Hypothalamic Leptin Gene Therapy Reduces Bone Marrow Adiposity in ob/ob Mice Fed Regular and High-Fat Diets.

Low bone mass is often associated with elevated bone marrow adiposity. Since osteoblasts and adipocytes are derived from the same mesenchymal stem cell (MSC) progenitor, adipocyte formation may increase at the expense of osteoblast formation. Leptin is an adipocyte-derived hormone known to regulate energy and bone metabolism. Leptin deficiency and high-fat diet-induced obesity are associated with increased marrow adipose tissue (MAT) and reduced bone formation. Short-duration studies suggest that leptin treatment reduces MAT and increases bone formation in leptin-deficient ob/ob mice fed a regular diet. Here, we determined the long-duration impact of increased hypothalamic leptin on marrow adipocytes and osteoblasts in ob/ob mice following recombinant adeno-associated virus (rAAV) gene therapy. Eight- to 10-week-old male ob/ob mice were randomized into four groups: (1) untreated, (2) rAAV-Lep, (3) rAAV-green fluorescent protein (rAAV-GFP), or (4) pair-fed to rAAV-Lep. For vector administration, mice were injected intracerebroventricularly with either rAAV-leptin gene therapy (rAAV-Lep) or rAAV-GFP (9 × 10(7) particles) and maintained for 30 weeks. In a second study, the impact of increased hypothalamic leptin levels on MAT was determined in mice fed high-fat diets; ob/ob mice were randomized into two groups and treated with either rAAV-Lep or rAAV-GFP. At 7 weeks post-vector administration, half the mice in each group were switched to a high-fat diet for 8 weeks. Wild-type (WT) controls included age-matched mice fed regular or high-fat diet. High-fat diet resulted in a threefold increase in MAT in WT mice, whereas MAT was increased by leptin deficiency up to 50-fold. Hypothalamic leptin gene therapy increased osteoblast perimeter and osteoclast perimeter with minor change in cancellous bone architecture. The gene therapy decreased MAT levels in ob/ob mice fed regular or high-fat diet to values similar to WT mice fed regular diet. These findings suggest that leptin plays an important role in regulating the differentiation of MSCs to adipocytes and osteoblasts, a process that may be dysregulated by high-fat diet. However, the results also illustrate that reducing MAT by increasing leptin levels does not necessarily result in increased bone mass.

Hypothalamic leptin gene therapy reduces body weight without accelerating age-related bone loss.

Excessive weight gain in adults is associated with a variety of negative health outcomes. Unfortunately, dieting, exercise, and pharmacological interventions have had limited long-term success in weight control and can result in detrimental side effects, including accelerating age-related cancellous bone loss. We investigated the efficacy of using hypothalamic leptin gene therapy as an alternative method for reducing weight in skeletally-mature (9 months old) female rats and determined the impact of leptin-induced weight loss on bone mass, density, and microarchitecture, and serum biomarkers of bone turnover (CTx and osteocalcin). Rats were implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either recombinant adeno-associated virus encoding the gene for rat leptin (rAAV-Leptin, n=7) or a control vector encoding green fluorescent protein (rAAV-GFP, n=10) and sacrificed 18 weeks later. A baseline control group (n=7) was sacrificed at vector administration. rAAV-Leptin-treated rats lost weight (-4±2%) while rAAV-GFP-treated rats gained weight (14±2%) during the study. At study termination, rAAV-Leptin-treated rats weighed 17% less than rAAV-GFP-treated rats and had lower abdominal white adipose tissue weight (-80%), serum leptin (-77%), and serum IGF1 (-34%). Cancellous bone volume fraction in distal femur metaphysis and epiphysis, and in lumbar vertebra tended to be lower (P<0.1) in rAAV-GFP-treated rats (13.5 months old) compared to baseline control rats (9 months old). Significant differences in cancellous bone or biomarkers of bone turnover were not detected between rAAV-Leptin and rAAV-GFP rats. In summary, rAAV-Leptin-treated rats maintained a lower body weight compared to baseline and rAAV-GFP-treated rats with minimal effects on bone mass, density, microarchitecture, or biochemical markers of bone turnover.

