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

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.