Effects of Fat and Carnitine on the Expression of Carnitine Acetyltransferase and Enoyl-CoA Hydratase Short-Chain 1 in the Liver of Juvenile GIFT (Oreochromis niloticus).

Carnitine acetyltransferase (CAT) and Enoyl-CoA hydratase short-chain 1 (ECHS1) are considered key enzymes that regulate the ß-oxidation of fatty acids. However, very few studies have investigated their full length and expression in genetically improved farmed tilapia (GIFT, Oreochromis niloticus), an important aquaculture species in China. Here, we cloned CAT and ECHS1 full-length cDNA via the rapid amplification of cDNA ends, and the expressions of CAT and ECHS1 in the liver of juvenile GIFT were detected in different fat and carnitine diets, as were the changes in the lipometabolic enzymes and serum biochemical indexes of juvenile GIFT in diets with different fat and carnitine levels. CAT cDNA possesses an open reading frame (ORF) of 2167 bp and encodes 461 amino acids, and the ECHS1 cDNA sequence is 1354 bp in full length, the ORF of which encodes a peptide of 391 amino acids. We found that juvenile GIFT had higher lipometabolic enzyme activity and lower blood CHOL, TG, HDL-C, and LDL-C contents when the dietary fat level was 2% or 6% and when the carnitine level was 500 mg/kg. We also found that the expression of ECHS1 and CAT genes in the liver of juvenile GIFT can be promoted by a 500 mg/kg carnitine level and 6% fat level feeding. These results suggested that CAT and ECHS1 may participate in regulating lipid metabolism, and when 2% or 6% fat and 500 mg/kg carnitine are added to the feed, it is the most beneficial to the liver and lipid metabolism of juvenile GIFT. Our results may provide a theoretical basis for GIFT feeding and treating fatty liver disease.

CRAT links cholesterol metabolism to innate immune responses in the heart.

Chronic inflammation is associated with increased risk and poor prognosis of heart failure; however, the precise mechanism that provokes sustained inflammation in the failing heart remains elusive. Here we report that depletion of carnitine acetyltransferase (CRAT) promotes cholesterol catabolism through bile acid synthesis pathway in cardiomyocytes. Intracellular accumulation of bile acid or intermediate, 7α-hydroxyl-3-oxo-4-cholestenoic acid, induces mitochondrial DNA stress and triggers cGAS-STING-dependent type I interferon responses. Furthermore, type I interferon responses elicited by CRAT deficiency substantially increase AIM2 expression and AIM2-dependent inflammasome activation. Genetic deletion of cardiomyocyte CRAT in mice of both sexes results in myocardial inflammation and dilated cardiomyopathy, which can be reversed by combined depletion of caspase-1, cGAS or AIM2. Collectively, we identify a mechanism by which cardiac energy metabolism, cholesterol homeostasis and cardiomyocyte-intrinsic innate immune responses are interconnected via a CRAT-mediated bile acid synthesis pathway, which contributes to chronic myocardial inflammation and heart failure progression.

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.

L-carnitine exerts a nutrigenomic effect via direct modulation of nuclear receptor signaling in adipocytes, hepatocytes and SKMC, demonstrating its nutritional impact.

L-carnitine is an indispensable metabolite facilitating the transport of fatty acids into the mitochondrial matrix and has been previously postulated to exert a nutrigenomic effect. However, the underlying molecular mechanisms remain mostly unclear. We hypothesized that L-carnitine interacts with nuclear receptors involved in metabolic regulation, thereby modulating downstream targets of cellular metabolism. Therefore, we investigated the effect of L-carnitine supplementation on protein activity, mRNA expression, and binding affinities of nuclear receptors as well as mRNA expression of downstream targets in skeletal muscle cells, hepatocytes, and differentiated adipocytes. L-carnitine supplementation to hepatocytes increased the protein activity of multiple nuclear receptors (RAR, RXR, VDR, PPAR, HNF4, ER, LXR). Diverging effects on the mRNA expression of PPAR-α, PPAR-δ, PPAR-γ, RAR-ß, LXR-α, and RXR-α were observed in adipocytes, hepatocytes, and skeletal muscle cells. mRNA levels of PPAR-α, a key regulator of lipolysis and ß-oxidation, were significantly upregulated, emphasizing a role of L-carnitine as a promoter of lipid catabolism. L-carnitine administration to hepatocytes modulated the transcription of key nuclear receptor target genes, including ALDH1A1, a promoter of adipogenesis, and OGT, a contributor to insulin resistance. Electrophoretic mobility shift assays proved L-carnitine to increase binding affinities of nuclear receptors to their promoter target sequences, suggesting a molecular mechanism for the observed transcriptional modulation. Overall, these findings indicate that L-carnitine modulates the activity and expression of nuclear receptors, thereby promoting lipolytic gene expression and decreasing transcription of target genes linked to adipogenesis and insulin resistance.

