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

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

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

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

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

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

Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus

Leptin, the protein product of the adipose tissue-specific ob (obese) gene (1), reduces the body weight, adiposity and food intake of obese ob/ob mice on peripheral or central injection (2, 3, 4). [125I]leptin binding has been detected in mouse choroid plexus (5), from which a leptin receptor gene was expression cloned (5). The gene has at least 6 splice variants (6, 7). Leptin receptor mRNA was localized in the hypothalamus by in situ hybridization being particularly abundantly expressed in the arcuate nucleus (8). There is evidence linking the physiological effects of injected leptin with hypothalamic neuropeptide Y (9, 10) (NPY), which has potent central effects on food intake and energy balance (11), and is also expressed in the arcuate nucleus. Here we report dual in situ hybridization studies for leptin receptor and NPY gene expression in the mouse arcuate nucleus, where the majority of cells examined expressed both genes. This provides the first direct evidence that leptin acts on cells that express NPY mRNA.     

Upregulation of leptin receptor mRNA expression in obese mouse brain

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

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

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

Neuropeptide Y and corticotropin-releasing hormone concentrations within specific hypothalamic regions of lean but not ob/ob mice respond to food-deprivation and refeeding

Leptin is proposed to control food intake at least in part by regulating hypothalamic neuropeptide Y (NPY), a stimulator of food intake, and corticotropin-releasing hormone (CRH), an inhibitor of food intake. Ob/ob mice are leptin-deficient and would thus be expected to exhibit alterations in hypothalamic NPY and CRH. We therefore measured concentrations of NPY and CRH in discrete regions of the hypothalamus (i.e., ARC, arcuate nucleus; PVN, paraventricular nucleus; VMH, ventromedial nucleus; DMH, dorsomedial nucleus; and SCN, suprachiasmatic nucleus) of 6.5-7-wk-old ob/ob and lean mice with free access to stock diet, 24 h after food deprivation, and 1 h after refeeding. Fed ob/ob mice had 55-75% higher concentrations of NPY in the ARC, VMH and SCN than lean mice. Food deprivation increased NPY concentrations approximately 70% in the ARC, PVN and VMH of lean mice, and refeeding lowered NPY concentrations approximately 70% in the PVN of these mice. NPY in these hypothalamic regions of ob/ob mice was unresponsive to food deprivation or refeeding. The most pronounced change in CRH concentrations within the regions examined (i.e., ARC, PVN and VMH) occurred in the ARC of lean mice where refeeding lowered CRH concentrations by 75% without influencing ARC CRH concentrations in ob/ob mice. The hypothalamic concentrations of two neuropeptides involved in body weight regulation (i.e., NPY and CRH) in leptin-deficient ob/ob mice respond abnormally to abrupt changes in nutritional status.     

Anisotropy measurements obtained by fractal analysis of trabecular bone at the calcaneus and radius

Antidepressant drugs have in common a delayed onset of clinical efficacy. In rats, long-term, daily administration of four different types of clinically effective antidepressant drugs results in decreased corticotropin releasing hormone (CRH) mRNA expression levels in the hypothalamic paraventricular nucleus (PVN). Because a subpopulation of neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (Arc) projects to the PVN, we measured NPY and POMC mRNA expression in the Arc using in situ hybridization histochemistry at several time points following daily administration of four different antidepressant drugs. After 14 and 56 days of imipramine treatment, Arc NPY mRNA levels are decreased to 85% and 75% of control levels, but are unchanged compared to control after one or five days of treatment. Arc POMC mRNA levels are unchanged compared to controls at 1, 5, 14, or 56 days following imipramine treatment. Unlike after imipramine, Arc NPY and POMC mRNA levels are increased significantly to 134-172% of control following 56-day treatment with the antidepressant drugs fluoxetine, phenelzine, or idazoxan. The divergent effects of imipramine vs the other 3 antidepressant drugs on Arc NPY mRNA expression are similar to the pattern of changes in tyrosine hydroxylase (TH) mRNA expression levels in the locus coeruleus (LC) using the same experimental paradigm, but are different from the unidirectional depressive effects of all four drugs on CRH mRNA expression in the PVN. Thus, the Arc NPY and LC noradrenergic systems may act coordinately in mediating antidepressant effects. The present data are consistent with the delayed onset of clinical efficacy for antidepressant drugs, and suggest that Arc NPY and POMC neurotransmitter systems play a role in the pathophysiology of depression.     

