Effects Of Nutritional Stress On Muscle Autophagy Of Siniperca Chuatsi And Tilapia And Its Regulatory Mechanism

Autophagy is a general term for lysosomal degradation of intracellular materials and is a relatively conservative mechanism unique to eukaryotic cells.Autophagy refers to the formation of autophagosomes from the bilayer membrane on the non-ribosome attachment area of the rough endoplasmic reticulum and some other cellular organelles,which leads to the formation of autophagic lysosomes to degrade cell contents and to achieve the metabolic needs of cells and some organelles.Autophagy has been observed in m any different types of cells,but its role in skeletal muscle protein degradation has not been thoroughly studied,especially in aquatic species.Under conditions of sufficient cell nutrition,autophagy can be effectively inhibited,while autophagy is acti vated when nutrition is deficient.Autophagy and ROS do have a strong correlation,not through simple activation or inhibition but through complex mechanisms.The activation of autophagy can increase the level of ROS,but the production of ROS can trigger autophagy when ROS excess or clearance is suppressed.The rhythmic research on autophagy has only been reported in zebrafish liver and brain at different nutritional conditions.To date,there is no report focused on the rhythmic changes of fish skeletal m uscle autophagy upon nutritional deprivation.In order to explore the molecular signal transduction mechanism of autophagy regulating the metabolic homeostasis of skeletal muscle under starvation,the effects of short-term starvation on the antioxidant cap acity,ROS content,autophagy genes and autophagosomes formation of skeletal muscle of Siniperca chuatsi were studied,and the circadian rhythm of autophagy gene response to starvation in juvenile tilapia was alsoassayed.The obtained major results are as follows:(1)Eight autophagy genes from the Siniperca chuatsi have been successfully cloned.And their structural domains,UBQ,atg14 and serine/threonine protease,are characterized.The m RNA expression levels of the ten important autophagy genes including Atg4 b,Atg4c,Atg4 d,Atp6v1d,Becn1,Bnip3,Gabarapl1,LC3 a,LC3b and Ulk1 expression levels were significantly expressed in intestine,liver and muscle tissues,respectively.(2)Under the starvation condition,the ROS content in juvenile Siniperca chuatsi muscles increased significantly.Compared with the control group,the activity of antioxidant enzymes,SOD,CAT,GPx,and GST,and the contents of MDA,ASA,and AHRFR all significantly increased after 2 days of starvation.The expression levels of GPx,GSTA,GS T4 A,GS TT1,Mn SOD,Zn S OD,and CAT genes were significantly up-regulated.The expression levels of antioxidant signal molecules Nrf2,S6K1 and m TOR m RNA were also increased,but Keap1 gene m RNA expression level was down-regulated,which improved the muscle’s ability to resist injury.However,upon further starvation for 5 days,the expression level of antioxidant signal factor genes and regulating antioxidant enzyme genes were down-regulated,and the antioxidant enzyme activity we re decreased,thus caused the antioxidant capacity decliling.(3)The autophagy gene expressions(except Bnip3)and autophagosomes did not change significantly in the muscles of Siniperca chuatsi juveniles upon starved for 2days.There was no significant difference in LC3-II/LC3-I protein levels,and the levels of autophagy remained basically unchanged.When the starvation time was prolonged to the 5 days,the expression level of autophagy gene m RNA was significantlyup-regulated,and the number of autophag osomes and the level of LC3-II/LC3-I protein were also apparantly increased by electron microscopy obervation.Thus,autophagy was significantly enhanced in juvenile Siniperca chuatsi muscle.(4)A working pattern of ROS mediated oxidative stress on autoph agy of muscle cells under starvation was constructed.ROS mediates the activation of antioxidant enzyme genes by regulating the antioxidant signal molecule Nrf2-Keap1,thereby increasing the activity of antioxidant enzymes.However,when oxidative stress i s intolerable,muscle autophagy occurs.(5)Using Dual-Luciferase reporter assay,we found that miR-181a-5p and miR-194 a could inhibit the expression of Nrf2.The other miRNAs,such as miR-1,miR-16 a and miR-29 a can also inhibit the expression of Keap1.The results demonstrate that miRNAs have a regulatory effect on autophagy.(6)Using biological cosine analysis,we found that starvation stress also changed the genes involved in maintaining muscle autophagy rhythmicity.At the same time,only LC3(Lc3a,Lc3 b,and Lc3c),as a marker of autophagy,is still rhythmic regardless of the normal feeding or fastingtreatment,and the COSINOR waveforms are similar.By comparing the m RNA expression levels of autophagy genes in the muscles of the fasting and the normal feeding treatment,it was found that Atg4 b,Becn1,Bnip3 la,Bnip3lb,Lc3 a,and Ulk1 b were significantly upregulated compared with the normal group.Electron microscopy analysis also revealed that autophagosome increased significantly after starvation.(7)GSK4112,the activator of Nr1d1,caused the disorder of Nr1d1,which also caused the rhythmic change of autophagy gene.It also showed that when Nr1d1 was significantly overexpressed,the autophagy genes Atg4 c,Atp6v1d,LC3 a and Ulk1 a expression was significantly down-regulated.Thus,the biological clock could involve in regulating the autophagy rhythmicity.