Carnitine o-octanoyltransferase is a p53 target that promotes oxidative metabolism and cell survival following nutrient starvation.

Whereas it is known that p53 broadly regulates cell metabolism, the specific activities that mediate this regulation remain partially understood. Here, we identified carnitine o-octanoyltransferase (CROT) as a p53 transactivation target that is upregulated by cellular stresses in a p53-dependent manner. CROT is a peroxisomal enzyme catalyzing very long-chain fatty acids conversion to medium chain fatty acids that can be absorbed by mitochondria during ß-oxidation. p53 induces CROT transcription through binding to consensus response elements in the 5'-UTR of CROT mRNA. Overexpression of WT but not enzymatically inactive mutant CROT promotes mitochondrial oxidative respiration, while downregulation of CROT inhibits mitochondrial oxidative respiration. Nutrient depletion induces p53-dependent CROT expression that facilitates cell growth and survival; in contrast, cells deficient in CROT have blunted cell growth and reduced survival during nutrient depletion. Together, these data are consistent with a model where p53-regulated CROT expression allows cells to be more efficiently utilizing stored very long-chain fatty acids to survive nutrient depletion stresses.

Carnitine-acylcarnitine translocase deficiency caused by SLC25A20 gene heterozygous variants in twins: a case report.

The current case report describes the clinical, biochemical and genetic characteristics of carnitine-acylcarnitine translocase deficiency (CACTD) in infant male and female twins that presented with symptoms shortly after elective caesarean delivery. The clinical manifestations were neonatal hypoglycaemia, arrhythmia and sudden death. The age of onset was 1.5 days and the age of the death was 1.5-3.5 days. Dried blood filter paper analysis was used for the detection of acylcarnitine. Peripheral venous blood and skin samples were used for next-generation sequencing. The twins and their parents underwent gene analysis and whole exome sequencing analyses of the solute carrier family 25 member 20 (SLC25A20; also known as carnitine-acylcarnitine translocase) gene. Both infants carried compound heterozygous variants of the SLC25A20 gene: variant M1:c.706_707insT:p.R236L fs*12 and variant M2:c.689C>G:p.P230R. The M1 variant was paternal and had not been previously reported regarding CACTD. The M2 variant was maternal. CACTD has severe clinical manifestations and a poor prognosis, which is manifested as hypoketotic hypoglycaemia, hyperammonaemia, liver function damage and elevated creatine kinase.

Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells.

In eukaryotes, carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for ß-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for posttranslational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood. To explain how exogenous LAC influences mammalian cell metabolism, we synthesized isotope-labeled forms of LAC and its analogs. In cultures of glucose-limited U87MG glioma cells, exogenous LAC contributed more robustly to intracellular acetyl-CoA pools than did ß-hydroxybutyrate, the predominant circulating ketone body in mammals. The fact that most LAC-derived acetyl-CoA is cytosolic is evident from strong labeling of fatty acids in U87MG cells by exogenous 13C2-acetyl-L-carnitine. We found that the addition of d3-acetyl-L-carnitine increases the supply of acetyl-CoA for cytosolic posttranslational modifications due to its strong kinetic isotope effect on acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis. Surprisingly, whereas cytosolic carnitine acetyltransferase is believed to catalyze acetyl group transfer from LAC to coenzyme A, CRAT-/- U87MG cells were unimpaired in their ability to assimilate exogenous LAC into acetyl-CoA. We identified carnitine octanoyltransferase as the key enzyme in this process, implicating a role for peroxisomes in efficient LAC utilization. Our work has opened the door to further biochemical investigations of a new pathway for supplying acetyl-CoA to certain glucose-starved cells.

The Regulators of Peroxisomal Acyl-Carnitine Shuttle CROT and CRAT Promote Metastasis in Melanoma.

