alpha-adrenergic stimulation mediates glucose uptake through phosphatidylinositol 3-kinase in rat heart

We examined whether insulin and catecholamines share common pathways for their stimulating effects on glucose uptake. We perfused isolated working rat hearts with Krebs-Henseleit buffer containing [2-3H]glucose (5 mmol/L, 0.05 microCi/mL) and sodium oleate (0.4 mmol/L). In the absence or presence of the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin (3 micromol/L), we added insulin (1 mU/mL), epinephrine (1 micromol/L), phenylephrine (100 micromol/L) plus propranolol (10 micromol/L, selective alpha-adrenergic stimulation), or isoproterenol (1 micromol/L) plus phentolamine (10 micromol/L, selective beta-adrenergic stimulation) to the perfusate. Cardiac power was found to be stable in all groups (between 8.07+/-0.68 and 10.7+/-0. 88 mW) and increased (25% to 47%) with addition of epinephrine, but not with selective alpha- and beta-adrenergic stimulation. Insulin and epinephrine, as well as selective alpha- and beta-receptor stimulation, increased glucose uptake (the following values are in micromol/[min. g dry weight]: basal, 1.19+/-0.13; insulin, 3.89+/-0.36; epinephrine, 3.46+/-0.27; alpha-stimulation, 4.08+/-0.40; and beta-stimulation, 3.72+/-0.34). Wortmannin completely inhibited insulin-stimulated and selective alpha-stimulated glucose uptake, but it did not affect the epinephrine-stimulated or selective beta-stimulated glucose uptake. Sequential addition of insulin and epinephrine or insulin and alpha-selective stimulation showed additive effects on glucose uptake in both cases. Wortmannin further blocked the effects of insulin on glycogen synthesis. We conclude that alpha-adrenergic stimulation mediates glucose uptake in rat heart through a PI3-K-dependent pathway. However, the additive effects of alpha-adrenergic stimulation and insulin suggest 2 different isoforms of PI3-K, compartmentation of PI3-K, potentiation, or inhibition by wortmannin of another intermediate of the alpha-adrenergic signaling cascade. The stimulating effects of both the alpha- and the beta-adrenergic pathways on glucose uptake are independent of changes in cardiac performance.     

Reduction of insulin-stimulated glucose uptake in L6 myotubes by the protein kinase inhibitor SB203580 is independent of p38MAPK activity

Insulin increases glucose uptake through translocation of the glucose transporter GLUT4 to the plasma membrane. We previously showed that insulin activates p38MAPK, and inhibitors of p38MAPKalpha and p38MAPKbeta (e.g. SB203580) reduce insulin-stimulated glucose uptake without affecting GLUT4 translocation. This observation suggested that insulin may increase GLUT4 activity via p38alpha and/or p38beta. Here we further explore the possible participation of p38MAPK through a combination of molecular strategies. SB203580 reduced insulin stimulation of glucose uptake in L6 myotubes overexpressing an SB203580-resistant p38alpha (drug-resistant p38alpha) but barely affected phosphorylation of the p38 substrate MAPK-activated protein kinase-2. Expression of dominant-negative p38alpha or p38beta reduced p38MAPK phosphorylation by 70% but had no effect on insulin-stimulated glucose uptake. Gene silencing via isoform-specific small interfering RNAs reduced expression of p38alpha or p38beta by 60-70% without diminishing insulin-stimulated glucose uptake. SB203580 reduced photoaffinity labeling of GLUT4 by bio-LC-ATB-BMPA only in the insulin-stimulated state. Unless low levels of p38MAPK suffice to regulate glucose uptake, these results suggest that the inhibition of insulin-stimulated glucose transport by SB203580 is likely not mediated by p38MAPK. Instead, changes experienced by insulin-stimulated GLUT4 make it susceptible to inhibition by SB203580.     

Chronic testosterone treatment induces selective insulin resistance in subcutaneous adipocytes of women

Adipose tissue plays a central role in determining whole body insulin sensitivity. Several aspects of adipose cell function are regulated by androgens. Given that high androgen levels and insulin resistance are linked in women, we proposed that androgens may influence insulin-mediated glucose metabolism in adipose cells. Preadipocytes harvested from s.c. adipose tissue of healthy women aged 37 +/- 5 years were differentiated in vitro, then treated with testosterone (T) and/or androgen receptor (AR) antagonists (cyproterone acetate, flutamide) for 48 h. Maximal insulin-stimulated glucose uptake (insulin 10 nM) and increment following insulin stimulation were significantly impaired in cells treated with T 10 and 100 nmol/l. This defect was abolished by cyproterone acetate and partially reversed by flutamide. The effect of T could not be accounted for by altered differentiation status of the adipocytes. In the glucose metabolic pathway of insulin signaling, treatment of cells with T 10 nmol/l did not alter insulin-stimulated phosphorylation of insulin receptor substrate-1 or Akt, but insulin-stimulated phosphorylation of protein kinase C (PKC) zeta was impaired. Insulin signaling via the mitogenic/gene regulatory pathway, as assessed by extracellular signal-regulated kinase phosphorylation, was unchanged. We conclude that (1) T, or an androgenic metabolite of T, induces insulin resistance in adipocytes of women, selective for metabolic signaling pathways; (2) this defect is via AR; and (3) the defect in signaling is independent of phosphatidylinositol 3-kinase activation and involves impaired phosphorylation of PKCzeta. These findings are relevant to understanding the pathogenesis of insulin resistance in hyperandrogenic women.     

