The Role And Mechanism Of Apolipoprotein M In Responding To Glucose Metabolism

Objective: To develop a real-time quantitative RT-PCR method with the TaqMan probe tomeasure apolipoprotein M (apoM) mRNA levels. Methods: The primers and TaqManprobe targeted at signature and conserved sequence of apoM gene were designed by thesoftware “Primer Express Versions2.0” and applied to detect the apoM mRNA levels. TheapoM recombined plasmid constructed by conventional molecular biological techniqueswas applied as the standards of apoM quantitative detection. The reaction system wasoptimized, and the sensitivity, specificity as well as reproducibility were evaluated.Results: The PCR amplification products were verified by the direct DNA sequencing.The results showed the sensitivity, linear rang, the interassay coefficient of variation, thecorrelation coefficient and the amplification efficiency were4101copies/μl,4.00×101～4.00×108copies/μl,6.38%～17.9%,1.00and90%，respectively. Conclusion: This methodrepresents a high sensitivity, specificity and stability and it can be applied forquantification of the apoM mRNA levels. Objective: To explore whether elevated plasma free fatty acid (FFA) downregulates apoMexpressions in vivo and in vitro. Methods: Human hepatoma cell line, HepG2cells weretreated with experiment medium containing different concentrations of palmitic acid. Also,rats were infused with Intralipid via carotid or tail vein. Both real-time PCR and PCR Array were applied for analyzing the effects of plasma FFA on apoM expressions.Results: As expected, FFA concentration increased by17.6-fold, and glucose infusionrates (GIR) were reduced by27%in the Intralipid group compared to the controls thoseinfused by5%glucose solution. apoM gene expression in rat livers was significantlydecreased after Intralipid infusion compared to the5%glucose infusion followed byhyperinsulinemic-euglycemic clamp (HEC). Meanwhile, in an additional experiment, HECtests were omitted to avoid the effects of20%glucose. ApoM mRNA levels were alsosignificantly lower in the Intralipid group than that in the control group. One-way ANOVAindicated a significant difference among palmitic acid treated HepG2cells groups (P<0.01),due to a significant reduction of apoM mRNA levels in the presence of1mM compared to0mM palmitic acid. The PCR Array analysis demonstrated that certain genes those wereassociated with the PI-3kinase (PI-3K), mitogen-activated protein kinase (MAPK),peroxisome proliferator-activated receptor gamma (PPARg), insulin or sterol regulatoryelement-binding protein-1(SREBP1) pathway were significantly increased. In this array,only secondary effector target gene for insulin signaling, solute carrier family2(SLC2A1)whose gene name is glucose transporter type4(GLUT4) was significantly decreased. Wealso detected expression of84genes those relating to the insulin-responsive genes in liversof rats infused with Intralipid using SABiosciences’ RT2Profiler PCR Array System. Somegenes those associating with the PI-3K, MAPK and etc. were also increased. Conclusion:ApoM, probably, could be down-regulated by FFA via PI-3K and/or PPARβ/δpathway(s). Objective: To investigate whether short-term infusion of25%glucose coulddown-regulate apoM expression via the FFA pathway. Methods: The rats were infusedwith glucose and/or rosiglitazone via carotid or tail vein. A modified colorimetric method, HEC test and real-time PCR were employed to analyze plasma FFA concentrations, GIRand apoM expression levels, respectively. Results: Plasma FFA concentration in groupinfused with25%glucose was lower than that in group infused with5%glucose (P<0.001).Rosiglitazone had no effect on plasma FFA levels in healthy rats (P>0.05). Two-WayANOVA showed that there was no interaction between rosiglitazone and glucose onplasma FFA. The GIR was reduced in25%glucose treated rats (P<0.0001) and it could bereversed by rosiglitazone (P<0.0001). The interaction between rosiglitazone