Circulating tumor cells are the key link between a primary tumor and distant metastases, but once in the bloodstream, loss of adhesion induces cell death. To identify the mechanisms relevant for melanoma circulating tumor cell survival, we performed RNA sequencing and discovered that detached melanoma cells and isolated melanoma circulating tumor cells rewire lipid metabolism by upregulating fatty acid (FA) transport and FA beta-oxidation‒related genes. In patients with melanoma, high expression of FA transporters and FA beta-oxidation enzymes significantly correlates with reduced progression-free and overall survival. Among the highest expressed regulators in melanoma circulating tumor cells were the carnitine transferases carnitine O-octanoyltransferase and carnitine acetyltransferase, which control the shuttle of peroxisome-derived medium-chain FAs toward mitochondria to fuel mitochondrial FA beta-oxidation. Knockdown of carnitine O-octanoyltransferase or carnitine acetyltransferase and short-term treatment with peroxisomal or mitochondrial FA beta-oxidation inhibitors thioridazine or ranolazine suppressed melanoma metastasis in mice. Carnitine O-octanoyltransferase and carnitine acetyltransferase depletion could be rescued by medium-chain FA supplementation, indicating that the peroxisomal supply of FAs is crucial for the survival of nonadherent melanoma cells. Our study identifies targeting the FA-based cross-talk between peroxisomes and mitochondria as a potential therapeutic opportunity to challenge melanoma progression. Moreover, the discovery of the antimetastatic activity of the Food and Drug Administration‒approved drug ranolazine carries translational potential.

Proline/Glycine residues of the PG-levels guide conformational changes along the transport cycle in the mitochondrial carnitine/acylcarnitine carrier (SLC25A20).

Mitochondrial carnitine/acylcarnitine carrier (CAC) is a member of the mitochondrial carrier (MC) family and imports acylcarnitine into the mitochondrial matrix in exchange for carnitine, playing a pivotal role in carnitine shuttle, crucial for fatty acid oxidation. The crystallized structure of CAC has not been solved yet, however, the availability of several in vitro/in silico studies, also based on the crystallized structures of the ADP/ATP carrier in the cytosolic-conformation and in the matrix-conformation, has made possible to confirm the hypothesis of the single-binding centered-gated pore mechanism for all the members of the MC family. In addition, our recent bioinformatics analyses allowed quantifying in silico the importance of protein residues of MC substrate binding region, of those involved in the formation of the matrix and cytosolic gates, and of those belonging to the Pro/Gly (PG) levels, proposed to be crucial for the tilting/kinking/bending of the six MC transmembrane helices, funneling the substrate translocation pathway. Here we present a combined in silico/in vitro analysis employed for investigating the role played by a group of 6 proline residues and 6 glycine residues, highly conserved in CAC, belonging to MC PG-levels. Residues of the PG-levels surround the similarly located MC common substrate binding region, and were proposed to lead conformational changes and substrate translocation, following substrate binding. For our analysis, we employed 3D molecular modeling approaches, alanine scanning site-directed mutagenesis and in vitro transport assays. Our analysis reveals that P130 (H3), G268 (H6) and G220 (H5), mutated in alanine, affect severely CAC transport activity (mutant catalytic efficiency lower than 5 % compared to the wild type CAC), most likely due to their major role in triggering CAC conformational changes, following carnitine binding. Notably, P30A (H1) and G121A (H3) CAC mutants, increase the carnitine uptake up to 217 % and 112 %, respectively, compared to the wild type CAC.

Low carnitine palmitoyltransferase 1 activity is a risk factor for narcolepsy type 1 and other hypersomnia.

