AMP-activated protein kinase signaling in metabolic regulation (original) (raw)
Role of AMPK in exercise-induced glucose uptake in skeletal muscle. Skeletal muscle is the major site of insulin-stimulated glucose disposal (16), and insulin resistance in this target tissue has long been viewed as a contributing factor in the pathogenesis of T2D. Therefore, alternative pathways that stimulate skeletal muscle glucose uptake independently of insulin signaling could potentially improve glycemic control in T2D subjects. Although exercise stimulates glucose uptake in skeletal muscle independently of the insulin pathway, the underlying molecular mechanism remains largely elusive.
The finding that glucose transport can be increased in isolated skeletal muscle in response to contraction in vitro suggests that the signaling pathways elicited by internal cellular energy deficiency are partly accountable for contraction-induced glucose uptake. AMPK is considered as an attractive candidate for contraction-induced skeletal muscle glucose uptake because it is activated in parallel with elevation in AMP and a reduction in creatine phosphate energy stores (17). Furthermore, AICAR-induced activation of AMPK increases glucose uptake (18), concomitantly with glucose transporter 4 (GLUT4)fusion with plasma membrane (19). The AICAR effect on glucose uptake is not affected by inhibition of the insulin-dependent PI3K pathway and is additive to insulin-stimulated glucose uptake, reminiscent of the exercise effect. However, in rat slow-twitch (oxidative) soleus muscle, AICAR does not induce glucose uptake, although phosphorylation of AMPK is increased (20). Furthermore, in slow-twitch muscle, contraction induces glucose uptake without any apparent increase in AMPK activity when there is a high glycogen content. (21). Given that AICAR induces AMPK phosphorylation and glucose uptake in rat fast-twitch (glycolytic) epitrochlearis muscle, the role of AMPK in mediating muscle glucose uptake is likely to be dependent on fiber type.
Earlier studies linking AMPK signaling with glucose metabolism were correlative in nature. The introduction of transgenic and genetic knockout mouse models to study AMPK action has enhanced our understanding of AMPK signaling in contraction-induced glucose uptake (Table 1). Overexpression of an α2 kinase–dead subunit in skeletal muscle abolishes AICAR, but only partially impairs contraction-stimulated glucose uptake (22). Furthermore, ablation of skeletal muscle AMPK α2 and γ3 subunit expression does not affect glucose uptake stimulated by contraction, although the AICAR effect is blunted (23–25). In the skeletal muscle–specific LKB1 knockout, however, glucose uptake in response to AICAR and contraction is impaired (26). These findings suggest that additional pathways mediate contraction-induced glucose uptake.
In vivo validation of the AMPK pathway in mouse models
Despite extensive study on upstream stimuli that activate AMPK, investigation on the downstream substrate(s) of AMPK-mediated glucose uptake is lacking. Recent reports revealed that Akt substrate of 160 kDa (AS160) is an important substrate downstream of Akt that is involved in insulin-stimulated glucose uptake. In cultured adipocytes, insulin-stimulated GLUT4 translocation requires phosphorylation of AS160 (27). Phosphorylation of AS160 by Akt inhibits its GTPase-activating protein (GAP) activity, which leads to an elevation in the GTP form of a Rab protein, which in turn increases GLUT4 vesicle translocation to the plasma membrane (27–29). In addition to insulin, contraction and activation of AMPK by AICAR is associated with increased phosphorylation of AS160 in rodent skeletal muscle. Likewise, in human skeletal muscle, insulin (30) and exercise (31) stimulate phosphorylation of AS160. Phosphorylation of AS160 is impaired or abolished in skeletal muscle from AMPK α2 knockout, γ3 knockout, and α2 kinase–dead mice in response to AICAR treatment (32). This corroborates findings ofimpaired AICAR-stimulated glucose uptake in skeletal muscle of these mice (22, 24, 25). Therefore, AS160 appears to be a downstream target of AMPK in mediating glucose uptake in skeletal muscle. Moreover, AMPK complexes containing the α2 and γ3 subunits are required for AS160 phosphorylation. However, AMPK heterotrimers containing the AMPK α2 subunit are essential for AS160 phosphorylation in response to muscle contraction, whereas complexes containing the γ3 subunit are dispensable. Despite abolished contraction-stimulated AS160 phosphorylation in skeletal muscle of AMPK α2 knockout and α2 kinase–dead mice, contraction-mediated glucose uptake is unaltered or slightly impaired in these mice (22, 24), which again challenges the role of AMPK in exercise-induced glucose uptake.
