Use of a novel impermeable biotinylated photolabeling reagent to assess insulin- and hypoxia-stimulated cell surface GLUT4 content in skeletal muscle from type 2 diabetic patients

Cell surface GLUT4 levels in skeletal muscle from nine type 2 diabetic subjects and nine healthy control subjects have been assessed by a new technique that involves the use of a biotinylated photo-affinity label. A profound impairment in GLUT4 translocation to the skeletal muscle cell surface in response to insulin was observed in type 2 diabetic patients. Levels of insulin-stimulated cell surface GLUT4 above basal in type 2 diabetic patients were only approximately 10% of those observed in healthy subjects. The magnitude of the defect in GLUT4 translocation in type 2 diabetic patients was greater than that observed for glucose transport activity, which was approximately 50% of that in healthy subjects. Reduced GLUT4 translocation is therefore a major contributor to the impaired glucose transport activity in skeletal muscle from type 2 diabetic subjects. When a marked impairment in GLUT4 translocation occurs, the contribution of other transporters to transport activity becomes apparent. In response to hypoxia, marked reductions in skeletal muscle cell surface GLUT4 levels were also observed in type 2 diabetic patients. Therefore, a defect in a common late stage in signal transduction and/or a direct impairment in the GLUT4 translocation process accounts for reduced glucose transport in type 2 diabetic patients.     

5-amino-imidazole carboxamide riboside increases glucose transport and cell-surface GLUT4 content in skeletal muscle from subjects with type 2 diabetes

AMP-activated protein kinase (AMPK) activation by AICAR (5-amino-imidazole carboxamide riboside) is correlated with increased glucose transport in rodent skeletal muscle via an insulin-independent pathway. We determined in vitro effects of insulin and/or AICAR exposure on glucose transport and cell-surface GLUT4 content in skeletal muscle from nondiabetic men and men with type 2 diabetes. AICAR increased glucose transport in a dose-dependent manner in healthy subjects. Insulin and AICAR increased glucose transport and cell-surface GLUT4 content to a similar extent in control subjects. In contrast, insulin- and AICAR-stimulated responses on glucose transport and cell-surface GLUT4 content were impaired in subjects with type 2 diabetes. Importantly, exposure of type 2 diabetic skeletal muscle to a combination of insulin and AICAR increased glucose transport and cell-surface GLUT4 content to levels achieved in control subjects. AICAR increased AMPK and acetyl-CoA carboxylase phosphorylation to a similar extent in skeletal muscle from subjects with type 2 diabetes and nondiabetic subjects. Our studies highlight the potential importance of AMPK-dependent pathways in the regulation of GLUT4 and glucose transport activity in insulin-resistant skeletal muscle. Activation of AMPK is an attractive strategy to enhance glucose transport through increased cell surface GLUT4 content in insulin-resistant skeletal muscle.     

Glucose and insulin chronically regulate insulin action via different mechanisms in BC3H1 myocytes. Effects on glucose transporter gene expression.

We previously reported that, in primary cultured adipocytes, chronic exposure to glucose plus insulin impairs the insulin-responsive glucose transport system. In this study, we examined regulation of glucose transport in BC3H1 myocytes as a model for muscle and found important differences between BC3H1 cells and adipocytes. In myocytes, chronic glucose exposure per se (25 mM) decreased basal glucose transport activity by 78% and insulin's acute ability to maximally stimulate transport by 68% (ED50 approximately 2.5 mM; T1/2 approximately 4 h). D-Mannose and 3-O-methyl-glucose diminished transport rates with approximately 100 and 50% of the potency of D-glucose, respectively, whereas L-glucose, D-fructose, and D-galactose were inactive. Chronic glucose exposure also reduced cell surface insulin binding by 30% via an apparent decrease in receptor affinity, and this effect was associated with a comparable rightward shift in the insulin-glucose transport dose-response curve. In other studies, persistent stimulation with 15 nM insulin also decreased maximally stimulated glucose transport activity, which was independent and additive to the regulatory effect of glucose. Moreover, glucose and insulin-induced insulin resistance via different mechanisms. Glucose (25 mM) reduced the number of cellular glucose transporter proteins by 84% and levels of GLUT1 transporter mRNA by 50% (whether normalized to total RNA or CHO-B mRNA). In contrast, chronic insulin exposure led to a 2.1-fold increase in GLUT1 mRNA but did not alter cellular levels of transporter protein. Cotreatment with glucose prevented the insulin-induced rise in GLUT1 mRNA. BC3H1 cells did not express GLUT4 mRNA that encodes the major transporter isoform in skeletal muscle. In conclusion, in BC3H1 myocytes 1) glucose diminished insulin sensitivity by decreasing insulin receptor binding affinity and decreased basal and maximally insulin-stimulated glucose transport rates via cellular depletion of glucose transporters and suppression of GLUT1 mRNA; 2) chronic insulin exposure exerted an independent and additive effect to reduce maximal transport activity; however, insulin increased levels of GLUT1 mRNA and did not alter the cellular content of glucose transporters; and 3) although BC3H1 cells are commonly used as a model for skeletal muscle, studies examining glucose transport should be interpreted cautiously due to the absence of GLUT4 expression. Nevertheless, the data generally support the idea that, in non-insulin-dependent diabetes mellitus, hyperglycemia and hyperinsulinemia can induce or exacerbate insulin resistance in target tissues.     

