Inhibitory effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport and On 125I-labelled insulin binding by rat soleus muscle

These experiments examined the effects of N-ethylmaleimide on insullin- and oxidant-stimulated sugar transport in soleus muscle in terms of the Thiol-Redox model for insulin-stimulated adipocyte sugar transport (Czech, M.P. (1976) J. Cell. Physiol. 89, 661–668). Brief exposure (1 min) to N-ethylmaleimide (0.3?10 nM) inhibited the stimulatory effect of insulin (0.1 U/ml) on D-[U-¹⁴C]xylose uptake by rat soleus muscle. N-Ethylmaleimide also inhibited the stimulatory effects of H₂O₂ (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin was paralleled by the inhibition of ¹²⁵I-labelled insulin binding by the muscle. N-ethylmaleimide lowered muscle ATP; however, its effects on sugar transport and ¹²⁵I-labelled insulin binding could be dissociated from its effect on ATP. Exposing muscles to insulin prior to N-ethylmaleimide did not abolish the inhibitory effect of sulphydryl blockae on insulin-stimulated sugar transport, but did reduce the effect of the inhibitor by 20–30%. Conversely, when muscles were first allowed to bind ¹²⁵I-labelled insulin and then exposed to the inhibitor, there was no effect of N-ethylmaleimide on pre-bound insulin. Exposure to diamide or vitamin K-5 before N-ethylmaleimide (1 mM) attenuated the inhibitory effet of sulphydryl blockade but no protective effect was observed with H₂O₂. None of the oxidants protected against the inhibitory effect of 3 nM N-ethylmaleimide. It is concluded that there are two N-ethylmaleimide-sensitive sites involved in the activation of muscle sugar transport at the post-receptor level. One of these would appear to be similar to the Thiol-Redox site described in the adipocyte; the other site appears to be an essential sulphydryl group whose function does not involve oxidation to a disulphide.     

Multiple effects of sulphydryl reagents on sugar transport by rat soleus muscle

Iodoacetate, over the range 0.2-2 mM, stimulated the uptake of D-xylose by rat soleus muscle and inhibited anaerobic lactate production by soleus muscle. Stimulation of sugar transport is considered to be due to the resultant fall in ATP. p-Chloromercuribenzene sulphonate (0.5-2 mM) stimulated xylose uptake to a lesser extent than iodoacetate and induced a proportionately smaller fall in ATP, consistent with the inhibitory effect of p-chloromercuribenzene sulphonate on lactate production. Under certain conditions, p-chloromercuribenzene sulphonate stimulated sugar transport without affecting the ATP level. This suggests that whereas p-chloromercuribenzene sulphonate can be expected to stimulate sugar transport through the lowering of muscle ATP, it may also act through some other mechanism. No stimulatory effect on xylose uptake was observed when muscles were exposed to N-ethylmaleimide (0.02-2 mM) either for brief (1 min) or more prolonged (30 min) periods. Because N-ethylmaleimide induced a marked fall in muscle ATP, it is surprising that N-ethylmaleimide did not stimulate sugar transport; in most experiments this inhibitor actually inhibited sugar transport. N-Ethylmaleimide inhibited the stimulation of sugar transport by 2,4-dinitrophenol and anoxia; this inhibitory effect appears to explain why N-ethylmaleimide itself did not stimulate sugar transport. p-Chloromercuribenzene sulphonate also inhibited 2,4-dinitrophenol-stimulated xylose uptake by a mechanism which seems similar to that of N-ethylmaleimide; this could explain in part the modest stimulatory effect of this inhibitor on muscle sugar transport.     

Effect of protein synthesis inhibitors on xylose uptake and 125I-insulin binding by rat soleus muscle

Prolonged exposure (90–180 min) to cycloheximide (0.2 mg/ml), puromycin (0.2 mg/ml) or chloramphenicol (0.1 mg/ml) did not affect ¹²⁵I-insulin binding by rat soleus muscle. Chloramphenicol (2 mg/ml) depressed insulin binding and insulin-stimulated xylose uptake; these effects were attributed to the “toxic” effect of chloramphenicol on muscle ATP levels. Cycloheximide and puromycin inhibited insulin-stimulated xylose uptake without affecting ATP. Puromycin and chloramphenicol, but not cycloheximide, also inhibited basal sugar transport. This difference, and the rapid onset of all these inhibitory effects, suggest that they are not due to the inhibition of protein synthesis, but rather to some more direct effect on sugar transport itself.     

