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Developing a high-efficiency benzylamine oxidation reaction (BOR) to replace the sluggish oxygen evolution reaction (OER) is an attractive pathway to promote H2 production and concurrently realize organic conversion. However, the electrochemical BOR performance is still far from satisfactory. Herein, we present a self-supported CuO nanorod array with abundant oxygen vacancies on copper foam (Vo-rich CuO/CF) as a promising anode for selective electro-oxidation of benzylamine (BA) to benzonitrile (BN) coupled with cathodic H2 generation. In situ infrared spectroscopy demonstrates the selective conversion of BA into BN on Vo-rich CuO. Furthermore, in situ Raman spectroscopy discloses a direct electro-oxidation mechanism of BA driven by electroactive hydroxyl species (OH*) over the Vo-rich CuO catalyst. Theoretical and experimental studies verify that the presence of oxygen vacancies is more favorable for the adsorption of OH* and BA molecules, enabling accelerated kinetics for the BOR. As expected, the Vo-rich CuO/CF electrode delivers outstanding BOR activity and stability, giving a high faradaic efficiency (FE) of over 93% for BN production at a potential of 0.40 V vs. Ag/AgCl. Impressively, almost 100% FE for H2 production can be further achieved at the NiSe cathode by integrating BA oxidation in a two-electrode electrolyzer.

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Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in developed countries. NAFLD describes a wide range of liver pathologies from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. NASH is distinguished from simple steatosis by inflammation, cell death and fibrosis. In this study we found that mice lacking triacylglycerol hydrolase (TGH, also known as carboxylesterase 3 or carboxylesterase 1d) are protected from high-fat diet (HFD) - induced hepatic steatosis via decreased lipogenesis, increased fatty acid oxidation and improved hepatic insulin sensitivity. To examine the effect of the loss of TGH function on the more severe NAFLD form NASH, we ablated Tgh expression in two independent NASH mouse models, Pemt(-/-) mice fed HFD and Ldlr(-/-) mice fed high-fat, high-cholesterol Western-type diet (WTD). TGH deficiency reduced liver inflammation, oxidative stress and fibrosis in Pemt(-/-) mice. TGH deficiency also decreased NASH in Ldlr(-/-) mice. Collectively, these findings indicate that TGH deficiency attenuated both simple hepatic steatosis and irreversible NASH.

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BACKGROUND & AIMS: Very low density lipoproteins (VLDLs) are triacylglycerol (TG)-rich lipoproteins produced by the human liver. VLDLs derive the majority of their TG cargo from the lipolysis of TG stored in hepatocellular lipid droplets (LDs). Important roles for LDs and the VLDL secretory pathway in the cell culture production of infectious hepatitis C virus (HCV) have been established. We hypothesized that TG lipolysis and VLDL production are impaired during HCV infection so that these cellular processes can be diverted towards HCV production. METHODS: We used an HCV permissive cell culture system (JFH-1/HuH7.5 cells) to examine the relationship between TG lipolysis, VLDL assembly, and the HCV lifecycle using standard biochemical approaches. RESULTS: Lipolysis of cellular TG and VLDL production were impaired in HCV infected cells during the early peak of viral infection. This was partially explained by an apparent deficiency of a putative TG lipase, arylacetamide deacetylase (AADAC). The re-introduction of AADAC to infected cells restored cellular TG lipolysis, indicating a role for HCV-mediated downregulation of AADAC in this process. Defective lipolysis of cellular TG stores and VLDL production were also observed in HuH7.5 cells stably expressing a short hairpin RNA targeting AADAC expression, proving AADAC deficiency contributes to these defective pathways. Finally, impaired production of HCV was observed with AADAC knockdown cells, demonstrating a role for AADAC in the HCV lifecycle. CONCLUSIONS: This insight into the biology of HCV infection and possibly pathogenesis identifies AADAC as a novel and translationally relevant therapeutic target.

