Genes Carnitine Palmitoyltransferase Research Articles

SGMS1-AS1/MicroRNA-106a-5p/CPT2 Axis as a Novel Target for Regulating Lactate Metabolism in Colon Cancer.

The malignant transformation of cells can lead to aerobic glycolysis, an important form of metabolic reprogramming in colon cancer cells, which can cause the accumulation of lactate and accelerate the proliferation of tumor cells also enhance their chemotherapy drug resistance. The aim of this study was to investigate the possible molecular mechanisms responsible for the increased lactate expression in colon cancer. Several bioinformatics methods, including differential analysis, gene ontology enrichment, univariate and multivariate Cox regression analysis were used to find the lactic acid-related gene carnitine palmitoyltransferase 2. We analyzed the relationship between carnitine palmitoyltransferase 2 and clinical features as well as immune microenvironment. To further explore the mechanism of carnitine palmitoyltransferase 2 in colon cancer, we performed methylation analysis and constructed a competitive endogenous RNA network, which was validated in cell lines and clinical specimens. We used bioinformatics to select the lactic acid-related gene carnitine palmitoyltransferase 2 and found low expression of carnitine palmitoyltransferase 2 was associated with poor prognosis in colon cancer. An inhibitory tumor microenvironment was created when carnitine palmitoyltransferase 2 expression was reduced, with decreased CD4 T cells, CD8 T cells, dendritic cells, and B cells but increased cancer-associated fibroblasts. Methylation analysis showed that the abnormal decrease in carnitine palmitoyltransferase 2 might be caused by hypermethylation. We constructed a network of SGMS1-AS1/microRNA-106a-5p/carnitine palmitoyltransferase 2 and verified their expression in cell lines and clinical specimens. Our work revealed the possible mechanism of lactate accumulation in colon cancer and explored a new potential treatment for colon cancer by cutting off aerobic glycolysis in tumor cells.

Optimization of the extract from flower of Citrus aurantium L. var. amara Engl. and its inhibition of lipid accumulation.

Flower of Citrus aurantium L. var. amara Engl. (CAVA) has been confirmed to have promising anti-obesity effects. However, the regulation of alkaloid extracts from flower of CAVA (Al) on lipid metabolism remain unknown. In this study, Al was optimized by ultrasound-assisted extraction using response surface methodology. The optimal conditions were ultrasonic time 72 min, ethanol concentration 78% and liquid/solid ratio 30 ml/g with the maximum alkaloid yield 5.66%. LC-MS assay indicated that the alkaloid compounds were enriched in Al after optimization. Nine alkaloid compounds were identified in Al by LC-MS assay and stachydrine, caffeine and cathine appeared as the major alkaloid compounds. Bioactivity assay showed that Al treatment significantly increased superoxide dismutase (SOD) activity, and reduced malonaldehyde (MDA) and reactive oxygen species (ROS) levels. Al administration also reversed oleic acid-induced hepatic steatosis in Hep G2 cells by inhibiting the expression of lipogenesis-signaling genes including fatty acid synthase (FAS), peroxisome proliferator-activated receptor subtype γ (PPARγ), uncoupling protein 2 (UCP2), and retinol binding protein (RBP4). However, OA-induced reduction of lipolysis-related gene carnitine palmitoyl transferase 1A (CPT1A) in Hep G2 cells was not improved by Al supplementation. Moreover, the increased SOD activity and decreased MDA and ROS contents were also observed in Caenorhabditis elegans by Al addition. Al intervention exhibited the ability to inhibit lipid accumulation in C. elegans by suppressing expression of lipid metabolism-related genes. These results suggested that the alkaloid extracts from the flower of CAVA showed great potential to regulate lipid metabolism. PRACTICAL APPLICATIONS: The extraction of alkaloid extracts from the flower of CAVA was optimized with a maximum yield of 5.66%. The regulatory effects and mechanisms of Al on lipid metabolism of Hep G2 cells and Caenorhabditis elegans were also investigated. More clinical studies are required to evaluate the potential of using alkaloids from the flower of CAVA as therapeutic agents against lipid metabolic disorders.

