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Reply to: Absence of evidence that Slc12a8 encodes a nicotinamide mononucleotide transporter

Nature Metabolism volume 1, pages 662–665 (2019)Cite this article

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The Original Article was published on 12 July 2019

replying to Mark S. Schmidt and Charles Brenner Nature Metabolism https://doi.org/10.1038/s42255-019-0085-0 (2019)

Second, they argue that “incubation of cells with 100 μM NMN does not produce a time-dependent increase in intracellular NMN” (see Fig. 1d in ref. 2). They also argue that “the assay for internalization of 3H-NMN is merely an assay for cell association”. However, experiments with 3H-NMN were performed not only with intact cells, but also with proteoliposomes. It is correct that NMN levels in mouse primary hepatocytes and Slc12a8-overexpressing (Slc12a8-OE) NIH3T3 cells do not keep increasing in the presence of NMN at high concentrations in the media (see Figs. 1d and 2b). On the other hand, in the proteoliposome experiments, NMN levels transported into the proteoliposomes made from Slc12a8-OE NIH3T3 cells reached a plateau by 10.5 min and did not decrease (see Supplementary Fig. 2b in ref. 2). Indeed, in this assay, we also subtracted 3H-NMN radioactivity due to non-specific/non-kinetic transport and/or non-specific binding, as described in the Methods section2. Thus, the 3H-NMN radioactivity we detected after this subtraction must be due to specific transport of NMN into proteoliposomes. Although we do not understand the exact reason for this phenomenon in live cells, we currently suspect that the Slc12a8 NMN transporter function might be regulated in a time-dependent and/or intracellular NMN-dependent manner in cells. Investigation addressing this possibility is currently ongoing. Regarding a possible cell association, this is why we conducted isotopic tracing experiments using both doubly labelled, 3 Da-heavier isotopic NMN and nicotinamide riboside (O18-D-NMN and O18-D-NR, respectively) in Slc12a8-OE NIH3T3 cells and primary hepatocytes from Slc12a8 knockout mice (see Figs. 2f and 3i in ref. 2) As described in our article, both results clearly demonstrate unequivocal NMN uptake without any conversion of NMN to nicotinamide riboside outside of cells.

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Fig. 1: Analysis of NMN using HPLC and LC–MS/MS.
Fig. 2: Hypercarb HPLC chromatograms of NMN in extracts from control and FK866-treated NIH3T3 cells and mouse primary hepatocytes.

References

  1. Schmidt, M. S. & Brenner, C. Absence of evidence that Slc12a8 encodes a nicotinamide mononucleotide transporter. Nat. Metab. https://doi.org/10.1038/s42255-019-0085-0 (2019).

  2. Grozio, A. et al. Slc12a8 is a nicotinamide mononucleotide transporter. Nat. Metab. 1, 47–57 (2019).

    Article  Google Scholar 

  3. Yoshino, J. & Imai, S. Accurate measurement of nicotinamide adenine dinucleotide (NAD+) with high-performance liquid chromatography. Methods Mol. Biol. 1077, 203–215 (2013).

    Article  CAS  Google Scholar 

  4. Yoshino, J., Mills, K. F., Yoon, M. J. & Imai, S. Nicotinamide mononucleotide, a key NAD+ intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice. Cell Metab. 14, 528–536 (2011).

    Article  CAS  Google Scholar 

  5. Trammell, S. A. & Brenner, C. Targeted, LCMS-based metabolomics for quantitative measurement of NAD+ metabolites. Comput. Struct. Biotechnol. J. 4, e201301012 (2013).

    Article  Google Scholar 

  6. Ramsey, K. M. et al. Circadian clock feedback cycle through NAMPT-mediated NAD+ biosynthesis. Science 324, 651–654 (2009).

    Article  CAS  Google Scholar 

  7. Ratajczak, J. et al. NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells. Nat. Commun. 7, 13103 (2016).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA

    Alessia Grozio, Kathryn Mills, Kyohei Tokizane, Hanyue Cecilia Lei & Shin-ichiro Imai

  2. Center for Human Nutrition, Division of Geriatrics and Nutritional Science, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA

    Jun Yoshino

  3. Department of Experimental Medicine, Section of Biochemistry, and Center of Excellence for Biomedical Research, University of Genova, Genova, Italy

    Santina Bruzzone & Giovanna Sociali

  4. Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA

    Yo Sasaki

  5. Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA

    Marie Migaud

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  1. Alessia Grozio
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  10. Shin-ichiro Imai
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Contributions

S.I. and A.G. mainly wrote the correspondence. J.Y. conducted the experiments and analyses discussed in Fig. 1. A.G. performed the experiments and analyses discussed in Fig. 2. K.M., S.B., G.S., K.T., H.C.L., M.M. and Y.S. contributed to the preparation of the correspondence.

Corresponding author

Correspondence to Shin-ichiro Imai.

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Competing interests

A.G. and S.I. are inventors on a patent (PCT/US18/46233) about the Slc12a8 NMN transporter, whose applicant is Washington University; it has been licensed by Teijin Limited. The other authors declare no competing interests.

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Grozio, A., Mills, K., Yoshino, J. et al. Reply to: Absence of evidence that Slc12a8 encodes a nicotinamide mononucleotide transporter. Nat Metab 1, 662–665 (2019). https://doi.org/10.1038/s42255-019-0086-z

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