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UNLABELLED: Carboxylesterase 3/triacylglycerol hydrolase (Ces3/TGH) participates in hepatic very low-density lipoprotein (VLDL) assembly and in adipose tissue basal lipolysis. Global ablation of Ces3/Tgh expression decreases serum triacylglycerol (TG) and nonesterified fatty acid levels and improves insulin sensitivity. To understand the tissue-specific role of Ces3/TGH in lipid and glucose homeostasis, we generated mice with a liver-specific deletion of Ces3/Tgh expression (L-TGH knockout [KO]). Elimination of hepatic Ces3/Tgh expression dramatically decreased plasma VLDL TG and VLDL cholesterol concentrations but only moderately increased liver TG levels in mice fed a standard chow diet. Significantly reduced plasma TG and cholesterol without hepatic steatosis were also observed in L-TGH KO mice challenged with a high-fat, high-cholesterol diet. L-TGH KO mice presented with increased plasma ketone bodies and hepatic fatty acid oxidation. Intrahepatic TG in L-TGH KO mice was stored in significantly smaller lipid droplets. Augmented hepatic TG levels in chow-fed L-TGH KO mice did not affect glucose tolerance or glucose production from hepatocytes, but impaired insulin tolerance was observed in female mice. CONCLUSION: Our data suggest that ablation of hepatic Ces3/Tgh expression decreases plasma lipid levels without causing severe hepatic steatosis.

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Lipid droplets (LDs) form from the endoplasmic reticulum (ER) and grow in size by obtaining triacylglycerols (TG). Triacylglycerol hydrolase (TGH), a lipase residing in the ER, is involved in the mobilization of TG stored in LDs for the secretion of very-low-density lipoproteins. In this study, we investigated TGH-mediated changes in cytosolic LD dynamics. We have found that TGH deficiency resulted in decreased size and increased number of LDs in hepatocytes. Using fluorescent fatty acid analogues to trace LD formation, we observed that TGH deficiency did not affect the formation of nascent LDs on the ER. However, the rate of lipid transfer into preformed LDs was significantly slower in the absence of TGH. Absence of TGH expression resulted in increased levels of membrane diacylglycerol and augmented phospholipid synthesis, which may be responsible for the delayed lipid transfer. Therefore, altered maturation (growth) rather than nascent formation (de novo synthesis) may be responsible for the observed morphological changes of LDs in TGH-deficient hepatocytes.

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Excessive accumulation of triacylglycerol in peripheral tissues is tightly associated with obesity and has been identified as an independent risk factor for insulin resistance, type 2 diabetes, and cardiovascular complications. Here we show that ablation of carboxylesterase 3 (Ces3)/triacylglycerol hydrolase (TGH) expression in mice (Tgh(-/-)) results in decreased plasma triacylglycerol, apolipoprotein B, and fatty acid levels in both fasted and fed states. Despite the attenuation of very low-density lipoprotein secretion, TGH deficiency does not increase hepatic triacylglycerol levels. Tgh(-/-) mice exhibit increased food intake, respiratory quotient, and energy expenditure without change in body weight. These metabolic changes are accompanied by improved insulin sensitivity and glucose tolerance. Tgh(-/-) mice have smaller sized pancreatic islets but maintain normal glucose-stimulated insulin secretion. These studies demonstrate the potential of TGH as a therapeutic target for lowering blood lipid levels.

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Apolipoprotein B (apoB)-containing lipoproteins play a critical role in whole body lipid homeostasis and the pathogenesis of atherosclerosis. The assembly of hepatic apoB-containing lipoproteins, VLDL, is governed by the availability of lipids, including triacylglycerol (TG). The majority of TG associated with VLDL is derived from the hepatic cytoplasmic lipid stores by a process involving lipolysis followed by reesterification. Microsomal triacylglycerol hydrolase (TGH) has been demonstrated to play a role in the lipolysis/reesterification process. To evaluate the potential regulatory role of TGH in hepatic VLDL assembly, we developed inducible transgenic mice expressing a human TGH minigene under the control of the mouse metallothionein promoter. Induction of human TGH by zinc resulted in liver-specific expression of the enzyme associated with 3- to 4-fold increases in lipolytic activity that could be attenuated with a TGH-specific inhibitor. Augmented TGH activity led to increased secretion of newly synthesized apoB and plasma TG levels. These results suggest that increased hepatic expression of TGH leads to a more proatherogenic plasma lipid and apoB profile.

