The Implications Of Serpins Family Members PEDF And KBP In Fat Metabolism And Anti-inflammation

Chapter1The Role of PEDF on Triglyceride Degradation of Hepatocytes1.1BackgroundHepatic steatosis provides the setting for nonalcoholic fatty liver disease (NAFLD).NAFLD is a worldwide disease frequently associated with obesity, insulin resistance,metabolic syndrome and type2diabetes. The accumulation of lipids in the liver results froma disturbance among uptake, synthesis, oxidation and output of fatty acids. Thus, research onthe regulation of hepatic triglyceride degradation may identify a novel therapeutic target forhepatic steatosis. Pigment epithelium-derived factor (PEDF) is a well known angiogenesisinhibitor and neuroprotective factor and has been implicated in oncotherapy. Clinical studieshave demonstrated that plasma PEDF was elevated in an obese population, as well as inmetabolic syndrome and type2diabetes. Adipose triglyceride lipase (ATGL) is the maintriglyceride hydrolase, PEDF was shown to directly interact with ATGL in the retina. PEDFknockout-mice displayed hepatic steatosis and that PEDF bound to ATGL and regulatedtriglyceride content in an ATGL dependent manner, but the underlying mechanism of ATGLregulation by PEDF remains unclear. In the present study, we constructed signal peptidedeletion PEDF(mPEDF) overexpressing vector which sequestered PEDF only in cytoplasm.We also study the role of intracellular PEDF on triglyceride degradation, and the level of hepatic PEDF in steatosis state.1.2ObjectiveThis chapter mainly investigates the expression pattern of hepatic PEDF during hepaticsteatosis development, the role and molecular mechanism of intracellular PEDF ontriglyceride degradation.1.3Results(1) HFD induced obese mice with hepatic steatosis model was successfullyestablished. The body weigh of HFD induced mice at8weeks was increased by54%.H&E and Sudan III staining examination of liver showed that apparent hepatic steatosiswas found in HFD-fed mice at8weeks. Hepatic triglyceride of HFD-fed mice was3foldhigher than that of chow-fed mice.(2) Hepatic PEDF of HFD-fed mice and db/db mice were downregulated compared tothe control group mice. qPCR assay and Western Blot analysis showed that mRNA leveland protein level of hepatic PEDF were increased in HFD-fed mice at4,8and12weeks,while decreased at16and20weeks. Hepatic PEDF of db/db mice was downregulatedcompared to that of control mice. Our in vitro study demonstrated that oleic aciddownregulated PEDF expression in hepatocytes.(3) PEDF has an intracellular distribution, and exogenous PEDF could be absorbedby cells. Immunofluorescent assay and confocal assay confirmed that PEDF was mainlydistributed in cytoplasm, and FITC labeling assay demonstrated that exogenousrecombinant PEDF protein could transport into cells.(4) Intracellular PEDF promotes triglyceride degradation. mPEDF and PEDFoverexpressing HeLa cells were established. Nile red staining and confocal assay showedthat both mPEDF and PEDF promoted triglyceride degradation, decreased lipid dropletassociated protein Perilipin2, and also promoted glycerol release.(5) Both mPEDF and PEDF bind to ATGL. Co-Immunoprecipitation assay showed thatATGL antibody precipitated with PEDF in mPEDF and PEDF expressing cells. On the other hand, rPEDF protein pulled down ATGL, which indicated signal peptide of PEDFdid not interfere the binding between PEDF and ATGL.(6) ATGL mediates the function of PEDF on triglyceride degradation, G0S2overexpression inhibits the lipolytic activity of PEDF. Both ATGL gene silencing andG0S2overexpression inhibited the glycerol release of PEDF, while ATGL overexpressionincreased.(7) Both mPEDF and PEDF increases binding between ATGL and G0S2, andpromotes their translocation onto lipid droplet. BiFc assay showed that both mPEDFand PEDF promoted ATGL-G0S2translocation onto lipid droplet. BiFC assay andimmunoprecipitation assay demonstrated that mPEDF and PEDF increased ATGL bindingto G0S2.(8) PEDF binds to C terminal of ATGL(aa268-504), and PEDF could interacts withATGL and G0S2simultaneously. ATGL fragment deletion vector overexpression andimmnuoprecipitation assay demonstrated that PEDF binded to C terminal of ATGL andsimultanenously binded to ATGL and G0S2.