the impacts of slc22a1 slc47a1downloads.hindawi.com/journals/ije/2016/4350712.pdf · research...

Research ArticleThe Impacts of SLC22A1 rs594709 and SLC47A1 rs2289669Polymorphisms on Metformin Therapeutic Efficacy in ChineseType 2 Diabetes Patients

Di Xiao123 Yu Guo123 Xi Li123 Ji-Ye Yin123 Wei Zheng123

Xin-Wen Qiu4 Ling Xiao123 Rang-Ru Liu123 Sai-Ying Wang123

Wei-Jing Gong123 Hong-Hao Zhou123 and Zhao-Qian Liu123

1Department of Clinical Pharmacology Xiangya Hospital Central South University Changsha 410008 China2Institute of Clinical Pharmacology Central South University and Hunan Key Laboratory of PharmacogeneticsChangsha 410078 China3Hunan Province Cooperation Innovation Center for Molecular Target New Drug Study Hengyang 421001 China4Changsha Medical University Teaching Hospital The Peoplersquos Hospital of Liuyang Liuyang 410300 China

Correspondence should be addressed to Zhao-Qian Liu liuzhaoqian63126com

Received 29 September 2015 Revised 2 January 2016 Accepted 6 January 2016

Academic Editor Franco Veglio

Copyright copy 2016 Di Xiao et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background We aimed to investigate the distributive characteristics of SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphismsand their influence onmetformin efficacy inChinese T2DMpatientsMethodsThe distributions of SLC22A1 rs594709 and SLC47A1rs2289669 polymorphisms were determined in 267 T2DM patients and 182 healthy subjects Subsequently 53 newly diagnosedpatients who receivedmetforminmonotherapy were recruited to evaluate metformin efficacy Results No significant difference wasfound between T2DM patients and healthy subjects in SLC22A1 rs594709 and SLC47A1 rs2289669 allele frequencies and genotypefrequencies After metformin treatment SLC22A1 rs594709 GG genotype patients showed a higher increase in FINS (119901 = 0015)and decrease in HOMA-IS (119901 = 0001) and QUICKI (119901 = 0002) than A allele carriers SLC47A1 rs2289669 GG genotype patientshad a higher decrease in TChol (119901 = 0030) and LDL-C (119901 = 0049) than A allele carriers Among SLC22A1 rs594709 AA genotypepatients with SLC47A1 rs2289669 AA genotype showed a higher decrease in FBG (119901 = 0015) PINS (119901 = 0041) and HOMA-IR(119901 = 0014) than G allele carriers However among SLC22A1 rs594709 G allele carriers SLC47A1 rs2289669 AA genotype patientsshowed a higher decrease in TChol (119901 = 0013) than G allele carriers Conclusion Our data suggest that SLC22A1 rs594709 andSLC47A1 rs2289669 polymorphisms may influence metformin efficacy together in Chinese T2DM patients

1 Introduction

Metformin is the most widely used oral antidiabetic drugwhich could effectively lower blood glucose and reduce therisk of cardiovascular diseases [1] The classical functionalmechanism of metformin is to decrease the hepatic energystatus by inhibiting the mitochondrial respiratory-chaincomplex 1 thus activating the adenosine 51015840-monophosphate-activated protein kinase (AMPK) a cellularmetabolic sensorto finally lower the blood glucose level [2] In recent years fur-ther probable mechanisms involved in cAMP the phospho-rylation of Acc1 and Acc2 mitochondrial glycerophosphate

dehydrogenase and the activated duodenal AMPK werefound to affect metforminrsquos effect in different ways [3ndash6]With its significant glycemic benefits and neutral effects onweight metformin remains the recommended first-line oraltherapy in the guidelines of the American Diabetes Associa-tion and European Association of the Study of Diabetes [7]However during its clinical usage the individual differencesand drug failure widely existed [8 9]

Metformin is not metabolized in vivo The gastrointesti-nal absorption hepatic uptake and renal excretion of met-formin are mediated greatly by organic cation transporters(OCTs) andmultidrug and toxin extrusion proteins (MATEs)

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2016 Article ID 4350712 7 pageshttpdxdoiorg10115520164350712

2 International Journal of Endocrinology

[10] It was widely reported that genetic variants in codinggenes of transporters mentioned above played importantroles in pharmacokinetics and pharmacodynamics of met-formin [9 11ndash13] Among these SLC22A1 rs628031 (tagged tors594709 in Asian) was reported to affect imatinib uptake inchronic myeloid leukemia (CML) cell line [14] As we knowimatinib is also transported byOCT1 in the human organismSLC47A1 rs2289669 was reported to be associated withmetformin excretion [15] as well as its therapeutic response[16 17] Besides a gene-gene interaction between geneticvariants in SLC47A1 and SLC22A1 SLC47A1 and SLC22A2had a significant influence on metforminrsquos efficacy [18 19]It was found that there was a SNP-SNP interaction betweenSLC47A1 rs2289669 and SLC22A1 rs622342 [18] Howeverthe effect of SLC22A1 rs594709 and SLC47A1 rs2289669polymorphisms and SNP-SNP interaction on metforminefficacy in Chinese T2DM patients has not been widelyevaluated

In this study we aimed to investigate the distributivecharacteristics of SLC22A1 rs594709 and SLC47A1 rs2289669between T2DM patients and healthy subjects and the influ-ences of SLC22A1 rs594709 and SLC47A1 rs2289669 poly-morphisms on metforminrsquos therapeutic efficacy in glucoselowering serum lipids and insulin sensitivity Moreover weare interested in whether there is a SNP-SNP interactionbetween SLC22A1 rs594709 and SLC47A1 rs2289669 onmetforminrsquos therapeutic efficacy

