Determination of the half-life of circulating leptin in the mouse.

BACKGROUND: The adipokine hormone, leptin, is a major component of body weight homeostasis. Numerous studies have been performed administering recombinant mouse leptin as an experimental reagent; however, the half-life of circulating leptin following exogenous administration of recombinant mouse leptin has not been carefully evaluated. METHODS: Exogenous leptin was administered (3 mg leptin per kg body weight) to 10-week-old fasted non-obese male mice and plasma was serially collected at seven time points; plasma leptin concentration was measured by enzyme-linked immunosorbent assay at each time point to estimate the circulating half-life of mouse leptin. RESULTS: Under the physiological circumstances tested, the half-life of mouse leptin was 40.2 (±2.2) min. Circulating leptin concentrations up to 1 h following exogenous leptin administration were 170-fold higher than endogenous levels at fasting. CONCLUSIONS: The half-life of mouse leptin was determined to be 40.2 min. These results should be useful in planning and interpreting experiments employing exogenous leptin. The unphysiological elevations in circulating leptin resulting from widely used dosing regimens for exogenous leptin are likely to confound inferences regarding some aspects of the hormone's clinical biology.

Metabolically healthy and unhealthy obese--the 2013 Stock Conference report.

Obesity is closely associated with cardiovascular diseases and type 2 diabetes, but some obese individuals, despite having excessive body fat, exhibit metabolic health that is comparable with that of lean individuals. The 'healthy obese' phenotype was described in the 1980s, but major advancements in its characterization were only made in the past five years. During this time, several new mechanisms that may be involved in health preservation in obesity were proposed through the use of transgenic animal models, use of sophisticated imaging techniques and in vivo measurements of insulin sensitivity. However, the main obstacle in advancing our understanding of the metabolically healthy obese phenotype and its related long-term health risks is the lack of a standardized definition. Here, we summarize the proceedings of the 13th Stock Conference of the International Association of the Study of Obesity. We describe the current research and highlight the unanswered questions and gaps in the field. Better understanding of metabolic health in obesity will assist in therapeutic decision-making and help identify therapeutic targets to improve metabolic health in obesity.

Effects of chronic leptin infusion on subsequent body weight and composition in mice: Can body weight set point be reset?

Circulating leptin concentrations correlate with fat mass and signal the status of somatic energy stores to the brain. Previous studies suggest that diet-induced elevations of body weight increase body weight "set-point". To assess whether chronic hyperleptinemia is responsible for this shift in defended body weight, we elevated circulating leptin concentrations in lean mice to those comparable to diet-induced obese mice for eighteen weeks. We hypothesized that following cessation of leptin infusion, a higher body weight would be defended. Compared to saline-infused controls, leptin-infused mice had elevated circulating leptin concentrations, gained less weight, yet had similar metabolic rates. Following cessation of leptin administration, leptin-infused mice gained some weight yet plateaued at 5-10% below controls. These results suggest that, unlike mice rendered hyperleptinemic by diet-induced weight gain, leptin-infused mice do not subsequently "defend" a higher body weight, suggesting that hyperleptinemia per se does not mimic the CNS consequences of chronic weight gain.

Genetic association analysis of 30 genes related to obesity in a European American population.

