Ciliary neurotrophic factor injected icv induces adipose tissue apoptosis in rats

Recent findings show that ciliary neurotrophic factor (CNTF) and leptin have similar effects on food intake and body weight, suggesting possible overlapping mechanisms. Intracerebroventricular (icv) injection of leptin results in adipose tissue apoptosis. To determine if CNTF has similar activity, male Sprague Dawley rats implanted with lateral cerebroventricular cannulas were randomly assigned to four treatment groups ( N = 8), including control (aCSF), 10 microg/day leptin, 1 microg/day CNTF, and 5 microg/day CNTF. Rats received daily icv injections for 4 successive days. Both leptin and CNTF (5 microg) decreased BW (8.6% and 11.77%, respectively, p <.05) and cumulative food intake was decreased 43% by leptin ( p <.05). Leptin and CNTF (5 microg) reduced adipose tissue mass in epididymal adipose (Epi) by 30 and 33.5%, ( p <.05), in inguinal adipose (Ing) by 51 and 55% ( p <.05), in retroperitoneal adipose (Rp) by 65 and 64% ( p <.05), and in intrascapular brown adipose (iBAT) by 34 and 25% ( p <.05), respectively. Gastrocnemius muscle was not affected. Leptin and CNTF (5 microg) increased apoptosis in Epi by 84 and 150%, respectively ( p <.05) and in Rp by 121 and 146%, respectively ( p <.05). Loss of adipocytes by apoptosis may provide an explanation for the unexpected delay in return to initial energy status following CNTF treatments.     

Effects of leptin replacement on hypothalamic-pituitary growth hormone axis function and circulating ghrelin levels in ob/ob mice

Leptin-deficient obese mice (ob/ob) have decreased circulating growth hormone (GH) and pituitary GH and ghrelin receptor (GHS-R) mRNA levels, whereas hypothalamic GH-releasing hormone (GHRH) and somatostatin (SST) expression do not differ from lean controls. Given the fact that GH is suppressed in diet-induced obesity (a state of hyperleptinemia), it remains to be determined whether the absence of leptin contributes to changes in the GH axis of ob/ob mice. Therefore, to study the impact of leptin replacement on the hypothalamic-pituitary GH axis of ob/ob mice, leptin was infused for 7 days (sc), resulting in circulating leptin levels that were similar to wild-type controls (approximately 1 ng/ml). Leptin treatment reduced food intake, body weight, and circulating insulin while elevating circulating n-octanoyl ghrelin concentrations. Leptin treatment did not alter hypothalamic GHRH, SST, or GHS-R mRNA levels compared with vehicle-treated controls. However, leptin significantly increased pituitary GH and GHRH-R expression and tended to enhance circulating GH levels, but this latter effect did not reach statistical significance. In vitro, leptin (1 ng/ml, 24 h) did not affect pituitary GH, GHRH-R, or GHS-R mRNA but did enhance GH release. The in vivo effects of leptin on circulating hormone and pituitary mRNA levels were not replicated by pair feeding ob/ob mice to match the food intake of leptin-treated mice. However, leptin did prevent the fall in hypothalamic GHRH mRNA and circulating IGF-I levels observed in pair-fed mice. These results demonstrate that leptin replacement has positive effects on multiple levels of GH axis function in ob/ob mice.     

Differential Modulation of Arcuate Nucleus and Mesolimbic Gene Expression Levels by Central Leptin in Rats on Short-Term High-Fat High-Sugar Diet

Objective

Leptin resistance is a common hallmark of obesity. Rats on a free-choice high-fat high-sugar (fcHFHS) diet are resistant to peripherally administered leptin. The aim of this study was to investigate feeding responses to central leptin as well as the associated changes in mRNA levels in hypothalamic and mesolimbic brain areas.

Design and Methods

Rats on a CHOW or fcHFHS diet for 8 days received leptin or vehicle intracerebro(lateral)ventricularly (ICV) and food intake was measured 5 h and 24 h later. Four days later, rats were sacrificed after ICV leptin or vehicle and mRNA levels were quantified for hypothalamic pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) and for preproenkephalin (ppENK) in nucleus accumbens and tyrosine hydroxylase (TH) in ventral tegmental area (VTA).

