Metformin Plus Vitamin D3 Shows Strongest Metabolic Effects in Diabetic Mice

Metformin and vitamin D3, given together, produced the largest observed improvements in blood glucose, body weight, blood lipids and liver enzyme levels among treated groups of streptozotocin-induced diabetic mice, according to a new experimental study published in Health Science Reports. The combination also showed the lowest blood glucose readings during oral glucose tolerance testing, falling to 3.66 ± 0.36 mmol/L at 180 minutes, compared with 5.22 ± 0.51 mmol/L for metformin alone and 6.18 ± 1.15 mmol/L for vitamin D3 alone. Researchers caution, however, that the findings are preliminary and do not establish a confirmed synergistic interaction between the two agents.

The study, conducted at the University of Information Technology and Sciences in Bangladesh, used 70 healthy adult male Swiss albino mice weighing 28 ± 3 grams, procured from the International Centre for Diarrhoeal Disease Research, Bangladesh. Male mice were chosen in line with previous experimental diabetes protocols and to reduce biological variability, a design choice that prevents any assessment of sex-related differences in metabolic responses. The animals were housed at 22°C ± 2°C with 55% ± 5% relative humidity under a 12-hour light/dark cycle, fed a standard pellet diet, and acclimatised for seven days before the experiment began.

The work unfolded in three phases. First, 30 normoglycaemic mice underwent an oral glucose tolerance test to establish baseline glucose handling. Second, mice were fasted and injected with streptozotocin, a selective beta-cell cytotoxic agent, at 45 mg/kg body weight intraperitoneally for three consecutive days to induce experimental diabetes. Of 32 streptozotocin-treated mice, 25, or 78.1%, successfully developed diabetes, defined as fasting blood glucose above 9 mmol/L. The induced animals showed sustained hyperglycaemia typical of streptozotocin-driven pancreatic beta-cell dysfunction. The authors note that this model predominantly reflects beta-cell injury and insulin deficiency and does not fully reproduce the complex pathophysiology of human Type 2 diabetes, particularly insulin resistance.

In the treatment phase, 24 diabetic mice were randomly divided into four groups of six. Group I, the normal control, received vehicle only. Group II, the diabetic control, also received vehicle. Group III received metformin at 250 mg/kg body weight, a dose chosen from prior mouse studies, and served as the positive control. Group IV received metformin plus vitamin D3 at 100 μg/kg body weight, and Group V received vitamin D3 alone. All treatments were given orally by gavage for 21 consecutive days. No formal a priori sample-size calculation was performed before the experiment began.

During the oral glucose tolerance test, all groups started with similar baseline glucose, ranging from 6.23 ± 0.87 to 7.10 ± 1.10 mmol/L. After a 2 g/kg oral glucose load, blood glucose peaked at 30 minutes in every group, with the negative control reaching 12.85 ± 3.85 mmol/L and the treated groups peaking between 12.55 and 13.36 mmol/L. From 60 minutes onward, the combination group consistently recorded the lowest values, dropping to 7.52 ± 0.84 mmol/L at 60 minutes, 5.26 ± 0.44 at 90, 4.54 ± 0.33 at 120, 3.80 ± 0.30 at 150 and 3.66 ± 0.36 mmol/L at 180 minutes. The authors report that the combination showed a significantly greater reduction in blood glucose during the later phases of the test compared with the monotherapy groups, at p < 0.05.

Over the 21-day treatment period, fasting blood glucose declined in all treated groups while the diabetic control remained hyperglycaemic, rising from 11.4 ± 1.36 mmol/L at baseline to 12.9 ± 0.93 mmol/L by Day 20. By the end of treatment, the combination group had the lowest fasting glucose at 6.1 ± 0.41 mmol/L, compared with 6.9 ± 0.59 mmol/L for metformin and 7.3 ± 0.88 mmol/L for vitamin D3 alone. Relative to their own baseline values, the combination, metformin and vitamin D3 groups achieved reductions of about 53.4%, 44.8% and 40.2%, respectively. Fasting glucose in all treatment groups was significantly lower than in the diabetic control throughout the study period, at p < 0.05.

Body weight followed a similar pattern. Diabetic control mice lost roughly 15.8% of their baseline weight, falling from 27.9 ± 1.90 g to 23.5 ± 1.03 g by Day 20. The metformin group lost about 8.6%, the vitamin D3 group about 10.4%, and the combination group only about 5.6%, ending at 27.2 ± 1.21 g. The authors state that metformin and vitamin D3, especially in combination, reduced diabetes-associated weight loss and improved overall metabolic status compared with untreated diabetic mice, at p < 0.05.