Extreme Acetylation of the Cardiac Mitochondrial Proteome Does Not Promote Heart Failure.

RATIONALE: Circumstantial evidence links the development of heart failure to posttranslational modifications of mitochondrial proteins, including lysine acetylation (Kac). Nonetheless, direct evidence that Kac compromises mitochondrial performance remains sparse. OBJECTIVE: This study sought to explore the premise that mitochondrial Kac contributes to heart failure by disrupting oxidative metabolism. METHODS AND RESULTS: A DKO (dual knockout) mouse line with deficiencies in CrAT (carnitine acetyltransferase) and Sirt3 (sirtuin 3)-enzymes that oppose Kac by buffering the acetyl group pool and catalyzing lysine deacetylation, respectively-was developed to model extreme mitochondrial Kac in cardiac muscle, as confirmed by quantitative acetyl-proteomics. The resulting impact on mitochondrial bioenergetics was evaluated using a respiratory diagnostics platform that permits comprehensive assessment of mitochondrial function and energy transduction. Susceptibility of DKO mice to heart failure was investigated using transaortic constriction as a model of cardiac pressure overload. The mitochondrial acetyl-lysine landscape of DKO hearts was elevated well beyond that observed in response to pressure overload or Sirt3 deficiency alone. Relative changes in the abundance of specific acetylated lysine peptides measured in DKO versus Sirt3 KO hearts were strongly correlated. A proteomics comparison across multiple settings of hyperacetylation revealed ≈86% overlap between the populations of Kac peptides affected by the DKO manipulation as compared with experimental heart failure. Despite the severity of cardiac Kac in DKO mice relative to other conditions, deep phenotyping of mitochondrial function revealed a surprisingly normal bioenergetics profile. Thus, of the >120 mitochondrial energy fluxes evaluated, including substrate-specific dehydrogenase activities, respiratory responses, redox charge, mitochondrial membrane potential, and electron leak, we found minimal evidence of oxidative insufficiencies. Similarly, DKO hearts were not more vulnerable to dysfunction caused by transaortic constriction-induced pressure overload. CONCLUSIONS: The findings challenge the premise that hyperacetylation per se threatens metabolic resilience in the myocardium by causing broad-ranging disruption to mitochondrial oxidative machinery.

A genetic screen identifies Crat as a regulator of pancreatic beta-cell insulin secretion.

OBJECTIVES: Glucose-stimulated insulin secretion is a critical function in the regulation of glucose homeostasis, and its deregulation is associated with the development of type 2 diabetes. Here, we performed a genetic screen using islets isolated from the BXD panel of advanced recombinant inbred (RI) lines of mice to search for novel regulators of insulin production and secretion. METHODS: Pancreatic islets were isolated from 36 RI BXD lines and insulin secretion was measured following exposure to 2.8 or 16.7 mM glucose with or without exendin-4. Islets from the same RI lines were used for RNA extraction and transcript profiling. Quantitative trait loci (QTL) mapping was performed for each secretion condition and combined with transcriptome data to prioritize candidate regulatory genes within the identified QTL regions. Functional studies were performed by mRNA silencing or overexpression in MIN6B1 cells and by studying mice and islets with beta-cell-specific gene inactivation. RESULTS: Insulin secretion under the 16.7 mM glucose plus exendin-4 condition was mapped significantly to a chromosome 2 QTL. Within this QTL, RNA-Seq data prioritized Crat (carnitine O-acetyl transferase) as a strong candidate regulator of the insulin secretion trait. Silencing Crat expression in MIN6B1 cells reduced insulin content and insulin secretion by ∼30%. Conversely, Crat overexpression enhanced insulin content and secretion by ∼30%. When islets from mice with beta-cell-specific Crat inactivation were exposed to high glucose, they displayed a 30% reduction of insulin content as compared to control islets. We further showed that decreased Crat expression in both MIN6B1 cells and pancreatic islets reduced the oxygen consumption rate in a glucose concentration-dependent manner. CONCLUSIONS: We identified Crat as a regulator of insulin secretion whose action is mediated by an effect on total cellular insulin content; this effect also depends on the genetic background of the RI mouse lines. These data also show that in the presence of the stimulatory conditions used the insulin secretion rate is directly related to the insulin content.

CRAT missense variants cause abnormal carnitine acetyltransferase function in an early-onset case of Leigh syndrome.

Leigh syndrome, or subacute necrotizing encephalomyelopathy, is one of the most severe pediatric disorders of the mitochondrial energy metabolism. By performing whole-exome sequencing in a girl affected by Leigh syndrome and her parents, we identified two heterozygous missense variants (p.Tyr110Cys and p.Val569Met) in the carnitine acetyltransferase (CRAT) gene, encoding an enzyme involved in the control of mitochondrial short-chain acyl-CoA concentrations. Biochemical assays revealed carnitine acetyltransferase deficiency in the proband-derived fibroblasts. Functional analyses of recombinant-purified CRAT proteins demonstrated that both missense variants, located in the acyl-group binding site of the enzyme, severely impair its catalytic function toward acetyl-CoA, and the p.Val569Met variant also toward propionyl-CoA and octanoyl-CoA. Although a single recessive variant in CRAT has been recently associated with neurodegeneration with brain iron accumulation (NBIA), this study reports the first kinetic analysis of naturally occurring CRAT variants and demonstrates the genetic basis of carnitine acetyltransferase deficiency in a case of mitochondrial encephalopathy.

Disruption of Acetyl-Lysine Turnover in Muscle Mitochondria Promotes Insulin Resistance and Redox Stress without Overt Respiratory Dysfunction.

This study sought to examine the functional significance of mitochondrial protein acetylation using a double knockout (DKO) mouse model harboring muscle-specific deficits in acetyl-CoA buffering and lysine deacetylation, due to genetic ablation of carnitine acetyltransferase and Sirtuin 3, respectively. DKO mice are highly susceptible to extreme hyperacetylation of the mitochondrial proteome and develop a more severe form of diet-induced insulin resistance than either single KO mouse line. However, the functional phenotype of hyperacetylated DKO mitochondria is largely normal. Of the >120 measures of respiratory function assayed, the most consistently observed traits of a markedly heightened acetyl-lysine landscape are enhanced oxygen flux in the context of fatty acid fuel and elevated rates of electron leak. In sum, the findings challenge the notion that lysine acetylation causes broad-ranging damage to mitochondrial quality and performance and raise the possibility that acetyl-lysine turnover, rather than acetyl-lysine stoichiometry, modulates redox balance and carbon flux.

In vivo and in vitro knockout system labelled using fluorescent protein via microhomology-mediated end joining.

Gene knockout is important for understanding gene function and genetic disorders. The CRISPR/Cas9 system has great potential to achieve this purpose. However, we cannot distinguish visually whether a gene is knocked out and in how many cells it is knocked out among a population of cells. Here, we developed a new system that enables the labelling of knockout cells with fluorescent protein through microhomology-mediated end joining-based knock-in. Using a combination with recombinant adeno-associated virus, we delivered our system into the retina, where the expression of Staphylococcus aureus Cas9 was driven by a retina ganglion cell (RGC)-specific promoter, and knocked out carnitine acetyltransferase (CAT). We evaluated RGCs and revealed that CAT is required for RGC survival. Furthermore, we applied our system to Keap1 and confirmed that Keap1 is not expressed in fluorescently labelled cells. Our system provides a promising framework for cell type-specific genome editing and fluorescent labelling of gene knockout based on knock-in.

