Expression Of OCT1 Research Articles

Systematic Investigation of the Effects of Long-Term Administration of a High-Fat Diet on Drug Transporters in the Mouse Liver, Kidney and Intestine.

Long-term intake of a high-fat diet is a crucial factor contributing to obesity, which has become a global public health problem. Progressive obesity subsequently leads to hepatic injury, renal damage and intestinal atrophy. Transporters expressed in the liver, kidney and intestine play important roles in the deposition of nutrients and drugs, but researchers have not clearly determined whether/how the expression of transporters changes after long-term administration of a High-Fat Diet (HFD). This study aims to explore the effects of the long-term administration of a HFD on the expression of drug transporters in the liver, kidney and intestine in mice and to provide useful information for medical applications in the clinic. Male C57BL/6J mice were fed either a basal diet or HFD for 24 weeks, and oral glucose tolerance tests were performed after 3, 11 and 23 weeks. Serum was obtained to measure lipid metabolism, inflammatory mediators, renal function and hepatic function. Adipose tissues, kidney, pancreas and liver were collected for hematoxylin and eosin (H&E) staining after 4, 12 and 24 weeks. The mRNA and proteins expression of drug transporters in the liver, kidney and intestine were detected using real-time PCR and western blot, respectively. Compared with the control group, long-term HFD administration significantly increased the adipose index. The serum lipid levels, including Total Cholesterol (TC), Triglyceride (TG), and Low-Density Lipoprotein Cholesterol (LDL-C), as well as the levels of the inflammatory cytokines Interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) were significantly elevated in HFD-induced obese mice. H&E staining revealed pathological changes in the adipose cells, liver, kidney and pancreas from the obese group following the long-term administration of the HFD. The liver of the obese group presented increased mRNA expression of the efflux transporter Mrp2 and uptake transporter Oat2 at 24 weeks. The relative expression of Oat2 increased 4.08-fold and the protein expression of Oat2 was upregulated at 24 weeks in HFD-fed mice, while the mRNA expression of the uptake transporters Oct1, Oatp1b2 and Oatp1a4 decreased by 79%, 61% and 19%, respectively. The protein expression of Oct1 was significantly downregulated in obese mice at 12 weeks. The mRNA expression of the efflux transporter Mdr1a was significantly reduced in HFD-fed mice compared with the control group at 24 weeks. Western blot showed that the trend of protein level of Mdr1 was consistent with the mRNA expression. In the kidney, the level of the Oct2 mRNA increased 1.92- and 2.46-fold at 4 and 12 weeks in HFD-fed mice, respectively. The expression of the Oat1 and Oat3 mRNAs was markedly downregulated in the kidneys of mice with HFD-induced obesity at 4 weeks. The decrease of 72% and 21% in Mdr1a mRNA expression was observed in the obese model at 4 weeks and 12 weeks, respectively. Western blot showed that the protein levels of Mdr1 and Oat1 were consistent with the mRNA expression. The qPCR experiments showed a 2.87-fold increase in Bcrp mRNA expression at 24 weeks, and the expression of the Pept1 mRNA increased 2.84-fold in intestines of obese mice subjected to long-term administration of the HFD compared with control mice at 12 weeks. Western blot showed that the trend of protein levels of Mdr1 and Mrp2 were consistent with the mRNA expression. The expression of uptake and efflux transporters mRNAs and protein levels were altered in obese mice compared with control mice, providing scientific evidence for future medical applications in the clinic.

Metformin has a direct effect on ovarian cells that is dependent on organic cation transporters

Metformin (MET) is the most widely prescribed hypoglycemic drug in type 2 diabetes and Polycystic Ovary Syndrome. Besides its effects on glucose metabolism, MET exerts beneficial effects on these patients’ fertility. However, the exact mechanisms of action of MET on female fertility are still unclear. In this work, we analyzed a possible direct effect of MET on ovarian cells. We found expression of the organic cation transporters OCT1, OCT2 and OCT3, responsible for MET uptake into the cells, in rat granulosa cells and human cumulus cells. Furthermore, MET increased pAMPK and decreased VEGF levels both in vivo and in rat granulosa cells in culture. These last effects were reversed when OCTs were inhibited. Our results suggest that MET acts directly on ovarian cells regulating cell metabolism and VEGF expression. Our findings are relevant to optimize PCOS fertility treatment and to explore ovarian MET actions in other female pathologies.

