Cut Down on Carbohydrate in the Dietary Therapy of Type 2 Diabetes - The Meal Box Study (CutDM-MealBox)

University of Copenhagen, University of Aarhus, The Danish Dairy Research Foundation, Denmark, Arla Foods

The cornerstone in the initial treatment of type 2 diabetes (T2D) is lifestyle modification, involving-among other things-a healthy diet. However, scientific evidence regarding optimal nutrition therapy for patients with T2D is insufficient. This clinical study will examine the effect of a carbohydrate-reduced high-protein (CRHP) diet compared to a conventional diabetes (CD) diet for 12 months on metabolic function and body weight in patients with T2D. The hypothesis of the study is that the CRHP diet will improve metabolic control and the cardiovascular risk profile of patients with T2D to a greater extent than the CD diet. In particular, the expectation is that, compared with the CD diet, the CRHP diet will: Reduce diurnal and postprandial glycemia measured by continuous glucose monitoring (CGM) and thereby facilitate a significant reduction of glycated hemoglobin (HbA1c) Reduce body weight Reduce ectopic fat deposition in the liver and the pancreas Improve the blood lipid profile Reduce or not affect blood pressure with no adverse effect on heart rate variability Increase insulin sensitivity and secretion Decrease inflammatory markers in the blood Improve satiety Reduce or not affect the need for antidiabetic, antihypertensive and/or lipid-lowering medications

Methods: This is a 12-month investigator-initiated, randomized, controlled, open-label, superiority trial with two parallel groups. The study examines the effect of a CRHP diet (which is reduced in carbohydrate and increased in protein and fat) compared with a CD diet (which follows the currently recommended macronutrient intake for patients with T2D). The study will include 100 T2D patients with overweight or obesity. The macronutrient intake in the CD diet and the CRHP diet is 50 percentage of energy (E%) and 30 E% from carbohydrate, 17 E% and 30 E% from protein, and 33 E% and 40 E% from fat, respectively. The CD diet and the CRHP diet do not principally differ in the quality of carbohydrate, protein, and fat; they both comprise of nutritious, organic, and sustainable food items. Participants will be randomized in a 1:1 ratio to either the CD diet or the CRHP diet for 12 months. About 2/3 of the total calculated energy requirements on both diets will be delivered to the participants free of charge, as meal box solutions containing breakfast meals, snack meals, and dinner meals to optimize compliance and adherence to the assigned diet. The daily energy requirements for body weight maintenance will be calculated by multiplying resting energy expenditure using the Mifflin-St Jeor equation, with an estimated physical activity level (estimated by using a physical activity questionnaire at the beginning of the study). Based on the calculated daily energy requirements, participants will be divided into one of three energy level groups; in all groups, the amount of recommended total daily energy intake will exceed the amount required for weight maintenance and subjects will be instructed to consume the diets ad libitum until satiety is achieved. The dietary interventions are implemented under the guidance of registered clinical dietitians (RCDs) in a free-living setting without any instruction or requirement for weight loss or increased physical activity level. Participants will be instructed to eat until satiety is achieved and allowed to consume alcoholic beverages within the recommendations from the Danish Health Authorities. Dietary advice and counselling regarding food choices and preparation of food concerning the allocated diet, especially the self-prepared lunch meals and how to navigate at special occasions, will be given under the guidance of RCDs. Medication will be kept unchanged during the study, if possible. Rescue medication will be commenced if a HbA1c target of 58 mmol/mol is not reached after six months. If study participants obtain a HbA1c below 48 mmol/mol antidiabetic medication will still be kept constant. Diurnal urine samples, fasting blood samples, dietary records and questionnaire responses will be collected every third month of the study. In addition, at baseline and 12 months, participants will undergo a standardized test battery including magnetic resonance imaging (MRI) and spectroscopy (MRS) for measurement of abdominal subcutaneous and visceral adipose tissue, and ectopic fat in the liver and pancreas, dual X-ray absorptiometry (DXA) scans for body composition, handgrip strength and 30-second chair-stand for muscle strength, oral glucose tolerance test (OGTT), continuous glucose monitoring (CGM), Holter-recording, diurnal blood pressure measurement, and dietary records. Analysis: The primary outcome will be tested by a constrained linear mixed model (CLMM) with inherent baseline-adjustment. Diet group, time and their interaction will be included as fixed effects and participants as random effect. Secondary analyses will furthermore adjust for potential confounders in multivariate CLMMs. The secondary and explorative outcomes will be tested for significance using CLMMs for continuous data (including changes in blood lipid profiles, body weight and hepatic fat content) and Chi-squared tests for proportions (including proportion of the participants who increase their need for glucose lowering medications from baseline to 12 months). Secondary and explorative outcomes will be tested at a significance level at 0.05 without correction for multiplicity, but interpreted according to their secondary and explorative nature. Missing data will be evaluated in regards to whether they can be assumed to be missing at random, and in that case handled implicitly by maximum likelihood estimation in the CLMMs, which is the optimal approach statistically. Intention-to-treat analyses will be performed including all available data. Per protocol analyses of the diet groups will be performed to support the primary analysis. Ethics and dissemination: The National Committee on Health Research Ethics of the Capitol Region of Denmark has approved the trial (H-21057605). The study will be conducted in accordance with the Declaration of Helsinki II. Results will be submitted for publication in international peer-reviewed scientific journals, regardless of being positive, negative or inconclusive.

