Melanocortin signaling is decreased during neurotoxin-induced transient hyperphagia and increased body-weight gain

Hypothalamic neuropeptides play critical roles in the regulation of feeding behavior and body weight (BW). Disruption of signaling in the ventromedial nucleus by microinjection of the neurotoxin, colchicine (COL), produces transient hyperphagia with corresponding BW gain lasting for 4 days. Because the melanocortin system exerts an inhibitory control on food intake, we hypothesized that hyperphagia in COL-treated rats is due to decreased melanocortin-induced restraint on feeding. Melanocortin restraint is exerted through alpha-melanocortin-stimulating hormone derived from proopiomelanocortin (POMC) and is antagonized by agouti-related peptide produced in neurons located in the arcuate nucleus (ARC). COL (4 microg/0.5 microl saline) or saline was microinjected bilaterally into the ventromedial nucleus of adult male rats. In conjunction with BW gain, blood leptin levels were elevated, whereas POMC mRNA in the ARC was significantly decreased in COL-injected rats. Levels of alpha-melanocortin-stimulating hormone were also decreased in the micropunched paraventricular nucleus, dorsomedial nucleus, and perifornical hypothalamus, sites implicated in the control of food intake. That diminution in melanocortin signaling underlies hyperphagia was supported by the observation that intracerebroventricular injection of the MC3/MC4 melanocortin receptor agonist, MTII, prevented the hyperphagia and BW gain. Surprisingly, however, mRNA levels of the orexigenic peptide agouti-related peptide in the ARC were decreased perhaps due to the action of elevated leptin. These results show that transient hyperphagia and BW gain induced by disruption of signaling in the ventromedial nucleus results from two neurochemical rearrangements: development of leptin resistance in POMC neurons and diminution in melanocortin signaling as reflected by decreased POMC gene expression in the ARC and decreased availability of alpha-melanocortin-stimulating hormone for release in feeding relevant sites.     

Defense of Elevated Body Weight Setpoint in Diet-Induced Obese Rats on Low Energy Diet Is Mediated by Loss of Melanocortin Sensitivity in the Paraventricular Hypothalamic Nucleus

Some animals and humans fed a high-energy diet (HED) are diet-resistant (DR), remaining as lean as individuals who were naïve to HED. Other individuals become obese during HED exposure and subsequently defend the obese weight (Diet-Induced Obesity- Defenders, DIO-D) even when subsequently maintained on a low-energy diet. We hypothesized that the body weight setpoint of the DIO-D phenotype resides in the hypothalamic paraventricular nucleus (PVN), where anorexigenic melanocortins, including melanotan II (MTII), increase presynaptic GABA release, and the orexigenic neuropeptide Y (NPY) inhibits it. After prolonged return to low-energy diet, GABA inputs to PVN neurons from DIO-D rats exhibited highly attenuated responses to MTII compared with those from DR and HED-naïve rats. In DIO-D rats, melanocortin-4 receptor expression was significantly reduced in dorsomedial hypothalamus, a major source of GABA input to PVN. Unlike melanocortin responses, NPY actions in PVN of DIO-D rats were unchanged, but were reduced in neurons of the ventromedial hypothalamic nucleus; in PVN of DR rats, NPY responses were paradoxically increased. MTII-sensitivity was restored in DIO-D rats by several weeks’ refeeding with HED. The loss of melanocortin sensitivity restricted to PVN of DIO-D animals, and its restoration upon prolonged refeeding with HED suggest that their melanocortin systems retain the ability to up- and downregulate around their elevated body weight setpoint in response to longer-term changes in dietary energy density. These properties are consistent with a mechanism of body weight setpoint.     

Downregulation of melanocortin-4 receptor during refeeding and its modulation by adrenalectomy in rats

Melanocortin system and corticotropin releasing hormone (CRH) are implicated in the control of feeding behavior. Besides its anorexigenic effect on food intake, CRH is one of the most important regulators of hypothalamic-pituitary-adrenal (HPA) axis activity. Therefore, there could be an interplay between HPA axis activity and melanocortin system. We investigated the expression of melanocortin-4 receptor (MC4-R) mRNA in the hypothalamus of rats after 14 days of food restriction or after a fasting-refeeding regimen, in sham or adrenalectomized rats. Male Wistar rats were subjected to free access to food or food ingestion restricted for 2 h a day (8-10 AM) during 14 d, when plasma corticosterone, ACTH, insulin, leptin concentrations, and MC4-R mRNA expression were determined before and after refeeding. Another set of rats was fasted for 48 h, followed by refeeding during 2 or 4 h on the seventh day after adrenalectomy (ADX) or sham surgery. On the day of the experiment, rats were anesthetized and perfused and the brain processed for MC4-R mRNA by in situ hybridization. Long-term reduction of food intake, either secondary to food restriction or adrenalectomy, reduced body weight gain and also leptin and insulin plasma concentrations. Food ingestion reduced MC4-R expression in the paraventricular nucleus in naive rats subjected to food restriction and also in sham rats fasted for 48 h. However, after ADX, MC4-R expression was not changed by refeeding. In conclusion, the present data indicate that MC4-R expression is downregulated by food ingestion and this response could be modulated by glucocorticoid withdrawal.     

