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The role of central leptin in regulating the heart from lipid accumulation in lean leptin-sensitive animals has not been fully elucidated. Herein, we investigated the effects of central leptin infusion on the expression of genes involved in cardiac metabolism and its role in the control of myocardial triacylglyceride (TAG) accumulation in adult Wistar rats. Intracerebroventricular (icv) leptin infusion (0.2 µg/day) for 7 days markedly decreased TAG levels in cardiac tissue. Remarkably, the cardiac anti-steatotic effects of central leptin were associated with the selective upregulation of gene and protein expression of peroxisome proliferator-activated receptor β/δ (PPARβ/δ, encoded by Pparb/d ) and their target genes, adipose triglyceride lipase (encoded by Pnpla2 , herefater referred to as Atgl ), hormone sensitive lipase (encoded by Lipe , herefater referred to as Hsl ), pyruvate dehydrogenase kinase 4 ( Pdk4 ) and acyl CoA oxidase 1 ( Acox1 ), involved in myocardial intracellular lipolysis and mitochondrial/peroxisomal fatty acid utilization. Besides, central leptin decreased the expression of stearoyl-CoA deaturase 1 ( Scd1 ) and diacylglycerol acyltransferase 1 ( Dgat1 ) involved in TAG synthesis and increased the CPT-1 independent palmitate oxidation, as an index of peroxisomal β-oxidation. Finally, the pharmacological inhibition of PPARβ/δ decreased the effects on gene expression and cardiac TAG content induced by leptin. These results indicate that leptin, acting at central level, regulates selectively the cardiac expression of PPARβ/δ, contributing in this way to regulate the cardiac TAG accumulation in rats, independently of its effects on body weight., (© 2018 Society for Endocrinology.)

Starvation induces low bone mass and high bone marrow adiposity in humans, but the underlying mechanisms are poorly understood. The adipokine leptin falls in starvation, suggesting that hypoleptinemia may be a link between negative energy balance, bone marrow fat accumulation, and impaired skeletal acquisition. In that case, treating mice with leptin during caloric restriction (CR) should reduce marrow adipose tissue (MAT) and improve bone mass. To test this hypothesis, female C57Bl/6J mice were fed a 30% CR or normal (N) diet from 5 to 10 weeks of age, with daily injections of vehicle (VEH), 1mg/kg leptin (LEP1), or 2mg/kg leptin (LEP2) (N=6-8/group). Outcomes included body mass, body fat percentage, and whole-body bone mineral density (BMD) via peripheral dual-energy X-ray absorptiometry, cortical and trabecular microarchitecture via microcomputed tomography (μCT), and MAT volume via μCT of osmium tetroxide-stained bones. Overall, CR mice had lower body mass, body fat percentage, BMD, and cortical bone area fraction, but more connected trabeculae, vs N mice (P<0.05 for all). Most significantly, although MAT was elevated in CR vs N overall, leptin treatment blunted MAT formation in CR mice by 50% vs VEH (P<0.05 for both leptin doses). CR LEP2 mice weighed less vs CR VEH mice at 9-10 weeks of age (P<0.05), but leptin treatment did not affect body fat percentage, BMD, or bone microarchitecture within either diet. These data demonstrate that once daily leptin bolus during CR inhibits bone marrow adipose expansion without affecting bone mass acquisition, suggesting that leptin has distinct effects on starvation-induced bone marrow fat formation and skeletal acquisition., Competing Interests: Declaration of Interest The authors declare no conflicts of interest., (© 2016 Society for Endocrinology.)

Mammals meet the increased nutritional demands of lactation through a combination of increased feed intake and a collection of adaptations known as adaptive metabolism (e.g., glucose sparing via insulin resistance, mobilization of endogenous reserves, and increased metabolic efficiency via reduced thyroid hormones). In the modern dairy cow, adaptive metabolism predominates over increased feed intake at the onset of lactation and develops concurrently with a reduction in plasma leptin. To address the role of leptin in the adaptive metabolism of early lactation, we asked which adaptations could be countered by a constant 96-h intravenous infusion of human leptin (hLeptin) starting on day 8 of lactation. Compared to saline infusion (Control), hLeptin did not alter energy intake or milk energy output but caused a modest increase in body weight loss. hLeptin reduced plasma glucose by 9% and hepatic glycogen content by 73%, and these effects were associated with a 17% increase in glucose disposal during an insulin tolerance test. hLeptin attenuated the accumulation of triglyceride in the liver by 28% in the absence of effects on plasma levels of the anti-lipolytic hormone insulin or plasma levels of free fatty acids, a marker of lipid mobilization from adipose tissue. Finally, hLeptin increased the plasma concentrations of T4 and T3 by nearly 50% without affecting other neurally regulated hormones (i.e., cortisol and luteinizing hormone (LH)). Overall these data implicate the periparturient reduction in plasma leptin as one of the signals promoting conservation of glucose and energy at the onset of lactation in the energy-deficient dairy cow., (© 2016 Society for Endocrinology.)

