Critical role for peptide YY in protein-mediated satiation and bodyweight

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1 Cell Metabolism, Volume 4 Supplemental data Critical role for peptide YY in protein-mediated satiation and bodyweight regulation Rachel L. Batterham, Helen Heffron, Saloni Kapoor, Joanna E. Chivers, Keval Chandarana, Herbert Herzog, Carel W. Le Roux, E. Louise Thomas, Jimmy D. Bell, Dominic J. Withers Supplemental experimental procedures Human studies and assays Test meals were prepared using conventional pasta (Sainsbury, London UK) or highprotein pasta (Atkins, Shelton, CT, USA) with a tomato based sauce. Additional protein, carbohydrate and fat supplements were added (Maxipro, UK, Maxijuel and Calogen, SHS International, Merseyside, UK) to the tomato sauce and dessert. Blood was collected into EDTA tubes containing 5000 kallikrein inhibitor units of aprotonin (Bayer, Newbury, Berks, U.K.) per ml. Plasma samples were separated immediately by centrifugation at 4 C. For subsequent PYY (total and PYY3-36) and active GLP-1 DPPIV inhibitor (Linco Research Inc., St Louis, MO, USA) was added to plasma samples to give a final concentration of 100 µm. Samples for analysis of active ghrelin had 100 µl of 1M HCL added per ml of plasma. ELISA kits were used to quantify desacyl-ghrelin, active-ghrelin, active-glp 1 and leptin (Linco Research Inc, St Louis, MO, USA). Total PYY and PYY3-36 were quantified by radioimmunoassay (Linco Research Inc., St Louis, MO, USA). Insulin was analyzed by microparticle enzyme immunoassay (AxSYM, Abbott laboratories, UK). Plasma glucose was measured using a hexokinase method (Olympus AU640 analyser, Olympus, Germany)

2 Murine studies Blood was taken by cardiac puncture under terminal anaesthesia and collected as above. The mediobasal hypothalamus, the stomach, a 3 cm section of the terminal ileum and a 3 cm section of colon were dissected, snap frozen in liquid nitrogen and then stored at -80 C. For assays, insulin and leptin ELISAs (Crystal Chem, Chicago, USA), active ghrelin and desacyl ghrelin ELISAs (Linco Research Inc, MO, USA) and total PYY ELISAs (DSL, Texas, USA) were used. RT-PCR for hypothalamic and gut peptide mrna RT-PCR was performed as previously described (Choudhury et al., 2005) using proprietary sequence Taqman Gene Expression assay FAM/TAMRA primers. For Pyy, pancreatic polypeptide (Ppy) and ghrelin mrna expression relative to transcription elongation factor A (SII) 1 (Tcea1) was calculated. For Npy, Pomc and Agrp expression relative to hypoxanthine phosphoribosyl transferase-encoding gene (Hprt1) was calculated. Magnetic resonance imaging (MRI) MRI was performed on terminally anaesthetised mice as previously described (Choudhury et al., 2005). Targeting of the mouse Pyy gene. The mouse Pyy gene locus was cloned from a 129Sv mouse genomic PAC library. A targeting vector was constructed with an frt site-flanked neomycin cassette placed 5 of the

3 Pyy gene which was itself flanked by loxp sites (Figure 3). ES cell targeting, generation of chimaeric mice and breeding to germ-line transmission was performed as previously described with Southern blotting used to identify homologous recombination and germ-line transmission (Choudhury et al., 2005). In vivo deletion of the neomycin cassette and the Pyy locus was achieved through crossing these mice with eflp-expressing mice (Dymecki, 1996) and with EIIa-Cre transgenic mice (Lakso et al., 1996) both of which were maintained on a pure C57Bl/6 background. These events were identified using PCR primers as indicated in Figure 3C and D. Mice were backcrossed after each stage of these crosses onto pure C57Bl/6 to remove the eflp and EIIa-Cre alleles. The resulting mice, heterozygote for the Pyy null allele, were then intercrossed to obtain Pyy null, heterozygote and wild type littermate mice. The breeding strategy undertaken to delete the neomycin cassette and Pyy locus as described above effectively resulted in four backcrosses into pure C57Bl/6 mice and therefore the predominant (>90%) genetic background for the animals used in these studies was C57Bl/6. Male and female Pyy null mice both displayed the energy homeostasis phenotype. We also maintained in the homozygous state and on the same genetic background Pyy lox/lox mice which lacked the neomycin cassette but retained the loxp flanked Pyy locus. Genotyping of mice was performed by PCR amplification of tail DNA (Figure 3E). ELISA for PYY confirmed the absence of detectable plasma PYY in fed Pyy null mice (Figure 3F). Ppy mrna expression was normal in pancreatic tissue (Figure 3G). The generation and genotyping of Npy2r null mice has been described previously (Baldock et al., 2002).