Melanocortin receptors mediate the excitatory effects of blood-borne murine leptin on hypothalamic paraventricular neurons in rat

The central pathways and mediators involved in sympathoexcitatory responses to circulating leptin are not well understood, although the arcuate-paraventricular nucleus (ARC-PVN) pathway likely plays a critical role. In urethane-anesthetized rats, ipsilateral intracarotid artery (ICA) injection of murine leptin (100 microg/kg) activated most PVN neurons tested. These responses were reduced by intracerebroventricular injection of the melanocortin subtype 3 and 4 receptor (MC3/4-R) antagonist SHU-9119 (0.6 nmol). The MC3/4-R agonist MTII (0.6 nmol icv) activated PVN neurons. Some PVN neurons that were excited by ICA leptin were inhibited by local application of neuropeptide Y (NPY, 2.5 ng). ICA leptin (100 microg/kg) excited presympathetic rostral ventrolateral medulla neurons and renal sympathetic nerve activity without significant change in blood pressure or heart rate; these effects were mimicked by intracerebroventricular injection of MTII (0.6 nmol). These data provide in vivo electrophysiological evidence to support the hypothesis that circulating leptin activates the sympathetic nervous system by stimulating the release of alpha-melanocyte-stimulating hormone in the vicinity of PVN neurons that are inhibited by the orexogenic peptide NPY.     

Alpha-melanocyte stimulating hormone and ghrelin: central interaction in feeding control

Alpha-melanocyte stimulating hormone (alpha-MSH) and ghrelin play significant yet opposite roles in the regulation of feeding: alpha-MSH inhibits, whereas ghrelin stimulates consumption. The two peptidergic systems may interact in the process of food intake control. A single report published thus far has shown that a synthetic agonist of the melanocortin receptors, MTII, injected in the hypothalamic paraventricular nucleus (PVN) decreases feeding generated by ghrelin. We found that very low doses of alpha-MSH and MTII administered ICV significantly reduced ghrelin-dependent hyperphagia. However, an endogenous molecule, alpha-MSH, infused in the PVN did not exert an inhibitory effect on ghrelin-induced consumption, whereas the effective dose of PVN MTII exceeded that necessary to decrease short-term deprivation-induced feeding. We conclude that it is likely that in feeding regulation alpha-MSH and ghrelin "interact" at the central nervous system level, but the involvement of the PVN in this interaction appears questionable.     

Altered Hypothalamic Signaling and Responses to Food Deprivation in Rats Fed a Low‐Carbohydrate Diet

Objective: To model how consuming a low‐carbohydrate (LC) diet influences food intake and body weight. Research Methods and Procedures: Food intake and body weight were monitored in rats with access to chow (CH), LC‐high‐fat (HF), or HF diets. After 8 weeks, rats received intracerebroventricular injections of a melanocortin agonist (melanotan‐II) and antagonist (SHU9119), and feeding responses were measured. At sacrifice, plasma hormones and hypothalamic expression of mRNA for proopiomelanocortin (POMC), melanocortin‐4 receptor, neuropeptide Y (NPY), and agouti related protein (AgRP) were assessed. A second set of rats had access to diet (chow or LC‐HF) for 4 weeks followed by 24 h food deprivation on two occasions, after which food intake and hypothalamic POMC, NPY, and AgRP mRNA expression were measured. Results: HF rats consumed more food and gained more weight than rats on CH or LC‐HF diets. Despite similar intakes and weight gains, LC‐HF rats had increased adiposity relative to CH rats. LC‐HF rats were more sensitive to melanotan‐II and less sensitive to SHU9119. LC‐HF rats had increased plasma leptin and ghrelin levels and decreased insulin levels, and patterns of NPY and POMC mRNA expression were consistent with those of food‐deprived rats. LC‐HF rats did not show rebound hyperphagia after food deprivation, and levels NPY, POMC, and AgRP mRNA expression were not affected by deprivation. Discussion: Our results demonstrate that an LC diet influences multiple systems involved in the controls of food intake and body weight. These data also suggest that maintenance on an LC‐HF diet affects food intake by reducing compensatory responses to food deprivation.     

