Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats

Hypothalamic neuropeptides play a role in appetite and weight regulation. Food restriction for 2 weeks and food deprivation for 4 days were used as models to characterize the effects of weight loss on hypothalamic peptide gene expression in male and female rats. We used in situ hybridization to examine the mRNA levels of hypothalamic peptides which stimulate and inhibit food intake and found selective effects primarily in the arcuate nucleus. Neuropeptide Y (NPY) mRNA was increased and pro-opiomelanocortin (POMC) and galanin (GAL) mRNA were decreased in the hypothalamic arcuate nucleus and corticotropin-releasing hormone (CRH) mRNA was decreased in the hypothalamic paraventricular nucleus in male and female food-restricted and food-deprived rats. Food restriction produced larger changes in peptide mRNA expression than did food deprivation. Changes in NPY, POMC and CRH gene expression induced by food restriction were greater in male than female rats. Elevated NPY and reduced CRH gene expression may be a compensatory physiological response to restore food intake in food-restricted and food-deprived animals. The discrete changes in NPY, POMC, GAL and CRH gene expression in food-restricted and food-deprived animals suggest the involvement of these peptides in abnormal appetitive behavior and weight loss associated with human eating disorders.     

Diurnal changes in hypothalamic neuropeptide and SOCS-3 expression: effects of lactation and relationship with serum leptin and food intake

Rats normally eat about 85% of their food at night. Lactation increases food intake 3- to 4-fold, but the diurnal pattern of food intake persists. The mechanisms responsible for the diurnal and lactation-induced changes in food intake are still unresolved, hence we have further investigated the possible roles of serum leptin and hypothalamic expression of neuropeptide Y (NPY), agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) in rats. Suppressor of cytokine signalling-3 (SOCS-3) acts as a feedback inhibitor of leptin signalling in the hypothalamus, hence changes in expression of SOCS-3 were also investigated. Changes in expression of NPY, AgRP or POMC alone could not account for the diurnal changes in intake and their alteration by lactation. However, there were increased AgRP mRNA:POMC mRNA ratios at night and also during lactation, which were very similar to estimated changes in food intake. Such changes in expression may result in dominance of the orexigenic AgRP peptide over the appetite-suppressing POMC-derived peptides, and so could contribute to the hyperphagia in these states. Diurnal and lactation-related changes in the AgRP mRNA:POMC mRNA ratio and food intake are not due to changes in leptin alone. However, hypoleptinaemia, possibly through increased expression of NPY, may contribute to the hyperphagia of lactation. In the dark, expression of SOCS-3 was decreased in non-lactating rats; lactation decreased SOCS-3 expression in both light and dark phases. However, such changes are likely to enhance the ability of leptin-responsive neurones to transmit the leptin signal, and so are unlikely to contribute to either the nocturnal increase in appetite or the hyperphagia of lactation.     

Corticotropin-releasing hormone (CRH) transgenic mice display hyperphagia with increased Agouti-related protein mRNA in the hypothalamic arcuate nucleus

Although glucocorticoid-induced hyperphagia is observed in the patients with glucocorticoid treatment or Cushing's syndrome, its molecular mechanism is not clear. We thus explored the expression of neuropeptide mRNAs in the hypothalamus related to appetite regulation in CRH over-expressing transgenic mice (CRH-Tg), a model of Cushing's syndrome. We measured food intake, body weight (including body fat weight) and plasma corticosterone levels in CRH-Tg and their wild-type littermates (WT) at 6 and 14 weeks old. We also examined neuropeptide Y (NPY), proopiomelanocortin (POMC) and Agouti-related protein (AgRP) mRNAs in the arcuate nucleus (ARC) using in situ hybridization. Circulating corticosterone levels in CRH-Tg were markedly elevated at both 6 and 14 weeks old. Body fat weight in CRH-Tg was significantly increased at 14 weeks old, which is considered as an effect of chronic glucocorticoid excess. At both 6 and 14 weeks old, CRH-Tg mice showed significant hyperphagia compared with WT (14w old: WT 3.9±0.1, CRH-Tg 5.1±0.7 g/day, p<0.05). Unexpectedly, NPY mRNA levels in CRH-Tg were significantly decreased at 14 weeks old (WT: 1571.5±111.2, CRH-Tg: 949.1±139.3 dpm/mg, p<0.05), and there were no differences in POMC mRNA levels between CRH-Tg and WT. On the other hand, AgRP mRNA levels in CRH-Tg were significantly increased compared with WT at both ages (14w old: WT 365.6±88.6, CRH-Tg 660.1±87.2 dpm/ mg, p<0.05). These results suggest that glucocorticoid-induced hyperphagia is associated with increased hypothalamic AgRP. Our results also indicate that hypothalamic NPY does not have an essential role in the increased food intake during glucocorticoid excess.     

