Effect Of Theacrine On Lipid Metabolism Mediated By SirT3 / AMPK Signaling Pathway And Its Mechanism

Objective：Simple fatty liver and restraint stress mouse model wereestablished respectively by fat diet-induced method. The effect of purinealkaloid theacrine on improving liver fat and lipid metabolism disordersin mice were studied carefully. The underlying mechanisms of those effectswere investigated. Researches on evaluation the biological activity andmechanism of theacrine were developed in order to confirm the applicationsof theacrine in lipid metabolism.Materials and methods：Part Ⅰ: Theaceine activated SirT3/AMPK/ACC signal pathway and reducedthe hepatic steatosis in high-fat diet mice.1. C57BL/6J mice were fed17weeks to induce as simple fat model， thendevided into standard diet group and high-fat group. Each group wasadministrated standard or high-fat diet respectively. Mice with similarweight were randomly divided into3groups， with8mice in each group，and treated as HF， low dose of theacrine (10mg/kg， HF+TL) and high doseof theacrine (20mg/kg， HF+TH). There were no significant differencesbetween groups in body weight (P>0.05). After the last administration，the mice were anesthetized with ether. Liver was rapidly removed and TGcontent was detected. About1cm3part of liver was cut and fixed in4%paraformaldehyde. Then paraffin sections were made to observe the degreeof fat tissue accumulation after Sudan IV staining. Western blot was usedto detect the protein expression in liver SirT3/AMPK/ACC.2. HepG2cells were cultured in monolayer and digested with trypsin. Then，inoculated in6-well plates with4×105per well and grouped into normalcontrol group， compound C group (15μmol/L)， theacrine group(2 mmol/L)， theacrine+compound C group(15μmol/L compound C+2mmol/Ltheacrine)。After cultured for24h， cells were collected to study theSirT3/AMPK/ACC protein expression and phosphorylation levels of AMPK andACC.Part Ⅱ: Effects and mechanisms of theacrine on lipid metabolism inrestraint stress mice were investigated systematically.1. Mice were randomly devided into groups according to body weight: normalcontrol group， the restraint stress group， low dose of theacrine group(10mg/kg，TL)， high dose of theacrine (20mg/kg，TH)， with10mice ineach group. Experimental groups were administrated once daily for7days，except the normal control group， other groups were experienced restraintstress after7days’ administration. Relevant indicators were detectedafter the corresponding experiment for20h. After30min of the restraintstress，5mice in each group were intravenously injected fat emulsionthrough tail veil. In the condition of anesthesia， blood was taken fromthe heart and analyzed the content of neural fat. The remaining5micein each group were anesthetized with ether30min after the end ofrestraint. Blood was collected from the heart. Commercial analysis kitwas used to detect plasma levels of lipoprotein lipase (lipoprteinlipase，LPL)， malondialdehyde (malonaldehyde，MDA) and superoxide dismutase(superoxide dismutase，SOD). Protein expression in liver SirT3/AMPK/ACCwas analysed by western blot. The mRNA expression of genes， LPL/GPIHBP1/ANGPTL4， which were related to lipid metabolism in heart ’s were alsodetected by RT-PCR. Changes of plasma corticosterone content weredetermined by high performance liquid chromatography.2. Experimental animals were devided in restraint stress group，10mg/kgcompound C group，20mg/kg theacrine+10mg/kg compound C group，25mg/kg Ru486group，20mg/kg theacrine+25mg/kg Ru486group， with10mice in each group. Theacrine group was administrated continuously for 7days， group of restraint stress and comound C were given the same doseof saline. In the administration of the first seven days， compound Calone group and combined with theacrine group were given10mg/kg compoundC abdominally， while Ru486alone and combination group were treatedintravenously with Ru486. Finally， indicators were measured after20-hour restraint stress of all the animals. The protein expressions ofSirT3and phosphorylation levels of ACC and AMPK in liver were analyzedby western blot. The mRNA expression of lipid metabolism-related genesin cardiac were detected by RT-PCR.Results:1. Theaceine activated SirT3/AMPK/ACC signal pathway and reduced thehepatic steatosis in high-fat diet mice.1.1The impact of theacrine on liver triglycerides in high-fat diet mouse.Compared with standard diet group， the content of triglyceride inthe liver of mice was significantly increased (P <0.01) in high-fat dietgroup， and triglyceride levels in the liver of high-fat diet mice weresignificantly reduced by threacrine which showed dose dependently (P<0.01).1.2The effect of theacrine on liver histopathology in high-fat dietmouse.Compared with the lobular structure of the liver in mice treated withstandard diet， fat vacuole was increased significantly around the liverportal area and interlobular veins in high-fat diet mouse， both low-doseand high-dose of theacrine can reduce fat vacuoles in liver tissuesections.1.3The effect of theacrine on protein expression in high-fat diet mouseCompared with normal condition， long-term high-fat diet causedhepatic steatosis， protein expression of SirT3was reduced in livermitochondria (P <0.05)， AMPK phosphorylation levels were significantly decreased (P <0.01)， while ACC protein phosphorylation was reduced toa certain degree. Compared with the normal group， theacrine treated groupincreased SirT3protein expression in liver and phosphorylation of AMPKand ACC.1.4The impact of theacrine on relative protein expression after AMPKsuppressionAfter