Function And Regulatory Role Of Cholesterol In The Nutrient Metabolism Of Fish

Cholesterol is an important component of cell membranes and a precursor to the synthesis of biological molecules such as bile acids,vitamin D and steroid hormones.In recent years,studies in mammals clarify that cholesterol also act as a signaling molecule in the regulation of energy metabolism,such as involed in the regulation of cellular mTORC1signaling pathway.In aquaculture,it is supposed that fish can grow without exogenous cholesterol supplementation,and dietary cholesterol supplementation increases the risk of atherosclerosis and fatty liver disease in fish.In addition,many studies reported that fish fed with high-fat diet often showed obvious cholesterol accumulation.However,there are few studies on the metabolic physiological function of cholesterol in fish.Therefore,it is very important to elucidate the function and regulation mechanism of cholesterol in nutrient metabolism of fish.In this study,we analyzed the growth performance,physiology,biochemistry,molecular biology,isotope tracer and multiomics of Oreochromis niloticus and Danio rerio,and the metabolic physiological function and regulation mechanism of cholesterol in fish were systematically and comprehensively discussed.In our sutdy,the important metabolic function of cholesterol in fish metabolism was firstly discussed:1)the adaptive metabolic characteristics of cholesterol-bile acid were showen in Nile tilapia after high-fat diet intake.To a certain extent,this result clarified the physiological function of cholesterol in lipid metabolism of fish.In addition,we used the lysosomal acid lipase（LAL,which can hydrolyze cholesterol esters and triglycerides in lysosomes）gene knockout of zebrafish and zebrafish liver cell lines to elucidate the important role of cholesterol in energy metabolism of fish at the molecular level,and the result reveals the regulatory mechanism of LAL in mTORC1 signaling pathway.Furthermore,in order to explore how fishes respond to the accumulation of cholesterol in the body,we established the high-cholesterol Nile tilapia model,and systematically clarified the role of high cholesterol intake in remodeling cholesterol metabolism and energy homeostasis.Finally,the effects and mechanisms of atorvastatin（cholesterol synthesis inhibitor）and trehalose（autophagy activator）on cholesterol accumulation in fish were explored.The main results and conclusions of our study are presented as follows:1.The adaptive characteristics of cholesterol and bile acid metabolism in Nile tilapia fed a high-fat dietSince high-fat diet（HFD）intake elevates liver cholesterol,and enhanced cholesterol-bile acids flux alleviates its lipid deposition,we assumed that the promoted cholesterol-bile acid flux is an adaptive metabolism in fish when fed an HFD.The present study investigated the characteristic of cholesterol and fatty acid metabolism in Nile tilapia（Oreochromis niloticus）after feeding an HFD（13%lipid level）for four and eight weeks.Visually healthy Nile tilapia fingerlings（average weight 3.50±0.05 g）were randomly distributed into four treatments（4-week control diet or HFD and 8-week control diet or HFD）.The liver lipid deposition and health statue,cholesterol/bile acid and fatty acid metabolism were analyzed in fish after short-term and long-term HFD intake.The results showed 4-week HFD feeding did not change serum alanine transaminase（ALT）and aspartate transferase（AST）enzyme activities,along with comparable liver malondialdehyde（MDA）content.But higher serum ALT and AST enzyme activities and liver MDA content were observed in fish fed 8-week HFD.Intriguingly,remarkably accumulated total cholesterol（mainly cholesterol ester,CE）was observed in the liver of fish fed 4-week HFD,along with slightly elevated free fatty acids（FFA）and comparable TG contents.Further molecular analysis in liver showed that obvious accumulation of CE and total bile acids（TBA）in fish fed 4-week HFD was mainly attributed to the enhancement of cholesterol synthesis,esterification,and bile acid synthesis.Furthermore,the increased protein expressions of acyl-Co A oxidase 1/2（Acox1 and Acox2）,which serve as peroxisomal fatty acidβ-oxidation（FAO）rate-limiting enzymes and play key roles in the transformation of cholesterol into bile acids,were found in fish after 4-week HFD intake.Notably,8-week HFD intake remarkably elevated FFA content（about 1.7-fold increase）and unaltered TBA were found in fish liver,accompanied by suppressed Acox2protein level and cholesterol/bile acid synthesis.Therefore,the robust cholesterol-bile acid flux serves as an adaptive metabolism in Nile tilapia when fed a short-term HFD,and is possibly via stimulating peroxisomal FAO.This finding enlightens our understanding on the adaptive characteristics of cholesterol metabolism in fish fed an HFD,and provides a new possible treatment