ATP depletion promotes deactivation of insulin-stimulated sugar transport in rat soleus muscle

It has been reported that deactivation of insulin-stimulated sugar transport in adipocytes is an energy-dependent process (F. V. Vega, R. J. Key, J. E. Jordan, and T. Kono (1980) Arch. Biochem. Biophys. 203, 167-173). The stimulatory effect of insulin (0.1 U/ml) on the uptake of D-[U-14C]xylose by rat soleus muscle was rapidly reversed when muscle ATP was depleted by exposure to 2,4-dinitrophenol (0.5 mM). Insulin action was not completely eliminated by ATP depletion; there was a small, residual stimulatory effect of the hormone which persisted for about 30 min after muscle ATP had been lowered to an unmeasurable level. The extent of deactivation was not altered when the rate of ATP depletion was accelerated, either by increasing the 2,4-dinitrophenol concentration, or by inducing leakiness by incubating muscles for 90 min at 37 degrees C prior to the addition of the uncoupler. 2,4-Dinitrophenol lowered steady-state 125I-insulin binding. These differences between the effect of ATP depletion on insulin-stimulated sugar transport in muscle and adipose tissue may be related to the action of the uncoupler in lowering steady-state insulin binding in muscle. Such a fall in bound insulin could be expected to promote deactivation during the period of ATP depletion. However, at present the possibility that these differences may represent some more fundamental difference in deactivation between muscle and adipose tissue cannot be excluded.     

Stimulatory and inhibitory effects of EDTA on sugar transport by rat soleus muscle

The uptake of D-[¹⁴C]xylose by rat soleus muscle was stimulated rapidly and transiently by brief exposure to EDTA (0.1–20 mM). EDTA also stimulated xylose uptake in the presence of insulin (0.1 U/ml). Prolonged exposure to EDTA (60 min) inhibited insulin-stimulated xylose uptake and depressed ¹²⁵I-insulin binding; these effects were associated with the lowering of muscle ATP. The stimulatory effect was abolished by the substitution of Ca-EDTA (or Mg-EDTA) for EDTA; Ca-EDTA did not eliminate the inhibitory effect. There was no inhibitory effect when Ca²⁺ (5 mM) was added along with Ca-EDTA, or when Zn-EDTA was used instead. There was no effect of EGTA (5 mM) on xylose uptake measured in the presence or absence of insulin. It is concluded (1) that the stimulatory effect of EDTA is most likely due to the chelation of Mg²⁺, (2) that the inhibitory effects of EDTA are due to the chelation of some metal ion whith a higher affinity for the chelator than either Ca²⁺ or Mg²⁺.     

Differential effects of sulfhydryl reagents on activation and deactivation of the fat cell hexose transport system.

A rapid filtration method was used to measure initial rates of 3-O-[3H]methylglucose uptake and thus estimate hexose transport system activity in isolated white fat cells. Insulin markedly stimulated the transport system activity and its effect was rapidly and completely reversible. In addition, such oxidants as vitamin K5 (50 muM), hydrogen peroxide (4mM), methylene blue (50 muM), and diamide (20 mM) also maximally activated 3-O-methylglucose transport and their effects were not additive to those of maximal concentrations of insulin. These oxidants had no effect on total cellular ATP levels under these conditions. Hexose transport system activity in either the presence or absence of these stimulatory agents was uniformly sensitive to inhibition by cytochalasin B. Treatment of fat cells with either 0.5 mM N-ethylmaleimide or 3 mM dithio(bis)nitrobenzoic acid abolished the ability of insulin or oxidants to activate hexose transport system activity. Control transport activity was not significantly influenced by these agents. Fat cells treated with dithio(bis)nitrobenzoic acid completely regained the ability to respond to insulin or vitamin K5 after removal of the agent by washing in low concentrations of reductant. Elevated rates of transport due to prior incubation of cells with insulin or vitamin K5 were completely resistant to inhibition by subsequent addition of N-ethylmaleimide or dithio(bis)nitrobenzoic acid. Deactivation of the hormone-stimulated transport system could be achieved by washing cells free of insulin or by destruction of insulin-receptor interaction by trypsin. N-Ethylmaleimide effectively blocked deactivation of insulin-stimulated transport system activity, while dithio(bis)nitrobenzoic acid was without effect. These results suggest that distinct cellular components mediate activation versus deactivation of the fat cell hexose transport system. N-Ethylmaleimide, which effectively penetrates fat cells, inhibits both processes while the layer, more polar dithio(bis)nitrobenzoic acid blocks activation but not deactivation of this transport system.     