and glucoseon insulin sensitivity was extremely significant (P<0.0001). ApoM mRNA levels in ratliver were significantly decreased by infusion of25%glucose (P<0.05), whereas increasedby rosiglitazone (P<0.0001). However, the interaction on apoM mRNA expressionbetween rosiglitazone and glucose was not considered significant (P>0.05). Conclusion:Both high concentration glucose and rosiglitazone could regulate expression ofapolipoprotein M, which was not via the FFA pathway. Objective: To understand the mechanism of down-regulation of apolipoprotein Mmediated by free fatty acid (FFA) and hyperglycemia, respectively. Methods: Humanhepatoma cell line, HepG2cells were treated with experiment medium containing palmiticacid (1mM) and/or PI-3K inhibitor LY294002(10μM), palmitic acid (1mM) and/or proteinkinase C inhibitor GF109203X (GFX,2μM) and palmitic acid (1mM) and/or PPARβ/δantagonist GSK3787(10μM), respectively. The rats were infused with Intralipid, glucoseand/or rosiglitazone from tail vein. Real-time PCR were applied for analyzing expressionlevels of the related genes. Results: Analysis of the data showed significant reductions ofapoM mRNA levels in the presence of1mM palmitic acid compared to without palmiticacid treatment (P<0.01). LY had no effect on expression of apoM gene in HepG2cells (P>0.05). Two-Way ANOVA showed that there was no interaction between palmitic acidand LY on apoM expression in HepG2cells. GFX significantly decreased apoM mRNAlevels (P<0.05), but it couldn’t reverse palmitic acid induced down-regulation of apoMexpression.20%Intralipid had no influence on PPARβ/δmRNA expression in rat liver(P>0.05). In HepG2cells, PPARβ/δantagonist GSK3787had no effect on apoM mRNAlevels (P>0.05), but it could significantly reverse the effect of palmitic acid induceddown-regulation of apoM expression. The interaction between palmitic acid and GSK3787on apoM mRNA levels was significant (P<0.05).25%glucose significantly inhibitedmRNA levels of LXRβ(P<0.001), SHP1(P<0.0001), LRH1(P<0.01), ABCA1(P<0.01)and PPARβ/δ(P<0.01) in rat liver, while rosiglitazone merely decreased mRNA levels ofSHP1(P<0.01) and ABCA1(P<0.05). Further study indicated that rosiglitazone coulddown-regultate ABCA1mRNA expression (P<0.05) in normal rat liver, but it significantlyincreased ABCA1mRNA levels in the presence of25%glucose (P<0.01). Two-WayANOVA showed that the interaction between rosiglitazone and25%glucose on ABCA1mRNA expression was extremely significant (P<0.001). Conclusion: The palmitic aciddown-regulated apoM gene expression via PPARβ/δpathway in HepG2cells. And, highconcentration glucose might inhibit apoM mRNA expression through LRH1and/orABCA1pathway(s). Objective: To explore whether a transfection of human apoM gene may improve insulinsensitivity in the Goto-Kakizaki（GK）rats. Methods: Human apoM gene was transferredinto the293T cells and GK rats using lentiviral vectors as delivery vehicles. PCR Array,real-time PCR and insulin tolerance test (ITT) were applied for analyzing genes thoserelated to the type2diabetes, regulating apoM expression and/or in relation to the insulin sensitivities. Results: The gene related to insulin resistance, MAPK8was down-regulatedto2.11-fold (P<0.05), while apoM mRNA levels in293T cells transferred with humanapoM gene increased79.43-fold (P<0.001). It was demonstrated that human apoM mRNAwas significantly expressed in lung of GK rat after injecting5×108TU lentiviral vectorswith human apoM gene. At14th day, there was no difference on fasting blood glucoselevels between the control group and the group transferred with human apoM gene.Interestingly, ITT analysis showed that the time of blood glucose dropped to50%frombaseline value in GK rats transferred with human apoM gene (60min) was much less thanthat of control rats (90min). Conclusion: Overexpression of apoM gene could improveinsulin sensitivity of individual with type2diabetes, the detail mechanism needs futherinvestigation.