AMP-activated Protein Kinase Plays a Role in the Control of Food Intake

Abstract

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that acts as an intracellular energy sensor maintaining the energy balance within the cell. The finding that leptin and adiponectin activate AMPK to alter metabolic pathways in muscle and liver provides direct evidence for this role in peripheral tissues. The hypothalamus is a key regulator of food intake and energy balance, coordinating body adiposity and nutritional state in response to peripheral hormones, such as leptin, peptide YY-(3–36), and ghrelin. To date the hormonal regulation of AMPK in the hypothalamus, or its potential role in the control of food intake, have not been reported. Here we demonstrate that counter-regulatory hormones involved in appetite control regulate AMPK activity and that pharmacological activation of AMPK in the hypothalamus increases food intake. In vivo administration of leptin, which leads to a reduction in food intake, decreases hypothalamic AMPK activity. By contrast, injection of ghrelin in vivo, which increases food intake, stimulates AMPK activity in the hypothalamus. Consistent with the effect of ghrelin, injection of 5-amino-4-imidazole carboxamide riboside, a pharmacological activator of AMPK, into either the third cerebral ventricle or directly into the paraventricular nucleus of the hypothalamus significantly increased food intake. These results suggest that AMPK is regulated in the hypothalamus by hormones which regulate food intake. Furthermore, direct pharmacological activation of AMPK in the hypothalamus is sufficient to increase food intake. These findings demonstrate that AMPK plays a role in the regulation of feeding and identify AMPK as a novel target for anti-obesity drugs. AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that acts as an intracellular energy sensor maintaining the energy balance within the cell. The finding that leptin and adiponectin activate AMPK to alter metabolic pathways in muscle and liver provides direct evidence for this role in peripheral tissues. The hypothalamus is a key regulator of food intake and energy balance, coordinating body adiposity and nutritional state in response to peripheral hormones, such as leptin, peptide YY-(3–36), and ghrelin. To date the hormonal regulation of AMPK in the hypothalamus, or its potential role in the control of food intake, have not been reported. Here we demonstrate that counter-regulatory hormones involved in appetite control regulate AMPK activity and that pharmacological activation of AMPK in the hypothalamus increases food intake. In vivo administration of leptin, which leads to a reduction in food intake, decreases hypothalamic AMPK activity. By contrast, injection of ghrelin in vivo, which increases food intake, stimulates AMPK activity in the hypothalamus. Consistent with the effect of ghrelin, injection of 5-amino-4-imidazole carboxamide riboside, a pharmacological activator of AMPK, into either the third cerebral ventricle or directly into the paraventricular nucleus of the hypothalamus significantly increased food intake. These results suggest that AMPK is regulated in the hypothalamus by hormones which regulate food intake. Furthermore, direct pharmacological activation of AMPK in the hypothalamus is sufficient to increase food intake. These findings demonstrate that AMPK plays a role in the regulation of feeding and identify AMPK as a novel target for anti-obesity drugs. AMP-activated protein kinase (AMPK) 1The abbreviations used are: AMPK, AMP-activated protein kinase; PVN, paraventricular nucleus; NPY, neuropeptide Y; PYY, peptide YY-(3–36); ICV, intracerebroventricular; ACC, acetyl-CoA carboxylase. plays a pivotal role in the regulation of energy metabolism and has been dubbed a cellular fuel gauge (1Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1147) Google Scholar). AMPK is activated following an increase in the AMP:ATP ratio within the cell that occurs following a decrease in ATP levels (2Hardie D.G. Scott J.W. Pan D.A. Hudson E.R. FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (720) Google Scholar, 3Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1281) Google Scholar). Once activated, AMPK switches on ATP-generating (catabolic) pathways, e.g. fatty acid oxidation, and switches off ATP-using pathways (anabolic) pathways, e.g. fatty acid synthesis, allowing the cell to restore its energy balance (2Hardie D.G. Scott J.W. Pan D.A. Hudson E.R. FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (720) Google Scholar, 3Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1281) Google Scholar). In addition to acute effects on metabolism, AMPK has more long term effects, altering both gene (4Foretz M. Carling D. Guichard C. Ferre P. Foufelle F. J. Biol. Chem. 1998; 272: 14767-14771Abstract Full Text Full Text PDF Scopus (215) Google Scholar) and protein expression (5Winder W. Holmes B. Rubink D. Jensen E. Chen M. Holloszy J. J. Appl. Physiol. 2000; 88: 2219-2226Crossref PubMed Scopus (605) Google Scholar, 6Fryer L.G.D. Foufelle F. Barnes K. Baldwin S.A. Woods A. Carling D. Biochem. J. 2002; 363: 167-174Crossref PubMed Scopus (157) Google Scholar). Recent results have demonstrated activation of AMPK in the absence of changes in adenine nucleotide levels, indicating that there may be multiple pathways upstream of AMPK (7Fryer L.G. Patel A.P. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 8Hawley S.A. Gadalla A.E. Olsen G.S. Hardie D.G. Diabetes. 2002; 51: 2420-2425Crossref PubMed Scopus (582) Google Scholar). The molecular mechanisms leading to activation of AMPK have not been fully elucidated, but it is clear that activation of AMPK requires phosphorylation of threonine 172 (Thr172) within the activation loop segment of the catalytic (α) subunit (9Crute B.E. Seefeld K. Gamble J. Kemp B.E. Witters L.A. J. Biol. Chem. 1998; 273: 35347-35354Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 10Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (499) Google Scholar). Very recently, LKB1, a protein kinase that is inactivated in a hereditary form of cancer termed Peutz-Jeghers syndrome, was shown to account for most of the AMPK kinase activity in cell extracts (11Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar, 12Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G.D. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1352) Google Scholar) raising the possibility that AMPK could link metabolism with cell proliferation. Until fairly recently, most of the studies examining the role of AMPK have focused on its response to acute changes in energy levels within individual cells. However, there is emerging evidence that AMPK also plays an important role in the regulation of whole-body energy metabolism, responding to adipocyte-derived hormones such as leptin (13Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1691) Google Scholar) and adiponectin (14Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3483) Google Scholar). Leptin activates AMPK in skeletal muscle increasing fatty acid oxidation (13Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1691) Google Scholar), while adiponectin activates AMPK in both liver and skeletal muscle, increasing glucose utilization and fatty acid oxidation, and inhibiting glucose production in the liver (14Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3483) Google Scholar). The hypothalamus and the dorsal vagal complex appear to be the main regions within the central nervous system directly regulating appetite. The arcuate nucleus and the paraventricular nucleus (PVN) of the hypothalamus have been shown to play an integrative role in appetite regulation (15Spiegelman B.M. Flier J.S. Cell. 2001; 104: 531-543Abstract Full Text Full Text PDF PubMed Scopus (1958) Google Scholar). Neurones within the hypothalamus respond to the different neuro-endocrine and metabolic signals coordinating the body's response to changes in energy intake and energy expenditure. The mechanisms involved are complex but depend at least in part on hormones derived from either adipose tissue, e.g. leptin or the gastrointestinal tract, e.g. ghrelin. Leptin, a hormone derived from adipocytes, acts on neurones within the arcuate nucleus of the hypothalamus, decreasing the release of orexigenic neuropeptides and