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FP Dominici, D Cifone, A Bartke, and D Turyn

Growth hormone (GH) excess is associated with secondary hyperinsulinemia, but the molecular mechanism and consequences of this alteration are poorly understood. To address this problem we have examined the levels and phosphorylation state of the insulin receptor (IR) and the insulin receptor substrate-1 (IRS-1), the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) as well as the PI 3-kinase activity in the livers of GH-transgenic mice. As expected, IR levels were reduced in the liver of GH-transgenic mice (55% of normal values) as determined by immunoblotting with an anti-IR beta-subunit antibody. IR and IRS-1 phosphorylation as determined by immunoblotting with antiphosphotyrosine antibody were increased in basal conditions by 315% and 560% respectively. After a bolus administration of insulin in vivo, IR phosphorylation increased by 40% while IRS-1 phosphorylation did not change. Insulin administration to control (normal) mice produced 670% and 300% increases in the IR and IRS-1 phosphorylation respectively. In the GH-transgenic animals, basal association of PI 3-kinase with IRS-1 as well as PI 3-kinase activity in liver was increased by 200% and 280% respectively, and did not increase further after administration of insulin in vivo, indicating a complete insensitivity to insulin at these levels. In conclusion, GH excess and the resulting secondary hyperinsulinemia were associated with alterations at the early steps of insulin action in liver. IR concentration was reduced, while IR and IRS-1 phosphorylation, IRS-1/PI 3-kinase association, and PI 3-kinase activity appeared to be maximally activated under basal conditions, thus making this tissue insensitive to further stimulation by exogenous insulin in vivo.

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FP Dominici, A Balbis, A Bartke, and D Turyn

Overexpression of bovine growth hormone (bGH) in transgenic (PEPCK-bGH) mice induces resistance to insulin, which is compensated by a major increase in insulin levels. In these animals, hepatic insulin receptors (InsRs) are downregulated while tyrosine kinase activity of wheat germ agglutinin (WGA)-purified InsRs towards exogenous substrates is unexpectedly increased. By normalizing insulinemia, we attempted to determine whether the alterations detected in the early steps of insulin signal transduction are due to exposure to chronically high GH levels or are secondary to hyperinsulinemia. Transgenic PEPCK-bGH animals were treated with a single intraperitoneal administration of streptozotocin (STZ) or were deprived of food for 48 h, to normalize insulin levels. Both fasting and STZ treatment were effective in reducing insulin blood levels to control values or below, while GH levels remained unchanged (STZ treatment) or increased (fasted animals). In the liver of untreated transgenic mice, the number of InsRs as determined by 125I-insulin binding was significantly diminished (65+/-5% and 60+/-6% of normal values in microsomes and solubilized membranes respectively;P<0.01 vs control mice). In treated transgenic mice, the number of InsRs increased to values similar to or slightly higher than those found in normal control mice (STZ-treated: 139+/-26% and 126+/-8%; fasted: 128+/-5% (P<0.05) and 102+/-1.5%, for microsomes and solubilized membranes respectively). Neither treatment altered InsR affinity. InsR concentration in liver as determined by immunoblotting using an antibody against the beta-subunit of the insulin receptor was found to be reduced in transgenic mice (69+/-3% of normal values,P<0.001) and was normalized after both STZ treatment (105+/-4%) and fasting (109+/-4%). Insulin-stimulated autophosphorylation activity of InsRs in transgenic mice was increased (154+/-13%,P<0.01 compared with the control group), essentially normalized by STZ treatment (96+/-14%), and reduced by fasting, to below the values measured in normal control mice (56+/-15%,P<0.05). The potential influence of basal serine/threonine (Ser/Thr) phosphorylation of the InsR beta-subunit on the regulation of the InsRs from transgenic mice was also investigated. The autophosphorylation activity of WGA-purified InsRs from all groups of mice studied was essentially unchanged after dephosphorylation with alkaline phosphatase or mild trypsinization. Consequently, our results suggest that the observed changes in InsR number and autophosphorylation activity in the liver of bGH transgenic mice are directly related to changes in insulin blood levels, and that Ser/Thr phosphorylation is apparently not involved in the regulation of the InsR autophosphorylation activity in this model of insulin resistance.

