Probiotics have beneficial metabolic effects in patients with type 2 diabetes mellitus: a meta-analysis of randomized clinical trials

Probiotics have been reported to have a positive impact on the metabolic control of patients with type 2 diabetes. We aimed to systematically evaluate the effects of probiotics on cardiometabolic parameters in type 2 diabetes based on randomized controlled studies. MEDLINE, Embase, and CENTRAL databases were reviewed to search for randomized controlled trials that examined the effects of probiotic supplementation on cardiometabolic parameters in patients with type 2 diabetes. 32 trials provided results suitable to be included in the analysis. The effects of probiotics were calculated for the following parameters: BMI, total cholesterol levels, LDL, triglycerides, HDL, CRP, HbA1c levels, fasting plasma glucose, fasting insulin levels, systolic and diastolic blood pressure values. Data analysis showed a significant effect of probiotics on reducing total cholesterol, triglyceride levels, CRP, HbA1c, fasting plasma glucose, fasting insulin levels, and both systolic and diastolic blood pressure values. Supplementation with probiotics increased HDL levels however did not have a significant effect on BMI or LDL levels. Our data clearly suggest that probiotics could be a supplementary therapeutic approach in type 2 diabetes mellitus patients to improve dyslipidemia and to promote better metabolic control. According to our analysis, probiotic supplementation is beneficial in type 2 diabetes mellitus.

Search strategy. Meta-analysis was performed using the PICO format: whether an intervention with probiotic supplementation (I) compared with placebo (C) has any effect on metabolic parameters (body mass index (BMI), total-cholesterol, low density lipoprotein (LDL), triglycerides (TG), high density lipoprotein (HDL), high sensitivity C-reactive protein (hs-CRP), haemoglobin A1c (HbA1c), fasting plasma glucose and insulin levels, systolic and diastolic blood pressure (SBP, DBP) (O) in patients with diabetes mellitus type 2 (P). In general, the following search terms were used in all databases: diabetes mellitus type 2 AND (probiotic* OR lactobacillus OR saccharomyces OR enterococcus OR escherichia coli OR streptococcus OR bifidobacterium) AND random*. Trials were identified by searching MEDLINE (via PubMed), EMBASE and CENTRAL databases up to 5th of April 2019. No filters or restrictions were applied. We included human trials without any restriction to language or year of publication.
Eligibility criteria and study selection. Duplicates were removed by the EndNote software first automatically, then manually. Randomized controlled trials in which probiotics in the form of any pharmaceutical formulations or dairy products administered to adult patients with type 2 diabetes were included after title and abstract screening. Combination therapy was not an exclusion criterion. Subsequently, full texts of the articles were reviewed for inclusion of eligible studies. Two review authors (TK and BM) selected the articles fulfilling the inclusion criteria independently, and any disagreement was resolved by consensus. Data collection. At the end of the screening process, relevant data were independently extracted from studies by the two review authors and any disagreement was resolved by consensus. Data were extracted into a standardized excel sheet form. Data extracted from the papers included: number of participants, dosage, the intervention used, study duration and the outcome parameters including BMI changes as primary outcome and changes in the total-cholesterol, LDL, TG, HDL, hs-CRP, HbA1C, fasting plasma glucose and insulin levels, SBP and DBP as secondary outcomes. The authors of the studies and year of publication were also recorded. Mean values for control and intervention groups, along with the measure of dispersion were extracted.
Risk of bias assessment. Two review authors assessed the risk of bias of the studies independently, and any disagreement was resolved by consensus. The assessment was performed using the updated version of the Cochrane risk-of-bias tool for randomized trials (RoB 2) with the following domains: bias arising from the randomization process, bias due to deviations from intended interventions, bias due to missing outcome data, bias in measurement of the outcome, bias in selection of the reported result, and overall bias 21 .
Quality of evidence. We used the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach to rate the quality of evidence on our primary outcomes.