Aldosterone suppresses insulin signaling via the downregulation of insulin receptor substrate-1 in vascular smooth muscle cells

Clinical reports indicate that patients with primary aldosteronism commonly have impaired glucose tolerance; however, the relationship between aldosterone and insulin signaling pathway has not been clarified. In this study, we examined the effects of aldosterone treatment on insulin receptor substrate-1 expression and insulin signaling pathway including Akt phosphorylation and glucose uptake in rat vascular smooth muscle cells. Insulin receptor substrate-1 protein expression and Akt phosphorylation were determined by Western blot analysis with anti-insulin receptor substrate-1 and phosphorylated-Akt antibodies, respectively. Glucose metabolism was evaluated using (3)H-labeled 2-deoxy-d-glucose uptake. Aldosterone (1-100 nmol/L) dose-dependently decreased insulin receptor substrate-1 protein expression with a peak at 18 hours (n=4). Aldosterone-induced degradation of insulin receptor substrate-1 was markedly attenuated by treatment with the selective mineralocorticoid receptor antagonist eplerenone (10 micromol/L; n=4). Furthermore, degradation was blocked by the Src inhibitor PP1 (20 micromol/L; n=4). Treatment with antioxidants, N-acetylcysteine (10 mmol/L), or ebselen (40 micromol/L) also attenuated aldosterone-induced insulin receptor substrate-1 degradation (n=4). In addition, proteasome inhibitor MG132 (1 micromol/L) prevented insulin receptor substrate-1 degradation (n=4). Aldosterone treatment abolished insulin-induced Akt phosphorylation (100 nmol/L; 5 minutes; n=4). Furthermore, aldosterone pretreatment decreased insulin-stimulated (100 nmol/L; 60 minutes; n=4) glucose uptake by 50%, which was reversed by eplerenone (10 micromol/L; n=4). These data indicate that aldosterone decreases insulin receptor substrate-1 expression via Src and reactive oxygen species stimulation by proteasome-dependent degradation in vascular smooth muscle cells; thus, aldosterone may be involved in the pathogenesis of vascular insulin resistance via oxidative stress.     

Dexamethasone impairs insulin signalling and glucose transport by depletion of insulin receptor substrate-1, phosphatidylinositol 3-kinase and protein kinase B in primary cultured rat adipocytes

OBJECTIVE: Glucocorticoid excess leads to insulin resistance. This study explores the effects of glucocorticoids on the glucose transport system and insulin signalling in rat adipocytes. The interaction between glucocorticoids and high levels of insulin and glucose is also addressed. DESIGN AND METHODS: Isolated rat adipocytes were cultured for 24 h at different glucose concentrations (5 and 15 mmol/l) with or without the glucocorticoid analogue dexamethasone (0.3 micromol/l) and insulin (10(4) microU/ml). After the culture period, the cells were washed and then basal and insulin-stimulated glucose uptake, insulin binding and lipolysis as well as cellular content of insulin signalling proteins (insulin receptor substrate-1 (IRS-1), IRS-2, phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (PKB)) and glucose transporter isoform GLUT4 were measured. RESULTS: Dexamethasone in the medium markedly decreased both basal and insulin-stimulated glucose uptake at both 5 and 15 mmol/l glucose (by approximately 40-50%, P<0.001 and P<0.05 respectively). Combined long-term treatment with insulin and dexamethasone exerted additive effects in decreasing basal, and to a lesser extent insulin-stimulated, glucose uptake capacity (P<0.05) compared with dexamethasone alone, but this was seen only at high glucose (15 mmol/l). Insulin binding was decreased (by approximately 40%, P<0.05) in dexamethasone-treated cells independently of surrounding glucose concentration. Following dexamethasone treatment a approximately 75% decrease (P<0.001) in IRS-1 expression and an increase in IRS-2 (by approximately 150%, P<0.001) was shown. Dexamethasone also induced a subtle decrease in PI3-K (by approximately 20%, P<0.01) and a substantial decrease in PKB content (by approximately 45%, P<0.001). Insulin-stimulated PKB phosphorylation was decreased (by approximately 40%, P<0.01) in dexamethasone-treated cells. Dexamethasone did not alter the amount of total cellular membrane-associated GLUT4 protein. The effects of dexamethasone per se on glucose transport and insulin signalling proteins were mainly unaffected by the surrounding glucose and insulin levels. Dexamethasone increased the basal lipolytic rate (approximately 4-fold, P<0.05), but did not alter the antilipolytic effect of insulin. CONCLUSIONS: These results suggest that glucocorticoids, independently of the surrounding glucose and insulin concentration, impair glucose transport capacity in fat cells. This is not due to alterations in GLUT4 abundance. Instead dexamethasone-induced insulin resistance may be mediated via reduced cellular content of IRS-1 and PKB accompanied by a parallel reduction in insulin-stimulated activation of PKB.     

