Study design

The study was designed as a 6 + 6 week open label, randomised, crossover-controlled trial. Inclusion criteria were: men and women with type 2 diabetes [14], HbA 1c 48–97 mmol/mol (6.5–11%), age >18 years, haemoglobin >6/>7 mmol/l (women/men) and eGFR >30 ml min−1 (1.73 m)−2. Range of eGFR was 56 ml min−1 (1.73 m)−2 to >90 ml min−1 (1.73 m)−2. The lower HbA 1c inclusion criterion was adjusted from the initial 58 mmol/mol (7.5%) to 48 mmol/mol (6.5%) to facilitate recruitment. Exclusion criteria were: critical illness, systemic corticosteroid treatment, severe food allergy or intolerance, severe gut disease, alcohol dependence syndrome, injectable diabetes medication, repeated fasting plasma glucose >13.3 mmol/l, urine albumin/creatinine ratio > 300 mg/g, lactation, pregnancy or planning of pregnancy during the study and inability, physically or mentally, to comply with the procedures required by the study protocol. Examinations were initiated in April 2016 and terminated according to the pre-specified study plan in November 2017. All physical examination procedures were performed at Copenhagen University Hospital, Bispebjerg, except the MRI and spectroscopy, which were performed at Copenhagen University Hospital, Herlev. A third-party study nurse randomised the participants by drawing blinded ballots. Block randomisation was applied with block sizes of two and balanced to ensure allocation in a 1:1 ratio.

All participants signed informed consent prior to any study-related procedures. The study was approved by the local Scientific Ethical Committee and carried out in accordance with the Declaration of Helsinki. The study was registered at ClinicalTrials.gov (registration no. NCT02764021).

Dietary interventions

For 12 weeks, participants were provided, free of charge, with five daily meals to fully cover daily energy needs (breakfast 25% of total energy expenditure [TEE%], lunch 30 TEE%, pre-dinner snack 7.5 TEE%, dinner 30 TEE%, post-dinner snack 7.5 TEE%). Consistent with diabetes dietary guidelines provided by the Diabetes and Nutrition Study Group (DNSG) of the EASD [2], the conventional diabetes (CD) diet consisted of 50% energy (E%) carbohydrate, 17 E% protein and 33 E% fat. Macronutrient composition of the carbohydrate-reduced high-protein (CRHP) diet was 30 E% carbohydrate, 30 E% protein and 40 E% fat (electronic supplementary material [ESM] Table 1). Meals were prepared as a 7 day rotational procedure and produced and distributed twice weekly by trained personnel at the Department of Nutrition, Exercise and Sports, Copenhagen University. Participants were restricted to consume study meals and beverages only according to assigned intervention. Ad libitum intake of non-caloric beverages was allowed.

Provision of foods for maintenance of body weight

Daily total energy expenditure (TEE) was estimated by an algorithm based on participant characteristics and body composition measured by dual-energy x-ray absorptiometry (Lunar iDXA, General Electric Healthcare, GE Medical Systems, Belgium), as previously described [9, 15]. Body weight was measured twice weekly. If changes occurred, energy intake was adjusted accordingly, by lowering/increasing portion sizes and/or by adding/retracting with additional 0.5 MJ macronutrient-adjusted meal snacks, to ensure weight stability. Participants were repeatedly instructed to maintain a constant physical activity level. Furthermore, participants were instructed to keep records of any leftovers and were interviewed twice per week to ensure adherence to study diets.

Urine samples

Twenty-four-hour urine samples were collected after 4 weeks on each diet to evaluate compliance to interventions unconfounded by the investigational programme. We used quantification of 24 h urinary urea excretion, based on an enzymatic ultraviolet test (ABX Pentra 400 Urea CP, Horiba ABX SAS, France), which provides a validated measure of protein intake [16]. Moreover, samples were used to assess 24 h urinary glucose excretion by colorimetry (ABX Pentra 400 Glucose PAP CP, Horiba ABX SAS), 24 h urinary albumin excretion by an immunoturbidimetric test (ABX Pentra 400 Micro Albumin 2 CP, Horiba ABX SAS) and 24 h urinary creatinine excretion based on the reaction between creatinine and sodium picrate (ABX Pentra 400 Creatinine 120 CP, Horiba ABX SAS) to calculate albumin/creatinine ratio.

Blood samples

Blood samples were collected after a 12 h overnight fast. Serum was stored at room temperature for 30 min to ensure coagulation before centrifugation. Plasma was collected in EDTA-treated test tubes and immediately centrifuged after sampling. Serum was used for analysis of insulin, C-peptide, NEFA, total cholesterol, HDL-cholesterol, triacylglycerol, apolipoprotein A1 (ApoA-1) and apolipoprotein B (ApoB). IMMULITE 2000 Immunoassay System (Siemens Healthcare, Erlangen, Germany) was used to analyse insulin and C-peptide. Wako NEFA-HR(2) (ASC-ACOD method, Wako Chemicals, Neuss, Germany) was used for fasting NEFA concentrations. Enzymatic colorimetric assays (CHOL2, HDLC4 and TRIGL, Cobas 6000, Roche Diagnostics, Mannheim, Germany) were used to analyse total cholesterol, HDL-cholesterol and triacylglycerol, respectively. LDL-cholesterol was calculated by using the Friedewald equation [17]. Non-HDL-cholesterol was calculated by subtracting HDL-cholesterol from total cholesterol. ApoA-1 and ApoB were analysed using an immunoturbidimetric test (APOAT and APOBT, Cobas 6000, Roche Diagnostics).

