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Context: Rabson-Mendenhall syndrome (RMS) is caused by biallelic pathogenic variants in the insulin receptor gene (INSR) leading to insulin-resistant diabetes, microvascular complications, and growth hormone resistance with short stature. Small, uncontrolled studies suggest that 1-year treatment with recombinant leptin (metreleptin) improves glycemia in RMS., Objective: This study aimed to determine effects of long-term metreleptin in RMS on glycemia, anthropometrics, the growth hormone axis, and kidney function., Methods: We compared RMS patients during nonrandomized open-label treatment with metreleptin (≥ 0.15 mg/kg/day) vs no metreleptin over 90 months (5 subjects in both groups at different times, 4 only in metreleptin group, 2 only in control group). Main outcome measures were A1c; glucose; insulin; 24-hour urine glucose; standard deviation scores (SDS) for height, weight, body mass index (BMI), and insulin-like growth factor 1 (IGF-1); growth hormone; and estimated glomerular filtration rate., Results: Over time, metreleptin-treated subjects maintained 1.8 percentage point lower A1c vs controls (P = 0.007), which remained significant after accounting for changes in insulin doses. Metreleptin-treated subjects had a reduction in BMI SDS, which predicted decreased A1c. Growth hormone increased after metreleptin treatment vs control, with no difference in SDS between groups for IGF-1 or height. Reduced BMI predicted higher growth hormone, while reduced A1c predicted higher IGF-1., Conclusion: Metreleptin alters the natural history of rising A1c in RMS, leading to lower A1c throughout long-term follow-up. Improved glycemia with metreleptin is likely attributable to appetite suppression and lower BMI SDS. Lower BMI after metreleptin may also worsen growth hormone resistance in RMS, resulting in a null effect on IGF-1 and growth despite improved glycemia., (Published by Oxford University Press on behalf of the Endocrine Society 2021.)

Context: Leptin is an adipokine that signals energy sufficiency. In rodents, leptin deficiency decreases energy expenditure (EE), which is corrected following leptin replacement. In humans, data are mixed regarding leptin-mediated effects on EE., Objective: To determine the effects of metreleptin on EE in patients with lipodystrophy., Design, Setting, and Patients: Nonrandomized crossover study of 25 patients with lipodystrophy (National Institutes of Health, 2013-2018)., Intervention: The initiation cohort consisted of 17 patients without prior exposure to metreleptin, studied before and after 14 days of metreleptin. The withdrawal cohort consisted of 8 previously metreleptin-treated patients, studied before and after 14 days of metreleptin withdrawal., Main Outcomes: 24-h total energy expenditure (TEE), resting energy expenditure (REE), autonomic nervous system activity [heart rate variability (HrV)], plasma-free triiodothyronine (T3), free thyroxine (T4), epinephrine, norepinephrine, and dopamine., Results: In the initiation cohort, TEE and REE decreased by 5.0% (121 ± 152 kcal/day; P = 0.006) and 5.9% (120 ± 175 kcal/day; P = 0.02). Free T3 increased by 19.4% (40 ± 49 pg/dL; P = 0.01). No changes in catecholamines or HrV were observed. In the withdrawal cohort, free T3 decreased by 8.0% (P = 0.04), free T4 decreased by 11.9% (P = 0.002), and norepinephrine decreased by 34.2% (P = 0.03), but no changes in EE, epinephrine, dopamine, or HrV were observed., Conclusions: Metreleptin initiation decreased EE in patients with lipodystrophy, but no changes were observed after metreleptin withdrawal. Thyroid hormone was higher on metreleptin in both initiation and withdrawal cohorts. Decreased EE after metreleptin in lipodystrophy may result from reductions in energy-requiring metabolic processes that counteract increases in EE via adipose tissue-specific neuroendocrine and adrenergic signaling., (Published by Oxford University Press on behalf of the Endocrine Society 2021.)

Context: Human milk contains hormones that regulate metabolism. Extrauterine growth restriction remains common among preterm infants, but the effect of ingesting milk hormones on preterm infant growth is poorly understood., Objective: To quantify associations of longitudinal exposure to leptin, adiponectin, and insulin in milk with physical growth of preterm infants., Design/methods: In 50 preterm neonates (median gestational age 29.4 weeks), we sampled maternal milk on day-of-life 7, 14, 21, and 28 and measured hormone levels in whole milk by ELISA. Milk leptin levels were available for a subset of 18 infants. We calculated milk hormone doses by multiplying the hormone level by the milk volume ingested on each day and estimated the area under the curve (AUC) to reflect longitudinal exposure. We analyzed associations of milk hormone exposure with growth outcomes in generalized estimated equations., Main Outcome Measures: Weight gain velocity and z-scores in weight, length, head circumference, and body mass index at 36 weeks' postmenstrual age (PMA)., Results: Higher leptin intake was associated with greater weight gain (2.17g/kg/day [95% CI, 1.31, 3.02]) and weight z-score at 36 weeks' PMA (0.30 [0.08, 0.53] higher z-score per tertile). Higher adiponectin intake was associated with greater length z-score (0.41 [0.13, 0.69]), however, this association was nullified after adjustment of protein and calorie intake. Higher adiponectin was associated with smaller head circumference z-score (-0.36 [-0.64, -0.07]). Insulin was not associated with growth outcomes., Conclusions: Milk leptin and adiponectin exposures may affect growth of preterm infants. The long-term effects of milk hormones warrant further investigation., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: While severe obesity due to congenital leptin deficiency is rare, studies in patients before and after treatment with leptin can provide unique insights into the role that leptin plays in metabolic and endocrine function., Objective: The aim of this study was to characterize changes in peripheral metabolism in people with congenital leptin deficiency undergoing leptin replacement therapy, and to investigate the extent to which these changes are explained by reduced caloric intake., Design: Ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) was used to measure 661 metabolites in 6 severely obese people with congenital leptin deficiency before, and within 1 month after, treatment with recombinant leptin. Data were analyzed using unsupervised and hypothesis-driven computational approaches and compared with data from a study of acute caloric restriction in healthy volunteers., Results: Leptin replacement was associated with class-wide increased levels of fatty acids and acylcarnitines and decreased phospholipids, consistent with enhanced lipolysis and fatty acid oxidation. Primary and secondary bile acids increased after leptin treatment. Comparable changes were observed after acute caloric restriction. Branched-chain amino acids and steroid metabolites decreased after leptin, but not after acute caloric restriction. Individuals with severe obesity due to leptin deficiency and other genetic obesity syndromes shared a metabolomic signature associated with increased BMI., Conclusion: Leptin replacement was associated with changes in lipolysis and substrate utilization that were consistent with negative energy balance. However, leptin's effects on branched-chain amino acids and steroid metabolites were independent of reduced caloric intake and require further exploration., (© Endocrine Society 2020.)

Leptin has been proposed to modulate viscerosensory information directly at the level of vagal afferents. In support of this view, broad expression for the leptin receptor (Lepr) has previously been reported in vagal afferents. However, the exact identity and distribution of leptin-sensitive vagal afferents has not been elucidated. Using quantitative PCR, we found that the whole mouse nodose ganglion was predominantly enriched in the short form of Lepr, rather than its long form. Consistent with this observation, the acute administration of leptin did not stimulate JAK-STAT signaling in the nodose ganglion. Using chromogenic in situ hybridization in wild-type mice and several reporter mouse models, we demonstrated that Lepr mRNA was restricted to nonneuronal cells in the epineurium and parenchyma of the nodose ganglion and a subset of vagal afferents, which accounted for only 3% of all neuronal profiles. Double labeling studies further established that Lepr-expressing vagal afferents were Nav1.8-negative fibers that did not supply the peritoneal cavity. Finally, double chromogenic in situ hybridization revealed that many Lepr-expressing neurons coexpressed the angiotensin 1a receptor (At1ar), which is a gene expressed in baroreceptors. Taken together, our data challenge the commonly held view that Lepr is broadly expressed in vagal afferents. Instead, our data suggest that leptin may exert a previously unrecognized role, mainly via its short form, as a direct modulator of a very small group of At1ar-positive vagal fibers., (Copyright © 2019 Endocrine Society.)

Study Objectives: Obesity hypoventilation and obstructive sleep apnea are common complications of obesity linked to defects in respiratory pump and upper airway neural control. Leptin-deficient ob/ob mice have impaired ventilatory control and inspiratory flow limitation during sleep, which are both reversed with leptin. We aimed to localize central nervous system (CNS) site(s) of leptin action on respiratory and upper airway neuroventilatory control., Methods: We localized the effect of leptin to medulla versus hypothalamus by administering intracerbroventricular leptin (10 μg/2 μL) versus vehicle to the lateral (n = 14) versus fourth ventricle (n = 11) of ob/ob mice followed by polysomnographic recording. Analyses were stratified for effects on respiratory (nonflow-limited breaths) and upper airway (inspiratory flow limitation) functions. CNS loci were identified by (1) leptin-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and (2) projections of respiratory and upper airway motoneurons with a retrograde transsynaptic tracer (pseudorabies virus)., Results: Both routes of leptin administration increased minute ventilation during nonflow-limited breathing in sleep. Phrenic motoneurons were synaptically coupled to the nucleus of the solitary tract, which also showed STAT3 phosphorylation, but not to the hypothalamus. Inspiratory flow limitation and obstructive hypopneas were attenuated by leptin administration to the lateral but not to the fourth cerebral ventricle. Upper airway motoneurons were synaptically coupled with the dorsomedial hypothalamus, which exhibited STAT3 phosphorylation., Conclusions: Leptin relieves upper airway obstruction in sleep apnea by activating the forebrain, possibly in the dorsomedial hypothalamus. In contrast, leptin upregulates ventilatory control through hindbrain sites of action, possibly in the nucleus of the solitary tract., (© 2016 Associated Professional Sleep Societies, LLC.)

