euglycaemic–hyperinsulinaemic clamp. This was attributable to improved skeletal muscle glucose uptake. Despite the reduction of Trib3 expression, AKT2 activity was not increased. Trib3 ASO increased white adipose tissue mass by 70%, and expression of Ppar-γ and its key target genes, raising the possibility that Trib3 ASO improves insulin sensitivity primarily in a PPAR-γ-dependent manner. Co-treatment with BADGE blunted the expansion of white adipose tissue and abrogated the insulin-sensitising effects of Trib3 ASO. Finally, Trib3 ASO also increased plasma HDL-cholesterol, a change that persisted with BADGE co-treatment. Conclusions/interpretation These data suggest that TRIB3 inhibition improves insulin sensitivity in vivo primarily in a PPAR-γ-dependent manner and without any change in AKT2 activity. PMID:21190014

  1. Natural anti-diabetic compound 1,2,3,4,6-penta-O-galloyl-D-glucopyranose binds to insulin receptor and activates insulin-mediated glucose transport signaling pathway.

    PubMed

    Li, Yunsheng; Kim, Jaekyung; Li, Jing; Liu, Fang; Liu, Xueqing; Himmeldirk, Klaus; Ren, Yulin; Wagner, Thomas E; Chen, Xiaozhuo

    2005-10-21

    Insulin mimetics from natural sources are potential therapeutics that can act alone or supplement insulin and other anti-diabetic drugs in the prevention and treatment of diabetes. We recently reported the insulin-like glucose transport stimulatory activity of tannic acid (TA) in 3T3-L1 adipocytes. In this study, we find that chemically synthesized 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose (beta-PGG), one of the components of TA, as well as its natural anomer alpha-PGG possess activity. Mechanistic studies in adipocytes with alpha-PGG, the more potent of the two anomers, reveal that inhibitors that block the insulin-mediated glucose transport, including one that inhibits the insulin receptor (IR), also completely abolish the glucose transport activated by alpha-PGG. In addition, alpha-PGG induces phosphorylation of the IR and Akt, activates PI 3-kinase, and stimulates membrane translocation of GLUT 4. Receptor binding studies indicate that alpha-PGG binds to the IR and affects the binding between insulin and IR by reducing the maximum binding of insulin to IR without significantly altering the binding affinity of insulin to IR. Western blotting analysis of the products of a cross-linking reaction suggests that alpha-PGG may bind to IR at a site located on the alpha-subunit of the receptor. Animal studies demonstrate that PGG reduces blood glucose levels and improves glucose tolerance in diabetic and obese animals. Our results suggest that PGG may serve as a model for the development of new types of anti-diabetic and anti-metabolic syndrome therapeutics.

  2. 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.

  3. 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…

  4. Insulin and Insulin-like Growth Factor II Differentially Regulate Endocytic Sorting and Stability of Insulin Receptor Isoform A*

    PubMed Central

    Morcavallo, Alaide; Genua, Marco; Palummo, Angela; Kletvikova, Emilia; Jiracek, Jiri; Brzozowski, Andrzej M.; Iozzo, Renato V.; Belfiore, Antonino; Morrione, Andrea

    2012-01-01

    The insulin receptor isoform A (IR-A) binds both insulin and insulin-like growth factor (IGF)-II, although the affinity for IGF-II is 3–10-fold lower than insulin depending on a cell and tissue context. Notably, in mouse embryonic fibroblasts lacking the IGF-IR and expressing solely the IR-A (R−/IR-A), IGF-II is a more potent mitogen than insulin. As receptor endocytosis and degradation provide spatial and temporal regulation of signaling events, we hypothesized that insulin and IGF-II could affect IR-A biological responses by differentially regulating IR-A trafficking. Using R−/IR-A cells, we discovered that insulin evoked significant IR-A internalization, a process modestly affected by IGF-II. However, the differential internalization was not due to IR-A ubiquitination. Notably, prolonged stimulation of R−/IR-A cells with insulin, but not with IGF-II, targeted the receptor to a degradative pathway. Similarly, the docking protein insulin receptor substrate 1 (IRS-1) was down-regulated after prolonged insulin but not IGF-II exposure. Similar results were also obtained in experiments using [NMeTyrB26]-insulin, an insulin analog with IR-A binding affinity similar to IGF-II. Finally, we discovered that IR-A was internalized through clathrin-dependent and -independent pathways, which differentially regulated the activation of downstream effectors. Collectively, our results suggest that a lower affinity of IGF-II for the IR-A promotes lower IR-A phosphorylation and activation of early downstream effectors vis à vis insulin but may protect IR-A and IRS-1 from down-regulation thereby evoking sustained and robust mitogenic stimuli. PMID:22318726

  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. Syndecan-1 couples the insulin-like growth factor-1 receptor to inside-out integrin activation

    PubMed Central

    Beauvais, DeannaLee M.; Rapraeger, Alan C.

    2010-01-01

    Syndecan-1 (Sdc1) engages and activates the αvβ3 (and/or αvβ5) integrin when clustered in human carcinoma and endothelial cells. Although the engagement is extracellular, the activation mechanism is cytoplasmic. This talin-dependent, inside-out signaling pathway is activated downstream of the insulin-like growth factor-1 receptor (IGF1R), whose kinase activity is triggered by Sdc1 clustering. In vitro binding assays using purified receptors suggest that association of the Sdc1 ectodomain with the integrin provides a ‘docking face’ for IGF1R. IGF1R docking and activation of the associated integrin is blocked by synstatin (SSTN92–119), a peptide derived from the integrin engagement site in Sdc1. IGF1R colocalizes with αvβ3 integrin and Sdc1 in focal contacts, but fails to associate with or activate the integrin in cells either lacking Sdc1 or expressing Sdc1Δ67–121, a mutant that is unable to form the Sdc1–integrin–IGF1R ternary complex. Integrin activation is also blocked by IGF1R inhibitors or by silencing IGF1R or talin expression with small-interfering RNAs (siRNAs). In both cases, expression of the constitutively active talin F23 head domain rescues integrin activation. We recently reported that SSTN92–119 blocks angiogenesis and impairs tumor growth in mice, therefore this Sdc1-mediated integrin regulatory mechanism might be a crucial regulator of disease processes known to rely on these integrins, including tumor cell metastasis and tumor-induced angiogenesis. PMID:20971705

  7. 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

  8. Insulin action is blocked by a monoclonal antibody that inhibits insulin receptor kinase

    SciTech Connect

    Morgan, D.O.; Ho, L.; Korn, L.J.; Roth, R.A.

    1986-01-01

    Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor ..beta.. 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 ..beta.. 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.

  9. 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.

  10. Peroxisome proliferator-activated receptor gamma (PPARG) modulates free fatty acid receptor 1 (FFAR1) dependent insulin secretion in humans

    PubMed Central

    Wagner, Robert; Hieronimus, Anja; Lamprinou, Apostolia; Heni, Martin; Hatziagelaki, Erifili; Ullrich, Susanne; Stefan, Norbert; Staiger, Harald; Häring, Hans-Ulrich; Fritsche, Andreas

    2014-01-01

    Genetic variation in FFAR1 modulates insulin secretion dependent on non-esterified fatty acid (NEFA) concentrations. We previously demonstrated lower insulin secretion in minor allele carriers of PPARG Pro12Ala in high-NEFA environment, but the mode of action could not been revealed. We tested if this effect is mediated by FFAR1 in humans. Subjects with increased risk of diabetes who underwent oral glucose tolerance tests were genotyped for 7 tagging SNPs in FFAR1 and PPARG Pro12Ala. The FFAR1 SNPs rs12462800 and rs10422744 demonstrated interactions with PPARG on insulin secretion. FFAR1 rs12462800 (p = 0.0006) and rs10422744 (p = 0.001) were associated with reduced insulin secretion in participants concomitantly carrying the PPARG minor allele and having high fasting FFA. These results suggest that the minor allele of the PPARG SNP exposes its carriers to modulatory effects of FFAR1 on insulin secretion. This subphenotype may define altered responsiveness to FFAR1-agonists, and should be investigated in further studies. PMID:25161890

  11. Human biliverdin reductase-based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor

    PubMed Central

    Gibbs, Peter E. M.; Lerner-Marmarosh, Nicole; Poulin, Amelia; Farah, Elie; Maines, Mahin D.

    2014-01-01

    Insulin binding changes conformation of the insulin receptor kinase (IRK) domain and initiates glucose uptake through the insulin, IGF-1, phosphatidyl inositol 3-kinase (PI3K), and MAPK pathways; human biliverdin reductase (hBVR) is an IRK substrate and pathway effector. This is the first report on hBVR peptide-mediated IRK activation and conformational change. 290KYCCSRK, which increased IRK Vmax without changing Km, stimulated glucose uptake and potentiated insulin and IGF-1 stimulation in 4 cell lines. KYCCSRK in native hBVR was necessary for the hBVR and IRK cross-activation. Peptide treatment also activated PI3K downstream effectors, Akt and ERK, phosphorylation, and Elk transcriptional activity. In cells transfected with CMV-regulated EGFP-VP-peptide plasmid, C292→A mutant did not stimulate glucose uptake; K296→A decreased uptake and kinase activity. KEDQYMKMTV, corresponding to hBVR's SH2-binding domain, was a potent inhibitor of glucose uptake and IRK. The mechanism of action of peptides was examined using cells expressing IRK (aa 988–1263) activated by coexpressed KYCCSRK. Three active cys-mutants of IRK, with fluorophore coupled to cysteines, C1056, C1138, or C1234, were examined for changes in fluorescence emission spectra in the presence of peptides. KYCCSRK and KEDQYMKMTV bound to different sites in IRK. The findings identify novel agents for activating or inhibiting insulin signaling and offer a new approach for treatment of type 2 diabetes and hypoglycemia.—Gibbs, P. E. M., Lerner-Marmarosh, N., Poulin, A., Farah, E., Maines, M. D. Human biliverdin reductase-based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor. PMID:24568842

  12. 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

    2015-01-02

    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.

  13. 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

  14. Clarification of signaling pathways mediated by insulin and insulin-like growth factor I receptors in fibroblasts from patients with specific defect in insulin receptor.

    PubMed

    Sasaoka, T; Kobayashi, M; Takata, Y; Ishibashi, O; Iwasaki, M; Shigeta, Y; Goji, K; Hisatomi, A

    1988-11-01

    Receptor binding and biological action of insulin and insulin-like growth factor I (IGF-I) were studied in fibroblasts from a patient with leprechaunism and a patient with type A syndrome of insulin resistance. Insulin binding was reduced to 18.8 and 27.7% of control value, respectively. In contrast, IGF-I binding was normal in both patients. In competitive binding studies, IGF-I had 0.2% of the ability of insulin to compete with 125I-labeled insulin binding, and insulin had 0.1% of the ability of IGF-I to compete with 125I-labeled IGF-I binding in control subjects and patient fibroblasts. The dose-response curves of insulin stimulation assessed by glucose incorporation and alpha-aminoisobutyric acid uptake showed normal responsiveness, and ED50 was significantly shifted to the right in fibroblasts from both patients. However, normal responsiveness and sensitivity were observed in thymidine incorporation studies. For IGF-I, dose-response curves of glucose incorporation, alpha-aminoisobutyric acid uptake, and thymidine incorporation were all normal in both patients. These results indicate that 1) the defect is specific to the insulin-receptor binding in these patients, 2) insulin and IGF-I activate glucose incorporation and alpha-aminoisobutyric acid uptake mainly through their own specific receptors, but 3) the IGF-I receptor appears to have a more important role in stimulating thymidine incorporation than the insulin receptor in physiological condition or, alternatively, an unknown postreceptor process with cascade signal transmission may overcome the decreased insulin-receptor binding to produce a normal dose-response curve.

  15. Leptin down-regulates insulin action through phosphorylation of serine-318 in insulin receptor substrate 1.

    PubMed

    Hennige, Anita M; Stefan, Norbert; Kapp, Katja; Lehmann, Rainer; Weigert, Cora; Beck, Alexander; Moeschel, Klaus; Mushack, Joanne; Schleicher, Erwin; Häring, Hans-Ulrich

    2006-06-01

    Insulin resistance in skeletal muscle is found in obesity and type 2 diabetes. A mechanism for impaired insulin signaling in peripheral tissues is the inhibition of insulin action through serine phosphorylation of insulin receptor substrate (Irs) proteins that abolish the coupling of Irs proteins to the activated insulin receptor. Recently, we described serine-318 as a protein kinase C (PKC)-dependent phosphorylation site in Irs1 (Ser-318) activated by hyperinsulinemia. Here we show in various cell models that the adipose hormone leptin, a putative mediator in obesity-related insulin resistance, promotes phosphorylation of Ser-318 in Irs1 by a janus kinase 2, Irs2, and PKC-dependent pathway. Mutation of Ser-318 to alanine abrogates the inhibitory effect of leptin on insulin-induced Irs1 tyrosine phosphorylation and glucose uptake in L6 myoblasts. In C57Bl/6 mice, Ser-318 phosphorylation levels in muscle tissue were enhanced by leptin and insulin administration in lean animals while in diet-induced obesity Ser-318 phosphorylation levels were already up-regulated in the basal state, and further stimulation was diminished. In analogy, in lymphocytes of obese hyperleptinemic human subjects basal Ser-318 phosphorylation levels were increased compared to lean individuals. During a hyperinsulinemic euglycemic clamp, the increment in Ser-318 phosphorylation observed in lean individuals was absent in obese. In summary, these data suggest that phosphorylation of Ser-318 in Irs1 mediates the inhibitory signal of leptin on the insulin-signaling cascade in obese subjects.

  16. 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.

  17. 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

    2015-04-09

    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.

  18. Hepatocyte insulin receptor is a calmodulin binding protein and is functionally inhibited by calmidazolium

    SciTech Connect

    Arnold, T.P.; Pollet, R.J.

    1986-05-01

    Insulin-induced autophosphorylation of the insulin receptor and changes in intracellular Ca/sup + +/ have been proposed as possible mediators of insulin action in target tissues. The authors have investigated the association of the 17kD calcium binding protein calmodulin with the insulin receptor solubilized from rat liver plasma membranes. Insulin receptors solubilized in 0.1% Triton X-100 exhibited strong binding to calmodulin-agarose affinity columns in the presence of 100..mu..M calcium and could be eluded with 100..mu..M ethelene glycol-bis (amino ethel ether) Tetra Acetic Acid (EGTA) with an 80% yield in insulin binding activity. In addition, /sup 125/I-Calmodulin was shown to bind to wheat germ agglutinin purified solubilized receptors, was specifically inhibited by EGTA (100 ..mu..M) and/or calmidazolium (10 ..mu..M) and was found to be insulin-dependent (max 10/sup -10/ M insulin). SDS-polyacrylamide gel electrophoresis data suggests that /sup 125/I-calmodulin may be associated with the 92 kD beta-subunit of the insulin receptor, consistent with the cytoplasmic domain of this subunit. While they have confirmed previous reports that the addition of calcium and calmodulin to solubilized insulin receptors preparations produces no demonstrable change in receptor phosphorylation, the addition of the calmodulin inhibitor calmidazolium did show more than 50% inhibition of insulin stimulated receptor phosphorylation, suggesting that a domain of the calmodulin molecule may be very tightly associated with the insulin receptor. These results indicate that calmodulin binds tightly and specifically to the insulin receptor of the hepatocyte and is insulin dependent. The findings also suggest that this interaction may be functionally significant in mediating insulin-induced receptor phosphorylation as well as other insulin actions. Thus, calmodulin may play a major role as an intracellular contributor to insulin action.

  19. High-affinity insulin binding to an atypical insulin-like growth factor-I receptor in human breast cancer cells.

    PubMed Central

    Milazzo, G; Yip, C C; Maddux, B A; Vigneri, R; Goldfine, I D

    1992-01-01

    We studied the nature of insulin receptor binding in MCF-7 breast cancer cells. In both intact cells and solubilized receptor preparations, high-affinity insulin binding was seen. However, unlabeled insulin-like growth factor-I (IGF-I) was five-fold more potent in inhibiting 125I-insulin binding than insulin itself. With monoclonal antibodies to the insulin receptor, 30% of 125I-insulin binding was inhibited. In contrast when alpha-IR3, a monoclonal antibody that recognizes typical IGF-I receptor, was employed over 60% of 125I-insulin binding was inhibited. The B29-MAB-125I-insulin photoprobe was then cross-linked to MCF-7 membranes. Cross-linking was inhibited by both unlabeled insulin and IGF-I. Further, the B29-MAB-125I-insulin photoprobe cross-linked to MCF-7 membranes was strongly immunoprecipitated by alpha-IR3. Employing sequential affinity chromatography with insulin-Affi-gel followed by insulin receptor monoclonal antibody agarose, atypical insulin binding activity was separated from insulin receptor binding activity. This atypical receptor had intrinsic tyrosine kinase activity. Both insulin and IGF-I stimulated the phosphorylation of the receptor's beta subunit. In MCF-7 cells both IGF-I and insulin stimulated [3H]thymidine incorporation; alpha-IR3 blocked all of the IGF-I effect but only 50-60% of the insulin effect. This study demonstrates in MCF-7 cells that, in addition to typical insulin and IGF-I receptors, there is another receptor that binds both insulin and IGF-I with high affinity. Images PMID:1311720

  20. Recombinant canine single chain insulin analogues: insulin receptor binding capacity and ability to stimulate glucose uptake.

    PubMed

    Adams, Jamie P; Holder, Angela L; Catchpole, Brian

    2014-12-01

    Virtually all diabetic dogs require exogenous insulin therapy to control their hyperglycaemia. In the UK, the only licensed insulin product currently available is a purified porcine insulin preparation. Recombinant insulin is somewhat problematic in terms of its manufacture, since the gene product (preproinsulin) undergoes substantial post-translational modification in pancreatic β cells before it becomes biologically active. The aim of the present study was to develop recombinant canine single chain insulin (SCI) analogues that could be produced in a prokaryotic expression system and which would require minimal processing. Three recombinant SCI constructs were developed in a prokaryotic expression vector, by replacing the insulin C-peptide sequence with one encoding a synthetic peptide (GGGPGKR), or with one of two insulin-like growth factor (IGF)-2 C-peptide coding sequences (human: SRVSRRSR; canine: SRVTRRSSR). Recombinant proteins were expressed in the periplasmic fraction of Escherichia coli and assessed for their ability to bind to the insulin and IGF-1 receptors, and to stimulate glucose uptake in 3T3-L1 adipocytes. All three recombinant SCI analogues demonstrated preferential binding to the insulin receptor compared to the IGF-1 receptor, with increased binding compared to recombinant canine proinsulin. The recombinant SCI analogues stimulated glucose uptake in 3T3-L1 adipocytes compared to negligible uptake using recombinant canine proinsulin, with the canine insulin/cIGF-2 chimaeric SCI analogue demonstrating the greatest effect. Thus, biologically-active recombinant canine SCI analogues can be produced relatively easily in bacteria, which could potentially be used for treatment of diabetic dogs.

  1. Blueberry intake alters skeletal muscle and adipose tissue peroxisome proliferator-activated receptor activity and reduces insulin resistance in obese rats.

    PubMed

    Seymour, E Mitchell; Tanone, Ignasia I; Urcuyo-Llanes, Daniel E; Lewis, Sarah K; Kirakosyan, Ara; Kondoleon, Michael G; Kaufman, Peter B; Bolling, Steven F

    2011-12-01

    Metabolic syndrome can precede the development of type 2 diabetes and cardiovascular disease and includes phenotypes such as obesity, systemic inflammation, insulin resistance, and hyperlipidemia. A recent epidemiological study indicated that blueberry intake reduced cardiovascular mortality in humans, but the possible genetic mechanisms of this effect are unknown. Blueberries are a rich source of anthocyanins, and anthocyanins can alter the activity of peroxisome proliferator-activated receptors (PPARs), which affect energy substrate metabolism. The effect of blueberry intake was assessed in obesity-prone rats. Zucker Fatty and Zucker Lean rats were fed a higher-fat diet (45% of kcal) or a lower-fat diet (10% of kcal) containing 2% (wt/wt) freeze-dried whole highbush blueberry powder or added sugars to match macronutrient and calorie content. In Zucker Fatty rats fed a high-fat diet, the addition of blueberry reduced triglycerides, fasting insulin, homeostasis model index of insulin resistance, and glucose area under the curve. Blueberry intake also reduced abdominal fat mass, increased adipose and skeletal muscle PPAR activity, and affected PPAR transcripts involved in fat oxidation and glucose uptake/oxidation. In Zucker Fatty rats fed a low-fat diet, the addition of blueberry also significantly reduced liver weight, body weight, and total fat mass. Finally, Zucker Lean rats fed blueberry had higher body weight and reduced triglycerides, but all other measures were unaffected. In conclusion, whole blueberry intake reduced phenotypes of metabolic syndrome in obesity-prone rats and affected PPAR gene transcripts in adipose and muscle tissue involved in fat and glucose metabolism.

  2. Differential hepatic distribution of insulin receptor substrates causes selective insulin resistance in diabetes and obesity

    PubMed Central

    Kubota, Naoto; Kubota, Tetsuya; Kajiwara, Eiji; Iwamura, Tomokatsu; Kumagai, Hiroki; Watanabe, Taku; Inoue, Mariko; Takamoto, Iseki; Sasako, Takayoshi; Kumagai, Katsuyoshi; Kohjima, Motoyuki; Nakamuta, Makoto; Moroi, Masao; Sugi, Kaoru; Noda, Tetsuo; Terauchi, Yasuo; Ueki, Kohjiro; Kadowaki, Takashi

    2016-01-01

    Hepatic insulin signalling involves insulin receptor substrates (Irs) 1/2, and is normally associated with the inhibition of gluconeogenesis and activation of lipogenesis. In diabetes and obesity, insulin no longer suppresses hepatic gluconeogenesis, while continuing to activate lipogenesis, a state referred to as ‘selective insulin resistance'. Here, we show that ‘selective insulin resistance' is caused by the differential expression of Irs1 and Irs2 in different zones of the liver. We demonstrate that hepatic Irs2-knockout mice develop ‘selective insulin resistance', whereas mice lacking in Irs1, or both Irs1 and Irs2, develop ‘total insulin resistance'. In obese diabetic mice, Irs1/2-mediated insulin signalling is impaired in the periportal zone, which is the primary site of gluconeogenesis, but enhanced in the perivenous zone, which is the primary site of lipogenesis. While hyperinsulinaemia reduces Irs2 expression in both the periportal and perivenous zones, Irs1 expression, which is predominantly in the perivenous zone, remains mostly unaffected. These data suggest that ‘selective insulin resistance' is induced by the differential distribution, and alterations of hepatic Irs1 and Irs2 expression. PMID:27708333

  3. 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

  4. Signal transduction through the IL-4 and insulin receptor families.

    PubMed

    Wang, L M; Keegan, A; Frankel, M; Paul, W E; Pierce, J H

    1995-07-01

    Activation of tyrosine kinase-containing receptors and intracellular tyrosine kinases by ligand stimulation is known to be crucial for mediating initial and subsequent events involved in mitogenic signal transduction. Receptors for insulin and insulin-like growth factor 1 (IGF-1) contain cytoplasmic tyrosine kinase domains that undergo autophosphorylation upon ligand stimulation. Activation of these receptors also leads to pronounced and rapid tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells of connective tissue origin. A related substrate, designated 4PS, is similarly phosphorylated by insulin and IGF-1 stimulation in many hematopoietic cell types. IRS-1 and 4PS possess a number of tyrosine phosphorylation sites that are within motifs that bind specific SH2-containing molecules known to be involved in mitogenic signaling such as PI-3 kinase, SHPTP-2 (Syp) and Grb-2. Thus, they appear to act as docking substrates for a variety of signaling molecules. The majority of hematopoietic cytokines bind to receptors that do not possess intrinsic kinase activity, and these receptors have been collectively termed as members of the hematopoietin receptor superfamily. Despite their lack of tyrosine kinase domains, stimulation of these receptors has been demonstrated to activate intracellular kinases leading to tyrosine phosphorylation of multiple substrates. Recent evidence has demonstrated that activation of different members of the Janus family of tyrosine kinases is involved in mediating tyrosine phosphorylation events by specific cytokines. Stimulation of the interleukin 4 (IL-4) receptor, a member of the hematopoietin receptor superfamily, is thought to result in activation of Jak1, Jak3, and/or Fes tyrosine kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Human blood-brain barrier insulin receptor.

    PubMed

    Pardridge, W M; Eisenberg, J; Yang, J

    1985-06-01

    A new model system for characterizing the human brain capillary, which makes up the blood-brain barrier (BBB) in vivo, is described in these studies and is applied initially to the investigation of the human BBB insulin receptor. Autopsy brains were obtained from the pathologist between 22-36 h postmortem and were used to isolate human brain microvessels which appeared intact on both light and phase microscopy. The microvessels were positive for human factor 8 and for a BBB-specific enzyme marker, gamma-glutamyl transpeptidase. The microvessels avidly bound insulin with a high-affinity dissociation constant, KD = 1.2 +/- 0.5 nM. The human brain microvessels internalized insulin based on acid-wash assay, and 75% of insulin was internalized at 37 degrees C. The microvessels transported insulin to the medium at 37 degrees C with a t1/2 = approximately 70 min. Little of the 125I-insulin was metabolized by the microvessels under these conditions based on the elution profile of the medium extract over a Sephadex G-50 column. Plasma membranes were obtained from the human brain microvessels and these membranes were enriched in membrane markers such as gamma-glutamyl transpeptidase or alkaline phosphatase. The plasma membranes bound 125I-insulin with and ED50 = 10 ng/ml, which was identical to the 50% binding point in intact microvessels. The human BBB plasma membranes were solubilized in Triton X-100 and were adsorbed to a wheat germ agglutinin Sepharose affinity column, indicating the BBB insulin receptor is a glycoprotein. Affinity cross-linking of insulin to the plasma membranes revealed a 127K protein that specifically binds insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action

    PubMed Central

    Eldar-Finkelman, Hagit; Krebs, Edwin G.

    1997-01-01

    The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glycogen synthase kinase 3 (GSK-3) is capable of phosphorylating IRS-1 and that this modification converts IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. Expression of wild-type GSK-3 or an “unregulated” mutant of the kinase (S9A) in CHO cells overexpressing IRS-1 and IR, resulted in increased serine phosphorylation levels of IRS-1, suggesting that IRS-1 is a cellular target of GSK-3. Furthermore, insulin-induced tyrosine phosphorylation of IRS-1 and IR was markedly suppressed in cells expressing wild-type or the S9A mutant, indicating that expression of GSK-3 impairs IR tyrosine kinase activity. Taken together, our studies suggest a new role for GSK-3 in attenuating insulin signaling via its phosphorylation of IRS-1 and may provide new insight into mechanisms important in insulin resistance. PMID:9275179

  7. A Novel Partial Agonist of Peroxisome Proliferator-Activated Receptor γ with Excellent Effect on Insulin Resistance and Type 2 Diabetes.

    PubMed

    Liu, Hui-juan; Zhang, Cheng-yu; Song, Fei; Xiao, Ting; Meng, Jing; Zhang, Qiang; Liang, Cai-li; Li, Shan; Wang, Jing; Zhang, Bo; Liu, Yan-rong; Sun, Tao; Zhou, Hong-gang

    2015-06-01

    Partial agonists of peroxisome proliferator-activated receptor γ (PPARγ) reportedly reverse insulin resistance in patients with type 2 diabetes mellitus. In this work, a novel non-thiazolidinedione-partial PPARγ ligand, MDCCCL1636 [N-(4-hydroxyphenethyl)-3-mercapto-2-methylpropanamide], was investigated. The compound displayed partial agonist activity in biochemical and cell-based transactivation assays and reversed insulin resistance. MDCCCL1636 showed a potential antidiabetic effect on an insulin-resistance model of human hepatocarcinoma cells (HepG2). High-fat diet-fed streptozotocin-induced diabetic rats treated with MDCCCL1636 for 56 days displayed reduced fasting serum glucose and reversed dyslipidemia and pancreatic damage without significant weight gain. Furthermore, MDCCCL1636 had lower toxicity in vivo and in vitro than pioglitazone. MDCCCL1636 also potentiated glucose consumption and inhibited the impairment in insulin signaling targets, such as AKT, glycogen synthase kinase 3β, and glycogen synthase, in HepG2 human hepatoma cells. Overall, our results suggest that MDCCCL1636 is a promising candidate for the prevention and treatment of type 2 diabetes mellitus.

  8. Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing U2AF65 binding and splicing of the upstream intron.

    PubMed

    Echeverria, Gloria V; Cooper, Thomas A

    2014-02-01

    Alternative splicing regulates developmentally and tissue-specific gene expression programs, disruption of which have been implicated in numerous diseases. Muscleblind-like 1 (MBNL1) regulates splicing transitions, which are disrupted on loss of MBNL1 function in myotonic dystrophy type 1 (DM1). One such event is MBNL1-mediated activation of insulin receptor exon 11 inclusion, which requires an intronic enhancer element downstream of exon 11. The mechanism of MBNL1-mediated activation of exon inclusion is unknown. We developed an in vitro splicing assay, which robustly recapitulates MBNL1-mediated splicing activation of insulin receptor exon 11 and found that MBNL1 activates removal of the intron upstream of exon 11 upon binding its functional response element in the downstream intron. MBNL1 enhances early spliceosome assembly as evidenced by enhanced complex A formation and binding of U2 small nuclear ribonucleoprotein auxiliary factor 65 kDa subunit (U2AF65) on the upstream intron. We demonstrated that neither the 5' splice site nor exon 11 sequences are required for MBNL1-activated U2AF65 binding. Interestingly, the 5' splice site is required for MBNL1-mediated activation of upstream intron removal, although MBNL1 has no effect on U1 snRNA recruitment. These results suggest that MBNL1 directly activates binding of U2AF65 to enhance upstream intron removal to ultimately activate alternative exon inclusion.

  9. Prolonged Activation of the Htr2b Serotonin Receptor Impairs Glucose Stimulated Insulin Secretion and Mitochondrial Function in MIN6 Cells

    PubMed Central

    Cataldo, Luis Rodrigo; Mizgier, María L.; Bravo Sagua, Roberto; Jaña, Fabián; Cárdenas, César; Llanos, Paola; Busso, Dolores; Olmos, Pablo; Galgani, José E.; Santos, José L.; Cortés, Víctor A.

    2017-01-01

    Aims Pancreatic β-cells synthesize and release serotonin (5 hydroxytryptamine, 5HT); however, the role of 5HT receptors on glucose stimulated insulin secretion (GSIS) and the mechanisms mediating this function is not fully understood. The aims of this study were to determine the expression profile of 5HT receptors in murine MIN6 β-cells and to examine the effects of pharmacological activation of 5HT receptor Htr2b on GSIS and mitochondrial function. Materials and Methods mRNA levels of 5HT receptors in MIN6 cells were quantified by RT qPCR. GSIS was assessed in MIN6 cells in response to global serotonergic activation with 5HT and pharmacological Htr2b activation or inhibition with BW723C86 or SB204741, respectively. In response to Htr2b activation also was evaluated the mRNA and protein levels of PGC1α and PPARy by RT-qPCR and western blotting and mitochondrial function by oxygen consumption rate (OCR) and ATP cellular content. Results We found that mRNA levels of most 5HT receptors were either very low or undetectable in MIN6 cells. By contrast, Htr2b mRNA was present at moderate levels in these cells. Preincubation (6 h) of MIN6 cells with 5HT or BW723C86 reduced GSIS and the effect of 5HT was prevented by SB204741. Preincubation with BW723C86 increased PGC1α and PPARy mRNA and protein levels and decreased mitochondrial respiration and ATP content in MIN6 cells. Conclusions Our results indicate that prolonged Htr2b activation in murine β-cells decreases glucose-stimulated insulin secretion and mitochondrial activity by mechanisms likely dependent on enhanced PGC1α/PPARy expression. PMID:28129327

  10. 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

  11. Berberine inhibits PTP1B activity and mimics insulin action.

    PubMed

    Chen, Chunhua; Zhang, Yuebo; Huang, Cheng

    2010-07-02

    Type 2 diabetes patients show defects in insulin signal transduction that include lack of insulin receptor, decrease in insulin stimulated receptor tyrosine kinase activity and receptor-mediated phosphorylation of insulin receptor substrates (IRSs). A small molecule that could target insulin signaling would be of significant advantage in the treatment of diabetes. Berberine (BBR) has recently been shown to lower blood glucose levels and to improve insulin resistance in db/db mice partly through the activation of AMP-activated protein kinase (AMPK) signaling and induction of phosphorylation of insulin receptor (IR). However, the underlying mechanism remains largely unknown. Here we report that BBR mimics insulin action by increasing glucose uptake ability by 3T3-L1 adipocytes and L6 myocytes in an insulin-independent manner, inhibiting phosphatase activity of protein tyrosine phosphatase 1B (PTP1B), and increasing phosphorylation of IR, IRS1 and Akt in 3T3-L1 adipocytes. In diabetic mice, BBR lowers hyperglycemia and improves impaired glucose tolerance, but does not increase insulin release and synthesis. The results suggest that BBR represents a different class of anti-hyperglycemic agents.

  12. Characterization of insulin receptors in chicken kidneys: effect of nutritional status.

    PubMed

    Bisbis, S; Derouet, M; Simon, J

    1994-10-01

    In chickens, the kidneys actively contribute to gluconeogenesis. A cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) is present in this tissue but is absent in liver. Cytosolic renal PEPCK is nutritionally and hormonally controlled which indicates a likely contribution of insulin in the control of this enzyme (and other renal functions). The present studies characterize renal insulin receptors in the chicken. The effects of the following nutritional conditions were examined: fed, 48 hr fasted, and 24 hr refed following a 48-hr fast. PEPCK activity was increased by the 48-hr fast and returned to normal after refeeding. Specific binding of 125I-insulin to renal membranes was time-, temperature-, and protein-dependent. Unlabeled insulin was more potent than IGF-1 in inhibiting 125I-insulin binding; the ratio of potencies for insulin and IGF-1, however, was dependent upon the nutritional state. Insulin binding was significantly higher (P < 0.05) following 48 hr fasting and lower (P < 0.05) following refeeding compared to ad libitum feeding. Receptor affinity was similar irrespective of the nutritional state. Solubilized and wheat germ agglutinin purified renal insulin receptors were devoid of ATPase activity in contrast to hepatic receptors. The sizes of alpha- and beta-subunits of renal receptors were similar to those of hepatic receptors: 135 and 95 kDa, respectively. Insulin-stimulated autophosphorylation of the beta-subunit was decreased, although not significantly, by prolonged fasting. Phosphorylation of artificial substrate: poly(Glu-Tyr) 4:1 was significantly decreased by the 48-hr fast at high insulin concentrations (10 and 100 nM). Kinase activities of renal insulin receptors from fed or refed chickens were very similar. In conclusion, typical insulin receptors are present in chicken kidneys. These receptors exhibit a regulation at the level of their number and kinase activity in a fashion similar to that found for hepatic receptors. The present

  13. Metabolites related to gut bacterial metabolism, peroxisome proliferator-activated receptor-alpha activation, and insulin sensitivity are associated with physical function in functionally-limited older adults

    PubMed Central

    Lustgarten, Michael S; Price, Lori L; Chalé, Angela; Fielding, Roger A

    2014-01-01

    Identification of mechanisms underlying physical function will be important for addressing the growing challenge that health care will face with physical disablement in the expanding aging population. Therefore, the goals of the current study were to use metabolic profiling to provide insight into biologic mechanisms that may underlie physical function by examining the association between baseline and the 6-month change in serum mass spectrometry-obtained amino acids, fatty acids, and acylcarnitines with baseline and the 6-month change in muscle strength (leg press one repetition maximum divided by total lean mass, LP/Lean), lower extremity function [short physical performance battery (SPPB)], and mobility (400 m gait speed, 400-m), in response to 6 months of a combined resistance exercise and nutritional supplementation (whey protein or placebo) intervention in functionally-limited older adults (SPPB ≤ 10; 70–85 years, N = 73). Metabolites related to gut bacterial metabolism (cinnamoylglycine, phenol sulfate, p-cresol sulfate, 3-indoxyl sulfate, serotonin, N-methylproline, hydrocinnamate, dimethylglycine, trans-urocanate, valerate) that are altered in response to peroxisome proliferator-activated receptor-alpha (PPAR-α) activation (α-hydroxyisocaproate, α-hydroxyisovalerate, 2-hydroxy-3-methylvalerate, indolelactate, serotonin, 2-hydroxypalmitate, glutarylcarnitine, isobutyrylcarnitine, cinnamoylglycine) and that are related to insulin sensitivity (monounsaturated fatty acids: 5-dodecenoate, myristoleate, palmitoleate; γ-glutamylamino acids: γ-glutamylglutamine, γ-glutamylalanine, γ-glutamylmethionine, γ-glutamyltyrosine; branched-chain amino acids: leucine, isoleucine, valine) were associated with function at baseline, with the 6-month change in function or were identified in backward elimination regression predictive models. Collectively, these data suggest that gut microbial metabolism, PPAR-α activation, and insulin sensitivity may be involved in

  14. Functional characterization of insulin receptor gene mutations contributing to Rabson-Mendenhall syndrome - phenotypic heterogeneity of insulin receptor gene mutations.

    PubMed

    Jiang, Shan; Fang, Qichen; Zhang, Feng; Wan, Hui; Zhang, Rong; Wang, Congrong; Bao, Yuqian; Zhang, Lei; Ma, Xiaojing; Lu, Junxi; Gao, Fei; Xiang, Kunsan; Jia, Weiping

    2011-01-01

    Rabson-Mendenhall syndrome (RMS) is a rare disorder that presents as severe insulin resistance as a result of mutations present in the insulin receptor (INSR). A Chinese girl with RMS presented with profound diabetes, hyperinsulinemia, acanthosis nigricans, hirsutism, and abnormalities of teeth and nails. Direct sequencing of the patient's INSR detected heterozygote mutations at Arg83Gln (R83Q) and Ala1028Val (A1028V), with the former representing a novel mutation. Functional studies of Chinese hamster ovary (CHO) cells transfected with wild-type (WT) and mutant forms of INSR were performed to evaluate the effects of these mutations on receptor expression and activation. Receptor expression, insulin binding activity, and phosphorylation of the R83Q variant were comparable to WT. In contrast, expression of the A1028V receptor was much lower than that of WT INSR, and impairment of insulin binding and autophosphorylation were nearly commensurate with the decrease in expression detected. Reductions in the phosphorylation of IRS-1, Akt, and Erk1/2 (60%, 40%, and 50% of WT, respectively) indicate that the A1028V receptor contributes to impaired signal transduction. In conclusion, INSR mutations associated with RMS were identified. Moreover, the A1028V mutation associated with a decrease in expression of INSR potentially accounts for loss of function of the INSR.

  15. G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway.

    PubMed

    Law, Nathan C; White, Morris F; Hunzicker-Dunn, Mary E

    2016-12-30

    G protein-coupled receptors (GPCRs) activate PI3K/v-AKT thymoma viral oncoprotein (AKT) to regulate many cellular functions that promote cell survival, proliferation, and growth. However, the mechanism by which GPCRs activate PI3K/AKT remains poorly understood. We used ovarian preantral granulosa cells (GCs) to elucidate the mechanism by which the GPCR agonist FSH via PKA activates the PI3K/AKT cascade. Insulin-like growth factor 1 (IGF1) is secreted in an autocrine/paracrine manner by GCs and activates the IGF1 receptor (IGF1R) but, in the absence of FSH, fails to stimulate YXXM phosphorylation of IRS1 (insulin receptor substrate 1) required for PI3K/AKT activation. We show that PKA directly phosphorylates the protein phosphatase 1 (PP1) regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) to activate PP1 associated with the IGF1R-IRS1 complex. Activated PP1 is sufficient to dephosphorylate at least four IRS1 Ser residues, Ser(318), Ser(346), Ser(612), and Ser(789), and promotes IRS1 YXXM phosphorylation by the IGF1R to activate the PI3K/AKT cascade. Additional experiments indicate that this mechanism also occurs in breast cancer, thyroid, and preovulatory granulosa cells, suggesting that the PKA-dependent dephosphorylation of IRS1 Ser/Thr residues is a conserved mechanism by which GPCRs signal to activate the PI3K/AKT pathway downstream of the IGF1R.

  16. 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...

  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 receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance

    PubMed Central

    Petersen, Max C.; Madiraju, Anila K.; Gassaway, Brandon M.; Marcel, Michael; Nasiri, Ali R.; Butrico, Gina; Marcucci, Melissa J.; Zhang, Dongyan; Abulizi, Abudukadier; Zhang, Xian-Man; Philbrick, William; Hubbard, Stevan R.; Samuel, Varman T.; Rinehart, Jesse

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet–induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance. PMID:27760050

  19. Adipose-specific peroxisome proliferator-activated receptor gamma knockout causes insulin resistance in fat and liver but not in muscle.

    PubMed

    He, Weimin; Barak, Yaacov; Hevener, Andrea; Olson, Peter; Liao, Debbie; Le, Jamie; Nelson, Michael; Ong, Estelita; Olefsky, Jerrold M; Evans, Ronald M

    2003-12-23

    Syndrome X, typified by obesity, insulin resistance (IR), dyslipidemia, and other metabolic abnormalities, is responsive to antidiabetic thiazolidinediones (TZDs). Peroxisome proliferator-activated receptor (PPAR) gamma, a target of TZDs, is expressed abundantly in adipocytes, suggesting an important role for this tissue in the etiology and treatment of IR. Targeted deletion of PPARgamma in adipose tissue resulted in marked adipocyte hypocellularity and hypertrophy, elevated levels of plasma free fatty acids and triglyceride, and decreased levels of plasma leptin and ACRP30. In addition, increased hepatic glucogenesis and IR were observed. Despite these defects, blood glucose, glucose and insulin tolerance, and insulin-stimulated muscle glucose uptake were all comparable to those of control mice. However, targeted mice were significantly more susceptible to high-fat diet-induced steatosis, hyperinsulinemia, and IR. Surprisingly, TZD treatment effectively reversed liver IR, whereas it failed to lower plasma free fatty acids. These results suggest that syndrome X may be comprised of separable PPARgamma-dependent components whose origins and therapeutic sites may reside in distinct tissues.

  20. 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

  1. Insulin-Dependent Activation of MCH Neurons Impairs Locomotor Activity and Insulin Sensitivity in Obesity.

    PubMed

    Hausen, A Christine; Ruud, Johan; Jiang, Hong; Hess, Simon; Varbanov, Hristo; Kloppenburg, Peter; Brüning, Jens C

    2016-12-06

    Melanin-concentrating-hormone (MCH)-expressing neurons (MCH neurons) in the lateral hypothalamus (LH) are critical regulators of energy and glucose homeostasis. Here, we demonstrate that insulin increases the excitability of these neurons in control mice. In vivo, insulin promotes phosphatidylinositol 3-kinase (PI3K) signaling in MCH neurons, and cell-type-specific deletion of the insulin receptor (IR) abrogates this response. While lean mice lacking the IR in MCH neurons (IR(ΔMCH)) exhibit no detectable metabolic phenotype under normal diet feeding, they present with improved locomotor activity and insulin sensitivity under high-fat-diet-fed, obese conditions. Similarly, obesity promotes PI3 kinase signaling in these neurons, and this response is abrogated in IR(ΔMCH) mice. In turn, acute chemogenetic activation of MCH neurons impairs locomotor activity but not insulin sensitivity. Collectively, our experiments reveal an insulin-dependent activation of MCH neurons in obesity, which contributes via distinct mechanisms to the manifestation of impaired locomotor activity and insulin resistance.

  2. Human insulin prepared by recombinant DNA techniques and native human insulin interact identically with insulin receptors.

    PubMed Central

    Keefer, L M; Piron, M A; De Meyts, P

    1981-01-01

    Human insulin synthesized from A and B chains separately produced in Escherichia coli from cloned synthetic genes (prepared by the Eli Lilly Research Laboratories, Indianapolis, IN) was characterized by examining its interaction with human cultured lymphocytes, human circulating erythrocytes in vitro, and isolated rat fat cells. The binding behavior of the biosynthetic insulin with human cells was indistinguishable from that of native human or porcine insulins, with respect to affinity, association and dissociation kinetics, negative cooperativity, and the down-regulation of lymphocyte receptors. Similarly, the biosynthetic insulin was as potent as the native insulins in stimulating lipogenesis in isolated rat fat cells. We also examined the receptor binding characteristics of 125I-labeled human and porcine insulins monoiodinated solely at Tyr-A14, which were obtained by means of high-performance liquid chromatography of the iodination reaction mixture (this material was prepared by B. Frank, Eli Lilly Research Laboratories). In all aspects studied, the pure [TyrA14-125I]iodoinsulins were superior as tracers to the monoiodoinsulin purified by the more conventional method of gel filtration. PMID:7015337

  3. Amplification and analysis of promoter region of insulin receptor gene in a patient with leprechaunism associated with severe insulin resistance.

    PubMed

    Haruta, T; Imamura, T; Iwanishi, M; Egawa, K; Goji, K; Kobayashi, M

    1995-04-01

    A patient with leprechaunism associated with severe insulin resistance was studied to identify the molecular and genetic basis for insulin resistance. Insulin binding and surface labeling of transformed lymphocytes prepared from the patient showed a significantly decreased insulin receptor number on the cell surface. Southern blot analysis of the insulin receptor gene showed no evidence of large insertions or deletions. Furthermore, direct sequencing of all 22 exons and exon-intron junctions of the insulin receptor gene failed to show any missense mutations, nonsense mutations, or mutations at exon-intron junctions. However, Northern blot analysis indicated significantly decreased insulin receptor mRNA expression in the patient's cells. Moreover, restriction endonuclease digestion of the amplified cDNA suggested that the expression levels of one allele were less efficient than the other. These findings suggested that the regulatory region of the insulin receptor gene might have abnormalities. Therefore, we examined the 5' flanking region of the insulin receptor gene. Southern blot analysis showed no major deletions or insertions between positions -1,823 and -2 relative to the translation initiation site. A 5' flanking region of the insulin receptor gene spanning positions -881 approximately +7 was amplified by polymerase chain reaction (PCR) and introduced into a reporter plasmid carrying the human growth hormone (hGH) gene. The nucleotide sequence of the amplified fragment showed two polymorphic sites at positions -603 and -500 in the patient, as well as in normal subjects. No other abnormal sequence was found in the patient. Promoter activity measured by hGH expression in transfected mouse L cells was not influenced by the polymorphism at position -603 located in a cluster of GC boxes.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

    SciTech Connect

    Wong, K.Y.; Hawley, D.; Vigneri, R.; Goldfine, I.D.

    1988-01-12

    Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor ..cap alpha.. subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-(..gamma..-/sup 32/P)triphosphate, a protein of 95 kilodaltons representing the insulin receptor ..beta.. subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins.

  5. 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.

  6. Berberine-improved visceral white adipose tissue insulin resistance associated with altered sterol regulatory element-binding proteins, liver x receptors, and peroxisome proliferator-activated receptors transcriptional programs in diabetic hamsters.

    PubMed

    Li, Guo-Sheng; Liu, Xu-Han; Zhu, Hua; Huang, Lan; Liu, Ya-Li; Ma, Chun-Mei; Qin, Chuan

    2011-01-01

    The diabetic "lipotoxicity" hypothesis presents that fat-induced visceral white adipose tissue insulin resistance plays a central role in the pathogenesis of type 2 diabetes. Berberine, a hypolipidemic agent, has been reported to have antidiabetic activities. The molecular mechanisms for this property are, however, not well clarified. Therefore in this study type 2 diabetic hamsters were induced by high-fat diet with low-dose streptozotocin. Then, we investigated the gene expression alterations and explored the molecular mechanisms underlying the therapeutic effect of berberine on fat-induced visceral white adipose tissue insulin resistance in diabetic hamsters by microarray analysis followed by real-time reverse transcription-polymerase chain reaction (RT-PCR) confirmation. Type 2 diabetic hamsters exhibited hyperglycemia and relative hyperinsulinemia, glucose intolerance, insulin resistance, intra-adipocyte lipid accumulation, significant increase in body weight and visceral white adipose tissue weight, abnormal serum adipokines levels, and deleterious dyslipidemia. Furthermore, they had increased sterol regulatory element-binding proteins (SREBPs) expression and decreased liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) expression in visceral white adipose tissue. After 9-week berberine treatment, fat-induced insulin resistance and diabetic phenotype in type 2 diabetic hamsters were significantly improved. Compared with diabetic hamsters, expression of LXRs and PPARs significantly increased and SREBPs significantly decreased in visceral white adipose tissue from berberine-treated diabetic hamsters. These results suggest that altered visceral white adipose tissue LXRs, PPARs, and SREBPs transcriptional programs are involved in the therapeutic mechanisms of berberine on fat-induced visceral white adipose tissue insulin resistance in type 2 diabetic hamsters.

  7. 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.

  8. p75 neurotrophin receptor regulates glucose homeostasis and insulin sensitivity

    PubMed Central

    Baeza-Raja, Bernat; Li, Pingping; Le Moan, Natacha; Sachs, Benjamin D.; Schachtrup, Christian; Davalos, Dimitrios; Vagena, Eirini; Bridges, Dave; Kim, Choel; Saltiel, Alan R.; Olefsky, Jerrold M.; Akassoglou, Katerina

    2012-01-01

    Insulin resistance is a key factor in the etiology of type 2 diabetes. Insulin-stimulated glucose uptake is mediated by the glucose transporter 4 (GLUT4), which is expressed mainly in skeletal muscle and adipose tissue. Insulin-stimulated translocation of GLUT4 from its intracellular compartment to the plasma membrane is regulated by small guanosine triphosphate hydrolases (GTPases) and is essential for the maintenance of normal glucose homeostasis. Here we show that the p75 neurotrophin receptor (p75NTR) is a regulator of glucose uptake and insulin resistance. p75NTR knockout mice show increased insulin sensitivity on normal chow diet, independent of changes in body weight. Euglycemic-hyperinsulinemic clamp studies demonstrate that deletion of the p75NTR gene increases the insulin-stimulated glucose disposal rate and suppression of hepatic glucose production. Genetic depletion or shRNA knockdown of p75NTR in adipocytes or myoblasts increases insulin-stimulated glucose uptake and GLUT4 translocation. Conversely, overexpression of p75NTR in adipocytes decreases insulin-stimulated glucose transport. In adipocytes, p75NTR forms a complex with the Rab5 family GTPases Rab5 and Rab31 that regulate GLUT4 trafficking. Rab5 and Rab31 directly interact with p75NTR primarily via helix 4 of the p75NTR death domain. Adipocytes from p75NTR knockout mice show increased Rab5 and decreased Rab31 activities, and dominant negative Rab5 rescues the increase in glucose uptake seen in p75NTR knockout adipocytes. Our results identify p75NTR as a unique player in glucose metabolism and suggest that signaling from p75NTR to Rab5 family GTPases may represent a unique therapeutic target for insulin resistance and diabetes. PMID:22460790

  9. A novel partial agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma) recruits PPARgamma-coactivator-1alpha, prevents triglyceride accumulation, and potentiates insulin signaling in vitro.

