Prenatal fat exposure and hypothalamic PPAR ß/δ: Possible relationship to increased neurogenesis of orexigenic peptide neurons.

Gestational exposure to a fat-rich diet, while elevating maternal circulating fatty acids, increases in the offspring's hypothalamus and amygdala the proliferation and density of neurons that express neuropeptides known to stimulate consummatory behavior. To understand the relationship between these phenomena, this study examined in the brain of postnatal offspring (day 15) the effect of prenatal fat exposure on the transcription factor, peroxisome proliferator-activated receptor (PPAR) ß/δ, which is sensitive to fatty acids, and the relationship of PPAR ß/δ to the orexigenic neuropeptides, orexin, melanin-concentrating hormone, and enkephalin. Prenatal exposure to a fat-rich diet compared to low-fat chow increased the density of cells immunoreactive for PPAR ß/δ in the hypothalamic paraventricular nucleus (PVN), perifornical lateral hypothalamus (PFLH), and central nucleus of the amygdala (CeA), but not the hypothalamic arcuate nucleus or basolateral amygdaloid nucleus. It also increased co-labeling of PPAR ß/δ with the cell proliferation marker, BrdU, or neuronal marker, NeuN, and the triple labeling of PPAR ß/δ with BrdU plus NeuN, indicating an increase in proliferation and density of new PPAR ß/δ neurons. Prenatal fat exposure stimulated the double-labeling of PPAR ß/δ with orexin or melanin-concentrating hormone in the PFLH and enkephalin in the PVN and CeA and also triple-labeling of PPAR ß/δ with BrdU and these neuropeptides, indicating that dietary fat increases the genesis of PPAR ß/δ neurons that produce these peptides. These findings demonstrate a close anatomical relationship between PPAR ß/δ and the increased proliferation and density of peptide-expressing neurons in the hypothalamus and amygdala of fat-exposed offspring.

Effects of embryonic ethanol exposure at low doses on neuronal development, voluntary ethanol consumption and related behaviors in larval and adult zebrafish: Role of hypothalamic orexigenic peptides.

Embryonic exposure to ethanol is known to affect neurochemical systems in rodents and increase alcohol drinking and related behaviors in humans and rodents. With zebrafish emerging as a powerful tool for uncovering neural mechanisms of numerous diseases and exhibiting similarities to rodents, the present report building on our rat studies examined in zebrafish the effects of embryonic ethanol exposure on hypothalamic neurogenesis, expression of orexigenic neuropeptides, and voluntary ethanol consumption and locomotor behaviors in larval and adult zebrafish, and also effects of central neuropeptide injections on these behaviors affected by ethanol. At 24h post-fertilization, zebrafish embryos were exposed for 2h to ethanol, at low concentrations of 0.25% and 0.5%, in the tank water. Embryonic ethanol compared to control dose-dependently increased hypothalamic neurogenesis and the proliferation and expression of the orexigenic peptides, galanin (GAL) and orexin (OX), in the anterior hypothalamus. These changes in hypothalamic peptide neurons were accompanied by an increase in voluntary consumption of 10% ethanol-gelatin and in novelty-induced locomotor and exploratory behavior in adult zebrafish and locomotor activity in larvae. After intracerebroventricular injection, these peptides compared to vehicle had specific effects on these behaviors altered by ethanol, with GAL stimulating consumption of 10% ethanol-gelatin more than plain gelatin food and OX stimulating novelty-induced locomotor behavior while increasing intake of food and ethanol equally. These results, similar to those obtained in rats, suggest that the ethanol-induced increase in genesis and expression of these hypothalamic peptide neurons contribute to the behavioral changes induced by embryonic exposure to ethanol.

Prenatal exposure to ethanol stimulates hypothalamic CCR2 chemokine receptor system: Possible relation to increased density of orexigenic peptide neurons and ethanol drinking in adolescent offspring.

