Mechanisms Of Fasting Enhance Cold Tolerance In Zebrafish(Danio Rerio)

Temperature is the most important environmental factor in fish.Cold stress is common in fish life and causes various effects on the reproduction,growth,behavior and even survival of fish.Moreover,cold stress has been a serious threat in aquaculture industry.While the mechanism of cold tolerance in fish is not well revealed.Target to reveal more physiological mechanisms related to cold resistance in fish and provide theories foundation for the industry,we tightly focused on the phenotype that fasting enhanced cold tolerance in zebrafish,and systematically studied the underlying mechanism.By using many research skills and approaches include transcriptomic analysis,gene knockout,qPCR,Western blot analysis,electron microscope,isotope tracking and so on,we found lipid catabolism,reduction of oxidative stress and autophagy were important mechanism of fasting-enhanced cold resistance,and mTOR signaling pathway largely participated the regulation of this process.The main contents of this thesis are summarized as below:1.Fasting enhances cold tolerance in zebrafish and the underlying mechanismDifferent from many homeothermic animals,which will increase food intake and thermogenesis to maintain body temperature in cold environment,fish will reduce even stop food intake when subjected to cold stress.Researches in other animals have revealed that suitable fasting could enhance stress resistance and delay aging through optimizing energy metabolism and reducing oxidative stress.To explore how fasting would affect cold resistance in fish,we firstly tested the cold tolerances of zebrafish with different fasting durations,and found that 48–96 h fasting enhanced significantly the survival rate of the zebrafish exposed to acute cold stress.Then we performed liver transcriptomic analysis to explore the underlying mechanisms.The transcriptomic results showed fasting significantly decreased the cold-induced cell damages and increased the expression of genes related to energy metabolism.Then we further tested the utilization of energy source in zebrafish during fasting.The results showed zebrafish mainly used carbohydrates as energy source at the beginning of fasting（within 24h）,and utilized lipid for energy after fasting for 48h-96h,but showed on utilization of protein.These results indicated lipid was most like the energy source used to resist cold stress in fasting zebrafish.UCPs are important proteins in helping mammals resist cold stress,so we also studied the effects of fasting and cold stress on the expressions of UCPs in zebrafish.The activations of UCPs by fasting and cold stress were transient and showed no regularity,indicated UCPs played minor roles in the fasting-enhanced cold tolerance in fish.Above all,we conclude that suitable fasting could help zebrafish to resist cold stress through improving energy supply and decreasing cell damages,and lipid is the probably energy source.2.Fasting enhances cold tolerance by inducing lipid catabolismBoth carbohydrate and lipid are important energy source in animals,and the accurate mechanism how fasting enhanced energy supply in fish exposed to cold stress is unknown.So we separately verified the roles of carbohydrate utilization or lipid utilization in cold tolerance in zebrafish in this part.Firstly,we used glucose to take the place of starch in fish diet,which largely enhanced the carbohydrate utilization of zebrafish at the suitable temperature.When exposed to cold stress,the replacement of dietary starch with glucose showed no increase in the carbohydrate utilization,energy supply or fish survival rate.These results indicated enhanced carbohydrate utilization was not correlated with cold resistance in the zebrafish.Then,we performed two experiments to verify the correlation between lipid utilization and cold tolerance in zebrafish.In the metabolic modulators experiment,lipid catabolism activator Fenofibrate enhanced the lipid catabolism and increased the fish survival under cold stress;On the contrary,lipid catabolism inhibitor Mildronate inhibited the lipid catabolism and decreased the cold tolerance in zebrafish.In the fatty acid diets experiments,diet rich in 18:2 fatty acid showed higher lipid utilization and higher cold tolerance.These two experiments indicated enhancing lipid utilization could improve the cold resistance in zebrafish.To further verify the role of lipid catabolism in the fasting-enhanced cold tolerance,we knocked out