Quercetin Attenuates High-Fat Diet-Induced Excessive Fat Deposition of Spotted Seabass (Lateolabrax maculatus) Through the Regulatory for Mitochondria and Endoplasmic Reticulum

This study aimed to investigate the effects of quercetin (QUE) on fat deposition and the underlying mechanism. Fish were fed four test diets: normal fat diet (NFD), high-fat diet (HFD), and HFD supplemented with 0.5 or 1.0 g/kg quercetin (QUE0.5 or QUE1.0). The results showed that HFD feeding resulted in poor growth and feed utilization while QUE treatment reversed this. The fat contents of serum and liver were increased by HFD and QUE supplementation significantly decreased fat content. Furthermore, gene expressions and ultrastructure observation showed that mitochondrial biogenesis and mitophagy were inhibited and endoplasmic reticulum stress (ERS) in the HFD group. QUE can activate the biogenesis and autophagy of mitochondria and suppress ERS, which is related to its fat-lowering effect.


INTRODUCTION
Fat plays a key role in metabolism as it supplies energy, essential fatty acids, and phospholipids for animals (Watanabe, 1982). It is reported that the increase of dietary lipid level moderately can improve feed efficiency and protein deposition (Du et al., 2006). Therefore, high-fat feed is widely used in fish farming. However, recent studies have indicated that the intake of high fat diet (HFD) often induces excess fat deposition, metabolic disorders, and immune dysfunction (Jin et al., 2013;Lu et al., 2014;Xie et al., 2021a;Yin et al., 2021).
It is well-known that fat deposition in the liver represents complex processes, such as uptake, oxidation, transport, and so on (Tilg and Moschen, 2010;Rocha et al., 2020). As the main state of fatty acid oxidation, mitochondrial damage caused by oxidation retardation is the immediate reason for excessive hepatic fat deposition (Rector et al., 2010;Tilg and Moschen, 2010). The lower hepatic fat secretion means an attenuated capacity for transporting lipids out of the liver of fish fed an HFD, which largely contributes to fat deposition in the liver (Lu et al., 2014(Lu et al., , 2017. Moreover, the damaged mitochondria often generate excess reactive oxygen species (ROS) and lead to oxidative stress thereby perpetuating the vicious cycle (Wallace, 2007;Shadel and Horvath, 2015). When the endoplasmic reticulum is damaged by ROS, it will generate superfluous abnormal protein folding and then endoplasmic reticulum stress (ERS) (Malhotra and Kaufman, 2007). Further, there is crosstalk and/or interaction between mitochondrial damage and ERS. Based on the above, more researchers suggest that the therapies acting on mitochondria and ER are novel strategies for the prevention and protection against excess fat deposition Dong et al., 2020).
Quercetin (QUE), a flavonoid, is widely present in various plants, such as tea, fruitage, and vegetables (Formica and Regelson, 1995;Boots et al., 2008). In mammals, many studies evidenced the regulatory effects of QUE on mitochondria and ER under metabolism diseases (Porras et al., 2017;Liu et al., 2018). In addition, the previous studies showed the antioxidant effects of QUE in fish (Pês et al., 2016(Pês et al., , 2018. Furthermore, QUE could alleviate HFD-induced fat accumulation in blunt snout bream (Megalobrama amblycephala) (Jia et al., 2019). However, the molecular mechanism of QUE on fat deposition still needs further study.
Spotted seabass (Lateolabrax maculatus) is a carnivorous species that has been largely cultured in East Asia, particularly in China. The lipid requirement of this fish is ∼10%, while the lipid content is often >15% in commercial feeds (Xu et al., 2011;Xie et al., 2021b). Compared to other cultured fish species, its artificial rearing is often associated with fatty liver probably due to its carnivorous instinct and high dietary lipid level. Hence, this fish model was used to investigate the effects of QUE on fat deposition, mitochondria, and endoplasmic reticulum status.

Ethics Statement
The feeding trial was conducted according to the principles of the Basel Declaration and Recommendations of Animal Research Institute Committee guidelines, Jimei University, China.