Leptin (Lep) is an anorexigenic hormone and regulates appetite-related neuropeptides in mammals. A number of neuropeptides have also been linked to appetite regulation in teleost fish, but Lep signaling activation and effects on appetite-regulating neurons are poorly elucidated in early vertebrates. This study uses cellular, tissue and organismal approaches to elucidate the acute, central Lep action in rainbow trout. The results demonstrate that Lep activates phosphorylation of protein kinase B (Akt) and signal transducer and activator of transcription 3 in rainbow trout hypothalamus-derived cells, and that the phosphatidylinositol-3-kinase (Pi3k) inhibitor LY294002 can suppress the Lep-induced Akt phosphorylation. Intracerebroventricular (ICV) Lep administration strongly suppresses food intake at the doses of 0.05 and 0.5 µg Lep fish(-1) At low dose, Lep stimulates hypothalamic transcription of anorexigenic cocaine- and amphetamine-regulated transcript (Cart) and orexigenic neuropeptide Y. At high dose, Lep stimulates hypothalamic transcription of anorexigenic proopiomelanocortin (Pomc) A1, A2, and B, while coinjection with LY294002 reverses this upregulation. The data suggest that the anorexigenic action of Lep in rainbow trout is mediated through stimulation of the anorexigenic neuropeptides Pomc and Cart. Furthermore, ICV Lep treatment increases phosphor-Akt-immunoreactive cells in the nucleus lateralis tuberis, periventricular zone along infundibulum, and lateral recess surrounded by nucleus anterior tuberis, while LY294002 inhibits this effect. Lep receptor-immunoreactive cells are also predominant in these regions. These results demonstrate that Lep activates the Pi3k-Akt pathway in the lateral tuberal hypothalamus of rainbow trout for acute appetite regulation, indicating the conservation of anorexigenic Lep action in the mediobasal hypothalamus., (© 2016 Society for Endocrinology.)

Ghrelin, a stomach-derived orexigenic peptide, transmits starvation signals to the hypothalamus via the vagus afferent nerve. Peripheral administration of ghrelin does not induce food intake in high fat diet (HFD)-induced obese mice. We investigated whether this ghrelin resistance was caused by dysfunction of the vagus afferent pathway. Administration (s.c.) of ghrelin did not induce food intake, suppression of oxygen consumption, electrical activity of the vagal afferent nerve, phosphorylation of ERK2 and AMP-activated protein kinase alpha in the nodose ganglion, or Fos expression in hypothalamic arcuate nucleus of mice fed a HFD for 12 weeks. Administration of anti-ghrelin IgG did not induce suppression of food intake in HFD-fed mice. Expression levels of ghrelin receptor mRNA in the nodose ganglion and hypothalamus of HFD-fed mice were reduced. Inflammatory responses, including upregulation of macrophage/microglia markers and inflammatory cytokines, occurred in the nodose ganglion and hypothalamus of HFD-fed mice. A HFD blunted ghrelin signaling in the nodose ganglion via a mechanism involving in situ activation of inflammation. These results indicate that ghrelin resistance in the obese state may be caused by dysregulation of ghrelin signaling via the vagal afferent., (© 2015 The authors.)

