Ghrelin.

BACKGROUND: The gastrointestinal peptide hormone ghrelin was discovered in 1999 as the endogenous ligand of the growth hormone secretagogue receptor. Increasing evidence supports more complicated and nuanced roles for the hormone, which go beyond the regulation of systemic energy metabolism. SCOPE OF REVIEW: In this review, we discuss the diverse biological functions of ghrelin, the regulation of its secretion, and address questions that still remain 15 years after its discovery. MAJOR CONCLUSIONS: In recent years, ghrelin has been found to have a plethora of central and peripheral actions in distinct areas including learning and memory, gut motility and gastric acid secretion, sleep/wake rhythm, reward seeking behavior, taste sensation and glucose metabolism.

Palmitoylethanolamide prevents metabolic alterations and restores leptin sensitivity in ovariectomized rats.

It has been suggested a role of fatty acid ethanolamides in control of feeding behavior. Among these, palmitoylethanolamide (PEA) has not been directly implicated in appetite regulation and weight gain. The aim of this study was to investigate the effect of PEA on food intake and body weight and the interaction between PEA and hypothalamic leptin signaling in ovariectomized rats. Ovariectomy produced hyperphagia and increased weight gain, making it an useful model of mild obesity. Ovariectomized rats were treated with PEA (30 mg/kg sc) for 5 weeks. Then, blood was collected, and hypothalamus and adipose tissue were removed for histological, cellular, and molecular measurements. We showed that PEA caused a reduction of food intake, body weight, and fat mass. The mechanisms underlying PEA effects involved an improvement in hypothalamic leptin signaling, through a raise in signal transducer and activator of transcription 3 phosphorylation. We also reported that PEA reduced AMP-activated protein kinase-α phosphorylation and modulated transcription of anorectic and orexigenic neuropeptides in the hypothalamus. Moreover, PEA increased AMP-activated protein kinase-α phosphorylation and carnitine palmitoyltransferase 1 transcription in adipose tissue, suggesting an increase in ATP-producing catabolic pathway. PEA also polarized adipose tissue macrophages to M2 lean phenotype, associated to a reduction of inflammatory cytokines/adipokines. To demonstrate the direct effect of PEA on leptin sensitivity without interference of adiposity loss, we obtained consistent data in PEA-treated sham-operated animals and in vitro in SH-SY5Y neuroblastoma cell line. Therefore, our data provide a rationale for the therapeutic use of PEA in obese postmenopausal woman.

Influence of a novel inhibitor (UM8190) of prolylcarboxypeptidase (PRCP) on appetite and thrombosis.

Preclinical pharmacological characterization of a novel inhibitor (UM8190) of prolylcarboxypeptidase (PRCP) was investigated. We synthesized and evaluated a library of proline-based analogs as prospective recombinant PRCP (rPRCP) inhibitors and inhibitors of PRCP-dependent prekallikrein (PK) activation on human pulmonary artery endothelial cells (HPAEC). Among the newly synthesized compounds, UM8190 was further characterized in vivo using methods that encompassed a mouse carotid artery thrombosis model and animal model of food consumption. (S)-N-dodecyl-1-((S)-pyrrolidine-2-carbonyl) pyrrolidine-2-carboxamide [Compound 3 (UM8190)] was selected for further evaluation from the initial assessment of its PRCP inhibitory action (K(i)= 43 µM) coupled with its ability to block PRCP-dependent PK activation on HPAEC (K(i)= 34 µM). UM8190 demonstrated excellent selectivity against a panel of carboxypeptidases and serine proteases and blocked bradykinin (BK) generation and BK-induced permeability by 100%, suggesting that it may be useful in preventing the local production of large amounts of BK. Furthermore, UM8190 showed an anorexigenic effect when systemically administered to fasted mice, reducing food intake in a dose- and time-dependent manner. In a mouse carotid artery thrombosis model, it also demonstrated an antithrombotic effect. UM8190 is a selective PRCP inhibitor and it may represent a new anorexigenic, and antithrombotic drug, that works by inhibiting PRCP-mediated mechanisms.