Diets, Feeding Test, and Sampling
According to the nutrition requirements of spotted seabass, a normal fat diet (NFD) with 11% crude lipid and an HFD with 17% crude lipid were formulated, named as NFD and HFD, respectively. QUE treatments were prepared by the supplementary of 0.5 or 1.0 g/kg QUE (Shanghai Aladdin Biochemical Technology Co., Ltd., China) to HFD. The formulation and proximate composition of the experimental diets are presented in Table 1. The method of feed manufacturing referred to the previous study (Cai et al., 2020).
For the feeding test, fish were obtained from a fish hatchery in Zhangzhou (Fujian, China). First, the fish were kept in a 1,500-L tank to adapt to the experimental facilities and conditions. During this period, fish were fed a commercial diet two times a day (8:00 and 17:00). At the end of the acclimation period, 240 healthy fish averaging 12.02 ± 0.17 g were randomly distributed into 12 circular fiberglass tanks (200 L) in a recirculating aquaculture system. The experiment was conducted using three replicates for each dietary treatment, and fish were fed the diets to apparent satiation two times a day (8:00 and 17:00) for 8 weeks. During the feeding test, the water conditions were maintained as following: water temperature 26-27 • C; pH 6.9-7.2; dissolved oxygen > 5.5 mg/L. After 8 weeks of feeding, fish were starved 24 h and weighed for calculation of growth parameters. Six fish per tank were anesthetized with 100 mg/L MS-222 (Sigma, Ronkonkoma, NY, USA), randomly selected, and sampled for analysis. Blood was collected from the caudal vein and centrifuged to gain serum. The liver was sampled on ice, flash frozen in liquid nitrogen, and then stored at −80 • C. Moreover, liver samples for transmission electron microscope examination were fixed in the 2.5% glutaraldehyde solution.

Measures of Biochemical Parameters
The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and the contents of triacylglycerols (TG), total cholesterol (T-CHO), and non-esterified-free fatty acids (NEFA) were measured by commercial kits (Nanjing JianCheng Bioengineering Institute, Nanjing, China).
Total lipid level in the liver was detected by Folch's method (Folch, 1957). Malondialdehyde (MDA) content was measured by the thiobarbituric acid method and superoxide dismutase (SOD) activity was determined by the hydroxylamine method, according to our previous study . The reduced glutathione (GSH) content, catalase (CAT) activity, and total antioxidant capacity (T-AOC) were measured by commercial kits (Nanjing JianCheng Bioengineering Institute, Nanjing, China). The protein level in the liver homogenate was determined by the BCA protein assay kit (LABLEAD Inc., Beijing, China).  (Cai et al., 2020). b Reference gene.

Analysis of Mitochondrial Status
Mitochondria were isolated from a fresh liver sample by a tissue mitochondria isolation kit (C3606, Beyotime Biotechnology, China). Then, the T-AOC and MDA contents of isolation mitochondria were determined. Another fresh liver was used to made single-cell suspension by mechanical method for mitochondrial ROS measurement. The hepatocyte was incubated with 5-µM MitoSOX Red (M36008, Invitrogen, Waltham, MA, USA) at 28 • C for 10 min, and the fluorescence was determined by a microplate reader (Thermo Scientific, Waltham, MA, USA).

Histology Examination
For transmission electron microscopy analysis, liver samples were fixed in cold 2.5% glutaraldehyde solution for 24 h and post-fixed in 1% OsO 4 at 4 • C for 2 h. Then, the ultrathin slices were cut into 60-nm thickness after being embedded in epoxy resin, stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (Hitachi H-7650, Tokyo, Japan).

Statistical Analysis
One-way ANOVA was used by the IBM SPSS 20 program. Tukey's test was used in significant difference analysis and P < 0.05 was considered as significant level. All data were shown as means ± SE with three replicates.

Growth Performance and Fat Deposition
Fish-fed HFD showed the lowest weight gain (WG) and specific growth rate (SGR; P < 0.05). WG and SGR were significantly increased in the two QUE groups (P < 0.05), and fish in the QUE1.0 group exhibited the highest WG. The feed conversion rate (FCR) in the HFD group was higher than that of the NFD group, and the tendency of protein efficiency ratio (PER) was the opposite. QUE supplementation decreased FCR and increased PER, and the QUE1.0 group exhibited the lowest FCR and the highest PER among all groups (Table 3). Fish-fed HFD showed excess fat deposition in the liver comparing to other groups. The contents of TG, T-CHO, and NEFA in the liver of fish-fed HFD were significantly higher than those of fish-fed NFD. The supplementation of 1 g/kg QUE  significantly decreased the contents of total lipid, TG, T-CHO, and NEFA (Figure 1).

Blood Chemistry
Both ALT and AST activities of the NFD group were significantly higher than the HFD group, while the QUE application decreased these two transaminase activities. For fat deposition, TG, T-CHO, and NEFA contents were all remarkably higher in fish-fed NFD compared to the NFD group. Meanwhile, the QUE application decreased these contents (Figure 2).

