Interaction between OCT1 and LPIN1 polymorphisms and response to pioglitazone-metformin tablets in patients with polycystic ovary syndrome.

To the Editor: Polycystic ovary syndrome (PCOS) is a common endocrine disorder among women of reproductive age. PCOS is characterized by infrequent or absent ovulation, high androgen levels, and hyperinsulinaemia. Androgenic excess is the main pathophysiological mechanism of the disease. Metformin can lower insulin and androgen levels, increase estradiol levels, and induce ovulation.[1] Pioglitazone can increase the sensitivity of peripheral tissues to insulin, improve insulin resistance, reduce androgen levels, restore the menstrual cycle, and promote ovulation.[2] Despite extensive research, the molecular mechanisms that mediate the beneficial effects of metformin or pioglitazone in the treatment of PCOS are still unclear. There are also differences in individual patient responses to drugs. Reportedly, genetic factors may contribute to this variability.[3] The effects of coding single nucleotide polymorphisms (SNPs) in organic cation transporter 1 (OCT1) on the uptake and thus efficacy of metformin have been extensively investigated previously.[4,5] Moreover, lipin-1 is required at an early step in adipocyte differentiation for the induction of the adipogenic gene transcription program, including the key regulator peroxisome proliferator-activated receptor γ (PPARγ).[6] To date, LPIN1 polymorphisms have been reported to be associated with type 2 diabetes and metabolic syndrome.[7] Therefore, in this study, we aimed to determine the influence of genetic polymorphisms in OCT1 and LPIN1 on the response to pioglitazone-metformin tablets in 30 patients with PCOS. We conducted the observational study in the Department of Endocrinology and Metabolism, Second Affiliated Hospital of Nanchang University. The patients were selected from amongst those who came to our hospital for the first time and were diagnosed as having PCOS and were enrolled between January 1, 2019 and January 31, 2020. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang Univer and all patients gave written informed consent. All patients took pioglitazone-metformin tablets (specification: 15 mg/500 mg [Sino-American East China Co., Ltd., Hangzhou, China]), at a dosage of one tablet two times per day. All subjects completed a unified questionnaire, recorded detailed medical history, and were primarily asked about personal habits (diet and exercise), menstrual status, work status, and adverse drug reactions. The inclusion criteria were as follows: (1) women aged 18 years and above; (2) meet the diagnostic criteria for PCOS, namely (i) oligomenorrhea or amenorrhea or irregular uterine bleeding, (ii) clinical manifestation of high androgen or high androgen blood symptoms, (iii) the ovary under ultrasound (number of follicles with an inner diameter of 2–9 mm ≥12, and/or ovarian volume ≥10 mL, (iv) absence of other diseases that may cause high androgen and abnormal ovulation (among these, diagnosis was necessary for [i] and [iv], and then one of [ii] or [iii] must be met); (3) did not take any hormones for 3 months before the consultation; (4) volunteered to participate in this study and signed the informed consent form. All of these conditions were required for selection. The exclusion criteria were as follows: pregnancy, hyperprolactinemia, diabetes, thyroid disease, severe liver and kidney insufficiency, and active peptic ulcer at the time of enrollment. DNA was performed using DP348 (Tiangen Biochemical, Beijing, China) according to standard procedures. For the LPIN1 rs10192566 polymorphism, the following primers were used: 5′-TGAAACAGCCCAGGGATACC-3′ (forward) and 5′-CCACTGTGTCTTGTCTCCAGCA-3′ (reverse). For the OCT1 rs683369 polymorphism, the following primers were used: 5′-GGAAGCCCTCGCACCTCTC-3′ (forward) and 5′-GCTATGCCTACATTCCACATTCTC-3′ (reverse). The PCR conditions were as follows: denaturation for 2 min at 95°C, 32 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 60°C, extension for 30 s at 72°C, and a final extension for 5 min at 72°C. Continuous variables are expressed as mean (standard deviation), and an independent sample t-test was used for analysis. Data with a non-normal distribution are presented as the median (interquartile range), and non-parametric tests of two independent samples were used. Multiple linear regression was used to analyze the association between the number of variant alleles and differences in testosterone levels. We tested interactions between rs10192566 and rs683369 genotypes in the