Intrauterine growth restriction (IUGR) may be accompanied by inadequate thermoregulation, especially in piglets that are not considered to possess any brown adipose tissue (BAT) and are thus entirely dependent on shivering thermogenesis in order to maintain body temperature after birth. Leptin can stimulate heat production by promoting non-shivering thermogenesis in BAT, but whether this response occurs in piglets is unknown. Newborn female piglets that were characterised as showing IUGR (mean birth weight of approximately 0.98 kg) were therefore administered injections of either saline or leptin once a day for the first 5 days of neonatal life. The dose of leptin was 0.5 mg/kg, which is sufficient to increase plasma leptin by approximately tenfold and on the day of birth induced a rapid increase in body temperature to values similar to those of normal-sized 'control' piglets (mean birth weight of ∼1.47 kg). Perirenal adipose tissue was then sampled from all offspring at 21 days of age and the presence of the BAT-specific uncoupling protein 1 (UCP1) was determined by immunohistochemistry and immunoblotting. UCP1 was clearly detectable in all samples analysed and its abundance was significantly reduced in the IUGR piglets that had received saline compared with controls, but was raised to the same amount as in controls in those IUGR females given leptin. There were no differences in gene expression between primary markers of brown and white adipose tissues between groups. In conclusion, piglets possess BAT that when stimulated exogenously by leptin can promote increased body temperature., (© 2014 Society for Endocrinology.)

Obesity is a risk factor for sepsis morbidity and mortality, whereas the hypothalamic-pituitary-adrenal (HPA) axis plays a protective role in the body's defence against sepsis. Sepsis induces a profound systemic immune response and cytokines serve as excellent markers for sepsis as they act as mediators of the immune response. Evidence suggests that the adipokine leptin may play a pathogenic role in sepsis. Mouse endotoxaemic models present with elevated leptin levels and exogenously added leptin increased mortality whereas human septic patients have elevated circulating levels of the soluble leptin receptor (Ob-Re). Evidence suggests that leptin can inhibit the regulation of the HPA axis. Thus, leptin may suppress the HPA axis, impairing its protective role in sepsis. We hypothesised that leptin would attenuate the HPA axis response to sepsis. We investigated the direct effects of an i.p. injection of 2 mg/kg leptin on the HPA axis response to intraperitoneally injected 25 μg/kg lipopolysaccharide (LPS) in the male Wistar rat. We found that LPS potently activated the HPA axis, as shown by significantly increased plasma stress hormones, ACTH and corticosterone, and increased plasma interleukin 1β (IL1β) levels, 2 h after administration. Pre-treatment with leptin, 2 h before LPS administration, did not influence the HPA axis response to LPS. In turn, LPS did not affect plasma leptin levels. Our findings suggest that leptin does not influence HPA function or IL1β secretion in a rat model of LPS-induced sepsis, and thus that leptin is unlikely to be involved in the acute-phase endocrine response to bacterial infection in rats.

Suppressors of cytokine signalling (SOCS) negatively regulate cytokine-induced signalling pathways and may be involved in leptin and prolactin (PRL) interactions. Herein, we examined the effect of PRL on SOCS-3 mRNA expression in pituitary explants and investigated whether leptin could modify the expression of SOCS-3 mRNA in pituitary explants. In the first experiment, we used pituitaries isolated from 16 ewes decapitated in March, May, July and October (four per month). Tissues were cut into 50 mg explants, which were treated with control or medium containing PRL (100 or 300 ng/ml). Incubation was maintained for different time intervals: 0, 60, 120, 180, 240 or 300 min. Real-time PCR was used to measure SOCS-3 mRNA levels. In the second study, we used 24 ewes surgically fitted with third ventricle cannulas (12 were used during the long-day period, and 12 were used during the short-day (SD) period). Each ewe was administered an i.c.v. injection of Ringer-Locke buffer or leptin (0.5 or 1.0 μg/kg body weight). Explants of anterior pituitaries were collected and snap frozen 1 h after injection. Semi-quantitative expression of SOCS-3 mRNA was performed using reverse transcription-PCR. PRL stimulated SOCS-3 expression in the pituitaries collected in March (P<0.05) and May (P<0.01 and P<0.05 for lower and higher doses respectively), inhibited SOCS-3 expression in pituitaries collected in July (P<0.01) and had no effect in pituitaries collected in October. Treatment with leptin increased SOCS-3 expression during the SDs in a dose-dependent manner (P<0.01). The results demonstrated that photoperiod may be involved in leptin and PRL effects on SOCS-3 expression in sheep.