2 Material and Methods

21 Subjects Distributive characteristics of SLC22A1rs594709 and SLC47A1 rs2289669 polymorphisms weredetermined in 267 unrelated patients with T2DM and182 healthy subjects 267 T2DM patients (143 males and124 females) were recruited from the Second XiangyaHospital of Central South University the Peoplersquos Hospitalof Liuyang and the First Hospital of Changsha 182 healthysubjects (86 males and 96 females) were recruited fromPhysical Examination Center of Xiangya Hospital of CentralSouth University T2DM was diagnosed according to DMdiagnostic criteria of WHO 1999 Patients with type 1diabetes gestational diabetes uncontrollable hypertensionsevere liver and kidney disease were not included in thisstudy

Furthermore 53 of 267 T2DM patients who were newlydiagnosed and drug naıve (31 males and 22 females averageage 49 years range 29ndash73 years) were enrolled to evaluatethe effect of metformin among different SLC22A1 rs594709and SLC47A1 rs2289669 genotype individuals Newly diag-nosed type 2 diabetes was defined as being diagnosed withT2DM within 6 months before enrollment All patientshad not been treated with any insulin secretagogue andreceived metformin monotherapy for 3 consecutive monthsMetformin doses ranged from 1000mgday to 2000mgdayamong individuals The study was approved by the EthicsCommittee of Xiangya School of Medicine Central SouthUniversity (Changsha Hunan China) (CTXY-110002-5) andwas performed in accordance with the Helsinki DeclarationWritten informed consent was provided by all subjects

22 Clinical Laboratory Tests Detailed clinical informationwas collected from each patient Bodymass index (BMI) fast-ing blood glucose (FBG) postprandial blood glucose (PBG)fasting insulin (FINS) postprandial insulin (PINS) gly-cosylated haemoglobin (HbA1c) total cholesterol (TChol)triglycerides (TG) low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) wereevaluated before and after metformin treatment as previouslydescribed [20 21] Homeostasis model assessment of insulinresistance (HOMA-IR) homeostasis model assessment ofinsulin sensitivity (HOMA-IS) homeostasis model assess-ment of 120573-cell function (HOMA-B) and quantitative insulinsensitivity check index (QUICKI) were calculated accordingto previous formulas [22 23]

23 DNA Sampling and Genotyping Genomic DNA wasisolated from peripheral blood leukocytes using PromegaDNA purification kit (Promega Wisconsin USA) accordingto the manufacturerrsquos protocol and stored at minus20∘C until useGenotyping was done using direct sequencing The primersused to amplify SLC22A1 rs594709 polymorphism were51015840GGCGCTTCCCACACTCAT31015840 (sense) and 51015840GAGGAA-AGCTCCACATGTAACC31015840 (antisense) and the primersfor SLC47A1 rs2289669 polymorphism were 51015840CCTGAC-CTCTGTCCCTATT31015840 (sense) and 51015840GACTTCCGTGCC-TGCT31015840 (antisense)The PCR products were then sequencedusing ABI 3700 automated sequencer (ABI Foster City CAUSA)

24 Statistical Analysis Independent-samples t-test was usedto compare baseline characteristics in T2DM patients andhealthy subjects The observed genotype frequencies inpatients and controls compared with expected frequenciesunder Hardy-Weinberg equilibrium were evaluated by 1205942test Comparisons of frequencies of genotypes and alleleswere also analyzed by 1205942 test

We evaluated associations between each SNP and thechange of clinical index before and after metformin therapyusing multivariate linear regression by adjusting for sex ageand BMI in additive model recessive model and dominantmodel respectively Besides the analysis for the SLC22A1rs594709 genotype was stratified for the SLC47A1 rs2289669genotype and vice versa Multivariate linear regression wasused to analyze the interaction between SLC22A1 rs594709and SLC47A1 rs2289669 genotype on metformin response inChinese T2DM patients All of the analyses were performedwith SPSS software (version 160 SPSS Chicago IllinoisUSA)

3 Results

31 Clinical Characteristics of Subjects A total of 267 T2DMpatients and 182 healthy subjects were enrolled to explorethe allele frequencies and genotype frequencies of SLC22A1rs594709 and SLC47A1 rs2289669 polymorphisms The clin-ical characteristics of T2DM patients and healthy subjectswere shown in Table 1 BMI (2510 plusmn 274 kgm2 versus2398 plusmn 294 kgm2 119901 lt 0001) waist circumference (8740 plusmn856 cm versus 8172 plusmn 952 cm 119901 lt 0001) waist-to-hip

International Journal of Endocrinology 3

Table 1 The clinical characteristics of T2DM patients and healthy subjects

Parameter Healthy controls (119899 = 182) T2DM patients (119899 = 267) 119901 valueGender

Male 86 143Female 96 124 0189

Age (years) 4904 plusmn 810 4982 plusmn 1011 0370BMI (kgm2) 2398 plusmn 294 2510 plusmn 274 0000

lowastlowastlowast

Waist circumference (cm) 8172 plusmn 952 8740 plusmn 856 0000lowastlowastlowast

Hip circumference (cm) 9532 plusmn 646 9649 plusmn 736 0097

Waist-hip ratio 085 plusmn 007 091 plusmn 006 0000lowastlowastlowast

FBG (mmolL) 511 plusmn 057 891 plusmn 340 0000lowastlowastlowast

TG (mmolL) 185 plusmn 682 312 plusmn 371 0025lowast

TChol (mmolL) 488 plusmn 097 505 plusmn 181 0255

LDL-C (mmolL) 282 plusmn 078 290 plusmn 101 0388

HDL-C (mmolL) 151 plusmn 045 132 plusmn 064 0000lowastlowastlowast

BMI body mass index FBG fasting blood glucose TG triglycerides TChol total cholesterol LDL-C low-density lipoprotein cholesterol HDL-C high-density lipoprotein cholesterol lowast119901 lt 005 lowastlowastlowast119901 lt 0001

Table 2 Comparisons of allelic frequencies of SLC22A1 rs594709and SLC47A1 rs2289669 polymorphisms in T2DM patients andhealthy subjects

