Histamine Receptor and its Regulation of Energy Metabolism

In a series of studies on brain functions of histamine, probes to manipulate activities of histaminergic neuronal systems were applied to assess histaminergic function in non-obese normal, and lean and obese Zucker rats. Food intake was suppressed by both activation of H₁-receptors and inhibition of H₃-receptors in the ventromedial hypothalamic nucleus (VMH) and the paraventricular nucleus, each of which is a satiety center. Feeding circadian rhythm was decreased in its amplitude through histaminergic modulation in the hypothalamus. Histamine neurons in the mesencephalic trigeminal nucleus (Me5) were involved in regulation of masticatory functions, particularly eating speed, while histamine-containing neurons in the VMH controlled intake volume of meals. Energy deficiency in the brain enhanced satiation through histaminergic activation of VMH neurons, which in turn produced glycogenolysis in the hypothalamus to maintain homeostatic control of glucose supply. A very-low-calorie conventional Japanese diet, which is a fiber rich and low energy food source, enhanced satiation by increased mastication and because of the low energy supply of the diet. Hypothalamic histamine neurons were activated by high ambient temperature and also by interleukin-1β, an endogenous pyrogen, to maintain homeostatic thermoregulation. Behavioral and metabolic abnormalities of Zucker obese rats were mediated by a deficit in hypothalamic neuronal histamine, and the Zucker rat was evaluated as an animal model of histamine deficiency. Transplantation of the lean fetal hypothalamus into the third cerebroventricle of host obese Zuckers attenuated the abnormalities.     

Expressions of the prepro-orexin and orexin type 2 receptor genes in obese rat

We examined the expressions of the prepro-orexin gene in the lateral hypothalamic area (LHA), the genes of the neuropeptide Y (NPY) and proopiomelanocortin (POMC) in the arcuate nucleus (ARC), the orexin type 1 receptor (OX1R) gene in the ventromedial hypothalamic nucleus (VMH) and the orexin type 2 receptor (OX2R) gene in the paraventricular nucleus (PVN) in 6-, 12- and 18-week-old male lean (Fa/?) and obese (fa/fa) Zucker rats, using in situ hybridization histochemistry. The fa/fa rats showed hyperglycemia at 12- and 18-week-old. The prepro-orexin mRNA level in fa/fa rats at 18-week-old and the OX2R mRNA level in fa/fa rats at 12- and 18-week-old were significantly decreased compared to controls. The NPY mRNA levels in fa/fa rats at each time point were significantly increased compared to controls, but the POMC mRNA levels were decreased. Prepro-orexin and OX2R mRNA levels in fa/fa rats pretreated with insulin normalized to the levels found in Fa/? rats. These results suggest that the regulation of prepro-orexin gene expression might be independent of the regulation of the NPY and POMC genes in the ARC in fa/fa rats.     

下丘脑LHA-VMH Ghrelin信号通路对肥胖大鼠摄食选择、胃肠道运动及自发活动的影响及机制研究

目的:探究下丘脑外侧区(LHA)-腹内侧核(VMH)ghrelin信号通路对肥胖大鼠的摄食选择、胃肠道运动及自发活动的影响。方法:采用免疫组织化学方法检测大鼠LHA中ghrelin受体的表达;观察LHA注射ghrelin对大鼠摄食选择胃肠道运动及自发活动的影响;电损毁VMH,观察LHA注射ghrelin对大鼠摄食的影响。结果:免疫组化结果显示,大鼠下丘脑LHA中存在ghrelin受体,且LHA-VMH之间存在纤维投射;大鼠LHA微量注射ghrelin后,肥胖(DIO)大鼠及肥胖抵抗(DR)大鼠的正常饮食、高脂饮食及高糖饮食均高于正常大鼠,但预注射ghrelin受体拮抗剂[D-Lys3]-GHRP-6 (DLS)能够阻断这种作用;而电损毁大鼠VMH,显著减弱了ghrelin对正常大鼠、DIO大鼠及DR大鼠的促摄食作用。大鼠LHA微量注射ghrelin后,正常大鼠、DIO大鼠及DR大鼠的自发活动中,X轴、Y轴方向上的活动增加,且总活动增加,但Z轴方向上活动无明显改变;此外,LHA注射ghrelin,DIO大鼠及DR大鼠的胃肠道转运速率明显加快,且DR大鼠胃肠道转运速率增加更为明显,而预注射ghrelin受体拮抗剂[D-Lys3]-GHRP-6(DLS)显著阻断ghrelin的促胃肠道转运作用。结论:下丘脑LHA-VMH ghrelin信号通路参与调节正常大鼠、DIO及DR大鼠的摄食选择、胃肠道运动及自发活动。     