Diosgenin ameliorates palmitic acid-induced lipid accumulation via AMPK/ACC/CPT-1A and SREBP-1c/FAS signaling pathways in LO2 cells.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is characterized by excessive hepatic lipid accumulation. Many studies have suggested that lipid overload is the key initial factor that contributes to hepatic steatosis. Our previous study indicated that diosgenin (DSG) has a beneficial effect on energy metabolism, but the underlying mechanism remains unclear. METHODS: Human normal hepatocytes (LO2 cells) were incubated with palmitic acid to establish the cell model of nonalcoholic fatty liver. The effects of DSG on lipid metabolism, glucose uptake and mitochondrial function were evaluated. Furthermore, the mechanism of DSG on oxidative stress, lipid consumption and lipid synthesis in LO2 cells was investigated. RESULTS: The results indicated that palmitic acid induced obvious lipid accumulation in LO2 cells and that DSG treatment significantly reduced the intracellular lipid content. DSG treatment upregulated expression of lipolysis proteins, including phospho-AMP activated protein kinase (p-AMPK), phospho-acetyl-coA carboxylase (p-ACC) and carnitine acyl transferase 1A (CPT-1A), and inhibited expression of lipid synthesis-related proteins, including sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase (FAS). Additionally, DSG-treated cells displayed a marked improvement in mitochondrial function, with less production of reactive oxygen species and a higher mitochondrial membrane potential compared with the model group. CONCLUSION: This study suggests that DSG can reduce intracellular lipid accumulation in LO2 cells and that the underlying mechanism may be related to the improving oxidative stress, increasing fatty acid ß-oxidation and decreasing lipid synthesis. The above changes might be mediated by the activation of the AMPK/ACC/CPT-1A pathway and inhibition of the SREBP-1c/FAS pathway.

Proximal Tubular Cell-Specific Ablation of Carnitine Acetyltransferase Causes Tubular Disease and Secondary Glomerulosclerosis.

Proximal tubular epithelial cells are highly energy demanding. Their energy need is covered mostly from mitochondrial fatty acid oxidation. Whether derailments in fatty acid metabolism and mitochondrial dysfunction are forerunners of tubular damage has been suggested but is not entirely clear. Here we modeled mitochondrial overload by creating mice lacking the enzyme carnitine acetyltransferase (CrAT) in the proximal tubules, thus limiting a primary mechanism to export carbons under conditions of substrate excess. Mice developed tubular disease and, interestingly, secondary glomerulosclerosis. This was accompanied by increased levels of apoptosis regulator and fibrosis markers, increased oxidative stress, and abnormal profiles of acylcarnitines and organic acids suggesting profound impairments in all major forms of nutrient metabolism. When mice with CrAT deletion were fed a high-fat diet, kidney disease was more severe and developed faster. Primary proximal tubular cells isolated from the knockout mice displayed energy deficit and impaired respiration before the onset of pathology, suggesting mitochondrial respiratory abnormalities as a potential underlying mechanism. Our findings support the hypothesis that derailments of mitochondrial energy metabolism may be causative to chronic kidney disease. Our results also suggest that tubular injury may be a primary event followed by secondary glomerulosclerosis, raising the possibility that focusing on normalizing tubular cell mitochondrial function and energy balance could be an important preventative strategy.

A variant at 9q34.11 is associated with HLA-DQB1*06:02 negative essential hypersomnia.

Essential hypersomnia (EHS) is a lifelong disorder characterized by excessive daytime sleepiness without cataplexy. EHS is associated with human leukocyte antigen (HLA)-DQB1*06:02, similar to narcolepsy with cataplexy (narcolepsy). Previous studies suggest that DQB1*06:02-positive and -negative EHS are different in terms of their clinical features and follow different pathological pathways. DQB1*06:02-positive EHS and narcolepsy share the same susceptibility genes. In the present study, we report a genome-wide association study with replication for DQB1*06:02-negative EHS (408 patients and 2247 healthy controls, all Japanese). One single-nucleotide polymorphism, rs10988217, which is located 15-kb upstream of carnitine O-acetyltransferase (CRAT), was significantly associated with DQB1*06:02-negative EHS (P = 7.5 × 10-9, odds ratio = 2.63). The risk allele of the disease-associated SNP was correlated with higher expression levels of CRAT in various tissues and cell types, including brain tissue. In addition, the risk allele was associated with levels of succinylcarnitine (P = 1.4 × 10-18) in human blood. The leading SNP in this region was the same in associations with both DQB1*06:02-negative EHS and succinylcarnitine levels. The results suggest that DQB1*06:02-negative EHS may be associated with an underlying dysfunction in energy metabolic pathways.

