To investigate the effect of Gegen Qinlian Decoction(GQD) on enzyme activity, gene expression and methylation level of fatty acid synthase(FASN) in adipose tissue from rats with insulin resistance induced by high-fat diet. The 60% fat-powered high-fat diet was continuously given to male SD rats to induce the insulin resistance model. Then, they were divided into five groups randomly and administrated by gavage every day for 16 weeks with following drugs respectively: 10 mL·kg~(-1)water for control group(C) and insulin resistance model control group(IR), 1.65 g·kg~(-1)GQD per day for low-dose group(GQDL), 4.95 g·kg~(-1)GQD per day for medium-dose group(GQDM), 14.85 g·kg~(-1)GQD per day for high-dose group(GQDH), and 5 mg·kg~(-1) rosiglitazone per day for rosiglitazone group(RGN). Epididymal adipose tissue was taken to determine enzyme activity of FASN by colorimetric method, mRNA expression level of Fasn by quantitative Real-time PCR(Q-PCR) and CpGs methylation level between +313 and +582 by bisulfite sequencing PCR(BSP). These results showed that Fasn expression was significantly lowered in IR model rats compared with the control rats(P<0.01). Enzymatic activity and CpGs methylation level of Fasn in IR group showed downward trends. Low and medium-dose GQD can increase enzyme activity of FASN(P<0.05). Moreover, low-dose GQD increased the total CpGs methylation level of Fasn fragment between +313 and +582 in insulin resistance rats(P<0.05). For GQDM group, the methylation frequency of CpGs at positions +506 and +508(P<0.01) as well as the methylation frequency of CpGs on the binding sites of transcription factorzinc finger protein 161(P<0.05) were significantly increased. The methylation frequency of CpG at +442 position was positively correlated with Fasn expression(P<0.01, r=0.735), and methylation frequencies of CpGs at +345 and +366 positions were positively associated to enzyme activity of FASN respectively(P<0.05, r=0.479; P<0.01, r=0.640). In conclusion, GQD can reverse enzyme activity of FASN and methylation level of Fasn in adipose tissue of insulin resistant rats, and CpG sites at positions +506 and +508 may be the targets of GQD. The methylation level of CpGs at + 345 and + 366 sites were possibly related to FASN activity, while methylation of CpG at + 442 site may be closely correlated with mRNA level of Fasn. In addition, GQD did not significantly change mRNA expression level of Fasn, but effectively reversed enzymatic activity, suggesting that GQD may regulate the post transcriptional expression of Fasn.
Adipose Tissue ; Animals ; Drugs, Chinese Herbal ; Fatty Acid Synthases/genetics* ; Gene Expression ; Insulin Resistance/genetics* ; Male ; Methylation ; Rats ; Rats, Sprague-Dawley

Tumorigenesis involves metabolic reprogramming and abnormal lipid metabolism, which is manifested by increased endogenous fat mobilization, hypertriglyceridemia, and increased fatty acid synthesis. Fatty acid synthase (FASN) is a key enzyme for the de novo synthesis of fatty acids, and monoacylglycerol esterase (MGLL) is an important metabolic enzyme that converts triglycerides into free fatty acids. Both enzymes play an important role in lipid metabolism and are associated with tumor-related signaling pathways, the most common of which is the PI3K-AKT signaling pathway. They can also regulate the immune microenvironment, participate in epithelial-mesenchymal transition, and then regulate tumor invasion and metastasis. Current literature have shown that these two genes are abnormally expressed in many types of tumors and are highly correlated with tumor migration and invasion. This article introduces the structures and functions of FASN and MGLL, their relationship with abnormal lipid metabolism, and the mechanism of the regulation of tumor invasion and metastasis and reviews the research progress of the relationship of FASN and MGLL with tumor invasion and metastasis.
Cell Line, Tumor ; Fatty Acid Synthase, Type I/metabolism* ; Humans ; Lipid Metabolism ; Monoacylglycerol Lipases/metabolism* ; Neoplasms ; Phosphatidylinositol 3-Kinases ; Signal Transduction ; Tumor Microenvironment

