C75, a Fatty Acid Synthase Inhibitor, Modulates AMP-activated Protein Kinase to Alter Neuronal Energy Metabolism
2004; Elsevier BV; Volume: 279; Issue: 5 Linguagem: Inglês
10.1074/jbc.m310991200
ISSN1083-351X
AutoresLeslie E. Landree, Andrea L. Hanlon, David W. Strong, Gavin Rumbaugh, Ian M. Miller, Jagan N. Thupari, Erin C. Connolly, Richard L. Huganir, Christine Richardson, Lee A. Witters, Francis P. Kuhajda, Gabriele V. Ronnett,
Tópico(s)Regulation of Appetite and Obesity
ResumoC75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior. C75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior. Obesity has become a worldwide health issue, affecting children and adults in developed and emerging countries (1Hill J.O. Wyatt H.R. Reed G.W. Peters J.C. Science. 2003; 299: 853-855Google Scholar, 2Mokdad A.H. Bowman B.A. Ford E.S. Vinicor F. Marks J.S. Koplan J.P. Jama. 2001; 286: 1195-1200Google Scholar, 3Kopelman P. Nature. 2000; 404: 635-643Google Scholar). Obesity is a disorder of both energy metabolism and appetite regulation and must be understood as a dysfunction of energy balance (3Kopelman P. Nature. 2000; 404: 635-643Google Scholar). The central nervous system (CNS) plays a critical role in the regulation of energy balance by coordinating peripheral and central signals to assess energy status and regulate feeding behavior (4Schwartz M. Woods S. Porte D.J. Seeley R. Baskin D. Nature. 2000; 404: 661-671Google Scholar, 5Spiegelman B. Flier J. Cell. 2001; 104: 531-543Google Scholar, 6Woods S. Seeley R. Nutrition. 2000; 16: 894-902Google Scholar). While it is well known that fatty acid metabolism is an important component of the peripheral regulation of energy metabolism, recent studies indicate that neurons in the hypothalamus may monitor flux through the fatty acid synthesis pathway to ascertain energy balance (7Obici S. Feng Z. Arduini A. Conti R. Rossetti L. Nat. Med. 2003; 9: 756-761Google Scholar, 8Loftus T. Jaworsky D. Frehywot G. Townsend C. Ronnett G. Lane M. Kuhajda F. Science. 2000; 288: 2379-2381Google Scholar, 9Kim E.K. Miller I. Landree L.E. Borisy-Rudin F.F. Brown P. Tihan T. Townsend C.A. Witters L.A. Moran T.H. Kuhajda F.P. Ronnett G.V. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E867-E879Google Scholar, 10Thupari J.N. Landree L.E. Ronnett G.V. Kuhajda F.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9498-9502Google Scholar). We and others have demonstrated that C75, a synthetic fatty acid synthase (FAS) 1The abbreviations used are: FASfatty acyl synthaseHPLChigh performance liquid chromatographyMe2SOdimethyl sulfoxideGFAPglia fibrillary acidic proteinFITCfluorescein isothiocyanateACCacetyl-CoA carboxylaseCPT-1carnitine palmitoyltransferase-1NSTneuron-specific tubulinAMPKAMP-activated protein kinasePBSphosphate-buffered salinemEPSCminiature excitatory postsynaptic current. inhibitor (11Kuhajda F.P. Pizer E.S. Li J.N. Mani N.S. Frehywot G.L. Townsend C.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3450-3454Google Scholar), decreases food intake and results in profound and reversible weight loss (8Loftus T. Jaworsky D. Frehywot G. Townsend C. Ronnett G. Lane M. Kuhajda F. Science. 2000; 288: 2379-2381Google Scholar, 12Shimokawa T. Kumar M.V. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 66-71Google Scholar, 13Kumar M.V. Shimokawa T. Nagy T.R. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1921-1925Google Scholar, 14Clegg D.J. Wortman M.D. Benoit S.C. McOsker C.C. Seeley R.J. Diabetes. 2002; 51: 3196-3201Google Scholar). Although inhibition of peripheral fatty acid synthesis might easily explain some of these effects, anorexia was also achieved by central (intracerebroventricular) administration of C75 (8Loftus T. Jaworsky D. Frehywot G. Townsend C. Ronnett G. Lane M. Kuhajda F. Science. 2000; 288: 2379-2381Google Scholar, 14Clegg D.J. Wortman M.D. Benoit S.C. McOsker C.C. Seeley R.J. Diabetes. 