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Fatty acids released from adipose triacylglycerol stores by lipolysis provide vertebrates with an important source of energy. We investigated the role of microsomal triacylglycerol hydrolase (TGH) in the mobilization of adipocyte triacylglycerols through inactivation of the TGH activity by RNA interference or chemical inhibition. Attenuation of TGH activity resulted in decreased basal but not isoproterenol-stimulated efflux of fatty acids from 3T3-L1 adipocytes. Lack of TGH activity was accompanied by accumulation of cellular triacylglycerols and cholesteryl esters without any changes in the expression of enzymes catalyzing triacylglycerol synthesis (diacylglycerol acyltransferases 1 and 2) or degradation (adipose triglyceride lipase and hormone-sensitive lipase). Inhibition of TGH-mediated lipolysis also did not affect insulin-stimulated Glut4 translocation from intracellular compartments to the plasma membrane or glucose uptake into adipocytes. These data suggest that TGH plays a role in adipose tissue triacylglycerol metabolism and may be a suitable pharmacological target for lowering fatty acid efflux from adipose tissue without altering glucose import.

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TGH (triacylglycerol hydrolase) catalyses the lipolysis of intracellular stored triacylglycerol. To explore the mechanisms that regulate TGH expression in adipose tissue, we studied the expression of TGH during the differentiation of 3T3-L1 adipocytes. TGH mRNA and protein levels increased dramatically in 3T3-L1 adipocytes compared with pre-adipocytes. Electrophoretic mobility shift assays demonstrated enhanced binding of nuclear proteins of adipocytes to the distal murine TGH promoter region (-542/-371 bp), yielding one adipocyte-specific migrating complex. Competitive and supershift assays demonstrated that the distal TGH promoter fragment bound C/EBPalpha (CCAAT/enhancer-binding protein alpha). Transient transfections of different mutant TGH promoter-luciferase constructs into 3T3-L1 adipocytes and competitive electromobility shift assays showed that the C/EBP-binding elements at positions -470/-459 bp and -404/-390 bp are important for transcriptional activation. Co-transfection with C/EBPalpha cDNA and TGH promoter constructs in 3T3-L1 pre-adipocytes demonstrated that C/EBPalpha increased TGH promoter activity. Ectopic expression of C/EBPalpha in NIH 3T3 cells activated TGH mRNA expression without causing differentiation into adipocytes. These experiments directly link increased TGH expression in adipocytes to transcriptional regulation by C/EBPalpha. This is the first evidence that C/EBPalpha participates directly in the regulation of an enzyme associated with lipolysis.

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Lecithin cholesterol acyltransferase (LCAT) is the major enzyme producing most plasma cholesterol esters(CE) and a key participant in the process of reverse cholesterol transfer (RCT). The aim of this research is to co-express LCAT and it's natural activator apoA-I, with the recombinant adeno-associated virus vectors in the skeletal muscle cells, in order to pave a new way for gene therapy of the primary or secondary LCAT deficiency. 293T cells was cotransfected with pDG and rAAVAIL/rAAVL plasmids to produce infectious rAAV, and non-ionic iodixanol gradient centrifugation, followed by heparin affinity chromatography, were performed for separation, purification and concentration of rAAV. The particle numbers of rAAV, assayed by dot blot, were 7 x 10(14)/L (rAAVAIL) and 1 x 10(14)/L (rAAVL). These vectors were then transduced into C2C12 myoblasts. The results of ELISA and Western blot for human apoA-I, and [3H]-cholesterol-labeled radiochemical methods for LCAT activity, showed that the expression of human apoA-I cDNA and/or human LCAT cDNA in transduced C2C12 cells lasted for 30 days, even after myoblasts were differentiated into myotubes. PCR products for the transgene indicated the long-term persistence of transduced vector sequences. The results indicate that the methods used for production and purification of rAAV is efficient, and rAAV vector mediated the expression and secretion of LCAT and apoA-I gene in C2C12 myoblasts successfully. It suggests that the use of rAAV vectors mediating the high efficiency, long-term expression of human LCAT cDNA and/or apoA-I cDNA in skeletal muscle in vivo can be a safe and fesible strategy for the gene therapy of LCAT deficiency.

