Insulin-stimulated Tyrosine Kinase Activity Research Articles

Compartmentalization and in vivo insulin‐induced translocation of the insulin‐signaling inhibitor Grb14 in rat liver

The molecular adaptor Grb14 binds in vitro to the activated insulin receptor (IR) and inhibits IR signaling. In this study, we have used rat liver subcellular fractionation to analyze in vivo insulin effects on Grb14 compartmentalization and IR phosphorylation and activity. In control rats, Grb14 was recovered mainly in microsomal and cytosolic fractions, but was also detectable at low levels in plasma membrane and Golgi/endosome fractions. Insulin injection led to a rapid and dose-dependent increase in Grb14 content, first in the plasma membrane fraction, and then in the Golgi/endosome fraction, which paralleled the increase in IR beta-subunit tyrosine phosphorylation. Upon sustained in vivo IR tyrosine phosphorylation induced by high-affinity insulin analogs, in vitro IR dephosphorylation by endogenous phosphatases, and in vivo phosphorylation of the IR induced by injection of bisperoxo(1,10 phenanthroline)oxovanadate, a phosphotyrosine phosphatase inhibitor, we observed a striking correlation between IR phosphorylation state and Grb14 content in both the plasma membrane and Golgi/endosome fractions. In addition, coimmunoprecipitation experiments provided evidence that Grb14 was associated with phosphorylated IR beta-subunit in these fractions. Altogether, these data support a model whereby insulin stimulates the recruitment of endogenous Grb14 to the activated IR at the plasma membrane, and induces internalization of the Grb14-IR complex in endosomes. Removal of Grb14 from fractions of insulin-treated rats by KCl treatment led to an increase of in vivo insulin-stimulated IR tyrosine kinase activity, indicating that endogenous Grb14 exerts a negative feedback control on IR catalytic activity. This study thus demonstrates that Grb14 is a physiological regulator of liver insulin signaling.

Reversal of insulin resistance postpartum is linked to enhanced skeletal muscle insulin signaling.

The restoration of maternal insulin sensitivity postpartum represents an important physiological and metabolic adaptation in a woman's reproductive lifespan. The present study was conducted to examine the potential cellular mechanisms underlying the changes in insulin sensitivity from late pregnancy to postpartum in human skeletal muscle. Nine nonobese women (age, 32 +/- 2 yr; body mass index, 21.2 +/- 0.8 kg/m(2)) with normal glucose tolerance were studied during late pregnancy (30-36 wk) and again approximately 1 yr postpartum using a euglycemic-hyperinsulinemic clamp (5 mm glucose, 40 mU/m(2).min insulin) to determine insulin sensitivity. Biopsies of the vastus lateralis muscle were obtained in the basal state before each clamp. Insulin sensitivity improved by 74% from late pregnancy to 1 yr postpartum (5.5 +/- 0.6 vs. 9.6 +/- 0.9 mg/kg fat-free mass.min; P < 0.005). Skeletal muscle insulin receptor (IR) protein expression increased by 42% postpartum, as measured by ELISA (4.0 +/- 0.6 vs. 5.7 +/- 0.6 ng/g protein; P < 0.05) and by Western blotting of the IR beta-subunit (28.7 +/- 4.7 vs. 42.0 +/- 4.8 arbitrary units; P < 0.003). However, in vitro studies showed that when adjusted for IR concentration, maximal insulin-stimulated (100 nm) IR tyrosine phosphorylation (0.75 +/- 0.06 vs. 0.92 +/- 0.08 U) and IR tyrosine kinase activity (183.8 +/- 27.0 vs. 204.3 +/- 23.7 fmol ATP/ng IR) were unchanged. There was a 69% increase in IR substrate-1 (IRS-1) protein expression (P = 0.05) in muscle postpartum. In addition, the p85alpha regulatory subunit of phosphatidylinositol 3-kinase was markedly reduced by 55% (P < 0.02) postpartum. The change in insulin sensitivity from late pregnancy to postpartum correlated highly with the corresponding change in IRS-1 protein (r = 0.84; P < 0.007). Downstream signaling proteins, including total Akt and p70s6 kinase, and the glucose transporter protein GLUT-4, were similar at both time points. These data suggest that reduced IR tyrosine kinase activity is not a major factor in the IR of pregnancy in lean women with normal glucose tolerance. Rather, the reversal of insulin resistance 1 yr postpartum is accompanied by increased skeletal muscle IRS-1 along with a down-regulation of the p85alpha subunit of phosphatidylinositol 3-kinase. These changes may allow for greater p85/p110 binding to IRS-1 and play a significant physiological role in the underlying metabolic adaptation to normal human pregnancy and restoration of insulin sensitivity postpartum.