Direct neural effect of lateral hypothalamic stimulation on insulin secretion by pancreases of normal and obese rats

Perfusion of CNS intact pancreases with 200 mg/dl glucose with concomitant lateral hypothalamic area (LHA) stimulation significantly inhibited insulin secretion both in normal and obese rats. Sprague-Dawley, Zucker lean (FaFa) and Zucker obese (fafa) rats all responded in a similar manner, suggesting a general effect unrelated to metabolic state. Insulin secretion during mins 25-40 of perfusion was inhibited in Sprague Dawley, lean Zucker and obese Zucker rats by 31%, 42% and 33%, even though LHA stimulation took place from mins 20-25. Thus, the duration of inhibition was greater than the period of LHA stimulation, indicating that this pathway can induce prolonged changes in the responsiveness of the pancreas. The data presented in this study demonstrate that LHA stimulation, in the absence of humoral factors, results in a direct CNS-mediated suppression of insulin secretion which is relatively long lasting. This effect may illustrate a basic control mechanism by the CNS to regulate the endocrine pancreas.     

Intraventricular (i3vt) ghrelin increases food intake in fatty Zucker rats

Ghrelin is an orexigenic peptide secreted from the stomach and also made in the brain. Ghrelin receptors are expressed on hypothalamic cells important in appetite and energy balance. We determined that intra-3rd-ventricular (i3vt) ghrelin dose-dependently increases acute (1 and 2 h) food intake in lean and fatty Zucker rats (0, 0.01, 0.1 and 1.0 nmol ghrelin). The percentage increase of food intake in fatty Zucker rats was significantly greater than that in lean rats. Fatty Zucker rats had 4.5 times more ghrelin receptor mRNA in the hypothalamus than lean Zucker rats, suggesting a possible mechanism for the increased sensitivity.     

Lateral hypothalamic serotonergic responsiveness to food intake in rat obesity as measured by microdialysis.

The hypothalamic serotonergic system is involved in the regulation of food ingestion and energy metabolism. Since disturbances of both energy intake and expenditure can contribute to obesity, the objective of the present study was to evaluate the serotonergic response stimulated by food ingestion in two different models of obesity: the hyperphagic Zucker and the hypophagic and hypometabolic, monosodium glutamate (MSG) obese Wistar rat. For this we used microdialysis to examine the release of 5-hydroxytryptamine (serotonin, 5HT) and 5-hydroxyindoleacetic acid (5HIAA) in the lateral hypothalamus. Daily intake of MSG-obese rats was 40% lower while that of Zucker obese rats was 60% higher than that of the respective lean controls. In overnight-fasted animals, 20-min microdialysate samples were collected before (basal release) and during a 2-h period of access to a balanced palatable food mash. The animals began to eat during the first 20 min of food access, and food consumption was similar among the four groups in all six individual 20-min periods recorded. Ingestion of food increased 5HT release in all groups. In MSG-obese and lean Wistar rats, 5HT levels were similarly elevated during the whole experimental period. In the Zucker strain, 5HT increments of basal release tended to be higher in obese than in lean rats at 20 and 40 min, and a significantly higher increment was observed at 60 min after food access (40 and 135% for lean and obese, respectively). The area under the curve relating serotonin levels to the 120 min of food availability was significantly higher in Zucker obese (246.7 +/- 23.3) than MSG-obese (152.7 +/- 13.4), lean Wistar (151.9 +/- 11.1), and lean Zucker (173.5 +/- 24.0) rats. The present observation, of a food-induced serotonin release in the lateral hypothalamus of lean Wistar and Zucker rats, evidences that 5HT in the lateral hypothalamus is important in the normal response to feeding. In obese animals, the serotonin response was similar to (in the hypophagic-hypometabolic MSG model) or even higher than (in the hyperphagic Zucker model) that seen in the respective lean controls. This result indicates that the energy homeostasis disturbances of both these obesity models may not be ascribed to an impairment of the acute lateral hypothalamic serotonin response to a dietary stimulus.     

