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. 2010 Mar 4;151(5):2087–2096. doi: 10.1210/en.2009-1043

The Lipoprivic Control of Feeding Is Governed by Fat Metabolism, Not by Leptin or Adipose Depletion

Bryan D Hudson 1, Alan J Emanuel 1, Michael F Wiater 1, Sue Ritter 1
PMCID: PMC2869253  PMID: 20203155

Abstract

A lipoprivic control of feeding has been proposed based on the finding that appetite is stimulated by drugs such as β-mercaptoacetate (MA) that reduce fatty acid oxidation. The adipose-derived hormone, leptin, has effects on feeding and fat oxidation that are opposite those produced by MA. However, effects of this hormone on MA-induced feeding are not known. Here we examined the effects of endogenous leptin levels and of acute central and peripheral leptin administration on MA-induced feeding. We also examined leptin-induced changes in feeding, body weight, and plasma fuels after capsaicin-induced deletion of the lipoprivic control. MA-induced feeding was not altered under any of these conditions, and leptin’s effects were not altered by capsaicin. We then examined MA-induced feeding during chronic leptin treatment. Because chronic leptin produces several distinct metabolic states as body adiposity is reduced, we tested MA before, during, and after leptin treatment at times that coincided with these states. MA-induced feeding was unchanged on d 3 of leptin treatment when rats were in a lipolytic state and rapidly metabolizing body fat stores but reduced on d 10 when they were adipose deplete and their level of fat oxidation was reduced. Together results suggest that the lipoprivic control is normally less active in the fat deplete state than during states associated with fat availability. If so, its insensitivity to leptin would enable the lipoprivic control to operate when dietary fat, adiposity, and leptin levels are elevated. The role played by the lipoprivic control under such conditions remains uncertain.

Leptin does not antagonize lipoprivation-induced feeding, permitting the lipoprivic control to function across a wide range of metabolic states.

The metabolic pathways governing fat oxidation and synthesis are a source of multiple signals that are pivotal for control of food intake. Acute blockade of fatty acid oxidation (lipoprivation) is a potent stimulus for feeding. This feeding response (lipoprivic feeding) can be activated experimentally by drugs that reduce fatty acid oxidation, including β-mercaptoacetate (MA) (1), which blocks mitochondrial acyl coenzyme-A dehydrogenases necessary for β-oxidation of fatty acids (2,3), methylpalmoxirate (4), and etomoxir (5), which block carnitine palmitoyltransferase 1, necessary for transport of long-chain fatty acids into mitochondrial oxidation sites (6). Etomoxir has also been shown to enhance hunger sensations and increase the amount of food consumed in humans (5). It has been clearly demonstrated in rats using a number of approaches that lipoprivation-induced feeding requires vagal sensory neurons (7,8,9). Although the lipoprivic control appears to be an important control of food intake that is linked to availability of a major metabolic fuel, very little is currently known about the physiological contexts in which the lipoprivic control is active or about the ways in which reduced fatty acid oxidation interacts with other controls of feeding.

Leptin, a circulating hormone secreted by adipose tissue (10,11), has also been shown to exert important control of food intake and metabolism. However, leptin has effects on feeding and metabolism that are opposite to those reported for MA. Elevated leptin signals an excess of body fat, whereas MA-induced blockade of fat oxidation presumably signals reduced availability of fat for cellular metabolism. Leptin stimulates fatty acid oxidation (12,13), whereas MA inhibits it (2,3). Leptin decreases food intake and increases energy metabolism (14,15,16,17,18), whereas MA stimulates food intake (1). Surprisingly, leptin’s effect on MA-induced feeding has not been examined. Therefore, in the present experiment, we tested the effects of acute and chronic leptin administration on MA-induced feeding.

To examine the interaction of MA with endogenous leptin, we measured the effect of MA on endogenous leptin levels and examined MA-induced feeding in two groups of rats with differing degrees of adiposity, which is well known to be positively correlated with endogenous leptin levels (10,15,19,20). Next, we examined the effect of exogenous peripheral leptin, administered ip for 2 d, on MA-induced feeding. Third, we tested feeding in rats given acute lateral ventricular leptin injections paired with ip administration of MA.

We also examined MA-induced feeding before, during, and after chronic leptin administration. Chronic leptin treatment produces a reproducible sequence of distinct metabolic states (21,22,23,24,25). To examine each of these conditions, we scheduled our MA-induced feeding tests to coincide with these different states: with the fat replete baseline condition (d −5), the period of leptin-induced hypophagia and lipolysis (d 3), the period of sustained fat depletion associated with simultaneously increasing food intake (d 10), and the postleptin period of hyperphagia and lipogenesis associated with recovering body weight (after leptin d 6).

