Edited by: Kang-le Lu, Jimei University, China
Reviewed by: Xian Li, Institute of Oceanology (CAS), China; Changhong Cheng, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, South China Sea Fisheries Research Institute (CAFS), China
This article was submitted to Marine Fisheries, Aquaculture and Living Resources, a section of the journal Frontiers in Marine Science
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Hepatic steatosis is the most common phenomenon of lipid metabolism disorder in farmed fish, but its molecular mechanism is poorly understood. Therefore, the present study was aimed to investigate hepatic steatosis induced by high-fat diet (HFD) and explore underlying mechanism in tilapia. The fish were fed on control diet or HFD for 90 days. The blood and liver tissues were collected to determine biochemical parameter, gene expression and protein level after 30, 60, and 90 days, and analyzed lipid accumulation, endoplasmic reticulum (ER) stress and autophagy. After 30 days of feeding, the plasmatic and hepatic lipid content (TG, TCH, LDL-C, and HDL-C) and fatty acid (FA) transportation (
Steatosis is the most common phenomenon of lipid metabolism disorder in liver of cultured fish, which results in reduction of growth, feed utilization rate, immunity, stress tolerance, etc. (
In fish, the adverse effects induced by HFD have garnered much attention from researchers in recent years. Dietary high fat (15 and 16%) suppressed growth performance, reduced immune capacity and altered lipid metabolism in grass carp (
Autophagy, a basic cellular process, is involved in the degradation of cell constituent, such as unfolded protein and damaged organelle. In the past decade, the key molecular pathways that regulate autophagy have be proposed, which consist mainly of autophagy (Atg) proteins (
Tilapia (
In this study, we evaluated possible molecular mechanism of hepatic steatosis in HFD-fed tilapia, and explored relationship between lipid accumulation and ER stress and autophagy after HFD feeding for 30, 60, and 90 days. Meanwhile, we analyzed multiple signaling pathways which mediated ER stress and autophagy including inositol-requiring enzyme 1 (IRE1), TFEB and AMPK pathways. Our results constituted novel insights into lipid accumulation induced by HFD in tilapia, which might be helpful for prevention and treatment of fatty liver injury in fish.
The experiment was performed taking into consideration the welfare of animal, and the use of fish was approved by the Freshwater Fisheries Research Centre (FFRC) of the Chinese Academy of Fishery Sciences, Wuxi, China.
Juvenile tilapias (weight 35 ± 1.2 g) were provided by the farm in Freshwater Fish Research Center of Chinese Academy of Fishery Sciences (Wuxi, China). The fish were acclimated to lab condition in a recirculation system (temperature 29 ± 2°C; dissolved oxygen > 6 mg/L; pH 7.4–8.1) for 2 weeks, and fed on a control diet twice per day.
After acclimation, the fish were weighed and randomly distributed into two groups: control group and HFD group. Each group contained 40 fish tested in duplicate. The fish were fed on control diet (6% fat) or HFD (21% fat) at approximately 4% of their body weight twice per day (9:00 and 16:00) for 90 days. The formation of experimental diet is presented in
After 30, 60, and 90 days of feeding, all fish were weighed and 10 fish were randomly netted from each group to get liver and blood tissues under anesthesia (100 mg/L MS-222, Sigma Diagnostics INS, St. Louis, MO, United States). The plasma was separated from blood by centrifugation (5000 rpm, 4°C, and 10 min) for analysis of blood biochemical parameters. The liver samples were flash-frozen in liquid nitrogen for measurement of enzymatic activity, gene and protein levels.
Plasma lipid parameters including triacylglycerol (TG), total cholesterol (TCH), free fatty acid (FFA), low density lipoprotein cholesterol (LDL-C), and high density lipoprotein cholesterol (HDL-C) were measured using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The TG, TCH and free CH were also tested in liver.