Clinical and animal studies indicate that maternal consumption of ethanol during pregnancy increases alcohol drinking in the offspring. Possible underlying mechanisms may involve orexigenic peptides, which are stimulated by prenatal ethanol exposure and themselves promote drinking. Building on evidence that ethanol stimulates neuroimmune factors such as the chemokine CCL2 that in adult rats is shown to colocalize with the orexigenic peptide, melanin-concentrating hormone (MCH) in the lateral hypothalamus (LH), the present study sought to investigate the possibility that CCL2 or its receptor CCR2 in LH is stimulated by prenatal ethanol exposure, perhaps specifically within MCH neurons. Our paradigm of intraoral administration of ethanol to pregnant rats, at low-to-moderate doses (1 or 3g/kg/day) during peak hypothalamic neurogenesis, caused in adolescent male offspring twofold increase in drinking of and preference for ethanol and reinstatement of ethanol drinking in a two-bottle choice paradigm under an intermittent access schedule. This effect of prenatal ethanol exposure was associated with an increased expression of MCH and density of MCH(+) neurons in LH of preadolescent offspring. Whereas CCL2(+) cells at this age were low in density and unaffected by ethanol, CCR2(+) cells were dense in LH and increased by prenatal ethanol, with a large percentage (83-87%) identified as neurons and found to colocalize MCH. Prenatal ethanol also stimulated the genesis of CCR2(+) and MCH(+) neurons in the embryo, which co-labeled the proliferation marker, BrdU. Ethanol also increased the genesis and density of neurons that co-expressed CCR2 and MCH in LH, with triple-labeled CCR2(+)/MCH(+)/BrdU(+) neurons that were absent in control rats accounting for 35% of newly generated neurons in ethanol-exposed rats. With both the chemokine and MCH systems believed to promote ethanol consumption, this greater density of CCR2(+)/MCH(+) neurons in the LH of preadolescent rats suggests that these systems function together in promoting alcohol drinking during adolescence.

Model of voluntary ethanol intake in zebrafish: effect on behavior and hypothalamic orexigenic peptides.

Recent studies in zebrafish have shown that exposure to ethanol in tank water affects various behaviors, including locomotion, anxiety and aggression, and produces changes in brain neurotransmitters, such as serotonin and dopamine. Building on these investigations, the present study had two goals: first, to develop a method for inducing voluntary ethanol intake in individual zebrafish, which can be used as a model in future studies to examine how this behavior is affected by various manipulations, and second, to characterize the effects of this ethanol intake on different behaviors and the expression of hypothalamic orexigenic peptides, galanin (GAL) and orexin (OX), which are known in rodents to stimulate consumption of ethanol and alter behaviors associated with alcohol abuse. Thus, we first developed a new model of voluntary intake of ethanol in fish by presenting this ethanol mixed with gelatin, which they readily consume. Using this model, we found that individual zebrafish can be trained in a short period to consume stable levels of 10% or 20% ethanol (v/v) mixed with gelatin and that their intake of this ethanol-gelatin mixture leads to pharmacologically relevant blood ethanol concentrations which are strongly, positively correlated with the amount ingested. Intake of this ethanol-gelatin mixture increased locomotion, reduced anxiety, and stimulated aggressive behavior, while increasing expression of GAL and OX in specific hypothalamic areas. These findings, confirming results in rats, provide a method in zebrafish for investigating with forward genetics and pharmacological techniques the role of different brain mechanisms in controlling ethanol intake.

Common effects of fat, ethanol, and nicotine on enkephalin in discrete areas of the brain.