Insulin induces the translocation of GLUT4 from a unique intracellular organelle to transverse tubules in rat skeletal muscle.

Skeletal muscle surface membrane is constituted by the PM domain and its specialized deep invaginations known as TTs. We have shown previously that insulin induces a rapid translocation of GLUT4s from an IM pool to the PM in rat skeletal muscle (6). In this study, we have investigated the possibility that insulin also stimulates the translocation of GLUT4 proteins to TTs, which constitute the largest area of the cell surface envelope. PM, TTs, and IM components of control and insulinized skeletal muscle were isolated by subcellular fractionation. The TTs then were purified further by removing vesicles of SR origin by using a Ca-loading procedure. Ca-loading resulted in a five- to sevenfold increase in the purification of TTs in the unloaded fraction relative to the loaded fraction, assessed by immunoblotting with an anti-DHP-receptor monoclonal antibody. In contrast, estimation of the content of Ca(2+)-ATPase protein (a marker of SR) with a specific polyclonal antibody revealed that most, if not all, SR vesicles were recovered in the Ca-loaded fraction. Western blotting with an anti-COOH-terminal GLUT4 protein polyclonal antibody revealed that acute insulin injection in vivo (30 min) increased the content of GLUT4 (by 90%) in isolated PMs and markedly enhanced (by 180%) GLUT4 content in purified TTs. Importantly, these insulin-dependent changes in GLUT4 content of PM and purified TTs were seen in the absence of changes in the alpha 1-subunit of the Na(+)-K(+)-ATPase, a surface membrane marker.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skeletal muscle of stroke-prone spontaneously hypertensive rats exhibits reduced insulin-stimulated glucose transport and elevated levels of caveolin and flotillin

Insulin resistance is of major pathogenic importance in several common human disorders, but the underlying mechanisms are unknown. The stroke-prone spontaneously hypertensive (SHRSP) rat is a model of human insulin resistance and is characterized by reduced insulin-mediated glucose disposal and defective fatty acid metabolism in isolated adipocytes (Collison et al. [Diabetes 49:2222-2226, 2000]). In this study, we have examined skeletal muscle and cultured skeletal muscle myoblasts for defects in insulin action in the male SHRSP rat model compared with the normotensive, insulin-sensitive control strain, Wistar-Kyoto (WKY). We show that skeletal muscle from SHRSP animals exhibits a marked decrease in insulin-stimulated glucose transport compared with WKY animals (fold increase in response to insulin: 1.4 +/- 0.15 in SHRSP, 2.29 +/- 0.22 in WKY; n = 4, P = 0.02), but the stimulation of glucose transport in response to activation of AMP-activated protein kinase was similar between the two strains. Similar reductions in insulin-stimulated glucose transport were also evident in myoblast cultures from SHRSP compared with WKY cultures. These differences were not accounted for by a reduction in cellular GLUT4 content. Moreover, analysis of the levels and subcellular distribution of insulin receptor substrates 1 and 2, the p85alpha subunit of phosphatidylinositol 3'-kinase, and protein kinase B (PKB)/cAKT in skeletal muscle did not identify any differences between the two strains; the insulin-dependent activation of PKB/cAKT was not different between the two strains. However, the total cellular levels of caveolin and flotillin, proteins implicated in insulin signal transduction/compartmentalization, were markedly elevated in skeletal muscles from SHRSP compared with WKY animals. Increased cellular levels of the soluble N-ethylmaleimide attachment protein receptor (SNARE) proteins syntaxin 4 and vesicle-associated membrane protein (VAMP)-2 were also observed in the insulin-resistant SHRSP strain. Taken together, these data suggest that the insulin resistance observed in the SHRSP is manifest at the level of skeletal muscle, that muscle cell glucose transport exhibits a blunted response to insulin but unchanged responses to activation of AMP-activated protein kinase, that alterations in key molecules in both GLUT4 trafficking and insulin signal compartmentalization may underlie these defects in insulin action, and that the insulin resistance of these muscles appears to be of genetic origin rather than a paracrine or autocrine effect, since the insulin resistance is also observed in cultured myoblasts over several passages.     

