Distributed Control of Muscle Glucose Uptake: A Tribute to the Late Dr. David H. Wasserman by Revisiting a 2004 Diabetes Classic by Fueger etal.

Contraction and insulin promote glucose uptake in skeletal muscle through GLUT4 translocation to cell surface membranes. Although the signaling mechanisms leading to GLUT4 translocation have been extensively studied in muscle, the cellular transport machinery is poorly understood. Myo1c is an actin-based motor protein implicated in GLUT4 translocation in adipocytes; however, the expression profile and role of Myo1c in skeletal muscle have not been investigated. Myo1c protein abundance was higher in more oxidative skeletal muscles and heart. Voluntary wheel exercise (4 weeks, 8.2 ± 0.8 km/day), which increased the oxidative profile of the triceps muscle, significantly increased Myo1c protein levels by ∼2-fold versus sedentary controls. In contrast, high fat feeding (9 weeks, 60% fat) significantly reduced Myo1c by 17% in tibialis anterior muscle. To study Myo1c regulation of glucose uptake, we expressed wild-type Myo1c or Myo1c mutated at the ATPase catalytic site (K111A-Myo1c) in mouse tibialis anterior muscles in vivo and assessed glucose uptake in vivo in the basal state, in response to 15 min of in situ contraction, and 15 min following maximal insulin injection (16.6 units/kg of body weight). Expression of wild-type Myo1c or K111A-Myo1c had no effect on basal glucose uptake. However, expression of wild-type Myo1c significantly increased contraction- and insulin-stimulated glucose uptake, whereas expression of K111A-Myo1c decreased both contraction-stimulated and insulin-stimulated glucose uptake. Neither wild-type nor K111A-Myo1c expression altered GLUT4 expression, and neither affected contraction- or insulin-stimulated signaling proteins. Myo1c is a novel mediator of both insulin-stimulated and contraction-stimulated glucose uptake in skeletal muscle.

The following letters are in response to the Point:Counterpoint series “Glucose phosphorylation is/is not a significant barrier to muscle glucose uptake by the working muscle” that appears in this issue. To the Editor : The Point:Counterpoint debate ([4][1], [6][2]) provides important insights

The Regulation of Glucose Uptake in Muscle: the Work of Charles R. Park

Novel regulatory mechanisms in muscle metabolism during exercise.

Previous work suggests that normal GLUT4 content is sufficient for increases in muscle glucose uptake (MGU) during exercise because GLUT4 overexpression does not increase exercise-stimulated MGU. Instead of glucose transport, glucose phosphorylation is a primary limitation of exercise-stimulated MGU. It was hypothesized that a partial ablation of GLUT4 would not impair exercise-stimulated MGU when glucose phosphorylation capacity is normal but would do so when glucose phosphorylation capacity was increased. Thus, C57BL/6J mice with hexokinase II (HKII) overexpression (HK(Tg)), a GLUT4 partial knock-out (G4(+/-)), or both (HK(Tg) + G4(+/-)) and wild-type (WT) littermates were implanted with carotid artery and jugular vein catheters for sampling and infusions at 4 months of age. After a 7-day recovery, 5-h fasted mice remained sedentary or ran on a treadmill at 0.6 mph for 30 min (n = 9-12 per group) and received a bolus of 2-deoxy[3H]glucose to provide an index of MGU (Rg). Arterial blood glucose and plasma insulin concentrations were similar in WT, G4(+/-), HKTg, and HKTg + G4(+/-) mice. Sedentary Rg values were the same in all genotypes in all muscles studied, confirming that glucose transport is a significant barrier to basal glucose uptake. Gastrocnemius and soleus Rg were greater in exercising compared with sedentary mice in all genotypes. During exercise, G4(+/-) mice had a marked increase in blood glucose that was corrected by the addition of HK II overexpression. Exercise Rg (micromol/100g/min) was not different between WT and G4(+/-) mice in the gastrocnemius (24 +/- 5 versus 21 +/- 2) or the soleus (54 +/- 6 versus 70 +/- 7). In contrast, the enhanced exercise Rg observed in HKTg mice compared with that in WT mice was absent in HKTg + G4(+/-) mice in both the gastrocnemius (39 +/- 7 versus 22 +/- 6) and the soleus (98 +/- 13 versus 65 +/- 13). Thus, glucose transport is not a significant barrier to exercise-stimulated MGU despite a 50% reduction in GLUT4 content when glucose phosphorylation capacity is normal. However, when glucose phosphorylation capacity is increased by HK II overexpression, GLUT4 availability becomes a marked limitation to exercise-stimulated MGU.