Histopathology of hepatic steatosis induced by HFD was evaluated by observing pathological section of hematoxylin and eosin (H&E) staining, Oil red O staining and transmission electron microscope (TEM). The H&E staining was performed as previously described by
Liver fatty acids (FAs) were prepared as described by
Total RNA was extracted from liver using RNAiso Plus kit (Takara, Beijing, China). Complementary DNA (cDNA) was synthesized from total RNA (1 μg) by PrimeScript RT reagent Kit with gDNA Eraser (Takara). The relative expression of specific genes was detected using quantitative real-time PCR (qPCR) kit (TB Green Premix Ex Taq II, Takara). The reaction condition was as follows: one cycle 95°C for 30 s, then 40 cycles of 95°C for 5 s and 60–64°C for 1 min. Specific primers used for qPCR were listed in
The procedure of western blotting was performed as previous method described by
All data in this study are expressed as mean ± SEM (standard error of mean), calculated by SPSS software (version 20.0). Two-tail Student’s
To investigate steatosis in liver of tilapia fed on HFD, we observed liver appearance (
Gross phenotype and histopathology in tilapia fed on control diet and HFD at 90 day.
In plasma, the levels of TG, TCH, FFA, LDL-C, and HDL-C were significantly increased in HFD-fed fish from 30 days of feeding onward, except FFA at 30 days (
Plasma lipid parameters of tilapia fed on control diet and HFD for 90 days.
Growth performance and liver lipid content in control diet and HFD-fed tilapia.
Fatty acid composition in the livers of control diet or HFD-fed tilapia after 90 days.
Fatty acid (g/100 g) | Control | HFD | |
C12:0 | 0.0074 ± 0.0019 | 0.005 ± 0.0007 | 0.293 |
C14:0 | 0.4422 ± 0.0761 | 0.2702 ± 0.0485 | 0.129 |
C14:1 | 0.0104 ± 0.0032 | 0.0077 ± 0.0023 | 0.537 |
C15:0 | 0.0158 ± 0.0023 | 0.0431 ± 0.0017 | 0.001 |
C16:0 | 2.5454 ± 0.3274 | 4.8858 ± 0.1637 | 0.003 |
C16:1 | 0.3355 ± 0.0948 | 0.5354 ± 0.1214 | 0.264 |
C17:0 | 0.0247 ± 0.0015 | 0.0577 ± 0.0062 | 0.007 |
C17:1 | 0.0201 ± 0.0040 | 0.0523 ± 0.0021 | 0.002 |
C18:0 | 0.4553 ± 0.1313 | 0.1691 ± 0.0389 | 0.105 |
C18:1n9c | 3.2821 ± 0.3099 | 10.775 ± 0.1946 | <0.001 |
C18:2n6t | 0.0023 ± 0.0003 | 0.0094 ± 0.0013 | 0.007 |
C18:2n6c | 1.8105 ± 0.2133 | 5.9322 ± 0.2471 | <0.001 |
C20:0 | 0.0344 ± 0.0018 | 0.0535 ± 0.0096 | 0.125 |
C18:3n6 | 0.1582 ± 0.0380 | 0.2718 ± 0.0553 | 0.166 |
C20:1n9 | 0.1198 ± 0.0219 | 0.3605 ± 0.1828 | 0.261 |
C18:3n3 | 0.0967 ± 0.0143 | 0.5317 ± 0.1982 | 0.094 |
C21:0 | 0.0033 ± 0.0004 | 0.041 ± 0.00442 | 0.001 |
C20:2 | 0.1154 ± 0.0226 | 0.6186 ± 0.0634 | 0.002 |
C22:0 | 0.0078 ± 0.0008 | 0.0318 ± 0.0110 | 0.095 |
C20:3n6 | 0.108 ± 0.02212 | 0.3942 ± 0.0451 | 0.005 |
C22:1n9 | 0.0031 ± 0.0006 | 0.027 ± 0.00248 | 0.001 |
C20:3n3 | 0.019 ± 0.00298 | 0.137 ± 0.02433 | 0.009 |
C20:4n6 | 0.393 ± 0.07291 | 0.7509 ± 0.0513 | 0.016 |
C22:2n6 | 0.0038 ± 0.0009 | 0.0336 ± 0.0051 | 0.005 |
C20:5n3 | 0.0104 ± 0.0011 | 0.0131 ± 0.0017 | 0.274 |
C24:0 | 0.0075 ± 0.0027 | 0.0126 ± 0.0007 | 0.147 |
C24:1n9 | 0.0022 ± 0.0001 | 0.0061 ± 0.0005 | 0.003 |
C22:6n3 | 0.3313 ± 0.0417 | 0.5388 ± 0.1082 | 0.148 |
Total SFA | 3.0942 ± 0.2297 | 5.2945 ± 0.1756 | <0.001 |
Total MUFA | 3.7731 ± 0.4226 | 11.764 ± 0.4962 | <0.001 |
Total PUFA | 3.0487 ± 0.3897 | 9.2313 ± 0.2097 | 0.022 |
n-3 PUFA | 0.4575 ± 0.0520 | 1.2206 ± 0.2025 | <0.001 |
