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J. Biol. Chem., Vol. 279, Issue 5, 3817-3827, January 30, 2004
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From the
Departments of Neuroscience, ¶¶Neurology, ¶Pathology, Oncology, and Biological Chemistry, The Johns Hopkins University School of Medicine and The Howard Hughes Medical Institute, Baltimore, Maryland 21205 and the ||Endocrine-Metabolism Division, the Departments of Medicine and Biochemistry, Dartmouth Medical School and the **Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755
Received for publication, October 6, 2003 , and in revised form, November 6, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We and others have demonstrated that C75, a synthetic fatty acid synthase (FAS)1 inhibitor (11), decreases food intake and results in profound and reversible weight loss (8, 12-14). Although inhibition of peripheral fatty acid synthesis might easily explain some of these effects, anorexia was also achieved by central (intracerebroventricular) administration of C75 (8, 14). 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.
FAS is a lipogenic enzyme that catalyzes the condensation of acetyl-CoA and malonyl-CoA to generate long-chain fatty acids (15). 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 
-ketoacyl synthase domain of FAS, thus inhibiting long-chain fatty acid elongation (16, 17). Although it is established that FAS mRNA levels are high in lipogenic tissues such as liver, lung, and adipose (18), FAS was only recently described in brain (19, 9). 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 (9), thus positioning FAS to play a role in neuronal energy metabolism.
In addition to inhibiting FAS (11), C75 stimulates carnitine palmitoyltransferase-1 (CPT-1) activity in cultured adipocytes and hepatocyes by preventing malonyl-CoA-mediated inhibition of CPT-1 (10). CPT-1 catalyzes the esterification of long-chain acyl-CoAs to L-carnitine for transport into the mitochondria for fatty acid oxidation (20). 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 (20, 21). 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 (
) and two regulatory subunits ( and 
) (22, 23). AMPK is most commonly activated by metabolic stresses that deplete cellular ATP and lead to an elevation of the AMP/ATP ratio (22, 23), such as heat shock, exercise, ischemia/hypoxia, low glucose, and metabolic or excitotoxic insults in neurons (24).
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 (22, 25). Additionally, AMPK stimulates catabolic processes by activating glucose uptake, glycolysis, and fatty acid oxidation in an attempt to restore cellular ATP levels (27-30). AMPK has chronic effects on these pathways via alterations in gene expression (for review see Ref. 25). Although AMPK has been shown to be expressed the brain (24, 31, 32), 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 (9, 19, 24, 32). 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.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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; AMPK (2-20) 1:500; and FAS (9) 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 (34). 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 (35). 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 (36). 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 -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 (37). 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 (38). 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 x 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-pAMPK, AMPK (Cell Signaling, Beverly, MA), and sheep anti-FAS (39). 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 (40).
Measurement of AMP-activated Protein Kinase Activity—AMPK activity was determined by performing SAMS peptide assays as previously described (41). 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 AMPK was immunoprecipitated in the presence anti-AMPK (2-20) antibody coupled to protein A/G beads (Santa Cruz Biotechnology). Immunoprecipitates were then washed and resuspended in 4x 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 recording configuration was achieved, neurons were voltage clamped and passive properties were monitored throughout. In the event of a change in Rs or Ri greater than 15% during the course of a recording the data were excluded from the set. mEPSCs were acquired through an Axopatch 200B amplifier (Axon Instruments, Union City, CA), filtered at 2 kHz and digitized at 5 kHz. Sweeps (20 s) with zero latency were acquired until a sufficient number of events were recorded (minimum of 5 min). Data was continuously recorded only after a period of 1-2 min where the cell was allowed to stabilize. mEPSCs were manually detected with MiniAnalysis (Synaptosoft Inc, Decatur, GA) by setting the amplitude threshold to 
RMS * 3 (usually 4 pA). Once a minimum of 100 events was collected from a neuron, the amplitude, frequency, rise time (time to peak), decay time (10-90%), and passive properties were measured. In all electrophysiological experiments, a similar amount of data (n) was acquired from each experimental group (i.e. Me2SO, Drug). Data from each group was then averaged and statistical significance determined by the Student's t test. Data were never reused or transferred from one experimental group to another (Me2SO controls were exclusive).