This study was conducted to examine the contributions of central and peripheral leptin to hyperphagia in lactation. Lactating rats were mated at 7-8 weeks of age and housed singly with their litters. In experiment 1, food intakes were significantly (P<0.01) greater (350% on average) in lactation than in non-lactation throughout a day. Cerebrospinal fluid (CSF) leptin levels remained constant despite plasma leptin levels being significantly (P<0.05) greater in non-lactation than in lactation. In experiment 2, CSF leptin levels were not altered by i.v. injections of leptin (0.2 and 0.4 mg/kg body weight) despite that plasma leptin levels were dose dependently (P<0.01) increased. Moreover, i.v. administration of leptin significantly (P<0.05) decreased food intake in non-lactating rats but not in lactating rats. In experiment 3, nocturnal food intakes were temporarily (P<0.05) reduced in non-lactating and lactating rats. I.c.v. administration of a leptin antagonist (15 μg) blocked the reductions of food intakes. I.c.v. administration of leptin (10 μg) significantly (P<0.05) decreased cumulative food intakes during 24 h in both the physiological states. In conclusion, this study has presented new evidence that the hyperphagia of lactating rats could be partly due to depressed sensitivity of neurons contacting blood leptin. In contrast, the responsiveness of leptin receptors contacting CSF leptin may not differ between non-lactating and lactating rats. Furthermore, the levels of CSF leptin remained constant independent of those of blood leptin. Therefore, the expression of hypothalamic leptin receptors contacting CSF could be involved in the difference in food intake between non-lactating and lactating rats.

In the central nervous system of mammals, the gene encoding diazepam-binding inhibitor (DBI) is exclusively expressed in glial cells. Previous studies have shown that central administration of a DBI processing product, the octadecaneuropeptide ODN, causes a marked inhibition of food consumption in rodents. Paradoxically, however, the effect of food restriction on DBI gene expression has never been investigated. Here, we show that in mice, acute fasting dramatically reduces DBI mRNA levels in the hypothalamus and the ependyma bordering the third and lateral ventricles. I.p. injection of insulin, but not of leptin, selectively stimulated DBI expression in the lateral ventricle area. These data support the notion that glial cells, through the production of endozepines, may relay peripheral signals to neurons involved in the central regulation of energy homeostasis.

The adipose tissue hormone leptin and homocysteine (Hcy)-thiolactone are linked to the pathogenesis of atherosclerosis through their interactions with the anti-atherogenic enzyme paraoxonase 1 that has the ability to hydrolyze Hcy-thiolactone and minimizes protein N-homocysteinylation. Here we examined the relationships between hyperleptinemia, Hcy-thiolactonase, and protein N-homocysteinylation in rats. Hyperleptinemia was induced in adult rats by administration of leptin for 7 days (0.25 mg/kg twice daily s.c). We found that serum Hcy-thiolactonase was lower in hyperleptinemic than in control animals (-41.0%, P<0.001). Leptin administration increased the level of N-linked Hcy in plasma proteins (+92.9%, P<0.01), but had no effect on plasma total Hcy. These effects were not reproduced by pair-feeding. We also found that the synthetic liver X receptor (LXR) agonist, T0901317 (1 mg/kg per day) normalized Hcy-thiolactonase and protein N-homocysteinylation levels in leptin-treated rats. However, leptin-induced increase in plasma isoprostane levels (a marker of oxidative stress) was not normalized by T0901317. The NADPH oxidase inhibitor apocynin prevented leptin-induced increase in isoprostane levels but did not normalize Hcy-thiolactonase and protein N-homocysteinylation levels. These results suggest that the decreased capacity to metabolize Hcy-thiolactone and concomitant increase in protein N-homocysteinylation contribute to pro-atherogenic effect of chronic hyperleptinemia, independently of oxidative stress. LXR agonists normalize Hcy-thiolactonase levels and decrease protein N-homocysteinylation, especially under conditions associated with excess leptin such as metabolic syndrome.

Leptin, administered either into the ventricles of the brain or systemically, has been shown to normalize blood glucose concentrations in streptozotocin (STZ)-induced diabetic rats. We hypothesized that an intact sympathetic nervous system is necessary for centrally administered leptin to normalize or attenuate high blood glucose concentrations in STZ-induced diabetic rats. Young male Wistar rats (approximately 50 g) were treated every other day with either s.c. guanethidine (100 mg/kg) or vehicle for 2 weeks. Rats were then implanted with an intracerebroventricular cannula directed to the lateral ventricle and made diabetic with an i.v. injection of STZ (50 mg/kg). Half of the animals in each group were given daily injections of leptin (10 microl), while the remaining animals received vehicle injections. Blood glucose concentrations were measured daily and tissue norepinephrine content was determined by high performance liquid chromatography at the end of the study. Guanethidine pretreatment did not block the ability of centrally administered leptin to decrease blood glucose concentrations in diabetic rats. This suggests that the sympathetic nervous system does not mediate the leptin-induced attenuation of high blood glucose concentrations observed in diabetic rats.

