Ablation of XP-V causes adipose tissue PNAS PLUS senescence and metabolic abnormalities

Yih-Wen Chena, Robert A. Harrisb, Zafer Hatahetc, and Kai-ming Choua,1

aDepartment of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202; bRichard Roudebush Veterans Affairs Medical Center and the Department of and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202; and cDepartment of Biological and Physical Sciences, Northwestern State University of Louisiana, Natchitoches, LA 71497

Edited by James E. Cleaver, University of California, San Francisco, CA, and approved June 26, 2015 (received for review April 12, 2015) Obesity and the metabolic syndrome have evolved to be major DNA η (pol η) is a specialized lesion bypass poly- health issues throughout the world. Whether loss of genome merase that faithfully replicates across UV-induced cyclobutane integrity contributes to this epidemic is an open question. DNA dimers (9) to rescue stalled DNA replication forks polymerase η (pol η), encoded by the from potential breakages and mutations. Defects in the gene (XP-V) gene, plays an essential role in preventing cutaneous cancer encoding pol η produce a variant form of the autosomal recessive caused by UV radiation-induced DNA damage. Herein, we demon- disease xeroderma pigmentosum (XP-V) (9). Patients with XP-V strate that pol η deficiency in mice (pol η−/−) causes obesity with are highly sensitive to sunlight and prone to cutaneous cancer (9). visceral fat accumulation, hepatic steatosis, hyperleptinemia, In addition to skin, pol η is expressed in most tissues (10). The hyperinsulinemia, and glucose intolerance. In comparison to WT expression of pol η correlates with the effectiveness of anticancer − − mice, adipose tissue from pol η / mice exhibits increased DNA therapeutic agents that exert their activity by introducing DNA damage and a greater DNA damage response, indicated by up- lesions that block the progression of DNA replication (11). In regulation and/or phosphorylation of ataxia telangiectasia mu- addition to exogenous introduced DNA lesions, pol η contributes tated (ATM), phosphorylated H2AX (γH2AX), and poly[ADP-ribose] to genomic stability during unperturbed DNA replication (12) and polymerase 1 (PARP-1). Concomitantly, increased cellular senes- replicates across reactive oxygen species (ROS)-induced oxidative cence in the adipose tissue from pol η−/− mice was observed and DNA lesions 8-oxoG (7,8-dihydro-8-oxoguanine), glycol, GENETICS measured by up-regulation of senescence markers, including p53, and lipid peroxidation DNA adducts generated during endoge- p16Ink4a, p21, senescence-associated (SA) β-gal activity, and SA se- nous metabolic processes (13, 14). ROS generated from endoge- cretion of proinflammatory cytokines interleukin 6 (IL-6) and tu- nous metabolism or exogenous sources has been associated with mor necrosis factor α (TNF-α) as early as 4 wk of age. Treatment of cancer, aging, and the metabolic syndrome. − − pol η / mice with a p53 inhibitor, pifithrin-α, reduced adipocyte Therefore, the initial hypothesis for our studies was that pol η senescence and attenuated the metabolic abnormalities. Further- contributes to reduce tumorigenesis by managing oxidative DNA more, elevation of adipocyte DNA damage with a high-fat diet or lesions in other organs. However, in a 2-y study, no increase in the − − sodium arsenite exacerbated adipocyte senescence and metabolic incidence of tumors of any type was found with pol η KO (pol η / ) − − − − abnormalities in pol η / mice. In contrast, reduction of adipose mice (15). Instead, we found pol η / mice develop metabolic DNA damage with N-acetylcysteine or metformin ameliorated cel- abnormalities that induce obesity. lular senescence and metabolic abnormalities. These studies indi- cate that elevated DNA damage is a root cause of adipocyte Results and Discussion senescence, which plays a determining role in the development Metabolic Abnormalities in Pol η Mice. Compared