Effects of leptin on corticotropin-releasing factor (CRF) synthesis and CRF neuron activation in the paraventricular hypothalamic nucleus of obese (ob/ob) mice

The effects of leptin on the levels of CRF messenger RNA (mRNA) in the paraventricular hypothalamic nucleus (PVN), on the activation of the PVN CRF cells, and on the plasma levels of corticosterone were investigated in lean (+/?) and obese (ob/ob) C57BL/6J male mice. Murine leptin was s.c. infused using osmotic minipumps. The treatment period extended to 7 days, and the daily dose of leptin delivered was 100 microg/kg. The mice were killed either in a fed state or following 24 h of total food deprivation. The starvation paradigm was employed to enhance the activity of the hypothalamic-pituitary-adrenal axis in obese mice. In situ hybridization histochemistry was performed to determine the PVN levels of CRF mRNA and the arcuate nucleus levels of neuropeptide Y mRNA. The activity of the PVN CRF cells was estimated from the number of PVN cells colocalizing CRF mRNA and the protein Fos. Leptin led to a reduction in body weight gain and fat deposition. These effects were seen in both +/? and ob/ob mice and were observed to be particularly striking in obese mutants, in which leptin also caused an important reduction in food intake. Leptin also was found to affect plasma levels of corticosterone. It lowered the high corticosterone levels of obese mutants, an effect that appeared more evident in food-deprived than in fed mice. Finally, leptin prevented the induction of CRF synthesis in the PVN and the activation of the PVN CRF neurons observed in food-deprived ob/ob mice and hindered the elevation of arcuate nucleus neuropeptide Y synthesis in ob/ob mice. Together these results suggest a role for leptin in the excessive response of the hypophysiotropic CRF system of the ob/ob mouse.     

Induction of uncoupling protein expression in brown and white adipose tissue by leptin

Deposition of excess body fat occurs when energy intake chronically exceeds energy expenditure. In ob/ob mice, the absence of leptin affects both components of the energy balance equation, and the mice become morbidly obese after weaning. Treatment of ob/ob mice with exogenous leptin reduces body weight by decreasing food intake and stimulating energy utilization, but even when saline- and leptin-injected ob/ob mice are pair-fed, mice receiving leptin lose significantly more weight. Therefore, the purpose of the present study was to test the hypotheses that uncoupling protein-1 (UCP1) expression is reduced in adipose tissue from ob/ob mice and is restored by treatment with exogenous leptin. Lean and ob/ob mice (5-6 weeks old) were housed at 23 C and treated with leptin (20 microg/g BW x day) for 3 days before they were killed. Compared with levels in lean littermates, UCP1 messenger RNA (mRNA) and protein levels were lower in brown adipose tissue (BAT) and retroperitoneal white adipose tissue (WAT) from ob/ob mice. Treatment of ob/ob mice with leptin reduced body weight and produced a 4- to 5-fold increase in UCP1 mRNA levels in both interscapular BAT and retroperitoneal WAT. The increases in UCP1 mRNA were accompanied by comparable increases in UCP1 protein in mitochondrial preparations from each tissue. Given that the sole known function of UCP1 is to uncouple oxidative phosphorylation, the present results are consistent with the conclusion that leptin stimulates energy utilization in ob/ob mice by increasing thermogenic activity and capacity (UCP1). In addition, the present results suggest that decreased UCP1 expression in BAT and WAT of ob/ob mice is in part responsible for their increased metabolic efficiency and propensity to become obese.     

In vitro lipolytic effect of leptin on mouse adipocytes: evidence for a possible autocrine/paracrine role of leptin

The present study has examined the effects of the adipocyte-derived hormone, leptin, on lipolysis in fat cells of different types of mice. Exposure to leptin (1.25.10(-6) M to 1.25.10(-12) M) increased (P < 0.01) the lipolytic activity of fat cells obtained from lean mice. A greater stimulation was observed when adipocytes from ob/ob mice were examined. Throughout the concentrations tested, the leptin-induced lipolysis observed in fat cells of lean animals was smaller than that obtained in ob/ob mice. The maximal lipolytic effect in obese animals was observed with 10(-8) M of OB protein. The lipolytic activity following the addition of 1.25.10(-10) M to 1.25.10(-6) M was significantly increased (P < 0.01) in ob/ob mice compared to lean animals. Adipocytes from ob/ob mice responded in a dose-dependent manner to the OB protein, while the leptin-induced lipolysis observed in lean animals was dose-independent. In contrast to lean and ob/ob mice, leptin did not stimulate lipolysis in adipocytes from db/db mice, which have a mutation in the leptin receptor gene. These in vitro studies suggest an autocrine/paracrine action of leptin on white fat cells and envisages the involvement of the OB protein, not only in centrally mediated pathways, but also in physiological functions which take place peripherally.     

Regulation of ob gene and overexpression in obesity

The ob gene product, called leptin, is a recently discovered hormone secreted by the adipose cells. By acting as a satiety factor and increasing energy expenditure, leptin plays a major role in body weight homeostasis in mice. Ob gene and leptin production by the adipose cells are under the control of various hormonal and metabolic factors. Ob mRNA levels are markedly reduced by fasting and restored to normal by refeeding. High-fat feeding increases ob gene and plasma leptin, and induces a state of resistance to leptin. Two hormones, insulin and corticosterone, increase leptin production in rodent and human adipose cells. In contrast, the activity of the sympathetic nervous system exerts an opposite effect, mainly through activation of the adipose beta 3-adrenergic receptors. Leptin synthesis is also decreased by thiazolidinediones, a new class of antidiabetic drugs. The obese Zucker fa/fa rats bear a mutation in the leptin receptor gene (OB-R) and are leptin resistant. In these rats, ob mRNA levels are increased early in life and are not reduced by fasting. This suggests that functional OB-Rs are required for the generation of the signal(s) that downregulates ob gene expression in the adipose cell. The extent to which this is relevant to human obesities, which are characterized by increased leptin levels, remains to be determined.     

Effects of leptin adipose tissue lipoprotein lipase in the obese ob/ob mouse

OBJECTIVE: To characterize the adaptations of lipid metabolism, with special emphasis on tissue lipoprotein lipase, to negative energy balance brought by chronic treatment of obese ob/ob mice with leptin. DESIGN: According to a 2 x 2 factorial analysis, lean and obese C57BL/6J mice were subcutaneously infused with leptin (100 micrograms.kg-1.day-1) or vehicle (phosphate-buffered saline) during seven days. RESULTS: Cumulative food intake and final body weight of vehicle-infused obese mice were twofold higher than in lean controls. Leptin decreased cumulative food intake and body weight of obese, but not lean mice. Lipoprotein lipase (LPL) activity in white inguinal and epididymal and brown interscapular adipose tissues of control obese mice was at least twofold higher than in lean mice, but comparable in the vastus lateralis muscle. Leptin treatment of obese mice significantly lowered LPL activity to that of lean mice in all tissues examined. Vehicle-infused obese mice had higher liver triglyceride content and were hypertriglyceridemic compared to lean mice, and triglyceride concentrations in plasma and liver were decreased proportionally after leptin treatment. Leptin lowered glycemia and insulinemia of obese mice to lean levels and decreased plasma corticosterone. Leptin infusion had no notable effect on tissue lipoprotein lipase nor plasma variables of lean mice. CONCLUSIONS: Leptin infusion abolished hyperinsulinemia in the ob/ob mouse, an effect that was probably responsible for the concomitant normalization of adipose LPL activity. This study shows that decreased LPL activity, plasma triglyceride concentrations and hepatic triglyceride production constitute some of the adaptive peripheral adaptations of lipid metabolism, which accompany the reduction in fat mass accretion brought by leptin treatment of the obese ob/ob mouse.     

Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice

Correction of the obese state induced by genetic leptin deficiency reduces elevated levels of both blood glucose and hypothalamic neuropeptide Y (NPY) mRNA in ob/ob mice. To determine whether these responses are due to a specific action of leptin or to the reversal of the obese state, we investigated the specificity of the effect of systemic leptin administration to ob/ob mice (n = 8) on levels of plasma glucose and insulin and on hypothalamic expression of NPY mRNA. Saline-treated controls were either fed ad libitum (n = 8) or pair-fed to the intake of the leptin-treated group (n = 8) to control for changes of food intake induced by leptin. The specificity of the effect of leptin was further assessed by 1) measuring NPY gene expression in db/db mice (n = 6) that are resistant to leptin, 2) measuring NPY gene expression in brain areas outside the hypothalamus, and 3) measuring the effect of leptin administration on hypothalamic expression of corticotropin-releasing hormone (CRH) mRNA. Five daily intraperitoneal injections of recombinant mouse leptin (150 micrograms) in ob/ob mice lowered food intake by 56% (P < 0.05), body weight by 4.1% (P < 0.05), and levels of NPY mRNA in the hypothalamic arcuate nucleus by 42.3% (P < 0.05) as compared with saline-treated controls. Pair-feeding of ob/ob mice to the intake of leptin-treated animals produced equivalent weight loss, but did not alter expression of NPY mRNA in the arcuate nucleus. Leptin administration was also without effect on food intake, body weight, or NPY mRNA levels in the arcuate nucleus of db/db mice. In ob/ob mice, leptin did not alter NPY mRNA levels in cerebral cortex or hippocampus or the expression of CRH mRNA in the hypothalamic paraventricular nucleus (PVN). Leptin administration to ob/ob mice also markedly reduced serum glucose (8.3 +/- 1.2 vs. 24.5 +/- 3.8 mmol/l; P < 0.01) and insulin levels (7,263 +/- 1,309 vs. 3,150 +/- 780 pmol/l), but was ineffective in db/db mice. Pair-fed mice experienced reductions of glucose and insulin levels that were < 60% of the reduction induced by leptin. The results suggest that in ob/ob mice, systemic administration of leptin inhibits NPY gene overexpression through a specific action in the arcuate nucleus and exerts a hypoglycemic action that is partly independent of its weight-reducing effects. Furthermore, both effects occur before reversal of the obesity syndrome. Defective leptin signaling due to either leptin deficiency (in ob/ob mice) or leptin resistance (in db/db mice) therefore leads directly to hyperglycemia and the overexpression of hypothalamic NPY that is implicated in the pathogenesis of the obesity syndrome.     

Hypothalamic agouti-related protein messenger ribonucleic acid is inhibited by leptin and stimulated by fasting

Agouti-related protein (AGRP) is a homologue of the agouti gene product and, when overexpressed, promotes obesity. Like neuropeptide Y (NPY) messenger RNA (mRNA), AGRP mRNA is produced in the hypothalamic arcuate nucleus and is elevated in leptin-deficient ob/ob and leptin-resistant db/db mice. These data suggest that AGRP mRNA might be affected by leptin and nutritional status in parallel with NPY mRNA. To test this hypothesis, we examined if AGRP mRNA would be, like NPY mRNA, inhibited by leptin injections and stimulated by fasting. AGRP mRNA was elevated in ob/ob mice about 5-fold compared with wild-type controls and was significantly inhibited by leptin treatment, as assessed by Northern blot analysis. In wild-type mice, AGRP mRNA was increased at least 13-fold by a 2-day fast, as assessed both by Northern blot analysis and in situ hybridization. In ad lib fed db/db mice, AGRP mRNA was elevated about 8-fold compared with ad lib fed wild-type controls, and was further increased by fasting in db/db mice. These data suggest that AGRP mRNA and NPY mRNA respond similarly to leptin and fasting.     

