Chromium and vanadate combination increases insulin-induced glucose uptake by 3T3-L1 adipocytes

Insulin activates signaling pathways in target tissues through the insulin receptor and Tyr phosphorylation of intracellular proteins. Vanadate mimics insulin and enhances its actions through inhibition of protein Tyr phosphatases. Chromium is a micronutrient that enhances insulin action to normalize blood glucose, but the mechanism is not understood. Here we show that either vanadate or chromium stimulates Tyr phosphorylation of insulin receptor in mouse 3T3-L1 adipocytes compared to insulin alone, but a combination of vanadate and chromium is not additive. Phosphorylation of MAPK or 4E-BP1 as markers for insulin signaling is stimulated by vanadate plus insulin, and chromium does not enhance the effects. Vanadate robustly activates glucose uptake by 3T3-L1 adipocytes even without added insulin and increases insulin-stimulated glucose uptake. Chromium pretreatment of adipocytes slightly enhances glucose uptake in response to insulin, but significantly increases glucose uptake above that induced by insulin plus vanadate. These data show that chromium enhances glucose uptake even when Tyr phosphorylation levels are elevated by vanadate plus insulin, suggesting separate mechanisms of action for vanadate and chromium.     

Effects of high-fat diet and exercise training on intracellular glucose metabolism in rats

We examined the effects of high-fat diet (HFD) and exercise training on insulin-stimulated whole body glucose fluxes and several key steps of glucose metabolism in skeletal muscle. Rats were maintained for 3 wk on either low-fat (LFD) or high-fat diet with or without exercise training (swimming for 3 h per day). After the 3-wk diet/exercise treatments, animals underwent hyperinsulinemic euglycemic clamp experiments for measurements of insulin-stimulated whole body glucose fluxes. In addition, muscle samples were taken at the end of the clamps for measurements of glucose 6-phosphate (G-6-P) and GLUT-4 protein contents, hexokinase, and glycogen synthase (GS) activities. Insulin-stimulated glucose uptake was decreased by HFD and increased by exercise training (P < 0.01 for both). The opposite effects of HFD and exercise training on insulin-stimulated glucose uptake were associated with similar increases in muscle G-6-P levels (P < 0.05 for both). However, the increase in G-6-P level was accompanied by decreased GS activity without changes in GLUT-4 protein content and hexokinase activities in the HFD group. In contrast, the increase in G-6-P level in the exercise-trained group was accompanied by increased GLUT-4 protein content and hexokinase II (cytosolic) and GS activities. These results suggest that HFD and exercise training affect insulin sensitivity by acting predominantly on different steps of intracellular glucose metabolism. High-fat feeding appears to induce insulin resistance by affecting predominantly steps distal to G-6-P (e.g., glycolysis and glycogen synthesis). Exercise training affected multiple steps of glucose metabolism both proximal and distal to G-6-P. However, increased muscle G-6-P levels in the face of increased glucose metabolic fluxes suggest that the effect of exercise training is quantitatively more prominent on the steps proximal to G-6-P (i.e., glucose transport and phosphorylation).     

Divergent molecular mechanisms for insulin-resistant glucose transport in muscle and adipose cells in vivo

Glucose homeostasis depends on regulated changes in glucose transport in insulin-responsive tissues (e.g. muscle and adipose cells). This transport is mediated by at least two distinct glucose transporters: "adipose-muscle" and "erythrocyte-brain." To understand the molecular basis for in vivo insulin resistance we investigated the effects of fasting and refeeding on the expression of these two glucose transporters in adipose cells and skeletal muscle. In vivo insulin resistance seen with fasting and hyperresponsiveness seen with refeeding influence glucose transporter expression in a transporter-specific and tissue-specific manner. In adipose cells only the adipose-muscle glucose transporter mRNA and protein decrease dramatically with fasting and increase above control levels with refeeding, changes that parallel effects on insulin-stimulated glucose transport. In contrast, in muscle expression of both glucose transporters increase with fasting and return to control levels with refeeding, also in accordance with changes in glucose uptake in vitro. Although expression of the adipose-muscle glucose transporter predicts the physiological response at the tissue level, factors in the hormonal/metabolic milieu appear to override its increased expression in muscle resulting in insulin-resistant glucose uptake in this tissue in vivo.     