Alterations in the binding characteristics of glucocorticoid receptors from obese Zucker rats

Obese Zucker rats appear to lack a circadian rhythm of serum corticosterone and maintain relatively high concentrations throughout the 24-h day. The binding characteristics of glucocorticoid receptors in lean and obese Zucker rats were examined in three tissues suggested to be involved in the feedback inhibition of corticosterone: the anterior pituitary, hypothalamus and hippocampus. Hepatic glucocorticoid receptors were also examined to determine if receptor alterations exist in a peripheral tissue. The dissociation constant (Kd) of glucocorticoid receptors in the anterior pituitary of obese rats was 50% greater than the Kd of receptors derived from lean rats. This suggests a decrease in the affinity of these receptors and could indicate a reduced feedback inhibition of corticosterone at the anterior pituitary. Hepatic glucocorticoid receptors of obese rats also showed an increase (150%) in the Kd of binding and a reduction (40%) in the number of receptors. No difference was observed in the Kd or maximal binding of receptors from the hypothalamus or hippocampus of lean and obese rats. It appears that glucocorticoid receptor alterations exist in obese Zucker rats and that these alterations may affect the drive of the pituitary-adrenal axis and possibly the expression of obesity.     

Corticosterone-dependent metabolic and neuroendocrine abnormalities in obese Zucker rats in relation to feeding

The obese Zucker (fa/fa) rat is characterized by hyperphagia, hyperinsulinemia, an increase in fat deposition, and a hyperactivity in the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis in fa/fa rats is hypersensitive to stressful experimental conditions. Food deprivation even leads to a stress reaction in obese fa/fa rats. The present study was conducted to investigate the role of corticosterone in obese rats on the basal, fasting, and postprandial metabolic rate as well as on the central expression of the thyrotropin-releasing hormone (TRH) in these conditions. In addition, the study was aimed at clarifying whether the high levels of corticosterone in obese rats are responsible for the induction of the stress reaction to food deprivation in these animals. The present results demonstrate that whole body fat oxidation and postprandial metabolic responses in obese Zucker rats were improved by adrenalectomy (ADX). At the level of the central nervous system, ADX reversed a decrease in TRH mRNA expression in the paraventricular hypothalamus (PVH) detected in fasting animals. Considering all feeding conditions, the obese rats demonstrated lower TRH mRNA levels compared with lean animals. ADX resulted in an enhanced postprandial activation of the parvocellular PVH. In contrast, the magnocellular part of the PVH was less responsive to refeeding in ADX animals. Finally, ADX failed to prevent the stress response of obese rats to food deprivation. The present results provide evidence that the removal of adrenals resolve some of the metabolic defects encountered in obese Zucker rats. They also demonstrate that not all the abnormalities of the obese Zucker rats are attributable to the hyperactivity of the HPA axis.     

