Central and peripheral immunoreactivity of melanin-concentrating hormone in hypothalamic obese and lactating rats

Melanin-concentrating hormone (MCH) is believed to be an important orexigenic peptide mainly localized in the lateral hypothalamic area. Its involvement in the hyperphagia induced by hypothalamic lesions and lactation remains unclear. In this study, we investigated MCH immunoreactivity in the hypothalamus using immunohistochemistry and MCH concentration in the peripheral circulation using an enzyme immunoassay in rats with a lesion in the ventromedial hypothalamus or the paraventricular nucleus, and in lactating rats. Bilateral lesions of the ventromedial or paraventricular nuclei were performed using an electrolytic method. Quantification of immunoreactivity by image analysis revealed that the number and mean staining intensity of MCH-immunoreactive neurones in the lateral hypothalamic area and the zona incerta were significantly decreased by both types of lesions compared to sham controls, whereas circulating MCH concentration was not significantly different on day 7 postlesion. By contrast, in lactating rats on days 11-12 postpartum, the expression of MCH in the lateral hypothalamic area and the zona incerta was significantly increased compared to nonlactating controls. Circulating MCH concentration was not changed in lactating rats. These results suggest that hyperphagia induced by lactation, but not hypothalamic lesion, might be induced by excessive expression of MCH in the lateral hypothalamic area and the zona incerta.     

Effect of Nitric Oxide Synthase Inhibition on Fos Expression in the Hypothalamus of Female Rats Following Central Oxytocin and Systemic Urethane Administration

Three experiments were carried out to investigate the pattern of neuronal activation induced by central oxytocin administration and its modulation by nitric oxide (NO). First, we compared the induction of Fos-like immunoreactivity (lir) in the supraoptic (SON) and paraventricular (PVN) nuclei and medial preoptic area (MPOA) after central oxytocin administration between nonlactating and lactating rats. Next, we investigated whether NO modulated Fos induction following central oxytocin administration using a nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME). Finally, to determine whether the effects of NOS inhibition on Fos induction would generalize to stimuli other than oxytocin, we compared Fos-lir in the SON and PVN of lactating and nonlactating rats following L-NAME and urethane administration. In the first two experiments, oxytocin (50 ng in 2 microl) or vehicle was administered into the third ventricle. L-NAME (50 mg/kg) was given by an intraperitoneal (i.p.) injection 30 min before oxytocin administration (experiment 2) or an i.p. injection of urethane (1.4 g/kg) (experiment 3). In all experiments, lactating rats were tested on day 12 or 13 postpartum and nonlactating females at least 11 days after surgery or the start of the experiment. Central oxytocin infusion induced Fos expression in the SON and PVN in lactating and nonlactating rats and in the MPOA and bed nucleus of the stria terminalis in lactating rats. Overall, lactating rats that received L-NAME and oxytocin had a greater number of cells showing Fos-lir in both the SON and PVN. Conversely, L-NAME administration reduced Fos-lir in the SON and PVN in oxytocin-stimulated nonlactating rats. In urethane-treated rats, L-NAME administration did not change Fos-lir in lactating rats but reduced Fos-lir in nonlactating rats. These data suggest that the role of NO in modulating the activity of neurones in discrete nuclei in the hypothalamus varies across reproductive state and with the stimulus presented.     

Resistance to the satiety action of leptin following chronic central leptin infusion is associated with the development of leptin resistance in neuropeptide Y neurones

