Generation of cell lines for drug discovery through random activation of gene expression: application to the human histamine H3 receptor.

Target-based high-throughput screening (HTS) plays an integral role in drug discovery. The implementation of HTS assays generally requires high expression levels of the target protein, and this is typically accomplished using recombinant cDNA methodologies. However, the isolated gene sequences to many drug targets have intellectual property claims that restrict the ability to implement drug discovery programs. The present study describes the pharmacological characterization of the human histamine H3 receptor that was expressed using random activation of gene expression (RAGE), a technology that over-expresses proteins by up-regulating endogenous genes rather than introducing cDNA expression vectors into the cell. Saturation binding analysis using [125I]iodoproxyfan and RAGE-H3 membranes revealed a single class of binding sites with a K(D) value of 0.77 nM and a B(max) equal to 756 fmol/mg of protein. Competition binding studies showed that the rank order of potency for H3 agonists was N(alpha)-methylhistamine approximately (R)-alpha- methylhistamine > histamine and that the rank order of potency for H3 antagonists was clobenpropit > iodophenpropit > thioperamide. The same rank order of potency for H3 agonists and antagonists was observed in the functional assays as in the binding assays. The Fluorometic Imaging Plate Reader assays in RAGE-H3 cells gave high Z' values for agonist and antagonist screening, respectively. These results reveal that the human H3 receptor expressed with the RAGE technology is pharmacologically comparable to that expressed through recombinant methods. Moreover, the level of expression of the H3 receptor in the RAGE-H3 cells is suitable for HTS and secondary assays.

Development of homogeneous high-affinity agonist binding assays for 5-HT2 receptor subtypes.

The serotonin (5-hydroxytryptamine) 5-HT2 receptor subfamily consists of three members, 5-HT2A, 5-HT2B, and 5-HT2C. These receptors share high homology in their amino acid sequence, have similar signaling pathways, and have been indicated to play important roles in feeding, anxiety, aggression, sexual behavior, mood, and pain. Subtype-selective agonists and antagonists have been explored as drugs for hypertension, Parkinson's disease, sleep disorders, anxiety, depression, schizophrenia, and obesity. In this study, we report the development of homogeneous agonist binding assays in a scintillation proximity assay (SPA) format to determine the high-affinity binding state of agonist compounds for the human 5-HT2C, 5-HT2A, and 5-HT2B receptors. The 5-HT2 agonist 1-(4- [125I]iodo-2,5-dimethoxyphenyl)-2-aminopropane ([125I]DOI) was used to label the high-affinity sites for the 5-HT2A and 5-HT2C receptors. The high-affinity sites for the 5-HT2B receptor were labeled with [3H]lysergic acid diethylamide. Total receptor expression was determined with the 5-HT2 antagonist [3H]mesulergine for the 5-HT2B and 5-HT2C receptors, and [3H]ketanserin for the 5-HT2A receptor. The agonist high-affinity binding sites accounted for 2.3% (5-HT(2C) receptor), 4.0% (5-HT2A receptor), and 22% (5-HT2B receptor) of the total receptor population. Competition binding studies using known agonists indicated high Z' values of the agonist binding assays in SPA format (Z' > 0.70). The Ki values of 5-HT, (R)(-)DOI, and VER-3323 for the 5-HT2A, 5-HT2B, and 5-HT2C receptors by SPA format were equivalent to published data determined by filtration binding assays. These results indicate that agonist binding assays in SPA format can be easily adapted to a high throughput assay to screen for selective 5-HT2C receptor agonists, as well as for selectivity profiling of the compounds.

Resistance to the orexigenic effect of ghrelin in dietary-induced obesity in mice: reversal upon weight loss.

