Introduction

Diabetes mellitus (DM) has been a critical global public health problem since the 20th century. It is reported in the literature that the number of DM patients is expected to reach 578 million in 2030, and continue to increase to about 700 million by 2045, of which type 2 diabetes mellitus (T2DM) patients accounts for about 90%.^1–3 At present, the pathogenesis of T2DM has been relatively clear, and insulin resistance (IR) is the core link that leads to abnormal glucose metabolism.⁴ Recent studies have shown that gut microbiota disorder is an essential factor in the pathogenesis of T2DM, and abnormal blood glucose and hypoglycemic agents change the composition of gut microbiota and its metabolites, thus affecting blood glucose control. Therefore, probiotic adjuvant therapy for T2DM is beneficial to improve the distribution of gut microbiota in patients, and it exerts a positive effect to improve the compliance rate of blood glucose levels.^5–7 However, owing to diverse hypoglycemic agents commonly used in clinical practice, there are still many disputations about the effects of hypoglycemic agents on the gut microbiota of T2DM patients and the corresponding probiotic treatment regimens in different reports. Metformin is the first choice of hypoglycemic agents for treating T2DM, and previous studies have shown that metformin treatment of T2DM is beneficial to regulate gut microbiota, and it effectively increases the flora abundance of short-chain fatty acids (SCFA) produced by Bacteroides species and lessens intestinal inflammation, thereby lowering IR and reducing blood glucose levels. However, the effect of combined use with probiotics remains to be explored.⁸ In this study, an in-depth study was carried out on the effect of metformin on gut microbiota imbalance in T2DM patients and the value of probiotic supplementation, described in detail.

Materials and Methods

General data

A total of 84 newly diagnosed T2DM patients at Affiliated Renhe Hospital of China were selected as samples for a prospective randomized controlled study. Inclusion criteria were as follows: (1) Patients who met the diagnostic criteria of T2DM;⁹ (2) age range 18–65 years; (3) body mass index (BMI): 18.5–30.0 kg/m²; and (4) all patients were aware of the details of this study and voluntarily signed the consent form. Exclusion criteria were as follows: (1) Patients who had been treated with hypoglycemic agents, probiotics, antibiotics, or weight-loss drugs in the past 1 month; (2) patients with inflammation, ulcers, or tumors and other gastrointestinal lesions; (3) patients who have undergone gastrointestinal surgery in the past 3 months; (4) patients with hyperthyroidism, hypothyroidism, or hypercortisolism (HCM) and other endocrine lesions; (5) patients with ketoacidosis, diabetic retinopathy, or diabetic foot and other complications; (6) patients with infectious diseases or malignant tumors; (7) patients with severe liver and kidney dysfunction; (8) patients with a history of mental illness or alcohol dependence; and (9) patients with contraindications regarding metformin or probiotics treatment.

The selected patients were equally divided via simple randomization, with 28 cases in each group, into the following three groups: probiotic group, metformin group, and control group. The probiotic group had 15 males and 13 females, aged 46–65 years, with a mean age of 55.32 ± 4.38 years; BMI was 17.1–27.8 kg/m², and mean BMI was 21.87 ± 2.82 kg/m². The metformin group had 17 males and 11 females, aged 48–65 years, with a mean age of 55.25 ± 5.23 years; BMI was 17.3 ~ 28.2 kg/m², and mean BMI was 22.09 ± 2.48 kg/m². The control group had 12 males and 16 females, aged 46–65 years, with a mean age of 55.64 ± 4.86 years; BMI was 18.1–26.3 kg/m², and mean BMI was 22.24 ± 2.46 kg/m². No significant difference was discovered in baseline data between the three groups (P > 0.05).

The study was approved by the Ethics Committee of Affiliated Renhe Hospital of China, Three Gorges University. All participants were informed and their consents were obtained in accordance with the Declaration of Helsinki.¹⁰ Also, written informed consent was obtained from a legally authorized representative(s) for anonymized patient information to be published in this article.

Study protocol

All three groups actively accomplished health education on T2DM-related knowledge. The patients were asked to quit smoking and alcohol and adjust dietary patterns. Carbohydrate accounted for 50–65% of dietary energy, and protein accounted for 15–20%, with more than one-third were high-quality proteins. Salt intake was limited, and dietary fiber and trace elements were supplemented. Moderate-intensity exercises, such as brisk walking, Tai Chi, or badminton, were performed for 5–7 times a week for 30 min daily. Resistance exercises were performed twice or thrice a week with intervals of ≥48 h if allowed. On this basis, the probiotic group was prescribed metformin tablets, 0.25 g (Disha Pharmaceutical, Weihai, Shandong, China; State Medical Permit Number: H20103615) orally, 1 tablet three times a day (tds); and Bifidobacterium quadruple viable bacteria tablets, 0.5 g (Hangzhou Grand Biologic Pharmaceutical, INC, Bejing, China; State Medical Permit Number: S20060010) orally, 3 tablets tds. The metformin group was prescribed metformin tablet orally (1 tablet tds). In both groups, the intervention course was of 3 months. The control group consisted of patients who did not take any hypoglycemic drugs or had not received any hypoglycemic therapy.

Outcome measures

1. Effect of blood glucose control: In all groups, fasting plasma glucose (FPG) and 2-h postprandial plasma glucose (2-h PG) were measured by Biosen C-line glucometer (EKF, Germany) prior to and after the treatment. In addition, HLC-723G8 glycosylated hemoglobin A1c (HbA1c) analyzer (Tosoh Corporation, Japan) was used to measure fasting HbA1c prior to and after the treatment.

2. Islet function: Fasting peripheral venous blood (3 mL) was collected from the three groups prior to and after the treatment, centrifuged at 3000 revolutions/min for 10 min, and the supernatant was stored at -20°C in two parts for future use. Fasting insulin (FINS) levels were measured using a Cobas C501 automatic biochemical analyzer (Roche, Switzerland). Then, islet β cell (HOMA-β) function was calculated according to the following formula:

   Islet β cell function index (HOMA-β) = 20 × FINS/(FPG-3.5).

   A steady-state model was established to assess insulin-resistance islet β cells (HOMA-IR).

3. Gut microbiota: Fresh feces were collected from the three groups prior to and after the treatment in a closed sterile container; 0.1-g fresh feces was diluted 100-fold with normal saline; then 10^-2–10^-8 dilutions at different concentrations were created according to a 10-fold gradient, and 10^-4, 10^-5, 10^-6, 10^-7, and 10^-8 dilution samples of bifidobacteria and lactobacilli were inoculated in De Man–Rogosa–Sharpe (MRS) agar medium (Thermo Fisher, MA, USA) in an anaerobic environment at 37°C for 48 h; 10^-3, 10^-4, 10^-5, and 10^-6 dilution samples of Enterobacteriaceae and Enterococcus were cultured at 37°C for 24 h. Colony analysis and counting were performed using a MicroScan WAwayalk-40 automatic microorganism analyzer (SIMENS, Germany).

4. Inflammatory factors: Serum samples were collected from the three groups before and after treatment, and C-reactive protein (CRP), lipopolysaccharide (LPS), and interleukin 6 (IL-6) levels were measured by enzyme-linked immunosorbent serologic assay (ELISA). The kits were purchased from Shanghai Laifeng Biotechnology Co. Ltd (Shanghai, China). All operative steps were completed according to the given instructions.

Statistical analysis

Enumeration data were expressed as n (%). Chi-squared (χ²) test was performed for group comparison, and measurement data were expressed in the form of (x± s). One-way analysis of variance was performed for comparison of multiple groups, and independent sample t-test was performed for comparison between two groups; paired sample t-test was performed for comparison between the same group prior to and after the treatment, and the SPSS 23.0 software was used for data analysis, with P < 0.05 showing statistical significance.

Results

Comparison of blood glucose control effect between three groups

After 3 months of treatment, FPG, 2-h PG, and HbA1c levels were notably decreased in both probiotic and metformin groups (P < 0.05), and were lower than that in the control group prior to the treatment. Besides, FPG, 2-h PG, and HbA1c were lower in both probiotic and metformin groups than that in the control group, and FPG, 2-h PG, and HbA1c were further lower in the probiotic group than in the metformin group, and the differences were statistically significant (P < 0.05). The results are shown in Table 1.

