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Article

Effect of Oral Vitamin D Overdose in Male and Female Wistar Rats with Induced MASLD

by
Silvia Muller de Moura Sarmento
1,
Gênifer Erminda Schreiner
2,
Laura Smolski dos Santos
2,
Camila Berny Pereira
2,
Elizandra Gomes Schmitt
2,
Vinicius Tejada Nunes
2,
Rafael Tamborena Malheiros
1,
Clóvis Klock
3,
Chaline Casanova Petry
3,
Itamar Luís Gonçalves
4 and
Vanusa Manfredini
1,*
1
Program in Multicentric Graduate Studies in Physiological Sciences, Federal University of Pampa, Uruguaiana 97501-970, Brazil
2
Graduate Program in Biochemistry, Federal University of Pampa, BR-472, Km 585, Uruguaiana 97501-970, Brazil
3
Medicina Diagnóstica, Rua Pedro Álvares Cabral, 21, Erechim 99700-296, Brazil
4
Department of Health Sciences, Regional University of Alto Uruguai and Missions, Erechim Campus, Avenida Sete de Setembro, 1621, Erechim 99709-910, Brazil
*
Author to whom correspondence should be addressed.
Livers 2025, 5(4), 52; https://doi.org/10.3390/livers5040052 (registering DOI)
Submission received: 11 August 2025 / Revised: 22 September 2025 / Accepted: 11 October 2025 / Published: 23 October 2025
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Abstract

Background/Objectives: Vitamin D is recognized as a key modulator of metabolic diseases, including metabolic-dysfunction-associated steatotic liver disease (MASLD), in which its deficiency contributes to both disease onset and progression. Despite the widespread and often prolonged use of vitamin D supplementation, optimal serum levels in individuals with MASLD remain unclear and warrant further investigation. Methods: In this study, hepatic steatosis was induced in male and female Wistar rats over a 45-day period. The animals were then divided into five groups (control, 2500, 7000, 14,000, and 21,000 IU/kg/week of cholecalciferol). After four weeks of treatment, the animals were euthanized, and blood samples were collected for biochemical, hormonal, inflammatory, oxidative stress analyses and liver architecture evaluation. Results: High-dose vitamin D supplementation in rats with MASLD induced dose-dependent metabolic, inflammatory, and oxidative changes, with some sex-specific differences. Urea and alanine aminotransferase levels increased at higher doses in both sexes, suggesting potential nephrotoxic and hepatotoxic effects, while creatinine and aspartate aminotransferase remained stable. Adiponectin levels decreased consistently, and leptin levels rose across all doses, indicating a shift toward a pro-adipogenic profile. Pro-inflammatory molecules (IL-1β, IL-6, IL-8, TNF, C-reactive protein) increased progressively with dose, while IL-10 followed a U-shaped curve. Oxidative stress markers showed elevated protein carbonylation only at the highest dose, a slight reduction in TBARS, and a peak in total antioxidant status at 7000 IU/kg/week. Conclusions: High-dose vitamin D triggers antioxidant responses but drives harmful inflammatory and metabolic shifts in MASLD.

1. Introduction

Vitamin D was initially classified as a vitamin because it was believed to be obtainable only through exogenous sources, such as natural foods (e.g., fish, organ meats, mushrooms) or fortified foods (e.g., starchy products) and supplements [1]. However, it is now established as a fat-soluble steroid hormone, which can be synthesized endogenously through cutaneous production when the epidermis is exposed to ultraviolet B (UV-B) radiation. This process depends on multiple factors, including the intensity of UV-B radiation, duration of exposure, time of day, geographical latitude, season, and the degree of air pollution [2,3].
In recent years, debates have emerged regarding the cutoff values for defining vitamin D sufficiency and deficiency, as well as disagreements concerning the optimal serum concentration of 25-hydroxyvitamin D [25(OH)D]. These controversies extend to discussions about the appropriate 25(OH)D levels in various pathological conditions, including autoimmune diseases, cardiovascular disorders, and metabolic syndromes. They also involve consideration of tissues involved in hydroxylation processes, particularly the liver and kidneys [4].
Among metabolic disorders influenced by vitamin D status, metabolic-dysfunction-associated steatotic liver disease (MASLD) is of particular relevance. Deficiency of this hormone has been associated with both the development and progression of MASLD [5,6,7]. Furthermore, recently, vitamin D supplementation in MASLD patients reduced markers of liver fibrosis and improved metabolic health [8]. Vitamin D plays crucial roles in regulating oxidative processes, modulating the production of pro-inflammatory cytokines, and controlling hepatocyte apoptosis [9].
Given these associations, supplementation with vitamin D represents a complex clinical decision, and serum 25(OH)D concentrations should be monitored by qualified healthcare professionals. With the increasing availability of over-the-counter supplements, the incidence of hypervitaminosis D has risen, raising concerns regarding the potential consequences of bioaccumulation and its short- and long-term effects on health [10,11].
Recently, a comprehensive study reported the role of vitamin D overdose (doses of 2500 to 21,000 IU/kg/week) in rats; however, the effects of these specific doses in MASLD models are not yet available in the literature [12]. In this context, the present study aimed to evaluate the dose–response effects of increasing vitamin D supplementation on biochemical, inflammatory, and antioxidant markers, as well as liver histology, in male and female Wistar rats with MASLD.

