Increased leptin expression selectively in the hypothalamus suppresses inflammatory markers CRP and IL-6 in leptin-deficient diabetic obese mice

Low-grade systemic inflammation, as indicated by increased circulating levels of inflammatory markers CRP and IL-6, is linked to increased risks for cardiovascular diseases (CVD) and diabetes mellitus in obese subjects. Whereas hyperleptinemia in obesity are associated with increased CRP and IL-6 release, the hypothalamic versus peripheral site of leptin action has not been ascertained. The effects of increased leptin supply selectively in the hypothalamus by gene therapy on pro-inflammatory CRP and IL-6 levels and on markers of diabetes in the circulation of ob/ob mice displaying either age-related or dietary obesity were assessed. A recombinant adeno-associated viral vector encoding either green-fluorescent protein (control) or leptin gene was injected intracerebroventricularly. Five weeks later, one-half of each of the vector groups was switched to high-fat diet consumption and the other half continued to consume regular low-fat chow diet. Body weight and visceral white adipose tissue were drastically reduced and hyperinsulinemia and hyperglycemia were abrogated by leptin gene therapy, independent of the dietary fat content. The elevated plasma CRP and IL-6 levels seen in obese ob/ob mice receiving the control vector, regardless of the fat content of the diet, were markedly suppressed by increased hypothalamic leptin in both groups. The results show for the first time that leptin deficiency elevates and reinstatement of leptin selectively in the hypothalamus suppresses the release of pro-inflammatory biomarkers, a response likely to alleviate CVD associated with obesity.     

Central LIF gene therapy suppresses food intake,body weight,serum leptin and insulin for extended periods

Leukemia inhibitory factor (LIF) overexpression, induced by the intracerebroventricular (i.c.v.) injection of an recombinant adeno-associated viral vector encoding LIF (rAAV-LIF), resulted in a dose-dependent reduction in body weight (BW) gain, food intake (FI) and adiposity, evidenced by suppression of serum leptin and free fatty acids for an extended period in outbred adult female rats. A dose-dependent reduction in serum insulin levels and unchanged serum glucose, energy expenditure through thermogenesis as indicated by uncoupling protein-1 (UCP-1) mRNA expression in brown adipose tissue (BAT), and metabolism as indicated by serum T3 and T4, accompanied the blockade of weight gain. Thus, central rAAV-LIF therapy is a viable strategy to voluntarily reduce appetite and circumvent leptin resistance, a primary factor underlying age-dependent weight gain and obesity in rodents and humans.     

Central leptin gene therapy blocks ovariectomy-induced adiposity

Objective: In this study, we tested the hypothesis that insufficiency of leptin restraint in the hypothalamus is responsible for promoting weight gain and adiposity after ovariectomy (ovx). Whether increasing leptin transgene expression can overcome the diminution in leptin restraint was evaluated in ovx rats. Research Methods and Procedures: Enhanced leptin or green fluorescent protein (GFP; control) transgene expression was induced by a single intracerebroventricular injection of recombinant adeno‐associated viral vector encoding either leptin gene (rAAV‐lep) or GFP gene (rAAV‐GFP; control) in acutely and chronically ovx rats. Body weight and food intake responses were monitored weekly. White adipose tissue (WAT) mass and serum levels of WAT‐derived hormones, leptin, and adiponectin were analyzed at termination of the experiments. Results and Discussion: An increase in leptin transgene expression in the hypothalamus initiated soon after ovx blocked hyperphagia and body weight gain and markedly suppressed WAT mass and adipokines, leptin, and adiponectin. Similar suppression of weight gain and adiposity and serum leptin and adiponectin levels after intracerebroventricular rAAV‐lep injection in chronically ovx rats were observed concomitant with unchanged daily food intake. These findings are consistent with the hypothesis that in the absence of ovarian steroids, the existent insufficiency of leptin restraint at the hypothalamic level can be overcome with ectopic leptin expression, thereby reinstating central control on weight and adiposity.     

