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Background: Plasma leptin in humans is subject to both long- and short-term regulation; it correlates with indexes of body fat that can only change slowly. However, short-term fasting causes large and rapid decreases., Objective: We tested the interactions between energy intake and fat loss on plasma leptin during prolonged moderate and severe energy restriction, with a view to understanding mechanisms of control., Design: Postabsorptive leptin was measured with an enzyme-linked immunosorbent assay specific for the human peptide in 21 obese women aged 41 +/- 3 y (weight: 102 +/- 4 kg; 48 +/- 1% body fat) after 1 wk of a weight-maintaining diet and then weekly for 4 wk during a total fast (group 1); a 1.9-MJ/d all-protein, very-low-energy diet (VLED) (group 2); or a low-energy, balanced-deficit diet (BDD) providing 50% of maintenance energy (group 3). In groups 1 and 2, leptin was also measured after 1 wk of refeeding with a diet equivalent to the BDD., Results: Mean leptin decreased markedly by up to 66% (P < 0.001) at week 1 of energy restriction and then gradually thereafter. The change in leptin per kilogram fat mass correlated with that in glucose concentrations [r = 0.538 (P = 0.012) at week 1 and r = 0.447 (P = 0.042) at week 4] but not with that in fat mass. During refeeding postfasting, leptin increased (P = 0.008), despite an ongoing loss of fat mass and correlated positively with changes in resting energy expenditure. At times with comparable cumulative energy restriction and fat loss between diets, the percentage change in leptin paralleled that in glucose., Conclusions: In obesity, changes in energy intake over days to weeks are a primary modulator of plasma leptin concentrations that are related to the change in glycemia and are able to override the regulatory influence of fat mass.

To investigate whether brain leptin involves neuropeptidergic pathways influencing ingestion, metabolism, and gastrointestinal functioning, leptin (3.5 micrograms) was infused daily into the third cerebral ventricular of rats for 3 days. To distinguish between direct leptin effects and those secondary to leptin-induced anorexia, we studied vehicle-infused rats with food available ad libitum and those that were pair-fed to leptin-treated animals. Although body weight was comparably reduced (-8%) and plasma glycerol was comparably increased (142 and 17%, respectively) in leptin-treated and pair-fed animals relative to controls, increases in plasma fatty acids and ketones were only detected (132 and 234%, respectively) in pair-fed rats. Resting energy expenditure (-15%) and gastrointestinal fill (-50%) were reduced by pair-feeding relative to the ad libitum group, but they were not reduced by leptin treatment. Relative to controls, leptin increased hypothalamic mRNA for corticotropin-releasing hormone (CRH; 61%) and for proopiomelanocortin (POMC; 31%) but did not reduce mRNA for neuropeptide Y. These results suggest that CNS leptin prevents metabolic/gastrointestinal responses to caloric restriction by activating hypothalamic CRH- and POMC-containing pathways and raise the possibility that these peripheral responses to CNS leptin administration contribute to leptin's anorexigenic action.

Objective: The effects of short-term moderate physiological changes in energy flux and energy balance, by exercise and over- or underfeeding, on a 24h plasma leptin profile, were investigated., Design: Subjects were studied over 24h in four randomized conditions: no exercise/energy balance (energy intake (EI)=energy expenditure (EE)=11.8+/-0.8 MJ); exercise/energy balance (EI=EE=15.1+/-0.6 MJ); exercise/negative energy balance (EI=11.8+/-0.8 MJ, EE=15.1+/-0.8 MJ); exercise/positive energy balance (El=18.6+/-0.7 MJ, EE=15.1+/-0.6 MJ)., Subjects: Eight healthy, lean men (age: 23.5+/-7.0y, body fat 14.1+/-5.4%, body mass index (BMI): 21.4+/-2.3 kg/m2)., Measurements: Blood was sampled every hour during the daytime (09.00-23.00h) and every two hours during the night (01.00-09.00h) for analysis of plasma leptin, insulin, glucose, FFA and catecholamines., Results: Plasma leptin levels were highest around 01.00h (mean+/-s.e.m. 4.9+/-2.0 ng/ml) and lowest around 11.00 h. (2.3+/-0.7 ng/ml). An increased 24h EE, induced by exercise under conditions of energy balance, significantly decreased the peak and average 24h plasma leptin concentration. A positive energy balance, by overfeeding, resulted in a significantly higher amplitude of the 24h plasma leptin curve, compared to a condition of energy balance., Conclusion: Exercise decreases peak and average 24h plasma leptin concentration and a moderately positive energy balance increases the amplitude of the 24h plasma leptin profile. These effects are not acute, but are manifest within 24h. The variations of average 24h FFA and average 24h glucose concentrations almost fully explained the variation in average 24h leptin concentration across trials.

