Gastric bypass and banding equally improve insulin sensitivity and β cell function (original) (raw)

Selection and description of participants

20 consecutive, eligible patients who were scheduled to undergo RYGB (n = 10; 2 men, 8 women; 43 ± 7 years old) or LAGB (n = 10; 1 man, 9 women; 47 ± 14 years old) procedures at Barnes-Jewish Hospital (St. Louis, Missouri, USA) participated in this study, which was approved by the Washington University Institutional Review Board. All participants provided written informed consent and completed a comprehensive medical evaluation. Potential participants who smoked cigarettes, had previous malabsorptive or restrictive intestinal surgery, had a history of inflammatory intestinal disease, had evidence of severe organ dysfunction, had uncontrolled hypertension, or had a serum triglyceride concentration of 400 mg/dl or higher were excluded. Potential participants who had diabetes were excluded to avoid the confounding effects of differences in baseline glycemic control, glucose toxicity, and postsurgical changes in diabetes medications on our outcome measures.

Study design and experimental procedures

Each subject completed body composition analyses, a hyperinsulinemic-euglycemic clamp procedure, and a mixed-meal metabolic study before bariatric surgery and after 20% surgery-induced weight loss.

Body composition assessments. Body FM and FFM, IAAT volume, and IHTG content were determined using dual-energy X-ray absorptiometry, magnetic resonance imaging, and magnetic resonance spectroscopy, respectively (26, 27).

Hyperinsulinemic-euglycemic clamp procedure. Subjects were admitted to the Washington University School of Medicine Clinical Research Unit (CRU) and consumed a standard evening meal (12 kcal/kg FFM; 50% of calories as carbohydrate, 30% as fat, 20% as protein). The following morning, a catheter was inserted into a forearm vein for infusion, and a second catheter was inserted into a radial artery to obtain blood samples. At 6:00 am, a primed, continuous infusion of [6,6-2H2]glucose (priming dose, 22 μmol/kg; infusion rate, 0.22 μmol/kg/min) was started and maintained until the end of the study. At 9:30 am, insulin was infused at a rate of 50 mU × m2 body surface area × min–1 for 4 hours. Euglycemia (plasma glucose ∼100 mg/dl) was maintained by infusion of 20% dextrose enriched to 2.5% with [6,6-2H2]glucose. Blood samples were obtained immediately before starting the tracer infusion and during the final 30 minutes of the basal period and the clamp procedure to determine plasma substrate and insulin concentrations as well as glucose tracer/tracee ratios (TTRs).

Subcutaneous abdominal adipose tissue and vastus lateralis muscle tissue samples were obtained by percutaneous biopsy 60 minutes after starting the glucose tracer infusion during basal conditions. Biopsy sites were cleaned and draped. After anesthetizing the skin and underlying tissues with lidocaine, adipose tissue was aspirated from the periumbilical area using a 14-gauge needle, and skeletal muscle was obtained using Tilley-Henkel forceps (Sontec Instruments). Tissue samples were immediately rinsed with ice-cold saline and frozen in liquid nitrogen before being stored at –80°C.

Mixed meal metabolic study. Subjects were admitted to the CRU and consumed a standard evening meal. The following morning, a catheter was inserted into a forearm vein for infusion, and a second catheter was inserted into a radial artery to obtain blood samples. A primed, continuous infusion of [6,6-2H2]glucose (priming dose, 22 μmol/kg; infusion rate, 0.22 μmol/kg/min) was started and continued until the end of the study. After 3.5 hours of tracer infusion, subjects ingested a liquid meal (containing 46 g glucose mixed with 0.9 g [U-13C]glucose, 9 g fat, and 9 g protein), which was provided in 7 equally divided aliquots given every 5 minutes over a 30-minute period. Blood samples were obtained immediately before starting the glucose tracer infusion, every 10 minutes for 30 minutes just before starting the meal ingestion, and then every 15 minutes for the first hour and every 20 minutes for the subsequent 5 hours after starting the meal, to determine plasma substrate and hormone concentrations as well as glucose TTRs.

