apoB Expression Research Articles

PSXIII-41 Fibroblast growth factor 21 promotes lipid oxidation and transportation while inhibits lipogenesis via AMP-activated protein kinase signaling pathway in bovine hepatocytes

Abstract Background: Fibroblast growth factor 21 (FGF21) plays essential roles in the regulation of whole body energy metabolism. However, it is not entirely clear for dairy cows whether FGF21 activates AMPK signaling pathway and what will be affected for lipid metabolism in bovine hepatocytes. Methods: Bovine hepatocytes were isolated from caudate lobes by using three-step of perfusion and collagenase digestion method. The accumulation of TG and the secretion of VLDL were examined in hepatocytes and supernatant, respectively. The expressions of the metabolic key factors were detected by Real-time PCR and Western Blot.Results: The 4th hour is the optimal time that FGF21 activates AMPK. FGF21 has significant dose-dependent inhibition of TG in bovine hepatocytes; and high concentration (1800 pg/mL) significantly promoted VLDL secretion at 4 h. The protein expression of APOB 100, APOE and MTTP, which are components of VLDL, were stimulated by FGF21, and all the mRNA expression reached the peak point (P < 0.01) at medium concentration (900 pg/mL). Interestingly, the proteins associated with lipid transportation were promoted too, such as LDLR, CD36, L-FABP. To some extent, meanwhile, it could be observed that some genes regulating lipid oxidation were strengthened following FGF21 treatment. In detail, ACOX1 and SIRT1 were very sensitive to the concentration of FGF21, showing remarkable difference at low concentration (P < 0.01); PGC-α, PPAR-α and CPT-1 showed significant changes at 900 pg/mL (P < 0.01); CPT-2 required a higher concentration to achieve significant enhancement. However, the results had a negative impact on lipogenesis. SREBP1c, ACC, FASN and ACLY were inhibited after treatment with low or medium doses of FGF21 (P < 0.01). Conclusion: FGF21 can promote lipid oxidation and transport, while inhibit lipid synthesis via activating AMPK signalling pathway in primary hepatocytes of dairy cows, thereby participation in the adaptive regulation of energy metabolism.

In Utero Dexamethasone Exposure Exacerbates Hepatic Steatosis in Rats That Consume Fructose During Adulthood.

Distinct environmental insults might interact with fructose consumption and contribute to the development of metabolic disorders. To address whether in utero glucocorticoid exposure and fructose intake modulate metabolic responses, adult female Wistar rats were exposed to dexamethasone (DEX) during pregnancy, and the offspring were administered fructose at a later time. Briefly, dams received DEX during the third period of pregnancy, while control dams remained untreated. Offspring born to control and DEX-treated mothers were defined as CTL-off and DEX-off, respectively, while untreated animals were designated CTL-off-CTL and DEX-off-CTL. CLT-off and DEX-off treated with 10% fructose in the drinking water for 8 weeks are referred to as CTL-off-FRU and DEX-off-FRU. We found that fructose promoted glucose intolerance and whole-body gluconeogenesis in both CTL-off-FRU and DEX-off-FRU animals. On the other hand, hepatic lipid accumulation was significantly stimulated in DEX-off-FRU rats when compared to the CTL-off-FRU group. The DEX-off-FRU group also displayed impaired very-low-density lipoprotein (VLDL) production and reduced hepatic expression of apoB, mttp, and sec22b. DEX-off-FRU has lower hepatic levels of autophagy markers. Taken together, our results support the unprecedented notion that in utero glucocorticoid exposure exacerbates hepatic steatosis caused by fructose consumption later in life.

Clinical features of aflatoxin B1-exposed patients with liver cancer and the molecular mechanism of aflatoxin B1 on liver cancer cells

