Down-regulated expression of agouti-related protein (AGRP) mRNA in the hypothalamic arcuate nucleus of hyperphagic and obese tub/tub mice

A mutation in the mouse tub gene causes a phenotype characterized by maturity-onset obesity, blindness and deafness. The role of the intact tubby protein and the pathogenesis resulting in the phenotype of tub/tub mice remain largely unknown. In this study, we have investigated whether obese tub/tub mice exhibit altered expression levels for agouti-related protein (AGRP) or glutamic acid decarboxylase-65 (GAD65) in body weight-regulating neurons of the hypothalamic arcuate nucleus. In situ hybridization revealed that AGRP, but not GAD65 mRNA levels, were significantly lower in obese tub/tub mice as compared to tub/+ mice. The lower levels of AGRP mRNA in the arcuate nucleus of tub/tub mice were paralleled by lower fluorescence intensity and numbers of AGRP- and neuropeptide Y (NPY)-immunoreactive (ir) nerve fibers and terminals in the arcuate, ventromedial, dorsomedial hypothalamic nuclei and perifornical and lateral hypothalamic areas. No obvious differences in GAD65-ir nerve fibers and terminals could be detected. Measurements of daily food intake revealed that tub/tub mice displayed progressively higher food consumption as compared to lean tub/+ littermates over a 15-day observation period. When moved to an unfamiliar environment, e.g. a novel cage, daily food intake was initially lower in tub/tub mice than in tub/+ mice suggesting that tub/tub mice may be more sensitive to psychogenic stress. The results together show that tub/tub mice are hyperphagic and exhibit, within the hypothalamic arcuate nucleus, a depressed expression of neuropeptides involved in the regulation of feeding behavior.     

Decreased 5-HT transporter mRNA in neurons of the dorsal raphe nucleus and behavioral depression in the obese leptin-deficient ob/ob mouse

The neurotransmitter serotonin (5-hydroxytryptamine; 5-HT) is an important regulator of feeding behavior. A hypothalamic site of action for 5-HT in body weight control is supported by the presence of 5-HT receptors in hypothalamic regions which are intimately associated with regulation of food intake. In the present study we have investigated whether there may be an interaction between the hormone leptin, an adipose tissue-derived cytokine signaling factor that inhibits food intake and lowers body weight, and the brain serotonergic system. Immunohistochemical analysis of colchicine-treated rats showed colocalization of 5-HT transporter- and leptin receptor-immunoreactivity in cell bodies of the dorsal raphe nucleus, suggesting that dorsal raphe neurons are targets for circulating leptin. Levels of 5-HT transporter mRNA expression were compared in neurons of the dorsal raphe nucleus of obese leptin-deficient ob/ob mice and their lean littermates using in situ hybridization. 5-HT transporter mRNA levels were significantly down-regulated in neurons of the dorsal raphe nucleus of obese ob/ob mice as compared to lean control mice. Behavioral analysis showed that obese ob/ob mice had significantly lower locomotor activity and exhibited increased immobility in Porsolt's test, a model for depression. Taken together, these results suggest that serotonergic cell bodies in the rodent dorsal raphe nucleus possess leptin receptors and that the serotonergic system, as reflected by expression levels of 5-HT transporter mRNA, is down-regulated in the obese behaviorally depressed ob/ob mouse.     

Hypothalamic neurohistochemistry of the murine anorexia (anx/anx) mutation: Altered processing of neuropeptide Y in the arcuate nucleus

Neuropeptide Y is one of the most powerful neurochemical stimulants of food intake known. The neuronal substrate for this action is believed to be the neuropeptide Y-expressing cell population in the hypothalamic arcuate nucleus. In this study, mice homozygous for the anorexia mutation (anx) were investigated histochemically; anx is a recessive mutation that causes decreased food intake and starvation, leading to death 22 days after birth. We were interested to see whether any hypothalamic neurochemical abnormalities could be detected in this genetic model of starvation. By using immunohistochemistry and in situ hybridization, the hypothalamic distributions of neuropeptide Y, cholecystokinin, galanin, and serotonin, all messenger molecules postulated to be involved in the regulation of food intake and energy metabolism, were investigated. Immunoreactivities for somatostatin, the excitatory amino acid aspartate, and acetylcholinesterase were also studied. Neuropeptide Y-like immunoreactivity was increased markedly in arcuate cell bodies and decreased in terminals in the arcuate nucleus and other hypothalamic regions of anx/anx mice compared with normal litter mates. In situ hybridization for neuropeptide Y mRNA, however, showed no significant difference in gene expression in the arcuate nucleus. In addition, immunoreactivities for aspartate, acetylcholinesterase, and somatostatin in the arcuate nucleus were decreased in anx/anx mice. For cholecystokinin, galanin, and serotonin, no certain differences in hypothalamic immunoreactivity could be seen. These data suggest that a defect in neuropeptide Y-ergic signalling in the arcuate neurons may contribute to the failure to thrive in anx/anx mice. J. Comp. Neurol. 387:124–135, 1997. © 1997 Wiley-Liss, Inc.     

