Analysis of the effect of high doses of vitamin D on gene and protein expression in the intestine of pigs (Sus scrofa).

Vitamin D is one of the most popular supplements worldwide, yet its appropriate dosage and full impact on health of humans and animals are still debatable. In this study, 30 pigs were divided into three groups, differing in the amount of vitamin D in the diet (Group A - no supplementation, group B-5000 IU/Kg of vitamin D, and group C 10000 IU/Kg). After 3 months of fattening, animals were slaughtered, and samples of jejunum (the longest part of the small intestine in pigs) and colon were collected for transcriptome analysis. Comparison of the transcriptomes between jejunum and colon identified 3872 Differentially Expressed Genes (DEGs). In contrast, transcriptomic changes under the influence of vitamin D were subtle in both parts of the intestine. RNA-seq results showed that vitamin D supplementation with 5000 IU/Kg enhanced the expression of 7 genes in the jejunum and one gene (MEP1B) in the colon (FDR < 0.05, base mean > 10, and log2fold change>0.6), while supplementation with 10,000 IU/Kg increased the expression of one gene (OASL) in the jejunum. No DEGs with FDR < 0.05 were identified after supplementation with 10,000 IU/kg of vitamin D in the colon, however qPCR analysis showed that genes connected to cell cycle control (PLK1, PLK3, KIF4A, KIFC1, AURKB) are upregulated in this group. Gene Set enrichment analysis of the whole RNA-seq dataset revealed that among the most affected by vitamin D processes are that connected to immunity, especially antiviral response in the jejunum, and that connected to cell cycle control in the colon. Despite the use of very high dietary vitamin D doses, no evidence of overt intestinal toxicity was observed at the transcriptomic level. Nevertheless, the activation of molecular pathways involved in calcium handling and cell cycle regulation suggests that prolonged exposure to supraphysiological vitamin D levels may trigger adaptive responses whose long-term consequences remain unknown.