Periodic Reporting for period 1 - MEAL fish (Role of selective autophagy processes in regulating the glucose tolerance of carnivorous fish: seeking greater replacement of fishmeal by vegetal ingredients in aquafeeds)
Période du rapport: 2022-09-01 au 2024-08-31
Providing sustainable food supply to the rapidly growing human population is one of the biggest challenges of the planet. The aquaculture industry plays an important role as by 2018 it was already responsible for 52% of fish production for human consumption, but improving its blue growth requires further optimizations, including the replacement of fishmeal and fish oil by vegetal ingredients in aquafeeds. Rainbow trout (Oncorhynchus mykiss) represented in 2016 up to 78% of freshwater production in Europe, so this species deserves special attention. In this sense, formulating diets to replace fishmeal with plant-based ingredients is limited due to the physiology of carnivorous species. One of the reasons for this limitation in most carnivorous aquaculture species, including rainbow trout, is the low ability to use dietary carbohydrates (CBH) naturally abundant in plant products. These animals present a phenotype of glucose intolerance, characterized by reduced growth and prolonged postprandial hyperglycemia when CBH inclusion exceeds 20% of the diet. The identification and removal of “metabolic locks” limiting the use of dietary CBH would thus allow for increasing the range of plant raw materials that can be used in feeds.
Several studies suggest that in carnivorous fish, a diet rich in CBH is accompanied by oxidative stress. Previous findings in mammals demonstrated that free radicals induce the selective Chaperone-Mediated Autophagy (CMA). CMA is one of the main pathways of lysosomal catabolism involved in the degradation of key enzymes of different metabolic pathways (e.g. glycolysis, Krebs cycle or lipid storage), and is critical for the control of cellular homeostasis and intermediary metabolism. In this regard, we hypothesized that the oxidative stress observed after a CBH-rich meal in carnivorous fish may cause an over-activation of CMA as well, and thus an abnormal degradation of glycolysis-related enzymes, leading to the observed postprandial hyperglycemia. In addition to CMA, oxidative stress has also been shown to induce endosomal microautophagy (eMI), a recently discovered selective autophagic route, which shares substrates and some traits with the mechanism of action of CMA. Thus, we could expect that eMI may be involved in the control of intermediary metabolism, and possibly, in the glucose intolerance phenotype observed in trout. Dr. Seiliez's lab recently discovered that medaka fish performs CMA activity. Besides, the genetic blockage of CMA in medaka induced severe alterations in both glucose and lipid metabolism, similar to what has been observed in the liver of mice deficient for CMA. In addition, although the existence of eMI in fish is suspected but still remains unclear, these works suggested that fish lacking the CMA receptor present a compensatory mechanism that could correspond to an eMI-like activity. Altogether, these results constitute a breakthrough in our understanding of the regulatory pathways of nutrient use in fish, and suggested the existence of complex interactions between the different selective autophagic routes that remain to be discovered and understood.
With these in mind, in the MEAL fish project, we aimed to deepen our knowledge of the regulation and the role of eMI and CMA in trout to identify limiting factors for the use of dietary CBH in carnivorous farmed fish species. The overall objectives of the project were to explore the regulation of both eMI and CMA processes by glucose levels and to study their implications in the tolerance of rainbow trout to high-CBH diets. Together, the MEAL fish project seeks to make an intensive contribution to pursue the EU's blue growth goals.
The results obtained alongside the MEAL fish project have demonstrated the existence of CMA and eMI in trout, and have revealed the interplay between both autophagic pathways in situations of cellular stress to maintain cellular homeostasis, notably in the face of oxidative stress generated by exposure to high levels of glucose. Taken together, we can conclude that CMA and eMI are two protective processes that are involved to some extent in carbohydrate utilization in trout and that both processes should be taken into consideration when formulating new dietary strategies for improving carbohydrate utilization in those fish.
Several studies suggest that in carnivorous fish, a diet rich in CBH is accompanied by oxidative stress. Previous findings in mammals demonstrated that free radicals induce the selective Chaperone-Mediated Autophagy (CMA). CMA is one of the main pathways of lysosomal catabolism involved in the degradation of key enzymes of different metabolic pathways (e.g. glycolysis, Krebs cycle or lipid storage), and is critical for the control of cellular homeostasis and intermediary metabolism. In this regard, we hypothesized that the oxidative stress observed after a CBH-rich meal in carnivorous fish may cause an over-activation of CMA as well, and thus an abnormal degradation of glycolysis-related enzymes, leading to the observed postprandial hyperglycemia. In addition to CMA, oxidative stress has also been shown to induce endosomal microautophagy (eMI), a recently discovered selective autophagic route, which shares substrates and some traits with the mechanism of action of CMA. Thus, we could expect that eMI may be involved in the control of intermediary metabolism, and possibly, in the glucose intolerance phenotype observed in trout. Dr. Seiliez's lab recently discovered that medaka fish performs CMA activity. Besides, the genetic blockage of CMA in medaka induced severe alterations in both glucose and lipid metabolism, similar to what has been observed in the liver of mice deficient for CMA. In addition, although the existence of eMI in fish is suspected but still remains unclear, these works suggested that fish lacking the CMA receptor present a compensatory mechanism that could correspond to an eMI-like activity. Altogether, these results constitute a breakthrough in our understanding of the regulatory pathways of nutrient use in fish, and suggested the existence of complex interactions between the different selective autophagic routes that remain to be discovered and understood.
With these in mind, in the MEAL fish project, we aimed to deepen our knowledge of the regulation and the role of eMI and CMA in trout to identify limiting factors for the use of dietary CBH in carnivorous farmed fish species. The overall objectives of the project were to explore the regulation of both eMI and CMA processes by glucose levels and to study their implications in the tolerance of rainbow trout to high-CBH diets. Together, the MEAL fish project seeks to make an intensive contribution to pursue the EU's blue growth goals.
