The role of AMPK in controlling metabolism and mitochondrial biogenesis during exercise Katarina Marcinko1 and Gregory R. Steinberg1,2* 1
Division of Endocrinology and Metabolism, Department of Medicine and 2Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main St. W., Hamilton, Ontario, L8N 3Z5, Canada.
*Correspondence to: Gregory R. Steinberg, Division of Endocrinology and Metabolism, Department of Medicine, McMaster University, HSC 4N63, 1280 Main St. West, Hamilton, Ontario, L8N 3Z5, Canada. Tel: 905.521.2100 ext. 21691, Fax: 905.777.7856, E-mail: [email protected] Keywords: exercise training, endurance training, AMP-activated protein kinase, insulin resistance, insulin sensitivity, dysglycemia, skeletal muscle, peroxisome proliferator activated receptor co-activator-1 (PGC-1), mitohormesis New findings - What is the topic of this review? The topic of this review is the metabolic effects of AMP-activated protein kinase (AMPK) on glucose and fatty acid (FA) uptake, FA oxidation, and mitochondrial biogenesis in skeletal muscle at rest and during exercise/muscle contractions. - What advances does it highlight? This review describes recent studies examining the molecular mechanisms by which AMPK regulates muscle metabolism. It specifically discusses the role of exercise on acetyl-CoA carboxylase (ACC) and malonyl-CoA in the regulation of FA oxidation. It also discusses the role of AMPK in regulating glucose and FA uptake during exercise. Finally, the review describes the interaction between AMPK, peroxisome proliferator activated receptor co-activator-1 (PGC-1) and SIRT1 in controlling mitochondrial biogenesis.
This is an Accepted Article that has been peer-reviewed and approved for publication in the Experimental Physiology, but has yet to undergo copy-editing and proof correction. Please cite this article as an Accepted Article; doi: 10.1113/expphysiol.2014.1540. This article is protected by copyright. All rights reserved.
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Abstract Insulin resistance is associated with defects in skeletal muscle fatty acid (FA) metabolism that contribute to the development of type 2 diabetes (T2D). Endurance exercise increases fatty acid and glucose metabolism, muscle mitochondrial content and insulin sensitivity. In skeletal muscle, basal rates of FA oxidation are dependent on AMP-activated protein kinase (AMPK) phosphorylation of acetyl-CoA carboxylase 2 (ACC2), the rate-limiting enzyme controlling the production of the metabolic intermediate malonyl-CoA. Similarly, AMPK is essential for maintaining muscle mitochondrial content in untrained mice; effects that may be mediated through regulation of the peroxisome proliferator activated receptor co-activator1 (PGC-1). However, the importance of AMPK in regulating glucose and FA uptake, FA oxidation, and mitochondrial biogenesis during and following endurance exercise training is not fully understood. A better understanding of the mechanisms by which endurance exercise regulates substrate utilization and mitochondrial biogenesis may lead to improved therapeutic and preventative strategies for the treatment of insulin resistance and T2D.
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Introduction Reduced levels of daily physical activity increase the incidence of type 2 diabetes (T2D). Skeletal muscle insulin resistance is an important cause of T2D since it is the primary site of insulin-stimulated glucose disposal. The accumulation of intramuscular lipid intermediates such as diacylglycerol, ceramide and incompletely oxidized FAs are considered causal factors in the pathogenesis of skeletal muscle insulin resistance. Because endurance exercise enhances glucose and FA utilization, understanding the mechanisms mediating these effects may be important for the treatment of T2D. The AMP-activated protein kinase (AMPK) has gained significant attention as a possible therapeutic target for the treatment of T2D because it is activated by muscle contractions and exercise in an intensity and time dependent manner (reviewed in (Boule et al., 2001)). While some studies have observed that basal AMPK activity and responses to endurance exercise are reduced in individuals with insulin resistance or T2D, this has not been observed in all studies (reviewed in (Steinberg & Jorgensen, 2007). AMPK is required for endurance exercise as mice lacking AMPK subunits in skeletal muscle have a dramatically impaired ability to perform muscle contractions, forced treadmill running and also display reduced voluntary wheel running (Steinberg & Jorgensen, 2007). Despite these impairments, surprisingly, genetic removal of skeletal muscle AMPK does not promote the development of insulin resistance (O'Neill et al., 2011; Lantier et al., 2014).