Does exercise increase insulin sensitivity through angiotensin 1-7?

The aim of our work was to investigate a possible role of protein kinase C (PKC) in insulin-stimulated glucose uptake in mouse skeletal muscle, and to search for a defect in PKC activation in insulin resistance found in obesity. In isolated soleus muscle of lean mice, insulin (100 nM) and 12-O-tetradecanoylphorbol 13-acetate (TPA) (1 microM) acutely stimulated glucose uptake 3- and 2-fold respectively. The effects of insulin and TPA were not additive. When PKC activity was down-regulated by long-term (24 h) TPA pretreatment, before measurement of glucose transport, the TPA effect was abolished, but in addition insulin-stimulated glucose transport returned to basal values. Furthermore, polymyxin B, which inhibits PKC in muscle extracts, prevented insulin-stimulated glucose uptake in muscle. In muscle of obese insulin-resistant mice, glucose uptake evoked by insulin was decreased, whereas the TPA effect, expressed as a fold increase, was unaltered. Thus both agents stimulated glucose transport to the same extent. Furthermore, no difference was observed when PKC activation by TPA was measured in muscle from lean and obese mice. These results suggest that: (1) PKC is involved in the insulin effect on glucose transport in muscle; (2) PKC activation explains only part of the insulin stimulation of glucose transport; (3) the defect in insulin response in obese mice does not appear to be due to an alteration in the PKC-dependent component of glucose transport. We propose that insulin stimulation of glucose uptake occurs by a sequential two-step mechanism, with first translocation of transporters to the plasma membrane, which is PKC dependent, and second, activation of the glucose transporters. In obesity only the activation step was decreased, whereas the translocation step was unaltered.

Exercise increases hexokinase II mRNA, but not activity in obesity and type 2 diabetes

Muscle glucose uptake (MGU) is determined by glucose delivery, transport, and phosphorylation. C57Bl/6J mice overexpressing GLUT4, hexokinase II (HK II), or both were used to determine the barriers to MGU. A carotid artery and jugular vein were catheterized for arterial blood sampling and venous infusions. Experiments were conducted in conscious mice approximately 7 days after surgery. 2-Deoxy-[3H]glucose was administered during rest or treadmill exercise to calculate glucose concentration-dependent (Rg) and -independent (Kg) indexes of MGU. Compared with wild-type controls, GLUT4-overexpressing mice had lowered fasting glycemia (165 +/- 6 vs. 115 +/- 6 mg/dl) and increased Rg by 230 and 166% in the gastrocnemius and superficial vastus lateralis (SVL) muscles under sedentary conditions. GLUT4 overexpression was not able to augment exercise-stimulated Rg or Kg. Whereas HK II overexpression had no effect on fasting glycemia (170 +/- 6 mg/dl) or sedentary Rg, it increased exercise-stimulated Rg by 82, 60, and 169% in soleus, gastrocnemius, and SVL muscles, respectively. Combined GLUT4 and HK II overexpression lowered fasting glycemia (106 +/- 6 mg/dl), increased nonesterified fatty acids, and increased sedentary Rg. Combined GLUT4 and HK II overexpression did not enhance exercise-stimulated Rg compared with HK II-overexpressing mice because of the reduced glucose concentration. GLUT4 combined with HK II overexpression resulted in a marked increase in exercise-stimulated Kg. In conclusion, control of MGU shifts from membrane transport at rest to phosphorylation during exercise. Glucose transport is not normally a significant barrier during exercise. However, when the phosphorylation barrier is lowered by HK II overexpression, glucose transport becomes a key site of control for regulating MGU during exercise.