Leptin rapidly suppresses insulin release from insulinoma cells, rat and human islets and, in vivo, in mice

Obesity is associated with diabetes, and leptin is known to be elevated in obesity. To investigate whether leptin has a direct effect on insulin secretion, isolated rat and human islets and cultured insulinoma cells were studied. In all cases, mouse leptin inhibited insulin secretion at concentrations within the plasma range reported in humans. Insulin mRNA expression was also suppressed in the cultured cells and rat islets. The long form of the leptin receptor (OB-Rb) mRNA was present in the islets and insulinoma cell lines. To determine the significance of these findings in vivo, normal fed mice were injected with two doses of leptin. A significant decrease in plasma insulin and associated rise in glucose concentration were observed. Fasted normal and leptin receptor-deficient db/db mice showed no response to leptin. A dose of leptin, which mimicked that found in normal mice, was administered to leptin-deficient, hyperinsulinemic ob/ob mice. This caused a marked lowering of plasma insulin concentration and a doubling of plasma glucose. Thus, leptin has a powerful acute inhibitory effect on insulin secretion. These results suggest that the action of leptin may be one mechanism by which excess adipose tissue could acutely impair carbohydrate metabolism.     

Effects of pneumoperitoneum with carbon dioxide, argon, or helium on hemodynamic and respiratory function

In this study we investigated ob gene expression and plasma leptin levels in Psammomys obesus (the Israeli Sand Rat), a polygenic animal model of obesity and non-insulin-dependent diabetes mellitus. The ob gene was expressed exclusively in adipocytes of Psammomys obesus. DNA sequencing revealed a high degree of homology with other species (90% with mouse, 88% with rat and 79% with human). No ob gene sequence differences were found between lean and obese Psammomys obesus, and the codon 105 mutation found in ob/ob mice was not detected. Ob gene expression in Psammomys obesus correlated with body weight (r = 0.436, p < 0.001), percent body fat (r = 0.645, p < 0.001) and plasma insulin concentration (r = 0.651, p < 0.001). This is the first time that ob gene expression has been shown to increase steadily over a continuous wide range of body weight or plasma insulin in an animal model of obesity. Ob gene expression was significantly elevated in obese compared with lean Psammomys obesus (p < 0.05). No significant difference in ob gene expression was found between the four adipose tissue depots tested. Psammomys obesus plasma leptin levels correlated with body weight (r = 0.36, p < 0.05), percent body fat (r = 0.702, p < 0.01) and plasma insulin concentration (r = 0.735, p < 0.001). Plasma leptin concentrations were significantly increased in insulin-resistant animals independent of body weight. These results show that Psammomys obesus is an excellent animal model in which to study the ob gene and leptin, and confirm the importance of insulin as a significant factor in the regulation of leptin and ob gene expression.     

Abnormal regulation of the leptin gene in the pathogenesis of obesity

A subset of obese humans has relatively low plasma levels of leptin. This finding has suggested that in some cases abnormal regulation of the leptin gene in adipose tissue is etiologic in the pathogenesis of the obese state. The possibility that a relative decrease in leptin production can lead to obesity was tested by mating animals carrying a weakly expressed adipocyte specific aP2-human leptin transgene to C57BL/6J ob/ob mice (which do not express leptin). The transgene does not contain the regulatory elements of the leptin gene and is analogous to a circumstance in which the cis elements and/or trans factors regulating leptin RNA production are abnormal. The ob/ob mice carrying the transgene had a plasma leptin level of 1. 78 ng/ml, which is approximately one-half that found in normal, nontransgenic mice (3.72 ng/ml, P < 0.01). The ob/ob animals expressing the leptin transgene were markedly obese though not as obese as ob/ob mice without the transgene. The infertility as well as several of the endocrine abnormalities generally evident in ob/ob mice were normalized in the ob/ob transgenic mice. However, the ob/ob transgenic mice had an abnormal response when placed at an ambient temperature of 4 degreesC, suggesting that different thresholds exist for the different biologic effects of leptin. Leptin treatment of the ob/ob transgenic mice resulted in marked weight loss with efficacy similar to that seen after treatment of wild-type mice. In aggregate these data suggest that dysregulation of leptin gene can result in obesity with relatively normal levels of leptin and that this form of obesity is responsive to leptin treatment.     

