Two experiments were carried out to examine the impacts of hydroxytyrosol (HT) on lipid metabolism and mitochondrial function in Megalobrama amblycephala. Triplicate groups of fish were fed four test diets: (1) low-fat diet (LFD, 5% fat), (2) high-fat diet (HFD, 15% fat), (3) LFD + 100 mg/kg HT (LFD + HT), and (4) HFD + 100 mg/kg HT (HFD + HT) (in vivo). Hepatocytes from the same batch were exposed to three media including L-15 medium (L15), oleic acid (OA) medium [L15 + 400 μM OA], and OA + HT medium [L15 + 400 μM OA + 10 μM HT] to explore the roles of HT in mitochondrial function (in vitro). Fish fed HFD had excessive fat deposition in the liver, and HT inclusion in the HFD decreased hepatic fat deposition. Transmission electron microscopy revealed that the HFD triggers loss of cristae and metrical density and hydropic changes in mitochondria and that HT supplementation attenuates the ultrastructural alterations of mitochondria. The in vitro test showed that HT decreases fat deposition in hepatocytes, suppresses the reactive oxygen species formation, and facilitates the expression of phospho-AMPK protein and the genes involved in mitochondria biogenesis (PGC-1, NRF-1, TFAM) and autophagy (PINK1, Mul1, Atg5). These findings suggest the lipid-lowering effect of HT mediated by activation of mitochondrial biogenesis and autophagy through the AMPK pathway.

Emerging evidence suggests that mitochondrial dysfunction mediates the pathogenesis for non-alcoholic fatty liver disease (NAFLD). Hydroxytyrosol (HT) is a key component of extra virgin olive oil which can exert beneficial effects on NAFLD through modulating mitochondria. However, the mechanism of the impacts of HT still remains elusive. Thus, an in vivo and a series of in vitro experiments were carried out to examine the impacts of hydroxytyrosol (HT) on lipid metabolism and mitochondrial function in fish. For the in vivo experiment, two diets were produced to contain 10% and 16% fat as normal-fat and high-fat diets (NFD and HFD) and two additional diets were prepared by supplementing 200 mg/kg of HT to the NFD and HFD. The test diets were fed to triplicate groups of spotted seabass (Lateolabrax maculatus) juveniles for 8 weeks. The results showed that feeding HFD leads to increased fat deposition in the liver and induces oxidative stress, both of which were ameliorated by HT application. Furthermore, transmission electron microscopy revealed that HFD destroyed mitochondrial cristae and matrix and induced severe hydropic phenotype, while HT administration relieved these alterations. The results of in vitro studies using zebrafish liver cell line (ZFL) showed that HT promotes mitochondrial function and activates PINK1-mediated mitophagy. These beneficial effects of HT disappeared when the cells were treated with cyclosporin A (Csa) as a mitophagy inhibitor. Moreover, the PINK1-mediated mitophagy activation by HT was blocked when compound C (CC) was used as an AMPK inhibitor. In conclusion, our findings demonstrated that HT alleviates fat accumulation, oxidative stress and mitochondrial dysfunction, and its effects are deemed to be mediated via activating mitophagy through the AMPK/PINK1 pathway.

Oxidative stress is a common phenomenon in aquaculture, which can be induced by nutritional or environmental factors. Generally, oxidative stress causes poor growth performance, metabolic dysregulation, and even the death of aquatic animals. To identify a nutritional intervention strategy, high-fat diet (HFD) feeding (Experiment I) and acute ammonia nitrogen challenge (Experiment II) tests were carried out. In Experiment I, HFD feeding significantly decreased the growth performance concomitantly with excessive fat deposition in the liver and abdomen. The addition of 4-PBA in the diet improved the excessive fat accumulation. The activities of antioxidative enzymes were suppressed, and the levels of lipid and protein peroxidation were increased, indicating that HFD feeding induced oxidative stress. The endoplasmic reticulum stress (ERs) related genes were downregulated in the HFD group. Under a transmission electron microscope (TEM), more swollen and dilated ER lumen could be observed. These results indicated that the HFD induced ERs activation. Although 4-PBA acted as a potent ERs inhibitor, as evidenced by the alleviated alterations of ERs molecules and the ER ultrastructure, the oxidative stress was also attenuated by 4-PBA. In Experiment II, dietary 4-PBA improved the tolerance to the acute ammonia nitrogen challenge, as lower mortality and serum aminotransferase activity was found. Further results showed that 4-PBA decreased the peroxidation content and attenuated ERs, thus confirming the correlation between oxidative stress and ERs. Our findings showed that dietary 4-PBA supplementation can attenuate oxidative stress induced by a HFD or acute ammonia challenge; the mechanism is related to its potent inhibition effect for ERs.

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.