1. In freshly isolated rat hepatocytes, the activity of the AMP-activated protein kinase is high, but decreases by 5 -10-fold during incubation of the cells for 60 min. The expressed activity of acetyl-CoA carboxylase is initially very low, then rises in a reciprocal manner to the AMP-activated protein kinase activity. For both enzymes, treatment of partially purified preparations under dephosphorylating conditions abolishes the difference in activity between freshly isolated and preincubated cells. Thus, both the high activity of the AMP-activated protein kinase and the low activity of acetyl-CoA carboxylase in freshly isolated cells can be explained by phosphorylation.2. Immediately after isolation, the hepatocytes have AMP/ATP ratios that are unphysiologically high (z 1 : 1 S). During incubation of the cells for 60 min, AMP levels fall and ATP levels rise so that the ratio becomes about 1 : 15, close to previous estimates of the ratio in freeze-clamped liver. The fall in AMP/ATP ratio precedes the decrease in AMP-activated protein kinase activity.3. In cells which have been incubated for 60 min, treatment with 20 mM fructose, which causes a large but transient increase in the AMP/ATP ratio, also causes concomitant activation of the AMP-activated protein kinase and inactivation of acetyl-CoA carboxylase.4. In all cases described above, the increases in activity of acetyl-CoA carboxylase were blocked by treatment with the cell-permeable protein phosphatase inhibitor, okadaic acid. However, the decreases in activity of the AMP-activated protein kinase were not blocked by this inhibitor. This is consistent with the finding that okadaicacid-insensitive protein phosphatase 2C is the most effective at dephosphorylating the kinase in cell-free assays.5. The results above suggested that AMP either promotes phosphorylation, or inhibits dephosphorylation, of the kinase. Studies in a partially purified cell-free system suggested that the former hypothesis was correct; reactivation of dephosphorylated AMP-activated protein kinase by kinase kinase was completely dependent on the presence of AMP.6. Our results, obtained in both intact cells and a cell-free system, suggest that rises in the AMP/ATP ratio promote phosphorylation of the AMP-activated protein kinase by the kinase kinase, as well as causing direct allosteric activation. This represents a very sensitive system for switching off lipid biosynthetic pathways when ATP levels are limiting. The results with okadaic acid also suggest that protein phosphatase 2C is mainly responsible for dephosphorylation of the AMP-activated protein kinase in intact hepatocytes. Abbreviations. Fmoc, fluorenylmethoxycarbonyl; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; TosLysCH2C1, tosyllysinechloromethane; TosPheCH2C1, tosylphenylalaninechloromethane.Enzymes. AMP-activated protein kinase (EC 2.7.1.109); acetylCoA carboxylase (EC 6.4.1.2); HMG-CoA reductase (EC 1.1.1.34); hormone-sensitive lipase (EC 3.1.1.3; 3.1.1.13); adenylate kinase (EC 2.7.4.3); cyclic-AMP-dependent protein kina...

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AMP-activated protein kinase (AMPK) functions as a α/β/γ heterotrimer to preserve ATP levels and so cell viability during stressful conditions. However, its role in aiding survival of adult skeletal muscle precursor cells is unclear. Using the differentiating mouse C2C12 postnatal skeletal muscle myoblast cell line, we have determined that proteins for the AMPK subunit isoforms α2 and γ2 are constitutively expressed, while those for α1, β1 and β2 are undetectable in undifferentiated myoblasts but increasingly expressed with differentiation to myotubes. Although the γ3 subunit is expressed at a low level in myoblasts, it too is expressed increasingly with differentiation to myotubes. The p50 but not the p72 isoform of the embryonic α subunit homologue MELK is expressed only in proliferating myoblasts, while the ARK5 α subunit homologue is increasingly expressed with differentiation. Myotubes displayed higher basal and stimulated α1/α2 AMPK activation than myoblasts. Furthermore, serum starvation resulted in less apoptosis of differentiated myotubes than of undifferentiated myoblasts. This reflects, in part, the increased expression of functional AMPK in the myotubes, since specific inhibition of AMPK activity with 6-[4-(2-piperidin-1-ylethoxy)-phenyl]-3-pyridin-4-ylpyrazolo[1,5-α] pyrimidine (Compound C) exacerbated the apoptosis resulting from serum withdrawal. If these in vitro events can also occur in vivo, they could have implications for pathologies such as muscle wasting, in which undifferentiated satellite stem cells may be easier apoptotic targets than their differentiated counterparts. Furthermore, these results suggest that when interpreting results from in vitro or in vivo experiments on AMPK, the subunit expression profile should be taken into account.

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