The B2 receptor of bradykinin is not essential for the post-exercise increase in glucose uptake by insulin-stimulated mouse skeletal muscle

Bradykinin can enhance skeletal muscle glucose uptake (GU), and exercise increases both bradykinin production and muscle insulin sensitivity, but bradykinin's relationship with post-exercise insulin action is uncertain. Our primary aim was to determine if the B2 receptor of bradykinin (B2R) is essential for the post-exercise increase in GU by insulin-stimulated mouse soleus muscles. Wildtype (WT) and B2R knockout (B2RKO) mice were sedentary or performed 60 minutes of treadmill exercise. Isolated soleus muscles were incubated with [3H]-2-deoxyglucose +/-insulin (60 or 100 microU/ml). GU tended to be greater for WT vs. B2RKO soleus with 60 microU/ml insulin (P=0.166) and was significantly greater for muscles with 100 microU/ml insulin (P<0.05). Both genotypes had significant exercise-induced reductions (P<0.05) in glycemia and insulinemia, and the decrements for glucose (approximately 14 %) and insulin (approximately 55 %) were similar between genotypes. GU tended to be greater for exercised vs. sedentary soleus with 60 microU/ml insulin (P=0.063) and was significantly greater for muscles with 100 microU/ml insulin (P<0.05). There were no significant interactions between genotype and exercise for blood glucose, plasma insulin or GU. These results indicate that the B2R is not essential for the exercise-induced decrements in blood glucose or plasma insulin or for the post-exercise increase in GU by insulin-stimulated mouse soleus muscle.     

Polymyxin B inhibits contraction-stimulated glucose uptake in rat skeletal muscle

Glucose transport in muscle is activated by contractile activity, an effect that persists in the postexercise state. Polymyxin B, a cyclic decapeptide antibiotic, inhibits the stimulation of glucose uptake in isolated muscle by contractile activity but also decreases tension development in electrically stimulated muscle. The purpose of this study was to determine whether polymyxin B also inhibits contraction-stimulated glucose uptake after in vivo administration of the drug and to examine the relationship between the effects of polymyxin B on tension development and its effects on contraction-stimulated glucose uptake. When polymyxin B was administered to rats in vivo, glucose uptake in muscle after electrical stimulation was decreased, despite the same amount of tension developed as in control rats, indicating an effect of polymyxin B on glucose transport independent of tension development. Our results also indicate that the postexercise increase in glucose uptake is a function of the tension developed by prior contractions. When muscles were perfused with medium containing polymyxin B, this relationship was disrupted. These results provide evidence that polymyxin B causes a decrease in muscle glucose uptake independent of its effects on tension development. The extent to which its effects on glucose uptake are also the result of a diminution in contractile force is uncertain.     

Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) reportedly activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake by skeletal muscle cells. In this study, we investigated the role of AMPK in AICAR-induced glucose uptake by 3T3-L1 adipocytes and rat soleus muscle cells by overexpressing wild-type and dominant negative forms of the AMPKalpha2 subunit by use of adenovirus-mediated gene transfer. Overexpression of the dominant negative mutant had no effect on AICAR-induced glucose transport in adipocytes, although AMPK activation was almost completely abolished. This suggests that AICAR-induced glucose uptake by 3T3-L1 adipocytes is independent of AMPK activation. By contrast, overexpression of the dominant negative AMPKalpha2 mutant in muscle markedly suppressed both AICAR-induced glucose uptake and AMPK activation, although insulin-induced uptake was unaffected. Overexpression of the wild-type AMPKalpha2 subunit significantly increased AMPK activity in muscle but did not enhance glucose uptake. Thus, although AMPK activation may not, by itself, be sufficient to increase glucose transport, it appears essential for AICAR-induced glucose uptake in muscle.     

