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Ellen E Ladenheim Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA Abstract: The prevalence of obesity worldwide has nearly doubled since 1980 with current estimates of 2.1 billion in 2013. Overweight and obesity lead to numerous adverse conditions including type 2 diabetes, cardiovascular disease, stroke, and certain cancers. The worldwide spread of obesity and associated comorbidities not only threatens quality of life but also presents a significant economic burden. While bariatric surgery has proven to be a viable treatment option for the morbidly obese, there is clearly a need for less invasive alternatives. Recent research has suggested that long-acting analogs of the gut hormone, glucagon-like peptide 1 (GLP-1), may have potential as an antiobesity treatment. The GLP-1 receptor agonist, liraglutide (trade name Saxenda), was recently approved by the US Food and Drug Administration as an obesity treatment option and shown in clinical trials to be effective in reducing and sustaining body weight loss. This review presents the basis for GLP-1-based therapies with a specific focus on animal and human studies examining liraglutide’s effects on food intake and body weight. Keywords: glucagon-like peptide 1, obesity, liraglutide, exenatide

Obstructive sleep apnea (OSA) is characterized by recurrent upper airway obstruction during sleep. OSA leads to high cardiovascular morbidity and mortality. The pathogenesis of OSA has been linked to a defect in neuromuscular control of the pharynx. There is no effective pharmacotherapy for OSA. The objective of this study was to determine whether upper airway patency can be improved using chemogenetic approach by deploying designer receptors exclusively activated by designer drug (DREADD) in the hypoglossal motorneurons. DREADD (rAAV5-hSyn-hM3(Gq)-mCherry) and control virus (rAAV5-hSyn-EGFP) were stereotactically administered to the hypoglossal nucleus of C57BL/6J mice. In 6-8 weeks genioglossus EMG and dynamic MRI of the upper airway were performed before and after administration of the DREADD ligand clozapine-N-oxide (CNO) or vehicle (saline). In DREADD-treated mice, CNO activated the genioglossus muscle and markedly dilated the pharynx, whereas saline had no effect. Control virus treated mice showed no effect of CNO. Our results suggest that chemogenetic approach can be considered as a treatment option for OSA and other motorneuron disorders.

Multiple physiologic and neural systems contribute to the controls over what and how much we eat. These systems include signaling involved in the detection and signaling of nutrient availability, signals arising from consumed nutrients that provide feedback information during a meal to induce satiation, and signals related to the rewarding properties of eating. Each of these has a separate neural representation, but important interactions among these systems are critical to the overall controls of food intake., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Study Objectives: Obesity hypoventilation and obstructive sleep apnea are common complications of obesity linked to defects in respiratory pump and upper airway neural control. Leptin-deficient ob/ob mice have impaired ventilatory control and inspiratory flow limitation during sleep, which are both reversed with leptin. We aimed to localize central nervous system (CNS) site(s) of leptin action on respiratory and upper airway neuroventilatory control., Methods: We localized the effect of leptin to medulla versus hypothalamus by administering intracerbroventricular leptin (10 μg/2 μL) versus vehicle to the lateral (n = 14) versus fourth ventricle (n = 11) of ob/ob mice followed by polysomnographic recording. Analyses were stratified for effects on respiratory (nonflow-limited breaths) and upper airway (inspiratory flow limitation) functions. CNS loci were identified by (1) leptin-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and (2) projections of respiratory and upper airway motoneurons with a retrograde transsynaptic tracer (pseudorabies virus)., Results: Both routes of leptin administration increased minute ventilation during nonflow-limited breathing in sleep. Phrenic motoneurons were synaptically coupled to the nucleus of the solitary tract, which also showed STAT3 phosphorylation, but not to the hypothalamus. Inspiratory flow limitation and obstructive hypopneas were attenuated by leptin administration to the lateral but not to the fourth cerebral ventricle. Upper airway motoneurons were synaptically coupled with the dorsomedial hypothalamus, which exhibited STAT3 phosphorylation., Conclusions: Leptin relieves upper airway obstruction in sleep apnea by activating the forebrain, possibly in the dorsomedial hypothalamus. In contrast, leptin upregulates ventilatory control through hindbrain sites of action, possibly in the nucleus of the solitary tract., (© 2016 Associated Professional Sleep Societies, LLC.)

