Gastric mucosal devitalization reduces adiposity and improves lipid and glucose metabolism in obese rats  Andreas Oberbach, MD, PhD, DrPH, MPH, Nadine Schlichting, PhD, Marco Heinrich, PhD, Yvonne Kullnick, MSc, Ulf Retschlag, MSc, Stefanie Lehmann, PhD, MPH, Mouen A. Khashab, MD, Anthony N. Kalloo, MD, Vivek Kumbhari, MD  Gastrointestinal Endoscopy  Volume 87, Issue 1, Pages 288-299.e6 (January 2018) DOI: 10.1016/j.gie.2017.04.038 Copyright © 2018 American Society for Gastrointestinal Endoscopy Terms and Conditions

Figure 1 Regeneration of rat gastric mucosa after treatment with gastric mucosa devitalization (GMD). A, Image of untreated rat stomach illustrates a thick layer of mucosa. B, Immediately after GMD, the mucosa was essentially completely devitalized (arrowhead). C-E, Regeneration of the mucosal layer over the 8-week follow-up period. GMD, gastric mucosal devitalization. (H&E, orig. mag. ×10). Gastrointestinal Endoscopy 2018 87, 288-299.e6DOI: (10.1016/j.gie.2017.04.038) Copyright © 2018 American Society for Gastrointestinal Endoscopy Terms and Conditions

Figure 2 Expression of ghrelin-positive cells in rat stomach mucosa after gastric mucosa devitalization (GMD) treatment. A, Immunofluorescence image of untreated rat stomach mucosa against ghrelin (red) displays moderate expression (arrows). B, Immediately after GMD was performed, there was complete devitalization of the mucosa with no detectable ghrelin-positive cells. C, Two weeks after GMD, the mucosa was nearly completely devitalized with a paucity of detectable ghrelin-positive cells. D, At 4 weeks, the mucosa illustrated signs of regeneration and the presence of a slightly increased number of ghrelin-positive cells. E, At 8 weeks, the mucosa was even more reorganized, with a further increased number of ghrelin-positive cells. Nuclei are stained with DAPI (blue). GMD, gastric mucosa devitalization (DAPI, orig. mag. ×10). Gastrointestinal Endoscopy 2018 87, 288-299.e6DOI: (10.1016/j.gie.2017.04.038) Copyright © 2018 American Society for Gastrointestinal Endoscopy Terms and Conditions

Figure 3 Gastric mucosal devitalization (GMD) produces a significant reduction in body weight and visceral and subcutaneous adiposity compared with sham surgery rats. Animals were killed after 11 weeks of feeding (chow diet, high-fat diet) to measure body composition at 15 weeks. After intervention (GMD, vertical sleeve gastrectomy [VSG], sham surgery), animals were killed at 2, 4, and 8 weeks. A, GMD rats had a significant reduction in body weight at 2, 4, and 8 weeks. B, There was a significant reduction in visceral adiposity in GMD rats compared with sham surgery rats at 2, 4, and 8 weeks. Similar changes were noted when VSG rats were compared with sham surgery rats. C, There was a significant reduction in subcutaneous adiposity in GMD rats compared with sham surgery rats at 2, 4 and 8 weeks. Similar changes were noted when VSG rats were compared with sham surgery rats. GMD, gastric mucosal devitalization; VSG, vertical sleeve gastrectomy; SH, sham surgery; HFD, high-fat diet. Gastrointestinal Endoscopy 2018 87, 288-299.e6DOI: (10.1016/j.gie.2017.04.038) Copyright © 2018 American Society for Gastrointestinal Endoscopy Terms and Conditions

Figure 4 Gastric mucosal devitalization (GMD) resulted in a significant reduction in liver lipid content and size. A, Liver lipid content was measured at 2, 4, and 8 weeks after surgery. GMD rats had significantly reduced liver lipid content at 4 and 8 weeks compared with sham surgery rats. The increase in lipid content at 2 weeks was likely a consequence of liver metabolism of fat as a result of rapid reduction in visceral adiposity after intervention. B, Hematoxylin & eosin staining demonstrates the increased intracellular amount of lipid droplets in high-fat diet rats compared with chow diet rats (H&E, orig. mag. ×40). Immunofluorescence illustrates lipid droplet concentration and size at 4 weeks (Nile red stain, orig. mag. ×40). GMD results in a dramatic reduction in the concentration and size of lipid droplets. CD, chow diet; HFD, high-fat diet; GMD, gastric mucosa devitalization; VSG, vertical sleeve gastrectomy; SH, sham surgery. Gastrointestinal Endoscopy 2018 87, 288-299.e6DOI: (10.1016/j.gie.2017.04.038) Copyright © 2018 American Society for Gastrointestinal Endoscopy Terms and Conditions