STUDY OBJECTIVES: Narcolepsy type 1 (NT1) is associated with metabolic abnormalities but their etiology remains largely unknown. The gene for carnitine palmitoyltransferase 1B (CPT1B) and abnormally low serum acylcarnitine levels have been linked to NT1. To elucidate the details of altered fatty acid metabolism, we determined levels of individual acylcarnitines and evaluated CPT1 activity in patients with NT1 and other hypersomnia. METHODS: Blood samples from 57 NT1, 51 other hypersomnia patients, and 61 healthy controls were analyzed. The levels of 25 major individual acylcarnitines were determined and the C0/(t[C16] + t[C18]) ratio was used as a CPT1 activity marker. We further performed transcriptome analysis using independent blood samples from 42 NT1 and 42 healthy controls to study the relevance of fatty acid metabolism. NT1-specific changes in CPT1 activity and in expression of related genes were investigated. RESULTS: CPT1 activity was lower in patients with NT1 (p = 0.00064) and other hypersomnia (p = 0.0014) than in controls. Regression analysis revealed that CPT1 activity was an independent risk factor for NT1 (OR: 1.68; p = 0.0031) and for other hypersomnia (OR: 1.64; p = 0.0042). There was a significant interaction between obesity (BMI <25, ≥25) and the SNP rs5770917 status such that nonobese NT1 patients without risk allele had better CPT1 activity (p = 0.0089). The expression levels of carnitine-acylcarnitine translocase (CACT) and CPT2 in carnitine shuttle were lower in NT1 (p = 0.000051 and p = 0.00014, respectively). CONCLUSIONS: These results provide evidences that abnormal fatty acid metabolism is involved in the pathophysiology of NT1 and other hypersomnia.

NMR Characterization of Long-Chain Fatty Acylcarnitine Binding to the Mitochondrial Carnitine/Acylcarnitine Carrier.

The mitochondrial carnitine/acylcarnitine carrier (CAC) transports short-, medium- and long-carbon chain acylcarnitines across the mitochondrial inner membrane in exchange for carnitine. How CAC recognizes the substrates with various fatty acyl groups, especially long-chain fatty acyl groups, remains unclear. Here, using nuclear magnetic resonance (NMR) technology, we have shown that the CAC protein reconstituted into a micelle system exhibits a typical six transmembrane structure of the mitochondrial carrier family. The chemical shift perturbation patterns of different fatty acylcarnitines suggested that the segment A76-G81 in CAC specifically responds to the long-chain fatty acylcarnitine. Molecular dynamics (MD) simulations of palmitoyl-L-carnitine inside the CAC channel showed the respective interaction and motion of the long-chain acylcarnitine in CAC at the cytosol-open state and matrix-open state. Our data provided a molecular-based understanding of CAC structure and transport mechanism.

Lipid emulsions attenuate the inhibition of carnitine acylcarnitine translocase induced by toxic doses of local anesthetics in rat cardiomyoblasts.

The aim of this study was to examine the effects of lipid emulsions on carnitine palmitoyltransferase I (CPT-I), carnitine acylcarnitine translocase (CACT), carnitine palmitoyltransferase II (CPT-II), and the mitochondrial dysfunctions induced by toxic doses of local anesthetics in H9c2 rat cardiomyoblasts. The effects of local anesthetics and lipid emulsions on the activities of CPT-I, CACT, and CPT-II, and concentrations of local anesthetics were examined. The effects of lipid emulsions, N-acetyl-L-cysteine (NAC), and mitotempo on the bupivacaine-induced changes in cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), and intracellular calcium levels were examined. CACT, without significantly altering CPT-I and CPT-II, was inhibited by toxic concentration of local anesthetics. The levobupivacaine- and bupivacaine-induced inhibition of CACT was attenuated by all concentrations of lipid emulsion, whereas the ropivacaine-induced inhibition of CACT was attenuated by medium and high concentrations of lipid emulsion. The concentration of levobupivacaine was slightly attenuated by lipid emulsion. The bupivacaine-induced increase of ROS and calcium and the bupivacaine-induced decrease of MMP were attenuated by ROS scavengers NAC and mitotempo, and the lipid emulsion. Collectively, these results suggested that the lipid emulsion attenuated the levobupivacaine-induced inhibition of CACT, probably through the lipid emulsion-mediated sequestration of levobupivacaine.

Low Molecular Pectin Inhibited the Lipid Accumulation by Upregulation of METTL7B.