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Disease pathogenesis derives, at least in part, from the long polyglutamine tract encoded by mutant HTT. Therefore, considerable effort has been dedicated to the development of therapeutic strategies that significantly reduce the expression of the mutant HTT protein. Antisense oligonucleotides (ASOs) targeted to the CAG repeat region of HTT transcripts have been of particular interest due to their potential capacity to discriminate between normal and mutant HTT transcripts. Here, we focus on phosphorodiamidate morpholino oligomers (PMOs), ASOs that are especially stable, highly soluble and non-toxic. We designed three PMOs to selectively target expanded CAG repeat tracts (CTG22, CTG25 and CTG28), and two PMOs to selectively target sequences flanking the HTT CAG repeat (HTTex1a and HTTex1b). In HD patient-derived fibroblasts with expanded alleles containing 44, 77 or 109 CAG repeats, HTTex1a and HTTex1b were effective in suppressing the expression of mutant and non-mutant transcripts. CTGn PMOs also suppressed HTT expression, with the extent of suppression and the specificity for mutant transcripts dependent on the length of the targeted CAG repeat and on the CTG repeat length and concentration of the PMO. PMO CTG25 reduced HTT-induced cytotoxicity in vitro and suppressed mutant HTT expression in vivo in the N171-82Q transgenic mouse model. Finally, CTG28 reduced mutant HTT expression and improved the phenotype of Hdh(Q7/Q150) knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aims: The pathogenesis of obesity remains incompletely understood and the exploration of the role of novel proteins in obesity may provide important insights into its causes and treatments. Here, we report a previously unidentified role for synphilin-1 in the control of food intake and body weight. Synphilin-1, a cytoplasmic protein, was initially identified as an interaction partner of alpha-synuclein, and has implications in Parkinson's disease pathogenesis related to protein aggregation., Subjects and Methods: To study the in vivo role of synphilin-1, we characterized a human synphilin-1 transgenic mouse (SP1) by assessing synphilin-1 expression, plasma parameters, food intake and spontaneous activity to determine the major behavioral changes and their consequences in the development of the obesity phenotype., Results: Expression of human synphilin-1 in brain neurons in SP1 mice resulted in increased food intake, body weight and body fat. SP1 mice also displayed hyperinsulinemia, hyperleptinemia and impaired glucose tolerance. Pair-feeding SP1 mice to amounts consumed by non-transgenic mice prevented the increased body weight, adiposity, hyperinsulinemia and hyperleptinemia demonstrating that these were all the consequences of increased food intake. Transgenic expression of synphilin-1 was enriched in hypothalamic nuclei involved in feeding control, and fasting-induced elevated endogenous synphilin-1 levels at these sites, suggesting that synphilin-1 is an important player in the hypothalamic energy balance regulatory system., Conclusion: These studies identify a novel function of synphilin-1 in controlling food intake and body weight, and may provide a unique obesity model for future studies of obesity pathogenesis and therapeutics.

Endogenous cannabinoid signaling, mediated predominately by CB1 receptor activation, is involved in food intake control and body weight regulation. Despite advances in determining the role of the CB1 receptor in obesity, its involvement in the driven nature of eating pathologies has received little attention. The present study examined CB1 receptor alterations as a consequence of dietary-induced binge eating in female Sprague Dawley rats. Four control groups were used to control for calorie restriction and highly palatable food variables characterizing this behavioral model. All groups were kept on their respective feeding schedules for 6-weeks and were given a uniform 33% calorie restriction (~22 h food deprivation) prior to sacrifice. Our findings indicate that regional CB1 mRNA and density were influenced by dietary conditions, but were not specific to the dietary-induced binge eating paradigm used. An increase of approximately 50% (compared with naive controls) in CB1 receptor mRNA levels in the nucleus of the solitary tract as measured by in situ hybridization was found in animals receiving continuous access to a highly palatable food (i.e., vegetable shortening with 10% sucrose). This group also had a significant increase in body weight and adiposity. An approximate 20% reduction in CB1 mRNA was observed in the cingulate cortex (areas 1 and 2) in animals exposed to an intermittent schedule of feeding, compared with groups that had ad libitum feeding schedules (i.e., continuous access and naive controls). Receptor density as measured by [(3)H]CP55,940 autoradiography, was reduced by approximately 30% in the nucleus accumbens shell region in groups receiving repeated access to the highly palatable food. Taken together, these findings indicate that dietary conditions can differentially influence CB1 receptors in forebrain and hindbrain regions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose of Review: This review focuses on recent advances in understanding the multiple roles of gastrointestinal peptides in the control of food intake and body weight with specific emphasis on ghrelin, amylin and glucagon-like peptide 1., Recent Findings: Recent studies support a role for ghrelin, amylin and glucagon-like peptide 1 in short-term and long-term effects on food intake and body weight. Apart from contributing to energy homeostasis, ghrelin's participation in reward and sensory processing has been the focus of much recent work. New findings on amylin's effects on food intake and energy balance provide further support for its role in meal-related food intake and suggest that it may also function as an adiposity signal. New investigations on the role of central and peripheral glucagon-like peptide 1 receptors in mediating the anorexic effects of glucagon-like peptide 1 have suggested that they differentially contribute to short-term and long term effects on food intake., Summary: Gastrointestinal peptides can influence food intake through mechanisms that involve short-term meal-related effects or through activation of central pathways involved in energy balance. An appreciation of the multiple actions of gastrointestinal peptides on food intake will aid in developing new strategies for weight management.

