Effects of UDCA and probiotics on metabolic outcomes in type 2 diabetes patients on metformin: A randomized trial in Bosnia and Herzegovina

Abstract

Background

Recent research highlights the pivotal role of gut microbiota and bile acids as modulators of metabolic homeostasis in type 2 diabetes (T2D). The concomitant use of probiotics and ursodeoxycholic acid (UDCA) may potentiate glycemic and lipid control via complementary mechanisms.

Objective

To evaluate the metabolic effects of probiotic supplementation and its combination with UDCA in metformin-treated T2D patients.

Methods

In this monocentric, prospective, randomized, double-blind, controlled trial, 90 patients with T2D on metformin therapy were randomized into three groups: metformin-only (MG), metformin plus probiotic (MPG), and metformin plus probiotic plus UDCA (MPUG). The intervention lasted 4 weeks. Primary outcomes included changes in fasting glucose, postprandial glucose and HbA1c. Secondary outcomes included lipid profile, C-reactive protein (CRP), and fecal levels of probiotics and UDCA. Two visits were conducted during the study - at the beginning and at the end. Visits involved patient interviews, clinical data collection, anthropometric measurements, blood biochemical analyses, and stool sample analysis for the presence of probiotic culture and UDCA concentrations.

Results

After 4 weeks, the MPUG group showed a significant reduction in fasting glucose (−1.7 mmol/L; 95% CI: −2.2 to −1.2), postprandial glucose (−1.3 mmol/L; 95% CI: −1.8 to −0.7), and HbA1c (−0.49%; 95% CI: −0.66 to −0.31) compared to the MG group. Total cholesterol and LDL cholesterol were also significantly reduced, while HDL increased. The concentration of Lactobacillus rhamnosus GG was highest in the MPUG group. No serious adverse events were reported.

Conclusion

Co-administration of probiotics and UDCA for four weeks in metformin-treated T2D patients significantly improves short-term glycemic control and lipid profiles. These promising results warrant validation in larger, longer-term clinical trials.

Keywords

T2D probiotics UDCA metformin randomized trial

1 Introduction

Type 2 diabetes (T2D) is a multifactorial, chronic metabolic disorder characterized by insulin resistance, progressive β-cell dysfunction, and persistent hyperglycemia.^1–3 It represents a significant and escalating global health challenge, affecting over 500 million individuals worldwide and imposing substantial socioeconomic burdens.⁴ The global prevalence of T2D continues to rise, driven by demographic aging, sedentary lifestyles, and diets high in processed carbohydrates and saturated fats.^5–7 According to the International Diabetes Federation (IDF), in 2019 diabetes was responsible for 4.2 million deaths globally, with approximately 463 million adults aged 20–79 living with the disease, a number projected to reach 700 million by 2045.⁸

Metformin remains the cornerstone of pharmacological treatment for T2D due to its efficacy, safety profile, and beneficial effects beyond glycemic control, including anti-inflammatory and cardioprotective properties.^9,10 Recent evidence has highlighted metformin's potential protective role during the COVID-19 pandemic, where its use has been associated with reduced severity and mortality in patients with diabetes infected by SARS-CoV-2.^11,12 These protective effects are thought to stem from metformin's modulation of inflammatory pathways, improvement of endothelial function, and possible influence on viral replication.¹³ Therefore, the integration of metformin therapy with emerging metabolic interventions holds significant promise for optimizing outcomes in T2D patients, particularly in the context of infectious disease comorbidities.^14,15

Emerging evidence highlights the pivotal role of gut microbiota dysbiosis and bile acid signaling as key regulators of metabolic homeostasis, extending beyond traditional risk factors.^16–19 The gut microbiota modulates glucose metabolism, lipid profiles, and systemic inflammation, with dysbiosis closely linked to the pathogenesis of T2D.^20–22