Although it is apparent that AMPK mediates the effects of AICAR on glucose uptake, its role in contraction-induced glucose uptake remains unclear. Nonetheless, the therapeutic potential of AMPK activationin skeletal muscle to achieve whole-body glycemic control is apparent, given that genetic ablation of AMPK α2 activity abolishes the acute glucose-lowering effect of AICAR in mice (22–24). Further, activation of AMPK with metformin is associated with increased glucose uptake in rat skeletal muscle (33). Metformin also acts as an insulin sensitizer in isolated skeletal muscle from insulin-resistant humans (34). Therefore, the metabolic effects of metformin in T2D patients may be partly mediated by the activation of skeletal muscle AMPK (Figure 2).
Role of AMPK in the regulation of whole-body glucose homeostasis. Activation of AMPK turns on ATP-generating processes, while switching off ATP-consuming processes. In skeletal muscle, acute activation of AMPK increases glucose uptake and lipid oxidation, while chronic activation of AMPK is associated with mitochondrial biogenesis. Activation of AMPK inhibits glucose and lipid synthesis in the liver but increases lipid oxidation. Lipolysis and lipogenesis in adipose tissue are also reduced by AMPK activation. Collectively, activation of AMPK in skeletal muscle, liver, and adipose tissue results in a favorable metabolic milieu for the prevention or treatment of T2D, i.e., decreased circulating glucose, reduced plasma lipid, and ectopic fat accumulation, as well as enhanced insulin sensitivity. Activation of pancreatic AMPK is associated with decreased insulin secretion, likely a protective measure to prevent hypoglycemia during food deprivation, although this effect needs to be considered in pharmaceutical targeting of AMPK for the treatment of T2D.
Regulation of fatty acid oxidation by AMPK. The AMPK pathway has profound effects on the regulation of lipid metabolism. Fatty acid oxidation in skeletal muscle involves a rate-controlling step that is regulated by carnitine palmitoyltransferase 1 (CPT1). CPT1 transfers long-chain acyl-CoA into the mitochondria, and this process is inhibited allosterically by malonyl-CoA (35), synthesized by acetyl-CoA carboxylase (ACC) (36). The activity of ACC is regulated by reversible phosphorylation, and AMPK directly phosphorylates and inactivates this downstream target (37, 38). During exercise (37) and skeletal muscle contraction (17), activated AMPK inhibits ACC to reduce malonyl-CoA concentration, thereby driving the entry of long-chain acyl-CoA into the mitochondria for β-oxidation to restore energy balance (18, 39). The ability of AMPK to induce lipid oxidation and thus lower skeletal muscle (40) and liver (33) lipid deposition is considered an important feature for the insulin-sensitizing effect of AMPK activation. Indeed, when an activating form of AMPK γ3(R225Q) subunit is expressed in skeletal muscle via genetic manipulation, the transgenic mice are protected against the development of diet-induced skeletal muscle insulin resistance (25). This effect is associated with lower skeletal muscle triglyceride stores as a result of increased fatty acid oxidation (25).
AMPK activation, mitochondrial biogenesis, and insulin sensitivity in skeletal muscle. Exercise has been long recognized as an essential element of clinical management of T2D. Long-term exercise training enhances insulin sensitivity and glucose uptake in skeletal muscle (41, 42) and promotes mitochondrial biogenesis, which leads to improved oxidative metabolism (43). Decreased mitochondrial density and activity have been proposed to partly account for the development of skeletal muscle insulin resistance (44–46). Given that activation of AMPK recapitulates some of the exercise-induced adaptations, it is likely to mediate some beneficial effects of exercise on glucose homeostasis (41, 42).
Activation of AMPK by hypoxia and AICAR mimics contraction-enhanced insulin sensitivity of glucose transport in isolated skeletal muscle (47). In long-term treatment studies, in vivoAICAR administration increases GLUT4 and hexokinase II expression (48, 49) and enhances insulin-stimulated muscle glucose transport (50, 51) and GLUT4 translocation (51). In genetic mouse models, an activating mutant AMPK γ3(R225Q) subunit enhanced expression of genes essential for skeletal muscle lipid and oxidative metabolism, whereas ablation of the AMPK γ3 subunit impaired the expression of these genes (52, 53). In response to exercise, mutant γ3(R225Q) mice demonstrated enhanced skeletal muscle expression of GLUT4 and hexokinase II, whereas expression of these genes during recovery was blunted in γ3 knockout mice (54). The mechanisms for such effects are unclear. Nonetheless, several studies revealed candidate downstream players; AMPK activation is associated with increased expression of myocyte enhancer factor 2A (MEF2A) and MEF2D (55), transcription factors that regulates the GLUT4 gene promoter. Furthermore, diet-induced chronic energy deprivation in rodents activates AMPK and increases skeletal muscle mitochondrial density (56, 57), concomitantly with an elevation in nuclear respiratory factor 1 (NRF-1) activity (57) and PPARγ coactivator 1 (PGC-1) content (56), which are key regulators of mitochondrial gene expression (58). These effects, however, are essentially abolished in the AMPK α2 kinase–dead mice (56). Nevertheless, activation of AMPK by chronic AICAR treatment increases some, but not all, mitochondrial enzyme activity in rat skeletal muscle (59), and exercise-induced gene expression is unaltered in α1 and α2 knockout mice (60). Therefore, AMPK may mediate some, but not all, exercise-induced gene expression, although chronic exercise effects in the AMPK mouse genetic models remain uncharacterized.
Regulation of liver metabolism by AMPK. Glucose homeostasis is maintained by a balance between hepatic glucose production and glucose uptake by peripheral tissues. Elevated glucose production by the liver is a major cause of fasting hyperglycemia in T2D (61, 62). Gluconeogenesis in the liver is regulated by multiple enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) (63). Activation of AMPK suppresses the transcription of these genes in hepatoma cells (64), providing clues for the role of AMPK in gluconeogenesis. Furthermore, AMPK α2 liver-specific knockout mice are glucose intolerant and display fasting hyperglycemia, presumably because of elevated gluconeogenesis associated with increased PEPCK and G6Pase activity (65). Recent data have provided evidence that inhibition of the gluconeogenic program by AMPK involves a transcriptional coactivator, transducer of regulated CREB activity 2 (TORC2) (66). Glucagon and fasting conditions induce hepatic TORC2 nuclear translocation, which enhances CREB-dependent transcription of the gluconeogenic program, including PGC-1α. Conversely, AMPK activation causes TORC2 phosphorylation and sequesters the coactivator in the cytoplasm, thus blunting the expression of the gluconeogenic program. Deletion of hepatic LKB1 (an upstream kinase of AMPK) abolishes AMPK activation and leads to nuclear accumulation of TORC2, which in turn drives gluconeogenesis (67). Consistently, liver LKB1 knockout mice on a high-fat diet exhibit fasting hyperglycemia and glucose intolerance and are unresponsive to metformin treatment (67).
In addition to gluconeogenesis, AMPK action has been implicated in regulation of liver lipogenesis, lipid oxidation, and cholesterol synthesis (4, 6). AMPK suppresses glucose-induced expression of lipogenesis-associated genes such as fatty acid synthase (33, 68), ACC, and pyruvate kinase (69). In rat primary hepatocytes, activation of AMPK by AICAR or metformin increases fatty acid oxidation via inhibition of ACC (33). Consistently, in rats treated with metformin, triglyceride levels are reduced, concomitantly with an increase in β-hydroxybutyrate, suggesting elevated hepatic lipid oxidation (33). These enhanced metabolic profiles are accompanied by downregulation of lipogenic genes such as SREBP-1, fatty acid synthase, and S14 (33). Moreover, the lipid-lowering effects of metformin in cultured hepatocytes require AMPK activity (70). Taken together, the studies provide evidence that AMPK suppresses liver gluconeogenesis and lipid production, while decreasing hepatic lipid deposition via increased lipid oxidation, thus improving the glucose and lipid profile in T2D.