increasing the release of anorexigenic neuropeptides, resulting in decreased food intake (15Spiegelman B.M. Flier J.S. Cell. 2001; 104: 531-543Abstract Full Text Full Text PDF PubMed Scopus (1958) Google Scholar). Ghrelin is synthesized in the stomach and stimulates food intake, acting at least in part via the same neuronal circuits involved in the response to anorexigenic neuropeptides (15Spiegelman B.M. Flier J.S. Cell. 2001; 104: 531-543Abstract Full Text Full Text PDF PubMed Scopus (1958) Google Scholar). Thus, leptin and ghrelin have counter-regulatory effects on food intake, although the cellular mechanisms by which they act are poorly understood. The aim of the current study was to determine whether AMPK in the hypothalamus plays a role in the regulation of food intake. Materials—Ghrelin, neuropeptide Y (NPY), and peptide YY-(3–36) (PYY) were all purchased from Bachem UK Ltd. (Merseyside, UK). Leptin was purchased from R&D Systems (Abingdon, Oxford, United Kingdom). 5-Amino-4-imidazole carboxamide (AICA) riboside was from Sigma. Animals—Male Wistar rats (180–220 g) were maintained in individual cages under controlled temperature (21–23 °C) and light (12 h light (7 a.m. to 7 p.m.), 12 h dark) conditions with ad libitum access to food (RM1 diet, SDS UK Ltd.) and water. Animals were handled daily following recovery from surgery until the completion of the studies. All animal procedures undertaken were approved by the 1986 British Home Office Animals Scientific Procedures Act. Intraperitoneal Injection—Rats were accustomed to the intraperitoneal injection procedure by injection of 0.5 ml of saline and the measurement of food intake 2 days prior to the study. Where appropriate, rats received an intraperitoneal injection of saline, leptin (1.1 mg/kg), ghrelin (30 nmol/animal), or PYY (25 nmol/animal) in a total volume of 0.5 ml. Injections were given at the start of the light phase and in the case of leptin, and PYY animals were fasted for 12 h prior to injection. Intracerebroventricular (ICV) and Inter-PVN Cannulation and Injection—Animal surgical procedures and handling were carried out as described previously (16Abbott C.R. Rossi M. Wren A.M. Murphy K.G. Kennedy A.R. Stanley S.A. Zollner A.N. Morgan D.G. Morgan I. Ghatei M.A. Small C.J. Bloom S.R. Endocrinology. 2001; 142: 3457-3463Crossref PubMed Scopus (130) Google Scholar, 17Wren A.M. Small C.J. Abbott C.R. Dhillo W.S. Seal L.J. Cohen M.A. Batterham R.L. Taheri S. Stanley S.A. Ghatei M.A. Bloom S.R. Diabetes. 2001; 50: 2540-2547Crossref PubMed Google Scholar). Animals were anesthetized by intraperitoneal injection of a mixture of Ketalar (60 mg/kg ketamine HCl, Parke-Davis, Pontypool, UK) and Rompun (12 mg/kg xylazine, Bayer UK Ltd., Bury St. Edmunds, UK) and placed in a Kopf stereotaxic frame. Permanent 26-gauge stainless steel guide cannulae (Plastics One Inc., Roanoke, VA) were stereotactically placed 1.8 mm posterior to bregma, 0.5 mm lateral from the mid-sagittal line, and implanted 7 mm below the outer surface of the skull into the paraventricular nucleus of the hypothalamus. The third cerebral ventricle was cannulated with a permanent 22-gauge stainless steel guide cannula (Plastics One Inc.) stereotactically placed 0.8 mm posterior to bregma on the mid-line and implanted 6.5 mm below the outer surface of the skull. All animals used in the study were mock-injected on two occasions to acclimatize them to the procedure prior to the first study day. Substances were administered via a stainless steel injector placed in, and projecting 1 mm below, the tip of the guide cannulae. All compounds were dissolved in 0.9% saline and injected in a volume of 1 μl (PVN) and 5 μl (ICV). The entire injection process lasted under 2 min, and the rats were returned to their cages with the minimum of disruption. Correct placement of the cannula into the third cerebral ventricle was confirmed by injection of angiotensin II (150 ng) as described previously (18O'Shea D. Morgan D.G. Meeran K. Edwards C.M. Turton M.D. Choi S.J. Heath M.M. Gunn I. Taylor G.M. Howard J.K. Bloom C.I. Small C.J. Haddo O. Ma J.J. Callinan W. Smith D.M. Ghatei M.A. Bloom S.R. Endocrinology. 