Fat, ethanol, and nicotine share a number of properties, including their ability to reinforce behavior and produce overconsumption. To test whether these substances act similarly on the same neuronal populations in specific brain areas mediating these behaviors, we administered the substances short-term, using the same methods and within the same experiment, and measured their effects, in areas of the hypothalamus (HYPO), amygdala (AMYG), and nucleus accumbens (NAc), on mRNA levels of the opioid peptide, enkephalin (ENK), using in situ hybridization and on c-Fos immunoreactivity (ir) to indicate neuronal activity, using immunofluorescence histochemistry. In addition, we examined for comparison another reinforcing substance, sucrose, and also took measurements of stress-related behaviors and circulating corticosterone (CORT) and triglycerides (TG), to determine if they contribute to these substances' behavioral and physiological effects. Adult Sprague-Dawley rats were gavaged three times daily over 5 days with 3.5 mL of water, Intralipid (20% v/v), ethanol (12% v/v), nicotine (0.01% w/v) or sucrose (22% w/v) (approximately 7 kcal/dose), and tail vein blood was collected for measurements of circulating CORT and TG. On day five, animals were sacrificed, brains removed, and the HYPO, AMYG, and NAc processed for single- or double-labeling of ENK mRNA and c-Fos-ir. Fat, ethanol, and nicotine, but not sucrose, increased the single- and double-labeling of ENK and c-Fos-ir in precisely the same brain areas, the middle parvocellular but not lateral area of the paraventricular nucleus, central but not basolateral nucleus of the AMYG, and core but not shell of the NAc. While having little effect on stress-related behaviors or CORT levels, fat, ethanol, and nicotine all increased circulating levels of TG. These findings suggest that the overconsumption of these three substances and their potential for abuse are mediated by the same populations of ENK-expressing neurons in specific nuclei of the hypothalamus and limbic system.

Prenatal ethanol exposure stimulates neurogenesis in hypothalamic and limbic peptide systems: possible mechanism for offspring ethanol overconsumption.

Exposure to ethanol during the prenatal period contributes to increased alcohol consumption and preference in rodents and increased risk for alcoholism in humans. With studies in adult animals showing the orexigenic peptides, enkephalin (ENK), galanin (GAL) and orexin (OX), to stimulate ethanol consumption, the question addressed here is whether prenatal ethanol alters the development in utero of specific neurons that express these peptides. With reports describing suppressive effects of high doses of ethanol, we examined the offspring of dams gavaged from embryonic day 9 to parturition with a control solution or lower ethanol doses, 1 and 3g/kg/day, known to promote ethanol consumption in the offspring. To understand underlying mechanisms, measurements were taken in postnatal offspring of the expression of ENK in the hypothalamic paraventricular nucleus (PVN) and nucleus accumbens (NAc), GAL in the PVN, and OX in the perifornical lateral hypothalamus (PFLH) using real-time qPCR and in situ hybridization, and also of the cell proliferation marker, 5-bromo-2-deoxyuridine (BrdU), and its double-labeling with either neuronal nuclei (NeuN), a marker of mature neurons, or the peptides. On postnatal day 15 (P15), after two weeks without ethanol, the offspring showed increased expression of ENK in the PVN and NAc core but not shell, GAL in the PVN, and OX in the PFLH. In these same areas, prenatal ethanol compared to control increased the density at birth (P0) of neurons expressing these peptides and at P0 and P15 of neurons double-labeling BrdU and NeuN, indicating increased neurogenesis. These BrdU-positive neurons were found to express ENK, GAL and OX, indicating that prenatal ethanol promotes neurogenesis in these specific peptide systems. There were no changes in gliogenesis or apoptosis. This increase in neurogenesis and density of peptide-expressing neurons suggests the involvement of these hypothalamic and accumbal peptide systems in mediating the increased alcohol consumption observed in prenatal ethanol-exposed offspring.

Circulating triglycerides after a high-fat meal: predictor of increased caloric intake, orexigenic peptide expression, and dietary obesity.

Recent studies in normal-weight rats have linked circulating triglycerides (TG), when elevated by a high-fat (HF) compared to equicaloric low-fat (LF) meal, to an increase in subsequent food intake and hypothalamic expression of orexigenic peptides. The present study tested whether natural variations between rats in their TG levels after a small HF meal can also be related to their individual patterns of eating and peptide expression. In tail vein blood collected on three separate days 60 min after a HF meal, levels of TG were found to be strongly, positively correlated within rats from day to day but were highly variable between rats (75-365 mg/dl), allowing distinct subgroups (33% lowest or highest) to be formed. Compared to "Low-TG responders" with post-meal levels averaging 109 mg/dl, "High-TG responders" with 240 mg/dl showed in two separate experiments a significant increase in caloric intake in a subsequent laboratory chow meal. Before this larger meal, these rats with elevated TG consistently exhibited higher expression levels and synthesis of the orexigenic peptides, enkephalin, orexin and melanin-concentrating hormone, as revealed using real-time quantitative PCR, radiolabeled in situ hybridization, and immunofluorescence histochemistry. Over the long-term, the High-TG responders also showed an increased propensity to overeat, gain weight and accumulate excess body fat on a chronic HF diet. This simple measure of TG levels after a HF meal may offer a useful tool for identifying subpopulations with increased risk for overeating and dietary obesity and detecting early signs of brain disturbances that may contribute to this high-risk phenotype.

