Thioredoxin-Interacting Protein Deficiency Protects against Diabetic Nephropathy.

Expression of thioredoxin-interacting protein (TxNIP), an endogenous inhibitor of the thiol oxidoreductase thioredoxin, is augmented by high glucose (HG) and promotes oxidative stress. We previously reported that TxNIP-deficient mesangial cells showed protection from HG-induced reactive oxygen species, mitogen-activated protein kinase phosphorylation, and collagen expression. Here, we investigated the potential role of TxNIP in the pathogenesis of diabetic nephropathy (DN) in vivo. Wild-type (WT) control, TxNIP(-/-), and TxNIP(+/-) mice were rendered equally diabetic with low-dose streptozotocin. In contrast to effects in WT mice, diabetes did not increase albuminuria, proteinuria, serum cystatin C, or serum creatinine levels in TxNIP(-/-) mice. Whereas morphometric studies of kidneys revealed a thickened glomerular basement membrane and effaced podocytes in the diabetic WT mice, these changes were absent in the diabetic TxNIP(-/-) mice. Immunohistochemical analysis revealed significant increases in the levels of glomerular TGF-ß1, collagen IV, and fibrosis only in WT diabetic mice. Additionally, only WT diabetic mice showed significant increases in oxidative stress (nitrotyrosine, urinary 8-hydroxy-2-deoxy-guanosine) and inflammation (IL-1ß mRNA, F4/80 immunohistochemistry). Expression levels of Nox4-encoded mRNA and protein increased only in the diabetic WT animals. A significant loss of podocytes, assessed by Wilms' tumor 1 and nephrin staining and urinary nephrin concentration, was found in diabetic WT but not TxNIP(-/-) mice. Furthermore, in cultured human podocytes exposed to HG, TxNIP knockdown with siRNA abolished the increased mitochondrial O2 (-) generation and apoptosis. These data indicate that TxNIP has a critical role in the progression of DN and may be a promising therapeutic target.

Oltipraz upregulates the nuclear factor (erythroid-derived 2)-like 2 [corrected](NRF2) antioxidant system and prevents insulin resistance and obesity induced by a high-fat diet in C57BL/6J mice.

AIMS/HYPOTHESIS: We investigated whether oltipraz, a nuclear respiratory factor 2 alpha subunit (NRF2) activator, improves insulin sensitivity and prevents the development of obesity in mice. METHODS: C57BL/6J mice were fed with a low-fat diet (10% of energy as fat), a high-fat diet (HFD) (45% of energy as fat) or a HFD with oltipraz for 28 weeks. The effects of oltipraz on body weight, fat content, glucose disposal, insulin signalling, metabolic profiles and endogenous NRF2 functional status in the three groups of mice were investigated. RESULTS: Oltipraz prevented or significantly attenuated the effect of HFD on glucose disposal, body weight and fat gain. Impairment of protein kinase B/Akt phosphorylation in this HFD-fed mouse model in response to intraperitoneal insulin injection was observed in adipose tissue, but not in the muscles, accompanied by inhibition of AMP-activated protein kinase signalling and activation of p70S6 kinase, as well as reduced GLUT4 content. These defects were attenuated by oltipraz administration. Nuclear content of NRF2 in adipose tissue was reduced by HFD feeding, associated with increased Keap1 mRNA expression and reduced production of haem oxygenase-1 and superoxide dismutase, increased protein oxidation, decreased plasma reduced:oxidised glutathione ratio and the appearance of macrophage marker F4/80. These defects were also restored by oltipraz. Finally, oltipraz attenuated HFD-induced inducible nitric oxide synthase overproduction. CONCLUSIONS/INTERPRETATION: Impairment of the endogenous redox system is important in the development of obesity and insulin resistance in chronic HFD feeding. NRF2 activation represents a potential novel approach in the treatment and prevention of obesity and diabetes.

Free fatty acid-induced muscle insulin resistance and glucose uptake dysfunction: evidence for PKC activation and oxidative stress-activated signaling pathways.

