OCTN2 mRNA Research Articles

Protective Effect of Ergothioneine against 7-Ketocholesterol-Induced Mitochondrial Damage in hCMEC/D3 Human Brain Endothelial Cells.

Recent findings have suggested that the natural compound ergothioneine (ET), which is synthesised by certain fungi and bacteria, has considerable cytoprotective potential. We previously demonstrated the anti-inflammatory effects of ET on 7-ketocholesterol (7KC)-induced endothelial injury in human blood-brain barrier endothelial cells (hCMEC/D3). 7KC is an oxidised form of cholesterol present in atheromatous plaques and the sera of patients with hypercholesterolaemia and diabetes mellitus. The aim of this study was to elucidate the protective effect of ET on 7KC-induced mitochondrial damage. Exposure of human brain endothelial cells to 7KC led to a loss of cell viability, together with an increase in intracellular free calcium levels, increased cellular and mitochondrial reactive oxygen species, a decrease in mitochondrial membrane potential, reductions in ATP levels, and increases in mRNA expression of TFAM, Nrf2, IL-1β, IL-6 and IL-8. These effects were significantly decreased by ET. Protective effects of ET were diminished when endothelial cells were coincubated with verapamil hydrochloride (VHCL), a nonspecific inhibitor of the ET transporter OCTN1 (SLC22A4). This outcome demonstrates that ET-mediated protection against 7KC-induced mitochondrial damage occurred intracellularly and not through direct interaction with 7KC. OCTN1 mRNA expression itself was significantly increased in endothelial cells after 7KC treatment, consistent with the notion that stress and injury may increase ET uptake. Our results indicate that ET can protect against 7KC-induced mitochondrial injury in brain endothelial cells.

PGC-1α and MEF2 Regulate the Transcription of the Carnitine Transporter OCTN2 Gene in C2C12 Cells and in Mouse Skeletal Muscle.

OCTN2 (SLC22A5) is a carnitine transporter whose main function is the active transport of carnitine into cells. In skeletal muscle and other organs, the regulation of the SLC22A5 gene transcription has been shown to depend on the nuclear transcription factor PPAR-α. Due to the observation that the muscle OCTN2 mRNA level is maintained in PPAR-α knock-out mice and that PGC-1α overexpression in C2C12 myoblasts increases OCTN2 mRNA expression, we suspected additional regulatory pathways for SLC22A5 gene transcription. Indeed, we detected several binding sites of the myocyte-enhancing factor MEF2 in the upstream region of the SLC22A5 gene, and MEF2C/MEF2D stimulated the activity of the OCTN2 promoter in gene reporter assays. This stimulation was increased by PGC-1α and was blunted for a SLC22A5 promoter fragment with a mutated MEF2 binding site. Further, we demonstrated the specific binding of MEF2 to the SLC22A5 gene promoter, and a supershift of the MEF2/DNA complex in electrophoretic mobility shift assays. In immunoprecipitation experiments, we could demonstrate the interaction between PGC-1α and MEF2. In addition, SB203580, a specific inhibitor of p38 MAPK, blocked and interferon-γ stimulated the transcriptional activity of the SLC22A5 gene promoter. Finally, mice with muscle-specific overexpression of OCTN2 showed an increase in OCTN2 mRNA and protein expression in skeletal muscle. In conclusion, we detected and characterized a second stimulatory pathway of SLC22A5 gene transcription in skeletal muscle, which involves the nuclear transcription factor MEF2 and co-stimulation by PGC-1α and which is controlled by the p38 MAPK signaling cascade.

