Substance P as a Novel Anti-obesity Target

Background & Aims: Substance P (SP) is an 11-amino acid peptide that belongs to the tachykinin family of peptides. SP acts in the brain and in the periphery as a neuropeptide, neurotransmitter, and hormone affecting diverse physiologic pathways, mainly via its high-affinity neurokinin-1 receptor (NK-1R). Its presence in the hypothalamus and other areas of the brain that regulate feeding as well as in the stomach and small intestine prompted us to investigate its role on appetite control and energy balance. Methods: CJ 012,255 (CJ), a SP antagonist that binds to NK-1R, was injected into lean, diet-induced obese (DIO), and genetically obese (ob/ob) mice, and its effects on body weight, adiposity, and insulin sensitivity were investigated. Results: CJ administration prevented weight gain and accumulation of fat after 2 weeks of high-fat feeding, whereas similar CJ treatment in obese mice (following 3 months of high-fat diet) resulted in weight loss, reduction in adiposity, and improvement of insulin sensitivity, in part because of inhibition of food intake. The effects of SP in the control of energy balance are, at least in part, leptin independent because CJ treatment was also effective in leptin-deficient mice. Peripheral SP administration resulted in a mild, dose-dependent increase in food intake, evident 3 hours post-SP injection. Conclusions: CJ reduces appetite and promotes weight loss in mice. We speculate that NK-1R antagonists, already tested in clinical trials for various diseases, may represent a potential target against obesity. Background & Aims: Substance P (SP) is an 11-amino acid peptide that belongs to the tachykinin family of peptides. SP acts in the brain and in the periphery as a neuropeptide, neurotransmitter, and hormone affecting diverse physiologic pathways, mainly via its high-affinity neurokinin-1 receptor (NK-1R). Its presence in the hypothalamus and other areas of the brain that regulate feeding as well as in the stomach and small intestine prompted us to investigate its role on appetite control and energy balance. Methods: CJ 012,255 (CJ), a SP antagonist that binds to NK-1R, was injected into lean, diet-induced obese (DIO), and genetically obese (ob/ob) mice, and its effects on body weight, adiposity, and insulin sensitivity were investigated. Results: CJ administration prevented weight gain and accumulation of fat after 2 weeks of high-fat feeding, whereas similar CJ treatment in obese mice (following 3 months of high-fat diet) resulted in weight loss, reduction in adiposity, and improvement of insulin sensitivity, in part because of inhibition of food intake. The effects of SP in the control of energy balance are, at least in part, leptin independent because CJ treatment was also effective in leptin-deficient mice. Peripheral SP administration resulted in a mild, dose-dependent increase in food intake, evident 3 hours post-SP injection. Conclusions: CJ reduces appetite and promotes weight loss in mice. We speculate that NK-1R antagonists, already tested in clinical trials for various diseases, may represent a potential target against obesity. Obesity-related pathology has surpassed tobacco use as a cause of death in the United States1Mokdad A.H. Marks J.S. Stroup D.F. et al.Actual causes of death in the United States, 2000.JAMA. 2004; 291: 1238-1245Crossref PubMed Scopus (4326) Google Scholar and has become an alarming public health problem worldwide. According to the World Health Organization, more than 1 billion adults are overweight (body mass index [BMI] >25), and among them, 300 million are truly obese (BMI >30).2Kopelman P.G. 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Tachykinins in the gut Part I. Expression, release and motor function.Pharmacol Ther. 1997; 73: 173-217Crossref PubMed Scopus (307) Google Scholar mucosal permeability,18Pothoulakis C. Castagliuolo I. LaMont J.T. et al.CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin.Proc Natl Acad Sci U S A. 1994; 91: 947-951Crossref PubMed Scopus (253) Google Scholar and epithelial ion transport and proliferation19Riegler M. Castagliuolo I. So P.T. et al.Effects of substance P on human colonic mucosa in vitro.Am J Physiol. 