Transcriptinal Of ATGL And Its Role In Maintaining Adipocyte Lipid Homeostasis In Grass Carp (Ctenopharyngodon Idellus) Under Nutritional Restriction

Food deprivation is a major challenge faced by many animals in natural habitats.In order to survive,vertebrates evolved unique adipose tissue to store energy in the form of triglycerides within lipid droplets.Under nutritional stress,fatty acids released from adipocytes via lipolysis are subsequently transported to other target tissues and utilized as substrates for energy production by fatty acid oxidation.Interestingly,fish show an impressive capacity to withstand prolonged periods of starvation compared with mammals.However,the lack of knowledge about lipolysis in fish restricts the exploration of the mechanism underlying this phenomenon.In addition,excessive fat is deposited in farmed fish.The research of fish lipolysis could shed light on mechanisms that control metabolic disorders in farmed fish,but the current knowledge of teleost lipolytic enzymes is very limited.Based on the current situation,we summarized the following two scientific questions: 1.What is the mechanism underlying initiation of adipocytes lipolysis in response to nutritional restrictions in fish? 2.Increased concentrations of non-esterified FAs disrupt the integrity of biological membranes,impair membrane function and inducing endoplasmic reticulum(ER)stress,and celldeath.In this context,how do fish avoid the toxicity of free fatty acids produced by lipolysis and maintain the lipid homeostasis in adipocytes.In this study,grass carp(Ctenopharyngodon idellus)was chosen as a model fish and molecular cloning,quantitative real-time PCR,western blot,protein purification,histology analysis,and other cell biology approaches were used to address these questions.The main results obtained as follows:1.ATGL is a metabolic mediator linking environmental nutritional restrictions to metabolic adaptations.The genome of grass carp and other teleosts codes for one ATGL,two HSLs,and one MGL isoforms.Two isoforms of HSL gene,HSLa and HSLb,derived from paralogous genes that could be originated from teleost-specific genome duplication(TSGD)event.The genes encoding for fish ATGL and MGL were conserved and contained nine and seven coding exons,respectively.However,two isoforms of HSL gene had a remarkable variation in gene structure,such as HSLa gene contained ten and HSLb contained thirteen exons.All three enzymes,including two isoforms of HSL,were expressed in a wide range of tissues.The expression of ATGL in grass carp adipocytes responded strongly to nutritional stress,while the expression of HSL and MGL and the phosphorylation level of HSL did not change.2.ATGL-catalyzed lipolysis is coupled with PPAR?-mediated fatty acid re-esterificationGrass carp ATGL is a lipid droplet protein,most of which are located on the lipid droplets of adipocytes.It can cause larger lipid droplets to diffuse small lipid droplets in adipocytes,promoting the hydrolysis of TG.ATGL can affect the partitioning of free fatty acids.Adipocytes were treated with purified ATGL recombinant protein.ATGL had no effect on the de novo synthesis.The elevation of CPT1 b,fatty acid beta oxidation gene,indicated that free fatty acids produced by ATGL-mediated lipolysis could enter the mitochondria and were used as energy substrates.The activation of PPAR? and PPAR? indicated that free fatty acids produced by ATGL-mediated lipolysis could function asintracellular signaling molecules.ATGL-catalyzed lipolysis is coupled with PPAR?-mediated fatty acid re-esterification and this can prevent the endoplasmic reticulum stress and excessive loss of TG in adipocytes during lipolysis.Hence,the coupling of lipolysis and the re-esterification is a protective mechanism in adipocytes.3.The transcriptional expression of ATGL under nutritional restrictions is regulated by cAMP/PKA/CREB.The transcriptional regulation of ATGL is an important mechanism intheinitiation of lipolysis in response to nutritional stress in grass carp adipocytes.The minimum essential promoter region of grass carp ATGL gene is located in-299 to-261.The location of PPAR?a and CREB in this region affected the activity of grass carp ATGL gene promoter.However,inhibition of PPAR? could not reverse the increased lipolysis induced by Forskolin.There are two modes of transcriptional regulation of grass carp ATGL gene: PPAR?a regulates its transcription in basal lipolysis,and cAMP/PKA/CREB regulates its transcription in stimulated lipolysis.Inhibiting CREB can block ATGL-mediated lipolysis under nutritional restrictions.4.cAMP/PKA/CREB/ATGL preventes endoplasmic reticulum stress in adipocytes and excessive loss of TG via PPAR?-mediated fatty acid re-esterificationIn response to nutritional restrictions,free fatty acids produced by cAMP/PKA/CREB/ATGL-mediated adipocytes lipolysis will firstly enter the mitochondria to satisfy their own energetic requirements.However,with the prolongation of starvation time,grass carp adipocytes will synthesize TG through PPAR?/GyK,ensuring that intracellular TG will not be excessively lost.During the whole process of starvation,the increase of PPAR?/DGAT1 prevented endoplasmic reticulum stressduring lipolysis,thus protecting adipocytes from the toxicity of excessive concentration of free fatty acids.Therefore,characteristic of grass carp adipocytes,"egoistic-altruistic-egoistic",may be a potential mechanism to ensure energy homeostasis during long-term nutritional stress.In conclusion,(1)grass carp adipocytes triggered lipolysis through the transcription of ATGL via cAMP/PKA/CREB signaling pathway under nutritional stress.(2)In response to nutritional restrictions,cAMP/PKA/CREB/ATGL regulates PPAR?-mediated fatty acid re-esterification: 1.Prevent endoplasmic reticulum stress in adipocytes during lipolysis,which is a protective mechanism of adipocytes in the process of starvation;2.Prevent excessive loss of TG and prolong the time in providing energy substrates for other tissues to help the body withstand long-term starvation.It may be a potential mechanism by which fish can tolerate long-term starvation.Thus,the coupling of lipolysis initiated by cAMP/PKA/CREB/ATGL and fatty acid re-esterification mediated by PPAR? is an adaptive strategy for adapting to long-term starvation and maintain their energy homeostasis in grass carp adipocytes.