The Interaction Between Lipophagy And Lipolysis In Regulation Of Lipid Metabolism In Fish

Intracellular lipid droplets(LDs)are dynamic and complex organelles consisting of core neutral lipids,including triacylglycerol(TAG)and cholesterol esters(CE),encircled by a phospholipid monolayer.The LDs perform essential functions in energy metabolism by regulating lipid storage and release.At present,lipolysis and lipophagy are currently recognized as the two main processes for LDs degradation in mammalian cells.The former works through adipose triglyceride lipase(ATGL)-hormone sensitive lipase(HSL)-monoacylglycerol lipase(MGL)cascade pathway to break down TAGs.The latter is a more complicated biological processes: 1)LDs are engulfed by doublemembrane vesicles(phagophores: LC3-II positive membranes)to form autophagosomes;2)autophagosomes fuse with lysosomes to form autolysosomes;3)acidic lipases hydrolyse lipids to glycerol and non-esterified free fatty acids(NEFFA).The fatty acids then undergo further metabolism,such as mitochondrial ?-oxidation for provision of energy.In recent years,these two pathways have been confirmed during the lipid metabolism study on fish,but their roles in systemic metabolism and energy supply are not clear yet.At the same time,even in mammals,the comparison between lipolysis and lipophagy has not been discussed in detail,especially at the in vivo level,and their individual roles in LDs decomposition are still to be investigated.In addition,it has been reported that there is a certain interaction between lipolysis and lipophagy in some cell types or under specific physiological conditions,but similar interaction studies in fish are still unclear and seldom dealt.Therefore,this study first explored the importance of autophagy/lipolysis to the growth and metabolism in tilapia or zebrafish by treating them with corresponding inhibitors.Then,established key enzyme gene for lipolysis(atgl/pnpla2)and important lysosomal lipase gene(lal/lipf)knockout zebrafish models respectively,by using CRISPR/Cas9 gene-editing technology.Next,through analyzing the tissues expression,larval phenotype,biochemical measurement data,whole fish transcriptome,and using several detetion methods including thin-layer chromatography(TLC),gas chromatography(GC),the liver H&E staining,immunofluorescence,transmission electron microscopy(TEM)observation,RT-PCR,Western blot and ELISA kit,attempting to explore the specific functions of lipolysis and lipophagy in lipid metabolism of fish from the perspective of neutral/acid lipase deficiency,and the compensatory effects of lipolysis or lipophagy when one of them is blocked.Besides,the present study inhibited autophagy and lipolysis by using 3-Methyladenine(3MA)and Atglistatin(AI),respectively,in AKO and LKO zebrafish,to further explore the metabolic responses of the fish when the two processes are simultaneously inhibited.The main results of this thesis are as follows:1.Systemic metabolism changes of Tilapia with autophagy inhibition In our laboratory,we have demonstrated for the first time the existence of lipophagy in fasting zebrafish(Danio rerio)liver using TEM and measuring autophagy marker proteins(LC3-I,LC3-II),and it was showed that inhibition of different lipophagic steps in vivo and in vitro could cause TAG accumulation and downregulation of lipid metabolism activities during both fed and starved states.However,the relationship between autophagy and metabolic diseases in fish,especially commercial fishes,has not been well understood.In order to test the hypothesis that impaired autophagy may be an important cause of excess fat accumulation in fish,a one-week pre-experiment was first conducted in this study.Three concentrations were set to evaluate the reasonable dose of chloroquine(CQ,an autophagy inhibitor),and the final dose was100mg/kg diet.Subsequently,an 8-week tilapia feeding trial was conducted.Healthy Nile tilapias with similar weights were selected and randomly distributed into two groups,one was fed with control diet,the other group used experimental diet supplemented with CQ.At the end of the experiment,autophagy-related proteins and genes were measured in fish liver or muscle to confirm the efficiency of the CQ reagent.Then lipid metabolism-related genes in three tissues were tested,the molecular parameters of glycometabolism,protein synthesis and decomposition were also investigated to better understand the role of autophagy in nutrient metabolism in fish.The results showed that CQ treatment significantly inhibited autophagy and impaired fish growth and protein synthesis.In terms of glucose metabolism,autophagy inhibition promoted glycogen decomposition in fish.Besides,genetic data from the three tissues showed that glycolysis,elevated by CQ treatment,took place mainly in muscle,and CQ also improved glycolysis in adipose tissue;however,CQ mostly reduced the capacity of gluconeogenesis in liver.Therefore,CQ-induced autophagy inhibition seemed to reduce the portion of energy sourced from lipid metabolism,yet enhanced glycolysis compensated on the whole.In addition,inhibition of autophagy will worsen the metabolic profiles of otherwise healthy fish,as demonstrated by lipid accumulation,weaker antioxidant capacity and increased inflammation.Specific analysis of lipid metabolism data in different tissues revealed that compared with the