Increased expression of GLUT-4 and hexokinase in rat epitrochlearis muscles exposed to AICAR in vitro.

Exercise acutely stimulates muscle glucose transport and also brings about an adaptive increase in the capacity of muscle for glucose uptake by inducing increases in GLUT-4 and hexokinase.(1) Recent studies have provided evidence that activation of AMP protein kinase (AMPK) is involved in the stimulation of glucose transport by exercise. The purpose of this study was to determine whether activation of AMPK is also involved in mediating the adaptive increases in GLUT-4 and hexokinase. To this end, we examined the effect of incubating rat epitrochlearis muscles in culture medium for 18 h in the presence or absence of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which enters cells and is converted to the AMP analog ZMP, thus activating AMPK. Exposure of muscles to 0.5 mM AICAR in vitro for 18 h resulted in an approximately 50% increase in GLUT-4 protein and an approximately 80% increase in hexokinase. This finding provides strong evidence in support of the hypothesis that the activation of AMPK that occurs in muscle during exercise is involved in mediating the adaptive increases in GLUT-4 and hexokinase.     

Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle.

Muscle contraction causes an increase in activity of 5'-AMP-activated protein kinase (AMPK). This study was designed to determine whether chronic chemical activation of AMPK will increase mitochondrial enzymes, GLUT-4, and hexokinase in different types of skeletal muscle of resting rats. In acute studies, rats were subcutaneously injected with either 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mg/g body wt) in 0.9% NaCl or with 0.9% NaCl alone and were then anesthetized for collection and freezing of tissues. AMPK activity increased in the superficial, white region of the quadriceps and in soleus muscles but not in the deep, red region of the quadriceps muscle. Acetyl-CoA carboxylase (ACC) activity, a target for AMPK, decreased in all three muscle types in response to AICAR injection but was lowest in the white quadriceps. In rats given daily, 1 mg/g body wt, subcutaneous injections of AICAR for 4 wk, activities of citrate synthase, succinate dehydrogenase, and malate dehydrogenase were increased in white quadriceps and soleus but not in red quadriceps. Cytochrome c and delta-aminolevulinic acid synthase levels were increased in white, but not red, quadriceps. Carnitine palmitoyl-transferase and hydroxy-acyl-CoA dehydrogenase were not significantly increased. Hexokinase was markedly increased in all three muscles, and GLUT-4 was increased in red and white quadriceps. These results suggest that chronic AMPK activation may mediate the effects of muscle contraction on some, but not all, biochemical adaptations of muscle to endurance exercise training.     

AMP-activated protein kinase activation prevents denervation-induced decline in gastrocnemius GLUT-4.

This study was designed to determine whether the reductions in GLUT-4 seen in 3-day-denervated muscles can be prevented through chemical activation of 5'-AMP-activated protein kinase (AMPK). Muscle AMPK can be chemically activated in rats using subcutaneous injections with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). In this study, the tibial nerve was sectioned on one side; the other was sham operated but without nerve section. Acute injections of AICAR resulted in significantly increased AMPK activity in denervated gastrocnemius but not soleus muscles. Acetyl-CoA carboxylase activity, a reporter of AMPK activation, declined in both gastrocnemius and soleus in both denervated and contralateral muscles. Three days after denervation, GLUT-4 levels were significantly decreased by approximately 40% in gastrocnemius muscles and by approximately 30% in soleus muscles. When rats were injected with AICAR (1 mg/g body wt) for 3 days, the decline in GLUT-4 levels was prevented in denervated gastrocnemius muscles but not in denervated soleus muscles. The extent of denervation-induced muscle atrophy was similar in AICAR-treated vs. saline-treated rats. These studies provide evidence that some effects of denervation may be prevented by chemical activation of the appropriate signaling pathways.     

