Etomoxir

Etomoxir, a fatty acid oxidation inhibitor, increases food intake and reduces hepatic energy status in rats

Charles C. Horn*, Hong Ji, Mark I. Friedman
Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104, USA
Received 5 September 2003; received in revised form 8 January 2004; accepted 20 January 2004

Abstract

Etomoxir, an inhibitor of fatty acid oxidation, increases food intake and reported hunger in humans. Work with animal models suggests that other inhibitors of fatty acid oxidation stimulate feeding behavior by acting on the liver. In the following study, we assessed whether etomoxir would increase food intake in rats and to what degree the effects of etomoxir on feeding were associated with changes in hepatic energy status. The effects of etomoxir on hepatic energy status were assessed by measuring liver ATP, ADP, phosphorylation potential, and glycogen content. Blood glucose, free fatty acids, and ketone bodies were also measured to determine the availability of circulating fuels following etomoxir treatment. Etomoxir and methyl palmoxirate (MP; another inhibitor of fatty acid oxidation) increased food intake. Etomoxir, like MP, also reduced hepatic ATP/ADP ratio and phosphorylation potential. In combination with 2,5-anhydro-D-mannitol (an analogue of fructose that produces an increase in feeding by action on the liver), etomoxir synergistically increased food intake and reduced hepatic ATP/ADP ratio. In summary, etomoxir increased food intake and decreased hepatic energy status in the rat. This suggests that etomoxir stimulates feeding by action on the liver.

Keywords: Etomoxir; Metabolic inhibitor; Fatty acid oxidation; Feeding; Food intake; 2,5-Anhydro-D-mannitol; Methyl palmoxirate; Liver metabolism; ATP

1. Introduction

Inhibition of hepatic fatty acid oxidation stimulates food intake in rats (e.g., Refs. [4,13]). Administration of either mercaptoacetate (MA), which suppresses fatty acid oxidation by inhibiting the acyl-CoA dehydrogenases that catalyze mitochondrial h-oxidation (see Ref. [21]), or methyl palmoxirate (MP), which reduces fatty acid oxida- tion by inhibiting carnitine palmitoyltransferase I (CPT I; the rate-limiting enzyme for the transport of long-chain fatty acids into mitochondria) [3,24–26], stimulates feed- ing behavior in rats (e.g., Refs. [5,6,16]). MA-induced feeding is prevented or attenuated by total vagotomy, visceral nerve deafferentation induced by systemic capsa- icin, or hepatic branch vagotomy [13,18,19]. MP produced a dose-related increase in food intake that tracked dose- related reductions in hepatic energy status (liver ATP content, ATP/ADP ratio, and phosphorylation potential) [4]. These findings suggest that a decrease in fatty acid oxidation stimulates feeding behavior by reducing hepatic energy status that is signaled to the brain via the vagus nerve.
Etomoxir, like MP, inhibits CPT I and decreases fatty acid oxidation [28]. Human food intake and reported hunger increased following etomoxir treatment [10]. How- ever, little is known about the mechanism by which etomoxir stimulates feeding. In the following study, we determined whether etomoxir increases food intake in rats and assessed to what degree the effects of etomoxir on feeding were associated with changes in hepatic energy status. The effects of etomoxir on hepatic metabolism were examined by measuring liver ATP, ADP, phosphorylation potential, and glycogen content. Blood glucose, free fatty acids (FFAs), and ketone bodies were also measured to determine the availability of circulating fuels following etomoxir treatment. Etomoxir was compared with MP in its effects on food intake and metabolic parameters. Addi- tionally, etomoxir treatment was combined with the admin- istration of 2,5-anhydro-D-mannitol (2,5-AM; an analogue of fructose that produces an increase in feeding by its action on the liver) [23] to determine if combined treatment would produce a synergistic effect on feeding and hepatic energy status as does combined treatment of MP and 2,5- AM [7,16].

2. Methods

2.1. Animals

Adult male Sprague– Dawley rats (Charles River, Wil- mington, MA) were housed individually in a temperature- controlled (22 jC) vivarium maintained on a 12:12-h light– dark cycle (lights on at 07:00 h). Rats weighed 300 – 450 g at the time of testing and were fed a high-fat/low-carbohy- drate diet (67% kcal from fat, 12% kcal from carbohydrate, and 21% kcal from protein; Dyets, Bethlehem, PA) [5] because diet fat content modulates the effect of metabolic inhibitors on feeding (e.g., MP produces a robust stimula- tion of feeding when animals are fed a high-fat diet) [5,8]. Rats were maintained on the diet for at least 2 weeks prior to behavioral testing or sacrifice. Food and tap water were available ad libitum throughout the studies, unless otherwise noted. Rats were handled frequently and were adapted to test procedures by giving them at least two mock trials prior to testing, in which the gavage tube or injection needle was inserted with no injection.

