Leptin increases osteoblast-specific osteocalcin release through a hypothalamic relay.

Enhanced long-term expression of leptin by gene therapy selectively in the hypothalamus, without leakage to the systemic circulation, abrogated skeletal abnormalities and reinstated weight and insulin-glucose homeostasis in leptin-deficient ob/ob mice. Whether increases in osteocalcin, a hormone produced by osteoblasts and known to play a role in bone growth and recently in glucose-insulin homeostasis, may link these benefits of central leptin was assessed. The effects of a single intraventricular injection of non-immunogenic, non-pathogenic recombinant adeno-associated virus vector encoding leptin gene (rAAV-lep) or green fluorescent protein gene (rAAV-GFP, control) were studied in three genotypes, wild type (wt), obese diabetic, hyperinsulinemic ob/ob and non-obese, diabetic insulinopenic Akita mice. Selective hypothalamic leptin expression with central rAAV-lep treatment decreased weight, fat mass, food intake, suppressed insulin levels in ob/ob and wt mice, and conferred euglycemia by suppressing blood glucose in all three genotypes. Contemporaneously, rAAV-lep treatment also augmented blood osteocalcin levels. In wt mice, osteocalcin rose by 51% and, whereas, basal osteocalcin levels in ob/ob and Akita mice were significantly lower as compared to those in wt mice (26% and 55%, respectively), gene therapy reinstated levels to the control range in ob/ob mice, and raised 40% above the wt range even in the absence of insulin in Akita mice. These findings demonstrate that the central beneficial effects of leptin on bone growth involve increased hypothalamic relay of signals that augment osteocalcin efflux from osteoblasts into the general circulation, a response that, in turn, may also modulate glucose-insulin and weight homeostasis.

Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis.

Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcA/cytochrome-c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome-c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification.

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.

Low abundance of NPY in the hypothalamus can produce hyperphagia and obesity.

States of increased metabolic demand are associated with up-regulation of NPY and hyperphagia. However, we present some instances of hyperphagia in which NPY is not up-regulated. Ablation or functional disruption of specific sites in the hypothalamus, such as the ventromedial or paraventricular nuclei, or transection of inputs to the hypothalamus from the hindbrain results in hyperphagia and excess body weight gain. However, NPY expression and concentration in these experimental models is either decreased or unchanged. While there is no up-regulation of NPY in these models, there is increased sensitivity to the orexigenic effects of NPY. This enhanced responsiveness to NPY may more than compensate for the reduced levels of NPY and result in hyperphagia and excess body weight gain. The hyper-responsiveness may be due either to an increase in NPY receptors or to other changes in target cells and response pathways that may result from the treatments used in these models.

Leptin gene transfer in the hypothalamus enhances longevity in adult monogenic mutant mice in the absence of circulating leptin.

Leptin, a product of the ob gene, is a pleiotropic signal implicated in regulation of multiple physiological functions in the periphery and centrally, including hypothalamic integration of energy homeostasis. Recessive mutations of ob gene result in early onset of hyperphagia, morbid obesity, metabolic disorders, early mortality and shortened life-span. Intracerebroventricular injection of recombinant adeno-associated virus vector (rAAV) encoding the leptin gene in adult obese ob/ob mice enhanced leptin transgene expression only in the hypothalamus, normalized food intake, body weight and more than doubled the life-span as compared to control cohorts and extended it to near that of normal wild type mice. These life-extending benefits were associated with drastic reductions in visceral fat, and blood glucose and insulin levels, but elevated ghrelin levels, the anti-aging biomarkers. Thus, bioavailability of leptin transduced by ectopic gene in the hypothalamus alone is both necessary and sufficient to normalize life-span. Evidently, site-specific ectopic gene expression with rAAV is durable and safe for alleviating neural disorders that stem from missing or functional disruption of a single gene.

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.

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.

The hypothalamic paraventricular nucleus is not essential for orexigenic NPY or anorexigenic melanocortin action.

