Recent studies show that brain-derived neurotrophic factor (BDNF) decreases feeding and body weight after peripheral and ventricular administration. BDNF mRNA and protein, and its receptor TrkB, are widely distributed in the hypothalamus and other brain regions. However, there are few reports on specific brain sites of actions for BDNF. We evaluated the effect of BDNF, given into the ventromedial nucleus of the hypothalamus (VMH), on normal and deprivation- and neuropeptide Y (NPY)-induced feeding behavior and body weight. BDNF injected unilaterally or bilaterally into the VMH of food-deprived and nondeprived rats significantly decreased feeding and body weight gain within the 0- to 24-h and the 24- to 48-h postinjection intervals. Doses effectively producing inhibition of feeding behavior did not establish a conditioned taste aversion. BDNF-induced feeding inhibition was attenuated by pretreatment of the TrkB-Fc fusion protein that blocks binding between BDNF and its receptor TrkB. VMH-injected BDNF significantly decreased VMH NPY-induced feeding at 1, 2, and 4 h after injection. In summary, BDNF in the VMH significantly decreases food intake and body weight gain, by TrkB receptor-mediated actions. Furthermore, the anorectic effects of BDNF in this site appear to be mediated by NPY. These data suggest that the VMH is an important site of action for BDNF in its effects on energy metabolism.
- food intake
- body weight
the ventromedial nucleus of the hypothalamus (VMH) is a brain area important to the regulation of energy metabolism. Early studies indicated that VMH lesions resulted in hyperphagia and obesity (28, 38, 66), whereas electrical stimulation of the VMH immediately suppressed feeding and induced lipolysis (65). Glucose-sensing neurons (7, 27, 55, 74, 75) and receptors for neuropeptides important to energy metabolism have been identified in the VMH, including leptin (13, 17, 20, 22, 51), melanocortin (26), neuropeptide Y (NPY) (43), corticotrophin-releasing hormone (49), CCK (12), insulin (33), and orexin (44) receptors. Many biological agents given into the VMH have been demonstrated to affect feeding. Food intake is inhibited by administration of histamine (4, 47), glucagon-like peptide 1 (70), serotonin agonists (25), urocortin (58), CCK (79), leptin (51), and insulin (78), whereas thyroid hormone (3,5,3′-triiodothyronine) (40), GABA or GABA agonists (32, 34, 35), norepinephrine (73), orexin (74), and NPY induce feeding after administration into the VMH (5, 9, 24, 31, 43, 56, 76).
Anatomically, the VMH receives inputs from regions important to the regulation of energy metabolism, including the hypothalamic arcuate nucleus (ARC) (2, 14, 23), lateral hypothalamus (18, 67, 80), amygdala (45, 50), and lateral septum. The VMH also projects to ARC (77), paraventricular nucleus (PVN) (42, 52), lateral hypothalamus (72, 80), dorsomedial nucleus of the hypothalamus (46, 80), amygdala (6, 68), lateral septum (68), ventral tegmental area (68), nucleus accumbens (6), and nucleus of the solitary tract (6). These behavioral and neuroanatomic data provide evidence for the importance of the VMH in regulation of energy metabolism.
Brain-derived neurotrophic factor (BDNF) has recently been reported to affect energy metabolism. Intracerebroventricular (ICV) BDNF decreased feeding and body weight gain in animals (62). BDNF+/− heterozygous animals displayed hyperphagia and obesity (36), and exogenous BDNF reversed the phenotype. Human patients with BDNF receptor (TrkB) defects exhibit hyperphagia and obesity (91). Low levels of plasma BDNF have been found in obese patients with Type 2 diabetes and metabolic syndrome (21). This evidence indicates that BDNF is important in the regulation of energy metabolism, but information on sites of BDNF action in the central nervous system is limited. Low BDNF expression was observed in the hypothalamus of BDNF+/− heterozygous animals, including the PVN and VMH (36). Bariohay et al. (3) reported significant decreases in feeding and body weight gain during chronic infusion of BDNF in the dorsal ventral complex, and our previous work (85, 86) indicates that the PVN is an important site of BDNF action.