Finally, we examined the effect of capsaicin on leptin-induced changes in feeding, body weight, and plasma fuels. Capsaicin lesions vagal sensory neurons (26,27) and thereby eliminates the MA-induced feeding response (8,28), which requires these neurons. Thus, if the lipoprivic control influences daily food intake during chronic leptin treatment, then absence of this control should alter the response to leptin. In particular, if the lipoprivic control is activated by adipose depletion and mediates the increased feeding that is initiated at the nadir of body weight during chronic leptin treatment, then recovery of feeding will be impaired.

Materials and Methods

Subjects and housing conditions

Adult male Sprague Dawley rats were obtained from Simonsen Laboratories (Gilroy, CA) and individually housed in suspended wire-mesh cages, under standard Association Assessment and Accreditation of Laboratory Animal Care-approved conditions, in a temperature-controlled room (21 ± 1 C), illuminated between 0700 and 1900 h. Throughout the experiment, the rats had ad libitum access to pelleted rat chow (F6 Rodent Diet; Harlan Teklad, Madison, WI) and tap water, except as noted below. None of the rats used in any of the experiments describe here were fed fat-supplemented diets either before or during experimentation. Before experimentation, rats were handled and habituated to the laboratory environment and testing procedures. The Washington State University Institutional Animal Care and Use Committee, which conforms to National Institute of Health rules and regulations, approved all experimental animal protocols.

Implantation and screening of lateral ventricle cannulas

Stainless steel guide cannulas (26-gauge tubing), occluded with removable obturators (33-gauge wire), were stereotaxically implanted into the lateral ventricle and allowed to recover 1 wk. For injections, the obturator was removed from the guide cannula and replaced with an injector made of 33-gauge stainless steel tubing fashioned to extend 0.5 mm beyond the tip of the guide cannula. The injector was connected by polyethylene tubing to a 0.2-ml micrometer syringe (Gilmont Instruments, Barrington, IL). To verify correct positioning of cannulas within the ventricle, all cannulated rats were tested for angiotensin II (50 ng/3 μl; Chemicon, Phoenix Pharmaceuticals, Burlingame, CA)-induced drinking (29). Rats that failed to drink at least 5 ml of water in a 30-min test were excluded from the experiment.

Central and peripheral leptin administration

Although leptin has peripheral effects, its most potent metabolic effects are mediated by its central actions (13,23,30,31,32). Therefore, in all but one experiment, leptin was administered into the lateral cerebroventricle. For intracerebroventricular (ICV) injections, the drug delivery system was filled with either mouse recombinant leptin (Calbiochem, San Diego, CA; 2.5 μg/3 μl) or artificial cerebrospinal fluid (aCSF; 3 μl). Solutions were delivered over a 1-min period. Drug delivery was monitored by observing movement of an indicator bubble in the calibrated infusion line. The injector remained in place for 1 min after the injection after which the rat was returned immediately to its home cage. Peripheral leptin was administered ip (1 mg/kg, 1 mg/ml).

Feeding in response to mercaptoacetate

Because MA acts peripherally, requiring vagal afferents for its effects on feeding, MA was administered ip in all cases. At the start of each test, rats were injected with MA (68 mg/kg, thioglycolic acid, T0632; Sigma Chemical Co., St. Louis, MO) or control sterile saline (0.9%, 2 ml/kg) and returned immediately to its cage. Injection time was noon except as noted. Consumption of the standard pelleted rodent diet was measured 2 and 4 h after injection. Spillage was subtracted from the total.

Experiment 1: effect of body fat on MA-induced feeding in nonleptin-treated rats

The amount of body fat is known to be positively correlated with circulating leptin levels (10,15,19,20). We determined whether differences in body fat and endogenous leptin levels alter MA-induced feeding. To avoid alteration of diet, which could be a confounding factor, two groups of rats of different ages with body weight differentials of about 125 g and maintained on the standard pelleted rat chow, were used for measurement of leptin levels, for dual-energy-x-ray absorptiometry (DEXA) and for MA-induced feeding tests. The lean rats weighed 369 ± 3.4 g and fat rats weighed 490 ± 2.6 g. Feeding tests were conducted, as described above, using MA (68 mg/kg, ip) and saline as the control. DEXA scans were made with the QDR 4500A machine (Hologic, Bedford, MA) using 11.1:3 software. Rats were lightly anesthetized with the ketamine-xylazine-acepromazine cocktail before DEXA scans. DEXA has been validated by our work (33) and other investigators as a useful method for the direct determination of adiposity levels in the rat (34,35). Plasma leptin levels were measured from saphenous blood samples using an ELISA kit (Millipore, Billerica, MA) and rats were then euthanized. Retroperitoneal and epididymal fat pads were removed unilaterally and weighed.

