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The membrane impermeability of a drug poses a significant challenge in drug research and development, preventing effective drug delivery to the target site. Specifically, the blood-brain barrier (BBB) presents a formidable obstacle to the delivery of drugs targeting the central nervous system (CNS) into the brain, whereas the blood-retinal barrier (BRB) presents a tremendous obstacle to the delivery of drugs targeting the ocular diseases into the eyes. The development of drugs for Alzheimer’s or Parkinson’s disease targeting the CNS and for diabetic retinopathy and age-related macular degeneration targeting the eyes remains an unmet medical need for patients. Transporters play a crucial physiological role in maintaining homeostasis in metabolic organs. Various types of solute carrier (SLC) transporters are expressed in the capillary endothelial cells of the BBB, facilitating the delivery of nutrients from the blood flow to the brain. Therefore, carrier-mediated transport across the BBB can be achieved using SLC transporters present in capillary endothelial cells. It is well-known that CNS drugs typically incorporate N-containing groups, indicating that cation transporters facilitate their transport into the brain. In fact, carrier-mediated transport across the BBB can be accomplished using glucose transporter type 1 (Glut1) as a glucose transporter, L-type amino acid transporter 1 (LAT1) as a large neutral amino acid transporter, and H+/cation antiporter as a cation transporter. Surprisingly, although organic cation transporter novel type 2 (OCTN2) is expressed in the capillary endothelial cells, there has been limited investigation into OCTN2-mediated substance delivery into the brain across the BBB. Furthermore, it is suggested that OCTN2 is expressed at the BRB. In this prospective review, I present the advantages and possibilities of substance delivery into the brain across the BBB or into the eyes across the BRB, mediated by OCTN2 via carrier-mediated transport or receptor-mediated transcytosis.

The membrane impermeability of a drug poses a significant challenge in drug research and development, preventing effective drug delivery to the target site. Specifically, the blood-brain barrier (BBB) presents a formidable obstacle to the delivery of drugs targeting the central nervous system (CNS) into the brain, whereas the blood-retinal barrier (BRB) presents a tremendous obstacle to the delivery of drugs targeting the ocular diseases into the eyes. The development of drugs for Alzheimer's or Parkinson's disease targeting the CNS and for diabetic retinopathy and age-related macular degeneration targeting the eyes remains an unmet medical need for patients. Transporters play a crucial physiological role in maintaining homeostasis in metabolic organs. Various types of solute carrier (SLC) transporters are expressed in the capillary endothelial cells of the BBB, facilitating the delivery of nutrients from the blood flow to the brain. Therefore, carrier-mediated transport across the BBB can be achieved using SLC transporters present in capillary endothelial cells. It is well-known that CNS drugs typically incorporate N-containing groups, indicating that cation transporters facilitate their transport into the brain. In fact, carrier-mediated transport across the BBB can be accomplished using glucose transporter type 1 (Glut1) as a glucose transporter, L-type amino acid transporter 1 (LAT1) as a large neutral amino acid transporter, and H+/cation antiporter as a cation transporter. Surprisingly, although organic cation transporter novel type 2 (OCTN2) is expressed in the capillary endothelial cells, there has been limited investigation into OCTN2-mediated substance delivery into the brain across the BBB. Furthermore, it is suggested that OCTN2 is expressed at the BRB. In this prospective review, I present the advantages and possibilities of substance delivery into the brain across the BBB or into the eyes across the BRB, mediated by OCTN2 via carrier-mediated transport or receptor-mediated transcytosis. [ABSTRACT FROM AUTHOR]

OCTN1 and OCTN2 are membrane transport proteins encoded by the SLC22A4 and SLC22A5 genes, respectively. Even though several transcripts have been predicted by bioinformatics for both genes, only one functional protein isoform has been described for each of them. Both proteins are ubiquitous, and depending on the physiopathological state of the cell, their expression is regulated by well-known transcription factors, although some aspects have been neglected. A plethora of missense variants with uncertain clinical significance are reported both in the dbSNP and the Catalogue of Somatic Mutations in Cancer (COSMIC) databases for both genes. Due to their involvement in human pathologies, such as inflammatory-based diseases (OCTN1/2), systemic primary carnitine deficiency (OCTN2), and drug disposition, it would be interesting to predict the impact of variants on human health from the perspective of precision medicine. Although the lack of a 3D structure for these two transport proteins hampers any speculation on the consequences of the polymorphisms, the already available 3D structures for other members of the SLC22 family may provide powerful tools to perform structure/function studies on WT and mutant proteins. [ABSTRACT FROM AUTHOR]

Objective: A prominent, safe and efficient therapy for patients with chronic myeloid leukemia (CML) is inhibiting oncogenic protein BCR::ABL1 in a targeted manner with imatinib, a tyrosine kinase inhibitor. A substantial part of patients treated with imatinib report skeletomuscular adverse events affecting their quality of life. OCTN2 membrane transporter is involved in imatinib transportation into the cells. At the same time, the crucial physiological role of OCTN2 is cellular uptake of carnitine which is an essential co-factor for the mitochondrial β-oxidation pathway. This work investigates the impact of imatinib treatment on carnitine intake and energy metabolism of muscle cells. Methods: HTB-153 (human rhabdomyosarcoma) cell line and KCL-22 (CML cell line) were used to study the impact of imatinib treatment on intracellular levels of carnitine and vice versa. The energy metabolism changes in cells treated by imatinib were quantified and compared to changes in cells exposed to highly specific OCTN2 inhibitor vinorelbine. Mouse models were used to test whether in vitro observations are also achieved in vivo in thigh muscle tissue. The analytes of interest were quantified using a Prominence HPLC system coupled with a tandem mass spectrometer. Results: This work showed that through the carnitine-specific transporter OCTN2, imatinib and carnitine intake competed unequally and intracellular carnitine concentrations were significantly reduced. In contrast, carnitine preincubation did not influence imatinib cell intake or interfere with leukemia cell targeting. Blocking the intracellular supply of carnitine with imatinib significantly reduced the production of most Krebs cycle metabolites and ATP. However, subsequent carnitine supplementation rescued mitochondrial energy production. Due to specific inhibition of OCTN2 activity, the influx of carnitine was blocked and mitochondrial energy metabolism was impaired in muscle cells in vitro and in thigh muscle tissue in a mouse model. Conclusions: This preclinical experimental study revealed detrimental effect of imatinib on carnitine-mediated energy metabolism of muscle cells providing a possible molecular background of the frequently occurred side effects during imatinib therapy such as fatigue, muscle pain and cramps.

Background: Fatty acid oxidation (FAO) is a major energy-generating process in the mitochondria and supports proliferation, growth, and survival of cancer cells. l-Carnitine is an essential co-factor for carrying long-chain fatty acids into the mitochondria. The entry of l-carnitine across cell membrane is regulated by OCTN2 (SLC22A5). Thus, it can plays a significant role in the mitochondrial fatty acid oxidation. This study aimed to evaluate the OCTN2 expression and its association with clinicopathological characteristics in breast cancer. Methods: In this work, OCTN2 was examined in 54 pairs of fresh samples of breast cancer (BC) and adjacent noncancerous tissue using quantitative real-time polymerase chain reaction and immunohistochemistry (IHC). The IHC approach was also used to investigate the expression of additional clinicopathological features. Results: The present research findings revealed that the relative expression of OCTN2 in BC tissues was substantially higher than the adjacent normal tissues. This up-regulation was correlated positively with tumor size and Ki-67 and negatively with the progesterone receptor (PR) status, providing evidence of the opposite effects of OCTN2 and PR on tumor development. Conclusion: The study shows that the OCTN2 expression in BC patients may be used as a prognostic biomarker and a tumor oncogene. As a result, it could be considered a possible therapeutic target. Nevertheless, the significance of the findings needs to be confirmed by further studies. [ABSTRACT FROM AUTHOR]

