Prolonged exposure to GH impairs insulin signaling in the heart

Acromegaly is associated with cardiac hypertrophy, which is believed to be a direct consequence of chronically elevated GH and IGF1. Given that insulin is important for cardiac growth and function, and considering that GH excess induces hyperinsulinemia, insulin resistance, and cardiac alterations, it is of interest to study insulin sensitivity in this tissue under chronic conditions of elevated GH. Transgenic mice overexpressing GH present cardiomegaly and perivascular and interstitial fibrosis in the heart. Mice received an insulin injection, the heart was removed after 2 min, and immunoblotting assays of tissue extracts were performed to evaluate the activation and abundance of insulin-signaling mediators. Insulin-induced tyrosine phosphorylation of the insulin receptor (IR) was conserved in transgenic mice, but the phosphorylation of IR substrate 1 (IRS1), its association with the regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), and the phosphorylation of AKT were decreased. In addition, total content of the glucose transporter GLUT4 was reduced in transgenic mice. Insulin failed to induce the phosphorylation of the mammalian target of rapamycin (mTOR). However, transgenic mice displayed increased basal activation of the IR/IRS1/PI3K/AKT/mTOR and p38 signaling pathways along with higher serine phosphorylation of IRS1, which is recognized as an inhibitory modification. We conclude that GH-overexpressing mice exhibit basal activation of insulin signaling but decreased sensitivity to acute insulin stimulation at several signaling steps downstream of the IR in the heart. These alterations may be associated with the cardiac pathology observed in these animals.     

Sam68 is a docking protein linking GAP and PI3K in insulin receptor signaling

The 68 kDa Src substrate associated during mitosis (Sam68) is an RNA binding protein with Src homology (SH) 2 and 3 domain binding sites. We have recently found that Sam68 is a substrate of the insulin receptor (IR) and that Tyr-phosphorylated Sam68 associates with the SH2 domains of p85 PI3K. In the present work, using HTC-IR cells, we have found that insulin stimulation promotes the relocalization of Sam68 from the nucleus to the cytoplasm, and we have further studied the role of Sam68 in insulin receptor signaling complexes, by co-precipitating experiments. Thus, Sam68 is co-precipitated with p85 PI3K, IRS-1 and IR. The association of Sam68 with these complexes is mediated by the SH2 domains of PI3K. Moreover, we have found that Sam68 is a p120GAP associated protein after Tyr-phosphorylation by the IR. This association is mediated by the SH2 domains of GAP (preferentially the C-terminal SH2). Thus, Sam68 is linking p120GAP to PI3K signaling pathway. In fact, PI3K activity was increased in both anti-Sam68 and anti-GAP immmunoprecipitates upon insulin stimulation. We propose that the recruitment of the docking protein Sam68 to the PI3K pathway may serve to allow the association of other signaling molecules, i.e. p120GAP. In this way, these signaling complexes may modulate other signaling cascades of IR, such as p21Ras pathway.     

The novel roles of liver for compensation of insulin resistance in human growth hormone transgenic rats

Chronic excess of GH is known to cause hyperinsulinemia and insulin resistance. We developed human GH transgenic (TG) rats, which were characterized by high plasma levels of human GH and IGF-I. These TG rats showed higher levels of plasma insulin, compared with control littermates, whereas plasma glucose concentrations were normal. Insulin-dependent glucose uptake into adipocytes and muscle was impaired, suggesting that these rats developed insulin resistance. In contrast, insulin-independent glucose uptake into hepatocytes from TG rats was significantly increased, and glycogen and lipid levels in livers of TG rats were remarkably high. Because the role of liver in GH-induced insulin resistance is poorly understood, we studied insulin signaling at early stages and insulin action in liver and primary cultures of hepatocytes prepared from TG rats. There was no difference in insulin receptor kinase activity induced by insulin between TG and control rats; however, insulin-dependent insulin receptor substrate-2 tyrosine phosphorylation, glycogen synthase activation, and expression of enzymes that induce lipid synthesis were potentiated in hepatocytes of TG rats. These results suggest that impairment of insulin-dependent glucose uptake by GH excess in adipose tissue and muscle is compensated by up-regulation of glucose uptake in liver and that potentiation of insulin signaling through insulin receptor substrate-2 in liver experiencing GH excess causes an increase in glycogen and lipid synthesis from incorporated glucose, resulting in accumulation of glycogen and lipids in liver. This novel mechanism explains normalization of plasma glucose levels at least in part in a GH excess model.     