Receptor-mediated insulin degradation and insulin-stimulated glycogenesis in cultured foetal hepatocytes.

Insulin-stimulated glycogenesis and insulin degradation were studied simultaneously at 37 degrees C in cultured foetal hepatocytes grown for 2-3 days in the presence of cortisol. Degradation of cell-associated insulin, as measured by trichloroacetic acid precipitation, was significant after 4 min in the presence of 1-3 nM-125I-labelled insulin. This process became maximal (30% of insulin degraded) after 20 min, a time when binding-state conditions were achieved. No insulin-degradative activity was detected in a medium that had been exposed to cells. At steady-state, the appearance of insulin degradation products in the medium was linearly dependent on time (1.5 fmol/min per 10(6) cells at 1nM-125I-labelled insulin). Chloroquine (3-50 microM), bacitracin (0.1-10 mM) and NH4Cl (1-10 mM) inhibited insulin degradation as soon as this became detectable and caused an increase in the association of insulin to hepatocytes after 20 min. Lidocaine and dansylcadaverine had similar effects, whereas N-ethylmaleimide, aprotinin, phenylmethanesulphonyl fluoride and leupeptin were found to be ineffective. Chloroquine, and also bacitracin, at concentrations that inhibited insulin degradation, decreased the insulin-stimulated incorporation of [14C]glucose into glycogen over 2 h. This effect of chloroquine was specific, since it did not modify the basal glycogenesis, or the glycogenic effect of a glucose load in the absence of insulin. It therefore appears that the receptor-mediated insulin degradation (or some associated pathway) is functionally related to the glycogenic effect of insulin in foetal hepatocytes.     

Muscle glycogen content affects insulin-stimulated glucose transport and protein kinase B activity

We investigated the possible regulatory role of glycogen in insulin-stimulated glucose transport and insulin signaling in skeletal muscle. Rats were preconditioned to obtain low (LG), normal, or high (HG) muscle glycogen content, and perfused isolated hindlimbs were exposed to 0, 100, or 10,000 microU/ml insulin. In the fast-twitch white gastrocnemius, insulin-stimulated glucose transport was significantly higher in LG compared with HG. This difference was less pronounced in the mixed-fiber red gastrocnemius and was absent in the slow-twitch soleus. In the white gastrocnemius, insulin activation of insulin receptor tyrosine kinase and phosphoinositide 3-kinase was unaffected by glycogen levels, whereas protein kinase B activity was significantly higher in LG compared with HG. In additional incubation experiments on fast-twitch epitrochlearis muscles, insulin-stimulated cell surface GLUT-4 content was significantly higher in LG compared with HG. The data indicate that, in fast-twitch muscle, the effect of insulin on glucose transport and cell surface GLUT-4 content is modulated by glycogen content, which does not involve initial but possibly more downstream signaling events.     

Effect of leptin on insulin resistance of muscle--direct or indirect?

We examined the effect of leptin on the insulin resistance in skeletal muscles by measuring glucose transport. Male Wistar rats were fed rat chow or high-fat diets for 30 days. Before sacrifice, rats fed high-fat diet were subcutaneously injected with leptin (1 mg/kg b.w.) for 3 days. The glucose transport in epitrochlearis and soleus muscles did not differ in the experimental groups under basal conditions, however these values decreased significantly in the rats fed high-fat diet under insulin stimulation (p<0.01). Leptin treatment recovered the decreased glucose transport in epitrochlearis (p<0.05) and soleus muscles (p=0.08). Triglyceride concentrations in soleus muscles were increased significantly in the rats fed high-fat diet as compared to rats fed chow diet (p<0.01), and were decreased significantly by leptin treatment (p<0.01). The glucose transport was measured under basal conditions and after 60 microU/ml of insulin treatment with or without 50 ng/ml of leptin. Leptin had no direct stimulatory effect on glucose transport under both basal and insulin-stimulated conditions in vitro. These results demonstrate that leptin injection to rats fed high-fat diet recovered impaired insulin responsiveness of skeletal muscles and muscle triglyceride concentrations. However, there was no direct stimulatory effect of leptin on insulin sensitivity of skeletal muscles in vitro.     