n-6 PUFA | 2.4758 ± 0.3267 | 7.3921 ± 0.26289 | 0.002 |
n-3:n-6 ratio | 0.1847 ± 0.01223 | 0.1651 ± 0.01125 | 0.125 |
Total lipid | 12.533 ± 1.6374 | 33.033 ± 2.8013 | 0.003 |
To examine the effects of HFD on lipid metabolism in tilapia liver, we assessed the expression of lipid metabolism-related genes including hepatic TG metabolism, and FA uptake, synthesis and β-oxidation (
Expression of fatty acids metilapia fed on control diet and HFD for 90 days.
In FHD-treated tilapia, the cholesterol synthesis and uptake in liver were inhibited, as validated by lower mRNA levels of hepatic lipase (
Expression of cholesterol metabolism-related genes in liver of tilapia fed on control diet and HFD for 90 days.
ER stress participates in multiple physiological process including lipid metabolism (
Expression of endoplasmic reticulum stress-related genes or proteins in liver of tilapia fed on control diet and HFD for 90 days.
Lysosome-mediated autophagy plays an important role in hepatic physiology and pathogenesis of fatty liver. Compared with control group, HFD feeding caused a marked downregulation of
Expression of autophagy-related genes in liver of tilapia fed on control diet and HFD for 90 days.
Correlation analysis showed that hepatic TG accumulation was positively related to IRE1-XBP1s pathways, but negatively related to AMPK and TFEB pathways (
Correlation analysis and principal component analysis (PCA).
Liver is the central organ that regulates the uptake, synthesis, secretion, catabolism and storage of lipid. The dysregulation of FAs and TG is a major reason of steatosis (
Another reason for steatosis is lower FA β-oxidation. In tilapia, our data displayed that HFD feeding downregulated genes expression of β-oxidation rate-limiting enzymes (CPT-1and ACOX-1), and
In liver, FAs can synthesize TG for storage in the form of lipid droplets or export in combination with VLDL. The high hepatic TG content occurred more frequently in HFD-induced NAFLD model (
Apart from FAs and TG, the CH, more specifically, free CH is also a major lipotoxic molecule in the fatty liver injury. Hepatic free CH accumulation can induce ER stress, mitochondrial dysfunction, oxidative damage, inflammation and liver injury (Reviewed by
In hepatocytes, CH homeostasis is maintained through a complex pathway including cholesterol uptake,
Hepatic CH efflux is associated with multiple pathways, including conversion to bile acids via catalysis of CYP7A1, export incorporation into VLDL, and secretion combination with nascent HDL particles with the assistance of Apoa1 (
The ER is pivotal organelle with major function in hepatic lipid metabolism including lipid synthesis, storage and export. Unfolded protein response (UPR), a highly conserved pathway in ER, monitors the status of ER protein assembly and lipid metabolism, and serves to restore ER homeostasis (
Autophagy plays important roles in regulation of hepatic lipid metabolism. In hepatocyte, LDs can be engulfed to form autophagosomes, and then generate autolysosomes by fusing to lysosome (
TFEB is a master regulator in lysosome biogenesis and function. Impaired TFEB inhibited lysosome biogenesis and autophagy, which deteriorated liver injury and steatosis (
