Type 2 diabetes mellitus (T2DM) is a progressive metabolic disorder characterized by chronic hyperglycemia and insulin resistance, with systemic low-grade inflammation playing a central role in the pathophysiology and progression of its complications (1). Mitochondrial dysfunction and innate immune activation are increasingly recognized as key contributors to this inflammatory milieu. In particular, circulating cell-free mitochondrial DNA (ccf-mtDNA), a damage-associated molecular pattern (DAMP), can activate the NLRP3 inflammasome, a cytosolic protein complex responsible for the cleavage and secretion of interleukin (IL)-1β and IL-18, further amplifying inflammatory responses (2–4).

Imeglimin is a first-in-class oral antidiabetic agent belonging to the tetrahydrotriazine-containing compound family. Chemically, it is a small heteroaromatic molecule with a unique tetrahydrotriazine ring structure. While structurally distinct, it shares some similarities with metformin, as both are guanidine derivatives with a biguanide-like pharmacophore that contributes to their mitochondrial-targeted actions. This structural resemblance partly explains overlapping mechanisms, such as inhibition of mitochondrial complex I and modulation of hepatic glucose production. However, unlike metformin, Imeglimin exhibits a pleiotropic “double action” by not only improving insulin sensitivity but also directly enhancing pancreatic β-cell function and survival. Beyond its glycemic control, emerging evidence suggests its potential to attenuate mitochondrial-derived oxidative stress and inflammation. However, clinical data evaluating the impact of Imeglimin on key mitochondrial and inflammasome-related biomarkers in T2DM patients remain limited. In 2021, it became the first drug of its class to receive regulatory approval in Japan for the treatment of type 2 diabetes, following the TIMES clinical trial program (5).

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is gaining attention as a sensitive indicator of mitochondrial dysfunction, particularly in metabolic diseases such as type 2 diabetes. Released into the bloodstream from damaged or stressed cells, ccf-mtDNA reflects disturbances in mitochondrial integrity and cellular homeostasis (6, 7). In diabetic individuals, elevated ccf-mtDNA levels are commonly associated with increased oxidative stress, impaired mitochondrial respiration, and systemic inflammation.m (8, 9) Circulating cell-free mitochondrial DNA (ccf-mtDNA) functions as a damage-associated molecular pattern (DAMP), capable of activating innate immune responses, including the NLRP3 inflammasome pathway. This activation promotes the release of pro-inflammatory cytokines, such as IL-1β and IL-18, which are implicated in sustaining the low-grade inflammation characteristic of diabetes (10–12). Therefore, measuring both ccf-mtDNA and serum NLRP3 levels can provide complementary insights into mitochondrial distress and inflammasome activation. A positive correlation between mtDNA and NLRP3 suggests that mitochondrial dysfunction may directly fuel inflammatory signaling in diabetes, offering potential targets for early detection and therapeutic intervention.

This prospective 6-month study aims to evaluate the effect of Imeglimin therapy on serum levels of ccf-mtDNA and NLRP3 inflammasome expression in patients with T2DM. Additionally, we investigate the association of these novel biomarkers with conventional inflammatory markers including and interleukins, to better understand the inflammatory modulation profile of Imeglimin and its potential therapeutic implications in diabetes-related inflammation.