Feeding, body weight, and sensitivity to non-ingestive reward stimuli during and after 12-day continuous central infusions of melanocortin receptor ligands

The brain melanocortin system mediates downstream effects of hypothalamic leptin and insulin signaling. Yet, there have been few studies of chronic intracerebroventricular (i.c.v.) melanocortin receptor (MCR) agonist or antagonist infusion. Although there is evidence of interaction between melanocortin and dopamine (DA) systems, effects of chronic MCR ligand infusion on behavioral sensitivity to non-ingestive reward stimuli have not been investigated. The objective of this study was to investigate effects of chronic i.c.v. infusion of the MCR agonist, MTII, and the MCR antagonist, SHU9119, on food intake, body weight, and sensitivity to rewarding lateral hypothalamic electrical stimulation (LHSS) and the reward-potentiating (i.e., threshold-lowering) effect of D-amphetamine. The MCR antagonist, SHU9119 (0.02 microg/h) produced sustained hyperphagia and weight gain during the 12-day infusion period, followed by compensatory hypophagia and an arrest of body weight gain during the 24-day post-infusion period. At no point during the experiment was sensitivity to LHSS or D-amphetamine (0.25mg/kg, i.p.) altered. The MCR agonist, MTII (0.02 microg/h) produced a brief hypophagia (3 days) followed by a return to control levels of daily intake, but with body weight remaining at a reduced level throughout the 12-day infusion period. This was followed by compensatory hyperphagia and weight gain during the 24-day post-infusion period. There was no change in sensitivity to non-ingestive reward stimuli during the infusion of MTII. However, sensitivity to D-amphetamine was increased during the 24-day post-infusion period. It therefore seems that changes in ingestive behavior that occur during chronic MCR ligand infusion may not affect the response to non-ingestive reward stimuli. However, it is possible that the drive to re-feed and restore body weight following MCR agonist treatment includes neuroadaptations that enhance the incentive effects of drug stimuli.     

Paraventricular hypothalamic alpha-melanocyte-stimulating hormone and MTII reduce feeding without causing aversive effects

alpha-Melanocyte-stimulating hormone (alpha-MSH) appears to play a tonic inhibitory role in feeding and energy storage. MTII, a specific synthetic MC3-R/MC4-R agonist, has similar effects on feeding in rats. The current studies demonstrate that PVN administration of alpha-MSH or MTII decreases nocturnal and NPY-stimulated food intake without causing aversive effects. Co-administration with NPY of 600 pmol alpha-MSH or 1 pmol MTII into the PVN caused a significant decrease in NPY-induced feeding. PVN administration of MTII or alpha-MSH at doses effective to suppress feeding did not cause conditioned taste aversion (CTA). ICV administration of alpha-MSH, however, did cause weak CTA. These results indicate that the potent effects on feeding of MC3-R and MC4-R agonists when injected into the PVN are not due to aversive effects.     

Sustained NPY Overexpression in the PVN Results in Obesity via Temporarily Increasing Food Intake

Increasing neuropeptide Y (NPY) signaling in the paraventricular nucleus (PVN) by recombinant adeno‐associated virus (rAAV)‐mediated overexpression of NPY in rats, results in hyperphagia and obesity in rats. To determine the importance of hyperphagia in the observed obesity phenotype, we pair‐fed a group of AAV‐NPY‐injected rats to AAV‐control‐injected rats and compared parameters of energy balance to ad libitum fed AAV‐NPY‐injected rats. For 3 weeks, AAV‐NPY‐injected rats, received the same amount of food as ad libitum‐fed rats injected with control rAAV They did not gain more body weight than these controls. When allowed access to food ad libitum, these AAV‐NPY‐injected rats increased food intake, which subsequently decreased when rats reached the same body weight as AAV‐NPY‐injected rats that were fed ad libitum for the entire study. These data indicate that overexpression of NPY in the PVN results in obesity by increasing food intake until a certain body weight is achieved.     

Hypothalamic NPY status during positive energy balance and the effects of the NPY antagonist, BW 1229U91, on the consumption of highly palatable energy-rich diet.