In human and ovine fetuses, glucocorticoids stimulate leptin secretion, although the extent to which leptin mediates the maturational effects of glucocorticoids on pulmonary development is unclear. This study investigated the effects of leptin administration on indices of lung structure and function before birth. Chronically catheterized singleton sheep fetuses were infused iv for 5 days with either saline or recombinant ovine leptin (0.5 mg/kg · d leptin (LEP), 0.5 LEP or 1.0 mg/kg · d, 1.0 LEP) from 125 days of gestation (term ∼145 d). Over the infusion, leptin administration increased plasma leptin, but not cortisol, concentrations. On the fifth day of infusion, 0.5 LEP reduced alveolar wall thickness and increased the volume at closing pressure of the pressure-volume deflation curve, interalveolar septal elastin content, secondary septal crest density, and the mRNA abundance of the leptin receptor (Ob-R) and surfactant protein (SP) B. Neither treatment influenced static lung compliance, maximal lung volume at 40 cmH2O, lung compartment volumes, alveolar surface area, pulmonary glycogen, protein content of the long form signaling Ob-Rb or phosphorylated signal transducers and activators of transcription-3, or mRNA levels of SP-A, C, or D, elastin, vascular endothelial growth factor-A, the vascular endothelial growth factor receptor 2, angiotensin-converting enzyme, peroxisome proliferator-activated receptor γ, or parathyroid hormone-related peptide. Leptin administration in the ovine fetus during late gestation promotes aspects of lung maturation, including up-regulation of SP-B.

Body weight loss of Lep(ob/ob) mice in response to leptin is larger than expected from the reduction in energy intake alone, suggesting a thermogenic action of unknown magnitude. We exploited the superior pharmacological properties of a novel long-acting leptin prepared via PASylation to study the contribution of its anorexigenic and thermogenic effects. PASylation, the genetic fusion of leptin with a conformationally disordered polypeptide comprising 600 Pro/Ala/Ser (PAS) residues, provides a superior way to increase the hydrodynamic volume of the fusion protein, thus retarding kidney filtration and extending plasma half-life. Here a single PAS(600)-leptin injection (300 pmol/g) resulted in a maximal weight reduction of 21% 6 days after application. The negative energy balance of 300 kJ/(4 d) was driven by a decrease in energy intake, whereas energy expenditure remained stable. Mice that were food restricted to the same extent showed an energy deficit of only 220 kJ/(4 d) owing to recurring torpor bouts. Therefore, the anorexigenic effect of PAS(600)-leptin contributes 75% to weight loss, whereas the thermogenic action accounts for 25% by preventing hypometabolism. In a second experiment, just four injections of PAS(600)-leptin (100 pmol/g) administered in 5- to 6-day intervals rectified the Lep(ob/ob) phenotype. In total, 16 nmol of PAS(600)-leptin per mouse triggered a weight loss of 43% within 20 days and normalized hypothermia and glucose homeostasis as well as hepatic steatosis. The beneficial properties of PAS(600)-leptin are substantiated by a comparison with previous studies in which approximately 400 nmol (∼25-fold) unmodified leptin was mandatory to achieve similar improvements.

Background: Leptin, an adipose-derived hormone, has been implicated in emotional regulation. We have previously shown that systemic administration of leptin produces anxiolytic-like effects and deletion of the leptin receptor, LepRb, in midbrain dopamine neurons leads to an anxiogenic phenotype. This study investigated whether activation or deletion of LepRb in the ventral tegmental area of adult mice is capable of inducing anxiolytic and anxiogenic effects, respectively., Methods: Mice were cannulated in the ventral tegmental area and received bilateral intra-ventral tegmental area infusions of leptin or the JAK2/STAT3 inhibitor AG490. Anxiety-like behaviors were assessed using the elevated plus-maze, light-dark box, and novelty suppressed feeding tests. Deletion of LepRb in the ventral tegmental area was achieved by bilateral injection of AAV-Cre into the ventral tegmental area of adult Lepr(flox/flox) mice. Anxiety-related behaviors were evaluated 3 weeks after viral injection., Results: Intra-ventral tegmental area infusions of leptin reduced anxiety-like behaviors, as indicated by increased percent open-arm time and open-arm entries in the elevated plus-maze test, increased time spent in the light side and decreased latency to enter the light side of the light-dark box, and decreased latency to feed in the novelty suppressed feeding test. Blockade of JAK2/STAT3 signaling in the ventral tegmental area by AG490 attenuated the anxiolytic effect produced by systemic administration of leptin. Lepr(flox/flox) mice injected with AAV-Cre into the ventral tegmental area showed decreased leptin-induced STAT3 phosphorylation and enhanced anxiety-like behaviors in the elevated plus-maze test and the novelty suppressed feeding test., Conclusions: These findings suggest that leptin-LepRb signaling in the ventral tegmental area plays an important role in the regulation of anxiety-related behaviors., (© The Author 2015. Published by Oxford University Press on behalf of CINP.)

There is uncertainty about the relationship between plasma leptin and sweet taste in mice. Whereas 2 studies have reported that elevations in plasma leptin diminish responsiveness to sweeteners, another found that they enhanced responsiveness to sucrose. We evaluated the impact of plasma leptin on sweet taste in C57BL/6J (B6) and leptin-deficient ob/ob mice. Although mice expressed the long-form leptin receptor (LepRb) selectively in Type 2 taste cells, leptin failed to activate a critical leptin-signaling protein, STAT3, in taste cells. Similarly, we did not observe any impact of intraperitoneal (i.p.) leptin treatment on chorda tympani nerve responses to sweeteners in B6 or ob/ob mice. Finally, there was no effect of leptin treatment on initial licking responses to several sucrose concentrations in B6 mice. We confirmed that basal plasma leptin levels did not exceed 10ng/mL, regardless of time of day, physiological state, or body weight, suggesting that taste cell LepRb were not desensitized to leptin in our studies. Furthermore, i.p. leptin injections produced plasma leptin levels that exceeded those previously reported to exert taste effects. We conclude that any effect of plasma leptin on taste responsiveness to sweeteners is subtle and manifests itself only under specific experimental conditions., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: There is an inverse relationship between triglycerides and high-density lipoprotein cholesterol (HDL-C) in insulin resistance, such that improvement in insulin resistance decreases triglycerides and increases HDL-C. Patients with lipodystrophy have extreme insulin resistance with high triglycerides and low HDL-C. Leptin replacement in lipodystrophy leads to a marked decrease in triglycerides (∼60%)., Objective: Our objective was to study the effects of metreleptin on triglycerides and HDL-C in lipodystrophy in contrast to changes in triglycerides and HDL-C in interventions for the obesity-associated metabolic syndrome., Design, Setting, and Patients: This open-label nonrandomized study at the National Institutes of Health included 82 patients with various forms of lipodystrophy., Intervention: Metreleptin (0.06-0.24 mg/kg/d) was administered for 24 months in lipodystrophy., Main Outcome Measures: Serum triglycerides and HDL-C were measured., Results: At baseline, lipodystrophy patients had low HDL-C (30 ± 1 mg/dL) and high triglycerides (961 ± 220 mg/dL) with an inverse relationship between the two (R = -0.37, P = .0006). There was no change in HDL-C with metreleptin despite major improvement in triglycerides, and individual changes in triglycerides only weakly predicted HDL-C change. On linear regression, in obesity, a decrease of 0.1 mg/dL in log(triglycerides) was associated with a 4.2 mg/dL rise in HDL-C, whereas in lipodystrophy, a decrease of 0.1 mg/dL in log(triglycerides) was associated with only a 0.6 mg/dL rise in HDL-C., Conclusions: The normal reciprocal relationship between triglyceride and HDL-C change seen in response to interventions for the obesity-associated metabolic syndrome is quantitatively different from that seen in lipodystrophy in response to metreleptin. Further work is needed to understand HDL-C regulation in this condition.

Context: Rabson-Mendenhall syndrome (RMS) is caused by mutations of the insulin receptor and results in extreme insulin resistance and dysglycemia. Hyperglycemia in RMS is very difficult to treat, and patients are at risk for early morbidity and mortality from complications of diabetes., Objective: Our objective was to study 1-year effects of recombinant human methionyl leptin (metreleptin) in 5 patients with RMS and 10-year effects in 2 of these patients., Design and Setting: We conducted an open-label nonrandomized study at the National Institutes of Health., Patients: Patients were adolescents with RMS and poorly controlled diabetes., Intervention: Two patients were treated with escalating doses (0.02 up to 0.22 mg/kg/d) of metreleptin for 10 years, including 3 cycles of metreleptin withdrawal and reinitiation. In all 5 patients, 1-year effects of metreleptin (0.22 mg/kg/d) were studied., Outcome Measures: Hemoglobin A1c (HbA1c) and body mass index (BMI) z-scores were evaluated every 6 months., Results: HbA1c decreased from 11.4% ± 1.1% at baseline to 9.3% ± 1.9% after 6 months and 9.7% ± 1.6% after 12 months of metreleptin (P = .007). In patients treated for 10 years, HbA1c declined with each cycle of metreleptin and rose with each withdrawal. BMI z-scores declined from -1.4 ± 1.8 at baseline, to -2.6 ± 1.6 after 12 months of metreleptin (P = .0006). Changes in BMI z-score correlated with changes in HbA1c (P < .0001)., Conclusions: Metreleptin treatment for 12 months was associated with a 1.7% reduction in HbA1c; part of this improvement was likely mediated via decreased BMI. Metreleptin is a promising treatment option for RMS, but additional therapies are needed to achieve HbA1c targets.