Counterregulatory responses to hypoglycemia in patients with maturity-onset diabetes of the young caused by HNF-1alpha gene mutations (MODY3)

Mutations of HNF-1alpha lead to severe beta cell dysfunction, resulting in decreased glucose-induced insulin secretion. HNF-1alpha is also expressed in liver, kidney and pancreatic alpha cells, but the functional consequences of HNF-1alpha mutations in these organs remain unknown. We therefore assessed the counterregulatory responses to hypoglycemia in six patients with HNF-1alpha mutations (MODY3), five patients with non-insulin-dependent diabetes mellitus (NIDDM) and in nine healthy controls. Plasma glucagon concentrations and endogenous glucose production were measured every 15 min during a hyperinsulinemic clamp with progressive hypoglycemia. Plasma glucagon concentrations were similar at basal glycemia (73+/-6, 69+/-5 and 69+/-7 ng/l) and reached peak values of 88+/-9, 88+/-11 and 89+/-7 ng/l at a glycemia of 3.6 mmol/l in MODY3 patients, patients with NIDDM and controls respectively (NS). Suppression of endogenous glucose production by insulin was blunted in MODY3 patients (3.3+/-1.2 micromol/kg per min) and in patients with NIDDM (4.4+/-0.6 micromol/kg per min) compared with controls (1.7+/-0.5 micromol/kg per min, P<0.05 compared with both MODY3 patients and patients with NIDDM). During hypoglycemia, endogenous glucose production increased to 8.6+/-2.1, 8.8+/-0.7 and 7.0+/-1.0 micromol/kg per min in MODY3 patients, patients with NIDDM and controls respectively (all NS). These data indicate that mutations of HNF-1alpha in MODY3 do not result in a decreased glucagon secretion or alterations of glucose production during hypoglycemia.     

Activation of the mammalian target of rapamycin pathway acutely inhibits insulin signaling to Akt and glucose transport in 3T3-L1 and human adipocytes

The mammalian target of rapamycin (mTOR) pathway has recently emerged as a chronic modulator of insulin-mediated glucose metabolism. In this study, we evaluated the involvement of this pathway in the acute regulation of insulin action in both 3T3-L1 and human adipocytes. Insulin rapidly (t(1/2) = 5 min) stimulated the mTOR pathway, as reflected by a 10-fold stimulation of 70-kDa ribosomal S6 kinase 1 (S6K1) activity in 3T3-L1 adipocytes. Inhibition of mTOR/S6K1 by rapamycin increased insulin-stimulated glucose transport by as much as 45% in 3T3-L1 adipocytes. Activation of mTOR/S6K1 by insulin was associated with a rapamycin-sensitive increase in Ser636/639 phosphorylation of insulin receptor substrate (IRS)-1 but, surprisingly, did not result in impaired IRS-1-associated phosphatidylinositol (PI) 3-kinase activity. However, insulin-induced activation of Akt was increased by rapamycin. Insulin also activated S6K1 and increased phosphorylation of IRS-1 on Ser636/639 in human adipocytes. As in murine cells, rapamycin treatment of human adipocytes inhibited S6K1, blunted Ser636/639 phosphorylation of IRS-1, leading to increased Akt activation and glucose uptake by insulin. Further studies in 3T3-L1 adipocytes revealed that rapamycin prevented the relocalization of IRS-1 from the low-density membranes to the cytosol in response to insulin. Furthermore, inhibition of mTOR markedly potentiated the ability of insulin to increase PI 3,4,5-triphosphate levels concomitantly with an increased phosphorylation of Akt at the plasma membrane, low-density membranes, and cytosol. However, neither GLUT4 nor GLUT1 translocation induced by insulin were increased by rapamycin treatment. Taken together, these results indicate that the mTOR pathway is an important modulator of the signals involved in the acute regulation of insulin-stimulated glucose transport in 3T3-L1 and human adipocytes.     

Epinephrine suppresses rap1B.GAP-activated GTPase activity in human platelets.

