Autophagy is an intracellular bulk degradation process, induced under nutrient starvation. Failure of autophagy has been recognized as a contributor to aging and multiple age related neurodegenerative diseases. Improving autophagy is a beneficial anti-aging strategy, however very few physiological regulators have been identified. Here, we demonstrate that vitamin C is a nutritional stimulator of autophagy. Supplementation of fresh hepatocytes with vitamin C increased autophagic proteolysis significantly in the presence of amino acids in a dose- and time-dependent manner, although no effect was observed in the absence of amino acids. In addition, inhibitor studies with 3-methyladenine, chloroquine, leupeptin and β-lactone confirmed that vitamin C is active through the lysosomal autophagy and not the proteasome pathway. Furthermore, the autophagy marker LC3 protein was significantly increased by vitamin C, suggesting its possible site of action is at the formation step. Both the reduced (ascorbic acid, AsA) and oxidized form (dehydroascorbic acid, DHA) of vitamin C exhibited equal enhancing effect, indicating that the effect does not depend on the anti-oxidation functionality of vitamin C. To understand the mechanism, we established that the effective dose (50 μM) was 15× lower than the intracellular content suggesting these would be only a minor influx from the extracellular pool. Moreover, transporter inhibitor studies in an AsA deficient ODS model rat revealed more accurately that the enhancing effect on autophagic proteolysis still existed, even though the intracellular influx of AsA was blocked. Taken together, these results provide evidence that vitamin C can potentially act through extracellular signaling.
Bibliographical noteFunding Information:
This work was supported in part by a Grant-in-Aid for Scientific Research (23380074 to M. Kadowaki) from the Japan Society for the Promotion of Science (23380074), and a Grant-in-Aid for Young Scientists (B) (21780123 to M.R. Karim) from the Ministry of Education, Culture, Sports, Science and Technology (21780123), Japan. We are thankful to Professor Noriko Miyake and Professor Tadao Kurata (Niigata University of Pharmacy and Applied Life Sciences) for their kind help to allow us using their HPLC facilities regarding AsA measurement. The authors would like to acknowledge Professor Deborah A. Ferrington (University of Minnesota) for critical discussion and suggestions regarding manuscript preparation.
© 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)
- Amino acids
- Ascorbic acid
- Cell signaling