Volume 138, Issue 7, Pages 2468-2476.e1 (June 2010) The Hyperphagic Effect of Ghrelin Is Inhibited in Mice by a Diet High in Fat  James V. Gardiner, Daniel Campbell, Michael Patterson, Aysha Kent, Mohammed A. Ghatei, Stephen R. Bloom, Gavin A. Bewick  Gastroenterology  Volume 138, Issue 7, Pages 2468-2476.e1 (June 2010) DOI: 10.1053/j.gastro.2010.02.012 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Excess ghrelin protects against diet-induced obesity by reducing food intake. Male mice (n = 6) were fed either RC or an HFD from 5 weeks of age. (A) Cumulative energy intake; (B) body weight curves; (C) body composition measured at 16 weeks of age; (D) plasma leptin concentration measured at 16 weeks of age; (E) cumulative food intake measured by the comprehensive laboratory animal monitoring system (CLAMS); (F) average light and dark phase food intake measured by the CLAMS. Data represent mean ± standard error of mean. *P < .05 compared with TG HF. Gastroenterology 2010 138, 2468-2476.e1DOI: (10.1053/j.gastro.2010.02.012) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 High-fat feeding does not exacerbate ghrelin-induced impairment of glucose tolerance. Male mice (n = 6) were fed either RC or HFD from 5 weeks of age. (A) Fasting plasma glucose; (B) fasting insulin levels measured at 16 weeks of age; (C) plasma glucose disposal during a glucose tolerance test at 14 weeks of age; (D) area under the curve for C *P < .05 compared with RC Wt; (E) plasma glucose during an insulin tolerance test at 15 weeks of age; (F) area under the curve for E. Data represent mean ± standard error of mean. *P < .05. Gastroenterology 2010 138, 2468-2476.e1DOI: (10.1053/j.gastro.2010.02.012) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Food preference, hypothalamic neuropeptide expression, and acylation substrate are unchanged by high-fat feeding. (A) Average daily food intake in male mice (n = 8) fed a choice of RC and HFD from 5 weeks of age; (B) proopiomelanocortin (POMC) and neuropeptide Y (NPY) messenger RNA expression in whole hypothalamus extracts from male mice (n = 6) fed an HFD. Acylated species of ghrelin in Wt (C and D) and Tg (E and F) plasma from mice fed RC HFD resolved using FPLC. Data represent mean ± standard error of mean. *P < .05. Gastroenterology 2010 138, 2468-2476.e1DOI: (10.1053/j.gastro.2010.02.012) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Chronic administration of ghrelin does not increase food intake under high-fat feeding conditions. Male mice (n = 8) were implanted with subcutaneous osmotic minipumps, which delivered 0.6 nmol/g/day of ghrelin or saline intraperotoneally. Mice were fed either regular or HFD. (A) Plasma active ghrelin (*P < .05); (B) body weight; (C) cumulative food intake; (D) cumulative energy intake. Data represent mean ± standard error of mean. (*P < .05 between saline and ghrelin-treated mice fed regular chow.) Gastroenterology 2010 138, 2468-2476.e1DOI: (10.1053/j.gastro.2010.02.012) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 A circulating acylated ghrelin measured in male mice fed an HFD n = 9. Male mice n = 6 were fed either RC or an HFD from 5 weeks of age and placed in the CLAMS. The ClAMS measured B Lococmotor activity, C energy expenditure and D respiratory quotient. E Brown adipose tissue uncoupling protein mRNA was measured at 16 weeks of age in male mice n = 6 fed either RC or HFD from 5 weeks of age. Data are represented mean ± SEM, (*P < 0.05). Gastroenterology 2010 138, 2468-2476.e1DOI: (10.1053/j.gastro.2010.02.012) Copyright © 2010 AGA Institute Terms and Conditions