Neonatal MSG reduces hypothalamic DA, beta-endorphin, and delays weight gain in genetically obese (A viable yellow/alpha) mice

Neonatal treatment with monosodium glutamate (MSG) decreases proopiomelanocortin (POMC) peptides and results in obesity. The yellow mouse is a model of obesity induced by the viable yellow (Avy) gene at the agouti locus on Chromosome 2, which results in overproduction of a POMC receptor antagonist. Thus we hypothesized that MSG, when imposed on the genetically susceptible model, would alter the development of obesity. Both yellow obese (Avy) and black lean (alpha/alpha) males were injected on Postnatal Days 1, 3, 5, 7, and 9 with 2.0 mg/g body weight MSG or saline SC. Their food intake, growth parameters, and neurochemical status were examined. Paradoxically, MSG interacted with the yellow phenotype to delay the rapid rate of weight gain characteristic of this model (p < 0.05). Food intake was decreased (p < 0.05) in both phenotypes treated with MSG, as was hypothalamic content of dopamine (p < 0.05) and of the POMC peptide, beta-endorphin (p < 0.001). The yellow obese phenotype was more sensitive than the black lean phenotype to the neurochemical effect of early postnatal MSG administration. Recent reports suggest the agouti locus protein is an antagonist of the receptor for another POMC peptide, melanocyte-stimulating hormone (MSH). Therefore, the balance of functional activity between various POMC peptides appears to be an important factor in the development of both acquired and genetic obesity.     

The ob gene product (leptin)--a new hormone of adipose tissue

Obesity--an important problem in modern societies--is caused by energy balance dysregulation and produces numerous adverse effects on health. Recently a particular attention has been paid to molecular and physiological mechanisms in the development of obesity and to the signalling role of adipose tissue in energy stores maintenance on the hypothalamic level. Leptin, the obese gene product discovered in 1995, may play a key role in the feedback system between adipose tissue and the ventromedial nucleus of the hypothalamus (satiety centre). The level of ob gene expression in adipose tissue and plasma leptin concentrations in humans are highly correlated with BMI. So far no mutations in the ob gene in obese subjects have been reported therefore leptin molecule could be active. Despite markedly increased leptin levels found in obesity its central action decreasing food intake and increasing energy expenditure is hindered. Defective ob protein signalling to the brain may be due to receptor and post-receptor defects. Neuropeptide Y, the hypothalamic neurotransmitter involved in the maintaining of energy homeostasis, is a likely candidate for mediating leptin afferent signals. In adipose tissue, the level of ob mRNA is regulated by insulin and glucocorticoids--hormones responsible for glucose homeostasis as well as for the central regulation of feeding behaviour. Until now the character of interactions between leptin and other hormones that regulate energy balance is not known, neither is the exact nature of leptin hypothalamic receptor defect. Defining of the role of leptin in the regulation of satiety and energy expenditure will undoubtedly contribute to a better understanding of the pathogenesis of obesity and its related metabolic complications and may lead to a new treatment approach to human obesity based on leptin or its analogues. At present research work focuses on leptin receptor studies and on ob gene polymorphism and its expression in feeding disorders including obesity and anorexia nervosa. The ob gene is one of a few genes involved in energy balance, however, very promising one.     

