The function and survival of all organisms is dependent on the dynamic control of energy metabolism, when energy demand is matched to energy supply. The AMP-activated protein kinase (AMPK) αβγ heterotrimer has emerged as an important integrator of signals that control energy balance through the regulation of multiple biochemical pathways in all eukaryotes. In this review, we begin with the discovery of the AMPK family and discuss the recent structural studies that have revealed the molecular basis for AMP binding to the enzyme's γ subunit. AMPK's regulation involves autoinhibitory features and phosphorylation of both the catalytic α subunit and the β-targeting subunit. We review the role of AMPK at the cellular level through examination of its many substrates and discuss how it controls cellular energy balance. We look at how AMPK integrates stress responses such as exercise as well as nutrient and hormonal signals to control food intake, energy expenditure, and substrate utilization at the whole body level. Lastly, we review the possible role of AMPK in multiple common diseases and the role of the new age of drugs targeting AMPK signaling.

؊/؊ murine embryo fibroblasts. These data indicate that the CaMKKs function in intact cells as AMPKKs, predicting wider roles for these kinases in regulating AMPK activity in vivo.

The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK coimmunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca 2+ -calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca 2+ -calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaMbinding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.z 1999 Federation of European Biochemical Societies.

Although interleukin-6 (IL-6) has been associated with insulin resistance, little is known regarding the effects of IL-6 on insulin sensitivity in humans in vivo. Here, we show that IL-6 infusion increases glucose disposal without affecting the complete suppression of endogenous glucose production during a hyperinsulinemic-euglycemic clamp in healthy humans. Because skeletal muscle accounts for most of the insulin-stimulated glucose disposal in vivo, we examined the mechanism(s) by which IL-6 may affect muscle metabolism using L6 myotubes. IL-6 treatment increased fatty acid oxidation, basal and insulin-stimulated glucose uptake, and translocation of GLUT4 to the plasma membrane. Furthermore, IL-6 rapidly and markedly increased AMP-activated protein kinase (AMPK). To determine whether the activation of AMPK mediated cellular metabolic events, we conducted experiments using L6 myotubes infected with dominant-negative AMPK ␣-subunit. The effects described above were abrogated in AMPK dominant-negative-infected cells. Our results demonstrate that acute IL-6 treatment enhances insulin-stimulated glucose disposal in humans in vivo, while the effects of IL-6 on glucose and fatty acid metabolism in vitro appear to be mediated by AMPK. Diabetes

The obesity epidemic has led to an increased incidence of non–alcoholic fatty liver disease (NAFLD) and type 2 diabetes. AMP–activated protein kinase (Ampk) regulates energy homeostasis and is activated by cellular stress, hormones and the widely prescribed anti–type 2 diabetic drug metformin1,2. Ampk phosphorylates murine acetyl–CoA carboxylase3,4 (Acc) 1 at Ser79 and Acc2 at Ser212, inhibiting the conversion of acetyl–CoA to malonyl–CoA, a precursor in fatty acid synthesis5 as well as an allosteric inhibitor of fatty acid transport into mitochondria for oxidation6. To test the physiological impact of these phosphorylation events we generated mice with alanine knock–in mutations in both Acc1 (Ser79) and Acc2 (Ser212) (Acc double knock–in, AccDKI). These mice have elevated lipogenesis and lower fatty acid oxidation compared to wild–type (WT) mice, which contribute to the progression of insulin resistance, glucose intolerance and NAFLD, but not obesity. Remarkably, AccDKI mice made obese by high–fat feeding, are refractory to the lipid–lowering and insulin–sensitizing effects of metformin. These findings establish that inhibitory phosphorylation of Acc by Ampk is essential for the control of lipid metabolism, and in the setting of obesity, for metformin–induced improvements in insulin action.

The regulatory domain of protein kinase C contains an amino acid sequence between residues 19 and 36 that resembles a substrate phosphorylation site in its distribution of basic residue recognition determinants. The corresponding synthetic peptide (Arg19-Phe-Ala-Arg-Lys-Gly-Ala25-Leu-Arg-Gln-Lys-Asn-Val-His -Glu-Val-Lys-Asn36) acts as a potent substrate antagonist with an inhibitory constant of 147 +/- 9 nM. It is a specific inhibitor of protein kinase C and inhibits both autophosphorylation and protein substrate phosphorylation. Substitution of Ala25 with serine transforms the pseudosubstrate into a potent substrate. These results demonstrate that the conserved region of the regulatory domain (residues 19 to 36) of protein kinase C has the secondary structural features of a pseudosubstrate and may be responsible for maintaining the enzyme in the inactive form in the absence of allosteric activators such as phospholipids.

Abstract-The activity of the endothelial nitric oxide synthase (eNOS) can be regulated independently of an increase in Ca 2ϩ by the phosphorylation of Ser 1177 but results only in a low nitric oxide (NO) output. In the present study, we assessed whether the agonist-induced (Ca 2ϩ -dependent, high-output) activation of eNOS is associated with changes in the phosphorylation of

Salicylate, a plant product, has been in medicinal use since ancient times. More recently it has been replaced by synthetic derivatives such as aspirin and salsalate, both rapidly broken down to salicylate in vivo. At concentrations reached in plasma following administration of salsalate, or aspirin at high doses, salicylate activates adenosine monophosphate-activated protein kinase (AMPK), a central regulator of cell growth and metabolism. Salicylate binds at the same site as the synthetic activator, A-769662, to cause allosteric activation and inhibition of dephosphorylation of the activating phosphorylation site, Thr172. In AMPK knockout mice, effects of salicylate to increase fat utilization and lower plasma fatty acids in vivo were lost. Our results suggest that AMPK activation could explain some beneficial effects of salsalate and aspirin in humans.The medicinal effects of willow bark have been known since the time of Hippocrates. The active component is salicylate, a hormone produced by plants in response to pathogen infection (1). For medicinal use it was largely replaced by aspirin (acetyl salicylate), which is rapidly broken down to salicylate in vivo (2, 3). Salicylate can also be administered as salsalate, which shows promise for treatment of insulin resistance and type 2 diabetes (4, 5). Aspirin and salicylate inhibit cyclo-oxygenases and hence prostanoid biosynthesis (6), as well as the protein kinase IKKβ in the NF-κB pathway (7). However, some effects of these drugs are still observed in mice deficient in these pathways (8).Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor conserved throughout eukaryotes. This heterotrimeric enzyme is composed of catalytic α Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts subunits and regulatory β and γ subunits (9, 10). Once activated in response to metabolic stress, AMPK phosphorylates targets that switch off adenosine triphosphate (ATP) consuming processes, while switching on catabolic pathways that generate ATP. AMPK is activated >100-fold by phosphorylation at Thr172 in the α subunit by the tumour suppressor protein kinase, LKB1, or the Ca 2+ -dependent kinase, CaMKKβ (9, 10). Binding of AMP or adenosine diphosphate (ADP) to the γ subunit triggers a conformational change that promotes phosphorylation and inhibits dephosphorylation (11-15), causing a switch to the active form. Binding of AMP (but not ADP) to a second site (15) causes further allosteric activation, leading to >1,000-fold activation overall (16). Most drugs or xenobiotics that activate AMPK work by inhibiting mitochondrial ATP synthesis and increasing the concentration of AMP and ADP (17). However, a synthetic activator, A-769662 (18), which also causes allosteric activation and inhibits Thr172 dephosphorylation, binds directly to AMPK at distinct site(s) (19-21).Salicylate, but not aspirin, activated AMPK when applied to HEK-293 cells, with its effects being significant at 1 mM and above ( Fig. 1A; it appears that the estera...