Adipogenic and orexigenic effects of the ghrelin-receptor ligand tabimorelin are diminished in leptin-signalling-deficient ZDF rats

OBJECTIVE: The aim was to investigate the possible interactions of the two peripheral hormones, leptin and ghrelin, that regulate the energy balance in opposite directions. METHODS: Leptin-receptor mutated Zucker diabetic fatty (ZDF) and lean control rats were treated with the ghrelin-receptor ligand, tabimorelin (50 mg/kg p.o.) for 18 days, and the effects on body weight, food intake and body composition were investigated. The level of expression of anabolic and catabolic neuropeptides and their receptors in the hypothalamic area were analysed by in situ hybridization. RESULTS: Tabimorelin treatment induced hyperphagia and adiposity (increased total fat mass and gain in body weight) in lean control rats, while these parameters were not increased in ZDF rats. Treatment with tabimorelin of lean control rats increased hypothalamic mRNA expression of the anabolic neuropeptide Y (NPY) mRNA and decreased hypothalamic expression of the catabolic peptide pro-opiomelanocortin (POMC) mRNA. In ZDF rats, the expression of POMC mRNA was not affected by treatment with tabimorelin, whereas NPY mRNA expression was increased in the hypothalamic arcuate nucleus. CONCLUSION: This shows that tabimorelin-induced adiposity and hyperphagia in lean control rats are correlated with increased hypothalamic NPY mRNA and decreased POMC mRNA expression. The elimination of tabimorelin-induced adiposity and hyperphagia in ZDF rats may be due to lack of POMC mRNA downregulation. In conclusion, we suggest that ghrelin-receptor ligands exert their adipogenic and orexigenic effects via hypothalamic mechanisms that are dependent on intact leptin-receptor signalling.     

Decreased type 2 corticotropin-releasing hormone receptor mRNA expression in the ventromedial hypothalamus during repeated immobilization stress.

Chronic or repeated stress results in reduction of food intake and body weight in rats. Stress-induced anorexia has been attributed to increased corticotropin-releasing hormone (CRH) function in the central nervous system. To explore possible roles of other neuropeptides and peripheral hormones involved in food intake and energy utilization during continuing stress, we examined the impact of repeated immobilization stress on expression of mRNAs coding for CRH, neuropeptide Y (NPY), galanin and pro-opiomelanocortin (POMC) mRNAs in such hypothalamic nuclei as the paraventricular nucleus (PVN), arcuate nucleus (ARC) and dorsomedial hypothalamus (DMH), as well as plasma insulin and leptin concentrations. Changes in type 2 CRH receptor (CRHR-2) mRNA in the ventromedial hypothalamus (VMH), a possible target of anorectic CRH effect, were also examined. Rats were immobilized for 2 h daily for 6 days and sacrificed 24 h after the last immobilization. Immobilized rats had lower food intake and body weight and higher levels of PVN CRH mRNA than controls. Repeated immobiliza tion also lowered plasma insulin and leptin concentrations and VMH CRHR-2 mRNA levels. These results provide additional evidence linking VMH CRHR-2 mRNA levels to plasma leptin concentration. ARC NPY and DMH galanin mRNAs increased following repeated immobilization, while ARC POMC mRNA decreased. DMH NPY mRNA and ARC galanin mRNA were unaltered by immobilization. Since NPY and galanin are considered orexigenic, while the POMC-melanocortin-4 receptor system is apparently anorexigenic, the changes in neuropeptide mRNAs and VMH CRHR-2 mRNA may play counterregulatory roles against anorectic CRH effects.     

Chronic blockade of the melanocortin 4 receptor subtype leads to obesity independently of neuropeptide Y action, with no adverse effects on the gonadotropic and somatotropic axes

