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. 2020 Dec 17;533(4):1347-1351.
doi: 10.1016/j.bbrc.2020.10.017. Epub 2020 Oct 14.

BDNF secretion from C2C12 cells is enhanced by methionine restriction

Ryan Antony  1 , Yifan Li  2
Affiliations

BDNF secretion from C2C12 cells is enhanced by methionine restriction

Ryan Antony et al. Biochem Biophys Res Commun. .

Abstract

Brain derived neurotrophic factor (BDNF) is produced in skeletal muscle as a myokine that plays a role in muscle metabolism. However, how metabolic changes affect skeletal muscle BDNF expression and release remains to be fully understood. Amino acid restrictions such as methionine restriction (MR) are considered as an alternative fasting approach. Here we reported that in C2C12 myotubes, MR enhanced BDNF release, which was measured using ELISA, RT-qPCR, cell immunostaining, and Western blot. Inhibition of protein transport pathway blocked the MR enhanced BDNF release, confirming that MR-induced BDNF release involved classic protein secretory pathway. MR increased l-lactate product in media, suggesting that MR promoted glycolysis. Treatment with 2-deoxy glucose (2-DG) attenuated lactate production as well as BDNF release, suggesting that glycolysis is involved in the enhanced BDNF release induced by MR. Moreover, treatment with l-Lactate, the end-product of glycolysis, enhanced BDNF gene expression and release in control cells in a dose dependent manner, suggesting lactate produced by glycolysis may mediate the enhanced BDNF release by MR. Overall, the results of this study suggest that MR promotes BDNF secretion from C2C12 myotubes at least partially via enhancing glycolysis and lactate production.

Keywords: Brain derived neurotrophic factor; C2C12; Lactate; Methionine restriction; Myokine.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1,
Fig. 1,
MR enhanced BDNF release: A: BDNF ELISA with media of cells in control and MR media for 8 to 16 hours. n=8. B: Relative BDNF gene expression by RT-qPCR in cells in control or MR media for 8 hours. n=5. C: BDNF immunostaining cells in control and MR media for 8 hours. The bar is 200 μM. The bottom images were the enlarged views of the boxed areas in the top images. Arrows point to the enhanced BDNF distribution in plasma membrane. D: Western blot: showing pro- and mature-BDNF bands and beta actin in cell lysate from MR and control cells.
Fig. 2,
Fig. 2,
Inhibition of protein secretory pathway blocked BDNF release. A: ELISA assay of BDNF in media of control and MR with or without protein transport inhibitor cocktail (1:500 dilution) for 16 hours. n=4. B: BDNF immunostaining of cells in control or MR media with or without protein secretion inhibitor cocktail for 16 hours. The bars are 100 μM. Arrows indicate the accumulation of BDNF in ER area.
Fig. 3,
Fig. 3,
Glycolysis is involved in MR-enhanced BDNF release. A: ELISA of BDNF level in media of cells in control media with or without addition of L-methionine (50ug/ml) for 8 hours. B: L-lactate level in media of control and MR cells with or without 2-DG (1 mM) for 8 hours. n=4. C: ELISA of BDNF in media of control and MR with or without 2-DG for 8 hours. n=4. D: BDNF immunostaining in control or MR cells with or without 2-DG for 8 hours. The bars are 200 μM.
Fig. 4,
Fig. 4,
Lactate stimulated BDNF release: A: ELISA of BDNF in media of control cells with L-lactate at 0, 5 and 10 mM for 8 hours. n=4. B: Relative BDNF gene expression measured by RT-qPCR from cells treated with control or L-lactate for 8 hours. n=5. C: Immunostaining of BDNF in cells in control media with or without L-lactate for 8 hours. The bars are 200 μM. D: ELISA of BDNF in media of MR cells with or without lactate. n=4.
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