The Effect of Single Probiotic on Metabolic Control in Type 2 Diabetes

Study Overview
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Brief Summary
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Recent studies indicate that dysbiosis of intestinal microbiota and low grade inflammation are important pathogenic determinants of type 2 diabetes (T2DM), which has increased in epidemic size over the last 20 years. Probiotics have been used in T2DM for the modification of IM and anti-inflammatory effects. However, effect of probiotics on metabolic control in T2DM are inconsistent. Present study will be designed to determine the effects of Lactobacillus GG (LGG) on glycemic control, lipid profile, inflammation parameters and expression of certain genes linked to T2DM. This study will be conducted at the Istanbul Faculty of Medicine, a tertiary care diabetes outpatient clinic and should involve 34 T2DM subjects. Subjects will be randomly assign to receive either LGG probiotic drop or a placebo.In this placebo controlled trial, effect of single strain probiotic vs. placebo on metabolic control and certain genes linked to T2DM will be assessed.

Detailed Description
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Evidence-based data showed that intestinal microbiota (IM) plays a role in the development of metabolic diseases. Recent studies have reported that dysbiosis of IM and low-grade inflammation is effective in pathogenesis of type 2 diabetes mellitus (T2DM), which has increased in epidemic size over the last 20 years. Firmicutes, Bacteroidetes and Proteobacteria's ratios in obese and T2DM patients were found to be different than healthy subjects. In these cases, there is an association between increasing the proportion of gram-negative bacteria in the intestines and subclinical inflammation. Probiotics are live microorganisms that are intended to have health benefits by regulating mucosal and systemic immunity, when consumed as a nutritional supplement. There are studies investigating the effects of probiotic use on insulin sensitivity, glycemic control, lipid profile and inflammatory parameters in patients with T2DM. However, several probiotic strains were used frequently in these studies, or probiotics and prebiotics were given as cocktails. Their effects might be together or even synergistic. Lactobacillus rhamnosus GG (or Lactobacillus GG: LGG) is a widely used probiotic microorganism. Studies have shown that LGG prevents diarrhea and atopic dermatitis, provides antitumor activity, improves the immune system, and lowers serum cholesterol levels. However, there is a limited data about the effects of LGG on the glycemic control of diabetic animal models but human studies are scarce. Therefore, present study is designed to determine the effects of LGG on glycemic control, lipid profile, inflammation parameters, and expression of certain genes linked to T2DM. Subjects will be randomly assign to receive probiotic Lactobacillus Rhamnosus GG (ATCC 53103) or placebo for 8-weeks administered as a drop formulation. Patients in the intervention group receive 10 probiotic drops (1x1010 cfu LGG) once daily with breakfast. Subjects will be contacted via telephone every week for an assessment of adverse events and probiotic/placebo compliance. Fasting blood samples will be taken at baseline and post treatment to measure carbohydrate metabolism (glucose, insulin, fructosamine and HbA1c), lipid profile (triglycerides; total, HDL- and LDL-cholesterol) and biomarkers of inflammation (hs-CRP and IL-6). TLR2, TLR4, MUC2 and MUC3A genes expressions will be investigated on stool samples at baseline and post treatment. Stool samples will be stored at -80°C until RNA isolation.The gene expression levels will be determined by Quantitative Real Time PCR method using the determined cDNA samples. Dietary intake will be evaluated by the 3-day food record. During the 4th and 8th week of the study, a 3 day food consumption records will be taken. Diabetics will be given detailed oral and written instructions regarding the completion of food record, consisting of 2 midweek days and 1 weekend day. In order to determine the amounts of consumed foods correctly, information will be given about measuring cups such as water glass, tea glass, teaspoon, tablespoon, serving spoon, bowl. Dietary intake will be assessed using a food composition database of BEBIS programme including specific Turkish foods. All anthropometric measures will be conducted in a fasting state taken at baseline and following an 8-week intervention by experienced examiner (dietitian). Body weight and body composition will be assessed by bioelectrical impedance analysis device (Tanita BC-420 MA). Body mass index (BMI) will be calculated as weight (kg) divided by height squared (m2). Waist circumference (measured midway between lowest rib and iliac crest) will be measured using a non-stretchable measuring tape. All analysis will be performed using the Statistical Package for Social Sciences (SPSS) 21.0 package program and significance will defined as p<0.05. Descriptive statistics will be given as mean, standard deviation and median (minimum to maximum) for continuous measures. Categorical variables will be expressed as case numbers and percentage values. The Shapiro-Wilk tests will used to determine whether the distribution of continuous measures are normal. Student's t test and Mann-Whitney-U test will used for the two groups comparisons according to whether the variables showed normal distribution. Comparisons of changes in groups within themselves (before and after probiotic or placebo administration) will made using the t-test if the variances is normal, and if the Wilcoxon test is not normal in the cohort. The web-based RT2 Profiler PCR Array Data Analysis program will used to determine the change of ΔCt values obtained from the Real Time-PCR gene expression study (before and after probiotic and placebo administration). p<0.05 will be considered statistically significant.

