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.

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.

Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor.

Mice harboring mutations in the obese (ob) and diabetes (db) genes display similar phenotypes, and it has been proposed that these genes encode the ligand and receptor, respectively, for a physiologic pathway that regulates body weight. The cloning of ob, and the demonstration that it encodes a secreted protein (leptin) that binds specifically to a receptor (OB-R) in the brain, have validated critical aspects of this hypothesis. Here it is shown by genetic mapping and genomic analysis that mouse db, rat fatty (a homolog of db), and the gene encoding the OB-R are the same gene.

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.

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.

Transgenic complementation of leptin-receptor deficiency. I. Rescue of the obesity/diabetes phenotype of LEPR-null mice expressing a LEPR-B transgene.

Mice homozygous for the Leprdb3J (db3J) mutation are null for all known isoforms of the leptin receptor (LEPR). These animals are obese, hyperphagic, cold intolerant, insulin resistant, and infertile. Mice homozygous for the Leprdb (db) mutation (lacking the B isoform only) have the same phenotype as db3J animals. To better understand the function(s) of the LEPR isoforms in vivo, we generated db3J/db3J and db/db mice bearing a transgene (neuron-specific enolase [NSE]-Rb) expressing the B isoform of LEPR, the isoform capable of activating the signal transducer and activator of transcription (STAT) pathway, under the control of the neuron-specific enolase enhancer/promoter. The NSE-Rb transgene was expressed in the brain, with low levels of expression in adrenals, testis, and white adipose tissue. LEPR-B transgene expression in NSE-Rb db3J/db3J mice partially corrected the increased fat mass, hyperphagia, and glucose intolerance while restoring fertility in males and rescuing the cold intolerance in both sexes. The body weights of NSE-Rb transgenic mice that possessed the full complement of short LEPR isoforms (NSE-Rb db/db mice) were similar to those of NSE-Rb db3J/db3J mice, suggesting that the short LEPR isoforms play little role in body weight regulation. Based on quantitative analysis of hypothalamic neuropeptide gene expression in the transgenic animals, we infer full restoration of leptin sensitivity to proopiomelanocortin (POMC) neurons, partial correction of leptin sensitivity in agouti gene-related protein (AGRP)/neuropeptide Y (NPY) neurons, and a lack of effect on leptin sensitivity of melanin concentrating hormone neurons. Thus, hypothalamic POMC and AGRP/NPY neurons are primary candidates as the mediators of the effects of the NSE-Rb transgene on energy homeostasis, ingestive behavior, the neuroendocrine system, and glucose metabolism.

Phenotype of the obese Koletsky (f) rat due to Tyr763Stop mutation in the extracellular domain of the leptin receptor (Lepr): evidence for deficient plasma-to-CSF transport of leptin in both the Zucker and Koletsky obese rat.

The obese phenotypes of the diabetes (db) mouse and fatty fa) rat are due to functional null mutations of the leptin receptor (Lepr). The recessive mutation in the Koletsky (f) obese rat maps to the same genetic intervals as db and fa and fails to complement the fa mutation. Comparison of the sequence of brain Lepr cDNA from +/+ and f/f animals reveals a T2349A transversion resulting in a Tyr763Stop nonsense mutation in the gene just before the transmembrane domain. Virtual absence of Lepr mRNA in whole brain from f/f animals is consistent with the presence of a null mutation. The predicted reduced cerebrospinal fluid (CSF) transport of leptin in both f/f and fa/fa mutants is reflected in the approximately 10-fold lower ratio of CSF/plasma leptin concentration in the obese versus lean animals. However, equivalent CSF leptin concentration between lean and obese rats (fa/fa, f/f) indicates that leptin can enter the CSF through a non-Lepr-mediated mechanism, which may be saturated at normal physiological plasma leptin concentration.

Absence of linkage of obesity and energy metabolism to markers flanking homologues of rodent obesity genes in Pima Indians.

The homologues of single genes that cause obesity in rodents are suggested as candidate genes for modulation of body composition in humans. Among these genes are the four mouse mutations-diabetes (db), obesity (ob), tubby (tub), and yellow agouti (Ay). Variation in the human counterparts to these genes (OB, DB, TUB, and ASP, respectively) may contribute to human obesity, which is thought to have a substantial genetic component. To initially assess the potential contribution of these genes to human obesity, we examined polymorphic DNA markers that, by virtue of syntenic relationships to appropriate regions of the mouse genome, should be closely linked to the human counterparts of these genes. Using combined data from 716 Pima Indians comprising 217 nuclear families, we have tested a number of polymorphic microsatellite markers (three at DB, two at OB, five at TUB, and three at ASP) for sib-pair linkage to BMI, percentage body fat, resting metabolic rate, 24-h energy expenditure, and 24-h respiratory quotient. No significant linkages were found in an analysis of all sibships or in an analysis restricted to discordant sib pairs.

