Abstract
In the present study, we test the hypothesis that AMP-activated protein kinase (AMPK) initiates metabolic rate suppression in isolated goldfish hepatocytes. To accomplish this, we attempted to pharmacologically activate AMPK in goldfish hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the thienopyridone, A769662, to examine the effects of AMPK activation on eukaryotic elongation factor-2 (eEF2), protein synthesis, and cellular oxygen consumption rate (\( \dot{M}_{{{\text{O}}_{ 2} }} \)). Goldfish hepatocytes treated with 1 mM AICAR under normoxic conditions (>200 μM O2) showed a modest but significant 1.1-fold increase in AMPK phosphorylation, a 7.5-fold increase in AMPK activity, a 1.4-fold increase in eEF2 phosphorylation, and a 24% decrease in \( \dot{M}_{{{\text{O}}_{ 2} }} \). At physiologically relevant [O2] (<40 μM O2), the addition of 1 mM AICAR resulted in only a 13% decrease in cellular \( \dot{M}_{{{\text{O}}_{ 2} }} \) with no change in sensitivity to [O2] as assessed by estimates of cellular P50 and P90 values. The addition of compound C, a general protein kinase inhibitor, after AICAR incubation did not reverse the effects of AICAR on \( \dot{M}_{{{\text{O}}_{ 2} }} \) in normoxia. Treatment of hepatocytes with ≤200 μM A769662 did not affect AMPK activity, AMPK phosphorylation, eEF2 phosphorylation, or cellular \( \dot{M}_{{{\text{O}}_{ 2} }} \). These data suggest that A769662 is not an activator of AMPK in goldfish hepatocytes. Although our study provides support for the hypothesis that AMPK plays a role in initiating metabolic rate suppression in goldfish hepatocytes, this support must be viewed cautiously because of the known off-target effects of the pharmacological agents used.
Similar content being viewed by others
References
Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, Klevernic I, Arthur JS, Alessi DR, Cohen P (2007) The selectivity of protein kinase inhibitors: a further update. Biochem J 408:297–315
Bartrons M, Ortega E, Obach M, Calvo MN, Navarro-Sabate A, Bartrons R (2004) Activation of AMP-dependent protein kinase by hypoxia and hypothermia in the liver of frog Rana perezi. Cryobiology 49:190–194
Benziane B, Bjornholm M, Lantier L, Viollet B, Zierath JR, Chibalin AV (2009) AMP-activated protein kinase activator A-769662 is an inhibitor of the Na+-K+-ATPase. Am J Physiol 297:C1554–C1566
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Browne GJ, Finn SG, Proud CG (2004) 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. J Biol Chem 279:12220–12231
Buck LT, Hochachka PW (1993) Anoxic suppression of Na+-K+-ATPase and constant membrane potential in hepatocytes: support for channel arrest. Am J Physiol 265:R1020–R1025
Burggren W (1982) "Air gulping" improves blood oxygen transport during aquatic hypoxia in the goldfish Carassius auratus. Physiol Zool 55:327–334
Carling D, Hardie DG (1989) The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase. Biochim Biophys Acta 1012:81–86
Carling D, Clarke PR, Zammit VA, Hardie DG (1989) Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities. Eur J Biochem 186:129–136
Cool B, Zinker B, Chiou W, Kifle L, Cao N, Perham M, Dickinson R, Adler A, Gagne G, Iyengar R et al (2006) Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab 3:403–416
Corton JM, Gillespie JG, Hawley SA, Hardie DG (1995) 5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells? Eur J Biochem 229:558–565
Emerling BM, Viollet B, Tormos KV, Chandel NS (2007) Compound C inhibits hypoxic activation of HIF-1 independent of AMPK. FEBS Lett 581:5727–5731
Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543:39–55
Goransson O, McBride A, Hawley SA, Ross FA, Shpiro N, Foretz M, Viollet B, Hardie DG, Sakamoto K (2007) Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase. J Biol Chem 282:32549–32560
Guigas B, Taleux N, Foretz M, Detaille D, Andreelli F, Viollet B, Hue L (2007) AMP-activated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside. Biochem J 404:499–507
Guigas B, Sakamoto K, Taleux N, Reyna SM, Musi N, Viollet B, Hue L (2009) Beyond AICA riboside: in search of new specific AMP-activated protein kinase activators. IUBMB Life 61:18–26