BDNF in the VMH may also play an important role in the regulation of energy metabolism. Both mRNA and protein for BDNF and its receptor TrkB have been identified in the VMH (8, 36, 87, 90). Low levels of BDNF in the VMH are accompanied by hyperphagia and obesity (36), and animals lacking the orphan nuclear steroidogenic factor-1 receptor develop BDNF deficiency and VMH abnormalities and also display hyperphagia and obesity (11, 48, 82, 92). Recent studies indicate that expression of BDNF in the VMH is regulated by leptin (39) and melanocortins (89), two key mediators of energy metabolism. On the basis of these data, we set out to determine whether the VMH is an important site of BDNF action.
We first tested the effect of a single injection of BDNF in the VMH on normal feeding and deprivation-induced feeding. We then determined whether these effects could be reversed by preadministration of the TrkB-Fc fusion protein, which functionally blocks binding between BDNF and the TrkB receptor. We also performed a conditional taste aversion (CTA) study to determine whether BDNF into this brain area results in feelings of malaise, which could potentially explain reductions in feeding. Finally, we tested the interaction between BDNF and NPY by measuring the effect of BDNF on NPY-induced feeding. Our study demonstrated that 1) a single injection of BDNF in the VMH significantly reduces feeding and body weight gain in normal and food-deprived animals for up to 48 h; 2) pretreatment with TrkB-Fc significantly blocks BDNF-induced anorexia; 3) BDNF, at doses that effectively inhibit feeding (0.1–1 μg), does not result in a conditioned taste aversion; and 4) BDNF significantly blocks NPY-induced feeding.
Male Sprague-Dawley rats (Harlan, Madison, WI) weighing 280–320 g were housed individually in cages with a 12:12-h light-dark photocycle (lights on at 7:00 AM) in a room at 21–22°C. Teklad lab Chow and water were allowed ad libitum, except where noted. All experimental study protocols were approved by the Veterans Affairs Medical Center Institutional Animal Care and Use Committee before commencement of procedures.
Cannulation and Verification of Placement
Rats were anesthetized with intramuscular xylazine (3.5 mg/kg) and ketamine (20 mg/kg) and were fitted with 28-gauge stainless steel guide cannulae placed just above the VMH either unilaterally or bilaterally. Stereotaxic coordinates were determined from the rat brain atlas of Paxinos and Watson (61) and are as follows: 0.6 mm lateral and 2.5 mm posterior to bregma and 8.5 mm below the skull surface. The injector extended 1 mm further than the end of the guide cannula. The animals were given at least 1 wk to recover after surgery before experimental trials. After terminal experiments, the rats were decapitated and whole brain tissues were removed and soaked in 10% formalin solution for at least 48 h. The brain tissues were sectioned by cryostat at a thickness of 50 μm, mounted on gelatin-coated slides, stained with 0.1% thionin, and treated with ethanol (from 30% to 100%) and clearing agent (Electron Microscopy Sciences, Hatfield, PA). After the slides had dried, injection placement was determined microscopically at ×10, using the brain atlas of Paxinos and Watson (61) as a reference. Placements of the unilateral injection sites for study of BDNF effects on normal and deprivation-induced feeding and bilateral injection sites for study of BDNF effects on normal feeding are summarized in Fig. 1, and photomicrographs of representative histologies with bilateral cannulation are shown in Fig. 2. A cannula was deemed correct if the histological examination indicated that the injection was within a 0.25 mm diameter from the targeted site. This rationale is based on diffusion coefficients for the injection volume delivered (57) and work showing effectiveness within this range (85, 86). Data from animals with misplaced cannulae were excluded from data analyses. Histological examination of brain tissue to verify injection site was not possible in some studies because rats were undergoing additional studies. In this situation, we used feeding response to injected NPY as a behavioral assay to determine correct cannula placement, as verified in previous studies (5, 9, 24, 31, 43, 56, 76). According to this assay, cannula placement is deemed correct if the animal consumes more than 2 g of chow within 2 h after 100 pmol NPY. Animals not responding to NPY are excluded from the study.