In the present study, we examined the effects of free fatty acids (FFAs) on insulin sensitivity and signaling cascades in the C2C12 skeletal muscle cell culture system. Our data clearly manifested that the inhibitory effects of PKC on insulin signaling may at least in part be explained by the serine/threonine phosphorylation of IRS-1. Both oleate and palmitate treatment were able to increase the Serine(307) phosphorylation of IRS-1. IRS-1 Serine(307) phosphorylation is inducible which causes the inhibition of IRS-1 tyrosine phosphorylation by either IkappaB-kinase (IKK) or c-jun N-terminal kinase (JNK) as seen in our proteomic kinases screen. Furthermore, our proteomic data have also manifested that the two FFAs activate the IKKalpha/beta, the stress kinases S6 kinase p70 (p70SK), stress-activated protein kinase (SAPK), JNK, as well as p38 MAP kinase (p38MAPK). On the other hand, the antioxidant, Taurine at 10mM concentrations was capable of reversing the oleate-induced insulin resistance in myocytes as manifested from the glucose uptake data. Our current data point out the importance of FFA-induced insulin resistance via multiple signaling mechanisms.

Links between enhanced fatty acid flux, protein kinase C and NFkappaB activation, and apoB-lipoprotein production in the fructose-fed hamster model of insulin resistance.

In the current study, we show evidence, in a fructose-fed hamster model of insulin resistance, that free fatty acid (FFA) can induce hepatic insulin resistance in part via PKC activation leading to increased production of atherogenic apoB100-containing lipoproteins. Interestingly, IkappaB-kinase beta (IKKbeta)-dependent NF-kappaB was activated in hepatocytes from the fructose-fed hamster as an indication for PKC activation. Treatment of hepatocytes with oleate for 16h showed the activation of the PKC isoforms, PKCalpha/betaII, in a dose dependent manner. Strikingly, the general PKC inhibitor, bisindolylmaleimide-I, Bis-I (5 microM) was found to ameliorate fructose-induced insulin resistance, restoring the phosphorylation status of PKB and suppressing apoB100 overproduction in ex vivo and in vivo. The data suggest that hepatic PKC activation, induced by increased circulating FFA may be an important factor in the development of insulin resistance and dyslipidemia seen in the fructose-fed hamster model.

Glucosamine activates the plasminogen activator inhibitor 1 gene promoter through Sp1 DNA binding sites in glomerular mesangial cells.

Increased flux through the hexosamine biosynthetic pathway is associated with altered gene expression. To investigate the underlying mechanisms, we treated glomerular mesangial cells with glucosamine and studied the regulation of the plasminogen activator inhibitor (PAI)-1 gene. Incubating mesangial cells with 2 mmol/l glucosamine for 4 days resulted in a 3.1+/-0.4-fold increase in PAI-1 mRNA levels (P < 0.01) and a 33+/-9-fold increase in the activity of a transiently transfected PAI-1 promoter-luciferase reporter gene (P < 0.01). Cotransfection of an expression vector for a dominant-negative type II TGF-beta receptor with the PAI-1 promoter-reporter gene did not interfere with this effect of glucosamine. However, mutation of 2 putative Sp1 sites in the PAI-1 promoter, at -76 to -71 and -44 to -39, markedly reduced induction of PAI-1 luciferase activity by glucosamine, from 8.9+/-1.9-fold to 1.7+/-0.5-fold (P < 0.01). An electrophoretic mobility shift assay demonstrated that glucosamine increased Sp1 DNA binding by 31+/-11% (P < 0.05), implying that the effects of glucosamine were explained, in part, by changes in Sp1 DNA binding. High glucose (20 mmol/l) also activated the transiently transfected PAI-1 promoter (2.5+/-0.4-fold). This effect was diminished by mutation of both the PAI-1 promoter Sp1 sites (1.2+/-0.3-fold, P < 0.05). In addition, 6-diazo-5-oxo-L-norleucine, a glutamine:fructose-6-phosphate-amidotransferase inhibitor, blocked the induction by high glucose (4.7+/-0.8- to 0.9+/-0.1-fold, P < 0.01). These results indicate that stimulation of the PAI-1 promoter by both high glucose and glucosamine involves Sp1 and that the hexosamine pathway may be involved in the regulation of gene expression by high glucose in glomerular mesangial cells.

Vanadate augments insulin-stimulated insulin receptor kinase activity and prolongs insulin action in rat adipocytes. Evidence for transduction of amplitude of signaling into duration of response.