Experiment 2: effect of MA on plasma leptin levels

Food was removed from the cages and the rats (n = 6) were injected with 0.9% saline on one day and with MA the next day. Blood was collected from the tail 1 h after MA or control injections for measurement of leptin levels. This sampling time was selected to correspond with the time when the feeding response to MA is typically reaching its peak to test the hypothesis that MA-induced reduction of leptin levels is responsible for the lipoprivic feeding response. Leptin levels were determined from plasma using an ELISA kit (EZRL-83K, Millipore).

Experiment 3: effect of acute central leptin treatment on MA-induced feeding

In this experiment (n = 6), leptin or control aCSF solution was administered into the lateral ventricles 1 or 24 h before ip injection of 0.9% saline or MA using a counterbalanced design. MA or saline was injected at 1000 h, and food intake was measured 2 and 4 h after the injection, as described above.

Experiment 4: effect of peripheral leptin on MA-induced feeding

Leptin is known to have effects directly on the vagus nerve (36) and MA-induced feeding requires vagal sensory neurons (8,9). Therefore, to determine whether leptin and MA interact when both are administered peripherally, leptin (1 mg/kg; n = 7) or saline (0.9%; n = 10) were injected daily for 2 d into rats having similar body weights (346 ± 8 g and 335 ± 6 g, respectively) at the start of testing. MA (68 mg/kg) was injected on d 3 and food intake was measured 2 and 4 h later.

Experiment 5: effect of chronic central leptin on feeding in response to MA-induced blockade of fatty acid oxidation

Body weight and food intake measurements (corrected for spillage) were taken between 0900 and 1000 h every day, beginning 3 d before the onset of leptin (n = 6) or aCSF (n = 8) injection and continuing for 21 d after leptin injections began. The average of the 3-d preleptin food intake was used as the baseline food intake to which the leptin-induced changes were compared. Leptin or control aCSF solution was then administered into the lateral cerebroventricle (ICV) daily at 1400 h for 13 consecutive days. To limit possible brain infection over such a long injection series, a fresh autoclaved injector was used for each rat and each injection.

MA-induced feeding was tested 5 d before the beginning of leptin/aCSF treatment (d −5), on d 3, and d 10 of leptin or aCSF treatment and 6 d after leptin and aCSF treatments were discontinued (i.e. d 19 of the experiment). Feeding in response to ip saline control injections was tested on the day before each MA test (d −6, 2, 9, and 18 of the experiment). Each feeding test began at 1000 h with an ip injection of saline or MA (68 mg/kg). Chow consumption with spillage subtracted was measured 2 and 4 h after injection.

Experiment 6: effect of capsaicin on leptin-induced changes in body weight, food intake, and plasma triglycerides

Capsaicin treatment

Capsaicin, which lesions vagal C-fiber afferents, was administered as described previously (8,26). Briefly, rats were given atropine sulfate (3 mg, sc), anesthetized 30 min later with isoflurane (Webster Veterinary Supply, Inc., Sterling, MA), and injected ip with capsaicin (90%, M-1022; Sigma) or vehicle [10% alcohol in 10% Tween 80 in 0.9% saline (AT)] solutions (n = 10 and 12 per group, respectively). The total capsaicin dose (125 mg/kg) was administered as a series of three injections (25, 50, and 50 mg/kg, each 1 ml/kg), all made within a 24-h period (0, 18, and 24 h, respectively). AT was administered on a similar schedule. Artificial ventilation was provided, as required, during the brief period of respiratory arrest that typically occurs within a few minutes after the first capsaicin injection. To assess the effectiveness of the capsaicin treatment, rats were tested as described above for MA-induced feeding, a response known to depend on capsaicin-sensitive vagal sensory neurons (8) and for the corneal chemosensory response (eye wipe) to mild corneal irritation, mediated by the capsaicin-sensitive trigeminal innervation of the cornea (37,38). Number of eye wipes were measured during the 15 sec immediately after application of 1% NH4OH to the corneal surface of one eye (39). This test was conducted at the beginning and end of experimentation. All capsaicin-treated rats demonstrated loss of both responses in both tests.

Leptin treatment

After the screening tests, capsaicin (cap) and AT-treated rats were divided into four groups: aCSF-cap (n = 5), leptin-cap (n = 5), aCSF-AT (n = 6), and leptin-AT (n = 6) and implanted with lateral ventricle cannulas for chronic leptin and aCSF administration, as described above. Body weight and food intake measurements, taken at 1000 h every day, began 6 d before the onset of leptin or aCSF injection. Leptin or aCSF was administered daily, as described for experiment 5, for 18 d.