Starvation increases gastrointestinal somatostatin in normal and obese Zucker rats: a possible regulatory mechanism

Normal, male Sprague-Dawley (S-D) rats and female, lean and obese Zucker rats were studied in the fed state and after 48 hours of food deprivation. Somatostatin-like immunoreactivity (SLI) was measured from acetic acid extracts of oesophagus-cardia, stomach, small and large intestine, pancreas, hypothalamus, pituitary and cerebellum. Within the CNS, the highest levels of SLI were found in the hypothalamus, while in the gut, these levels were highest in the stomach and pancreas. All Zucker rats displayed higher hypothalamic levels of SLI than did S-D rats. Obese Zucker rats in the fed state differed from their lean littermates in that SLI levels were lower in oesophagus-cardia, stomach and hypothalamus, while being higher in pancreas and pituitary. The response to starvation in both obese and lean Zucker rats was qualitatively similar, and included significant increases in stomach and oesophagus-cardia SLI, but with a significant fall hypothalamic SLI. We have concluded that the increase in gastrointestinal SLI with starvation in Zucker as well as in S-D rats may represent a significant regulatory mechanism in nutrient homeostasis. We postulate that gastric SLI may decrease the availability of intestinal insulin secretagogues in the fasting state. This adaptive mechanism appears to be intact in the obese Zucker rat.     

Ghrelin and body weight regulation in the obese Zucker rat in relation to feeding state and dark/light cycle

Ghrelin is a new orexigenic peptide primarily produced by the stomach but also present in the hypothalamus. It has adipogenic effects when it is chronically injected in rodents but in obese humans, its plasma concentration is decreased. It can reverse the anorectic effects of leptin when it is co-injected with this peptide in the brain ventricles. The Zucker fa/fa rat is a genetic model of obesity related to a default in the leptin receptor. It is characterized by a large dysregulation of numerous hypothalamic peptides but the ghrelin status of this rat has not yet been determined. Through several experiments, we determine in lean and obese Zucker rats its circulating form in the plasma, its tissue levels and/or expression, and studied the influence of different feeding conditions and its light/dark variations. Ghrelin expression was higher in the obese stomach and hypothalamus (P < 0.05 and P < 0.02, respectively). The ratio of [Octanoyl-Ser3]-ghrelin (active form) to [Des-Octanoyl-Ser3]-ghrelin (inactive form) was approximately 1:1 in the stomach and 2:1 in the plasma in lean and obese rats (no differences). After fasting, plasma ghrelin concentrations increased significantly in lean (+ 64%; P < 0.001) and obese (+ 60%; P < 0.02) rats. After 24 hours of refeeding, they returned to their initial ad lib levels. Ghrelin concentrations were higher in obese rats by 69% (P < 0.005), 65% (P < 0.02), and 73% (P < 0.005) in the ad libitum, fast, and refed states respectively. These results indicate that the obese Zucker rat is characterized by increases in the stomach mRNA expression and in peptide release in the circulation. They clearly support a role for ghrelin in the development of obesity in the absence of leptin signaling.     

Brain insulin binding is decreased in Wistar Kyoto rats carrying the 'fa' gene

We have previously reported that insulin binding is decreased in the olfactory bulb of both heterozygous (Fa/fa) and obese (fa/fa) Zucker rats. In the present study, we measured insulin binding in membranes prepared from the olfactory bulb, cerebral cortex, and hypothalamus of control (Fa/Fa) Wistar Kyoto rats; "fatty" (fa/fa) Wistar Kyoto rats; and phenotypically lean (Fa/?) Wistar Kyoto rats. Insulin binding was decreased in all brain regions, as well as the liver of the obese Wistar Kyoto fa/fa rats. Additionally, insulin binding was decreased in the liver and brain membranes from the Fa/? Wistar Kyoto rats. As most of the Fa/? rats were probably carriers of one 'fa' gene, but the population was only slightly hyperinsulinemic, we conclude that--as in the Zucker rat--it is the presence and expression of the 'fa' gene rather than downregulation which results in the decreased insulin binding. Thus, regulation of the brain insulin receptor appears to be independent of plasma or cerebrospinal fluid insulin levels.     