In this study, baseline characteristics were well-matched across the four intervention groups, ensuring comparability for evaluating the effects of Imeglimin and combination therapies. Our findings demonstrated that combination therapies involving Imeglimin or Metformin with other oral hypoglycemic agents (OHAs) led to significant improvements in glycemic control, as evidenced by reductions in HbA1c, whereas Imeglimin monotherapy and the Imeglimin + Metformin group did not achieve statistically significant changes. Singh et al. found that imeglimin (1000 mg BID) effectively reduced HbA1c in T2D patients across Phase 2 and 3 trials. It showed comparable efficacy to metformin, SUs, AGIs, DPP-4is, and SGLT-2is, but lower than TZDs. Combination therapy with metformin and TZDs enhanced HbA1c reduction, whereas no significant benefit was observed with GLP-1RAs. Imeglimin demonstrated good tolerability, fewer gastrointestinal side effects than metformin, and no cases of lactic acidosis, suggesting a favorable safety and efficacy profile (16). The INDI-TIMES study, India’s largest real-world analysis of imeglimin in T2DM (n=8301), showed that imeglimin 1000 mg twice daily significantly reduced HbA1c (−1.12%), FPG, PPG, improved lipid profiles, and lowered serum creatinine. Results were consistent across monotherapy and combination therapy groups. Compared to TIMES studies in Japanese patients, a greater HbA1c reduction was seen, possibly due to higher baseline glycemia in Indian patients. Weight loss and improved liver enzymes were also observed without major safety concerns, supporting imeglimin’s effectiveness in routine clinical practice (17). Imeglimin add-on therapy to metformin acutely reduced 24-hour mean glucose levels and improved glycemic variability in patients with type 2 diabetes. Higher baseline HDL cholesterol levels correlated with greater improvements. Continuous glucose monitoring confirmed significant increases in time in range and decreases in glucose variability after imeglimin initiation (18). Yang et al. (2020) observed that metformin treatment over one year significantly reduced peripheral mitochondrial DNA copy number (mtDNA-CN) in women with polycystic ovary syndrome. The reduction in mtDNA-CN correlated with decreased serum testosterone but not with metabolic factors, suggesting a link between mitochondrial function improvement and androgen regulation (19). Wang et al. (2023) found that mitochondrial DNA copy number (mtDNA-CN) may help guide treatment choice between metformin and acarbose in newly diagnosed type 2 diabetes. Higher mtDNA-CN was associated with better β-cell function protection with metformin but worse with acarbose, although no association with glycemic response was observed (20). Liu et al. (21) demonstrated that circulating cell-free mitochondrial DNA (ccf-mtDNA) levels were significantly elevated in coronary heart disease (CHD) patients with type 2 diabetes compared to those without. Plasma ccf-mtDNA showed strong diagnostic value (AUC 0.907), and levels correlated with fasting glucose, suggesting its potential as a biomarker (21). Bae et al. (9) reported that plasma ccf-mtDNA levels were significantly elevated in patients with type 2 diabetes compared to healthy controls. Elevated ccf-mtDNA correlated with IL-1β levels and activated the AIM2 inflammasome in macrophages, suggesting that ccf-mtDNA contributed to chronic inflammation through AIM2 inflammasome-mediated pathways in type 2 diabetes (9). Previous studies have demonstrated that metformin modulates mitochondrial DNA copy number and mitochondrial function (19, 20), and that elevated circulating cell-free mitochondrial DNA is associated with diabetic complications such as coronary heart disease, considering imeglimin’s structural similarity to metformin and its targeting of mitochondrial bioenergetics, we hypothesize that imeglimin could exert a similar modulatory effect on mitochondrial health, with this study being the first to explore this possibility. This study represents the first attempt to explore this potential effect with imeglimin.

Interestingly, significant reductions in circulating cell-free mitochondrial DNA (ccf-mtDNA) were observed in the Imeglimin + Metformin and Imeglimin + Other OHA groups, suggesting a potential synergistic effect on mitochondrial stress or turnover when Imeglimin is combined with other agents. Figure 2 illustrates changes in serum ccf-mtDNA (A) and NLRP3 (B) concentrations across different therapeutic regimens in T2DM patients over 6 months. The combination of Imeglimin with other OHAs showed the most significant reductions in both ccf-mtDNA (–18.5 ± 3.2 copies/μL, p < 0.001) and NLRP3 levels (–7.9 ± 1.6 ng/mL, p = 0.042), indicating improved mitochondrial integrity and reduced inflammation. Metformin + other OHA also significantly reduced ccf-mtDNA (–9.4 ± 1.9, p = 0.002), while changes in other groups were not statistically significant. Notably, Imeglimin monotherapy led to a non-significant increase in NLRP3, suggesting limited anti-inflammatory effect when used alone ( Figure 2 ).

The scatter plots in Figure 3 illustrate significant positive correlations between markers of systemic inflammation and mitochondrial stress in patients with type 2 diabetes mellitus (T2DM). Panel 2A shows a positive association between serum IL-6 levels and circulating cell-free mitochondrial DNA (ccf-mtDNA), suggesting that increased systemic inflammation may be linked to elevated mitochondrial stress or damage. Panel 2B reveals a positive correlation between IL-1β and NLRP3 concentrations, indicating the activation of the NLRP3 inflammasome pathway in response to pro-inflammatory stimuli. Furthermore, Panel 2C demonstrates a mild positive correlation between serum NLRP3 and ccf-mtDNA concentrations, implying a potential crosstalk between mitochondrial dysfunction and innate immune activation. Collectively, these findings suggest that mitochondrial-derived danger signals, such as ccf-mtDNA, may contribute to the activation of inflammatory pathways in T2DM, potentially playing a role in the progression of metabolic inflammation. These findings imply that Imeglimin exerts optimal anti-inflammatory and mitochondrial protective effects when used in combination therapy, especially with Metformin or other OHAs. Alfadul et al. (2023) found that six months of lifestyle modification in prediabetic individuals significantly modulated serum NLRP3 inflammasome activity and related interleukins. Glycemic control led to reductions in NLRP3, IL-33, and IL-1α, suggesting that early intervention could reverse inflammatory changes and reduce progression to type 2 diabetes mellitus (22). Zhang et al. demonstrated that metformin corrected glycometabolic reprogramming and suppressed NLRP3 inflammasome-induced pyroptosis in trophoblasts by inhibiting the TLR4/NF-κB/PFKFB3 signaling pathway. Their findings suggested that metformin mitigated oxidative stress and inflammatory pyroptosis (23). Several other animal and preclinical studies have demonstrated the therapeutic effects of metformin in inhibiting NLRP3 inflammasome activation, and due to the structural and mechanistic similarities between metformin and imeglimin, it is hypothesized that imeglimin may exert a similar modulatory effect on NLRP3-mediated inflammation (24, 25). However, despite moderate correlations between inflammatory markers and metabolic parameters, In the current study no significant longitudinal changes in serum NLRP3 or other inflammatory cytokines were detected, indicating that a 6-month intervention period may be insufficient to produce measurable anti-inflammatory effects, or that mitochondrial and glycemic improvements precede detectable changes in systemic inflammation. The positive correlations between changes in ccf-mtDNA and glycemic and lipid parameters underscore the intertwined relationship between mitochondrial health and metabolic control. In contrast to our findings, Berezina et al. (2023) demonstrated that circulating cell-free mitochondrial DNA (cf-mtDNA) levels were significantly lower in T2DM patients with heart failure compared to those without. Their results suggest a depletion of cf-mtDNA in advanced cardiac dysfunction, diverging from the elevated cf-mtDNA levels observed in our cohort (26).