    PubMed

    Burgermeister, Elke; Schnoebelen, Astride; Flament, Angele; Benz, Jörg; Stihle, Martine; Gsell, Bernard; Rufer, Arne; Ruf, Armin; Kuhn, Bernd; Märki, Hans Peter; Mizrahi, Jacques; Sebokova, Elena; Niesor, Eric; Meyer, Markus

    2006-04-01

    Partial agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma), also termed selective PPARgamma modulators, are expected to uncouple insulin sensitization from triglyceride (TG) storage in patients with type 2 diabetes mellitus. These agents shall thus avoid adverse effects, such as body weight gain, exerted by full agonists such as thiazolidinediones. In this context, we describe the identification and characterization of the isoquinoline derivative PA-082, a prototype of a novel class of non-thiazolidinedione partial PPARgamma ligands. In a cocrystal with PPARgamma it was bound within the ligand-binding pocket without direct contact to helix 12. The compound displayed partial agonism in biochemical and cell-based transactivation assays and caused preferential recruitment of PPARgamma-coactivator-1alpha (PGC1alpha) to the receptor, a feature shared with other selective PPARgamma modulators. It antagonized rosiglitazone-driven transactivation and TG accumulation during de novo adipogenic differentiation of murine C3H10T1/2 mesenchymal stem cells. The latter effect was mimicked by overexpression of wild-type PGC1alpha but not its LXXLL-deficient mutant. Despite failing to promote TG loading, PA-082 induced mRNAs of genes encoding components of insulin signaling and adipogenic differentiation pathways. It potentiated glucose uptake and inhibited the negative cross-talk of TNFalpha on protein kinase B (AKT) phosphorylation in mature adipocytes and HepG2 human hepatoma cells. PGC1alpha is a key regulator of energy expenditure and down-regulated in diabetics. We thus propose that selective recruitment of PGC1alpha to favorable PPARgamma-target genes provides a possible molecular mechanism whereby partial PPARgamma agonists dissociate TG accumulation from insulin signaling.

  10. Antidepressants activate the lysophosphatidic acid receptor LPA(1) to induce insulin-like growth factor-I receptor transactivation, stimulation of ERK1/2 signaling and cell proliferation in CHO-K1 fibroblasts.

    PubMed

    Olianas, Maria C; Dedoni, Simona; Onali, Pierluigi

    2015-06-15

    Different lines of evidence indicate that the lysophosphatidic acid (LPA) receptor LPA1 is involved in neurogenesis, synaptic plasticity and anxiety-related behavior, but little is known on whether this receptor can be targeted by neuropsychopharmacological agents. The present study investigated the effects of different antidepressants on LPA1 signaling. We found that in Chinese hamster ovary (CHO)-K1 fibroblasts expressing endogenous LPA1 tricyclic and tetracyclic antidepressants and fluoxetine induced the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and CREB. This response was antagonized by either LPA1 blockade with Ki16425 and AM966 or knocking down LPA1 with siRNA. Antidepressants induced ERK1/2 phosphorylation in human embryonic kidney (HEK)-293 cells overexpressing LPA1, but not in wild-type cells. In PathHunter™ assay measuring receptor-β-arrestin interaction, amitriptyline, mianserin and fluoxetine failed to induce activation of LPA2 and LPA3 stably expressed in CHO-K1 cells. ERK1/2 stimulation by antidepressants and LPA was suppressed by pertussis toxin and inhibition of Src, phosphatidylinositol-3 kinase and insulin-like growth factor-I receptor (IGF-IR) activities. Antidepressants and LPA induced tyrosine phosphorylation of IGF-IR and insulin receptor-substrate-1 through LPA1 and Src. Prolonged exposure of CHO-K1 fibroblasts to either mianserin, mirtazapine or LPA enhanced cell proliferation as indicated by increased [(3)H]-thymidine incorporation and Ki-67 immunofluorescence. This effect was inhibited by blockade of LPA1- and ERK1/2 activity. These data provide evidence that different antidepressants induce LPA1 activation, leading to receptor tyrosine kinase transactivation, stimulation of ERK1/2 signaling and enhanced cell proliferation.

  11. Role of the intra-A-chain disulfide bond of insulin-like peptide 3 in binding and activation of its receptor, RXFP2.

    PubMed

    Zhang, Suode; Hughes, Richard A; Bathgate, Ross A D; Shabanpoor, Fazel; Hossain, M Akhter; Lin, Feng; van Lierop, Bianca; Robinson, Andrea J; Wade, John D

    2010-09-01

    INSL3 is a member of the insulin-IGF-relaxin superfamily and plays a key role in male fetal development and in adult germ cell maturation. It is the cognate ligand for RXFP2, a leucine-rich repeat containing G-protein coupled receptor. To date, and in contrast to our current knowledge of the key structural features that are required for the binding of INSL3 to RXFP2, comparatively little is known about the key residues that are required to elicit receptor activation and downstream cell signaling. Early evidence suggests that these are contained principally within the A-chain. To further explore this hypothesis, we have undertaken an examination of the functional role of the intra-A-chain disulfide bond. Using solid-phase peptide synthesis together with regioselective disulfide bond formation, two analogs of human INSL3 were prepared in which the intra-chain disulfide bond was replaced, one in which the corresponding Cys residues were substituted with the isosteric Ser and the other in which the Cys were removed altogether. Both of these peptides retained nearly full RXFP2 receptor binding but were devoid of cAMP activity (receptor activation), indicating that the intra-A-chain disulfide bond makes a significant contribution to the ability of INSL3 to act as an RXFP2 agonist. Replacement of the disulfide bond with a metabolically stable dicarba bond yielded two isomers of INSL3 that each exhibited bioactivity similar to native INSL3. This study highlights the critical structural role played by the intra-A-chain disulfide bond of INSL3 in mediating agonist actions through the RXFP2 receptor.

  12. Cannabinoids Inhibit Insulin Receptor Signaling in Pancreatic β-Cells

    PubMed Central

    Kim, Wook; Doyle, Máire E.; Liu, Zhuo; Lao, Qizong; Shin, Yu-Kyong; Carlson, Olga D.; Kim, Hee Seung; Thomas, Sam; Napora, Joshua K.; Lee, Eun Kyung; Moaddel, Ruin; Wang, Yan; Maudsley, Stuart; Martin, Bronwen; Kulkarni, Rohit N.; Egan, Josephine M.

    2011-01-01

    OBJECTIVE Optimal glucose homeostasis requires exquisitely precise adaptation of the number of insulin-secreting β-cells in the islets of Langerhans. Insulin itself positively regulates β-cell proliferation in an autocrine manner through the insulin receptor (IR) signaling pathway. It is now coming to light that cannabinoid 1 receptor (CB1R) agonism/antagonism influences insulin action in insulin-sensitive tissues. However, the cells on which the CB1Rs are expressed and their function in islets have not been firmly established. We undertook the current study to investigate if intraislet endogenous cannabinoids (ECs) regulate β-cell proliferation and if they influence insulin action. RESEARCH DESIGN AND METHODS We measured EC production in isolated human and mouse islets and β-cell line in response to glucose and KCl. We evaluated human and mouse islets, several β-cell lines, and CB1R-null (CB1R−/−) mice for the presence of a fully functioning EC system. We investigated if ECs influence β-cell physiology through regulating insulin action and demonstrated the therapeutic potential of manipulation of the EC system in diabetic (db/db) mice. RESULTS ECs are generated within β-cells, which also express CB1Rs that are fully functioning when activated by ligands. Genetic and pharmacologic blockade of CB1R results in enhanced IR signaling through the insulin receptor substrate 2-AKT pathway in β-cells and leads to increased β-cell proliferation and mass. CB1R antagonism in db/db mice results in reduced blood glucose and increased β-cell proliferation and mass, coupled with enhanced IR signaling in β-cells. Furthermore, CB1R activation impedes insulin-stimulated IR autophosphorylation on β-cells in a Gαi-dependent manner. CONCLUSIONS These findings provide direct evidence for a functional interaction between CB1R and IR signaling involved in the regulation of β-cell proliferation and will serve as a basis for developing new therapeutic interventions to

  13. 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.

  14. Angiotensin Receptor Blockade Recovers Hepatic UCP2 Expression and Aconitase and SDH Activities and Ameliorates Hepatic Oxidative Damage in Insulin Resistant Rats

    PubMed Central

    Montez, Priscilla; Vázquez-Medina, José Pablo; Rodríguez, Rubén; Thorwald, Max A.; Viscarra, José A.; Lam, Lisa; Peti-Peterdi, Janos; Nakano, Daisuke; Nishiyama, Akira

    2012-01-01

    Metabolic syndrome (MetS) is commonly associated with elevated renin-angiotensin system, oxidative stress, and steatohepatitis with down-regulation of uncoupling proteins (UCPs). However, the mechanisms linking renin-angiotensin system, steatosis, and UCP2 to hepatic oxidative damage during insulin resistance are not described. To test the hypothesis that angiotensin receptor activation contributes to decreased hepatic UCP2 expression and aconitase activity and to increased oxidative damage after increased glucose intake in a model of MetS, lean and obese Long Evans rats (n = 10/group) were randomly assigned to the following groups: 1) untreated Long Evans Tokushima Otsuka (lean, strain control), 2) untreated Otsuka Long Evans Tokushima Fatty (OLETF) (MetS model), 3) OLETF + angiotensin receptor blocker (ARB) (10 mg olmesartan/kg·d × 6 wk), 4) OLETF + high glucose (HG) (5% in drinking water × 6 wk), and 5) OLETF + ARB + HG (ARB/HG × 6 wk). HG increased body mass (37%), plasma triglycerides (TGs) (35%), plasma glycerol (87%), plasma free fatty acids (28%), and hepatic nitrotyrosine (74%). ARB treatment in HG decreased body mass (12%), plasma TG (15%), plasma glycerol (23%), plasma free fatty acids (14%), and hepatic TG content (42%), suggesting that angiotensin receptor type 1 (AT1) activation and increased adiposity contribute to the development of obesity-related dyslipidemia. ARB in HG also decreased hepatic nitrotyrosine and increased hepatic UCP2 expression (59%) and aconitase activity (40%), as well as antioxidant enzyme activities (50-120%), suggesting that AT1 activation also contributes to protein oxidation, impaired lipid metabolism, and antioxidant metabolism in the liver. Thus, in addition to promoting obesity-related hypertension, AT1 activation may also impair lipid metabolism and antioxidant capacity, resulting in steatosis via decreased UCP2 and tricarboxylic acid cycle activity. PMID:23087176

  15. Angiotensin receptor blockade recovers hepatic UCP2 expression and aconitase and SDH activities and ameliorates hepatic oxidative damage in insulin resistant rats.

    PubMed

    Montez, Priscilla; Vázquez-Medina, José Pablo; Rodríguez, Rubén; Thorwald, Max A; Viscarra, José A; Lam, Lisa; Peti-Peterdi, Janos; Nakano, Daisuke; Nishiyama, Akira; Ortiz, Rudy M

    2012-12-01

    Metabolic syndrome (MetS) is commonly associated with elevated renin-angiotensin system, oxidative stress, and steatohepatitis with down-regulation of uncoupling proteins (UCPs). However, the mechanisms linking renin-angiotensin system, steatosis, and UCP2 to hepatic oxidative damage during insulin resistance are not described. To test the hypothesis that angiotensin receptor activation contributes to decreased hepatic UCP2 expression and aconitase activity and to increased oxidative damage after increased glucose intake in a model of MetS, lean and obese Long Evans rats (n = 10/group) were randomly assigned to the following groups: 1) untreated Long Evans Tokushima Otsuka (lean, strain control), 2) untreated Otsuka Long Evans Tokushima Fatty (OLETF) (MetS model), 3) OLETF + angiotensin receptor blocker (ARB) (10 mg olmesartan/kg·d × 6 wk), 4) OLETF + high glucose (HG) (5% in drinking water × 6 wk), and 5) OLETF + ARB + HG (ARB/HG × 6 wk). HG increased body mass (37%), plasma triglycerides (TGs) (35%), plasma glycerol (87%), plasma free fatty acids (28%), and hepatic nitrotyrosine (74%). ARB treatment in HG decreased body mass (12%), plasma TG (15%), plasma glycerol (23%), plasma free fatty acids (14%), and hepatic TG content (42%), suggesting that angiotensin receptor type 1 (AT1) activation and increased adiposity contribute to the development of obesity-related dyslipidemia. ARB in HG also decreased hepatic nitrotyrosine and increased hepatic UCP2 expression (59%) and aconitase activity (40%), as well as antioxidant enzyme activities (50-120%), suggesting that AT1 activation also contributes to protein oxidation, impaired lipid metabolism, and antioxidant metabolism in the liver. Thus, in addition to promoting obesity-related hypertension, AT1 activation may also impair lipid metabolism and antioxidant capacity, resulting in steatosis via decreased UCP2 and tricarboxylic acid cycle activity.

  16. Insulin-Dependent Regulation of Insulin Receptor Concentrations: A Direct Demonstration in Cell Culture

    PubMed Central

    Gavin, James R.; Roth, Jesse; Neville, David M.; De Meyts, Pierre; Buell, Donald N.

    1974-01-01

    Chronic (5-16 hr) exposure of cultured human lymphocytes to 10-8 M insulin at 37° in vitro produced a decrease in insulin receptor concentrations unaccounted for by simple occupancy of sites; acute exposure (0-2 hr) was without effect. These results reproduced observations in vivo where chronic hyperinsulinemia (e.g., 10-8 M insulin in the circulation of obese insulinresistant hyperglycemic mice) is associated with a substantial reduction in the concentration of insulin receptors per cell, while acute hyperinsulinemia in vivo has no effect on receptor concentration. These data suggest a reciprocal relationship between insulin in the extracellular fluid and the concentration of insulin receptors per cell, which is mediated at the target cell itself by intracellular insulin-sensitive regulatory processes and directly affects target-cell sensitivity to hormone. PMID:4359334

  17. Changes of insulin effect on lipogenesis and insulin binding receptors during hypokinesia

    NASA Astrophysics Data System (ADS)

    Macho, L.; Fickova, M.; Zorad, S.

    The effect of hypokinesia on insulin action and insulin binding to specific receptors in fat cells was studied. Male Wistar rats were exposed to hypokinesia in special adjustable plastic cages for 1, 7, 21 and 60 days, and the stimulatory effect of insulin (10 and 100 mU) on the incorporation of radiocarbon labelled glucose into lipids of fat tissue and the binding of insulin to receptors of isolated adipocytes was estimated. The stimulation of lipogenesis by insulin was slightly diminished after hypokinesia for 1 day, however, an important increase of insulin action was found in rats exposed to hypokinesia for 60 days. The decrease of insulin binding capacity of the number of binding sites per cell and of the insulin receptor density was found after 1 day of hypokinesia. In rats exposed to hypokinesia for 60 days, in agreement with the higher stimulatory affect of insulin, an increase of insulin receptor density was observed. These results showed that hypokinesia has an important influence on stimulatory action of insulin and on insulin receptors in adipocytes.

  18. 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.

  19. [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.

  20. Cellular mechanisms of insulin resistance: role of stress-regulated serine kinases and insulin receptor substrates (IRS) serine phosphorylation.

    PubMed

    Tanti, Jean-François; Jager, Jennifer

    2009-12-01

    Insulin receptor substrates (IRS) serine phosphorylation is a time-controlled physiological feedback mechanism in insulin signaling that is hijacked by metabolic and inflammatory stresses to promote insulin resistance. Kinases, including IKKbeta, JNK, ERK, mTOR, and S6K, activated by the inducers of insulin resistance induce uncontrolled IRS serine phosphorylation. Studies with genetically modified mice reveal that these kinases integrate signals from metabolic and inflammatory stresses in adipose tissue, liver, and hypothalamus leading to peripheral and central insulin resistance. Moreover, IKKbeta/NF-kappaB and JNK1 pathways in myeloid cells represent a core mechanism involved in inflammation linked to obesity. These kinases are thus potential drug targets against insulin resistance and the targeting of the IKKbeta/NF-kappaB or the JNK pathway may evolve into future diabetes medication.

  1. A peptide from Porphyra yezoensis stimulates the proliferation of IEC-6 cells by activating the insulin-like growth factor I receptor signaling pathway.

    PubMed

    Lee, Min-Kyeong; Kim, In-Hye; Choi, Youn-Hee; Nam, Taek-Jeong

    2015-02-01

    Porphyra yezoensis (P. yezoensis) is the most noteworthy red alga and is mainly consumed in China, Japan and Korea. In the present study, the effects of a P. yezoensis peptide (PY‑PE) on cell proliferation and the associated signaling pathways were examined in IEC‑6 rat intestinal epithelial cells. First, the MTS assay showed that PY‑PE induced cell proliferation in a dose‑dependent manner. Subsequently, the mechanism behind the proliferative activity induced by PY‑PE was determined. The insulin‑like growth factor‑I receptor (IGF‑IR) signaling pathway was the main focus as it plays an important role in the regulation of cell growth and proliferation. PY‑PE increased the protein and mRNA expression of IGF‑IR, insulin receptor substrate‑1, Shc and PY‑99. In addition, PY‑PE stimulated extracellular signal‑regulated kinase phosphorylation and phosphatidylinositol 3‑kinase/Akt activation but inhibited p38 and c‑Jun N‑terminal kinase phosphorylation. Furthermore, PY‑PE treatment increased protein and mRNA expression levels of activator protein‑1, which regulates cell proliferation and survival, in the nuclear fraction. These results have significant implications for understanding the role of cell proliferation signaling pathways in intestinal epithelial cells.

  2. Decreased autophosphorylation of EGF receptor in insulin-deficient diabetic rats

    SciTech Connect

    Okamoto, M.; Kahn, C.R.; Maron, R.; White, M.F. )

    1988-04-01

    The authors have previously reported that despite an increase in receptor concentration, there is a decrease in autophosphorylation and tyrosine kinase activity of the insulin receptor in insulin-deficient diabetic rats. To determine if other tyrosine kinases might be altered, they have studied the epidermal growth factor (EGF) receptor kinase in wheat germ agglutinin-purified, Triton X-100-solubilized liver membranes from streptozotocin (STZ)-induced diabetic rats and the insulin-deficient BB rat. They find that autophosphorylation of EGF receptor is decreased in proportion to the severity of the diabetic state in STZ rats with a maximal decrease of 67%. A similar decrease in autophosphorylation was observed in diabetic BB rats that was partially normalized by insulin treatment. Separation of tryptic phosphopeptides by reverse-phase high-performance liquid chromatography revealed a decrease in labeling at all sites of autophosphorylation. A parallel decrease in EGF receptor phosphorylation was also found by immunoblotting with an antiphosphotyrosine antibody. EGF receptor concentration, determined by Scatchard analysis of {sup 125}I-labeled EGF binding, was decreased by 39% in the STZ rat and 27% in the diabetic BB rat. Thus autophosphorylation of EGF receptor, like that of the insulin receptor, is decreased in insulin-deficient rat liver. In the case of EGF receptor, this is due in part to a decrease in receptor number and in part to a decrease in the specific activity of the kinase.

  3. Berberine reduces insulin resistance through protein kinase C-dependent up-regulation of insulin receptor expression.

    PubMed

    Kong, Wei-Jia; Zhang, Hao; Song, Dan-Qing; Xue, Rong; Zhao, Wei; Wei, Jing; Wang, Yue-Ming; Shan, Ning; Zhou, Zhen-Xian; Yang, Peng; You, Xue-Fu; Li, Zhuo-Rong; Si, Shu-Yi; Zhao, Li-Xun; Pan, Huai-Ning; Jiang, Jian-Dong

    2009-01-01

    Natural product berberine (BBR) has been reported to have hypoglycemic and insulin-sensitizing activities; however, its mechanism remains unclear. This study was designed to investigate the molecular mechanism of BBR against insulin resistance. Here, we identify insulin receptor (InsR) as a target of BBR to increase insulin sensitivity. In cultured human liver cells, BBR increased InsR messenger RNA (mRNA) and protein expression in a dose- and time-dependent manner. Berberine increased InsR expression in the L6 rat skeletal muscle cells as well. Berberine-enhanced InsR expression improved cellular glucose consumption only in the presence of insulin. Silencing InsR gene with small interfering RNA or blocking the phosphoinositol-3-kinase diminished this effect. Berberine induced InsR gene expression through a protein kinase C (PKC)-dependent activation of its promoter. Inhibition of PKC abolished BBR-caused InsR promoter activation and InsR mRNA transcription. In animal models, treatment of type 2 diabetes mellitus rats with BBR lowered fasting blood glucose and fasting serum insulin, increased insulin sensitivity, and elevated InsR mRNA as well as PKC activity in the liver. In addition, BBR lowered blood glucose in KK-Ay type 2 but not in NOD/LtJ type 1 diabetes mellitus mice that were insulin deficient. Our results suggest that BBR is a unique natural medicine against insulin resistance in type 2 diabetes mellitus and metabolic syndrome.

  4. Peroxisome proliferator-activated receptoractivation enhances insulin-stimulated glucose disposal by reducing ped/pea-15 gene expression in skeletal muscle cells: evidence for involvement of activator protein-1.

    PubMed

    Ungaro, Paola; Mirra, Paola; Oriente, Francesco; Nigro, Cecilia; Ciccarelli, Marco; Vastolo, Viviana; Longo, Michele; Perruolo, Giuseppe; Spinelli, Rosa; Formisano, Pietro; Miele, Claudia; Beguinot, Francesco

    2012-12-14

    The gene network responsible for inflammation-induced insulin resistance remains enigmatic. In this study, we show that, in L6 cells, rosiglitazone- as well as pioglitazone-dependent activation of peroxisome proliferator-activated receptor-γ (PPARγ) represses transcription of the ped/pea-15 gene, whose increased activity impairs glucose tolerance in mice and humans. Rosiglitazone enhanced insulin-induced glucose uptake in L6 cells expressing the endogenous ped/pea-15 gene but not in cells expressing ped/pea-15 under the control of an exogenous promoter. The ability of PPARγ to affect ped/pea-15 expression was also lost in cells and in C57BL/6J transgenic mice expressing ped/pea-15 under the control of an exogenous promoter, suggesting that ped/pea-15 repression may contribute to rosiglitazone action on glucose disposal. Indeed, high fat diet mice showed insulin resistance and increased ped/pea-15 levels, although these effects were reduced by rosiglitazone treatment. Both supershift and ChIP assays revealed the presence of the AP-1 component c-JUN at the PED/PEA-15 promoter upon 12-O-tetradecanoylphorbol-13-acetate stimulation of the cells. In these experiments, rosiglitazone treatment reduced c-JUN presence at the PED/PEA-15 promoter. This effect was not associated with a decrease in c-JUN expression. In addition, c-jun silencing in L6 cells lowered ped/pea-15 expression and caused nonresponsiveness to rosiglitazone, although c-jun overexpression enhanced the binding to the ped/pea-15 promoter and blocked the rosiglitazone effect. These results indicate that PPARγ regulates ped/pea-15 transcription by inhibiting c-JUN binding at the ped/pea-15 promoter. Thus, ped/pea-15 is downstream of a major PPARγ-regulated inflammatory network. Repression of ped/pea-15 transcription might contribute to the PPARγ regulation of muscle sensitivity to insulin.

  5. 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.

  6. 4PS/insulin receptor substrate (IRS)-2 is the alternative substrate of the insulin receptor in IRS-1-deficient mice.

    PubMed

    Patti, M E; Sun, X J; Bruening, J C; Araki, E; Lipes, M A; White, M F; Kahn, C R

    1995-10-20

    Insulin receptor substrate-1 (IRS-1) is the major cytoplasmic substrate of the insulin and insulin-like growth factor (IGF)-1 receptors. Transgenic mice lacking IRS-1 are resistant to insulin and IGF-1, but exhibit significant residual insulin action which corresponds to the presence of an alternative high molecular weight substrate in liver and muscle. Recently, Sun et al. (Sun, X.-J., Wang, L.-M., Zhang, Y., Yenush, L. P., Myers, M. G., Jr., Glasheen, E., Lane, W.S., Pierce, J. H., and White, M. F. (1995) Nature 377, 173-177) purified and cloned 4PS, the major substrate of the IL-4 receptor-associated tyrosine kinase in myeloid cells, which has significant structural similarity to IRS-1. To determine if 4PS is the alternative substrate of the insulin receptor in IRS-1-deficient mice, we performed immunoprecipitation, immunoblotting, and phosphatidylinositol (PI) 3-kinase assays using specific antibodies to 4PS. Following insulin stimulation, 4PS is rapidly phosphorylated in liver and muscle, binds to the p85 subunit of PI 3-kinase, and activates the enzyme. Insulin stimulation also results in the association of 4PS with Grb 2 in both liver and muscle. In IRS-1-deficient mice, both the phosphorylation of 4PS and associated PI 3-kinase activity are enhanced, without an increase in protein expression. Immunodepletion of 4PS from liver and muscle homogenates removes most of the phosphotyrosine-associated PI 3-kinase activity in IRS-1-deficient mice. Thus, 4PS is the primary alternative substrate, i.e. IRS-2, which plays a major role in physiologic insulin signal transduction via both PI 3-kinase activation and Grb 2/Sos association. In IRS-1-deficient mice, 4PS/IRS-2 provides signal transduction to these two major pathways of insulin signaling.

  7. Concerted transcriptional activation of the low density lipoprotein receptor gene by insulin and luteinizing hormone in cultured porcine granulosa-luteal cells: possible convergence of protein kinase a, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways.

    PubMed

    Sekar, N; Veldhuis, J D

    2001-07-01

    Insulin and insulin-like growth factor I (IGF-I) can amplify gonadotropin-stimulated steroidogenesis by augmenting the expression of key sterol regulatory genes in ovarian cells, viz. low density lipoprotein (LDL) receptor, steroidogenic acute regulatory protein, and P450 cholesterol side-chain cleavage enzyme (CYP11A). The mechanisms underlying the foregoing bihormonal interactions are not known. Accordingly, in relation to the LDL receptor gene, the present study tests the hypothesis that insulin/IGF-I and LH can act via concerted transcriptional control of promoter expression. To this end, we transiently transfected primary monolayer cultures of porcine granulosa-luteal cells with a reporter vector containing the putative 5'-upstream full-length (pLDLR1076/luc) regulatory region (-1076 to +11 bp) of the homologous LDL receptor gene driving firefly luciferase in the presence or absence of insulin (or IGF-I) and/or LH (each 100 ng/ml). Combined exposure to LH and insulin (or IGF-I) stimulated LDL receptor transcriptional activity maximally at 4 h by 8- to 20-fold, as normalized by coexpression of Renilla luciferase. Further analysis of multiple 5'-nested deletional constructs of the LDL receptor gene promoter showed that deletion of -139 bp upstream of the transcriptional start site virtually abolished basal expression and promoter responsiveness to LH and insulin/IGF-I. In contrast, full basal activity and 60-80% of maximal monohormonal and bihormonal drive were retained by the -255 to +11 bp fragment. As LDL receptor gene expression in other tissues is negatively regulated by the abundance of intracellular free cholesterol, we assessed the impact of concomitant pretreatment of granulosa-luteal cells with an exogenous soluble sterol (25-hydroxycholesterol, 1 and 10 microM). Excess sterol markedly (50-70%) attenuated bihormonally and, in lesser measure, LH-stimulated and basal LDL receptor promoter expression, thus affirming a feedback-sensitive sterol

  8. Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by the insulin receptor is necessary for insulin metabolic signaling.

    PubMed

    Fiory, Francesca; Alberobello, Anna Teresa; Miele, Claudia; Oriente, Francesco; Esposito, Iolanda; Corbo, Vincenzo; Ruvo, Menotti; Tizzano, Barbara; Rasmussen, Thomas E; Gammeltoft, Steen; Formisano, Pietro; Beguinot, Francesco

    2005-12-01

    In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.

  9. Oestrogen requires the insulin-like growth factor-I receptor for stimulation of prolactin synthesis via mitogen-activated protein kinase.

    PubMed

    Arroba, A I; Frago, L M; Argente, J; Chowen, J A

    2005-02-01

    Sex steroids and growth factors interact at the intracellular level in a variety of tissues to control numerous physiological functions. Oestrogen is known to stimulate prolactin synthesis and secretion, but the effect of insulin-like growth factor (IGF)-I is less clear. We used GH3 cells, a somatolactotroph cell line, to study the interaction of 17beta-oestradiol (E(2)) and IGF-I on prolactin protein levels and the intracellular mechanisms involved. Cell cultures were treated with E(2) (10 nM) and/or IGF-I (10 ng/ml) for 8 h. The real-time reverse transcriptase-polymerase chain reaction, Western blot and enzyme-immunoassay were used to determine changes in prolactin mRNA and protein levels. At this time-point, there were no significant changes in cell number, prolactin mRNA expression, or the amount of secreted prolactin. However, E(2) increased intracellular prolactin concentrations. IGF-I alone had no effect, but blocked the stimulatory effect of E(2). MAPK (ERK1/2) activation, as determined by Western blot analysis, increased with both E(2) and IGF-I, but not with the combination of these factors. The MAPK inhibitor PD98059 blocked the ability of E(2) to increase intracellular prolactin concentrations. Similarly, the IGF-I receptor antagonist, JB1, blocked the effect of E(2) on prolactin synthesis and MAPK activation, as did the oestrogen receptor antagonist ICI182 780. These results suggest that, to stimulate prolactin synthesis, E(2) activates the MAPK cascade and that this requires the presence of both oestrogen and IGF-I receptors.

  10. Insulin phosphorylates calmodulin in preparations of solubilized rat hepatocyte insulin receptors

    SciTech Connect

    Sacks, D.B.; McDonald, J.M.

    1987-05-01

    It has previously been shown that insulin stimulates the phosphorylation of calmodulin in adipocyte insulin receptor preparations. Here they demonstrate that insulin also stimulates the phosphorylation of calmodulin in wheat germ lectin-enriched insulin receptor preparations obtained from rat hepatocytes. Standard phosphorylation assays were performed at 30C in the presence of 50mM Tris-HCl (pH 7.5), 0.1% (v/v) Triton X-100, 1mM EGTA, 50 M (el-TSP)ATP, 5mM MgCl2, 0.25 M polylysine, 1.2 M calmodulin and various CaS and insulin concentrations. The phosphorylation of calmodulin was determined by SDS-PAGE and autoradiography. Phosphorylation of calmodulin had an absolute requirement for insulin receptors, insulin and certain basic proteins. Phosphorylation was maximal above 13 nM insulin and at submicromolar CaS concentrations, whereas supramicromolar CaS concentrations were inhibitory. As was observed in the adipocyte insulin receptor system, calmodulin phosphorylation was dependent upon the presence of co-factors, such as polylysine, histone H/sub f/2b and protamine sulfate. The role played by these co-factors has not yet been established. These data suggest that both CaS and calmodulin participate in post receptor insulin events in hepatocytes.

  11. Insulin response sequence-dependent and -independent mechanisms mediate effects of insulin on glucocorticoid-stimulated insulin-like growth factor binding protein-1 promoter activity.

    PubMed

    Gan, Lixia; Pan, Haiyun; Unterman, Terry G

    2005-10-01

    IGF binding protein-1 (IGFBP-1) gene expression is stimulated by glucocorticoids and suppressed by insulin in the liver. Insulin response sequences (IRSs) mediate effects of insulin on basal promoter function, whereas glucocorticoids stimulate promoter activity through a contiguous glucocorticoid response element. Here we examined the role of IRS-dependent and -independent mechanisms in mediating insulin and glucocorticoids effects on IGFBP-1 promoter activity. Dexamethasone (Dex) stimulates IGFBP-1 promoter activity in HepG2 cells, and mutation of IRSs reduces this effect, indicating that IRS-associated factors enhance glucocorticoid effects on promoter function. Conversely, insulin inhibits basal promoter activity by 40% and Dex-stimulated promoter activity by 65%, indicating that glucocorticoids enhance the ability of insulin to suppress promoter activity. Mutation of IRSs completely disrupts the insulin effect on basal promoter activity and reduces but does not abolish inhibition of Dex-stimulated promoter activity, indicating that insulin suppresses glucocorticoid-stimulated promoter activity through both IRS-dependent and -independent mechanisms. IRS-independent effects of insulin are context dependent because insulin does not suppress glucocorticoid-stimulated activity of a promoter containing multiple glucocorticoid response elements. Cotransfection studies indicate that suppression of peroxisomal proliferator-activated receptor-gamma coactivator-1alpha, an insulin-regulated coactivator of the glucocorticoid receptor, is not required for this effect of insulin. Studies with pharmacological inhibitors indicate that both phosphatidylinositol-3' kinase and mitogen-activated kinase kinase pathways contribute to IRS-independent effects. These studies indicate that glucocorticoids and IRS-associated factors function together to mediate effects of insulin and glucocorticoids on promoter activity and that glucocorticoid treatment creates a complex environment in

  12. [A case of leprechaunism with extreme insulin resistance due to a primary defect in insulin receptors].

    PubMed

    Goji, K; Takata, Y; Kobayashi, M

    1985-09-20

    This report describes a 3-month-old female infant with the typical physical features of leprechaunism. The patient demonstrated glucose intolerance and marked hyperinsulinemia (4600 microU/ml). Since an intravenous insulin injection (actrapid insulin: 0.15 U/kg) caused no significant decrease in the blood glucose level, the presence of insulin resistance was suggested. Neither insulin antibodies nor insulin receptor antibodies were were found in the patient's plasma, and other circulating insulin antagonists such as glucagon, growth hormone, and cortisol were within normal limits. [125I]Insulin binding to the erythrocytes from the patient was as low as 1.02% (control infants: 4.89 +/- 1.08% [mean +/- SD]). [125I]Insulin binding to the cultured transformed lymphocytes from the patient was similarly reduced to 3.58% (control: 20.9 +/- 2.71% [mean +/- SD]). From these findings we concluded that the insulin resistance was due to a primary defect in insulin receptors. Interestingly, transient remissions of the patient's glucose intolerance and hyperinsulinemia were observed during a year of follow-up study. The insulin tolerance test which was performed at the remission period showed an improvement in insulin resistance. However, the insulin binding defect to erythrocytes remained unchanged even at the remission period. The exact cause of these remissions was not clear and remained to be elucidated.

  13. Myeloid cell-restricted insulin receptor deficiency protects against obesity-induced inflammation and systemic insulin resistance.

    PubMed

    Mauer, Jan; Chaurasia, Bhagirath; Plum, Leona; Quast, Thomas; Hampel, Brigitte; Blüher, Matthias; Kolanus, Waldemar; Kahn, C Ronald; Brüning, Jens C

    2010-05-06

    A major component of obesity-related insulin resistance is the establishment of a chronic inflammatory state with invasion of white adipose tissue by mononuclear cells. This results in the release of pro-inflammatory cytokines, which in turn leads to insulin resistance in target tissues such as skeletal muscle and liver. To determine the role of insulin action in macrophages and monocytes in obesity-associated insulin resistance, we conditionally inactivated the insulin receptor (IR) gene in myeloid lineage cells in mice (IR(Deltamyel)-mice). While these animals exhibit unaltered glucose metabolism on a normal diet, they are protected from the development of obesity-associated insulin resistance upon high fat feeding. Euglycemic, hyperinsulinemic clamp studies demonstrate that this results from decreased basal hepatic glucose production and from increased insulin-stimulated glucose disposal in skeletal muscle. Furthermore, IR(Deltamyel)-mice exhibit decreased concentrations of circulating tumor necrosis factor (TNF) alpha and thus reduced c-Jun N-terminal kinase (JNK) activity in skeletal muscle upon high fat feeding, reflecting a dramatic reduction of the chronic and systemic low-grade inflammatory state associated with obesity. This is paralleled by a reduced accumulation of macrophages in white adipose tissue due to a pronounced impairment of matrix metalloproteinase (MMP) 9 expression and activity in these cells. These data indicate that insulin action in myeloid cells plays an unexpected, critical role in the regulation of macrophage invasion into white adipose tissue and in the development of obesity-associated insulin resistance.

  14. 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

  15. Insulin receptor: Interaction with nonreceptor glycoprotein from liver cell membranes

    PubMed Central

    Maturo, Joseph M.; Hollenberg, Morley D.

    1978-01-01

    In crude receptor preparations (either particulate or soluble) of rat liver membranes, the insulin receptor exhibits complicated binding kinetics (two binding plateaus, half-saturated at approximately 60 pM and 700 pM insulin) and an apparent chromatographic heterogeneity, suggested by the presence of two detectable, soluble insulin-binding components with apparent Stokes radii of 72 Å and 38 Å. In contrast, the insulin receptor isolated by affinity chromatography exhibits a simple binding isotherm (half-maximal saturation of binding at 700 pM insulin) without evidence for negative cooperativity and behaves as a single component (apparent Stokes radius of 38 Å) upon chromatography on Sepharose 6B. The apparent discrepancies between the properties of the unpurified insulin receptor and the affinity-purified receptor can be attributed to the presence in crude preparations of a nonreceptor constituent(s) having properties consistent with those of a membrane glycoprotein. A glycoprotein fraction from such crude soluble membrane preparations, freed from insulin receptor and subsequently partially purified using concanavalin-A-agarose, when combined with affinity-purified insulin receptor, causes both a reappearance of the complicated binding kinetics and an increase in the receptor's apparent Stokes radius from 38 Å to 72 Å. Similar results are observed for a glycoprotein fraction obtained from rat adipocyte membranes but are not observed for an identical fraction isolated from human erythrocyte membranes. We conclude that the insulin receptor in rat liver membranes can interact with another nonreceptor membrane glycoprotein that may represent either a nonrecognition moiety of the receptor oligomer or an effector molecule to the biological action of insulin. PMID:277909

  16. 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

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

    PubMed

    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.

  18. 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

  19. 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.

  20. 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.

  1. Replacement of insulin receptor tyrosine residues 1162 and 1163 does not alter the mitogenic effect of the hormone

    SciTech Connect

    Debant, A.; Clauser, E.; Ponzio, G.; Filloux, C.; Auzan, C.; Contreres, J.O.; Rossi, B. )

    1988-11-01

    Chinese hamster ovary transfectants that express insulin receptors in which tyrosine residues 1162 and 1163 were replaced by phenylalanine exhibit a total inhibition of the insulin-mediated tyrosine kinase activity toward exogenous substrates; this latter activity is associated with total inhibition of the hypersensitivity reported for insulin in promoting 2-deoxyglucose uptake. The authors now present evidence that the twin tyrosines also control the insulin-mediated stimulation of glycogen synthesis. Surprisingly, this type of Chinese hamster ovary transfectant is as hypersensitive to insulin for its mitogenic effect as are Chinese hamster ovary cells expressing many intact insulin receptors. Such data suggest that (i) the insulin mitogenic effect routes through a different pathway than insulin uses to activate the transport and metabolism of glucose and (ii) the mitogenic effect of insulin is not controlled by the twin tyrosines. At the molecular level, the solubilized mutated receptor has not insulin-dependent tyrosine kinase activity, whereas this receptor displays measurable insulin-stimulated phosphorylation of its {beta} subunit in {sup 32}P-labeled cells. The authors therefore propose that the autocatalytic phosphorylating activity of the receptor reports a cryptic tyrosine kinase activity that cannot be visualized by the use of classical exogenous substrates.

  2. Insulin-induced myosin light-chain phosphorylation during receptor capping in IM-9 human B-lymphoblasts.

    PubMed Central

    Majercik, M H; Bourguignon, L Y

    1988-01-01

    We have examined further the interaction between insulin surface receptors and the cytoskeleton of IM-9 human lymphoblasts. Using immunocytochemical techniques, we determined that actin, myosin, calmodulin and myosin light-chain kinase (MLCK) are all accumulated directly underneath insulin-receptor caps. In addition, we have now established that the concentration of intracellular Ca2+ (as measured by fura-2 fluorescence) increases just before insulin-induced receptor capping. Most importantly, we found that the binding of insulin to its receptor induces phosphorylation of myosin light chain in vivo. Furthermore, a number of drugs known to abolish the activation properties of calmodulin, such as trifluoperazine (TFP) or W-7, strongly inhibit insulin-receptor capping and myosin light-chain phosphorylation. These data imply that an actomyosin cytoskeletal contraction, regulated by Ca2+/calmodulin and MLCK, is involved in insulin-receptor capping. Biochemical analysis in vitro has revealed that IM-9 insulin receptors are physically associated with actin and myosin; and most interestingly, the binding of insulin-receptor/cytoskeletal complex significantly enhances the phosphorylation of the 20 kDa myosin light chain. This insulin-induced phosphorylation is inhibited by calmodulin antagonists (e.g. TFP and W-7), suggesting that the phosphorylation is catalysed by MLCK. Together, these results strongly suggest that MLCK-mediated myosin light-chain phosphorylation plays an important role in regulating the membrane-associated actomyosin contraction required for the collection of insulin receptors into caps. Images Fig. 2. Fig. 4. PMID:3048249

  3. Short-term in vivo inhibition of insulin receptor substrate-1 expression leads to insulin resistance, hyperinsulinemia, and increased adiposity.

    PubMed

    Araújo, Eliana P; De Souza, Cláudio T; Gasparetti, Alessandra L; Ueno, Mirian; Boschero, Antonio C; Saad, Mário J A; Velloso, Lício A

    2005-03-01

    Insulin receptor substrate-1 (IRS-1) has an important role as an early intermediary between the insulin and IGF receptors and downstream molecules that participate in insulin and IGF-I signal transduction. Here we employed an antisense oligonucleotide (IRS-1AS) to inhibit whole-body expression of IRS-1 in vivo and evaluate the consequences of short-term inhibition of IRS-1 in Wistar rats. Four days of treatment with IRS-1AS reduced the expression of IRS-1 by 80, 75, and 65% (P < 0.05) in liver, skeletal muscle, and adipose tissue, respectively. This was accompanied by a 40% (P < 0.05) reduction in the constant of glucose decay during an insulin tolerance test, a 78% (P < 0.05) reduction in glucose consumption during a hyperinsulinemic-euglycemic clamp, and a 90% (P < 0.05) increase in basal plasma insulin level. The metabolic effects produced by IRS-1AS were accompanied by a significant reduction in insulin-induced [Ser (473)] Akt phosphorylation in liver (85%, P < 0.05), skeletal muscle (40%, P < 0.05), and adipose tissue (85%, P < 0.05) and a significant reduction in insulin-induced tyrosine phosphorylation of ERK in liver (20%, P < 0.05) and skeletal muscle (30%, P < 0.05). However, insulin-induced tyrosine phosphorylation of ERK was significantly increased (60%, P < 0.05) in adipose tissue of IRS-1AS-treated rats. In rats treated with IRS-1AS for 8 d, a 100% increase (P < 0.05) in relative epididymal fat weight and a 120% (P < 0.05) increase in nuclear expression of peroxisome proliferator-activated receptor-gamma were observed. Thus, acute inhibition of IRS-1 expression in rats leads to insulin resistance accompanied by activation of a growth-related pathway exclusively in white adipose tissue.

  4. 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.

  5. 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

  6. Restoration of insulin secretion in pancreatic islets of protein-deficient rats by reduced expression of insulin receptor substrate (IRS)-1 and IRS-2.

    PubMed

    Araujo, E P; Amaral, M E C; Filiputti, E; De Souza, C T; Laurito, T L; Augusto, V D; Saad, M J A; Boschero, A C; Velloso, L A; Carneiro, E M

    2004-04-01

    Autocrine and paracrine insulin signaling may participate in the fine control of insulin secretion. In the present study, tissue distribution and protein amounts of the insulin receptor and its major substrates, insulin receptor substrate (IRS)-1 and IRS-2, were evaluated in a model of impaired glucose-induced insulin secretion, the protein-deficient rat. Immunoblot and RT-PCR studies showed that the insulin receptor and IRS-2 expression are increased, whilst IRS-1 protein and mRNA contents are decreased in pancreatic islets of protein-deficient rats. Immunohistochemical studies revealed that the insulin receptor and IRS-1 and -2 are present in the great majority of islet cells; however, the greatest staining was localized at the periphery, suggesting a co-localization with non-insulin-secreting cells. Exogenous insulin stimulation of isolated islets promoted higher insulin receptor and IRS-1 and -2 tyrosine phosphorylation in islets from protein-deficient rats, as compared with controls. Moreover, insulin-induced IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activity are increased in islets of protein-deficient rats. The reduction of IRS-1 and IRS-2 protein expression in islets isolated from protein-deficient rats by the use of antisense IRS-1 or IRS-2 phosphorthioate-modified oligonucleotides partially restored glucose-induced insulin secretion. Thus, the impairment of insulin cell signaling through members of the IRS family of proteins in isolated rat pancreatic islets improves glucose-induced insulin secretion. The present data reinforced the role of insulin paracrine and autocrine signaling in the control of its own secretion.

  7. JAK1 kinase forms complexes with interleukin-4 receptor and 4PS/insulin receptor substrate-1-like protein and is activated by interleukin-4 and interleukin-9 in T lymphocytes.

    PubMed

    Yin, T; Tsang, M L; Yang, Y C

    1994-10-28

    Interleukin (IL)-4 and IL-9 regulate the proliferation of T lymphocytes through interactions with their receptors. Previous studies have shown that unknown tyrosine kinases are involved in the proliferative signaling triggered by IL-4 and IL-9. Here we show that IL-4 and IL-9 induce overlapping (170, 130, and 125 kilodalton (kDa)) and distinct (45 and 88/90 kDa, respectively) protein tyrosine phosphorylation in T lymphocytes. We further identify the 170-kDa tyrosine-phosphorylated protein as 4PS/insulin receptor substrate-1-like (IRS-1L) protein and 130-kDa protein as JAK1 kinase. Furthermore, we demonstrate for the first time that JAK1 forms complexes with the IL-4 receptor and 4PS/IRS-1L protein following ligand-receptor interaction. In addition, we demonstrate that IL-9, but not IL-4, induced tyrosine phosphorylation of Stat 91 transcriptional factor. The overlapping and distinct protein tyrosine phosphorylation and activation of the same JAK1 kinase in T lymphocytes strongly suggests that IL-4 and IL-9 share the common signal transduction pathways and that the specificity for each cytokine could be achieved through the unique tyrosine-phosphorylated proteins triggered by individual cytokines.

  8. 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

  9. 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

    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.

  10. 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.

  11. 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

  12. 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...

  13. Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells

    SciTech Connect

    Saperstein, R.; Vicario, P.P.; Strout, H.V.; Brady, E.; Slater, E.E.; Greenlee, W.J.; Onedyka, D.L.; Patchett, A.A.; Hangauer, D.G. )

    1989-06-27

    An inhibitor of the insulin receptor tyrosine kinase (IRTK), (hydroxy-2-napthalenylmethyl)phosphonic acid, was designed and synthesized and was shown to be an inhibitor of the biological effects of insulin in vitro. With a wheat germ purified human placental insulin receptor preparation, this compound inhibited the insulin-stimulated autophosphorylation of the 95-kDa {beta}-subunit of the insulin receptor. The ability of the kinase to phosphorylate an exogenous peptide substrate, angiotensin II, was also inhibited. Half-maximal inhibition of basal and insulin-stimulated human placental IRTK activity was found at concentrations of 150 and 100 {mu}M, respectively, with 2 mM angiotensin II as the peptide substrate. The inhibitor was found to be specific for tyrosine kinases over serine kinases and noncompetitive with ATP. The inhibitor was converted into various (acyloxy)methyl prodrugs in order to achieve permeability through cell membranes. These prodrugs inhibited insulin-stimulated autophosphorylation of the insulin receptor 95-kDa {beta}-subunit in intact CHO cells transfected with human insulin receptor. Inhibition of insulin-stimulated glucose oxidation in isolated rat adipocytes and 2-deoxyglucose uptake into CHO cells was observed with these prodrugs. The data provide additional evidence for the involvement of the insulin receptor tyrosine kinase in the regulation of glucose uptake and metabolism. These results and additional data reported herein suggest that this class of prodrugs and inhibitors will be useful for modulating the activity of a variety of tyrosine kinases.

  14. The insulin receptor concept and its relation to the treatment of diabetes.

    PubMed

    Ward, G M

    1987-02-01

    The initial step in insulin action is binding to specific receptors. Two covalent receptor modifications possibly involved in producing pharmacodynamic effects as a result of insulin receptor binding are autophosphorylation and disulphide insulin binding. Insulin receptor numbers are 'down regulated' by insulin, but this effect may be minimised by pulsatile insulin secretion. Insulin receptor affinity is modulated rapidly by fasting, exercise and dietary composition. In non-insulin-dependent diabetes coupling of receptor binding to bioeffects is impaired. Binding is also reduced in those subjects with hyperinsulinaemia and non-insulin-dependent diabetes. Insulin-dependent diabetics have reduced insulin sensitivity, which is only partially reversed by conventional insulin therapy. 'Post-binding defects' in some diabetics could be related to defective covalent receptor modifications resulting from genetic receptor defects. High carbohydrate diets improve diabetes control through effects on the binding and coupling defects. In addition to stimulating insulin secretion, oral hypoglycaemics stimulate post-binding insulin action in vivo and in vitro. Insulin therapy in diabetes also tends to reverse post-binding defects. Pulsatile insulin delivery is more effective in lowering blood sugar than continuous administration, and produces less 'down regulation' of receptors. Combined insulin and sulphonylurea drugs reduce insulin requirements only in insulin-dependent diabetics with some endogenous insulin secretion, whereas metformin reduces insulin requirement in C-peptide negative insulin-dependent diabetes mellitus.

  15. 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

  16. Defect in cooperativity in insulin receptors from a patient with a congenital form of extreme insulin resistance.

    PubMed Central

    Taylor, S I; Leventhal, S

    1983-01-01

    Previously, we have described a novel qualitative defect in insulin receptors from a patient with a genetic form of extreme insulin resistance (leprechaunism). Receptors from this insulin-resistant child are characterized by two abnormalities: (a) an abnormally high binding affinity for insulin, and (b) a markedly reduced sensitivity of 125I-insulin binding to alterations in pH and temperature. In this paper, we have investigated the kinetic mechanism of this abnormality in steady-state binding. The increased binding affinity for 125I-insulin results from a decrease in the dissociation rate of the hormone-receptor complex. In addition, the cooperative interactions among insulin binding sites are defective with insulin receptors from this child with leprechaunism. With insulin receptors on cultured lymphocytes from normal subjects, both negative and positive cooperativity may be observed. Porcine insulin accelerates the dissociation of the hormone-receptor complex (negative cooperativity). In contrast, certain insulin analogs such as desoctapeptide-insulin and desalanine-desasparagine-insulin retard the dissociation of the hormone-receptor complex (positive cooperativity). With insulin receptors from the leprechaun child, positive cooperativity could not be demonstrated, although negative cooperativity appeared to be normal. It seems likely that the same genetic defect may be responsible for the abnormalities in both insulin sensitivity and positive cooperativity. PMID:6345588

  17. Impaired activation of phosphoinositide 3-kinase by insulin in fibroblasts from patients with severe insulin resistance and pseudoacromegaly. A disorder characterized by selective postreceptor insulin resistance.