Recent studies have demonstrated photoperiodic changes in leptin sensitivity of seasonal mammals. Herein, we examined the interaction of season (long days (LD) versus short days (SD)) and recombinant ovine leptin (roleptin) on secretion of melatonin and prolactin (PRL) and on mRNA expression of suppressor of cytokine signaling-3 (SOCS-3) in the medial basal hypothalamus (MBH) in sheep. Twenty-four Polish Longwool ewes, surgically fitted with third ventricle (IIIV) cannulas, were utilized in a replicated switchback design involving 12 ewes per season. Within-season and replicate ewes were assigned randomly to one of three treatments (four ewes/treatment) and infused centrally three times at 0, 1 and 2 h beginning at sunset. Treatments were 1) control, Ringer-Locke buffer; 2) L1, roleptin, 0.5 microg/kg BW; and 3) L2, roleptin, 1.0 microg/kg BW. Jugular blood samples were collected at 15-min intervals beginning immediately before the start of infusions and continued for 6 h. At the end of blood sampling, a washout period of at least 3 days elapsed before ewes were re-randomized and treated with one of the treatments described above (four ewes/treatment). Ewes were then killed and brains were collected for MBH processing. Leptin treatments increased (P<0.001) circulating leptin concentrations compared with controls during both seasons in a dose-dependent manner. Overall, mean plasma concentrations of melatonin were greater (P<0.001) during LD than SD. However, leptin treatments increased melatonin concentrations during SD in a dose-dependent manner and decreased it during LD. Similarly, plasma concentrations of PRL were greater (P<0.001) during LD than SD. However, unlike changes in melatonin, circulating PRL decreased (P<0.001) in response to leptin during LD. Semi-quantitative PCR revealed that leptin increased (P<0.001) SOCS-3 expression in the MBH region during LD in a dose-dependent manner. Data provide evidence that secretion of photoperiodic hormones such as melatonin and PRL are inversely regulated by leptin during SD and LD. However, the increase in expression of SOCS-3 in the MBH during LD compared with SD fails to fully explain these effects.

Leptin was initially identified as a regulator of appetite and weight control centers in the hypothalamus, but appears to be involved in a number of physiological processes. This study was carried out to examine the possible role of leptin in regulating prolactin (PRL) release using the teleost pituitary model system. This advantageous system allows isolation of a nearly pure population of lactotropes in their natural, in situ aggregated state. The rostral pars distalis were dissected from tilapia pituitaries and exposed to varying concentrations of leptin (0, 1, 10, 100 nM) for 1 h. Release of PRL was stimulated by leptin in a potent and concentration-dependent manner. A time-course experiment showed that the strongest response in PRL release with leptin occurs within the first hour (approximately sixfold), and stimulation was sustained after 16 h (approximately twofold). Many of the actions of leptin are mediated by the activation of extracellular signal-regulated kinase (ERK1/2) but nothing is known about the cellular mechanisms by which leptin might regulate PRL secretion in vertebrates. We therefore tested whether ERK1/2 might be involved in the leptin PRL response and found that the ERK inhibitor, PD98059, hindered leptin-induced PRL release. We further analyzed leptin response by quantifying tyrosine and threonine phosphorylation of ERK1/2 using western blots. One hour incubation with leptin induced a concentration-dependent increase in phosphorylated, and thus active, ERK1/2. Our data show that leptin is a powerful stimulator of in vitro PRL release and that its actions occur in part through stimulation of ERK1/2.

