Introduction

AMP-activated protein kinase (AMPK) was first discovered as an activity that inhibited preparations of acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase, HMGR) and was induced by AMP. AMPK induces a cascade of events within cells in response to the ever changing energy charge of the cell. The role of AMPK in regulating cellular energy charge places this enzyme at a central control point in maintaining energy homeostasis. More recent evidence has shown that AMPK activity can also be regulated by physiological stimuli, independent of the energy charge of the cell, including hormones and nutrients.

Once activated, AMPK-mediated phosphorylation events switch cells from active ATP consumption (e.g. fatty acid and cholesterol biosynthesis) to active ATP production (e.g. fatty acid and glucose oxidation). These events are rapidly initiated and are referred to as short-term regulatory processes. The activation of AMPK also exerts long-term effects at the level of both gene expression and protein synthesis. Other important activities attributable to AMPK are regulation of insulin synthesis and secretion in pancreatic islet β-cells and modulation of hypothalamic functions involved in the regulation of satiety. How these latter two functions impact obesity and diabetes will be discussed below.
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Structure of AMPK

The mammalian AMPK is a trimeric enzyme composed of a catalytic α subunit and the non-catalytic β and γ subunits. There are two genes encoding isoforms of both the α and β subunits (α1, α2, β1 and β2) and three genes encoding isoforms of the γ subunit (γ1-γ3). The α2 isoform is the subunit of AMPK found predominantly within skeletal and cardiac muscle, whereas, approximately equal distribution of both the α1 and α2 isoforms are present in hepatic AMPK. Within pancreatic islet β-cells the α1 isoform predominates.

The N-terminal half of the α subunits contains a typical serine/threonine kinase catalytic domain. Interaction with the β and γ subunits occurs via the C-terminal half of the α subunits. The yeast AMPK β subunits are lipid modified with myristic acid. Myristoylation may account for the membrane association mammalian AMPK. The core of the β subunits have a glycogen-binding domain (GBD). This domain is closely related to the isoamylase N domain subfamily and weakly related to domains in the glycogen-targeting phosphatase subunits and several starch-binding proteins. The close proximity of AMPK to cellular glycogen stores allows it to rapidly affect changes in glycogen metabolism in response to changes in metabolic demands. The γ subunits of AMPK have been shown to contain nucleotide binding sites with similarity to cystathionine β-synthase (CBS) domains. Indeed, direct AMP-binding studies have shown that AMP is bound to the γ subunits by a pair of CBS domains. Of clinical significance is the observation that mutations in the CBS domains of the γ2 subunit are associated with Wolff-Parkinson-White syndrome and familial hypertrophic cardiomyopathy.
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Regulation of AMPK

AMPK is activated by phosphorylation by one or more upstream AMPK kinases (AMPKKs). In the absence of phosphorylation there is no detectable activity of AMPK towards any substrates. Phosphorylation of AMPK occurs in the α subunit at threonine 172 (Thr-172) which lies in the activation loop. One kinase activator of AMPK is Ca²⁺-calmodulin-dependent kinase kinase β (CAMKKβ) which phosphorylates and activates AMPK in response to increased calcium. Recent evidence has demonstrated that the threonine kinase, LKB1, encoded by the Peutz-Jegher syndrome tumor suppressor gene, is required for activation of AMPK in response to stress. Loss of LKB1 activity in adult mouse liver leads to near complete loss of AMPK activity and is associated with hyperglycemia. The hyperglycemia is, in part, due to an increase in the transcription of gluconeogenic genes. Of particular significance is the increased expression of the peroxisome proliferator-activated receptor-γ (PPAR-γ) coactivator 1α (PGC-1α) which drives gluconeogenesis. Reduction in PGC-1α activity results in normalized blood glucose levels in LKB1-deficient mice.

As the name implies, AMPK is also regulated by AMP. The effects of AMP are two-fold: a direct allosteric activation and making AMPK a poorer substrate for dephosphorylation. Because AMP affects both the rate of AMPK phoshorylation in the positive direction and dephosphorylation in the negative direction, the cascade is ultrasensitive. This means that a very small rise in AMP levels can induce a dramatic increase in the activity of AMPK. The activity of adenylate kinase, catalyzing the reaction shown below, ensures that AMPK is highly sensitive to small changes in the intracellular [ATP]/[ADP] ratio.