BACKGROUND: Ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R), is known to increase food intake in lean humans and rodents. In addition, ghrelin levels are increased by fasting in lean rodents and are elevated before meals in humans, suggesting an important role for ghrelin in meal initiation. However, in obese human, circulating ghrelin levels were found to be significantly reduced as compared to lean individuals. OBJECTIVES: To evaluate whether circulating ghrelin levels, as well as ghrelin sensitivity, are decreased in obese individuals in order to limit its effect on food intake. DESIGN: : Lean C57BL/6J mice fed a chow, a low- (LFD) or a high-fat diet (HFD) were used to determine ghrelin regulation and secretion as well as ghrelin sensitivity. MEASUREMENTS: Plasma ghrelin levels were measured in low- and high-fat fed mice. Ghrelin-induced food intake was measured in chow, low- and high-fat fed mice. RESULTS: We measured ghrelin levels in lean and diet-induced obese mice, fed on an LFD or an HFD, respectively. We observed that not only ghrelin secretion was reduced in obese mice but its diurnal regulation was also lost. In addition, we failed to observe any change in ghrelin secretion upon fasting and refeeding. Moreover, we observed that the sensitivity to the orexigenic effects of exogenous ghrelin was reduced in obese mice when compared to lean mice fed a chow or a LFD. The insensitivity of obese mice to ghrelin was improved upon weigh loss. CONCLUSION: : Altogether, these results indicate that ghrelin secretion and regulation is impaired in dietary-induced obesity in mice and suggest that ghrelin inhibition could prevent weight regain after weight loss.

Up-regulation of neuropeptide Y receptors in the hypothalamus of monosodium glutamate-lesioned Sprague-Dawley rats.

Monosodium-glutamate (MSG) is neurotoxic for brain regions devoid of blood-brain barrier when it is injected at high doses during the neonatal period. Neuropeptide Y (NPY) neurons in the arcuate nucleus are particularly sensitive to MSG treatment. But, despite of the large decrease of this potent orexigenic peptide, feeding behavior is only slightly affected. We hypothesized that the hypothalamic NPY receptor system might be modified in these rats. The present study characterizes hypothalamic NPY and NPY receptors in normal and MSG-treated rats. MSG-treated rats were lighter (p < 0.01) and ate 17% less than the control rats (p < 0.01). NPY levels in the mediobasal and mediodorsal hypothalamus were reduced in MSG-treated rats compared to normal rats (-26% and -43%, p < 0.05 and p < 0.01, respectively). Combined hypothalamic Y1 and Y5 NPY receptor density was increased in MSG-treated rats compared to normal rats (+25%, p < 0.04), but affinity remained unaltered. Blockade with a selective Y1 antagonist showed that the Y1 receptor subtype represented more than 90% of the combined Y1 and Y5 receptor populations. The up-regulation of the NPY receptors is an element necessary to maintain food intake at a sufficient level to allow survival and growth of the lesioned rats.

Obesity is associated with decreasing levels of the circulating soluble leptin receptor in humans.

OBJECTIVE: Leptin plays a major role in the regulation of body weight. It circulates in both free and bound form. One of the leptin receptor isoforms exists in a circulating soluble form that can bind leptin. In the present study, we measured the soluble leptin receptor (SLR) levels in lean and obese humans. We investigated the relationship between plasma SLR levels, plasma leptin levels and the degree of obesity. We also examined whether SLR concentrations could be modulated by fat mass loss induced by a 3 month weight-reducing diet. SUBJECTS: A total of 112 obese (age 18-50 y; body mass index (BMI) 30-44 kg/m2; 23 men and 89 women), 38 overweight (age 19-48 y; BMI 25-29 kg/m2; 10 men and 28 women) and 63 lean (age 18-50 y; BMI 17-24 kg/m2; 16 men and 47 women) humans. MEASUREMENTS: A direct double monoclonal sandwich enzyme-linked immunosorbent assay (ELISA) was used for the quantitative measurement of the soluble human leptin receptor. Leptin was measured by radioimmunoassay (RIA). Body composition was assessed by biphotonic absorptiometry DEXA (dual energy X-ray absorptiometry). RESULTS: We observed that the SLR is present in human plasma (range 10-100 ng/ml). SLR levels were lower in obese and overweight than lean subjects (28.7+/-8.8, 40.2+/-14.9, 51.2+/-12.5 ng/ml, respectively) and were inversely correlated to leptin and percentage of body fat (r=-0.74 and r=-0.76; respectively; P<0.0001). The ratio of circulating leptin to SLR was strongly related to the percentage of body fat (r=0.91; P<0.0001). Interestingly a gender difference was observed in SLR levels, which were higher in obese and overweight men than in obese and overweight women. In obese subjects after a 3 month low-calorie diet, SLR levels increased in proportion to the decrease in fat mass. In the gel filtration profile, SLR coeluted exactly with the bound leptin fractions. CONCLUSION: Obesity, in humans is associated with decreasing levels of the circulating soluble leptin receptor (SLR). The relationship of SLR with the degree of adiposity suggests that high SLR levels may enhance leptin action in lean subjects more than in obese subjects.