Carnitine uptake defect due to a 5′UTR mutation in a pedigree with false positives and false negatives on Newborn screening

Carnitine Uptake Defect (CUD) is an autosomal recessive disorder due to mutations in the SLC22A5 gene. Classically patients present in infancy with profound muscle weakness and cardiomyopathy with characteristic EKG findings. Later presentations include recurrent hypoketotic hypoglycemia, proximal limb girdle myopathy,and/or recurrent muscle pain. Newborn screening detects most of these clinical variants but in addition has identified maternal CUD often in asymptomatic women. We describe a family ascertained through 3 newborn screening (NBS) positive infants found to be unaffected themselves but in whom the mothers (sisters) were affected. There were also two affected children born to an affected male and his heterozygous wife who were false negatives on NBS but had increased fractional excretion of free carnitine in the urine. Analysis on a Next Generation Sequencing panel specifically designed to fully cover newborn screening disease targets showed a homozygous change in the five probands (SLC22A5; NM_003060:c.-149G > A; p.?). The mutation segregates with the CUD within the family. It is in the 5′ UTR and has a frequency within the gnomAd database of 0.001198. Plasma carnitine was decreased and fractional excretion of free carnitine was increased in all affected individuals. Functional carnitine uptake studies in cultured skin fibroblasts of one proband showed carnitine uptake at the 5 μM concentration to be 6% of controls. Relative expression of OCTN2 mRNA to beta-actin mRNA by qRT-PCR was increased in a proband relative to controls by a factor of 465-fold. Western blotting revealed a 120 kDa protein band, as well as a weaker 240 kDa band in the proband, the significance of which is unknown at this time.

Developmental Regulation of Drug-Processing Genes in Livers of Germ-Free Mice.

Very little is known about the effect of gut microbiota on the ontogeny of drug-processing genes (DPGs) in liver. In this study, livers were harvested from conventional (CV) and germ-free (GF) male and female mice from 1 to 90 days of age. RNA-Seq in livers of 90-day-old male mice showed that xenobiotic metabolism was the most downregulated pathway within the mRNA transcriptome in absence of intestinal bacteria. In male livers, the mRNAs of 67 critical DPGs partitioned into 4 developmental patterns (real-time-quantitative polymerase chain reaction): Pattern-1 gradually increased to adult levels in livers of CV mice and were downregulated in livers of GF mice, as exemplified by the major drug-metabolizing enzymes cytochrome 3a (Cyp3a) family, which are prototypical pregnane X receptor (PXR)-target genes. Genes in Pattern-2 include Cyp1a2 (aryl hydrocarbon receptor-target gene), Cyp2c family, and Cyp2e1, which were all upregulated mainly at 90 days of age; as well as the peroxisome proliferator-activated receptor α (PPARα)-target genes Cyp4a family and Aldh3a2, which were upregulated not only in 90-days adult age, but also between neonatal and adolescent ages (from 1 to 30 days of age). Genes in Pattern-3 were enriched predominantly in livers of 15-day-old mice, among which the sterol-efflux transporter dimers Abcg5/Abcg8 were downregulated in GF mice. Genes in Pattern-4 were neonatal-enriched, among which the transporter Octn1 mRNA tended to be lower in GF mice at younger ages but higher in adult GF mice as compared with age-matched CV mice. Protein assays confirmed the downregulation of the PXR-target gene Cyp3a protein (Western-blot and liquid chromatography tandem mass spectroscopy), and decreased Cyp3a enzyme activities in male GF livers. Increased microsomal-Cyp4a proteins and nuclear-PPARα were also observed in male GF livers. Interestingly, in contrast to male livers, the mRNAs of Cyp2c or Cyp4a were not readily upregulated in female GF livers approaching adult age, suggesting the maturation of female-specific hormones interferes with the interactions between intestinal microbiota and DPG ontogeny. In conclusion, intestinal microbiota markedly impacts the ontogeny of many hepatic DPGs in a gender-specific manner.

