| Turbot and tiger puffer are important aquaculture species.They both are also becoming important fish models for lipid research due to the relatively special lipid storage patterns.This thesis,with turbot and tiger puffer,was conducted to investigate the tissue distribution of key lipid-metabolism genes,the necessity of dietary cholesterol supplementation,the effects of dietary cholesterol on lipid metabolism and the functional role of lipid metabolism-related enzyme MID1 interacting protein 1(Mid1ip1).The results and conclusions are summarized as follows:1.Tissue distribution of transcription for key lipid metabolism-related genes in turbot and tiger puffer2.The tissue distribution pattern of lipid is highly diverse among different fish species.Turbot and tiger puffer store their lipids mainly in the subcutaneous adipose tissue around the fin and in the liver,respectively.The present study preliminarily investigated the tissue distribution of transcription for partial key lipid metabolism-related genes in turbot and tiger puffer,which are involved in lipogenesis,fatty acid oxidation,biosynthesis and hydrolysis of glycerides,lipid transport,and relevant transcription regulation.The tissue expression distribution of lipid metabolism-related genes in different tissues of 30 turbot(10 as a replicate pool)and 15 tiger puffer(5 as a replicate pool)was analyzed by q RT-PCR,respectively.The results of this study indicate that the intestine and brain have high transcription of lipogenic genes,whereas the liver and muscle have low expression levels.Peroxisomal acyl-coenzyme A oxidases(ACOX1 and ACOX3)were highly expressed in the pyloric caecum of turbot and in the liver and intestine of both species,however,the expression of carnitine O-palmitoyltransferase-1(CPT1)was low in these tissues.In addition,the expression of acetyl Co A carboxylase(ACACβ),ACOX1 and ACOX3 in turbot decreases from the foregut to the cecum.For the biosynthesis of glycerides,glycerol-3-phosphate acyltransferase(GPAT)was lowly expressed in the intestine of turbot and nearly undetectable in the intestine of tiger puffer,whereas diacylglycerol O-acyltransferase 1(DGAT)was highly expressed in the intestine of both species.Pancreatic lipase(PL)and bile salt activated lipase(BSAL)were highly expressed in the pyloric caecum of turbot and in the liver of tiger puffer.Adipose triacylglyceride lipase(ATGL),hepatic lipase(HL)and lipoprotein lipase(LPL)were primarily expressed in the liver,whereas the diacylglycerol lipase,alpha(DAGLα)and monoglyceride lipase(MGLL)were highly expressed in the brain.In addition,most apolipoproteins and lipid metabolism-related transcription factors had high expression in the intestine.The transcription of fatty acidβ-oxidation-related genes was low in the muscle.In conclusion,intestine could be a center of lipid metabolism in turbot and tiger puffer.Moreover,lipid metabolism is more active in the foregut of turbot.The peroxisomal fatty acid oxidation may occupy an important position relative to mitochondrialβ-oxidation in the liver and intestine of turbot and tiger puffer.The monoacylglycerol(MAG)pathway probably occupies an important position relative to the glycerol-3-phosphate(G3P)pathway in the re-acylation of absorbed lipids in the intestine.Effects of dietary cholesterol on growth performance and lipid metabolism in turbot and tiger pufferThis study aims to evaluate the necessity of dietary cholesterol supplementation in turbot and tiger puffer and the effect of dietary cholesterol supplementation on lipid metabolism.Graded levels(0,0.5,1.0,2.0 and 4.0%of dry matter)of cholesterol were added into the basal diet(with 30%fishmeal but no fish oil)to make five experimental diets,which were designated as Control,CHO-0.5,CHO-1.0,CHO-2.0 and CHO-4.0,respectively.In both turbot and tiger puffer trials,each dietary group had triplicate tanks,and each tank was stocked with 30 fish.Due to the difference in digestive characteristics,turbot were hand-fed to apparent satiation twice daily,whereas tiger puffer were fed three times a day to apparent satiation.All the