Results

ICV leptin decreased caloric intake both in CHOW and fcHFHS rats. In fcHFHS, leptin preferentially decreased chow and fat intake. Leptin increased POMC and decreased NPY mRNA in CHOW, but not in fcHFHS rats. In CHOW rats, leptin had no effect on ppENK mRNA and decreased TH mRNA. In fcHFHS, leptin decreased ppENK mRNA and increased TH mRNA.

Conclusion

Despite peripheral and arcuate leptin resistance, central leptin suppresses feeding in fcHFHS rats. As the VTA and nucleus accumbens are still responsive to leptin, these brain areas may therefore, at least partly, account for the leptin-induced feeding suppression in rats on a fcHFHS diet.     

Acute food deprivation and chronic food restriction differentially affect hypothalamic NPY mRNA expression

Although acute food deprivation and chronic food restriction both result in body weight loss, they produce different metabolic states. To evaluate how these two treatments affect hypothalamic peptide systems involved in energy homeostasis, we compared patterns of hypothalamic neuropeptide Y (NPY), agouti-related protein (AgRP), proopiomelanocotin (POMC), and leptin receptor gene expression in acutely food-deprived and chronically food-restricted rats. Both acute food deprivation and chronic food restriction reduced body weight and circulating leptin levels and resulted in increased arcuate NPY and decreased arcuate POMC gene expression. Arcuate AgRP mRNA levels were only elevated in acutely deprived rats. NPY gene expression was increased in the compact subregion of the dorsomedial hypothalamus (DMH) in response to chronic food restriction, but not in response to acute food deprivation. Leptin receptor expression was not affected by either treatment. Double in situ hybridization histochemistry revealed that, in contrast to the situation in the arcuate nucleus, NPY and leptin receptor mRNA-expressing neurons were not colocalized in the DMH. Together, these data suggest that arcuate and DMH NPY gene expression are differentially regulated. DMH NPY-expressing neurons do not appear to be under the direct control of leptin signaling.     

Hypothalamic response to leptin changes during a hormonally induced estrous cycle in rats

The leptin system regulates body fat mass through a feedback loop between adipose tissue and the hypothalamus. To test if leptin responsiveness may be regulated we assayed hypothalamic response to leptin during the estrous cycle; when changes in food intake are known to occur. Immature rats were treated with pregnant mare’s serum gonadotropin (PMSG) to induce synchronized follicular development, ovulation and corpus luteum formation. Leptin response was estimated by measuring the in vitro induction of tis11, a primary response gene activated by STAT3-dependent cytokines in hypothalamic explants after leptin stimulation. In addition, mRNA levels of the suppressor cytokine signaling-3 (SOCS-3), a possible mediator of leptin resistance, were analyzed. Serum leptin levels did not change between days 2 and day 3 (corresponding to proestrus and estrus, respectively) but the response to leptin was higher on day 2 than on day 3 (p=0.05). Food intake displayed a tendency towards downregulation between day 1 and day 2 (p=0.057), and a tendency towards upregulation between day 2 and day 3 (p=0.072), although the body weight increased on day of the study (p<0.0001). There was no significant difference in hypothalamic expression of SOCS-3 between day 2 and day 3. In conclusion, we have shown that leptin responsiveness changes during a hormonally induced estrous cycle in rats. Our data suggest that a change in the hypothalamic response to leptin may cause the cyclic feeding behavior seen in rats.     

