Prediabetes is when an individual has above-average blood sugar levels but does not yet match the diagnostic criteria for diabetes. Prediabetes is not a disease in and of itself but rather an indicator of future health problems, including diabetes and cardiovascular disease (1). The World Health Organization instead called it “Intermediate Hyperglycemia.” At the same time, the American Diabetes Association referred to it as a “High-Risk State of Developing Diabetes” (2). Prediabetes is characterized by hyperinsulinemia which leads to insulin resistance. Eventually, chronic hyperinsulinemia will lead to beta cell dysfunction and favor the development of type 2 diabetes mellitus (2–4). Several strategies have been suggested for preventing diabetes in the prediabetic population. However, many few have been proven effective. No pharmacological intervention has been used explicitly to treat insulin resistance (4–6).

Recently, studies have linked vitamin D deficiency to diabetes pathogenesis (7–9). Several studies have shown that vitamin D deficiency may have a role in insulin resistance, yet the findings are still controversial. In some in-vivo and clinical studies, the lack of vitamin D levels has been associated with increased insulin resistance and impaired insulin production (7, 10–12). Vitamin D is suggested to promote insulin sensitivity and optimizes the activity of beta cells through several pathways. Vitamin D directly affects pancreatic beta cells by activating beta-cell calcium-dependent endopeptidases to release insulin (10, 12, 13).

In addition to vitamin D deficiency, a high-fat diet and sedentary lifestyle may produce adipocyte hypertrophy and hyperplasia, which aggravates hyperglycemia and hyperinsulinemia (14, 15). In a previous study in a mouse model with a high-fat diet, inflammatory insulin signaling markers were dysregulated. Chronic high fat intake will then be attributed to the development of insulin resistance (16).

Multiple studies have consistently shown reduced serum 25OHD concentrations in diabetic individuals. An analysis of the collective findings coming from multiple studies conducted to investigate the effectiveness of vitamin D supplementation in preventing type 2 diabetes revealed that compared to placebo, vitamin D supplementation reduced the risk of developing type 2 diabetes in people with prediabetes (17, 18). Despite the encouraging benefits of vitamin D, vitamin D supplementation in prediabetic and diabetic individuals has shown inconclusive outcomes in several studies (10, 19–21). Vitamin D supplementation’s mechanism of inhibiting insulin resistance in prediabetes has yet to be well understood. Thus, in the present study, we aimed to investigate the effect and mechanism of vitamin D supplementation in prediabetic rats on a high-fat, high-glucose diet.

In the present study, we examined the modulating effects of vitamin D supplementation on the molecular mechanism of insulin resistance in prediabetic rats. Our study showed that vitamin D3 administration improved glucose control and ameliorated insulin resistance in prediabetic rats. The modulation of the insulin signaling pathway and improved balance between proinflammatory, and anti-inflammatory cytokines contribute to reducing insulin resistance in prediabetic rats.

The prediabetic conditions in our study were induced by the chronic administration of a high-fat and high-glucose diet (HFD-G) in combination with a small dose of streptozotocin (30 mg/kg BW). The present model of prediabetes demonstrates impaired glucose homeostasis, as indicated by variations in glucose tolerance such as fasting blood glucose, OGTT, and insulin levels. No specific criteria for prediabetic conditions are currently available for rodents. However, the criteria used in the study paradigm are consistent with other studies (25, 26). HFD-G is widely used in the animal model of diabetes induction by inducing hyperglycemia and insulin resistance. Studies have shown that prolonged administration of a high-fat diet may inhibit the insulin receptor signaling pathway and trigger insulin resistance (27, 28). A high-fat diet will increase the formation of diacylglycerol (DAG) in the liver and muscles over time. Increased DAG in the liver activates protein kinase C (PKC), which phosphorylates serine residues on IRS1, blocking the phosphorylation of tyrosine residues. Insulin resistance develops because of a reduction in insulin-PI3K-Akt signaling. Reduced insulin-PI3K-Akt in muscle promotes reduced glucose absorption and a decrease in GLUT-4, both of which contribute to insulin resistance (28). In adipocytes, studies revealed a distinct connection between decreased insulin receptor expression and impairment of insulin signaling in adipocytes. One of the most critical roles is the activation of a miRNA (miR-128) in adipocytes, which causes mRNA instability of the insulin receptor (29, 30).