Insulin resistance plays a major role in the pathogenesis of type 2 diabetes. Insulin regulates blood glucose levels primarily by promoting glucose uptake from the blood into multiple tissues and by suppressing glucose production from the liver. The glucose transporter, GLUT4, mediates insulin-stimulated glucose uptake in muscle and adipose tissue. Decreased GLUT4 expression in adipose tissue is a common feature of many insulin resistant states. GLUT4 expression is preserved in skeletal muscle in many insulin resistant states. However, functional defects in the intracellular trafficking and plasma membrane translocation of GLUT4 result in impaired insulin-stimulated glucose uptake in muscle. Tissue-specific genetic knockout of GLUT4 expression in adipose tissue or muscle of mice has provided new insights into the pathogenesis of insulin resistance. We recently determined that the expression of serum retinol binding protein (RBP4) is induced in adipose tissue as a consequence of decreased GLUT4 expression. We found that RBP4 is elevated in the serum of insulin resistant humans and mice. Furthermore, we found that increasing serum RBP4 levels by transgenic overexpression or by injection of purified RBP4 protein into normal mice causes insulin resistance. Therefore, RBP4 appears to play an important role in mediating adipose tissue communication with other insulin target tissues in insulin resistant states.

Skeletal muscle glucose uptake requires delivery of glucose to the sarcolemma, transport across the sarcolemma, and the irreversible phosphorylation of glucose by hexokinase (HK) inside the cell. Here, a novel method was used in the conscious rat to address the roles of these three steps in controlling the rate of glucose uptake in soleus, a muscle comprised of type I fibers, and two muscles comprised of type II fibers. Experiments were performed on conscious rats under basal conditions or during hyperinsulinemic euglycemic clamps. Rats received primed, constant infusions of 3-O-methyl-[3H]glucose (3-O-MG) and [1-14C]mannitol. Total muscle glucose concentration and the steady-state ratio of intracellular to extracellular 3-O-MG concentration, which distributes based on the transsarcolemmal glucose gradient (TSGG), were used to calculate glucose concentrations at the inner and outer sarcolemmal surfaces ([G](im) and [G](om), respectively) in muscle. Muscle glucose uptake was much lower in muscle comprised of type II fibers than in soleus under both basal and insulin-stimulated conditions. Under all conditions, the TSGG in type II muscle exceeded that in soleus, indicating that glucose transport plays a more important role to limit glucose uptake in type II muscle. Although hyperinsulinemia increased [G](im) in soleus, indicating that phosphorylation was a limiting factor, type II muscle was limited primarily by glucose delivery and glucose transport. In conclusion, the relative importance of glucose delivery, transport, and phosphorylation in controlling the rate of insulin-stimulated muscle glucose uptake varies between muscle fiber types, with glucose delivery and transport being the primary limiting factors in type II muscle.

Thiazolidinediones have been shown to up-regulate adiponectin expression in white adipose tissue and plasma adiponectin levels, and these up-regulations have been proposed to be a major mechanism of the thiazolidinedione-induced amelioration of insulin resistance linked to obesity. To test this hypothesis, we generated adiponectin knock-out (adipo-/-) ob/ob mice with a C57B/6 background. After 14 days of 10 mg/kg pioglitazone, the insulin resistance and diabetes of ob/ob mice were significantly improved in association with significant up-regulation of serum adiponectin levels. Amelioration of insulin resistance in ob/ob mice was attributed to decreased glucose production and increased AMP-activated protein kinase in the liver but not to increased glucose uptake in skeletal muscle. In contrast, insulin resistance and diabetes were not improved in adipo-/-ob/ob mice. After 14 days of 30 mg/kg pioglitazone, insulin resistance and diabetes of ob/ob mice were again significantly ameliorated, which was attributed not only to decreased glucose production in the liver but also to increased glucose uptake in skeletal muscle. Interestingly, adipo-/-ob/ob mice also displayed significant amelioration of insulin resistance and diabetes, which was attributed to increased glucose uptake in skeletal muscle but not to decreased glucose production in the liver. The serum-free fatty acid and triglyceride levels as well as adipocyte sizes in ob/ob and adipo-/-ob/ob mice were unchanged after 10 mg/kg pioglitazone but were significantly reduced to a similar degree after 30 mg/kg pioglitazone. Moreover, the expressions of TNFalpha and resistin in adipose tissues of ob/ob and adipo-/-ob/ob mice were unchanged after 10 mg/kg pioglitazone but were decreased after 30 mg/kg pioglitazone. Thus, pioglitazone-induced amelioration of insulin resistance and diabetes may occur adiponectin dependently in the liver and adiponectin independently in skeletal muscle.

