Carnitine Palmitoyltransferase 1A mRNA Research Articles

MiR-21-3p inhibitor exerts myocardial protective effects by altering macrophage polarization state and reducing excessive mitophagy

Chronic heart failure (CHF) is closely associated with inflammation and mitochondrial dysfunction in cardiomyocytes. This study attempts to investigate the effects of microRNA-21-3p (miR-21-3p) on macrophage polarization and mitophagy in CHF. Here we found miR-21-3p was upregulated in CHF and negatively correlated with carnitine palmitoyl transferase 1A (CPT1A). L-palmitoyl carnitine (L-PC) exacerbated isoproterenol (ISO)-induced myocardial structural disruption and fibrosis in rats, which was exacerbated by miR-21-3p. Mechanistically, miR-21-3p accelerated M1 macrophage polarization. Both miR-21-3p inhibitor and CPT1A overexpression suppressed mitophagy. The inhibition of CPT1A on mitophagy was reversed by miR-21-3p. MiR-21-3p targeted CPT1A mRNA and co-localized with CPT1A protein in cardiomyocytes. In the co-culture system of M1 macrophages and H9c2 cells, miR-21-3p mimics in H9c2 cells promoted M1 polarization, whereas miR-21-3p inhibitor reduced M1 phenotype. M1 macrophages exacerbated H9c2 cell damage. These findings support the potential therapeutic targeting of miR-21-3p to regulate inflammation and mitophagy by inducing CPT1A in CHF.

Hypoxic preconditioning accelerates the healing of ischemic intestinal injury by activating HIF-1α/PPARα pathway-mediated fatty acid oxidation

Hypoxic preconditioning (HPC) has been shown to improve organ tolerance to subsequent severe hypoxia or ischemia. However, its impact on intestinal ischemic injury has not been well studied. In this study, we evaluated the effects of HPC on intestinal ischemia in rats. Intestinal rehabilitation, levels of fatty acid oxidation (FAO) by-products, intestinal stem cells (ISCs), levels of hypoxia-inducible factor 1 subunit α (HIF-1α) and its downstream genes such as peroxisome proliferator-activated receptor α (PPARα), and carnitine palmitoyltransferase 1a (CPT1A) were assessed at distinct time intervals following intestinal ischemia with or without the interference of HIF-1α. Our data showed that HPC facilitates the restoration of the intestinal structure and enhances the FAO, by boosting intestinal stem cells. Additionally, HIF-1α, PPARα, and CPT1A mRNA and their protein levels were generally up-regulated in the small intestine of HPC rats as compared to the control group. Our vitro experiment also shows low-oxygen induces highly levels of HIF-1α and its downstream genes, with a concurrent increase in FAO products in IEC-6 cells. Furthermore, the above phenomenon could be reversed by silencing HIF-1α. In conclusion, we hypothesize that HPC can stimulate the activation of intestinal stem cells via HIF-1α/PPARα pathway-mediated FAO, thereby accelerating the healing process post ischemic intestinal injury.

Carbohydrate and fat intake associated with risk of metabolic diseases through epigenetics of CPT1A