Signaling from energy stores provides feedback on overall nutrient availability to influence food intake. Beginning with seminal studies by Woods and colleagues identifying insulin as an adiposity signal, it has become clear that such factors affect food intake by modulating the efficacy of within meal feedback satiety signals. More recent work with leptin has revealed actions of the hormone in modulating the efficacy of multiple gut feedback signals, identified the dorsal hindbrain as a site of signal integration and suggested both local and descending hypothalamic to hindbrain actions in mediating these effects. The original work by Woods and colleagues provided the necessary experimental paradigms for these advances., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Fusion proteins made up of glucagon-like peptide 1 (GLP-1) and exendin-4 (EX-4) fused to a nonglycosylated form of human transferrin (GLP-1-Tf or EX-4-Tf) were produced and characterized. GLP-1-Tf activated the GLP-1 receptor, was resistant to inactivation by peptidases, and had a half-life of approximately 2 days, compared with 1 to 2 min for native GLP-1. GLP-1-Tf retained the acute, glucose-dependent insulin-secretory properties of native GLP-1 in diabetic animals and had a profound effect on proliferation of pancreatic beta-cells. In addition, Tf and the fusion proteins did not cross the blood-brain-barrier but still reduced food intake after peripheral administration. EX-4-Tf proved to be as effective as EX-4 but had longer lived effects on blood glucose and food intake. This novel transferrin fusion technology could improve the pharmacology of various peptides.

Gastrin-releasing peptide (GRP) is a bombesin-like peptide widely distributed in the gastrointestinal tract and central nervous system. In the brain, GRP mRNA is located in the hypothalamic paraventricular nucleus (PVN), a region that receives neural input from the arcuate nucleus and plays a critical role in food intake and energy balance. Because GRP neurons are localized in the vicinity of projection sites in the PVN for peptides that participate in energy homeostasis, we investigated whether GRP mRNA expression in the PVN may be sensitive to challenges imposed by either 38 h food deprivation or stimulation of the melanocortin system by the melanocortin 3/4 receptor agonist, melanotan II (MTII). We found that food deprivation significantly decreased GRP mRNA expression, whereas lateral ventricular MTII administration increased GRP mRNA expression in ad libitum-fed rats 4 h after administration. Furthermore, administration of MTII at a dose that reduces 24 h food intake and body weight prevented the decrease in GRP mRNA expression observed in animals that were pair fed to the amount of food consumed by those injected with MTII. These results demonstrate that food deprivation and stimulation of the melanocortin system produce opposing changes in GRP gene expression in the PVN, suggesting that GRP-containing neurons in the PVN may be part of the hypothalamic signaling pathway controlling food intake and energy balance.

Mice with a targeted disruption of bombesin receptor subtype-3 (BRS-3 KO) develop hyperphagia, obesity, hypertension, and impaired glucose metabolism. However, the factors contributing to their phenotype have not been clearly established. To determine whether their obesity is a result of increased food intake or a defect in energy regulation, we matched the caloric intake of BRS-3 KO mice to wild-type (WT) ad libitum (ad lib)-fed controls over 21 wk. Although BRS-3 KO ad lib-fed mice were 29% heavier, the body weights of BRS-3 KO pair-fed mice did not differ from WT ad lib-fed mice. Pair-feeding BRS-3 KO mice normalized plasma insulin but failed to completely reverse increased adiposity and leptin levels. Hyperphagia in ad lib-fed KO mice was due to an increase in meal size without a compensatory decrease in meal frequency resulting in an increase in total daily food intake. An examination of neuropeptide Y, proopiomelanocortin, and agouti-related peptide gene expression in the arcuate nucleus revealed that BRS-3 KO mice have some deficits in their response to energy regulatory signals. An evaluation of the satiety effects of cholecystokinin, bombesin, and gastrin-releasing peptide found no differences in feeding suppression by these peptides. We conclude that hyperphagia is a major factor leading to increased body weight and hyperinsulinemia in BRS-3 KO mice. However, our finding that pair-feeding did not completely normalize fat distribution and plasma leptin levels suggests there is also a metabolic dysregulation that may contribute to, or sustain, their obese phenotype.

