Intraperitoneal injections of low doses of C75 elicit a behaviorally specific and vagal afferent-independent inhibition of eating in rats.

Central and intraperitoneal C75, an inhibitor of fatty acid synthase and stimulator of carnitine palmitoyl-transferase-1, inhibits eating in mice and rats. Mechanisms involved in feeding inhibition after central C75 have been identified, but little is yet known about how systemic C75 might inhibit eating. One issue is whether intraperitoneal C75 reduces food intake in rats by influencing normal physiological controls of food intake or acts nonselectively, for example by eliciting illness or aversion. Another issue relates to whether intraperitoneal C75 acts centrally or, similar to some other peripheral metabolic controls of eating, activates abdominal vagal afferents to inhibit eating. To further address these questions, we investigated the effects of intraperitoneal C75 on spontaneous meal patterns and the formation of conditioned taste aversion (CTA). We also tested whether the eating inhibitory effect of intraperitoneal C75 is vagally mediated by testing rats after either total subdiaphragmatic vagotomy (TVX) or selective subdiaphragmatic vagal deafferentations (SDA). Intraperitoneal injection of 3.2 and 7.5 mg/kg of C75 significantly reduced food intake 3, 12, and 24 h after injection by reducing the number of meals without affecting meal size, whereas 15 mg/kg of C75 reduced both meal number and meal size. The two smaller doses of C75 failed to induce a CTA, but 15 mg/kg C75 did. The eating inhibitory effect of C75 was not diminished in either TVX or SDA rats. We conclude that intraperitoneal injections of low doses of C75 inhibit eating in a behaviorally specific manner and that this effect does not require abdominal vagal afferents.

Dissociation of mercaptoacetate's effects on feeding and fat metabolism by dietary medium- and long-chain triacylglycerols in rats.

OBJECTIVE AND METHODS: Mercaptoacetate (MA) inhibits hepatic fatty acid oxidation (FAO) and stimulates feeding in rats fed fat-rich diets. To test whether the feeding stimulation by MA depends on hepatic FAO, we compared the effects of intraperitoneally injected MA (45.6 mg/kg body weight) with saline in rats fed diets containing 18% predominately long-chain triacylglycerols (LCTs; > or =90% 16 C) or 18% medium-chain triacylglycerols (MCTs; 51% 10-12 C). We hypothesized that, because medium-chain fatty acids reach the liver and are oxidized faster than long-chain fatty acids, if MA's feeding-stimulatory effect depends on hepatic FAO, MA should stimulate feeding more in MCT-fed rats than in LCT-fed rats. RESULTS: Although MA injected in mid-light phase stimulated feeding similarly in MCT- and LCT-fed rats, MA injected at light onset initially stimulated food intake (1 h) only in LCT- and not in MCT-fed rats. To investigate MA's metabolic effects during the initial hour, rats were sacrificed 30 min after light-onset injections. At this time plasma beta-hydroxybutyrate appeared to be higher in MCT- than in LCT-fed rats and to be increased by MA. In a final experiment, MA did not affect fatty acid content in liver and duodenum tissues but increased fatty acids in duodenal tissue mitochondria from 12 h-fasted rats fed chow. CONCLUSION: In light-onset tests, adaptation to the MCT diet increased hepatic FAO but not the feeding-stimulatory effect of MA in comparison with adaptation to the LCT diet, suggesting that at this time MA does not act in the liver to stimulate feeding or that this effect is not due to FAO inhibition. Inhibition of duodenal mitochondrial FAO may be another metabolic process through which MA stimulates feeding.

Vagal afferents mediate the feeding response to mercaptoacetate but not to the beta (3) adrenergic receptor agonist CL 316,243.