In vivo effects of vanadate on hepatic glycogen metabolizing and lipogenic enzymes in insulin-dependent and insulin-resistant diabetic animals

The insulin-mimetic action of vanadate is well established but the exact mechanism by which it exerts this effect is still not clearly understood. The role of insulin in the regulation of hepatic glycogen metabolizing and lipogenic enzymes is well known. In our study, we have, therefore, examined the effects of vanadate on these hepatic enzymes using four different models of diabetic and insulin-resistant animals. Vanadate normalized the blood glucose levels in all animal models. In streptozotocin-induced diabetic rats, the amount of liver glycogen and the activities of the active-form of glycogen synthase, both active and inactive-forms of phosphorylase, and lipogenic enzymes like glucose 6-phosphate dehydrogenase and malic enzyme were decreased and vanadate treatment normalized all of these to near normal levels. The other three animal models (db/db mouse, sucrose-fed rats and fa/fa obese Zucker rats) were characterized by hyperinsulinemia, hypertriglyceridemia, increases in activities of lipogenic enzymes, and marginal changes in glycogen metabolizing enzymes. Vanadate treatment brought all of these values towards normal levels. It should be noted that vanadate shows differential effects in the modulation of lipogenic enzymes activities in type I and type II diabetic animals. It increases the activities of lipogenic enzymes in streptozotocin-induced diabetic animals and prevents the elevation of activities of these enzymes in hyperinsulinemic animals. The insulin-stimulated phosphorylation of insulin receptor subunit and its tyrosine kinase activity was increased in streptozotocin-induced diabetic rats after treatment with vanadate. Our results support the view that insulin receptor is one of the sites involved in the insulin-mimetic actions of vanadate.     

Effects of vanadate and insulin on glucose 1,6-P2 and fructose 2,6-P2 levels in rat skeletal muscle

Levels of glucose 1,6-P2 but not fructose 2,6-P2 were found decreased in skeletal muscle of alloxan-diabetic ketotic rats. Administration of both insulin and vanadate restored the altered values without affecting fructose 2,6-P2 concentrations. In normal rats, insulin increased muscle levels of both sugars, and vanadate decreased glucose 1,6-P2 without changing fructose 2,6-P2 levels. Enzymatic activities involved in glucose 1,6-P2 and fructose 2,6-P2 metabolism were not affected under any experimental condition.     

Insulin-like effect of vanadate on malic enzyme and glucose-6-phosphate dehydrogenase activities in streptozotocin-induced diabetic rat liver

The effect of oral administration of sodium orthovanadate on hepatic malic enzyme (EC 1.1.1.40) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activities was investigated in nondiabetic and diabetic rats. Streptozotocin-induced diabetic rats were characterized by 4.7-fold increase in plasma glucose and 82% decrease in plasma insulin levels. The activities of hepatic malic enzyme and glucose-6-phosphate dehydrogenase were also diminished (P less than 0.001). Vanadate treatment in diabetic rats led to a significant decrease (P less than 0.001) in plasma glucose levels and to the normalization of enzyme activities, but it did not alter plasma insulin levels. In nondiabetic rats vanadate decreased the plasma insulin level by 64% without altering the enzyme activities. Significant correlation was observed between plasma insulin and hepatic lipogenic enzyme activities in untreated and vanadate-treated rats. Vanadate administration caused a shift to left in this correlation suggesting improvement in insulin sensitivity.     

Insulin-like and non-insulin-like action of vanadate in hepatocytes cultured in vitro

The aim of this study was to determine possible insulin-mimetic effects of vanadate on amino acid transport, glycogen synthesis and glucose production in hepatocytes cultured in vitro. Vanadate, like insulin, caused a significant (100%) increase of amino acid transport. However, vanadate produced a significant (31%) decrease of 14C-glucose incorporation into glycogen, in contrast to insulin which caused a 54% increase. Vanadate also caused a significant decrease (24%) of basal glucose production but had no effect on glucagon-stimulated glucose production. Vanadate and insulin given together reduced more strongly the basal glucose production (68%) and also effectively prevented the glucagon-stimulated effect on glucose production. In hepatocytes cultured in vitro vanadate had insulin-like and non-insulin-like action. Our results clearly demonstrated that non-insulin-like effects of vanadate could have a beneficial influence on the therapy of diabetes type 2 causing a decrease of glucose production from the liver.     