DLX6-AS1/miR-204-5p/OCT1 positive feedback loop promotes tumor progression and epithelial-mesenchymal transition in gastric cancer.

Accumulating evidence indicates that long non-coding RNAs (lncRNAs) participate in progression of gastric cancer (GC). Nevertheless, the function and expression level of DLX6-AS1 in GC remain unknown. We explored the sequencing data of DLX6-AS1 downloaded from The Cancer Genome Atlas. The expression of DLX6-AS1, miR-204-5p and OCT1 in 56 GC patients and GC cell lines was quantified by qRT-PCR and western blotting. Furthermore, we performed in vitro functional assays to assess proliferation, invasion and migration of GC cells by knockdown of DLX6-AS1. The expression level of epithelial-mesenchymal transition (EMT)-related genes was also determined by qRT-PCR and western blotting. Actin remodeling was detected by F-actin phalloidin staining. The luciferase reporter assay and chromatin immunoprecipitation assay was utilized to confirm the bioinformatic prediction. The function of the DLX6-AS1/miR-204-5p/OCT1 axis in GC proliferation was clarified by rescue assays. We first demonstrated that DLX6-AS1 was upregulated in GC tissues and cell lines and was associated with T3/T4 invasion, distant metastasis and poor clinical prognosis. Further functional analysis showed that downregulation of DLX6-AS1 inhibited GC cell proliferation, migration, invasion and EMT in vitro. Mechanistic investigation indicated that DLX6-AS1 acted as a cancer-promoting competing endogenous RNA (ceRNA) by binding miR-204-5p and upregulating OCT1. Moreover, the transcription factor OCT1 was confirmed to enhance DLX6-AS1 expression by targeting the promoter region. This study revealed that OCT1-induced DLX6-AS1 promoted GC progression and the EMT via the miR-204-5p/OCT1 axis, suggesting that this lncRNA might be a promising prognostic biomarker and therapeutic target for GC.

Effects of salinomycin and niclosamide on small cell lung cancer and small cell lung cancer circulating tumor cell lines

SummaryTumor dissemination and recurrence is attributed to highly resistant cancer stem cells (CSCs) which may constitute a fraction of circulating tumor cells (CTCs). Small cell lung cancer (SCLC) constitutes a suitable model to investigate the relation of CTCs and CSCs due to rapid tumor spread and a high number of CTCs. Expansion of five SCLC CTC lines (BHGc7, 10, 16, 26 and UHGc5) in vitro at our institution allowed for the analysis of CSC markers and cytotoxicity of the CSC-selective drugs salinomycin and niclosamide against CTC single cell suspensions or CTC spheroids/ tumorospheres (TOS). Salinomycin exerted dose-dependent cytotoxicity against the SCLC lines but, with exception of BHGc7 TOS, there was no markedly enhanced activity against TOS. Similarly, niclosamide exhibits high activity against BHGc7 TOS and UHGc5 TOS but not against the other CTC spheroids. High expression of the CSC marker CD133 was restricted to three SCLC tumor lines and the BHGc10 CTC line. All SCLC CTCs are CD24-positive but lack expression of CD44 and ABCG2 in contrast to the SCLC tumor lines which show a phenotype more similar to that of CSCs. The stem cell marker SOX2 was found in all CTC lines and SCLC GLC14/16, whereas elevated expression of Oct-3/4 and Nanog was restricted to BHGc26 and UHGc5. In conclusion, the SCLC CTCs established from patients with relapsed disease lack a typical CSC phenotype in respect to chemosensitivity to CSC-selective drugs, surface markers, expression of pluripotent stem cell and transcription factors.