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AIM: To study the effects of praeruptorin C (pra-C) on proliferation of cattle aortic smooth muscle cells (SMC). METHODS: The DNA synthesis of SMC was measured using the incorporation of [3H]thymidine([3H]TdR). Cell cycle phase was evaluated by flow cytometry and cytotoxicity was evaluated by measuring lactic dehydrogenase (LDH) activity. RESULTS: Whether or not treated with angiotensin II (Ang-II), SMC proliferation was suppressed by pra-C in a concentration-dependent manner at range from 0.001 micromol/L to 10 micromol/L. The inhibitory effects appeared to be related to G1-S block in cell cycle traverse while the LDH activities did not change dramatically. CONCLUSION: Pra-C can completely inhibit SMC proliferation induced by Ang II and partly inhibit the growth of SMC- induced by bovine serum, which is important in prevention and treatment of vascular hyperplastic disease.

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AIM: To investigate the effects of ginkgolide B (GB) on proliferation of bovine aortic smooth muscle cells (SMC) and its related mechanisms. METHODS: After pretreating with GB or the mixture of ginkgolide A and B (GA:GB) at 37 degrees C for 0.5 h, the VSMC were treated with or without angiotensin II (Ang II) for 24 h. The proliferation of SMC was evaluated by 3H-thymidine incorporation and the cell cycle phase was measured by flow cytometry. Cytotoxicity was reflected by MTT and lactate dehydrogenase (LDH) activity of the supernatant. RESULTS: Whether or not treated with Ang II, GB and GA:GB were shown to suppress SMC proliferation in concentration-dependent fashion at concentrations ranging from 10(-9) mol.L-1 to 10(-5) mol.L-1. The inhibitory effects appeared to be related to a G1-->S block in cell cycle traverse. CONCLUSION: The suppression of SMC proliferation by GB might not only be contributed by blockage of the PAF receptor activity.

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In order to investigate the relationship between scavenger receptor type A and cell signal transduction, human U937 macrophages were treated with tyrosine protein kinase inhibitor genistein, then the cells were incubated with [(125)I]ox-LDL or ox-LDL, and the cellular degradation of [(125)I]ox-LDL or binding were measured separately. Then the effect of the drug on cell surface expression of SR-A were measured by means of autoradiography, it was found that genistein could reduce cellular SR-A mRNA transcription by RT-PCR. The results indicated that genistein could reduce U937 macrophages to bind lipids and reduce SR-A expression by suppression of transcription, and could reduce degradation of lipids by U937 macrophage and accumulation of cholesterol within the cells. It suggests that the function of scavenger receptors may be correlated with cell tyrosine protein kinase, the mechanism is transcriptional and it also suggests that SR-A may participate in the signal transduction directly.

