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.     

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.     

Collective and individual functions of leptin receptor modulated neurons controlling metabolism and ingestion

Two known types of leptin-responsive neurons reside within the arcuate nucleus: the agouti gene-related peptide (AgRP)/neuropeptide Y (NPY) neuron and the proopiomelanocortin (POMC) neuron. By deleting the leptin receptor gene (Lepr) specifically in AgRP/NPY and/or POMC neurons of mice, we examined the several and combined contributions of these neurons to leptin action. Body weight and adiposity were increased by Lepr deletion from AgRP and POMC neurons individually, and simultaneous deletion in both neurons (A+P LEPR-KO mice) further increased these measures. Young (periweaning) A+P LEPR-KO mice exhibit hyperphagia and decreased energy expenditure, with increased weight gain, oxidative sparing of triglycerides, and increased fat accumulation. Interestingly, however, many of these abnormalities were attenuated in adult animals, and high doses of leptin partially suppress food intake in the A+P LEPR-KO mice. Although mildly hyperinsulinemic, the A+P LEPR-KO mice displayed normal glucose tolerance and fertility. Thus, AgRP/NPY and POMC neurons each play mandatory roles in aspects of leptin-regulated energy homeostasis, high leptin levels in adult mice mitigate the importance of leptin-responsiveness in these neurons for components of energy balance, suggesting the presence of other leptin-regulated pathways that partially compensate for the lack of leptin action on the POMC and AgRP/NPY neurons.     

Changes in hypothalamic corticotropin-releasing hormone, neuropeptide Y, and proopiomelanocortin gene expression during chronic rapid eye movement sleep deprivation of rats

Chronic rapid eye movement (paradoxical) sleep deprivation (REM-SD) of rats leads to two conspicuous pathologies: hyperphagia coincident with body weight loss, prompted by elevated metabolism. Our goals were to test the hypotheses that 1) as a stressor, REM-SD would increase CRH gene expression in the hypothalamus and that 2) to account for hyperphagia, hypothalamic gene expression of the orexigen neuropeptide Y (NPY) would increase, but expression of the anorexigen proopiomelanocortin (POMC) would decrease. Enforcement of REM-SD of adult male rats for 20 d with the platform (flowerpot) method led to progressive hyperphagia, increasing to approximately 300% of baseline; body weight steadily declined by approximately 25%. Consistent with changes in food intake patterns, NPY expression rapidly increased in the hypothalamic arcuate nucleus by d 5 of REM-SD, peaking at d 20; by contrast, POMC expression decreased progressively during REM-SD. CRH expression was increased by d 5, both in mRNA and ability to detect neuronal perikaryal staining in paraventricular nucleus with immunocytochemistry, and it remained elevated thereafter with modest declines. Taken together, these data indicate that changes in hypothalamic neuropeptides regulating food intake are altered in a manner consistent with the hyperphagia seen with REM-SD. Changes in CRH, although indicative of REM-SD as a stressor, suggest that the anorexigenic actions of CRH are ineffective (or disabled). Furthermore, changes in NPY and POMC agree with current models of food intake behavior, but they are opposite to their acute effects on peripheral energy metabolism and thermogenesis.     

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.     

Loss of agouti-related peptide does not significantly impact the phenotype of murine POMC deficiency