The gut microbiota contributes to fat mass and the susceptibility to obesity. However, the underlying mechanisms are not completely understood. To investigate whether the gut microbiota affects hypothalamic and brainstem body fat-regulating circuits, we compared gene expression of food intake-regulating neuropeptides between germ-free and conventionally raised (CONV-R) mice. We found that CONV-R mice had decreased expression of the antiobesity neuropeptide glucagon-like peptide-1 (GLP-1) precursor proglucagon (Gcg) in the brainstem. Moreover, in both the hypothalamus and the brainstem, CONV-R mice had decreased expression of the antiobesity neuropeptide brain-derived neurotrophic factor (Bdnf). CONV-R mice had reduced expression of the pro-obesity peptides neuropeptide-Y (Npy) and agouti-related protein (Agrp), and increased expression of the antiobesity peptides proopiomelanocortin (Pomc) and cocaine- and amphetamine-regulated transcript (Cart) in the hypothalamus. The latter changes in neuropeptide expression could be secondary to elevated fat mass in CONV-R mice. Leptin treatment caused less weight reduction and less suppression of orexigenic Npy and Agrp expression in CONV-R mice compared with germ-free mice. The hypothalamic expression of leptin resistance-associated suppressor of cytokine signaling 3 (Socs-3) was increased in CONV-R mice. In conclusion, the gut microbiota reduces the expression of 2 genes coding for body fat-suppressing neuropeptides, Gcg and Bdnf, an alteration that may contribute to fat mass induction by the gut microbiota. Moreover, the presence of body fat-inducing gut microbiota is associated with hypothalamic signs of Socs-3-mediated leptin resistance, which may be linked to failed compensatory body fat reduction.

Objective: Chemerin is an adipocyte-secreted hormone and has recently been associated with obesity and the metabolic syndrome. Although studies in rodents have outlined the aspects of chemerin's function and expression, its physiology and expression patterns are still to be elucidated in humans., Methods: To evaluate for any day/night variation in chemerin secretion, we analyzed hourly serum samples from six females in the fed state. To examine whether energy deprivation affects chemerin levels, and whether this could be mediated through leptin, we analyzed samples from the same subjects in the fasting state while administering either placebo or leptin. To evaluate for any potential dose-effect relationship between leptin and chemerin, we administered increasing metreleptin doses to five females. A tissue array was used to study the expression of chemerin in different human tissues. Ex vivo treatment of human fat explants from three subjects with leptin was carried out to evaluate for any direct effect of leptin on adipocyte chemerin secretion., Results: Chemerin does not display a day/night variation, while acute energy deprivation resulted in a significant drop in circulating chemerin levels by ∼42%. The latter was unaltered by metreleptin administration, and leptin administration did not affect the secretion of chemerin by human adipose tissue studied ex vivo. Chemerin was expressed primarily in the pancreas and liver. Chemerin receptor showed increased expression in the lymph nodes and the spleen., Conclusions: We outline for the first time chemerin expression and physiology in humans, which are different from those in mice.

In rodent models of type 1 diabetes, leptin administration into brain ventricles normalizes blood glucose at doses that have no effect when given peripherally. The ventromedial nucleus of the hypothalamus (VMN) is a potential target for leptin's antidiabetic effects because leptin-sensitive neurons in this brain area are implicated in glucose homeostasis. To test this hypothesis, we injected leptin directly into the bilateral VMN of rats with streptozotocin-induced uncontrolled diabetes mellitus. This intervention completely normalized both hyperglycemia and the elevated rates of hepatic glucose production and plasma glucagon levels but had no effect on tissue glucose uptake in the skeletal muscle or brown adipose tissue as measured using tracer dilution techniques during a basal clamp. To determine whether VMN leptin signaling is required for leptin-mediated normalization of diabetic hyperglycemia, we studied mice in which the leptin receptor gene was deleted in VMN steroidogenic factor 1 neurons using cre-loxP technology. Our findings indicate leptin action within these neurons is not required for the correction of diabetic hyperglycemia by central leptin infusion. We conclude that leptin signaling in the VMN is sufficient to mediate leptin's antidiabetic action but may not be necessary for this effect. Leptin action within a distributed neuronal network may mediate its effects on glucose homeostasis.

Leptin receptors (ObRs) in the forebrain and hindbrain have been independently recognized as important mediators of leptin responses. It is unclear how leptin activity in these areas is integrated. We tested whether both forebrain and hindbrain ObRs have to be activated simultaneously to change energy balance and to maintain metabolic homeostasis. Previous studies used acute leptin injections in either the third ventricle (1-5 μg) or the fourth ventricle (3-10 μg); here we used 12-day infusions of low doses of leptin in one or both ventricles (0.1 μg/24 h in third, 0.6 μg/24 h in fourth). Male Sprague Dawley rats were fitted with third and fourth ventricle cannulas, and saline or leptin was infused from Alzet pumps for 6 or 12 days. Rats that received leptin into only the third or the fourth ventricle were not different from controls that received saline in both ventricles. By contrast, rats with low-dose leptin infusions into both the third and fourth ventricle showed a dramatic 60% reduction in food intake that was reversed on day 6, a 20% weight loss that stabilized on day 6, and a 50% decrease in body fat at day 12 despite the correction of food intake. They displayed normal activity and maintained energy expenditure despite weight loss, indicating inappropriately high thermogenesis that coincided with increased signal transducer and activator of transcription 3 (STAT3) phosphorylation in the brainstem. Altogether, these findings show that with low doses of leptin, chronic activation of both hypothalamic and brainstem ObRs is required to reduce body fat.

Context: Leptin has nitric oxide (NO)-dependent vasodilator actions, but hyperleptinemia is an independent risk factor for cardiovascular disease., Objective: The objective of the study was to investigate whether, in the human circulation, properties of leptin to release NO are opposed by stimulation of vasculotoxic substances, such as endothelin (ET)-1, and whether this mechanism might contribute to vascular damage in hyperleptinemic states like obesity., Methods: Forearm blood flow responses (plethysmography) to ETA receptor antagonism (BQ-123, 10 nmol/min) and NO synthase inhibition [N(G)-monomethyl L-arginine (L-NMMA), 4 μmol/min] were assessed before and after intraarterial administration of leptin (2 μg/min) in lean controls (n = 8) and patients with obesity-related metabolic syndrome (MetS; n = 8)., Results: Baseline plasma leptin was higher in patients than in controls (P < .001). Before infusion of leptin, the vasodilator response to BQ-123 was greater in patients than in controls (P < .001), whereas infusion of L-NMMA induced higher vasoconstriction in controls than in patients (P = .04). In lean subjects, hyperleptinemia resulted in increased vasodilator response to ETA receptor antagonism (P < .001 vs before) and enhanced vasoconstrictor effect of L-NMMA during ETA receptor blockade (P = .015 vs before). In patients with the MetS, by contrast, vascular responses to both BQ-123 and L-NMMA were not modified by exogenous leptin (both P > .05 vs before)., Conclusions: These findings indicate that, under physiological conditions, leptin stimulates both ET-1 and NO activity in the human circulation. This effect is absent in hyperleptinemic patients with the MetS who are unresponsive to additional leptin. In these patients, therefore, hyperleptinemia may be a biomarker of vascular dysfunction, rather than a mediator of vascular damage.

Several studies have demonstrated that maternal undernutrition or overnutrition during pregnancy can have negative consequences for the health of children born to these pregnancies, but the physiological mechanisms by which this occurs are not completely understood. During periods of food restriction, concentrations of leptin decline, whereas leptin is elevated in obesity, suggesting that it may play a role in the response to altered nutrition during pregnancy. This study compares placental development and global placental gene expression profiles at Day 11.5 in pregnant control mice, mice that were undernourished, and mice that were undernourished but given leptin. Placentas from mothers exposed to food restriction preserved the placental labyrinth zone at the expense of the junctional zone, an effect abrogated in the restricted plus leptin group, which had a significant decrease in the labyrinth zone area compared with controls. Similarly, there were more significant differences in gene expression between placentas from control and restricted plus leptin mothers (1128 differentially expressed genes) than between placentas of control and restricted mothers (281 differentially expressed genes). We conclude that the presence of high concentrations of circulating leptin during food restriction disrupts the normal adaptive response of the placenta to reduced energy availability.

Context: Lipodystrophy is a disease characterized by a paucity of adipose tissue and low circulating concentrations of adipocyte-derived leptin. Leptin-replacement therapy improves eating and metabolic disorders in patients with lipodystrophy., Objective: The aim of the study was to clarify the pathogenic mechanism of eating disorders in lipodystrophic patients and the action mechanism of leptin on appetite regulation., Subjects and Interventions: We investigated food-related neural activity using functional magnetic resonance imaging in lipodystrophic patients with or without leptin replacement therapy and in healthy controls. We also measured the subjective feelings of appetite., Results: Although there was little difference in the enhancement of neural activity by food stimuli between patients and controls under fasting, postprandial suppression of neural activity was insufficient in many regions of interest including amygdala, insula, nucleus accumbens, caudate, putamen, and globus pallidus in patients when compared with controls. Leptin treatment effectively suppressed postprandial neural activity in many of these regions of interest, whereas it showed little effect under fasting in patients. Consistent with these results, postprandial formation of satiety feeling was insufficient in patients when compared with controls, which was effectively reinforced by leptin treatment., Conclusions: This study demonstrated the insufficiency of postprandial suppression of food-related neural activity and formation of satiety feeling in lipodystrophic patients, which was effectively restored by leptin. The findings in this study emphasize the important pathological role of leptin in eating disorders in lipodystrophy and provide a clue to understanding the action mechanism of leptin in human, which may lead to development of novel strategies for prevention and treatment of obesity.