Statistical Analysis—Data are presented as means ± S.E. of the mean from multiple determinations (n>4). Unless otherwise noted, data were analyzed by one-way analysis of variance with Dunnett post-test to compare treated samples with controls. Differences from post-tests were considered statistically significant at *, p < 0.05; **, p < 0.01; ***, p < 0.001. For the analysis of AMPK activity results (Fig. 5, C and D) each time point was compared with control samples by performing unpaired one-tailed Student's t tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1 and 2) have been localized to neurons throughout the developing and adult rat brain (24, 32). Neuronal expression of AMPK was confirmed in the cortical cultures by double immunolabeling (Fig. 1B).
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C75 Inhibits FAS and Stimulates CPT-1 and Fatty Acid Oxidation in Neuronal Cultures—C75 effectively inhibits the activity of purified FAS (11), and the activity of FAS in adipose tissue (8). The IC50 of C75 for FAS activity in cortical cultures was determined. Primary cultures of cortical neurons were prepared as described under "Experimental Procedures," and assayed for FAS activity by measuring the incorporation of [3H]acetic acid into lipid (Fig. 2A). C75 treatment caused a dose-dependent decrease in acetate incorporation, with an approximate IC50 of 40 µg/ml. These results indicate that C75 can inhibit FAS activity in primary cortical neurons.
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Based on RT-PCR results, the primary cortical neurons used here contain both the liver and muscle CPT-1 isoforms (data not shown). To determine the effect of C75 on neuronal CPT-1 activity, CPT-1 activity assays were performed by measuring acylcarnitine formation. C75 significantly increased CPT-1 activity (217% of control), whereas malonyl-CoA inhibited activity to 
30% of control as expected (Fig. 2B). Although previous studies indicated that minimal fatty acid oxidation occurs in neurons as compared with other tissues (46), experiments were performed to determine whether C75 could alter the rate of fatty acid oxidation in cortical neurons, as it did in peripheral tissues (10). The level of total fatty acid oxidation was determined by combining the 14C-labeled acid soluble and CO2 products obtained after the addition of [1-14C]palmitate. Though the average basal rate of fatty acid oxidation is low in these neurons, (0.316 nmol/h/mg), C75 does significantly increase the total fatty acid oxidation to 146% of control after 2 h (Fig. 2C). The increase seen with C75 in both CPT-1 activity and fatty acid oxidation suggests that C75, through modulation of these metabolic pathways, may influence cellular energy balance.
C75 Increases ATP Levels in Primary Cortical Neuronal Cultures—FAS utilizes acetyl-CoA, malonyl-CoA (synthesized through the action of acetyl-CoA carboxylase (ACC) in an ATP-dependent step), and NADPH to generate palmitate. We hypothesized that C75-induced alterations in fatty acid metabolism might affect neuronal energy balance, which could signal a change in energy status. To investigate this, the effect of C75 on ATP levels was determined (Fig. 3). Treatment of cortical neuronal cultures with C75 for 2 h showed a significant dose-dependent increase in cellular ATP levels, up to 267.7% of control (Fig. 3A), consistent with the effect of C75 on other cell types, including 3T3-L1 adipocytes (10). ATP levels then decreased with higher doses of C75, possibly through compensatory mechanisms. Neurons can be very sensitive to sudden changes in cellular ATP, and sudden changes in ATP levels may be a signal of cell stress prior to cell death (47). Therefore, viability assays were performed to demonstrate that even at extremely high doses of C75, minimum loss of neuronal viability is observed (Fig. 3B). The effect of 40 µg/ml of C75 on neuronal ATP levels was sustained, maintaining ATP levels above control values for at least 15 h after treatment (Fig. 3C), with no significant loss of viability (Fig. 3D), suggesting that, although this alteration in neuronal energy status is significant and prolonged, it does not negatively impact neuronal viability. For the remainder of these studies, 40 µg/ml C75 was used. These results demonstrate that a single dose of C75 significantly affects neuronal ATP levels for a prolonged time course.