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.
Apolipoprotein B-100 ; metabolism ; Cells, Cultured ; Emodin ; pharmacology ; Fatty Acid Synthase, Type I ; metabolism ; Hepatocytes ; drug effects ; metabolism ; Humans ; Inflammation ; Interleukin-6 ; metabolism ; Lipid Metabolism ; Lipids ; NF-kappa B ; metabolism ; Sterol Regulatory Element Binding Protein 2 ; metabolism

To understand molecular modules related to polyunsaturated fatty acids (PUFA) synthesis and eventually produce PUFA at high efficiency, we developed a protein complex analysis technology in Synechocystis sp. PCC 6803, and applied it to identify possible partner proteins interacting with the key enzymes that catalyze PUFA biosynthesis. We first constructed a recombinant expression of protein of slr1609 encoding the fatty acid activation enzyme, by fusing 3xFLAG tag with the target protein. Then we verified its expression by Western blotting targeting 3xFLAG tag. To maximize purification of Slr1609 protein complex, we optimized the protein expression conditions of Slr1609 in Synechocystis in a 5 L fermenter by monitoring its gene expression using RT-qPCR. The purification of the Slr1609 protein complexes was demonstrated by a Native-PAGE analysis. Finally, LC-MS/MS proteomic analysis allowed identification of the possible partner proteins interacting with Slr1609.
Bacterial Proteins ; chemistry ; Chromatography, Liquid ; Fatty Acid Synthases ; chemistry ; Fatty Acids, Unsaturated ; biosynthesis ; Proteome ; chemistry ; Proteomics ; Synechocystis ; enzymology ; Tandem Mass Spectrometry

The Escherichia coli fadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ-proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensis fabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensis fabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of Shewanella fabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid synthesis in the marine bacteria Shewanella genus.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; metabolism ; Base Sequence ; Binding Sites ; DNA, Bacterial ; metabolism ; Escherichia coli ; genetics ; metabolism ; Fatty Acid Synthase, Type II ; genetics ; metabolism ; Fatty Acids ; biosynthesis ; Gene Expression Regulation, Bacterial ; drug effects ; Molecular Sequence Data ; Oleic Acid ; pharmacology ; Protein Binding ; drug effects ; Regulon ; genetics ; Repressor Proteins ; chemistry ; metabolism ; Shewanella ; genetics ; metabolism

OBJECTIVE: To explore the eff ect of silibinin on β cells in C57BL/6J mice fed a high-fat diet and the possible mechanisms. METHODS: A total of 18 male C57BL/6J mice at 3 weeks old were divided into a normal chow group (n=6), a high-fat diet group (n=6) and a high-fat diet plus silibinin group (n=6). Aft er intervention for 10 weeks, fasting blood glucose (FBG), fasting insulin (FINS), triglycerides (TG), alanine aminotransferase (ALT), creatinine (Cr) and blood urea nitrogen (BUN), lipid metabolism, antioxidant enzyme activities and apoptosis were evaluated. Pancreatic tissues were isolated to examine insulin-induced gene-1 (Insig-1), sterol regulatory element binding protein-1c (SREBP-1c) and fatty acid synthetase (FAS) mRNA and protein expression. RESULTS: Compared with the high-fat diet group, the function of insulin secretion was improved, and the level of blood glucose was decreased in the high-fat diet plus silibinin group (P<0.05). The levels of lipid content and oxidative stress and the rates of β cell apoptosis were lower in high-fat diet plus silibinin group than those in the high-fat diet group (both P<0.05). Simultaneously, the silibinin could promote the expression of Insig-1 and depress the expression of SREBP-1c and FAS (all P<0.05). Moreover, there was no significant difference in the levels of serum ALT, Cr and BUN among the 3 groups (all P>0.05). CONCLUSION Silibinin can protect β cells of mice fed a high-fat diet, and this effect might be related to, at least partially, increase in its antioxidative ability through regulation of insig-1/SREBP-1c pathway. Moreover, silibinin is safe for long-term treatment.
Alanine Transaminase ; blood ; Animals ; Apoptosis ; Blood Glucose ; analysis ; Blood Urea Nitrogen ; Creatinine ; blood ; Diet, High-Fat ; Fatty Acid Synthases ; metabolism ; Insulin ; blood ; Insulin-Secreting Cells ; cytology ; drug effects ; Lipid Metabolism ; Lipids ; Male ; Membrane Proteins ; metabolism ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; Silybin ; Silymarin ; pharmacology ; Sterol Regulatory Element Binding Protein 1 ; metabolism ; Triglycerides ; blood