BDNF was kindly provided by Regeneron Pharmaceuticals (Tarrytown, NY) and stored at −70°C in 10 mg/ml of 150 mM NaCl, 10 mM NaHPO3 buffer, and 0.004% Tween 20 until use. Just before use, BDNF was diluted with artificial cerebrospinal fluid (aCSF). NPY was purchased from Phoenix Pharmaceuticals (Mountain View, CA) and was diluted with aCSF just before use. TrkB-Fc fusion protein was purchased from R&D System (Minneapolis, MN), dissolved in PBS (pH 7.4) as stock, and diluted to a final concentration of 1.0 μg/μl in a mixture of PBS and aCSF (1:1 vol/vol) containing 0.1% BSA.
Volume of 0.5 μl was injected slowly over 2 min for TrkB-Fc and over 30 s for vehicle and other compounds, with injector left in place for an additional 15 s to ensure extrusion from the tip and to minimize distribution of drug upward on the cannula tract. In total, animals received <10 injections. Injection sites were examined by light microscopy for tissue damage in the present studies, and none was found.
Effect of BDNF on normal feeding.
Food was allowed ad libitum before and during the experimental period. Eighteen rats were injected unilaterally with aCSF and 0.1, 0.3, or 0.5 μg of BDNF just before the dark phase (7:00 PM). Food intake was measured at 1, 2, 4, 24, and 48 h after injection. Body weight was measured at 0 h and at 24 and 48 h after injection. Each animal received each treatment once with at least 72 h between treatments to allow for clearance of BDNF from the central nervous system and for normal feeding patterns to be reestablished. Treatments were given in a randomly selected Latin square design to avoid a treatment-order confound. Sixteen rats had correct cannula placement (verified by histology) and therefore were included in the final statistical analyses. Another set of 17 rats with bilateral cannulae were injected with 0.1, 0.3, 0.5, 1.0, and 3.0 μg of BDNF on each side at 2:00 PM. Food intake and body weight change were measured at 24 and 48 h after injection. After 3 rats were excluded because of incorrect cannula placement (verified by histology), 14 rats were included in the final statistical analyses.
Effect of BDNF on deprivation-induced feeding.
Eighteen rats were injected unilaterally with aCSF or BDNF (0.1, 0.3, or 0.5 μg) after 18 h of food deprivation and were given food immediately after injection. Food intake was measured at 1, 2, 4, 24, and 48 h after injection. Body weight was measured at 0 h and at 24 and 48 h after injection. Each animal received each treatment once with at least 72–96 h between treatments. Sixteen rats had correct cannula placement (verified by histology) and were included in the statistical analyses. Another set of 20 rats was bilaterally injected with 0.1, 0.3, 0.5, or 1.0 μg BDNF on each side. Each animal received each treatment once with at least 72–96 h between treatments. Correct placement was determined by response to NPY. All rats were deemed to have correct placement and were included in the statistical analyses.
Effect of TrkB-Fc fusion protein on BDNF-suppressed feeding.
TrkB-Fc is a fusion protein that consists of an extracellular domain of the TrkB receptor and an Fc domain of IgG. TrkB-Fc binds to BDNF with high affinity and thus functionally blocks BDNF binding to its receptor. Twenty-four rats were injected bilaterally with one of the following combined treatments on each side: 1) aCSF + aCSF, 2) aCSF + 0.5 μg BDNF, 3) 0.5 μg TrkB-Fc + 0.5 μg BDNF, or 4) 0.5 μg TrkB-Fc + aCSF. The second injection was given 10–15 min after the first injection. Food intake and body weight were measured at 0, 24, and 48 h after the second injection. Each animal received each treatment once with at least 72 h between treatments. The dose of TrkB-Fc was chosen based on effective dose ranges from published in vivo studies (30, 41, 64, 69) and pilot studies in our laboratory. After 3 rats were excluded because they did not respond to NPY, 21 rats were included in the statistical analysis.
The effect of VMH-injected BDNF on preference for saccharin solution.
