Early Enhancements of Hepatic and Later of Peripheral Insulin Sensitivity Combined With Increased Postprandial Insulin Secretion Contribute to Improved Glycemic Control After Roux-en-Y Gastric Bypass

Subjects

We recruited 10 obese patients with T2D (age 43.6 ± 3.4 years; median duration of diabetes 2.5 years [range 1–11]) and 10 obese normal glucose-tolerant (NGT) subjects (age 40.1 ± 2.8 years) who were scheduled for laparoscopic RYGB at Hvidovre Hospital (Hvidovre, Denmark). Before enrollment in the study, all participants fulfilled the inclusion criteria for bariatric surgery in Denmark and had completed a preoperative diet-induced total body weight loss of at least 8% as required by health authorities. After the preoperative weight loss, all patients in the T2D group had a 2-h plasma glucose (P-glucose) of ≥11.1 mmol/L, and diabetes was controlled with diet alone (n = 2), metformin alone (n = 4), or metformin combined with liraglutide (n = 2) or NPH insulin (n = 2). Liraglutide was discontinued ≥10 days before the first study day, and all other antidiabetic agents were discontinued ≥3 days before each study day; all antidiabetic medication was discontinued from the time of surgery. One patient with diabetes for 11 years required metformin at 4–11 months postoperatively. In the NGT group, all had a 2-h P-glucose level <7.8 mmol/L and HbA_1c <6% (42 mmol/mol). Written informed consent was obtained from all participants, and the study was approved by the Municipal Ethical Committee of Copenhagen in accordance with the Declaration of Helsinki II and by the Danish Data Protection Agency.

Study Design

On separate days before and 1 week, 3 months, and 1 year after RYGB, we performed hyperinsulinemic- euglycemic clamps and IV glucose-glucagon tests. Before and 3 months and 1 year after RYGB, oral glucose tolerance tests (OGTTs) and whole-body dual-energy X-ray absorptiometry (DEXA) scans were performed on an additional study day. All participants completed the preoperative and 3 months visits; four subjects did not complete the 1-week visit because of postoperative complications and two did not wish to participate at the 1-year visit.

Before the experiments, participants were instructed to refrain from strenuous physical activity and alcohol for 3 days and to fast overnight (10–12 h). On the day of the experiment, subjects were weighed and placed in a reclining position in a hospital bed, allowing no physical activity.

Hyperinsulinemic-Euglycemic Clamp

Catheters were placed in an antecubital vein for infusion and in a dorsal vein in the hand for blood sampling, with the hand placed in a heated box for arterialization. After collecting three fasting samples, a primed continuous basal infusion (0.036 mg/kg/min) of [6,6-²H₂]-glucose (99 atom percent enrichment; Cambridge Isotope Laboratories, Andover, MA; sterilized and packed in vials at the Central Pharmacy of the Capital Region, Herlev, Denmark) was started and continued for 120 min using a precision infusion pump (P2000; IVAC Medical Systems, Hampshire, U.K.). The priming dose was adjusted for the ambient fasting P-glucose (3.6 mg/min × fasting P-glucose [mmol/L] × 1/5). Urine was sampled during the basal infusion period in the T2D group and tested for traces of glucose (Multistix7; Siemens, Berlin, Germany). After 120 min, the clamp was initiated (4-h primed continuous insulin infusion of 40 mU/m²/min Actrapid; Novo Nordisk, Bagsværd, Denmark). Insulin was dissolved in saline, to which was added blood from the participant. P-glucose was allowed to drop to 5.5 mmol/L before initiation of a variable infusion of 20% glucose (w/v) enriched with [6,6-²H₂]-glucose (median enrichment 2.16% [interquartile range 2.10–2.24]), while glucose infusion was initiated at clamp start if P-glucose was ≤5.5 mmol/L. The basal infusion of [6,6-²H₂]-glucose was decreased to 25% upon initiation of glucose infusion. Blood was sampled every 10 min during the last 30 min of the basal and clamp periods and every 20 min during the remainder of the clamp, whereas P-glucose was assessed every 5 min. Stable tracer-to-tracee ratios (TTRs) were obtained in the basal and clamp periods with mean coefficient of variation (CV) ± SD of 4.8 ± 2.2% and 2.9 ± 2.1%, respectively, and P-glucose was kept stable for the last 30 min of the clamp (CV 3.1 ± 1.4%); there were no differences in CVs between pre- and postoperative days or between groups. Biopsies of abdominal subcutaneous fat and tissue from the vastus lateralis muscle were obtained during the basal period and after 4 h of insulin infusion using a modified Bergström needle with suction under local anesthesia. Results from biopsies will be presented elsewhere.

