The activity and surface presence of organic cation/carnitine transporter OCTN2 (SLC22A5) in breast cancer cells depends on AKT kinase.

l-carnitine is indispensable for transfer of fatty acids to mitochondria for the process of ß-oxidation, a process, whose significance in cancer has drawn attention in recent years. In humans majority of carnitine is delivered by diet and enters the cell due to activity of solute carriers (SLCs), mainly by ubiquitously expressed organic cation/carnitine transporter (OCTN2/SLC22A5). In control and cancer human breast epithelial cell lines the major fraction of OCTN2 is present as a not matured non-glycosylated form. Studies on overexpressed OCTN2 demonstrated an exclusive interaction with SEC24C, as the cargo-recognizing subunit of coatomer II in transporter exit from endoplasmic reticulum. Co-transfection with SEC24C dominant negative mutant completely abolished presence of the mature form of OCTN2, pointing to a possibility of trafficking regulation. SEC24C was previously shown to be phosphorylated by serine/threonine kinase AKT, known to be activated in cancer. Further studies on breast cell lines showed that inhibition of AKT with MK-2206 in control and cancer lines decreased level of OCTN2 mature form. Proximity ligation assay showed that phosphorylation of OCTN2 on threonine was significantly abolished by AKT inhibition with MK-2206. Carnitine transport was positively correlated with the level of OCTN2 phosphorylated by AKT on threonine moiety. The observed regulation of OCTN2 by AKT places this kinase in the center of metabolic control. This points to both proteins, AKT and OCTN2, as druggable targets, in particular in a combination therapy of breast cancer.

Regulation of SLC6A14 trafficking in breast cancer cells by heat shock protein HSP90ß.

SLC6A14 is a plasma membrane transporter specific for neutral and basic amino acids, upregulated in many tumors. This study focused on breast cancer cell lines, showing the fully glycosylated band, known to be at the cell surface, in estrogen receptor positive lines. Inhibition of heat shock protein 90ß (HSP90ß) decreased the level of this band, what correlated with a decrease of SLC6A14 transport activity. A direct interaction between SLC6A14 and HSP90ß was confirmed in proximity ligation assay, pointing to the role of HSP90 in folding control in endoplasmic reticulum and affecting farther transporter trafficking to the cell surface. Either inhibitor of SLC6A14 (α-methyltryptophan) or of HSP90 (radicicol) had the cytotoxic effect, when added alone, while treatment with both compounds had a synergistic effect. This points to SLC6A14 as a druggable target in breast cancer and a combination therapy being more efficient in killing cancer cells.

Trafficking to the Cell Surface of Amino Acid Transporter SLC6A14 Upregulated in Cancer Is Controlled by Phosphorylation of SEC24C Protein by AKT Kinase.

Cancer cells need a constant supply of nutrients. SLC6A14, an amino acid transporter B0,+ (ATB0,+) that is upregulated in many cancers, transports all but acidic amino acids. In its exit from the endoplasmic reticulum (ER), it is recognized by the SEC24C subunit of coatomer II (COPII) for further vesicular trafficking to the plasma membrane. SEC24C has previously been shown to be phosphorylated by protein kinase B/AKT, which is hyper-activated in cancer; therefore, we analyzed the influence of AKT on SLC6A14 trafficking to the cell surface. Studies on overexpressed and endogenous transporters in the breast cancer cell line MCF-7 showed that AKT inhibition with MK-2206 correlated with a transient increase of the transporter in the plasma membrane, not resulting from the inhibition of ER-associated protein degradation. Two-dimensional electrophoresis demonstrated the decreased phosphorylation of SLC6A14 and SEC24C upon AKT inhibition. A proximity ligation assay confirmed this conclusion: AKT inhibition is correlated with decreased SLC6A14 phosphothreonine and SEC24C phosphoserine. Augmented levels of SLC6A14 in plasma membrane led to increased leucine transport. These results show that the inactivation of AKT can rescue amino acid delivery through SLC6A14 trafficking to the cell surface, supporting cancer cell survival. The regulation of the ER export of the amino acid transporter seems to be a novel function of AKT.

Glioma cells survival depends both on fatty acid oxidation and on functional carnitine transport by SLC22A5.