OB protein (or leptin) administration causes a long-lasting reduction in food intake and body weight in obese ob/ob mice. Neuropeptide Y, a stimulator of feeding, has been proposed to be a major mediator of the biological actions of OB protein. To test this hypothesis, the interaction of brain administration of exogenous OB protein and NPY on the feeding behavior of ob/ob mice was examined. Human OB protein, in a dose-dependent manner, partially or completely blocked feeding induced by exogenous NPY. These results demonstrate that OB protein can functionally antagonize and dominate the actions of exogenous NPY on feeding.

The mechanisms by which OB protein controls food intake and energy balance are unknown. Therefore, we investigated the effects of a novel modified human recombinant OB protein (Mod-OB) on spontaneous feeding patterns, body weight, running wheel activity, and ovarian cycling in female rats. Mod-OB or vehicle was injected (4 mg . kg-1 . day-1 sc) for 2 ovarian cycles (8 days) using a within-subjects design. Observations were continued for five ovarian cycles after injections; treatments were then reversed. Mod-OB reduced food intake approximately 20% from injection day 1 to postinjection day 2. Body weight was reduced from injection day 3 to postinjection day 15 (maximum decrease, 25 +/- 4 g, postinjection days 3 and 4). Food intake was reduced due to decreases in nocturnal meal size, which appeared to be superimposed on the normal pattern of spontaneous feeding (i.e., reductions in meal size at estrus). Mod-OB did not significantly affect diurnal food intake or meal patterns, failed to alter wheel running, and did not disrupt the rats' ovarian cycles. We conclude that chronically administered Mod-OB reduces food intake in female rats by selectively affecting the mechanisms controlling meal size.

The potent hypophagic effect of OB protein (OB) is well established, but the mechanism of this effect is largely unknown. We investigated the effects of chronic administration of a novel modified recombinant human OB (Mod-OB) with a prolonged half-life (>48 h) on ad libitum food intake, spontaneous meal patterns, and body weight in 24 adult, male Sprague-Dawley rats (body weight at study onset: 292 g). Single daily subcutaneous injections of Mod-OB (4 mg/kg daily) for 8 consecutive days significantly reduced ad libitum food intake compared with vehicle injections from injection day 3 through postinjection day 3. Mod-OB-injected rats ate between 4.5 and 7.1 g (or 13-20%) per day less than controls, with the reduction primarily occurring during the dark period. Body weight gain was significantly decreased in response to Mod-OB from injection day 8 until postinjection day 4, with a maximum difference of 24 g on postinjection day 3. The reduction of food intake by Mod-OB was mainly due to a 21-34% decrease in nocturnal spontaneous meal size. There was no significant effect of Mod-OB on nocturnal meal frequency or duration. Mod-OB also did not reliably affect the size, duration, or frequency of diurnal meals. Mod-OB-injected rats displayed no compensatory hyperphagia after the injection period. These results indicate that chronically administered OB selectively affects the mechanisms controlling meal size in male rats.

Objective: Prader-Willi syndrome (PWS) is an autosomal dominant disorder involving the proximal long arm of chromosome 15, in which obesity is common. However, there is limited information on the underlying physiological mechanisms promoting obesity in this population. We tested whether there was a significant positive association between leptin and total body fat (TBF) in subjects with PWS, and whether this association was stronger among subjects with than without PWS., Research Methods and Procedures: We studied 21 PWS patients and 64 non-PWS controls on whom we measured serum leptin, total body fat, glucose, insulin, and resting energy expenditure. We tested whether the slope of the regression line between leptin and TBF (in kg), measured by dual energy X-ray absorptiometry, was the same for PWS patients and non-PWS controls., Results: Regression analyses indicated that the leptin-TBF association was significantly stronger among PWS patients. In contrast, the slope of the leptin-body mass index association did not significantly differ between PWS patients and non-PWS controls. None of the other outcome variables showed associations with leptin., Discussion: Results suggest that the role of leptin in promoting obesity may be greater among subjects with PWS than among non-PWS controls.