Effect of Sustained Physiological Hyperinsulinaemia on Hypothalamic Neuropeptide Y and NPY mRNA Levels in the Rat

Neuropeptide Y (NPY) synthesized in the arcuato-paraventricular projection in the rat hypothalamus is thought to play an important role in controlling energy homeostasis. The factors that regulate hypothalamic NPY are not known but, amongst others, insulin has been postulated as an inhibitory modulatory agent. To test this hypothesis, normal male rats were given either insulin (2 units/day) or saline via subcutaneous osmotic minipumps for 3 days. Euglycaemia was maintained by a concomitant glucose infusion in insulin-infused rats which had peripheral insulin levels 5–8 times higher than saline-infused controls. Hyperinsulinaernic rats ate 42% less than controls, but their total energy intake (food intake plus glucose infusion) was higher than that of controls, and they gained more weight than controls during the experimental period. Hyperinsulinaemia had no significant effect on hypothalamic NPY mRNA or NPY levels in the arcuate nucleus. NPY concentrations in the paraventricular nucleus were, however, significantly increased by 73% in hyperinsulinaemic rats, but were closely similar to controls in all other areas. Insulin may act as a satiety factor in that hyperinsulinaemic rats ate less, but the fact that these animals had increased total energy intake and gained excessive weight suggests that insulin may not function as an overall regulator of energy balance. In addition, physiological hyperinsulinaemia does not apparently inhibit NPY gene expression in the arcuate nucleus. Due to the lack of effect of hyperinsulinaemia on NPY synthesis in the arcuate nucleus, the elevated NPY concentrations in the paraventricular nucleus could result from a reduction of its release, which would be in keeping with the reduction in food intake.     

Anorectic Effect of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) in Rats: Lack of Evidence for Involvement of Hypothalamic Neuropeptide Gene Expression

We investigated the effect of centrally administered pituitary adenylate cyclase activating polypeptide (PACAP) on feeding in rats, and the involvement of hypothalamic neuropeptide gene expression using in situ hybridization. lntracerebroventricular injection of PACAP (1000  pmol/rat) significantly decreased food intake in a dose-dependent manner. In PACAP-treated rats, neuropeptide Y (NPY) mRNA levels in the arcuate nucleus and galanin mRNA levels in the paraventricular nucleus increased, and corticotropin-releasing hormone (CRH) mRNA levels in the paraventricular nucleus decreased. In rats fasted for 72  h, NPY mRNA levels increased, and CRH mRNA levels decreased, but galanin mRNA levels were unchanged. These results indicate that the anorectic function of PACAP is not mediated by NPY or CRH, and that PACAP increases galanin synthesis.     

A potential role for the secretogranin II‐derived peptide EM66 in the hypothalamic regulation of feeding behaviour

EM66 is a conserved 66‐amino acid peptide derived from secretogranin II (SgII), a member of the granin protein family. EM66 is widely distributed in secretory granules of endocrine and neuroendocrine cells, as well as in hypothalamic neurones. Although EM66 is abundant in the hypothalamus, its physiological function remains to be determined. The present study aimed to investigate a possible involvement of EM66 in the hypothalamic regulation of feeding behaviour. We show that i.c.v. administration of EM66 induces a drastic dose‐dependent inhibition of food intake in mice deprived of food for 18 hours, which is associated with an increase of hypothalamic pro‐opiomelanocortin (POMC) and melanocortin‐3 receptor mRNA levels and c‐Fos immunoreactivity in the POMC neurones of the arcuate nucleus. By contrast, i.c.v. injection of EM66 does not alter the hypothalamic expression of neuropeptide Y (NPY), or that of its Y1 and Y5 receptors. A 3‐month high‐fat diet (HFD) leads to an important decrease of POMC and SgII mRNA levels in the hypothalamus, whereas NPY gene expression is not affected. Finally, we show that a 48 hours of fasting in HFD mice decreases the expression of POMC and SgII mRNA, which is not observed in mice fed a standard chow. Taken together, the present findings support the view that EM66 is a novel anorexigenic neuropeptide regulating hypothalamic feeding behaviour, at least in part, by activating the POMC neurones of the arcuate nucleus.     