Gliomas are the most common primary malignant brain tumor in adults, but current treatment for glioblastoma multiforme (GBM) is insufficient. Even though glucose is the primary energetic substrate of glioma cells, they are capable of using fatty acids to generate energy. Fatty acid oxidation (FAO) in mitochondria requires L-carnitine for the formation of acylcarnitines by carnitine palmitoylotransferase 1 (CPT1) and further transport of acyl carnitine esters to mitochondrial matrix. Carnitine can be delivered to the cell by an organic cation/carnitine transporter-SLC22A5/OCTN2. In this study, we show that SLC22A5 is up-regulated in glioma cells and that they vary in the amount of SLC22A5 in the plasma membrane. Research on glioma cells (lines U87MG, LN229, T98G) with various expression levels of SLC22A5 demonstrated a correlation between the FAO rate, the level of the transporter, and the carnitine transport. Inhibition of carnitine transport by chemotherapeutics, such as vinorelbine and vincristine, led to inhibition of FAO, which was further intensified by etomoxir-a CPT1 inhibitor. This led to reduced viability and increased apoptosis in glioma cells. Modulation of SLC22A5 level by either silencing or up-regulation of SLC22A5 also affected glioma cell survival in a FAO-dependent way. These observations suggest that the survival of glioma cells is heavily reliant on both FAO and SLC22A5 activity, as well as that CPT1 and SLC22A5 might be possible drug targets.

Amino Acid Transporter SLC6A14 (ATB0,+) - A Target in Combined Anti-cancer Therapy.

Cancer cells are characterized by quick growth and proliferation, demanding constant supply of various nutrients. Several plasma membrane transporters delivering such compounds are upregulated in cancer. Solute carrier family 6 member 14 (SLC6A14), known as amino acid transporter B0,+ (ATB0,+) transports all amino acids with exception of the acidic ones: aspartate and glutamate. Its malfunctioning is correlated with several pathological states and it is upregulated in solid tumors. The high expression of SLC6A14 is prognostic and unfavorable in pancreatic cancer, while in breast cancer it is expressed in estrogen receptor positive cells. As many plasma membrane transporters it resides in endoplasmic reticulum (ER) membrane after translation before further trafficking through Golgi to the cell surface. Transporter exit from ER is strictly controlled. The proper folding of SLC6A14 was shown to be controlled from the cytoplasmic side by heat shock proteins, further exit from ER and formation of coatomer II (COPII) coated vesicles depends on specific interaction with COPII cargo-recognizing subunit SEC24C, phosphorylated by kinase AKT. Inhibition of heat shock proteins, known to be upregulated in cancer, directs SLC6A14 to degradation. Targeting proteins regulating SLC6A14 trafficking is proposed as an additional pharmacological treatment of cancer.

SLC22A5 (OCTN2) Carnitine Transporter-Indispensable for Cell Metabolism, a Jekyll and Hyde of Human Cancer.

Oxidation of fatty acids uses l-carnitine to transport acyl moieties to mitochondria in a so-called carnitine shuttle. The process of ß-oxidation also takes place in cancer cells. The majority of carnitine comes from the diet and is transported to the cell by ubiquitously expressed organic cation transporter novel family member 2 (OCTN2)/solute carrier family 22 member 5 (SLC22A5). The expression of SLC22A5 is regulated by transcription factors peroxisome proliferator-activated receptors (PPARs) and estrogen receptor. Transporter delivery to the cell surface, as well as transport activity are controlled by OCTN2 interaction with other proteins, such as PDZ-domain containing proteins, protein phosphatase PP2A, caveolin-1, protein kinase C. SLC22A5 expression is altered in many types of cancer, giving an advantage to some of them by supplying carnitine for ß-oxidation, thus providing an alternative to glucose source of energy for growth and proliferation. On the other hand, SLC22A5 can also transport several chemotherapeutics used in clinics, leading to cancer cell death.

Amino acid transporter SLC6A14 depends on heat shock protein HSP90 in trafficking to the cell surface.

Plasma membrane transporter SLC6A14 transports all neutral and basic amino acids in a Na/Cl - dependent way and it is up-regulated in many types of cancer. Mass spectrometry analysis of overexpressed SLC6A14-associated proteins identified, among others, the presence of cytosolic heat shock proteins (HSPs) and co-chaperones. We detected co-localization of overexpressed and native SLC6A14 with HSP90-beta and HSP70 (HSPA14). Proximity ligation assay confirmed a direct interaction of overexpressed SLC6A14 with both HSPs. Treatment with radicicol and VER155008, specific inhibitors of HSP90 and HSP70, respectively, attenuated these interactions and strongly reduced transporter presence at the cell surface, what resulted from the diminished level of the total transporter protein. Distortion of SLC6A14 proper folding by both HSPs inhibitors directed the transporter towards endoplasmic reticulum-associated degradation pathway, a process reversed by the proteasome inhibitor - bortezomib. As demonstrated in an in vitro ATPase assay of recombinant purified HSP90-beta, the peptides corresponding to C-terminal amino acid sequence following the last transmembrane domain of SLC6A14 affected the HSP90-beta activity. These results indicate that a plasma membrane protein folding can be controlled not only by chaperones in the endoplasmic reticulum, but also those localized in the cytosol.