Increased caloric intake after a high-fat preload: relation to circulating triglycerides and orexigenic peptides.

To investigate mechanisms that mediate the greater food intake induced by a fat-rich diet, the present study tested an acute "preload-to-test meal" paradigm in normal-weight rats. In this paradigm, the rats were given a small high-fat (HF) compared to low-fat (LF) preload and, after an intermeal interval, allowed to consume freely on a subsequent test meal. Modified versions of this paradigm were tested to determine the robustness of the greater caloric intake induced by the HF preload while standardizing the test protocol. A HF preload of 10-15 kcals, compared to an equicaloric LF preload, significantly increased food intake by 40-50% in the subsequent test meal. This effect, a 4-6 kcal increase, was observed with HF preloads equal in energy density and palatability to the LF preloads. It was evident with preloads or test meals that were liquid or solid, preloads that were injected, test meals that had variable fat content, and natural intermeal intervals of 60-120 min. This overeating after a HF preload was invariably associated with a 2- to 3-fold increase in circulating levels of triglycerides (TG), with no change in leptin or insulin. It was also accompanied by increased expression of the orexigenic peptides, galanin in the paraventricular nucleus and orexin in the perifornical lateral hypothalamus. Moreover, if given repeatedly over several days, the HF compared to equicaloric LF preload significantly increased 24-h food intake. These results establish a protocol for studying the phenomenon of increased feeding on a HF diet under controlled conditions and suggest possible underlying mechanisms involving circulating lipids and orexigenic peptides.

Diacylglycerol kinase zeta in hypothalamus interacts with long form leptin receptor. Relation to dietary fat and body weight regulation.

Leptin and its long form receptor, Ob-Rb, in hypothalamic nuclei play a key role in regulating energy balance. The mutation of Ob-Rb into one of its natural variants, Ob-Ra, results in severe obesity in rodents. We demonstrate here that diacylglycerol kinase zeta (DGKzeta) interacts, via its ankyrin repeats, with the cytoplasmic portion of Ob-Rb in yeast two-hybrid systems, in protein precipitation experiments in vitro and in vivo. It does not interact, however, with the short form, Ob-Ra, which mediates the entry of leptin into the brain. Furthermore, we show by in situ hybridization that DGKzeta is expressed in neurons of hypothalamic nuclei known to synthesize Ob-Rb and to participate in energy homeostasis. The mutant ob-/ob- and db-/db- mice exhibit increased hypothalamic DGKzeta mRNA level compared with their wild-type controls, suggesting a role for the leptin/OB-Rb system in regulating DGKzeta expression. Further experiments show that hypothalamic DGKzeta mRNA level is stimulated by the consumption of a high-fat diet. In addition, DGKzeta mRNA is statistically significantly lower in rats and inbred mice that become obese on a high-fat diet compared with their lean counterparts. In fact, it is strongly, negatively correlated with both body fat and circulating levels of leptin. Taken together, our evidence suggests that DGKzeta constitutes a downstream component of the leptin signaling pathway and that reduced hypothalamic DGKzeta mRNA, and possibly activity, is associated with obesity.

Rapid changes in hypothalamic neuropeptide Y produced by carbohydrate-rich meals that enhance corticosterone and glucose levels.

