Ogg1−/− mice have an Increased Propensity to Adiposity, Especially Upon High-fat Diet Feeding
In a previous study examining potassium bromate-induced carcinogenesis in Ogg1−/−
mice, data were also presented that suggested a trend towards increased body weights in Ogg1−/−
mice, relative to wild-type (WT) counterparts that were maintained on a chow diet 
. In order to determine if body weights and body composition are indeed significantly altered in Ogg1−/−
animals, male mice were individually housed at 12 weeks of age and given ad libitum
access to either chow or a hypercaloric high-fat diet (HFD). Over the duration of the 10-week study, chow-fed WT and Ogg1−/−
mice displayed similar body weights (). HFD-feeding significantly increased weight gain in all mice, with WT mice gaining 17.5 g on average and Ogg1−/−
counterparts gaining 20.4 g over 10 weeks of feeding (). However, total weight gain over the 10-week feeding period was not significantly different between WT and Ogg1−/−
mice on either chow or HFD (). Although chow-fed Ogg1−/−
mice were not significantly heavier at the end of the feeding study, in a separate cohort of animals allowed to age to 12–15 months on a chow diet, Ogg1−/−
mice weighed 45.1±1.40 g (n
5) on average, compared to WT counterparts that weighed 34.5±0.87 g (n
In addition to body weight, body composition was also measured by NMR before and 4 weeks after the start of feeding. Fat accumulation was significantly higher in HFD-fed Ogg1−/− mice, compared to WT counterparts (). While WT mice gained 9.14 g of fat (a 23% increase) upon HFD-feeding, Ogg1−/− mice gained 12.95 g of fat (a 28% increase) in the same period (). By 12–15 months of age, body fat in the aged cohort maintained on chow diet was also significantly different, with aged Ogg1−/− animals having 32.2% of their body weight as fat mass, compared to 19.5% body fat in WT counterparts (p<0.05). These data indicate that Ogg1−/− mice have an increased propensity to adiposity with age or in response to HFD-feeding, compared to WT controls.
At the end of the feeding period, visceral and subcutaneous adipose depots were collected and weighed. Both visceral (2.66±0.12 in WT vs. 2.91±0.19 g in Ogg1−/−; p>0.05) and subcutaneous (0.92±0.09 in WT vs. 1.28±0.06 g in Ogg1−/−; p<0.05 ) adipose depots tended to be larger in HFD-fed Ogg1−/− mice, relative to WT counterparts, indicating a generalized increase in fat accumulation in these mice. Voluntary activity () and food intake were not significantly different between WT and Ogg1−/− mice either on chow (4.14±0.19 g in WT vs. 4.10±0.18 g in Ogg1−/−) or HFD (2.91±0.15 g in WT vs. 3.21±0.19 g in Ogg1−/−).
While a HFD is known to induce oxidative stress, the effect of extended high-fat feeding on expression of BER glycosylases has not been characterized. Therefore, we measured the expression of three key BER glycosylases, Ogg1, Neil1, and Nth1, in livers of chow- and HFD-fed mice (). HFD-feeding increased expression of all three glycosylases in WT mice, suggesting that the BER pathway is upregulated in response to HFD-feeding. Neil1 and Nth1 expression were increased by HFD-feeding in Ogg1−/− mice, as well. As expected, Ogg1 expression was undetectable in Ogg1−/− animals.
Ogg1−/− mice have Increased Hepatic Lipid Accumulation and Impaired Glucose Tolerance
Hepatic lipid accumulation was visualized by H&E staining, and total hepatic TG was quantified. Under chow-fed conditions, there were no significant differences in hepatic TG levels between WT and Ogg1−/− mice (). However, HFD-fed Ogg1−/− mice had a more than 2-fold higher accumulation of hepatic TG, compared to WT animals ().
Hepatic lipid accumulation and triglyceride content.
