Environmental enrichment accentuates glucose-induced feeding suppression and glial cell line-derived neurotrophic factor gene expression in the hypothalamus of mice.

The hypothalamus controls food intake by integrating nutrient signals, of which one of the most important is glucose. Consequently, impairments in hypothalamic glucose-sensing mechanisms are associated with hyperphagia and obesity. Environmental enrichment (EE) is an animal housing protocol that provides complex sensory, motor, and social stimulations and has been proven to reduce adiposity in laboratory mice. However, the mechanism by which EE promotes adiposity-suppressing effect remains incompletely understood. Neurotrophic factors play an important role in the development and maintenance of the nervous system, but they are also involved in the hypothalamic regulation of feeding. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) are expressed in the hypothalamus and their expression is stimulated by glucose. EE is associated with increased expression of Bdnf mRNA in the hypothalamus. Therefore, we hypothesized that EE potentiates the anorectic action of glucose by altering the expression of neurotrophic factor genes in the hypothalamus. Male C57BL/6 mice were maintained under standard or EE conditions to investigate the feeding response to glucose and the associated expression of feeding-related neurotrophic factor genes in the hypothalamus. Intraperitoneal glucose injection reduced food intake in both control and EE mice with a significantly greater reduction in the EE group compared to the control group. EE caused a significantly enhanced response of Gdnf mRNA expression to glucose without altering basal Gdnf mRNA expression and Bdnf mRNA response to glucose. These findings suggest that EE enhances glucose-induced feeding suppression, at least partly, by enhancing hypothalamic glucose-sensing ability that involves GDNF.

Nitric oxide treatment attenuates muscle atrophy during hind limb suspension in mice.

Debilitating muscle-disuse atrophy in aging or obesity has huge socioeconomic impact. Since nitric oxide (NO) mediates muscle satellite cell activation and induces hypertrophy with exercise in old mice, we tested whether treatment with the NO donor, isosorbide dinitrate (ISDN), during hind limb suspension would reduce atrophy. Mice were suspended 18 days, with or without daily ISDN (66mg/kg). Muscles were examined for atrophy (weight, fiber diameter); regulatory changes in atrogin-1 (a negative regulator of muscle mass), myostatin (inhibits myogenesis), and satellite cell proliferation; and metabolic responses in myosin heavy chains (MyHCs), liver lipid, and hypothalamic gene expression. Suspension decreased muscle weight and weight relative to body weight between 25-55%, and gastrocnemius fiber diameter vs. CONTROLS: In young-adult mice, ISDN attenuated atrophy by half or more. In quadriceps, ISDN completely prevented the suspension-induced rise in atrogin-1 and drop in myostatin precursor, and attenuated the changes in MyHCs 1 and 2b observed in unloaded muscles without treatment. Fatty liver in suspended young-adult mice was also reduced by ISDN; suspended young mice had higher hypothalamic expression of the orexigenic agouti-related protein, Agrp than controls. Notably, a suspension-induced drop in muscle satellite cell proliferation by 25-58% was completely prevented (young mice) or attenuated (halved, in young-adult mice) by ISDN. NO-donor treatment has potential to attenuate atrophy and metabolic changes, and prevent regulatory changes during disuse and offset/prevent wasting in age-related sarcopenia or space travel. Increases in precursor proliferation resulting from NO treatment would also amplify benefits of physical therapy and exercise.

Xenin-induced feeding suppression is not mediated through the activation of central extracellular signal-regulated kinase signaling in mice.