A nutrient-sensing pathway regulates leptin gene expression in muscle and fat

Leptin, the protein encoded by the obese (ob) gene, is synthesized and released in response to increased energy storage in adipose tissue. However, it is still not known how incoming energy is sensed and transduced into increased expression of the ob gene. The hexosamine biosynthetic pathway is a cellular 'sensor' of energy availability and mediates the effects of glucose on the expression of several gene products. Here we provide evidence for rapid activation of ob gene expression in skeletal muscle by glucosamine. Increased tissue concentrations of the end product of the hexosamine biosynthetic pathway, UDP-N-acetylglucosamine (UDP-GlcNAc), result in rapid and marked increases in leptin messenger RNA and protein levels (although these levels were much lower than those in fat). Plasma leptin levels and leptin mRNA and protein levels in adipose tissue also increase. Most important, stimulation of leptin synthesis is reproduced by either hyperglycaemia or hyperlipidaemia, which also increase tissue levels of UDP-N-acetylglucosamine in conscious rodents. Finally, incubation of 3T3-L1 pre-adipocytes and L6 myocytes with glucosamine rapidly induces ob gene expression. Our findings are the first evidence of inducible leptin expression in skeletal muscle and unveil an important biochemical link between increased availability of nutrients and leptin expression.     

Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin

Reduction in the activity of the alpha-melanocyte-stimulating hormone (alpha-MSH) system causes obesity, and infusions of alpha-MSH can produce satiety, raising the possibility that alpha-MSH may mediate physiological satiety signals. Since alpha-MSH is coded for by the pro-opiomelanocortin (POMC) gene, we examined if POMC gene expression would be inhibited by fasting in normal mice or in models of obesity characterized by leptin insufficiency (ob/ob) or leptin insensitivity (db/db). In wild-type mice, hypothalamic POMC mRNA was decreased > 60% after a 2-day fast and was positively correlated with leptin mRNA. Similarly, compared with controls, POMC mRNA was decreased by at least 60% in both db/db and ob/ob mice. POMC mRNA was negatively correlated with both neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) mRNA. Finally, treatment of both male and female ob/ob mice with leptin stimulated hypothalamic POMC mRNA by about threefold. These results suggest that impairment in production, processing, or responsiveness to alpha-MSH may be a common feature of obesity and that hypothalamic POMC neurons, stimulated by leptin, may constitute a link between leptin and the melanocortin system.     

Upregulation of leptin receptor mRNA expression in obese mouse brain

Leptin receptor gene expression in the brains of lean (+/+) and obese (ob/ob) C57Bl/6 mice was examined using a non-radioactive in situ hybridization detection method. Significant increases in leptin receptor mRNA expression were found in the ventromedial and arcuate hypothalamic nuclei, piriform and olfactory cortices and medial habenular nucleus. There were very minor changes in the amount of leptin receptor mRNA expression in hippocampus proper (CA1-3). Results indicated that leptin receptor is upregulated when there is a lack of functional leptin, as in hereditary obese (ob/ob) mice. It is also suggested that leptin receptor may be an autoreceptor.     

Total energy expenditure and the level of physical activity correlate with plasma leptin concentrations in five-year-old children