Surgical procedures

Bariatric surgeries were performed using standard laparoscopic approaches. The RYGB procedure involved creating a small (∼30 ml) proximal gastric pouch and a stapled gastrojejunostomy. A 100-cm Roux limb was constructed by transecting the jejunum 30 cm distal to the ligament of Treitz and performing a stapled jejunojejunostomy at this site (28). The standard pars flaccid technique was used for LAGB (Lap-Band; Allergan; ref. 29).

Weight management after surgery

Subjects participated in a supervised dietary weight loss program to help subjects in both groups consume a similar energy-deficit diet and achieve a 20% weight loss within 4–6 months after surgery. All subjects were instructed to consume a clear liquid diet (400–600 kcal/d) for the first 2 days after surgery, a full liquid diet (400–600 kcal/d) on postoperative day 3, a pureed diet (2–3 oz/meal providing 700–800 kcal/d) on days 4–7, and a soft diet (3–4 oz/meal providing 800–1,000 kcal/d) on days 8–29, followed by a regular-food diet containing 1,000–1,200 kcal/d and 1.0 g protein/kg body weight/d. After subjects achieved a 20% weight loss, a balanced weight maintenance diet was prescribed, and subjects maintained a stable body weight (<2% change) for at least 2 weeks before repeat studies were performed.

Analyses of blood samples

Plasma glucose concentration was determined using an automated glucose analyzer (YSI 2300 STAT plus; Yellow Spring Instrument Co.). Plasma C-peptide, insulin, glucagon, active GLP-1, leptin, adiponectin, and CRP concentrations were measured using enzyme-linked immunosorbent assays (Millipore). Plasma glucose TTRs were determined by using gas chromatography–mass spectrometry (30).

Analyses of adipose tissue samples

Isolation of mRNA and quantitative PCR. Frozen adipose tissue samples were homogenized in RNAzol Bee (Tel-Test Inc.), and RNA was isolated according to the manufacturer’s protocol. Gene expression was determined using quantitative real-time PCR. cDNA was synthesized by using SuperScript VILO (Life Technologies), and cDNA samples were amplified by using SybrGreen reagent and the ABI 7500 thermal cycler (Life Technologies). The expression of each gene (EMR1, CD11B, CCL2, CSF3, IL6, TNFA, IL10, and LEP) was determined by correcting the threshold crossing (Ct) of each sample to the housekeeping control gene, acidic ribosomal phosphoprotein P0 (36B4), calculated as 2–ΔCt. The following primer sequences were used: EMR1, GGAAGGGCACATAAGACCCAG and GGGCACAAGGTACTGTCTCTA; CD11B, CAGCCTTGACCTTATGTCATGG and CCTGTGCTGTAGTCGCACT; CCL2, ATAGCAGCCACCTTCATTCC and GCTTCTTTGGGACACTTGCT; CSF3, GCGGCTTGAGCCAACTCCATA and GAACGCGGTACGACACCTC; IL6, CCTGAACCTTCCAAAGATGG and TGGCTTGTTCCTCACTACTCTC; TNFA, GGAAAGGACACCATGAGCA and CAGAGGGCTCATTAGAGAGAGG; IL10, TCAAGGCGCATGTGAA and GATGTCAAACTCACTC; LEP, GTGGCTTTGGCCCTATCTT and GCATACTGGTGAGGATCTGTTG; 36B4, GTGATGTGCAGCTGATCAAGACT and GATGACCAGCCCAAAGGAGA.

