The Carnitine Palmitoyltransferase 1A Inhibitor Teglicar Shows Promising Antitumour Activity against Canine Mammary Cancer Cells by Inducing Apoptosis.

Canine mammary tumours (CMTs) are the most common cancer in intact female dogs. In addition to surgery, additional targeted and non-targeted therapies may offer survival benefits to these patients. Therefore, exploring new treatments for CMT is a promising area in veterinary oncology. CMT cells have an altered lipid metabolism and use the oxidation of fatty acids for their energy needs. Here we investigated the tumoricidal effects of teglicar, a reversible inhibitor of carnitine palmitoyl transferase 1A (CPT1A), the rate-limiting enzyme for fatty acid import into mitochondria, on two CMT cells, P114 and CMT-U229. Viability and apoptosis were examined in CMT cells using the crystal violet assay, trypan blue assay, and flow cytometry analysis. The expression of mediators of apoptosis signalling (e.g., caspase-9, caspase-8, and caspase-3) was assessed by quantitative real-time polymerase chain reaction and western blot analyses. Teglicar was able to decrease cell viability and induce apoptosis in P114 and CMT-U229 cells. At the molecular level, the effect of teglicar was associated with an upregulation of the mRNA expression levels of caspase-9, caspase-8, and caspase-3 and an increase in their protein levels. In summary, our results show that teglicar has a potential effect against CMTs through the induction of apoptotic cell death, making it a promising therapeutic agent against CMTs.

Buffalo Milk Whey Activates Necroptosis and Apoptosis in a Xenograft Model of Colorectal Cancer.

Recent pharmacological research on milk whey, a byproduct of the dairy industry, has identified several therapeutic properties that could be exploited in modern medicine. In the present study, we investigated the anticancer effects of whey from Mediterranean buffalo (Bubalus bubalis) milk. The antitumour effect of delactosed milk whey (DMW) was evaluated using the HCT116 xenograft mouse model of colorectal cancer (CRC). There were no discernible differences in tumour growth between treated and untreated groups. Nevertheless, haematoxylin and eosin staining of the xenograft tissues showed clearer signs of different cell death in DMW-treated mice compared to vehicle-treated mice. Detailed biochemical and molecular biological analyses revealed that DMW was able to downregulate the protein expression levels of c-myc, phospho-Histone H3 (ser 10) and p-ERK. Moreover, DMW also activated RIPK1, RIPK3, and MLKL axis in tumour tissues from xenograft mice, thus, suggesting a necroptotic effect. The necroptotic pathway was accompanied by activation of the apoptotic pathway as revealed by increased expression of both cleaved caspase-3 and PARP-1. At the molecular level, DMW-induced cell death was also associated with (i) upregulation of SIRT3, SIRT6, and PPAR-γ and (ii) downregulation of LDHA and PPAR-α. Overall, our results unveil the potential of whey as a source of biomolecules of food origin in the clinical setting of novel strategies for the treatment of CRC.

Metabolic Flexibility in Canine Mammary Tumors: Implications of the Carnitine System.

Deregulation of fatty acid catabolism provides an alternative energy source to glycolysis for cancer cell survival and proliferation. The regulator enzymes of the carnitine system (CS), responsible for the transport of fatty acids across mitochondrial membranes for ß-oxidation are deregulated in tumorigenesis. Recently, we found that Carnitine Palmitoyl Transferase 1 (CPT1), a crucial regulator of CS components, is expressed and dysregulated in canine mammary tumor (CMT) tissues and cells. In this study, we examined the protein expression of the three remaining enzymes of CS (Carnitine Acylcarnitine Translocase (CACT), Carnitine Palmitoyl Transferase 2 (CPT2), Carnitine O-acetyltransferase (CrAT), in canine mammary cells and tissues by Western blot and immunohistochemistry. Protein expression of the components of CS was found in normal mammary glands and a concomitant deregulation of expression in CMT tissues that inversely correlated with the degree of tumor differentiation. Moreover, the expression and a different deregulation of CS-related proteins was also observed in CF33, CMT-U27, CMT-U309, and P114 cell lines used as in vitro model. These results demonstrate for the first time the expression of CS components in CMT tissues and cancer cells; however, further studies are needed to elucidate their roles in dogs as well.

Carnitine palmitoyltransferase 1 A expression profile in canine mammary tumors.