Leptin regulates food intake and body weight by acting primarily in the hypothalamus. In humans and rodents, obesity is associated with hyperleptinaemia, suggesting a possible state of leptin resistance. Thus, to begin to examine the mechanisms of leptin resistance, we developed a rat model in which chronic central leptin infusion results in the development of resistance to leptin's satiety action. Adult male rats were infused chronically into the lateral cerebroventricle with leptin (160 ng/h) or phosphate-buffered saline via Alzet pumps for 28 days, followed by artificial cerebrospinal fluid infusion for 3 weeks. After the initial decrease in food intake, rats developed resistance to the satiety action of leptin, and withdrawal of the chronic leptin infusion resulted in hyperphagia. During leptin infusion, body weight was gradually decreased to reach a nadir on day 12, and thereafter, body weight was sustained at a reduced level throughout the entire 28-day infusion, despite normalization in food intake. Body weight was mostly normalized by day 22 postleptin. Since neuropeptide Y (NPY) neurones are one of the targets of leptin signalling in the hypothalamus, we next examined whether the development of resistance to the satiety action of leptin was due to altered NPY gene expression. On day 3-4 of infusion, hypothalamic NPY mRNA levels, as determined by RNAse protection assay (RPA), were significantly decreased in leptin treated rats compared to controls. By contrast, on day 16 of infusion, NPY mRNA levels in the leptin treated group had returned to control levels. In situ hybridization study confirmed the results obtained with RPA and showed further that the effect of chronic leptin infusion on NPY mRNA levels was restricted to the rostral and middle parts of the arcuate nucleus. Overall, the finding that the action of continuous leptin exposure on NPY neurones was not sustained suggests that NPY neurones may be involved in the development of leptin resistance to the satiety action of leptin in the hypothalamus.     

Galanin-like peptide promotes feeding behaviour via activation of orexinergic neurones in the rat lateral hypothalamus

Galanin-like peptide (GALP) is produced in neurones in the hypothalamic arcuate nucleus and is implicated in the neural control of feeding behaviour. Previously, we have reported that GALP immunoreactive fibres were in direct contact with orexin/hypocretin immunoreactive neurones in the rat lateral hypothalamus using double-immunofluorescence. Centrally administered GALP is known to stimulate feeding behaviour. However, the target neurones of this action have not been clarified. The present study aimed to determine features of the GALP-mediated neuronal feeding pathway in rat. Accordingly, at the ultrastructural level, GALP-immunoreactive axon terminals were found to make synapses on orexin/hypocretin immunoreactive cell bodies and dendritic processes in the lateral hypothalamus. c-Fos immunoreactivity was expressed in orexin/hypocretin-immunoreactive neurones but not in melanin concentrating hormone-immunoreactive neurones in the lateral hypothalamus at 90 min after the application of GALP by i.c.v. infusion. Furthermore, to determine whether GALP regulates feeding behaviour via orexin/hypocretin neurones, the feeding behaviour of rats was studied following GALP i.c.v. injection with or without anti-orexin A and B immunoglobulin (IgG) pretreatment. The anti-orexin IgGs markedly inhibited GALP-induced hyperphagia. These results suggest that orexin/hypocretin-containing neurones in the lateral hypothalamus are targeted by GALP, and that GALP-induced hyperphagia is mediated via orexin/hypocretin neurones in the rat hypothalamus.     

Differential regulation of leptin receptor but not orexin in the hypothalamus of the lactating rat

During lactation, hypothalamic levels of neuropeptide Y (NPY) and agouti related protein (AGRP) mRNA are increased, while pro-opiomelanocortin (POMC) mRNA is decreased. Serum leptin levels are also decreased during lactation. These changes may underlie the large increases of both food and water intake that occur in concert with milk production. However, additional hypothalamic substances, such as the novel peptide, orexin, may be involved. In addition, in the presence of chronically suppressed levels of serum leptin, there may be a change in leptin receptor expression in the hypothalamus. The objectives of the present study were to determine if orexin and leptin receptor mRNA levels were changed during lactation. Rats were studied on dioestrus of the oestrous cycle or on day 10 postpartum (the lactating animals were suckling eight pups). Orexin mRNA levels in the lateral hypothalamus did not differ between dioestrus and lactation. There was a significant increase in leptin receptor mRNA levels in the supraoptic nucleus during lactation compared to dioestrus. Furthermore, leptin receptor protein, as determined by immunocytochemistry, was colocalized in virtually all vasopressin and oxytocin cells in the supraoptic nucleus. Lactating animals exhibited a decrease in leptin receptor mRNA in the ventromedial hypothalamic nucleus whereas no change was apparent in other hypothalamic areas compared to the dioestrus animals. These results demonstrate that changes in orexin do not appear to contribute to the increase in food intake during lactation. It is likely that the increases in NPY and ARGP, coupled with the decrease in POMC, are primarily responsible for sustaining the chronic hyperphagia of lactation. The changes observed in leptin receptor expression in the hypothalamus, along with the suppression of serum leptin levels, also suggest that the leptin signalling system may play a significant role in the regulation of food and water intake during lactation.     