Analyses of skeletal muscle lipid metabolites

Intramyocellular DAG and ceramide concentrations were determined using mass spectrometry (31). Muscle tissue samples were weighed and lipids were extracted as we previously described (32). About 10% of the lipid extract was used to evaluate ceramide content. Quantitation of ceramide molecular species was performed by using a Thermo triple quadrupole (TSQ) Vantage mass spectrometer and electrospray ionization (ESI) (33). Neutral lipids were extracted from the rest of the lipid extract by hexane and the free hydroxyl group in DAG was protected with 2,4-diflourophenyl isocyanate to quantitate DAG molecular species using an Agilent 1100 LC system connected to an Agilent 6460 TSQ mass spectrometer operated in ESI and positive ion mode (34). The LC system allows separation of DAG species from other neutral lipids and resolution of 1,2-_sn_-DAG and 1,3-_sn_-DAG species. Total DAG and ceramide contents were calculated by summation of the individual 1,2-_sn_-DAG (C22:0, C23:0, C24:0, C25:0, C26:0, C27:0, C28:0, C29:0, C30:1, C30:0, C31:0, C30:0, C31:0, C32:2, C32:1, C32:0, C33:1, C33:0, C34:3, C34:2, C34:1, C34:0, C35:1, C35:0, C36:4, C36:3, C36:2, C36:1, C36:0, C38:5, C38:4, C38:3, C38:2, C40:5) and 1,3-_sn_-DAG (C22:0, C24:0, C26:0, C28:0, C30:0, C32:1, C32:0, C34:2, C34:1, C34:0, C36:3, C36:2, C36:1, C36:0) species and the individual ceramide (C16:0, C18:1, C18:0, C19:0, C20:0, C22:0, C23:0, C24:2, C24:1, C24:0, C24:1OH, C24:0OH) species.

Calculations

Insulin sensitivity. HOMA-IR was used to provide an index of whole-body insulin resistance (35). Glucose Ra into plasma was calculated by dividing the tracer infusion rate by the average plasma glucose TTR during the last 30 minutes of the basal and insulin infusion periods (36). Glucose disposal rate was equal to endogenous glucose Ra plus the rate of exogenously infused dextrose and glucose tracer. Insulin-stimulated glucose uptake was used as an index of skeletal muscle insulin sensitivity.

β Cell function. Minimal model analysis was used to calculate Φtotal, an overall index of postprandial insulin secretion in response to plasma glucose, using plasma C-peptide as a function of glucose concentration (37). The minimal model can also resolve the total ISR into 2 components representing the release of insulin from 2 distinct pools of insulin secretory granules within the β cell: a dynamic component that represents the rapid release of a “readily releasable pool,” which usually accounts for less than 10% of total insulin secretory granules, and a static component that represents the slower release of a “reserve pool” of insulin secretory granules (38). The DI, which provides an assessment of insulin secretion in relationship to insulin sensitivity (39), was calculated as the product of Φtotal and the change in glucose Rd per kg FFM per change in insulin concentration during the hyperinsulinemic-euglycemic clamp procedure.

Metabolic response to the mixed meal. Plasma glucose, insulin, and C-peptide concentration AUCs for 6 hours after initiating meal consumption were calculated using the trapezoid method (40). Total glucose Ra into the systemic circulation during the meal was calculated using Steele’s equation for non–steady-state conditions (36). Glucose Ra into the systemic circulation from ingested glucose and from EGP were calculated as previously described (41).

Statistics

Data were examined for normality according to the Shapiro-Wilks criteria, and non–normally distributed data sets were log-transformed before statistical analyses were performed. A 2-way repeated measures analysis of variance with Tukey’s post-hoc procedure was used to compare the effects of RYGB and LAGB surgery on study outcome measures (except for postprandial metabolic outcomes) in the 2 groups. For postprandial metabolic outcomes, mixed-model repeated-measures analysis of variance was used to evaluate differences in weight loss–induced changes between LAGB and RYGB groups. A P value of 0.05 or less was considered statistically significant. All data are presented as means ± SD unless otherwise indicated.

Based on the interindividual variability of glucose rate of disappearance (Rd) during a hyperinsulinemic-euglycemic clamp procedure in a large cohort of nondiabetic obese subjects we previously studied (42), we estimated that 10 subjects in each group (RYGB and LAGB) would be needed to detect a 40% difference in insulin-stimulated glucose Rd between the RYGB and the LAGB group with a β value of 0.20 (i.e., 80% power) and an α value of 0.05. This proposed difference between groups is a conservative estimate based on the results from the only 2 previous studies comparing the effect of gastric restriction versus RYGB procedures on insulin sensitivity using the hyperinsulinemic clamp procedure (43, 44). In both studies, insulin-stimulated glucose disposal was more than 200% greater after RYGB than after restrictive surgical procedures.

Study approval

This study was approved by the Human Research Protection Office at Washington University School of Medicine. All study subjects provided written informed consent before screening and participation in the study.