Lactate induces insulin resistance in skeletal muscle by suppressing glycolysis and impairing insulin signaling

Elevation of plasma lactate levels induces peripheral insulin resistance, but the underlying mechanisms are unclear. We examined whether lactate infusion in rats suppresses glycolysis preceding insulin resistance and whether lactate-induced insulin resistance is accompanied by altered insulin signaling and/or insulin-stimulated glucose transport in skeletal muscle. Hyperinsulinemic euglycemic clamps were conducted for 6 h in conscious, overnight-fasted rats with or without lactate infusion (120 micromol x kg(-1) x min(-1)) during the final 3.5 h. Lactate infusion increased plasma lactate levels about fourfold. The elevation of plasma lactate had rapid effects to suppress insulin-stimulated glycolysis, which clearly preceded its effect to decrease insulin-stimulated glucose uptake. Both submaximal and maximal insulin-stimulated glucose transport decreased 25-30% (P < 0.05) in soleus but not in epitrochlearis muscles of lactate-infused rats. Lactate infusion did not alter insulin's ability to phosphorylate the insulin receptor, the insulin receptor substrate (IRS)-1, or IRS-2 but decreased insulin's ability to stimulate IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activities and Akt/protein kinase B activity by 47, 75, and 55%, respectively (P < 0.05 for all). In conclusion, elevation of plasma lactate suppressed glycolysis before its effect on insulin-stimulated glucose uptake, consistent with the hypothesis that suppression of glucose metabolism could precede and cause insulin resistance. In addition, lactate-induced insulin resistance was associated with impaired insulin signaling and decreased insulin-stimulated glucose transport in skeletal muscle.     

Effect of hydrocortisone and dexamethasone on xylose uptake by isolated rat soleus muscle.

To determine the effects of glucocorticoids on sugar uptake, xylose uptake by isolated rat soleus muscle of bilaterally adrenalectomized animals was studied. The results indicate that in vitro addition of 10-4 M hydrocortisone, dexamethasone or hydrocortisone sodium succinate had no inhibitory effect on basal xylose uptake. In the presence of both low and high medium insulin, the above steroids failed to inhibit insulin-stimulated uptake. When the concentration of hydrocortisone sodium succinate was increased to 10-2 M, insulinstimulated uptake was decreased. The results thus indicate that glucocorticoids at concentrations observed under physiological or pathological conditions do not inhibit basal or insulin-stimulated sugar uptake.     

Topiramate is an insulin-sensitizing compound in vivo with direct effects on adipocytes in female ZDF rats

We have studied the in vivo and in vitro effects of Topiramate (TPM) in female Zucker diabetic fatty (ZDF) rats. After weight matching, drug treatment had a marked effect to lower fasting glucose levels of relatively normoglycemic animals as well as during an oral glucose tolerance test. The glucose clamp studies revealed a approximately 30% increased glucose disposal, increased hepatic glucose output (HGO) suppression from approximately 30 to 60%, and an increased free fatty acid suppression from 40 to 75%. Therefore, TPM treatment led to enhanced insulin sensitivity at the level of tissue glucose disposal (increased ISGDR), liver (increased inhibition of HGO), and adipose tissue (enhanced suppression of lipolysis). When soleus muscle strips of control or TPM-treated ZDF rats were studied ex vivo, insulin-stimulated glucose transport was not enhanced in the drug-treated animals. In contrast, when isolated adipocytes were studied ex vivo, a marked increase (+55%) in insulin-stimulated glucose transport was observed. In vitro treatment of muscle strips and rat adipocytes showed no effect on glucose transport in muscle with a 40% increase in insulin-stimulated adipocyte glucose transport. In conclusion, 1) TPM treatment leads to a decrease in plasma glucose and increased in vivo insulin sensitivity; 2) insulin sensitization was observed in adipocytes, but not muscle, when tissues were studied ex vivo or in vitro; and 3) TPM directly enhances insulin action in insulin-resistant adipose cells in vitro. Thus the in vivo effects of TPM treatment appear to be exerted through adipose tissue.     

Effect of hydrocortisone and dexamethasone on xylose uptake by isolated rat soleus muscle

To determine the effects of glucocorticoids on sugar uptake, xylose uptake by isolated rat soleus muscle of bilaterally adrenalectomized animals was studied. The results indicate that in vitro addition of 10⁻⁴ M hydrocortusine, dexamethasone or hydrocortisone sodium succinate had no inhibitory effect on basal xylose uptake. In the presence of both low and high medium insulin, the above steroids failed to inhibit insulin-stimulated uptake. When the concentration of hydrocortisone sodium succinate was increased to 10⁻² M, insulin-stimulated uptake was decreased. The results thus indicate that glucocorticoids at concentrations observed under physiological or pathological conditions do not inihibit basal or insulin-stimulated sugar uptake.     