2. Methodology

2.1. Ethical Aspects

All procedures performed in this study were approved by the Ethics Committee on the Use of Animals of the Federal University of Pampa (CEUA-Unipampa), under protocol number 016/2020.

2.2. Chemicals

All chemicals used were of analytical grade. Unless otherwise stated, reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The vitamin D suspension was prepared in a certified compounding pharmacy from Purifarma® (code: 081300.000005, batch: B-151-M201124), with a concentration of 40,576 IU/g (anhydrous basis). The compounded solutions contained 500 IU, 1000 IU, 2000 IU, or 3000 IU of vitamin D (cholecalciferol) in each 300 µL aliquot.

2.3. Animals

Sixty Wistar rats (30 males and 30 females), aged 45 days, were used. The animals were obtained from the Central Animal Facility of the Federal University of Pelotas (UFPel), Rio Grande do Sul, Brazil. Upon arrival, they were acclimated for 15 days in the vivarium (BIOPAMPA) of the Federal University of Pampa (UNIPAMPA), under controlled environmental conditions (12-h light/dark cycle, controlled temperature and humidity) with free access to commercial chow and water. Both sexes were included, and all procedures were performed equivalently and concomitantly.

2.4. Experimental Design

After MASLD induction, 30 male and 30 female rats were randomly assigned according to sex and subsequently subdivided into five experimental groups (n = 6 per group) as follows: (1) MASLD + 0.5 mL saline; (2) MASLD + 2500 IU/kg/week vitamin D; (3) MASLD + 7000 IU/kg/week; (4) MASLD + 14,000 IU/kg/week; and (5) MASLD + 21,000 IU/kg/week. The supplement was administered once weekly for 4 weeks via gavage, always at the same time of day. The administered dose was calculated according to each animal’s body weight on the day of administration to minimize handling. The doses were cumulative over the seven-day interval (2500, 7000, 14,000, and 21,000 IU/kg).
At the end of the experimental period, following a 12 h fast, animals were anesthetized intraperitoneally with ketamine (80 mg/kg) and xylazine (10 mg/kg). Cardiac puncture was performed to collect whole blood, and liver samples were obtained for subsequent histological analyses. Blood was collected in tubes containing EDTA for plasma separation and in tubes with separator gel for serum separation. Samples were immediately centrifuged at 2500 rpm (plasma) or 3000 rpm (serum) for 10 min. The obtained plasma and serum were stored for subsequent biochemical, inflammatory, hormonal, and oxidative stress analyses.

2.5. Diet for Induction of Hepatic Steatosis

A high-fat and high-sucrose diet [13] was provided for 45 days to induce hepatic steatosis. The high-fat diet consisted of commercial rodent chow supplemented with 7.5% thermolyzed lard and 2.5% commercial corn oil (per 100 g of feed), adapted from another investigation [14]. Additionally, 45% sucrose was added to the drinking water. This diet model (high-fat diet combined with sucrose) has been previously validated for the induction of MASLD [15].

2.6. Analysis of Laboratory Parameters

Biochemical parameters, including glycemic and lipid profiles, as well as liver and pancreatic markers (ALT, AST, alkaline phosphatase) and kidney markers (creatinine and urea), were determined in serum using Labtest® kits on the ChemWell-T analyzer. Bone metabolism markers (calcium and phosphorus) were measured using LabMax®1000 equipment (Guangzhou Max Laboratory Equipment Co., Ltd., Guangzhou, China) and commercial kits [16]. Oxidative stress was evaluated by measuring total antioxidant status (TAS) in serum according to the manufacturer’s instructions (Randox Laboratory®). Lipid peroxidation was assessed via thiobarbituric acid reactive substances (TBARS) assay [17], and protein oxidation was determined by quantifying protein carbonyls [18]. Vitamin D, hormones (leptin, adiponectin), cytokines (IL-1β, IL-6, IL-8, IL-10, TNF), and ultrasensitive C-reactive protein (CRP) were measured by ELISA using commercial kits from Abbott® (Architect, Abbott Laboratories, Abbott Park, IL, USA) and Thermo Fisher Scientific® (Waltham, MA, USA) [16].

2.7. Histological Analysis

Liver tissue was processed for histology according to classical methodologies [19]. Sections were examined and photographed under an optical microscope (Leica® DM500, Wetzlar, Germany) at 400× magnification. Hepatic steatosis was determined by the presence of lipid droplets in hepatocyte cytoplasm, and the percentage of affected hepatocytes was quantified.