Xenin is a gut hormone that reduces food intake by partly acting through the hypothalamus via neurotensin receptor 1 (Ntsr1). However, specific signaling pathways that mediate xenin-induced feeding suppression are not fully understood. Activation of Ntsr1 leads to the activation of the extracellular signal-regulated kinase (ERK). Hypothalamic ERK participates in the regulation of food intake by mediating the effect of hormonal signals. Therefore, we hypothesized that the anorectic effect of xenin is mediated by hypothalamic ERK signaling. To address this hypothesis, we compared levels of phosphorylation of ERK1/2 (pERK1/2) in the hypothalamus of both control and xenin-treated mice. The effect of xenin on ERK1/2 phosphorylation was also examined in mouse hypothalamic neuronal cell lines with or without Ntsr1. We also examined the effect of the blockade of central ERK signaling on xenin-induced feeding suppression in mice. The intraperitoneal (i.p.) injection of xenin caused a significant increase in the number of pERK1/2-immunoreactive cells in the hypothalamic arcuate nucleus. The majority of pERK1/2-positive cells expressed neuronal nuclei (NeuN), a marker for neurons. Xenin treatment increased pERK1/2 levels in one cell line expressing Ntsr1 but not another line without Ntsr1 expression. Both i.p. and intracerebroventricular (i.c.v.) injections of xenin reduced food intake in mice. The i.c.v. pre-treatment with U0126, a selective inhibitor of ERK1/2 upstream kinases, did not affect xenin-induced reduction in food intake. These findings suggest that although xenin activates ERK signaling in subpopulations of hypothalamic neurons, xenin does not require the activation of hypothalamic ERK signaling pathway to elicit feeding suppression.

Impaired suppression of feeding by the gut hormone xenin in type I interleukin-1 receptor-deficient mice.

Xenin is a gut hormone that reduces food intake partly by acting through the hypothalamus. However, the mechanism of hypothalamic xenin action is not fully understood. To identify xenin-regulated genes in the hypothalamus, we compared expression levels of metabolism-related genes in the hypothalamus between saline-treated control and xenin-treated mice. Intraperitoneal injection of xenin caused a significant increase in hypothalamic interleukin 1 beta (IL-1ß) mRNA levels without causing a significant change in hypothalamic IL-1α mRNA levels. To further examine the possible contribution of IL-1 signaling to xenin's anorexigenic action, the effect of intraperitoneal injection of xenin on food intake was compared between wild-type and type I IL-1 receptor (IL-1RI)-deficient mice. Intraperitoneal administration of xenin (7.5 µg/g b.w.) caused a significant reduction of food intake in wild-type mice, while it failed to reduce food intake in pre-obese IL-1RI-deficient mice. These findings support the role of hypothalamic IL-1ß-IL-1RI signaling in the mediation of the anorexigenic effect of xenin.

Mediation of glucose-induced anorexia by central nervous system interleukin 1 signaling.

Hypothalamic glucose sensing plays a critical role in the regulation of food intake and metabolism. Glucose injection, either centrally or peripherally suppresses food intake. However, the mechanism of glucose-induced feeding suppression is not fully understood. It has been demonstrated that hypothalamic interleukin 1 beta (IL-1ß) mRNA levels are altered by metabolic states and IL-1 signaling participates in the regulation of food intake. Therefore, we hypothesized that hypothalamic IL-1 gene expression is regulated by glucose and glucose-induced feeding suppression is mediated via hypothalamic IL-1 signaling. To address this hypothesis, we examined the effect of glucose on IL-1α and IL-1ß mRNA expression in the hypothalamus. We also examined the effect of intraperitoneal injection of glucose on food intake in wild-type and type I IL-1 receptor (IL-1RI)-deficient mice. Levels of IL-1α and IL-1ß mRNA in the hypothalamus were increased in response to feeding and intraperitoneal injection of glucose, and were positively correlated with blood glucose levels in mice. Exposure of hypothalamic explants to high glucose (10 mM) media increased IL-1α and IL-1ß mRNA levels compared to low glucose (1 mM) media. Intraperitoneal glucose administration reduced food intake in wild-type mice, while the feeding-suppressing effect of glucose was attenuated in IL-1RI-deficient mice. These findings support the role for hypothalamic IL-1 signaling in the mediation of the anorectic effect of glucose.

Xenin, a gastrointestinal peptide, regulates feeding independent of the melanocortin signaling pathway.

OBJECTIVE: Xenin, a 25-amino acid peptide, was initially isolated from human gastric mucosa. Plasma levels of xenin rise after a meal in humans, and administration of xenin inhibits feeding in rats and chicks. However, little is known about the mechanism by which xenin regulates food intake. Signaling pathways including leptin and melanocortins play a pivotal role in the regulation of energy balance. Therefore, we addressed the hypothesis that xenin functions as a satiety factor by acting through the melanocortin system or by interacting with leptin. RESEARCH DESIGN AND METHODS: The effect of intracerebroventricular and intraperitoneal administration of xenin on food intake was examined in wild-type, agouti, and ob/ob mice. The effect of intracerebroventricular injection of SHU9119, a melanocortin receptor antagonist, on xenin-induced anorexia was also examined in wild-type mice. To determine whether the hypothalamus mediates the anorectic effect of xenin, we examined the effect of intraperitoneal xenin on hypothalamic Fos expression. RESULTS: Both intracerebroventricular and intraperitoneal administration of xenin inhibited fasting-induced hyperphagia in wild-type mice in a dose-dependent manner. The intraperitoneal injection of xenin also reduced nocturnal intake in ad libitum-fed wild-type mice. The intraperitoneal injection of xenin increased Fos immunoreactivity in hypothalamic nuclei, including the paraventricular nucleus and the arcuate nucleus. Xenin reduced food intake in agouti and ob/ob mice. SHU9119 did not block xenin-induced anorexia. CONCLUSIONS: Our data suggest that xenin reduces food intake partly by acting through the hypothalamus but via signaling pathways that are independent of those used by leptin or melanocortins.

Tail suspension increases energy expenditure independently of the melanocortin system in mice.

Space travelers experience anorexia and body weight loss in a microgravity environment, and microgravity-like situations cause changes in hypothalamic activity. Hypothalamic melanocortins play a critical role in the regulation of metabolism. Therefore, we hypothesized that microgravity affects metabolism through alterations in specific hypothalamic signaling pathways, including melanocortin signaling. To address this hypothesis, the microgravity-like situation was produced by an antiorthostatic tail suspension in wild-type and agouti mice, and the effect of tail suspension on energy expenditure and hypothalamic gene expression was examined. Energy expenditure was measured using indirect calorimetry before and during the tail suspension protocol. Hypothalamic tissues were collected for gene expression analysis at the end of the 3 h tail suspension period. Tail suspension significantly increased oxygen consumption, carbon dioxide production, and heat production in wild-type mice. Tail suspension-induced increases in energy expenditure were not attenuated in agouti mice. Although tail suspension did not alter hypothalamic proopiomelanocortin (POMC) and agouti-related protein (AGRP) mRNA levels, it significantly increased hypothalamic interleukin 6 (Il-6) mRNA levels. These data are consistent with the hypothesis that microgravity increases energy expenditure and suggest that these effects are mediated through hypothalamic signaling pathways that are independent of melanocortins, but possibly used by Il-6.

Impaired anorectic effect of leptin in neurotensin receptor 1-deficient mice.

Neurotensin plays a role in regulating feeding behavior. Central injection of neurotensin reduces food intake and the anorectic effect of neurotensin is mediated through neurotensin receptor 1 (Ntsr1). Ntsr1-deficient mice are characterized by mild hyperphagia and overweight without hyperleptinemia. The mechanism by which Ntsr1-deficient mice develop these metabolic abnormalities is not well understood. Leptin, secreted by adipocytes, regulates food intake by acting on hypothalamic neurons including neurotensin-producing neurons. Since the anorectic effect of leptin is blocked by neurotensin receptor antagonist, we hypothesized that the anorectic effect of leptin is mediated through Ntsr1 in the central nervous system and that decreased sensitivity to the anorectic effect of leptin contributes to metabolic perturbations in Ntsr1-deficient mice. To address this hypothesis, we examined the effect of intracerebroventricular (i.c.v.) administration of leptin on food intake in Ntsr1-deficient mice. A single i.c.v. injection of leptin caused robust reductions in food intake in wild-type mice. These effects were markedly attenuated in Ntsr1-deficient mice. These data are consistent with our hypothesis that the anorectic effect of leptin is at least partly mediated through central Ntsr1 and that the leptin-Ntsr1 signaling pathway is involved in the regulation of food intake. Our data also suggest that the lack of Ntsr1 reduces sensitivity to the anorectic action of leptin, causing hyperphagia and abnormal weight gain.

The physiological function of the agouti-related peptide gene: the control of weight and metabolic rate.

