The Effect Of Mangiferin Berberine Salt On The Regulation Of Glycolipid Metabolism In HepG2 Cells

Natural product mangiferin(M)is a xanthone glycoside and is reported to have favorable hypolipidemic,hypoglycemic,anti-obesity,antioxidative,and anti-inflammatory activities.Natural product berberine(B)is an isoquinoline alkaloid which has promising activities in modulating lipid and glucose metabolisms.In this study,we chemically synthesize the compound of mangiferin-berberine(MB)salt by ionic bonding of M and B at an equal molecular ratio to form a stable single molecule.Then,we investigate the activities and mechanisms of MB salt in modulating lipid and glucose metabolisms in HepG2 cells in detail and compare them with those of M or B alone.The MTT method was used to test the in vitro toxicities of M,B,and MB salt to detennine the concentrations the compounds for the following experiments.Western blot was used to determine the influences of the compounds on the phosphorylation of AMP-activated protein kinase a(AMPKa)(threonine(Thr)172)and acetyl-CoA carboxylase(ACC)(serine(Ser)79);a commercially available kit was used to determine the activities of carnitine palmitoyl transferase 1(CPT1).For the study of glucose metabolism,commercially available kits were used to determine the stimulating activities of the compounds on basal and insulin-stimulated glucose consumption and the inhibiting activities of the compounds on gluconeogenesis of HepG2 cells.Quantitative real-time RT-PCR was used to determine the mRNA expression levels of the key enzymes of gluconeogenesis,which included phosphoenolpyruvate carboxykinase(PEPCK)and glucose-6-phosphatase(G6Pase).For the study of lipid metabolism,quantitative real-time RT-PCR was used to determine the influences of the compounds on the mRNA expression level of low-density lipoprotein receptor(LDLR).Then,oleic acid(OA)was used to treat the cells to induce steatosis,oil red O(ORO)staining and triglyceride(TG)quantification were used to determine the influences of the compounds on intracellular lipids.Accordingly,quantitative real-time RT-PCR was used to determine the mRNA expression levels of key transcription factors for lipid synthesis,such as sterol regulatory element-binding protein-lc(SREBP-lc)and carbohydrate-responsive element-binding protein,(ChREBP),as well as their target genes like fatty acid synthetase,(FAS)and stearoyl-CoA desaturase-1,(SCD1).An AMPK specific inhibitor,compound C(CC),was used in the blocking experiments.Our results showed that in HepG2 cells,the 50%inhibiting concentration(IC50)of M is larger than 400?M,and those of B and MB salt were 133.9±10.6?M,and 131.0±9.4?M,respectively.Our results also showed that the studying compounds dose-dependently stimulated the phosphorylation of AMPKa(Thr172)and ACC(Ser79),and increased the activity of CPT1,which could be completely blocked by CC pretreatment.Importantly,the stimulating activity of MB salt on the AMPK pathway was significantly superior to those of M or B when administered alone at an equal molar concentration.In modulating glucose metabolism,the 3 compounds enhanced both the basal and the insulin-stimulated glucose consumption,and the efficacy of MB salt was significantly superior to those of M or B when administered alone at an equal molar concentration.B and MB salt stimulated the release of lactate in HepG2 cells,while M had no effect.M of the MB salt stimulated cellular glucose consumption in an AMPK-dependent manner,while that of B was AMPK-independent;the stimulating activity of MB salt on cellular glucose consumption was partially dependent on the AMPK pathway,while its effect on lactate release was AMPK-independent.In addition,MB salt inhibited cellular gluconeogenesis significantly in an AMPK-dependent manner,and its efficacy was superior to those of M or B when administered alone.When inhibiting gluconeogenesis,MB salt down-regulated the mRNA expression levels of key enzymes such as PEPCK and G6Pase.In modulating lipid metabolism,MB salt inhibited OA-induced steatosis,reduced intracellular TG content,and down-regulated the mRNA expression levels of SREBP-1c,ChREBP,Fas,and SCD1 significantly.The lipid-modulating activities of MB salt was AMPK dependent and was significantly superior to those of M or B when administered alone at an equal molar concentration.In vitro,the modulating activities of MB salt on glucose and lipid metabolisms were comparable to those of combination of M and B(M+B)at equal molar concentrations.However,as a stable single molecule,MB salt may be absorbed more easily in vivo,which means that this compound may have a better pharmacokinetic profile.In summary,our results indicate that MB salt reduces intracellular TG through activating the AMPK/ACC/CPT1 pathway and inhibiting the expression levels of lipogenic genes;suppresses gluconeogenesis through activating AMPK and down-regulating the expression levels of PEPCK and G6Pase;and it stimulates glucose consumption through AMPK activation(M)and promoting lactate release(B),respectively.In HepG2 cells,the modulating effects of MB salt on glucose and lipid metabolisms are significantly superior to those of M or B alone.This compound has combined advantages of M and B;it is a promising agent which can be developed for the treatment of glucose and lipid metabolism disorders in the future.