strategy against metabolic disease induced by HFD in aquatic animals.2.The regulatory role of Lal in the mTORC1 signaling pathwayCholesterol,as a signaling molecule,is also involved in the recruitment and activation of mTORC1 by lysosomes.Lysosome acid lipase（Lal）is the only known acid lipase that hydrolyzes cholesterol ester（CE）and triglyceride（TAG）in lysosome.Therefore,whether it is involved in the regulation of mTORC1 signaling pathway is still not clear.The present study explored the regulatory role of Lal in mTORC1 signaling pathway by constructing lal gene knockout zebrafish in vivo and knocking down lal or pharmacological inhibiting Lal protein in vitro zebrafish hepatocytes.The results showed that lal knockout zebrafish exhibited the retarded growth,reduced body protein and inhibited mTORC1 signaling pathway protein.Furthermore,in vitro zebrafish hepatocytes,knockdown or drug inhibition of Lal showed that a large amount of cholesterol accumulated in the lysosomes,and inhibited the recruitment of mTORC1 to the lysosomes and the activity of mTORC1 signaling pathway.In addition,cholesterol or LDL treatment in zebrafish hepatocytes indeed promoted the recruitment of mTORC1 to the lysosome and activated the mTORC1 signaling pathway,thus promoting the growth and proliferation of hepatocytes.When Slc38a9 was knocked down on zebrafish hepatocytes and then treated with cholesterol or LDL,the recruitment and activation of mTORC1 were significantly inhibited.Therefore,Slc38a9 also acts as a conserved cholesterol sensing protein in zebrafish.Furthermore,knockdown or drug inhibition of Lal also enhanced the interaction between P14 and Rag A/C,indicating that the functional deficiency of Lal sensed cholesterol level through Slc38a9 protein and regulated the recruitment and activation of mTORC1 through Ragulator complex.3.High cholesterol intake remodels cholesterol turnover and energy homeostasis in Nile tilapiaThe roles of dietary cholesterol in fish physiology are currently contradicting.The dispute is attributed by limited studies on the metabolic consequences of cholesterol intake in fish.The present study investigated the metabolic responses to high cholesterol intake in fish.Nile tilapia（Oreochromis niloticus）were fed with four cholesterol-contained diets（0.8,1.6,2.4 and 3.2%）and a control diet for eight weeks.Fish fed cholesterol diets all showed increased body weight but accumulated cholesterol（the peak level in 1.6%cholesterol group）.Thus,we then selected 1.6%cholesterol and control diets for further analysis.The high cholesterol diet impaired liver function and reduced mitochondria number in fish.Furthermore,high cholesterol intake triggered protective adaptation via 1)inhibiting endogenous cholesterol synthesis,2)elevating expression of genes related to cholesterol esterification and efflux,and 3)promoting chenodeoxycholic acid synthesis and efflux.Accordingly,high cholesterol intake reshaped fish gut microbiome by increasing the abundance of Lactobacillus spp.and Mycobacterium spp.,which are involved in cholesterol and/or bile acids catabolism.Moreover,high cholesterol intake inhibited lipid catabolic activities,through mitochondrialβ-oxidation and lysosome-mediated lipophagy,and depressed insulin signaling sensitivity.Protein catabolism was elevated as a compulsory response to maintain energy homeostasis.Therefore,although high cholesterol intake promoted growth,it caused metabolic disorders in fish.Our study provides the systemic metabolic response to high cholesterol intake in fish for the first time.This knowledge helps to understand the metabolic syndromes caused by high cholesterol intake or deposition in fish.4.Atorvastatin remodels lipid distribution between liver and adipose tissues through blocking lipoprotein efflux in fishThe regulation of cholesterol metabolism in fish is still unclear.Statins play important roles in promoting cholesterol metabolism development in mammals.However,studies on the role of statins in cholesterol metabolism in fish are currently limited.The present study evaluated the effects of statins on cholesterol metabolism in fish.Nile tilapia（Oreochromis niloticus）were fed on control diets supplemented with three atorvastatin levels（0,12 and 24mg/kg diet,ATV0,ATV12 and ATV24,respectively）for four weeks.Intriguingly,the results showed that both atorvastatin treatments increased hepatic cholesterol and triglyceride contents mainly through inhibiting bile acid synthesis and efflux,and compensatorily enhancing cholesterol synthesis in fish liver（P<0.05）.Moreover,atorvastatin treatment significantly inhibited hepatic very-low-density lipoprotein（VLDL）assembly and thus decreased serum VLDL content（P<0.05）.However,fish treated with atorvastatin significantly reduced cholesterol and triglycerides contents in adipose tissue（P<0.05）.Further molecular analysis showed that atorvastatin