Increasing skeletal muscle carnitine content can manipulate fuel metabolism and improve exercise performance. Intravenous insulin infusion during hypercarnitinemia increases plasma carnitine clearance and Na+‐dependent muscle carnitine accretion, likely via stimulating Na+/K+ ATPase pump activity. We hypothesized that the ingestion of high‐dose caffeine, also known to stimulate Na+/K+ ATPase activity, would stimulate plasma carnitine clearance during hypercarnitinemia in humans. In a randomized placebo‐controlled study, six healthy young adults (aged 24 ± 5 years, height 175 ± 8 cm, and weight 70 ± 13 kg) underwent three 5‐h laboratory visits involving the primed continuous intravenous infusion of l‐carnitine (CARN and CARN + CAFF) or saline (CAFF) in parallel with ingestion of caffeine (CARN + CAFF and CAFF) or placebo (CARN) at 0, 2, 3, and 4 h. Regular blood samples were collected to determine concentrations of blood Na+ and K+, and plasma carnitine and caffeine, concentrations. Caffeine ingestion (i.e., CAFF and CARN + CAFF conditions) and l‐carnitine infusion (i.e., CARN and CARN + CAFF) elevated steady‐state plasma caffeine (to ~7 μg·mL−1) and carnitine (to ~400 μmol·L−1) concentrations, respectively, throughout the 5 h infusions. Plasma carnitine concentration was ~15% lower in CARN + CAFF compared with CARN during the final 90 min of the infusion (at 210 min, 356 ± 96 vs. 412 ± 94 μmol·L−1; p = 0.0080: at 240 min, 350 ± 91 vs. 406 ± 102 μmol·L−1; p = 0.0079: and at 300 min, 357 ± 91 vs. 413 ± 110 μmol·L−1; p = 0.0073, respectively). Blood Na+ concentrations were greater in CAFF and CARN + CAFF compared with CARN. Ingestion of high‐dose caffeine stimulates plasma carnitine clearance during hypercarnitinemia, likely via increased Na+/K+ ATPase activity. Carnitine co‐ingestion with caffeine may represent a novel muscle carnitine loading strategy in humans, and therefore manipulate skeletal muscle fuel metabolism and improve exercise performance. [ABSTRACT FROM AUTHOR]

A prominent, safe and efficient therapy for patients with chronic myeloid leukemia (CML) is inhibiting oncogenic protein BCR::ABL1 in a targeted manner with imatinib, a tyrosine kinase inhibitor. A substantial part of patients treated with imatinib report skeletomuscular adverse events affecting their quality of life. OCTN2 membrane transporter is involved in imatinib transportation into the cells. At the same time, the crucial physiological role of OCTN2 is cellular uptake of carnitine which is an essential co-factor for the mitochondrial β-oxidation pathway. This work investigates the impact of imatinib treatment on carnitine intake and energy metabolism of muscle cells. HTB-153 (human rhabdomyosarcoma) cell line and KCL-22 (CML cell line) were used to study the impact of imatinib treatment on intracellular levels of carnitine and vice versa. The energy metabolism changes in cells treated by imatinib were quantified and compared to changes in cells exposed to highly specific OCTN2 inhibitor vinorelbine. Mouse models were used to test whether in vitro observations are also achieved in vivo in thigh muscle tissue. The analytes of interest were quantified using a Prominence HPLC system coupled with a tandem mass spectrometer. This work showed that through the carnitine-specific transporter OCTN2, imatinib and carnitine intake competed unequally and intracellular carnitine concentrations were significantly reduced. In contrast, carnitine preincubation did not influence imatinib cell intake or interfere with leukemia cell targeting. Blocking the intracellular supply of carnitine with imatinib significantly reduced the production of most Krebs cycle metabolites and ATP. However, subsequent carnitine supplementation rescued mitochondrial energy production. Due to specific inhibition of OCTN2 activity, the influx of carnitine was blocked and mitochondrial energy metabolism was impaired in muscle cells in vitro and in thigh muscle tissue in a mouse model. This preclinical experimental study revealed detrimental effect of imatinib on carnitine-mediated energy metabolism of muscle cells providing a possible molecular background of the frequently occurred side effects during imatinib therapy such as fatigue, muscle pain and cramps. [Display omitted] • Imatinib therapy of chronic myeloid leukemia outcompetes carnitine for OCTN2 cell intake. • Reduced carnitine cell intake impairs energy metabolism and harming muscle tissues. • Carnitine supplementation doesn't affect imatinib's influx or BCR::ABL1 inhibition. • High-dose carnitine rescued impaired mitochondrial energy metabolism after imatinib treatment. [ABSTRACT FROM AUTHOR]

Abstract Increasing skeletal muscle carnitine content can manipulate fuel metabolism and improve exercise performance. Intravenous insulin infusion during hypercarnitinemia increases plasma carnitine clearance and Na+‐dependent muscle carnitine accretion, likely via stimulating Na+/K+ ATPase pump activity. We hypothesized that the ingestion of high‐dose caffeine, also known to stimulate Na+/K+ ATPase activity, would stimulate plasma carnitine clearance during hypercarnitinemia in humans. In a randomized placebo‐controlled study, six healthy young adults (aged 24 ± 5 years, height 175 ± 8 cm, and weight 70 ± 13 kg) underwent three 5‐h laboratory visits involving the primed continuous intravenous infusion of l‐carnitine (CARN and CARN + CAFF) or saline (CAFF) in parallel with ingestion of caffeine (CARN + CAFF and CAFF) or placebo (CARN) at 0, 2, 3, and 4 h. Regular blood samples were collected to determine concentrations of blood Na+ and K+, and plasma carnitine and caffeine, concentrations. Caffeine ingestion (i.e., CAFF and CARN + CAFF conditions) and l‐carnitine infusion (i.e., CARN and CARN + CAFF) elevated steady‐state plasma caffeine (to ~7 μg·mL−1) and carnitine (to ~400 μmol·L−1) concentrations, respectively, throughout the 5 h infusions. Plasma carnitine concentration was ~15% lower in CARN + CAFF compared with CARN during the final 90 min of the infusion (at 210 min, 356 ± 96 vs. 412 ± 94 μmol·L−1; p = 0.0080: at 240 min, 350 ± 91 vs. 406 ± 102 μmol·L−1; p = 0.0079: and at 300 min, 357 ± 91 vs. 413 ± 110 μmol·L−1; p = 0.0073, respectively). Blood Na+ concentrations were greater in CAFF and CARN + CAFF compared with CARN. Ingestion of high‐dose caffeine stimulates plasma carnitine clearance during hypercarnitinemia, likely via increased Na+/K+ ATPase activity. Carnitine co‐ingestion with caffeine may represent a novel muscle carnitine loading strategy in humans, and therefore manipulate skeletal muscle fuel metabolism and improve exercise performance.

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. [ABSTRACT FROM AUTHOR]

The disadvantage of a traditional dosage regimen is the inability to deliver a sufficient drug concentration to the lesion site, which can result in adverse side effects due to nonspecific drug delivery. Actively targeting hepatic cells is a promising therapeutic strategy for liver disease. In this study, l-carnitine and a targeting peptide derived from the hepatitis B virus large envelope protein were used to modify liposomes for drug delivery to the liver through the sodium taurocholate cotransporting polypeptide (NTCP) and the organic cation/carnitine transporter 2 (OCTN2) receptors. Silybin was selected as the model drug. The solubility of silybin can reach 0.3 mg/mL after encapsulation in liposomes. The NTCP-specific and OCTN2-accelerated Myrcludex B and l-carnitine dual-modified liposomes were validated in vitro . The uptake of coumarin-6 in dual ligand-modified liposomes by hepatocytes was up to 2.36 μg/mg compared with unmodified liposomes (1.05 μg/mg). The pharmacokinetics and targeting abilities of various liposome formulations were evaluated in Kunming mice. Targeted liposomes increased the concentration of silybin and prolonged the drug's retention time in the liver. The area under the liver's pharmacokinetic curve of targeted liposomes was twice that of silybin injection, suggesting the promising application potential of silybin-loaded hepatotropic nanovesicles.