OBJECTIVE: Obesity, which is frequently associated with diabetes, hypertension and cardiovascular diseases, is primarily the result of a net excess of caloric intake over energy expenditure. Human obesity is highly heritable, but the specific genes mediating susceptibility in non-syndromic obesity remain unclear. We tested candidate genes in pathways related to food intake and energy expenditure for association with body mass index (BMI). METHODS: We reanalyzed 355 common genetic variants of 30 candidate genes in seven molecular pathways related to obesity in 1982 unrelated European Americans from the New York Cancer Project. Data were analyzed by using a Bayesian hierarchical generalized linear model. The BMIs were log-transformed and then adjusted for covariates, including age, age(2), gender and diabetes status. The single-nucleotide polymorphisms (SNPs) were modeled as additive effects. RESULTS: With the stipulated adjustments, nine SNPs in eight genes were significantly associated with BMI: ghrelin (GHRL; rs35683), agouti-related peptide (AGRP; rs5030980), carboxypeptidase E (CPE; rs1946816 and rs4481204), glucagon-like peptide-1 receptor (GLP1R; rs2268641), serotonin receptors (HTR2A; rs912127), neuropeptide Y receptor (NPY5R;Y5R1c52), suppressor of cytokine signaling 3 (SOCS3; rs4969170) and signal transducer and activator of transcription 3 (STAT3; rs4796793). We also found a gender-by-SNP interaction (rs1745837 in HTR2A), which indicated that variants in the gene HTR2A had a stronger association with BMI in males. In addition, NPY1R was detected as having a significant gene effect even though none of the SNPs in this gene was significant. CONCLUSION: Variations in genes AGRP, CPE, GHRL, GLP1R, HTR2A, NPY1R, NPY5R, SOCS3 and STAT3 showed modest associations with BMI in European Americans. The pathways in which these genes participate regulate energy intake, and thus these associations are mechanistically plausible in this context.

Molecular basis for the regulation of islet beta cell mass in mice: the role of E-cadherin.

AIMS/HYPOTHESIS: In rodents and humans, the rate of beta cell proliferation declines rapidly after birth; formation of the islets of Langerhans begins perinatally and continues after birth. Here, we tested the hypothesis that increasing levels of E-cadherin during islet formation mediate the decline in beta cell proliferation rate by contributing to a reduction of nuclear ß-catenin and D-cyclins. METHODS: We examined E-cadherin, nuclear ß-catenin, and D-cyclin levels, as well as cell proliferation during in vitro and in vivo formation of islet cell aggregates, using ß-TC6 cells and transgenic mice with green fluorescent protein (GFP)-labelled beta cells, respectively. We tested the role of E-cadherin using antisense-mediated reductions of E-cadherin in ß-TC6 cells, and mice segregating for a beta cell-specific E-cadherin knockout (Ecad [also known as Cdh1] ßKO). RESULTS: In vitro, pseudo-islets of ß-TC6 cells displayed increased E-cadherin but decreased nuclear ß-catenin and cyclin D2, and reduced rates of cell proliferation, compared with monolayers. Antisense knockdown of E-cadherin increased cell proliferation and levels of cyclins D1 and D2. After birth, beta cells showed increased levels of E-cadherin, but decreased levels of D-cyclin, whereas islets of Ecad ßKO mice showed increased levels of D-cyclins and nuclear ß-catenin, as well as increased beta cell proliferation. These islets were significantly larger than those of control mice and displayed reduced levels of connexin 36. These changes correlated with reduced insulin response to ambient glucose, both in vitro and in vivo. CONCLUSIONS/INTERPRETATION: The findings support our hypothesis by indicating an important role of E-cadherin in the control of beta cell mass and function.

Estimating energy expenditure in mice using an energy balance technique.

OBJECTIVE: To compare, in mice, the accuracy of estimates of energy expenditure (EE) using an energy balance technique (TEEbal: food energy intake and body composition change) vs indirect calorimetry (TEEIC). SUBJECTS: In 32 male C57BL/6J mice, EE was estimated using an energy balance (caloric intake minus change in body energy stores) method over a 37-day period. EE was also measured in the same animals by indirect calorimetry. These measures were compared. RESULTS: The two methods were highly correlated (r(2)=0.87: TEEbal=1.07*TEEIC-0.22, P<0.0001). By Bland-Altman analysis, TEEbal estimates were slightly higher (4.6±1.5%; P<0.05) than TEEIC estimates (Bias=0.55 kcal per 24 h). CONCLUSION: TEEbal can be performed in 'home cages' and provides an accurate integrated long-term measurement of EE while minimizing potentially confounding stress that may accompany the use of indirect calorimetry systems. The technique can also be used to assess long-term energy intake.

Neurofunctional imaging of ß-cell dynamics.

Here, we outline how islet cells use autocrine and paracrine 'circuits' of classical neurotransmitters and their corresponding receptors and transporters to communicate with vicinal ß-cells to regulate glucose-stimulated insulin secretion. Many of these same circuits operate in the central nervous system and can be visualized by molecular imaging. We discuss how these techniques might be applied to measuring the dynamics of ß-cell function in real time.

Effects of chronic weight perturbation on energy homeostasis and brain structure in mice.

Maintenance of reduced body weight in lean and obese human subjects results in the persistent decrease in energy expenditure below what can be accounted for by changes in body mass and composition. Genetic and developmental factors may determine a central nervous system (CNS)-mediated minimum threshold of somatic energy stores below which behavioral and metabolic compensations for weight loss are invoked. A critical question is whether this threshold can be altered by environmental influences and by what mechanisms such alterations might be achieved. We examined the bioenergetic, behavioral, and CNS structural responses to weight reduction of diet-induced obese (DIO) and never-obese (CON) C57BL/6J male mice. We found that weight-reduced (WR) DIO-WR and CON-WR animals showed reductions in energy expenditure, adjusted for body mass and composition, comparable (-10-15%) to those seen in human subjects. The proportion of excitatory synapses on arcuate nucleus proopiomelanocortin neurons was decreased by ∼50% in both DIO-WR and CON-WR mice. These data suggest that prolonged maintenance of an elevated body weight (fat) alters energy homeostatic systems to defend a higher level of body fat. The synaptic changes could provide a neural substrate for the disproportionate decline in energy expenditure in weight-reduced individuals. This response to chronic weight elevation may also occur in humans. The mouse model described here could help to identify the molecular/cellular mechanisms underlying both the defense mechanisms against sustained weight loss and the upward resetting of those mechanisms following sustained weight gain.

Adaptive thermogenesis in humans.

The increasing prevalence of obesity and its comorbidities reflects the interaction of genes that favor the storage of excess energy as fat with an environment that provides ad libitum availability of energy-dense foods and encourages an increasingly sedentary lifestyle. Although weight reduction is difficult in and of itself, anyone who has ever lost weight will confirm that it is much harder to keep the weight off once it has been lost. The over 80% recidivism rate to preweight loss levels of body fatness after otherwise successful weight loss is due to the coordinate actions of metabolic, behavioral, neuroendocrine and autonomic responses designed to maintain body energy stores (fat) at a central nervous system-defined 'ideal'. This 'adaptive thermogenesis' creates the ideal situation for weight regain and is operant in both lean and obese individuals attempting to sustain reduced body weights. Much of this opposition to sustained weight loss is mediated by the adipocyte-derived hormone 'leptin'. The multiple systems regulating energy stores and opposing the maintenance of a reduced body weight illustrate that body energy stores in general and obesity in particular are actively 'defended' by interlocking bioenergetic and neurobiological physiologies. Important inferences can be drawn for therapeutic strategies by recognizing obesity as a disease in which the human body actively opposes the 'cure' over long periods of time beyond the initial resolution of symptomatology.

Molecular physiology of weight regulation in mice and humans.

Evolutionary considerations relating to efficiency in reproduction, and survival in hostile environments, suggest that body energy stores are sensed and actively regulated, with stronger physiological and behavioral responses to loss than gain of stored energy. Many physiological studies support this inference, and suggest that a critical axis runs between body fat and the hypothalamus. The molecular cloning of leptin and its receptor-projects based explicitly on the search for elements in this axis-confirmed the existence of this axis and provided important tools with which to understand its molecular physiology. Demonstration of the importance of this soma-brain reciprocal connection in body weight regulation in humans has been pursued using both classical genetic approaches and studies of physiological responses to experimental weight perturbation. This paper reviews the history of the rationale and methodology of the cloning of leptin (Lep) and the leptin receptor (Lepr), and describes some of the clinical investigation characterizing this axis.

Association of K121Q polymorphism in ENPP1 (PC-1) with BMI in Caucasian and African-American adults.

OBJECTIVE: To test for association of the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) K121Q polymorphism with body mass index (BMI) and diabetes in a large sample of Caucasians and African-Americans by selectively genotyping individuals at the extremes of the phenotypic distribution. SUBJECTS: Subsets comprising the extremes of the BMI distribution (10th-20th and above the 90th BMI percentile for Caucasians and between the 10th-30th and above the 80th percentile for African-Americans) from a group of 10,260 Caucasian and 2268 African-American adults participating in New York Cancer Project were studied. METHODS: Subjects were genotyped for the ENPP1 K121Q polymorphism by pyrosequencing and tested for association with BMI and diabetes by regression analysis. RESULTS: Regression analysis with BMI as the dependent variable demonstrated a significant association (P = 0.02) of genotype at K121Q with BMI, with no significant race-by-genotype interaction (P = 0.30). Compared with Q/Q or Q/K individuals, the K/K individuals had a BMI approximately 1.3 kg/m2 higher, without effects of age, gender or race. By logistic regression analysis, the K121Q alleles had no significant effect on diabetes status (P = 0.37) in obese subjects. CONCLUSION: In both Caucasians and African-Americans, the K121 polymorphism in ENPP1 was associated with increased BMI, but not with diabetes.

Metabolic and hemodynamic responses to exercise in subcutaneous adipose tissue and skeletal muscle.

This study evaluated the effect of standardized bicycle exercise on metabolism and blood flow in abdominal ( aSAT) and femoral subcutaneous adipose tissue ( fSAT) and skeletal muscle in eleven women and nine men. Using microdialysis, the respective tissues were perfused with Ringer's solution (+ 50 mM ethanol) and dialysate [ethanol], [glycerol], [lactate] and [pyruvate] were measured in order to estimate blood flow (ethanol dilution technique), lipolysis and glycolysis, respectively. At rest, blood flow tended to be higher in the respective tissues of women when compared to men. During exercise, blood flow was increased significantly in fSAT and muscle, but not in aSAT. Dialysate [glycerol] was increased two- to three-fold in aSAT and fSAT, similarly in men and women. However, in muscle, dialysate [glycerol] was increased five-fold in women and four-fold in men without reaching a steady state in women. Corrected for blood flow, the increase in lipolysis was greater in muscle than in fSAT, and greater in fSAT than in aSAT, and in muscle the increase was greater for women compared with men. Dialysate [lactate] and [lactate]/[pyruvate] ratio were much more increased in muscle compared with aSAT and fSAT. It is concluded that lipids stored in muscle are rather used than lipids stored in adipose tissue for fueling the energy metabolism of muscle during exercise. During exercise, lipid mobilization is much greater in women than in men.

A meta-analytic investigation of linkage and association of common leptin receptor (LEPR) polymorphisms with body mass index and waist circumference.

METHODS: We analyzed data pooled from nine studies on the human leptin receptor (LEPR) gene for the association of three alleles (K109R, Q223R and K656N) of LEPR with body mass index (BMI; kg/m(2)) and waist circumference (WC). A total of 3263 related and unrelated subjects from diverse ethnic backgrounds including African-American, Caucasian, Danish, Finnish, French Canadian and Nigerian were studied. We tested effects of individual alleles, joint effects of alleles at multiple loci, epistatic effects among alleles at different loci, effect modification by age, sex, diabetes and ethnicity, and pleiotropic genotype effects on BMI and WC. RESULTS: We found that none of the effects were significant at the 0.05 level. Heterogeneity tests showed that the variations of the non-significant effects are within the range of sampling variation. CONCLUSIONS: We conclude that, although certain genotypic effects could be population-specific, there was no statistically compelling evidence that any of the three LEPR alleles is associated with BMI or WC in the overall population.

Leptin regulation of Agrp and Npy mRNA in the rat hypothalamus.