with littermate WT − − of obesity and insulin resistance. mice, pol η / mice gained more weight with age, particularly after 48 wk, with no genotype difference in food or water consumption DNA polymerase η | obesity | senescence | DNA damage | adipose tissue Significance he is constantly challenged by exogenous and Tendogenous DNA damaging agents. To ensure genome in- The metabolic syndrome has evolved to be a major health issue tegrity, human cells have faithful DNA replication machinery globally. The association between genome integrity and meta- and DNA repair systems that are coordinated by DNA damage bolic abnormalities is not well understood. Our results indicate response networks. In response to different extents or types of that increased DNA damage and persistent activation of the DNA lesions, the DNA damage response activates appropriate DNA damage response induce adipocyte senescence in DNA cellular responses, including transient or permanent (senes- polymerase η knockout (pol η−/−) mice. Suppression of adipocyte cence) cell cycle arrest, or apoptosis, to minimize the detrimental senescence with a p53 inhibitor, pifithrin-α, alleviated metabolic − − effects of DNA lesions (1). Reduction or deficiency in DNA abnormalities in pol η / mice.AnincreaseordecreaseinDNA repair/replication enzyme activity is well documented to increase damage affected the senescence status of adipocytes accord- vulnerability for the development of cancer, neurodegenerative ingly, which was also in concordance with the severity of met- −/− diseases, and aging (2). In addition, defective DNA repair en- abolic abnormalities in the pol η mice. Our current results zymes are associated with the metabolic symptom; for example, indicate that reduced genome integrity plays a causative role in DNA glycosylase (Neil1)- and OGG1-deficient mice are obese provoking adipocyte senescence that leads to development of (3–5), and excision repair ERCC1-XPF de- obesity and insulin resistance. ficiency causes lipodystrophy (6). Furthermore, DNA damage Author contributions: Y.-W.C., R.A.H., Z.H., and K.-m.C. designed research; Y.-W.C., Z.H., response protein ataxia telangiectasia mutated (ATM) sup- and K.-m.C. performed research; Y.-W.C., R.A.H., and K.-m.C. analyzed data; and Y.-W.C., presses JNK activity through p53 signaling and mediates an an- R.A.H., and K.-m.C. wrote the paper. tioxidant action that has been suggested to be relevant to the The authors declare no conflict of interest. metabolic syndrome (7). Nucleotide excision repair XP-A pro- This article is a PNAS Direct Submission. tein may affect metabolism by altering mitochondrial function 1To whom correspondence should be addressed. Email: [email protected]. (8). To date, the mechanisms that link genome instability and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. metabolic dysregulation have not been elucidated. 1073/pnas.1506954112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1506954112 PNAS Early Edition | 1of9 Downloaded by guest on September 27, 2021 Fig. 1. Pol η−/− mice develop metabolic abnormalities. (A) Autopsy of WT and pol η−/− mice at 4 (4W) and 72 (72W) wk of age; (B) body weight gain (n = 12); (C) body fat percentages (n = 12); (D) circulating leptin and (E) insulin levels; (F) HOMA-IR (n = 8); (G) glucose tolerance test (GTT) with area under curve (AUC) + + (n = 8); (H) liver stained with Oil Red O (Scale bars: 50 μM); (I) H&E staining of adipose macrophage infiltration; (J) F4/80 /CD11b -positive cells (n = 6) in adipocytes from mice at 72 wk (w) of age; (K) IL-6; (L) TNF-α;(M) adiponectin in plasma (n = 8); (N) H&E staining of visceral fat histological sections; (O) quantitation of adipocyte size (n = 400); and (P) expression of PPAR-γ, SREBP1-p125, and SREBP1-p68 subunits. Histological pictures and blots are repre- sentative of six independent experiments. The graphical data, which are shown as the mean ± SEM, were analyzed by the Student t test (*P < 0.05).

