Administration Of Recombinant Leptin Research Articles

POS-043 DEVELOPMENT AND MANIFESTATION OF CHRONIC KIDNEY DISEASE IN GENERALIZED CONGENITAL LIPODYSTROPHY: REPORT OF A CHILEAN CASE AND REVIEW OF THE LITERATURE

POS-043 DEVELOPMENT AND MANIFESTATION OF CHRONIC KIDNEY DISEASE IN GENERALIZED CONGENITAL LIPODYSTROPHY: REPORT OF A CHILEAN CASE AND REVIEW OF THE LITERATURE

Interplay of adipocyte and hepatocyte: Leptin upregulates hepcidin

Conflicting evidence concerning leptin, an adipocyte-derived hormone, in atherogenesis and non-alcoholic fatty liver disease (NAFLD) has been reported. Iron metabolism and iron-mediated oxidative stress should be taken into consideration for the clarification of the pathogenesis of these diseases. In this study, we demonstrate that leptin receptor activation directly affects iron metabolism by the finding that serum levels of hepcidin, the master regulator of iron in the whole body, were significantly lower in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice. The administration of recombinant leptin to ob/ob mice for two weeks showed a significant increase in serum hepcidin and hepatic Hamp mRNA levels. Hamp mRNA levels were significantly correlated with hepatic iron content and BMP6 mRNA levels. Hepatic iron content was associated with the increase in mRNA levels of divalent metal transporter 1 and transferrin receptor. Our data provide evidence that the interplay of these two hormones could help improve the understanding of the pathogenesis of atherosclerosis and NAFLD.

Protein and Calorie Restriction Contribute Additively to Protection from Renal Ischemia Reperfusion Injury Partly via Leptin Reduction in Male Mice1–3

Background: Short-term dietary restriction (DR) without malnutrition preconditions against surgical stress in rodents; however, the nutritional basis and underlying nutrient/energy-sensing pathways remain poorly understood.Objectives: We investigated the relative contribution of protein restriction (PR) vs. calorie restriction (CR) to protection from renal ischemia reperfusion injury (IRI) and changes in organ-autonomous nutrient/energy-sensing pathways and hormones underlying beneficial effects.Methods: Mice were preconditioned on experimental diets lacking total calories (0–50% CR) or protein/essential amino acids (EAAs) vs. complete diets consumed ad libitum (AL) for 1 wk before IRI. Renal outcome was assessed by serum markers and histology and integrated over a 2-dimensional protein/energy landscape by geometric framework analysis. Changes in renal nutrient/energy-sensing signal transduction and systemic hormones leptin and adiponectin were also measured. The genetic requirement for amino acid sensing via general control non-derepressible 2 (GCN2) was tested with knockout vs. control mice. The involvement of the hormone leptin was tested by injection of recombinant protein vs. vehicle during the preconditioning period.Results: CR-mediated protection was dose dependent up to 50% with maximal 2-fold effect sizes. PR benefits were abrogated by EAA re-addition and additive with CR, with maximal benefits at any given amount of CR occurring with a protein-free diet. GCN2 was not required for functional benefits of PR. Activation and repression of nutrient/energy-sensing kinases, AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1), respectively, on PR reflected a state of negative energy balance, paralleled by 13% weight loss and an 87% decrease in leptin, independent of calorie intake. Recombinant leptin administration partially abrogated benefits of dietary preconditioning against renal IRI.Conclusions: In male mice, PR and CR both contributed to the benefits of short-term DR against renal IRI independent of GCN2 but partially dependent on reduced circulating leptin and coincident with AMPK activation and mTORC1 repression.

Reanalysis of parabiosis of obesity mutants in the age of leptin

In this study we set out to explain the differing effects of parabiosis with genetically diabetic (db) mice versus administration of recombinant leptin. Parabiosis of db mutant, which overexpress leptin, to wildtype (WT) or genetically obese (ob) mice has been reported to cause death by starvation, whereas leptin infusions do not produce lethality at any dose or mode of delivery tested. Leptin is not posttranslationally modified other than a single disulphide bond, raising the possibility that it might require additional factor(s) to exert the maximal appetite-suppressing effect. We reconfirmed the lethal effect of parabiosis of db mutant on WT mice and further showed that this lethality could not be rescued by administration of ghrelin or growth hormone. We then initiated a biochemical fractionation of a high-molecular-weight leptin complex from human plasma and identified clusterin as a major component of this leptin-containing complex. However, in contrast to previous reports, we failed to observe a leptin-potentiating effect of either exogenous or endogenous clusterin, and parabiosis of db clusterin(-/-) double-mutant to WT mice still caused lethality. Intriguingly, in parabiotic pairs of two WT mice, leptin infusion into one of the mice led to an enhanced starvation response during calorie restriction as evidenced by increased plasma ghrelin and growth-hormone levels. Moreover, leptin treatment resulted in death of the parabiotic pairs. These data suggest that the appetite suppression in WT mice after parabiosis to db mutants is the result of induced hyperleptinemia combined with the stress or other aspect(s) of the parabiosis procedure.

Hepatic endothelin-1 and endocannabinoids-dependent effects of hyperleptinemia in nonalcoholic steatohepatitis-cirrhotic rats

Leptin, the ob gene product, is a protein released from adipocytes and has been detected in fibrotic and cirrhotic livers. Leptin in brain has an inhibitory effect on food intake. Nonalcoholic steatohepatitis (NASH) is characterized by hyperleptinemia. This study explores the possible mechanisms of hyperleptinemia in relation to increased intrahepatic resistance (IHR) and portal hypertension in NASH cirrhotic rats. NASH cirrhotic rats with hyperleptinemia were induced in Zucker (fa/fa) and lean rats by feeding the animals a high fat/methionine-choline-deficient (HF/MCD) diet with and without exogenous administration of recombinant leptin. Portal venous pressure (PVP), IHR, plasma and hepatic levels of various substances, histopathology of the liver, the hepatic hydroxyproline content, and the expression of various hepatic protein and messenger RNA (mRNA) were measured. Hepatic microcirculatory dysfunction and the vasoconstrictive response to endothelin-1 were also observed using a liver perfusion system and intravital microscopy. Finally, the effect of leptin on hepatic stellate cells (HSCs) was evaluated. Both in HF/MCD-Zucker and HF/MCD+leptin lean rats, significant hepatic fibrogenesis and cirrhosis, marked portal hypertension, microcirculatory dysfunction, an enhanced vasoconstrictive response to endothelin-1, and an increased IHR were found to be associated with higher levels of hepatic endothelin-1 and endocannabinoids, expression levels of the cannabinoid type 1 receptor, endothelin-1 type A receptor (ET(A) R), activator protein-1, transforming growth factor beta (TGF-β)(1), osteopontin, tumor necrosis factor alpha (TNF-α), leptin, and the leptin receptor (OBRb). Interestingly, acute incubation of leptin directly increases the expression of ET(A) R, OBRb and activator protein-1 in HSCs. An HF/MCD diet and hyperleptinemia increase hepatic endocannabinoids production, promote hepatic fibrogenesis, enhance the hepatic vasoconstrictive response to endothelin-1, and aggravate hepatic microcirculatory dysfunction; these events subsequently increase IHR and portal hypertension in NASH cirrhotic rats.

Human Recombinant Leptin Administration as a Potential Obesity Therapy

Obesity is an escalating global health problem and established cardiovascular risk factor for the development of cardiovascular and metabolic diseases. Leptin derived from the adipose tissue acts centrally to suppress appetite and increase energy expenditure, therefore leptin resistance is considered as an important mechanism for the onset and maintenance of obesity; Leptin analogues as well as leptin receptor blockers have been candidates for the treatment of obesity, however clinical trials in obese populations have yielded variable results. Given specific circumstances in obese subjects could optimize the beneficial actions of the hormone and minimize its deleterious effects. In this review, human recombinant leptin (pegylated polyethylene glycol recombinant human leptin (PEG-OB) and recombinant methionyl human leptin (r-metHuLeptin) is developed to control resistant and morbid obesity will be discussed. Keywords: Obesity, leptin, pegylated human recombinant leptin, recombinant methionyl human leptin, myocardial hypertrophy, dyslipidemia, hyperleptinemia, hypothalamic pro-opiomelanocortin, anorectic effects, neuroendocrine, hypothalamus, thermogenesis, sympathoectomy, retinopathy, nephropathy, genetic mutation, myocardium, atherosclerosis, thrombosis, arterial hypertension, neointinal hyperplasia, blood-brain barrier, norepinephrine spillover, melanocortin system, neuropeptide mediators, Pegylated Polyethylene Glycol, pramlintide, triglyceride, somatotropic axes, plasminogen activator, adiponectin, ghrelin