Neuropeptide Y (NPY) is a powerful orexigenic factor, and alphaMSH is a melanocortin (MC) peptide that induces satiety by activating the MC4 receptor subtype. Genetic models with disruption of MC4 receptor signaling are associated with obesity. In the present study, a 7-day intracerebroventricular infusion to male rats of either the MC receptor antagonist SHU9119 or porcine NPY (10 nmol/day) was shown to strongly stimulate food and water intake and to markedly increase fat pad mass. Very high plasma leptin levels were found in NPY-treated rats (27.1 +/- 1.8 ng/ml compared with 9.9 +/- 0.9 ng/ml in SHU9119-treated animals and 2.1 +/- 0.2 ng/ml in controls). As expected, NPY infusion induced hypogonadism, characterized by an impressive decrease in seminal vesicle and prostate weights. No such effects were seen with the SHU9119 infusion. Similarly, whereas the somatotropic axis of NPY-treated rats was fully inhibited, this axis was normally activated in the obese SHU9119-treated rats. Chronic infusion of SHU9119 strikingly reduced hypothalamic gene expression for NPY (65.2 +/- 3.6% of controls), whereas gene expression for POMC was increased (170 +/- 19%). NPY infusion decreased hypothalamic gene expression for both POMC and NPY (70 +/- 9% and 75.4 +/- 9.5%, respectively). In summary, blockade of the MC4 receptor subtype by SHU9119 was able to generate an obesity syndrome with no apparent side-effects on the reproductive and somatotropic axes. In this situation, it is unlikely that hyperphagia was driven by increased NPY release, because hypothalamic NPY gene expression was markedly reduced, suggesting that hyperphagia mainly resulted from loss of the satiety signal driven by MC peptides. NPY infusion produced hypogonadism and hyposomatotropism in the face of markedly elevated plasma leptin levels and an important reduction in hypothalamic POMC synthesis. In this situation NPY probably acted both by exacerbating food intake through Y receptors and by reducing the satiety signal driven by MC peptides.     

Chronic administration of neuropeptide Y into the lateral ventricle of C57BL/6J male mice produces an obesity syndrome including hyperphagia, hyperleptinemia, insulin resistance, and hypogonadism.

Neuropeptide Y (NPY) is involved in the central regulation of appetite, sexual behavior, and reproductive function. We have previously shown that chronic infusion of NPY into the lateral ventricle of normal rats produced an obesity syndrome characterized by hyperphagia, hyperinsulinism and collapse of reproductive function. We further demonstrated that acute inhibition of LH secretion in castrated rats was preferentially mediated by the NPY receptor subtype 5 (Y(5)). In the present study, the effects of chronic, central infusion of NPY, or the mixed Y2-Y5 agonist PYY(3-36), were evaluated both in normal male C57BL/6J mice and Sprague-Dawley rats. After a 7-day infusion to male mice, both NPY and PYY(3-36) at 5 nmol per day, induced marked hyperphagia leading to significant increases in body and fat pad weights. Furthermore, both compounds markedly reduced several markers of the reproductive axis. In the rat study, PYY(3-36) was more active than NPY to inhibit the pituitary-testicular axis, confirming the importance of the Y5 subtype for such effects. In the mouse, chronic NPY infusion induced a sustained increase in corticosterone and insulin secretion. Plasma leptin levels were also markedly increased possibly explaining the observed reduction in gene expression for hypothalamic NPY. Gene expression for hypothalamic POMC was reduced in the NPY- or PYY(3-36)-infused mice, suggesting that NPY exacerbated food intake by both acting through its own receptor(s), and reducing the satiety signal driven by the POMC-derived alpha-MSH. The present study in the mouse suggests in analogy with available rat data, that constant exposure to elevated NPY in the hypothalamic area unabatedly enhances food intake leading to an obesity syndrome including increased adiposity, insulin resistance, hypercorticism, and hypogonadism, reminiscent of the phenotype of the ob/ob mouse, that displays elevated hypothalamic NPY secondary to lack of leptin negative feedback action.     

Effects of leptin and corticosterone on the expression of corticotropin-releasing hormone, agouti-related protein, and proopiomelanocortin in the brain of ob/ob mouse

The present study was conducted to assess the effect of leptin and corticosterone on the expression of corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC) and agouti-related protein (AGRP) in the mouse brain. To this end, a 3 x 3 factorial experiment was designed in which adrenalectomized (ADX) ob/ob mice were treated with leptin and corticosterone. Leptin and corticosterone downregulated CRH expression in the paraventricular hypothalamic nucleus (PVH). Leptin prevented the stimulating effects of ADX on the expression of CRH and the combination of small doses of leptin and corticosterone was as potent as the high dose of corticosterone in suppressing CRH mRNA expression in the PVH. Leptin and corticosterone enhanced the expression of CRH in the central nucleus of amygdala and in the bed nucleus of the stria terminalis. In addition, the present results confirmed the downregulating effects of leptin on the expression of AGRP mRNA in the hypothalamic arcuate nucleus (ARC), and demonstrated that this effect was more apparent in ADX mice treated with corticosterone than in ADX mice not supplemented with corticosterone. Also, leptin and corticosterone had opposite effects on the expression of POMC in the ARC. The opposite effect of leptin and corticosterone on the expression of POMC and AGRP seems consistent with the reported effects that these hormones and peptides have on food intake and thermogenesis, suggesting that the modulation of POMC and AGRP expression can be a mechanism whereby leptin and corticosterone exert their effects in the regulation of energy balance. In contrast, the similarity in the action of leptin and corticosterone is not a priori consistent with a role of CRH in the effects of these hormones in the regulation of energy balance. The downregulating effect of leptin on the expression of CRH in the PVH strongly suggests that leptin can be a potent regulator of hypothalamic-pituitary-adrenal axis activity. Finally, the present results suggest that the effects of leptin on the expression of CRH, POMC and AGRP are not curbed by glucocorticoids.     

