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The aim of this study was to evaluate the effect of leptin administration on the ventral prostate lobe of adult rat. Twenty adult male rats were divided into 2 groups: L-animals were daily injected with 50 μL of leptin (8 μg/100 g BW, subcutaneous) for four days and C-animals received the same volume of saline solution. Lipid profile and testosterone serum levels were evaluated. The prostate ventral lobe was processed for histomorphometric analysis. Gene expression of aromatase, androgen, leptin and estrogen receptors isoforms was evaluated by real-time PCR. Cell proliferation was evaluated by PCNA immunohistochemistry. Data were expressed as mean±standard error and analyzed by student's t-test. Serum levels of cholesterol (C=39.7±4.2;L=55.2±4.2, mg/dL; P<0.02) increased and testosterone (C=1.6±0.43;L=0.6±0.15, ng/dL; P<0.03) decreased in L group. The histomorphometric analysis showed a reduction in cell density (C=8868±242; L=8211±210, mm(2); P<0.04), in total (C=0.24±0.026; L=0.10±0.009, mm(2); P<0.001) and in the internal acini areas (C=0.16±0.009; L=0.08±0.006, mm(2); P<0.0002). On the other hand, there was an increase in the epithelial height (C=17.3±0.3; L=22.8±0.2, μm; P<0.0001) and in the number of acini (C=7.0±0.2; L=8.7±0.1, mm(2); P<0.0002). The histomorphometric analyses together with PCNA immunohistochemistry results suggest that leptin increases cell proliferation. In relation to the gene expression, leptin treatment increased the expression of all genes, but ER-α, in more than 200 times compared to the expression in C group. In conclusion, in this paper we showed that leptin has a direct effect on the prostate gland of adult rats leading to an increase in proliferation and in the gene expression of aromatase, androgen, leptin and estrogen receptors isoforms that are important for the physiology of the prostate gland., (Copyright © 2012. Published by Elsevier B.V.)

Centrally acting leptin induces the activation of the sympathetic nervous system with a pressor effect in normotensive rats. The purpose of the study was to examine central leptin-evoked action in critical haemorrhagic hypotension. In anaesthetized male Wistar rats subjected for irreversible haemorrhagic shock with mean arterial pressure (MAP) 20-25 mmHg haemodynamic parameters and plasma concentrations of adrenaline and noradrenaline were measured. Leptin given intracerebroventricularly (20 μg) evoked long-lasting rises in MAP and heart rate (HR), with a subsequent increase in renal, mesenteric and hindquarters blood flows and a 100% survival at 2 h. MAP and peripheral blood flow changes were inhibited by a pre-treatment with α(1)- and α(2)-adrenoceptor antagonists prazosin (0.5 mg/kg) and yohimbine (1 mg/kg), while β-adrenoceptor antagonist propranolol (1 mg/kg) blocked leptin-induced HR changes, without influence on MAP, peripheral blood flows and survival. Twenty min after leptin treatment, there were higher plasma concentrations of noradrenaline, but not adrenaline, in comparison with the saline-treated control group. In conclusion, centrally acting leptin induces a long-lasting pressor effect with an improvement in the survival rate in haemorrhage-shocked rats. The effect may be associated with the activation of the sympathetic nervous system., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We showed that neonatal leptin treatment programmes for hyperleptinemia and central leptin resistance both at 30days-old and adulthood, while programmes for lower serum T3 at 30days-old, but higher thyroid hormones (TH) at adulthood. As in these animals, acute cold at 30days-old normalized leptinemia and restored the expression of hypothalamic leptin receptor (OBR), here we evaluate the effect of cold exposure on the thyroid function and OBR in adult rats programmed by neonatal hyperleptinemia. Pups were divided into 2 groups: Lep-injected with leptin (8μg/100g/BW, sc) for the first 10days of lactation, and C-injected with saline. At 150days, both groups were subdivided into: LepC and CC, which were exposed to 8°C for 12h. Serum leptin, TH, TSH, liver type I and brown adipose tissue (BAT) type II deiodinases (D1 and D2) activities, liver mitochondrial alpha-glycerol-3-phosphate dehydrogenase (mGPD) activity and adrenal catecholamine content were measured. Hypothalamic and thyroid OBR protein contents were evaluated. Differences were significant when p<0.05. Lep group had hyperleptinemia (+19%), higher T4 (+20%) and T3 (+30%) with lower TSH (-55%), higher liver D1 (1.4 fold-increase), lower BAT D2 (-44%) and liver mGPD activities (-55%), higher adrenal catecholamines (+44%), lower hypothalamic OBR (-51%) and normal thyroid OBR. Cold exposure normalized leptinemia, D1, mGPD, catecholamine and hypothalamic OBR. However, cold exposure further increased TH and decreased D2. Thus, cold exposure normalizes most of the changes programmed by neonatal hyperleptinemia, at the expense of worsening the hyperthyroidism and BAT thermogenesis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Using a synthetic peptide strategy, we localized an active domain in mouse leptin to a sequence between amino acids 106 and 140. Intraperitoneal (ip) administration of a number of synthetic peptide amides encompassed by this domain reduced body weight gain, food and water intake, blood glucose levels, and increased insulin sensitivity in genetically obese mice. In the present study, we examined the pharmacokinetics of mouse [D-Leu-4]OB3, our most potent peptide, in male Swiss Webster mice following ip, subcutaneous (sc), and intramuscular (im) injection, and after intranasal administration with Intravail, a new class of patented transmucosal delivery enhancement agents. Total uptake (1,072,270, 1,182,498; 1,481,060; ng/ml/min), serum half-life (48.8; 34.0; 30.0 min) and relative bioavailability (1.0, 1.1; 1.4;) of mouse [D-Leu-4]OB3 were similar when the peptide was given by ip, sc, or im injection, respectively. Total uptake and relative bioavailability were enhanced following intranasal delivery (4,336,963 ng/ml/min and 4.0, respectively), and serum half-life was 41.1 min. These results indicate that intranasal delivery of mouse [D-Leu-4]OB3 with Intravail is a more effective method of peptide administration than injection methods, and suggest that it may have potential as a novel, non-invasive approach to the treatment of obesity and its associated metabolic dysfunctions in humans.