Single intracerebroventricular bolus injection of a recombinant adenovirus expressing leptin results in reduction of food intake and body weight in both lean and obese Zucker fa/fa rats

Leptin acts as a satiety factor within the central nervous system by binding to its receptor located in the hypothalamus. A missense mutation of the leptin receptor induces hyperphagia and obesity in the obese Zucker fa/fa rat. Since the CNS is an important target of leptin action, we hypothesized that leptin gene transfer into the lateral cerebral ventricle could efficiently lead to inhibition of food intake and reduction of body weight in obese fa/fa rats as well as in lean animals. A single intracerebroventricular injection of an adenoviral vector containing a cDNA encoding leptin resulted in the expression of leptin in the ependymal cells lining the ventricle and the secretion of leptin into the cerebrospinal fluid (CSF). During the first week after injection, when high concentrations of leptin were produced in the CSF, the reducing effects of leptin on food intake and body weight were comparable in lean and in obese fa/fa rats. The subsequent decline in CSF leptin levels, that was similar in lean and obese fa/fa rats, resulted in the faster resumption of food intake and body weight gain in obese than in lean animals, confirming a reduced sensitivity to leptin in the obese group. The results of this study show that leptin gene delivery into the cerebral ventricles allows for the production of elevated leptin concentrations in CSF, and they support the hypothesis that the impaired sensitivity to leptin may be overcome in obese fa/fa rats.     

Decreased epoxygenase and increased epoxide hydrolase expression in the mesenteric artery of obese Zucker rats

Previous studies suggest that epoxyeicosatrienoic acids (EETs) are vasodilators of the mesenteric artery; however, the production and regulation of EETs in the mesenteric artery remain unclear. The present study was designed 1) to determine which epoxygenase isoform may contribute to formation of EETs in mesenteric arteries and 2) to determine the regulation of mesenteric artery cytochrome P-450 (CYP) enzymes in obese Zucker rats. Microvessels were incubated with arachidonic acid, and CYP enzyme activity was determined. Mesenteric arteries demonstrate detectable epoxygenase and hydroxylase activities. Next, protein and mRNA expressions were determined in microvessels. Although renal microvessels express CYP2C23 mRNA and protein, mesenteric arteries lacked CYP2C23 expression. CYP2C11 and CYP2J mRNA and protein were expressed in mesenteric arteries and renal microvessels. In addition, mesenteric artery protein expression was evaluated in lean and obese Zucker rats. Compared with lean Zucker rats, mesenteric arterial CYP2C11 and CYP2J proteins were decreased by 38 and 43%, respectively, in obese Zucker rats. In contrast, soluble epoxide hydrolase mRNA and protein expressions were significantly increased in obese Zucker rat mesenteric arteries. In addition, nitric oxide-independent dilation evoked by acetylcholine was significantly attenuated in mesenteric arteries of obese Zucker rats. These data suggest that the main epoxygenase isoforms expressed in mesenteric arteries are different from those expressed in renal microvessels and that decreased epoxygenases and increased soluble epoxide hydrolase are associated with impaired mesenteric artery dilator function in obese Zucker rats.     

Insulin and glucose transporter gene expression in obesity and diabetes.

In order to determine the role of insulin and glucose transporter gene expression in the development of diabetes in obesity, we examined insulin and GLUT2-liver type and GLUT4-muscle-fat type glucose transporter mRNA levels in obese and diabetic rats. Ventromedial hypothalamus-lesioned (VMH), Zucker fatty (ZF), and Wistar fatty (WF) rats were used as models. VMH and ZF rats are most frequently used as models for simple obesity. In contrast, WF rats, which have been established by transferring the fa gene of ZF rats to Wistar Kyoto rats, develop both obesity and diabetes. Pancreatic insulin content of VMH rats at 10 weeks after the operation and of ZF rats at 5 and 14 weeks of age was significantly higher than that of controls. On the other hand, insulin content of WF rats at 5 and 14 weeks of age was not significantly different from that of lean littermates. The insulin mRNA levels of VMH rats were increased progressively and were significantly higher than those in sham-operated animals at 4 and 10 weeks after the operation. In ZF rats, the insulin mRNA levels at 5 and 14 weeks of age were significantly higher than those of their lean littermates. In WF rats, by contrast, the insulin mRNA levels were similar to those of lean littermates at 5 and 14 weeks of age. The insulin mRNA levels of WF rats were about 40% of that of ZF rats at 14 weeks of age. On the other hand, at 14 weeks of age, the GLUT2 mRNA levels of liver were significantly higher in ZF and WF rats than those in their respective littermates, but not at 5 weeks of age. The GLUT4 mRNA levels of skeletal muscle in both ZF and WF rats were not significantly different from those of controls. It is suggested that the inability of WF rats to augment insulin gene expression in response to a large demand for insulin is associated with the occurrence of diabetes, and that the activation of GLUT2 mRNA without the activation of GLUT4 mRNA is common to obesity with and without diabetes.     

