AMP-activated protein kinase

5'AMP-activated protein kinase or AMPK consists of three proteins (subunits) that together make a functional enzyme, conserved from yeast to humans, that plays a role in cellular energy homeostasis. It is expressed in a number of tissues, including the liver, brain, and skeletal muscle. The net effect of AMPK activation is stimulation of hepatic fatty acid oxidation and ketogenesis, inhibition of cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibition of adipocyte lipolysis and lipogenesis, stimulation of skeletal muscle fatty acid oxidation and muscle glucose uptake, and modulation of insulin secretion by pancreatic beta-cells (4).

Structure
The heterotrimeric protein AMPK is formed by α, β, and γ subunits. Each of these three subunits takes on a specific role for both the stability and activity of AMPK (11). Specifically, the γ subunit includes four particular Cystathione Beta Synthase (CBS) domains giving AMPK its ability to sensitively detect shifts in the AMP:ATP ratio. The four CBS domains create two binding sites for AMP commonly referred to as Bateman domains. Binding of one AMP to a Bateman domain cooperatively increases the binding affinity of the second AMP to the other Bateman domain (49). As AMP binds both Bateman domains the γ subunit undergoes a conformational change which exposes the catalytic domain found on the α subunit. It is in this catalytic domain where AMPK becomes activated when phosphorylation takes place at threonine-172 by an upstream AMPK kinase (AMPKK) (19). The α, β, and γ subunits can also be found in different isoforms: the γ subunit can exist as either the γ1, γ2 or γ3 isoform; the β subunit can exist as either the β1 or β2 isoform; and the α subunit can exist as either the α1 or α2 isoform. Although the most common isoforms expressed in most cells are the α1, β1, and γ1 isoforms, it has been demonstrated that the α2, β2, γ2, and γ3 isoforms are also expressed in cardiac and [skeletal muscle (11, 12, 13).

Function
AMPK acts as a metabolic master switch regulating several intracellular systems including the cellular uptake of glucose, the β-oxidation of fatty acids and the biogenesis of glucose transporter 4 (GLUT4) and mitochondria (1, 2, 4, 5, 36). The energy-sensing capability of AMPK can be attributed to its ability to detect and react to fluctuations in the AMP:ATP ratio that take place during rest and exercise (muscle stimulation). During muscle stimulation, AMP increases while ATP decreases, which changes AMPK into a good substrate for activation via an upstream kinase complex, AMPKK. AMPKK is a complex of three proteins, STE-related adaptor (STRAD), mouse protein 25 (MO25), and LKB1 (a serine/threonine kinase). During a bout of exercise, AMPK activity increases while the muscle cell experiences metabolic stress brought about by an extreme cellular demand for ATP. Upon activation, AMPK increases celluar energy levels by inhibiting anabolic energy consuming pathways (fatty acid synthesis, protein synthesis, etc.) and stimulating energy producing, catabolic pathways (fatty acid oxidation, glucose transport, etc.).

Recent research on mice at Harvard University has shown that when the activity of AMPK was inhibited, the mice ate less and lost weight, but these data are controversial. When AMPK levels were artificially raised the mice ate more and gained weight. Research in Britain has shown that the appetite-stimulating hormone ghrelin also affects AMPK levels.

A 2001 study (Zhou G et al) has indicated that the antidiabetic drug metformin (Glucophage®) acts by stimulating AMPK, leading to reduced insulin resistance in the liver. Metformin usually causes weight loss and reduced appetite, not weight gain and increased appetite, which is opposite of what might be expected given the Harvard mouse study results.

AMPK Activation
Triggering the activation of AMPK can be carried out provided that two conditions are met. First, the γ subunit of AMPK must undergo a conformational change so as to expose the active site (Thr-172) on the α subunit. The conformational change of the γ subunit of AMPK can be accomplished under increased concentrations of AMP. Increased concentrations of AMP will give rise to the conformational change on the γ subunit of AMPK as two AMP bind the two Bateman domains located on that subunit. It is this conformational change brought about by increased concentrations of AMP that exposes the active site (Thr-172) on the α subunit. This critical role of AMP is further substantiated in experiments that demonstrate AMPK activation via an AMP analogue 5-amino-4-imidazolecarboxamide ribotide (ZMP) which is derived from the familiar 5-amino-4-imidazolecarboxamide riboside (AICAR) (14, 15, 16, 17). The second condition that must be met is the phosphorylation and consequent activation of AMPK on its activating loop at Thr-172 of the α subunit brought about by an upstream kinase (AMPKK) (19, 20). The complex formed between LKB1 (STK 11), mouse protein 25 (MO25), and the pseudokinase STE-related adaptor protein (STRAD) has of late been identified as the major upstream kinase responsible for phosphorylation of AMPK on its activating loop at Thr-172 (21, 22, 23). Although AMPK must be phosphorylated by the LKB1/MO25/STRAD complex, it can also be regulated by allosteric modulators which directly increase general AMPK activity and modify AMPK to make it a better substrate for AMPKK and a worse substrate for phosphatases (19, 20, 24). It has recently been found that 3-phosphoglycerate (glycolysis intermediate) acts to further pronounce AMPK activation via AMPKK. Muscle contraction is the main method carried out by the body that can provide the conditions mentioned above needed for AMPK activation (25). As muscles contract, ATP is hydrolyzed, forming ADP. ADP then helps to replenish cellular ATP by donating a phosphate group to another ADP, forming an ATP and an AMP. As more AMP is produced during muscle contraction, the AMP:ATP ratio dramatically increases, leading to the allosteric activation of AMPK (10, 26, 27). This fact is further authenticated with studies, such as those sited above, that used electrical stimuli as a means to contract muscle to facilitate AMPK activation (1, 6, 18, 28, 29).