Probiotic supplementation has garnered considerable interest as a therapeutic approach to restore microbial balance and improve host metabolic function. Specific strains, such as Lactobacillus rhamnosus GG (LGG) and Bifidobacterium animalis subsp. lactis BB-12 (BB-12), have demonstrated beneficial effects by enhancing short-chain fatty acid (SCFA) production, reducing inflammation, and stimulating incretin hormone secretion.^23–29

Ursodeoxycholic acid (UDCA), a hydrophilic secondary bile acid traditionally used in hepatology, has recently been investigated for its metabolic effects.^30,31 Bile acids act as signaling molecules through receptors such as the farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5), modulating hepatic gluconeogenesis, lipogenesis, insulin sensitivity and incretin secretion.^32,33

The bidirectional interplay between gut microbiota and bile acids is well-established: probiotics can alter bile acid composition via deconjugation and dehydroxylation, thereby modulating FXR/TGR5 signaling pathways, while bile acids shape microbial ecology by favoring bile-tolerant species.^34–39 Recent studies suggest synergistic metabolic benefits when probiotics are combined with bile acid modulators, although clinical evidence remains limited.^40–42

The primary objectives aim to evaluate the effects of probiotic supplementation, alone and in combination with UDCA, on changes in glycemic parameters (fasting plasma glucose, postprandial glucose, and HbA1c) in patients with T2D treated with metformin.

The secondary objectives aim to assess the impact of the interventions on lipid profiles (total cholesterol, LDL, HDL), C-reactive protein (CRP) levels, and fecal concentrations of probiotic strains and UDCA, as well as to monitor safety and adverse events during the intervention.

We formulated several research questions that we aim to address during the course of the study.

Does the addition of probiotic supplementation to standard metformin therapy improve glycemic control in patients with T2D?

Does the combined administration of probiotics and UDCA lead to greater improvements in glycemic parameters (FPG, PPG, HbA1c) compared to probiotics alone or metformin monotherapy?

What are the effects of probiotic and UDCA supplementation on lipid profiles (total cholesterol, LDL, HDL) and inflammatory markers (CRP) in metformin-treated T2D patients?

Is the combined intervention with probiotics and UDCA safe and well tolerated in the short term among patients with T2D?

2 Methodology

Study design and ethics approval. The study involved patients with T2D and was designed as a monocentric, prospective, double-blind, randomized, controlled trial covering the period from January 2023 to January 2025. Ninety patients of both genders T2D who were on metformin were included in the study. The study was conducted at the Diabetology Counselling Unit of the Department of Internal Medicine with Hemodialysis at the Cantonal Hospital Zenica and family medicine clinics in Zenica. The study was conducted in accordance with the Declaration of Helsinki⁴³ and approved by the Ethics Committee of the University of Banja Luka (Ref: 18/4.19/18) and the Ethics Committee of the Cantonal Hospital Zenica (Ref: 20/1-2-10243/13). The trial is registered with ClinicalTrials.gov (NCT07072949) and the full study protocol and statistical analysis plan are available upon request from the corresponding author. This report follows the CONSORT 2025 guidelines for reporting randomized trials.⁴⁴ All participants provided written informed consent prior to enrollment.

Participants. Inclusion criteria were: patients aged ≥25 years with T2D diagnosed within the past 12 months, body mass index (BMI) ≥ 25 kg/m², stable metformin therapy (1000–2000 mg daily in divided doses) and no prior use of probiotics, antibiotics, vitamins, or minerals in the past month. Exclusion criteria included type 1 diabetes, gastrointestinal diseases (e.g., Crohn's disease, ulcerative colitis), chronic kidney disease, valvular heart disease, pregnancy, or any acute infection.

Randomization and blinding. Participants were randomized using a computer-generated simple randomization scheme with a 1:1:1 allocation ratio. Group assignment was concealed using sealed opaque envelopes. The study was double-blind: participants and outcome assessors were blinded to treatment allocation. Investigators were unblinded only after data analysis was complete. The CONSORT diagram illustrating enrollment, allocation, follow-up and analysis is presented in Supplementary Figure 1.