Effects of acute bouts of running and swimming exercise on PGC-1alpha protein expression in rat epitrochlearis and soleus muscle

The purpose of this study was to elucidate the mechanisms underlying low-intensity exercise-induced peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) protein expression in rat skeletal muscles. Rats (5-6 wk old) swam without a load and ran on the treadmill at a speed of 13 m/min, respectively, in two 3-h sessions separated by 45 min of rest. PGC-1alpha content in epitrochlearis muscle (EPI) was increased by 75 and 95%, immediately and 6 h after swimming, respectively, with no increase in PGC-1alpha content in the soleus (SOL). After running, PGC-1alpha content in EPI was unchanged, whereas a 107% increase in PGC-1alpha content was observed in SOL 6 h after running. Furthermore, in EPI and SOL as well as other muscles (triceps, plantaris, red and white gastrocnemius), PGC-1alpha expression was enhanced concomitant with reduced glycogen postexercise, suggesting that expression of PGC-1alpha occurs in skeletal muscle recruited during exercise. PGC-1alpha content in EPI was increased after 18-h in vitro incubation with 0.5 mM 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and 4 mM caffeine. However, AICAR incubation did not affect PGC-1alpha content in the SOL, whereas caffeine incubation increased it. These results suggest that exercise-induced PGC-1alpha expression in skeletal muscle may be mediated by at least two exercise-induced signaling factors: AMPK activation and Ca2+ elevation. The number of factors involved (both AMPK and Ca2+, or Ca2+ only) in exercise-induced PGC-1alpha expression may differ among muscles.     

Effect of fiber type and nutritional state on AICAR- and contraction-stimulated glucose transport in rat muscle

AMP-activated protein kinase (AMPK) may mediate the stimulatory effect of contraction and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on glucose transport in skeletal muscle. In muscles with different fiber type composition from fasted rats, AICAR increased 2-deoxyglucose transport and total AMPK activity approximately twofold in epitrochlearis (EPI), less in flexor digitorum brevis, and not at all in soleus muscles. Contraction increased both transport and AMPK activity more than AICAR did. In EPI muscles, the effects of AICAR and contractions on glucose transport were partially additive despite a lower AMPK activity with AICAR compared with contraction alone. In EPI from fed rats, glucose transport responses were smaller than what was seen in fasted rats, and AICAR did not increase transport despite an increase in AMPK activity. AICAR and contraction activated both alpha(1)- and alpha(2)-isoforms of AMPK. Expression of both isoforms varied with fiber types, and alpha(2) was highly expressed in nuclei. In conclusion, AICAR-stimulated glucose transport varies with muscle fiber type and nutritional state. AMPK is unlikely to be the sole mediator of contraction-stimulated glucose transport.     

Effect of acute activation of 5'-AMP-activated protein kinase on glycogen regulation in isolated rat skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been implicated in glycogen metabolism in skeletal muscle. However, the physiological relevance of increased AMPK activity during exercise has not been fully clarified. This study was performed to determine the direct effects of acute AMPK activation on muscle glycogen regulation. For this purpose, we used an isolated rat muscle preparation and pharmacologically activated AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR). Tetanic contraction in vitro markedly activated the alpha(1)- and alpha(2)-isoforms of AMPK, with a corresponding increase in the rate of 3-O-methylglucose uptake. Incubation with AICAR elicited similar enhancement of AMPK activity and 3-O-methylglucose uptake in rat epitrochlearis muscle. In contrast, whereas contraction stimulated glycogen synthase (GS), AICAR treatment decreased GS activity. Insulin-stimulated GS activity also decreased after AICAR treatment. Whereas contraction activated glycogen phosphorylase (GP), AICAR did not alter GP activity. The muscle glycogen content decreased in response to contraction but was unchanged by AICAR. Lactate release was markedly increased when muscles were stimulated with AICAR in buffer containing glucose, indicating that the glucose taken up into the muscle was catabolized via glycolysis. Our results suggest that AMPK does not mediate contraction-stimulated glycogen synthesis or glycogenolysis in skeletal muscle and also that acute AMPK activation leads to an increased glycolytic flux by antagonizing contraction-stimulated glycogen synthesis.     