Should leptin replace insulin as a lifetime monotherapy for diabetes type 1 and 2?

Evidence accumulated during the last decade has affirmed that adipocyte leptin insufficiency in the hypothalamus is the primary etiological factor in the pathogenesis of diabetes type 1 and 2 and related metabolic morbidities. Leptin insufficiency disrupts the relay of hypothalamic regulatory information along three descending pathways to the organs in the periphery that normally participate in maintenance of glucose homeostasis on a minute-to-minute basis throughout lifetime. Reinstatement of leptin sufficiency in the hypothalamus by either systemic or central injections, or its provision selectively in the hypothalamus with the aid of gene therapy extinguished hyperglycemia and normalized blood glucose stably during the entire course of treatment in a variety of animal models of diabetes type 1 and 2. In follow-up clinical trials, twice daily leptin treatment in leptinopenic and insulinopenic type 1 diabetics and leptinopenic and hyperinsulinemic type 2 diabetics with congenital lipodystrophy or acquired lipoatrophy normalized blood glucose without any discernible adverse effects during the extended course of treatment. Taken together, these findings have amply endorsed the efficacy of leptin therapy to restore glucose homeostasis in insulin-deficient as well as hyperinsulinemic diabetic patients. Consequently, restoration of optimal hypothalmic signaling to reinstate glucose homeostasis with leptin is a highly suitable new therapeutic strategy to ameliorate diabetes type 1 and 2 for the lifetime and to replace the currently in vogue insulin monotherapy. In view of the relentless challenges posed by the worldwide epidemic of diabetes and soaring treatment costs, taken together with the well-known shortcomings of therapies based on restoring insulin signaling, it is highly critical and timely to undertake new clinical trials that ascertain appropriate dosage and route of leptin delivery to the hypothalamus capable of safely sustaining stable glycemia for lifetime.

Peripheral leptin regulates bone formation.

Substantial evidence does not support the prevailing view that leptin, acting through a hypothalamic relay, decreases bone accrual by inhibiting bone formation. To clarify the mechanisms underlying regulation of bone architecture by leptin, we evaluated bone growth and turnover in wild-type (WT) mice, leptin receptor-deficient db/db mice, leptin-deficient ob/ob mice, and ob/ob mice treated with leptin. We also performed hypothalamic leptin gene therapy to determine the effect of elevated hypothalamic leptin levels on osteoblasts. Finally, to determine the effects of loss of peripheral leptin signaling on bone formation and energy metabolism, we used bone marrow (BM) from WT or db/db donor mice to reconstitute the hematopoietic and mesenchymal stem cell compartments in lethally irradiated WT recipient mice. Decreases in bone growth, osteoblast-lined bone perimeter and bone formation rate were observed in ob/ob mice and greatly increased in ob/ob mice following subcutaneous administration of leptin. Similarly, hypothalamic leptin gene therapy increased osteoblast-lined bone perimeter in ob/ob mice. In spite of normal osteoclast-lined bone perimeter, db/db mice exhibited a mild but generalized osteopetrotic-like (calcified cartilage encased by bone) skeletal phenotype and greatly reduced serum markers of bone turnover. Tracking studies and histology revealed quantitative replacement of BM cells following BM transplantation. WT mice engrafted with db/db BM did not differ in energy homeostasis from untreated WT mice or WT mice engrafted with WT BM. Bone formation in WT mice engrafted with WT BM did not differ from WT mice, whereas bone formation in WT mice engrafted with db/db cells did not differ from the low rates observed in untreated db/db mice. In summary, our results indicate that leptin, acting primarily through peripheral pathways, increases osteoblast number and activity.

Hypothalamic leptin gene therapy prevents weight gain without long-term detrimental effects on bone in growing and skeletally mature female rats.