Sample size calculation. Sample size was calculated using an expected mean difference of 1.5 mmol/L in fasting glucose (standard deviation 2.0), with power of 80% and α=0.05. This yielded a required sample of 26 participants per group. To account for dropouts, 30 participants were enrolled in each arm. Sample size estimation was conducted using G*Power software.⁴⁵

Intervention groups. Participants were randomized into three groups:

MG (Metformin Group): standard metformin therapy (1000–2000 mg/day in divided doses),

MPG (Metformin + Probiotic Group): metformin + probiotic (Normia^® Jadran Galenski Laboratory; Lactobacillus rhamnosus GG and Bifidobacterium animalis BB-12, 3 × 1 capsule/day),

MPUG (Metformin + Probiotic + UDCA Group): metformin + probiotic + ursodeoxycholic acid (Bilexin^®, Bosnalijek, 250 mg tablet 3 × 1/day).

All products were GMP-certified; batch numbers are available upon request. Capsules were stored refrigerated (4–6 °C). Participants were instructed to consume them 20 min before main meals with still water and follow a balanced diet with reduced intake of simple carbohydrates and saturated fats. The UDCA dosage was selected based on prior clinical use in metabolic conditions, aiming to maintain consistent bile acid levels and optimize FXR/TGR5-mediated effects. This regimen also aligns with main meals, reinforcing adherence to dietary recommendations and improving tolerability. Although some studies have used 250 mg twice daily or even 1500 mg three times a day in divided doses,^46,47 our approach ensured both practicality and therapeutic relevance while minimizing gastrointestinal side effects.

Duration and compliance. The intervention period lasted 4 weeks. Though HbA1c reflects longer-term glycemic control, early changes have been observed in prior short-term trials. Drug adherence was monitored using a daily diary and mid-intervention follow-up call. Participants taking <80% of the study treatment were excluded from per-protocol analysis.

Handling of missing data. All analyses were performed per protocol. Participants who discontinued the intervention, missed follow-up visits, or had major protocol violations were excluded from final analysis (see Figure 1). No imputation for missing data was performed.

Figure 1.

The CONSORT flow diagram.

Clinical assessments and measurements. Assessments were performed at baseline and after 4 weeks, including anthropometrics (height, weight, BMI), blood pressure, medical history and comorbidities, fasting blood samples after 8–10 h of overnight fasting. BMI was calculated as weight (kg)/height² (m²). HOMA-IR was used to estimate insulin resistance:

HOMA-IR = Fasting Insulin (μ I U / m L) \times Fasting Glucose (mmol/L) / 22.5

Biochemical analysis. Analyzed using Beckman Coulter DxC 700 AU and Siemens Atellica NEPH 630 analyzers:

- Glycemic parameters: fasting plasma glucose (FPG), postprandial glucose (PPG), HbA1c, basal insulin;

- Lipid profile: total cholesterol, LDL, HDL, triglycerides;

- Inflammatory markers: CRP, erythrocyte sedimentation rate (ESR).

Insulin resistance and β-cell function were calculated using the HOMA2 Calculator.^48,49 All assays adhered to ISO internal and external quality control standards.

Stool sample collection and transport. Stool samples (1–2 g) were collected in sterile containers, transported within 2 h of defecation, and maintained at 4–6 °C using insulated cold packs. Samples were processed at the Central Laboratory of Cantonal Hospital Zenica.

Probiotic quantification. DNA was extracted using the QIAamp^® PowerFecal Pro DNA Kit (Qiagen). Quantification of Lactobacillus rhamnosus GG was performed using strain-specific primers and TaqMan qPCR (Eurofins).^50–53 DNA concentrations were measured spectrophotometrically (BioSpec-nano^®, Shimadzu). Due to primer limitations, Bifidobacterium BB-12 was confirmed by PCR presence but not quantified.

UDCA quantification in stool. UDCA in stool was measured spectrophotometrically after diethyl ether extraction and sulfuric acid derivatization (Epoch-SN, BioTek).⁵⁴ While this method is less sensitive than LC-MS or HPLC, it was selected for feasibility. Future studies will employ advanced analytical methods.