2002; 51: 3196-3201Google Scholar). These results suggest that the FAS pathway may play a role in energy homeostasis in the brain and that modulation of flux through this pathway in neurons or glia could alter energy levels. fatty acyl synthase high performance liquid chromatography dimethyl sulfoxide glia fibrillary acidic protein fluorescein isothiocyanate acetyl-CoA carboxylase carnitine palmitoyltransferase-1 neuron-specific tubulin AMP-activated protein kinase phosphate-buffered saline miniature excitatory postsynaptic current. FAS is a lipogenic enzyme that catalyzes the condensation of acetyl-CoA and malonyl-CoA to generate long-chain fatty acids (15Wakil S. Biochemistry. 1989; 28: 4523-4530Google Scholar). In the fed state, long-chain fatty acids are synthesized and stored as triglycerides, which are broken down for energy during periods of energy deficiency. C75 interferes with the binding of malonyl-CoA to the β-ketoacyl synthase domain of FAS, thus inhibiting long-chain fatty acid elongation (16Kuhajda F.P. Jenner K. Wood F.D. Hennigar R.A. Jacobs L.B. Dick J.D. Pasternack G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6379-6383Google Scholar, 17Kuhajda F. Pizer E. Mani N. Pinn M. Han F. Chrest F. Freywot G. Townsend C. Proc. Am. Assoc. of Cancer Res. 1999; 40: 121Google Scholar). Although it is established that FAS mRNA levels are high in lipogenic tissues such as liver, lung, and adipose (18Jayakumar A. Tai M.H. Huang W.Y. al-Feel W. Hsu M. Abu-Elheiga L. Chirala S.S. Wakil S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8695-8699Google Scholar), FAS was only recently described in brain (19Kusakabe T. Maeda M. Hoshi N. Sugino T. Watanabe K. Kukuda T. Suzuki T. J. Histochem. Cytochem. 2000; 48: 613-622Google Scholar, 9Kim E.K. Miller I. Landree L.E. Borisy-Rudin F.F. Brown P. Tihan T. Townsend C.A. Witters L.A. Moran T.H. Kuhajda F.P. Ronnett G.V. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E867-E879Google Scholar). We have demonstrated that FAS, and other enzymes required for long-chain fatty acid synthesis, are highly expressed in neurons in many brain regions, including hypothalamic neurons that regulate feeding behavior (9Kim E.K. Miller I. Landree L.E. Borisy-Rudin F.F. Brown P. Tihan T. Townsend C.A. Witters L.A. Moran T.H. Kuhajda F.P. Ronnett G.V. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E867-E879Google Scholar), thus positioning FAS to play a role in neuronal energy metabolism. In addition to inhibiting FAS (11Kuhajda F.P. Pizer E.S. Li J.N. Mani N.S. Frehywot G.L. Townsend C.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3450-3454Google Scholar), C75 stimulates carnitine palmitoyltransferase-1 (CPT-1) activity in cultured adipocytes and hepatocyes by preventing malonyl-CoA-mediated inhibition of CPT-1 (10Thupari J.N. Landree L.E. Ronnett G.V. Kuhajda F.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9498-9502Google Scholar). CPT-1 catalyzes the esterification of long-chain acyl-CoAs to l-carnitine for transport into the mitochondria for fatty acid oxidation (20McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Google Scholar). When energy reserves and fatty acid synthesis are high, elevated levels of malonyl-CoA, the endogenous inhibitor of CPT-1, prevent the oxidation of newly formed fatty acids (20McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Google Scholar, 21Eaton S. Bartlett K. Quant P.A. Biochem. Biophys. Res. Commun. 2001; 285: 537-539Google Scholar). Conversely, during starvation, when energy levels are low, malonyl-CoA levels fall, releasing the inhibition on CPT-1, allowing fatty acids to enter the mitochondria where they are broken down for energy. While C75 can affect FAS and CPT-1 activities in peripheral tissues, its effects on neurons are unknown. The effects of C75 on FAS and CPT-1 activities could alter energy flux through metabolic pathways in neurons. One important sensor of cellular energy balance is mammalian AMP-activated protein kinase (AMPK), a heterotrimeric protein kinase present in most mammalian tissues, composed of a catalytic subunit (α) and two regulatory subunits (β and γ) (22Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Google Scholar, 23Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.P. Witters L.A. Trends Biochem. Sci. 1999; 24: 22-25Google Scholar). AMPK is most commonly activated by metabolic stresses that deplete cellular ATP and lead to an elevation of the AMP/ATP ratio (22Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Google Scholar, 23Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.P. Witters L.A. Trends Biochem. Sci. 1999; 24: 22-25Google Scholar), such as heat shock, exercise, ischemia/hypoxia, low glucose, and metabolic or excitotoxic insults in neurons (24Culmsee C. Monnig J. Kemp B.E. Mattson M.P. J. Mol. Neurosci. 2001; 17: 45-58Google Scholar). The regulation of AMPK activity is complex, and once activated, AMPK modulates many aspects of metabolism. Acutely, AMPK phosphorylates and inactivates certain enzymes involved in biosynthetic pathways, such as HMG-CoA reductase and acetyl-CoA carboxylase, thereby preventing further ATP utilization (22Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Google Scholar, 25Hardie D.G. Hawley S.A. Bioessays. 2001; 23: 1112-1119Google Scholar). Additionally, AMPK stimulates catabolic processes by activating glucose uptake, glycolysis, and fatty acid oxidation in an attempt to restore cellular ATP levels (27Abbud W. Habinowski S. Zhang J.Z. Kendrew J. Elkairi F.S. Kemp B.E. Witters L.A. Ismail-Beigi F. Arch. Biochem. Biophys. 2000; 380: 347-352Google Scholar, 28Kurth-Kraczek E.J. Hirshman M.F. Goodyear L.J. Winder W.W. Diabetes. 1999; 48: 1667-1671Google Scholar, 29Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Google Scholar, 30Winder W.W. Hardie D.G. Am. J. Physiol. 1996; 270: E299-E304Google Scholar). AMPK has chronic effects on these pathways via alterations in gene expression (for review see Ref. 25Hardie D.G. Hawley S.A. Bioessays. 2001; 23: 1112-1119Google Scholar). Although AMPK has been shown to be expressed the brain (24Culmsee C. Monnig J. Kemp B.E. Mattson M.P. J. Mol. Neurosci. 2001; 17: 45-58Google Scholar, 31Davies S.P. Carling D. Hardie D.G. Eur. J. Biochem. 1989; 186: 123-128Google Scholar, 32Turnley A.M. Stapleton D. Mann R.J. Witters L.A. Kemp B.E. Bartlett P.F. J. Neurochem. 1999; 72: 1707-1716Google Scholar), its function in neurons is unknown. We have chosen AMPK as a candidate neuronal metabolic sensor that may be affected by C75 based on its well established roles in energy sensing and the control of fatty acid metabolism in the periphery. In this study, we explored the role of FAS, CPT-1, and AMPK in neuronal energy metabolism. We utilized primary cortical neuronal cultures as a model system after demonstrating high levels of expression and activity of these key proteins, consistent with their in vivo expression patterns (9Kim E.K. Miller I. Landree L.E. Borisy-Rudin F.F. Brown P. Tihan T. Townsend C.A. Witters L.A. Moran T.H. Kuhajda F.P. Ronnett G.V. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E867-E879Google Scholar, 19Kusakabe T. Maeda M. Hoshi N. Sugino T. Watanabe K. Kukuda T. Suzuki T. J. Histochem. Cytochem. 2000; 48: 613-622Google Scholar, 24Culmsee C. Monnig J. Kemp B.E. Mattson M.P. J. Mol. Neurosci. 2001; 17: 45-58Google Scholar, 32Turnley A.M. Stapleton D. Mann R.J. Witters L.A. Kemp B.E. Bartlett P.F. J. Neurochem. 1999; 72: 1707-1716Google Scholar). The effects of C75 on ATP levels and AMPK activity were then investigated, and the consequences of altered AMPK activity to energy production were determined. The work presented here demonstrates that C75, through both direct and indirect actions, alters neuronal energy metabolism in a biphasic fashion. These alterations are functionally important and suggest a role for AMPK in neuronal energy perception. Primary Cortical Neuronal Cultures—All experimental protocols were approved by the Johns Hopkins University Institutional Animal Care and Use Committee, and all applicable guidelines from the National Institutes of Health Guide for the care and use of laboratory animals were followed. Cortices were removed from E17 Sprague-Dawley rats (Harlan, Indianapolis, IN), and were dissociated by mild trypsinization and trituration as described (33Dawson V.L. Dawson T.M. Bartley D. Uhl G. Snyder S. J. Neurosci. 1993; 13: 2651-2661Google Scholar). Cells were plated on poly-d-lysine coated plastic Nunclon culture dishes at a density of 5 × 105 cells/cm2 in Minimum Essential Media (MEM) supplemented with horse serum, fetal bovine serum, glutamine, and the antibiotics gentamycin and kanamycin. Cells were plated onto vessels as required for each type of experiment: T-25 flasks for oxidation assays; 6-well plates for Western blots, SAMS peptide assays, and HPLC analysis; 24 well plates for FAS and CPT-1 activity assays; 4 well chamber slides for immunocytochemistry; and 96 well plates for the determination of ATP levels and cell viability assays. For standard cultures cells were treated with cytosine arabinoside on day 4, and were assayed after 7-10 days in vitro. For cultures overgrown with glia, cells were not treated with cytosine arabinoside and were used for immunocytochemistry on day 6. Drug treatments were performed with vehicle or C75 (obtained from and characterized by Craig Townsend and Jill McFadden, the Department of Chemistry at the Johns Hopkins University, and from FASgen, Inc.), resuspended in RPMI; cerulenin (Sigma) resuspended in RPMI; and 5-(tetradecyloxy)-2 Furoic Acid (TOFA) (Craig Townsend and Jill McFadden) resuspended in 100% Me2SO. Immunocytochemistry—Cortical neurons were grown as described and harvested 7 days after plating for immunocytochemistry. Cells were fixed with 4% PFA and 20% sucrose for 20 min at 4 °C, and permeated with 0.2% Triton X-100 in PBS for 10 min at 4 °C. As these cultures normally contain less than 1% glial cells, cultures were also prepared in which glia were allowed to overgrow, as described, to better evaluate the expression of FAS and AMPK in glia. Cells were incubated in blocking solution (PBS containing 4% normal serum) for 1 h at 4 °C. Primary antibodies against the following antigens were diluted in blocking solution overnight at 4 °C: glia fibrillary acidic protein (GFAP) (Chemicon International Temecula, CA) 1:1000; neuron-specific tubulin (NST) (Bacbo, Richmond VA) 1:1000; AMPKα (2Mokdad A.H. Bowman B.A. Ford E.S. Vinicor F. Marks J.S. Koplan J.P. Jama. 2001; 286: 1195-1200Google Scholar, 3Kopelman P. Nature. 2000; 404: 635-643Google Scholar, 4Schwartz M. Woods S. Porte D.J. Seeley R. Baskin D. Nature. 2000; 404: 661-671Google Scholar, 5Spiegelman B. Flier J. Cell. 2001; 104: 531-543Google Scholar, 6Woods S. Seeley R. Nutrition. 2000; 16: 894-902Google Scholar, 7Obici S. Feng Z. Arduini A. Conti R. Rossetti L. Nat. Med. 2003; 9: 756-761Google Scholar, 8Loftus T. Jaworsky D. Frehywot G. Townsend C. Ronnett G. Lane M. Kuhajda F. Science. 2000; 288: 2379-2381Google Scholar, 9Kim E.K. Miller I. Landree L.E. Borisy-Rudin F.F. Brown P. Tihan T. Townsend C.A. Witters L.A. Moran T.H. Kuhajda F.P. Ronnett G.V. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E867-E879Google Scholar, 10Thupari J.N. Landree L.E. Ronnett G.V. Kuhajda F.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9498-9502Google Scholar, 11Kuhajda F.P. Pizer E.S. Li J.N. Mani N.S. Frehywot G.L. Townsend C.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3450-3454Google Scholar, 12Shimokawa T. Kumar M.V. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 66-71Google Scholar, 13Kumar M.V. Shimokawa T. Nagy T.R. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1921-1925Google Scholar, 14Clegg D.J. Wortman M.D. Benoit S.C. McOsker C.C. Seeley R.J. Diabetes. 