Study design
The study was performed as an acute randomised, single-blinded, intervention with four test meals consisting of breads made with a mix of hulless barley flour (50% or 75%) or AmOn flour (50%) with wheat flour (50% or 25%) and compared to 100% wheat flour bread. The study participants were randomised to the order of visits using RedCap®.
The primary outcome, postprandial glucose response, was evaluated by changes in the incremental area under the glucose response curve. The secondary outcomes were postprandial changes in insulin, glucagon, triglyceride (TG), free fatty acids (FFA), glucagon like peptide 1 (GLP-1), and gastric inhibitory polypeptide (GIP) responses given as either iAUC or total AUC (tAUC).
Study participants
Twenty Caucasian subjects with T2D were recruited through local newspapers and electronic advertisement to participate in the study. The study was conducted at the Department of Endocrinology and Internal Medicine, Aarhus University Hospital, between March 2021 and October 2021. After receiving written and oral information, all subjects gave their written informed consent before participating in the study. Subjects were screened on the basis of their medical history and a physical examination. The inclusion criteria were as follows: Adults ≥18 years of age. Diabetes based on International Diabetes Federation criteria (https://apps.who.int/iris/bitstream/handle/10665/43588/9241594934_eng.pdf) with on-treatment haemoglobin A1c (HbA1c) between 42 and 78. All anti-diabetic medications were allowed except the below mentioned.
The exclusion criteria were: type 1 diabetes, insulin treated T2D, use of weekly administrated GLP-1 agonist, use of acarbose, significant cardiovascular, kidney, liver or endocrine comorbidity, significant psychiatric history, treatment with steroids, alcohol or drug abuse, pregnancy or breastfeeding, or legal incompetence.
Treatment with drugs for hypertension or high cholesterol was allowed if the treatment dose was stable throughout the study period.
The study protocol was carried out in accordance with the Helsinki Declaration of 1975 as revised in 1983 and was approved by the Central Denmark Region Committees on Health Research Ethics (1-10-72-299-20). The study was registered at clinicaltrials.gov as NCT04646746.
Production of dietary supplements and dietary assessment
All participants were provided with a standard meal for dinner the night before each study day. The meals were commercially produced ‘chili con carne’ (Salling Group A/S, Denmark). The women’s meal contained 536 kJ and the men’s meal contained 638 kJ.
Bread with 100% wheat flour (standard commercial Manitoba wheat flour, HavneMøllen, Vejle, Denmark) and bread mixed of wheat and hulless barley flour were produced by ‘P.A. Andersen bakery’ (Vejle, Denmark), whereas bread made from a mixture of wheat and AmOn flour was produced at ‘Plantcarb ApS’ (Slagelse, Denmark), since genetically modified flour is not allowed in Danish commercial bakeries. The grains were grown in field plots over the summer of the year 2020 at Aarhus University (Flakkebjerg, Denmark). Whole grains of AmOn were ground into flour by a Komo Fidibus 21 benchtop mill (KOMO GMBH & CO. KG, Hopfgarten, Germany). The breeding and characterisation of starch from the AmOn barley variety has previously been described [14]. In brief, the AmOn was based on a hulled barley variant (H. vulgare var. golden promise) and was bred to contain more than 99% amylose of the starch fraction by introducing a chimeric RNAi hairpin DNA construct that simultaneously reduced the expression levels of the genes SBEIIa, SBEIIb and SBEI by more than 90%. The hulless barley (H. vulgare var. PS3) used in this study was developed by the breeder Agrologica (Mariager, Denmark).
Similar recipes were used for the four breads. The amount of total carbohydrate was calculated using ‘Vitakost ApS’ (Kolding, Denmark) and the amount of bread corresponding to 50 g of total carbohydrate was estimated. All four bread types were packed in individual sealed bags containing either 114 g (100% wheat), 119 g (50% hulless barley), 122 g (75% hulless barley) or 128 g (50% AmOn) of bread corresponding to 50 g of estimated total carbohydrate. Hereafter the bread was frozen at –20 °C. At the study days the bread was taken out of the freezer and defrosted without being heated.