AMPK Regulation of Glucose Uptake during Exercise/Muscle Contractions. Over the last decade, the importance of AMPK on glucose uptake during muscle contractions has been examined extensively in genetically modified mouse models. It is now established that contraction-stimulated glucose uptake can occur via an AMPK223 (the major heterotrimer found in muscle) independent pathway (O'Neill et al., 2011). However, a
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concern of studies in which only a single AMPK isoform is genetically altered/deleted is that there may be residual AMPK activity that is sufficient to increase glucose uptake during muscle contractions. Consistent with this idea, male mice lacking the AMPK 2 subunit specifically in skeletal muscle had normal glucose uptake (despite reductions in AMPK activity of greater than 90%) but deletion of both AMPK 1 and 2 subunits simultaneously, results in reduced muscle (soleus and extensor digitorium longus (EDL)) glucose uptake during treadmill exercise and muscle contractions (O’Neill et al. 2011). Mice lacking both AMPK subunits in skeletal muscle have also recently been characterized and male mice have been found to have a reduction in contraction-stimulated glucose uptake in the soleus but not EDL muscle (Thomas et al., 2014). The reason for the difference between studies is not currently known but may be related to the intensity of muscle contractions or the Crepromoters used to drive deletion of AMPK subunits. Future studies investigating the mechanisms by which AMPK regulates glucose uptake during muscle contractions are warranted.
AMPK and the Regulation of FA uptake Increased FA uptake with muscle contractions is associated with relocation of the FA transporter FAT/CD36 to the plasma membrane (Jeppesen et al. 2011). The AMPK activator AICAR increases FAT/CD36 translocation via an AMPK dependent pathway; however, with muscle contraction, increases in FA uptake occur via an AMPK independent mechanism (Jeppesen et al. 2011). Recent studies have suggested calcium/calmodulin-dependent protein kinases may be required (O'Neill et al., 2011; Bujak et al., 2014; Lantier et al., 2014); an idea which is consistent with the very rapid translocation of CD36 to the plasma membrane at the onset of muscle contractions before any detectable increases in AMPK activity (Jeppesen et al. 2011).
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AMPK and Fatty Acid Oxidation Increases in FA uptake into skeletal muscle with exercise and muscle contractions is accompanied by a reduction in the activity of acetyl-CoA carboxylase (ACC), the enzyme that catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. Malonyl-CoA is a metabolic intermediate that is used for the synthesis of FAs and is an allosteric inhibitor of carnitine palmitoyl-transferase 1 (CPT1). Since rates of FA synthesis are low in skeletal muscle, the primary role of malonyl-CoA in this tissue is thought to involve regulation of CPT1 and therefore FA oxidation. ACC exists as two distinct isoforms, ACC1 and ACC2. Skeletal muscle is enriched in the ACC2 isoform which is phosphorylated at Ser221 in response to muscle contractions; an effect associated with reductions in ACC activity and malonyl-CoA and increased rates of skeletal muscle FA oxidation (Barnes et al., 2004; Jorgensen et al., 2004; Fujii et al., 2005; Steinberg et al., 2010). Surprisingly, mouse models with genetically induced reductions in skeletal muscle AMPK have normal rates of basal and AICARstimulated FA oxidation, which may be due to the presence of residual ACC phosphorylation (Abbott et al., 2009; Lantier et al., 2014). The reason for this remaining ACC phosphorylation and preserved FA oxidation in skeletal muscle but not other tissues such as liver (Abu-Elheiga et al., 2001) or macrophages (Olson et al., 2010) is not known and has raised the idea that skeletal muscle may contain an alternative ACC kinase (Hoehn et al., 2010). To directly assess the role of ACC phosphorylation in controlling FA oxidation, our group has recently generated mice with Serine-Alanine knock-in mutations in the AMPK phosphorylation sites on ACC1 (Ser79) and ACC2 (Ser212, the equivalent to ACCSer221 in humans).
These single amino acid substitutions resulted in ACC enzymes that were
constitutively active and led to elevated levels of malonyl-CoA (Vavvas et al., 1997).