OBJECTIVETo establish nonalcoholic fatty liver disease (NAFLD) model induced by free fatty acid (FFA) and iron, and to explore the synergistic effect of FFA and Fe(2+) on the pathogenesis of NAFLD and mechanisms.

METHODSHuman liver carcinoma cell HepG2 was respectively treated with 0.250, 0.500, 1.000 mmol/L oleic acid, 0.500 mmol/L oleic acid+0.125 mmol/L Fe(2+), 0.500 mmol/L oleic acid+0.250 mmol/L Fe(2+), and 0.500 mmol/L oleic acid+0.500 mmol/L Fe(2+). Human liver carcinoma cell HepG2 was normally cultured in the control group. Lipid accumulation of cells were observed by oil red O staining and the determination of the triglyceride (TG) contents by GPO-PAP, then the expression of key genes involved in fatty acid β-oxidation (fatty acyl CoA synthetase-1 (ACSL-1), carnitine acyl transferase 1 (CPT-1a), fatty acid synthetase (FAS)) was determined using RT-PCR. The differences of TG content and ACSL-1, CPT-1a, FAS, mRNA relative value were analyzed among different groups.

RESULTSThe results of oil red O staining indicated that the contents of lipid droplets were obviously elevated with the increase of Fe(2+) concentration in human liver carcinoma cell HepG2 treated with 0.500 mmol/L oleic acid and different concentrations of Fe(2+). The TG contents of HepG2 cell in control group, 0.250, 0.500, 1.000 mmol/L oleic acid groups, 0.500 mmol/L oleic acid+0.125 mmol/L Fe(2+) group, 0.500 mmol/L oleic acid+0.250 mmol/L Fe(2+) group, 0.500 mmol/L oleic acid+0.500 mmol/L Fe(2+) group respectively were (90.0 ± 1.6), (131.7 ± 5.4), (153.7 ± 3.0), (254.1 ± 4.0), (164.5 ± 6.0), (180.1 ± 7.7), (235.6 ± 4.5) nmol/mg (F = 396.00, P < 0.05). The expression levels of ACSL-1 mRNA in 0.500 mmol/L oleic acid group, 0.500 mmol/L oleic acid+0.125 mmol/L Fe(2+) group, 0.500 mmol/L oleic acid +0.250 mmol/L Fe(2+) group, 0.500 mmol/L oleic acid +0.500 mmol/L Fe(2+) group respectively were (0.94 ± 0.02), (0.89 ± 0.04), (0.85 ± 0.02), (0.74 ± 0.04) (F = 50.00, P < 0.05); the mRNA levels of CPT-1a were (0.89 ± 0.03), (0.79 ± 0.05), (0.67 ± 0.04), (0.51 ± 0.05) (F = 79.00, P < 0.05); the mRNA levels of FAS were (1.31 ± 0.05) , (1.44 ± 0.03), (1.51 ± 0.05), (1.56 ± 0.06 ) (F = 79.70, P < 0.05).

CONCLUSIONThe NAFLD liver cell model could be established by oleic acid and Fe(2+) in HepG2 cells. FFA and iron might be involved in the pathogenesis of NAFLD through the intervention of fatty acid β-oxidation.