2 ADP ----> ATP + AMP

Negative allosteric regulation of AMPK also occurs and this effect is exerted by phosphocreatine. As indicated above, the β subunits of AMPK have a glycogen-binding domain, GBD. In muscle, a high glycogen content represses AMPK activity and this is likely the result of interaction between the GBD and glycogen, although this has not been shown directly. As suggested above, the GBD of AMPK allows association of the enzyme with the regulation of glycogen metabolism by placing AMPK in close proximity to one of its substrates glycogen synthase.

AMPK has also been shown to be activated by receptors that are coupled to phospholipase C-γ (PLC-γ) and by cytokines secreted by adipose tissue (adipocytokines) such as leptin and adiponectin.
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Targets of AMPK

The signaling cascades initiated by the activation of AMPK exert effects on glucose and lipid metabolism, gene expression and protein synthesis. These effects are most important for regulating metabolic events in the liver, skeletal muscle, heart, adipose tissue, and pancreas.

Demonstration of the central role of AMPK in the regulation of metabolism in response to events such as nutrient- or exercise-induced stress. Several of the known physiologic targets for AMPK are included as well as several pathways whose flux is affected by AMPK activation. Arrows indicate positive effects of AMPK, whereas, T-lines indicate inhibitory effects. See text for definition of enzyme abbreviations.

The uptake, by skeletal muscle, accounts for >70% of the glucose removal from the serum in humans. Therefore, it should be obvious that this event is extremely important for overall glucose homeostasis, keeping in mind, of course, that glucose uptake by cardiac muscle and adipocytes cannot be excluded from consideration. An important fact related to skeletal muscle glucose uptake is that this process is markedly impaired in individuals with type 2 diabetes. The uptake of glucose increases dramatically in response to stress (such as ischemia) and exercise and is stimulated by insulin-induced recruitment of glucose transporters to the plasma membrane, primarily GLUT4. Insulin-independent recruitment of glucose transporters also occurs in skeletal muscle in response to contraction (exercise). The activation of AMPK plays an important, albeit not an exclusive, role in the induction of GLUT4 recruitment to the plasma membrane. In addition, there is some demonstration that AMPK may regulate glucose transport through GLUT1. Increased glucose uptake will result in an increase in glycolysis and ATP production.

Under ischemic conditions in the heart the activation of AMPK leads to the phosphorylation and activation of phosphofructokinase-2, PFK-2 (6-phosphofructo-2-kinase). PFK-2 is one of the most potent regulators of the rate of flux through glycolysis and gluconeogenesis. It is important to note that like many enzymes, there are multiple isoforms of PFK-2 and neither the liver nor the skeletal muscle isoforms contain the AMPK phosphorylation sites of the inducible form of PFK-2. Of particular significance is the fact that the inducible form of PFK-2 is commonly found in tumor cells and this may allow AMPK to play an important role in protecting tumor cells from hypoxic stress. Indeed, techniques for depleting AMPK in tumor cells have shown that these cells become sensitized to nutritional stress upon loss of AMPK activity.

Whereas, stress and exercise are powerful inducers of AMPK activity in skeletal muscle, additional regulators of its activity have been identified. Insulin-sensitizing drugs of the thiazolidinedione family (activators of PPAR-γ, see below) as well as the hypoglycemia drug metformin exert a portion of their effects through regulation of the activity of AMPK. As indicated above, the activity of the AMPK activating kinase, LKB1, is critical for regulation of gluconeogenic flux and consequent glucose homeostasis. The action of metformin in reducing blood glucose levels requires the activity of LKB1 in the liver for this function. Also, several adipocytokines either stimulate or inhibit AMPK activation: leptin and adiponectin have been shown to stimulate AMPK activation, whereas, resistin inhibits AMPK activation.