The aim of this study was to investigate the contributions of multiple transport mechanisms to the intestinal absorption of metformin, focusing on OCT3, PMAT, THTR2, SERT and OCTN2. We also assessed the impact of these transporters on the nonlinear absorption of metformin. Uptake studies with MDCKII cells expressing OCT3, PMAT, THTR2 or SERT confirmed that metformin is a substrate of these transporters. Decynium22 strongly inhibited metformin uptake mediated by all the transporters. 7-Cyclopentyl inhibited OCT3- and THTR2-mediated uptake of metformin. AG835, thiamine and paroxetine specifically inhibited PMAT-, THTR2- and SERT-mediated uptake of metformin, respectively. Using these inhibitors, the relative contributions of OCT3, PMAT, THTR2, SERT, OCTN2 and others to the intestinal permeation of metformin across Caco-2 cells were estimated to be 9.77%, 9.68%, 22.2%, 1.52%, 0% and 0.66%, respectively. Concentration-dependent analysis of metformin uptake by Caco-2 cells revealed nonlinear kinetics with the similar K m(app) value to the value for THTR2. Further in situ absorption study demonstrated that rat intestinal permeability of metformin was significantly decreased in the presence of decynium22, 7-cyclopentyl and thiamine. The present study indicated that THTR2 is the major determinant of the nonlinear absorption of metformin, although multiple transport mechanisms contribute to its intestinal absorption. [ABSTRACT FROM AUTHOR]

Abstract Background Primary carnitine deficiency due to mutations in the SLC22A5 gene is a rare but well-treatable metabolic disorder that puts patients at risk for metabolic decompensations, skeletal and cardiac myopathy and sudden cardiac death. Results We report on a 7-year-old boy diagnosed with primary carnitine deficiency 2 years after successful heart transplantation thanks his younger sister’s having been identified via expanded newborn screening during a pilot study evaluating an extension of the German newborn screening panel. Conclusion As L-carnitine supplementation can prevent and mostly reverse clinical symptoms of primary carnitine deficiency, all patients with cardiomyopathy should be investigated for primary carnitine deficiency even if newborn screening results were unremarkable.

Targeted nanocarriers have shown great promise in drug delivery because of optimized drug behavior and improved therapeutic efficacy. How to improve the targeting efficiency of nanocarriers for the maximum possible drug delivery is a critical issue. Here we developed L-carnitine-conjugated nanoparticles targeting the carnitine transporter OCTN2 on enterocytes for improved oral absorption. As a variable, we introduced various lengths of the polyethylene glycol linker (0, 500, 1000, and 2000) between the nanoparticle surface and the ligand (CNP, C5NP, C10NP and C20NP) to improve the ligand flexibility, and consequently for more efficient interaction with the transporter, to enhance the oral delivery of the cargo load into cells. An increased absorption was observed in cellular uptake in vitro and in intestinal perfusion assay in situ when the polyethylene glycol was introduced to link L-carnitine to the nanoparticles; the highest absorption was achieved with C10NP. In contrast, the linker decreased the absorption efficiency in vivo. As the presence or absence of the mucus layer was the primary difference between in vitro/in situ versus in vivo, the presence of this layer was the likely reason for this differential effect. In summary, the size of the polyethylene glycol linker improved the absorption in vitro and in situ, but interfered with the absorption in vivo. Even though this strategy of increasing the ligand flexibility with the variable size of the polyethylene glycol failed to increase oral absorption in vivo, this approach is likely to be useful for enhanced cellular uptake following intravenous administration of the nanocarriers.

Esophageal cancer is the ninth most common malignancy worldwide, ranking sixth in mortality. Platinum-based chemotherapy is commonly used for treating locally advanced esophageal cancer, yet it is ineffective in a large portion of patients. There is a need for reliable molecular markers with direct clinical application for a prospective selection of patients who can benefit from chemotherapy and patients in whom toxicity is likely to outweigh the benefit. The cytotoxic activity of platinum derivatives largely depends on the uptake and accumulation into cells, primarily by organic cation transporters (OCTs). The aim of the study was to investigate the impact of OCT expression on the clinical outcome of patients with esophageal cancer treated with oxaliplatin. Twenty patients with esophageal squamous cell carcinoma (SCC) were prospectively enrolled and surgical specimens used for screening OCT expression level by western blotting and/or immunostaining, and for culture of cancer cells. Sixty-seven patients with SCC who received oxaliplatin and for whom follow-up was available were retrospectively assessed for organic cation/carnitine transporter 2 (OCTN2) expression by real time RT-PCR and immunostaining. OCTN2 staining was also performed in 22 esophageal adenocarcinomas. OCTN2 function in patient-derived cancer cells was evaluated by assessing L-carnitine uptake and sensitivity to oxaliplatin. The impact of OCTN2 on oxaliplatin activity was also assessed in HEK293 cells overexpressing OCTN2. OCTN2 expression was higher in tumor than in normal tissues. In patient-derived cancer cells and HEK293 cells, the expression of OCTN2 sensitized to oxaliplatin. Patients treated with oxaliplatin who had high OCTN2 level in the tumor tissue had a reduced risk of recurrence and a longer survival time than those with low expression of OCTN2 in tumor tissue. In conclusion, OCTN2 is expressed in esophageal cancer and it is likely to contribute to the accumulation and cytotoxic activity of oxaliplatin in patients with esophageal carcinoma treated with oxaliplatin.

Esophageal cancer is the ninth most common malignancy worldwide, ranking sixth in mortality. Platinum-based chemotherapy is commonly used for treating locally advanced esophageal cancer, yet it is ineffective in a large portion of patients. There is a need for reliable molecular markers with direct clinical application for a prospective selection of patients who can benefit from chemotherapy and patients in whom toxicity is likely to outweigh the benefit. The cytotoxic activity of platinum derivatives largely depends on the uptake and accumulation into cells, primarily by organic cation transporters (OCTs). The aim of the study was to investigate the impact of OCT expression on the clinical outcome of patients with esophageal cancer treated with oxaliplatin. Twenty patients with esophageal squamous cell carcinoma (SCC) were prospectively enrolled and surgical specimens used for screening OCT expression level by western blotting and/or immunostaining, and for culture of cancer cells. Sixty-seven patients with SCC who received oxaliplatin and for whom follow-up was available were retrospectively assessed for organic cation/carnitine transporter 2 (OCTN2) expression by real time RT-PCR and immunostaining. OCTN2 staining was also performed in 22 esophageal adenocarcinomas. OCTN2 function in patient-derived cancer cells was evaluated by assessing L-carnitine uptake and sensitivity to oxaliplatin. The impact of OCTN2 on oxaliplatin activity was also assessed in HEK293 cells overexpressing OCTN2. OCTN2 expression was higher in tumor than in normal tissues. In patient-derived cancer cells and HEK293 cells, the expression of OCTN2 sensitized to oxaliplatin. Patients treated with oxaliplatin who had high OCTN2 level in the tumor tissue had a reduced risk of recurrence and a longer survival time than those with low expression of OCTN2 in tumor tissue. In conclusion, OCTN2 is expressed in esophageal cancer and it is likely to contribute to the accumulation and cytotoxic activity of oxaliplatin in patients with esophageal carcinoma treated with oxaliplatin. [ABSTRACT FROM AUTHOR]

Abstract Background Several inhaled drugs are dependent on organic cation transporters to cross cell membranes. To further evaluate their potential to impact on inhaled drug disposition, the localization of MATE1, P-gp, OCTN1 and OCTN2 were investigated in human lung. Methods Transporter proteins were analysed by immunohistochemistry in lung tissue from healthy subjects and COPD patients. Transporter mRNA was analysed by qPCR in lung tissue and in bronchoalveolar lavage (BAL) cells from smokers and non-smokers. Results We demonstrate for the first time MATE1 protein expression in the lung with localization to the apical side of bronchial and bronchiolar epithelial cells. Interestingly, MATE1 was strongly expressed in alveolar macrophages as demonstrated both in lung tissue and in BAL cells, and in inflammatory cells including CD3 positive T cells. P-gp, OCTN1 and OCTN2 were also expressed in the alveolar epithelial cells and in inflammatory cells including alveolar macrophages. In BAL cells from smokers, MATE1 and P-gp mRNA expression was significantly lower compared to cells from non-smokers whereas no difference was observed between COPD patients and healthy subjects. THP-1 cells were evaluated as a model for alveolar macrophages but did not reflect the transporter expression observed in BAL cells. Conclusions We conclude that MATE1, P-gp, OCTN1 and OCTN2 are expressed in pulmonary lung epithelium, in alveolar macrophages and in other inflammatory cells. This is important to consider in the development of drugs treating pulmonary disease as the transporters may impact drug disposition in the lung and consequently affect pharmacological efficacy and toxicity.