AMPK regulates behavior and physiological plasticity of Haliotis discus hannai under different spectral compositions

In natural environments, the spectral composition of incident light is often subject to drastic changes due to the abundance of suspended particles, floating animals, and plants in coastal waters. In this study, after four months of culturing under blue light (NB), orange light (NY), dark environment (ND), and natural light (NN), the shell length and weight-specific growth rate in Pacific abalone, Haliotis discus hannai, were ranked in the following order: NY > NN > ND > NB. To understand the growth differences in abalone under these different light environments, we first performed 24-h video monitoring and found that the cumulative movement distance and duration were lowest in group NB, whereas the cumulative movement distance and duration were significantly higher in group ND than in any other group (P < 0.05). In group NB, the time spent hidden underneath the attachment substrate accounted for 81% of the resting time, but this ratio was lowest in group ND, at only 37% (P < 0.05). Next, LC-MS metabolomics identified 201 and 105 metabolites in NB vs. NN, ND vs. NN, and NY vs. NN under the positive and negative ion modes, respectively. According to the fold changes and annotations for differential metabolites in the KEGG enrichment pathways, adenosine, NAD+, cGMP, and arachidonic acid were used as differential metabolism markers, and the AMPK signaling pathway was enriched in every comparison group, and thus investigated further. The gene sequences of three subtypes of AMPK were obtained by cloning and we found that the expression levels of AMPKα and AMPKγ, and the AMP content were significantly higher in group NB than in any other group (P < 0.05). In addition, the ATP contents and adenylate energy charge values were ranked in the following order: NY > NN > ND > NB. According to in situ hybridization analysis, the three subtype genes were widely expressed in the hepatopancreas. Finally, the contents of many lipid metabolites differed significantly among groups and the expression levels of the triglyceride hydrolysis-related gene hormone sensitive lipase and fatty acid oxidation-related gene carnitine palmitoyltransferase 1 were higher in groups ND and NB than in groups NN and NY according to fluorescence quantification PCR (P < 0.05). The expression levels of fatty acid synthase and acetyl-CoA carboxylase were significantly lower in groups ND and NB than in groups NN and NY (P < 0.05). These findings indicated that differences in the spectral composition of incident light could reshape the behavior and physiological metabolism in abalone by influencing the “energy switch” AMPK, thereby providing some insights into the mechanisms that allow nocturnal marine organisms to adapt to different lighting environments.

Effects of Replacement of Dietary Fishmeal by Cottonseed Protein Concentrate on Growth Performance, Liver Health, and Intestinal Histology of Largemouth Bass (Micropterus salmoides).

An 8-week feeding trial was conducted to explore the effects of replacement of dietary fishmeal by cottonseed protein concentrate (CPC) on growth performance, liver health, and intestine histology of largemouth bass. Four isoproteic and isolipidic diets were formulated to include 0, 111, 222, and 333 g/kg of CPC, corresponding to replace 0% (D1), 25% (D2), 50% (D3), and 75% (D4) of fishmeal. Two hundred and forty largemouth bass (15.11 ± 0.02 g) were randomly divided into four groups with three replicates per group. During the experiment, fish were fed to apparent satiation twice daily. Results indicated that CPC could replace up to 50% fishmeal in a diet for largemouth bass without significant adverse effects on growth performance. However, weight gain rate (WGR), specific growth rate (SGR), feed efficiency (FE), and condition factor (K) of the largemouth bass were significantly decreased when 75% of dietary fishmeal that was replaced by CPC. The whole body lipid content was increased with the increasing of dietary CPC levels. Oil red O staining results indicated that fish fed the D4 diet showed an aggravated fat deposition in the liver. Hepatocytes exhibited serious degeneration, volume shrinkage, and inflammatory cells infiltration in the D4 group. Intestinal villi appeared shorter and sparse with severe epithelial damage in the D4 group. The transcription levels of anti-inflammatory cytokines, such as transforming growth factor β (tgf-β), interleukin 10 (il-10), and interleukin 11 β (il-11β), were downregulated in the D4 group. The lipid metabolism-related genes carnitine palmitoyl transferase 1 (cpt1), peroxisome proliferator-activated receptor α (pparα), and target of rapamycin (TOR) pathway were also significantly downregulated in the D4 group. It was concluded that suitable replacement of fishmeal by less than 222 g CPC/kg diet had a positive effect on growth performance of fish, but an excessive substitution of 75% fishmeal by CPC would lead to the suppressed growth, liver inflammation, and intestinal damage of largemouth bass.

Gynostemma pentaphyllum extract and Gypenoside L enhance skeletal muscle differentiation and mitochondrial metabolism by activating the PGC-1α pathway in C2C12 myotubes.

