The effect of dietary vitamin D supplementation on sodium-dependent phosphate uptake and expression of NaPi-IIb in the small intestine of weanling pigs

Abstract

Two experiments were conducted to investigate the effects of 1,25(OH)2D3 to stimulate Na+-dependent phosphate uptake in Caco-2 cells, and the effects of dietary vitamin D supplementation to vitamin D-deficient nursery pigs on Na+-dependent nutrient uptake and mRNA expression of NaPi-IIb cotransporter and calbindin D9k in the jejunum. In Exp. 1, 250,000 Caco-2 cells were seeded on Costar 12 mm Snapwell inserts with a 0.40 µm polycarbonate filter and a seeding density of 0.25 × 106 and studied at 15 d postconfluence. Cells were treated with 10 nM of either 1,25(OH)2D3 or vehicle for 48 h and then mounted in modified Ussing chambers for transepithelial measurements. In Exp. 2, pigs (n = 32) were removed from sows at 3 d of age, placed on a vitamin D-deficient milk replacer diet and housed in a room devoid of sunlight and UV light in the range of 280 to 300 nm. On day 28, serum 25(OH)D3 concentrations were measured to verify low vitamin D status. Pigs (BW 10.10 ± 0.38 kg) were then individually housed day 28 postweaning and allotted to 1 of 2 dietary treatments. Dietary treatments consisted of corn-soybean-based diets with vitamin D supplementations of 0 or 1,500 IU/kg diet for 12 d. Blood samples were taken from the brachiocephalic vein on the initial (day 0) and final day (day 10, 11, or 12) of the study for analysis of serum 25(OH)D3, P, and Ca. Pigs were euthanized and jejunal segments were harvested and used in modified Ussing chambers and for RNA isolation and subsequent quantitative RT-PCR analysis. In Exp. 1, treating Caco-2 cells with 10 nM 1,25(OH)2D3 resulted in a 52% increase (P < 0.005) in Na+-dependent phosphate uptake compared with cells treated with a vehicle. In Exp. 2, Na+-dependent phosphate and glucose transport did not differ (P > 0.10) among treatment groups. Additionally, NaPi-IIb and calbindin D9k mRNA expression were not different (P > 0.10) between treatment groups. No differences (P > 0.10) were detected in final serum P or 25(OH)D3 concentrations between treatments. However, serum Ca linearly increased (P < 0.05) as the concentration of supplemental vitamin D increased in the diet. Overall, while 1,25(OH)2D3 stimulated Na+-dependent phosphate uptake in Caco-2 cells, supplementing diets with 1,500 IU/kg vitamin D3 from cholecalciferol did not increase jejunal Na+-dependent phosphate uptake or NaPi-IIb mRNA expression over that of pigs fed diets with no supplemental cholecalciferol.

Keywords: phosphate; small intestine; swine; vitamin D.

Figure 1.

The effect of 10 nM 1,25(OH)₂D₃ or vehicle on active ion transport in Caco-2 cells. Cells were challenged in modified Ussing chambers with 10 mM phosphate on the mucosal side and osmotically balanced with 10 mM mannitol on the serosal side. Baseline I_sc values were subtracted from I_sc values obtained 15 min post-challenge to calculate the change in I_sc. Values are reported as means ± SE. Treatment effect (P < 0.005).

Figure 2.

The effect of dietary vitamin D supplementation on (A) serum Ca concentrations; time effect, P = 0.016, Trt effect, P = 0.057, (B) serum P concentrations; time effect, P = 0.065, Trt effect, P = 0.10, and (C) serum 25(OH)D₃; time effect, P < 0.001, Trt effect, P = 0.938 in weanling pigs. Values are reported as means ± SE.

Figure 3.

The effect of dietary vitamin D supplementation on active ion transport in the small intestine of weanling pigs. Tissues were challenged in modified Ussing chambers with 10 mM phosphate and glucose on the mucosal side and osmotically balanced with 10 mM mannitol on the serosal side. Baseline I_sc values were subtracted from I_sc values obtained 15 min post-challenge to calculate the change in I_sc. Values are reported as means ± SE. Differences were not detected between dietary treatments (P > 0.10).

Figure 4.

The effect of dietary vitamin D supplementation on gene expression in the small intestine of weanling pigs. Gene expression is reported as amount relative to the housekeeper gene, GAPDH. Values are reported as means ± SE. Differences were not detected between dietary treatments (P > 0.10).