Susceptibility to a parkinsonian toxin varies during primate development.

Symptoms of Parkinson's disease typically emerge later in life when loss of nigrostriatal dopamine neuron function exceeds the threshold of compensatory mechanisms in the basal ganglia. Although nigrostriatal dopamine neurons are lost during aging, in Parkinson's disease other detrimental factors must play a role to produce greater than normal loss of these neurons. Early development has been hypothesized to be a potentially vulnerable period when environmental or genetic abnormalities may compromise central dopamine neurons. This study uses a specific parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to probe the relative vulnerability of nigrostriatal dopamine neurons at different stages of primate development. Measures of dopamine, homovanillic acid, 1-methyl-pyridinium concentrations and tyrosine hydroxylase immunoreactive neurons indicated that at mid-gestation dopamine neurons are relatively vulnerable to MPTP, whereas later in development or in the young primate these neurons are resistant to the neurotoxin. These studies highlight a potentially greater risk to the fetus of exposure during mid-gestation to environmental agents that cause oxidative stress. In addition, the data suggest that uncoupling protein-2 may be a target for retarding the progressive loss of nigrostriatal dopamine neurons that occurs in Parkinson's disease and aging.

Effects of chronic weight perturbation on energy homeostasis and brain structure in mice.

Maintenance of reduced body weight in lean and obese human subjects results in the persistent decrease in energy expenditure below what can be accounted for by changes in body mass and composition. Genetic and developmental factors may determine a central nervous system (CNS)-mediated minimum threshold of somatic energy stores below which behavioral and metabolic compensations for weight loss are invoked. A critical question is whether this threshold can be altered by environmental influences and by what mechanisms such alterations might be achieved. We examined the bioenergetic, behavioral, and CNS structural responses to weight reduction of diet-induced obese (DIO) and never-obese (CON) C57BL/6J male mice. We found that weight-reduced (WR) DIO-WR and CON-WR animals showed reductions in energy expenditure, adjusted for body mass and composition, comparable (-10-15%) to those seen in human subjects. The proportion of excitatory synapses on arcuate nucleus proopiomelanocortin neurons was decreased by ∼50% in both DIO-WR and CON-WR mice. These data suggest that prolonged maintenance of an elevated body weight (fat) alters energy homeostatic systems to defend a higher level of body fat. The synaptic changes could provide a neural substrate for the disproportionate decline in energy expenditure in weight-reduced individuals. This response to chronic weight elevation may also occur in humans. The mouse model described here could help to identify the molecular/cellular mechanisms underlying both the defense mechanisms against sustained weight loss and the upward resetting of those mechanisms following sustained weight gain.

Hypothalamic and pituitary expression of ghrelin receptor message is increased during lactation.

In the lactating rat there is a dramatic increase in food intake that peaks at around day 15 postpartum, a time when pups are near weaning age, yet still fully dependant on maternal nourishment. We examined whether the orexigenic hormone ghrelin plays a role in increasing food intake during lactation. To do this, we compared plasma levels ghrelin, as well as brain and pituitary expression of the growth hormone secretagogue receptor (GHS-R 1a) rats in one of three groups: (1) dams whose litters were removed the day after giving birth (non-lactating); (2) dams whose litters were removed on day 13 postpartum (litter removed), and dams allowed keeping their litters (lactating). On day 15 postpartum, all dams were decapitated and trunk blood collected for plasma analysis of active ghrelin levels. Also, brain and pituitaries were collected and snap frozen using liquid nitrogen and stored at -80 degrees C before mRNA extraction and RT-PCR analysis. Results show no differences in ghrelin concentrations between lactating and non-lactating rats. Hypothalamic and pituitary expression of GHS-R 1a, however, was significantly increased in lactating animals compared to non-lactating animals. Interestingly, litter removed dams had higher levels of plasma ghrelin concentrations than either lactating or non-lactating females. Furthermore, GHS-R mRNA expression in these animals remained elevated in the pituitary but not the hypothalamus. These data suggest that the hypothalamus and pituitary of lactating rats are more sensitive to the effects of ghrelin, and that hypothalamic sensitivity to ghrelin depends on the presence of a suckling litter.