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1506954112 Chen et al. Downloaded by guest on September 27, 2021 (Fig. 1 A–C and Fig. S1A). Autopsies revealed increased visceral fat SREBP1 and PPAR-γ (18) (Fig. 1P and Fig. S2B). The PNAS PLUS − − encasing the abdominal organs of pol η / mice (Fig. 1A). By 72 wk, administration of diets high in fat or fructose promoted greater − − − − pol η / mice had gained an average of 55% more weight than WT obesity and exacerbated the metabolic abnormalities in the pol η / littermates (14.0 g vs. 9.0 g, respectively; Fig. 1B), with 37.5% more mice relative to their WT littermates (Fig. S3), which further in- − − − − body fat (38.5% vs. 28.0%, respectively; Fig. 1C). Pol η / mice also dicated that pol η / mice are prone to the development of meta- developed hyperleptinemia (Fig. 1D), hyperinsulinemia (Fig. 1E), bolic abnormalities. Together, these characteristics of obesity with higher blood glucose (Fig. S1B), higher homeostasis model assess- concomitant development of glucose intolerance and reduced − − ment of insulin resistance (HOMA-IR) (Fig. 1F), reduced energy insulin sensitivity in the pol η / mice suggest a preventive role expenditure (Fig. S1C), and reduced glucose tolerance (Fig. 1G) for pol η in the onset of metabolic abnormalities. compared with WT littermates. Hepatic steatosis was also appar- − − − − ent in the pol η / mice, as evidenced by enlarged and light- Increased DNA Breaks and DNA Damage Response in Pol η / Mice. colored livers (Fig. 1A), increased micro- and macrovesicles (Fig. To our knowledge, there is no information in the literature as to S1D), and increased Oil Red O staining (Fig. 1H). how a deficiency of a DNA lesion bypass polymerase causes Infiltration of macrophages into adipose tissue is an established metabolic abnormalities. Autopsy and histopathological analysis characteristic of obesity (16), which was apparent in the adipose indicated that visceral adipose tissue accumulation was the most − − − − tissue of pol η / mice, as evidenced by crown-like structures in prominent change in major organs between pol η / and WT + histological sections (Fig. 1I) and an increased number of F4/80 / mice (Fig. 1 A–C). In addition to its vital role in energy storage, + CD11b -positive cells compared with WT mice (17) (Fig. 1J). adipose tissue plays essential roles in the immune response and Consistent with these findings, the inflammatory cytokines IL-6, the secretion of hormones and cytokines. Adipose tissue is TNF-α, and monocyte chemoattractant protein-1 (MCP-1) were closely associated with metabolic dysfunction, inflammation, and increased and the antiinflammatory cytokine adiponectin (16) was aging-related health issues. decreased in the plasma and adipocytes (Fig. 1 K–M and Fig. S2A) Previous studies demonstrated that ROS are selectively in- − − of pol η / mice. creased in the adipose tissue in obese mice (19), which implies a − − The adipocytes from pol η / mice developed hypertrophy with higher amount of DNA damage in adipose tissue and a potential age (4.6-fold larger than WT at 72 wk old; Fig. 1 N and O) and linkage between oxidative DNA damage, fat accumulation, and exhibited elevated expression of the master adipogenic regulator obesity. However, it is not clear whether increased DNA damage GENETICS

