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 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.

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.

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.

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.

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.

Nitric oxide inhibits the mitochondrial carnitine/acylcarnitine carrier through reversible S-nitrosylation of cysteine 136.

S-nitrosylation of the mitochondrial carnitine/acylcarnitine transporter (CACT) has been investigated on the native and the recombinant proteins reconstituted in proteoliposomes, and on intact mitochondria. The widely-used NO-releasing compound, GSNO, strongly inhibited the antiport measured in proteoliposomes reconstituted with the native CACT from rat liver mitochondria or the recombinant rat CACT over-expressed in E. coli. Inhibition was reversed by the reducing agent dithioerythritol, indicating a reaction mechanism based on nitrosylation of Cys residues of the CACT. The half inhibition constant (IC50) was very similar for the native and recombinant proteins, i.e., 74 and 71µM, respectively. The inhibition resulted to be competitive with respect the substrate, carnitine. NO competed also with NEM, correlating well with previous data showing interference of NEM with the substrate transport path. Using a site-directed mutagenesis approach on Cys residues of the recombinant CACT, the target of NO was identified. C136 plays a major role in the reaction mechanism. The occurrence of S-nitrosylation was demonstrated in intact mitochondria after treatment with GSNO, immunoprecipitation and immunostaining of CACT with a specific anti NO-Cys antibody. In parallel samples, transport activity of CACT measured in intact mitochondria, was strongly inhibited after GSNO treatment. The possible physiological and pathological implications of the post-translational modification of CACT are discussed.

The contribution of the citrate pathway to oxidative stress in Down syndrome.

Inflammatory conditions and oxidative stress have a crucial role in Down syndrome (DS). Emerging studies have also reported an altered lipid profile in the early stages of DS. Our previous works demonstrate that citrate pathway activation is required for oxygen radical production during inflammation. Here, we find up-regulation of the citrate pathway and down-regulation of carnitine/acylcarnitine carrier and carnitine palmitoyl-transferase 1 genes in cells from children with DS. Interestingly, when the citrate pathway is inhibited, we observe a reduction in oxygen radicals as well as in lipid peroxidation levels. Our preliminary findings provide evidence for a citrate pathway dysregulation, which could be related to some phenotypic traits of people with DS.

Detection of miR-33 Expression and the Verification of Its Target Genes in the Fatty Liver of Geese.

BACKGROUND: miRNAs are single-stranded, small RNA molecules with a length of 18-25 nucleotides. They bind to the 3' untranslated regions of mRNA transcripts to reduce the translation of these transcripts or to cause their degradation. The roles of these molecules differ in biological processes, such as cell differentiation, proliferation, apoptosis and tumor genesis. miRNA-33 is encoded by the gene introns of proteins that bind sterol-regulatory elements. This molecule cooperates with these proteins to control cholesterol homeostasis, fatty acid levels and the genes that are related to the expression of fat metabolism. The examination of miR-33 expression and its target genes can promote the in-depth study of the miRNA regulation mechanism in the formation process of goose fatty liver and can lay a foundation for research into human fatty liver. METHODOLOGY/PRINCIPAL FINDINGS: (1) Through real-time fluorescent quantitative polymerase chain reaction (TaqMan MicroRNA Assay), we detected the expression of miR-33 during the feeding of Landes geese. The expression level of miR-33 increases significantly in the liver after 19 days in comparison with the control group; (2) By using the bioinformatics software programs TargetScan, miRDB and miRCosm to predict the target genes of miR-33 according to laboratory prophase transcriptome results and references, we screen nine target genes: adenosine triphosphate binding cassette transporters A1, adenosine triphosphate binding cassette transporters G1, Neimann Pick C, carnitine O-octanoyltransferase (CROT), cyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, beta subunit (HADHB), AMP-activated protein kinase, alpha subunit 1 (AMPKα1), insulin receptor substrate 2, glutamic pyruvate transaminase and adipose differentiation-related protein. The dual luciferase reporter gene system in the CHO cell line verifies that CROT, HADHB and NPC1 are the target genes of miR-33 in geese. The inhibition rate of CROT is highest and reaches 70%; (3) The seed sequence (5' 2-8 bases) is the acting site of miR-33. The two predicted target sites of CROT are the target sites of miR-33. Moreover, the predicted target site of HADHB and NPC1 is the target site of miR-33. CONCLUSIONS/SIGNIFICANCE: (1) After 19 days of overfeeding, the expression level of miR-33 increases significantly in the livers of geese; (2) CROT, HADHB and NPC1 are the target genes of miR-33 in geese. These genes determine the combined target site.

