Establishment And Application Of Cell Models Stably Overexpressing HMATE1 Or Coexpressing With HOCT1/2

Transporters play an important role in drug disposition in vivo,and they also mediate numerous drug-drug interaction.So they catch more and more attention in interaction with drugs.Metformin is one of the most commonly used drugs for treatment of type ?diabetes.It is usually orally administrated,and mainly excreted by the kidney in original form.After oral administrated,metformin accesses into hepatocytes,and subsquently causes blood glucose reduction.Extra metformin accumulated in the kidney can lead to renal injury.It has been demonstrated that human organic cation transporters(hOCTs)and multidrug and toxin extrusion family(hMATEs)play important roles in the disposition of metformin in the liver and kidney,and the clinical curative effect and side effect are related to the gene polymorphism of hOCTs and hMATEs.Hyperlipidemia is a common complication of diabetes and the earliest complication in old diabetic.Thus,it is likely to administrate with products of lotus leaf during metformin therapy.Akaloids,which might be inhibitors or substrates of hOCTs and hMATE1 according to their structures,are the main lipid-decreasing ingredients in lotus leaf.But whether it is safe to combine metformin and product of lotus is unknown,even whether they have influence on the efficacy of metformin and induce side effect via OCTs and MATEs is unknown.Because immortalized cell lines from the liver or kidney express transporters in lower level,moreover expression of transporters on primary cells will decrease with the culture period increase.Herein,cell models stably expressing hMATE1 or coexpressing hMATEl with hOCTl or hOCT2 have been established and applied to study the effect of alkaloids from lotus leaf on metformin uptake and transportation via hOCTs and hMATE1,which can provide information for further study on whether alkaloids in lotus leaf affect the curative effect and side effect of metformin in vivo.1.Construction of pcDNA3.1(+)-hMATE1 recombined plasmidmRNA has been extracted from human kidney tissue,and used to amplify hMATE1 cDNA by reverse transcript PCR and primer specific PCR.The PCR product has been purified and recovered by agarose gel electrophoresis,and then the PCR product has been sequenced and contrasted with SLC47A1 mRNA coding sequence.The A-T clone has been processed using PCR product which matching 100%with SLC47A1 mRNA coding sequence to construct pMD19-T-hMATE1,and the connection product has been transformed into E.coli 5? to amplify more pMD19-T-hMATE1.Both pcDNA3.1(+)and pMD19-T-hMATE1 have been digested with Hind ? and Kpn ?,and then digest product of both pcDNA3.1(+)and hMATE1 cDNA have been purified before mixed together to produce pcDNA3.1(+)-hMATE1.After having been amplified in E.coli 5?,recombined plasmid has been sequenced again to make sure that correct wild type hMATEl cDNA has been inserted.The alignment result revealed that pcDNA3.1(+)-hMATE1 containing correct wild type hMATE1 cDNA is successfully constructed.2.Establishment of MDCK cell models stably expressing hMATE1 and coexpressing hMATE1 with hOCT1 or hOCT2pcDNA3.1(+)-hMATE1 has been transfected into MDCK,MDCK-hOCT1 or MDCK-hOCT2 cell using Lipofectamine TM 2000.After 14-day's screening with hygromycin B,monoclones accumulated more DAPI and MPP+ than negative control cells have been selected.Furthermore,the hMATE1 mRNA in those three cell models has been quantified.The accumulation of metofrmin on MDCK-hMATE1,and the net efflux ratio of cimetidine on polarized MDCK-hOCT1/hMATE1 and MDCK-hOCT1/hMATE1 cell monolayer have been determined to validate the function of hMATE1.The results showed that hMATE1 mRNA expressed in the above three cell models were 7300,27600 and 2400 folds of their control cells.The accumulation of metfromin in MDCK-hMATE1 cells was 17.6 folds of that in control cells,and the accumulation could be significantly reduced by cimetidine.The efflux ratio of cimetidine on MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 were 34.0 and 17.5 respectively,while the net efflux ratio were 17.5 and 3.65 respectively.The above results demonstrate that MDCK-hMATE1,MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 cell models are successfully established,and can be used to study interaction between alkaloids in lotus leaf and metformin via hOCTs and MATEs.3.The effect of four alkaloids from lotus leaf on metformin transportation via hOCTs and hMATE1Aporphine alkaloid,including nuciferine,N-nornuciferine,N-methylasimilobine and asimilobine,is the most abundant alkaloid class in lotus leaf.Based on the sturctures of aporphine alkaloids they might the substrates or inhibitors of organic cation transporters(hOCTs)and MATE1,however whether there are drug-drug interaction between the above alkaloids and metformin via hOCTs and MATEs has not been reported.The effect of aporphine alkaloids on metformin uptake on single-transfected cells including MDCK-hOCT1,MDCK-hOCT2,MDCK-hOCT3 and MDCK-hMATE1 has been studied in the first place.The results showed that all four aporphine alkaloids inhibited metformin transport via those transporters,the inhibition extent hOCT1>hMATE1>hOCT3>hOCT2.Considering that hOCT1 and hOCT2,are the most abundant OCT transporters in liver and kidney,respectively,while hMATEl is enrich in both liver and kidney,moreover all four aporphine alkaloids reduced metformin(10?mol/L)transportation on hOCT2 very slightly(<50%)even at concentration of 100?mol/L,thus hOCT1 and hMATE1 have been selected to further study the effect on metformin transportation,The half maximal inhibitory concentration(IC50)of nuciferine,N-nornuciferine,N-methylasimilobine and asimilobine on MDCK-hOCT1 were 13.6± 1.8,14.9 ± 1.2,6.68±0.44 and 16.6 ± 3.1?mol/L separately,while the IC50 values on MDCK-hMATE1 were 16.9±0.47,19.0±1.5,16.4±0.54 and 21.4±0.81?mol/L,respectively.Because the IC50 values of the above four alkaloids on hOCT1 and hMATE1 were adjacent,nuciferine was selected to further study the effect on metformin transportation and accumulation on polarized MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 cell monolayers.The results showed that nuciferine concentration dependently inhibited metformin transportation from baslateral to apicial on polarized MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 cell monolayer and reduced the cellular accumulation of metformin in MDCK-hOCT1/hMATE1 but increased the accumulation in MDCK-hOCT2/hMATE1 cells.Our study demonstrated that aporphine alkaloids,such as nuciferine,inhibit transportation of metfromin mediated by hOCT1/2/3 and hMATE1,and influence its accumulation in cells coexpressing hMATE1 and hOCT1 or hOCT2.