control,CQ supplementation generated lipometabolic disturbance,and the order of reaction sensitivity from strong to weak was liver,muscle and adipose tissue.This study suggested that impaired autophagy might be at least one cause of the metabolic diseases which has been commonly seen in aquaculture.These results indicate that inhibition of autophagy could significantly affect the metabolism of lipid,carbohydrate and protein in fish.Hence,autophagy could play important roles in maintaining homeostasis of nutrient metabolism in cultured fish,and the more intricate mechanism is possibly associated with the AMPK and m TOR signalling pathways.2.The energy metabolism mechanism of zebrafish with deficient lipolysis Previous studies have confirmed that the genes encoding for ATGL are conserved in teleost and contain the patatin domain with “GXSXG” motif as well,which are conservative structures of lipases.Supportably,the TG lipolysis in fish also occur through the ATGL-HSL-MGL enzyme system,like mammals.However,the exact roles of ATGL in fish metabolism has not been clarified.In addition,fish generally have a lower utilization of carbohydrates than mammals,and lots of studies have shown that the modified lipid metabolism affect glucose and protein metabolism in order to maintain energy homeostasis.However,the exact roles of ATGL-mediated lipolysis in energy homeostasis have not been reported in fish.Atglistatin(AI)is the first specific synthetic ATGL inhibitor,and its inhibitory effect on lipolysis of fish remains unclear.In this study,zebrafish were choosen as the experimental object.A one-week preliminary experiment was carried out to find out that the ultimate concentration of AI was 80mg/kg diet(no obvious toxic effect).Then,male zebrafish were selected and randomly divided into two groups and fed with Atglistatin or not for five weeks.After the feeding trial,lipid deposition and composition,blood glucose and organs glycogen,oxygen consumption rate(OCR),and activities of FA ?-oxidation and lipases in tissues were measured.The results showed that Atglistatin-treated fish also increased condition factor,whole fish total lipid and plasma TG than control.Accordingly,the Atglistatin-treated fish had conspicuous white adipose tissue while the control zebrafish did not contain them.In addition,Atglistatin-treated fish also showed less OCR and reduced ?-oxidation activities in liver and muscle,the contents of TG and PL in total lipid were significantly increased in AI group,while the proportions of FFA,DG and MG were reduced.Altogether,inhibition of ATGL resulted in severe fat accumulation,altered lipid profile,decreased lipid catabolism,and induced oxidative stress and inflammatory responses to a certain extent in zebrafish.In addition,the partial increased HSL failed to reverse the effects caused by ATGL inhibition,and the compensatory autophagy increase was not obtained.Whole fish transcriptome data further revealed that inhibition of ATGL changed the nutrient metabolism of zebrafish.From the perspective of nutrient utilization,the ATGL inhibition also accelerated glycogenolysis and protein turnover,without affecting the fish protein content,however,reduced the expressions of glycolysis-related genes.Therefore,ATGL is a crucial enzyme in metabolic homeostasis,and its inhibition causes loss of lipid-sourced energy production,which cannot be compensated by activation of HSL,autophagy and utilization of other nutrients.3.Comparative study on metabolic characteristics of Atgl and Lal knockout zebrafish In order to exclude the target uncertainty caused by inhibitors,this study constructed whole-body Atgl and Lal knockout zebrafish homozygous lines(AKO or LKO)using CRISPA/Cas9 gene editing technology,and detected the expression of these two genes in different tissues of wild-type zebrafish.The results showed that atgl(pnpla2)and lal(lipf)were widely expressed in zebrafish,and the expression levels in different tissues were not significantly different.It should be noted that,in mammals,the expression of atgl was extremely high in adipose tissue or mammary gland tissue,which differed from fish.Phenotypic exploration of juvenile zebrafish showed that,compared with WT,AKO zebrafish were more active,and at the 16 th day,whole fish already showed significant fat accumulation.However,the locomotor activity of LKO zebrafish was significantly reduced,and the effect of fat deposition was not as obvious as that of Atgl knockout homozygotes,although with good appetite.In addition,after a 7-week feeding experiment,despite the absence of Atgl or Lal causes restrained growth,decreased protein,sluggish mobility and reduced OCR,they had completely different contents of total lipid and triglyceride,as well as lipid profile.To put it more specifically,compared with WT,the total lipid of AKO fish was remarkably increased,together with a higher proportion of triglycerides but less PE,PC in the total lipids than WT.Additionally,the AKO zebrafish had higher percentages of C16:1,C18:1n-9 and C18:3n-6 but lower percentages of C24:1 and C22:6n-3 than the WT fish.On the other hand,the LKO mutants decreased significantly the total lipid and TAG compared to the WT,without affecting PL content.Accordingly,the LKO fish decreased the percentage of TAG while the CE,PE and PC percentages were increased in the total lipids.Moreover,the