Preventing the calorie restriction-induced increase in insulin-stimulated Akt2 phosphorylation eliminates calorie restriction's effect on glucose uptake in skeletal muscle

Calorie restriction (CR; ~60% of ad libitum, AL, consumption) improves insulin-stimulated glucose uptake in skeletal muscle. The precise cellular mechanism for this healthful outcome is unknown, but it is accompanied by enhanced insulin-stimulated activation of Akt. Previous research using Akt2-null mice demonstrated that Akt2 is essential for the full CR-effect on insulin-stimulated glucose uptake by muscle. However, because Akt2-null mice were completely deficient in Akt2 in every cell throughout life, it would be valuable to assess the efficacy of transient, muscle-specific Akt inhibition for attenuation of CR-effects on glucose uptake. Accordingly, we used a selective Akt inhibitor (MK-2206) to eliminate the CR-induced elevation in insulin-stimulated Akt2 phosphorylation and determined the effects on Akt substrates and glucose uptake. We incubated isolated epitrochlearis muscles from 9-month-old AL and CR (~60-65% of AL intake for 6months) rats with or without MK-2206 and measured insulin-stimulated (1.2nM) glucose uptake and phosphorylation of the insulin receptor (Tyr1162/1163), pan-Akt (Thr308 and Ser473), Akt2 (Thr308 and Ser473), AS160/TBC1D4 (Thr642), and Filamin C (Ser2213). Incubation of isolated skeletal muscles with a dose of a selective Akt inhibitor that eliminated the CR-induced increases in Akt2 phosphorylation prevented CR's effects on insulin-stimulated glucose uptake, pAS160(Thr642) and pFilamin C(Ser2213) without altering pIR(Tyr1162/1163). These data provide compelling new evidence linking the CR-induced increase in insulin-stimulated Akt2 phosphorylation to CR's effects on insulin-mediated phosphorylation of Akt substrates and glucose uptake in skeletal muscle.     

Role of Akt2 in contraction-stimulated cell signaling and glucose uptake in skeletal muscle

The serine/threonine kinase Akt/PKB plays diverse roles in cells, and genetic studies have indicated distinct roles for the three Akt isoforms expressed in mammalian cells and tissues. Akt2 is a key signaling intermediate for insulin-stimulated glucose uptake and glycogen synthesis in skeletal muscle. Akt2 has also been shown to be activated by exercise and muscle contraction in both rodents and humans. In this study, we used Akt2 knockout mice to explore the role of Akt2 in exercise-stimulated glucose uptake and glycogen synthesis as well as intracellular signaling pathways that regulate glycogen metabolism in skeletal muscle. We found that Akt2 deficiency does not affect basal or exercise-stimulated glucose uptake or intracellular glycogen content in the soleus muscle. In addition, lack of Akt2 did not result in alterations in basal Akt Thr(308) or basal and contraction-stimulated glycogen synthase kinase-3beta (GSK-3beta) Ser(9) phosphorylation, glycogen synthase phosphorylation, or glycogen synthase activity. In contrast, in situ contraction failed to elicit normal increases in Akt T-loop Thr(308) phosphorylation and GSK-3alpha Ser(21) phosphorylation in tibialis anterior muscles from Akt2-deficient animals. Our data establish a key role for Akt2 in the regulation of GSK-3alpha Ser(21) phosphorylation with contraction and add genetic evidence to support the separation of the intracellular pathways regulated by insulin and exercise that converge on glucose uptake and glycogen synthesis in skeletal muscle.     

Metformin can activate imidazoline I-2 receptors to lower plasma glucose in type 1-like diabetic rats

Metformin is widely used in clinic for handling the diabetic disorders. However, action mechanisms of metformin remain obscure. It has recently been indicated that guanidinium derivatives are ligands to activate type-2 imidazoline receptors (I-2 receptors) that can improve diabetes through increment in skeletal muscle glucose uptake. Also, activation of I-2 receptors can increase the release of ?-endorphin in diabetic animals. Because metformin is a guanidinium derivative, we were interested in the effect of metformin on I-2 receptors. In the present study, the marked blood glucose-lowering action of metformin in streptozotocin-induced type-1 like diabetes rats was blocked by specific I-2 receptor antagonist, BU224, in a dose-dependent manner. Also, the increase of ?-endorphin release by metformin was blocked by BU224 in same manner. A specific competition between metformin and BU224 was observed in isolated adrenal medulla. Otherwise, amiloride at the dose sufficient to block I-2A receptor abolished the metformin-induced ?-endorphin release, but only the blood glucose-lowering action of metformin was markedly reduced. In addition, the blood glucose-lowering action of metformin in bilateral adrenalectomized rats was diminished by amiloride at higher doses. These results suggest that metformin might activate imidazoline I-2 receptors while I-2A receptors link the increase of ?-endorphin release and I-2B receptors couple to the other actions for lowering of blood glucose in type-1 like diabetic rats.     

Myo1c regulates glucose uptake in mouse skeletal muscle

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.     