Leptin Expression in Hypothalamic PVN Reverses Dietary Obesity and Hyperinsulinemia but Stimulates Ghrelin*

Objective: In order to circumvent the multiple peripheral effects of hyperleptinemia and leptin resistance, the efficacy of leptin transgene expression in the hypothalamic paraventricular nucleus (PVN) to reinstate the central energy homeostasis in obesity was examined. Research Methods and Procedures: A recombinant adeno‐associated viral vector encoding either leptin (rAAV‐lep) or green fluorescent protein (rAAV‐GFP) was microinjected into the PVN of obesity‐prone rats consuming a high‐fat diet (HFD). Results: rAAV‐lep, and not rAAV‐GFP, microinjection significantly reduced energy intake and enhanced energy expenditure, thereby resulting in normalization of weight and blood levels of leptin, insulin, free fatty acids, and glucose concomitant with enhanced ghrelin secretion during the extended period of observation. Discussion: Thus, we show, for the first time, that amelioration of leptin insufficiency with enhanced localized leptin availability in the PVN alone can reverse dietary obesity and the attendant hyperinsulinemia and concurrently block the central stimulatory effects of elevated endogenous ghrelin on food intake and adiposity.     

Neuromedin U has a novel anorexigenic effect independent of the leptin signaling pathway

Neuromedin U (NMU) is a hypothalamic neuropeptide that regulates body weight and composition. Here we show that mice lacking the gene encoding NMU (Nmu(-/-) mice) develop obesity. Nmu(-/-) mice showed increased body weight and adiposity, hyperphagia, and decreased locomotor activity and energy expenditure. Obese Nmu(-/-) mice developed hyperleptinemia, hyperinsulinemia, late-onset hyperglycemia and hyperlipidemia. Notably, however, treatment with exogenous leptin was effective in reducing body weight in obese Nmu(-/-) mice. In addition, central leptin administration did not affect NMU gene expression in the hypothalamus of rats. These results indicate that NMU plays an important role in the regulation of feeding behavior and energy metabolism independent of the leptin signaling pathway. These characteristic functions of NMU may provide new insight for understanding the pathophysiological basis of obesity.     

Obesity reduced the gene expressions of leptin receptors in hypothalamus and liver.

The expression of leptin receptor (OB-R) is downregulated by leptin in some cell lines. This study investigated the expressions of leptin receptors at central nerve system and peripheral site in a dietary model of obesity. Rats in the 8 week high-diet and control group were classified based on body weight gain into obese and control groups. Serum leptin and insulin concentrations were measured and gene expressions of short form of leptin receptor (OB-Ra) and long form (OB-Rb) in hypothalamus and liver were detected by RT-PCR. The levels of serum leptin in obese rats were increased compared with control rats (p<0.05). The levels of OB-Ra and OB-Rb gene expressions in both hypothalamus and liver in obese rats were reduced significantly (p<0.01). Serum leptin concentrations of obese rats had a significant negative relationship with both of OB-Ra or OB-Rb gene expression levels in hypothalamus and liver (p<0.01). On the other hand, serum insulin levels had no relationship with OB-Ra or OB-Rb gene expression levels in neither liver nor hypothalamus. Rats with diet-induced obesity have hyperleptinemia and reduced expressions of leptin receptors in hypothalamus and liver. The results suggest that a leptin downregulated OB-R expression is one of leptin resistant mechanisms for maintaining obesity.     

AAV-mediated leptin receptor installation improves energy balance and the reproductive status of obese female Koletsky rats

Leptin is a hormone secreted primarily by white adipocytes that regulates energy homeostasis and reproduction via CNS receptors. Koletsky (f/f) rats with a leptin receptor (OB-Rb) gene mutation are obese, diabetic and infertile. We employed recombinant adeno-associated viral (rAAV) vectors to transfer the human OB-Rb gene into the brains of female Koletsky rats to identify sites of leptin action in the brain. rAAV-OB-Rb was microinjected into the medial preoptic area (MPOA), the paraventricular nucleus (PVN), the ventromedial hypothalamus, the arcuate nucleus (ARC), or the dorsal vagal complex in the brainstem. Food intake and body weight were monitored bi-weekly for 55 days. Vaginal cytology was examined daily to assess estrous cyclicity. After sacrifice, uncoupling protein-1 (UCP-1) mRNA in brown adipose tissue and serum concentrations of leptin, insulin, glucose, estradiol and progesterone were measured. Expression of OB-Rb was documented by RT-PCR and site specificity of microinjection was verified by immunohistochemical detection of green fluorescent protein following a control microinjection of rAAV-GFP. OB-Rb installation in the ARC reduced food intake, however, energy expenditure, assessed by UCP-1 mRNA expression, was increased by OB-Rb installation in all sites except the PVN. When injected into the MPOA and ARC, rAAV-OB-Rb stimulated the reproductive axis as evidenced by normalization of estrous cycle length and increased luteinizing hormone releasing hormone concentrations in the hypothalamus. These studies show that long-term installation of a functional leptin receptor in the CNS is achievable using rAAV vectors and further show that leptin acts on specific sites in the brain to produce differential effects on food intake, energy expenditure and reproduction.     