Aflatoxin B1 (AFB1) induces hepatocellular carcinoma (HCC) through consumption of contaminated food in Southern China. Aldo-keto reductase-7A (AKR7A) functionally plays a potent role in the biodetoxification in the liver. In addition, hepatocellular lipid disorder has found to be closely linked to the development of HCC. This study was, therefore, designed to investigate the potent bioeffect of AKR7A on the lipid metabolism in AFB1-exposed hepatocellular carcinoma cells through assaying human cancerous samples and cell culture. In the baseline data, the HCC patients showed increased contents of AFB1 in sera and cancerous samples. In the clinical parameters, the HCC patients demonstrated changed lipid settings in sera. As revealed by immunostaining and immunoblotting, AFB1-elevated HCC sections showed marked down-regulation of AKR7A expression, accompanied with reduced ApoB expression and increased CD36, S6K1 expressions in the HCC. Studies in the human hepatocarcinoma line HepG2 also showed AFB1-exposure to increase ApoA1, LDL, TC, and TG contents; induce cell proliferation; and reduce hepatocellular AKR7A expression. Furthermore, AKR7A bioactivity was inactivated after treatment with perfluorooctane sulfonate (PFOS), an ApoB activator, in AFB1-dosed HepG2 cells. Collectively, our current findings suggest that hepatocellular AKR7A has a protective role against AFB1-induced cytotoxicity through the regulation of CD36, S6K1 and ApoB expression through the reduction of lipid utilization in malignant liver cells.

Interruption of Autophagosome Formation in Cardiovascular Disease, an Evidence for Protective Response of Autophagy

ABSTRACTBackground: A heart attack occurs when coronary artery blockage interrupts the blood supply to the heart such as is seen in cardiovascular disease (CVD). Importantly, autophagy is commonly regarded as a host defense mechanism against microbial invaders. Methods: A total of 50 blood samples were obtained from cardiovascular (CV) patients in addition to 30 samples that were obtained from healthy individuals and served as controls. Macrophages were isolated in vitro and propagated from the blood samples. Autophagosome formation, cytokine secretion, and apolipoprotein B (ApoB) gene expression were monitored in patient samples and their derived macrophages. Results: The results showed that autophagy-related (Atg) LC3 and Atg5 genes were significantly down-regulated in all samples obtained from CV patients. Furthermore, the relative gene expression of ApoB, which plays the major role in lipoprotein metabolism, was significantly increased in CV patients. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels were increased in these blood samples. Interestingly, targeting of ApoB by small interference RNA (siRNA) reduced the production levels of low-density lipoprotein (LDL), IL-6 and TNF-α in patient-derived macrophages. Further, treatment of patient-derived macrophages with rapamycin, an autophagy inducer agent, successfully regulated the production of LDL, IL-6, TNF-α, and ApoB expression via activation of autophagosome formation. Conclusion: The current data reveal the potential disturbance of autophagy in CV patients that accompanied ApoB over-expression. Furthermore, our findings provide evidence for the protective role of autophagy in accumulation of pro-inflammatory cytokines and intracellular LDL degradation in CV patient-derived macrophages.

Abstract 3474: Loss of function mutations in APOB and their clinical significance in hepatocellular carcinoma

Abstract APOB is major lipid binding proteins of low-density lipoprotein (LDL) particles and responsible for carrying fat molecules to cells within all tissues. High levels of APOB are known to be related to heart disease and vascular disease. Interestingly, recent The Cancer Genome Atlas (TCGA) study revealed that APOB is one of frequently mutated genes in multiple cancers including melanoma, liver cancer, stomach, esophageal, head and neck, uterine, and lung cancers (from >20 to 10%). Majority mutations are considered to cause loss of function as they are truncation mutations, suggesting that APOB might have tumor suppressive activity. However, because of its physiological roles in circulating fat molecules in our body, APOB has never been studied in cancer-related setting and thus its clinical relevance or biological roles of APOB in cancer development is currently unknown. Here we show that loss of APOB in hepatocellular carcinoma (HCC) is significantly associated with poor survival of HCC patients by applying comparative genomics approach that integrate genomic data from mouse and human HCC tumors. We further show that loss of APOB leads to shifting balance of lipid metabolisms favoring for tumor growth. For development of genomic signature reflecting hepatic APOB activity and test and validation of its association with prognosis, we used unsupervised approach combined with supervised prediction models by integrating gene expression data from mouse models and human HCC. When HCC patients were stratified into APOB-active and APOB-inactive subgroups, overall survival (OS) rate and recurrence free survival (RFS) rate of patients in APOB-inactive group is significantly lower than those in APOB-active (P < 0.001), indicating that APOB activity in HCC is significantly associated with prognosis of patients. This association was validated in 4 independent cohorts of HCC patients (n=88, 240, 242, and 371 in total of 941 patients). Gene network analysis revealed that many of the regulators involved in cell growth were activated in Apob-silenced livers. Many of the upregulated genes in Apob-silenced livers are direct downstream targets of Erbb2, Vegfa, and regulator of metastasis and cancer initiation cells Cd44. Consistent with activation of many tumor-promoting regulators, Tp53, a major tumor suppressor, was inactivated in Apob-silenced livers, suggesting that loss of Apob in the liver may provoke initiation of tumor development. More interestingly, proliferation of three liver cancer cells was significantly increased when expression of APOB was silenced by siRNAs, further suggesting functional roles of APOB in tumor development. For the first time, we showed that APOB inactivation has a clinical impact in HCC patients with significant alteration in regulators associated with tumorigenesis. Citation Format: Gena Lee, Yun Seong Jeong, Min Jun Kwak, Kim DoWon, Ju-Seog Lee, Yim Sun Young. Loss of function mutations in APOB and their clinical significance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3474.