The central melanocortin system plays an important role in the regulation of energy homeostasis both in rodents and humans, and melanocortin receptors appear to be the core of this system. Alpha-melanocyte-stimulating hormone (alpha-MSH) inhibits feeding through melanocrtin 3 and 4 receptors (MC3-R and MC4-R) as an endogenous agonist. Although mutations in the agouti gene cause an over-expression of agouti peptide which antagonizes effects of alpha-MSH at MC4-R in the brain and causes obese phenotypes, there was no evidence for the presence of an endogenous antagonist for MC3-R and MC4-R until agouti related protein (AGRP) was identified. AGRP is expressed primarily in the hypothalamic arcuate nucleus and central administration of AGRP stimulates feeding and weight gain, and decreases metabolic rate. Although a complete deletion of the AGRP gene does not produce any significant metabolic phenotypes, reduction in AGRP expression by RNA interference is associated with increased metabolic rate along with reduced weight gain. The currently available data suggest that elevated AGRP mRNA along with reduced proopiomelanocortin (POMC) mRNA is associated with many types of obesity and agents antagonizing the effect of AGRP may be a potential therapeutic target in treating obesity and obesity-associated disorders in which endogenous hypothalamic AGRP is elevated.

The fatty acid synthase inhibitor cerulenin and feeding, like leptin, activate hypothalamic pro-opiomelanocortin (POMC) neurons.

Hypothalamic POMC neurons mediate catabolic responses such as decreased food intake and increased energy expenditure by, in part, monitoring levels of metabolic factors such as glucose, insulin and leptin. Recently, fatty acid synthase inhibitors were reported to reduce body weight, inhibit food intake, and increase metabolic rate, possibly by acting on hypothalamic neurons through a mechanism involving malonyl-CoA accumulation. Given the observation that leptin mediates similar catabolic effects by, in part, activating hypothalamic POMC neurons, it is possible that other catabolic signals such as feeding and fatty acid synthase inhibition may also activate POMC neurons. To test this hypothesis, hypothalamic sections from mice that were fed or injected with the fatty acid synthase inhibitor cerulenin were examined for Fos (a marker for neuronal activation) and POMC product immunoreactivity and compared with similarly processed sections from leptin-injected mice. Feeding increased Fos immunoreactivity in the lateral peri-arcuate area of the hypothalamus of both wild-type and leptin-deficient ob/ob mice (P<0.05), indicating that nutritional activation of the hypothalamus can be leptin-independent. Furthermore, feeding significantly induced Fos immunoreactivity in neurons expressing POMC (P<0.003), indicating that feeding, like leptin, activates POMC neurons. Injection with cerulenin, like feeding and leptin, also increased Fos immunoreactivity in the lateral peri-arcuate area (P<0.03) and, more specifically, in neurons expressing POMC. In contrast, injection with cerulenin had no grossly observable effects on cortical Fos immunoreactivity and appeared to suppress fasting-induced Fos immunoreactivity by about 35% (although the decrease did not reach statistical significance) in the medial arcuate nucleus, an area associated with anabolic responses such as increased food intake. Injection with cerulenin also decreased Fos immunoreactivity in the granular layer of the dentate gyrus of the hippocampus by about 30% (P<0.05), further suggesting that cerulenin does not non-specifically activate wide varieties of neurons. These results suggest that activation of hypothalamic POMC neurons may help to mediate some of the catabolic effects associated with feeding, cerulenin and leptin.

Role of glucocorticoids in mediating effects of fasting and diabetes on hypothalamic gene expression.