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 leptin's 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 leptin's 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 leptin's 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 leptin's anorectic signaling cascade.

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.

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). Leptin's 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.

Extract: During and following a meal, ingested nutrients come into contact with multiple sites in the gastrointestinal tract that have the potential to alter peptide and neural signaling. Such signals can serve as feedback mediators influencing current or subsequent food intake. Ingested nutrients accumulate within the stomach, activating gastric mechanoreceptors and providing a signal of gastric fullness. Even during a meal, some ingested nutrients pass from the stomach and contact intestinal receptors. Such contact results in gastrointestinal peptide release and the activation of neural fibers producing reflex alterations in gastrointestinal motor and secretory activity and providing feedback information about the volume and nature of ingested nutrients that could alter the size of the current meal or affect subsequent eating. Recent work has characterized the ability of multiple gut peptides to affect eating and, consistent with their different patterns of release around meals, various roles for these peptides in overall eating control have been suggested. With the current rapid increase in rates of obesity, peripheral peptides with the ability to affect food intake provide attractive targets for antiobesity drug development.

Leptin amplifies feeding inhibition and neural activation produced by either cholecystokinin or intragastric preloads, suggesting that leptin may increase the efficacy of gastrointestinal meal-related signals. To determine whether leptin would similarly potentiate the feeding inhibitory actions of another putative satiety peptide, we evaluated the effects of third ventricular leptin administration on food intake and c-Fos activation in response to systemically administered bombesin (BN). Leptin (3.5 microg) was administered 1 h before either 0.9% saline or BN (0.32 and 1.0 nmol/kg) followed by 30-min access to Ensure liquid diet. Although neither leptin nor 0.32 nmol/kg BN alone suppressed Ensure intake, the combination reduced intake by 28%. The higher BN dose (1.0 nmol/kg) produced a significant suppression by itself but was further enhanced in the presence of leptin. Consistent with the behavioral results, c-Fos activation in the nucleus of the solitary tract was increased by combined dosages of leptin and 0.32 nmol/kg BN beyond the individual response to either peptide. In the presence of leptin, BN produced a 3.4- to 5.2-fold increase in the number of c-Fos-positive cells in the nucleus of the solitary tract compared with when BN was given alone. These data provide further support for the hypothesis that the effect of leptin on food intake may be mediated, in part, by modulating meal-related satiety signals.

Otsuka Long-Evans Tokushima fatty (OLETF) rats lacking cholecystokinin-A receptors are hyperphagic, obese, and diabetic. Although exercise attenuates OLETF rats' obesity, the mechanisms underlying the effects of exercise are unclear. In this study, we determined the effects of running wheel activity on patterns of body weight gain, food intake, and hypothalamic gene expression. We demonstrate that voluntary running activity beginning at 8 wk of age normalized meal patterns, food intake, body weight, and plasma levels of glucose and leptin in OLETF rats. During the initial exercise period, corticotropin-releasing factor (CRF) mRNA expression was significantly elevated in the dorsomedial hypothalamus (DMH) but not in the paraventricular nucleus in both OLETF and control Long-Evans Tokushima rats. In response to long-term exercise, arcuate nucleus (Arc) neuropeptide Y (NPY), and proopiomelanocortin as well as DMH NPY and CRF mRNA expression were increased in Long-Evans Tokushima rats. In contrast, whereas exercising OLETF rats had increased Arc NPY and DMH CRF expression, Arc proopiomelanocortin and DMH NPY mRNA levels were not elevated. Finally, we demonstrate that the effects of exercise on body weight in OLETF rats were long lasting. Although food intake and body weight were increased in OLETF rats when running wheels were locked, weights did not return to those of sedentary OLETF rats. Together, these data suggest that the elevation of DMH CRF expression may mediate the short-term feeding inhibitory effects of exercise and that exercise limits the elevation of DMH NPY expression to account for the overall prevention of OLETF rats' obesity.