Leptin Regulation of Agrp and Npy mRNA in the Rat Hypothalamus

Agouti-related protein (AGRP) is synthesized in the same neurones in the arcuate nucleus as neuropeptide Y (NPY), another potent orexigenic peptide. AGRP antagonizes the action of alpha-melanocyte stimulating hormone, a derivative of pro-opiomelanocortin (POMC) at the hypothalamic MC4 receptor to increase food intake. Although leptin has been shown to regulate Agrp/Npy and Pomc-expressing neurones, there are differences with respect to Agrp regulation in leptin receptor-deficient mice and rats. Unlike the obese leptin receptor-deficient db/db mouse, which exhibits upregulation of Agrp mRNA expression in the medial basal hypothalamus (MBH) compared to lean controls, the obese leptin receptor-deficient (faf; Koletsky) rat does not exhibit upregulation of Agrp expression. To determine whether this represents a general difference between leptin receptor-deficient mice and rats, neuropeptide gene expression was analysed in the MBH of lean and obese rats segregating for a different leptin receptor mutation, Leprfa (Zucker). Fasting in lean rats (+/fa) for 72 h significantly increased Agrp and Npy mRNA expression, and decreased Pomc mRNA expression as detected by a sensitive solution hybridization/S1 nuclease protection assay. Npy mRNA levels were significantly increased in fed obese fa/fa compared to lean rats, and further increased in the obese animals after fasting. In contrast, Agrp mRNA levels did not differ between fed lean and fed obese rats, and fasting did not significantly change Agrp levels in obese rats. To determine whether the change in Agrp expression that occurs with food deprivation in lean rats could be prevented by leptin replacement, Sprague-Dawley rats were fasted and infused via subcutaneous osmotic micropumps for 48 h with either saline or recombinant mouse leptin. Fasting significantly increased Agrp and Npy, and decreased Pomc mRNA levels. Leptin infusion almost completely reversed these changes such that there was no significant difference between the levels in the fasted rats and those that were fed ad libitum. Thus, in fasted lean rats, Agrp and Npy are upregulated in parallel when leptin levels fall and are downregulated by leptin infusion. By contrast, the absence of a functional leptin receptor results in the upregulation of Npy but not Agrp mRNA.     

Effects of Cholecystokinin in the Supraoptic Nucleus and Paraventricular Nucleus are Negatively Modulated by Leptin in 24‐h Fasted Lean Male Rats

Cholecystokinin (CCK) and leptin are two important satiety factors that are considered to act in synergy to reduce meal size. Peripheral injection of CCK activates neurones in several hypothalamic nuclei, including the supraoptic (SON) and paraventricular (PVN) nuclei and neurones in the brainstem of fed rats. We investigated whether peripheral leptin would modulate the effects of CCK on neuronal activity in the hypothalamus and brainstem of fasted rats by investigating Fos expression in the PVN, SON, arcuate nucleus, ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), area postrema (AP) and the nucleus tractus solitarii (NTS). Male rats, fasted for 24 h, received either one i.p. injection of vehicle, leptin or CCK‐8 alone, or received one injection of vehicle or leptin before an i.p. injection of CCK‐8. We found that CCK increased Fos expression in the PVN and SON as well as in the NTS and AP, but had no effect on Fos expression in the arcuate nucleus, VMH or DMH compared to vehicle. Leptin injected alone significantly increased Fos expression in the arcuate nucleus but had no effect on Fos expression in the VMH, DMH, SON, PVN, AP or NTS compared to vehicle. Fos expression was significantly increased in the AP in rats injected with both leptin and CCK compared to rats injected with vehicle and CCK. Unexpectedly, there was significantly less Fos expression in the PVN and SON of fasted rats injected with leptin and CCK than in rats injected with vehicle and CCK, suggesting that leptin attenuated CCK‐induced Fos expression in the SON and PVN. However, Fos expression in the NTS was similar in fasted rats injected with vehicle and CCK or with leptin and CCK. Taken together, these results suggest that leptin dampens the effects of CCK on Fos expression in the SON and PVN, independently from NTS pathways, and this may reflect a direct action on magnocellular neurones.     