Reduced L-Carnitine Transport in Aortic Endothelial Cells from Spontaneously Hypertensive Rats

Impaired L-carnitine uptake correlates with higher blood pressure in adult men, and L-carnitine restores endothelial function in aortic rings from spontaneously hypertensive rat (SHR). Thus, endothelial dysfunction in hypertension could result from lower L-carnitine transport in this cell type. L-Carnitine transport is mainly mediated by novel organic cation transporters 1 (Octn1, Na+-independent) and 2 (Octn2, Na+-dependent); however, their kinetic properties and potential consequences in hypertension are unknown. We hypothesize that L-carnitine transport kinetic properties will be altered in aortic endothelium from spontaneously hypertensive rats (SHR). L-Carnitine transport was measured at different extracellular pH (pHo 5.5–8.5) in the absence or presence of sodium in rat aortic endothelial cells (RAECs) from non-hypertensive Wistar-Kyoto (WKY) rats and SHR. Octn1 and Octn2 mRNA relative expression was also determined. Dilation of endothelium-intact or denuded aortic rings in response to calcitonine gene related peptide (CGRP, 0.1–100 nmol/L) was measured (myography) in the absence or presence of L-carnitine. Total L-carnitine transport was lower in cells from SHR compared with WKY rats, an effect due to reduced Na+-dependent (Na+ dep) compared with Na+-independent (Na+ indep) transport components. Saturable L-carnitine transport kinetics show maximal velocity (V max), without changes in apparent K m for Na+ indep transport in SHR compared with WKY rats. Total and Na+ dep component of transport were increased, but Na+ indep transport was reduced by extracellular alkalization in WKY rats. However, alkalization reduced total and Na+ indep transport in cells from SHR. Octn2 mRNA was higher than Octn-1 mRNA expression in cells from both conditions. Dilation of artery rings in response to CGRP was reduced in vessels from SHR compared with WKY rats. CGRP effect was endothelium-dependent and restored by L-carnitine. All together these results suggest that reduced L-carnitine transport (likely via Na+-dependent Octn2) could limit this compound's potential beneficial effects in RAECs from SHR.

Functional Expression of Organic Cation/carnitine Transporter 2 (OCTN2/SLC22A5) in Human Brain Capillary Endothelial Cell Line hCMEC/D3, a Human Blood-brain Barrier Model

The aim of this study was to examine whether organic cation/carnitine transporter 2 (OCTN2/SLC22A5) plays a role in the human blood-brain barrier (BBB) by evaluating its functional activity in human brain endothelial cells (hCMEC/D3), which are considered to be a model of the BBB. The uptake of [(3)H]L-carnitine by hCMEC/D3 cells was time-, extracellular sodium- and concentration-dependent, with a Km value of 15.7 µM. These results are consistent with the properties of OCTN2-mediated L-carnitine transport. hCMEC/D3 cells showed relatively high expression of OCTN2 mRNA, and this expression was effectively decreased at 24-72 h after lipofection of cells with OCTN2 siRNA under optimized conditions. [(3)H]L-Carnitine uptake was dramatically suppressed by silencing of the OCTN2 gene. The inhibitory effect of OCTN2 gene silencing was similar to that of an excess amount of unlabeled L-carnitine. These results indicate that OCTN2 is involved in L-carnitine transport at the human BBB.

Functional expression of carnitine/organic cation transporter OCTN1 in mouse brain neurons: Possible involvement in neuronal differentiation

The aim of the present study is to clarify the functional expression and physiological role in brain neurons of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring antioxidant ergothioneine (ERGO) as a substrate in vivo. After intracerebroventricular administration, the distribution of [3H]ERGO in several brain regions of octn1−/− mice was much lower than that in wild-type mice, whereas extracellular marker [14C]mannitol exhibited similar distribution in the two strains. The [3H]ERGO distribution in wild-type mice was well correlated with the amount of ERGO derived from food intake and the OCTN1 mRNA level in each brain region. Immunohistochemical analysis revealed colocalization of OCTN1 with neuronal cell markers microtubule-associated protein 2 (MAP2) and βIII-tubulin in mouse brain and primary cultured cortical neurons, respectively. Moreover, cultured cortical neurons exhibited time-dependent and saturable uptake of [3H]ERGO. These results demonstrate that OCTN1 is functionally expressed in brain neurons. The addition of ERGO simultaneously with serum to culture medium of cortical neurons attenuated mRNA and protein expressions of MAP2, βIII-tubulin and synapse formation marker synapsin I, and induced those of sex determining region Y-box 2 (Sox2), which is required to maintain the properties of undifferentiated neural stem cells. In neuronal model Neuro2a cells, knockdown of OCTN1 by siRNA reduced the uptake of [3H]ERGO with concomitant up-regulation of oxidative stress marker HO-1 and Sox2, and down-regulation of neurite outgrowth marker GAP43. Interestingly, the siRNA knockdown decreased the number of differentiated Neuro2a cells showing long neurites, but increased the total number of cells. Thus, OCTN1 is involved in cellular differentiation, but inhibits their proliferation, possibly via the regulation of cellular oxidative stress. This is the first evidence that OCTN1 plays a role in neuronal differentiation and proliferation, which are required for brain development.

