Investigation Of The Effect And Regulatory Mechanism Of Mitochondrial Fatty Acid β-oxidation Inhibition On Protein Synthesis And Deposition In Fish

Fish are an important source of dietary protein,and improving their capacity for protein synthesis to increase their protein supply is one of the main goals in the current aquatic research field.The mechanistic target of rapamycin(mTOR),a key regulator of protein synthesis,plays a critical role in protein synthesis and is closely associated with protein anabolism mediated via fish energy metabolism regulation.Studies in both mammals and fish have found that inhibition of mitochondrial fatty acid β-oxidation(mFAO)activates mTORC1 and promotes protein synthesis and deposition,but the exact regulatory mechanism is unknown.In addition,studies in recent years have found that protein acetylation modification mechanisms are very closely linked to both energy metabolism and mTORC1 activity regulation,and are an important bridge connecting energy metabolism and the mTORC1 pathway,but no studies have yet confirmed this.In this study,the model fish zebrafish was used as the research object,and the experiments were conducted by isotope-labeled nutrient tracing,modification-specific proteomics,real-time fluorescence quantitative PCR,western blot,coimmunoprecipitation,high performance liquid chromatography detection,immunofluorescence,and gene manipu Lation.First,we constructed zebrafish mFAO inhibition models in vivo and in vitro by Mildronate(MD)treatment or cpt1 ab knockout,and confirmed the effects of mFAO inhibition on mTORC1 pathway activation as well as protein synthesis.Then,we explored the effect of mFAO inhibition on protein acetylation modification in zebrafish and verified the relationship between cytoplasmic protein acetylation modification and mTORC1 pathway activation.On this basis,this study further elucidated the effect of Raptor protein acetylation modification on mTORC1 pathway activation.Furthermore,the present study carried out a systematic screening for regulatory enzymes related to the Raptor protein acetylation modification.A final validation trial was performed in the economic fish Nile tilapia.The main results and conclusions of this study are as follows:1.Effects of mFAO inhibition on mTORC1 pathway and protein deposition in zebrafishmTORC1 is a key regulator of protein anabolism and plays an important role in the regulation of cell proliferation and protein synthesis.Studies in mammals and fish have found that mFAO inhibition triggers metabolic remodeling,activates the mTORC1 pathway and promotes protein synthesis.To further clarify the effects of mFAO inhibition on nutrient metabolism,mTORC1 pathway and protein anabolism.This study was conducted in zebrafish,and mFAO inhibition models were constructed in vivo and in vitro by dietary feeding of 1 g/1 kg of Mildronate(MD),cpt1 ab gene knockout(cpt1ab-/-)or treatment of muscle or liver cells with 1 m M MD,respectively.The results showed that MD treatment could effectively inhibit mFAO,enhance glucose oxidative catabolism,and promote protein synthesis and zebrafish growth.Both MDtreated and cpt1ab-/-zebrafish had significantly elevated carcass protein content and carcass ratio.Also,MD treatment significantly promoted the proliferation of zebrafish liver and muscle cells.In addition,both in vivo and in vitro studies revealed that mFAO inhibition significantly activated the mTORC1 pathway.Taken together,mFAO inhibition could activate the mTORC1 pathway,increase protein synthesis and deposition,and promote cell proliferation and zebrafish growth.2.Effect of mFAO inhibition on protein acetylation modification in zebrafishProtein acetylation modification can affect the protein properties and thus mediate the protein function regulation.Studies revealed that acetyl-CoA,a central product of nutrient metabolism,can influence protein acetylation modification and thus participate in the physiological metabolic regulation,and there is a close association between protein acetylation modification and mTORC1 pathway activity regulation.Currently,studies related to protein acetylation modifications in fish are still lagging behind.Therefore,this study was conducted to investigate the fish protein acetylation modification and its effect on mTORC1 pathway activity in different zebrafish mFAO inhibition models.The results showed that mFAO inhibition significantly increased the production of glucose-derived acetyl-CoA and elevated the global protein acetylation modification level.Acetylation modification proteomics revealed that a large number of proteins in zebrafish are capable of lysine acetylation modification,and the metabolic enrichment pathways closely linked to mFAO inhibition are glycolysis and TCA cycle pathways.Immunofluorescence results showed that protein acetylation induced by mFAO inhibition occurred mainly in the cytoplasm,while inhibition of cytoplasmic protein acetylation significantly reduced mTORC1 pathway activity.The results of the present study suggest that the protein acetylation modification mechanism is conserved between mammals and fish,and that cytoplasmic protein