The endocrinologists responsible for the pharmacological regulations during the study will be blinded to the allocation of the participants. In addition, the bio-analysts performing laboratory analyses, the MR physicist responsible for evaluating MRI/MRS scans, and the individual performing DXA scans, handgrip strength and 30-second chair-stand tests will also be blinded to the allocated diet group.

Carbohydrate-reduced high-protein (CRHP) dietary intervention. Intervention: Therapeutic Diet: CRHP Diet.

Macronutrient intake of 30 percentage of energy from carbohydrate, 30 percentage of energy from protein and 40 percentage of energy from fat.

Macronutrient intake of 50 percentage of energy from carbohydrate, 17 percentage of energy from protein and 33 percentage of energy from fat.

HbA1c will be measured in fasting blood samples at baseline, 3, 6, 9 and 12 months. HbA1c will be expressed in mmol/mol.

Total body weight will be measured at baseline, 3, 6, 9 and 12 months. Total body weight will be expressed in kilograms (kg).

Hepatic fat content will be assessed by magnetic resonance imaging/spectroscopy (MRI/MRS) at baseline and 12 months.

GV will be assessed using 7-day continuous glucose monitoring (CGM) (FreeStyle Libre Pro from Abbott) at baseline and 12 months.

Diurnal glycemia will be assessed using 7-day continuous glucose monitoring (CGM) (FreeStyle Libre Pro from Abbott) at baseline and 12 months. Diurnal glycemia will be expressed as mmol/L and evaluated as the area under the curve (AUC).

Change in abdominal subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) after 12 months on the CRHP diet compared with the CD diet

SAT and VAT will be assessed by magnetic resonance imaging/spectroscopy (MRI/MRS) at baseline and 12 months.

Pancreatic fat content will be assessed by magnetic resonance imaging/spectroscopy (MRI/MRS) at baseline and 12 months.

LBM will be assessed by dual-energy X-ray absorptiometry (DXA) at baseline and 12 months. LBM will be expressed in kilograms (kg).

ALM will be assessed by dual-energy X-ray absorptiometry (DXA) at baseline and 12 months. ALM will be expressed in kilograms (kg).

FM will be assessed by dual-energy X-ray absorptiometry (DXA) at baseline and 12 months. FM will be expressed in kilograms (kg).

Change in fasting serum triglycerides (TG) after 12 months on the CRHP diet compared with the CD diet

TG concentration will be measured in fasting blood samples at baseline, 3, 6, 9 and 12 months. TG will be expressed in mmol/L.