The effects of leptin on bone are controversial. Although in vitro studies have shown that leptin stimulates osteoblast differentiation and mineralization and inhibits osteoclastogenesis, some rodent studies have shown that leptin administered centrally might result in decreased bone formation. In the present study we have investigated the skeletal effects of supraphysiological concentrations of leptin administered sc to rats. Female Fischer rats were given leptin 100 μg/d, 200 μg/d, or saline by continuous infusion for 9 wk. Bone mineral density (BMD) was measured by dual energy x-ray absorptiometry, bone microarchitecture was analyzed by micro-computed tomography, and biomechanical properties were tested by three-point bending experiments. At the end of the study, the body weight was significantly lower in rats receiving leptin compared with controls (-10.8% and -12.0% in low- and high-dose leptin groups, respectively). The high-dose leptin group also significantly lost weight compared with baseline. The plasma leptin concentration was 14- and 33-fold increased in the low- and high-dose groups, respectively. No significant differences in femoral BMD were observed. Whole-body BMD was significantly lower in the low-dose leptin group, whereas there was no difference between the high-dose leptin group and the control. Mechanical strength and microarchitecture were similar in the high-dose and the control group. The low-dose group, however, had decreased cortical volume in the femoral metaphysis, lowered bone strength, and altered moment of inertia. In conclusion, leptin given at very high doses maintains BMD, microarchitecture, and mechanical strength in female rats, despite a significant decrease in body weight.

Context: Leptin replacement therapy improves metabolic complications in patients with lipodystrophy and severe hypoleptinemia (SH), but whether the response is related to the degree of hypoleptinemia remains unclear., Objective: The aim of the study was to compare efficacy of leptin therapy in familial partial lipodystrophy, Dunnigan variety (FPLD) patients with SH (serum leptin<7th percentile of normal) vs. those with moderate hypoleptinemia (MH; serum leptin in 7th to 20th percentiles)., Design, Setting, and Patients: We conducted an open-label, parallel group, observational study in 14 SH (mean±sd, serum leptin, 1.9±1.1 ng/ml) and 10 MH (serum leptin, 5.3±1.0 ng/ml) women with FPLD., Intervention: Patients received 0.08 mg/kg·d of metreleptin by twice daily sc injections for 6 months., Main Outcome Measures: The primary outcome variable was change in fasting serum triglycerides. Other secondary variables were fasting plasma glucose and insulin, insulin sensitivity, hemoglobin A1c, and hepatic triglyceride content., Results: Median fasting serum triglycerides decreased from 228 to 183 mg/dl in the SH group (P=0.04) and from 423 to 339 mg/dl in the MH group (P=0.02), but with no difference between the groups (P value for interaction=0.96). Hepatic triglyceride levels similarly declined significantly from 8.8 to 4.9% in the SH group and from 23.7 to 9.2% in the MH group (P value for interaction=0.9). Loss of body weight and body fat occurred in both groups. Fasting glucose, insulin, glucose tolerance, and hemoglobin A1c levels did not change. K value on insulin tolerance test improved slightly in the SH group (0.98 to 1.24%; P=0.01), but not in the MH group (1.1 to 1.27%; P=0.4)., Conclusion: Metreleptin replacement therapy is equally effective in FPLD patients with both SH and MH in reducing serum and hepatic triglyceride levels, but did not improve hyperglycemia.

Resistance to brain-mediated effects of leptin is a characteristic feature of obesity, resulting from alterations in leptin receptor signaling in hypothalamic neurons and/or transport across the blood-brain-barrier. We have shown previously, that the latter can be circumvented by intranasal (i.n.) application of leptin in lean rats. This prompted us to test i.n. leptin in animals with diet-induced obesity (DIO) as a basis for future human administration. DIO was induced in male Wistar rats by feeding a cafeteria diet for 25 or 32 wk, respectively. Consecutively, these DIO animals (seven to eight per treatment) and standard diet rats (lean) (14-15 per treatment, matched for age and diet duration) were treated with 0.1, 0.2 mg/kg leptin, or control solution i.n. daily for 4 wk before onset of dark period. Energy intake and body weight were measured daily; blood glucose, serum insulin, and leptin were measured before and after treatment. Expression of hypothalamic neuropeptides was assessed by quantitative real-time PCR. We demonstrate, for the first time, that i.n. leptin reduces appetite and induces weight loss in DIO to the same extent as in lean rats. Our findings are supported accordingly by an altered expression pattern of anorexigenic and orexigenic neuropeptides in the hypothalamus, e.g. proopiomelanocortin, cocaine and amphetamine-related transcript, neuropeptide Y, agouti-related protein. It now appears clear that i.n. leptin is effectively acting in obese animals in the same fashion as in their lean counterparts. These findings now clearly warrant studies in humans and may open new perspectives in the treatment of obesity.

Context: Activin A, myostatin, and follistatin have recently emerged as important regulatory molecules of reproduction and the musculoskeletal system. Little is known, however, about their day/night patterns of secretion and their physiological regulation by energy availability., Objective: The objective of the study was to explore day/night patterns of secretion and assess whether energy deprivation alters circulating levels of activin A, myostatin, follistatin, and cortisol and to examine whether leptin may mediate this effect. DESIGN, SETTING AND PATIENTS, AND INTERVENTIONS: Seven healthy lean men (aged 23.2 ± 3.7 yr, body mass index 23.6 ± 1.7 kg/m(2)) were studied for 72 h under three different conditions: on their baseline/isocaloric diet and in a complete fasting state with administration of either placebo or metreleptin. The two fasting studies were randomized and double blinded. Blood samples were obtained every 15 min from 0800 h on d 3 until 0800 h on d 4 and pooled hourly., Main Outcome Measures: Serum concentrations of activin A, myostatin, follistatin, cortisol, and leptin were measured., Results: In contrast to cortisol, we demonstrated no day/night pattern of activin A, myostatin, and follistatin secretion. Activin A concentrations decreased significantly in response to energy deprivation (P < 0.01). Follistatin and cortisol concentrations increased significantly (P < 0.01 and P < 0.01, respectively). Myostatin remained unaffected (P = 0.40). Leptin administration reversed cortisol response (P < 0.01) but failed to alter activin A, follistatin, or myostatin concentrations., Conclusions: Unlike cortisol, there is no day/night variation in the concentrations of activin A, myostatin, and follistatin in healthy young males. Although energy deprivation-induced cortisol changes are leptin mediated, the changes in follistatin and activin A concentrations occur through a leptin-independent pathway.

Although acute leptin administration in the hypothalamus decreases food intake and increases peripheral energy metabolism, the peripheral actions of central chronic leptin administration are less understood. In this study, we investigated what effects chronic (7 d) intracerebroventricular (ICV) administration of leptin has on energy metabolism and insulin sensitivity in diet-induced obese mice. C57/BL mice were fed a low-fat diet (LFD; 10% total calories) or high-fat diet (HFD; 60% total calories) for 8 wk after which leptin was administered ICV for 7 consecutive days. Mice fed a HFD showed signs of insulin resistance, as evidenced by an impaired glucose tolerance test. Chronic leptin treatment resulted in a decrease in food intake and body weight and normalization of glucose clearance but no improvement in insulin sensitivity. Chronic ICV leptin increased hypothalamic signal transducer and activator of transcription-3 and AMP-activated protein kinase phosphorylation but did not change hypothalamic malonyl CoA levels in HFD fed and LFD-fed mice. In the gastrocnemius muscles, the levels of malonyl CoA in both leptin-treated groups were lower than their respective control groups, suggesting an increase in fatty acid oxidation. However, only in the muscles of ICV leptin-treated LFD mice was there a decrease in lipid metabolites including diacylglycerol, triacylglycerol, and ceramide. Our results suggest that chronic ICV leptin decreases food consumption and body weight via a mechanism different from acute ICV leptin administration. Although chronic ICV leptin treatment in HFD mice improves glucose tolerance, this occurs independent of changes in insulin sensitivity in the muscles of HFD mice.

Context: Leptin affects neurogenesis, neuronal growth, and viability. We previously reported that leptin supplementation increased gray matter (GM) concentration in the anterior cingulate gyrus (ACG), cerebellum, and inferior parietal lobule, areas that are also involved in food intake., Objective: The aim of this study was to report the changes in brain structure at different states of leptin supplementation., Design: We conducted a nonrandomized trial., Setting and Patients: We studied three adults with congenital leptin deficiency due to a mutation in the leptin gene., Intervention: Patients received treatment with recombinant methionyl human leptin, with annual 11- to 36-d periods of treatment withholding followed by treatment restoration over 3 yr., Main Outcome Measures: GM concentration (by voxel-based morphometry analysis of magnetic resonance scans) was correlated with body mass index (BMI) and leptin supplementation., Results: Annually withholding leptin supplementation for several weeks increased BMI and reversed the original effects of leptin in the cerebellum and ACG. The changes in the ACG were consistent with an indirect effect of leptin mediated through increased BMI. In the cerebellum, where leptin receptors are most dense, GM changes appeared to be direct effects of leptin. Leptin restoration did not lead to recovery of GM in the short term but did lead to an unexpected GM increase in the posterior half of the left thalamus, particularly the pulvinar nucleus., Conclusion: These findings provide the first in vivo evidence of remarkably plastic, reversible, and regionally specific effects of leptin on human brain morphology. They suggest that leptin may have therapeutic value in modulating plasticity-dependent brain functions.

