Developmental maturation of the colonic uptake process of the microbiota-generated thiamin pyrophosphate

Abstract

The water-soluble vitamin B1 is essential for normal human health and physiology. In its main biologically active form, i.e., thiamin pyrophosphate (TPP), the vitamin plays many critical roles in cell metabolism; thus, its deficiency leads to a variety of adverse effects. Humans/mammals obtain vitamin B1 from two exogenous sources: diet and gut microbiota. Considerable amount of the microbiota-generated vitamin B1 exists in the form of TPP, and colonocytes can efficiently absorb this TPP via a high-affinity and specific carrier-mediated mechanism that involves the recently cloned colonic TPP transporter (cTPPT; product of SLC44A4 gene). There is nothing currently known about colonic uptake of TPP during early stages of life and whether the process undergoes developmental regulation. We addressed this issue using the mouse as animal model. Our results showed that colonic uptake of TPP undergoes developmental upregulation as the animal moves from the suckling period to weanling and adulthood. This upregulation in uptake was found to be associated with a parallel induction in level of expression of the cTPPT protein, mRNA, and heterogeneous nuclear RNA, suggesting possible involvement of transcriptional mechanism(s). We also found a parallel upregulation in the level of expression of the two nuclear factors that drive activity of the SLC44A4 promoter (i.e., CREB-1 and Elf-3) with maturation. These results demonstrate, for the first time, to our knowledge, that colonic TPP uptake process and cTPPT expression are developmentally upregulated and that this upregulation is likely driven via transcriptional mechanism(s).NEW & NOTEWORTHY The colonic carrier-mediated uptake process of the microbiota-generated and phosphorylated form of vitamin B1, i.e., thiamin pyrophosphate, undergoes ontogenic changes that parallel the development of the gut microbiota (and their ability to generate vitamins) during early stages of life.

Keywords: colonic uptake; developmental maturation; gut microbiota; ontogenic regulation; thiamin pyrophosphate.

Graphical abstract

Figure 1.

Colonic carrier-mediated TPP uptake in suckling, weanling, and adult mice. [³H]-TPP (0.46 µM) uptake was examined using colonic sheets as described in materials and methods. Data are means ± SE of 4 mice from each group. Statistical analysis was done by one-way ANOVA test using GraphPad Prism software. **P < 0.01. TPP, thiamin pyrophosphate.

Figure 2.

Level of expression of the cTPPT protein, mRNA, and hnRNA in colonic mucosa of suckling, weanling, and adult mice. A: protein levels of TPPT were determined by Western blot analysis and data were normalized relative to β-actin expression. The graphs show relative protein expression of TPPT in colonic mucosa of suckling, weanling, or adult mice [Note: jejunal mucosal samples (which do not express TPPT) were run in parallel for comparison purposes]. mRNA (B) and hnRNA (C) levels of TPPT (Slc44a4) were determined by RT-qPCR from colonic mucosa; data were normalized relative to β-actin. Data are means ± SE of 4 mice from each group. Statistical analysis was done as in Fig. 1. **P < 0.01. cTPPT, colonic thiamin pyrophosphate transporter; hnRNA, heterogeneous nuclear RNA; RT-qPCR, quantitative real-time PCR; TPPT, thiamin pyrophosphate transporter.

Figure 3.

Expression of the transcription factors CREB-1 and Elf-3 at the protein and mRNA levels in colonic mucosa of suckling, weanling, and adult mice. A: protein levels of CREB-1 (I) and Elf-3 (II) were determined by Western blot analysis; data normalized relative to Lamin-B. Data are means ± SE of 3 mice from each group. B: mRNA levels of CREB-1 and Elf-3 were determined by RT-qPCR; normalization was relative to β-actin. Data are means ± SE of 4 mice from each group. Statistical analysis was done as in Fig. 1. *P < 0.05, **P < 0.01. RT-qPCR, quantitative real-time PCR.

Figure 4.

Expression of the mitochondrial TPPT (MTPPT) protein, mRNA, and hnRNA in colonic mucosa of suckling, weanling, and adult mice. A: protein levels of MTPPT were normalized relative to pyruvate dehydrogenase (PDH). mRNA (B) and hnRNA (C) levels of MTPPT (Slc25a19) were determined by RT-qPCR; data were normalized relative to β-actin. Data are means ± SE of 4 mice from each group. Statistical analysis as in Fig. 1. *P < 0.05; **P < 0.01. hnRNA, heterogeneous nuclear RNA; RT-qPCR, quantitative real-time PCR; TPPT, thiamin pyrophosphate transporter.