Epigenome-wide association studies identified the cg00574958 DNA methylation site at the carnitine palmitoyltransferase-1A (CPT1A) gene to be associated with reduced risk of metabolic diseases (hypertriglyceridemia, obesity, type 2 diabetes, hypertension, metabolic syndrome), but the mechanism underlying these associations is unknown. We aimed to elucidate whether carbohydrate and fat intakes modulate cg00574958 methylation and the risk of metabolic diseases. We examined associations between carbohydrate (CHO) and fat (FAT) intake, as percentages of total diet energy, and the CHO/FAT ratio with CPT1A-cg00574958, and the risk of metabolic diseases in 3 populations (Genetics of Lipid Lowering Drugs and Diet Network, n=978; Framingham Heart Study, n=2331; and REgistre GIroní del COR study, n=645) while adjusting for confounding factors. To understand possible causal effects of dietary intake on the risk of metabolic diseases, we performed meta-analysis, CPT1A transcription analysis, and mediation analysis with CHO and FAT intakes as exposures and cg00574958 methylation as the mediator. We confirmed strong associations of cg00574958 methylation with metabolic phenotypes (BMI, triglyceride, glucose) and diseases in all 3 populations. Our results showed that CHO intake and CHO/FAT ratio were positively associated with cg00574958 methylation, whereas FAT intake was negatively correlated with cg00574958 methylation. Meta-analysis further confirmed this strong correlation, with β=58.4±7.27, P=8.98 x 10-16 for CHO intake; β = -36.4±5.95, P=9.96 x 10-10 for FAT intake; and β=3.30±0.49, P=1.48 x 10-11 for the CHO/FAT ratio. Furthermore, CPT1A mRNA expression was negatively associated with CHO intake, and positively associated with FAT intake, and metabolic phenotypes. Mediation analysis supports the hypothesis that CHO intake induces CPT1A methylation, hence reducing the risk of metabolic diseases, whereas FAT intake inhibits CPT1A methylation, thereby increasing the risk of metabolic diseases. Our results suggest that the proportion of total energy supplied by CHO and FAT can have a causal effect on the risk of metabolic diseases via the epigenetic status of CPT1A.Study registration at https://www.clinicaltrials.gov/: the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN)-NCT01023750; and the Framingham Heart Study (FHS)-NCT00005121.

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.

Effect of divergence in residual feed intake on expression of lipid metabolism-related genes in the liver of meat-type ducks1

Lipid metabolism is considered one of the important factors affecting residual feed intake (RFI). However, the relationship between RFI and expression of lipid metabolism-related genes is unknown in meat-type ducks. To address this issue, a total of 1,000 male meat-type ducks with similar body weight were randomly selected to measure body weight gain and feed intake from 21 to 42 d of age to estimate RFI. The 8 greatest- (high RFI [HRFI]) and lowest- (low RFI [LRFI]) ranking birds were then selected for the present study. Relative expressions of key genes, namely sirtuin 1 (Sirt1), forkhead box O1 (Foxo1), peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding transcription factor 1c (SREBP-1c), fas cell surface death receptor (FAS), acetyl-CoA carboxylase alpha (ACC), carnitine palmitoyltransferase 1A (CPT1A), and acyl-CoA oxidase 1 (ACOX1), were then determined in the HRFI and LRFI ducks by quantitative PCR. The results showed that RFI, feed conversion ratio (FCR), and average daily feed intake (ADFI) were significantly lower (P < 0.05) in LRFI ducks than in HRFI ducks. In addition, expression of Sirt1, Foxo1, CPT1A, and ACOX1 were significantly higher in LRFI ducks than in HRFI ducks (P < 0.05), whereas PPARγ and FAS expression levels were significantly lower in LRFI ducks than in HRFI ducks (P < 0.01). Correlation analysis showed that Sirt1, CPT1A, and ACOX1 expressions were significantly negatively correlated with FCR (r = -0.81 to -0.93; P < 0.01), whereas PPARγ and FAS expressions were significantly positively correlated with FCR (r = 0.74 to 0.87; P < 0.01). PPARγ expression was significantly positively correlated with RFI (r = 0.83; P < 0.01), whereas CPT1A and ACOX1 expressions were significantly negatively correlated with RFI (r = -0.84 to -0.89; P < 0.01). Sirt1 mRNA expression was positively correlated with Foxo1, CPT1A, and ACOX1 mRNA expression (r = 0.78 to 0.92; P < 0.01). Association of Foxo1 with CPT1A and ACOX1 was positive (r = 0.88 to 0.96; P < 0.01). These results suggest that genes related to fatty acid oxidation are upregulated in the liver of ducks with high feed efficiency, while genes associated with lipid synthesis are downregulated. Furthermore, the inclusion of lipid metabolism-related genes in future breeding programs might be beneficial for selecting ducks with greater feed efficiency phenotype.