Comparison of islet function between three groups

After 3 months of treatment, FINS, HOMA-β, and HOMA-IR in the control group were not conspicuously different from the values prior to treatment (P > 0.05). FINS and HOMA-β in both probiotic and metformin groups were evidently higher than the values prior to treatment (P < 0.05); however, HOMA-IR was significantly lower in both groups compared to the values before treatment (P < 0.05). FINS and HOMA-β in both groups were significantly higher than that in the control group prior to treatment (P < 0.05); however, HOMA-IR was dramatically lower than that in the control group prior to treatment (P < 0.05), and the same in the probiotic group was significantly lower than that in the metformin group (P < 0.05). FINS and HOMA-β values in the probiotic group were compared to that in the metformin group, with differences demonstrating non-statistical significance (P > 0.05). The results are displayed in Table 2.

Comparison of gut microbiota between three groups

After 3 months of treatment, no significant difference was observed in the number of lactobacilli, bifidobacteria, enterobacteria, and enterococci in the control group, compared to the numbers prior to treatment (P > 0.05). The number of lactobacilli and bifidobacteria in probiotic and metformin groups was substantially increased (P < 0.05), but the number of enterobacteria and enterococci was decreased (P < 0.05) in both probiotic and metformin groups. The number of lactobacilli and bifidobacteria was higher and the number of enterobacteria and enterococci was lower in both groups, compared to the control group prior to treatment. The probiotic group had higher numbers of lactobacilli and bifidobacteria and lower numbers of enterobacteria and enterococci than that in the metformin group. The differences were statistically significant (P < 0.05). The results are shown in Table 3.

Comparison of inflammatory factors between three groups

After 3 months of treatment, no significant differences were observed in serum LPS, IL-6, and CRP levels in the control group, compared to pretreatment values (P > 0.05); the serum LPS, IL-6, and CRP levels in both probiotic and metformin groups were evidently lower than that prior to treatment (P < 0.05). The serum LPS, IL-6, and CRP levels in both groups were lower than that in the control group prior to treatment. The serum LPS, IL-6, and CRP levels in the probiotic group were lower than that in the metformin group after treatment. The differences were statistically significant (P < 0.05). The results are shown in Table 4.

DISCUSSION

Gut microbiota is a microecosystem that comprises more than 500 bacteria involved in maintaining physiological functions, such as intestinal mucosal barrier, immune regulation, and intestinal metabolism.¹¹ Previous studies have shown that the structure of gut microbiota in T2DM patients is different from that in healthy individuals, and is mainly manifested as reduced beneficial flora. Increase in opportunistic pathogens triggers chronic intestinal inflammation, thus promoting the progression of IR and T2DM. Regulating gut microbiota is an important developmental direction of T2DM treatment.¹²

Metformin is the first-line drug for the treatment of newly diagnosed T2DM patients, but its hypoglycemic mechanism has not been fully clarified. In addition, to promote directly the absorption and metabolism of glucose in the liver and skeletal muscles, and improve insulin sensitivity, metformin participates in mediating bile acid cycle. Some studies have suggested that its mechanism is related to the regulation of gut microbiota.^13–15 In the current study, the effect of metformin was analyzed in newly diagnosed T2DM patients as samples. The results showed that FPG, 2-h PG, and HbA1c levels were evidently reduced after 3 months of treatment whereas FINS and HOMA-β were significantly increased, and HOMA-IR was significantly reduced. Besides, improvement of each index was superior to that of the control group, compared to the values of the same group prior to treatment, indicating that metformin has a good effect in treating newly diagnosed T2DM. Metformin improves HOMA-β cell function, promotes insulin secretion and improves its sensitivity, reduces IR levels, and produces good hypoglycemic effect; this was consistent with the results reported in the literature.¹⁶ Additionally, the results of this study demonstrated that the number of intestinal lactobacilli and bifidobacteria, as well as the number of enterobacteria and enterococci, was notably reduced in the metformin group, with significantly reduced serum LPS, IL-6, and CRP levels. A significant difference was observed between the control group without any hypoglycemic therapy and the metformin group, which implied that metformin in the treatment of T2DM regulates the distribution of gut microbiota and lessens body’s chronic inflammatory status, thus lowering IR and blood glucose.