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65% of control at 2.5 min (Fig. 4A). By 30 min, ATP levels began to recover, returning to control levels by 1 h, after which ATP levels increased (Fig. 3C). Though short lived, this transient decrease in ATP that occurs prior to the prolonged increase in ATP may play an important role in the downstream effects of C75.
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To test this hypothesis, ATP levels were determined following treatment with TOFA and cerulenin, inhibitors of ACC and FAS, respectively (48, 49). Cerulenin is a natural product FAS inhibitor, and does not directly affect CPT-1 activity (50). Concentrations of inhibitors were tested for their abilities to inhibit the incorporation of [3H]acetic acid into lipid to a similar extent as 40 µg/ml C75 (data not shown). The cerulenin and TOFA concentrations that were chosen corresponded to or exceeded the IC50 for FAS inhibition compared with the dose of C75 employed. These concentrations were found to have no significant effect on neuronal viability for the treatment duration used in these studies (data not shown).
Treatment of neuronal cultures for 2 h with increasing concentrations of cerulenin caused no significant increase in ATP levels above control (Fig. 4C); however, a decrease in ATP (to 70% of control), comparable to that seen with C75, was observed at early time points (5 min to 1 h) (Fig. 4D). Similar to C75, full recovery of ATP levels to control values was seen by 2 h. In contrast, inhibition of ACC with increasing concentrations of TOFA increased ATP levels significantly to 240% of control (Fig. 4E). However, unlike the inhibition of FAS with C75 or cerulenin, no decrease in ATP was observed from 5 min to 1 h with 20 µg/ml TOFA (Fig. 4F). These data demonstrated that FAS inhibition by either C75 or cerulenin transiently, but significantly, reduced ATP levels. In contrast, TOFA, which acts proximally in the pathway prior to ATP consumption, did not cause a decrease in ATP levels. Collectively, these data support a futile cycling hypothesis and indicate that manipulation of the fatty acid synthesis pathway can affect energy levels in neurons.
To confirm the changes in ATP levels with C75 treatment, and to determine AMP levels, HPLC was performed on lysates prepared from primary cortical neuronal cultures. In most cases, it is the combined rise in AMP and the fall in ATP levels that activates AMPK (25). AMP and ATP levels were found to change reciprocally in response to C75 treatment (Fig. 4G). At 5 min following C75 treatment, AMP was increased significantly above control. The converse was seen after 2 h of treatment, a time at which ATP levels are just beginning to increase. Since AMP and ATP levels display a reciprocal relationship with C75 treatment, it is likely that ATP was generated through catabolic reactions, not through the action of adenylate kinase (25, 51).
C75 Treatment Modulates AMPK Phosphorylation and Activity—The long term increase in ATP levels seen with C75 might alter the activity of proteins that function to monitor energy levels. To determine whether the alteration in neuronal energy levels observed with C75 treatment was physiologically important, the effect of C75 on a potential downstream effector, AMPK, was examined. The sensitivity of neurons to changes in energy status and the well established role of AMPK as an energy sensor that activates catabolic pathways when energy states are low made AMPK an attractive target for the consequences of the effect of C75. To be fully activated AMPK must be phosphorylated once bound to AMP (25). To assess the phosphorylation status of AMPK, Western blot analysis was performed on extracts from C75- and vehicle-treated cultures. AMPK phosphorylation initially increased compared with control (at 5 and 30 min), after which phosphorylation decreased (Fig. 5A). No changes in the overall level of AMPK or FAS protein were observed with C75 treatment. As anticipated, the level of AMPK phosphorylation was inversely related to the level of ATP in cells in culture (Fig. 5B).