Leptin acts centrally to induce the prepubertal secretion of luteinizing hormone in the female rat

Recent data generated from adult male and female rats indicates that leptin is capable of stimulating luteinizing hormone (LH) secretion via a hypothalamic action. Consequently, we hypothesized that this peptide may similarly play a role in controlling LH secretion during late juvenile and peripubertal development; hence, contributing to hypothalamic-pituitary function during sexual maturation. Therefore, this study was conducted to determine if leptin is capable of stimulating LH release during this critical time of development and, if so, to determine whether this action is due to an effect at the hypothalamic level. Results showed that leptin, when administered directly into the brain third ventricle (3V), can stimulate (P < 0. 01) LH release in late juvenile animals at doses of 0.01-1.0 microg. A higher dose of 10 microg was ineffective in stimulating LH release. Immunoneutralization of luteinizing hormone-releasing hormone (LHRH) via 3V administration of LHRH antiserum to late juvenile animals indicated a hypothalamic site of action, since the leptin-induced LH release was blocked in the animals that received anti-LHRH, but not in the control animals that received normal rabbit serum. Leptin did not significantly stimulate LH release from animals in first proestrus, estrus, or diestrus. We also report that the serum levels of leptin increase (P < 0.05) during the late juvenile period of development, then decrease (P < 0.05) once the animal enters the peripubertal period. Collectively, our results show that leptin is capable of acting centrally to stimulate LH release, but only during late juvenile development; thus, we suggest the peptide likely plays a facilitatory role on late juvenile LH secretion, but does not drive the LHRH/LH releasing system to first ovulation and hence, sexual maturity.     

Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons

The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB₁ receptor activity. Here we investigated the possibility of a negative regulation by CB₁ receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2′-chloroethylamide (ACEA) in a CB₁-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL^−/− mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB₁ activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.     

Effects of central and systemic administration of leptin on neurotransmitter concentrations in specific areas of the hypothalamus

Leptin, a hormone produced by adipocytes, has been shown to affect a number of central functions, such as regulation of the hypothalamo-pituitary-adrenal axis, feeding, and body weight regulation. Because hypothalamic monoamines are intricately involved in the regulation of these functions, we hypothesized that leptin may produce its effects by altering the activity of these neurotransmitters. To test this hypothesis, male rats received peripheral (0, 100, or 500 microg ip), or central (0 or 5 microg icv) injections of leptin. The animals were killed 5 h later, and their brains were removed, frozen, and sectioned. Serum was collected to measure leptin and corticosterone by RIA. The paraventricular nucleus (PVN), arcuate nucleus (AN), ventromedial hypothalamus (VMH), dorsomedial dorsal nucleus (DMD), median eminence (ME), and medial preoptic area (MPA) were obtained using Palkovits' microdissection technique, and monoamine concentrations in these areas were determined using HPLC-EC. Intraperitoneal administration of leptin increased serum leptin concentrations in a dose-dependent manner (P < 0.05). Both intraperitoneal and intracerebroventricular administration of leptin decreased serum corticosterone significantly (P < 0.05). Norepinephrine (NE) concentration decreased significantly in the PVN, AN, and VMH after both intraperitoneal and intracerebroventricular administration of leptin (P < 0.05). NE concentrations decreased significantly in the DMN after intracerebroventricular administration of leptin (P < 0.05). Leptin treatment (both ip and icv) decreased dopamine concentrations significantly in the PVN. Serotonin (5-HT) concentration decreased significantly in the PVN after both intraperitoneal and intracerebroventricular injections of leptin and decreased in the VMH only with intracerebroventricular treatment of leptin. Leptin did not affect any of the monoamines in the ME and MPA. These results indicate that both central and systemic administration of leptin can affect hypothalamic monoamines in a region-specific manner, which, in turn, could mediate many of leptin's central and neuroendocrine effects.     

Chronic leptin administration decreases fatty acid uptake and fatty acid transporters in rat skeletal muscle.

