Individuals with type 1 diabetes who contacted the National Institutes of Health (NIH) recruitment office were asked to complete a questionnaire. At the NIH clinical center, 47 patients were selected for testing and 20 were enrolled (; ). Inclusion criteria were 1) age 18–60 years, 2) C-peptide ≥0.3 ng/ml (0.1 nmol/l) at baseline or after arginine stimulation, 3) positive antibodies (GAD65, ICA-512) and/or a typical history of type 1 diabetes, 4) duration of disease ≥5 years, and 5) BMI 20–30 kg/m2. Exclusion criteria were gastroparesis, diabetic nephropathy, insulin requirement ≥0.8 units · kg−1 · day−1, and hypoglycemia unawareness. To determine reference values, we performed mixed-meal testing in three healthy individuals (all female, mean age 33.3 ± 11.1 years, BMI 21.7 ± 1.8 kg/m2). This study (NCT00064714) was approved by the institutional review board of the National Institute of Diabetes and Digestive and Kidney Diseases. All patients provided written informed consent.
Clinical and immunological characteristics of the 20 enrolled patients at screening
Study design (A) and timeline for testing procedures (B): 20 patients entered the optimization period, 16 were randomized, and 14 completed the entire trial. B: A, arginine stimulation test; M, mixed-meal test; T, T-cell proliferation test.
The study followed a prospective randomized open-label crossover design (). It consisted of an optimization period of 2–4 months during which the NIH study team improved the patients' glucose control. Patients then entered the run-in period, a 4-month period during which no major changes in diabetes management were made. At screening and during the run-in period, patients underwent a total of four arginine stimulation tests and two mixed-meal tests to determine baseline C-peptide secretion (B). Thereafter, subjects were randomized to one of four treatment arms (insulin only, insulin plus exenatide, insulin plus exenatide plus daclizumab, or insulin plus daclizumab). After 6 months (period A), patients treated with exenatide discontinued its administration, whereas patients not previously treated with exenatide initiated it. The treatment assignment regarding daclizumab remained unchanged during this second 6-month interval (period B). In both periods A and B, three arginine stimulation tests and one mixed-meal test were performed. Finally, enrollees entered a 3-month extension period, in which they continued the assigned treatment of period B. Special attention was paid to prevent or treat vitamin D deficiency, hyperlipidemia, and hypertension.
Exenatide (Byetta; Amylin, San Diego, CA) was administered subcutaneously via a noncommercial pen that allowed dose adjustments in 2.5-μg increments. Dose and frequency were gradually increased from 2.5 μg twice daily to 10 μg four times daily. At the onset of exenatide therapy, insulin bolus doses were reduced by 50%, whereas basal insulin (for insulin pump users) and long-acting insulin doses (for multiple–daily injection users) remained unchanged. Insulin bolus doses were gradually titrated upward according to blood glucose values. Daclizumab (Zenapax; Roche, Nutley, NJ) was infused intravenously at 2 mg/kg over 30 min once monthly.
Arginine and mixed-meal tests were performed after an overnight fast. With pretest blood glucose levels of ~150 mg/dl (8.3 nmol/l), two intravenous lines were placed. Baseline blood samples were obtained at −10 and 0 min before intravenous infusion of 5 g arginine (10% solution). Samples were collected from the contralateral cephalic vein at 2, 3, 4, 5, 7, 9, and 10 min and immediately placed on ice until centrifuged at 2,000 rpm for 10 min at 4°C. Samples were then aliquoted and frozen at −20°C.
Mixed-meal tests were performed 30 min after the arginine stimulation tests. Patients assigned to exenatide received medication 15 min prior to testing. Blood samples were obtained at −10, 0, 15, 30, 45, 60, 90, 120, 150, 180, and 240 min. At time point 0 min, patients consumed Boost High Protein (7 ml/kg body wt, maximally 400 ml) in ≤10 min. Blood samples were processed and stored as described above.
C-peptide was determined with a solid-phase, two-site immunochemiluminescent assay (Esoterix, lower limit of detection 0.05 ng/ml [0.02 nmol/l]). Cross-reactivity with insulin was <0.01% at 100 and 200 μU/ml human insulin. The glucose oxidase method was used for serum glucose measurements. A1C was determined by high-performance liquid chromatography (normal 4.8–6.4%). Glucagon was measured using a double-antibody radioimmunoassay (Esoterix, lower limit of detection 10 pg/ml). Acetaminophen was determined by a particle-enhanced turbidimetric inhibition immunoassay with the analytical range 10–300 μg/ml (lowest detectable dose 2.5 μg/ml). Total GLP-1 was measured with a radioimmunoassay (Millipore, lower limit of detection 3.0 pmol/l).
T-cell assays were performed at three time points (B
) in two different laboratories (M. Dosch, Toronto, Canada; and B. Brooks-Worrell and J. Palmer, Seattle, WA) (12
). At both sites, T-cell proliferation in response to diabetes-related antigens was determined by direct 3
H-thymidine incorporation. Samples were labeled as positive for autoimmunity if the T-cells proliferated to ≥3 (Toronto) or ≥4 (Seattle) of the test antigens compared with responses against unspecific control antigens. The Toronto laboratory used a panel of 12 predefined diabetes-related peptide or protein antigens and included exogenous interleukin-2 in the assay medium. The Seattle laboratory used mixtures of antigen eluted from gel-fractionated human islet cell proteins with 18 size fractions separated by SDS polyacrylamide gel. HLA haplotypes were determined using sequence-specific primers at the Department of Transfusion Medicine at the NIH Clinical Center.
Sample size calculations were based on data from 1) NIH patients having undergone islet transplantation with no C-peptide before transplantation but measurable C-peptide after transplantation and 2) data on the variability of individual patient values in periods similar to the run-in period of this study. Sample size estimates were designed to detect a 50% increase of C-peptide levels on exenatide treatment, resulting in a sample size estimate of 12 patients.
Primary outcome measures were based on basal (−10 and 0 min) and stimulated (single highest value during test procedure) C-peptide levels. These were measured monthly before and during arginine stimulation during the last 3 months of each treatment period (B
). C-peptide values were averaged across these three time points. Because 50% of patients also received daclizumab, two-way analyses of variance (ANOVAs) were run to assess daclizumab treatment and its possible interaction with exenatide: daclizumab usage was a second factor and the period-sequence by daclizumab interaction term was included. Because the P
values for the interaction term were highly nonsignificant (basal P
= 0.74; stimulated P
= 0.81), as were the P
values for the overall effect of daclizumab (basal P
= 0.87; stimulated P
= 0.84), the daclizumab and no-daclizumab groups were combined in the main analyses of exenatide. To eliminate confounding due to a possible period effect (e.g., due to prolonged intensive insulin treatment), the test based on the two-sample t
approach was used (13
). Two-sided P
values ≤0.05 were considered significant and results were presented as means ± SD if not indicated otherwise.