Effects of emodin on lipid accumulation and inflammation in hepatocytes

The aim of this study was to explore the effect of emodin on lipid accumulation and inflammation in hepatocytes. The cell morphology was observed by microscopy. LDH release was detected by the kit. Levels of intracellular lipid droplets were observed by oil red O staining. The contents of TC and TG in cells were detected by the kit. Western blot was used to determine protein expressions of FASN,SREBF2,APOB,IL-6 and p-NF-κB in hepatocytes. The results showed that the levels of L02 cell LDH were significantly increased after being treated with emodin,and the cells showed shrinkage,volume reduction,decrease in quantity with the increase of dose. Red lipid droplets were observed in L02 hepatocytes. Intracellular TC and TG contents of L02 cell increased in a concentrationdependent manner,with significant differences between medium and high-dose groups( P < 0. 05). Protein expressions of FASN,SREBF2,IL-6 and p-NF-κB were significantly higher than those of the control group,and the expression level of APOB was significantly lower than that of the control group( P<0. 05). In conclusion,emodin could induce lipid accumulation and inflammatory damage in hepatocytes in a dose-dependent manner,which in turn could damage liver cells. This process was related to the up-regulation of FASN,SREBF2,IL-6,p-NF-κB,as well as the down-regulation of the protein expression of APOB.

Short-chain Fatty Acids, 4-phenylbutyric Acid and R-α-lipoic Acid, Downregulate mTORC1-dependent Intestinal Fat Absorption (P08-115-19)

ObjectivesKnowing that (i) high-fat diets upregulate inflammatory biomarkers in the small intestine and activate the mechanistic target of rapamycin complex 1 (mTORC1, a conserved protein kinase) and (ii) mTORC1 drives hepatic VLDL secretion, we investigated the role of intestinal mTORC1 in fat absorption and the lipid-regulating properties of 4-phenylbutyric acid (PBA) and R-α-lipoic acid (LA) in relation to mTORC1. MethodsLentivirus shRNA-based gene editing was utilized to create three stable Caco-2 cell lines that exhibit low, normal or high mTORC1 activity. Fat absorption was investigated in differentiated stable Caco-2 cells grown with oleate/albumin in Transwell permeable inserts. ResultsGenetic ablation of mTORC1 revealed that mTORC1 is required for the production and secretion of apoB-containing chylomicrons in Caco-2 cells. Rapamycin, an inhibitor of mTORC1, confirmed this finding as it lowered the secretion of apoB48 (−75%) and apoB100 (−47%) (P < 0.05). mTORC1 deletion was accompanied by the downregulation of APOB (−87%), MTTP (−94%), FASN (−76%), DGAT1 (−77%) and DGAT2 (−81%) genes (all P < 0.05); lower cellular abundance of apoB100 (−89%), MTTP (−63%) and FASN (−80%) (all P < 0.05); and lower media abundance of apoB48 (−66%), apoB100 (−51%) and triacylglycerols (−69%) (all P < 0.05). In Caco-2 cells harboring high mTORC1 activity, PBA or LA decreased the amounts of secreted apoB48 (−58% or −22%, respectively), apoB100 (−54% or −43%), and triacylglycerols (−46% or −38%), and the gene expression of APOB (−48% or −33%), MTTP (−54% or −31%), SREBF1 (−39% or −37%), FASN (−84% or −38%), and DGAT2 (−76% or −47%) (all P < 0.05). ConclusionsA functional intestinal mTORC1 is indispensable for chylomicron production. Intake of short-chain fatty acid, PBA or LA, may be useful to prevent chylomicron overproduction-associated hypertriglyceridemia and postprandial inflammation. Funding SourcesUSDA Hatch Act, USDA-AFRI.