Change in triglyceride (TG) response during 4-hour oral glucose tolerance test after 12 months on the CRHP diet compared with the CD diet

TG response will be measured during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. TG will be expressed in mmol/L.

Change in lipid profile: total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein (HDL-C), non-high-density lipoprotein cholesterol (non-HDL-C) after 12 months on the CRHP diet compared with the CD diet

TC, LDL-C, HDL-C. and non-HDL-C will be measured in fasting blood samples at baseline, 3, 6, 9 and 12 months. TC, LDL-C, HDL-C and non-HDL-C will be expressed as mmol/L.

Apolipoprotein A-1 and B will be measured in fasting blood samples at baseline, 3, 6, 9 and 12 months. Apolipoprotein A-1 and B will be expressed as g/L.

Change in lipoprotein subfractions: low-density lipoprotein subclasses 1 to 5 (LDL1-5), high-density lipoprotein subclasses 1 to 5 (HDL1-5), and triacylglycerol-rich lipoprotein (TRL) after 12 months on the CRHP diet compared with the CD diet

Change in non-esterified fatty acids (NEFA) metabolism after 12 months on the CRHP diet compared with the CD diet

NEFA metabolism will be assessed by using the minimal model technique based on a oral glucose tolerance test (OGTT) at baseline and 12 months. NEFA concentrations will be expressed in umol/L.

Change in diurnal systolic blood pressure (BP) and diastolic BP after 12 months on the CRHP diet compared with the CD diet

Diurnal systolic and diastolic BP will be measured over 24 hours by a BP monitor (Ontrak 90227 from Spacelabs Healthcare) at baseline and 12 months. Diurnal systolic and diastolic BP will be expressed in mmHg.

HRV will be assessed by 48-hour Holter-monitoring (Faros 360 from Bittium) at baseline and 12 months.

Glucose will be measured at fasting and during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. Glucose concentration will be expressed in mmol/L and evaluated as the area under the curve (AUC).

Insulin sensitivity will be assessed based on a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. Insulin sensitivity will be expressed as the Matsuda Index.

Insulin secretion will be measured during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months, using C peptide deconvolution. Insulin concentration will be expressed in pmol/L.

C-peptide concentrations will be measured at fasting and during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. C-peptide will be expressed in pmol/L.

Beta-cell function will be assessed based on a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. Beta-cell function will be expressed by the Insulinogenic Index and Disposition Index.

Change in Homeostasis Model Assessment (HOMA) of insulin resistance and beta-cell function after 12 months on the CRHP compared with the CD diet

HOMA will be calculated at baseline and 12 months based on fasting glucose and insulin concentrations, and expressed as HOMA-IR (insulin resistance) and HOMA-beta (beta-cell function).

Glucagon concentration will be measured at fasting and during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. Glucagon will be expressed in pmol/L.

GLP-1 concentration will be measured at fasting and during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. GLP-1 will be expressed in pmol/L.

Change in glucose-dependent insulinotropic polypeptide (GIP) after 12 months on the CRHP diet compared with the CD diet

GIP will be measured at fasting and during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. GIP will be expressed in pmol/L.

GH will be measured at fasting and during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. GH will be expressed in ng/mL.

Change in insulin-like growth factor-binding protein 1 (IGFBP-1) after 12 months on the CRHP diet compared with the CD diet

IGFBP-1 will be measured at fasting and during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. IGFBP-1 will be expressed in ng/mL.

Change in insulin-like growth factor-1 (IGF-1) after 12 months on the CRHP diet compared with the CD diet

IGF-1 will be measured in fasting blood samples at baseline and 12 months. IGF-1 will be expressed in ng/mL.

PYY will be measured at fasting and during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. PYY will be expressed in pmol/L.

CKK will be measured at fasting and during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. CKK will be expressed in pmol/L.

Gastrin will be measured at fasting and during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. Gastrin will be expressed in pmol/L.

Ghrelin will be measured at fasting and during a 4-hour oral glucose tolerance test (OGTT) at baseline and 12 months. Ghrelin will be expressed in pmol/L.