We examined the possibility that chronic, low-dose peripheral leptin infusion would inhibit food intake but not increase blood pressure. Male Fisher Brown Norway (FBNF1) and Sprague-Dawley (SD) rats were instrumented for cardiovascular telemetry, housed in metabolic chambers, and given leptin (LEP: 600 microg/kg/day) or vehicle (SAL: 10 microl/h) via a subcutaneous osmotic pump for seven days. Leptin infusion increased plasma leptin levels to about 40 ng/ml, decreased food intake by 25-35% and stimulated lipolysis in both strains of rats. Leptin infusion for one week decreased mean arterial pressure from baseline. The reduction developed slowly, was generally about 3 to 7 mm Hg, and observed in both strains. The peripheral, hypotensive effect of chronic leptin in FBNF1 rats was prevented by blockade of nitric oxide production with L-NAME treatment. These results indicate that peripheral leptin treatment, at a level which inhibits food intake and induces lipolysis, produces nitric oxide-dependent decreases in blood pressure.

Leptin, a hormone mainly generated by adipocytes, acts centrally in the hypothalamus to regulate body weight and energy expenditure. However, there is strong evidence that leptin is also involved in cell-mediated immunity and cytokine crosstalk. In the present study the effects of diet-induced obesity and central and peripheral leptin treatment on leukocyte subsets and cytokine production was investigated. Leptin was injected either intravenously (i.v.) or intracerebroventricularly (i.c.v.) in male endotoxaemic or vehicle-treated healthy LEW-rats. Numbers of blood leukocyte subsets were analysed by FACS and cytokines (TNF-alpha and IL-6) by ELISA. Results showed that peripheral rather than central leptin treatment was able to significantly increase numbers of granulocytes, NK cells and monocytes. Three-colour staining revealed that the increase of ED9(+) monocytes was most likely due to the mobilization of two distinct monocyte subsets, predominantly ED9(+)CD4(-)NKR-P1A(+) and ED9(+)CD4(+)NKR-P1A(+). ELISA analysis revealed significantly elevated TNF-alpha levels in obese animals compared to their lean littermates, while IL-6 failed to show notable changes. In conclusion, the data of the present study revealed that leptin application induces a nutrition- and application-site dependent increase of circulating NK cells, granulocytes and specific monocyte subsets.