In a separate group of animals, the two-bottle preference test was used to determine whether BDNF results in aversive consequences after administration into the VMH. The procedures for the experiment have been previously described (84). In brief, drinking water was allowed only between 10:30 and 11:00 AM for 30 bilaterally VMH-cannulated rats each day for 7 days. During water access, two bottles of water were presented. The rats were then randomly divided into five groups (with an even distribution of body weight) and given 15 ml of 0.1% saccharin; this was immediately followed by injection on each side with either aCSF or BDNF (0.1, 0.3, 0.5, or 1.0 μg). This conditioned stimulation was repeated once after 2 days, and the animals were then given the choice of water or saccharin in the absence of drug administration 2 days later. To avoid the possible confound of bottle placement, initial position and order of presentation of the saccharin bottle were counterbalanced across subjects, and the two-bottle test was repeated 2 days later in which the two bottle positions were reversed. Intake of water and saccharin during 24 h was measured, and the mean of the two tests was calculated. One rat was excluded because it did not respond to VMH NPY; thus 29 rats were included in the statistical analyses.
Effect of BDNF on NPY-induced feeding.
Food was allowed ad libitum before and during the experimental period. Ten VMH bilaterally cannulated rats were randomly assigned to one of six treatments: 1) aCSF + aCSF, 2) aCSF + NPY (100 pmol), 3) BDNF (0.1 μg) + NPY (100 pmol), 4) BDNF (0.3 μg) + NPY (100 pmol), 5) BDNF (0.5 μg) + NPY (100 pmol), or 6) BDNF (0.5 μg) + aCSF. The first injection was made bilaterally at 7:00 AM, and the second injection was made unilaterally 4 h later (11:00 AM) on the alternate side on each experimental day. The 4-h delay was used in an effort to match the delayed effect of BDNF on feeding behavior in food-deprived animals. Each animal received each treatment once with at least 72 h between treatment to allow for clearance of BDNF from the central nervous system and for normal feeding patterns to be reestablished. Food intake was measured at 1, 2, and 4 h after the second injection. All 10 rats responded to NPY and were included in the statistical analyses.
For all feeding experiments, data were analyzed with the use of StatView 5.0 (Cary, NC) and are expressed as means ± SE. Repeated-measures ANOVA was used to analyze food intake and body weight change. Thus each rat served as its own control. When main effects were observed, post hoc analysis was performed using multiple-comparison contrasts. For the conditioned taste aversion experiment, data were analyzed by a one-factor ANOVA followed by Fisher's least-significant difference t-test to compare means.
Effect of BDNF on Normal Feeding
Unilateral injections of BDNF did not significantly affect feeding within the first 4 h postinjection (Fig. 3A), but 0.5 μg BDNF significantly decreased feeding in the 4- to 24-h postinjection interval by 21.6% (P = 0.0056; Fig. 3B), by 17.4% (P = 0.0084) in the 0- to 24-h interval, and by 13.2% (P = 0.0061; Fig. 3B) in the 0- to 48-h interval. BDNF at 0.1 and 0.5 μg significantly reduced body weight gain in the 0- to 24-h postinjection interval (P = 0.0449 and P = 0.0005, respectively) and in the 0- to 48-h postinjection interval, and the 0.5-μg BDNF dose significantly reduced body weight gain (P = 0.0170; Fig. 3C).
Bilaterally injected BDNF dose-dependently inhibited normal feeding (Fig. 4A). At 24 h after injection, BDNF at the 1- and 3-μg doses significantly inhibited normal feeding by 10.3% (P = 0.024) and by 16.3% (P = 0.0005), respectively. In the 24- to 48-h interval, BDNF at 0.5, 1, and 3 μg significantly inhibited normal feeding by 9.5% (P = 0.0073), 7.5% (P = 0.0329), and 9.7% (P = 0.0066), respectively. In the 0- to 48-h postinjection interval, BDNF at 1 and 3 μg significantly inhibited normal feeding by 8.9% (P = 0.0112) and by 12.9% (P = 0.0003), respectively. In the 0- to 24-h postinjection interval, BDNF at 0.5, 1, and 3 μg significantly inhibited body weight gain by 53.9% (P = 0.0471), 60.1% (P = 0.0295), and 98.8% (P = 0.0004), respectively (Fig. 4B). In the 0- to 48-h interval, BDNF at 0.5, 1, and 3 μg inhibited body weight gain (P = 0.0412, P = 0.0074, and P = 0.0004, respectively; Fig. 4B).
