The Interaction Between Protein Intake, Gut Microbiota and Type 2 Diabetes in Subjects With Different Ethnic Backgrounds

Study Overview
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Brief Summary
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Context and justification: There is growing evidence that the gut microbiota is a key element in the pathophysiology of cardio-metabolic diseases (CMD) such as Type 2 Diabetes (T2D). One hypothesis is that gut-derived metabolites (from diet) have an important role in the host metabolism. Preliminary results show that imidazole propionate (ImP), a degradation product of the essential amino acid histidine, is produced by the gut microbiota of T2D patients, but not healthy subjects. The gut microbiota itself is strongly influenced by diet and ethnicity. However, most dietary intervention studies have focused on the role of fiber intake and the effect of dietary protein on the gut microbiota composition and metabolite production is not well known. Our hypothesis is that, depending on the baseline gut microbiome composition, a diminution in protein intake could decrease the microbial production of metabolites such as ImP and improve the metabolism of the host. We also hypothesize that the effects of such an intervention could depend the ethnic background. Objective: To study the effects of a high protein (HP) vs a low protein (LP) diet on gut microbiota composition and production of pro-diabetic metabolites in type 2 diabetes (T2D) patients from Caucasian and Caribbean ethnicity depending on baseline metagenomics richness. Study design: Randomized controlled three months dietary intervention study Study Population: T2D patients from Caucasian (N=80) and Caribbean (N=40) background who are on a stable dose of metformin and do not use insulin or proton-pump inhibitors. Intervention: Subjects will be randomized to either a high protein (HP) or low protein (LP) diet for three months. Individuals of Caucasian ethnicity, will also be stratified according to either a high or low gut microbiota gene richness. All subjects will receive pre-cooked meals 6 days per week and daily food packages. Subjects are required to keep food diaries three days a week and will also have weekly contact with an Pitié-Salpêtrière dietician. Outcome measures: Primary endpoint is the change in glycemic excursion (area under the curve) after a mixed meal test between baseline and 12 weeks after the beginning of the intervention. Furthermore, we will study oral and fecal microbiota composition changes as well as serum levels of intestinal metabolites, such as ImP, body weight and body composition at baseline and after 12 weeks. Sample Size: It is calculated that a total of 20 patients per arm are needed so 120 patients in total.

Detailed Description
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Context and justification: There is growing evidence that the gut microbiota is a key element in the pathophysiology of cardio-metabolic diseases (CMD) such as Type 2 Diabetes (T2D). One hypothesis is that gut-derived metabolites (from diet) have an important role in the host metabolism. Preliminary results show that imidazole propionate (ImP), a degradation product of the essential amino acid histidine, is produced by the gut microbiota of T2D patients, but not healthy subjects. The gut microbiota itself is strongly influenced by diet and ethnicity. However, most dietary intervention studies have focused on the role of fiber intake and the effect of dietary protein on the gut microbiota composition and metabolite production is not well known. Moreover, it has been shown that the response to a dietary intervention may depend on the baseline gut microbiome richness. Main hypothesis: Depending on the baseline gut microbiome composition, a diminution in protein intake could decrease the microbial production of metabolites such as ImP and improve the metabolism of the host. We also hypothesize that the effects of such an intervention could depend the ethnic background. Study population: Individuals with type 2 diabetes (T2D), of Caucasian or Caribbean origin, 120 patients will be included in total Intervention: Assignment after randomization to one of the following 2 diets: HP diet: high protein (High Protein, HP) diet with 30% protein, 40% carbohydrate and 30% fat (as% of total energy intake) LP diet: low protein diet (Low Protein, LP) with 10% protein, 55% carbohydrate and 35% fat (as% of total energy intake) Food boxes adapted to each diet (HP or LP pre-cooked meals, HP or LP breads and snacks) will be provided to the participants throughout the study reaching 40-50% of their prescribed daily energy intake for 6 days per week. Subjects are required to keep food diaries three days a week and will also have weekly contact with a dietician. Visits: - Inclusion visit V0 (maximum 1 month before V1): Participants will first be recruited from the diabetic population of the French cohort of the European project METACARDIS. Individuals eligible for the study are screened for inclusion. Baseline phenotyping is performed (metabolic, inflammatory blood markers, stool and oral microbiota sampling, body composition by DXA, questionnaires) - Randomization visit V1 (T0 - start of the intervention): Randomization into 2 parallel groups (High Protein or Low protein diet) will be stratified based on metagenomics richness (obtained from Metacardis results), age (< or ≥ 60), gender, ethnic background (Caribbean or Caucasian). Meal tolerance test, anthropometric measures, resting energy expenditure measure, one week CGMS, 24h urinary urea measure are performed. - Follow-up visit V2 (T42 +/- 7 days): Mid protocol visit with anthropometric measures, one week CGMS, 24h urinary urea measures, stool sampling. - End of study visit V3 (T84 +/- 7 days): Phenotyping is performed (metabolic, inflammatory blood markers, stool and oral microbiota sampling, body composition by DXA, questionnaires, meal tolerance test, anthropometric measures, resting energy expenditure measure, one week CGMS, 24h urinary urea measure) Statistical analysis: There are no multiple hypotheses since our study has only one primary objective (AUC delta of the glycemic excursion after a mixed meal tolerance test (MMT) between the beginning of study and 3 months post intervention). Thus, the problem of the type 1 error will not arise. The primary endpoint will be analyzed to compare changes in AUC for glycemic excursion versus diet (rich vs. low protein), based on initial metagenomic richness (high vs. low) and ethnicity (Caucasian vs. Caribbean). AUC changes after dietary intervention between the different groups will be tested using linear regression models for repeated measurements with adjustment for initial levels. The effect of diet composition within the groups will be tested using Bonferroni's post-hoc covariance analysis (ANCOVA) analyzes. For secondary endpoints, the same approaches will be used for analysis of postprandial metabolites (AUC, AUC, post-MMT variation). Differences in relative abundance of bacterial species and functional modules (generated by metagenomic sequencing) and quality of life questionnaires will also be analyzed by subgroups using uni / multivariate analyzes. Correlations will be sought between changes in bio-clinical variables and changes in measurements of different metabolites. Funding: European Program (Join Program Initiative, JPI HDHL / ERA-NET cofund HDHL-INTIMIC, -Edition 2017) through the National Agency for Research (ANR) Leducq Foundation (Research Grant 17CVD01 titled Gut Microbiome as a Target for the Treatment of Cardiometabolic Diseases). K Santé Society providing meals for the study. Nutrisens Society providing snacks for the study.