Genetic alterations and/or epigenetic modifications occur frequently in the majority of cancer cells. In addition to playing a crucial role as promoters of tumorigenesis, these processes can also generate metabolic pathways that are different from those in normal cells. Besides the Warburg effect, an alteration in lipid metabolism is also found in cancer cells. Thus, elucidation of the regulators involved in this metabolic reprogramming might provide tools for diagnosis, prognosis, and ultimately treatment of canine mammary tumours (CMTs) in particular. One such regulator is carnitine palmitoyltransferase 1A (CPT1A), which is involved in transportation of long-chain fatty acids into the mitochondrial matrix for beta-oxidation, thereby providing an alternative pathway for the generation of energy for tumour growth and development. In this study, the canine cell lines MDCK, CMT-U309, CMT-U27, and P114 were used as in vitro models for western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Furthermore, western blot and immunohistochemistry were carried out to evaluate CPT1A protein expression in the CMT specimens. The CPT1A protein and mRNA expression levels were increased in the CMT cell lines relative to their levels in normal epithelial cells. Moreover, increased CPT1A expression levels were found in the CMT tissues, being inversely correlated with the tumour differentiation grade. However, additional studies are required to further specify the role of CPT1A in CMTs.

Ginkgo biloba Prevents Oxidative Stress-Induced Apoptosis Blocking p53 Activation in Neuroblastoma Cells.

Oxidative stress has been associated to neuronal cell loss in neurodegenerative diseases. Neurons are post-mitotic cells that are very sensitive to oxidative stress-especially considering their limited capacity to be replaced. Therefore, reduction of oxidative stress, and inhibiting apoptosis, will potentially prevent neurodegeneration. In this study, we investigated the neuroprotective effect of Ginkgo biloba extract (EGb 761) against H2O2 induced apoptosis in SK-N-BE neuroblastoma cells. We analysed the molecular signalling pathway involved in the apoptotic cell death. H2O2 induced an increased acetylation of p53 lysine 382, a reduction in mitochondrial membrane potential, an increased BAX/Bcl-2 ratio and consequently increased Poly (ADP-ribose) polymerase (PARP) cleavage. All these effects were blocked by EGb 761 treatment. Thus, EGb 761, acting as intracellular antioxidant, protects neuroblastoma cells against activation of p53 mediated pathway and intrinsic mitochondrial apoptosis. Our results suggest that EGb 761, protecting against oxidative-stress induced apoptotic cell death, could potentially be used as nutraceutical for the prevention and treatment of neurodegenerative diseases.

Bioactive Polyphenols and Neuromodulation: Molecular Mechanisms in Neurodegeneration.

The interest in dietary polyphenols in recent years has greatly increased due to their antioxidant bioactivity with preventive properties against chronic diseases. Polyphenols, by modulating different cellular functions, play an important role in neuroprotection and are able to neutralize the effects of oxidative stress, inflammation, and apoptosis. Interestingly, all these mechanisms are involved in neurodegeneration. Although polyphenols display differences in their effectiveness due to interindividual variability, recent studies indicated that bioactive polyphenols in food and beverages promote health and prevent age-related cognitive decline. Polyphenols have a poor bioavailability and their digestion by gut microbiota produces active metabolites. In fact, dietary bioactive polyphenols need to be modified by microbiota present in the intestine before being absorbed, and to exert health preventive effects by interacting with cellular signalling pathways. This literature review includes an evaluation of the literature in English up to December 2019 in PubMed and Web of Science databases. A total of 307 studies, consisting of research reports, review articles and articles were examined and 146 were included. The review highlights the role of bioactive polyphenols in neurodegeneration, with a particular emphasis on the cellular and molecular mechanisms that are modulated by polyphenols involved in protection from oxidative stress and apoptosis prevention.

Castanea sativa Mill. Shells Aqueous Extract Exhibits Anticancer Properties Inducing Cytotoxic and Pro-Apoptotic Effects.