To evaluate the role of subdiaphragmatic vagal afferents in the anorectic response to peripheral administration of the highly selective beta(3)-adrenergic receptor agonist CL 316,243 (CL), we tested the ability of intraperitoneal (IP) injections of CL to inhibit feeding in rats with subdiaphragmatic vagal deafferentation (SDA, n=13) or sham surgeries (SHAM, n=13). Doses of 10, 100 and 1000 ng/kg CL significantly reduced feeding by statistically similar amounts in SHAM and SDA rats. One hour after IP injection, each dose of CL also significantly increased plasma concentrations of free fatty acids and beta-hydroxybutyrate, an indicator of hepatic fatty acid oxidation (FAO), whereas 6h after injection only the two highest CL doses increased plasma beta-hydroxybutyrate. In contrast, peripheral administration of the FAO inhibitor mercaptoacetate (MA, 45.6 mg/kg IP) stimulated feeding in SHAM but not in SDA rats, extending previous data suggesting a necessary role of vagal afferents in the feeding-stimulatory effect of FAO inhibition. We conclude that subdiaphragmatic vagal afferents are essential for the feeding-stimulatory action of MA but not for the anorectic action of peripheral CL and that CL-induced increase in hepatic FAO is not essential for its feeding-inhibitory effect.

Beta-adrenergic-mediated inhibition of feeding by mercaptoacetate in food-deprived rats.

This study investigated the effect of intraperitoneal (IP) injections of the fatty acid oxidation (FAO) inhibitor mercaptoacetate (MA, 45.6 mg/kg) on feeding in food-deprived rats. As previously, MA significantly stimulated feeding in ad libitum-fed rats. MA, however, reduced feeding in 18 and 36 h-fasted rats despite apparently antagonizing the fasting-induced increase in hepatic FAO. To test whether this anorectic effect involves beta-adrenergic stimulation, 36 h-fasted rats were IP injected with the nonspecific beta-adrenergic receptor antagonist propranolol (PROP, 0.5 mg/kg) just before MA injection. PROP attenuated MA's feeding-inhibitory effect, suggesting that MA anorexia is at least partially mediated by beta-adrenergic stimulation. Finally, we evaluated the role of subdiaphragmatic vagal afferent fibers in MA's feeding-inhibitory effect by testing the ability of MA to inhibit food intake in fasted rats after subdiaphragmatic vagal deafferentation (SDA). MA inhibited feeding similarly in SDA rats and sham-operated rats. These data demonstrate that subdiaphragmatic vagal afferents are not necessary for the feeding-inhibitory effect of peripheral MA. These results suggest that the FAO inhibitor MA elicits a feeding-inhibitory effect in fasted rats that is mediated by a different mechanism than its feeding-stimulatory effect.

Hepatic-portal oleic acid inhibits feeding more potently than hepatic-portal caprylic acid in rats.

In several human and animal studies, medium-chain triglycerides decreased food intake more than did long-chain triglycerides. It is possible that faster uptake and metabolism of medium-chain fatty acids in the liver is responsible for this difference. To test this hypothesis we compared the feeding effects of hepatic portal vein (HPV) infusion of the medium-chain fatty acid caprylic acid (CA) with those of the long-chain fatty acid oleic acid (OA). Contrary to our expectation, six-h HPV infusion of 14 microg/min (50 nmol/min) OA robustly inhibited feeding, whereas infusion of 22 or 220 microg/min (150 and 1500 nmol/min) CA failed to have any effect on feeding. Only a much larger dose of CA, 1100 microg/min (7500 nmol/min) inhibited feeding similarly to 14 microg/min OA. The increased feeding-inhibitory potency of OA did not appear to be due to differences in stimulation of hepatic fatty acid oxidation because equimolar (50 nmol/min) doses of OA (14 microg/min) and CA (7 microg/min) did not differentially affect post-infusion levels of beta-hydroxybutyrate. Stress, inflammation, acute hepatotoxicity or oxidative stress also do not appear to account for the increased feeding-inhibitory potency of HPV OA because plasma concentrations of the stress hormones corticosterone and epinephrine, the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha, the liver enzymes gamma-glutamyl transferase and alanine aminotransferase and as well as hepatic levels of malondialdehyde and glutathione were all similar after HPV infusion of saline or of 50 nmol/min OA or CA.

