Effect of Probiotics on Pre-diabetes and Diabetes in China-the Role of Gut Microbiota Composition
Phase: NA
Conditions: Type 2 Diabetes Mellitus
Sponsor: Chinese PLA General Hospital

STUDY PROTOCOL 
 
Effect of probiotics on T2DM and pre-diabetes in China ---the role of 
gut microbiota composition  
 
Background 
According to recent surveyed，diabetes prevalence rate in Chinese adults 
had gone up to 11.6%，the pre-diabetics diabetes prevalence rate was 
50.1%. Effective intervention is urgently needed. Study of relationship 
between intestinal flora and diabetes is a hot topic at present.Gut microbiota 
disorders may be involved in the development of diabetes by means of 
increasing the number of inflammatory mediators, chronic inflammation, 
insulin resistance, increased energy intake, and obesity ect. In view of the 
correlation between gut microbiota and diabetes, gut microbiota can be taken 
as an important target for diabetes intervention. In this study, we aim to 
explore whether probiotics intervention affect blood glucose level and Risk 
factors for diabetes in T2DM and pre-diabetes via modification of gut 
microbiota composition and other post-interventional effects.  
 
Objectives： 
1. To investigate the effects of probiotics intervention on blood glucose 
metabolism (blood glucose, insulin sensitivity) and diabetes risk factors 
(blood lipid, weight, body composition, inflammatory state) in diabetes and 
pre-diabetes. 
2. To investigate the effect of probiotics intervention on gut microbiota 
composition and its correlated metabolite. 
3. To investigate the mediating effect of gut microbiota on glucose control in 
diabetes and pre-diabetes. 
 
Methods/design 
Trial design   

This is a four-arm randomized controlled trial（Diabetes-intervention n=30，
Diabetes-control n=30，prediabetes-intervention n=80，prediabetes-control 
n=80）. Participants are recruited from the outpatient registration pool of those 
who have participated in annual health checks for diabetes during 2017 to 2018 
at Health care Center, Southwest Hospital, Chongqing, China. After an initial 
screening, potential subjects will be assessed for inclusion in the run-in trial. 
Those subjects who meet the inclusion criteria will be then invited to the 
intervention study for a total duration of 3 month. Those participants who are 
qualified for the intervention study will be randomized into intervention or control 
group after baseline assessments. 
 
Eligibility criteria 
Inclusion criteria: 
1. The patient was diagnosed with pre-diabetes or diabetes by  
OGTT   
2. Age: 18-60y  
3.  BMI:18-35kg/m2 
 
Exclusion criteria: 
1. Patients who have diabetes，hyperlipemia , and need regular use of drugs.  
2. Secondary obesity or diabetes. 
3. Serious systematic diseases, such as digestive diseases, tumor, cardiac, 
pulmonary, renal, rheumatic and immune system diseases. 
4. Pregnant women, women ready for pregnancy, and nursing mothers. 
5. Take antibiotics or bacterial agents within 1 month 
6. Diarrhea or abscess in 1 month or blood or other abnormal feces 
7. Diabetics history more than 2 years  
8. The OGTT experiment FG≥9mmol/L or 2h≥14mmol/L in diabetic patients 
 
Study settings 
The interventions and laboratory tests and performed and managed at the 
Health Care Center, Southwest Hospital, Chongqing, and Health 
management institute, PLA General hospital, Beijing, China.  
 

Trial design 
Interventions 
1. Probiotics group: Diabetes/prediabetes participants take four packs of 
probiotics a day on an empty stomach with 200ml water every morning. The 
probiotics is called KAWAI, each packs contains 880 billion dead 
Lactobacillales（S. thermophilus）. 
2. Placebo group: 
Diabetes/prediabetes participants take four packs of probiotics a day on an 
empty stomach with 200ml water every morning. 
 
