Ellen E. Ladenheim

Central leptin modulates behavioral and neural responsivity to CCK

The mechanisms through which leptin, the protein product of the ob gene, affects food intake remain to be determined. To assess whether the actions of leptin depend on modulation of within-meal satiety signals, we measured the effect of third ventricular leptin administration on the satiety actions of CCK. Leptin (10 micrograms) administered 1 h before 30-min access to a liquid diet had no effect on intake when administered alone, but doses of 3.5 or 10 micrograms dose dependently increased the suppression of intake produced by 1 nmol/kg CCK. Examination of patterns of c-Fos activation induced by 3.5 micrograms leptin and 1 nmol/kg CCK revealed that the combination produced significant c-Fos activation within the area postrema and the caudal and medial nucleus of the solitary tract (NST) compared with either leptin or CCK treatments alone. The leptin-CCK combination also resulted in increased c-Fos activation within the paraventricular nucleus of the hypothalamus above that produced by leptin alone. These data suggest that the actions of leptin in food intake are mediated through its ability to modulate responsivity to within-meal satiety signals.The mechanisms through which leptin, the protein product of the ob gene, affects food intake remain to be determined. To assess whether the actions of leptin depend on modulation of within-meal satiety signals, we measured the effect of third ventricular leptin administration on the satiety actions of CCK. Leptin (10 μg) administered 1 h before 30-min access to a liquid diet had no effect on intake when administered alone, but doses of 3.5 or 10 μg dose dependently increased the suppression of intake produced by 1 nmol/kg CCK. Examination of patterns of c-Fos activation induced by 3.5 μg leptin and 1 nmol/kg CCK revealed that the combination produced significant c-Fos activation within the area postrema and the caudal and medial nucleus of the solitary tract (NST) compared with either leptin or CCK treatments alone. The leptin-CCK combination also resulted in increased c-Fos activation within the paraventricular nucleus of the hypothalamus above that produced by leptin alone. These data suggest that the actions of leptin in food intake are mediated through its ability to modulate responsivity to within-meal satiety signals.

Leptin activates hypothalamic acetyl-CoA carboxylase to inhibit food intake

Hypothalamic fatty acid metabolism has recently been implicated in the controls of food intake and energy homeostasis. We report that intracerebroventricular (ICV) injection of leptin, concomitant with inhibiting AMP-activated kinase (AMPK), activates acetyl-CoA carboxylase (ACC), the key regulatory enzyme in fatty acid biosynthesis, in the arcuate nucleus (Arc) and paraventricular nucleus (PVN) in the hypothalamus. Arc overexpression of constitutively active AMPK prevents the Arc ACC activation in response to ICV leptin, supporting the hypothesis that AMPK lies upstream of ACC in leptins Arc intracellular signaling pathway. Inhibiting hypothalamic ACC with 5-tetradecyloxy-2-furoic acid, a specific ACC inhibitor, blocks leptin-mediated decreases in food intake, body weight, and mRNA level of the orexigenic neuropeptide NPY. These results show that hypothalamic ACC activation makes an important contribution to leptins anorectic effects. Furthermore, we find that ICV leptin up-regulates the level of malonyl-CoA (the intermediate of fatty acid biosynthesis) specifically in the Arc and increases the level of palmitoyl-CoA (a major product of fatty acid biosynthesis) specifically in the PVN. The rises of both levels are blocked by 5-tetradecyloxy-2-furoic acid along with the blockade of leptin-mediated hypophagia. These data suggest malonyl-CoA as a downstream mediator of ACC in leptins signaling pathway in the Arc and imply that palmitoyl-CoA, instead of malonyl-CoA, could be an effector in relaying ACC signaling in the PVN. Together, these findings highlight site-specific impacts of hypothalamic ACC activation in leptins anorectic signaling cascade.

Leptin amplifies the feeding inhibition and neural activation arising from a gastric nutrient preload.

