Type 2 diabetes is one of the most prevalent chronic diseases worldwide, significantly impacting patients' quality of life. Current treatment options like metformin (MET) effectively counteract hyperglycemia but fail to alleviate diabetes-associated complications such as retinopathy, neuropathy, nephropathy, hepatopathy, and cardiovascular diseases.

To propose the supplementation of cholecalciferol (CHO) and taurine (TAU) to enhance MET efficacy in controlling diabetes while minimizing the risk of associated complications.

The study involved sixty rats, including ten non-diabetic control rats and fifty experimental rats with type 2 diabetes induced by streptozotocin. The experimental rats were further subdivided into positive control and treatment subgroups. The four treatment groups were randomly allocated to a single MET treatment or MET combined with supplements either CHO, TAU, or both.

Diabetic rats exhibited elevated levels of glucose, insulin, Homeostasis Model Assessment of Insulin Resistance (HOMA-IR), glycated hemoglobin percentage, lipid markers, aspartate aminotransferase, and malondialdehyde, along with reduced levels of antioxidant enzymes (catalase and superoxide dismutase). The administration of CHO and TAU supplements alongside MET in diabetic rats led to a noticeable recovery of islet mass. The antioxidative, anti-inflammatory, and anti-apoptotic properties of the proposed combination therapy significantly ameliorated the aforementioned abnormalities.

The supplementation of CHO and TAU with MET showed the potential to significantly improve metabolic parameters and protect against diabetic complications through its antioxidative, anti-inflammatory, and anti-apoptotic effects.

Diabetes mellitus (DM) poses a substantial global health threat because of the high prevalence with different etiologies and increased risk of mortality[1]. This metabolic disorder’s hallmarks are chronic hyperglycemia, hyperlipidemia, and insulin resistance, contributing to macrovascular and microvascular events[2]. Atherosclerotic cardiovascular disease is a prominent global cause of morbidity and mortality, with approximately 70% to 80% of deaths in individuals with diabetes attributed to vascular disorders[3]. Other serious diabetic complications may damage many vital organs, which can result in more severe and irreversible pathological illnesses, including nephropathy, retinopathy, vasculopathy, neuropathy, cardiovascular diseases, and hepatopathy[4].

The hyperglycemic state leads to complications, especially atherosclerotic cardiovascular disease, by affecting lipoproteins, vascular matrix components, and cellular metabolism. This results in the excessive formation of free radicals due to glucose autoxidation and non-enzymatic protein glycation[5]. Over time, it damages cellular organelles and enzymes, leading to increased lipid peroxidation[6]. A complete understanding of the precise mechanisms underlying oxidative stress in diabetes requires further exploration, which may involve multiple pathways. In diabetes, antioxidant defenses are compromised, and free radical and reactive oxygen species (ROS) levels are elevated, suggesting the potential antioxidant supplements[7]. Various ROS, originating mainly from mitochondrial electron transport chains, cause damage to vascular endothelial cells, disrupt junctions, and increase permeability, which in turn accelerates microvascular disease progression[8].

Despite advances in DM research, insights into its etiology, effects, and progression, and developments in insulin and its analogs, optimal glycemic control remains challenging. For patients with type 2 DM (T2DM), lifestyle modifications like diet and exercise are important, yet they are challenging to follow consistently[9]. There are currently two existing primary treatment protocols for T2DM: Injections of insulin or insulin-like peptides and oral administration of hypoglycemic agents. Although these therapies are essential for the treatment of T2DM, they also bear adverse effects[10]. For instance, several prescribed medications, such as metformin (MET), sulfonylureas, GLP-1 receptor agonists, and SGLT2 inhibitors, are involved with gastrointestinal issues, risk of hypoglycemia, weight gain, and increased susceptibility to infection[11]. Aside from this, these medications can become less effective over time as the disease progresses, necessitating the need for dose adjustments or combination therapy, which complicates treatment regimens and, in turn, leads to poor adherence. Additionally, MET has been a standard treatment for T2DM due to its remarkable ability to lower plasma glucose levels[12]. Despite this, MET suffers from the lowest compliance of all oral antidiabetic medications due to minor to severe adverse effects that impair patient compliance. While MET controls diabetes and provides hepatoprotection, patients are still experiencing liver-related complications, necessitating a more effective therapeutic option[13].

