Effects of increased hypothalamic leptin gene expression on ovariectomy-induced bone loss in rats.

Estrogen deficiency results in accelerated bone turnover with a net increase in bone resorption. Subcutaneous administration of leptin attenuates bone loss in ovariectomized (ovx) rats by reducing bone resorption. However, in addition to its direct beneficial effects, leptin has been reported to have indirect (central nervous system-mediated) antiosteogenic effects on bone, which may limit the efficacy of elevated serum leptin to prevent estrogen deficiency-associated bone loss. The present study evaluated the long-term effects of increased hypothalamic leptin transgene expression, using recombinant adeno-associated virus-leptin (rAAV-Lep) gene therapy, on bone mass, architecture, and cellular endpoints in sexually mature ovx Sprague-Dawley rats. Ovx rats were implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP (control vector encoding green fluorescent protein) and maintained for 10 weeks. Additional controls consisted of ovary-intact rats and ovx rats pair-fed to rAAV-Lep rats. Lumbar vertebrae were analyzed by micro-computed tomography and tibiae by histomorphometry. Cancellous bone volume was lower and osteoclast perimeter, osteoblast perimeter, and bone marrow adipocyte density were greater in ovx rats compared to ovary-intact controls. In contrast, differences among ovx groups were not detected for any endpoint evaluated. In conclusion, whereas estrogen deficiency resulted in marked cancellous osteopenia, increased bone turnover and marrow adiposity, increasing hypothalamic leptin transgene expression in ovx rats had neither detrimental nor beneficial effects on bone mass, architecture, or cellular endpoints. These findings demonstrate that the antiresorptive effects of subcutaneous leptin administration in ovx rats are mediated through leptin targets in the periphery.

Pivotal role of leptin-hypothalamus signaling in the etiology of diabetes uncovered by gene therapy: a new therapeutic intervention?

The incidence of diabetes mellitus has soared to epidemic proportion worldwide. The debilitating chronic hyperglycemia is caused by either lack of insulin as in diabetes type 1 or its ineffectiveness as in diabetes type 2. Frequent replacement of insulin with or without insulin analogs for optimum glycemic control are the conventional cumbersome therapies. Recent application of leptin gene transfer technology has uncovered the participation of adipocytes-derived leptin-dependent hypothalamic neural signaling in glucose homeostasis and demonstrated that a breakdown in this communication due to leptin insufficiency in the hypothalamus underlies the etiology of chronic hyperglycemia. Reinstatement of central leptin sufficiency by hyperleptinemia produced either by intravenous leptin infusion or a single systemic injection of recombinant adenovirus vector encoding leptin gene suppressed hyperglycemia and evoked euglycemia only transiently in rodent models of diabetes type 1. In contrast, stable restoration of leptin sufficiency, solely in the hypothalamus, with biologically active leptin transduced by an intracerebroventicular injection of recombinant adeno-associated virus vector encoding leptin gene (rAAV-lep) abolished hyperglycemia and imposed euglycemia through the extended duration of experiment by stimulating glucose disposal in the periphery in models of diabetes type 1. Further, similar hypothalamic leptin transgene expression abrogated chronic hyperglycemia and hyperinsulinemia, the predisposing risk factors of the age and environmentally acquired diabetes type 2, and instituted euglycemia by independently activating relays that stimulate glucose metabolism and repress hyperinsulinemia and improve insulin sensitivity in the periphery. Consequently, this durable antidiabetic efficacy of one time rAAV-lep neurotherapy offers a potential novel substitute for insulin therapy following preclinical trials in subhuman primates and humans.

Body mass influences cortical bone mass independent of leptin signaling.