Identification of key genes and molecular mechanisms associated with low egg production of broiler breeder hens in ad libitum

BackgroundOverfeeding reduces laying performance in broiler breeder hens, which is associated with obesity, hepatic steatosis and systemic inflammation. To unravel the underlying mechanisms governing the effect of feeding regimes on energy metabolism and egg production, a transcriptomics approach was carried out for screening differentially expressed genes (DEGs) in ovary, liver and adipose tissues of broiler chickens under ad libitum and restricted feeding.ResultsIt showed that 289, 388 and 204 DEGs were identified in the adipose, liver and ovary, respectively. These DEGs were significantly enriched in phagosome pathway, lipid transport, activity and nutrient reservoir activity in ovary; steroid hormone biosynthesis and metabolism of xenobiotics by cytochrome P450 pathways in adipose tissue; and the metabolic pathways, peroxisome proliferator-activated receptor (PPAR) and Jak-STAT signaling pathway in liver. Estrogen receptor 1, identified as one of important hubs by constructing PPI network, was up-regulated in ad libitum group, which would make more apolipoproteins be transferred to ovary.ConclusionsHigh expression of VTGs, APOB, CYBB and CTSS in ovary would induce excess lipid deposit, oxidative stress and potential damage to ovulation. Our results contribute to understanding effects of feeding regimes on metabolic regulation during egg production of broiler breeder hens and also provide new evidence of metabolic regulation from integrated multi-tissue processes.

Exploring the effects of FATP2 gene deletion on fatty acid‐responsive gene expression in liver

Fatty Acid Transport Protein 2 (FATP2) is highly expressed in the small intestine, liver, kidney, pancreas, and placenta. FATP2 has two activities: [1] it facilitates the uptake of long chain fatty acids into the cell and [2] it activates very long chain fatty acids destined for incorporation into cellular lipids or β‐oxidation. Previous work from this laboratory implicates FATP2 activity in the promotion of lipotoxicity that may lead to cell death. The present studies employ mice lacking the FATP2 gene (FATP2−/−) to more fully understand the function and effects of FATP2‐dependent fatty acid uptake on liver metabolism and lipotoxic disease. We hypothesize a reduction in uptake and metabolism of fatty acids dependent upon FATP2 may lead to alterations in expression of genes that are responsive to exogenous fatty acids. Gene expression was measured in wild type and FATP2−/− livers using quantitative PCR and validated using western blots to assess the impact of genotype, diet, and fasting period. Preliminary data shows that expression of FABP1, CD36, Cyp4A10, and ACOX are elevated in livers of FATP2−/− mice independent of dietary fat levels. In contrast, expression of NPC1L1 was decreased. Differences between diets alone resulted in an increase in FABP2 and FABP4, FAS, Serpinb6 and ApoB expression, while FABP1, CD36, and ACOX expression was dependent on both genotype and diet. These results further suggest deletion of FATP2 increases PPARa signaling, perhaps through increases in a native fatty acid ligand. Overall, these studies further demonstrate an important role for FATP2 in liver lipid homeostasis during high fat dietary challenge.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of a fatty liver model using oleic acid in primary liver cells isolated from Atlantic salmon and the prevention of lipid accumulation using metformin

The following study aimed to develop a fatty liver model in primary hepatocytes isolated from Atlantic salmon. In order to induce the fatty liver, oleic acid (OA) at 0.2 or 0.4 mM was used. Metformin, known to prevent and cure fatty liver in mammalian cells, was used at 1 or 10 mM for 24 hr before addition of OA to test possible prevention effect of metformin on the OA-induced fatty liver phenotype. Cells grown in 0.2 mM OA did not increase the mean number of lipid droplets, while cells grown in 0.4 mM OA increased the number of lipid droplets within the liver cells (p < 0.0001). Metformin pretreatment prior to OA supplementation reduced the mean number of lipid droplets. Gene expression of ApoB100, CD36 and PPARa increased in cells treated with metformin and most so at 10 mM. On the other hand, gene expression of LXR, SREBP2 and CPT-1 decreased at both concentrations of metformin, while OA treatment did not affect these genes. Gene expression of IL-8 increased by 0.4 mM OA (p = 0.002). Metformin reduced the gene expression of IL-8. Thus, metformin efficiently enhanced the expression of genes related to transport and oxidation of lipids in hepatic cells of salmon, but required higher concentrations of OA and metformin than those required in rodent models to increase and prevent lipid accumulation, respectively.