Mercaptoacetate fails to block the feeding-inhibitory effect of the beta3-adrenergic receptor agonist CGP 12177A.

Peripherally administered beta3-adrenergic receptor (beta3-AR) agonists stimulate lipolysis and inhibit food intake. To test the hypothesis that this inhibition of feeding is due to a substrate-driven increase in hepatic fatty acid oxidation (FAO), we assessed the ability of the FAO inhibitor mercaptoacetate (MA) to reverse the feeding-inhibitory effect of the beta3-AR agonist CGP 12177A (CGP). Adult male Sprague-Dawley rats received intraperitoneal injections of 1 mg/kg CGP, of 45.6 mg/kg MA, or of both drugs, and the effects on food intake, plasma free fatty acids (FFA), and plasma beta-hydroxybutyrate (BHB), an indicator for hepatic FAO, were assessed. Control rats received saline injections. CGP significantly reduced food intake after 0.5 and 6 h and increased plasma FFA and BHB at 0.5 h, suggesting increased lipolysis and hepatic FAO. MA completely reversed the increase in plasma BHB and thus appeared to effectively abolish CGP's effect on hepatic FAO, but MA failed to affect CGP's feeding-inhibitory action. These findings do not support the hypothesis that the beta3-AR agonist CGP inhibits feeding by enhancing hepatic FAO or ketogenesis. Although the beta3-AR agonist CGP reduced saccharin intake in a one-bottle condition taste aversion test, it seems unlikely that the hypophagic effect of CGP is elicited by malaise.

Beneficial and deleterious effects of hydroxycitrate in rats fed a high-fructose diet.

OBJECTIVE: The present study assessed whether long-term supplementation of a high-fructose diet with hydroxycitrate (HCA), an inhibitor of de novo lipogenesis that is widely used as a non-prescription dietary aid, decreases food intake, visceral fat accumulation, hypertriglyceridemia, and hyperinsulinemia in rats. METHODS: We examined the effects of HCA (1.8% of diet) on food intake, body weight gain, visceral fat accumulation, hypertriglyceridemia, and hyperinsulinemia in rats during a 4-wk period of ad libitum access to a 50% fructose diet after a 3-wk period of food restriction in which they lost about 20% of their body weight. RESULTS: HCA decreased food intake and weight gain throughout the test and reduced visceral fat accumulation compared with control rats fed ad libitum (CON). Rats that were pair-fed (PF) to the HCA rats showed similar decreases in weight gain and visceral fat. HCA did not ameliorate the hypertriglyceridemia induced by high-fructose feeding. HCA improved insulin sensitivity on day 10 in comparison with CON rats, but by day 27 insulin levels were similarly higher and liver glycogen levels were similarly lower in HCA and CON rats in comparison with PF rats. Liver lipid content was elevated in HCA rats compared with CON and PF rats. CONCLUSION: These findings indicate that, although HCA attenuates body weight gain and visceral fat accumulation by reducing food intake under these conditions, it has no lasting beneficial effects on hypertriglyceridemia and hyperinsulinemia and leads to the accumulation of liver lipids.

Caprylic acid infusion acts in the liver to decrease food intake in rats.