Substrate regulation of the glucose transport system in rat skeletal muscle. Characterization and kinetic analysis in isolated soleus muscle and skeletal muscle cells in culture

A self-regulatory mechanism of the glucose transport in rat skeletal muscle cells is described. In isolated rat soleus muscles and rat skeletal myocytes and myotubes in culture, pre-exposure to varying glucose concentrations modulated the rate of 2-deoxyglucose uptake. Maximal uptake was observed at glucose concentrations below 3 mM. Between 2.5 and 4.0 mM glucose it was reduced by 25-35%; further elevation of the glucose concentration resulted in a gradual decrease of the transport rate by approximately 2% for each millimolar glucose. The effect of glucose was time-dependent and fully reversible. Insulin rapidly increased the 2-deoxyglucose uptake in the soleus muscle; however, the insulin effect depended on the glucose concentration of the preincubation. Insulin was totally ineffective in muscles pre-exposed to 1.0-3.0 mM glucose, whereas its stimulatory action increased with increasing glucose concentrations above 4 mM. The effect of low glucose and insulin were not additive, and the maximal 2-deoxyglucose uptake rates induced by both conditions were of identical magnitude. It is postulated that glucose may "up- and down-regulate" its transport by affecting the number of active glucose transporters in the plasma membrane, and that insulin exerts its stimulatory effect only when the extracellular glucose reaches a threshold concentration.     

Effects in adipocytes of diamide on GSH levels,glucose uptake and cell integrity

Concentrations of insulin and chemical agents (H₂O₂, vitamin K-5) which stimulate hexose transport in fat cells do not alter the cellular levels of glutathione (reduced form; GSH). Diamide, another agent used in studies of insulin action, markedly reduces GSH levels and increases the movement of sugar into the cell. However, unlike insulin, H₂O₂ or vitamin K-5, diamide causes a change in the permeability of fat cells that allows entry of compounds (inulin, sucrose, l-glucose) which are normally excluded by the plasma membrane. Moreover, the accelerated rate of methylglucose uptake produced by diamide treatment is not inhibited by cytochalasin B, an agent that blocks basal and insulin-stimulated methylglucose transport. These results indicate that diamide does not cause a stimulation of the glucose transport system and should not be used (or used with caution) in transport studies. Furthermore, oxidation of GSH does not appear to be necessary for the stimulation of hexose transport in adipocytes by insulin, H₂O₂ or vitamin K-5.     

Effects of islet-activating protein on insulin- and isoprenaline-stimulated glucose transport in isolated rat adipocytes

The effects of islet-activating protein (IAP), a Bordetella pertussis toxin, on insulin- and isoprenaline-stimulated glucose transport were studied in isolated rat adipocytes. Basal as well as insulin-stimulated glucose transport were not affected when cells were pretreated with IAP. In contrast, IAP pretreatment abolished the stimulatory effect of isoprenaline. When IAP-pretreated cells were exposed to a combination of insulin and isoprenaline, the catecholamine significantly reduced the stimulatory effect of insulin. Since IAP is supposed to specifically block the inhibitory component Ni of adenylate cyclase, the results suggest that: (a) the effect of insulin is unrelated to the regulation of adenylate cyclase; (b) isoprenaline may exert both stimulatory and inhibitory effects depending on activation of Ni. The inhibitory regulation of adenylate cyclase may thus be a pivotal link in the regulation of glucose transport.     

Resistance of the intact and reconstituted adipocyte hexose transport system to irreversible inhibition by sulfhydryl and amino reagents

Sensitivity of the adipocyte D-glucose transport system in intact plasma membranes or following solubilization and reconstitution into phospholipid vesicles to several protein-modifying reagents was investigated. When intact plasma membranes were incubated with N-ethylmaleimide (20 mM) or fluorodinitrobenzene (4 mM), D-glucose transport activity was virtually abolished. However, washing the membranes free of unreacted reagents restored transport activity, indicating that covalent interaction with the membranes did not mediate the transport inhibition. Reaction of [³H] N-ethylmaleimide with plasma membranes under similar conditions resulted in extensive labeling of all protein fractions resolved on dodecyl sulfate gels. Similarly, addition of N-ethyl-maleimide to cholate-solubilized membrane protein had no effect on transport activity in artifical phospholipid vesicles reconstituted under conditions where the membrane protein was free of unreacted N-ethylmaleimide. Transport activity in plasma membranes was also inhibited by both reduced and oxidized dithiothreitol or glutathione (15 mM) in a readily reversible manner, consistent with a noncovalent mode of inhibition. Thus, the insulin-responsive adipocyte D-glucose transport system differs from the red cell hexose transport system in its remarkable insensitivity to modulation by covalent blockade of sulfhydryal or amino groups by the reagents studied.     

