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Pharmacological and nutritional modulation of autophagy in a rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1

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Abstract

Autophagy is involved in the modulation of nutrition, immunity, and disease in humans and animals. To understand the impact of autophagy modulation on a rainbow trout gill cell line, RTgill-W1, treatments including reduced nutrition (2% fetal bovine serum compared with 10% control), rapamycin, 3-methyladenine, deoxynivalenol, and chloroquine were tested. Western blot and immunofluorescence were used to detect microtubule-associated protein 1A/1B-light chain protein and quantitative polymerase chain reaction was used to detect the expression of 10 autophagy-related genes. At 3-d post-treatment, reduced nutrition significantly (p < 0.05) increased autophagy while deoxynivalenol significantly (p < 0.01) suppressed it compared to controls. These phenomena were confirmed by using immunofluorescence to detect the number of autophagosomes in RTgill-W1. Chloroquine is critical to allow observation of microtubule-associated protein 1A/1B-light chain protein in this model. The commonly used autophagy-modulating chemicals rapamycin and 3-methyladenine either activated or suppressed microtubule-associated protein 1A/1B-light chain protein, respectively, as expected from the literature, but did not act in a consistently significant manner. Expression of five of the 10 Atg genes, including lc3, gabarap, atg4, atg7, and atg12, were altered in a similar pattern to microtubule-associated protein 1A/1B-light chain protein. The consistent trend of autophagy-related gene upregulation including becn1, lc3, gabarap, and atg9 following treatment with 3-methyladenine and chloroquine is suggestive of a novel feedback regulation in the autophagy machinery.

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Acknowledgments

The authors acknowledge Dr. P. Pham for technical advice and for review of the manuscript and Dr. R. Horricks for statistical advice. RTgill-W1 was a generous gift from Dr. NC. Bols.

Funding

Funding was provided by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant to Lumsden), Ontario Ministry of Agriculture, Food and Rural Affairs, Education Ministry of Taiwan Government (scholarship to Liu). Balmori-Cedeno was supported by an Ontario Veterinary College Scholarship.

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  1. Jaramar Balmori-Cedeno

    Present address: MOWI West Canada, Campbell River, BC, V9W 8C9, Canada

  2. Ehab Misk

    Present address: Huntsman Marine Science Center, Saint Andrews, NB, E5B 2L7, Canada

Authors and Affiliations

  1. Department of Pathobiology, University of Guelph, Guelph, ON, N1G 2W1, Canada

    Juan-Ting Liu, Jaramar Balmori-Cedeno, Ehab Misk & John S. Lumsden

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  1. Juan-Ting Liu
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Correspondence to John S. Lumsden.

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Liu, JT., Balmori-Cedeno, J., Misk, E. et al. Pharmacological and nutritional modulation of autophagy in a rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1. In Vitro Cell.Dev.Biol.-Animal 56, 659–669 (2020). https://doi.org/10.1007/s11626-020-00490-1

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