2002; 51: 3196-3201Google Scholar, 15Wakil S. Biochemistry. 1989; 28: 4523-4530Google Scholar, 16Kuhajda F.P. Jenner K. Wood F.D. Hennigar R.A. Jacobs L.B. Dick J.D. Pasternack G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6379-6383Google Scholar, 17Kuhajda F. Pizer E. Mani N. Pinn M. Han F. Chrest F. Freywot G. Townsend C. Proc. Am. Assoc. of Cancer Res. 1999; 40: 121Google Scholar, 18Jayakumar A. Tai M.H. Huang W.Y. al-Feel W. Hsu M. Abu-Elheiga L. Chirala S.S. Wakil S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8695-8699Google Scholar, 19Kusakabe T. Maeda M. Hoshi N. Sugino T. Watanabe K. Kukuda T. Suzuki T. J. Histochem. Cytochem. 2000; 48: 613-622Google Scholar, 20McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Google Scholar) 1:500; and FAS (9Kim E.K. Miller I. Landree L.E. Borisy-Rudin F.F. Brown P. Tihan T. Townsend C.A. Witters L.A. Moran T.H. Kuhajda F.P. Ronnett G.V. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E867-E879Google Scholar) 1:1000. Cells were incubated for 1 h at room temperature with secondary antibodies conjugated with FITC for NST and GFAP staining, or with rhodamine for FAS and AMPK staining. Measurement of Acetate Incorporation—Cells were pretreated with the indicated concentrations of vehicle or C75 for 15 min in conditioned media, and then labeled with 100 μm [3H]acetic acid (PerkinElmer Life Sciences) for an additional 1.75 h as previously described (34Pizer E.S. Wood F.D. Pasternack G.R. Kuhajda F.P. Cancer Res. 1996; 1996: 745-751Google Scholar). Lipids were extracted with chloroform/methanol, dried under N2 and counted using a liquid scintillation counter. Measurement of ATP—Neurons were lysed on ice using TE buffer (100 mm Tris and 4 mm EDTA) and removed from the plate. ATP levels were then measured in the linear range using the ATP Bioluminescence kit CLS II (Roche Applied Science, Indianapolis, IN) by following the manufacturer's protocol, and results were read by a PerkinElmer Victor2 1420. Cell Viability Assay—Cortical neurons were treated for the indicated times with the indicated doses of drug, and viability was determined using the Live/Dead Viability/Cytotoxicity kit (Molecular Probes, Eugene, OR). The conversion of the cell permeant non-fluorescent calcein AM dye to the intensely fluorescent calcein dye is catalyzed by intracellular esterase activity in live cells and is measured by detecting the absorbance at 485 nm/535 nm using the PerkinElmer Victor2 1420. HPLC—Adenine nucleotide levels in primary cortical neuron lysates were determined by HPLC analysis as previously described (35Stocchi V. Cucchiarini L. Canestrari F. Piacentini M.P. Fornaini G. Anal. Biochem. 1987; 167: 181-190Google Scholar). Briefly, each well of a 6-well plate was washed with 2 ml of ice cold PBS, and lysed with 70 μl of ice-cold 0.5 m KOH and scraped. 140 μl of H20 were added to lysates and incubated on ice for 5 min, and the pH was then adjusted to 6.5 by addition of 1 m KH2PO4. Cell lysates were spun through Microcon YM-50 centrifugal filters and stored at -80 °C for subsequent HPLC analysis. The HPLC used was an Agilent 1100 LC with a variable wavelength detector. The analysis was done using Chemstation A.10.01 software. Measurement of Fatty Acid Oxidation—Fatty acid oxidation was measured as previously described (36Watkins P.A. Ferrell Jr., E.V. Pedersen J.I. Hoefler G. Arch. Biochem. Biophys. 1991; 289: 329-336Google Scholar). Briefly, primary cortical neurons adherent to the flask were treated in triplicate with C75 at the indicated doses for the indicated times in of HAM-F10 media supplemented with 10% fetal bovine serum. 