Evaluation of the role of AMP-activated protein kinase and its downstream targets in mammalian hibernation.

Mammalian hibernation requires an extensive reorganization of metabolism that typically includes a greater than 95% reduction in metabolic rate, selective inhibition of many ATP-consuming metabolic activities and a change in fuel use to a primary dependence on the oxidation of lipid reserves. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in this reorganization. AMPK activity and the phosphorylation state of multiple downstream targets were assessed in five organs of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) comparing euthermic animals with squirrels in deep torpor. AMPK activity was increased 3-fold in white adipose tissue from hibernating ground squirrels compared with euthermic controls, but activation was not seen in liver, skeletal muscle, brown adipose tissue or brain. Immunoblotting with phospho-specific antibodies revealed an increase in phosphorylation of eukaryotic elongation factor-2 at the inactivating Thr56 site in white adipose tissue, liver and brain of hibernators, but not in other tissues. Acetyl-CoA carboxylase phosphorylation at the inactivating Ser79 site was markedly increased in brown adipose tissue from hibernators, but no change was seen in white adipose tissue. No change was seen in the level of phosphorylation of the Ser565 AMPK site of hormone-sensitive lipase in adipose tissues of hibernating animals. In conclusion, AMPK does not appear to participate in the metabolic re-organization and/or the metabolic rate depression that occurs during ground squirrel hibernation.

[1]  D. Carling,et al.  Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia , 2000, Current Biology.

[2]  M. Carlson,et al.  The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? , 1998, Annual review of biochemistry.

[3]  W. Kolch,et al.  Raf-1-associated Protein Phosphatase 2A as a Positive Regulator of Kinase Activation* , 2000, The Journal of Biological Chemistry.

[4]  M. Lowe,et al.  Expression of a chimeric retroviral-lipase mRNA confers enhanced lipolysis in a hibernating mammal. , 2001, American journal of physiology. Regulatory, integrative and comparative physiology.

[5]  A. Sim,et al.  Location and function of three sites phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase. , 1990, European journal of biochemistry.

[6]  C. Proud,et al.  ATP depletion increases phosphorylation of elongation factor eEF2 in adult cardiomyocytes independently of inhibition of mTOR signalling , 2002, FEBS letters.

[7]  Jérôme Boudeau,et al.  Complexes between the LKB1 tumor suppressor, STRADα/β and MO25α/β are upstream kinases in the AMP-activated protein kinase cascade , 2003, Journal of biology.

[8]  D. Hardie,et al.  The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase. , 1989, Biochimica et biophysica acta.

[9]  K. Storey,et al.  Enzymes of adenylate metabolism and their role in hibernation of the white-tailed prairie dog, Cynomys leucurus. , 2000, Archives of biochemistry and biophysics.

[10]  D. Bolster,et al.  AMP-activated Protein Kinase Suppresses Protein Synthesis in Rat Skeletal Muscle through Down-regulated Mammalian Target of Rapamycin (mTOR) Signaling* , 2002, The Journal of Biological Chemistry.

[11]  R. Boutilier,et al.  Mechanisms of cell survival in hypoxia and hypothermia. , 2001, The Journal of experimental biology.

[12]  B. Kemp,et al.  Mammalian AMP-activated Protein Kinase Subfamily (*) , 1996, The Journal of Biological Chemistry.

[13]  P. Dubbelhuis,et al.  Hepatic amino acid‐dependent signaling is under the control of AMP‐dependent protein kinase , 2002, FEBS letters.

[14]  David Carling,et al.  Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[15]  G. Krause,et al.  Suppression of protein synthesis in brain during hibernation involves inhibition of protein initiation and elongation. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[16]  C. Wernstedt,et al.  Identification of Novel Phosphorylation Sites in Hormone-sensitive Lipase That Are Phosphorylated in Response to Isoproterenol and Govern Activation Properties in Vitro * , 1998, The Journal of Biological Chemistry.

[17]  D. Hardie,et al.  Phosphorylation control of cardiac acetyl-CoA carboxylase by cAMP-dependent protein kinase and 5'-AMP activated protein kinase. , 1999, European journal of biochemistry.

[18]  A. Prescott,et al.  AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the alpha2 isoform. , 1998, The Biochemical journal.

[19]  K. Storey,et al.  Glycolysis and energetics in organs of hibernating mice (Zapus hudsonius) , 1995 .

[20]  C. Proud,et al.  Regulation of peptide-chain elongation in mammalian cells. , 2002, European journal of biochemistry.

[21]  David Carling,et al.  The AMP-activated protein kinase cascade--a unifying system for energy control. , 2004, Trends in biochemical sciences.

[22]  K. Storey Mammalian hibernation. Transcriptional and translational controls. , 2003, Advances in experimental medicine and biology.

[23]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[24]  D. Hittel,et al.  The translation state of differentially expressed mRNAs in the hibernating 13-lined ground squirrel (Spermophilus tridecemlineatus). , 2002, Archives of biochemistry and biophysics.