Adaptive response of hypertrophied skeletal muscle to endurance training

The response of hypertrophied soleus and plantaris muscle of rats to endurance training was studied. Hypertrophy was produced by bilateral extirpation of the gastrocnemius muscle. A 13-wk training program of treadmill running initiated 30 days after removal of the gastrocnemius muscle accentuated (P less than 0.01) the hypertrophy. Succinate dehydrogenase activities of the enlarged muscles of sedentary rats were similar to those of normal animals, as were the increases associated with training. Phosphorylase and hexokinase activities were unaltered as a result of the experimental perturbations. Rates of glycogen depletion during exercise were lower (P less than 0.01) in the liver and soleus and plantaris muscles of endurance-trained animals. No difference existed in the rate of glycogen depletion of normal and hypertrophied muscle within the sedentary or trained groups. These data demonstrate that extensively hypertrophied muscle responds to training and exercise in a manner similar to that of normal muscle.     

Energy-sensing and signaling by AMP-activated protein kinase in skeletal muscle.

AMP-activated protein kinase (AMPK) is emerging as an important energy-sensing/signaling system in skeletal muscle. This kinase is activated allosterically by 5'-AMP and inhibited allosterically by creatine phosphate. Phosphorylation of AMPK by an upstream kinase, AMPK kinase (also activated allosterically by 5'-AMP), results in activation. It is activated in both rat and human muscle in response to muscle contraction, the extent of activation depending on work rate and muscle glycogen concentration. AMPK can also be activated chemically in resting muscle with 5-aminoimidazole-4-carboxamide-riboside, which enters the muscle and is phosphorylated to form ZMP, a nucleotide that mimics the effect of 5'-AMP. Once activated, AMPK is hypothesized to phosphorylate proteins involved in triggering fatty acid oxidation and glucose uptake. Evidence is also accumulating for a role of AMPK in inducing some of the adaptations to endurance training, including the increase in muscle GLUT-4, hexokinase, uncoupling protein 3, and some of the mitochondrial oxidative enzymes. It thus appears that AMPK has the capability of monitoring intramuscular energy charge and then acutely stimulating fat oxidation and glucose uptake to counteract the increased rates of ATP utilization during muscle contraction. In addition, this system may have the capability of enhancing capacity for ATP production when the muscle is exposed to endurance training.     

5-Aminoimidazole-4-carboxamide 1-beta -D-ribofuranoside (AICAR) stimulates myocardial glycogenolysis by allosteric mechanisms

We tested the hypothesis that activation of AMP-activated protein kinase (AMPK) promotes myocardial glycogenolysis by decreasing glycogen synthase (GS) and/or increasing glycogen phosphorylase (GP) activities. Isolated working hearts from halothane-anesthetized male Sprague-Dawley rats perfused in the absence or presence of 0.8 or 1.2 mM 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR), an adenosine analog and cell-permeable activator of AMPK, were studied. Glycogen degradation was increased by AICAR, while glycogen synthesis was not affected. AICAR increased myocardial 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranotide (ZMP), the active intracellular form of AICAR, but did not alter the activity of GS and GP measured in tissue homogenates or the content of glucose-6-phosphate and adenine nucleotides in freeze-clamped tissue. Importantly, the calculated intracellular concentration of ZMP achieved in this study was similar to the K(m) value of ZMP for GP determined in homogenates of myocardial tissue. We conclude that the data are consistent with allosteric activation of GP by ZMP being responsible for the glycogenolysis caused by AICAR in the intact rat heart.     

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.     

Effects of low-intensity prolonged exercise on PGC-1 mRNA expression in rat epitrochlearis muscle

We previously reported that the peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) mRNA in rat epitrochlearis muscle was increased after swimming exercise training. In the present study, we demonstrated further that PGC-1 mRNA expression in the epitrochlearis muscle of 4-5-week-old male Sprague-Dawley rats was increased after a 6-h acute bout of low-intensity swimming exercise. With this increase, the expression level was approximately 8-fold of control and immersion group rats that stayed for 6-h in warm water, maintained at the identical temperature of the swimming barrel (35 degrees C) (p<0.01). Second, PGC-1 mRNA expression in the muscle was found to have increased 6-h after 30 10-s tetani contractions were induced by in vitro electrical stimulation. Finally, PGC-1 mRNA expression in the muscle incubated for 18-h with 0.5mM 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR: a 5' AMP-activated protein kinase (AMPK) activator) was elevated to approximately 3-fold of the control muscle (n=6, p<0.001). AMPK activity in epitrochlearis muscle after the swimming was also found to be elevated to approximately 4-fold of the pre-exercise value (p<0.001). These results may suggest that an acute bout of low-intensity prolonged swimming exercise directly enhances the PGC-1 mRNA expression in the activated muscle during exercise, possibly through, at least in part, an AMPK-related mechanism.     