Trafficking of the amino acid transporter B0,+ (SLC6A14) to the plasma membrane involves an exclusive interaction with SEC24C for its exit from the endoplasmic reticulum.

A plasma membrane amino acid transporter B0,+ (ATB0,+), encoded by the SLC6A14 gene, is specific for neutral and basic amino acids. It is up-regulated in several types of malignant cancers. Neurotransmitter transporters of the SLC6 family interact with specific SEC24 proteins of the COPII complex along their pathway from the endoplasmic reticulum (ER) to Golgi. This study focused on the possible role of SEC24 proteins in ATB0,+ trafficking. Rat ATB0,+ was expressed in HEK293 cells, its localization and trafficking were examined by Western blot, deglycosylation, immunofluorescence (co-localization with ER and trans-Golgi markers) and biotinylation. The expression of ATB0,+ at the plasma membrane was decreased by dominant negative mutants of SAR1, a GTPase, whose activity triggers the formation of the COPII complex. ATB0,+ co-precipitated with SEC24C (but not with the remaining isoforms A, B and D). This interaction was confirmed by immunocytochemistry and the proximity ligation assay. Co-localization of SEC24C with endogenous ATB0,+ was also observed in MCF-7 breast cancer cells. Contrary to the endogenous transporter, part of the overexpressed ATB0,+ is directed to proteolysis, a process significantly reversed by a proteasome inhibitor bortezomib. Co-transfection with a SEC24C dominant negative mutant attenuated ATB0,+ expression at the plasma membrane, due to proteolytic degradation. These results support a hypothesis that lysine at position +2 downstream of the ER export "RI" motif on the cargo protein is crucial for SEC24C binding and for further trafficking to the Golgi. Moreover, there is an equilibrium between ER export and degradation mechanisms in case of overexpressed transporter.

Is fat tasty for the brain?: An Editorial Comment for 'Developmental regulation and localization of Carnitine Palmitoyltransferases (CPTs) in rat brain'.

Read the commented article 'Developmental regulation and localization of Carnitine Palmitoyltransferases (CPTs) in rat brain' on page 407.

Tight junction protein ZO-1 controls organic cation/carnitine transporter OCTN2 (SLC22A5) in a protein kinase C-dependent way.

OCTN2 (SLC22A5) is an organic cation/carnitine transporter belonging to the solute carrier transporters (SLC) family. OCTN2 is ubiquitously expressed and its presence was shown in various brain cells, including the endothelial cells forming blood-brain barrier, where it was mainly detected at abluminal membrane and in proximity of tight junctions (TJ). Since OCTN2 contains a PDZ-binding domain, the present study was focused on a possible role of transporter interaction with a TJ-associated protein ZO-1, containing PDZ domains and detected in rat Octn2 proteome. We showed previously that activation of protein kinase C (PKC) in rat astrocytes regulates Octn2 surface presence and activity. Regulation of a wild type Octn2 and its deletion mutant without a PDZ binding motif were studied in heterologous expression system in HEK293 cells. Plasma membrane presence of overexpressed Octn2 did not depend on either PKC activation or presence of PDZ-binding motif, anyhow, as assayed in proximity ligation assay, the truncation of PDZ binding motif resulted in a strongly diminished Octn2/ZO-1 interaction and in a decreased transporter activity. The same effects on Octn2 activity were detected upon PKC activation, what correlated with ZO-1 phosphorylation. It is postulated that ZO-1, when not phosphorylated by PKC, keeps Octn2 in an active state, while elimination of this binding in ΔPDZ mutant or after ZO-1 phosphorylation leads to diminution of Octn2 activity.

Solute Carriers in the Blood-Brain Barier: Safety in Abundance.

