AMP-activated protein kinase phosphorylates and inactivates liver glycogen synthase

2012 ◽  
Vol 443 (1) ◽  
pp. 193-203 ◽  
Author(s):  
Laurent Bultot ◽  
Bruno Guigas ◽  
Alexander Von Wilamowitz-Moellendorff ◽  
Liliane Maisin ◽  
Didier Vertommen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glycogen Synthase ◽  
Phosphorylation Site ◽  
Rat Hepatocytes ◽  
Liver Glycogen ◽  
Enzyme Inactivation ◽  
Dependent Manner ◽  
Isolated Rat Hepatocytes ◽  
Amp Activated Protein Kinase ◽  
Specific Deletion

Recombinant muscle GYS1 (glycogen synthase 1) and recombinant liver GYS2 were phosphorylated by recombinant AMPK (AMP-activated protein kinase) in a time-dependent manner and to a similar stoichiometry. The phosphorylation site in GYS2 was identified as Ser7, which lies in a favourable consensus for phosphorylation by AMPK. Phosphorylation of GYS1 or GYS2 by AMPK led to enzyme inactivation by decreasing the affinity for both UDP-Glc (UDP-glucose) [assayed in the absence of Glc-6-P (glucose-6-phosphate)] and Glc-6-P (assayed at low UDP-Glc concentrations). Incubation of freshly isolated rat hepatocytes with the pharmacological AMPK activators AICA riboside (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) or A769662 led to persistent GYS inactivation and Ser7 phosphorylation, whereas inactivation by glucagon treatment was transient. In hepatocytes from mice harbouring a liver-specific deletion of the AMPK catalytic α1/α2 subunits, GYS2 inactivation by AICA riboside and A769662 was blunted, whereas inactivation by glucagon was unaffected. The results suggest that GYS inactivation by AMPK activators in hepatocytes is due to GYS2 Ser7 phosphorylation.

scholarly journals Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins

2005 ◽  
Vol 386 (2) ◽  
pp. 237-244 ◽  
Author(s):  
Hamid R. SAMARI ◽  
Michael T. N. MØLLER ◽  
Lise HOLDEN ◽  
Tonje ASMYHR ◽  
Per O. SEGLEN
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Rat Hepatocytes ◽  
Calyculin A ◽  
Β Subunit ◽  
Isolated Rat Hepatocytes ◽  
Α Subunit ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

Autophagic activity in isolated rat hepatocytes is strongly suppressed by OA (okadaic acid) and other PP (protein phosphatase)-inhibitory toxins as well as by AICAR (5-aminoimidazole-4-carboxamide riboside), a direct activator of AMPK (AMP-activated protein kinase). To investigate whether AMPK is a mediator of the effects of the toxin, a phosphospecific antibody directed against the activation of phosphorylation of the AMPK α (catalytic)-subunit at Thr172 was used to assess the activation status of this enzyme. AICAR as well as all the toxins tested (OA, microcystin-LR, calyculin A, cantharidin and tautomycin) induced strong, dose-dependent AMPKα phosphorylation, correlating with AMPK activity in situ (in intact hepatocytes) as measured by the AMPK-dependent phosphorylation of acetyl-CoA carboxylase at Ser79. All treatments induced the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK α-subunit, consistent with phosphorylation at several sites other than Thr172. The flavonoid naringin, an effective antagonist of OA-induced autophagy suppression, inhibited the AMPK phosphorylation and mobility shifting induced by AICAR, OA or microcystin, but not the changes induced by calyculin A or cantharidin. AMPK may thus be activated both by a naringin-sensitive and a naringin-resistant mechanism, probably involving the PPs PP2A and PP1 respectively. Neither the Thr172-phosphorylating protein kinase LKB1 nor the Thr172-dephosphorylating PP, PP2C, were mobility-shifted after treatment with toxins or AICAR, whereas a slight mobility shifting of the regulatory AMPK β-subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the β-subunit revealed a naringin-sensitive phosphorylation induced by OA, microcystin and AICAR and a naringin-resistant phosphorylation induced by calyculin A and cantharidin, suggesting that β-subunit phosphorylation could play a role in AMPK activation. Naringin antagonized the autophagy-suppressive effects of AICAR and OA, but not the autophagy suppression caused by cantharidin, consistent with AMPK-mediated inhibition of autophagy by toxins as well as by AICAR.

scholarly journals Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes

2003 ◽  
Vol 373 (2) ◽  
pp. 505-513 ◽  
Author(s):  
Michael T. N. MØLLER ◽  
Hamid R. SAMARI ◽  
Monica FENGSRUD ◽  
Per E. STRØMHAUG ◽  
Anne C. ØSTVOLD ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Limited Region ◽  
Flight Mass Spectrometry ◽  
Methylation Capacity ◽  
Dependent Protein ◽  
Amp Activated Protein Kinase ◽  
Isolated Rat

Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S-adenosylmethionine (AdoMet), into S-adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of 32P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and 32P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet–AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.

scholarly journals Control of gluconeogenesis and of enzymes of glycogen metabolism in isolated rat hepatocytes. A parallel study of the effect of phenylephrine and of glucagon

1978 ◽  
Vol 176 (3) ◽  
pp. 791-797 ◽  
Author(s):  
Louis Hue ◽  
Juan Emilio Felíu ◽  
Henri-Géry Hers
Keyword(s):  
Protein Kinase ◽  
Pyruvate Kinase ◽  
Incubation Medium ◽  
Glycogen Phosphorylase ◽  
Glycogen Synthase ◽  
Rat Hepatocytes ◽  
Glycogen Metabolism ◽  
Parallel Study ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

