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Activation of TRPV2 negatively regulates the differentiation of mouse brown adipocytes

  • Ion channels, receptors and transporters
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Abstract

Transient receptor potential vanilloid 2 (TRPV2) acts as a Ca2+-permeable non-selective cation channel that has been reported to be sensitive to temperature, mechanical force, and some chemicals. We recently showed that TRPV2 is critical for maintenance of the thermogenic function of brown adipose tissue in mice. However, the involvement of TRPV2 in the differentiation of brown adipocytes remains unexplored. We found that the expression of TRPV2 was dramatically increased during the differentiation of brown adipocytes. Non-selective TRPV2 agonists (2-aminoethoxydiphenyl borate and lysophosphatidylcholine) inhibited the differentiation of brown adipocytes in a dose-dependent manner during the early stage of differentiation of brown adipocytes. The inhibition was rescued by a TRPV2-selective antagonist, SKF96365 (SKF). Mechanical force, which activates TRPV2, also inhibited the differentiation of brown adipocytes in a strength-dependent manner, and the effect was reversed by SKF. In addition, the inhibition of adipocyte differentiation by either TRPV2 ligand or mechanical stimulation was significantly smaller in the cells from TRPV2KO mice. Moreover, calcineurin inhibitors, cyclosporine A and FK506, partially reversed TRPV2 activation-induced inhibition of brown adipocyte differentiation. Thus, we conclude that TRPV2 might be involved in the modulation of brown adipocyte differentiation partially via a calcineurin pathway.

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Acknowledgments

We are grateful to Drs. Yasuhiko Minokoshi, Kazuhiro Ikenaka (National Institute for Physiological Sciences), and Toshihiko Yada from Jichi Medical University for their kind discussion. We also thank Dr. Minji Kim from Kyoto University for the technical assistance.

Author contributions

W.S., K.U., and M.T. designed experiments and wrote the manuscript. W.S. performed experiments. W.S., K.U., N.T., T.G., T.K., and M.T. discussed and interpreted the data. K.U., N.T., and T.G. gave technical assistance. Y.I. and S.W. supplied TRPV2KO mice. K.U. and M.T. supervised this work.

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Authors and Affiliations

  1. Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki, Aichi, 444-8787, Japan

    Wuping Sun, Kunitoshi Uchida & Makoto Tominaga

  2. Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan

    Wuping Sun, Kunitoshi Uchida & Makoto Tominaga

  3. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

    Nobuyuki Takahashi, Tsuyoshi Goto & Teruo Kawada

  4. Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan

    Yuko Iwata & Shigeo Wakabayashi

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  1. Wuping Sun
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  2. Kunitoshi Uchida
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Correspondence to Kunitoshi Uchida or Makoto Tominaga.

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Funding

This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology (#15H02501) (M.T.) and Takeda Science Foundation (K.U.).

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Fig. A1

TRPV2 is expressed in differentiated mouse brown adipocytes a and b Mean responses evoked by a TRPV2 agonist, 500 μM 2APB (a) or 30 μM LPC- (b) were blocked by a selective TRPV2 antagonist (10 μM SKF). The 1 μM NE-evoked response indicates that the examined brown adipocytes have differentiated. Iono was used to confirm cell viability. Ratio values correspond to the actual [Ca2+]i of differentiated mouse brown adipocytes. c A representative whole-cell current evoked by a TRPV2 agonist, 2APB, was inhibited by SKF in differentiated mouse brown adipocytes. A voltage ramp-pulse protocol (300 ms) is shown in the inset (upper left). Current–voltage curves of basal currents (black), currents in the presence of 2APB + SKF (blue) and currents in the presence of 2APB alone (red) at the time points of i, ii and iii, respectively, in differentiated mouse brown adipocytes are shown in the inset (upper right). d Responses of individual differentiated brown adipocytes that were evoked by a TRPV2 agonist (500 μM 2APB) in the presence or absence of 2 mM calcium. (GIF 54 kb)