Chronic leptin administration reduces triacylglycerol content in skeletal muscle. We hypothesized that chronic leptin treatment, within physiologic limits, would reduce the fatty acid uptake capacity of red and white skeletal muscle due to a reduction in transport protein expression (fatty acid translocase (FAT/CD36) and plasma membrane-associated fatty acid-binding protein (FABPpm)) at the plasma membrane. Female Sprague-Dawley rats were infused for 2 weeks with leptin (0.5 mg/kg/day) using subcutaneously implanted miniosmotic pumps. Control and pair-fed animals received saline-filled implants. Leptin levels were significantly elevated (approximately 4-fold; p < 0.001) in treated animals, whereas pair-fed treated animals had reduced serum leptin levels (approximately -2-fold; p < 0.01) relative to controls. Palmitate transport rates into giant sarcolemmal vesicles were reduced following leptin treatment in both red (-45%) and white (-84%) skeletal muscle compared with control and pair-fed animals (p < 0.05). Leptin treatment reduced FAT mRNA (red, -70%, p < 0.001; white, -48%, p < 0.01) and FAT/CD36 protein expression (red, -32%; p < 0.05) in whole muscle homogenates, whereas FABPpm mRNA and protein expression were unaltered. However, in leptin-treated animals plasma membrane fractions of both FAT/CD36 and FABPpm protein expression were significantly reduced in red (-28 and -34%, respectively) and white (-44 and -56%, respectively) muscles (p < 0.05). Across all experimental treatments and muscles, palmitate uptake by giant sarcolemmal vesicles was highly correlated with the plasma membrane FAT/CD36 protein (r = 0.88, p < 0.01) and plasma membrane FABPpm protein (r = 0.94, p < 0.01). These studies provide the first evidence that protein-mediated long chain fatty acid transport is subject to long term regulation by leptin.     

Hypothalamic neuronal histamine in genetically obese animals: its implication of leptin action in the brain

Leptin regulates feeding behavior and energy metabolism by affecting hypothalamic neuromodulators. The present study was designed to examine hypothalamic neuronal histamine, a recently identified mediator of leptin signaling in the brain, in genetic obese animals. Concentrations of hypothalamic histamine and tele-methylhistamine (t-MH), a major histamine metabolite, were significantly lower in obese (ob/ob) and diabetic (db/db) mice, and Zucker fatty (fa/fa) rats, leptin-deficient and leptin-receptor defective animals, respectively, relative to lean littermates (P < 0.05 for each). A bolus infusion of leptin (1.0 microg) into the lateral ventricle (ilvt) significantly elevated the turnover rate of hypothalamic neuronal histamine, as assessed by pargyline-induced accumulation of t-MH, in ob/ob mice compared with phosphate-buffered saline (PBS) infusions (P < 0.05). However, this same treatment did not affect hypothalamic histamine turnover in db/db mice. In agouti yellow (A(y)/a) mice, animals defective in pro-opiomelanocortin (POMC) signaling, normal levels of histamine, and t-MH were seen in the hypothalamus at 4 weeks of age when obesity had not yet developed. These amine levels in A(y)/a mice showed no change until 16 weeks of age, although the mice were remarkably obese by this time. Infusions of corticotropin releasing hormone (CRH), one of neuropeptide related to leptin signaling, into the third ventricle (i3vt) increased histamine turnover in the hypothalamus of Wistar King A rats (P < 0.05 versus PBS infusion). Infusion of neuropeptide Y (NPY) or alpha-melanocyte stimulating hormone (MSH), a POMC-derived peptide failed to increase histamine turnover. These results indicate that lowered activity of hypothalamic neuronal histamine in ob/ob and db/db mice, and fa/fa rats may be due to insufficiency of leptin action in the brains of these animals. These results also suggest that disruption of POMC signaling in A(y)/a mice may not impact on neuronal histamine. Moreover, CRH but neither POMC-derived peptide nor NPY may act as a signal to neuronal histamine downstream of the leptin signaling pathway.     