Studies showed impaired insulin signaling and secretion are linked to reduced 25-hydroxy-vitamin D3 concentrations in the blood in prediabetic individuals (7, 31). The current study showed that vitamin D3 supplementation might prevent the decrease of 25-hydroxy-vitamin D3 levels in the prediabetic group. Previous studies showed that a high-fat diet might increase vitamin D3 storage in the liver and adipose tissue, contributing to low serum 25(OH) D3 levels (32).

Based on findings linked to the role of vitamin D in insulin generation and glucose homeostasis, studies have demonstrated a causal relationship between vitamin D deficiency and diabetes mellitus (8, 9, 20, 21, 33, 34). Compared to the prediabetes group, vitamin D supplementation at 1000 IU/kg BW successfully lowered fasting blood glucose, plasma insulin, and insulin resistance, as shown by HOMA IR. In diabetic individuals, vitamin D levels were negatively associated with insulin resistance (HOMA-IR). Vitamin D deficiency is hypothesized to cause insulin resistance via several pathways, including increased proinflammatory cytokines, reduced insulin production by pancreatic beta cells, and decreased glucose absorption in peripheral tissues (20). Another study of diabetic rats given vitamin D supplementation (1000 IU and 2000 IU) for 45 days showed better glucose control and insulin resistance (35). Vitamin D indirectly impacts insulin secretion and interacts via β-cells to modulate extracellular calcium or calcium flow (21). Vitamin D may also activate calcium-dependent endopeptidase, which aids in the conversion of proinsulin to insulin (36).

Our findings were supported by histological examination using hematoxylin and eosin staining, which revealed a decrease in Langerhans islets as well as fatty pancreatic acinar cell atrophy while increasing the number of fatty acinar cells. For the histopathology analysis, we utilized the same strategy as earlier research that effectively reported pancreatic histology (37). The morphological differences between the negative and positive control groups, as well as the treatment group, were clearly visible using hematoxylin and eosin (H&E) staining. However, it will be beneficial for future research to add scan and image-based phenotypic analysis utilizing the cell painting approach.

Regarding to the vitamin D doses utilized in the study, were well below the hazardous quantity. Vitamin D toxicity is highly uncommon. The most common way to get vitamin D intoxication is by continuing to take very high dosages of vitamin D over an extended period. More than 150 µg/L may cause vitamin D intoxication and hypercalcemia in humans (38). In rats, the toxicity of vitamin D3 has been documented at extremely high dosages. Ali et al. reported vitamin D toxicity at a dose of 6,750 IU/rat/day, or equivalent to 27,000 IU/kg BW/day, while Chavhan et al. demonstrated toxicity at 2 mg/kg BW/day, or equal to 80,000 IU/kg BW/day (39, 40). In our study, the highest dose used was 1,000 IU/kg BW/daily, lower than those demonstrated in the studies of Ali et al. (39) and Chavhan et al. (40). Additionally, the highest plasma concentrations of vitamin D3 after treatment with vitamin D3 1,000 IU/kg BW were 31 µg/L.

In a rat model of type 2 diabetes mellitus with no vitamin D deficiency, vitamin D therapy was shown to reduce blood glucose levels by 40% (41). In addition to modulating calcium regulation in pancreatic beta cells, vitamin D3 directly impacts pancreatic beta cells via the binding to the vitamin D receptor (VDR) in the active form 1.25-hydroxy-vitamin D3. After binding to 1.25-hydroxyvitamin D3, VDR will interact with the vitamin D response element (VDRE), consequently leading to the insulin gene’s induced activation (11).