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Consistent with increases in malonyl-CoA, ACC knock-in mice show reductions in skeletal muscle and liver FA oxidation, elevated liver FA synthesis and impairments in insulin sensitivity, an effect associated with the accumulation of diacylglycerol (Vavvas et al., 1997). An important finding from this study was that both ACC1 and ACC2 could contribute to the control of FA synthesis and oxidation in hepatocytes; suggesting that malonyl-CoA produced by the two different ACC isoforms does not have distinct metabolic functions (i.e. FA synthesis for ACC1 and FA oxidation for ACC2), as originally proposed. Reductions in skeletal muscle FA oxidation and insulin sensitivity are also observed in mice with a knockin mutation only to ACC2, indicating that AMPK phosphorylation of ACC2 is sufficient to regulate FA oxidation (Dzamko et al., 2008). In addition, a lack of ACC2 phosphorylation blocks AICAR-induced increases in FA oxidation, suggesting that ACC2 S212 phosphorylation is the primary point of AMPK regulation of skeletal muscle FA oxidation (O'Neill et al., 2011; Jeppesen et al., 2013). Collectively, these studies establish that ACC2 phosphorylation is sufficient for controlling malonyl-CoA content, FA oxidation and insulin sensitivity in resting skeletal muscle. The role of AMPK and ACC in regulating FA oxidation during exercise/muscle contractions has been studied in both rodents and humans. In contrast to the observations detailed above, there appears to be a mismatch between AMPK activation, ACC2 phosphorylation, malonyl-CoA levels and rates of FA oxidation. For example, it is well established that rates of FA oxidation increase until ~65% of maximal oxygen uptake; however, AMPK and ACC phosphorylation are only partially increased at these moderate exercise intensities and malonyl-CoA content is unchanged or only modestly reduced (Dzamko et al., 2010). In contrast, during high-intensity exercise where carbohydrates are preferentially utilized and absolute rates of FA oxidation decline, AMPK is activated and ACC phosphorylation increased, while malonyl-CoA levels do not change (Galic et al.,
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2011). These findings suggest that during muscle contractions and/or exercise, the concentration of malonyl-CoA is not vital for controlling mitochondrial FA flux; a concept observed in a recent report detailing insensitivity of mitochondrial FA oxidation to malonylCoA in permeabilized muscle fibers (Dzamko et al., 2008). Consistent with a potential AMPK/ACC2 independent pathway during muscle contractions/exercise, AMPK 2 kinase dead (Fullerton et al., 2013), AMPK 2 (Fullerton et al., 2013) and 2 null (O'Neill et al., 2014) mice display normal FA oxidation during exercise/muscle contractions. However, a caveat of these studies is that these mice with partial AMPK deficiencies maintain the ability to phosphorylate and inhibit ACC2 during muscle contractions (O'Neill et al., 2014). As such future studies in AMPK double null and ACC2 KI mice are warranted before it can be conclusively established that an AMPK-ACC2malonyl-CoA independent pathway regulates FA oxidation during muscle contractions and exercise.
AMPK and Mitochondrial Biogenesis Pharmacological activation of skeletal muscle AMPK enhances mitochondrial biogenesis, an effect shown to be largely dependent on expression of the AMPK 2 subunit (O'Neill et al., 2014) and peroxisome proliferator activated receptor co-activator-1 (PGC1). The overexpression of constitutively active skeletal muscle AMPK also increases PGC1 and mitochondrial function, further supporting a critical role for this pathway in regulating mitochondrial biogenesis (Odland et al., 1998; Roepstorff et al., 2005). Consistent with these findings, mice lacking AMPK subunits or LKB1 (Roepstorff et al., 2005) have reduced muscle mitochondrial contents. Many studies have investigated the mechanisms by which AMPK may regulate mitochondrial biogenesis and this has been shown to involve both direct phosphorylation and acetylation (via SIRT1) of PGC-1 (reviewed in (Smith et This article is protected by copyright. All rights reserved.
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al., 2012)). However, with exercise, mice lacking PGC-1 (Jeppesen et al., 2013) or SIRT1 have normal increases in mitochondrial biogenesis (Dzamko et al., 2008). Interestingly, in LKB1 muscle null mice, exercise training does not increase components of the mitochondrial electron transport chain (Steinberg et al., 2010). Future studies investigating the effects of exercise on mitochondrial biogenesis in mice lacking both AMPK subunits in muscle will be important to establish if factors other than the AMPK/PGC-1/SIRT1 pathway are necessary for mediating adaptations to exercise.