Mitochondrial carnitine/acylcarnitine translocase: insights in structure/ function relationships. Basis for drug therapy and side effects prediction.

The mitochondrial carnitine/acylcarnitine translocase has been identified, purified and reconstituted in liposomes in 1990. Since that time it has been object of studies aimed to characterize its function and to define the molecular determinants of the translocation pathway. Thanks to these tenacious studies the molecular map of the amino acids involved in the catalysis has been constructed and the roles of critical residues in the translocation pathway have been elucidated. This has been possible through the combination of transport assay in reconstituted liposomes, site-directed mutagenesis, chemical labeling and bioinformatics. Recently some molecules which modulate CACT activity have been identified, such as glutathione and hydrogen peroxide, constituting some of the few cases of control mechanisms of mitochondrial carriers. The vast knowledge on the carnitine/acylcarnitine translocase is essential both as a progress in basic science and as instrument to foresee therapeutic or toxic effects of xenobiotics and drugs. Such studies have been already started pointing out the inhibitory action of drugs such as K(+)/H(+)-ATPase inhibitors (omeprazole) or antibiotics (ß-lactams) on the carnitine/acylcarnitine translocase, which can explain some of their adverse effects.

Monocarboxylate transporters 1 and 4: expression and regulation by PPARα in ovine ruminal epithelial cells.

In the intact rumen epithelium, isoforms 1 and 4 of the monocarboxylate transporter (MCT1 and MCT4) are thought to play key roles in mediating transcellular and intracellular permeation of short-chain fatty acids and their metabolites and in maintaining intracellular pH. We examined whether both MCT1 and MCT4 are expressed at mRNA and protein levels in ovine ruminal epithelial cells (REC) maintained in primary culture and whether they are regulated by peroxisome proliferator-activated receptor-α (PPARα). Because both transporters have been characterized to function coupled to protons, the influence of PPARα on the recovery of intracellular pH after l-lactate exposure was evaluated by spectrofluorometry. MCT1 and MCT4 were detected using immunocytochemistry both at the cell margins and intracellularly in cultured REC. To test regulation by PPARα, cells were exposed to WY 14.643, a selective ligand of PPARα, for 48 h. The subsequent qPCR analysis resulted in a dose-dependent upregulation of MCT1 and PPARα target genes, whereas response of MCT4 was not uniform. Protein expression of MCT1 and MCT4 quantified by Western blot analysis was not altered by WY 14.643 treatment. l-Lactate-dependent proton export was blocked almost completely by pHMB, a specific inhibitor of MCT1 and MCT4. However, l-lactate-dependent, pHMB-inhibited proton export in WY 14.643-treated cells was not significantly altered compared with cells not treated with WY 14.643. These data suggest that PPARα is particularly regulating MCT1 but not MCT4 expression. Extent of lactate-coupled proton export indicates that MCT1 is already working on a high level even under unstimulated conditions.

Expression of non-neuronal cholinergic system in maxilla of rat in vivo.