2.8. Statistical Analysis

Data are presented as mean ± standard deviation (SD). Normality was assessed using the Shapiro–Wilk test prior to applying one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons or the Kruskal–Wallis test when appropriate. Principal component analysis (PCA) was performed to identify clustering of the groups according to the profile of the investigated variables. All variables were correlated with vitamin D levels using Pearson’s or Spearman’s correlation coefficients, as appropriate. Differences were considered statistically significant at p < 0.05. Statistical analyses were conducted using GraphPad Prism 9.2 software.

3. Results

3.1. Biochemical Parameters

Vitamin D supplementation had no significant effect on glucose levels in female rats with MASLD. In male rats, a significant reduction in glucose was observed only at the two highest doses (p = 0.0002 and p < 0.0001). Total cholesterol levels were significantly affected only at the highest dose in female rats (p = 0.0003), whereas in male rats, marked changes were observed at all doses tested (p < 0.0001). A consistent decrease in HDL-cholesterol levels was found in females at 21,000 IU/kg/week (p = 0.0080). In males, significant reductions in HDL-cholesterol compared to the control group were observed at 7000, 14,000, and 21,000 IU/kg/week (p < 0.0001). Vitamin D supplementation at higher doses also led to a deleterious increase in LDL-cholesterol levels in both female and male rats at 7000, 14,000, and 21,000 IU/kg/week (p < 0.0001). Triglyceride levels increased significantly only in female rats at 21,000 IU/kg/week (p = 0.0067), with no marked effects observed in males. The effects of vitamin D supplementation on glucose and lipid profiles in rats with MASLD induction are illustrated in Figure 1a–e.
Regarding renal function, supplementation with the highest dose of vitamin D resulted in a significant increase in urea levels in female rats compared to the control group (p = 0.0013). In male rats, both the 14,000 and 21,000 IU/kg/week doses led to a significant elevation in urea levels (p < 0.0001), as shown in Figure 1f. No statistically significant effect was observed on creatinine levels in either female or male rats (Figure 1g). Vitamin D supplementation at doses of 7000, 14,000, and 21,000 IU/kg/week led to a significant increase in ALT levels in both sexes compared to the control group (p < 0.0001). In contrast, AST levels were less sensitive to vitamin D overdose: no significant changes were observed in females, while in males, a significant increase occurred only at the 21,000 IU/kg/week dose (p < 0.0001), as illustrated in Figure 1h,i. Alkaline phosphatase, phosphorus, and calcium levels remained unaltered across all experimental conditions (Figure 1h–j).
Vitamin D supplementation at all tested doses induced a metabolically unfavorable state in both female and male rats with MASLD, characterized by alterations in adipokine profiles. In females, leptin levels were significantly elevated at 7000 and 14,000 IU/kg/week (p < 0.0001), while in males, all three doses (7000, 14,000, and 21,000 IU/kg/week) produced a similar increase in leptin levels (p < 0.0001), as shown in Figure 1m. With respect to adiponectin, all doses resulted in a significant reduction in both sexes (p < 0.0001; Figure 1n). Finally, confirming the effectiveness of vitamin D administration, all doses led to a significant increase in serum vitamin D levels in both female and male rats (p < 0.0001), as shown in Figure 1o.

3.2. Inflammatory Parameters

Regarding the inflammatory profile, vitamin D supplementation resulted in a dose-dependent increase in proinflammatory markers in both female and male rats (Figure 2a–c,e,f). In females, a significant elevation was observed for IL-1β, IL-6, IL-8, TNF, and C-reactive protein (CRP) at all doses (p < 0.0001), as shown in Figure 2a1,b1,c1,e1,f1. Interestingly, IL-10 exhibited a U-shaped response in females, with a significant decrease at intermediate doses followed by a marked increase at the highest dose (Figure 2d1). In male rats, a similar proinflammatory pattern was observed. IL-1β, IL-6, IL-8, TNF, and CRP levels increased significantly across all doses (p < 0.0001; Figure 2a2,b2,c2,e2,f2). IL-10 levels in males also exhibited a response pattern very similar to that observed in females, characterized by an initial decrease followed by a significant increase at the highest dose (Figure 2d2). These findings indicate that high doses of vitamin D exacerbate systemic inflammation in both sexes.

3.3. Oxidative Stress Parameters

Protein carbonylation remained unchanged at doses of 2500, 7000, and 14,000 IU/kg/week in both female and male rats. A significant increase was observed only at the dose of 21,000 IU/kg/week when compared to the control group (p = 0.0255 for females; p = 0.0082 for males), as shown in Figure 3a. With respect to TBARS levels, a slight decrease was noted with increasing doses in both sexes (Figure 3b). For TAS levels, an inverted U-shaped dose–response profile was observed, with the peak value occurring at 7000 IU/kg/week, as represented in Figure 3c.