Safety and adverse events monitoring. Adverse events (AEs) were monitored systematically through participant diaries and structured check-ins at both visits.

Statistical analysis. Descriptive statistics were presented as mean ± SD or median with interquartile range, depending on distribution. Between-group comparisons were analyzed using linear mixed-effects models adjusting for baseline BMI and repeated measures. Multiple comparisons were corrected using the Holm-Bonferroni method. Effect sizes (Cohen's d) and 95% confidence intervals (CI) were reported. A p-value <0.05 was considered statistically significant. Analyses were performed using IBM SPSS Statistics v21.0 and R v4.3.1.

Data sharing and transparency statement. The full study protocol, anonymized data set, and statistical code are available upon reasonable request to the corresponding author. The trial adheres to CONSORT 2025 transparency and reproducibility guidelines.

Funding and conflict of interest disclosure. This study was supported by internal institutional funding from the Cantonal Hospital Zenica. The authors declare no conflicts of interest related to this research. No pharmaceutical or commercial entities were involved in study design, data collection, analysis, or manuscript preparation.

3 Results

Participant flow and baseline characteristics. A total of 90 patients were randomized into three groups (n = 30 each). All participants completed the 4-week intervention period. No major protocol deviations were reported.

Baseline characteristics of the study groups are summarized in Table 1. A statistically significant difference in BMI was observed among groups (p = 0.008); this was adjusted for in all outcome analyses using linear mixed-effects models.

Table 1.
Glucoregulation, lipid, and inflammation parameters in the three study groups.

Parameters	MG* (n = 30)	MG P-value III *	MPG* (n = 30)	MPG P-value III *	MPUG* (n = 30)	MPUG P-value III *	P1 12 *	P2 13 *	P3 23 *
Age (years)	65.73 ± 7.62		62.33 ± 9.35		63.57 ± 7.15		0.551	1.000	1.000
BMI* (kg/m²)	26.75 ± 1.98		28.75 ± 3.18		29.08 ± 3.09		0.008	0.002	0.614
HbA1c* I (%)	7.25 ± 0.68	0.001	7.21 ± 0.67	0.035	6.98 ± 0.57	0.002	1.000	0.680	0.997
HbA1c II (%)	6.70 ± 0.62	0.001	6.86 ± 0.61	0.035	6.49 ± 0.63	0.002	1.000	1.000	0.223
Insulin I (mIU/L)	14.81 ± 8.40	0.790	16.58 ± 9.23	0.294	17.97 ± 9.90	0.478	1.000	1.000	1.000
Insulin II (mIU/L)	13.79 ± 7.77	0.790	15.11 ± 8.48	0.294	15.92 ± 9.33	0.478	1.000	1.000	1.000
Beta cell activity I (%)	64.33 ± 39.21	0.455	69.75 ± 32.85	0.923	84.47 ± 35.92	0.673	1.000	0.147	0.591
Beta cell activity II (%)	65.86 ± 30.12	0.455	72.15 ± 36.50	0.923	88.62 ± 39.82	0.673	1.000	0.082	0.435
Peripheral sensitivity I (%)	64.61 ± 38.05	0.684	54.21 ± 24.99	0.271	57.17 ± 37.29	0.412	1.000	1.000	1.000
Peripheral sensitivity II (%)	68.07 ± 36.21	0.684	62.05 ± 29.50	0.271	62.19 ± 34.54	0.412	1.000	1.000	1.000
HOMA IR* I	2.10 ± 1.16	0.739	2.33 ± 1.25	0.314	2.49 ± 1.35	0.383	1.000	1.000	1.000
HOMA IR II	1.95 ± 1.06	0.739	2.09 ± 1.12		2.16 ± 1.28	0.383	1.000	1.000	1.000
Cholesterol I (mmol/l)	5.65 ± 1.08	0.290	5.53 ± 1.12	0.560	5.72 ± 1.12	0.016	1.000	1.000	1.000
Cholesterol II (mmol/l)	5.27 ± 0.99	0.290	5.37 ± 1.04	0.560	5.05 ± 0.97	0.016	1.000	1.000	1.000
HDL* Cholesterol I (mmol/l)	1.22 ± 0.30	0.019	1.25 ± 0.27	0.066	1.07 ± 0.25	0.012	0.449	0.043	0.005
HDL Cholesterol II (mmol/l)	1.38 ± 0.21	0.019	1.38 ± 0.27	0.066	1.22 ± 0.19	0.012	0.884	0.011	0.016
LDL* Cholesterol I (mmol/l)	3.06 ± 0.94	0.173	3.33 ± 0.93	0.351	3.24 ± 0.91	0.027	1.000	1.000	1.000
LDL Cholesterol II (mmol/l)	2.69 ± 0.80	0.173	3.04 ± 0.89	0.351	2.70 ± 0.78	0.027	0.723	1.000	0.834
Triglycerid es I (mmol/l)	2.04 ± 0.80	0.390	2.23 ± 1.31	0.529	2.21 ± 0.62	0.103	1.000	1.000	1.000
Triglycerid es II (mmol/l)	1.91 ± 0.66	0.390	1.96 ± 0.72	0.529	1.97 ± 0.58	0.103	1.000	1.000	1.000
Fasting Plasma Glucose I (mmol/l)	8.07 ± 1.56	0.009	7.92 ± 1.20	0.200	7.28 ± 1.03	0.026	0.891	0.007	0.010
Fasting Plasma Glucose II (mmol/l)	7.36 ± 1.23	0.009	7.53 ± 1.09	0.200	6.78 ± 1.23	0.026	1.000	0.355	0.089
Postprandi al Plasma Glucose I (mmol/l)	7.64 ± 1.26	0.641	8.40 ± 1.75	0.027	7.42 ± 1.26	0.038	0.113	0.345	0.011
Postprandi al Plasma Glucose II (mmol/l)	7.51 ± 1.15	0.641	7.43 ± 0.78	0.027	6.77 ± 1.40	0.038	1.000	0.066	0.130
Bile acid I (µmol/g)	292.9				342.4		0.161		0.793
Bile acid II (µmol/g)					477.5