2.2. Food intake tests

2.2.1. Experiment 1: comparison between etomoxir- and MP-induced food intake
Rats received gastric intubations of vehicle (0.5% methyl cellulose; n=7), etomoxir (10 mg/kg/3 ml in vehicle; n=8), or MP (10 mg/kg/3 ml in vehicle; n=8) on the test day. Food intakes were measured to the nearest 0.1 g and corrected for spillage at 1, 2, 3, 5, 7, and 24 h after treatment.

2.2.2. Experiment 3: combined effect of etomoxir and 2,5-AM on food intake

Rats were intubated with either saline (0.9% NaCl) or etomoxir (10 mg/kg/3 ml in saline) at 09:00 h. Three hours later, animals were intraperitoneally injected with saline (0.9% NaCl) or 2,5-AM (200 mg/kg/4 ml in saline). Etomoxir was injected 3 h before 2,5-AM treatment for two reasons: (1) It was hypothesized that etomoxir, like MP, would have a delayed effect on food intake compared with 2,5-AM; therefore, a close temporal alignment of the physiological effects of etomoxir and 2,5-AM would pro- duce the largest synergistic effect on feeding and metabo- lism. (2) To allow a direct comparison with prior studies using combined treatment of MP and 2,5-AM [7,16] in which the same temporal protocol was used, MP treatment was injected 3 h prior to 2,5-AM treatment in prior studies. The separate groups consisted of animals given saline+sa- line (n=7), etomoxir+saline (n=7), saline+2,5-AM (n=7), and etomoxir+2,5-AM (n=7). Food intakes were measured to the nearest 0.1 g and corrected for spillage every hour for 7 h and at 24 h starting at 09:00 h.

2.3. Metabolic measures

2.3.1. Experiment 2: comparison between etomoxir- and MP-induced metabolic changes

Rats received gastric intubations of vehicle (0.5% methyl cellulose; n=8), etomoxir (10 mg/kg/3 ml in vehicle; n=8), or MP (10 mg/kg/3 ml in vehicle; n=8) on the test day. Before each intubation beginning at 09:00 h, rats were weighed and food cups were removed. Rats were sacrificed 5 h after intubation. Food was removed in Experiments 2 and 4 because eating would have confounded the metabolic measures.

2.3.2. Experiment 4: combined effect of etomoxir and 2,5-AM on metabolic parameters

At 09:00 h, rats were weighed and food was removed from cages. Rats were intubated with either saline (0.9% NaCl) or etomoxir (10 mg/kg/3 ml in saline) at 09:00 h. Before each intubation beginning at 09:00 h, rats were weighed and food cups were removed. Three hours later, rats were intraperitoneally injected with saline or 2,5-AM (200 mg/kg/4 ml in saline). The separate groups consisted of animals given saline+saline (n=12), etomoxir+saline (n=9), saline+2,5-AM (n=13), and etomoxir+2,5-AM (n=9). Rats were sacrificed at 5 h after intubation with saline or etomoxir.

2.3.3. Collection and analysis of liver tissue and blood

Each rat was anesthetized by an intramuscular injection (0.4 ml) of ketamine HCl (100 mg/ml)+acepromazine male- ate (1 mg/ml). A piece of liver (2 – 3 g) was excised through a midline abdominal incision and immediately freeze clamped using a pair of aluminum tongs prechilled in liquid nitrogen. The samples were stored at 80 jC and analyzed for ATP and ADP by HPLC [12,20] and inorganic phosphate (Pi) using a commercial kit (kit 360-3; Sigma). Phosphorylation potential was calculated as ATP/(ADP×Pi). Liver glycogen was mea- sured using an enzymatic method [11]. Blood was collected in heparinized tubes from the heart at the time of sacrifice. Plasma obtained after centrifugation was analyzed for glu- cose (glucose oxidase; kit 510; Sigma) and (unesterified) FFAs (Wako NEFA, Wako, Dallas, TX) using commercial kits. Total ketone bodies (h-hydroxybutyrate+acetoacetate) were analyzed by enzymatic assay with fluorometric detec- tion (see Ref. [15]).

