The Impacts of SLC22A1 rs594709 and SLC47A1 rs2289669 Polymorphisms on Metformin Therapeutic Efficacy in Chinese Type 2 Diabetes Patients

Background. We aimed to investigate the distributive characteristics of SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms and their influence on metformin efficacy in Chinese T2DM patients. Methods. The distributions of SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms were determined in 267 T2DM patients and 182 healthy subjects. Subsequently, 53 newly diagnosed patients who received metformin monotherapy were recruited to evaluate metformin efficacy. Results. No significant difference was found between T2DM patients and healthy subjects in SLC22A1 rs594709 and SLC47A1 rs2289669 allele frequencies and genotype frequencies. After metformin treatment, SLC22A1 rs594709 GG genotype patients showed a higher increase in FINS (p = 0.015) and decrease in HOMA-IS (p = 0.001) and QUICKI (p = 0.002) than A allele carriers. SLC47A1 rs2289669 GG genotype patients had a higher decrease in TChol (p = 0.030) and LDL-C (p = 0.049) than A allele carriers. Among SLC22A1 rs594709 AA genotype, patients with SLC47A1 rs2289669 AA genotype showed a higher decrease in FBG (p = 0.015), PINS (p = 0.041), and HOMA-IR (p = 0.014) than G allele carriers. However, among SLC22A1 rs594709 G allele carriers, SLC47A1 rs2289669 AA genotype patients showed a higher decrease in TChol (p = 0.013) than G allele carriers. Conclusion. Our data suggest that SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms may influence metformin efficacy together in Chinese T2DM patients.


Introduction
Metformin is the most widely used oral antidiabetic drug, which could effectively lower blood glucose and reduce the risk of cardiovascular diseases [1]. The classical functional mechanism of metformin is to decrease the hepatic energy status by inhibiting the mitochondrial respiratory-chain complex 1, thus activating the adenosine 5 -monophosphateactivated protein kinase (AMPK), a cellular metabolic sensor, to finally lower the blood glucose level [2]. In recent years, further probable mechanisms involved in cAMP, the phosphorylation of Acc1 and Acc2, mitochondrial glycerophosphate dehydrogenase, and the activated duodenal AMPK were found to affect metformin's effect in different ways [3][4][5][6]. With its significant glycemic benefits and neutral effects on weight, metformin remains the recommended first-line oral therapy in the guidelines of the American Diabetes Association and European Association of the Study of Diabetes [7]. However, during its clinical usage, the individual differences and drug failure widely existed [8,9].
Metformin is not metabolized in vivo. The gastrointestinal absorption, hepatic uptake, and renal excretion of metformin are mediated greatly by organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) 2 International Journal of Endocrinology [10]. It was widely reported that genetic variants in coding genes of transporters mentioned above played important roles in pharmacokinetics and pharmacodynamics of metformin [9,[11][12][13]. Among these, SLC22A1 rs628031 (tagged to rs594709 in Asian) was reported to affect imatinib uptake in chronic myeloid leukemia (CML) cell line [14]. As we know, imatinib is also transported by OCT1 in the human organism. SLC47A1 rs2289669 was reported to be associated with metformin excretion [15] as well as its therapeutic response [16,17]. Besides, a gene-gene interaction between genetic variants in SLC47A1 and SLC22A1, SLC47A1 and SLC22A2 had a significant influence on metformin's efficacy [18,19]. It was found that there was a SNP-SNP interaction between SLC47A1 rs2289669 and SLC22A1 rs622342 [18]. However, the effect of SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms and SNP-SNP interaction on metformin efficacy in Chinese T2DM patients has not been widely evaluated.
In this study, we aimed to investigate the distributive characteristics of SLC22A1 rs594709 and SLC47A1 rs2289669 between T2DM patients and healthy subjects and the influences of SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms on metformin's therapeutic efficacy in glucose lowering, serum lipids, and insulin sensitivity. Moreover, we are interested in whether there is a SNP-SNP interaction between SLC22A1 rs594709 and SLC47A1 rs2289669 on metformin's therapeutic efficacy. Furthermore, 53 of 267 T2DM patients who were newly diagnosed and drug naïve (31 males and 22 females; average age 49 years, range 29-73 years) were enrolled to evaluate the effect of metformin among different SLC22A1 rs594709 and SLC47A1 rs2289669 genotype individuals. Newly diagnosed type 2 diabetes was defined as being diagnosed with T2DM within 6 months before enrollment. All patients had not been treated with any insulin secretagogue and received metformin monotherapy for 3 consecutive months. Metformin doses ranged from 1000 mg/day to 2000 mg/day among individuals. The study was approved by the Ethics Committee of Xiangya School of Medicine, Central South University (Changsha, Hunan, China) (CTXY-110002-5), and was performed in accordance with the Helsinki Declaration. Written informed consent was provided by all subjects.