BACKGROUND/OBJECTIVESPeroxisome proliferator-activated receptor-gamma co-activator-1α (PGC-1α) has a central role in regulating muscle differentiation and mitochondrial metabolism. PGC-1α stimulates muscle growth and muscle fiber remodeling, concomitantly regulating lactate and lipid metabolism and promoting oxidative metabolism. Gynostemma pentaphyllum (Thumb.) has been widely employed as a traditional herbal medicine and possesses antioxidant, anti-obesity, anti-inflammatory, hypolipemic, hypoglycemic, and anticancer properties. We investigated whether G. pentaphyllum extract (GPE) and its active compound, gypenoside L (GL), affect muscle differentiation and mitochondrial metabolism via activation of the PGC-1α pathway in murine C2C12 myoblast cells.MATERIALS/METHODSC2C12 cells were treated with GPE and GL, and quantitative reverse transcription polymerase chain reaction and western blot were used to analyze the mRNA and protein expression levels. Myh1 was determined using immunocytochemistry. Mitochondrial reactive oxygen species generation was measured using the 2′7′-dichlorofluorescein diacetate assay.RESULTSGPE and GL promoted the differentiation of myoblasts into myotubes and elevated mRNA and protein expression levels of Myh1 (type IIx). GPE and GL also significantly increased the mRNA expression levels of the PGC-1α gene (Ppargc1a), lactate metabolism-regulatory genes (Esrra and Mct1), adipocyte-browning gene fibronectin type III domain-containing 5 gene (Fndc5), glycogen synthase gene (Gys), and lipid metabolism gene carnitine palmitoyltransferase 1b gene (Cpt1b). Moreover, GPE and GL induced the phosphorylation of AMP-activated protein kinase, p38, sirtuin1, and deacetylated PGC-1α. We also observed that treatment with GPE and GL significantly stimulated the expression of genes associated with the anti-oxidative stress response, such as Ucp2, Ucp3, Nrf2, and Sod2.CONCLUSIONSThe results indicated that GPE and GL enhance exercise performance by promoting myotube differentiation and mitochondrial metabolism through the upregulation of PGC-1α in C2C12 skeletal muscle.

Herbal Combination of Phyllostachys pubescens and Scutellaria baicalensis Inhibits Adipogenesis and Promotes Browning via AMPK Activation in 3T3-L1 Adipocytes.

To investigate the anti-obesity effects and underlying mechanism of BS21, a combination of Phyllostachys pubescens leaves and Scutellaria baicalensis roots was used to investigate the effects of BS21 on adipogenesis, lipogenesis, and browning in 3T3-L1 adipocytes. The expression of adipocyte-specific genes was observed via Western blot, and the BS21 chemical profile was analyzed using ultra-performance liquid chromatography (UPLC). BS21 treatment inhibited adipocyte differentiation and reduced the expression of the adipogenic proteins peroxisome proliferator-activated receptor γ (PPAR-γ), CCAAT/enhancer-binding protein (C/EBP-α), and adipocyte protein 2 (aP2), as well as the lipogenic proteins sterol regulatory element-binding protein 1c (SREBP-1c) and fatty-acid synthase (FAS). BS21 enhanced protein levels of the beta-oxidation genes carnitine palmitoyltransferase (CPT1) and phospho-acetyl-coA carboxylase (p-ACC). BS21 also induced protein expressions of the browning marker genes PR domain containing 16 (PRDM16), peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC1α), and uncoupling protein (UCP) 1, and it induced the expression of the thermogenic gene UCP2. Furthermore, BS21 increased adenosine monophosphate-activated protein kinase (AMPK) activation. UPLC analysis showed that BS21 contains active constituents such as chlorogenic acid, orientin, isoorientin, baicalin, wogonoside, baicalein, tricin, wogonin, and chrysin. Our findings demonstrate that BS21 plays a modulatory role in adipocytes by reducing adipogenesis and lipogenesis, increasing fat oxidation, and inducing browning.

Long-term wet precipitation of PM2.5 disturbed the gut microbiome and inhibited the growth of marine medaka Oryzias melastigma

Wet precipitation, as an important process of geochemical cycling and the most effective way of cleaning fine atmospheric particles (PM2.5), can introduce the toxic substances in the atmosphere into the water environment. The adverse effect of wet precipitation of PM2.5 on marine fish is still unclear. In this study, PM2.5 samples were collected from six locations along coastal areas of the south China sea for 30 days and used to simulate the impacts of multiday discontinuity wet precipitation of PM2.5 on marine medaka (Oryzias melastigma) in the case of 30 days discontinuity heavy rain (rainfall ≥ 7.6 mm/h and persist 1 h each day). Results showed that wet precipitation of PM2.5 significantly inhibited the body weight gain of fish. In accordance, the size and number of lipid droplets in liver of the exposed groups were lower than those in normal control (NC) group. The expressions of genes involving in lipid degradation including lipoprotein lipase gene (LPL) and carnitine palmitoyltransferase gene (CPT) were up-regulated after exposure. The composition, diversity and function of gut microbiome were affected by wet precipitation of PM2.5. PM2.5 from industrial areas that have higher concentrations of metal profiles show more obvious impacts than PM2.5 from agricultural leisure areas that possessed lower concentrations. All together, the results indicated that wet precipitation of PM2.5 can decrease the diversity of gut microbiome, affect the lipid metabolism, and finally suppress the growth of marine medaka. It confirmed the potential ecological risks of long-term rainfall in air pollution areas to the aquatic organisms.

