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.     

AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing protein kinase responsible for coordinating metabolism and energy demand. In rodents, exercise accelerates fatty acid metabolism, enhances glucose uptake, and stimulates nitric oxide (NO) production in skeletal muscle. AMPK phosphorylates and inhibits acetyl-coenzyme A (CoA) carboxylase (ACC) and enhances GLUT-4 translocation. It has been reported that human skeletal muscle malonyl-CoA levels do not change in response to exercise, suggesting that other mechanisms besides inhibition of ACC may be operating to accelerate fatty acid oxidation. Here, we show that a 30-s bicycle sprint exercise increases the activity of the human skeletal muscle AMPK-alpha1 and -alpha2 isoforms approximately two- to threefold and the phosphorylation of ACC at Ser(79) (AMPK phosphorylation site) approximately 8.5-fold. Under these conditions, there is also an approximately 5.5-fold increase in phosphorylation of neuronal NO synthase-mu (nNOSmu;) at Ser(1451). These observations support the concept that inhibition of ACC is an important component in stimulating fatty acid oxidation in response to exercise and that there is coordinated regulation of nNOSmu to protect the muscle from ischemia/metabolic stress.     

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside-induced AMP-activated protein kinase phosphorylation inhibits basal and insulin-stimulated glucose uptake, lipid synthesis, and fatty acid oxidation in isolated rat adipocytes

The objective of this study was to investigate the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-induced AMP-activated protein kinase (AMPK) activation on basal and insulin-stimulated glucose and fatty acid metabolism in isolated rat adipocytes. AICAR-induced AMPK activation profoundly inhibited basal and insulin-stimulated glucose uptake, lipogenesis, glucose oxidation, and lactate production in fat cells. We also describe the novel findings that AICAR-induced AMPK phosphorylation significantly reduced palmitate (32%) and oleate uptake (41%), which was followed by a 50% reduction in palmitate oxidation despite a marked increase in AMPK and acetyl-CoA carboxylase phosphorylation. Compound C, a selective inhibitor of AMPK, not only completely prevented the inhibitory effect of AICAR on palmitate oxidation but actually caused a 2.2-fold increase in this variable. Compound C also significantly increased palmitate oxidation in the presence of inhibitory concentrations of malonyl-CoA and etomoxir indicating an increase in CPT1 activity. In contrast to skeletal muscle in which AMPK stimulates fatty acid oxidation to provide ATP as a fuel, we propose that AMPK activation inhibits lipogenesis and fatty acid oxidation in adipocytes. Inhibition of lipogenesis would conserve ATP under conditions of cellular stress, although suppression of intra-adipocyte oxidation would spare fatty acids for exportation to other tissues where their utilization is crucial for energy production. Additionally, the stimulatory effect of compound C on long chain fatty acid oxidation provides a novel pharmacological approach to promote energy dissipation in adipocytes, which may be of therapeutic importance for obesity and type II diabetes.     

Alpha-lipoic acid increases insulin sensitivity by activating AMPK in skeletal muscle

Triglyceride accumulation in skeletal muscle contributes to insulin resistance in obesity. We recently showed that alpha-lipoic acid (ALA) reduces body weight and prevents the development of diabetes in diabetes-prone obese rats by reducing triglyceride accumulation in non-adipose tissues. AMP-activated protein kinase (AMPK) is a major regulator of cellular energy metabolism. We examined whether ALA lowers triglyceride accumulation in skeletal muscle by activating AMPK. Alpha2-AMPK activity was decreased in obese rats compared to control rats. Administration of ALA to obese rats increased insulin-stimulated glucose disposal in whole body and in skeletal muscle. ALA also increased fatty acid oxidation and activated AMPK in skeletal muscle. Adenovirus-mediated administration of dominant negative AMPK into skeletal muscle prevented the ALA-induced increases in fatty acid oxidation and insulin-stimulated glucose uptake. These results suggest that ALA-induced improvement of insulin sensitivity is mediated by activation of AMPK and reduced triglyceride accumulation in skeletal muscle.     

Dual regulation of the AMP-activated protein kinase provides a novel mechanism for the control of creatine kinase in skeletal muscle.