Oestrogen-dependent suppression of pulsatile luteinising hormone secretion and kiss1 mRNA expression in the arcuate nucleus during late lactation in rats

Follicular development and ovulation are strongly suppressed during lactation in mammals via a profound suppression of gonadotrophin secretion. The present study aimed to examine the role of oestrogen feedback action in suppressing luteinising hormone (LH) secretion and hypothalamic kisspeptin expression during the latter half of lactation. Plasma LH concentrations kept at low levels throughout the lactating period in intact and oestrogen‐replaced ovariectomised (OVX) lactating rats, whereas plasma LH concentrations gradually elevated from day 10 postpartum in lactating OVX rats. OVX lactating rats showed frequent LH pulses at late lactation, although the LH pulses were significantly inhibited by an oestrogen replacement, which is much less effective on LH release in nonlactating rats. Oestrogen replacement in lactating OVX rats significantly reduced the number of Kiss1 mRNA‐expressing cells in the arcuate nucleus (ARC) at late lactation, although the same oestrogen treatment did not affect the number of Kiss1‐expressing cells in nonlactating controls. Exogenous kisspeptin challenge (0.2 nmol) into the third cerebroventricle significantly increased LH secretion in lactating OVX, lactating OVX + subcutaneous 17β‐oestradiol and intact lactating rats at day 16 postpartum. These results suggest that LH pulse suppression during late lactation could be a result of the enhanced oestrogen‐dependent suppression of ARC kisspeptin expression.     

Down-regulation of orexin gene expression by severe obesity in the rats: studies in Zucker fatty and zucker diabetic fatty rats and effects of rosiglitazone

Orexins (hypocretins) are lateral hypothalamic neuropeptides implicated in regulating feeding and the sleep-wake cycle. To study their possible relevance to obesity and diabetes, we measured hypothalamic prepro-orexin mRNA levels in obese, normoglycemic Zucker fatty (fa/fa) and in hyperglycemic, non-obese Zucker diabetic fatty (ZDF) rats. Hypothalamic prepro-orexin mRNA concentrations in Zucker fatty rats were 31% lower than those in lean controls (0. 69+/-0.06 vs. 1.00+/-0.10 arbitrary units, P<0.05), but did not differ between ZDF diabetic rats and non-diabetic controls. Treatment of ZDF diabetic rats with rosiglitazone (1 or 3 mg/kg body weight daily for 13 weeks) normalized plasma glucose and significantly reduced plasma insulin, while leptin levels were 67% higher than in untreated ZDF rats (20.2+/-0.5 vs. 12.1+/-2.5, P<0. 001). Rosiglitazone treatment markedly enhanced weight gain compared with untreated ZDF rats (final weight 732+/-13 g vs. 409+/-13 g, P<0. 001) even though they were restricted to the same food intake. Rosiglitazone-treated ZDF rats had significantly lower hypothalamic prepro-orexin mRNA levels (0.68+/-0.07 arbitrary units) than both non-diabetic lean controls (1.00+/-0.10, P=0.02) and untreated diabetics (1.03+/-0.14, P=0.03). Our data suggest that prepro-orexin gene expression may be suppressed by substantial weight gain. Obesity-related signals that might mediate this effect have not been identified, but plasma leptin, insulin and glucose are not obviously involved.     