Leptin will be measured in fasting blood samples at baseline and 12 months. Leptin will be expressed in ng/mL.

Adiponectin will be measured in fasting blood samples at baseline and 12 months. Adiponectin will be expressed in ug/mL.

Change in tumor necrosis factor alpha (TNF-alpha) after 12 months on the CRHP diet compared with the CD diet

TNF-alpha will be measured in fasting blood samples at baseline and 12 months. TNF-alpha will be expressed in pg/mL.

IL-6 will be measured in fasting blood samples at baseline and 12 months. IL-6 will be expressed in pg/mL.

CRP will be measured by high-sensitivity CRP test in fasting blood samples at baseline and 12 months. CRP will be expressed in mg/L.

Change in renal function assessed from eGFR after 12 months on the CRHP diet compared with the CD diet

Change in renal function assessed diurnal excretion of albumin after 12 months on the CRHP diet compared with the CD diet

Diurnal excretion of albumin will be assessed at baseline and 12 months. Diurnal excretion of albumin will be expressed in mg/day.

Urinary urea excretion will be assessed at baseline, 3, 6, 9 and 12 months. Urinary urea excretion will be expressed in mmol/day.

Change in metabolic syndrome severity Z-score (MetS-Z) after 12 months on the CRHP diet compared with the CD diet

Higher MetS-Z-scores indicate increased severity of MetS. A score ≥1 will be yielded if: 1) waist circumference >94 cm (in men) and >80 cm (in women), 2) HDL-C <1,0 mmol/L (in men) and <1,3 mmol/L (in women), 3) triglycerides ≥1,7 mmol/L, 4) fasting glucose ≥5,6 mmol/L, 5) systolic blood pressure ≥130 mmHg, or 6) diastolic blood pressure ≥85 mmHg. Participants will be classified as having MetS if three or more component thresholds were exceeded. MetS-Z will be assessed at baseline and 12 months.

Change in antidiabetic, antihypertensive and lipid-lowering medications after 12 months on the CRHP diet compared with the CD diet

Antidiabetic, antihypertensive and lipid-lowering medications will be assessed at baseline and after 3, 6, 9 and 12 months.

Handgrip strength will be measured with a SAEHAN hand dynamometer in both the right and the left hand at baseline and 12 months.

Change in muscle strength measured by 30-second chair-stand after 12 months on the CRHP diet compared with the CD diet

Muscle strength will be measured by 30-second chair-stand at baseline and 12 months. Participants will be asked to fold arms across the chest and to stand up and sit down on a chair as many times as possible for 30 seconds.

Difference between calculated and advised energy intake in the CRHP diet compared with the CD diet after 12 months

Energy intake will be calculated from a three-day dietary record entered in the online diet calculation tool "MADLOG" at the beginning of the intervention and at 3, 6, 9, and 12 months. Energy intake will be expressed in kilocalories (kcal).

Difference between calculated and advised macronutrient intake expressed in kilocalories (kcal) in the CRHP diet compared with the CD diet after 12 months

Macronutrient (carbohydrate, added sugar, protein, fat and fatty acids) intake will be calculated from a three-day dietary record entered in the online diet calculation tool "MADLOG" at the beginning of the intervention and at 3, 6, 9, and 12 months. Macronutrients will be expressed in kilocalories (kcal).

Difference between calculated and advised macronutrient intake expressed in percentage of energy (E%) in the CRHP diet compared with the CD diet at beginning after 12 months

Macronutrient (carbohydrate, added sugar, protein, fat and fatty acids) intake will be calculated from a three-day dietary record entered in the online diet calculation tool "MADLOG" at the beginning of the intervention and at 3, 6, 9, and 12 months. Macronutrients will be expressed in percentage of energy (E%).

Differences in dietary intake expressed in kilocalories (kcal) after 12 months on the CRHP diet compared with the CD diet

Dietary intake will be calculated from a three-day dietary record entered in the online diet calculation tool "MADLOG" at the beginning of the intervention and at 3, 6, 9, and 12 months. Dietary intake will be expressed in kilocalories (kcal).