BACKGROUND: Fasting and diabetes are characterized by elevated glucocorticoids and reduced insulin, leptin, elevated hypothalamic AGRP and NPY mRNA, and reduced hypothalamic POMC mRNA. Although leptin replacement can reverse changes in hypothalamic gene expression associated with fasting and diabetes, leptin also normalizes corticosterone; therefore the extent to which the elevated corticosterone contributes to the regulation of hypothalamic gene expression in fasting and diabetes remains unclear. To address if elevated corticosterone is necessary for hypothalamic responses to fasting and diabetes, we assessed the effects of adrenalectomy on hypothalamic gene expression in 48-hour-fasted or diabetic mice. To assess if elevated corticosterone is sufficient for the hypothalamic responses to fasting and diabetes, we assessed the effect of corticosterone pellets implanted for 48 hours on hypothalamic gene expression. RESULTS: Fasting and streptozotocin-induced diabetes elevated plasma glucocorticoid levels and reduced serum insulin and leptin levels. Adrenalectomy prevented the rise in plasma glucocorticoids associated with fasting and diabetes, but not the associated reductions in insulin or leptin. Adrenalectomy blocked the effects of fasting and diabetes on hypothalamic AGRP, NPY, and POMC expression. Conversely, corticosterone implants induced both AGRP and POMC mRNA (with a non-significant trend toward induction of NPY mRNA), accompanied by elevated insulin and leptin (with no change in food intake or body weight). CONCLUSION: These data suggest that elevated plasma corticosterone mediate some effects of fasting and diabetes on hypothalamic gene expression. Specifically, elevated plasma corticosterone is necessary for the induction of NPY mRNA with fasting and diabetes; since corticosterone implants only produced a non-significant trend in NPY mRNA, it remains uncertain if a rise in corticosterone may be sufficient to induce NPY mRNA. A rise in corticosterone is necessary to reduce hypothalamic POMC mRNA with fasting and diabetes, but not sufficient for the reduction of hypothalamic POMC mRNA. Finally, elevated plasma corticosterone is both necessary and sufficient for the induction of hypothalamic AGRP mRNA with fasting and diabetes.

Adrenalectomy stimulates hypothalamic proopiomelanocortin expression but does not correct diet-induced obesity.

BACKGROUND: Elevated glucocorticoid production and reduced hypothalamic POMC mRNA can cause obese phenotypes. Conversely, adrenalectomy can reverse obese phenotypes caused by the absence of leptin, a model in which glucocorticoid production is elevated. Adrenalectomy also increases hypothalamic POMC mRNA in leptin-deficient mice. However most forms of human obesity do not appear to entail elevated plasma glucocorticoids. It is therefore not clear if reducing glucocorticoid production would be useful to treat these forms of obesity. We hypothesized that adrenalectomy would increase hypothalamic POMC mRNA and reverse obese phenotypes in obesity due to a high-fat diet as it does in obesity due to leptin deficiency. RESULTS: Retired breeder male mice were placed on a high-fat diet or a low-fat diet for two weeks, then adrenalectomized or sham-adrenalectomized. The high-fat diet increased body weight, adiposity, and plasma leptin, led to impaired glucose tolerance, and slightly stimulated hypothalamic proopiomelanocortin (POMC) expression. Adrenalectomy of mice on the high-fat diet significantly reduced plasma corticosterone and strikingly increased both pituitary and hypothalamic POMC mRNA, but failed to reduce body weight, adiposity or leptin, although slight improvements in glucose tolerance and metabolic rate were observed. CONCLUSION: These data suggest that neither reduction of plasma glucocorticoid levels nor elevation of hypothalamic POMC expression is effective to significantly reverse diet-induced obesity.

Reducing hypothalamic AGRP by RNA interference increases metabolic rate and decreases body weight without influencing food intake.

BACKGROUND: Several lines of evidence strongly suggest that agouti-related peptide (AGRP) plays a key role in the regulation of metabolic function but ablation of the AGRP gene has no apparent effect on metabolic function. Since specific pharmacological antagonists of AGRP do not presently exist, we assessed if reduction of hypothalamic AGRP mRNA by RNA interference (RNAI) would influence metabolic function, an outcome suggesting that pharmacological antagonists might constitute useful reagents to treat obesity. RESULTS: The RNAI protocol specifically reduced hypothalamic expression of AGRP mRNA by 50% and resulted in reduction of AGRP peptide immunoreactivity. Physiologically, the reduction in AGRP levels was associated with increased metabolic rate and reduced body weight without changes in food intake. CONCLUSION: AGRP can function to increase body weight and reduce metabolic rate without influencing food intake. The present study demonstrates that RNAI protocols can be used to assess physiological function of neuronal genes in vivo.