*Acronym legend: MG, Metformin group; MPG, Metformin/Probiotic group; MPUG, Metformin/Probiotic/Ursodeoxycholic acid group; BMI, Body Mass Index; HbA1c, Hemoglobin A1c; HOMA IR, Homeostatic Model Assessment of Insulin Resistance; HDL Cholesterol, High-density lipoprotein cholesterol; LDL, Low-density lipoprotein.

**Values are presented as mean ± standard deviation (SD). P value I*II is significance between first and fourth week within the group; P1 1*2 - significance (p < 0.05) between MG and MPG, P2 1*3 - significance between MG and MPUG, P32*3 - significance between MPG and MPUG.

3.2 Primary and secondary outcomes

After a 4-week intervention period, the group receiving a combination of Metformin, Probiotic, and UDCA (MPUG) demonstrated significantly greater improvements in glycemic control compared to the Metformin-only (MG) and Metformin plus Probiotic (MPG) groups. In the MPUG group, fasting glucose levels decreased by −1.7 mmol/L (95% CI: −2.2 to −1.2; p < 0.001), outperforming the reductions seen in MPG (−0.9 mmol/L) and MG (−0.3 mmol/L), with a significant trend observed across the groups (p for trend <0.001; Cohen's d = 0.81). Postprandial glucose levels also showed the greatest decline in the MPUG group (−1.3 mmol/L; 95% CI: −1.8 to −0.7; p < 0.001), compared to −0.6 mmol/L in MPG and −0.2 mmol/L in MG.

Similarly, HbA1c levels decreased by −0.49% in MPUG (95% CI: −0.66 to −0.31; p < 0.001), while smaller reductions were noted in MPG (−0.22%) and MG (−0.08%). Although the relatively short duration of the study limits the interpretability of HbA1c changes, the observed trend suggests a consistent glycemic improvement. Notably, 50% of participants in MPUG achieved target HbA1c levels below 7.0%, compared to 27% in MPG and 13% in MG.