Blood-brain barrier formed by brain capillary endothelial cells, being in contact with astrocytes endfeet and pericytes, separates extracellular fluid from plasma. Supply of necessary nutrients and removal of certain metabolites takes place due to the activity of transporting proteins from ABC (ATP binding cassette) and SLC (solute carrier) superfamilies. This review is focused on the SLC families involved in transport though the blood-brain barrier of energetic substrates (glucose, monocarboxylates, creatine), amino acids, neurotransmitters and their precursors, as well as organic ions. Members of SLC1, SLC2, SLC3/SLC7, SLC5, SLC6, SLC16, SLC22, SLC38, SLC44, SLC47 and SLCO (SLC21), whose presence in the blood-brain barriers has been demonstrated are characterized with a special emphasis put on polarity of transporters localization in a luminal (blood side) versus an abluminal (brain side) membrane.

Protein phosphatase PP2A - a novel interacting partner of carnitine transporter OCTN2 (SLC22A5) in rat astrocytes.

l-Carnitine is essential for translocation of fatty acids for their mitochondrial ß-oxidation, a process shown in the brain to take place in astrocytes. Organic cation and carnitine plasma membrane transporter OCTN2 (SLC22A5) is present in astrocytes. OCTN2 activity and localization were previously shown to be regulated by protein kinase C (PKC), although no phosphorylation of the transporter was detected. In this study, mass spectrometry was used to identify rOctn2-interacting partners in astrocytes: several cytoskeletal, ribosomal, mitochondrial, heat-shock proteins, as well as proteins involved in trafficking and signaling pathways. The analysis of signaling proteins shows that Octn2 co-precipitated with PP2A phosphatase catalytical (C) and structural (A) subunits, and with its regulatory B"' subunits - striatin, SG2NA, and zinedin. The Octn2/PP2A complex is mainly detected in endoplasmic reticulum. PKC activation increases both, carnitine transport and, as shown by immunofluorescence and surface biotinylation, transporter presence in plasma membrane. It also results in phosphorylation of SG2NA, zinedin, and catalytical subunit, although co-precipitation, immunocytochemistry, and proximity ligation assay experiments showed that only the amount of SG2NA decreased in the complex with Octn2. PP2A inhibition with okadaic acid does not lead to Octn2 phosphorylation; however, it abolishes observed effects of PKC activation. We postulate that PKC phosphorylates SG2NA, resulting in its dissociation from the complex and transfer of Octn2 to the plasma membrane, leading to increased transporter activity. The observed interaction could affect brain functioning in vivo, both in fatty acid metabolism and in control of carnitine homeostasis, known to change in certain brain pathologies.

[Carnitine - mitochondria and beyond]. / Karnityna - mitochondria i nie tylko.

Carnitine [(3R)-3-hydroxy-4-(trimethylazaniumyl)butanoate] in mammals is mainly delivered with diet. It enters the cell due to the activity of organic cation/carnitine transporter OCTN2 (SLC22A5), it can be as well transported by CT2 (SLC22A16) and a transporter of neutral and basic amino acids ATB0, + (SLC6A14). The hydroxyl group of carnitine is able to form esters with organic acids (xenobiotics, fatty acids) due to the activity of acylcarnitine transferases. Carnitine is necessary for transfer of fatty acids to mitochondria: in functioning of the so-called carnitine shuttle an essential role is fulfilled by palmitoylcarnitine transferase 1, carnitine carrier (SLC25A20) in the inner mitochondrial membrane and palmitoylcarnitine transferase 2. Oxidation of fatty acids takes also place in peroxisomes. The produced medium-chain acyl derivatives are exported as acylcarnitines, most probably by OCTN3 (Slc22a21). It has been postulated that acylcarnitines can cross the outer mitochondrial membrane through the voltage-dependent anion channel (VDAC) and/or through the palmitoycarnitine transferase 1 oligomer. Mutations of genes coding carnitine plasma membrane transporters result in the primary carnitine deficiency, with symptoms affecting normal functioning of muscles (including heart) and brain. Mechanisms regulating functioning of these transporters have been presented with emphasis on their role as potential therapeutic targets.

Protein kinase C restricts transport of carnitine by amino acid transporter ATB(0,+) apically localized in the blood-brain barrier.