Vanadate, a protein tyrosine phosphatase inhibitor, preserves insulin-stimulated lipogenesis after removal of insulin. To investigate the mechanism of this action of vanadate, lipogenesis was studied in isolated rat adipocytes exposed to vanadate for 60 min followed by insulin for 15 min at 37 degrees C. Vanadate (10-50 microM) prolonged insulin-stimulated lipogenesis. The half-time (t1/2) of the decay in insulin (0.34 nM)-stimulated lipogenesis after removal of insulin by washing in pH 7.0 followed by pH 7.6 buffer was 21 min in the absence and 59 min in the presence of vanadate. During these conditions, vanadate did not alter insulin binding nor the removal of insulin by the series of washes. In contrast to lipogenesis, the t1/2 of the decay in insulin receptor tyrosine kinase (IRK) activity, assayed with the artificial substrate Poly[Glu:Tyr] (4:1), was not significantly prolonged by vanadate (6 vs. 6.8 min). However, insulin-stimulated IRK activity was markedly augmented by vanadate to 319 +/- 19% of insulin alone, associated with a similar augmentation of phosphotyrosine incorporation into the insulin receptor beta-subunit determined by Western blotting with antiphosphotyrosine antibodies. To determine the relationship between prolongation of lipogenesis and the increase in IRK, adipocytes were exposed to 17.2 nM insulin to activate the IRK to the same extent as insulin (0.34 nM) plus vanadate (maximum activation). During these two conditions, the decay of lipogenesis was similar and after stimulation with 17.2 nM insulin was not prolonged by vanadate. We conclude that vanadate prolongs insulin action at insulin concentrations that do not maximally activate the IRK by augmenting IRK activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine phosphatase inhibitors, vanadate and pervanadate, stimulate glucose transport and GLUT translocation in muscle cells by a mechanism independent of phosphatidylinositol 3-kinase and protein kinase C.

Vanadate and pervanadate (pV) are protein tyrosine phosphatase (PTP) inhibitors that mimic insulin to stimulate glucose transport. To determine whether phosphatidylinositol (PI) 3-kinase is required for vanadate and pV, as it is for insulin, cultured L6 myotubes were treated with vanadate and pV. The two compounds stimulated glucose transport to levels similar to those stimulated by insulin; however, while PI 3-kinase activity and the increase in the lipid products PI 3,4-bisphosphate and PI 3,4,5-trisphosphate were inhibited by wortmannin after stimulation by all three agents--insulin, vanadate, and pV--wortmannin blocked glucose transport stimulated by insulin but not vanadate or pV. Vanadate and pV stimulated the translocation of GLUTs from an intracellular compartment to the plasma membrane; this stimulation was not blocked by wortmannin, but insulin-induced GLUT translocation was inhibited. Similar results were obtained in cultured H9c2 cardiac muscle cells in which wortmannin did not inhibit glucose transport or the vanadate-induced translocation of GLUT4 in c-myc-GLUT4 transfected cells. The ser/thr kinase PKB (Akt/PKB/RAC-PK) is activated by insulin, lies downstream of PI 3-kinase, and has been implicated in signaling of glucose transport. Insulin and pV stimulated PKB activity, and both were inhibited by wortmannin. In contrast, vanadate, at concentrations that maximally stimulated glucose transport, did not significantly increase PKB activity. To determine the potential role of protein kinase C (PKC), L6 cells were incubated chronically with phorbol myristate acetate (PMA) or acutely with the PKC inhibitors calphostin C and bisindolylmaleimide. There was no inhibition of glucose transport stimulation by insulin, vanadate, or pV, and a combination of wortmannin and PKC inhibitors also failed to block the effect of vanadate and pV. In contrast, disassembly of the actin network with cytochalasin D blocked the stimulation of glucose transport by all three agents. In conclusion, vanadate and pV are able to stimulate glucose transport and GLUT translocation by a mechanism independent of PI 3-kinase and PKC. Similar to that by insulin, glucose transport stimulation by vanadate and pV requires the presence of an intact actin network.

The insulin-mimetic agent vanadate promotes receptor endocytosis and inhibits intracellular ligand-receptor degradation by a mechanism distinct from the lysosomotropic agents.