The AMP-activated protein kinase (AMPK) is activated by a fall in the ATP:AMP ratio within the cell in response to metabolic stresses. Once activated, it phosphorylates and inhibits key enzymes in energy-consuming biosynthetic pathways, thereby conserving cellular ATP. The creatine kinase-phosphocreatine system plays a key role in the control of ATP levels in tissues that have a high and rapidly fluctuating energy requirement. In this study, we provide direct evidence that these two energy-regulating systems are linked in skeletal muscle. We show that the AMPK inhibits creatine kinase by phosphorylation in vitro and in differentiated muscle cells. AMPK is itself regulated by a novel mechanism involving phosphocreatine, creatine and pH. Our findings provide an explanation for the high expression, yet apparently low activity, of AMPK in skeletal muscle, and reveal a potential mechanism for the co-ordinated regulation of energy metabolism in this tissue. Previous evidence suggests that AMPK activates fatty acid oxidation, which provides a source of ATP, following continued muscle contraction. The novel regulation of AMPK described here provides a mechanism by which energy supply can meet energy demand following the utilization of the immediate energy reserve provided by the creatine kinase-phosphocreatine system.     

Regulation of nutrient uptake by AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that is a key regulator of energy balance at both the cellular and whole-body level. AMPK acts to stimulate ATP production and reduce ATP consumption when cellular ATP levels fall, thereby normalizing energy balance. Given the central role of AMPK in cellular carbohydrate and lipid metabolism, AMPK activation has been proposed to be a therapeutic target for conditions associated with dysfunctional nutrient metabolism including obesity, type 2 diabetes, hepatic steatosis, cardiovascular diseases and cancer. One way by which increased ATP production can be achieved is by increasing the supply of nutrient substrates. In the 1990s, AMPK activation was demonstrated to stimulate glucose uptake in striated muscle, thereby improving substrate supply for ATP production. Subsequently AMPK activation was postulated to underlie the increase in glucose uptake that occurs during muscle contraction. More recently, however, several lines of evidence have demonstrated that AMPK activation is unlikely to be required for contraction-mediated glucose uptake. Furthermore, despite the importance of AMPK in cellular and whole-body metabolism, far fewer studies have investigated either the role of AMPK in glucose uptake by non-muscle tissues or whether AMPK regulates the uptake of fatty acids. In the present review, we discuss the role of AMPK in nutrient uptake by tissues, focusing on glucose uptake out with muscle and fatty acid uptake.     

Role of AMP kinase and PPARdelta in the regulation of lipid and glucose metabolism in human skeletal muscle

The peroxisome proliferator-activated receptor (PPAR)delta has been implicated in the regulation of lipid metabolism in skeletal muscle. Furthermore, activation of PPARdelta has been proposed to improve insulin sensitivity and reduce glucose levels in animal models of type 2 diabetes. We recently demonstrated that the PPARdelta agonist GW501516 activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake in skeletal muscle. However, the underlying mechanism remains to be clearly identified. In this study, we first confirmed that incubation of primary cultured human muscle cells with GW501516 induced AMPK phosphorylation and increased fatty acid transport and oxidation and glucose uptake. Using small interfering RNA, we have demonstrated that PPARdelta expression is required for the effect of GW501516 on the intracellular accumulation of fatty acids. Furthermore, we have shown that the subsequent increase in fatty acid oxidation induced by GW501516 is dependent on both PPARdelta and AMPK. Concomitant with these metabolic changes, we provide evidence that GW501516 increases the expression of key genes involved in lipid metabolism (FABP3, CPT1, and PDK4) by a PPARdelta-dependent mechanism. Finally, we have also demonstrated that the GW501516-mediated increase in glucose uptake requires AMPK but not PPARdelta. In conclusion, the PPARdelta agonist GW501516 promotes changes in lipid/glucose metabolism and gene expression in human skeletal muscle cells by PPARdelta- and AMPK-dependent and -independent mechanisms.     