Tyrosine phosphorylation of specific protein kinase C isoenzymes participates in insulin stimulation of glucose transport in primary cultures of rat skeletal muscle.

Several reports indicate that protein kinase C (PKC) plays a role in insulin-induced glucose transport in certain cells. The precise effects of insulin on specific PKC isoforms are as yet unknown. Utilizing primary cultures of rat skeletal muscle, we investigated the possibility that insulin may influence the activation state of PKC isoenzymes by inducing their translocation and tyrosine phosphorylation. This, in turn, may mediate insulin effects on glucose transport. We identified and determined the glucose transporters and PKC isoforms affected by insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA). Insulin and TPA each caused an increase in glucose uptake. Insulin translocated GLUT3 and GLUT4 without affecting GLUT1. In contrast, TPA translocated GLUT1 and GLUT3 without affecting GLUT4. Insulin translocated and tyrosine phosphorylated and activated PKC-beta2 and -zeta; these effects were blocked by phosphatidylinositol 3-kinase (PI3K) inhibitors. TPA translocated and activated PKC-alpha, -beta2, and -delta; these effects were not noticeably affected by PI3K inhibitors. Furthermore, wortmannin significantly inhibited both insulin and TPA effects on GLUT translocation and glucose uptake. Finally, insulin-induced glucose transport was blocked by the specific PKC-beta2 inhibitor LY379196. These results indicate that specific PKC isoenzymes, when tyrosine-phosphorylated, are implicated in insulin-induced glucose transport in primary cultures of skeletal muscle.     

Platelet-derived growth factor stimulates glucose transport in skeletal muscles of transgenic mice specifically expressing platelet-derived growth factor receptor in the muscle, but it does not affect blood glucose levels

Insulin stimulates the disposal of blood glucose into skeletal muscle and adipose tissues by the translocation of GLUT4 from intracellular pools to the plasma membrane, and consequently the concentration of blood glucose levels decreases rapidly in vivo. Phosphatidylinositol (PI) 3-kinase and Akt play a pivotal role in the stimulation of glucose transport by insulin, but detailed mechanisms are unknown. We and others reported that not only insulin but also platelet-derived growth factor (PDGF) and epidermal growth factor facilitate glucose uptake through GLUT4 translocation by activation of PI 3-kinase and Akt in cultured cells. However, opposite results were also reported. We generated transgenic mice that specifically express the PDGF receptor in skeletal muscle. In these mice, PDGF stimulated glucose transport into skeletal muscle in vitro and in vivo. Thus, PDGF apparently shares with insulin some of the signaling molecules needed for the stimulation of glucose transport. The degree of glucose uptake in vivo reached approximately 60% of that by insulin injection in skeletal muscle, but blood glucose levels were not decreased by PDGF in these mice. Therefore, PDGF-induced disposal of blood glucose into skeletal muscle is insufficient for rapid decrease of blood glucose levels.     

Reversal of insulin resistance in diabetic rat adipocytes by insulin therapy. Restoration of pool of glucose transporters and enhancement of glucose-transport activity