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Summary This report discusses the role of skeletal muscle AMPK in regulating glucose and FA uptake, FA oxidation and mitochondrial biogenesis at rest and with exercise/contractions. The regulation of FA uptake appears to be largely independent of AMPK. The importance of AMPK in controlling glucose uptake under different exercise/muscle contraction intensities and the mechanisms involved requires further clarification based on conflicting reports in mouse models lacking skeletal muscle AMPK. Under resting conditions AMPK regulation of ACC2 and PGC-1 in skeletal muscle has important roles in the regulation of FA oxidation and mitochondrial biogenesis; however, future studies are needed to determine whether AMPK is required with exercise/muscle contractions. As disturbances in AMPK control of the above metabolic pathways are implicated in insulin resistance, a better understanding of these processes during exercise may lead to improved therapeutic and preventative strategies for T2D.
Acknowledgements: We apologize to colleagues whose work could not be cited due to a 30 reference limit. We thank Adam Bujak for critical review of the manuscript. Research in the Steinberg laboratory is supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council and the Canadian Diabetes Association. GRS is a Canada Research Chair in Metabolism and Obesity.
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Garcia-Roves PM, Osler ME, Holmstrom MH & Zierath JR (2008). Gain-of-function R225Q mutation in AMP-activated protein kinase gamma3 subunit increases mitochondrial biogenesis in glycolytic skeletal muscle. J Biol Chem 283, 35724-35734. Jeppesen J, Maarbjerg SJ, Jordy AB, Fritzen AM, Pehmoller C, Sylow L, Serup AK, Jessen N, Thorsen K, Prats C, Qvortrup K, Dyck JR, Hunter RW, Sakamoto K, Thomson DM, Schjerling P, Wojtaszewski JF, Richter EA & Kiens B (2013). LKB1 regulates lipid oxidation during exercise independently of AMPK. Diabetes 62, 1490-1499. Jorgensen SB, Treebak JT, Viollet B, Schjerling P, Vaulont S, Wojtaszewski JF & Richter EA (2007). Role of AMPKalpha2 in basal, training-, and AICAR-induced GLUT4, hexokinase II, and mitochondrial protein expression in mouse muscle. Am J Physiol Endocrinol Metab 292, E331-339. Jorgensen SB, Viollet B, Andreelli F, Frosig C, Birk JB, Schjerling P, Vaulont S, Richter EA & Wojtaszewski JF (2004). Knockout of the alpha2 but not alpha1 5'-AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-1-beta-4ribofuranosidebut not contraction-induced glucose uptake in skeletal muscle. J Biol Chem 279, 1070-1079. Lantier L, Fentz J, Mounier R, Leclerc J, Treebak JT, Pehmoller C, Sanz N, Sakakibara I, Saint-Amand E, Rimbaud S, Maire P, Marette A, Ventura-Clapier R, Ferry A, Wojtaszewski JF, Foretz M & Viollet B (2014). AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity. FASEB J 28, 32113224. O'Neill HM, Holloway GP & Steinberg GR (2012). AMPK regulation of fatty acid metabolism and mitochondrial biogenesis: implications for obesity. Mol Cell Endocrinol 366, 135-151. O'Neill HM, Lally JS, Galic S, Thomas M, Azizi PD, Fullerton MD, Smith BK, Pulinilkunnil T, Chen Z, Samaan MC, Jorgensen SB, Dyck JR, Holloway GP, Hawke TJ, van Denderen BJ, Kemp BE & Steinberg GR (2014). AMPK phosphorylation of ACC2 is required for skeletal muscle fatty acid oxidation and insulin sensitivity in mice. Diabetologia 57, 1693-1702. O'Neill HM, Maarbjerg SJ, Crane JD, Jeppesen J, Jorgensen SB, Schertzer JD, Shyroka O, Kiens B, van Denderen BJ, Tarnopolsky MA, Kemp BE, Richter EA & Steinberg GR (2011). AMP-activated protein kinase (AMPK) beta1beta2 muscle null mice reveal an essential role for AMPK in maintaining mitochondrial content and glucose uptake during exercise. Proc Natl Acad Sci U S A 108, 16092-16097. Odland LM, Howlett RA, Heigenhauser GJ, Hultman E & Spriet LL (1998). Skeletal muscle malonyl-CoA content at the onset of exercise at varying power outputs in humans. Am J Physiol 274, E1080-1085. Philp A, Chen A, Lan D, Meyer GA, Murphy AN, Knapp AE, Olfert IM, McCurdy CE, Marcotte GR, Hogan MC, Baar K & Schenk S (2011). Sirtuin 1 (SIRT1) deacetylase activity is not required for mitochondrial biogenesis or peroxisome proliferator-