Maintenance of skeletal muscle energy homeostasis during prolonged wintertime fasting in the raccoon dog (<Emphasis Type="Italic">Nyctereutes procyonoides</Emphasis>)

The raccoon dog (Nyctereutes procyonoides) is a canid species with autumnal fattening and prolonged wintertime fasting. Nonpathological body weight cycling and the ability to tolerate food deficiency make this species a unique subject for studying physiological mechanisms in energy metabolism. AMP-activated protein kinase (AMPK) is a cellular energy sensor regulating energy homeostasis. During acute fasting, AMPK promotes fatty acid oxidation and enhances glucose uptake. We evaluated the effects of prolonged fasting on muscle energy metabolism in farm-bred raccoon dogs. Total and phosphorylated AMPK and acetyl-CoA carboxylase (ACC), glucose transporter 4 (GLUT 4), insulin receptor and protein kinase B (Akt) protein expressions of hind limb muscles were determined by Western blot after 10 weeks of fasting. Plasma insulin, leptin, ghrelin, glucose and free fatty acid levels were measured, and muscle myosin heavy chain (MHC) isoform composition analyzed. Fasting had no effects on AMPK phosphorylation, but total AMPK expression decreased in m. rectus femoris, m. tibialis anterior and m. extensor digitorum longus resulting in a higher phosphorylation ratio. Decreased total expression was also observed for ACC. Fasting did not influence GLUT 4, insulin receptor or Akt expression, but Akt phosphorylation was lower in m. flexor digitorum superficialis and m. extensor digitorum longus. Three MHC isoforms (I, IIa and IIx) were detected without differences in composition between the fasted and control animals. The studied muscles were resistant to prolonged fasting indicating that raccoon dogs have an effective molecular regulatory system for preserving skeletal muscle function during wintertime immobility and fasting.     

Glucose deprivation accelerates VLDL receptor-mediated TG-rich lipoprotein uptake by AMPK activation in skeletal muscle cells

Glucose and fatty acids are major energy sources in skeletal muscle. Very low-density lipoprotein receptor (VLDL-R), which is highly expressed in heart, skeletal muscle and adipose tissue, plays a crucial role in metabolism of triglyceride (TG)-rich lipoproteins. To explore energy switching between glucose and fatty acids, we studied expression of VLDL-R and lipoprotein uptake in rat L6 myoblasts. l-Glucose or d-glucose deprivation in the medium noticeably induced the AMPK (AMP-activated protein kinase) activation and VLDL-R expression. Dose-dependent induction of VLDL-R expression was observed when d-glucose was less than 4.2 mM. The same phenomenon was also observed in rat primary skeletal myoblasts and cultured vascular smooth muscle cells. The uptake of β-VLDL but not LDL was accompanied by induction of VLDL-R expression. Our study suggests that the VLDL-R-mediated uptake of TG-rich lipoproteins might compensate for glucose shortfall through AMPK activation in skeletal muscle.     

The PPARdelta agonist, GW501516, promotes fatty acid oxidation but has no direct effect on glucose utilisation or insulin sensitivity in rat L6 skeletal muscle cells

Peroxisome proliferator-activated receptor-delta (PPARdelta) activation enhances skeletal muscle fatty acid oxidation and improves whole body glucose homeostasis and insulin sensitivity. Recently, GW501516, a selective PPARdelta agonist, was reported to increase glucose uptake in human skeletal myotubes by an AMPK-dependent mechanism that may contribute to the improved glucose tolerance. Here, we demonstrate that whilst GW501516 increases expression of PGC-1alpha and CPT-1 and stimulates fatty-acid oxidation in L6 myotubes, it fails to enhance insulin sensitivity, AMPK activity or glucose uptake and storage. Our findings exclude sarcolemmal glucose transport as a potential target for the therapeutic action of PPARdelta agonists in skeletal muscle.     

The 5′ adenosine monophosphate-activated protein kinase: Regulating the ebb and flow of cellular energetics

The 5′ adenosine monophosphate-activated protein kinase (AMPK) is a heterotrimeric, evolutionary conserved enzyme which has emerged as a critical regulator of skeletal muscle cellular bioenergetics. AMPK is activated by both chemical (adipokines) and mechanical (stretch, contraction) stimuli leading to metabolic changes within muscle cells that include increased fatty acid oxidation, glucose uptake and glycolysis, as well as the stimulation and regulation of mitochondrial biogenesis. Collectively these acute responses and chronic adaptations act to reduce cellular disturbances, resulting in tighter metabolic control and maintenance of energy homeostasis. This brief review will describe the structure, function and activation of AMPK in skeletal muscle and how this ubiquitous molecule may be a plausible target for the treatment of several lifestyle-related metabolic disorders.     

