The Roles Of CIDE-3 In Differentiation And Metabolism Of Adipocyte

Adipose tissue is an important place for energy storage and expenditure in the body and the differentiation and metabolism disorders of adipocytes can lead to obesity. These years obesity is becoming a worldwide health problem, while obesity is the risk factor of a variety of chronic diseases such as diabetes, cardio-cerebral vascular diseases, et al. Therefore, it is of great significance to study the pathogenesis for the prevention and treatment of obesity and its related diseases.CIDE(cell death-inducing DFF45-like effector) family is a group of genes found in the late 20th century, which could induce apoptosis. However, it was recently found that these family members and the occurrence of obesity were closely related. The member Fsp27 of CIDE family is a fat-specific protein in mice, and it is only expressed in the mature adipocytes. Fsp27 located in lipid droplets and could inhibite the accumulation of fat and promote lipolysis. CIDE-3 in the human being is the homolog of Fsp27, and it is mainly distributed in the small intestine, heart, colon and stomach and is expressed at low level in the brain, kidney and liver. So CIDE-3 is not a fat specific potein. Nevertheless, the biological function of CIDE-3 in the the human body is not very clear.In this study, we constructed EGFP-CIDE-3 and DsRed1-CIDE-3 fusion gene eukaryotic expression vectors and determined the subcellular localization of CIDE-3 protein in COS-7 cells by transfecting these recombinant vectors. Meanwhile we constructed the recombinant adenoviruses respectively carrying EGFP-CIDE-3 and DsRed1-CIDE-3 fusion gene. In addition, the preadipocyte isolation and primary culture and the over-expression CIDE-3 by recombinant adenovirus were performed in order to explore mechanism of the adipocyte differentiation and metabolism and the role of CIDE-3 in this process.【Objectives】1. To determine the subcellular localization of CIDE-3 protein;2. To construct the recombinant adenovirus carrying fusion gene EGFP- CIDE-3 or DsRed1-CIDE-3;3. To study the role of CIDE-3 in differentiation and metabolism of adipocyte【Methods】1. Recombinant eukaryotic expression vector pShuttle-CMV-EGFP- CIDE-3 or pShuttle-CMV-DsRed1-CIDE-3 was constructed and then was transfected into COS-7 cells by lipofectamine 2000 determine the subcellular localization of CIDE-3 protein;2. Recombinant adenovirus carrying EGFP-CIDE-3 and DsRed1-CIDE-3 were constructed by AdEasyTM XL system;3. Human preadipocytes were isolated and cultured from fat tissue obtained by liposuction;4. The growth curve of preadipocytes was determined by MTT; 5. The surface markers of preadipocytes were measured using flow cytometry to identify the cell type;6. The over-expression of CIDE-3 was performed by recombinant adenovirus carrying CIDE-3 to explore the role of CIDE-3 in differentiation and metabolism of adipocytes.【Results】1. CIDE-3 protein mainly located in the surface of lipid droplets, and partly in endoplasmic reticulum.At the beginning, recombinant plasmid pShuttle-CMV-EGFP-CIDE-3 and pShuttle-CMV-DsRed1-CIDE-3 was constructed and was confirmed by digestion and sequencing analysis. In order to study the subcellular localization of CIDE-3 protein, we transfected the recombinant plasmids to COS-7 cells by lipofectamine. And GFP-CB5 (Cytochrome B5, known to the endoplasmic reticulum targeting) was used as the ER marker and GFP-ADRP as the marker of lipid droplets. Neutral fat was stained by Bodipy 493/503, and mitochondria were stained by MitoTracker Red CMXRos, and nucleus was stained by Hoechst 33258. It was observed that: CIDE-3 and endoplasmic reticulum markers CB5 partly showed co-localization and CIDE-3 located in the mesh structure of GFP-CB5; CIDE-3 located in the surface of lipid droplets and co-located with ADRP; CIDE-3 was not found the distribution in mitochondria.2. Recombinant adenovirus carrying fusion genes containing CIDE-3 was successfully constructedWe used AdEasyTM XL system to construct recombinant adenovirus carrying the gene CIDE-3. The main procedures included cloning the interesting gene, constructing the recombinant shuttle plasmid, obtaining the recombinant adenovirus plasmid by homologous recombination in E. coli BJ5183 and transfecting the recombinant adenovirus plasmid into packaging cells such as AD293 to produce recombinant adenovirus carrying CIDE-3 gene. It was confirmed that recombinant adenovirus Ad-EGFP-CIDE-3 and Ad-DsRed1- CIDE-3 was constructed successfully by PCR amplification of CIDE-3 gene and observing the expression of fusion protein under the inverted fluorescence microscope.3. CIDE-3 could inhibit the oxidation of fatty acids and promote the maturation of lipid dropletsWe isolated and cultured preadipocytes from the fat emulsion obtained by liposuction and measured the growth curve of these preadipocytes. They were confirmed as preadipocyte according to three typical characteristics proposed by the literatures: 1) They are isolated from the adipose tissue, spindle-shaped, and have sparse cytoplasmic lipid inclusions; 2) They have similar replicative rate with fibroblasts; 3) Upon reaching monolayer confluency, they acquire many lipid inclusions and become rounder. At the same time, we found that CD markers detected by flow cytometry of the isolated cells were consistent with the previous reports.In the subsequent experiment, we induced the differentiation of preadipocytes towards adipocytes. And we observed the number and size of the lipid droplet under fluorescence microscope after adding recombinant adenovirus Ad-EGFP-CIDE-3 and control adenovirus Ad-EGFP into the culture medium. It was found that the cells infected by Ad-EGFP-CIDE-3 accumulated more and larger lipid droplets and showed higher rate of triglyceride synthesis than those infected by Ad-EGFP. So CIDE-3 could inhibit the secretion and promote the accumulation of triglyceride to accelerate the maturation of lipid droplets, which was similar as FSP 27.【Conclusions】 The study found that CIDE-3 protein located in the surface of lipid droplets and partly in endoplasmic reticulum. So it is a surface protein of lipid droplets. Human CIDE-3 protein could promote the maturation of lipid droplets by inhibiting the oxidation of fatty acids during the preadipocyte differentiation.