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activated receptor-gamma coactivator-1alpha (PGC-1alpha) deacetylation following endurance exercise. J Biol Chem 286, 30561-30570. Roepstorff C, Halberg N, Hillig T, Saha AK, Ruderman NB, Wojtaszewski JF, Richter EA & Kiens B (2005). Malonyl-CoA and carnitine in regulation of fat oxidation in human skeletal muscle during exercise. Am J Physiol Endocrinol Metab 288, E133-142. Smith BK, Perry CG, Koves TR, Wright DC, Smith JC, Neufer PD, Muoio DM & Holloway GP (2012). Identification of a novel malonyl-CoA IC(50) for CPT-I: implications for predicting in vivo fatty acid oxidation rates. Biochem J 448, 13-20. Steinberg GR & Jorgensen SB (2007). The AMP-activated protein kinase: role in regulation of skeletal muscle metabolism and insulin sensitivity. Mini Rev Med Chem 7, 519526. Steinberg GR, O'Neill HM, Dzamko NL, Galic S, Naim T, Koopman R, Jorgensen SB, Honeyman J, Hewitt K, Chen ZP, Schertzer JD, Scott JW, Koentgen F, Lynch GS, Watt MJ, van Denderen BJ, Campbell DJ & Kemp BE (2010). Whole body deletion of AMP-activated protein kinase {beta}2 reduces muscle AMPK activity and exercise capacity. J Biol Chem 285, 37198-37209. Tanner CB, Madsen SR, Hallowell DM, Goring DM, Moore TM, Hardman SE, Heninger MR, Atwood DR & Thomson DM (2013). Mitochondrial and performance adaptations to exercise training in mice lacking skeletal muscle LKB1. Am J Physiol Endocrinol Metab 305, E1018-1029. Thomas MM, Wang DC, D'Souza DM, Krause MP, Layne AS, Criswell DS, O'Neill HM, Connor MK, Anderson JE, Kemp BE, Steinberg GR & Hawke TJ (2014). Musclespecific AMPK beta1beta2-null mice display a myopathy due to loss of capillary density in nonpostural muscles. FASEB J 28, 2098-2107. Vavvas D, Apazidis A, Saha AK, Gamble J, Patel A, Kemp BE, Witters LA & Ruderman NB (1997). Contraction-induced changes in acetyl-CoA carboxylase and 5'-AMPactivated kinase in skeletal muscle. J Biol Chem 272, 13255-13261.
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Figure 1: Effects of AMPK on glucose and FA uptake, FA oxidation, and mitochondrial biogenesis in skeletal muscle during exercise/muscle contractions. (A) AMPK likely has a direct role in regulating glucose uptake during exercise and low-intensity muscle contractions. (B) Muscle contraction increases in FA uptake occur via an AMPK independent mechanism and calcium/calmodulin-dependent protein kinases may be required. Under resting conditions AMPK has a direct role in regulating fatty acid oxidation and mitochondrial biogenesis, effects which are dependent on the control of (C) ACC2 and (D) PGC-1 although, future studies are needed to determine whether AMPK is required for regulating these pathways during exercise/muscle contractions.
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Division of Endocrinology and Metabolism, Department of Medicine and 2Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main St. W., Hamilton, Ontario, L8N 3Z5, Canada.
*Correspondence to: Gregory R. Steinberg, Division of Endocrinology and Metabolism, Department of Medicine, McMaster University, HSC 4N63, 1280 Main St. West, Hamilton, Ontario, L8N 3Z5, Canada. Tel: 905.521.2100 ext. 21691, Fax: 905.777.7856, E-mail: [email protected] Keywords: exercise training, endurance training, AMP-activated protein kinase, insulin resistance, insulin sensitivity, dysglycemia, skeletal muscle, peroxisome proliferator activated receptor co-activator-1 (PGC-1), mitohormesis New findings - What is the topic of this review? The topic of this review is the metabolic effects of AMP-activated protein kinase (AMPK) on glucose and fatty acid (FA) uptake, FA oxidation, and mitochondrial biogenesis in skeletal muscle at rest and during exercise/muscle contractions. - What advances does it highlight? This review describes recent studies examining the molecular mechanisms by which AMPK regulates muscle metabolism. It specifically discusses the role of exercise on acetyl-CoA carboxylase (ACC) and malonyl-CoA in the regulation of FA oxidation. It also discusses the role of AMPK in regulating glucose and FA uptake during exercise. Finally, the review describes the interaction between AMPK, peroxisome proliferator activated receptor co-activator-1 (PGC-1) and SIRT1 in controlling mitochondrial biogenesis.