Abstract B42: Identification of CPT1A as a novel driver of proliferation in luminal breast cancer

Abstract Clinically, approximately two-thirds of the nearly 250,000 breast cancer cases diagnosed each year in the United States are hormone receptor-positive, luminal breast tumors. Slower-growing luminal breast tumors are often successfully treated using endocrine-based therapies, resulting in a relatively good prognosis for these patients. However, more highly proliferative luminal tumors, even with the recent approval of CDK4/6 inhibitors, are often resistant, or become resistant, to current therapies, leading to a worse outcome for these women. Therefore, it is necessary to identify genetic events responsible for tumorigenesis and to identify potential druggable genetic alterations and/or pathways in order to improve clinical outcome. To address this question, we recently developed and used an innovative genomics-based strategy to interrogate orthogonal genome-wide data from more than 2,500 patients from the TCGA and METABRIC studies. By using an 11-gene mRNA-based gene expression signature of proliferation as a conceptual framework, we identified DNA copy number alterations and somatic mutations associated with luminal breast cancer proliferation. In order to identify the subset of amplified genes that are essential for cell viability, we analyzed a dataset of 27 breast cancer cell lines with available mRNA expression and genome-wide shRNA proliferation data. By integrating DNA copy number and shRNA analyses, we identified amplified carnitine palmitoyltransferase 1A (CPT1A) as a novel driver of proliferation in luminal breast cancer. CPT1A was found to be amplified in 40.2% of highly proliferative (top quartile) luminal tumors compared to 24.7% of all other samples and CPT1A DNA copy number status correlated with mRNA expression levels. Importantly, CPT1A was shown to be essential in luminal breast cancer cell lines with a high proliferation signature score. By analyzing RPPA data from luminal breast tumors, we confirmed CPT1A amplified tumors have increased expression of protein markers of proliferation. Since CPT1A is the rate-limiting enzyme responsible for fatty acid import into the mitochondria during fatty acid β oxidation (FAO), these data indicate that highly proliferative luminal tumors may utilize FAO as a prominent energy source. Interestingly, our analyses of RNA-seq data (n=1,031) demonstrated that highly proliferative luminal tumors have significantly higher CPT1A mRNA expression than either basal-like, which are largely synonymous with TNBC, or less proliferative luminal tumors, suggesting that these tumors may have a greater dependency on CPT1A activity. CPT1A has two major variants: cytoplasmic Variant 1 is the rate-limiting enzyme responsible for fatty acid import into the mitochondria during FAO while nuclear Variant 2 is purported to mediate histone deacetylase activity. Analyses of RNAseq data (n=819) demonstrated that Variant 1 represents &amp;gt;99.2% of CPT1A mRNA transcripts in a given tumor, suggesting the FAO-associated role of CPT1A is the more critical function of this protein in breast cancer. Consistent with this observation, we determined that breast cancer cell lines with high CPT1A protein expression are significantly more sensitive to etomoxir, a CPT1A-specific inhibitor known to repress FAO, when compared to cell lines with low CPT1A protein levels. Finally, we determined that under glucose-depleted conditions, cell lines with high CPT1A expression showed no difference in etomoxir response whereas cells with low CPT1A become significantly more sensitive. Collectively, these data suggest that CPT1A amplification and overexpression contribute to proliferation. Given that a number of approved or experimental drugs target CPT1A or FAO, our data suggest that CPT1A and/or FAO may represent a novel therapeutic target for luminal breast cancer. Citation Format: Kristina Mastrioanni, Kimberly Parker, Michael L. Gatza. Identification of CPT1A as a novel driver of proliferation in luminal breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B42.

Effects of hypoxic exercise training on microRNA expression and lipid metabolism in obese rat livers.