Ventromedial hypothalamic lesions produced by gold thioglucose do not impair induction of NPY mRNA in the arcuate nucleus by fasting

There is increasing evidence that neuropeptide Y (NPY) plays an important role in the regulation of food intake. Neuropeptide Y mRNA in the arcuate nucleus increases after fasting and it has been proposed that this increase in NPY activity occurs as a result of the decreased circulating levels of both insulin and glucose associated with a fast. Glucose-responsive neurons in the ventromedial nucleus (VMN) of the hypothalamus alter their activity in response to changes in circulating glucose levels and these neurons have been proposed to be involved in the regulation of feeding behavior and metabolism. However, it is not known if these glucose-responsive neurons are involved in the response of NPY mRNA in the arcuate nucleus to fasting. To address this relationship, mice were injected with either saline or gold thioglucose (GTG), which appears to act on glucose-responsive neurons, and killed 6 weeks later after a 72 h fast or under ad lib fed conditions. In situ hybridization histochemistry for NPY mRNA was performed on hypothalamic sections containing the arcuate nucleus. The number of labelled cells was counted and the density of autoradiographic silver grains overlying the cells was also quantified (i.e. pixels per cell). Fasting resulted in increased levels of total NPY mRNA (number of labelled cells multiplied by the pixels per cell) in the arcuate nucleus of both control and GTG-treated mice. In addition, the relative fasting-induced increase (i.e. the fasted to fed ratio) in number of cells detected, number of pixels per cell, and total NPY mRNA was similar in both the control and GTG-treated mice. These data suggest that GTG-sensitive VMN neurons play little role in the induction of NPY mRNA by fasting in the arcuate nucleus.     

Effects of repeated administration of mifepristone and 8-OH-DPAT on expression of preproneuropeptide Y mRNA in the arcuate nucleus of obese Zucker rats.

Neuropeptide Y (NPY) is an important hypothalamic regulator of feeding behavior. In this study we have investigated the regulation of the expression of preproNPY mRNA in male obese and lean Zucker rats by in situ hybridization. These animals represent a model of genetic obesity with hyperphagia, hyperinsulinemia and altered endocrine functions. Obese Zucker rats, treated for 12 days with 0.9% saline, had about 210% higher level of basal preproNPY mRNA expression in the arcuate nucleus when compared to their lean littermate controls. Repeated administrations of 8-hydroxy-dipropylaminotetralin (8-OH-DPAT), a serotonergic 5-HT1A agonist, or mifepristone, a glucocorticoid receptor antagonist, did not modify the basal expression of preproNPY mRNA in the Zucker phenotypes. The 8-OH-DPAT treatment significantly reduced hyperinsulinemia in obese Zucker rats without changing plasma glucose levels. The mifepristone treatment significantly increased plasma corticosterone levels in lean animals, but not in obese animals. The present study demonstrates enhanced expression of preproNPY mRNA in the arcuate nucleus in obese Zucker rats suggesting an involvement of NPY in the pathophysiology of the hyperphagic syndrome and genetically determined obesity in Zucker rats. Neither the antagonism of glucocorticoid receptors by mifepristone, nor repeated treatment with 8-OH-DPAT resulting in reduced insulin levels in obese Zucker rats, modified the basal expression of preproNPY mRNA in the arcuate nucleus.     

Oestrogenic regulation of pro-opiomelanocortin, neuropeptide Y and corticotrophin-releasing hormone mRNAs in mouse hypothalamus

It is well documented that oestrogen suppresses food intake by an action at the hypothalamic level. Using in situ hybridisation, we studied the effect of castration (CX) and short-term administration of oestradiol (E2) in CX female mice for three neuropeptides involved in feeding behaviour: two anorexigenic peptides, (i) the pro-opiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone and (ii) corticotrophin-releasing hormone (CRH), and the orexigenic peptide, (iii) neuropeptide Y (NPY). POMC-expressing neurones were mostly laterally located in the arcuate nucleus. POMC mRNA expression was decreased following CX and a single injection of E2 induced an increase in mRNA levels at 12- and 24-h time intervals. In the parvocellular area of the paraventricular nucleus, CRH mRNA levels were similarly decreased after CX and completely restored to normal levels at 12 and 24 h following E2 injection. On the other hand, the levels of NPY mRNA expressed in neurones located in the inner zone of the arcuate nucleus were increased by CX and decreased to the levels observed in intact animals by E2 injection (3-24 h). The present data suggest that oestrogen might exert an anorexigenic action by stimulating POMC and CRH mRNA expression and decreasing NPY mRNA expression in the hypothalamus.     