This is an Accepted Article that has been peer-reviewed and approved for publication in the Experimental Physiology, but has yet to undergo copy-editing and proof correction. Please cite this article as an Accepted Article; doi: 10.1113/expphysiol.2014.1540. This article is protected by copyright. All rights reserved.
1
Abstract Insulin resistance is associated with defects in skeletal muscle fatty acid (FA) metabolism that contribute to the development of type 2 diabetes (T2D). Endurance exercise increases fatty acid and glucose metabolism, muscle mitochondrial content and insulin sensitivity. In skeletal muscle, basal rates of FA oxidation are dependent on AMP-activated protein kinase (AMPK) phosphorylation of acetyl-CoA carboxylase 2 (ACC2), the rate-limiting enzyme controlling the production of the metabolic intermediate malonyl-CoA. Similarly, AMPK is essential for maintaining muscle mitochondrial content in untrained mice; effects that may be mediated through regulation of the peroxisome proliferator activated receptor co-activator1 (PGC-1). However, the importance of AMPK in regulating glucose and FA uptake, FA oxidation, and mitochondrial biogenesis during and following endurance exercise training is not fully understood. A better understanding of the mechanisms by which endurance exercise regulates substrate utilization and mitochondrial biogenesis may lead to improved therapeutic and preventative strategies for the treatment of insulin resistance and T2D.
This article is protected by copyright. All rights reserved.
2
Introduction Reduced levels of daily physical activity increase the incidence of type 2 diabetes (T2D). Skeletal muscle insulin resistance is an important cause of T2D since it is the primary site of insulin-stimulated glucose disposal. The accumulation of intramuscular lipid intermediates such as diacylglycerol, ceramide and incompletely oxidized FAs are considered causal factors in the pathogenesis of skeletal muscle insulin resistance. Because endurance exercise enhances glucose and FA utilization, understanding the mechanisms mediating these effects may be important for the treatment of T2D. The AMP-activated protein kinase (AMPK) has gained significant attention as a possible therapeutic target for the treatment of T2D because it is activated by muscle contractions and exercise in an intensity and time dependent manner (reviewed in (Boule et al., 2001)). While some studies have observed that basal AMPK activity and responses to endurance exercise are reduced in individuals with insulin resistance or T2D, this has not been observed in all studies (reviewed in (Steinberg & Jorgensen, 2007). AMPK is required for endurance exercise as mice lacking AMPK subunits in skeletal muscle have a dramatically impaired ability to perform muscle contractions, forced treadmill running and also display reduced voluntary wheel running (Steinberg & Jorgensen, 2007). Despite these impairments, surprisingly, genetic removal of skeletal muscle AMPK does not promote the development of insulin resistance (O'Neill et al., 2011; Lantier et al., 2014).
AMPK Regulation of Glucose Uptake during Exercise/Muscle Contractions. Over the last decade, the importance of AMPK on glucose uptake during muscle contractions has been examined extensively in genetically modified mouse models. It is now established that contraction-stimulated glucose uptake can occur via an AMPK223 (the major heterotrimer found in muscle) independent pathway (O'Neill et al., 2011). However, a
This article is protected by copyright. All rights reserved.
3
concern of studies in which only a single AMPK isoform is genetically altered/deleted is that there may be residual AMPK activity that is sufficient to increase glucose uptake during muscle contractions. Consistent with this idea, male mice lacking the AMPK 2 subunit specifically in skeletal muscle had normal glucose uptake (despite reductions in AMPK activity of greater than 90%) but deletion of both AMPK 1 and 2 subunits simultaneously, results in reduced muscle (soleus and extensor digitorium longus (EDL)) glucose uptake during treadmill exercise and muscle contractions (O’Neill et al. 2011). Mice lacking both AMPK subunits in skeletal muscle have also recently been characterized and male mice have been found to have a reduction in contraction-stimulated glucose uptake in the soleus but not EDL muscle (Thomas et al., 2014). The reason for the difference between studies is not currently known but may be related to the intensity of muscle contractions or the Crepromoters used to drive deletion of AMPK subunits. Future studies investigating the mechanisms by which AMPK regulates glucose uptake during muscle contractions are warranted.