Results
Characteristics of the included studies. A flow chart of selection for the meta-analysis is shown in 32 eligible studies were included in the meta-analysis 12,23-52 . Main characteristics of the studies included are shown in Table 1. Fifteen studies administered one bacterial species, while the rest of the studies used a combination of more than one strain: seventeen studies administered two to seven bacterial species. In one article, the flora of the probiotic yoghurt of the intervention group was enriched with specific strains, however placebo yoghurt also contained bacterial flora 12 . In three articles, probiotics were co-administered with chromium 35,51,52 , in one article with selenium 42 , and in one article with vitamin D 43 . The duration of intervention ranged from four to 34 weeks. Seventeen of the 32 articles were published from Iran, two from Saudi Arabia, two from Ukraine, two from Brazil, two from India, and the remaining ones from Malaysia, Denmark, Taiwan, Poland, Sweden, Japan, and Greek. Summary of findings. Data of outcome parameters are summarized in Table 2. The summary of findings table provides a synopsis of the analysis ( Table 3).

Risk of bias within the individual studies.
One study had high risk overall 40 . In seven studies, some concerns were detected; however, we found no articles with any concern about missing outcome data. The quality of the included studies is shown in detail in Fig. 2. Generally, the quality of the studies was good, in most cases with published pre-study protocols. We found three studies that were single-blind 37,51,52 , three more studies without blinding 26,46,48 , all the other articles contained double-blind studies.
Probiotics did not change body mass index. Seventeen studies reported BMI changes. Pooled data showed no difference between the probiotic and placebo group. Considerable heterogeneity (I 2 : 86.6%, p < 0.001) was detected.  We found a significant difference between these two sub-groups (p = 0.001). Sub-group analysis according to the number of bacterial strains (single or multiple, Fig. 4) did not change heterogeneity, either (multiple: I 2 : 91.5%, p < 0.001, single: I 2 : 81.6%, p < 0.001). The beneficial effect of multiple strains probiotics on total cholesterol was significant (− 11.70 mg/dL, 95% CI − 18.60, − 4.79, p = 0.001), however no difference was observed in single bacteria probiotic sub-group (p = 0.611) with significant difference between the two sub-groups (p < 0.001).
If we excluded the six articles where probiotics were co-supplemented with either vitamin D or chromium or selenium, and the article where the placebo group also got yoghurt with some bacteria, the heterogeneity did not change, nor the direction of the association ( Figure S1).
Twenty studies reported data about LDL levels. No significant difference in LDL levels was observed between probiotic and placebo users (− 3.77 mg/dL, 95% CI − 8.47, 0.93, p = 0.116) with a considerable heterogeneity (I 2 : 88.6%, p < 0.001). Sub-group analysis according to the length of treatment ( Figure S2) did not decrease the heterogeneity (short: I 2 : 88.9%, p < 0.001, long: I 2 : 89.5%, p < 0.001). Pooled studies with 8 weeks treatment period or shorter (p = 0.167) and studies of 12 weeks or longer showed no change of total cholesterol level (p = 0.493). We found no significant difference between these two groups (p = 0.555). Sub-group analysis according to the number of bacteria used (single or multiple, Figure S3 did not change heterogeneity, either (multiple: I 2 : 90.6%, p < 0.001, single: I 2 : 86.0%, p < 0.001). We found no effect of multiple-strain probiotics on total cholesterol (p = 0.139), and no difference was observed in single bacterium containing probiotic sub-group (p = 0.985), while there was no difference between the two sub-groups (p = 0.119). If we excluded the six articles where probiotics were co-supplemented with either vitamin D or chromium or selenium, and the article where the placebo group also got yoghurt with some bacteria, heterogeneity did not change, either ( Figure S4). However, based on our trial sequential analysis, a number of 3,442 observations would be needed to provide sufficient statistical power (vs. the 1,090 patients in the current analysis) ( Figure S5).
The meta-analysis of twenty-two trials showed a significant increase of HDL by 1.62 mg/dL (95% CI 0.21, 3.04, p = 0.025). I 2 test (57.4%, p < 0.001) may represent moderate heterogeneity due to the differences between the interventions.