We have previously shown that the activity of a synthetic peptide amide corresponding to amino acid residues 116-130 of mouse leptin is contained in a restricted sequence at the amino terminus of the peptide, between residues 116 and 122 (Ser-Cys-Ser-Leu-Pro-Glu-Thr). This peptide was named mouse OB3. The potency of OB3 was improved by replacing the L-leucine residue at position four with its D-isomer. Intraperitoneal administration (i.p.) of mouse OB3 or [D-Leu-4]OB3 to ob/ob and db/db mice reduces food intake, body weight gain and serum glucose levels, and enhances insulin sensitivity. These effects of OB3 and [D-Leu-4]OB3 are very pronounced in young mice and diminish with age. In the present study, we measured uptake, serum half-life, and bioavailability of mouse [D-Leu-4]OB3 in mice of different ages. Groups of male C57BL/6J mice, six and 25 weeks of age, were given a single i.p. injection of 1 mg mouse [D-Leu-4]OB3 in PBS. Five, 10, 20, 40, 60, 120, or 180 min after injection, the mice were anesthetized and exsanguinated. Serum samples were prepared and assayed for mouse [D-Leu-4]OB3 content by competitive ELISA. In six week-old mice, the maximum concentration of mouse [D-Leu-4]OB3 was reached in 10 min, and the serum half-life was approximately 52.5 min. In 25 week-old mice, however, mouse [D-Leu-4]OB3 peaked in 5 min, and the serum half-life was approximately 30.6 min. The relative bioavailability of mouse [D-Leu-4]OB3 in six and 25 week-old mice was determined by measuring the area under the uptake curves. Bioavailability of mouse [D-Leu-4]OB3 was approximately 20% greater in six week-old mice than in 25 week-old mice. The results of this study indicate that at least some pharmacokinetic parameters of peptide uptake change as mice age. They also suggest that differences in uptake, serum half-life, and relative bioavailability of mouse [D-Leu-4]OB3 may contribute, at least in part, to the reduced efficacy of bioactive leptin-related peptides we have consistently observed in ob/ob and db/db mice as they age.

Leptin presents an important role in energy balance and neuroendocrine control in mammals. In an attempt to identify regions of the leptin molecule responsible for its bioactivity, we have synthesized six peptides based on the protein three-dimensional structure. Fragments were synthesized by the solid-phase methodology, purified by reverse-phase high-performance liquid chromatography (RP-HPLC), and characterized by liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS). They were injected intravenously and their ability to induce Fos immunoreactivity (Fos-ir) in rat hypothalamus was compared with that of the recombinant human leptin and saline. Fragment Ac-[Ser117]Lep116-140-NH2 (V) induced Fos-ir in hypothalamic nuclei that express leptin receptor long form. No similar ability was observed for the other five fragments. To investigate whether Fos-ir was induced in the same neuronal group activated by leptin, we proceeded with a dual-label immunohistochemistry for cocaine- and amphetamine-regulated transcript (CART), a neuropeptide related to leptin action in rat hypothalamus. We found that Ac-[Ser117]Lep116-140-NH2 (V) differentially activates CART neurons through the rostrocaudal extension of the arcuate nucleus. These results suggest that this fragment acts in the same group of neurons that mediate leptin response. This approach may offer the basis for the development of leptin-related compounds, having potential application in human or veterinary medicine.

Because of connections between CART peptide containing neurons and the sympathetic nervous system (SNS) and the possible role of the SNS in leptin-induced adipose apoptosis, CART may act as a downstream effector of leptin-induced adipose apoptosis. Male Sprague-Dawley rats received continuous intracerebroventricular (i.c.v.) infusion for 4 days of either artificial cerebrospinal fluid (aCSF, 12 microl/day), leptin (15 microg/day), or CART55-102 at 2.4 microg/day (CART2.4) or 9.6 microg/day (CART9.6). Food intake (FI) was decreased 10.8% for CART2.4, 41.9% for CART9.6 and 33.4% for leptin (p<0.05). CART9.6 and leptin reduced meal size and meal number. Body weight (BW) was reduced by CART9.6 (14.6%) and leptin (11.6%) (p<0.05), but not by CART2.4. CART9.6 and CART2.4, but not leptin, caused hypothermia, and CART9.6 inhibited physical activity (p<0.05). Epididymal, inguinal and retroperitoneal fat pad weights were reduced (p<0.05) by both CART treatments and leptin; CART9.6 also reduced gastrocnemius muscle weight (18.1%, p<0.05). Leptin, but not CART, increased serum free fatty acid concentrations by 31.1% (p<0.05) and increased adipose apoptosis by 48% (p<0.05). These data show that although leptin and CART55-102 have some similar actions, CART55-102 is probably not a mediator for leptin-induced adipose apoptosis in the brain., (Copyright 2004 Elsevier B.V.)

