Animals and rodent diabetes model
The animal experiments and animal care were performed in compliance with and were approved by the ethics committee of the third affiliated hospital to the Third Military Medical University. Non-obese male Sprague Dawley (SD) rats, C57BL/6 J mice were used in this study. Rats and mice were housed under a 12-hour day-night cycle and had free access to food and water. Rats were randomly assigned to the sham-treated or metabolic surgery groups. The protocol is provided in the text. After an overnight fast, SD rats were intraperitoneally injected with vehicle (0.1 M sodium citrate, pH 4.5) or 45 mg/kg STZ (Sigma-Aldrich Co LLC, St. Louis, MO) according to the protocol from the Animal Models of Diabetes Complications Consortium (AMDCC). After three days, rats were described as diabetic if their fasting blood glucose levels were greater than 300 mg/dL (16.7 mmol/L) for three consecutive days52,53,54.
Oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance tests (IPGTT)
Animals were fasted overnight before all the tests. After fasting, animals were given the glucose solution (2 g glucose/kg body weight) orally for OGTT, or glucose solution (2 g glucose/kg body weight or 4 g glucose/kg body weight) injected intraperitoneally for IPGTT55,56. OGTT and IPGTT were performed in separate days. Blood glucose were monitored by the glucose telemetric transmitter before and after the test, or using the Nova StatStrip Xpress glucometer (Nova Biomedical, Waltham, MA) by cutting the tail and gently massaging blood onto a glucose test strip.
Surgery and post-surgery procedures
After an overnight fast, animals were deeply anesthetized with isoflurane (4% induction and 1.5% maintenance; VIP-3000 Vaporizer; Matrix, Orchard Park, NY), and the abdominal organs were exposed by laparotomy. Animals were fasted for 12 h after surgery. After evaluating the health status and behavior of each animal, a limited liquid diet was reintroduced 12–72 h after the operation. Three days after surgery, normal chow was provided ad libitum to the animals11,44. Animals used in this study underwent operations in different cohorts, and STZ was injected 2 weeks before surgery57.
The total length of the small intestine was measured, the ligament of Treitz was identified, and the small intestine was divided at the appropriate distance (8–10 cm) downstream of this ligament11. A gastrojejunostomy and an end-to-side jejuno-jejunostomy (18–20 cm from the ligament of Treitz) were created with a 6–0 silk interrupted suture57. A gastric pouch consisting of 10% of the total stomach volume was created using scissors, and the distal cut end of the stomach was sutured with 6–0 silk. The intestine remained hydrated during this procedure. The gastric artery was preserved, and only the small vascular branches were cauterized with electrocoagulation. The laparotomy was closed with a 4–0 Prolene suture in two layers.
The upper gastrointestinal tract was exposed, and the length of the small intestine was measured. The intestine was cut at a distance of 10–15% of the intestinal length from the pylorus. The stomach was gently dissected from the spleen and liver by using cotton swabs, and the gastro-esophageal junction was fully externalized. The left gastric vessels and vagal fibers were removed to create a small pouch (approximately 2% of the stomach capacity). The stomach remnant was closed using a continuous stitch with an 8–0 Prolene suture (Ethicon US, LLC, USA). The stomach pouch was then anastomosed with a distal jejunum, and a jejuno-jejunostomy was performed using an interrupted stitch with an 8–0 Prolene suture at a distance of 10–15% of the intestinal length from the gastrojejunostomy. Warm saline was regularly added to wet the tissue. The abdominal muscle and skin were closed using an interrupted stitch with a 6–0 silk suture26.
Vertical sleeve gastrectomy (VSG)
The stomach was isolated outside the abdominal cavity and placed on warm saline-soaked gauze pads. It was then freed from the spleen and liver, and the lateral 80% of the stomach was cut, leaving a tubular gastric remnant (closed using an interrupted stitch with a 6–0 silk suture) while keeping the proximal and distal ends intact. Finally, the abdominal wall was closed in layers using an interrupted stitch with a 4–0 silk suture26.
Duodenal-jejunal bypass (DJB)
The stomach was fully externalized with a VSG surgical procedure, and the jejunum was transected using the previously described rat RYGB procedure. The pyloric sphincter was ligated with a 4–0 silk suture. An incision was created on the lateral wall of the stomach, and the distal jejunum was anastomosed to the stomach. A jejuno-jejunostomy was performed with an RYGB procedure. The abdominal wall was closed in layers using an interrupted stitch with a 4–0 silk suture10.
Restoration of the gastroduodenal passage after RYGB surgery
A loop of the jejunum (1 cm) was isolated and transposed between the oesophagus and the stomach to restore the gastroduodenal passage of nutrients after RYGB surgery58. After the operation, nutrients flowed from the esophagus through the jejunal loop to the stomach in a latero-terminal fashion involving the duodenum, jejunum, and remaining intestinal tract. Anastomoses were performed with a 6–0 silk interrupted suture11.
In all experiments, animals undergoing a sham operation were used as controls. The sham operation was consisted of laparotomy, jejunal transection, and sutured; and was performed in animals that were matched (age- and weight-) to rats underwent RYGB or restoration.