A Novel Role for the DNA Repair Enzyme 8-Oxoguanine DNA Glycosylase in Adipogenesis

Abstract

Cells sustain constant oxidative stress from both exogenous and endogenous sources. When unmitigated by antioxidant defenses, reactive oxygen species damage cellular macromolecules, including DNA. Oxidative lesions in both nuclear and mitochondrial DNA are repaired via the base excision repair (BER) pathway, initiated by DNA glycosylases. We have previously demonstrated that the BER glycosylase 8-oxoguanine DNA glycosylase (OGG1) plays a novel role in body weight maintenance and regulation of adiposity. Specifically, mice lacking OGG1 (Ogg1^-/-) are prone to increased fat accumulation with age and consumption of hypercaloric diets. Conversely, transgenic animals with mitochondrially-targeted overexpression of OGG1 (Ogg1^Tg) are resistant to age- and diet-induced obesity. Given these phenotypes of altered adiposity in the context of OGG1 genotype, we sought to determine if OGG1 plays a cell-intrinsic role in adipocyte maturation and lipid accumulation. Here, we report that preadipocytes from Ogg1^-/- mice differentiate more efficiently and accumulate more lipids than those from wild-type animals. Conversely, OGG1 overexpression significantly blunts adipogenic differentiation and lipid accretion in both pre-adipocytes from Ogg1^Tg mice, as well as in 3T3-L1 cells with adenovirus-mediated OGG1 overexpression. Mechanistically, changes in adipogenesis are accompanied by significant alterations in cellular PARylation, corresponding with OGG1 genotype. Specifically, deletion of OGG1 reduces protein PARylation, concomitant with increased adipogenic differentiation, while OGG1 overexpression significantly increases PARylation and blunts adipogenesis. Collectively, these data indicate a novel role for OGG1 in modulating adipocyte differentiation and lipid accretion. These findings have important implications to our knowledge of the fundamental process of adipocyte differentiation, as well as to our understanding of lipid-related diseases such as obesity.

Keywords: DNA repair; adipocyte differentiation; base excision repair; lipid accretion; obesity.

Figure 1

OGG1 expression and activity are regulated during adipogenesis. 3T3-L1 cells were differentiated using MDI. Cells were harvested, and RNA and proteins were isolated at indicated time points. (A) Gene expression of Ogg1 was measured by qRT-PCR using 18S rRNA as a control. (B) OGG1 protein expression was analyzed by Western blotting. (C,D) Proteins isolated at the indicated time points were used for the OGG1 activity assay. Data are expressed as average ±  SEM and represent at least 3 independent replicates per time point. * p  <  0.05 vs. 0 h.

Figure 2

Primary adipocytes from Ogg1^−/− mice accumulate more lipid than WT cells. Preadipocytes isolated from WT and Ogg1^−/− mice were differentiated using MDI. (A–C) On the 6th day of differentiation, cells were stained with Oil Red O, and RNA and protein were isolated for (D) gene expression analyses and (E) immunoblotting. The scale bar in Figure 2B represents 130 µm. Data are expressed as average ±  SEM and represent at least 3 independent replicates per time point. * p  <  0.05 vs. 0 h.

Figure 3

Primary adipocytes from Ogg1^Tg mice accumulate less lipid than WT cells. Preadipocytes isolated from WT and Ogg1^Tg mice were differentiated using MDI. On the 8th day of differentiation, (A–C) cells were stained with Oil Red O, and RNA and protein were isolated for (D) gene expression analyses and (E) immunoblotting. The scale bar in Figure 3B represents 130 µm. Data are expressed as average ±  SEM and represent at least 3 independent replicates per time point. * p  <  0.05 vs. 0 h.

Figure 4

Overexpression of MTS-hOGG1a impairs adipogenic differentiation in 3T3-L1 cells. 3T3-L1-CARΔ cells were transduced with adenovirus particles to overexpress either GFP or MTS-hOGG1-1a and differentiated using MDI. (A) OGG1 expression was assessed by immunoblotting at the indicated time points. On the 8th day of differentiation, (B,C) cells were stained with oil Red O, and RNA was isolated for (D) gene expression analyses. (E) Proteins were isolated at the indicated time points for immunoblotting. Data are expressed as average ±  SEM and represent at least 3 independent replicates per time point. * p  <  0.05 vs. 0 h.

Figure 5

OGG1 genotype alters PARylation of cellular proteins in adipocytes and adipose tissue. Proteins were isolated from 3T3-L1-CARΔ cells transduced with GFP or MTS-hOGG1-1a for detection of (A) PARP1 and (B) total cellular PARylation by immunoblotting. Cellular PARylation was assessed in protein extracts from differentiated primary adipocytes and adipose tissue from (C) WT vs. Ogg1^Tg animals and (D) from WT vs. Ogg1^−/− mice. Data are representative of at least 3 independent replicates for cell studies and 4–6 age- and sex-matched animals per genotype for tissue extracts. * p  <  0.05 vs. 0 h.