1999; 276: G1473-G1483PubMed Google Scholar, 20Castagliuolo I. Valenick L. Liu J. et al.Epidermal growth factor receptor transactivation mediates substance P-induced mitogenic responses in U-373 MG cells.J Biol Chem. 2000; 275: 26545-26550Crossref PubMed Scopus (101) Google Scholar via its high-affinity neurokinin-1 receptor (NK-1R). NK-1R is present in several cell types including neurons, epithelial cells, and various types of immune cells,21Pothoulakis C. Castagliuolo I. Leeman S.E. et al.Substance P receptor expression in intestinal epithelium in Clostridium difficile toxin A enteritis in rats.Am J Physiol. 1998; 275: G68-G75PubMed Google Scholar, 22Tsuchida K. Shigemoto R. Yokota Y. et al.Tissue distribution and quantitation of the mRNAs for three rat tachykinin receptors.Eur J Biochem. 1990; 193: 751-757Crossref PubMed Scopus (167) Google Scholar, 23Stewart-Lee A. Burnstock G. Actions of tachykinins on the rabbit mesenteric artery: substance P and [Glp6,L-Pro9]SP6-11 are potent agonists for endothelial neurokinin-1 receptors.Br J Pharmacol. 1989; 97: 1218-1224Crossref PubMed Scopus (33) Google Scholar and its expression is up-regulated in numerous inflammatory conditions.21Pothoulakis C. Castagliuolo I. Leeman S.E. et al.Substance P receptor expression in intestinal epithelium in Clostridium difficile toxin A enteritis in rats.Am J Physiol. 1998; 275: G68-G75PubMed Google Scholar, 24Schafer M.K. Nohr D. Krause J.E. et al.Inflammation-induced up-regulation of NK1 receptor mRNA in dorsal horn neurones.Neuroreport. 1993; 4: 1007-1010Crossref PubMed Scopus (79) Google Scholar, 25O’Connor T.M. O’Connell J. O’Brien D.I. et al.The role of substance P in inflammatory disease.J Cell Physiol. 2004; 201: 167-180Crossref PubMed Scopus (602) Google Scholar However, there is no evidence that SP is involved in the regulation of food intake and energy homeostasis.SP binds to 3 G-protein-coupled receptors, neurokinin (NK) 1, 2, and 3, with NK-1R being the receptor with the highest affinity.26Quartara L. Maggi C.A. The tachykinin NK1 receptor Part I: ligands and mechanisms of cellular activation.Neuropeptides. 1997; 31: 537-563Abstract Full Text PDF PubMed Scopus (188) Google Scholar NK-1R mediates several physiologic and pathophysiologic responses,27Quartara L. Maggi C.A. The tachykinin NK1 receptor Part II: Distribution and pathophysiological roles.Neuropeptides. 1998; 32: 1-49Abstract Full Text PDF PubMed Scopus (263) Google Scholar and pharmacologic antagonists of NK-1R have been used so far for treating diverse conditions, such as depression, anxiety, and stress, nausea associated with chemotherapy, rheumatoid arthritis, and inflammatory bowel disease.28Duffy R.A. Potential therapeutic targets for neurokinin-1 receptor antagonists.Expert Opin Emerg Drugs. 2004; 9: 9-21Crossref PubMed Scopus (70) Google Scholar, 29Furness J.B. TAK-637 Takeda.Curr Opin Investig Drugs. 2001; 2: 1437-1440PubMed Google Scholar In the intestine, SP produced by several cell types may circulate in the blood as a hormone or act locally in a paracrine fashion.14Severini C. Improta G. Falconieri-Erspamer G. et al.The tachykinin peptide family.Pharmacol Rev. 2002; 54: 285-322Crossref PubMed Scopus (507) Google Scholar Recently, accumulating evidence points to an important role of gut-derived peptides in obesity, by means of conveying meal-related signals to appetite-regulating centers, mainly in the hypothalamus and the nucleus tractus solitarius, and/or by directly influencing insulin production or sensitivity.4Badman M.K. Flier J.S. The gut and energy balance: visceral allies in the obesity wars.Science. 2005; 307: 1909-1914Crossref PubMed Scopus (388) Google ScholarCJ 012,255 (CJ; Pfizer, Groton, CT) is a selective NK-1R antagonist30Snider R.M. Constantine J.W. Lowe III, J.A. et al.A potent nonpeptide antagonist of the substance P (NK1) receptor.Science. 1991; 251: 435-437Crossref PubMed Scopus (826) Google Scholar that has been used to reduce ileal pouch inflammation31Stucchi A.F. Shebani K.O. Leeman S.E. et al.A neurokinin 1 receptor antagonist reduces an ongoing ileal pouch inflammation and the response to a subsequent inflammatory stimulus.Am J Physiol Gastrointest Liver Physiol. 