| IntroductionDiabetes is an increasing problem throughout the world and a major contributor to the growing burden of chronic disease. The International Diabetes Federation (IDF) estimated that in 2014,387 million people had diabetes, and by 2035 this number will rise to 592 million. It is associated with significant economic burden and mortality due to its complications, which include heart disease, stroke, diabetic foot, diabetic retinopathy and diabetic nephropathy. Diabetes caused at least 612 billion dollars in health expenditure in 2014, represents 11% of total spending on adults. Additionally, diabetes caused 4.9 million deaths in 2014, means that every seven seconds a person dies from diabetes. Therefore, searching for new and efficient treatments for diabetes is of great importance and urgency.Type 2 diabetes mellitus (T2DM), characterized by deteriorative insulin secretion function in pancreatic islets and detrimental insulin resistance in insulin-sensitive target organs, comprises about 90% of all cases of diabetes. Currently, the dominating methods for T2DM treatment include lifestyle interventions, pharmacotherapy, and bariatric surgery. A growing body of evidence indicates that bariatric surgery is the most effective treatment for obese T2DM, leads to rapid and sustained weight loss and significant improvement or complete remission of diabetes. Along with the rising prevalence of diabetes, there has been a constant increase in the total number of bariatric surgery performed worldwide over the past 10 years. The most commonly performed bariatric procedure in the world in 2013 was Roux-en-Y gastric bypass (RYGB) represented 45% of all bariatric procedures, followed by sleeve gastrectomy (SG) represented 37%, and adjustable gastric banding (AGB) represented 10%. RYGB surgery with a gastric pouch draining into the mid-jejunum and pancreatico-biliary flow diverted to the distal jejunum, is considered the standard bariatric procedure with evidenced long-term effectiveness for weight loss and resolution of diabetes and its comorbidities, such as dyslipidemia, hypertension, hepatic steatosis, diabetic nephropathy and retinopathy. However, the underlying mechanisms that mediate the anti-diabetic effect after RYGB are still poorly understood.Some of the potential mechanisms of action of RYGB include alterations in hormones secretion, gut microbiota, and bile acids recycling. Accumulating evidences suggest that the small intestine displays morphological adaptation and reprogramming of glucose metabolism after RYGB due to early exposure to undigested nutrients, which may explain the therapeutic effect of bariatric surgery. Recently, a novel function of intestinal gluconeogenesis in the control of glucose homeostasis has also emerged to explain the therapeutic effect of bariatric surgery. Although gluconeogenesis has been well demonstrated and investigated in the liver and intestine, the role of gluconeogenesis after RYGB is still inconclusive. This study was designed to investigate the effects of RYGB on glucose homeostasis, lipid metabolism, fasting serum total bile acids level, intestinal morphological adaption, as well as the hepatic and intestinal gluconeogenesis in a T2DM rat model. The results of this investigation might help to provide new insight in elucidating the underlying metabolic mechanisms of diabetes remission after RYGB.Materials and Methods1. Animals and Diet ProtocolsThis animal study was approved by the Animal Care and Utilization Committee of Shanghai Sixth People’s Hospital. Thirty 6-week-old male Sprague-Dawley (SD) rats were purchased from Sino-British Sippr/BK Lab Animal Ltd (Shanghai, China). All animals were kept in individual cages under controlled ambient temperature (24±2 ℃) and humidity in a 12-h light/dark cycle. All the rats had free accesses to tap water and food unless otherwise stated. After 2 weeks of acclimatization, thirty rats were fed a high-fat diet (HFD) (60% fat,20% carbohydrate,20% protein, as a total percentage of calories; Research Diets, Inc., USA) for 4 weeks to induce insulin resistance. After fasted for 12 h, the rats were injected intraperitoneally with 1% of streptozotocin (STZ) (35 mg/kg) (Sigma, USA) to induce a diabetic state. Seventy-two hours after STZ injection, non-fasting blood glucose was measured in duplicate from tail vein blood with a glucometer (Sinocare Inc., China). Rats with non-fasting blood glucose≥16.7 mmol/1 were considered diabetic and randomly assigned to the sham group (n=10) or RYGB group (n=10). All rats had a standard rodent chow (10% fat,70% carbohydrate,20% protein, as a total percentage of calories; Research Diets, Inc., USA) after STZ injection. One week after STZ injection, sham or RYGB surgery was performed, respectively. In all groups, body weight and food intake were measured daily during the first 2 weeks after surgery and then once a week for the following time. Fasting plasma glucose was measured by a glucometer during the first 2 weeks after surgery and then biweekly for the following time.2. Surgical ProceduresAfter a 24-h fast, the rats were anaesthetised with an intraperitoneal injection of 1% sodium pentobarbital solution (5 ml/kg). RYGB