AMPK and the Regulation of FA uptake Increased FA uptake with muscle contractions is associated with relocation of the FA transporter FAT/CD36 to the plasma membrane (Jeppesen et al. 2011). The AMPK activator AICAR increases FAT/CD36 translocation via an AMPK dependent pathway; however, with muscle contraction, increases in FA uptake occur via an AMPK independent mechanism (Jeppesen et al. 2011). Recent studies have suggested calcium/calmodulin-dependent protein kinases may be required (O'Neill et al., 2011; Bujak et al., 2014; Lantier et al., 2014); an idea which is consistent with the very rapid translocation of CD36 to the plasma membrane at the onset of muscle contractions before any detectable increases in AMPK activity (Jeppesen et al. 2011).
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AMPK and Fatty Acid Oxidation Increases in FA uptake into skeletal muscle with exercise and muscle contractions is accompanied by a reduction in the activity of acetyl-CoA carboxylase (ACC), the enzyme that catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. Malonyl-CoA is a metabolic intermediate that is used for the synthesis of FAs and is an allosteric inhibitor of carnitine palmitoyl-transferase 1 (CPT1). Since rates of FA synthesis are low in skeletal muscle, the primary role of malonyl-CoA in this tissue is thought to involve regulation of CPT1 and therefore FA oxidation. ACC exists as two distinct isoforms, ACC1 and ACC2. Skeletal muscle is enriched in the ACC2 isoform which is phosphorylated at Ser221 in response to muscle contractions; an effect associated with reductions in ACC activity and malonyl-CoA and increased rates of skeletal muscle FA oxidation (Barnes et al., 2004; Jorgensen et al., 2004; Fujii et al., 2005; Steinberg et al., 2010). Surprisingly, mouse models with genetically induced reductions in skeletal muscle AMPK have normal rates of basal and AICARstimulated FA oxidation, which may be due to the presence of residual ACC phosphorylation (Abbott et al., 2009; Lantier et al., 2014). The reason for this remaining ACC phosphorylation and preserved FA oxidation in skeletal muscle but not other tissues such as liver (Abu-Elheiga et al., 2001) or macrophages (Olson et al., 2010) is not known and has raised the idea that skeletal muscle may contain an alternative ACC kinase (Hoehn et al., 2010). To directly assess the role of ACC phosphorylation in controlling FA oxidation, our group has recently generated mice with Serine-Alanine knock-in mutations in the AMPK phosphorylation sites on ACC1 (Ser79) and ACC2 (Ser212, the equivalent to ACCSer221 in humans).
These single amino acid substitutions resulted in ACC enzymes that were
constitutively active and led to elevated levels of malonyl-CoA (Vavvas et al., 1997).
This article is protected by copyright. All rights reserved.
5
Consistent with increases in malonyl-CoA, ACC knock-in mice show reductions in skeletal muscle and liver FA oxidation, elevated liver FA synthesis and impairments in insulin sensitivity, an effect associated with the accumulation of diacylglycerol (Vavvas et al., 1997). An important finding from this study was that both ACC1 and ACC2 could contribute to the control of FA synthesis and oxidation in hepatocytes; suggesting that malonyl-CoA produced by the two different ACC isoforms does not have distinct metabolic functions (i.e. FA synthesis for ACC1 and FA oxidation for ACC2), as originally proposed. Reductions in skeletal muscle FA oxidation and insulin sensitivity are also observed in mice with a knockin mutation only to ACC2, indicating that AMPK phosphorylation of ACC2 is sufficient to regulate FA oxidation (Dzamko et al., 2008). In addition, a lack of ACC2 phosphorylation blocks AICAR-induced increases in FA oxidation, suggesting that ACC2 S212 phosphorylation is the primary point of AMPK regulation of skeletal muscle FA oxidation (O'Neill et al., 2011; Jeppesen et al., 2013). Collectively, these studies establish that ACC2 phosphorylation is sufficient for controlling malonyl-CoA content, FA oxidation and insulin sensitivity in resting skeletal muscle. The role of AMPK and ACC in regulating FA oxidation during exercise/muscle contractions has been studied in both rodents and humans. In contrast to the observations detailed above, there appears to be a mismatch between AMPK activation, ACC2 phosphorylation, malonyl-CoA levels and rates of FA oxidation. For example, it is well established that rates of FA oxidation increase until ~65% of maximal oxygen uptake; however, AMPK and ACC phosphorylation are only partially increased at these moderate exercise intensities and malonyl-CoA content is unchanged or only modestly reduced (Dzamko et al., 2010). In contrast, during high-intensity exercise where carbohydrates are preferentially utilized and absolute rates of FA oxidation decline, AMPK is activated and ACC phosphorylation increased, while malonyl-CoA levels do not change (Galic et al.,
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2011). These findings suggest that during muscle contractions and/or exercise, the concentration of malonyl-CoA is not vital for controlling mitochondrial FA flux; a concept observed in a recent report detailing insensitivity of mitochondrial FA oxidation to malonylCoA in permeabilized muscle fibers (Dzamko et al., 2008). Consistent with a potential AMPK/ACC2 independent pathway during muscle contractions/exercise, AMPK 2 kinase dead (Fullerton et al., 2013), AMPK 2 (Fullerton et al., 2013) and 2 null (O'Neill et al., 2014) mice display normal FA oxidation during exercise/muscle contractions. However, a caveat of these studies is that these mice with partial AMPK deficiencies maintain the ability to phosphorylate and inhibit ACC2 during muscle contractions (O'Neill et al., 2014). As such future studies in AMPK double null and ACC2 KI mice are warranted before it can be conclusively established that an AMPK-ACC2malonyl-CoA independent pathway regulates FA oxidation during muscle contractions and exercise.
