Function Of Hormone-sensitive Lipase In Maintenance Of Nutrient Metabolism Hemeostasis In Zebrafish

Lipid metabolism plays an important role in maintaining energy homeostasis,and its disorder would cause some metabolic diseases such as fatty liver,type 2 diabetes and cardiovascular diseases.Lipolysis,also called hydrolysis of triacylglycerols（TG）,is the core process of lipid metabolism,it is defined as the biochemical process by which TG is broken down into non-esterified fatty acid（NEFAs）and glycerol under the function of multiple enzymes.Hormone-sensitive lipase（HSL）is mainly responsible for the decomposition of TG and diacylglycerol（DG）in this process.Lipolysis is a critical process controlling the mobilization and storage of TG.Unlike adipose triglyceride lipase（ATGL）and monoglyceride lipase（MGL）,HSL is the only lipase directly regulated by neuro-humoral signaling.In addition,HSL shows a wide range of substrates as it effectively hydrolyses TG,DG and monoacylglycerol（MG）,even cholesterol,and other substrates.All these features give HSL a key role in basal and stimulated lipolysis.In addition,the fish with excessive fat deposition was usually accompanied by abnormal hsl expression.Therefore,using zebrafish as a model to explore the functions of Hsl in fish could also help to understand the regulatory mechanisms of fish lipid metabolism.In the present study,we used either hsla-knockout or Hsl enzyme activity inhibitor to construct zebrafish models with Hsl dysfunction.By measuring the growth,swimming activity,oxygen consumption rate,biochemical changes,and the expressions of key genes and proteins in lipid,glucose and protein metabolism,the present study is the first to examine the roles of Hsl in systemic nutrient metabolism in fish.1.Effects of inhibiting Hsl enzyme activity on metabolic homeostasis of zebrafishPrevious studies have shown that energy metabolism is always in a dynamic balance,and the three metabolic pathways of lipid metabolism,protein metabolism and glucose metabolism interact with each other to maintain the energy homeostasis.If nutrient metabolism homeostasis is unbalanced,it will cause metabolic diseases such as serious accumulation of fat.Abnormal expression of hsl gene is usually found in fish with excessive fat deposition in aquatic animals.However,the role of Hsl in the regulation of nutrient metabolism in fish is still rarely reported.Therefore,male zebrafish were randomly selected and divided into two groups（control group and inhibitor group treated with HSL-IN-1）for a 5-week breeding experiment.Tissues were sampled and stored after culture.We detected growth,movement ability,biochemical indexes related to lipid metabolism,protein metabolism and glucose metabolism,H&E and Oil Red stained sections of the liver,expression levels of genes and proteins related to the three metabolic pathways,and expression levels of genes related to antioxidant enzyme activity and inflammation and apoptosis of zebrafish.The results showed that the condition factor,total body fat and plasma TG of zebrafish in the HSL-IN-1 treated group were significantly increased,and significant enlargement of adipose tissue was found in the group during the body anatomy,while no similar phenomenon was observed in the control group.In addition,the oxygen consumption rate of zebrafish in HSL-IN-1 treated group was lower,but the movement ability was not affected;In the lipid composition of whole fish,the contents of TG and DG increased significantly,while the contents of MG and phospholipid（PL）decreased significantly.In terms of glucose metabolism,the muscle glycogen content of zebrafish in HSL-IN-1 treated group was significantly decreased,and gene expressions related to glycogen phosphorylation and glycolysis in liver and muscle were significantly increased.In protein metabolism,the total protein content of whole fish of zebrafish in the HSL-IN-1 treated group was significantly decreased,and the protein expression content of m TOR,the core protein regulating protein synthesis,was significantly decreased,while the expression levels of genes related to amino acid metabolism were significantly up-regulated.These results indicate that biochemical inhibition of Hsl enzyme activity in zebrafish will lead to lipid catabolism disorder,resulting in excessive fat deposition,and high levels of inflammation and antioxidant reduction in body tissues,affecting body health.2.Construction of hsla-KO zebrafish modelAlthough the use of small molecule inhibitors can effectively inhibit target proteins,there may be some negative effects at the same time,such as producing active metabolites to produce toxicological effects on tissues and bodies.With the development of gene editing technology,CRISPR-Cas system has been widely used in life science research due to its high efficiency,convenience and speed.It has become the mainstream trend to use multiple models and multiple techniques to explore specific scientific problems.Therefore,we constructed the hsla^-/-zebrafish model using CRISPR-Cas9 technology.As the results show that:The first exon of the CDS sequence of zebrafish encoding hsla gene had a 7bp base deletion,which resulted in a pipework mutation.The Hsl protein translation process was terminated from 696 amino acids to51 amino acids in advance,and only 43 amino acids were retained.Therefore,the mutation was identified as an effective mutation.According to the expression of hsla gene in different tissues,hsla was highly expressed in liver,muscle and visceral mass of core metabolic tissues of zebrafish.The results of oil-red staining experiment showed that hsla^-/-zebrafish had more obvious fat deposition in the abdomen compared with15-day-old WT zebrafish.The above results indicate that the hsla-KO zebrafish model is successfully constructed,which can be used for further analysis in subsequent experiments.3.Study on Maintenance mechanism