The results obtained alongside the MEAL fish project have demonstrated the existence of CMA and eMI in trout, and have revealed the interplay between both autophagic pathways in situations of cellular stress to maintain cellular homeostasis, notably in the face of oxidative stress generated by exposure to high levels of glucose. Taken together, we can conclude that CMA and eMI are two protective processes that are involved to some extent in carbohydrate utilization in trout and that both processes should be taken into consideration when formulating new dietary strategies for improving carbohydrate utilization in those fish.
Alongside the project, we have stablished RTH-149 rainbow trout liver cells expressing fluorescent reporters to track in vitro CMA or eMI activities, and generated tools to silence the gene expression of key factors for those processes using siRNA or morpholino oligos. Using advanced fluorescence microscopy and molecular biology techniques, in combination with different experimental conditions, drug treatments, and incubations times, we have characterized the regulation of eMI and CMA activities. Using proteomics analysis, we have explored the consequences of invalidating CMA in cells exposed to high-glucose levels. By formulating and manufacturing fish diets and in vivo experimentation, we have studied the effect of carbohydrates on glycaemia, oxidative stress, and the regulation of CMA and eMI in the rainbow trout, as well as the effects of antioxidants supplementation.
The main results obtained can be divided in two parts;
CMA: we have demonstrated for the first time the existence of CMA in the rainbow trout. We have observed that exposure to high-glucose levels enhances CMA through the generation of mitochondrial reactive oxygen species and the activation of the transcription factor NRF2. We have demonstrated that CMA plays a key protective role against hyperglycemic stress. These results have been presented to different audiences [e.g. Aquaexcel3.0 annual meeting (Heraklion, Greece, 10/2022), SEFAGIA 2022 meeting (Toledo, Spain, 11/2022), the Séminaire Poissons INRAE (Rennes, France, 01/2023), MetaBoDay 2023 conference (Talence, France, 05/2023), the Journee Portes Ouvertes of Aquapole INRAE (Saint-Pée-sur-Nivelle, France, 06/2023), Aquaculture Europe 2023 conference (Vienna, Austria, 09/2023), the CFATG11 – 11th Scientific Days on Autophagy conference (Lyon, France, 11/2023)], published in the journal Autophagy (Vélez et al., 2023. Autophagy, 20(4), 752–768. https://doi.org/10.1080/15548627.2023.2267415(s’ouvre dans une nouvelle fenêtre)) and featured in the media, including an interview in Le Monde (https://bit.ly/LeMondeCMA(s’ouvre dans une nouvelle fenêtre)).
eMI: we have identified for the first time an eMI-like process in a fish species that is induced for different stressors, including hyperglycemic stress. The findings suggest eMI's stimulus-specific induction and its potential compensatory role when CMA is impaired. These results have been presented so far in the Journee Portes Ouvertes of Aquapole INRAE (Saint-Pée-sur-Nivelle, France, 06/2023), the CFATG11 – 11th Scientific Days on Autophagy conference (Lyon, France, 11/2023), during the wrapping up meeting of the project at the NuMeA monthly scientific seminars (St-pee, September 2024), and deposited as preprint (Vélez et al., 2024. bioRxiv 2024.09.26.615173 https://doi.org/0.1101/2024.09.26.615173(s’ouvre dans une nouvelle fenêtre)).
The main results obtained can be divided in two parts;
CMA: we have demonstrated for the first time the existence of CMA in the rainbow trout. We have observed that exposure to high-glucose levels enhances CMA through the generation of mitochondrial reactive oxygen species and the activation of the transcription factor NRF2. We have demonstrated that CMA plays a key protective role against hyperglycemic stress. These results have been presented to different audiences [e.g. Aquaexcel3.0 annual meeting (Heraklion, Greece, 10/2022), SEFAGIA 2022 meeting (Toledo, Spain, 11/2022), the Séminaire Poissons INRAE (Rennes, France, 01/2023), MetaBoDay 2023 conference (Talence, France, 05/2023), the Journee Portes Ouvertes of Aquapole INRAE (Saint-Pée-sur-Nivelle, France, 06/2023), Aquaculture Europe 2023 conference (Vienna, Austria, 09/2023), the CFATG11 – 11th Scientific Days on Autophagy conference (Lyon, France, 11/2023)], published in the journal Autophagy (Vélez et al., 2023. Autophagy, 20(4), 752–768. https://doi.org/10.1080/15548627.2023.2267415(s’ouvre dans une nouvelle fenêtre)) and featured in the media, including an interview in Le Monde (https://bit.ly/LeMondeCMA(s’ouvre dans une nouvelle fenêtre)).
eMI: we have identified for the first time an eMI-like process in a fish species that is induced for different stressors, including hyperglycemic stress. The findings suggest eMI's stimulus-specific induction and its potential compensatory role when CMA is impaired. These results have been presented so far in the Journee Portes Ouvertes of Aquapole INRAE (Saint-Pée-sur-Nivelle, France, 06/2023), the CFATG11 – 11th Scientific Days on Autophagy conference (Lyon, France, 11/2023), during the wrapping up meeting of the project at the NuMeA monthly scientific seminars (St-pee, September 2024), and deposited as preprint (Vélez et al., 2024. bioRxiv 2024.09.26.615173 https://doi.org/0.1101/2024.09.26.615173(s’ouvre dans une nouvelle fenêtre)).
The present project has contributed to advancing the state of the art on both the regulation and the role of the two autophagy pathways CMA and eMI during glucose-related metabolic disorders. This nowledge has the potential to be used to refine the formulation of aquaculture diets with a higher proportion of carbohydrates, but it could also have an impact in the field of biomedicine.