0.5 μCi/ml (20 nmol) of [1-14C]palmitic acid (Moravek Biochemicals, Brea, CA) resuspended in α-cyclodextran (10 mg/ml in 10 mm Tris) and 2 μm carnitine was added for the last 30 min of each treatment. Flasks were fitted with serum stoppers and plastic center wells (Kontes, Vineland, NJ) containing glass microfiber filters (presoaked in 10 μl of 20% KOH). Following the incubation, 200 μl of 2.6 N HClO4 was injected into the flasks and the 14CO2 was trapped for 2 h at 37 °C. The filters were removed and quantified by liquid scintillation counting. The contents of the flasks were then hydrolyzed with 200 μl of 4 n KOH and neutralized using H2SO4. The water soluble products were extracted using CHCl3/MeOH and H2O and quantified by liquid scintillation counting. The total amount of fatty acid oxidation was obtained by addition of the 14CO2 and water soluble products and represented as percent of control or as a specific activity (nmol/h/mg). Measurement of Glucose Oxidation—Glucose oxidation assays were based on previously described work (37Rubi B. Antinozzi P.A. Herrero L. Ishihara H. Asins G. Serra D. Wollheim C.B. Maechler P. Hegardt F.G. Biochem. J. 2002; 364: 219-226Google Scholar). Neurons adherent to the flask were treated in triplicate with C75 at the indicated doses for the indicated times in Krebs-Ringer bicarbonate HEPES buffer (KRBH buffer: 135 mm NaCl, 3.6 mm KCl, 0.5 mm NaH2PO4, 0.5 mm MgCl2, 1.5 mm CaCl2, 5 mm NaHO3 and 10 mm HEPES) containing 1% bovine serum albumin and 10 mm d-glucose. 0.5 μCi/ml [U-14C]glucose (PerkinElmer Life Sciences) was added for the last 30 min of each treatment and flasks were fitted as described for fatty acid oxidation assays. Reactions were stopped with the injection of 7% perchloric acid into the flask, and then 400 μl of benzethonium hydroxide was injected into the center well. After 2 h at 37 °C, complete oxidation was quantified by measuring the amount of 14CO2 in the center well by liquid scintillation counting, and represented as percent of control or as a specific activity (pmol/h/mg). Measurement of CPT-1 Activity—CPT-1 activity was measured using digitonin permeabilization (38Sleboda J. Risan K.A. Spydevold O. Bremer J. Biochim. Biophy. Acta. 1999; 1436: 541-549Google Scholar). Drugs and vehicle controls were added as indicated for each experiment. After 2 h, the medium was removed, cells were washed with PBS, and incubated with 700 μl of assay medium consisting of: 50 mm imidazole, 70 mm KCl, 80 mm sucrose, 1 mm EGTA, 2 mm MgCl2, 1 mm dithiothreitol, 1 mm KCN, 1 mm ATP, 0.1% fatty acid free bovine serum albumin, 70 μm palmitoyl-CoA, 0.25 μCi l-[methyl-14C]carnitine (Amersham Biosciences), 40 μg of digitonin, with or without 100 μm malonyl-CoA. After incubation for 6 min at 37 °C, the reaction was stopped by the addition of 500 μl of ice-cold 4 m perchloric acid. Cells were then harvested and centrifuged at 13,000 × g for 5 min. The pellet was washed with 500 μl of ice-cold 2 mm perchloric acid and centrifuged again. The resulting pellet was resuspended in 800 μl of dH2O and extracted with 400 μl of butyl alcohol. The butyl alcohol phase, representing the acylcarnitine derivative, was measured by liquid scintillation counting. Western Blot Analysis—Cells were lysed using an identical method and buffer as described for the AMPK activity assay. Samples were boiled and run on 10% polyacrylamide gels, and transferred to polyvinylidene difluoride membrane. Blots were successively probed, stripped, and reprobed with the following antibodies: anti-pAMPKα, AMPKα (Cell Signaling, Beverly, MA), and sheep anti-FAS (39Salles J. Sargueil F. Knoll-Gellida A. Witters L.A. Shy M. Jiang H. Cassagne C. Garbay B. Brain. Res. Mol. Brain Res. 2002; 101: 52-58Google Scholar). Samples for the ACC/pACC westerns were run on separate 5% polyacrylamide gels and probed with either anti-ACC or anti-phospho-Ser-79 ACC antibodies (40Habinowski S.A. Hirshman M. Sakamoto K. Kemp B.E. Gould S.J. Goodyear L.J. Witters L.A. Arch. Biochem. Biophys. 