Specific [125I]leptin receptor binding sites have been identified in choroid plexus (CP), but have eluded regional localization within the brain parenchyma. To optimize specific [125I]leptin binding in brain loci, we ran experiments varying the pH of incubation buffers. We found that specific [125I]leptin binding in CP was strikingly pH dependent with the most acidic buffer, pH 5.5, resulting in a greater than 100% increase over the amount of specific binding measured at pH 7.5. While low pH permitted detection of specific binding in parenchymal loci, clear pH dependency was only observed in the CP. In the caudate putamen (CauP), a locus with low specific binding, values for specific binding did not differ significantly across the range of pH conditions tested. Using incubation buffers at pH 6.0 in subsequent binding experiments, we localized specific [125I]leptin binding in several brain loci including thalamus and hypothalamus. In CP and thalamus, where the range of OD permitted analysis of binding parameters, [125I]leptin binding was saturable with increasing concentrations of unlabelled leptin. In all loci, specific [125I]leptin binding was insensitive to competition by high concentrations of other unlabelled compounds. Our results varying pH conditions of the incubation buffer suggest leptin receptors may be divided into subclassifications based on pH sensitivity of the specific binding. Furthermore, our results suggest that although densities are low, high affinity leptin receptors are present in neural loci implicated in food intake and energy balance, and are more widespread in the forebrain than previously determined.

The genes encoding the mature forms of mouse (mOB) and human OB (hOB) protein (also called leptin) were fused to the secretion signal coding sequence of the Escherichia coli outer membrane protein A (sOMP A). The hybrid genes were preceded by a ribosome binding site (RBS) and were expressed under transcriptional control of both the lipoprotein promoter (Plpp) and the lac promoter-operator (POlac). The recombinant fusion proteins were efficiently expressed and exported into the periplasmic compartment of E. coli cells from where they were recovered by osmotic shock as soluble mature polypeptides with the sOMP A precisely removed. Recombinant mOB and hOB proteins were also produced in Sf9 insect cells using the baculovirus expression system. Milligram quantities of both proteins were purified to homogeneity using ion-exchange, hydrophobic interaction chromatography and gel filtration and were found to be biologically active and to have antiobesity effects upon testing in genetically obese ob/ob mice.

Melanocortins are peptides, cleaved from the pro-opiomelanocortin (POMC) precursor, that act in the brain to reduce food intake and are potential mediators of leptin action. In the forebrain, melanocortins are derived from POMC-containing neurons of the hypothalamic arcuate nucleus. To test the hypothesis that these POMC neurons are regulated by leptin, we used in situ hybridization to determine whether reduced leptin signaling (as occurs in fasting), genetic leptin deficiency (in obese ob/ob mice), or genetic leptin resistance (in obese db/db mice) lower expression of POMC mRNA. We further hypothesized that leptin administration would raise hypothalamic POMC mRNA levels in leptin-deficient animals, but not in mice with defective leptin receptors. In wild-type mice (n = 12), fasting for 48 h lowered POMC mRNA levels in the rostral arcuate nucleus by 53%, relative to values in fed controls (n = 8; P < 0.001). Similarly, arcuate nucleus POMC mRNA levels were reduced by 46 and 70% in genetically obese ob/ob (n = 6) and db/db mice (n = 6), respectively, as compared with wild-type mice (n = 5) (P < 0.01 for both comparisons). Five daily intraperitoneal injections of recombinant murine leptin (150 microg) raised levels of POMC mRNA in the rostral arcuate nucleus of ob/ob mice (n = 8) by 73% over saline-treated ob/ob control values (n = 8; P < 0.01), but was without effect in db/db mice (n = 6). In normal rats, two injections of a low dose of leptin (3.5 microg) into the third cerebral ventricle (n = 15) during a 40-h period of fasting also increased POMC mRNA levels in the rostral arcuate nucleus to values 39% greater than those in vehicle-treated controls (n = 14; P = 0.02). We conclude that reduced central nervous system leptin signaling owing to fasting or to genetic defects in leptin or its receptor lower POMC mRNA levels in the rostral arcuate nucleus. The finding that leptin reverses this effect in ob/ob, but not db/db, mice suggests that leptin stimulates arcuate nucleus POMC gene expression via a pathway involving leptin receptors. These findings support the hypothesis that leptin signaling in the brain involves activation of the hypothalamic melanocortin system.