tanks were supplied with flow-through filtered seawater and the feeding trials lasted 70 days.The results showed that compared to the control,dietary cholesterol supplementation had no significant effect on the growth of both turbot and tiger puffer.Nevertheless,excess dietary cholesterol(CHO-2.0 and CHO-4.0)led to significantly lower weight gain of tiger puffer compared to CHO-1.0.With cholesterol supplementation,the feed intake and the crude lipid content in liver was decreased in both species.However,0.5%cholesterol supplementation in diet tended to increase the crude lipid content in muscle.Dietary cholesterol supplementation significantly regulated the lipid-related biochemical parameters in the serum,liver and muscle,and increased the C20:4n-6 content in the liver.Dietary cholesterol supplementation significantly down-regulated the gene expression of 3-hydroxy-3-methylglutaryl Co A reductase(HMG-COAr),up-regulated the cholesterol 7α-hydroxylase(CYP7A1)expression,down-regulated the expression of lipid absorption and biosynthesis genes,but had no significant effect on gene expression of carnitine O-palmitoyltransferase-1(CPT1)and lipid transport-related genes(Apo A1,Apo A4,Apo B100 and Apo Eα)in the liver.In conclusion,under the present experimental condition,dietary cholesterol supplementation had no significant effect on the growth of turbot and tiger puffer.Compared to turbot,tiger puffer body composition has a higher buffering capacity in response to changes in dietary cholesterol level.The results of this study will provide data to support the reasonable application of cholesterol in fish diets.3.Cloning,characterization and m RNA expression of Mid1ip1 from turbot and tiger puffer in response to dietary nutrients.Mid1ip1 plays important roles in lipogenesis and microtubule stabilization in mammals,but little relevant information has been reported in fish.In present study,to explore new genes regulating lipid metabolism in fish,the core fragments of Mid1ip1 of turbot and tiger puffer were cloned and their sequences were analyzed.Subsequently,samples from previous feeding trials were used to investigate the response of turbot Mid1ip1 to dietary lysophosphatidylcholine(LPC)and the response of tiger puffer Mid1ip1 to dietary bile acid and lipid levels.Graded levels(0.1%,0.25%and 0.5%of dry matter)of LPC were added into the turbot basal diet to make three experimental diets,which were designated as LPC-0.1,LPC-0.25 and LPC-0.5,respectively.Experimental diets with suitable(8.5%)or high lipid level(12.5%),with or without bile acid supplementation(0.02%and 0.10%in the suitable-lipid diet;0.02%in the high-lipid diet)were used in the trial of tiger puffer.The results of this study indicate that,similar to Mid1ip1αof turbot,the tiger puffer Mid1ip1,which is putative Mid1ip1 subtype B,shares moderate identity to its known orthologs of other teleost and much lower identity to human Mid1ip1.The Mid1ip1βshared moderate identity to its known orthologs of zebrafish and carp.Mid1ip1αof turbot and Mid1ip1 of tiger puffer had high transcription in the muscle and skin,but low expression levels in the liver.However,turbot Mid1ip1βhad low expression levels in all tissues.Dietary LPC supplementation had no significant effect on Mid1ip1 gene expression in liver and muscle of turbot.The high dietary bile acid level(0.1%)significantly reduced the hepatic m RNA expression of mid1ip1,but unexpectedly the dietary lipid level did not significantly affect the gene expression of tiger puffer Mid1ip1.Significantly positive correlation(R~2=0.812,P=0.000)was observed between hepatic m RNA expression of Mid1ip1 and Tubulin-α,but not between the gene expression of mid1ip1 and acetyl-Co A carboxylaseβ(R~2=-0.434,P=0.106).In summary,Mid1ip1 may have a limited role in the lipid metabolism in turbot and tiger puffer,but may play an important role in maintaining microtubule stability in tiger puffer.To our knowledge,this research is the first study investigating the regulation of fish mid1ip1 expression by dietary nutrients. |
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