Vanadate (sodium orthovanadate) is an insulin-mimetic agent and phosphotyrosine phosphatase inhibitor that has been proposed as a potential therapeutic agent for diabetes. We previously reported that vanadate decreased the number of cell-surface insulin receptors but inhibited receptor degradation in cultured lymphocytes (IM-9) (1). To determine whether vanadate affected receptors without intrinsic tyrosine kinase activity, its effects on LDL and transferrin receptors and their ligands were examined. Vanadate exposure resulted in a dose- and time-dependent decrease in LDL binding to cultured human fibroblasts associated with a decrease in cell surface receptor number while total solubilized cell LDL receptors increased. Vanadate also inhibited the LDL-mediated downregulation of total cellular LDL receptors in the absence and presence of cycloheximide consistent with an inhibition of LDL receptor degradation. In the case of the ligand, vanadate augmented the accumulation of intact 125I-LDL associated with an inhibition of up to 80% of the ability of LDL to decrease cholesterol synthesis. Since these actions were similar to the effects of lysosomotropic agents, we examined the effect of vanadate on intraendosomal pH using the fluorescent probe acridine orange. In contrast with chloroquine and NH4Cl, vanadate did not neutralize the pH of the acidic intracellular compartment. Furthermore, after a transient insulin-like effect, chronic exposure to vanadate diminished 125I-diferric transferrin binding to rat adipocytes. In contrast with the inhibitory action of NH4Cl, intracellular 59Fe uptake remained unaffected and was proportional to cell-surface binding capacity in the presence of vanadate. These data demonstrate a chronic effect of vanadate to promote the accumulation of intracellular receptors and to inhibit ligand and receptor degradation. The latter effect is not mediated by pH changes, appears to be localized to a late endosomal/lysosomal compartment, and suggests a possible role for tyrosine dephosphorylation in the regulation of receptor-ligand degradation.

High glucose-enhanced mesangial cell extracellular signal-regulated protein kinase activation and alpha1(IV) collagen expression in response to endothelin-1: role of specific protein kinase C isozymes.

High glucose (HG) stimulates glomerular mesangial cell (MC) expression of extracellular matrix, a process involving protein kinase C (PKC) isozymes and enhanced signaling by autocrine peptides such as endothelin-1 (ET-1). The purpose of this study was to identify the specific PKC isozymes mediating the effects of HG on MC extracellular signal-regulated protein kinase (ERK1/2) signaling and alpha1(IV) collagen expression in response to ET-1. HG (30 mmol/l for 72 h) enhanced ET-1-stimulated alpha1(IV) collagen mRNA expression from 1.2 +/- 0.1-fold to 1.9 +/- 0.2-fold (P < 0.05 vs. normal glucose [NG] + ET-1), and the effect was significantly reduced by Calphostin C or the MEK (mitogen-activated protein kinase kinase) inhibitor PD98059. In transiently transfected MCs, dominant-negative (DN)-PKC-delta, -epsilon, or -zeta inhibited ET-1 activation of ERK1/2. Likewise, downstream of ERK1/2, ET-1 stimulated Elk-1-driven GAL4 luciferase activity to 11 +/- 1-fold (P < 0.002 vs. NG + ET-1) in HG, and DN-PKC-delta, -epsilon, or -zeta attenuated this response to NG levels. HG enhanced ET-1-stimulated intracellular alpha1(IV) collagen protein expression, assessed by confocal immunofluorescence imaging, showed that individual DN-PKC-delta, -epsilon, -zeta, as well as DN-PKC-alpha and -beta, attenuated the response. Thus, HG-enhanced ET-1 stimulation of alpha1(IV) collagen expression requires PKC-delta, -epsilon, and -zeta to act through an ERK1/2-dependent pathway and via PKC-alpha and -beta, which are independent of ERK1/2.

Insulin binding and glucose transport in adipocytes in neonatal streptozocin-injected rat model of diabetes mellitus.

The neonatal streptozocin (STZ)-injected rat (NSIR) model of diabetes mellitus resembles human non-insulin-dependent diabetes mellitus (NIDDM) with respect to abnormalities in insulin secretory responses. The suggestion that insulin deficiency leads to insulin resistance, a prominent feature of human NIDDM, led us to examine insulin binding and glucose transport in the NSIR during the development of hyperglycemia. Male Wistar rats were injected at 2 days of age with STZ (90 mg/kg i.p.) or vehicle alone. Mild insulin deficiency, reflected by minimally decreased fed plasma insulin concentrations, was apparent at 4 wk (mean +/- SE, control vs. NSIR, 2.32 +/- 0.19 vs. 1.75 +/- 0.21 ng/ml) and at 8 wk. Pancreatic insulin content was dramatically reduced in NSIR to 12 and 5% of control values at 4 and 8 wk, respectively (P less than .001). Fed plasma glucose concentrations increased in the NSIR between 4 and 5 wk and were significantly elevated at 8 wk (251 +/- 25 vs. 527 +/- 52 mg/dl, P less than .001). 125l-labeled insulin binding showed a progressive increase as a function of adipocyte volume in control and NSIR. Epididymal fat pad weights and adipocyte volumes were significantly decreased in the NSIR. Thus, insulin binding did not differ when expressed per cell number but was increased in NSIR when corrected for cell size (percent specific binding X 10(2), 8.49 +/- 0.96 vs. 11.56 +/- 1.08/microliter cell vol; P less than .05, all ages combined).(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased in situ insulin receptor dephosphorylation in hyperglycemia-induced insulin resistance in rat adipocytes.