We have studied the hypothalamic activity of the neuropeptide Y (NPY) system in dietary-induced obese male Wistar rats and examined whether the NPY antagonist, BW1229U91, can inhibit the hyperphagia during positive energy balance associated with feeding rats an energy-rich, highly palatable diet. Rats given a highly palatable, high-fat diet became obese after 8 weeks and exhibited hyperinsulinemia and hyperleptinemia, as compared to lean rats fed on standard pellet laboratory diet. Hypothalamic NPY mRNA concentrations were significantly reduced by approximately 70% in dietary-obese rats compared with lean controls, and the former were hypersensitive to intracerebroventricular injections of NPY, possibly as a result of NPY receptor up-regulation. Intracerebroventricular injections of BW 1229U91, that inhibits food intake in starved rats, did not alter food intake in either control or obese rats fed either standard pellet diet or the highly palatable diet, respectively. We conclude that dietary-obese rats have underactive hypothalamic NPYergic neurons compared to lean controls, possibly as a result of increased plasma concentrations of leptin and/or insulin that directly inhibit the NPY neuronal activity. The lack of effect of BW1229U91 on the increased caloric intake of dietary-obese rats suggests that the hyperphagia is not NPY-driven and supports the data indicating reduced synaptic activity of the hypothalamic NPY system.     

CNS melanocortin and leptin effects on stearoyl-CoA desaturase-1 and resistin expression

The objective of this study was to determine whether centrally administered leptin decreased liver and adipose SCD1 expression or adipose resistin expression, and whether these effects were mediated by central melanocortin receptors. Male Sprague-Dawley rats were injected into the lateral cerebral ventricle (i.c.v.) once daily for 4 days with artificial cerebrospinal fluid (aCSF, 5 microl), leptin (10 microg) or MTII (0.1 nmol); two other groups were pretreated icv with the melanocortin antagonist, SHU9119 (1.0 nmol), followed by leptin or MTII. Epididymal and inguinal adipose tissue and liver were collected after rats were killed and mRNA expression of SCD1 and resistin was measured. Both leptin and MTII reduced SCD1 expression and pretreatment with SHU9119 reversed their effects. Neither leptin nor MTII affected resistin expression, but it was increased by SHU9119. These results show that CNS melanocortin receptors are likely mediators of leptin's effects on SCD1 expression in liver and adipose tissue, The findings were inconclusive concerning the effects of leptin and melanocortins on adipose resistin expression.     

Unchanged hypothalamic neuropeptide Y concentrations in hyperphagic, hypoglycemic rats: evidence for specific metabolic regulation of hypothalamic NPY

Hypothalamic concentrations of neuropeptide Y (NPY), a potent central appetite stimulant, increase dramatically in food-restricted and insulin-deficient diabetic rats. This suggest that NPY may drive hyperphagia in these conditions, which are characterized by weight loss and insulin deficiency. To test the hypothesis that insulin deficiency and weight loss are specific stimuli to hypothalamic NPY, we measured NPY concentrations in individual hypothalamic regions in rats with hyperphagia caused by insulin-induced hypoglycemia. Groups of 8 male Wistar rats were injected with ultralente insulin (20-60 U/kg) to induce either acute hypoglycemia (7 h after a single injection) or chronic hypoglycemia (8 days with daily injections). In hypoglycemic rats, plasma insulin concentrations were increased 6- to 7-fold compared with saline-injected controls; food intake was significantly increased with acute and chronic hypoglycemia and weight gain was significantly increased in the chronically hypoglycemic group. NPY concentrations were measured by radioimmunoassay in 8 hypothalamic regions microdissected from fresh brain slices. NPY concentrations were not increased in any region in either acute or chronic hypoglycemia. NPY therefore seems unlikely to mediate hyperphagia in hyperinsulinemia-induced hypoglycemia, supporting the hypothesis that weight loss is a specific stimulus to hypothalamic NPY and that insulin deficiency may be the metabolic signal responsible.     

Centrally administered MTII affects feeding, drinking, temperature, and activity in the Sprague-Dawley rat.

MTII, an agonist of melanocortinergic receptors, is a well-documented anorexigenic agent in rats. Many investigators have reported its effects on feeding without considering concurrent alterations in other behaviors. Accordingly, we performed studies to simultaneously measure nocturnal feeding, drinking, activity, and temperature of rats after intracerebroventricular (third ventricle) administration of a wide dose range of MTII (0.05-500 ng). We observed that MTII modulates these physiological parameters in a dose-dependent manner. Low doses of MTII (0.05 ng) caused reductions in feeding without alterations in body temperature, drinking, or activity. In contrast, hyperthermia and disrupted drinking patterns, along with food intake reductions, were evident at doses exceeding 50 ng. The fact that low doses altered only feeding, whereas higher doses affected a range of parameters, suggests that certain melanocortin-induced behavioral changes may be mediated by distinct populations of melanocortin receptors with varying affinities or that those changes seen at higher doses may be nonspecific in nature.     