Inhibition of lipid accumulation is the key step to prevent nonalcoholic fatty liver (NAFL) progressing to nonalcoholic steatohepatitis. We aimed to study the effect of low-molecular-weight citrus pectin (LCP) against lipid accumulation and the underlying mechanism. Oleic acid (OA)-induced lipid deposition in HepG2 cells was applied to mimic in vitro model of lipid accumulation. Oil Red O (ORO) stain result showed lipid accumulation was significantly reduced, and levels of adipose triglyceride lipase (ATGL) and carnitine palmitoyltransferase-1 (CPT-1), involved in triacylglycerol catabolism and fatty acid ß-oxidation, detected by RT-qPCR were increased after OA-stimulated HepG2 cells treated with LCP. RNA sequencing analysis identified 740 differentially expressed genes (DEGs) in OA-stimulated HepG2 cells treated with the LCP group (OA+LCP group), and bioinformatics analysis indicated that some DEGs were enriched in lipid metabolism-related processes and pathways. The expression of the top 8 known DEGs in the OA+LCP group was then verified by RT-qPCR, which showed that fold change (abs) of METTL7B was the highest among the 8 candidates. In addition, overexpression of METTL7B in HepG2 cells significantly inhibited the lipid accumulation and enhanced levels of ATGL and CPT-1. In conclusion, LCP inhibited lipid accumulation through the upregulation of METTL7B, and further enhancement of ATGL and CPT-1 levels. LCP is expected to develop as a promising agent to ameliorate fat accumulation in NAFL.

CROT (Carnitine O-Octanoyltransferase) Is a Novel Contributing Factor in Vascular Calcification via Promoting Fatty Acid Metabolism and Mitochondrial Dysfunction.

OBJECTIVE: Vascular calcification is a critical pathology associated with increased cardiovascular event risk, but there are no Food and Drug Administration-approved anticalcific therapies. We hypothesized and validated that an unbiased screening approach would identify novel mediators of human vascular calcification. Approach and Results: We performed an unbiased quantitative proteomics and pathway network analysis that identified increased CROT (carnitine O-octanoyltransferase) in calcifying primary human coronary artery smooth muscle cells (SMCs). Additionally, human carotid artery atherosclerotic plaques contained increased immunoreactive CROT near calcified regions. CROT siRNA reduced fibrocalcific response in calcifying SMCs. In agreement, histidine 327 to alanine point mutation inactivated human CROT fatty acid metabolism enzymatic activity and suppressed SMC calcification. CROT siRNA suppressed type 1 collagen secretion, and restored mitochondrial proteome alterations, and suppressed mitochondrial fragmentation in calcifying SMCs. Lipidomics analysis of SMCs incubated with CROT siRNA revealed increased eicosapentaenoic acid, a vascular calcification inhibitor. CRISPR/Cas9-mediated Crot deficiency in LDL (low-density lipoprotein) receptor-deficient mice reduced aortic and carotid artery calcification without altering bone density or liver and plasma cholesterol and triglyceride concentrations. CONCLUSIONS: CROT is a novel contributing factor in vascular calcification via promoting fatty acid metabolism and mitochondrial dysfunction, as such CROT inhibition has strong potential as an antifibrocalcific therapy.

Effect of Copper on the Mitochondrial Carnitine/Acylcarnitine Carrier Via Interaction with Cys136 and Cys155. Possible Implications in Pathophysiology.

The effect of copper on the mitochondrial carnitine/acylcarnitine carrier (CAC) was studied. Transport function was assayed as [3H]carnitine/carnitine antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Cu2+ (as well as Cu+) strongly inhibited the native transporter. The inhibition was reversed by GSH (reduced glutathione) or by DTE (dithioerythritol). Dose-response analysis of the inhibition of the native protein was performed from which an IC50 of 1.6 µM for Cu2+ was derived. The mechanism of inhibition was studied by using the recombinant WT or Cys site-directed mutants of CAC. From the dose-response curve of the effect of Cu2+ on the recombinant protein, an IC50 of 0.28 µM was derived. Inhibition kinetics revealed a non-competitive type of inhibition by Cu2+. However, a substrate protection experiment indicated that the interaction of Cu2+ with the protein occurred in the vicinity of the substrate-binding site. Dose-response analysis on Cys mutants led to much higher IC50 values for the mutants C136S or C155S. The highest value was obtained for the C136/155S double mutant, indicating the involvement of both Cys residues in the interaction with Cu2+. Computational analysis performed on the WT CAC and on Cys mutants showed a pattern of the binding energy mostly overlapping the binding affinity derived from the dose-response analysis. All the data concur with bridging of Cu2+ with the two Cys residues, which blocks the conformational changes required for transport cycle.