of Nutrient metabolism by hsla-KO in ZebrafishWe constructed the hsla^-/-zebrafish model using CRISPR-Cas9 technology.Combined with the results of Hsl inhibitor treatment experiment,we jointly explored the important function of Hsl in the maintenance of nutritional and metabolic functions of fish.Male zebrafish were randomly selected and divided into two groups（WT group and hsla-KO group）for a 5-week breeding experiment.Tissue samples were taken and stored at the end of culture.We examined growth,movement ability,biochemical indexes related to the metabolism of three major nutrients,expression levels of related genes and proteins,expression levels of autophagy related genes,expression levels of antioxidant enzyme activity and genes related to inflammation and apoptosis,fatty acid composition changes in whole fish,muscle and liver,and expression of fatty acid modification related genes in liver and muscle of zebrafish.The results showed that the growth of hsla-KO zebrafish was delayed,oxygen consumption rate was significantly decreased,but there was no significant difference in movement capacity.The viscerosomatic index of hsla-KO zebrafish increased significantly,and it was found that there was more obvious adipose tissue in the visceral mass after dissection,and according to the staining results of H&E and Oil Red sections,the volume and number of adipose vacuoles in the liver and viscera tissue were larger and significantly increased,and fat or TG was significantly accumulated in the whole fish,liver and muscle tissue.Corresponding to the significantly down-regulated lipid catabolism related genes;Impaired lipid catabolism induced by hsla-KO leads to up-regulation of autophagy pathway,which compensatively enhances the decomposition of lipid droplets.The glycogen content in liver and muscle and the total protein content of whole fish were significantly decreased in hsla-KO,indicating that the utilization of glycogen and protein was enhanced after hsla-KO weakened the supply of lipid source energy.The resistance to cold stress and antioxidant capacity of zebrafish were significantly weakened by hsla-KO,and the tissues showed higher expression of inflammatory and apoptotic genes.hsla-KO zebrafish resulted in remodeling of fatty acid profile in whole fish,muscle and liver,significantly increasing the content of saturated fatty acid but significantly decreasing the content of PUFA.These results indicate that hsla-KO can seriously weaken the lipid catabolic ability of zebrafish,thereby leading to the accumulation of fat and TG in the whole body and tissues,and reshaping the fatty acid spectrum composition of liver and muscle tissue,reducing the anti-cold stress ability and antioxidant capacity,and at the same time,along with the high expression of tissue inflammation and apoptosis,seriously affecting the body health.However,the weakening of lipid metabolism can lead to a certain degree of compensatory enhancement of other pathways such as autophagy,glucose metabolism and protein metabolism.Based on the results of the above two Hsl inhibition models with different principles,we compared the two Hsl dysfunction models.The results showed that both HSL-IN-1 treatment and hsla-KO could lead to lipid metabolism disorders of zebrafish,and fat and TG were deposited significantly in whole fish and multiple tissues.Both HSL-IN-1 treatment and hsla-KO enhanced the utilization of glycogen and protein to compensate for the lipid-derived energy deficiency.Both HSL-IN-1 treatment and hsla-KO can lead to high levels of inflammation and apoptosis expression in zebrafish tissues,which affects the body health.However,the two experimental models also had some different phenotypes:HSL-IN-1 treatment did not affect the weight gain of zebrafish,while hsla-KO zebrafish showed obvious growth retardation;HSL-IN-1treatment significantly decreased the expressions of liver adipose-de novo synthesis pathway related genes srebp1c,acc,fasn and dgat1a,while hsla-KO significantly increased the expressions of these genes.HSL-IN-1 treatment resulted in increased liver glycogen content and down-regulated expression of related glycogen phosphorylation and glycolysis genes,while liver glycogen content was significantly decreased and glycogen phosphorylation and glycolysis related genes were significantly enhanced in hsla-KO zebrafish.These results indicate that either gene-knockout or biochemical inhibition of Hsl will cause damage to lipid metabolism and decomposition ability of zebrafish,thus causing fat accumulation and metabolic disorders.At the same time,there are still some phenotypic differences between the two models that need to be further verified in subsequent studies.These results indicate that the results of biochemical inhibition and gene knockout treatment are not completely consistent,so it is necessary to use different models in combination to verify the same experimental results.In conclusion,Hsl functional deficiency leads to excessive fat deposition and serious disorder of lipid catabolism in the whole zebrafish and all tissues,and changes lipid composition in tissues,which directly leads to a higher level of inflammatory response and oxidative stress.In addition,from the perspective of maintaining the homeostasis of nutritional metabolism,Hsl dysfunction also leads to the promotion of glycogenolysis and the utilization of protein,which may be used to maintain the body’s energy balance.Therefore,Hsl plays a very important role in the maintenance of nutritional metabolic homeostasis of fish,and the imbalance of lipid metabolism caused by Hsl functional defect will lead to serious fat deposition in the body and damage the body health of fish.In addition,this study also illustrates the necessity of using different models for multiple tests when studying protein function through Hsl functional inhibition models of different principles.