Blood collection

On d 8 and 17 of leptin treatment, at 1100 h, rats were removed from their cages for collection of tail blood (0.2 ml/rat). Plasma was separated by centrifugation (10,000 rpm for 8 min), aliquoted, and stored at −80 C for later assay. Plasma was assayed for glucose, triglycerides, nonesterified free fatty acids, 3-hydroxybutyrate (β-HB), and blood urea nitrogen (BUN) were performed using a GM7 analyzer Analox instrument (Analox Instruments Ltd., London, UK).

Statistical analyses

Data were analyzed using Student’s t test or the appropriate ANOVA. Post hoc analyses were conducted using a Fisher’s least significant differences (LSD) test for ANOVAs with significant F values. Differences were considered significant if P < 0.05.

Results

Experiment 1: effect of body fat on MA-induced feeding in nonleptin-treated rats

Figure 1 shows that body weights (Fig. 1A) and body fat content, as determined by DEXA scan (Fig. 1B), differed significantly in the rats designated as fat and lean (32.2 ± 3.3 vs. 19.0 ± 2.7 g of fat, respectively, P < 0.05; n = 7/group). MA significantly increased feeding (P < 0.05) in both fat and lean groups, but the responses did not differ between groups either when evaluated as grams of food consumed (Fig. 1C) or as food intake proportional to body weight (P = 0.084, data not shown). Leptin levels in the leaner rats were significantly lower than those of the fatter rats [1.9 ± 0.3 and 4.9 ± 0.6 ng/ml, respectively (t = −4.5, df = 10, P < 0.001)]. The mean combined weights of the dissected epididymal and retroperitoneal fat pads were 2.0 ± 0.2 g in leaner and 4.2 ± 0.3 g in fatter rats. The fat pad weights as percent of body weight were 0.6 + 0.0% for lean and 0.9 ± 0.0% for fat rats. Two-tailed Student’s t tests were significant (P < 0.001) for both adipose measures.

Figure 1.

Figure 1

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Effect of adiposity and endogenous leptin levels on MA-induced feeding. Body weight in groups of fat and lean rats used for DEXA analysis (dark gray, n = 7/group) and MA tests (light gray, n = 7 lean, n = 5 fat) were comparable (A). Fat rats were slightly older than lean rats. All rats were maintained throughout their lives on standard rodent chow. DEXA analysis revealed a significant difference in body fat in the fat rat group compared with the lean rat group (B). However, whereas leptin levels (n = 6/group, not shown) were significantly elevated in fat rats, MA-induced feeding did not differ between the fat and the lean rats (C).

Experiment 2: effect of MA on plasma leptin levels

MA injections did not lower endogenous leptin levels, as would be expected if this mechanism were responsible for MA-induced feeding. Rather, endogenous leptin levels were nonsignificantly elevated by MA (2.3 ± 0.6 ng/ml), compared with levels in the same rats given saline control injections (1.3 ± 0.3 ng/ml; P = 0.18, two tailed t test, n = 6).

Experiment 3: effect of acute central treatment on MA-induced feeding

Figure 2 shows that feeding in response to MA was increased above peripheral saline control in rats given lateral ventricular injections of leptin either 1 h (Fig. 2A) or 24 h (Fig. 2B) before MA (n = 6/group). At both test times, the amount of food eaten in response to MA did not differ between rats treated centrally with aCSF and those treated centrally with leptin. Despite having no effect on MA-induced feeding, leptin significantly (P < 0.05) reduced daily feeding (Fig. 2C) and body weight (Fig. 2D) during the 24-h period between the leptin injection and the MA test.

Figure 2.

Figure 2

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MA-induced feeding in rats pretreated 1 h (A) or 24 h (B) before MA injection with lateral ventricular aCSF (3 μl) or leptin (2.5 μg per 3 μl). MA (68 mg/kg, ip) increased food intake in both the leptin-pretreated groups, and the MA response did not differ between ICV leptin- and aCSF-pretreated rats (n = 6/group) at either time point. Despite having no effect on MA-induced feeding, leptin significantly (P < 0.05) reduced daily feeding (C) and body weight (D) during the 24 h before the MA test.