The regulation of insulin receptor (IR) tyrosine (tyr) phosphorylation is a key step in the control of insulin signaling. Augmented IR tyr dephosphorylation by protein tyrosine phosphatases (PTPs) may contribute to insulin resistance. To investigate this possibility in hyperglycemia-induced insulin resistance, primary cultured rat adipocytes were rendered insulin-resistant by chronic exposure (18 h) to 15 mmo/l glucose combined with 10(-7) mol/l insulin. Insulin-resistant adipocytes showed a decrease in insulin sensitivity and a maximum response of 2-deoxyglucose uptake, which was associated with a decrease in maximum insulin-stimulated IR tyr phosphorylation in situ. To assess tyr dephosphorylation, IRs of insulin-stimulated permeabilized adipocytes were labeled with [gamma-32P]ATP and chased for 2 min with unlabeled ATP in the presence of EDTA. In a nonradioactive protocol, insulin-stimulated adipocytes were permeabilized and exposed to EDTA and erbstatin for 2 min, and IRs were immunoblotted with anti-phosphotyrosine (pY) antibodies. Both methods showed a similar diminished extent of IR tyr dephosphorylation in resistant cells. Immunoblotting of four candidate IR-PTPs demonstrated no change in PTP1B or the SH2 domain containing phosphatase-2 (SHP-2), whereas a significant decrease in leukocyte antigen-related phosphatase (LAR) (51 +/- 3% of control) and an increase in PTP-alpha (165 +/- 16%) were found. Activity of immunoprecipitated PTPs toward a triple tyr phosphorylated IR peptide revealed a correlation with protein content for PTP1B, SHP-2, and LAR but a decrease in apparent specific activity of PTP-alpha. The data indicate that decreased IR tyr phosphorylation in hyperglycemia-induced insulin resistance is not due to enhanced dephosphorylation. The diminished IR tyr dephosphorylation observed in this model is associated with decreased LAR protein content and activity.

Vanadium compounds biological actions and potential as pharmacological agents.

Vanadium is an element found in low concentrations in mammals, for which a function remains to be discovered. Over the past century, vanadium compounds have been suggested anecdotally as therapeutic agents for a variety of diseases. The discovery that vanadate inhibits various enzymes, in particular protein tyrosine phosphatases, and mimics many of the biological actions of insulin suggested a potential role in the therapy of diabetes mellitus. Successful use and an enhancement of insulin sensitivity in rodents and human diabetic subjects, as well as the finding that these agents are capable of stimulating metabolic effects while bypassing the insulin receptor and the early steps in insulin action, target these agents preferentially toward type II diabetes mellitus. Long-term safety remains a major concern, as tissue accumulation and relative nonspecificity of enzyme inhibition may result in adverse effects. Continued research into mechanism of action, consequences of chronic administration, and improvement of specificity is warranted. Regardless of their ultimate success or failure as therapeutic agents, vanadium compounds continue to be useful probes of enzyme structure and function in various biological processes. (Trends Endocrinol Metab 1997;8:51-58). (c) 1997, Elsevier Science Inc.

Vanadate augments insulin binding and prolongs insulin action in rat adipocytes.