Peptide YY3-36 [PYY(3-36)], a gastrointestinal peptide that is released into the circulation in response to ingesting a meal, has recently been suggested to play a role in controlling food intake. PYY(3-36) has been reported to inhibit food intake following peripheral administration in rodents and in human subjects. To more fully characterize the potential feeding actions of PYY(3-36), we examined the ability of a dose range of PYY(3-36) (0.3-3.0 nmol/kg) to affect liquid gastric emptying and daily 6-h food intake in male rhesus monkeys. Intramuscular PYY(3-36) produced a dose-related inhibition of saline gastric emptying that was maximal at a dose of 3 nmol/kg. Intramuscular PYY(3-36) administered before daily 6-h food access produced significant feeding reductions at doses of 1 and 3 nmol/kg. Analyses of the patterns of food intake across the 6-h period of food access revealed that PYY(3-36) increased the latency to the first meal and reduced average meal size without altering meal number. Although single doses of PYY(3-36) reduced intake, a suppressive effect on food intake was not sustained over multiple administrations across successive days. Together, these data suggest that PYY(3-36) has the ability to reduce food intake in acute test situations in nonhuman primates. Whether this is a physiological action of the endogenous peptide remains to be determined.

Heterozygous brain-derived neurotrophic factor (BDNF) (+/-) mice display abnormalities in central serotonergic neurotransmission, develop decrements in serotonergic innervation of the forebrain, and exhibit enhanced intermale aggressiveness. As disturbances of serotonin neurotransmission are implicated in alcohol abuse and aggression, we have examined in BDNF (+/-) mice alcohol drinking behavior, as well as central 5-hydroxytryptamine (5-HT)1A receptor function at the level of 5-HT1A receptor-G protein interaction. BDNF (+/-) mice displayed increased ethanol intake in a two-bottle choice procedure. There was no difference in the preference ratio for non-alcoholic tastants (i.e. quinine or saccharin) between genotypes. In the brains of alcohol-naive mice, we measured [35S]GTP gamma S binding stimulated by the 5-HT1A receptor agonist (+/-)-8-hydroxy-2-dipropyl-aminotetralin hydrobromide (8-OH-DPAT; 1 microM). In BDNF (+/-) versus wild-type (WT) mice, 5-HT1A receptor-stimulated [35S]GTP gamma S binding was significantly attenuated in the median raphe nucleus. There was a decrease in (+/-)8-OH-DPAT-stimulated [35S]GTP gamma S binding in the dorsal raphe, which did not reach statistical significance. In the hippocampus, 5-HT1A receptor-stimulated [35S]GTP gamma S binding was significantly attenuated in BDNF (+/-) mice. 5-HT1A receptor-stimulated [35S]GTP gamma S binding was attenuated in the anterior cingulate cortex and lateral septum, although these reductions did not reach statistical significance. 5-HT1A receptor number was not different between genotypes in any area of brain examined, suggesting that 5-HT1A receptor function, specifically the capacity of the 5-HT1A receptor to activate G proteins, is attenuated in BDNF (+/-) mice.

Cocaine- and amphetamine-regulated transcript peptide (CART) reduces rats' intake of liquid diet if the peptide reaches the 4th ventricle (4V). To test for specificity of 4V CART effects on feeding, the authors compared its ability to reduce intakes of liquid diet and water and tested for conditioned taste aversion (CTA). CART reduced 30-min intakes of both water and Ensure at a threshold of 1 microg. Lithium chloride (0.15 M, 20 ml/kg i.p.) and 4V CART (1 microg) paired with novel saccharin solution reduced saccharin preferences similarly in subsequent 2-bottle tests, compared with saline. Thus, CART can produce CTA. These data demonstrate that 4V CART's actions in ingestive behavior are not specific to nutrients and suggest that aspects of 4V CART's actions in reducing intake may be secondary to the production of an aversive state.

Otsuka Long-Evans Tokushima Fatty (OLETF) rat lacking CCK-A receptors are hyperphagic and obese. Previous work has demonstrated alterations in neuropeptide Y (NPY) and proopiomelanocortin (POMC) mRNA expression in ad libitum fed OLETF rats compared to lean Long-Evans Tokushima Otsuka (LETO) controls. In order to determine whether alterations in sensitivity to central peptides involved in overall feeding control may contribute to the hyperphagia and obesity in OLETF rats, we assessed OLETF and LETO rats feeding responses to lateral ventricular infusions of NPY (1 and 3.2 nmol), the melanocortin 3/4 agonist MTII (0.1 and 0.32 nmol) and the melanocortin receptor antagonist SHU-9119 (0.25 and 0.5 nmol). At a 3-h time point, NPY increased food intake in both OLETF and LETO rats. OLETF rats were more sensitive, having significant increases at both NPY doses and a greater increase at the higher dose. The melanocortin agonist MTII decreased intake in both LETO and OLETF rats. At the 20-h time point, the magnitude of suppression was greater in OLETF rats. SHU-9119 increased food intake in both groups. OLETF rats were more sensitive with larger relative increase and longer-lasting effects at the lower dose. These results are consistent with demonstrated alterations in neuropeptide gene expression in OLETF rats and indicate that alterations in responsivity to NPY and melanocortin signaling are unlikely to contribute to their hyperphagia and obesity.