operations were initiated by a 4 cm midline incision. The stomach was divided 5 mm below the gastro-oesophageal junction from the lesser to greater curvature horizontally. The proximal stomach was closed by 4-0 silk suture (Ningbo medical needle, China) in a simple interrupted suture technique to create a small gastric pouch, and the distal stomach was closed in a similar fashion. Then jejunum was transected 10 cm distal to the ligament of Treitz and the stump was ligated with 4-0 silk suture. The distal limb of jejunum was brought up to the small gastric pouch, and a 7 mm incision was made on the antimesenteric border of the bowel wall and anterior gastric wall along with greater curvature, respectively. The distal limb of jejunum was anastomosed to the small gastric pouch with a side-to-side gastrojejunostomy. The proximal limb of jejunum carrying the biliopancreatic juices was reconnected downward to the Roux limb at a distance of 15 cm from the gastrojejunostomy with a side-to-side jejunojejunostomy. Both the gastrojejunostomy and jejunojejunostomy were performed in a simple interrupted varus suture technique with an about 7 mm anastomosis using 5-0 silk suture (Ningbo medical needle, China). Sham surgeries involved the same abdominal incisions and gastrointestinal transections as those in the RYGB group, and reanastomosis was performed at the same sites, in order to produce a similar degree of surgical and anesthetic stress.Surgical rats were given glucose and sodium chloride solution postoperatively at 24 h and followed by standard rodent chow (10% fat,70% carbohydrate,20% protein, as a total percentage of calories; Research Diets, Inc., USA) at 4 postoperative days, until study ended.All rats were starved overnight and euthanatized at 8 weeks postoperatively. Jejunum (the proximal bowel about 17 cm below Treitz ligament in sham group or middle bowel of the Roux limb in RYGB group), ileum (about 10 cm above ileocecal junction in both groups), and liver biopsies were fixed in formalin for routine histology examination or stored at-80 ℃ after flash frozen in liquid nitrogen for quantitative real-time RT-PCR and Western blotting.3. Oral Glucose Tolerance TestOral glucose tolerance test (OGTT) was performed preoperatively and at 2 and 8 weeks postoperatively. Rats were administrated with 20% of glucose (1 g/kg) by oral gavage after an overnight fast. Blood glucose was measured using a glucometer at baseline as well as at 30,60,90, and 120 min after the glucose administration.4. Insulin Tolerance TestInsulin tolerance test (ITT) was performed preoperatively and at 2 and 8 weeks postoperatively. After an overnight fast, the rats were injected intraperitoneally with human insulin (0.5 IU/kg). Blood glucose of the tail vein samples was measured in conscious rats at baseline,30,60,90, and 120 min after injection.5. Serum Lipid Profiles and Total Bile AcidsAfter an overnight fast, blood samples were collected from the tail vein of conscious rats into chilled tubes containing EDTA solution preoperatively and at postoperative week 2 and 8. After centrifugation (1,006 g) at 4 ℃ for 15 min, serum was immediately extracted and stored at-80 ℃. Serum total cholesterol (TC), triglycerides (TG), free fatty acids (FFAs) and total bile acids were measured using enzymatic colorimetric assays kits according to the manufacture’s protocol.6. Tissue Histological AnalysisThe jejunum and ileum of each rat were embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin (H&E) for light microscopic examination. For assessment of morphological adaptation of intestine, a pathologist who was blinded to other details evaluated all histological sections under microscopy (100).7. Quantitative Real-time RT-PCRTotal RNA was extracted with TRIzol (Invitrogen, USA) according to the manufacture’s protocol, and the RNA concentration was measured at 260 nm. Quantitative real-time reverse transcription polymerase chain reaction (quantitative real-time RT-PCR) was performed in a Roche LightCycler 480 (Roche Diagnostics, Germany). Analyses were performed on 1 μg cDNA using the SYBR(?) Premix Ex Taq Master Mix (Takara, China), in a total PCR reaction volume of 10 μl, containing 0.2-0.6 μM of each primer. After the reaction, each PCR product was verified for its single amplification. All samples were measured in triplicate and the mean value was considered for comparative analysis. Quantitative measurements were determined using the △△Ct method andβ-actin expression was used as the internal control.8. Western BlottingTotal protein of the liver and intestine tissue was extracted by RIPA lysis buffer containing protease inhibitors (Beyotime, China), and the concentration of protein was determined by BCA Kit (Beyotime, China). Equal amount of protein was subjected to 10% SDS-PAGE gels(Beyotime, China) and separated by electrophoresis. Then the separated proteins were transferred onto polyvinylidene fluoride membranes (Millipore, USA). Proteins were detected using antibodies against the following: phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) (Santa Cruz Biotechnology, USA), and β-actin (Cell Signaling Technology, USA). After incubation at 4 ℃ overnight with primary