Uncoupling proteins-2 and 3 influence obesity and inflammation in transgenic mice.

OBJECTIVE: To test the hypothesis that either uncoupling protein-2 UCP2 or UCP3 or both together influence obesity and inflammation in transgenic mice. DESIGN: We generated 12 lines of transgenic mice for both human UCP2 and 3 using native promoters from a human bacterial artificial chromosome (BAC) clone. The BAC expresses no genes other than UCP2 and 3. Mice used for experiments are N4 or higher of backcross to C57BL/6J (B6). Each experiment used transgenic mice and their nontransgenic littermates. RESULTS: Northern blots confirmed expression on human UCP2 in adipose and spleen, while human UCP3 expression was detectable in gastrocnemius muscle. Western blots demonstrated a four-fold increase of UCP2 protein in spleens of Line 32 transgenic animals. Heterozygous mice of four lines showing expression of human UCP2 in spleen were examined for obesity phenotypes. There were no significant differences between Lines 1 and 32, but female transgenics of both lines had significantly smaller femoral fat depots than the control (littermate) mice (P=0.015 and 0.005, respectively). In addition, total fat of transgenic females was significantly less in Line 1 (P=0.05) and almost significantly different in Line 32 (P=0.06). Male Line 1 mice were leaner (P=0.04) while male Line 32 mice were almost significantly leaner (P=0.06). Heterozygous mice of Lines 35 and 44 showed no significant differences from the nontransgenic littermate controls. Effects of the UCP2/UCP3 transgene on obesity in Line 32 mice were confirmed by crossing transgenic mice with the B6.Cg-Ay agouti obese mice. B6.Cg-Ay carrying the UCP2/UCP3 transgene from Line 32 were significantly leaner than nontransgenic B6.Cg-Ay mice. Line 32 UCP2/UCP3 transgenics showed increased hypothalamic Neuropeptide (NPY) levels and food intake, with reduced spontaneous physical activity. Transgenic baseline interleukin4 (IL-4) and interleukin6 (IL-6) levels were low with lower or later increases after endotoxin injection compared to wild-type littermates. Endotoxin-induced fever was also diminished in transgenic male animals. Low-density lipoprotein (LDL) cholesterol levels were significantly higher in both Line 1 and 32 transgenics (P=0.05 and 0.001, respectively) after they had been placed on a moderate fat-defined diet containing 32% of calories from fat for 5 weeks. CONCLUSION: Moderate overexpression of UCP2 and 3 reduced fat mass and increased LDL cholesterol in two independent lines of transgenic mice. Thus, the reduced fat mass cannot be due to insertional mutagenesis since virtually identical fat pad weights and masses were observed with the two independent lines. Line 32 mice also have altered inflammation and mitochondrial function. We conclude that UCP2 and/or 3 have small but significant effects on obesity in mice, and that their mechanism of action may include alterations of metabolic rate.

Leptin uptake by serotonergic neurones of the dorsal raphe.

The effects of leptin on food intake, metabolism, sleep patterns and reproduction may be mediated, in part, by the midbrain serotonergic systems. Here, we report on the distribution of neurones that accumulate leptin in the raphe nuclei of male and female rats after intracerebroventricular administration of mouse recombinant leptin labelled with digoxigenin. Direct leptin-targeted cells were present in the periventricular grey, pontine and raphe nuclei. Confocal microscopy revealed that raphe neurones which accumulated leptin were predominantly serotonergic. The temporal pattern of leptin accumulation by raphe neurones showed a marked gender difference: 6 h after leptin administration, all male and female rats showed massive leptin binding in the dorsal raphe, while 30 min after leptin treatment, only 10% of male rats exhibited leptin-labelled cells in contrast to 50% of females. The present observations reveal that leptin can be selectively accumulated by serotonergic neurones in the raphe nuclei and that this mechanism is gender specific. These findings support the idea that the midbrain serotonergic system is an important mediator of the effects of leptin on brain function and may provide an explanation for gender differences in metabolism regulation and its coordination with higher functions of the brain.

