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Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080
Address all correspondence and requests for reprints to: John C. Lin, Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, California 94080. E-mail: john.lin{at}rinat.pfizer.com.
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Abstract |
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Introduction |
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Brain-derived neurotrophic factor (BDNF) and its receptor trkB were originally studied for their function in sensory neuron development (3), but are also implicated in the neuroendocrine control of mammalian feeding behavior and energy homeostasis (4). Mice (5) or humans (6) with a hypomorphic or loss-of-function allele of the tyrosine kinase receptor trkB display excessive appetite, reduced energy expenditure, and morbid obesity. The expression of BDNF in the ventromedial hypothalamus (VMH), an important component of the central nervous system (CNS) energy homeostasis circuitry, is reduced after food deprivation (5, 7), and mice with genetically reduced BDNF expression (7, 8, 9) or with postnatal forebrain excitatory neuron-selective BDNF deficiency (10) also develop severe obesity. TrkB is thought to act downstream of the melanocortin 4 receptor (Mc4r), a well established hypothalamic signaling system that mediates control of appetite in the arcuate nucleus by hormones such as leptin and insulin (4, 11). Thus, the Mc4r-deficient Ay lethal yellow mice, which are hyperphagic and obese, display reduced expression of BDNF in the VMH, and their obese phenotype is rescued by intracerebroventricular injections of BDNF (5).
The CNS feeding circuits receive hormonal signals such as leptin that reflect long-term energy status as well as vagally transmitted neural signals that sense short-term accumulation and movement of food in the gastrointestinal tract. Interestingly, mice deficient in neurotrophin-4 (NT4), the second trkB ligand, have 55% loss of nodose ganglion neurons and 80–90% of vagal intraganglionic mechanoreceptors in the small intestine. These mice suffer alterations in short-term meal patterns but display normal daily food intake and body weight because of compensatory changes in other meal parameters (12). One explanation for the lack of overt hyperphagia and obesity phenotype in the NT4-deficient mice is that BDNF, but not NT4, is expressed in the CNS regions critical for long-term feeding behavior and body weight homeostasis, such as the VMH. Alternatively, the phenotypic difference between BDNF–/– and NT4–/– mice regarding feeding and obesity may result from quantitative or qualitative differences in their ability to activate the common trkB signals. Support for the second hypothesis is provided by the finding that the trkBshc/trkBshc mutant mice exhibited a nearly complete loss of the NT4-dependent sensory neurons but only a modest loss of the BDNF-dependent neurons (13).
The mechanism by which trkB regulates feeding behavior and weight is not well understood. In addition, it is not known whether the central anorexigenic trkB system can regulate obesity of different causes, including monogenic leptin receptor deficiency, polygenic obesity, and high-fat diet-induced obesity (DIO). Leptin, for example, is effective in treating obesity due to leptin deficiency but not in high-fat DIO. Moreover, because BDNF and NT4 bind and signal also through the low-affinity neurotrophin receptor p75NTR, it is not known whether trkB activation alone is sufficient to mediate antiobesity effects. In addition, because trkB is implicated in a nociceptive pathway (14, 15), and severe pain can lead to anorexia and weight loss, it is important to establish whether the metabolic and nociceptive functions of trkB are dissociable. Finally, it would be important to determine whether the trkB system plays any role in taste preference, an important aspect of feeding behavior.
To expand our insight into the role of trkB agonists in energy homeostasis, we conducted a series of gain-of-function experiments using NT4 and a trkB-specific agonist antibody as pharmacological tools. We found that exogenously applied trkB agonists effectively reduce food intake and body weight and modify taste preference to high-calorie liquid food. TrkB agonists did not induce starvation-related stress response, LiCl-like visceral discomfort, or nerve growth factor (NGF)-like pain sensitization and were effective in several mouse models, including DIO, polygenic obesity, and db/db mice. Our pharmacological studies, together with the previous genetic evidence, strengthen the notion that trkB signaling is a key mediator of energy homeostasis that acts downstream of both the melanocortin and the leptin cascades. Given the prevalence of human obesity caused by Mc4r dysfunction and widespread leptin resistance in the human population, trkB modulators could emerge as attractive therapeutics for metabolic disorders.