Insulin-independent pathways mediating glucose uptake in hindlimb-suspended skeletal muscle.

Insulin resistance accompanies atrophy in slow-twitch skeletal muscles such as the soleus. Using a rat hindlimb suspension model of atrophy, we have previously shown that an upregulation of JNK occurs in atrophic muscles and correlates with the degradation of insulin receptor substrate-1 (IRS-1) (Hilder TL, Tou JC, Grindeland RF, Wade CE, and Graves LM. FEBS Lett 553: 63-67, 2003), suggesting that insulin-dependent glucose uptake may be impaired. However, during atrophy, these muscles preferentially use carbohydrates as a fuel source. To investigate this apparent dichotomy, we examined insulin-independent pathways involved in glucose uptake following a 2- to 13-wk hindlimb suspension regimen. JNK activity was elevated throughout the time course, and IRS-1 was degraded as early as 2 wk. AMP-activated protein kinase (AMPK) activity was significantly higher in atrophic soleus muscle, as were the activities of the ERK1/2 and p38 MAPKs. As a comparison, we examined the kinase activity in solei of rats exposed to hypergravity conditions (2 G). IRS-1 phosphorylation, protein, and AMPK activity were not affected by 2 G, demonstrating that these changes were only observed in soleus muscle from hindlimb-suspended animals. To further examine the effect of AMPK activation on glucose uptake, C2C12 myotubes were treated with the AMPK activator metformin and then challenged with the JNK activator anisomycin. While anisomycin reduced insulin-stimulated glucose uptake to control levels, metformin significantly increased glucose uptake in the presence of anisomycin and was independent of insulin. Taken together, these results suggest that AMPK may be an important mediator of insulin-independent glucose uptake in soleus during skeletal muscle atrophy.     

Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.     

Limitations to basal and insulin-stimulated skeletal muscle glucose uptake in the high-fat-fed rat

Rats fed a high-fat diet display blunted insulin-stimulated skeletal muscle glucose uptake. It is not clear whether this is due solely to a defect in glucose transport, or if glucose delivery and phosphorylation are also impaired. To determine this, rats were fed standard chow (control rats) or a high-fat diet (HF rats) for 4 wk. Experiments were then performed on conscious rats under basal conditions or during hyperinsulinemic euglycemic clamps. Rats received primed constant infusions of 3-O-methyl-[(3)H]glucose (3-O-MG) and [1-(14)C]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 soleus. Total muscle glucose was also measured in two fast-twitch muscles. Muscle glucose uptake was markedly decreased in HF rats. In control rats, hyperinsulinemia resulted in a decrease in soleus TSGG compared with basal, due to increased [G](im). In HF rats during hyperinsulinemia, [G](im) also exceeded zero. Hyperinsulinemia also decreased muscle glucose in HF rats, implicating impaired glucose delivery. In conclusion, defects in extracellular and intracellular components of muscle glucose uptake are of major functional significance in this model of insulin resistance.     

Hydroxylamine enhances glucose uptake in C2C12 skeletal muscle cells through the activation of insulin receptor substrate 1

Diabetes mellitus is a global disease, and the number of patients with it is increasing. Of various agents for treatment, those that directly act on muscle are currently attracting attention because muscle is one of the main tissues in the human body, and its metabolism is decreased in type II diabetes. In this study, we found that hydroxylamine (HA) enhances glucose uptake in C2C12 myotubes. Analysis of HA’s mechanism revealed the involvement of IRS1, PI3 K and Akt that is related to the insulin signaling pathway. Further investigation about the activation mechanism of insulin receptor or IRS1 by HA may provide a way to develop a novel anti-diabetic agent alternating to insulin.     

Hypoglycaemia induced by Trichinella infection is due to the increase of glucose uptake in infected muscle cells

The present study investigates how Trichinella infection induces host hypoglycaemia and explores a potential relationship between infection and the insulin signalling pathway. The results showed that mice infected with Trichinella spiralis or Trichinella pseudospiralis exhibited a temporary decrease in blood glucose level between 8 and 28 days p.i. and the kinetics of the glucose levels corresponded to the process of muscle larval growth and development. Histochemical results showed that glycogen accumulation increased in infected muscle cells during the period of hypoglycaemia. Analysis of gene expression profiles with quantitative PCR demonstrated that insulin signalling pathway-related genes, such as insulin receptor (IR), insulin receptor substance 1 (IRS-1), IRS-2, phosphatidylinositol 3-kinase (PI3-K) and V-akt murine thymoma viral oncogene homologue 2 (Akt2) were up-regulated in infected muscle cells during infection and these expression changes correlated with the kinetics of blood glucose level, glycogen accumulation and the process of larval growth and development in infected muscle cells. Western blot analysis clarified that the expression of IR and Akt2 proteins increased in muscle tissues infected with both species of Trichinella. This study suggests that hypoglycaemia induced by Trichinella infection is the result of an increase in glucose uptake by infected muscle cells via up-regulation of insulin signalling pathway factors.     

AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle

The AMP-activated protein kinase (AMPK) has been hypothesized to mediate contraction and 5-aminoimidazole-4-carboxamide 1-beta-D-ribonucleoside (AICAR)-induced increases in glucose uptake in skeletal muscle. The purpose of the current study was to determine whether treadmill exercise and isolated muscle contractions in rat skeletal muscle increase the activity of the AMPK alpha 1 and AMPK alpha 2 catalytic subunits in a dose-dependent manner and to evaluate the effects of the putative AMPK inhibitors adenine 9-beta-D-arabinofuranoside (ara-A), 8-bromo-AMP, and iodotubercidin on AMPK activity and 3-O-methyl-D-glucose (3-MG) uptake. There were dose-dependent increases in AMPK alpha 2 activity and 3-MG uptake in rat epitrochlearis muscles with treadmill running exercise but no effect of exercise on AMPK alpha1 activity. Tetanic contractions of isolated epitrochlearis muscles in vitro significantly increased the activity of both AMPK isoforms in a dose-dependent manner and at a similar rate compared with increases in 3-MG uptake. In isolated muscles, the putative AMPK inhibitors ara-A, 8-bromo-AMP, and iodotubercidin fully inhibited AICAR-stimulated AMPK alpha 2 activity and 3-MG uptake but had little effect on AMPK alpha 1 activity. In contrast, these compounds had absent or minimal effects on contraction-stimulated AMPK alpha 1 and -alpha 2 activity and 3-MG uptake. Although the AMPK alpha 1 and -alpha 2 isoforms are activated during tetanic muscle contractions in vitro, in fast-glycolytic fibers, the activation of AMPK alpha 2-containing complexes may be more important in regulating exercise-mediated skeletal muscle metabolism in vivo. Development of new compounds will be required to study contraction regulation of AMPK by pharmacological inhibition.     

Contraction-induced increase in Vmax of palmitate uptake and oxidation in perfused skeletal muscle

To evaluate the effects of contractions on thekinetics of uptake and oxidation of palmitate in a physiological musclepreparation, rat hindquarters were perfused with glucose (6 mmol/l),albumin-bound [1-¹⁴C]palmitate, andvarying amounts of albumin-bound palmitate (200-2,200 µmol/l) atrest and during muscle contractions. When plotted against the unboundpalmitate concentration, palmitate uptake and oxidation displayedsimple Michaelis-Menten kinetics with estimated maximal velocity(V_max)and Michaelis-Menten constant(K_m) values of42.8 ± 3.8 (SE)nmol · min¹ · g¹and 13.4 ± 3.4 nmol/l for palmitate uptake and 3.8 ± 0.4 nmol · min¹ · g¹and 8.1 ± 2.9 nmol/l for palmitate oxidation, respectively, at rest.Whereas muscle contractions increased theV_maxfor both palmitate uptake and oxidation to 91.6 ± 10.1 and 16.5 ± 2.3 nmol · min¹ · g¹,respectively, theK_m remainedunchanged.V_maxand K_m estimates obtained from Hanes-Woolf plots (substrate concentration/velocity vs.substrate concentration) were not significantly different. In theresting perfused hindquarter, an increase in palmitate delivery from31.9 ± 0.9 to 48.7 ± 1.2 µmol · g¹ · h¹by increasing perfusate flow was associated with a decrease in thefractional uptake of palmitate so that the rates of uptake andoxidation of palmitate remained unchanged. It is concluded that therates of uptake and oxidation of long-chain fatty acids (LCFA) saturatewith an increase in the concentration of unbound LCFA in perfusedskeletal muscle and that muscle contractions, but not an increase inplasma flow, increase theV_maxfor LCFA uptake and oxidation. The data are consistent with the notion that uptake of LCFA in muscle may be mediated in part by a transport system.