Metabolic interactions between peroxisomes and mitochondria with a special focus on acylcarnitine metabolism.

Carnitine plays an essential role in mitochondrial fatty acid ß-oxidation as a part of a cycle that transfers long-chain fatty acids across the mitochondrial membrane and involves two carnitine palmitoyltransferases (CPT1 and CPT2). Two distinct carnitine acyltransferases, carnitine octanoyltransferase (COT) and carnitine acetyltransferase (CAT), are peroxisomal enzymes, which indicates that carnitine is not only important for mitochondrial, but also for peroxisomal metabolism. It has been demonstrated that after peroxisomal metabolism, specific intermediates can be exported as acylcarnitines for subsequent and final mitochondrial metabolism. There is also evidence that peroxisomes are able to degrade fatty acids that are typically handled by mitochondria possibly after transport as acylcarnitines. Here we review the biochemistry and physiological functions of metabolite exchange between peroxisomes and mitochondria with a special focus on acylcarnitines.

Tryptophan 224 of the rat mitochondrial carnitine/acylcarnitine carrier is crucial for the antiport mechanism.

The mitochondrial carnitine/acylcarnitine carrier (CACT) catalyzes an antiport of carnitine and acylcarnitines and also a uniport reaction with a rate of about one tenth with respect to the antiport rate. The antiport process results from the coupling of the two uniport reactions in opposite directions. In this mechanism, the transition of the carrier from the outward open conformation to the inward open one (or vice versa) is much faster for the carrier-substrate complex than for the unbound carrier. To investigate the molecular determinants that couple the binding of the substrate with the conformational transitions, site directed mutagenesis has been employed. The antiport or the uniport reaction was followed as [3H]carnitine uptake in or efflux from proteoliposomes reconstituted with the WT or Trp mutants of the rat CACT. Substitution of each the three Trp residues led to different results. Nearly no variations were observed upon substitution of W192 and/or W296 with Ala. While, substantial alteration of the transport function was observed in the mutants W224A, W224Y and W224F. Mutation of W224 led to the loss of the antiport function while the uniport function was unaltered. In these mutants impairment of the substrate affinity on the external side was also observed. The data highlights that W224 is involved in the coupling of the substrate binding with the matrix gate opening. The experimental data are in line with predictions by homology modeling of the CACT in its cytosolic (c-state) or matrix (m-state) opened conformations.

Resveratrol shifts energy metabolism to increase lipid oxidation in healthy old mice.

OBJECTIVES: The objective of this work was to determine the specific mechanisms by which resveratrol inhibits lipogenesis and stimulates lipolysis. METHODS: Twelve male mice were individually introduced into a metabolic cage for 24 h to measure basal metabolic rate, prior to intervention. They were randomly divided into two groups, resveratrol (RSV) and control (C), and administered resveratrol intraperitoneally or vehicle, respectively, for two consecutive days. After 24 h, the metabolic energy expenditure was again determined for 24 h, before mice were sacrificed. Protein and gene expression of different enzymes related to metabolism in the hepatic tissue, adipose tissue and gastrocnemius of mice were analyzed by RT-PCR, western blot or ELISA. RESULTS: We report that resveratrol lowers the respiratory quotient in old mice and that this may be due to the activation of fatty acid mobilization from white adipose tissue (because hormone-activated lipase expression is increased) and fatty acid transport into mitochondria and eventual oxidation in muscle and liver (because transport enzymes and beta oxidation enzymes are also increased). Indeed, we have observed that resveratrol in vivo causes an increase in the expression and phosphorylation of AMPKα in liver, muscle and adipose tissue and an increase in the expression of acyl-CoA synthetase, of carnitine palmitoyl transferase 1 and of carnitine acylcarnitine translocase, all enzymes involved in lipid catabolism. On the other hand, the levels of acetyl-CoA carboxylase as well as those its product, i.e. malonyl CoA, are decreased. CONCLUSIONS: We conclude that a controlled dose of resveratrol activates fatty acid mobilization and degradation and inhibits fatty acid synthesis in old mice. This is the first time that these effects of resveratrol in lipid metabolism in healthy old (non-obese) animals are reported.