Akt mediates the cross-talk between beta-adrenergic and insulin receptors in neonatal cardiomyocytes

Upregulation of the sympathetic nervous system plays a key role in the pathogenesis of insulin resistance. Although the heart is a target organ of insulin, few studies have examined the mechanisms by which beta-adrenergic stimulation affects insulin sensitivity in cardiac muscle. In this study, we explored the molecular mechanisms involved in the regulation of the cross-talk between beta adrenergic and insulin receptors in neonatal rat cardiomyocytes and in transgenic mice with cardiac overexpression of a constitutively active mutant of Akt (E40K Tg). The results of this study show that beta-adrenergic receptor stimulation has a biphasic effect on insulin-stimulated glucose uptake. Short-term stimulation induces an additive effect on insulin-induced glucose uptake, and this effect is mediated by phosphorylation of Akt in threonine 308 through PKA/Ca2+-dependent and PI3K-independent pathway, whereas insulin-evoked threonine phosphorylation of Akt is exclusively PI3K-dependent. On the other hand, long-term stimulation of beta-adrenergic receptors inhibits both insulin-stimulated glucose uptake and insulin-induced autophosphorylation of the insulin receptor, and at the same time promotes threonine phosphorylation of the insulin receptor. This is mediated by serine 473 phosphorylation of Akt through PKA/Ca2+ and PI3K-dependent pathways. Under basal conditions, E40K Tg mice show increased levels of threonine phosphorylation of the beta subunit of the insulin receptor and blunted tyrosine autophosphorylation of the beta-subunit of the insulin receptor after insulin stimulation. These results indicate that, in cardiomyocytes, beta-adrenergic receptor stimulation impairs insulin signaling transduction machinery through an Akt-dependent pathway, suggesting that Akt is critically involved in the regulation of insulin sensitivity.     

Loss of sensitivity to insulin at early events of the insulin signaling pathway in the liver of growth hormone-transgenic mice.

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.     

Blockade of class IB phosphoinositide-3 kinase ameliorates obesity-induced inflammation and insulin resistance

Obesity and insulin resistance, the key features of metabolic syndrome, are closely associated with a state of chronic, low-grade inflammation characterized by abnormal macrophage infiltration into adipose tissues. Although it has been reported that chemokines promote leukocyte migration by activating class IB phosphoinositide-3 kinase (PI3Kγ) in inflammatory states, little is known about the role of PI3Kγ in obesity-induced macrophage infiltration into tissues, systemic inflammation, and the development of insulin resistance. In the present study, we used murine models of both diet-induced and genetically induced obesity to examine the role of PI3Kγ in the accumulation of tissue macrophages and the development of obesity-induced insulin resistance. Mice lacking p110γ (Pik3cg(-/-)), the catalytic subunit of PI3Kγ, exhibited improved systemic insulin sensitivity with enhanced insulin signaling in the tissues of obese animals. In adipose tissues and livers of obese Pik3cg(-/-) mice, the numbers of infiltrated proinflammatory macrophages were markedly reduced, leading to suppression of inflammatory reactions in these tissues. Furthermore, bone marrow-specific deletion and pharmacological blockade of PI3Kγ also ameliorated obesity-induced macrophage infiltration and insulin resistance. These data suggest that PI3Kγ plays a crucial role in the development of both obesity-induced inflammation and systemic insulin resistance and that PI3Kγ can be a therapeutic target for type 2 diabetes.     