Objective: Short-term energy deprivation reduces leptin concentrations and alters the levels of circulating hormones of the hypothalamic-pituitary-peripheral axis in lean subjects. Whether the reduction in leptin concentration during long-term weight loss in obese individuals is linked to the same neuroendocrine changes seen in lean, leptin-sensitive subjects remains to be fully clarified., Methods: In this study, 24 overweight and obese adults (16 women and eight men; body mass index (BMI): 27.5-38.0 kg/m(2)) were prescribed a hypocaloric diet (-500 kcal/day) and were randomized to receive recombinant methionyl leptin (n=18, metreleptin, 10 mg/day self-injected s.c.) or placebo (n=6, same volume and time as metreleptin) for 6 months., Results: Metreleptin administration did not affect weight loss beyond that induced by hypocaloric diet alone (P for interaction=0.341) but increased the serum concentrations of total leptin by six- to eight-fold (P<0.001) and led to the generation of anti-leptin antibodies. Despite free leptin concentration (P for interaction=0.041) increasing from 9±1 ng/ml at baseline to 43±15 and 36±12 ng/ml at 3 and 6 months, respectively, changes in circulating hormones of the thyroid and IGF axes at 3 and 6 months were not significantly different in the placebo- and metreleptin-treated groups., Conclusions: Leptin does not likely mediate changes in neuroendocrine function in response to weight loss induced by a mild hypocaloric diet in overweight and obese subjects.

Recent epidemiological studies indicate that obesity increases the incidence of depression. We examined the implication of leptin for obesity-associated depression. Leptin induced antidepressive behavior in normal mice in a forced swimming test (FST), and leptin-overexpressing transgenic mice with hyperleptinemia exhibited more antidepressive behavior in the FST than nontransgenic mice. In contrast, leptin-deficient ob/ob mice showed more severe depressive behavior in the FST than normal mice, and leptin administration substantially ameliorated this depressive behavior. Diet-induced obese (DIO) mice fed a high-fat diet showed more depressive behavior in the FST and in a sucrose preference test compared with mice fed a control diet (CD). In DIO mice, leptin induced neither antidepressive action nor increment of the number of c-Fos immunoreactive cells in the hippocampus. Diet substitution from high-fat diet to CD in DIO mice ameliorated the depressive behavior and restored leptin-induced antidepressive action. Brain-derived neurotrophic factor concentrations in the hippocampus were significantly lower in DIO mice than in CD mice. Leptin administration significantly increased hippocampal brain-derived neurotrophic factor concentrations in CD mice but not in DIO mice. The antidepressant activity of leptin in CD mice was significantly attenuated by treatment with K252a. These findings demonstrated that leptin induces an antidepressive state, and DIO mice, which exhibit severe depressive behavior, did not respond to leptin in both the FST and the biochemical changes in the hippocampus. Thus, depression associated with obesity is due, at least in part, to impaired leptin activity in the hippocampus.

Leptin acts on the brain to increase postprandial heat production in skeletal muscle of sheep. To determine a mechanism for this effect, we examined the role of mitochondrial uncoupling and AMP-activated protein kinase (AMPK). Ovariectomized ewes (n=4/group) received infusion lines into the lateral cerebral ventricle, and leptin (10 μg/h) was infused to increase heat production in skeletal muscle. In animals that were program fed (1100-1600 h), skeletal muscle biopsies were taken after either central infusion of leptin or vehicle to measure the expression of uncoupling protein (UCP) mRNA and the phosphorylation status of AMPK. Respiratory function was also quantified in mitochondria isolated from skeletal muscle. Leptin infusion increased the expression of UCP2 and UCP3 mRNA as well as UCP3 protein but not UCP1 mRNA in muscle. Leptin also increased substrate-driven, coupled (ADP-driven), and uncoupled (oligomycin) respiration but had no effect on the total respiratory capacity. The respiratory control ratio was lower in leptin-treated (vs. vehicle-treated) animals, indicating a predominant effect on uncoupled respiration. There was no effect of central leptin treatment on AMPK phosphorylation. We then infused 5-aminoimidazole-4-carboxamide-1β-riboside (AICAR) (10 mg/h for 6 h) directly into the femoral artery and measured skeletal muscle temperature; muscle was also collected for isolated mitochondria studies. AICAR had no effect on heat production or substrate-driven, uncoupled, or total respiratory states in skeletal muscle mitochondria. However, AICAR increased ADP-driven (coupled) respiration in mitochondria. In conclusion, leptin acts at the brain to increase heat production in muscle through altered mitochondrial function, indicative of adaptive thermogenesis.

The efficacy of exercise as primary prevention of obesity is the subject of intense investigation. Here, we show that voluntary exercise in a mouse strain susceptible to diet-induced obesity (C57B6J) decreases fat mass and increases energy expenditure. In addition, exercise attenuates obesity in mice fed a high-fat diet (HFD). Using FosB immunoreactivity as a marker of chronic neuronal activation, we found that exercise activates leptin receptor-positive neurons in the ventromedial hypothalamic nucleus, involved in homeostatic control of energy balance. FosB immunoreactivity in the ventromedial hypothalamic nucleus is decreased in sedentary mice exposed to HFD but is increased in exercised mice independent of adiposity. To determine whether the antiobesity effects of voluntary exercise improve central nervous system (CNS) leptin action, we measured the anorectic and weight reducing effects of intracerebroventricular (ICV) leptin in sedentary and exercised mice exposed to HFD (EH), as well as in sedentary mice that have been calorie restricted (SR) to match the fat mass of EH mice. ICV leptin was ineffective in lowering food intake and body weight (BW) in sedentary mice exposed to HFD mice. The anorectic potency of leptin was partially restored in EH and SR groups. However, ICV leptin significantly lowered BW in EH but not SR mice. Thus, exercise leads to the maintenance of a lower BW and leaner composition, as well as to improved CNS leptin action, independent of fat mass. These results support the notion that physical exercise directly influences the responsiveness of the CNS circuits involved in energy homeostasis by allowing the defense of a lowered BW.

Astrocytes participate in neuroendocrine functions partially through modulation of synaptic input density in the hypothalamus. Indeed, glial ensheathing of neurons is modified by specific hormones, thus determining the availability of neuronal membrane space for synaptic inputs, with the loss of this plasticity possibly being involved in pathological processes. Leptin modulates synaptic inputs in the hypothalamus, but whether astrocytes participate in this action is unknown. Here we report that astrocyte structural proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, are induced and astrocyte morphology modified by chronic leptin administration (intracerebroventricular, 2 wk), with these changes being inversely related to modifications in synaptic protein densities. Similar changes in glial structural proteins were observed in adult male rats that had increased body weight and circulating leptin levels due to neonatal overnutrition (overnutrition: four pups/litter vs. control: 12 pups/litter). However, acute leptin treatment reduced hypothalamic GFAP levels and induced synaptic protein levels 1 h after administration, with no effect on vimentin. In primary hypothalamic astrocyte cultures leptin also reduced GFAP levels at 1 h, with an induction at 24 h, indicating a possible direct effect of leptin. Hence, one mechanism by which leptin may affect metabolism is by modifying hypothalamic astrocyte morphology, which in turn could alter synaptic inputs to hypothalamic neurons. Furthermore, the responses to acute and chronic leptin exposure are inverse, raising the possibility that increased glial activation in response to chronic leptin exposure could be involved in central leptin resistance.

The purpose of this work was to study the central mechanisms involved in food intake regulation and leptin resistance during gestation in the rat. Sprague Dawley rats of 7, 13, and 18 d of pregnancy [days of gestation (G) 7, G13, and G18] were used and compared with nonpregnant animals in diestrus-1. Food intake was already increased in G7, before hyperleptinemia and central leptin resistance was established in midpregnancy. Leptin resistance was due to a reduction in leptin transport through the blood-brain barrier (BBB) and to alterations in leptin signaling within the hypothalamus based on an increase in suppressor of cytokine signaling 3 levels and a blockade of signal transducer and activator of transcription-3 phosphorylation (G13), followed by a decrease in LepRb and of Akt phosphorylation (G18). In early gestation (G7), no change in hypothalamic neuropeptide Y (NPY), agouti-related peptide (AgRP), or proopiomelanocortin (POMC) expression was shown. Nevertheless, an increase in NPY and AgRP and a decrease in POMC mRNA were observed in G13 and G18 rats, probably reflecting the leptin resistance. To investigate the effect of maternal vs. placental hormones on these mechanisms, we used a model of pseudogestation. Rats of 9 d of pseudogestation were hyperphagic, showing an increase in body and adipose tissue weight, normoleptinemia, and normal responses to iv/intracerebroventricular leptin on hypothalamic leptin signaling, food intake, and body weight. Leptin transport through the BBB, and hypothalamic NPY, AgRP and POMC expression were unchanged. Finally, the transport of leptin through the BBB was assessed using a double-chamber culture system of choroid plexus epithelial cells or brain microvascular endothelial cells. We found that sustained high levels of prolactin significantly reduced leptin translocation through the barrier, whereas progesterone and β-estradiol did not show any effect. Our data demonstrate a dual mechanism of leptin resistance during mid/late-pregnancy, which is not due to maternal hormones and which allows the maintenance of hyperphagia in the presence of hyperleptinemia driven by an increase in NPY and AgRP and a decrease in POMC mRNA. By contrast, in early pregnancy maternal hormones induce hyperphagia without the regulation of hypothalamic NPY, AgRP, or POMC and in the absence of leptin resistance.