a key gene related to fatty acidβ-oxidation（CPT1b）.Knockout of CPT1b significantly reduced theβ-oxidation,the cold tolerance as well as the fasting-enhanced cold tolerance in zebrafish.So we conclude that lipid is an essential energy source for resisting coldness and lipid catabolism plays an important role in the fasting-induced cold resistance.3.Fasting improves cold tolerance through reducing oxidative stress in zebrafishOxidative stress is an important reason in causing cell damages even death.Previous researches have showed cold stress could induce oxidative stress in various fishes.And researches in many other species also indicated fasting could reduce oxidative stress.So,we hypothesized fasting could enhance cold tolerance in fish by reducing oxidative stress.In this study,we firstly demonstrate that acute cold exposure induced high oxidative stress in zebrafish liver,which may lead to mortality.While fasting significantly reduced the cold-induced oxidative stress.Then we performed in vitro and in vivo experiments to determine the effects of oxidative state on cold tolerance.In the experiment using zebrafish cells,increasing oxidative stress by H₂O₂decreased the cellular cold tolerance,and cold tolerance was partly recovered when oxidative stress was reduced by addition of Vitamin C.In the in vivo study,the zebrafish which were fed withα-lipoic acid or reduced glutathione had lower cold-caused oxidative stress and tissues damage,and showed higher cold tolerance.Taken together,we conclude the reduction of oxidative stress increase cold tolerance,and fasting may improve cold tolerance by reducing oxidative stress in fish.4.Fasting induces cold tolerance through activating autophagyAutophagy is an important process to clean cell damages and improve stress resistance,and fasting could activate autophagy in many species.But the role of autophagy in the fasting-enhanced cold tolerance in fish was never reported before.To answer this question,we performed three in vitro and in vivo experiments.In the experiment performed in ZFL,RAPA induced higher autophagy level,and led to lower cell damages and higher cold tolerance;while CQ inhibited the autophagy in cell,and led to higher cell damages and lower cold resistance.In the vivo experiment,feeding CQ largely decreased the autophagy in fish liver and reduced its resistance to cold stress.Then,we further knocked out the autophagy-related protein 12（ATG12）in zebrafish to verified the role of autophagy in fasting-enhanced cold tolerance.The knockout of ATG12 significantly decreased the autophagy level,and reduced both the cold tolerance and the fasting-enhanced cold tolerance in zebrafish.Taken together,autophagy level in fish could largely affect its cold tolerance.Furthermore,autophagy plays an essential role in fasting-induced cold resistance in zebrafish.5.mTOR pathway participates the fasting-enhanced cold tolerancemTOR pathway plays a center role in sensing nutrition and energy signaling,and regulating various functions in cell.Among these functions,lipid catabolism,autophagy and regulation of oxidative stress were also found participated in the fasting-enhanced cold resistance in our previous study.So,mTOR may play an important role in the regulation of fasting-enhanced cold tolerance in fish.To confirm this hypothesis,we firstly verified that fasting inhibited the mTOR signaling pathway in zebrafish.Then zebrafish were fed with RAPA diets and high leucine diet respectively,zebrafish fed with RAPA had lower mTOR pathway activity and higher fish survival under cold stress;while high leucine diet activated the mTOR pathway and decreased the cold tolerance of fish.We further compared the effects of Rapamycin-induced mTOR inhibition and fasting on the mechanisms of cold tolerance in zebrafish.The results showed mTOR inhibition and fasting had similar effects on fish survival,mTOR pathway activity,autophagy level,oxidative stress level and the efficiency of lipid utilization,and the transcriptomic analysis also indicated they had similar global gene patterns in fish under cold stress.So we concluded these results that mTOR may largely participated in the regulation of fasting-enhanced cold tolerance in fish.Through exploring the mechanisms of how fasting enhance cold tolerance in zebrafish,we found lipid catabolism,autophagy and mTOR pathway paly important ole in resisting cold stress in fish,and firstly demonstrated that cold-induced fasting may be the strategy of resisting cold stress in fish.