Leptin regulates appetite-related neuropeptides in the hypothalamus of developing rats without affecting food intake

Leptin regulates food intake in adult mammals by stimulating hypothalamic anorexigenic pathways and inhibiting orexigenic ones. In developing rodents, fat stores are low, yet circulating leptin levels are high and do not appear to regulate food intake. We determined whether two appetite-related neuropeptides [neuropeptide Y (NPY) and proopiomelanocortin (POMC)] and food intake behavior are sensitive to leptin [3 mg/kg body weight (BW), ip] in neonates. We measured the effects of 1) acute leptin administration (3 mg/kg BW, ip, 3 h before testing) on food intake on postnatal day (PND) 5, 8, and 10; and 2) chronic leptin treatment (3 mg/kg BW, ip, daily PND3-PND10) on BW gain and fat pads weight on PND10. In addition to hypothalamic POMC and NPY expression, we determined the expression of suppressor of cytokine signaling-3, all subtypes of leptin receptors, and corticotropin-releasing factor receptor-2 mRNA in PND10 pups receiving either an acute (PND10) or a chronic (PND 3-10) leptin (3 mg/kg BW, ip) or vehicle treatment. Brains were removed 30 or 120 min after the last injection. Acute leptin administration did not affect food intake at any age tested. Chronic leptin treatment did not change BW but decreased fat pad weight significantly. In the arcuate nucleus (ARC), acute leptin increased SOCS-3 and POMC mRNA levels, but decreased NPY mRNA levels in the rostral part of ARC. Chronic leptin down-regulated all subtypes of leptin receptors mRNA and decreased NPY mRNA levels in the caudal ARC but had no further effect on POMC expression. Chronic leptin increased corticotropin-releasing factor receptor-2 mRNA levels in the ventromedial hypothalamus. We conclude that despite adult-like effects of leptin on POMC, NPY, and CRFR-2 expression in neonates, leptin does not regulate food intake during early development.     

Central dysregulation of the hypothalamic-pituitary-adrenal axis in neuron-specific proopiomelanocortin-deficient mice

Proopiomelanocortin (POMC) is synthesized predominantly in pituitary corticotrophs, melanotrophs, and arcuate hypothalamic neurons. Corticotroph-derived ACTH mediates basal and stress-induced glucocorticoid secretion, but it is uncertain whether POMC peptides produced in the brain also regulate the hypothalamic-pituitary-adrenal axis. To address this question, we generated neuron-specific POMC-deficient mice by transgenic (Tg) replacement of pituitary POMC in a global Pomc(-/-) background. Selective restoration of pituitary POMC prevented the adrenal insufficiency and neonatal mortality characteristic of Pomc(-/-) mice. However, adult Pomc(-/-)Tg/+ mice expressing the pituitary-specific transgene exhibited adrenal cortical hypertrophy, elevated basal plasma corticosterone, elevated basal but attenuated stress-induced ACTH secretion, and inappropriately elevated CRH expression in the hypothalamic paraventricular nucleus. In addition, Pomc(-/-)Tg/+, Pomc(+/-)Tg/+, and Pomc(+/-) mice, which all displayed varying degrees of elevated CRH, frequently developed melanotroph adenomas after 1 yr of age, whereas Pomc(-/-) mice, with maximal CRH expression and glucocorticoid disinhibition, developed corticotroph and melanotroph adenomas. These results indicate that neuronal POMC peptides are necessary to regulate CRH within physiological limits and that a chronic reduction or absence of hypothalamic POMC leads to trophic stimulation of pituitary cells directly or indirectly through elevated CRH levels.     