Leptin, the product of the ob gene, is a hormone secreted by adipocytes that is known to decrease food intake and increase energy expenditure in ob/ob mice. In humans, variants in the OB gene have not been detected and very little is known about the action of leptin on food intake and energy expenditure, although circulating leptin concentrations are positively correlated to body fat stores. The purpose of this study was to assess the relationship between fasting plasma leptin concentrations and energy expenditure in 123 5-yr-old Pima Indian children (67 males/76 females). Body composition was assessed by isotopic water dilution (18O) whereas total energy expenditure (TEE) and resting metabolic rate (RMR) were measured using doubly labeled water and indirect calorimetry, respectively. The physical activity level was calculated as the ratio of TEE:RMR. Plasma leptin concentrations were positively correlated to percent body fat (r = 0.84, P < 0.0001), but were similar in boys and girls after adjusting for percent body fat. Most importantly, we found that, independent of the percentage of body fat, plasma leptin concentrations correlated with TEE (in absolute values, r = 0.37, P < 0.0001, or adjusted for body size r = 0.42; P < 0.0001) and with physical activity level (r = 0.26, P < 0.01), but not RMR. These results suggest that, as in animal models, leptin plays a role in energy expenditure in humans.     

Multihormonal control of ob gene expression and leptin secretion from cultured human visceral adipose tissue: increased responsiveness to glucocorticoids in obesity

The direct role of hormones on leptin synthesis has not yet been studied in cultured adipose cells or tissue from lean and obese subjects. Moreover, this hormonal regulation has never been addressed in human visceral fat, although this site plays a determinant role in obesity-linked disorders. In this study, we investigated the hormonal control of ob expression and leptin production in cultured visceral adipose tissue from lean and obese subjects. We more particularly focused on the interactions between glucocorticoids and insulin. We also briefly tackled the role of cAMP, which is still unknown in man. Visceral (and subcutaneous) adipose tissues from eight obese (body mass index, 41 +/- 2 kg/m2) and nine nonobese (24 +/- 1 kg/m2) subjects were sampled during elective abdominal surgery, and explants were cultured for up to 48 h in MEM. The addition of dexamethasone to the medium increased ob gene expression and leptin secretion in a time-dependent manner. Forty-eight hours after dexamethasone (50 nmol/L) addition, the cumulative integrated ob messenger ribonucleic acid (mRNA) and leptin responses were, respectively, approximately 5- and 4-fold higher in obese than in lean subjects. These responses closely correlated with the body mass index. The stimulatory effect of the glucocorticoid was also concentration dependent (EC50 = approximately 10 nmol/L). Although the maximal response was higher in obese than in lean subjects, the EC50 values were roughly similar in both groups. Unlike dexamethasone, insulin had no direct stimulatory effect on ob gene expression and leptin secretion. Singularly, insulin even inhibited the dexamethasone-induced rise in ob mRNA and leptin release. This inhibition was observed in both lean and obese subjects, whereas the expected stimulation of insulin on glucose metabolism and the accumulation of mRNA species for the insulin-sensitive transporter GLUT4 and glyceraldehyde-3-phosphate dehydrogenase occurred in lean patients only. This inhibitory effect was already detectable at 10 nmol/L insulin and was also observed in subcutaneous fat. Although a lowering of intracellular cAMP concentrations is involved in some of the effects of insulin on adipose tissue, this cannot account for the present finding, because the addition of cAMP to the medium also decreased ob mRNA and leptin secretion (regardless of whether dexamethasone was present). In conclusion, glucocorticoids, at physiological concentrations, stimulated leptin secretion by enhancing the pretranslational machinery in human visceral fat. This effect was more pronounced in obese subjects due to a greater responsiveness of the ob gene and could contribute to the metabolic abnormalities associated with central obesity by para/endocrine actions of hyperleptinemia on adipocytes and liver. Unlike dexamethasone, insulin had no direct stimulatory effect on ob gene expression and leptin secretion, and even prevented the positive response to dexamethasone by a cAMP-independent mechanism that remained functional despite insulin resistance.     