3.4. Principal Component Analysis

To better understand the overall effects of the experimental protocols on the 24 monitored serum parameters, a Principal Component Analysis (PCA) was conducted. Given that female and male rats exhibited similar dose–response profiles in most situations, the datasets were merged to avoid redundant description. Additionally, when the clustering was assessed based on sex, no consistent pattern of distribution was identified (data no shown). The first two principal components (PC1 and PC2) together accounted for 60.49% of the total variance in the dataset. When PC1 scores were plotted against PC2, a clear dose-dependent distribution of the experimental groups emerged in the two-dimensional PCA plot (Figure 4a). Most of the separation occurred along the PC1 axis: control animals clustered on the left side of the graph, while increasing doses of vitamin D shifted progressively to the right, culminating in a distinct separation between the control group and the group receiving the highest dose (21,000 IU/kg/week).
As shown in the vector plot (Figure 4b), and consistent with previous findings, control animals (blue dots, low PC1 scores) were characterized by higher levels of HDL-cholesterol and adiponectin. In contrast, rats treated with the highest vitamin D dose exhibited elevated levels of LDL-cholesterol, triglycerides, urea, alanine aminotransferase, and inflammatory markers (IL-1β, TNF, IL-6, IL-8, and CRP), which contributed to their high and positive PC1 values and placement on the right side of the plot (Figure 4a). The vector plot also revealed biologically meaningful correlations among variables. Notably, there was a negative correlation between adiponectin and leptin, and between HDL- and LDL-cholesterol, as indicated by vectors pointing in opposite directions. Conversely, the proinflammatory cytokines clustered closely, suggesting a strong positive correlation among them, as reflected by their aligned vectors with minimal angular divergence. These findings reinforce the utility of PCA in integrating multiple biochemical and inflammatory parameters, enabling the identification of consistent, dose-related changes in serum profiles.
Finally, we investigated the correlations between vitamin D levels and each analyte quantified in the plasma of the rats by constructing a series of scatter plots, as shown in Figure 4c. Vitamin D levels showed strong positive correlations with total cholesterol (p < 0.0001), HDL cholesterol (p = 0.0003), urea (p < 0.0001), AST (p < 0.0001), ALT (p < 0.0001), and all pro-inflammatory markers (IL-1β: p < 0.0001; IL-6: p < 0.0001; IL-8: p < 0.0001; TNF: p < 0.0001; CRP: p < 0.0001). No significant correlations were observed between vitamin D and creatinine (p = 0.2831), alkaline phosphatase (p = 0.0865), calcium (p = 0.4020), phosphorus (p = 0.3198), or IL-10 (p = 0.7517). We observed weaker correlations between vitamin D and glucose (p = 0.0003), LDL cholesterol (p = 0.0003), as well as oxidative stress markers (carbonyl, TBARS, and TAS).

3.5. Histological Analysis

The histopathological evaluation of liver sections from female rats (Figure 5), stained with hematoxylin and eosin (H&E), revealed the presence of grade 1 hepatic steatosis in the control group (Figure 5a), characterized by the accumulation of fat droplets within hepatocytes. In animals treated with 2500 IU/kg/week and 7000 IU/kg/week—Figure 5b,c), a reduction in lipid accumulation was observed, suggesting a protective effect of the treatment. However, in females receiving 14,000 and 21,000 IU/kg/week (Figure 5d,e), histological analysis suggested an increased degree of hepatic fibrosis, an alteration not observed in the livers of male rats. Similarly, liver sections from male rats demonstrated that the control group (Figure 5f) exhibited fat accumulation within hepatocytes, consistent with grade 1 steatosis. A reduction in the number of lipid droplets was seen in animals treated with 2500 IU/kg/week (Figure 5g) and 7000 IU/kg/week (Figure 5h), again suggesting a protective effect of vitamin D at these doses. In contrast, animals receiving higher doses (14,000 and 21,000 IU/kg/week, Figure 5i,j) showed a progressive increase in hepatic lipid accumulation, particularly pronounced in the 21,000 IU/kg/week group.