Genotype T2DM patients(119899 = 267)

Healthy controls(119899 = 182) 119901 value

SLC22A1 rs594709AA 146 (5468) 95 (5220)

0927GA 99 (3708) 70 (3846)GG 22 (824) 17 (934)

AllelesA 391 (7322) 260 (7143) 0555G 143 (2678) 104 (2857)

SLC47A1 rs2289669AA 77 (2884) 49 (2692)

0669GA 128 (4794) 95 (5220)GG 62 (2322) 38 (2088)

AllelesA 282 (5281) 193 (5302) 0950G 252 (4719) 171 (4697)

ratio (091 plusmn 006 versus 085 plusmn 007 119901 lt 0001) FBG(891 plusmn 340mmolL versus 511 plusmn 057mmolL 119901 lt 0001)and TG (312 plusmn 371mmolL versus 185 plusmn 682mmolL119901 lt 005) in T2DM patients were significantly higher thanthose of healthy subjects Moreover HDL-C level (132 plusmn064mmolL versus 151plusmn045mmolL 119901 lt 0001) in T2DMpatients was significantly lower than that in healthy subjectsNo significant differences were found in hip circumferenceTChol and LDL-C level between two groups

32 Genotype Analysis and Allelic Frequencies The frequen-cies of the SLC22A1 rs594709 and SLC47A1 rs2289669 poly-morphisms in T2DM patients and healthy subjects wereshown in Table 2 Both SNPs were in agreement with Hardy-Weinberg equilibrium in each group (119901 gt 005) Minor allele

frequencies (MAFs) of SLC22A1 rs594709 in T2DM groupand healthy control were 2678 and 2857 respectivelyMAFs of SLC47A1 rs2289669 were respectively 4719 and4697 inT2DMpatients and healthy subjects No significantdifferences were found in allele frequencies (119901 = 0555 forrs594709119901 = 0950 for rs2289669) and genotype frequencies(119901 = 0927 for rs594709 119901 = 0669 for rs2289669) betweenT2DM patients and healthy controls

33 Influence of SLC22A1 rs594709 and SLC47A1 rs2289669Polymorphisms onTherapeutic Efficacy ofMetformin inT2DMPatients 53 newly diagnosed T2DM patients who had takenthe first prescription of metformin for 3 consecutive monthswere enrolled to evaluate metformin efficacy As shown inTable 3 SLC22A1 rs594709 GG genotype patients had ahigher increase in FINS (119901 = 0015) and greater decrease inHOMA-IS (119901 = 0001) and QUICKI (119901 = 0002) than thoseof A allele carriers (Table 3) However SLC47A1 rs2289669polymorphism was associated with changes in TChol andLDL-C level patients carrying SLC47A1 rs2289669 A allelehad a relatively minor decrease in TChol (119901 = 0030) andLDL-C (119901 = 0049) level compared to GG genotype patients(Table 3)

Additionally we found a significant SNP-SNP interac-tion between SLC47A1 rs2289669 and SLC22A1 rs594709which affected the improvement of insulin resistance andblood lipid induced by metformin therapy Among SLC22A1rs594709 AA genotypes patients with SLC47A1 rs2289669AA genotype showed significantly higher decrease in FBG(119901 = 0015) PINS (119901 = 0041) and HOMA-IR (119901 = 0014)than GA or GG genotypes (Table 4) while among SLC22A1rs594709 G allele carriers patients with SLC47A1 rs2289669AA genotype showed a greater decrease in TChol (119901 = 0013)than GA or GG genotypes (Table 4)

4 Discussion

In the current study we evaluated the distributive charac-teristics of SLC22A1 rs594709 and SLC47A1 rs2289669 poly-morphisms and the separate as well as combined influence of


Table 3 Comparisons of differential values (post-administration minus pre-administration) in T2DM patients with different SLC22A1rs594709 and SLC47A1 rs2289669 genotypes before and after metformin treatment

Parameter SLC22A1 rs594709119901 value SLC47A1 rs2289669

119901 valueAAGA (119899 = 50) GG (119899 = 3) AAGA (119899 = 37) GG (119899 = 16)

FBG (mmolL) minus202 plusmn 284 minus010 plusmn 062 0112 minus215 plusmn 311 minus127 plusmn 172 0668PBG (mmolL) minus528 plusmn 603 minus173 plusmn 266 0171 minus561 plusmn 617 minus384 plusmn 532 0692FINS (mUL) 390 plusmn 540 1135 plusmn 446 0015

a407 plusmn 606 519 plusmn 447 0501

PINS (mUL) minus977 plusmn 2558 minus2058 plusmn 2272 0259 minus1369 plusmn 2973 minus353 plusmn 681 0457HbA1c () minus283 plusmn 482 minus060 plusmn 053 0227 minus289 plusmn 538 minus221 plusmn 264 1000TG (mmolL) minus084 plusmn 371 minus074 plusmn 128 0434 minus072 plusmn 371 minus110 plusmn 344 0163TChol (mmolL) minus060 plusmn 146 005 plusmn 156 0224 minus039 plusmn 140 minus096 plusmn 155 0030b

LDL-C (mmolL) minus014 plusmn 100 minus009 plusmn 078 0451 minus008 plusmn 110 minus028 plusmn 063 0049b

HDL-C (mmolL) minus003 plusmn 018 minus005 plusmn 015 0399 minus002 plusmn 019 minus008 plusmn 014 0254HOMA-IR 060 plusmn 287 306 plusmn 072 0081 055 plusmn 327 131 plusmn 131 0357HOMA-IS minus0003 plusmn 003 minus008 plusmn 010 0001

aminus0009 plusmn 005 minus0009 plusmn 002 0382

HOMA-B 090 plusmn 170 096 plusmn 070 0493 086 plusmn 148 101 plusmn 206 0295QUICKI minus003 plusmn 017 minus043 plusmn 046 0002