Carnitine (3-hydroxy-4-trimethylammoniobutyrate) is necessary for transfer of fatty acids through the inner mitochondrial membrane. Carnitine, not synthesized in the brain, is delivered there through the strongly polarized blood-brain barrier (BBB). Expression and presence of two carnitine transporters - organic cation/carnitine transporter (OCTN2) and amino acid transporter B(0,+) (ATB(0,+)) have been demonstrated previously in an in vitro model of the BBB. Due to potential protein kinase C (PKC) phosphorylation sites within ATB(0,+) sequence, the present study verified effects of this kinase on transporter function and localization in the BBB. ATB(0,+) can be regulated by estrogen receptor α and up-regulated in vitro, therefore its presence in vivo was verified with the transmission electron microscopy. The analyses of brain slices demonstrated ATB(0,+) luminal localization in brain capillaries, confirmed by biotinylation experiments in an in vitro model of the BBB. Brain capillary endothelial cells were shown to control carnitine gradient. ATB(0,+) was phosphorylated by PKC, what correlated with inhibition of carnitine transport. PKC activation did not change the amount of ATB(0,+) present in the apical membrane of brain endothelial cells, but resulted in transporter exclusion from raft microdomains. ATB(0,+) inactivation by a lateral movement in plasma membrane after transporter phosphorylation has been postulated.

Caveolin-1--a novel interacting partner of organic cation/carnitine transporter (Octn2): effect of protein kinase C on this interaction in rat astrocytes.

OCTN2--the Organic Cation Transporter Novel family member 2 (SLC22A5) is known to be a xenobiotic/drug transporter. It transports as well carnitine--a compound necessary for oxidation of fatty acids and mutations of its gene cause primary carnitine deficiency. Octn2 regulation by protein kinase C (PKC) was studied in rat astrocytes--cells in which ß-oxidation takes place in the brain. Activation of PKC with phorbol ester stimulated L-carnitine transport and increased cell surface presence of the transporter, although no PKC-specific phosphorylation of Octn2 could be detected. PKC activation resulted in an augmented Octn2 presence in cholesterol/sphingolipid-rich microdomains of plasma membrane (rafts) and increased co-precipitation of Octn2 with raft-proteins, caveolin-1 and flotillin-1. Deletion of potential caveolin-1 binding motifs pointed to amino acids 14-22 and 447-454 as the caveolin-1 binding sites within Octn2 sequence. A direct interaction of Octn2 with caveolin-1 in astrocytes upon PKC activation was detected by proximity ligation assay, while such an interaction was excluded in case of flotillin-1. Functioning of a multi-protein complex regulated by PKC has been postulated in rOctn2 trafficking to the cell surface, a process which could be important both under physiological conditions, when carnitine facilitates fatty acids catabolism and controls free Coenzyme A pool as well as in pathology, when transport of several drugs can induce secondary carnitine deficiency.

Palmitoylcarnitine affects localization of growth associated protein GAP-43 in plasma membrane subdomains and its interaction with Gα(o) in neuroblastoma NB-2a cells.

Palmitoylcarnitine was observed previously to promote differentiation of neuroblastoma NB-2a cells, and to affect protein kinase C (PKC). Palmitoylcarnitine was also observed to increase palmitoylation of several proteins, including a PKC substrate, whose expression augments during differentiation of neural cells-a growth associated protein GAP-43, known to bind phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. Since palmitoylated proteins are preferentially localized in sphingolipid- and cholesterol-rich microdomains of plasma membrane, the present study has been focused on a possible effect of palmitoylcarnitine on GAP-43 localization in these microdomains. Palmitoylcarnitine treatment resulted in GAP-43 appearance in floating fractions (rafts) in sucrose gradient and increased co-localization with cholesterol and with PI(4,5)P(2), although co-localization of both lipids decreased. GAP-43 disappeared from raft fraction upon treatment with 2-bromopalmitate (an inhibitor of palmitoylating enzymes) and after treatment with etomoxir (carnitine palmitoyltransferase I inhibitor). Raft localization of GAP-43 was completely abolished by treatment with methyl-ß-cyclodextrin, a cholesterol binding agent, while there was no change upon sequestration of PI(4,5)P(2) with neomycin. GAP-43 co-precipitated with a monomeric form of Gα(o), a phenomenon diminished after palmitoylcarnitine treatment and paralleled by a decrease of Gα(o) in the raft fraction. These observations point to palmitoylation of GAP-43 as a mechanism leading to an increased localization of this protein in microdomains of plasma membrane rich in cholesterol, in majority different, however, from microdomains in which PI(4,5)P(2) is present. This localization correlates with decreased interaction with Gα(o) and suppression of its activity-an important step regulating neural cell differentiation.

Protein kinase C regulates amino acid transporter ATB(0,+).