4. Discussion

The evolution of modern society has brought substantial changes to both the environment and human lifestyles, including the adoption of diets rich in ultra-processed foods, a predominantly indoor lifestyle, and reduced exposure to sunlight. These factors have contributed to a marked decline in vitamin D levels [10]. As a pleiotropic molecule involved in numerous metabolic and catabolic pathways, vitamin D plays a critical role in maintaining the body’s homeostasis [4].
In this experimental model, oral vitamin D was administered at doses of 2500 IU, 7000 IU, 14,000 IU, and 21,000 IU, resulting in serum 25(OH)D concentrations of 152.8 ng/mL, 175.6 ng/mL, 193.6 ng/mL, and 275.5 ng/mL, respectively, in male Wistar rats, with a baseline value of 52.2 ng/mL in the control group. Female rats presented serum 25(OH)D levels of 44.9 ng/mL in the control group, followed by 115.3 ng/mL (2500 IU), 117.6 ng/mL (7000 IU), 138.4 ng/mL (14,000 IU), and 184.3 ng/mL (21,000 IU). The literature provides different perspectives on the optimal serum vitamin D [25(OH)D] range, leading to considerable variation in recommendations for target serum concentrations and daily supplementation. The Institute of Medicine, for instance, recommends a daily intake of 400–1000 IU of vitamin D to maintain serum 25(OH)D levels not exceeding 150 ng/mL [4,20].
Among systemic metabolic disorders, MASLD is the most prevalent chronic liver condition worldwide, with its progression primarily associated with metabolic dysfunction and increased oxidative and inflammatory stress in hepatic tissue [21,22]. Evidence suggests a correlation between hypovitaminosis D and MASLD, and vitamin D supplementation has been proposed as a potential therapeutic strategy [23].
In a randomized, double-blind clinical trial involving individuals with type 2 diabetes mellitus and MASLD, oral supplementation with 2000 IU of vitamin D for 24 weeks did not improve hepatic steatosis or metabolic (AST, ALT, GGT) and cardiovascular parameters [24]. Another trial in overweight or obese individuals found no significant changes in serum liver enzyme levels following oral vitamin D supplementation [25]. In the present study, analysis of ALT and AST data (Figure 1) revealed an increase in both enzyme levels after vitamin D supplementation in males and females, with a more pronounced effect on ALT.
Data analysis revealed a progressive increase in total cholesterol, LDL cholesterol, and triglyceride levels, accompanied by a reduction in HDL cholesterol in both sexes (Figure 1). Asano et al. (2017) reported that vitamin D can directly inhibit the activity of sterol regulatory element-binding proteins, providing experimental evidence that the association between vitamin D status and the lipid profile may be attributed to the hormone’s regulatory role in lipid metabolism and synthesis [26].
Serum deficiency of 25(OH)D has been associated with low HDL levels and increased total cholesterol, LDL cholesterol, and triglycerides [27]. However, when examined in the context of cardiovascular diseases, there is no conclusive evidence supporting a direct link between low vitamin D status and elevated serum lipid levels [28,29]. Longitudinal data from a 13-year follow-up study indicate a correlation between higher vitamin D levels and reduced triglyceride concentrations [30].
The role of vitamin D in renal function is well established, particularly regarding metabolic dysfunctions that lead to both skeletal and non-skeletal alterations [31,32]. In a preclinical study, administration of an acute vitamin D dose (3000 IU/kg) in obese mice increased insulin sensitivity and reduced body mass but was also associated with significant kidney damage [33].
In vivo and in vitro studies indicate that vitamin D influences lipid metabolism, with potential effects on both the type and distribution of adipose tissue. Among the adipose tissue types, white adipose tissue is characterized by a higher protein content and secretes two key adipokines (adiponectin and leptin) which play essential roles in energy homeostasis [34].
Adiponectin improves insulin sensitivity by promoting glucose uptake in skeletal muscle, inhibiting hepatic gluconeogenesis, and exerting anti-inflammatory effects in adipose tissue [35]. It has been reported that vitamin D deficiency reduces adiponectin levels [36], whereas supplementation shows inconsistent effects [37]. In the present study, adiponectin levels decreased with increasing vitamin D doses, without significant changes in serum glucose. These results differ from human trials in type 2 diabetes mellitus and MASLD, where vitamin D supplementation increased adiponectin [24].
With respect to leptin (Figure 1m), a sex-specific pattern was observed. In females, serum leptin levels increased progressively up to 14,000 IU of vitamin D, returning to values similar to the control at 21,000 IU. Leptin regulates lipid metabolism by promoting lipolysis and inhibiting lipogenesis, with synthesis stimulated by insulin and TNF and inhibited by growth hormone and free fatty acids [38].
Vitamin D and leptin exhibit a bidirectional relationship: calcitriol suppresses leptin expression in mouse adipocytes, while leptin inhibits vitamin D biotransformation through FGF-23–mediated suppression of CYP27B1 (1-hydroxylase). In vitro, primary adipocyte cultures from VDR-positive animals produce leptin in a receptor-dependent manner, whereas VDR knockout mice display a lean phenotype and hypoleptinemia [34,39]. The present findings suggest that vitamin D saturation relative to VDR at 3000 IU in female rats may impair leptin synthesis at higher doses.