Lysate from quiescent platelets promotes rapid hydrolysis of [gamma-32P]GTP bound to rap1B. Various platelet agonists, including platelet-activating factor, phorbol 12,13-dibutyrate, alpha-thrombin, epinephrine, ADP, and iloprost, that affect platelet metabolism by different signal transduction pathways were used to stimulate intact platelets and study their effects on rap1B.GAP-activated GTPase activity (GAP, GTPase-activating protein). Only epinephrine was found to dramatically decrease not only the rate but also the amount of hydrolysis of rap1B-bound GTP activated by rap1B.GAP. This effect was dose dependent and occurred rapidly. The suppression of GTPase activity was specific for rap1B.GAP in that ras.GAP- and rap2B.GAP-activated GTPase activity were not affected by epinephrine stimulation. This effect appears to be mediated by the alpha 2-adrenergic receptor, as evidenced by a similar suppression of GTPase activity by stimulating platelets with the synthetic alpha 2-adrenergic receptor agonist UK14304 (bromoxidine). Furthermore, the selective alpha 2-adrenergic receptor antagonist yohimbine blocked the suppression of GTPase activity expressed in epinephrine-stimulated cell lysates. No apparent changes in the patterns of protein expression or tyrosine phosphorylation were observed. Although the migration characteristics upon anion-exchange chromatography of rap1B.GAP and ras.GAP activities were unaffected by epinephrine stimulation, the specific activity of rap1B.GAP was noticeably decreased with 250 and 500 microM epinephrine. These results suggest a possible role for rap1B and rap1B.GAP in epinephrine-stimulated signal transduction.     

Nuclear receptor agonists improve insulin responsiveness in cultured cardiomyocytes through enhanced signaling and preserved cytoskeletal architecture

Insulin resistance is the failure of insulin to stimulate the transport of glucose into its target cells. A highly regulatable supply of glucose is important for cardiomyocytes to cope with situations of metabolic stress. We recently observed that isolated adult rat cardiomyocytes become insulin resistant in vitro. Insulin resistance is combated at the whole body level with agonists of the nuclear receptor complex peroxisome proliferator-activated receptor gamma (PPARgamma)/retinoid X receptor (RXR). We investigated the effects of PPARgamma/RXR agonists on the insulin-stimulated glucose transport and on insulin signaling in insulin-resistant adult rat cardiomyocytes. Treatment of cardiomyocytes with ciglitazone, a PPARgamma agonist, or 9-cis retinoic acid (RA), a RXR agonist, increased insulin- and metabolic stress-stimulated glucose transport, whereas agonists of PPARalpha or PPARbeta/delta had no effect. Stimulation of glucose transport in response to insulin requires the phosphorylation of the signaling intermediate Akt on the residues Thr308 and Ser473 and, downstream of Akt, AS160 on several Thr and Ser residues. Phosphorylation of Akt and AS160 in response to insulin was lower in insulin-resistant cardiomyocytes. However, treatment with 9-cis RA markedly increased phosphorylation of both proteins. Treatment with 9-cis RA also led to better preservation of microtubules in cultured cardiomyocytes. Disruption of microtubules in insulin-responsive cardiomyocytes abolished insulin-stimulated glucose transport and reduced phosphorylation of AS160 but not Akt. Metabolic stress-stimulated glucose transport also involved AS160 phosphorylation in a microtubule-dependent manner. Thus, the stimulation of glucose uptake in response to insulin or metabolic stress is dependent in cardiomyocytes on the presence of intact microtubules.     

Polycystic ovary syndrome is associated with tissue-specific differences in insulin resistance

OBJECTIVE: The potential differential contributions of skeletal muscle and adipose tissue to whole body insulin resistance were evaluated in subjects with polycystic ovary syndrome (PCOS). Research Design and METHODS: Forty-two PCOS subjects and 15 body mass index-matched control subjects were studied. Insulin action was evaluated by the hyperinsulinemic/euglycemic clamp procedure. Isolated adipocytes and cultured muscle cells were analyzed for glucose transport activity; adipocytes, muscle tissue, and myotubes were analyzed for the expression and phosphorylation of insulin-signaling proteins. RESULTS: Fifty-seven per cent of the PCOS subjects had impaired glucose tolerance and the lowest rate of maximal insulin-stimulated whole body glucose disposal compared to controls (P < 0.01). PCOS subjects with normal glucose tolerance had intermediate reduction in glucose disposal rate (P < 0.05 vs. both control and impaired glucose tolerance subjects). However, rates of maximal insulin-stimulated glucose transport (insulin responsiveness) into isolated adipocytes were comparable between all three groups, whereas PCOS subjects displayed impaired insulin sensitivity. In contrast, myotubes from PCOS subjects displayed reduced insulin responsiveness for glucose uptake and normal sensitivity. There were no differences between groups in the expression of glucose transporter 4 or insulin-signaling proteins or maximal insulin stimulation of phosphorylation of Akt in skeletal muscle, myotubes, or adipocytes. CONCLUSIONS: Individuals with PCOS display impaired insulin responsiveness in skeletal muscle and myotubes, whereas isolated adipocytes display impaired insulin sensitivity but normal responsiveness. Skeletal muscle and adipose tissue contribute differently to insulin resistance in PCOS. Insulin resistance in PCOS cannot be accounted for by differences in the expression of selected signaling molecules or maximal phosphorylation of Akt.     