Decreased orexigenic response to neuropeptide Y in rats with obstructive cholestasis

Neuropeptide Y (NPY) is a key factor in the neurochemical control of food intake, and obstructive cholestasis can be associated with disturbances in food intake. Our aim in this study was to determine whether obstructive cholestasis in the rat is associated with defective central responsiveness to NPY. Cholestasis was induced in rats by surgical bile duct resection. Rats with obstructive cholestasis exhibited a 20% reduction in food intake 2 days after laparotomy (compared with sham-resected controls) that had resolved by 4 days after surgery. Responsiveness to the orexigenic action of NPY was tested by measuring food intake after intracerebroventricular injection of NPY. In sham-resected rats, NPY infusion strikingly increased food intake, whereas bile duct-resected (BDR) rats showed a consistent significantly impaired feeding response to NPY at postlaparotomy days 2, 4, and 7. Separate experiments measured specific binding of [(3)H]NPY to hypothalamic receptors. Fos protein expression was measured in the hypothalamic paraventricular nucleus (PVN) as a marker of NPY-induced neuronal activation. The decreased orexigenic responsiveness to NPY was not caused by altered NPY binding at hypothalamic receptors or its ability to activate neurons in the PVN. Therefore, cholestatic rats demonstrate an attenuated NPY-induced orexigenic drive that occurs early after biliary obstruction, when cholestatic rats exhibit reduced food intake, and persists despite the return of food intake to normal levels and the presence of intact central NPY-related neuronal pathways.     

Effect of the melanocortin receptor stimulation or inhibition on ethanol intake in alcohol-preferring rats

It has been recently reported that acute intracerebroventricular injection of 1 nmol/rat of the non-selective melanocortin 3 and 4 receptor (MC3/4) agonist MTII reduces ethanol intake in female AA alcohol-preferring rats and alters opioid peptide levels in the ventral tegmental area of rats. To better understand the role of the MC system in the control of ethanol intake, we tested the acute and chronic effects of lateral ventricular (LV) injections of 0.01-1 nmol MTII, of 0.1-1 nmol of the MC3/4R receptor antagonist agouti related peptide (AgRP), and 0.1-0.5 nmol of the MC3/4R receptor antagonist SHU9119 on food, water, and 10% ethanol intake in Marchigian-Sardinian alcohol-preferring (msP) rats, which spontaneously ingest pharmacologically relevant quantities of ethanol both under short and long term access conditions. The data showed that with 2h/day ethanol access, LV MTII injections reduced intake of food and ethanol intakes. When food, water, and ethanol were available ad libitum and 0.01 nmol MTII was given by daily LV injection, however, ethanol intake was reduced for only the first 2 days, whereas food intake was reduced for all 5 days of treatment. Finally, acute LV injection of neither AgRP nor SHU9119 affected ethanol intake under ad libitum conditions, although both antagonists significantly increased food and water intake. In conclusion, these data fail to support a role for endogenous MC3/4R in the control of spontaneous ethanol intake in the msP rat. MC3/4R agonism, however, reduced ethanol intake in association with reduced food intake, suggesting that MTII might reduce nutrient-related controls of ethanol intake rather than, or in addition to, reward-related controls of ethanol intake.     

Amylin blunts hyperphagia and reduces weight and fat gain during recovery in socially stressed rats

During recovery from social stress in a visible burrow system (VBS), during which a dominance hierarchy is formed among the males, rats display hyperphagia and gain weight preferentially as visceral adipose tissue. By proportionally increasing visceral adiposity, social stress may contribute to the establishment of metabolic disorder. Amylin was administered to rats fed ad libitum during recovery from VBS stress in an attempt to prevent hyperphagia and the resultant gain in body weight and fat mass. Amylin treatment reduced food intake, weight gain, and accumulation of fat mass in male burrow rats, but not in male controls that spent time housed with a single female rather than in the VBS. Amylin did not alter neuropeptide Y (NPY), agouti-related peptide (AgRP), or proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus of the hypothalamus as measured at the end of the recovery period, nor did it affect plasma corticosterone or leptin. Amylin exerted most of its effect on food intake during the first few days of recovery, possibly through antagonism of NPY and/or increasing leptin sensitivity. The potential for chronic social stress to contribute to metabolic disorder is diminished by amylin treatment, though the neuroendocrine mechanisms behind this effect remain elusive.