Relevant domestic and foreign studies have already discussed the effect of metformin on gut microbiota. Zhang et al. confirmed through an animal experiment that metformin, acarbose, and sitagliptin showed different effects on gut microbiota composition, and the hypoglycemic effect could be improved by selectively increasing the abundance of beneficial bacteria.¹⁷ Mueller et al. conducted a randomized controlled study with overweight/obese patients as samples, and discovered that metformin affected composition of gut microbiota and upgraded SCFA and acetate levels.¹⁸ Imbalance in gut microbiota and increased pathogenic bacteria lead to the disruption of intestinal mucosal barrier function. Endotoxin enters blood circulation and causes chronic inflammation, giving rise to IR and islet dysfunction. Thus, improving gut microbiota has a positive effect on controlling blood glucose. The results of the current study showed that metformin treatment of newly diagnosed T2DM increased the abundance of probiotics Lactobacillus and Bifidobacterium. It is conducive to increase the synthesis of endogenous metabolites, such as branched-chain amino acids, bile acids, and SCFA, and regulate insulin function and glucose metabolism through pathways such as G protein-coupled receptors.^19–21 Therefore, it exerts a positive effect on the regulation of gut microbiota, and increases the number of probiotics as well as improves glycemic control.

The effect of probiotics combined with metformin in the treatment of T2DM is contentious. Seicaru et al. concluded that the addition of intestinal microbiota modulators, such as probiotics or prebiotics, to metformin was beneficial to enhancing its hypoglycemic effect and reducing adverse reactions.²² Hata et al. utilized bifidobacterium G9-1 combined with metformin to treat T2DM, and the results showed that it effectively improved gastrointestinal manifestations but had no significant effect on blood glucose control.²³

In the current study, the probiotic group was treated with Bifidobacterium quadruple viable bacteria integrated with metformin in newly diagnosed T2DM patients. The results demonstrated that FPG, 2-h PG, and HbA1c were evidently lower than that in the metformin group following 3 months of treatment, indicating that the addition of probiotics was beneficial to improving glycemic control, which could be used as an effective treatment of T2DM. Meanwhile, this study revealed no obvious difference in FINS and HOMA-β between both probiotic and metformin groups following 3 months of treatment. However, HOMA-IR was notably lower, suggesting that the mechanism by which probiotics improved hypoglycemic effect was mainly by reducing IR, and the relationship with pancreatic β-cell function and insulin secretion was to be confirmed. Previous studies demonstrated that systemic chronic inflammatory response was a critical cause of IR and abnormal glucose metabolism. Inflammatory factors, such as IL-6, block the insulin signaling pathway and lead to IR through a variety of pathways. The imbalance of gut microbiota structure and increase in pathogenic bacteria destroy intestinal mucosal barrier function, causing endotoxins to enter the blood, leading to a systemic chronic inflammatory response. Therefore, regulating the gut microbiota structure has a positive effect on lowering chronic inflammation and IR; therefore, probiotics could be added to the treatment of T2DM.

The results of the present study showed that the use of Bifidobacterium quadruple viable bacteria combined with metformin in treating newly diagnosed T2DM was beneficial to increase the number of lactobacilli and bifidobacteria in gut microbiota, thus reducing the number of enterobacteria and enterococci and promoting the recovery of flora structure. Besides, it reduces intestinal mucosal injury and the levels of inflammatory factors, such as LPS, IL-6, and CRP, in blood circulation, and also lowers the chronic inflammatory status and IR of the body, thereby improving the effect of blood glucose control.

Conclusion

Metformin treatment of T2DM is beneficial by reducing gut microbiota imbalance. The supplementation of probiotic further regulates gut microbiota, reduces chronic inflammation, and improves insulin function as well as blood glucose control.