Hypothalamic leptin gene therapy normalizes the mosaic skeletal phenotype of leptin-deficient ob/ob mice. However, it is not clear whether increased hypothalamic leptin alters bone metabolism in animals already producing the hormone. The objective of this study was to evaluate the long duration effects of recombinant adeno-associated virus-rat leptin (rAAV-Lep) hypothalamic gene therapy on weight gain and bone metabolism in growing and skeletally mature leptin-replete female Sprague-Dawley rats. Rats were either unoperated or implanted with cannulas in the third ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP (control vector encoding green fluorescent protein) and maintained on standard rat chow fed ad libitum for either 5 or 10 weeks (starting at 3 months of age) or 18 weeks (starting at 9 months of age). Tibias, femurs, or lumbar vertebrae were analyzed by micro-computed tomography and/or histomorphometry. In comparison with age-matched rAAV-GFP rats, rAAV-Lep rats maintained a lower body weight for the duration of studies. At 5 weeks after vector administration, rAAV-Lep rats had lower cancellous bone volume and bone marrow adiposity but higher osteoblast perimeter compared with nonoperated controls. However, these values did not differ between the two groups at 10 weeks after vector administration. Differences in cancellous bone volume and architecture were not detected between the rAAV-Lep and rAAV-GFP groups at either time point. Also, rAAV-Lep had no negative effects on bone in the 9-month-old skeletally mature rats at 18 weeks after vector administration. We hypothesize that the transient reductions in bone mass and bone marrow adiposity at 5 weeks after vector administration were due to hypothalamic surgery. We conclude that increased hypothalamic leptin, sufficient to prevent weight gain, has minimal specific effects (rAAV-Lep versus rAAV-GFP) on bone metabolism in normal female rats.

Neuroendocrine control of energy homeostasis: update on new insights.

Recent upsurge in research has uncovered distinct circuitries that regulate appetite, energy expenditure and fat accrual under the supervision of hormonal feedback signalling of adipocyte leptin and gastric ghrelin in the hypothalamic integration of energy homeostasis. A host of messenger molecules of diverse chemical composition and origin mediate the crosstalk between the three circuitries. Leptin is now recognized as the mandatory afferent signal in maintenance of weight homeostasis. Leptin insufficiency in the hypothalamus due to diminished transport of leptin across the blood-brain barrier (BBB) imposed by environmental causes, such as consumption of energy-enriched diets and diminished energy expenditure, orchestrates unregulated fat accrual and the attendant disease cluster of metabolic syndrome. Bioavailability of leptin selectively in the hypothalamic targets with the aid of gene therapy successfully averted the environmentally induced metabolic afflictions and normalized lifespan. Thus, sustenance of optimal sufficiency in leptin signalling solely in the hypothalamus is a novel strategy to combat the worldwide epidemic of obesity and metabolic syndrome.

Central leptin gene therapy ameliorates diabetes type 1 and 2 through two independent hypothalamic relays; a benefit beyond weight and appetite regulation.

Although its role in energy homeostasis is firmly established, the evidence accumulated over a decade linking the adipocyte leptin-hypothalamus axis in the pathogenesis of diabetes mellitus has received little attention in the contemporary thinking. In this context various lines of evidence are collated here to show that (1) under the direction of leptin two independent relays emanating from the hypothalamus restrain insulin secretion from the pancreas and mobilize peripheral organs--liver, skeletal muscle and brown adipose tissue--to upregulate glucose disposal, and (2), leptin insufficiency in the hypothalamus produced by either leptinopenia or restriction of leptin transport across the blood brain barrier due to hyperleptinemia of obesity and aging, initiate antecedent pathophysiological sequalae of diabetes type 1 and 2. Further, we document here the efficacy of leptin replenishment in vivo, especially by supplying it to the hypothalamus with the aid of gene therapy, in preventing the antecedent pathophysiological sequalae--hyperinsulinemia, insulin resistance and hyperglycemia--in various animal models and clinical paradigms of diabetes type 1 and 2 with or without attendant obesity. Overall, the new insights on the long-lasting antidiabetic potential of two independent hypothalamic relays engendered by central leptin gene therapy and the preclinical safety indicators in rodents warrant further validation in subhuman primates and humans.