BMI body mass index FBG fasting blood glucose PBG postprandial blood glucose FINS fasting insulin PINS postprandial insulin HbA1c glycosylatedhaemoglobin TG triglycerides TChol total cholesterol LDL-C low-density lipoprotein cholesterol HDL-C high-density lipoprotein cholesterol HOMA-IRhomeostasis model assessment of insulin resistance HOMA-IS homeostasis model assessment of insulin sensitivity HOMA-B homeostasis model assessmentof 120573-cell function QUICKI quantitative insulin sensitivity check indexa119901 lt 005 for comparison of GG genotype with GAAA genotype of SLC22A1 rs594709 polymorphism adjusted for age sex and BMI in recessive model

b119901 lt 005 for comparison of GG genotype with GAAA genotype of SLC47A1 rs2289669 polymorphism adjusted for age sex and BMI in recessive model

Table 4 The differential values (DV) of clinical characteristics per SLC22A1 rs594709 and SLC47A1 rs2289669 genotypes

SLC47A1 rs2289669 SLC22A1 rs594709 AA (119899 = 32)119901 value SLC22A1 rs594709 GAGG (119899 = 21)

119901 valueAA (119899 = 6) GA + GG (119899 = 26) AA (119899 = 7) GA + GG (119899 = 14)

FBG (mmolL) minus394 plusmn 625 minus163 plusmn 195 0015a minus156 plusmn 261 minus181 plusmn 237 0641PBG (mmolL) minus963 plusmn 583 minus456 plusmn 584 0067 minus332 plusmn 646 minus532 plusmn 579 0310FINS (mUL) 517 plusmn 689 471 plusmn 428 0252 325 plusmn 835 417 plusmn 641 0865PINS (mUL) minus3168 plusmn 5656 minus461 plusmn 2091 0041a minus1372 plusmn 2571 minus1493 plusmn 2225 0799HbA1c () minus325 plusmn 311 minus333 plusmn 630 0487 minus184 plusmn 143 minus176 plusmn 213 0778TG (mmolL) minus415 plusmn 697 minus105 plusmn 270 0061 minus077 plusmn 220 069 plusmn 405 0466TChol (mmolL) minus128 plusmn 156 minus065 plusmn 146 0197 minus133 plusmn 115 032 plusmn 126 0013b

LDL-C (mmolL) 038 plusmn 135 minus009 plusmn 076 0148 minus066 plusmn 049 minus011 plusmn 138 0228HDL-C (mmolL) 009 plusmn 025 minus004 plusmn 020 0134 minus010 plusmn 015 minus003 plusmn 011 0205HOMA-IR minus202 plusmn 750 122 plusmn 136 0014a 034 plusmn 233 110 plusmn 277 0841HOMA-IS minus001 plusmn 002 minus001 plusmn 002 0440 minus002 plusmn 008 0003 plusmn 005 0345HOMA-B 111 plusmn 074 081 plusmn 157 0441 162 plusmn 290 057 plusmn 086 0149QUICKI minus007 plusmn 013 minus007 plusmn 012 0447 minus010 plusmn 042 00003 plusmn 025 0437BMI body mass index FBG fasting blood glucose PBG postprandial blood glucose FINS fasting insulin PINS postprandial insulin HbA1c glycosylatedhaemoglobin TG triglycerides TChol total cholesterol LDL-C low-density lipoprotein cholesterol HDL-C high-density lipoprotein cholesterol HOMA-IRhomeostasis model assessment of insulin resistance HOMA-IS homeostasis model assessment of insulin sensitivity HOMA-B homeostasis model assessmentof 120573-cell function QUICKI quantitative insulin sensitivity check indexa119901 lt 005 for comparison of AA genotype with GAGG genotype of SLC47A1 rs2289669 polymorphism in SLC22A1 rs594709 AA genotyped T2DM patientsadjusted for age sex and BMI in dominant modelb119901 lt 005 for comparison of AA genotype with GAGG genotype of SLC47A1 rs2289669 polymorphism in SLC22A1 rs594709 GA or GG genotyped T2DMpatients adjusted for age sex and BMI in dominant model

their polymorphisms onmetforminrsquos effect in Chinese T2DMpatients Our results showed that there were no significantdifferences in SLC22A1 rs594709 and SLC47A1 rs2289669allele frequencies and genotype frequencies between T2DMpatients and healthy subjects SLC22A1 rs594709 A allelecarriers showed a better metformin efficacy in insulinsensitivity compared with GG genotype patients SLC47A1

rs2289669 GG genotype patients showed a better efficacyin improving blood lipid Furthermore we found that therewas a significant interaction between SLC47A1 rs2289669and SLC22A1 rs594709 which might function together inmetformin efficacy

OCTs mediate the interactions between cells and theirenvironment and are involved in transporting a broad


spectrum of endogenous substrates and the detoxification ofxenobiotics [24] Nutrient transporters including OCT1 andMATE1 could affect nutrient homeostasis through differentways [25] Polymorphisms in OCT1 or MATE1 have beencorrelated with the susceptibility of type 2 diabetes diabeticnephropathy hypertension and primary biliary cirrhosis[26ndash28] SLC47A1 rs2453583 and SLC22A1 rs651154 havebeen reported to be nominally associated with diabetesincidence in the DPP (Diabetes Prevention Program) [26]Therefore we investigated the distributive characteristics ofthe two SNPs that we are concerned with namely SLC22A1rs594709 and SLC47A1 rs2289669 in T2DM patients andhealthy subjects However no statistical difference was foundbetween two groups