Official Title
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Modulation of Protein Intake to Target Gut-microbiota Derived Metabolites of Amino Acids in Individuals With Type 2 Diabetes From Varying Ethnic Backgrounds

Conditions
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Type 2 Diabetes

Intervention / Treatment
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* Other: Diet HP
* Other: Diet LP


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Age ≥ 40 years and <70 years 2. Type 2 Diabetic Subjects (T2D) Treated with stable dose of metformin (no dose change in the last 3 months) BMI ≥ 25 kg / m2 Caucasian or Caribbean origin Written and oral comprehension of the French language Patient affiliated to health care. Patient having been informed of the study and having given written consent to participation Exclusion Criteria: Pregnancy or breastfeeding Insulin treatment HbA1c ≥ 9% (<3 months) Recent antibiotic treatment (<3 months) Recent treatment with proton pump inhibitor (<3 months) Food allergies or documented intolerances Patient not willing to eat the foods provided in the protocol Neuromuscular or neurological disease History of digestive cancer and / or abdominal radiotherapy History of gastrointestinal surgery with gastrointestinal resection Acute or chronic inflammatory or infectious disease (including HIV, HCV, HBV) Organ Transplantation, Immunosuppressive drugs Severe chronic renal insufficiency (creatinine> 150 μmol / l or eDFG <50 ml / min per 1.73 m2 body surface area) Patient currently included in an interventional clinical study (patients included in an observational study may be included) Patient who received an experimental treatment in a research involving the human person in the last 2 months Subject taking a dietary supplement (> 100kcal / d) Subject with severe eating disorders (anorexia, bulimia, binge eating disorders, etc.) History of bariatric surgery Subject practicing an intense sport activity (more than 10 hours of sport per week) Subject unwilling to maintain an alcohol consumption of less than 50g per week (eg 5 glasses of wine) and less than 10g per day (eg 1 glass of wine) Patient under tutorship or curatorship