Different effects of IGF-I on insulin-stimulated glucose uptake in adipose tissue and skeletal muscle

The effect of insulin-like growth factor I (IGF-I) on insulin-stimulated glucose uptake was studied in adipose and muscle tissues of hypophysectomized female rats. IGF-I was given as a subcutaneous infusion via osmotic minipumps for 6 or 20 days. All hypophysectomized rats received L-thyroxine and cortisol replacement therapy. IGF-I treatment increased body weight gain but had no effect on serum glucose or free fatty acid levels. Serum insulin and C-peptide concentrations decreased. Basal and insulin-stimulated glucose incorporation into lipids was reduced in adipose tissue segments and isolated adipocytes from the IGF-I-treated rats. In contrast, insulin treatment of hypophysectomized rats for 7 days increased basal and insulin-stimulated glucose incorporation into lipids in isolated adipocytes. Pretreatment of isolated adipocytes in vitro with IGF-I increased basal and insulin-stimulated glucose incorporation into lipids. These results indicate that the effect of IGF-I on lipogenesis in adipose tissue is not direct but via decreased serum insulin levels, which reduce the capacity of adipocytes to metabolize glucose. Isoproterenol-stimulated lipolysis, but not basal lipolysis, was enhanced in adipocytes from IGF-I-treated animals. In the soleus muscle, the glycogen content and insulin-stimulated glucose incorporation into glycogen were increased in IGF-I-treated rats. In summary, IGF-I has opposite effects on glucose uptake in adipose tissue and skeletal muscle, findings which at least partly explain previous reports of reduced body fat mass, increased body cell mass, and increased insulin responsiveness after IGF-I treatment.     

Effect of vanadate on the glucose-1,6-bisphosphate synthase and glucose-1,6-bisphosphatase activities of phosphoglucomutase

Phosphoglucomutase, in addition to catalyzing the interconversion of glucose 1-P and glucose 6-P, catalyzes both the synthesis of glucose 1,6-P2 from glucose monophosphate and either fructose 1,6-P2 or glycerate 1,3-P2, and the hydrolysis of glucose 1,6-P2. Vanadate inhibits the mutase activity, activates the synthase activities, and does not affect the phosphatase activity. These effects suggest that the "exchange" step postulated for the phosphoglucomutase pathway is specifically inhibited by vanadate.     

Microdialysis and 2-[18F]fluoro-2-deoxy-D-glucose (FDG): a study on insulin action on FDG transport,uptake and metabolism in rat muscle,liver and adipose tissue

A combination of microdialysis (MD) and 2-[18F ]fluoro-2-deoxy-D-glucose (FDG) was used to assess FDG uptake, phosphorylation and the glucose metabolic index (Rg') in certain tissues of fed and fasting anesthetized Sprague-Dawley rats which received an i.v. bolus injection of insulin or saline during the course of the study. The relative recovery for FDG for the MD probes was also measured as a function of flow rate and temperature. The elimination half-life (T(1/2 FDG)) of FDG from the plasma and the extracellular fluid of muscle and liver was studied with MD. The phosphorylation of FDG in muscle, liver, subcutaneous fat and mesenteric fat from homogenates of these tissues was analyzed by a radioHPLC-method and the Rg' was calculated. The results show that the nutritional status does not affect the T(1/2 FDG), the total uptake of FDG 6-phosphate or the Rg' values in the studied tissues at ambient glucose. Insulin stimulation decreased T(1/2 FDG), and increased the total FDG 6-P accumulation and Rg' in the muscle of fed and fasted rats. In adipose tissues the insulin stimulation enhanced the phosphorylation but in muscle the proportion of FDG 6-P remained unchanged. Rg' in adipose tissue was higher after insulin administration in fed rats than without insulin but with fasted rats there were no differences in Rg' values with or without insulin, although the proportion of FDG 6-P did increase. The Rg' values for the livers were unaffected by any of the manipulations, but fasted rats accumulated proportionately more FDG 6-P after insulin administration than did fed rats. These results indicate that the combination of MD and FDG is a valuable and reliable tool when studying glucose metabolism in physiological and pathological models in vivo.     