Although NLRP3 expression showed only a falling trend without statistical significance, reductions in IL-6 and IL-1β suggest partial suppression of inflammasome activity. Prior studies support such effects, as Kim et al. showed SGLT2 inhibition lowers IL-1β via ketone- and insulin-mediated mechanisms, and Birnbaum et al. reported SGLT2 ± DPP4 inhibition attenuates inflammasome activation and renal injury (1,2). In our cohort, “other OHAs” comprised sulfonylureas and DPP4 inhibitors, which may provide anti-inflammatory benefits. The weak correlation and modest changes observed could also be attributed to our relatively low sample size, potentially underpowering statistical significance (27, 28). Collectively, these findings highlight the greater efficacy of combination therapies in improving glycemic and mitochondrial parameters, while suggesting that more prolonged or intensified interventions may be required to influence inflammatory pathways.

In our study, the HbA1c reduction observed in the Imeglimin + OHA group (Δ=–0.5%) was lower than that reported in the INDI-TIMES study (Δ=–1.12%) (17). Several factors may explain this difference. First, the baseline HbA1c levels in our cohort were slightly lower (7.9%) compared to those in INDI-TIMES (8.07%). Since the degree of HbA1c reduction is strongly influenced by initial glycemic status, a lower starting HbA1c generally results in a smaller absolute decrease following therapy. Second, differences in background therapy composition could have influenced the glycemic effect. INDI-TIMES enrolled a heterogeneous population on a variety of oral hypoglycemic agents, including sulfonylureas, while our cohort included only patients on DPP-4 inhibitors and SGLT2 inhibitors in combination with Imeglimin. Both of these classes are associated with modest HbA1c reductions and may offer less additive benefit when combined with Imeglimin, compared to agents with stronger glucose-lowering potential such as sulfonylureas. Third, the study population size was considerably larger in INDI-TIMES (n=7821) compared to our sample, increasing the precision of effect size estimates and possibly capturing a greater range of treatment responses.

Together, these factors likely contributed to the smaller HbA1c reduction observed in our study. This highlights the importance of considering baseline glycemia, background therapy type, and study design differences when comparing across trials, and suggests that the magnitude of Imeglimin’s effect may vary according to patient and treatment characteristics.

While this study provides novel insights into the effects of Imeglimin on ccf-mtDNA and NLRP3 inflammasome activity in T2DM, several limitations should be noted. The relatively small sample size limits the statistical power and generalizability of our findings, and therefore the results should be interpreted in an explorative context. The 6-month duration may be insufficient to capture long-term effects, and the modest sample size limits the power for subgroup analyses. The study focused on circulating biomarkers without assessing tissue-specific or functional mitochondrial parameters. Future research should involve longer follow-up, larger and more diverse cohorts, and incorporation of functional mitochondrial assessments. Exploring tissue-specific pathways and combining Imeglimin with lifestyle or adjunctive therapies could further clarify its therapeutic potential in T2DM management. Although preclinical evidence suggests that Imeglimin, similar to metformin, may modulate NLRP3 inflammasome activity, our clinical findings (greater effectiveness with Imeglimin plus other OHAs compared to Imeglimin plus metformin) do not directly support this hypothesis. The efficacy against NLRP3 may also vary depending on the specific combination drug used with Imeglimin, which could influence the overall anti-inflammatory effect. This highlights the need for further mechanistic studies to clarify Imeglimin’s role in NLRP3-mediated inflammation and pyroptosis.

Imeglimin-based therapies, particularly in combination with non-metformin oral hypoglycemic agents, demonstrate significant benefits in glycemic control and may alleviate mitochondrial stress by reducing ccf mtDNA. However, direct assessment of mitochondrial function was not performed, which represents a limitation of using surrogate biomarkers. Although NLRP3 inflammasome levels did not show consistent reductions, positive correlations with glycemic and lipid changes suggest an underlying inflammatory component influenced by therapy. The findings support the use of imeglimin combinations over monotherapy for enhanced metabolic outcomes in type 2 diabetes mellitus. Further long-term studies are needed to confirm these observations and explore the mechanistic pathways linking mitochondrial function, inflammation, and glycemic control in patients undergoing imeglimin-based interventions.