Bilateral electrolytic lesions of the paraventricular nucleus of the hypothalamus (PVN) produce hyperphagia with excess weight gain. The orexigenic neuropeptide Y (NPY) system and the anorexigenic melanocortin system act in the PVN to regulate food intake, and participate in mediating the anorexic effects of leptin. We hypothesized that changes in the responsiveness of these systems may contribute to the hyperphagia observed in PVN-lesioned rats. Adult female Sprague-Dawley rats received either sham or electrolytic lesions in the PVN immediately followed by implantation of a guide cannula into the third cerebroventricle. Twenty-five days following surgery groups of sham and hyperphagic PVN-lesioned rats were injected intracerebroventricularly (i.c.v.) with either 118 pmole or 470 pmole of NPY and food intake was measured for 3 h. Food intake in response to NPY was nearly three-fold higher in PVN-lesioned rats as compared to sham rats. However, the response to 5 microg leptin i.c.v. was not different in lesioned versus sham rats. The effect of the melanocortin agonist MTII on food intake was tested in additional rats beginning either 7-14 days or 30-40 days following surgery. Doses of 0.1 nmole or 1.0 nmole of MTII were injected immediately before lights-off and food intake was measured at 2 h, 24 h and 48 h post-injection. Suppression of food intake in PVN-lesioned rats was not different from that in sham-lesioned rats. These data suggest that hyper-responsiveness to NPY may account in part for the hyperphagia observed in PVN-lesioned rats. Furthermore, based on the similarities of responses of PVN-lesioned and sham control rats to the anorexigenic agents MTII and leptin and the hypersensitivity of lesioned rats to NPY, we conclude that the PVN is not essential for NPY stimulation of food intake or for melanocortin suppression of food intake and that NPY and melanocortin receptors outside of the PVN are sufficient to produce these effects.

Perigestational suppression of weight gain with central leptin gene therapy results in lower weight F1 generation.

The efficacy of central leptin therapy on weight homeostasis through various phases of reproduction, pregnancy outcome and postnatal, prepubertal and pubertal growth of offspring was assessed. Enhanced leptin transgene expression after a single intracerebroventricular injection of recombinant adeno-associated virus vector encoding the leptin gene (rAAV-lep) decreased calorie intake and weight in adult nulliparous female rats. rAAV-lep treated rats conceived normally, displayed unremarkable pregnancy rate, parturition and delivered normal sized litters. Significantly lower weight was maintained through gestation, lactation, and post-lactation periods. The maintenance of a modest weight reduction was accompanied by voluntarily reduced calorie intake, increased thermogenic energy expenditure, decreased adiposity as reflected by drastically reduced leptin levels, and suppressed insulin and insulin-like growth factor 1 levels through lactation and post-lactation in rAAV-lep treated dams. The offspring at birth weighed significantly less than those of controls and this lower weight range was sustained during postnatal, prepubertal, pubertal and adult (3 months old) periods, contemporaneous with metabolic circulating hormones in the normal range. For the first time we show the persistent efficacy of central leptin gene therapy to suppress weight gain through all phases of reproduction, lactation and post-lactation in dams and reveal the potential imprinting link to producing lower weight in the F1 generation.

Central leptin differentially modulates ultradian secretory patterns of insulin, leptin and ghrelin independent of effects on food intake and body weight.

We tested the hypothesis that leptin acts centrally to differentially modulate the ultradian communication of leptin, insulin and ghrelin with the hypothalamus. The ultradian fluctuation of these hormones in plasma after central leptin gene therapy was analyzed. Increased leptin transgene expression in the hypothalamus significantly decreased energy intake and body weight concomitant with severe hypoleptinemia and hypoinsulinemia resulting from drastically suppressed peak heights with unchanged frequency discharge of these hormones. Ghrelin secretion was, however, increased solely due to increased pulse amplitude. In pair-fed control rats leptin and ghrelin secretion was unchanged. In conclusion, independent of restraint on caloric intake and weight, leptin acting centrally modulates only the pulse amplitude of ultradian rhythmicity of the three afferent signals involved in the hypothalamic integration of energy balance. Since rhythmic discharge patterns dictate target response of hormones, these findings reveal a novel hypothalamic action of leptin in the pathophysiology of the obesity-dependent metabolic syndrome.

High-fat diet-induced ultradian leptin and insulin hypersecretion are absent in obesity-resistant rats.

OBJECTIVE: Sprague-Dawley rats fed a high-fat diet (HFD) are either obesity prone (OP) or obesity resistant (OR). We tested the hypothesis that differences in the ultradian rhythmic patterns of insulin and ghrelin in OP vs. OR rats promote obesity in OP rats. RESEARCH METHODS AND PROCEDURES: Rats were fed regular chow or an HFD, and ultradian fluctuations in leptin, insulin, and ghrelin were analyzed in blood samples collected at 5-minute intervals from intrajugular cannulae of freely moving rats. RESULTS: Regular chow feeding resulted in a slow weight gain accompanied by small increases in insulin and leptin and a decrease in ghrelin discharge, with only the pulse amplitude significantly altered. Similar changes were observed in OR rats, despite HFD consumption. In contrast, OP rats exhibited a high rate of weight gain and marked hyperinsulinemia, hyperleptinemia, and hypoghrelinemia; amplitude was altered, but frequency was stable. In a short-term experiment, HFD elicited similar secretory patterns of smaller magnitude even in the absence of weight gain. DISCUSSION: We showed that three hormonal signals of disparate origin involved in energy homeostasis were secreted in discrete episodes, and only the pulse amplitude component was vulnerable to age and HFD consumption. Increases in insulin and leptin and decreases in ghrelin pulse amplitude caused by HFD were exaggerated in OP rats relative to OR rats and preceded the weight increase. These findings show that a distinct genetic predisposition in the endocrine organs of OR rats confers protection against high-fat intake-induced ultradian hypersecretion of obesity-promoting hormonal signals.

Leptin modulates orexigenic effects of ghrelin and attenuates adiponectin and insulin levels and selectively the dark-phase feeding as revealed by central leptin gene therapy.

We tested the hypothesis that leptin acts centrally and peripherally by different mechanisms to control peripheral hormones that normally regulate weight homeostasis. The paradigm of selectively increasing leptin transgene expression with a single intracerebroventricular injection of adeno-associated viral vectors encoding leptin (rAAV-lep) or green fluorescent protein (control) in the hypothalamus of mutant leptin-deficient ob/ob and wild-type (wt) mice was employed in these experiments. rAAV-lep injection increased hypothalamic leptin expression in the complete absence of peripheral leptin in ob/ob mice; suppressed body weight and adiposity; voluntarily decreased dark-phase food intake; suppressed plasma levels of adiponectin, TNFalpha, free fatty acids and insulin, concomitant with normoglycemia; and elevated ghrelin levels for extended period. Body weight and plasma levels of leptin and metabolic variables were suppressed to a lesser extent in rAAV-lep wt mice without decreasing food intake. The sustained high leptin transgene expression decreased only the dark-phase phagia in both genotypes, but wt mice escaped from leptin restraint during the lights-on phase, resulting in normal overall food intake. Leptin administration rapidly decreased plasma gastric ghrelin and adipocyte adiponectin but not TNFalpha levels, thereby demonstrating a peripheral restraining action of leptin on the secretion of hormones of varied origins. Whereas ghrelin administration readily stimulated feeding in controls, it was completely ineffective in rAAV-lep-treated wt mice. Thus, leptin expressed locally in the hypothalamus counteracted the central orexigenic effects of peripheral ghrelin. Cumulatively, these results identify newer central and peripheral modulatory influences of leptin on hormonal signals of disparate origin implicated in weight homeostasis and metabolic disorders.

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.

Decreased hypothalamic concentration of neuropeptide Y correlates with onset of hyperphagia in fa/fa rats on postnatal day 12.

An increased action of hypothalamic neuropeptide Y (NPY) has been proposed as a major factor in the pathophysiology of the obesity syndrome in Zucker (fa/fa) rats. Using a developmental strategy to test this hypothesis, we showed previously that significantly more arcuate NPY was expressed in fa/fa pups than in lean littermates on postnatal day (P) 2 and throughout the preweaning period [Physiol. Behav. 67 (1999) 521], and that hyperphagia first appeared on P12 [Am. J. Physiol. 275 (1998) R1106]. To test the hypothesis further, we used a specific radioimmunoassay to measure the concentration of hypothalamic NPY peptide in lean (+/+ and +/fa) and obese fa/fa Zucker rat pups on P9, P10, and P12. The concentration of NPY in fa/fa pups was not significantly different from that of the other genotypes. There was, however, a significant decrease in NPY concentration from P9 to P12 in fa/fa pups, but not in lean pups. The combination of increased NPY message and decreasing concentration of NPY peptide in fa/fa pups with age is consistent with, but does not prove, increased release of hypothalamic NPY in fa/fa pups just before and on P12 when hyperphagia emerges. These results provide further support for the importance of hypothalamic NPY in the phenotypic expression of hyperphagia in the fa/fa pups during the second postnatal week.

