The Mechanism Of PKD1 Conditional Knockout Contributions To De Novo Lipogenesis In Adipose Tissue

BackgroundIn recent years,obesity has become a worldwide epidemic disease.It causes a series of complications such as arthritis,cholelithiasis,obstructive sleep apnea,hypopnea syndrome and infertility.What's more,it can accelerate cardiovascular disease and increased cancer risk.It is well known that excessive carbohydrate intake and less caloric requirements contribute to the development of obesity.The redundant glucose stored in liver and adipose must be converted into lipids by the progress of de novo lipogenesis for subsequent storage.The triglycerides derived from liver can undergo hydrolysis and be transferred to adipose and muscle by very low density lipoproteins(VLDL).White adipose tissue(WAT)can reesterify the free fatty acids from diets and liver and stores the triglycerides.De novo lipogenesis(DNL)is a complex metabolic pathway that regulate synthesis of triglycerides with excess glucose as substrate.This progress usually occurs in both liver and adipose tissue after treated with high carbohydrate or fat diets.While most of the serum TG content is obtained from dietary sources.However,some studies suggest that hepatic DNL could make significant contribution to serum lipid content in individuals on high carbohydrate diet.The flow of carbons from glucose to fatty acids,which is modulated by the lipogenic pathway,includes a coordinated series of enzymatic reactions.The first step of this series of reactions is the conversion of citrate to acetyl CoA by ATP-citrate lyase(ACLY).The resulting acetyl-CoA is carboxylated to malonyl-CoA by acetyl-CoA carboxylase(ACC).Fatty acid synthase(FASN)is the key rate-limiting enzyme that brings about the conversion of malonyl-CoA into palmitate.After a series of reactions palmitate is further converted into complicated fatty acids.The main product of DNL is palmitate but stearate and shorter fatty acids are also generated.Sterol regulatory element binding protein(SREBP)is a key lipogenic transcription factor that controls several rate-limiting enzymes of de novo lipogenesis pathway.The SREBPs are consist of three members(SREBP-la,SREBP-1c and SREBP-2)that share the same structure of helix-loop-helix-leucine zipper.SREBP-1a can activate the pathway of cholesterol and fatty acid synthesizing.SREBP-lc mainly regulate fatty acid synthesis and adipocyte differentiation.SREBP-2,primarily expressed in liver,relatively specific control cholesterol biosynthesis and uptake.The isoforms of SREBP-1 and SREBP-2 are synthesized as precursors binding to the endoplasmic reticulum(ER).Under the regulation of insulin and sterols,the SERBPs precursor could transfer from ER to Golgi combining with sterol-sensing domain of SREBP cleavage-activating protein(SCAP).On the Golgi complex,SREBPs are cleaved by two membrane bound proteases Site-1 protease(SIP)and Site-2 protease(S2P)and release the transcriptionally active N-terminal basic-helix-loophelix(bHLH)-Zip domain(68kDa).The mature form SREBP will translocate into the nuclear and bing to sterol regulatory element(SRE).At last the nuclear form SREBP can activates the transcription of relative genes such as ATP-citrate lyase(ACL),acetyl-CoA carboxylase(ACC),fatty acid synthase(FAS)and stearoyl-CoA desaturase(SCD).Protein kinase D 1(PKD1)belongs to a serine/threonine protein kinases family(PKD1(PKC?),PKD2,and PKD3(PKCv))within the Ca2+/calmodulin-dependent protein kinase(CAMK)group.PKD1 has a catalytic domain that is distantly related to Ca2+-regulated kinases and a pleckstrin homology(PH)domain that regulates enzyme activity.The N-terminal region of PKD1 contains two cysteine-rich DAG-binding(CRD)domains,cysl and cys2,which confer high binding affinity for phorbol esters and play a role in the regulation of catalytic kinase activity.PKD family is reported to localize to cytosol,nucleus,Golgi complex and plasma membrane.Recent researches had shown that PKD1 can bind to Golgi membranes through its aminoterminal domain in a diacyl glycerol(DAG)dependent manner involved in protein fission from TGN to the cell surface.PKD is recruited from the cytosol to the TGN via the interaction of an amino-terminal zinc finger domain with the lipid diacylglycerol.Once recruited to the membrane,PKD is activated by PKC?-mediated phosphorylation,which itself is activated by the trimeric G-protein subunits G?1?2 and G?3?2.Active PKD at the TGN is then thought to phosphorylate downstream targets to bring about the fission of transport carriers destined for the plasma membrane.Recent researches have shown that PKD mediates negative regulation of the adiponectin gene expression by AP-2.Adiponectin is a protein hormone that modulates a number of metabolic processes,including glucose regulation and fatty acid oxidation.PKD1 was highly phosphorylated in the re-feeding condition compared to the fasting condition in mouse adipose tissue,while other PKD isoforms remained unchanged.Furthermore,overexpression of PKD1 in 3T3-L1 adipocytes,but not other PKD isoforms,positively regulated the mRNA expression and promoter activity of McP-1 and IL-6,and negatively regulated those of adiponectin.These researches