Cellular mechanism of insulin resistance: potential links with inflammation

Insulin resistance is a pivotal feature in the pathogenesis of type 2 diabetes, and it may be detected 10-20 y before the clinical onset of hyperglycemia. Insulin resistance is due to the reduced ability of peripheral target tissues to respond properly to insulin stimulation. In particular, impaired insulin-stimulated muscle glycogen synthesis plays a significant role in insulin resistance. Glucose transport (GLUT4), phosphorylation (hexokinase) and storage (glycogen synthase) are the three potential rate-controlling steps regulating insulin-stimulated muscle glucose metabolism, and all three have been implicated as being the major defects responsible for causing insulin resistance in patients with type 2 diabetes. Using (13)C/(31)P magnetic resonance spectroscopy (MRS), we demonstrate that a defect in insulin-stimulated muscle glucose transport activity is the rate-controlling defect. Using a similar (13)C/(31)P MRS approach, we have also demonstrated that fatty acids cause insulin resistance in humans due to a decrease in insulin-stimulated muscle glucose transport activity, which could be attributed to reduced insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity, a required step in insulin-stimulated glucose transport into muscle. Furthermore, we have recently proposed that this defect in insulin-stimulated muscle glucose transport activity may be due to the activation of a serine kinase cascade involving protein kinase C theta and IKK-beta, which are key downstream mediators of tissue inflammation. Finally, we propose that any perturbation that leads to an increase in intramyocellular lipid (fatty acid metabolites) content such as acquired or inherited defects in mitochondrial fatty acid oxidation, defects in adipocyte fat metabolism or simply increased fat delivery to muscle/liver due to increased energy intake will lead to insulin resistance through this final common pathway. Understanding these key cellular mechanisms of insulin resistance should help elucidate new targets for treating type 2 diabetes.     

Inhibition of lipolysis during acute GH exposure increases insulin sensitivity in previously untreated GH-deficient adults

OBJECTIVE: Previous studies evaluating the lipolytic effect of GH have in general been performed in subjects on chronic GH therapy. In this study we assessed the lipolytic effect of GH in previously untreated patients and examined whether the negative effect of enhanced lipolysis on glucose metabolism could be counteracted by acute antilipolysis achieved with acipimox. METHODS: Ten GH-deficient (GHD) adults participated in four experiments each, during which they received in a double-blind manner: placebo (A); GH (0.88+/-0.13 mg) (B); GH+acipimox 250 mg b.i.d. (C); and acipimox b.i.d. (no GH) (D), where GH was given the night before a 2 h euglycemic, hyperinsulinemic clamp combined with infusion of [3-(3)H]glucose and indirect calorimetry. RESULTS: GH increased basal free fatty acid (FFA) levels by 74% (P=0.0051) and insulin levels by 93% (P=0.0051). This resulted in a non-significant decrease in insulin-stimulated glucose uptakes (16.61+/-8.03 vs 12.74+/-5.50 micromol/kg per min (s.d.), P=0.07 for A vs B). The rates of insulin-stimulated glucose uptake correlated negatively with the FFA concentrations (r=-0.638, P<0.0001). However, acipimox caused a significant improvement in insulin-stimulated glucose uptake in the GH-treated patients (17.35+/-5.65 vs 12.74+/-5.50 micromol/kg per min, P=0.012 for C vs B). The acipimox-induced enhancement of insulin-stimulated glucose uptake was mainly due to an enhanced rate of glucose oxidation (8.32+/-3.00 vs 5.88+/-2.39 micromol/kg per min, P=0.07 for C vs B). The enhanced rates of glucose oxidation induced by acipimox correlated negatively with the rate of lipid oxidation in GH-treated subjects both in basal (r=-0.867, P=0.0093) and during insulin-stimulated (r=-0.927, P=0.0054) conditions. GH did not significantly impair non-oxidative glucose metabolism (6.86+/-5.22 vs 8.67+/-6.65 micromol/kg per min, P=NS for B vs A). The fasting rate of endogenous glucose production was unaffected by GH and acipimox administration (10.99+/-1.98 vs 11.73+/-2.38 micromol/kg per min, P=NS for B vs A and 11.55+/-2.7 vs 10.99+/-1.98 micromol/kg per min, P=NS for C vs B). On the other hand, acipimox alone improved glucose uptake in the untreated GHD patients (24.14+/-8.74 vs 16.61+/-8.03 micromol/kg per min, P=0.0077 for D vs A) and this was again due to enhanced fasting (7.90+/-2.68 vs 5.16+/-2.28 micromol/kg per min, P=0.01 for D vs A) and insulin-stimulated (9.78+/-3.68 vs 7.95+/-2.64 micromol/kg per min, P=0.07 for D vs A) glucose oxidation. CONCLUSION: The study of acute administration of GH to previously untreated GHD patients provides compelling evidence that (i) GH-induced insulin resistance is mainly due to induction of lipolysis by GH; and (ii) inhibition of lipolysis can prevent the deterioration of insulin sensitivity. The question remains whether GH replacement therapy should, at least at the beginning of therapy, be combined with means to prevent an excessive stimulation of lipolysis by GH.     