Diurnal expression of ABC and SLC transporters in jejunum is modulated by adrenalectomy

The circadian clock system drives many physiological processes, including plasma concentration of glucocorticoids and epithelial transport of some ions and nutrients. As glucocorticoids entrain the circadian rhythms in various peripheral organs, we examined whether adrenalectomy affects the expression and circadian rhythmicity of intestinal transporters of the solute carrier (SLC) and ATP-binding cassette (ABC) families, which participate in intestinal barriers for absorption of nutrients, nonnutrients and oral drugs. The rat jejunum showed rhythmic circadian profiles of Sglt1, Pept1, Nhe3, Mdr1 and Mrp2 but not Mct1, Oct1, Octn1, Oatp1, Cnt1 and Bcrp. With the exception of Pept1 and Mct1, adrenalectomy decreased the expression of all rhythmic and arrhythmic transporters including the amplitude of Sglt1 and Nhe3 rhythms but minimally affected the phases of rhythmic transporters except of Nhe3. Similarly, adrenalectomy downregulated the expression of rhythmic (Pparα, Hlf, Pgc1α) and arrhythmic (Hnf1β, Hnf4α) transcription factors, which are known to regulate the expression of transporters. We conclude that endogenous corticosteroids have a profound effect on the expression of intestinal SLC and ABC transporters and their nuclear transcription factors. The circulating corticosteroids are necessary for maintaining upregulated expression of Sglt1, Oct1, Octn1, Oatp1, Cnt1, Nhe3, Mdr1, Bcrp, Mrp2, Pparα, Pgc1α, Hnf1β, Hnf4α and Hlf and for maintaining the high amplitude of Sglt1, Nhe3, Pparα, Pgc1α and Hlf circadian rhythms. The study demonstrates that signals from the adrenal gland are necessary for maintaining the expression of arrhythmic and rhythmic intestinal transporters and that changes in the secretion of corticosteroids associated with stress might reorganize intestinal transport barriers.

Role of OCT1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers causing death worldwide. It is difficult to detect at an early stage and most patients with advanced HCC rarely achieve satisfying therapeutic results. Accordingly, researchers have been trying to find new biomarkers for diagnosis and new methods of treatment. OCT1, a member of solute carrier super family, is highly expressed in normal liver tissues, and predominantly transports endogenous and exogenous substances, such as metabolites, drugs and toxins to hepatocytes. Studies have demonstrated that the expression of OCT1 is related to the progression and survival of HCC patients. Furthermore, sorafenib, which is regarded as the only effective molecular targeting drug for advanced HCC, is affected by OCT1 variants. In the current review, we summarized the reports about OCT1 and HCC in order to present a comprehensive overview of the relationship between OCT1 and HCC.

An in vitro study on interaction of anisodine and monocrotaline with organic cation transporters of the SLC22 and SLC47 families.

Current study systematically investigated the interaction of two alkaloids, anisodine and monocrotaline, with organic cation transporter OCT1, 2, 3, MATE1 and MATE2-K by using in vitro stably transfected HEK293 cells. Both anisodine and monocrotaline inhibited the OCTs and MATE transporters. The lowest IC50 was 12.9 µmol·L-1 of anisodine on OCT1 and the highest was 1.8 mmol·L-1 of monocrotaline on OCT2. Anisodine was a substrate of OCT2 (Km = 13.3 ± 2.6 µmol·L-1 and Vmax = 286.8 ± 53.6 pmol/mg protein/min). Monocrotaline was determined to be a substrate of both OCT1 (Km = 109.1 ± 17.8 µmol·L-1, Vmax = 576.5 ± 87.5 pmol/mg protein/min) and OCT2 (Km = 64.7 ± 14.8 µmol·L-1, Vmax = 180.7 ± 22.0 pmol/mg protein/min), other than OCT3 and MATE transporters. The results indicated that OCT2 may be important for renal elimination of anisodine and OCT1 was responsible for monocrotaline uptake into liver. However neither MATE1 nor MATE2-K could facilitate transcellular transport of anisodine and monocrotaline. Accumulation of these drugs in the organs with high OCT1 expression (liver) and OCT2 expression (kidney) may be expected.

