was established, which allowed for direct quantitation of the unphosphorylated and phosphorylated artificial peptide substrate, as well as the adenine nucleotides. Our results show a 1000-fold activation of AMPK by the combined effects of upstream kinase and saturating concentrations of AMP. The two AMPK isoforms exhibit similar specific activities (6 mol/ min/mg) and do not differ significantly by their responsiveness to AMP. Due to the inherent instability of ATP and ADP, it proved impossible to assay AMPK activity in the absolute absence of AMP. However, the half-maximal stimulatory effect of AMP is reached below 2 M. AMP does not appear to augment phosphorylation by upstream kinases in the purified in vitro system, but deactivation by dephosphorylation of AMPK ␣-subunits at Thr-172 by protein phosphatase 2C␣ is attenuated by AMP. Furthermore, it is shown that neither purified NAD ؉ nor NADH alters the activity of AMPK in a concentration range of 0 -300 M, respectively. Finally, evidence is provided that ZMP, a compound formed in 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside-treated cells to activate AMPK in vivo, allosterically activates purified AMPK in vitro, but compared with AMP, maximal activity is not reached. These data shed new light on physiologically important aspects of AMPK regulation.

The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by b-adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormonesensitive lipase (HSL). TAG resynthesis is associated with high-energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA-mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKa1 at Ser-173 to impede threonine (Thr-172) phosphorylation and thus activation of AMPKa1 by LKB1 in response to lipolytic signals. Activation of AMPKa1 by LKB1 is also blocked by PKA-mediated phosphorylation of AMPKa1 in vitro. Functional analysis of an AMPKa1 species carrying a non-phosphorylatable mutation at Ser-173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKAactivating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.

Abstract-AMP-activated protein kinase (AMPK) is emerging as an important signaling protein during myocardial ischemia. AMPK is a heterotrimeric complex containing an ␣ catalytic subunit and ␤ and ␥ regulatory subunits. Phosphorylation of Thr 172 in the activation loop of the ␣ subunit by upstream AMPK kinase(s) (AMPKK) is a critical determinant of AMPK activity. However, the mechanisms regulating AMPK phosphorylation in the ischemic heart remain uncertain and were therefore investigated. In the isolated working rat heart, low-flow ischemia rapidly activated AMPKK activity when measured using recombinant AMPK (rAMPK) as substrate. The addition of AMP (10 to 200 mol/L) augmented the ability of heterotrimeric ␣ 1 ␤ 1 ␥ 1 or ␣ 2 ␤ 1 ␥ 1 rAMPK to be phosphorylated by heart AMPKK in vitro, whereas physiologic concentrations of ATP inhibited rAMPK phosphorylation. However, neither AMP nor ATP directly influenced AMPKK activity: they had no effect on AMPKK-mediated phosphorylation of rAMPK substrates lacking normal AMP-binding ␥ subunits (isolated truncated ␣ 1 1-312 or ␣ 1 ␤ 1 ␥ 1 rAMPK containing an R70Q mutation in the ␥ 1 AMP-binding site). Regional ischemia in vivo also increased AMPKK activity and AMPK phosphorylation in the rat heart. AMPK phosphorylation could also be induced in vivo without activating AMPKK: AICAR infusion increased AMPK phosphorylation without activating AMPKK; however, the AMP-mimetic AICAR metabolite ZMP enhanced the ability of heterotrimeric rAMPK to be phosphorylated by AMPKK. Thus, heart AMPKK activity is increased by ischemia and its ability to phosphorylate AMPK is highly modulated by the interaction of AMP and ATP with the heterotrimeric AMPK complex, indicating that dual mechanisms regulate AMPKK action in the ischemic heart. (Circ Res. 2005;96:337-345.) Key Words: AMP-activated protein kinase Ⅲ AMPK kinase Ⅲ ischemia A MP-activated protein kinase (AMPK) regulates energy generating metabolic and biosynthetic pathways during physiologic and pathologic cellular stress. AMPK activation stimulates fatty acid oxidation, 1 promotes glucose transport, 2,3 accelerates glycolysis, 4 and inhibits triglyceride 5 and protein synthesis. 6 By increasing ATP synthesis and decreasing ATP utilization, AMPK functions to maintain normal cellular energy stores during ischemia. Chronic activation of AMPK also phosphorylates transcription factors altering gene expression 7 and modulates muscle mitochondrial biogenesis. 8 AMPK is a heterotrimer consisting of an ␣ catalytic subunit and ␤ and ␥ regulatory subunits. The primary mechanism responsible for AMPK activation involves phosphorylation of the Thr 172 residue located within the activation loop of the ␣ catalytic subunit. 9 Additional phosphorylation sites have been identified on the ␣ and ␤ subunits, but their functional roles remain uncertain. 10,11 Activation of AMPK during myocardial ischemia, 1,12 exercise, 13 hypoglycemia, 14 and hypoxia 15 is associated with ATP breakdown and increases in intracellular AMP. However, AMPK is also phosphorylated thr...

Background: AMP-activated protein kinase (AMPK) is a current drug target. AMPK can attach to glycogen granules.Results: Autophosphorylation of AMPK prevents its association with glycogen.Conclusion: Subcellular localization of AMPK is affected by the kinase autophosphorylation status.Significance: Understanding the regulation of AMPK at subcellular level is crucial for the currently pursued drug targeting approach.