Leptin affects food intake by reducing meal size, suggesting that it may modulate the efficacy of within-meal satiety signals. To assess whether leptin would amplify the feeding inhibitory actions of a nutrient gastric preload, we compared liquid diet food intake and patterns of c-Fos activation in response to intraventricular leptin (3.5 microg), intragastric Ensure (10 ml over 10 min), or their combination. Leptin alone did not affect Ensure intake but significantly increased the suppression of intake produced by the intragastric preload. Within the nucleus of the solitary tract (NTS), leptin alone did not stimulate c-Fos but significantly elevated the number of c-Fos positive cells in response to intragastric Ensure at medial and rostral levels. Within the paraventricular nucleus (PVN), both leptin and the gastric load stimulated c-Fos expression, but the combination resulted in significantly greater number of c-Fos positive cells. These data demonstrate that leptin modulates the feeding inhibition produced by meal-related signals and suggest that this modulation occurs at the levels of the NTS and PVN.

Distinct distribution of two bombesin receptor subtypes in the rat central nervous system

Abstract We have previously demonstrated the presence of two distinct bombesin receptor subtypes in the rat CNS and distinguished them as bombesin/gastrin-releasing peptide (BBS/GRP) and neuromedin B (NMB)-preferring binding sites. In the present study, we conducted a complete evaluation of the distribution of these binding sites throughout the rat brain using in vitro receptor autoradiography. The BBS/GRP-preferring binding sites were characterized as those that bound 125I-(Tyr4)BBS but not 125I-( d -Tyr0)NMB. At these sites 125I-(Tyr4)BBS binding was inhibited in the presence of 100 nM BBS but not by the same concentration of NMB. In contrast, NMB-preferring sites bound both radioligands and binding at these sites was inhibited in the presence of 100 nM NMB. Our results indicate that the distributions of BBS/GRP and NMB-preferring binding sites are widespread and distinct at all levels of the rat brain suggesting these peptides mediate separate functions in the rat central nervous system.

Receptor heterogeneity for bombesin-like peptides in the rat central nervous system

As an initial characterization of bombesin binding sites in the rat central nervous system, we examined the pharmacological specificity of binding of bombesin (BBS) and several BBS analogs to rat cortex and compared these results to those for rat pancreas. In addition, we used in vitro receptor autoradiography to evaluate binding of 125I-[Tyr4]bombesin and 125I-Bolton-Hunter neuromedin B (NMB) to several regions of the rat brain. The results of the pharmacological study indicated that the pancreas and cortex had different binding affinities for BBS-like peptides. While cortical binding sites had a high affinity for NMB, pancreatic binding sites had almost no affinity for NMB. Results from the autoradiographic study demonstrated that BBS receptor heterogeneity exists in individual nuclei in the rat brain. Some nuclei have a high affinity for NMB, similar to cortical BBS binding sites, other regions have a low affinity similar to pancreatic BBS binding sites. These results provide evidence that subtypes of BBS receptors are present in different tissues and within discrete regions of the rat central nervous system.

Receptor subtype mediation of feeding suppression by bombesin-like peptides

Bombesin (BN) and the related mammalian peptides gastrin-releasing peptide (GRP), neuromedin C (NMC), and neuromedin B (NMB) suppress food intake in rats. Recent studies show two distinct receptor subtypes, GRP-preferring and NMB-preferring. BN interacts equally with both subtypes raising the possibility that one or both subtypes mediate the reduction of feeding by BN. To examine this issue, we compared suppression of intake produced by dose ranges (0-100 nmol/kg) of BN, GRP, NMC, and NMB and acetylated NMC and NMB. We found that all peptides elicited dose-dependent reductions of intake with overall differences in potency and efficacy. At intermediate doses, the rank order of potency for suppression was BN = AcNMC > NMC = GRP > NMB = AcNMB; however BN, GRP, and NMC were equipotent at the lowest and highest doses. Coadministration of NMC or GRP and NMB produced suppressions above that of either peptide alone and equivalent to BN. Taken together, these data support a role for both receptor subtypes in the suppression of food intake by BN and BN-like peptides.