    PubMed Central

    Dib, K; Whitehead, J P; Humphreys, P J; Soos, M A; Baynes, K C; Kumar, S; Harvey, T; O'Rahilly, S

    1998-01-01

    Some patients with severe insulin resistance develop pathological tissue growth reminiscent of acromegaly. Previous studies of such patients have suggested the presence of a selective postreceptor defect of insulin signaling, resulting in the impairment of metabolic but preservation of mitogenic signaling. As the activation of phosphoinositide 3-kinase (PI 3-kinase) is considered essential for insulin's metabolic signaling, we have examined insulin-stimulated PI 3-kinase activity in anti-insulin receptor substrate (IRS)-1 immunoprecipitates from cultured dermal fibroblasts obtained from pseudoacromegalic (PA) patients and controls. At a concentration of insulin (1 nM) similar to that seen in vivo in PA patients, the activation of IRS-1-associated PI 3-kinase was reduced markedly in fibroblasts from the PA patients (32+/-7% of the activity of normal controls, P < 0.01). Genetic and biochemical studies indicated that this impairment was not secondary to a defect in the structure, expression, or activation of the insulin receptor, IRS-1, or p85alpha. Insulin stimulation of mitogenesis in PA fibroblasts, as determined by thymidine incorporation, was indistinguishable from controls, as was mitogen-activated protein kinase phosphorylation, confirming the integrity of insulin's mitogenic signaling pathways in this condition. These findings support the existence of an intrinsic defect of postreceptor insulin signaling in the PA subtype of insulin resistance, which involves impairment of the activation of PI 3-kinase. The PA tissue growth seen in such patients is likely to result from severe in vivo hyperinsulinemia activating intact mitogenic signaling pathways emanating from the insulin receptor. PMID:9486982

  18. 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.

  19. New concept for long-acting insulin: spontaneous conversion of an inactive modified insulin to the active hormone in circulation: 9-fluorenylmethoxycarbonyl derivative of insulin.

    PubMed

    Gershonov, E; Shechter, Y; Fridkin, M

    1999-07-01

    Insulin is a short-lived species in the circulatory system. After binding to its receptor sites and transmission of its biological signals, bound insulin undergoes receptor-mediated endocytosis and consequent degradation. An inactive insulin derivative that is not recognized by the receptor has a longer circulation life, but obviously is biologically impotent. (Fmoc)2 insulin is an insulin derivative purified through high-performance liquid chromatography in which two 9-fluorenylmethoxycarbonyl (Fmoc) moieties are covalently linked to the (alpha-amino group of phenylalanine B1 and the epsilon-amino group of lysine B29. It has 1-2% of the biological potency and receptor binding capacity of the native hormone. After incubation, (Fmoc)2 insulin undergoes a time-dependent spontaneous conversion to fully active insulin in aqueous solution at 37 degrees C and a pH range of 7-8.5. At pH 7.4, the conversion proceeds slowly (t1/2 = 12 +/- 1 days) and biological activity is generated gradually. A single subcutaneous administration of (Fmoc)2 insulin to streptozocin-treated diabetic rats normalized their blood glucose levels and maintained the animals in an anabolic state over 2-3 days. A broad shallow peak of immunoreactive insulin was found to persist in circulation over this period. To confirm further that the long-acting effect of (Fmoc)2 insulin proceeds via slow release in the blood circulation itself, we administered native insulin, NPH insulin, or the (Fmoc)2 derivative intraperitoneally. The rats recovered from hypoglycemia at t1/2 = 8.0 +/- 0.3 and 10 +/- 0.4 h after administration of native and NPH insulin, respectively. In contrast, (Fmoc)2 insulin was active for a significantly longer time, with an extended onset of t1/2 = 26 +/- 1h, and a glucose-lowering effect even 40 h after administration. (Fmoc)2 insulin was also found to be more resistant to proteolysis. Finally, we found that (Fmoc)2 insulin does not induce antigenic effects. In summary, we present here a

  20. β-Adrenergic Receptor and Insulin Resistance in the Heart.

    PubMed

    Mangmool, Supachoke; Denkaew, Tananat; Parichatikanond, Warisara; Kurose, Hitoshi

    2017-01-01

    Insulin resistance is characterized by the reduced ability of insulin to stimulate tissue uptake and disposal of glucose including cardiac muscle. These conditions accelerate the progression of heart failure and increase cardiovascular morbidity and mortality in patients with cardiovascular diseases. It is noteworthy that some conditions of insulin resistance are characterized by up-regulation of the sympathetic nervous system, resulting in enhanced stimulation of β-adrenergic receptor (βAR). Overstimulation of βARs leads to the development of heart failure and is associated with the pathogenesis of insulin resistance in the heart. However, pathological consequences of the cross-talk between the βAR and the insulin sensitivity and the mechanism by which βAR overstimulation promotes insulin resistance remain unclear. This review article examines the hypothesis that βARs overstimulation leads to induction of insulin resistance in the heart.

  1. β-Adrenergic Receptor and Insulin Resistance in the Heart

    PubMed Central

    Mangmool, Supachoke; Denkaew, Tananat; Parichatikanond, Warisara; Kurose, Hitoshi

    2017-01-01

    Insulin resistance is characterized by the reduced ability of insulin to stimulate tissue uptake and disposal of glucose including cardiac muscle. These conditions accelerate the progression of heart failure and increase cardiovascular morbidity and mortality in patients with cardiovascular diseases. It is noteworthy that some conditions of insulin resistance are characterized by up-regulation of the sympathetic nervous system, resulting in enhanced stimulation of β-adrenergic receptor (βAR). Over-stimulation of βARs leads to the development of heart failure and is associated with the pathogenesis of insulin resistance in the heart. However, pathological consequences of the cross-talk between the βAR and the insulin sensitivity and the mechanism by which βAR over-stimulation promotes insulin resistance remain unclear. This review article examines the hypothesis that βARs over-stimulation leads to induction of insulin resistance in the heart. PMID:28035081

  2. 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.

  3. Intracellular presence of insulin and its phosphorylated receptor in non-small cell lung cancer.

    PubMed

    Mattarocci, Stefano; Abbruzzese, Claudia; Mileo, Anna M; Visca, Paolo; Antoniani, Barbara; Alessandrini, Gabriele; Facciolo, Francesco; Felsani, Armando; Radulescu, Razvan T; Paggi, Marco G

    2009-12-01

    Insulin has been known for a long time to influence the growth and differentiation of normal and transformed cells. In order to delineate the role of insulin specifically in non-small cell lung cancer (NSCLC), we have now searched by immunohistochemistry (IHC) for the presence of insulin in NSCLC samples. Among the 112 samples we studied, 30 were found to contain insulin, which was detected in the form of intracytoplasmic granula. Moreover, its expression significantly correlated with (a) the morphological/histopathological subtype of NSCLC, being more frequent in adenocarcinomas; (b) the grade of tumor differentiation, displaying an increase in low-grade carcinomas; (c) tumor size, occurring predominantly in smaller tumors; (d) the presence of phosphorylated, activated insulin receptor; (e) the median patient age, being present in relatively younger individuals. Furthermore and interestingly, surrounding atypical adenomatous hyperplastic areas and normal alveolar pneumocytes scored insulin-positive in some of the insulin-negative tumors. In addition, PCR exploration for insulin transcripts in some samples positive for immunoreactive insulin was negative, indicating a possibly exogenous origin for the intracellular insulin in our NSCLC cohort. Taken together, our data suggest that an intracellular insulin activity is important for the progression of low-grade human lung adenocarcinomas.

  4. Knockout of Vasohibin-1 Gene in Mice Results in Healthy Longevity with Reduced Expression of Insulin Receptor, Insulin Receptor Substrate 1, and Insulin Receptor Substrate 2 in Their White Adipose Tissue

    PubMed Central

    Takeda, Eichi; Suzuki, Yasuhiro; Yamada, Tetsuya; Katagiri, Hideki

    2017-01-01

    Vasohibin-1 (Vash1), originally isolated as an endothelium-derived angiogenesis inhibitor, has a characteristic of promoting stress tolerance in endothelial cells (ECs). We therefore speculated that the lack of the vash1 gene would result in a short lifespan. However, to our surprise, vash1−/− mice lived significantly longer with a milder senescence phenotype than wild-type (WT) mice. We sought the cause of this healthy longevity and found that vash1−/− mice exhibited mild insulin resistance along with reduced expression of the insulin receptor (insr), insulin receptor substrate 1 (irs-1), and insulin receptor substrate 2 (irs-2) in their white adipose tissue (WAT) but not in their liver or skeletal muscle. The expression of vash1 dominated in the WAT among those 3 organs. Importantly, vash1−/− mice did not develop diabetes even when fed a high-fat diet. These results indicate that the expression of vash1 was required for the normal insulin sensitivity of the WAT and that the target molecules for this activity were insr, irs1, and irs2. The lack of vash1 caused mild insulin resistance without the outbreak of overt diabetes and might contribute to healthy longevity. PMID:28367331

  5. A 3-basepair in-frame deletion ({Delta}Leu{sup 999}) in exon 17 of the insulin receptor gene in a family with insulin resistance

    SciTech Connect

    Awata, T.; Matsumoto, C.; Iwamoto, Y.

    1994-12-01

    We studied a woman with acanthosis nigricans and insulin resistance. The patient`s Epstein-Barr virus-transformed lymphocytes revealed slightly decreased insulin binding and markedly decreased insulin-stimulated autophosphorylation of the insulin receptor. The nucleotide sequence analysis of the patient`s genomic DNA revealed a 3-basepair in-frame deletion of one allele, resulting in the loss of leucine at position 999 of the insulin receptor ({Delta}Leu{sup 999}). The messenger ribonucleic acid transcripts from the mutant allele in the patient`s lymphocytes were not decreased. Insulin-stimulated autophosphorylation of the insulin receptor from cells expressing {Delta}Leu{sup 999} mutant insulin receptor complementary DNA was markedly decreased. The proband, her mother, elder brother, and younger brother, who were heterozygous for this mutation, showed moderate or marked hyperinsulinemia during oral glucose tolerance tests. Although fasting glucose levels were normal and fasting insulin values were preserved in all subjects with the mutation for the 8-yr period of observation, a tendancy of progressive increase in postload glucose levels were observed. These results suggest that the {Delta}Leu{sup 999} mutation, which reduces tyrosine kinase activity, was responsible for insulin resistance and contributed to postload hyperglycemia. 27 refs., 3 figs., 1 tab.

  6. Comparison the relationship between the levels of insulin resistance, hs-CRP, percentage of body fat and serum osteoprotegerin/receptor activator of nuclear factor κβ ligand in prediabetic patients.

    PubMed

    Bilgir, Oktay; Yavuz, Mehmet; Bilgir, Ferda; Akan, Ozden Y; Bayindir, Aslı G; Calan, Mehmet; Bozkaya, Giray; Yuksel, Arif

    2017-01-31

    BACKGROUND Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPN) are soluble members of the tumor necrosis factor superfamily. Growing evidence suggest that there is link between inflammation, insulin resistance and OPG, sRANKL. We aimed to ascertain whether OPG and sRANKL levels are altered in prediabetic subjects and there is association between OPG, sRANKL and metabolic parameters.

  7. Peroxisome proliferator-activated receptor gene expression in human tissues. Effects of obesity, weight loss, and regulation by insulin and glucocorticoids.

    PubMed Central

    Vidal-Puig, A J; Considine, R V; Jimenez-Liñan, M; Werman, A; Pories, W J; Caro, J F; Flier, J S

    1997-01-01

    The peroxisome proliferator activated receptor (PPAR gamma) plays a key role in adipogenesis and adipocyte gene expression and is the receptor for the thiazolidinedione class of insulin-sensitizing drugs. The tissue expression and potential for regulation of human PPAR gamma gene expression in vivo are unknown. We have cloned a partial human PPAR gamma cDNA, and established an RNase protection assay that permits simultaneous measurements of both PPAR gamma1 and PPAR gamma2 splice variants. Both gamma1 and gamma2 mRNAs were abundantly expressed in adipose tissue. PPAR gamma1 was detected at lower levels in liver and heart, whereas both gamma1 and gamma2 mRNAs were expressed at low levels in skeletal muscle. To examine the hypothesis that obesity is associated with abnormal adipose tissue expression of PPAR gamma, we quantitated PPARgamma mRNA splice variants in subcutaneous adipose tissue of 14 lean and 24 obese subjects. Adipose expression of PPARgamma 2 mRNA was increased in human obesity (14.25 attomol PPAR gamma2/18S in obese females vs 9.9 in lean, P = 0.003). This increase was observed in both male and females. In contrast, no differences were observed in PPAR gamma1/18S mRNA expression. There was a strong positive correlation (r = 0.70, P < 0.001) between the ratio of PPAR gamma2/gamma1 and the body mass index of these patients. We also observed sexually dimorphic expression with increased expression of both PPAR gamma1 and PPAR gamma2 mRNAs in the subcutaneous adipose tissue of women compared with men. To determine the effect of weight loss on PPAR gamma mRNA expression, seven additional obese subjects were fed a low calorie diet (800 Kcal) until 10% weight loss was achieved. Mean expression of adipose PPAR gamma2 mRNA fell 25% (P = 0.0250 after a 10% reduction in body weight), but then increased to pretreatment levels after 4 wk of weight maintenance. Nutritional regulation of PPAR gamma1 was not seen. In vitro experiments revealed a synergistic effect of

  8. Mitochondrial H2O2 as an enable signal for triggering autophosphorylation of insulin receptor in neurons

    PubMed Central

    2013-01-01

    Background Insulin receptors are widely distributed in the brain, where they play roles in synaptic function, memory formation, and neuroprotection. Autophosphorylation of the receptor in response to insulin stimulation is a critical step in receptor activation. In neurons, insulin stimulation leads to a rise in mitochondrial H2O2 production, which plays a role in receptor autophosphorylation. However, the kinetic characteristics of the H2O2 signal and its functional relationships with the insulin receptor during the autophosphorylation process in neurons remain unexplored to date. Results Experiments were carried out in culture of rat cerebellar granule neurons. Kinetic study showed that the insulin-induced H2O2 signal precedes receptor autophosphorylation and represents a single spike with a peak at 5–10 s and duration of less than 30 s. Mitochondrial complexes II and, to a lesser extent, I are involved in generation of the H2O2 signal. The mechanism by which insulin triggers the H2O2 signal involves modulation of succinate dehydrogenase activity. Insulin dose–response for receptor autophosphorylation is well described by hyperbolic function (Hill coefficient, nH, of 1.1±0.1; R2=0.99). N-acetylcysteine (NAC), a scavenger of H2O2, dose-dependently inhibited receptor autophosphorylation. The observed dose response is highly sigmoidal (Hill coefficient, nH, of 8.0±2.3; R2=0.97), signifying that insulin receptor autophosphorylation is highly ultrasensitive to the H2O2 signal. These results suggest that autophosphorylation occurred as a gradual response to increasing insulin concentrations, only if the H2O2 signal exceeded a certain threshold. Both insulin-stimulated receptor autophosphorylation and H2O2 generation were inhibited by pertussis toxin, suggesting that a pertussis toxin-sensitive G protein may link the insulin receptor to the H2O2-generating system in neurons during the autophosphorylation process. Conclusions In this study, we demonstrated for

  9. Insulin-like Receptor and Insulin-like Peptide Are Localized at Neuromuscular Junctions in Drosophila

    PubMed Central

    Gorczyca, Michael; Augart, Carolyn; Budnik, Vivian

    2015-01-01

    Insulin and insulin-like growth factor (IGF) receptors are members of the tyrosine kinase family of receptors, and are thought to play an important role in the development and differentiation of neurons. Here we report the presence of an insulin-like peptide and an insulin receptor (dlnsR) at the body wall neuromuscular junction of developing Drosophila larvae. dlnsR-like immunoreactivity was found in all body wall muscles at the motor nerve branching regions, where it surrounded synaptic boutons. The identity of this immunoreactivity as a dlnsR was confirmed by two additional schemes, in vivo binding of labeled insulin and immunolocalization of phosphotyrosine. Both methods produced staining patterns markedly similar to dlnsR-like immunoreactivity. The presence of a dlnsR in whole larvae was also shown by receptor binding assays. This receptor was more specific for insulin (>25-fold) than for IGF II, and did not appear to bind IGF I. Among the 30 muscle fibers per hemisegment, insulin-like immunoreactivity was found only on one fiber, and was localized to a subset of morphologically distinct synaptic boutons. Staining in the CNS was limited to several cell bodies in the brain lobes and in a segmental pattern throughout most of the abdominal ganglia, as well as in varicosities along the neuropil areas of the ventral ganglion and brain lobes. Insulin-like peptide and dlnsR were first detected by early larval development, well after neuromuscular transmission begins. The developmental significance of an insulin-like peptide and its receptor at the neuromuscular junction is discussed. PMID:8366341

  10. 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

  11. Evidence that insulin and guanosine triphosphate regulate dephosphorylation of the beta-subunit of the insulin receptor in sarcolemma membranes isolated from skeletal muscle.

    PubMed Central

    Horn, R S; Lystad, E; Adler, A; Walaas, O

    1986-01-01

    When sarcolemma membranes isolated from rat skeletal muscle were incubated with [gamma-32P]ATP, a membrane protein of apparent Mr 95,000 was rapidly phosphorylated, with the 32P content reaching a maximum within 2 s. On the basis of immunoprecipitation with anti-insulin-receptor antiserum, phosphoamino acid analysis and Mr, this protein probably represents the beta-subunit of the insulin receptor. Similarly, on incubation of the membrane with adenosine 5'-[gamma-[35S]thio] triphosphate the 95 kDa protein was thiophosphorylated, indicating thiophosphorylation of the beta-subunit of the insulin receptor on the basis of immunoprecipitation studies. The effect of insulin on the phosphorylation of this protein in the membrane was studied. Insulin induced a 20% decrease in the 32P labelling of the protein when the membranes were phosphorylated for 10 s. This insulin effect was dose-dependent, with half-maximal effect obtained at 2-3 nM-insulin. Addition of GTP, but not GDP or guanosine 5'-[beta, gamma-imido]triphosphate, enhanced the effect to 35% inhibition, with half-maximal effect of GTP obtained at 0.5 microM. GTP had no effect on the phosphorylation of the protein in the absence of insulin. Analysis of this insulin effect showed that insulin increased the rate of dephosphorylation of the 95 kDa protein in the membrane. In contrast, insulin had no effect on thiophosphorylation of the 95 kDa membrane protein after incubation with adenosine 5'-[gamma-[35S]thio]triphosphate. Since thiophosphorylated proteins are less sensitive to phosphatase action, these investigations suggest that insulin stimulated a protein phosphatase activity in a GTP-dependent manner. The possibility that GTP-regulatory proteins are involved in the action of insulin on the phosphorylation of the insulin receptor and other membrane proteins is discussed. Images Fig. 1. Fig. 3. PMID:3521589

  12. Importance of the type I insulin-like growth factor receptor in HER2, FGFR2 and MET-unamplified gastric cancer with and without Ras pathway activation

    PubMed Central

    Saisana, Marina; Griffin, S. Michael; May, Felicity E.B.

    2016-01-01

    Amplification of seven oncogenes: HER2, EGFR, FGFR1, FGFR2, MET, KRAS and IGF1R has been identified in gastric cancer. The first five are targeted therapeutically in patients with HER2-positivity, FGFR2- or MET-amplification but the majority of patients are triple-negative and require alternative strategies. Our aim was to evaluate the importance of the IGF1R tyrosine kinase in triple-negative gastric cancer with and without oncogenic KRAS, BRAF or PI3K3CA mutations. Cell lines and metastatic tumor cells isolated from patients expressed IGF1R, and insulin-like growth factor-1 (IGF-1) activated the PI3-kinase/Akt and Ras/Raf/MAP-kinase pathways. IGF-1 protected triple-negative cells from caspase-dependent apoptosis and anoikis. Protection was mediated via the PI3-kinase/Akt pathway. Remarkably, IGF-1-dependent cell survival was greater in patient samples. IGF-1 stimulated triple-negative gastric cancer cell growth was prevented by IGF1R knockdown and Ras/Raf/MAP-kinase pathway inhibition. The importance of the receptor in cell line and metastatic tumor cell growth in serum-containing medium was demonstrated by knockdown and pharmacological inhibition with figitumumab. The proportions of cells in S-phase and mitotic-phase, and Ras/Raf/MAP-kinase pathway activity, were reduced concomitantly. KRAS-addicted and BRAF-impaired gastric cancer cells were particularly susceptible. In conclusion, IGF1R and the IGF signal transduction pathway merit consideration as potential therapeutic targets in patients with triple-negative gastric cancer. PMID:27437872

  13. Hepatic structural enhancement and insulin resistance amelioration due to AT1 receptor blockade

    PubMed Central

    Souza-Mello, Vanessa

    2017-01-01

    Over the last decade, the role of renin-angiotensin system (RAS) on the development of obesity and its comorbidities has been extensively addressed. Both circulating and local RAS components are up-regulated in obesity and involved in non-alcoholic fatty liver disease onset. Pharmacological manipulations of RAS are viable strategies to tackle metabolic impairments caused by the excessive body fat mass. Renin inhibitors rescue insulin resistance, but do not have marked effects on hepatic steatosis. However, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers (ARB) yield beneficial hepatic remodeling. ARBs elicit body mass loss and normalize insulin levels, tackling insulin resistance. Also, this drug class increases adiponectin levels, besides countering interleukin-6, tumoral necrosis factor-alpha, and transforming growth factor-beta 1. The latter is essential to prevent from liver fibrosis. When conjugated with peroxisome proliferator-activated receptor (PPAR)-alpha activation, ARB fully rescues fatty liver. These effects might be orchestrated by an indirect up-regulation of MAS receptor due to angiotensin II receptor type 1 (AT1R) blockade. These associations of ARB with PPAR activation and ACE2-angiotensin (ANG) (1-7)-MAS receptor axis deserve a better understanding. This editorial provides a brief overview of the current knowledge regarding AT1R blockade effects on sensitivity to insulin and hepatic structural alterations as well as the intersections of AT1R blockade with peroxisome proliferator-activated receptor activation and ACE2-ANG (1-7) - MAS receptor axis. PMID:28144388

  14. Hepatic structural enhancement and insulin resistance amelioration due to AT1 receptor blockade.

    PubMed

    Souza-Mello, Vanessa

    2017-01-18

    Over the last decade, the role of renin-angiotensin system (RAS) on the development of obesity and its comorbidities has been extensively addressed. Both circulating and local RAS components are up-regulated in obesity and involved in non-alcoholic fatty liver disease onset. Pharmacological manipulations of RAS are viable strategies to tackle metabolic impairments caused by the excessive body fat mass. Renin inhibitors rescue insulin resistance, but do not have marked effects on hepatic steatosis. However, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers (ARB) yield beneficial hepatic remodeling. ARBs elicit body mass loss and normalize insulin levels, tackling insulin resistance. Also, this drug class increases adiponectin levels, besides countering interleukin-6, tumoral necrosis factor-alpha, and transforming growth factor-beta 1. The latter is essential to prevent from liver fibrosis. When conjugated with peroxisome proliferator-activated receptor (PPAR)-alpha activation, ARB fully rescues fatty liver. These effects might be orchestrated by an indirect up-regulation of MAS receptor due to angiotensin II receptor type 1 (AT1R) blockade. These associations of ARB with PPAR activation and ACE2-angiotensin (ANG) (1-7)-MAS receptor axis deserve a better understanding. This editorial provides a brief overview of the current knowledge regarding AT1R blockade effects on sensitivity to insulin and hepatic structural alterations as well as the intersections of AT1R blockade with peroxisome proliferator-activated receptor activation and ACE2-ANG (1-7) - MAS receptor axis.

  15. Insulin-receptor kinase is enhanced in placentas from non-insulin-dependent diabetic women with large-for-gestational-age babies.

    PubMed

    Takayama-Hasumi, S; Yoshino, H; Shimisu, M; Minei, S; Sanaka, M; Omori, Y

    1994-01-01

    The function of insulin receptor and IGF-1 receptor was investigated in placentas from 10 healthy control mothers, 8 diabetic mothers with appropriate-for-gestational-age babies (AGA group) and 9 diabetic mothers with large-for-gestational-age babies (LGA group). None of the diabetic mothers were obese before pregnancy; their blood glucose was well controlled during pregnancy and glycosylated HbA1c was 6.52 +/- 0.71% (M +/- S.E.). Insulin and IGF-1 receptors were partially purified from placentas using wheat germ agglutinin chromatography. The insulin-binding capacity was significantly increased in both the AGA and the LGA groups compared to the control, whereas the IGF-1 binding capacity was similar in the three groups. Autophosphorylation studies were performed with partially purified receptors equalized for similar binding capacity, then immunoprecipitated with anti-insulin receptor antibody or anti-IGF-1 receptor antibody. Insulin-stimulated 32P-incorporation into the insulin receptor beta-subunit was increased by 133% in the LGA group versus the control, whereas incorporation in the AGA group was equivalent to the control. Insulin-stimulated tyrosine kinase activity of the receptor preparation for histone H2B phosphorylation was also significantly increased in the LGA group compared to the control. 32P-incorporation into beta-subunit IGF-1 receptor and IGF-1-stimulated tyrosine kinase activity did not show any significant differences among the three groups. The data in the present study suggest that elevated insulin receptor kinase might be involved in fetal overgrowth in diabetic mothers.

  16. 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

  17. Rab5 activity regulates GLUT4 sorting into insulin-responsive and non-insulin-responsive endosomal compartments: a potential mechanism for development of insulin resistance.

    PubMed

    Tessneer, Kandice L; Jackson, Robert M; Griesel, Beth A; Olson, Ann Louise

    2014-09-01

    Glucose transporter isoform 4 (GLUT4) is the insulin-responsive glucose transporter mediating glucose uptake in adipose and skeletal muscle. Reduced GLUT4 translocation from intracellular storage compartments to the plasma membrane is a cause of peripheral insulin resistance. Using a chronic hyperinsulinemia (CHI)-induced cell model of insulin resistance and Rab5 mutant overexpression, we determined these manipulations altered endosomal sorting of GLUT4, thus contributing to the development of insulin resistance. We found that CHI induced insulin resistance in 3T3-L1 adipocytes by retaining GLUT4 in a Rab5-activity-dependent compartment that is unable to equilibrate with the cell surface in response to insulin. Furthermore, CHI-mediated retention of GLUT4 in this non-insulin-responsive compartment impaired filling of the transferrin receptor (TfR)-positive and TfR-negative insulin-responsive storage compartments. Our data suggest that hyperinsulinemia may inhibit GLUT4 by chronically maintaining GLUT4 in the Rab5 activity-dependent endosomal pathway and impairing formation of the TfR-negative and TfR-positive insulin-responsive GLUT4 pools. This model suggests that an early event in the development of insulin-resistant glucose transport in adipose tissue is to alter the intracellular localization of GLUT4 to a compartment that does not efficiently equilibrate with the cell surface when insulin levels are elevated for prolonged periods of time.

  18. 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

  19. All-atom structural models of insulin binding to the insulin receptor in the presence of a tandem hormone-binding element.

    PubMed

    Vashisth, Harish; Abrams, Cameron F

    2013-06-01

    Insulin regulates blood glucose levels in higher organisms by binding to and activating insulin receptor (IR), a constitutively homodimeric glycoprotein of the receptor tyrosine kinase (RTK) superfamily. Therapeutic efforts in treating diabetes have been significantly impeded by the absence of structural information on the activated form of the insulin/IR complex. Mutagenesis and photo-crosslinking experiments and structural information on insulin and apo-IR strongly suggest that the dual-chain insulin molecule, unlike the related single-chain insulin-like growth factors, binds to IR in a very different conformation than what is displayed in storage forms of the hormone. In particular, hydrophobic residues buried in the core of the folded insulin molecule engage the receptor. There is also the possibility of plasticity in the receptor structure based on these data, which may in part be due to rearrangement of the so-called CT-peptide, a tandem hormone-binding element of IR. These possibilities provide opportunity for large-scale molecular modeling to contribute to our understanding of this system. Using various atomistic simulation approaches, we have constructed all-atom structural models of hormone/receptor complexes in the presence of CT in its crystallographic position and a thermodynamically favorable displaced position. In the "displaced-CT" complex, many more insulin-receptor contacts suggested by experiments are satisfied, and our simulations also suggest that R-insulin potentially represents the receptor-bound form of hormone. The results presented in this work have further implications for the design of receptor-specific agonists/antagonists.

  20. 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

  1. Disruption of Growth Hormone Receptor Prevents Calorie Restriction from Improving Insulin Action and Longevity

    PubMed Central

    Bonkowski, Michael S.; Dominici, Fernando P.; Arum, Oge; Rocha, Juliana S.; Al Regaiey, Khalid A.; Westbrook, Reyhan; Spong, Adam; Panici, Jacob; Masternak, Michal M.; Kopchick, John J.; Bartke, Andrzej

    2009-01-01

    Most mutations that delay aging and prolong lifespan in the mouse are related to somatotropic and/or insulin signaling. Calorie restriction (CR) is the only intervention that reliably increases mouse longevity. There is considerable phenotypic overlap between long-lived mutant mice and normal mice on chronic CR. Therefore, we investigated the interactive effects of CR and targeted disruption or knock out of the growth hormone receptor (GHRKO) in mice on longevity and the insulin signaling cascade. Every other day feeding corresponds to a mild (i.e. 15%) CR which increased median lifespan in normal mice but not in GHRKO mice corroborating our previous findings on the effects of moderate (30%) CR on the longevity of these animals. To determine why insulin sensitivity improves in normal but not GHRKO mice in response to 30% CR, we conducted insulin stimulation experiments after one year of CR. In normal mice, CR increased the insulin stimulated activation of the insulin signaling cascade (IR/IRS/PI3K/AKT) in liver and muscle. Livers of GHRKO mice responded to insulin by increased activation of the early steps of insulin signaling, which was dissipated by altered PI3K subunit abundance which putatively inhibited AKT activation. In the muscle of GHRKO mice, there was elevated downstream activation of the insulin signaling cascade (IRS/PI3K/AKT) in the absence of elevated IR activation. Further, we found a major reduction of inhibitory Ser phosphorylation of IRS-1 seen exclusively in GHRKO muscle which may underpin their elevated insulin sensitivity. Chronic CR failed to further modify the alterations in insulin signaling in GHRKO mice as compared to normal mice, likely explaining or contributing to the absence of CR effects on insulin sensitivity and longevity in these long-lived mice. PMID:19234595

  2. Insulin and epidermal growth factor receptors in rat liver after administration of the hepatocarcinogen 2-acetylaminofluorene: ligand binding and autophosphorylation

    SciTech Connect

    Hwang, D.L.; Roitman, A.; Carr, B.I.; Barseghian, G.; Lev-Ran, A.

    1986-04-01

    The livers of male F344 rats which were fed 0.02% 2-acetylaminofluorene (2-AAF) for two days or more had decreased binding of insulin and epidermal growth factor (EGF) to their hepatic receptors in microsomal and Golgi fractions. Hepatic receptors which were partially purified from carcinogen-fed rats by Triton X-100 solubilization and wheat germ agglutinin affinity column chromatography also had decreased binding activity compared to receptors from normal rats. Scatchard analysis indicated that the decrease in insulin receptor binding was due to decreased receptor number whereas the change in EGF receptor binding was attributed to decreased receptor affinity. Insulin receptor phosphokinase activity was also decreased in 2-AAF-fed rats and correlated with the decrease in receptor binding. EGF receptor phosphokinase activity was unchanged in 2-AAF-fed rats when stimulated with a high concentration (1 microM) of EGF but was decreased when stimulated with low concentrations (0.01-0.1 microM) of EGF. No EGF or insulin competing activity for receptor binding was found using acid-ethanol extracts of 2-AAF-altered liver. These results suggest that 2-AAF causes different alterations in the insulin and EGF receptors of the rat liver.

  3. Amyloid beta oligomers induce impairment of neuronal insulin receptors.

    PubMed

    Zhao, Wei-Qin; De Felice, Fernanda G; Fernandez, Sara; Chen, Hui; Lambert, Mary P; Quon, Michael J; Krafft, Grant A; Klein, William L

    2008-01-01

    Recent studies have indicated an association between Alzheimer's disease (AD) and central nervous system (CNS) insulin resistance. However, the cellular mechanisms underlying the link between these two pathologies have not been elucidated. Here we show that signal transduction by neuronal insulin receptors (IR) is strikingly sensitive to disruption by soluble Abeta oligomers (also known as ADDLs). ADDLs are known to accumulate in AD brain and have recently been implicated as primary candidates for initiating deterioration of synapse function, composition, and structure. Using mature cultures of hippocampal neurons, a preferred model for studies of synaptic cell biology, we found that ADDLs caused a rapid and substantial loss of neuronal surface IRs specifically on dendrites bound by ADDLs. Removal of dendritic IRs was associated with increased receptor immunoreactivity in the cell body, indicating redistribution of the receptors. The neuronal response to insulin, measured by evoked IR tyrosine autophosphorylation, was greatly inhibited by ADDLs. Inhibition also was seen with added glutamate or potassium-induced depolarization. The effects on IR function were completely blocked by NMDA receptor antagonists, tetrodotoxin, and calcium chelator BAPTA-AM. Downstream from the IR, ADDLs induced a phosphorylation of Akt at serine473, a modification associated with neurodegenerative and insulin resistance diseases. These results identify novel factors that affect neuronal IR signaling and suggest that insulin resistance in AD brain is a response to ADDLs, which disrupt insulin signaling and may cause a brain-specific form of diabetes as part of an overall pathogenic impact on CNS synapses.

  4. Insulin receptor kinase-independent signaling via tyrosine phosphorylation of phosphatase PHLPP1.

    PubMed

    Zhang, Manchao; Riedel, Heimo

    2009-05-01

    Most insulin responses correlate well with insulin receptor (IR) Tyr kinase activation; however, critical exceptions to this concept have been presented. Specific IR mutants and stimulatory IR antibodies demonstrate a lack of correlation between IR kinase activity and specific insulin responses in numerous independent studies. IR conformation changes in response to insulin observed with various IR antibodies define an IR kinase-independent signal that alters the C-terminus. IR-related receptors in lower eukaryotes that lack a Tyr kinase point to an alternative mechanism of IR signaling earlier in evolution. However, the implied IR kinase-independent signaling mechanism remained obscure at the molecular level. Here we begin to define the molecular basis of an IR-dependent but IR kinase-independent insulin signal that is equally transmitted by a kinase-inactive mutant IR. This insulin signal results in Tyr phosphorylation and catalytic activation of phosphatase PHLPP1 via a PI 3-kinase-independent, wortmannin-insensitive signaling pathway. Dimerized SH2B1/PSM is a critical activator of the IR kinase and the resulting established insulin signal. In contrast it is an inhibitor of the IR kinase-independent insulin signal and disruption of SH2B1/PSM dimer binding to IR potentiates this signal. Dephosphorylation of Akt2 by PHLPP1 provides an alternative, SH2B1/PSM-regulated insulin-signaling pathway from IR to Akt2 of opposite polarity and distinct from the established PI 3-kinase-dependent signaling pathway via IRS proteins. In combination, both pathways should allow the opposing regulation of Akt2 activity at two phosphorylation sites to specifically define the insulin signal in the background of interfering Akt-regulating signals, such as those controlling cell proliferation and survival.

  5. Effect of H1- and H2-histamine receptor blockade on postexercise insulin sensitivity

    PubMed Central

    Pellinger, Thomas K; Dumke, Breanna R; Halliwill, John R

    2013-01-01

    Following a bout of dynamic exercise, humans experience sustained postexercise vasodilatation in the previously exercised skeletal muscle which is mediated by activation of histamine (H1 and H2) receptors. Skeletal muscle glucose uptake is also enhanced following dynamic exercise. Our aim was to determine if blunting the vasodilatation during recovery from exercise would have an adverse effect on blood glucose regulation. Thus, we tested the hypothesis that insulin sensitivity following exercise would be reduced with H1- and H2-receptor blockade versus control (no blockade). We studied 20 healthy young subjects (12 exercise; eight nonexercise sham) on randomized control and H1- and H2-receptor blockade (fexofenadine and ranitidine) days. Following 60 min of upright cycling at 60% VO2 peak or nonexercise sham, subjects consumed an oral glucose tolerance beverage (1.0 g/kg). Blood glucose was determined from “arterialized” blood samples (heated hand vein). Postexercise whole-body insulin sensitivity (Matsuda insulin sensitivity index) was reduced 25% with H1- and H2-receptor blockade (P < 0.05), whereas insulin sensitivity was not affected by histamine receptor blockade in the sham trials. These results indicate that insulin sensitivity following exercise is blunted by H1- and H2-receptor blockade and suggest that postexercise H1- and H2-receptor–mediated skeletal muscle vasodilatation benefits glucose regulation in healthy humans. PMID:24303118

  6. Tissue localization of Drosophila melanogaster insulin receptor transcripts during development.

    PubMed Central

    Garofalo, R S; Rosen, O M

    1988-01-01

    The Drosophila melanogaster insulin receptor (Drosophila insulin receptor homolog [dIRH]) is similar to its mammalian counterpart in deduced amino acid sequence, subunit structure, and ligand-stimulated protein tyrosine kinase activity. The function of this receptor in D. melanogaster is not yet known. However, a role in development is suggested by the observations that levels of insulin-stimulated kinase activity and expression of dIRH mRNA are maximal during Drosophila midembryogenesis. In this study, a 2.9-kilobase (kb) cDNA clone corresponding to both the dIRH tyrosine kinase domain and some of the 3' untranslated sequence was used to determine the tissue distribution of dIRH mRNA during development. Two principal mRNAs of 11 and 8.6 kb hybridized with the dIRH cDNA in Northern (RNA) blot analysis. The abundance of the 8.6-kb mRNA increased transiently in early embryos, whereas the 11-kb species was most abundant during midembryogenesis. A similar pattern of expression was previously determined by Northern analysis, using a dIRH genomic clone (L. Petruzzelli, R. Herrera, R. Arenas-Garcia, R. Fernandez, M. J. Birnbaum, and O. M. Rosen, Proc. Natl. Acad. Sci. USA 83:4710-4714, 1986). In situ hybridization revealed dIRH transcripts in the ovaries of adult flies, in which the transcripts appeared to be synthesized by nurse cells for eventual storage as maternal RNA in the mature oocyte. Throughout embryogenesis, dIRH transcripts were ubiquitously expressed, although after midembryogenesis, higher levels were detected in the developing nervous system. Nervous system expression remained elevated throughout the larval stages and persisted in the adult, in which the cortex of the brain and ganglion cells were among the most prominently labeled tissues. In larvae, the imaginal disk cells exhibited comparatively high levels of dIRH mRNA expression. The broad distribution of dIRH mRNA in embryos and imaginal disks is compatible with a role for dIRH in anabolic processes

  7. Insulin receptor changes in type 2 diabetes after short term insulin treatment.

    PubMed

    Rizkalla, S W; Weissbrodt, P; Tchobroutsky, G; Slama, G

    1985-10-01

    We have studied erythrocyte insulin receptor changes before and after 8 days of continuous subcutaneous insulin infusion by a pump in 11 uncontrolled obese non-insulin-dependent diabetics (type 2), diet and drug resistant for at least three months previously. All the patients were hospitalized. On day 1 of the study, their oral hypoglycemic agents were stopped and hypocaloric diet (1000 Kcal/day) was maintained (strictly reinforced). This period of reinforced treatment was not accompanied by correction of hyperglycemia. On day 9 patients were placed for 12 hours on artificial pancreas in order to bring their fasting blood glucose levels down to normal values. Then they were submitted to a continuous subcutaneous insulin infusion (CSII) for the following 8 days. There was a significant decrease in mean fasting plasma glucose (P less than 0.001) and a rise in insulin (P less than 0.05) levels after insulin treatment. Mean specific insulin binding was also significantly increased (P less than 0.01). The increase in binding (with insulin therapy) correlated with the fall in fasting hyperglycemia (r = 0.786, P less than 0.01). In addition, the increase in binding correlated negatively with changes in fasting plasma insulin levels (r = -0.867, P less than 0.01), under treatment, on one hand and with the dose of exogenous insulin administered (r = -0.681, P less than 0.05) on the other hand. There was no correlation between binding and fasting plasma insulin levels (before and after insulin therapy), or between diabetes duration and any of the previous parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. B Cell Receptor Affinity for Insulin Dictates Autoantigen Acquisition and B Cell Functionality in Autoimmune Diabetes

    PubMed Central

    Packard, Thomas A.; Smith, Mia J.; Conrad, Francis J.; Johnson, Sara A.; Getahun, Andrew; Lindsay, Robin S.; Hinman, Rochelle M.; Friedman, Rachel S.; Thomas, James W.; Cambier, John C.

    2016-01-01

    B cells have been strongly implicated in the development of human type 1 diabetes and are required for disease in the NOD mouse model. These functions are dependent on B cell antigen receptor (BCR) specificity and expression of MHC, implicating linked autoantigen recognition and presentation to effector T cells. BCR-antigen affinity requirements for participation in disease are unclear. We hypothesized that BCR affinity for the autoantigen insulin differentially affects lymphocyte functionality, including tolerance modality and the ability to acquire and become activated in the diabetogenic environment. Using combined transgenic and retrogenic heavy and light chain to create multiple insulin-binding BCRs, we demonstrate that affinity for insulin is a critical determinant of the function of these autoreactive cells. We show that both BCR affinity for insulin and genetic background affect tolerance induction in immature B cells. We also find new evidence that may explain the enigmatic ability of B cells expressing 125 anti-insulin BCR to support development of TID in NOD mice despite a reported affinity beneath requirements for binding insulin at in vivo concentrations. We report that when expressed as an antigen receptor the affinity of 125 is much higher than determined by measurements of the soluble form. Finally, we show that in vivo acquisition of insulin requires both sufficient BCR affinity and permissive host/tissue environment. We propose that a confluence of BCR affinity, pancreas environment, and B cell tolerance-regulating genes in the NOD animal allows acquisition of insulin and autoimmunity. PMID:27834793

  9. 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

  10. 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.

  11. Protein kinase C-alpha regulates insulin action and degradation by interacting with insulin receptor substrate-1 and 14-3-3 epsilon.

    PubMed

    Oriente, Francesco; Andreozzi, Francesco; Romano, Chiara; Perruolo, Giuseppe; Perfetti, Anna; Fiory, Francesca; Miele, Claudia; Beguinot, Francesco; Formisano, Pietro

    2005-12-09

    Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKCalpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKCalpha co-precipitation with IRS-1, but not with IRS-2, and with 14-3-3epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKCalpha activity, without altering IRS-1/PKCalpha co-precipitation. 14-3-3epsilon overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKCalpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKCalpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3epsilon depletion on insulin signaling. Moreover, PKCalpha inhibition was accompanied by a >2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKCalpha, and 14-3-3epsilon. The presence of 14-3-3epsilon in the complex is not necessary for IRS-1/PKCalpha interaction but modulates PKCalpha activity, thereby regulating insulin signaling and degradation.

  12. Naturally occurring amino acid substitutions at Arg1174 in the human insulin receptor result in differential effects on receptor biosynthesis and hybrid formation, leading to discordant clinical phenotypes.

    PubMed

    Rau, H; Kocova, M; O'Rahilly, S; Whitehead, J P

    2000-07-01

    Missense mutations in the tyrosine kinase domain of the human insulin receptor frequently result in a dominantly inherited form of insulin resistance. We noted a marked disparity in the clinical phenotypes of our study subjects with different missense mutations at the same residue (Arg1174) of the insulin receptor. Subjects with a tryptophan substitution (W) were only moderately hyperinsulinemic, whereas those with a glutamine substitution (Q) had severe clinical and biochemical insulin resistance. Studies were undertaken to explore the molecular mechanisms underlying these differences. Both W and Q mutant receptors bound insulin normally but were kinase inactive. The W mutation resulted in more rapid degradation of newly synthesized mutant receptor, which contrasted with the near-normal biosynthesis of the Q receptor. The propensity of the W receptor to form hybrids with the cotransfected wild-type (WT) receptor was also markedly impaired compared with the Q receptor, to an extent greater than could be explained by lower steady-state expression. Thus, the more clinically benign consequences of the heterozygous W mutant receptor are likely to relate to its impaired biosynthesis and/or reduced capacity to form hybrids with WT receptors. In addition to providing an explanation for the milder phenotype of 1174W versus 1174Q carriers, these studies provide further support for the notion that the dominant-negative effect of insulin receptor tyrosine kinase mutations involves the competition between inactive mutant homodimers and WT/mutant hybrids with active WT homodimers for both ligands and intracellular substrates.

  13. G Protein–Coupled Receptor Kinase 2 Plays a Relevant Role in Insulin Resistance and Obesity

    PubMed Central

    Garcia-Guerra, Lucia; Nieto-Vazquez, Iria; Vila-Bedmar, Rocio; Jurado-Pueyo, María; Zalba, Guillermo; Díez, Javier; Murga, Cristina; Fernández-Veledo, Sonia; Mayor, Federico; Lorenzo, Margarita

    2010-01-01

    OBJECTIVE Insulin resistance is associated with the pathogenesis of metabolic disorders as type 2 diabetes and obesity. Given the emerging role of signal transduction in these syndromes, we set out to explore the possible role that G protein–coupled receptor kinase 2 (GRK2), first identified as a G protein–coupled receptor regulator, could have as a modulator of insulin responses. RESEARCH DESIGN AND METHODS We analyzed the influence of GRK2 levels in insulin signaling in myoblasts and adipocytes with experimentally increased or silenced levels of GRK2, as well as in GRK2 hemizygous animals expressing 50% lower levels of this kinase in three different models of insulin resistance: tumor necrosis factor-α (TNF-α) infusion, aging, and high-fat diet (HFD). Glucose transport, whole-body glucose and insulin tolerance, the activation status of insulin pathway components, and the circulating levels of important mediators were measured. The development of obesity and adipocyte size with age and HFD was analyzed. RESULTS Altering GRK2 levels markedly modifies insulin-mediated signaling in cultured adipocytes and myocytes. GRK2 levels are increased by ∼2-fold in muscle and adipose tissue in the animal models tested, as well as in lymphocytes from metabolic syndrome patients. In contrast, hemizygous GRK2 mice show enhanced insulin sensitivity and do not develop insulin resistance by TNF-α, aging, or HFD. Furthermore, reduced GRK2 levels induce a lean phenotype and decrease age-related adiposity. CONCLUSIONS Overall, our data identify GRK2 as an important negative regulator of insulin effects, key to the etiopathogenesis of insulin resistance and obesity, which uncovers this protein as a potential therapeutic target in the treatment of these disorders. PMID:20627936

  14. Glucose regulates insulin mitogenic effect by modulating SHP-2 activation and localization in JAr cells.

    PubMed

    Bifulco, Giuseppe; Di Carlo, Costantino; Caruso, Matilde; Oriente, Francesco; Di Spiezio Sardo, Attilio; Formisano, Pietro; Beguinot, Francesco; Nappi, Carmine

    2002-07-05

    The glucose effect on cell growth has been investigated in the JAr human choriocarcinoma cells. When JAr cells were cultured in the presence of 6 mm glucose (LG), proliferation and thymidine incorporation were induced by serum, epidermal growth factor, and insulin-like growth factor 1 but not by insulin. In contrast, at 25 mm glucose (HG), proliferation and thymidine incorporation were stimulated by insulin, serum, epidermal growth factor, and insulin-like growth factor 1 to a comparable extent, whereas basal levels were 25% lower than those in LG. HG culturing also enhanced insulin-stimulated insulin receptor and insulin receptor substrate 1 (IRS1) tyrosine phosphorylations while decreasing basal phosphorylations. These actions of glucose were accompanied by an increase in cellular tyrosine phosphatase activity. The activity of SHP-2 in HG-treated JAr cells was 400% of that measured in LG-treated cells. SHP-2 co-precipitation with IRS1 was also increased in HG-treated cells. SHP-2 was mainly cytosolic in LG-treated cells. However, HG culturing largely redistributed SHP-2 to the internal membrane compartment, where tyrosine-phosphorylated IRS1 predominantly localizes. Further exposure to insulin rescued SHP-2 cytosolic localization, thereby preventing its interaction with IRS1. Antisense inhibition of SHP-2 reverted the effect of HG on basal and insulin-stimulated insulin receptor and IRS1 phosphorylation as well as that on thymidine incorporation. Thus, in JAr cells, glucose modulates insulin mitogenic action by modulating SHP-2 activity and intracellular localization.

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

    PubMed

    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.