2.4. Data analysis

Food intakes and metabolic measures were analyzed by analysis of variance (ANOVA) and were followed by planned comparisons of means using least significant dif- ference tests (LSD tests; i.e., t tests using the error variance of all groups). Cumulative food intake data at different time points in Experiment 1 (Fig. 1) were compared with saline treatment using t tests. A level of P<.05 was used to determine statistical significance. Fig. 1. Cumulative food intake produced by etomoxir (10 mg/kg) and MP (10 mg/kg) treatment. Rats received a gastric intubation of vehicle (0.5% methyl cellulose), etomoxir, or MP at 09:00 h. Comparisons (t tests on cumulative data) revealed that etomoxir treatment produced significantly greater food intake than vehicle treatment by 2, 3, and 7 h, and MP treatment resulted in greater food intake than saline treatment at 3, 5, and 7 h. Values are meansFS.E.M. *P<.5, t test, etomoxir or MP versus vehicle. 3. Results 3.1. Experiment 1: etomoxir increased food intake Food intake increased significantly after etomoxir and MP treatments for the initial 7-h test period [ F(8,80)=2.6, P<.05, interaction effect of treatment by time on noncumu- lative food intake data]. Cumulative food intake is plotted in Fig. 1. Comparisons (t tests on cumulative data) revealed that etomoxir treatment produced significantly greater food in- take than saline treatment by 2, 3, and 7 h, and MP treatment stimulated greater food intake than the saline condition at 3, 5, and 7 h (see Fig. 1). By 7 h, MP-treated animals had eaten more food than etomoxir-treated rats ( P<.05, t test; see Fig. 1). Twenty-four-hour food intakes (meanFS.E.M.) were less in etomoxir-treated (22.6F1.7 g) and MP-treated (17.6F1.1 g) animals compared with rats treated with saline [27.8F1.1 g; P<.05, LSD tests; F(2,20)=13.8, P<.0005, one-way ANOVA]. 3.2. Experiment 2: etomoxir reduced hepatic energy status There was no significant effect of etomoxir or MP treatment on liver ATP levels ( P=.08, one-way ANOVA; see Fig. 2A). The hepatic ATP/ADP ratio, phosphorylation potential, and glycogen levels were lower in etomoxir- and MP-treated animals compared with saline treatment [ Ps<.05, LSD tests; F’s(2,21)z31.0, PsV.0001, one-way ANOVAs; see Fig. 2A]. Blood glucose was lowered by both etomoxir and MP treatment [ Ps<.05, LSD tests; F(2,21)=8.6, P<.005, one-way ANOVA; see Fig. 2B]. There were no significant effects of etomoxir or MP treatment on blood levels of FFA ( P=.09, one-way ANOVA; see Fig. 2B) or ketone bodies ( P=.4, one-way ANOVA; see Fig. 2B). 3.3. Experiment 3: etomoxir +2,5-AM synergistically increased food intake There were no significant differences between treatment groups for the 3 h following etomoxir treatment [i.e., before intraperitoneal injection of saline or 2,5-AM; Psz.08, interaction effects; 2 (saline or etomoxir)×2 (saline or 2,5-AM) ANOVAs using cumulative food intake at 1-, 2-, and 3-h time points]. Food intake was synergistically increased with combined treatment of etomoxir and 2,5-AM at 4 and 5 h [1 and 2 h after intraperitoneal injection of 2,5-AM; F(1,24)z4.2, PsV.05; 2×2 interaction effects on cumula- tive food intake at 4- and 5-h time points]. Comparisons revealed that only combined treatment with etomoxir and 2,5-AM produced a significant increase in food intake versus saline treatment at 4 and 5 h (1 and 2 h after intraperitoneal injection with 2,5-AM; Ps<.05, LSD tests; see Fig. 3 for 5-h mean food intakes). There was no significant effect of etomoxir and 2,5-AM on 24-h food intake ( P=.14, interaction effect; 2×2 ANOVA). Fig. 2. Effects of etomoxir (10 mg/kg) and MP (10 mg/kg) on metabolic parameters. Rats received a gastric intubation of vehicle (0.5% methyl cellulose), etomoxir, or MP at 09:00 h. (A) Hepatic energy status. There were no effects of etomoxir or MP treatment on hepatic ATP levels. The ATP/ADP ratio, phosphorylation potential, and glycogen levels were lower in etomoxir- and MP-treated animals compared with vehicle treatment. (B) Blood levels of metabolic fuels. Blood glucose was lowered by etomoxir or MP treatment. There were no significant effects of etomoxir or MP treatment on blood levels of FFA or ketone bodies. Values are meansFS.E.M. *P<.5, LSD test, etomoxir or MP versus vehicle. 