High resolution image (TIF 595 kb)

Fig. A2

Expression of Trpm7 mRNA in adipocytes and representative images of mouse brown adipocytes at different stages a RT-PCR analysis of Trpm7 mRNA expression in differentiated mouse brown adipocytes. Control lane indicates the results obtained with plasmid DNA as a template. Reverse transcription (RT) lanes indicate that the samples were treated with (+) or without (−) RT. b Real-time PCR analysis of Trpm7 mRNA expression in pre-adipocytes and 6-day-differentiated brown adipocytes. Mean ± SEM, n = 6; **, P < 0.01 vs. pre-adipocytes. Unpaired Student’s t-test. c Representative phase-contrast images of mouse brown adipocytes at different stages. Scale bar indicates 100 μm. (GIF 218 kb)

High resolution image (TIF 3687 kb)

Fig. A3

Ligand-evoked responses in mouse pre-adipocytes and 6-day-differentiated brown adipocytes Individual traces of changes in [Ca2+]i in response to 1 μM capsaicin (a and b), 250 μM 2APB + 500 μM carvacrol (Carva) (c and d), 300 nM GSK (e and f) or 1 mM menthol (g and h) in pre-adipocytes (a, c, e and g) and 6-day-differentiated brown adipocytes (b, d, f and h). One μM NE was used to confirm differentiation. Five μM Iono was used to confirm cell viability. (GIF 160 kb)

High resolution image (TIF 2084 kb)

Fig. A4

Ionomycin reduced the number of differentiated mouse brown adipocytes in a dose-dependent manner a Representative images of differentiated mouse brown adipocytes after a 6-day-differentiation in differentiation medium containing different doses of Iono. Scale bar indicates 100 μm. b Dose-dependency of the Iono-induced reduction in the number of differentiated mouse brown adipocytes. Mean ± SEM, n = 6; * P <0.05 and ** P < 0.01 vs. control group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. (GIF 122 kb)

High resolution image (TIF 2104 kb)

Fig. A5

Lack of TRPV2 facilitated brown adipocyte differentiation a Oil red O staining of 6-day-differentiated mouse brown adipocytes from wild-type (WT) and TRPV2 knockout (TRPV2KO) mice with indicated differentiation media. 1/3 suppl. indicates differentiation medium was diluted 3-fold with DMEM. Scale bar indicates 100 μm. b, c and d Comparison of the numbers of 6-day-differentiated mouse brown adipocytes (b), triglyceride levels (c) and adipocyte diameters (d) in cells from WT and TRPV2KO mice with indicated differentiation media. Mean ± SEM, n = 8; *, P < 0.05 and **, P < 0.01 vs. control group; ##, P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. (GIF 126 kb)

High resolution image (TIF 1862 kb)

Fig. A6

Calcineurin inhibitors reversed TG-induced inhibition of mouse brown adipocyte differentiation a Representative images of Oil Red O staining of differentiated mouse brown adipocytes after 6-day-differentiation in differentiation medium containing indicated compounds. Scale bar indicates 100 μm. b and c Effects of FK506 and CsA on the numbers of 6-day-differentiated mouse brown adipocytes (b) and triglyceride levels (c) after 10 nM TG treatment. Mean ± SEM, n = 6; *, P < 0.05 and **, P < 0.01 vs. control group; #, P < 0.05 and ##, P < 0.01 vs. TG group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. d Real-time PCR analysis of calcineurin mRNA expression in pre-adipocytes and 6-day-differentiated brown adipocytes. Mean ± SEM, n = 6. Unpaired Student’s t-test. (GIF 136 kb)

High resolution image (TIF 2017 kb)

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Sun, W., Uchida, K., Takahashi, N. et al. Activation of TRPV2 negatively regulates the differentiation of mouse brown adipocytes. Pflugers Arch - Eur J Physiol 468, 1527–1540 (2016). https://doi.org/10.1007/s00424-016-1846-1

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