4 Supplemental Table S1: Test meal composition for human diet studies. Isocaloric meals with high-protein (HP), high-carbohydrate (HC) and high-fat (HF) were used. HP HC HF kcals % kcals % kcals % CHO Pasta Fat Protein Total CHO Dessert Fat Protein Total CHO Total meal Fat Protein Total

5 Normal weight Obese Diet HC HF HP HC HF HP 8,052 ± ,258 1 ± ,246 3 ± ,388 8 ± ,202 3 ± ,899 1 ± Total PYY AUC pmol/l.180 min HP vs. HC P < HP vs. HF P < 0 05 HF vs. HC P < 0 01 Active ghrelin AUC pmol/l.180 min GLP-1 AUC pmol/l.180 min Leptin ng/ml Time 0 Leptin ng/ml Time 180 Insulin AUC pmol/l.180 min 4, ,838 1 ± ± HP vs. HC P > ,208 9 ± ,241 5 ± HP vs. HC P > ± 0 31 ± ± 0 22 ± HP vs. HC P > ,205 ± 17, 160 7,173 ± 2,274 HP vs. HC P < HF vs. HC P < ,347 ± ,306 3 ± ± ± ,074 ± 2,679 HP vs. HC P < 0 05 HP vs. HF P < , ,165 1 ± 340 ± HP vs. HC P > ± ± HP vs. HC P > ± 3 27 ± ± 2 90 ± 2 58 HP vs. HC P > ,313 49,092 ± 26,791 ± 5,889 HP vs. HC P < HP vs. HF P > 0 5 HF vs. HC P < ,842 3 ± ± ± ± ,400 ± 11,061 Supplemental Table S2. Metabolic characteristics in human subjects during studies with high-protein (HP), high-carbohydrate (HC) and high-fat (HF) meals. Values represent mean

6 ± SEM or integrated area under the curve (AUC) profiles, n = 9 for obese group and n = 10 for normal weight group HFNP HFHP LFNP LFHP CHO (% energy) Fat (% energy) Protein (% energy) Energy/g kcal Supplemental Table S3. Mouse diet composition. Composition of the high-fat normalprotein (HFNP), high-fat high-protein (HFHP), low-fat normal-protein (LFNP) and low-fat high-protein (LFHP) diets used in mouse studies.

7 Figure S1. Active ghrelin and active GLP-1 levels in normal weight and obese human subjects following high-protein, high-fat and high carbohydrate meals. A and B) Time course of plasma active ghrelin in normal weight subjects (A) and obese subjects (B) after ingestion at time 0 of isocaloric meals. C and D) Time course of plasma active GLP-1 in normal weight subjects (C) and obese subjects (D) after ingestion at time 0 of isocaloric meals. PROT = high-protein. CHO = high-carbohydrate. FAT = high-fat. All

8 values represent group mean ± SEM, n = 10 for normal weight subjects and n = 9 for obese subjects.

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10 Figure S2. Effect of high-protein diets on plasma ghrelin profiles, leptin, gut and hypothalamic peptide expression in mice. For all panels high-fat normal-protein (HFNP: blue symbols), high-fat high-protein (HFHP: green symbols), low-fat normal-protein (LFNP: black symbols) and low-fat high-protein (LFHP: red symbols) diets. A) Plasma active ghrelin profiles in 10-week-old male C57Bl/6 mice after ingestion of isocaloric meals, n = 6 per diet. B) Plasma desacyl ghrelin levels in 10-week-old male C57Bl/6 mice after ingestion of isocaloric meals, n = 6 per diet C) Plasma leptin levels in male C57Bl/6 mice exposed to 16 weeks of different diets, n = 10 per diet. HFNP vs. HFHP, ** p < 0.01; LFNP vs. LFHP # p < D) Fasted (fast) and fed ileal Pyy mrna levels in male C57Bl/6 mice after 16 weeks of different diets, n = 10 per diet (HFNP vs. HFHP, * p < 0.05; LFNP vs. LFHP, # p < 0.05). Pyy mrna levels are expressed as relative to Tcea1. E) Fasted (fast) and fed plasma active ghrelin levels in male C57Bl/6 mice after 16 weeks on different diets, n = 10 per diet. F) Fasted (fast) and fed stomach ghrelin mrna levels in male C57Bl/6 mice after 16 weeks on different diets, n = 10 per diet. Ghrelin mrna levels are expressed as relative to Tcea1. G) Fasted (fast) and fed hypothalamic Pomc mrna levels in male C57Bl/6 mice after 16 weeks of different diets, n = 10 per diet. Pomc mrna levels are expressed as relative to Hprt. All values represent group mean ± SEM.

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12 Figure S3. Pyy null mice have increased body fat, leptin and normal hypothalamic Pomc mrna levels and PYY lox/lox mice have normal body weight. A and B) Volume of internal adipose tissue (A) and subcutaneous adipose tissue (B) in 10- week-old male wild type control (WT) and Pyy null mice determined by MRI, n = 6 per group, *** p < C) Plasma leptin levels in 10-week-old male WT and Pyy null mice, n = 10 per group, ** p < D) Body weights in 8 and 26-week-old male WT and PYY lox/lox mice, n = 15 per genotype. E) Fasted (fast) and fed hypothalamic Pomc mrna levels in 10-week-old male WT and Pyy null mice. Pomc mrna levels are expressed as relative to Hprt. All values represent group mean ± SEM. Supplemental references Baldock, P.A., Sainsbury, A., Couzens, M., Enriquez, R.F., Thomas, G.P., Gardiner, E.M., and Herzog, H. (2002). Hypothalamic Y2 receptors regulate bone formation. J. Clin. Invest. 109, Choudhury, A.I., Heffron, H., Smith, M.A., Al-Qassab, H., Xu, A.W., Selman, C., Simmgen, M., Clements, M., Claret, M., Maccoll, G., et al. (2005). The role of insulin receptor substrate 2 in hypothalamic and beta cell function. J. Clin. Invest. 115, Dymecki, S.M. (1996). Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice. Proc. Natl. Acad. Sci. USA 93, Lakso, M., Pichel, J.G., Gorman, J.R., Sauer, B., Okamoto, Y., Lee, E., Alt, F.W., and Westphal, H. (1996). Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl. Acad. Sci. USA 93,