Peptides from cocaine- and amphetamine-regulated transcript (CART) reduce food intake in rats when injected into the lateral ventricle. Hypothalamic and hindbrain sites important in the control of feeding contain CART-immunoreactive fibers. To further define the site of CART's anorectic action, we compared feeding and other behavioral responses to third or fourth ventricular (3V, 4V) CART-(55-102) in 6-h food-deprived rats, both before and after cerebral aqueduct occlusion. 3V CART reduced the volume of Ensure consumed and resulted in fewer observations of eating and grooming within the 30-min test session. These reductions were significantly attenuated by aqueduct obstruction. 4V CART suppressed Ensure intake and resulted in decreased observations of feeding both with and without aqueduct blockade. 3V CART produced flat-backed postures and movement-associated tremors that were prevented by aqueduct obstruction. 4V CART also produced these signs, both with and without aqueduct blockade. We conclude that the major hypophagic effect of intracerebroventricular CART is mediated at a hindbrain site. The association of CART-induced feeding suppression with altered motor behavior questions the specificity of intracerebroventricular CART for actions on feeding.

People with anorexia (AN) and bulimia nervosa (BN) have altered patterns of eating. It is possible that alterations of the neuropeptide gastrin releasing peptide (GRP), a bombesin (BBS) -like peptide with potent central anorexigenic activity, could contribute to disturbed eating behavior. To avoid the confounding effects of pathologic eating behavior, we measured cerebrospinal fluid (CSF) GRP concentrations in women who were long-term recovered (>1 year, normal weight, and regular menstrual cycles, no binging or purging) from AN (REC AN, N=12) or BN (REC BN, N=21) compared to healthy control women (NC, N=15). CSF GRP was significantly lower (chi(2)=9.41(3), p<0.01) in REC BN (9.6+/-3.1 pg/ml) compared to NC (13.4+/-5.5 pg/ml) and REC AN (11.6+/-2.9 pg/ml). Persistent GRP abnormalities after recovery from BN raise the possibility that this alteration might be trait-related and contribute to episodic hyperphagia in BN., (Copyright 2000 Academic Press.)

Otsuka Long-Evans Tokushima Fatty (OLETF) rats lacking CCK-A receptors are hyperphagic, obese, and diabetic. We have previously demonstrated that these rats have a peripheral satiety deficit resulting in increased meal size. To examine the potential role of hypothalamic pathways in the hyperphagia and obesity of OLETF rats, we compared patterns of hypothalamic neuropeptide Y (NPY), proopiomelanocortin (POMC), and leptin receptor mRNA expression in ad libitum-fed Long-Evans Tokushima (LETO) and OLETF rats and food-restricted OLETF rats that were pair-fed to the intake of LETO controls. Pair feeding OLETF rats prevented their increased body weight and elevated levels of plasma insulin and leptin and normalized their elevated POMC and decreased NPY mRNA expression in the arcuate nucleus. In contrast, NPY expression was upregulated in the dorsomedial hypothalamus (DMH) in pair-fed OLETF rats. A similar DMH NPY overexpression was evident in 5-wk-old preobese OLETF rats. These findings suggest a role for DMH NPY upregulation in the etiology of OLETF hyperphagia and obesity.

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.

The amphibian peptide bombesin (BN) and the related mammalian peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) inhibit gastric emptying in rats. Exogenous administration of BN stimulates the release of cholecystokinin (CCK), a gastrointestinal peptide that also potently inhibits gastric emptying. To determine whether the inhibition of gastric emptying by BN-like peptides is mediated by a CCK-dependent mechanism, we examined the ability of the CCK-A receptor antagonist, devazepide, to block the inhibition of saline gastric emptying produced by BN, GRP18-27 and NMB. Using the same dosages as in the gastric emptying experiment, we also evaluated the effect of devazepide on feeding suppression produced by systemically administered BN. Our results showed that devazepide completely blocked the suppression of gastric emptying produced by BN, GRP18-27 and NMB but had no effect on BN-induced suppression of food intake. These results suggest that BN-like peptides inhibit gastric emptying through an indirect mechanism that is dependent upon CCK-A receptor activation. In contrast, the suppression of food intake by BN, in this experimental paradigm, is independent of CCK-A receptors.