3.4. Experiment 4: etomoxir+2,5-AM reduced hepatic energy status There were no significant interaction effects [2 (saline or etomoxir)×2 (saline or 2,5-AM) ANOVA] of etomoxir and 2,5-AM treatment on ATP ( P=.41), ATP/ADP ratio ( P=.24), phosphorylation potential ( P=.21), and glycogen level ( P=.78). However, there was a significant decrease of ATP content by 2,5-AM treatment [ F(1,39)=38.7, P<.0001, main effect of 2,5-AM treatment], and this was observed in the 2,5-AM-only and etomoxir+2,5-AM groups ( Ps<.05, LSD tests; see Fig. 4A). The ATP/ADP ratio was reduced by etomoxir treatment [ F(1,39)=6.8, P<.05, main effect of etomoxir treatment], but this only occurred with combined treatment of etomoxir and 2,5-AM ( P<.05, LSD test; see both conditions in Fig. 4A). There were no significant effects of etomoxir or 2,5-AM treatment on the phosphor- ylation potential ( Psz.08, main effects of etomoxir and 2,5- AM treatment; see Fig. 4A). Hepatic glycogen content was reduced by etomoxir treatment [ F(1,39)=70.6, P<.0001, main effect of etomoxir treatment], and etomoxir-treated animals and those treated with etomoxir+2,5-AM showed a decrease in glycogen levels ( Ps<.05, LSD tests; see Fig. 4A). 2,5-AM also produced a significant increase in liver glycogen content [ F(1,39)=6.4, P<.02, main effect of 2,5- AM treatment]. Fig. 3. Combined effect of etomoxir (10 mg/kg) and 2,5-AM (200 mg/kg) on food intake at 5 h (2 h after intraperitoneal injection of saline or 2,5-AM). Saline or etomoxir was gastrically intubated at 09:00 h and saline or 2,5-AM was intraperitoneally injected at 12:00 h. There were four separate treatment groups: control (saline+saline), etomoxir (etomoxir+saline), 2,5-AM (saline+2,5-AM), and both (etomoxir+2,5-AM). Combined treatment of etomoxir and 2,5-AM produced a synergistic effect on food intake [ P<.05, 2 (saline or etomoxir)×2 (saline or 2,5-AM) interaction effect]. Mean comparisons revealed that only combined treatment with etomoxir and 2,5-AM treatment produced a significant increase in food intake versus control (saline+saline) treatment (*P<.05, control versus both conditions, LSD test). Values are meansFS.E.M. Fig. 4. Combined effect of etomoxir (10 mg/kg) and 2,5-AM (200 mg/kg) on metabolic parameters. Saline or etomoxir was gastrically intubated at 09:00 h and saline or 2,5-AM was intraperitoneally injected at 12:00 h. There were four separate treatment groups: control (saline+saline), etomoxir (etomoxir+saline), 2,5-AM (saline+2,5-AM), and both (eto- moxir+2,5-AM). (A) Hepatic energy status. There were significant effects of 2,5-AM on hepatic ATP levels and etomoxir on liver glycogen content. Combined treatment with etomoxir and 2,5-AM reduced the ATP/ADP ratio. (B) Blood levels of metabolic fuels. Blood glucose was lowered and FFA levels were increased by etomoxir and 2,5-AM treatment. Etomoxir reduced the level of ketone bodies. Values are meansFS.E.M. *P<.5, LSD test, treatments versus control condition. Blood glucose and FFAs were affected in an additive fashion by etomoxir and 2,5-AM treatments [ F’s(1,39)z 15.9, Ps<.001, main effects of etomoxir and 2,5-AM treat- ment]. Animals treated with etomoxir, 2,5-AM, or eto- moxir+2,5-AM showed a decrease in blood glucose and an increase in FFA levels compared with saline-treated rats ( Ps<.05, LSD tests; see Fig. 4B). Etomoxir treatment lowered the level of ketone bodies [ F(1,39)=29.9, P<.001, main effect of etomoxir treatment], and animals treated with etomoxir or etomoxir+2,5-AM showed a decrease in ketone bodies compared with saline-treated rats ( Ps<.05, LSD tests; see Fig. 4B). 4. Discussion Etomoxir produced similar effects on food intake and hepatic energy status in rats as did MP treatment [4,5,7,16]. Etomoxir, like MP, increased food intake (see Fig. 1). Also similar to MP, etomoxir reduced the hepatic ATP/ADP ratio and the phosphorylation potential (see Fig. 2). In combination with 2,5-AM, etomoxir synergistically increased food intake (see Fig. 3) and reduced hepatic energy status (the ATP/ADP ratio; see Fig. 4A), effects that are also observed after combined treatment of MP and 2,5-AM [9]. Like MP, etomoxir has a higher affinity for the liver isoform of CPT I than for the muscle isoform of CPT I [24,28]. Evidence suggests that fatty acid oxidation inhibitors act in liver to stimulate food intake [9,13]. Etomoxir also affects the metabolism of cardiac, adipose, and intestinal tissues (e.g., Refs. [1,2,27]), but it is unclear how these effects might contribute to increased food intake. The current data are consistent with the idea that, like other inhibitors of fatty acid oxidation, etomoxir induces rats to eat by its action in the liver [4]. Although ATP level was only marginally reduced by etomoxir in