Decreased NPY innervation of the hypothalamic nuclei in rats with cancer anorexia

Whether the decrease in food intake that occurs at the onset of anorexia in tumor bearing (TB) rats is related to a change in the hypothalamic neuropeptide Y (NPY) system was tested by comparing NPY expression in sham operated Fischer Control and anorectic TB rats. Coronal cryocut sections of their fixed brain were processed by the peroxidase-antiperoxidase method with NPY polyclonal antibodies. NPY-immunoreactive fibers were widely distributed throughout the forebrain, but were most prominent in the hypothalamic paraventricular, suprachiasmatic, arcuate and periventricular nuclei. NPY-immunoreactive neurons were visualized in Control and anorectic TB rats in the preoptic region, the arcuate nucleus, and occasionally in the lateral hypothalamus. Semiquantitative image analysis showed a significant decrease in the NPY immunostaining in some hypothalamic nuclei of the anorectic TB rats, most prominently in the supraoptic nucleus, the parvocellular portion of the paraventricular nucleus, and, to a lesser extent, the suprachiasmatic and arcuate nuclei. No changes in NPY innervation were seen in the ventromedial nucleus and the lateral hypothalamus. The data support the hypothesis of an altered hypothalamic NPY system at the onset of anorexia in TB rats and also reveal the hypothalamic nuclei through which NPY influences food intake.     

Elevated hypothalamic neuropeptide Y levels in rats with dorsomedial hindbrain lesions

Lesions centered on the area postrema (AP) and adjacent nucleus of the solitary tract (AP/mNTS-lesions) are reported to result in increased consumption of highly palatable diets. Recent studies suggest that neuropeptide Y (NPY) may cause a preference for carbohydrate-rich diets. Thus, it is possible that NPY may play a role in the enhanced intake of highly palatable diets by AP/mNTS-lesioned rats. In the studies reported here, we found that lesions centered on the AP result in increased levels of NPY-immunoreactivity in the paraventricular nucleus of the hypothalamus. Additionally, steady-state NPY mRNA in the basomedial hypothalamus including the arcuate nucleus was elevated. Enhanced NPY was not found throughout the hypothalamus however, as NPY-immunoreactivity was not elevated in the lateral hypothalamus or the tissue bordering the anteroventral third ventricle. These data suggest the possibility that elevated hypothalamic NPY, particularly in the arcuate and paraventricular nuclei, may contribute to the altered food intake and energy balance observed in rats with lesions centered on the AP.     

Differential regulation of neuropeptide Y mRNA expression in the arcuate nucleus and locus coeruleus by stress and antidepressants

In rats, circulating corticosterone and insulin are involved in regulation of the hypothalamic neuropeptide Y (NPY) system, which in turn, is involved in regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Since the HPA axis and stress responsivity is altered in diseases such as depression, we investigated interactions between the effects of stress and antidepressant drug treatment on arcuate nucleus and locus coeruleus NPY mRNA expressions using in-situ hybridization histochemistry. After acute (2 h) and repeated immobilization (2 h daily, for 14 days), plasma concentrations of corticosterone increased, and those of insulin decreased. The expression of NPY mRNA was significantly increased in the arcuate nucleus, but was unchanged in the locus coeruleus following acute and repeated immobilization. Adrenalectomized rats with systemic corticosterone replacement (ADX+CORT), whose corticosterone concentration was maintained at approximately 50-100 ng/ml during repeated stress, showed a decrease in plasma insulin and an increase in arcuate nucleus NPY mRNA similar to that observed in sham rats, suggesting that changes in NPY mRNA levels are more closely tied to circulating insulin than to circulating corticosterone. In contrast, locus coeruleus NPY mRNA expressions in ADX+CORT rats were significantly higher than those in sham rats after repeated stress. Desmethylimipramine (DMI) treatment for 24 days did not affect basal plasma concentrations of corticosterone or insulin, or arcuate nucleus NPY mRNA expressions, but significantly decreased basal levels of locus coeruleus NPY mRNA compared to saline-treated rats. After repeated immobilization (2 h daily, for 4 days), DMI significantly reduced the stress-induced rise in locus coeruleus NPY mRNA levels, but potentiated the stress-induced rise in arcuate nucleus NPY mRNA expression. These results demonstrate that: (1) the increase in arcuate nucleus NPY mRNA expressions in stressed rats closely follows the decrease in plasma concentrations of insulin; (2) increases in NPY mRNA expressions occur in the absence of changes in plasma corticosterone; and (3) desipramine treatment potentiated the effect of stress on arcuate nucleus NPY mRNA expressions, but blocked the repeated stress-induced increase in locus coeruleus NPY mRNA expressions. Thus, NPY mRNA expression in the arcuate nucleus and the locus coeruleus is sensitive to the effects of stress and to the antidepressant drug desipramine, but the arcuate nucleus NPY system is regulated by different mechanisms than the locus coeruleus NPY system. The results provide further evidence for the importance of circulating insulin in the regulation of the arcuate nucleus NPY system.     