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Materials and Methods |
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TrkB agonist monoclonal antibody (mAb)
TrkB antibodies were generated by immunizing BALB/c mice with the extracellular domain of recombinant trkB protein. Hybridomas were screened for trkB binding and agonist activity (experimental details will be described elsewhere). The ascites obtained from a single BALB/C mouse was purified batchwise with protein A resin. Approximately 1 ml resin equilibrated with PBS was incubated with ascites fluid overnight at 4 C with gentle agitation. The resin was spun down and washed twice with PBS. Antibody was eluted from the resin using 5 vol 50 mM sodium citrate-phosphate buffer (pH 3). Eluates were immediately neutralized with 1 M HEPES buffer (pH 7) and then dialyzed into PBS, concentrated, and sterile filtered. The antibody was shown to be a specific TrkB agonist by a cell-based receptor tyrosine kinase activation assay as shown in supplemental Fig. S4 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) (18).
Animals
All animal use and procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Rinat Laboratories. All animals were housed in a temperature- and humidity-controlled room with a 12-h light, 12-h dark cycle, with ad libitum access to water and food unless otherwise specified for the pair-fed study. The high-fat DIO mice were produced as follows. Male C57BL/6J mice (JAX West, West Sacramento, CA) were weaned at 4 wk of age; immediately upon weaning, they were put on a 58% high-fat diet (D12331i; Research Diets, New Brunswick, NJ). The db/db mice (BKS.Cg-m+/+Leprdb/J strain) and the NONcNZO-10 mice were obtained from Jackson Laboratory (Bar Harbor, ME). They were fed on the regular mouse diet (PicoLab Rodent Diet 20, containing 4.5–5.4% fat). For intracranial injections, male C57B6 retired breeder mice (aged 8–12 months) were obtained from Charles River Laboratories (Hollister, CA). Sprague Dawley rats weighing 150–170 g were obtained from Charles River. Unless otherwise specified, d 1 of a given study in the time axis denotes the first day when a treatment or compound was given. For the delivery of compounds, all the injections were given on the indicated days between 0900 and 1100 h.
Metabolic cage and body composition
C57BL/6J mice that were fed a high-fat diet (DIO mice) were housed three per cage in ventilated cages with ad libitum access to food and water. After acclimation for 1 wk, mice were measured by dual-energy x-ray absorptiometry (DEXA) scanning (PIXImus; GE LUNAR, Madison, WI) and then placed in the Comprehensive Cage Monitoring System (Columbus Instruments, Columbus, OH) and allowed to acclimate for 2 d. Then DIO mice were dosed once per day for 5 d by sc injection with vehicle or 10 mg/kg NT4. Body weight was measured once per day. CO2 production (VCO2), O2 consumption (VO2), food intake, water intake, and locomotor activity were monitored continuously for the entire 7 d, and the respiratory quotient (RQ) was calculated by RQ = VCO2/VO2. At the end of metabolic testing, mice were measured by DEXA scanning.
Theoretical estimates of fat utilization as a percentage of total energy consumption were made according to the following steps. Complete oxidation of carbohydrates yields RQ (VCO2/VO2) = 1. Complete oxidation of triglycerides with long-chain fatty acids yields RQ of approximately 0.67. Assuming that the animals primarily derived their energy from carbohydrates and triglycerides under our experimental conditions, we can calculate the average percentage of fat as the fuel source with vehicle treatment (X%) and with NT4 treatment (Y%) by solving the following equations: vehicle treatment, 1 x (100% – X%) + 0.67 x (X%) = 0.846 
X% = 46.6%; and NT4 treatment, 1 x (100% – Y%) + 0.67 x (Y%) = 0.806 Y% = 58.8%.