Central and peripheral dysregulation of melanin-concentrating hormone in obese Zucker rats.

Melanin concentrating hormone (MCH) is a peptide synthesized in the lateral hypothalamus which stimulates food ingestion and leptin secretion in rodents. In this experiment, we measured the expressions of MCH as well as of its receptor (SLC-1) in the hypothalamus of obese hyperphagic and lean Zucker rats by quantitative real time RT-PCR. MCH mRNA expression in the obese rats was significantly increased by a factor of five (P<0.01) whereas expression of SLC-1 was decreased by more than 50% (P<0.05). Circulating levels of leptin and MCH were increased in the plasma of obese Zucker rats when compared to lean rats (38-fold and 1.7-fold, respectively, P<0.001 and P<0.01). However, individual MCH levels were not directly correlated to leptin levels in the lean (functional leptin receptor) or in the obese (non-functional leptin receptor) Zucker rats. These results indicate that the absence of leptin signaling in rats is associated with an increased hypothalamic expression and circulating release of MCH, contributing to their obesity syndrome.

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.

Plasma leptin and hypothalamic neuropeptide Y and galanin levels in Long-Evans rats with marked dietary preferences.

Neuropeptides present in the hypothalamus and new messengers in the periphery such as leptin modulate food intake in mammals. Neuropeptide Y (NPY) and galanin in microdissected brain areas and plasma leptin levels were measured by specific radioimmunoassays during the resting period in rats selected for their strong preference either for carbohydrate or fat, but with identical energy intake. NPY concentrations were 23% lower (p <.02) in carbohydrate-preferring (CP) than in fat-preferring (FP) rats in the parvocellular part of the paraventricular nucleus (PVN), which is one of the main areas involved in the regulation of feeding behavior. On the other hand, galanin was significantly (+25%, p = .03) higher in CP rats than in FP rats in the magnocellular part of the PVN. Plasma leptin was more than 50% higher in FP rats than in CP rats (p < .01) and highly correlated with the fat preference (r = 0.57; p = .003) and body weight gain. We conclude that the rats with a spontaneous and marked dietary preference have a characteristic peptidergic profile. Due to their anatomical relationships, neuropeptide Y could act in conjunction with galanin in a peptidergic balance located in the paraventricular nucleus. This model integrates information provided by the energy stores and translated by peripheral messengers such as leptin which could act in a counterregulatory manner in order to limit the overweight induced by the ingestion of unbalanced diets.

Identification of leptin-induced transcripts in the mouse hypothalamus.

Long-form leptin receptor (OB-R(L)) is a signal-transducing member of the cytokine receptor superfamily that is essential for mediating the effects of leptin on mammalian body weight homeostasis. At present, the range of transcriptional targets responsive to OB-R(L) activation, and consequently, the likely mediators of leptin action, remain undefined. In this report, we have used cDNA subtractive hybridization to identify transcripts induced by leptin in immortalized hypothalamic neurons expressing OB-R(L). Differential expression of the identified transcripts in these cells was confirmed by both array technology and Northern blotting. In situ hybridization studies indicate that these transcripts are expressed in the mouse central nervous system, including nuclei of the hypothalamus that coexpress OB-R(L). Comparative in situ analysis of slices of hypothalami generated from control and leptin-injected ob/ob mice demonstrates that a subset of the identified transcripts is induced in vivo after leptin injection. The potential role of the proteins encoded by these transcripts in mediating the effects of leptin on body weight and energy homeostasis are discussed.

Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice.

Protein-tyrosine phosphatase 1B (PTP-1B) is a major protein-tyrosine phosphatase that has been implicated in the regulation of insulin action, as well as in other signal transduction pathways. To investigate the role of PTP-1B in vivo, we generated homozygotic PTP-1B-null mice by targeted gene disruption. PTP-1B-deficient mice have remarkably low adiposity and are protected from diet-induced obesity. Decreased adiposity is due to a marked reduction in fat cell mass without a decrease in adipocyte number. Leanness in PTP-1B-deficient mice is accompanied by increased basal metabolic rate and total energy expenditure, without marked alteration of uncoupling protein mRNA expression. In addition, insulin-stimulated whole-body glucose disposal is enhanced significantly in PTP-1B-deficient animals, as shown by hyperinsulinemic-euglycemic clamp studies. Remarkably, increased insulin sensitivity in PTP-1B-deficient mice is tissue specific, as insulin-stimulated glucose uptake is elevated in skeletal muscle, whereas adipose tissue is unaffected. Our results identify PTP-1B as a major regulator of energy balance, insulin sensitivity, and body fat stores in vivo.