Leptin increases thromboxane A<sub>2</sub> formation in the rat

Chronic hyperleptinemia may contribute to various complications of obesity including atherosclerosis, however, the underlying mechanisms are incompletely clear. We examined the effect of leptin on platelet activity by measuring stable metabolites of thromboxane A 2 (TXA 2 ), TXB 2 , 11-dehydro-TXB 2 and 2,3-dinor-TXB 2 , in plasma and urine. In vitro, leptin stimulated TXB2 formation by platelet-rich plasma (PRP). In vivo, leptin (1 mg/kg ip.) increased urinary excretion of 11-dehydro-TXB 2 and 2,3-dinor-TXB 2 . Urinary excretion of these metabolites was also elevated in rats made hyperleptinemic by administration of recombinant leptin (0.5 mg/kg/day) for 8 days. The stimulatory effect of leptin on TXB 2 formation in PRP isolated from hyperleptinemic animals was impaired in comparison to the control group. In rats made obese, hyperleptinemic and hyperinsulinemic/insulin resistant by cafeteria diet administered for 3 months, acute stimulatory effect of leptin on TXB 2 formation by PRP was not impaired. In rats made insulin resistant by fructose feeding for 8 weeks, stimulatory effect of leptin on TXB 2 formation in PRP was augmented in comparison to the control group. Insulin sensitizer, rosiglitazone, decreased insulin level and attenuated the stimulatory effect of leptin on TXB 2 formation in obese and fructose-fed animals. In contrast, rosiglitazone had no effect on insulin level or leptin-induced TXB 2 formation in control rats and rats receiving recombinant leptin for 8 days. These results indicate that: (i) leptin stimulates platelet TXA 2 formation both in vitro and in vivo, (ii) chronic hyperleptinemia impairs acute stimulatory effect of leptin on platelet activity if insulin sensitivity is normal, (iii) insulin resistance/hyperinsulinemia augments the stimulatory effect of leptin on TXA 2 formation, which results in normal platelet sensitivity to leptin in obesity associated with both hyperleptinemia and hyperinsulinemia, and (iv) PPAR-γ agonists such as rosiglitazone decrease platelet sensitivity to leptin by reducing insulin resistance. Adipobiology 2009; 1: 77-85.

Regulation of energy balance by leptin.

The high prevalence of obesity and its well documented association with the cardiovascular risk factors diabetes mellitus, dyslipidemia and hypertension represents a major problem for the general health status of industrialized societies. Although numerous studies have shown that genetic factors have a major influence on the regulation of energy homeostasis and the susceptibility to obesity, the genes and predisposing mutations involved are insufficiently understood. Among several known rodent models of obesity due to single gene mutations, mice homozygous for the obese (ob) gene exhibit massive early-onset obesity, hyperphagia, non-insulin-dependent diabetes mellitus, defective thermoregulation and infertility. Recently the ob gene was identified by positional cloning and shown to be mutated in ob/ob mice. Leptin, the product of the ob gene, is a 167-amino acid secreted protein that is synthesized exclusively in adipose tissue. With the exception of ob/ob mice, circulating plasma leptin is elevated in obesity. Administration of recombinant leptin to ob/ob mice reduces fat mass, food intake, hyperglycemia and hyperinsulinemia. The various effects of the hormone are mediated by leptin receptors expressed at high levels in the hypothalamus, but also in several other non-neuronal tissues. A mutation in the leptin receptor gene is responsible for the obese phenotype of db/db mice. Plasma leptin in humans is positively correlated with body fat mass, suggesting that leptin resistance rather than leptin deficiency is a common feature of human obesity. This review briefly summarizes the current status of the rapidly growing evidence that leptin plays an important role in the regulation of body weight and fat deposition.