Leptin, a hormone secreted by adipose tissue in proportion to body adiposity, is proposed to be involved in the central nervous regulation of food intake and body weight. In addition, evidence is emerging that leptin regulates neuroendocrine and metabolic functions as well, presumably via its action in the central nervous system (CNS). To investigate this regulatory effect of leptin, we infused 3.5 microg of human leptin directly into the third cerebral ventricle (i3vt) of lean male Long-Evans rats, 90 min before the onset of their dark phase. Before and after infusion, blood samples were withdrawn through indwelling catheters for assessment of hormonal (plasma corticosterone, insulin, leptin), autonomic (plasma norepinephrine, epinephrine), and metabolic (plasma glucose) parameters. I3vt leptin caused an increase in plasma corticosterone and plasma leptin levels relative to the control condition. The effects of i3vt leptin on corticosterone secretion became particularly apparent after the onset of the dark phase. The results of the present study indicate that i3vt leptin stimulates the hypothalamo-pituitary-adrenal (HPA) axis, particularly when rats normally encounter their largest meals. These results are consistent with the possibility that high circulating leptin levels may underlie the increased activity of the HPA axis that is generally characteristic of human obesity and most animal models of obesity.

1. Adipocyte-derived leptin is postulated to represent the afferent hormonal signal to the hypothalamus in a feedback mechanism that regulates fat mass. In this proposed feedback mechanism, increased fat mass leads to an elevated plasma leptin level that eventually induces a decrease in appetite and an increase in energy expenditure, and vice versa. 2. As anorexia and hypermetabolism play a role in the development of cancer cachexia, we investigated the hypothesis that underlying abnormalities in the leptin feedback mechanism (in particular relatively high plasma leptin levels or, on the other hand, a hypothalamic insensitivity to a fall in leptin levels) might be involved. For this purpose, total plasma leptin, body composition (fat mass and fat-free mass), appetite and resting energy expenditure were assessed in 21 male lung-cancer patients. 3. Total leptin was detectable in six patients and non-detectable in 15. In comparison with the latter, the patients with detectable leptin were characterized by a trend towards less weight loss (3.4% compared with 11.0%, P = 0.07), as being less underweight (body mass index 23.8 kg/m2 compared with 19.4 kg/m2, P = 0.004) and by a higher fat mass (21.4 kg compared with 9.7 kg, P = 0.001). Significant between-group differences in appetite and resting energy expenditure were lacking. 4. Based on these findings, we conclude that in cancer the afferent part of the leptin feedback mechanism functions normally and that, in particular, elevated leptin levels are not involved in the development of cachexia. Since the absence of plasma leptin was not associated with an increased appetite and decreased energy expenditure, disturbances in the hypothalamic part of the feedback mechanism are hypothesized.

Leptin (ob protein) and glucagon-like peptide-1-(7-36) amide (GLP-1) are peptides recently proposed to be involved in the regulation of food intake. Although the ability of exogenous leptin and GLP-1 to modulate consummatory behavior is consistent with the suggestion that these peptides are endogenous regulatory agents, central administration of these peptides may have aversive side effects, which could explain the anorexia. In the present experiment, exposure to a saccharine taste was immediately followed by central administration of leptin or GLP-1 to determine if these drugs could produce a conditioned taste aversion (CTA) in rats. At doses equated for producing comparable reductions in short-term food intake, GLP-1, but not leptin, generated a robust CTA. Although leptin caused no aversion, this peptide was the only drug to cause relatively long-term reductions in food consumption (16 h) and body weight (24 h). Hence, the results indicate that central GLP-1 produces aversive side effects, and it is argued that these nonspecific effects may explain the anorectic actions of GLP-1.