• This study reported the largest series of patients with PCD detected by NBS. • The incidence of newborns with PCD in Zhejiang province was 1:30,182. • Thirty-seven distinct variants were identified in SLC22A5 , of which 10 were novel. • c.1400C>G (p.S467C) was the most common variant in newborns and mothers with PCD. • c.760C>T (p.R254*) was rarely detected in the maternal PCD patients. Testing for primary carnitine deficiency (PCD) has been implemented in many newborn screening (NBS) programs, but few large-scale studies on NBS for PCD have been reported in China. This study aimed to assess the incidence and biochemical, clinical, and genetic characteristics of PCD discovered by NBS. Dried blood spots from newborns were analyzed by tandem mass spectrometry (MS/MS) and suspected positive patients were further tested using molecular genetic analysis. Infants who carried two variants in SLC22A5 or those with extremely low free carnitine levels during recall were referred for follow-up and treatment. Over 3.4 million newborns were screened and 113 newborns were diagnosed with PCD, yielding a positive predictive value of 1.93%. In addition, 63 mothers with PCD were identified. The incidence of PCD in newborns and mothers in Zhejiang was 1:30,182 and 1:54,137, respectively. Thirty-seven distinct variants were identified in SLC22A5 of which 10 were novel. c.1400C > G (p.S467C) was the most prevalent variant in both newborns and mothers with PCD, while c.760C > T (p.R254*), which is reportedly common in other Chinese regions, was rarely detected in maternal PCD patients. This study reports the largest series of patients with PCD detected by NBS and identifies 10 novel variants, expanding the variant spectrum of SLC22A5. [ABSTRACT FROM AUTHOR]

Background: Primary carnitine deficiency due to mutations in the SLC22A5 gene is a rare but well-treatable metabolic disorder that puts patients at risk for metabolic decompensations, skeletal and cardiac myopathy and sudden cardiac death.Results: We report on a 7-year-old boy diagnosed with primary carnitine deficiency 2 years after successful heart transplantation thanks his younger sister's having been identified via expanded newborn screening during a pilot study evaluating an extension of the German newborn screening panel.Conclusion: As L-carnitine supplementation can prevent and mostly reverse clinical symptoms of primary carnitine deficiency, all patients with cardiomyopathy should be investigated for primary carnitine deficiency even if newborn screening results were unremarkable. [ABSTRACT FROM AUTHOR]

[Display omitted] As a bioactive saponin derived from the seeds of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow, jujuboside B (JuB) shows great potential in anti-anxiety, anti-depression and improving learning and memory function. However, its oral bioavailability is very poor. In this study, a novel drug-loading nanoparticles system was prepared with polyethylene glycol and polylactic-co-glycolic acid copolymer (PEG-PLGA), and further modified with L-carnitine (LC) to target intestinal organic cation/carnitine transporter 2 (OCTN2) to improve the oral absorption of JuB. Under the optimized preparation conditions, the particle sizes of obtained JuB-PEG-PLGA nanoparticles (B-NPs) and LC modified B-NPs (LC-B-NPs) were 110.67 ± 11.37 nm and 134.00 ± 2.00 nm with the entrapment efficiency (EE%) 73.46 ± 1.26 % and 76.01 ± 2.10 %, respectively. The pharmacokinetics in SD rats showed that B-NPs and LC-B-NPs increased the bioavailability of JuB to 134.33 % and 159.04 % respectively. In Caco-2 cell model, the prepared nanoparticles significantly increased cell uptake of JuB, which verified the pharmacokinetic results. The absorption of LC-B-NPs mainly depended on OCTN2 transporter, and Na+ played an important role. Caveolin and clathrin were involved in the endocytosis of the two nanoparticles. In conclusion, both B-NPs and LC-B-NPs can improve the oral absorption of JuB, and the modification of LC can effectively target the OCTN2 transporter. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX) is a widely used antitumor agent; however, its clinical application is limited by dose-related organ damage. Because organic cation/carnitine transporters (OCTN1 and OCTN2), which are critical for DOX uptake, are highly expressed in hepatocytes, we aimed to elucidate the role of these transporters in hepatic DOX uptake. The results indicated that inhibitors and RNA interference both significantly reduced DOX accumulation in HepG2 and HepaRG cells, suggesting that OCTN1/2 contribute substantially to DOX uptake by hepatocytes. To determine whether metformin (MET, an inhibitor of OCTN1 and OCTN2) ameliorates DOX-induced hepatotoxicity, we conducted in vitro and in vivo studies. MET (1–100 μM) inhibited DOX (500 nM) accumulation and cytotoxicity in vitro in a concentration-dependent manner. Furthermore, intravenous MET administration at 250 or 500 mg/kg or by gavage at 50, 100, or 200 mg/kg reduced DOX (8 mg/kg) accumulation in a dose-dependent manner in the mouse liver and attenuated the release of alanine aminotransferase, aspartate aminotransferase, and carboxylesterase 1. Additionally, MET reduced the distribution of DOX in the heart, liver, and kidney and enhanced the urinary elimination of DOX; however, it did not increase the nephric toxicity of DOX. In conclusion, our study demonstrated that MET alleviates DOX hepatotoxicity by inhibiting OCTN1- and OCTN2-mediated DOX uptake in vitro (mouse hepatocytes and HepaRG or HepG2 cells) and in mice. [Display omitted] • OCTN1/2 contributed to the uptake of DOX in liver cell lines. • MET decreased the hepatic DOX accumulation and improved hepatotoxicity. • MET decreased the distribution of DOX in the heart, liver and kidney. • MET increased the urinary elimination of DOX without nephrotoxicity increase. [ABSTRACT FROM AUTHOR]

l-Carnitine, obligatory for oxidation of fatty acids, is transported into cells by the Na+-coupled transporter OCTN2 and the Na+/Cl–-coupled transporter ATB0,+. Here we investigated the potential of L-carnitine-conjugated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (LC-PLGA NPs) to deliver chemotherapeutic drugs into cancer cells by targeting the nanoparticles to both OCTN2 and ATB0,+. The cellular uptake of LC-PLGA NPs in the breast cancer cell line MCF7 and the colon cancer cell line Caco-2 was increased compared to unmodified nanoparticles, but decreased in the absence of co-transporting ions (Na+ and/or Cl–) or in the presence of competitive substrates for the two transporters. Studies with fluorescently labeled nanoparticles showed their colocalization with both OCTN2 and ATB0,+, confirming the involvement of both transporters in the cellular uptake of LC-PLGA NPs. As the expression levels of OCTN2 and ATB0,+ are higher in colon cancer cells than in normal colon cells, LC-PLGA NPs can be used to deliver chemotherapeutic drugs selectively into cancer cells for colon cancer therapy. With 5-fluorouracil-loaded LC-PLGA NPs, we were able to demonstrate significant increases in the uptake efficiency and cytotoxicity in colon cancer cells that were positive for OCTN2 and ATB0,+. In a 3D spheroid model of tumor growth, LC-PLGA NPs showed increased uptake and enhanced antitumor efficacy. These findings indicate that dual-targeting LC-PLGA NPs to OCTN2 and ATB0,+ has great potential to deliver chemotherapeutic drugs for colon cancer therapy. Dual targeting LC-PLGA NPs to OCTN2 and ATB0,+ can selectively deliver chemotherapeutics to colon cancer cells where both transporters are overexpressed, preventing targeting to normal cells and thus avoiding off-target side effects.

