SLC44A4 Is the Predominant Transport System for Uptake of the Microbiota-Generated Phosphorylated Form of Vitamin B1 (Thiamin Pyrophosphate; TPP) in Colonocytes: Role in Colonocyte's Physiology

Abstract

Background: Humans/mammals obtain vitamin B1 from diet and the gut-microbiota. Considerable amount of the microbiota-generated vitamin B1 exists in the form of TPP. We have previously shown that colonocytes are capable of absorbing intact TPP via a specific and effcient carrier-mediated process ( JBC 289:4405-16, 2014); we have also identified the SLC44A4 as a colonic TPP transporter (cTPPT) in both human and mouse and have characterized different aspects of its regulation and cell biology ( Plos One 14: e0224234, 2019). Little, however, is known about the relative contribution of the cTPPT toward total colonic carrier-mediated TPP uptake. Aims: To determine the relative contribution of cTPPT toward total colonic carrier-mediated TPP uptake, and to investigate the effect of its knockout/down on colonocyte's physiology. Methods: An Slc44a4 knockout (KO) mouse model was generated by crossing homozygous Slc44a4 LoxP (+/+) animals with CMV promoter-driven Cre-transgenic mice. Knocking down the SLC44A4 in human colonic epithelial CCD841 cells and in human primary differentiated colonocyte monolayers (from organ donors) was achieved using gene-specific siRNAs. [3H]-TPP was used in uptake studies. Results: Body weight and colonic length of the Slc44a4 KO mice were significantly lower than those of WT-littermates. Near complete (and specific) inhibition in colonic carrier-mediated [3H]-TPP uptake was observed in Slc44a4 KO mice compared to WT-littermates. Similarly, knocking down SLC44A4 of human CCD841 cells and that of human primary colonocyte monolayers led to significant reduction in [3H]-TPP uptake. Since colonocytes have limited ability to synthesize TPP endogenously [they express a very low level of the enzyme thiamin pyrophosphokinase; BBA 1517:320-2, 2001] and that recent genomic studies have suggested that the SLC44A4 gene could be a potential “ulcerative colitis susceptibility” gene ( Genes Immun 17:105-9, 2016), we also examined the effect of Slc44a4 KO on colonocytes physiology. Results of our RNA-seq and Ingenuity Pathway Analysis showed an increase in several genes related to chronic colitis and intestinal inflammation, and changes in genes involved in energy metabolism. Also, the Slc44a4 KO mice were found to be more susceptible to developing colitis when exposed to a low dose of dextran sodium sulfate compared to their WT-littermates. Conclusion: The SLC44A4 is the predominant/only transport system for uptake of the microbiota-generated TPP and that its loss compromise normal colonocyte's physiology. Supported by NIH grants DK-56061 & AA-018071, and VA grants 101BX001142 & IK6BX006189. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.