All animal experiments complied with the ARRIVE guidelines and the study was approved by the Animal Research Ethics Board at the University of Alberta (AUP00003000). All methods were performed in accordance with the relevant guidelines and regulations. Thirty-six male Wistar rats were randomly assigned to four cohorts based on procedure and planned date of euthanasia: 2-week RYGB, 14-week RYGB, 2-week sham, or 14-week sham. Male rats were chosen to avoid estrous cycles of female rats which may affect hormone outcomes in the 2-week cohorts. The rats were doubly housed until 6 weeks of age after which they were separated into single cages to avoid cage effects biasing microbial analysis. Sterile HFD (Bio-Serv S3282, 60% calories from fat) was introduced at 6 weeks of age and continued throughout the experiment, except during the perioperative period. Body weight was monitored weekly. RYGB or sham surgery were performed at 16 weeks of age.
Importantly, MedicGLP Official this study was a model of severe obesity and not of diabetes. Rats were euthanized at 2 and 14 weeks after surgery to evaluate early and late post-RYGB L-cell and enteroendocrine changes. Euthanasia occurred with deep isoflurane anesthesia via cardiac puncture. The 2-week time point was chosen to evaluate early changes to allow for a 1-week washout from postoperative liquid diet and to assess for metabolic changes while on HFD. The 14-week timepoint was chosen to allow for evaluation of late RYGB-adaptive L-cell changes. One week prior to euthanasia, intraperitoneal glucose tolerance testing (IPGTT) was performed. Rats were then euthanized with collection of blood for postprandial gut hormones, ileal tissue and ileal enteric contents. Ileal tissue underwent immunofluorescence staining to quantify the number of L and K cells. Intestinal morphology quantification was conducted with direct microscopy. Reverse transcription polymerase chain reaction (RT-PCR) was performed on ileal tissue for gene expression of GLP-1 and gastric inhibitory polypeptide (GIP) relevant genes. Ileal enteric contents underwent 16S rRNA sequencing for microbial composition and liquid chromatography-mass spectrometry for bile acid analysis.
The primary objective of this study was to determine early and late changes to L-cell quantity and GLP-1 gene expression of ileal L-cells between RYGB and sham surgery, defined as 2 weeks and 14 weeks, respectively. Secondary objectives included determining changes in villi morphology, bile acid composition, and microbial composition within the ileum and identifying pathways in which these changes affect L-cell gene expression and quantity. Additionally, serum GLP-1 and glucose tolerance were compared between groups to determine the effects of RYGB on glucose metabolism. Sample size calculations were designed to ensure GLP-1 changes induced by surgery would be adequately captured. In prior literature, rats had significantly increased GLP-1 after RYGB (25 vs. 30)43. Powering to detect a 25% GLP-1 difference between cohorts, with an alpha of 0.05 and a beta of 0.80, would require 8 rats per arm. 10%, this would require 9 rats per arm for a total of 36 rats. After an overnight fast, RYGB (Supplementary Fig. 7) or sham surgery were performed based on previously published protocols44.
In summary, anesthesia was induced using isoflurane and rats were given subcutaneous buprenorphine sustained release (1 mg/kg). A midline incision was made sharply, and the stomach was mobilized by dividing the gastric attachments using a combination of electrocautery and ligation with 6-0 polypropylene. A window was made in the gastrohepatic ligament superior to the left gastric artery to allow for stapling. The ligament of Treitz was located and the jejunum was divided 7 cm distally. The stomach was divided with a 45 mm laparoscopic linear stapler (Ethicon, ETS45) with 3.5 mm blue load staplers. Hemostasis at the staple line was achieved with pressure and suture ligation. A second stapler was deployed to resect the gastric fundus (forestomach) to prevent retained food within a large pouch. A gastrotomy was created in the distal pouch. A circular gastrojejunostomy was created with 6-0 polypropylene (continuous on anterior side, interrupted on posterior). A leak check was performed by gently compressing enteric contents through the anastomosis.
