OBJECTIVES: To investigate the effect of BRD4 inhibition on migration of esophageal squamous cell carcinoma (ESCC) cells with low acetyl-CoA carboxylase 1 (ACC1) expression. METHODS: ESCC cell lines with lentivirus-mediated ACC1 knockdown or transfected with a negative control sequence (shNC) were treated with DMSO, JQ1 (a BRD4 inhibitor), co-transfection with shNC-siBRD4 or siNC with additional DMSO or C646 (an ahistone acetyltransferase inhibitor) treatment, or JQ1combined with 3-MA (an autophagy inhibitor). BRD4 mRNA expression in the cells was detected using RT-qPCR. The changes in cell proliferation, migration, autophagy, and epithelial-mesenchymal transition (EMT) were examined with CCK8 assay, Transwell migration assay, and Western blotting. RESULTS: ACC1 knockdown did not significantly affect BRD4 expression in the cells but obviously increased their sensitivity to JQ1. JQ1 treatment at 1 and 2 μmol/L significantly inhibited ESCC cell proliferation, while JQ1 at 0.2 and 2 μmol/L promoted cell migration. The cells with ACC1 knockdown and JQ1 treatment showed increased expresisons of vimentin and Slug and decreased expression of E-cadherin. BRD4 knockdown promoted migration of ESCC cells, and co-transfection with shACC1 and siBRD4 resulted in increased vimentin and Slug expressions and decreased E-cadherin expression in the cells. C646 treatment of the co-transfected cells reduced acetylation levels, decreased vimentin and Slug expressions, and increased E-cadherin expression. Treatment with JQ1 alone obviously increased LC3A/B-II levels in the cells either with or without ACC1 knockdown. In the cells with ACC1 knockdown and JQ1 treatment, additional 3-MA treatment significantly decreased the expressions of vimentin, Slug and LC3A/B-II and increased the expression of E-cadherin. CONCLUSIONS BRD4 inhibition promotes autophagy of ESCC cells via a histone acetylation-dependent mechanism, thereby enhancing EMT and ultimately increasing cell migration driven by ACC1 deficiency.
Humans ; Cell Movement ; Transcription Factors/metabolism* ; Esophageal Neoplasms/metabolism* ; Cell Line, Tumor ; Cell Cycle Proteins ; Azepines/pharmacology* ; Epithelial-Mesenchymal Transition ; Carcinoma, Squamous Cell/metabolism* ; Esophageal Squamous Cell Carcinoma ; Triazoles/pharmacology* ; Nuclear Proteins/genetics* ; Cell Proliferation ; Acetyl-CoA Carboxylase/genetics* ; Transfection ; Autophagy ; Bromodomain Containing Proteins

BACKGROUNDCurrently it is unclear whether lipid accumulation occurs in a particular sequence and its relationship with whole body insulin resistance (IR). This study aimed to answer this question.

METHODSMale Sprague-Dawley (SD) rats were fed on a normal or a high-fat diet for 20 weeks. Serum triglycerides (TG), serum free fatty acids (FFA), fasting plasma glucose (FPG), and liver and skeletal muscle TG were measured. The glucose infusion rate (GIR) and mRNA levels of acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase-1 (CPT-1) in the liver and skeletal muscle were determined at different stages.

RESULTSCompared with rats fed on the normal diet, serum FFA was not significantly increased in rats fed on the high-fat diet until 20 weeks. In contrast, liver TG was significantly increased by the high-fat diet by four weeks (20-fold; P < 0.01), and remained elevated until the end of the study. However, skeletal muscle TG was not significantly increased by the high-fat diet until 20 weeks (10.6-fold; P < 0.01), and neither was the FPG. The GIR was significantly reduced (1.6-fold; P < 0.01) by the high-fat diet after 8 weeks. The mRNA levels of ACC gradually increased over time and CPT-1 decreased over time, in both the liver and skeletal muscle in rats fed the high-fat diet.

CONCLUSIONSLipid accumulation in the liver occurs earlier than lipid accumulation in the skeletal muscle. Fatty liver may be one of the early markers of whole body IR. Changes in the gene expression levels of ACC and CPT-1 may have important roles in the process of IR development.

Acetyl-CoA Carboxylase ; genetics ; Animals ; Blood Glucose ; analysis ; Carnitine O-Palmitoyltransferase ; genetics ; Fatty Acids, Nonesterified ; blood ; Fatty Liver ; etiology ; Insulin Resistance ; Lipid Metabolism ; Liver ; metabolism ; Male ; Muscle, Skeletal ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Triglycerides ; metabolism

BACKGROUNDAs an agonist of peroxisome proliferator-activated receptor-gamma (PPARγ), rosiglitazone can prevent acute fatty acid-induced insulin resistance in rats, however, the precise mechanisms by which rosiglitazone alleviates insulin resistance induced by high-fat diet need to be further investigated.