Central leptin gene therapy fails to overcome leptin resistance associated with diet-induced obesity

The objective of this study was to determine if central overexpression of leptin could overcome the leptin resistance caused by 100 days of high-fat feeding. Three-month old-F344XBN male rats were fed either control low fat chow (Chow), which provides 15% of energy as fat, or a high-fat/high-sucrose diet (HF), which provides 59% of energy as fat. Over several weeks, the HF-fed animals spontaneously split into two groups of animals: those that became obese on the HF diet (DIO) and those that did not gain extra weight on the HF diet [diet resistant (DR)]. After 100 days of HF feeding, animals were given a single intracerebroventricular injection containing 5.75E10 particles of rAAV encoding leptin (rAAV-leptin) or control virus (rAAV-con). Chow animals responded robustly to rAAV-leptin, including significant anorexia, weight loss, and lipopenia. In contrast, DIO were completely unresponsive to rAAV-leptin. DR rats responded to rAAV-leptin, but in a more variable fashion than Chow. Unlike what was observed in Chow, the anorectic response to rAAV-leptin rapidly attenuated and was no longer significant by day 14 postvector delivery. Both DIO and DR animals were found to have reduced long-form leptin receptor expression and enhanced basal P-STAT-3 in the hypothalamus with respect to Chow. rAAV-leptin caused an increase in STAT3 phosphorylation and proopiomelanocortin expression in the hypothalamus and an increase in uncoupling protein-1 in brown adipose tissue in both Chow and DR animals, but failed to do so in DIO. This suggests that central overexpression of leptin is not a viable strategy to reverse diet-induced obesity.     

Single intracerebroventricular bolus injection of a recombinant adenovirus expressing leptin results in reduction of food intake and body weight in both lean and obese Zucker fa/fa rats

Leptin acts as a satiety factor within the central nervous system by binding to its receptor located in the hypothalamus. A missense mutation of the leptin receptor induces hyperphagia and obesity in the obese Zucker fa/fa rat. Since the CNS is an important target of leptin action, we hypothesized that leptin gene transfer into the lateral cerebral ventricle could efficiently lead to inhibition of food intake and reduction of body weight in obese fa/fa rats as well as in lean animals. A single intracerebroventricular injection of an adenoviral vector containing a cDNA encoding leptin resulted in the expression of leptin in the ependymal cells lining the ventricle and the secretion of leptin into the cerebrospinal fluid (CSF). During the first week after injection, when high concentrations of leptin were produced in the CSF, the reducing effects of leptin on food intake and body weight were comparable in lean and in obese fa/fa rats. The subsequent decline in CSF leptin levels, that was similar in lean and obese fa/fa rats, resulted in the faster resumption of food intake and body weight gain in obese than in lean animals, confirming a reduced sensitivity to leptin in the obese group. The results of this study show that leptin gene delivery into the cerebral ventricles allows for the production of elevated leptin concentrations in CSF, and they support the hypothesis that the impaired sensitivity to leptin may be overcome in obese fa/fa rats.     