Experiment 4: effect of peripheral leptin on MA-induced feeding

Figure 3 shows the mean 24-h food intake (Fig. 3A), body weight change (Fig. 3B) in response to daily ip leptin (1 mg/kg, 1 ml/kg, n = 7), or 0.9% saline injection (n = 10). Both food intake and body weight were significantly reduced by leptin. Feeding responses to MA and saline were tested 24 h after the second leptin injection. Food intake was significantly increased by MA in both leptin- and saline-pretreated groups (Fig. 3C). Body weights (335 ± 6 and 346 ± 8 g) did not differ between saline- and leptin-treated groups at the start of the experiment (Fig. 3D).

Figure 3.

Figure 3

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The mean 24-h food intake (A) and body weight change (B) in response to daily ip leptin (1 mg/kg. 1 ml/kg) or 0.9% saline (Sal; 1 ml/kg) injection. Both food intake and body weight were significantly reduced by leptin. Feeding in response to MA (68 mg/kg, ip) and saline was tested 24 h after the second leptin injection. Food intake was significantly increased by MA in both leptin- (n = 7) and saline (n = 10)-pretreated groups (C). Body weights (335 ± 6 and 346 ± 8 g) did not differ between saline- and leptin-treated groups at the start of the experiment (D).

Experiment 5: effect of chronic central leptin on food intake, body weight, and feeding responses to MA-induced blockade of fatty acid oxidation

Repeated-measure analysis demonstrated a significant main effect of leptin on weight loss [F (1, 306) = 122.6]. The rats that received injections of ICV leptin (n = 6) lost significant amounts of weight by the end of d 1 of leptin treatment (P = 0.0002) relative to the rats that received injections of ICV artificial aCSF (n = 8). Leptin induced significant weight loss for each of the 13 d of treatment compared with baseline or aCSF controls. After leptin treatment was stopped on d 13, body weight began to increase but remained significantly below control levels through d 21, when the experiment was terminated (Fig. 4, top panel). Repeated-measure analysis of food intake indicated a significant main effect for leptin treatment [F (1, 306) = 37.9], day [F (21, 306) = 21.8], and their interaction [F (21, 306) = 14.9]. Fisher’s LSD post hoc analysis revealed that food intake for leptin-treated rats was significantly different from food intake for aCSF-treated rats on all days, except d 16 and 17. On d 1–13 of leptin treatment, food intake was decreased and on d 18–22, after discontinuation of leptin, food intake was increased (Fig. 4, bottom panel).

Figure 4.

Figure 4

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Mean percent change in body weight (top panel) and food intake (bottom panel) of rats injected daily into the lateral ventricle with leptin (2.5 μg per 3.0 μl, n = 6) or aCSF (n = 8) for 13 d followed by 8 d of recovery from injections. MA feeding test days are indicated by arrows (see Fig. 5 for MA induced feeding data). Discontinuation of leptin treatment is indicated by the vertical line at d 13. Body weight was significantly reduced by leptin across all treatment days compared with control and returned rapidly toward control levels after discontinuation of leptin treatment. Leptin-treated rats consumed significantly less chow than aCSF controls during the 13 d of leptin administration, although a trend toward control levels is apparent after d 7. Five to 8 d after cessation of treatments, the postleptin group consumed significantly more chow than the post-aCSF group.

Results of the MA tests before, during, and after chronic leptin treatment are shown in Fig. 5A. Only the 4-h food intake is shown in the figure. Two-hour food intake followed the same pattern as the 4-h food intake. The saline tests were pooled across all time points until the end of leptin treatment because they were not significantly different from each other. Two-way ANOVA indicated a significant stimulatory effect of MA on food intake [F (1, 55) = 30.8, P < 0.001]. Fisher’s LSD post hoc planned comparison tests indicated that MA-induced feeding was significantly reduced on d 10 in leptin-treated rats, compared with intakes on d −5 and 3 of leptin treatment (within groups) and was significantly reduced on d 10 compared with aCSF-treated rats (between groups).

Figure 5.

Figure 5

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A, Mean 4-h food intake induced by MA (68 mg/kg, ip, top panel) or saline (1 ml/kg, bottom panel) during chronic lateral ventricular leptin (2.5 μg/d, n = 6) or aCSF (3 μl; n = 8) treatment for 13 d. MA-induced feeding was tested on d −5 (preleptin baseline), 3, and 10 of leptin treatment. Saline baseline tests were conducted on d −6, 2, and 9. There were no differences between saline baseline intakes, so they were pooled, as shown. MA (68 mg/kg) significantly increased food intake in both leptin and aCSF groups, compared with saline baseline. Responses of aCSF- and leptin-treated rats did not differ. However, responses of the leptin-treated group were significantly reduced on d 10 compared with the response to the aCSF group and baseline intake. *, P < 0.05. B, Mean 4-h food intake induced by saline (1 ml/kg) and MA (68 mg/kg, ip) on d 5 and 6 (respectively) after discontinuation of chronic aCSF or leptin treatment. Postleptin hyperphagic rats ate significantly more in response to MA than in their previous tests and more than aCSF-treated rats. *, P < 0.05.