Since increased adiposity and hepatic lipid accumulation are risk factors for insulin resistance, glucose tolerance was assessed after 7 weeks of chow or HFD-feeding. After an intraperitoneal injection of glucose, chow-fed mice showed an increase in plasma glucose that returned to baseline levels by 180 minutes after injection (). As anticipated, HFD-feeding resulted in greater elevations in plasma glucose after glucose injection. While starting plasma glucose levels were comparable to WT values in HFD-fed Ogg1−/− mice, glucose clearance was significantly delayed in these animals. Plasma glucose levels at 90 and 180 minutes after glucose injection were significantly higher in HFD-fed Ogg1−/− mice, relative to WT counterparts (). Fasting plasma insulin levels were comparable in chow-fed WT and Ogg1−/− mice (). HFD-feeding increased fasting plasma insulin levels by 2.2-fold in WT mice and by 5.8-fold in Ogg1−/− animals (). In conjunction with the delayed glucose clearance (), the elevated plasma insulin levels in HFD-fed Ogg1−/− mice () indicate a significant impairment in insulin sensitivity in these animals, relative to WT controls.
Glucose tolerance and plasma insulin.
Resting Respiratory Exchange Ratio, Fasting Plasma Ketones, and Hepatic Glycogen Content are Altered in Ogg1−/− mice
In order to determine if basal metabolic rates were altered due to OGG1 deficiency, O2 consumption and CO2 production were measured in chow and HFD-fed mice. There were no significant differences in O2 consumption or CO2 production between chow-fed WT and Ogg1−/− mice (). After HFD-feeding, CO2 production during the resting phase was significantly higher in Ogg1−/− mice, relative to WT animals (). Consistently, the respiratory exchange ratio (RER), an indicator of substrate utilization, was also significantly increased during the resting phase in HFD-fed Ogg1−/− mice (), relative to WT animals, indicating a slight, but significant decrease in reliance on fatty acid oxidation for energy needs during the resting phase in these animals.
Indirect calorimetry, plasma ketones, and hepatic glycogen.
Fasting plasma ketones were measured after an overnight fast to obtain an additional measure of in vivo fatty acid oxidation. Interestingly, in chow-fed Ogg1−/− mice, fasting plasma ketones were reduced by 39% (p<0.05), relative to WT controls (). HFD-fed Ogg1−/− mice also had a similar 34% decrease (p<0.05) in fasting plasma ketones, indicating reduced rates of fatty acid oxidation in Ogg1−/− mice.
Based on the significant increase in RER (), we hypothesized that Ogg1−/− mice may preferentially utilize carbohydrate stores to meet energy needs. Therefore, hepatic glycogen content was measured and found to be diminished by 27% (p<0.05) in both chow-fed and HFD-fed Ogg1−/− mice, relative to WT counterparts (). Concomitantly, we also observed a slight, but significant increase in gene expression of two rate-limiting glycolytic enzymes, glucokinase (32%; p<0.05) and phosphofructokinase (23%; p<0.05), in livers of HFD-fed Ogg1−/− mice. Taken together with the reduced fasting plasma ketones and the significant increase in resting phase RER, these data are suggestive of a decreased reliance on fatty acids as a fuel source in Ogg1−/− mice.
Since OGG1 has both nuclear and mitochondrial localization, we sought to determine if mtDNA content or mitochondrial structure was altered in Ogg1−/−
mice. mtDNA abundance was measured by PCR and was not consistently reduced in all Ogg1−/−
mice (Supporting Figure S1A
). TEM analysis of mitochondrial density and ultrastructure in liver did not reveal differences between WT and Ogg1−/−
mice (Supporting Figure S1B
Hepatic Lipogenic Genes are not Upregulated in Ogg1−/− Livers
To gain further insight into the mechanistic changes underlying the metabolic phenotype of Ogg1−/−
animals, hepatic gene expression was assessed through a combination of high-throughput DNA microarray and quantitative real-time PCR (qPCR). Analysis of differentially expressed probe sets (DEPs) by GeneSifter revealed 26 probesets (10 upregulated, 16 downregulated) to be altered by at least 1.5 fold in chow-fed Ogg1−/−
livers, compared to WT livers (Supporting Table S1
). After 10 weeks of HFD feeding, 1572 probesets were differentially expressed (132 upregulated and 1440 downregulated) in Ogg1−/−