Compensatory alterations of insulin signal transduction in liver of growth hormone receptor knockout mice

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.     

Exercise training eliminates age-related differences in skeletal muscle insulin receptor and IRS-1 abundance in rats

Insulin resistance is common in old age, and exercise training can improve insulin sensitivity. The purpose of this study was to determine the influence of age (6 vs 26 months) and exercise training (10 weeks of treadmill running) on insulin signaling protein abundance in skeletal muscle from male Fisher 344 rats. Muscle levels of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and Akt1, a serine-threonine kinase, were determined. IRS-1 was reduced with aging, IR and PI3K abundance was greater in old rats, and Akt1 was unchanged. IRS-1 was increased by training in old but not young rats, and IR was increased by training in young but not old rats. PI3K tended to increase and Akt1 did not change with training, regardless of age. Aging does not uniformly affect insulin signaling protein abundance, and exercise differentially alters IR and IRS-1 in young and old rats, thereby eliminating age-related differences in these proteins.     

The G972R variant of the insulin receptor substrate-1 gene impairs insulin signaling and cell differentiation in 3T3L1 adipocytes; treatment with a PPARgamma agonist restores normal cell signaling and differentiation

The insulin receptor substrate-1 (IRS-1) plays a central role in insulin sensitivity, and association studies have shown that the IRS-1 G972R variant is a risk factor for insulin resistance. However, how this mutation may lead to impaired insulin sensitivity is still to be determined. Our study aimed to evaluate, after transfection of the IRS-1 G972R variant in 3T3L1 adipocytes, the effect of this mutation on insulin signaling and on cell differentiation. The 3T3L1 cells were transfected with pcDNA3 expression vector containing either the human wild-type IRS-1 or the G972R variant. After induction of differentiation, the 3T3L1 transfected with wild-type IRS-1 differentiated in 6-8 days, while the cells transfected with G972R variant did not differentiate. To determine whether the defect in IRS-1 was responsible for this, we analyzed the expression of several genes involved in the insulin signaling pathway. Results showed that PPARgamma expression was significantly reduced in cells transfected with the mutated IRS-1, together with a significant decrease in binding of phosphatidylinositol-3 kinase (PI 3-kinase) to IRS-1 G972R and in PI 3-kinase activity. In addition, we observed that the interaction between the insulin receptor (IR) and the IRS-1 G972R protein was increased and that the autophosphorylation of the IR was significantly inhibited in 3T3L1-G972R cells compared with 3T3L1-WT. Treatment of the 3T3L1-G972R cells with pioglitazone (PIO), a PPARgamma agonist, restored differentiation with higher level of PPARgamma expression and restoration of PI 3-kinase binding to IRS-1 G972R and PI 3-kinase activity. IR autophosphorylation was also increased. Withdrawal of PIO in fully differentiated 3T3L1-G972R cells determined the reappearance of the insulin signaling defect. Finally, we observed higher levels of IRS-2 expression, suggesting that IRS-2 may play a more important role in adipocyte insulin signaling. In conclusion, IRS-1 G972R variant impairs insulin signaling, and treatment with PPARgamma agonist restores the normal phenotype of 3T3L1 cells.     

Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation

Interaction of GH with the cell-surface GH receptor (GHR) causes activation of the GHR-associated tyrosine kinase, JAK2, and consequent triggering of signaling cascades including the STAT, Ras/Raf/MEK1/MAP kinase, and insulin receptor substrate-1(IRS-1)/PI3kinase pathways. We previously showed that IRS- and GHR-deficient 32D cells that stably express the rabbit GHR and rat IRS-1 (32D-rbGHR-IRS-1) exhibited markedly enhanced GH-induced proliferation and MAP kinase (ERK1 and ERK2) activation compared with cells expressing only the GHR (32D-rbGHR). We now examine biochemical mechanism(s) by which IRS-1 augments GH-induced MAP kinase activation. Time-course experiments revealed a similarly transient (maximal at 15 min) GH-induced ERK1 and ERK2 activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells, but, consistent with our prior findings, substantially greater activation was seen in the IRS-1-containing cells. In both cells, GH-induced MAP kinase activation was markedly blunted by the MEK1 inhibitor, PD98059, but not by the PKC inhibitor, GF109203X. Interestingly, pretreatment with the PI3K inhibitor, wortmannin (EC50 approximately 10 nM), significantly reduced GH-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. This same pattern in both cells of IRS-1-dependent augmentation and IRS-1-independent wortmannin sensitivity was also observed for GH-induced activation of Akt and MEK1 (using state-specific antibody blotting for both), despite the lack of difference in GHR, JAK2, SHP-2, p85, Akt, Ras, Raf-1, MEK1, ERK1, or ERK2 abundance between the two cells. A different PI3K inhibitor, LY294002 (50 microM), substantially inhibited (roughly 72%) GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells, but only marginally (and statistically insignificantly) inhibited GH-induced MAP kinase activation in 32D-rbGHR cells. Because GH-induced Akt activation was completely inhibited in both cells by the same concentration of LY294002, these findings indicate that the wortmannin sensitivity of both the IRS-1-independent and -dependent GH-induced MAP kinase activation may reflect the activity of another wortmannin-sensitive target(s) in addition to PI3K in mediation of GH-induced MAP kinase activation in these cells. Notably, GH-induced STAT5 tyrosine phosphorylation, unlike Akt or MAPK activation, did not differ between the cells. Finally, while GH promoted accumulation of activated Ras in both cells, both basal and GH-induced activated Ras levels were greater in cells expressing IRS-1 than in 32D-rbGHR cells. These data indicate that while GH induces tyrosine phosphorylation of STAT5 and activation of the Ras/Raf/MEK1/MAPK and PI3K pathways, IRS-1 expression augments the latter two more than the former.     

Continually high insulin levels impair Akt phosphorylation and glucose transport in human myoblasts

Chronic hyperinsulinemia is both a marker and a cause for insulin resistance. This study analyzes the effect of long-term exposure to high insulin levels on the insulin-signaling pathway and glucose transport in cultured human myoblasts. Human myoblasts were grown in the presence of low (107 pmol/L, SkMC-L) or high (1430 pmol/L, SkMC-H) insulin concentrations for 3 weeks. Glucose transport, insulin receptor (IR), and IR substrate 1 (IRS1) phosphorylation, phosphatidylinositol 3'-kinase (PI3K) activity, as well as Akt-Ser473 phosphorylation have been investigated at the end of the incubation period and after a further short-term insulin stimulation. At the end of the incubation period, IR, IRS1, p85/PI3K, Akt, and GLUT4 protein expression levels were similar in both culture conditions. Basal glucose transport was similar in SkMC-L and SkMC-H, but after short-term insulin stimulation significantly increased (P < .01) only in SkMC-L. IR binding was down-regulated in SkMC-H (P < .01), but IR and IRS1 tyrosine phosphorylation and PI3K activity were significantly higher (P < .01) in SkMC-H than SkMC-L. Despite increased PI3K activation, Akt-Ser473 phosphorylation was similar in SkMC-L and SkMC-H. After a short-term insulin stimulation (10 nmol/L insulin for 10 minutes), IR and IRS1 tyrosine phosphorylation, PI3K activation, and Akt-Ser473 phosphorylation significantly increased (P < .01 and P < .05 for Akt) in SkMC-L but not in SkMC-H. Serine phosphorylation of IRS1 was similar in SkMC-L and SkMC-H. Moreover, in the SkMC-H, insulin stimulation was associated with the inhibition of IRS1 tyrosine dephosphorylation (P < .05). In summary, continuous exposure of cultured myoblasts to high insulin levels induces a persistent up-regulation of IR, IRS1, and PI3K activity associated with the demodulation of insulin signaling. Moreover, the impairment of the insulin-signaling steps between PI3K and Akt is concomitant with the desensitization of glucose transport. These alterations may contribute to the derangement insulin-signaling pathway states of hyperinsulinemia such as obesity and type 2 diabetes.     