• Olanzapine up-regulates hepatic FABP1 and FATP2 expression in livers of female mice. • Olanzapine down-regulates OCTN2 expression in livers of male mice. • Olanzapine inhibits hepatic OCTN2 activity and decreases hepatic L-carnitine level. • L-carnitine supplementation attenuates olanzapine-induced simple steatosis. Olanzapine, a representative of antipsychotics, is a first-line drug for treatment of schizophrenia. However, olanzapine-induced liver steatosis limits its clinical utilization. This study is to explore the mechanism of liver steatosis induced by olanzapine based on the regulation of transporters involved in uptake and oxidation of fatty acids. Our results revealed that 12-week oral administration of olanzapine increased hepatic triglyceride(TG), caused liver steatosis. Our further studies showed that the expression of fatty acid transporter 2(FATP2) and fatty acid binding protein 1(FABP1) were up-regulated in liver of female mice after 12-week olanzapine exposure, as well as in primary mouse hepatocytes treated with olanzapine. Olanzapine treatment also reduced hepatic β-hydroxybutyrate level (indicator of fatty acid β-oxidation), meanwhile, the L-carnitine (L-Car) concentration in liver of olanzapine group was significantly lower than that in control group. Further study demonstrated that both mRNA and protein expression of hepatic OCTN2 (carnitine/organic cation transporter 2) were obviously down-regulated in male mice after 12-week olanzapine treatment. Also, olanzapine markedly inhibited L-Car uptake in MDCK-hOCTN2 cells (1.06 μM of IC 50), HepG2 cells and primary mouse hepatocytes. Supplementation of L-Car attenuated hepatic TG rise and improved simple steatosis in olanzapine treatment mice. Taken together, up-regulation of FATP2/FABP1 and down-regulation/inhibition of hepatic OCTN2 probably contribute to olanzapine-induced liver steatosis. Supplementation of L-Car is a promising strategy to attenuate olanzapine-induced simple steatosis. [ABSTRACT FROM AUTHOR]

Glucocorticoid (GC) resistance is associated with poor response to the following chemotherapy in lymphoid malignancies, such as lymphoma and leukemia. However, it remains unclear whether GCs interfere with the cytotoxic effects of anti-cancer drugs on GC-resistant cells. In this study, we examined whether GCs affected the sensitivities to vincristine (VCR)/doxorubicin (DOX) and the expression of drug transporters in GC-resistant cells. The dexamethasone (DEX)/prednisolone (PSL)-resistant lymphoid and non-lymphoid cell lines Raji and HL60 were cultured with DEX for 7 days and then treated with VCR or DOX for 3 days. Seven days of DEX treatment increased the IC50s of both VCR and DOX in Raji cells but not in HL60 cells. The mRNA and protein expression levels of organic cation/carnitine transporter (OCTN) 2, one of the drug uptake transporters expressed in both cell lines, were decreased only in Raji cells. When Raji cells were cultured with PSL, the IC50 of DOX but not VCR increased as the expression of OCTN2 decreased. No significant increases in efflux transporter expression were induced by DEX or PSL. When siRNA against OCTN2 was introduced into Raji cells, the IC50 of DOX but not VCR increased significantly. These data suggested that both DEX and PSL decreased the sensitivity of the DEX/PSL-resistant Raji cells to DOX, a change that was at least partially due to reductions in OCTN2. Thus, the continuous usage of GCs may interfere with the effects of chemotherapy on GC-resistant lymphoid cells. [ABSTRACT FROM AUTHOR]

Abstract Clozapine, an atypical antipsychotic drug, is widely utilized for the treatment of schizophrenia; however, clozapine-induced metabolic disorders, such as fatty liver and weight gain, warrant increased attention. Considering the crucial role of l -carnitine (L-Car) in fatty acid oxidation and carnitine/organic cation transporter (OCTN) 2 in renal reabsorption of L-Car, we aimed to study whether clozapine-induced liver lipid metabolic disorder is associated with L-Car dysregulation via inhibition/down-regulation of renal OCTN2. Our results reveal that clozapine inhibits L-Car uptake in MDCK-hOCTN2 cells with an IC 50 value of 1.78 μM. Additionally, clozapine significantly reduces the uptake of L-Car in HK-2 cells, mouse primary cultured proximal tubular (mPCPT) cells and HepG2 cells. Acute (intraperitoneal injection) and 21-day successive oral administration of clozapine at 12.5, 25, and 50 mg/kg to mice resulted in 2–3-fold greater renal excretion of L-Car than in the vehicle group, and the concentration of L-Car in plasma and liver was significantly decreased. Concomitantly, mRNA and protein levels of mOctn2 in the kidney were markedly down regulated. Additionally, 28-day oral administration of clozapine induced increased triglyceride (TG) and total cholesterol (TCHO) levels in mouse livers, while L-Car (40 mg/kg - 1 g/kg) attenuated clozapine-induced liver TG and TCHO increase in a dose-dependent manner. These results indicate that clozapine-induced reduction of L-Car reabsorption via inhibition/down-regulation of renal OCTN2 contributes to liver lipid metabolic disorder. L-Car supplementation is probably an effective strategy to attenuate clozapine-induced abnormal lipid metabolism. Highlights • Clozapine inhibits OCTN2 activity and down-regulates OCTN2 expression in the kidney. • Clozapine decreases renal reabsorption and increases urinary excretion of L-Car. • Clozapine reduces plasma and liver L-Car levels and induces fatty liver in mice. • L-Car supplementation attenuates clozapine-induced lipid accumulation in the liver. [ABSTRACT FROM AUTHOR]