Physiological regulation of hypothalamic IL-1beta gene expression by leptin and glucocorticoids: implications for energy homeostasis

Interleukin-1beta (IL-1beta) is synthesized in a variety of tissues, including the hypothalamus, where it is implicated in the control of food intake. The current studies were undertaken to investigate whether hypothalamic IL-1beta gene expression is subject to physiological regulation by leptin and glucocorticoids (GCs), key hormones involved in energy homeostasis. Adrenalectomy (ADX) increased hypothalamic IL-1beta mRNA levels twofold, measured by real-time PCR (P < 0.05 vs. sham-operated controls), and this effect was blocked by subcutaneous infusion of a physiological dose of corticosterone. Conversely, hypothalamic IL-1beta mRNA levels were reduced by 30% in fa/fa (Zucker) rats, a model of genetic obesity caused by leptin receptor mutation (P = 0.01 vs. lean littermates), and the effect of ADX to increase hypothalamic IL-1beta mRNA levels in fa/fa rats (P = 0.02) is similar to that seen in normal animals. Moreover, fasting for 48 h (which lowers leptin and raises corticosterone levels) reduced hypothalamic IL-1beta mRNA levels by 30% (P = 0.02), and this decrease was fully reversed by refeeding for 12 h. Thus leptin and GCs exert opposing effects on hypothalamic IL-1beta gene expression, and corticosterone plays a physiological role to limit expression of this cytokine in both the presence and absence of intact leptin signaling. Consistent with this hypothesis, systemic leptin administration to normal rats (2 mg/kg ip) increased hypothalamic IL-1beta mRNA levels twofold (P < 0.05 vs. vehicle), an effect similar to that of ADX. These data support a model in which expression of hypothalamic IL-1beta is subject to opposing physiological regulation by corticosterone and leptin.     

Regulation of leptin mRNA and protein expression in pituitary somatotropes.

Leptin, the ob protein, regulates food intake and satiety and can be found in the anterior pituitary. Leptin antigens and mRNA were studied in the anterior pituitary (AP) cells of male and female rats to learn more about its regulation. Leptin antigens were found in over 40% of cells in diestrous or proestrous female rats and in male rats. Lower percentages of AP cells were seen in the estrous population (21 +/- 7%). During peak expression of antigens, co-expression of leptin and growth hormone (GH) was found in 27 +/- 4% of AP cells. Affinity cytochemistry studies detected 24 +/- 3% of AP cells with leptin proteins and growth hormone releasing hormone (GHRH) receptors. These data suggested that somatotropes were a significant source of leptin. To test regulatory factors, estrous and diestrous AP populations were treated with estrogen (100 pM) and/or GHRH (2 nM) to learn if either would increase leptin expression in GH cells. To rule out the possibility that the immunoreactive leptin was bound to receptors in somatotropes, leptin mRNA was also detected by non-radioactive in situ hybridization in this group of cells. In estrous female rats, 39 +/- 0.9% of AP cells expressed leptin mRNA, indicating that the potential for leptin production was greater than predicted from the immunolabeling. Estrogen and GHRH together (but not alone) increased percentages of cells with leptin protein (41 +/- 9%) or mRNA (57 +/- 5%). Estrogen and GHRH also increased the percentages of AP cells that co-express leptin mRNA and GH antigens from 20 +/- 2% of AP cells to 37 +/- 5%. Although the significance of leptin in GH cells is not understood, it is clearly increased after stimulation with GHRH and estrogen. Because GH cells also have leptin receptors, this AP leptin may be an autocrine or paracrine regulator of pituitary cell function.     

Stimulation by eicosapentaenoic acids of leptin mRNA expression and its secretion in mouse 3T3-L1 adipocytes in vitro

Recent evidence indicates that both leptin and eicosapentaenoic acids (EPA) improve insulin sensitivity. In the present study, we examined the effect of EPA on endogenous leptin expression in 3T3-L1 adipocytes to clarify whether the EPA's effect is exerted through leptin expression. EPA caused a time- and dose-dependent increase of leptin mRNA levels in 3T3-L1 adipocytes. Leptin mRNA expression was significantly increased up to 309.4 +/- 17.0% of the control by 24 h (P < 0.01; n = 6). Leptin secretion was also significantly increased up to 193.3 +/- 12.1% of the control by 24 h (P < 0.01; n = 6). EPA is a ligand for peroxisome proliferator-activated receptors (PPARs) with the highest affinity to PPARalpha. We examined the effect on leptin expression of clofibrate, a ligand for PPARalpha, bezafibrate, for PPARbeta, or troglitazone, for PPARgamma, to clarify whether these ligands for PPARs could mimic EPA-induced stimulation of leptin expression. Neither clofibrate nor bezafibrate affected leptin mRNA expression, whereas troglitazone significantly suppressed leptin mRNA expression. On the other hand, inhibition by 6-diazo-5-oxo-l-norleucine of the rate-limiting enzyme in hexosamine biosynthesis blunted EPA-induced stimulation of leptin mRNA expression and its secretion. These data suggest that EPA up-regulates leptin gene expression and its secretion probably through a hexosamine biosynthetic pathway.     