Neuronal PTP1B regulates body weight, adiposity and leptin action

Obesity is a major health problem and a risk factor for type 2 diabetes. Leptin, an adipocyte-secreted hormone, acts on the hypothalamus to inhibit food intake and increase energy expenditure. Most obese individuals develop hyperleptinemia and leptin resistance, limiting the therapeutic efficacy of exogenously administered leptin. Mice lacking the tyrosine phosphatase PTP1B are protected from diet-induced obesity and are hypersensitive to leptin, but the site and mechanism for these effects remain controversial. We generated tissue-specific PTP1B knockout (Ptpn1(-/-)) mice. Neuronal Ptpn1(-/-) mice have reduced weight and adiposity, and increased activity and energy expenditure. In contrast, adipose PTP1B deficiency increases body weight, whereas PTP1B deletion in muscle or liver does not affect weight. Neuronal Ptpn1(-/-) mice are hypersensitive to leptin, despite paradoxically elevated leptin levels, and show improved glucose homeostasis. Thus, PTP1B regulates body mass and adiposity primarily through actions in the brain. Furthermore, neuronal PTP1B regulates adipocyte leptin production and probably is essential for the development of leptin resistance.     

Melanocortin activation of nucleus of the solitary tract avoids anorectic tachyphylaxis and induces prolonged weight loss

To examine the role of the brain stem melanocortin system in long-term energy regulation, we assessed the effects of overproduction of proopiomelanocortin (POMC) in the caudal brain stem of F344xBN rats with adult-onset obesity. Recombinant adeno-associated viral vector encoding POMC gene was delivered to the nucleus of solitary tract (NTS) in the hindbrain, and food intake, body weight, glucose and fat metabolism, brown adipose tissue thermogenesis, and mRNA levels of neuropeptides and melanocortin receptors were assessed. POMC delivery resulted in sustained reduction in food intake and body weight over 42 days and improved insulin sensitivity. At death, in recombinant adeno-associated viral vector-POMC-treated rats vs. control rats, alpha-melanocyte-stimulating hormone in NTS increased nearly 21-fold, whereas hypothalamic alpha-melanocyte-stimulating hormone remained unchanged. Visceral adiposity decreased by 37%; tissue triglyceride content diminished by 26% and 47% in liver and muscle, respectively; serum triglyceride and nonesterified fatty acids were reduced by 35% and 34%, respectively; phosphorylation of acetyl-CoA carboxylase was elevated by 63% in soleus muscle; brown adipose tissue uncoupling protein 1 increased by 30%; and melanocortin 3 receptor expression declined by 60%, whereas neuropeptide Y, agouti-related protein, and MC4 receptor mRNA levels were unchanged in the NTS. In conclusion, POMC overexpression in the NTS produces a characteristic unabated hypophagia that is uniquely different from the anorexic tachyphylaxis following POMC overexpression in the hypothalamus. The sustained anorectic response may result from absence of compensatory elements in the NTS, such as increased agouti-related protein expression, suggesting melanocortin activation of the brain stem may be a viable strategy to alleviate obesity.     

Hypothalamic clamp on insulin release by leptin-transgene expression

The effects of sustained leptin action locally in the hypothalamus on the functional link between fat accrual and insulin secretion after chronic high fat diet (HFD) consumption in leptin-deficient ob/ob mice, and on the post-prandial insulin response in rats consuming regular chow diet (RCD), was examined in this study. A single intracerebroventricular (icv) injection of recombinant adeno-associated virus vector encoding leptin gene (rAAV-lep) enhanced hypothalamic leptin-transgene expression in ob/ob mice consuming RCD and suppressed the time-related weight gain and fat accumulation concomitant with abrogation of hyperinsulinemia and enhanced glucose tolerance. This increased hypothalamic leptin-transgene expression continued to impose insulinopenia and increased glucose tolerance but was ineffective in suppressing weight gain and fat accumulation after these mice were switched to chronic HFD consumption. A similar icv rAAV-lep pretreatment in rats consuming RCD markedly attenuated the post-prandial rise in insulin release concomitant with suppressed weight and fat depots. These results show for the first time that a sustained hypothalamic leptin action can stably clamp pancreatic insulin secretion independent of the status of fat accrual engendered by diets of varying caloric enrichment. Thus, the efficacy of increased leptin afferent signaling in the hypothalamus to persistently restrain pancreatic insulin release and insulin resistance can be explored as an adjunct therapeutic modality to alleviate pathophysiological derrangements that confer type 2 diabetes.     