ATB(0,+) (SLC6A14) is a transporter specific towards neutral and cationic amino acids, known to be up-regulated in malignant tumor cells. We cloned cDNA for rATB(0,+) and expressed it in HEK 293 cells. The ATB(0,+) over-expression correlated with increased l-leucine transport, stimulated by protein kinase C (PKC) activator and attenuated by PKC inhibitors. Transport stimulation was correlated with phosphorylation on serine moiety of the transporter and its augmented plasma membrane presence. Immunoprecipitation experiments demonstrated ATB(0,+) interaction with PKCα, but not with other classical or novel PKC isoforms. Immunocytochemistry experiments showed a transfer of PKCα to plasma membrane upon phorbol ester activation and co-localization with ATB(0,+). The observed regulation of ATB(0,+) by PKC correlates with high activity of both proteins reported for cancer cells.

Regulation of amino acid/carnitine transporter B 0,+ (ATB 0,+) in astrocytes by protein kinase C: independent effects on raft and non-raft transporter subpopulations.

Neutral and basic amino acid transporter B(0,+) belongs to a Na,Cl-dependent superfamily of proteins transporting neurotransmitters, amino acids and osmolytes, known to be regulated by protein kinase C (PKC). The present study demonstrates an increased phosphorylation of B(0,+) on serine moiety after treatment of rat astrocytes with phorbol 12-myristate 13-acetate, a process correlated with an augmented activity of l-leucine transport and an enhanced presence of the transporter at the cell surface. After solubilization with Triton X-100 and sucrose gradient centrifugation, B(0,+) was detected in non-raft as well as in detergent-resistant raft fractions under control conditions, while phorbol 12-myristate 13-acetate treatment resulted in a complete disappearance of the transporter from the raft fraction. B(0,+) was observed to interact with caveolin-1 and flotillin-1 (reggie-2) proteins, the markers of detergent-resistant microdomains of plasma membrane. As verified in immunocytochemistry and immunoprecipitation experiments, modification of PKC activity did not affect these interactions. It is proposed that PKC reveals different effects on raft and non-raft subpopulations of B(0,+). Phorbol ester treatment results in trafficking of the transporter from the intracellular pool to non-raft microdomains and increased activity, while B(0,+) present in raft microdomains undergoes either internalization or is transferred laterally to non-raft domains.

High affinity carnitine transporters from OCTN family in neural cells.

Neurons are known to accumulate L-carnitine--a compound necessary for transfer of acyl moieties through biological membranes, apart from very low beta-oxidation of fatty acids in adult brain. Present study demonstrates expression of octn2 and octn3 genes coding high affinity carnitine transporters, as well as presence of both proteins in neurons obtained from suckling and adult rats, and also in mouse transformed neural cells. Measurements of carnitine transport show activity of both transporters in neural cells, pointing to their importance in physiological processes other than beta-oxidation.

Organic cation/carnitine transporter OCTN3 is present in astrocytes and is up-regulated by peroxisome proliferators-activator receptor agonist.

In the brain beta-oxidation, which takes place in astrocytes, is not a major process of energy supply. Astrocytes synthesize important lipid metabolites, mainly due to the processes taking place in peroxisomes. One of the compounds necessary in the process of mitochondrial beta-oxidation and export of acyl moieties from peroxisomes is l-carnitine. Two Na-dependent plasma membrane carnitine transporters were shown previously to be present in astrocytes: a low affinity amino acid transporter B(0,+) and a high affinity cation/carnitine transporter OCTN2. The expression of OCTN2 is known to increase in peripheral tissues upon the stimulation of peroxisome proliferators-activator receptor alpha (PPARalpha), a nuclear receptor known to up-regulate several enzymes involved in fatty acid metabolism. The present study was focused on another high affinity carnitine transporter-OCTN3, its presence, regulation and activity in astrocytes. Experiments using the techniques of real-time PCR, Western blot and immunocytochemistry analysis demonstrated the expression of octn3 in rat astrocytes and, out of two rat sequences ascribed as similar to mouse OCTN3, XM_001073573 was found in these cells. PPARalpha activator-2-[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl]thio]acetic acid (WY-14,643) stimulated by 50% expression of octn3, while, on the contrary to peripheral tissues, it did not change the expression of octn2. This observation was correlated with an increased Na-independent activity of carnitine transport. Analysis by transmission electron microscopy showed an augmented intracellular localization of OCTN3 upon PPARalpha stimulation, mainly in peroxisomes, indicating a physiological role of OCTN3 as peroxisomal membrane transporter. These observations point to an important role of OCTN3 in peroxisomal fatty acid metabolism in astrocytes.