The hypothalamic melanocortin system is unique among neuropeptide systems controlling energy homeostasis, in that both anorexigenic proopiomelanocortin (POMC)-derived and orexigenic Agouti related-peptide (AgRP)-derived ligands act at the same receptors, namely melanocortin 3 and 4 receptors (MC3/4R). AgRP clearly acts as a competitive antagonist at MC3R and MC4R but may also have an inverse agonist action at these receptors. The physiological relevance of this remains uncertain. We generated a mouse lacking both POMC and AgRP [double knockout (DKO) mouse]. Phenotyping was performed in the absence and presence of glucocorticoids, and the response to central peptide administration was studied. The phenotype of DKO mice is indistinguishable from that of mice lacking Pomc alone, with both exhibiting highly similar degrees of hyperphagia and increased body length, fat, and lean mass compared with wild-type controls. After a 24-h fast, there was no difference in the refeeding response between Pomc(-/-) and DKO mice. Similarly, corticosterone supplementation caused an equivalent increase in food intake and body weight in both genotypes. Although the central administration of [Nle?, d-Phe?]-α-MSH to DKO mice caused a decrease in food intake and an increase in brown adipose tissue Ucp1 expression, both of which could be antagonized with the coadministration of AgRP, there was no effect of AgRP alone. These data suggest AgRP acts predominantly as a melanocortin antagonist. If AgRP has significant melanocortin-independent actions, these are of insufficient magnitude in vivo to impact any of the detailed phenotypes we have measured under a wide variety of conditions.     

Effects of lifelong moderate caloric restriction on levels of neuropeptide Y, proopiomelanocortin, and galanin mRNA

We are interested in how neuropeptides that regulate both food intake and reproductive function change with age and how life-prolonging moderate caloric restriction may influence the expression of these neuropeptides. We measured neuropeptide Y (NPY) and proopiomelanocortin (POMC) mRNA in the arcuate nucleus (AN), and galanin (GAL) mRNA in the AN, paraventricular nucleus (PVN) and medial septum-diagonal band of Broca in young, middle-aged and old female rats on a controlled feeding regimen. Female Sprague-Dawley rats (7 weeks old) were placed on caloric restriction (CR; n = 70) which was 60% of ad libitum (AL) intake measured in control rats (n = 70). Rats were rapidly decapitated 2.5 weeks following ovariectomy, when they were 4, 12, or 18 months old. Brains were frozen and coronal sections (12 microns) were cut at -20 degrees C using a cryostat. Relative levels of NPY, POMC and GAL mRNA were measured using in situ hybridization histochemistry. cDNA clones complementary to rat NPY, mouse POMC and rat GAL were used to synthesize 35S-UTP-labeled cRNA probes. Slides were dipped in photographic emulsion. Silver grains were quantified using computer-assisted image analysis. Caloric restriction increased NPY mRNA/cell, decreased POMC mRNA/cell in the AN, and did not influence GAL mRNA/cell. Age and caloric restriction did not influence the number of cells with detectable NPY mRNA, POMC mRNA or GAL mRNA. Reproductive status at the time of decapitation influenced both the level and the number of cells expressing GAL mRNA in the PVN. In summary, CR differentially affects levels of NPY, POMC and GAL mRNA in the AN. In this animal model, there was no statistically significant effect of age nor any interaction between age and diet on expression of any of these genes between 4 and 18 months of age.     

Agouti related protein (AgRP) is upregulated in Cushing's syndrome.

Alpha melanocyte-stimulating hormone (alpha-MSH) is an agonist at the melanocortin 3 (MC3-R) and melanocortin 4 (MC4-R) receptors. Alpha-MSH stimulates corticosterone release from rat and human adrenal cells. Patients with Cushing's syndrome have elevated levels of serum alpha-MSH. Agouti related protein (AgRP) is an endogenous antagonist at the MC3-R and MC4-R and is expressed in the rat adrenal cortex. AgRP antagonises alpha-MSH-induced corticosterone release from rat and bovine adrenal cells. This suggests that AgRP may have an inhibitory paracrine role in the adrenal gland. We measured adrenal AgRP mRNA expression and circulating AgRP in 2 patients with Cushing's syndrome and controls. Adrenal AgRP mRNA expression and plasma AgRP were higher in the patients with Cushing's syndrome compared to controls. Plasma AgRP in the patients with Cushing's syndrome following bilateral adrenalectomy and hydrocortisone replacement were similar to the levels seen in controls. Our results suggest that AgRP may have a novel inhibitory paracrine role in the human adrenal gland.     