Fig. 2. Elevated DNA damage and DNA damage responses in the adipocytes from pol η−/− mice. (A) Alkaline comet assay. (Scale bars: 100 μM.) (B) Quan-

titation of comet assay (n = 200). (C) Immunofluorescence staining of phosphorylated γH2AX. (Scale bars: 25 μM.) DNA damage protein expression (D)and − − − − quantitation (E) in WT, pol η / adipocyte control (Ctl), and pol η / adipocytes treated with NAC or Met (n = 6). The data, which are shown as the mean ± SEM, were analyzed by one-way ANOVA (*P < 0.05).

Chen et al. PNAS Early Edition | 3of9 Downloaded by guest on September 27, 2021 can be a causal factor for obesity development. We therefore 20 wk compared with the regular chow diet after 72 wk in mice of postulated that increased genome instability in adipose tissues, both genotypes (Fig. S6). due to pol η ablation, leads to the development of the observed Adipose tissue consists of a complex mixture of adipocytes, − − metabolic disorders in pol η / mice. To test this hypothesis, an preadipocytes, macrophages, neutrophils, lymphocytes, endothe- alkaline comet assay was performed, and, indeed, the results lial cells, and stem cells (25). The differentiation of preadipocytes − − indicated greater DNA damage in pol η / adipocytes as early as into mature adipocytes plays a key role in the development of − − 4 wk of age compared with pol η / adipocytes from WT mice of obesity (25). Because the mature adipocytes are responsible for the same age (Fig. 2 A and B). These results were further sup- lipid storage, we hypothesized that pol η deficiency induces ported by increased staining of 8-oxoG (13) and a higher amount senescence mainly in the mature adipocytes of adipose tissue. of DNA double-strand breaks, as indicated by phosphorylated To test this hypothesis, we monitored the SA–β-gal activity −/− γH2AX (20) in the adipose tissue from pol η mice (Fig. 2 C–E through the differentiation of preadipocytes to mature adipocytes. and Fig. S4). Concurrently, up-regulation of DNA damage re- Whereas no apparent SA–β-gal activity was detected in pre- sponse , including ATM, poly[ADP-ribose] polymerase 1 adipocytes from mice of both genotypes, differentiation of these (PARP-1), p53, and p21, at the protein, mRNA, and phos- cells into mature adipocytes resulted in increases in SA–β-gal ac- − − − − phorylation levels, was observed in adipocytes from pol η / mice tivity and lipid accumulation with preadipocytes from pol η / (Fig. 2 D and E and Fig. S5A). mice (Fig. 3 E and F). These findings suggest that mature adi- Activation of the DNA damage response has been demon- pocytes are the cells that become senescent and accumulate lipid − − strated to up-regulate the inflammatory response through the in the adipose tissue of pol η / mice, suggesting senescent adi- master regulator of inflammation gene NF-κB (21). The ob- pocytes play a causative role in the development of obesity in − − − − served up-regulation of ATM, p53, and PARP-1 in the pol η / pol η / mice. mice may induce up-regulation of NF-κB via IL-6 and TNF-α (21), which would provide an explanation for the elevated in- Suppression of Adipocyte Senescence Alleviates Metabolic Abnormalities. − − flammatory responses observed in pol η / mice (Fig. 1 J and K). Pol η has the ability to handle various oxidative DNA lesions, in- Indeed, expression of NF-κB (p65) at both the protein and cluding lipid peroxidation products that impede the progression of − − mRNA levels was higher in adipocytes from pol η / mice (Fig. 2 DNA replication (13, 14). Both pol η and p53 are required for re- D and E and Fig. S5A). Concomitantly, the expression of IκBα, covery from DNA damage-stalled DNA replication forks (27). In an inhibitor of NF-κB (22), was reduced (Fig. 2 D and E). These the pol η-deficient XP-V cells, p53 plays a major role in mediating findings indicate that ablation of pol η increases the amount of the downstream cellular events in response to DNA damage (27). DNA damage and DNA damage response, which contributes to The observed up-regulation and activation of p53 (Fig. 2 D and E)in − − − − the elevated inflammatory responses of pol η / mice. In addi- the adipose tissue of pol η / mice also support adipose senescence tion, IL-6 and TNF-α increase oxidative stress, which is likely to results (Fig. 3 A and B). More importantly, p53 activation is essential induce additional DNA damage (16), and subsequently propa- for the establishment