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Diet-induced Obesity Alters AMP Kinase Activity in Hypothalamus and Skeletal Muscle

2006; Elsevier BV; Volume: 281; Issue: 28 Linguagem: Inglês

10.1074/jbc.m512831200

1083-351X

Tonya Martin, Thierry Alquier, Kenji Asakura, Noboru Furukawa, Frédéric Preitner, Barbara B. Kahn,

Metabolism, Diabetes, and Cancer

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance and of the effects of leptin on food intake and fatty acid oxidation. Obesity is usually associated with resistance to the effects of leptin on food intake and body weight. To determine whether diet-induced obesity (DIO) impairs the AMPK response to leptin in muscle and/or hypothalamus, we fed FVB mice a high fat (55%) diet for 10–12 weeks. Leptin acutely decreased food intake by ∼30% in chow-fed mice. DIO mice tended to eat less, and leptin had no effect on food intake. Leptin decreased respiratory exchange ratio in chow-fed mice indicating increased fatty acid oxidation. Respiratory exchange ratio was low basally in high fat-fed mice, and leptin had no further effect. Leptin (3 mg/kg intraperitoneally) increased α2-AMPK activity 2-fold in muscle in chow-fed mice but not in DIO mice. Leptin decreased acetyl-CoA carboxylase activity 40% in muscle from chow-fed mice. In muscle from DIO mice, acetyl-CoA carboxylase activity was basally low, and leptin had no further effect. In paraventricular, arcuate, and medial hypothalamus of chow-fed mice, leptin inhibited α2-AMPK activity but not in DIO mice. In addition, leptin increased STAT3 phosphorylation 2-fold in arcuate of chow-fed mice, but this effect was attenuated because of elevated basal STAT3 phosphorylation in DIO mice. Thus, DIO in FVB mice alters α2-AMPK in muscle and hypothalamus and STAT3 in hypothalamus and impairs further effects of leptin on these signaling pathways. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states. AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance and of the effects of leptin on food intake and fatty acid oxidation. Obesity is usually associated with resistance to the effects of leptin on food intake and body weight. To determine whether diet-induced obesity (DIO) impairs the AMPK response to leptin in muscle and/or hypothalamus, we fed FVB mice a high fat (55%) diet for 10–12 weeks. Leptin acutely decreased food intake by ∼30% in chow-fed mice. DIO mice tended to eat less, and leptin had no effect on food intake. Leptin decreased respiratory exchange ratio in chow-fed mice indicating increased fatty acid oxidation. Respiratory exchange ratio was low basally in high fat-fed mice, and leptin had no further effect. Leptin (3 mg/kg intraperitoneally) increased α2-AMPK activity 2-fold in muscle in chow-fed mice but not in DIO mice. Leptin decreased acetyl-CoA carboxylase activity 40% in muscle from chow-fed mice. In muscle from DIO mice, acetyl-CoA carboxylase activity was basally low, and leptin had no further effect. In paraventricular, arcuate, and medial hypothalamus of chow-fed mice, leptin inhibited α2-AMPK activity but not in DIO mice. In addition, leptin increased STAT3 phosphorylation 2-fold in arcuate of chow-fed mice, but this effect was attenuated because of elevated basal STAT3 phosphorylation in DIO mice. Thus, DIO in FVB mice alters α2-AMPK in muscle and hypothalamus and STAT3 in hypothalamus and impairs further effects of leptin on these signaling pathways. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states. Obesity has reached epidemic proportions worldwide and currently affects one in three Americans (1Friedman J.M. Nature. 2000; 404: 632-634Crossref PubMed Scopus (629) Google Scholar, 2Kopelman P.G. Nature. 2000; 404: 635-643Crossref PubMed Scopus (3663) Google Scholar). Most obese people are resistant to the actions of insulin. Obesity is a major risk factor for developing type 2 diabetes, cardiovascular disease, and some forms of cancer (1Friedman J.M. Nature. 