Hepatic portal vein (HPV) infusion of the medium chain fatty acid caprylic acid (CA; 2.3 mg/min, 40 microl/min) for 90 min beginning at dark onset in 18-h food-deprived male rats reduced the size of the first nocturnal meal about 40% (P < 0.01) and reduced 24-h food intake by about 15% (P < 0.001). Identical infusions into the vena cava affected neither initial meal size nor food intake. HPV CA infusion attenuated the postprandial decreases in plasma free fatty acids (P < 0.01) and beta-hydroxybutyrate (P < 0.01). HPV CA infusions did not significantly reduce nocturnal saccharine intake in a two-bottle conditioned taste aversion test, and there was no association between the saccharine intake on the test day and the feeding-inhibitory effect of CA on the conditioning day. HPV CA infusion did not affect plasma concentrations of corticosterone or of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha. HPV CA infusion did not increase plasma concentration of the liver enzyme alanine aminotransferase, but did increase plasma concentration of gamma-glutamyl transferase, although not into the pathophysiological range. These data indicate that CA acts in the liver to produce a signal that inhibits feeding and that this inhibitory effect may be related to increases in hepatic fatty acid oxidation rather than be the result of aversion or toxicity.

CPT1alpha over-expression increases long-chain fatty acid oxidation and reduces cell viability with incremental palmitic acid concentration in 293T cells.

To test the cellular response to an increased fatty acid oxidation, we generated a vector for an inducible expression of the rate-limiting enzyme carnitine palmitoyl-transferase 1alpha (CPT1alpha). Human embryonic 293T kidney cells were transiently transfected and expression of the CPT1alpha transgene in the tet-on vector was activated with doxycycline. Fatty acid oxidation was measured by determining the conversion of supplemented, synthetic cis-10-heptadecenoic acid (C17:1n-7) to C15:ln-7. CPT1alpha over-expression increased mitochondrial long-chain fatty acid oxidation about 6-fold. Addition of palmitic acid (PA) decreased viability of CPT1alpha over-expressing cells in a concentration-dependent manner. Both, PA and CPT1alpha over-expression increased cell death. Interestingly, PA reduced total cell number only in cells over-expressing CPT1alpha, suggesting an effect on cell proliferation that requires PA translocation across the mitochondrial inner membrane. This inducible expression system should be well suited to study the roles of CPT1 and fatty acid oxidation in lipotoxicity and metabolism in vivo.

Fatty acid oxidation and control of food intake.

Fatty acid oxidation is thought to play a role in the control of food intake, and a low postprandial oxidation of ingested fat may contribute to the overeating on a high-fat diet. Evidence for a role of fatty acid oxidation in control of food intake is mainly derived from the stimulation of feeding in response to administration of the acyl-CoA-dehydrogenase inhibitor mercaptoacetate (MA) and other inhibitors of fatty acid oxidation in different species (rat, mouse, man). Denervation studies suggest that this "lipoprivic feeding" is related to changes in hepatic fatty acid oxidation. In contrast to the strong case for a feeding stimulatory effect of an inhibition of fatty acid oxidation, the evidence for a feeding suppressive effect of a stimulation of fatty acid oxidation is inconsistent and comparatively weak. Ingestion of medium-chain fatty acids (MCFA) and peripheral administration of substances known to increase fatty acid oxidation, such as the fatty acid synthase inhibitor C75 and beta3-adrenergic agonists, decrease feeding. Yet, these substances also reduce the rats' usual preference for saccharin solution, indicating that the feeding suppressive effect is not only due to a stimulation of fatty acid oxidation. A possible approach to answer the question of whether a stimulation of hepatic fatty acid oxidation enhances satiety is to selectively increase expression and activity of the enzyme CPT 1alpha in the liver. CPT 1alpha transfers long-chain fatty acids in the cytosol from CoA to carnitine, which is the precondition for the entry of long-chain fatty acids into mitochondria and the rate-controlling step in mitochondrial fatty acid oxidation. The generation of rats with inducible, liver-specific overexpression of CPT 1alpha should permit to critically examine the putative contribution of hepatic fatty acid oxidation to the control of food intake.

Effect of hydroxycitrate on respiratory quotient, energy expenditure, and glucose tolerance in male rats after a period of restrictive feeding.