Context: Recently, in a 4-month proof-of-concept trial, beneficial metabolic effects were reported in non-diabetic children with Berardinelli-Seip congenital lipodystrophy (BSCL); this information prompted us to hypothesize that long-term leptin-replacement therapy might improve or reverse the early complications of the disease in these patients., Patients and Methods: A 28-month trial was implemented in eight patients. Efficacy assessment was based on a decrease in serum triglyceride concentrations, and/or a decrease in liver volume and/or an increase in insulin sensitivity of at least 30% respectively. The response was defined as follows: total (3/3 positive criteria), partial (1 or 2/3), or negative (0/3). Anti-leptin antibodies were measured with a radiobinding assay, and a neutralizing effect was assessed in primary cultures of embryonic neurons incubated with an apoptotic agent (N-methyl-D-aspartate) and the patient serum, with or without leptin., Results: A negative or partial response to treatment was observed in five of eight patients even when leptin dosages were increased. A displaceable leptin binding was detectable in all patients after 2 months of treatment. At 28 months, binding was higher in the patients with a negative response than in the total responders, and it paralleled both the increase in leptin dosage and serum leptin concentrations. Co-incubation of embryonic neurons with serum from two patients with a negative response inhibited the neuroprotective effect of leptin., Conclusion: Under leptin therapy, patients with BSCL may develop a resistance to leptin, which could be partly of immunological origin, blunting the previously reported beneficial effects.

Kisspeptin stimulates reproduction, and kisspeptin cells in the arcuate nucleus (ARC) express Ob-Rb in the mouse. Herein we report studies in ewes to determine whether kisspeptin cells express Ob-Rb and respond to leptin and whether reciprocal connections exist between kisspeptin cells and proopiomelanocortin (POMC) or neuropeptide Y (NPY) cells to modulate reproduction and metabolic function. Kiss1 mRNA was measured by in situ hybridization in ovariectomized ewes that were normal body weight, lean, or lean with leptin treatment by intracerebroventricular (icv) infusion (4 microg/h, 3 d). Kiss1 expression in the ARC and the preoptic area was lower in hypogonadotropic lean animals than animals of normal weight, and icv infusion of leptin partially restored Kiss1 expression in lean animals. Single-cell laser capture microdissection coupled with real-time PCR showed that Kiss1 cells in the preoptic area and ARC express Ob-Rb. Double-label fluorescent immunohistochemistry showed that reciprocal connections exist between kisspeptin cells and NPY and POMC cells. Accordingly, we treated ovariectomized ewes with kisspeptin (5 microg/h, icv) or vehicle for 20 h and examined POMC and NPY gene expression by in situ hybridization. Kisspeptin treatment reduced POMC and increased NPY gene expression. Thus, kisspeptin neurons respond to leptin and expression of Kiss1 mRNA is affected by leptin status. Kisspeptin cells communicate with NPY and POMC cells, altering expression of the relevant genes in the target cells; reciprocal connections also exist. This network between the three cell types could coordinate brain control of reproduction and metabolic homeostatic systems.

It is widely accepted that leptin acts on first-order neurons in the arcuate nucleus (ARC) with information then relayed to other hypothalamic centers. However, the extent to which leptin mediates its central actions solely, or even primarily, via this route is unclear. We used a model of hypothalamo-pituitary disconnection (HPD) to determine whether leptin action on appetite-regulating systems requires the ARC. This surgical preparation eliminates the ARC. We measured effects of iv leptin to activate hypothalamic neurons (Fos labeling). In ARC-intact animals, leptin increased the percentage of Fos-positive melanocortin neurons and reduced percentages of Fos-positive neuropeptide Y neurons compared with saline-treated animals. HPD itself increased Fos labeling in the lateral hypothalamic area (LHA). Leptin influenced Fos labeling in the dorsomedial nucleus (DMH), ventromedial nucleus, and paraventricular nucleus (PVN) in HPD and normal animals, with effects on particular cell types varying. In the LHA and DMH, leptin decreased orexin cell activation in HPD and ARC-intact sheep. HPD abolished leptin-induced expression of Fos in melanin-concentrating hormone cells in the LHA and in CRH cells in the PVN. In contrast, HPD accentuated activation in oxytocin neurons. Our data from sheep with lesions encompassing the ARC do not suggest a primacy of action of leptin in this nucleus. We demonstrate that first order to second order signaling may not represent the predominant means by which leptin acts in the brain to generate integrated responses. We provide evidence that leptin exerts direct action on cells of the DMH, ventromedial nucleus, and PVN.

Acute leptin increase as well as endogenous hyperleptinemia evoked by high-fat diets (HF) activate fatty acid metabolism in nonadipose tissues. This supports the notion that hyperleptinemia is pivotal to prevent/delay steatosis during periods of positive energy balance. We have previously shown that long-term HF spares ectopic accumulation of lipids specifically in the miocardium. Because carnitine palmitoyltransferase I (CPT-I) allows mitochondrial uptake/oxidation of fatty acids, we have hypothesized that leptin drives cardiac CPT-I activity. In the current study, hyperleptinemia was induced in C57BL/6J mice either by exogenous leptin administration or by means of HF, and the ability of malonyl-coenzyme A (malonyl-CoA) (the main endogenous inhibitor of CPT-I) to inhibit cardiac CPT was analyzed. IC(50) values of malonyl-CoA were 8.1 +/- 1.5 micromol/liter in controls vs. 69.3 +/- 5.2 micromol/liter (P < 0.01) in leptin-treated mice. This effect was also observed in cardiac explants incubated with leptin and was blocked by triciribine, a compound shown to inhibit protein kinase B (Akt) phosphorylation (pAkt). In accordance, acute leptin evoked an increase of cardiac pAkt levels, which correlated with CPT sensitivity to malonyl-CoA. Otherwise, the inhibitory effect of malonyl-CoA was hindered in HF hyperleptinemic mice, and in this case, pAkt levels also correlated with CPT sensitivity to malonyl-CoA. Our data show that leptin reduces the sensitivity of cardiac CPT-I to malonyl-CoA and suggest the involvement of an Akt-related signaling pathway in this effect. This mechanism appears to be sensitive to both acute and chronic hyperleptinemia. We conclude that this action of leptin is pivotal to drive cardiac metabolism under situations associated to hyperleptinemia.

The adipose-derived hormone leptin communicates information about metabolic status to the hypothalamic GnRH neuronal system. It is unclear whether leptin can act directly on GnRH neurons. To examine this, we used three approaches. First, the presence of leptin-induced signal transducer and activator of transcription-3 activation was examined in GnRH neurons in male and female rats. Intracerebroventricular treatment with 4 mug leptin-induced robust signal transducer and activator of transcription-3 expression within the anteroventral periventricular nucleus but not in GnRH neurons. Second, fertility was assessed in male and female CRE-loxP transgenic mice with conditional leptin receptor (Lepr) deletion from either all forebrain neurons or GnRH neurons only. Forebrain neuron LEPR deletion prevented the onset of puberty resulting in infertility in males and females and blocked estradiol-induced LH surge. However, mice with GnRH neuron-selective Lepr deletion exhibited normal fertility apart from a slight puberty delay in males. Lastly, the highly sensitive technique of single-cell nested PCR was used to test for Lepr transcript presence in individual GnRH neurons, identified in situ using GnRH-green fluorescent protein transgenics. Whereas 75% of positive control (proopiomelanocortin) neurons contained Lepr mRNA, no (none of 18) GnRH neurons were Lepr mRNA positive. Collectively, these results show that leptin does not act directly on GnRH neurons in rats and mice. Leptin appears to regulate GnRH function via forebrain neurons that are afferent to GnRH because forebrain neuronal LEPR deletion caused infertility. The location and phenotype of these leptin-responsive neurons remains to be elucidated.

Of the central nervous system receptors that could mediate the energy balance effects of leptin, those of the hypothalamic arcuate nucleus receive the greatest attention. Melanocortin receptors (MC-Rs) contribute to the feeding and energetic effects of hypothalamically delivered leptin. Energy balance effects of leptin are also mediated by extrahypothalamic neurons including the hindbrain nucleus tractus solitarius. Hindbrain leptin receptors play a role in leptin's anorectic effects, but their contribution to its energetic effects and their functional interaction with melanocortin systems within the hindbrain remains unexplored. Here rats implanted with telemetric devices for recording energetic/cardiovascular responses were examined to determine whether: 1) hindbrain (fourth ventricular) leptin receptor stimulation triggers energetic and cardiovascular effects, 2) these effects are altered by a 6-wk high-fat diet maintenance, and 3) hindbrain MC-Rs mediate the thermogenic, cardiovascular, and anorexic effects of hindbrain leptin delivery. Results show that hindbrain leptin receptor stimulation produced long-lasting (>6 h) increases in core temperature and heart rate and also decreased food intake and body weight. These responses were not altered by high-fat maintenance, in contrast to what has been reported for forebrain leptin delivery. Fourth ventricular pretreatment with MC-R antagonist SHU 9119 completely abolished the hyperthermia, anorexia, and body weight loss seen with hindbrain-directed leptin but had no effects of its own. These data highlight a role for hindbrain leptin receptors in the initiation of energetic and anorexic responses and show that MCRs are part of the downstream mediation of hindbrain leptin-induced energy balance effects, paralleling effects observed for hypothalamic leptin receptors.