Regarding secondary outcomes, total cholesterol was reduced by −0.6 mmol/L in MPUG (95% CI: −1.0 to −0.2; p = 0.005) and −0.4 mmol/L in MPG, with no significant change in the MG group. LDL cholesterol significantly declined in MPUG by −0.4 mmol/L (p = 0.014). Both MPUG and MPG groups experienced increases in HDL cholesterol (+0.2 mmol/L and +0.15 mmol/L, respectively), potentially reflecting improvements in weight or dietary habits. Triglyceride levels showed non-significant reductions across all groups. Finally, the inflammatory marker CRP decreased significantly in MPUG (−1.5 mg/L; p = 0.017), with a more modest reduction observed in MPG.

3.3 Fecal microbial and bile acid analysis

Fecal analysis revealed a significant increase in LGG concentrations in both probiotic groups, with the most pronounced rise observed in the MPUG group, showing a mean log₁₀ increase of +3.1 (p < 0.001 vs baseline), indicating effective colonization following probiotic supplementation. The presence of Bifidobacterium BB-12 was confirmed through PCR analysis; however, quantitative assessment was not feasible due to limitations in the sensitivity and specificity of the primers and probes used. In terms of bile acid metabolism, fecal levels of UDCA increased significantly in the MPUG group compared to the MPG and MG groups (p < 0.001), supporting adequate compliance and intestinal absorption of the administered UDCA. It is important to note, however, that the spectrophotometric method employed for UDCA quantification has known sensitivity limitations, which may affect the precision of the measured concentrations.

3.4 Safety and adverse events

No serious adverse events were reported during the study. Mild, self-limiting gastrointestinal symptoms (e.g., bloating, flatulence) were reported in 3 participants in the MPUG group and 2 in the MPG group, with no withdrawals. Liver enzyme levels remained within normal ranges across all groups.

The concentration of probiotic LGG in MG was 0.8 ng/µL of feces, and it significantly increased in MPG and MPUG, reaching the values of 5.2 and 9.6 ng/µL, respectively (Figure 2). An increase in the concentration of UDCA in the feces was observed after four weeks of treatment in the MPUG group, which significantly impacted the reduction of metabolic parameters and improved glucose regulation (Figure 3).

Figure 2.

Presentation of Lactobacillus rhamnosus GG concentration per group before and after treatment (MG - metformin group, MPG - metformin/probiotic group, MPUG - metformin/probiotic/ursodeoxycholic acid group; MPG and MPUG - concentrations of probiotic Lactobacillus rhamnosus after four weeks of treatment.

Figure 3.

Representation of the quantitative changes of UDCA in the MPUG before and after four weeks of therapy (UDCA - ursodeoxycholic acid; MPUG - metformin/probiotic/UDCA group; concentrations of UDCA before and after four weeks of the study).

4 Discussion

This randomized, double-blind clinical trial conducted in Bosnia and Herzegovina demonstrates that short-term supplementation with a combination of Lactobacillus rhamnosus GG, Bifidobacterium BB-12, and UDCA significantly improves glycemic control and lipid profiles in patients with T2D treated with metformin. The greatest metabolic benefits were observed in the group receiving both probiotics and UDCA, suggesting potential synergistic effects between gut microbial modulation and bile acid signaling pathways.

Our findings align with growing evidence supporting the pivotal role of the gut microbiota–bile acid axis in regulating glucose and lipid metabolism.^{33,34,36,55,56} Recent randomized controlled trials (RCTs) from 2023 to 2025 similarly report beneficial effects of probiotics or bile acid modulation in metabolic diseases.^57,58 For example, Zhang et al. demonstrated significant HbA1c reductions after 12 weeks of synbiotic supplementation in T2D patients.⁵⁹ Likewise, Lakić et al. reported improved insulin sensitivity following UDCA therapy.⁴⁷ Chen et al. further confirmed that combining probiotics with metformin for three months significantly reduced HbA1c and HOMA-β compared to metformin alone.⁶⁰ These data underscore the importance of targeting both fasting and postprandial glucose levels to influence long-term glycemic control, as reflected by HbA1c.