Catalpol Attenuates Hepatic Steatosis by Regulating Lipid Metabolism via AMP-Activated Protein Kinase Activation.

The increased prevalence of nonalcoholic fatty liver disease (NAFLD), which develops from hepatic steatosis, represents a public health challenge. Catalpol, a natural component extracted from the roots of Radix Rehmanniae, has several pharmacological activities. The present study is aimed at examining whether catalpol prevents hepatic steatosis in cell and animal experiments and elucidating the possible mechanisms. HepG2 cells were treated with 300 μM palmitate (PA) and/or catalpol for 24 h in vitro, and male C57BL/6J mice fed a high-fat diet (HFD) were administered catalpol for 18 weeks in vivo. The results revealed that catalpol significantly decreased lipid accumulation in PA-treated HepG2 cells. Moreover, catalpol drastically reduced body weight and lipid accumulation in the liver, whereas it ameliorated hepatocyte steatosis in HFD-fed mice. Notably, catalpol remarkably promoted the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase. Subsequently, catalpol repressed the expressions of lipogenesis-associated genes such as sterol regulatory element-binding protein 1c and fatty acid synthase but promoted the expressions of genes associated with fatty acid β-oxidation such as peroxisome proliferator-activated receptor α together with its target genes carnitine palmitoyltransferase 1 and acyl-CoA oxidase 1 (ACOX1). However, the preincubation of the HepG2 cells with compound C (10 μM), an AMPK inhibitor, prevented catalpol-mediated beneficial effects. These findings suggest that catalpol ameliorates hepatic steatosis by suppressing lipogenesis and enhancing fatty acid β-oxidation in an AMPK-dependent manner. Therefore, catalpol has potential as a novel agent in the treatment of NAFLD.

High-altitude chronic hypoxia ameliorates obesity-induced non-alcoholic fatty liver disease in mice by regulating mitochondrial and AMPK signaling

AimsHigh-fat intake induces obesity and non-alcoholic fatty liver disease (NAFLD). However, high-altitude chronic hypoxia might alleviate NAFLD progression through improved mitochondrial function and AMP-activated protein kinase (AMPK) signaling. We hypothesized that high-altitude chronic hypoxia would have protective effects against NAFLD development. Main methodsC57BL/6J mice were randomly divided into control (normal diet and altitude 50 m), CHH (normal diet and altitude 4300 m), HFD (high-fat diet and altitude 50 m), and HFD-CHH (high-fat diet and altitude 4300 m) groups. After being maintained for 8 weeks under the appropriate conditions, mice were evaluated. Key findingsThe degree of liver lipid accumulation and expression of the lipid synthesis-related genes acetyl-CoA carboxylase1 (ACC1), fatty acid synthesis (FAS), and sterol regulatory element binding protein-1c (SREBP-1c) were reduced in the HFD-CHH group; however, expression of the lipolysis-related gene carnitine palmitoyl transferase 1 (CPT1) was increased. Furthermore, in addition to increased expression of mitochondrial biogenesis-related genes, mitochondrial respiratory function and mitochondrial DNA content were elevated in the HFD-CHH group compared to those in the HFD group. The HFD-CHH group also exhibited significantly increased antioxidation activity and decreased reactive oxygen species production (P < 0.05). Finally, AMPK signaling in the liver was activated and the expression of phosphorylated-AMPK (P-AMPK) was significantly increased in the HFD-CHH group. SignificanceCollectively, our findings suggest that high altitude-induced hypoxia might improve impaired mitochondrial function and activate AMPK signaling in obesity-induced NAFLD. High-altitude chronic hypoxia could be a new treatment strategy for obesity-induced NAFLD.

Eggshell membrane powder lowers plasma triglyceride and liver total cholesterol by modulating gut microbiota and accelerating lipid metabolism in high-fat diet-fed mice.