of cell senescence in adipose tissue and re- gate a vicious cycle between DNA damage and inflammation. quired for the development of insulin resistance (28). We therefore hypothesize that suppression of p53 activity will be a reasonable − − DNA Damage-Mediated Adipocyte Senescence in Pol η / Mice. We approach to examine the importance of adipocyte senescence in the next addressed how elevated DNA damage and inflammatory development of metabolic abnormalities. This hypothesis was tested responses lead to the observed metabolic abnormalities in pol with the p53 inhibitor pifithrin-α (PFT-α) (29). PFT-α treatment − − − − η / mice. As shown in Fig. 2, adipocytes from pol η / mice exhibited a more significant inhibitory effect on senescence in the − − exhibited a persistently higher level of DNA damage and DNA adipose tissue of pol η / mice (Fig. 3 G–I) compared with WT mice. damage response in contrast to WT mice. Persistent DNA Similarly, PFT-α also has a more profound impact in reducing the damage is causally involved in cellular senescence (23) and in the body weight, body fat accumulation, and circulating levels of insulin, − − senescence-associated secretory phenotype (SASP) that results and it caused a marked improvement in glucose tolerance in pol η / in the secretion of inflammatory cytokines IL-6, TNF-α, and mice (Fig. 3 J–M). These results demonstrate the importance of p53 MCP-1 (24). The observed adipocyte hypertrophy; up-regulation activation in mediating adipocyte senescence and suggest a critical of p53 and p21; and higher circulating levels of IL-6, TNF-α, and role of adipocyte senescence in the observed metabolic abnormali- − − − − MCP-1 observed with pol η / mice are established characteris- ties in pol η / mice. tics of senescence (Figs. 1 J, K, M, and N and 2 D and E and Fig. S5A). A previous report indicated that obese human subjects Magnitude of the DNA Damage Affects Adipocyte Senescence have more senescent cells in their s.c. adipose tissue (25), im- and Severity of Metabolic Abnormalities. To test further whether plying a correlation between adipocyte senescence and obesity. DNA damage is causative for the development of metabolic − − To determine the senescent status in the adipose tissues, two abnormalities in pol η / mice, the antioxidant N-acetylcysteine cellular senescence markers, senescence-associated (SA) β-gal (NAC) was administered to mice as a way to reduce oxidative activity and p16Ink4a expression, were examined (25). Notably, as stress and oxidative DNA lesions (30). NAC significantly re- early as 4 wk of age, which is before the onset of obesity, and up duced the amount of DNA damage, as measured by the comet −/− to 72 wk of age, the adipose tissue of pol η mice displayed assay and the activation of ATM, γ-H2AX, p53, p21, and NF-κB. significantly greater amounts of SA–β-gal activity and p16Ink4a NAC also attenuated adipocyte senescence and secretion of expression compared with WT mice (Fig. 3 A–D). These data SASP IL-6, TNF-α, and MCP-1 (Fig. 2 B, D, and E and Fig. indicate that senescence in adipose tissue precedes the devel- S5A), and reduced adipocyte hypertrophy. Moreover, with sim- − − opment of metabolic abnormalities in pol η / mice. Concomi- ilar uptake (Fig. S5B), NAC attenuated and suppressed the − − tantly, reduced expression of the proliferation marker Ki-67 and metabolic abnormalities in pol η / mice with no apparent im- apoptosis markers Bax, Noxa, and Puma (26) was observed (Fig. pact on the WT mice (Fig. S5 C–H). The latter is likely due to a 3 C and D), suggesting decreased proliferation and decreased low amount of endogenous DNA damage in the WT mice fed a − − apoptosis in the adipose tissue from pol η / mice compared with regular chow diet. Similarly, administration of metformin (Met), WT. The role of DNA damage and adipose tissue cell senes- the drug used to treat diabetes (31), also suppressed the activation cence on metabolic dysfunction was further supported by the of the DNA damage response, the increase in inflammatory fac- − − exacerbation of the metabolic abnormalities with a high-fat diet tors, and the metabolic abnormalities in the pol η / mice, perhaps study. The high-calorie diet produced a similar amount of DNA because it enhances antioxidant defenses and reduces inflamma- damage and prominent elevation of SA–β-gal activity within tion (32) (Fig. 2 D and E and Fig. S5). Furthermore, both NAC