1997; 138: 196-202Crossref PubMed Scopus (0) Google Scholar). Animals not displaying a prompt and sustained drinking response were excluded from further study. This was ∼5% of cannulated animals. For intranuclear cannulated rats correct cannula placement was confirmed histologically at the end of the study period, as described previously (16Abbott C.R. Rossi M. Wren A.M. Murphy K.G. Kennedy A.R. Stanley S.A. Zollner A.N. Morgan D.G. Morgan I. Ghatei M.A. Small C.J. Bloom S.R. Endocrinology. 2001; 142: 3457-3463Crossref PubMed Scopus (130) Google Scholar, 19Morgan D.G. Small C.J. Abusnana S. Turton M. Gunn I. Heath M. Rossi M. Goldstone A.P. O'Shea D. Meeran K. Ghatei M.A. Smith D.M. Bloom S.R. Regul. Pep. 1998; 75–76: 377-382Crossref PubMed Scopus (48) Google Scholar). Following injection of black ink, animals were decapitated, the guide cannulae removed, and the brains immediately frozen in liquid nitrogen and stored at –70 °C. Brains were sliced on a cryostat (Bright Instruments, Huntingdon, Cambridgeshire, UK) into 15-μm coronal sections and stained with cresyl violet. Sections were compared with the corresponding section from the rat brain atlas. The ink remained localized at the injection site at the guide tip without significant diffusion. Data from an animal were excluded if its injection site extended more than 0.2 mm outside the nucleus or if any ink was detected in the cerebral ventricular system. Consistent with previously reported studies (16Abbott C.R. Rossi M. Wren A.M. Murphy K.G. Kennedy A.R. Stanley S.A. Zollner A.N. Morgan D.G. Morgan I. Ghatei M.A. Small C.J. Bloom S.R. Endocrinology. 2001; 142: 3457-3463Crossref PubMed Scopus (130) Google Scholar, 19Morgan D.G. Small C.J. Abusnana S. Turton M. Gunn I. Heath M. Rossi M. Goldstone A.P. O'Shea D. Meeran K. Ghatei M.A. Smith D.M. Bloom S.R. Regul. Pep. 1998; 75–76: 377-382Crossref PubMed Scopus (48) Google Scholar) ∼12% of intranuclear cannulated animals were excluded for incorrect cannula placement. Isolation of Tissues—Animals were sacrificed by decapitation, brains immediately dissected, and the hypothalamus removed and snap-frozen in liquid nitrogen. Frozen tissues (∼50 mg) were homogenized in 0.2 ml of ice-cold 50 mm Tris/HCl, pH 7.5, 50 mm NaF, 5 mm sodium pyrophosphate, 250 mm sucrose, 1 mm EDTA, 1 mm dithiothreitol, 1 mm benzamidine, 0.1% (w/v) phenylmethylsulfonyl fluoride using an UltraTurax homogenizer (3 × 30-s bursts). Insoluble material was removed by centrifugation and the resulting supernatant used for immunoprecipitation of AMPK and Western blot analysis. AMPK Assay—AMPK was immunoprecipitated from 100 μg of total protein using an anti-pan β antibody (20Woods A. Cheung P.C.F. Smith F.C. Davison M.D. Scott J. Beri R.K. Carling D. J. Biol. Chem. 1996; 271: 10282-10290Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar) bound to protein A-Sepharose and activity measured by phosphorylation of the SAMS synthetic peptide (21Davies S.P. Carling D. Hardie D.G. Eur. J. Biochem. 1989; 186: 123-128Crossref PubMed Scopus (373) Google Scholar). Western Blot Analysis—Tissue lysates (40 μg) were resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Phosphorylation of AMPK was determined by blotting with a phosphothreonine 172-specific antibody (Cell Signaling Technologies). Total AMPK was estimated by blotting with an anti-pan β antibody (20Woods A. Cheung P.C.F. Smith F.C. Davison M.D. Scott J. Beri R.K. Carling D. J. Biol. Chem. 1996; 271: 10282-10290Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Acetyl-CoA carboxylase (ACC) phosphorylation was measured with a phospho-ACC specific antibody (Upstate Biotechnology). In each case, anti-rabbit antibody linked to horseradish peroxidase (Bio-Rad) was used as the secondary antibody. Total ACC was determined using streptavidin linked to horseradish peroxidase (Bio-Rad). Blots were developed using enhanced chemiluminescence (Pierce), and quantification was performed using NIH Image 1.62. Intraperitoneal injection of leptin (1.1 mg/kg body weight) caused a time-dependent decrease in AMPK activity in the hypothalamus (Fig. 1a). Forty minutes following injection there was no decrease in AMPK activity, but 60 