Plasma glucose concentrations were analysed immediately after sampling by use of YSI 2300 STAT Plus (Yellow Springs Instruments, Yellow Springs, OH, USA). Whole blood was collected and handled in accordance with local operating procedure for analysis of HbA 1c using the Tosoh Automated Glycohemoglobin Analyser HLC-723G8 (G8; Tosoh Bioscience, Japan). Insulin resistance (HOMA2-IR) was calculated by use of the HOMA2 calculator (version 2.2.3; www.dtu.ox.ac.uk/homacalculator/, accessed 14 January 2019) based on fasting plasma glucose and C-peptide.

Mixed meal testing

As displayed in ESM Fig. 1, a CRHP mixed meal test (MMT) was performed after 6 weeks of CRHP intervention and a CD MMT was performed after 6 weeks of CD intervention in order to test the postprandial responses of the two study diets, i.e. differences in macronutrient composition. Solid phase mixed test meals (ESM Table 1) were produced in five energy levels ranging from 2.25 MJ (598 kcal) to 3.25 MJ (837 kcal) to approximately match 25% of TEE for each participant. All ingredients were weighed out with an accuracy of 0.5 g by trained personnel at the research kitchen, Department of Nutrition, Exercise and Sports, University of Copenhagen. Participants were instructed to avoid any strenuous physical activity 48 h prior to meal tests. Alcohol consumption was prohibited during the entire study. Meal tests were performed after consumption of a standardised CD or CRHP dinner according to assigned intervention and a subsequent 12 h overnight fast. A peripheral i.v. cannula was inserted in an antecubital vein before a solid CRHP or iso-energetic CD meal was served at time 0 and consumed within 25 min. Participants remained sedentary throughout the examination.

Magnetic resonance examination

Magnetic resonance measurements were performed at baseline and at the end of each dietary intervention period using a 3.0 T Ingenia MRI system (Philips Medical Systems, Best, the Netherlands) with a dStream torso coil. Magnetic resonance scanning protocol included planning scans, single voxel spectroscopy (Point RESolved Spectroscopy [PRESS]) for measuring liver fat content and muscle fat content [18, 19], and chemical shift encoding-based water-fat imaging (mDixon) for measuring pancreatic fat content, visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT) and waist perimeter. Means of duplicate analyses of pancreas fat fractions were used to reduce intra-observer variability. VAT and SAT volumes and waist perimeter were measured using a single transverse section of 10 mm thickness acquired at the middle of the third lumbar vertebra, as described in detail previously [20]. All magnetic resonance data were analysed by a research technician blinded to treatment.

Diurnal blood pressure

Twenty-four-hour ambulatory blood pressure (ambulatory blood pressure monitor: Model 90217, Spacelabs Healthcare, WA, USA) was measured at home at baseline and at the end of each dietary treatment period.

Statistical analysis

An a priori calculation of sample size was performed based on earlier published effect size [7]. With 90% power, a sample size of six participants was sufficient to detect a 12.1 mmol/mol (~1.1%) between-diet difference in the primary outcome, HbA 1c , in this crossover setting, provided an SD of ±6.6 mmol/mol (~0.6%) and a significance level of 5%. To account for possible dropouts, smaller than expected effect size and multiple pre-specified exploratory analyses of secondary endpoints, a total of 30 participants were included.

The study includes a total of 37 pre-specified secondary outcomes, of which nine, i.e. glycaemic variables, ectopic fat content and 24 h blood pressure, are presented in this paper (for details on all outcomes, please refer to ESM Table 2). Other pre-specified outcomes, i.e. beta cell function, NEFA metabolism, low-grade inflammation, growth hormone axis, heart rate variability, gut and satiety hormones and subjective satiety will, following analyses, be reported subsequently. The statistical analysis of the primary outcome and the secondary outcomes was performed with a significance level of p < 0.05 (two-tailed test). Due to multiple comparisons, a risk of false-positive results may inherently exist.

Results are presented as means (±SEM) or as medians (interquartile range) if normal distribution assumptions were not met as assessed by visual examination. AUC was calculated using the trapezoidal rule. Net AUC was calculated by subtracting area below baseline values from the AUC. A linear mixed effects model was used to evaluate differences in treatment effects between diets adjusted for body weight change for all variables reported, except body weight and BMI, for which a paired sample t test was applied. All linear mixed effects models included body weight change and an interaction between diet and period as fixed effects. To account for inter-participant variability caused by the paired design, participants were applied as random effects. Residuals were visually examined for linearity and normality. If appropriate, data were log transformed to meet model assumptions for the mixed effects model. Depending on the distribution of data, a paired sample t test or a Wilcoxon signed-rank test was used to evaluate differences between baseline circulating and imaging biomarkers. Baseline values were defined as means of the initial measurements at the beginning of the two CRHP and the two CD dietary treatment periods. Due to the crossover design, the final measure of the first 6 week period was used as the baseline for the second 6 week period. Considering the low dropout rate (2 out of 30), only per-protocol analyses were carried out. All available data were included, i.e. n = 28, except for 24 h ambulatory blood pressure monitoring and hepatic and pancreatic fat fractions, which, due to technical failure and problems with diurnal urine sampling, were obtained in 27 participants only. Statistical analyses were performed by use of RStudio (Version 0.99.473; RStudio, Boston, MA, USA) and graphical presentations were made using GraphPad Prism (Version 7.02; GraphPad Software, San Diego, CA, USA).