The Metabolic reprogramming of tumor cells plays a vital role in the progression of hepatocellular carcinoma. Organic cation/carnitine transporter 2 (OCTN2), a sodium-ion dependent carnitine transporter and a sodium-ion independent tetraethylammonium (TEA) transporter, has been reported to contribute tumor malignancies and metabolic dysregulation in renal and esophageal carcinoma. However, the role of lipid metabolism deregulation mediated by OCTN2 in HCC cells has not been clarified. Bioinformatics analyses and immunohistochemistry assay were employed to identify OCTN2 expression in HCC tissues. The correlation between OCTN2 expression and prognosis was elucidated through K-M survival analysis. The expression and function of OCTN2 were examined via the assays of western blotting, sphere formation, cell proliferation, migration and invasion. The mechanism of OCTN2-mediated HCC malignancies was investigated through RNA-seq and metabolomic analyses. Furthermore, xenograft tumor models based on HCC cells with different OCTN2 expression levels were conducted to analyze the tumorigenic and targetable role of OCTN2 in vivo. We found that gradually focused OCTN2 was significantly upregulated in HCC and tightly associated with poor prognosis. Additionally, OCTN2 upregulation promoted HCC cells proliferation and migration in vitro and augmented the growth and metastasis of HCC. Moreover, OCTN2 promoted the cancer stem-like properties of HCC by increasing fatty acid oxidation and oxidative phosphorylation. Mechanistically, PGC-1α signaling participated in the HCC cancer stem-like properties mediated by OCTN2 overexpression, which is confirmed by in vitro and in vivo analyses. Furthermore, OCTN2 upregulation may be transcriptionally activated by YY1 in HCC. Particularly, treatment with mildronate, an inhibitor of OCTN2, showed a therapeutic influence on HCC in vitro and in vivo. Our findings demonstrate that OCTN2 plays a critical metabolic role in HCC cancer stemness maintenance and HCC progression, providing evidence for OCTN2 as a promising target for HCC therapy. [Display omitted] • OCTN2 is frequently upregulated in HCC and tightly associated with poor prognosis. • OCTN2 upregulation promotes HCC cells proliferation and migration in vitro and in vivo. • OCTN2 promotes the cancer stem-like properties of HCC by increasing fatty acid oxidation (FAO) and OXPHOS. • PGC-1α signaling participated in HCC cancer stem-like properties mediated by OCTN2 overexpression. • Mildronate, an inhibitor of OCTN2, showed a therapeutic influence on HCC in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Overcoming blood-brain barrier (BBB) and targeting tumor cells are two key steps for glioma chemotherapy. By taking advantage of the specific expression of Na+-coupled carnitine transporter 2 (OCTN2) on both brain capillary endothelial cells and glioma cells, L-carnitine conjugated poly(lactic-co-glycolic acid) nanoparticles (LC-PLGA NPs) were prepared to enable enhanced BBB permeation and glioma-cell targeting. Conjugation of L-carnitine significantly enhanced the uptake of PLGA nanoparticles in the BBB endothelial cell line hCMEC/D3 and the glioma cell line T98G. The uptake was dependent on Na+ and inhibited by the excessive free L-carnitine, suggesting involvement of OCTN2 in the process. In vivo mouse studies showed that LC-PLGA NPs resulted in high accumulation in the brain as indicated by the biodistribution and imaging assays. Furthermore, compared to Taxol and paclitaxel-loaded unmodified PLGA NPs, the drug-loaded LC-PLGA NPs showed improved anti-glioma efficacy in both 2D-cell and 3D-spheroid models. The PEG spacer length of the ligand attached to the nanoparticles was optimized, and the formulation with PEG1000 (LC-1000-PLGA NPs) showed the maximum targeting efficiency. We conclude that L-carnitine-mediated cellular recognition and internalization via OCTN2 significantly facilitate the transcytosis of nanoparticles across BBB and the uptake of nanoparticles in glioma cells, resulting in improved anti-glioma efficacy. [ABSTRACT FROM AUTHOR]

Carnitine is well recognized for transporting fatty acids across the mitochondrial membrane; however, studies conducted in the last decade have highlighted another role of carnitine in buffering the excess of mitochondrial acetyl-CoA, an intermediate metabolite of betaoxidation and glycolytic metabolism. Although acetyl-CoA is an essential metabolite, excess accumulation of acetyl-CoA inhibits pyruvate dehydrogenase, resulting in negative regulation of glucose uptake. In this situation, carnitine binds to acetyl-CoA and is converted to acetylcarnitine, resulting in a decrease in acetyl-CoA levels. It has been demonstrated that carnitine acetylation is essential for glucose homeostasis, and that its dysfunction, caused by aging and high-fat feeding, induces metabolic failure. To analyze carnitine dynamics in skeletal muscle, we have used imaging mass spectrometry to visualize the distribution of acetylcarnitine in rodent skeletal muscle and performed tracing experiments using isotopic labeled carnitine. It was shown that carnitine uptake and acetylation were elevated in oxidative muscles, and that they were dynamically controlled by muscle contraction. Recent studies using cell culture experiments demonstrated that acetylcarnitine is exported from skeletal muscle cells. It is hypothesized that muscle carnitine acetylation in skeletal muscle is not only beneficial for buffering the excess acetyl-CoA, but also plays in the endocrine system. In fact, previous work has shown that plasma acetylcarnitine concentration increases during exercise in humans, suggesting that the acetylcarnitine produced during muscle contraction may be released from muscle cells to serve different functions. In this article, we reviewed the novel roles of carnitine in skeletal muscle by analyzing carnitine dynamics. [ABSTRACT FROM AUTHOR]

Introduction Mitochondrial dysfunction, oxidative stress and energy production have been implicated in the etiology of Parkinson’s disease (PD). Several agents are under investigation for potential neuroprotective effects including acetyl- l -carnitine (ALC). Objective To investigate whether low carnitine levels and mutations in the SLC22A5 gene encoding the carnitine transporter are associates with PD risk in the Faroe Islands where the prevalence of both PD and carnitine transporter deficiency (CTD) is high. Methods We conducted a case-control study with 121 cases and 235 randomly selected controls, matched by gender and age. Blood spots were analyzed for free and total carnitine levels by QUATTRO LC triple quadrupole mass spectrometry (MS/MS) and sequencing performed for five genetic mutations in the SLC22A5 gene with ABI PRISM 3130. Results PD cases had significantly lower levels of free and total carnitine levels compared with controls (P < .001). However, stratifying according to mutation status, the lower levels of carnitine was only evident among the non-mutation carriers. Specifically, no difference was found in c.95A > G mutation frequency in the SLC22A5 gene among cases and controls (p = .70). Conclusion Low carnitine levels seem to be associated with PD, but only in individuals without the c.95A > G mutation rendering the carnitine transporter less efficient. Thus, the difference in carnitine levels is not caused by a higher frequency of c.95A > G mutation carriers in cases. The cause may be dietary or due to different gut microbiota among cases. [ABSTRACT FROM AUTHOR]

Systemic primary carnitine deficiency (SPCD) is an autosomal recessive inborn error of fatty acid metabolism caused by a defect in the transporter responsible for moving carnitine across plasma membrane. The clinical features of SPCD vary widely based on the age of onset and organs involved. During infancy, patients might show episodes of hypoketotic hypoglycemia, hepatomegaly, elevated transaminases, and hyperammonemia. Skeletal myopathy, elevated creatine kinase, and cardiomyopathy are the main manifestations in children with SPCD, while in adults, the disorder is usually manifested as cardiomyopathy, arrhythmias, or fatigability. Here, we report a 5-year-old boy with SPCD that presented as dilated cardiomyopathy with atypical features, such as anemia, respiratory distress, and proximal muscle weakness. This report supports considering carnitine deficiency treatment in the work-up of unexplained pediatric dilated cardiomyopathy. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX) has been widely used to treat various tumors; however, DOX-induced cardiotoxicity limits its utilization. Since high accumulation of DOX in cardiomyocytes/mitochondria is the key reason, we aimed to clarify the mechanisms of DOX uptake and explore whether selectively inhibiting DOX uptake transporters would attenuate DOX accumulation and cardiotoxicity. Our results demonstrated that OCTN1/OCTN2/PMAT (organic cation/carnitine transporter 1/2 or plasma membrane monoamine transporter), especially OCTN2, played crucial roles in DOX uptake in cardiomyocytes, while OCTN2 and OCTN1 contributed to DOX transmembrane transport in mitochondria. Metformin (1–100 μM) concentration-dependently reduced DOX (5 μM for accumulation, 500 nM for cytotoxicity) concentration and toxicity in cardiomyocytes/mitochondria via inhibition of OCTN1-, OCTN2- and PMAT-mediated DOX uptake but did not affect its efflux. Furthermore, metformin (iv : 250 and 500 mg/kg or ig : 50, 100 and 200 mg/kg) could dose-dependently reduce DOX (8 mg/kg) accumulation in mouse myocardium and attenuated its cardiotoxicity. In addition, metformin (1–100 μM) did not impair DOX efficacy in breast cancer or leukemia cells. In conclusion, our study clarified the role of multiple transporters, especially OCTN2, in DOX uptake in cardiomyocytes/mitochondria; metformin alleviated DOX-induced cardiotoxicity without compromising its antitumor efficacy by selective inhibition of multiple transporters mediated DOX accumulation in myocardium/mitochondria. [Display omitted] • OCTN1, OCTN2 and PMAT, especially OCTN2 mediate DOX uptake in myocardium. • OCTN2 and OCTN1 contribute to DOX uptake in myocardial mitochondria. • Metformin alleviates DOX cardiotoxicity via inhibiting its uptake both in myocardium and mitochondria. • Metformin attenuates DOX-induced cardiotoxicity without impairing its antineoplastic effect. [ABSTRACT FROM AUTHOR]