Prior studies have demonstrated that chronic consumption over several weeks of a high-carbohydrate (65%) diet, compared to a moderate-carbohydrate (45%) or low-carbohydrate (15%) diet, potentiates the expression, synthesis and release of hypothalamic NPY. This effect occurs specifically in neurons of the arcuate nucleus (ARC) which project to the paraventricular nucleus (PVN). In the present experiments, tests involving acute manipulations were conducted to determine whether such diet-induced changes in NPY can occur rapidly, perhaps within 1-2 h, and whether these effects can be linked to specific changes in circulating glucoregulatory hormones or glucose itself., In adult, albino rats maintained on lab chow, the acute manipulations included the presentation of either a high-carbohydrate, moderate-carbohydrate or high-fat diet for 90 min at the onset of the natural feeding cycle. They also involved manipulations of glucose itself, either through the ingestion of a glucose (20%) solution in a drinking tube or intraperitoneal injection of a glucose solution (10%). After a high-carbohydrate meal compared to a moderate-carbohydrate or high-fat meal, NPY gene expression examined via in situ hybridization is found to be significantly enhanced in the ARC. The high-carbohydrate meal also potentiates NPY immunoreactivity in the ARC and PVN but has little effect on NPY in other hypothalamic areas examined and actually causes a reduction in the feeding-stimulatory peptide, galanin, specifically in the PVN. The meal-induced increase in NPY is associated with specific endocrine patterns, as revealed by measurements in serum collected from trunk blood or from rats implanted with a chronic jugular catheter. After a high-carbohydrate meal, levels of glucose, together with corticosterone and insulin, are significantly elevated, while non-esterified fatty acids are reduced. A possible effect of circulating glucose on hypothalamic NPY is further suggested by the finding that the consumption or a single injection of a glucose solution at the onset of the feeding cycle similarly elevates NPY mRNA and peptide immunoreactivity in the ARC and PVN. These results demonstrate that hypothalamic NPY can change rapidly in response to dietary carbohydrate. They also suggest that this effect may be related to changes in circulating CORT as well as to the availability or utilization of glucose.

Hypothalamic serotonin in control of eating behavior, meal size, and body weight.

Serotonin (5-HT) has been implicated in the control of eating behavior and body weight. Stimulants of this monoamine reduce food intake and weight gain and increase energy expenditure, both in animals and in humans. This article reviews evidence that supports a role for hypothalamic serotonergic receptor mechanisms in the mediation of these effects. A variety of studies in rodents indicate that, at low doses, 5-HT or drugs that enhance the release of this neurotransmitter preferentially inhibit the ingestion of carbohydrate, more than fat or protein. This phenomenon is mediated, in part, by 5-HT receptors located in various medial hypothalamic nuclei. A negative feedback loop exists between the consumption of this macronutrient and the turnover of 5-HT in the hypothalamus. That is, carbohydrate ingestion enhances the synthesis and release of hypothalamic 5-HT, which in turn serves to control the size of carbohydrate-rich meals. A model is described that proposes the involvement of circulating hormones and glucose in this feedback process. These hormones, including insulin, corticosterone, and the adipose tissue-derived hormone, leptin, have impact on serotonergic function as well as satiety. This model further suggests that 5-HT exerts its strongest effect on appetite at the start of the natural feeding cycle, when carbohydrate is normally preferred. Clinical studies provide evidence that is consistent with the proposed model and that implicates 5-HT in disturbances of eating and body weight disorders.

Hypothalamic galanin: control by signals of fat metabolism.

The peptide, galanin (GAL), is known to stimulate eating behavior, reduce energy expenditure and affect the release of metabolic hormones. Further, the activity of this peptide in the hypothalamus is modulated, in turn, by these hormones as well as by the ingestion of nutrients. The focus of this investigation is on signals related to nutrient metabolism that may also affect GAL production and, through these neurochemical events, control the ingestion of specific nutrients. Three experiments were performed in normal-weight male, Sprague-Dawley rats. In Experiment 1, the impact of food deprivation (24 and 48 h) was examined. Experiment 2 tested the effects of the compound, 2-deoxy-D-glucose (2-DG, 200 and 400 mg/kg), which blocks glucose utilization, whereas Experiment 3 studied mercaptoacetate (MA, 200 and 600 micromol/kg), which blocks fatty acid oxidation. Eating behavior was examined in some rats, whereas hypothalamic GAL activity was measured in others using radioimmunoassay, immunohistochemistry and in situ hybridization. Both food deprivation and MA (600 micromol/kg), but not 2-DG, affected GAL in the hypothalamus, in one specific area. This is the anterior parvocellular region of the paraventricular nucleus (aPVN), which has a dense concentration of GAL-containing neurons and terminals. GAL gene expression and peptide immunoreactivity in this area is enhanced by food deprivation; in contrast, it is reduced by injection of MA. Other hypothalamic sites with dense concentrations of GAL-containing neurons or fibers are unaffected by food deprivation or MA, and the antimetabolite 2-DG has no impact on GAL in any area. Behavioral measurements indicate that these shifts in GAL activity are accompanied by specific changes in eating behavior. Food deprivation which enhances aPVN GAL produces a marked increase in fat ingestion, whereas MA which reduces aPVN GAL causes a specific reduction in fat ingestion along with a stimulation of protein intake. In contrast, 2-DG preferentially enhances ingestion of carbohydrate. These findings suggest a possible relationship between GAL activity in the aPVN and the metabolic and behavioral processes of fat metabolism and ingestion.