2003; 285: G1259-G1267Crossref PubMed Scopus (38) Google Scholar and postoperative peritoneal adhesion formation in rats.32Reed K.L. Fruin A.B. Gower A.C. et al.A neurokinin 1 receptor antagonist decreases postoperative peritoneal adhesion formation and increases peritoneal fibrinolytic activity.Proc Natl Acad Sci U S A. 2004; 101: 9115-9120Crossref PubMed Scopus (76) Google Scholar We recently reported that human preadipocytes bear functional NK-1Rs that signal to proinflammatory pathways.33Karagiannides I. Kokkotou E. Tansky M. et al.Induction of colitis causes inflammatory responses in fat depots: evidence for substance P pathways in human mesenteric preadipocytes.Proc Natl Acad Sci U S A. 2006; 103: 5207-5212Crossref PubMed Scopus (74) Google Scholar This observation along with the notion that obesity represents a low-grade, chronic, subacute inflammation in the adipose tissue34Xu H. Barnes G.T. Yang Q. et al.Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance.J Clin Invest. 2003; 112: 1821-1830Crossref PubMed Scopus (5110) Google Scholar prompted us to evaluate the role of CJ treatment in the development of obesity. The fact that SP is abundant in the stomach, duodenum, and jejunum, all important areas for digestion and nutrient uptake, as well as in hypothalamic areas implicated in feeding behavior, such as the arcuate and ventromedial nuclei, along with the presence of NK-1R in the hypothalamus35Mantyh P.W. Hunt S.P. Maggio J.E. Substance P receptors: localization by light microscopic autoradiography in rat brain using [3H]SP as the radioligand.Brain Res. 1984; 307: 147-165Crossref PubMed Scopus (191) Google Scholar and adipose tissue,33Karagiannides I. Kokkotou E. Tansky M. et al.Induction of colitis causes inflammatory responses in fat depots: evidence for substance P pathways in human mesenteric preadipocytes.Proc Natl Acad Sci U S A. 2006; 103: 5207-5212Crossref PubMed Scopus (74) Google Scholar led us also to investigate a potential role for SP in the regulation of energy balance. In the present study, we describe the effect of NK-1R antagonism in the feeding behavior and weight of the C57BL6 models of diet-induced obesity (DIO) and high-fat (HF) feeding. We report that SP positively influences energy balance and that administration of the NK-1R pharmacologic inhibitor CJ ameliorates the weight gain induced by feeding with HF/high-caloric content diets, and improves the ability to remove glucose from the blood and respond to insulin.Materials and MethodsAnimals and TreatmentsLean miceTwelve-week-old male C57BL6 mice (Taconic, Hudson, NY) were housed individually and fed a HF diet (45 kcal percent fat, 4.73 kcal/g, No. D12451 Research Diets, New Brunswick, NJ) for 5 days before CJ (Pfizer) administration for acclimatization and during the study. Mice (2 separate experiments, n = 10 mice per group per cohort) received daily intraperitoneal (IP) injections of 300 μg CJ in 200 μL saline or saline alone for a week, 1 hour before initiation of the dark cycle. Mouse body weight and food intake were monitored daily at the time of injection. To investigate whether the CJ-mediated effects on body weight were attributed to caloric intake or increases in energy expenditure, we included a third group of mice (pair fed) that had access to restricted food equal in grams to the food consumed by CJ-treated mice during the previous day.DIO modelEight-week-old male C57BL6 mice were placed in single cages and fed a HF diet for 12 weeks. Mice were then divided into the following groups (2 separate experiments, n = 8 mice per group) matched by weight: group C: mice receiving saline injections; group J: mice receiving a low dose of CJ (150 μg/mouse); group JJ: mice receiving a high dose of CJ (300 μg/mouse); group PF: mice pair fed to those receiving the high dose of CJ. Mice were treated for a total of 18 days, and their food consumption and weight were monitored daily 2 hours before the dark cycle.Leptin-deficient (ob/ob) miceOb/ob mice (2 separate experiments, n = 8 per group per cohort) (Jackson Laboratories, Bar Harbor, ME) were housed separately and treated with 300 μg CJ for 7 days, followed by 600 μg CJ for 10 additional days. Food intake and body weight were monitored daily 2 hours before the dark cycle. The control group received mock IP injections of 200 μL saline.Acute effectMale C57BL6 mice (n = 8 per group) were injected IP with CJ (300μg) and either with a low (36 μg/mouse) or a high (72 μg/mouse) dose of SP (Bachem, King of Prussia, PA), 1 hour before the start of the dark cycle (0 hour). Mice were monitored for food intake up to 24 hours and compared with saline-injected controls.Glucose and Insulin Tolerance TestsMice were fasted overnight (20:00–08:00) and tested for glucose and insulin tolerance at the initial and final days of each experiment. Dextrose and insulin (Humulin; Eli Lilly, Indianapolis, IN) were injected IP (1 g/kg and 2 U/kg, respectively), and serum glucose was measured at 0, 15, 30, 60, and 120 minutes via tail blood collection. Glucose levels were measured using standard glucose test strips with an ACCU-CHECK Advantage meter (Roche, Nutley, NJ). The groups were selected before CJ administration and matched for weight.Real-time Reverse-Transcription Polymerase Chain ReactionRNA was isolated from tissues kept at −80°C using the RNeasy kit followed by DNAseQ treatment (Qiagen, Valencia, CA). Fifty nanograms RNA were subjected to reverse-transcription polymerase chain reaction (RT-PCR) amplification using TaqMan 1-step predeveloped assays in a GeneAmp 5700 sequence detection system (Applied Biosystems, Foster City, CA). Results were normalized by TATA-binding protein expression and presented as mRNA arbitrary units (AU).Dual-Energy X-ray AbsorptiometryMice were scanned, using the Lunar PIXImus2 mouse densitometer (GE Medical Systems, Madison, WI), and total body fat and lean body mass were determined using the analysis program as described by the manufacturer.Serum AnalysisSerum insulin and leptin were measured by EIA (Linco, St. Charles, MO) and serum alanine aminotrasferase by Ani Lytics (Gaithersgurg, MD).Statistical AnalysisResults were analyzed by protected analysis of variance (ANOVA) factorial with Dunn/Bonferroni correction, ANOVA repeated measures, and Student t test using Statview (SAS Institute, Cary, NC) and expressed as mean ± SEM; *P < .05 and **P < .001, unless indicated otherwise.ResultsEffects of the NK-1R Antagonist CJ on Weight Gain in Lean MiceCJ was well tolerated with no apparent toxicity as evaluated by serum alanine aminotransferase and tumor necrosis factor (TNF) α levels and gross pathology of all organs, except for a mild tissue reaction at the site of injection. However, CJ-treated mice showed an immediate hyperkinetic response that lasted 30 seconds after the injection with no other apparent unusual behavior relative to the controls. In contrast to vehicle-treated mice that gained on average 8% of their initial body weight, CJ-treated mice lost 3% of their weight within a week of treatment (Figure 1A,P < .001 for weight gain between control and CJ-treated mice). Upon discontinuation of treatment, weight gain (Figure 1B) and food intake (Figure 1C) returned gradually to pretreatment levels, indicating that CJ does not induce a persistent state of wasting. Weight loss was due to a reduction in fat, but not in lean mass, as assessed by epididymal fat pad weight (Figure 2A, 1.5 ± 0.12 g in control mice vs 1.23 ± 0.7 g in CJ-treated mice and 1.27 ± 0.95 g in PF mice, P < .05) and dual-energy x-ray absorptiometry (DEXA) analysis (Figure 2B). Weight loss was also associated with 50% lower serum leptin levels in these mice (Figure 2C, 20.7 ± 1.5 vs 47.1 ± 2.5 vs 28.3 ± 1.5 ng/mL for CJ, vehicle-treated and pair-fed mice, respectively, P < .01). Interestingly, pair-fed mice had 25% higher leptin levels compared with CJ-treated mice (P < .05), which may suggest a potential direct SP effect in the adipose tissue.33Karagiannides I. Kokkotou E. Tansky M. et al.Induction of colitis causes inflammatory responses in fat depots: evidence for substance P pathways in human mesenteric preadipocytes.Proc Natl Acad Sci U S A. 2006; 103: 5207-5212Crossref PubMed Scopus (74) Google ScholarFigure 2CJ-treated