Biotin and pantothenic acid oversupplementation to conditional SLC5A6 KO mice prevents the development of intestinal mucosal abnormalities and growth defects

Abstract

Intestinal absorption of the water-soluble vitamins biotin and pantothenic acid is carrier mediated and involves the sodium-dependent multivitamin transporter (SMVT; product of the SLC5A6 gene). We recently observed that intestinal-specific (conditional) knockout of the mouse Slc5a6 gene (SMVT-cKO) is associated with growth retardation, the development of spontaneous and severe inflammation, abnormal histology in the large intestine, altered gut permeability, and early death. Our aim in this study was to examine the possibility that biotin and pantothenic acid oversupplementation (BPS) of the SMVT-cKO mice could reverse the above-described abnormalities. BPS was provided in the drinking water to mice before conception, to dams during pregnancy and lactation, and to the SMVT-cKO mice throughout their life. Our findings showed that such a regimen prevents early death, as well as normalizes the growth rate, intestinal integrity, pathology, and inflammation in SMVT-cKO mice. These findings provide clear evidence for a role for biotin and/or pantothenic acid in the maintenance of normal intestinal integrity and health.

Keywords: SMVT; biotin; mucosal inflammation; mucosal integrity; pantothenic acid.

Fig. 1.

Effects of BPS to SMVT-cKO mice on growth rate, bone density, and biotin level. Ai: representative image of a SMVT-cKO mouse (left) and its sex-matched WT littermate (right) showing clear phenotypic differences in size and length that we have seen previously (12). Aii: representative image of BPS SMVT-cKO mouse (left) and its sex-matched BPS WT littermate (right) showing no difference in size and length. Bi: representative X-ray image of a SMVT-cKO mouse (left) and its sex-matched WT littermate (right) showing distinct difference in bone length. Bii: representative X-ray image of BPS SMVT-cKO mice (left) and its BPS WT littermate (right) showing no difference in bone size and length. C: growth chart showing no difference in weight gain of BPS SMVT-cKO mice compared with their BPS WT littermate. D: level of total biotinylated proteins in the liver (a measure of biotin status) of BPS SMVT-cKO mice and their BPS WT littermates. Data are means ± SE of at least three separate sets of mice. Abbreviations: BPS, biotin and pantothenic acid supplementation; cKO, conditional knockout of the mouse Slc5a6 gene; NS, not significant; SMVT, sodium-dependent multivitamin transporter; WT, wild type.

Fig. 2.

A: histology of the cecum of BPS SMVT-cKO mice and their age- and sex-matched BPS WT littermates. A: representative section of the cecum of BPS WT littermate (i) and BPS SMVT-cKO mouse (ii) (hematoxylin and eosin, × 40). Animals of both groups showed normal cecal morphology. B: effect of BPS to SMVT-cKO mice on intestinal permeability. Intestinal permeability was determined using 4-kDa FITC-dextran method. Data are means ± SE of three pairs of BPS SMVT-cKO and WT littermates. C: effect of BPS to SMVT-cKO mice on the level of mRNA expression of TJ proteins and MLCK in the cecum. mRNA levels were determined by real-time RT-PCR, and data were normalized relative to β-actin. Data are means ± SE of at least three pairs of BPS SMVT-cKO mice and WT littermates. D: effect of BPS to SMVT-cKO mice on the level of protein expression of TJ proteins and MLCK in the cecum. Protein levels were determined by Western blot analysis, and data were normalized relative to β-actin expression as described in Methods. The graphs show relative protein expression of claudin-1 (i), claudin-2 (ii), ZO-1 (iii), and MLCK (iv) in cecum samples of SMVT-cKO mice and their WT littermates. Data are means ± SE of at least three separate sets of mice. E: expression of mRNA of mucin genes in the cecum of SMVT-cKO mice and their WT littermates. mRNA levels were determined by real-time RT-PCR, and data were normalized relative to villin. Data are means ± SE of at least three separate sets of mice (*P < 0.05; **P < 0.01; NS, not significant). F: effect of BPS to SMVT-cKO mice and their WT littermates on mRNA expression of mucins in the cecum. Level of mRNA expression of MUC-1 (i), MUC-2 (ii), and MUC-3 (iii) in cecum of BPS SMVT-cKO mice and their sex-matched BPS WT littermates. Data are means ± SE of at least three sets of mice. Abbreviations: BPS, biotin and pantothenic acid supplementation; cKO, conditional knockout of the mouse Slc5a6 gene; MLCK, myosin light chain kinase; MUC, mucin; SMVT, sodium-dependent multivitamin transporter; TJ, tight junction; WT, wild type; ZO-1, zonula occludens 1.

Fig. 3.

A: effect of BPS of SMVT-cKO mice on the level of mRNA expression of proinflammatory cytokines in the cecum. mRNA levels were determined by real-time RT-PCR, and data were normalized relative to β-actin. Data are means ± SE of at least three separate sets of mice (NS, not significant). B: expression of mRNA of oxidative stress-responsive genes in the cecum of SMVT-cKO mice and their WT littermates. mRNA levels were determined by real-time RT-PCR and data were normalized relative to β-actin. Data are means ± SE of at least three separate sets of mice (**P < 0.01; NS, not significant). C: effect of BPS to SMVT-cKO mice and their WT littermates on mRNA expression of oxidative stress-responsive genes in the cecum. mRNA levels were determined by real-time RT-PCR, and data were normalized relative to β-actin. Data are means ± SE of at least three separate sets of mice. Abbreviations: BPS, biotin and pantothenic acid supplementation; cKO, conditional knockout of the mouse Slc5a6 gene; FMO, flavin-containing monooxygenase; LPO, lipid peroxidation; NOS, nitric oxide synthase; SMVT, sodium-dependent multivitamin transporter; SOD, superoxide dismutase; WT, wild type.