Obesity is a major global lifestyle disorder associated with gut microbiota. The health benefits of eggshell membrane (ESM) have been shown in previous reports, particularly as regards gut microbiota composition. Here, we investigated whether ESM improves lipid metabolism and alters gut microbiota in high‐fat diet‐fed mice. A total of 20 C57BL/6J mice aged 6 weeks were given either a control diet (CON), high‐fat diet (HFD), or high‐fat diet + 8% ESM powder (HESM) for 20 weeks. ESM supplementation in HFD‐fed mice reduced plasma triglycerides (TG) and liver total cholesterol (TC) and upregulated the expression of lipid metabolism genes carnitine palmitoyltransferase 1A and suppressor of cytokine signaling 2. Microbiota analysis showed increased relative abundance of the anti‐obesity bacterium, Lactobacillus reuteri, at 4, 12, and 16 weeks and reduced the abundance of inflammation‐related Blautia hydrogenotrophica, Roseburia faecis, and Ruminococcus callidus at 12 and 20 weeks. ESM‐supplemented mice had increased cecal isobutyrate, negatively correlated with B. hydrogenotrophica and Parabacteroides goldsteinii abundance. The results indicate that ESM supplementation in HFD‐fed mice reduced plasma TG and liver TC, possibly through alteration of lipid metabolism gene expression and gut microbiota composition, suggesting that ESM may be effective in obesity management.

Modulation effects of black-vinegar-based supplement against a high-fat dietary habit: antiobesity/hypolipidemic, antioxidative, and energy-metabolism effects.

An imbalanced fat or excess energy intake always results in obesity and increased serum/liver lipids, thus leading to metabolic syndromes. Given the bioactive components in black vinegar (BV), such as branched amino acids, phenolic profile, and mineral contents, we investigated the antiobesity effects of BV-based supplements in rats fed a high-fat diet (HFD). HFD (30% fat, w/w) feeding increased (P < 0.05) body weight, weight gains, weights of livers and mesenteric, epididymal, and perirenal adipose tissues, and serum/liver triglyceride levels relative to those of rats fed a normal diet (4% fat, w/w; CON). These increased values were ameliorated (P < 0.05) by supplementing with BV-based supplements but were still higher (P < 0.05) than those of CON rats. The increased areas of perirenal adipocytes in rats fed with an HFD were also decreased (P < 0.05) by supplementing with BV-based supplements, which might result from an upregulation (P < 0.05) of 5'-adenosine monophosphate-activated protein kinase (AMPK), carnitine palmitoyltransferase-1 (CPT1), and uncoupling protein-2 (UCP2) in the perirenal adipose tissues. A similar effect was observed for AMPK, peroxisome proliferator-activated receptor alpha, retinoid X receptor alpha, CPT1, and UCP2 gene and protein levels in livers (P < 0.05). Generally, BV-based supplements increased the fecal triglyceride, cholesterol, and bile acid levels of rats fed with an HFD, which partially contribute to the lipid-lowering effects. Furthermore, BV-based supplements increased (P < 0.05) hepatic Trolox equivalent antioxidant capacity and lowered (P < 0.05) serum/liver thiobarbituric acid reactive substances values in HFD-fed rats. In a chronic high-fat dietary habit, the food-grade BV-based supplement is a good daily choice to ameliorate obesity and its associated comorbidities. © 2020 Society of Chemical Industry.

Chronic Microcystin-LR Exposure Induces Abnormal Lipid Metabolism via Endoplasmic Reticulum Stress in Male Zebrafish

In order to explore effects of low levels of continuous microcystin-LR (MC-LR) (a cyanotoxin) exposure on hepatic lipid metabolism on the basis of the endoplasmic reticulum stress (ERS) pathway, we exposed adult male zebrafish to MC-LR (0, 1, 5, and 25 μg/L) for 60 days, and hepatic histopathology as well as lipid metabolic parameters were determined with mRNA levels of ERS signal molecules and downstream factors, along with genes associated with lipid metabolism in zebrafish liver. The results revealed that prolonged exposure to MC-LR remarkably altered the levels of hepatic total cholesterol and triglyceride and led to hepatic steatosis, which was also confirmed by hepatic cytoplasmic vacuolization in Hematoxylin/eosin (H&E) stain and lipid droplet accumulation in Oil Red O stain. The severity of hepatic damage and lipidation was increased in a dose-related manner. MC-LR exposure significantly upregulated transcriptional levels of ERS markers including hspa5, mapk8, and chop, indicating the occurrence of ERS in the liver of zebrafish. Concurrently, MC-LR significantly improved mRNA expression of unfolded protein response (UPR) pathway-related genes including atf6, eif2ak3, ern1, and xbp1s, suggesting that all of the three UPR branches were activated by MC-LR. MC-LR also induced significant upregulation of downstream lipid metabolism-related factors and genes including srebf1, srebf2, fatty acid synthase (fasn), acetyl-CoA carboxylase (acaca), stearoyl-CoA desaturase (scd), HMG CoA reductase (hmgcra), and HMG CoA synthase (hmgcs1), and downregulation of genes associated with lipolysis such as triglyceride hydrolase gene (atgl), hormone-sensitive enzyme gene (hsla), and carnitine palmitoyltransferase gene (cpt1aa). Our present results indicated that the cause of hepatic lipid accumulation by MC-LR was mainly by upregulating lipogenic and cholesterol genes but downregulating the expression of lipolytic genes through the induction of srebf1 and srebf2, which were involved in the activation of ERS signal pathways.