Corticotropin-releasing hormone-mediated pathway of leptin to regulate feeding,adiposity, and uncoupling protein expression in mice

To examine the functional role of CRH in the regulation of energy homeostasis by leptin, we measured the effects of the CRH antagonist, alpha-helical CRH 8-41 (alphaCRH) on a number of factors affected by leptin activity. These included food intake, body weight, hypothalamic c-fos-like immunoreactivity (c-FLI), weight and histological characterization of white adipose tissue, and mRNA expressions of uncoupling protein (UCP) in brown adipose tissue (BAT) in C57Bl/6 mice. Central infusion of leptin into the lateral cerebroventricle (icv) caused significant induction of c-FLI in the paraventricular nucleus (PVN), ventromedial hypothalamic nucleus (VMH), dorsomedial hypothalamic nucleus, and arcuate nucleus. In all these nuclei, the effect of leptin on expression of cFLI in the PVN and VMH was decreased by treatment with alphaCRH. Administration of leptin markedly decreased cumulative food intake and body weight with this effect being attenuated by pretreatment with alphaCRH. In peripheral tissue, leptin up-regulated BAT UCP1 mRNA expression and reduced fat depositions in this tissue. Those changes in BAT were also decreased by treatment with alphaCRH. As a consequence of the effects on food intake or energy expenditure, treatment with alphaCRH attenuated the leptin-induced reduction of body adiposity, fat cell size, triglyceride contents, and ob mRNA expression in white adipose tissue. Taken together, these results indicate that CRH neurons in the PVN and VMH may be an important mediator for leptin that contribute to regulation of feeding, adiposity, and UCP expression.     

Daily changes in hypothalamic gene expression of neuropeptide Y, galanin, proopiomelanocortin, and adipocyte leptin gene expression and secretion: effects of food restriction.

The participation of hypothalamic neuropeptide Y (NPY)-, galanin (GAL)-, and opioid-producing neurons in the restraint on food intake exerted by adipocyte leptin has recently been recognized. To further understand the interplay between the central appetite-stimulating- and peripheral appetite-inhibiting signals in the management of daily food intake, we have examined the daily patterns in expression of the hypothalamic neuropeptides and leptin receptor (R) and adipocyte leptin gene expression and secretion in freely feeding (FF) rats. These analyses were extended to determine the impact of food restriction (FR) to 4 h daily for 4 weeks. Groups of FF and FR rats were killed at 4-h intervals during a 24-h period, and hypothalamic NPY, GAL, POMC, and leptin-R gene expression and leptin gene expression were evaluated by RNase protection assays and serum leptin and corticosterone (CORT) levels were estimated by RIA. The following new findings emerged: 1) In FF rats, hypothalamic NPY messenger RNA (mRNA) levels fluctuated during the course of 24 h with high levels at 0700 h and 1100 h followed by a decrease at 1500 h during the lights-on phase that was sustained throughout the dark phase (1900 h-0500 h) of the light-dark cycle. Hypothalamic GAL and POMC mRNA also displayed daily patterns but with a different time course; GAL and POMC gene expression were elevated 4 h later than NPY mRNA at 1100 h and 1500 h. 2) Although FR to 4 h between 1100 h and 1500 h resulted in maintenance of body weight compared with a steady weight gain in FF rats, the daily patterns of fluctuations in hypothalamic neuropeptide gene expression were abolished. 3) In FF rats, hypothalamic leptin-R and adipocyte leptin gene expression and serum leptin levels displayed a daily pattern temporally different from that of hypothalamic neuropeptide gene expression. Adipocyte leptin mRNA remained low during the lights-on phase but increased at the onset of the lights-off phase (1900 h) and remained elevated through the dark phase. 4) Hypothalamic leptin-R gene expression, like that of adipocyte leptin gene expression, rose abruptly at the onset of nocturnal feeding behavior but receded progressively to low range thereafter. 5) On the other hand, a dichotomy in the daily rise in adipocyte leptin gene expression and leptin secretion was observed in FF rats. Unlike adipocyte leptin mRNA, serum leptin increased at 2300 h, 4 h after initiation of ingestive behavior. 6) In FR rats, adipocyte leptin gene expression fluctuated little over the 24-h period but, as in FF rats, leptin hypersecretion peaked 4 h after initiation of food intake. 7) In both FF and FR rats, increased serum CORT levels preceded serum leptin rise. Overall, these results show that in FF rats, gene expression of hypothalamic appetite stimulating peptides first rise and then fall to nadir during the lights-on phase when leptin levels are in low range; adipocyte leptin mRNA rises before impending ingestive behavior and increased leptin secretion reaching peak manifests itself during nocturnal feeding. The FR regimen, which curtailed the normal body weight gain, abolished these daily fluctuations in gene expression of hypothalamic orexigenic peptides and adipocyte leptin but permitted feeding-associated increased leptin secretion. Thus, it may be important to consider the daily patterns of gene expression and availability of hypothalamic orexigenic peptides in investigations aimed at elucidating the central mechanisms underlying the feedback action of the normal and altered leptin secretion patterns.