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Abstract
Murine 3T3-L1 adipocytes share many similarities with primary fat cells and represent a reliable in vitro model of adipogenesis. The aim of this study was to probe the effect of S-nitrosoglutathione (GSNO) on adipocyte differentiation. Adipogenesis was induced with a mixture of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine in the absence and presence of increasing GSNO concentrations. Biochemical analysis after 7 days of differentiation showed a prominent anti-adipogenic effect of GSNO which was evident as reduced cellular triglycerides and total protein content as well as decreased mRNA and protein expression of late transcription factors (e.g. peroxisome proliferator activated receptor γ) and markers of terminal differentiation (e.g. leptin). By contrast, the nitrosothiol did not affect mRNA and protein expression of CCAAT/enhancer-binding protein β (C/EBPβ), which represents a pivotal early transcription factor of the adipogenic cascade. Differentiation was also inhibited by the NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate. Biotin switch experiments showed significantly increased S-nitrosation of C/EBPβ variants indicating that posttranslational S-nitrosative modification of this transcription factor accounts for the observed anti-adipogenic effect of NO. Our results suggest that S-nitrosation might represent an important physiological regulatory mechanism of fat cell maturation.
Highlights
Murine 3T3-L1 adipocytes share many similarities with primary fat cells and represent a reliable in vitro model of adipogenesis
MRNA levels of the proinflammatory cytokine interleukin 6 (IL-6), which was reported to be higher in preadipocytes as compared to differentiated adipocytes[16], were upregulated in a concentration-dependent manner: At the highest GSNO concentration tested, a ~10-fold increase of IL-6 mRNA was observed compared to untreated cells
While protein levels of sterol regulatory element binding protein 1 (SREBP-1), adipose triglyceride lipase (ATGL), and hormone-sensitive lipase (HSL) were decreased upon treatment of cells with the thionitrite, cellular comparative gene identification-58 (CGI-58) expression was increased in the presence of increasing GSNO concentrations
Summary
Murine 3T3-L1 adipocytes share many similarities with primary fat cells and represent a reliable in vitro model of adipogenesis. Upon experimental challenge with mitotic and adipogenic inducers (insulin, glucocorticoids, cAMP elevating compounds, and growth hormones) cells synchronously re-enter the G1 phase of the cell cycle and undergo several rounds of mitosis This so-called mitotic clonal expansion is driven by expression of oncogenes c-jun, c-fos, c-myc as well as early transcription factor CCAAT/ enhancer-binding protein subtypes β and δ (C/EBPβ and C/EBPδ)[9]. Activation of PPARγ induces expression of C/EBPα which acts anti-mitotically on the one hand and stimulates PPARγ protein biosynthesis on the other hand[10] Via this positive feedback loop adipocytes generate high levels of PPARγ and C/EBPα, which synergistically promote the terminal phase of adipogenesis that is characterized by expression of a broad range of proteins that are required for maintenance of the mature adipogenic phenotype (e.g. lipogenic and lipolytic enzymes, fatty acid binding proteins, and leptin). The aim of the present study was to investigate the effect of S-nitrosation on the adipogenic cascade and to identify additional targets of S-nitrosation in greater detail by probing the effect of S-nitrosoglutathione (GSNO) on differentiation of 3T3-L1 cells
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