mice develop less adiposity when fed a high fat diet. (A) Epididymal fat pad weight in control, CJ-treated and pair-fed mice. (B) CJ treatment affects fat, but not lean, mass in mice, as assessed by DEXA scanning. (C) Reduced serum leptin levels in CJ-treated mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The similar weight loss among CJ-treated and pair-fed mice suggested that CJ might affect appetite. Indeed, CJ administration resulted in 25% reduction in cumulative food intake in the HF fed lean mice (Figure 3, 18.8 ± 0.7 g vs 13.4 ± 0.3 g food intake in, vehicle vs CJ-treated mice, respectively, P < .0001). Consistent with reduced food intake and lower adiposity, CJ-treated mice had also lower serum insulin levels (Figure 4A, 1.4 ± 0.1 vs 1.1 ± 0.1 vs 3.7 ± 0.6 ng/mL, in CJ, pair- fed and controls, respectively, P < .001) and improved responses to glucose (Figure 4B) and insulin tolerance tests (Figure 4C), suggesting increased insulin sensitivity compared with control mice.Figure 3Weight loss in CJ-treated (300 μg) mice is associated with a significant reduction in food intake.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4CJ-treated (300 μg) mice showed significantly improved insulin sensitivity at the end of their treatment. (A) Serum insulin levels. (B) Glucose tolerance test. (C) Insulin tolerance test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To exclude the possibility that the CJ effects on food intake and weight loss are due to illness or malaise, we examined whether CJ administration was associated with a taste aversion response. Our results show that while LiCl administration resulted in a significant decrease (Supplemental Figure 1,P < .0001) in saccharine preference, even a high dose of CJ administration did not have any significant effect. Thus, it does not appear that CJ reduces food intake as a result of malaise or illness. Furthermore, we have observed that CJ administration reduced acutely water intake, like food intake, during the hours of activity by approximately 40%, when compared with saline-injected controls (data not shown, P < .0001).Supplemental Figure 1Saccharin preference in mice during a conditioned taste aversion test. LiCl treatment, but not CJ treatment, significantly reduced saccharin intake. ***P < .0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Acute Effects of CJ and SP on AppetiteCJ treatment before the start of the dark cycle, when mice consume the majority of their food, resulted in reduction of food intake evident as early as 1 hour after injection (Figure 5A, 2.3 ± 0.2 vs 3.4 ± 0.1 g, cumulative food intake of CJ-treated mice vs control respectively, P < .0001). Likewise, during 6 hours of refeeding after an overnight fast, CJ-treated mice consumed 1.1 ± 0.1 g vs 1.9 ± 0.1 g of vehicle-treated control mice (Figure 5B, P < .01). Similar CJ-mediated anorectic effects were also evident when mice were fed standard chow (data not shown). We next treated control mice with 2 different doses of SP (36 or 72 μg/mouse) IP and found that peripheral SP administration resulted in a mild, dose-dependent increase in food intake (Figure 5C, 1.8 ± 0.2, 2.1 ± 0.2, and 2.4 ± 0.2 g for vehicle, SP low-dose-, and SP high-dose-treated mice, respectively, P < .05), evident 3 hours after injection. It has been reported that in rats, intravenous administration of SP induced acute (5–30 minutes) hypoinsulinemia, hyperglucagonemia, and hyperglycemia.36Brown M. Vale W. Effects of neurotensin and substance P on plasma insulin, glucagon and glucose levels.Endocrinology. 1976; 98: 819-822Crossref PubMed Scopus (161) Google Scholar We injected IP random-fed mice with SP (72 μg/mouse), in the absence of food, and found no differences in blood glucose levels up to 4 hours posttreatment (Figure 5D).Figure 5Acute effects of CJ on appetite. (A) In an acute feeding paradigm, mice were treated IP with a single dose of CJ (300 μg) at 30 minutes prior to the start of their feeding cycle (dark cycle), and food intake was monitored at intervals up to 24 hours. (B) CJ also inhibits re-feeding. Mice were food deprived overnight and then treated as above. (C) Mice were treated with a single low (36 μg) or high (72 μg) dose of SP as in (A) and their food intake was monitored acutely. (D) Mice were injected IP with SP (100 μg) at the start of their light cycle and food was removed from the cages. Glucose levels were measured at various time points in tail vein blood by a glucometer. (E) Mice were treated IP with a single dose of SP (100 μg) at the start of the light cycle in the absence of food, and hypothalamic expression of the appetite regulating neuropeptides POMC and NPY was measured after 3 hrs by real-time RT-PCR.