Human SLC22A1 gene is highly polymorphic and severalpolymorphisms have been described to affect metforminresponses [29 30] including polymorphisms located in theintron regions [31] SLC22A1 rs594709 locates in intron 4Pairwise linkage disequilibrium (LD) analysis indicated thatthere were strong LDs between rs594709 and rs2282143rs628031 rs3798168 and rs683369 Polymorphisms men-tioned above could more or less affect the metabolizingandor efficacy of OCT1 substrates SLC22A1 rs2282143variation led to a decrease of transport activity but notcompletely abolished [32] SLC22A1 rs628031 (Met408Val)was identified in multiethnic groups worldwide [33] andhas been characterized to have normal metformin uptakein vitro [9 11] However it has been recognized as a weakpositive predictor of metformin response in clinical study[8 34 35] though this genetic variant did not alter proteinexpression [34 36] Recent studies reported that SLC22A1rs3798168 rs683369 and rs628031 could affect the responseto imatinib therapy in chronic myeloid leukemia (CML)patients [37 38] As SLC22A1 rs594709 is the tagger SNP ofrs3798168 rs683369 and rs628031 (1199032 gt 08) it is possiblethat the structural changes in SLC22A1 caused by any of thesepolymorphisms or even a combination effect of them mayaffect its transport ability of metformin

Previously Caucasians population-based study in dia-betic patients revealed that rs2289669 GA in SLC47A1gene was associated with the therapeutic efficacy of met-formin [16 17] The decrease in HbA1c was greater inAA homozygotes compared with the GA + GG genotypepatients after metformin treatment The effect of SLC47A1rs2289669 polymorphism on pharmacodynamics (119899 = 202)and pharmacokinetics (119899 = 28) of metformin was alsoevaluated in Chinese patients and showed that AA genotypepatients had a better glucose lowering effect after one-yearfollow-up As for pharmacokinetics parameters AA genotypepatients had higher AUC

12h and lower renal clearance andrenal clearance by secretion [15] Thus it is possible thatthe effect of the rs2289669 polymorphism on metforminglucose lowering efficacy was caused partly or completely bya reduced renal excretion and increased metformin plasmalevels [39] However in our study we did not find SLC47A1rs2289669 polymorphism was correlated with metforminglucose lowering effect but was associated with the reductionof TChol and LDL-C This result was similar to long-termmetformin and sulphonylurea combination therapy study

carried out in Slovenia [40] Their results suggested thatSLC47A1 rs2289669 genotype was not associated with HbA1clevels but significantly associated with cholesterol levels inmetformin-treated T2DM patients even after adjustment forrenal function In contrast to the previous studies the samplesize period of metformin treatment and different races wereall possible reasons leading to different results Replicationstudies in a relatively large population in a multi-ethnicdiabetic cohort are needed to confirm SLC47A1 rs2289669polymorphism effect on metformin efficacy both in glycemiccontrol and in lipid profiles

Furthermore we found that there was an interactionbetween SLC47A1 rs2289669 and SLC22A1 rs594709 whichaffected the blood glucose insulin level insulin resistanceimprovement and blood lipid after metformin treatmentAmong SLC22A1 rs594709 AA genotype patients SLC47A1rs2289669 AA genotype patients showed a better glycemiccontrol compared with G allele carriers while amongSLC22A1 rs594709 G allele carriers patients with SLC47A1rs2289669 AA genotype showed a better metformin effi-cacy in blood lipid than GA or GG genotypes Interac-tion between SLC22A1 and SLC47A1 polymorphisms andmetformin response had been reported previously [18] theeffect of the SLC47A1 rs2289669 polymorphism on thedecrease of HbA1c by metformin was larger in incidentusers with the SLC22A1 rs622342 CC genotype than inincident users with the AA or AC genotype patients Bothof the rs622342 and rs594709 are located in intron regionsof SLC22A1 gene The interaction is possibly due to thegenetic mutations in SLC22A1 and SLC47A1 altering thefunction of OCT1-mediated influx and MATE1-mediatedefflux As OCT1 transports metformin into the hepatocyteand MATE1 transports metformin out of the hepatocyte intothe bile altered OCT1 and MATE1 function may be ableto influence the intracellular metformin concentrations andglucose lowering function of metformin Further replicationandmechanism studies are needed to illustrate the influencesof SLC22A1 and SLC47A1 polymorphisms on metforminefficacy

In summarywe found that SLC22A1 594709 and SLC47A1rs2289669 polymorphisms may interact to affect the ther-apeutic efficacy of metformin in Chinese T2DM patientsFrom our data T2DM patients with AA genotype of bothSLC22A1 rs594709 and SLC47A1 rs2289669 may get maxi-mum glucose lowering benefit from metformin monother-apyThe SLC22A1 and SLC47A1 gene-gene interaction furthersuggests the complexity of metformin efficacy in T2DMpatients

Conflict of Interests

No potential conflict of interests was disclosed

Acknowledgments

The authors thank the study participants and supportedgrants of the National High-Tech RampD Program of China(863 Program) (2009AA022704 2012AA02A517)


References

[1] G Anfossi I Russo K Bonomo and M Trovati ldquoThe cardio-vascular effects ofmetformin further reasons to consider an olddrug as a cornerstone in the therapy of type 2 diabetes mellitusrdquoCurrent Vascular Pharmacology vol 8 no 3 pp 327ndash337 2010

[2] X Stephenne M Foretz N Taleux et al ldquoMetformin activatesAMP-activated protein kinase in primary human hepatocytesby decreasing cellular energy statusrdquo Diabetologia vol 54 no12 pp 3101ndash3110 2011

[3] R A Miller Q Chu J Xie M Foretz B Viollet and M JBirnbaum ldquoBiguanides suppress hepatic glucagon signallingby decreasing production of cyclic AMPrdquo Nature vol 494 no7436 pp 256ndash260 2013

[4] M D Fullerton S Galic KMarcinko et al ldquoSingle phosphory-lation sites in Acc1 and Acc2 regulate lipid homeostasis and theinsulin-sensitizing effects of metforminrdquo Nature Medicine vol19 no 12 pp 1649ndash1654 2013