Carnitine O-Palmitoyltransferase ; Coenzyme A Ligases ; Fatty Acid Synthase, Type I ; Fatty Acids ; Fatty Acids, Nonesterified ; adverse effects ; Hep G2 Cells ; Humans ; Iron ; adverse effects ; Non-alcoholic Fatty Liver Disease ; chemically induced ; Oleic Acid ; RNA, Messenger ; Triglycerides

Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.
Acetyltransferases ; genetics ; metabolism ; Arachidonic Acid ; biosynthesis ; Docosahexaenoic Acids ; biosynthesis ; Eicosapentaenoic Acid ; biosynthesis ; Fatty Acid Desaturases ; genetics ; metabolism ; Fatty Acid Synthases ; genetics ; metabolism ; Fatty Acids, Unsaturated ; biosynthesis ; Genetic Vectors ; HEK293 Cells ; Humans ; Transfection

Fatty acid synthase (FAS) catalyses the reaction between acetyl-CoA and malonyl-CoA to produce fatty acids. It is one of the most important enzyme in lipid biosynthesis. FAS of the oleaginous yeast Rhodosporidium toruloides has two acyl carrier protein (ACP) domains and a distinct subunit composition compared with FASs of other species. As ACP is a protein cofactor crucial for fatty acid chain elongation, more ACPs in the FAS may facilitate the reaction. To study the biochemical and structural properties of this novel FAS from R. toruloides, plasmids were constructed and transformed into Escherichia coli BL21 (DE3). The strain ZWE06 harboring plasmids pET22b-FAS1 and pET24b-FAS2 could co-overexpress the two subunits. The recombinant FAS was purified by sequentially using ammonium sulphate precipitation, sucrose density gradient centrifugation and anion exchange chromatography. The specific activity of the recombinant FAS was 548 mU/mg. The purified complex would be used to study enzyme kinetics and protein structure of FAS, and heterogeneous expression and purification will facilitate revealing the mechanism of this novel FAS with double ACPs.
Acyl Carrier Protein ; Basidiomycota ; enzymology ; Chromatography ; Escherichia coli ; metabolism ; Fatty Acid Synthases ; biosynthesis ; genetics ; Fatty Acids ; biosynthesis ; Plasmids ; Recombinant Proteins ; biosynthesis ; genetics

Escherichia coli (E. coli) FadR regulator plays dual roles in fatty acid metabolism, which not only represses the fatty acid degradation (fad) system, but also activates the unsaturated fatty acid synthesis pathway. Earlier structural and biochemical studies of FadR protein have provided insights into interplay between FadR protein with its DNA target and/or ligand, while the missing knowledge gap (esp. residues with indirect roles in DNA binding) remains unclear. Here we report this case through deep mapping of old E. coli fadR mutants accumulated. Molecular dissection of E. coli K113 strain, a fadR mutant that can grow on decanoic acid (C10) as sole carbon sources unexpectedly revealed a single point mutation of T178G in fadR locus (W60G in FadRk113). We also observed that a single genetically-recessive mutation of W60G in FadR regulatory protein can lead to loss of its DNA-binding activity, and thereby impair all the regulatory roles in fatty acid metabolisms. Structural analyses of FadR protein indicated that the hydrophobic interaction amongst the three amino acids (W60, F74 and W75) is critical for its DNA-binding ability by maintaining the configuration of its neighboring two β-sheets. Further site-directed mutagenesis analyses demonstrated that the FadR mutants (F74G and/or W75G) do not exhibit the detected DNA-binding activity, validating above structural reasoning.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase ; genetics ; metabolism ; Amino Acid Sequence ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; metabolism ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; Fatty Acid Synthase, Type II ; genetics ; metabolism ; Fatty Acids ; metabolism ; Gene Expression Regulation, Bacterial ; Hydro-Lyases ; genetics ; metabolism ; Hydrophobic and Hydrophilic Interactions ; Lipid Metabolism ; Models, Molecular ; Molecular Sequence Data ; Mutation ; Protein Binding ; Protein Structure, Secondary ; Repressor Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; Signal Transduction