Opposite regulation of hypothalamic orexin and neuropeptide Y receptors and peptide expressions in obese Zucker rats

Many hyothalamic neuropeptides are involved in the regulation of food intake and body weight. The orexins (OX) which are synthesized in the lateral hypothalamus are among the most recently characterized whereas neuropeptide Y (NPY) belongs to a group of "older" peptides extensively studied for their effects on feeding behavior. Both stimulate food ingestion in rodents. In this experiment, we measured the expressions of these peptides as well as of their receptors (OX1-R and OX2-R, Y1 and Y5) in the hypothalamus of obese hyperphagic and lean Zucker rats by real-time RT-PCR using the TaqMan apparatus. NPY mRNA expression in the obese rats was significantly increased by a factor of 10 (P < 0.002) whereas expressions of the Y1 and Y5 receptors were decreased by 25% (P < 0.01) and 50% (P < 0.002), respectively. Their prepro-orexin mRNA expression was more than twofold decreased (P < 0.01) and expressions of their OX receptors 1 and 2 mRNA were five- and fourfold increased (P < 0.05), respectively. An inverse phenomenon was therefore noted between the two peptides: for NPY, increased levels and downregulation of receptors; and for OX, diminished levels with upregulation of receptors. The reasons for these changes might be linked to the absence of leptin signaling as similar profiles are found in the ob/ob mice. For orexins at least, other factors such as hyperglycemia might be involved. Based on anatomical considerations, a direct effect of NPY or of other brain peptides such as CRH cannot be excluded. We conclude that the diminution in the OX tone might participate in a counterregulatory system necessary to limit the noxious effects of NPY on food intake and body weight.     

Effects of intracerebroventricular leptin administration on feeding and sexual behaviors in lean and obese female Zucker rats

Obese Zucker rats (fa/fa) are characterized by inadequate leptin signaling caused by a mutation in the leptin receptor gene. Obese Zucker females are infertile and hyporesponsive to the inductive effects of ovarian hormones on sexual behaviors. Leptin treatment reverses aspects of reproductive dysfunction due to perturbations in energy balance in other animal models. Our first experiment tested the hypothesis that intracerebroventricular (icv) leptin administration would enhance the display of sexual behaviors in obese Zucker females. A second experiment compared lean and obese Zucker females' responses to leptin, during fed and fasted conditions. Ovariectomized (OVX) Zucker rats were implanted with lateral ventricular cannulae. In Experiment 1, fasted, obese females received estradiol benzoate, progesterone, and icv injections of 3, 18, or 36 microg murine leptin or vehicle. Leptin administration reduced food intake, but did not enhance sexual behaviors. In Experiment 2, steroid-replaced, OVX lean and obese females (from a different source than those in Experiment 1) received icv injections of vehicle or 3 or 36 microg leptin under fed and fasted conditions. Leptin treatment reduced food intake and weight gain in the fed, but not the fasted, condition in both genotypes. Sexual receptivity and locomotion were not affected, but icv leptin injections reduced proceptive behaviors in ad libitum-fed rats. These data confirm previous reports that centrally administered leptin decreases food intake and weight gain in obese Zucker rats; results from Experiment 2 suggest that lean and obese females are similarly responsive to these actions of leptin. Contrary to our hypothesis, leptin treatment did not stimulate sexual behaviors; rather, the hormone appears to inhibit the display of sexual proceptivity in ad libitum-fed lean and obese Zucker female rats.