Results of the postleptin MA test are shown in Fig. 5B. A one-way ANOVA was significant [F(3, 24) = 14.5, P < 0.001], and Fisher’s LSD post hoc planned comparison test revealed that MA-induced feeding was significantly increased above saline baseline in both postleptin and post-aCSF groups, but the response was significantly greater in postleptin rats than in post-aCSF groups. These groups did not differ with respect to baseline intakes.

Experiment 6: effect of capsaicin on leptin-induced changes in body weight, food intake, and plasma triglycerides

Capsaicin caused a significant reduction of MA-induced feeding, indicating that the capsaicin treatment destroyed vagal sensory neurons with C-fiber afferent processes. AT-treated animals significantly increased their feeding after ip MA compared with saline control (5.5 ± 0.6 vs. 0.3 ± 0.1 g, respectively, P < 0.001), whereas capsaicin-lesioned rats did not (0.9 ± 0.3 vs. 0.4 ± 0.2 g, respectively, P = 0.85). The eye-wipe response to corneal irritation was present in AT-treated rats (12.7 ± 1.22 wipes per 15 sec) but was absent in all capsaicin-treated rats (0.0 ± 0.0 wipes per 15 sec), providing another indicator of the effectiveness of the capsaicin lesion.

Body weight

At the beginning of the experiment, body weights did not differ among the four groups [F(3, 21) = 1.5, P = 0.2]. The main effect of leptin over time on body weight was significant [F(1, 630) = 57.5, P < 0.001, Fig. 6, top panel]. Daily ICV leptin administration rats significantly decreased body weight after only 1 d [F(1, 22) = 15.5, P = 0.001], and body weight remained significantly reduced through d 35 [F(1, 22) = 8.0, P = 0.01]. There was no significant main effect of the capsaicin lesion.

Figure 6.

Figure 6

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Mean percent change in body weight (top panel) and food intake (bottom panel) of capsaicin- (Cap; n = 5/group) and AT vehicle (n = 6/group)-treated rats injected daily into the lateral ventricle with leptin (2.5 μg per 3.0 μl) or aCSF for 18 d followed by 17 d of recovery. The vertical line at d 18 indicates the end of leptin or aCSF treatment. Body weight and food intake were significantly reduced by leptin at all time points in Cap- and AT-treated rats, but there were no differences due to capsaicin treatment. After leptin treatment was discontinued, body weight remained significantly suppressed, although increasing rapidly toward normalization. During this same time, food intake was significantly increased beginning 2 d after leptin withdrawal and continuing through d 32. These postleptin effects were similar in both Cap and AT groups.

Food intake

Baseline food intake was not significantly different between the four groups [F(3, 21) = 0.91, P = 0.46]. Leptin reduced food intake in both capsaicin and AT-treated rats (Fig. 6, bottom panel), but capsaicin pretreatment did not significantly alter leptin’s effect on feeding. The main effect of leptin on food intake was significant [F(1, 630) = 36.3, P < 0.001]. Food intake was reduced significantly by leptin from d 1 [F(1, 22) = 94.4, P < 0.01] through d 19 [F(1, 22) = 9.2, P = 0.007]. Food intake began to increase toward control on about d 7 of leptin treatment, even though body weight remained at its nadir. Two days after leptin withdrawal, leptin-treated animals significantly increased their food intake above aCSF baseline levels [F(1, 22) = 70.1, P < 0.001] and remained hyperphagic through d 32 [F(1, 21) = 4.9, p = 0.04].

Plasma factors

There was no significant main effect of leptin on plasma glucose, BUN, or β-HB on either d 8 or 17 (Fig. 7), indicating that the animals were euglycemic, not ketotic, and were not in negative protein balance. However, circulating triglyceride levels were significantly reduced in leptin-treated rats on both d 8 [F(3, 21) = 12.8, P < 0.001] and d 17 [F(3, 21) = 6.2, P = 0.004], consistent with the loss of body weight and adiposity and indicating a reduction in availability of fat for energy metabolism. Capsaicin-treated rats did not differ from AT controls at any time with respect to these plasma factors.

Figure 7.

Figure 7

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Mean plasma levels of glucose (upper left panel), triglycerides (upper right panel), (β-HB lower left panel), and BUN (lower right panel) in capsaicin (Cap) and vehicle (AT)-treated rats injected daily into the lateral ventricle with leptin or aCSF. Circulating triglycerides were significantly reduced in leptin-treated rats on d 8 and 17 in both the Cap and AT groups. There were no significant differences due to Cap treatment. *, P < 0.05.