Sini Decoction Intervention on Atherosclerosis via PPARγ-LXRα-ABCA1 Pathway in Rabbits.

AimSini decoction (SND) is a commonly used herbal formula showing lipid-lowering effects and is applied in traditional Chinese medicine (TCM) for the treatment of cardiovascular disease (CVD) and atherosclerosis (AS). However, the mechanisms behind its anti-atherosclerotic effects are still unknown, and will be investigated in this study.MethodsAS was induced in rabbits by high fat diet (HFD) and treated with solvent (HFD group), atorvastatin (Ator group), or SND (SND group) for 12 weeks. Healthy rabbits (Chow group) were used as control. Serum and liver homogenates were collected, and lipid profiles as well as serum ApoA-I and ApoB were examined. Histopathological changes and lipid deposition in the proximal aorta and liver were detected by Oil red O staining. Western blot was used to detect the expression of ABCA1, ApoA-I, ApoB, PPARγ, and LXRα in liver, peritoneal macrophages, peripheral mononuclear cells (PMC), and adipose tissues.ResultsSND significantly attenuated the levels of total cholesterol (TC), triglyceride (TG), and low density lipoprotein cholesterol(LDL-C) in serum and liver. However, high density lipoprotein cholesterol (HDL-C) dramatically increased. SND treatment also decreased lipid deposition and improved the structure of the liver and aorta. Furthermore, SND enhanced the expression levels of ABCA1, PPARγ, and LXRα in liver, adipose tissues, PMC, and peritoneal macrophages. It also upregulated hepatic and serum ApoA-I expression and serum ApoA-I/ApoB ratio. CONCLUSIONS:SND treatment relieved AS, improved lipid profiles, and increased serum HDL-C level. The potential mechanism behind this might be the improvement of reverse cholesterol transport (RCT) involved with enhanced expression of ABCA1, ApoA-I, PPARγ, and LXRα.

Fc Gamma Receptor IIb Expressed in Hepatocytes Promotes Lipid Accumulation and Gluconeogenesis

Non-alcoholic fatty liver disease (NAFLD) is characterized by ectopic lipid accumulation in the liver, usually combined with hepatic insulin resistance. Fc-gamma receptor-IIb (FcγRIIb) and its ligand are reported to be associated with obesity and type 2 diabetes mellitus (T2DM). As knowledge about FcγRIIb in the literature is mostly generated from studies on skeletal muscle tissue, the expression and function of FcγRIIb in the liver and hepatocytes are largely unknown. In this study, we identified the expression of FcγRIIb in primary cultured mouse hepatocytes: FcγRIIb was upregulated in response to oleic acid (OA) in a dose dependent manner. FcγRIIb knockdown using shRNA suppressed the lipid and triglyceride accumulation, and mRNA expression of ACC1, FASn, CD36, MTTP, and ApoB in OA-treated HepG2 cells. FcγRIIb deficiency mice fed with high fat diet (HFD) had significantly lower liver weight and liver to body weight ratio, as well as less triglyceride accumulation in the livers. In glycometabolism, FcγRIIb hindered insulin-induced phosphorylation of AKT and FOXO1, and in turn upregulated G6Pase and PEPCK mRNA expression, suggesting that FcγRIIb promotes gluconeogenesis by suppressing the AKT/FOXO1/G6Pase/PEPCK pathway in hepatocytes. This study reveals a novel role for FcγRIIb in regulating lipid metabolism and glycometabolism, and provides a new therapeutic target to improve NAFLD.

Liver lipid metabolism disruption in cancer cachexia is aggravated by cla supplementation -induced inflammation