treatment promoted cholesterol synthesis and lipogenesis pathways,but inhibited lipid catabolism and low-density lipoprotein（LDL）uptake in the adipose tissue of fish（P<0.05）.In general,atorvastatin induced the remodeling of lipid distribution between liver and adipose tissues through blocking VLDL efflux from the liver to adipose tissue of fish.Our results provide a novel regulatory pattern of cholesterol metabolism response caused by atorvastatin in fish,which is distinct from mammals:cholesterol inhibition by atorvastatin activates hepatic cholesterol synthesis and inhibits its efflux to maintain cholesterol homeostasis,consequently reduces cholesterol storage in fish adipose tissue.5.Trehalose alleviated hepatic cholesterol accumulation via inhibiting transformation from glucose-derived acyl-Co A to cholesterol synthesis in Nile tilapiaTrehalose has been identified as an autophagy enhancer with atheroprotective properties in mammals that reduces cholesterol accumulation in the body.However,such effects have been rarely explored in aquatic animals.This study examined the effects of trehalose supplementation in a high-cholesterol diet on liver cholesterol accumulation and the related mechanisms in Nile tilapia（Oreochromis niloticus）.The fish were fed high cholesterol（1.6%）diets supplemented with different levels（0,1.5,3 and 4.5%）of trehalose for eight weeks.The results showed that inclusion of 3%trehalose in high-cholesterol diet led to the significant reduction of viscerosomatic index（VSI）,hepatosomatic index（HSI）and total and free cholesterol（TC and FC）accumulation in the serum and liver of fish.Moreover,3%trehalose supplementation decreased serum alanine aminotransferase（ALT）and aspartate aminotransferase（AST）activities,indicating the alleviation of high-cholesterol diet-induced liver impairment.Surprisingly,such beneficial effects did not correlate with autophagy activity since dietary inclusion of 3%trehalose inhibited the expression of autophagy-related genes and proteins（atg5/12,autophagy related gene 5/12;Lc3,microtubule-associated protein 1 light chain 3 and Beclin protein）.Also,the same group inhibited the expression of genes related to hepatic cholesterol synthesis,glycogen metabolism,glycolysis,and glucose oxidative phosphorylation.These results indicated that trehalose treatment reduced the capability of glucose catabolism in the liver of high-cholesterol diet-fed fish.In the primary hepatocytes of Nile tilapia,which were cultured in a medium containing high cholesterol concentration,the[1-¹⁴C]-glucose tracking test showed that trehalose treatment resulted in decreased cellular retention of ¹⁴C-glucose and unaltered release of ¹⁴CO₂.Likewise,the trehalose treatment inhibited the expression of genes associated with glucose uptake and catabolism,cholesterol synthesis.Also,the inhibited autophagy activities were found in the primary hepatocytes treated with trehalose.Overall,trehalose treatment alleviated high-cholesterol diet-induced hepatic and serum cholesterol accumulation in Nile tilapia,but such effect was not associated with the autophagy activities.Alternatively,our results suggested that trehalose exerted a cholesterol-lowering effect via inhibiting the transformation from glucose-mediated acyl-Co A to cholesterol synthesis in the liver of fish.This is the first study revealing the cholesterol-lowering function of trehalose in fish and could contribute to developing new strategies for maintaining liver health in fish.In conclusion,cholesterol plays an important role in the nutritional metabolism of fish:on the one hand,the enhancement of liver cholesterol-bile acid flow in Nile tilapia under short-term HFD is an adaptive metabolic characteristic in response to HFD,which may be related to the activation of peroxisome FAO.On the other hand,cholesterol,as a nutritional signaling molecule,is involved in the regulation of energy metabolism in animals.In zebrafish,LAL dysfunction senses the decrease of free cholesterol level through lysosomal cholesterol receptor Slc38a9,and mediates Rag-Ragulator complex to inhibit the recruitment and activation of mTORC1 protein.However,cholesterol accumulation can damage liver health and metabolic disorders in fish.High cholesterol intake inhibits lipid catabolism by damaging mitochondrialβoxidation and lysosomal mediated lipotophagy in fish liver,and also inhibites insulin sensitivity in liver.The enhancement of protein catabolism is a compensation mechanism for maintaining energy homeostasis.Furthermore,we explored an effective way to alleviate cholesterol accumulation in fish,and found that,atorvastatin remodels lipid distribution between liver and adipose tissuees by inhibiting VLDL transport from liver to adipose tissue,whereas trehalose plays a cholesterol-lowering role by inhibiting the synthesis of cholesterol by acetyl-co A from glucose in fish liver.