Effect of BDNF on Deprivation-Induced Feeding
Unilateral injections of BDNF did not significantly affect feeding within the first 4 h postinjection. In the 4- to 24-h postinjection interval, BDNF at 0.3 and 0.5 μg significantly decreased feeding by 12.3% (BDNF, 18.37 ± 0.69 g vs. aCSF, 21.29 ± 0.81 g; P = 0.0163) and by 15.8% (BDNF, 17.93 ± 0.85 g vs. aCSF, 21.29 ± 0.81 g; P = 0.0025), respectively. In the 0- to 24-h postinjection interval, BDNF at 0.1, 0.3, and 0.5 μg significantly reduced feeding by 7.8% (BDNF, 29.16 ± 0.96 g vs. aCSF, 31.61 ± 0.91 g; P = 0.0196), 8.1% (BDNF, 29.04 ± 0.81 g vs. aCSF, 31.61 ± 0.91 g; P = 0.0147), and 9.3% (BDNF, 28.66 ± 0.72 g vs. aCSF, 31.61 ± 0.91 g; P = 0.0056). In the 0- to 48-h postinjection interval, BDNF reduced feeding and body weight gain in a dose-dependent pattern, but these changes did not reach statistical significance (P > 0.05, data not shown).
Similar to the data from unilateral BDNF administration, bilaterally injected BDNF did not inhibit feeding at 4 h after injection (Fig. 5A). In the 4- to 24-h postinjection interval, BDNF at 0.3, 0.5, and 0.1 μg dose dependently inhibited deprivation-induced feeding by 13.4% (P = 0.0077), 18.1% (P = 0.0004), and 22.1% (P < 0.0001), respectively (Fig. 5B). BDNF at 0.1, 0.3, 0.5, and 1 μg in the 0- to 24-h postinjection interval significantly reduced feeding by 7.0% (P = 0.0234), 11.8% (P = 0.0002), 17.5% (P < 0.0001), and 17.0% (P < 0.0001), respectively. BDNF-induced reductions in feeding in the 24- to 48-h postinjection interval did not reach statistical significance. During the 0- to 48-h postinjection interval, BDNF at 0.3, 0.5, and 1 μg significantly decreased feeding by 6.8% (P = 0.0111), 13.3% (P < 0.0001), and 14.3% (P < 0.0001), respectively (Fig. 5B). In the 0- to 24-h postinjection interval, BDNF at 0.1, 0.3, 0.5, and 1 μg dramatically decreased body weight gain by 16.4% (P = 0.0551), 27.1% (P = 0.0019), 42.9% (P < 0.0001), and 45.4%, (P < 0.0001), respectively (Fig. 5C). In the 0- to 48-h postinjection interval, BDNF at 0.3, 0.5, and 1 μg significantly and dose dependently decreased body weight gain by 34.5% (P = 0.0028), 43.6% (P = 0.0002), and 58.5%, (P < 0.0001), respectively (Fig. 5C).
Effect of TrkB-Fc Fusion Protein on BDNF-Induced Feeding Inhibition
In this experiment, the second injection (BDNF or aCSF) was made 10–15 min after the first injection (TrkB-Fc or aCSF). During the 0- to 24-h postinjection interval, BDNF at 0.5 μg significantly decreased feeding by 19.4% (P < 0.0001; Fig. 6A) and significantly decreased body weight gain (P < 0.0001; Fig. 6B). Preadministration of 0.5 μg of TrkB-Fc significantly attenuated BDNF-induced anorexia by 13.7% (P = 0.0011; Fig. 6A) and body weight decrease (P = 0.0426; Fig. 6B). During the 24- to 48-h postinjection interval, BDNF significantly decreased feeding by 6.2% (P = 0.0077; Fig. 6A) and significantly decreased body weight gain (P = 0.0028; Fig. 6B); preadministration of TrkB-Fc attenuated BDNF-induced anorexia by 4.7% and body weight loss, but this did not reach statistical significance (P = 0.2122 and P = 0.2147, respectively). During the 0- to 48-h postinjection interval, BDNF significantly decreased feeding by 14.3% (P < 0.0001; Fig. 6A) and decreased body weight gain (P < 0.0001; Fig. 6B), whereas pretreatment with TrkB-Fc significantly attenuated BDNF-induced feeding inhibition by 9% (P = 0.0073; Fig. 6A) and attenuated BDNF-induced body weight loss (P < 0.0191; Fig. 6B).