We investigated the effect of leptin on systemic nitric oxide (NO) production, arterial pressure, renal hemodynamics and renal excretory function in the rat. Leptin (1 mg/kg) was injected intravenously and mean arterial pressure (MAP), heart rate (HR), renal blood flow (RBF) and renal cortical blood flow (RCBF), were measured for 210 min after injection. Urine was collected for seven consecutive 30-min periods and blood samples were withdrawn at 15, 45, 75, 105, 135, 165 and 195 min after leptin administration. Leptin had no effect on MAP, HR, RBF, RCBF and creatinine clearance, but increased urine output by 37.8% (0-30 min), 32.4% (31-60 min) and 27.0% (61-90 min), as well as urinary sodium excretion by 175.8% (0-30 min), 136.4% (31-60 min) and 124.2% (61-90 min). In contrast, leptin had no effect on potassium and phosphate excretion. Plasma concentration of NO metabolites, nitrites + nitrates (NOx), increased following leptin injection at 15, 45, 75 and 105 min by 27.7%, 178.1%, 156.4% and 58.7%, respectively. Leptin increased urinary NOx excretion by 241.6% (0-30 min), 552.6% (31-60 min), 430.7% (61-90 min) and 88.9% (91-120 min). This was accompanied by increase in plasma and urinary cyclic GMP. These data indicate that leptin stimulates systemic NO production but has no effect on arterial pressure and renal hemodynamics.

We have recently shown that the activity of a synthetic peptide corresponding to amino acid residues 116-130 of secreted mouse leptin is contained in a restricted sequence at the amino terminus of the peptide, between residues 116-122 (Ser-Cys-Ser-Leu-Pro-Gln-Thr, OB3). Substitution of the Leu residue at position 4 of OB3 with its D-isomer ([D-Leu-4]-OB3) enhanced the ability of OB3 (1 mg/day, ip, 7 days) to reduce body weight gain, food and water intake, and serum glucose in female C57BL/6J ob/ob mice. In the present study, we have utilized a pair-feeding approach to demonstrate that the antihyperglycemic action of [D-Leu-4]-OB3 is not solely due to its effects on caloric intake. One group of female C57BL/6J ob/ob mice (n=6) was fed ad libitum, and two additional groups (n=6 per group) were allowed 3.0 g food/mouse daily, an amount previously determined to satisfy [D-Leu-4]-OB3-treated mice. At the end of the 7-day test period, vehicle-injected mice fed ad libitum were approximately 10% heavier than their initial body weights, while pair-fed mice injected with vehicle and [D-Leu-4]-OB3-treated mice lost 5% of their initial body weights. After 1 day of treatment, blood glucose was reduced by 20% in pair-fed vehicle-injected mice, and by 40% in mice given [D-Leu-4]-OB3. Food restriction reduced blood glucose throughout the 7-day study, but not to levels seen in wild-type nonobese C57BL/6J mice of the same sex and age. After 2 days of treatment with [D-Leu-4]-OB3, however, blood glucose was reduced to levels comparable to those seen in wild-type nonobese mice. [D-Leu-4]-OB3 also lowered serum insulin levels by 53% when compared to mice fed ad libitum. Neither pair-feeding nor [D-Leu-4]-OB3 treatment had any apparent effect on thermogenesis. These results suggest that [D-Leu-4]-OB3 exerts its effects on serum glucose not only by suppressing caloric intake, but also through a separate effect on glucose metabolism which may involve increased tissue sensitivity to insulin.

The weight-reducing effects of leptin are predominantly mediated through the hypothalamus in the brain. Gene therapy strategies designed for weight control have so far tested the short-term effect of peripherally delivered viral vectors encoding the leptin gene. In order to circumvent the multiple peripheral effects of hyperleptinemia and to overcome the age-related development of leptin resistance due to multiple factors, including defective leptin transport across the blood brain barrier, we determined whether delivery of viral vectors directly into the brain is a viable therapeutic strategy for long-term weight control in normal wild-type rats. A recombinant adeno-associated virus (rAAV) vector encoding rat leptin (Ob) cDNA was generated (rAAV-betaOb). When administered once intracerebroventricularly (i.c.v.), rAAV-betaOb suppressed the normal time-related weight gain for extended periods of time in adult Sprague-Dawley rats. The vector expression was confirmed by immunocytochemical localization of GFP and RT-PCR analysis of leptin in the hypothalamus. This sustained restraint on weight gain was not due to shifts in caloric consumption because food-intake was similar in rAAV-betaOb-treated and rAAV-GFP-treated control rats throughout the experiment. Weight gain suppression, first apparent after 2 weeks, was a result of reduced white fat depots and was accompanied by drastically reduced serum leptin and insulin concentrations in conjunction with normoglycemia. Additionally, there was a marked increase in uncoupling protein-1 (UCP1) mRNA expression in brown adipose tissue, thereby indicating increased energy expenditure through thermogenesis. Seemingly, a selective enhancement in energy expenditure following central delivery of the leptin gene is a viable therapeutic strategy to control the age-related weight gain and provide protection from the accompanying multiple peripheral effects of hyperleptinemia and hyperinsulinemia.