To determine the role of insulin in reversing the insulin resistance associated with depletion of the intracellular pool of glucose transporters, streptozocin-induced diabetic rats were treated with 5 U/day s.c. of insulin for 0, 8, or 14 days. At each time point, adipose cells were isolated, and 3-O-methylglucose transport was measured in the absence and presence of 1000 microU/ml insulin. With the cytochalasin B-binding assay, concentrations of glucose transporters in the plasma and the low-density microsomal membrane fractions were determined. Eight-day insulin therapy enhanced glucose transport rate (mean +/- SE) from 0.2 +/- 0.0 to 1.1 +/- 0.1 fmol X cell-1 X min-1 in the basal state and from 0.8 +/- 0.1 to 5.5 +/- 0.4 fmol X cell-1 X min-1 in the insulin-stimulated state in untreated and treated diabetic rats, respectively; this is a 3-fold increment of glucose transport rate in both states compared with control rats. After 14-day insulin therapy, glucose-transport activity declined toward normal but still remained approximately 1.5- and 4-fold higher than control and diabetic rats, respectively. Despite the persistent enhancement of glucose transport rate, concentration of glucose transporters in the intracellular pool was restored only to its prediabetic state. Likewise, the increased concentration of glucose transporters in the plasma membranes after insulin stimulation was similar to that of control rats. Thus, we suggest that 8-14 days of insulin therapy reversed the insulin resistance in diabetic rat adipocytes by at least two mechanisms: restoration of the intracellular pool of glucose transporters and enhancement of glucose-transport activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence against altered expression of GLUT1 or GLUT4 in skeletal muscle of patients with obesity or NIDDM

Studies of experimental diabetes in rodents induced by the beta-cell toxin streptozocin have shown that the insulin-resistant glucose transport of peripheral tissues (muscle and adipose) in these animals can be ascribed in part to a pretranslational reduction of the major insulin-sensitive glucose transporter (GLUT4) in these tissues. Because a central feature of non-insulin-dependent diabetes mellitus (NIDDM) is an imparied ability of insulin to enhance glucose disposal in skeletal muscle, we examined the hypothesis that reduced expression of GLUT4 is a characteristic finding in the skeletal muscle of subjects with NIDDM. Biopsies of skeletal muscles were obtained from 17 patients with NIDDM and 10 lean and 9 obese nondiabetic subjects. Among the diabetic subjects, 7 were newly diagnosed and untreated. Compared with age-matched and body-weight-matched healthy control subjects, there was no significant alteration in the level of GLUT4 mRNA demonstrated by Northern blot and slot blot or GLUT4 protein determined by immunoblotting muscle membranes. Neither GLUT4 mRNA nor protein concentration correlated with the degree of glycemic control, fasting plasma insulin or glucose, diabetes duration, body mass index, sex, or age. GLUT1 mRNA and protein levels were also not significantly different between diabetic and matched control subjects. Thus, unlike streptozocin-induced diabetes in rodents, there is no evidence that impaired expression of the major insulin-responsive glucose transporter is responsible for insulin-resistant glucose transport in the skeletal muscle of these lean and moderately obese NIDDM patients.     

Role of glucose transporters in glucocorticoid-induced insulin resistance. GLUT4 isoform in rat skeletal muscle is not decreased by dexamethasone.

The diabetogenic effects of glucocorticoid excess are due in part to peripheral resistance to insulin. To test the hypothesis that glucocorticoid-induced peripheral insulin resistance might be attributable to a decreased number of glucose transporters, we examined the effects of dexamethasone treatment on the expression of the GLUT4 (insulin regulatable) glucose transporter in skeletal muscle, the major site of insulin-mediated glucose uptake. Dexamethasone treatment of rats (1 mg/day for 1 wk) induced hyperglycemia and hyperinsulinemia. At dosages of either 0.1 or 1 mg/day, insulin-stimulated 2-deoxyglucose uptake in isolated soleus muscle was reduced by greater than or equal to 50%, demonstrating the presence of insulin resistance in skeletal muscle. Immunoblots of crude membranes from deep quadriceps muscle showed that dexamethasone treatment (1 mg/day) increased the amount of GLUT4 protein by 84%. GLUT4 mRNA abundance was similarly increased when expressed per unit RNA but was unchanged when expressed on a DNA basis because the tissue RNA content was decreased by dexamethasone. In contrast to quadriceps, GLUT4 protein concentration in soleus and extensor digitorum longus extracts was not significantly increased by dexamethasone treatment. Because glucocorticoids cause selective atrophy of type IIb muscle fibers, which express relatively less GLUT4 protein, the apparent increase in GLUT4 content in quadriceps muscle from dexamethasone-treated animals may have resulted from inadvertent increased sampling of GLUT4-enriched type I and IIA fibers, caused by a glucocorticoid-induced decrease in the relative mass of the GLUT4-poor type IIb fibers. We conclude that glucocorticoids do not decrease GLUT4 content in skeletal muscle and that glucocorticoid-induced insulin resistance in this tissue is not due to suppression of glucose transporter gene expression.     