Development of mazF-based markerless genome editing system and metabolic pathway engineering in Candida tropicalis for producing long-chain dicarboxylic acids.

Candida tropicalis can grow with alkanes or plant oils as the sole carbon source, and its industrial application thus has great potential. However, the choice of a suitable genetic operating system can effectively increase the speed of metabolic engineering. MazF functions as an mRNA interferase that preferentially cleaves single-stranded mRNAs at ACA sequences to inhibit protein synthesis, leading to cell growth arrest. Here, we constructed a suicide plasmid named pPICPJ-mazF that uses the mazF gene of Escherichia coli as a counterselectable marker for the markerless editing of C. tropicalis genes to increase the rate of conversion of oils into long-chain dicarboxylic acids. To reduce the ß-oxidation of fatty acids, the carnitine acetyltransferase gene (CART) was deleted using the gene editing system, and the yield of long-chain acids from the strain was increased to 8.27 g/L. By two homologous single exchanges, the promoters of both the cytochrome P450 gene and the NADPH-cytochrome P450 reductase gene were subsequently replaced by the constitutively expressed promoter pGAP, and the production of long-chain dicarboxylic acids by the generated strain (C. tropicalis PJPP1702) reached 11.39 g/L. The results of fed-batch fermentation showed that the yield of long-chain acids from the strain was further increased to 32.84 g/L, which was 11.4 times higher than that from the original strain. The results also showed that the pPICPJ-mazF-based markerless editing system may be more suited for completing the genetic editing of C. tropicalis.

Carnitine Acetyltransferase in AgRP Neurons Is Required for the Homeostatic Adaptation to Restricted Feeding in Male Mice.

Behavioral adaptation to periods of varying food availability is crucial for survival, and agouti-related protein (AgRP) neurons have been associated with entrainment to temporal restricted feeding. We have shown that carnitine acetyltransferase (Crat) in AgRP neurons enables metabolic flexibility and appropriate nutrient partitioning. In this study, by restricting food availability to 3 h/d during the light phase, we examined whether Crat is a component of a food-entrainable oscillator (FEO) that helps link behavior to food availability. AgRP Crat knockout (KO) mice consumed less food and regained less body weight but maintained blood glucose levels during the 25-day restricted feeding protocol. Importantly, we observed no difference in meal latency, food anticipatory activity (FAA), or brown adipose tissue temperature during the first 13 days of restricted feeding. However, as the restricted feeding paradigm progressed, we noticed an increased FAA in AgRP Crat KO mice. The delayed increase in FAA, which developed during the last 12 days of restricted feeding, corresponded with elevated plasma levels of corticosterone and nonesterified fatty acids, indicating it resulted from greater energy debt incurred by KO mice over the course of the experiment. These experiments highlight the importance of Crat in AgRP neurons in regulating feeding behavior and body weight gain during restricted feeding but not in synchronizing behavior to food availability. Thus, Crat within AgRP neurons forms a component of the homeostatic response to restricted feeding but is not likely to be a molecular component of FEO.

Organelle-Derived Acetyl-CoA Promotes Prostate Cancer Cell Survival, Migration, and Metastasis via Activation of Calmodulin Kinase II.