Obesity in humans is associated with increased bone mass. Leptin, a hormone produced by fat cells, functions as a sentinel of energy balance, and may mediate the putative positive effects of body mass on bone. We performed studies in male C57Bl/6 wild type (WT) and leptin-deficient ob/ob mice to determine whether body mass gain induced by high fat intake increases bone mass and, if so, whether this requires central leptin signaling. The relationship between body mass and bone mass and architecture was evaluated in 9-week-old and 24-week-old WT mice fed a regular mouse diet. Femora and lumbar vertebrae were analyzed by micro computed tomography. In subsequent studies, slowly and rapidly growing ob/ob mice were injected in the hypothalamus with a recombinant adeno-associated virus containing the leptin gene (rAAV-lep) or a control vector, rAAV-GFP (green fluorescent protein). The mice were maintained on a regular control diet for 5 or 7 weeks and then subdivided into groups and either continued on the control diet or fed a high fat diet (45% of kcal from fat) for 8 weeks. In the WT mice, femoral and vertebral bone mass was positively correlated with body mass (Pearson's r=0.65-0.88 depending on endpoint). rAAV-lep therapy dramatically decreased body mass (-61%) but increased femur length. However, in the distal femur and lumbar vertebra, rAAV-lep therapy reduced cancellous bone volume/tissue volume, trabecular number and trabecular thickness, and increased trabecular spacing. The high fat diet increased body mass, irrespective of vector treatment. Total femur bone volume, length, cross-sectional volume, and cortical volume and thickness were increased in mice with increased body mass, independent of rAAV treatment. In the distal femur, increased body mass had no effect on cancellous architecture and there were no vector x body mass interactions. In WT mice, increased body mass resulted in increased (+33%) vertebral cancellous bone volume/tissue volume. Increased body mass had minimal independent effect on cancellous vertebral bone mass in ob/ob mice. Taken together, these findings suggest that increased body mass has a positive effect on femur cortical bone mass that is independent of leptin signaling.

Neuroendocrine actions and regulation of hypothalamic neuropeptide Y during lactation.

The expression of neuropeptide Y (NPY) and its co-messenger, agouti-related peptide (AgRP), in arcuate neurons of the hypothalamus is increased during lactation in rats. Our research has been addressing the questions of the physiological actions of these peptides during lactation and the physiological signals associated with lactation that result in increased expression of their genes. Our studies indicate that NPY and AgRP exert pleiotropic actions during lactation that help integrate neuroendocrine regulation of energy balance with controls over anterior and posterior pituitary hormone secretion. Further, reciprocal signaling to the NPY/AgRP system by leptin and ghrelin is responsible for the changes in expression of these hypothalamic peptides in lactating animals, and thus, may contribute to regulation of food intake and the various neuroendocrine adaptations of lactation.

Circulating adiponectin levels, body composition and obesity-related variables in Prader-Willi syndrome: comparison with obese subjects.

BACKGROUND: People with obesity and/or the metabolic syndrome have an increased risk for developing diabetes and cardiovascular disease and may have low adiponectin levels. The obesity associated with Prader-Willi syndrome (PWS) would be expected to have similar complications. However, it was recently reported that, despite their adiposity, people with PWS have reduced visceral fat and are less likely to develop diabetes mellitus or the metabolic syndrome compared with people with simple obesity. OBJECTIVE: To determine if plasma adiponectin levels and other variables relevant to diabetes and cardiovascular risk are different in a cohort of PWS subjects with known genetic subtypes compared with age-, sex- and weight-matched control subjects. RESULTS: Fasting plasma glucose, C-peptide, triglycerides, leptin and cholesterol levels were similar in PWS and obese subjects. Our 20 PWS subjects (mean age = 27.7 years) had higher percent body fat (54.1 vs 48.5%) determined by DEXA measurements and lower percent lean mass (45.9 vs 51.5%) compared with 14 obese controls (mean age = 26.9 year). Plasma adiponectin levels were significantly higher in PWS (15.5 +/- 8.2 microg/ml) than in obese controls (7.5 +/- 2.7 microg/ml). A significant positive correlation was found with insulin sensitivity in PWS subjects (r = 0.75, P = 0.0003) but not in obese controls (r = 0.36, P = 0.20). DISCUSSION: Our study confirmed an earlier observation of higher adiponectin levels in PWS subjects and less insulin resistance proportionate to their obesity status than found in subjects with simple obesity. Furthermore, no differences were seen in PWS subjects with the chromosome 15 deletion or maternal disomy 15. The reported excessive visceral adiposity in subjects with simple obesity compared with PWS may be associated with decreased production and lower circulating levels of adiponectin.