Effect of VMH-Injected BDNF on Preference for Saccharin Solution
In the conditioned taste aversion experiment, the percentage of fluid intake attributed to saccharin was 91.4 ± 1.8% for aCSF, 78.1 ± 8.2% for 0.1 μg BDNF, 86.8 ± 7.2% for 0.3 μg BDNF, 80.8 ± 6.1% for 0.5 μg BDNF, and 71.3 ± 8.9% for 1.0 μg BDNF. There was no main effect of BDNF on the intake of saccharin solution (P = 0.3013).
Effect of BDNF on NPY-Induced Feeding
Injection of NPY into the VMH significantly increased food intake at 1 h after injection (P = 0.0018; Fig. 7), and preadministration of BDNF (0.5 μg) significantly decreased NPY-induced feeding by 67.4% (P = 0.0139; Fig. 7). In the 1- to 2-h and 2- to 4-h postinjection intervals, there was no main effect on feeding (Fig. 7). However, in the 0- to 2-h post-NPY injection interval, NPY significantly induced feeding (P = 0.0066; Fig. 7), which was decreased 50.1% by 0.5 μg BDNF, but this did not reach statistical significance (P = 0.0645; Fig. 7). In the 0- to 4-h interval, NPY significantly increased feeding (P = 0.0131; Fig. 7), which was significantly inhibited by 0.5 μg BDNF (54.9%, P = 0.0091; Fig. 7).
In the present study, a single injection of BDNF in the VMH significantly inhibited normal feeding, deprivation- and NPY-induced feeding, and body weight gain. The conditioned taste aversion experiment suggests that BDNF at doses effective in reducing feeding did not cause taste aversion, suggesting that BDNF effects on feeding are not the result of malaise.
Unilateral injections of BDNF did not affect normal feeding in the first 4 h after injection (Fig. 3A), but it decreased feeding and body weight gain at the 24- and 48-h postinjection intervals (Fig. 3, B and C). To determine whether bilateral injection of BDNF would effectively inhibit feeding, we injected BDNF bilaterally, using a wider range of doses. Bilateral injection of BDNF dose dependently inhibited normal feeding and body weight gain (Fig. 4, A and B), but in general the bilateral BDNF injections were no more effective than unilateral BDNF injections in reducing feeding or body weight gain.
We also tested unilateral and bilateral administration approaches in deprivation-induced feeding. With both unilateral and bilateral injection, BDNF did not significantly affect feeding in the first 4-h postinjection interval (see results and Fig. 5A). Both unilateral and bilateral BDNF decreased feeding at 24 h postinjection, and bilateral BDNF (same dose on each side) did not create further inhibition of feeding. At 48 h postinjection, unilateral BDNF did not significantly inhibit feeding and body weight gain (results), whereas bilateral BDNF at 0.1–1 μg dramatically decreased feeding and body weight gain (Fig. 5, B and C), suggesting that in this case the bilateral approach was more effective.
Several studies have reported inhibitory effects of BDNF on feeding after peripheral and central administration. Pelleymounter et al. (62) in the mid 1990s reported that chronic and continuous ICV BDNF decreased feeding and body weight gain, and Kernie et al. (36) reported that ICV BDNF reversed hyperphagia and obesity in BDNF heterozygous mice. Chronic, intermittent subcutaneous injections of BDNF reduced body weight (81), suggesting peripheral sites of action for BDNF, as a molecule of this size (13.6 kDa) cannot cross the blood-brain barrier (59, 60). In a recent study in which the dorsal vagal complex was specifically targeted, chronic administration of BDNF significantly decreased feeding and body weight gain at doses of 0.1 and 1.0 μg (3). Because these studies relied on a chronic infusion method, the time course of BDNF anorectic effects is unknown. In our previous study of BDNF in the PVN (86), a single BDNF injection significantly decreased feeding and body weight gain during normal, deprivation-induced, and NPY-induced feeding for up to 48 h after injection. Similarly, BDNF in the VMH also shows inhibitory effects on feeding and body weight gain during this time frame, implicating the VMH as an additional site of action for central BDNF.