Vitamin D has been reported to exert protective effects against oxidative and inflammatory stress, particularly in tissues with fatty acid accumulation [40]. In MASLD, the hepatic balance between anti- and pro-inflammatory cytokines is mediated by IL-10, with anti-inflammatory action, and by inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF [41,42]. IL-8 is considered an important mediator of hepatic fibrosis, whereas TNF plays a key role in the pathogenesis of MASLD [43,44]. In the present study, increased levels of inflammatory mediators were observed in both sexes when comparing treated groups to the control (Figure 2), with a strong correlation as shown in with vitamin D (Figure 4).
Vitamin D is recognized as a negative regulator of inflammation, modulating cytokines such as TNF, IL-1, IL-6, and IL-8 in mononuclear cells [34]. In mice on a high-fat diet stimulated with lipopolysaccharides, co-treatment with vitamin D (2000 IU/day/kg of diet) and vitamin E (0.9 g/day/kg of diet) increased IL-10 and reduced IL-6 levels, suggesting that supplementation can mitigate inflammation in obesity [45]. However, in obese humans treated with vitamin D (7000 IU/day) for 26 weeks, no changes in circulating IL-6 or IL-8 were observed, indicating that oral vitamin D may not reduce systemic inflammation in this population [46].
Preclinical and clinical studies indicate that vitamin D can improve liver and kidney function, either via endocrine mechanisms, reducing hormones such as homocysteine and inflammatory proteins such as CRP [47], or by enhancing antioxidant status [48], potentially lowering cardiovascular and hepatic disease risk. However, renal effects vary depending on dose and duration, with high or chronic doses potentially impairing function.
In MASLD, oxidative stress from fatty acid metabolism amplifies inflammatory signaling in the liver [23]. In rats with MASLD, vitamin D treatment reduced hepatic senescence, inflammation, and oxidative stress by blocking cell cycle signaling and increasing VDR expression [49]. In the present study, protein carbonylation and lipid oxidation (TBARS) were similar between sexes. Protein oxidation increased in the 21,000 IU group compared with controls, while lipid oxidation was reduced in all vitamin D treated groups (Figure 3). TBARS, produced by ROS interaction with polyunsaturated fatty acids, are elevated in obese individuals and inversely associated with serum vitamin D levels [50]. Our findings align with this relationship, as vitamin D supplementation at different doses reduced TBARS compared to controls.
Regarding protein oxidative stress, vitamin D protects muscle tissue from proteolysis by increasing SOD and glutathione peroxidase activity [51,52]. In liver injury models, vitamin D reduced glycooxidant stress and protein carbonylation [53]. Notably, the present study suggests greater protection against lipid oxidative stress, evident in TAS measurements (Figure 3), with antioxidant protection up to 7000 IU of vitamin D, but it is reduced at higher doses (14,000 and 21,000 IU). Similar patterns were reported in MASLD models treated with different intraperitoneal doses of vitamin D, where higher doses (10 µg/kg) reduced TAS compared to lower doses (5 µg/kg) [49]. A clinical trial in breast cancer patients also found increased TAS with vitamin D supplementation versus placebo [54].
Histopathological analysis (Figure 5) revealed fat droplets in control animals of both sexes (Figure 5a,f). Steatosis improved in the 2500 and 7000 IU vitamin D groups but worsened at 14,000 and 21,000 IU/week, with structural abnormalities suggesting loss of protection against MASLD, differing between males and females. Consistent with this, vitamin D reduced steatosis scores in alcoholic and non-alcoholic models in previous studies [53].
Vitamin D is a pleiotropic biomolecule that regulates gene expression and modulates lipid synthesis by controlling sterol regulatory element-binding protein target genes, thereby reducing lipogenesis [26]. It also coordinates transcriptional mechanisms via interaction with the vitamin D receptor, influencing glucose metabolism in adipocytes and suppressing pro-inflammatory genes [55]. However, the association between vitamin D and MASLD remains unclear, highlighting the need for further research to elucidate its role in disease progression and modulation. One of the most promising molecular targets explored in MASLD is the thyroid hormone receptor-β, for which an agonist (resmetirom) was approved by the FDA in 2024 [56].
This study provides the first comprehensive report on the effects of oral vitamin D overdose in both male and female rats with MASLD, including four different doses and the concomitant monitoring of more than 20 biochemical, hormonal, inflammatory, oxidative, and histological parameters. Furthermore, the study demonstrated for the first time that vitamin D supplementation can exert beneficial effects at low doses, but also deleterious outcomes at higher doses, particularly by aggravating the inflammatory profile and altering lipid metabolism. Nevertheless, some limitations must be acknowledged, such as, only a single time point was assessed, which may not capture the dynamics of long-term supplementation. To provide other perspectives, future investigations should evaluate longitudinal effects, explore molecular mechanisms underlying the dual role of vitamin D, and expand analyses to other liposoluble vitamins in the context of MASLD.

