Effect Of Acylation Stimulating Protein On The Expression Of Lipid Droplet Associated Proteins During 3T3-L1 Preadipocytes Differentiation And Related Signaling Pathway

Partâ… Effect of acylation stimulating protein on adipophilin, perilipin andTIP47 expression during 3T3-L1 preadipocyte differentiationBACKGROUD: Over the past few years, the discovery of numerous adipokines, haverevolutionized the conception of its biological function,consolidating the idea that whiteadipose tissue is not just a supplier and storer of energy, but a dynamic organ and central tometabolic regulation.The novel adipokine acylation stimulating protein (ASP) is involvedin lipid metabolism and obesity-related disorders. ASP binds to its cell surface receptorC5L2 to increase triglyceride synthesis (fatty acid esterification), glucose transport anddecrease hormone sensitive lipase activity. Through the effects of ASP on intracellular fattyacid re-esterification, ASP increases the lipolytic efficiency of lipoprotein lipase. Increasesin glucose transport aremediated through increased activity and translocation of both Glut4 and Glutl in bothadipocytes and preadipocytes. In vivo, ASP appears to be closely related to postprandialtriglyceride (TG) clearance. In ASP deficient (C3 knockout) mice and C5L2 knockout mice,the absence of a functional ASP-C5L2 pathway leads to delayed postprandial TG. This isfurther enhanced in diet-induced obese mice or the genetically obese ob/ob mice.Postprandial chylomicrons, stimulate ASP production in human adipocytes in vitro, and thishas been documented in vivo in humans as well. Further, fasting plasma ASP is increased inobesity, insulin resistance, coronary artery disease and diabetes, even in the absence ofobesity, while weight loss and exercise decrease ASP. During differentiation to matureadipocytes, young adipocytes contain multiple small lipid droplets, which coalesce to forma single lipid inclusion as the cell matures. Adipophilin, perilipin and TIP47, three mostimportant members of the lipid droplet specific protein family, participate in ectopic lipid deposition in the form of cytoplasmic triglyceride droplets to provide efficient fatmobilization within many types of mammalian cells. However, mechanisms controlling thesynthesis and turnover of these lipid droplet proteins are only partially understood, themechanisms regulating gene/protein expression as yet unidentified. Thus a potentialapproach to regulate lipid accumulation in adipocytes may be to seek a possible keyhormonal factor regulating synthesis and expression of lipid droplet-coated proteins.AIM: Our aim in this study was to elucidate(1) whether ASP rather than insulin is a powerful potentiator which could physiologicallyand directly influence TG synthesis (TGS) during 3T3-L1 preadipocyte differentiation;(2) whether ASP exposure at indicated time point during 3T3-L1 preadipocytedifferentiation could influence the intracellular gene expression of adipophilin, perilipin andTIP47;(3) whether ASP exposure at indicated time point during 3T3-L1 preadipocytedifferentiation could influence the intracellular protein expression of adipophilin, perilipinand TIP47.METHOD: 3T3-L1 preadipocytes were differentiated by traditional hormone cocktail anddivided into control, ASP and insulin groups according to the treatment of ASP or insulin.ASP-stimulated and insulin-stimulated TGS rate at indicated time points (0 d, 3 d, 6 d, 9 d)were assayed by measuring the incorporation of [³H]-oleic acid into TG, and thecorresponding glucose transport by [³H]-2-DG uptake. The effect of ASP or insulin ongene/protein expression of adipophilin, perilipin and TIP47 at indicated time points wasevaluated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)and Western blot.RESULTS:(1) on the 3rd and 6th day of differentiation, ASP dramatically enhanced TGS ratecompared with control group (P＜0.05, P＜0.01); There was no significant difference in TGS ratio between insulin group and control group;(2) on the 6th and 9th day of differentiation, both ASP and insulin promoted glucose uptake(P＜0.05, P＜0.01), and the promoting effect in ASP group was more obvious than that ininsulin group;(3) ASP elevated adipophilin gene and protein expression at the very early stage ofdifferentiation (P＜0.05, P＜0.001) and had no significant effect from