Vanadate has been documented to inhibit tyrosine phosphatase activity and to have insulin-mimetic effects. However, oral administration to hypoinsulinemic diabetic rats in vivo lowers blood glucose at serum concentrations of vanadate that have minimal insulin-like effects in vitro. We, therefore, investigated the effect of low concentrations of vanadate on insulin binding, processing, and action. Preincubation of rat adipocytes for 2 h at 37 C with 10-200 microM vanadate resulted in a dose-dependent increase in [125I]insulin binding at 37 C to a maximum of 45% above the control value. Total cell-associated radioactivity and internalized (acid-resistant) hormone were similarly increased. Binding studies at 15 C in the presence of potassium cyanide revealed that this effect was associated with an increase in insulin receptor affinity. Consistent with these results, vanadate affected binding at 37 C only at low concentrations of insulin. Preloading adipocytes for 8 min with 0.4 ng/ml [125I]insulin revealed that vanadate slowed the rate of release of internalized hormone (50% release; 9.0 min vs. 12.5 min). The proportion of [125I]insulin released in intact form (trichloroacetic acid precipitable) was significantly increased by vanadate up to 15 min. Preincubation of adipocytes with vanadate resulted in an apparent increased sensitivity, with a shift to the left in the dose-response curve of insulin-stimulated lipogenesis (ED50, 0.2 vs. 0.08 ng/ml). Furthermore, vanadate maintained maximum insulin-stimulated lipogenesis after extensive washing to remove insulin. These effects could not be accounted for by the insulin-mimetic effect of vanadate alone. We conclude that 1) low concentrations of vanadate (less than 200 microM) increase insulin receptor affinity and consequent insulin uptake in rat adipocytes; 2) the excess cell-associated insulin exists largely as intact hormone; and 3) the increased binding at low insulin concentrations results in an apparent increase in insulin sensitivity. Vanadate at low concentrations also prolongs insulin action. Whether tyrosine phosphatase inhibition is the basic biochemical mechanism remains to be determined.

Enhancement of cytosolic tyrosine kinase activity by propylthiouracil-induced hyperplasia in the rat thyroid.

Hyperplasia of the thyroid gland induced by propylthiouracil (PTU) is a well established model of rapid cell proliferation in vivo. Recent evidence indicates that tyrosine kinase activity is associated with growth factor receptors and oncogene protein products and may have an important regulatory action in the control of cell growth. Thus, we examined tyrosine kinase activity in rat thyroid membrane and cytosol preparations at rest and during PTU-induced hyperplasia. Although kinase activity was present in a crude microsomal membrane preparation, no change was observed during thyroid growth. In contrast, tyrosine kinase activity assayed with the artificial substrate poly(Glu,Na:Tyr) 4:1 was present in normal rat thyroid cytosol and increased 2- to 6-fold during the rapid phase of hyperplasia in the first 5-10 days of PTU treatment. It declined to control values by day 15, when the size and DNA content of the thyroid reached a plateau. Preincubation of the cytosolic preparations with several peptides known to bind to and activate growth factor receptor tyrosine kinases failed to enhance the activity, suggesting, along with the cytosolic localization, that the activity was distinct from these receptors. By gel filtration chromatography and polyacrylamide gel electrophoresis, tyrosine kinase activity was associated with a 55 kDa protein. Partial purification over a poly(Glu,Na:Tyr)4:1-Sepharose column, yielded a protein that appeared capable of autophosphorylation. It is suggested that this tyrosine kinase plays a role in mediating the growth-promoting effects of this model of thyroid cell hyperplasia.

Stimulation of glucose uptake and increased plasma membrane content of glucose transporters in L6 skeletal muscle cells by the sulfonylureas gliclazide and glyburide.

Many studies suggest that sulfonylureas (SUs) have direct extrapancreatic actions. The action of gliclazide, a new SU, was examined and compared to that of glyburide in L6 myotubes, a model of skeletal muscle. Gliclazide and glyburide increased 2-deoxy-D-glucose (2DG) uptake in a time- and dose-dependent fashion after 24 h to a maximum of 179% and 202% of the basal value, respectively (P < 0.001). Acute (30-min) insulin (10(-7) M) stimulated 2DG uptake to similar levels (203% of basal), but this effect was absent after maximum stimulation by SU. SU action did not require insulin and was not blocked by the protein synthesis inhibitor cycloheximide. To investigate the mechanism of stimulation of 2DG uptake, cells were fractionated, and total plasma membrane and internal membrane levels of glucose transporter (GLUT) isoforms were determined by immunoblotting. Both drugs significantly increased the total content (1.7-fold) and plasma membrane level (1.8-fold) of GLUT1, with no change in internal membrane. Total content and plasma membrane levels of GLUT4 and GLUT3 did not change or showed a small decrease. We conclude that the stimulation of glucose uptake in L6 cells by gliclazide and glyburide is associated not with a redistribution but, rather, with an increase in the total membrane content and plasma membrane level of GLUT1, which is independent of protein synthesis. These data suggest a novel action of SU to stabilize GLUT1 protein at the plasma membrane.

Mechanism of action of metformin: insulin receptor and postreceptor effects in vitro and in vivo.