Single Cell Gene Co-Expression Network Reveals FECH/CROT Signature as a Prognostic Marker.

Aberrant activation of signaling pathways is frequently observed and reported to be associated with the progression and poor prognosis of prostate cancer (PCa). We aimed to identify key biological processes regulated by androgen receptor (AR) using gene co-expression network from single cell resolution. The bimodal index was used to evaluate whether two subpopulations exist among the single cells. Gene expression among single cells revealed averaging pitfalls and bimodality pattern. Weighted gene co-expression network analysis (WGCNA) was used to identify modules of highly correlated genes. Twenty-nine gene modules were identified and AR-regulated modules were screened by significantly overlapping reported androgen induced differentially expressed genes. The biological function "generation of precursor metabolites and energy" was significantly enriched by AR-regulated modules with bimodality, presenting differential androgen response among subpopulations. Integrating with public ChIP-seq data, two genes FECH, and CROT has AR binding sites. Public in vitro studies also show that androgen regulates FECH and CROT. After receiving androgen deprivation therapy, patients lowly express FECH and CROT. Further survival analysis indicates that FECH/CROT signature can predict PCa recurrence. We reveal the heterogeneous function of "generation of precursor metabolites and energy" upon androgen stimulation from the perspective of single cells. Inhibitors targeting this biological process will facilitate to prevent prostate cancer progression.

Human mitochondrial carnitine acylcarnitine carrier: Molecular target of dietary bioactive polyphenols from sweet cherry (Prunus avium L.).

The effect of polyphenols, recognized as the principal antioxidant and beneficial molecules introduced with the diet, extracted from sweet cherry (Prunus avium L.) on the recombinant human mitochondrial carnitine/acylcarnitine transporter (CACT) has been studied in proteoliposomes. CACT transport activity, which was strongly impaired after oxidation by atmospheric O2 or H2O2, due to the formation of a disulfide bridge between cysteines 136 and 155, was restored by externally added polyphenols. CACT reduction by polyphenols was time dependent. Spectroscopic analysis of polyphenolic extracts revealed eight most represented compounds in four cultivars. Molecular docking of CACT structural omology model with the most either abundant and arguably bio-available phenolic compound (trans 3-O-feruloyl-quinic acid) of the mix, is in agreement with the experimental data since it results located in the active site close to cysteine 136 at the bottom of the translocation aqueous cavity.

Physiology of L-carnitine in plants in light of the knowledge in animals and microorganisms.

L-carnitine is present in all living kingdoms where it acts in diverse physiological processes. It is involved in lipid metabolism in animals and yeasts, notably as an essential cofactor of fatty acid intracellular trafficking. Its physiological significance is poorly understood in plants, but L-carnitine may be linked to fatty acid metabolism among other roles. Indeed, carnitine transferases activities and acylcarnitines are measured in plant tissues. Current knowledge of fatty acid trafficking in plants rules out acylcarnitines as intermediates of the peroxisomal and mitochondrial fatty acid metabolism, unlike in animals and yeasts. Instead, acylcarnitines could be involved in plastidial exportation of de novo fatty acid, or importation of fatty acids into the ER, for synthesis of specific glycerolipids. L-carnitine also contributes to cellular maintenance though antioxidant and osmolyte properties in animals and microbes. Recent data indicate similar features in plants, together with modulation of signaling pathways. The biosynthesis of L-carnitine in the plant cell shares similar precursors as in the animal and yeast cells. The elucidation of the biosynthesis pathway of L-carnitine, and the identification of the enzymes involved, is today essential to progress further in the comprehension of its biological significance in plants.