A peptide leptin antagonist reduces food intake in rodents.

OBJECTIVE: The purpose of the present study was to investigate the continuing validity of the hypothesis that leptin is a physiologically important regulator of food intake, using the human leptin mutant R128Q leptin. DESIGN: In a cellular proliferation assay, based on BAF-3 cells transfected with the murine ObRb receptor, R128Q leptin was shown to be devoid of agonistic activity and to competitively inhibit the proliferative effects of leptin. To determine whether R128Q leptin was also an antagonist of leptin in vivo, the leptin mutant was injected intracerebroventricularly (i.c.v.) into rats in the absence and presence of leptin. R128Q was also injected intraperitoneally (i.p.) into ob/ob and into db/db mice expressing, respectively, either normal or defective ObRb receptors. RESULTS: R128Q was shown to be a competitive antagonist of leptin induced cellular proliferation in vitro. Surprisingly, in vivo R128Q leptin produced a strong dose-dependent decrease in food intake, and was only slightly less potent than leptin itself. In fasted rats, the inhibitory effects of leptin and R128Q leptin (i.c.v.) on post-fast refeeding were additive. Finally, R128Q leptin produced the same inhibition of food intake as leptin when injected i.p. in ob/ob mice and, like leptin, was inactive after i.p. injection to db/db mice. CONCLUSION: R128Q leptin is a leptin agonist in vivo, but behaves as an antagonist against leptin induced proliferation in vitro. The data demonstrate that the human leptin mutant R128Q leptin is not a suitable tool for investigating the physiological actions of leptin.

Induction of obesity and hyperleptinemia by central glucocorticoid infusion in the rat.

It has been claimed that factors favoring the development or maintenance of animal or human obesity may include increases in glucocorticoid production or hyperresponsiveness of the hypothalamic-pituitary-adrenal axis. In normal rats, glucocorticoids have been shown to be necessary for chronic intracerebroventricular infusion of neuropeptide Y to produce obesity and related abnormalities. Conversely, glucocorticoids inhibited the body weight-lowering effect of leptin. Such dual action of glucocorticoids may occur within the central nervous system, since both neuropeptide Y and leptin act within the hypothalamus. The aim of this study was to determine the effects of glucocorticoids (dexamethasone) given intracerebroventricularly to normal rats on body weight homeostasis and hypothalamic levels of neuropeptide Y and corticotropin-releasing hormone. Continuous central glucocorticoid infusion for 3 days resulted in marked sustained increases in food intake and body weight relative to saline-infused controls. The infusion abolished endogenous corticosterone output and produced hyperinsulinemia, hypertriglyceridemia, and hyperleptinemia, three salient abnormalities of obesity syndromes. Central glucocorticoid infusion also produced a marked decrease in the expression of uncoupling protein (UCP)-1 and UCP-3 in brown adipose tissue and UCP-3 in muscle. Finally, chronic central glucocorticoid administration increased the hypothalamic levels of neuropeptide Y and decreased those of corticotropin-releasing hormone. When the same dose of glucocorticoids was administered peripherally, it resulted in decreases in food intake and body weight, in keeping with the decrease in hypothalamic neuropeptide Y levels. These results suggest that glucocorticoids induce an obesity syndrome in rodents by acting centrally and not peripherally.

Evidence that hypothalamic neurotensin signals leptin effects on feeding behavior in normal and fat-preferring rats.