Differential effects of methamphetamine on expression of neuropeptide Y mRNA in hypothalamus and on serum leptin and ghrelin concentrations in ad libitum-fed and schedule-fed rats.

Relatively little is known concerning the interaction of psychostimulants with hypothalamic neuropeptide systems or metabolic hormones implicated in regulation of energy balance. The present studies tested whether methamphetamine alters the expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP), two important orexigenic neuropeptides, or proopiomelanocortin (POMC), the precursor for the anorexigenic peptide alpha-melanocyte-stimulating hormone, or the secretion of leptin, insulin and ghrelin, concomitant with inhibition of food intake. Female rats were either fed ad libitum (AL) or placed on a scheduled feeding (SF) regimen, with access to food limited to 4 h/day. Administration of (+/-)-methamphetamine (7.5 mg/kg, i.p.) 2 h prior to food presentation significantly inhibited food intake in SF animals, but did not affect intake in AL animals. In a separate study, AL and SF animals were killed just prior to expected food presentation, and expression of NPY, AgRP and POMC mRNAs in hypothalamus was determined using in situ hybridisation; concentrations of leptin, insulin and ghrelin in serum were determined with radioimmunoassays. In saline-treated, SF controls, NPY and AgRP mRNA expression in arcuate nucleus and serum ghrelin were significantly elevated, and serum leptin and insulin were significantly reduced. Methamphetamine reversed the up-regulation of NPY mRNA expression observed in the SF condition, without affecting AgRP mRNA or the serum concentrations of metabolic hormones. However, in AL animals, NPY mRNA expression in arcuate and dorsomedial nuclei was significantly increased by methamphetamine, which also reduced serum leptin and insulin and increased serum ghrelin concentrations. These findings suggest that the inhibition of NPY expression in SF animals may be a mechanism underlying the anorexigenic effect of methamphetamine seen in this condition. The increase in NPY expression produced by methamphetamine in AL animals may be mediated by the ability of this drug to decrease secretion of leptin and insulin and increase secretion of ghrelin.

NPY and cohorts in regulating appetite, obesity and metabolic syndrome: beneficial effects of gene therapy.

Neuropeptide Y is the most potent physiological appetite transducer known. The NPY network is the conductor of the hypothalamic appetite regulating orchestra in the arcuate nucleus-paraventricular nucleus (ARC-PVN) of the hypothalamus. NPY and cohorts, AgrP, GABA and adrenergic transmitters, initiate appetitive drive directly through Y1, Y5, GABAA and alpha1 receptors, co-expressed in the magnocellular PVN (mPVN) and ARC neurons and by simultaneously repressing anorexigenic melanocortin signaling in the ARC-PVN axis. The circadian and ultradian rhythmicities in NPY secretion imprint the daily circadian and episodic feeding patterns. Although a number of afferent hormonal signals from the periphery can directly modulate NPYergic signaling, the reciprocal circadian and ultradian rhythmicities of anorexigenic leptin from adipocytes and orexigenic ghrelin from stomach, encode a corresponding pattern of NPY discharge for daily meal patterning. Subtle and progressive derangements produced by environmental and genetic factors in this exquisitely intricate temporal relationship between the two opposing humoral signals and the NPY network promote hyperphagia and abnormal rate of weight gain culminating in obesity and attendant metabolic disorders. Newer insights at cellular and molecular levels demonstrate that a breakdown of the integrated circuit due both to high and low abundance of NPY at target sites, underlies hyperphagia and increased adiposity. Consequently, interruption of NPYergic signaling at a single locus with NPY receptor antagonists may not be the most efficacious therapy to suppress hyperphagia and obesity. Central leptin gene therapy in rodents has been shown to subjugate, i.e. bring under homeostatic control, NPYergic signaling and suppress the age-related and dietary obesity for extended periods and thus shows promise as a newer treatment modality to curb the pandemic of obesity and metabolic syndrome.

Role of leptin in orexigenic neuropeptide expression during lactation in rats.

The expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP), both of which are important neuropeptides involved in regulation of energy balance and hormone secretion, is up-regulated in the arcuate nucleus during lactation in rodents. The present study tested whether reductions in circulating insulin and/or leptin that occur in lactation provide the critical signals to these systems. Lactating female rats received 3-day infusions of either bovine insulin or recombinant rat leptin via Alzet Osmotic minipumps implanted subcutaneously in regimens designed to restore serum concentrations of these hormones to the higher non-lactating level. Compared to non-lactating rats in diestrus, lactating rats displayed significantly lower serum concentrations of insulin and leptin, and significantly increased NPY peptide concentrations in the paraventricular nucleus (PVN) and median eminence, and AgRP mRNA in the arcuate nucleus. Infusion of leptin in lactating females significantly increased serum concentrations of leptin and significantly reduced NPY concentrations in the PVN and median eminence, and decreased NPY and AgRP mRNAs in the arcuate nucleus. The same effects were produced by infusion of insulin in lactating rats, which restored both insulin and leptin concentrations in serum. The levels of pro-opiomelanocortin mRNA in the arcuate nucleus were not different in non-lactating and lactating females, and were not altered by leptin or insulin treatment. These findings support the hypothesis that the reduction in circulating leptin during lactation contributes to increased expression of NPY and AgRP in hypothalamic systems involved in the behavioural and neuroendocrine adaptations to lactation.

Day Care Surgery in Otolaryngology : a three year Prospective Study.

Day care surgery is now an integral part of otolaryngology and is widely practised in the West. But in our military setup it still has to gain popularity. A Day Care Surgical Centre was established at Armed Forces Clinic in 1996. In the period May 1998 to Apr 2001, a total of 752 otolaryngology cases were operated at the centre, of which 474 cases were major and 278 cases were minor. All cases were performed under local anaesthesia supplemented by sedation. Strict patient selection criteria were adopted resulting in a low immediate post-operative complication rate (1.46%). None required admission to hospital for the management of complications. The feasibility of performing otolaryngological surgery on a day care basis is demonstrated, especially in nasal and aural cases. The experience gained shows that high medical standards can be achieved with low resource expenditure. Patient selection criteria and guidelines are suggested.

Evidence for the existence of distinct central appetite, energy expenditure, and ghrelin stimulation pathways as revealed by hypothalamic site-specific leptin gene therapy.

To identify the specific hypothalamic sites in which leptin acts to decrease energy intake and/or increase energy expenditure, recombinant adeno-associated virus vector-encoding leptin was microinjected bilaterally into one of four hypothalamic sites in female rats. Leptin transgene expression in the ventromedial nucleus and paraventricular nucleus induced comparable decreases in daily food intake (FI; 18-20%) and body weight (BW; 26-29%), accompanied by drastic reductions in serum leptin (81-97%), insulin (92-93%), free fatty acids (35-36%), and normoglycemia. Leptin transgene expression in the arcuate nucleus (ARC) decreased BW gain (21%) and FI (11%) to a lesser range, but the metabolic hormones were suppressed to the same extent. Leptin transgene expression in the medial preoptic area (MPOA) decreased BW and metabolic hormones without decreasing FI. Finally, leptin transgene expression in all four sites augmented serum ghrelin and thermogenic energy expenditure, as shown by uncoupling protein-1 mRNA expression in brown adipose tissue. Proopiomelanocortin gene expression in the ARC was up-regulated by leptin expression in all four sites, but neuropeptide Y gene expression in the ARC was suppressed by leptin transgene expression in the ARC but not in the MPOA. Thus, whereas leptin expression in the paraventricular nucleus, ventromedial nucleus, or ARC suppresses adiposity and insulin by decreasing energy intake and increasing energy expenditure, in the MPOA it suppresses these variables by increasing energy expenditure alone.

Capillary liquid chromatography of multiple peptides with on-line capillary electrophoresis immunoassay detection.