Although emerging evidence suggests a potential role of calcium/calmodulin-dependent kinase II (CaMKII) in prostate cancer, its role in prostate cancer tumorigenesis is largely unknown. Here, we examine whether the acetyl CoA-CaMKII pathway, first described in frog oocytes, promotes prostate cancer tumorigenesis. In human prostate cancer specimens, metastatic prostate cancer expressed higher levels of active CaMKII compared with localized prostate cancer. Correspondingly, basal CaMKII activity was significantly higher in the more tumorigenic PC3 and PC3-mm2 cells relative to the less tumorigenic LNCaP and C4-2B4 cells. Deletion of CaMKII by CRISPR/Cas9 in PC3-mm2 cells abrogated cell survival under low-serum conditions, anchorage-independent growth and cell migration; overexpression of constitutively active CaMKII in C4-2B4 cells promoted these phenotypes. In an animal model of prostate cancer metastasis, genetic ablation of CaMKII reduced PC3-mm2 cell metastasis from the prostate to the lymph nodes. Knockdown of the acetyl-CoA transporter carnitine acetyltransferase abolished CaMKII activation, providing evidence that acetyl-CoA generated from organelles is a major activator of CaMKII. Genetic deletion of the ß-oxidation rate-limiting enzyme ACOX family proteins decreased CaMKII activation, whereas overexpression of ACOXI increased CaMKII activation. Overall, our studies identify active CaMKII as a novel connection between organelle ß-oxidation and acetyl-CoA transport with cell survival, migration, and prostate cancer metastasis.Significance: This study identifies a cell metabolic pathway that promotes prostate cancer metastasis and suggests prostate cancer may be susceptible to ß-oxidation inhibitors. Cancer Res; 78(10); 2490-502. ©2018 AACR.

Cytosolic carnitine acetyltransferase as a source of cytosolic acetyl-CoA: a possible mechanism for regulation of cardiac energy metabolism.

The role of carnitine acetyltransferase (CrAT) in regulating cardiac energy metabolism is poorly understood. CrAT modulates mitochondrial acetyl-CoA/CoA (coenzyme A) ratios, thus regulating pyruvate dehydrogenase activity and glucose oxidation. Here, we propose that cardiac CrAT also provides cytosolic acetyl-CoA for the production of malonyl-CoA, a potent inhibitor of fatty acid oxidation. We show that in the murine cardiomyocyte cytosol, reverse CrAT activity (RCrAT, producing acetyl-CoA) is higher compared with the liver, which primarily uses ATP-citrate lyase to produce cytosolic acetyl-CoA for lipogenesis. The heart displayed a lower RCrAT Km for CoA compared with the liver. Furthermore, cytosolic RCrAT accounted for 4.6 ± 0.7% of total activity in heart tissue and 12.7 ± 0.2% in H9C2 cells, while highly purified heart cytosolic fractions showed significant CrAT protein levels. To investigate the relationship between CrAT and acetyl-CoA carboxylase (ACC), the cytosolic enzyme catalyzing malonyl-CoA production from acetyl-CoA, we studied ACC2-knockout mouse hearts which showed decreased CrAT protein levels and activity, associated with increased palmitate oxidation and acetyl-CoA/CoA ratio compared with controls. Conversely, feeding mice a high-fat diet for 10 weeks increased cardiac CrAT protein levels and activity, associated with a reduced acetyl-CoA/CoA ratio and glucose oxidation. These data support the presence of a cytosolic CrAT with a low Km for CoA, favoring the formation of cytosolic acetyl-CoA, providing an additional source to the classical ATP-citrate lyase pathway, and that there is an inverse relation between CrAT and the ratio of acetyl-CoA/CoA as evident in conditions affecting the regulation of cardiac energy metabolism.

l-carnitine supplementation during vitrification or warming of in vivo-produced ovine embryos does not affect embryonic survival rates, but alters CrAT and PRDX1 expression.