Minireview: ghrelin and the regulation of energy balance--a hypothalamic perspective.

The recently discovered hormone, ghrelin, has been recognized as an important regulator of GH secretion and energy homeostasis. Orexigenic and adipogenic ghrelin is produced by the stomach, intestine, placenta, pituitary, and possibly in the hypothalamus. The concentration of circulating ghrelin, principally derived from the stomach, is influenced by acute and chronic changes in nutritional state. To date, most studies focused on the role of ghrelin in GH secretion or its function in complementing leptin action to prevent energy deficits. The potential significance of ghrelin in the etiology of obesity and cachexia as well as in the regulation of growth processes is the subject of ongoing discussions. A large quantity of information based on clinical trials and experimental studies with ghrelin and previously available synthetic ghrelin receptor agonists (GH secretagogues) must now be integrated with a rapidly increasing amount of data on the central regulation of metabolism and appetite. In this overview, we summarize recent findings and strategies on the integration of ghrelin into neuroendocrine networks that regulate energy homeostasis.

Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus.

The administration of leptin to leptin-deficient humans, and the analogous Lepob/Lepob mice, effectively reduces hyperphagia and obesity. But common obesity is associated with elevated leptin, which suggests that obese humans are resistant to this adipocyte hormone. In addition to regulating long-term energy balance, leptin also rapidly affects neuronal activity. Proopiomelanocortin (POMC) and neuropeptide-Y types of neurons in the arcuate nucleus of the hypothalamus are both principal sites of leptin receptor expression and the source of potent neuropeptide modulators, melanocortins and neuropeptide Y, which exert opposing effects on feeding and metabolism. These neurons are therefore ideal for characterizing leptin action and the mechanism of leptin resistance; however, their diffuse distribution makes them difficult to study. Here we report electrophysiological recordings on POMC neurons, which we identified by targeted expression of green fluorescent protein in transgenic mice. Leptin increases the frequency of action potentials in the anorexigenic POMC neurons by two mechanisms: depolarization through a nonspecific cation channel; and reduced inhibition by local orexigenic neuropeptide-Y/GABA (gamma-aminobutyric acid) neurons. Furthermore, we show that melanocortin peptides have an autoinhibitory effect on this circuit. On the basis of our results, we propose an integrated model of leptin action and neuronal architecture in the arcuate nucleus of the hypothalamus.

A GABA-neuropeptide Y (NPY) interplay in LH release.

Neuropeptide Y (NPY) stimulates and gamma-amino butyric acid (GABA) inhibits LH release in the rat. Since a sub-population of NPY-producing neurons in the arcuate nucleus (ARC) of the hypothalamus co-express GABA, the possibility of an interplay between NPY and GABA in the release of LH was investigated in two ways. First by employing light and electron microscopic double staining for NPY and GABA, using pre and post-immunolabeling on rat brain sections, we detected GABA in NPY immunoreactive axon terminals in the MPOA, one of the primary sites of action of these neurotransmitters/neuromodulators in the regulation of LH release. These morphological findings raised the possibility that inhibitory GABA co-released with NPY may act to restrain the excitatory effects of NPY on LH release. Muscimol (MUS, 0.44 or 1.76 nmol/rat), a GABA(A) receptor agonist, administered intracerebroventricularly (icv), alone failed to affect LH release, but NPY (0.47 nmol/rat icv) alone stimulated LH release in ovarian steroid-primed ovariectomized rats. On the other hand, administration of MUS blocked the NPY-induced stimulation of LH release in a dose-dependent manner. Similarly, administration of MUS abolished the excitatory effects on LH release of 1229U91, a selective NPY Y4 receptor agonist. These results support the possibility that in the event of co-release of these neurotransmitters/neuromodulators, GABA may act to restrain stimulation of LH release by NPY during the basal episodic and cyclic release of LH in vivo.

Mitochondrial uncoupling protein 2 (UCP2) in the nonhuman primate brain and pituitary.