[5] A K Madiraju D M Erion Y Rahimi et al ldquoMetforminsuppresses gluconeogenesis by inhibiting mitochondrial glyc-erophosphate dehydrogenaserdquo Nature vol 510 no 7506 pp542ndash546 2014

[6] F A Duca C D Cote B A Rasmussen et al ldquoMetformin acti-vates a duodenal Ampk-dependent pathway to lower hepaticglucose production in ratsrdquo Nature Medicine vol 21 no 5 pp506ndash511 2015

[7] S E Inzucchi R M Bergenstal J B Buse et al ldquoManagementof hyperglycemia in type 2 diabetes 2015 a patient-centeredapproach update to a position statement of the AmericanDiabetes Association and the European Association for theStudy of Diabetesrdquo Diabetes Care vol 38 no 1 pp 140ndash1492015

[8] L Tarasova I Kalnina K Geldnere et al ldquoAssociation ofgenetic variation in the organic cation transporters OCT1OCT2 and multidrug and toxin extrusion 1 transporter proteingenes with the gastrointestinal side effects and lower BMI inmetformin-treated type 2 diabetes patientsrdquo Pharmacogeneticsand Genomics vol 22 no 9 pp 659ndash666 2012

[9] Y Shu S A Sheardown C Brown et al ldquoEffect of genetic vari-ation in the organic cation transporter 1 (OCT1) on metforminactionrdquo The Journal of Clinical Investigation vol 117 no 5 pp1422ndash1431 2007

[10] G G Graham J Punt M Arora et al ldquoClinical pharmacoki-netics of metforminrdquo Clinical Pharmacokinetics vol 50 no 2pp 81ndash98 2011

[11] Y Shu C Brown R A Castro et al ldquoEffect of genetic variationin the organic cation transporter 1 OCT1 on metforminpharmacokineticsrdquo Clinical Pharmacology and Therapeuticsvol 83 no 2 pp 273ndash280 2008

[12] W Hou D Zhang W Lu et al ldquoPolymorphism of organiccation transporter 2 improves glucose-lowering effect of met-formin via influencing its pharmacokinetics in chinese type 2diabetic patientsrdquoMolecular Diagnosis ampTherapy vol 19 no 1pp 25ndash33 2015

[13] H Yoon H-Y Cho H-D Yoo S-M Kim and Y-B LeeldquoInfluences of organic cation transporter polymorphisms on thepopulation pharmacokinetics ofmetformin in healthy subjectsrdquoThe AAPS Journal vol 15 no 2 pp 571ndash580 2013

[14] A Giannoudis L Wang A L Jorgensen et al ldquoThe hOCT1SNPs M420del and M408V alter imatinib uptake and M420delmodifies clinical outcome in imatinib-treated chronic myeloidleukemiardquo Blood vol 121 no 4 pp 628ndash637 2013

[15] R He D Zhang W Lu et al ldquoSLC47A1 gene rs2289669 GgtAvariants enhance the glucose-lowering effect of metformin viadelaying its excretion in Chinese type 2 diabetes patientsrdquoDiabetes Research and Clinical Practice vol 109 no 1 pp 57ndash63 2015

[16] M L Becker L E Visser R H N van Schaik A HofmanA G Uitterlinden and B H C Stricker ldquoGenetic variationin the multidrug and toxin extrusion 1 transporter proteininfluences the glucose-lowering effect of metformin in patientswith diabetes a preliminary studyrdquo Diabetes vol 58 no 3 pp745ndash749 2009

[17] I Tkac L Klimcakova M Javorsky et al ldquoPharmacogenomicassociation between a variant in SLC47A1 gene and therapeuticresponse tometformin in type 2 diabetesrdquoDiabetes Obesity andMetabolism vol 15 no 2 pp 189ndash191 2013

[18] M L Becker L E Visser R H N van Schaik A HofmanA G Uitterlinden and B H C Stricker ldquoInteraction betweenpolymorphisms in the OCT1 and MATE1 transporter andmetformin responserdquo Pharmacogenetics and Genomics vol 20no 1 pp 38ndash44 2010

[19] M M H Christensen R S Pedersen T B Stage et al ldquoAgene-gene interaction between polymorphisms in the OCT2andMATE1 genes influences the renal clearance of metforminrdquoPharmacogenetics and Genomics vol 23 no 10 pp 526ndash5342013

[20] H-L Liu Y-G Lin J Wu et al ldquoImpact of genetic polymor-phisms of leptin and TNF-alpha on rosiglitazone response inChinese patients with type 2 diabetesrdquo European Journal ofClinical Pharmacology vol 64 no 7 pp 663ndash671 2008

[21] H Sun Z-C Gong J-Y Yin et al ldquoThe association ofadiponectin allele 45TG and -11377CG polymorphisms withtype 2 diabetes and rosiglitazone response in Chinese patientsrdquoBritish Journal of Clinical Pharmacology vol 65 no 6 pp 917ndash926 2008

[22] A Katz S S Nambi K Mather et al ldquoQuantitative insulinsensitivity check index a simple accurate method for assessinginsulin sensitivity in humansrdquo Journal of Clinical Endocrinologyand Metabolism vol 85 no 7 pp 2402ndash2410 2000

[23] D R Matthews J P Hosker A S Rudenski B A Naylor DF Treacher and R C Turner ldquoHomeostasis model assessmentinsulin resistance and beta-cell function from fasting plasmaglucose and insulin concentrations in manrdquo Diabetologia vol28 no 7 pp 412ndash419 1985

[24] A Lautem M Heise A Grasel et al ldquoDownregulation oforganic cation transporter 1 (SLC22A1) is associated with tumorprogression and reduced patient survival in human cholangio-cellular carcinomardquo International Journal of Oncology vol 42no 4 pp 1297ndash1304 2013

[25] Y Taketani H Yamanaka-Okumura H Yamamoto and ETakeda ldquoRole of nutrient transporters in lifestyle-related dis-easesrdquo The Journal of Physical Fitness and Sports Medicine vol2 no 4 pp 409ndash416 2013