Neuropeptide Y in relation to carbohydrate intake, corticosterone and dietary obesity.

Neuropeptide Y (NPY) is known to stimulate eating behavior and to be related to behavioral patterns of carbohydrate ingestion. The present report investigates this relationship further to: (1) characterize the specific NPY projection activated in different dietary paradigms; (2) understand associated changes in circulating hormones that may mediate dietary effects on NPY neurons; and (3) determine whether endogenous NPY in conditions with macronutrient diets can be linked to body fat. Male albino Sprague-Dawley rats were tested in two feeding paradigms, one in which the rats were given a choice of the macronutrients, carbohydrate, fat or protein, or the other involving a single diet varying in carbohydrate of fat content. These studies consistently demonstrated a close association between the ingestion of carbohydrate and NPY levels, specifically in the arcuate nucleus (ARC) and medial portion of the paraventricular nucleus (PVN) of the hypothalamus. In addition to revealing increased NPY activity in animals that naturally select high carbohydrate when given a choice of macronutrients, a single diet with 65% carbohydrate (10% fat), compared to a control diet with 45% carbohydrate (30% fat), significantly potentiates NPY gene expression and NPY-immunoreactivity, as determined by in situ hybridization and immunohistochemistry. A further lowering of carbohydrate to 15% has little effect on NPY. Studies of medial hypothalamic fragments in vitro also reveal enhanced NPY release from hypothalamic tissue taken from rats maintained on high-carbohydrate diet. Together with NPY, circulating corticosterone (CORT) levels are also highest in a high-carbohydrate condition and positively correlated with NPY in the ARC. An association between NPY and adiposity in these dietary conditions is indicated by significantly higher levels of NPY in the medial PVN in rats with high body fat, whether consuming a high-carbohydrate of high-fat diet. This evidence, linking NPY to carbohydrate intake and circulating CORT, suggests a role for this peptide in glucose homeostasis that is normally exhibited under conditions when carbohydrate stores are low. Disturbances in this homeostatic process, associated with hyperinsulinemia and higher levels of NPY, become evident with only a moderate rise in body fat on a high-carbohydrate as well as high-fat diet.

Gonadal steroids and hypothalamic galanin and neuropeptide Y: role in eating behavior and body weight control in female rats.

The neuropeptides, galanin (GAL) and neuropeptide Y (NPY), based on studies in male rodents, are believed to have a role in controlling energy balance, both nutrient ingestion and metabolism. Whereas these peptides are also involved in reproduction, little is known about their specific function in energy balance in females. In rats consuming lab chow or macronutrient diets, measurements across the estrous cycle were taken of hypothalamic GAL and NPY, using RIA and immunohistochemistry; of the circulating hormones, estradiol, progesterone, and LH; and also of food intake and body weight. Levels of GAL and NPY peak during the proestrous phase of the female cycle when circulating estradiol and progesterone also rise. As previously reported for GAL, this peak is detected in two areas, the medial preoptic area (MPOA; +110%; P < 0.05) and the external zone of the median eminence (+57%; P < 0.05). In addition, this proestrous peak is seen in the paraventricular nucleus (PVN), specifically the anterior parvocellular portion (+35%; P < 0.05). Similarly, NPY rises during proestrous in the medial region of the PVN (+21%; P < 0.05) in addition to the MPOA (+78%; P < 0.05) and arcuate nucleus (+35%; P < 0.05). This peak in peptide levels is accompanied by an increase in caloric intake in rats receiving the lab chow diet and a specific increase in preference for fat in rats receiving macronutrient diets. Animals showing a preference for a fat-rich diet exhibit higher levels of GAL in the MPOA as well as the PVN and median eminence and also of NPY specifically in the MPOA. These peptides in the MPOA are similarly enhanced in animals with greater body fat, independent of diet. This evidence suggests that in the female rat, both GAL and NPY in the MPOA may contribute to the overeating and increased weight gain that occur during a fat-rich diet.