  16. Biphasic modulation of insulin receptor substrate-1 during goitrogenesis.

    PubMed

    Grozovsky, R; Morales, M M; Carvalho, D P

    2007-05-01

    Insulin receptor substrate-1 (IRS-1) is the main intracellular substrate for both insulin and insulin-like growth factor I (IGF-I) receptors and is critical for cell mitogenesis. Thyrotropin is able to induce thyroid cell proliferation through the cyclic AMP intracellular cascade; however, the presence of either insulin or IGF-I is required for the mitogenic effect of thyroid-stimulating hormone (TSH) to occur. The aim of the present study was to determine whether thyroid IRS-1 content is modulated by TSH in vivo. Strikingly, hypothyroid goitrous rats, which have chronically high serum TSH levels (control, C = 2.31 +/- 0.28; methimazole (MMI) 21d = 51.02 +/- 6.02 ng/mL, N = 12 rats), when treated with 0.03% MMI in drinking water for 21 days, showed significantly reduced thyroid IRS-1 mRNA content. Since goiter was already established in these animals by MMI for 21 days, we also evaluated IRS-1 expression during goitrogenesis. Animals treated with MMI for different periods of time showed a progressive increase in thyroid weight (C = 22.18 +/- 1.21; MMI 5d = 32.83 +/- 1.48; MMI 7d = 31.1 +/- 3.25; MMI 10d = 33.8 +/- 1.25; MMI 14d = 45.5 +/- 2.56; MMI 18d = 53.0 +/- 3.01; MMI 21d = 61.9 +/- 3.92 mg, N = 9-15 animals per group) and serum TSH levels (C = 1.57 +/- 0.2; MMI 5d = 9.95 +/- 0.74; MMI 7d = 10.38 +/- 0.84; MMI 10d = 17.72 +/- 1.47; MMI 14d = 25.65 +/- 1.23; MMI 18d = 35.38 +/- 3.69; MMI 21d = 31.3 +/- 2.7 ng/mL, N = 9-15 animals per group). Thyroid IRS-1 mRNA expression increased progressively during goitrogenesis, being significantly higher by the 14th day of MMI treatment, and then started to decline, reaching the lowest values by the 21st day, when a significant reduction was detected. In the liver of these animals, however, a significant decrease of IRS-1 mRNA was detected after 14 days of MMI treatment, a mechanism probably involved in the insulin resistance that occurs in hypothyroidism. The increase in IRS-1 expression during goitrogenesis may represent

  17. Tyrosine-specific phosphorylation of calmodulin by the insulin receptor kinase purified from human placenta.

    PubMed Central

    Sacks, D B; Fujita-Yamaguchi, Y; Gale, R D; McDonald, J M

    1989-01-01

    It has previously been demonstrated that calmodulin can be phosphorylated in vitro and in vivo by both tyrosine-specific and serine/threonine protein kinase. We demonstrate here that the insulin receptor tyrosine kinase purified from human placenta phosphorylates calmodulin. The highly purified receptors (prepared by insulin-Sepharose chromatography) were 5-10 times more effective in catalysing the phosphorylation of calmodulin than an equal number of partially purified receptors (prepared by wheat-germ agglutinin-Sepharose chromatography). Phosphorylation occurred exclusively on tyrosine residues, up to a maximum of 1 mol [0.90 +/- 0.14 (n = 5)] of phosphate incorporated/mol of calmodulin. Phosphorylation of calmodulin was dependent on the presence of certain basic proteins and divalent cations. Some of these basic proteins, i.e. polylysine, polyarginine, polyornithine, protamine sulphate and histones H1 and H2B, were also able to stimulate the phosphorylation of calmodulin via an insulin-independent activation of the receptor tyrosine kinase. Addition of insulin further increased incorporation of 32P into calmodulin. The magnitude of the effect of insulin was dependent on the concentration and type of basic protein used, ranging from 0.5- to 9.0-fold stimulation. Maximal phosphorylation of calmodulin was obtained at an insulin concentration of 10(-10) M, with half-maximal effect at 10(-11) M. Either Mg2+ or Mn2+ was necessary to obtain phosphorylation, but Mg2+ was far more effective than Mn2+. In contrast, maximal phosphorylation of calmodulin was observed in the absence of Ca2+. Inhibition of phosphorylation was observed as free Ca2+ concentration exceeded 0.1 microM, with almost complete inhibition at 30 microM free Ca2+. The Km for calmodulin was approx. 0.1 microM. To gain further insight into the effects of basic proteins in this system, we examined the binding of calmodulin to the insulin receptor and the polylysine. Calmodulin binds to the insulin

  18. Insilico docking study of compounds elucidated from helicteres isora fruits with ampkinase- insulin receptor

    PubMed Central

    Vennila, Subramanium; Bupesh, Giridharan; Saravanamurali, Krishnan; SenthilKumar, Viajayan; SenthilRaja, Ramalingam; Saran, Natarajan; Magesh, Sachidanandam

    2014-01-01

    Insulin receptor (IR) proteins were essential intracellular signaling peptides in the insulin action cascade. Insulin receptor substrate proteins (IRS-1and IRS-2) serve and regulate the insulin level in the normal insulin action. The broad role of IRS-1 and IRS-2 in cell growth and survival reveals a common regulatory pathway linking development, somatic growth, fertility, neuronal proliferation, and aging to the core mechanisms used by vertebrates for nutrient sensing. Such type of proteins were cyclic adenosine monophosphate-activated protein kinase, this proteins play a key role in the insulin response and regulation. Type -2 Diabetes mellitus occurs during prolonged periods of peripheral insulin resistance due to inactivation of IRS proteins. The compounds isolated from the medicinal plants were safer than synthetic drugs and possess high bio activity. In the present study, four compounds were elucidated from fruits of Helicteres isora. The elucidated compounds were evaluated for the antidiabetic activity using in silico docking study. The receptor was analyzed for the active site and pocket finder tools. The aminoacids such as Phenylalanine, Lysine, Glutamic acid and Asparigine were predicted as active site binding residues. Docking studies were done through Autodock 4 software. All the compounds from fruits of Helicteres isora showed good docking profiles with AMP Kinase, except compound-3 (1,2,3,4-tetrahydro-1,5,6,8-tetramethyl-7-(2-methylprop-1-enylnaphthalene-4-ylpivalate). Finally the result from the study demonstrates that the HS-1, HS-2 and HS-4 posses potent anti diabetic activity against type-2 diabetes mellitus through drug action on AMP kinase cascade system. PMID:24966532

  19. Insilico docking study of compounds elucidated from helicteres isora fruits with ampkinase- insulin receptor.

    PubMed

    Vennila, Subramanium; Bupesh, Giridharan; Saravanamurali, Krishnan; SenthilKumar, Viajayan; SenthilRaja, Ramalingam; Saran, Natarajan; Magesh, Sachidanandam

    2014-01-01

    Insulin receptor (IR) proteins were essential intracellular signaling peptides in the insulin action cascade. Insulin receptor substrate proteins (IRS-1and IRS-2) serve and regulate the insulin level in the normal insulin action. The broad role of IRS-1 and IRS-2 in cell growth and survival reveals a common regulatory pathway linking development, somatic growth, fertility, neuronal proliferation, and aging to the core mechanisms used by vertebrates for nutrient sensing. Such type of proteins were cyclic adenosine monophosphate-activated protein kinase, this proteins play a key role in the insulin response and regulation. Type -2 Diabetes mellitus occurs during prolonged periods of peripheral insulin resistance due to inactivation of IRS proteins. The compounds isolated from the medicinal plants were safer than synthetic drugs and possess high bio activity. In the present study, four compounds were elucidated from fruits of Helicteres isora. The elucidated compounds were evaluated for the antidiabetic activity using in silico docking study. The receptor was analyzed for the active site and pocket finder tools. The aminoacids such as Phenylalanine, Lysine, Glutamic acid and Asparigine were predicted as active site binding residues. Docking studies were done through Autodock 4 software. All the compounds from fruits of Helicteres isora showed good docking profiles with AMP Kinase, except compound-3 (1,2,3,4-tetrahydro-1,5,6,8-tetramethyl-7-(2-methylprop-1-enylnaphthalene-4-ylpivalate). Finally the result from the study demonstrates that the HS-1, HS-2 and HS-4 posses potent anti diabetic activity against type-2 diabetes mellitus through drug action on AMP kinase cascade system.

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

    PubMed Central

    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

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

    PubMed Central

    Schmidt, Vanessa; Schulz, Nadja; Yan, Xin; Schürmann, Annette; Kempa, Stefan; Kern, Matthias; Blüher, Matthias; Poy, Matthew N.

    2016-01-01

    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

  2. A potential role for lamellar insulin-like growth factor-1 receptor in the pathogenesis of hyperinsulinaemic laminitis.

    PubMed

    de Laat, Melody A; Pollitt, Christopher C; Kyaw-Tanner, Myat T; McGowan, Catherine M; Sillence, Martin N

    2013-08-01

    The reason why a sustained high concentration of insulin induces laminitis in horses remains unclear. Cell proliferation occurs in the lamellae during insulin-induced laminitis and in other species high concentrations of insulin can activate receptors for the powerful cell mitogen, insulin-like growth factor (IGF)-1. The first aim of this study was to determine if IGF-1 receptors (IGF-1R) are activated in the hoof during insulin-induced laminitis. Gene expression for IGF-1R and the insulin receptor (InsR) was measured using qRT-PCR, in lamellar tissue from control horses and from horses undergoing a prolonged euglycaemic, hyperinsulinaemic clamp (p-EHC), during the mid-developmental (24h) and acute (46 h) phases of insulin-induced laminitis. Gene expression for both receptors was decreased 13-32-fold (P<0.05) at both time-points in the insulin-treated horses. A second aim was to determine if the down-regulation of the receptor genes could be accounted for by an increase in circulating IGF-1. Serum IGF-1 was measured at 0, 10, 25 and 46 h post-treatment in horses given a p-EHC for approximately 46 h, and in matched controls administered a balanced, electrolyte solution. There was no increase in serum IGF-1 concentrations during the p-EHC, consistent with down-regulation of both receptors by insulin. Stimulation of the IGF-1R by insulin may lead to inappropriate lamellar epidermal cell proliferation and lamellar weakening, a potential mechanism for hyperinsulinaemic laminitis. Targeting this receptor may provide insights into the pathogenesis or identify a novel therapy for hyperinsulinaemic laminitis.

  3. Insulin activates Erk1/2 signaling in the dorsal vagal complex to inhibit glucose production.

    PubMed

    Filippi, Beatrice M; Yang, Clair S; Tang, Christine; Lam, Tony K T

    2012-10-03

    Insulin activates PI3-kinase (PI3K)/AKT to regulate glucose homeostasis in the peripheral tissues and the mediobasal hypothalamus (MBH) of rodents. We report that insulin infusion into the MBH or dorsal vagal complex (DVC) activated insulin receptors. The same dose of insulin that activated MBH PI3K/AKT did not in the DVC. DVC insulin instead activated Erk1/2 and lowered glucose production in rats and mice. Molecular and chemical inhibition of DVC Erk1/2 negated, while activation of DVC Erk1/2 recapitulated, the effects of DVC insulin. Circulating insulin failed to inhibit glucose production when DVC Erk1/2 was inhibited in normal rodents, while DVC insulin action was disrupted in high-fat-fed rodents. Activation of DVC ATP-sensitive potassium channels was necessary for insulin-Erk1/2 and sufficient to inhibit glucose production in normal and high-fat-fed rodents. DVC is a site of insulin action where insulin triggers Erk1/2 signaling to inhibit glucose production and of insulin resistance in high-fat feeding.

  4. 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.

  5. A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin-resistant diabetes.

    PubMed Central

    Accili, D; Frapier, C; Mosthaf, L; McKeon, C; Elbein, S C; Permutt, M A; Ramos, E; Lander, E; Ullrich, A; Taylor, S I

    1989-01-01

    Insulin binds to a receptor on the cell surface, thereby triggering a biological response within the target cell. Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. We have studied a family in which two sisters have a genetic form of insulin-resistant diabetes mellitus. The technique of homozygosity mapping has been used to demonstrate that the mutation causing diabetes in this consanguineous family is genetically linked to the insulin receptor gene. The two insulin-resistant sisters are homozygous for a mutation encoding substitution of valine for phenylalanine at position 382 in the alpha-subunit of the insulin receptor. Transfection of mutant insulin receptor cDNA into NIH3T3 cells demonstrated that the Val382 mutation impaired post-translational processing and retarded transport of the insulin receptor to the plasma membrane. Thus, the mutation causes insulin resistance by decreasing the number of insulin receptors on the surface of the patients' cells. Images PMID:2573522

  6. Estrogen Signaling via a Linear Pathway Involving Insulin-Like Growth Factor I Receptor, Matrix Metalloproteinases, and Epidermal Growth Factor Receptor to Activate Mitogen-Activated Protein Kinase in MCF-7 Breast Cancer Cells

    PubMed Central

    Song, Robert X.-D.; Zhang, Zhenguo; Chen, Yucai; Bao, Yongde; Santen, Richard J.

    2009-01-01

    We present an integrated model of an extranuclear, estrogen receptor-α (ERα)-mediated, rapid MAPK activation pathway in breast cancer cells. In noncancer cells, IGF-I initiates a linear process involving activation of the IGF-I receptor (IGF-IR) and matrix metalloproteinases (MMP), release of heparin-binding epidermal growth factor (HB-EGF), and activation of EGF receptor (EGFR)-dependent MAPK. 17β-Estradiol (E2) rapidly activates IGF-IR in breast cancer cells. We hypothesize that E2 induces a similar linear pathway involving IGF-IR, MMP, HB-EGF, EGFR, and MAPK. Using MCF-7 breast cancer cells, we for the first time demonstrated that a sequential activation of IGF-IR, MMP, and EGFR existed in E2 and IGF-I actions, which was supported by evidence that the selective inhibitors of IGF-IR and MMP or knockdown of IGF-IR all inhibited E2- or IGF-I-induced EGFR phosphorylation. Using the inhibitors and small inhibitoryRNA strategies,we also demonstrated that the same sequential activation of the receptors occurred in E2-, IGF-I-, but not EGF-induced MAPK phosphorylation. Additionally, a HB-EGF neutralizing antibody significantly blocked E2-induced MAPK activation, further supporting our hypothesis. The biological effects of sequential activation of IGF-IR and EGFR on E2 stimulation of cell proliferation were also investigated. Knockdown or blockade of IGF-IR significantly inhibited E2- or IGF-I-stimulated but not EGF-induced cell growth. Knockdown or blockade of EGFR abrogated cell growth induced by E2, IGF-I, and EGF, indicating that EGFR is a downstream molecule of IGF-IR in E2 and IGF-I action. Together, our data support the novel view that E2 can activate a linear pathway involving the sequential activation of IGF-IR, MMP, HB-EGF, EGFR, and MAPK. PMID:17525128

  7. 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

  8. Insulin resistance influences central opioid activity in polycystic ovary syndrome.

    PubMed

    Berent-Spillson, Alison; Love, Tiffany; Pop-Busui, Rodica; Sowers, MaryFran; Persad, Carol C; Pennington, Kathryn P; Eyvazaddeh, Aimee D; Padmanabhan, Vasantha; Zubieta, Jon-Kar; Smith, Yolanda R

    2011-06-30

    This pilot study describes a relationship between insulin resistance and μ-opioid neurotransmission in limbic appetite and mood-regulating regions in women with polycystic ovary syndrome (PCOS), suggesting that insulin-opioid interactions may contribute to behavioral and reproductive pathologies of PCOS. We found that [1] patients with PCOS who are insulin-resistant (n = 7) had greater limbic μ-opioid receptor availability (nondisplaceable binding potential) than controls (n = 5); [2] receptor availability was correlated with severity of insulin resistance; and [3] receptor availability normalized after insulin-regulating treatment.

  9. 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

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

    PubMed

    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

    2015-10-27

    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.

  11. A peroxovanadium compound induces Xenopus oocyte maturation: inhibition by a neutralizing anti-insulin receptor antibody.

    PubMed

    Cummings, C; Zhu, L; Sorisky, A; Liu, X J

    1996-05-01

    Synthetic peroxovanadium compounds are a new class of potent inhibitors of protein phosphotyrosine phosphatases. These compounds exhibit insulin-like activity both in vitro and in experimental animals. However, the molecular mechanism by which these compounds exert their biological effect is not well defined. We demonstrate here that several of these compounds induce Xenopus oocyte maturation in vitro, as indicated by germinal vesicle breakdown. Using one of these compounds for further studies, we show that the induction is dose-dependent and is accompanied by activation of maturation promoting factor as well as activation of Xenopus MAP kinase. Like insulin, bpV(pic) causes an acute accumulation of PI(3,4,5)P3 (phosphotidylinositol-3,4,5-trisphosphate), a product of PI 3-kinase. More importantly, bpV(pic)-induced oocyte maturation was abolished by microinjection of a neutralizing monoclonal anti-insulin receptor antibody (17A3) into oocytes or preincubation of oocytes with a PI 3-kinase inhibitor (wortmannin). These results suggest that bpV(pic) acts upstream of the Xenopus IGF-1 receptor in the induction of meiotic maturation, presumably by neutralizing an inhibitory protein tyrosine phosphatase(s) that may regulate the receptor. Finally, using an oocyte-follicle cell complex that responded to human chorionic gonadotropin (hCG) to undergo GVBD, we showed that injection of 17A3 anti-insulin receptor antibody into oocytes did not affect hCG-induced oocyte maturation.

  12. APS, an adapter protein with a PH and SH2 domain, is a substrate for the insulin receptor kinase.

    PubMed Central

    Ahmed, Z; Smith, B J; Kotani, K; Wilden, P; Pillay, T S

    1999-01-01

    APS (adapter protein with a PH and SH2 domain) is the newest member of a family of tyrosine kinase adapter proteins including SH2-B and Lnk. We previously identified SH2-B as an insulin-receptor-binding protein and substrate [Kotani, Wilden and Pillay (1998) Biochem J. 335, 103-109]. Here we show that APS interacts with the insulin receptor kinase activation loop through its SH2 domain and insulin stimulates the tyrosine-phosphorylation of APS. Furthermore, the phosphorylation of activation-loop tyrosine residues 1158 and 1162 are required for this interaction. PMID:10417330

  13. Fucosterol activates the insulin signaling pathway in insulin resistant HepG2 cells via inhibiting PTP1B.

    PubMed

    Jung, Hyun Ah; Bhakta, Himanshu Kumar; Min, Byung-Sun; Choi, Jae Sue

    2016-10-01

    Insulin resistance is a characteristic feature of type 2 diabetes mellitus (T2DM) and is characterized by defects in insulin signaling. This study investigated the modulatory effects of fucosterol on the insulin signaling pathway in insulin-resistant HepG2 cells by inhibiting protein tyrosine phosphatase 1B (PTP1B). In addition, molecular docking simulation studies were performed to predict binding energies, the specific binding site of fucosterol to PTP1B, and to identify interacting residues using Autodock 4.2 software. Glucose uptake was determined using a fluorescent D-glucose analogue and the glucose tracer 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose, and the signaling pathway was detected by Western blot analysis. We found that fucosterol enhanced insulin-provoked glucose uptake and conjointly decreased PTP1B expression level in insulin-resistant HepG2 cells. Moreover, fucosterol significantly reduced insulin-stimulated serine (Ser307) phosphorylation of insulin receptor substrate 1 (IRS1) and increased phosphorylation of Akt, phosphatidylinositol-3-kinase, and extracellular signal- regulated kinase 1 at concentrations of 12.5, 25, and 50 µM in insulin-resistant HepG2 cells. Fucosterol inhibited caspase-3 activation and nuclear factor kappa B in insulin-resistant hepatocytes. These results suggest that fucosterol stimulates glucose uptake and improves insulin resistance by downregulating expression of PTP1B and activating the insulin signaling pathway. Thus, fucosterol has potential for development as an anti-diabetic agent.

  14. Localization of the mosquito insulin receptor homolog (MIR) in reproducing yellow fever mosquitoes (Aedes aegypti).

    PubMed

    Helbling, P; Graf, R

    1998-12-01

    The female mosquito takes a blood meal to produce a batch of eggs. Initiation of egg maturation and growth of oocytes is governed by several endocrine factors. Peptide factors from the brain are involved in this process and some are also responsible for the induction of ecdysone secretion. The latter appears to be required to maintain a high rate of vitellogenin synthesis. By analogy with the known functions of insulin-like molecules (e.g. bombyxins) which in insects activate the secretion of ecdysteroids, we have postulated that there is an insulin receptor homolog responsible for activation of endysone secretion in the ovary. We have recently cloned the mosquito homolog (MIR) and are now investigating its spatial and temporal distribution. Here, we have localized the insulin receptor (MIR) both at the mRNA and protein level using in situ-hybridization and immunocytochemistry. The receptor is expressed before a blood meal mainly in the nurse cells of ovaries. After a meal, follicle and nurse cells contain mRNA coding for the receptor. The intensity of expression rises in the follicle cells until they degenerate during choriogenesis. Immunocytochemical localization confirms the in situ data: the protein is present before and after a meal. Both methods confirm our previous findings by Northern blot analysis, in which the ovary was found to be the main source of the receptor mRNA. The dynamics of receptor mRNA are related to the dynamics of ecdysone secretion and its action on physiological processes.

  15. Targeting Insulin Receptor with a Novel Internalizing Aptamer

    PubMed Central

    Iaboni, Margherita; Fontanella, Raffaela; Rienzo, Anna; Capuozzo, Maria; Nuzzo, Silvia; Santamaria, Gianluca; Catuogno, Silvia; Condorelli, Gerolama; de Franciscis, Vittorio; Esposito, Carla Lucia

    2016-01-01

    Nucleic acid-based aptamers are emerging as therapeutic antagonists of disease-associated proteins such as receptor tyrosine kinases. They are selected by an in vitro combinatorial chemistry approach, named Systematic Evolution of Ligands by Exponential enrichment (SELEX), and thanks to their small size and unique chemical characteristics, they possess several advantages over antibodies as diagnostics and therapeutics. In addition, aptamers that rapidly internalize into target cells hold as well great potential for their in vivo use as delivery tools of secondary therapeutic agents. Here, we describe a nuclease resistant RNA aptamer, named GL56, which specifically recognizes the insulin receptor (IR). Isolated by a cell-based SELEX method that allows enrichment for internalizing aptamers, GL56 rapidly internalizes into target cells and is able to discriminate IR from the highly homologous insulin-like growth factor receptor 1. Notably, when applied to IR expressing cancer cells, the aptamer inhibits IR dependent signaling. Given the growing interest in the insulin receptor as target for cancer treatment, GL56 reveals a novel molecule with great translational potential as inhibitor and delivery tool for IR-dependent cancers. PMID:27648925

  16. 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

  17. 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

  18. Assay of phosphotyrosyl protein phosphatase using synthetic peptide 1142-1153 of the insulin receptor.

    PubMed

    King, M J; Sale, G J

    1988-09-12

    Synthetic peptide 1142-1153 of the insulin receptor was phosphorylated on tyrosine by the insulin receptor and found to be a potent substrate for dephosphorylation by rat liver particulate and soluble phosphotyrosyl protein phosphatases. Apparent Km values were approximately 5 microM. Vm values (nmol phosphate removed/min per mg protein) were 0.62 (particulate) and 0.2 (soluble). This corresponds to 80% of total activity being membrane-associated, indicating that membrane-bound phosphatases are important receptor phosphatases. The phosphatase activities were distinct from acid and alkaline phosphatase. In conclusion peptide 1142-1153 provides a useful tool for the further study and characterization of phosphotyrosyl protein phosphatases.

  19. Insulin Receptor Isoform Variations in Prostate Cancer Cells

    PubMed Central

    Perks, Claire M.; Zielinska, H. A.; Wang, Jing; Jarrett, Caroline; Frankow, A.; Ladomery, Michael R.; Bahl, Amit; Rhodes, Anthony; Oxley, Jon; Holly, Jeff M. P.

    2016-01-01

    Men who develop prostate cancer (PCa) increasingly have one of the co-morbidities associated with a Western lifestyle that are characterized by hyperinsulinemia, hyperglycemia and increased expression of insulin-like growth factors-I (IGF-I) and IGF-II. Each have been associated with poor prognosis and more aggressive cancers that exhibit increased metabolism and increased glucose uptake. The insulin receptor (IR) has two splice isoforms IR-A and IR-B: IR-A has a higher affinity for IGF-II comparable to that for insulin, whereas the IR-B isoform predominantly just binds to insulin. In this study, we assessed alterations in the IR-A and IR-B isoform ratio and associated changes in cell proliferation and migration of PCa cell lines following exposure to altered concentrations of glucose and treatment with IGF-II and insulin. We observed that where IR-B predominated insulin had a greater effect on migration than IGF-II and IGF-II was more effective when IR-A was the main isoform. With regard to proliferation IGF-II was more effective than insulin regardless of which isoform was dominant. We assessed the abundance of the IR isoforms both in vivo and in vitro and observed that the majority of the tissue samples and cell lines expressed more IR-A than IR-B. Alterations in the isoforms in response to changes in their hormonal milieu could have a profound impact on how malignant cells behave and play a role in promoting carcinogenesis. A greater understanding of the mechanisms underlying changes in alternative splicing of the IR may provide additional targets for future cancer therapies. PMID:27733843

  20. 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.

  1. 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.

  2. Dual pathways for the intracellular processing of insulin. Relationship between retroendocytosis of intact hormone and the recycling of insulin receptors.

    PubMed

    Marshall, S

    1985-11-05

    Adipocytes process insulin through either of two pathways: a retroendocytotic pathway that culminates in the release of intact insulin, and a degradative pathway that terminates in the intracellular catabolism and release of degraded ligand. Mechanistically, these pathways were found to differ in several ways. First, temporal differences were found in the rate at which intact and degraded products were extruded. After 125I-insulin was preloaded into the cell interior, intact ligand was completely released during the first 10 min (t 1/2 = 2 min), whereas degraded insulin was released at a much slower rate over 1 h (t 1/2 greater than 8 min). Secondly, it was found that chloroquine profoundly inhibited the insulin degradative pathway, resulting in the intracellular accumulation of intact ligand and a reduction in the release of degraded products. In contrast, however, chloroquine was without effect on the retroendocytotic processing of insulin. Based on the known actions of chloroquine, it appears that retroendocytosis of insulin does not involve vesicular acidification or dissociation of the insulin-receptor complex and that insulin is most likely carried to the cell exterior in the same vesicles (either receptor-bound or free) as those mediating recycling receptors. Interestingly, accumulation of undergraded insulin within chloroquine-treated cells did not result in the release of additional intact ligand, suggesting that once insulin enters the degradative compartment it is committed to catabolism and cannot exit the cell through the retroendocytotic pathway. A third difference was revealed by the finding that extracellular unlabeled insulin (100 ng/ml) markedly accelerated the rate at which preloaded 125I-insulin was released from adipocytes (t 1/2 of 3 min versus 7 min in controls cells). Analysis of the composition of the released products revealed that extracellular insulin rapidly augmented (over 10 min) in a dose-dependent manner (5-200 ng/ml) the amount of

  3. Deletion of exon 3 of the insulin receptor gene in a kindred with a familial form of insulin resistance

    SciTech Connect

    Wertheimer, E.; Barbetti, F.; Accili, D.; Taylor, S.I.; Litvin, Y.; Ebstein, R.P.; Bennet, E.R.

    1994-05-01

    Molecular scanning techniques, such as denaturing gradient gel electrophoresis (DGGE), greatly facilitate screening candidate genes for mutations. The authors have used DGGE to screen for mutations in the insulin receptor gene in a family in which four of five daughters were affected by type A insulin resistance in association with acanthosis nigricans and hyperandrogenism. DGGE did not detect mutations in any of the 22 exons of the insulin receptor gene. Nevertheless, Southern blot analysis suggested that there was a deletion of exon 3 in the other paternal allele of the insulin receptor gene. Analysis of the father`s cDNA confirmed that exon 3 was deleted from mRNA molecules derived from one of his two alleles of the insulin receptor gene. Furthermore, the father was found to be hemizygous for a polymorphic sequence (GAC{sup Asp} at codon 234) in exon 3 that was not inherited by any of the five daughters. Instead, all five daughters inherited the paternal allele with the deletion mutation. They did not detect mutations in the mother`s insulin receptor gene. Furthermore, the clinical syndrome did not segregate with either of the mother`s two alleles of the insulin receptor gene. Although the youngest daughter inherited the mutant allele from her father, she was not clinically affected. The explanation for the incomplete penetrance is not known. These results emphasize the importance of specifically searching for deletion mutations when screening candidate genes for mutations. Furthermore, the existence of apparently asymptomatic carriers of mutations in the insulin receptor gene, such as the father in the present study, suggests that the prevalence of mutations in the insulin receptor gene may be higher than would be predicted on the basis of the observed prevalence of patients with extreme insulin resistance. 34 refs., 6 figs., 1 tab.

  4. 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.

  5. 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.

  6. APPL1 potentiates insulin sensitivity by facilitating the binding of IRS1/2 to the insulin receptor.

    PubMed

    Ryu, Jiyoon; Galan, Amanda K; Xin, Xiaoban; Dong, Feng; Abdul-Ghani, Muhammad A; Zhou, Lijun; Wang, Changhua; Li, Cuiling; Holmes, Bekke M; Sloane, Lauren B; Austad, Steven N; Guo, Shaodong; Musi, Nicolas; DeFronzo, Ralph A; Deng, Chuxia; White, Morris F; Liu, Feng; Dong, Lily Q

    2014-05-22

    Binding of insulin receptor substrate proteins 1 and 2 (IRS1/2) to the insulin receptor (IR) is essential for the regulation of insulin sensitivity and energy homeostasis. However, the mechanism of IRS1/2 recruitment to the IR remains elusive. Here, we identify adaptor protein APPL1 as a critical molecule that promotes IRS1/2-IR interaction. APPL1 forms a complex with IRS1/2 under basal conditions, and this complex is then recruited to the IR in response to insulin or adiponectin stimulation. The interaction between APPL1 and IR depends on insulin- or adiponectin-stimulated APPL1 phosphorylation, which is greatly reduced in insulin target tissues in obese mice. appl1 deletion in mice consistently leads to systemic insulin resistance and a significant reduction in insulin-stimulated IRS1/2, but not IR, tyrosine phosphorylation, indicating that APPL1 sensitizes insulin signaling by acting at a site downstream of the IR. Our study uncovers a mechanism regulating insulin signaling and crosstalk between the insulin and adiponectin pathways.

  7. Effect of insulin and glucose on the activity of insulin-degrading enzymes in rat liver.

    PubMed

    Jurcovicová, J; Németh, S; Vigas, M

    1977-09-01

    The degradation of insulin by insulin protease and glutathion-insulin transhydrogenase (glutathioneproteindisulphide oxidoreductase--EC 1.8.4.2, GIT) was measured in rat liver either after replacing food and water by 15% glucose solution, or after daily insulin administration 8 U daily for 3 days or after fasting. The breakdown of radioiodinated insulin was followed by measuring the increase of TCA soluble radioactivity during incubation of cell fractions with 125I insulin at 37 degrees C. The highest GIT activity was observed in liver microsomes of rats after glucose feeding and after insulin administration, whereas enzyme activity of fasted animals did not essentially differ from corresponding values of normally fed controls. The insulin protease in cytosol of liver cells remained unchanged after these procedures. The important role of GIT in insulin degradation seems to be conclusively demonstrated.

  8. The role of transplanted visceral fat from the long-lived growth hormone receptor knockout mice on insulin signaling.

    PubMed

    Bennis, Mohammed T; Schneider, Augusto; Victoria, Berta; Do, Andrew; Wiesenborn, Denise S; Spinel, Lina; Gesing, Adam; Kopchick, John J; Siddiqi, Shadab A; Masternak, Michal M

    2017-02-01

    Growth hormone receptor knockout mice (GHRKO) are characterized by high insulin sensitivity and extended lifespan. Interestingly, the secretory activity of visceral fat in GHRKO mice is altered, stimulating whole body insulin sensitivity. In this study, we transplanted normal (N) mice with visceral fat pads from GHRKO or N mice to determine the role of visceral fat on the insulin signaling. We found that the transplant of visceral fat from GHRKO mice to N mice (N-GHRKO) improved whole body insulin sensitivity when comparing with sham-operated mice (N-S) and with mice that received visceral fat from N mice (N-N). This was associated with increased hepatic insulin sensitivity as observed by the increased phosphorylated insulin receptor and increased hepatic expression of Pparα and Pparγ. In conclusion, we demonstrated that visceral fat transplant from GHRKO mice into normal mice enhanced insulin sensitivity and glucose tolerance. These results further confirm the differential physiological role played by visceral adipose tissue from GH receptor deficient mice, indicating that the increase of this fat depot can be associated with beneficial effects on insulin signaling and longevity.

  9. 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

  10. 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

  11. Vitamin D3 restores altered cholinergic and insulin receptor expression in the cerebral cortex and muscarinic M3 receptor expression in pancreatic islets of streptozotocin induced diabetic rats.

    PubMed

    Kumar, Peeyush T; Antony, Sherin; Nandhu, Mohan S; Sadanandan, Jayanarayanan; Naijil, George; Paulose, Chiramadathikudiyil S

    2011-05-01

    Nutritional therapy is a challenging but necessary dimension in the management of diabetes and neurodegenerative changes associated with it. The study evaluates the effect of vitamin D(3) in preventing the altered function of cholinergic, insulin receptors and GLUT3 in the cerebral cortex of diabetic rats. Muscarinic M3 acetylcholine receptors in pancreas control insulin secretion. Vitamin D(3) treatment in M3 receptor regulation in the pancreatic islets was also studied. Radioreceptor binding assays and gene expression was done in the cerebral cortex of male Wistar rats. Immunocytochemistry of muscarinic M3 receptor was studied in the pancreatic islets using specific antibodies. Y-maze was used to evaluate the exploratory and spatial memory. Diabetes induced a decrease in muscarinic M1, insulin and vitamin D receptor expression and an increase in muscarinic M3, α7 nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 expression. Vitamin D(3) and insulin treatment reversed diabetes-induced alterations to near control. Diabetic rats showed a decreased Y-maze performance while vitamin D(3) supplementation improved the behavioural deficit. In conclusion, vitamin D(3) shows a potential therapeutic effect in normalizing diabetes-induced alterations in cholinergic, insulin and vitamin D receptor and maintains a normal glucose transport and utilisation in the cortex. In addition vitamin D(3) modulated muscarinic M3 receptors activity in pancreas and plays a pivotal role in controlling insulin secretion. Hence our findings proved, vitamin D(3) supplementation as a potential nutritional therapy in ameliorating diabetes mediated cortical dysfunctions and suggest an interaction between vitamin D(3) and muscarinic M3 receptors in regulating insulin secretion from pancreas.

  12. 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

  13. G protein–coupled receptor 21 deletion improves insulin sensitivity in diet-induced obese mice

    PubMed Central

    Osborn, Olivia; Oh, Da Young; McNelis, Joanne; Sanchez-Alavez, Manuel; Talukdar, Saswata; Lu, Min; Li, PingPing; Thiede, Lucinda; Morinaga, Hidetaka; Kim, Jane J.; Heinrichsdorff, Jan; Nalbandian, Sarah; Ofrecio, Jachelle M.; Scadeng, Miriam; Schenk, Simon; Hadcock, John; Bartfai, Tamas; Olefsky, Jerrold M.

    2012-01-01

    Obesity-induced inflammation is a key component of systemic insulin resistance, which is a hallmark of type 2 diabetes. A major driver of this inflammation/insulin resistance syndrome is the accumulation of proinflammatory macrophages in adipose tissue and liver. We found that the orphan GPCR Gpr21 was highly expressed in the hypothalamus and macrophages of mice and that whole-body KO of this receptor led to a robust improvement in glucose tolerance and systemic insulin sensitivity and a modest lean phenotype. The improvement in insulin sensitivity in the high-fat diet–fed (HFD-fed) Gpr21 KO mouse was traced to a marked reduction in tissue inflammation caused by decreased chemotaxis of Gpr21 KO macrophages into adipose tissue and liver. Furthermore, mice lacking macrophage expression of Gpr21 were protected from HFD-induced inflammation and displayed improved insulin sensitivity. Results of in vitro chemotaxis studies in human monocytes suggested that the defect in chemotaxis observed ex vivo and in vivo in mice is also translatable to humans. Cumulatively, our data indicate that GPR21 has a critical function in coordinating macrophage proinflammatory activity in the context of obesity-induced insulin resistance. PMID:22653059

  14. Dysregulation of Insulin Secretion in Children With Congenital Hyperinsulinism due to Sulfonylurea Receptor Mutations

    PubMed Central

    Grimberg, A.; Ferry, R.J.; Kelly, A.; Koo-McCoy, S.; Polonsky, K.; Glaser, B.; Permutt, M.A.; Aguilar-Bryan, L.; Stafford, D.; Thornton, P.S.; Baker, L.; Stanley, Charles A.

    2012-01-01

    Mutations in the high-affinity sulfonylurea receptor (SUR)-1 cause one of the severe recessively inherited diffuse forms of congenital hyperinsulinism or, when associated with loss of heterozygosity, focal adenomatosis. We hypothesized that SUR1 mutations would render the β-cell insensitive to sulfonylureas and to glucose. Stimulated insulin responses were compared among eight patients with diffuse hyperinsulinism (two mutations), six carrier parents, and ten normal adults. In the patients with diffuse hyperinsulinism, the acute insulin response to intravenous tolbutamide was absent and did not overlap with the responses seen in either adult group. There was positive, albeit significantly blunted, acute insulin response to intravenous dextrose in the patients with diffuse hyperinsulinism. Graded infusions of glucose, to raise and then lower plasma glucose concentrations over 4 h, caused similar rises in blood glucose but lower peak insulin levels in the hyperinsulinemic patients. Loss of acute insulin response to tolbutamide can identify children with diffuse SUR1 defects. The greater response to glucose than to tolbutamide indicates that ATP-sensitive potassium (KATP) channel–independent pathways are involved in glucose-mediated insulin release in patients with diffuse SUR1 defects. The diminished glucose responsiveness suggests that SUR1 mutations and lack of KATP channel activity may contribute to the late development of diabetes in patients with hyperinsulinism independently of subtotal pancreatectomy. PMID:11272143

  15. Brain Deletion of Insulin Receptor Substrate 2 Disrupts Hippocampal Synaptic Plasticity and Metaplasticity

    PubMed Central

    Costello, Derek A.; Claret, Marc; Al-Qassab, Hind; Plattner, Florian; Irvine, Elaine E.; Choudhury, Agharul I.; Giese, K. Peter; Withers, Dominic J.; Pedarzani, Paola

    2012-01-01

    Objective Diabetes mellitus is associated with cognitive deficits and an increased risk of dementia, particularly in the elderly. These deficits and the corresponding neurophysiological structural and functional alterations are linked to both metabolic and vascular changes, related to chronic hyperglycaemia, but probably also defects in insulin action in the brain. To elucidate the specific role of brain insulin signalling in neuronal functions that are relevant for cognitive processes we have investigated the behaviour of neurons and synaptic plasticity in the hippocampus of mice lacking the insulin receptor substrate protein 2 (IRS-2). Research Design and Methods To study neuronal function and synaptic plasticity in the absence of confounding factors such as hyperglycaemia, we used a mouse model with a central nervous system- (CNS)-restricted deletion of IRS-2 (NesCreIrs2KO). Results We report a deficit in NMDA receptor-dependent synaptic plasticity in the hippocampus of NesCreIrs2KO mice, with a concomitant loss of metaplasticity, the modulation of synaptic plasticity by the previous activity of a synapse. These plasticity changes are associated with reduced basal phosphorylation of the NMDA receptor subunit NR1 and of downstream targets of the PI3K pathway, the protein kinases Akt and GSK-3β. Conclusions These findings reveal molecular and cellular mechanisms that might underlie cognitive deficits linked to specific defects of neuronal insulin signalling. PMID:22383997

  16. Impairment of the liver insulin receptor autoactivation cascade at full-term pregnancy in the rat.

    PubMed

    Martinez, C; Molero, J C; Ruiz, P; Del Arco, A; Andres, A; Carrascosa, J M

    1995-10-15

    Partially purified liver insulin receptors from full-term pregnant rats show decreased autophosphorylation rates if compared with receptors from virgins. We studied the molecular mechanism of this phenomenon, looking at possible structural and functional changes of several domains. The ATP-binding domain seems to be unaltered in receptors from pregnant rats since Km for ATP was similar to that observed in virgins. In contrast, the Vmax. is decreased some 45%, suggesting changes in the kinase domain. Truncation of a fragment of 10 kDa from the C-terminal tail does not normalize the kinase activity in receptors from pregnant rats, suggesting that this domain is not involved in the inhibitory regulation. Treatment with alkaline phosphatase increases the [32P]Pi incorporation into receptors from pregnant rats; however, the autophosphorylation remains lower than that observed in virgin rats. Tryptic phosphopeptide maps of phosphorylated receptors show that the same phosphopeptides are present in receptors from virgin and pregnant rats. However, the progression through the autoactivation cascade in the kinase domain is impaired in receptors from pregnant rats. Differences in the cleavage by trypsin at the two alternative sites in the kinase domain were observed, indicating possible structural changes in receptors from pregnant rats that could be related to the impairment of the autoactivation cascade. Integrity of the alpha- and beta-subunits, as well as differential expression of the two receptor isotypes, were shown to be unaltered. We conclude that (1) the decreased autophosphorylation rate of the liver insulin receptor from pregnant rats is associated with the impairment of its autoactivation cascade, probably as a consequence of the basal Ser/Thr phosphorylation; and (2) the inhibition of the autoactivation cascade does not account for the overall inhibition of autophosphorylation observed in receptors from pregnant rats.

  17. Insulin receptor substrate 2 is a negative regulator of memory formation

    PubMed Central

    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, K. Peter

    2015-01-01

    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 deleted Irs2, either in the whole brain or selectively in the forebrain, using the nestin Cre- or D6 Cre- deleter mouse lines respectively. We show that brain- and forebrain-specific Irs2 knockout mice have enhanced hippocampal spatial reference memory. Furthermore, NesCreIrs2KO mice have enhanced spatial working memory and contextual- and cued-fear memory. Deletion of Irs2 in the brain also increases PSD-95 expression and the density of dendritic spines in hippocampal area CA1, possibly reflecting an increase in the number of excitatory synapses per neuron in the hippocampus that can become activated during memory formation. This increase in activated excitatory synapses might underlie the improved hippocampal memory formation observed in NesCreIrs2KO mice. Overall, these results suggest that Irs2 acts as a negative regulator on memory formation by restricting dendritic spine generation. PMID:21597043

  18. Basal Insulin Use With GLP-1 Receptor Agonists.

    PubMed

    Anderson, Sarah L; Trujillo, Jennifer M

    2016-08-01

    IN BRIEF The combination of basal insulin and a glucagon-like peptide 1 receptor agonist is becoming increasingly common and offers several potential benefits to patients with type 2 diabetes. Clinical studies have demonstrated improved glycemic control and low risks of hypoglycemia and weight gain with the combination, which provides a safe and effective alternative to basal-bolus insulin with less treatment burden. Fixed-ratio combination products that administer both agents in a single injection are in the pipeline and will offer additional options for clinicians and patients. This review focuses on the rationale for, clinical evidence on, and implications of using this combination of therapies in the treatment of type 2 diabetes.

  19. Pharmacologic inhibition of ghrelin receptor signaling is insulin sparing and promotes insulin sensitivity.

    PubMed

    Longo, Kenneth A; Govek, Elizabeth K; Nolan, Anna; McDonagh, Thomas; Charoenthongtrakul, Soratree; Giuliana, Derek J; Morgan, Kristen; Hixon, Jeffrey; Zhou, Chaoseng; Kelder, Bruce; Kopchick, John J; Saunders, Jeffrey O; Navia, Manuel A; Curtis, Rory; DiStefano, Peter S; Geddes, Brad J

    2011-10-01

    Ghrelin influences a variety of metabolic functions through a direct action at its receptor, the GhrR (GhrR-1a). Ghrelin knockout (KO) and GhrR KO mice are resistant to the negative effects of high-fat diet (HFD) feeding. We have generated several classes of small-molecule GhrR antagonists and evaluated whether pharmacologic blockade of ghrelin signaling can recapitulate the phenotype of ghrelin/GhrR KO mice. Antagonist treatment blocked ghrelin-induced and spontaneous food intake; however, the effects on spontaneous feeding were absent in GhrR KO mice, suggesting target-specific effects of the antagonists. Oral administration of antagonists to HFD-fed mice improved insulin sensitivity in both glucose tolerance and glycemic clamp tests. The insulin sensitivity observed was characterized by improved glucose disposal with dramatically decreased insulin secretion. It is noteworthy that these results mimic those obtained in similar tests of HFD-fed GhrR KO mice. HFD-fed mice treated for 56 days with antagonist experienced a transient decrease in food intake but a sustained body weight decrease resulting from decreased white adipose, but not lean tissue. They also had improved glucose disposal and a striking reduction in the amount of insulin needed to achieve this. These mice had reduced hepatic steatosis, improved liver function, and no evidence of systemic toxicity relative to controls. Furthermore, GhrR KO mice placed on low- or high-fat diets had lifespans similar to the wild type, emphasizing the long-term safety of ghrelin receptor blockade. We have therefore demonstrated that chronic pharmacologic blockade of the GhrR is an effective and safe strategy for treating metabolic syndrome.

  20. Characterization of the growth of murine fibroblasts that express human insulin receptors. II. Interaction of insulin with other growth factors

    SciTech Connect

    Randazzo, P.A.; Jarett, L. )

    1990-09-01

    The effects of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin on DNA synthesis were studied in murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental NIH 3T3 cells. In NIH 3T3/HIR cells, individual growth factors in serum-free medium stimulated DNA synthesis with the following relative efficacies: insulin greater than or equal to 10% fetal calf serum greater than PDGF greater than IGF-1 much greater than EGF. In comparison, the relative efficacies of these factors in stimulating DNA synthesis by NIH 3T3 cells were 10% fetal calf serum greater than PDGF greater than EGF much greater than IGF-1 = insulin. In NIH 3T3/HIR cells, EGF was synergistic with 1-10 ng/ml insulin but not with 100 ng/ml insulin or more. Synergy of PDGF or IGF-1 with insulin was not detected. In the parental NIH 3T3 cells, insulin and IGF-1 were found to be synergistic with EGF (1 ng/ml), PDGF (100 ng/ml), and PDGF plus EGF. In NIH 3T3/HIR cells, the lack of interaction of insulin with other growth factors was also observed when the percentage of cells synthesizing DNA was examined. Despite insulin's inducing only 60% of NIH 3T3/HIR cells to incorporate thymidine, addition of PDGF, EGF, or PDGF plus EGF had no further effect. In contrast, combinations of growth factors resulted in 95% of the parental NIH 3T3 cells synthesizing DNA. The independence of insulin-stimulated DNA synthesis from other mitogens in the NIH 3T3/HIR cells is atypical for progression factor-stimulated DNA synthesis and is thought to be partly the result of insulin receptor expression in an inappropriate context or quantity.

  1. Interaction mechanism of insulin-like peptide 5 with relaxin family peptide receptor 4.

    PubMed

    Hu, Meng-Jun; Wei, Dian; Shao, Xiao-Xia; Wang, Jia-Hui; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

    2017-04-01

    Insulin-like peptide 5 (INSL5) is a gut peptide hormone belonging to the insulin/relaxin superfamily. It is implicated in the regulation of food intake and glucose homeostasis by activating relaxin family peptide receptor 4 (RXFP4). Previous studies have suggested that the B-chain is important for INSL5 activity against RXFP4. However, functionalities of the B-chain residues have not yet been systematically studied. In the present work, we conducted alanine-scanning mutagenesis of the B-chain residues of human INSL5 to obtain an overview of their contributions. Binding and activation assays of these INSL5 mutants with human RXFP4 identified two essential exposed B-chain C-terminal residues (B23Arg and B24Trp) and one important exposed central B-chain residue (B16Ile). These three determinant residues together with the C-terminal carboxylate moiety probably constitute a central receptor-binding patch that forms critical hydrophobic and electrostatic interactions with RXFP4 during INSL5 binding. Some other exposed residues, including B10Glu, B12Ile, B13Arg, B17Tyr, B21Ser, and B22Ser, made minor contributions to INSL5 function. These auxiliary residues are scattered around the edge of the central receptor-binding patch, and thus form a peripheral receptor-binding patch on the surface of INSL5. Our present work provides new insights into the interaction mechanism of INSL5 with its receptor RXFP4.

  2. Down-regulation of cell surface insulin receptor and insulin receptor substrate-1 phosphorylation by inhibitor of 90-kDa heat-shock protein family: endoplasmic reticulum retention of monomeric insulin receptor precursor with calnexin in adrenal chromaffin cells.

    PubMed

    Saitoh, Tomokazu; Yanagita, Toshihiko; Shiraishi, Seiji; Yokoo, Hiroki; Kobayashi, Hideyuki; Minami, Shin-Ichi; Onitsuka, Toshio; Wada, Akihiko

    2002-10-01

    Treatment (>/=6 h) of cultured bovine adrenal chromaffin cells with geldanamycin (GA) or herbimycin A (HA), an inhibitor of the 90-kDa heat-shock protein (Hsp90) family, decreased cell surface (125)I-insulin binding. The effect of GA was concentration (EC(50) = 84 nM)- and time (t(1/2) = 8.5 h)-dependent; GA (1 microM for 24 h) lowered the B(max) value of (125)I-insulin binding by 80%, without changing the K(d) value. Western blot analysis showed that GA (>/=3 h) lowered insulin receptor (IR) level by 83% (t(1/2) = 7.4 h; EC(50) = 74 nM), while raising IR precursor level by 100% (t(1/2) = 7.9 h; EC(50) = 300 nM). Pulse-label followed by reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that monomeric IR precursor (~190 kDa) developed into the homodimeric IR precursor (approximately 380 kDa) and the mature alpha(2)beta(2) IR (~410 kDa) in nontreated cells, but not in GA-treated cells; in GA-treated cells, the homodimerization-incompetent form of monomeric IR precursor was degraded via endoplasmic reticulum (ER)-associated protein degradation. Immunoprecipitation followed by immunoblot analysis showed that IR precursor was associated with calnexin (CNX) to a greater extent in GA-treated cells, compared with nontreated cells. GA had no effect on IR mRNA levels and internalization rate of cell surface IRs. In GA-treated cells, insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was attenuated by 77%, with no change in IRS-1 level. Thus, inhibition of the Hsp90 family by GA or HA interrupts homodimerization of monomeric IR precursor in the ER and increases retention of monomeric IR precursor with CNX; this event retards cell surface expression of IR and attenuates insulin-induced activation of IRS-1.

  3. Chronic Exposure to Nicotine Enhances Insulin Sensitivity through α7 Nicotinic Acetylcholine Receptor-STAT3 Pathway

    PubMed Central

    Wang, Pei; Song, Jie; Le, Ying-Ying; Viollet, Benoit; Miao, Chao-Yu

    2012-01-01

    This study was to investigate the effect of nicotine on insulin sensitivity and explore the underlying mechanisms. Treatment of Sprague-Dawley rats with nicotine (3 mg/kg/day) for 6 weeks reduced 43% body weight gain and 65% blood insulin level, but had no effect on blood glucose level. Both insulin tolerance test and glucose tolerance test demonstrated that nicotine treatment enhanced insulin sensitivity. Pretreatment of rats with hexamethonium (20 mg/kg/day) to antagonize peripheral nicotinic receptors except for α7 nicotinic acetylcholine receptor (α7-nAChR) had no effect on the insulin sensitizing effect of nicotine. However, the insulin sensitizing effect but not the bodyweight reducing effect of nicotine was abrogated in α7-nAChR knockout mice. Further, chronic treatment with PNU-282987 (0.53 mg/kg/day), a selective α7-nAChR agonist, significantly enhanced insulin sensitivity without apparently modifying bodyweight not only in normal mice but also in AMP-activated kinase-α2 knockout mice, an animal model of insulin resistance with no sign of inflammation. Moreover, PNU-282987 treatment enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) in skeletal muscle, adipose tissue and liver in normal mice. PNU-282987 treatment also increased glucose uptake by 25% in C2C12 myotubes and this effect was total abrogated by STAT3 inhibitor, S3I-201. All together, these findings demonstrated that nicotine enhanced insulin sensitivity in animals with or without insulin resistance, at least in part via stimulating α7-nAChR-STAT3 pathway independent of inflammation. Our results contribute not only to the understanding of the pharmacological effects of nicotine, but also to the identifying of new therapeutic targets against insulin resistance. PMID:23251458

  4. Inhibition of insulin receptor gene expression and insulin signaling by fatty acid: interplay of PKC isoforms therein.