Within skeletal muscle and heart, activation of AMPK leads to the phosphorylation and inhibition of acetyl-CoA carboxylase (ACC). This inhibition results in a drop in the level of malonyl-CoA which itself is an inhibitor of carnitine palmitoyltransferase I (CPT I). With a drop in the inhibition of CPT I a concomitant increase in β-oxidation of fatty acid will occur within the mitochondria. An increase in fatty acid oxidation, like increases in glycolysis, will lead to increases in ATP production. In addiiton to ACC, AMPK has been shown to phosphorylate and thus regulate the activities of HMG-CoA reductase, (HMGR); hormone-sensitive lipase, (HSL); glycerophosphate acyltransferase, (GPAT); malonyl-CoA decarboxylase, (MCD); glycogen synthase, (GS) and creatine kinase, (CK). Therefore, the effects of AMPK activation are exerted on not only glucose homeostasis and fatty acid metabolism but overall energy homeostasis including glycogen metabolism, cholesterol metabolism and phosphocreatine metabolism.

Cardiac effects exerted by activation of AMPK also include phosphorylation of endothelial nitric oxide synthase, eNOS in cardiac endothelium. AMPK-mediated phosphorylation of eNOS leads to increased activity and consequent NO production and provides a link between metabolic stresses and cardiac function. In platelets, insulin action leads to an increase in eNOS activity that is due to its phosphorylation by AMPK. Activation of NO production in platelets leads to a decrease in thrombin-induced aggregation, thereby, limiting the pro-coagulant effects of platelet activation. The response of platelets to insulin function clearly indicates why disruption in insulin action is a major contributing factor in the development of the metabolic syndrome.

Not only does activation of AMPK exerts effects on enzyme activity through phosphorylation, there are marked effects on the expression of a number of glycolytic and lipogenic enzymes in the liver and adipose tissue. Included in this list are the genes for the liver isoform of pyruvate kinase (L-PK), fatty acid synthase (FAS), and ACC. Activation of AMPK leads to a reduction in the level of SREBP a transcription factor that is a key regulator of the expression of numerous lipogenic enzymes. Another transcription factor reduced in response to AMPK activation is hepatocyte nuclear factor 4α, HNF4α which is a member of the steroid/thyroid hormone superfamily. HNF4α is known to regulate the expression of several liver and pancreatic β-cell genes such as GLUT2, L-PK and preproinsulin. Of clinical significance is that mutations in HNF4α are responsible for maturity-onset diabetes of the young, MODY-1. Recent evidence indicates that the newly discovered carbohydrate-response-element-binding protein (ChREBP) is a target for AMPK in the liver. ChREBP is implicated in the transcriptional regulation of L-PK.

The target of the thiazolidinedione class of drugs used to treat type 2 diabetes is the peroxisome proliferator-activated receptor γ, PPARγ which itself may be a target for the action of AMPK. The transcription co-activator, p300, is phosphorylated by AMPK which inhibits interaction of p300 with not only PPARγ but also the retinoic acid receptor, retinoid X receptor, and thyroid receptor. Another transcription factor target of AMPK is the forkhead protein, FKHR (now referred to as FoxO1a). FoxO1a is involved in the activation of glucose-6-phosphatase expression and, therefore, loss of FoxO1a activity in response to AMPK activation will lead to reduced hepatic output of glucose.

AMPK activation in response to hypoxia exerts effects on rates of protein synthesis. Hepatic translation elongation factor, eEF2 is a target for phosphorylation in repsonse to AMPK activation. AMPK phosphorylates and activates the kinase that phosphorylates eEF2 (eEF2K) leading to inhibition of protein synthesis. Another indirect substrate for AMPK that plays a role in protein synthesis is the mammalian target of rapamycin, mTOR. A detailed description of the role of mTOR in regulating protein synthesis can be found on the Protein Synthesis page.
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Relevance of AMPK to Type 2 Diabetes

An impairment in fuel metabolism occurs in obesity and this impairment is a leading pathogenic factor in type 2 diabetes. The insulin resistance associated with type 2 diabetes is most profound at the level of skeletal muslce as this is the primary site of glucose and fatty acid utilization. Therefore, an understanding of how to activate AMPK in skeletal muscle would offer significant pharmacologic benefits in the treatment of type 2 diabetes. As indicated above, it has already been shown that metformin and the thiazolidinedione drugs exert some of their effects via activation of AMPK. In the non-pharmacologic context, activation of AMPK occurs in response to exercise, an activity known to have significant benefit for type 2 diabetics. Future targets for type 2 diabetes treatments will likely be those that can effect beneficial changes in the activity of AMPK.
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Michael W. King, Ph.D / IU School of Medicine / miking at iupui.edu