Roux-en-Y gastric bypass surgery but not vertical sleeve gastrectomy decreases bone mass in male rats

Transcription

1 SUPPLEMENTAL DATA Roux-en-Y gastric bypass surgery but not vertical sleeve gastrectomy decreases bone mass in male rats 1 Kerstin Stemmer, 2 Maximilian Bielohuby, 3 Bernadette E. Grayson, 3 Denovan P. Begg, 3 Adam P. Chambers, 1 Christina Neff, 3 Stephen C. Woods, 4 Reinhold G. Erben, 1 Matthias H. Tschöp, 2 Martin Bidlingmaier, 5 Thomas L. Clemens and 3* Randy J. Seeley 1 Institute for Diabetes and Obesity, Helmholtz Centre Munich, Neuherberg, Germany 2 Endocrine Research Unit, Medizinische Klinik und Poliklinik IV, Klinikum der LMU, Munich, Germany. 3 Metabolic Diseases Institute, University of Cincinnati, Cincinnati, Ohio, USA 4 Department of Biomedical Sciences, University of Veterinary Medicine Vienna, Vienna, Austria 5 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Corresponding Author

2 Supplemental Methods: Gene expression analysis: RNA from snap frozen livers or RNAlater (Qiagen, CA) fixed femoral bone marrow was isolated using RNeasy Lipid Tissue mini kit (Qiagen, CA). RNA was transcribed into cdna by using Superscript III reverse transcriptase, according to the manufacturer s instructions (Invitrogen, CA). Quantitative real-time PCR (Q-PCR) reactions were performed in an ABI Prism 7900HT Sequence Detection System by using TaqMan probes specific for rat fibroblast growth factor 21 (FGF21; Rn _g1), osteocalcin (BGLAP, Rn _g1), osteoclast-associated receptor (OSCAR; Rn _m1) and receptor activator of NF-κB ligand (RANKL, Rn _m1). Relative expression levels were normalized to the housekeeping genes ß-actin (Rn _s1) or hypoxanthine phosphoribosyltransferase 1 (HPRT-1; Rn _m1) and expression changes were evaluated using the delta-delta Ct (ΔΔCt) method.

3 Supplemental Figure 1: A B Liver FGF21 / ß-actin Osteocalcin / HPRT-1 C D OSCAR / HPRT RANKL / HPRT Supplemental Figure 1: Gene expression changes in liver and bone marrow following and surgery. (A) Liver fibroblast growth factor 21 (FGF21) expression levels were similar at day 60 post surgery in operated rats and sham controls and tended to be decreased in operated rats. (B) RNA expression levels of the bone formation marker osteocalcin showed a non-significant decrease in the bone marrow of but not operated rats. (C-D) Bone resorption markers osteoclast-associated receptor (OSCAR) (C) and receptor activator of NF-κB ligand (RANKL) (D) showed a trend towards an increased expression in but not rats. All data are expressed as mean ± standard error of mean (SEM). Statistical differences between surgical groups were analyzed using one-way ANOVA followed by a Tukey post hoc test. Analyses were done using GraphPad Prism 6.0 software. A p-value < 5 was considered statistically significant.

4 Supplemental Figure 2:

5 Supplemental Figure 2: Dietary supplementation following roux-en-y gastric bypass () has no influence on body composition, food intake, glucose tolerance and lipid profiles. Following sham surgery or, groups of rats (N = 10) were either fed a regular high fat diet (HFD, 40% fat; 4.54 kcal/g, D , Research Diets, New Brunswick, NJ) or an isocaloric HFD diet, modified by the addition of 2200 IU vitamin D per kg, 2% calcium (calcium phosphate and calcium carbonate), 1.25% phosphorus and the replacement of 20% of carbohydrates from corn starch, maltodextrin 10 and sucrose with lactose (Research Diets D ) for 60 days. (A) Dietary supplementation with calcium, vitamin D, phosphate and lactose did not affect the significant body weight reduction (***p < 01) in operated rats. (B) Cumulative food intake was significantly lower following surgery (**p < 1), but did not differ in the supplemented vs. non-supplemented groups. (C) Similar and significant fat mass change in (***p < 01) and supplemented (***p < 01) rats (D) No significant changes in lean tissue mass were observed in any treatment group. (E) Blood glucose levels before (time 0) and after an oral mixed-meal tolerance test indicate that glucose excursions were decreased to a similar extend in supplemented and nonsupplemented -operated rats compared to the respective sham-operated controls. (F) Area under the curve (AUC) for plasma glucose across the 2 hours glucose tolerance test showed a significant decrease in - (**p < 1) and supplemented -operated (*p < 5) rats compared to sham controls. (G) surgery produced a significant increase in postprandial GIP release 15 min following a mixed meal tolerance test (*p < 5 and ***p < 01) which was not affected by dietary supplementation. (F) Similar significantly improved levels of serum triglyceride (**p < 1) in supplemented and non-supplemented operated rats, vs. the respective sham controls.