treated with15μmol/L compound C， a AMPK inhibitor， proteinexpression of SirT3unchanged， and levels of ACC phosphorylationdecreased. After the suppression of intracellular AMPK phosphorylation，theacrine could still activate SirT3， but inhibited phosphorylation ofACC.2. Research on the effects of theacrine on the restraint stress andmechanism of fatty acid metabolism in mice2.1The effect of theacrine on neutral fat tolerance in plasma in restraintstress mice.Compared with normal control mice， plasma TG elimination rate wasslowed down after treated with fat emulsion in mice20h restraint stress，plasma triglyceride levels were significantly increased. Both10mg/kgand20mg/kg dose of theacrine could significantly improve TG plasmaelimination rate in restraint mouse and decrease plasma triglyceridelevels.2.2The impact of theacrine on palsma lipoprotein lipase LPL activity inrestraint stress mouse.The activity of fatty acid lipase wae decreased in mice caused byrestraint(P <0.05)， while LPL activity in the blood of mice wassignificantly improved by theacrine administration.2.3The effect of theacrine on plasma lipid peroxide levels in restraintstress mice.After the stress load， compared with the normal group， the restraint load significantly increased plasma lipid peroxidation product MDA inmice. Either dose of theacrine could significantly improve the contentof MDA in plasma.2.4The effect of theacrine on plasma superoxide dismutase levels inrestraint stress miceCompared with the normal group， plasma level of SOD was significantlyreduced in restraint mice (P <0.01). Superoxide dismutase activity canbe significantly increased by treated with theacrine， then relieve thecondition of body oxidative stress.2.5The impact of theacrine on protein expression in restraint stressmice.SirT3protein expression was significantly decreased in stress-loadmodel mice， while either dose of theacrine group could significantlyincrease the expression of SirT3protein in restraint stress mice. Afteradministration， compared with the model group， p-AMPK levels increasedsignificantly， indicating both dose of theacrine could significantlypromote the transformation from AMPK to P-AMPK.2.6The effect of theacrine on gene expression in restraint stress miceCompared with the normal group， mRNA expressions of LPL and GPIHBP1did not change significantly after restraint stress (P>0.05). Aftergiving theacrine， there were also no significant changes (P>0.05).Compared with the normal group， mRNA levels of ANGPTL4were significantlyup-regulated after the restraint stress (P <0.05). In the effect of bothhigh and low dose of theacrine， up-regulation of mRNA of ANGPTL4weresignificantly reversed (P <0.05，P <0.01).2.7The impact of theacrine on plasma corticosterone levels in restraintstress miceAfter restraint stress， the plasma corticosterone levels weresignificantly increased in mice. While， after giving theacrine， plasma corticosterone levels can be reduced in a dose-dependent manner. Theactivation effect of theacrine on LPL may be associated with decreasingplasma corticosterone levels.2.8After glucocorticoid receptor inhibited， the impact of theacrine onplasma LPL， TG clearance rate， genes and proteins in the heart ofrestraint stress mouseRU486was given in restraint stress mice， plasma levels of LPL wasimproved (P <0.05)， while clearance rate of plasma TG was enhancedsimultaneously， the above results depicted that RU486inhibiteddownstream effectors which generated from corticosterone binding toglucocorticoid receptor. Theacrine could further improve the plasma LPLlevel and TG clearance efficiency. Therefore， the role of theacrineplayed on LPL activation may not be relevant to the glucocorticoidreceptor.2.9The effect of theacrine on plasma LPL， TG clearance rate， genes andproteins expressions in the heart of restraint stress mouse after AMPKsuppression.In the effect of restraint stress， mice were given compound C. PlasmaLPL levels and clearance rates were unchanged， indicating that LPLactivity was not affcted after the inhibition of AMPK. The same resultwas observed in the theacrine group， plasma levels of LPL and TG removalefficiency were not increased either. But interestingly， SirT3proteinexpression increased， mRNA ANGPTL4expression decreased. Thus， theactivation effect of theacrine on LPL may be through reducing SirT3/AMPKmediated downstream ANGPTL4and the inactivation of LPL.Conclusions：In the study， high-fat-induced fatty liver model and oxidative stressmodel after restraint stress-induced were used， the effect of purinealkaloid theacrine on fatty acid metabolism and mechanism were investigated and discussed. According to the findings， conclusions areas followings:1. Theacrine may reduce ACC activity by activating the pathway of SirT3/AMPK/ACC pathway， thereby reducing fatty acid synthesis， inhibiting thetriglyceride accumulation of hepatic fat， improving the status of liverfat in high-fat diet-induced mice.2. Theacrine could significantly increase the elimination rate of plasmatriglycerides in mice and activate plasma LPL activity in stress mice.Oxidative stress in the body was also significantly alleviated， whichmay be one reason of why theacrine could improve lipid metabolismdisorders in the condition of stress.3. The activation effect of theacrine on LPL may through reducingSirT3/AMPK mediated downstream ANGPTL4and the inactivation of LPL.4. Theacrine could reduce the plasma levels of corticosterone. but theactivation effect of theacrine on LPL may not be relevant with the bindingof corticosterone and glucocorticoid receptor.