In vitro effects of sulfonylurea on glucose transport and translocation of glucose transporters in adipocytes from streptozocin-induced diabetic rats

The in vitro effects of the sulfonylurea glyburide on insulin binding and action were compared in adipocytes from control and nonketotic streptozocin-induced diabetic rats. Adipose tissue from control and diabetic animals was maintained in the absence or presence of 2 micrograms/ml glyburide for 20 h. Insulin binding and insulin-stimulated glucose transport were examined in adipocytes prepared from this tissue. As expected, insulin binding was increased in adipocytes from diabetic animals. Exposure of tissue to glyburide did not influence insulin binding in either control or diabetic cells. Glucose transport activity of diabetic cells, assessed with 2-deoxyglucose, was decreased 30-40% in both the absence (basal) and presence of insulin compared with controls. Glyburide potentiated insulin's effects in both control (15-20%) and diabetic (30-40%) adipocytes. As a result, glucose transport activity in glyburide-treated diabetic cells was restored to a level similar to that of control cells not exposed to the drug. The mechanism by which glyburide potentiated glucose transport activity was examined with the D-glucose-displaceable cytochalasin B-binding technique to measure glucose-transporter concentration in membranes prepared from control and diabetic adipocytes exposed to the drug. Adipocytes from this model of diabetes are known to have a decreased cellular content of glucose transporters. The concentration of glucose transporters was decreased by 31% in plasma membranes from insulin-treated diabetic cells. There were corresponding decreases in diabetic microsomal and total membrane fractions. There was also a 40% decrease in the translocation of transporters from the microsomes to the plasma membrane in response to insulin in diabetic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

GLUT4 overexpression in db/db mice dose-dependently ameliorates diabetes but is not a lifelong cure

We previously reported that overexpression of GLUT4 in lean, nondiabetic C57BL/KsJ-lepr(db/+) (db/+) mice resulted in improved glucose tolerance associated with increased basal and insulin-stimulated glucose transport in isolated skeletal muscle. We used the diabetic (db/db) litter mates of these mice to examine the effects of GLUT4 overexpression on in vivo glucose utilization and on in vitro glucose transport and GLUT4 translocation in diabetic mice. We examined in vivo glucose disposal by oral glucose challenge and hyperinsulinemic-hyperglycemic clamps. We also evaluated the in vitro relationship between glucose transport activity and cell surface GLUT4 levels as assessed by photolabeling with the membrane-impermeant reagent 2-N-(4-(1-azi-2,2,2-trifluoroethyl)benzoyl)-1,3-bis(D-mannose-4-yloxy)-2-propylamine in extensor digitorum longus (EDL) muscles. All parameters were examined as functions of animal age and the level of GLUT4 overexpression. In young mice (age 10-12 weeks), both lower (two- to threefold) and higher (four- to fivefold) levels of GLUT4 overexpression were associated with improved glucose tolerance compared to age-matched nontransgenic (NTG) mice. However, glucose tolerance deteriorated with age in db/db mice, although less rapidly in transgenic mice expressing the higher level of GLUT4. Glucose infusion rates during hyperinsulinemic-hyperglycemic clamps were increased with GLUT4 overexpression, compared with NTG mice in both lower and higher levels of GLUT4 overexpression, even in the older mice. Surprisingly, isolated EDL muscles from diabetic db/db mice did not exhibit alterations in either basal or insulin-stimulated glucose transport activity or cell surface GLUT4 compared to nondiabetic db/+ mice. Furthermore, both GLUT4 overexpression levels and animal age are associated with increased basal and insulin-stimulated glucose transport activities and cell surface GLUT4. However, the observed increased glucose transport activity in older db/db mice was not accompanied by an equivalent increase in cell surface GLUT4 compared to younger animals. Thus, although in vivo glucose tolerance is improved with GLUT4 overexpression in young animals, it deteriorates with age; in contrast, insulin responsiveness as assessed by the clamp technique remains improved with GLUT4 overexpression, as does in vitro insulin action. In summary, despite an impairment in whole-body glucose tolerance, skeletal muscle of the old transgenic GLUT4 db/db mice is still insulin responsive in vitro and in vivo.     