OGTT

Catheters were placed in antecubital veins in both arms. At t = 0 min, a bolus of 50% glucose (w/v) was injected over 1 min. The volume of the bolus was fixed for the individual patient throughout the study: (20 – fasting preoperative P-glucose [mmol/L]) × (height² [m²]) × (2.1 [mL/m²/mmol/L]). At t = 2 min, a bolus of 1 mg glucagon (Novo Nordisk) was injected. Blood was sampled at t = −10, −5, 0, 2, 6, 8, 10, and 12 min.

Oral Glucose Tolerance Test

Participants ingested 75 g of glucose dissolved in 250 mL of water within 5 min. Blood samples were obtained from a catheter in an antecubital vein at t = 0, 15, 30, 60, 90, and 120 min. Two participants did not complete the postoperative OGTTs because of vomiting. Otherwise, the test was well tolerated after surgery, except for mild degrees of nausea during the first hour.

DEXA

Body composition was assessed by DEXA scanning (Discovery A, S/N 83487; Hologic Inc., Bedford, MA) using the Apex 2.3 software package.

Surgical Procedure

Surgery was performed as previously described (9).

Preoperative Weight Loss

Data on preoperative weight loss was collected retrospectively from patient records.

Postoperative Diet

From the day after the operation patients were on a liquid diet of approximately 1,200 kcal/day until 14 days postoperatively, when the diet gradually changed toward solid foods.

Analytic Procedures

Blood collected in prechilled EDTA tubes (for analysis of glucagon and fatty acids [FAs]), EDTA tubes with dipeptidyl peptidase-4 inhibitor (valine-pyrrolidide; final concentration 0.01 mmol/L, for GLP-1, glucose-dependent insulinotropic polypeptide [GIP], and glucagon from OGTTs), and heparin tubes (for TTR) was immediately centrifuged, while clot-activator tubes (for insulin and C-peptide) were left for 30 min before centrifugation. Eppendorph tubes containing EDTA were immediately centrifuged and used for analysis of P-glucose using YSI model 2300 STAT plus (YSI, Yellow Springs, OH). Serum C-peptide and insulin were analyzed using AutoDELFIA fluoroimmunoassay (Wallac OY, Turku, Finland), HbA_1c was measured using high-pressure liquid chromatography (Tosoh Bioscience, Tokyo, Japan), and plasma FAs (NEFA C kit; Wako Chemicals GmbH, Neuss, Germany) were measured using enzymatic colorimetric methods (Hitachi 912 automatic analyzer; Boehringer, Mannheim, Germany). Glucagon, total GLP-1, and total GIP were analyzed as previously described (9). TTR was analyzed using mass spectrometry as previously described (27).

Calculations and Statistical Analysis

Rate of appearance (Ra) and rate of disappearance (Rd) of glucose were calculated from the last 30 min of the basal and clamp periods using Steele’s equation (28). One patient was excluded from analysis of basal Ra because of preoperative glucosuria. Mean serum C-peptide concentration in the basal period was used to assess basal hepatic insulin sensitivity (HISI_basal = 10⁶/[Ra_basal ×C-peptide_basal]), whereas mean serum insulin in the clamp period was used to calculate insulin-adjusted glucose disposal (Rd_clamp/insulin_clamp). Suppression of Ra, FAs, and glucagon was calculated as the difference between basal and clamp levels and expressed as a percentage of the basal level. Hepatic insulin clearance at fasting was calculated as the ratio of fasting serum C-peptide to insulin, whereas clearance during the clamp was adjusted for endogenous insulin secretion (CI_clamp = insulin infusion rate/(insulin_clamp – [C-peptide_clamp × insulin_basal/C-peptide_basal]) (29).

Incremental areas under the curve (iAUCs) were calculated using the trapezoidal rule subtracting fasting levels. Early insulin secretion was estimated using insulinogenic index (IGI) from OGTTs (IGI = ΔC-peptide_0–30/Δglucose_{0–30 min}), and the acute insulin response from IV glucose-glucagon tests was calculated as the mean serum C-peptide from 6–12 min subtracting fasting levels. Indices of insulin secretion (IGI and acute insulin response, respectively) were related to insulin sensitivity (Rd_clamp/insulin_clamp) by calculating disposition indices (oral and IV, respectively) (26).

Data are expressed as means ± SEMs unless otherwise specified. Postoperative changes were analyzed by ANOVA in a linear mixed effects model using time from surgery and group as fixed effects and individual subjects as random effect. Logarithmic transformation was used if distribution was skewed. Post hoc comparisons of group differences at a given point in the study were performed using unpaired t tests. Pearson test was used to evaluate correlations. Before the study, we estimated that eight patients would allow detection of within-group postoperative changes in insulin sensitivity of 20% (power 0.80, level of significance 0.05) based on data from patients with T2D (30). Statistical analyses were performed in R software version 2.11.1 (www.r-project.org).