Differential functions of hypothalamic galanin cell grows in the regulation of eating and body weight.

Evidence suggests that hypothalamic galanin (GAL) has a variety of functions related to energy and nutrient balance, reproduction, water balance, and neuroendocrine regulation. The focus of this chapter is the role of GAL in eating and body weight regulation. Findings described herein demonstrate that GAL, in a cell group of the anterior region of the paraventricular nucleus (aPVN) that projects to the median eminence, has a role in the control of fat intake, fat metabolism, and body fat. This function of aPVN GAL neurons is carried out in close relation to circulating insulin and glucose. Galanin-expressing perikarya in the medial preoptic area (MPOA) have a similar function, although GAL here operates in association with the female steroids estrogen and progesterone. These GAL cell groups of the aPVN and MPOA contrast with those in the arcuate nucleus as well as the magnocellular vasopressin-containing neurons of the PVN and supraoptic nucleus, which show no relation to fat balance. This evidence reveals differential functions for the distinct GAL neuronal cell groups of the hypothalamus.

Hypothalamic galanin gene expression and peptide levels in relation to circulating insulin: possible role in energy balance.

The peptide galanin (GAL) exists in dense concentrations within the medial hypothalamus and is synthesized in a population of neurons within the paraventricular nucleus (PVN). This peptide has been linked to energy homeostasis through its behavioral, metabolic and endocrine actions, including pancreatic insulin secretion. This investigation examined whether circulating insulin, in turn, has impact on hypothalamic GAL production, GAL mRNA and peptide concentrations in the hypothalamus. Streptozotocin (STZ)-induced diabetic rats, compared to control subjects, were tested with or without insulin replacement. After STZ treatment, the rats exhibited hyperglycemia, increased food and water intake, and decreased weight gain compared to controls. These changes were reversed by daily, subcutaneous injections of insulin. Measurements of GAL mRNA, via solution hybridization/nuclease protection assay, revealed a 6-fold elevation after STZ treatment compared to controls, accompanied by a similar rise in GAL peptide levels. This increase in GAL message and peptide was reversible by insulin and was detected in a mediodorsal hypothalamic (MDH) dissection which contains the PVN. It was not seen in a dissection of the mediobasal hypothalamus that includes the GAL-synthesizing neurons of the arcuate nucleus. Measurements of GAL in discrete hypothalamic nuclei of STZ diabetic rats showed a 100% increase in peptide concentrations (p < 0.05) in the PVN that was insulin responsive. Other hypothalamic areas examined failed to exhibit any change in peptide. These findings are consistent with other evidence indicating an inverse association, between circulating insulin and GAL message or peptide in the PVN, that may have physiological relevance in the control of energy balance.

Hypothalamic neuropeptide Y, its gene expression and receptor activity: relation to circulating corticosterone in adrenalectomized rats.