Hepatocytes isolated from the livers of fed rats were used for a comparative study of the effects of phenylephrine, vasopressin and glucagon on gluconeogenesis and on enzymes of glycogen metabolism. When hepatocytes were incubated in the presence of Ca2+, phenylephrine stimulated gluconeogenesis from pyruvate less than did glucagon, but, in contrast with this hormone, it did not affect the activities of protein kinase and pyruvate kinase, nor the concentration of phosphoenolpyruvate, and it did not decrease the release of 3H2O from [6-3H]glucose. The effects of vasopressin were similar to those of phenylephrine. Gluconeogenesis from fructose was also stimulated by phenylephrine and, more markedly, by glucagon at the expense of the conversion of fructose into lactate. Insulin was able to antagonize the stimulatory effect of phenylephrine on gluconeogenesis from pyruvate. When Ca2+ was removed from the incubation medium, phenylephrine still stimulated gluconeogenesis from pyruvate, but it also caused an activation of protein kinase and an inactivation of pyruvate kinase; accordingly, the concentration of phosphoenolpyruvate was increased, and, in contrast, vasopressin had no effect on all these parameters. The property of phenylephrine to cause the activation of glycogen phosphorylase was decreased by glucose or by the absence of Ca2+; it was abolished when these two conditions were combined. Glycogen synthase was inactivated by phenylephrine in the presence or the absence of Ca2+, although presumably by different mechanisms.

scholarly journals Control of glycogen synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon

1984 ◽  
Vol 142 (3) ◽  
pp. 511-520 ◽  
Author(s):  
Carlos CIUDAD ◽  
Marcella CAMICI ◽  
Zafeer AHMAD ◽  
Yuhuan WANG ◽  
Anna A. DePAOLI-ROACH ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

scholarly journals The roles of different protein kinases and of calmodulin in the effects of Ca2+ mobilization on 3-hydroxy-3-methylglutaryl-CoA reductase activity in isolated rat hepatocytes

1991 ◽  
Vol 273 (2) ◽  
pp. 485-488 ◽  
Author(s):  
V A Zammit ◽  
A M Caldwell
Keyword(s):  
Protein Kinase ◽  
Reductase Activity ◽  
Rat Hepatocytes ◽  
Total Activity ◽  
Isolated Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Hmg Coa Reductase ◽  
Lysosomal Proteolysis ◽  
Dependent Protein ◽  
Coa Reductase

The roles of protein kinase C, Ca2+/calmodulin-dependent protein kinase and AMP-activated protein kinase in the phosphorylation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase induced by Ca2(+)-mobilizing conditions in isolated hepatocytes were investigated. Only partial evidence for the involvement of AMP-activated kinase was found. Antagonism of calmodulin action prolonged the decrease in expressed/total activity ratio induced by vasopressin plus glucagon. Protease inhibitors active against Ca2(+)-dependent cytosolic proteases or lysosomal proteolysis did not attenuate the loss of total HMG-CoA reductase induced by glucagon plus vasopressin, but calmodulin antagonists largely prevented this effect.

scholarly journals Regulation of Glycogen Synthase by Glucose and Glycogen: A Possible Role for AMP-Activated Protein Kinase

Diabetes ◽  
2003 ◽  
Vol 52 (1) ◽  
pp. 9-15 ◽  
Author(s):  
R. Halse ◽  
L. G.D. Fryer ◽  
J. G. McCormack ◽  
D. Carling ◽  
S. J. Yeaman
Keyword(s):  
Protein Kinase ◽  
Glycogen Synthase ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle

2001 ◽  
Vol 280 (5) ◽  
pp. E677-E684 ◽  
Author(s):  
Nicolas Musi ◽  
Tatsuya Hayashi ◽  
Nobuharu Fujii ◽  
Michael F. Hirshman ◽  
Lee A. Witters ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Muscle Contractions ◽  
Treadmill Running ◽  
Dependent Manner ◽  
Rat Skeletal Muscle ◽  
Amp Activated Protein Kinase ◽  
Dose Dependent

The AMP-activated protein kinase (AMPK) has been hypothesized to mediate contraction and 5-aminoimidazole-4-carboxamide 1-β-d-ribonucleoside (AICAR)-induced increases in glucose uptake in skeletal muscle. The purpose of the current study was to determine whether treadmill exercise and isolated muscle contractions in rat skeletal muscle increase the activity of the AMPKα1 and AMPKα2 catalytic subunits in a dose-dependent manner and to evaluate the effects of the putative AMPK inhibitors adenine 9-β-d-arabinofuranoside (ara-A), 8-bromo-AMP, and iodotubercidin on AMPK activity and 3- O-methyl-d-glucose (3-MG) uptake. There were dose-dependent increases in AMPKα2 activity and 3-MG uptake in rat epitrochlearis muscles with treadmill running exercise but no effect of exercise on AMPKα1 activity. Tetanic contractions of isolated epitrochlearis muscles in vitro significantly increased the activity of both AMPK isoforms in a dose-dependent manner and at a similar rate compared with increases in 3-MG uptake. In isolated muscles, the putative AMPK inhibitors ara-A, 8-bromo-AMP, and iodotubercidin fully inhibited AICAR-stimulated AMPKα2 activity and 3-MG uptake but had little effect on AMPKα1 activity. In contrast, these compounds had absent or minimal effects on contraction-stimulated AMPKα1 and -α2 activity and 3-MG uptake. Although the AMPKα1 and -α2 isoforms are activated during tetanic muscle contractions in vitro, in fast-glycolytic fibers, the activation of AMPKα2-containing complexes may be more important in regulating exercise-mediated skeletal muscle metabolism in vivo. Development of new compounds will be required to study contraction regulation of AMPK by pharmacological inhibition.

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