For over a decade it has been known that calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) can phosphorylate and thereby activate AMPK, but it was not the main AMPKK in liver (26). Richter et al (27) found that CaMKK inhibitors strongly inhibited AMPK phosphorylation in mouse soleus and EDL muscles after 2 minutes of contraction, but much less as time of contraction increased. CaMKK inhibitors had no effect on 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) phosphorylation and activation of AMPK (27). AICAR is taken into the cell and converted to ZMP, an AMP analog that has been shown to activate AMPK (28). Recent LKB1 knockout studies have shown that without LKB1, electrical and AICAR stimulation of muscle results in very little phosphorylation of AMPK and of ACC, providing evidence that LKB1-STRAD-MO25 is the major AMPKK in muscle (29).

AMPK and Exercise/Training
Many biochemical adaptations of skeletal muscle that take place during a single bout of exercise or an extended duration of training, such as increased mitochondrial biogenesis and capacity (31, 32), increased muscle glycogen (37), and an increase in enzymes which specialize in glucose uptake in cells such as GLUT4 and Hexokinase II (33, 35, 37) are thought to be mediated in part by AMPK when it is activated (2, 34). Additionally, recent discoveries can conceivably suggest a direct AMPK role in increasing blood supply to exercised/trained muscle cells by stimulating and stabilizing both vasculogenesis and angiogenesis (30). Taken together, these adaptations most likely transpire as a result of both temporary and maintained increases in AMPK activity brought about by increases in the AMP:ATP ratio during single bouts of exercise and long-term training. During a single acute exercise bout, AMPK takes on immediate roles to allow the contracting muscle cells adapt to the energy challenges taking place by increasing Hexokinase II expression (37), GLUT4 translocation to the plasma membrane (28, 40, 41, 42) for glucose uptake, and by stimulating glycolysis (50). If exercise bouts continue through a long-term training regimen, AMPK and other signals will facilitate contracting muscle adaptations by escorting muscle cell activity to a metabolic transition resulting in an oxidative dependent approach to energy metabolism as opposed to a glycolytic approach. AMPK accomplishes this transition to the oxidative mode of metabolism by upregulating and activating oxidative enzymes such as GLUT4, Hexokinase II, PPARalpha, PGC-1, UCP-3, Cytochrome C and TFAM, just to name a few (2, 35, 37, 38, 39, 43).

AMPKK and Exercise/Training
AMPK activity increases with exercise and the LKB1/MO25/STRAD complex is considered to be the major upstream AMPKK of the 5’-AMP-activated protein kinase phosphorylating the α subunit of AMPK at Thr-172 (19, 20, 21, 22). This fact is puzzling considering that although AMPKK protein abundance has been shown to increase in skeletal tissue with endurance training, its level of activity has been shown to decrease with endurance training in both trained and untrained tissue (18, 29, 45, 46). Currently, the activity of AMPKK immediately following a 2-hr bout of exercise of an endurance trained rat is unclear. It is possible that there exists a direct link between the observed decrease in AMPKK activity in endurance trained skeletal muscle and the apparent decrease in the AMPK response to exercise with endurance training.