Chronic exercise lowers the defended body weight gain and adiposity in diet-induced obese rats

The effects of running wheel exercise and caloric restriction on the regulation of body weight, adiposity, and hypothalamic neuropeptide expression were compared in diet-induced obese male rats over 6 wk. Compared with sedentary controls, exercising rats had reduced body weight gain (24%), visceral (4 fat pads; 36%) and carcass (leptin; 35%) adiposity but not insulin levels. Hypothalamic arcuate nucleus (ARC) proopiomelanocortin (POMC) mRNA expression was 25% lower, but ARC neuropeptide Y (NPY), agouti- related peptide, dorsomedial nucleus (DMN) NPY, and paraventricular nucleus (PVN) corticotropin- releasing hormone (CRH) expression was comparable to controls. Sedentary rats calorically restricted to 85% of control body weight reduced their visceral adiposity (24%), leptin (64%), and insulin (21%) levels. ARC NPY (23%) and DMN NPY (60%) were increased, while ARC POMC (40%) and PVN CRH (14%) were decreased. Calorically restricted exercising rats an half as much as ad libitum-fed exercising rats and had less visceral obesity than comparably restricted sedentary rats. When sedentary restricted rats were refed after 4 wk, they increased intake and regained the weight gain and adiposity of sedentary controls. While refed exercising rats and sedentary rats ate comparable amounts, refed exercising rats regained weight and adiposity only to the level of ad libitum-fed exercising rats. Thus exercise lowers the defended level of weight gain and adiposity without a compensatory increase in intake and with a very different profile of hypothalamic neuropeptide expression from calorically restricted rats. This may be due to exercise-related factors other than plasma insulin and leptin.     

AAV-mediated leptin receptor installation improves energy balance and the reproductive status of obese female Koletsky rats

Leptin is a hormone secreted primarily by white adipocytes that regulates energy homeostasis and reproduction via CNS receptors. Koletsky (f/f) rats with a leptin receptor (OB-Rb) gene mutation are obese, diabetic and infertile. We employed recombinant adeno-associated viral (rAAV) vectors to transfer the human OB-Rb gene into the brains of female Koletsky rats to identify sites of leptin action in the brain. rAAV-OB-Rb was microinjected into the medial preoptic area (MPOA), the paraventricular nucleus (PVN), the ventromedial hypothalamus, the arcuate nucleus (ARC), or the dorsal vagal complex in the brainstem. Food intake and body weight were monitored bi-weekly for 55 days. Vaginal cytology was examined daily to assess estrous cyclicity. After sacrifice, uncoupling protein-1 (UCP-1) mRNA in brown adipose tissue and serum concentrations of leptin, insulin, glucose, estradiol and progesterone were measured. Expression of OB-Rb was documented by RT-PCR and site specificity of microinjection was verified by immunohistochemical detection of green fluorescent protein following a control microinjection of rAAV-GFP. OB-Rb installation in the ARC reduced food intake, however, energy expenditure, assessed by UCP-1 mRNA expression, was increased by OB-Rb installation in all sites except the PVN. When injected into the MPOA and ARC, rAAV-OB-Rb stimulated the reproductive axis as evidenced by normalization of estrous cycle length and increased luteinizing hormone releasing hormone concentrations in the hypothalamus. These studies show that long-term installation of a functional leptin receptor in the CNS is achievable using rAAV vectors and further show that leptin acts on specific sites in the brain to produce differential effects on food intake, energy expenditure and reproduction.