Hyperprolactinemia and hyperleptinemia occur during gestation and lactation with marked hyperphagia associated with leptin resistance. Prolactin (PRL) induces the expression of orexigenic neuropeptide Y (NPY) through the activation of JAK-2/STAT-3 signaling pathway in hypothalamic paraventricular nucleus (PVN) leading to hyperphagia. PRL may also act through the inhibition of anorexigenic effect of leptin via induction of suppressor of cytokine signaling 3 (SOCS-3). This paper aimed to co-localize PRL (PRL-R) and leptin (ObRb) receptors in the hypothalamus of female rats and investigate the possible cross-desensitization between PRL-R and ObRb. We showed that: 1) PRL-R and ObRb are expressed in the PVN and co-localized in the same neurons; 2) in lactating females leptin failed to activate JAK-2/STAT-3 signaling pathway; 3) in Chinese Hamster Ovary (CHO) stably co-expressing PRL-R and ObRb, overexposure to PRL did not affect leptin signaling but totally abolished PRL-dependent STAT-5 phosphorylation. The overexposure to leptin produces similar results with strong alteration of leptin-dependent STAT-3 phosphorylation, whereas PRL-dependent STAT-5 was not affected; and 4) CHO-ObRb/PRL-R cells overexposure to leptin or PRL induces the expression of negative regulators SOCS-3 and PTP-1B. Thus, we conclude that these negative regulators affect specifically the inducer signaling pathway; for instance, SOCS-3 induced by PRL will affect PRL-R signaling but not ObRb signaling and vice versa. Finally, the lack of cross-desensitization between PURL-R and ObRb suggests that hyperphagia observed during gestation and lactation may be attributed to a direct effect of PRL on NPYexpression, and is most likely exacerbated by the physiological leptin resistance state.

We had previously shown that neonatal leptin treatment programs thyroid function in adulthood. As both thyroid hormones (TH) and leptin increased thermogenesis, it was interesting to evaluate the effect of cold exposure on the thyroid function of neonate rats treated with leptin. Pups were divided into two groups: Lep, injected with leptin (8 mug/100 g/BW, s.c.) for the first 10 days of lactation and control (C), injected with saline. When they were 30 days old, the groups were subdivided into two subgroups: LepC and CC, which were exposed to 8 degrees C for 12 h and compared with C and Lep groups, maintained at 25 +/- 1 degrees C. Serum leptin, TH, and TSH were measured by RIA. Type I liver deiodinase (D1) and mitochondrial alpha-glycerol-3-phosphate dehydrogenase (mGPD) activities were assayed by the release of (125)I from (125)I-reverse and colorimetric method respectively. Leptin receptor (OB-Rb) was evaluated by western blot. Lep group had hyperleptinemia (+22%) and lower free tri-iodothyronine (FT(3); -33%). Cold exposure increased TH both in LepC and CC groups compared with respective controls free thyroxine (FT(4):+63 and +39%; FT(3):+75 and +40%). Liver D1 activity was lower in Lep (-22%) and increased with cold exposure (LepC +51% and CC +22%). The mGPD activity was lower in Lep (-34%) and increased (fourfold) when this group is cold exposed. Hypothalamic and thyroidal OB-Rb receptors were lower in Lep group (-47 and -36% respectively) and they were restored to normal levels after cold exposure. Leptin-programmed rats had higher TH response after cold exposure. OB-Rb had a fast response to cold exposure normalizing the lower levels observed in the leptin-programmed animals and may contribute to the higher TH cold responses.

Bombesin is one of the most powerful substances showing anorexic effects in the hypothalamus (Moody TW & Merali Z 2004 Bombesin-like peptides and associated receptors within the brain: distribution and behavioral implications. Peptides 25 511-520). In mammals, neuromedin B (NMB) is one member of a family of bombesin-like peptides, which have been shown to reduce food intake when administered systemically. Using Taqman real-time PCR with specific primers, we report the expression of NMB mRNA in both human and rodent adipose tissue. Expression of NMB in rodent epididymal adipose tissue was higher than in other tissues studied. Expression of NMB in adipose tissue appears to be regulated by changes in energy balance and leptin. It is decreased fourfold in the epididymal fat depot of ob/ob mice when compared with the same fat depot in lean mice. It is further decreased with the intra-peritoneally (i.p.) administration of leptin in both lean and obese ob/ob mice. This may relate to its function in food intake regulation or to changes in energy expenditure. We demonstrate that NMB expression in rodent adipose tissue is decreased in cold exposed animals. However, when we investigated the effects of NMB on resting metabolic rate by i.p. injection, there was no effect on oxygen consumption, RQ or physical activity when compared with saline-treated controls. In conclusion, NMB is expressed in both human and rodent adipose tissue and appears to be regulated by changes in energy balance. Given its anorexic effects centrally, it may form part of a new adipose tissue--hypothalamic axis regulating food intake.