min after injection AMPK activity was reduced by 25–30% (n = 10, p < 0.005). The reduction in AMPK activity persisted for up to min < but returned to control after Consistent with the decrease in AMPK activity, the phosphorylation state of ACC, a downstream target of AMPK (13Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1691) Google Scholar), from the hypothalamus was reduced at both 60 and min following leptin administration (Fig. AMPK is activated by phosphorylation of threonine 172 (Thr172) within the activation loop of the catalytic (α) subunit of AMPK S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (499) Google Scholar). This phosphorylation is by an upstream which was as LKB1, a kinase that is inactivated in a hereditary cancer termed Peutz-Jeghers (11Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar, 12Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G.D. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1352) Google Scholar). In vivo administration of leptin to a reduction in activity, although this not (Fig. and a reduction in the phosphorylation state of (Fig. In to the effects of leptin, intraperitoneal injection of ghrelin (30 nmol/animal) activated AMPK in the hypothalamus (Fig. The of ghrelin we used has previously been shown to food intake 1 h after administration A.M. Small C.J. Abbott C.R. Dhillo W.S. Seal L.J. Cohen M.A. Batterham R.L. Taheri S. Stanley S.A. Ghatei M.A. Bloom S.R. Diabetes. 2001; 50: 2540-2547Crossref PubMed Google Scholar). to the effect of leptin, min following injection of ghrelin there was no in AMPK activity, 60 min after injection AMPK activity was increased by (n = 10, p < AMPK activity returned to control levels after In with the activation of AMPK there was an increase in the phosphorylation state of ACC 60 min following injection but no after min (Fig. activity in the hypothalamus was but this not (Fig. and an increase in the phosphorylation state of was (Fig. PYY is a gastrointestinal hormone that is in to the of and leads to a reduction in food intake in and R.L. M.A. Small C.J. H. Cohen M.A. Wren A.M. A.E. Ghatei M.A. Bloom S.R. Nature. 2002; PubMed Scopus Google Scholar). However, leptin and ghrelin we not an effect of PYY on AMPK activity or phosphorylation or on ACC phosphorylation not that PYY not regulate AMPK in the hypothalamus. the effect of altering AMPK activity in the hypothalamus on food intake. riboside be up by and into or which acts as an and activates AMPK K.J. A.E. F. Carling D. Beri R.K. FEBS Lett. PubMed Scopus Google Scholar). were injected with riboside either into the third cerebral ventricle (ICV) or into the and food intake measured the following to a significant increase in food intake (Fig. there has been that AMPK play a role in the central nervous system regulation of energy metabolism for Endocrinology. 2003; PubMed Scopus Google Scholar). results the first evidence that pharmacological activation of AMPK in the hypothalamus stimulates food intake. Furthermore, AMPK activity in the hypothalamus is regulated by leptin and ghrelin, hormones that have effects on food intake. In to its effect in skeletal muscle, leptin decreased AMPK activity in the hypothalamus, ghrelin increased AMPK activity. The by which leptin and ghrelin regulate AMPK are although results suggest that this may changes in the activity of LKB1, the kinase immediately upstream of with their effects on AMPK, leptin decreased ACC phosphorylation and ghrelin increased ACC of effects is that the of be increased by leptin and decreased by ghrelin. of a synthetic of fatty acid into leads to reduced food intake and reduced body a that is to M.D. 2000; PubMed Scopus Google Scholar). demonstrated that of 1 which is by food intake S. A. R. L. Nat. Med. 2003; PubMed Scopus Google Scholar). results are with a that hormonal changes in AMPK activity leading to changes in ACC phosphorylation activity, which in alter levels to regulate food intake. In of such a is finding that pharmacological activation of AMPK in the hypothalamus increases food intake. results the first that AMPK is directly involved in regulating food intake and it as a potential target for at body