Characterization of glucose uptake by cultured rat podocytes

The nonmetabolizable glucose analogue [(3)H]-2-deoxy-D-glucose ((3)H-2DG) was used to study glucose transport in cultured rat podocytes. Intracellular accumulation of (3)H-2DG was linear up to 20 min and was inhibited by cytochalasin B (80% inhibition) and by phlorizin (20% inhibition). Pretreatment with insulin stimulated the (3)H-2DG uptake 1.5-fold. A Hill analysis of the rate of glucose transport yielded a V(max) value of approximately 10 mM and S(0.5)of 7.8 mM. The value h = 1.0 for a Hill coefficient confirmed that glucose uptake exhibited a Michaelis-Menten kinetics. Transporters GLUT2 and GLUT4 were expressed in over 90% podocytes. Of the GLUT2- and GLUT4-expressing cells, approximately one-fourth expressed the membrane-bound fraction. We conclude that cultured rat podocytes possess a differentiated glucose transport system consisting chiefly of facilitative GLUT2 and GLUT4 transporters. It seems likely that a sodium-dependent glucose cotransporter may also be present in these cells.     

Muscle fiber type-specific defects in insulin signal transduction to glucose transport in diabetic GK rats

To determine whether defects in the insulin signal transduction pathway to glucose transport occur in a muscle fiber type-specific manner, post-receptor insulin-signaling events were assessed in oxidative (soleus) and glycolytic (extensor digitorum longus [EDL]) skeletal muscle from Wistar or diabetic GK rats. In soleus muscle from GK rats, maximal insulin-stimulated (120 nmol/l) glucose transport was significantly decreased, compared with that of Wistar rats. In EDL muscle from GK rats, maximal insulin-stimulated glucose transport was normal, while the submaximal response was reduced compared with that of Wistar rats. We next treated diabetic GK rats with phlorizin for 4 weeks to determine whether restoration of glycemia would lead to improved insulin signal transduction. Phlorizin treatment of GK rats resulted in full restoration of insulin-stimulated glucose transport in soleus and EDL muscle. In soleus muscle from GK rats, submaximal and maximal insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation and IRS-1-associated phosphatidylinositol (PI) 3-kinase activity were markedly reduced, compared with that of Wistar rats, but only submaximal insulin-stimulated PI 3-kinase was restored after phlorizin treatment. In EDL muscle, insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI-3 kinase were not altered between GK and Wistar rats. Maximal insulin-stimulated Akt (protein kinase B) kinase activity is decreased in soleus muscle from GK rats and restored upon normalization of glycemia (Krook et al., Diabetes 46:2100-2114, 1997). Here, we show that in EDL muscle from GK rats, maximal insulin-stimulated Akt kinase activity is also impaired and restored to Wistar rat levels after phlorizin treatment. In conclusion, functional defects in IRS-1 and PI 3-kinase in skeletal muscle from diabetic GK rats are fiber-type-specific, with alterations observed in oxidative, but not glycolytic, muscle. Furthermore, regardless of muscle fiber type, downstream steps to PI 3-kinase (i.e., Akt and glucose transport) are sensitive to changes in the level of glycemia.     

Immunolocalization of glucose transporter GLUT4 within human skeletal muscle

To investigate the cellular and subcellular distribution of glucose transporters in skeletal muscle, the glucose transporter isoform GLUT4 was localized in human muscle by electron microscopy via immunogold labeling with monoclonal (1F8) or COOH-terminal peptide polyclonal (ECU4) antibody and in isolated rat membranes by Western blot. There was no labeling of GLUT4 in endothelial cells of the capillaries. There also was no labeling of GLUT4 on the surface plasma membrane (sarcolemma) under either basal or insulin-stimulated conditions. Specific labeling for GLUT4 was clearly observed in two compartments: within the triad (on terminal cisternae and transverse tubules) and on an intracellular compartment, possibly sarcoplasmic tubules. Isolated triad membranes from rat muscle also contained substantial quantities of GLUT4 transporter, but there was no detectable GLUT4 protein in isolated sarcolemmal membranes. These data suggest a possible mechanism that involves glucose transport across the muscle cell at the transverse tubule membrane, not the sarcolemma.     

Does hyperoxia affect glucose regulation and transport in the newborn?