In this study, chestnut shells (CS) were used in order to obtain bioactive compounds through different extraction procedures. The aqueous extracts were chemically characterized. The highest extraction yield and total phenolic content was obtained by conventional liquid extraction (CLE). Gallic and protocatechuic acids were the main simple phenols in the extract, with 86.97 and 11.20 mg/g chestnut shells dry extract (CSDE), respectively. Six tumor cell lines (DU 145, PC-3, LNCaP, MDA-MB-231, MCF-7, and HepG2) and one normal prostate epithelial cell line (PNT2) were exposed to increasing concentration of CSDE (1-100 µg/mL) for 24 h, and cell viability was evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide MTT assay. A reduced rate in cell viability was observed in DU 145, PC-3, LNCaP, and MCF-7 cells, while viability of the other assessed cells was not affected, except for PNT2 cells at a concentration of 100 µg/mL. Furthermore, CSDE-at concentrations of 55.5 and 100 µg/mL-lead to a significant increase of apoptotic cells in DU 145 cells of 28.2% and 61%, respectively. In conclusion, these outcomes suggested that CS might be used for the extraction of several polyphenols that may represent good candidates for alternative therapies or in combination with current chemotherapeutics.

High grade glioblastoma is associated with aberrant expression of ZFP57, a protein involved in gene imprinting, and of CPT1A and CPT1C that regulate fatty acid metabolism.

The diagnosis of glioblastoma is still based on tumor histology, but emerging molecular diagnosis is becoming an important part of glioblastoma classification. Besides the well-known cell cycle-related circuitries that are associated with glioblastoma onset and development, new insights may be derived by looking at pathways involved in regulation of epigenetic phenomena and cellular metabolism, which may both be highly deregulated in cancer cells. We evaluated if in glioblastoma patients the high grade of malignancy could be associated with aberrant expression of some genes involved in regulation of epigenetic phenomena and lipid metabolism. We measured the mRNA levels of ZFP57, TRIM28, CPT1A, CPT1B, and CPT1C in a cohort of 80 patients divided in two groups: grade II and grade IV. We evidenced that high grade glioblastoma is associated with increased level of ZFP57, a protein involved in gene imprinting, and aberrant expression of CPT1A and CPT1C, regulators of fatty acid oxidation. Our study may pave the way to identify new markers that could be potentially useful for diagnosis and/or prognosis of glioblastoma.

Effect of layered double hydroxide intercalated with fluoride ions on the physical, biological and release properties of a dental composite resin.

OBJECTIVES: The aim of this work was the preparation of a new fluoride-releasing dental material characterized by a release of fluoride relatively constant over time without any initial toxic burst effect. This type of delivery is obtained by a matrix controlled elution and elicits the beneficial effect of a low amount of fluoride on human dental pulp stem cells (hDPSCs) towards mature phenotype. METHODS: The modified hydrotalcite intercalated with fluoride ions (LDH-F), used as filler, was prepared via ion exchange procedure and characterized by X-ray diffraction and FT-IR spectroscopy. The LDH-F inorganic particles (0.7, 5, 10, 20wt.%) were mixed with a photo-activated Bis-GMA/TEGDMA (45/55wt/wt) matrix and novel visible-light cured composites were prepared. The dynamic thermo-mechanical properties were determined by dynamic mechanical analyzer. The release of fluoride ions in physiological solution was determined using a ionometer. Total DNA content was measured by a PicoGreen dsDNA quantification kit to assess the proliferation rate of hDPSCs. Alkaline phosphatase activity (ALP) was measured in presence of fluoride resins. RESULTS: Incorporation of even small mass fractions (e.g. 0.7 and 5wt.%) of the fluoride LDH in Bis-GMA/TEGDMA dental resin significantly improved the mechanical properties of the pristine resin, in particular at 37°C. The observed reinforcement increases on increasing the filler concentration. The release of fluoride ions resulted very slow, lasting months. ALP activity gradually increased for 28 days in hDPSCs cell grown, demonstrating that low concentrations of fluoride contributed to the cell differentiation. CONCLUSIONS: The prepared composites containing different amount of hydrotalcite filler showed improved mechanical properties, slow fluoride release and promoted hDPSCs cell proliferation and cell differentiation.

Enhanced in vitro antitumor activity of a titanocene complex encapsulated into polycaprolactone (PCL) electrospun fibers.

PURPOSE: The purpose of this work was to achieve detailed biomaterials characterization of a drug delivery system for local cancer treatment based on electrospun titanocene trichloride-loaded resorbable polycaprolactone (PCL) fibers. METHODS: The PCL fibers were characterized for their structural, morphologic and physical properties. The drug release kinetics of the titanocene complex was investigated at different concentrations, to obtain a set of correlations between structure and tuneable release. After exposing cancer cells directly onto the surface of PCL fibers, the anti-proliferative effects of titanocene-loaded PCL were assessed by: (i) counting viable cells via live/dead staining methods, and (ii) analyzing cell apoptosis. RESULTS AND CONCLUSIONS: Titanocene concentration influenced fiber diameters reduced for PCL filled with titanocene. X-ray analysis suggested that the titanocene, encapsulated into the PCL fibers, is not allowed to crystallize and exists as amorphous aggregates into the fibers. The titanocene release curves presented two stages unrelated to PCL degradation: an initial burst release followed by a release linear with time, extending for a very long time. All of the titanocene-loaded fibers revealed sustained drug release properties suggesting their potential clinical applicability for the treatment of local cancer diseases.