LKO fish had higher percentages of very long-chain polyunsaturated fatty acid(VLCPUFA),such as C20:5n-3 and C22:6n-3 but lower percentages of C16:0,C18:1n-9 and C18:2 than the WT fish.Aside from the biochemical distinction,histological differences existed between AKO and LKO zebrafish.For example,after vivisection,the liver of AKO zebrafish was white,and its histological image showed much more vacuoles,indicating fat accumulation,than the bright red liver in WT fish,while LKO zebrafish had yellow liver with small and dense vacuoles surprisingly.What's more,transcriptome analysis showed that lipid metabolism was the most influenced biological process,and the loss of Atgl hindered the holistic TAG metabolism,which corresponded to the severe TAG accumulation in AKO zebrafish.Besides,Atgl deletion decreased cholesterol synthesis,but enhanced cholesterol utilization,and down-regulated several genes involved in phospholipids,especially PE and PC synthesis,compared to the WT fish.As for LKO fish,genes related to TAG and phospholipids syntheses were not much affected,while lipid catabolism-related genes,such as lipca,acsbg1 and cpt-1,were significantly up-regulated.Moreover,the LKO fish increased cholesterol synthesis and weakened synthesis of steroid hormones.These transcriptome data further revealed that AKO and LKO zebrafish perform different lipid metabolism patterns,and lipolysis and lipophagy are both important to maintain lipid metabolism homeostasis across vertebrates.4.The proposed relationship between lipolysis and lipophagy in zebrafish liver Considering the striking characteristic differences in the liver of AKO and LKO zebrafish after anatomy,and given the high expression of lipolysis related genes in fish livers(compared to mammals),subsequent studies on the relationship between lipolysis and lipophagy mainly focused on the tissue of liver.This research firstly used AKO male zebrafish and corresponding WT(F3 generation)to carry out feeding trial for 4weeks,and autophagy inhibitor 3-methyl adenine(3MA)was added in the daily diet to suppress the autophagy in AKO zebrafish,hoping to answer the question that whether Atgl defects would affect lipophagy.The current conclusion is that Atgl deficiency seriously impeded lipolysis in fish liver,but lipophggy was not activated,and 3MA treatment further increased triglyceride accumulation in the liver of AKO zebrafish,which means that when lipolysis is depressed,lipophagy still plays a basic role in lipid degradation.Transmission electron microscopy(TEM)images and biochemical data also illustrated that Atgl-mediated lipolysis has a stronger effect to degrade large LDs enriched with TAG.Secondly,LKO male zebrafish and its corresponding WT were also fed for 4weeks,in an attempt to detect the changes of lipolysis and lipophagy in liver under the condition of acid lipase deficiency.At this time,diet was supplemented with AI to measure whether Lal deficiency would affect the function of fish lipolysis.Results found that hepatocytes of LKO zebrafish accumulated large amounts of small LDs,which contained high cholesterol esters rather than triglycerides.It was verified that the function of Lal in cholesterol breakdown is conserved across vertebrates,and also indicated that lipolysis and lipophagy have different roles in regulating TAG and cholesterol metabolism,respectively.In addition,compared to WT,lipolysis related genes were significantly upregulated in the liver of LKO zebrafish,indicating that the loss of Lal activated lipolysis,especially the enhancement of Atgl function.Surprisingly,additional AI treatment induced Lal-independent autophagy in the liver of LKO zebrafish,and TEM images suggested that mitochondrial autophagy may be triggered when both lipolysis and lipophagy were blocked.Then,liver samples from six groups(WT/ AKO/AKO + 3MA;WT/LKO/LKO+AI)mentioned above were used to test liver damage and mitochondrial related indicators.The data showed that AKO +3MA fish obviously increased the number and size of mitochondria compared with AKO fish,accompanied with higher expressions of mt DNA copy number(nd1 and atp6)and mark genes related to mt DNA proliferation(ssbp1)than the AKO fish.On the other hand,the LKO + AI hepatocytes specifically showed obvious swelling mitochondria and mitophagy characteristic,marked with the common appearance of mitochondria in the cavity of APL which contains many undegraded LDs.Accordingly,the LKO + AI fish had higher expression of Lamp1(lysosomal marker protein)and more obvious colocalization of Lamp1 and Hsp60(mitochondrial marker protein)than WT or LKO fish,confirming the occurrence of mitophagy.Besides,the LKO and LKO+AI fish similarly increased nearly all liver injury-related genes,including DNA damage(ddit3,gadd45?a),apoptosis(casp3a,casp9)and inflammation(il6,il1?,tnf?),while LKO+AI fish tended to show stronger effects than LKO in the majority of these genes.Therefore,when the two intracellular LD degrading pathways are blocked synchronously,the removal of damaged mitochondria through mitophagy may be a cellular protective strategy to prevent cell death.On the other hand,when lipolysis and lipophagy were both cut off,the increased mitophagy may also be an alternative process to supply energy.This interaction among cellular organelles under lipid-sourced energy exhaustion should be further investigated.