Leptin has been proposed to be a sensor of energy storage in adipose tissues, and is capable of mediating a feedback signal to the hypothalamus, which is involved in the regulation of energy homeostasis and body weight. In order to investigate the issue of whether resistance to the activity of leptin on insulin sensitivity is observed in young Otsuka Long-Evans Tokushima Fatty (OLETF) rats at 8 weeks of age, leptin (50 nmol/kg/h) was administered intravenously for 16 h to OLETF and Long-Evans Tokushima Otsuka (LETO) (lean controls) rats, followed by a measurement of insulin-stimulated glucose uptake in hindlimb muscles during hyperinsulinemic euglycemic clamp technique. In the case of LETO rats, the administration of leptin significantly decreased plasma insulin levels prior to the clamp test, but did not change plasma glucose levels. Furthermore, leptin led to an increase in insulin-stimulated glucose uptake in hindlimb muscles. However, in the case of OLETF rats, leptin administration changed neither plasma insulin levels nor insulin-stimulated glucose uptake. These data demonstrate that OLETF rats at 8 weeks of age have already become resistant to high concentration of peripheral leptin.

Leptin administered (either intracerebroventricularly, icv, or intraperitoneally, ip) acts in synergy with CCK to suppress food intake and body weight in lean mice or rats. The potentiating effect induced by the co-injection of ip CCK and leptin to inhibit food consumption in mice is mediated by the CCK-A receptor and capsaicin sensitive afferents. In vitro, studies in rats showed that a subset of gastric vagal afferent fibers responded to leptin injected directly into the gastric artery only after a prior intra-arterial CCK injection. Moreover, the tonic activity of gastric-related neurons in the nucleus tractus solitarius (NTS) increased when leptin was delivered into the gastric chamber of an in vitro stomach-brainstem preparation. CCK co-injected with leptin potentiated Fos expression selectively in the area postrema, NTS and paraventricular nucleus of the hypothalamus (PVN), which points to the PVN as part of the afferent and efferent limbs of the circuitry involved in the synergistic interaction between leptin and CCK. The dampening of CCK or leptin inhibitory action on ingestive behavior when either factor is not present or their receptors are non functional supports the notion that such leptin-CCK interaction may have a physiological relevance. These observations provide a mean through which leptin and CCK integrate short- and mid-term meal-related input signals into long-term control of energy balance.

Leptin, the product of the obese gene, is a circulating hormone secreted primarily from adipocytes. The lack of leptin in ob/ob mice, who are homozygous for the obese gene, results in hyperglycemia, hyperinsulinemia, hyperphagia, obesity, infertility, decreased brain size and decreased stature. To this end, we investigated the role of leptin as a hormonal regulator of bone growth. Leptin administration led to a significant increase in femoral length, total body bone area, bone mineral content and bone density in ob/ob mice as compared to vehicle treated controls. The increase in total body bone mass was a result of an increase in both trabecular and cortical bone mass. These results suggest that the decreased stature of the ob/ob mouse is due to a developmental defect that is readily reversible upon leptin administration. Our demonstration that the signalling or long form (Ob-Rb) of the leptin receptor is present in both primary adult osteoblasts and chondrocytes suggests that the growth promoting effects of leptin could be direct. In summary, these results indicate a novel role for leptin in skeletal bone growth and development.