Effects of chronic AICAR administration on the metabolic and contractile phenotypes of rat slow- and fast-twitch skeletal muscles

The present study examined the effects of chronic activation of 5'-AMP-activated protein kinase (AMPK) on the oxidative capacity and myosin heavy chain (MHC) based fibre phenotype of rodent fast- and slow-twitch muscles. Sprague-Dawley rats received daily injections for 4 weeks of the known AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) or vehicle (control). The AICAR group displayed increases in hexokinase-II (HXK-II) activity, expression, and phosphorylation in fast-twitch muscles (P<0.001) but not in the slow-twitch soleus (SOL). In the AICAR group, citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA-dehydrogenase (EC 1.1.1.35) were elevated 1.6- and 2.1-fold (P<0.05), respectively, in fast-twitch medial gastrocnemius (MG), and by 1.2- and 1.4-fold (P<0.05) in the slower-twitch plantaris (PLANT). No changes were observed in the slow-twitch SOL. In contrast, the activity of glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12) remained unchanged in all muscles. AICAR treatment did not alter the MHC-based fibre type composition in fast- or slow-twitch muscles, as determined by immunohistochemical and electrophoretic analytical methods or by RT-PCR. We conclude that chronic activation of AMPK mimics the metabolic changes associated with chronic exercise training (increased oxidative capacity) in the fast-twitch MG and PLANT, but does not coordinately alter MHC isoform content or mRNA expression.     

AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling

AMP-activated protein kinase (AMPK) is viewed as an energy sensor that acts to modulate glucose uptake and fatty acid oxidation in skeletal muscle. Given that protein synthesis is a high energy-consuming process, it may be transiently depressed during cellular energy stress. Thus, the intent of this investigation was to examine whether AMPK activation modulates the translational control of protein synthesis in skeletal muscle. Injections of 5-aminoimidazole-4-carboxamide 1-beta-d-ribonucleoside (AICAR) were used to activate AMPK in male rats. The activity of alpha1 AMPK remained unchanged in gastrocnemius muscle from AICAR-treated animals compared with controls, whereas alpha2 AMPK activity was significantly increased (51%). AICAR treatment resulted in a reduction in protein synthesis to 45% of the control value. This depression was associated with decreased activation of protein kinases in the mammalian target of rapamycin (mTOR) signal transduction pathway as evidenced by reduced phosphorylation of protein kinase B on Ser(473), mTOR on Ser(2448), ribosomal protein S6 kinase on Thr(389), and eukaryotic initiation factor eIF4E-binding protein on Thr(37). A reduction in eIF4E associated with eIF4G to 10% of the control value was also noted. In contrast, eIF2B activity remained unchanged in response to AICAR treatment and therefore would not appear to contribute to the depression in protein synthesis. This is the first investigation to demonstrate changes in translation initiation and skeletal muscle protein synthesis in response to AMPK activation.     

Chronic Treatment with the AMP-Kinase Activator AICAR Increases Glycogen Storage and Fatty Acid Oxidation in Skeletal Muscles but Does Not Reduce Hyperglucagonemia and Hyperglycemia in Insulin Deficient Rats