5. Conclusions

In contrast to previous studies that associate hypervitaminosis D or vitamin D toxicity mainly with calcium/phosphorus metabolism and renal pathophysiology, the present study found that oral vitamin D administration reduced hepatic steatosis and lipid peroxidation at low doses (2500 and 7000 IU). At higher doses, however, a potential pro-oxidant and inflammatory effect were observed. Further research is needed to clarify this finding, as few studies have addressed the relationship between vitamin D overdose, supplementation duration, and metabolic diseases such as MASLD. In summary, oral vitamin D supplementation should be approached with caution, considering the physiological context of each individual. Factors such as sex, age, genetic background, and underlying metabolic conditions must be taken into account when determining optimal dosage and duration.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/livers5040052/s1, Table S1. Planilha1.

Author Contributions

S.M.d.M.S. performed the experimental work, conducted statistical analyses, and wrote the initial draft of the manuscript. G.E.S., L.S.d.S., C.B.P., E.G.S., V.T.N. and R.T.M. assisted with the experimental work. C.K. and C.C.P. carried out the histological analyses. I.L.G. and V.M. contributed to manuscript writing, performed statistical analyses, and revised the manuscript for important intellectual content. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

All experimental procedures were conducted in accordance with the guidelines of the Ethics Committee on the Use of Animals of the Federal University of Pampa (CEUA–Unipampa, protocol no. 016/2020, 9 July 2020).

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its Supplementary Information Files.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Biochemical parameters in female (red) and male (blue) rats treated with increasing doses of vitamin D (x-axis: 103 IU/kg/week). Data are expressed as mean ± standard deviation. In (a) glucose; in (b) total cholesterol; in (c) HDL cholesterol; in (d) LDL cholesterol; in (e) triglycerides; in (f) urea; in (g) creatinine; in (h) AST; in (i) ALT; in (j) alkaline phosphatase; in (k) phosphorous; in (l) calcium; in (m) leptin; in (n) adiponectin and in (o) vitamin D. Asterisks indicate statistically significant differences compared to the control group (** p < 0.01; *** p < 0.001; **** p < 0.0001). Abbreviations: glu: glucose; TC: total cholesterol; HDL: HDL-cholesterol; LDL: LDL-cholesterol; TG: triglycerides; cre: creatinine; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; P: phosphorus; Ca: calcium; adpn: adiponectin; VD: vitamin D.
Figure 1. Biochemical parameters in female (red) and male (blue) rats treated with increasing doses of vitamin D (x-axis: 103 IU/kg/week). Data are expressed as mean ± standard deviation. In (a) glucose; in (b) total cholesterol; in (c) HDL cholesterol; in (d) LDL cholesterol; in (e) triglycerides; in (f) urea; in (g) creatinine; in (h) AST; in (i) ALT; in (j) alkaline phosphatase; in (k) phosphorous; in (l) calcium; in (m) leptin; in (n) adiponectin and in (o) vitamin D. Asterisks indicate statistically significant differences compared to the control group (** p < 0.01; *** p < 0.001; **** p < 0.0001). Abbreviations: glu: glucose; TC: total cholesterol; HDL: HDL-cholesterol; LDL: LDL-cholesterol; TG: triglycerides; cre: creatinine; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; P: phosphorus; Ca: calcium; adpn: adiponectin; VD: vitamin D.
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Figure 2. Inflammatory markers in female (red) and male (blue) rats after vitamin D supplementation at increasing doses (x-axis: 103 IU/kg/week). Data are expressed as mean ± standard deviation. In (a) IL-1β; in (b) IL-6; in (c) IL-8; in (d) IL-10; in (e) TNF; and in (f) C-reactive protein (CRP). Asterisks denote statistically significant differences when compared to the control group (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
Figure 2. Inflammatory markers in female (red) and male (blue) rats after vitamin D supplementation at increasing doses (x-axis: 103 IU/kg/week). Data are expressed as mean ± standard deviation. In (a) IL-1β; in (b) IL-6; in (c) IL-8; in (d) IL-10; in (e) TNF; and in (f) C-reactive protein (CRP). Asterisks denote statistically significant differences when compared to the control group (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
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Figure 3. Oxidative stress markers in female (red) and male (blue) rats treated with increasing doses of vitamin D (x-axis: 103 IU/kg/week). Data are expressed as mean ± standard deviation. In (a) protein carbonyl content (CAR); in (b) TBARS levels; and in (c) total antioxidant status (TAS). Asterisks represent statistically significant differences relative to the control group (* p < 0.05; ** p < 0.01; **** p < 0.0001).
Figure 3. Oxidative stress markers in female (red) and male (blue) rats treated with increasing doses of vitamin D (x-axis: 103 IU/kg/week). Data are expressed as mean ± standard deviation. In (a) protein carbonyl content (CAR); in (b) TBARS levels; and in (c) total antioxidant status (TAS). Asterisks represent statistically significant differences relative to the control group (* p < 0.05; ** p < 0.01; **** p < 0.0001).
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Figure 4. Principal component analysis (PCA) of the dataset obtained from male and female rats treated with increasing doses of vitamin D (2500, 7000, 14,000, and 21,000 IU/kg/week, corresponding to groups 2, 3, 4, and 5, respectively; the control group is represented by blue circles). In panel (a), the distribution of the rats according to PC1 and PC2 is shown, while panel (b) displays the contribution of each variable to the clustering pattern. In (c) correlation profiles of all measured parameters with plasma vitamin D levels. The x-axis in each plot represents the vitamin D concentration. Correlation coefficients (r) are color-coded: blue for p < 0.0001, green for p < 0.001, orange for p < 0.01, and black for non-significant correlations (p ≥ 0.01). Abbreviations: GLU, glucose; TC: total cholesterol; HDL: HDL-cholesterol; LDL: LDL-cholesterol; TG: triglycerides; CRE: creatinine; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; P: phosphorus; Ca: calcium; VD: vitamin D; adpn: adiponectin; IL-1β: interleukin-1 beta; IL-6: interleukin-6; IL-8: interleukin-8; IL-10: interleukin-10; TNF: tumor necrosis factor alpha; CRP: C-reactive protein; CAR: protein carbonylation; TBARS: thiobarbituric acid reactive substances; TAS: total antioxidant status.
Figure 4. Principal component analysis (PCA) of the dataset obtained from male and female rats treated with increasing doses of vitamin D (2500, 7000, 14,000, and 21,000 IU/kg/week, corresponding to groups 2, 3, 4, and 5, respectively; the control group is represented by blue circles). In panel (a), the distribution of the rats according to PC1 and PC2 is shown, while panel (b) displays the contribution of each variable to the clustering pattern. In (c) correlation profiles of all measured parameters with plasma vitamin D levels. The x-axis in each plot represents the vitamin D concentration. Correlation coefficients (r) are color-coded: blue for p < 0.0001, green for p < 0.001, orange for p < 0.01, and black for non-significant correlations (p ≥ 0.01). Abbreviations: GLU, glucose; TC: total cholesterol; HDL: HDL-cholesterol; LDL: LDL-cholesterol; TG: triglycerides; CRE: creatinine; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; P: phosphorus; Ca: calcium; VD: vitamin D; adpn: adiponectin; IL-1β: interleukin-1 beta; IL-6: interleukin-6; IL-8: interleukin-8; IL-10: interleukin-10; TNF: tumor necrosis factor alpha; CRP: C-reactive protein; CAR: protein carbonylation; TBARS: thiobarbituric acid reactive substances; TAS: total antioxidant status.
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Figure 5. Liver histology of female rats (top row, panels (a–e)) and male rats (bottom row, panels (f–j)) treated with increasing doses of vitamin D. Each column represents either the control group or one of the vitamin D doses. All images were obtained at 400× magnification.
Figure 5. Liver histology of female rats (top row, panels (a–e)) and male rats (bottom row, panels (f–j)) treated with increasing doses of vitamin D. Each column represents either the control group or one of the vitamin D doses. All images were obtained at 400× magnification.
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Muller de Moura Sarmento, S.; Erminda Schreiner, G.; Smolski dos Santos, L.; Berny Pereira, C.; Gomes Schmitt, E.; Tejada Nunes, V.; Tamborena Malheiros, R.; Klock, C.; Casanova Petry, C.; Gonçalves, I.L.; et al. Effect of Oral Vitamin D Overdose in Male and Female Wistar Rats with Induced MASLD. Livers 2025, 5, 52. https://doi.org/10.3390/livers5040052