Increased leptin expression in the dorsal vagal complex suppresses adiposity without affecting energy intake and metabolic hormones

Objective: Increased leptin transgene expression locally in hypothalamic sites suppresses weight and energy intake, enhances thermogenic energy expenditure, and differentially modulates metabolic hormones for an extended period. We evaluated whether a similar localized expression of leptin transgene in the dorsal vagal complex (DVC) in the caudal brain stem that also displays the biologically relevant leptin receptor would reproduce these varied responses and thus demonstrate functional connectivity between the hypothalamus and DVC. Research Methods and Procedures: Adult female rats were microinjected with a recombinant adeno‐associated virus encoding either rat leptin or green fluorescent protein gene (control) in the DVC. Food intake and body weight were monitored weekly, and metabolic variables were analyzed at the end of 10 weeks. Results and Discussion: Increased leptin transgene expression in the DVC suppressed the time‐related increase in body weight accompanied by a transient decrease in food intake at week 1 post‐injection and little effect on thermogenic energy expenditure. That suppression of weight was due to decreased adiposity is shown by the markedly suppressed white adipose tissue‐derived hormones, leptin and adiponectin. Circulating concentrations of pancreatic insulin, gastric ghrelin, and glucose levels were unchanged. This segregation of the varied effects of leptin expression in hypothalamic sites vs. DVC endorses the view that among the various endocrine organs under sympathetic nervous system control, only those leptin‐activated neural circuits in the hypothalamus that suppress weight and adiposity on a long‐term basis transverse through DVC en route to white adipose tissue.     

Long-term consequences of maternal high-fat feeding on hypothalamic leptin sensitivity and diet-induced obesity in the offspring

Epidemiological and animal studies suggest that the alteration of hormonal and metabolic environment during fetal and neonatal development can contribute to development of metabolic syndrome in adulthood. In this paper, we investigated the impact of maternal high-fat (HF) diet on hypothalamic leptin sensitivity and body weight gain of offspring. Adult Wistar female rats received a HF or a control normal-fat (C) diet for 6 wk before gestation until the end of the suckling period. After weaning, pups received either C or HF diet during 6 wk. Body weight gain and metabolic and endocrine parameters were measured in the eight groups of rats formed according to a postweaning diet, maternal diet, and gender. To evaluate hypothalamic leptin sensitivity in each group, STAT-3 phosphorylation was measured in response to leptin or saline intraperitoneal bolus. Pups exhibited similar body weights at birth, but at weaning, those born to HF dams weighed significantly less (-12%) than those born to C dams. When given the HF diet, males and females born to HF dams exhibited smaller body weight and feed efficiency than those born to C dams, suggesting increased energy expenditure programmed by the maternal HF diet. Thus, maternal HF feeding could be protective against adverse effects of the HF diet as observed in male offspring of control dams: overweight (+17%) with hyperleptinemia and hyperinsulinemia. Furthermore, offspring of HF dams fed either C or HF diet exhibited an alteration in hypothalamic leptin-dependent STAT-3 phosphorylation. We conclude that maternal high-fat diet programs a hypothalamic leptin resistance in offspring, which, however, fails to increase the body weight gain until adulthood.     

Hormonal and metabolic effects of olanzapine and clozapine related to body weight in rodents

Objective: To characterize a model of atypical antipsychotic drug‐induced obesity and evaluate its mechanism. Research Methods and Procedures: Chronically, olanzapine or clozapine was self‐administered via cookie dough to rodents (Sprague‐Dawley or Wistar rats; C57Bl/6J or A/J mice). Chronic studies measured food intake, body weight, adiponectin, active ghrelin, leptin, insulin, tissue wet weights, glucose, clinical chemistry endpoints, and brain dopaminergic D2 receptor density. Acute studies examined food intake, ghrelin, leptin, and glucose tolerance. Results: Olanzapine (1 to 8 mg/kg), but not clozapine, increased body weight in female rats only. Weight changes were detectable within 2 to 3 days and were associated with hyperphagia starting ～24 hours after the first dose. Chronic administration (12 to 29 days) led to adiposity, hyperleptinemia, and mild insulin resistance; no lipid abnormalities or changes in D2 receptor density were observed. Topiramate, which has reversed weight gain from atypical antipsychotics in humans, attenuated weight gain in rats. Acutely, olanzapine, but not clozapine, lowered plasma glucose and leptin. Increases in glucose, insulin, and leptin following a glucose challenge were also blunted. Discussion: A model of olanzapine‐induced obesity was characterized which shares characteristics of patients with atypical antipsychotic drug‐induced obesity; these characteristics include hyperphagia, hyperleptinemia, insulin resistance, and weight gain attenuation by topiramate. This model may be a useful and inexpensive model of uncomplicated obesity amenable to rapid screening of weight loss drugs. Olanzapine‐induced weight gain may be secondary to hyperphagia associated with acute lowering of plasma glucose and leptin, as well as the inability to increase plasma glucose and leptin following a glucose challenge.     