Regulation of the insulin receptor by protein kinase C isoenzymes: preferential interaction with β isoenzymes and interaction with the catalytic domain of βII

We analysed the effects of high glucose in rat1 cells overexpressing insulin receptor. High (25 mM) glucose inhibited insulin-stimulated tyrosine kinase activity completely at insulin concentrations of 1 and 5 ng/ml. Decapeptides modelled on insulin receptor sequences surrounding serines 1035 and 1270 were found to inhibit protein kinase C activity in vitro and after microinjection into cells blocked the inhibition of mitogenesis induced by glucose. Purification of receptor from 3T3L1 adipocytes revealed that only the isoenzymes β1, βII and δ were detected. The site of the interaction was mapped to the catalytic domain of βII. These results demonstrate that the inhibition of insulin receptor tyrosine kinase activity can be ameliorated using insulin receptor peptide sequences and there is constitutive and differential interaction of individual PKC isoenzymes with the insulin receptor, and in the case of βII, this interaction maps to the catalytic domain rather than the regulatory domain.

Dietary zinc supplementation attenuates hyperglycemia in db/db mice.

Although zinc (Zn) deficiency has been associated with insulin resistance, and altered Zn metabolism (e.g., hyperzincuria, low-normal plasma Zn concentrations) may be present in diabetes, the potential effects of Zn on modulation of insulin action in Type II diabetes have not been established. The objective of this study was to compare the effects of dietary Zn deficiency and Zn supplementation on glycemic control in db/db mice. Weanling db/db mice and lean littermate controls were fed Zn-deficient (3 ppm Zn; dbZD and InZD groups), Zn-adequate control (30 ppm Zn; dbC and InC groups) or Zn-supplemented (300 ppm Zn; dbZS and InZS groups) diets for 6 weeks. Mice were assessed for Zn status, serum and urinary indices of diabetes, and gastrocnemius insulin receptor concentration and tyrosine kinase activity. Fasting serum glucose concentrations were significantly lower in the dbZS group compared with the dbZD group (19.3 +/- 2.9 and 27.9 +/- 4.1 mM, respectively), whereas the dbC mice had an intermediate value. There was a negative correlation between femur Zn and serum glucose concentrations (r = -0.59 for lean mice, P = 0.007). The dbZS group had higher pancreatic Zn and lower circulating insulin concentrations than dbZC mice. Insulin-stimulated tyrosine kinase activity in gastrocnemius muscle was higher in the db/db genotype, and insulin receptor concentration was not altered. In summary, dietary Zn supplementation attenuated hyperglycemia and hyperinsulinemia in db/db mice, suggesting that the roles of Zn in pancreatic function and peripheral tissue glucose uptake need to be further investigated.

Bradykinin enhances insulin receptor tyrosine kinase in 32D cells reconstituted with bradykinin and insulin signaling pathways

We have previously shown that bradykinin potentiated insulin-induced glucose uptake through GLUT4 translocation in canine adipocytes and skeletal muscles. The aim of this study was to determine the molecular mechanism of bradykinin enhancement of the insulin signal. For this purpose, 32D cells, which express a limited number of insulin receptors and lack endogenous bradykinin B2 receptor (BK2R) or insulin receptor substrate (IRS)-1 were transfected with BK2R cDNA and/or insulin receptor cDNA and/or IRS-1 cDNA, and analyzed. In 32D cells that expressed BK2R and insulin receptor (32D-BKR/IR), bradykinin alone had no effect on the phosphorylation of the insulin receptor, but it enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor. In 32D cells that expressed BK2R, insulin receptor and IRS-1 (32D-BKR/IR/IRS1), bradykinin also enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1. An increase in insulin-stimulated phosphorylation of IRS-1 by treatment with bradykinin in 32D-BKR/IR/IRS1 cell was associated with increased binding of 85 kD subunit of phosphatidylinositol 3 (PI 3)-kinase and increased IRS-1 associated PI 3-kinase activity. These effects of bradykinin were not observed in 32D cells which lack the expression of BK2R (32D-IR/IRS1) or insulin receptor (32D-BKR/IRS1). Furthermore, tyrosine phosphatase activity against insulin receptor β-subunit in plasma membrane fraction of 32D-BKR/IR cells was significantly reduced by bradykinin, suggesting that the effect of bradykinin was in part mediated by inhibition of protein tyrosine phosphatase(s). Our results clearly demonstrated that bradykinin enhanced insulin-stimulated tyrosine kinase activity of the insulin receptor and downstream insulin signal cascade through the BK2R mediated signal pathway.