The altered phosphorylation status suggested that C75 affected the activity of AMPK. To directly measure AMPK activity, SAMS peptide activity assays were performed (Fig. 5C). In control cultures, AMPK activity was 0.3446 pmol/min/mg. AMPK activity was altered by C75 treatment in agreement with AMPK phosphorylation. Thus, at 5 min, when ATP levels were decreased, AMPK activity was increased to 125% above control (Fig. 5C). As ATP levels began to increase after 30 min of C75 treatment, AMPK activity decreased, and was significantly reduced to 25% of control levels at 2 h. AMPK activity remained decreased for at least 4 h.
All three reagents, C75, cerulenin, and TOFA, affected ATP levels to some degree, albeit with different profiles (Fig. 4). Thus, a biphasic effect on ATP levels (an initial reduction followed by an increase) occurred with C75 and cerulenin, but not with TOFA. TOFA caused an increase in ATP levels, but did not cause an initial decrease. While both cerulenin and C75 caused transient decreases in ATP levels, only C75 treatment resulted in an increase in ATP above basal levels. We wished to determine whether all reagents also affected AMPK activity, or whether only certain profiles of ATP level fluctuations correlated with changes in AMPK activity. This is a relevant point; while C75 and cerulenin influence food intake, TOFA treatment alone does not, but TOFA reverses the anorexic effect of C75 in mice (8). SAMS peptide assay was performed on cortical cultures treated with cerulenin or TOFA (Fig. 5D). Although cerulenin treatment significantly decreased AMPK activity, this effect was not as great as that of C75. Surprisingly, TOFA, which had caused a significant increase in ATP levels, did not affect AMPK activity. These results suggested that it was not the absolute level of ATP that influences AMPK activity, but a fluctuation (a decrease followed by an increase) in ATP levels that affected AMPK activity.
C75 Affects Both Metabolic Pathways Downstream of AMPK and Neuronal Activity—To determine whether the changes seen with C75 regarding ATP levels and AMPK activity were physiologically relevant to neurons, the activities of pathways that are regulated by AMPK were examined after C75 treatment. It is known that when activated, AMPK inactivates ACC via phosphorylation (30, 52). No change in ACC phosphorylation status could be detected at early time points, when AMPK was activated at 5 min (data not shown); however, at 2 h of C75 treatment, when AMPK is unphosphorylated and inactive, the level of phosphorylated ACC present was drastically reduced, suggesting that the status of AMPK is affecting downstream ACC activity (Fig. 6A).
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We next questioned whether C75 would have an effect on neuronal activity based on the fact that it appeared to alter neuronal energy metabolism, and hypothalamic c-Fos expression (54). To investigate potential effects of FAS inhibitors on synaptic transmission, we applied either C75 or cerulenin to cortical neurons in culture and measured AMPA-mediated miniature excitatory postsynaptic currents (mEPSCs). Applying cerulenin over a 30-min period resulted in little change in frequency or amplitude (n = 4) of mEPSCs (Fig. 6C). However, application of C75 over the same time period resulted in a dramatic increase in mEPSC frequency, while having no effect on mEPSC amplitude (n = 4) (Fig. 6, C and D). In addition to acute application of FAS inhibitors, cerulenin and C75 were also applied chronically (2 h), and the AMPA-mediated mEPSCs relative to Me2SO control neurons determined. Neurons treated with C75 exhibited a 15-fold increase in mEPSC frequency, while neurons treated with cerulenin showed a slight, but not significant increase in frequency (2-fold)(Fig. 6E). In contrast, application of either C75 or cerulenin to cultured neurons did not affect mEPSC amplitude.