This study tested whether the glycogen-accumulating effect of chronic in vivo pharmacological 5′AMP-activated protein kinase (AMPK) activation could improve glycemic control under conditions of insulin deficiency. Male Wistar rats were rendered diabetic through the administration of streptozotocin (STZ) and then treated for 7 consecutive days with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Subsequently, glycogen content and synthesis, glucose oxidation, and fatty acid oxidation (FAO) were determined in oxidative and glycolytic skeletal muscles. Glycemia, insulinemia, glucagonemia, and circulating triglycerides (TG) and non-esterified fatty acids (NEFAs) were measured after AICAR treatment. Insulin was almost undetectable in STZ rats and these animals were severely hyperglycemic. Glycogen content was markedly low mainly in glycolytic muscles of STZ rats and AICAR treatment restored it to control values. No differences were found among all muscles studied with regards to the content and phosphorylation of Akt/protein kinase B and glycogen synthase kinase 3. Even though glycogen synthase content was reduced in all muscles from STZ rats, insulin-induced dephosphorylation/activation of this enzyme was preserved and unaffected by AICAR treatment. Glucagon and NEFAS were 2- and 7.4-fold fold higher in STZ rats than controls, respectively. AICAR did not affect hyperglycemia and hyperglucagonemia in STZ rats; however, it normalized circulating NEFAs and significantly increased FAO in glycolytic muscles. In conclusion, even though AICAR-induced AMPK activation enhanced glycogen accumulation in glycolytic muscles and normalized circulating NEFAs and TG levels, the hyperglycemic effects of glucagon likely offset the potentially glucose-lowering effects of AICAR, resulting in no improvement of glycemic control in insulin-deficient rats.     

Insulin stimulation of glucose uptake fails to decrease palmitate oxidation in muscle if AMPK is activated.

Fatty acid oxidation in muscle has been reported to be diminished when insulin and glucose levels are elevated. This study was designed to determine whether activation of AMP-activated protein kinase (AMPK) will prevent inhibitory effects of insulin and glucose on the rate of fatty acid oxidation. Rat hindlimbs were perfused with medium containing 0, 0.3, or 60 nM insulin with or without 2 mM 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). Glucose uptake was stimulated four- to fivefold by inclusion of insulin in the medium. Insulin attenuated the increase in AMPK caused by AICAR both in perfused hindlimbs and in isolated epitrochlearis muscles. The activation constant for citrate activation of acetyl-CoA carboxylase (ACC) was significantly increased in response to AICAR, and the increase was slightly attenuated if insulin was present in the perfusion medium. Insulin stimulated an increase in malonyl-CoA content of the muscles in the absence of AICAR. Malonyl-CoA was decreased to approximately the same value in AICAR-perfused muscle, regardless of insulin concentration. Muscle glucose 6-phosphate and citrate were significantly increased in response to AICAR and insulin. The rate of palmitate oxidation tended to decrease in response to insulin and in the absence of AICAR. AICAR increased palmitate oxidation to approximately the same level regardless of the insulin concentration or the rate of glucose uptake into the muscle. The rate of palmitate oxidation showed a curvilinear relationship as a function of muscle malonyl-CoA content, with half-maximal inhibition at approximately 0.6 nmol/g. We conclude that AMPK activation can prevent high rates of glucose uptake and glycolytic flux from inhibiting palmitate oxidation in predominantly fast-twitch muscle under these conditions.     

Effects of AICAR and exercise on insulin-stimulated glucose uptake, signaling, and GLUT-4 content in rat muscles.

Physical activity is known to increase insulin action in skeletal muscle, and data have indicated that 5'-AMP-activated protein kinase (AMPK) is involved in the molecular mechanisms behind this beneficial effect. 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) can be used as a pharmacological tool to repetitively activate AMPK, and the objective of this study was to explore whether the increase in insulin-stimulated glucose uptake after either long-term exercise or chronic AICAR administration was followed by fiber-type-specific changes in insulin signaling and/or changes in GLUT-4 expression. Wistar rats were allocated into three groups: an exercise group trained on treadmill for 5 days, an AICAR group exposed to daily subcutaneous injections of AICAR, and a sedentary control group. AMPK activity, insulin-stimulated glucose transport, insulin signaling, and GLUT-4 expression were determined in muscles characterized by different fiber type compositions. Both exercised and AICAR-injected animals displayed a fiber-type-specific increase in glucose transport with the most marked increase in muscles with a high content of type IIb fibers. This increase was accompanied by a concomitant increase in GLUT-4 expression. Insulin signaling as assessed by phosphatidylinositol 3-kinase and PKB/Akt activity was enhanced only after AICAR administration and in a non-fiber-type-specific manner. In conclusion, chronic AICAR administration and long-term exercise both improve insulin-stimulated glucose transport in skeletal muscle in a fiber-type-specific way, and this is associated with an increase in GLUT-4 content.