AMPK and Adipocytokine Relations
Adipokines, also known as adipocytokines, are secreted by adipose tissue to take on several different but important physiological roles in the body including the regulation of appetite, metabolism, fatty acid catabolism, coagulation and systemic inflammation, for example. Collectively, the adipokines are in essence cytokines (cell-to-cell signaling proteins) which, when secreted, act on other cells, usually resulting in a biochemical and metabolic response. Two particular adipokines, adiponectin and leptin, have even been demonstrated to regulate AMPK. It has been known for some time now that among many of the metabolic roles of leptin, one of its main functions in skeletal muscle is the upregulation of fatty acid oxidation. Recently, a study revealed that leptin is able to do this by way of the AMPK signaling pathway (44). A similar study showed that much like leptin, adiponectin also stimulates the oxidation of fatty acids via the AMPK pathway, and that it also stimulates the uptake of glucose in skeletal muscle (45). As of yet, the metabolic roles of leptin and adiponectin pertaining to biochemical adaptations to long-term endurance training remain unclear. Certainly future studies will involve an investigation of leptin and adiponectin activities and their respective relationships with the AMPK signaling pathway immediately following a high-intensity endurance training protocol.

AMPK and Maximum Life Span
The C.elegans homologue of AMPK, aak-2, has been shown by Michael Ristow and colleagues to be required for extension of life span in states of glucose restriction mediating a process named mitohormesis.

AMPK and Lipid Metabolism
One of the effects of exercise is an increase in fatty acid metabolism, which provides more energy for the cell. One of the key pathways in AMPK’s regulation of fatty acid oxidation is the phosphorylation and inactivation of acetyl-CoA carboxylase (30). Acetyl-CoA carboxylase (ACC) converts acetyl-CoA to malonyl-CoA, an inhibitor of carnitine parmitoyltransferase 1 (CPT-1). CPT-1 transports fatty acids into the mitochondria for oxidation. Inactivation of ACC, therefore, results in increased fatty acid transport and subsequent oxidation. It is also thought that the decrease in malonyl-CoA occurs as a result of malonyl-CoA decarboxylase (MCD), which may be regulated by AMPK (31). MCD is an antagonist to ACC, decarboxylating malonyl-CoA to acetyl-CoA, resulting in decreased malonyl-CoA and increased CPT-1 and fatty acid oxidation. AMPK also plays an important role in lipid metabolism in the liver. It has long been known that hepatic ACC has been regulated in the liver by phosphorylation (32). AMPK also phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), a key enzyme in cholesterol synthesis (28). HMGR converts 3-hydroxy-3-methylglutaryl-CoA, which is made from acetyl-CoA, into mevalonic acid, which then travels down several more metabolic steps to become cholesterol. AMPK, therefore, helps regulate fatty acid oxidation and cholesterol synthesis.

AMPK and Glucose Transport
Insulin is a hormone which helps regulate glucose levels in the body. When blood glucose is high, insulin is released from the Islets of Langerhans. Insulin, among other things, will then facilitate the uptake of glucose into cells via increased expression and translocation of glucose transporter GLUT-4 (34). Under conditions of exercise, however, blood sugar levels are not necessarily high, and insulin is not necessarily activated, yet muscles are still able to bring in glucose. AMPK seems to be responsible in part for this exercise-induced glucose uptake. Goodyear et al. (33) observed that with exercise, the concentration of GLUT-4 was increased in the plasma membrane, but decreased in the microsomal membranes, suggesting that exercise facilitates the translocation of vesicular GLUT-4 to the plasma membrane. While acute exercise increases GLUT-4 translocation, endurance training will increase the total amount of GLUT-4 protein available (35). It has been shown that both electrical contraction and AICAR treatment increase AMPK activation, glucose uptake, and GLUT-4 translocation in perfused rat hindlimb muscle, linking exercise-induced glucose uptake to AMPK (36, 37, 38). Chronic AICAR injections, simulating some of the effects of endurance training, also increase the total amount of GLUT-4 protein in the muscle cell (39). Two proteins are essential for the regulation of GLUT-4 expression at a transcriptional level – myocyte enhancer factor 2 (MEF2) and GLUT-4 enhancer factor (GEF). Mutations in the DNA binding regions for either of these proteins results in ablation of transgene GLUT-4 expression (40, 41). These results prompted a study in 2005 which showed that AMPK directly phosphorylates GEF, but it doesn’t seem to directly activate MEF2 (42). AICAR treatment has been shown, however, to increase transport of both proteins into the nucleus, as well as increase the binding of both to the GLUT-4 promoter region (42). There is another protein involved in carbohydrate metabolism that is worthy of mention along with GLUT-4. The enzyme hexokinase phosphorylates a six-carbon sugar, most notably glucose, which is the first step in glycolysis. When glucose is transported into the cell it is phosphorylated by hexokinase. This phosphorylation keeps glucose from leaving the cell, and by changing the structure of glucose through phosphorylation, it decreases the concentration of glucose molecules, allowing a gradient for more glucose to be transported into the cell. Hexokinase II transcription is increased in both red and white skeletal muscle upon treatment with AICAR (43). With chronic injections of AICAR, total protein content of hexokinase II increases in rat skeletal muscle (44).