Dephosphorylation increases insulin-stimulated receptor kinase activity in skeletal muscle of obese Zucker rats.

Serine/threonine phosphorylation of insulin receptor has been implicated in the development of insulin resistance. To investigate whether dephosphorylation of serine/threonine residues of the insulin receptor may restore the decreased insulin-stimulated receptor tyrosine kinase activity in skeletal muscle of obese Zucker rats, insulin receptor tyrosine kinase activity was measured before and after alkaline phosphatase treatment. Compared to lean controls, insulin-stimulated glucose transport was depressed by 61% (p < 0.05) in obese Zucker rats. The insulin receptor and insulin receptor substrate-1 contents were decreased by 14% (p < 0.05) and 16% (p < 0.05), respectively, in skeletal muscle of obese Zucker rats. In vivo insulin-induced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 was depressed by 82% (p < 0.05) and 86% (p < 0.05), respectively. In the meantime, in vitro insulin-stimulated receptor tyrosine kinase activity in obese rats was decreased by 39% (p < 0.05). Dephosphorylation of the insulin receptor by prior alkaline phosphatase treatment increased insulin-stimulated receptor tyrosine kinase activity in both lean and obese Zucker rats, but the increase was three times greater in obese Zucker rats (p < 0.05). These findings suggest that excessive serine/threonine phosphorylation of the insulin receptor in obese Zucker rats may be a cause for insulin resistance in skeletal muscle.

Pinealectomy causes glucose intolerance and decreases adipose cell responsiveness to insulin in rats.

Although the pineal gland influences several physiological systems, only a few studies have investigated its role in the intermediary metabolism. In the present study, male Wistar rats, pinealectomized or sham-operated 6 wk before the experiment, were submitted to both intravenous glucose tolerance tests (IVGTT) and insulin binding as well as glucose transport assays in isolated adipocytes. The insulin receptor tyrosine kinase activity was assessed in liver and muscle. The insulin secretory response during the IVGTT was impaired, particularly in the afternoon, and the glucose transport responsiveness was 33% lower in pinealectomized rats. However, no difference was observed in the insulin receptor number of adipocytes between groups as well as in insulin-stimulated tyrosine kinase activity, indicating that the initial steps in the insulin signaling were well conserved. Conversely, a 40% reduction in adipose tissue GLUT-4 content was detected. In conclusion, pinealectomy is responsible for both impaired insulin secretion and action, emphasizing the influence of the pineal gland on glucose metabolism.

Insulin receptor signaling in the beta-cell influences insulin gene expression and insulin content: evidence for autocrine beta-cell regulation.