Obesity and type 2 diabetes are world wide health issues and their incidence is rapidly increasing. Currently the biological factors responsible for the development of obesity are only partially understood. Recent research has shown that maternal nutrition during pregnancy may have long-term metabolic consequences in offspring. In the present study we investigated interactions between prenatal and postnatal nutrition on leptin sensitivity and obesity development. Wistar rats were time-mated and randomly assigned to either ad-libitum (AD) or to 30% of ad-libitum (UN) food intake throughout pregnancy. After weaning, female offspring were fed standard chow, a high-fat diet or a calorie restricted diet. Female offspring of UN dams were growth retarded at birth and showed increased susceptibility to diet-induced obesity on a high-fat diet. At 142 +/- 5 days of age, leptin sensitivity was measured as a response to 14 days of leptin treatment (2.5 microg/g/day, s.c.). In UN offspring fed chow, leptin treatment failed to reduce food intake and weight loss was diminished. This leptin resistance observed in UN offspring was independent of diet-induced obesity and was associated with fasting hyperinsulinemia and hypertriglyceridemia. Our study suggests that prenatal nutrition can shape future susceptibility to obesity through alterations in leptin sensitivity and changes in energy metabolism during adult life.

Orexins (A and B) have been implicated in feeding behavior, energy balance and state of vigilance. During pregnancy, their involvement in feeding regulation and reproduction are poorly understood. In this study, we investigated orexin-A immunoreactivity in the hypothalamus and serum in pregnant rats and women by immunofluorescence staining, image analysis and radioimmunoassay, examined the correlation of serum orexin-A and leptin with gestational age in pregnant women by regression analysis, and explored the effect of leptin injected intracerebroventricularly (i.c.v.) on orexin-A immunoreactivity in the hypothalamus of normal rats by immunohistochemistry. The results showed that pregnant rats had significantly greater daily food intake on days 15 and 20 of pregnancy than virgin ones (+27.3%, P< 0.01 and +38.6%, P< 0.001 respectively), with significantly fewer number and lower mean staining intensity of orexin-A-immunoreactive (ir) neurons on days 16 (both P< 0.05) and 21 (both P< 0.01) of pregnancy. Moreover, serum levels of orexin-A exhibited 2.0-fold and 2.2-fold increases (both P< 0.001) in rats on days 16 and 21 of pregnancy compared with those in virgin rats, and 1.9-fold and 2.0-fold increases (both P< 0.001) in mid (13-26 weeks) and late pregnant women (27-40 weeks) compared with those in non-pregnant women. Simultaneously, serum levels of leptin showed a 2.3-fold and 2.2-fold increase (both P< 0.001) in rats on days 16 and 21 of pregnancy, and a 3.3-fold and 4.3-fold increase (both P< 0.001) in mid and late pregnant women. Serum levels of both orexin-A and leptin correlated positively with gestational age in pregnant women. Leptin injected i.c.v. significantly decreased the number (P< 0.01) and mean staining intensity (P< 0.01) of orexin-A-ir neurons in the hypothalamus, food intake (P< 0.01) and body weight gain (P< 0.001) compared with vehicle injection in normal rats. These results suggested that central and serum orexin-A might be involved in the regulation of feeding and energy metabolism during pregnancy. The change in central orexin-A immunoreactivity might be related to the increased serum leptin concentrations.