livers, compared to WT counterparts (Supporting Table S2
). In addition to data analysis by GeneSifter, the Affymetrix data was simultaneously submitted to Ingenuity Systems for analysis via Ingenuity iReport for Gene Expression Analysis, the results of which were over 75% concordant with the GeneSifter analyses (Supporting Tables S3
). To investigate possible biological interactions and commonalities between the differentially regulated genes, DEPs identified by GeneSifter analyses were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology terms in GeneSifter, and pathways with a z-score greater than or equal to 2.0 and less than or equal to −2.0 were considered to be enriched or underrepresented, respectively (Supporting Tables S5
Given the observations of metabolic dysfunction in Ogg1−/−
mice, the microarray data were queried for potential mechanisms that may explain these phenotypes. Since increased hepatic TG can occur secondary to increased de novo
lipogenesis, we sought to determine if hepatic lipogenic genes were differentially regulated in livers of Ogg1−/−
mice. The master regulator of lipogenic genes, sterol regulatory element binding protein-1c (SREBP-1c, gene ID: Srebf
), which also regulates its own gene expression, was increased by 1.9-fold in livers of HFD-fed Ogg1−/−
mice (Supporting Tables S2
). Interestingly, none of the classical target genes of SREBP-1c, including acetyl-CoA carboxylase (Acc
), fatty acid synthase (Fas
) or stearoyl-CoA desaturase-1 (Scd1
) were found to be upregulated by either microarray or by qPCR analyses (). In fact, all these genes were significantly lower in livers of HFD-fed Ogg1−/−
mice, relative to WT counterparts. The expression of PPAR gamma co-activator -1β (Pgc-1β
), a requisite co-activator of SREBP-1c, was also significantly lower in HFD-fed Ogg1−/−
mice, relative to WT mice (). This downregulation of Pgc1-β
may explain the lack of increase in hepatic lipogenic gene expression, despite a significant induction of Srebp-1c
. Nevertheless, based on these results, the increase in hepatic lipids in Ogg1−/−
mice () does not appear to be a consequence of increased hepatic lipogenesis.
Hepatic lipogenic and fatty acid oxidation gene expression.
Fatty Acid Oxidation and TCA Cycle Gene Expression is Reduced in Ogg1−/− Livers
Since resting phase RER was significantly higher and plasma ketones were reduced in HFD-fed Ogg1−/− mice, expression of genes involved in fat oxidation was examined. Consistent with a reduction in fatty acid oxidation, the microarray data revealed that several genes involved in fatty acid oxidation were significantly downregulated in HFD-fed Ogg1−/− livers, relative to WT counterparts (). Calcium-binding protein 39-like (Cab39l; also termed mouse protein 25 beta), electron-transferring-flavoprotein dehydrogenase (Etfdh), long-chain acyl-coenzymeA (CoA) synthetase isoforms 5 (Acsl5), AMP-activated protein kinase alpha subunit 2 (AMPKα2), PPAR-gamma coactivator -1 alpha (Pgc-1α), medium chain acyl-Coenzyme A dehydrogenase (Acadm), and peroxisomal enoyl-CoA delta isomerase (Peci), were all significantly downregulated by at least 1.5-fold in HFD-fed Ogg1−/− livers (). Furthermore, Pgc-1α, a key regulator of fatty acid oxidation gene expression in liver, was significantly lower in livers of chow-fed (), as well as HFD-fed ( and ) Ogg1−/− mice. Concomitant with the reduction in Pgc-1α, additional key genes of fatty acid oxidation, including carnitine palmitoyl transferase-1 (Cpt-1), acyl CoA oxidase (Aox), and peroxisome proliferator-activated receptor alpha (Pparα)? were also decreased in HFD-fed Ogg1−/− mice, relative to WT counterparts ().
In addition to fatty acid oxidation, PGC-1α also regulates genes involved in TCA cycle metabolism 
. Consistent with the reduction in Pgc-1α
levels ( and ), several genes involved in the TCA cycle, including dihydrolipoamide dehydrogenase (Dld
), malate dehydrogenase 1 (Mdh1
), and fumarate hydratase (Fh) were all reduced by at least 1.5 fold in HFD-fed Ogg1−/−
mice, as indicated by microarray analysis (). Additional genes involved in TCA cycle metabolism including isocitrate dehydrogenase 3 (Idh3a
), pyruvate dehydrogenase E1 alpha 1 (Pdha1
), succinate-Coenzyme A ligase (Sucla2
), aconitase 1 (Aco1
), succinate dehydrogenase complex, subunit A (Sdha
) were all downregulated by at least 20% in HFD-fed Ogg1−/−