METHODSWistar rats aged 23-24 weeks were divided into three groups: (1) aged control group (OC), (2) high-fat diet (HF) group and (3) high-fat diet plus rosiglitazone maleate tablets (HF + Rosi) treatment group (n = 20 in each group). Insulin sensitivity was evaluated by conscious hyperinsulinemic-euglycemic clamp technique. mRNA levels of fatty acid translocase (FAT/CD36), AMP-activated protein kinase α1 (AMPKα1), AMPKα2 and acetyl CoA carboxylase (ACC) of rat skeletal muscle were determined using real-time PCR, while muscle carnitine palmitoyltransferase-1 (CPT-1β) was determined using semi-quantitative PCR. Protein expression levels of FAT/CD36, AMPK phosphorylation (reflecting AMPK activity), P-ACC (inversely related with ACC activity) and muscle CPT-1M in rat skeletal muscles were measured using Western blotting.

RESULTSAged rats fed by diet rich in fat for more than 8 weeks led to significant increases of plasma lipids, skeletal muscle intramuscular triglyceride and long-chain fatty acyl-CoA (LCACoA) compared to aged rats fed by normal chow diet (OC) (P < 0.05), which might correlate with the lower (reduced by 42.4%) whole body insulin sensitivity in HF rats. FAT/CD36 protein concentrations and mRNA levels increased in untreated HF aged rats (P < 0.01) and high-fat diet induced a significant decrease in P-AMPK, P-ACC, CPT-1M protein concentrations and AMPKα2 and CPT-1β mRNA levels in rat skeletal muscles (P < 0.05). No change in AMPKα1 mRNA levels was observed in the HF group.

CONCLUSIONHigh-fat diet in aged rats results in a lipid accumulation and subsequent insulin resistance, while rosiglitazone can alleviate the insulin resistance by reducing fatty acid uptake as well as enhancing lipometabolism.

AMP-Activated Protein Kinases ; genetics ; Acetyl-CoA Carboxylase ; genetics ; Animals ; CD36 Antigens ; genetics ; Dietary Fats ; administration & dosage ; Hypoglycemic Agents ; pharmacology ; Insulin Resistance ; Male ; Muscle, Skeletal ; enzymology ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Thiazolidinediones ; pharmacology

Mammals have two major isoforms of acetyl-CoA carboxyase (ACC). The 275 kDa beta-form (ACC beta) is predominantly in heart and skeletal muscle while the 265 kDa alpha-form (ACC alpha) is the major isoform in lipogenic tissues such as liver and adipose tissue. ACC alpha is thought to control fatty acid oxidation by means of the ability of malonyl-CoA to inhibit carnitine palmitoyl-CoA transferase-1 (CPT-1), which is a rate-limiting enzyme of fatty acid oxidation in mitochondria. Previously, it was reported that MyoD and other muscle regulating factors (MRFs) up-regulate the expression of ACC beta by interactions between these factors and several cis-elements of ACC beta promoter. We described here that ACC beta expression mediated by MRFs is regulated by retinoic acids. Endogenous expression of ACCb in differentiated H9C2 myotube was significantly increased by retinoic acid treatment. However, on transient transfection assay in H9C2 myoblast, ACC beta promoter activity was suppressed by RXRa and more severely by RAR alpha. These effects on ACCb expression in myoblasts and myotubes by RXR alpha and RAR alpha seem to be mediated by their interactions with MRFs because no consensus sequence for RXR alpha and RAR alpha has been found in ACC beta promoter and retinoic acid receptors did not affect this promoter activities by itself. In transient transfection in NIH3T3 fibroblast, the activation of ACC beta promoter by MyoD, main MRF in myoblast, was significantly suppressed by RAR alpha and to a less extent by RXR alpha while the RXR alpha drastically augmented the activation by MRF4, major MRF in myotube. These results explained that retinoic acids differentially affected the action of MRFs according to their types and RXR alpha specially elevates the expression of muscle specific genes by stimulating the action of MRF4.
3T3 Cells ; Acetyl-CoA Carboxylase/genetics/*metabolism ; Animals ; Cell Differentiation ; Cells, Cultured ; Gene Expression Regulation, Enzymologic/drug effects ; Mice ; MyoD Protein/*metabolism ; Myoblasts/drug effects/metabolism ; Myogenic Regulatory Factors/*metabolism ; Promoter Regions (Genetics)/drug effects ; Receptors, Retinoic Acid/genetics/*metabolism ; Trans-Activation (Genetics) ; Transcription Factors/genetics/*metabolism ; Tretinoin/pharmacology