Discussion

The first aim of these experiments was to determine whether leptin and MA-induced blockade of fat oxidation interact in controlling food intake. Our results failed to reveal significant interactions between these two fat-based controls. Rats with different levels of adiposity and different endogenous leptin levels ate similar amounts in response to MA, as did rats given acute ventricular or ip leptin doses sufficient to reduce body weight and daily feeding. We also asked whether MA might stimulate feeding by reducing endogenous leptin levels. However, 1 h after MA injection, at a time when the feeding response is normally maximal, endogenous leptin levels were elevated, not reduced, compared with saline injected rats. On the basis of these data sets, we conclude that MA activates an orexigenic signal that is insensitive to leptin’s suppressive effects on food intake. Furthermore, elimination of the lipoprivic control by capsaicin administration did not alter food intake, body weight, or blood metabolites at any time during or after chronic leptin treatment, suggesting that the lipoprivic control and vagal afferent neurons are not critical for these responses to leptin treatment.

During chronic leptin treatment, MA-induced food intake was significantly reduced on d 10, but the response on d 3 did not differ from the response of aCSF controls on that day or from the preleptin MA response (d −5). Because the same dose of leptin was administered on d 3 and 10 and because leptin and MA did not interact in earlier experiments, it seems likely that the suppression of the MA response on d 10 was not a direct effect of leptin but due to other factors, the most obvious of these being fat depletion. On d 10, rats had lost approximately 20% of their starting body weight (compared with only 6% on d 3) and had reached a stable nadir of body weight. Our previous work (25) using DEXA scans and fat pad dissection in chronic leptin-treated rats has shown that body fat is depleted at the nadir point of the body weight loss curve. Because MA-induced feeding was decreased and not increased on d 10, our results argue against the hypothesis that the lipoprivic control of food intake is an emergency control elicited by depletion of adipose reserves. Furthermore, the fact that elimination of the lipoprivic control by capsaicin administration did not impair the recovery of feeding initiated at the nadir of body weight during chronic leptin treatment also indicates that the lipoprivic control is not stimulated by depletion of fat stores and does not mediate this increased feeding.

An alternative hypothesis regarding the lipoprivic control is that its efficacy in stimulating food intake is positively correlated with the level of ongoing fat oxidation, i.e., an animal that is more dependent on fat as a metabolic fuel will be more sensitive to a lipoprivic challenge. This hypothesis is based on reports by a number of investigators that MA-induced feeding is enhanced when the experimental animals are maintained on fat-enriched diets (1,40,41). MA test results for d 3 and 10 support this hypothesis. MA-induced feeding was reduced on d 10 of leptin treatment, when body weight was at its nadir point, approximately 80% of preleptin levels. Given their depleted fat stores, their low fat maintenance diet, and their reduced intake of this diet, it is safe to assume that leptin-treated rats were using very little fat for energy metabolism on d 10. The fact that plasma triglyceride levels were greatly reduced under these same conditions (Fig. 7, d 8 of leptin treatment) supports this assumption. Thus, reduced availability of fat as a fuel source for ongoing energy metabolism was associated with a significant reduction in the effectiveness of MA in stimulating feeding. Conversely, on d 3 of leptin treatment, rats were largely dependent on mobilized fat as a metabolic fuel source: body weight was reduced by only 6%, and rats were hypophagic and in a state of lipolysis, mobilizing and using stored fat as their primary energy substrate. On d 3, MA elicited a robust feeding response, also supporting the hypothesis that the level of fat metabolism determines the effectiveness of the lipoprivic control.

The response to MA was enhanced 6 d after discontinuation of leptin. At this point, rats that had been treated previously with leptin were hyperphagic and lipogenic, gaining body weight rapidly. How these conditions facilitated the response to MA will require further investigation. For example, it will be important to determine the level of fat oxidation prevailing during this postleptin recovery period and whether the enhanced MA response is related specifically to fat oxidation, lipogenesis, or general up-regulation of appetitive feeding responses during this period. However, it is interesting to note that rats that are genetically hyperphagic and predisposed to obesity (lipogenesis) (40), and rats that are lipogenic due to increased dietary fat (41), also exhibit enhanced feeding responses to MA.