[26] K A Jablonski J B McAteer P I W de Bakker et alldquoCommon variants in 40 genes assessed for diabetes incidenceand response to metformin and lifestyle intervention in thediabetes prevention programrdquoDiabetes vol 59 no 10 pp 2672ndash2681 2010

[27] R Sallinen M A Kaunisto C Forsblom et al ldquoAssociation ofthe SLC22A1 SLC22A2 and SLC22A3 genes encoding organiccation transporters with diabetic nephropathy and hyperten-sionrdquo Annals of Medicine vol 42 no 4 pp 296ndash304 2010


[28] Y Ohishi M Nakamuta N Ishikawa et al ldquoGenetic polymor-phisms of OCT-1 confer susceptibility to severe progressionof primary biliary cirrhosis in Japanese patientsrdquo Journal ofGastroenterology vol 49 no 2 pp 332ndash342 2014

[29] H Takane E Shikata K Otsubo S Higuchi and I IeirildquoPolymorphism in human organic cation transporters andmetformin actionrdquo Pharmacogenomics vol 9 no 4 pp 415ndash422 2008

[30] M-K Choi and I-S Song ldquoOrganic cation transporters andtheir pharmacokinetic and pharmacodynamic consequencesrdquoDrug Metabolism and Pharmacokinetics vol 23 no 4 pp 243ndash253 2008

[31] M L Becker L E Visser R H N van Schaik A HofmanA G Uitterlinden and B H C Stricker ldquoGenetic variation inthe organic cation transporter 1 is associated with metforminresponse in patients with diabetes mellitusrdquo PharmacogenomicsJournal vol 9 no 4 pp 242ndash247 2009

[32] A Takeuchi H Motohashi M Okuda and K Inui ldquoDecreasedfunction of genetic variants Pro283Leu and Arg287Gly inhuman organic cation transporter hOCT1rdquo Drug Metabolismand Pharmacokinetics vol 18 no 6 pp 409ndash412 2003

[33] H-J Kang I-S Song H J Shin et al ldquoIdentification and func-tional characterization of genetic variants of human organiccation transporters in a Korean populationrdquo Drug Metabolismand Disposition vol 35 no 4 pp 667ndash675 2007

[34] A T Nies H Koepsell S Winter et al ldquoExpression of organiccation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3)is affected by genetic factors and cholestasis in human liverrdquoHepatology vol 50 no 4 pp 1227ndash1240 2009

[35] E Shikata R Yamamoto H Takane et al ldquoHuman organiccation transporter (OCT1 andOCT2) gene polymorphisms andtherapeutic effects of metforminrdquo Journal of Human Geneticsvol 52 no 2 pp 117ndash122 2007

[36] M V Tzvetkov S V Vormfelde D Balen et al ldquoThe effectsof genetic polymorphisms in the organic cation transportersOCT1 OCT2 and OCT3 on the renal clearance of metforminrdquoClinical Pharmacology andTherapeutics vol 86 no 3 pp 299ndash306 2009

[37] D H Kim L Sriharsha W Xu et al ldquoClinical relevance of apharmacogenetic approach using multiple candidate genes topredict response and resistance to imatinib therapy in chronicmyeloid leukemiardquo Clinical Cancer Research vol 15 no 14 pp4750ndash4758 2009

[38] O Singh J Y Chan K Lin C C T Heng and B ChowbayldquoSLC22A1-ABCB1 haplotype profiles predict imatinib pharma-cokinetics in Asian patients with chronic myeloid leukemiardquoPLoS ONE vol 7 no 12 Article ID e51771 2012

[39] M Tsuda T Terada T Mizuno T Katsura J Shimakura andK-I Inui ldquoTargeted disruption of the multidrug and toxinextrusion 1 (Mate1) gene in mice reduces renal secretion ofmetforminrdquo Molecular Pharmacology vol 75 no 6 pp 1280ndash1286 2009

[40] J Klen K Goricar A Janez and V Dolzan ldquoThe role of geneticfactors and kidney and liver function in glycemic control in type2 diabetes patients on long-term metformin and sulphonylureacotreatmentrdquo BioMed Research International vol 2014 ArticleID 934729 7 pages 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


[10] It was widely reported that genetic variants in codinggenes of transporters mentioned above played importantroles in pharmacokinetics and pharmacodynamics of met-formin [9 11ndash13] Among these SLC22A1 rs628031 (tagged tors594709 in Asian) was reported to affect imatinib uptake inchronic myeloid leukemia (CML) cell line [14] As we knowimatinib is also transported byOCT1 in the human organismSLC47A1 rs2289669 was reported to be associated withmetformin excretion [15] as well as its therapeutic response[16 17] Besides a gene-gene interaction between geneticvariants in SLC47A1 and SLC22A1 SLC47A1 and SLC22A2had a significant influence on metforminrsquos efficacy [18 19]It was found that there was a SNP-SNP interaction betweenSLC47A1 rs2289669 and SLC22A1 rs622342 [18] Howeverthe effect of SLC22A1 rs594709 and SLC47A1 rs2289669polymorphisms and SNP-SNP interaction on metforminefficacy in Chinese T2DM patients has not been widelyevaluated

In this study we aimed to investigate the distributivecharacteristics of SLC22A1 rs594709 and SLC47A1 rs2289669between T2DM patients and healthy subjects and the influ-ences of SLC22A1 rs594709 and SLC47A1 rs2289669 poly-morphisms on metforminrsquos therapeutic efficacy in glucoselowering serum lipids and insulin sensitivity Moreover weare interested in whether there is a SNP-SNP interactionbetween SLC22A1 rs594709 and SLC47A1 rs2289669 onmetforminrsquos therapeutic efficacy