Clinical biochemistry
All animals except db/db mice were bled retroorbitally, and a fresh drop of whole blood was applied to the OneTouch Ultra glucose strip (LIFESCAN, Milpitas, CA) or the PTS PANELS triglyceride test strips and BioScanner 2000 (Polymer Technology Systems, Inc., Indianapolis, IN). The serum glucose, triglyceride, nonesterified fatty acids, aspartate aminotransferase, alanine aminotransferase, total cholesterol, and high-density lipoprotein cholesterol levels of db/db mice were measured by IDEXX Laboratories (West Sacramento, CA). The glycosylated hemoglobin (HbA1c) level in the whole blood was measured by DCA 2000+ Analyzer (Bayer, Tarrytown, NY) with the HbA1c reagent kit. The serum samples of various mouse models were prepared and subjected to the following assays as indicated: mouse insulin and leptin ELISA (Crystal Chem Inc., Downers Grove, IL), TNF-
(Linco Research Inc., St. Charles, MO), adiponectin (B-Bridge International, Sunnyvale, CA), rat corticosterone enzyme immunoassay (Diagnostic Systems Laboratories, Inc., Webster, TX).
Intracranial injections
Male C57BL/6J mice at the age of 8–12 months, and DIO mice at the age of 10–12 months, were allowed to acclimate in a temperature- and humidity-controlled environment, with a 12-h light, 12-h dark cycle, with ad libitum access to food and water, for at least 5 d before injection. The mouse was first anesthetized with 5% vaporized isoflurane in conjunction with oxygen (1 liter/min flow rate) and immobilized onto the stereotaxic surgery instrument (Kopf model 900; Kopf, Tujunga, CA). Throughout the surgery, each mouse was maintained on 2% isoflurane with oxygen (1 liter/min flow rate) and kept warm with an electric heating pad. The coordinates for a single, unilateral, intrahypothalamic injection were as follows: 1.30 mm posterior to the bregma; –0.5 mm from midline; depth, 5.70 mm on a flat skull according to the mouse brain atlas (19). A small hole was drilled through the skull, avoiding contact with the brain. The drill was replaced with a beveled 26-gauge needle attached to a Hamilton syringe (model 84851; Hamilton Co., Reno, NV) and returned to the same coordinates. Two microliters of compound were injected into the hypothalamus incrementally over the course of 2 min. Mice were dosed with vehicle (PBS), 2.6 mg/ml mouse IgG control (ChromPure whole molecule; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), 0.072 mg/ml NT4 (in DIO mice), 0.72 mg/ml NT4 (in C57BL/6J), or 2.65 mg/ml of a trkB receptor-specific mAb (TrkB mAb). The needle was kept at this position for 30 sec after injection and then raised 1 mm. After another 30 sec, the needle was slowly removed at a rate of 1 mm/20 sec. The incision was then closed and held together with two 9-mm Autoclip wound clips (Braintree Scientific, Inc., Braintree, MA). Body weight and food intake were monitored daily before and after the injection.
Conditioned taste aversion
Male Sprague Dawley rats were acclimated to the presence of two water bottles over a week. For the next 3 d, animals were trained to drink water during the day (instead of night) by being subjected to water deprivation for 23 h and exposed to water for 1 h each day. The following day, for conditioning, animals were first exposed to 10% (wt/vol) sucrose solution and 1 h later injected ip with lithium chloride [0.15 M stock solution given at 2% volume (milliliters)/body weight (grams)], vehicle (PBS), or NT4. After dosing, rodents were water deprived for 24 h before presentation of one bottle of water and one bottle of sucrose. The volumes of each solution consumed over the defined periods of 6, 24, and 72 h were recorded. The taste preference ratio (TPR) was calculated as follows: TPR = (volume of sucrose consumed)/[(volume of water consumed) + (volume of sucrose consumed)]. This ratio reflects the preference of a given animal to sucrose solution over water. Normal rats usually greatly prefer sucrose over water, with TPR closer to 1 (see for example the vehicle control in Fig. 6C
, open squares with solid line). On the other hand, if their first exposure to sucrose was immediately followed by a noxious stimulus or an emetic agent such as LiCl, rats would develop an aversion to further sucrose intake resulting in a TPR closer to 0 (see Fig. 6C, triangles with dotted line).
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Alternatively, on the conditioning day, rats were injected with LiCl or NT4 1 h before their first exposure to sucrose solution. The reverse order of noxious stimulus (i.e. LiCl) and novel or sweet taste (sucrose solution) presentation failed to elicit the classical conditioning response.