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.

The genotoxicity of PEI-based nanoparticles is reduced by acetylation of polyethylenimine amines in human primary cells.

The ultrasmall size and unique properties of polymeric nanoparticles (NPs) have led to raising concerns about their potential cyto- and genotoxicity on biological systems. Polyethylenimine (PEI) is a highly positive charged polymer and is known to have varying degree of toxic effect to cells based on its chemical structure (i.e., amount of primary and secondary amine). Herein, drug delivery carriers such as PEI-PLGA nanoparticles (PEI-NPs) and acetylated PEI-PLGA nanoparticles (AcPEI-NPs) were utilized to examine the effect of acetylation on NPs biocompatibility and genotoxicity, using human primary cells as in vitro model. Cell uptake of NPs was characterized along with their effects on cellular viability. The results indicate that both NPs showed an equivalent behavior in terms of uptake and biocompatibility. In depth analysis of NP uptake on cell biology evidenced that these nanoparticles induced dose dependant genotoxic effects. This phenomenon was significantly reduced by PEI acetylation. Endocytosed PEI-NPs trigger an oxidative stress on cells by inducing the production of reactive oxygen species (ROS), which cause DNA damage without apparently affecting cell viability. Thus, the genotoxicity of nanoparticles, that could be used as non-viral drug carriers, should be evaluated based on the intracellular level of ROS generation and DNA damage even in absence of a significant cell death.

Mutant huntingtin regulates EGF receptor fate in non-neuronal cells lacking wild-type protein.

Huntingtin (htt) is a scaffold protein localized at the subcellular level and is involved in coordinating the activity of several protein for signaling and intracellular transport. The emerging properties of htt in intracellular trafficking prompted us to study the role of mutant htt (polyQ-htt) in the intracellular fate of epidermal growth factor receptor (EGFR), whose activity seems to be strictly regulated by htt. In particular, to evaluate whether protein trafficking dysfunction occurs in non-neuronal cells in the absence of functional htt, we monitored the EGFR protein in fibroblasts from homozygotic HD patients and their healthy counterpart. We found that polyQ-htt controls EGFR degradation and recycling. Lack of wild-type htt caused alteration of the ubiquitination cycle, formation of EGFR-incorporating high-molecular weight protein aggregates and abnormal EGFR distribution in endosomes of the degradation and recycling pathways after EGF stimulation. PolyQ-htt-induced alteration of EGFR trafficking affected cell migration and proliferation, at least in part, through inhibition of ERK signaling. To our knowledge the data here reported represent the first signaling and phenotypic characterization of polyQ-htt involvement in the modulation of growth factor stimulation in non-neuronal cells.

Colon OCTN2 gene expression is up-regulated by peroxisome proliferator-activated receptor gamma in humans and mice and contributes to local and systemic carnitine homeostasis.

In the large intestine organic cation transporter type-2 (OCTN2) is recognized as a transporter of compounds such as carnitine and colony sporulation factor, promoting health of the colon intestinal epithelium. Recent reports suggest that OCTN2 expression in small intestine is under control of peroxisome proliferator-activated receptor-alpha (PPARalpha). However, PPARalpha contribution to colonic OCTN2 expression remains controversial. Here we examined the transcriptional regulation of colon OCTN2 gene by PPARgamma. To exclude any additional modulation of other PPAR to OCTN2 expression, we used both in vivo and in vitro PPAR-null models and specific PPAR inhibitors. The PPARgamma agonists thiazolidinediones increased both OCTN2 mRNA and protein expression in colonic epithelial cell lines independently by PPARalpha expression. The induction was blocked only by PPARgamma antagonists or by gammaORF4, a PPARgamma isoform with dominant negative activity, suggesting a PPARgamma-dependent mechanism. A conserved noncanonical PPAR-responsive element was found by computational analysis in the first intron of human OCTN2 gene and validated by EMSA assay. Promoter-reporter assays further confirmed transcriptional functionality of the putative PPAR response element, whereas selective mutation caused complete loss of responsiveness to PPARgamma activation. Finally, adenovirus-mediated overexpression of constitutively active PPARgamma mutant increased colon OCTN2 expression in PPARalpha(-/-) mice. Interestingly, animals overexpressing colon PPARgamma showed a significant increase in plasma carnitine, thus demonstrating the functional contribution of large intestine to systemic carnitine homeostasis. This study reveals a PPARgamma-dependent absorption machinery in colon that is likely involved in the health of colon epithelium, in the microbiota-host interactions and in the absorption of nutraceuticals and drugs.

