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Quantitative RT-PCR was applied to measure the relative expression levels of the adipose differentiation-related protein (ADFP) gene, fatty acid transport protein 1 (FATP1) gene and apolipoprotein B (ApoB) gene in subcutaneous fat, abdominal fat, liver and muscle at five growth stages (28, 49, 70, 91 and 112 d) to determine the effect of the expression ofthese genes on fat deposition in Daweishan Mini chickens.The relative expression of ADFP gene mRNA in the abdominal fat andthe liver was significantly different between 49 d and 70 d (p

Adipose differentiation-related protein (ADFP) is a fatty acid-binding protein that can promote the absorption of long-chain fatty acids. However, few results have been published regarding its role in Yunnan Native chicken breeds. The aim of this study was to determine ADFP gene tissue-specific expression in Piao chickens (PC) and Wuliangshan black-bone chickens (WBC) by RT-qPCR. The ontogenetic expression levels of the ADFP gene were significantly different during growth and development phases in the subcutaneous fat, liver, and pectoralis muscle of PC, and in the subcutaneous fat, liver, and pectoralis muscle of WBC (p0.05). The results suggest ADFPdifferential expression in the liver and pectoral muscles of PC and WBC during the growth and development phases (p

// Zhongwei Xu 1, 7 , Linghui Zhai 1 , Tailong Yi 1, 4 , Huiying Gao 1 , Fengxu Fan 1, 4 , Yanchang Li 1 , Youliang Wang 2 , Ning Li 1 , Xiaohua Xing 1 , Na Su 1 , Feilin Wu 1 , Lei Chang 1 , Xiuli Chen 6 , Erhei Dai 6 , Chao Zhao 5 , Xiao Yang 2 , Chunping Cui 1 , Ping Xu 1, 3, 4 1 State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences Beijing, Institute of Radiation Medicine, Beijing, 102206, P.R. China 2 Beijing Institute of Bioengineering, Beijing, 100071, P. R. China 3 Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and Wuhan University School of Pharmaceutical Sciences, Wuhan, 430072, P. R. China 4 Anhui Medical University, Hefei, 230032, China 5 Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, and Research Center on Aging and Medicine, Fudan University, Shanghai, 200032, China 6 The Fifth Hospital of Shijiazhuang City, Shijiazhuang, 050021, China 7 Central Laboratory, Logistics University of Chinese People’s Armed Police Force, Tianjin, 300309, China Correspondence to: Ping Xu, email: xuping@mail.ncpsb.org Keywords: HBx, SILAM, CDC42, CFL1, ADFP Received: March 10, 2016 Accepted: September 13, 2016 Published: September 30, 2016 ABSTRACT Hepatitis B virus X protein (HBx) participates in the occurrence and development processes of hepatocellular carcinoma (HCC) as a multifunctional regulation factor. However, the underlying molecular mechanism remains obscure. Here, we describe the use of p21 HBx/+ mouse and SILAM (Stable Isotope Labeling in Mammals) strategy to define the pathological mechanisms for the occurrence and development of HBx induced liver cancer. We systematically compared a series of proteome samples from regular mice, 12- and 24-month old p21 HBx/+ mice representing the inflammation and HCC stages of liver disease respectively and their nontransgenic wild-type (WT) littermates. Totally we identified 22 and 97 differentially expressed proteins out of a total of 2473 quantified proteins. Bioinformatics analysis suggested that the lipid metabolism and CDC42-induced cytoskeleton remodeling pathways were strongly activated by the HBx transgene. Interestingly, the protein-protein interaction MS study revealed that HBx directly interacted with multiple proteins in these two pathways. The same effect of up-regulation of cytoskeleton and lipid metabolism related proteins, including CDC42, CFL1, PPARγ and ADFP, was also observed in the Huh-7 cells transfected with HBx. More importantly, CFL1 and ADFP were specifically accumulated in HBV-associated HCC (HBV-HCC) patient samples, and their expression levels were positively correlated with the severity of HBV-related liver disease. These results provide evidence that HBx induces the dysregulation of cytoskeleton remodeling and lipid metabolism and leads to the occurrence and development of liver cancer. The CFL1 and ADFP might be served as potential biomarkers for prognosis and diagnosis of HBV-HCC.

Although neutral lipid storage droplets are ubiquitous in eukaryotic cells, very little is known about how their synthesis and turnover are controlled. Adipocyte differentiation-related protein (ADRP; also known as adipophilin) is found on the surface of lipid droplets in most mammalian cell types. To learn how ADRP affects lipid storage, we stably expressed the protein in human embryonic kidney 293 (HEK 293) cells, which express little endogenous ADRP. As expected, ADRP was targeted to the surface of lipid droplets and caused an increase in triacylglycerol (TAG) mass under both basal and oleate-supplemented conditions. At least part of the increased mass resulted from a 50% decrease in the rate of TAG hydrolysis in ADRP-expressing cells. Furthermore, ADRP expression increased the fraction of total cellular TAG that was stored in lipid droplets. ADRP expression induced a striking decrease in the association of adipose triglyceride lipase (ATGL) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins. Transient expression of ADRP in two other cell lines also reduced the lipid droplet association of catalytically inactive ATGL. We conclude that the reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing HEK 293 cells.

Accumulation of neutral lipids, in particular triglycerides, in non-adipocytes is highly related to the development of insulin resistance and its consequences, type-2 diabetes and cardiovascular diseases. The accumulation of triglycerides occurs in so-called lipid droplets in the cytosol. The lipid droplet is a highly dynamic organelle consisting of a core of neutral lipids surrounded by a monolayer of amphipathic lipids and proteins. The mechanism of assembly of these droplets is poorly understood and the main aim of this thesis was to investigate this mechanism at the molecular level. Another aim was to determine the relationship between lipid storage and insulin sensitivity of the cell. In paper I, gain- and loss-of-function experiments showed that phospholipase D1 promotes the formation of lipid droplets. In addition, a cytosolic protein required for assembly of the droplets was isolated and identified as the extracellular regulated kinase 2 (ERK2). The importance of ERK2 in the formation of lipid droplets was confirmed in intact cells using gain- and loss-of-function experiments. Both PLD1 and ERK2 were shown to be necessary for the effect of insulin on lipid droplet biosynthesis. Finally, ERK2 was shown to exert its effects through phosphorylation of the motor protein dynein. Lipid droplets are formed as primordial structures with a diameter of 0.1–0.4 µm. In paper II, it was found that these primordial droplets grow in size by a fusion process that is independent of triglyceride biosynthesis. This conclusion was based on investigations in a cell-free system, on pulse-chase experiments in intact cells, and by 3D reconstructions of time-lapse studies of fluorescent droplets in intact cells. Intact microtubules and dynein were found to be essential for fusion between the droplets. The mechanism behind the fusion process was investigated further in paper III. The SNARE proteins SNAP23, VAMP4, and syntaxin5 were shown to be present on lipid droplets and to mediate their fusion. Previously described co-factors for SNARE-mediated fusion events (NSF and -SNAP) were also found to be present on droplets. It is well known that SNAP23 also mediates the insulin-stimulated fusion between transport vesicles containing the glucose transporter 4 (GLUT4) and the plasma membrane—a process that is essential for insulin-stimulated glucose uptake. Treatment of cells with oleic acid caused massive accumulation of lipid droplets, and also translocation of SNAP23 from the plasma membrane to sites within the cell, including lipid droplets. This was paralleled by an ablation of insulin-stimulated glucose uptake—an effect that was totally reversed by overexpression of SNAP23. Thus, SNAP23 may be a molecular link between insulin resistance and neutral lipid storage.