Conditional knockout of the Slc5a6 gene in mouse intestine impairs biotin absorption

Abstract

The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.

Fig. 1.

Generation of the sodium-dependent multivitamin transporter (SMVT) conditional knockout (KO) mice and genotyping. A: schematic representation of the homologous recombination to introduce the loxP sites at the upstream and downstream regions of exons 4 and 6 of the slc5a6 gene, respectively. A 5′ loxP recombination site was inserted into intron 3 of the Slc5a6 gene together with a Neo cassette, and a second 3′ loxP recombination site was inserted into intron 6 of the gene. Exons 4, 5, and 6 of the Slc5a6 gene were then conditionally removed when animals were crossed with transgenic mice expressing the Cre-recombinase only in intestinal epithelial cells under the villin promoter. B: PCR analysis of mouse ear DNA samples using 2 primers, showing presence of loxP (i) (+/+) and Cre transgenes (ii). Conditional deletion of Slc5a6, exon 4 to exon 6, was confirmed in enterocytes (iii).

Fig. 2.

Effect of intestine-specific (conditional) SMVT KO on mouse body weight, bone density, and bone length. A: representative image of a KO mouse (right) and its sex-matched wild-type littermate (left) showing distinct size differences. B: representative X-ray image of a KO mouse (right) and its sex-matched wild-type littermate (left) showing clear phenotypic differences in size and bone length. C: plot showing clear reduction in body weight of KO mice compared with their sex-matched wild-type littermates (P < 0.01). D: plot showing significant decrease of bone density in KO mice (expressed as % relative to wild-type littermates). E: plot showing significant decrease in bone length in KO mice (expressed as % relative to wild-type littermates). Data are means ± SE of 4 separate sets of mice. *P < 0.05, **P < 0.01.

Fig. 3.

Histology of the small bowel (A–C) and the cecum (D–F) of the SMVT conditional KO mice and their sex-matched littermates. Small bowel: representative section of small bowel of sex-matched wild-type (WT) littermate (A) and KO mouse (B), hematoxylin and eosin (H&E), ×40, inset ×200. A: normal small bowel morphology of the WT littermate. B: significant shortening of the villi and focal dysplastic changes of the surface epithelium (inset). C: semiquantitative analysis of small bowel length of villi in mm (left y-axis) and total area of dysplasia in percent (right y-axis). (*P < 0.01, n = 5). Cecum: representative section of cecum of sex-matched wild-type littermate (D) and KO mouse (E), H&E, ×40, inset ×200. D: normal morphology of the WT littermate cecum. E: significant submucosal edema (open arrow) and acute inflammation involving surface (closed arrows) and crypts (D, inset). F: semiquantitative analysis of number of neutrophils in 10 high-power fields (×400, left y-axis), and total area of dysplasia and submucosal edema in percent (right y-axis). (*P < 0.01, n = 5).

Fig. 4.

Expression of SMVT mRNA and protein in intestinal mucosa, liver, and kidney of SMVT conditional KO mice and their sex-matched wild-type littermates. A: quantitative real-time PCR from reverse-transcribed total RNA of jejunal mucosal scrapings (i), liver (ii), and kidney (iii) of KO mice and their sex-matched wild-type controls. B: Western blot analysis of SMVT protein in jejunal mucosal scrapings (i), liver (ii), and kidney (iii) of KO mice and their sex-matched wild-type controls. Data are means ± SE of at least 3 separate sets of mice. (*P < 0.01).

Fig. 5.

Effect and specificity of conditional KO of the intestinal SMVT on intestinal biotin uptake in vivo and in vitro. A: carrier-mediated biotin uptake in vivo using intact jejunal loops of KO mice and their sex-matched wild-type littermates. B: carrier-mediated biotin uptake in vitro using freshly isolated and suspended jejunal epithelial cells of KO mice and their sex-matched wild-type littermates. C: carrier-mediated pantothenic acid uptake in vivo using intact jejunal loops of KO mice and their sex-matched wild-type littermates. D: carrier-mediated ascorbic acid uptake in vivo using intact jejunal loops of KO mice and their sex-matched wild-type littermates. E: carrier-mediated biotin uptake by kidney brush-border membrane vesicle (BBMV) of KO mice and their sex-matched wild-type littermates. Data are means ± SE of 3 to 6 independent observations from 3 to 6 different sets of mice. *P < 0.01.