Leptin resistance of adipocytes in obesity: role of suppressors of cytokine signaling

Liver-derived hyperleptinemia induced in normal rats by adenovirus-induced gene transfer causes rapid disappearance of body fat, whereas the endogenous adipocyte-derived hyperleptinemia of obesity does not. Here we induce liver-derived hyperleptinemia in rats with adipocyte-derived hyperleptinemia of acquired obesity caused by ventromedial hypothalamus lesioning (VMH rats) or by feeding 60% fat (DIO rats). Liver-derived hyperleptinemia in obese rats caused only a 5-7% loss of body weight, compared to a 13% loss in normoleptinemic lean animals; but in actual grams of weight lost there was no significant difference between obese and lean groups, suggesting that a subset of cells remain leptin-sensitive in obesity. mRNA and protein of a putative leptin-resistance factor, suppressor of cytokine signaling (SOCS)-1 or -3, were both increased in white adipose tissues (WAT) of VMH and DIO rats. Since transgenic overexpression of SOCS-3 in islets reduced the lipopenic effect of leptin by 75%, we conclude that the increased expression of SOCS-1 and -3 in WAT of rats with acquired obesity could have blocked leptin's lipopenic action in the leptin-resistant WAT population.     

Maternal obesity and fetal programming: effects of a high-carbohydrate nutritional modification in the immediate postnatal life of female rats

Our earlier studies have shown that the artificial rearing of newborn rat pups [first generation high carbohydrate (1-HC)] on an HC milk formula resulted in chronic hyperinsulinemia and adult-onset obesity (HC phenotype). Offspring [second-generation HC (2-HC)] of 1-HC female rats spontaneously acquired the HC phenotype in the postweaning period. In this study, we have characterized the development of the abnormal intrauterine environment in the 1-HC female rats and the effects on fetal development under such pregnancy conditions for the offspring. 1-HC female rats demonstrated hyperphagia on laboratory chow and increased body weight gain beginning from the immediate postweaning period along with hyperinsulinemia and hyperleptinemia. During pregnancy, 1-HC female rats showed several metabolic alterations including increased body weight gain and increased plasma levels of insulin, leptin, proinflammatory markers, and lipid peroxidation products. Although there were no significant changes in the body weights or litter size of term 2-HC fetuses, the plasma levels of insulin and leptin were significantly higher compared with those of control term fetuses. Quantitation of mRNA levels by real-time RT-PCR indicated significant increases in the mRNA levels of orexigenic neuropeptides in the hypothalamus of 2-HC term fetuses. Collectively, these results indicate that the HC diet in infancy results in an adverse pregnancy condition in female rats with deleterious consequences for the offspring.     

To subjugate NPY is to improve the quality of life and live longer

The interactive network of neuropeptide Y (NPY) and cohorts is necessary for integrating the hypothalamic regulation of appetite and energy expenditure with the endocrine and neuroendocrine systems on a daily basis. Genetic and environmental factors that produce an insufficiency of leptin restraint on NPY and cognate receptors deregulate the homeostasis to engender various life-threatening risk factors. Recent studies from our laboratory show that neurotherapy consisting of a single central administration of recombinant adeno-associated virus vector encoding the leptin gene can repress the hypothalamic NPY system for the lifetime of rodents. A major benefit of this stable tonic restraint is deceleration of pathophysiologic sequalae that shorten life span. These include suppression of weight gain, fat accumulation, circulating adipokines, amelioration of major symptoms of metabolic syndrome, improved reproduction and bone health. Thus, sustained repression of NPY signaling in the hypothalamus by leptin transgene expression can improve the quality of life and extend longevity.