the 4th day ofdifferentiation. Perilipin gene and protein expression increased throughout preadipocytedifferentiation and its expression was up-regulated following ASP stimulation from the 3rdday of differentiation (P＜0.05, P＜0.001) to complete differentiation (P＜0.05). Exposure toASP for certain time resulted in the obvious increase of TIP47 gene and protein expressionin 3T3-L1 cells from the beginning of differentiation, compared with the control group, butthe effect of ASP was not significant after 48h of differentiation;(4) Insulin did not affect gene and protein pattern of adipophilin and perilipin. Whileinsulin increased gene and protein expression of TIP47 in 3T3-L1 cells when differentiationinitiated, compared with the control group, but it had not action after then.CONCLUSION: This study provides evidence that ASP-revoked change in gene andprotein expression of adipophilin, perilipin and TIP47 correlate with ASP-stimulated TGS,and adipophilin, perilipin and TIP47 are involved in the molecular mechanisms ofASP-induced adipogenesis and LD formation.Partâ…¡Acylation Stimulating Protein regulates expression of Adipophilin,Perilipin and TIP47 through a Phospholipase C-dependent pathwayduring 3T3-L1 preadipocyte differentiation. BACKGROUD: Acylation stimulating protein (ASP; C3adesArg) stimulates triglyceridesynthesis (TGS) and glucose transport in preadipocytes/adipocytes through C5L2, aG-proteincoupled receptor. After ASP-C5L2 receptor activation, theGαsubunit dissociatesfrom the Gβ/γsubunit, which leads to PLC and PI3K activation. PLC activation (which inthis case could occur through the Gαq or Gβ/γsubunit) results in the release of inositol1,4,5-triphosphate and DAG. Inositol 1,4,5-triphosphate stimulates the release ofintracellular Ca²⁺ from the endoplasmic reticulum, which further serves as a signalingmolecule, activating a number of intracellular enzymes, including PKC, PI3K, Akt, andcPLA2, all of which leads to an effective and prolonged stimulation of TGS.AIM: Our study was to elucidate:(1) whether PLC signaling pathway was involved in ASP-triggered alteration of adipophilinexpression at indicated time point during 3T3-L1 differentiation.(2) whether PLC signaling pathway was involved in ASP-triggered alteration of perilipinexpression at indicated time point during 3T3-LI differentiation.(3) whether PLC signaling pathway was involved in ASP-triggered alteration of TIP47expression at indicated time point during 3T3-L1 differentiation.METHOD: U73122 was used to evaluate the involvement of the PLC in ASP action.Confluent cells were pretreated with serum-free medium containing 5μM U73122 for 30min and were then stimulated with 1μM ASP for an additional 2h. The inhibitor was presentthroughout the experiment. Cells were lysed, and total cell protein was subjected to WesternBlot analysis using antibodies to adipophilin, perilipin and TIP47 respectively.RESULTS:(1) PLC inhibitor U73122 decreased gene/protein expression of adipophilin, perilipin andTIP47 respectively at indicated time point during 3T3-L1 differentiationâ‘ adipophilin mRNA levels at 0d, 3d, 6d and 9d were all significantly reducedwhen PLC was inhibited in cells by U73122 (5μM, 2.5 hours) (P＜0.05, P＜0.01, P＜0.001). Further, U73122 decreased the protein level ofadipophilin (P＜0.05) at 0d, 1d, 3d and 4d, but this effect was no longerdetectable at 6d (P＞0.05)â‘¡perilipin mRNA level in U73122 group was less than that in the control group at 3d,6d and 9d(P＜0.05), and perilipin protein amount in U73122 group vs controlgroup was decreased at 3d, 4d, 6d and 9d(P＜0.05, P＜0.01)â‘¢exposure to U73122 which is blocking agent of PLC signal pathway for certaintime resulted in the decrease of TIP47 mRNA and protein expression in 3T3-L1cells, compared with the control group(P＜0.05, P＜0.01)(2) Pre-exposure of 3T3-L1 cells at indicated time points during differentiation to thephospholipase C inhibitor U73122 followed by stimulation with ASP resulted in notablydecreased gene/protein expression of adipophilin, perilipin and TIP47.â‘ adipophilin mRNA level in U73122+ASP group was substantially decreased vsASP alone on days 0, 3, 6, and 9 respectively(P＜0.05, P＜0.01), adipophilinprotein amount in U73122+ASP group vs ASP alone was decreased at 0d, 1d, 3d,4d and 6d(P＜0.05).