To investigate the effects of hypoxic exercise training on microRNA (miRNA) expression and the role of miRNA expression in regulating lipid metabolism, 20 dietary-induced obese SD rats were divided into a normoxic sedentary group (N, n=10) and a hypoxic exercise training group (H, n=10). After four weeks, measurements were taken of body weight, body length, fat mass, serum lipid concentration, miRNAs differentially expressed in rat liver, and gene and protein expression levels of peroxisome proliferator activated receptor α (PPARα), fatty acid synthetase (FAS), and carnitine palmitoyl transferase 1A (CPT1A) in rat liver. Body weight, Lee's index, fat mass, fat/weight ratio, and serum levels of total cholesterol (TC) and high density lipoprotein cholesterol (HDL-C) were all significantly lower in the H group than in the N group (P<0.01). Six miRNAs expressed significantly differently in the liver (P<0.05). Specifically, expression levels of miR-378b were significantly lower in the H group than in the N group (P<0.05). Compared with the normoxic sedentary group, hypoxic exercise training resulted in a lower ratio of FAS mRNA to CPT1A mRNA (P<0.05), as well as lower CPT1A protein levels (P<0.01), while a higher ratio of FAS to CPT1A protein levels (P<0.01) was observed. In conclusion, hypoxic training may elevate the resistance of high fat diet induced obesity in rats by reducing the expression of miR-378b, and decrease the fatty acid mitochondrial oxidation in obese rat livers by decreasing the protein expression of CPT1A and increasing the protein expression ratio of FAS/CPT1A.

Structure of the Carnitine Palmitoyltransferase‐1A Gene in Rats, Mice and Humans

Carnitine Palmitoyltransferase‐1A (CPT1A) is expressed in all tissues of mammals except skeletal muscle and adipose tissue. CPT1A is the key regulatory enzyme of long‐chain fatty acid oxidation, and it is especially important in liver and kidney where it is important for controlling ketogenesis. When inhibited by its physiological inhibitor, malonyl‐CoA, CPT1A is also important for directing fatty acids into pathways of esterification resulting in increased secretion of lipoproteins. Since knowledge of the structure and control of the CPT1A gene will be important for understanding diabetes, altered thyroid states, and other aberrant energy states, we examined gene sequence data, genomic structure and mRNA expression data in rats, mice and human cell lines. We initially used RACE reactions to produce the full‐length rat cDNA and discovered that the initially published sequence for the rat cDNA did not include the most upstream exons. In experiments reported here, we used real‐time PCR to examine the structure of genes encoding CPT1A in these three species to see which exons were expressed in each species. The human CPT1A gene expresses 2 alternative exons (1a and 1b) upstream of the exon containing the start site for transcription. We found that the rat gene expresses a similar but larger exon (340 bp vs. 163 bp) that is approximately the same distance upstream (28 kbp) of the ATG. Furthermore, we found that rat gene expresses 3 additional intervening exons, all of which are different, both in size and location, from the human exon 1b. The mouse liver also expresses an initial exon in the same position as the human and rat gene, and it has 89% similarity with the rat exon 1a. The mouse also has sequences that are approx. 90% similar to the two additional rat exons, but they were not found to be expressed in mature mouse mRNA. In conclusion, the rat liver expresses at least 4 CPT1A mRNA species, whereas there are only 2 species expressed in humans. In mouse liver, 2 species of mRNA are expressed, but they are substantially different from the human mRNA species.