Background: The prevalence of primary carnitine deficiency (PCD) in the Faroe Islands is the highest reported in the world (1:300). Serious symptoms related to PCD, e.g. sudden death, have previously only been associated to the c.95A > G/c.95A > G genotype in the Faroe Islands. We report and characterize novel mutations associated with PCD in the Faroese population and report and compare free carnitine levels and OCTN2 transport activities measured in fibroblasts from PCD patients with different genotypes. Methods: Genetic analyses were used to identify novel mutations, and carnitine uptake analyses in cultured skin fibroblasts from selected patients were used to examine residual OCTN2 transporter activities of the various genotypes. Results: Four different mutations, including the unpublished c.131C > T (p.A44V), the novel splice mutation c.825-52G > A and a novel risk-haplotype (RH) were identified in the Faroese population. The two most prevalent genotypes were c.95A > G/RH (1:600) and c.95A > G/c.95A > G (1:1300). Patients homozygous for the c.95A > G mutation had both the significantly (p G/c.95A > G genotype had the significantly lowest mean free carnitine level and residual OCTN2 transporter activity.

The carnitine transporter OCTN2 is associated with asthma and other inflammatory diseases. The aims of this work were (i) to determine carnitine uptake into freshly isolated human alveolar type I (ATI)-like epithelial cells in primary culture, (ii) to compare the kinetics of carnitine uptake between respiratory epithelial in vitro cell models, and (iii) to establish whether any cell line was a suitable model for studies of carnitine transport at the air-blood barrier. Levels of time-dependent [3H]-acetyl-l-carnitine uptake were similar in ATI-like, NCl-H441, and Calu-3 epithelial cells, whereas uptake into A549 cells was ~5 times higher. Uptake inhibition was more pronounced by OCTN2 modulators, such as l-Carnitine and verapamil, in ATI-like primary epithelial cells compared to NCl-H441 and Calu-3 epithelial cells. Our findings suggest that OCTN2 is involved in the cellular uptake of acetyl-l-carnitine at the alveolar epithelium and that none of the tested cell lines are optimal surrogates for primary cells.

Primary carnitine deficiency is caused by a defect in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. It can cause hypoketotic hypoglycemia or cardiomyopathy in children, and sudden death in children and adults. Fibroblasts from affected patients have reduced carnitine transport. We evaluated carnitine transport in fibroblasts from 358 subjects referred for possible carnitine deficiency. Carnitine transport was reduced to 20% or less of normal in fibroblasts of 140 out of 358 subjects. Sequencing of the 10 exons and flanking regions of the SLC22A5 gene in 95 out of 140 subjects identified causative variants in 84% of the alleles. The missense variants identified in our patients and others previously reported ( n = 92) were expressed in CHO cells. Carnitine transport was impaired by 73 out of 92 variants expressed. Prediction algorithms (Polyphen-2, SIFT) correctly predicted the functional effects of expressed variants in about 80% of cases. These results indicate that mutations in the coding region of the SLC22A5 gene cannot be identified in about 16% of the alleles causing primary carnitine deficiency. Prediction algorithms failed to determine the functional effects of amino acid substitutions in this transmembrane protein in about 20% of cases. Therefore, functional studies in fibroblasts remain the best strategy to confirm or exclude a diagnosis of primary carnitine deficiency. [ABSTRACT FROM AUTHOR]

Carnitine/organic cation transporter 2 (OCTN2) is localized at the basolateral membrane of epididymal epithelial cells, and mainly serves to reabsorb carnitine as an essential factor for sperm maturation; however, its functional features in epididymal epithelial cells have remained unclear. We isolated primary epididymal epithelial cells from rat epididymides and verified their phenotype by detecting the presence of cytokeratin-19 (CK-19, an epithelial cell marker) and the absence vimentin (an interstitial cell marker). We found that cultured epididymal epithelial cells isolated from rat epididymides expressed high levels of CK-19 but barely expressed vimentin. Gain-of-function assays, which included the CCK-8 assay and EdU flow cytometry assay, indicated that overexpression of OCTN2 significantly promoted epididymal epithelial cell growth and proliferation. Moreover, forced expression of OCTN2 inhibited the cell apoptosis process, and at the same time increased expression of the pro-apoptosis factor BAX, and decreased expression of the anti-apoptosis factors BCL-2 and Survivin. Furthermore, we also found that OCTN2 overexpression dramatically increased the levels of biomarkers associated with spermatogenesis, including azoospermia-like (DAZL), phosphoglycerate kinase 2 (PGK2), and protamine 2 (PRM2). These results demonstrate that OCTN2 plays a positive role in epididymal epithelial cells, and might be useful in the clinical treatment of male infertility by serving as a key regulatory factor. [ABSTRACT FROM AUTHOR]

l-Carnitine, obligatory for oxidation of fatty acids, is transported into cells by the Na+-coupled transporter OCTN2 and the Na+/Cl–-coupled transporter ATB0,+. Here we investigated the potential of L-carnitine-conjugated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (LC-PLGA NPs) to deliver chemotherapeutic drugs into cancer cells by targeting the nanoparticles to both OCTN2 and ATB0,+. The cellular uptake of LC-PLGA NPs in the breast cancer cell line MCF7 and the colon cancer cell line Caco-2 was increased compared to unmodified nanoparticles, but decreased in the absence of co-transporting ions (Na+and/or Cl–) or in the presence of competitive substrates for the two transporters. Studies with fluorescently labeled nanoparticles showed their colocalization with both OCTN2 and ATB0,+, confirming the involvement of both transporters in the cellular uptake of LC-PLGA NPs. As the expression levels of OCTN2 and ATB0,+are higher in colon cancer cells than in normal colon cells, LC-PLGA NPs can be used to deliver chemotherapeutic drugs selectively into cancer cells for colon cancer therapy. With 5-fluorouracil-loaded LC-PLGA NPs, we were able to demonstrate significant increases in the uptake efficiency and cytotoxicity in colon cancer cells that were positive for OCTN2 and ATB0,+. In a 3D spheroid model of tumor growth, LC-PLGA NPs showed increased uptake and enhanced antitumor efficacy. These findings indicate that dual-targeting LC-PLGA NPs to OCTN2 and ATB0,+has great potential to deliver chemotherapeutic drugs for colon cancer therapy. Dual targeting LC-PLGA NPs to OCTN2 and ATB0,+ can selectively deliver chemotherapeutics to colon cancer cells where both transporters are overexpressed, preventing targeting to normal cells and thus avoiding off-target side effects. [ABSTRACT FROM AUTHOR]

l-Carnitine is essential for translocation of fatty acids for their mitochondrial β-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, SG2 NA, 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 SG2 NA, zinedin, and catalytical subunit, although co-precipitation, immunocytochemistry, and proximity ligation assay experiments showed that only the amount of SG2 NA 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 SG2 NA, 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. [ABSTRACT FROM AUTHOR]

Carnitine is essential for the transfer of long-chain fatty acids across the inner mitochondrial membrane for subsequent β-oxidation. It can be synthesized by the body or assumed with the diet from meat and dairy products. Defects in carnitine biosynthesis do not routinely result in low plasma carnitine levels. Carnitine is accumulated by the cells and retained by kidneys using OCTN2, a high affinity organic cation transporter specific for carnitine. Defects in the OCTN2 carnitine transporter results in autosomal recessive primary carnitine deficiency characterized by decreased intracellular carnitine accumulation, increased losses of carnitine in the urine, and low serum carnitine levels. Patients can present early in life with hypoketotic hypoglycemia and hepatic encephalopathy, or later in life with skeletal and cardiac myopathy or sudden death from cardiac arrhythmia, usually triggered by fasting or catabolic state. This disease responds to oral carnitine that, in pharmacological doses, enters cells using the amino acid transporter B 0,+ . Primary carnitine deficiency can be suspected from the clinical presentation or identified by low levels of free carnitine (C0) in the newborn screening. Some adult patients have been diagnosed following the birth of an unaffected child with very low carnitine levels in the newborn screening. The diagnosis is confirmed by measuring low carnitine uptake in the patients' fibroblasts or by DNA sequencing of the SLC22A5 gene encoding the OCTN2 carnitine transporter. Some mutations are specific for certain ethnic backgrounds, but the majority are private and identified only in individual families. Although the genotype usually does not correlate with metabolic or cardiac involvement in primary carnitine deficiency, patients presenting as adults tend to have at least one missense mutation retaining residual activity. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou. [ABSTRACT FROM AUTHOR]