Twenty-four studies reported data about fasting plasma glucose. Pooled data showed a significant effect of probiotics in reducing fasting plasma glucose levels with a mean difference of − 16.52 mg/dL, (95% CI − 23.28; − 9.76, p < 0.001) with a substantial heterogeneity (I 2 : 66.2%, p < 0.001). Sub-group analysis according to the Table 2. Summary data of outcome parameters. Bold values indicate statistically significant weighted mean differences between the intervention and control groups, where p < 0.05. N number of RCTs, WMD weighted mean difference, CI confidence interval, BMI body mass index, T-chol total cholesterol, LDL low-density lipoprotein, TG triglyceride, HDL high-density lipoprotein, CRP C-reactive protein, HbA1c hemoglobin A1c, FPG fasting plasma glucose, SBP systolic blood pressure, DBP diastolic blood pressure. www.nature.com/scientificreports/ length of investigation (Fig. 5) did not change the heterogeneity in the long-term treatment sub-group (I 2 : 80.6, p < 0.001), however it decreased significantly in the short period therapy sub-group (I 2 : 25.8%, p = 0.183). Short studies with 8 weeks or shorter showed significant decrease of fasting plasma glucose level (− 15.35 mg/ dL, 95% CI − 24.83, − 5.87, p = 0.002), and studies of 12 weeks or longer also showed a significant decrease (− 18.82 mg/dL, 95% CI − 28.58, − 9.06, p < 0.001). We found no significant difference between these two subgroups (p = 0.723). Sub-group analysis according to the number of applied bacteria strains (single or multiple, Fig. 6) showed an increased heterogeneity in the single strain sub-group, and there was some minor decrease in the multiple strains sub-group (single: I 2 : 74.5%, p < 0.001; multiple: I 2 : 60.6%, p < 0.001).
The beneficial effect on fasting glucose level was significant both in the multiple strains probiotics subgroup (− 19.84 mg/dL, 95% CI − 31.45, − 8.23, p = 0.001) and in the single bacteria probiotic sub-group (− 16.07 mg/ dL, 95% CI − 25.88, − 6.26, p = 0.001) with no significant difference between the two sub-groups (p = 0.892). If we excluded the six articles where probiotics were co-supplemented with either vitamin D or chromium or selenium, and the article where the placebo group also got yoghurt with some bacteria, the heterogeneity did not change, either ( Figure S6).

Discussion
In the present meta-analysis, we aimed to evaluate the effects of probiotics on BMI and metabolic parameters in patients with type 2 diabetes mellitus. Data analysis showed a significant effect of probiotics in reduction of total cholesterol, triglyceride levels, CRP, HbA1c, fasting plasma glucose, fasting insulin levels and both systolic and diastolic blood pressure values. Supplementation with probiotics increased HDL levels however did not have a significant effect on BMI or LDL levels.
Such an evaluation is of high potential importance, as this patient group has especially high risk of cardiovascular diseases. It is crucial to reduce all the modifiable risk factors with efficient and multifactorial therapeutic methods and probiotic supplementation could be a complementary approach. www.nature.com/scientificreports/ High total cholesterol levels, high blood pressure and type 2 diabetes mellitus are major risk factors of cardiovascular diseases. Reduction of the high total cholesterol and LDL levels in order to reduce the risk of major cardiovascular events is essential 53 . Every 1 mmol/L increment in total cholesterol levels increases the risk of cardiovascular diseases by 20% in women and by 24% in men 54 . Our results show that the consumption of probiotics has a decreasing effect on serum cholesterol levels. The mechanisms behind this reduction are that probiotics seem to be able to reduce serum cholesterol levels by reducing cholesterol absorption in the intestines 55 and by the inhibition of HMG-CoA reductase enzyme thereby inhibiting endogenous cholesterol synthesis 56 .
