Supplemental methods. Total RNA was extracted from the stomach, liver, pancreas, pituitary, and

Transcription

1 Supplemental methods Real-time quantitative RT-PCR and Semi-quantitative PCR Total RNA was extracted from the stomach, liver, pancreas, pituitary, and hypothalamus as previously described (). Primers and taqman probes are shown in supplemental table 1. Real-time quantitative PCR was performed using an ABI PRISM Sequence Detection System as described () (Applied Biosystems, Foster City, CA). For each gene, mrna expression levels were normalized to the levels of 1S ribosomal RNA. Semi-quantitative PCR was performed using sense '-GCATGCTCTGGATGGACATG-' and antisense '-TGGTGGCTTCTTGGATTCCT -' primers. Thirty-five cycles were performed at C for s, C for s, and C 11 for s. 1 Glucose and Insulin Tolerance Test (GTT and ITT) 1 After a 1h fast, glucose tolerance testing was performed in which mice were 1 injected intra-peritoneally (i.p.) with 1. g/kg (-week-old mice) or. g/kg 1 (-week-old mice) glucose. Insulin tolerance testing was performed after a h fast; 1 mice were injected i.p. with. units/kg (-week-old mice) or 1. units/kg 1 (-week-old mice) human regular insulin (Hummulin R; Eli Lilly Japan, Japan). Blood 1 glucose levels were measured at,,,, and min after injection. 1

2 Insulin release After a 1h fast, mice were injected i.p. with 1. g/kg glucose. Insulin was measured from blood samples collected,, and min after injection. Measurement of plasma ghrelin, des-acyl ghrelin, and Trp -ghrelin levels and Reverse phase HPLC Blood samples were collected from the inferior vena cavas of mice under anesthesia with diethyl ether. Samples were immediately transferred to polypropylene tubes containing 1 mg/ml Na EDTA and kallikrein inactivator U/ml aprotinin (Ohkura Pharmaceutical, Inc., Kyoto, Japan) and centrifuged at C. For N-RIA, hydrogen chloride was added to samples at a final concentration of.1 N immediately 11 after separating plasma. Plasma ghrelin was measured as previously reported (). 1 Briefly, samples were applied to Sep-Pak C1 cartridges prior to performing C-RIA and 1 N-RIA. Plasma samples were also subjected to reverse phase HPLC as described (1). 1 Measurements of body weights and length 1 Body weights were measured weekly beginning at two weeks of age. Body 1 lengths of -week-old mice were represented by measuring a single individual with 1 manual immobilization and extension of mice to determine the nose to-anus length. 1 Measurement of daily food intake

3 Eight-week-old mice were placed in individual cages; food intake was monitored every 1 hours (-) for a two-week period, facilitating the calculation of average daily food intake. Measurement of biochemical and hormonal parameters The levels of serum GH, insulin-like growth factor-i (IGF-I), insulin, NEFA, T.Cho, and TG levels were determined using a rat growth hormone EIA kit (SPI bio, Massy Cedes, France), a mouse IGF-I immunoassay kit (R&D Systems, Inc., Minneapolis, MN), a rat insulin ELISA kit (Morinaga, Yokohama, Japan), a NEFA C Assay kit, (Wako Pure Chemical Industries, Ltd., Osaka, Japan), Cholesterol E Assay kit, (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and Triglyceride E Assay kit 11 (Wako Pure Chemical Industries, Ltd., Osaka, Japan), respectively. We measured blood 1 glucose levels by the glucose oxidase method using a Glutest sensor (Sanwa Kagaku, 1 Kyoto, Japan). 1 Measurement of total body fat percentage and lean body mass 1 Mice were anesthetized with pentobarbital. Total body fat percentages and lean 1 body masses were measured by computed tomography (Latheta LTC-, ALOKA, 1 Tokyo, Japan). 1 Statistical analyses

4 Results were expressed as means ± SEM. Statistical significance for Fig1A-C, FigB, D, F, FigA-D, Supplemental Fig1B-E, and Supplemental FigA, B were determined by two-sided Student s t test. Statistical significance for FigA, C, E, and Supplemental Fig1A, E, F were determined by repeated measures analysis of variance. P values less than. were considered to be statistically significant. All statistical analyses were performed by StatView (version.; SAS Institute, Inc., Cary, NC)

