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Author Affiliations: Nanjing Medical University Affiliated Nanjing Maternal & Children Health Hospital, Nanjing 210004, China (Fan HQ, Guo XR, Zhang M); Institute of Pediatrics, Nanjing Medical University, Nanjing 210029, China (Fan HQ, Guo XR, Chen RH, Ni YH, Wang B, Liu F, Gu N, Qiu J)
Corresponding Author: Xi-Rong Guo, PhD, Nanjing Medical University Affiliated Nanjing Maternal & Children Health Hospital, Nanjing 210004, China; Institute of Pediatrics, Nanjing Medical University, Nanjing 210029, China (Tel: 86-25-8686-2996; Email: xrguo@njmu.edu.cn)
Background: The size and number of adipocytes are believed to play a key role in the pathophysiology of obesity. The former mainly refers to the differentiation of adipocytes and the latter principally refers to the proliferation and apoptosis of adipocytes. Our cDNA microarray assay had, compared to normal rats, suggested that apoptotic protease activating factor 1 (APAF1) gene expression level was lower in the adipose tissue of diet-induced obese (DIO) rats. This study was undertaken to investigate the changes of APAF1 gene expression during the differentiation of 3T3-L1 preadipocytes (0-10 days), and to analyze the regulative role of tumor necrosis factor-alpha (TNF-¦Á) on APAF1 gene expression in matured 3T3-L1 adipocytes.
Methods: 3T3-L1 preadipocytes were cultured in vitro and differentiated into matured adipocytes by insulin, 3-isobutyl-1-methyxanthine (MIX), and dexamethasone (DEX). Oil red O dyeing was used to identify the differentiation of 3T3-L1 preadipocytes. To further evaluate the linkage between the APAF1 gene and 3T3-L1 cells differentiation/de-differentiation, TNF-¦Á which can promote the matured adipocytes to de-differentiation was incubated with 3T3-L1 matured adipocytes. TNF-alpha (1.0 ng/ml) was added into the culture medium of differentiated 3T3-L1 cells (day 10) for various periods (2, 6, 12, 24 hours). Total RNA and protein of these cells were then extracted. The level of APAF1 gene mRNA was evaluated by RT-PCR. Western blot was used to detect the protein level of APAF1.
Results: During the differentiation of 3T3-L1 preadipocytes, the down-regulation of APAF1 gene expression was significant (P<0.05). TNF-¦Á treatment significantly up-regulated the expression of the APAF1 gene in adipocytes in a time-dependent way.
Conclusions: The APAF1 gene may play a role in the differentiation of preadipocytes and TNF-¦Á related adipocytes and this finding highlights that the APAF1 gene has a potential role in the pathophysiology of obesity.
Key words: APAF1; preadipocyte; differentiation; TNF-¦Á
World J Pediatr 2007;3(4):276-281
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