AMPK regulates lipid metabolism in adipocytes. A common characteristic of T2D is high circulating lipid levels of lipids, partly accounted for by impaired insulin-mediated suppression of lipolysis in adipose tissue (62, 71). Elevated circulating FFAs released by adipocytes are associated with ectopic fat accumulation and can cause insulin resistance in skeletal muscle and liver, as well as impaired insulin secretion by β cells (72). Insulin controls the release of FFAs from adipose tissue by inhibition of hormone-sensitive lipase, a rate-limiting enzyme of lipolysis. However, AMPK has also been implicated in the regulation of lipolysis through direct phosphorylation of hormone-sensitive lipase, which leads to inhibition of subsequent activation by protein kinase A (73). AMPK is thought to inhibit lipolysis, since treatment of isolated rat adipocytes with AICAR antagonizes isoprenaline-induced lipolysis (13, 74). Furthermore, expression of a constitutively active form of AMPK reduces isoproterenol-stimulated lipolysis, whereas reduced AMPK activity increases lipolysis in adipocytes (75). AMPK inhibits lipogenesis in isolated adipocytes via increased ACC phosphorylation in response to AICAR stimulation (74). AMPK also appears to control whole-body adiposity; when subjected to a high-fat diet, AMPK α2 knockout mice developed increased body weight and fat mass as compared with the wild-type mice (76). Although an in vivo antilipolytic effect of AICAR has been demonstrated (77), AMPK activity in adipocytes is paradoxically augmented in response to fasting (75), a condition when lipolysis is elevated. Furthermore, there is also evidence that activation of AMPK promotes lipolysis (78). Therefore, the physiological relevance and role of AMPK in the regulation of lipolysis in vivo remain to be addressed.
AMPK and insulin release by pancreatic β cells. The SNF1 complex (a yeast ortholog of AMPK) in S. cerevisiae responds to changes in the glucose concentration in the medium and is essential for transcriptional activation of glucose-repressed genes (3–5). This glucose-sensing property is also found in mammalian pancreatic β cells. Glucose increases the intracellular ATP/ADP ratio in β cells (79, 80), which induces closure of ATP-sensitive potassium channels (81), and influx of calcium ions (82), a critical step in exocytosis of insulin (83). Moreover, AMP and ADP concentrations in β cells decrease in response to elevations in glucose concentration (84, 85), and this suggests that AMPK could play a role in insulin release by acting as a fuel sensor. An increase in glucose levels represses AMPK activity in β cell lines (85–88), whereas AICAR-induced activation of AMPK markedly reduced glucose-stimulated insulin release from primary pancreatic islets (85, 87) and β cell lines (87, 89). Furthermore, overexpression of a constitutively active form of AMPK reduces calcium influx in response to depolarizing agents and results in repressed glucose-induced insulin release from β cell lines (87, 90). Conversely, overexpression of a dominant-negative form of AMPK leads to increases in insulin release without apparent changes in glucose metabolism and calcium influx (87). Despite its profound effect on insulin release, the role of AMPK in β cell vesicle trafficking is unclear, and downstream targets of AMPK that mediate these physiological processes remain to be identified.
Given that antidiabetic drugs such as metformin activate AMPK, AMPK activators have been proposed to counter effects on insulin release in T2D patients (91). Incubation of either human islets or cultured β cells with metformin activates AMPK and inhibits glucose-stimulated insulin secretion (88). Although seemingly undesirable in the treatment of T2D, AMPK-mediated suppression of insulin release may be physiologically relevant for maintaining glucose homeostasis through inhibition of insulin secretion during glucose deficiency. The role of AMPK in the regulation of β cell function is clearly an unresolved question that requires further investigation.