2000; 404: 632-634Crossref PubMed Scopus (629) Google Scholar, 2Kopelman P.G. Nature. 2000; 404: 635-643Crossref PubMed Scopus (3663) Google Scholar). Leptin (Ob), a hormone secreted by the adipocyte in proportion to fat stores, plays a major role in regulating energy homeostasis by decreasing food intake and increasing energy expenditure. Although these effects are primarily through actions in the hypothalamus (3Friedman J.M. Halaas J.L. Nature. 1998; 395: 763-770Crossref PubMed Scopus (4523) Google Scholar), peripheral actions of leptin have also been described (4Muoio D.M. Dohm G.L. Fiedorek Jr., F.T. Tapscott E.B. Coleman R.A. Diabetes. 1997; 46: 1360-1363Crossref PubMed Google Scholar, 5Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1679) Google Scholar). Rodents with diet-induced obesity (DIO) 3The abbreviations used are: DIO, diet-induced obesity; ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; AMPK, AMP-activated protein kinase; ARC, arcuate nucleus; GTT, glucose tolerance test; MH, medial hypothalamus; PTP-1B, protein tyrosine phosphatase 1B; PVN, paraventricular nucleus; RER, respiratory exchange ratio; SOCS-3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3; ANOVA, analysis of variance. and most obese humans are resistant to the effects of leptin (6Van Heek M. Compton D.S. France C.F. Tedesco R.P. Fawzi A.B. Graziano M.P. Sybertz E.J. Strader C.D. Davis Jr., H.R. J. Clin. Investig. 1997; 99: 385-390Crossref PubMed Scopus (660) Google Scholar, 7Heymsfield S.B. Greenberg A.S. Fujioka K. Dixon R.M. Kushner R. Hunt T. Lubina J.A. Patane J. Self B. Hunt P. McCamish M. J. Am. Med. 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One proposed mechanism for leptin resistance in DIO is decreased leptin transport across the blood-brain barrier (14Hileman S.M. Pierroz D.D. Masuzaki H. Bjorbaek C. El-Haschimi K. Banks W.A. Flier J.S. Endocrinology. 2002; 143: 775-783Crossref PubMed Scopus (189) Google Scholar, 15Banks W.A. Coon A.B. Robinson S.M. Moinuddin A. Shultz J.M. Nakaoke R. Morley J.E. Diabetes. 2004; 53: 1253-1260Crossref PubMed Scopus (393) Google Scholar). However, this appears to account for only part of the defect in leptin action. Although intracerebroventricular leptin administration induces STAT3 phosphorylation in DIO, this phosphorylation is dramatically reduced when compared with chow-fed mice (13Prpic V. Watson P.M. Frampton I.C. Sabol M.A. Jezek G.E. Gettys T.W. Endocrinology. 2003; 144: 1155-1163Crossref PubMed Scopus (66) Google Scholar, 16El-Haschimi K. Pierroz D.D. Hileman S.M. Bjorbaek C. Flier J.S. J. Clin. Investig. 2000; 105: 1827-1832Crossref PubMed Scopus (714) Google Scholar). 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AMPK stimulates fatty acid oxidation through phosphorylation of acetyl-CoA carboxylase (ACC) thereby decreasing malonyl-CoA levels, which disinhibits carnitine palmitoyl transferase-1 and increases fatty acid entry into mitochondria. In addition, AMPK phosphorylates target proteins involved in a number of metabolic pathways, including lipolysis (adipocytes), lipid metabolism (liver and muscle), glucose transport (muscle and adipocytes), and glycogen metabolism (muscle and liver) (34Kahn B.B. Alquier T. Carling D. Hardie D.G. Cell Metab. 2005; 1: 15-25Abstract Full Text Full Text PDF PubMed Scopus (2330) Google Scholar). We demonstrated a direct, transient effect of leptin on AMPK activation in oxidative muscle and a more sustained effect that is mediated through the hypothalamus and sympathetic nervous system (5Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1679) Google Scholar). 