Molecular characterization and tissue distribution of carnitine palmitoyltransferases in Chinese mitten crab Eriocheir sinensis and the effect of dietary fish oil replacement on their expression in the hepatopancreas.

The carnitine palmitoyltransferase (CPT) family includes CPT 1 and CPT 2 that transport long-chain fatty acids into the mitochondrial compartment for β-oxidation. In this study, three isoforms (CPT 1α, CPT 1β and CPT 2) of the CPT family were cloned from Chinese mitten crab (Eriocheir sinensis) and their complete coding sequences (CDS) were obtained. Sequence analysis revealed deduced amino acid sequences of 915, 775 and 683 amino acids, respectively. Gene expression analysis revealed a broad tissue distribution for all three isoforms, with high CPT 1α and CPT 2 mRNA levels in the hepatopancreas of males and females. In males, CPT 1β was highly expressed in gill, heart, brain ganglia and muscle, while in females, CPT 1β-mRNA levels were relatively high in muscle, hepatopancreas and ovary tissue. The effects of dietary fish oil replacement on the expression of the three CPT isoforms in the hepatopancreas during gonadal development were investigated using five experimental diets formulated with replacement of 0, 25, 50, 75 and 100% fish oil by 1:1 rapeseed oil: soybean oil. The results showed that Diets 2# and 5# yielded higher CPT 1α and CPT 2 mRNA expression in males (P < 0.05), while in females, expression of all three CPT isoforms increased then declined in the hepatopancreas with increasing dietary fish oil replacement. The observed changes in CPT gene expression varied in different isoforms and gender, suggesting the three CPT genes might play different roles in fatty acid β-oxidation in E. sinensis.

Carnitine palmitoyltransferase gene upregulation by linoleic acid induces CD4+ T cell apoptosis promoting HCC development

Hepatocellular carcinoma (HCC) is a common cause of cancer-related death worldwide. As obesity and diabetes become more prevalent, the contribution of non-alcoholic fatty liver disease (NAFLD) to HCC is rising. Recently, we reported intrahepatic CD4+ T cells are critical for anti-tumor surveillance in NAFLD. Lipid accumulation in the liver is the hallmark of NAFLD, which may perturb T cell function. We sought to investigate how the lipid-rich liver environment influences CD4+ T cells by focusing on carnitine palmitoyltransferase (CPT) family members, which control the mitochondrial β-oxidation of fatty acids and act as key molecules in lipid catabolism. Linoleic acid (C18:2) co-localized within the mitochondria along with a corresponding increase in CPT gene upregulation. This CPT upregulation can be recapitulated by feeding mice with a high-C18:2 diet or the NAFLD promoting methionine-choline-deficient (MCD) diet. Using an agonist and antagonist, the induction of CPT genes was found to be mediated by peroxisome proliferator-activated receptor alpha (PPAR-α). CPT gene upregulation increased mitochondrial reactive oxygen species (ROS) and led to cell apoptosis. In vivo, using liver-specific inducible MYC transgenic mice fed MCD diet, blocking CPT with the pharmacological inhibitor perhexiline decreased apoptosis of intrahepatic CD4+ T cells and inhibited HCC tumor formation. These results provide useful information for potentially targeting the CPT family to rescue intrahepatic CD4+ T cells and to aid immunotherapy for NAFLD-promoted HCC.

P38\u03b1 MAPK antagonizing JNK to control the hepatic fat accumulation in pediatric patients onset intestinal failure

The p38α mitogen-activated protein kinase (MAPK) has been related to gluconeogenesis and lipid metabolism. However, the roles and related mechanisms of p38α MAPK in intestinal failure (IF)-associated liver steatosis remained poor understood. Here, our experimental evidence suggested that p38α MAPK significantly suppressed the fat accumulation in livers of IF patients mainly through two mechanisms. On the one hand, p38α MAPK increased hepatic bile acid (BA) synthesis by upregulating the expression of the rate-limiting enzyme cholesterol 7-α-hydroxylase (CYP7A1) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which in turn activated the transcription of the CYP7A1. On the other hand, p38α MAPK promoted fatty acid (FA) β-oxidation via upregulating peroxisome proliferator-activated receptor alpha (PPARα) and its transcriptional target genes carnitine palmitoyltransferase 1A (CPT1A) and peroxisomal acyl-coenzyme aoxidase 1 (ACOX1). Dual luciferase assays indicated that p38α MAPK increased the transcription of PPARα, PGC-1α and CYP7A1 by upregulating their promoters’ activities. In addition, in vitro and in vivo assays indicated p38α MAPK negatively regulates the hepatic steatosis by controlling JNK activation. In conculsion, our findings demonstrate that hepatic p38α MAPK functions as a negative regulator of liver steatosis in maintaining BA synthesis and FAO by antagonizing the c-Jun N-terminal kinase (JNK).