In a separate study, sucrose solution was substituted with saccharin solution (Sweet’N Low, 0.1% wt/vol) with essentially the same results.
Behavioral test of thermal hyperalgesia (Hargreaves test)
Male Sprague Dawley rats were acclimated in the colony 7 d before use and were between 220 and 250 g at the time of testing. A single dose of 2 mg/kg NGF, NT4, or vehicle was administered sc into adult Sprague Dawley rats to determine the sensitivity to noxious heat using the modified Hargreaves test (University of California, San Diego, San Diego, CA). The latency to withdraw the hind paw (seconds) from the radiant heat source was measured in three trials and averaged for both hind paws at 2.5, 4.5, and 24 h after the time of injection. The time of 22 sec served as the cutoff for each trial to prevent damage to the tissue.
Statistics
Statistical analyses were performed by using PRISM (GraphPad Software Inc., San Diego, CA). All data and graphs are expressed in mean ± SEM.
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Results |
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NT4 reduces food intake and body fat in mice with DIO
Mice with high-fat DIO were treated with daily sc injections of vehicle or NT4 (2 and 10 mg/kg·d) for 16 d with dosing starting on d 1. Food intake and body weight were reduced by as much as 30–50% and 12–19%, respectively, after NT4 treatment in a dose-dependent manner (Fig. 1, A and B). Similar results were obtained after treatment with BDNF (supplemental Fig. S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) or trkB-specific activating antibodies trkB (TrkB mAb) (data not shown) but not with the agonistic antibodies that activate the structurally related tyrosine kinase receptors trkA and trkC (data not shown). In a separate experiment, indirect calorimetry by a metabolic chamber showed that treatment of NT4 at 10 mg/kg·d over the course of 5 d significantly reduced the RQ (RQ = VCO2/VO2, Fig. 1C), reflecting an increase in fat utilization from 46.6 to 58.8% as the energy source. There was no significant change in overall rate of oxygen consumption (Fig. 1D) during the same period. Body composition analysis using DEXA revealed that body fat mass was preferentially reduced to 22.7% in the NT4-treated mice compared with 27.9% of the vehicle group (Fig. 1E), whereas the lean body mass was relatively preserved compared with the vehicle group (Fig. 1F). Thus, NT4 modifies body weight and body composition through a number of mechanisms, including decrease in food intake and increase in fat oxidation.
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). There was no rebound hyperphagia associated with the discontinuation of NT4 therapy (Fig. 1B). The DIO mice did not exhibit hyperglycemia or hypertriglyceridemia under nonfasting conditions. Administrations of NT4 at 2 or 10 mg/kg·d over 16 d did not affect nonfasting glucose, glucose tolerance, or triglyceride concentration (data not shown).
NT4 is efficacious in the polygenic obese model NONcNZO-10
We then sought to evaluate the effects of NT4 in the polygenic obesity model named NONcNZO-10, a recombinant congenic strain generated and characterized by Leiter and Reifsnyder (20) at the Jackson Laboratory. Previously, both leptin and leptin receptor loci have been characterized and shown to be functional in NONcNZO-10 mice (20). We found that 2 and 10 mg/kg·d sc injections of NT4 to NONcNZO-10 mice produced a dose-dependent reduction in body weight and food intake (2 and 10 mg/kg·d with dosing start on d 1, dosing duration underlined, Fig. 2, A and B) very similar to that of DIO mice (Fig. 1, A and B). Importantly, the body weight and food intake recovery curves of the polygenic NONcNZO-10 mice resemble those of DIO mice. There was no rebound hyperphagia or weight gain.
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, C and D). Although the pair-fed group also lost weight, the magnitude of its weight loss was smaller than that of the NT4-treated group (Fig. 2D). Thus, NT4 does not reduce body weight solely by reducing food intake.