Dibenzoylmethane Exerts Metabolic Activity through Regulation of AMP-Activated Protein Kinase (AMPK)-Mediated Glucose Uptake and Adipogenesis Pathways

Dibenzoylmethane (DBM) has been shown to exert a variety of beneficial effects on human health. However, the mechanism of action is poorly understood. In this study, DBM increased phosphorylation of AMP-activated protein kinase (AMPK) and stimulated glucose uptake in a skeletal muscle cell line. Both knockdown of AMPK with siRNA and inhibition with AMPK inhibitor blocked DBM-induced glucose uptake. DBM increased the concentration of intracellular calcium and glucose uptake due to DBM was abolished by STO-609 (a calcium/calmodulin-dependent protein kinase inhibitor). DBM stimulated phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which was blocked by pretreatment with compound C, an AMPK inhibitor. The expression of glucose transporter type 4 (GLUT4) was increased by DBM. The translocation of GLUT4 to the plasma membrane was also increased by DBM in AMPK dependently. In addition, DBM suppressed weight gain and prevented fat accumulation in the liver and abdomen in mice fed a high-fat diet. In pre-adipocyte cells, DBM decreased the activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis. Expression of the adipogenic gene, fatty acid synthase (FAS), was suppressed by DBM in an AMPK-dependent manner. These results showed that the beneficial metabolic effects of DBM might be due to regulation of glucose uptake via AMPK in skeletal muscle and inhibition of adipogenesis in pre-adipocytes.     

The effect of AMP-activated protein kinase and its activator AICAR on the metabolism of human umbilical vein endothelial cells.

In several non-vascular tissues in which it has been studied, AMP-activated protein kinase (AMPK) appears to modulate the cellular response to stresses such as ischemia. In liver and muscle, it phosphorylates and inhibits acetyl CoA carboxylase (ACC), leading to an increase in fatty acid oxidation; and in muscle, its activation is associated with an increase in glucose transport. Here we report the presence of both AMPK and ACC in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with 2 mM AICAR, an AMPK activator, caused a 5-fold activation of AMPK, which was accompanied by a 70% decrease in ACC activity and a 2-fold increase in fatty acid oxidation. Surprisingly, glucose uptake and glycolysis, the dominant energy-producing pathway in HUVEC, were diminished by 40-60%. Despite this, cellular ATP levels were increased by 35%. Thus activation of AMPK by AICAR is associated with major alterations in endothelial cell energy balance. Whether these alterations protect the endothelium during ischemia or other stresses remains to be determined.     

Alpha2-AMPK activity is not essential for an increase in fatty acid oxidation during low-intensity exercise

A single bout of exercise increases glucose uptake and fatty acid oxidation in skeletal muscle, with a corresponding activation of AMP-activated protein kinase (AMPK). While the exercise-induced increase in glucose uptake is partly due to activation of AMPK, it is unclear whether the increase of fatty acid oxidation is dependent on activation of AMPK. To examine this, transgenic mice were produced expressing a dominant-negative (DN) mutant of alpha(1)-AMPK (alpha(1)-AMPK-DN) in skeletal muscle and subjected to treadmill running. alpha(1)-AMPK-DN mice exhibited a 50% reduction in alpha(1)-AMPK activity and almost complete loss of alpha(2)-AMPK activity in skeletal muscle compared with wild-type littermates (WT). The fasting-induced decrease in respiratory quotient (RQ) ratio and reduced body weight were similar in both groups. In contrast with WT mice, alpha(1)-AMPK-DN mice could not perform high-intensity (30 m/min) treadmill exercise, although their response to low-intensity (10 m/min) treadmill exercise was not compromised. Changes in oxygen consumption and the RQ ratio during sedentary and low-intensity exercise were not different between alpha(1)-AMPK-DN and WT. Importantly, at low-intensity exercise, increased fatty acid oxidation in response to exercise in soleus (type I, slow twitch muscle) or extensor digitorum longus muscle (type II, fast twitch muscle) was not impaired in alpha(1)-AMPK-DN mice, indicating that alpha(1)-AMPK-DN mice utilize fatty acid in the same manner as WT mice during low-intensity exercise. These findings suggest that an increased alpha(2)-AMPK activity is not essential for increased skeletal muscle fatty acid oxidation during endurance exercise.     