(9) Endogenous PEDF binds with ATGL and is involved in hepatocytes triglyceridedegradation. siRNA-mediated PEDF knockdown and mPEDF, PEDF overexpressiondemonstrated that intracellular PEDF regulated hepatocyte triglycerdie degradation.Immunofluorescent assay and Co-immunoprecipitation assay showed that hepatic PEDFcolocalized and binded with ATGL.(10) PEDF downregulated hepatic G0S2expression. Our in vivo and in vitro studiesshowed PEDF decreased G0S2transcription.1.4ConclusionHepatic PEDF was downregulated in obese mice with hepatic steatosis. PEDF has acytoplasmic distribution, and promotes ATGL and G0S2translocation onto lipid droplet viabinding to C terminal of ATGL, finally enhances triglyceride degradation. PEDF alsodecreases hepatic G0S2expression, which inhibits triglyceride accumulation. Our study suggests a direct functional role of intracellular PEDF in triglyceride homeostasis andidentifies a therapeutic strategy for attenuating obesity-associated hepatic steatosis.Chapter2The Role of PEDF on adipose lipid regulation2.1BackgroundAdipose tissue represents the pool of excess energy in the form of triglycerides depositedin lipid droplets. During starvation and exercise, adipose tissue mobilizes to meet the energydemand of body by hydrolysing triglyceride to free fatty acids (FFA) and glycerol. Over thepast several years, adipose tissue has been emphasised as the master regulatory organdistributing whole-body lipid flux. Deregulation of adipose tissue function may lead toexcessive circulating FFA and ectopic fat accumulation in non-adipose tissues, such as liverand skeletal muscle, which results in obesity and type2diabetes. Hence, studies regarding toadipocyte lipid regulation may provide therapeutic potential for obesity and type2diabetes.ATGL is the rate limiting enzyme controlling the release of cellular FFA. Both plasma FFAand cellular FFA level are correlated positively with increased insulin resistance and obesity.Elevated plasma FFA comes from enhancing basal lipolysis in obesity. However,downregulation of adipose ATGL, the key player of lipolysis, is often observed in obese/insulin resistance humans and rodents. The reasons for the paradoxical phenomena are stillunknown. Investigation on the mechanism of decreased adipose ATGL and elevated plasmaFFA will contribute to the prevention and cure for obesity and insulin resistance.PEDF is wellknown as an multifunctional protein. Clinical studies elucidated that plasma PEDF waselevated in the population of obesity and metabolic syndrome and type2diabetes patients.Recent work reported that PEDF souring from adipose tissue contributed to the pathogenesisof insulin resistance in obesity, and enhanced adipocyte lipolysis via ATGL. In the presentstudy, we investigated the expression pattern of adipose PEDF and ATGL in HFD-fed mice,and then examined the downregulation of ATGL by PEDF and the underlying mechanisms.2.2ObjectiveThis chapter mainly investigates the expression pattern of adipose PEDF and ATGL duringobesity development, the downregulation and molecular mechanism of PEDF on ATGL protein.2.3Results(1) Adipose PEDF was upregulated in HFD-fed mice, while adipose ATGL wasdownregulated. QPCR and Western Blot analysis demonstrated that adipose PEDFmRNA and protein levels and plasma PEDF of HFD-fed mice were increased from4weeks to20weeks, while adipose ATGL protein was decreased.(2) Adipose ATGL was downregulated by PEDF. Western blot found that prolonged andacute PEDF treatment downregulated adipose ATGL protein, and PEDF antibody restoredATGL protein in obese mice. Our in vitro studies showed that PEDF decreased ATGLprotein in differentiated3T3-L1cells without altering ATGL transcription. siRNAmediated PEDF knockdown and antibody blockage increased ATGL levels.(3) PEDF promoted ATGL protein undergoing ubiquitin-mediated proteasomaldegradation pathway. Cycloheximide blockage found that the half-life of ATGL proteinwas69minutes, and MG132incubation demonstrated that ATGL degradation was viaproteasome mediated pathway. Immunopricipitation assay found that ATGL waspolyubiquitination and enhanced by PEDF treatment.