    PubMed

    Dey, Debleena; Mukherjee, Mohua; Basu, Dipanjan; Datta, Malabika; Roy, Sib Sankar; Bandyopadhyay, Arun; Bhattacharya, Samir

    2005-01-01

    Fatty acids are known to play a key role in promoting the loss of insulin sensitivity causing insulin resistance and type 2 diabetes. However, underlying mechanism involved here is still unclear. Incubation of rat skeletal muscle cells with palmitate followed by I(125)- insulin binding to the plasma membrane receptor preparation demonstrated a two-fold decrease in receptor occupation. In searching the cause for this reduction, we found that palmitate inhibition of insulin receptor (IR) gene expression effecting reduced amount of IR protein in skeletal muscle cells. This was followed by the inhibition of insulin-stimulated IRbeta tyrosine phosphorylation that consequently resulted inhibition of insulin receptor substrate 1 (IRS 1) and IRS 1 associated phosphatidylinositol-3 kinase (PI3 Kinase), phosphoinositide dependent kinase-1 (PDK 1) phosphorylation. PDK 1 dependent phosphorylation of PKCzeta and Akt/PKB were also inhibited by palmitate. Surprisingly, although PKCepsilon phosphorylation is PDK1 dependent, palmitate effected its constitutive phosphorylation independent of PDK1. Time kinetics study showed translocation of palmitate induced phosphorylated PKCepsilon from cell membrane to nuclear region and its possible association with the inhibition of IR gene transcription. Our study suggests one of the pathways through which fatty acid can induce insulin resistance in skeletal muscle cell.

  5. 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.

  6. Hepatic protein phosphotyrosine phosphatase. Dephosphorylation of insulin and epidermal growth factor receptors in normal and alloxan diabetic rats.

    PubMed Central

    Gruppuso, P A; Boylan, J M; Posner, B I; Faure, R; Brautigan, D L

    1990-01-01

    Polypeptide hormone signal transmission by receptor tyrosine kinases requires the rapid reversal of tyrosine phosphorylation by protein phosphotyrosine phosphatases (PPTPases). We studied hepatic PPTPases in the rat with emphasis on acute and chronic regulation by insulin. PPTPase activity with artificial substrates ([32P]Tyr-reduced, carboxyamidomethylated, and maleylated lysozyme and [32P]Tyr-poly[glutamic acid:tyrosine] 4:1) was present in distinct membrane, cytoskeletal, and cytosolic fractions. These PPTPase activities were unaffected by alloxan diabetes. Acute administration of insulin to normal animals also did not change PPTPase activity in liver plasma membranes or endosomal membranes. Although alloxan diabetes did not affect PPTPase activity measured with artificial substrates or with epidermal growth factor receptors, a decrease in insulin receptor dephosphorylation was noted. Dephosphorylation of hepatic receptors from normal and diabetic rats by membrane PPTPase from control rats was similar. These results indicate that alloxan diabetes does not lead to a generalized effect on hepatic PPTPase activity, although a substrate-specific decrease in activity with the insulin receptor may occur. Images PMID:2161429

  7. Apoptosis induced by an anti-epidermal growth factor receptor monoclonal antibody in a human colorectal carcinoma cell line and its delay by insulin.

    PubMed Central

    Wu, X; Fan, Z; Masui, H; Rosen, N; Mendelsohn, J

    1995-01-01

    Both EGF and insulin, or IGF, stimulate the growth of many cell types by activating receptors that contain tyrosine kinase activities. A monoclonal antibody (mAb 225) against the EGF receptor produced in this laboratory has been shown to competitively inhibit EGF binding and block activation of receptor tyrosine kinase. Here we report that a human colorectal carcinoma cell line, DiFi, which expresses high levels of EGF receptors on plasma membranes, can be induced to undergo G1 cell cycle arrest and programmed cell death (apoptosis) when cultured with mAb 225 at concentrations that saturate EGF receptors. Addition of IGF-1 or high concentrations of insulin can delay apoptosis induced by mAb 225, while the G1 arrest cannot be reversed by either IGF-1 or insulin. Insulin/IGF-1 cannot activate EGF receptor tyrosine kinase that has been inhibited by mAb 225. Moreover, an mAb against the IGF-1 receptor, which has little direct effect on DiFi cell growth, can block the capacity of insulin/IGF-1 to delay apoptosis induced by mAb 225, suggesting that the insulin/IGF-1-mediated delay of apoptosis is acting through the IGF-1 receptor. In contrast, insulin/IGF-1 cannot delay the apoptosis caused by the DNA damaging agent, cisplatin. The results indicate that EGF receptor activation is required both for cell cycle progression and for prevention of apoptosis in DiFi cells, and that a signal transduction pathway shared by receptors for insulin/IGF-1 and EGF may be involved in regulating apoptosis triggered by blockade of the EGF receptor. Images PMID:7706497

  8. Increased abundance of insulin/insulin-like growth factor-I hybrid receptors in skeletal muscle of obese subjects is correlated with in vivo insulin sensitivity.

    PubMed

    Federici, M; Porzio, O; Lauro, D; Borboni, P; Giovannone, B; Zucaro, L; Hribal, M L; Sesti, G

    1998-08-01

    We reported that in noninsulin-dependent diabetes melitus (NIDDM) patients expression of insulin/insulin-like growth factor I (IGF-I) hybrid receptors is increased in insulin target tissues. Whether this is a defect associated with NIDDM or represents a generalized abnormality associated with insulin resistant states is still unsettled. To address this, we applied a microwell-based immunoassay to measure abundance of insulin receptors, type 1 IGF receptors, and hybrid receptors in muscle of eight normal and eight obese subjects. Maximal insulin binding to insulin receptors was lower in obese than in control subjects (B/T = 1.8 +/- 0.20 and 2.6 +/- 0.30; P < 0.03, respectively) and was negatively correlated with insulinemia (r = -0.60; P < 0.01). Maximal IGF-I binding to type 1 IGF receptors was higher in obese than in controls (B/T = 1.9 +/- 0.20 and 0.86 +/- 0.10; P < 0.0001, respectively) and was negatively correlated with plasma IGF-I levels (r = -0.69; P < 0.003). Hybrid receptor abundance was higher in obese than in normal subjects (B/T = 1.21 +/- 0.14 and 0.44 +/- 0.06; P < 0.0003, respectively) and was negatively correlated with insulin binding (r = -0.60; P < 0.01) and positively correlated with IGF-I binding (r = 0.92; P < 0.0001). Increased abundance of hybrids was correlated with insulinemia (r = 0.70; P < 0.002) and body mass index (r = 0.71; P < 0.0019), whereas it was negatively correlated with in vivo insulin sensitivity measured by ITT (r = -0.67; P < 0.016). These results indicate that downregulation of insulin receptors or upregulation of type 1 IGF receptors because of changes in plasma insulin and IGF-I levels may result in modifications in hybrid receptor abundance.

  9. Novel Small Molecule Glucagon-Like Peptide-1 Receptor Agonist Stimulates Insulin Secretion in Rodents and From Human Islets

    PubMed Central

    Sloop, Kyle W.; Willard, Francis S.; Brenner, Martin B.; Ficorilli, James; Valasek, Kathleen; Showalter, Aaron D.; Farb, Thomas B.; Cao, Julia X.C.; Cox, Amy L.; Michael, M. Dodson; Gutierrez Sanfeliciano, Sonia Maria; Tebbe, Mark J.; Coghlan, Michael J.

    2010-01-01

    OBJECTIVE The clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor. RESEARCH DESIGN AND METHODS Screening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo. RESULTS Novel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment. CONCLUSIONS These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization. PMID:20823098

  10. [Insulin and glucocorticoid binding by blood cell receptors after hydrocortisone administration in rabbits].

    PubMed

    Tikhonova, N E; Tatarinova, G Sh

    1989-07-01

    Repeated i.v. administration of hydrocortisone (10 mg/kg) revealed an increase in the resistance against insulin although endogenous corticosterone was decreased in 33 male rabbits. The insulin- and dexamethasone-binding receptors of erythrocytes and mononuclear leucocytes. changed after 3-7 hydrocortisone injections, the binding increasing for insulin and diminishing for dexamethasone.

  11. 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…

  12. Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus.

    PubMed Central

    Folli, F; Saad, M J; Backer, J M; Kahn, C R

    1993-01-01

    Insulin stimulates tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), which in turn binds to and activates phosphatidylinositol 3-kinase (PI 3-kinase). In the present study, we have examined these processes in animal models of insulin-resistant and insulin-deficient diabetes mellitus. After in vivo insulin stimulation, there was a 60-80% decrease in IRS-1 phosphorylation in liver and muscle of the ob/ob mouse. There was no insulin stimulation of PI 3-kinase (85 kD subunit) association with IRS-1, and IRS-1-associated PI 3-kinase activity was reduced 90%. Insulin-stimulated total PI 3-kinase activity was also absent in both tissues of the ob/ob mouse. By contrast, in the streptozotocin diabetic rat, IRS-1 phosphorylation increased 50% in muscle, IRS-1-associated PI 3-kinase activity was increased two- to threefold in liver and muscle, and there was a 50% increase in the p85 associated with IRS-1 after insulin stimulation in muscle. In conclusion, (a) IRS-1-associated PI 3-kinase activity is differentially regulated in hyperinsulinemic and hypoinsulinemic diabetic states; (b) PI 3-kinase activation closely correlates with IRS-1 phosphorylation; and (c) reduced PI 3-kinase activity may play a role in the pathophysiology of insulin resistant diabetic states, such as that seen in the ob/ob mouse. Images PMID:7691886

  13. Changes in erythrocyte insulin receptors in normal dogs and keeshond dogs with inheritable, early onset, insulin dependent diabetes mellitus

    SciTech Connect

    Klaassen, J.K.

    1986-01-01

    Validation of a procedure to evaluate insulin receptors on erythrocytes (RBC-IR) in dogs is described. The specific binding of (/sup 125/I)iodoinsulin to RBC-IR of normal dogs is significantly greater than binding in keeshonds with an inheritable form of early onset diabetes mellitus. This decreased binding was due to a significant decrease in RBC-IR affinity in the diabetic keeshonds. To determine the effect on RBC-IR, normal dogs were treated with either dexamethasone (0.1 mg/kg) or prednisone (0.3 mg/kg) for 10 days: concentrations of plasma cortisol, glucose, and insulin, plus binding characteristics of RBC-IR were determined. In the dexamethasone treated group, plasma glucose concentrations were elevated significantly by day 6 and continued through day 10. Insulin concentrations were elevated significantly by day 3 and remained elevated through day 10. In the prednisone treated group, glucose concentrations were elevated significantly by day 3, while insulin concentrations were elevated significantly by day 8. Maximum binding of RBC-IR was unaffected by prednisone and neither affinities nor receptor numbers were significantly different from day 1. No changes in plasma cortisol concentration were seen. Diabetic keeshonds on daily insulin treatment were removed from exogenous insulin therapy for 48 hours. Significant increases in glucose concentrations were observed, but no significant changes in cortisol, insulin, average receptor binding affinity, or RBC-IR number per cell occurred.

  14. 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.

  15. Sweet-taste receptors, low-energy sweeteners, glucose absorption and insulin release.

    PubMed

    Renwick, Andrew G; Molinary, Samuel V

    2010-11-01

    The present review explores the interactions between sweeteners and enteroendocrine cells, and consequences for glucose absorption and insulin release. A combination of in vitro, in situ, molecular biology and clinical studies has formed the basis of our knowledge about the taste receptor proteins in the glucose-sensing enteroendocrine cells and the secretion of incretins by these cells. Low-energy (intense) sweeteners have been used as tools to define the role of intestinal sweet-taste receptors in glucose absorption. Recent studies using animal and human cell lines and knockout mice have shown that low-energy sweeteners can stimulate intestinal enteroendocrine cells to release glucagon-like peptide-1 and glucose-dependent insulinotropic peptide. These studies have given rise to major speculations that the ingestion of food and beverages containing low-energy sweeteners may act via these intestinal mechanisms to increase obesity and the metabolic syndrome due to a loss of equilibrium between taste receptor activation, nutrient assimilation and appetite. However, data from numerous publications on the effects of low-energy sweeteners on appetite, insulin and glucose levels, food intake and body weight have shown that there is no consistent evidence that low-energy sweeteners increase appetite or subsequent food intake, cause insulin release or affect blood pressure in normal subjects. Thus, the data from extensive in vivo studies in human subjects show that low-energy sweeteners do not have any of the adverse effects predicted by in vitro, in situ or knockout studies in animals.

  16. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT

    PubMed Central

    Fishwick, Katherine J.; Rylett, R. Jane

    2015-01-01

    Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer’s Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization. PMID:26161852

  17. Elucidating the Activation Mechanism of the Insulin-Family Proteins with Molecular Dynamics Simulations.

    PubMed

    Papaioannou, Anastasios; Kuyucak, Serdar; Kuncic, Zdenka

    2016-01-01

    The insulin-family proteins bind to their own receptors, but insulin-like growth factor II (IGF-II) can also bind to the A isoform of the insulin receptor (IR-A), activating unique and alternative signaling pathways from those of insulin. Although extensive studies of insulin have revealed that its activation is associated with the opening of the B chain-C terminal (BC-CT), the activation mechanism of the insulin-like growth factors (IGFs) still remains unknown. Here, we present the first comprehensive study of the insulin-family proteins comparing their activation process and mechanism using molecular dynamics simulations to reveal new insights into their specificity to the insulin receptor. We have found that all the proteins appear to exhibit similar stochastic dynamics in their conformational change to an active state. For the IGFs, our simulations show that activation involves two opening locations: the opening of the BC-CT section away from the core, similar to insulin; and the additional opening of the BC-CT section away from the C domain. Furthermore, we have found that these two openings occur simultaneously in IGF-I, but not in IGF-II, where they can occur independently. This suggests that the BC-CT section and the C domain behave as a unified domain in IGF-I, but as two independent domains in IGF-II during the activation process, implying that the IGFs undergo different activation mechanisms for receptor binding. The probabilities of the active and inactive states of the proteins suggest that IGF-II is hyperactive compared to IGF-I. The hinge residue and the hydrophobic interactions in the core are found to play a critical role in the stability and activity of IGFs. Overall, our simulations have elucidated the crucial differences and similarities in the activation mechanisms of the insulin-family proteins, providing new insights into the molecular mechanisms responsible for the observed differences between IGF-I and IGF-II in receptor binding.

  18. Elucidating the Activation Mechanism of the Insulin-Family Proteins with Molecular Dynamics Simulations

    PubMed Central

    Papaioannou, Anastasios; Kuyucak, Serdar; Kuncic, Zdenka

    2016-01-01

    The insulin-family proteins bind to their own receptors, but insulin-like growth factor II (IGF-II) can also bind to the A isoform of the insulin receptor (IR-A), activating unique and alternative signaling pathways from those of insulin. Although extensive studies of insulin have revealed that its activation is associated with the opening of the B chain-C terminal (BC-CT), the activation mechanism of the insulin-like growth factors (IGFs) still remains unknown. Here, we present the first comprehensive study of the insulin-family proteins comparing their activation process and mechanism using molecular dynamics simulations to reveal new insights into their specificity to the insulin receptor. We have found that all the proteins appear to exhibit similar stochastic dynamics in their conformational change to an active state. For the IGFs, our simulations show that activation involves two opening locations: the opening of the BC-CT section away from the core, similar to insulin; and the additional opening of the BC-CT section away from the C domain. Furthermore, we have found that these two openings occur simultaneously in IGF-I, but not in IGF-II, where they can occur independently. This suggests that the BC-CT section and the C domain behave as a unified domain in IGF-I, but as two independent domains in IGF-II during the activation process, implying that the IGFs undergo different activation mechanisms for receptor binding. The probabilities of the active and inactive states of the proteins suggest that IGF-II is hyperactive compared to IGF-I. The hinge residue and the hydrophobic interactions in the core are found to play a critical role in the stability and activity of IGFs. Overall, our simulations have elucidated the crucial differences and similarities in the activation mechanisms of the insulin-family proteins, providing new insights into the molecular mechanisms responsible for the observed differences between IGF-I and IGF-II in receptor binding. PMID

  19. Bis(hinokitiolato)zinc complex ([Zn(hkt)2]) activates Akt/protein kinase B independent of insulin signal transduction.

    PubMed

    Naito, Yuki; Yoshikawa, Yutaka; Masuda, Kazufumi; Yasui, Hiroyuki

    2016-07-01

    Since many Zn complexes have been developed to enhance the insulin-like activity and increase the exposure and residence of Zn in the animal body, these complexes are recognized as one of the new candidates with action mechanism different from existing anti-diabetic drugs. However, the molecular mechanism by which Zn complexes exert an anti-DM effect is unknown. Therefore, we evaluated the activity of Zn complexes, especially related to the phosphorylation of insulin signaling pathway components. We focused on the insulin-like effects of the bis(hinokitiolato)zinc complex, [Zn(hkt)2], using 3T3-L1 adipocytes. [Zn(hkt)2] was taken up by cells and induced Akt phosphorylation in a time-dependent manner. Additionally, it showed inhibitory activity against PTP1B and PTEN, which are major negative regulators of insulin signaling. It did not promote the phosphorylation of IR (insulin receptor)-β or IRS (insulin receptor substrate)-1 by itself, but in combination with insulin, it enhanced the phosphorylation of IRβ. We conclude that [Zn(hkt)2] has effects on the proteins of insulin signaling pathway without insulin receptor mediation, and [Zn(hkt)2] promotes insulin function and shows the anti-DM effects. Thus, [Zn(hkt)2] may be the basis for improved DM treatments.

  20. Mechanisms of insulin action on sympathetic nerve activity

    NASA Technical Reports Server (NTRS)

    Muntzel, Martin S.; Anderson, Erling A.; Johnson, Alan Kim; Mark, Allyn L.

    1996-01-01

    Insulin resistance and hyperinsulinemia may contribute to the development of arterial hypertension. Although insulin may elevate arterial pressure, in part, through activation of the sympathetic nervous system, the sites and mechanisms of insulin-induced sympathetic excitation remain uncertain. While sympathoexcitation during insulin may be mediated by the baroreflex, or by modulation of norepinephrine release from sympathetic nerve endings, it has been shown repeatedly that insulin increases sympathetic outflow by actions on the central nervous system. Previous studies employing norepinephrine turnover have suggested that insulin causes sympathoexcitation by acting in the hypothalamus. Recent experiments from our laboratory involving direct measurements of regional sympathetic nerve activity have provided further evidence that insulin acts in the central nervous system. For example, administration of insulin into the third cerebralventricle increased lumbar but not renal or adrenal sympathetic nerve activity in normotensive rats. Interestingly, this pattern of regional sympathetic nerve responses to central neural administration of insulin is similar to that seen with systemic administration of insulin. Further, lesions of the anteroventral third ventricle hypothalamic (AV3V) region abolished increases in sympathetic activity to systemic administration of insulin with euglycemic clamp, suggesting that AV3V-related structures are critical for insulin-induced elevations in sympathetic outflow.

  1. Combining a GLP-1 receptor agonist and basal insulin: study evidence and practical considerations.

    PubMed

    Carris, Nicholas W; Taylor, James R; Gums, John G

    2014-12-01

    Most patients with diabetes mellitus require multiple medications to achieve glycemic goals. Considering this and the increasing incidence of type 2 diabetes worldwide, the need for effective combination therapy is pressing. Basal insulin and glucagon-like peptide 1 (GLP-1) receptor agonists are frequently used to treat type 2 diabetes. Though both classes of medication are exclusively injectable, which may cause initial hesitation from providers, evidence for their combined use is substantial. This review summarizes the theoretical benefit, supporting evidence, and implementation of a combined basal insulin-GLP-1 receptor agonist regimen. Basal insulin added to a GLP-1 receptor agonist reduces hemoglobin A1c (HbA1c) without weight gain or significantly increased hypoglycemia. A GLP-1 receptor agonist added to basal insulin reduces HbA1c and body weight. Compared with the addition of meal-time insulin to basal insulin, a GLP-1 receptor agonist produces similar or greater reduction in HbA1c, weight loss instead of weight gain, and less hypoglycemia. Gastrointestinal adverse events are common with GLP-1 receptor agonists, especially during initiation and titration. However, combination with basal insulin is not expected to augment expected adverse events that come with using a GLP-1 receptor agonist. Basal insulin can be added to a GLP-1 receptor agonist with a slow titration to target goal fasting plasma glucose. In patients starting a GLP-1 receptor agonist, the dose of basal insulin should be decreased by 20 % in patients with an HbA1c ≤8 %. The evidence from 15 randomized prospective studies supports the combined use of a GLP-1 receptor agonist with basal insulin in a broad range of patients with uncontrolled type 2 diabetes.

  2. Differences in the sites of phosphorylation of the insulin receptor in vivo and in vitro

    SciTech Connect

    White, M.F.; Takayama, S.; Kahn, C.R.

    1985-08-05

    Phosphorylation of the insulin receptor was studied in intact well differentiated hepatoma cells (Fao) and in a solubilized and partially purified receptor preparation obtained from these cells by affinity chromatography on wheat germ agglutinin agarose. Tryptic peptides containing the phosphorylation sites of the beta-subunit of the insulin receptor were analyzed by reverse-phase high performance liquid chromatography. Phosphoamino acid content of these peptides was determined by acid hydrolysis and high voltage electrophoresis. Separation of the phosphopeptides from unstimulated Fao cells revealed one major and two minor phosphoserine-containing peptides and a single minor phosphothreonine-containing peptide. Insulin (10(-7) M) increased the phosphorylation of the beta-subunit of the insulin receptor 3- to 4-fold in the intact Fao cell. After insulin stimulation, two phosphotyrosine-containing peptides were identified. Tyrosine phosphorylation reached a steady state within 20 s after the addition of insulin and remained nearly constant for 1 h. Under our experimental conditions, no significant change in the amount of (TSP)phosphoserine or (TSP)phosphothreonine associated with the beta-subunit was found during the initial response of cells to insulin. When the insulin receptor was extracted from the Fao cells and incubated in vitro with (gamma-TSP)ATP and MnS , very little phosphorylation occurred in the absence of insulin.

  3. Tyrosine phosphorylation of the insulin receptor is not required for receptor internalization: studies in 2,4-dinitrophenol-treated cells

    SciTech Connect

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

    1989-05-01

    The relation between insulin-stimulated autophosphorylation of the insulin receptor and internalization of the receptor was studied in Fao rat hepatoma cells. Treatment of Fao cells with 2,4-dinitrophenol for 45 min depleted cellular ATP by 80% and equally inhibited insulin-stimulated receptor autophosphorylation, as determined by immunoprecipitation of surface-iodinated or (/sup 32/P)phosphate-labeled cells with anti-phosphotyrosine antibody. In contrast, internalization of the insulin receptor and internalization and degradation of /sup 125/I-labeled insulin by 2,4-dinitrophenol-treated cells were normal. These data show that autophosphorylation of the insulin receptor is not required for the receptor-mediated internalization of insulin in Fao cells and suggest that insulin receptor recycling is independent of autophosphorylation.

  4. Arg972 insulin receptor substrate-1 enhances tumor necrosis factor-α-induced apoptosis in osteoblasts.

    PubMed

    You, Yunhui; Liu, Shiqing; Peng, Lijuan; Long, Mei; Deng, Hongxiang; Zhao, Hongjun

    2015-07-01

    The presence of Arg972 insulin receptor substrate-1 (IRS-1) is associated with impaired insulin/IRS-1 signaling to activate phosphatidylinositol-3 kinase (PI3K). Tumor necrosis factor-α (TNF-α), an inflammatory cytokine with a central role in the pathogenesis of rheumatoid arthritis (RA), induces apoptosis in osteoblasts, which are the principal cell type responsible for bone loss in RA. In our previous study, an association between Arg972 IRS-1 and a high risk and severity of RA was identified. In the present study, the effects of Arg972 IRS-1 and IRS-1 on TNF-α-induced apoptosis in human osteoblasts were examined. Normal and RA osteoblasts were stably transfected with Arg972 IRS-1 and IRS-1. In addition, cells were stably transduced with IRS-1-shRNA to knock down IRS1. Following stimulation with 10 nM insulin for 30 min, the stable overexpression of Arg972 IRS-1 and knock down of IRS-1 significantly decreased IRS-1-associated PI3K activity and Akt activation/phosphorylation at serine 473 (ser473) and enhanced TNF-α-induced apoptosis in normal and in RA osteoblasts. By contrast, the stable overexpression of IRS-1 significantly increased the levels of IRS-1-associated PI3K activity and Akt phosphorylation (ser473) and inhibited TNF-α-induced apoptosis, which was eliminated by pretreatment with 50 µn BJM120, a selective PI3K inhibitor, for 30 min. In conclusion, the present study provided the first evidence, to the best of our knowledge, that insulin stimulation of Arg972 IRS-1 and IRS-1 enhanced and inhibited TNF-α-induced apoptosis, respectively in normal and RA osteoblasts by a PI3K‑dependent mechanism. These findings suggest that insulin/IRS-1 signaling is important in the pathogenesis of RA.

  5. Novel roles for insulin receptor (IR) in adipocytes and skeletal muscle cells via new and unexpected substrates.

    PubMed

    Ramalingam, Latha; Oh, Eunjin; Thurmond, Debbie C

    2013-08-01

    The insulin signaling pathway regulates whole-body glucose homeostasis by transducing extracellular signals from the insulin receptor (IR) to downstream intracellular targets, thus coordinating a multitude of biological functions. Dysregulation of IR or its signal transduction is associated with insulin resistance, which may culminate in type 2 diabetes. Following initial stimulation of IR, insulin signaling diverges into different pathways, activating multiple substrates that have roles in various metabolic and cellular processes. The integration of multiple pathways arising from IR activation continues to expand as new IR substrates are identified and characterized. Accordingly, our review will focus on roles for IR substrates as they pertain to three primary areas: metabolism/glucose uptake, mitogenesis/growth, and aging/longevity. While IR functions in a seemingly pleiotropic manner in many cell types, through these three main roles in fat and skeletal muscle cells, IR multi-tasks to regulate whole-body glucose homeostasis to impact healthspan and lifespan.

  6. Domain-dependent effects of insulin and IGF-1 receptors on signalling and gene expression

    PubMed Central

    Cai, Weikang; Sakaguchi, Masaji; Kleinridders, Andre; Gonzalez-Del Pino, Gonzalo; Dreyfuss, Jonathan M.; O'Neill, Brian T.; Ramirez, Alfred K.; Pan, Hui; Winnay, Jonathon N.; Boucher, Jeremie; Eck, Michael J.; Kahn, C. Ronald

    2017-01-01

    Despite a high degree of homology, insulin receptor (IR) and IGF-1 receptor (IGF1R) mediate distinct cellular and physiological functions. Here, we demonstrate how domain differences between IR and IGF1R contribute to the distinct functions of these receptors using chimeric and site-mutated receptors. Receptors with the intracellular domain of IGF1R show increased activation of Shc and Gab-1 and more potent regulation of genes involved in proliferation, corresponding to their higher mitogenic activity. Conversely, receptors with the intracellular domain of IR display higher IRS-1 phosphorylation, stronger regulation of genes in metabolic pathways and more dramatic glycolytic responses to hormonal stimulation. Strikingly, replacement of leucine973 in the juxtamembrane region of IR to phenylalanine, which is present in IGF1R, mimics many of these signalling and gene expression responses. Overall, we show that the distinct activities of the closely related IR and IGF1R are mediated by their intracellular juxtamembrane region and substrate binding to this region. PMID:28345670

  7. Growth hormone, but not insulin, activates STAT5 proteins in adipocytes in vitro and in vivo.

    PubMed

    Zvonic, Sanjin; Story, David J; Stephens, Jacqueline M; Mynatt, Randall L

    2003-03-07

    STAT 5 proteins are latent transcription factors which have been shown to be activated by growth hormone (GH) in many cell types. However, some recent studies also suggest that STAT 5B is a physiological substrate of the insulin receptor. In our studies, we have shown that physiological levels of insulin do not induce STAT 5 tyrosine phosphorylation or affect the nuclear distribution of STATs 5A or 5B in 3T3-L1 adipocytes. Moreover, we did not observe the activation of STAT 5 in the adipose tissue or skeletal muscle of mice following an acute intraperitoneal injection of insulin. However, acute GH administration, both in vitro and in vivo, resulted in the activation of STAT 5 proteins. In summary, our results indicate that STAT 5 proteins are not activated by physiological levels of insulin in adipose tissue.

  8. Nuclear Receptors Resolve Endoplasmic Reticulum Stress to Improve Hepatic Insulin Resistance

    PubMed Central

    2017-01-01

    Chronic endoplasmic reticulum (ER) stress culminating in proteotoxicity contributes to the development of insulin resistance and progression to type 2 diabetes mellitus. Pharmacologic interventions targeting several different nuclear receptors have emerged as potential treatments for insulin resistance. The mechanistic basis for these antidiabetic effects has primarily been attributed to multiple metabolic and inflammatory functions. Here we review recent advances in our understanding of the association of ER stress with insulin resistance and the role of nuclear receptors in promoting ER stress resolution and improving insulin resistance in the liver. PMID:28236381

  9. Insulin and IGF-1 receptors regulate FoxO-mediated signaling in muscle proteostasis

    PubMed Central

    O’Neill, Brian T.; Lee, Kevin Y.; Klaus, Katherine; Softic, Samir; Krumpoch, Megan T.; Stanford, Kristin I.; Robinson, Matthew M.; Cai, Weikang; Kleinridders, Andre; Pereira, Renata O.; Hirshman, Michael F.; Accili, Domenico; Goodyear, Laurie J.; Nair, K. Sreekumaran

    2016-01-01

    Diabetes strongly impacts protein metabolism, particularly in skeletal muscle. Insulin and IGF-1 enhance muscle protein synthesis through their receptors, but the relative roles of each in muscle proteostasis have not been fully elucidated. Using mice with muscle-specific deletion of the insulin receptor (M-IR–/– mice), the IGF-1 receptor (M-IGF1R–/– mice), or both (MIGIRKO mice), we assessed the relative contributions of IR and IGF1R signaling to muscle proteostasis. In differentiated muscle, IR expression predominated over IGF1R expression, and correspondingly, M-IR–/– mice displayed a moderate reduction in muscle mass whereas M-IGF1R–/– mice did not. However, these receptors serve complementary roles, such that double-knockout MIGIRKO mice displayed a marked reduction in muscle mass that was linked to increases in proteasomal and autophagy-lysosomal degradation, accompanied by a high-protein-turnover state. Combined muscle-specific deletion of FoxO1, FoxO3, and FoxO4 in MIGIRKO mice reversed increased autophagy and completely rescued muscle mass without changing proteasomal activity. These data indicate that signaling via IR is more important than IGF1R in controlling proteostasis in differentiated muscle. Nonetheless, the overlap of IR and IGF1R signaling is critical to the regulation of muscle protein turnover, and this regulation depends on suppression of FoxO-regulated, autophagy-mediated protein degradation. PMID:27525440

  10. Two steps of insulin receptor internalization depend on different domains of the beta-subunit [published erratum appears in J Cell Biol 1993 Nov;123(4):1047

    PubMed Central

    1993-01-01

    The internalization of signaling receptors such as the insulin receptor is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin receptor and to establish which receptor domains are implicated in each of these by the use of receptors possessing in vitro mutations. We find that kinase activation and autophosphorylation of all three regulatory tyrosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the COOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the receptor from microvilli where initial binding occurs to the nonvillous domain of the cell. Early intracellular steps in insulin signal transduction involving the activation of phosphatidylinositol 3'-kinase are not required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin- coated pits. In contrast to the first ligand specific step, this step is common to many receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but requires a juxta-membrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent mechanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit. PMID:8376461

  11. Regulation of insulin-like growth factor II receptors by growth hormone and insulin in rat adipocytes

    SciTech Connect

    Loennroth, P.; Assmundsson, K.; Eden, S.; Enberg, G.; Gause, I.; Hall, K.; Smith, U.

    1987-06-01

    The acute and long-term effects of growth hormone (GH) on the binding of insulin-like growth factor II (IGF-II) were evaluated in adipose cells from hypophysectomized rats given replacement therapy with thyroxine and hydrocortisone and in cells from their sham-operated littermates. After the cells were incubated with insulin and/or GH, the recycling of /sup 125/I-labeled IGF-II receptors was metabolically inhibited by treating the cells with KCN. IGF-II binding was 100 +/- 20% higher in cells from GH-deficient animals when compared with sham-operated controls. These GH-deficient cells also showed an increased sensitivity for insulin as compared with control cells (the EC/sub 50/ for insulin was 0.06 ng/ml in GH-deficient cells and 0.3 ng/ml in control cells.). However, the maximal incremental effect of insulin on IGH-II binding was reduced approx. = 27% by hypophysectomy. GH added to the incubation medium increased the number of IGF-II binding sites by 100 +/- 18% in cells from hypophysectomized animals. This increase was rapidly induced, but the time course was slower than that for the stimulatory effect of insulin. Half-maximal effect of GH on IGF-II binding was obtained at approx. = 30 ng/ml. Thus, GH added in vitro exerted a rapid insulin-like effect on the number of IGH-II receptors. GH also appears to play a regulating role for maintaining the cellular number of IGH-II receptors and, in addition, modulates the stimulatory effect of insulin on IGF-II binding.

  12. Treating Diabetes Mellitus: Pharmacophore Based Designing of Potential Drugs from Gymnema sylvestre against Insulin Receptor Protein

    PubMed Central

    Hossain, Mohammad Uzzal; Khan, Md. Arif; Rakib-Uz-Zaman, S. M.; Ali, Mohammad Tuhin; Islam, Md. Saidul; Keya, Chaman Ara; Salimullah, Md.

    2016-01-01

    Diabetes mellitus (DM) is one of the most prevalent metabolic disorders which can affect the quality of life severely. Injectable insulin is currently being used to treat DM which is mainly associated with patient inconvenience. Small molecules that can act as insulin receptor (IR) agonist would be better alternatives to insulin injection. Herein, ten bioactive small compounds derived from Gymnema sylvestre (G. sylvestre) were chosen to determine their IR binding affinity and ADMET properties using a combined approach of molecular docking study and computational pharmacokinetic elucidation. Designing structural analogues were also performed for the compounds associated with toxicity and less IR affinity. Among the ten parent compounds, six were found to have significant pharmacokinetic properties with considerable binding affinity towards IR while four compounds were associated with toxicity and less IR affinity. Among the forty structural analogues, four compounds demonstrated considerably increased binding affinity towards IR and less toxicity compared with parent compounds. Finally, molecular interaction analysis revealed that six parent compounds and four analogues interact with the active site amino acids of IR. So this study would be a way to identify new therapeutics and alternatives to insulin for diabetic patients. PMID:27034931

  13. Treating Diabetes Mellitus: Pharmacophore Based Designing of Potential Drugs from Gymnema sylvestre against Insulin Receptor Protein.

    PubMed

    Hossain, Mohammad Uzzal; Khan, Md Arif; Rakib-Uz-Zaman, S M; Ali, Mohammad Tuhin; Islam, Md Saidul; Keya, Chaman Ara; Salimullah, Md

    2016-01-01

    Diabetes mellitus (DM) is one of the most prevalent metabolic disorders which can affect the quality of life severely. Injectable insulin is currently being used to treat DM which is mainly associated with patient inconvenience. Small molecules that can act as insulin receptor (IR) agonist would be better alternatives to insulin injection. Herein, ten bioactive small compounds derived from Gymnema sylvestre (G. sylvestre) were chosen to determine their IR binding affinity and ADMET properties using a combined approach of molecular docking study and computational pharmacokinetic elucidation. Designing structural analogues were also performed for the compounds associated with toxicity and less IR affinity. Among the ten parent compounds, six were found to have significant pharmacokinetic properties with considerable binding affinity towards IR while four compounds were associated with toxicity and less IR affinity. Among the forty structural analogues, four compounds demonstrated considerably increased binding affinity towards IR and less toxicity compared with parent compounds. Finally, molecular interaction analysis revealed that six parent compounds and four analogues interact with the active site amino acids of IR. So this study would be a way to identify new therapeutics and alternatives to insulin for diabetic patients.

  14. A Novel Non-agonist Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand UHC1 Blocks PPARγ Phosphorylation by Cyclin-dependent Kinase 5 (CDK5) and Improves Insulin Sensitivity*

    PubMed Central

    Choi, Sun-Sil; Kim, Eun Sun; Koh, Minseob; Lee, Soo-Jin; Lim, Donghyun; Yang, Yong Ryoul; Jang, Hyun-Jun; Seo, Kyung-ah; Min, Sang-Hyun; Lee, In Hee; Park, Seung Bum; Suh, Pann-Ghill; Choi, Jang Hyun

    2014-01-01

    Thiazolidinedione class of anti-diabetic drugs which are known as peroxisome proliferator-activated receptor γ (PPARγ) ligands have been used to treat metabolic disorders, but thiazolidinediones can also cause several severe side effects, including congestive heart failure, fluid retention, and weight gain. In this study, we describe a novel synthetic PPARγ ligand UNIST HYUNDAI Compound 1 (UHC1) that binds tightly to PPARγ without the classical agonism and which blocks cyclin-dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation. We modified the non-agonist PPARγ ligand SR1664 chemically to improve its solubility and then developed a novel PPARγ ligand, UHC1. According to our docking simulation, UHC1 occupied the ligand-binding site of PPARγ with a higher docking score than SR1664. In addition, UHC1 more potently blocked CDK5-mediated PPARγ phosphorylation at Ser-273. Surprisingly, UHC1 treatment effectively ameliorated the inflammatory response both in vitro and in high-fat diet-fed mice. Furthermore, UHC1 treatment dramatically improved insulin sensitivity in high-fat diet-fed mice without causing fluid retention and weight gain. Taken together, compared with SR1664, UHC1 exhibited greater beneficial effects on glucose and lipid metabolism by blocking CDK5-mediated PPARγ phosphorylation, and these data indicate that UHC1 could be a novel therapeutic agent for use in type 2 diabetes and related metabolic disorders. PMID:25100724

  15. Rhodopsin-Regulated Insulin Receptor Signaling Pathway in Rod Photoreceptor Neurons

    PubMed Central

    Rajala, Raju V.S.; Anderson, Robert E.

    2010-01-01

    The retina is an integral part of the central nervous system and retinal cells are known to express insulin receptors (IR), although their function is not known. This article describes recent studies that link the photoactivation of rhodopsin to tyrosine phosphorylation of the IR and subsequent activation of phosphoinositide 3-kinase (PI3K), a neuron survival factor. Our studies suggest that the physiological role of this process is to provide neuroprotection of the retina against light-damage by activating proteins that protect against stress-induced apoptosis. We focus mainly on our recently identified regulation of the IR pathway through the G-protein-coupled receptor rhodopsin. Various mutant and knockout proteins of phototransduction cascade have been used to study the light-induced activation of the retinal IR. Our studies suggest that rhodopsin may have additional previously uncharacterized signaling functions in photoreceptors. PMID:20407846

  16. Insulin Dissociates the Effects of Liver X Receptor on Lipogenesis, Endoplasmic Reticulum Stress, and Inflammation*

    PubMed Central

    Sun, Xiaowei; Haas, Mary E.; Miao, Ji; Mehta, Abhiruchi; Graham, Mark J.; Crooke, Rosanne M.; de Barros, Jean-Paul Pais; Wang, Jian-Guo; Aikawa, Masanori; Masson, David; Biddinger, Sudha B.

    2016-01-01

    Diabetes is characterized by increased lipogenesis as well as increased endoplasmic reticulum (ER) stress and inflammation. The nuclear hormone receptor liver X receptor (LXR) is induced by insulin and is a key regulator of lipid metabolism. It promotes lipogenesis and cholesterol efflux, but suppresses endoplasmic reticulum stress and inflammation. The goal of these studies was to dissect the effects of insulin on LXR action. We used antisense oligonucleotides to knock down Lxrα in mice with hepatocyte-specific deletion of the insulin receptor and their controls. We found, surprisingly, that knock-out of the insulin receptor and knockdown of Lxrα produced equivalent, non-additive effects on the lipogenic genes. Thus, insulin was unable to induce the lipogenic genes in the absence of Lxrα, and LXRα was unable to induce the lipogenic genes in the absence of insulin. However, insulin was not required for LXRα to modulate the phospholipid profile, or to suppress genes in the ER stress or inflammation pathways. These data show that insulin is required specifically for the lipogenic effects of LXRα and that manipulation of the insulin signaling pathway could dissociate the beneficial effects of LXR on cholesterol efflux, inflammation, and ER stress from the negative effects on lipogenesis. PMID:26511317

  17. The effects of digestive enzymes on characteristics of placental insulin receptor. Comparison of particulate and soluble receptor preparations.

    PubMed Central

    Clark, S; DeLuise, M; Larkins, R G; Melick, R A; Harrison, L C

    1978-01-01

    The role of the surrounding membrane structure on the binding characteristics of the insulin receptor was studied by using several digestive enzymes. The effects observed with particulate membrane preparations are compared with those from soluble receptor preparations. beta-Galactosidase and neuraminidase had no effect on insulin binding to either particulate or soluble receptors from human placentae. Exposure to 2 units of phospholipase C/ml increased insulin binding to particulate membranes, but was without effect on the soluble receptor preparation. The increase in binding to particulate membranes was shown to be due to an increase in apparent receptor number. After 5 min exposure to 500 microgram of trypsin/ml there was an increase in insulin binding to the particulate membrane fraction, owing to an increase in receptor affinity. After 15 min exposure to this amount of trypsin, binding decreased, owing to a progressive decrease in receptor availability. In contrast, this concentration of trypsin had no effect on the solubilized receptor preparation. Because of the differential effects of phospholipase C and trypsin on the particulate compared with the solubilized receptor preparations, it is concluded that the effects of these enzymes were due to an effect on the surrounding membrane structure. Changes in receptor configuration due to alterations within the adjoining membrane provide a potential mechanism for mediating short-term alterations in receptor function. PMID:100106

  18. Cholinergic and glutamergic receptor functional regulation in long-term, low dose somatotropin and insulin treatment to ageing rats: rejuvenation of brain function.

    PubMed

    Balakrishnan, Savitha; Mathew, Jobin; Paulose, C S

    2010-01-15

    The role of somatotropin and insulin treatment in the regulation of neurotransmitter levels in the ageing brain is not fully established. We evaluated the long-term, low dose effects of somatotropin and insulin on acetylcholine and glutamate receptor subtypes functional regulation in the cerebral cortex of young (4-16 weeks) and old rats (60-90 weeks). Somatotropin and insulin treated young rats showed significant upregulation in muscarinic M1 and M3 expression whereas in old rats, somatotropin and insulin treatment downregulated M1 and M3 expression. N-methyl-D-aspartate and metabotropic glutamate receptor gene expression were significantly downregulated with somatotropin treatment while insulin treatment showed upregulation in both young and old rats. Acetylcholine esterase activity showed a decrease with age and after somatotropin and insulin treatment, the activity increased in both young and old rats. Electroencephalogram studies confirmed the brain wave activity in both young and old somatotropin and insulin treated rats. The results highlight long-term low dose somatotropin and insulin treatment in regulating cholinergic and glutamergic receptors subtypes in ageing rats and rejuvenation of brain function.

  19. Insulin receptor substrates Irs1 and Irs2 coordinate skeletal muscle growth and metabolism via the Akt and AMPK pathways.

    PubMed

    Long, Yun Chau; Cheng, Zhiyong; Copps, Kyle D; White, Morris F

    2011-02-01

    Coordination of skeletal muscle growth and metabolism with nutrient availability is critical for metabolic homeostasis. To establish the role of insulin-like signaling in this process, we used muscle creatine kinase (MCK)-Cre to disrupt expression of insulin receptor substrates Irs1 and Irs2 in mouse skeletal/cardiac muscle. In 2-week-old mice, skeletal muscle masses and insulin responses were slightly affected by Irs1, but not Irs2, deficiency. In contrast, the combined deficiency of Irs1 and Irs2 (MDKO mice) severely reduced skeletal muscle growth and Akt→mTOR signaling and caused death by 3 weeks of age. Autopsy of MDKO mice revealed dilated cardiomyopathy, reflecting the known requirement of insulin-like signaling for cardiac function (P. G. Laustsen et al., Mol. Cell. Biol. 27:1649-1664, 2007). Impaired growth and function of MDKO skeletal muscle were accompanied by increased Foxo-dependent atrogene expression and amino acid release. MDKO mice were resistant to injected insulin, and their isolated skeletal muscles showed decreased insulin-stimulated glucose uptake. Glucose utilization in MDKO mice and isolated skeletal muscles was shifted from oxidation to lactate production, accompanied by an elevated AMP/ATP ratio that increased AMP-activated protein kinase (AMPK)→acetyl coenzyme A carboxylase (ACC) phosphorylation and fatty acid oxidation. Thus, insulin-like signaling via Irs1/2 is essential to terminate skeletal muscle catabolic/fasting pathways in the presence of adequate nutrition.

  20. Insulin Receptor Substrate-1 Associates with Small Nucleolar RNA Which Contributes to Ribosome Biogenesis

    PubMed Central

    Ozoe, Atsufumi; Sone, Meri; Fukushima, Toshiaki; Kataoka, Naoyuki; Chida, Kazuhiro; Asano, Tomoichiro; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2014-01-01

    Insulin receptor substrates (IRSs) are well known to play crucial roles in mediating intracellular signals of insulin-like growth factors (IGFs)/insulin. Previously, we showed that IRS-1 forms high molecular mass complexes containing RNAs. To identify RNAs in IRS-1 complexes, we performed ultraviolet (UV) cross-linking and immunoprecipitation analysis using HEK293 cells expressing FLAG–IRS-1 and FLAG–IRS-2. We detected the radioactive signals in the immunoprecipitates of FLAG–IRS-1 proportional to the UV irradiation, but not in the immunoprecipitates of FLAG–IRS-2, suggesting the direct contact of RNAs with IRS-1. RNAs cross-linked to IRS-1 were then amplified by RT-PCR, followed by sequence analysis. We isolated sequence tags attributed to 25 messenger RNAs and 8 non-coding RNAs, including small nucleolar RNAs (snoRNAs). We focused on the interaction of IRS-1 with U96A snoRNA (U96A) and its host Rack1 (receptor for activated C kinase 1) pre-mRNA. We confirmed the interaction of IRS-1 with U96A, and with RACK1 pre-mRNA by immunoprecipitation with IRS-1 followed by Northern blotting or RT-PCR analyses. Mature U96A in IRS-1−/− mouse embryonic fibroblasts was quantitatively less than WT. We also found that a part of nuclear IRS-1 is localized in the Cajal body, a nuclear subcompartment where snoRNA mature. The unanticipated function of IRS-1 in snoRNA biogenesis highlights the potential of RNA-associated IRS-1 complex to open a new line of investigation to dissect the novel mechanisms regulating IGFs/insulin-mediated biological events. PMID:24624118

  1. Hepatocyte Toll-like receptor 4 regulates obesity-induced inflammation and insulin resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic low-grade inflammation is a hallmark of obesity and thought to contribute to the development of obesity-related insulin resistance. Toll-like receptor 4 (Tlr4) is a key mediator of pro-inflammatory responses. Mice lacking Tlr4s are protected from diet-induced insulin resistance and inflammat...

  2. BACE2 plays a role in the insulin receptor trafficking in pancreatic ß-cells.

    PubMed

    Casas, Silvia; Casini, Paola; Piquer, Sandra; Altirriba, Jordi; Soty, Maud; Cadavez, Lisa; Gomis, Ramon; Novials, Anna

    2010-12-01

    BACE1 (β-site amyloidogenic cleavage of precursor protein-cleaving enzyme 1) is a β-secretase protein that plays a central role in the production of the β-amyloid peptide in the brain and is thought to be involved in the Alzheimer's pathogenesis. In type 2 diabetes, amyloid deposition within the pancreatic islets is a pathophysiological hallmark, making crucial the study in the pancreas of BACE1 and its homologous BACE2 to understand the pathological mechanisms of this disease. The objectives of the present study were to characterize the localization of BACE proteins in human pancreas and determine their function. High levels of BACE enzymatic activity were detected in human pancreas. In normal human pancreas, BACE1 was observed in endocrine as well as in exocrine pancreas, whereas BACE2 expression was restricted to β-cells. Intracellular analysis using immunofluorescence showed colocalization of BACE1 with insulin and BACE2 with clathrin-coated vesicles of the plasma membrane in MIN6 cells. When BACE1 and -2 were pharmacologically inhibited, BACE1 localization was not altered, whereas BACE2 content in clathrin-coated vesicles was increased. Insulin internalization rate was reduced, insulin receptor β-subunit (IRβ) expression was decreased at the plasma membrane and increased in the Golgi apparatus, and a significant reduction in insulin gene expression was detected. Similar results were obtained after specific BACE2 silencing in MIN6 cells. All these data point to a role for BACE2 in the IRβ trafficking and insulin signaling. In conclusion, BACE2 is hereby presented as an important enzyme in β-cell function.

  3. Effects of heparin on insulin binding and biological activity

    SciTech Connect

    Kriauciunas, K.M.; Grigorescu, F.; Kahn, C.R.

    1987-02-01

    The effect of heparin, a polyanionic glycosaminoglycan known to alter the function of many proteins, on insulin binding and bioactivity was studied. Cultured human lymphocytes (IM-9) were incubated with varying concentrations of heparin, then extensively washed, and /sup 125/I-labeled insulin binding was measured. Heparin at concentrations used clinically for anticoagulation (1-50 U/ml) inhibited binding in a dose-dependent manner; 50% inhibition of binding occurred with 5-10 U/ml. Scatchard analysis indicated that the decrease in binding was due to a decrease in both the affinity and the apparent number of available insulin receptors. The effect occurred within 10 min at 22 degrees C and persisted even after the cells were extensively washed. Inhibition of insulin binding also occurred when cells were preincubated with heparinized plasma or heparinized serum but not when cells were incubated with normal serum or plasma from blood anticoagulated with EDTA. By contrast, other polyanions and polycations, e.g., poly-L-glutamic acid, poly-L-lysine, succinylated poly-L-lysine, and histone, did not inhibit binding. Heparin also inhibited insulin binding in Epstein-Barr (EB) virus-transformed lymphocytes but had no effect on insulin binding to isolated adipocytes, human erythrocytes, or intact hepatoma cells. When isolated adipocytes were incubated with heparin, there was a dose-dependent inhibition of insulin-stimulated glucose oxidation and, to a lesser extent, of basal glucose oxidation. Although heparin has no effect on insulin binding to intact hepatoma cells, heparin inhibited both insulin binding and insulin-stimulated autophosphorylation in receptors solubilized from these cells.

  4. A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells.

    PubMed

    Dai, Feihan F; Bhattacharjee, Alpana; Liu, Ying; Batchuluun, Battsetseg; Zhang, Ming; Wang, Xinye Serena; Huang, Xinyi; Luu, Lemieux; Zhu, Dan; Gaisano, Herbert; Wheeler, Michael B

    2015-10-09

    GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H(+)-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca(2+) influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion.

  5. Functionally significant insulin-like growth factor I receptor mutations in centenarians

    PubMed Central

    Suh, Yousin; Atzmon, Gil; Cho, Mi-Ook; Hwang, David; Liu, Bingrong; Leahy, Daniel J.; Barzilai, Nir; Cohen, Pinchas

    2008-01-01

    Rather than being a passive, haphazard process of wear and tear, lifespan can be modulated actively by components of the insulin/insulin-like growth factor I (IGFI) pathway in laboratory animals. Complete or partial loss-of-function mutations in genes encoding components of the insulin/IGFI pathway result in extension of life span in yeasts, worms, flies, and mice. This remarkable conservation throughout evolution suggests that altered signaling in this pathway may also influence human lifespan. On the other hand, evolutionary tradeoffs predict that the laboratory findings may not be relevant to human populations, because of the high fitness cost during early life. Here, we studied the biochemical, phenotypic, and genetic variations in a cohort of Ashkenazi Jewish centenarians, their offspring, and offspring-matched controls and demonstrated a gender-specific increase in serum IGFI associated with a smaller stature in female offspring of centenarians. Sequence analysis of the IGF1 and IGF1 receptor (IGF1R) genes of female centenarians showed overrepresentation of heterozygous mutations in the IGF1R gene among centenarians relative to controls that are associated with high serum IGFI levels and reduced activity of the IGFIR as measured in transformed lymphocytes. Thus, genetic alterations in the human IGF1R that result in altered IGF signaling pathway confer an increase in susceptibility to human longevity, suggesting a role of this pathway in modulation of human lifespan. PMID:18316725

  6. Characterization of a second ligand binding site of the insulin receptor

    SciTech Connect

    Hao Caili; Whittaker, Linda; Whittaker, Jonathan . E-mail: jonathan.whittaker@case.edu

    2006-08-18

    Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the {alpha} subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K {sub d} of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site.

  7. Alternative (M2) activation of Kupffer cells by PPARδ ameliorates obesity-induced insulin resistance

    PubMed Central

    Odegaard, Justin I.; Ricardo-Gonzalez, Roberto R.; Eagle, Alex Red; Vats, Divya; Morel, Christine R.; Goforth, Matthew H.; Subramanian, Vidya; Mukundan, Lata; Ferrante, Anthony W.; Chawla, Ajay

    2008-01-01

    SUMMARY Macrophage infiltration and activation in metabolic tissues underlie obesity-induced insulin resistance and type 2 diabetes. While inflammatory activation of resident hepatic macrophages potentiates insulin resistance, the functions of alternatively activated Kupffer cells in metabolic disease remain unknown. Here we show that, in response to the Th2 cytokine interleukin-4 (IL-4), peroxisome proliferator activated receptor δ (PPARδ) directs expression of the alternative phenotype in Kupffer cells and adipose tissue macrophages of lean mice. However, adoptive transfer of PPARδ null bone marrow into wild type mice only diminishes alternative activation of hepatic macrophages, causing hepatic dysfunction and systemic insulin resistance. Suppression of hepatic oxidative metabolism is recapitulated by treatment of primary hepatocytes with conditioned media from PPARδ null macrophages, indicating direct involvement of Kupffer cells in liver lipid metabolism. Taken together, these data suggest an unexpected beneficial role for alternatively activated Kupffer cells in metabolic syndrome and type 2 diabetes. PMID:18522831

  8. How IGF-1 activates its receptor

    PubMed Central

    Kavran, Jennifer M; McCabe, Jacqueline M; Byrne, Patrick O; Connacher, Mary Katherine; Wang, Zhihong; Ramek, Alexander; Sarabipour, Sarvenaz; Shan, Yibing; Shaw, David E; Hristova, Kalina; Cole, Philip A; Leahy, Daniel J

    2014-01-01

    The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation. DOI: http://dx.doi.org/10.7554/eLife.03772.001 PMID:25255214

  9. Decreased Insulin Receptors but Normal Glucose Metabolism in Duchenne Muscular Dystrophy

    NASA Astrophysics Data System (ADS)

    de Pirro, Roberto; Lauro, Renato; Testa, Ivano; Ferretti, Ginofabrizio; de Martinis, Carlo; Dellantonio, Renzo

    1982-04-01

    Compared to matched controls, 17 patients with Duchenne muscular dystrophy showed decreased insulin binding to monocytes due to decreased receptor concentration. These patients showed no signs of altered glucose metabolism and retrospective analysis of the clinical records of a further 56 such patients revealed no modification in carbohydrate metabolism. These data suggest that reduced insulin receptor number does not produce overt modifications of glucose metabolism in Duchenne muscular dystrophy.

  10. Nigella sativa Relieves the Altered Insulin Receptor Signaling in Streptozotocin-Induced Diabetic Rats Fed with a High-Fat Diet

    PubMed Central

    El-Zeftawy, Marwa; Taha, Nabil; Mandour, Abdel Wahab

    2016-01-01

    The black cumin (Nigella sativa) “NS” or the black seeds have many pharmacological activities such as antioxidant, anticarcinogenic, antihypertensive, and antidiabetic properties. In this work, streptozotocin-induced diabetic rats fed with a high-fat diet were treated daily with NS oil (NSO) in order to study the effect on the blood glucose, lipid profile, oxidative stress parameters, and the gene expression of some insulin receptor-induced signaling molecules. This treatment was combined also with some drugs (metformin and glimepiride) and the insulin receptor inhibitor I-OMe-AG538. The administration of NSO significantly induced the gene expression of insulin receptor compared to rats that did not receive NSO. Also, it upregulated the expression of insulin-like growth factor-1 and phosphoinositide-3 kinase, whereas the expression of ADAM-17 was downregulated. The expression of ADAM-17 is corroborated by the analysis of TIMP-3 content. In addition, the NSO significantly reduced blood glucose level, components of the lipid profile, oxidative stress parameters, serum insulin/insulin receptor ratio, and the tumor necrosis factor-α, confirming that NSO has an antidiabetic activity. Thus, the daily NSO treatment in our rat model indicates that NSO has a potential in the management of diabetes as well as improvement of insulin-induced signaling. PMID:27579151

  11. Structure of the insulin receptor ectodomain reveals a folded-over conformation.

    PubMed

    McKern, Neil M; Lawrence, Michael C; Streltsov, Victor A; Lou, Mei-Zhen; Adams, Timothy E; Lovrecz, George O; Elleman, Thomas C; Richards, Kim M; Bentley, John D; Pilling, Patricia A; Hoyne, Peter A; Cartledge, Kellie A; Pham, Tam M; Lewis, Jennifer L; Sankovich, Sonia E; Stoichevska, Violet; Da Silva, Elizabeth; Robinson, Christine P; Frenkel, Maurice J; Sparrow, Lindsay G; Fernley, Ross T; Epa, V Chandana; Ward, Colin W

    2006-09-14

    The insulin receptor is a phylogenetically ancient tyrosine kinase receptor found in organisms as primitive as cnidarians and insects. In higher organisms it is essential for glucose homeostasis, whereas the closely related insulin-like growth factor receptor (IGF-1R) is involved in normal growth and development. The insulin receptor is expressed in two isoforms, IR-A and IR-B; the former also functions as a high-affinity receptor for IGF-II and is implicated, along with IGF-1R, in malignant transformation. Here we present the crystal structure at 3.8 A resolution of the IR-A ectodomain dimer, complexed with four Fabs from the monoclonal antibodies 83-7 and 83-14 (ref. 4), grown in the presence of a fragment of an insulin mimetic peptide. The structure reveals the domain arrangement in the disulphide-linked ectodomain dimer, showing that the insulin receptor adopts a folded-over conformation that places the ligand-binding regions in juxtaposition. This arrangement is very different from previous models. It shows that the two L1 domains are on opposite sides of the dimer, too far apart to allow insulin to bind both L1 domains simultaneously as previously proposed. Instead, the structure implicates the carboxy-terminal surface of the first fibronectin type III domain as the second binding site involved in high-affinity binding.

  12. Toll-like receptor 4-induced endoplasmic reticulum stress contributes to impairment of vasodilator action of insulin

    PubMed Central

    Jang, Hyun-Ju; Hwang, Daniel H.

    2015-01-01

    Impairment of vasodilator action of insulin is associated with endothelial dysfunction and insulin resistance. Activation of Toll-like receptor 4 (TLR4) induces proinflammatory response and endoplasmic reticulum (ER) stress. Saturated fatty acids (SFA) activate TLR4, which induces ER stress and endothelial dysfunction. Therefore, we determined whether TLR4-mediated ER stress is an obligatory step mediating SFA-induced endothelial dysfunction. Palmitate stimulated proinflammatory responses and ER stress, and this was suppressed by knockdown of TLR4 in primary human aortic endothelial cells (HAEC). Next, we examined the role of TLR4 in vasodilatory responses in intact vessels isolated from wild-type (WT, C57BL/6) and TLR4-KO mice after feeding high-fat (HFD) or normal chow diet (NCD) for 12 wk. Arterioles isolated from HFD WT mice exhibited impaired insulin-stimulated vasodilation compared with arterioles isolated from NCD WT mice. Deficiency of TLR4 was protective from HFD-induced impairment of insulin-stimulated vasodilation. There were no differences in acetylcholine (Ach)- or sodium nitroprusside (SNP)-stimulated vasodilation between the two groups. Furthermore, we examined whether ER stress is involved in SFA-induced impairment of vasodilator actions of insulin. Infusion of palmitate showed the impairment of vasodilatory response to insulin, which was ameliorated by coinfusion with tauroursodeoxycholic acid (TUDCA), an ER stress suppressor. Taken together, the results suggest that TLR4-induced ER stress may be an obligatory step mediating the SFA-mediated endothelial dysfunction. PMID:26522062

  13. Distinct growth hormone receptor signaling modes regulate skeletal muscle development and insulin sensitivity in mice.

    PubMed

    Mavalli, Mahendra D; DiGirolamo, Douglas J; Fan, Yong; Riddle, Ryan C; Campbell, Kenneth S; van Groen, Thomas; Frank, Stuart J; Sperling, Mark A; Esser, Karyn A; Bamman, Marcas M; Clemens, Thomas L

    2010-11-01

    Skeletal muscle development, nutrient uptake, and nutrient utilization is largely coordinated by growth hormone (GH) and its downstream effectors, in particular, IGF-1. However, it is not clear which effects of GH on skeletal muscle are direct and which are secondary to GH-induced IGF-1 expression. Thus, we generated mice lacking either GH receptor (GHR) or IGF-1 receptor (IGF-1R) specifically in skeletal muscle. Both exhibited impaired skeletal muscle development characterized by reductions in myofiber number and area as well as accompanying deficiencies in functional performance. Defective skeletal muscle development, in both GHR and IGF-1R mutants, was attributable to diminished myoblast fusion and associated with compromised nuclear factor of activated T cells import and activity. Strikingly, mice lacking GHR developed metabolic features that were not observed in the IGF-1R mutants, including marked peripheral adiposity, insulin resistance, and glucose intolerance. Insulin resistance in GHR-deficient myotubes derived from reduced IR protein abundance and increased inhibitory phosphorylation of IRS-1 on Ser 1101. These results identify distinct signaling pathways through which GHR regulates skeletal muscle development and modulates nutrient metabolism.

  14. Neuronal Androgen Receptor Regulates Insulin Sensitivity via Suppression of Hypothalamic NF-κB–Mediated PTP1B Expression

    PubMed Central

    Yu, I-Chen; Lin, Hung-Yun; Liu, Ning-Chun; Sparks, Janet D.; Yeh, Shuyuan; Fang, Lei-Ya; Chen, Lumin; Chang, Chawnshang

    2013-01-01

    Clinical investigations highlight the increased incidence of metabolic syndrome in prostate cancer (PCa) patients receiving androgen deprivation therapy (ADT). Studies using global androgen receptor (AR) knockout mice demonstrate that AR deficiency results in the development of insulin resistance in males. However, mechanisms by which AR in individual organs coordinately regulates insulin sensitivity remain unexplored. Here we tested the hypothesis that functional AR in the brain contributes to whole-body insulin sensitivity regulation and to the metabolic abnormalities developed in AR-deficient male mice. The mouse model selectively lacking AR in the central nervous system and AR-expressing GT1-7 neuronal cells were established and used to delineate molecular mechanisms in insulin signaling modulated by AR. Neuronal AR deficiency leads to reduced insulin sensitivity in middle-aged mice. Neuronal AR regulates hypothalamic insulin signaling by repressing nuclear factor-κB (NF-κB)–mediated induction of protein-tyrosine phosphatase 1B (PTP1B). Hypothalamic insulin resistance leads to hepatic insulin resistance, lipid accumulation, and visceral obesity. The functional deficiency of AR in the hypothalamus leads to male mice being more susceptible to the effects of high-fat diet consumption on PTP1B expression and NF-κB activation. These findings suggest that in men with PCa undergoing ADT, reduction of AR function in the brain may contribute to insulin resistance and visceral obesity. Pharmacotherapies targeting neuronal AR and NF-κB may be developed to combat the metabolic syndrome in men receiving ADT and in elderly men with age-associated hypogonadism. PMID:23139353

  15. Chronic renin inhibition with aliskiren improves glucose tolerance, insulin sensitivity, and skeletal muscle glucose transport activity in obese Zucker rats.

    PubMed

    Marchionne, Elizabeth M; Diamond-Stanic, Maggie K; Prasonnarong, Mujalin; Henriksen, Erik J

    2012-01-01

    We have demonstrated previously that overactivity of the renin-angiotensin system (RAS) is associated with whole body and skeletal muscle insulin resistance in obese Zucker (fa/fa) rats. Moreover, this obesity-associated insulin resistance is reduced by treatment with angiotensin-converting enzyme inhibitors or angiotensin receptor (type 1) blockers. However, it is currently unknown whether specific inhibition of renin itself, the rate-limiting step in RAS functionality, improves insulin action in obesity-associated insulin resistance. Therefore, the present study assessed the effect of chronic, selective renin inhibition using aliskiren on glucose tolerance, whole body insulin sensitivity, and insulin action on the glucose transport system in skeletal muscle of obese Zucker rats. Obese Zucker rats were treated for 21 days with either vehicle or aliskiren (50 mg/kg body wt ip). Renin inhibition was associated with a significant lowering (10%, P < 0.05) of resting systolic blood pressure and induced reductions in fasting plasma glucose (11%) and free fatty acids (46%) and homeostatic model assessment for insulin resistance (13%). Glucose tolerance (glucose area under the curve) and whole body insulin sensitivity (inverse of the glucose-insulin index) during an oral glucose tolerance test were improved by 15% and 16%, respectively, following chronic renin inhibition. Moreover, insulin-stimulated glucose transport activity in isolated soleus muscle of renin inhibitor-treated animals was increased by 36% and was associated with a 2.2-fold greater Akt Ser(473) phosphorylation. These data provide evidence that chronic selective inhibition of renin activity leads to improvements in glucose tolerance and whole body insulin sensitivity in the insulin-resistant obese Zucker rat. Importantly, chronic renin inhibition is associated with upregulation of insulin action on skeletal muscle glucose transport, and it may involve improved Akt signaling. These data support the

  16. Phosphatidylinositol 3'-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase.

    PubMed Central

    Cengel, K A; Kason, R E; Freund, G G

    1998-01-01

    Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3'-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase-IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate. PMID:9761740

  17. The Macrophage A2b Adenosine Receptor Regulates Tissue Insulin Sensitivity

    PubMed Central

    Koupenova, Milka; Carroll, Shannon; Ravid, Katya

    2014-01-01

    High fat diet (HFD)-induced type 2 diabetes continues to be an epidemic with significant risk for various pathologies. Previously, we identified the A2b adenosine receptor (A2bAR), an established regulator of inflammation, as a regulator of HFD-induced insulin resistance. In particular, HFD was associated with vast upregulation of liver A2bAR in control mice, and while mice lacking this receptor showed augmented liver inflammation and tissue insulin resistance. As the A2bAR is expressed in different tissues, here, we provide the first lead to cellular mechanism by demonstrating that the receptor's influence on tissue insulin sensitivity is mediated via its expression in macrophages. This was shown using a newly generated transgenic mouse model expressing the A2bAR gene in the macrophage lineage on an otherwise A2bAR null background. Reinstatement of macrophage A2bAR expression in A2bAR null mice fed HFD restored insulin tolerance and tissue insulin signaling to the level of control mice. The molecular mechanism for this effect involves A2bAR-mediated changes in cyclic adenosine monophosphate in macrophages, reducing the expression and release of inflammatory cytokines, which downregulate insulin receptor-2. Thus, our results illustrate that macrophage A2bAR signaling is needed and sufficient for relaying the protective effect of the A2bAR against HFD-induced tissue inflammation and insulin resistance in mice. PMID:24892847

  18. Modularity and hormone sensitivity of the Drosophila melanogaster insulin receptor/target of rapamycin interaction proteome.

    PubMed

    Glatter, Timo; Schittenhelm, Ralf B; Rinner, Oliver; Roguska, Katarzyna; Wepf, Alexander; Jünger, Martin A; Köhler, Katja; Jevtov, Irena; Choi, Hyungwon; Schmidt, Alexander; Nesvizhskii, Alexey I; Stocker, Hugo; Hafen, Ernst; Aebersold, Ruedi; Gstaiger, Matthias

    2011-11-08

    Genetic analysis in Drosophila melanogaster has been widely used to identify a system of genes that control cell growth in response to insulin and nutrients. Many of these genes encode components of the insulin receptor/target of rapamycin (InR/TOR) pathway. However, the biochemical context of this regulatory system is still poorly characterized in Drosophila. Here, we present the first quantitative study that systematically characterizes the modularity and hormone sensitivity of the interaction proteome underlying growth control by the dInR/TOR pathway. Applying quantitative affinity purification and mass spectrometry, we identified 97 high confidence protein interactions among 58 network components. In all, 22% of the detected interactions were regulated by insulin affecting membrane proximal as well as intracellular signaling complexes. Systematic functional analysis linked a subset of network components to the control of dTORC1 and dTORC2 activity. Furthermore, our data suggest the presence of three distinct dTOR kinase complexes, including the evolutionary conserved dTTT complex (Drosophila TOR, TELO2, TTI1). Subsequent genetic studies in flies suggest a role for dTTT in controlling cell growth via a dTORC1- and dTORC2-dependent mechanism.

  19. Modularity and hormone sensitivity of the Drosophila melanogaster insulin receptor/target of rapamycin interaction proteome

    PubMed Central

    Glatter, Timo; Schittenhelm, Ralf B; Rinner, Oliver; Roguska, Katarzyna; Wepf, Alexander; Jünger, Martin A; Köhler, Katja; Jevtov, Irena; Choi, Hyungwon; Schmidt, Alexander; Nesvizhskii, Alexey I; Stocker, Hugo; Hafen, Ernst; Aebersold, Ruedi; Gstaiger, Matthias

    2011-01-01

    Genetic analysis in Drosophila melanogaster has been widely used to identify a system of genes that control cell growth in response to insulin and nutrients. Many of these genes encode components of the insulin receptor/target of rapamycin (InR/TOR) pathway. However, the biochemical context of this regulatory system is still poorly characterized in Drosophila. Here, we present the first quantitative study that systematically characterizes the modularity and hormone sensitivity of the interaction proteome underlying growth control by the dInR/TOR pathway. Applying quantitative affinity purification and mass spectrometry, we identified 97 high confidence protein interactions among 58 network components. In all, 22% of the detected interactions were regulated by insulin affecting membrane proximal as well as intracellular signaling complexes. Systematic functional analysis linked a subset of network components to the control of dTORC1 and dTORC2 activity. Furthermore, our data suggest the presence of three distinct dTOR kinase complexes, including the evolutionary conserved dTTT complex (Drosophila TOR, TELO2, TTI1). Subsequent genetic studies in flies suggest a role for dTTT in controlling cell growth via a dTORC1- and dTORC2-dependent mechanism. PMID:22068330

  20. Physical activity, insulin action, and diabetes prevention and control.

    PubMed

    Colberg, Sheri R

    2007-08-01

    Control of blood glucose levels in individuals with diabetes mellitus (DM) is directly affected by the balance between insulin and glucose-raising endocrine hormones, along with other metabolic factors, including fuel use and availability, exercise intensity and duration, training status, and visceral fat levels, all of which can impact the effect of physical activity on insulin action in diabetic or prediabetic individuals. Current research suggests that type 2 DM can be prevented and controlled with increased physical activity, largely through improvements in the muscles' sensitivity to insulin that are affected by changes in both glucose and fat metabolism. In addition, abnormal insulin action in the body is associated with a host of other health conditions, including cardiovascular disease and hypertension, which can be better controlled when their associations are fully understood. This article discusses the importance of varying types of physical activity on insulin action to enhance metabolic control and how they can be undertaken safely by all diabetic individuals.

  1. In skeletal muscle advanced glycation end products (AGEs) inhibit insulin action and induce the formation of multimolecular complexes including the receptor for AGEs.

    PubMed

    Cassese, Angela; Esposito, Iolanda; Fiory, Francesca; Barbagallo, Alessia P M; Paturzo, Flora; Mirra, Paola; Ulianich, Luca; Giacco, Ferdinando; Iadicicco, Claudia; Lombardi, Angela; Oriente, Francesco; Van Obberghen, Emmanuel; Beguinot, Francesco; Formisano, Pietro; Miele, Claudia

    2008-12-26

    Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Calpha (PKCalpha). Here we show that HGA-induced PKCalpha activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCalpha in HGA-treated L6 cells. A direct interaction of PKCalpha with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCalpha co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCalpha activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCalpha. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCalpha.

  2. Regulation of Gene33 expression by insulin requires MEK-ERK activation.

    PubMed

    Keeton, Adam B; Xu, Jie; Franklin, J Lee; Messina, Joseph L

    2004-09-17

    Gene33 and its human homologue, mitogen inducible gene-6/receptor-associated late transducer (mig-6, RALT), is a 53-kDa soluble protein that was identified as a hepatic gene regulated by glucocorticoids and insulin. Its mRNA is expressed in numerous tissues in addition to the liver. Mitogen inducibility of Gene33 mRNA has been described in several experimental systems. Recent reports have suggested a role for Gene33 in inhibition of proliferation induced by factors that bind to members of the ErbB family of receptors. In the present work, we examine the regulation of Gene33 protein by insulin in hepatoma cells of rat (H4IIE) and human (HepG2/Hep3B) origin. Inhibition of MEK1 significantly inhibited extracellularly regulated kinase (ERK)1/2 activation and insulin-regulated Gene33 transcription and protein levels in H4IIE cells. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity alone did not significantly alter transcription of Gene33. In Hep3B and HepG2 cells, insulin did not significantly induce either ERK1/2 activation or Gene33 expression. This work suggests that the MEK-ERK, but not the phosphatidylinositol 3-kinase (PI3-K), pathway plays a direct role in insulin regulation of Gene33 transcription and protein expression.

  3. Insulin activates EGFR by stimulating its interaction with IGF-1R in low-EGFR-expressing TNBC cells.

    PubMed

    Shin, Miyoung; Yang, Eun Gyeong; Song, Hyun Kyu; Jeon, Hyesung

    2015-06-01

    The expression of epidermal growth factor receptor (EGFR) is an important diagnostic marker for triple-negative breast cancer (TNBC) cells, which lack three hormonal receptors: estrogen and progesterone receptors as well as epidermal growth factor receptor 2. EGFR transactivation can cause drug resistance in many cancers including TNBC, but the mechanism underlying this phenomenon is poorly defined. Here, we demonstrate that insulin treatment induces EGFR activation by stimulating the interaction of EGFR with insulin-like growth factor receptor 1 (IGF-1R) in the MDA-MB-436 TNBC cell line. These cells express low levels of EGFR, while exhibiting high levels of IGF-1R expression and phosphorylation. Low-EGFRexpressing MDA-MB-436 cells show high sensitivity to insulinstimulated cell growth. Therefore, unexpectedly, insulin stimulation induced EGFR transactivation by regulating its interaction with IGF-1R in low-EGFR-expressing TNBC cells.

  4. Fatty acid represses insulin receptor gene expression by impairing HMGA1 through protein kinase C{epsilon}

    SciTech Connect

    Dey, Debleena; Bhattacharya, Anirban; Roy, SibSankar; Bhattacharya, Samir . E-mail: smrbhattacharya@gmail.com

    2007-06-01

    It is known that free fatty acid (FFA) contributes to the development of insulin resistance and type2 diabetes. However, the underlying mechanism in FFA-induced insulin resistance is still unclear. In the present investigation we have demonstrated that palmitate significantly (p < 0.001) inhibited insulin-stimulated phosphorylation of PDK1, the key insulin signaling molecule. Consequently, PDK1 phosphorylation of plasma membrane bound PKC{epsilon} was also inhibited. Surprisingly, phosphorylation of cytosolic PKC{epsilon} was greatly stimulated by palmitate; this was then translocated to the nuclear region and associated with the inhibition of insulin receptor (IR) gene transcription. A PKC{epsilon} translocation inhibitor peptide, {epsilon}V1, suppressed this inhibitory effect of palmitate, suggesting requirement of phospho-PKC{epsilon} migration to implement palmitate effect. Experimental evidences indicate that phospho-PKC{epsilon} adversely affected HMGA1. Since HMGA1 regulates IR promoter activity, expression of IR gene was impaired causing reduction of IR on cell surface and that compromises with insulin sensitivity.

  5. A Novel Approach to Identify Two Distinct Receptor Binding Surfaces of Insulin-like Growth Factor II*S⃞

    PubMed Central

    Alvino, Clair L.; McNeil, Kerrie A.; Ong, Shee Chee; Delaine, Carlie; Booker, Grant W.; Wallace, John C.; Whittaker, Jonathan; Forbes, Briony E.

    2009-01-01

    Very little is known about the residues important for the interaction of insulin-like growth factor II (IGF-II) with the type 1 IGF receptor (IGF-1R) and the insulin receptor (IR). Insulin, to which IGF-II is homologous, is proposed to cross-link opposite halves of the IR dimer through two receptor binding surfaces, site 1 and site 2. In the present study we have analyzed the contribution of IGF-II residues equivalent to insulin's two binding surfaces toward the interaction of IGF-II with the IGF-1R and IR. Four “site 1” and six “site 2” analogues were produced and analyzed in terms of IGF-1R and IR binding and activation. The results show that Val43, Phe28, and Val14 (equivalent to site 1) are critical to IGF-1R and IR binding, whereas mutation to alanine of Gln18 affects only IGF-1R and not IR binding. Alanine substitutions at Glu12, Asp15, Phe19, Leu53, and Glu57 analogues resulted in significant (>2-fold) decreases in affinity for both the IGF-1R and IR. Furthermore, taking a novel approach using a monomeric, single-chain minimized IGF-1R we have defined a distinct second binding surface formed by Glu12, Phe19, Leu53, and Glu57 that potentially engages the IGF-1R at one or more of the FnIII domains. PMID:19139090

  6. Insulin Receptor Substrate-4 Binds to Slingshot-1 Phosphatase and Promotes Cofilin Dephosphorylation*

    PubMed Central

    Homma, Yuta; Kanno, Shin-ichiro; Sasaki, Kazutaka; Nishita, Michiru; Yasui, Akira; Asano, Tomoichiro; Ohashi, Kazumasa; Mizuno, Kensaku

    2014-01-01

    Cofilin plays an essential role in cell migration and morphogenesis by enhancing actin filament dynamics via its actin filament-severing activity. Slingshot-1 (SSH1) is a protein phosphatase that plays a crucial role in regulating actin dynamics by dephosphorylating and reactivating cofilin. In this study, we identified insulin receptor substrate (IRS)-4 as a novel SSH1-binding protein. Co-precipitation assays revealed the direct endogenous binding of IRS4 to SSH1. IRS4, but not IRS1 or IRS2, was bound to SSH1. IRS4 was bound to SSH1 mainly through the unique region (amino acids 335–400) adjacent to the C terminus of the phosphotyrosine-binding domain of IRS4. The N-terminal A, B, and phosphatase domains of SSH1 were bound to IRS4 independently. Whereas in vitro phosphatase assays revealed that IRS4 does not directly affect the cofilin phosphatase activity of SSH1, knockdown of IRS4 increased cofilin phosphorylation in cultured cells. Knockdown of IRS4 decreased phosphatidylinositol 3-kinase (PI3K) activity, and treatment with an inhibitor of PI3K increased cofilin phosphorylation. Akt preferentially phosphorylated SSH1 at Thr-826, but expression of a non-phosphorylatable T826A mutant of SSH1 did not affect insulin-induced cofilin dephosphorylation, and an inhibitor of Akt did not increase cofilin phosphorylation. These results suggest that IRS4 promotes cofilin dephosphorylation through sequential activation of PI3K and SSH1 but not through Akt. In addition, IRS4 co-localized with SSH1 in F-actin-rich membrane protrusions in insulin-stimulated cells, which suggests that the association of IRS4 with SSH1 contributes to localized activation of cofilin in membrane protrusions. PMID:25100728

  7. Venus Kinase Receptors at the Crossroads of Insulin Signaling: Their Role in Reproduction for Helminths and Insects.

    PubMed

    Dissous, Colette

    2015-01-01

    Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (TKs) first discovered in the human parasite Schistosoma. They contain an extracellular Venus FlyTrap module similar to the ligand-binding domain of G protein-coupled receptors of class C and an intracellular TK domain similar to that of insulin receptors. VKRs are present from cnidarians to echinoderms. They were shown to be activated by amino-acids, to induce insulin-like intracellular pathways, and to be highly expressed in larvae and in gonads of helminths and insects. The function of VKR in gametogenesis was demonstrated in schistosomes by VKR silencing and recent studies in Aedes aegypti have confirmed the importance of VKR in mosquito egg formation. AaeVKR was shown to bind to ovary ecdysteroidogenic hormone and to activate the production of ecdysteroids by the ovary, independently of signaling mediated by insulin-like peptides. These new data confirm and specify the function of VKRs in the reproduction of helminths and insects and they open interesting perspectives for elucidating the role of VKRs in other models. VKR targeting would also provide opportunities for the control of parasites and various vector-borne infectious diseases.

  8. Semisynthesis and biological activity of porcine [LeuB24]insulin and [LeuB25]insulin.

    PubMed Central

    Tager, H; Thomas, N; Assoian, R; Rubenstein, A; Saekow, M; Olefsky, J; Kaiser, E T

    1980-01-01

    Two analogs of porcine insulin with substitutions of leucine for phenylalanine in the COOH-terminal region of the insulin B chain have been prepared by a combination of solid-phase synthesis and semisynthesis. Solid-phase synthesis of the substituted octapeptides B23-B30 bearing the trifluoracetyl group on lysine-B29, enzymatic coupling of the octapeptides to bis(tertiary-butyloxycarbonyl)desoctapeptide insulin by trypsin, and deprotection of the corresponding adducts in formic acid and piperidine resulted in two insulin derivatives, one with leucine at position B24 and the other with leucine at position B25. These analogs had only about 10% and 1%, respectively, of the activity of porcine insulin in competing for the binding of [125I]iodoinsulin to both rat adipocytes and human IM-9 lymphocytes. The relative potencies of the analogs in stimulating glucose oxidation by rat adipocytes decreased in the order porcine insulin > [LeuB24]insulin > [LeuB25]insulin. However, at high concentrations both analogs had full agonists activity. Experiments in which the semisynthetic insulins were mixed with the native hormone showed that [LeuB24]insulin, but not [LeuB25]insulin, was an active antagonist of insulin action. These results suggest that the antagonistic activity of a human insulin variant having leucine at position B24 or B25 can be assigned to the molecule with the sequence Gly-Leu-Phe-Tyr (residues B23-B26) in its active site. Images PMID:6997872

  9. Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling.

    PubMed

    Kuo, Taiyi; Lew, Michelle J; Mayba, Oleg; Harris, Charles A; Speed, Terence P; Wang, Jen-Chywan

    2012-07-10

    Glucocorticoids elicit a variety of biological responses in skeletal muscle, including inhibiting protein synthesis and insulin-stimulated glucose uptake and promoting proteolysis. Thus, excess or chronic glucocorticoid exposure leads to muscle atrophy and insulin resistance. Glucocorticoids propagate their signal mainly through glucocorticoid receptors (GR), which, upon binding to ligands, translocate to the nucleus and bind to genomic glucocorticoid response elements to regulate the transcription of nearby genes. Using a combination of chromatin immunoprecipitation sequencing and microarray analysis, we identified 173 genes in mouse C2C12 myotubes. The mouse genome contains GR-binding regions in or near these genes, and gene expression is regulated by glucocorticoids. Eight of these genes encode proteins known to regulate distinct signaling events in insulin/insulin-like growth factor 1 pathways. We found that overexpression of p85α, one of these eight genes, caused a decrease in C2C12 myotube diameters, mimicking the effect of glucocorticoids. Moreover, reducing p85α expression by RNA interference in C2C12 myotubes significantly compromised the ability of glucocorticoids to inhibit Akt and p70 S6 kinase activity and reduced glucocorticoid induction of insulin receptor substrate 1 phosphorylation at serine 307. This phosphorylation is associated with insulin resistance. Furthermore, decreasing p85α expression abolished glucocorticoid inhibition of protein synthesis and compromised glucocorticoid-induced reduction of cell diameters in C2C12 myotubes. Finally, a glucocorticoid response element was identified in the p85α GR-binding regions. In summary, our studies identified GR-regulated transcriptional networks in myotubes and showed that p85α plays a critical role in glucocorticoid-induced insulin resistance and muscle atrophy in C2C12 myotubes.

  10. Identification of common ligand binding determinants of the insulin and insulin-like growth factor 1 receptors. Insights into mechanisms of ligand binding.

    PubMed

    Mynarcik, D C; Williams, P F; Schaffer, L; Yu, G Q; Whittaker, J

    1997-07-25

    Insulin and insulin-like growth factor 1 (IGF-1) are peptides that share nearly 50% sequence homology. However, although their cognate receptors also exhibit significant overall sequence homology, the affinity of each peptide for the non-cognate receptor is 2-3 orders of magnitude lower than for the cognate receptor. The molecular basis for this discrimination is unclear, as are the molecular mechanisms underlying ligand binding. We have recently identified a major ligand binding site of the insulin receptor by alanine scannning mutagenesis. These studies revealed that a number of amino acids critical for insulin binding are conserved in the IGF-1 receptor, suggesting that they may play a role in ligand binding. We therefore performed alanine mutagenesis of these amino acids to determine whether this is the case. cDNAs encoding alanine-substituted secreted recombinant IGF-1 receptors were expressed in 293 EBNA cells, and the ligand binding properties of the expressed proteins were evaluated. Mutation of Phe701 resulted in a receptor with undetectable IGF-1 binding; alanine substitution of the corresponding amino acid of the insulin receptor, Phe714, produces a 140-fold reduction in affinity for insulin. Mutation of Asp8, Asn11, Phe58, Phe692, Glu693, His697, and Asn698 produces a 3.5-6-fold reduction in affinity for IGF-1. In contrast, alanine mutation of the corresponding amino acids of the insulin receptor with the exception of Asp12 produces reductions in affinity that are 50-fold or greater. The affinity of insulin for these mutants relative to wild type receptor was similar to that of their relative affinity for IGF-1 with two exceptions; the IC50 values for insulin binding to the mutants of Arg10, which has normal affinity for IGF-1, and His697, which has a 6-fold reduction in affinity for IGF-1, were both at least 2 orders of magnitude greater than for wild type receptor. The Kd values for insulin of the corresponding alanine mutants of the insulin receptor

  11. Validation of Algorithms for Basal Insulin Rate Reductions in Type 1 Diabetic Patients Practising Physical Activity

    ClinicalTrials.gov

    2013-04-19

    Type 1 Diabetes With a Subcutaneous Insulin Pump; Adjustment of the Recommended Basal Insulin Flow Rate in the Event of Physical Activity; Adjustment of the Recommended Prandial Insulin in the Event of Physical Activity

  12. Removal of melatonin receptor type 1 induces insulin resistance in the mouse.

    PubMed

    Contreras-Alcantara, Susana; Baba, Kenkichi; Tosini, Gianluca

    2010-09-01

    The incidence of obesity, insulin resistance, and type 2 diabetes (T2D) is increasing at an alarming rate worldwide. Emerging experimental evidence suggests that the hormone melatonin plays an important role in the regulation of glucose metabolisms. In this study, we report that removal of melatonin receptor type 1 (MT1) significantly impairs the ability of mice to metabolize glucose and such inability is probably due to an increased insulin resistance in these mice. Our data suggest that MT1 receptors are implicated in the pathogenesis of T2D and open the door for a detailed exploration on the mechanisms by which MT1 receptors signaling may affect glucose metabolism.

  13. Reduced insulin-receptor mediated modulation of striatal dopamine release by basal insulin as a possible contributing factor to hyperdopaminergia in schizophrenia

    PubMed Central

    Caravaggio, Fernando; Hahn, Margaret; Nakajima, Shinichiro; Gerretsen, Philip; Remington, Gary; Graff-Guerrero, Ariel

    2017-01-01

    Schizophrenia is a severe and chronic neuropsychiatric disorder which affects 1% of the world population. Using the brain imaging technique positron emission tomography (PET) it has been demonstrated that persons with schizophrenia have greater dopamine transmission in the striatum compared to healthy controls. However, little progress has been made as to elucidating other biological mechanisms which may account for this hyperdopaminergic state in this disease. Studies in animals have demonstrated that insulin receptors are expressed on midbrain dopamine neurons, and that insulin from the periphery acts on these receptors to modify dopamine transmission in the striatum. This is pertinent given that several lines of evidence suggest that insulin receptor functioning may be abnormal in the brains of persons with schizophrenia. Post-mortem studies have shown that persons with schizophrenia have less than half the number of cortical insulin receptors compared to healthy persons. Moreover, these post-mortem findings are unlikely due to the effects of antipsychotic treatment; studies in cell lines and animals suggest antipsychotics enhance insulin receptor functioning. Further, hyperinsulinemia – even prior to antipsychotic use – seems to be related to less psychotic symptoms in patients with schizophrenia. Collectively, these data suggest that midbrain insulin receptor functioning may be abnormal in persons with schizophrenia, resulting in reduced insulin-mediated regulation of dopamine transmission in the striatum. Such a deficit may account for the hyperdopaminergic state observed in these patients and would help guide the development of novel treatment strategies. We hypothesize that, (i) insulin receptor expression and/or function is reduced in midbrain dopamine neurons in persons with schizophrenia, (ii) basal insulin should reduce dopaminergic transmission in the striatum via these receptors, and (iii) this modulation of dopaminergic transmission by basal

  14. Antihyperglycemic and Insulin Secretagogue Activities of Abrus precatorius Leaf Extract

    PubMed Central

    Umamahesh, Balekari; Veeresham, Ciddi

    2016-01-01

    Aim: Abrus precatorius leaves methanolic extract (APME) was evaluated for in vivo antihyperglycemic activity and in vitro insulinotropic effect. Materials and Methods: In vivo antihyperglycemic and insulin secretagogue activities were assessed in streptozotocin-induced diabetic rats by oral administration of APME (200 mg/kg body weight [bw]) for 28 days. In vitro insulin secretion mechanisms were studied using mouse insulinoma beta cells (MIN6-β). In vivo body weight and blood glucose and in vivo and in vitro insulin levels were estimated. Results: In diabetic rats, APME treatment significantly restored body weight (26.39%), blood glucose (32.39%), and insulin levels (73.95%) in comparison to diabetic control rats. In MIN6-β cells, APME potentiated insulin secretion in a dependent manner of glucose (3–16.7 mM) and extract (5–500 μg/mL) concentration. Insulin secretagogue effect was demonstrated in the presence of 3-isobutyl-1-methyl xanthine, glibenclamide, elevated extracellular calcium, and K+ depolarized media. Insulin release was reduced in the presence of nifedipine, ethylene glycol tetra acetic acid (calcium blocking agents), and diazoxide (potassium channel opener). Conclusion: The study suggests that APME antihyperglycemic activity might involve the insulin secretagogue effect by pancreatic beta cells physiological pathways via K+-ATP channel dependent and independently, along with an effect on Ca2+ channels. SUMMARY Abrus precatorius leaves methanolic extract (APME) showed a significant anti hyperglycemic and insulin secretagogue activities in streptozotocin induced diabetic rats. Also demonstrated a potent In vitro insulin secretagogue effect in mouse insulinoma beta cells (MIN6-β)APME treatment significantly restored body weight (26.39%), reduced blood glucose (32.39%) and enhanced circulatory insulin levels (73.95%) in diabetic ratsAPME demonstrated glucose and extract dose dependent insulin secretionInsulin secretagogue effect was demonstrated

  15. The insulin receptor juxtamembrane region contains two independent tyrosine/beta-turn internalization signals

    PubMed Central

    1992-01-01

    We have investigated the role of tyrosine residues in the insulin receptor cytoplasmic juxtamembrane region (Tyr953 and Tyr960) during endocytosis. Analysis of the secondary structure of the juxtamembrane region by the Chou-Fasman algorithms predicts that both the sequences GPLY953 and NPEY960 form tyrosine-containing beta-turns. Similarly, analysis of model peptides by 1-D and 2-D NMR show that these sequences form beta-turns in solution, whereas replacement of the tyrosine residues with alanine destabilizes the beta-turn. CHO cell lines were prepared expressing mutant receptors in which each tyrosine was mutated to phenylalanine or alanine, and an additional mutant contained alanine at both positions. These mutations had no effect on insulin binding or receptor autophosphorylation. Replacements with phenylalanine had no effect on the rate of [125I]insulin endocytosis, whereas single substitutions with alanine reduced [125I]insulin endocytosis by 40-50%. Replacement of both tyrosines with alanine reduced internalization by 70%. These data suggest that the insulin receptor contains two tyrosine/beta-turns which contribute independently and additively to insulin-stimulated endocytosis. PMID:1500426

  16. Role of β-Adrenergic Receptor Regulation of TNF-α and Insulin Signaling in Retinal Müller Cells

    PubMed Central

    Walker, Robert J.; Anderson, Nancy M.; Jiang, Youde; Bahouth, Suleiman

    2011-01-01

    Purpose. The goal of this study was to determine the relationship of TNF-α and the downregulation of insulin receptor signaling in retinal Müller cells cultured under hyperglycemic conditions and the role of β-adrenergic receptors in regulating these responses. Methods. Retinal Müller cells were cultured in normal (5 mM) or high (25 mM) glucose until 80% confluent and then were reduced to 2% serum for 18 to 24 hours. The cells were then treated with 10 μM salmeterol followed by Western blot analysis or ELISA. For TNF-α inhibitory studies, the cells were treated with 5 ng/mL of TNF-α for 30 minutes or by a 30-minute pretreatment with TNF-α followed by salmeterol for 6 hours. In the TNF-α short hairpin (sh)RNA experiments, the cells were cultured until 90% confluent, followed by transfection with TNF-α shRNA for 18 hours. Results. TNF-α-only treatments of Müller cells resulted in significant decreases of tyrosine phosphorylation of the insulin receptor and Akt in high-glucose conditions. Salmeterol (10 μM), a β-2-adrenergic receptor agonist, significantly increased phosphorylation of both insulin receptor and Akt. TNF-α shRNA significantly decreased phosphorylation of IRS-1Ser307, which was further decreased after salmeterol+TNF-α shRNA. Both TNF-α shRNA and salmeterol significantly reduced death of the retinal Müller cells. Conclusions. These studies demonstrate that β-adrenergic receptor agonists in vitro can restore the loss of insulin receptor activity noted in diabetes. By decreasing the levels of TNF-α and decreasing the phosphorylation of IRS-1Ser307 while increasing tyrosine phosphorylation of insulin receptor, these results suggest a possible mechanism by which restoration of β-adrenergic receptor signaling may protect the retina against diabetes-induced damage. PMID:22110065

  17. Deficiency of the leukotriene B4 receptor, BLT-1, protects against systemic insulin resistance in diet-induced obesity.

    PubMed

    Spite, Matthew; Hellmann, Jason; Tang, Yunan; Mathis, Steven P; Kosuri, Madhavi; Bhatnagar, Aruni; Jala, Venkatakrishna R; Haribabu, Bodduluri

    2011-08-15

    Chronic inflammation is an underlying factor linking obesity with insulin resistance. Diet-induced obesity promotes an increase in circulating levels of inflammatory monocytes and their infiltration into expanding adipose tissue. Nevertheless, the endogenous pathways that trigger and sustain chronic low-grade inflammation in obesity are incompletely understood. In this study, we report that a high-fat diet selectively increases the circulating levels of CD11b(+) monocytes in wild-type mice that express leukotriene B(4) receptor, BLT-1, and that this increase is abolished in BLT-1-null mice. The accumulation of classically activated (M1) adipose tissue macrophages (ATMs) and the expression of proinflammatory cytokines and chemokines (i.e., IL-6 and Ccl2) was largely blunted in adipose tissue of obese BLT-1(-/-) mice, whereas the ratio of alternatively activated (M2) ATMs to M1 ATMs was increased. Obese BLT-1(-/-) mice were protected from systemic glucose and insulin intolerance and this was associated with a decrease in inflammation in adipose tissue and liver and a decrease in hepatic triglyceride accumulation. Deletion of BLT-1 prevented high fat-induced loss of insulin signaling in liver and skeletal muscle. These observations elucidate a novel role of chemoattractant receptor, BLT-1, in promoting monocyte trafficking to adipose tissue and promoting chronic inflammation in obesity and could lead to the identification of new therapeutic targets for treating insulin resistance in obesity.

  18. Membrane receptor cross talk in gonadotropin-, IGF-I-, and insulin-mediated steroidogenesis in fish ovary: An overview.

    PubMed

    Mukherjee, Dilip; Majumder, Suravi; Roy Moulik, Sujata; Pal, Puja; Gupta, Shreyasi; Guha, Payel; Kumar, Dhynendra

    2017-01-01

    Gonadal steroidogenesis is critical for survival and reproduction of all animals. The pathways that regulate gonadal steroidogenesis are therefore conserved among animals from the steroidogenic enzymes to the intracellular signaling molecules and G protein-coupled receptors (GPCRs) that mediate the activity of these enzymes. Regulation of fish ovarian steroidogenesis in vitro by gonadotropin (GtH) and GPCRs revealed interaction between adenylate cyclase and calcium/calmodulin-dependent protein kinases (CaMKs) and also MAP kinase pathway. Recent studies revealed another important pathway in GtH-induced fish ovarian steroidogenesis: cross talk between GPCRs and membrane receptor tyrosine kinases. Gonadotropin binding to Gαs-coupled membrane receptor in fish ovary leads to production of cAMP which in turn trans-activate the membrane-bound epidermal growth factor receptor (EGFR). This is followed by activation of ERK1/2 signaling that promotes steroid production. Interestingly, GtH-induced trans-activation of EGFR in the fish ovary uniquely requires matrix-metalloproteinase-mediated release of EGF. Inhibition of these proteases blocks GtH-induced steroidogenesis. Increased cAMP production in fish ovarian follicle upregulate follicular cyp19a1a mRNA expression and aromatase activity leading to increased biosynthesis of 17β-estradiol (E2). Evidence for involvement of SF-1 protein in inducing cyp19a1a mRNA and aromatase activity has also been demonstrated. In addition to GtH, insulin-like growth factor (IGF-I) and bovine insulin can alone induced steroidogenesis in fish ovary. In intact follicles and isolated theca cells, IGF-I and insulin had no effect on GtH-induced testosterone and 17a,hydroxysprogeaterone production. GtH-stimulated E2 and 17,20bdihydroxy-4-pregnane 3-one production in granulosa cells however, was significantly increased by IGF-I and insulin. Both IGF-I and insulin mediates their signaling via receptor tyrosine kinases leading to activation of PI3

  19. Insulin-releasing and insulin-like activity of Agaricus campestris (mushroom).

    PubMed

    Gray, A M; Flatt, P R

    1998-05-01

    Agaricus campestris (mushroom) has been documented as a traditional treatment for diabetes. Here the administration of mushroom in the diet (62.5 g/kg) and drinking water (2.5 g/l) countered the hyperglycaemia of streptozotocin-diabetic mice. An aqueous extract of mushroom (1 mg/ml) stimulated 2-deoxyglucose transport (2.0-fold), glucose oxidation (1.5-fold) and incorporation of glucose into glycogen (1.8-fold) in mouse abdominal muscle. In acute 20 min tests, 0.25-1 mg/ml aqueous extract of mushroom evoked a stepwise 3.5- to 4.6-fold stimulation of insulin secretion from the BRIN-BD11 pancreatic B-cell line. This effect was abolished by 0.5 mM diazoxide and prior exposure to extract did not affect subsequent stimulation of insulin secretion by 10 mM L-alanine, thereby negating a detrimental effect on cell viability. The effect of extract was potentiated by 16.7 mM glucose, L-alanine (10 mM) and IBMX (1 mM), and a depolarising concentration of KCl (25 mM) did not augment the insulin-releasing activity of mushroom. Activity of the extract was found to be heat stable, acetone soluble and unaltered by exposure to alkali, but decreased with exposure to acid. Dialysis to remove components with molecular mass < 2000 Da caused a 40% reduction in activity. Sequential extraction with solvents revealed insulin-releasing activity to be greatest in polar fractions. Lack of haemagglutinin activity with extract activity indicated that activity was unlikely to be due to a lectin-mediated event. These results demonstrate the presence of antihyperglycaemic, insulin-releasing and insulin-like activity in A. campestris.

  20. Insulin receptor aggregation and autophosphorylation in the presence of cationic polyamino acids

    SciTech Connect

    Kohanski, R.A. )

    1989-12-15

    Aggregation and autophosphorylation of the insulin receptor-protein kinase, from cultured 3T3-L1 adipocytes, were studied in the presence of cationic polyamino acids. Poly-L-lysine and poly-L-arginine produced the following effects with the purified receptor: first, the autophosphorylation rate was increased by polycations. Half-maximal stimulation was proportional to polymer length. The rate enhancement was greater at lower ATP concentrations. Second, near-endpoint (equilibrium) autophosphorylation was greater in the presence of the polycations. Polycations inhibited the reverse reaction: ADP + phosphoreceptor yielding ATP + aporeceptor. Third, the (32P)phosphopeptides generated by trypsin digestion of the 32P-beta-subunit, showed that no new autophosphorylation sites resulted from the presence of polycations. Fourth, the polycations, but not insulin, promoted receptor aggregation, and phosphoreceptor aggregated more readily than aporeceptor. Insulin receptor enriched through the wheat germ agglutinin eluate step was compared with purified receptor. Higher concentrations of poly-L-arginine were required to stimulate autophosphorylation and to promote aggregation. Finally, several polycation-dependent substrates present in the wheat germ agglutinin eluate co-aggregated with the insulin receptor. Polycation-stimulated receptor autophosphorylation is linked to a lower KM,app for ATP, but substrate phosphorylation may require the aggregation.

  1. Transient receptor potential vanilloid type-1 channel regulates diet-induced obesity, insulin resistance, and leptin resistance.