BACKGROUND: Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, which is also produced in a variety of non-neuronal tissues and cell. The existence of ACh in maxilla in vivo and potential regulation role for osteogenesis need further study. RESULTS: Components of the cholinergic system (ACh, esterase, choline acetyltransferase, high-affinity choline uptake, n- and mAChRs) were determined in maxilla of rat in vivo, by means of Real-Time PCR and immunohistochemistry. Results showed RNA for CarAT, carnitine/acylcarnitine translocase member 20 (Slc25a20), VAChT, OCTN2, OCT1, OCT3, organic cation transporter member 4 (Slc22a4), AChE, BChE, nAChR subunits α1, α2, α3, α5, α7, α10, ß1, ß2, ß4, γ and mAChR subunits M1, M2, M3, M4, M5 were detected in rat's maxilla. RNA of VAChT, AChE, nAChR subunits α2, ß1, ß4 and mAChR subunits M4 had abundant expression (2(-ΔCt) > 0.03). Immunohistochemical staining was conducted for ACh, VAChT, nAChRα7 and AChE. ACh was expressed in mesenchymal cells, chondroblast, bone and cartilage matrix and bone marrow cells, The VAChT expression was very extensively while ACh receptor α7 was strongly expressed in newly formed bone matrix of endochondral and bone marrow ossification, AchE was found only in mesenchymal stem cells, cartilage and bone marrow cells. CONCLUSIONS: ACh might exert its effect on the endochondral and bone marrow ossification, and bone matrix mineralization in maxilla.

Mildronate, the inhibitor of L-carnitine transport, induces brain mitochondrial uncoupling and protects against anoxia-reoxygenation.

The preservation of mitochondrial function is essential for normal brain function after ischaemia-reperfusion injury. l-carnitine is a cofactor involved in the regulation of cellular energy metabolism. Recently, it has been shown that mildronate, an inhibitor of l-carnitine transport, improves neurological outcome after ischaemic damage of brain tissues. The aim of the present study was to elucidate the mitochondria targeted neuroprotective action of mildronate in the model of anoxia-reoxygenation-induced injury. Wistar rats were treated daily with mildronate (per os; 100mg/kg) for 14 days. The acyl-carnitine profile was determined in the brain tissues. Mitochondrial respiration and the activities of carnitine acetyltransferase (CrAT) and tricarboxylic acid (TCA) cycle enzymes were measured. To assess tolerance to ischaemia, isolated mitochondria were subjected to anoxia followed by reoxygenation. The mildronate treatment significantly reduced the concentrations of free l-carnitine (FC) and short-chain acyl-carnitine (AC) in brain tissue by 40-76%, without affecting the AC:FC ratio. The activities of CrAT and TCA cycle enzymes were slightly increased after mildronate treatment. Despite partially induced uncoupling, mildronate treatment did not affect mitochondrial bioenergetics function under normoxic conditions. After exposure to anoxia-reoxygenation, state 3 respiration and the respiration control ratio were higher in the mildronate-treated group. The results obtained demonstrate that mildronate treatment improves tolerance against anoxia-reoxygenation due to an uncoupling preconditioning-like effect. Regulating l-carnitine availability provides a potential novel target for the treatment of cerebral ischaemia and related complications.

Expression of non-neuronal cholinergic system in maxilla of rat in vivo

BACKGROUND: Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, which is also produced in a variety of non-neuronal tissues and cell. The existence of ACh in maxilla in vivo and potential regulation role for osteogenesis need further study. RESULTS: Components of the cholinergic system (ACh, esterase, choline acetyltransferase, high-affinity choline uptake, n- and mAChRs) were determined in maxilla of rat in vivo, by means of Real-Time PCR and immunohistochemistry. Results showed RNA for CarAT, carnitine/acylcarnitine translocase member 20 (Slc25a20), VAChT, OCTN2, OCT1, OCT3, organic cation transporter member 4 (Slc22a4), AChE, BChE, nAChR subunits α1, α2, α3, α5, α7, α10, ß1, ß2, ß4, γ and mAChR subunits M1, M2, M3, M4, M5 were detected in rat's maxilla. RNA of VAChT, AChE, nAChR subunits α2, ß1, ß4 and mAChR subunits M4 had abundant expression (2(-ΔCt) > 0.03). Immunohistochemical staining was conducted for ACh, VAChT, nAChRα7 and AChE. ACh was expressed in mesenchymal cells, chondroblast, bone and cartilage matrix and bone marrow cells, The VAChT expression was very extensively while ACh receptor α7 was strongly expressed in newly formed bone matrix of endochondral and bone marrow ossification, AchE was found only in mesenchymal stem cells, cartilage and bone marrow cells. CONCLUSIONS: ACh might exert its effect on the endochondral and bone marrow ossification, and bone matrix mineralization in maxilla.