â‘¡perilipin mRNA level in U73122+ASP group was less than that in the ASP groupat 3d, 6d and 9d(P＜0.05, P＜0.01), As well, perilipin protein level inU73122+ASP group was reduced at 3, 4, 6, and 9 days, although significant onlyfor 4d and 9d(p＜0.05).â‘¢mRNA and protein level of TIP47 in 3T3-L1 cells treated with both U73122 andASP was significant lower than that in adipocytes only treated with ASP(P＜0.05,P＜0.01).CONCLUSION: PLC signaling pathway was involved in ASP-induced up-regulation ofadipophilin,perilipin and TIP47 mRNA and protein. Partâ…¢Acylation Stimulating Protein regulates expression of Adipophilin,Perilipin and TIP47 through a Phosphoinositide 3-kinasesdependentpathway during 3T3-L1 preadipocyte differentiation.BACKGROUD: Phosphoinositide 3-kinases (PI 3-Ks) are a subfamily of lipidkinase that catalyze the addition of a phosphate molecule specifically to the 3-position ofthe inositol ring of phosphoinositides. PI3Ks, which could be activated by lots ofphysico-chemical factors or cytokines, is involved in regulation of many cellular activities,such as, cell proliferation, survival, glucose transport, and cytoskeletal organization, etc.While Wortmannin, a kind of fungal metabolites separated by Penicillium-wortmannii atthe first time, is also a specific inhibitor of PI3K. The results of past work have illustrateddown-regulation of adipophilin expression by PI3K inhibitor wortmannin or LY294002 andinvolvement of PI3K in ASP/C5L2 signal cascades.AIM: Our study was to elucidate:(1) whether PI3K signaling pathway was involved in ASP-triggered alteration ofadipophilin expression at indicated time point during 3T3-L1 differentiation.(2) whether PI3K signaling pathway was involved in ASP-triggered alteration of perilipinexpression at indicated time point during 3T3-L1 differentiation.(3) whether PI3K signaling pathway was involved in ASP-triggered alteration of TIP47expression at indicated time point during 3T3-L1 differentiation.METHOD: METHOD: Wortmannin was used to evaluate the involvement of the PI3K inASP action. Confluent cells were pretreated with serum-free medium containing 100nMwortmannin for 30 min and were then stimulated with 1μM ASP for an additional 2h. The inhibitor was present throughout the experiment. Cells were lysed, and total cell proteinwas subjected to Western Blot analysis using antibodies to adipophilin, perilipin and TIP47respectively.RESULTS:(1) PI3K inhibitor wortmannin decreased gene/protein expression of adipophilin, perilipinand TIP47 respectively at indicated time point during 3T3-L1 differentiationâ‘ adipophilin mRNA levels at 0d, 3d, 6d and 9d were all significantly reducedwhen PLC was inhibited in cells by wortmannin (100nM, 2.5 hours)(P＜0.01, P＜0.001). Further, wortmannin decreased the protein level ofadipophilin at 0d, 1d, 3d and 4d (P＜0.05, P＜0.01).â‘¡perilipin mRNA level in wortmannin group was less than that in the control group at0d, 3d, 6d and 9d(P＜0.05), and perilipin protein amount in wortmannin group vscontrol group was decreased at 3d, 4d, 6d and 9d(P＜0.05)â‘¢exposure to wortmannin which is blocking agent of PI3K signal pathway for certaintime resulted in the decrease of TIP47 mRNA and protein expression in 3T3-L1cells, compared with the control group(P＜0.05, P＜0.01, P＜0.001)(2) Pre-exposure of 3T3-L1 cells at indicated time points during differentiation to thephosphoinositide-3 kinase inhibitor wortmannin followed by stimulation with ASPresulted in notably decreased gene/protein expression of adipophilin, perilipin andTIP47.â‘ adipophilin mRNA level in wortmannin +ASP group was substantially decreased vsASP alone on days 0, 3, 6, and 9 respectively (P＜0.01, P＜0.001), adipophilinprotein amount in wortmannin +ASP group vs ASP alone was decreased at 0d, 1d,3d, 4d and 6d(P＜0.05, P＜0.01).â‘¡perilipin mRNA level in wortmannin +ASP group was less than that in the ASPgroup at 3d, 6d and 9d(P＜0.05, P＜0.01), As well, perilipin protein level in wortmannin +ASP group was reduced from 4th day (p＜0.05).â‘¢mRNA and protein level of TIP47 in 3T3-L1 cells treated with both wortmannin andASP was significant lower than that in adipocytes only treated with ASP(P＜0.05,P＜0.01 ). From 2nd day, there was no significant difference of mRNA level betweenthe two groups.CONCLUSION: PI3K signaling pathway was involved in ASP-induced up-regulation ofadipophilin,perilipin and TIP47 mRNA and protein.