Yoshimichi Sai

Molecular and Functional Identification of Sodium Ion-dependent, High Affinity Human Carnitine Transporter OCTN2*

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 ofl-[3H]carnitine was strongly enhanced in a sodium-dependent manner with K m value of 4.34 μm, whereas typical substrates for previously known organic cation transporters, tetraethylammonium and guanidine, were not good substitutes. OCTN2-mediatedl-[3H]carnitine transport was inhibited by the d-isomer, acetyl-d,l-carnitine, and γ-butyrobetaine with high affinity and by glycinebetaine with lower affinity, whereas choline, β-hydroxybutyric acid, γ-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.

Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter

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 (jvs; ref. 6). 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 (ref. 9). 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.

Active secretion of drugs from the small intestinal epithelium in rats by P-glycoprotein functioning as an absorption barrier.

Because the significance of P‐glycoprotein in the in‐vivo secretion of β‐blockers in intestinal epithelial cells is unclear, the secretory mechanism for β‐blockers and other drugs has been evaluated.

Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter

Complementary DNA clones encoding the rat PepT1 small-intestinal oligopeptide transporter were isolated from a jejunal library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicates that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Northern blot analysis detected rat PepT1 mRNA in the small intestine and kidney. Intestinal PepT1 mRNA levels were highest in 4-day old rats, and then decreased reaching the adult level by day 28 after birth. These results indicate that the expressions of PepT1 gene change markedly during development.

CCR5 Plays a Critical Role in Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance by Regulating Both Macrophage Recruitment and M1/M2 Status

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.

PREDOMINANT CONTRIBUTION OF ORGANIC ANION TRANSPORTING POLYPEPTIDE OATP-B (OATP2B1) TO APICAL UPTAKE OF ESTRONE-3-SULFATE BY HUMAN INTESTINAL CACO-2 CELLS

Human organic anion transporting polypeptide OATP-B (OATP2B1) is a pH-sensitive transporter expressed in the apical membranes of small intestinal epithelial cells. In this study, we have examined the contribution of OATP-B to the uptake of [3H]estrone-3-sulfate in Caco-2 cells in comparison with those of its homologs OATP-D (OATP3A1) and OATP-E (OATP4A1). Immunocytochemical study revealed that OATP-B is expressed in the apical membranes of Caco-2 cells. The uptake of [3H]estrone-3-sulfate by Caco-2 cells was Na+-independent and inhibited by several organic anions. It showed biphasic saturation kinetics with Km values of 1.81 μM and 1.40 mM. The uptake of [3H]estrone-3-sulfate by human embryonic kidney (HEK) 293 cells stably expressing OATP-B (HEK293/OATP-B) was also Na+-independent and inhibited by several organic anions. The Km value for estrone-3-sulfate uptake by OATP-B (1.56 μM) was close to that for the high-affinity component observed in Caco-2 cells. The mRNA expression level of OATP-B was higher than that of OATP-D or OATP-E in Caco-2 cells and in human jejunum biopsies from healthy volunteers. The values of [3H]estrone-3-sulfate uptake normalized to OATP-B mRNA expression were similar in Caco-2 cells and HEK293/OATP-B cells. The specific activity of OATP-B per mRNA expression was much higher than that of OATP-D and OATP-E. [3H]Estrone-3-sulfate uptake by membrane vesicles prepared from HEK293/OATP-B cells exhibited an overshoot phenomenon in the presence of an inwardly directed H+ gradient, suggesting that an H+ gradient is the driving force of estrone-3-sulfate transport by OATP-B. These results suggest that OATP-B is predominantly responsible for the apical uptake of estrone-3-sulfate in Caco-2 cells.

Functional relevance of carnitine transporter OCTN2 to brain distribution of l‐carnitine and acetyl‐l‐carnitine across the blood–brain barrier

Transport of l‐[3H]carnitine and acetyl‐l‐[3H]carnitine at the blood–brain barrier (BBB) was examined by using in vivo and in vitro models. In vivo brain uptake of acetyl‐l‐[3H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl‐l‐carnitine and in the absence of sodium ions. Similar transport properties for l‐[3H]carnitine and/or acetyl‐l‐[3H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes of l‐[3H]carnitine and acetyl‐l‐[3H]carnitine by RBEC1 were sodium ion‐dependent, saturable with Km values of 33.1 ± 11.4 µm and 31.3 ± 11.6 µm, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT‐PCR method. Furthermore, the uptake of acetyl‐l‐[3H]carnitine by the BCECs of juvenile visceral steatosis (jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions of l‐[3H]carnitine and acetyl‐l‐[3H]carnitine in jvs mice were slightly lower than those of wild‐type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport of l‐carnitine and acetyl‐l‐carnitine from the circulating blood to the brain across the BBB.

Immunohistochemical and functional characterization of pH-dependent intestinal absorption of weak organic acids by the monocarboxylic acid transporter MCT1

The participation of the monocarboxylic acid transporter MCT1 in the intestinal absorption of weak organic acids has been clarified by functional characterization, by use of stably transfected cells, and by immunohistochemical location of the transporter in intestinal tissues.

The Predominant Contribution of Oligopeptide Transporter PepT1 to Intestinal Absorption of β‐Lactam Antibiotics in the Rat Small Intestine

Although recent evidence suggests that certain β‐lactam antibiotics are absorbed via a specific transport mechanism, its nature is unclear. To confirm whether peptide transport in the rat can be largely ascribed to the intestinal oligopeptide transporter PepT1, the transporter has been functionally characterized and its significance in the intestinal absorption of β‐lactam antibiotics was evaluated.

Transporter-mediated drug delivery: recent progress and experimental approaches

A comprehensive list of drug transporters has recently become available as a result of extensive genome analysis, as well as membrane physiology and molecular biology studies. This review covers recent progress in identification and characterization of drug transporters, illustrative cases of successful drug delivery to, or exclusion from, target organs via transporters, and novel experimental approaches to therapeutics using heterologously transduced transporters in tissues. We aim to provide clues that could lead to efficient strategies for the use of transporters to deliver drugs and/or to optimize lead compounds.