Human OCTN2 (SLC22A5) is down‐regulated in virus‐ and nonvirus‐mediated cancer

The expression of carnitine plasma membrane transporter OCTN2 was evaluated in virus and nonvirus-mediated cancer. Both OCTN2 mRNA and protein levels were reduced in keratinocytes retrotransduced with HPV16 E6 and E7 compared with the control. The OCTN2 expression was reduced also in keratinocytes retrotransduced with the sole HPV16 E6. A similar down-regulation of OCTN2 mRNA level was observed in a naturally HPV16-infected cancer cell line, CaSki, harbouring several copies of HPV16 whole genome. The mechanism of down-regulation is not related to p53 transcriptional activity because in SAOS (p53-null) cell line, the restoration of p53 expression did not rescue OCTN2 expression. The treatment of keratinocytes retrotransduced with HPV16 E6 and E7 with 5-aza-cytidine rescued the OCTN2 expression, indicating that the mechanism of down-regulation is linked to DNA methylation. Low levels of mRNA expression of OCTN2 were found also in several nonvirus-related epithelial cancer cell lines. The treatment of those cell lines with 5-aza-cytidine again rescued the expression of OCTN2 as well. These data demonstrate for the first time that the OCTN2 transporter is generally down-regulated in virus and nonvirus-mediated epithelial cancers, probably via methylation of its promoter region.

Inhibition of Gene Expression of Organic Cation/Carnitine Transporter and Antioxidant Enzymes in Oxazaphosphorines-Induced Acute Cardiomyopathic Rat Models

It is well documented that high therapeutic doses of oxazaphosphorines, cyclophosphamide (CP) and ifosfamide (IFO), are associated with cardiomyopathy. This study investigated whether oxazaphosphorines alter the expression of organic cation/carnitine transporter (OCTN2) and antioxidant genes and if so, whether these alterations contribute to CP and IFO-induced cardiotoxicity. Adult male Wistar albino rats were assigned to one of six treatment groups namely, control, L carnitine, CP, IFO, CP plus L carnitine and IFO plus L carnitine. In cardiac and kidney tissues, CP and IFO significantly decreased mRNA and protein expression of OCTN2. Oxazaphosphorines significantly increased serum acyl-carnitine/free carnitine ratio and urinary carnitine excretion and significantly decreased total carnitine in cardiac tissues. Interestingly, carnitine supplementation completely reversed the biochemical and gene expression changes-induced by oxazaphosphorines to the control values, except OCTN2 expression remained inhibited by IFO. Data from this study suggest that: (1) Oxazaphosphorines decreased myocardial carnitine content following the inhibition of OCTN2 mRNA and protein expression in cardiac tissues. (2) Oxazaphosphorine therapy increased urinary loss of carnitine secondary to the inhibition of OCTN2 mRNA and protein expression in proximal tubules of the kidney. (3) Carnitine supplementation attenuates CP but not IFO-induced inhibition of OCTN2 mRNA and protein expression in heart and kidney tissues.