The protein encoded by the obese (ob) gene, leptin, is secreted from adipose tissue and is proposed to act in the brain as an important regulator of food intake and body weight. To investigate the direct effects of leptin within the CNS, we injected 3.5 microg of either mouse or human leptin into the third ventricle (ICV) of lean Long-Evans rats or obese (fa/fa) Zucker rats, in which obesity results from a mutation in the leptin receptor gene. ICV administration of leptin reduced 4-h food intake in both deprived and non-deprived lean rats. In addition, repeated ICV administration produced a long-lasting reduction in body weight while peripheral administration of the same dose had no effect. ICV administration of the same dose of leptin into the third ventricle of obese Zucker rats did not reduce food intake. These results are consistent with the hypothesis that leptin has direct actions in the CNS as an afferent signal related to the state of energy stores in adipose tissue. Furthermore, insensitivity to these central effects of leptin may be an important determinant of obesity.

OB protein (also known as leptin), a previously unknown protein signal, is secreted from adipose tissue, circulates in the blood, probably bound to a family of binding proteins, and acts on central neural networks that regulate ingestive behavior and energy balance. OB protein provides a communication link from fat tissue and the brain. Rapidly accumulating evidence suggests that OB protein appears to play a major role in the control of body fat stores through coordinated regulation of feeding behavior, metabolism, autonomic nervous system and body energy balance in rodents, primates and humans. The field has rapidly moved from cloning of the ob gene to demonstration of complex regulation of ob gene expression in adipose tissue in rats and humans, and then the demonstration of potent biological activity of OB protein in ob/ob, diet-induced, and lean mice as well as obese and lean rats but not in db/db obese mice. A significant milestone was our demonstration that central administration of OB protein lead to reductions in food intake, body weight and alterations in metabolism consistent with activation of the autonomic nervous system. These findings were followed by the identification of a central binding site for labelled OB protein in the choroid plexus in ob/ob, db/db and lean mice as well as lean and obese Zucker rats. The expression cloning of a central receptor, OB-R, from the mouse choroid plexus soon followed. The OB-R receptor was found to be expressed in the choroid plexus, the hypothalamus as well as several peripheral tissues. OB-R exists in multiple forms; the two major forms are a short form (with a truncated intracellular domain) and long form (with the complete intracellular domain). The long form is thought to be the form that signals and mediates the biological effects of OB protein. Initial in situ hybridization studies have demonstrated the mRNA for the long form OB-R receptor to be localized to the hypothalamus as well as peripheral sites. Recently, it was demonstrated that the db gene encodes the OB-R receptor. Evidence has been provided for a specific transport system for OB protein to cross the blood-brain-barrier and enter the brain of mice, rats and humans. The rate of transport can be decreased by high plasma concentrations of OB protein. Thus, reduced entry of OB protein to the brain may be one of the mechanisms of reduced sensitivity of the OB protein pathway in obese individuals. OB protein appears to also play a role in the important neuroendocrine adaptive responses to fasting and in the control of reproduction. Therapeutic approaches to the treatment of obesity based on OB protein ranging from OB protein by injection to OB-R receptor agonists and to upregulation of OB signalling pathways are under intense investigation.

Recently, glucagon-like peptide-1-(7-36) amide (GLP-1) and leptin have been implicated in the regulation of food intake. In the present study, we compared the effects of third ventricular administration (i3vt) of leptin (3.5 micrograms) and GLP-1 (10.0 micrograms) on short-term food intake and c-Fos-like immunoreactivity (c-FLI) in hypothalamic, limbic, and hindbrain areas in the rat. Relative to controls, infusion of leptin or GLP-1 (3 h before lights off) significantly reduced food intake over the first 2 h in the dark phase (53 and 63%, respectively). In different rats, infusion of leptin or GLP-1 elevated c-FLI in the paraventricular hypothalamus and central amygdala. Furthermore, leptin selectively elevated c-FLI in the dorsomedial hypothalamus, whereas GLP-1 selectively elevated c-FLI in the nucleus of the solitary tract, area postrema, lateral parabrachial nucleus, and arcuate hypothalamic nucleus. The fact that most of the c-FLI after leptin or GLP-1 administration was observed in separate regions within the central nervous system (CNS) suggests different roles for leptin and GLP-1 in the CNS regulation of food intake and body weight.