Differences in dietary intake expressed in percentage of energy (E%) after 12 months on the CRHP diet compared with the CD diet

Dietary intake will be calculated from a three-day dietary record entered in the online diet calculation tool "MADLOG" at the beginning of the intervention and at 3, 6, 9, and 12 months. Dietary intake will be expressed in percentage of energy (E%).

Differences in dietary intake expressed in grams (g) after 12 months on the CRHP diet compared with the CD diet

Dietary intake will be calculated from a three-day dietary record entered in the online diet calculation tool "MADLOG" at the beginning of the intervention and at 3, 6, 9, and 12 months. Dietary intake will be expressed in grams (g).

Change in diabetes-related emotional distress after 12 months on the CRHP diet compared with the CD diet

Diabetes-related emotional distress will be measured by the Danish version of The Problem Areas in Diabetes (PAID) Questionnaire. PAID contains 20 items that describe negative emotions related to diabetes (e.g. fear, anger, frustration) experienced by patients with diabetes. Each question has five possible answers with a value from 0 to 4, with 0 representing "no problem" and 4 "a serious problem". The scores are added up and multiplied by 1.25, generating a total score between 0-100. Patients scoring 40 or higher may be at the level of "emotional burnout" and warrant special attention. PAID scores in these patients may drop 10-15 points in response to educational and medical interventions. An extremely low score (0-10) combined with poor glycaemic control may be indicative for denial. Diabetes-related emotional distress will be measured at baseline, 6 and 12 months.

Perceived dietary adherence will be measured by Perceived Dietary Adherence questionnaires which consists of questions structured to cover the CRHP diet and the CD diet, with responses on a seven-point Likert scale at 3, 6, 9 and 12 months.

Change in health-related quality of life (HRQoL) after 12 months on the CRHP diet compared with the CD diet

Scale title: 'SF-36 Spørgeskema om Helbredstilstand' (Eng: SF-36 questionnaire about health condition). HRQoL will be assessed using a self-reported Short-Form (SF-36) health survey with 36 questions regarding different domains of HRQoL. Questions are answered via a 2-6 point categorical scales at baseline, 6 and 12 months. The following minimum and maximum values are used: 2-point scale ranging from 1=Yes, 2=No 3-point scale ranging from 1=Yes, very limited, to 3=No, not at all limited, 5-point scales ranging from 1=Much better - 5=Much worse; 1=Not at all - 5=Very much; 1=All the time - 5=At no time; 1=Completely right - 5=Completely wrong 6-point scales ranging from 1= No pain - 6=Very heavy pain; 1= All the time - 6=At no time For all question the better outcome very depending on the question in focus.

Change in meal acceptance of 3 morning and 3 evening meals after 12 months on the CRHP diet compared with the CD diet

Scale title: 'Meal acceptance' (note: self-created scale). Meal acceptance will be measured by an 8-item self-reported questionnaire via visual analog scales (VAS), a 4-point categorical scale and an open-text reply field. Meal acceptance will be assessed at baseline, 3, 6, 9 and 12 months. The following minimum and maximum values are used: VAS scale with anchor points 'Dislike extremely' to 'Like Extremely', with 'dislike extremely meaning the better outcome 4 point categorical scale with no minimum and maximum value, and no better/worse outcome

Scale title: 'Well-being' (note: self-created scale). Well-being will be measured by a 9-item self-reported questionnaire using visual analog scales (VAS) and a categorical option. Well-being will be assessed at baseline, 3, 6, 9 and 12 months. The following minimum and maximum values are used: • VAS scale with anchor points 'Extremely little' to 'Extremely much', with the better outcome varying depending on the question in focus

Change in subjective satiating capacity of meals after 12 months on the CRHP diet compared with the CD diet

Scale title: 'Appetite' (note:self-created scale). Satiating capacity of meals will be measured after intake of 3 morning and 3 evening meals by an 18-item self-report questionnaire using visual analog scales (VAS) and a categorical option. Satiating capacity will be assessed at baseline, 3, 6, 9 and 12 months. The following minimum and maximum values are used: VAS scale with anchor points 'Not at all' to 'A very large amount', with no better/worse outcome VAS scale with anchor points 'Extremely little' to 'Extremely much', with no better/worse outcome