Understanding the neural substrates through which fat metabolism influences food intake constitutes an imposing and important challenge. To date, experimental observations suggest three aspects of fat metabolism that affect food intake. First, increased fat storage inhibits food intake at least in part via the action of leptin. Second, increased fat oxidation, induced pharmacologically by drugs such as C75, decreases food intake (42,43,44). Third, pharmacological blockade of fat oxidation by MA and methylpalmoxirate increases food intake. In addition to these aspects of fat metabolism that influence feeding, long-chain fatty acids in the intestinal lumen reduce food intake by releasing gastrointestinal peptides, some of which activate vagal sensory neurons (45). Conceivably, each of these aspects of fat metabolism comprises a separate facet of a unitary control of food intake. If this were the case, one might expect that all three would share a common neural substrate. However, a common neural substrate has not been identified, and the known neural substrates for these fat-related signals appear at present to be disparate.

MA increases food intake by a mechanism dependent on vagal afferent signaling (7,8,9), whereas vagal afferents do not appear essential for leptin-induced reduction of food intake or for C75 effects on food intake (46). Intracranial injection of leptin appears to reproduce all of the effects of systemic leptin with regard to reduction of food intake, whereas central MA injections do not increase food intake (our unpublished data). Lesion studies and analysis of Fos expression have shown that neurons in the nucleus of the solitary tract and lateral parabrachial and central nucleus of the amygdala are activated by systemic MA injection and are essential for MA-induced feeding (8,9,47,48,49). These sites may also be involved in leptin’s actions, but none are required for suppression of feeding by leptin (33,50,51). Similarly, the arcuate nucleus of the hypothalamus is a major site of action for leptin. Lesions in this area reduce or eliminate leptin-induced suppression of feeding (52,53,54) but do not reduce MA-induced feeding (52). MA does not alter hypothalamic expression of proopiomelanocortin, orexin, agouti gene-related protein, or neuropeptide Y mRNA (55), whereas leptin increases arcuate proopiomelanocortin/cocaine- and amphetamine-related transcript mRNA and decreases arcuate neuropeptide Y and agouti gene-related protein mRNA (56). Further anatomical analysis is essential for complete understanding of the role fat-related signals in central neural control of food intake, body weight, and energy homeostasis.

The present study indicates that the lipoprivic control is not directly influenced by leptin or adipose mass, suggesting that this control is not involved in maintenance of body adiposity per se. Rather, the potency of lipoprivation in controlling food intake appears to wax and wane, potentially on a moment-to-moment basis, under the influence of the dominant fuel source. This suggests that the lipoprivic control may be involved in functions such as the selection of appropriate metabolizable nutrients or in maximizing intake of calorically dense food sources. Such roles would be consistent with previously published data (57,58,59). The fact that leptin does not suppress lipoprivic feeding is also consistent with the observation that the potency of the lipoprivic control of feeding is maximized by lipid availability. Because metabolic conditions associated with an abundance of fatty acids are likely to be associated with elevated levels of endogenous leptin, the independent actions of MA and leptin would permit both controls to operate simultaneously under these conditions.

Finally, chronic leptin treatment, as used in these experiments, provides a model for studying certain controls of food intake. Obviously we do not claim that elevated central leptin levels used in this model represent a physiological condition, especially when combined with fat depletion. Elevated leptin and adipose depletion would not occur simultaneously under physiological conditions. Nevertheless, this model offers several unique advantages for behavioral and metabolic studies. The model is reproducible. It can be used to produce sequential metabolic states in the same animal. These metabolic states can be monitored in vivo by biochemical and behavioral measures during the course of the experiment. The model is unique in producing a healthy fatless rat. Unlike food-deprived rats, leptin-treated rats do not lose body protein (57). Furthermore, rats given chronic leptin treatment can be maintained in the fatless state while on an ad libitum feeding schedule and even while consuming calories at normal or near-normal levels (19). This avoids the confounding factors associated with prolonged food deprivation and the difficulty of testing stimulatory controls of feeding during food deprivation. Thus, this model may prove valuable in developing and testing additional hypotheses that will lead to a better understanding of the lipoprivic control and other controls of feeding and the influence of fat on the function of these controls.

Footnotes

This work was supported by National Institutes of Health Grants DK040498 and DK00345072 and Juvenile Diabetes Research Foundation Grant 1-2006-308 (to S.R.).

Current address for B.D.H.: Molecular Cardiovascular Research Program, University of Arizona, Tucson, Arizona 85724-5217.

Disclosure Summary: The authors have nothing to disclose.

First Published Online March 4, 2010

Abbreviations: aCSF, Artificial cerebrospinal fluid; AT, alcohol in Tween 80 in saline; BUN, blood urea nitrogen; DEXA, dual-energy-x-ray absorptiometry; β-HB, 3-hydroxybutyrate; ICV, intracerebroventricular; LSD, least significant differences; MA, β-mercaptoacetate.

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