5 Supplemental Table 1: PCR primers and Taqman probes ghrelin GOAT Insulin 1 GH GHRH somatostati n (SST) NPY AgRP Orexin GHS-R sense '-GCATGCTCTGGATGGACATG-' antisense '-TGGTGGCTTCTTGGATTCCT-' probe '-AGCCCAGAGCACCAGAAAGCCCA-' sense '-AGGGACTCTAGGAAGGACAG-' antisense '-CCCATCTGAAAGAAGAAGGT-' with power SYBR Green sense '-CAGCTATAATCAGAGACCATCAGCAA-' antisense '-GGGTAGGAAGTGCACCAACAG-' probe '-CAGGTCATTGTTTCAAC-' sense '-AAGAGTTCGAGCGTGCCTACA-' antisense '-GAAGCAATTCCATGTCGGTTC-' probe '-CCATTCAGAATGCCCAGGCTGCTTTC-' sense '-AGGATGCAGCGACACGTAGA-' antisense '-TCTCCCCTTGCTTGTTCATGA-' probe '-CCACCAACTACAGGAAACTCCTGAGCCA-' sense '-AGCTGAGCAGGACGAGATGAG-' antisense '-ACAGGATGTGAATGTCTTCCAGTT-' probe '-CGAACCCAGCAATGGCACCCC-' sense '-TCCGCTCTGCGACACTACAT-' antisense '-GGAAGGGTCTTCAAGCCTTGT-' probe '-CAAGGGCTGGATCTCTTGCCATATCTCTG-' sense '-GCTCCACTGAAGGGCATCA-' antisense '-TAGCACCTCCGCCAAAGCT-' probe '-TTCCCAGGTCTAAGTCTGAATGGCCTCA-' sense '-TCCAGATTACTCCCCTGAGC-' antisense '-TCACGGCGGCCCAGGGAACC-' probe '-CAGGCACCATGAACTTTCCTTCTACAAAAGTT- sense '-CACCAACCTCTATCCAGCAT-' antisense '-CTGACAAACTGGAAGCGTTTGCA-' probe '-TCCGATCTGCTCATCTTCCTGTGCATG-'

6 Supplemental Figure legends Supplemental Figure 1: The analysis of Trp -ghrelin transgenic mice in early life. A: Changes in body weight of Trp -ghrelin- mice (closed triangles) and non-transgenic littermates (open squares) from to weeks of age (n = 1-1). B: Body lengths (nose-to-anus length) of -week-old Trp -ghrelin- mice (closed bars) and non-transgenic littermates (open bars) (n = -1).) and non-transgenic littermates (open squares) (n = 1-1). C and D: Serum GH (C) and IGF-I (D) levels were assessed in -week-old Trp -ghrelin- mice (closed bars) and non-transgenic littermates (open bars) (n = ). E: Average daily food intake of Trp -ghrelin- mice (closed bars) and non-transgenic littermates (open bars) from eight to weeks of age (n = -). 11 F: Glucose tolerance test using injection of 1. g/kg glucose was performed in 1 -week-old Trp -ghrelin- mice (closed triangles) and non-transgenic littermates 1 (open squares) (n= 1-1). 1 G: Insulin tolerance test with injection of regular insulin. U/kg was performed in 1 -week-old Trp -ghrelin- mice (closed triangles) and non-transgenic littermates 1 (open squares) (n = 1-1). 1 Data are presented as the means ± SEM. 1

7 Supplemental Figure : Hypothalamic and pituitary mrna levels of factors involved in GH regulation and food intake. A: The mrna levels of GH and the GH secretagogue receptor (GHS-R) in the pituitaries of -week-old Trp -ghrelin- mice (closed bars) and non-transgenic littermates (open bars). B: mrna levels of GH-releasing hormone (GHRH), somatostatin (SST), neuropeptide Y (NPY), agouti-related protein (AgRP), orexin (OR), and GHS-R in the hypothalamus samples from -week-old Trp -ghrelin- mice (closed bars) and non-transgenic littermates (open bars). n =, Data are presented as the means ± SEM.

8 A Body weight (g) non- B Body length (cm) 1 Weeks non- C D E Serum GH (ng/ml) 1 Serum IGF-I (ng/ml) Food intake (g/day) non- non-. non- E F Blood glucose (mg/dl) non- Time (min) Glucose change (%) non- 1 Time (min) Supplemental Figure 1

9 A. B. Arbitrary unit Arbitrary unit non GH non GHS-R. non- non- non- non- non- non- GHRH SST NPY AgRP OR GHS-R Supplemental Figure