Neuropeptide Y facilitates activity-based-anorexia

The hypothesis that treatment with neuropeptide Y (NPY) can increase running activity and decrease food intake and body weight was tested. Female rats with a running wheel lost more weight than sedentary rats and ran progressively more as the availability of food was gradually reduced. When food was available for only 1h/day, the rats lost control over body weight. Correlatively, the level of NPY mRNA was increased in the hypothalamic arcuate nucleus. This phenomenon, activity-based-anorexia, was enhanced by intracerebroventricular infusion of NPY in rats which had food available during 2h/day. By contrast, NPY stimulated food intake but not wheel running in rats which had food available continuously. These findings are inconsistent with the prevailing theory of the role of the hypothalamus in the regulation of body weight according to which food intake is a homeostatic process controlled by "orexigenic" and "anorexigenic" neural networks. However, the finding that treatment with NPY, generally considered an "orexigen", can increase physical activity and decrease food intake and cause a loss of body weight is in line with the clinical observation that patients with anorexia nervosa are physically hyperactive and eat only little food despite having depleted body fat and up-regulated hypothalamic "orexigenic" peptides.     

Rapid and localized alterations of neuropeptide Y in discrete hypothalamic nuclei with feeding status

Neuropeptide Y (NPY) is believed to regulate the normal eating behavior and body weight in rats via central mechanisms. We have investigated whether NPY, which stimulates food intake, may in turn be modified by the nutritional state of the animals. Thus the impact of food deprivation (FD) (48 h) and subsequent refeeding on the levels of NPY in discrete hypothalamic areas was examined in this study. The results showed site specific change in only 3 of 7 hypothalamic sites. A 5-fold increment in NPY was reported in the paraventricular nucleus (PVN) and a 10-fold increase was observed in the arcuate nucleus-median eminence (ARC-ME). While subsequent refeeding for 6 h reversed the effect of FD in the ARC-ME, the levels of NPY in the PVN remained high in the refed rats. The perifornical lateral hypothalamus displayed a different pattern, namely, a significant increase in NPY content in refed as compared to satiated and deprived rats. The NPY levels in 4 other hypothalamic sites, namely, the dorsomedian, ventromedian, supraoptic and suprachiasmatic nuclei, and two extrahypothalamic sites, namely caudate nucleus and nucleus accumbens, showed total resistance to any change following deprivation and refeeding. These data emphasize the important and specific role of the paraventricular and arcuate nuclei in NPY's regulation of food intake and provide support for the idea that the variations of hypothalamic NPY after food deprivation reflect a specific physiological response of feeding regulatory system to alterations in the animal nutritional state and body weight.     

Effect of peripheral 2-DG on opioid and neuropeptide Y gene expression

It is well known that 2-Deoxy-d-glucose (2-DG) blocks intracellular utilization of glucose and increases food intake. The aim of the present study was to determine whether administration of 2-DG alters gene expression of the orexigenic peptides, neuropeptide Y (NPY) and endogenous opioids, in the arcuate nucleus of the hypothalamus (ARC). Male Sprague–Dawley rats were injected peripherally (i.p.) with 2-DG (200 or 400 mg/kg body weight) and were sacrificed at 2 or 6 h post injection. Half of the animals were given ad libitum access to food whereas the other half of the animals were food-deprived. 2-DG increased food intake fourfold compared to saline injected animals, but did not affect NPY mRNA levels after 2 h. Messenger RNA levels of ProDynorphin (proDYN), but not pro-opiomelanocortin (POMC) nor proEnkephalin (proENK) were significantly decreased 2 h after 2-DG injection. Administration of 400 mg/kg of 2-DG increased mRNA levels of NPY in the arcuate nucleus after six h, but only in those animals not receiving food.