Role of UBC9 in the Regulation of the Adipogenic Program in 3T3-L1 Adipocytes

Abstract

The small ubiquitin-like modifier-conjugating enzyme UBC9, involved in protein modification through covalent attachment of small ubiquitin-like modifier and other less defined mechanisms, has emerged as a key regulator of cell proliferation and differentiation. To explore the role of UBC9 in adipocyte differentiation, the UBC9 protein levels were examined in differentiating 3T3-L1 cells. UBC9 mRNA and protein levels were increased 2.5-fold at d 2 and then gradually declined to basal levels at d 8 of differentiation. In addition, UBC9 was expressed predominantly in the nucleus of preadipocytes but shifted to cytoplasmic compartments after d 4, after induction of differentiation. UBC9 knockdown was then achieved in differentiating 3T3-L1 preadipocytes using a specific small interfering RNA. Oil-Red-O staining demonstrated accumulation of large triglyceride droplets in approximately 90% of control cells, whereas lipid droplets were smaller and evident in only 30% of cells treated with the UBC9-specific small interfering RNA. CCAAT/enhancer-binding protein (C/EBP)-␦, peroxisome proliferator-activated receptor-␥, and C/EBP␣ mRNA levels were increased severalfold 2-6 d after induction of differentiation in control cells, whereas the expression of these transcription factors was significantly lower in the presence of UBC9 gene silencing. Adenovirus-mediated overexpression of a catalytically inactive mutant UBC9 protein in 3T3-L1 cells resulted in no changes in expression of adipogenic transcription factors and conversion to mature adipocytes as compared with control. In conclusion, UBC9 appears to play an important role in adipogenesis. The temporal profile of UBC9 induction and its ability to affect C/EBP␦ mRNA induction support a role for this protein during early adipogenesis. (Endocrinology 151: 5255-5266, 2010)

The SUMO Conjugating Enzyme Ubc9 Is Required for Inducing and Maintaining Stem Cell Pluripotency

“…Consistent with what we observed (Supporting Information Fig. S1A, S1B, S1E, S1F), two other groups have reported that Ubc9 is not essential for survival of C2C12 myoblasts [22] or 3T3-L1 preadipocytes [34]. MEFs and ESCs were transduced using lentivirus expressing the shRNA against mouse Ubc9 (shUbc9-4).…”

“…It has been shown that global sumoylation occurs during torpor in ground squirrels and that overexpression of Ubc9 in cell lines and cortical neurons protects against oxygen and glucose deprivation [37]. In addition, Ubc9 levels changes during adipocyte differentiation [34]. Therefore, these studies reiterate the dynamic and active roles of Ubc9 and sumoylation in specific but diverse cellular programs.…”

Ubc9 Impairs Activation of the Brown Fat Energy Metabolism Program in Human White Adipocytes

“…In addition, rosiglitazone stimulates robust induction of UCP1 (45,46) and acquisition of the beige phenotype in primary human subcutaneous adipocyte models, but not in 3T3-L1 cells. Therefore, a previous report indicating Ubc9 knockdown disrupts adipogenesis in 3T3-L1 cells (47) does not extrapolate to the human subcutaneous adipocytes used in our experiments. Importantly, our experiments establish that adipocyte function is likely enhanced following Ubc9 knockdown as demonstrated by the expression of adipocyte marker genes and key regulators of mitochondrial oxidative capacity.…”

Ubc9deletion in adipocytes causes lipoatrophy in mice

“…For reasons that remain unclear, Ubc9 expression peaks early in differentiation of 3T3-L1 adipocytes, when adiponectin remains low, to regulate expression of the key adipogenic transcription factors PPAR , C/EBP , and C/EBP [31]. Adiponectin expression occurs late in adipogenesis [32] and AdipoQ-Cre mediated ablation of Ubc9 occurs in relatively mature adipocytes.…”