Bread component analysis
The moisture content was determined by the weight loss after drying in a 120 °C oven for 24 hours. The total carbohydrate content was measured as the sum of the fibre and starch content. The Megazyme K-TSTA kit (Megazyme, Co. Wicklow, Ireland) was used to measure the total starch content of samples containing resistant starch following the manufacturer’s instructions. This method variant uses dimethyl sulfoxide and boiling bath, and dissolution in dimethyl sulfoxide at 100 °C is effective to solubilize all starches in the bread, including the resistant starch. The fibre content was determined using the Megazyme total fibre assay kit (K-TDFR-200A, Megazyme, Co. Wicklow, Ireland) following the manufacturer’s instructions. The K-TDFR-200A kit measure mainly the cell wall polysaccharides and some RS.
Visits
After signing the informed consent form, screening blood samples were analysed for alanine aminotransferase, HbA1c, fasting glucose, TG, thyroid-stimulating hormone, sodium, potassium, creatinine, and haemoglobin to rule out intercurrent disease and to ensure that the inclusion criteria of HbA1c was fulfilled.
After a standardised evening meal and an overnight fast (from midnight), the study participants arrived at the clinic between 07.00–07.30 AM at all four study days. A catheter was placed in a cubital vein for blood sampling. Baseline blood samples were drawn (at timepoints –10 and 0 min), and then the test bread was consumed within the next 10 min together with 250 ml of tap water.
During the following four hours blood samples were drawn at specified time points: glucose, insulin, and glucagon at –10, 0, 10, 20, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min; TG, FFA, GLP-1 and GIP at –10, 0, 30, 60, 120, 180, and 240 min. All blood samples were immediately centrifuged at 3000 g for 10 min at 4 °C; thereafter, the plasma samples were frozen at –20 °C and the next day stored at –80 °C.
Smoking was not allowed during the overnight fast or during the study visits. Alcohol consumption was not permitted for two days before the fasting visits and strenuous exercise was not permitted the day before the fasting visits. Anti-hypertensive, cholesterol-lowering drugs and anti-diabetic drugs were paused 24 hours before every study day. The four intervention days were separated by at washout period of minimum six days.
Blood analyses
Plasma glucose was measured by a glucose oxidase method with a GOD-PAP glucose kit (no. 11491253216; Roche Diagnostics GmbH, Germany). Serum insulin and glucagon were measured with ELISA (insulin no. 10-1113-01 and glucagon no. 10-1271-01; Mercodia AB, Sweden). Plasma TG and FFA concentrations were measured with enzymatic colorimetric assays by using commercial kits (No. 04657594190, Roche Diagnostics GmbH, Germany, for TG and code 270–7700, Wako Chemicals GmbH, Germany, for FFA). Measurements of both parameters were made on a Cobas c111 analyser (Roche Diagnostics GmbH, Germany). GLP-1 and GIP were measured with NL-ELISA techniques (GLP-1 no. 10-1278-01 and GIP no. 10-1258-01; Mercodia AB, Sweden).
Statistical analysis
The power calculation was based on previous results from our group and made to detect a difference in our primary outcome (i.e., postprandial glucose response, given as iAUC) of 20% between diets [11]. The number of participants needed to complete the study and achieve a statistical power of 80% was calculated to be 20 (a < 0.05, b = 0.80). A mixed model ANOVA was used to examine the effect of each bread type compared with the wheat bread (control). P < 0.05 was considered statistically significant. Results are given as mean ± 95% confidence interval (CI) in tables and as mean ± SEM in graphs, unless otherwise stated. Fasting values of the outcomes are presented in Table 3, whereas postprandial changes, given as changes in percentages from fasting value, are presented in graphs along with the corresponding iAUC (area above fasting value) for glucose, insulin, GLP-1 and GIP, and as tAUC (area above zero) for glucagon, TG and FFA. All statistical calculations were performed with STATA version 17 (StataCorp LP, Texas, USA) and GraphPad Prism 6 (GraphPad Software, Boston, USA) was used to generate the graphical elements.