Effects of streptozotocin-induced diabetes on glucose transport in skeletal muscle

Female Sprague-Dawley rats were injected with streptozotocin (45 mg/kg) to induce mild diabetes (glucose, greater than 13 mM). Half of the animals received daily insulin injections to reduce hyperglycemia. After 10 weeks, sarcolemmal membranes were isolated from hindlimb muscles to study glucose transport, and the number of glucose transporters was assessed by cytochalasin-beta binding. Both glucose transport (19.2 +/- 1.6 vs. 31.93 +/- 3.29 pmol/mg protein.15 sec) and cytochalasin-beta binding (3.06 +/- 0.28 vs. 6.14 +/- 0.59 pmol/mg protein) were significantly (P less than 0.05) reduced in the diabetic untreated rats compared to control values. Daily insulin injections restored both (P less than 0.05) basal transport (33.22 +/- 3.62 pmol/mg protein.15 sec) and cytochalasin-beta binding (5.52 +/- 0.66 pmol/mg protein) to control levels. Maximum insulin stimulation (1 U/kg, iv) significantly increased (P less than 0.05) both glucose transport (30.18 +/- 3.76 vs. 96.48 +/- 4.21 pmol/mg protein.15 sec) and cytochalasin-beta binding (4.38 +/- 0.29 vs. 9.40 +/- 0.42 pmol/mg protein) in the untreated diabetic and control rats. However, the stimulation in the untreated diabetic rats only reached basal control levels, which was significantly (P less than 0.05) below the insulin-stimulated value for the controls. In the rats receiving daily insulin injections, maximum insulin stimulation increased (P less than 0.05) both glucose transport (58.67 +/- 15.24 pmol/mg protein.15 sec) and cytochalasin-beta binding (6.4 +/- 0.7 pmol/mg protein), but both transport and binding were significantly (P less than 0.05) below insulin-stimulated values for the control rats. These data show that insulin deficiency adversely affected the glucose transport system in skeletal muscle. Both basal and maximum insulin-stimulated transport and the number of transport molecules were reduced. Daily insulin treatment corrected some of the defects, but maximum insulin stimulation was still significantly below values for control animals.     

High glucose and insulin in combination cause insulin receptor substrate-1 and -2 depletion and protein kinase B desensitisation in primary cultured rat adipocytes: possible implications for insulin resistance in type 2 diabetes

OBJECTIVE: The purpose of this study was to investigate the cellular effects of long-term exposure to high insulin and glucose levels on glucose transport and insulin signalling proteins. DESIGN AND METHODS: Rat adipocytes were cultured for 24 h in different glucose concentrations with 10(4) microU/ml of insulin or without insulin. After washing, (125)I-insulin binding, basal and acutely insulin-stimulated d-[(14)C]glucose uptake, and insulin signalling proteins and glucose transporter 4 (GLUT4) were assessed. RESULTS: High glucose (15 and 25 mmol/l) for 24 h induced a decrease in basal and insulin-stimulated glucose uptake compared with control cells incubated in low glucose (5 or 10 mmol/l). Twenty-four hours of insulin treatment decreased insulin binding capacity by approximately 40%, and shifted the dose-response curve for insulin's acute effect on glucose uptake 2- to 3-fold to the right. Twenty-four hours of insulin treatment reduced basal and insulin-stimulated glucose uptake only in the presence of high glucose (by approximately 30-50%). At high glucose, insulin receptor substrate-1 (IRS-1) expression was downregulated by approximately 20-50%, whereas IRS-2 was strongly upregulated by glucose levels of 10 mmol/l or more (by 100-400%). Insulin treatment amplified the suppression of IRS-1 when combined with high glucose and also IRS-2 expression was almost abolished. Twenty-four hours of treatment with high glucose or insulin, alone or in combination, shifted the dose-response curve for insulin's effect to acutely phosphorylate protein kinase B (PKB) to the right. Fifteen mmol/l glucose increased GLUT4 in cellular membranes (by approximately 140%) compared with 5 mmol/l but this was prevented by a high insulin concentration. CONCLUSIONS: Long-term exposure to high glucose per se decreases IRS-1 but increases IRS-2 content in rat adipocytes and it impairs glucose transport capacity. Treatment with high insulin downregulates insulin binding capacity and, when combined with high glucose, it produces a marked depletion of IRS-1 and -2 content together with an impaired sensitivity to insulin stimulation of PKB activity. These mechanisms may potentially contribute to insulin resistance in type 2 diabetes.     

Insulin- and glucose-induced phosphorylation of the Na(+),K(+)-adenosine triphosphatase alpha-subunits in rat skeletal muscle.