The liver is the main organ regulating metabolism. In spite of that, few studies examine liver metabolism in cachexia, a wasting syndrome associated with increased morbidity and mortality in cancer. Cachexia induces major metabolic disruption, inflammation and fat and lean mass loss. We have previously shown impairment of hepatic lipid metabolism in cancer cachexia that contributes to the aggravation of the symptoms. The present study addresses the effects of Conjugated Linoleic Acid supplementation upon liver lipid metabolism in cachectic rats. Male Wistar rats were randomly assigned to control groups (C) receiving 0.9NaCl (Placebo CP); or to groups supplemented with sunflower oil (CSF), supplemented with CLA (CCLA), or still, to tumour bearing animals (T) receiving NaCl (TP), sunflower oil (TSF), or CLA (TCLA). Supplementation (0.5ml) by gavage was carried out for 14 days. Body weight, dietary intake, glucose, cholesterol and triacylglycerol plasma content, liver glycogen and triacylglycerol content and mRNA expression of liver carnitine palmitoyltransferase I and II (CPT I and II), as well as microsomal triglyceride transfer protein (MTP), liver fatty acid-binding protein (L-FABP), peroxisome proliferator-activated receptor-alpha (PPAR-alpha), and apolipoprotein B (apoB), were assessed. Liver CPT II activity was reduced in all groups, when compared with CP. Hepatic mRNA expression of MTP, apoB and FABP was reduced in TCLA, when compared with all groups. TCLA also presented increased hepatic and plasma triacylglycerol content, when compared with all T groups. Adipose tissue-derived inflammatory factors were assessed. No differences among the groups were observed in regard to Retro Peritoneal Adipose Tissue cytokine (IL-1β, IL-6, and TNF-α) protein content and expression, with the exception of IL-10 in tumour-bearing animals. In the Epididymal Adipose Tissue, the inflammatory cytokines were augmented in TCLA, compared with all other groups. CLA supplementation fails to promote the re-establishment of hepatic lipid metabolism in tumour-bearing animals, and therefore is not recommended in cancer-related cachexia.

Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice.

Obesity and dyslipidemia are major risk factors for the development of cardiovascular diseases (CVD). Quercetin, a natural flavonoid, lowers plasma triglycerides (TG) in human intervention studies, and its intake is associated with lower CVD risk. The aim of this study was to elucidate the mechanism by which quercetin lowers plasma TG levels in diet-induced obesity. C57Bl/6J mice received a high-fat diet (45% of calories derived from fat) with or without quercetin (0.1% w/w) for 12 weeks. Quercetin decreased plasma TG levels from nine weeks onwards (−19%, p < 0.05), without affecting food intake, body composition, or energy expenditure. Mechanistically, quercetin did not reduce intestinal fatty acid (FA) absorption. Rather, quercetin induced a slight reduction in liver Apob expression (−13%, p < 0.05), which suggests decreased very-low density lipoprotein-TG production. Interestingly, quercetin also markedly increased the uptake of [3H]oleate, which was derived from glycerol tri[3H]oleate-labeled lipoprotein-like particles by subcutaneous white adipose tissue (sWAT, +60%, p < 0.05). Furthermore, quercetin also markedly increased mRNA expression of Ucp1 (+229%, p < 0.05) and Elovl3 (+138%, p < 0.05), specifically in sWAT. Accordingly, only quercetin-treated animals showed uncoupling protein-1 protein-positive cells in sWAT, which is fully compatible with increased browning. Taken together, the TG-lowering effect of quercetin may, at least in part, be due to increased TG-derived FA uptake by sWAT as a consequence of browning.

Loss-of-function mutations in Zn-finger DNA-binding domain of HNF4A cause aberrant transcriptional regulation in liver cancer.

Hepatocyte nuclear factors (HNF) are transcription factors that crucially regulate cell-specific gene expression in many tissues, including the liver. Of these factors, HNF4A acts both as a master regulator of liver organogenesis and a tumor suppressor in the liver. In our whole genome sequencing analysis, we found seven somatic mutations (three Zn-finger mutations, three deletion mutants, and one intron mutation) of HNF4A in liver cancers. Interestingly, three out of seven mutations were clustered in its Zn-finger DNA-binding domain; G79 and F83 are positioned in the DNA recognition helix and the sidechain of M125 is sticking into the core of domain. These mutations are likely to affect DNA interaction from a structural point of view. We then generated these mutants and performed in-vitro promoter assays as well as DNA binding assays. These three mutations reduced HNF4 transcriptional activity at promoter sites of HNF4A-target genes. Expectedly, this decrease in transcriptional activity was associated with a change in DNA binding. RNA-Seq analysis observed a strong correlation between HNF4A expression and expression of its target genes, ApoB and HNF1A, in liver cancers. Since knockdown of HNF4A caused a reduction in ApoB and HNF1A expression, possibly loss of HNF4 reduces the expression of these genes and subsequently tumor growth is triggered. Therefore, we propose that HNF4A mutations G79C, F83C, and M125I are functional mutations found in liver cancers and that loss of HNF4A function, through its mutation, leads to a reduction in HNF1A and ApoB gene expression with a concomitant increased risk of liver tumorigenesis.