Metformin (Met) is a biguanide oral hypoglycemic agent used in the treatment of noninsulin-dependent diabetes mellitus (NIDDM). To define whether the glucose-lowering effects are mediated via alterations of insulin receptors, the effects of Met in vitro in rat adipocytes and in vivo in patients with poorly controlled NIDDM were studied. In vitro exposure of rat adipose tissue to metformin for 20 h resulted in a significant increase in insulin binding (mean +/- SEM percent specific [125I]insulin bound per 10(5) adipocytes: control, 1.35 +/- 0.13; Met, 1.69 +/- 0.18; P less than 0.02). No change occurred after 2 h of exposure or less. In contrast, after only 1 h of preincubation. Met alone stimulated [U-14C]glucose oxidation by 58 +/- 15.5% (P less than 0.01). Met did not stimulate glucose oxidation in the presence of a high insulin concentration. For the in vivo studies, oral glucose tolerance tests and monocyte [125I]insulin binding assays were performed before and after 7 days of Met treatment (2 g/day) in 18 patients with poorly controlled NIDDM. All patients responded to Met with a decrease in fasting and postglucose plasma glucose concentrations, but no change in insulin concentrations [pre-Met vs. post-Met: fasting plasma glucose, 210 +/- 10 vs. 157 +/- 11 mg/dl (P less than 0.001); fasting plasma insulin, 20.3 +/- 3.1 vs. 18.4 +/- 2.0 microU/ml]. When insulin binding was examined, 8 patients with decreased binding each responded to Met with a 50% or greater increase (group 1), while 10 patients with normal binding had no increase after treatment (group 2). However, both groups had similar lowering of glucose concentrations [fasting plasma glucose: group 1, 205 +/- 19 vs. 153 +/- 20 (P less than 0.001); group 2, 214 +/- 11 vs. 160 +/- 13 (P less than 0.001)]. We conclude that 1) Met has an acute insulin-like effect in vitro independent of its ability to increase insulin binding; 2) Met acts in vivo predominantly at a postreceptor site to lower plasma glucose; 3) the glucose-lowering effect is independent of pretreatment insulin binding status; and 4) the increase in insulin binding after Met treatment in patients with NIDDM and low insulin binding occurs without changes in insulin concentrations.

Evidence for abnormal regulation of insulin receptors in Friedreich's ataxia.

Friedreich's ataxia is associated with a high incidence of diabetes mellitus. We have previously demonstrated that insulin resistance is present in nondiabetic patients with Friedreich's ataxia. This was associated with a reduction in the affinity of insulin receptors on freshly isolated monocytes. In this study we investigated the ability of the monocyte insulin receptor to acutely alter its affinity in response to oral glucose. Glucose and insulin concentrations were higher in the patients with Friedreich's ataxia after an oral glucose load, consistent with the presence of insulin resistance. The normal increase in the affinity of insulin receptors on monocytes 5 h after oral glucose was absent in the five patients with Friedreich's ataxia. Receptor affinity actually decreased in three of the five patients. These findings support the concept that a membrane abnormality that alters the binding function of the insulin receptor is present in these patients.

The effect of glucocorticoids on the insulin receptor: an in vivo and in vitro study.

We have studied the effect of glucocorticoid exposure on the insulin receptor of short term cultures of human lymphocytes (IM-9 cells) and the effect of short term administration of these agents to normal volunteers. When cultured human lymphocytes were exposed to 10(-5) M hydrocortisone for 18 h at 37 C, insulin binding increased due to an increase in the number of receptors per cell. The effect had appropriate specificity in terms of concentration and type of steroid used. By contrast, hGH binding to these cells was decreased under similar circumstances of incubation, due to a decrease in the number of hGH receptors per cell. When prednisone (40 mg/day) was given to normal subjects for 3 days, a moderate state of insulin resistance was induced characterized by a modest increase in blood glucose and a more pronounced increase in plasma insulin concentration. Under these circumstances there was no change in tracer insulin binding to peripheral monocytes nor changes in the concentration of insulin necessary to inhibit binding by 50%, the number of receptors per cell or the affinity of the receptor. We conclude that glucocorticoids increase insulin binding in vitro in cultured human lymphocytes but that competing influences in vivo such as increasing circulating insulin concentration, redistribution of cell types in the circulation, and possibly other influences prevent these changes from occurring in circulating monocytes. These findings emphasize the complexity of studying the effects of pharmacological agents on hormone binding.