Pancreatic enzymes: secretion and luminal nutrient digestion in health and disease

Leptin is a hormone secreted by the adipocytes that regulates food intake and energy expenditure. It is known that growth hormone (GH) secretion is markedly influenced by body weight, being suppressed in obesity and cachexia, and recent data have demonstrated that GH release is regulated by leptin levels. Although one of the sites of action of leptin is likely to be the hypothalamus, since leptin receptor mRNA is particularly abundant in several hypothalamic nuclei, the mechanisms by which leptin regulates GH secretion are not yet known. The aim of the present study was to investigate whether leptin could act at the hypothalamic level modulating somatostatin and GH-releasing hormone (GHRH) expression. The administration of anti-GHRH serum (500 microl, i.v.) completely blocked leptin-induced GH release in fasting rats. In contrast, the treatment with anti-somatostatin serum (500 microl, i.v.) significantly increased GH release in this condition. Furthermore, leptin administration (10 microg, i.c.v.) to intact fasting animals reversed the inhibitory effect produced by fasting on GHRH mRNA levels in the arcuate nucleus of the hypothalamus, and increased somatostatin mRNA content in the periventricular nucleus. Finally, leptin administration (10 microgram, i.c.v.) to hypophysectomized fasting rats increased GHRH mRNA levels, and decreased somatostatin mRNA content, indicating an effect of leptin on hypothalamic GHRH- and somatostatin-producing neurons. These findings suggest a role for GHRH and somatostatin as mediators of leptin-induced GH secretion.     

Behavioral and neurochemical effects of neonatal administration of monosodium {l}-glutamate in mice.

Studied the feeding behavior, activity level, and thermoregulatory ability of 117 CL-1 mice made obese by neonatal administration of monosodium {l}-glutamate (MSG). The degree of obesity and other characteristics of the syndrome depended on age, diet, and housing condition. Carcass fat determinations demonstrated the presence of obesity in all MSG Ss; however, body weight was elevated over control levels only in adult mice caged in groups. Group-housed MSG Ss also failed to increase food intake in response to food deprivation and were both hypoactive and hypothermic. Individually caged MSG Ss showed normal activity levels and body temperature, an attenuated response to food deprivation, and an enhanced response to a high-fat diet. Since MSG obesity may be the consequence of damage to the dopamine-rich arcuate nucleus of the hypothalamus, a 2nd goal was to measure central catecholamines and examine any changes in the MSG S's behavioral responses to catecholaminergic drugs. Ss treated with MSG sustained some loss of hypothalamic dopamine, but no systematic relation between central catecholamines and behavioral aspects of the syndrome could be discerned. (46 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Evidence suggesting that galanin (GAL), melanin-concentrating hormone (MCH), neurotensin (NT), proopiomelanocortin (POMC) and neuropeptide Y (NPY) are targets of leptin signaling in the hypothalamus

Leptin (OB protein) reduces food intake by acting at the hypothalamic level. The purpose of the present study was to identify potential targets of leptin signaling in the hypothalamus in ad-lib fed rats. Central administration of leptin (5 microg) for 3 days decreased food intake and body weight gain in association with a decrease in hypothalamic galanin (GAL), melanin-concentrating hormone (MCH), proopiomelanocortin (POMC) and neuropeptide Y (NPY) gene expression and with an increase in neurotensin (NT) gene expression. In pair-fed rats, NPY gene expression was increased and there was no change in either MCH, GAL, POMC or NT gene expression. This study identifies GAL, MCH, POMC and NT as non-NPY targets of leptin signaling and suggests that leptin's action on food intake and body weight is most likely mediated by inhibiting excitatory (e.g. NPY, MCH, GAL, POMC) and stimulating inhibitory (e.g., NT) signals in the feeding circuitry.     