Glucocorticoid regulation of leptin synthesis and secretion in humans: elevated plasma leptin levels in Cushing's syndrome

Leptin, the obese (ob) gene product, is an adipocyte-derived satiety factor that is involved in the regulation of food ingestion and body weight. To investigate glucocorticoid regulation of leptin synthesis and secretion in humans, we measured plasma leptin levels in patients with Cushing's syndrome with adrenal or pituitary adenoma and in patients with iatrogenic Cushing's syndrome. Plasma leptin levels in patients with Cushing's syndrome were significantly elevated compared to those in nonobese healthy subjects and obese subjects without any metabolic or endocrine diseases at a given percentage of body fat by analysis of covariance. In patients with adrenal or pituitary adenoma, after the tumor resection, plasma leptin levels were reduced, with a concurrent decrease in plasma cortisol levels. With no significant changes in body weight, plasma leptin levels were also elevated significantly in lean healthy volunteers 24 h after the administration of 1 mg dexamethasone. Dexamethasone potently induced ob gene expression and leptin secretion in the organ culture of human adipose tissue. The data demonstrate that glucocorticoids act, at least in part, directly on the adipose tissue and increase leptin synthesis and secretion in humans.     

Obesity and hyperleptinemia in metallothionein (-I and -II) null mice

Metallothionein (MT) has several putative roles in metal detoxification, in Zn and Cu homeostasis, in scavenging free radicals, and in the acute phase response. Mice of mixed 129/Ola and C57BL/6J background with targeted disruption of MT-I and MT-II genes are more sensitive to toxic metals and oxidative stress. We noted that these animals were larger than most strains of mice, and we systematically studied aspects of their physiology and biochemistry relating to energy metabolism. During the first 2 weeks after weaning, the growth rates of MT-null and C57BL/6J mice were similar, but the transgenic mice became significantly heavier at age 5-6 weeks. At age 14 weeks, the body weight and food intake of MT-null mice was 16 and 30% higher, respectively, compared with C57BL/6J mice. Most 22- to 39-week-old male MT-null mice were obese, as shown by increased fat accretion, elevated obese (ob) gene expression, and high plasma leptin levels, similar to those recorded in Zucker fatty (fa/fa) rats. Seven-week-old MT-null mice also had significantly higher levels of plasma leptin and elevated expression of ob, lipoprotein lipase, and CCAAT enhancer binding protein alpha genes as compared with age-matched C57BL/6J mice. These observations indicate that abnormal accretion of body fat and adipocyte maturation is initiated at 5-7 weeks of age, possibly coincident with sexual maturation. Targeted disruption of MT-I and MT-II genes seems to induce moderate obesity, providing a new obese animal model. A link between MT and the regulation of energy balance is implied.     

Leptin inhibits glycogen synthesis in the isolated soleus muscle of obese (ob/ob) mice

The ob gene product, leptin, causes significant and dose-dependent inhibition of basal and insulin-stimulated glycogen synthesis in isolated soleus muscle from ob/ob mice, and a smaller, non-significant inhibition in muscle from wild-type mice. Leptin had no inhibitory effect on glycogen synthesis in soleus muscle from the diabetic (db/db) mice, which lack the functional leptin receptor. The full-length leptin receptor (Ob-Rb), is expressed in soleus muscle of both ob/ob and wild-type mice, however with no detectable differences in expression level. These results suggest that hyperleptinaemia may attenuate insulin action on glucose storage in skeletal muscle.     

13C NMR study of the effects of leptin treatment on kinetics of hepatic intermediary metabolism

The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin's biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin's effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive 13C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring flux of 13C-labeled substrate through several multistep pathways. In perfused liver from either ob/ob or lean mice, we found that acute treatment with leptin in vitro modulates pathways controlling carbohydrate flux into 13C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of ob/ob liver also caused a rapid stimulation of long-chain fatty acid synthesis from 13C-labeled acetyl-CoA by the de novo synthesis route. Chronic leptin treatment in vivo induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-13C]pyruvate in ob/ob mouse liver perfused in the absence of the hormone. Consistent with the 13C NMR results, leptin treatment of the ob/ob mouse in vivo resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin in vivo exerted the opposite effect of acute treatment in vitro and markedly decreased hepatic de novo synthesis of fatty acids in ob/ob mouse liver. In agreement with the 13C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the ob/ob mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.