Objective: Leptin is an adipocyte secreted hormone and an important regulator of neuroendocrine, metabolic, and immune function. Both r-metHuLeptin and IGF1 administration result in reduced central adipose tissue in subjects with highly active antiretroviral therapy-induced metabolic syndrome (HAART-MS) but whether the effects of leptin are mediated through increasing IGF levels remains unknown., Methods: To assess whether r-metHuLeptin improves the HAART-MS by regulating circulating IGF and IGFBPs, we first conducted a cross-sectional study of 118 men and women with HIV infection and >6 months of exposure to antiretroviral medications to examine any association between circulating IGF1 and leptin levels. We also performed a randomized, double-blinded, placebo-controlled, crossover trial of recombinant human leptin (r-metHuLeptin) administration to seven HIV positive men with lipoatrophy and leptin deficiency (leptin <3 ng/ml) related to antiretroviral medication use., Results: In the observational study, leptin levels were inversely associated with circulating IGF1 levels after adjusting for age and gender (r=0.27 P=0.002), but this inverse association became non-significant after adjustment for % body fat and exercise. In the interventional leptin study, leptin levels increased significantly during r-metHuLeptin treatment (from 1.34+/-0.20 ng/ml at baseline to 17+/-5.05 ng/ml after 8 weeks P=0.046) and metabolic parameters improved including reduced fasting insulin levels and reduced homeostasis model assessment-insulin resistance (HOMA-IR). Despite the increase in circulating leptin levels, there was no change in IGF1, IGF2, free IGF1, or IGF-binding proteins during the 2-month treatment period., Conclusion: The effects of r-metHuLeptin in patients with HAART-MS are not mediated through increasing IGF or IGFBP levels.

Obesity and type 2 diabetes are associated with insulin and leptin resistance, and increased ceramide contents in target tissues. Because the adipose tissue has become a central focus in these diseases, and leptin-induced increases in insulin sensitivity may be related to effects of leptin on lipid metabolism, we investigated herein whether central leptin was able to regulate total ceramide levels and the expression of enzymes involved in ceramide metabolism in rat white adipose tissue (WAT). After 7 d central leptin treatment, the total content of ceramides was analyzed by quantitative shotgun lipidomics mass spectrometry. The effects of leptin on the expression of several enzymes of the sphingolipid metabolism, sterol regulatory element binding protein (SREBP)-1c, and insulin-induced gene 1 (INSIG-1) in this tissue were studied. Total ceramide levels were also determined after surgical WAT denervation. Central leptin infusion significantly decreased both total ceramide content and the long-chain fatty acid ceramide species in WAT. Concomitant with these results, leptin decreased the mRNA levels of enzymes involved in de novo ceramide synthesis (SPT-1, LASS2, LASS4) and ceramide production from sphingomyelin (SMPD-1/2). The mRNA levels of enzymes of ceramide degradation (Asah1/2) and utilization (sphingomyelin synthase, ceramide kinase, glycosyl-ceramide synthase, GM3 synthase) were also down-regulated. Ceramide-lowering effects of central leptin were prevented by local autonomic nervous system denervation of WAT. Finally, central leptin treatment markedly increased INSIG-1 mRNA expression and impaired SREBP-1c activation in epididymal WAT. These observations indicate that in vivo central leptin, acting through the autonomic nervous system, regulates total ceramide levels and SREBP-1c proteolytic maturation in WAT, probably contributing to improve the overall insulin sensitivity.

Previously, we reported that combination treatment with rat amylin (100 microg/kg.d) and murine leptin (500 microg/kg.d) elicited greater inhibition of food intake and greater body weight loss in diet-induced obese rats than predicted by the sum of the monotherapy conditions, a finding consistent with amylin-induced restoration of leptin responsiveness. In the present study, a 3 x 4 factorial design was used to formally test for a synergistic interaction, using lower dose ranges of amylin (0, 10, and 50 microg/kg.d) and leptin (0, 5, 25, and 125 microg/kg.d), on food intake and body weight after 4 wk continuous infusion. Response surface methodology analysis revealed significant synergistic anorexigenic (P < 0.05) and body weight-lowering (P < 0.05) effects of amylin/leptin combination treatment, with up to 15% weight loss at doses considerably lower than previously reported. Pair-feeding (PF) experiments demonstrated that reduction of food intake was the predominant mechanism for amylin/leptin-mediated weight loss. However, fat loss was 2-fold greater in amylin/leptin-treated rats than PF controls. Furthermore, amylin/leptin-mediated weight loss was not accompanied by the counterregulatory decrease in energy expenditure and chronic shift toward carbohydrate (rather than fat) utilization observed with PF. Hepatic gene expression analyses revealed that 28 d treatment with amylin/leptin (but not PF) was associated with reduced expression of genes involved in hepatic lipogenesis (Scd1 and Fasn mRNA) and increased expression of genes involved in lipid utilization (Pck1 mRNA). We conclude that amylin/leptin interact synergistically to reduce body weight and adiposity in diet-induced obese rodents through a number of anorexigenic and metabolic effects.

The metabolic effects of leptin may involve both centrally and peripherally mediated actions with a component of the central actions potentially independent of alterations in food intake. Ob/ob mice have significant abnormalities in lipid metabolism, correctable by leptin administration. We used ob/ob mice to study the relative importance of the subtypes of actions of leptin (central vs. peripheral; food intake dependent vs. independent) on lipid metabolism. Mice were treated for 3 d with leptin, either centrally [intracerebroventricular (icv)] or peripherally (ip), and compared with mice pair-fed to the leptin-treated mice (PF) and with ad libitum-fed controls (C). All treatment groups (icv, ip, PF) showed indistinguishable changes in liver weight; hepatic steatosis; hepatic lipidemic profile; and circulating free fatty acids, triglycerides, and cholesterol lipoprotein profile. Changes in the expression of genes involved in lipogenesis and fatty acid oxidation in liver, muscle, and white fat were broadly similar in ip, icv, and PF groups. Leptin (both icv and ip) stimulated expression of both mitochondrial and peroxisomal acyl-coenzyme A oxidase (liver) and peroxisomal proliferator-activated receptor-alpha (skeletal muscle) to an extent not replicated by pair feeding. Leptin had profound effects on peripheral lipid metabolism, but the majority were explained by its effects on food intake. Leptin had additional centrally mediated effects to increase the expression of a limited number of genes concerned with fatty acid oxidation. Whereas we cannot exclude direct peripheral effects of leptin on certain aspects of lipid metabolism, we were unable to detect any such effects on the parameters measured in this study.

Leptin acts not only as an anorexigenic hormone but also regulates cell-mediated immunity via leptin receptors (Ob-R) expressed on T and B lymphocytes. However, the impact of leptin on natural killer (NK) cells is currently elusive. We evaluated leptin effects on NK cells in relation to the body weight in rats using in vivo and in vitro approaches. Leptin was injected iv in male lean and diet-induced obese Lewis and F344 rats. NK cell numbers were analyzed in blood and spleen by fluorescence activated cell sorting and immunohistochemistry, and the activity of NK cells was measured by chromium release assay. Ob-R expression was investigated by confocal laser scanning and quantitative RT-PCR. To compare leptin-dependent intracellular signaling under basal and leptin- and tumor cell (MADB106)-stimulated conditions, intracellular target proteins of NK cells were evaluated by Western blotting. Number and distribution pattern of splenic NK cells were significantly different in lean and obese animals. Leptin administration resulted in a 4-fold higher stimulation of the NK activity in lean than obese animals. This was not due to a decreased expression of Ob-R because quantitative RT-PCR revealed significantly higher Ob-Rb mRNA levels in NK cells from obese rats. In contrast, postreceptor signaling is differentially abrogated in obese animals with significantly lower activation of postreceptor signaling components (Janus kinase-2p, protein kinase B pT308, AMPalphapT172) after an in vivo leptin challenge. In conclusion, the results for the first time assign leptin a central role as a modulator of NK cell number and activity only in lean but not obese subjects. The differential role of leptin has important implications for the influence of body weight in the response to systemic inflammations and in the immunological defense of cancer.

Leptin reduces adiposity and exerts antisteatotic effects on nonadipose tissues. However, the mechanisms underlying leptin effects on lipid metabolism in liver and white adipose tissue have not been fully clarified. Here, we have studied the effects of central leptin administration on key enzymes and transcription factors involved in lipid metabolism in liver and epididymal adipose tissue. Intracerebroventricular leptin infusion for 7 d did not change leptin plasma levels but decreased triacylglyceride content in liver, epididymal adipose tissue, and plasma. In both tissues this treatment markedly decreased the expression of key enzymes of the de novo fatty acid (FA) synthesis such as acetyl-coenzyme A-carboxylase, FA synthase, and stearoyl-coenzyme A desaturase-1, in parallel with a reduction in mRNA expression of sterol regulatory element binding protein-1c in liver and carbohydrate regulatory element binding protein in adipose tissue. In addition, leptin also decreased phosphoenol-pyruvate carboxykinase-C expression in adipose tissue, an enzyme involved in glyceroneogenesis in this tissue. Central leptin administration down-regulates delta-6-desaturase expression in liver and adipose tissue, in parallel with the decrease of the expression of sterol regulatory element binding protein-1c in liver and peroxisome proliferator activated receptor alpha in adipose tissue. Finally, leptin treatment, by regulating adipose triglyceride lipase/hormone sensitive lipase/diacylglycerol transferase 1 expression, also established a new partitioning in the FA-triacylglyceride cycling in adipose tissue, increasing lipolysis and probably the FA efflux from this tissue, and favoring in parallel the FA uptake and oxidation in the liver. These results suggest that leptin, acting at central level, exerts tissue-specific effects in limiting fat tissue mass and lipid accumulation in nonadipose tissues, preventing the development of obesity and type 2 diabetes.