Blockade of feeding inhibition by neuromedin B using a selective receptor antagonist

The ability of a selective neuromedin B receptor antagonist, D-Nal-cyclo[Cys-Tyr-D-Trp-Orn-Val-Cys]-Nal-NH2 (BIM-23127), to block suppression of food intake produced by the mammalian bombesin-like peptides neuromedin B and neuromedin C was examined. BIM-23127 completely blocked suppression of intake produced by neuromedin B but not by neuromedin C. These results suggest an independent role for neuromedin B receptors in suppression of food intake by bombesin-like peptides and demonstrate the utility of this group of antagonists for in vivo experiments.

Leptin modulation of peripheral controls of meal size.

Leptin reduces food intake through a specific effect on meal size. Investigations into how this within meal effect of leptin is mediated have demonstrated that leptin increases the ability of within meal inhibitory feedback signaling to limit intake and activate neurons within the nucleus of the solitary tract (NTS). Leptins effects on neural activation can be demonstrated both as an increase in c-fos activation and as increase in electrophysiological activity in response to peripheral stimuli. Leptin can exert these effects through interactions at hypothalamic sites and activation of a descending pathway. NPY has opposite effect suggesting a role for reduced NPY signaling in the actions of leptin. Forebrain ventricular administration of a melanocortin agonist does not mimic the actions of leptin. As well as modulating within meal signaling through a descending pathway leptin, NPY and melanocortins could work directly at hindbrain integrative sites suggesting the possibility of distributed controls of meal size by anorexigenic and orexigenic signaling.

Characterization of bombesin binding sites in the rat stomach

We characterized the bombesin receptor population in the rat stomach and determined the receptor subtype mediating the contractile effect of bombesin in the gastric fundus. Using in vitro receptor autoradiography, we evaluated the ability of the specific gastrin-releasing peptide-preferring receptor antagonist [D-F5,Phe6,D-Ala11]bombesin-(6-13) methyl ester to inhibit binding of 125I-[Tyr4]bombesin to the gastric fundus, corpus and antrum. Binding to these regions was completely inhibited by [D-F5,Phe6,D-Ala11]bombesin-(6-13) methyl ester suggesting that these receptors are the gastrin-releasing peptide-preferring subtype. We found that the rank order of potency for the contractile effect of bombesin, and the related mammalian peptides neuromedin C and neuromedin B, was bombesin > neuromedin C > neuromedin B. [D-F5,Phe6,D-Ala11]bombesin-(6-13) methyl ester was equipotent in antagonizing contractions produced by all three peptides. Furthermore, receptor tachyphylaxis to either neuromedin C or neuromedin B abolished the subsequent contractile response elicited by neuromedin C and neuromedin B, suggesting that one bombesin receptor subtype mediates rat gastric fundal contractions. Together, these results demonstrate that the bombesin receptor subtype in the rat stomach is gastrin-releasing peptide-preferring subtype and that this subtype is responsible for the effects of bombesin-like peptides on fundal smooth muscle contraction.

Capsaicin treatment differentially affects feeding suppression by bombesin-like peptides

Peripheral administration of bombesin (BN) and the related mammalian peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB), suppress food intake in rats. To examine whether all BN-like peptides utilize the same neural pathways to reduce feeding, rats were treated on postnatal day 2 with the injection vehicle or capsaicin, a neurotoxin that damages a subset of visceral afferent fibers. When rats reached adulthood, we compared the ability of a dose range of systemically administered BN, GRP18-27 and NMB to reduce intake of a 0.5 kcal/ml glucose solution in a short-term feeding test. Our results demonstrate that capsaicin treatment abolished or attenuated the suppression of glucose intake produced by BN and NMB but had no effect on the ability of GRP to reduce feeding. These results suggest that different neural substrates underlie the anorexic effects of peripherally administered BN-like peptides.