Oxidative Status in Liver and Mitochondrion
In the liver, the activities/levels of CAT, SOD, GSH, and T-AOC of fish-fed HFD were significantly decreased, while MDA content was significantly increased. Both 0.5 and  1 g/kg supplementary of QUE dramatically elevated SOD activity, GSH content, and T-AOC and reduced MDA content (Figure 3).
In mitochondrion, fish-fed HFD showed the lowest T-AOC and the highest contents of MDA and ROS. Meanwhile, the supplementation of 0.1% QUE significantly enhanced  T-AOC and reduced the contents of MDA and ROS (Figure 4).

Mitochondrial Status
The liver of fish-fed NFD showed normal ultrastructure. In these fish, the nucleus was round and the nucleolus was visible. Hepatocytes displayed dark and slender mitochondria. However, abnormal ultrastructure was found in mitochondria of fish-fed HFD, such as swell and nucleus atrophy. Further, the HFD-induced abnormal of mitochondria can be attenuated in QUE-fed fish (Figure 5).

Endoplasmic Reticulum Status
The expressions of ERS-related genes (chop, grp78, ire1, atf6, and perk) were significantly increased in the HFD group, and the application of QUE significantly downregulated the expressions of these genes (Figure 7).

DISCUSSION
Similar to some previous studies, fish-fed HFD showed poor growth, excess fat deposition, and increased transaminase activities (Dong et al., 2020;Yin et al., 2021). QUE supplementation mitigated the decreasing growth performance and excess fat deposition, which is in agreement with previous studies (Jia et al., 2019). NEFA in the liver is a primary cause of lipotoxicity (Brent, 2010), while QUE can decrease its level and thus attenuate lipotoxicity. Excess fat often induces more ROS in mitochondria and causes oxidative stress (Marí et al., 2006). It is reported that QUE can be served as an antioxidant FIGURE 7 | The expressions of ERS-related genes in the liver of fish fed with experimental diets. ERS, endoplasmic reticulum stress. Different letters indicate a significant difference among groups (P < 0.05).
due to its free radical scavenging capacity (Boots et al., 2008;Pês et al., 2016Pês et al., , 2018. Similarly, our study also showed the antioxidant ability of QUE. It is considered that oxidative stress is often associated with the dysfunction of the mitochondrion (Lu et al., 2014(Lu et al., , 2017. This was evidenced that the over ROS production of mitochondria in fish-fed HFD. Further, the HFD-induced abnormality of mitochondria and oxidative stress can be attenuated in QUE application. These findings indicate the regulatory effects of QUE on mitochondrion. The mitochondria network in metabolism tissues is a dynamic state (Auger et al., 2015). Under normal conditions, new mitochondria can be constantly produced and the aberrant ones should be removed by mitophagy (Palikaras and Tavernarakis, 2014). The suppression of biogenesis and/or elimination will cause the dysfunction of the mitochondrion (Greene et al., 2012). PGC-1 is a key factor for mitochondrial biogenesis due to its promotion for downstream genes transcription, such as nrf1 (Rector et al., 2010). PINK1 is an activator in the classical mitophagy pathway stimulating E3 ubiquitin ligases to ubiquitinate membrane proteins of dysfunctional mitochondria (Tolkovsky, 2009). This study indicated that HFD feeding arrested mitochondrial biogenesis and mitophagy by the downregulation of the genes. QUE can protect mitochondria through activation of the gene expressions. In addition, it was reported that QUE can play a key role in the mitochondrial homeostasis of mice .
It is considered that there is a crosstalk between the endoplasmic reticulum and mitochondrion (Daw et al., 2020). Previous studies have indicated that ERS can be caused by the dysfunction of the mitochondrion (Malhotra and Kaufman, 2007;Hafiz et al., 2016). The damaged endoplasmic reticulum induces ERS through unfolded proteins response by IRE1, ATF6, and PERK pathways (Walter and Ron, 2011). These unfolded proteins induce the release of molecular chaperones and then apoptosis and inflammation (Szegezdi et al., 2006;Kaplowitz et al., 2007;Urra et al., 2013). Many evidence indicate that ERS contributed to the liver damage induced by HFD (Cao et al., 2018;Jia et al., 2020). Our results also found that HFD upregulated the expression of key genes in ERS pathway. Furthermore, the application of QUE suppressed the activation of ERS by downregulating the expressions of related genes.

CONCLUSION
The supplementary of quercetin can attenuate excess fat deposition in fish tissues induced by the intake of HFD. Quercetin can activate the biogenesis and autophagy of mitochondria and suppress ERS, which is related with its fat-lowering effect.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
This animal study was reviewed and approved by the Animal Research Institute Committee of Jimei University.

AUTHOR CONTRIBUTIONS
Y-ZD and C-XZ designed the study. Y-ZD and TX conducted the experiments. Y-ZD wrote the manuscript. J-BL corrected the writing style. LW and KS provided technical support. All authors read and approved the final version of the manuscript.