    PubMed

    Lee, Eunjung; Jung, Dae Young; Kim, Jong Hun; Patel, Payal R; Hu, Xiaodi; Lee, Yongjin; Azuma, Yoshihiro; Wang, Hsun-Fan; Tsitsilianos, Nicholas; Shafiq, Umber; Kwon, Jung Yeon; Lee, Hyong Joo; Lee, Ki Won; Kim, Jason K

    2015-08-01

    Insulin resistance is a major characteristic of obesity and type 2 diabetes, but the underlying mechanism is unclear. Recent studies have shown a metabolic role of capsaicin that may be mediated via the transient receptor potential vanilloid type-1 (TRPV1) channel. In this study, TRPV1 knockout (KO) and wild-type (WT) mice (as controls) were fed a high-fat diet (HFD), and metabolic studies were performed to measure insulin and leptin action. The TRPV1 KO mice became more obese than the WT mice after HFD, partly attributed to altered energy balance and leptin resistance in the KO mice. The hyperinsulinemic-euglycemic clamp experiment showed that the TRPV1 KO mice were more insulin resistant after HFD because of the ∼40% reduction in glucose metabolism in the white and brown adipose tissue, compared with that in the WT mice. Leptin treatment failed to suppress food intake, and leptin-mediated hypothalamic signal transducer and activator of transcription (STAT)-3 activity was blunted in the TRPV1 KO mice. We also found that the TRPV1 KO mice were more obese and insulin resistant than the WT mice at 9 mo of age. Taken together, these results indicate that lacking TRPV1 exacerbates the obesity and insulin resistance associated with an HFD and aging, and our findings further suggest that TRPV1 has a major role in regulating glucose metabolism and hypothalamic leptin's effects in obesity.

  2. Defective insulin secretion in pancreatic β cells lacking type 1 IGF receptor

    PubMed Central

    Xuan, Shouhong; Kitamura, Tadahiro; Nakae, Jun; Politi, Katerina; Kido, Yoshiaki; Fisher, Peter E.; Morroni, Manrico; Cinti, Saverio; White, Morris F.; Herrera, Pedro L.; Accili, Domenico; Efstratiadis, Argiris

    2002-01-01

    Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase of β cell mass and impaired glucose-dependent insulin release. β cell proliferation and secretion are thought to be regulated by signaling through receptor tyrosine kinases. In this regard, we sought to examine the potential proliferative and/or antiapoptotic role of IGFs in β cells by tissue-specific conditional mutagenesis ablating type 1 IGF receptor (IGF1R) signaling. Unexpectedly, lack of functional IGF1R did not affect β cell mass, but resulted in age-dependent impairment of glucose tolerance, associated with a decrease of glucose- and arginine-dependent insulin release. These observations reveal a requirement of IGF1R-mediated signaling for insulin secretion. PMID:12370279

  3. Arsenite and insulin exhibit opposing effects on epidermal growth factor receptor and keratinocyte proliferative potential

    SciTech Connect

    Patterson, Timothy J.; Rice, Robert H. . E-mail: rhrice@ucdavis.edu

    2007-05-15

    Previous work has suggested that arsenic exposure contributes to skin carcinogenesis by preserving the proliferative potential of human epidermal keratinocytes, thereby slowing the exit of putative target stem cells into the differentiation pathway. To find a molecular basis for this action, present work has explored the influence of arsenite on keratinocyte responses to epidermal growth factor (EGF). The ability of cultured keratinocytes to found colonies upon passaging several days after confluence was preserved by arsenite and EGF in an additive fashion, but neither was effective when the receptor tyrosine kinase activity was inhibited. Arsenite prevented the loss of EGF receptor protein and phosphorylation of tyrosine 1173, preserving its capability to signal. The level of nuclear {beta}-catenin was higher in cells treated with arsenite and EGF in parallel to elevated colony forming ability, and expression of a dominant negative {beta}-catenin suppressed the increase in both colony forming ability and yield of putative stem cells induced by arsenite and EGF. As judged by expression of three genes regulated by {beta}-catenin, this transcription factor had substantially higher activity in the arsenite/EGF-treated cells. Trivalent antimony exhibited the same effects as arsenite. A novel finding is that insulin in the medium induced the loss of EGF receptor protein, which was largely prevented by arsenite exposure.

  4. Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

    NASA Astrophysics Data System (ADS)

    Geetha, Thangiah; Langlais, Paul; Luo, Moulun; Mapes, Rebekka; Lefort, Natalie; Chen, Shu-Chuan; Mandarino, Lawrence J.; Yi, Zhengping

    2011-03-01

    Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.

  5. Designed Inhibitors of Insulin-Degrading Enzyme Regulate the Catabolism and Activity of Insulin

    SciTech Connect

    Leissring, Malcolm A.; Malito, Enrico; Hedouin, Sabrine; Reinstatler, Lael; Sahara, Tomoko; Abdul-Hay, Samer O.; Choudhry, Shakeel; Maharvi, Ghulam M.; Fauq, Abdul H.; Huzarska, Malwina; May, Philip S.; Choi, Sungwoon; Logan, Todd P.; Turk, Benjamin E.; Cantley, Lewis C.; Manolopoulou, Marika; Tang, Wei-Jen; Stein, Ross L.; Cuny, Gregory D.; Selkoe, Dennis J.

    2010-09-20

    Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE), a structurally and evolutionarily distinctive zinc-metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are {approx} 10{sup 6} times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-a-vis conventional zinc-metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE's 'closed,' inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. Conclusions/Significance: The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE's active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes.

  6. High-Fat Diet Augments VPAC1 Receptor-Mediated PACAP Action on the Liver, Inducing LAR Expression and Insulin Resistance

    PubMed Central

    Nakata, Masanori; Zhang, Boyang; Yang, Yifei; Okada, Takashi; Shintani, Norihito; Hashimoto, Hitoshi

    2016-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) acts on multiple processes of glucose and energy metabolism. PACAP potentiates insulin action in adipocytes and insulin release from pancreatic β-cells, thereby enhancing glucose tolerance. Contrary to these effects at organ levels, PACAP null mice exhibit hypersensitivity to insulin. However, this apparent discrepancy remains to be solved. We aimed to clarify the mechanism underlying the antidiabetic phenotype of PACAP null mice. Feeding with high-fat diet (HFD) impaired insulin sensitivity and glucose tolerance in wild type mice, whereas these changes were prevented in PACAP null mice. HFD also impaired insulin-induced Akt phosphorylation in the liver in wild type mice, but not in PACAP null mice. Using GeneFishing method, HFD increased the leukocyte common antigen-related (LAR) protein tyrosine phosphatase in the liver in wild type mice. Silencing of LAR restored the insulin signaling in the liver of HFD mice. Moreover, the increased LAR expression by HFD was prevented in PACAP null mice. HFD increased the expression of VPAC1 receptor (VPAC1-R), one of three PACAP receptors, in the liver of wild type mice. These data indicate that PACAP-VPAC1-R signaling induces LAR expression and insulin resistance in the liver of HFD mice. Antagonism of VPAC1-R may prevent progression of HFD-induced insulin resistance in the liver, providing a novel antidiabetic strategy. PMID:28044141

  7. Calorie restriction minimizes activation of insulin signaling in response to glucose: potential involvement of the growth hormone-insulin-like growth factor 1 axis.

    PubMed

    Hayashi, Hiroko; Yamaza, Haruyoshi; Komatsu, Toshimitsu; Park, Seongjoon; Chiba, Takuya; Higami, Yoshikazu; Nagayasu, Takeshi; Shimokawa, Isao

    2008-09-01

    Calorie restriction (CR) may modulate insulin signaling in response to energy intake through suppression of the growth hormone (GH)-IGF-1 axis. We investigated the glucose-stimulated serum insulin response and subsequent alterations in insulin receptor (IR), Akt, and FoxO1 in the rat liver and quadriceps femoris muscle (QFM). Nine-month-old wild-type (W) male Wistar rats fed ad libitum (AL) or a 30% CR diet initiated at 6 weeks of age and GH-suppressed transgenic (Tg) rats fed AL were killed 15 min after intraperitoneal injection of glucose or saline. In W-AL rats, the serum insulin concentration was elevated by glucose injection. Concomitantly, the phosphorylated (p)-IR and p-Akt levels were increased in both tissues. The active FoxO1 level was decreased in the liver, but not significantly in the QFM. In W-CR and Tg-AL rats, the serum insulin response was lower, and no significant changes were noted for the p-IR, p-Akt, or active FoxO1 levels in the liver. In the QFM, the p-Akt level was increased in W-CR and Tg-AL rats with an insignificant elevation of p-IR levels. The phenotypic similarity of W-CR and Tg-AL rats suggest that CR minimizes activation of insulin signaling in response to energy intake mostly through the GH-IGF-1 axis.

  8. Context-dependent regulation of feeding behaviour by the insulin receptor, DAF-2, in Caenorhabditis elegans.

    PubMed

    Dillon, James; Holden-Dye, Lindy; O'Connor, Vincent; Hopper, Neil A

    2016-06-01

    Insulin signalling plays a significant role in both developmental programmes and pathways modulating the neuronal signalling that controls adult behaviour. Here, we have investigated insulin signalling in food-associated behaviour in adult C. elegans by scoring locomotion and feeding on and off bacteria, the worm's food. This analysis used mutants (daf-2, daf-18) of the insulin signalling pathway, and we provide evidence for an acute role for insulin signalling in the adult nervous system distinct from its impact on developmental programmes. Insulin receptor daf-2 mutants move slower than wild type both on and off food and showed impaired locomotory responses to food deprivation. This latter behaviour is manifest as a failure to instigate dispersal following prolonged food deprivation and suggests a role for insulin signalling in this adaptive response. Insulin receptor daf-2 mutants are also deficient in pharyngeal pumping on food and off food. Pharmacological analysis showed the pharynx of daf-2 is selectively compromised in its response to 5-HT compared to the excitatory neuropeptide FLP-17. By comparing the adaptive pharyngeal behaviour in intact worms and isolated pharyngeal preparations, we determined that an insulin-dependent signal extrinsic to the pharyngeal system is involved in feeding adaptation. Hence, we suggest that reactive insulin signalling modulates both locomotory foraging and pharyngeal pumping as the animal adapts to the absence of food. We discuss this in the context of insulin signalling directing a shift in the sensitivity of neurotransmitter systems to regulate the worm's response to changes in food availability in the environment.

  9. Berberine lowers blood glucose in type 2 diabetes mellitus patients through increasing insulin receptor expression.

    PubMed

    Zhang, Hao; Wei, Jing; Xue, Rong; Wu, Jin-Dan; Zhao, Wei; Wang, Zi-Zheng; Wang, Shu-Kui; Zhou, Zheng-Xian; Song, Dan-Qing; Wang, Yue-Ming; Pan, Huai-Ning; Kong, Wei-Jia; Jiang, Jian-Dong

    2010-02-01

    Our previous work demonstrated that berberine (BBR) increases insulin receptor (InsR) expression and improves glucose utility both in vitro and in animal models. Here, we study the InsR-up-regulating and glucose-lowering activities of BBR in humans. Our results showed that BBR increased InsR messenger RNA and protein expression in a variety of human cell lines, including CEM, HCT-116, SW1990, HT1080, 293T, and hepatitis B virus-transfected human liver cells. Accordingly, insulin-stimulated phosphorylations of InsR beta-subunit and Akt were increased after BBR treatment in cultured cells. In the clinical study, BBR significantly lowered fasting blood glucose (FBG), hemoglobin A(1c), triglyceride, and insulin levels in patients with type 2 diabetes mellitus (T2DM). The FBG- and hemoglobin A(1c)-lowering efficacies of BBR were similar to those of metformin and rosiglitazone. In the BBR-treated patients, the percentages of peripheral blood lymphocytes that express InsR were significantly elevated after therapy. Berberine also lowered FBG effectively in chronic hepatitis B and hepatitis C patients with T2DM or impaired fasting glucose. Liver function was improved greatly in these patients by showing reduction of liver enzymes. Our results confirmed the activity of BBR on InsR in humans and its relationship with the glucose-lowering effect. Together with our previous report, we strongly suggest BBR as an ideal medicine for T2DM with a mechanism different from metformin and rosiglitazone.

  10. Sam68 Mediates the Activation of Insulin and Leptin Signalling in Breast Cancer Cells

    PubMed Central

    Pérez-Pérez, Antonio; Sánchez-Jiménez, Flora; Vilariño-García, Teresa; de la Cruz, Luis; Virizuela, Juan A.; Sánchez-Margalet, Víctor

    2016-01-01

    Obesity is a well-known risk factor for breast cancer development in postmenopausal women. High insulin and leptin levels seem to have a role modulating the growth of these tumours. Sam68 is an RNA-binding protein with signalling functions that has been found to be overexpressed in breast cancer. Moreover, Sam68 may be recruited to insulin and leptin signalling pathways, mediating its effects on survival, growth and proliferation in different cellular types. We aimed to study the expression of Sam68 and its phosphorylation level upon insulin and leptin stimulation, and the role of Sam68 in the proliferative effect and signalling pathways that are activated by insulin or leptin in human breast adenocarcinoma cells. In the human breast adenocarcinoma cell lines MCF7, MDA-MB-231 and BT-474, Sam68 protein quantity and gene expression were increased upon leptin or insulin stimulation, as it was checked by qPCR and immunoblot. Moreover, both insulin and leptin stimulation promoted an increase in Sam68 tyrosine phosphorylation and negatively regulated its RNA binding capacity. siRNA was used to downregulate Sam68 expression, which resulted in lower proliferative effects of both insulin and leptin, as well as a lower activation of MAPK and PI3K pathways promoted by both hormones. These effects may be partly explained by the decrease in IRS-1 expression by down-regulation of Sam68. These results suggest the participation of Sam68 in both leptin and insulin receptor signaling in human breast cancer cells, mediating the trophic effects of these hormones in proliferation and cellular growth. PMID:27415018

  11. Mechanisms of estradiol-induced insulin secretion by the G protein-coupled estrogen receptor GPR30/GPER in pancreatic beta-cells.

    PubMed

    Sharma, Geetanjali; Prossnitz, Eric R

    2011-08-01

    Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes.

  12. Selection of an RNA molecule that mimics a major autoantigenic epitope of human insulin receptor.

    PubMed Central

    Doudna, J A; Cech, T R; Sullenger, B A

    1995-01-01

    Autoimmunity often involves the abnormal targeting of self-antigens by antibodies, leading to tissue destruction and other pathologies. This process could potentially be disrupted by small ligands that bind specifically to autoantibodies and inhibit their interaction with the target antigen. Here we report the identification of an RNA sequence that binds a mouse monoclonal antibody specific for an autoantigenic epitope of human insulin receptor. The RNA ligand binds specifically and with high affinity (apparent Kd congruent to 2 nM) to the anti-insulin receptor antibody and not to other mouse IgGs. The RNA can also act as a decoy, blocking the antibody from binding the insulin receptor. Thus, it probably binds near the combining site on the antibody. Strikingly, the RNA cross-reacts with autoantibodies from patients with extreme insulin resistance. One simple explanation is that the selected RNA may structurally mimic the antigenic epitope on the insulin receptor protein. These results suggest that decoy RNAs may be used in the treatment of autoimmune diseases. Images Fig. 1 Fig. 4 Fig. 5 PMID:7534420

  13. Association of the insulin-receptor variant Met-985 with hyperglycemia and non-insulin-dependent diabetes mellitus in the Netherlands: A population-based study

    SciTech Connect

    `t Hart, L.M.; Maassen, J.A.; Does, F.E.E. van der

    1996-11-01

    One of the characteristics of non-insulin-dependent diabetes mellitus (NIDDM) is the presence of insulin. Most NIDDM patients have a normal sequence of the insulin receptor, indicating that, if insulin-receptor mutations contribute to the development of NIDDM, they will be present only in a minor fraction of the NIDDM population. The goal of the present study was to examine whether insulin-receptor mutations contribute to the development of NIDDM. We examined 161 individuals with NIDDM and 538 healthy controls from the population-based Rotterdam study for the presence of mutations in the insulin-receptor gene by SSCP. A heterozygous mutation changing valine-985 into methionine was detected in 5.6% of diabetic subjects and in 1.3% of individuals with normal oral glucose tolerance test. Adjusted for age, gender, and body-mass index, this revealed a relative risk for diabetes of 4.49 (95% confidence interval 1.59-12.25) for Met-985 carriers. When the total study group was analyzed, the prevalence of the mutation increased with increasing serum glucose levels (test for trend P < .005). We conclude that the Met-985 insulin-receptor variant associates with hyperglycemia and represents a risk factor for NIDDM. 30 refs., 3 figs., 1 tab.

  14. Conjugated Linoleic Acids Mediate Insulin Release through Islet G Protein-coupled Receptor FFA1/GPR40*

    PubMed Central

    Schmidt, Johannes; Liebscher, Kathrin; Merten, Nicole; Grundmann, Manuel; Mielenz, Manfred; Sauerwein, Helga; Christiansen, Elisabeth; Due-Hansen, Maria E.; Ulven, Trond; Ullrich, Susanne; Gomeza, Jesús; Drewke, Christel; Kostenis, Evi

    2011-01-01

    Among dietary components, conjugated linoleic acids (CLAs) have attracted considerable attention as weight loss supplements in the Western world because they reduce fat stores and increase muscle mass. However, a number of adverse effects are also ascribed to the intake of CLAs such as aggravation of insulin resistance and the risk of developing diabetes. However, the mechanisms accounting for the effects of CLAs on glucose homeostasis are incompletely understood. Herein we provide evidence that CLAs specifically activate the cell surface receptor FFA1, an emerging therapeutic target to treat type 2 diabetes. Using different recombinant cellular systems engineered to stably express FFA1 and a set of diverse functional assays including the novel, label-free non-invasive dynamic mass redistribution technology (Corning® Epic® biosensor), both CLA isomers cis-9, trans-11-CLA and trans-10, cis-12-CLA were found to activate FFA1 in vitro at concentrations sufficient to also account for FFA1 activation in vivo. Each CLA isomer markedly increased glucose-stimulated insulin secretion in insulin-producing INS-1E cells that endogenously express FFA1 and in primary pancreatic β-cells of wild type but not FFA1−/− knock-out mice. Our findings establish a clear mechanistic link between CLAs and insulin production and identify the cell surface receptor FFA1 as a molecular target for CLAs, explaining their acute stimulatory effects on insulin secretion in vivo. CLAs are also revealed as insulinotropic components in widely used nutraceuticals, a finding with significant implication for development of FFA1 modulators to treat type 2 diabetes. PMID:21339298

  15. Structure and function of the type 1 insulin-like growth factor receptor.

    PubMed

    Adams, T E; Epa, V C; Garrett, T P; Ward, C W

    2000-07-01

    The type 1 insulin-like growth factor receptor (IGF-1R), a transmembrane tyrosine kinase, is widely expressed across many cell types in foetal and postnatal tissues. Activation of the receptor following binding of the secreted growth factor ligands IGF-1 and IGF-2 elicits a repertoire of cellular responses including proliferation, and the protection of cells from programmed cell death or apoptosis. As a result, signalling through the IGF-1R is the principal pathway responsible for somatic growth in foetal mammals, whereas somatic growth in postnatal animals is achieved through the synergistic interaction of growth hormone and the IGFs. Forced overexpression of the IGF-1R results in the malignant transformation of cultured cells: conversely, downregulation of IGF-1R levels can reverse the transformed phenotype of tumour cells, and may render them sensitive to apoptosis in vivo. Elevated levels of IGF-IR are observed in a variety of human tumour types, whereas epidemiological studies implicate the IGF-1 axis as a predisposing factor in the pathogenesis of human breast and prostate cancer. The IGF-1R has thus emerged as a therapeutic target for the development of antitumour agents. Recent progress towards the elucidation of the three-dimensional structure of the extracellular domain of the IGF-1R represents an opportunity for the rational assembly of small molecule antagonists of receptor function for clinical use.

  16. Modulatory effect of insulin on T cell receptor mediated calcium signaling is blunted in long lasting type 1 diabetes mellitus.

    PubMed

    Demkow, Urszula; Winklewski, Paweł; Ciepiela, Olga; Popko, Katarzyna; Lipińska, Anna; Kucharska, Anna; Michalska, Beata; Wąsik, Maria

    2012-01-01

    Insulin significantly influences Ca(2+) signals evoked by various stimulants. In type 1 recent onset diabetes mellitus the proliferative response of T cells is significantly decreased. The number of clinical trials exploring the role of anti-CD3 monoclonal antibodies (mAb) as a therapeutic agent in recent onset diabetes mellitus type 1 is increasing last years. Therefore, a better understanding of the interplay between T cell receptor (TCR) dependent Ca(2+) increase, and insulin is of vital clinical significance. The aim of the study was to assess the effect of insulin on TCR evoked Ca(2+) responses in T lymphocytes obtained from healthy volunteers and patients suffering from long lasting diabetes mellitus type 1. Analysis was performed with use of the flow cytometer. We demonstrated that T cells ability to mobilize Ca(2+) was significantly reduced in long lasting diabetes mellitus type 1. Ca(2+) decrease achieved by the long term incubation with anti-CD3 mAb in T cells from healthy volunteers was restored by insulin. Strong interrelationship between baseline Ca(2+) level and plateau phase response to TCR stimulation was observed in the cytoplasm of cells pre-incubated with insulin from both healthy subjects and diabetic patients (r = 0.95, p < 0.0001 and r = 0.94, p < 0.0001, respectively). We postulate the existence of the interplay between TCR mediated activation and insulin. The TCR-insulin interplay is blunted in long lasting diabetes mellitus type 1. These observations may have an important implication for future therapeutic options in diabetes.

  17. Artemisia extracts activate PPARγ, promote adipogenesis, and enhance insulin sensitivity in adipose tissue of obese mice

    PubMed Central

    Richard, Allison J.; Burris, Thomas P.; Sanchez-Infantes, David; Wang, Yongjun; Ribnicky, David M.; Stephens, Jacqueline M.

    2014-01-01

    Objective Studies have shown that the inability of adipose tissue to properly expand during the obese state or respond to insulin can lead to metabolic dysfunction. Artemisia is a diverse group of plants that has a history of medicinal use. This study examines the ability of ethanolic extracts of Artemisia scoparia (SCO) and Artemisia santolinifolia (SAN) to modulate adipocyte development in cultured adipocytes and white adipose tissue (WAT) function in vivo using a mouse model of diet-induced obesity. Research Design & Procedures Adipogenesis was assessed using Oil Red O staining and immunoblotting. A nuclear receptor specificity assay was used to examine the specificity of SCO- and SAN-induced PPARγ activation. C57BL/6J mice, fed a high-fat diet, were gavaged with saline, SCO, or SAN for 2 weeks. Whole-body insulin sensitivity was examined using insulin tolerance tests. WAT depots were assessed via immunoblotting for markers of insulin action and adipokine production. Results We established that SCO and SAN were highly specific activators of PPARγ and did not activate other nuclear receptors. After a one-week daily gavage, SCO- and SAN-treated mice had lower insulin-induced glucose disposal rates than control mice. At the end of the 2-week treatment period, SCO- and SAN-treated mice had enhanced insulin-responsive Akt serine-473 phosphorylation and significantly decreased MCP-1 levels in visceral WAT relative to control mice; these differences were depot specific. Moreover, plasma adiponectin levels were increased following SCO treatment. Conclusion Overall, these studies demonstrate that extracts from two Artemisia species can have metabolically favorable effects on adipocytes and WAT. PMID:24985103

  18. The insulin-like growth factor 1 receptor in cancer: old focus, new future.

    PubMed

    Hartog, Hermien; Wesseling, Jelle; Boezen, H Marike; van der Graaf, Winette T A

    2007-09-01

    The importance of insulin-like growth factor 1 receptor (IGF-1R) signalling in malignant behaviour of tumour cells is well established. Currently, development of drugs targeting the IGF-1R as anticancer treatment is emerging. Several IGF-1R targeting strategies are being investigated in phases I and II clinical trials. Interactions of IGF-1R with insulin receptor, however, might complicate efficiency and tolerability of such drugs. This review describes mechanisms, recent developments and potential limitations of IGF-1R antibodies and tyrosine kinase inhibitors.

  19. Analysis of in vitro interactions of protein tyrosine phosphatase 1B with insulin receptors.

    PubMed

    Wang, X Y; Bergdahl, K; Heijbel, A; Liljebris, C; Bleasdale, J E

    2001-02-28

    One strategy to treat the insulin resistance that is central to type II diabetes mellitus may be to maintain insulin receptors (IR) in the active (tyrosine phosphorylated) form. Because protein tyrosine phosphatase 1B (PTP1B) binds and subsequently dephosphorylates IR, inhibitors of PTP1B-IR binding are potential insulin 'sensitizers.' A Scintillation Proximity Assay (SPA) was developed to characterize and quantitate PTP1B-IR binding. Human IR were solubilized and captured on wheat germ agglutinin (WGA)-coated SPA beads. Subsequent binding of human, catalytically inactive [35S] PTP1B Cys(215)/Ser (PTP1B(C215S)) to the lectin-anchored IR results in scintillation from the SPA beads that can be quantitated. Binding of PTP1B to IR was pH- and divalent cation-sensitive. Ca(2+) and Mn(2+), but not Mg(2+), dramatically attenuated the loss of PTP1B-IR binding observed when pH was raised from 6.2 to 7.8. PTP1B binding to IR from insulin-stimulated cells was much greater than to IR from unstimulated cells and was inhibited by either an antiphosphotyrosine antibody or treatment of IR with alkaline phosphatase, suggesting that tyrosine phosphorylation of IR is required for PTP1B binding. Phosphopeptides modeled after various IR phosphotyrosine domains each only partially inhibited PTP1B-IR binding, indicating that multiple domains of IR are likely involved in binding PTP1B. However, competitive displacement of [35S]PTP1B(C215S) by PTP1B(C215S) fitted best to a single binding site with a K(d) in the range 100-1000 nM, depending upon pH and divalent cations. PNU-200898, a potent and selective inhibitor of PTP1B whose orientation in the active site of PTP1B has been solved, competitively inhibited catalysis and PTP1B-IR binding with equal potency. The results of this novel assay for PTP1B-IR binding suggest that PTP1B binds preferentially to tyrosine phosphorylated IR through its active site and that binding may be susceptible to therapeutic disruption by small molecules.

  20. Identification of the bioactive and consensus peptide motif from Momordica charantia insulin receptor-binding protein.

    PubMed

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

    2016-08-01

    Many food bioactive peptides with diverse functions have been discovered by studying plant proteins. We have previously identified a 68-residue insulin receptor (IR)-binding protein (mcIRBP) from Momordica charantia that exhibits hypoglycemic effects in mice via interaction with IR. By in vitro digestion, we found that mcIRBP-19, spanning residues 50-68 of mcIRBP, enhanced the binding of insulin to IR, stimulated the phosphorylation of PDK1 and Akt, induced the expression of glucose transporter 4, and stimulated both the uptake of glucose in cells and the clearance of glucose in diabetic mice. Furthermore, mcIRBP-19 homologs were present in various plants and shared similar β-hairpin structures and IR kinase-activating abilities to mcIRBP-19. In conclusion, our findings suggested that mcIRBP-19 is a blood glucose-lowering bioactive peptide that exhibits IR-binding potentials. Moreover, we newly identified novel IR-binding bioactive peptides in various plants which belonged to different taxonomic families.

  1. Nandinine, a Derivative of Berberine, Inhibits Inflammation and Reduces Insulin Resistance in Adipocytes via Regulation of AMP-Kinase Activity.

    PubMed

    Zhao, Wenwen; Ge, Haixia; Liu, Kang; Chen, Xiuping; Zhang, Jian; Liu, Baolin

    2017-02-01

    Nandinine is a derivative of berberine that has high efficacy for treating cardiovascular diseases. This study investigated the effects of berberine and nandinine on the regulation of insulin sensitivity in adipocytes. Through treatment with macrophage-derived conditioned medium in 3T3-L1 adipocytes, dysregulation of adipokine production and activation of the IκB kinase β/nuclear factor-kappa B pathway was induced. However, these phenomena were effectively reversed by berberine, nandinine, and salicylate pretreatments. Furthermore, both berberine and nandinine inhibited serine phosphorylation of insulin receptor substrate-1 induced by IκB kinase β and increased tyrosine phosphorylation of insulin receptor substrate-1 to activate the PI3K/Akt pathway, which finally led to insulin-mediated glucose uptake. In addition, berberine and nandinine significantly increased AMP-activated protein kinase activity, thereby contributing to their anti-inflammatory effect by inhibiting IκB kinase β activation. Finally, in vivo studies demonstrated that both berberine (100 or 200 mg/kg) and nandinine (100 or 200 mg/kg) effectively ameliorated glucose intolerance and induced the insulin sensitivity index in mice. In conclusion, berberine and nandinine attenuated insulin resistance in adipocytes by inhibiting inflammation in an AMP-activated protein kinase-dependent manner. Berberine and nandinine may be used as dietary supplements and nandinine is a new candidate for obesity treatment.

  2. Interleukin-6 signaling promotes alternative macrophage activation to limit obesity-associated insulin resistance and endotoxemia

    PubMed Central

    Mauer, Jan; Chaurasia, Bhagirath; Goldau, Julia; Vogt, Merly C.; Ruud, Johan; Nguyen, Khoa D.; Theurich, Sebastian; Hausen, A. Christine; Schmitz, Joel; Brönneke, Hella S.; Estevez, Emma; Allen, Tamara L.; Mesaros, Andrea; Partridge, Linda; Febbraio, Mark A.; Chawla, Ajay; Wunderlich, F. Thomas; Brüning, Jens C.

    2014-01-01

    Obesity and insulin resistance are closely associated with the development of low-grade inflammation. Interleukin 6 (IL-6) is linked to obesity-associated inflammation, however its role in this context remains controversial. Here, we show that mice with inactivated Il6ra gene in myeloid cells (Il6raΔmyel) displayed exaggerated deterioration of glucose homeostasis upon diet-induced obesity due to enhanced insulin resistance. Insulin target tissues showed increased inflammation and a shift in macrophage polarization. IL-6 induced IL-4-receptor expression and augmented the response to IL-4 in macrophages in a cell-autonomous manner. Il6raΔmyel mice were resistant to IL-4-mediated alternative macrophage polarization and exhibited increased susceptibility to LPS-induced endotoxemia. These results reveal IL-6 signaling as an important determinant for alternative macrophage-activation and assign IL-6 an unexpected homeostatic role to limit inflammation. PMID:24681566

  3. Adiponectin increases glucose-induced insulin secretion through the activation of lipid oxidation.

    PubMed

    Patané, G; Caporarello, N; Marchetti, P; Parrino, C; Sudano, D; Marselli, L; Vigneri, R; Frittitta, L

    2013-12-01

    The expression of adiponectin receptors has been demonstrated in human and rat pancreatic beta cells, where globular (g) adiponectin rescues rat beta cells from cytokine and fatty acid-induced apoptosis. The aim of our study was to evaluate whether adiponectin has a direct effect on insulin secretion and the metabolic pathways involved. Purified human pancreatic islets and rat beta cells (INS-1E) were exposed (1 h) to g-adiponectin, and glucose-induced insulin secretion was measured. A significant increase in glucose-induced insulin secretion was observed in the presence of g-adiponectin (1 nmol/l) with respect to control cells in both human pancreatic islets (n = 5, p < 0.05) and INS-1E cells (n = 5, p < 0.001). The effect of globular adiponectin on insulin secretion was independent of AMP-dependent protein kinase (AMPK) activation or glucose oxidation. In contrast, g-adiponectin significantly increased oleate oxidation (n = 5, p < 0.05), and the effect of g-adiponectin (p < 0.001) on insulin secretion by INS-1E was significantly reduced in the presence of etomoxir (1 μmol/l), an inhibitor of fatty acid beta oxidation. g-Adiponectin potentiates glucose-induced insulin secretion in both human pancreatic islets and rat beta cells via an AMPK independent pathway. Increased fatty acid oxidation rather than augmented glucose oxidation is the mechanism responsible. Overall, our data indicate that, in addition to its anti-apoptotic action, g-adiponectin has another direct effect on beta cells by potentiating insulin secretion. Adiponectin, therefore, in addition to its well-known effect on insulin sensitivity, has important effects at the pancreatic level.

  4. Synthesis and Evaluation of a Library of Trifunctional Scaffold-Derived Compounds as Modulators of the Insulin Receptor.

    PubMed

    Fabre, Benjamin; Pícha, Jan; Vaněk, Václav; Selicharová, Irena; Chrudinová, Martina; Collinsová, Michaela; Žáková, Lenka; Buděšínský, Miloš; Jiráček, Jiří

    2016-12-12

    We designed a combinatorial library of trifunctional scaffold-derived compounds, which were derivatized with 30 different in-house-made azides. The compounds were proposed to mimic insulin receptor (IR)-binding epitopes in the insulin molecule and bind to and activate this receptor. This work has enabled us to test our synthetic and biological methodology and to prove its robustness and reliability for the solid-phase synthesis and testing of combinatorial libraries of the trifunctional scaffold-derived compounds. Our effort resulted in the discovery of two compounds, which were able to weakly induce the autophosphorylation of IR and weakly bind to this receptor at a 0.1 mM concentration. Despite these modest biological results, which well document the well-known difficulty in modulating protein-protein interactions, this study represents a unique example of targeting the IR with a set of nonpeptide compounds that were specifically designed and synthesized for this purpose. We believe that this work can open new perspectives for the development of next-generation insulin mimetics based on the scaffold structure.

  5. Erythropoietin alleviates hepatic insulin resistance via PPARγ-dependent AKT activation

    PubMed Central

    Ge, Zhijuan; Zhang, Pengzi; Hong, Ting; Tang, Sunyinyan; Meng, Ran; Bi, Yan; Zhu, Dalong

    2015-01-01

    Erythropoietin (EPO) has beneficial effects on glucose metabolism and insulin resistance. However, the mechanism underlying these effects has not yet been elucidated. This study aimed to investigate how EPO affects hepatic glucose metabolism. Here, we report that EPO administration promoted phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation in palmitic acid (PA)-treated HepG2 cells and in the liver of high-fat diet (HFD)-fed mice, whereas adenovirus-mediated silencing of the erythropoietin receptor (EPOR) blocked EPO-induced AKT signalling in HepG2 cells. Importantly, a peroxisome proliferator-activated receptor γ (PPARγ) antagonist and PPARγ small interfering RNA (siRNA) abrogated the EPO-induced increase in p-AKT in HepG2 cells. Lentiviral vector-mediated hepatic PPARγ silencing in HFD-fed C57BL/6 mice impaired EPO-mediated increases in glucose tolerance, insulin sensitivity and hepatic AKT activation. Furthermore, EPO activated the AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) signalling pathway, and AMPKα and SIRT1 knockdown each attenuated the EPO-induced PPARγ expression and deacetylation and PPARγ-dependent AKT activation in HepG2 cells. In summary, these findings suggest that PPARγ is involved in EPO/EPOR-induced AKT activation, and targeting the PPARγ/AKT pathway via EPO may have therapeutic implications for hepatic insulin resistance and type 2 diabetes. PMID:26643367

  6. The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

    PubMed Central

    Lou, Meizhen; Garrett, Thomas P. J.; McKern, Neil M.; Hoyne, Peter A.; Epa, V. Chandana; Bentley, John D.; Lovrecz, George O.; Cosgrove, Leah J.; Frenkel, Maurice J.; Ward, Colin W.

    2006-01-01

    The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1–CR–L2) of human IR at 2.3 Å resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) β-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more α-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R. PMID:16894147

  7. The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity.

    PubMed

    Lou, Meizhen; Garrett, Thomas P J; McKern, Neil M; Hoyne, Peter A; Epa, V Chandana; Bentley, John D; Lovrecz, George O; Cosgrove, Leah J; Frenkel, Maurice J; Ward, Colin W

    2006-08-15

    The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1-CR-L2) of human IR at 2.3 A resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) beta-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more alpha-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R.

  8. Regression of acanthosis nigricans correlates with disappearance of anti-insulin receptor autoantibodies and achievement of euglycemia in type B insulin resistance syndrome.

    PubMed

    Fareau, Gilbert G; Maldonado, Mario; Oral, Elif; Balasubramanyam, Ashok

    2007-05-01

    Autoantibodies directed against specific epitopes in the insulin receptor are rarely the cause of either recurrent hypoglycemia or a severe form of insulin resistance (type B insulin resistance). Type B insulin resistance occurs more commonly in women of African heritage and is frequently associated with a history of other autoimmune diseases. We present the unusual case of a 61-year-old African American woman with a background of autoimmune hypothyroidism and autoimmune hepatitis who developed type 2 diabetes mellitus and marked facial acanthosis nigricans (AN) over a period of weeks. Despite treatment with multiple oral antidiabetic agents, she rapidly developed severe, recalcitrant hyperglycemia and ketoacidosis, requiring hospitalization and intravenous insulin administration for 4 weeks at rates of up to 180 U/h. Immunologic testing revealed a high titer of anti-insulin receptor autoantibodies of both immunoglobulin G and immunoglobulin A classes. After a recurrence of diabetic ketoacidosis despite aggressive management, the patient was treated with a short course of cyclophosphamide; within 10 weeks, she experienced striking improvement of her hyperglycemia as well as marked regression of the AN lesions. Subsequently, the patient also experienced episodes of fasting hypoglycemia, which resolved with a brief course of glucocorticoids. She has since remained euglycemic with no therapy for 5 years. We have documented, for the first time, regression of AN in temporal association with disappearance of circulating anti-insulin receptor autoantibodies and achievement of euglycemia in a patient with type B insulin resistance.

  9. Interferon γ Attenuates Insulin Signaling, Lipid Storage, and Differentiation in Human Adipocytes via Activation of the JAK/STAT Pathway*

    PubMed Central

    McGillicuddy, Fiona C.; Chiquoine, Elise H.; Hinkle, Christine C.; Kim, Roy J.; Shah, Rachana; Roche, Helen M.; Smyth, Emer M.; Reilly, Muredach P.

    2009-01-01

    Recent reports demonstrate T-cell infiltration of adipose tissue in early obesity. We hypothesized that interferon (IFN) γ, a major T-cell inflammatory cytokine, would attenuate human adipocyte functions and sought to establish signaling mechanisms. Differentiated human adipocytes were treated with IFNγ ± pharmacological inhibitors prior to insulin stimulation. [3H]Glucose uptake and AKT phosphorylation were assessed as markers of insulin sensitivity. IFNγ induced sustained loss of insulin-stimulated glucose uptake in human adipocytes, coincident with reduced Akt phosphorylation and down-regulation of the insulin receptor, insulin receptor substrate-1, and GLUT4. Loss of adipocyte triglyceride storage was observed with IFNγ co-incident with reduced expression of peroxisome proliferator-activated receptor γ, adiponectin, perilipin, fatty acid synthase, and lipoprotein lipase. Treatment with IFNγ also blocked differentiation of pre-adipocytes to the mature phenotype. IFNγ-induced robust STAT1 phosphorylation and SOCS1 mRNA expression, with modest, transient STAT3 phosphorylation and SOCS3 induction. Preincubation with a non-selective JAK inhibitor restored glucose uptake and Akt phosphorylation while completely reversing IFNγ suppression of adipogenic mRNAs and adipocyte differentiation. Specific inhibition of JAK2 or JAK3 failed to block IFNγ effects suggesting a predominant role for JAK1-STAT1. We demonstrate that IFNγ attenuates insulin sensitivity and suppresses differentiation in human adipocytes, an effect most likely mediated via sustained JAK-STAT1 pathway activation. PMID:19776010

  10. Combination therapy with GLP-1 receptor agonists and basal insulin: a systematic review of the literature

    PubMed Central

    Balena, R; Hensley, I E; Miller, S; Barnett, A H

    2013-01-01

    Treatment algorithms for type 2 diabetes call for intensification of therapy over time as the disease progresses and glycaemic control worsens. If diet, exercise and oral antihyperglycaemic medications (OAMs) fail to maintain glycaemic control then basal insulin is added and ultimately prandial insulin may be required. However, such an intensification strategy carries risk of increased hypoglycaemia and weight gain, both of which are associated with worse long-term outcomes. An alternative strategy is to intensify therapy by the addition of a short-acting glucagon-like peptide-1 receptor agonist (GLP-1 RA) rather than prandial insulin. Short-acting GLP-1 RAs such as exenatide twice daily are particularly effective at reducing postprandial glucose while basal insulin has a greater effect on fasting glucose, providing a physiological rationale for this complementary approach. This review analyzes the latest randomized controlled clinical trials of insulin/GLP-1 RA combination therapy and examines results from ‘real-world’ use of the combinations as reported through observational and clinical practice studies. The most common finding across all types of studies was that combination therapy improved glycaemic control without weight gain or an increased risk of hypoglycaemia. Many studies reported weight loss and a reduction in insulin use when a GLP-1 RA was added to existing insulin therapy. Overall, the relative degree of benefit to glycaemic control and weight was influenced by the insulin titration employed in conjunction with the GLP-1 RA. The greatest glycaemic benefits were observed in studies with structured titration of insulin to glycaemic targets while the greatest weight benefits were observed in studies with a protocol-specified focus on insulin sparing. The adverse event profile of GLP-1 RAs in the reviewed trials was similar to that reported with GLP-1 RAs as monotherapy or in combination with OAMs with gastrointestinal events being the most commonly

  11. Insulin receptor isoform A ameliorates long-term glucose intolerance in diabetic mice

    PubMed Central

    Diaz-Castroverde, Sabela; Gómez-Hernández, Almudena; Fernández, Silvia; García-Gómez, Gema; Di Scala, Marianna; González-Aseguinolaza, Gloria; Fernández-Millán, Elisa; González-Rodríguez, Águeda; García-Bravo, María; Chambon, Pierre; Álvarez, Carmen; Perdomo, Liliana; Beneit, Nuria; Benito, Manuel

    2016-01-01

    ABSTRACT Type 2 diabetes mellitus is a complex metabolic disease and its pathogenesis involves abnormalities in both peripheral insulin action and insulin secretion. Previous in vitro data showed that insulin receptor isoform A, but not B, favours basal glucose uptake through its specific association with endogenous GLUT1/2 in murine hepatocytes and beta cells. With this background, we hypothesized that hepatic expression of insulin receptor isoform A in a mouse model of type 2 diabetes could potentially increase the glucose uptake of these cells, decreasing the hyperglycaemia and therefore ameliorating the diabetic phenotype. To assure this hypothesis, we have developed recombinant adeno-associated viral vectors expressing insulin receptor isoform A (IRA) or isoform B (IRB) under the control of a hepatocyte­-specific promoter. Our results demonstrate that in the long term, hepatic expression of IRA in diabetic mice is more efficient than IRB in ameliorating glucose intolerance. Consequently, it impairs the induction of compensatory mechanisms through beta cell hyperplasia and/or hypertrophy that finally lead to beta cell failure, reverting the diabetic phenotype in about 8 weeks. Our data suggest that long-term hepatic expression of IRA could be a promising therapeutic approach for the treatment of type 2 diabetes mellitus. PMID:27562101

  12. Insulin receptor isoform A ameliorates long-term glucose intolerance in diabetic mice.

    PubMed

    Diaz-Castroverde, Sabela; Gómez-Hernández, Almudena; Fernández, Silvia; García-Gómez, Gema; Di Scala, Marianna; González-Aseguinolaza, Gloria; Fernández-Millán, Elisa; González-Rodríguez, Águeda; García-Bravo, María; Chambon, Pierre; Álvarez, Carmen; Perdomo, Liliana; Beneit, Nuria; Escribano, Oscar; Benito, Manuel

    2016-11-01

    Type 2 diabetes mellitus is a complex metabolic disease and its pathogenesis involves abnormalities in both peripheral insulin action and insulin secretion. Previous in vitro data showed that insulin receptor isoform A, but not B, favours basal glucose uptake through its specific association with endogenous GLUT1/2 in murine hepatocytes and beta cells. With this background, we hypothesized that hepatic expression of insulin receptor isoform A in a mouse model of type 2 diabetes could potentially increase the glucose uptake of these cells, decreasing the hyperglycaemia and therefore ameliorating the diabetic phenotype. To assure this hypothesis, we have developed recombinant adeno-associated viral vectors expressing insulin receptor isoform A (IRA) or isoform B (IRB) under the control of a hepatocyte--specific promoter. Our results demonstrate that in the long term, hepatic expression of IRA in diabetic mice is more efficient than IRB in ameliorating glucose intolerance. Consequently, it impairs the induction of compensatory mechanisms through beta cell hyperplasia and/or hypertrophy that finally lead to beta cell failure, reverting the diabetic phenotype in about 8 weeks. Our data suggest that long-term hepatic expression of IRA could be a promising therapeutic approach for the treatment of type 2 diabetes mellitus.

  13. Susceptibility to Apoptosis in Insulin-like Growth Factor-I Receptor-deficient Brown Adipocytes

    PubMed Central

    Valverde, Angela M.; Mur, Cecilia; Brownlee, Michael; Benito, Manuel

    2004-01-01

    Fetal brown adipocytes are insulin-like growth factor-I (IGF-I) target cells. To assess the importance of the IGF-I receptor (IGF-IR) in brown adipocytes during fetal life, we have generated immortalized brown adipocyte cell lines from the IGF-IR-/- mice. Using this experimental model, we demonstrate that the lack of IGF-IR in fetal brown adipocytes increased the susceptibility to apoptosis induced by serum withdrawal. Culture of cells in the absence of serum and growth factors produced rapid DNA fragmentation (4 h) in IGF-IR-/- brown adipocytes, compared with the wild type (16 h). Consequently, cell viability was decreased more rapidly in fetal brown adipocytes in the absence of IGF-IR. Furthermore, caspase-3 activity was induced much earlier in cells lacking IGF-IR. At the molecular level, IGF-IR deficiency in fetal brown adipocytes altered the balance of the expression of several proapoptotic (Bcl-xS and Bim) and antiapoptotic (Bcl-2 and Bcl-xL) members of the Bcl-2 family. This imbalance was irreversible even though in IGF-IR-reconstituted cells. Likewise, cytosolic cytochrome c levels increased rapidly in IGF-IR-deficient cells compared with the wild type. A rapid entry of Foxo1 into the nucleus accompanied by a rapid exit from the cytosol and an earlier activation of caspase-8 were observed in brown adipocytes lacking IGF-IR upon serum deprivation. Activation of caspase-8 was inhibited by 50% in both cell types by neutralizing anti-Fas-ligand antibody. Adenoviral infection of wild-type brown adipocytes with constitutively active Foxol (ADA) increased the expression of antiapoptotic genes, decreased Bcl-xL and induced caspase-8 and -3 activities, with the final outcome of DNA fragmentation. Up-regulation of uncoupling protein-1 (UCP-1) expression in IGF-IR-deficient cells by transduction with PGC-1α or UCP-1 ameliorated caspase-3 activation, thereby retarding apoptosis. Finally, insulin treatment prevented apoptosis in both cell types. However, the survival

  14. Quasi-Steady-State Analysis based on Structural Modules and Timed Petri Net Predict System’s Dynamics: The Life Cycle of the Insulin Receptor

    PubMed Central

    Scheidel, Jennifer; Lindauer, Klaus; Ackermann, Jörg; Koch, Ina

    2015-01-01

    The insulin-dependent activation and recycling of the insulin receptor play an essential role in the regulation of the energy metabolism, leading to a special interest for pharmaceutical applications. Thus, the recycling of the insulin receptor has been intensively investigated, experimentally as well as theoretically. We developed a time-resolved, discrete model to describe stochastic dynamics and study the approximation of non-linear dynamics in the context of timed Petri nets. Additionally, using a graph-theoretical approach, we analyzed the structure of the regulatory system and demonstrated the close interrelation of structural network properties with the kinetic behavior. The transition invariants decomposed the model into overlapping subnetworks of various sizes, which represent basic functional modules. Moreover, we computed the quasi-steady states of these subnetworks and demonstrated that they are fundamental to understand the dynamic behavior of the system. The Petri net approach confirms the experimental results of insulin-stimulated degradation of the insulin receptor, which represents a common feature of insulin-resistant, hyperinsulinaemic states. PMID:26694479

  15. Quasi-Steady-State Analysis based on Structural Modules and Timed Petri Net Predict System's Dynamics: The Life Cycle of the Insulin Receptor.

    PubMed

    Scheidel, Jennifer; Lindauer, Klaus; Ackermann, Jörg; Koch, Ina

    2015-12-17

    The insulin-dependent activation and recycling of the insulin receptor play an essential role in the regulation of the energy metabolism, leading to a special interest for pharmaceutical applications. Thus, the recycling of the insulin receptor has been intensively investigated, experimentally as well as theoretically. We developed a time-resolved, discrete model to describe stochastic dynamics and study the approximation of non-linear dynamics in the context of timed Petri nets. Additionally, using a graph-theoretical approach, we analyzed the structure of the regulatory system and demonstrated the close interrelation of structural network properties with the kinetic behavior. The transition invariants decomposed the model into overlapping subnetworks of various sizes, which represent basic functional modules. Moreover, we computed the quasi-steady states of these subnetworks and demonstrated that they are fundamental to understand the dynamic behavior of the system. The Petri net approach confirms the experimental results of insulin-stimulated degradation of the insulin receptor, which represents a common feature of insulin-resistant, hyperinsulinaemic states.

  16. Insulin receptor autophosphorylation in cultured myoblasts correlates to glucose disposal in Pima Indians.

    PubMed

    Youngren, J F; Goldfine, I D; Pratley, R E

    1999-05-01

    In a previous study [Youngren, J. F., I. D. Goldfire, and R. E. Pratley. Am. J. Physiol. 273 (Endocrinol. Metab. 36): E276-E283, 1997] of skeletal muscle biopsies from insulin-resistant, nondiabetic Pima Indians, we demonstrated that diminished insulin receptor (IR) autophosphorylation correlated with in vivo insulin resistance. In the present study, to determine whether decreased IR function is a primary trait of muscle, and not secondary to an altered in vivo environment, we cultured myoblasts from 17 nondiabetic Pima Indians in whom insulin-stimulated glucose disposal (M) was measured during hyperinsulinemic-euglycemic glucose clamps. Myoblast IR autophosphorylation was determined by a highly sensitive ELISA. IR autophosphorylation directly correlated with M (r = 0.56, P = 0.02) and inversely correlated with the fasting plasma insulin (r = -0.58, P < 0.05). The relationship between M and IR autophosphorylation remained significant after M was adjusted for the effects of percent body fat (partial r = 0.53, P < 0.04). The relationship between insulin resistance and the capacity for myoblast IR autophosphorylation in nondiabetic Pima Indians suggests that variations in IR-signaling capacity may be intrinsic characteristics of muscle that contribute to the genetic component determining insulin action in this population.

  17. Aptamer-based single-molecule imaging of insulin receptors in living cells

    NASA Astrophysics Data System (ADS)

    Chang, Minhyeok; Kwon, Mijin; Kim, Sooran; Yunn, Na-Oh; Kim, Daehyung; Ryu, Sung Ho; Lee, Jong-Bong

    2014-05-01

    We present a single-molecule imaging platform that quantitatively explores the spatiotemporal dynamics of individual insulin receptors in living cells. Modified DNA aptamers that specifically recognize insulin receptors (IRs) with a high affinity were selected through the SELEX process. Using quantum dot-labeled aptamers, we successfully imaged and analyzed the diffusive motions of individual IRs in the plasma membranes of a variety of cell lines (HIR, HEK293, HepG2). We further explored the cholesterol-dependent movement of IRs to address whether cholesterol depletion interferes with IRs and found that cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin reduces the mobility of IRs. The aptamer-based single-molecule imaging of IRs will provide better understanding of insulin signal transduction through the dynamics study of IRs in the plasma membrane.