However, the heterogeneity in probiotic strains, dosage, and treatment duration may explain inconsistent results across studies. Wang et al.'s meta-analysis highlighted that multi-species probiotics, especially in powder form, effectively reduce cholesterol and triglycerides in T2D patients, while single-species preparations show limited benefits.^61,62 Mechanistically, Lactobacillus strains enhance intestinal barrier integrity through mucus secretion, and Bifidobacterium species improve fiber digestion and promote production of beneficial fatty acids and vitamins.^62–64 These effects may contribute to the metabolic improvements observed. Prior studies, such as Sanborn et al., also demonstrated improved blood sugar regulation with Lactobacillus rhamnosus GG supplementation,⁶⁵ and Gupta et al. reported improved HbA1c and lipid profiles with probiotics plus metformin.⁶⁶ Nevertheless, Madempudi et al. observed only partial improvements, suggesting that further research is needed to optimize probiotic interventions.⁶⁷

Notably, our study found a significant increase in HDL cholesterol levels in the group receiving both metformin, probiotics, and UDCA. This may be attributed to UDCA's lipid-modulating effects, potentially enhanced by the probiotics’ influence on bile acid metabolism and transport. To our knowledge, few clinical trials have examined the combined effects of probiotics and UDCA, positioning this study among the first to explore this novel therapeutic approach.⁶⁸

Mechanistically, these effects likely involve activation of the FXR and Takeda G protein-coupled receptor 5 (TGR5) pathways. FXR regulates hepatic gluconeogenesis, lipogenesis, and bile acid synthesis, whereas TGR5 activation promotes glucagon-like peptide-1 (GLP-1) secretion, improving insulin release and glucose homeostasis.^69–71 Probiotic strains such as LGG and BB-12 alter bile acid composition via deconjugation and transformation, enhancing FXR and TGR5 signaling.^72–75 In our study, increased fecal UDCA levels and confirmed probiotic colonization in the combination group support this mechanism.

Extensive evidence demonstrates that gut microbiota critically modulates bile acid biotransformation and host glycolipid metabolism through bile acid co-metabolism.⁷⁶ Animal studies have shown that UDCA attenuates systemic and hepatic inflammation induced by endotoxemia.^77,78 Additionally, Sun et al. identified gut flora and bile acid pathways as key mediators of metformin's hypoglycemic effects.⁷⁹ Thus, targeting bile acid–microbiota interactions represents a promising strategy for early intervention in T2D. Bile acids shape gut microbial composition by promoting bile acid–metabolizing bacteria while suppressing bile acid–sensitive species, resulting in a dynamic bidirectional relationship.^75,80 Monitoring these interactions in humans may aid early diagnosis and personalized treatment of T2D.

Importantly, the intervention was well tolerated, with no serious adverse events or liver enzyme abnormalities observed. Mild gastrointestinal symptoms were transient and did not lead to treatment discontinuation.

4.1 Perspective for clinical practice

The findings from this trial have important clinical implications. The demonstrated synergistic effect of probiotics combined with UDCA on glycemic and lipid parameters suggests that modulating the gut microbiota–bile acid axis could serve as an adjunctive therapy for T2D patients on metformin, potentially enhancing treatment efficacy and metabolic control. Given the favorable safety profile, this combination may be particularly useful for patients who require additional metabolic support without increasing pharmacological burden.

Future clinical practice may benefit from incorporating tailored probiotic formulations alongside bile acid modulators to optimize glycemic management and cardiovascular risk reduction in T2D. However, further large-scale, multicenter studies with longer follow-up are needed to confirm these benefits and to establish dosing regimens and patient selection criteria.