Controlled delivery of the heparan sulfate/FGF-2 complex by a polyelectrolyte scaffold promotes maximal hMSC proliferation and differentiation.

Growth factors and other regulatory molecules are required to direct differentiation of bone marrow-derived human mesenchymal stem cells (hMSC) along specific lineages. However, the therapeutic use of growth factors is limited by their susceptibility to degradation, and the need to maintain prolonged local release of growth factor at levels sufficient to stimulate hMSC. The aim of this study was to investigate whether a device containing heparan sulfate (HS), which is a co-factor in growth factor-mediated cell proliferation and differentiation, could potentiate and prolong the delivery of fibroblast growth factor-2 (FGF-2) and thus enhance hMSC stimulation. To this aim, we synthesized cationic polyelectrolyte polymers covalently and non-covalently anchored to HS and evaluated their effect on hMSC proliferation. Polymers non-covalently bound to HS resulted in the release of an HS/FGF-2 complex rather than FGF-2 alone. The release of this complex significantly restored hMSC proliferation, which was abolished in serum-free medium and only partially restored by the release of FGF-2 alone as occurred with polymer covalently bound to HS. We also demonstrate that exposure to HS/FGF-2 during early growth but not during post-confluence is essential for hMSC differentiation down the fibroblast lineage, which suggests that both factors are required to establish the correct stem cell commitment that is necessary to support subsequent differentiation. In conclusion, the delivery platform described here is a step towards the development of a new class of biomaterial that enables the prolonged, non-covalent binding and controlled delivery of growth factors and cofactors without altering their potency.

Cationic copolymers nanoparticles for nonviral gene vectors: synthesis, characterization, and application in gene delivery.

The major aim of nonviral delivery systems for gene therapy is to mediate high levels of gene expression with low toxicity. Nowadays, one of the most successful synthetic polycations used in gene delivery research is poly(ethylenimine) (PEI) in its high-molecular weight (HMW) branched form. However, PEI is not the ideal transfection agent in vivo because of its overwhelming cytotoxicity. To overcome its toxic effects with a minimal impact on transfection efficiency, PEI has been conjugated with several nonionic biocompatible polymers. Here, we describe the synthesis of nanosized particles consisting of HMW PEI (25 kDa) crosslinked with poly(epsilon-caprolactone) (PCL, 50-60 kDa), a biodegradable aliphatic polyester. PCL was modified by the insertion of glycidyl groups able to condense with the amines of PEI to chemically bind PEI onto PCL. The nanoparticles obtained have been characterized in relation to their physicochemical and biological properties, and the results are extremely promising in terms of low cell toxicity and high transfection efficiency. These biological effects might be related to the peculiar DNA binding to covalently connected polymeric nanoparticles, without the formation of entangled DNA/polymer-soluble aggregates.

Compatibilized polymer blends based on PDLLA and PCL for application in bioartificial liver.

Porous scaffolds for tissue engineering applications based on poly(D,L-lactide)/poly(epsilon-caprolactone) compatibilized blends are described. The addition of a third polymer, namely poly( D, L-lactide-co-caprolactone) copolymer, has a profound effect on morphological properties of the blends scaffolds. In fact, the copolymer acts as compatibilizing agent and reduces the dimension of the dispersed phase of an order of magnitude. Such effect is function of the polymer composition. The efficiency of scaffolds obtained with poly( D, L-lactide) based blends containing 30% by weight of poly(epsilon-caprolactone) as dispersed phase toward hepatocytes has been tested by several biological assays and we found that they are able to promote a perfect adhesion, proliferation and growth of cells. Moreover, the addition of the copolymer significantly improves the biomedical performance of the scaffold.

The immunomodulatory protein SV-IV protects serum-deprived cells against apoptosis but not against G0/G1 arrest: possible implications for the survival of implanting embryo.