2 Material and Methods

21 Subjects Distributive characteristics of SLC22A1rs594709 and SLC47A1 rs2289669 polymorphisms weredetermined in 267 unrelated patients with T2DM and182 healthy subjects 267 T2DM patients (143 males and124 females) were recruited from the Second XiangyaHospital of Central South University the Peoplersquos Hospitalof Liuyang and the First Hospital of Changsha 182 healthysubjects (86 males and 96 females) were recruited fromPhysical Examination Center of Xiangya Hospital of CentralSouth University T2DM was diagnosed according to DMdiagnostic criteria of WHO 1999 Patients with type 1diabetes gestational diabetes uncontrollable hypertensionsevere liver and kidney disease were not included in thisstudy

Furthermore 53 of 267 T2DM patients who were newlydiagnosed and drug naıve (31 males and 22 females averageage 49 years range 29ndash73 years) were enrolled to evaluatethe effect of metformin among different SLC22A1 rs594709and SLC47A1 rs2289669 genotype individuals Newly diag-nosed type 2 diabetes was defined as being diagnosed withT2DM within 6 months before enrollment All patientshad not been treated with any insulin secretagogue andreceived metformin monotherapy for 3 consecutive monthsMetformin doses ranged from 1000mgday to 2000mgdayamong individuals The study was approved by the EthicsCommittee of Xiangya School of Medicine Central SouthUniversity (Changsha Hunan China) (CTXY-110002-5) andwas performed in accordance with the Helsinki DeclarationWritten informed consent was provided by all subjects

22 Clinical Laboratory Tests Detailed clinical informationwas collected from each patient Bodymass index (BMI) fast-ing blood glucose (FBG) postprandial blood glucose (PBG)fasting insulin (FINS) postprandial insulin (PINS) gly-cosylated haemoglobin (HbA1c) total cholesterol (TChol)triglycerides (TG) low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) wereevaluated before and after metformin treatment as previouslydescribed [20 21] Homeostasis model assessment of insulinresistance (HOMA-IR) homeostasis model assessment ofinsulin sensitivity (HOMA-IS) homeostasis model assess-ment of 120573-cell function (HOMA-B) and quantitative insulinsensitivity check index (QUICKI) were calculated accordingto previous formulas [22 23]

23 DNA Sampling and Genotyping Genomic DNA wasisolated from peripheral blood leukocytes using PromegaDNA purification kit (Promega Wisconsin USA) accordingto the manufacturerrsquos protocol and stored at minus20∘C until useGenotyping was done using direct sequencing The primersused to amplify SLC22A1 rs594709 polymorphism were51015840GGCGCTTCCCACACTCAT31015840 (sense) and 51015840GAGGAA-AGCTCCACATGTAACC31015840 (antisense) and the primersfor SLC47A1 rs2289669 polymorphism were 51015840CCTGAC-CTCTGTCCCTATT31015840 (sense) and 51015840GACTTCCGTGCC-TGCT31015840 (antisense)The PCR products were then sequencedusing ABI 3700 automated sequencer (ABI Foster City CAUSA)

24 Statistical Analysis Independent-samples t-test was usedto compare baseline characteristics in T2DM patients andhealthy subjects The observed genotype frequencies inpatients and controls compared with expected frequenciesunder Hardy-Weinberg equilibrium were evaluated by 1205942test Comparisons of frequencies of genotypes and alleleswere also analyzed by 1205942 test

We evaluated associations between each SNP and thechange of clinical index before and after metformin therapyusing multivariate linear regression by adjusting for sex ageand BMI in additive model recessive model and dominantmodel respectively Besides the analysis for the SLC22A1rs594709 genotype was stratified for the SLC47A1 rs2289669genotype and vice versa Multivariate linear regression wasused to analyze the interaction between SLC22A1 rs594709and SLC47A1 rs2289669 genotype on metformin response inChinese T2DM patients All of the analyses were performedwith SPSS software (version 160 SPSS Chicago IllinoisUSA)

3 Results

31 Clinical Characteristics of Subjects A total of 267 T2DMpatients and 182 healthy subjects were enrolled to explorethe allele frequencies and genotype frequencies of SLC22A1rs594709 and SLC47A1 rs2289669 polymorphisms The clin-ical characteristics of T2DM patients and healthy subjectswere shown in Table 1 BMI (2510 plusmn 274 kgm2 versus2398 plusmn 294 kgm2 119901 lt 0001) waist circumference (8740 plusmn856 cm versus 8172 plusmn 952 cm 119901 lt 0001) waist-to-hip




Male 86 143Female 96 124 0189


lowastlowastlowast













SLC22A1 rs594709AA 146 (5468) 95 (5220)

0927GA 99 (3708) 70 (3846)GG 22 (824) 17 (934)

AllelesA 391 (7322) 260 (7143) 0555G 143 (2678) 104 (2857)

SLC47A1 rs2289669AA 77 (2884) 49 (2692)

0669GA 128 (4794) 95 (5220)GG 62 (2322) 38 (2088)

AllelesA 282 (5281) 193 (5302) 0950G 252 (4719) 171 (4697)






4 Discussion







a407 plusmn 606 519 plusmn 447 0501



























Acknowledgments



References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Male 86 143Female 96 124 0189


lowastlowastlowast













SLC22A1 rs594709AA 146 (5468) 95 (5220)

0927GA 99 (3708) 70 (3846)GG 22 (824) 17 (934)

AllelesA 391 (7322) 260 (7143) 0555G 143 (2678) 104 (2857)

SLC47A1 rs2289669AA 77 (2884) 49 (2692)

0669GA 128 (4794) 95 (5220)GG 62 (2322) 38 (2088)

AllelesA 282 (5281) 193 (5302) 0950G 252 (4719) 171 (4697)






4 Discussion







a407 plusmn 606 519 plusmn 447 0501



























Acknowledgments



References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















a407 plusmn 606 519 plusmn 447 0501



























Acknowledgments



References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























Acknowledgments



References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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