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In this study, we sought to determine whether the impaired response of the AMPK pathway to leptin could contribute to the molecular pathogenesis of leptin resistance in mice on a high fat diet. We demonstrate that by 12 weeks of high fat feeding, DIO mice are resistant to the effects of leptin administration on AMPK activity in both muscle and hypothalamus. This may be due, at least in part, to constitutive alterations in the AMPK signaling pathway in the absence of leptin administration. Basal activity of AMPK tends to be increased, and basal ACC activity is decreased in muscle. In paraventricular nucleus (PVN) from DIO mice, AMPK activity is constitutively decreased, and in PVN, arcuate (ARC), and medial hypothalamus, leptin fails to suppress AMPK activity. These data suggest that the AMPK pathway is dysregulated in muscle and hypothalamus in obese states resulting from high fat feeding and that lack of dynamic responsiveness of this pathway may play a role in the pathophysiology of leptin resistance in diet-induced obesity. Mice and Diets—Male FVB mice were obtained from Taconic at approximately 3 weeks of age. After an acclimation period of 1 week, mice were randomly assigned to two groups, chow or high fat (DIO). Chow mice were fed Purina Chow diet 5008 (4.5% calories from fat), whereas DIO mice ate a diet high in fat (55% calories from lard; Harlan Teklad 93075) for 5–12 weeks. Mice were housed one per cage in a temperature-controlled room and were maintained on a 14/10-h light-dark cycle. Mice had ad libitum access to both food and water. Treatment and Tissue Harvesting—Mice were handled for 3–5 days prior to experiments to reduce stress during the experiment. After an overnight fast, mice were injected with saline (control) or leptin intraperitoneally (3 mg/kg; A. F. Parlow, National Hormone & Peptide Program, Torrance, CA). Five hours later, mice were anesthetized with ketamine/xylazine and killed by decapitation. Hypothalamic nuclei and peripheral tissues were rapidly dissected and frozen in liquid nitrogen. Each hypothalamic region was dissected from 1-mm-thick sagittal sections of fresh brain. PVN, ARC, ventromedial hypothalamus, and dorsomedial hypothalamus were dissected from the first sections from the midline of the brain (10Minokoshi Y. Alquier T. Furukawa N. Kim Y.B. Lee A. Xue B. Mu J. Foufelle F. Ferre P. Birnbaum M.J. Stuck B.J. Kahn B.B. Nature. 2004; 428: 569-574Crossref PubMed Scopus (1339) Google Scholar). All assays were performed on hypothalamic regions from individual mice. Metabolic Parameters—Body weights were measured at the same time each week. Random fed mice were bled prior to starting the diets and again a week before sacrifice. Plasma samples were centrifuged, and serum was stored at –20 °C until it was assayed. Plasma glucose was measured using the One-Touch Ultra glucometer. Plasma insulin and leptin levels were determined by their respective enzyme-linked immunosorbent assay kits (Crystal Chem Inc., Downers Grove, IL). For the glucose tolerance test (GTT), mice were fasted for 16 h, and 2 mg/g glucose was injected intraperitoneally. Blood glucose was measured at 0, 15, 30, 60, and 120 min after injection. For the insulin tolerance test, food was removed at 8 a.m. Four hours later, mice were injected with 1 unit/kg human insulin (Lilly) intraperitoneally. Blood was withdrawn from the tail vein at 0, 15, 30, 45, 60, and 90 min. Food Intake—After 10 weeks of DIO or chow diet, six mice from each group received an intraperitoneal injection of leptin (3 mg/kg body weight) or saline at the start of the dark cycle and again 14 h later. Body weight and food intake were measured 14 and 24 h after the first injection. Indirect Calorimetry—The metabolic rate of mice was measured by indirect calorimetry in eight opencircuit oxymax chambers that are a component of the Comprehensive Lab Animal Monitoring System (Columbus Instruments, Columbus, OH). Mice were housed singly and maintained at 24 °C under a 12-h light-dark cycle (dark period 20:00–8:00). Food and water were available ad libitum. All mice were acclimated to monitoring cages for 24 h prior to beginning the physiological recordings. Mice were injected with saline at 4 p.m. on day 1 and again at 8 a.m. on day 2. Mice were then injected with leptin (3mg/kg) at 4 p.m. on day 2 and at 8 a.m. and 4 p.m. on day 3. To calculate oxygen consumption (VO2), carbon dioxide production (VCO2), and RER (ratio of VCO2 to VO2), gas concentrations were measured at the inlet and outlet of the sealed chambers. Western Blot Analysis—Tissue lysates were prepared as described previously (10Minokoshi Y. Alquier T. Furukawa N. Kim Y.B. Lee A. Xue B. Mu J. Foufelle F. Ferre P. Birnbaum M.J. Stuck B.J. Kahn B.B. Nature. 