The role of leptin in neuroendocrine appetite and reproductive regulation remains to be fully resolved. A series of three experiments was conducted using adequately nourished oestradiol-implanted castrated male sheep. In a cross-over design (n=6), responses to a single i.c.v. (third ventricle) injection of leptin (0.5, 1.0 and 1.5 mg ovine leptin (oLEP) and 1.0 mg murine leptin (mLEP)), N-methyl-D-aspartate (NMDA, 20 micro g) or 0.9% saline (control) were measured in terms of LH secretion (4 h post-injection compared with 4 h pre-injection) and appetite (during 2 h post-injection) in autumn (Experiment 1). NMDA and 1.0 mg oLEP treatments were repeated in the same sheep in the following spring (Experiment 2). With an additional 12 sheep (n=18 in cross-over design), responses to low-dose 'physiological' i.c.v. infusion of leptin (8 ng/h for 12 h daily for 4 days), insulin (0.7 ng/h) and artificial cerebrospinal fluid were measured in the next spring (Experiment 3). LH was studied over 8 h and appetite over 1 h on days 1 and 4 of infusion. In Experiment 1 (autumn), oLEP overall increased LH pulse frequency by up to 110% (P<0.05), decreased LH pulse amplitude (P<0.05) and decreased appetite (P<0.05). mLEP reduced LH pulse amplitude (P<0.05) without significant effect on appetite, while NMDA reduced appetite (P<0.05) but had no effect on LH. In Experiment 2 (spring), LH responses were 'surge-like' with highly significant increases in the moving average LH concentration after 1.0 mg oLEP (P<0.001) and after NMDA (P<0.001). Compared with similar analysis of experiment 1 results, the LH response in spring was greater than that in autumn for both 1.0 mg oLEP (P<0.05) and NMDA (P<0.005). Conversely, unlike in autumn (Experiment 1), there was no effect of 1.0 mg oLEP or NMDA on appetite in the spring (Experiment 2). In Experiment 3 (spring), 'physiological' i.c.v. infusion of oLEP or insulin increased LH pulse frequency by up to 100% (P<0.001) compared with the control infusion on both days 1 and 4, but there were no effects on appetite. These results indicate that intracerebral leptin both stimulates reproductive neuroendocrine output and decreases appetite in adequately nourished sheep. However, the responses of these two axes were dose-dependent and differentially affected by the time of year, suggesting dissociation of the neural pathways involved.

Leptin is a peripheral immunoenhancing reagent that directly activates splenic lymphocytes in mice. We found that a 48 h fast in rats resulted in a decrease in serum leptin that was accompanied by a lower delayed-type hypersensitivity (DTH) response. Peripheral leptin replacement completely restored this response in fasted animals. We employed a recombinant adeno-associated virus (rAAV) system to deliver leptin gene directly into rat brain to assess the effect of sustained long-term central expression of leptin on immune responses. The rAAV-leptin rats had elevated central leptin over the 60 day duration of the experiment, whereas body fat and circulating leptin fell to near zero levels. The DTH response was significantly reduced by 10-20% in rats receiving rAAV-leptin compared with the control rats, and the difference was maintained for over 50 h. When the rats undergoing rAAV-leptin gene therapy were given either murine recombinant leptin or PBS s.c., rats receiving leptin had a 17% higher DTH response than rats receiving PBS. The isolated splenocytes from the former group also proliferated 34% more in vitro in response to the mitogen concanavalin A as compared with the latter group. These results suggest that peripheral leptin has a dominant role in maintaining T-cell-mediated immune responses in rats, and central leptin is unable to compensate for the immunosuppression associated with peripheral hypoleptinemia. Furthermore, preservation of normal cell-mediated immune responses does not require fat tissue as along as serum leptin levels are maintained.

Leptin can act as a satiety factor and exert neuroendocrine effects, but most studies have been performed in fasted animals. We aimed to determine the effect of chronic under-nutrition on the response to a 3-day intracerebroventricular infusion of leptin with regard to food intake and the secretion of pituitary hormones. Ovariectomised ewes (n=6) had a mean (+/-s.e.m. ) bodyweight of 56+/-0.8 kg on a diet available ad libitum (ad lib) or 33.4+/-1 kg on a restricted diet. The differential bodyweight was achieved by dietary means over a period of 6 months prior to the commencement of the study. Leptin (4 microg/h) or vehicle (artificial cerebrospinal fluid (aCSF)) was infused into the third cerebral ventricle for 3 days. Blood samples were taken prior to commencement and on day 3 of infusion for the assay of plasma hormone levels. The experiment was repeated one week later in a cross-over design. Food intake and metabolic status were monitored daily. The luteinising hormone (LH) pulse amplitude was lower (P<0.05) but plasma growth hormone (GH) levels were higher (P<0.05) in the food-restricted animals. Plasma levels of glucose, lactate, insulin, urea and triglycerides were similar in the two groups but non-esterified fatty acid levels were higher (P<0.01) in the animals on an ad lib diet. Leptin reduced (P<0.05) food intake only in the animals fed an ad lib diet. Leptin increased (P<0.05) the secretion of LH in the food-restricted group only and increased (P<0.05) GH irrespective of bodyweight. In conclusion, leptin does not alter food intake in animals on a restricted diet but can increase the secretion of LH in the same animals. The treatment of leptin was not sufficient to reduce plasma GH levels in the food-restricted animals, suggesting that other factors or mechanisms must be involved in the regulation of this axis.