Regulation of adiponectin secretion by insulin and amino acids in 3T3-L1 adipocytes

Adiponectin is a fat cell-derived hormone with insulin-sensitizing properties. Low plasma adiponectin levels are associated with insulin resistance as found in obesity. One of the mechanisms for this finding is hampered insulin signaling via phosphatidylinositol 3-kinase (PI3K) with concomitant decreased adiponectin secretion. Because insulin can also stimulate signaling at the level of mammalian target of rapamycin (mTOR) by a mechanism that is dependent on the presence of amino acids, the role of mTOR signaling in adiponectin secretion was studied. In view of the vesicular nature of adiponectin secretion, the role of lysosomes was explored as well. In 3T3-L1 adipocytes, both insulin and amino acids stimulated adiponectin secretion. The stimulation by insulin was PI3K dependent but mTOR independent. The stimulation by amino acids was independent of both PI3K and mTOR. Whereas the effect of insulin via PI3K was mainly on adiponectin secretion from adipocytes, the effect of amino acids was predominantly due to their role as substrates for adiponectin synthesis. The acidotropic agents ammonia and methylamine, but not the lysosomal protease inhibitor leupeptin and the autophagy inhibitor 3-methyladenine, strongly inhibited adiponectin secretion and increased the intracellular adiponectin pool. In conclusion, adiponectin production is substrate driven. Phosphatidylinositol 3-kinase and an acidic lysosomal pH, but not amino acid-mediated mTOR signaling or lysosomal breakdown, are involved in adiponectin secretion.     

Impaired insulin signaling in the liver of transgenic rats with low circulating growth hormone levels.

GH is known to regulate glucose and lipid metabolism as well as body growth. Controversy exists as to whether GH-deficient adults are indeed insulin sensitive or insulin resistant. In GH-deficient animal models, however, no clear observation indicating insulin resistance has been made, while increased insulin sensitivity has been reported in those animals. We have produced human GH (hGH) transgenic rats characterized by low circulating hGH levels and virtually no endogenous rat GH secretion. Although the body length of the transgenic rat is normal, they develop massive obesity and insulin resistance, indicating that the transgenic rat is a good model for the analysis of insulin resistance under GH deficiency. In this study, we have examined how GH deficiency affects the early steps of insulin signaling in the liver of the transgenic rat. Circulating glucose and insulin concentrations were significantly higher in the transgenic rats than in their littermates. In addition, impaired glucose tolerance was observed in the transgenic rat. The amount of insulin receptor was smaller in the liver of the transgenic rat, resulting in decreased tyrosine phosphorylation in response to insulin stimulation. The amounts of insulin receptor substrate-1 and -2 (IRS-1 and -2) and insulin-stimulated phosphorylation of IRSs were also smaller in the transgenic rat. Despite the decrease in tyrosine phosphorylation levels of IRSs being mild to moderate (45% for IRS-1 and 16% for IRS-2), associated phosphatidylinositol 3-kinase (PI3-kinase) activity was not increased by insulin stimulation at all in the transgenic rat. To elucidate whether this discrepancy resulted from the alteration in binding of the p85 subunit of PI3-kinase to phosphotyrosine residues of the IRSs, we determined the amount of p85 subunit in the immunocomplexes with anti-phosphotyrosine antibody. Insulin did not affect the amount of p85 subunit associated with phosphotyrosine in the transgenic rats, while it significantly increased in the controls, indicating that alteration may have occurred at the sites of phosphorylated tyrosine residues in IRSs. These results suggest that GH deficiency in the transgenic rat leads to impairment in at least the early steps of insulin signaling in the liver with a resultant defect in glucose metabolism.     