Effect of the Catechol-O-Methyltransferase Inhibitors Tolcapone and Entacapone on Fatty Acid Metabolism in HepaRG Cells.

Tolcapone and entacapone are catechol-O-methyltransferase inhibitors used in patients with Parkinson's disease. For tolcapone, patients with liver failure have been reported with microvesicular steatosis observed in the liver biopsy of 1 patient. We therefore investigated the impact of tolcapone and entacapone on fatty acid metabolism in HepaRG cells exposed for 24 h and on acutely exposed mouse liver mitochondria. In HepaRG cells, tolcapone induced lipid accumulation starting at 100 µM, whereas entacapone was ineffective up to 200 µM. In HepaRG cells, tolcapone-inhibited palmitate metabolism and activation starting at 100 µM, whereas entacapone did not affect palmitate metabolism. In isolated mouse liver mitochondria, tolcapone inhibited palmitate metabolism starting at 5 µM and entacapone at 50 µM. Inhibition of palmitate activation could be confirmed by the acylcarnitine pattern in the supernatant of HepaRG cell cultures. Tolcapone-reduced mRNA and protein expression of long-chain acyl-CoA synthetase 1 (ACSL1) and protein expression of ACSL5, whereas entacapone did not affect ACSL expression. Tolcapone increased mRNA expression of the fatty acid transporter CD36/FAT, impaired the secretion of ApoB100 by HepaRG cells and reduced the mRNA expression of ApoB100, but did not relevantly affect markers of fatty acid binding, lipid droplet formation and microsomal lipid transfer. In conclusion, tolcapone impaired hepatocellular fatty acid metabolism at lower concentrations than entacapone. Tolcapone increased mRNA expression of fatty acid transporters, inhibited activation of long-chain fatty acids and impaired very low-density lipoprotein secretion, causing hepatocellular triglyceride accumulation. The findings may be relevant in patients with a high tolcapone exposure and preexisting mitochondrial dysfunction.

High fat diet-induced oxidative stress blocks hepatocyte nuclear factor 4α and leads to hepatic steatosis in mice.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease with manifestation of over-accumulation of fat in liver. Increasing evidences indicate that NAFLD may be in part caused by malfunction of very low density lipoprotein (VLDL) secretion. Hepatocyte nuclear factor 4α (HNF4α), a nuclear receptor protein, plays an important role in sustain hepatic lipid homeostasis via transcriptional regulation of genes involved in secretion of VLDL, such as apolipoprotein B (ApoB). However, the exact functional change of HNF4α in NAFLD remains to be elucidated. In the present study, we found that high fat diet (HFD) induced cytoplasmic retention of HNF4α in hepatocytes, which led to down-regulation of hepatic ApoB expression and its protein level in serum, as well as reduced secretion of VLDL. We further revealed that oxidative stress, elevated in fatty liver, was the key factor inducing the cytoplasmic retention of HNF4α in hepatocytes by activating protein kinase C (PKC)-mediated phosphorylation in HNF4α. Thus, our findings reveal a novel mechanism underlying HFD-induced fatty liver that oxidative stress impairs function of HNF4α on ApoB expression and VLDL secretion via PKC activation, eventually promoting fat accumulation in the liver. Therefore, oxidative stress/PKC/HNF4α pathway may be a novel target to treat diet-induced fatty liver.

Effect of Quercus infectoria and Zataria multiflora extracts on the expression of Apo-B100 and PPAR-α in liver and adipose tissues in insulin resistant rats

Background: Insulin resistance can increase the risk of metabolic syndrome. Studies have shown that expression of PPAR alpha improved insulin function in patients with insulin resistance. Also ApoB100 is an essential ligand for the receptors of low-density lipoproteins (LDL). Increased plasma level of apoB100 is a risk factor for cardiovascular disease (CVD) and its increased production leads to insulin resistance. The aim of this study was to evaluate the effects of Q. Infectoria and Z. multiflora extracts on the expression of PPARα and Apo-B100 genes in adipose and hepatic tissues of insulin-resistant ratsMaterials and methods: Forty Wistar rats were divided into 1- healthy control, 2- high fat control, 3- fenofibrate,4- Q. Infectoria and 5- Z. multiflora groups. All groups were fed with high fat diet for 6 weeks expect for the healthy control. Glucose tolerance test was performed to confirm insulin resistance in rats. Then groups 3, 4, and 5 were treated by fenofibrate, Q. Infectoria and Z. multiflora extracts respectively. After sacrificing the rats, their liver and fat tissues were removed. Real-time PCR was used to assess PPARα and ApoB100 gene expressions. Results: All groups had significant weight gain after 8 weeks. Expression of PPAR-α and ApoB100 genes were the same in Q. Infectoria, Z. multiflora, fenofibrate and healthy control groups.Conclusion: In conclusion, Q. Infectoria and Z. multiflora extracts decreased ApoB100 and increased PPARα gene expressions but these changes were not statistically significant.