l-carnitine is an antioxidant and ß-oxidation stimulator substance commonly used to improve metabolic performance of oocytes and embryos in in vitro systems. However, few studies have evaluated its beneficial effects in embryos produced in vivo. This study aimed to evaluate the effect of l-carnitine supplementation into vitrification or warming solutions on the post-warming character of day 6-7 in vivo-produced ovine embryos. l-carnitine (3.72 mM) was added to vitrification (Experiment 1) or warming solutions (Experiment 2). In experiments 1 and 2, the embryos were vitrified using straw and cryo-tip protocols, respectively. In vitro culture (IVC) of warmed embryos was performed for 72 h in order to evaluate survival rates, reactive oxygen species (ROS) levels, total cell number (TCN), number of apoptotic cells, apoptotic index evaluation, and gene expression analysis of carnitine palmitoyltransferase I and 2 (CPT1 and CPT2), carnitine O-acetyltransferase (CrAT), and peroxiredoxin-1 (PRDX1). In experiment 1, survival rate, ROS levels after 24 h of IVC, total cell number at 24 h and 72 h, apoptotic cells and apoptotic index at 72 h of IVC were similar in embryos vitrified in medium supplemented with LC or not. Gene expression analysis showed no differences in CPT1 and CPT2 mRNA relative abundance in embryos of both experiments compared to fresh embryos (FE); however, CrAT was downregulated (p < 0.05) in C1, and PRDX1 was downregulated (p < 0.05) in both the control (C1) and l-carnitine (LC1) groups, compared to FE. Moreover, CrAT and PRDX1 were upregulated (p < 0.05) in C2, and CrAT was downregulated (p < 0.05) in LC2, in relation to FE. Although the short-term LC supplementation at 3.72 mM did not improve survival, and quality parameters of in vivo-produced ovine embryos, it could affect their quality at a molecular level. In conclusion, further investigations with different concentrations of LC and tools are needed for improvement of the efficiency of these strategies.

Carnitine acetyltransferase (CRAT) expression in macrophages is dispensable for nutrient stress sensing and inflammation.

OBJECTIVE: Fatty acid oxidation in macrophages is thought to regulate inflammatory status and insulin-sensitivity. An important unanswered question in this field is whether carnitine acetyl-transferase (CrAT) that regulates fatty acid oxidation and mitochondrial acetyl-CoA balance is required to integrate nutrient stress sensing to inflammatory response in macrophages. METHODS: Mice with myeloid lineage-specific Crat deletion were subjected to several metabolic stressors, including high-fat diet-induced obesity, fasting, and LPS-induced endotoxemia. Their metabolic homeostasis was compared to that of Crat-sufficient littermate controls. Inflammatory potential of Crat-deficient and Crat-sufficient macrophages were measured both in vitro and in vivo. RESULTS: Our studies revealed that ablation of CrAT in myeloid lineage cells did not impact glucose homeostasis, insulin-action, adipose tissue leukocytosis, and inflammation when animals were confronted with a variety of metabolic stressors, including high-fat diet, fasting, or LPS-induced acute endotoxemia. CONCLUSIONS: These findings demonstrate that unlike muscle cells, substrate switch mechanisms that control macrophage energy metabolism and mitochondrial short-chain acyl-CoA pools during nutrient stress are controlled by pathways that are not solely reliant on CrAT.

Influence of betaine and arginine supplementation of reduced protein diets on fatty acid composition and gene expression in the muscle and subcutaneous adipose tissue of cross-bred pigs.

The isolated or combined effects of betaine and arginine supplementation of reduced protein diets (RPD) on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig m. longissimus lumborum and subcutaneous adipose tissue (SAT) were assessed. The experiment was performed on forty intact male pigs (Duroc×Large White×Landrace cross-breed) with initial and final live weights of 60 and 93 kg, respectively. Pigs were randomly assigned to one of the following five diets (n 8): 16·0 % of crude protein (control), 13·0 % of crude protein (RPD), RPD supplemented with 0·33 % of betaine, RPD supplemented with 1·5 % of arginine and RPD supplemented with 0·33 % of betaine and 1·5 % of arginine. Data confirmed that RPD increase intramuscular fat (IMF) content and total fat content in SAT. The increased total fat content in SAT was accompanied by higher GLUT type 4, lipoprotein lipase and stearoyl-CoA desaturase mRNA expression levels. In addition, the supplementation of RPD with betaine and/or arginine did not affect either IMF or total fat in SAT. However, dietary betaine supplementation slightly affected fatty acid composition in both muscle and SAT. This effect was associated with an increase of carnitine O-acetyltransferase mRNA levels in SAT but not in muscle, which suggests that betaine might be involved in the differential regulation of some key genes of lipid metabolism in pig muscle and SAT. Although the arginine-supplemented diet decreased the mRNA expression level of PPARG in muscle and SAT, it did not influence fat content or fatty acid composition in any of these pig tissues.