Effect of vitamin D3 supplementation on blood parameters and liver gene expression in female rats.

BACKGROUND: To better understand the molecular mechanism responsible for the therapeutic potential of vitamin D, we conducted an analysis of the liver transcriptomes of adult female rats. METHODS: Adult female rats (n = 18) were divided into three groups, receiving different doses of vitamin D: group I, 0; group II, 1000 U/kg; and group III, 5000 U/kg. Growth, body weight, the weight of main organs, blood haematological and biochemical parameters were evaluated. Gene expression in the liver were analyzed using RNA-seq and qPCR techniques. RESULTS: We observed a lower platelet count (p < 0,008) and a significantly greater (p < 0.02) number of WBCs in rats supplemented with 1000 U/kg than in rats from group III (5000 U/kg). Moreover, we noted a trend (p < 0.06) in total cholesterol concentration, suggesting a linear decrease with increasing doses of vitamin D. RNA-seq analysis did not reveal any differentially expressed genes with FDR < 0.05. However, GSEA revealed significant activation of a number of processes and pathways, including: "metallothionein, and TspO/MBR family", and "negative regulation of tumor necrosis factor production". qPCR analysis revealed significant upregulation of the Mt1, Mt2 and Orm1 genes in animals receiving high doses of vitamin D (p < 0.025, p < 0.025, and p < 0009, respectively). Moreover, Srebp2 and Insig2 were significantly lower in both experimental groups than in the control group (p < 0.003 and p < 0.036, respectively). CONCLUSIONS: Our results support the anti-inflammatory, anitioxidant and anticholesterologenic potential of vitamin D but suggest that high doses of vitamin D are needed to obtain significant results in this regard.

Changes in miRNA expression in the lungs of pigs supplemented with different levels and forms of vitamin D.

BACKGROUND: Vitamin D is an immunomodulator, and its effects have been linked to many diseases, including the pathogenesis of cancer. However, the effect of vitamin D supplementation on the regulation of gene expression of the lungs is not fully understood. This study aims to determine the effect of the increased dose of cholecalciferol and a combination of cholecalciferol + calcidiol, as well as the replacement of cholecalciferol with calcidiol, on the miRNA profile of healthy swine lungs. METHODS AND RESULTS: The swine were long-term (88 days) supplemented with a standard dose (2000IU/kg) of cholecalciferol and calcidiol, the increased dose (3000 IU/kg) of cholecalciferol, and the cholecalciferol + calcidiol combination: grower: 3000 IU/Kg of vitamin D (67% of cholecalciferol and 33% of calcidiol), finisher 2500 IU/Kg of vitamin D (60% of cholecalciferol and 40% of calcidiol). Swine lung tissue was used for Next Generation Sequencing (NGS) of miRNA. Long-term supplementation with the cholecalciferol + calcidiol combination caused significant changes in the miRNA profile. They embraced altered levels of the expression of miR-150, miR-193, miR-145, miR-574, miR-340, miR-381, miR-148 and miR-96 (q-value < 0.05). In contrast, raising the dose of cholecalciferol only changed the expression of miR-215, and the total replacement of cholecalciferol with calcidiol did not significantly affect the miRNAome profile. CONCLUSIONS: The functional analysis of differentially expressed miRNAs suggests that the use of the increased dose of the cholecalciferol + calcidiol combination may affect tumorigenesis processes through, inter alia, modulation of gene regulation of the TGF- ß pathway and pathways related to metabolism and synthesis of glycan.

Effect of different doses of cholecalciferol and calcidiol on meat quality parameters and skeletal muscle transcriptome profiles in swine.

Skeletal muscle tissue is one of the potential targets for vitamin D actions. There are indications that vitamin D supplementation to swine has a positive effect on meat quality. However, these issues need further study, especially in terms of response to the use of different forms of vitamin D. We carried out a multi-purpose study to compare the effects of cholecalciferol and calcidiol on meat quality and muscle tissue transcriptome. Meat quality assessment and gene expression analysis were performed on longissimus dorsi samples collected from swine fed grower/finisher diets containing 2000 IU cholecalciferol/1500 IU cholecalciferol per kg (n = 8), 3000 IU cholecalciferol/2500 IU cholecalciferol per kg (n = 10), 2000 IU cholecalciferol +1000 IU calcidiol/1500 IU cholecaliferol +1000 IU calcidiol per kg (n = 8), and 2000 IU calcidiol/1500 IU calcidiol per kg (n = 8). The results suggest that increasing the dose of cholecalciferol and using calcidiol in the diet of finishers may improve meat texture parameters (shear force P = 0,014, toughness P = 0,048, cohesiveness P = 0,017, resilience P = 0,002). Shear force (68.46 N-51.42 N) and toughness (145.85 N-114.52 N) decreased the most under the effect of increasing cholecalciferol dosage. In turn, cohesiveness (0.60 N-0.65 N) and resilience (0.23 N-0.28 N) increased most strongly under the use of cholecalciferol+calcidiol. Moreover, the results indicate no significant effect of increasing cholecalciferol dose and use calcidiol in the swine diet on muscle tissue transcriptome.