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FP Dominici, G Arostegui Diaz, A Bartke, JJ Kopchick, and D Turyn

Growth hormone (GH) deficiency is associated with increased sensitivity to insulin, but the molecular mechanisms involved in this association are poorly understood. In the current work, we have examined the consequences of the absence of the biological effects of GH on the first steps of the insulin signaling system in vivo in liver of mice with targeted disruption of the GH receptor/GH binding protein gene (GHR-KO mice). In these animals, circulating insulin concentrations are less than 4 microIU/ml, and glucose concentrations are low, concordant with a state of insulin hypersensitivity. The abundance and tyrosine phosphorylation state of the insulin receptor (IR), the IR substrate-1 (IRS-1), and Shc, the association between IRS-1 and the p85 subunit of phosphatidylinositol (PI) 3-kinase, the IRS-1- and the phosphotyrosine-associated PI 3-kinase in liver were examined. We found that, in liver of GHR-KO mice, the lack of GHR and GH eff! ects is associated with: (1) increased IR abundance, (2) increased insulin-stimulated IR tyrosine phosphorylation, (3) normal efficiency of IRS-1 and Shc tyrosine phosphorylation and (4) normal activation of PI 3-kinase by insulin. These alterations could represent an adaptation to the low insulin concentrations displayed by these animals, and may account for their increased insulin sensitivity.

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FP Dominici, S Hauck, DP Argentino, A Bartke, and D Turyn

In the present study we have used hypopituitary Ames dwarf mice, which lack GH, prolactin and TSH, to investigate the consequences of the deficiency of these hormones on glucose homeostasis and on the initial components of the insulin signal transduction pathway in the liver. Ames dwarf mice displayed hypersensitivity to insulin since they maintained lower fasting glucose concentrations (73% of control values), had significantly reduced amounts of insulin (58% of control values), and exhibited an increased hypoglycemic response to exogenous insulin. Probably as a result of reduced insulin production, Ames dwarf mice displayed intolerance to glucose. The insulin-stimulated phosphorylation of the insulin receptor (IR) tended to be increased in the liver of Ames dwarf mice, while IR receptor protein content was increased by 38%. Insulin-stimulated phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 was increased by 61 and 72% respectively, while IRS-1 and IRS-2 protein levels were increased by 76 and 95%. The insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase with IRS-1 was increased by 28%, but unaltered with IRS-2. Interestingly, while the insulin-stimulated phosphotyrosine-derived PI 3-kinase activity was not changed, insulin-stimulated protein kinase B activation was increased by 41% in this tissue. These alterations may account for the insulin hypersensitivity exhibited by these animals. The present findings in long-lived Ames dwarf mice add to the evidence that insulin signaling is importantly related to the regulation of aging and life span.

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MP Coba, MC Munoz, FP Dominici, JE Toblli, C Pena, A Bartke, and D Turyn

Serine phosphorylation of the insulin receptor (IR) has been proposed to exert an inhibitory influence on its tyrosine kinase activity. Previous works using site-directed mutagenesis suggested that serine 994 of the IR (IR Ser 994) might be part of an inhibitory domain of the receptor. In this study we examined whether this residue is subjected to phosphorylation in vivo. We used a site-phosphospecific antibody to determine the extent of phosphorylation of IR Ser 994 in insulin target tissues from two animal models of insulin resistance with different IR kinase (IRK) activity: obese (fa/fa) Zucker rats and transgenic mice overexpressing bovine growth hormone (PEPCK-bGH mice).Phosphorylation at IR Ser 994 was markedly increased in liver of obese rats. This alteration appeared to be tissue-selective since no phosphorylation on Ser 994 was detected in IRs isolated from skeletal muscle of these animals. On the other hand, the phosphorylation level of IR Ser 994 was very low in liver of PEPCK-bGH mice and did not differ from that of the control group. We have also demonstrated that protein kinase (PK) C isoforms alpha, betaI and zeta are able to promote the in vitro phosphorylation of the IR on Ser 994. Differential findings in these two models of insulin resistance might thus reflect increased PKC activity resulting from increased lipid availability in obese Zucker rats. Our results suggest that Ser 994 is a novel in vivo IR phosphorylation site that might be involved in the regulation of the IRK in some states of insulin resistance.