Effects Of Hypoxia Stress On Glucose And Lipid Metabolism And Regulation Effect Of Salidroside In Nile Tilapia

Hypoxia stress is a common stress factor in aquaculture,and it will seriously affect the growth and survival of aquatic animals,reduce the production and benefits of aquaculture,thereby restricting the development of aquaculture industry.In order to explore thephysiologicalmetabolism change and nutritional mitigation methods of aquatic animals under hypoxia stress,Nile tilapia(Oreochromis niloticus)was selected as the research object.We explored the effects of acute hypoxia stress(DO=0.6~0.8mg/L)and long-term hypoxia stress(DO=1.0~1.2mg/L)on the glucose and lipid metabolism of Nile tilapia,and the effects of different levels of salidroside supplemented in diets on hypoxia stress of Nile tilapia.The main results and conclusions are as follows:(1)Effect of acute hypoxia stress(DO=0.6~0.8mg/L)on glucose,lipid metabolism and expression of hypoxia inducible factor in Nile tilapiaIn order to investigate the glucose and lipid metabolism patterns of Nile tilapia in acute hypoxia,the erperiment of 6 hourswas conducted by selecting tilapia with initial body weight of 6.3±1.2 g.The dissolved oxygen in the hypoxic group was 0.6-0.8 mg/L,while that of control group was 6.5-7.5 mg/L.Each group has three parallel.The results showed that the activity of the fish were significantly lower than the control group(P<0.05)in hypoxia stress.At the same time,the content of hemoglobin(HB)in blood was significantly increased than normoxic group(P<0.05).The content of blood glucose,hepatic glycogen,and muscle glycogen in the hypoxia-stressed group decreased significantly(P<0.05),and the activity of lactate dehydrogenase(LDH)in the liver increased significantly(P<0.05).The mRNA expression of pyruvate kinase(PK)and fructose 1,6 bisphosphatase(FBP)were significantly up-regulated(P<0.05),and the content of lactic acid(LA)in serum increased significantly in hypoxia than in normoxic group(P<0.05).There were no significant differences between hypoxia and normoxic group in content of triglyceride(TG)content in serum,liver,and muscle(P>0.05).Besides,the mRNA expression of monoglyceride lipase(MGL)and peroxisome proliferator-activated receptor(PPAR?)genes in liver were significantly up-regulated(P<0.05),and gene expression of hormone-sensitive lipase(HSL)and adipose triglyceride lipase(ATGL)did not change significantly(P<0.05).To explore the effects of hypoxia stress on tilapia,the genes of hypoxia inducible factor(HIF1?)in tilapia was cloned in our study.There are two transcripts in HIF1?,the full length of HIF1?X1 is 3658 bp,the open reading frame is 2310 bp,which encodes 769 amino acids.The full length of X2 is 3655 bp,and the open reading frame is 2307 bp,which encodes 768 amino acids.Fluorescent quantitative PCR results showed that the expression of HIF1? was significantly up-regulated in acute hypoxia stress(P<0.05),and the same variationoccurred in the downstream target genes glucose transporter(GLUT1),vascular endothelial growth factor(VEGF)and PK.In summary,acute hypoxia stress decreased the activity of Nile tilapia,and increased the HB content,together withincreasing sugar and anaerobic glycolysisability,and the decomposition of sugar.Lipogenic gene expression was upregulated but did not affect the content of TG in blood and tissues.Therefore,Nile tilapia would select carbohydrat emetabolismas the mainlyenergy source,when confronted with acute hypoxia stress.In the process ofadapting hypoxia stress,HIF1? activity is stimulated,which in turn regulates the activities of related downstream genes,so that tilapia could adapt to acute hypoxia stress easily.(2)Effects of chronic hypoxia stress(DO=1.0~1.2mg/L)on growth,immunity and glucose and lipid metabolism in tilapiaIn order to explore the effects of long-term hypoxia stress on Nile tilapia,the fish with initial body weight about 6.1±1.3g was selected.The study last for 4-weeks.In the hypoxia group,the DO was 1.0 to 1.2 mg/L,and the DO of the normoxic group was 6.5 to 7.5 mg/L.Each group was three parallels.The experimental results showed that tilapia activity and HB contentdecreased significantly(P<0.05)in chronic hypoxia stress,The ingestion rate,weight gain rate and whole fish water content in the hypoxic group were all significantly decreased(P<0.05),while the total fish protein content was significantly increased(P<0.05).The blood glucose and hepatic glycogen content were significantly increased in the hypoxia-stressed group(P<0.05).At the same time,hexokinase(HK)and PK activity were significantly decreased in the liver(P<0.05),while LDH activity was significantly increased(P<0.05).The LA content in serum was also significantly higher than that in normoxic group(P<0.05).In addition,the total lipids of whole body,together with the content of TG in serum and liver were significantly down-regulated in hypoxia-stressed group(P<0.05).Total lipase activity was significantly higher in the hypoxia-stressed group than that in the normoxic group(P<0.05).The ?-oxidation capacity in the mitochondria was significantly increased in the hypoxia-stressed group than in the normoxic group(P<0.05).Furthermore,the activity of aspartate aminotransferase(GOT)in serum in hypoxia group was significantly increased(P<0.05).The gene expression of(IL-1?)and transforming growth factor(TGF-?1)were significantly upregulated(P<0.05).In conclusion,the anaerobic glucose metabolism of Nile tilapia was up-regulated,and the ?-oxidation ability of fatty acid was increased markedly in chronic hypoxia stress.Which suggested that tilapia use the lipid as the mainly energy sourse and anaerobic glycolysisas auxiliary energy source.