Abstract 4391: OCTN1- and OCTN2-mediated uptake of oxaliplatin in rat dorsal root ganglion neurons and HEK293 cells over-expressing OCTN1 or OCTN2

Abstract Purpose: Organic cation transporters (OCTs) have been recently implicated in the uptake and toxicity of oxaliplatin. Peripheral neuropathy is the dose-limiting toxicity of oxaliplatin due to accumulation of platinum in the dorsal root ganglia (DRG) and damage to sensory neurons. This study sought to determine the role of OCTs in the DRG neuronal uptake of oxaliplatin. Experimental Design: [14C] Oxaliplatin uptake and cytotoxicity in HEK/rOctn1, HEK/rOctn2, HEK/hOCTN1, HEK/hOCTN2, and primary culture of DRG neurons was determined to evaluate the role of Octn1 and Octn2 in oxaliplatin transport and neurotoxicity. mRNA, protein and functional expression of organic cation transporters in rat lumbar DRG were characterised using RT-PCR, Western blotting and uptake of model OCT substrates [3H] MPP, [14C] TEA, [3H] ergothioneine and [3H] L-carnitine. Results: HEK293 cells over-expressing rOctn1, rOctn2, hOCTN1 and hOCTN2 showed increased uptake and cytotoxicity of oxaliplatin compared to Mock-transfected HEK293 controls, and Octn2-mediated uptake of oxaliplatin was significantly higher than Octn1 in both human and rat. Uptake of ergothioneine mediated by Octn1, and L-carnitine mediated by Octn2, was decreased during oxaliplatin exposure. Rat DRG showed high Octn1 and Octn2 mRNA levels, and uptake of ergothioneine and L-carnitine that was temperature- and sodium-dependent and significantly inhibited by model substrates of Octn1 and Octn2. In contrast, DRG had negligible functional activity and low or undetectable mRNA levels of Oct1, Oct2, and Oct3. [14C] Oxaliplatin uptake by DRG neurons was temperature- and sodium-dependent and inhibited by ergothioneine and L-carnitine. Ergothioneine and L-carnitine uptake by DRG neurons was inhibited in the presence of oxaliplatin. Conclusions: In conclusion, Octn1 and Octn2, but not Oct1, Oct2 or Oct3, are functionally expressed in rat DRG neurons. Oxaliplatin uptake by rat DRG neurons and HEK293 cells was mediated by Octn2 and to lesser extent by Octn1. Supported by Grant-in-Aid for Scientific Research (B) (I. T.), Research Activity Start-up (T. N.), Auckland Medical Research Foundation (Doctoral Scholarship) and Cancer Society of New Zealand (Research Grant). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4391. doi:10.1158/1538-7445.AM2011-4391

IL23R, ATG16L1, IRGM, OCTN1, and OCTN2 mRNA expression in inflamed and noninflamed mucosa of IBD patients

IL23R, ATG16L1, IRGM, OCTN1, and OCTN2 mRNA expression in inflamed and noninflamed mucosa of IBD patients

Hypoxia-Mediated Down-Regulation of OCTN2 and PPARα Expression in Human Placentas and in BeWo Cells

The objective of the present study was to investigate the effects of hypoxia on placental expression of OCTN2 and PPARα. OCTN2 and PPARα expression in the human placenta in the presence or absence of preeclampsia was examined by immunohistochemical (IHC) analysis, Western blotting, and quantitative polymerase chain reaction (qPCR). Effects of hypoxia on the expression of OCTN2 and PPARα in human placental explants and human choriocarcinoma BeWo cells were examined by Western blotting and qPCR analyses. IHC, Western blot, and qPCR studies showed that OCTN2 and PPARα protein and mRNA levels were lower in syncytiotrophoblasts from preeclamptic human placentas than in those from normal placentas. Hypoxic treatment caused a decrease in OCTN2 and PPARα expression in human placental explants and in BeWo cells. WY14643, a PPARα agonist, caused an increase in OCTN2 expression in BeWo cells under hypoxic conditions. In conclusion, under hypoxic conditions, placental OCTN2 is down-regulated through PPARα-mediated pathways.

Timing and Duration of Drug Exposure Affects Outcomes of a Drug-Nutrient Interaction During Ontogeny.