Phosphorylation of the alpha-subunits of Na(+),K(+)-adenosine triphosphatase in response to insulin, high extracellular glucose concentration, and phorbol 12-myristate 13-acetate was investigated in isolated rat soleus muscle. All three stimuli increased alpha-subunit phosphorylation approximately 3-fold. Phorbol 12-myristate 13-acetate- and high glucose-induced phosphorylation of the alpha-subunit was completely abolished by the PKC inhibitor GF109203X, whereas insulin-stimulated phosphorylation was only partially reduced. Notably, insulin stimulation resulted in phosphorylation of the alpha-subunit on serine, threonine, and tyrosine residues, whereas high extracellular glucose or phorbol 12-myristate 13-acetate stimulation mediated phosphorylation only on serine and threonine residues. Insulin stimulation resulted in translocation of Na(+),K(+)-adenosine triphosphatase alpha(2)-subunit to the plasma membrane and increased Na(+),K(+)-adenosine triphosphatase activity in the same membrane fraction. High glucose had no effect on alpha-subunits distribution. Immunoprecipitation with antiphosphotyrosine antibody and subsequent Western blot analysis with anti-alpha(1)- and -alpha(2)-subunit antibodies revealed that both alpha(1)- and alpha(2)-subunit isoforms underwent phosphorylation on tyrosine residues in response to insulin, although with different time course and magnitude. Thus, we show that insulin-stimulated phosphorylation of Na(+),K(+)-adenosine triphosphatase alpha-subunit occurs via a PKC- and tyrosine kinase-dependent mechanism, whereas high glucose-induced phosphorylation is only PKC-dependent. Phosphorylation of Na(+),K(+)-adenosine triphosphatase alpha-subunits may be involved in regulation of Na(+),K(+)-adenosine triphosphatase activity by insulin or high extracellular glucose in skeletal muscle.     

Gliclazide protects 3T3L1 adipocytes against insulin resistance induced by hydrogen peroxide with restoration of GLUT4 translocation

Increased oxidative stress under hyperglycemia may contribute to progressive deterioration of peripheral insulin sensitivity. In this study, we investigated whether gliclazide, a second-generation sulfonylurea, can protect 3T3L1 adipocytes from insulin resistance induced by oxidative stress, and whether gliclazide can restore insulin-stimulated glucose transporter 4 (GLUT4) translocation under oxidative stress. We incubated 3T3L1 adipocytes in hydrogen peroxide to produce oxidative stress, then administered various concentrations of gliclazide, N-acetylcystein (NAC), or glibenclamide. Cells treated with these drugs were next exposed to insulin, subsequent glucose uptake was measured, and the insulin-stimulated GLUT4 translocation was monitored in living cells. We found that hydrogen peroxide treatment alone suppressed glucose uptake by insulin stimulation to 65.9%+/-7.8% of the corresponding controls (P<.01). However, addition of 0.1 to 10 micromol/L gliclazide to hydrogen peroxide-treated cells dose-dependently restored glucose uptake, with 5 micromol/L gliclazide significantly restoring glucose uptake to 93.3+/-6.6% (P<.01) even under hydrogen peroxide. Treatment with the known anti-oxidant NAC also dose-dependently (0.1-10 mmol/L) restored insulin-induced glucose uptake in the presence of hydrogen peroxide. However, glibenclamide (0.1-10 micromol/L), another second-generation sulfonylurea, failed to improve glucose uptake. Similarly, treatment with 5 micromol/L gliclazide or 10 mmol/L NAC significantly overcome the reduction in insulin-stimulated GLUT4 translocation by hydrogen peroxide (P<.01), whereas 5 micromol/L glibenclamide did not. Therefore our data regarding gliclazide further characterize its mechanism of hypoglycemic effect: the observed improvements in insulin sensitivity and in GLUT4 translocation indicate that gliclazide counters the hydrogen peroxide-induced insulin resistance in 3T3L1 adipocytes and also would further augment the hypoglycemic effect of this drug as insulinotropic sulfonylurea.     

1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells.

Insulin stimulation drives the formation of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and 1-phosphatidylinositol 3-kinase (PI 3-kinase; ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137), a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits. This interaction takes place via the phosphorylated YMXM motifs of IRS-1 and the Src homology region 2 (SH2) domains of p85. In this study, the stable overexpression in a Chinese hamster ovary (CHO) cell line of a mutant p85 alpha (delta p85) protein, which lacks a binding site for p110, disrupted the complex formation between IRS-1 and the catalytic subunit of PI 3-kinase in intact cells during insulin stimulation. Activation of insulin receptor kinase and the tyrosine phosphorylation of IRS-1 remained unaffected. In this cell line, both insulin-stimulated accumulation of phosphatidylinositol 3,4,5-trisphosphate and the insulin-stimulated glucose uptake due to the translocation of GLUT1 glucose transporters were markedly impaired, whereas neither phorbol 12-myristate 13-acetate-stimulated glucose uptake nor the insulin-stimulated activation of RAS was impaired. These results suggest that PI 3-kinase is required for glucose transport in insulin signaling in CHO cells.     

