Primary carnitine deficiency, because of a defect of the tissue plasma membrane carnitine transporters, causes critical symptoms. However, the transporter has not been molecularly identified. In this study, we screened a human kidney cDNA library and assembled a cDNA-encoding OCTN2 as a homologue of the organic cation transporter OCTN1, and then we examined the function of OCTN2 as a carnitine transporter. OCTN2-cDNA encodes a polypeptide of 557 amino acids with 75.8% similarity to OCTN1. Northern blot analysis showed that OCTN2 is strongly expressed in kidney, skeletal muscle, heart, and placenta in adult humans. When OCTN2 was expressed in HEK293 cells, uptake of L-[ 3 H]carnitine was strongly enhanced in a sodium-dependent manner with K m value of 4.34 M, whereas typical substrates for previously known organic cation transporters, tetraethylammonium and guanidine, were not good substitutes. OCTN2-mediated L-[ 3 H]carnitine transport was inhibited by the D-isomer, acetyl-D,Lcarnitine, and ␥-butyrobetaine with high affinity and by glycinebetaine with lower affinity, whereas choline, -hydroxybutyric acid, ␥-aminobutyric acid, lysine, and taurine were not inhibitory. Because the observed tissue distribution of OCTN2 is consistent with the reported distribution of carnitine transport activity and the functional characteristics of OCTN2 coincide with those reported for plasma membrane carnitine transport, we conclude that OCTN2 is a physiologically important, high affinity sodium-carnitine cotransporter in humans.
cDNA for a novel proton/organic cation transporter, OCTN1, was cloned from human fetal liver and its transport activity was investigated. OCTN1 encodes a 551-amino acid protein with 11 transmembrane domains and one nucleotide binding site motif. It is strongly expressed in kidney, trachea, bone marrow and fetal liver and in several human cancer cell lines, but not in adult liver. When expressed in HEK293 cells, OCTN1 exhibited saturable and pH-dependent [ 3 H]tetraethyl ammonium uptake with higher activity at neutral and alkaline pH than at acidic pH. Furthermore, treatment with metabolic inhibitors reduced the uptake, which is consistent with the presence of the nucleotide binding site sequence motif. Although its subcellular localization and detailed functional characteristics are not clear at present, OCTN1 appears to be a novel proton antiporter that functions for active secretion of cationic compounds across the renal epithelial brush-border membrane. It may play a role in the renal excretion of xenobiotics and their metabolites.
Carnitine is essential for -oxidation of fatty acids, and a defect of cell membrane transport of carnitine leads to fatal systemic carnitine deficiency. We have already shown that a defect of the organic cation/carnitine transporter OCTN2 is a primary cause of systemic carnitine deficiency. In the present study, we further isolated and characterized new members of the OCTN family, OCTN1 and -3, in mice. All three members were expressed commonly in kidney, and OCTN1 and -2 were also expressed in various tissues, whereas OCTN3 was characterized by predominant expression in testis. When their cDNAs were transfected into HEK293 cells, the cells exhibited transport activity for carnitine and/or the organic cation tetraethylammonium (TEA). Carnitine transport by OCTN1 and OCTN2 was Na ؉ -dependent, whereas that by OCTN3 was Na ؉ -independent. TEA was transported by OCTN1 and OCTN2 but not by OCTN3. The relative uptake activity ratios of carnitine to TEA were 1.78, 11.3, and 746 for OCTN1, -2, and -3, respectively, suggesting high specificity of OCTN3 for carnitine and significantly lower carnitine transport activity of OCTN1. Thus, OCTN3 is unique in its limited tissue distribution and Na ؉ -independent carnitine transport, whereas OCTN1 efficiently transported TEA with minimal expression of carnitine transport activity and may have a different role from other members of the OCTN family.
Primary systemic carnitine deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. SCD has also been linked to sudden infant death syndrome. Membrane-physiological studies have suggested a defect of the carnitine transport system in the plasma membrane in SCD patients and in the mouse model, juvenile visceral steatosis. Although the responsible loci have been mapped in both human and mouse, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2. Our observation that OCTN2 has the ability to transport carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5. Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.