Leptin receptor is not essential for the antiobesity effect of NT4 but can influence weight recovery
Leptin is an important hormone regulating food intake and body weight in mice, and hyperleptinemia and leptin resistance are common characteristics in human obesity (1). We next asked whether the therapeutic effect of NT4 would require a functional leptin receptor. To address this question, we tested NT4 in 12-wk-old db/db mice, which are severely hyperphagic, obese, and diabetic due to the lack of leptin receptor function (Figs. 3 and 4). Daily sc administration of NT4 significantly reduced the food intake and body weight of db/db mice in a dose-dependent manner (2–20 mg/kg·d with dosing starting on d 1, Fig. 3, A and B). Thus, leptin receptor function is not essential for NT4 to exert its therapeutic effects in mice.
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). This is in sharp contrast to DIO mice (Fig. 1A), the polygenic obese strain NONcNZO-10 (Fig. 2A), and the wild-type C57BL/6 mice (supplemental Fig. S2), all of which recovered their body weight more gradually to approach, but never exceed, the control weight level.
Body wasting was evident in the vehicle-treated db/db mice during the latter part of the study (Fig. 3A). This could have contributed to the apparent overshoot of body weight recovery in NT4-treated animals. To eliminate the age-related wasting as a factor, we tested NT4 in younger db/db mice for a shorter period of time. A single dose of 2 mg/kg NT4 initially elicited a slight decrease in body weight in 9-wk-old db/db mice, followed by a remarkably extended rebound of body weight (Fig. 3C). At 24 d after dosing, the body weight of the NT4-treated group still exceeded that of the vehicle group by 15%, even though the latter did not exhibit wasting. Thus, proper leptin receptor function appears to be required for blocking the rebound weight gain after NT4 administration.
NT4 has long-lasting effects on glucose and triglyceride in db/db mice
In db/db mice, NT4 treatment also ameliorated their hypertriglyceridemia (Fig. 4A) and hyperglycemia (Fig. 4B). The long-lasting normalization of the nonfasting glucose level was confirmed by the significant reduction of HbA1c levels in all NT4-treated mice (Fig. 4C). The nonfasting glucose and triglyceride levels in db/db mice returned slowly and approached the control levels around 30 d after the termination of NT4 treatment (Fig. 4, A and 4B). In db/db mice, the therapeutic effects of NT4 on glucose and triglyceride levels are much longer lasting than those on body weight and food intake.
Despite the lack of leptin receptor function in the db/db mice, their serum leptin levels decreased after NT4 treatment (Fig. 4D). The reduction in leptin level was specific in that it was not accompanied by changes in other cytokines/hormones secreted by the adipose tissues, such as TNF- (Fig. 4E) or adiponectin (Fig. 4F). There were also no changes in nonesterified fatty acid, total cholesterol, high-density lipoprotein cholesterol, alanine aminotransferase, or aspartate aminotransferase after NT4 treatment in these mice (data not shown).
Direct NT4 injections to the hypothalamus reduce food intake and body weight
Because peripherally administered BDNF and NT4 have equivalent weight-reducing efficacy (supplemental Fig. S1) and because BDNF expressed in the VMH was implicated in the physiological regulation of feeding, we investigated whether the ventral hypothalamus is the target tissue for NT4’s antiobesity effects. Consistent with this idea, we found significant increase in TrkB receptor phosphorylation in the hypothalamic extracts of rats after peripheral administration of NT4 (supplemental Fig. S3). Next, we asked whether administration of NT4 into the hypothalamus would also affect food intake and body weight. A single injection of about 0.14 µg NT4 protein (equivalent to 0.0029–0.0035 mg/kg body weight) into the hypothalamus produced significant reductions in body weight and food intake in DIO mice (Fig. 5, A and B). Furthermore, a single intrahypothalamic injection of about 1.4 µg NT4 (equivalent to 0.040–0.051 mg/kg body weight) also significantly reduced body weight and food intake in C57BL/6J mice fed on normal chow (Fig. 5, C and D). If delivered systemically instead of intracranially, such small amounts of NT4 were not able to affect body weight or food intake in mice (data not shown).