PPARα regulates tumor cell proliferation and senescence via a novel target gene carnitine palmitoyltransferase 1C.

Carnitine palmitoyltransferase 1C (CPT1C), an enzyme located in the outer mitochondria membrane, has a crucial role in fatty acid transport and oxidation. It is also involved in cell proliferation and is a potential driver for cancer cell senescence. However, its upstream regulatory mechanism is unknown. Peroxisome proliferator activated receptor α (PPARα) is a ligand-activated transcription factor that regulates lipid metabolism and tumor progression. The current study aimed to elucidate whether and how PPARα regulates CPT1C and then affects cancer cell proliferation and senescence. Here, for the first time we report that PPARα directly activated CPT1C transcription and CPT1C was a novel target gene of PPARα, as revealed by dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Moreover, regulation of CPT1C by PPARα was p53-independent. We further confirmed that depletion of PPARα resulted in low CPT1C expression and then inhibited proliferation and induced senescence of MDA-MB-231 and PANC-1 tumor cell lines in a CPT1C-dependent manner, while forced PPARα overexpression promoted cell proliferation and reversed cellular senescence. Taken together, these results indicate that CPT1C is a novel PPARα target gene that regulates cancer cell proliferation and senescence. The PPARα-CPT1C axis may be a new target for the intervention of cancer cellular proliferation and senescence.

Molecular characterization and nutritional regulation of carnitine palmitoyltransferase (CPT) family in grass carp (Ctenopharyngodon idellus).

The carnitine palmitoyltransferase (CPT) gene family plays an essential role in fatty acid β-oxidation in the mitochondrion. We identified six isoforms of the CPT family in grass carp and obtained their complete coding sequences (CDS). The isoforms included CPT 1α1a, CPT 1α1b, CPT 1α2a, CPT 1α2b, CPT 1β, and CPT 2, which may have resulted from fish-specific genome duplication. Sequence analysis showed that the predicted protein structure was different among the CPT gene family members in grass carp. The N-terminal domain of grass carp CPT 1α1a, CPT 1α1b, CPT 1α2a, and CPT 1α2b contained two transmembrane region domains and two acyltransferase choActase domains that exist in human and mouse proteins also; however, only one acyltransferase choActase domain was found in grass carp CPT 1β. The grass carp CPT 2 had two acyltransferase choActase domains. The grass carp CPT 1α1b, CPT 1α2a, CPT 1α2b, and CPT 1β contained 18 coding exons, while CPT 1α1a and CPT 2 consisted of 17 coding exons and 5 coding exons, respectively. The mRNA of the six CPT isoforms was expressed in a wide range of tissues, but the mRNA abundance of each CPT showed tissue-dependent expression patterns. The expression of CPT 1α1a, CPT 1α2a, and CPT 1β at 48h post-feeding was significantly increased in the liver (P<0.01, P<0.05, and P<0.01, respectively). The diverse responses of multiple isoforms in the liver during nutritional limitation suggest that they may play different roles in fatty acid β-oxidation.

Abstract 301: Initial Increase of Klf5 and Ppara Expression After Myocardial Ischemia/Reperfusion in Mice Appears to be Critical for Survival

Krüppel-like factors (KLF) have important roles in metabolism. We previously found that KLF5 is a positive transcriptional regulator of peroxisome proliferator-activated receptor α ( Ppara) , a central regulator of cardiac fatty acid oxidation (FAO). Mice with cardiomyocyte-specific Klf5 ablation ( α MHC-Klf5 -/- ) had reduced cardiac Ppara expression and FAO. At age 6-12 months these mice develop distinct cardiac dysfunction. The role of PPARα activation in I/R injury is unclear as both beneficial and detrimental effects have been reported. We aimed to study if Ppara expression changes during I/R are driven by KLF5 and explore its protective or detrimental role. Wild type mice were subjected to in vivo I/R or sham surgery. I/R resulted in an initial increase in Ppara , and its target gene pyruvate dehydrogenase kinase 4 ( Pdk4) mRNA after 2h reperfusion, followed by decreased expression after 24h reperfusion. The Ppara expression is associated with parallel changes in cardiac Klf5 mRNA expression. Concurrent, there was a decrease of cardiac FAO-related genes carnitine palmitoyl-transferase 1β ( Cpt1b), very long chain acyl-CoA dehydrogenase (Vlcad), and acyl-CoA oxidase ( Aox) in mice with I/R. To define the cell type causing the temporal changes in Klf5 and Ppara after I/R we isolated primary cardiomyocytes and fibroblasts. Our data suggest a similar effect in primary isolated cardiomyocytes only. Klf5 mRNA expression is increased after 2 hour hypoxia and normalized after 4 hour re-oxygenation in cardiomyocytes, whereas there are no changes after hypoxia/normoxia in fibroblasts. To assess the importance of cardiomyocyte KLF5 in I/R we used α MHC-Klf5 -/- mice. Interestingly, despite reduced FAO, 7 month old αMHC-Klf5 -/- mice subjected to I/R had a marked increase in mortality; 4 of 7 αMHC-Klf5 -/- mice died within the first 24h of reperfusion while no mortality was observed in age-matched wild type mice that underwent I/R. In conclusion, I/R is associated with an increase in Klf5 and Ppara in the first hours of reperfusion followed by a decrease in Klf5 and Ppara , likely accounted for by cardiomyocytes. Increased mortality for α MHC-Klf5 -/- mice with I/R injury suggests that the initial increase may be an adaptive response that is critical for survival.