C-reactive protein impairs hepatic insulin sensitivity and insulin signaling in rats: role of mitogen-activated protein kinases

Plasma C-reactive protein (CRP) concentration is increased in the metabolic syndrome, which consists of a cluster of cardiovascular disease risk factors, including insulin resistance. It is not known, however, whether CRP is merely a marker of accompanying inflammation or whether it contributes causally to insulin resistance. The objective of this study is to investigate the role that CRP may play in the development of insulin resistance. We examined the effect of single-dose intravenous administration of purified human (h)CRP on insulin sensitivity in Sprague-Dawley rats using the euglycemic, hyperinsulinemic clamp technique. hCRP was associated with impaired insulin suppression of endogenous glucose production with no reduction in peripheral tissue glucose uptake, suggesting that hCRP mediated insulin resistance in the liver but not extrahepatic tissues. We further assessed components of the insulin signaling pathway and mitogen-activated protein kinases (MAPKs) in the liver. Liver tissues derived from hCRP-treated rats showed reduced insulin-stimulated insulin receptor substrate (IRS) tyrosine phosphorylation, IRS/phosphatidylinositol 3-kinase (PI3K) association, and Akt phosphorylation, consistent with hCRP-induced impairment of hepatic insulin signaling. Furthermore, hCRP enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 MAPK as well as IRS-1 Ser(612) . Finally, we observed in primary cultured rat hepatocytes that U0126 (a selective inhibitor of MAPK/ERK kinase1/2) corrected hCRP-induced impairment of insulin signaling. CONCLUSIONS: hCRP plays an active role in inducing hepatic insulin resistance in the rat, at least in part by activating ERK1/2, with downstream impairment in the insulin signaling pathway.     

Effect of chronic growth hormone treatment on insulin signal transduction in rat tissues

Growth hormone (GH) is known to produce insulin resistance, but the exact molecular mechanism remains unclear. We have chronically treated rats with GH and observed that the levels of insulin receptor in the liver or muscle were similar in both the GH-treated and non-treated rats. Insulin-stimulated receptor autophosphorylation was unaltered in the liver, but was reduced in the muscle of rats treated with GH. Insulin receptor substrate-1 (IRS-1) and phosphatidylinositol (PI) 3-kinase protein levels decreased in the liver but not muscle of GH-treated rats. There was no change in hepatic and muscle IRS-2 concentrations. A common finding in liver and muscle was the decrease in IRS-1 and IRS-2 tyrosine phosphorylation associated with a reduction in the interaction between these substrates and PI 3-kinase. These data suggest that changes in the early steps of insulin signal transduction may have a role in the insulin resistance observed in rats exposed to an excess of GH.     

Alterations in insulin binding induced by changes in vivo in the levels of glucocorticoids and growth hormone.

Previous studies have shown that the sensitivity of tissues to insulin is diminished in states of glucocorticoid and GH excess and is increased when these hormones are deficient. To evaluate the role of the insulin receptor in these states, we have studied [125I]insulin binding to purified liver plasma membranes obtained from rats with a variety of perturbations of both glucocorticoids and GH. Glucocorticoid excess was produced in rats by administration of ACTH (40 U/day for 4 days) or dexamethasone (1 mg/day for 4 days). This resulted in an insulin-resistant state. Associated with this insulin resistance, there was a 50-60% decrease in insulin binding to its specific receptors in liver. Conversely, adrenalectomy, which produces an increase in insulin sensitivity, was associated with an increase in insulin binding to liver. Computer-assisted Scatchard analysis using a negative cooperative model for the inulin-receptor interaction indicated that, in contrast to our findings with obesity, the changes in insulin binding in these states were most likely due entirely to changes in receptor affinity, with no change in receptor concentration. GH administration also produced mild insulin resistance and a decrease in receptor concentration. This was associated with a reciprocal increase in receptor affinity and thus, no major alteration in insulin binding occurred at low physiological insulin concentrations. Hypophysectomized rats, on the other hand, showed an increase in receptor concentration and a decrease in affinity, and GH treatment only partially corrected these changes. Rats implanted with the MtT tumor (which secretes ACTH, GH, and PRL) have the combined effects of excess glucocorticoids and GH and are very insulin resistant. Liver membranes prepared from these rats showed a decrease in insulin binding and receptor affinity similar to that observed in other states of glucocorticoid excess. Further, adrenalectomy of the tumor-bearing rats resulted in an increase in insulin binding despite the persistence of the elevated levels of GH, ACTH, and PRL. These findings suggest that alterations in insulin receptor affinity and number may play a major role in the states of altered insulin sensitivity which accompany glucocorticoid excess and deficiency, and follow hypophysectomy. In contrast, the insulin resistance associated with GH excess is mediated at either a site on the receptor distal to the insulin-binding site (i.e. transduction) or at one or more of the intracellular reactions important in insulin action.     