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To determine whether activation of trkB alone is sufficient to elicit antiobesity effects, we developed a trkB-specific agonist mAb (TrkB mAb). This TrkB mAb can activate trkB receptor in a CHO cell receptor tyrosine phosphorylation assay and support the trkB-dependent embryonic nodose neuronal survival (supplemental Fig. S4 and data not shown). Next we compared direct hypothalamic injections of NT4 with TrkB mAb. A single dose of TrkB mAb (
5.3 µg, molar equivalent to NT4), produced significant reductions in body weight and food intake in the C57BL/6J mice (Fig. 5, C and D). The body weight reduction was significantly reduced by 10–12% from 2–5 d after the TrkB mAb injection, whereas NT4 injections showed immediate weight loss on the first day, followed by weight recovery. These results suggest that activation of trkB receptor in the ventral hypothalamus alone is sufficient to mediate the appetite- and weight-reducing effects.
NT4 does not induce starvation stress
Reduction in food intake and body weight could be due to a number of untoward side effects, such as stress, visceral discomfort, or persistent pain. We conducted a series of experiments to address these possibilities.
Acute and chronic starvation is known to induce a stress response, including the elevation of serum corticosterone levels. Serum corticosterone levels in DIO mice were not significantly altered by daily treatment with either 1 or 5 mg/kg NT4 (Fig. 6A). Similarly, NONcNZO-10 mice treated daily with 2 or 10 mg/kg sc over a course of 11 d showed significant reduction in food intake and body weight (Fig. 3, A and B) without any significant increase in serum corticosterone levels relative to placebo controls (supplemental Fig. S5). On the other hand, pair-fed NONcNZO-10 mice exhibited substantially higher serum levels of corticosterone (150–200 ng/ml). Therefore, the reduction of food intake by NT4 does not induce the stress response typically associated with forced dieting.
NT4 elicits aversion to sweet taste distinct from the emetic agent LiCl
To evaluate other behavioral effects of NT4, such as visceral discomfort and pain response, we used rats instead of mice. First we tested NT4 in Sprague Dawley rats and confirmed that daily sc injection of 2 and 10 mg/kg NT4 led to significant reductions of food intake and body weight growth in Sprague Dawley rats (Fig. 6B and data not shown). Of note, 2 mg/kg sc NT4 appeared to have reached the maximal appetite-suppressing effects in the rats.
Visceral discomfort leading to nausea and/or vomiting should be evaluated as a potential mechanism for any appetite-suppressing compound. To study potential effects of visceral discomfort, we employed the Sprague Dawley rat model of conditioned taste aversion. In the classical (i.e. forward) conditioning paradigm, the known emetic agent LiCl induced taste aversion when temporally paired after the first presentation of 10% sucrose solution (Fig. 6C, triangles with dotted line). The suppression of sucrose ingestion by LiCl was long lasting (up to 72 h). By contrast, when LiCl was injected before the first presentation of sucrose (i.e. the reverse conditioning paradigm), it failed to suppress future ingestion of sucrose (Fig. 6C, inverted triangles with solid line).
Remarkably, rats avoided the sucrose taste no matter whether a single dose of 2 mg/kg NT4 was given before (Fig. 6C, solid circles with solid line) or after (Fig. 5C, diamonds with dotted line) the first exposure to sucrose. There were no changes in overall fluid intake levels by NT4 or LiCl (Fig. 6D). Furthermore, substituting sucrose with saccharin in the same experimental design yielded similar results (data not shown). Finally, we have checked the detailed time course of NT4 vs. LiCl in the forward conditioning paradigm and found that both agents have indistinguishable time of onset, as early as 1.5 h (supplemental Fig. S6). Thus, NT4 exerts a general suppressive effect on the ingestion of novel, high-calorific, or sweet-tasting fluid in a manner distinct from that of the emetic agent LiCl.
NT4, unlike NGF, does not induce thermal hyperalgesia
Acute and chronic pain can cause depression and lead to loss of appetite. Another member of the neurotrophin family, NGF, has been implicated in the induction and maintenance of pain states (22) leading to weight loss (data not shown). Thus, we were prompted to examine any potential hyperalgesic effects of NT4. A single injection of 2 mg/kg NGF induced a significant thermal hyperalgesia in the rats over several hours, whereas a single injection of NT4, at the same dose, did not induce thermal hyperalgesia (Fig. 6E). Therefore, suppression of food intake by NT4 at 2 mg/kg sc dose is not due to induction of pain.