1234 PRO-APOPTOTIC MIR-34A/SIRT1/P53 PATHWAY IN NAFLD PATIENTS AND TARGETING BY URSODEOXYCHOLIC ACID IN RAT LIVER

histology was graded and staged according to the Kleiner system. Hepatic triglycerides were quantified, and qPCR/Western blotting used to assess inflammation, fibrosis and lipid metabolism. Hepatic (oval) cell response was assessed by pan-cytokeratin (panCK) and Sox9 immunohistochemistry with morphometric analysis. Results: ALIOS fed mice developed lobular inflammation/ballooning by 6m and F3 fibrosis by 12m. By 6m, 5aR1 mice fed ALIOS diet demonstrated greater steatosis histologically (median Kleiner steatosis score; 3 vs 1), and biochemically (mean hepatic triglyceride; 93.1 vs 62.4 nmol/mg, p < 0.01) than WT mice fed ALIOS diet. There was no difference in inflammation or fibrosis. 5aR1 mice had reduced carnitine palmitoyl transferase-1a mRNA expression (p < 0.05), and increased acetyl-CoA carboxylase protein expression, consistent with decreased fatty acid b-oxidation and increased de novo lipogenesis. Focal hepatocellular lesions consistent with dysplasia/hepatocellular carcinoma developed after 12m ALIOS diet in 5/10 WT mice, with none developing in 5aR1 mice. Dysplastic/hepatocellular carcinoma lesions expressed the oval cell marker Sox9. Hepatic oval cell proliferation increased after ALIOS diet, but was attenuated in 5aR1 compared to WT mice (mean surface area panCK positive; 0.62% vs. 0.94%; Sox9 positive cells per view; 60 vs. 76/field of view respectively). Conclusions: 5aR1 deficiency promotes hepatic steatosis, but protects against the development of NASH-related dysplasia/hepatocellular carcinoma and reduces hepatic oval cell proliferation. Prolonged ALIOS diet is a unique murine model for studying NASH-related oval cell proliferation and hepatocarcinogenesis in the absence of cirrhosis. These data support a role of 5aR1 in hepatocarcinogenesis associated with NASH.

Effects of osthol on blood pressure and lipid metabolism in stroke-prone spontaneously hypertensive rats

Osthol, a coumarin compound, was isolated from the dried fruits of Cnidium monnieri (Umbelliferae) and the effect of dietary osthol on hypertension and lipid metabolism was examined in stroke-prone spontaneously hypertensive rats (SHRSP). Six-week-old male SHRSP were fed the experimental diet containing 0.05% osthol by weight for 4 weeks with free access to the diet and water. Elevation of systolic blood pressure was significantly suppressed on and after 3 weeks. In addition, significant decreases in cholesterol and triglyceride contents in the liver were recognized without any significant changes in serum lipids profiles. A comparative study on hepatic mRNA expression indicated that osthol induced a significant increase in 3-hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase mRNA expression, which may lead to decrease in hepatic cholesterol pool through inhibition of the enzyme activity. Moreover, osthol induced a significant increase in acyl-CoA oxidase mRNA expression associated with an increase in carnitine palmitoyl transferase 1a mRNA expression, which suggests the acceleration of β-oxidation of hepatic fatty acids. This may be responsible, at least in part, for the reduction of hepatic triglyceride content in SHRSP. These beneficial effects of osthol could be useful for both prevention of atherosclerosis and suppression of hepatic lipid accumulation.

Individual and combined effects of docosahexaenoic acid, stearic acid, trans‐vaccenic acid, and c9,t11‐conjugated linoleic acid on hepatic gene expression

Dietary long-chain fatty acids (LCFA), polyunsaturated fatty acids (PUFA) and their combinations alter metabolism by modulating expression of fatty acid synthase (FAS), carnitine palmitoyltransferase-1a (CPT-1a), and acyl-CoA oxidase (ACO). The effects of LCFA and PUFA on mRNA expression were determined in H4IIE rat hepatoma cells incubated with docosahexaenoic acid (DHA), c9, t11-conjugated linoleic acid (CLA), trans-vaccenic acid (TVA), and stearic acid (SA) or their combinations. Treatments were: 200 μM of individual fatty acids DHA, CLA, SA, or TVA; Replacement of 100 μM DHA with either 100 μM CLA or 100 μM SA; 100 μM CLA replaced by 100 μM TVA (final fatty acid concentration of 200 μM); 200 μM of DHA or CLA supplemented with 200μM of CLA, SA, or TVA. When CLA replaced DHA, FAS mRNA decreased by 53.7% (P< 0.05). Replacement of DHA with SA resulted in a 74.9% decrease (P< .05) in FAS mRNA. When 200 μM CLA was supplemented with 200 μM DHA or with 200 μM TVA, FAS mRNA decreased (P< 0.05) by 62.4% and 48.6% respectively. When CLA was replaced with TVA, CTP-1a mRNA tended (P= 0.05) to decrease by 47.5%. When CLA was supplemented with DHA there was a tendency (P< 0.10) for a 40.2% decrease in CPT-1a mRNA. Fatty acid concentration and mixture had no effect on ACO mRNA. The data suggest that individual and combined LCFA/PUFA exert different potencies on expression of genes for fatty acid metabolism in liver.

