Adults with type 1 diabetes were included in the analysis after giving informed consent. Investigations were carried out in accordance with the principles of the Declaration of Helsinki as revised in 2008. The entry criterion was diabetes onset after the age of 15 years. Participants (n = 29, 38% female, 100% white) had a median age of 33 years (interquartile range [IQR] 28.5–38.5 years) and a median age at diabetes onset of 24 years (IQR 19–26.5 years). After an overnight fast of at least 10 h, participants underwent a 2 h MMTT under conditions of pre-test plasma glucose between 4 and 12 mmol/l. Participants treated with an insulin pump were kept on their basal rate of insulin and those on injections had their basal insulin the night before the test. A standard liquid test of 4.4 g/100 ml fat, 17.6 g/100 ml carbohydrate and 10 g/100 ml protein (Delical HP-HC; Lactalis Nutrition Santé, Torcé, France) was given at a dose of 4 ml/kg i.e. 25.10 kJ/kg (6 kcal/kg) to a maximum of 360 ml taken in less than 2 min. Blood samples were drawn for measurements of plasma glucose, C-peptide, glucagon and GLP-1 before and 15, 30, 45, 60, 90 and 120 min after the test.
C-peptide was measured by ultrasensitive ELISA (Mercodia, Uppsala, Sweden) with a lower detection limit of 2.5 pmol/l. Intra-assay CVs were 6.2% and 4.6% and inter-assay CVs were 4.3% and 3.5% at 15 pmol/l and 54 pmol/l, respectively. Glucagon was measured by a solid phase two-site enzyme immunoassay (Mercodia) with a detection limit of 1 pmol/l. Glucagon ELISA intra-assay CVs were 5.1% and 3.3% and inter-assay CVs were 8.1% and 7.3% at 3 pmol/l and 21.9 pmol/l, respectively. GLP-1 was measured by a total GLP-1 ELISA kit (Epitope Diagnostics, San Diego, CA, USA) with a detection limit of 0.6 pmol/l. Intra-assay CVs were 3.7% and 4.7% at 3.0 pmol/l and 10.2 pmol/l, respectively. Inter-assay CVs were 6.2% and 9.5% at 4.2 pmol/l and 12.6 pmol/l, respectively.
Data are presented as median and IQR or mean ± SEM where appropriate. The sample size was determined to establish a reduction of 40% in peak glucagon levels during the MMTT, assuming an SD of 5 pmol/l, an alpha risk of 5% with 80% power, and a two-sided t test at a 5% significance level. The required sample size was calculated to be 20 (ten participants per arm), using the package epiR, version 0.9-87 (R Foundation for Statistical Computing, Vienna, Austria). Analysis was performed using Student’s t test or the Mann–Whitney U test where appropriate. Mean values from the three groups were compared by one-way ANOVA. AUCs were calculated using GraphPad Prism software (version 7.0, www.graphpad.com). Comparisons were performed using the two-sided Wilcoxon signed-rank test. A two-tailed p value <0.05 was considered statistically significant.