Many phytochemicals exert activities as agonists of peroxisome proliferator-activated receptor gamma (PPARγ). This study aims to investigate whether phytochemicals are agonists of the PPARγ/RXRα pathway and modulate the target gene OCTN2. In this study, a luciferase reporter gene system was used to screen novel OCTN2 activators from 39 phytochemicals. Kaempferol, curcumin, and puerarin were found to show the significant PPRE-mediated luciferase activities (> 150%) at 20 μM and showed a dose-dependent manner. Phytochemicals also elevated the mRNA and protein expression of OCTN2 in a dose-dependent fashion in colorectal cancer SW480 cells. These induction effects were gradually inhibited by PPARγ antagonist GW9662 in the luciferase reporter gene system and in SW480 cells. Moreover, the results of cell viability assay imply that three phytochemicals probably induce OCTN2 expression leading to the enhanced uptake of its substrate, oxaliplatin, thereby making cells more sensitive to oxaliplatin. The molecular docking study showed the possible binding sites of phytochemicals in PPARγ protein, and all of the docked phytochemicals fitted the same active pocket in PPARγ as troglitazone. All three phytochemicals exhibited hydrogen bonds between their polar moieties and the amino acid residues. Thus, we identified three phytochemicals as PPARγ ligands, which potentiated the expression and activity of OCTN2. [ABSTRACT FROM AUTHOR]

Background Organic cation transporters transfer solutes with a positive charge across the plasma membrane. The novel organic cation transporter 1 (OCTN1) and 2 (OCTN2) transport ergothioneine and carnitine, respectively. Mutations in the SLC22A5 gene encoding OCTN2 cause primary carnitine deficiency, a recessive disorders resulting in low carnitine levels and defective fatty acid oxidation. Variations in the SLC22A4 gene encoding OCTN1 are associated with rheumatoid arthritis and Crohn disease. Methods Here we evaluate the functional properties of the OCTN1 transporter using chimeric transporters constructed by fusing different portion of the OCTN1 and OCTN2 cDNAs. Their relative abundance and subcellular distribution was evaluated through western blot analysis and confocal microscopy. Results Substitutions of the C-terminal portion of OCTN1 with the correspondent residues of OCTN2 generated chimeric OCTN transporters more active than wild-type OCTN1 in transporting ergothioneine. Additional single amino acid substitutions introduced in chimeric OCTN transporters further increased ergothioneine transport activity. Kinetic analysis indicated that increased transport activity was due to an increased V max , with modest changes in K m toward ergothioneine. Conclusions Our results indicate that the OCTN1 transporter is tolerant to extensive amino acid substitutions. This is in sharp contrast to the OCTN2 carnitine transporter that has been selected for high functional activity through evolution, with almost all substitutions reducing carnitine transport activity. General significance The widespread tolerance of OCTN1 to amino acid substitutions suggests that the corresponding SLC22A4 gene may have derived from a recent duplication of the SLC22A5 gene and might not yet have a defined physiological role. [ABSTRACT FROM AUTHOR]

Carnitine plays a physiologically important role in the β-oxidation of fatty acids, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane. Distribution of carnitine within the body tissues is mainly performed by novel organic cation transporter (OCTN) family, including the isoforms OCTN1 (SLC22A4) and OCTN2 (SLC22A5) expressed in human. We performed here a characterization of carnitine transport in human airway epithelial cells A549, Calu-3, NCl-H441, and BEAS-2B, by means of an integrated approach combining data of mRNA/protein expression with the kinetic and inhibition analyses of L-[ 3 H]carnitine transport. Carnitine uptake was strictly Na + -dependent in all cell models. In A549 and BEAS-2B cells, carnitine uptake was mediated by one high-affinity component (K m < 2 μM) identifiable with OCTN2. In both these cell models, indeed, carnitine uptake was maximally inhibited by betaine and strongly reduced by SLC22A5/OCTN2 silencing. Conversely, Calu-3 and NCl-H441 exhibited both a high (K m ~ 20 μM) and a low affinity (K m > 1 mM) transport component. While the high affinity component is identifiable with OCTN2, the low affinity uptake is mediated by ATB 0,+ , a Na + , and Cl – -coupled transport system for neutral and cationic amino acids, as demonstrated by the inhibition by leucine and arginine, as well as by SLC6A14/ATB 0,+ silencing. The presence of this transporter leads to a massive accumulation of carnitine inside the cells and may be of peculiar relevance in pathologic conditions of carnitine deficiency, such as those associated to OCTN2 defects. [ABSTRACT FROM AUTHOR]

Primary carnitine deficiency is caused by the defective OCTN2 carnitine transporter encoded by the SLC22A5 gene. A lack of carnitine impairs fatty acid oxidation resulting in hypoketotic hypoglycemia, hepatic encephalopathy, skeletal and cardiac myopathy, and arrhythmia. This condition can be detected by finding low levels of free carnitine (C0) in neonatal screening. Mothers with primary carnitine deficiency can also be identified by low carnitine levels in their infant by newborn screening. Primary carnitine deficiency is rare (1:40,000–1:140,000 newborns) except in the Faroe Islands (1:300) due to a founder effect. A specific mutation (c.95A>G, p.N32S) is prevalent, but not unique, with three additional mutations (c.131C>T/p.A44V, a splice mutation c.825-52G>A, and a risk-haplotype) recently identified in the Faroese population. In the Faroe Islands, several adult patients suffered sudden death from primary carnitine deficiency leading to the implementation of a nationwide population screening (performed after 2 months of age) in addition to universal neonatal screening. While most affected infants can be identified at birth, some patients with primary carnitine deficiency might be missed by the current neonatal screening and could be better identified with a repeated test performed after 2 months of age.

Imazalil (IMZ) is a fungicide recommended by the Chinese ministry of agriculture. However, recent study was observed high level of IMZ by dietary exposure in pregnant women. To determine the cross-generational effects, C57BL/6 mice were exposed to IMZ at dietary levels of 0, 0.025‰, and 0.25‰ during the gestation and lactation periods. Then, we assessed the changes in growth phenotypes, carnitine levels, and gut microbiota in F 0 , F 1 or F 2 generations. The growth phenotypes of dams didn't observe significant difference, but there were significant changes in the offspring. Plasma samples revealed low levels of free carnitine (C0), long-chain acyl-carnitines and total carnitine. In particular, C0 may be regarded as relatively potential, specific markers by maternal IMZ exposure. Caco2 cell culture and animal experiment confirmed IMZ affected carnitine absorption through the organic cation transporter type-2 (OCTN2) protein encoded by solute carrier family 22A member 5 (SLC22A5) gene in colon. Maternal IMZ exposure also had a greater effect on gut microbiota in offspring, especially anaerobic bacteria, which positively correlated with C0 and acyl-carnitines. These results suggested that maternal IMZ exposure affected carnitine absorption through OCTN2 protein, which led to the decline of anaerobic bacteria and unbalanced intestinal homeostasis. [Display omitted] • Maternal IMZ exposure affected the growth phenotypes of offspring. • IMZ altered carnitine absorption via the OCTN2 in colon. • IMZ-induced changes of anaerobic bacteria were highly correlated with carnitine. [ABSTRACT FROM AUTHOR]