The Pathophysiology of Amenorrhea in the Adolescent

Menstrual irregularity is a common occurrence during adolescence, especially within the first 2-3 years after menarche. Prolonged amenorrhea, however, is not normal and can be associated with significant medical morbidity, which differs depending on whether the adolescent is estrogen-deficient or estrogen-replete. Estrogen-deficient amenorrhea is associated with reduced bone mineral density and increased fracture risk, while estrogen-replete amenorrhea can lead to dysfunctional uterine bleeding in the short term and predispose to endometrial carcinoma in the long term. In both situations, appropriate intervention can reduce morbidity. Old paradigms of whom to evaluate for amenorrhea have been challenged by recent research that provides a better understanding of the normal menstrual cycle and its variability. Hypothalamic amenorrhea is the most prevalent cause of amenorrhea in the adolescent age group, followed by polycystic ovary syndrome. In anorexia nervosa, exercise-induced amenorrhea, and amenorrhea associated with chronic illness, an energy deficit results in suppression of hypothalamic secretion of GnRH, mediated in part by leptin. Administration of recombinant leptin to women with hypothalamic amenorrhea has been shown to restore LH pulsatility and ovulatory menstrual cycles. The use of recombinant leptin may improve our understanding of the pathophysiology of hypothalamic amenorrhea in adolescents and may also have therapeutic possibilities.

Burn wound angiogenesis is increased by exogenously administered recombinant leptin in rats

BACKGROUND: Leptin is a potent direct angiogenic factor that stimulates endothelial cell migration and activation in vitro and angiogenesis in vivo. In addition, leptin has been discussed to play an important role in angiogenesis, as it promotes the formation of new blood vessels. PURPOSE: The effect of exogenously administered leptin on the healing process of a full tissue burn wound model. METHODS: Sixty-three Sprague-Dawley male rats were used. Full tissue burn wound was created by electrocautery. The width of the pin was 0.3 cm; its length was 2 cm and was used at the "cut" modulation. Rats were divided into seven groups of nine animals each. Burn wounds were injected with murine recombinant leptin and the rats were sacrificed 3, 7 and 9 days after surgery. Every group had obtained three animals for the three different days of sacrifice. Three different leptin doses of 250 pg/ml, 500 pg/ml and 1000 pg/ml were used in different animal groups (A, B and C). For every one of the three leptin doses used, another animal group was evaluated by using the combined injection of leptin and antileptin (A1, B1, and C1), in order to study the inhibitory effect to the leptin factor. Nine rats were served as controls. These were injected with 0.3 ml water for injection solution and sacrificed at the same time intervals. After sacrifice of the animals, the skin was grossly determined by its appearance, colour and texture. Full thickness burn wounds were dissected for histological examination. A qualitative analysis of angiogenesis in the burn wound was conducted following a standard hematoxylin and eosin stain. The wound tissue samples from each experimental group underwent immunohistochemical evaluation of microvessel density by endothelial cell staining with mouse anti-rat CD 34 monoclonal antibody. RESULTS: The most impressive growth of new blood vessels appeared seven and nine days after treatment with the highest leptin doses. There were no significant differences in microvessel density between the seventh and the ninth postoperative day among different groups treated with leptin. All wounds from the control group, as well as those from animal groups treated with the combined injection of leptin and antileptin did not develop any new vessels. CONCLUSION: Exogenous administration of recombinant leptin increases early tissue angiogenesis in the burn wound level of an experimental animal model.

Transplantation of wild-type white adipose tissue normalizes metabolic, immune and inflammatory alterations in leptin-deficient ob/ ob mice