AMPK and Mitochondrial Biogenesis Pharmacological activation of skeletal muscle AMPK enhances mitochondrial biogenesis, an effect shown to be largely dependent on expression of the AMPK 2 subunit (O'Neill et al., 2014) and peroxisome proliferator activated receptor co-activator-1 (PGC1). The overexpression of constitutively active skeletal muscle AMPK also increases PGC1 and mitochondrial function, further supporting a critical role for this pathway in regulating mitochondrial biogenesis (Odland et al., 1998; Roepstorff et al., 2005). Consistent with these findings, mice lacking AMPK subunits or LKB1 (Roepstorff et al., 2005) have reduced muscle mitochondrial contents. Many studies have investigated the mechanisms by which AMPK may regulate mitochondrial biogenesis and this has been shown to involve both direct phosphorylation and acetylation (via SIRT1) of PGC-1 (reviewed in (Smith et This article is protected by copyright. All rights reserved.
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al., 2012)). However, with exercise, mice lacking PGC-1 (Jeppesen et al., 2013) or SIRT1 have normal increases in mitochondrial biogenesis (Dzamko et al., 2008). Interestingly, in LKB1 muscle null mice, exercise training does not increase components of the mitochondrial electron transport chain (Steinberg et al., 2010). Future studies investigating the effects of exercise on mitochondrial biogenesis in mice lacking both AMPK subunits in muscle will be important to establish if factors other than the AMPK/PGC-1/SIRT1 pathway are necessary for mediating adaptations to exercise.
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Summary This report discusses the role of skeletal muscle AMPK in regulating glucose and FA uptake, FA oxidation and mitochondrial biogenesis at rest and with exercise/contractions. The regulation of FA uptake appears to be largely independent of AMPK. The importance of AMPK in controlling glucose uptake under different exercise/muscle contraction intensities and the mechanisms involved requires further clarification based on conflicting reports in mouse models lacking skeletal muscle AMPK. Under resting conditions AMPK regulation of ACC2 and PGC-1 in skeletal muscle has important roles in the regulation of FA oxidation and mitochondrial biogenesis; however, future studies are needed to determine whether AMPK is required with exercise/muscle contractions. As disturbances in AMPK control of the above metabolic pathways are implicated in insulin resistance, a better understanding of these processes during exercise may lead to improved therapeutic and preventative strategies for T2D.
Acknowledgements: We apologize to colleagues whose work could not be cited due to a 30 reference limit. We thank Adam Bujak for critical review of the manuscript. Research in the Steinberg laboratory is supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council and the Canadian Diabetes Association. GRS is a Canada Research Chair in Metabolism and Obesity.
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Figure 1: Effects of AMPK on glucose and FA uptake, FA oxidation, and mitochondrial biogenesis in skeletal muscle during exercise/muscle contractions. (A) AMPK likely has a direct role in regulating glucose uptake during exercise and low-intensity muscle contractions. (B) Muscle contraction increases in FA uptake occur via an AMPK independent mechanism and calcium/calmodulin-dependent protein kinases may be required. Under resting conditions AMPK has a direct role in regulating fatty acid oxidation and mitochondrial biogenesis, effects which are dependent on the control of (C) ACC2 and (D) PGC-1 although, future studies are needed to determine whether AMPK is required for regulating these pathways during exercise/muscle contractions.
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