Official Title
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The Effects of Single Probiotic on Glycemic Control, Lipid Profile, Some of Inflammatory Cytokines and Gene Expression Levels in Type 2 Diabetic Patients

Conditions
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Diabetes Mellitus, Type 2

Intervention / Treatment
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* Dietary Supplement: Lactobacillus rhamnosus GG (ATCC 53103)
* Dietary Supplement: Placebo


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Clinical diagnosis of Type 2 Diabetes Taking oral-antidiabetic medication Exclusion Criteria: Smokers, Alcohol drinkers, Inflammatory bowel or autoimmune disease, Immunodeficiency, Using anti-epileptic, incretin enhancer (DPP-4 inhibitor), insulin or insulin analogs, dietary supplements Systemic antibiotics within 6 weeks before inclusion Use of probiotics within 3 months before inclusion Breast-feeding or pregnancy

Ages Eligible for Study
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Minimum Age: 30 Years
Maximum Age: 60 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Supportive Care
Allocation: Non-Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: prospective, single center, interventional
Masking: Single


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Probiotic<br>For 8 weeks of interventional period, the patient received 10 probiotic drops (1x1010 Cfu LGG) once daily at breakfast. | Dietary Supplement: Lactobacillus rhamnosus GG (ATCC 53103)<br>* One probiotic drop contained a formulation of 1x109 Cfu Lactobacillus rhamnosus GG (LGG; ATCC 53103)<br>|
| Placebo Comparator: Placebo<br>For 8 weeks of interventional period, the patient received 10 probiotic drops (placebo) once daily at breakfast. | Dietary Supplement: Placebo<br>* Carrier material of probiotic product, not containing bacterial strain, similar appearance as the probiotic<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| HbA1c | HbA1c % | 8 weeks compared to baseline | 
| HOMA-IR | HOMA-IR= Fasting plasma glucose (mg/dL) x Fasting plasma insulin (μU/mL)/405 | 8 weeks compared to baseline | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| QUICKI | 1/ [log (fasting plasma insulin (μU/mL)+log (fasting blood glucose (mg/dL)] [22, 23]. | 8 weeks compared to baseline | 
| Fasting plasma glucose | FPG in mg/dL | 8 weeks compared to baseline | 
| Fructosamine | μmol/L | 8 weeks compared to baseline | 
| HDL-C | mg/dl, HDL cholesterol | 8 weeks compared to baseline | 
| LDL-C | mg/dl, LDL cholesterol | 8 weeks compared to baseline | 
| Triglycerides | mg/dl | 8 weeks compared to baseline | 
| hs-CRP | mg/dl, high sensitive c reactive protein | 8 weeks compared to baseline | 
| IL-6 | pg/mL, Interleukin 6 | 8 weeks compared to baseline | 
| TLR2 | Toll-like receptor 2 gene expression | 8 weeks compared to baseline | 
| TLR4 | Toll-like receptor 4 gene expression | 8 weeks compared to baseline | 
| MUC2 | Mucin 2 gene expression | 8 weeks compared to baseline | 
| MUC3A | Mucin 3A gene expression | 8 weeks compared to baseline | 
| Weight | body weight, kg | 8 weeks compared to baseline | 
| BMI | body mass index, kg/m2 | 8 weeks compared to baseline | 
| WHR | waist and hip ratio % | 8 weeks compared to baseline | 
| Fat mass | body fat mass, kg | 8 weeks compared to baseline | 
| Fat mass | body fat mass, % | 8 weeks compared to baseline | 
| Lean body mass | body lean body mass, kg | 8 weeks compared to baseline | 
| Muscle mass | body muscle mass, kg | 8 weeks compared to baseline | 
| Total body water | kg kg and % | 8 weeks compared to baseline | 
| Total body water | % kg and % | 8 weeks compared to baseline | 
| Bone mass | Body bone mass, kg | 8 weeks compared to baseline | 
| Basal metabolic rate | Acoording to bioelectrical impedance analysis device, kcal | 8 weeks compared to baseline | 
| Energy | Energy intake, kcal | During 4th and 8th weeks | 
| Carbohydrate | Carbohydrate intake, gram | During 4th and 8th weeks | 
| Carbohydrate | Carbohydrate intake, % | During 4th and 8th weeks | 
| Protein | Protein intake, gram | During 4th and 8th weeks | 
| Protein | Protein intake, % | During 4th and 8th weeks | 
| Fat | Fat intake, gram | During 4th and 8th weeks | 
| Fat | Fat intake, % | During 4th and 8th weeks | 
| Dietary fiber | Dietary fiber intake, gram | During 4th and 8th weeks | 
| Dietary cholesterol | Dietary cholesterol intake, gram | During 4th and 8th weeks | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Diabetes Mellitus, Type 2, Lactobacillus GG, probiotic, gene expression