multivariate linear regression model. Statistical significance was set at P < 0.05. All statistical analyses were performed using SPSS 23.0 (SPSS Inc., Chicago, IL, USA). A total of 35 PCOS patients who met the inclusion criteria between January 2019 and January 2020. We excluded five patients who were pregnant (n = 2), had gastrointestinal reactions (n = 1), or were lost to follow-up at the time of study (n = 2). Baseline characteristics of individuals with different LPIN1 rs10192566 and OCT1 rs683369 genotypes are shown in [Supplementary Table 1, https://links.lww.com/CM9/B611]. As shown in Supplementary Table 2, https://links.lww.com/CM9/B611, patients with OCT1 rs683369 G allele and LPIN1 rs10192566 G allele had reduced testosterone levels. The changes in testosterone levels are presented in Figure 1. In participants with the OCT1 rs683369 CC genotype, the effect of the LPIN1 rs10192566 genotype was lesser than that of the OCT1 rs683369 CG/GG genotype. In participants with the LPIN1 rs10192566 CC genotype, the effect of the OCT1 rs683369 genotype was lesser than that of the LPIN1 rs10192566 CG/GG genotype. Testing for the interaction between OCT1 rs683369 and LPIN1 rs10192566 polymorphisms revealed that the change in testosterone level with the number of LPIN1 rs10192566 G alleles differed significantly between patients with the OCT1 rs683369 CC and CG/GG genotypes (6.5, 95% CI: 2.1–28.8, P =0.03), and the change in testosterone level with the number of OCT1 rs683369 G alleles also differed significantly between patients with the LPIN1 rs10192566 CC and CG/GG genotypes (5.9, 95% CI: 3.4–27.8, P =0.02) [Supplementary Table 3, https://links.lww.com/CM9/B611].Figure 1: Average change in testosterone levels in 30 patients with the ITAL rs683369 genotype and the LPIN1 rs10192566 genotype. d-testosterone level is expressed as the level at 0-month pre-administration minus the level at 3-month post-administration. ITAL: Organic cation transporter 1.The results indicated a correlation between the interaction of OCT1 rs683369 and LPIN1 rs10192566 polymorphisms and changes in testosterone levels after pioglitazone-metformin tablet treatment in patients with PCOS. The polymorphisms of rs683369 and rs10192566 were significantly associated with changes in testosterone levels in all 30 PCOS patients treated with pioglitazone-metformin tablets for 3 months. Pioglitazone is a PPARγ agonist, and lipin-1 can regulate PPARγ activity by releasing PPARγ co-inhibitors and recruiting co-activators through mechanisms other than PPARα activation.[8]In vitro studies have shown that metformin is a substrate of the OCT transporter family and that liver-specific OCT1 can regulate the pharmacokinetics and pharmacodynamics of metformin.[9–11] In our previous study, we found that administering pioglitazone-metformin tablets for 12 weeks significantly reduced fasting insulin, blood glucose, luteinizing hormone (LH), testosterone levels, and homeostatic model assessment of insulin resistance(HOMA-IR) in PCOS patients. OCT1 rs683369 (G > C) and LPIN1 rs10192566 (G > C) polymorphisms are associated with the response to metformin and rosiglitazone, respectively.[12,13] Most of the previous studies have focused on the effects of genetic polymorphisms and single drugs on patients with PCOS. In this study, we describe the effect of the interaction between these two SNPs in the treatment of patients with PCOS using a combination of pioglitazone and metformin. We found that the OCT1 rs683369 G allele and the LPIN1 rs10192566 G allele had a greater effect on lowering testosterone levels in PCOS patients taking pioglitazone-metformin tablets. The gene-gene interaction between rs683369 and rs10192566 may have a synergistic effect on lowering testosterone levels. This interaction is most likely due to the OCT1 transporter transporting metformin into the hepatocytes, where the intracellular metformin concentrations are sufficiently high to reduce testosterone levels in most individuals. LPIN1 supports pioglitazone in reducing testosterone levels by regulating PPARγ activity. In conclusion, the effect of the LPIN1 rs10192566 polymorphism on the testosterone-lowering effect of pioglitazone-metformin tablets was higher in incident users with the OCT1 rs683369 CG/GG genotype than in incident users with the CC genotype. In our next study, we intend to use a larger sample size and further include coding SNPs in OCT1 and LPIN1 to analyze the complex associations among pioglitazone, metformin, metabolic indicators, menstrual cycle, and ovulation rate. Conflicts of interest None.