Central leptin gene therapy, a substitute for insulin therapy to ameliorate hyperglycemia and hyperphagia, and promote survival in insulin-deficient diabetic mice.

Long-term benefits of central leptin gene therapy in insulin-deficient diabetes are not known despite its therapeutic effects in obesity animal models such as ob/ob and diet-induced obese mice. Adult male mice were injected intraperitoneally with streptozotocin (STZ, 200mg/kg) to induce insulitis. A week later, only diabetic STZ-pretreated mice (blood glucose >350 mg/dl) received intracerebroventricularly (icv) an injection of recombinant adeno-associated virus vector (rAAV) encoding either green fluorescent protein (control), or leptin gene (rAAV-lep). Body weight (BW), food intake, blood glucose, insulin and survival rate responses were monitored post-icv injection at regular intervals for 52 weeks. The STZ pre-injected diabetic mice remained hyperphagic, gradually lost BW and died by week 6 after receiving control vector. In marked contrast, injection of rAAV-lep to raise hypothalamic leptin levels, rescued the STZ-pretreated mice from early mortality, gradually curbed hyperphagia to normalize intake by week 20, and maintained BW at significantly lower than the control range. Blood glucose levels in these mice started to recede dramatically by week 2-3 to normalize by week 8, and euglycemia was sustained during the remaining course of the experiment. rAAV-lep injected mice did not exhibit any discernible untoward gross behavioral changes and diabetic complications and showed a partial return of pancreatic beta-cell function. These results show for the first time that one time central leptin gene therapy is effective and durable in reinstating euglycemia and energy homeostasis for extended periods in the absence of insulin.

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.

Disruption in the leptin-NPY link underlies the pandemic of diabetes and metabolic syndrome: new therapeutic approaches.

Multidisciplinary research from my and my colleagues' laboratory has shown that disruption at various levels of leptin signaling to the interactive hypothalamic network of neuropeptide Y (NPY) and cohorts contributes to the antecedent pathophysiologic sequelae of the disease cluster of the metabolic syndrome. Disruptions in NPY signaling due to high or low abundance of NPY and cognate receptors dysregulate the homeostatic milieu to promote hyperinsulinemia, hyperglycemia, fat accrual, and overt diabetes. Hyperleptinemia induced by consumption of energy-rich diets inhibits leptin transport across the blood-brain barrier and thereby produces leptin insufficiency in the hypothalamus. Sustained leptin insufficiency results in loss of hypothalamic restraint on pancreatic insulin secretion and diminished glucose metabolism and energy expenditure. This chain of events culminates in hyperinsulinemia, hyperglycemia, and diabetes. Our recent studies have shown that increasing the supply of leptin centrally by gene therapy reinstates the restraint on hypothalamic NPY signaling and ameliorates diabetes and the attendant disease cluster of the metabolic syndrome. Thus, newer therapies that would enhance leptin transport across the blood-brain barrier in a timely manner or reinstate leptin restraint on NPY signaling through central leptin gene therapy or pharmacologically with leptin mimetics are likely to curtail the pathophysiologic sequelae of diabetes and related ailments of the metabolic syndrome.

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 leptin insufficiency syndrome: an interactive etiology for obesity, metabolic and neural diseases and for designing new therapeutic interventions.