Eicosapentaenoic Acid, Arachidonic Acid and Eicosanoid Metabolism in Juvenile Barramundi Lates calcarifer.

A two part experiment was conducted to assess the response of barramundi (Lates calcarifer; initial weight=10.3±0.03g; mean±S.D.) fed one of five diets with varying eicosapentaenoic acid (diets 1, 5, 10, 15 and 20g/kg) or one of four diets with varying arachidonic acid (1, 6, 12, 18g/kg) against a fish oil control diet. After 6weeks of feeding, the addition of EPA or ARA did not impact on growth performance or feed utilisation. Analysis of the whole body fatty acids showed that these reflected those of the diets. The ARA retention demonstrated an inversely related curvilinear response to either EPA or ARA. The calculated marginal utilisation efficiencies of EPA and ARA were high (62.1 and 91.9% respectively) and a dietary ARA requirement was defined (0.012g/kg(0.796)/day). The partial cDNA sequences of genes regulating eicosanoid biosynthesis were identified in barramundi tissues, namely cyclooxygenase 1 (Lc COX1a, Lc COX1b), cyclooxygenase 2 (Lc COX2) and lipoxygenase (Lc ALOX-5). Both Lc COX2 and Lc ALOX-5 expression in the liver tissue were elevated in response to increasing dietary ARA, meanwhile expression levels of LcCOX2 and the mitochondrial fatty acid oxidation gene carnitine palmitoyltransferase 1 (LcCPT1a) were elevated in the kidney. A low level of EPA increased the expression of Lc COX1b in the liver. Consideration should be given to the EPA to ARA balance for juvenile barramundi in light of nutritionally inducible nature of the cyclooxygenase and lipoxygenase enzymes.

Cpt1a gene expression in peripheral blood mononuclear cells as an early biomarker of diet-related metabolic alterations

BackgroundResearch on biomarkers that provide early information about the development of future metabolic alterations is an emerging discipline. Gene expression analysis in peripheral blood mononuclear cells (PBMC) is a promising tool to identify subjects at risk of developing diet-related diseases.ObjectiveWe analysed PBMC expression of key energy homeostasis-related genes in a time-course analysis in order to find out early markers of metabolic alterations due to sustained intake of high-fat (HF) and high-protein (HP) diets.DesignWe administered HF and HP diets (4 months) to adult Wistar rats in isocaloric conditions to a control diet, mainly to avoid overweight associated with the intake of hyperlipidic diets and, thus, to be able to characterise markers of metabolically obese normal-weight (MONW) syndrome. PBMC samples were collected at different time points of dietary treatment and expression of relevant energy homeostatic genes analysed by real-time reverse transcription-polymerase chain reaction. Serum parameters related with metabolic syndrome, as well as fat deposition in liver, were also analysed.ResultsThe most outstanding results were those obtained for the expression of the lipolytic gene carnitine palmitoyltransferase 1a (Cpt1a). Cpt1a expression in PBMC increased after only 1 month of exposure to both unbalanced diets, and this increased expression was maintained thereafter. Interestingly, in the case of the HF diet, Cpt1a expression was altered even in the absence of increased body weight but correlated with alterations such as higher insulin resistance, alteration of serum lipid profile and, particularly, increased fat deposition in liver, a feature characteristic of metabolic syndrome, which was even observed in animals fed with HP diet.ConclusionsWe propose Cpt1a gene expression analysis in PBMC as an early biomarker of metabolic alterations associated with MONW phenotype due to the intake of isocaloric HF diets, as well as a marker of increased risk of metabolic diseases associated with the intake of HF or HP diets.