Ages Eligible for Study
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Minimum Age: 40 Years
Maximum Age: 70 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Other: Diet High Protein (HP)<br>Diet High Protein (HP) : 30% protein, 40% carbohydrate and 30% fat | Other: Diet HP<br>* 2000kcal for men 1800 kcal for women. Food boxes (HP pre-cooked meals and meat/chicken/fish portions, HP breads and snacks) will be provided to the participants throughout the study reaching 40-50% of their prescribed daily energy intake for 6 days per week. In total 932 kcal are provided through this food boxes (54g of carbohydrate, 101g of protein, 34,6g of fat). The rest of the daily food intake will be guided by a dietician with a list of recommended high protein foods. Subjects are required to keep food diaries three days a week and will also have weekly contact with a dietician.<br>|
| Other: Diet Low Protein (LP)<br>Diet Low Protein (LP) : 10% protein, 55% carbohydrate and 35% fat | Other: Diet LP<br>* 2000kcal for men 1800 kcal for women. Food boxes (LP pre-cooked meals, LP breads and snacks) will be provided to the participants throughout the study<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Post meal tolerance test glycemic excursion (area under the curve) | After overnight fasting: Ingestion of 2x125ml de Fortimel® Compact (Nutricia) 600 kcal with 74g carbohydrates (50% of energy), 24g protein (16% of energy) et 23,2g fat (34% of energy). Blood glucose sampling à T0, 30, 60, 90, 120, 180, 240 min | Change between baseline (T0) and the end of the intervention (T12 weeks) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Post meal tolerance test insulin excursion (area under the curve) | After overnight fasting: Ingestion of 2x125ml de Fortimel® Compact (Nutricia) 600 kcal with 74g carbohydrates (50% of energy), 24g protein (16% of energy) et 23,2g fat (34% of energy). Blood glucose sampling à T0, 30, 60, 90, 120, 180, 240 min | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Matsuda index (from post meal tolerance test glucose and insulin levels) | After overnight fasting: Ingestion of 2x125ml de Fortimel® Compact (Nutricia) 600 kcal with 74g carbohydrates (50% of energy), 24g protein (16% of energy) et 23,2g fat (34% of energy). Blood glucose sampling à T0, 30, 60, 90, 120, 180, 240 min | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Insulinogenic index (from post meal tolerance test glucose and insulin levels) | After overnight fasting: Ingestion of 2x125ml de Fortimel® Compact (Nutricia) 600 kcal with 74g carbohydrates (50% of energy), 24g protein (16% of energy) et 23,2g fat (34% of energy). Blood glucose sampling à T0, 30, 60, 90, 120, 180, 240 min | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Disposition index (kahn) (from post meal tolerance test glucose and insulin levels) | After overnight fasting: Ingestion of 2x125ml de Fortimel® Compact (Nutricia) 600 kcal with 74g carbohydrates (50% of energy), 24g protein (16% of energy) et 23,2g fat (34% of energy). Blood glucose sampling à T0, 30, 60, 90, 120, 180, 240 min | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Serum concentration of glycated hemoglobin (HbA1c) | After overnight fasting | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Fasting concentration of glucose | After overnight fasting | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Insulin resistance index : HOMA 2 IR (based on fasting glucose and insulin concentration) | Fasting | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Insulin secretion index: HOMA 2 B (based on fasting glucose and insulin concentration) | Fasting | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| One week postprandial glucose excursions measured by continuous glucose monitoring sensors (CGMS) | Freestyle libre (Abbott) sensors placed for one week with continuous glucose monitoring | Evolution between T0 (baseline) T6 weeks and T12 weeks of intervention | 
| Weight (kg) | Measured with same scale | Evolution between T0 (baseline) T6 weeks and T12 weeks of intervention | 
| Waist circumference (cm) | Measured standing with a GULICK meter | Evolution between T0 (baseline) T6 weeks and T12 weeks of intervention | 
| Sagittal diameter (cm) | Measured lying down with measuring rod | Evolution between T0 (baseline) T6 weeks and T12 weeks of intervention | 
| Fat mass (DXA) | Measured by Dual-energy X-ray absorptiometry (DXA) | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Fat free mass (DXA) | Measured by Dual-energy X-ray absorptiometry (DXA) | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Visceral fat mass (DXA) | Measured by Dual-energy X-ray absorptiometry (DXA) | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Fat mass (BIA) | Measured by Body impedance analysis (Tanita scale) | Evolution between T0 (baseline) T6 weeks and T12 weeks of intervention | 
| Fat free mass (BIA) | Measured by Body impedance analysis (Tanita scale)aspiration on a subgroup of patients | Evolution between T0 (baseline) T6 weeks and T12 weeks of intervention | 
| Fasting concentration of Alanine transaminase (ALT) | After overnight fast | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Fasting concentration of Aspartate transaminase (AST) | After overnight fast | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Concentration of total cholesterol | After overnight fast | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Concentration of LDL cholesterol | After overnight fast | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Concentration of HDL cholesterol | After overnight fast | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Concentration of triglycerides | After overnight fast | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Gut microbiota changes | Shotgun metagenomic sequencing of DNA extracted from stool and saliva samples. | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Oral microbiota changes | Shotgun metagenomic sequencing of DNA extracted from stool and saliva samples. | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Concentration of Imidazole propionate | Targeted metabolomics | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Concentration of Trimethyl amine oxide (TMAO) | Targeted metabolomics | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Concentration of p cresol | Targeted metabolomics | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Concentration of indoxyl sulfate | Targeted metabolomics | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Concentration of C reactive protein (CRP) | Fasting serum levels measures | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Urinary urea excretion | 24h urinary sample measure | Evolution between T0 (baseline) T6 weeks and T12 weeks of intervention | 
| SF 36 score (short form 36 quality of life questionnaire) | SF-36 questionnaire | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| General self efficacy scale score (GSES questionnaire) | GSES questionnaire | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Patient health questionnaire 9 score (PHQ-9 questionnaire) | PHQ-9 questionnaire | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Gastro-intestinal discomfort changes | Rome IV criteria | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Resting energy expenditure changes | Indirect calorimetry (Cosmed Quark RMR) | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Epigenetic modifications | On serum isolated monocytes for a subgroup of patients | Change between baseline (T0) and the end of the intervention (T12 weeks) | 
| Adipose tissue gene expression modifications | RNA sequencing of RNA extracted from adipose tissue obtained from adipose tissue aspiration on a subgroup of patients | Change between baseline (T0) and the end of the intervention (T12 weeks) | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Type 2 diabetes, Diet, Protein, Gut microbiota, Metabolites