Leptin effects on the expression of type-2 CRH receptor mRNA in the ventromedial hypothalamus in the rat

The product of the ob gene, leptin, is thought to act in the hypothalamus to reduce food intake and body weight (b.w.) in rats and mice; however, the mechanisms of leptin action in the brain have not been fully elucidated. Corticotropin-releasing hormone (CRH) is a potent anorectic neuropeptide, and its type-2 receptor (CRHR-2) in the ventromedial hypothalamus (VMH) appears to play an important role in the expression of this anorectic effect. We explored here the impact of systemic leptin administration on CRH mRNA expression in the hypothalamic paraventricular nucleus (PVN) and CRHR-2 mRNA expression in the VMH in male rats, using in-situ hybridization histochemistry. The expression of CRH mRNA in the PVN and CRHR-2 mRNA in the VMH were increased at 2 h and 6 h, respectively, after a single intraperitoneal injection of leptin (1.0 mg/kg). Continuous subcutaneous infusion of leptin (1.2 mg/kg/day) via an osmotic minipump for 5 days increased the expression of CRHR-2 mRNA in the VMH, but not the expression of CRH mRNA in the PVN, compared with vehicle treatment. The rats that received the single or continuous administration of leptin showed reductions of food intake and b.w. compared with vehicle-treated rats. These results are consistent with our previous findings that the expression of CRHR-2 mRNA in the VMH is positively correlated with plasma leptin concentrations under various conditions, and highlight the importance of circulating leptin for the regulation of VMH CRHR-2 mRNA. The present results also raise the possibility that leptin reduces food intake and b.w. at least partially due to the enhancement of the anorectic effect of CRH via increased PVN CRH expression and/or VMH CRHR-2 expression.     

Characterisation and origins of melanin‐concentrating hormone immunoreactive fibres of the posterior lobe of the pituitary and median eminence during lactation in the Long‐Evans rat

Although the melanin‐concentrating hormone (MCH) and its coding mRNA are predominantly found in the tuberal hypothalamus, there is detectable synthesis of MCH in the preoptic hypothalamus exclusively in lactating dams, suggesting a participation of MCH in the alterations that take place after parturition. Also implicated in the dam physiology is oxytocin, a neurohormone released from the posterior pituitary that is necessary for milk ejection. Because the projection fields from oxytocin‐immunoreactive (‐IR) neurones and the mediobasal preoptic hypothalamus overlap and MCH‐IR neurones are found in proximity to oxytocin neurones, we investigated the spatial relationship between MCH and oxytocin fibres. Accordingly, we employed multiple immunohistochemistry labelling for MCH and oxytocin for light and electron microscopy techniques, in addition to i.v. tracer injection combined with in situ hybridisation to identify MCH neurones that project to neurosecretory areas. As described for other strains, lactating Long‐Evans dams also display immunoreactivity for MCH in the preoptic hypothalamus on days 12 and 19 of lactation. The appearance of these neurones is contemporaneous with an increase in MCH‐IR fibres in both the internal layer of the median eminence and the posterior pituitary. In both regions, MCH‐ and oxytocin‐IR fibres were found in great proximity, although there was no evidence for synaptic interaction between these two populations at the ultrastructural level. The tracer injection revealed that only mediobasal preoptic MCH neurones project to the posterior pituitary, suggesting a neuroendocrine‐modulatory role for this population. When taken together, the results obtained in the present study indicate that neuroplasticity events at the mediobasal preoptic hypothalamus that occur during late lactation may be part of a neuroendocrinology control loop involving both MCH and oxytocin.     