antibody, the membranes were incubated with horseradish peroxidase conjugated secondary antibody (Cell Signaling Technology, USA) for 60 min. Protein bands were detected using ECL reagents (Thermo Scientific, USA). Membrane was detected using ImageQuant LAS-4,000 mini (GE, USA). The bands intensity was assessed with ImageJ software (http://rsb.info.nih.gov/ij, National Institutes of Health, USA).9. Statistical AnalysisQuantitative data were presented as mean±standard deviation (SD). Areas under curves (AUC) for OGTT (AUCOGTT) and ITT (AUCITT) were calculated by trapezoidal integration. For repeated measurements made over time (such as body weight, food intake, fasting plasma glucose, OGTT and ITT), a two-way analysis of variance (ANOVA) with repeated measures was used. For measurements made at one time, a Student’s t test was used for unpaired comparison. P<0.05 represented statistically significant difference in all cases. SPSS Version 20.0 was used for statistical analysis.Results1. Fasting Plasma GlucoseThere were no significant between-group preoperative differences in fasting plasma glucose level. In the sham group, fasting plasma glucose level was increased gradually after surgery, while the increase in the RYGB group was blunted and kept steady from week 1 postoperatively. The postoperative fasting plasma glucose levels in the RYGB group were significantly lower than those in the sham group at week 4.2. Body Weight and Food IntakeThere were no significant between-group preoperative differences in body weight and food intake. In the sham and RYGB groups, body weight reached its lowest value at 1 week postoperatively. In the sham group, body weight was nearly restored to the preoperative values at 2 weeks postoperatively, while the body weight increase in the RYGB group was blunted from week 2 postoperatively. The postoperative body weights in the RYGB group were significantly lower than those in the sham group at week 3. Daily food intake in the RYGB group was significantly decreased postoperatively between weeks 2 and 8 compared with that in the sham group.3. OGTT and ITTThere were no significant between-group preoperative differences in OGTT and ITT. The rats in the RYGB group showed significant improvements in glucose tolerance at postoperative weeks 2 and 8 weeks, as demonstrated by the lower values of AUCOGTT-Compared with the sham group, the RYGB group demonstrated lower postoperative values of AUCrrr at weeks 2 and 8, indicating improved systemic insulin sensitivity.4. Serum Lipids ProfilesThere were no preoperative differences in the fasting serum TC, TG, and FFAs concentrations between the sham and RYGB groups. At 8 postoperative weeks, the rats in the RYGB group showed significantly lower fasting serum levels of TG and FFAs than those in the sham group. The fasting serum level of TC decreased slightly in both groups postoperatively, and there was no significant difference between the sham and RYGB groups.5. Fasting Serum Total Bile AcidsThere was no preoperative difference in the fasting serum total bile acids concentration between the sham and RYGB groups. At 2 and 8 postoperative weeks, the rats in the RYGB group showed significantly higher fasting serum level of fasting serum total bile acids than those in the sham group.6. Histological Changes in the Small IntestineThe jejunum in the RYGB group exhibited marked increases in the length and number of intestinal villi compared with the sham group. Similarly, the ileum in the RYGB group also exhibited marked increases in the length and number of intestinal villi compared with the sham group.7. Expression of Key Regulatory Enzymes of Gluconeogenesis in the Liver and IntestineThe mRNA expression levels of PEPCK and G6Pase in the liver were significantly decreased in the RYGB group compared with the sham group.In addition, the protein expression levels of PEPCK and G6Pase in the liver were also markedly reduced in the RYGB group compared with the sham group, which was consistent with the results of the corresponding mRNA expression in the liver.The mRNA expression levels of PEPCK and G6Pase in the jejunum and ileum were significantly increased in the RYGB group compared with the sham group. The protein expression levels of PEPCK and G6Pase in the jejunum and ileum were also markedly increased in the RYGB group compared with the sham group, which was consistent with the results of the corresponding mRNA expression in the jejunum and ileum.Conclusions1. RYGB could significantly reduce the body weight and food intake in a T2DM rat model.2. RYGB could significantly improve glucose homeostasis and lipid metabolism in a T2DM rat model.3. The fasting serum total bile acids elevated significantly after RYGB, which may involved in metabolic improvement.4. The small intestine displayed significant morphological adaption exhibited hyperplasia and hypertrophy after RYGB.5. RYGB surgery suppresses hepatic gluconeogenesis and stimulates intestinal gluconeogenesis, which may then improve glucose homeostasis. Further studies are needed to determine the role that morphological and metabolic adaption of the small intestine plays in glucose homeostasis after RYGB. |
PDF Full Text Request