OBJECTIVE: Hyperglycemia has been found to occur in children placed on cardiopulmonary bypass. Our laboratory demonstrated that hyperoxia plays a role in this hyperglycemic response and also occurs in the absence of cardiopulmonary bypass. The purpose of this study was to elucidate potential mechanisms underlying the hyperoxic-induced hyperglycemia by examining glucagon, insulin, and epinephrine, which are important in glucose regulation and skeletal and cardiac glucose transporters (GLUT1 and GLUT4), which facilitate glucose entry. METHODS: Three-day-old piglets were anesthetized, intubated, and ventilated to normoxia. Animals were then randomly allocated to either 5 hours of normoxia (n = 4) or hyperoxia (n = 6). Measurements of oxygen, blood glucose, plasma glucagon, insulin, and epinephrine levels were made. Total GLUT1 and GLUT4 content in cardiac and skeletal muscle was measured using Western blotting analysis. RESULTS: A sustained hyperglycemic response (P <.001) was seen throughout the 5-hour ventilatory period. A significant twofold elevation in glucagon levels (P <.001) and a threefold elevation (P <.003) in plasma insulin levels occurred, despite no significant changes in plasma epinephrine. Total GLUT1 and GLUT4 content were significantly reduced in skeletal muscle by 66% and 59%, respectively, while no significant changes occurred in cardiac muscle. CONCLUSION: This study demonstrates that significant elevations in glucagon and insulin and reductions in total skeletal muscle GLUT1 and GLUT4 content all contribute to hyperoxia-induced hyperglycemia seen in newborns. To optimize postoperative recovery of newborns, consideration should be given to the levels of oxygen used to avoid the potential development of insulin resistance and subsequent decrease in glucose entry.     

Glucose transporter levels in tissues of spontaneously diabetic Zucker fa/fa rat (ZDF/drt) and viable yellow mouse (Avy/a).

We used antibodies to the fat/muscle glucose transporter (GLUT4) and the liver glucose transporter (GLUT2) to measure levels of these proteins in various tissues of two rodent models of non-insulin-dependent (type II) diabetes mellitus: the obese spontaneously diabetic male Zucker fa/fa rat (ZDF/drt) and the male viable yellow Avy/a obese diabetic mouse. The ZDF/drt strain generally develops overt diabetes associated with decreased plasma insulin levels. Depending on the age of the animals, the ZDF/drt rats can be arbitrarily segregated into age-matched obese, mildly diabetic (blood glucose less than 11 mM) and obese, and severely diabetic (blood glucose greater than 20 mM) groups. Avy/a mice are comparably hyperglycemic but unlike the ZDF/drt rats are severely hyperinsulinemic. In both groups of diabetic animals, GLUT4 in adipose tissue, heart, and skeletal muscle was reduced 25-55%, and GLUT2 in liver was increased 30-40%, relative to lean, age-matched controls. However, when the mildly diabetic ZDF/drt rats were compared to the lean controls, the only significant difference was a 25% reduction of GLUT4 in heart. Within all of the ZDF/drt rats (excluding the lean controls), GLUT2 in liver and GLUT4 in adipose tissue, heart, and skeletal muscle correlated significantly with glycemia. These data suggest that, in these two models of type II diabetes, glucose transporter levels in muscle, adipose tissue, and liver are regulated in a tissue-selective manner in response to changes in insulin and glucose. Furthermore, at least in the ZDF/drt rat, alterations in GLUT2 and/or GLUT4 protein levels appear not to be associated with obesity per se but appear to be secondary to the severely diabetic state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opposite effects of acute hypoglycemia and acute hyperglycemia on glucose transport and glucose transporters in perfused rat skeletal muscle.

This study was undertaken to characterize the effects of glycemia per se (glucose effectiveness) on muscle glucose transport. Isolated rat hindlimbs were perfused in situ for 2 h with perfusate containing either low (2 mmol/l, n = 7), normal (6.5 mmol/l, n = 6), or high (20 mmol/l, n = 6) concentrations of glucose, without insulin, to simulate hypo-, eu-, and hyperglycemic conditions. The effect of varying glucose concentrations on muscle glucose transport was assessed by an ensuing 30-min perfusion with 5.5 mmol/l glucose perfusate without insulin. The 2-h of low glucose perfusion induced significant increases in both muscle glucose clearance (approximately 2.3-fold, P < 0.01) and plasma membrane GLUT4 content (approximately 20%, P < 0.05) relative to normal. In contrast, high glucose perfusion decreased glucose clearance (approximately 1.7-fold, P < 0.01) and plasma membrane GLUT4 content (approximately 20%, P < 0.05). Glucose extraction during the following 30-min perfusion was 2.5-fold greater (P < 0.0001) in the low group and threefold less (P < 0.0001) in the high group, relative to normal. 2-[3H]deoxyglucose-6-phosphate content in both red (soleus) and white (extensor digitorum longus) muscles increased approximately twofold after 2 h of low glucose perfusion (P < 0.0001) and decreased > or =2-fold after high glucose perfusion (P < 0.0001), relative to normal. It is concluded that glycemia regulates glucose transport in skeletal muscle independently of insulin, achieved at least partially via changes in plasma membrane GLUT4. We propose that high glucose levels can acutely downregulate GLUT4 and glucose clearance, thus limiting excessive glucose uptake in muscle. Conversely, low glucose-induced upregulation of muscle glucose clearance and GLUT4 can compensate for reduced glucose availability in the circulation.     