The aim of the present study was to determine whether the anorexic and thermogenic effects of leptin were attenuated in overweight aged rats following intracerebroventricular (i.c.v.) injection of murine leptin. Male F344/BN rats of two ages (6 months: young (n=20) and 24 months: old (n=18)) were divided into three groups (control, pair-fed and leptin) and were treated with either vehicle (artificial cerebrospinal fluid) or leptin (15.6 microgram/day) for 3 days. There was an age-related increase in basal food intake (20+/-2%), serum leptin levels (363+/-106%) and leptin (OB) mRNA (72+/-16%) in perirenal white adipose tissue (PWAT). In contrast, basal expression of hypothalamic NPY mRNA and brown adipose tissue (BAT) uncoupling protein 1 (UCP1) mRNA was reduced significantly (-35+/-4% and -51+/-5%, respectively) with age. I.c.v. leptin treatment had a significantly greater effect in reducing food intake (-42+/-5% vs. -23+/-4%), serum leptin levels (-55+/-7% vs. 10+/-2%) and PWAT OB mRNA (-46+/-2% vs. 10+/-5%) in young than in old rats. Similarly, central leptin treatment also had a greater effect in suppressing hypothalamic NPY mRNA expression in young (-23+/-4%) than in old (-8+/-4%) rats compared with their age-matched pair-fed treated rats. The stimulatory effect of i.c.v. leptin treatment on BAT UCP1 mRNA expression was also significantly greater in young rats (45+/-8%) than in old rats (10+/-6%) compared with age-matched pair-fed rats. Our previous report indicated that these overweight aged rats were resistant to peripheral administered leptin. The present data extend those findings and demonstrate that the impaired anorexic and metabolic effects of leptin are centrally mediated. This leptin resistance may be due to either the elevated obesity and serum leptin with age or due to age itself or both. The development of leptin resistance with age may contribute to the hyperphagia, hyperleptinemia and impaired energy balance with age.

Leptin is produced mainly by adipose tissue and has been shown to regulate feeding, energy balance and neuroendocrine function. Regulation of energy homeostasis by leptin is thought to be mediated by hypothalamic neuropeptides, at least in adult rodents. The neonatal period is a critical stage of development during which mammals have to optimize caloric intake to support growth and development, as well as maintain body temperature. It is likely that leptin is involved in the transition from preweaning to adult metabolism. To test this hypothesis, we compared the effect of leptin treatment on body weight and adiposity between neonatal and adult mice. We also determined whether well known hypothalamic neuropeptide targets, e.g. neuropeptide Y (NPY), proopiomelanocortin (POMC), agouti-related peptide (AGRP) and cocaine and amphetamine-regulated transcript (CART) were regulated in a pattern consistent with their presumed roles as mediators of leptin action. Once daily intraperitoneal leptin injection for 7 days did not alter body weight, fat content or expression of hypothalamic neuropeptide mRNAs in 10-day-old mice. In contrast, leptin decreased body weight and adiposity, increased CART and suppressed NPY and AGRP mRNA expression in adult mice. These results are consistent with previous studies showing that the timing of leptin's anorectic action develops after weaning. Furthermore, the association between leptin's ability to influence body weight in adult mice but not in neonates, and the regulation of hypothalamic neuropeptide mRNA expression, is consistent with the view that these peptides mediate leptin's effects on energy balance.

Leptin is a hormone synthesized and secreted from adipose tissue. To study the physiologic effects of chronic leptin treatment, normal adult female Sprague-Dawley rats were injected subcutaneously for 35 days. Twice daily injections (250 microgram/day, b.i.d.) resulted in a significant (P<0.05) decrease in food intake that was maintained for 10 days before gradually returning to control level by day 21. Leptin decreased body weight by a maximum of 12% of the initial body weight on day 22 and remained reduced for the duration of the treatment. After 35 days of treatment, visible peritoneal adipose tissue was not detected. Body composition analysis showed that chronic injection of leptin resulted in a dramatic decrease in fat content (28+/-2 to 4+/-2 g, P<0.05; mean+/-SEM) while the lean content remained unchanged. Rats pair-fed to the leptin-treated group but treated with vehicle had the same body composition (23+/-3 g fat mass) as that measured for the ad libitum fed controls. Using indirect calorimetry we observed that leptin decreased respiratory quotient and thus increased fat oxidation. Leptin also prevented energy expenditure reduction typically associated with food restriction. Leptin treatment for 35 days decreased plasma triglyceride (0.75+/-0.07 to 0.30+/-0.03 mM, P<0.05), free fatty acid (0.56+/-0.06 to 0.32+/-0.04 mM) and insulin (3.2+/-0.5 to 1. 4+/-0.4 ng/ml, P<0.05) concentrations despite the fact that food intake was normalized by day 35. Withdrawal of leptin triggered hyperphagia indicating that leptin biology remained throughout the duration of the chronic treatment. These data suggest that leptin reduces fat mass by initially decreasing appetite and by maintaining enhanced fat utilization even when food intake has returned to that of vehicle-treated control.