Exercise is a potent physiological stimulus to clear blood glucose from the circulation into skeletal muscle. The mammalian target of rapamycin complex 2 (mTORC2) is an important regulator of muscle glucose uptake in response to insulin stimulation. Here we report for the first time that the activity of mTORC2 in mouse muscle increases during exercise. We further show that glucose uptake during exercise is decreased in mouse muscle that lacks mTORC2 activity. We also provide novel identifications of new mTORC2 substrates during exercise in mouse muscle. Exercise increases glucose uptake into insulin-resistant muscle. Thus, elucidating the exercise signalling network in muscle may uncover new therapeutic targets. The mammalian target of rapamycin complex 2 (mTORC2), a regulator of insulin-controlled glucose uptake, has been reported to interact with ras-related C3 botulinum toxin substrate1 (Rac1), which plays a role in exercise-induced glucose uptake in muscle. Therefore, we tested the hypothesis that mTORC2 activity is necessary for muscle glucose uptake during treadmill exercise. We used mice that specifically lack mTORC2 signalling in muscle by deletion of the obligatory mTORC2 component Rictor (Ric mKO). Running capacity and running-induced changes in blood glucose, plasma lactate and muscle glycogen levels were similar in wild-type (Ric WT) and Ric mKO mice. At rest, muscle glucose uptake was normal, but during running muscle glucose uptake was reduced by 40% in Ric mKO mice compared to Ric WT mice. Running increased muscle phosphorylated 5' AMP-activated protein kinase (AMPK) similarly in Ric WT and Ric mKO mice, and glucose transporter type 4 (GLUT4) and hexokinase II (HKII) protein expressions were also normal in Ric mKO muscle. The mTORC2 substrate, phosphorylated protein kinase C α (PKCα), and the mTORC2 activity readout, phosphorylated N-myc downstream regulated 1 (NDRG1) protein increased with running in Ric WT mice, but were not altered by running in Ric mKO muscle. Quantitative phosphoproteomics uncovered several additional potential exercise-dependent mTORC2 substrates, including contractile proteins, kinases, transcriptional regulators, actin cytoskeleton regulators and ion-transport proteins. Our study suggests that mTORC2 is a component of the exercise signalling network that regulates muscle glucose uptake and we provide a resource of new potential members of the mTORC2 signalling network.

Activation of the ROS/TXNIP/NLRP3 pathway disrupts insulin-dependent glucose uptake in skeletal muscle of insulin-resistant obese mice

This study followed changes in the capacities of uptake and phosphorylation of glucose in response to contractile activity in low-frequency stimulated (10Hz, 24 h/d) rat fast-twitch muscle. We investigated the intracellular distribution of GLUT-4, the major glucose transporter isoform in muscle, changes in the amounts of its specific mRNA and total cellular protein, as well as changes in its relative synthesis rate. These analyses were complemented by measurements of total hexokinase activity and hexokinase II (HKII) expression at the levels of mRNA content and protein synthesis. Changes in protein synthesis were determined by in vivo labeling with [35S]methionine. Translocation of GLUT-4 into the sarcolemma was an immediate response to contractile activity, whereas changes in its total amount were observed only with ongoing stimulation (5 d and longer). A twofold increase in GLUT-4 content after 5 d and longer stimulation periods was preceded by elevations of its mRNA and by enhanced [35S]methionine incorporation. Conversely, increases in HKII expression with a rise in total hexokinase activity occurred soon after the onset of stimulation (30-fold elevations of HKII mRNA after 12 h and 20-fold increases in [35S]methionine incorporation after 24 h). With ongoing stimulation, HKII mRNA and synthesis returned to lower levels (fivefold elevations). Nevertheless, hexokinase activity continued to rise, stabilizing at fivefold-elevated levels after 3 d. These observation suggested that posttranscriptional mechanisms contributed to the upregulation of HKII, e.g. stabilization by elevated intracellular glucose and mitochondrial binding of the enzyme. This suggestion was supported by experiments with cessation after 24 h where hexokinase activity continued to increase, although the mRNA content and, especially, the [35S]methionine incorporation decayed steeply. The increase in HKII prior to GLUT-4 suggests that phosphorylation may be rate limiting in glucose utilization of glycolytic fibers under conditions of sustained contractile activity. Taken together, the changes in distribution and content of GLUT-4, as well as in HKII represent early metabolic adaptations. In addition, they are related to the overall process of stimulation-induced fiber type transformation.