Regulation of OCT2 transcriptional repression by histone acetylation in renal cell carcinoma

ABSTRACTRenal cell carcinoma (RCC) is a common malignant tumour affecting the urinary system, and multidrug resistance is one of the major reasons why chemotherapy for this type of cancer often fails. Previous studies have shown that loss of the human organic cation transporter OCT2 is the main factor contributing to oxaliplatin resistance in RCC, and that DNA hypermethylation and histone methylation play important roles in the transcriptional repression of OCT2 in RCC. In this study, we found that histone acetylation also regulates OCT2 repression in RCC and elucidated the underlying mechanisms. In normal renal cells, HDAC7 combines with MYC at the OCT2 promoter, resulting in a decrease in free HDAC7, which in turn increases the levels of H3K18ac and H3K27ac at the OCT2 promotor and activates OCT2 expression. In RCC cells, however, the interaction between HDAC7 and MYC does not occur, which leads a high abundance of HDAC7 and low levels of H3K18ac and H3K27ac at the OCT2 promoter, thereby resulting in the inhibition of OCT2 transcription. We found that combined treatment using the DNA methylation inhibitor decitabine and the histone deacetylase inhibitor vorinostat significantly increased the expression of OCT2 in RCC cell lines, which sensitized these cells to oxaliplatin. We accordingly propose that the combination of anticancer agents and epigenetic drugs can provide a novel chemotherapeutic regimen.

In vivo pharmacodynamic and pharmacokinetic effects of metformin mediated by the gut microbiota in rats

AimsThe gut microbiota plays a crucial role in the efficacy of metformin in T2DM treatment. We evaluated whether the pharmacodynamics and pharmacokinetics of metformin are mediated by gut microbiota. Main methodsWe used conventional diabetic and pseudo-germ-free rats. After 6 weeks of metformin treatment, pharmacodynamic indexes were determined. Metformin concentrations were measured with a validated HPLC-MS/MS method after the first oral administration. Key findingsMost endpoints were similar between vehicle-treated diabetic and vehicle-treated pseudo-germ-free diabetic rats. However, after 6 weeks of metformin treatment, compared with conventional diabetic rats, pseudo-germ-free diabetic rats exhibited significantly increased FBG, decreased oral glucose, reduced GSP, worsened insulin resistance, increased hyperlipidemia, and increased hepatic steatosis severity. Moreover, the Cmax of pseudo-germ-free rats increased significantly, while the t1/2α decreased significantly. These pharmacodynamic and pharmacokinetic changes were probably due to a decrease in Oct1 expression in the liver, resulting in altered hepatic uptake of metformin in vivo. SignificanceThese results implied that the gut microbiota may play an important role in the pharmacodynamics and pharmacokinetics of metformin and that the changes in these properties are probably due to Oct1 downregulation in the livers of pseudo-germ-free rats.

Epigenetic events involved in organic cation transporter 1-dependent impaired response of hepatocellular carcinoma to sorafenib.

The expression of the human organic cation transporter-1 (hOCT1, gene SLC22A1) is reduced in hepatocellular carcinoma (HCC). The molecular bases of this reduction and its relationship with the poor response of HCC to sorafenib were investigated. HCC transcriptomes from 366 samples available at TCGA were analysed. Alternative splicing was determined by RT-PCR. The role of miRNAs in SLC22A1 downregulation was investigated. Expression of Oct1 was measured in rodent HCC models (spontaneously generated in Fxr-/- mice and chemically-induced in rats). hOCT1 was overexpressed in human hepatoma cells (HuH7 and HepG2). Sorafenib and regorafenib uptake was determined by HPLC-MS/MS. hOCT1 overexpression enhanced sorafenib, but not regorafenib, quinine-inhibitable uptake by hepatoma cells. In rodent HCC, Oct1 was downregulated, which was accompanied by impaired sorafenib uptake. In mice with s.c.-implanted HCC, sorafenib inhibited the growth of hOCT1 overexpressing tumours. In human HCC, hOCT1 expression was inversely correlated with SLC22A1 promoter methylation, whereas demethylation with decitabine enhanced hOCT1 expression in hepatoma cells. Increased proportion of aberrant hOCT1 mRNA variants was found in HCC samples. In silico analysis identified six miRNAs as candidates to target hOCT1 mRNA. When overexpressed in HepG2 cells a significant hOCT1 mRNA decay was induced by hsa-miR-330 and hsa-miR-1468. Analysis of 39 paired tumour/adjacent samples from TCGA revealed that hsa-mir-330 was consistently upregulated in HCC. Impaired hOCT1 expression/function in HCC, in part due to epigenetic modifications, plays an important role in the poor pharmacological response of this cancer to sorafenib.