Carnitine-acyltransferase system inhibition, cancer cell death, and prevention of myc-induced lymphomagenesis.

BACKGROUND: The metabolic alterations of cancer cells represent an opportunity for developing selective antineoplastic treatments. We investigated the therapeutic potential of ST1326, an inhibitor of carnitine-palmitoyl transferase 1A (CPT1A), the rate-limiting enzyme for fatty acid (FA) import into mitochondria. METHODS: ST1326 was tested on in vitro and in vivo models of Burkitt's lymphoma, in which c-myc, which drives cellular demand for FA metabolism, is highly overexpressed. We performed assays to evaluate the effect of ST1326 on proliferation, FA oxidation, and FA mitochondrial channeling in Raji cells. The therapeutic efficacy of ST1326 was tested by treating Eµ-myc mice (control: n = 29; treatment: n = 24 per group), an established model of c-myc-mediated lymphomagenesis. Experiments were performed on spleen-derived c-myc-overexpressing B cells to clarify the role of c-myc in conferring sensitivity to ST1326. Survival was evaluated with Kaplan-Meier analyses. All statistical tests were two-sided. RESULTS: ST1326 blocked both long- and short-chain FA oxidation and showed a strong cytotoxic effect on Burkitt's lymphoma cells (on Raji cells at 72 hours: half maximal inhibitory concentration = 8.6 µM). ST1326 treatment induced massive cytoplasmic lipid accumulation, impairment of proper mitochondrial FA channeling, and reduced availability of cytosolic acetyl coenzyme A, a fundamental substrate for de novo lipogenesis. Moreover, treatment with ST1326 in Eµ-myc transgenic mice prevented tumor formation (P = .01), by selectively impairing the growth of spleen-derived primary B cells overexpressing c-myc (wild-type cells + ST1326 vs. Eµ-myc cells + ST1326: 99.75% vs. 57.5%, difference = 42.25, 95% confidence interval of difference = 14% to 70%; P = .01). CONCLUSIONS: Our data indicate that it is possible to tackle c-myc-driven tumorigenesis by altering lipid metabolism and exploiting the neoplastic cell addiction to FA oxidation.

Inhibition of mitochondrial carnitine/acylcarnitine transporter by H(2)O(2): molecular mechanism and possible implication in pathophysiology.

H(2)O(2) inhibits the [(3)H]carnitine/carnitine antiport catalysed by the mitochondrial carnitine/acylcarnitine transporter reconstituted in proteoliposomes. The inhibition was reversed by dithioerythritol, N-acetylcysteine and L-cysteine. Inhibition time-dependence revealed a faster and a slower reaction stages with orders of reaction of 1.0 and 1.9, respectively. Inhibition was tested on mutants in which one or more of the six Cys residues had been substituted with Ser or with Val. The four replacement mutant C23S/C58S/C89S/C283S containing C136 and C155 was inhibited as the wild-type. Mutants C23V/C58V/C155V/C89S/C283S and C23V/C58V/C136V/C89S/C283S containing only C136 or C155, respectively, were inhibited at a much lower extent respect to the wild-type, while the mutant C136S/C155S in which the two Cys were substituted and the C-less protein were virtually insensitive to inhibition. DTE reversed the inhibition of the H(2)O(2) sensitive proteins except that in the case of the mutants containing only C136 or C155 after long time of incubation with H(2)O(2). The IC(50) values obtained by dose-response curves of H(2)O(2) inhibition were 0.17 mM for the wild-type, 0.39 mM for the four replacement mutant containing C136 and C155, 2.23 or 1.8mM in the five replacement mutants containing the single C136 or C155, respectively. Carnitine and acetylcarnitine protected the protein from the inhibition by H(2)O(2). Inhibition kinetics showed a competitive behaviour of H(2)O(2) respect to carnitine. All the data concur to demonstrate that H(2)O(2) interacts with C136 and C155 and completely inactivates the transporter by inducing the formation of a disulphide.