A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle

Eukaryotic cells possess systems for sensing nutritional stress and inducing compensatory mechanisms that minimize the consumption of ATP while utilizing alternative energy sources. Such stress can also be imposed by increased energy needs, such as in skeletal muscle of exercising animals. In these studies, we consider the role of the metabolic sensor, AMP-activated protein kinase (AMPK), in the regulation of glucose transport in skeletal muscle. Expression in mouse muscle of a dominant inhibitory mutant of AMPK completely blocked the ability of hypoxia or AICAR to activate hexose uptake, while only partially reducing contraction-stimulated hexose uptake. These data indicate that AMPK transmits a portion of the signal by which muscle contraction increases glucose uptake, but other AMPK-independent pathways also contribute to the response.     

Dissociation of 5' AMP-activated protein kinase activation and glucose uptake stimulation by mitochondrial uncoupling and hyperosmolar stress: differential sensitivities to intracellular Ca2+ and protein kinase C inhibition

2,4-dinitrophenol (DNP) compromises ATP production within the cell by disrupting the mitochondrial electron transport chain. The resulting loss of ATP leads to an increase in glucose uptake for anaerobic generation of ATP. In L6 skeletal muscle cells, DNP increases the rate of glucose uptake by twofold. We previously showed that DNP increases cell surface levels of glucose transporter 4 (GLUT4) and hexose uptake via a Ca2+-sensitive and conventional protein kinase C (cPKC)-dependent mechanism. Recently, 5' AMP-activated protein kinase (AMPK) has been proposed to mediate the stimulation of glucose uptake by energy stressors such as exercise and hypoxia. Changes in Ca2+ and cPKC have also been invoked in the stimulation of glucose uptake by exercise and hypoxia. Here we examine whether changes in cytosolic Ca2+ or cPKC lead to activation of AMPK. We show that treatment of L6 cells with DNP (0.5 mM) or hyperosmolar stress (mannitol, 0.6 M) increased AMPK activity by 3.5-fold. AMPK activation peaked by 10-15 min prior to maximal stimulation of glucose uptake. Intracellular Ca2+ chelation and cPKC inhibition prior to treatment with DNP and hyperosmolarity significantly reduced cell surface GLUT4 levels and hexose uptake but had no effect on AMPK activation. These results illustrate a break in the relationship between AMPK activation and glucose uptake in skeletal muscle cells. Activation of AMPK does not suffice to stimulate glucose uptake in response to DNP and hyperosmolarity.     

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.     

Insulin receptor substrates Irs1 and Irs2 coordinate skeletal muscle growth and metabolism via the Akt and AMPK pathways

Coordination of skeletal muscle growth and metabolism with nutrient availability is critical for metabolic homeostasis. To establish the role of insulin-like signaling in this process, we used muscle creatine kinase (MCK)-Cre to disrupt expression of insulin receptor substrates Irs1 and Irs2 in mouse skeletal/cardiac muscle. In 2-week-old mice, skeletal muscle masses and insulin responses were slightly affected by Irs1, but not Irs2, deficiency. In contrast, the combined deficiency of Irs1 and Irs2 (MDKO mice) severely reduced skeletal muscle growth and Akt→mTOR signaling and caused death by 3 weeks of age. Autopsy of MDKO mice revealed dilated cardiomyopathy, reflecting the known requirement of insulin-like signaling for cardiac function (P. G. Laustsen et al., Mol. Cell. Biol. 27:1649-1664, 2007). Impaired growth and function of MDKO skeletal muscle were accompanied by increased Foxo-dependent atrogene expression and amino acid release. MDKO mice were resistant to injected insulin, and their isolated skeletal muscles showed decreased insulin-stimulated glucose uptake. Glucose utilization in MDKO mice and isolated skeletal muscles was shifted from oxidation to lactate production, accompanied by an elevated AMP/ATP ratio that increased AMP-activated protein kinase (AMPK)→acetyl coenzyme A carboxylase (ACC) phosphorylation and fatty acid oxidation. Thus, insulin-like signaling via Irs1/2 is essential to terminate skeletal muscle catabolic/fasting pathways in the presence of adequate nutrition.