A competitive immunoassay for neuropeptide Y (NPY) based on capillary electrophoresis (CE) with laser-induced fluorescence detection was developed utilizing polyclonal antisera as the immunoreagent and fluorescein-labeled NPY as the tracer. The assay was performed with on-line mixing of reagents, automated injections, and a 3 s separation time. The assay had a detection limit of 850 pM. To detect NPY at lower concentrations, the assay was coupled on-line to reversed-phase capillary liquid chromatography (LC). In this arrangement, 5 microL samples were preconcentrated by capillary LC and eluted by a gradient of isopropanol-containing mobile phase. The resulting chromatographic peaks were monitored by the CE immunoassay. With preconcentration, the concentration detection limit was improved to 40 microM and NPY could be measured in push-pull perfusion samples collected from the paraventricular nucleus of freely moving rats. The technique was extended to simultaneous detection of NPY and glucagon secretion from islets of Langerhans.

Viral vectors as probes to decipher brain circuitry for weight control.

Multidisciplinary research has recently identified an intrinsic appetite-regulating network (ARN) in the hypothalamus. The idea that viruses could help to chart this complex network has gained impetus owing to a combination of our improved understanding of virology and of genetic engineering. Recently, three groups have employed viral vectors as probes to: (1) trace the inflow of sensory information from the neocortex and limbic systems to the ARN; (2) trace the outflow of information from the ARN to the sympathetic nervous system to monitor adiposity and energy expenditure; and (3) decipher the mechanisms underlying leptin resistance, which is responsible for environmentally based obesity.

Dose-dependent effects of central leptin gene therapy on genes that regulate body weight and appetite in the hypothalamus.

We have examined the dose-dependent effects and central action of intraventricular administration of a recombinant adeno-associated virus encoding rat leptin (rAAV-leptin) in suppressing body weight (BW) gain in adult female rats. A low dose of rAAV-leptin (5x10(10) particles) suppressed weight gain (15%) without changing daily food intake (FI), but a twofold higher dose decreased BW by 30% along with a reduction in daily FI. Reduced BW was due to a loss in body adiposity because serum leptin was reduced. Serum insulin levels were decreased (96%) by only the high dose along with a slight reduction in glucose. Uncoupling protein-1 (UCP-1) mRNA expression in brown adipose tissue (BAT), reflecting energy expenditure through thermogenesis, was upregulated to the same magnitude by the two rAAV-leptin doses. We analyzed by in situ hybridization the expression in the hypothalamus of genes encoding the appetite-regulating neuropeptides. Only the high dose decreased expression of neuropeptide Y (NPY), the orexigenic peptide, and increased proopiomelanocortin (POMC), precursor of the an orexigenic peptide, alpha-MSH. Our studies show for the first time that increased availability of leptin within the hypothalamus through central leptin gene therapy dose-dependently decreases weight gain, adiposity, and serum insulin by increasing energy expenditure and decreasing FI. The decrease in FI occurs only when NPY is reduced and alpha-MSH is increased in the hypothalamus by the high dose of rAAV-leptin. Delivery of the leptin gene centrally through rAAV vectors is a viable therapeutic modality for long-term control of weight and metabolic hormones.

Central leptin gene therapy suppresses body weight gain, adiposity and serum insulin without affecting food consumption in normal rats: a long-term study.

The weight-reducing effects of leptin are predominantly mediated through the hypothalamus in the brain. Gene therapy strategies designed for weight control have so far tested the short-term effect of peripherally delivered viral vectors encoding the leptin gene. In order to circumvent the multiple peripheral effects of hyperleptinemia and to overcome the age-related development of leptin resistance due to multiple factors, including defective leptin transport across the blood brain barrier, we determined whether delivery of viral vectors directly into the brain is a viable therapeutic strategy for long-term weight control in normal wild-type rats. A recombinant adeno-associated virus (rAAV) vector encoding rat leptin (Ob) cDNA was generated (rAAV-betaOb). When administered once intracerebroventricularly (i.c.v.), rAAV-betaOb suppressed the normal time-related weight gain for extended periods of time in adult Sprague-Dawley rats. The vector expression was confirmed by immunocytochemical localization of GFP and RT-PCR analysis of leptin in the hypothalamus. This sustained restraint on weight gain was not due to shifts in caloric consumption because food-intake was similar in rAAV-betaOb-treated and rAAV-GFP-treated control rats throughout the experiment. Weight gain suppression, first apparent after 2 weeks, was a result of reduced white fat depots and was accompanied by drastically reduced serum leptin and insulin concentrations in conjunction with normoglycemia. Additionally, there was a marked increase in uncoupling protein-1 (UCP1) mRNA expression in brown adipose tissue, thereby indicating increased energy expenditure through thermogenesis. Seemingly, a selective enhancement in energy expenditure following central delivery of the leptin gene is a viable therapeutic strategy to control the age-related weight gain and provide protection from the accompanying multiple peripheral effects of hyperleptinemia and hyperinsulinemia.

A GABA-neuropeptide Y (NPY) interplay in LH release.

Neuropeptide Y (NPY) stimulates and gamma-amino butyric acid (GABA) inhibits LH release in the rat. Since a sub-population of NPY-producing neurons in the arcuate nucleus (ARC) of the hypothalamus co-express GABA, the possibility of an interplay between NPY and GABA in the release of LH was investigated in two ways. First by employing light and electron microscopic double staining for NPY and GABA, using pre and post-immunolabeling on rat brain sections, we detected GABA in NPY immunoreactive axon terminals in the MPOA, one of the primary sites of action of these neurotransmitters/neuromodulators in the regulation of LH release. These morphological findings raised the possibility that inhibitory GABA co-released with NPY may act to restrain the excitatory effects of NPY on LH release. Muscimol (MUS, 0.44 or 1.76 nmol/rat), a GABA(A) receptor agonist, administered intracerebroventricularly (icv), alone failed to affect LH release, but NPY (0.47 nmol/rat icv) alone stimulated LH release in ovarian steroid-primed ovariectomized rats. On the other hand, administration of MUS blocked the NPY-induced stimulation of LH release in a dose-dependent manner. Similarly, administration of MUS abolished the excitatory effects on LH release of 1229U91, a selective NPY Y4 receptor agonist. These results support the possibility that in the event of co-release of these neurotransmitters/neuromodulators, GABA may act to restrain stimulation of LH release by NPY during the basal episodic and cyclic release of LH in vivo.

The mPVN mediates blockade of NPY-induced feeding by a Y5 receptor antagonist: a c-FOS analysis.

To identify the site(s) of NPY Y5 receptor (Y5R) mediation of NPY-induced feeding, we employed c-Fos immunostaining and a selective Y5R antagonist (Y5R-A), CGP71683A, in adult male rats. Intracerebroventricular (icv) administration of NPY stimulated feeding and c-Fos-like immunoreactivity (FLI) in the dorsomedial hypothalamus, supraoptic nucleus and the two subdivision of the hypothalamic paraventricular nucleus (pPVN), the parvocellular (pPVN), and magnocellular (mPVN). Y5R-A on its own, injected either intraperitoneally or icv, neither affected feeding nor FLI in hypothalamic sites. However, Y5R-A pretreatment suppressed NPY-induced food intake and FLI selectively in the mPVN. Taken together with our previous similar finding of Y1R involvement, these results suggest that NPY receptor sites concerned with feeding behavior reside selectively in the mPVN and Y1 and Y5 receptors are either coexpressed or expressed separately in those target neurons that promote appetitive drive.

Pancytopenia--a six year study.

All cases of pancytopenia seen over a six year period in two hematology centres were analysed. Patients receiving myelotoxic chemotherapy or those with leukemic cells in peripheral smear were excluded. There were a total of 166 cases. The four major causes were: aplastic anaemia in 49, megaloblastic anaemia in 37, aleukemic leukemia or lymphoma in 30 and hypersplenism in 19 cases. Bone marrow aspiration was often unsuccessful in obtaining an adequate sample, while biopsy was usually diagnostic. Both bone marrow aspiration and biopsy should be performed simultaneously in pancytopenic patients when the diagnosis is elusive. Megaloblastic anaemia is a major cause of pancytopenia, and may present acutely in the critically ill. It is a rapidly correctable disorder and should not be missed.