Leptin inhibits food intake when it is injected in the periphery or in the central nervous system. It is likely that its action is not only mediated by the inhibition of orexigenic peptides such as neuropeptide Y. Therefore, we characterized the pharmacological and physiological relationships of leptin with neurotensin (NT), a central feeding inhibitor. Firstly, we investigated the central interactions of leptin and NT. Intracerebro-ventricular (ICV) injection were done in normal Long-Evans rats. NT had a short lasting (30 minutes; p<0.01) inhibitory effect on spontaneous food intake measured at the beginning of the dark phase whereas the effect of ICV leptin was observed after 24 hours (p<0.001). Co-injection with leptin potentiated NT effect at 30 minutes (p<0. 001) and prolonged it for 30 additional minutes (p<0.01). In addition, NT potentiated the effect of leptin at 30 and 60 minutes (p<0.02 and p<0.001 respectively) but not at 24 hours. Secondly, we observed that NT concentrations were augmented in selective brain areas in fat-preferring rats (+ 34% for hypothalamic NT; p<0.03). This increase was observed in the parvocellular part of the paraventricular nucleus (PVNp) only and was associated with an increase in circulating leptin levels (+ 75%; p<0.003). Interestingly, plasma leptin and NT in the PVNp were strongly correlated (r=0.57; p<0.003), suggesting changes of NT processing or release in this nucleus. These results strongly suggest that the short-term anorexigenic effects of leptin in normal rats are at least partly mediated by changes in NT processing or release. They also suggest that these processes take place in the hypothalamus, most probably in the PVNp and that they might be sensitive to fat ingestion. Therefore, the neurotensin increase observed in fat-preferring rats would limit the overconsumption of energy, a physiological mechanism translated by leptin.

Food intake in free-feeding and energy-deprived lean rats is mediated by the neuropeptide Y5 receptor.

The new neuropeptide Y (NPY) Y5 receptor antagonist CGP 71683A displayed high affinity for the cloned rat NPY Y5 subtype, but > 1, 000-fold lower affinity for the cloned rat NPY Y1, Y2, and Y4 subtypes. In LMTK cells transfected with the human NPY Y5 receptor, CGP 71683A was without intrinsic activity and antagonized NPY-induced Ca2+ transients. CGP 71683A was given intraperitoneally (dose range 1-100 mg/kg) to a series of animal models of high hypothalamic NPY levels. In lean satiated rats CGP 71683A significantly antagonized the increase in food intake induced by intracerebroventricular injection of NPY. In 24-h fasted and streptozotocin diabetic rats CGP 71683A dose-dependently inhibited food intake. During the dark phase, CGP 71683A dose-dependently inhibited food intake in free-feeding lean rats without affecting the normal pattern of food intake or inducing taste aversion. In free-feeding lean rats, intraperitoneal administration of CGP 71683A for 28 d inhibited food intake dose-dependently with a maximum reduction observed on days 3 and 4. Despite the return of food intake to control levels, body weight and the peripheral fat mass remained significantly reduced. The data demonstrate that the NPY Y5 receptor subtype plays a role in NPY-induced food intake, but also suggest that, with chronic blockade, counterregulatory mechanisms are induced to restore appetite.

The pharmacology of neuropeptide Y (NPY) receptor-mediated feeding in rats characterizes better Y5 than Y1, but not Y2 or Y4 subtypes.

Thirteen neuropeptide Y (NPY) agonists were administered intracerebroventricularly (i.c.v.) in rats (full dose-response curves) to estimate their half-effective dose (ED50) on feeding. These values were compared to their binding affinities (IC50) for rat NPY receptor subtypes Y1, Y2, Y4 and Y5 in vitro. Correlations between in vivo ED50 and in vitro IC50 were strong for the Y5 (r = 0.87; P < 0.01), weak for the Y1 (r = 0.48; P < 0.04) and non-significant for the Y2 and Y4 receptor subtypes. In vitro, h[D-Trp32]NPY was found to be a Y5-selective ligand and a full agonist in Y5-expressing cells. In vivo, it dose-dependently stimulated feeding, but failed to induce the full maximal response observed with pNPY. It did not antagonize pNPY-induced feeding and overfeeding in 24 h fasted rats. These findings demonstrate a role for the Y5, or possibly Y5 in combination with Y1, but not Y2 or Y4 receptor subtypes in feeding. No evidence was found for the existence of an additional, as yet undescribed, NPY feeding receptor.

Neuropeptide Y Y1 receptor antisense oligodeoxynucleotides enhance food intake in energy-deprived rats.