For each drug, we also measured various passive parameters of AMPA miniature events. For instance, both C75 (Me2SO = 2.21 ± 0.02 ms; C75 = 2.01 ± 0.02 ms) and cerulenin (Me2SO = 2.10 ± 0.01 ms; C75 = 1.91 ± 0.05 ms) significantly reduced the decay time of mEPSCs, while each also showed a trend toward a reduction in mEPSC rise time (data not shown). Further, passive properties from each group were also measured. We found no change in access resistance from either C75 or cerulenin groups, though we did find that C75 caused a significant increase in input resistance when compared with Me2SO control neurons (Me2SO = 427 ± 47 mOhms; C75 = 610 ± 70 mOhms), while cerulenin treatment resulted in a non-significant effect on this parameter (Me2SO = 476 ± 61 mOhms; cerulenin = 571 ± 59 mOhms). Together, these results demonstrate that the cellular changes induced by C75 affect many downstream processes, including neuronal firing and multiple downstream targets of AMPK, and are thus physiologically relevant.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We propose that it is the initial decrease in ATP following C75 treatment with the corresponding increase in AMP that activates AMPK. A similar decrease in ATP, without a transient activation of AMPK, is seen with cerulenin; however, both compounds cause a significant decrease in AMPK activity. This is consistent with the similar but less dramatic effect on weight loss and anorexia seen with cerulenin when compared with C75 (8, 14, 12). Of the fatty acid synthesis inhibitors tested, C75 is the most effective at weight loss in vivo and at modulating AMPK activity in vitro. These results suggest that it may be the fluctuations in the changes in cellular energy levels, not only the absolute values they attain, that are important for C75 action. There is precedent for such a mechanism in the regulation of feeding. It is the transient decrease and then rise of plasma glucose, as opposed to simply a change in absolute plasma glucose concentration, that is detected by the brain and important for triggering meal initiation (for review, see Ref. 56).
While TOFA, which inhibits ACC, increased ATP levels in this study, it did not affect AMPK activity. Similarly, TOFA had no effect on feeding behavior in vivo, whereas pretreatment with TOFA actually reversed the anorexic effect of C75 (8). A somewhat different result was obtained by Wakil and co-workers (57), who found that AccII-/- mutant mice had an increase in food intake compared with wild-type mice. These effects may be the result of compensatory responses observed in mice with chronic alterations in ACC activity.
AMPK, known for its role in peripheral tissues as an energy sensor, is predominantly expressed in neurons of the central nervous system, and may mediate at least some of the downstream changes in energy production that occur as a consequence of C75 treatment. The presence of AMPK in neurons and its relatively low level of expression or absence in glia as indicated by this study is consistent with published work demonstrating that AMPK is present in most peripheral tissues and neurons but is absent from unactivated glia (32). The absence of glial AMPK expression may be indicative of the differences in energy flux or monitoring between the two cell types. It has been estimated that 95% of brain energy usage is accounted for by neurons and is mainly due to excitatory signaling (58, 59), and that the glutamate released by neurons and its uptake by astrocytes during excitation serves to couple neuronal synaptic activity with astrocytic glucose usage and lactate production. The production of lactate by the glia generates 2 ATP, whereas the oxidation of 2 lactate molecules by neurons would provide 36 ATP. Therefore, the lesser energy flux through glia as compared with neurons may translate into a reduced need for energy sensing molecules such as AMPK.
This activation of AMPK triggers a cascade of potential downstream effects in an attempt to restore cellular energy levels, thus leading to an increase in ATP. To determine the physiological relevance of altering AMPK activity, two downstream pathways were examined. It has been shown that when energy stores are low, when fatty acid breakdown is favored over synthesis, ACC is phosphorylated and inactivated by AMPK (52). When AMPK is inactivated following C75 treatment, ACC phosphorylation is dramatically reduced, suggesting that ACC activity is affected by the change in AMPK activity. Additionally, AMPK is known to affect glucose metabolism, (60, 61), and therefore the effect of C75 on glucose oxidation was examined, and found to increase at early time points (30 min). C75 stimulation of glucose oxidation is consistent with the correction of hyperglycemia observed in ob/ob mice (8) and with a recent report that C75 increases glucose metabolism (60). We propose that it is the increase in both fatty acid and glucose oxidation that contributes to an energy rich state, and that once this energy rich state is obtained, compensatory mechanisms inhibit AMPK and return glucose oxidation to control levels.