Leptin-deficient ob/ ob mice exhibit several metabolic and immune abnormalities, including thymus atrophy and markedly reduced inflammatory responses. We evaluated whether transplantation of wild-type (WT) white adipose tissue (WAT) into ob/ ob mice could mimic the effect of recombinant leptin administration in normalizing metabolic, immune and inflammatory abnormalities. Female ob/ ob mice received a subcutaneous transplantation of WAT obtained from WT littermates. A separate group of ob/ ob mice was sham-operated. Despite raising leptin levels to only 15% of those observed in WT mice, WAT transplantation normalized metabolic abnormalities (glycemia, ALT, liver weight) in ob/ ob mice and prevented further body weight gain. The transplanted group demonstrated normalization of thymus and spleen cellularity, thymocyte subpopulations and rates of thymocyte apoptosis. In the model of dextran sulfate sodium-induced colitis, WAT transplantation restored inflammation to levels equivalent to those of WT mice. Colonic production of IL-6 and MIP-2 was markedly reduced in the non-transplanted ob/ ob group compared to transplanted ob/ ob and WT mice. Our data indicate that WAT transplantation is an effective way to normalize metabolic as well as immune and inflammatory parameters in ob/ ob mice. The threshold of leptin sufficient to normalize metabolic, immune and inflammatory function is significantly lower than levels present in lean WT mice. Finally, leptin derived exclusively from WAT is sufficient to normalize metabolic, immune and inflammatory parameters in ob/ ob mice.

Severe pulmonary metastasis in obese and diabetic mice

Although obesity is known as a risk factor for several human cancers, the association of obesity with cancer recurrence and metastasis remains to be characterized. Here, B16-BL6 melanoma and Lewis lung carcinoma cells were intravenously injected into diabetic (db/db) and obese (ob/ob) mice. The number of experimental lung colonies was markedly promoted in these mice when compared with C57BL/6 mice. In contrast, tumor growth at the implanted site was comparable when cells were inoculated orthotopically. The use of B16-BL6 cells stably transfected with the luciferase gene revealed that the increased metastasis reflected a difference mainly within 6 hr after the intravenous inoculation of tumor cells. Administration of recombinant leptin in ob/ob mice abolished the increase in metastasis early on as well as the decrease in the splenic NK cell number. In addition, depletion of NK cells by an anti-asialo-GM1 antibody abrogated the enhanced metastasis in db/db mice. These results demonstrate that metastasis is markedly promoted in diabetic and obese mice mainly because of decreased NK cell function during the early phase of metastasis.

Leptin and inhibin B as predictors of reproductive recovery in patients with anorexia nervosa during weight gain

Anorexia nervosa represents an eating disorder that is associated with substantial psychological, social and physiological abnormalities, involving 0.5-2% of female population. The secretion patterns of inhibin B, as marker of gonadal activity, and leptin, as an indicator of energy balance and body composition, were analyzed in our cross-sectional study in order to asses the restoration of reproductive function in patients with anorexia nervosa (AN) during gaining of normal weight. The study included 20 patients with low weight AN (BMI 14.3 +/- 0.3 kg/m2), 22 partially recovered AN (BMI 17.4 +/- 0.1 kg/m2), and 29 gained regular weight, out of whom 16 had no restoration of menstrual cycle (BMI 19.5 +/- 0.1 kg/m2), and 13 had at least six consecutive menstrual cycles (BMI 19.3 +/- 1.0 kg/m2). Nineteen eumenorrheic females with BMI 19.8 +/- 0.4 kg/m2 were the controls. Significant correlation between leptin and inhibin B (c = 0.446; p = 0.000), leptin and delta LH (c = 0.611; p < 0.001), and inhibin B and delta LH (c = 0.574; p < 0.001) was found in patients with anorexia nervosa during weight gain. Leptin (p = 0.0039), inhibin B (p = 0.0173), LH (p = 0.0323) and delta LH (p = 0.0087) were important predictors of reproductive recovery in patients with anorexia nervosa during gaining of normal weight. Among aforementioned parameters, leptin (p = 0.0057) appeared to be the most important. Leptin is the most important predictor of reproductive recovery in patients with anorexia nervosa during weight normalization. These findings suggest that decreased leptin levels may be responsible for several neuroendocrine abnormalities seen in anorexia nervosa. Thus, interventional studies involving administration of recombinant leptin are required to fully clarify the physiologic and potentially therapeutic role of leptin in anorexia nervosa.

