Effect of Probiotic Supplementation on Fecal Microbiota, Nutritional Status, Metabolic and Inflammatory Parameters in Patients With Type 2 Diabetes Mellitus

Study Overview
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Brief Summary
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The purpose of this study is to evaluate the effect of probiotic supplementation on fecal microbiota, nutritional status, metabolic and inflammatory parameters in patients with type 2 diabetes mellitus. Study hypothesis: Supplementation of multispecies probiotic (Bifidobacterium Lactis, B. brebe, B. longum, Lactobacillus gasseri, L. casei, L. rhamnosus) during 12 weeks improves the the fecal microbiota composition and promotes reduction of plasma/serum levels of acute phase proteins, cytokines, metabolic and anthropometric parameters in individuals with type 2 diabetes mellitus.

Detailed Description
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The purpose of this study is to evaluate the effect of multispecies probiotic supplementation (specifically designed for the present study) on fecal microbiota, nutritional status, metabolic and inflammatory parameters in patients with type 2 diabetes mellitus (T2DM). Adult individuals (35 to 75 years old) of both sexes with T2DM (diagnosed at least 1 year ago), body mass index from 25.00 kg/m² to 39.99 kg/m², glycated hemoglobin ≤ 9.0% and using metformin will be invited to participate in this randomized, placebo-controlled, triple-blind study. The participants will be randomized into two groups: G1 - probiotic group and G2 - control group (placebo). The study will consist of two experimental time points: M0 - baseline and start of supplementation; M1 - after 12 weeks of the first outpatient visit and start of supplementation. In the two experimental moments, individual fecal samples will be obtained for analysis of the fecal microbiota; The metabolic parameters will be assessed by determination of circulating levels of SCFA, FFA, insulin, fasting glucose and glycated hemoglobin, HOMA-IR; Total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides; the inflammatory response will be assessed by determination of plasma indicadors (LPS, Adiponectina, IL-10, IL-1, IL-6, TNF- α, Leptina, Resistina) ; besides the evaluation of indicators of nutritional status (bone densitometry with body composition and anthropometric measurements). The primary endpoint will be the fecal microbiota composition, SCFA concentrations and the inflammatory parameters.

Official Title
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Effect of Probiotic Supplementation on Fecal Microbiota, Nutritional Status, Metabolic and Inflammatory Parameters in Patients With Type 2 Diabetes Mellitus: Study Protocol for a Triple-blind Controlled Randomized Clinical Trial

Conditions
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Type 2 Diabetes Mellitus

Intervention / Treatment
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* Dietary Supplement: Probiotic
* Dietary Supplement: Placebo


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Age from 35 to 75 years old; Individuals diagnosed with type 2 diabetes mellitus (at least 1 year ago); Body mass index from 25.00 kg/m² to 39.99 kg/m²; Glycated hemoglobin ≤ 9.0% ; Using metformin, combined or not with other antidiabetic drugs Exclusion Criteria: Previous bowel diseases (inflammatory bowel disease and irritable bowel syndrome); or previous gastrointestinal surgery (eg, colectomy, gastrectomy) Intolerances and ∕ or food allergies with a previous medical diagnosis (eg lactose intolerance or celiac disease); Glomerular filtration rate <30 ml/min/1.73m²; inflammatory diseases and immunodeficiencies; Diagnosis of autonomic neuropathy with gastrointestinal involvement such as: diabetic gastroparesis, diabetic enteropathy (diarrhea) or colonic hypomotility (constipation); Hospital admission and/or use of anti-inflammatory drugs (non-hormonal and corticosteroids) up to 1 month before the study; and ∕or use of antibiotics up to 3 months before the study; Regular use of laxatives, opioid narcotic analgesics or appetite suppressants; Current or previous use (up to 1 month) of prebiotics, probiotics, symbiotics or products enriched with these food supplements; Intolerance to prebiotics, probiotics or symbiotics; Pregnant or breastfeeding; Follow-up of a diet, guided by a nutritionist, for weight loss or gain up to 1 month before the study or current follow-up of unusual diets (eg vegetarian, macrobiotic, paleolithic); Alcohol consumption (> 1 drink/day or 14g of alcohol for women; >2 drinks/day or 28 grams of alcohol for men); use of illicit drugs and smokers; Change of lipid-lowering and/or antidiabetic drugs in the last 3 months

Ages Eligible for Study
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Minimum Age: 35 Years
Maximum Age: 75 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: Quadruple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Placebo Comparator: Placebo<br>Maltodextrin (1 capsule/day) | Dietary Supplement: Placebo<br>* Maltodextrin (1 capsule/day)<br>|
| Active Comparator: Probiotic<br>Probiotic (Bifidobacterium animalis subsp. Lactis, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus rhamnosus) - 1 capsule/day | Dietary Supplement: Probiotic<br>* Probiotic (Bifidobacterium animalis subsp. Lactis, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus rhamnosus) - 1 capsule/day<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Fecal Microbiota | analysis method: 16s rRNA sequencing and bioinformatics analysis. Reported measure: taxonomic profiles of the microbial populations (operational taxonomic units / OTUs) | 12 weeks compared to baseline | 
| SCFA | Acetate, propionate, isobutyrate, butyrate and isovalerate (μmol/L) | 12 weeks compared to baseline | 
| Inflammatory parameters | Plasma LPS, adiponectin, leptin, resistin, IL-1, IL-6, IL-8, IL-10 and TNF-alpha concentrations (pg/mL) | 12 weeks compared to baseline | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Fasting insulin | μUI/mL | 12 weeks compared to baseline | 
| fasting blood glucose | mg/dL | 12 weeks compared to baseline | 
| HOMA-IR | HOMA-IR = [fasting blood glucose (mmol) x fasting insulin (UI/ml)] ÷ 22,5 | 12 weeks compared to baseline | 
| Glycated hemoglobin | percentage (%) | 12 weeks compared to baseline | 
| Free Fat Acids | µmol/L | 12 weeks compared to baseline | 
| Triglycerides | mg/dL. | 12 weeks compared to baseline | 
| Total cholesterol | mg/dL | 12 weeks compared to baseline | 
| LDL-c | (Friedewald equation) LDL-c = total colesterol - HDL-c - Triglicerídeos/5 Reported measure: mg/dL | 12 weeks compared to baseline | 
| HDL-c | mg/dL | 12 weeks compared to baseline | 
| Bone densitometry | Dual X-ray Absorptiometry (DXA). Reported measure: T-score and Z-score | 12 weeks compared to baseline | 
| Body weight (kg) | kilograms | 12 weeks compared to baseline | 
| Waist circumference (cm) | centimeters (cm) | 12 weeks compared to baseline | 
| Body mass index (BMI) | In metric units: BMI (kg/m²) = weight (kg) ÷ height² (meters). Reported measure: kg/m². | 12 weeks compared to baseline | 
| Total Body Fat Percentage (%BF) | Dual X-ray Absorptiometry (DXA). Reported measure: Total Body Fat Percentage (%BF) | 12 weeks compared to baseline | 
| Fat Mass Index (FMI) | Dual X-ray Absorptiometry (DXA) - Fat Mass Index (FMI) - the total amount of fat (in kilograms) relative to the height (in meters²) | 12 weeks compared to baseline | 
| Total Body lean mass Percentage (%) | Dual X-ray Absorptiometry (DXA) - Total Body lean mass Percentage (%): The percent of the body that is not composed of fat. | 12 weeks compared to baseline |