CMOS-compatible biosensor for L-carnitine detection.

A CMOS-compatible ISFET with a Ta2O5 sensitive surface was developed. The structure was optimized for achieving high sensitivity using a subthreshold operation mode and by reducing the influence of the capacitances on the value of subthreshold swing. The developed ISFET was used as a basis for a biosensor for L-carnitine detection. To this end, carnitine acetyltransferase was immobilized on the ISFET sensitive surface. The immobilized enzyme was active (0.082 U/g model plate). The complete microsystem, consisting of a packaged chip, an immobilized enzyme and a microfluidic channel, detected L-carnitine at a range of 0.2-100 µM with a LOD of 0.2 µM. The biosensor response was linear in the range of 0.2-50 µM of L-carnitine with sensitivity 18.0 ±â€¯1.7 mV/µM. An experiment with artificial urine containing 1.3 µM L-carnitine showed that the proposed biosensor could be used on a real sample. Therefore, a new sensor specially optimized for biosensing CMOS-compatible ISFET structures and direct determination of L-carnitine with immobilized carnitine acetyltransferase was developed.

Elevated mitochondrial SLC25A29 in cancer modulates metabolic status by increasing mitochondria-derived nitric oxide.

Warburg effect has been recognized as a hallmark of cancer cells for many years, but its modulation mechanism remains a great focus. Our current study found a member of solute carrier family 25 (SLC25A29), the main arginine transporter on mitochondria, significantly elevated in various cancer cells. Knockout of SLC25A29 by CRISPR/Cas9 inhibited proliferation and migration of cancer cells both in vitro and in vivo. SLC25A29-knockout cells also showed an altered metabolic status with enhanced mitochondrial respiration and reduced glycolysis. All of above impacts could be reversed after rescuing SLC25A29 expression in SLC25A29-knockout cells. Arginine is transported into mitochondria partly for nitric oxide (NO) synthesis. Deletion of SLC25A29 resulted in severe decrease of NO production, indicating that the mitochondria is a significant source of NO. SLC25A29-knockout cells dramatically altered the variation of metabolic processes, whereas addition of arginine failed to reverse the effect, highlighting the necessity of transporting arginine into mitochondria by SLC25A29. In conclusion, aberrant elevated SLC25A29 in cancer functioned to transport more arginine into mitochondria, improved mitochondria-derived NO levels, thus modulated metabolic status to facilitate increased cancer progression.

The carnitine system and cancer metabolic plasticity.

Metabolic flexibility describes the ability of cells to respond or adapt its metabolism to support and enable rapid proliferation, continuous growth, and survival in hostile conditions. This dynamic character of the cellular metabolic network appears enhanced in cancer cells, in order to increase the adaptive phenotype and to maintain both viability and uncontrolled proliferation. Cancer cells can reprogram their metabolism to satisfy the energy as well as the biosynthetic intermediate request and to preserve their integrity from the harsh and hypoxic environment. Although several studies now recognize these reprogrammed activities as hallmarks of cancer, it remains unclear which are the pathways involved in regulating metabolic plasticity. Recent findings have suggested that carnitine system (CS) could be considered as a gridlock to finely trigger the metabolic flexibility of cancer cells. Indeed, the components of this system are involved in the bi-directional transport of acyl moieties from cytosol to mitochondria and vice versa, thus playing a fundamental role in tuning the switch between the glucose and fatty acid metabolism. Therefore, the CS regulation, at both enzymatic and epigenetic levels, plays a pivotal role in tumors, suggesting new druggable pathways for prevention and treatment of human cancer.