The CTA experiment indicates that BDNF at effective feeding-inhibitory doses does not result in a CTA, suggesting that BDNF anorectic effects are not due to malaise or other aversive consequences of BDNF injection. Although it is possible that the temporal relationship between pairing of the novel solution exposure and potential BDNF injection-related malaise is not accurately matched, in our previous work (86) with BDNF in the PVN, matching the saccharin exposure with the delayed timing of the anorectic response did not result in a CTA. Although not absolute, these data provide strong support for the idea that BDNF anorectic effects are not via malaise or other aversive consequences of BDNF injections.
Leptin has been reported not only to activate nuclear groups in the ventromedial hypothalamus (10, 15, 16) but also to increase BDNF mRNA and protein in the VMH (39). ICV leptin rapidly reduces food intake within 0.5–1 h postinjection (71) (53), and VMH leptin also dramatically decreases feeding and body weight (29). Because leptin stimulates BDNF expression in the VMH, it is speculated that leptin decreases feeding by mediating VMH BDNF. However, as indicated above, the timing of BDNF inhibitory effects does not match that of leptin, suggesting that BDNF may not mediate leptin feeding inhibitory effects. BDNF has also been suggested as an important downstream effector of melanocortin-4 receptor signaling, and one study showed that melanocortin administration stimulates BDNF expression in the VMH (89). However, like leptin, the time course of melanocortin-induced anorexia (19, 88) does not match that of BDNF. Although these data do not exclude BDNF interaction with either leptin or melanocortins, the timing differences in behavioral responses suggest that BDNF likely does not mediate leptin- and melanocortin-induced feeding inhibition.
Several reports have indicated that the TrkB-Fc fusion protein is effective in blocking BDNF effects, such as BDNF-mediated N-methyl-d-aspartate-evoked responses (37), synaptic regulation (83), and noxious stimulation (63). To verify that BDNF decreases feeding and body weight gain via activation of TrkB, we used the TrkB-Fc fusion protein to block BDNF binding to its receptor. Although TrkB-Fc protein does not directly target the TrkB receptor, it contains the TrkB extracellular domain (BDNF binding site) and thus acts as a competitive inhibitor of TrkB receptor binding to BDNF and reduces BDNF-TrkB signaling. Therefore, this fusion protein can be used to test whether TrkB receptor binding is necessary for BDNF effects. On the basis of effective in vivo concentrations (1.6–2.5 μg/μl) and in vitro ED50 data available from the manufacturer indicating that relatively a large amount of the fusion protein is needed (ED50 reached when the ratio of TrkB-Fc to BDNF molecules was 6:1), we initially tested 1.5 μg of TrkB-Fc but found that this dose resulted in immediate hyperactivity, which endured for ∼10–15 min. The dose was reduced to 0.5 μg, which did not cause hyperactivity. TrkB-Fc at 0.5 μg significantly blocked BDNF-induced feeding inhibition and body weight loss (Fig. 6, A and B), indicating that BDNF in the VMH reduces feeding and body weight gain via its receptor TrkB.
ARC NPY neurons project to other areas of the hypothalamus, including the PVN and VMH. NPY has been reported to increase feeding not only after administration into the PVN but also after VMH injection (5, 9, 24, 31, 43, 56, 76). We tested interactions between BDNF and NPY on feeding behavior. VMH NPY significantly increased feeding, and this effect was significantly reduced by BDNF (Fig. 7). This finding is similar to that for PVN BDNF and NPY coinjection studies and suggests that both VMH and PVN BDNF effects may be due to blockade of NPY orexigenic pathways.
In conclusion, BDNF in the VMH significantly decreases normal and deprivation- and NPY-induced feedings and body weight, at doses not producing a condition taste aversion, and the effect of BDNF is mediated by its receptor TrkB in the VMH. The delayed effect on feeding inhibition suggests that BDNF in the VMH may trigger activation and/or inhibition of neural circuit(s) or may be transported to other sites for the action (1, 54), for which further studies are needed. Together, these data implicate the VMH as an important site of BDNF action to influence energy metabolism.
This work was supported by the Department of Veterans Affairs and by the Minnesota Obesity Center (pilot and feasibility program no. 14).
We thank Regeneron Pharmaceuticals (Tarrytown, NY) for providing BDNF and Mark R. Margosian, Mary Mullett, and Martha Grace for technical support.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society