Experiment 1, ATP level by itself does not necessarily reflect the availability or turnover rate of ATP. The ATP/ADP ratio or phosphorylation potential might be more indicative of changes in the dynamic process of ATP usage (for review of these metabolic effects as they relate to food intake, see Refs. [4,9]). A lack of ADP usage to produce more ATP might explain why etomoxir treatment led to an increase in ADP, which resulted in a decrease in the ATP/ADP ratio (see Fig. 1). Twenty-four-hour food intakes were suppressed by etomoxir and MP treatments in Experiment 1, which may indicate toxic effects of these treatments over the long term (at least at the doses used). Indeed, MP, as well as MA (an inhibitor of fatty acid metabolism), can produce conditioned taste aversions (CTA) in rats [22]. A CTA produced by these agents suggests that these treatments can cause malaise. There were differences between the potency of etomoxir to produce changes in food intake and hepatic energy status between Experiments 1 and 2 (comparing etomoxir to MP treatment) versus Experiments 3 and 4 (combining etomoxir with 2,5-AM treatment). In Experiments 3 and 4, etomoxir alone did not produce an increase in food intake by 5 h (see Fig. 3) or reductions in the ATP/ADP ratio and phosphorylation potential (see Fig. 4). These lack of effects in Experiments 3 and 4 are in contrast to the clear effects of etomoxir alone on food intake and hepatic energy status in Experiments 1 and 2 (see Figs. 1 and 2). However, despite the lesser potency of etomoxir in Experiments 3 and 4, etomoxir had clear effects on feeding and metabolism in these experiments when combined with 2,5-AM treatment (see Figs. 3 and 4). Although etomoxir was mixed at a concentration of 10 mg/3 ml in all cases, the vehicle (methyl cellulose or saline) that was used in each experiment to produce a suspension of etomoxir was the most likely cause of the difference in potency. Eto- moxir, despite reported use (e.g., Ref. [14]), was not observed to be easily soluble in water. It came out of solution in saline but could be suspended in methyl cellulose for a much longer time (several hours). There- fore, the mixture of saline and etomoxir (Experiments 3 and 4) likely produced a slightly lower concentration of etomoxir compared with etomoxir in methyl cellulose (Experiments 1 and 2). The behavioral response to combined treatment of etomoxir and 2,5-AM was accompanied by a decrease in hepatic energy status reflected in the reduction in liver ATP/ADP ratio. This result suggests that etomoxir and 2,5-AM stimulate eating via the same mechanism of action (i.e., by decreasing liver energy status) and that the increase in food intake seen after a combined treat- ment with these inhibitors is due to their combined effect of reducing liver energy status. Both etomoxir and 2,5- AM affect metabolic pathways in liver that are involved in the processing of metabolic fuels; etomoxir inhibits the oxidation of long-chain fatty acids [28], and 2,5-AM limits hepatic gluconeogenesis and glycogenolysis [17]. This is reflected in the changes in circulating fuels result- ing from etomoxir and 2,5-AM treatments. For example, both etomoxir and 2,5-AM produce a decrease in blood glucose concentration. However, these and other actions (e.g., increase in FFAs) of the inhibitors at the substrate level of metabolism do not appear to account for their effects on food intake or for the synergistic eating re- sponse to the combined treatment. That etomoxir and 2,5- AM act to produce a greater reduction in liver energy status than would be predicted from their additive effects suggests that the effect of these inhibitors on food intake depends on their actions at the level of energy production, beyond those pathways for the metabolism of specific fuels. In summary, these results suggest that etomoxir stimulates feeding by its action on energy production in the liver. Acknowledgements We thank Dr. Kunio Torii for supplying the 2,5-AM and Drs. Horst Wolf and Wolfgang Langhans for supplying the Etomoxir. MP was donated by the R.W. Johnson Pharma- ceutical Research Institute. This work was supported by grants from the National Institutes of Health (DK02894 and DK 36339). References [1] Cabrero A, Alegret M, Sanchez R, Adzet T, Laguna JC, Vazquez M. Etomoxir, sodium 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxyl- ate, up-regulates uncoupling protein-3 mRNA levels in primary cul- ture of rat preadipocytes. 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