(4) PEDF induced basal lipolysis in adipose tissue. Both plasma PEDF and FFA wereelevated during obese state. In vivo an in vitro studies demonstrated that PEDF enhancedbasal lipolysis instead of ISO-stimulated lipolysis, and PEDF antibody blockagedecreased circulating FFA in obese mice.(5) ATGL mediated the lipolytic activity of PEDF. ATGL inhibitor bromoenol lactonetreatment blocked the enhanced lipolysis by PEDF. PEDF downregulated ATGL inhibitorG0S2and lipid droplet associated protein Perilipin1expression.2.4ConclusionOur findings have elucidated the dual regulation of ATGL by PEDF: PEDF downregulatedATGL expression and activated lipolysis via ATGL in obesity. PEDF-induced ATGLdownregulation was via proteasome-degradation. Fat accumulation in adipose tissue resulted in increased adipose PEDF and plasma PEDF, which induced ATGL degradation and FFArelease in adipose tissue, and in turn led to progressive obesity and insulin resistance. Ourstudy elucidated that elevated adipose PEDF decreased ATGL protein expression in adiposetissue in obesity, as well as induced basal lipolysis, which explains the concurrent phenomenaof progressive fat accumulation and enhanced lipolysis in obesity, and probably contributedto the prevention and cure of obesity and insulin resistance.Chapter3The Anti-inflammatory Activity of KBP on Endotoxin Model2.1BackgroundKallikrein-binding protein (KBP), is firstly identified as a member of the serine proteinaseinhibitor family. Previous studies showed that KBP specifically formed a covalent complexwith tissue kallikrein, and inhibited proteolytic activities of kallkrein, therefore modulated thekinin formation in vivo. Importantly, KBP has other functions independent of binding tokallikrein. It has been showed that KBP had the activities of reducing blood pressure,inhibiting angiogenesis and anti-inflammation. Lipopolysaccharide (LPS), known asendotoxin, is the principal component of gram-negative bacterial outer membrane. It is ahighly immunogenic antigen with the ability to induce host inflammatory response and tissuedamage. Over-reacting to LPS may lead to septic shock. Investigation on LPS responsibleinflammatory cytokines may gain new insight into the drug discovery. In the present study,we obtained recombinant KBP using gene engineering technique, and investigated theprotective effects and the underlying mechanism of KBP on mice endotoxin shock.2.2ObjectiveThis chapter mainly investigates the anti-inflammatory activity and molecular mechanismof KBP on LPS-induced TNF-α expression and endotoxin shock.2.3Results(1) KBP inhibited LPS-induced TNF-α transcription and expression. RecombinantKBP was obtained using gene engineering technique, and qPCR and ELISA assaydemonstrated that KBP significantly inhibited LPS-induced TNF-α transcription and expression in macrophages. KBP overexpression also decreased TNF-α proteinexpression.(2) KBP improves survival of mice by endotoxin injection. KBP pretreatmentdownregulated plasma TNF-α protein in LPS injected mice, and protected mice fromendotoxin shock by injecting a lethal dose of LPS.(3) KBP increased SOCS3transcription and protein expression. QPCR and WesternBlot analysis demonstrated that KBP upregualted SOCS3mRNA levels and proteinexpression instead of SOCS1.(4) SOCS3mediated the activity of KBP on TNF-α inhibition. siRNA-mediatedSOCS3knockdown significantly blocked the effect of KBP on anti-inflammation.(5) KBP did not alter NF-κB subunit p65activity and β-catenin/TCF transcriptionalactivity. Luciferase assay and immunofluorescent assay showed that KBP had no effecton NF-κB subunit p65translocation and activity. Luciferase assay showed that β-catenin/TCF was not involved in anti-inflammatory activity of KBP.2.4ConclusionKBP-mediated TNF-α suppression is involved in up-regulating SOCS3transcription andexpression, which in turn exhibits negative regulation of the pro-inflammatory signaling. Viaattenuating acute inflammation, KBP could be implicated in therapy for septic shock.