Androgen response to endogenous insulin secretion during the frequently sampled intravenous glucose tolerance test in normal and hyperandrogenic women.

Women with ovarian hyperandrogenism frequently have insulin resistance, whose underlying mechanism remains to be determined. In the present study we have investigated the relationship between insulin sensitivity and the acute effect of endogenous insulin secretion on circulating androgen levels. Insulin sensitivity, glucose-mediated insulin release, and glucose/insulin-stimulated androgen responses were determined during a frequently sampled iv glucose tolerance test in a group of 19 women with clinical evidence of polycystic ovary syndrome (PCOS) and 9 age- and weight-matched controls. Insulin (I), glucose, androstenedione, testosterone (T), free T, and dehydroepiandrosterone (DHEA) levels were measured before and during the 3 h following iv administration of glucose (300 mg/kg). Intravenous tolbutamide (300-500 mg) was injected 20 min after the glucose injection. Insulin sensitivity (SI) was calculated by application of the minimal model of glucose kinetics. Fasting androstenedione, T, free T, and I concentrations were significantly higher in the women with PCOS than in controls (P less than 0.02). In PCOS subjects, fasting I was correlated with both T (r = 0.51; P less than 0.05) and DHEA (r = 0.706; P less than 0.01). SI was significantly lower in PCOS subjects [SI, 68.35 +/- 8.34 min-1/(nmol/mL] than in control subjects (SI, 133.36 +/- 21.7 min-1/(nmol/mL)]. A significant decline in DHEA levels was observed in control subjects 3 h after glucose administration (from 28.4 +/- 3.0; final, 16.2 +/- 2.4; P less than 0.02). PCOS women with normal insulin sensitivity [SI, greater than 75.0 min-1/(nmol/mL)] showed a similar fall in DHEA (from 20.3 +/- 2.5 to 12.8 +/- 1.8 nmol/L; P less than 0.02). No significant change occurred in insulin-resistant PCOS subjects [SI, less than 75.0 min-1/(nmol/mL)]. Other androgen levels showed a modest nonsignificant decline during the study in PCOS and control groups. These findings confirm the weight-independent insulin resistance of some hyperandrogenic women. The failure of glucose-stimulated endogenous insulin secretion to significantly depress DHEA levels in insulin-resistant women with PCOS may account in part for their androgen excess.

Circulating tumor necrosis factor-alpha concentrations in a native Canadian population with high rates of type 2 diabetes mellitus.

Recent research suggests that tumor necrosis factor-alpha (TNF alpha) may play an important role in obesity-associated insulin resistance and diabetes. We studied the relationship between TNF alpha and the anthropometric and physiological variables associated with insulin resistance and diabetes in an isolated Native Canadian population with very high rates of type 2 diabetes mellitus (DM). A stratified random sample (n = 80) of participants was selected from a population-based survey designed to determine the prevalence of type 2 DM and its associated risk factors. Fasting blood samples for glucose, insulin, triglyceride, leptin, and TNF alpha were collected; a 75-g oral glucose tolerance test was administered, and a second blood sample was drawn after 120 min. Insulin resistance was estimated using the homeostasis assessment (HOMA) model. Systolic and diastolic blood pressure (BP), height, weight, and waist and hip circumferences were determined, and percent body fat was estimated using biological impedance analysis. The relationship between circulating concentrations of TNF alpha and the other variables was assessed using Spearman correlation coefficients, analysis of covariance, and multiple linear regression. The mean TNF alpha concentration was 5.6 pg/mL (SD = 2.18) and ranged from 2.0-12.9 pg/mL, with no difference between men and women (P = 0.67). There were moderate, but statistically significant, correlations between TNF alpha and fasting insulin, HOMA insulin resistance (HOMA IR) waist circumference, fasting triglyceride, and systolic BP (r = 0.23-0.34; all P < 0.05); in all cases, coefficients for females were stronger than those for males. Individuals with normal glucose tolerance had lower log TNF alpha concentrations than those with impaired glucose tolerance or type 2 DM (both P = 0.03, adjusted for age and sex), although differences were not significant after adjustment for HOMA IR (both P > 0.25). Regression analysis indicated that log HOMA IR and log systolic BP were significant independent contributors to variations in log TNF alpha concentration (model r2 = 0.32). We conclude that in this homogeneous Native Canadian population, circulating TNF alpha concentrations are positively correlated with insulin resistance across a spectrum of glucose tolerance. The data suggest a possible role for TNF alpha in the pathophysiology of insulin resistance.