Changes in DNA Methylation and mRNA Expression in Lung Tissue after Long-Term Supplementation with an Increased Dose of Cholecalciferol.

Maintaining an appropriate concentration of vitamin D is essential for the proper functioning of the body, regardless of age. Nowadays, there are more and more indications that vitamin D supplementation at higher than standard doses may show protective and therapeutic effects. Our study identified differences in the body's response to long-term supplementation with cholecalciferol at an increased dose. Two groups of pigs were used in the experiment. The first group received a standard dose of cholecalciferol (grower, 2000 IU/kg feed, and finisher, 1500 IU/kg feed), and the second group received an increased dose (grower, 3000 IU/kg feed, and finisher, 2500 IU/kg feed). After slaughter, lung samples were collected and used for RRBS and mRNA sequencing. Analysis of the methylation results showed that 2349 CpG sites had significantly altered methylation patterns and 1116 (47.51%) identified DMSs (Differentially Methylated Sites) were related to genes and their regulatory sites. The mRNA sequencing results showed a significant change in the expression of 195 genes. The integrated analysis identified eleven genes with DNA methylation and mRNA expression differences between the analyzed groups. The results of this study suggested that an increased vitamin D intake may be helpful for the prevention of lung cancer and pulmonary fibrosis. These actions may stem from the influence of vitamin D on the expression of genes associated with collagen production, such as SHMT1, UGT1A6, and ITIH2.The anti-cancer properties of vitamin D are also supported by changes in KLHL3 and TTPA gene expression.

Analysis of the effect of vitamin D supplementation and sex on Vdr, Cyp2r1 and Cyp27b1 gene expression in Wistar rats' tissues.

Vitamin D supplementation is widely recommended for animals and humans. However, its effects on extra-skeletal disorders have not been well proven. Our research aimed to analyse the effect of supplementation with standard and increased doses of vitamin D on the health status, biochemical and haematological parameters of blood as well as on the level of expression of genes metabolising vitamin D and the Vdr gene in the kidney, liver, fat and brain of rats of different sexes. 36 Wistars rats (18 males and 18 females) divided into three supplementation groups (I-O vitamin D, II- 1000 U / kg of vitamin D, III - 5000 U / Kg of vitamin D) were analysed. Vitamin D supplementation had little effect on growth traits and biochemical blood indices; slight decrease in a heart size was observed in females supplemented with high doses of vitamin D (p = 0.0075), moreover male rats tended to have increased level of triglyceride (TG) (p = 0.0516)). In our study we observed that vitamin D supplementation did not change the expression of Vdr gene in any of analysed tissue. However, we noticed evident downregulation of Cyp27b1 gene by vitamin D supplementation in the liver and kidney in a dose-dependent manner. Additionally, we observed that in the female's liver, Vdr and Cyp2r1 were upregulated, in the female's kidney Vdr was upregulated while Cyp27b1 was downregulated compared to males. Moreover, in the female's brain and fat Cyp27b1 and Cyp2r1 tended to be upregulated compared to males. Our results confirm that Vdr and vitamin D metabolising genes are regulated by sex in the kidney and several extra-renal tissues.

3'quant mRNA-Seq of Porcine Liver Reveals Alterations in UPR, Acute Phase Response, and Cholesterol and Bile Acid Metabolism in Response to Different Dietary Fats.

Animal fats are considered to be unhealthy, in contrast to vegetable fats, which are rich in unsaturated fatty acids. However, the use of some fats, such as coconut oil, is still controversial. In our experiment, we divided experimental animals (domestic pigs) into three groups differing only in the type of fat used in the diet: group R: rapeseed oil (n = 5); group B: beef tallow (n = 5); group C: coconut oil (n = 6). After transcriptomic analysis of liver samples, we identified 188, 93, and 53 DEGs (differentially expressed genes) in R vs. B, R vs. C, and B vs. C comparisons, respectively. Next, we performed a functional analysis of identified DEGs with String and IPA software. We observed the enrichment of genes engaged in the unfolded protein response (UPR) and the acute phase response among genes upregulated in B compared to R. In contrast, cholesterol biosynthesis and cholesterol efflux enrichments were observed among genes downregulated in B when compared to R. Moreover, activation of the UPR and inhibition of the sirtuin signaling pathway were noted in C when compared to R. The most striking difference in liver transcriptomic response between C and B was the activation of the acute phase response and inhibition of bile acid synthesis in the latest group. Our results suggest that excessive consumption of animal fats leads to the activation of a cascade of mutually propelling processes harmful to the liver: inflammation, UPR, and imbalances in the biosynthesis of cholesterol and bile acids via altered organelle membrane composition. Nevertheless, these studies should be extended with analysis at the level of proteins and their function.