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 gastrin-releasing peptide receptor (GRP-R) is one of three members of the mammalian bombesin subfamily of seven-transmembrane G protein-coupled receptors that mediate diverse biological responses including secretion, neuromodulation, chemotaxis, and growth. The X chromosome-linked GRP-R gene is expressed widely during embryonic development and predominantly in gastrointestinal, neuronal, and neuroendocrine systems in the adult. Surprisingly, gene-targeted mice lacking a functional GRP-R gene develop and reproduce normally and show no gross phenotypic abnormalities. However, peripheral administration of bombesin at dosages up to 32 nmol/kg to such mice had no effect on the suppression of glucose intake, whereas normal mice showed a dose-dependent suppression of glucose intake. These data suggest that selective agonists for the GRP-R may be useful in inducing satiety.

The brain-gut peptides cholecystokinin (CCK) and the mammalian bombesin-like peptide gastrin-releasing peptide (GRP) suppress food intake. Vagotomy blocks CCK- but not bombesin (BN)-induced feeding suppression, demonstrating differential vagal contributions. We examined the relationship between the ability of CCK and the active fragment of GRP, GRP-(18-27), to stimulate gastric vagal afferent activity and their ability to elicit changes in gastric motility. We also examined ligated cervical vagal segments and revealed specific 125I-CCK vagal binding without evidence of radiolabeled BN binding sites. Both close arterial and intraperitoneal CCK and GRP-(18-27) produced dose-dependent increases in activity in gastric vagal mechanoreceptive afferents. CCK dose dependently decreased gastric pressure without altering antral wall tension, whereas GRP-(18-27) dose dependently increased both gastric pressure and peak antral wall muscle tension. These results suggest that GRP-(18-27) activates gastric vagal afferents secondary to its stimulation of gastric motor effects. CCK activates this same population of vagal afferents independent of changes in gastric tension, suggesting a direct action of CCK at functional vagal CCK receptors.

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.

Recent studies have identified two subtypes of bombesin (BN) receptors in the rat central nervous system: gastrin releasing-peptide (GRP) preferring and neuromedin B (NMB) preferring. To investigate a role for the NMB-preferring receptor subtype in feeding suppression elicited by fourth ventricular (4V) BN administration, we evaluated the ability of a selective NMB-preferring receptor antagonist, BIM-23127, to block suppression of glucose intake produced by 4V BN (10 pmol). Our results showed that 4V administration of BIM-23127 dose dependently antagonized the suppression of glucose intake produced by 4V BN. In addition, 4V administration of BIM-23127 alone increased glucose intake above that observed in the baseline condition. These results support a role for the NMB-preferring BN receptor subtype in the suppression of intake produced by 4V BN administration and suggest that endogenously released NMB participates in ingestive control.

The ability of dose combinations of cholecystokinin (CCK) and bombesin to inhibit liquid glucose (0.125 gm/ml) intake and affect microstructural components of ingestive behavior in rats was examined. Five minutes before access to the glucose solution, rats were injected with dose pairs of CCK and bombesin. Both CCK and bombesin inhibited intake in a dose-related fashion. The magnitude of the inhibition produced by dose combinations was never greater than the sum of the inhibitions produced by the individual doses. CCK and bombesin affected different microstructural components of ingestion. CCK reduced burst size and cluster size, whereas bombesin reduced burst number and cluster number. These effects of the 2 peptides were not altered by the presence of the other peptide. These results suggest that different mechanisms of action underlie the feeding inhibitory actions of the 2 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.

Bombesin (BN)-like peptides injected peripherally or centrally suppress food intake in rats. The relationship between the central and peripheral actions of BN is unknown. However, experimental evidence supports a critical role for the caudal hindbrain in mediating the feeding effects of BN. To investigate this relationship further, we examined the ability of fourth ventricular infusion of a specific gastrin-releasing peptide (GRP) antagonist, [D-F5, Phe6, D-Ala11]BN-(6-13) methyl ester (BN-ME), to block suppression of glucose intake (0.5 kcal/ml) produced by intraperitoneal administration of GRP-(18-27) in 5-h food-deprived male Sprague-Dawley rats (n = 10). We found that fourth ventricular administration of 10, 32, and 100 ng BN-ME blocked the suppression of glucose intake produced by peripherally administered 10 nmol/kg GRP-(18-27). The most effective dose of BN-ME (100 ng) blocked the ability of peripheral injection of GRP-(18-27) to inhibit glucose intake but had no effect on intake when given alone. These results demonstrate that the availability of caudal hindbrain GRP receptors is necessary for peripherally administered GRP-(18-27) to reduce food intake in rats.