Taurine (TAU), a semi-essential amino acid relatively abundant in the brain, the heart, and muscles, is active in several cellular processes and has anti-inflammatory and antioxidant properties[14,15]. Regarding health benefits, TAU controls glucose and lipid metabolism, promotes energy metabolism, and reduces inflammation while acting as an antioxidant by acting as a ROS scavenger[16]. TAU depletion causes numerous pathological changes, including severe cardiomyopathy, renal and pancreatic ailments, as well as loss of retinal photoreceptors. The use of TAU supplements has shown promise in preventing complications of diabetes, including endothelial dysfunction, nephropathy, retinopathy, and cardiomyopathy[17].

Earlier studies have demonstrated TAU's critical role in endocrine pancreas development and insulin production and secretion. A diet containing TAU was found to increase pancreatic islet mass while simultaneously reducing beta cell apoptosis, suggesting it protects beta cells from damage[18]. TAU also mitigates the effects of cytokine-induced apoptosis in pancreatic beta cells, which suggests it could be beneficial to the anti-inflammatory processes associated with autoimmune diabetes[19]. Considering TAU's potential for preventing or alleviating diabetes, particularly in early life stages, further research into its role in improving pancreatic function and mitigating diabetic complications is necessary. Taking into account the negative aspects, numerous studies have been conducted on the effects of high TAU intake but have not reported serious adverse effects[20,21]. There could be potential unwanted effects of TAU on the nervous system, cardiovascular system, and muscle emerging from its activity in regulating calcium release[22]. Despite that, TAU appears to be safe for humans at doses as high as 10 g/day for six months[23]. However, as with any dietary supplement, TAU should be consumed moderately. When TAU is overconsumed, side effects may arise as nausea, vomiting, diarrhea, dizziness, tremors, and headache[24]. Furthermore, caution must be exercised due to the potential interactions between TAU and cardiovascular and nervous system drugs, which may exacerbate the effects of such treatments and increase the risk of hypotension[25], altered heart rhythms, or neurological concerns[26].

In addition, cholecalciferol (CHO), the major form of vitamin D in humans, plays a significant role in bone health by increasing intestinal absorption of calcium, magnesium, and phosphate[27]. Aside from promoting bone health, research suggests that CHO also possesses anti-inflammatory and antioxidant properties[28]. This antioxidant role is relevant in insulin resistance and diabetes, where oxidative stress has been linked to their related complications[29]. Supplementation with CHO is unlikely to cause severe adverse reactions, even at high doses[30]. The excessive intake of this nutrient, on the other hand, may lead to hypercalcemia, which may cause calcium deposits in soft tissues or lead to kidney stones and cardiovascular complications[31,32]. Moreover, hypercalcemia can be exacerbated by CHO in individuals with high calcium levels, as well as hypertensive patients on thiazide diuretics[33]. In this regard, blood calcium levels are advised to be monitored to prevent CHO toxicity.

The current study aimed to determine if CHO and TAU supplements could enhance MET’s effectiveness in treating diabetes while minimizing the associated pathological risks, including hepatitis, dyslipidemia, and oxidative stress tissue damage. If so, it may be possible to anticipate improvements in patient compliance with MET.

T2DM exhibits a progressive nature with evolving pathological implications over time. Initial monotherapy becomes less effective, thereby requiring combination therapy for efficient management[51]. While several studies have introduced the beneficial effects of dietary supplements (TAU, CHO)[18,29], our study specifically aimed to explore their synergistic effects in enhancing the effectiveness of MET for T2DM treatment.