Central Leptin Acutely Reverses Diet-Induced Hepatic Insulin Resistance

Voluntary overfeeding rapidly induces resistance to the effects of systemic insulin and leptin on liver glucose metabolism. To examine whether central administration of recombinant leptin can restore leptin and insulin action on liver glucose fluxes, we infused leptin in the third cerebral ventricle of conscious overfed rats during pancreatic-insulin clamp studies. The effect of leptin on the phosphorylation of the signal transducer and activator of transcription-3 in the arcuate nuclei of the hypothalamus was similar in animals fed a regular diet or a high-fat diet for 3 days. The infusion of leptin in the third cerebral ventricle markedly inhibited glucose production in rats fed a high-fat diet mainly by decreasing glycogenolysis. The inhibition of glycogenolysis was sufficient to normalize glucose production and was accompanied by leptin-induced decreases in the hepatic expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. Thus central administration of leptin rescues the hepatic insulin resistance induced by short-term hyperphagia.

Leptin expression in human primary skeletal muscle cells is reduced during differentiation

We found leptin to be strongly expressed in undifferentiated human myoblasts derived from biopsies of the thigh (Musculus vastus lateralis). Both mRNA expression and secretion of leptin were reduced during in vitro differentiation into primary myotubes. However, the expression of the leptin receptor (OB-Rb) mRNA, was unchanged during differentiation of the muscle cells. Administration of recombinant leptin had no effect on leptin, myogenin, myoD, or GLUT4 mRNA expressions during the period of cellular differentiation. A functional leptin receptor was demonstrated by an acute leptin-induced 1.5-fold increase in ERK activity (P = 0.029). Although mRNA expression of regulation of suppressor of cytokine signaling-3 (SOCS-3) mRNA expression was unaltered, leptin significantly stimulated fatty acid oxidation after 6 h measured as acid soluble metabolites (ASM). Palmitic acid (PA), oleic acid (OA), and eicosapentaenoic acid (EPA), known to modulate leptin expression in other tissues, had no effect on mRNA expression or secretion of leptin from human myotubes. In conclusion, we demonstrate that leptin is highly expressed in undifferentiated human myoblasts and the expression is reduced during differentiation to mature myotubes. The role of leptin in these cells needs to be further characterized.

Effect of three treatment schedules of recombinant methionyl human leptin on body weight in obese adults: a randomized, placebo‐controlled trial

The aim of this study was to evaluate the effect on body weight and safety of subcutaneously administered recombinant leptin in obese adults and to evaluate whether the timing of recombinant leptin administration influences efficacy. A randomized, double-blind, placebo-controlled, multicentre study was designed, comprising of a 3-week dietary lead-in followed by a 12-week leptin or placebo treatment period. A total of 284 overweight and obese (body mass index 27-37.0 kg/m(2)) predominantly white (98%) women (66%) and men (34%) with a mean (+/-s.d.) 46.8+/-10.4 years of age were randomized into three treatment groups with three matching placebo groups. Recombinant leptin was administered by subcutaneous injection [10 mg/morning, 10 mg/evening or 20 mg/day (10 mg twice daily)]. Patients were counselled at baseline to reduce dietary intake by 2,100 kJ/day (500 kcal/day), and dietary advice was reinforced every 2-4 weeks. No statistically significant change in body weight occurred with recombinant leptin treatment compared with placebo treatment in any treatment group. No clinically significant adverse effects were observed with the exception of an increase in injection-site reactions in patients treated with recombinant leptin (83%) vs. placebo (36%). Administration of recombinant leptin to an overweight and obese population, in addition to a mildly energy-restricted diet, was not efficacious in terms of weight loss at the doses and schedules studied. The hypothesis that nocturnal administration of recombinant leptin might have a specific effect on weight loss was not supported.

Leptin and TNF-Alpha Levels in Patients with Chronic Obstructive Pulmonary Disease and Their Relationship to Nutritional Parameters