Insulin and (or) dexamethasone effects on leptin production and metabolic activities of ovine adipose tissue explants

The in vitro effects of insulin and/or dexamethasone (DEX) on leptin production were studied on adipose tissue (AT) from adult non-lactating, non-pregnant ewes. Perirenal AT explants were incubated for 2 or 4 days and leptin production was determined using a specific ovine RIA. The effects of these hormones were simultaneously measured on glucose and acetate utilisation and on lipogenic enzyme activities. A preliminary dose-response study showed a maximal leptin production by the addition in the incubation medium of 2 mIU x mL(-1) of insulin and 100 nM of DEX. By using these concentrations, insulin or DEX increased leptin production by ovine AT explants whatever the incubation duration and the effects of these two hormones were additive. Insulin also increased substrate utilisation as well as lipogenic enzyme activities while DEX decreased substrate utilisation and did not change the lipogenic enzyme activities. To conclude, leptin response to DEX is specific and largely independent of the overall metabolic or lipogenic activity.     

PID1 regulates insulin-dependent glucose uptake by controlling intracellular sorting of GLUT4-storage vesicles

The phosphotyrosine interacting domain-containing protein 1 (PID1) serves as a cytosolic adaptor protein of the LDL receptor-related protein 1 (LRP1). By regulating its intracellular trafficking, PID1 controls the hepatic, LRP1-dependent clearance of pro-atherogenic lipoproteins. In adipose and muscle tissues, LRP1 is present in endosomal storage vesicles containing the insulin-responsive glucose transporter 4 (GLUT4). This prompted us to investigate whether PID1 modulates GLUT4 translocation and function via its interaction with the LRP1 cytosolic domain. We initially evaluated this in primary brown adipocytes as we observed an inverse correlation between brown adipose tissue glucose uptake and expression of LRP1 and PID1. Insulin stimulation in wild type brown adipocytes induced LRP1 and GLUT4 translocation from endosomal storage vesicles to the cell surface. Loss of PID1 expression in brown adipocytes prompted LRP1 and GLUT4 sorting to the plasma membrane independent of insulin signaling. When placed on a diabetogenic high fat diet, systemic and adipocyte-specific PID1-deficient mice presented with improved hyperglycemia and glucose tolerance as well as reduced basal plasma insulin levels compared to wild type control mice. Moreover, the improvements in glucose parameters associated with increased glucose uptake in adipose and muscle tissues from PID1-deficient mice. The data provide evidence that PID1 serves as an insulin-regulated retention adaptor protein controlling translocation of LRP1 in conjunction with GLUT4 to the plasma membrane of adipocytes. Notably, loss of PID1 corrects for insulin resistance-associated hyperglycemia emphasizing its pivotal role and therapeutic potential in the regulation of glucose homeostasis.     

Vanadate induces the recruitment of glut-4 glucose transporter to the plasma membrane of rat adipocytes

Summary In rat adipocytes, the insulin stimulation of the rate of glucose uptake is due, at least partially, to the recruitment of glucose transporter proteins from an intracellular compartment to the plasma membrane.Vanadate is a known insulin mimetic agent and causes an increase in the rate of glucose transport in rat adipocytes similar to that seen with insulin. The objective of the present study was to determine whether vanadate exerts its effect through the recruitment of glucose transporters to the plasma membrane.We report that under conditions where vanadate stimulates the rate of 2-deoxyglucose uptake to the same extent as insulin, the concentration of GLUT-4 in the plasma membrane was increased similarly by both insulin and vanadate, and its concentration was decreased in the low density microsomal fraction. These results suggest that vanadate induces the recruitment of GLUT-4 to the plasma membrane. The effects of vanadate and insulin on the stimulation of 2-deoxyglucose uptake and recruitment of GLUT-4 were not additive.This is the first report of an effect of vanadate on the intracellular distribution of the glucose transporter.     