The insulin receptor (IR) is expressed by insulin-secreting beta-cells, but its cellular function is unknown. We transfected betaTC6-F7 beta-cells with cDNAs encoding either wild-type or mutant kinase-inactive (A/K1018) IRs, and by fluorescence-activated cell sorting generated polyclonal beta-cell lines that overexpressed each receptor type at levels two- to fourfold higher than the parental cells. Beta-cells overexpressing wild-type IRs had a proportional increase in insulin-stimulated tyrosine kinase activity; no change occurred in beta-cells expressing the mutant IR. We observed a threefold increase in cellular insulin content in beta-cells that overexpressed the wild-type IR, as determined by radioimmunoassay. This increase occurred despite a fivefold elevated rate of both basal and 10 mmol/l glucose-induced insulin secretion. Fractional insulin secretion (percentage of total cell insulin releasable at 10 mmol/l glucose) was unchanged in beta-cells overexpressing the wild-type IR compared with the parental beta-cell line. Insulin content and insulin secretion were unaffected by overexpression of kinase-inactive IRs. Steady-state insulin mRNA levels were elevated twofold in the beta-cells overexpressing the wild-type IR and unchanged in the beta-cells expressing the kinase-inactive receptor, as determined by Northern blot analysis. The rate of insulin mRNA degradation measured in the presence of 5 microg/ml actinomycin D was not significantly affected in either cell line. In the absence of glucose, the basal level of (pro)insulin biosynthesis in the beta-cells overexpressing the wild-type IR increased significantly (61%) compared with the beta-cells transfected with the kinase-inactive IR. These data indicate that IR signaling can regulate insulin gene transcription and can modulate the steady-state insulin content of beta-cells.

Bisoprolol and Captopril Effects on Insulin Receptor Tyrosine Kinase Activity in Essential Hypertension

Angiotension converting enzyme (ACE) inhibitors and β-blockers have been reported to possess disparate effects on insulin sensitivity. The aim of this study was to study the effects of the selective β-1 blocker bisoprolol and of the ACE inhibitor captopril on cellular insulin action in hypertensive individuals. After washout, 12 mild to moderate essential hypertensives were randomized in a double-blind manner to 5 mg bisoprolol daily or 25 mg captopril twice daily for 8 weeks. Erythrocyte insulin binding and insulin-stimulated tyrosine kinase (TK) activity were measured before and after therapy. Both agents decreased diastolic blood pressure significantly (bisoprolol 96.5 ± 0.9 to 87.8 ± 3.1 mm Hg; captopril 96.5 ± 0.9 to 91.5 ± 1.8 mm Hg; P < .05). Fasting plasma glucose, insulin, and insulin/glucose indices remained unchanged after both therapies, as did lipid profiles. Maximal insulin-stimulated TK activity, assessed by phosphorylation of the exogenous substrate poly-Glu 80Tyr 20, was significantly higher ( P < .05) after bisoprolol treatment, but not after captopril treatment, when compared to placebo (bisoprolol 8.5 ± 1.8; captopril 7.3 ± 1.5; placebo: 6.4 ± 1.3 pmol 32P-ATP/fmol bound insulin). However, captopril, but not bisoprolol, increased the sensitivity of the receptor TK activity, as measured by the half-maximal activity concentration (ED 50). Specific insulin binding was not affected by these two agents. Thus, both captopril and bisoprolol may have favorable but different effects on TK activity and insulin action at the cellular level.

Purification and characterization of an insulin-stimulated insulin receptor serine kinase.

In cells, insulin stimulates autophosphorylation of the insulin receptor on tyrosine and its phosphorylation on serine and threonine by poorly characterized kinases. Here we describe methods for the purification of an insulin-stimulated insulin receptor serine kinase from human placenta and rat liver by sequential chromatography of solubilized membranes on wheat germ agglutinin-agarose, Mono Q, phenyl-Superose, and Superose 12. On silver-stained SDS-polyacrylamide gels, the resulting kinase was homogeneous (human) or near-homogeneous (rat) and had an apparent M(r) of 40000. The apparent M(r) determined by gel filtration was also 40000, suggesting that the kinase exists as a monomer. The kinase could be reconstituted back to the insulin receptor stripped of the kinase to yield a high stoichiometry of serine phosphorylation of the insulin receptor in the presence of insulin (0.75 +/- 0.15 mol/mol of beta-subunit, mean +/- SEM, n = 3). The activity of the reconstituted kinase toward the insulin receptor was insulin-regulated, being stimulated > 5-fold by insulin. Insulin increased the catalytic activity of the reconstituted kinase. The purified kinase specifically phosphorylated serine 1078 of the insulin receptor, a major site of insulin-stimulated serine phosphorylation in vivo, showing that the purified kinase phosphorylated a physiologically relevant site on the insulin receptor. Phosphorylation of serine 1078 of the insulin receptor to high stoichiometry by the kinase did not affect insulin-stimulated exogenous protein tyrosine kinase activity of the insulin receptor. Similarly, insulin receptor phosphorylated with or without the purified kinase exhibited the same levels of tyrosine autophosphorylation and of the tyrosine kinase-activating tris-phosphorylated kinase domain species. Properties of the kinase distinguished it from kinases known to act on the insulin receptor and other kinases that are insulin-stimulated, indicating that the kinase is a novel entity. The serine kinase underwent autophosphorylation on serine and immunoprecipitated with the insulin receptor. The availability of the purified kinase should facilitate cloning of the kinase, determination of the mechanism of activation of the kinase, and study of the wider potential role of the kinase in insulin signalling, and the ability to be able to phosphorylate serine 1078 to high stoichiometry should facilitate further studies into the function of this serine phosphorylation site.