Significant drug-nutrient interactions are possible when drugs and nutrients share the same absorption and disposition mechanisms. During postnatal development, the outcomes of drug-nutrient interactions may change with postnatal age since these processes undergo ontogenesis through the postnatal period. Our study investigated the dependence of a significant drug-nutrient interaction (cefepime-carnitine) on the timing and duration of drug exposure relative to postnatal age. Rat pups were administered cefepime (5 mg/kg) twice daily subcutaneously according to different dosing schedules (postnatal day 1-4, 1-8, 8-11, 8-20, or 1-20). Cefepime significantly reduced serum and heart L-carnitine levels in postnatal day 1-4, 1-8 and 8-11 groups and caused severe degenerative changes in ventricular myocardium in these groups. Cefepime also altered the ontogeny of several key L-carnitine homeostasis pathways. The qualitative and quantitative changes in levels of hepatic γ-butyrobetaine hydroxylase mRNA and activity, hepatic trimethyllysine hydroxlase mRNA, intestinal organic cation/carnitine transporter (Octn) mRNA, and renal Octn2 mRNA depended on when during postnatal development the cefepime exposure occurred and duration of exposure. Despite lower levels of heart L-carnitine in earlier postnatal groups, levels of carnitine palmitoyltransferase mRNA and activity, heart Octn2 mRNA and ATP levels in all treatment groups remained unchanged with cefepime exposure. However, changes in other high energy phosphate substrates were noted and reductions in the phosphocreatine/ATP ratio were found in rat pups with normal serum L-carnitine levels. In summary, our data suggest a significant drug-nutrient transport interaction in developing neonates, the nature of which depends on the timing and duration of exposure relative to postnatal age.

Inhibition of gene expression of heart fatty acid binding protein and organic cation/carnitine transporter in doxorubicin cardiomyopathic rat model

This study examined whether doxorubicin therapy alters the expression of heart fatty acid binding protein (H-FABP) and organic cation/carnitine transporter (OCTN2) genes in cardiac tissues, and if so, whether these alterations contributes to doxorubicin-induced cardiotoxicity. Male Wistar albino rats were divided into six groups: group 1 rats were given daily intraperitoneal (i.p.) injections of normal saline for 10 consecutive days; groups 2, 3 and 4 rats were injected every other day with doxorubicin (3 mg/kg, i.p.), to obtain treatments with cumulative doses of 6, 12, and 18 mg/kg. Rats in the fifth group were injected with l-carnitine (200 mg/kg, i.p.) for 10 consecutive days. Animals in the sixth group received doxorubicin (18 mg/kg) and l-carnitine (200 mg/kg). Treatment with doxorubicin resulted in a significant and dose-dependent decrease in H-FABP and OCTN2 mRNA expression, total carnitine and ATP in cardiac tissues and a significant increase in cardiac enzymes. Moreover, doxorubicin treatment showed significant and dose-dependent increase in the expression of apoptotic genes namely P53 and CD95. Interestingly, carnitine supplementation restored doxorubicin-induced inhibition of gene expression of H-FABP and OCTN2, decrease in myocardial carnitine and ATP to the control values. In conclusion, data from this study suggest that: chronic doxorubicin therapy decreased the expression of H-FABP and OCTN2 mRNA expression in cardiac tissues. The progressive increase in cardiotoxicity enzymatic indices and the decrease in H-FABP and OCTN2 expression may point to the possible contribution of H-FABP and OCTN2 as a mechanism during development of doxorubicin cardiotoxicity.

The plasma carnitine concentration regulates renal OCTN2 expression and carnitine transport in rats

Previous findings in rats and in human vegetarians suggest that the plasma carnitine concentration and/or carnitine ingestion may influence the renal reabsorption of carnitine. We tested this hypothesis in rats with secondary carnitine deficiency following treatment with N-trimethyl-hydrazine-3-propionate (THP) for 2 weeks and rats treated with excess l-carnitine for 2 weeks. Compared to untreated control rats, treatment with THP was associated with an approximately 70% decrease in plasma carnitine and with a 74% decrease in the skeletal muscle carnitine content. In contrast, treatment with l-carnitine increased plasma carnitine levels by 80% and the skeletal muscle carnitine content by 50%. Treatment with l-carnitine affected neither the activity of carnitine transport into isolated renal brush border membrane vesicles, nor renal mRNA expression of the carnitine transporter OCTN2. In contrast, in carnitine deficient rats, carnitine transport into isolated brush border membrane vesicles was increased 1.9-fold compared to untreated control rats. Similarly, renal mRNA expression of OCTN2 increased by a factor of 1.7 in carnitine deficient rats, whereas OCTN2 mRNA expression remained unchanged in gut, liver or skeletal muscle. Our study supports the hypothesis that a decrease in the carnitine plasma and/or glomerular filtrate concentration increases renal expression and activity of OCTN2.