Scale title: 'Food-related Quality of Life'. Change in food-related quality of life will be measured by a 5-item self-reported questionnaire using a 5-point likert scale. Food-related quality of life will be assessed at baseline, 6 and 12 months. The following minimum and maximum values are used: • 5-point scale ranging from 1=Very much agree, to 5=Very much disagree, with 1 meaning the better outcome

Scale title: 'VARSEEK'. Variety-seeking behavior will be measured using the 8-item Variety-seeking likert scale at baseline, 6 and 12 months. The following minimum and maximum values are used: • 5-point scale ranging from 1=Very much agree, to 5=Very much disagree, with no better/worse outcome

Change in emotional, retained and external eating behaviors after 12 months on the CRHP diet compared with the CD diet

Scale title: 'Dutch Eating Behavior Questionnaire'. Emotional, retained and external eating behaviors will be measured using the 32-item categorical Dutch Eating Behavior scale at baseline, 6 and 12 months. The following minimum and maximum values are used: • 5-point scale ranging from 1=Never, to 5=Very often, with no better/worse outcome

Change in drivers of food-related pleasure after 12 months on the CRHP diet compared with the CD diet

Scale title: 'The Food Pleasure Scale'. Drivers of food-related pleasure will be measured using the 21-item categorical Food Pleasure Scale at baseline, 6 and 12 months. The following minimum and maximum values are used: 2-point scale ranging from 1=Yes, to 5=No, with no better/worse outcome 5-point scale ranging from 1=Not at all important, to 5=Extremely important, with no better/worse outcome

Inclusion Criteria: Men or postmenopausal women aged 18-75 years. Menopause is defined as >12 months without menses Overweight or obesity with Body Mass Index (BMI) >25 kg/m2 Type 2 diabetes with HbA1c between 48 mmol/mol and 75 mmol/mol (6.5%-9.0%) Treated with or without Metformin, dipeptidyl peptidase 4 (DPP-4) inhibitors, sodium-glucose cotransporter 2 (SGLT-2) inhibitors and/or glucagon-like peptide 1 receptor agonists (GLP-1RA) Nonsmokers or having quitted smoking >1 year before the study Acceptance of regulation of antidiabetic, antihypertensive, and lipid-lowering medications by Cut-DM endocrinologists only Exclusion Criteria: Ongoing insulin or insulin analog therapy Severe gut disease as evaluated by the principal investigator e.g. Crohn's disease, Ulcerative colitis, Celiac disease etc. Extensive surgery to the gut e.g. bariatric surgery, colectomy etc. Severe heart disease as evaluated by the principal investigator e.g. angina pectoris, coronary heart disease, congestive heart failure (NYHA III-IV) Severe renal impairment (eGFR<45 ml/min/1.73 m2 or urine albumin / creatinine ratio > 300 mg/g) Severe hepatic impairment as evaluated by the principal investigator (measure of alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) Cancer within the last 5 years (except basal cell skin cancer or squamous cell skin cancer) Psychiatric disease, e.g. a history of major depressive or other severe psychiatric disorders Systemic corticosteroid treatment, e.g. prednisolone Reported or documented food allergy, food intolerance, or strong food preferences Reported or documented alcohol consumption exceeding the recommendations from The Danish Health Authorities Ongoing treatment with sulfonylureas and/or thiazolidinediones due to the risk of hypoglycemia unless discontinuation is possible, in which case a >3-month wash-out is mandatory Hemoglobin <7 mmol/L for men and <6 mmol/L for women Inability, physically and/or mentally, to comply with the procedures required by the study protocol, as evaluated by the principal investigator Weight change ≥5% the preceding 3 months of screening Participation in other on-going clinical trials

Individual participant data (IPD) may be shared by request to the corresponding author in accordance with the Danish Data Protection Agency.