The Amino Acid Sequence GPLY Is Not Necessary for Normal Endocytosis of the Human Insulin Receptor B Isoform

The human insulin receptor (hIR) cytoplasmic juxtamembrane domain contains two tyrosine (Y) residues which exist in GPLY and NPEY motifs that have been implicated in endocytic function. We have previously shown that the NPEY motif is not necessary for endocytosis of the B isoform (exon 11+) of hIR. To examine the role of the GPLY sequence in transmembrane insulin signaling and endocytic functions of hIR-B, we constructed a mutant receptor, hIRΔGPLY, that lacks the GPLY sequence (residues 962-965), and stably expressed it in CHO cells. When compared to wild type hIR-B (hIR-WT) similarly expressed in CHO cells, the hIRΔGPLY mutant exhibited higher insulin binding affinity (EC50 of 1.0 vs 3.5 nM) and normal insulin-stimulated receptor tyrosine autophosphorylation and kinase activity towards the endogenous 185 kDa insulin receptor substrate. The hIRΔGPLY receptor also exhibited normal endocytic functions as hIR-WT in that: a) the internalization of surface photoaffinity labeled hIRΔGPLY was similar to that of hIR-WT, and b) the rate and extent of 125I-insulin internalization and degradation at 37°C were also unimpaired. Therefore, these results demonstrate that the GPLY sequence is not necessary for transmembrane insulin signaling and endocytic functions of the hIR-B isoform.

Membrane glycoprotein PC-1 and insulin resistance in non-insulin-dependent diabetes mellitus.

Most patients with non-insulin-dependent diabetes mellitus are resistant to both endogenous and exogenous insulin. Insulin resistance precedes the onset of this disease, suggesting that it may be an initial abnormality. Insulin-receptor kinase activity is impaired in muscle, fibroblasts and other tissues of many patients with non-insulin-dependent diabetes mellitus, but abnormalities in the insulin-receptor gene do not appear to be the cause of this decreased kinase activity. Skin fibroblasts from certain insulin-resistant patients contain an inhibitor of insulin-receptor tyrosine kinase. Here we show that this inhibitor is a membrane glycoprotein, termed PC-1 (refs 10, 11). We find that PC-1 activity is increased in fibroblasts from seven of nine patients with typical non-insulin-dependent diabetes mellitus. In addition, overexpression of PC-1 in transfected cultured cells reduces insulin-stimulated tyrosine kinase activity. These studies raise the possibility that PC-1 has a role in the insulin resistance of non-insulin-dependent diabetes mellitus.

Potentiation of insulin stimulation of phosphatidylinositol 3-kinase by thiazolidinedione-derived antidiabetic agents in Chinese hamster ovary cells expressing human insulin receptors and L6 myotubes.

Thiazolidinedione derivatives are insulin-sensitizing agents with proven antidiabetic activities in vivo. To explore the mechanism of action of this class of compounds, the effects of pioglitazone, CP-86,325, and AD-5075 on elements of the insulin signal transduction pathways were studied in Chinese hamster ovary cells overexpressing human insulin receptor (CHO.T) and L6 myotubes. In CHO.T cells, the binding of insulin to its receptor and the insulin-stimulated tyrosine kinase activity of the receptor were not altered by pioglitazone or CP-86,325. In contrast, treatment of CHO.T cells with the compounds resulted in significant increases in insulin-stimulated phosphatidylinositol (PI) 3-kinase activity. This insulin-enhancing effect was also observed in L6 myotubes treated with CP-86,325. The augmentations in kinase activity observed in CHO.T cells correlated with increases in the amount of PI 3-kinase (p85 subunit) in anti-phosphotyrosine immunoprecipitates of cell lysates. No gross changes in the tyrosine phosphorylation state of the insulin receptor substrate-1 were detected in insulin-stimulated CHO.T cells following treatment with the compounds. Furthermore, the compounds did not enhance insulin stimulation of mitogen-activated protein kinase or DNA synthesis in CHO.T cells. Thus, thiazolidinedione-derived antidiabetic agents may act as insulin sensitizers by augmenting insulin stimulation of PI 3-kinase activity in a rather specific manner.