DNA Sampling and Genotyping.
Genomic DNA was isolated from peripheral blood leukocytes using Promega DNA purification kit (Promega, Wisconsin, USA) according to the manufacturer's protocol and stored at −20 ∘ C until use. Genotyping was done using direct sequencing. The primers used to amplify SLC22A1 rs594709 polymorphism were 5 GGCGCTTCCCACACTCAT3 (sense) and 5 GAGGAA-AGCTCCACATGTAACC3 (antisense), and the primers for SLC47A1 rs2289669 polymorphism were 5 CCTGAC-CTCTGTCCCTATT3 (sense) and 5 GACTTCCGTGCC-TGCT3 (antisense). The PCR products were then sequenced using ABI 3700 automated sequencer (ABI, Foster City, CA, USA).

Statistical Analysis.
Independent-samples t-test was used to compare baseline characteristics in T2DM patients and healthy subjects. The observed genotype frequencies in patients and controls compared with expected frequencies under Hardy-Weinberg equilibrium were evaluated by 2 test. Comparisons of frequencies of genotypes and alleles were also analyzed by 2 test.
We evaluated associations between each SNP and the change of clinical index before and after metformin therapy using multivariate linear regression by adjusting for sex, age, and BMI in additive model, recessive model, and dominant model, respectively. Besides, the analysis for the SLC22A1 rs594709 genotype was stratified for the SLC47A1 rs2289669 genotype and vice versa. Multivariate linear regression was used to analyze the interaction between SLC22A1 rs594709 and SLC47A1 rs2289669 genotype on metformin response in Chinese T2DM patients. All of the analyses were performed with SPSS software (version 16.0; SPSS, Chicago, Illinois, USA).

Genotype Analysis and Allelic
Frequencies. The frequencies of the SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms in T2DM patients and healthy subjects were shown in Table 2. Both SNPs were in agreement with Hardy-Weinberg equilibrium in each group ( > 0.05). Minor allele frequencies (MAFs) of SLC22A1 rs594709 in T2DM group and healthy control were 26.78% and 28.57%, respectively. MAFs of SLC47A1 rs2289669 were, respectively, 47.19% and 46.97% in T2DM patients and healthy subjects. No significant differences were found in allele frequencies ( = 0.555 for rs594709; = 0.950 for rs2289669) and genotype frequencies ( = 0.927 for rs594709; = 0.669 for rs2289669) between T2DM patients and healthy controls.

Influence of SLC22A1 rs594709 and SLC47A1 rs2289669
Polymorphisms on Therapeutic Efficacy of Metformin in T2DM Patients. 53 newly diagnosed T2DM patients who had taken the first prescription of metformin for 3 consecutive months were enrolled to evaluate metformin efficacy. As shown in Table 3, SLC22A1 rs594709 GG genotype patients had a higher increase in FINS ( = 0.015) and greater decrease in HOMA-IS ( = 0.001) and QUICKI ( = 0.002) than those of A allele carriers (Table 3). However, SLC47A1 rs2289669 polymorphism was associated with changes in TChol and LDL-C level; patients carrying SLC47A1 rs2289669 A allele had a relatively minor decrease in TChol ( = 0.030) and LDL-C ( = 0.049) level compared to GG genotype patients (Table 3).