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Food intake is under the control of several hypothalamic neuropeptides, the orexigenic neuropetide-tyrosine (NPY) and the anorexigenic pro-opiomelanocortin among them.4Badman M.K. Flier J.S. The gut and energy balance: visceral allies in the obesity wars.Science. 2005; 307: 1909-1914Crossref PubMed Scopus (388) Google Scholar We thus investigated whether SP administration had an effect on the hypothalamic expression of appetite-regulating neuropeptides. Mice (n = 16/group) were injected IP with SP (100 μg/mouse) immediately after the end of their feeding cycle, and food was removed from their cages. After 3 hours, the hypothalami were extracted, and RNA was analyzed by real-time PCR. We found a small not statistically significant increase in NPY mRNA expression and reduced pro-opiomelanocortin (POMC) expression in 2 separate cohorts of mice (Figure 5E). Such a finding seems compatible with the food intake pattern in mice treated acutely with SP (Figure 5C).Effects of NK-1R Antagonism on Food Intake and Body Weight in Leptin-Deficient ob/ob MiceWe next investigated whether the effects of CJ in food intake and body weight were leptin mediated, using leptin-deficient ob/ob mice. During the study period, body weight was increased by 4.9 ± 0.53% in vehicle-treated mice and decreased by 8.0 ± 2.1% with CJ treatment (Figure 6A,P < .0001). The effect of CJ on body weight reduction was dose dependent and associated with 30% less adiposity (Figure 6B, 2.1 ± 0.2 vs 1.4 ± 0.1 g of epididymal fat in vehicle- and CJ-treated mice, respectively, P < .001). Consistent with our results with the HF fed lean mice (Figure 3), CJ-treated ob/ob mice had reduced appetite as measured by their food consumption (Figure 6C, 63.9 ± 1.3 vs 44.6 ± 2.3 g for vehicle- and CJ-treated mice, respectively, P < .0001), which could explain, at least in part, the weight loss evident in these mice (Figure 6A). We also examined whether hypothalamic SP expression is under the control of leptin, as was observed with the appetite-controlling peptide NPY.4Badman M.K. Flier J.S. The gut and energy balance: visceral allies in the obesity wars.Science. 2005; 307: 1909-1914Crossref PubMed Scopus (388) Google Scholar We found that fasting of wild-type mice, which results in a significant drop of leptin levels, did not affect hypothalamic SP mRNA expression, whereas it increased NPY mRNA expression in the same animals (Figure 6D).Figure 6CJ treatment reduces food intake and weight gain in leptin-deficient mice. (A) There was a significant reduction in the weight of leptin-deficient (ob/ob) mice treated with CJ (300 μg for 7 days followed by 600 μg for 10 additional days). (B) Epididymal fat pad weight and (C) food intake in mice treated as in (A). (D) Hypothalamic expression of SP and NPY (as a positive control) was evaluated in fed and overnight fasted wild-type mice (n=6/group) by real time RT-PCR.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of CJ Treatment on Body Weight and Adiposity in DIO MiceHaving established that NK-1R receptor antagonism prevents weight gain in lean mice on a HF diet, we next examined whether CJ treatment could stimulate weight loss in mice with DIO. We found that, at the end of the treatment period, mice treated with the higher dose of CJ (JJ) lost 14.5% ± 2.6% and pair-fed mice lost 11.1% ± 2.0% of their initial body weight. In contrast, control mice slightly increased their body weight by 2.2 ± 1.9% (Figure 7A,P < .0001). Interestingly, CJ-mediated weight loss was also reflected in a 30% reduction in epididymal fat mass, whereas pair-fed mice d