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Discussion |
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We have established here that both BDNF and NT4 are effective in reducing body weight in DIO mice (supplemental Fig. S1). We demonstrated that systemic and central administration of NT4 at pharmacological doses can reduce food intake and body weight of a wide range of mouse obesity models in a dose-dependent fashion. Thus, NT4 and BDNF exert qualitatively similar, antiobesity effects. Notably, in the previous studies, only extremely high doses (10–50 mg/kg·d) of BDNF were used to treat the obese/diabetic mouse models (25, 26). Such high doses of BDNF were impractical in clinical application, and it was not clear whether lower doses of BDNF or other trkB agonists would be efficacious. Here, we have demonstrated that at a dose as low as 2 mg/kg, NT4 can ameliorate obesity and its associated metabolic disturbances in mouse models. In fact, for some mouse models, concentrations as low as 1 mg/kg NT4 given once weekly can reduce body weight (unpublished observations).
NT4 is effective in essentially all mouse obesity models we have tested, including wild-type C57BL/6J on normal chow, high-fat DIO, polygenic obesity (NONcNZO-10 strain), and db/db mice. The weight-recovery curves between db/db mice and other mouse strains exhibit very different kinetics and endpoints. After NT4 treatment, the body weight of mouse strains with a normal leptin receptor gene recovered slowly and converged with that of the control group. In contrast, body weight rebounded quickly in db/db mice to reach a new, higher steady-state level than that of the control group. Significant weight rebound was also evident in younger db/db mice after just a single injection of NT4. Although these results clearly demonstrate that NT4 can exert its antiobesity effect in the absence of leptin receptor, the unusual rebound response of db/db mice after NT4 treatment may be worthy of future investigation.
It is noteworthy in this context that in db/db mice, NT4’s reducing effects on serum glucose and triglyceride levels are much longer lasting than its effects on body weight and food intake. Limited by the duration of our studies, we currently do not know whether the triglyceride or glucose levels in db/db mice would eventually rebound and exceed control levels, similar to the effects on body weight.
Where does NT4 exert its antiobesity effects in mice? In a previous study, bolus injection of 15 µg BDNF into the third ventricle of db/db mice once a day for 5 d did not elicit a reduction in food intake (27). In another study, continuous infusion of BDNF or NT4 at a rate of 1.2 µg/d for 14 d to the third ventricle of BDNF heterozygous knockout mice resulted in significant reductions of body weight after 8–10 d of continuous infusion (7). Because BDNF is highly expressed in the VMH, we asked whether the direct administration of a small dose of NT4 into the hypothalamus would produce the same effect as systemic NT4 injections. We observed a significant reduction in body weight and food intake in DIO mice with a single dose of 0.14 µg NT4 (Fig. 5, A and B). Likewise, we found a significant weight-reducing and anorectic effect in normal C57BL/6 mice after a single dose of 1.4 µg NT4 (Fig. 5, C and D) but not with 0.14 µg NT4 in our pilot studies (data not shown). These results suggest that NT4 and trkB exert their antiobesity effect primarily in the hypothalamus. The apparent higher efficacy in our intracranial injections compared with the previous study may be either due to the different mouse models used or due to the fact that direct injection into the hypothalamus is more effective than infusion to the third ventricle.
Injections with a single dose of the trkB receptor agonist mAb (TrkB mAb) in the hypothalamus also demonstrated significant reductions in body weight and food intake. The reductions in body weight and food intake by TrkB mAb were delayed compared with NT4, but the effects appear to be longer lasting, and the overall magnitude of body weight loss was greater than with NT4. This difference in the time of onset and duration of action may reflect a number of biophysical and biochemical differences between NT4 and TrkB mAb, including the size-dependent diffusion rates in the hypothalamus (molecular mass of NT4 is 28 kDa vs. TrkB mAb, which is 150 kDa) and the potential difference in their rate of clearance from the brain. Alternatively or additionally, the kinetic difference in their biological responses may be attributable to the receptor specificity. In particular, NT4 binds both trkB and the low-affinity receptor p75, whereas TrkB mAb interacts with trkB but not with p75 (data not shown). Although these possible explanations will require further investigation, our data are the first demonstrations that direct trkB activation in the hypothalamus can suppress food intake and reduce weight. Furthermore, NT4’s antiobesity effect is likely mediated, at least in part, through the activation of trkB receptor.