Figure 5 Gastric mucosal devitalization (GMD) rats had a lower body weight and reduced intrabdominal adiposity compared with sham surgery rats. The morphologic findings at 8 weeks postoperative were compared with those of sham surgery. GMD rats appeared smaller in size compared with sham surgery rats. Additionally, GMD rats had grossly diminished adiposity of the greater omentum and surrounding intestinal organs. Findings were quantified in Figure 2. HFD, high-fat diet; SH, sham surgery; GMD, gastric mucosal devitalization; VSG, vertical sleeve gastrectomy. Gastrointestinal Endoscopy 2018 87, 288-299.e6DOI: (10.1016/j.gie.2017.04.038) Copyright © 2018 American Society for Gastrointestinal Endoscopy Terms and Conditions

Figure 6 Serum and liver total bile acids and cholecystokinin after 2 hours oral glucose tolerance stimulation test. Bile acids are signalling molecules that regulate lipid, glucose, and energy metabolism. A, Gastric mucosa devitalization (GMD) resulted in an early increase in serum bile acid levels at 2 weeks (176%). B, GMD resulted in an early increase in liver bile acid levels at 2 weeks (75%). C, GMD resulted in a significant increase in cholecystokinin at 4 weeks compared with sham surgery (455%). CD, chow diet; HFD, high-fat diet; GMD, gastric mucosal devitalization; VSG, vertical sleeve gastrectomy; SH, sham surgery. Gastrointestinal Endoscopy 2018 87, 288-299.e6DOI: (10.1016/j.gie.2017.04.038) Copyright © 2018 American Society for Gastrointestinal Endoscopy Terms and Conditions

Supplemental Figure 1 Allocation of animals demonstrated as a flow diagram. CD, chow diet; HFD, high-fat diet; GMD, gastric mucosa devitalization; VSG, vertical sleeve gastrectomy; SH, sham surgery. Gastrointestinal Endoscopy 2018 87, 288-299.e6DOI: (10.1016/j.gie.2017.04.038) Copyright © 2018 American Society for Gastrointestinal Endoscopy Terms and Conditions

Supplemental Figure 2 Principle and histology of gastric mucosal devitalization (GMD) in rat stomach before (A), during (B), and 8 weeks after (C) the GMD procedure. A, A surgical entrance is created through a 1-cm ventral incision. The instruments were inserted into the mobilized stomach, which shows normal anatomic areas. Hematoxylin & eosin (H&E) staining revealed prominence of the mucosal layer. 1, mucosa; 2, muscularis mucosae; 3, submucosa; 4, muscularis propria. B, The GMD procedure was performed on the stomach while outside the abdominal cavity. The tissue changed from a pink to golden brown color after devitalization (arrows). H&E staining immediately after the procedure demonstrated devitalization of the mucosa. C, At 8 weeks after intervention, the rats showed significantly reduced amounts of abdominal fat. The stomach retained only small cicatrices from the access of instruments. GMD, gastric mucosal devitalization. Gastrointestinal Endoscopy 2018 87, 288-299.e6DOI: (10.1016/j.gie.2017.04.038) Copyright © 2018 American Society for Gastrointestinal Endoscopy Terms and Conditions

Supplemental Figure 3 Gross appearances of the explanted stomach at 8-week follow-up. There was no apparent difference in volume between sham surgery (SH) (A) and gastric mucosal devitalization (GMD) (B) stomachs. Vertical sleeve gastrectomy (VSG) stomachs (C) demonstrated a significantly reduced volume in relation to both GMD and SH. GMD, gastric mucosal devitalization; VSG, vertical sleeve gastrectomy; SH, sham surgery. Gastrointestinal Endoscopy 2018 87, 288-299.e6DOI: (10.1016/j.gie.2017.04.038) Copyright © 2018 American Society for Gastrointestinal Endoscopy Terms and Conditions

Supplemental Figure 4 Gastric mucosal devitalization (GMD) results in a progressive increase in concentration of fecal-free fatty acids (FFA) compared with sham surgery (SH). At 8 weeks, GMD rats had a significantly increased concentration of fecal FFA compared with SH rats. Similar findings were observed in vertical sleeve gastrectomy (VSG) rats compared with SH rats. FFA, free fatty acids; CD, chow diet; HFD, high-fat diet; GMD, gastric mucosa devitalization; VSG, vertical sleeve gastrectomy; SH, sham surgery. Gastrointestinal Endoscopy 2018 87, 288-299.e6DOI: (10.1016/j.gie.2017.04.038) Copyright © 2018 American Society for Gastrointestinal Endoscopy Terms and Conditions