Phorbol esters affect skeletal muscle glucose transport in a fiber type-specific manner

Recent evidence has shown that activation of lipid-sensitive protein kinase C (PKC) isoforms leads to skeletal muscle insulin resistance. However, earlier studies demonstrated that phorbol esters increase glucose transport in skeletal muscle. The purpose of the present study was to try to resolve this discrepancy. Treatment with the phorbol ester 12-deoxyphorbol-13-phenylacetate 20-acetate (dPPA) led to an approximately 3.5-fold increase in glucose transport in isolated fast-twitch epitrochlearis and flexor digitorum brevis muscles. Phorbol ester treatment was additive to a maximally effective concentration of insulin in fast-twitch skeletal muscles. Treatment with dPPA did not affect insulin signaling in the epitrochlearis. In contrast, phorbol esters had no effect on basal glucose transport and inhibited maximally insulin-stimulated glucose transport approximately 50% in isolated slow-twitch soleus muscle. Furthermore, dPPA treatment inhibited the insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the threonine and serine phosphorylation of PKB by approximately 50% in the soleus. dPPA treatment also caused serine phosphorylation of IRS-1 in the slow-twitch soleus muscle. In conclusion, our results show that phorbol esters stimulate glucose transport in fast-twitch skeletal muscles and inhibit insulin signaling in slow-twitch soleus muscle of rats. These findings suggest that mechanisms other than PKC activation mediate lipotoxicity-induced whole body insulin resistance.     

Denervation provokes greater reductions in insulin-stimulated glucose transport in muscle than severe diabetes

We have examined the independent and combined effects of insulin insufficiency (streptozotocin (STZ)-induced diabetes, 85 mg/kg i.p.) and reduced muscle activity (denervation) (7 days) on basal, insulin-stimulated and contraction-stimulated glucose transport in rat muscles (soleus, red and white gastrocnemius). There were four treatments: control, denervated, diabetic, and denervated + diabetic muscles. Contraction-stimulated glucose transport was lowered (~ 50%) (p < 0.05) to the same extent in all experimental groups. In contrast, there was a much smaller reduction insulin-stimulated glucose transport in muscles from diabetic animals (18-24% reduction, p < 0.05) than in denervated muscles (40-60% reduction, p < 0.05) and in denervated + diabetic muscles (40-60% reduction, p < 0.05). GLUT-4 mRNA reduction was greatest in denervated + diabetic muscles (~ -75%, p < 0.05). GLUT-4 protein was decreased (p < 0.05) to a similar extent in all three experimental conditions (~ -30-40%). In conclusion, (1) muscle inactivity (denervation) and STZ-induced diabetes had similar effects on reducing contraction-stimulated glucose transport, but (2) muscle inactivity (denervation), rather than severe diabetes, produced a 2-fold greater impairment in skeletal muscle insulin-stimulated glucose transport.     

Effect of N-ethylmaleimide on leucine transport in the Chang liver cell. II. Effect on the kinetics of Na+-independent transport

The Na+-independent leucine transport system is resolved into two components by their different affinity (Km about 44 microM and 8.0 mM) for leucine in the Chang liver cell. Treatment of the cells with N-ethylmaleimide (1 mM) specifically stimulates the high-affinity component of the Na+-independent system by greatly increasing its Vmax value, whereas the Vmax value of the low-affinity component is markedly lowered. The stimulatory effect of N-ethylmaleimide on leucine transport is reduced by prior treatment of the cells with 2,4-dinitrophenol, but this phenomenon seems to be irrelevant to the ATP-depleting action of the uncoupler. The treatment with 2,4-dinitrophenol has been found not to be inhibitory on the subsequent Na+-independent leucine uptake itself. Treatment with dibucaine, a phospholipid-interacting drug, also reduces to varying degrees (depending on its concentration) the stimulatory effect of N-ethylmaleimide on the subsequent leucine uptake, although pretreatment with dibucaine can stimulate the Na+-independent leucine uptake itself. We conclude that the stimulatory effect of N-ethylmaleimide on leucine transport is not correlated with the energy level of cell, but involves the perturbation of the membrane bilayer structures.