AMA Style

Muller de Moura Sarmento S, Erminda Schreiner G, Smolski dos Santos L, Berny Pereira C, Gomes Schmitt E, Tejada Nunes V, Tamborena Malheiros R, Klock C, Casanova Petry C, Gonçalves IL, et al. Effect of Oral Vitamin D Overdose in Male and Female Wistar Rats with Induced MASLD. Livers. 2025; 5(4):52. https://doi.org/10.3390/livers5040052

Chicago/Turabian Style

Muller de Moura Sarmento, Silvia, Gênifer Erminda Schreiner, Laura Smolski dos Santos, Camila Berny Pereira, Elizandra Gomes Schmitt, Vinicius Tejada Nunes, Rafael Tamborena Malheiros, Clóvis Klock, Chaline Casanova Petry, Itamar Luís Gonçalves, and et al. 2025. "Effect of Oral Vitamin D Overdose in Male and Female Wistar Rats with Induced MASLD" Livers 5, no. 4: 52. https://doi.org/10.3390/livers5040052

APA Style

Muller de Moura Sarmento, S., Erminda Schreiner, G., Smolski dos Santos, L., Berny Pereira, C., Gomes Schmitt, E., Tejada Nunes, V., Tamborena Malheiros, R., Klock, C., Casanova Petry, C., Gonçalves, I. L., & Manfredini, V. (2025). Effect of Oral Vitamin D Overdose in Male and Female Wistar Rats with Induced MASLD. Livers, 5(4), 52. https://doi.org/10.3390/livers5040052

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