Background: Leptin is a protein mainly secreted by adipocytes, and the major function of leptin was its role in body weight regulation. In humans, there was a strong correlation between leptin and nutritional parameters, such as body mass index (BMI) and fat mass (FM). Administration of recombinant leptin to ob/ob mice, which have a genetic defect in leptin production, reduces food intake, increases energy expenditure, and decreases body weight. It is suggested that increased levels of circulating leptin levels may contribute to anorexia and weight loss in pathologic conditions including chronic obstructive pulmonary disease (COPD). Recent studies have provided evidence for a link between leptin and proinflammatory cytokines such as TNF-α. Objective: This study aimed to detect serum leptin and TNF-α levels in COPD patients without weight loss during stable disease and acute exacerbation, and to investigate relationships between leptin, TNF-α and nutritional parameters at different stages of the disease. Material and Methods: 26 stable COPD patients, 16 COPD patients with acute exacerbation and 15 control subjects participated in this study. To eliminate the effects of sex differences, all patients and controls were male. BMI, percent ideal body weight, percent FM, sum of skinfold tickness and serum leptin and TNF-α levels were measured in all participants. Leptin and TNF-α levels were measured by ELISA. Results: Serum leptin and TNF-α levels were significantly higher in the patients experiencing exacerbation than in the stable patients and controls. Although leptin levels were lower and TNF-α levels were higher in the stable patient group than in the controls, these differences were not statistically different. Leptin levels were significantly correlated with the nutritional parameters in both control and stable groups. However, in patients with acute exacerbation, a correlation between leptin and nutritional parameters was not found. There was no significant relationship between TNF-α and nutritional parameters in the three groups. In addition, while there was no correlation between leptin and TNF-α levels in the stable and control groups, a significant positive correlation was observed in patients with exacerbation. Conclusion: In conclusion (1) elevated TNF-α levels may be related to increased inflammation in patients, (2) circulating TNF-α levels were associated with increased leptin levels and (3) although leptin and nutritional parameters were correlated in the stable COPD patients, the correlation was weaker compared to controls, and during an exacerbation it disappeared completely. Therefore, inappropriately increased levels of leptin and TNF-α noted during recurrent acute exacerbations in patients with COPD may lead to changes in nutritional parameters and body weight in the course of the disease.

Ghrelin levels are not regulated by recombinant leptin administration and/or three days of fasting in healthy subjects.

Ghrelin, a stomach-derived orexigenic peptide, and leptin, a fat-derived anorexigenic hormone, act primarily in the hypothalamus to regulate energy homeostasis and have been reported to be regulated in opposite directions by acute and chronic changes in nutritional state. Nutritional, anthropometric, and hormonal predictors of circulating ghrelin have not yet been fully elucidated, and whether ghrelin is regulated by leptin in humans remains unknown. To address these questions, we performed cross-sectional and interventional studies. In 120 healthy men and women, ghrelin was negatively associated with leptin as well as overall and central adiposity, but not with total energy or specific macronutrient intake. The sexual dimorphism in ghrelin levels (higher levels in women than in men) and the negative correlation between ghrelin and insulin are largely mediated by central adiposity. In six lean men, complete fasting for 3 d resulted in a low leptin state without a major change in fat mass and abolished the meal-related secretory pattern of ghrelin without increasing 24-h ghrelin levels. In addition, recombinant human leptin administration in physiological and pharmacological doses did not regulate ghrelin over several hours to a few days. These data do not support a role for regulation of circulating ghrelin by leptin levels independently of changes in adiposity and suggest that the leptin and ghrelin systems for energy homeostasis function independently of each other in healthy humans.

Essential role of STAT3 in body weight and glucose homeostasis.

STAT3 is a ubiquitous transcription factor that is indispensable during early embryogenesis. To study the functions of STAT3 postnatally, we generated conditional STAT3-deficient mice. To that end, STAT3(lox/lox) mice were crossed with mice expressing Cre under the control of rat insulin II gene promoter (RIP-Cre mice). Immunohistochemical and Western blot analyses showed that STAT3 is deleted from beta cells in the islets of Langerhans. Genomic DNA PCR revealed that STAT3 deletion also occurred in the hypothalamus. Hypothalamic Cre expression was further confirmed by crossing RIP-Cre/STAT3(lox/lox) mice with the ROSA26 Cre reporter strain and staining for lacZ activity. Double immunohistochemical staining confirmed that deletion of STAT3 occurred in leptin receptor (OB-Rb isoform)-positive neurons. RIP-Cre/STAT3(lox/lox) mice are mildly hyperglycemic and hyperinsulinemic at the time of weaning, become hyperphagic immediately after weaning, and exhibit impaired glucose tolerance. Body weight, body fat, and mRNA and protein levels of leptin are all significantly increased in RIP-Cre/STAT3(lox/lox) mice. Administration of recombinant leptin by intracerebroventricular infusion failed to cause complete loss of body fat in RIP-Cre/STAT3(lox/lox) mice. Transplantation of wild-type islets into RIP-Cre/STAT3(lox/lox) mice also failed to decrease adiposity or to correct other abnormalities in these mice. These data thus suggest that loss of STAT3 in the hypothalamus caused by RIP-Cre action likely interferes with normal body weight homeostasis and glucose metabolism.