Methionine-supplemented diet affects the expression of cardiovascular disease-related genes and increases inflammatory cytokines in mice heart and liver

ABSTRACTSome important environmental factors that influence the development of cardiovascular diseases (CVD) include tobacco, excess alcohol, and unhealthy diet. Methionine obtained from the diet participates in the synthesis of DNA, proteins, lipids and affects homocysteine levels, which is associated with the elevated risk for CVD development. Therefore, the aim of this study was to investigate the manner in which dietary methionine might affect cellular mechanisms underlying CVD occurrence. Swiss albino mice were fed either control (0.3% DL-methionine), methionine-supplemented (2% DL-methionine), or a methionine-deprived diet (0% DL-methionine) over a 10-week period. The parameters measured included plasma homocysteine concentrations, oxidative stress by reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, levels of inflammatory cytokines IL-1ß, TNF-α, and IL-6, as well as expression of genes associated with CVD. The levels of apolipoprotein A5 (APOA5), a regulator of plasma triglycerides, were measured. The methionine-supplemented diet increased oxidative stress by lowering the GSH/GSSG ratio in heart tissues and decreased expression of the genes Apob, Ctgf, Serpinb2, Spp1, Il1b, and Sell, but elevated expression of Thbs4, Tgfb2, Ccr1, and Vegfa. Methionine-deprived diet reduced expression of Col3a1, Cdh5, Fabp3, Bax, and Hbegf and increased expression of Sell, Ccl5, Itga2, Birc3, Msr1, Bcl2a1a, Il1r2, and Selp. Methionine-deprived diet exerted pro-inflammatory consequences as evidenced by elevated levels of cytokines IL-1ß, TNF-α, and IL-6 noted in liver. Methionine-supplemented diet increased hepatic IL-6 and cardiac TNF-α. Both methionine supplementation and deprivation lowered hepatic levels of APOA5. In conclusion, data demonstrated that a methionine-supplemented diet modulated important biological processes associated with high risk of CVD development.

A Novel Selective PPARα Modulator (SPPARMα), K-877 (Pemafibrate), Attenuates Postprandial Hypertriglyceridemia in Mice.

Aims: Fasting and postprandial hypertriglyceridemia (PHTG) are caused by the accumulation of triglyceride (TG)-rich lipoproteins and their remnants, which have atherogenic effects. Fibrates can improve fasting and PHTG; however, reduction of remnants is clinically needed to improve health outcomes. In the current study, we investigated the effects of a novel selective peroxisome proliferator-activated receptor α modulator (SPPARMα), K-877 (Pemafibrate), on PHTG and remnant metabolism.Methods: Male C57BL/6J mice were fed a high-fat diet (HFD) only, or an HFD containing 0.0005% K-877 or 0.05% fenofibrate, from 8 to 12 weeks of age. After 4 weeks of feeding, we measured plasma levels of TG, free fatty acids (FFA), total cholesterol (TC), HDL-C, and apolipoprotein (apo) B-48/B-100 during fasting and after oral fat loading (OFL). Plasma lipoprotein profiles after OFL, which were assessed by high performance liquid chromatography (HPLC), and fasting lipoprotein lipase (LPL) activity were compared among the groups.Results: Both K-877 and fenofibrate suppressed body weight gain and fasting and postprandial TG levels and enhanced LPL activity in mice fed an HFD. As determined by HPLC, K-877 and fenofibrate significantly decreased the abundance of TG-rich lipoproteins, including remnants, in postprandial plasma. Both K-877 and fenofibrate decreased intestinal mRNA expression of ApoB and Npc1l1; however, hepatic expression of Srebp1c and Mttp was increased by fenofibrate but not by K-877. Hepatic mRNA expression of apoC-3 was decreased by K-877 but not by fenofibrate.Conclusion: K-877 may attenuate PHTG by suppressing the postprandial increase of chylomicrons and the accumulation of chylomicron remnants more effectively than fenofibrate.