acetylation modification mediates the mTORC1 pathway activation induced by mFAO inhibition.3.Raptor acetylation modification mediates the mTORC1 pathway activation induced by mFAO inhibitionOur preceding studies have shown that intracellular protein acetylation modification is involved in the regulation of mTORC1 pathway activity.Studies in mammals have also found that the mTORC1 subunit Raptor can be acetylated to activate the mTORC1 pathway.Therefore,the present study first examined the Raptor acetylation modification level in different zebrafish mFAO inhibition models and further investigated whether Raptor acetylation modification mediates the mTORC1 pathway activation induced by mFAO inhibition at the cellular level.The results showed that mFAO inhibition significantly increased the acetylation modification of Raptor in vivo and in vitro,and that raptor knockdown blocked the mTORC1 pathway activation induced by mFAO inhibition;further inhibition of Raptor acetylation modification also significantly reduced the mTORC1 activity increased by mFAO inhibition.The results of this part of the study indicate that zebrafish Raptor protein acetylation modification mediates the mTORC1 pathway activation induced by mFAO inhibition.4.Inhibition of mFAO activates the mTORC1 pathway dependent on Gcn5-mediated Raptor acetylation modificationProtein acetylation modification usually requires the involvement of related acetylases.To elucidate the acetylases of zebrafish Raptor acetylation modification,we carried out a systematic screening study in ZFL cells.The results showed that mFAO inhibition significantly increased the gene and protein expression level of acetylase Gcn5,while inhibition of acetylase activity of Gcn5 significantly decreased the mTORC1 activity induced by mFAO inhibition.The protein interaction prediction analysis also revealed a direct protein-protein interaction between Gcn5 and Raptor,which was confirmed by subsequent co-immunoprecipitation experiments.Further studies showed that inhibition/knockdown of Gcn5 significantly inhibited the acetylation modification of Raptor and was accompanied by a decrease in mTORC1 pathway activity.In addition,we also explored the regulatory effects of other nonhistone acetylases P300 and MYST on the Raptor acetylation modification and found that they did not mediate the acetylation modification of Raptor.The study in this section show that the mTORC1 pathway activation induced by mFAO inhibition in zebrafish is mainly dependent on Raptor acetylation modification mediated by the acetylase Gcn5.5.Lysine deacetylase family HDACs mediate deacetylation modification of zebrafish RaptorLysine acetylation modification of proteins is dynamically reversible and regulated by both lysine acetylase and deacetylase.Therefore,in order to more comprehensively elucidate the mechanism of Raptor acetylation regulation,we continued our screening study of Raptor deacetylases in ZFL cells.The results showed that inhibition of the deacetylase activity of HDACs and Sirtuins using their inhibitors,TSA and NAM,respectively,could significantly enhance the global intracellular protein acetylation modification level.However,only TSA treatment significantly increased the level of Raptor acetylation modification and activated the mTORC1 pathway.Further,inhibition of mFAO significantly downregulated gene expression of hdac7(belonging to HDAC class IIa of the HDACs family)in ZFL cells.This part of the study shows that the deacetylation modification of Raptor in zebrafish is mediated by deacetylases HDACs rather than Sirtuins,of which Hdac7 is a potential key candidate enzyme.6.Dietary sodium acetate increases hepatic acetyl-CoA and Raptor acetylation level and promotes protein deposition and growth in Nile tilapiaShort-chain fatty acids(SCFAs)play an important role in host energy metabolism and physiological regulation.Acetate,as the most abundant SCFAs,can enter central metabolism by direct synthesis of acetyl-CoA through acetate metabolism.Therefore,acetate and its derivative salts are often used as precursors of acetyl-CoA to explore the role of acetyl-CoA in metabolism and physiological regulation.In this study,an 8-week culture trial was conducted on the economic fish Nile tilapia(2.0-3.0 g)by dietary supplementation with sodium acetate(1800 mg/kg).In order to elucidate whether the increase of exogenous acetyl-CoA can also mediate mTORC1 pathway regulation through Raptor acetylation modification mechanism and affect the protein anabolism and growth of Nile tilapia.The results showed that dietary sodium acetate addition significantly promoted Acss1/2 protein expression and increased acetyl-CoA content in liver,accompanied by increased Raptor acetylation modification level and mTORC1 pathway activity.Meanwhile,the addition of dietary sodium acetate also inhibited the amino acid catabolism and significantly promoted protein deposition and growth in Nile tilapia.This part of the study shows that Raptor,as an intracellular acetyl-CoA sensor,also receives exogenous acetyl-CoA signaling stimuli and mediates metabolic physiological regulation through activating the mTORC1 pathway.