In the literature, conflicting data on the effect of NPY Y1 antisense oligodeoxynucleotides (ODNs) on food intake have been reported, describing either an increase or a decrease in feeding in antisense-treated animals. In the present studies antisense oligodeoxynucleotides targeted to the Y1 receptor (Y1 antisense ODNs) were used to re-investigate the functional importance of this receptor subtype in vivo in the regulation of feeding in rats. We used phosphothioate-terminal protected derivatives of two ODN sequences used in previous reports. In addition, as one of these sequences was not tested in vitro, we demonstrated its efficacy in LMTK-cells transfected with the Y1 receptor subtype. In vivo, repeated intracerebroventricular (i.c.v.) injections of Y1 antisense ODNs did not affect basal food intake or the increase in food intake after i.c.v. injection of neuropeptide Y (NPY, 300 pmol). Y1 antisense ODNs given intracerebroventricularly enhanced food intake in energy-deprived rats (+175% and +60% vs. control scrambled and sense sequences, respectively after 2 h of refeeding). Analysis of the structure of feeding behaviour revealed that Y1 antisense ODNs enhanced fasting-induced food intake during the first hour of refeeding by inducing increases in meal size (+143% and +155% vs. sense and scrambled ODNs) but not meal duration. These data suggest that the NPY Y1 receptor is not directly implicated in feeding in the rat when calorie intake is normal but might be specifically activated during energy deprivation.

Stimulation of feeding in lean but not in obese Zucker rats by a selective neuropeptide Y Y5 receptor agonist.

Obese Zucker rats are characterized by a reduced hypothalamic NPY receptor density. We tested the effects of intracerebroventricular injections of human NPY (hNPY) and [D-Trp32]NPY, a weak but selective NPY Y5 receptor agonist, on food intake in lean and obese Zucker rats. The effect of a maximal dose of hNPY (10 microg) on feeding was more pronounced in lean than in obese rats. [D-Trp32]NPY (10 microg) stimulated feeding in lean but not in obese Zucker rats. It did not affect the feeding response to hNPY, excluding the activation of an inhibitory receptor. These results are in favor of a down-regulation of the NPY 'feeding' receptor in the obese rat, which is suggested to be the Y5 subtype.

Behavioral deficits in monosodium glutamate rats: specific changes in the structure of feeding behavior.

We studied the feeding rhythms and feeding patterns of adult Long-Evans rats treated with monosodium glutamate (MSG) in their early post-natal period. This treatment is known to induce neuronal degeneration in the arcuate nucleus (ARC), a major hypothalamic site implicated in the regulation of feeding. Neonatal rats were treated intraperitoneally with MSG or saline (controls) alone on the first days of life. At age of 6 months, male control and male MSG rats were placed in our automatic feeding system, and the structure of feeding behavior and diurnal feeding rhythms were analysed. On a 24 hours basis, MSG rats ate less than control rats (-24%). This hypophagia resulted from a mild diurnal hyperphagia (+6%) and a pronounced nocturnal hypophagia (-34%). This hypophagia was the main consequence of a decrease of meal size in MSG rats (-37%) and was associated with an increase in meal duration (+52%). It was also associated with a total disappearance of the two feeding peaks that normally occur at light and dark onset in the rat (-90% 2 h after dark onset and -49% 2 h before light onset). These results indicate that neonatal treatment with MSG induces important changes in feeding patterns and feeding rhythms in the adulthood. These changes might be related to the disappearance of neurotransmitters located in the arcuate nucleus.

Neuropeptide Y release in the paraventricular nucleus of Long-Evans rats treated with leptin.

Neuropeptide Y (NPY) and leptin are actually two of the most potent peptides involved in the regulation of food intake with their respective stimulatory and inhibitory actions. The infusion of each peptide has a significant influence on the mRNA expression of the other in the adipose tissue for leptin and in the arcuate nucleus of the hypothalamus for NPY. To confirm this functional interaction, we measured the in vivo release of hypothalamic neuropeptide Y in awake fasted and refed Long-Evans rats after intraperitoneal (I.P.) injection of leptin. For this purpose, we used the push-pull perfusion technique with the cannula placed above the right paraventricular nucleus. I.P. leptin significantly inhibited food intake during the two hours of food access (-50%; p < 0.02). NPY release was not modified by leptin alone. But, when food was present, it slightly but significantly increased (p < 0.03 or less) and remained at a sustained level in the leptin-treated rats whereas it decreased in the control saline-injected group (p < 0.04). Thus, leptin did not acutely regulate NPY release and other food-related factors are probably involved as mediators of its anorexigenic effects.