Free fatty acids increase basal hepatic glucose production and induce hepatic insulin resistance at different sites

To investigate the sites of the free fatty acid (FFA) effects to increase basal hepatic glucose production and to impair hepatic insulin action, we performed 2-h and 7-h Intralipid + heparin (IH) and saline infusions in the basal fasting state and during hyperinsulinemic clamps in overnight-fasted rats. We measured endogenous glucose production (EGP), total glucose output (TGO, the flux through glucose-6-phosphatase), glucose cycling (GC, index of flux through glucokinase = TGO - EGP), hepatic glucose 6-phosphate (G-6-P) content, and hepatic glucose-6-phosphatase and glucokinase activities. Plasma FFA levels were elevated about threefold by IH. In the basal state, IH increased TGO, in vivo glucose-6-phosphatase activity (TGO/G-6-P), and EGP (P < 0.001). During the clamp compared with the basal experiments, 2-h insulin infusion increased GC and in vivo glucokinase activity (GC/TGO; P < 0.05) and suppressed EGP (P < 0.05) but failed to significantly affect TGO and in vivo glucose-6-phosphatase activity. IH decreased the ability of insulin to increase GC and in vivo glucokinase activity (P < 0.01), and at 7 h, it also decreased the ability of insulin to suppress EGP (P < 0.001). G-6-P content was comparable in all groups. In vivo glucose-6-phosphatase and glucokinase activities did not correspond to their in vitro activities as determined in liver tissue, suggesting that stable changes in enzyme activity were not responsible for the FFA effects. The data suggest that, in overnight-fasted rats, FFA increased basal EGP and induced hepatic insulin resistance at different sites. 1) FFA increased basal EGP through an increase in TGO and in vivo glucose-6-phosphatase activity, presumably due to a stimulatory allosteric effect of fatty acyl-CoA on glucose-6-phosphatase. 2) FFA induced hepatic insulin resistance (decreased the ability of insulin to suppress EGP) through an impairment of insulin's ability to increase GC and in vivo glucokinase activity, presumably due to an inhibitory allosteric effect of fatty acyl-CoA on glucokinase and/or an impairment in glucokinase translocation.     

Glucose uptake in vivo in skeletal muscles of insulin-injected chicks

Glucose uptake across the plasma membrane in animal cells plays a crucial role in whole-body glucose homeostasis. Insulin-stimulated glucose transport activity in vivo in several tissues was estimated using the 2-deoxy-D-[1-(3)H]glucose ([(3)H]2DG) uptake determination method. A tracer dose of [(3)H]2DG was injected intravenously into 8-day-old chicks (Gallus gallus) administered simultaneously or previously with porcine insulin (40 microg/kg BW). After 10 or 20 min, several major tissues, including skeletal and cardiac muscle, were sampled and their 2-deoxy-D-[1-(3)H]glucose 6-phosphate content analyzed. Plasma glucose concentration and [(3)H]2DG radioactivity were lowered by insulin within 20 min of [(3)H]2DG administration, while the plasma [(3)H]2DG/glucose ratio was not significantly different between chicks injected with insulin and their control counterparts. A marked uptake of 2DG was observed in cardiac tissue and brain, followed by kidney and skeletal muscles. In skeletal muscles, insulin increased the 2DG uptake in soleus, extensor digitorum longus and pectoralis superficialis muscles. On the other hand, no significant increases in insulin-induced 2DG uptake were detected in cardiac muscle or adipose tissue compared to controls. The results show that glucose transport across the plasma membrane in vivo in most skeletal muscles tested, but not cardiac muscle, was increased by insulin administration to chicks. These findings suggest that an insulin-responsive glucose transport mechanism is present in chickens, even though they intrinsically lack GLUT4 homologous gene, the insulin-responsive glucose transporter in mammals.     

In vitro glucose and 2-aminoisobutyric acid uptake by rat interscapular brown adipose tissue

The dependence upon substrate and insulin concentrations, as well as on sodium and potassium concentrations in the medium of the uptake of glucose and 2-aminoisobutyric acid, was determined for fragments of brown and white adipose tissues incubated in vitro. Brown adipose tissue showed a high capacity for glucose uptake at high glucose concentrations, this uptake being dependent on both glucose and insulin concentration. White adipose tissue showed much more limited uptake capabilities. The presence of Na+ and K+ had little effect on the uptake. The uptake of 2-aminoisobutyric acid was similar in both adipose tissues, being enhanced by physiological levels of insulin and depressed by ouabain. This amino acid transport was dependent on Na+ and K+ concentrations, and the overall transporting capability was two to three orders of magnitude lower than that for glucose. It was concluded that amino acids could not play a significant role as bulk thermogenic substrates for brown adipose tissue, as their transporters lack the plasticity of response to high substrate and insulin concentrations which characterize brown adipose tissue uptake of glucose.     