2001; 396: 71-79Google Scholar). Measurement of AMP-activated Protein Kinase Activity—AMPK activity was determined by performing SAMS peptide assays as previously described (41Witters L.A. Kemp B.E. J. Biol. Chem. 1992; 267: 2864-2867Google Scholar). Neurons plated on 6-well culture dishes were lysed using 350 μl of per well of Triton X-100 lysis buffer: 20 mm Tris-HCl, pH 7.4, 50 mm NaCl, 1% Triton X-100, 250 mm sucrose, 50 mm NaF, 5 mm NaPPi, 1 mm dithiothreitol, 50 μg/ml leupeptin, 0.1 mm benzamidine, and 50 μg/ml trypsin inhibitor. Three wells were pooled per condition, and AMPKα was immunoprecipitated in the presence anti-AMPKα (2Mokdad A.H. Bowman B.A. Ford E.S. Vinicor F. Marks J.S. Koplan J.P. Jama. 2001; 286: 1195-1200Google Scholar, 3Kopelman P. Nature. 2000; 404: 635-643Google Scholar, 4Schwartz M. Woods S. Porte D.J. Seeley R. Baskin D. Nature. 2000; 404: 661-671Google Scholar, 5Spiegelman B. Flier J. Cell. 2001; 104: 531-543Google Scholar, 6Woods S. Seeley R. Nutrition. 2000; 16: 894-902Google Scholar, 7Obici S. Feng Z. Arduini A. Conti R. Rossetti L. Nat. Med. 2003; 9: 756-761Google Scholar, 8Loftus T. Jaworsky D. Frehywot G. Townsend C. Ronnett G. Lane M. Kuhajda F. Science. 2000; 288: 2379-2381Google Scholar, 9Kim E.K. Miller I. Landree L.E. Borisy-Rudin F.F. Brown P. Tihan T. Townsend C.A. Witters L.A. Moran T.H. Kuhajda F.P. Ronnett G.V. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E867-E879Google Scholar, 10Thupari J.N. Landree L.E. Ronnett G.V. Kuhajda F.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9498-9502Google Scholar, 11Kuhajda F.P. Pizer E.S. Li J.N. Mani N.S. Frehywot G.L. Townsend C.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3450-3454Google Scholar, 12Shimokawa T. Kumar M.V. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 66-71Google Scholar, 13Kumar M.V. Shimokawa T. Nagy T.R. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1921-1925Google Scholar, 14Clegg D.J. Wortman M.D. Benoit S.C. McOsker C.C. Seeley R.J. Diabetes. 2002; 51: 3196-3201Google Scholar, 15Wakil S. Biochemistry. 1989; 28: 4523-4530Google Scholar, 16Kuhajda F.P. Jenner K. Wood F.D. Hennigar R.A. Jacobs L.B. Dick J.D. Pasternack G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6379-6383Google Scholar, 17Kuhajda F. Pizer E. Mani N. Pinn M. Han F. Chrest F. Freywot G. Townsend C. Proc. Am. Assoc. of Cancer Res. 1999; 40: 121Google Scholar, 18Jayakumar A. Tai M.H. Huang W.Y. al-Feel W. Hsu M. Abu-Elheiga L. Chirala S.S. Wakil S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8695-8699Google Scholar, 19Kusakabe T. Maeda M. Hoshi N. Sugino T. Watanabe K. Kukuda T. Suzuki T. J. Histochem. Cytochem. 2000; 48: 613-622Google Scholar, 20McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Google Scholar) antibody coupled to protein A/G beads (Santa Cruz Biotechnology). Immunoprecipitates were then washed and resuspended in 4× assay buffer and kinase activity was assessed by measurement (for 20 min at 30 °C) of the incorporation of 32P into the synthetic SAMS peptide substrate, HMRSAMSGLHLVKRR, (Princeton Biomolecules). Samples were spotted on P81 phosphocellulose paper, washed extensively, and quantitated by Cerenkov counting. Each sample was corrected for protein concentration and reported either as percent of control or as pmol/min/mg. Electrophysiology and Miniature Excitatory Postsynaptic Current (mEPSC) Analysis—Whole cell patch clamp recordings were performed from cortical cultures at 14-21 days in vitro. To isolate AMPA-mediated mEPSCs, neurons were continuously perfused with artificial cerebral spinal fluid (aCSF) at a flow rate of <1 ml/min. The composition of aCSF was as follows (in mm): 150 NaCl, 3.1 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 0.1 dl-APV, 0.005 strychnine, 0.1 picrotoxin, and 0.001 tetrodotoxin (TTX). The osmolarity of the aCSF was adjusted to 305-310, pH was 7.3-7.4. Intracellular saline consisted of (in mm): 135 CsMeSo4, 10 CsCl, 10 HEPES, 5 EGTA, 2 MgCl2, 4 Na-ATP, and 0.1 Na-GTP. This saline was adjusted to 290-295 mOsm, pH 7.2. Once the whole cell r
Referência(s)