Annexin I as a Potential Inhibitor of Insulin Receptor Protein Tyrosine Kinase

Insulin receptor (IR) purified from human placenta by wheat germ agglutinin affinity chromatography was incubated in the presence of insulin, [γ-32P]ATP and annexin I. In parallel to its own tyrosine phosphorylation, annexin I promoted a dose-dependent inhibition of IR autophosphorylation (IC50 0.5 μM). This effect was specific for insulin-stimulated tyrosine kinase activity and required the N-terminal end of the protein containing the phosphorylatable Tyr21 residue. A pentadecapeptide encompassing residues 16-30 of human annexin I displayed a similar activity, but at higher concentrations. These data underscore a specific interaction of IR with annexin I, which should be considered as a potential physiological regulator of the effects of insulin on its target tissues.

Reduced insulin receptor expression and function in human colonic Caco-2 cells by ras and polyoma middle T oncogenes

Taking advantage of the potent mitogenic effect exerted by insulin in human colonic cells, we used Caco-2 cells transfected with an activated (Val-12) human Haras gene or the polyoma middle T (PyMT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity, to investigate the effect of oncogenic p21ras and PyMT/pp60c-src on insulin mitogenic signaling. As compared to vector control Caco-2 cells, both oncogene-transfected cells exhibited: 1) a loss of response to insulin's stimulatory effect on mitogen-activated protein (MAP) kinase activity and cell proliferation, both of which were constitutively increased; 2) a decrease in insulin receptor (IR) affinity and insulin-stimulated exogenous tyrosine kinase activity, which resulted from increased protein kinase C (PKC) activity (Delage, S., Chastre, E., Empereur, S., Wicek, D., Veissiére, D., Capeau, J., Gespach, C., and Cherqui, G. (1993) Cancer Res. 53, 2762-2770), since IR alterations were corrected by PKC down-regulation; and 3) a decrease in both IR mRNA level and IR number, which was independent of PKC since it persisted after PKC down-regulation. In conclusion, this is the first evidence that oncogenic p21ras and PyMT/pp60c-src abolish insulin mitogenic signaling in human colonic cells through mechanisms involving (i) constitutive activation of MAP kinase and (ii) marked decreases in both IR function and expression which are mediated by PKC-dependent and PKC-independent pathways, respectively.

Insulin-Receptor Tyrosine Kinase Activity is Decreased in Erythrocytes from Non-Obese Patients with NIDDM

We have examined insulin binding, and insulin receptor associated tyrosine kinase activity in detergent solubilized and Ricin II-agarose purified receptor preparations from erythrocytes of obese and non-obese subjects with normal glucose tolerance and non-obese patients with NIDDM. Insulin receptor activity, as assessed by [125I Tyr A14] insulin binding, was significantly lower in erythrocyte preparations from the obese group when compared with similar preparations from non-obese subjects, with either normal glucose tolerance or NIDDM. The affinity of the receptor for insulin, however, was reduced in both obese subjects and patients with NIDDM as compared to non-obese subjects with normal glucose tolerance. Insulin receptor tyrosine kinase activity, measured in the absence (basal) and presence of insulin (0.3-3000 nM), was decreased in obese and NIDDM subjects with normal glucose tolerance and in patients with NIDDM. Insulin sensitivity, measured as the dose of insulin required for half-maximal activation of kinase activity, however, was comparable among three groups. In contrast, insulin-stimulated tyrosine kinase activity, when normalized to insulin binding activity, was unchanged in both non-obese and obese subjects with normal glucose tolerance, but was reduced approximately 60% in the NIDDM group. These findings indicate that the functional behavior of insulin receptor-kinase signaling system is markedly impaired in non-obese patients with NIDDM. Furthermore, the insulin receptor-tyrosine kinase defect (i.e. decrease in activity) observed in patients with NIDDM is probably related to a reduction in coupling efficiency between insulin binding and the activation of the receptor tyrosine kinase activity.