Recent evidence suggests that the peptide hormone prolactin (PRL) modulates energy balance through a number of mechanisms, including acting in the brain to increase food intake. In the current studies, we first demonstrated that chronic infusions of PRL into the lateral ventricles increased food intake in cycling rats without disrupting estrous cyclicity. In subsequent experiments the hypothesis that at least part of PRL's ability to increase food intake resulted from PRL-induced leptin resistance was tested. Female rats given chronic infusions of PRL (5 microg/h) into the cerebral ventricles for 10 d did not show a reduction in food intake or body weight after a central injection of 4 microg murine leptin, whereas the expected reduction in both of these parameters was seen in vehicle-infused rats. Leptin injections were without effect on these parameters, whether they were administered to free feeding PRL-infused rats or after 24-h food deprivation. This lack of a behavioral response to leptin was accompanied by an attenuation in Fos induction and phosphorylation of signal transducer and activator of transcription 3 after leptin administration in PRL-infused rats in both the ventromedial hypothalamus and paraventricular hypothalamic nucleus.

Hypothalamic neurons expressing the long form of the leptin receptor (LRb) mediate important leptin actions. Although it has been suggested that leptin crosses the blood-brain barrier (BBB) via a specific transport system, we hypothesized the existence of a population of hypothalamic arcuate nucleus (ARC) neurons that senses leptin independently of this transport system. Indeed, endogenous circulating leptin results in detectable levels of baseline activated signal transducer and activator of transcription 3 (STAT3) phosphorylation in a population of ARC/LRb neurons, consistent with increased sensing of circulating leptin in these neurons compared with other LRb neurons. Furthermore, a population of ARC/LRb neurons that responds more rapidly and sensitively to circulating leptin compared with other hypothalamic LRb neurons detected by leptin activated phosphorylated STAT3. In addition, peripheral application of the BBB-impermeant retrograde tracer fluorogold revealed a population of ARC/LRb neurons that directly contact the circulation (e.g. via neuronal processes reaching outside the BBB). Taken together, these data suggest that a population of ARC/LRb neurons directly contacts the circulation and displays increased sensitivity to circulating leptin compared with neurons residing entirely behind the BBB elsewhere in the hypothalamus.

Peripheral infusions of physiological doses of leptin decrease body fat mass, but it is not known whether this results from direct effects on peripheral tissue or activation of central leptin receptors. In this study, we infused chronically decerebrate (CD) rats, in which the forebrain was surgically isolated from the caudal brainstem, with 60 microg leptin/d or PBS for 14 d from ip mini-osmotic pumps. The CD rats were tube fed an amount of food equivalent to the intake of ad libitum-fed intact controls or 75% of this amount to account for their reduced energy expenditure. Control rats fed ad libitum or tube fed 75, 100, or 125% of their ad libitum intake also were peripherally infused with leptin or PBS. CD rats had a lower serum testosterone, energy expenditure, and lean body mass compared with controls but had increased levels of adiponectin and leptin and were obese. Leptin increased body fat and decreased energy expenditure during the light period in 100%-fed CD rats, but not 75%-fed CD rats. Leptin decreased body fat of ad libitum- and 100%-fed but not 75%-fed or 125%-fed intact controls. Energy expenditure did not change in any control group. These results show that leptin can change body fat independent of a change in food intake or energy expenditure, that the forebrain normally prevents leptin from inhibiting energy expenditure through mechanisms initiated in the caudal brainstem or peripheral tissues, and that the leptin response in both intact and CD rats is determined by the energy status of the animal.

Published data describing leptin effects on bone are at variance with both positive and negative consequences reported. These findings are consistent with a bimodal threshold response to serum leptin levels. To test this theory, two groups of female rats (tail-suspended and unsuspended) were treated with ip leptin at two different doses or vehicle for 14 d. In tail-suspended rats, low-dose leptin compensated the decrease in serum leptin levels observed with suspension and was able to prevent the induced bone loss at both the trabecular and cortical level (assessed by three-dimensional microtomography). In contrast, high-dose leptin inhibited femoral bone growth and reduced bone mass by decreasing bone formation rate and increasing bone resorption in both tail-suspended and unsuspended groups. High- and low-dose leptin administration resulted in a reduced medullar adipocytic volume in all groups. High-dose leptin (but not low) induced a decrease in body-weight abdominal fat mass and serum IGF-I levels. Thus, the observed bone changes at high-dose leptin are at least partly mediated by a leptin-induced energy imbalance. In conclusion, a balance between negative and positive leptin effects on bone is dependent on a bimodal threshold that is triggered by leptin serum concentration. Also, the negative effects of high leptin levels are likely induced by reduced energy intake and related hormonal changes. The respective part of each pathway will be unraveled by additional studies.

Context: Recombinant human leptin (r-metHuLeptin) has demonstrated efficacy in improving hormonal and metabolic parameters in leptin-deficient states, but pharmacokinetic parameters after sc administration have not yet been published. In addition, the effect of potential variability across different leptin assays on concentration-dependent pharmacokinetic parameters remains unknown., Objective: The objective of the study was to characterize pharmacokinetic parameters after sc r-metHuLeptin administration using three commercially available leptin assays (Linco, Diagnostic Systems Laboratories, and Alpco)., Design, Setting, Patients, and Intervention: We analyzed pharmacokinetic profiles in five lean and five obese men after sc administration of physiological (0.01 mg/kg) and pharmacological (0.3 mg/kg) doses of r-metHuLeptin., Main Outcome Measures: Leptin pharmacokinetic parameters were measured., Results: Measurement of leptin produced typical pharmacokinetic profiles in all assays with time to maximal concentration and half-life of approximately 3 h. Diagnostic Systems Laboratories consistently measured leptin higher than Linco, with Alpco measuring intermediate between or similar to Linco. There was high correlation among assays (R(2) ranging from 0.89 to 0.98, all P < 0.01). Concentration-dependent parameters such as maximal concentration, area under the curve, and clearance were significantly different among assays, whereas concentration-independent parameters such as time to maximal concentration and half-life were generally not different., Conclusions: We report novel data on leptin pharmacokinetic parameters after sc administration, which will be relevant for the future therapeutic use of r-metHuLeptin. Although commercially available assays demonstrated high correlation, they can provide substantially different measures of leptin levels. This demonstrates the importance of standardizing leptin assays for diagnosing patients with relative leptin deficiency, determining appropriate doses of r-metHuLeptin for administration, and monitoring response to therapy.

A recombinantly produced murine leptin analog (MLA) antagonizes leptin-induced signaling in cell lines that express the long form of the leptin receptor. However, the effects of MLA on the activity of leptin-sensitive neurons and on central neural controls of food intake have not been reported. Here we report effects of MLA on food intake and body weight in adult rats and on the activity of cultured rat vagal afferent neurons. Daily intracerebroventricular coinjection of MLA with exogenous leptin significantly attenuated leptin-induced reduction of 48-h food intake and body weight. Coinjection of MLA with leptin also reduced leptin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3) in the hypothalamus. In addition, chronic intracerebroventricular MLA infusion over 14 d via osmotic minipumps significantly increased daily food intake, rate of body weight gain, fat-pad mass, and circulating plasma leptin concentrations. Surprisingly, however, MLA did not antagonize leptin-evoked increases in cytosolic calcium concentrations in vagal afferent neurons in primary culture. Rather, MLA itself produced acute activation selectively in leptin-responsive vagal afferent neurons. These data suggest that MLA is an antagonist for the central effects of leptin on food intake and body weight but an agonist at sites where leptin induces acute neuronal activation. This mixed antagonist/agonist action suggests either 1) that the coupling of a single leptin receptor (ObRb) to acute activation of neurons occurs by a signaling mechanism different from those that mediate centrally evoked reductions in food intake and body weight or 2) that acute neuronal activation and centrally induced reductions of food intake and body weight are mediated by different leptin receptor subtypes.

Background: Lack of leptin is implicated in insulin resistance and other metabolic abnormalities in generalized lipodystrophy; however, the efficacy, safety, and underlying mechanisms of leptin-replacement therapy in patients with generalized lipodystrophy remain unclear., Methods: Seven Japanese patients with generalized lipodystrophy, two acquired and five congenital type, were treated with the physiological replacement dose of recombinant leptin during an initial 4-month hospitalization followed by outpatient follow-up for up to 36 months., Results: The leptin-replacement therapy with the twice-daily injection dramatically improved fasting glucose (mean +/- SE, 172 +/- 20 to 120 +/- 12 mg/dl, P < 0.05) and triglyceride levels (mean +/- SE, 700 +/- 272 to 260 +/- 98 mg/dl, P < 0.05) within 1 wk. The leptin-replacement therapy reduced insulin resistance evaluated by euglycemic clamp method and augmented insulin secretion at glucose tolerance test with different responses between acquired and congenital types. Improvement of the fatty liver was also observed. The efficacy and safety of the once-daily injection were comparable to those of the twice-daily injection. The leptin-replacement therapy ameliorated macro- and microalbuminuria and showed no deterioration of neuropathy and retinopathy of these patients. The leptin-replacement therapy is beneficial to diabetic complications and lipodystrophic ones. Two patients developed antileptin antibodies but not neutralizing antibodies. The therapy was well tolerated, and its effects were maintained for up to 36 months without any notable adverse effects such as hypoglycemia, high blood pressure, or reduction of bone mineral density., Conclusions: The present study demonstrates the efficacy and safety of the long-term leptin-replacement therapy and possible mechanisms of leptin actions in patients with generalized lipodystrophy.