P.62 INFLIXIMAB MODULATES MUCOSAL CYTOKINE PROFILE IN PATIENTS WITH INFLAMMATORY BOWEL DISEASES

s / Digestive and Liver Disease 42S (2010) S61–S192 S125 was significantly reduced compared to non-inflamed areas, both in CD and UC patients, (CD: Median = 0.34, Q1-Q3 = 0.17-0.74, p=0.001; UC: Median = 0.47, Q1-Q3 = 0.24-0.77, p=0.0004), irrespective of the risk genotypes for the rs2631367 variant. The differences remained still significant when comparing the normalized quantities (CD: p=0.0001; UC: p=0.000056). Conclusions: The OCTN2 and IL23R mRNA levels are significantly reduced in inflamed colonic mucosa, in both CD and UC patients, irrespective of patients’ genotypes. # G. Inflammatory bowel diseases 1. Basic science

Characteristics of L-carnitine transport in cultured human hepatoma HLF cells.

The recently cloned organic cation transporter, OCTN2, isolated as a homologue of OCTN1, has been shown to be of physiological importance in the renal tubular reabsorption of filtered L-carnitine as a high-affinity Na+ carnitine transporter in man. Although the mutation of the OCTN2 gene has been proved to be directly related to primary carnitine deficiency, there is little information about the L-carnitine transport system in the liver. In this study, the characteristics of L-carnitine transport into hepatocytes were studied by use of cultured human hepatoma HLF cells, which expressed OCTN2 mRNA to a greater extent than OCTN1 mRNA. The uptake of L-carnitine into HLF cells was saturable and the Eadie-Hofstee plot showed two distinct components. The apparent Michaelis constant and the maximum transport rate were 6.59+/-1.85 microM (mean+/-s.d.) and 78.5+/-21.4 pmol/5 min/10(6) cells, respectively, for high-affinity uptake, and 590+/-134 microM and 1507+/-142 pmol/5 min/10(6) cells, respectively, for low-affinity uptake. The high affinity L-carnitine transporter was significantly inhibited by metabolic inhibitors (sodium azide, dinitrophenol, iodoacetic acid) and at low temperature (4 degrees C). Uptake of [3H]L-carnitine also required the presence of Na+ ions in the external medium. The uptake activity was highest at pH 7.4, and was significantly lower at acidic or basic pH. L-Carnitine analogues (D-carnitine, L-acetylcarnitine and gamma-butyrobetaine) strongly inhibited uptake of [3H] L-carnitine, whereas beta-alanine, glycine, choline, acetylcholine and an organic anion and cation had little or no inhibitory effect. In conclusion, L-carnitine is absorbed by hepatocytes from man by an active carrier-mediated transport system which is Na+-, energy- and pH-dependent and has properties very similar to those of the carnitine transporter OCTN2.

Expression of OCTN2 and OCTN3 in the apical membrane of rat renal cortex and medulla