In addition to altering multiple aspects of cellular metabolism, C75 treatment appears to alter neuronal activity. We have shown that C75 has a robust effect on the frequency of mEPSCs, while having no discernable effect on mEPSC amplitude. This effect appears to be specific to C75. In regards to the mechanism by which C75 may affect mEPSC frequency, it likely occurs via a change at the level of the presynaptic terminal. It is well accepted that changes in mEPSC frequency, especially robust changes over relatively short time periods, are due to an increase in release probability at the presynaptic terminal (62). However, there are many possibilities that could change terminal release probability. It is beyond the scope of this study to identify the precise molecular mechanism involved in C75 enhancement of synaptic transmission. The ability of C75 to affect these parameters, while cerulenin does not, may reflect the larger effect of C75 on ATP levels compared with that achieved with cerulenin. Recently published work demonstrates that C75 increases neuronal activity in several neuronal types tested in a slice preparation, leading the authors to conclude that C75 is a nonspecific neuronal activator (63). This study is inconsistent with a previous report, in which c-Fos expression was utilized to assess the effect of C75 on neuronal activity; investigators demonstrated that C75 induced c-Fos expression in specific regions of the hypothalamus and brainstem, not global neuronal activation (54).
Being inhibited by ATP and activated by long-chain acyl-CoAs, ATP-sensitive K+ channels (KATP) may also serve as metabolic sensors (64-66). It has been suggested that KATP channels in the hypothalamus are involved in the regulation of feeding, and that activation of KATP channels leads to an increase in feeding (26). The increase in neuronal activity described in this study could result from the inhibitory effect of increased ATP on KATP channels, leading to depolarization and increased firing, potentially affecting feeding behavior. The metabolic state of the cell is coupled to electrical activity via KATP channels in many tissues (53).
The regulation of brain FAS expression is different from that of peripheral tissues (9) and it is becoming apparent that, even though the brain does not use fats as fuel, as fatty acid oxidation is limiting (46, 55), neuronal energy balance may be regulated by modulating the breakdown of fatty acids. This is a model supported by the present study and by recent work described by Obici et al. (7) in which the inhibition of CPT-1 decreases food intake. We, however, propose that it is the breakdown rather than the build up of fatty acids through C75 stimulation of CPT-1 that leads to an energy positive state. This energy surplus could signal the neuron that it is in the fed state, and assuming the same mechanisms are in play in hypothalamic neurons, inhibit feeding. The apposing results seen by our group and those of Obici et al. suggest that perhaps it is the fluctuations in activities in lipid synthetic and degradative pathways that are important in regulating feeding behavior, not just the activities of these pathways.
Collectively, the results presented here demonstrate that C75 treatment has several effects on neuronal energy metabolism and on neuronal activity. By altering glucose and fatty acid metabolism, and thus neuronal energy levels, C75 may influence energy perception, at least partially through the modulation of AMPK activity. We believe that the effect of C75 on neuronal energy is physiologically relevant due to the robust effect on neuronal activity and on downstream energy sensing molecules such as AMPK, followed by alterations in pathways that are regulated by AMPK (ACC phosphorylation and glucose metabolism). This shift in the energy state of the neuron could signal the central nervous system, via altering neuronal activity, to inhibit feeding. Understanding the effect of modulating fatty acid metabolism on neuronal energy perception could lead to a better understanding of the mechanisms underlying feeding behavior and could potentially lead to new therapeutic targets for weight loss.
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FOOTNOTES |
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|||| To whom correspondence should be addressed: Dept. of Neuroscience, 1006B Preclinical Teaching Building, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. Tel.: 410-614-6482; Fax: 410-614-8033; E-mail: gronnett{at}jhmi.edu.
1 The abbreviations used are: FAS, fatty acyl synthase; HPLC, high performance liquid chromatography; Me2SO, dimethyl sulfoxide; GFAP, glia fibrillary acidic protein; FITC, fluorescein isothiocyanate; ACC, acetyl-CoA carboxylase; CPT-1, carnitine palmitoyltransferase-1; NST, neuron-specific tubulin; AMPK, AMP-activated protein kinase; PBS, phosphate-buffered saline; mEPSC, miniature excitatory postsynaptic current.
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