Expression of a dominant-negative mutant human insulin receptor in the muscle of transgenic mice.

To examine the in vivo effects of a kinase-deficient mutant human insulin receptor, we used the muscle creatine kinase promoter to express a putative dominant-negative receptor: Ala1134-->Thr (Moller, D. E., Yokota, A., White, M. F., Pazianos, A. G., and Flier, J. S. (1990) J. Biol. Chem. 265, 14979-14985) in transgenic mice. Two lines were generated, where receptor expression was restricted to striated muscle and was increased by 5-12-fold in skeletal muscle. Transgenic gluteal muscle insulin receptor kinase activity was reduced by approximately 80% after maximal in vitro insulin stimulation. Glycogen content in this muscle was reduced by 45% in transgenic mice. Insulin levels were approximately 2-fold higher, and glucose concentrations were 12% higher in transgenics fed ad libitum. Transgenic mice exhibited reduced in vivo sensitivity to low dose (0.1 milliunits/g) intravenous insulin. In isolated soleus muscles from transgenics, where mutant receptors were expressed at lower levels, insulin-stimulated receptor kinase activity was reduced by 42%, but insulin-stimulated 2-deoxyglucose uptake was unaffected. These results indicate that (i) overexpression of a kinase-deficient human insulin receptor in muscle causes dominant-negative effects at the level of receptor kinase activation, (ii) impairment of insulin-stimulated muscle receptor tyrosine kinase activity can cause decreased insulin sensitivity in vivo, (iii) kinase-defective receptor mutants may be used to create novel animal models of tissue-specific insulin resistance.

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

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 HbA 1c 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 β-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 β-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.

Characterization of the hepatic insulin receptor undergoing internalization through clathrin-coated vesicles and endosomes

Administration of insulin to rats caused a transient increase in the amount of hepatic insulin receptor present in clathrin-coated vesicles and endosomes. However, the total ‘in vitro’ insulin stimulated tyrosine kinase activity of the receptor present in endosomes did not vary when expressed per mg of protein and decreased when expressed per β-subunit content. A decrease in the endogenous phospho tyrosine content of the receptor β-subunit was observed in endosomes in response to insulin. This indicates that a fraction of the internalized receptor is dephosphorylated in endosomes, which renders it unable to become stimulated by insulin ‘in vitro’.

Decreased tyrosine kinase activity in partially purified insulin receptors from muscle of young, non-obese first degree relatives of patients with type 2 (non-insulin-dependent) diabetes mellitus.

Recently, we demonstrated insulin resistance due to reduced glucose storage in young relatives of Type 2 diabetic patients. To investigate whether this was associated with a defective insulin receptor kinase, we studied ten of these young (27 +/- 1 years old) non-obese glucose tolerant first degree relatives of patients with Type 2 diabetes and eight matched control subjects with no family history of diabetes. Insulin sensitivity was assessed by a hyperinsulinaemic, euglycaemic clamp. Insulin receptors were partially purified from muscle biopsies obtained in the basal and the insulin-stimulated state during the clamp. Insulin binding capacity was decreased by 28% in the relatives (p < 0.05) in the basal biopsy. Tyrosine kinase activity in the receptor preparation was decreased by 50% in both basal and insulin-stimulated biopsies from the relatives. After stimulation with insulin "in vitro", kinase activity was reduced in the relatives in basal (p < 0.005) and insulin-stimulated (p < 0.01) biopsies and also when expressed per insulin binding capacity (p approximately 0.05). Insulin stimulation of non-oxidative glucose metabolism correlated with "in vitro" insulin-stimulated tyrosine kinase activity (r = 0.61, p < 0.01) and also when expressed per binding capacity (r = 0.53, p < 0.025). We suggest that the marked defect in tyrosine kinase activity in partially purified insulin receptors from skeletal muscle is an early event in the development of insulin resistance and contributes to the pathophysiology of Type 2 diabetes.