Insulin-like effects of vanadate on glucose uptake and on maturation in Xenopus laevis oocytes.

Vanadate, an inhibitor of phosphotyrosyl phosphatases that exerts insulin-like effects in intact cells, stimulated both maturation and glucose uptake in isolated Xenopus laevis oocytes. Vanadate enhanced the effects of insulin/IGF-I and progesterone on maturation in a dose-dependent manner, with an effective concentration of 750 microM and a maximum at 2 mM, whereas, in the absence of hormone, activation of maturation was seen at 10 mM vanadate. Further, vanadate at 2 mM increased glucose uptake, but this effect was not additive to that of the hormone. In cell-free systems, vanadate caused a 12-fold stimulation of autophosphorylation of the oocyte IGF-I receptor in the absence, but not in the presence, of IGF-I and inhibited largely, but not totally, receptor dephosphorylation induced by an extract of oocytes rich in phosphotyrosyl phosphatase activities. These effects were dose dependent, with effective concentrations of 50-100 microM and maxima at 2 mM. Moreover, using an acellular assay to study the effect of vanadate on the activation of maturation promoting factor (MPF), we found that vanadate at 2 mM stimulated the activation of the MPF H1 kinase. This suggests that vanadate did not prevent dephosphorylation of p34cdc2 on tyrosine residues. Vanadate thus exerted insulin-like effects in oocytes, including stimulation of maturation. These effects might result from a direct or indirect action of vanadate on the IGF-I receptor kinase and on MPF activity.     

Hexokinase II-deficient mice. Prenatal death of homozygotes without disturbances in glucose tolerance in heterozygotes.

Type 2 diabetes is characterized by decreased rates of insulin-stimulated glucose uptake and utilization, reduced hexokinase II mRNA and enzyme production, and low basal levels of glucose 6-phosphate in insulin-sensitive skeletal muscle and adipose tissues. Hexokinase II is primarily expressed in muscle and adipose tissues where it catalyzes the phosphorylation of glucose to glucose 6-phosphate, a possible rate-limiting step for glucose disposal. To investigate the role of hexokinase II in insulin action and in glucose homeostasis as well as in mouse development, we generated a hexokinase II knock-out mouse. Mice homozygous for hexokinase II deficiency (HKII(-/-)) died at approximately 7.5 days post-fertilization, indicating that hexokinase II is vital for mouse embryogenesis after implantation and before organogenesis. HKII(+/-) mice were viable, fertile, and grew normally. Surprisingly, even though HKII(+/-) mice had significantly reduced (by 50%) hexokinase II mRNA and activity levels in skeletal muscle, heart, and adipose tissue, they did not exhibit impaired insulin action or glucose tolerance even when challenged with a high-fat diet.     

Effects of vanadate on glucose production in cultured hepatocytes isolated from rats on high saturated fat diet

Insulin resistance is a common phenomenon in obesity and Type 2 diabetes. Common factor important for development of diabetes and insulin resistance is intake of saturated fat. Vanadate treatment improves glucose homeostasis in vivo. The aim of this study was to find out changing of hepatic glucose output in dependence of saturated fat diet and possible direct action of vanadate in cultured hepatocytes. Hepatocytes were isolated by a collagenase perfusion technique and cultured for 24 h in M 199 serum-free medium. The glucose production in hepatocytes isolated from rats on high saturated fat diet was significantly 139% higher comparable to standard controls. Glucagon 100% increased glucose production in hepatocytes from rats on standard diet and 200% in hepatocytes on saturated high fat diet. The addition vanadate significantly decreased basic glucose production and did not influence glucagon stimulated glucose production. Presence of insulin did not influence either glucagon or vanadate effect. High saturated fat diet not only increases insulin resistance but also decreases chances of successful therapy of diabetes.