Disruption in neuropeptide Y and leptin signaling in obese ventromedial hypothalamic-lesioned rats

Electrolytic lesions placed in the ventromedial hypothalamus (VMH) of rats induce instant hyperphagia and excessive weight gain. Since neuropeptide Y (NPY) is a potent hypothalamic orexigenic signal, and leptin secreted by adipocytes regulates NPY output, we tested the hypothesis that altered NPYergic-leptin signaling may underlie hyperphagia in VMH-lesioned rats. VMH-lesioned rats exhibiting hyperphagia and excessive weight gain in a time-related fashion were sacrificed on days 2, 7, and 21 post-surgery. Quite unexpectedly, NPY concentrations in the hypothalamic paraventricular nucleus (PVN), a major site of NPY release for stimulation of feeding, and in other sites, such as the dorsomedial nucleus, lateral hypothalamic area and median eminence-arcuate nucleus decreased, with the earliest diminution occurring on day 2 in the PVN only. In vitro basal and K⁺-evoked NPY release from the PVN of VMH-lesioned rats was significantly lower than that of controls. Analysis of hypothalamic NPY gene expression showed that although the daily decrease in NPY mRNA from 0800 to 2200 h occurred as in control rats, NPY mRNA concentrations were markedly reduced at these times in the hypothalami of VMH-lesioned rats. Leptin synthesis in adipocytes as indicated by leptin mRNA levels was also profoundly altered in VMH-lesioned rats. The daily pattern of increase in adipocyte leptin mRNA at 2200 h from 0800 h seen in controls was abolished, higher levels of leptin gene expression at 2200 h were maintained at 0800 h. The pattern of increase in serum leptin and insulin levels diverged in VMH-lesioned rats. Serum insulin concentration increased to maximal on day 2 and remained at that level on day 21-post-lesion; serum leptin levels on the other hand, increased slowly in a time-related fashion during this period. These results demonstrate that hyperphagia and excessive weight gain in VMH-lesioned rats are associated with an overall decrease in hypothalamic NPY and augmented leptin signaling to the hypothalamus. The divergent time course of increases in serum leptin and insulin levels suggest independent mechanisms responsible for their augmented secretion, and neither these hormones nor VMH lesions altered the daily rhythm in NPY gene expression. These observations underscore the existence of an independent mechanism controlling the daily rhythm in hypothalamic NPY gene expression and suggest that leptin feedback action requires an intact VMH.     

Activation of Nesfatin‐1‐Containing Neurones in the Hypothalamus and Brainstem by Peripheral Administration of Anorectic Hormones and Suppression of Feeding via Central Nesfatin‐1 in Rats

Peripheral anorectic hormones, such as glucagon‐like peptide (GLP)‐1, cholecystokinin (CCK)‐8 and leptin, suppress food intake. The newly‐identified anorectic neuropeptide, nesfatin‐1, is synthesised in both peripheral tissues and the central nervous system, particularly by various nuclei in the hypothalamus and brainstem. In the present study, we examined the effects of i.p. administration of GLP‐1 and CCK‐8 and co‐administrations of GLP‐1 and leptin at subthreshold doses as confirmed by measurement of food intake, on nesfatin‐1‐immunoreactive (‐IR) neurones in the hypothalamus and brainstem of rats by Fos immunohistochemistry. Intraperitoneal administration of GLP‐1 (100 μg/kg) caused significant increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the supraoptic nucleus (SON), the area postrema (AP) and the nucleus tractus solitarii (NTS) but not in the paraventricular nucleus (PVN), the arcuate nucleus (ARC) or the lateral hypothalamic area (LHA). On the other hand, i.p. administration of CCK‐8 (50 μg/kg) resulted in marked increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the SON, PVN, AP and NTS but not in the ARC or LHA. No differences in the percentage of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the nuclei of the hypothalamus and brainstem were observed between rats treated with saline, GLP‐1 (33 μg/kg) or leptin. However, co‐administration of GLP‐1 (33 μg/kg) and leptin resulted in significant increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the AP and the NTS. Furthermore, decreased food intake induced by GLP‐1, CCK‐8 and leptin was attenuated significantly by pretreatment with i.c.v. administration of antisense nesfatin‐1. These results indicate that nesfatin‐1‐expressing neurones in the brainstem may play an important role in sensing peripheral levels of GLP‐1 and leptin in addition to CCK‐8, and also suppress food intake in rats.