Effects of morning cortisol replacement on glucose and lipid metabolism in GH-treated subjects

OBJECTIVE: Insulin resistance is a frequent consequence of GH replacement therapy but patients on GH replacement therapy often also have replacement of other hormone deficiencies which theoretically could modify the metabolic effects of GH. In particular, cortisol replacement if given in supra physiologic doses immediately before the evaluation of insulin sensitivity could influence insulin sensitivity. The aim of this study was thus to evaluate the effect of morning cortisol replacement given prior to a euglycaemic clamp combined with infusion of [3-(3)H]glucose and indirect calorimetry on glucose and lipid metabolism. METHODS: Ten GH/ACTH-deficient adults received, in a double-blind manner, either cortisol (A) or placebo (B) before the clamp whereas five GH-deficient-ACTH-sufficient adults participated in a control (C) clamp experiment. All subjects received GH replacement therapy. RESULTS: Serum cortisol levels were significantly higher after cortisol than after placebo (324+/-156 vs 132+/-136 mmol/l; P=0.006) and similar to controls (177+/-104 mmol/l). As a measure of the biological effect of cortisol, eosinophil leukocyte counts in peripheral blood decreased (164+/-91 x 10(9)/l vs 216+/-94 x 10(9)/l; P=0.04). Cortisol replacement had no significant effect on insulin-stimulated glucose uptake (11.8+/-1.8 vs 13.2+/-3.9 micromol/kg min), either on glucose oxidation or on glucose storage. There was also no significant effect of cortisol on fasting endogenous glucose production and no effect was seen on serum free fatty acid concentrations. CONCLUSION: Administration of cortisol in the morning before a clamp cannot explain the insulin resistance seen with GH replacement therapy.     

Impaired endothelial alpha-2 adrenergic receptor-mediated vascular relaxation in the fructose-fed rat.

To investigate the vascular endothelial dysfunction in the insulin resistance syndrome, muscarinic and alpha2-adrenergic mediated relaxations were studied in the fructose-fed rat. Male Sprague-Dawley rats were fed either fructose-rich chow (FFR, n=14) or normal chow (CNT, n=13) for 8 weeks. Systolic blood pressure (SBP) was measured by the tail-cuff method. A 3 mm segment of mesenteric artery was cannulated and pressurized, pretreated with prazosin (10(-6) mol/l) and propranolol (3x10(-6) mol/l), then pre-contracted with serotonin (10(-6) mol/l). Endothelium-dependent relaxation was induced by addition of acetylcholine (ACh, 10(-9)-10(-4) mol/l) or a selective alpha2-agonist, B-HT 920 (10(-9)-10(-5) mol/l), with or without the nitric oxide (NO) synthase inhibitor, L-NAME (10(-4) mol/l). SBP was significantly elevated in FFR but not in CNT. Plasma triglyceride in FFT (241+/-115 mg/dl) was significantly (p<0.01) higher than in CNT (84+/-34 mg/dl). Insulin and insulin/glucose ratio were higher but not significantly. Plasma glucose was not different between the two groups. In the dose-response curves to ACh, maximum relaxation and ED50 were similar between FFR and CNT. Moreover, L-NAME shifted the dose-response curves similarly to the right in both groups. Dose-response curves to B-HT 920, however, showed less relaxation in FFR than in CNT (p<0.05). B-HT 920-induced relaxations were mostly abolished by L-NAME. It is concluded that endothelial alpha2-adrenergic relaxation, predominantly mediated by NO, is likely more sensitive to the development of insulin resistance than muscarinic receptor relaxation in this 8-weeks FFR model. This early impairment of endothelial alpha2-adrenergic relaxation may contribute to the development of hypertension and insulin resistance in the FFR.     

Insulin signal transduction and glucose transport in human adipocytes: effects of obesity and low calorie diet

AIM/HYPOTHESIS: We examined insulin signal transduction at the level of insulin receptor substrates (IRS) 1 and 2, phosphatidylinositol (PI) 3-kinase and glucose transport in isolated subcutaneous adipocytes from obese and lean women. METHODS: Glucose transport and insulin signalling were investigated in isolated adipocytes from six obese women (BMI 36-43 kg/m(2)) (before and after 11 days of very low calorie diet) and from six lean women (BMI 22-26 kg/m(2)). RESULTS: Insulin sensitivity of glucose transport was reduced in adipocytes from obese women (p<0.05), with further reductions in basal and maximal insulin-stimulated glucose transport after a very low calorie diet (p<0.05). In obese women, IRS-1 associated PI 3-kinase activity was markedly impaired (p<0.05), whereas, IRS-2 associated PI 3-kinase activity was normal. IRS-1 associated PI 3-kinase activity remained blunted after a very low calorie diet, whereas IRS-2 associated PI 3-kinase activity was increased. GLUT4 protein was reduced by 37% in obese versus lean subjects (p<0.05), and decreased further after a very low calorie diet (from 19+/-4 to 14+/-4 arbitrary units; p<0.05). CONCLUSION/INTERPRETATION: IRS-1 signalling to PI 3-kinase is a site of insulin resistance in adipocytes from obese women, whereas insulin action on IRS-2 is normal. Thus, IRS-1 and IRS-2 undergo differential regulation in adipocytes from obese insulin resistant subjects. Finally, a very low calorie diet is associated with a further impairment in glucose transport in adipose tissue. The defect in glucose transport after a very low calorie diet occurs independent of further defects in insulin signalling at the level of the PI 3-kinase.