Discussion
In the current study, we evaluated the distributive characteristics of SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms and the separate as well as combined influence of 4 International Journal of Endocrinology  their polymorphisms on metformin's effect in Chinese T2DM patients. Our results showed that there were no significant differences in SLC22A1 rs594709 and SLC47A1 rs2289669 allele frequencies and genotype frequencies between T2DM patients and healthy subjects. SLC22A1 rs594709 A allele carriers showed a better metformin efficacy in insulin sensitivity compared with GG genotype patients. SLC47A1 rs2289669 GG genotype patients showed a better efficacy in improving blood lipid. Furthermore, we found that there was a significant interaction between SLC47A1 rs2289669 and SLC22A1 rs594709, which might function together in metformin efficacy. OCTs mediate the interactions between cells and their environment and are involved in transporting a broad International Journal of Endocrinology 5 spectrum of endogenous substrates and the detoxification of xenobiotics [24]. Nutrient transporters, including OCT1 and MATE1, could affect nutrient homeostasis through different ways [25]. Polymorphisms in OCT1 or MATE1 have been correlated with the susceptibility of type 2 diabetes, diabetic nephropathy, hypertension, and primary biliary cirrhosis [26][27][28]. SLC47A1 rs2453583 and SLC22A1 rs651154 have been reported to be nominally associated with diabetes incidence in the DPP (Diabetes Prevention Program) [26]. Therefore, we investigated the distributive characteristics of the two SNPs that we are concerned with, namely, SLC22A1 rs594709 and SLC47A1 rs2289669, in T2DM patients and healthy subjects. However, no statistical difference was found between two groups.
Human SLC22A1 gene is highly polymorphic, and several polymorphisms have been described to affect metformin responses [29,30], including polymorphisms located in the intron regions [31]. SLC22A1 rs594709 locates in intron 4. Pairwise linkage disequilibrium (LD) analysis indicated that there were strong LDs between rs594709 and rs2282143, rs628031, rs3798168, and rs683369. Polymorphisms mentioned above could more or less affect the metabolizing and/or efficacy of OCT1 substrates. SLC22A1 rs2282143 variation led to a decrease of transport activity, but not completely abolished [32]. SLC22A1 rs628031 (Met408Val) was identified in multiethnic groups worldwide [33] and has been characterized to have normal metformin uptake in vitro [9,11]. However, it has been recognized as a weak positive predictor of metformin response in clinical study [8,34,35], though this genetic variant did not alter protein expression [34,36]. Recent studies reported that SLC22A1 rs3798168, rs683369, and rs628031 could affect the response to imatinib therapy in chronic myeloid leukemia (CML) patients [37,38]. As SLC22A1 rs594709 is the tagger SNP of rs3798168, rs683369, and rs628031 ( 2 > 0.8), it is possible that the structural changes in SLC22A1 caused by any of these polymorphisms or even a combination effect of them may affect its transport ability of metformin.
Previously, Caucasians population-based study in diabetic patients revealed that rs2289669 G/A in SLC47A1 gene was associated with the therapeutic efficacy of metformin [16,17]. The decrease in HbA1c was greater in AA homozygotes compared with the GA + GG genotype patients after metformin treatment. The effect of SLC47A1 rs2289669 polymorphism on pharmacodynamics ( = 202) and pharmacokinetics ( = 28) of metformin was also evaluated in Chinese patients and showed that AA genotype patients had a better glucose lowering effect after one-year follow-up. As for pharmacokinetics parameters, AA genotype patients had higher AUC 12h and lower renal clearance and renal clearance by secretion [15]. Thus, it is possible that the effect of the rs2289669 polymorphism on metformin glucose lowering efficacy was caused partly or completely by a reduced renal excretion and increased metformin plasma levels [39]. However, in our study, we did not find SLC47A1 rs2289669 polymorphism was correlated with metformin glucose lowering effect but was associated with the reduction of TChol and LDL-C. This result was similar to long-term metformin and sulphonylurea combination therapy study carried out in Slovenia [40]. Their results suggested that SLC47A1 rs2289669 genotype was not associated with HbA1c levels but significantly associated with cholesterol levels in metformin-treated T2DM patients even after adjustment for renal function. In contrast to the previous studies, the sample size, period of metformin treatment, and different races were all possible reasons leading to different results. Replication studies in a relatively large population in a multi-ethnic diabetic cohort are needed to confirm SLC47A1 rs2289669 polymorphism effect on metformin efficacy both in glycemic control and in lipid profiles.
Furthermore, we found that there was an interaction between SLC47A1 rs2289669 and SLC22A1 rs594709, which affected the blood glucose, insulin level, insulin resistance improvement, and blood lipid after metformin treatment. Among SLC22A1 rs594709 AA genotype patients, SLC47A1 rs2289669 AA genotype patients showed a better glycemic control compared with G allele carriers, while, among SLC22A1 rs594709 G allele carriers, patients with SLC47A1 rs2289669 AA genotype showed a better metformin efficacy in blood lipid than GA or GG genotypes. Interaction between SLC22A1 and SLC47A1 polymorphisms and metformin response had been reported previously [18]; the effect of the SLC47A1 rs2289669 polymorphism on the decrease of HbA1c by metformin was larger in incident users with the SLC22A1 rs622342 CC genotype than in incident users with the AA or AC genotype patients. Both of the rs622342 and rs594709 are located in intron regions of SLC22A1 gene. The interaction is possibly due to the genetic mutations in SLC22A1 and SLC47A1 altering the function of OCT1-mediated influx and MATE1-mediated efflux. As OCT1 transports metformin into the hepatocyte and MATE1 transports metformin out of the hepatocyte into the bile, altered OCT1 and MATE1 function may be able to influence the intracellular metformin concentrations and glucose lowering function of metformin. Further replication and mechanism studies are needed to illustrate the influences of SLC22A1 and SLC47A1 polymorphisms on metformin efficacy.
In summary, we found that SLC22A1 594709 and SLC47A1 rs2289669 polymorphisms may interact to affect the therapeutic efficacy of metformin in Chinese T2DM patients. From our data, T2DM patients with AA genotype of both SLC22A1 rs594709 and SLC47A1 rs2289669 may get maximum glucose lowering benefit from metformin monotherapy. The SLC22A1 and SLC47A1 gene-gene interaction further suggests the complexity of metformin efficacy in T2DM patients.