Agouti-related protein (AGRP) is synthesized in the same neurones in the arcuate nucleus as neuropeptide Y (NPY), another potent orexigenic peptide. AGRP antagonizes the action of alpha-melanocyte stimulating hormone, a derivative of pro-opiomelanocortin (POMC) at the hypothalamic MC4 receptor to increase food intake. Although leptin has been shown to regulate Agrp/Npy and Pomc-expressing neurones, there are differences with respect to Agrp regulation in leptin receptor-deficient mice and rats. Unlike the obese leptin receptor-deficient db/db mouse, which exhibits upregulation of Agrp mRNA expression in the medial basal hypothalamus (MBH) compared to lean controls, the obese leptin receptor-deficient (faf; Koletsky) rat does not exhibit upregulation of Agrp expression. To determine whether this represents a general difference between leptin receptor-deficient mice and rats, neuropeptide gene expression was analysed in the MBH of lean and obese rats segregating for a different leptin receptor mutation, Leprfa (Zucker). Fasting in lean rats (+/fa) for 72 h significantly increased Agrp and Npy mRNA expression, and decreased Pomc mRNA expression as detected by a sensitive solution hybridization/S1 nuclease protection assay. Npy mRNA levels were significantly increased in fed obese fa/fa compared to lean rats, and further increased in the obese animals after fasting. In contrast, Agrp mRNA levels did not differ between fed lean and fed obese rats, and fasting did not significantly change Agrp levels in obese rats. To determine whether the change in Agrp expression that occurs with food deprivation in lean rats could be prevented by leptin replacement, Sprague-Dawley rats were fasted and infused via subcutaneous osmotic micropumps for 48 h with either saline or recombinant mouse leptin. Fasting significantly increased Agrp and Npy, and decreased Pomc mRNA levels. Leptin infusion almost completely reversed these changes such that there was no significant difference between the levels in the fasted rats and those that were fed ad libitum. Thus, in fasted lean rats, Agrp and Npy are upregulated in parallel when leptin levels fall and are downregulated by leptin infusion. By contrast, the absence of a functional leptin receptor results in the upregulation of Npy but not Agrp mRNA.

Pooling analysis of genetic data: the association of leptin receptor (LEPR) polymorphisms with variables related to human adiposity.

Analysis of raw pooled data from distinct studies of a single question generates a single statistical conclusion with greater power and precision than conventional metaanalysis based on within-study estimates. However, conducting analyses with pooled genetic data, in particular, is a daunting task that raises important statistical issues. In the process of analyzing data pooled from nine studies on the human leptin receptor (LEPR) gene for the association of three alleles (K109R, Q223R, and K656N) of LEPR with body mass index (BMI; kilograms divided by the square of the height in meters) and waist circumference (WC), we encountered the following methodological challenges: data on relatives, missing data, multivariate analysis, multiallele analysis at multiple loci, heterogeneity, and epistasis. We propose herein statistical methods and procedures to deal with such issues. With a total of 3263 related and unrelated subjects from diverse ethnic backgrounds such as African-American, Caucasian, Danish, Finnish, French-Canadian, and Nigerian, we tested effects of individual alleles; joint effects of alleles at multiple loci; epistatic effects among alleles at different loci; effect modification by age, sex, diabetes, and ethnicity; and pleiotropic genotype effects on BMI and WC. The statistical methodologies were applied, before and after multiple imputation of missing observations, to pooled data as well as to individual data sets for estimates from each study, the latter leading to a metaanalysis. The results from the metaanalysis and the pooling analysis showed that none of the effects were significant at the 0.05 level of significance. Heterogeneity tests showed that the variations of the nonsignificant effects are within the range of sampling variation. Although certain genotypic effects could be population specific, there was no statistically compelling evidence that any of the three LEPR alleles is associated with BMI or waist circumference in the general population.

Sexual dimorphism in circulating leptin concentrations is not accounted for by differences in adipose tissue distribution.