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.

We have previously demonstrated that bombesin (BBS) infused into the fourth cerebral ventricle in rats suppressed feeding at doses 10-1,000 times less than those required after lateral ventricular administration. These results suggest that centrally administered BBS acts in the caudal hindbrain to suppress food intake. To investigate the participation of caudal hindbrain structures in fourth-ventricular BBS-induced suppression of feeding, we made lesions that destroyed the following: 1) area postrema (AP) and 2) and medial nucleus of the solitary tract (NST). Additionally, we examined the role of the AP/NST in the suppression of food intake after peripheral BBS administration. Our experiments revealed that lesions of the AP/NST abolished the suppression of food intake by fourth-ventricular BBS and attenuated the suppression of food intake by peripheral BBS. Lesions restricted to the AP attenuated the suppression of food intake by fourth-ventricular BBS but had no effect on suppression of intake by peripherally administered BBS. These results suggest that the NST may be a common neural substrate for the inhibition of food intake by both centrally and peripherally administered BBS.

Previous studies have shown that various bombesin receptor antagonists can distinguish between bombesin receptor subtypes in peripheral tissues. To determine whether these antagonists would be useful in differentiating bombesin receptor subtypes in the rat central nervous system, we used in vitro receptor autoradiography to examine the binding affinities of several bombesin receptor antagonists for two brain regions we had characterized as possessing distinct bombesin receptor subtypes. Our results demonstrate that, consistent with peripheral bombesin receptors, bombesin receptor subtypes in the rat brain can be differentiated by various bombesin receptor antagonists.

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.

This study was conducted to evaluate the extent to which bombesin (BBS)-induced suppression of food intake relies on small, unmyelinated sensory neurons. Rats were pretreated intraperitoneally with capsaicin, a neurotoxin that destroys a subpopulation of small-diameter sensory neurons, and tested for suppression of food intake after intraperitoneal administration of 2, 4, 8, 16, and 32 micrograms/kg BBS. Capsaicin pretreatment attenuated suppression of food intake by BBS over a wide range of doses. The site of capsaicin action was not determined in this study. However, work by others indicates that vagal and nonvagal neural substrates participate in suppression of food intake by BBS. Afferents of both of these substrates are predominantly small and unmyelinated. Therefore, the deficits in BBS-induced suppression of food intake that we observed may reflect damage to both vagal and/or nonvagal neurons that participate in BBS-induced suppression of food intake.

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.

Cholecystokinin octapeptide (CCK-8) binding sites were examined in the caudal hindbrain of unilaterally nodosectomized and intact rats by in vitro autoradiography with 125I-CCK-8. Unilateral nodosectomy caused a significant reduction in CCK-8 binding in the caudal medial nucleus of the solitary tract (NST) on the side ipsilateral to the nodose ganglionectomy. Examination of other caudal hindbrain regions exhibiting CCK-8 binding did not reveal changes in binding in nodosectomized animals compared to controls. These findings suggest that the CCK receptors in this brain region may be located on terminals of vagal afferent neurons.

To study the role of the dorsomedial hindbrain in cholecystokinin (CCK)-induced satiety, lesions were produced in the region of the obex. We observed that lesions that were limited to the area postrema (AP) and immediately adjacent nucleus of the solitary tract (NST) did not attenuate the satietogenic effects of CCK. Similarly, lesions of the medial NST rostral to the AP had no effect on CCK-induced satiety. However, when the majority of the medial and commissural subnuclei of the NST as well as the AP were lesioned, there was a significant attenuation of the satietogenic effect of CCK. Since this lesion includes the bulk of the terminal field from the gastric branch of the vagus nerve, these results support the role of gastric afferent projections in the mediation of CCK-induced satiety. Although we do not rule out the possibility that the AP itself monitors circulating CCK, our data clearly show that the AP is not essential for induction of satiety by exogenous CCK.

Capsaicin, the pungent principal in red pepper, has been shown to damage small-diameter peptide-containing sensory neurons. Suppression of feeding by cholecystokinin octapeptide (CCK OP) was attenuated after systemic pretreatment with capsaicin. Capsaicin pretreatment did not attenuate suppression of food intake by intragastric preloads. Pretreatment of rats with microgram quantities of capsaicin injected into the fourth ventricle, near the sites of vagal afferent termination, also attenuated CCK OP-induced suppression of food intake. These results suggest that the satiety-inducing effects of CCK OP are mediated, at least in part, by capsaicin-sensitive afferent neurons.