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Viral and nonviral vectors containing apoAI, apoE or LCAT genes were constructed and transfected into myogenic cells in vitro or injected directly into mouse skeletal muscle. The expression efficiencies of these vectors were assaied to investigate the possibility of ectopic expression of these genes in skeletal muscle and to develop a safe and convenient gene therapy method for atherosclerosis. The results showed that the primary cultured mouse myoblasts, C2C12 cells transfected with pCMVapoE3 expressed human apoE3 successfully and the expressed product was secreted into the medium. Mouse skeletal muscle efficiently expressed apoE3 in vivo after direct plasmid injection. The expression level of Ad-RSV-apoAI in primary cultured mouse myoblasts was correlated with virus titer. Human apoAI was synthesized in mouse skeletal muscle by direct injection of recombinant virus and was secreted into blood continuously up to 30 days functional LCAT was expressed by C2C12 and 293 cells transfected with conventional vector or recombinant AAV plasmid DNA. The expression efficiency of recombinant AAV plasmid DNA was 2-5 times higher than that of conventional plasmid vector. The above results provided experimental data for further studying and developing a gene therapy method for atherosclerosis by enhancement of reverse cholesterol transport using skeletal muscle as target.

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Scavenger receptor A(SR-A)on the mouse peritoneal macrophages(MPM) mediates the endocytic uptake of oxidized low density lipoproteins(ox-LDL). However, using ligand blotting and immunoblotting, a novel macrophage membrane protein binding to ox-LDL with estimated molecular mass of 92 kD was found. This membrane protein could not bind to ac-LDL. Its binding to ligands was not affected by reducing reagents either. Preincubation with medium containing neuraminidase dramatically decreased binding of the 92 kD membrane protein to ox-LDL. Excess amounts of ox-LDL could completely block the binding of (125)I ox-LDL to 92 kD membrane protein, but polyI, dextran sulfate and fucoidin showed partial competitive inhibition effects to the binding. These results suggest that the novel 92 kD membrane protein plays important role in the MPM uptake of ox-LDL.

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In order to investigate effects of cell protein phosphorylation on scavenger receptor, human U937 macrophage-like cells were treated with protein kinase C inhibitor staurosporine, then the cells were incubated with (125)I ox-LDL or ox-LDL, and the cellular degradation of (125)I ox-LDL, its binding to receptor and the internalization of cell surface ox-LDL receptor complex as well as the accumulation of lipids within cells were measured separately. Moreover, the effects of the drug on expression of cell surface receptor were observed by means of autoradiography. The results indicated that staurosporine could enhance U937 cells to bind lipids and stimulate scavenger receptor expression, and could reduce degradation of lipids by U937 cells and the accumulation of cholesterol within the cells. It suggests that the function of scavenger receptors may be correlated with cell protein phorsphorylation.

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In order to observe the effect of type I collagen on the susceptibility of low density lipoprotein (LDL) to oxidation, the collagen gel system was established in vitro. The presence of collagen resulted in a protracted lag phase as well as a decreased production of the thiobarbituric acid reactive substance (TBARS) when LDL was oxidized by the addition of copper ion. The relative electrophoretic mobilities (REM) of LDL in the treated group were far lower than those in the control group; while during the 2,2'-amidinopropane hydrochloride(AAPH)-catalyzed oxidation of LDL, neither the lag phase nor the maximum content of the output of TBARS were significantly affected by the presence of collagen. These results suggest that collagen may decrease the susceptibility of LDL to copper-catalyzed oxidation the mechanism of which may not involve the capture of free radicals.

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In order to investigate the influence of collagen on the uptake of oxidized low density lipoprotein (ox-LDL) by macrophages, a type I collagen gel-macrophages system was established in vitro. The binding of ox-LDL, malondialdehyde (MDA)-modified LDL and acetyl LDL to collagen was higher but the binding of 4-hydroxynonenal (HNE)-modified LDL was lower than that of native LDL. When mouse peritoneal macrophages were cultivated on the collagen gel, the uptake of ox-LDL decreased dramatically. Most of the modified (125)I-LDL were bound to the collagen gel rather than degraded by macrophages. If the cells were treated with cytochalasin D (an inhibitor of non-specific phagocytosis of macrophages), the degradation of ox-LDL by cells decreased markedly in the absence of collagen. However, the degradation of ox-LDL by cells showed little change in the presence of collagen. These results suggest that the non-specific phagocytosis of ox-LDL by macrophages might be prevented when type I collagen is present.