Comparing adiposity profiles in three mouse models with altered GH signaling.

Three mouse lines with altered growth hormone (GH) signaling were used to study GH's role in adiposity. Dwarf GH receptor knockout mice (GHR -/-) and bovine GH antagonist expressing mice (GHA) had an increased percent body fat with most of the excess fat mass accumulating in the subcutaneous region. Giant bovine GH expressing mice (bGH) had a reduced percent body fat. Only GHA mice consumed significantly more food per body weight. Serum leptin levels were significantly increased in GHA mice and decreased in bGH mice but unchanged in the GHR -/- mice. Interestingly, serum adiponectin levels were significantly increased in the GHR -/- and GHA lines but decreased in bGH mice. These data suggest that suppression or absence of GH action and enhanced GH action indeed have opposite metabolic effects in terms of adiposity. Interestingly, adiponectin levels were positively correlated with previously reported insulin sensitivity of these mice, but also positively correlated with adiposity, which is contrary to findings in other mouse models. Thus, adiponectin levels were negatively correlated with GH function suggesting a role for adiponectin in GH-induced insulin resistance.     

Cell lineage-specific signaling of insulin and insulin-like growth factor I in rabbit blastocysts

The insulin/IGF system plays a critical role in embryo growth and development. We have investigated the expression of insulin receptor (IR) and IGF-I receptor (IGF-IR) and the activation of their downstream pathways in rabbit 6-d-old blastocysts. IR was expressed in embryoblast (Em, inner cell mass) and trophoblast (Tr) cells, whereas IGF-IR was localized mainly in Em. Isoform A (IR-A) represents the main insulin isoform in blastocysts and was found in Em and Tr cells. IR-B was detectable only in Tr. IR/IGF-IR signaling pathways were analyzed after stimulation with insulin (17 nm) or IGF-I (1.3 nm) in cultured blastocysts. Insulin stimulated Erk1/2 in Em and Tr and Akt in Tr but not in Em. IGF-I activated both kinases exclusively in Em. The target genes c-fos (for MAPK kinase-1/Erk signaling) and phosphoenolpyruvate carboxykinase (PEPCK, for PI3K/Akt signaling) were also specifically regulated. Insulin down-regulated PEPCK RNA amounts in Tr by activation of the phosphatidylinositol 3-kinase/Akt pathway. Expression of c-fos by insulin and IGF-I was different with respect to time and fortitude of expression, mirroring again the specific IR and IGF-IR expression patterns in Em and Tr. Taken together, we show that IGF-I acts primarily mitogenic, an effect that is cell lineage-specifically restricted to the Em. By contrast, insulin is the growth factor of the Tr stimulating mitogenesis and down-regulating metabolic responses. As soon as blastocyst differentiation in Em and Tr has been accomplished, insulin and IGF-I signaling is different in both cell lineages, implying a different developmental impact of both growth factors.