To experimentally validate the induction of T2DM, the group treated with STZ demonstrated a notable and significant rise in the mean random blood glucose levels throughout the six-week experiment. This increase was observed in comparison to the control non-diabetic group. Further evidence supporting these findings comes from histological examination, through which T2DM was evident by the cytotoxic effects of STZ on pancreatic β-cells.

Nevertheless, the proposed treatment groups exhibited a significant reduction in mean random blood glucose compared to the STZ and MET-treated groups throughout the experimental period. As seen in Figure 9, both su-pplements exerted an effect alone in counteracting the STZ impact on the glycemic profile. The effect of CHO on blood glucose was prominent and was in alignment with a previous report by Santos and Vianna, who reported the administration of 12.5 μg/kg⁻¹ of CHO to Wistar rats led to a reduction of 40% in blood glucose[52]. Also, Yousefi Rad et al[53] have reported the beneficial effect of CHO on the control of blood glucose levels as well as insulin resistance in T2DM patients. This could be attributed to the stimulating effect of CHO on insulin receptors in beta cells, leading to insulin gene expression, as well as its role in promoting intestinal transport of glucose molecules[54]. Aside from this, calcitriol, the active form of CHO, participates in calcium absorption, an essential step in insulin release from beta cells[55].

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Figure 9 Analysis of immunohistochemical results. A: The relative islet staining intensity of stained islets; B: The number and area of the stained islets. The data, presented as means ± SE (n = 6), indicate significant differences at P < 0.05. STZ: Streptozotocin; MET: Metformin; CHO: Cholecalciferol; ^aSignificance compared to the control group. ^bSignificance compared to the Streptozotocin diabetic group. ^cSignificance compared to the metformin-treated group.

Additionally, TAU also influenced blood glucose levels when combined with MET. This effect was also evident in earlier preclinical studies and clinical trials. For instance, in a trial by Maleki et al[14], TAU demonstrated a significant reduction in fasting blood glucose, insulin, and HOMA-IR. The cause of this may be linked to the reported effect it has on the liver, in which it reduces the phosphorylation of the insulin receptor β subunit (IRβ) and hepatic Akt, thereby reducing glucose production as well as glucagon activity[56,57]. Also, TAU was reported to improve beta-pancreatic cell function and insulin clearance while enhancing AMPK activity in muscles that help maintain glucose hemostasis[18,58]. Also, the immunohistochemical localization of insulin antibodies supported the microscopic H and E histopathological results of pancreas tissues (Figure 8). TAU treatment prominently restored insulin staining and islet size, with regular outlining comparable to the control. These findings were in line with those of Arany et al[18], who reported the increased pancreatic islet mass in TAU-supplemented rats, which could explain the augmented stain intensity for rats co-supplemented with CHO and TAU. Eventually, TAU and CHO held great potential on the glycemic profile, and as shown in Figures 1 and 9, the synergy resulted in a significant reduction in blood glucose levels, serum insulin, HOMA-IR, and HbA1C comparable to those of non-diabetic rats.

Considering the lipid profile evaluation, it can be concluded that MET intake contributes to ameliorating STZ effects on HDL levels (Figure 2). These effects stem from control over the hyperglycemia-induced ROS generation. As a result, the formation of advanced glycation end-products is reduced, and consequently, oxidative stress and inflammation[59]. The CHO supplement, on the other hand, was the primary supplement that affected the lipid profile in a positive way, specifically through reducing LDL-C and TG. However, CHO is thought to directly reduce lecithin-cholesterol acyltransferase activity, directly influencing TGs, total cholesterol, and LDL cholesterol. Also, as it enhances calcium absorption, CHO can be indirectly implicated by impacting cholesterol absorption and hepatic bile acid conversion by decreasing fat absorption[60].