Primary and secondary outcomes 
Primary outcomes: 
1. Blood glucose levels(FBG and OGTT)，insulin sensitivity(Insulin，C- 
peptide) 
2. Gut microbiota composition  
 
Secondary outcomes: 
1.  Blood inflammatory markers 
2.  Diabetes risk factors (blood pressure, blood lipids) 
3.  Weight, waist circumference，BMI, Body composition 
4.  Intestinal endocrine function（GPL-1） 
5.  Gut microbiota metabolites 
The primary and secondary outcome measures will be assessed at 
baseline, 1- and 3-month time points. 
 
Measurements procedures  
1. Screening: 
The potential subjects will be selected from the outpatient pool of those who 
have records of abnormal blood glucose level in 2017. Registered nurses from 
health care center will first contact the potential subjects and invite them to a 

study information meeting. Detailed information of the objectives of the study, 
its nature and constraints, the anticipated risks and the expected benefits are 
given by the investigating physician of endocrinology and professor of sport 
medicine in the recruitment information meetings. When the screening 
consent form has been signed, patients will be asked to fill in a screening 
questionnaire to check their health and medication background as well as 
alcohol consumption, and to undergo a glucose tolerance test (blood draw 
after overnight fasting, followed by samples of 2 hours after the intake of 75 g 
of glucose). 
 
Follow-up visits 
The purpose of the follow-up visits is to monitor the compliance, enhance 
retention of the subject in the study, and obtain information on factors that 
may affect outcomes of the study. A study coordinator will contact participants 
every month to check their oral probiotics records. The study coordinator will 
also give feedback. The participants will have follow-up visits to the laboratory 
of Health care Center, Southwest Hospital, Chongqing at months1, 3. Their 
height, weight, blood pressure, blood samples, fecal samples, and changes in 
health status and body composition will be checked at those time points.  
 
Methods of measurements 
1.Questionnaire, anthropometry and physical examinations 
Background information regarding lifestyle, behavioral and motivational 
characteristics as well as medical history will be collected by questionnaires. 
Data gathered from eligible subjects will then be used to describe the study 
populations, and individual results will be used to screen the changes and for 
monthly feedback. 
  Height will be determined using a wall-fixed measuring device, and weight 
using a calibrated scale. Height and weight will also be used to determine 
body mass index (BMI, weight(kg)/(height(m))2. Blood pressure will be 
measured after 5 min rest. A physician will examine the physical conditions of 
subjects and ensure that subjects meet the inclusion criteria. 
2.Blood samples 
Glucose tolerance tests will be performed after overnight fasting and 2 hours 

after the intake of 75 g glucose for the assessment of serum insulin, and 
glucose. Venous blood samples will be taken in standardized fasting conditions 
at 7–8 a.m. Total cholesterol, high-density lipoprotein (HDL), low-density 
lipoprotein (LDL), triglycerides, will be measured from serum sample by 
conventional methods. ELISA can be used to assess serum samples for 
adiponectin,tumor necrosis factor alpha (TNFα), C-reactive protein (CRP), 
interleukin (IL-6), interleukin 10 (IL-10), and lipopolysaccharides (LPS).Gut 
microbiota metabolites will be measured from serum sample by LC-MS/MS. 
3.Fecal samples collection 
The fecal samples (two samples per each time) will be collected in the health 
care center during the second day of the 3-day dietary records collection. The 
study will provide to subjects the materials for sample collection. The subjects 
are advised to sample approximately 2–3 grams of feces by spatula attached 
to the cover of the plastic specimen jar. Fecal samples are frozen after 
collection within 30 min , stored at −80°C. 16SrDNA sequencing will be 
performed. Different bacterial indices will be calculated to evaluate the 
interrelationship of different bacterial species and how they are associated 
with diabetes.  
4.Body composition 
Bioelectrical impedance analysis will be used to estimate lean tissue mass (LM), 
fat mass (FM) and bone mass (BM) of the whole body, legs, arms, and trunk 
as well as android and gynoid regions. 
 
Concomitant adherences and procedures 
To ensure good compliance in probiotics and placebo group, we will keep in 
contact with them via phone call monthly questionnaire to check on their 
physical condition and lifestyle at 1 and 3months follow-up during the 
intervention. 
 
Sample size estimation 
The sample size calculation with an estimate of how many participants will be 
needed for the primary outcome to be statistically, clinically and/or politically 
significant. For the specific primary outcome of blood glucose and microbiota, 
based on our plot study, 68 prediabetes subjects in each group would have 
85% power for mean comparison between the randomized groups for the fast 

blood glucose. We set the significance level at 0.05 and allowed for 10 mean 
comparisons with the Bonferroni correction between the groups. Taking into 
account the compliance and drop-out (15%), we increase our simple size to 
80 in each prediabetes group. With the same approach, sample size for each 
diabetes group is 60 subjects.  
 
Randomization methods 
1. Randomization Process: 
SPSS will be used for generating the randomization assignment for Diabetes 
groups and prediabetes groups respectively. Randomization numbers will be 
generated and sealed in an envelope and kept by the study coordinator. On the 
basis of subjects’ enrolment time when 20 qualified subjects are reached, the 
study coordinator will open one block to allocate subjects into each group. 
2.Blinding: 
Both subjects and investigators will be blinded with respect to the 
randomization assignment of each participant. The investigators will remain 
blinded until the completion of the study. The study coordinator will be blinded 
from which group the participants are signed in. The accuracy of the 
randomization log is the responsibility of the study coordinator. 
 
Statistical methods 
All data collected by questionnaires will be entered into database 
management software (EpiData), double checked by independent 
researchers, and exported to SPSS statistics version. If data is not normally 
distributed, their natural logarithms will be used for further analysis. 
Descriptive statistics will be used to present the background and 
anthropometric data at the baseline and follow-up assessments as mean and 
95% confidence interval (CI) unless otherwise stated. 
  An intention-to-treat (ITT) analysis will be performed to compare the 
Probiotics group to Placebo group. The effects of the interventions will be 
assessed using analysis of variance (ANCOVA) for repeated measures 
(treatment group × time) and baseline difference as a covariate. If the 
significance of the group by time interaction is p < 0.05, the effect will be 
localized utilizing Bonferroni for multiple comparisons. The level of statistical 
significance chosen for the contrasts will be p < 0.05. In addition to the ITT 

analysis, efficacy or active treatment analysis will be done when the 
compliance of the participation of the intervention is ≥60% of the whole trial 
[ANCOVA for repeated measures (treatment group × time) and baseline 
difference as a covariate.] The percentage differences (0–3month) will be 
calculated from duration between baseline and end point measurements for 
each individual. The comparison of percentage changes in different groups 
will be performed using ANCOVA (two factor interactions: compliance/ 
noncompliance × treatment group) controlled for the baseline value using 
Bonferroni for multiple comparisons. If the significance of the overall group 
difference is p < 0.05, then the effect will be localized by contrast to the Con 
group. When the 95% CI does not include zero, the difference is regarded as 
statistically significant at α = 0.05. 
  Linear Pearson’s correlation, partial correlation, Kendall Tau’s, and 
bivariate, logistic, and multivariate regression analysis will be used for 
analysing relationships. In addition, systems biology approaches will be used 
to develop models which integrate the different types of data and the high-
throughput data measured in terms of gut microbiota composition, as well as 
LPSs and inflammation. 
 
Ethical issues, research permits or information permit applications 
The current study will continue to adhere to all relevant guidelines for good 
scientific and clinical practice. All subjects in this study are volunteers. None 
of the measurements are known to have any significant health risk.  
The benefits and associated risks of the study will be carefully explained to 
the subjects and voluntary participation will be stressed. Informed consent will 
be obtained from all subjects. The study physician will be available during the 
laboratory tests. The study has been approved by Ethics Committee of 
PLAGH, ( S2016-068-01). 
All data will be handled and archived confidentially. All image scans and 
background information will be electronically transferred and stored in the 
specific database. The blood samples and fecal samples will be stored at -
80°C. If the analyses will be performed outside the data collection site, the 
sample transportation will be handled according to the relevant safety 
standards. The duration of the sample storage is generally 10 years. If the 
samples storage time exceeds the permitted time, a new permit will be 
obtained from both the subjects and the Ethical committee. If the subjects are 
deceased, permission will be sought from the relatives.