The exact mechanism of action for the beneficial effects of probiotics on glycemia-related parameters is not fully elucidated. The favorable effects may be due to the immunoregulatory properties of probiotics. Cani et al. demonstrated, that metabolic endotoxemia dysregulates the inflammatory tone and triggers body weight gain and diabetes. Alterations in glucose homeostasis are associated with low-grade inflammation promoted by gut microbiota-derived lipopolysaccharide or endotoxin in mice 57 . Therefore, lowering plasma lipopolysaccharide concentration could be a strategy for the control of metabolic diseases, such as diabetes mellitus. Naito et al. showed that oral administration of Lactobacillus casei strain to obese mice led to a better insulin resistance through decreasing plasma levels of lipopolysaccharide-binding protein, a marker of endotoxemia 58 .
In our meta-analysis, probiotics significantly reduced total cholesterol, triglyceride levels, CRP levels, HbA1c levels, fasting plasma glucose levels, fasting insulin, and blood pressure together with the increase of the HDL levels. The observed small changes may not seem to be clinically significant, however the beneficial changes in many parameters can add up leading to a reduction in the severity of type 2 diabetes-related complications, and, www.nature.com/scientificreports/ as a consequence in lower mortality. The main strength of our study is that we included exclusively randomized clinical trials for evaluation and the number of the included trials were much higher than in other meta-analyses in this field. Some of our outcomes (triglyceride levels, systolic and diastolic blood pressure values) included a homogenous data set, so confounding factors are unlikely to distort our results. Waist to hip ratio was not measured in most of the articles, so that we could not pool the data. We attempted to determine whether the observed heterogeneity in our outcomes was due to the differences in the length of treatment or in the number of probiotics used. However, according to our subgroup analyses high heterogeneity still remained unknown. We need more randomized clinical trials to be able to determine the most beneficial bacteria, the optimal dosage and treatment period. The identified significant heterogeneity is due to the significant differences between the intervention of the selected articles.
There are considerable limitations in our study. The diversified settings made it impossible to assess the effect of specific probiotic strains on the analyzed parameters. Many of the analyzed studies used probiotic mixtures or dairy products containing several probiotic strains. The data of diversity and richness of gut microbiota are absent in some of the included studies. The number of the probiotic species used in the included trials varied between the studies included in the analysis. The duration of probiotic intervention differed between the included trials. Consequently, substantial heterogeneity was observed between trials within this meta-analysis. No subgroup analysis was possible to assess which particular probiotic preparation could be the most effective to improve metabolic parameters in diabetic patients. Differences in population or differences in outcome were not considerable. The study aim was to test different cardiometabolic parameters in patients with diabetes mellitus type 2 in all included studies. However, differences in intervention were substantial, due to the fact, that different species www.nature.com/scientificreports/ or different probiotic combinations were used. This fact is worth to mention, because we are not able to have high quality evidence due to the very high indirectness.
In conclusion, according to our meta-analysis the administration of probiotics has a beneficial role in the management of type 2 diabetes regarding metabolic profile. We have shown a significant effect of probiotics in reducing total cholesterol, triglyceride levels, CRP, HbA1c, fasting plasma glucose, fasting insulin levels and both systolic and diastolic blood pressure values. Supplementation with probiotics increased HDL level and it did not had a significant effect on BMI or LDL levels. The practical implication of our study is that probiotic administration as a supportive intervention of type 2 diabetes could be incorporated into diabetes guidelines to beneficially modify cardiometabolic risk factors. Further studies are needed to investigate the combined effects of the different antidiabetic drugs and probiotic species.  Forest plot for the effect of probiotics on fasting plasma glucose (FPG) compared to controls in pooled analysis. The shaded diamonds indicate the effect of probiotics in a particular study (weighted difference in mean). The horizontal lines represent 95% confidence intervals (CIs). The big diamond data marker indicates the pooled effect. The figure shows the summary of studies overall and subdivided by the number of bacterial species used. "multiple": combination of bacteria, "single": one bacterial species used.