C-C motif chemokine receptor (CCR)2 and its ligand, monocyte chemoattractant protein (MCP)-1, are pivotal for adipose tissue macrophage (ATM) recruitment and the development of insulin resistance. However, other chemokine systems also may play a role in these processes. In this study, we investigated the role of CCR5 in obesity-induced adipose tissue inflammation and insulin resistance. We analyzed expression levels of CCR5 and its ligands in white adipose tissue (WAT) of genetically (ob/ob) and high-fat (HF) diet–induced obese (DIO) mice. Furthermore, we examined the metabolic phenotype of Ccr5−/− mice. CCR5 and its ligands were markedly upregulated in WAT of DIO and ob/ob mice. Fluorescence-activated cell sorter analysis also revealed that DIO mice had a robust increase in CCR5+ cells within ATMs compared with chow-fed mice. Furthermore, Ccr5−/− mice were protected from insulin resistance, glucose intolerance, and hepatic steatosis induced by HF feeding. The effects of loss of CCR5 were related to both reduction of total ATM content and an M2-dominant shift in ATM polarization. It is noteworthy that transplantation of Ccr5−/− bone marrow was sufficient to protect against impaired glucose tolerance. CCR5 plays a critical role in ATM recruitment and polarization and subsequent development of insulin resistance.
Genetic polymorphisms of human organic anion transporting polypeptides OATP-C (SLC21A6) and OATP-B (SLC21A9) in the Japanese population were analyzed. The allele frequencies of OATP-C*1a, OATP-C*1b (N130D), OATP-C*1c (R152K and D241N), and OATP-C*5 (V174A) were 35.2, 53.7, 0, and 0.7%, respectively, in 267 healthy Japanese subjects. In the OATP-C gene, we found a novel allele called OATP-C*15 possessing two single nucleotide polymorphisms (SNPs), N130D and V174A, simultaneously. The allele frequency of OATP-C*15 was 3.0%. The allele frequencies of OATP-B*1, OATP-B*2 (T392I), and OATP-B*3 (S486F) were 69.1, 0, and 30.9%, respectively. For functional analysis, each OATP-C and OATP-B allele was expressed in human embryonic kidney (HEK293) cells, and the kinetics of uptake of [ 3 H]estrone-3-sulfate was determined. In the case of OATP-C alleles, no significant alteration in K m or V max values of [ 3 H]estrone-3-sulfate uptake was observed, even when the V max values were corrected for the expression levels of OATP-C protein. In contrast, V max , corrected with the expression level of OATP-B*3, was decreased to 42.5% of OATP-B*1, whereas the K m values were comparable. Since the frequency of the OATP-B*3 allele was high (30.9%) in our subjects, the SNP of S486F may affect the physiological function and/or pharmacological effects of OATP-B substrates in vivo.
OCTN2 is an Na(+)-dependent transporter for carnitine, which is essential for fatty acid metabolism, and its functional defect leads to fatal systemic carnitine deficiency (SCD). It also transports the organic cation tetraethylammonium (TEA) in an Na(+)-independent manner. Here, we studied the multifunctionality of OCTN2, by examining the transport characteristics in cells transfected with mouse OCTN2 and in juvenile visceral steatosis (jvs) mice that exhibit a SCD phenotype owing to mutation of the OCTN2 gene. The physiological significance of OCTN2 as an organic cation transporter was confirmed by using jvs mice. The embryonic fibroblasts from jvs mice exhibited significantly decreased transport of [(14)C]TEA. Pharmacokinetic analysis of [(14)C]TEA disposition demonstrated that jvs mice showed decreased tissue distribution and renal secretory clearance. In transport experiments using OCTN2-expressing cells, TEA and carnitine showed mutual trans-stimulation effects in their transport, implying a carnitine/TEA exchange mechanism. In addition, Na(+) affected the affinity of carnitine for OCTN2, whereas Na(+) is unlikely to be involved in TEA transport. This is the first molecular and physiological demonstration of the operation of an organic cation transporter in renal apical membrane. The results are consistent with the physiological coupling of carnitine reabsorption with the secretion of organic cations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.