Our findings were consistent with those of Wahba et al., who discovered that vitamin D might improve oral glucose tolerance in prediabetic rats (42). The present study also showed that in both dosages studied, and vitamin D reduced glycated albumin levels. The link between vitamin D supplementation and glycated albumin levels in prediabetics has received little attention. Glycated albumin is a novel biomarker for monitoring short-term glycemic control due to its shorter half-life (2 to 3 weeks) compared to HbA1c (43).

Improvement of insulin resistance by vitamin D supplementation can be partially explained by reducing IRS1 phosphorylation at Serine 307. Insulin signaling is mediated by insulin receptor substrates 1 and 2 (IRS1 and IRS2), which regulate glucose homeostasis and energy metabolism. To date, the increased phosphorylation of IRS1 at serine 307 was considered the best available mechanism to understand the desensitization of insulin signaling (44). Phosphorylation of Ser307 in IRS1 limits insulin action by blocking connections with the insulin receptor (45). Our result was in line with a previous study in a diabetic rat model, which demonstrated that vitamin D supplementation for eight weeks and a high-fat diet reduced Ser307 phosphorylation of IRS1. Increased degradation of IRS1 causes impaired GLUT4 mobilization and decreased glucose uptake in the diabetic rat (46).

Multiple inflammatory responses are closely connected and play critical roles in developing insulin resistance and type 2 diabetes (47). Insulin resistance associated with obesity is characterized by chronic low-grade inflammation. There were increased proinflammatory cytokines and other bioactive compounds such as TNF-α, IL-1β, IL-6, or monocyte attractant protein-1 (MCP-1) (48). VDR, the receptor for 1.25-hydroxy-vitamin D3, is present in more than 38 different tissues and is known to regulate essential genes involved in bone metabolism, oxidative damage, chronic illnesses, and inflammation. Macrophages and dendritic cells express VDR constitutively, indicating that vitamin D likely plays a significant role in regulating the inflammatory response (49). Our study showed that vitamin D3 supplementation did not alter individual concentrations of proinflammatory cytokines (TNF-α, IL-6) and anti-inflammatory cytokines IL-10. Vitamin D3 may restore the balance between proinflammatory and anti-inflammatory cytokines, as shown by increasing the IL-10 levels and thus reducing the ratio of IL-6/IL-10.

The interaction between PPARγ and NF-κB is a signaling pathway that connects insulin resistance, metabolic syndrome, and inflammation (50, 51). PPARγ is a ligand-activated transcription factor that plays a crucial role in glucose homeostasis and adipocyte formation (52, 53). Several investigations have demonstrated that PPARγ may decrease inflammation by reducing NF-κB transcriptional activity by competing with p65 (51). The transcription factor NF-κB is an essential regulator of inflammation. It is necessary to produce proinflammatory cytokines such as IL-1β and IL-6 (54, 55). According to Ke et al., the inactivation of NF-κB p65 may modulate hepatic insulin sensitivity by elevating cAMP through PDE3B gene transcription suppression (55). Our data showed that 1000 IU/kg BW vitamin D3 supplementation enhanced PPARγ expression while decreasing NF-κB p65 phosphorylation at Ser536. NF-κB p65 phosphorylation at Ser536 is essential in inhibiting NF-κB transcription responses in toll-like receptor-activated macrophages, contributing to inflammation resolution (56).

Our study indicated that vitamin D supplementation improves insulin resistance in prediabetic rats. Additionally, the decreased phosphorylation of IRS1 increased expression of PPARγ and reduced phosphorylation of NF-κB could be attributed to the attenuation of insulin resistance of vitamin D3. Therefore, vitamin D supplementation in a prediabetic state may prevent the progression of insulin resistance to diabetes.

The original contributions presented in the study are included in the article/supplementary materials. Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the ethics committee of The Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia.

DK, ML, TT, VS, and EP: study design; DK, ML, and VS: data analysis; DK, CN, and ML: funding; HW: acquisition. All authors contributed to the article and approved the submitted version.