Positive regulation of hepatic carnitine palmitoyl transferase 1A (CPT1A) activities by soy isoflavones and L–carnitine

Genistein increases CPT1A, a rate-limiting enzyme in the beta-oxidation pathway, enzyme activity by increasing CPT1A transcription in HepG2 cells and, consequently, suppresses high fat induced obesity in C57BL/6J mice. Genistein and daidzein are the most abundant isoflavones in soy. To investigate the effect of co-treatment of genistein and L-carnitine on CPT1A enzyme activity and to determine whether daidzein also increases CPT1A activity and to establish a cell line that can be used to screen chemicals to regulate CPT1A transcription. The enzyme activities of CPT1A were determined after HepG2 cells were incubated with 10 microM genistein or 10 microM daidzein or 1 mM L-carnitine or in combination with 10 microM genistein and 1 mM L-carnitine or in combination with 10 microM daidzein and 1 mM L-carnitine. The mRNA expression levels of CPT1A were determined by real time PCR method after HepG2 cells were incubated with 10 microM genistein or 10 microM daidzein. A suggested CPT1A promoter region was cloned from human genomic DNA and the CPT1A promoter-luciferase reporter gene construct was made, and the promoter-reporter gene construct was transfected into human hepatoma cell line Huh7. The enzyme activity of CPT1A was at least 2.3- fold higher in L-carnitine and genistein co-treated HepG2 cells than either single-agent treated cells. Daidzein also significantly increased the mRNA expression of CPT1A as well as the enzyme activity of CPT1A. A stable Huh7 cell line, which was selected after Huh7 cells were transfected with CPT1A promoter luciferase reporter gene construct, was characterized by confirming that luciferase activity of the cell line can be regulated by genistein and daidzein as well as clofibrate, a well-known CPT1A mRNA up-regulating drug. Genistein and daidzein can up-regulate CPT1A enzyme activity through up-regulation of CPT1A transcription. Co-treatment of L-carnitine and genistein additively increases CPT1A enzyme activity in HepG2 cells. A stable Huh7 cell line transfected with the CPT1A promoter luciferase reporter gene was established and characterized.

Organization of the human liver carnitine palmitoyltransferase 1 gene ( CPT1A) and identification of novel mutations in hypoketotic hypoglycaemia.

Carnitine palmitoyltransferase 1A (CPT1A) deficiency is a rare autosomal recessive disorder of mitochondrial fatty acid oxidation. CPT1 controls the import of long-chain fatty acids into the mitochondria, where they are oxidized. Two CPT1 isoforms, the so-called "liver" and "muscle" CPT1s encoded by the CPT1Aand CPT1Bgenes, respectively, have been identified so far. While the cDNA sequences of both isoforms are known, only CPT1Bgene organization has yet been described. We took advantage of the working draft data to characterize the organization of the human CPT1A gene. We have shown the existence of 20 exons, spanning 60 kb of DNA. Two alternate promoters and numerous transcription factor-binding sites were identified within the 5' upstream region of the gene. In the 3' untranslated region, the major polyA signal was suggested to lie about 2 kb downstream of the stop codon. These data enabled us to characterize six novel mutations in four CPT1A-deficient patients; namely Q100X (exon 4), A414 V (exon 11), Y498C (exon 13), 1876-1G>A (intron 15), a 113-bp intronic insertion in the mature CPT1A mRNA (exon 13-14 junction), and a large 8-kb deletion encompassing intron 14 to exon 17. Thus, identification of the CPT1A gene organization contributes to improve the molecular screening in patients and provides tools for the study of the human CPT1A gene expression.