AMPK and Mitochondria
Mitochondria are often called the powerhouse of the cell. After pyruvate is formed from glucose during glycolysis in the cytoplasm, it is transported into the mitochondria, where it is oxidized to acetyl-CoA and enters the citric acid cycle. Oxidative phosphorylation is the process by which NADH and FADH2 are oxidized and oxygen is reduced. This process creates a proton gradient which is used to drive ATP synthase and produce ATP. This process creates energy for cellular properties, and since ATP is indispensable in the contraction process, so then, are mitochondria. Mitochondrial enzymes, such as cytochrome c, succinate dehydrogenase, malate dehydrogenase, α-ketoglutarate dehydrogenase, and citrate synthase, increase in expression and activity in response to exercise (45). AICAR stimulation of AMPK increases cytochrome c and δ-aminolevulinate synthase (ALAS), a rate-limiting enzyme involved in the production of heme. Malate dehydrogenase and succinate dehydrogenase also increase, as well as citrate synthase activity, in rats treated with AICAR injections (46). Conversely, in LKB1 knockout mice, there are decreases in cytochrome c and citrate synthase activity, even if the mice are “trained” by voluntary exercise (47). Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is a transcriptional regulator for genes involved in mitochondrial biogenesis, fatty acid oxidation, and gluconeogenesis (48). To do this, it enhances the activity of transcription factors like nuclear respiratory factor 1 (NRF-1), myocyte enhancer factor 2 (MEF2), host cell factor (HCF), and others (49, 50). It also has a positive feedback loop, enhancing its own expression (51). Both MEF2 and cAMP response element (CRE) are essential for contraction-induced PGC-1α promoter activity (50). AMPK is required for increased PGC-1α expression in skeletal muscle in response to creatine depletion (52). LKB1 knockout mice show a decrease in PGC-1α, as well as mitochondrial proteins (47).

Thyroid Hormone and AMPK
AMPK and thyroid hormone regulate some similar processes. Knowing these similarities, Winder and Hardie et al designed an experiment to see if AMPK was influenced by thyroid hormone (1). They found that all of the subunits of AMPK were increased in skeletal muscle, especially in the soleus and red quadriceps, with thyroid hormone treatment. There was also an increase in phospho-ACC, a marker of AMPK activity.

Controversy
A seemingly paradoxical role of AMPK occurs when we take a closer look at the energy-sensing enzyme in relation to exercise and long-term training. Similar to short-term acute training scale, long-term endurance training studies also reveal increases in oxidative metabolic enzymes and increases in GLUT-4, mitochondrial size and quantity, and an increased dependency on the oxidation of fatty acids; however, Winder et al. reported in 2002 that despite observing these increased oxidative biochemical adaptations to long-term endurance training (similar to those mentioned above), the AMPK response (activation of AMPK with the onset of exercise) to acute bouts of exercise decreased in red quadriceps (RQ) with training (3 – see Fig.1). Conversely, the study did not observe the same results in white quadriceps (WQ) and soleus (SOL) muscles that they did in RQ. The trained rats used for that endurance study ran on [[treadmills 5 days/wk in two 1-h sessions, morning and afternoon. The rats were also running up to 31m/min (grade 15%).  Finally, following training, the rats were sacrificed either at rest or following 10 min. of exercise. Because the AMPK response to exercise decreases with increased training duration, many questions arise that would challenge the AMPK role with respect to biochemical adaptations to exercise and endurance training.  This is due in part to the marked increases in the biogenesis and upregulation of mitochondria, GLUT-4, UCP-3, Hexokinase II and other metabolic and mitochondrial enzymes despite decreases in AMPK activity with training.  Questions also arise because skeletal muscle cells which express these decreases in AMPK activity in response to endurance training also seem to be maintaining an oxidative dependent approach to energy metabolism, which is likewise thought to be regulated to some extent by AMPK activity (38, 39). If the AMPK response to exercise is responsible in part for biochemical adaptations to training, how then can these adaptations to training be maintained if the AMPK response to exercise is being attenuated with training?  It is hypothesized that these adaptive roles to training are maintained by AMPK activity and that the increases in AMPK activity in response to exercise in trained skeletal muscle have not yet been observed due to biochemical adaptations that the training itself stimulated in the muscle tissue to reduce the metabolic need for AMPK activation.  In other words, AMPK will not become activated until it is "apparent" that the cell is in need of greater adaptation to exercise.  Until energy stores (ATP) are depleted (ATP low + AMP high), AMPK will remain inactivated.  Biochemical preparations for a high-intensity energy challenge must be exhausted before AMPK is to be activated in order to mediate further metabolic adaptations to exercise.