Phenotype of fatty due to Gln269Pro mutation in the leptin receptor (Lepr).

The rat fatty (fa) mutation produces profound obesity of early onset caused by hyperphagia, defective nonshivering thermogenesis, and preferential deposition of energy into adipose tissue. Genetic mapping studies indicate that fa and diabetes (db) are homologous loci in the rat and mouse genomes, respectively. It has been shown that db alleles carry mutations in the Lepr (leptin receptor) gene. This paper describes a point mutation in the fatty allele of Lepr. A nucleotide substitution at position 880 (A-->C) causes an amino acid substitution at position 269 (Gln-->Pro). The mutation generates a novel Msp I site that cosegregates with fa in 1,028 meioses examined in obese F2 progeny from two crosses (Bnx13M and WKYx13M) and is still segregating in three rat colonies. PCR-based mutagenesis was used to introduce the fa mutation into the mouse Lepr cDNA. Transient transfection studies indicate that the mutant Lepr cDNA has greatly reduced binding of leptin (Lep) at the cell surface. These data are strong evidence that the single nucleotide substitution in the fa allele of Lepr (Leprfa) is responsible for the obese phenotype.

Genetic and physiologic analysis of the role of uncoupling protein 3 in human energy homeostasis.

By virtue of its potential effects on rates of energy expenditure, uncoupling protein 3 (UCP3) is an obesity candidate gene. We identified nine sequence variants in UCP3, including Val9Met, Val102Ile, Arg282Cys, and a splice site mutation in the intron between exons 6 and 7. The splice mutation results in an inability to synthesize mRNA for the long isoform (UCP3L) of UCP3. Linkage (sib pair), association, and transmission disequilibrium testing studies on 942 African-Americans did not suggest a significant effect of UCP3 on body composition in this group. In vastus lateralis skeletal muscle of individuals homozygous for the splice mutation, no UCP3L mRNA was detectable; the short isoform (UCP3S) was present in an increased amount. In this muscle, we detected no alterations of in vitro mitochondrial coupling activity, mitochondrial respiratory enzyme activity, or systemic oxygen consumption or respiratory quotient at rest or during exercise. These genetic and physiologic data suggest the following possibilities: UCP3S has uncoupling capabilities equivalent to UCP3L; other UCPs may compensate for a deficiency of bioactive UCP3L; UCP3L does not function primarily as a mitochondrial uncoupling protein.

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.

Leptin: a molecule integrating somatic energy stores, energy expenditure and fertility.

The signaling of fat mass to central nervous system (CNS) regulators of food intake, energy expenditure and fertility has been inferred by experimental physiologists for over 75 years. The ability to modify such phenotypes based upon the status of body energy stores (fat) has critical survival value and, therefore, has been the object of potent selection pressure in evolution. The recent molecular cloning of the mouse ob mutation and the subsequent elucidation of the fundamentals of its regulatory physiology has identified a protein secreted by adipocytes, leptin, as a plausible candidate for a humoral signal with the requisite endocrinology and neurobiology.

Autosomal dominant growth hormone (GH) deficiency type II: the Del32-71-GH deletion mutant suppresses secretion of wild-type GH.

Familial isolated GH deficiency type II is an autosomal dominant form of short stature, associated in some families with mutations that result in missplicing to produce del32-71-GH, a protein that cannot fold normally. The mechanism by which this mutant suppresses the secretion of wild-type GH encoded by the normal allele is not known. Coexpression of del32-71-GH with wild-type human GH in transient transfections of the neuroendocrine cell lines GH4C1 and AtT20 suppressed accumulation of wild-type GH. The suppression of wild-type GH accumulation by del32-71-GH was a posttranslational effect on wild-type GH caused by decreased stability, rather than decreased synthesis, of wild-type GH. Coexpression of del32-71-GH with human PRL did not suppress accumulation of PRL, indicating that there was not a general suppression of secretory pathway function. Accumulation of del32-71-GH protein was not necessary for the suppression of wild-type GH, because del32-71-GH did not accumulate in the neuroendocrine cell lines in which suppression of accumulation of wild-type GH was observed. Del32-71-GH did accumulate in transfected COS and CHO cells, but did not suppress the accumulation of wild-type GH in these cells. These studies suggest that del32-71-GH may cause GH deficiency in somatotropes of heterozygotes expressing both wild-type and del32-71-GH by decreasing the intracellular stability of wild-type GH.