Hardie DG (2007) AMP-activated protein kinase as a drug target. Annu Rev Pharmacol Toxicol 47:185–210
Hardie DG, Hawley SA (2001) AMP-activated protein kinase: the energy charge hypothesis revisited. Bioessays 23:1112–1119
Hardie DG, Hawley SA, Scott JW (2006) AMP-activated protein kinase–development of the energy sensor concept. J Physiol 574:7–15
Henin N, Vincent MF, Gruber HE, Van den Berghe G (1995) Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase. FASEB J 9:541–546
Hochachka PW, Lutz PL (2001) Mechanism, origin, and evolution of anoxia tolerance in animals [small star, filled]. Comp Biochem Physiol 130:B435–B459
Hochachka PW, Buck LT, Doll CJ, Land SC (1996) Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack. Proc Natl Acad Sci USA 93:9493–9498
Horman S, Browne G, Krause U, Patel J, Vertommen D, Bertrand L, Lavoinne A, Hue L, Proud C, Rider M (2002) Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis. Curr Biol 12:1419–1423
Jibb LA, Richards JG (2008) AMP-activated protein kinase activity during metabolic rate depression in the hypoxic goldfish, Carassius auratus. J Exp Biol 211:3111–3122
Kahn BB, Alquier T, Carling D, Hardie DG (2005) AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1:15–25
Krumschnabel G, Wieser W (1994) Inhibition of the sodium pump does not cause a stoichiometric decrease of ATP-production in energy limited fish hepatocytes. Cell Mol Life Sci 50:483–485
Krumschnabel G, Malle S, Schwarzbaum PJ, Wieser W (1994a) Glycolytic function in goldfish hepatocytes at different temperatures: relevance for Na+ pump activity and protein synthesis. J Exp Biol 192:285–290
Krumschnabel G, Schwarzbaum P, Wieser W (1994b) Coupling of Energy Supply and Energy Demand in Isolated Goldfish Hepatocytes. Physiol Zool 67:438–448
Krumschnabel G, Schwarzbaum PJ, Lisch J, Biasi C, Wieser W (2000) Oxygen-dependent energetics of anoxia-tolerant and anoxia-intolerant hepatocytes. J Exp Biol 203:951–959
Krumschnabel G, Manzl C, Schwarzbaum PJ (2001) Importance of glycolysis for the energetics of anoxia-tolerant and anoxia-intolerant teleost hepatocytes. Physiol Biochem Zool 74:413–419
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
McBride A, Hardie DG (2009) AMP-activated protein kinase–a sensor of glycogen as well as AMP and ATP? Acta Physiol (Oxf) 196:99–113
McLeod LE, Proud CG (2002) ATP depletion increases phosphorylation of elongation factor eEF2 in adult cardiomyocytes independently of inhibition of mTOR signalling. FEBS Lett 531:448–452
Meley D, Bauvy C, Houben-Weerts JH, Dubbelhuis PF, Helmond MT, Codogno P, Meijer AJ (2006) AMP-activated protein kinase and the regulation of autophagic proteolysis. J Biol Chem 281:34870–34879
Mendelsohn BA, Kassebaum BL, Gitlin JD (2008) The zebrafish embryo as a dynamic model of anoxia tolerance. Dev Dyn 237:1780–1788
Merrill GF, Kurth EJ, Hardie DG, Winder WW (1997) AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. Am J Physiol 273:E1107–E1112
Mommsen T, Moon T, Walsh P (1994) Hepatocytes: isolation, maintenance and utilization. In: Hochachka PW, Mommsen T (eds) Biochemistry and molecular biology of fishes, analytical techniques, vol. 3. Elsevier Science Ltd, Amsterdam, pp 355–372
Moreno D, Knecht E, Viollet B, Sanz P (2008) A769662, a novel activator of AMP-activated protein kinase, inhibits non-proteolytic components of the 26S proteasome by an AMPK-independent mechanism. FEBS Lett 582:2650–2654
Ramnanan C, McMullen D, Groom A, Storey K (2010) The regulation of AMPK signaling in a natural state of profound metabolic rate depression. Mol Cell Biochem 335:91–105
Reznick RM, Shulman GI (2006) The role of AMP-activated protein kinase in mitochondrial biogenesis. J Physiol 574:33–39
Richards JG (2009) Metabolic and molecular responses of fish to hypoxia. In: Richards JG, Farrell AP, Brauner CJ (eds) Fish physiology, vol. 27. Elsevier, San Diego, pp 443–485
Rider M, Hussain N, Dilworth S, Storey K (2009) Phosphorylation of translation factors in response to anoxia in turtles, Trachemys scripta elegans: role of the AMP-activated protein kinase and target of rapamycin signalling pathways. Mol Cell Biochem 332:207–213