Immunological assays and transport measurements in apical membrane vesicles revealed that the apical membrane of rat kidney cortex and medulla presents OCTN2 and OCTN3 proteins and transports L-[(3)H]-carnitine in a Na(+)-dependent and -independent manner. OCTN2 mediates the Na(+)/L-carnitine transport activity measured in medulla because (i) the transport showed the same characteristics as the cortical Na(+)/L-carnitine transporter and (ii) the medulla expressed OCTN2 mRNA and protein. The Na(+)-independent L-carnitine transport activity appears to be mediated by both OCTN2 and OCTN3 since: (i) Na(+)-independent L-carnitine uptake was inhibited by both, anti-OCTN2 and anti-OCTN3 antibodies, (ii) kinetics studies revealed the involvement of a high- and a low-affinity transport systems, and (iii) Western and immunohistochemistry studies revealed that OCTN3 protein is located at the apical membrane of the kidney epithelia. The Na(+)-independent L-carnitine uptake exhibited trans-stimulation by intravesicular L-carnitine or betaine. This trans-stimulation was inhibited by anti-OCTN3 antibody, but not by anti-OCTN2 antibody, indicating that OCTN3 can function as an L-carnitine/organic compound exchanger. This is the first report showing a functional apical OCTN2 in the renal medulla and a functional apical OCTN3 in both renal cortex and medulla.

Urinary excretion of carnitine as a marker of proximal tubular damage associated with platin-based antineoplastic drugs

Patients treated with cisplatin or carboplatin show increased renal excretion of carnitine. It is currently unclear whether this is also the case for oxaliplatin and which are the responsible mechanisms. We investigated 22 patients treated either with a single dose of cisplatin, carboplatin or oxaliplatin. Carnitine and kidney function parameters were determined in plasma and urine. Inhibition and mRNA expression of OCTN2, the principle carnitine transporter, were assessed in L6 cells overexpressing OCTN2 and in 293-EBNA cells, respectively. Renal excretion of free and short-chain acylcarnitine increased already at the day of administration was maximal the day after and had normalized 1 week after administration of cisplatin, carboplatin or oxaliplatin. The renal excretion fractions for free carnitine and acylcarnitines increased 4-10 times during treatment with platin derivatives. Renal excretions of alpha1-microglobulin and other proximal tubular markers were also increased, compatible with a proximal tubular defect. Direct inhibition of OCTN2 expressed in L6 cells by cisplatin, oxaliplatin or platinum(2+) could not be demonstrated, and experiments using urine from patients treated with cisplatin inhibited OCTN2 activity no more than expected from the carnitine content in the respective urine sample. Cisplatin was associated with a time- and concentration-dependent decrease of OCTN2 mRNA and protein expression in 293-EBNA cells. All platin derivatives investigated are associated with renal tubular damage in humans without significantly affecting glomerular function. The rapid onset and complete reversibility of this effect favour a functional mechanism such as impaired expression of OCTN2 in proximal tubular cells.

Functional Genetic Variation in the Basal Promoter of the Organic Cation/Carnitine Transporters OCTN1 (SLC22A4) and OCTN2 (SLC22A5)

The organic cation/ergothioneine transporter OCTN1 (SLC22A4) and the high-affinity carnitine transporter OCTN2 (SLC22A5), play an important role in the disposition of xenobiotics and endogenous compounds. Here, we analyzed the sequence of the proximal promoter regions of OCTN1 and OCTN2 in four ethnic groups and determined the effects of the identified genetic variants on transcriptional activities and mRNA expression. Six variants were found in the proximal promoter of OCTN1, one of which showed high allele frequency ranging from 13 to 34% in samples from individuals with ancestries in Africa, Europe, China, and Mexico. OCTN1 haplotypes had similar activities as the reference in luciferase reporter assays. For OCTN2, three of the seven variants identified in the proximal promoter showed allele frequencies greater than 29.5% in all populations, with the exception of -207C>G (rs2631367) that was monomorphic in Asian Americans. OCTN2 haplotypes containing -207G, present in all populations, were associated with a gain of function in luciferase reporter assays. Consistent with reporter assays, OCTN2 mRNA expression levels in lymphoblastoid cell lines (LCLs) from gene expression analysis were greater in samples carrying a marker for -207G. This SNP seems to contribute to racial differences in OCTN2 mRNA expression levels in LCLs. Our study with healthy subjects (n = 16) homozygous for either -207C or -207G, showed no appreciable effect of this SNP on carnitine disposition. However, there were significant effects of gender on carnitine plasma levels (p < 0.01). Further in vivo studies of OCTN2 promoter variants on carnitine disposition and variation in drug response are warranted.