Previous evidence has suggested a possible relationship between the adrenal steroid, corticosterone (CORT) and neuropeptide Y (NPY) in the brain. To provide a more systematic analysis of this interaction, the present study employed a variety of techniques, including in situ hybridization to measure NPY gene expression, radioimmunoassay to examine peptide levels and radioligand [125I]peptide YY (PYY) binding for analysis of peptide receptors. The results show that adrenalectomy (ADX), which caused a decline in CORT to levels < 0.3 micrograms %, has generally little impact on the hypothalamic NPY projection system under normal, basal conditions. This includes peptide gene expression or content in the area of its cell bodies (arcuate nucleus, ARC), in addition to peptide binding at its receptor sites. While it also includes peptide content at most hypothalamic terminal sites, there are three notable exceptions, namely, the medial paraventricular (PVN) and dorsomedial nuclei and medial preoptic area, where NPY nerve terminals and glucocorticoid receptors are particularly dense and the decline in CORT through ADX markedly reduces NPY content. In contrast, evidence obtained from CORT replacement in ADX rats shows that this steroid has profound impact on all components of the hypothalamic NPY system. This peptide-steroid interaction is apparent at the level of the cell body (ARC), as well as at the nerve terminal or receptor site (PVN and ARC), where CORT levels > 10 micrograms % strongly potentiate NPY gene expression, peptide content and radioligand binding. These and other findings suggest that this CORT-NPY interaction in the hypothalamus occurs physiologically under conditions, e.g., at the onset of the active feeding cycle, when circulating CORT normally rises.

Adrenal steroid receptors: interactions with brain neuropeptide systems in relation to nutrient intake and metabolism.

The glucocorticoid, corticosterone (CORT), is believed to have an important function in modulating nutrient ingestion and metabolism. Recent evidence described in this review suggests that the effects of this adrenal hormone are mediated through two steroid receptor subtypes, the type I mineralocorticoid receptor and the type II glucocorticoid receptor. These receptors, which have different affinities for CORT, respond to different levels of circulating hormone. They mediate distinct effects of the steroid, which can be distinguished by the specific nutrient ingested and by the particular period of the circadian cycle. Under normal physiological conditions, the type I receptor is tonically activated, either by low basal levels of circulating CORT (0.5-2 microgram %) normally available across the circadian cycle or possibly by the mineralocorticoid aldosterone. This type I activation is required for the maintenance of fat ingestion and fat deposition that occurs during most meals of the feeding cycle. In contrast, the type II receptor is phasically activated by moderate levels of CORT (2-10 micrograms %) normally reached during the circadian peak. Activation of this receptor is required for the natural surge in carbohydrate ingestion and metabolism that is essential at the onset of the active feeding cycle when the body's glycogen stores are at their nadir, and gluconeogenesis is needed to maintain blood glucose levels. This receptor is also activated during periods of increased energy requirements, such as, after exercise and food restriction, when CORT levels rise further (> 10 micrograms %) and when its catabolic effects on fat and protein stores predominate to provide additional substrates for glucose homeostasis. These functions of CORT on fat and carbohydrate balance are mediated, in part, by type I and type II receptors located within the hypothalamic paraventricular nucleus, which is known to have key functions in controlling nutrient intake and metabolism, as well as circulating CORT levels. Moreover, the type II receptors within this nucleus, in addition to the arcuate nucleus, may interact positively with the peptide, neuropeptide Y, and the catecholamine, norepinephrine, both of which act to enhance natural carbohydrate feeding and CORT release at the onset of the natural feeding cycle. Thus, under normal conditions, endogenous CORT has a primary function in controlling nutrient ingestion and metabolism over the natural circadian cycle, through the coordinated action of the type I and type II steroid receptor systems. Through this action, CORT has impact on total caloric intake and body weight gain over the long term.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracerebroventricular injection of dibutyryl cyclic adenosine 3',5'-monophosphate increases hypothalamic levels of neuropeptide Y.

This investigation examined in vivo the relationship between the nucleotide cAMP and hypothalamic levels of two peptides, neuropeptide Y (NPY) and galanin (GAL), which are known to potentiate feeding behavior. In brain-cannulated rats, third ventricular injections of N6,2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate ((Bu)2cAMP, 25 micrograms), compared to saline, caused a significant increase in NPY levels in the arcuate nucleus (ARC) and medial parvocellular portion of the paraventricular nucleus (mPVN), while having no impact in other hypothalamic areas. These site-specific changes in NPY occurred in the absence of any alteration in circulating levels of insulin, corticosterone, aldosterone or glucose, or of changes in hypothalamic levels of GAL. These findings implicate cAMP as having regulatory functions within specific hypothalamic NPY-synthesizing neurons, projecting from the ARC to the mPVN, that are believed to be involved in energy homeostasis.