2004; 428: 569-574Crossref PubMed Scopus (1339) Google Scholar). Phosphorylation of STAT3 in hypothalamic regions was determined with 7.5% SDS-acrylamide gels using an antibody against phosphotyrosine705 (Cell Signaling) of STAT3. Phosphorylation of the α-subunit of AMPK in soleus lysates was determined with 10% SDS-acrylamide gels by using antibodies that recognize phospho-Thr172 of the α-subunit of human AMPK (Cell Signaling). Blots were re-probed with antibodies to α2-AMPK (generous gift from Dr. D. Carling) or ACC (streptavidin-horseradish peroxidase from Amersham Biosciences). Chemiluminescence (Western Lightning, PerkinElmer Life Sciences) was quantified by laser densitometry within the linear range (Amersham Biosciences) or GeneSnap. Activity Assays—AMPK activity was measured in soleus muscle or hypothalamic regions by immunoprecipitation of α2-AMPK from muscle lysates (100 μg of protein) or brain regions (40–50 μg) with specific antibodies against the catalytic α2-subunits bound to protein-G/Sepharose beads. Kinase activity was measured using synthetic "SAMS" peptide and [γ-32P]ATP as described previously (10Minokoshi Y. Alquier T. Furukawa N. Kim Y.B. Lee A. Xue B. Mu J. Foufelle F. Ferre P. Birnbaum M.J. Stuck B.J. Kahn B.B. Nature. 2004; 428: 569-574Crossref PubMed Scopus (1339) Google Scholar). The activity of ACC in red (slow twitch) muscle lysates was measured by 14CO2 fixation to acid-stable products in the presence of citrate (2 mm), an allosteric activator of ACC. Statistical Analyses—All data are expressed as means ± S.E. Significance is set at p < 0.05. For GTT, insulin tolerance test, and RER, statistical analyses were performed using repeated measures ANOVA with Bonferroni post-test. Comparisons of mean plasma insulin, plasma leptin, food intake, AMPK activity, phosphorylated AMPK, ACC activity, and phosphorylated STAT3 in DIO versus chow were made by one-way ANOVA with Bonferroni's post-test. Comparisons of total protein levels (AMPK, ACC, and STAT3) between two groups (Chow and DIO) were made using Student's t test. Male FVB mice were randomized to either chow or DIO so initial body weights were similar in both groups. By 2 weeks on the high fat diet, the DIO mice were heavier than their chow-fed counterparts, and the weights continued to diverge throughout 11 weeks on the high fat diet (p < 0.05) (Fig. 1A). After 1 week on the high fat diet, serum insulin levels were normal, but after 10 weeks the insulin levels were ∼2-fold higher in DIO mice than in chow-fed mice (Fig. 1B). After 1 week on the diet, plasma leptin levels in the fed state were not different in DIO mice compared with chow-fed mice. Serum leptin levels increased ∼3-fold in chow-fed mice between 1 and 11 weeks of the study, whereas serum leptin levels in DIO mice increased ∼7-fold during this same period. After 11 weeks on the diet, leptin levels in the fed state were 3-fold higher in DIO mice compared with chow-fed mice (Fig. 1C). An overnight fast decreased serum leptin levels by 51% in chow-fed mice and 78% in DIO mice. Thus, in the fasted state, serum leptin was not elevated in DIO mice compared with fasted chow-fed mice (Fig. 1C). Overnight fasted DIO mice had elevated blood glucose (chow 101 ± 13 versus DIO 145 ± 5 mg/dl, p < 0.05). Glucose tolerance tests revealed overt diabetes in DIO mice (Fig. 1D), and