Rhythmic, reciprocal ghrelin and leptin signaling: new insight in the development of obesity.

The hypothalamus integrates metabolic, neural and hormonal signals to evoke an intermittent appetitive drive in the daily management of energy homeostasis. Three major players identified recently in the feedback communication between the periphery and hypothalamus are leptin, ghrelin and neuropeptide Y (NPY). We propose that reciprocal circadian and ultradian rhythmicities in the afferent humoral signals, anorexigenic leptin from adipocytes and orexigenic ghrelin from stomach, encode a corresponding discharge pattern in the appetite-stimulating neuropeptide Y network in the hypothalamus. An exquisitely intricate temporal relationship among these signaling modalities with varied sites of origin is paramount in sustenance of weight control on a daily basis. Our model envisages that subtle and progressive derangements in temporal communication, imposed by environmental shifts in energy intake, impel a positive energy balance culminating in excessive weight gain and obesity. This conceptual advance provides a new target for designing pharmacologic or gene transfer therapies that would normalize the rhythmic patterns of afferent hormonal and efferent neurochemical messages.

Long-term suppression of weight gain, adiposity, and serum insulin by central leptin gene therapy in prepubertal rats: effects on serum ghrelin and appetite-regulating genes.

Intracerebroventricular administration of recombinant adeno-associated virus (rAAV) encoding the rat leptin gene (rAAV-lep) to 24-d-old female and male rats suppressed postpubertal weight gain for extended periods by decreasing food consumption and adiposity, as reflected by lowered serum leptin, insulin, and FFA. Serum ghrelin levels were increased in young but not older rats. Central rAAV-lep therapy also increased energy expenditure through nonshivering thermogenesis in younger rats as shown by expression of uncoupling protein mRNA in brown adipose tissue. The sustained decrease in appetite seemingly resulted from attenuation of appetite-stimulating neuropeptide Y and enhancement of appetite-inhibiting melanocortin signalings in the hypothalamus. Neither the onset of pubertal sexual maturation nor reproductive cyclicity in adult female rats was affected by the sustained reduction in energy consumption and weight gain. These findings demonstrate that central leptin gene therapy in prepubertal rats is a novel therapy to control postpubertal weight gain, adiposity, and hyperinsulinemia for extended periods.

Central leptin gene therapy blocks high-fat diet-induced weight gain, hyperleptinemia, and hyperinsulinemia: increase in serum ghrelin levels.

Recombinant adeno-associated virus (rAAV), encoding either rat leptin (rAAV-lep) or green fluorescent protein (rAAV-GFP, control), was injected intracerebroventricularly in rats consuming a high-fat diet (HFD; 45 kcal%). Caloric consumption and body weight were monitored weekly until the rats were killed at 9 weeks. Untreated control rats consuming regular rat diet (RCD; 11 kcal%) were monitored in parallel. Body weight gain was accelerated in rAAV-GFP + HFD control rats relative to those consuming RCD, despite equivalent kcal consumption. At 9 weeks, serum leptin, free fatty acids, triglycerides, and insulin were elevated in HFD control rats. In contrast, rAAV-lep treatment reduced intake and blocked the HFD-induced increase in weight, adiposity, and metabolic variables. Blood glucose was slightly reduced but within the normal range, and serum ghrelin levels were significantly elevated in rAAV-lep + HFD rats. Uncoupling protein-1 (UCP1) mRNA in brown adipose tissue (BAT), an index of energy expenditure through nonshivering thermogenesis, was decreased in rats consuming HFD. Treatment with rAAV-lep significantly augmented BAT UCP1 mRNA expression, indicating increased thermogenic energy expenditure. These findings demonstrate that central leptin gene therapy efficiently prevents weight gain, increased adiposity, and hyperinsulinemia in rats consuming an HFD by decreasing energy intake and increasing thermogenic energy expenditure.