This review critically reappraises recent scientific evidence concerning central leptin insufficiency versus leptin resistance formulations to explain metabolic and neural disorders resulting from subnormal or defective leptin signaling in various sites in the brain. Research at various fronts to unravel the complexities of the neurobiology of leptin is surveyed to provide a comprehensive account of the neural and metabolic effects of environmentally imposed fluctuations in leptin availability at brain sites and the outcome of newer technology to restore leptin signaling in a site-specific manner. The cumulative new knowledge favors a unified central leptin insufficiency syndrome over the, in vogue, central resistance hypothesis to explain the global adverse impact of deficient leptin signaling in the brain. Furthermore, the leptin insufficiency syndrome delineates a novel role of leptin in the hypothalamus in restraining rhythmic pancreatic insulin secretion while concomitantly enhancing glucose metabolism and non-shivering thermogenic energy expenditure, sequelae that would otherwise promote fat accrual to store excess energy resulting from consumption of energy-enriched diets. A concerted effort should now focus on development of newer technologies for delivery of leptin or leptin mimetics to specifically target neural pathways for remediation of diverse ailments encompassing the central leptin insufficiency syndrome.

Global life-long health benefits of repression of hypothalamic NPY system by central leptin gene therapy.

A minute-to-minute crosstalk between the hypothalamic neuropeptide Y (NPY) network and the hormone leptin is essential for energy homeostasis. Leptin insufficiency i.e. lack of leptin restraint due to genetic and environmental factors on the hypothalamic NPY system confers obesity, a cluster of metabolic afflictions and shorter lifespan. A state-of-the-art gene transfer technology using recombinant adeno-associated viral vector to overcome hypothalamic leptin insufficiency was employed in rodent models of obesity, metabolic syndrome and shorter lifespan. Our findings show that life-long tonic repression of NPY system with a stable increase in leptin availability in the hypothalamus prevented the age-related and high fat-diet-induced obesity, hyperinsulinemia and diabetes and extended lifespan. Additional health benefits include increased energy expenditure and normalization of neuroendocrine control on reproduction, and promotion of brain and bone growth. We propose that central leptin gene therapy or novel long-acting leptin mimetics should be tested clinically to decelerate the worldwide epidemic of obesity, diabetes and shortened lifespan.

Central leptin gene therapy corrects skeletal abnormalities in leptin-deficient ob/ob mice.

Skeletal growth is tightly coupled to energy balance via complex and incompletely understood mechanisms. Leptin-deficient ob/ob mice are obese and develop multiple pathologies associated with the metabolic syndrome. Additionally, ob/ob mice have skeletal abnormalities. The objective of this study was to evaluate the effects of leptin deficiency and long duration selective central leptin repletion via recombinant adeno-associated virus-leptin (rAAV-lep) gene therapy on bone in growing ob/ob mice. The ob/ob mice were injected in the hypothalamus with either rAAV-lep or rAAV-GFP (control vector). Treated ob/ob and untreated wild-type (WT) mice were then maintained on a normal diet for 15 weeks. In a second experiment, similarly treated mice along with a group of pair-fed mice were maintained for 30 weeks. Leptin was not detected in blood of either rAAV-lep- or rAAV-GFP-treated mice although rAAV-lep-treated mice displayed leptin transgene expression in the hypothalamus. As expected, rAAV-lep normalized body weight and food intake. Compared to WT mice, rAAV-GFP-treated ob/ob mice had decreased femoral length (by 1.6 mm or 10%, P<0.001), decreased total femur bone volume (by 3.3 mm(3) or 19%, P<0.001), but increased cancellous bone volume in the distal femur (by 0.04 mm(3) or 60%, P<0.09) and lumbar vertebrae (by 0.26 mm(3) or 118%, P<0.001). Treatment with rAAV-lep rescued the ob/ob skeletal phenotype by increasing femoral length and total bone volume, and decreasing femoral and vertebral cancellous bone volume, so that at 15 weeks post-rAAV-lep injection the ob/ob mice no longer differed from WT mice. No further skeletal changes in either the femur or lumbar vertebra were observed at 30 weeks post-rAAV-lep administration. The results suggest that hypothalamic leptin functions as an essential permissive factor for normal bone growth.

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.

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.

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.