PUBLICATION
Methodological advancements in organ-specific ectopic lipid quantitative characterization: Effects of high fat diet on muscle and liver intracellular lipids
- Authors
- Grepper, D., Tabasso, C., Aguettaz, A., Martinotti, A., Ebrahimi, A., Lagarrigue, S., Amati, F.
- ID
- ZDB-PUB-230116-1
- Date
- 2023
- Source
- Molecular metabolism 68: 101669 (Journal)
- Registered Authors
- Aguettaz, Axel, Amati, Francesca, Ebrahimi, Ammar, Grepper, Dogan, Lagarrigue, Silviane, Martinotti, Adrien, Tabasso, Cassandra
- Keywords
- Fatty liver, Intrahepatic lipids, Intramyocellular lipids, Lipid droplets, Lipid metabolism, Obesity, Zebrafish
- MeSH Terms
-
- Animals
- Diabetes Mellitus*/metabolism
- Diet, High-Fat*
- Dietary Fats/metabolism
- Humans
- Liver/metabolism
- Muscle, Skeletal/metabolism
- Obesity/metabolism
- Zebrafish
- PubMed
- 36642092 Full text @ Mol Metab
Citation
Grepper, D., Tabasso, C., Aguettaz, A., Martinotti, A., Ebrahimi, A., Lagarrigue, S., Amati, F. (2023) Methodological advancements in organ-specific ectopic lipid quantitative characterization: Effects of high fat diet on muscle and liver intracellular lipids. Molecular metabolism. 68:101669.
Abstract
Objective Ectopic lipid accumulation is a hallmark of metabolic diseases, linking obesity to non-alcoholic fatty liver disease, insulin resistance and diabetes. The use of zebrafish as a model of obesity and diabetes is raising due to the conserved properties of fat metabolism between humans and zebrafish, the homologous genes regulating lipid uptake and transport, the implementation of the '3R's principle and their cost-effectiveness. To date, a method allowing the conservation of lipid droplets (LDs) and organs in zebrafish larvae to image ectopic lipids is not available. Our objectives were to develop a novel methodology to quantitatively evaluate organ-specific LDs, in skeletal muscle and liver, in response to a nutritional perturbation.
Methods We developed a novel embedding and cryosectioning protocol allowing the conservation of LDs and organs in zebrafish larvae. To establish the quantitative measures, we used a three-arm parallel nutritional intervention design. Zebrafish larvae were fed a control diet containing 14% of nutritional fat or two high fat diets (HFDs) containing 25 and 36% of dietary fats. In muscle and liver, LDs were characterized using immunofluorescence confocal microscopy. In liver, intrahepatocellular lipids were discriminated from intrasinusoid lipids. To complete liver characteristics, fibrosis was identified with Masson's Trichrome staining. Finally, to confirm the conservation and effect of HFD, molecular players of fat metabolism were evaluated by RT-qPCR.
Results The cryosections obtained after setting up the embedding and cryopreservation method were of high quality, preserving tissue morphology and allowing the visualization of ectopic lipids. Both HFDs were obesogenic, without modifying larvae survival or development. Neutral lipid content increased with time and augmented dietary fat. Intramuscular LD volume density increased and was explained by an increase in LDs size but not in numbers. Intrahepatocellular LD volume density increased and was explained by an increased number of LDs, not by their increased size. Sinusoid area and lipid content were both increased. Hepatic fibrosis appeared with both HFDs. We observed an increase in the expression of genes associated with LD coating proteins, LD dynamics, lipogenesis, lipolysis and fatty acid oxidation.
Conclusions In this study, we propose a reproducible and fast method to image zebrafish larvae without losing LD quality and organ morphology. We demonstrate the impact of HFD on LD characteristics in liver and skeletal muscle accompanied by alterations of key players of fat metabolism. Our observations confirm the evolutionarily conserved mechanisms in lipid metabolism and reveal organ specific adaptations. The methodological advancements proposed in this work open the doors to study organelle adaptations in obesity and diabetes related research such as lipotoxicity, organelle contacts and specific lipid depositions.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping