Suppression of Glutamine:Fructose‐6‐phosphate amidotransferase‐1 inhibits adipogenesis in 3T3‐L1 adipocytes

Abstract

O-linked N-acetylglucosamine (O-GlcNAc) protein modification has been implicated in the regulation of signaling pathways, cell function, and gene expression. Glutamine:fructose-6-phosphate amidotransferase-1 (GFAT-1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway (HBP), which generates the sugar nucleotide UDP-GlcNAc, where this nucleotide acts as the donor for O-GlcNAc modification. In this study, we determined whether GFAT-1 regulates adipogenesis in adipocytes. 3T3-L1 preadipocytes were differentiated using medium containing high glucose, insulin, dexamethasone, and isobutylmethylxanthine. Cells were harvested 4, 8, and 12 h and 1, 2, 3, 4, 6, and 8 days after the initiation of differentiation. Global level of O-GlcNAc modification increased 4 h after induction and persisted for 8 days of observation. GFAT-1 mRNA and protein expression was also upregulated beginning 4 h after induction. Pharmacological inhibition of GFAT-1 or GFAT-1 siRNA treatment blocked the increase in O-GlcNAcylation and the formation of lipid droplets in adipocytes. GFAT-1 may regulate the expression of C/EBPβ, PPARγ, SREBP-1, fatty acid synthase, S3-12, perilipin, or adipophilin during adipogenesis. Our results suggest that GFAT-1 plays a critical role in modulating adipogenesis via the regulation of protein O-GlcNAcylation in adipocytes.

Functional O-GlcNAc modifications: Implications in molecular regulation and pathophysiology

“…As expected, an increase in OGT and GFAT-1 protein levels, as well as GFAT-1 mRNA, directly correlate with observed O-GlcNAc elevations (Hsieh et al , 2012; Ishihara et al , 2010). OGT and GFAT inhibition decreases O-GlcNAc levels and prevents preadipocyte differentiation in 3T3-L1 cells (Hsieh et al , 2012). …”

“…It has since been identified as one of the main transcriptional regulatory modifications dictating adipocyte differentiation. Studies using the 3T3-L1-adipocyte cell line reveal protein O-GlcNAcylation increases during adipocyte differentiation (Hsieh et al , 2012; Ishihara et al , 2010). As expected, an increase in OGT and GFAT-1 protein levels, as well as GFAT-1 mRNA, directly correlate with observed O-GlcNAc elevations (Hsieh et al , 2012; Ishihara et al , 2010).…”

“…Studies using the 3T3-L1-adipocyte cell line reveal protein O-GlcNAcylation increases during adipocyte differentiation (Hsieh et al , 2012; Ishihara et al , 2010). As expected, an increase in OGT and GFAT-1 protein levels, as well as GFAT-1 mRNA, directly correlate with observed O-GlcNAc elevations (Hsieh et al , 2012; Ishihara et al , 2010). OGT and GFAT inhibition decreases O-GlcNAc levels and prevents preadipocyte differentiation in 3T3-L1 cells (Hsieh et al , 2012).…”

“…C/EBPα and C/EBPβ are critically important for controlling adipocyte differentiation (Christy et al , 1991; Mandrup & Lane, 1997; Yeh et al , 1995) and respond directly to changes in O-GlcNAc (Figure 3) (Hsieh et al , 2012; Ishihara et al , 2010; Li et al , 2009b; Maury et al , 2013). Elevating O-GlcNAc levels increases C/EBPα expression along with another adipose-related mesoderm marker, PPARg, during differentiation (Maury et al , 2013).…”

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

“…Techniques such as over expression of OGT (Zachara et al 2004b), OGlcNAcase (Slawson et al 2005), and GFAT (Chen et al 1997;Marshall et al 2004;James et al 2000) by both transient transfection and viral transduction have been successful, as has reducing the expression of these enzymes using RNA interference (Zachara et al 2004b;Ngoh et al 2009a;Hsieh et al 2012). OGT has also been overexpressed using a tetracycline-inducible OGT stably expressed in HeLa cells, although for effective overexpression a histone deacetylase inhibitor was included with the tetracycline for appropriate regulation of OGT (Marshall et al 2003).…”