Energy dissipating mechanisms and their regulatory components represent key elements of metabolism and may offer novel targets in the treatment of metabolic disorders, such as obesity and diabetes. Recent studies have shown that a mitochondrial uncoupling protein (UCP2), which uncouples mitochondrial oxidation from phosphorylation, is expressed in the rodent brain by neurons that are known to regulate autonomic, metabolic, and endocrine processes. To help establish the relevance of these rodent data to primate physiology, we now examined UCP2 messenger RNA and peptide expressions in the brain and pituitary gland of nonhuman primates. In situ hybridization histochemistry showed that UCP2 messenger RNA is expressed in the paraventricular, supraoptic, suprachiasmatic, and arcuate nuclei of the primate hypothalamus and also in the anterior lobe of the pituitary gland. Immunocytochemistry revealed abundant UCP2 expression in cell bodies and axonal processes in the aforementioned nuclei as well as in other hypothalamic and brain stem regions and all parts of the pituitary gland. In the hypothalamus, UCP2 was coexpressed with neuropeptide Y, CRH, oxytocin, and vasopressin. In the pituitary, vasopressin and oxytocin-producing axonal processes in the posterior lobe and POMC cells in the intermediate and anterior lobes expressed UCP2. On the other hand, none of the GH-producing cells of the anterior pituitary was found to produce UCP2. The abundance and distribution pattern of UCP2 in the primate brain and pituitary suggest that this protein is evolutionary conserved and may relate to central autonomic, endocrine and metabolic regulation.

3,5-diiodo-L-thyronine regulates glucose-6-phosphate dehydrogenase activity in the rat.

Thyroid hormones influence the activity of lipogenic enzymes such as malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PD). The effect of T3 on ME is exerted at the transcriptional level, but it is unclear if its effect on G6PD is also nuclear mediated. Furthermore, other iodothyronines that have been shown to possess biological activity (such as diiodothyronines) could contribute to this enzyme's regulation. In this study the effects of 3,5-diiodothyronine (T2) on the aforementioned enzymes were examined and compared with those of T3. Rats made hypothyroid by propylthiouracil and iopanoic acid treatment were used throughout. Enzyme activities were determined spectrophotometrically, and G6PD messenger RNA (mRNA) expression was analyzed by Northern blotting using a human G6PD complementary DNA probe. Injections of T2 to hypothyroid animals significantly enhanced the activity of both enzymes. The effect of T2 on ME was nuclear mediated and mimicked the effect of T3. The effects of T2 and T3 on G6PD differed. Injection of T3 into hypothyroid rats induced an increase in both enzyme activity and G6PD mRNA expression, indicating a nuclear-mediated effect. The effect of T2 on G6PD activity, on the other hand, was not nuclear mediated. The injection of T2 into hypothyroid animals did not change G6PD mRNA expression, and the strong increase in the enzyme's activity (from +70% to +300%) was unaffected by simultaneous injection of protein synthesis inhibitors. As the lowest dose of 1 microg T2/100 g BW affects G6PD activity 3-5 times more than the same dose of T3, these data provide the first evidence that T2 is a factor capable of regulating G6PD activity.

Brain uncoupling protein 2: uncoupled neuronal mitochondria predict thermal synapses in homeostatic centers.

Distinct brain peptidergic circuits govern peripheral energy homeostasis and related behavior. Here we report that mitochondrial uncoupling protein 2 (UCP2) is expressed discretely in neurons involved in homeostatic regulation. UCP2 protein was associated with the mitochondria of neurons, predominantly in axons and axon terminals. UCP2-producing neurons were found to be the targets of peripheral hormones, including leptin and gonadal steroids, and the presence of UCP2 protein in axonal processes predicted increased local brain mitochondrial uncoupling activity and heat production. In the hypothalamus, perikarya producing corticotropin-releasing factor, vasopressin, oxytocin, and neuropeptide Y also expressed UCP2. Furthermore, axon terminals containing UCP2 innervated diverse hypothalamic neuronal populations. These cells included those producing orexin, melanin-concentrating hormone, and luteinizing hormone-releasing hormone. When c-fos-expressing cells were analyzed in the basal brain after either fasting or cold exposure, it was found that all activated neurons received a robust UCP2 input on their perikarya and proximal dendrites. Thus, our data suggest the novel concept that heat produced by axonal UCP2 modulates neurotransmission in homeostatic centers, thereby coordinating the activity of those brain circuits that regulate daily energy balance and related autonomic and endocrine processes.

Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system.

Hypocretin has been identified as a regulator of metabolic and endocrine systems. Several brain regions involved in the central regulation of autonomic and endocrine processes or attention are targets of extensive hypocretin projections. The most dense arborization of hypocretin axons in the brainstem was detected in the locus coeruleus (LC). Multiple labeling immunocytochemistry revealed a massive synaptic innervation of catecholaminergic LC cells by hypocretin axon terminals in rats and monkeys. In both species, all tyrosine hydroxylase-immunopositive cells in the LC examined by electron microscopy were found to receive asymmetrical (excitatory) synaptic contacts from multiple axons containing hypocretin. In parallel electrophysiological studies with slices of rat brain, all LC cells showed excitatory responses to the hypocretin-2 peptide. Hypocretin-2 uniformly increased the frequency of action potentials in these cells, even in the presence of tetrodotoxin, indicating that receptors responding to hypocretin were expressed in LC neurons. Two mechanisms for the increased firing rate appeared to be a reduction in the slow component of the afterhyperpolarization (AHP) and a modest depolarization. Catecholamine systems in other parts of the brain, including those found in the medulla, zona incerta, substantia nigra or olfactory bulb, received significantly less hypocretin input. Comparative analysis of lateral hypothalamic input to the LC revealed that hypocretin-containing axon terminals were substantially more abundant than those containing melanin-concentrating hormone. The present results provide evidence for direct action of hypothalamic hypocretin cells on the LC noradrenergic system in rats and monkeys. Our observations suggest a signaling pathway via which signals acting on the lateral hypothalamus may influence the activity of the LC and thereby a variety of CNSfunctions related to noradrenergic innervation, including vigilance, attention, learning, and memory. Thus, the hypocretin innervation of the LC may serve to focus cognitive processes to compliment hypocretin-mediated activation of autonomic centers already described.

Estrogen receptor beta and progesterone receptor mRNA in the intergeniculate leaflet of the female rat.

The present study was undertaken to explore the possibility that the integration of hormonal cues in the regulation of neuroendocrine mechanisms may occur outside of the hypothalamus at the level of the lateral geniculate body. In situ hybridization for mRNA encoding estrogen receptor beta and progesterone receptor was carried out on sections containing the lateral geniculate body using [35S]-labeled antisense riboprobes. Labeled cells were present in different limbic and hypothalamic sites as described previously. Populations of cells distributed homogeneously in the ventral lateral geniculate nucleus and intergeniculate leaflet were also found to express mRNA for estrogen receptor beta and progesterone receptor. The dorsal lateral geniculate nucleus lacked specific labeling for either type of gonadal steroid hormone receptor mRNA. The present observation together with the recent demonstration of a direct pathway between the intergeniculate leaflet and hypothalamic neuroendocrine cells indicate that integration of hormonal and photic stimuli in the central regulation of endocrine mechanisms occurs outside of the hypothalamus in the lateral geniculate body.

Aromatase and estrogen receptor immunoreactivity in the coronary arteries of monkeys and human subjects.

OBJECTIVE: The objective of this study was to determine whether estrogen could be formed locally in the coronary arteries. DESIGN: Coronary arteries were examined from monkeys (Macaca fascicularis, one male and one female) and human subjects (one premenopausal woman, one postmenopausal woman, and one man) by immunocytochemistry, using purified antisera against human placental estrogen synthetase (aromatase) and ER alpha. The arteries were graded for the amount of atherosclerosis. RESULTS: There was clear immunopositivity for both aromatase and estrogen receptors in all arteries studied. Although all endothelial cells (CD31 positive) stained for both antigens, the staining in macrophages, fibroblasts, and smooth muscle cells was irregular. CONCLUSION: The present results provide the first evidence for the local formation of estrogen in the coronary arteries. In addition to complementing the evidence of a cardioprotective effect of estrogen on the coronary circulation, our results highlight the potential importance of local regulation of estrogen formation and the role of available precursor androgens in maintaining the cardiovascular system.