A role for leptin in immune-mediated inflammatory diseases?

Leptin, the product of the Ob gene, is a polypeptide hormone that plays an important role in the regulation of body weight by inhibiting food intake and stimulating energy expenditure. Moreover, leptin exhibits a variety of other effects including the regulation of endocrine function, reproduction and hematopoiesis. Consistently, leptin-deficient mice are not only obese, but display major hormonal disturbances, including hypercorticosteronemia, diabetes, and infertility. In addition, it has been known for many years that leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice have an altered immune response. More recent studies have shown that T cells and B cells express the long signaling leptin receptor isoform (ObRb) and that leptin exerts direct effects on T lymphocytes, including the stimulation of cell proliferation, the promotion of Th1 responses, and the protection of thymocytes from corticosteroid-induced apoptosis. Consistently, ob/ob mice are protected from inflammation mediated by T cells and B cells in some models such as experimental colitis, experimental autoimmune encephalomyelitis, and concanavalin A-induced hepatitis. Conversely, the administration of recombinant leptin increased the severity of experimental autoimmune encephalomyelitis. Using a model of antigen-induced arthritis, we demonstrated that ob/ob and db/db mice have a milder form of arthritis and that cellular and humoral immune responses to the injected antigen are decreased as compared with wild-type mice. Furthermore, ex vivo stimulated lymph node cells from ob/ob and db/db mice produced less interferon gamma and more IL-10 than cells obtained from control mice. Leptin production is regulated by various proinflammatory cytokines during the acute-phase response, and leptin stimulates the production of both proinflammatory and antiinflammatory cytokines in vitro. ob/ob mice exhibit increased susceptibility to lipopolysaccharide-induced and tumor necrosis factor alpha-induced lethality, suggesting that leptin is also involved in regulating the innate immune response. We recently examined the role of leptin in zymosan-induced arthritis (ZIA), where joint inflammation is dependent on innate immunity. Our results demonstrate that, as opposed to antigen-induced arthritis, ZIA is not impaired in ob/ob and db/db mice. On the contrary, these mice exhibit a delayed resolution of the inflammatory process, with increased circulating levels of IL-6 and serum amyloid A, and a tendency to develop more severe joint damage. Leptin deficiency thus appears to interfere with adequate control of the inflammatory response in ZIA. Although data obtained in different models of inflammatory diseases suggest that leptin plays a role in immune response, the analysis of direct effects of leptin on lymphocytes in vivo is precluded by the important hormonal and metabolic alterations, which are linked to leptin deficiency. To our knowledge, it has never been thoroughly investigated to which extent immune defects in ob/ob or db/db animals are linked to leptin deficiency or, respectively, leptin receptor deficiency per se, as opposed to confounding factors such as hypercorticosteronemia. To examine the importance of direct effects of leptin on lymphopoiesis and immune response, we generated bone marrow chimeras by transplantation of db/db bone marrow cells into lethally irradiated normal recipient mice, which provide a normal environment for the grafted cells. Donor db/db mice display a marked atrophy of the thymus as compared with db/+ controls. In bone marrow chimeras, the size and cellularity of the thymus were not different between mice grafted with db/db or db/+ bone marrow 12 weeks after the graft, suggesting that the thymic atrophy observed in db/db donors is not due to direct effects of leptin on lymphocytes. Moreover, RT-PCR analyses suggest that CD4/CD8 double positive thymocytes do not express ObRb. Further studies are in progress in order to examine which cell types respond to leptin in the thymus and whether impaired leptin signaling in lymphocytes affects immune responses after antigen challenge in db/db bone marrow chimeras.