Onset of glucocorticoid responsiveness of anterior pituitary corticotrophs during development is scheduled by corticotropin-releasing factor

Expression of the CRF gene in the hypothalamus and that of the POMC gene in the anterior pituitary are reduced during the first week of life in the rat. During this so-called stress nonresponsive period (SNRP), stimuli such as ether vapors, electroshocks, and hypoxia do not elicit ACTH secretion from the pituitary, as occurs later in development. The current hypothesis to explain the SNRP is an increased negative glucocorticoid feedback on POMC and CRF synthesis and/or release during this time. To test this hypothesis we studied the effects of adrenalectomy (ADX) on anterior pituitary POMC mRNA expression. In 7-day-old rats POMC mRNA levels were increased only 3-fold 48 h post-ADX, compared to a 7-fold increase in 14-day-old animals. This blunted effect of endogenous glucocorticoid removal on pituitary POMC mRNA could be due to decreased up-regulation of CRF after removal of glucocorticoids or normal up-regulation of CRF but decreased pituitary responsiveness to CRF relative to those in 14-day-old animals. Therefore, we studied in vitro beta-endorphin release from pituitaries obtained from 7- and 14-day-old rats. CRF stimulated basal beta-endorphin release to the same extent in pituitaries from both groups. The inhibition by corticosterone of CRF-stimulated beta-endorphin secretion was also indistinguishable in pituitaries obtained from 7- or 14-day-old rats. Since the responsiveness of the 7-day-old pituitary was normal, the blunted enhancement of POMC biosynthesis after ADX must be mediated at the level of the hypothalamus. Indeed, in situ hybridization showed that while in 14-day-old rats ADX induced a significant increase [190 +/- 10% (+/- SE) of control; n = 5; P less than 0.0005] in hypothalamic mRNA levels, ADX did not change the expression of the CRF gene in the paraventricular nucleus of 7-day-old rats, indicating a lack of glucocorticoid modulation of hypothalamic CRF synthesis. Finally, we studied the effects of 48 h CRF treatment on the post-ADX increase in POMC mRNA levels in 7-day-old rats. Daily injections of 200 ng CRF/rat induced an increase in anterior pituitary POMC mRNA concentrations [669 +/- 139% (+/- SE) of control; n = 6; P less than 0.02 vs. adrenalectomized vehicle-treated rats] comparable to that in adrenalectomized untreated 14-day-old rats. In conclusion, our data indicate that the glucocorticoid regulation of hypothalamic CRF gene expression is not mature during the first week of life, i.e. within the so-called SNRP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pro-opiomelanocortin (POMC)-derived peptides and the regulation of energy homeostasis

Human genetic data indicate impaired synthesis or processing of POMC results in obesity. We have used a mouse model of POMC deficiency (Pomc null) to explore the role of POMC-derived peptides in energy homeostasis. The phenotype of Pomc null mice recapitulates the clinical syndrome seen in humans congenitally lacking POMC. Loss of only one copy of the Pomc gene is sufficient to render mice susceptible to the effects of high fat feeding, emphasizing an important gene-environment interaction predisposing to obesity. Our studies indicate that POMC-derived peptides have influences on the response to a high fat diet, including a major influence on the dietary preference for fat. Pomc null mice are unusual in that obesity and hyperphagia develop in the absence of circulating glucocorticoid (GC). To investigate the interaction between GCs and the melanocortin system, we administered corticosterone to Pomc null mice. They appear hypersensitive to the adverse metabolic effects of GCs, developing hypertension, an exacerbation of both hyperphagia and obesity and a profound insulin resistance. GC treatment of Pomc null mice significantly increases the expression of the melanocortin antagonist agouti-related protein (AgRP). On-going studies in mice lacking both AgRP and Pomc will determine whether the metabolic phenotype seen with this GC therapy is due to a lack of melanocortin peptide, the unopposed action of AgRP or a combination of both.