Interactions between neuropeptide Y and gamma-aminobutyric acid in stimulation of feeding: a morphological and pharmacological analysis.

Neuropeptide Y (NPY) produced in neurons in the arcuate nucleus and brain stem and released in the paraventricular nucleus (PVN) and surrounding areas is involved in stimulation of feeding in rats. We recently reported that gamma-aminobutyric acid (GABA) is coexpressed in a subpopulation of NPY neurons in the arcuate nucleus. To determine whether GABA is colocalized in NPY terminals in the PVN, the site of NPY action, light and electron microscopic double staining for NPY and GABA using pre- and postembedding immunolabeling was performed on rat brain sections. GABA was detected in NPY-immunopositive axons and axon terminals within both the parvocellular and magnocellular divisions of the PVN. These morphological findings suggested a NPY-GABA interaction in the hypothalamic control of feeding. Therefore, the effects of muscimol (MUS), a GABA(A) receptor agonist, on NPY-induced food intake were examined in sated rats. When injected intracerebroventricularly, both NPY and MUS elicited dose-dependent feeding responses that were blocked by the administration of 1229U91 (a putative Y1 receptor antagonist) or bicuculline (a GABA(A) receptor antagonist), respectively. Coadministration of NPY and MUS intracerebroventricularly amplified the feeding response over that evoked by NPY or MUS alone. Similarly, microinjection of either NPY or MUS into the PVN stimulated food intake in a dose-related fashion, and coinjection elicited a significantly higher response than that evoked by either individual treatment. These results suggest that GABA and NPY may coact through distinct receptors and second messenger systems in the PVN to augment food intake.

Synaptic interaction between hypocretin (orexin) and neuropeptide Y cells in the rodent and primate hypothalamus: a novel circuit implicated in metabolic and endocrine regulations.

Hypocretin (orexin) has recently been shown to increase feeding when injected into the brain. Using both rat and primate brains, we tested the hypothesis that a mechanism of hypocretin action might be related to synaptic regulation of the neuropeptide Y (NPY) system. Hypocretin-immunoreactive terminals originating from the lateral hypothalamus make direct synaptic contact with neurons of the arcuate nucleus that not only express NPY but also contain leptin receptors. In addition, hypocretin-containing neurons also express leptin receptor immunoreactivity. This suggests a potential mechanism of action for hypocretin in the central regulation of metabolic and endocrine processes. The excitatory actions of hypocretin could increase NPY release, resulting in enhanced feeding behavior and altered endocrine regulation, whereas leptin, released from adipose tissue as an indicator of fat stores, would have the opposite effect on the same neurons, leading to a decrease in NPY and NPY-mediated hypothalamic functions. On the other hand, the innervation of hypocretin cells by NPY boutons raises the possibility that NPY may exert an effect on hypothalamic functions, at least in part, via mediation or feedback action on these lateral hypothalamic cells. Our data indicate that a direct interaction between leptin, hypocretin, and NPY exists in the hypothalamus that may contribute to the central regulation of metabolic and endocrine processes in both rodents and primates.

Leptin receptors in estrogen receptor-containing neurons of the female rat hypothalamus.

This study was undertaken to reveal whether integration of the peripheral signals, leptin and estradiol, that convey information on the metabolic state and gonadal function, respectively, might occur in the same hypothalamic neuronal perikarya. Light and electron microscopic immunolabeling for leptin receptors (LRs) and estrogen receptors (ERs) was carried out on hypothalamic sections of female rats. In the medial preoptic area, periventricular regions, including the parvicellular paraventricular nucleus, the arcuate nucleus and the ventromedial hypothalamic nucleus, all of the cells that expressed immunoreactivity for ERs were also immunopositive for LR. On the other hand, only a subpopulation of LR-containing cells was found to express ERs. The extensive colocalization of receptors for leptin and estrogen in neuronal perikarya of all parts of the hypothalamus suggests a closely coupled interaction between these peripheral signals in the regulation of a variety of behavioral and neuroendocrine mechanisms.