Upregulation of miR-489-3p and miR-630 inhibits oxaliplatin uptake in renal cell carcinoma by targeting OCT2

Renal cell carcinoma (RCC) is one of the most common malignant tumors affecting the urogenital system, accounting for 90% of renal malignancies. Traditional chemotherapy options are often the front-line choice of regimen in the treatment of patients with RCC, but responses may be modest or limited due to resistance of the tumor to anticarcinogen. Downregulated expression of organic cation transporter OCT2 is a possible mechanism underlying oxaliplatin resistance in RCC treatment. In this study, we observed that miR-489-3p and miR-630 suppress OCT2 expression by directly binding to the OCT2 3′-UTR. Meanwhile, via 786-O-OCT2-miRNAs stable expression cell models, we found that miRNAs could repress the classic substrate 1-methyl-4-phenylpyridinium (MPP+), fluorogenic substrate N,N-dimethyl-4-(2-pyridin-4-ylethenyl) aniline (ASP+), and oxaliplatin uptake by OCT2 both in vitro and in xenografts. In 33 clinical samples, miR-489-3p and miR-630 were significantly upregulated in RCC, negatively correlating with the OCT2 expression level compared to that in adjacent normal tissues, using tissue microarray analysis and qPCR validation. The increased binding of c-Myc to the promoter of pri-miR-630, responsible for the upregulation of miR-630 in RCC, was further evidenced by chromatin immunoprecipitation and dual-luciferase reporter assay. Overall, this study indicated that miR-489-3p and miR-630 function as oncotherapy-obstructing microRNAs by directly targeting OCT2 in RCC.

Evaluation of MUC-1, MUC-4, CDX-2 and OCT-1 expression profiles in gastric carcinomas and precancerous lesions

Aim: We investigated the diagnostic and prognostic utility of MUC-1, MUC-4, CDX2 and OCT-1 expression profiles in high-mortality gastric carcinomas and their precancerous lesions.Material and Methods: Areas in proximity of dysplasias and gastric carcinomas that were suspected of having intestinal metaplasia were stained with PAS / Alcian Blue at pH 2.5 and pH 0.5; Giemsa staining was applied for evaluation of presence of H. Pylori.Results: Presence of MUC-1 expression may exclude the diagnosis of low-grade dysplasia. MUC-1 and MUC-4 overexpression may be used for determination of groups at high risk of development of carcinomas, primarily in cases of high-grade dysplasia, while overexpression of MUC-1 in gastric carcinomas may be used as a favorable prognostic factor. Presence of CDX2 expression in various histological types of carcinomas gives rise to a suggestion that intestinal differentiation occurs in all histological types at least to some degree. OCT-1 plays a role in CDX2-mediated carcinogenesis and its regulation. Conclusion: In cases of dysplasia occurring with intestinal metaplasia, CDX2 was found to be expressed at a significantly higher degree (95.5%, p=0.011). Expression of CDX2 and OCT-1 was observed in all high-grade dysplasia cases, and was significantly higher in comparison to low-grade dysplasia cases (p=0.042; p=0.006, respectively). MUC-1 and MUC-4 expression significantly increased with each progressing stage of precancerous lesions in all lesions following low-grade dysplasia (p=0.0001; p=0.027, respectively). MUC-1 expression was the lowest (53.6%) in weak cohesive type carcinomas. Positive immunoreactivity with MUC-1 and CDX2 was higher in cases with a smaller number of metastatic lymph nodes compared to the cases with a larger number thereof.