these mice were unresponsive to exogenous insulin during an insulin tolerance test (Fig. 1E), indicating marked insulin resistance. One of the primary biological indicators of leptin resistance is the inability of leptin to decrease food intake. After 10 weeks on the diets, mice were injected with leptin intraperitoneally, and 24-h food intake was measured. Two injections of leptin (3 mg/kg, each) over 24 h decreased food intake in the chow-fed mice by more than 30%. Saline-injected DIO mice tended to eat less than saline-injected chow-fed mice, and leptin did not reduce food intake in DIO mice (Fig. 2A). We also measured food intake over 48 h and saw no effect of leptin in DIO mice (not shown). These data suggest that the DIO mice are resistant to the effects of leptin administration on food intake. In addition to its ability to decrease food intake, leptin also increases energy expenditure and fatty acid oxidation. To determine whether DIO mice are resistant to the effects of leptin on fat utilization, mice received three intraperitoneal injections of leptin (3 mg/kg, each) over a 24-h period while in the indirect calorimeter. The RER is a ratio of carbohydrate oxidation to lipid oxidation assuming protein oxidation is negligible (39McLean J.A. Tobin G. Animal and Human Calorimetry. Cambridge University Press, New York1987Google Scholar). An RER of 1.0 indicates high utilization of carbohydrate for energy, and an RER of 0.7 indicates increased fatty acid oxidation (39McLean J.A. Tobin G. Animal and Human Calorimetry. Cambridge University Press, New York1987Google Scholar). Fig. 2, B and C, shows the RER after the third intraperitoneal injection of leptin. Prior to the third injection, RER was lower in the leptin-injected chow group compared with the saline-injected chow-fed mice because of the previous two leptin injections. The third injection of leptin led to a sustained decrease in RER in mice on the chow diet compared with saline-injected chow-fed mice (p < 0.01). Even though RER rose in both chow-fed groups as they ate more starting around 18:00, RER remained lower in the leptin-injected chow-fed mice throughout most of the dark cycle. DIO mice have a lower base-line RER because they utilize fat for energy. Leptin had no effect on RER in DIO mice. Fig. 2C is a quantitation of the RER from 18:00 to 6:00 h after three intraperitoneal leptin injections. Whether the inability of leptin to further decrease RER in DIO mice is because of leptin resistance or because DIO mice are already at the lower biological limit for RER from the high fat content of the diet is unknown. The lowest RER we are aware of is 0.7 and is seen in mice on a ketogenic diet with an extremely low carbohydrate content. 4E. Maratos-Flier, personal communication. Lower RER in DIO mice reflects increased fatty acid utilization, and we demonstrated previously that leptin increases fatty acid oxidation through activation of AMPK in muscle (5Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1679) Google Scholar). Thus, changes in AMPK and ACC activities in muscle in DIO mice might explain, at least in part, their lower base-line RER. We demonstrated previously that leptin activates AMPK in muscle both directly and through the hypothalamic-sympathetic nervous system (5Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1679) Google Scholar). Because the physiological contribution of direct leptin action on peripheral tissues to whole-body energy homeostasis is controversial, we focused on the effects of leptin on AMPK that are mediated by the sympathetic nervous system. The effects of leptin on AMPK in the hypothalamus are more pronounced after an overnight fast (10Minokoshi Y. Alquier T. Furukawa N. Kim Y.B. Lee A. Xue B. Mu J. Foufelle F. Ferre P. Birnbaum M.J. Stuck B.J. Kahn B.B. Nature. 2004; 428: 569-574Crossref PubMed Scopus (1339) Google Scholar). In fasted chow-fed mice, leptin increased AM