Serum deprivation induced in human lymphoblastoid Raji cells oxidative stress-associated apoptotic death and G0/G1 cell cycle arrest. Addition into culture medium of the immunomodulatory protein Seminal vesicle protein 4 (SV-IV) protected these cells against apoptosis but not against cycle arrest. The antiapoptotic activity was related to: (1) decrease of endocellular reactive Oxygen species (ROS) (2) increase of mRNAs encoding anti-oxidant enzymes (catalase, G6PD) and antiapoptotic proteins (survivin, cox-1, Hsp70, c-Fos); (3) decrease of mRNAs encoding proapoptotic proteins (c-myc, Bax, caspase-3, Apaf-1). The biochemical changes underlaying these effects were probably induced by a protein tyrosine kinase (PTK) activity triggered by the binding of SV-IV to its putative plasma membrane receptors. The ineffectiveness of SV-IV to abrogate the cycle arrest was accounted for by its downregulating effects on D1,3/E G1-cyclins and CdK2/4 gene expression, ppRb/pRb ratio, and intracellular ROS concentration. In conclusion, these experiments: (1) prove that SV-IV acts as a cell survival factor; (2) suggest the involvement of a PTK in SV-IV signaling; (3) point to cell cycle-linked enzyme inhibition as responsible for cycle arrest; (4) provide a model to dissect the cycle arrest and apoptosis induced by serum withdrawal; (5) imply a possible role of SV-IV in the survival of hemiallogenic implanting embryos.

Experimental colitis: decreased Octn2 and Atb0+ expression in rat colonocytes induces carnitine depletion that is reversible by carnitine-loaded liposomes.

Carnitine transporters have recently been implicated in susceptibility to inflammatory bowel disease (IBD). Because carnitine is required for beta-oxidation, it was suggested that decreased carnitine transporters, and hence reduced carnitine uptake, could lead to impaired fatty acid oxidation in intestinal epithelial cells, and to cell injury. We investigated this issue by examining the expression of the carnitine transporters OCTN2 and ATB0+, and butyrate metabolism in colonocytes in a rat model of IBD induced by trinitrobenzene sulfonic acid (TNBS). We found that Octn2 and Atb0+ expression was decreased in inflammatory samples at translational and functional level. Butyrate oxidation, evaluated based on CO2 production and acetyl-coenzyme A synthesis, was deranged in colonocytes from TNBS-treated rats. Treatment with carnitine-loaded liposomes corrected the butyrate metabolic alterations in vitro and reduced the severity of colitis in vivo. These results suggest that carnitine depletion in colonocytes is associated with the inability of mitochondria to maintain normal butyrate beta-oxidation. Our data indicate that carnitine is a rate-limiting factor for the maintenance of physiological butyrate oxidation in colonic cells. This hypothesis could also explain the contradictory therapeutic efficacy of butyrate supplementation observed in clinical trials of IBD.

Differential carnitine/acylcarnitine translocase expression defines distinct metabolic signatures in skeletal muscle cells.

Import of acylcarnitine into mitochondrial matrix through carnitine/acylcarnitine-translocase (CACT) is fundamental for lipid catabolism. To probe the effect of CACT down-expression on lipid metabolism in muscle, human myocytes were stably transfected with CACT-antisense construct. In presence of low concentration of palmitate, transfected cells showed decreased palmitate oxidation and acetyl-carnitine content, increased palmitoyl-carnitine level, and reduced insulin-dependent decrease of fatty acylcarnitine-to-fatty acyl-CoA ratio. The augmented palmitoyl-carnitine synthesis, also in the presence of insulin, could be related to an altered regulation of carnitine-palmitoyl-transferase 1 (CPT 1) by malonyl-CoA, whose synthesis is dependent by the availability of cytosolic acetyl-groups. Indeed, all the described effects were completely overcome by CACT neo-expression by recombinant adenovirus vector or by addition of acetyl-carnitine to cultures. Acetyl-carnitine effect was related to an increase of malonyl-CoA and was abolished by down-expression, via antisense RNA strategy, of acetyl-CoA carboxylase-beta, the mitochondrial membrane enzyme involved in the direct CPT 1 inhibition via malonyl-CoA synthesis. Thus, in our experimental model the modulation of CACT expression has consequences for CPT 1 activity, while the biologic effects of acetyl-carnitine are not associated with a generic supply of energy compounds but to the anaplerotic property of the molecule.