The hypothesis that leptin (OB protein) acts in the hypothalamus to reduce food intake and body weight is based primarily on evidence from leptin-deficient, ob/ob mice. To investigate whether leptin exerts similar effects in normal animals, we administered leptin intracerebroventricularly (icv) to Long-Evans rats. Leptin administration (3.5 microg icv) at the onset of nocturnal feeding reduced food intake by 50% at 1 h and by 42% at 4 h, as compared with vehicle-treated controls (both P < 0.05). To investigate the basis for this effect, we used in situ hybridization (ISH) to determine whether leptin alters expression of hypothalamic neuropeptides involved in energy homeostasis. Two injections of leptin (3.5 microg icv) during a 40 h fast significantly decreased levels of mRNA for neuropeptide Y (NPY, which stimulates food intake) in the arcuate nucleus (-24%) and increased levels of mRNA for corticotrophin releasing hormone (CRH, an inhibitor of food intake) in the paraventricular nucleus (by 38%) (both P < 0.05 vs. vehicle-treated controls). To investigate the anatomic basis for these effects, we measured leptin receptor gene expression in rat brain by ISH using a probe complementary to mRNA for all leptin receptor splice variants. Leptin receptor mRNA was densely concentrated in the arcuate nucleus, with lower levels present in the ventromedial and dorsomedial hypothalamic nuclei and other brain areas involved in energy balance. These findings suggest that leptin action in rat hypothalamus involves altered expression of key neuropeptide genes, and implicate leptin in the hypothalamic response to fasting.

Binding studies were conducted to identify the anatomical location of brain target sites for OB protein, the ob gene product. 125I-labeled recombinant mouse OB protein or alkaline phosphatase-OB fusion proteins were used for in vitro and in vivo binding studies. Coronal brain sections or fresh tissue from lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats were probed to identify potential central OB protein-binding sites. We report here that recombinant OB protein binds specifically to the choroid plexus. The binding of OB protein (either radiolabeled or the alkaline phosphatase-OB fusion protein) and its displacement by unlabeled OB protein was similar in lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats. These findings suggest that OB protein binds with high affinity to a specific receptor in the choroid plexus. After binding to the choroid plexus receptor, OB protein may then be transported across the blood-brain barrier into the cerebrospinal fluid. Alternatively, binding of OB protein to a specific receptor in the choroid plexus may activate afferent neural inputs to the neural network that regulates feeding behavior and energy balance or may result in the clearance or degradation of OB protein. The identification of the choroid plexus as a brain binding site for OB protein will provide the basis for the construction of expression libraries and facilitate the rapid cloning of the choroid plexus OB receptor.

The ob gene product, leptin, is an important circulating signal for the regulation of body weight. To identify high affinity leptin-binding sites, we generated a series of leptin-alkaline phosphatase (AP) fusion proteins as well as [125I]leptin. After a binding survey of cell lines and tissues, we identified leptin-binding sites in the mouse choroid plexus. A cDNA expression library was prepared from mouse choroid plexus and screened with a leptin-AP fusion protein to identify a leptin receptor (OB-R). OB-R is a single membrane-spanning receptor most related to the gp130 signal-transducing component of the IL-6 receptor, the G-CSF receptor, and the LIF receptor. OB-R mRNA is expressed not only in choroid plexus, but also in several other tissues, including hypothalamus. Genetic mapping of the gene encoding OB-R shows that it is within the 5.1 cM interval of mouse chromosome 4 that contains the db locus.

The recent positional cloning of the mouse ob gene and its human homology has provided the basis to investigate the potential role of the ob gene product in body weight regulation. A biologically active form of recombinant mouse OB protein was overexpressed and purified to near homogeneity from a bacterial expression system. Peripheral and central administration of microgram doses of OB protein reduced food intake and body weight of ob/ob and diet-induced obese mice but not in db/db obese mice. The behavioral effects after brain administration suggest that OB protein can act directly on neuronal networks that control feeding and energy balance.