Effect of high uric acid on the disposition of metformin: in vivo and in vitro studies.

Metformin is always used as the baseline antidiabetic therapy for patients with type 2 diabetes mellitus (T2DM) and hyperuricemia. Metformin is excreted into urine through active secretion mediated by rOCTs and rMATE1.The aim of this study was to identify the effects of high uric acid on the disposition and its mechanism. For the in vivo study, a hyperuricemic animal model was induced by intraperitoneal injection of potassium oxonate (250mg/kg) in rats. Metformin (100mg/kg) was administered orally to investigate the pharmacokinetics in control and hyperuricemic rats, respectively. For the in vitro study, HEK293 and HepaRG cells were used to investigate the effect of uric acid (15mg/dl) on the expression of OCT1, OCT2 and MATE1 and the disposition of metformin, respectively. The in vivo study showed that the AUC0→600 of metformin was significantly decreased by 33.3%, whereas the cumulative urinary excretion of metformin was increased by 25.4% in hyperuricemic rats compared with that in control rats. The renal rOCT1, rOCT2 and rMATE1 and hepatic rMATE1 levels were increased in hyperuricemic rats compared with those in control rats, respectively. The in vitro study showed that uric acid could upregulate the expression of OCT2 and MATE1 in HEK293 cells and MATE1 in HepaRG cells and increase the intracellular metformin concentration in these two cell lines. These results demonstrated that a high uric acid level promoted urinary metformin excretion and decreased the plasma metformin concentration; the in vivo and in vitro studies provided a possible explanation being that high uric acid could upregulate the expression of renal metformin transporters OCTs and MATE1.

Impact of Promoter Polymorphisms on the Transcriptional Regulation of the Organic Cation Transporter OCT1 (SLC22A1).

The organic cation transporter 1 (OCT1, SLC22A1) is strongly expressed in the human liver and facilitates the hepatic uptake of drugs such as morphine, metformin, tropisetron, sumatriptan and fenoterol and of endogenous substances such as thiamine. OCT1 expression is inter-individually highly variable. Here, we analyzed SNPs in the OCT1 promoter concerning their potential contribution to the variability in OCT1 expression. Using electrophoretic mobility shift and luciferase reporter gene assays in HepG2, Hep3B, and Huh7 cell lines, we identified the SNPs −1795G>A (rs6935207) and −201C>G (rs58812592) as having effects on transcription factor binding and/or promoter activity. The A-allele of the −1795G>A SNP showed allele-specific binding of the transcription factor NF-Y leading to 2.5-fold increased enhancer activity of the artificial SV40 promoter. However, the −1795G>A SNP showed no significant effects on the native OCT1 promoter activity. Furthermore, the −1795G>A SNP was not associated with the pharmacokinetics of metformin, fenoterol, sumatriptan and proguanil in healthy individuals or tropisetron efficacy in patients undergoing chemotherapy. Allele-dependent differences in USF1/2 binding and nearly total loss in OCT1 promoter activity were detected for the G-allele of −201C>G, but the SNP is apparently very rare. In conclusion, common OCT1 promoter SNPs have only minor effects on OCT1 expression.