Sanders MJ, Ali ZS, Hegarty BD, Heath R, Snowden MA, Carling D (2007a) Defining the mechanism of activation of AMP-activated protein kinase by the small molecule A-769662, a member of the thienopyridone family. J Biol Chem 282:32539–32548
Sanders MJ, Grondin PO, Hegarty BD, Snowden MA, Carling D (2007b) Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochem J 403:139–148
Smith RW, Houlihan DF, Nilsson GE, Brechin JG (1996) Tissue-specific changes in protein synthesis rates in vivo during anoxia in crucian carp. Am J Physiol 271:R897–R904
Speers-Roesch B, Sandblom E, Lau GY, Farrell AP, Richards JG (2010) Effects of environmental hypoxia on cardiac energy metabolism and performance in tilapia. Am J Physiol 298:R104–R119
Staples JF, Buck LT (2009) Matching cellular metabolic supply and demand in energy-stressed animals. Comp Biochem Physiol 153:A95–A105
Stenslokken KO, Ellefsen S, Stecyk JA, Dahl MB, Nilsson GE, Vaage J (2008) Differential regulation of AMP-activated kinase and AKT kinase in response to oxygen availability in crucian carp (Carassius carassius). Am J Physiol 295:R1803–R1814
Suter M, Riek U, Tuerk R, Schlattner U, Wallimann T, Neumann D (2006) Dissecting the role of 5’-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase. J Biol Chem 281:32207–32216
Takeda T (1990) Ventilation, cardiac output and blood respiratory parameters in the carp, Cyprinus carpio, during hyperoxia. Respir Physiol 81:227–239
Treebak JT, Birk JB, Hansen BF, Olsen GS, Wojtaszewski JF (2009) A-769662 activates AMPK beta1-containing complexes but induces glucose uptake through a PI3-kinase-dependent pathway in mouse skeletal muscle. Am J Physiol 297:C1041–C1052
van Ginneken VJT, Snelderwaard P, van der Linden R, van der Reijden N, van den Thillart GEEJM, Kramer K (2004) Coupling of heart rate with metabolic depression in fish: a radiotelemetric and calorimetric study. Thermochimica Acta 414:1–10
Van Waversveld J, Addink A, Van den Thillart G (1989) Simultaneous direct and indirect calorimetry on normoxic and anoxic goldfish. J Exp Biol 142:325–335
Vassault A (1983) Lactate dehydrogenase. UV-method with pyruvate and NADH. In: Bergmeyer H (ed) Methods of enzymatic analysis. enzymes I: oxidoreductases, transferase, vol. 3. Weinheim, F.R.G: Verlag Chemie, Academic Press, New York, pp 118–126
Vincent MF, Marangos PJ, Gruber HE, Van den Berghe G (1991) Inhibition by AICA riboside of gluconeogenesis in isolated rat hepatocytes. Diabetes 40:1259–1266
Vincent MF, Bontemps F, Van den Berghe G (1992) Inhibition of glycolysis by 5-amino-4-imidazolecarboxamide riboside in isolated rat hepatocytes. Biochem J 281:267–272
Viollet B, Foretz M, Guigas B, Horman S, Dentin R, Bertrand L, Hue L, Andreelli F (2006) Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders. J Physiol 574:41–53
Wang X, Proud CG (2006) The mTOR pathway in the control of protein synthesis. Physiology (Bethesda) 21:362–369
Whitmer JT, Idell-Wenger JA, Rovetto MJ, Neely JR (1978) Control of fatty acid metabolism in ischemic and hypoxic hearts. J. Biol. Chem. 253:4305–4309
Wieser W, Krumschnabel G (2001) Hierarchies of ATP-consuming processes: direct compared with indirect measurements, and comparative aspects. Biochem J 355:389–395
Zhang H, Gao P, Fukuda R, Kumar G, Krishnamachary B, Zeller KI, Dang CV, Semenza GL (2007) HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient renal cell carcinoma by repression of C-MYC activity. Cancer Cell 11:407–420
Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N et al (2001) Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108:1167–1174
Acknowledgments
This work was funded by a Natural Science and Engineering Research Council (NSERC) of Canada Discovery Grant to JGR.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by H.V. Carey.
Rights and permissions
About this article
Cite this article
Lau, G.Y., Richards, J.G. AMP-activated protein kinase plays a role in initiating metabolic rate suppression in goldfish hepatocytes. J Comp Physiol B 181, 927–939 (2011). https://doi.org/10.1007/s00360-011-0575-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00360-011-0575-1