[25]  S. Deeb,et al.  Seasonal changes in hormone-sensitive and lipoprotein lipase mRNA concentrations in marmot white adipose tissue. , 1992, The American journal of physiology.

[26]  D. Vertommen,et al.  Myocardial Ischemia and Increased Heart Work Modulate the Phosphorylation State of Eukaryotic Elongation Factor-2* , 2003, Journal of Biological Chemistry.

[27]  C. Proud,et al.  Regulation of elongation factor-2 by multisite phosphorylation. , 1993, European journal of biochemistry.

[28]  M. Rider,et al.  Regulation by noradrenaline of the mitochondrial and microsomal forms of glycerol phosphate acyltransferase in rat adipocytes. , 1983, The Biochemical journal.

[29]  P. Cohen,et al.  Primary structure of the site on bovine hormone‐sensitive lipase phosphorylated by cyclic AMP‐dependent protein kinase , 1988, FEBS letters.

[30]  K. Inoki,et al.  TSC2 Mediates Cellular Energy Response to Control Cell Growth and Survival , 2003, Cell.

[31]  B. Viollet,et al.  Anti-lipolytic Action of AMP-activated Protein Kinase in Rodent Adipocytes* , 2005, Journal of Biological Chemistry.

[32]  G. Lopaschuk,et al.  Acetyl-CoA carboxylase control of fatty acid oxidation in hearts from hibernating Richardson's ground squirrels. , 1998, Biochimica et biophysica acta.

[33]  C. Proud,et al.  Activity of protein phosphatases against initiation factor-2 and elongation factor-2. , 1990, The Biochemical journal.

[34]  C. Proud,et al.  Stimulation of the AMP-activated Protein Kinase Leads to Activation of Eukaryotic Elongation Factor 2 Kinase and to Its Phosphorylation at a Novel Site, Serine 398* , 2004, Journal of Biological Chemistry.

[35]  P. Strålfors,et al.  Hormonal regulation of hormone-sensitive lipase in intact adipocytes: identification of phosphorylated sites and effects on the phosphorylation by lipolytic hormones and insulin. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[36]  A. Nairn,et al.  Mechanisms for increased levels of phosphorylation of elongation factor-2 during hibernation in ground squirrels. , 2001, Biochemistry.

[37]  M. Desmadril,et al.  Regulation of the skeletal muscle metabolism during hibernation of Jaculus orientalis. , 1990, Comparative biochemistry and physiology. B, Comparative biochemistry.

[38]  D. Carling,et al.  Tissue distribution of the AMP-activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide assay. , 1989, European journal of biochemistry.

[39]  R. Colbran,et al.  Phosphorylation of bovine hormone-sensitive lipase by the AMP-activated protein kinase. A possible antilipolytic mechanism. , 1989, European journal of biochemistry.

[40]  D. Grahame Hardie,et al.  Possible involvement of AMP‐activated protein kinase in obesity resistance induced by respiratory uncoupling in white fat , 2004, FEBS letters.

[41]  J. Scott,et al.  The alpha1 and alpha2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro. , 1996, FEBS letters.

[42]  D. Hardie,et al.  The α1 and α2 isoforms of the AMP‐activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro , 1996 .

[43]  A. Jaeschke,et al.  Mammalian TOR: A Homeostatic ATP Sensor , 2001, Science.

[44]  K. Storey,et al.  Metabolic rate depression in animals: transcriptional and translational controls , 2004, Biological reviews of the Cambridge Philosophical Society.

[45]  A. Ryazanov,et al.  Regulation of elongation factor-2 kinase by pH. , 2002, Biochemistry.

[46]  B. Kemp,et al.  Dealing with energy demand: the AMP-activated protein kinase. , 1999, Trends in biochemical sciences.

[47]  S. Hawley,et al.  Characterization of the AMP-activated Protein Kinase Kinase from Rat Liver and Identification of Threonine 172 as the Major Site at Which It Phosphorylates AMP-activated Protein Kinase* , 1996, The Journal of Biological Chemistry.

[48]  D. Vertommen,et al.  Activation of AMP-Activated Protein Kinase Leads to the Phosphorylation of Elongation Factor 2 and an Inhibition of Protein Synthesis , 2002, Current Biology.

[49]  David Carling,et al.  Thr2446 Is a Novel Mammalian Target of Rapamycin (mTOR) Phosphorylation Site Regulated by Nutrient Status* , 2004, Journal of Biological Chemistry.

[50]  D. Hardie,et al.  Management of cellular energy by the AMP‐activated protein kinase system , 2003, FEBS letters.

[51]  G. Shulman,et al.  Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. , 1999, The American journal of physiology.

[52]  L. Bertrand,et al.  Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytes. , 2002, European journal of biochemistry.

[53]  K. Storey,et al.  Regulation of ground squirrel Na+K+-ATPase activity by reversible phosphorylation during hibernation. , 1999, Biochemical and biophysical research communications.

[54]  D. Hardie,et al.  AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. , 1997, American journal of physiology. Endocrinology and metabolism.