Mitigation of cell apoptosis induced by ochratoxin A (OTA) is possibly through organic cation transport 2 (OCT2) knockout

Ochratoxin A (OTA) is a secondary metabolite of fungi such as Aspergillus ochraceus, A. niger and A. carbonarius, Penicillium verrucosum, and various other Penicillium, Petromyces, and Neopetromyces species. Various foods can be contaminated with OTA, potentially causing several toxic effects such as nephrotoxicity, hepatotoxicity and neurotoxicity. Typically, OTA is excreted by organic anion transporters (OATs). There is no research indicating organic cation transporters (OCTs) are involved in OTA nephrotoxicity.In our study, NRK-52E cells and rats were treated with OTA. OTA changed the expression of OCT1, OCT2 and OCT3 in NRK-52E cells and rat kidneys. TEA alleviated OTA-induced cell death, apoptosis, and DNA damage, and increased ROS. The OCT2 knockout cell line was constructed by the CRISPR/Cas 9 system. OCT2 knockout did not change the gene expression of OCT1, OAT1 and OAT3. OCT2 knockout alleviated the increase of Caspase 3 and CDK1 induced by OTA, leading to a reduction of apoptosis. In addition, OCT2 overexpression increased cell toxicity and expression of Caspase 3. In short, our findings indicate that OCT2 knockout possibly mitigate OTA-induced apoptosis by preventing the increase of Caspase 3 and CDK1.

The Importance of Incorporating OCT2 Plasma Membrane Expression and Membrane Potential in IVIVE of Metformin Renal Secretory Clearance.

Transporter expression, determined by quantitative proteomics, together with PBPK models is a promising approach for in vitro-to-in vivo extrapolation (IVIVE) of transporter-mediated drug clearance. OCT2-expressing HEK293 and MDCKII cells were used to predict in vivo renal secretory clearance (CLr,sec) of metformin. [14C]-Metformin uptake clearance in OCT2-expressing cells was determined and scaled to in vivo CLr,sec by using OCT2 expression in the cells versus the human kidney cortex. Through quantitative targeted proteomics, the total expression of OCT2 in HEK293, MDCKII cells, and human kidney cortex was 369.4 ± 26.8, 19 ± 1.1, and 7.6 ± 3.8 pmol/mg cellular protein, respectively. The expression of OCT2 in the plasma membrane of HEK293 and MDCKII cells, measured using an optimized biotinylation method followed by quantitative proteomics, was 30.2% and 51.6%, respectively. After correcting for percent of OCT2 expressed in the plasma membrane and the resting membrane potential (millivolts) difference between the OCT2-expressing cells and the renal epithelial cells, the predicted CLr,sec of metformin was 250.7 ml/min, a value within the range of the observed CLr,sec of metformin. These data demonstrate the promise of using quantitative proteomics for IVIVE of transporter-mediated drug clearance and highlight the importance of quantifying plasma membrane expression of transporters and utilizing cells that mimic the in vivo mechanism(s) of transport of drugs.

Involvement of organic cation transporter 2 in the metformin-associated increased lactate levels caused by contrast-induced nephropathy

The application of iodinated contrast medium has become a risk factor for metformin-associated lactic acidosis (MALA), which leads to the accumulation of metformin in vivo is one of the principal reasons for MALA. However, the molecular mechanism of the adverse event is not yet clear. In this study, iohexol injection was used as a model drug. The contrast agent acute kidney injury rat model, in vivo rat pharmacokinetic study, kidney slices and HK-2 cells were performed to elucidate the pharmacokinetic molecular mechanism of accumulation of metformin caused by contrast-induced nephropathy (CIN). Plasma exposure of metformin was increased significantly in the CIN group compared with that in the normal and control groups. The AUC of metformin was from 2791 ± 382 μg min mL−1 to 4784 ± 767 μg min mL−1. The cumulative urinary excretion of metformin was also reduced in the CIN group. The uptake of metformin decreased in kidney slices in the CIN group. Compared with the normal and control groups, the blood lactate concentration was increased after intravenous administration of metformin in the CIN group followed a similar trend to the plasma concentrations of metformin. After treatment with contrast medium, the expression of OCT2 was reduced in rat kidney and HK-2 cells. These findings highlight that OCT2 deficiency was associated with increased lactate levels during metformin treatment caused by CIN.