  18. Insulin

    MedlinePlus

    ... container that can be closed like a laundry detergent bottle. Check the expiration date on the insulin ... in a hard container like an empty laundry detergent bottle or a metal coffee can. Make sure ...

  19. A new EcoRI polymorphism for the insulin receptor gene

    SciTech Connect

    Accili, D.; Elbein, S.; McKeon, C.; Taylor, S.I. )

    1989-01-25

    A 550 bp BamHI-Pst I fragment encompassing bp 1,926-2,476 of the human insulin receptor cDNA was obtained. EcoRI identifies a two allele polymorphism, with bands of 5.8 and 5.5 kb. Co-dominant segregation was demonstrated in one Venezuelan pedigree.

  20. Characterization of IRA/IRB hybrid insulin receptors using bioluminescence resonance energy transfer.

    PubMed

    Blanquart, Christophe; Achi, Josepha; Issad, Tarik

    2008-10-01

    The insulin receptor (IR) is composed of two alpha-chains that bind ligands and two beta-chains that possess an intracellular tyrosine kinase activity. The IR is expressed in cells as two isoforms containing or not exon 11 (IRB and IRA, respectively). Several mRNA studies have demonstrated that the two isoforms are co-expressed in different tissues and in several cancer cells. IRA/IRB hybrid receptors, constituting of an alphabeta-chain from IRA and an alphabeta-chain from IRB, are likely to occur in cells co-expressing both isoforms, but their study has been hampered by the lack of specific tools. In previous work, we used BRET to study IR and IGF1R homodimers and heterodimers. Here, we have used BRET to characterize IRA/IRB hybrids. BRET saturation experiments showed that IRA/IRB hybrids are randomly formed in cells. Moreover, by co-transfecting HEK-293 cells with a luciferase-tagged kinase-dead version of one isoform and a wild-type untagged version of the other isoform, we showed that IRA/IRB hybrids can recruit, upon ligand stimulation, a YFP-tagged intracellular partner. Finally, using BRET, we have studied ligand-induced conformational changes within IRA/IRB hybrids. Dose-response experiments showed that hybrid receptors bind IGF-2 with the same affinity than IRA homodimers, whereas they bind IGF-1 with a lower affinity. Altogether, our data indicate that IRA/IRB hybrid receptors can form in cells co-expressing both IR isoforms, that they are capable of recruiting intracellular partners upon ligand stimulation, and that they have pharmacological properties more similar to those of IRA than those of IRB homodimers with regards to IGF-2.

  1. The Addition of Liquid Fructose to a Western-Type Diet in LDL-R(-/-) Mice Induces Liver Inflammation and Fibrogenesis Markers without Disrupting Insulin Receptor Signalling after an Insulin Challenge.

    PubMed

    Sangüesa, Gemma; Baena, Miguel; Hutter, Natalia; Montañés, José Carlos; Sánchez, Rosa María; Roglans, Núria; Laguna, Juan Carlos; Alegret, Marta

    2017-03-15

    A high consumption of fat and simple sugars, especially fructose, has been related to the development of insulin resistance, but the mechanisms involved in the effects of these nutrients are not fully understood. This study investigates the effects of a Western-type diet and liquid fructose supplementation, alone and combined, on insulin signalling and inflammation in low-density lipoprotein (LDL) receptor-deficient mice (LDL-R(-/-)). LDL-R(-/-) mice were fed chow or Western diet ±15% fructose solution for 12 weeks. Plasma glucose and insulin, and the expression of genes related to inflammation in the liver and visceral white adipose tissue (vWAT), were analysed. V-akt murine thymoma viral oncogene homolog-2 (Akt) activation was measured in the liver of the mice after a single injection of saline or insulin. None of the dietary interventions caused inflammation in vWAT, whereas the Western diet induced hepatic inflammation, which was further enhanced by liquid fructose, leading also to a significant increase in fibrogenesis markers. However, there was no change in plasma glucose or insulin, or insulin-induced Akt phosphorylation. In conclusion, hepatic inflammation and fibrogenesis markers induced by a Western diet supplemented with liquid fructose in LDL-R(-/-) mice are not associated with a significant impairment of hepatic insulin signalling.

  2. The Addition of Liquid Fructose to a Western-Type Diet in LDL-R−/− Mice Induces Liver Inflammation and Fibrogenesis Markers without Disrupting Insulin Receptor Signalling after an Insulin Challenge

    PubMed Central

    Sangüesa, Gemma; Baena, Miguel; Hutter, Natalia; Montañés, José Carlos; Sánchez, Rosa María; Roglans, Núria; Laguna, Juan Carlos; Alegret, Marta

    2017-01-01

    A high consumption of fat and simple sugars, especially fructose, has been related to the development of insulin resistance, but the mechanisms involved in the effects of these nutrients are not fully understood. This study investigates the effects of a Western-type diet and liquid fructose supplementation, alone and combined, on insulin signalling and inflammation in low-density lipoprotein (LDL) receptor-deficient mice (LDL-R−/−). LDL-R−/− mice were fed chow or Western diet ±15% fructose solution for 12 weeks. Plasma glucose and insulin, and the expression of genes related to inflammation in the liver and visceral white adipose tissue (vWAT), were analysed. V-akt murine thymoma viral oncogene homolog-2 (Akt) activation was measured in the liver of the mice after a single injection of saline or insulin. None of the dietary interventions caused inflammation in vWAT, whereas the Western diet induced hepatic inflammation, which was further enhanced by liquid fructose, leading also to a significant increase in fibrogenesis markers. However, there was no change in plasma glucose or insulin, or insulin-induced Akt phosphorylation. In conclusion, hepatic inflammation and fibrogenesis markers induced by a Western diet supplemented with liquid fructose in LDL-R−/− mice are not associated with a significant impairment of hepatic insulin signalling. PMID:28294959

  3. Insulin binding and glycolytic activity in erythrocytes from dialyzed and nondialyzed uremic patients.

    PubMed

    Weisinger, J R; Contreras, N E; Cajias, J; Bellorin-Font, E; Amair, P; Guitierrez, L; Sylva, V; Paz-Martínez, V

    1988-01-01

    Insulin resistance in uremia has been attributed to impaired hormone-receptor binding or to postbinding defects. Oral glucose tolerance tests, insulin binding, and in vitro glycolytic activity were studied in purified red blood cells from normal control subjects (C) and from uremic patients belonging to three groups: nondialyzed (U), on chronic hemodialysis (HD), and on continuous ambulatory peritoneal dialysis (CAPD). Glucose intolerance and hyperinsulinemia were demonstrated in all groups of patients. Maximal specific binding of 125I-insulin to erythrocytes, kinetically derived receptor numbers per cell, and affinity constants for insulin binding did not differ between control and patient groups. No correlation was found between the degree of glucose intolerance and insulin binding parameters. Basal lactate production by erythrocytes incubated in vitro was significantly higher in U and HD patients than in C, whereas CAPD patients did not differ from C in this respect. Addition of 1 mM dibutyryl-cAMP and 0.5 mM isobutyl-methyl-xanthine during incubation of erythrocytes caused an increase in the rate of lactate production that was similar in magnitude in the U, HD and C groups, whereas cells from CAPD subjects showed a significantly larger absolute response to these compounds after 1 h of incubation. There was no evidence of impairment of glycolytic capacity in red blood cells from uremic patients. In addition, no correlation was found between the degree of glucose intolerance and basal or stimulated lactate production by erythrocytes. Our results obtained in human erythrocytes suggest that the insulin resistance observed in uremia does not involve a defect in hormone binding or in the intracellular capacity to utilize glucose through glycolysis.

  4. Targeting colorectal cancer via its microenvironment by inhibiting IGF-1 Receptor-insulin receptor substrate and STAT3 signaling

    PubMed Central

    Sanchez-Lopez, Elsa; Flashner-Abramson, Efrat; Shalapour, Shabnam; Zhong, Zhenyu; Taniguchi, Koji; Levitzki, Alexander; Karin, Michael

    2015-01-01

    The tumor microenvironment (TME) exerts critical pro-tumorigenic effects through cytokines and growth factors that support cancer cell proliferation, survival, motility and invasion. Insulin-like growth factor-1 (IGF-1) and Signal transducer and activator of transcription 3 (STAT3) stimulate colorectal cancer (CRC) development and progression via cell autonomous and microenvironmental effects. Using a unique inhibitor, NT157, which targets both IGF-1 receptor (IGF-1R) and STAT3, we show that these pathways regulate many TME functions associated with sporadic colonic tumorigenesis in CPC-APC mice, in which cancer development is driven by loss of the Apc tumor suppressor gene. NT157 causes a substantial reduction in tumor burden by affecting cancer cells, cancer-associated fibroblasts (CAF) and myeloid cells. Decreased cancer cell proliferation and increased apoptosis were accompanied by inhibition of CAF activation and decreased inflammation. Furthermore, NT157 inhibited expression of pro-tumorigenic cytokines, chemokines and growth factors, including IL-6, IL-11 and IL-23 as well as CCL2, CCL5, CXCL7, CXCL5, ICAM1 and TGFβ; decreased cancer cell migratory activity and reduced their proliferation in the liver. NT157 represents a new class of anti-cancer drugs that affect both the malignant cell and its supportive microenvironment. PMID:26364612

  5. Characterization of the insulin sensitivity of ghrelin receptor KO mice using glycemic clamps

    PubMed Central

    2011-01-01

    Background We and others have demonstrated previously that ghrelin receptor (GhrR) knock out (KO) mice fed a high fat diet (HFD) have increased insulin sensitivity and metabolic flexibility relative to WT littermates. A striking feature of the HFD-fed GhrR KO mouse is the dramatic decrease in hepatic steatosis. To characterize further the underlying mechanisms of glucose homeostasis in GhrR KO mice, we conducted both hyperglycemic (HG) and hyperinsulinemic-euglycemic (HI-E) clamps. Additionally, we investigated tissue glucose uptake and specifically examined liver insulin sensitivity. Results Consistent with glucose tolerance-test data, in HG clamp experiments, GhrR KO mice showed a reduction in glucose-stimulated insulin release relative to WT littermates. Nevertheless, a robust 1st phase insulin secretion was still achieved, indicating that a healthy β-cell response is maintained. Additionally, GhrR KO mice demonstrated both a significantly increased glucose infusion rate and significantly reduced insulin requirement for maintenance of the HG clamp, consistent with their relative insulin sensitivity. In HI-E clamps, both LFD-fed and HFD-fed GhrR KO mice showed higher peripheral insulin sensitivity relative to WT littermates as indicated by a significant increase in insulin-stimulated glucose disposal (Rd), and decreased hepatic glucose production (HGP). HFD-fed GhrR KO mice showed a marked increase in peripheral tissue glucose uptake in a variety of tissues, including skeletal muscle, brown adipose tissue and white adipose tissue. GhrR KO mice fed a HFD also showed a modest, but significant decrease in conversion of pyruvate to glucose, as would be anticipated if these mice displayed increased liver insulin sensitivity. Additionally, the levels of UCP2 and UCP1 were reduced in the liver and BAT, respectively, in GhrR KO mice relative to WT mice. Conclusions These results indicate that improved glucose homeostasis of GhrR KO mice is characterized by robust

  6. Insulin Receptor Processing and Lipid Composition of Erythrocyte Membrane in Patients with Hyperlipidemia.

    PubMed

    Masella, R.; Cantafora, A.; Maffì, D.; Volpe, R.; Ginnetti, M.G.; Ricci, G.; Mace, N.L.; Buxton, G.M.; Peterson, S.W.

    1995-08-01

    The aim of this study was to determine whether the common forms of dyslipidemia could affect either the lipid composition or insulin receptor processing (down-regulation) of erythrocytes. The study included 22 patients with type IIa hypercholesterolemia, 15 patients with type IV hypertriglyceridemia and 12 patients with type IIb hyperlipidemia. Ten normolipidemic subjects were used as controls. Their erythrocyte membranes were analyzed for lipid composition and insulin receptor down-regulation. The results show that all the hyperlipidemias investigated were characterized by significant increases in the cholesterol to phospholipid molar ratio (0.56 +/- 0.08 in controls and 1.11 +/- 0.13, 1.09 +/- 0.14, 1.04 +/- 0.15, p < 0.001, in types IIa, IIb and IV, respectively). Surface insulin receptors of type IIa and IIb patients did not appear to down-regulate when compared to normal subjects, but rather up-regulated (+65.2% in controls, -1.0% and -8.7%, p < 0.001, in type IIa and IIb patients, respectively). Patients with type IV hypertriglyceridemia showed a residual capacity for insulin receptor internalization (10.7% down-regulation). Membranes of all the patients contained a higher proportion of phosphatidylethanolamine; the molar ratio of sphingomyelin to phosphatidylcholine was significantly higher in types IIb than in controls (1.22 +/- 0.11 and 1.12 +/- 0.10, p < 0.05, respectively); all the patients showed a lower content of polyunsaturated fatty acids in the major glycerophospholipid classes. However, type IV hypertriglyceridemics showed less variations, especially in the phosphatidylserine fraction. These results indicate that the alterations in lipoprotein pattern may affect both the lipid membrane equilibria and the processing ability of surface insulin receptors. Copyright 1995 S. Karger AG, Basel

  7. Regulation of gonadotropin receptors, gonadotropin responsiveness, and cell multiplication by somatomedin-C and insulin in cultured pig Leydig cells

    SciTech Connect

    Bernier, M.; Chatelain, P.; Mather, J.P.; Saez, J.M.

    1986-11-01

    The author have investigated the effects of insulin and somatomedin-C/insulin like growth factor I(Sm-C) in purified porcine Leydig cells in vitro on gonadotrophins (hCG) receptor number, hCG responsiveness (cAMP and testosterone production), and thymidine incorporation into DNA. Leydig cells cultured in a serum-free medium containing transferrin, vitamin E, and insulin (5 ..mu..g/ml) maintained fairly constant both hCG receptors and hCG responsiveness. When they were cultured for 3 days in the same medium without insulin, there was a dramatic decline (more than 80%) in both hCG receptor number and hCG responsiveness. However the cAMP but not the testosterone response to forskolin was normal. Both insulin and Sm-C at nanomolar concentrations prevent the decline of both hCG receptors and hCG-induced cAMP production. At nanomolar concentrations, Sm-C and insulin enhanced hCG-induced testosterone production but the effect of Sm-C was significantly higher than that of insulin. However, the effect of insulin at higher concentrations (5 ..mu..g/ml) was significantly higher than that of Sm-C at 50 ng/ml. In contrast, at nanomolar concentrations only Sm-C stimulated (/sup 3/H)-thymidine incorporation into DNA and cell multiplication, the stimulatory effect of insulin on these parameters, was seen only at micromolar concentrations. These results indicate that both Sm-C and insulin acting through the receptors increase Leydig cell steroidogenic responsiveness to hCG by increasing hCG receptor number and improving some step beyond cAMP formation. In contrast, the mitogenic effects of insulin are mediated only through Sm-C receptors.

  8. 5'AMP-activated protein kinase activity is increased in adipose tissue of northern elephant seal pups during prolonged fasting-induced insulin resistance.

    PubMed

    Viscarra, Jose A; Champagne, Cory D; Crocker, Daniel E; Ortiz, Rudy M

    2011-06-01

    Northern elephant seals endure a 2- to 3-month fast characterized by sustained hyperglycemia, hypoinsulinemia, and increased plasma cortisol and free fatty acids, conditions often seen in insulin-resistant humans. We had previously shown that adipose Glut4 expression and 5'AMP-activated protein kinase (AMPK) activity increase and plasma glucose decreases in fasting seals suggesting that AMPK activity contributes to glucose regulation during insulin-resistant conditions. To address the hypothesis that AMPK activity increases during fasting-induced insulin resistance, we performed glucose tolerance tests (GTT) on early (n=5) and late (n=8)-fasted seal pups and compared adipose tissue expression of insulin signaling proteins, peroxisome proliferator-activated receptor γ (PPARγ), and AMPK, in addition to plasma adiponectin, leptin, cortisol, insulin, and non-esterified fatty acid (NEFA) levels. Fasting was associated with decreased glucose clearance, plasma insulin and adiponectin, and intracellular insulin signaling, as well as increased plasma cortisol and NEFAs, supporting the suggestion that seals develop insulin resistance late in the fast. The expression of Glut4 and VAMP2 increased (52 and 63% respectively) with fasting but did not change significantly during the GTT. PPARγ and phosphorylated AMPK did not change in the early fasted seals, but increased significantly (73 and 50% respectively) in the late-fasted seals during the GTT. Increased AMPK activity along with the reduction in the activity of insulin-signaling proteins supports our hypothesis that AMPK activity is increased following the onset of insulin resistance. The association between increased AMPK activity and Glut4 expression suggests that AMPK plays a greater role in regulating glucose metabolism in mammals adapted to prolonged fasting than in non-fasting mammals.

  9. Single-Nucleotide Polymorphism on Exon 17 of Insulin Receptor Gene Influences Insulin Resistance in PCOS: A Pilot Study on North Indian Women.

    PubMed

    Gangopadhyay, Sukanya; Agrawal, Nitin; Batra, Aruna; Kabi, Bhaskar Charan; Gupta, Akash

    2016-04-01

    Polycystic ovarian syndrome (PCOS), a major cause of infertility, is also strongly associated with insulin resistance. Defects in insulin receptor signaling are considered as one of the major molecular pathogeneses for insulin resistance. To investigate the possible mechanism of this signaling defect at genetic level, single-nucleotide polymorphism (SNP) [His 1085 C/T] at the exon 17 of insulin receptor gene (INSR) was studied in this pilot study. Polymerase chain reaction was performed on leucocytic DNA of women diagnosed with PCOS, selected from the outpatient department of Safdarjung Hospital, New Delhi, using suitable primer to amplify a region on INSR. An equal number of age-matched healthy women were selected as controls. SNP analysis was performed with restriction enzyme length polymorphism technique using Pm II enzyme. Serum insulin level was measured by ELISA kit and HOMA-IR was calculated mathematically. A higher frequency of the CC genotype was observed in PCOS women than in controls. Also, HOMA-IR, a tool for estimating insulin resistance, was significantly high in PCOS women with the CC genotype. C1008T SNP at exon 17 of INSR is associated with insulin resistance in Indian women with PCOS. Presence of CC genotype (C1085T) could be developed as a marker for insulin resistance and metabolic complications in PCOS women.

  10. Insulin enhances RANKL-induced osteoclastogenesis via ERK1/2 activation and induction of NFATc1 and Atp6v0d2.

    PubMed

    Oh, Ju Hee; Lee, Jae Yoon; Joung, Seung Hee; Oh, Yoon Taek; Kim, Hong Sung; Lee, Na Kyung

    2015-12-01

    Insulin is one of the main factors affecting bone and energy metabolism, however, the direct effect of insulin on osteoclast differentiation remains unclear. Thus, in order to help elucidate that puzzle, the authors investigated the roles and regulatory mechanisms of insulin on osteoclasts differentiation. Co-stimulation with insulin and RANKL significantly enhanced the number of larger (>100 μm) osteoclastic cells and of TRAP-positive multinucleated cells compared with treatment by RANKL alone. Conversely, the insulin receptor shRNA markedly decreased osteoclast differentiation induced by insulin and RANKL. Insulin treatment significantly activated ERK1/2 MAP kinase as well as markedly induced the expression of NFATc1, an osteoclast marker gene, and Atp6v0d2, an osteoclast fusion-related gene. The pretreatment of PD98059, an ERK1/2 inhibitor, or insulin receptor shRNA effectively suppressed osteoclast differentiation and, in addition, blocked the expression of NFATc1 and Atp6vod2 induced by insulin stimulation. These data reveal insights into the regulation of osteoclast differentiation and fusion through ERK1/2 activation and the induction of NFATc1 and Atp6v0d2 by insulin.

  11. Delayed puberty but normal fertility in mice with selective deletion of insulin receptors from Kiss1 cells.

    PubMed

    Qiu, Xiaoliang; Dowling, Abigail R; Marino, Joseph S; Faulkner, Latrice D; Bryant, Benjamin; Brüning, Jens C; Elias, Carol F; Hill, Jennifer W

    2013-03-01

    Pubertal onset only occurs in a favorable, anabolic hormonal environment. The neuropeptide kisspeptin, encoded by the Kiss1 gene, modifies GnRH neuronal activity to initiate puberty and maintain fertility, but the factors that regulate Kiss1 neurons and permit pubertal maturation remain to be clarified. The anabolic factor insulin may signal nutritional status to these neurons. To determine whether insulin sensing plays an important role in Kiss1 neuron function, we generated mice lacking insulin receptors in Kiss1 neurons (IR(ΔKiss) mice). IR(ΔKiss) females showed a delay in vaginal opening and in first estrus, whereas IR(ΔKiss) males also exhibited late sexual maturation. Correspondingly, LH levels in IR(ΔKiss) mice were reduced in early puberty in both sexes. Adult reproductive capacity, body weight, fat composition, food intake, and glucose regulation were comparable between the 2 groups. These data suggest that impaired insulin sensing by Kiss1 neurons delays the initiation of puberty but does not affect adult fertility. These studies provide insight into the mechanisms regulating pubertal timing in anabolic states.

  12. The Novel Endocrine Disruptor Tolylfluanid Impairs Insulin Signaling in Primary Rodent and Human Adipocytes through a Reduction in Insulin Receptor Substrate-1 Levels

    PubMed Central

    Sargis, Robert M.; Neel, Brian A.; Brock, Clifton O.; Lin, Yuxi; Hickey, Allison T.; Carlton, Daniel A.; Brady, Matthew J.

    2012-01-01

    Emerging data suggest that environmental endocrine disrupting chemicals (EDCs) may contribute to the pathophysiology of obesity and diabetes. In prior work, the phenylsulfamide fungicide tolylfluanid (TF) was shown to augment adipocyte differentiation, yet its effects on mature adipocyte metabolism remain unknown. Because of the central role of adipose tissue in global energy regulation, the present study tested the hypothesis that TF modulates insulin action in primary rodent and human adipocytes. Alterations in insulin signaling in primary mammalian adipocytes were determined by the phosphorylation of Akt, a critical insulin signaling intermediate. Treatment of primary murine adipose tissue in vitro with 100 nM TF for 48 h markedly attenuated acute insulin-stimulated Akt phosphorylation in a strain- and species-independent fashion. Perigonadal, perirenal, and mesenteric fat were all sensitive to TF-induced insulin resistance. A similar TF-induced reduction in insulin-stimulated Akt phosphorylation was observed in primary human subcutaneous adipose tissue. TF-treatment led to a potent and specific reduction in insulin receptor substrate-1 (IRS-1) mRNA and protein levels, a key upstream mediator of insulin’s diverse metabolic effects. In contrast, insulin receptor-β, phosphatidylinositol 3-kinase, and Akt expression were unchanged, indicating a specific abrogation of insulin signaling. Additionally, TF-treated adipocytes exhibited altered endocrine function with a reduction in both basal and insulin-stimulated leptin secretion. These studies demonstrate that TF induces cellular insulin resistance in primary murine and human adipocytes through a reduction of IRS-1 expression and protein stability, raising concern about the potential for this fungicide to disrupt metabolism and thereby contribute to the pathogenesis of diabetes. PMID:22387882

  13. Deletion of Macrophage Vitamin D Receptor Promotes Insulin Resistance and Monocyte Cholesterol Transport to Accelerate Atherosclerosis in Mice

    PubMed Central

    Oh, Jisu; Riek, Amy E.; Darwech, Isra; Funai, Katsuhiko; Shao, JianSu; Chin, Kathleen; Sierra, Oscar L.; Carmeliet, Geert; Ostlund, Richard E.; Bernal-Mizrachi, Carlos

    2015-01-01

    Summary Intense effort has been devoted to understanding predisposition to chronic systemic inflammation as this contributes to cardiometabolic disease. We demonstrate that deletion of the macrophage vitamin D receptor (VDR) in mice (KODMAC) is sufficient to induce insulin resistance by promoting M2 macrophage accumulation in the liver, as well as increase cytokine secretion and hepatic glucose production. Moreover, VDR deletion increases atherosclerosis by enabling lipid-laden M2 monocytes to adhere, migrate, and carry cholesterol into the atherosclerotic plaque, and by increasing macrophage cholesterol uptake and esterification. Increased foam cell formation results from lack of VDR-SERCA2b interaction, causing SERCA dysfunction, activation of ER stress-CaMKII-JNKp-PPARγ signaling, and induction of the scavenger receptors CD36 and SR-A1. BM transplant of VDR-expressing cells into KODMAC mice improved insulin sensitivity, suppressed atherosclerosis, and decreased foam cell formation. The immunomodulatory effects of vitamin D in macrophages are thus critical in diet-induced insulin resistance and atherosclerosis in mice. Graphical Abstract PMID:25801026

  14. Functional properties of an isolated. cap alpha beta. heterodimeric human placenta insulin-like growth factor 1 receptor complex

    SciTech Connect

    Feltz, S.M.; Swanson, M.L.; Wemmie, J.A.; Pessin, J.E.

    1988-05-03

    Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment of pH 7.6, resulted in the formation of a functional ..cap alpha beta.. heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state. The membrane-bound ..cap alpha beta.. heterodimeric complex displayed similar curvilinear /sup 125/I-IGF-1 equilibrium binding compared to the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric complex. /sup 125/I-IGF-1 binding to both the isolated ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of ..cap alpha beta.. heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric state. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of human placenta membranes results in the formation of an ..cap alpha beta.. heterodimeric IGF-1 receptor complex, (ii) unlike the insulin receptor, high-affinity homogeneous IGF-1 binding occurs in both the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes, and (iii) IGF-1-dependent autophosphorylation of the ..cap alpha beta.. heterodimeric IGF-1 receptor complex correlates wit an IGF-1 dependent covalent reassociation into an ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state.

  15. Identification and evolution of two insulin receptor genes involved in Tribolium castaneum development and reproduction.

    PubMed

    Sang, Ming; Li, Chengjun; Wu, Wei; Li, Bin

    2016-07-10

    The insulin and insulin-like signaling (IIS) pathway exists in a wide range of organisms from mammals to invertebrates and regulates several vital physiological functions. A phylogenetic analysis have indicated that insulin receptors have been duplicated at least twice among vertebrates, whereas only one duplication occurred in insects before the differentiation of Coleoptera, Hymenoptera, and Hemiptera. Thus, we cloned two putative insulin receptor genes, T.cas-ir1 and T.cas-ir2, from T. castaneum and determined that T.cas-ir1 is most strongly expressed during the late adult and early pupal stages, whereas T.cas-ir2 is most strongly expressed during the late larval stage. We found that larval RNAi against T.cas-ir1 and T.cas-ir2 causes 100% and 42.0% insect death, respectively, and that parental RNAi against T.cas-ir1 and T.cas-ir2 leads to 100% and 33.3% reductions in beetle fecundity, respectively. The hatching rate of ds-ir2 insects was 66.2%. Moreover, RNAi against these two genes increased the expression of the pkc, foxo, jnk, cdc42, ikk, and mekk genes but decreased erk gene expression. Despite these similarities, these two genes act via distinct regulatory pathways. These results indicate that these two receptors have functionally diverged with respect to the development and reproduction of T. castaneum, even though they retain some common regulatory signaling pathways.

  16. Signal regulatory protein-α interacts with the insulin receptor contributing to muscle wasting in chronic kidney disease.

    PubMed

    Thomas, Sandhya S; Dong, Yanjun; Zhang, Liping; Mitch, William E

    2013-08-01

    Insulin resistance from chronic kidney disease (CKD) stimulates muscle protein wasting but mechanisms causing this resistance are controversial. To help resolve this, we used microarray analyses to identify initiators of insulin resistance in the muscles of mice with CKD, glucose intolerance, and insulin resistance. CKD raised mRNAs of inflammatory cytokines in muscles and there was a 5.2-fold increase in signal regulatory protein-α (SIRP-α), a transmembrane glycoprotein principally present in muscle membranes. By immunoprecipitation we found it interacts with the insulin receptor and insulin receptor substrate-1 (IRS-1). Treatment of myotubes with a mixture of inflammatory cytokines showed that SIRP-α expression was increased by a NF-κB-dependent pathway. Blockade of NF-κB using a small-molecule chemical inhibitor or a dominant-negative IKKβ reduced cytokine-induced SIRP-α expression. The overexpression of SIRP-α in myotubes impaired insulin signaling and raised proteolysis while SIRP-α knockdown with siRNAs in skeletal muscle cells increased tyrosine phosphorylation of the insulin receptor and IRS-1 despite inclusion of cytokines. This led to increased p-Akt and suppression of protein degradation. Thus, SIRP-α is part of a novel mechanism for inflammation-mediated insulin resistance in muscle. In catabolic conditions with impaired insulin signaling, targeting SIRP-α may improve insulin sensitivity and prevent muscle atrophy.

  17. Insulin

    NASA Technical Reports Server (NTRS)

    2004-01-01

    The manipulation of organic materials--cells, tissues, and even living organisms--offers many exciting possibilities for the future from organic computers to improved aquaculture. Commercial researchers are using the microgravity environment to produce large near perfect protein crystals Research on insulin has yielded crystals that far surpass the quality of insulin crystals grown on the ground. Using these crystals industry partners are working to develop new and improved treatments for diabetes. Other researchers are exploring the possibility of producing antibiotics using plant cell cultures which could lead to both orbital production and the improvement of ground-based antibiotic production.

  18. One week treatment with the IL-1 receptor antagonist anakinra leads to a sustained improvement in insulin sensitivity in insulin resistant patients with type 1 diabetes mellitus.

    PubMed

    van Asseldonk, Edwin J P; van Poppel, Pleun C M; Ballak, Dov B; Stienstra, Rinke; Netea, Mihai G; Tack, Cees J

    2015-10-01

    Inflammation associated with obesity is involved in the development of insulin resistance. We hypothesized that anti-inflammatory treatment with the Interleukin-1 receptor antagonist anakinra would improve insulin sensitivity. In an open label proof-of-concept study, we included overweight patients diagnosed with type 1 diabetes with an HbA1c level over 7.5%. Selecting insulin resistant patients with longstanding type 1 diabetes allowed us to study the effects of anakinra on insulin sensitivity. Patients were treated with 100mg anakinra daily for one week. Insulin sensitivity, insulin need and blood glucose profiles were measured before, after one week and after four weeks of follow-up. Fourteen patients completed the study. One week of anakinra treatment led to an improvement of insulin sensitivity, an effect that was sustained for four weeks. Similarly, glucose profiles, HbA1c levels and insulin needs improved. In conclusion, one week of treatment with anakinra improves insulin sensitivity in patients with type 1 diabetes.

  19. Bavachin from Psoralea corylifolia Improves Insulin-Dependent Glucose Uptake through Insulin Signaling and AMPK Activation in 3T3-L1 Adipocytes.

    PubMed

    Lee, Hyejin; Li, Hua; Noh, Minsoo; Ryu, Jae-Ha

    2016-04-08

    The fruit of Psoralea corylifolia L. (Fabaceae) (PC), known as "Bo-Gol-Zhee" in Korea has been used as traditional medicine. Ethanol and aqueous extracts of PC have an anti-hyperglycemic effect by increasing plasma insulin levels and decreasing blood glucose and total plasma cholesterol levels in type 2 diabetic rats. In this study, we purified six compounds from PC and investigated their anti-diabetic effect. Among the purified compounds, bavachin most potently accumulated lipids during adipocyte differentiation. Intracellular lipid accumulation was measured by Oil Red-O (ORO) cell staining to investigate the effect of compounds on adipogenesis. Consistently, bavachin activated gene expression of adipogenic transcriptional factors, proliferator-activated receptorγ (PPARγ) and CCAAT/enhancer binding protein-α (C/EBPα). Bavachin also increased adiponectin expression and secretion in adipocytes. Moreover, bavachin increased insulin-induced glucose uptake by differentiated adipocytes and myoblasts. In differentiated adipocytes, we found that bavachin enhanced glucose uptake via glucose transporter 4 (GLUT4) translocation by activating the Akt and 5'AMP-activated protein kinase (AMPK) pathway in the presence or absence of insulin. These results suggest that bavachin from Psoralea corylifolia might have therapeutic potential for type 2 diabetes by activating insulin signaling pathways.

  20. Bavachin from Psoralea corylifolia Improves Insulin-Dependent Glucose Uptake through Insulin Signaling and AMPK Activation in 3T3-L1 Adipocytes

    PubMed Central

    Lee, Hyejin; Li, Hua; Noh, Minsoo; Ryu, Jae-Ha

    2016-01-01

    The fruit of Psoralea corylifolia L. (Fabaceae) (PC), known as “Bo-Gol-Zhee” in Korea has been used as traditional medicine. Ethanol and aqueous extracts of PC have an anti-hyperglycemic effect by increasing plasma insulin levels and decreasing blood glucose and total plasma cholesterol levels in type 2 diabetic rats. In this study, we purified six compounds from PC and investigated their anti-diabetic effect. Among the purified compounds, bavachin most potently accumulated lipids during adipocyte differentiation. Intracellular lipid accumulation was measured by Oil Red-O (ORO) cell staining to investigate the effect of compounds on adipogenesis. Consistently, bavachin activated gene expression of adipogenic transcriptional factors, proliferator-activated receptorγ (PPARγ) and CCAAT/enhancer binding protein-α (C/EBPα). Bavachin also increased adiponectin expression and secretion in adipocytes. Moreover, bavachin increased insulin-induced glucose uptake by differentiated adipocytes and myoblasts. In differentiated adipocytes, we found that bavachin enhanced glucose uptake via glucose transporter 4 (GLUT4) translocation by activating the Akt and 5′AMP-activated protein kinase (AMPK) pathway in the presence or absence of insulin. These results suggest that bavachin from Psoralea corylifolia might have therapeutic potential for type 2 diabetes by activating insulin signaling pathways. PMID:27070585

  1. CK2 acts as a potent negative regulator of receptor-mediated insulin release in vitro and in vivo

    PubMed Central

    Rossi, Mario; Ruiz de Azua, Inigo; Barella, Luiz F.; Sakamoto, Wataru; Zhu, Lu; Cui, Yinghong; Lu, Huiyan; Rebholz, Heike; Matschinsky, Franz M.; Doliba, Nicolai M.; Butcher, Adrian J.; Tobin, Andrew B.; Wess, Jürgen

    2015-01-01

    G protein-coupled receptors (GPCRs) regulate virtually all physiological functions including the release of insulin from pancreatic β-cells. β-Cell M3 muscarinic receptors (M3Rs) are known to play an essential role in facilitating insulin release and maintaining proper whole-body glucose homeostasis. As is the case with other GPCRs, M3R activity is regulated by phosphorylation by various kinases, including GPCR kinases and casein kinase 2 (CK2). At present, it remains unknown which of these various kinases are physiologically relevant for the regulation of β-cell activity. In the present study, we demonstrate that inhibition of CK2 in pancreatic β-cells, knockdown of CK2α expression, or genetic deletion of CK2α in β-cells of mutant mice selectively augmented M3R-stimulated insulin release in vitro and in vivo. In vitro studies showed that this effect was associated with an M3R-mediated increase in intracellular calcium levels. Treatment of mouse pancreatic islets with CX4945, a highly selective CK2 inhibitor, greatly reduced agonist-induced phosphorylation of β-cell M3Rs, indicative of CK2-mediated M3R phosphorylation. We also showed that inhibition of CK2 greatly enhanced M3R-stimulated insulin secretion in human islets. Finally, CX4945 treatment protected mice against diet-induced hyperglycemia and glucose intolerance in an M3R-dependent fashion. Our data demonstrate, for the first time to our knowledge, the physiological relevance of CK2 phosphorylation of a GPCR and suggest the novel concept that kinases acting on β-cell GPCRs may represent novel therapeutic targets. PMID:26598688

  2. The insulin-like growth factor I receptor regulates glucose transport by astrocytes.

    PubMed

    Hernandez-Garzón, Edwin; Fernandez, Ana M; Perez-Alvarez, Alberto; Genis, Laura; Bascuñana, Pablo; Fernandez de la Rosa, Ruben; Delgado, Mercedes; Angel Pozo, Miguel; Moreno, Estefania; McCormick, Peter J; Santi, Andrea; Trueba-Saiz, Angel; Garcia-Caceres, Cristina; Tschöp, Matthias H; Araque, Alfonso; Martin, Eduardo D; Torres Aleman, Ignacio

    2016-11-01

    Previous findings indicate that reducing brain insulin-like growth factor I receptor (IGF-IR) activity promotes ample neuroprotection. We now examined a possible action of IGF-IR on brain glucose transport to explain its wide protective activity, as energy availability is crucial for healthy tissue function. Using (18) FGlucose PET we found that shRNA interference of IGF-IR in mouse somatosensory cortex significantly increased glucose uptake upon sensory stimulation. In vivo microscopy using astrocyte specific staining showed that after IGF-IR shRNA injection in somatosensory cortex, astrocytes displayed greater increases in glucose uptake as compared to astrocytes in the scramble-injected side. Further, mice with the IGF-IR knock down in astrocytes showed increased glucose uptake in somatosensory cortex upon sensory stimulation. Analysis of underlying mechanisms indicated that IGF-IR interacts with glucose transporter 1 (GLUT1), the main facilitative glucose transporter in astrocytes, through a mechanism involving interactions with the scaffolding protein GIPC and the multicargo transporter LRP1 to retain GLUT1 inside the cell. These findings identify IGF-IR as a key modulator of brain glucose metabolism through its inhibitory action on astrocytic GLUT1 activity. GLIA 2016;64:1962-1971.

  3. Insulin continues to induce plasminogen activator inhibitor 1 gene expression in insulin-resistant mice and adipocytes.

    PubMed Central

    Samad, F.; Pandey, M.; Bell, P. A.; Loskutoff, D. J.

    2000-01-01

    BACKGROUND: Although the association between insulin resistance and cardiovascular risk is well established, the underlying molecular mechanisms are poorly understood. The antifibrinolytic molecule plasminogen activator inhibitor 1 (PAI-1) is a cardiovascular risk factor that is consistently elevated in insulin-resistant states such as obesity and non-insulin-dependent diabetes mellitus (NIDDM). The strong positive correlation between this elevated PAI-1 and the degree of hyperinsulinemia not only implicates insulin itself in this increase, but also suggests that PAI-1 is regulated by a pathway that does not become insulin resistant. The data in this report supports this hypothesis. MATERIALS AND METHODS: We show that insulin stimulates PAI-1 gene expression in metabolically insulin-resistant ob/ob mice and in insulin-resistant 3T3-L1 adipocytes. Moreover, we provide evidence that glucose transport and PAI-1 gene expression are mediated by different insulin signaling pathways. These observations suggest that the compensatory hyperinsulinemia that is frequently associated with insulin-resistant states, directly contribute to the elevated PAI-1. CONCLUSIONS: These results provide a potential mechanism for the abnormal increases in cardiovascular risk genes in obesity, NIDDM, and polycystic ovary disease. PMID:11055587

  4. Diabetes mellitus caused by mutations in human insulin: analysis of impaired receptor binding of insulins Wakayama, Los Angeles and Chicago using pharmacoinformatics.

    PubMed

    Islam, Md Ataul; Bhayye, Sagar; Adeniyi, Adebayo A; Soliman, Mahmoud E S; Pillay, Tahir S

    2017-03-01

    Several naturally occuring mutations in the human insulin gene are associated with diabetes mellitus. The three known mutant molecules, Wakayama, Los Angeles and Chicago were evaluated using molecular docking and molecular dynamics (MD) to analyse mechanisms of deprived binding affinity for insulin receptor (IR). Insulin Wakayama, is a variant in which valine at position A3 is substituted by leucine, while in insulin Los Angeles and Chicago, phenylalanine at positions B24 and B25 is replaced by serine and leucine, respectively. These mutations show radical changes in binding affinity for IR. The ZDOCK server was used for molecular docking, while AMBER 14 was used for the MD study. The published crystal structure of IR bound to natural insulin was also used for MD. The binding interactions and MD trajectories clearly explained the critical factors for deprived binding to the IR. The surface area around position A3 was increased when valine was substituted by leucine, while at positions B24 and B25 aromatic amino acid phenylalanine replaced by non-aromatic serine and leucine might be responsible for fewer binding interactions at the binding site of IR that leads to instability of the complex. In the MD simulation, the normal mode analysis, rmsd trajectories and prediction of fluctuation indicated instability of complexes with mutant insulin in order of insulin native insulin < insulin Chicago < insulin Los Angeles < insulin Wakayama molecules which corresponds to the biological evidence of the differing affinities of the mutant insulins for the IR.

  5. A Tale of Two Receptors: Insulin and Insulin-Like Growth Factor Signaling in Cancer

    PubMed Central

    Yee, Douglas

    2014-01-01

    Summary Inhibition of the type I IGF receptor (IGF1R) has been the focus of numerous clinical trials. Two reports in this issue describe the results of phase I trials of an IGF1R tyrosine kinase inhibitor OSI-906. This commentary will describe the complex endocrine changes induced by these types of agents. PMID:25303978

  6. The human insulin receptor substrate-1 gene (IRS1) is localized on 2q36

    SciTech Connect

    Nishiyama, Masaki; Matsufuji, Senya; Hayashi, Shin-ichi; Furusaka, Akihiro; Tanaka, Teruji ); Inazawa, J.; Nakamura, Yusuke ); Ariyama, Takeshi ); Wands, J.R. )

    1994-03-01

    The chromosomal localization of some of the genes participating in the insulin signaling pathway is known. The insulin and insulin receptor genes have been mapped to chromosomes 11 and 19, respectively. To identify the chromosomal localization of the human IRS1 gene, the fluorescence in situ hybridization technique was employed with Genomic Clone B-10. A total of 50 metaphase cells exhibiting either single or double spots of hybridization signals were examined. Among them, 32 showed the specific signals on 2q36. Therefore, the authors assigned the human IRS1 gene to 2q36. The genes for homeobox sequence (HOX4), fibronectin 1, alkaline phosphatase (intestinal), transition protein 1, villin 1, collagen (type IV), Waardenburg syndrome (type 1), alanine-glyoxylate aminotransferase, and glucagon have been localized in the vicinity of the IRS1 gene.

  7. The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

    PubMed

    Isakoff, S J; Taha, C; Rose, E; Marcusohn, J; Klip, A; Skolnik, E Y

    1995-10-24

    Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.

  8. Bcl10 links saturated fat overnutrition with hepatocellular NF-kB activation and insulin resistance.

    PubMed

    Van Beek, Matthew; Oravecz-Wilson, Katherine I; Delekta, Phillip C; Gu, Shufang; Li, Xiangquan; Jin, Xiaohong; Apel, Ingrid J; Konkle, Katy S; Feng, Yongjia; Teitelbaum, Daniel H; Ruland, Jürgen; McAllister-Lucas, Linda M; Lucas, Peter C

    2012-05-31

    Excess serum free fatty acids (FFAs) are fundamental to the pathogenesis of insulin resistance. With high-fat feeding, FFAs activate NF-kB in target tissues, initiating negative crosstalk with insulin signaling. However, the mechanisms underlying FFA-dependent NF-kB activation remain unclear. Here, we demonstrate that the saturated FA, palmitate, requires Bcl10 for NF-kB activation in hepatocytes. Uptake of palmitate, metabolism to diacylglycerol, and subsequent activation of protein kinase C (PKC) appear to mechanistically link palmitate with Bcl10, known as a central component of a signaling complex that, along with CARMA3 and MALT1, activates NF-kB downstream of selected cell surface receptors. Consequently, Bcl10-deficient mice are protected from hepatic NF-kB activation and insulin resistance following brief high-fat diet, suggesting that Bcl10 plays a major role in the metabolic consequences of acute overnutrition. Surprisingly, while CARMA3 also participates in the palmitate response, MALT1 is completely dispensable, thereby revealing an apparent nonclassical role for Bcl10 in NF-kB signaling.

  9. Human glycated albumin affects glucose metabolism in L6 skeletal muscle cells by impairing insulin-induced insulin receptor substrate (IRS) signaling through a protein kinase C alpha-mediated mechanism.

    PubMed

    Miele, Claudia; Riboulet, Audrey; Maitan, Maria Alessandra; Oriente, Francesco; Romano, Chiara; Formisano, Pietro; Giudicelli, Jean; Beguinot, Francesco; Van Obberghen, Emmanuel

    2003-11-28

    Nonenzymatic glycation is increased in diabetes and leads to increased levels of glycated proteins. Most studies have focused on the role of glycation products in vascular complications. Here, we have investigated the action of human glycated albumin (HGA) on insulin signaling in L6 skeletal muscle cells. Exposure of these cells to HGA inhibited insulin-stimulated glucose uptake and glycogen synthase activity by 95 and 80%, respectively. These effects were time- and dose-dependent, reaching a maximum after 12 h incubation with 0.1 mg/ml HGA. In contrast, exposure of the cells to HGA had no effect on thymidine incorporation. Further, HGA reduced insulin-stimulated serine phosphorylation of PKB and GSK3, but did not alter ERK1/2 activation. HGA did not affect either insulin receptor kinase activity or insulin-induced Shc phosphorylation on tyrosine. In contrast, insulin-dependent IRS-1 and IRS-2 tyrosine phosphorylation was severely reduced in cells preincubated with HGA for 24 h. Insulin-stimulated association of PI3K with IRS-1 and IRS-2, and PI3K activity were reduced by HGA in parallel with the changes in IRS tyrosine phosphorylation, while Grb2-IRS association was unchanged. In L6 myotubes, exposure to HGA increased PKC activity by 2-fold resulting in a similar increase in Ser/Thr phosphorylation of IRS-1 and IRS-2. These phosphorylations were blocked by the PKC inhibitor bisindolylmaleimide (BDM). BDM also blocked the action of HGA on insulin-stimulated PKB and GSK3 alpha. Simultaneously, BDM rescued insulin-stimulation of glucose uptake and glycogen synthase activity in cells exposed to HGA. The use of antibodies specific to PKC isoforms shows that this effect appears to be mediated by activated PKC alpha, independent of reactive oxygen species production. In summary, in L6 skeletal muscle cells, exposure to HGA leads to insulin resistance selectively in glucose metabolism with no effect on growth-related pathways regulated by the hormone.

  10. Embryonic morphogenesis in Caenorhabditis elegans integrates the activity of LET-502 Rho-binding kinase, MEL-11 myosin phosphatase, DAF-2 insulin receptor and FEM-2 PP2c phosphatase.

    PubMed

    Piekny, A J; Wissmann, A; Mains, P E

    2000-12-01

    let-502 rho-binding kinase and mel-11 myosin phosphatase regulate Caenorhabditis elegans embryonic morphogenesis. Genetic analysis presented here establishes the following modes of let-502 action: (i) loss of only maternal let-502 results in abnormal early cleavages, (ii) loss of both zygotic and maternal let-502 causes elongation defects, and (iii) loss of only zygotic let-502 results in sterility. The morphogenetic function of let-502 and mel-11 is apparently redundant with another pathway since elimination of these two genes resulted in progeny that underwent near-normal elongation. Triple mutant analysis indicated that unc-73 (Rho/Rac guanine exchange factor) and mlc-4 (myosin light chain) act in parallel to or downstream of let-502/mel-11. In contrast mig-2 (Rho/Rac), daf-2 (insulin receptor), and age-1 (PI3 kinase) act within the let-502/mel-11 pathway. Mutations in the sex-determination gene fem-2, which encodes a PP2c phosphatase (unrelated to the MEL-11 phosphatase), enhanced mutations of let-502 and suppressed those of mel-11. fem-2's elongation function appears to be independent of its role in sexual identity since the sex-determination genes fem-1, fem-3, tra-1, and tra-3 had no effect on mel-11 or let-502. By itself, fem-2 affects morphogenesis with low penetrance. fem-2 blocked the near-normal elongation of let-502; mel-11 indicating that fem-2 acts in a parallel elongation pathway. The action of two redundant pathways likely ensures accurate elongation of the C. elegans embryo.

  11. Insulin-independent, MAPK-dependent stimulation of NKCC activity in skeletal muscle.

    PubMed

    Wong, J A; Gosmanov, A R; Schneider, E G; Thomason, D B

    2001-08-01

    Na(+)-K(+)-Cl(-) cotransporter (NKCC) activity in quiescent skeletal muscle is modest. However, ex vivo stimulation of muscle for as little as 18 contractions (1 min, 0.3 Hz) dramatically increased the activity of the cotransporter, measured as the bumetanide-sensitive (86)Rb influx, in both soleus and plantaris muscles. This activation of cotransporter activity remained relatively constant for up to 10-Hz stimulation for 1 min, falling off at higher frequencies (30-Hz stimulation for 1 min). Similarly, stimulation of skeletal muscle with adrenergic receptor agonists phenylephrine, isoproterenol, or epinephrine produced a dramatic stimulation of NKCC activity. It did not appear that stimulation of NKCC activity was a reflection of increased Na(+)-K(+)-ATPase activity because insulin treatment did not stimulate NKCC activity, despite insulin's well-known stimulation of Na(+)-K(+)-ATPase activity. Stimulation of NKCC activity could be blocked by pretreatment with inhibitors of mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) activity, indicating that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) MAPKs may be required. These data indicate a regulated NKCC activity in skeletal muscle that may provide a significant pathway for potassium transport into skeletal muscle fibers.

  12. Leptin receptor polymorphisms interact with polyunsaturated fatty acids to augment risk of insulin resistance and metabolic syndrome in adults

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The leptin receptor (LEPR) is associated with insulin resistance, a key feature of metabolic syndrome (MetS). Gene-fatty acid interactions may affect MetS risk. The objective was to investigate the relationship among LEPR polymorphisms, insulin resistance, andMetSrisk and whether plasma fatty acids,...

  13. DPP4-inhibitor improves neuronal insulin receptor function, brain mitochondrial function and cognitive function in rats with insulin resistance induced by high-fat diet consumption.

    PubMed

    Pipatpiboon, Noppamas; Pintana, Hiranya; Pratchayasakul, Wasana; Chattipakorn, Nipon; Chattipakorn, Siriporn C

    2013-03-01

    High-fat diet (HFD) consumption has been demonstrated to cause peripheral and neuronal insulin resistance, and brain mitochondrial dysfunction in rats. Although the dipeptidyl peptidase-4 inhibitor, vildagliptin, is known to improve peripheral insulin sensitivity, its effects on neuronal insulin resistance and brain mitochondrial dysfunction caused by a HFD are unknown. We tested the hypothesis that vildagliptin prevents neuronal insulin resistance, brain mitochondrial dysfunction, learning and memory deficit caused by HFD. Male rats were divided into two groups to receive either a HFD or normal diet (ND) for 12 weeks, after which rats in each group were fed with either vildagliptin (3 mg/kg/day) or vehicle fo