MET treatment appears to be the contributor to the reversal of STZ effects on HDL levels (Figure 2). Treatment with CHO, TAU, MET, and their combination resulted in significant improvements in levels of lipid markers, supporting Malek et al[61] and Asemi et al[62], who revealed that CHO, TAU, and MET together have therapeutic roles in modification of lipid abnormalities via lipid peroxidation prevention and reduction of the atherosclerotic inflammatory process, which in turn may help to prevent the onset of plaque and all subsequent disorders.

Elevated levels of ALT and AST in the STZ group are related to insulin resistance and fatty liver, while elevated LDH levels interfere with glucose metabolism and insulin production and play a role in increasing cytosolic free ATP and Ca^{2 +} levels within islet beta cells[63]. These effects were exacerbated by MET treatment, which is in line with previous hepatotoxicity reports[64]. It is clear from Figure 3 that the hepatocellular necrosis induced by STZ was reversed by CHO, as evidenced by the decline in AST, ALT, and LDH levels. The findings were in accordance with those reported by Lorvand Amiri et al[65], who found that vitamin D led to a significant reduction in liver aminotransferase levels. Additionally, histopathological examination of liver tissues in the STZ diabetic group revealed lobular inflammation, sinusoidal dilatation, and fibrous portal expansion. The combination therapy (CHO + TAU + MET) demonstrated preserved hepatocyte arrangement with no inflammation or fibrosis, surpassing the effects of MET alone.

A significant part of the protective action of CHO is to enhance the activities of key antioxidant enzymes, SOD, and CAT (Figure 4). Also, CHO exerted a significant negative effect on MDA levels, which were in alignment with a meta-analysis of clinical data[66]. This can be explained by the fact that the CHO supplement interacts with Vitamin D receptors, which in turn activates the Vitamin D Response Elements in the nucleus, leading to the transcription of antioxidant genes[66]. Moreover, CHO also stimulates antioxidant pathways such as nuclear factor erythroid-2-related factor 2 (Nrf2)/Keap1[67] and ROS-scavenging enzymes (Glucose-6-phosphate dehydrogenase)[68] while reducing ROS-generating enzymes (NADPH oxidase)[69], thus indirectly reducing the risk of oxidative damage.

Despite the significance of preclinical studies, animal models offer limited predictive power[70]. This is attributed to the biological and physiological differences due to the discrepancy in how disease manifests and how treatment responds between rodents and humans. In contrast to animal models, the high genetic variation in drug metabolism and immune activity between individuals potentially affects estimates of treatment efficacy and toxicity[71]. Even so, it may still be necessary to conduct animal studies of TAU interaction prior to clinical studies to detect any potential pharmacokinetic interactions between the suggested combination therapy. Also, more parameters can be included in future glucose-insulin dynamic studies to directly assess insulin sensitivity (hyperinsulinemic-euglycemic clamp) or indirectly (glucose tolerance tests). It will, therefore, be crucial to conduct dose-finding trials to identify effective and well-tolerated dosages of CHO and TAU to minimize MET's adverse effects and optimize therapeutic outcomes in patients with diabetes. Furthermore, conducting clinical trials on diabetic patients can offer insight into the long-term benefits, safety profiles, and complications associated with the proposed co-therapy.

The study highlighted hyperglycemia, oxidative stress, and insulin resistance as contributors to pathological tissue changes and abnormal biochemical parameters in diabetic rats. MET, while possessing mild antioxidant and anti-inflammatory properties, was deemed insufficient to prevent diabetic complications. The combined therapy involving MET, CHO, and TAU proved more effective in mitigating apoptosis and ameliorating the biochemical, histological, and immunohistochemical changes associated with diabetes. This supplementation approach effectively restored these parameters to normal levels. The current study findings demonstrate the importance of a synergistic therapeutic approach to alleviating diabetes-related complications through treating oxidative stress-induced tissue damage and achieving metabolic balance.

The authors acknowledge their research institutes for the technical support.