OBJECTIVE: Recently we demonstrated that hydroxycitrate (HCA) suppresses food intake and body weight regain in male rats after substantial body weight loss. However, it is not known whether HCA also affects the respiration quotient (RQ), energy expenditure (EE), and glucose tolerance in this animal model. METHODS: Twenty-four male rats (initial body weight, 378 +/- 3 g) were fed restrictively (10 g/d) for 10 d and then given ad libitum access to a high-glucose diet supplemented with 3% HCA for 6 d. Controls received the same diet without the supplement. RQ and EE were measured during ad libitum days 1, 2, and 6. An oral glucose tolerance test was performed on ad libitum day 4 or 5. RESULTS: HCA decreased RQ and EE during ad libitum days 1 and 2. In all probability, these findings reflect a decrease in de novo lipogenesis. On ad libitum day 6, RQ and EE did not differ between treatment groups. HCA suppressed food intake during the first 3 d ad libitum, but overall body weight regain was not decreased in the HCA group. The oral glucose tolerance test showed that HCA significantly decreased the increase in plasma glucose from baseline (Deltaglucose) and tended to decrease the area under the curve for glucose. Deltainsulin and area under the curve for insulin did not differ between groups. CONCLUSIONS: These results indicate that, in this animal model, HCA suppresses de novo lipogenesis. Moreover, HCA may improve glucose tolerance.

Subdiaphragmatic vagal deafferentation fails to block the anorectic effect of hydroxycitrate.

We investigated the neural mediation of the feeding suppression through orally administered hydroxycitrate (HCA) in male rats that were fed a high-glucose diet (about 48% glucose). Ten-day ad libitum food intake and body weight regain after previous body weight loss (13% of initial body weight) due to restrictive feeding were measured in rats with sham deafferentation (SHAM; n = 6), subdiaphragmatic vagal deafferentation (SDA; n = 7), and SDA plus celiac-superior mesenteric ganglionectomy (SDA/CGX; n = 9). HCA suppressed the 10-day cumulative food intake in all surgical groups and body weight regain in SDA and SDA/CGX groups. Independent of HCA, SDA and SDA/CGX rats consumed less food and gained less weight compared to SHAM rats. These results demonstrate that all vagal afferents from below the diaphragm and vagal efferents of the dorsal trunk as well as splanchnic nerves (afferents and efferents) are not necessary for the feeding-suppressive effect of HCA in this animal model. Vagal afferents, however, appear to play a role in the control of intake when a high-glucose diet is consumed after a period of restrictive feeding.

Hydroxycitrate has long-term effects on feeding behavior, body weight regain and metabolism after body weight loss in male rats.

We examined the long-term effect of hydroxycitrate (HCA) on food intake, meal patterns, body weight regain and energy conversion ratio as well as on different blood and liver variables in rats after substantial body weight loss. Rats were fed a 1% (g/100 g) fat diet (81% carbohydrate, 10% protein, 1% fat) or a 12% (g/100 g) fat diet (76% carbohydrate, 9% protein, 12% fat) in two separate experiments. Supplementing both diets with 3% HCA after 10 d of restrictive feeding reduced body weight regain over the whole subsequent period of ad libitum consumption (22 d) and decreased the energy conversion ratio [body weight regain (g)/energy intake (MJ)] at the end of the experiment. Only in rats fed the 12% fat diet, HCA had a long-term suppression of food intake. The anorectic effect occurred predominately during the light phase, and was due mainly to a reduction in numbers of meals. In rats fed the 12% fat diet, HCA had no effect on plasma beta-hydroxybutyrate (BHB), but reduced plasma triacylglycerol and increased liver fat concentration. In rats fed the 1% fat diet, HCA did not affect any metabolic variable examined. Thus, the suppressive effect of HCA on body weight regain, which was maintained for at least 3 wk, appears to be independent of the dietary fat content. Yet, the fat content of the diet seemed to be important for the long-term suppressive effect of HCA on feeding. The fact that HCA did not change plasma BHB concentration does not support a role for increased hepatic fatty acid oxidation in the anorectic effect of HCA.