Autophagy can be regulated through different pathways including the classic AMPK pathway. Activation of AMPK induced autophagy to attenuate HFD-induced liver steatosis, but inhibition of AMPK abolished the improvement of liver steatosis and autophagy activation (
In summary, the current study demonstrated that HFD feeding could induce steatosis via increasing FAs uptake, impairing FAs β-oxidation, blocking VLDL assembly and enhancing HDL particles formation (
Possible mechanisms of lipotoxicity induced by HFD in tilapia. HFD feeding increased fatty acids (FAs) and free CH (cholesterol) via uptake from plasma. Meanwhile, suppression of FAs β-oxidation, VLDL assembly and CH efflux induced by HFD feeding also enhanced the hepatic FAs and CH content. Increased FAs and CH promoted TG formation which induced hepatic steatosis. Excessive FFA and free CH could cause ER stress and further accelerated steatosis by IRE1-XBP1s signaling pathway. In HFD-induced fatty liver, the decreased AMPK pathway inhibited formation of UKL1 and Atg13 complex, hampered autophagy proteins expression, and ultimately resulted in low autophagy. Moreover, the low autophagy was related to depressed lysosomal biogenesis mediated by TFEB pathway. Suppressed autophagy blocked decomposition of lipid droplets and worsened steatosis.
All datasets generated for this study are included in the article/
The animal study was reviewed and approved by Freshwater Fisheries Research Centre (FFRC) of the Chinese Academy of Fishery Sciences.
RJ designed the study and wrote the manuscript. L-PC, and J-LD provided technical assistance and analyzed the data for the study. QH and Z-YG conducted the experiments. GJ corrected writing and grammar mistakes. PX and G-JY directed the project and edited the manuscript. All authors read and approved the final version of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at:
acetyl-co-enzyme A acetyltransferase 2
acetyl-CoA carboxylase
acyl-CoA oxidase
AMP-activated protein kinase
apolipoprotein A1
apolipoprotein
AMP-dependent transcription factor 6
autophagy
adipose triglyceride lipase
cluster of differentiation 36
C/EBP-homologous protein
carnitine palmitoyltransferase 1
sterol 7a hydroxylase
diacylglycerol acyltransferase 1
eukaryotic translation initiation factor 2 α
endoplasmic reticulum
fatty acid binding protein 1
fatty acids
FA synthase
free fatty acid
farnesoid X receptor
glycerol-3-phosphate acyltransferase 1
78 kDa glucose-regulated protein
hematoxylin and eosin
high density lipoprotein cholesterol
high-fat diet
3-Hydroxy-3-methylglutaryl-CoA reductase
hormone sensitive lipase
including inositol-requiring enzyme 1
microtubule-associated protein 1 light chain-3B
low density lipoprotein cholesterol
low density lipoprotein receptor
hepatic lipase
lipoprotein
liver X receptor
mucolipin-1
mammalian target of rapamycin
microsomal triglyceride transfer protein
monounsaturated fatty acid
non-alcoholic fatty liver disease
protein kinase R (PKR)-like endoplasmic reticulum kinase
peroxisome proliferator-activated receptor γ
polyunsaturated fatty acid
polyvinylidene
saturated fatty acid
sterol regulatory element binding protein 2
total cholesterol
transmission electron microscope
transcription factor EB
triacylglycerol
ubiquitin-conjugating enzyme
unc-51 like autophagy activating kinase 1
UV radiation resistance associated gene
very low-density lipoproteins
spliced X box binding protein 1.