The effect of the volatile oils of Chenopodium ambrosioides and Thymus vulgaris against the larvae of Lucilia sericata (Meigen)

We have isolated a stable, transplantable, and small glucagonoma (MSL-G-AN) associated with abrupt onset of severe anorexia occurring 2-3 wk after subcutaneous transplantation. Before onset of anorexia, food consumption is comparable to untreated controls. Anorexia is followed by adipsia and weight loss, and progresses rapidly in severity, eventually resulting in reduction of food and water intake of 100 and 80%, respectively. During the anorectic phase, the rats eventually become hypoglycemic and hypothermic. The tumor-associated anorexia shows no sex difference, and is not affected by bilateral abdominal vagotomy, indicating a direct central effect. The adipose satiety factor leptin, known to suppress food intake by reducing hypothalamic neuropeptide Y (NPY) levels, was not found to be expressed by the tumor, and circulating leptin levels were reduced twofold in the anorectic phase. A highly significant increase in hypothalamic (arcuate nucleus) NPY mRNA levels was found in anorectic rats compared with control animals. Since elevated hypothalamic NPY is among the most potent stimulators of feeding and a characteristic of most animal models of hyperphagia, we conclude that the MSL-G-AN glucagonoma releases circulating factor(s) that overrides the hypothalamic NPY-ergic system, thereby eliminating the orexigenic effect of NPY. We hypothesize a possible central role of proglucagon-derived peptides in the observed anorexia.     

Withdrawal of [corrected] estrogen increases hypothalamic neuropeptide Y (NPY) mRNA expression in ovariectomized obese rat

We determined the changes in neuropeptide Y (NPY) mRNA expression of the arcuate nucleus (ARC) in sham-operated (SHAM) and bilaterally ovariectomized (OVX) rats with estradiol (E2) supplement. Ovariectomy increases body weight gain for 3 weeks, accompanied by an increase of daily food intake. Ovariectomy significantly reduced serum corticosterone levels. E2 supplement reversed the effects of ovariectomy on body weight gain, food intake and serum corticosterone levels. Ovariectomy significantly increased NPY mRNA expression in the ARC. E2 supplement decreased NPY mRNA expression in the ARC of OVX rats. The present findings indicated that hypothalamic NPY mRNA expression, which involves the regulation of feeding behavior, are in parallel with circulating estrogen levels. Hypothalamic NPY mRNA expression may be important in the induction of hyperphagia after the withdrawal of estrogen by bilateral ovariectomy.     

The role of neuropeptide Y in the antiobesity action of the obese gene product

Recently Zhang et al. cloned a gene that is expressed only in adipose tissue of the mouse. The obese phenotype of the ob/ob mouse is linked to a mutation in the obese gene that results in expression of a truncated inactive protein. Human and rat homologues for this gene are known. Previous experiments predict such a hormone to have a hypothalamic target. Hypothalamic neuropeptide Y stimulates food intake, decreases thermogenesis, and increases plasma insulin and corticosterone levels making it a potential target. Here we express the obese protein in Escherichia coli and find that it suppresses food intake and decreases body weight dramatically when administered to normal and ob/ob mice but not db/db (diabetic) mice, which are thought to lack the appropriate receptor. High-affinity binding was detected in the rat hypothalamus. One mechanism by which this protein regulated food intake and metabolism was inhibition of neuropeptide-Y synthesis and release.     

Modulation of neuropeptide Y overflow by leptin in the rat hypothalamus, cerebral cortex and medulla

This study examined whether leptin can exert inhibitory actions on brain NPY overflow in Sprague-Dawley and in lean and obese Zucker rats, and tested the site-specificity of the effect. Slices of rat hypothalamus, cerebral cortex and medulla oblongata were perfused with modified Krebs buffer containing either leptin or KCl. Depolarization of tissues with 40 mM KCl elicited a significant doubling of NPY overflow in all brain regions tested. At 1 microM, leptin significantly reduced NPY overflow only in the rat hypothalamus, while at 3 microM, leptin reduced NPY overflow from all regions. However, no effect of 1 microM leptin was observed in the hypothalamus of obese Zucker rats: this insensitivity to leptin is in keeping with their genetic defect. In conclusion, the inhibitory effect of leptin on hypothalamic NPY overflow provides further evidence for important modulatory actions between these two feeding mediators. Moreover, the effect of leptin observed in the cerebral cortex and medulla oblongata supports a role of leptin in brain regions other than the hypothalamus.