Human adenovirus type 36 enhances glucose uptake in diabetic and nondiabetic human skeletal muscle cells independent of insulin signaling

OBJECTIVE—Human adenovirus type 36 (Ad-36) increases adiposity but improves insulin sensitivity in experimentally infected animals. We determined the ability of Ad-36 to increase glucose uptake by human primary skeletal muscle (HSKM) cells.RESEARCH DESIGN AND METHODS—The effect of Ad-36 on glucose uptake and cell signaling was determined in HSKM cells obtained from type 2 diabetic and healthy lean subjects. Ad-2, another human adenovirus, was used as a negative control. Gene expression and proteins of GLUT1 and GLUT4 were measured by real-time PCR and Western blotting. Role of insulin and Ras signaling pathways was determined in Ad-36–infected HSKM cells.RESULTS—Ad-36 and Ad-2 infections were confirmed by the presence of respective viral mRNA and protein expressions. In a dose-dependent manner, Ad-36 significantly increased glucose uptake in diabetic and nondiabetic HSKM cells. Ad-36 increased gene expression and protein abundance of GLUT1 and GLUT4, GLUT4 translocation to plasma membrane, and phosphatidylinositol 3-kinase (PI 3-kinase) activity in an insulin-independent manner. In fact, Ad-36 decreased insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and IRS-1–and IRS-2–associated PI 3-kinase activities. On the other hand, Ad-36 increased Ras gene expression and protein abundance, and Ras siRNA abrogated Ad-36–induced PI 3-kinase activation, GLUT4 protein abundance, and glucose uptake. These effects were not observed with Ad-2 infection.CONCLUSIONS—Ad-36 infection increases glucose uptake in HSKM cells via Ras-activated PI 3-kinase pathway in an insulin-independent manner. These findings may provide impetus to exploit the role of Ad-36 proteins as novel therapeutic targets for improving glucose handling.Increasing prevalence of type 2 diabetes and insulin resistance is a major health and economic concern (1,2) and necessitates more effective prevention and treatment strategies. Intense search for identifying novel agents that may provide therapeutic targets for better management of diabetes is underway (3,4). Human adenovirus Ad-36 is such a novel candidate that increases adiposity but enhances insulin sensitivity in experimentally infected rats (5), an effect that is robust and reminiscent of the thiozolinediones (6,7). After a single inoculation of Ad-36, fat depot weight increased by >60%, but the fasting insulin levels and homeostasis model assessment (HOMA) index were ∼50% lower in rats up to 7 months later (5). Therefore, we postulated that Ad-36 increases glucose uptake in infected tissue, which may contribute in enhancing whole-body insulin sensitivity.This study investigated the ability of Ad-36 to enhance glucose uptake by skeletal muscle. Skeletal muscle is the largest organ of the human body and is a major site of glucose disposal and insulin action (8). Therefore, exploiting the ability of Ad-36 to enhance glucose uptake by skeletal muscle may provide a novel therapeutic target to treat glycemic disregulation in humans.In a stepwise approach, we investigated how Ad-36 influences the biomarkers of insulin sensitivity and glucose uptake. First, we determined whether Ad-36 increases glucose uptake in primary skeletal muscle cells from healthy lean and diabetic subjects. Next, the effect of Ad-36 on glucose transporters and their upstream cellular signaling, including phosphatidylinositol 3-kinase (PI 3-kinase) and its activators, was determined. Adenovirus type 2, a human adenovirus that is not adipogenic in animals (9), was used as a negative control. The following experiments showed that Ad-36 activates PI 3-kinase and increases glucose uptake in nondiabetic and diabetic human skeletal muscle (HSKM) cells. Activation of PI 3-kinase by Ad-36 requires Ras signaling but not insulin signaling pathway.