Endotoxin Modulates the Expression of Renal Drug Transporters in HIV-1 Transgenic Rats

Bacterial co-infections and low grade endotoxemia are common in HIV patients. Inflammation due to endotoxin or HIV may influence the expression and activity of transporters. Kidney transporters influence renal drug clearances including many antiretroviral agents. Our objective was to study the effect of endotoxin and HIV on the renal expression of drug transporters in an HIV-transgenic (HIV-Tg) rat model. These rats develop immune dysfunction and AIDS-associated conditions like humans. Endotoxin or saline was administered intraperitoneally to HIV-Tg or wild type (WT) littermates and kidneys were collected 18 hours later. Expression of transporters and cytokines were measured by qRT-PCR and Western blots. Serum cytokine levels were measured by ELISA. Endotoxin induced serum levels of IL-6, TNF-α and IFN-γ in both HIV-Tg and WT animals. The basal mRNA expression of Oct2, Oct3, Octn1, Mate1, Urat1 and Ent1was significantly lower (33-60%) and the expression of Ent2 and Pept2 was significantly higher (33-45%) in HIV-Tg as compared to WT. While endotoxin significantly downregulated the mRNA expression of Mdra1 and Pept2 in both HIV and WT groups (69-78%), it imposed a significant reduction on the mRNA expression of Oct2, Oct3, Octn1, Mate1, Oat2, urat1, and Ent1 (54-83%) only in the WT group. Endotoxin significantly increased the mRNA expression of Pept1 (140%) in both WT and HIV groups. HIV and endotoxin each imposed alterations in the expression of many clinically important renal drug transporters although co-infection did not augment this effect. Viral and/or bacterial infections may impact the renal clearance of drug substrates in patients and could potentially be a source of drug-disease interactions.

Metformin targets brown adipose tissue in vivo and reduces oxygen consumption in vitro.

To test the hypothesis that brown adipose tissue (BAT) is a metformin target tissue by investigating in vivo uptake of [11 C]-metformin tracer in mice and studying in vitro effects of metformin on cultured human brown adipocytes. Tissue-specific uptake of metformin was assessed in mice by PET/CT imaging after injection of [11 C]-metformin. Human brown adipose tissue was obtained from elective neck surgery and metformin transporter expression levels in human and murine BAT were determined by qPCR. Oxygen consumption in metformin-treated human brown adipocyte cell models was assessed by Seahorse XF technology. Injected [11 C]-metformin showed avid uptake in the murine interscapular BAT depot. Metformin exposure in BAT was similar to hepatic exposure. Non-specific inhibition of the organic cation transporter (OCT) protein by cimetidine administration eliminated BAT exposure to metformin, demonstrating OCT-mediated uptake. Gene expression profiles of OCTs in BAT revealed ample OCT3 expression in both human and mouse BAT. Incubation of a human brown adipocyte cell models with metformin reduced cellular oxygen consumption in a dose-dependent manner. These results support BAT as a putative metformin target.

Boron enhances early embryonic gene expressions and improves fetal development of rats

Boron is present as several different components in nature. Besides its industrial use, it is an essential element and is playing a very important role in the metabolism. In this study, it was aimed to determine the in vivo effects of boron on mRNA expression of HEX, NANOG, and OCT-3/4 genes in embryo and histological changes during fetal development. Therefore, totally 60 female rats were allocated into 5 equal groups. Experimental groups are as the followings; positive control (fed with standart rat diet), negative control (fed with boron free diet), low boron group (fed with boron free diet and given 0.04 μg boron/ml via gastric gavage), marginal boron group (fed with boron free diet and given 0.3 μg boron/ml via gastric gavage) and normal boron group (fed with boron free diet and given 2 μg boron/ml via gastric gavage). Experimental period was performed for 14 days. Embryos were collected after 4 days of mating and the expression and protein levels of early embryonic genes namely HEX, NANOG, and OCT-3/4 were determined by using Real-Time PCR. Also, 10–20 day embryo and fetus development were histologically determined. According to the results, mRNA expression and protein levels of early embryonic genes were increased in boron groups while decreased in boron deficient group. Histopathologically, tissue and organ developments were definitely observed in the boron groups. In conclusion, mRNA expression levels of early embryonic genes decreased in boron deficient group and boron has an important role for fetal development.