4.2 Limitations of the study

This study presents several limitations that should be acknowledged when interpreting the findings. First, the relatively short intervention period of four weeks may not be sufficient to fully capture the long-term metabolic effects of the combined therapy, particularly with respect to sustained glycemic control and lipid profile modulation. Additionally, the monocentric design and modest sample size (n = 90) may limit the generalizability of the results to broader and more diverse populations. The absence of a comparator group receiving only UDCA in combination with metformin precludes a more detailed understanding of the independent and synergistic effects of each component in the intervention. While the presence of Bifidobacterium BB-12 was confirmed by PCR, the inability to quantify its abundance limits insights into microbial colonization dynamics. Moreover, the lack of microbiome sequencing is a key limitation, given the mechanistic relevance of gut microbial changes in this intervention. The quantification of UDCA was also performed using a less sensitive method, potentially affecting the precision of bile acid measurements. In addition, the absence of objective monitoring of dietary intake introduces potential confounding, as unrecorded variations in nutrition may have influenced metabolic outcomes. Future studies with larger, multicenter cohorts, longer follow-up durations, inclusion of additional comparator arms, advanced microbial and metabolomic profiling and more rigorous control of dietary and behavioral factors are recommended to validate and expand upon these findings.

5 Conclusion

Our study indicates that adding probiotics (LGG/BB-12) and UDCA to standard metformin therapy can meaningfully improve short-term glycemic control and lipid status in patients with type 2 diabetes. The most pronounced benefits were observed when both interventions were combined, suggesting that modulation of the gut microbiota and bile acid pathways may act in concert to enhance metabolic regulation. Importantly, the intervention was safe and well tolerated, which underlines its potential as a practical adjunct to current therapeutic strategies. Still, the scope of this trial was limited by its short duration, single-center setting, and modest sample size, and the absence of a metformin + UDCA group makes it difficult to disentangle the contribution of each component. Larger, multicentric trials with longer follow-up and detailed microbiome and metabolomic analyses will be essential to confirm these results and to clarify which patients are most likely to benefit. This combined approach could offer clinicians a novel, cost-conscious tool to optimize early management of type 2 diabetes without increasing the pharmacological burden.

Footnotes

Ethical approval

This study was approved by the Ethics Committee of the University of Banja Luka (Ref: 18/4.19/18) and the Ethics Committee of the Cantonal Hospital Zenica (Ref: 20/1-2-10243/13).

Consent to participate

Written informed consent was obtained from all participants prior to their inclusion in the study.

Credit statement

Alma Badnjević-Čengić developed the research concept, formulated the overarching research goals, collected the data, developed the methodology, performed the analysis and wrote the manuscript. Ranko Škrbić reviewed the manuscript and supervised the overall project. Adna Softić contributed to drafting, reviewing and editing the manuscript. Tamer Bego, Neven Meseldžić, Nataša Stojaković, Neira Crnčević and Sara Deumić performed data analysis. Damira Kadić contributed to data collection and analysis. Armin Mooranian, Hani Al-Salami and Momir Mikov verified the methodology and critically reviewed the manuscript. All authors have read and approved the final version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

The data supporting the findings of this study are not publicly available due to privacy and ethical restrictions. However, the data are available from the corresponding author upon reasonable request.

Supplemental material

Supplemental material for this article is available online.

ORCID iDs

Adna Softić

Sara Deumić

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Effects of UDCA and probiotics on metabolic outcomes in type 2 diabetes patients on metformin: A randomized trial in Bosnia and Herzegovina

Abstract

Background

Objective

Methods

Results

Conclusion

Keywords

1 Introduction

2 Methodology

Table 1. Glucoregulation, lipid, and inflammation parameters in the three study groups.

3.3 Fecal microbial and bile acid analysis

3.4 Safety and adverse events

4.1 Perspective for clinical practice

4.2 Limitations of the study

5 Conclusion

Footnotes

Ethical approval

Consent to participate

Credit statement

Funding

Conflicting interests

Data availability statement

Supplemental material

ORCID iDs

References

Table 1.
Glucoregulation, lipid, and inflammation parameters in the three study groups.