(3)Liver transcriptome analysis of Nile tilapia responses to chronic hypoxia stressThetiter analysis of tilapia livers was performed to investigate the response mechanism of Nile tilapia under chronic hypoxia stress.A total of 348.2 million sequences were obtained by splicing,of which 179.6 million sequencesin normoxic group,and hypoxic group obtained 168.6 million sequences.Through analysised the sequencing results by edgeR software,there were 451 differentially expressed genes in the hypoxia stress group,in which 282 genes were up-regulated and 169 genes were down-regulated compared with the control group.Besides,the KOBAS analysis pathway,there were 73 pathways changed,in which 14 pathways had significant changes,including 4 pathways related to signal transduction(AMPK,FOXO,PPAR,and adipocytokine signaling pathways)and 8 pathways related to metabolic(tyrosine,glycine,serine,threonine,methane,sulfur,fatty acids,linoleic acid,glycerides,and vitamin B6 metabolic pathways).The rest pathways were associated with inflammatory factors and circadian rhythms.By analysis of differential carbohydrate and lipid metabolism genes,we found that genes related to glycosynthesis(GS,PEPCK)and lipolysis(ATGL,PPAR,CPT1,CYP7AL)were significantly up-regulated,lipid synthesis related genes(FASN,ACACA,ACLYA)and glycolysis related genes(GCK,PDHB,ACLYA)were significantly downregulated.In conclusion,under long-term hypoxia stress,tilapia changes the signaling pathway in vivo,which promotes glycogenogenesis and lipolysis,reduces amino acid metabolism.Compared with normoxic group,in order to cope with hypoxia stress well,tilapia changes the nutritional and energy metabolism mode.(4)Effects of different levels of salidroside on Nile tilapia in diets under chronic hypoxia stressThe initial weight(4.98±0.57g)of tilapia were selected to explore the relief effect of salidroside on Nile tilapia in response to hypoxia stress.Experimental diets were formulated containing the same level of lipid(5%).There were 4 groups in the experiment,including normoxic control group,hypoxic control group,and low salidroside group(hypoxia added 30?g/kg salidroside)and high salidroside group(hypoxia added 60 ?g/kg salidroside).After 4-week-trial,the results showed that there was no significant difference in the feeding rate among the three hypoxic groups(P>0.05),but they were all lower than those in the normoxic group(P<0.05).Compared with the hypoxic control group,the addition of salidroside had no significant effect on the weight gain rate of tilapia(P>0.05),but the weight gain rate of the high salidrosided group in the 2nd week was significant higher than the hypoxic control group(P<0.05),but no significant difference with the normoxic group(P> 0.05).Behavioral analysis showed that the resting time of three hypoxic groups was significantly longer than that of normoxic group(P<0.05).The addition of salidroside did not affect the tilapia activity time(P>0.05).The hepatic body ratio of high salidroside group was significantly higher than that of low salidroside group and hypoxia control group(P<0.05),and there was no significant difference with normoxic group(P>0.05).The HB content in the three hypoxia groups was significantly lower than that in the normoxic group(P<0.05).There was no significant difference in the total lipid content between the three hypoxic groups(P>0.05),but they were all significantly lower than the normoxic group(P<0.05);The total protein of whole body in the low salidroside group was significantly higher than that in the normoxic control group and high salidroside group(P<0.05).The content of blood glucose and hepatic glycogen in lowsalidroside group were significantly lower than those in hypoxia control group and high salidroside group(P<0.05).However,there was no significant difference with the normoxic(P>0.05).The activity of LDH in liver of the hypoxic control group was significantly higher than that in other three groups(P<0.05),while there was no significant difference in LDH activity between the addition of salidroside group and normoxic group(P>0.05).The content of LA in hypoxia control group and high salidroside group were significantly higher than those in normoxic group and low salidroside group(P<0.05),whereas the content of LA in serum in low salidroside group was higher than in normoxic group(P<0.05).However,both the low salidroside group and the high salidroside group were significantly reduced the LA content in liver(P<0.05),and was significantly higher in hypoxic control group than that in normoxic group(P<0.05).There was no significant difference in the activity of HK amongfour groups(P>0.05).On the other hand,there was no significant difference in serum TG content among high salidroside group and other three groups(P>0.05),but the TG content in low salidroside group was significantly lower than that in normoxic group(P<0.05).However,the content of TG in the hypoxic control group was significantly lower than that in other three groups(P<0.05).Compared with the normoxic group,the ?-oxidation ability of the three hypoxic groups was significantly increased(P<0.05).The peroxisomal oxidation ability in low salidroside group and high salidroside group were significantly increased compared with the normoxic group and the hypoxic control group.In contrast to the normoxic group and the high salidroside group,low salidroside group,the mitochondrial oxidative capacity were increased in hypoxia control group(P<0.05).Furthermore,the amount of two levels of salidroside significantly decreased the activity of aspartate aminotransferase(GOT)in blood(P<0.05)and the expression of IL-1 in liver tissue(P<0.05).In conclusion,adding salidroside in diets can not only reduce serum LDH activity,LA accumulation,and increase TG content and hepatic peroxisomal oxidative capacity in tilapia livers under chronic hypoxia stress,but can reduce the liver damage and the expression of inflammatory factors under the hypoxia stress.