Serum lipid and liver enzyme measurements, performed in three animals per group via terminal cardiac puncture, revealed marked diabetic dyslipidaemia. Total cholesterol in the diabetic control reached 161.33 ± 9.03 mg/dL versus 87.66 ± 5.03 mg/dL in normal controls. The combination lowered total cholesterol to 93.06 ± 2.89 mg/dL, a 42.3% reduction, while metformin achieved a 30.6% decrease and vitamin D3 an 11.4% decrease. LDL cholesterol fell from 102.33 mg/dL in diabetic controls to 28.66 mg/dL with the combination, a 72.0% reduction, and HDL rose to 40.33 ± 2.10 mg/dL, about 39.1% above diabetic control levels and comparable to normal controls. Triglycerides dropped to 120.33 ± 2.10 mg/dL with the combination, and the LDL/HDL ratio fell by 80.0%, reaching 0.71 ± 0.08, close to the normal control value of 0.60 ± 0.04. The total cholesterol/HDL ratio, an indicator of cardiovascular risk, fell by 58.7% with the combination, though no direct cardiovascular outcomes were assessed.

Serum ALT activity, a marker of hepatic stress, was significantly elevated in diabetic controls at 89.60 ± 5.68 IU/L versus 40.30 ± 4.16 IU/L in normal controls. The combination produced the greatest observed reduction, lowering ALT to 52.60 ± 3.05 IU/L, a decrease of about 41.3%, compared with 29.4% for metformin and 20.8% for vitamin D3 alone. The authors emphasise that because only ALT was measured, without additional hepatic biomarkers or liver histology, this finding should be interpreted as an improvement in diabetes-associated ALT elevation rather than definitive evidence of hepatoprotection. Qualitative histopathological examination of pancreatic tissue, based on one representative animal per group and not formally blinded, showed that the combination group had comparatively better-preserved islet architecture than the diabetic control and monotherapy groups, though these observations are descriptive and cannot establish quantitative beta-cell preservation or regeneration.

The study carries substantial limitations that the authors detail extensively. Only three animals per group were used for serum lipid and ALT analyses, which may have limited statistical power, and no a priori power calculation was performed. The histopathological assessment was qualitative, unblinded, and based on a single animal per group. The 21-day treatment period was short and did not allow assessment of long-term efficacy or safety. Serum insulin, HbA1c, circulating vitamin D status, oxidative stress biomarkers, calcium and renal safety parameters, food and water intake, inflammatory mediators and molecular markers of glucose-signalling pathways were not measured. The study was not designed to formally distinguish additive, synergistic or antagonistic interactions, and direct statistical superiority of the combination over metformin monotherapy was not consistently established across all endpoints. The original detailed statistical output and individual-level datasets were not retained, so some exact p-values could not be reconstructed retrospectively. The authors conclude that the findings represent preliminary experimental evidence and that larger studies with adequate sample sizes, quantitative histopathology, insulin and HbA1c measurements, formal drug-interaction analysis, longer treatment periods and insulin-resistant or other Type 2 diabetes models are needed before the therapeutic relevance of vitamin D3 as an adjunct to metformin can be established.

Subject of Research: Experimental evaluation of metformin and vitamin D3 combination therapy in streptozotocin-induced diabetic mice

Article Title: Exploring the Therapeutic Potential of Metformin and Vitamin D3 Combination in the Management of Diabetes Mellitus

Article References: Azam, M. F., Jami, M. A. B. S., Suprio, N. Y., Kabir, M. I., Oishi, S. T., Ekra, J. F., Islam, N., Zaman, S., Hossain, M. M., & Al Amin, M. (2026). Exploring the Therapeutic Potential of Metformin and Vitamin D 3 Combination in the Management of Diabetes Mellitus. Endocrinology, Diabetes & Metabolism, 9(6), Article e70384. https://doi.org/10.1002/edm2.70384

Image Credits: AI Generated

DOI: 10.1002/edm2.70384

Keywords: metformin, vitamin D3, diabetes mellitus, streptozotocin, hyperglycaemia, dyslipidaemia, ALT, oral glucose tolerance test, body weight, pancreatic islets, Swiss albino mice, experimental pharmacology