BACKGROUND: Circulating concentrations of leptin normalized to total adipose tissue mass are significantly greater in females than in males. Rates of leptin expression (per gram of adipose tissue) are significantly greater in subcutaneous (SAT) than visceral (VAT) adipose tissue and the relative amount of fat stored as SAT vs VAT is significantly greater in pre-menopausal females than in males. Gender-related differences in the relative amounts of SAT and VAT may account for the greater circulating leptin concentration relative to fat-mass in females than males. METHODS: We examined body composition and anatomic fat distribution by dual energy X-ray-absorptiometry (DEXA) and magnetic resonance imaging (MRI), and post-absorptive circulating concentrations of leptin and insulin in 58 subjects (26 females, 32 males). Stepwise multiple linear regression analyses, treating gender as a dichotomous variable, were performed to determine inter-relationships among leptin concentrations and insulin concentrations, VAT and SAT. RESULTS: Body composition by DEXA and MRI were highly correlated (r(2)=0.97, P<0.0001). There were significant gender effects on leptin/total fat mass (males, 0.17+/-0.01 ng/ml/kg; females, 0.49+/-0.05 ng/ml/kg; P<0.0001) and relative amounts of fat in SAT and VAT depots (ratio of SAT/VAT; males, 12.3+/-1.5; females, 32.9+/-3.2; P<0.0001). Circulating leptin concentration was significantly correlated with insulin concentration (P=0.001), SAT (P<0.0001) and gender (P=0.033). Circulating concentrations of insulin were significantly correlated with VAT, but not SAT, in males and with SAT, but not VAT, in females. CONCLUSIONS: The sexual dimorphism in the relationship between leptin and adipose tissue mass cannot be explained by differences in the relative amounts of VAT and SAT. Thus, the sexual dimorphism in plasma leptin concentration appears to reflect, at least in part, effects of circulating concentrations of gonadal steroids (especially androgens) and/or primary genetic differences that are independent of amounts of VAT or SAT.

Effects of leptin deficiency and short-term repletion on hepatic gene expression in genetically obese mice.

By supplying most organs of the body with metabolic substrates, the liver plays a central role in maintaining energy balance. Hepatic metabolism of glucose, fatty acids, and lipoproteins is disrupted in the leptin-deficient obese (Lep(ob)/Lep(ob)) mouse, leading to hyperglycemia, steatosis, and hypercholesterolemia. Microarray expression profiles were used to identify transcriptional perturbations that underlie the altered hepatic physiology of Lep(ob)/Lep(ob) mice. A wide variety of genes involved in fatty acid metabolism are altered in expression, which suggests that both fatty acid synthesis and oxidation programs are activated in obese mice. The expression of a small subset of genes is upregulated by leptin deficiency, not modulated by caloric restriction, and markedly suppressed by short-term leptin treatment. Among these leptin-regulated genes, apolipoprotein A-IV is a strong candidate for mediating the atherogenic-resistant phenotype of Lep(ob)/Lep(ob) mice.

Synergy of sibutramine and low-dose leptin in treatment of diet-induced obesity in rats.

Tachyphylaxis to the effects of anorexigenic agents, such as sibutramine (S), may be due, in part, to counterregulatory decreases in energy expenditure (EE) and increases in hunger that result from reduced circulating leptin (L) due to loss of body fat and lowered L production/adipocyte. The present study was conducted to test the hypothesis that L administered at low doses sufficient to restore ambient L to preweight loss concentrations would enhance the intercurrent efficacy of S by reducing the strength of physiologic counterregulation to weight loss. Forty male Sprague-Dawley rats were fed a high-fat (HF) diet (45% energy) to induce obesity. After 8 weeks, the obese rats (600 +/- 58 g) were weight-matched into 4 groups (N = 8/group) and implanted subcutaneously (SC) with 2 mL, 7-day Alzet mini-pumps that provided: vehicle (V, saline), L (0.5 mg/kg/d), S (3 mg/kg/d), or L+S. Food intake (FI) on the HF diet was measured daily. On day 7, 24-hour EE was measured by indirect calorimetry, and the animals then killed for body composition analysis. Compared with vehicle, treatment with S alone, but not L alone, produced significant weight loss (-23 +/- 26 v -6 +/- 16 g, P <.01). L alone, or with S, increased fat oxidation (decreased respiratory quotient [RQ]) compared with V (P <.05). The lack of decline in EE with S may be due to its documented effect to stimulate thermogenesis. Administration of L with S synergistically decreased FI and increased weight loss and fractional fat loss. A reduction in plasma L concentration may contribute to the "plateau phenomenon" observed in studies of weight loss therapies. Replacement doses of L during S administration increased weight loss and fractional fat loss by (1) decreasing food intake and (2) by increasing fat oxidation. Such drug combinations may be useful in the treatment of human obesity.