The anorectic effect of NT4 can be due to emotional stress, acute or chronic pain, or induction of nausea. A recent study showed that intracerebroventricular infusion of 12 µg/d BDNF led to an increase of plasma corticosterone in rats, implicating a potential role of the hypothalamic-pituitary-adrenal axis in the anorectic effect of BDNF (28). We found, in contrast, that serum corticosterone levels were not altered by systemic administration of NT4 at doses that were sufficient to suppress feeding and weight. Our result does not support the involvement of hypothalamic-pituitary-adrenal stress hormones in the anorectic effect by systemic NT4. Likewise, NT4 did not appear to exert its anorectic effect through pain induction or sensitization.
We also studied the potential effect of NT4 on nausea or visceral sickness by employing the experimental paradigm of conditioned taste aversion (29). A classical emetic, LiCl, can reduce the ingestion of sweet-tasting fluid in forward, but not backward, conditioning. This temporal directionality is consistent with the interpretation that LiCl does not generally suppress the ingestion of sweet-tasting liquid, but rather it does so by inducing visceral illness that acts as an unconditional stimulus in the classical Pavlovian conditioning. During the training sessions, the unconditional stimulus has to be preceded by a conditional stimulus (i.e. sweet taste in this case) for the conditional stimulus to elicit the conditional response (i.e. taste aversion in this case) in the future. The backward conditioning usually is ineffective, as was the case with LiCl preceding the initial sucrose presentation (Fig. 6C). By contrast, NT4 reduces the intake of sweet-tasting fluid irrespective of the temporal sequence of conditioning. This is inconsistent with the notion that NT4 induces visceral discomfort similar to the emetic agent LiCl in rodents. Whether NT4 can or cannot induce visceral discomfort such as nausea or vomiting is best tested directly in an animal species in which the emetic reflex exists (e.g. ferrets). Our unpublished results showed that even as high as 10 mg/kg NT4 did not induce retching or vomiting in the ferrets (Lin, J. C., and K. Sharkey, manuscript in preparation). The data together suggest that NT4 is likely to exert a general suppressive effect on the intake of sweet-tasting fluid, at least in rodents. At the same time, it raises the intriguing possibility that NT4’s modulation on taste preference may contribute to its anorectic effect.
In summary, we found that NT4 can effectively reduce food intake and body weight in several mouse models of obesity. Leptin receptor is not required for NT4’s antiobesity effect but is required for resetting body weight during recovery from NT4 treatment. Although it was formally possible that the anorexic effects of NT4, BDNF, and the TrkB mAb are mediated by mechanisms not related to trkB activation, the receptor specificity of these agents combined with the genetic evidence for the role of trkB in energy homoeostasis strongly support the hypothesis that the trkB agonists used here mediated their actions through trkB activation in the hypothalamus. Appetite suppression by NT4 is independent of stress or pain induction, but instead it may be related to NT4’s potent effect on sweet taste preference. Modulators of trkB signal therefore represent attractive therapeutic agents for metabolic disorders such as obesity and its sequelae.
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Footnotes |
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First Published Online December 6, 2007
Abbreviations: BDNF, Brain-derived neurotrophic factor; CNS, central nervous system; DEXA, dual-energy x-ray absorptiometry; DIO, diet-induced obesity; DTT, dithiothreitol; HbA1c, glycosylated hemoglobin; mAb, monoclonal antibody; Mc4r, melanocortin 4 receptor; NGF, nerve growth factor; NT4, neurotrophin-4; RQ, respiratory quotient; TPR, taste preference ratio; VCO2, CO2 production; VO2, O2 consumption.
Received August 22, 2007.
Accepted for publication November 26, 2007.
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