indicate that PKD1 may participate in regulating metabolic process in adipose tissue.PKD1 is highly expressed in mice adipose tissue(white adipose tissue and brown adipose tissue)but is low expressed in other organs.Interestingly,the preadipocyte 3T3-L1 cell has an extremely rarely expression level of PKD1 but dramatically increase after differentiation.However,seldom researches can explain this phenomenon.In order to figure out it,we generate adipose-specific PKD1 KO(PKD1ad-/-)mice.Our study hypothesis that PKD1 is essential for the pathway of de novo lipogenesis by regulate the process of proteolytic cleavage of SREBP-lc via binging with SCAP in mice adipose.ObjectivesThe purpose of this study is to investigate the relationship between PKD1 and metabolism of adipose tissue and figure out the mechanism underlying the interaction of PKD1,SREBP1 and SCAP which not only contribute to a better understanding of PKD1 function in white adipose tissue,but also give us new ideas about obesity treatment.MethodsFirstly,The model of adipose specific knockout of PKD1 in mouse was generated to confirm the effects and related phenotypes of lacking PKD1.RNA-sequence and GO analyze of adipose tissue provide us correlation genes with significant changes between wild type group and knockout group.By real-time quantitative PCR,Western blot,immunofluorescence,immunoprecipitation methods confirmed the mechanisms of PKD1 regulating the process of de novo lipogenesis by interacting with SREBP1 and SCAP.Results1.PKD1 adipose-specific knockout mice is resistant to high fat diet induced obesity.To elucidate the role of PKD1 in adipose tissue,we utilized the cre/loxP system to generate mice in which PKD1 is deleted exclusively in adipose tissue.As a first analysis of the effect of PKD1ad-/-on adipose tissue,we monitored the weight of KO mice and littermate controls(PKD1fl/fl)under high-fat diet over 20 weeks between the ages of 8 and 28 weeks.Results show that PKD1ad-/-mice is not sensitive to the effect of high-fat diet.Control group(PKD1fl/fl)gain more weight than KO group(PKD1ad-/-).All internal organs of mice were weighed and there was no difference indicated that the effect of the knockout was specific to adipose tissue.The experimental group(PKD1ad-/-)and control group(PKD1fl/fl)mice white adipose was stained by hematoxylin-Eosin.The results suggested that the adipocytes from PKD1 ad-/-mice are smaller than those from PKD1fl/fl mice under high fat diet(n=4,p<0.01).PKD1 is required to maintain adipose tissue.2.RNA-sequence and GO analysis results showed that knockout of PKD1 in the adipose tissue have an effect on de novo lipogenesis process.The RNA-sequencing and GO analysis indicated that rate-limiting enzymes of de novo lipogenesis like acetyl-CoA carboxylase(ACC),fatty acid synthase(FAS),stearoyl-CoA desaturase(SCD)dramatically decrease in the experimental group(PKD1ad-/-).While the genes like LPL,CD36 which involved in facilitating fatty acid uptake displayed no significant change.Thses results suggest that the weight loss of PKD1ad-/-mice is caused by the de novo lipogenesis(DNL)lipogenesis process.3.Knockouting of PKD1 in adipose tissue can reduced expression of the rate-limiting enzyme proteins of de novo synthesis.Real time quantitative PCR and Western blot showed that knockouting of PKD1 in adipose tissue could reduce the expression of related rate-limiting enzymes of lipid synthesis such as fatty acid synthase(FASN),ATP-Citrate lyase(ACLY),Acetyl Coenzyme Carboxylase(ACC)and stearoyl-CoA desaturasel(SCD1).These results suggested that PKD1 have an effect on lipid synthesis in adipose tissue by stimulating the progress of de novo lipogenesis(DNL).4.PKD1 Promotes SREBP-1 Cleavage Processing and De Novo Lipogenesis in adipose tissueResearch has shown that AMPK,via Ser372 phosphorylation,inhibits SREBP-1 c cleavage processing,thereby blocking lipogenic gene transcription.Western blot show us that in adipose tissue PKD1 deficiency can lead to elevate phosphorylation levels of Ser372 Sites of SREBP-lc.That will inhibit SREBP-1 cleavage processing and de novo lipogenesis.The amount of cleaved nuclear form of SREBP-1c,NH2-Terminal,68kDa,is reduced.It can be concluded that PKD1 can promote the processing of SREBP-1c cleavage and translocate into the nucleus.5.PKD1 can interacte with SREBP1 and SCAP and knockdown of PKD1 expression can reduce the formation of mature SREBP-lcKnockdown of PKD1 in differentiation 3T3-L1 adipocytes can promote the phosphorylation levelof SREBP-lc-Ser372 Sites,down-regulate the expression of DNL related proteins Immunoprecipitation experiments imply that PKD1 can interact with SREBP1 and SCAP.We further searched PKD1 recognition consensus sites on the mouse SREBP-1c codingsequence(CDS)but failed.However,we got the recognition consensus sites on mouse SCAP codingsequence..ConclusionThe results of this study suggest that in adipose tissue PKD1 can interact with the SCAP and promote SREBP-lc cleavage processing and translocate into the nucleus,promoting expression of genes related to lipid synthesis like FASN,ACC.The research provide us a new horizon about the factors contribute to lipid accumlation and new directions for obesity treatment.