Introduction
Sustainable aquaculture production requires a greater reduction in the use of fish-based ingredients, and one avenue investigated over the past years is the increase in the proportion of plant-derived digestible carbohydrates in aquafeeds. However, this strategy presents a number of drawbacks for high trophic level teleost fish such as rainbow trout (RT, Oncorhynchus mykiss), which show, for still unknown reasons, reduced growth associated with persistent postprandial hyperglycemia and hepatomegaly, when fed diets containing more than 20% of carbohydrates (1,2). Among the factors that may be involved in the apparent glucose intolerance of RTs, one of the major pathways of lysosomal catabolism known as Chaperone-Mediated Autophagy (CMA) attracted our attention. In mammals, CMA is described as a critical player in the turnover of glucose and lipid metabolism-related enzymes, and dysregulation of this function has been shown to lead to significant alterations in liver metabolic homeostasis (3). However, CMA has only recently been identified in fish (4) and no data are currently available regarding its regulation as well as its role in the metabolic specificities of RT. In this work, we first assessed in vitro the existence of functional CMA in the RT. We then studied its regulation upon high glucose treatment and identified the underlying mechanisms. Finally, we studied the impact of silencing either of the two paralogous genes encoding the CMA-essential factor lysosome-associated membrane protein type 2A (LAMP2A) on the overall proteostasis of RT.
Material and methods
First, we established a trout hepatoma cell line (RTH-149) stably expressing a fluorescent reporter (KFERQ-PA-mCherry1) previously used to track CMA in mammalian cells (5), and more recently validated in medaka fibroblasts (4). Then, we exposed the cells either to mild-oxidative stress (H2O2, 25 µM), which activates CMA in mammals (6), or to high glucose (HG, 25 mM). We monitored by advanced imaging both the cellular localization and the half-life of the reporter. Subsequently, we used OxyBlot to analyze the oxidative stress status in cells incubated with HG or H2O2, and we explored the relationship between oxidative stress and CMA using antioxidants and specific mitochondrial inhibitors. Besides, we investigated the role of the nuclear factor erythroid-derived 2, like 2 (NRF2) on the effects of HG on CMA in RTH-149 cells using immunofluorescences, gene expression, in silico analysis, and siRNA-mediated knockdown approaches. Finally, to gain insight into the specific contribution of each of the two RT LAMP2As to the overall proteostasis of RTH-149 cells under an HG condition, we performed comparative quantitative proteomics after transfection with siRNAs designed to specifically target each of the lamp2a paralogs (hereafter referred to as si14 and si31, respectively).
Results
Our results showed that upon HG or H2O2 exposure, the KFERQ-CMA reporter accumulated in characteristic CMA-puncta that colocalized with lysosomes (Fig. 1A). Moreover, the half-life of the reporter was substantially shortened under these conditions compared to the CT, suggesting an active CMA-flux and supporting the existence of functional CMA in the RT. Then we observed that these mechanisms underlying the effects of HG on CMA in RTH-149 cells depended on the generation of reactive oxygen species (ROS) (Fig. 1B) at the mitochondrial level. In addition, the results showed that the NRF2 transcription factor mediates the upregulation of CMA by increasing the mRNA levels of both Lamp2a.
Finally, proteomics analysis reveals no significant alteration of any biological process in the cells transfected with si14 and exposed to HG, whereas different processes associated with cellular metabolism and its regulation were identified in si31 transfections (Fig. 1C), supporting a functional divergence between the two LAMP2As during hyperglycemia.
Discussion
These sets of experiments univocally revealed that RT, like medaka, exhibits functional CMA activity, and emphasized, for the first time, the strong responsiveness of RT to HG exposure via the NRF2 pathway. Besides, the result highlighted the role of CMA, especially mediated through the LAMP2A paralogue 31, on the regulation of cellular metabolism upon glucose overload. Altogether, this work underlines the importance of considering this selective autophagy process to understand carbohydrate intolerance in carnivorous fish. Future studies will consider novel approaches for exploiting CMA metabolic impacts through aquafeed formulation to, in the last term, optimize the use of plant-derived carbohydrates and contribute to more sustainable aquaculture.
References
1. Kamalam BS, et al. Aquaculture (2017) 467:3–27. 2. Kostyniuk DJ, et al. Physiol Genomics (2019) 51:411. 3. Schneider JL, et al. Cell Metab (2014) 20:417–432. 4. Lescat L, et al. Mol Biol Evol (2020) 37:2887–2899. 5. Koga H, et al. Nat Commun (2011) 2:386. 6. Kiffin R, et al. Mol Biol Cell (2004) 15:4829–4840.