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− − Fig. 3. Cellular senescence in adipose tissues and metabolic abnormalities. (A and B)SA–β-gal activity and quantitation from WT or pol η / mice at different ages (n = 8). (Scale bar: 1 cm.) (C and D) Expression of senescent and apoptotic proteins and quantitation (n = 6). PFT-α suppressed adipose senescence and metabolic abnormalities. (E and F)SA–β-gal activity (Top) and Oil Red O staining (Bottom) of SVF (mainly preadipocytes) and mature adipocytes through the differentiation process. (Scale bars: 200 μM.) SA–β-gal activity and quantitation (n = 6) (G and H), IL-6 (I), body weight gain (J), body fat (K), plasma insulin (L), and GTT analysis (n = 6) (M) are shown. C, control; HF, high fat; P, PFT-α.The graphical data were shown as the mean ± SEM. The data were analyzed by one- way ANOVA (*P < 0.05).

Chen et al. PNAS Early Edition | 5of9 Downloaded by guest on September 27, 2021 and Met reduced the DNA lesions and metabolic abnormalities pression, but they exhibited reduced expression of Ki-67 (Fig. 4A − − that were induced by a high-fat diet in both WT and pol η / mice and Fig. S9C), increased secretion of IL-6 (Fig. 4B), and greater (Fig. S6). activity of SA–β-gal than 3T3L1-NTC-shRNA–transfected cells In contrast to the positive effects of antioxidants, sodium ar- (Fig. 4C). Furthermore, the elevated SA–β-gal activity in the senite, a well-established oxidative stress inducer (33), increased 3T3L1-shPol η cells was suppressed by PFT-α (Fig. S9 D and E). DNA lesions, elevated adipocyte senescence, and promoted The 3T3L1-pol η-shRNA–transfected cells also displayed up- − − metabolic dysfunction in pol η / mice (Figs. S7 and S8). These regulated levels of SREBP1 and PPAR-γ and accumulated more findings support the notion that endogenous DNA damage is lipid droplets (Fig. 4 A and D). Second, the effect of pol η was causative of the observed obesity and metabolic abnormalities of examined with a salvage study by independently transfecting − − − − pol η / mice. The opposite effects of antioxidants (NAC or Met) pol η / mouse embryo fibroblast (MEF) cells with vectors that (Figs. S5 and S8) and oxidative stress inducers (a high-fat diet or express human WT pol η or an R61F mutant form of human sodium arsenite) (Figs. S6–S8) reinforce the importance of adi- pol η with reduced polymerase activity (34). The expression of pocyte senescence in responding to DNA damage and the onset WT-pol η reduced the DNA damage response, secretion of IL-6, − − of metabolic abnormalities in pol η / mice. Notably, NAC and SA–β-gal activity, and accumulation of lipid droplets (Fig. 4 E–H Met also suppressed DNA damage and the metabolic changes and Fig. S9E). By contrast, the expression of the less active pol η induced by a high-fat diet in WT mice, suggesting that metabolic mutant R61F (34) only partially reduced these parameters (Fig. abnormalities may be a general response to elevated DNA 4 E–H and Fig. S9F). These findings suggest that the DNA lesion damage (Fig. S6B). In addition, because cell senescence pro- bypass activity of pol η protects adipocytes from early adipocyte motes aging (25) and Met improves life span (32), our data senescence and lipid accumulation. In addition, because MEF support the evidence that Met defers the aging process (32). cells were derived from embryos, the elevated amount of DNA − − damage and SA–β-gal activity in the primary pol η / MEF cells Impact of Pol η on Cellular Senescence and Adipogenesis Using compared with WT cells further supports the notion that chronic Knockdown and Salvage Studies with Cells in Culture. To confirm DNA damage-mediated senescence, which occurs before the further that lack of pol η activity is responsible for the cell se- accumulation of body fat, creates conditions that lead to obesity − − nescence and metabolic changes observed in pol η / mice, two and metabolic abnormalities. additional systems were used. First, pol η expression was down- The association between tumorigenesis and reduced genome regulated by the transfection of pol η-specific shRNA into NIH integrity, due to exposure to exogenous or endogenous DNA 3T3L1 cells (3T3L1-shPol η)(Fig. S9A). The 3T3L1-shPol η cells damaging agents or genetic defects in DNA repair/replication exhibited elevated amounts of DNA damage (Fig. S9B) and in- enzymes, is well documented for many tissues (35). However, Ink4a creased activation of ATM, p53, p21, γ-H2AX, and p16 ex- the potential impact of genomic integrity on adipose tissue has

Fig. 4. Correlation between pol η expression, DNA damage, cell senescence, and lipid accumulation in 3T3L1 and MEF cell systems. (A–D) 3T3L1cells. (A) Quantitation of expression and activation of DNA damage, senescence, and adipogenic protein markers. (B) IL-6 secretion. (C)SA–β-gal activity. (D) Oil Red O staining (n = 6). (E–H) MEF cells. (E) Quantitation of expression and activation of DNA damage, senescence, and adipogenic protein markers in WT, pol η−/−, pol η−/−-hPol η WT, and pol η−/−-hPol η R61F MEF cells. (F) IL-6 secretion (n = 6). (G)SA–β-gal activity. (H) Oil Red O staining. The images presented were in the same scale. The graphical data are shown as the mean ± SEM. The data were analyzed by the Student t test (*P < 0.05).

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1506954112 Chen et al. Downloaded by guest on September 27, 2021 received little attention, at least in part because adipose tissue repair enzyme-deficient mice that develop obesity, such as Neil1 PNAS PLUS tumors are usually benign. Adipose tissue is vital for energy and OGG1 (3, 4). − − homeostasis and serves as an essential endocrine and immune Studies have been conducted with pol η / mice to determine organ (25). Adipose tissue is the main site for lipid peroxidation the importance of pol η on mutation frequency, carcinogenesis, and acts as a major “sink” to store and handle free radical- and of Ig genes (42–44). Most of the induced peroxidation products (36). Oxidative DNA lesions pro- studies were performed with mice at an age (2–6 mo) at which duced by lipid peroxidation products block DNA replicative the metabolic abnormalities observed in the present study could and stall the progression of DNA replication (13, have been easily missed (42–44). Mice were followed for 24 mo 14), which triggers cell cycle arrest and leads to different cellular in one study, but the focus was on , and neither body responses, depending on the magnitude of DNA damage. Mild weights nor metabolic abnormalities were reported (45). The − − DNA damage causes temporary pausing of the cell cycle to allow metabolic abnormalities that occur in pol η / mice develop with the DNA repair system to repair the damage. Medium or severe age, and the phenotype is less severely affected than in the DNA damage induces cellular senescence or cell death (apo- leptin-deficient (ob/ob) or leptin-receptor–mutated (db/db) − − ptosis), which also depends on the tissue type. Our results sup- mouse model (46, 47). The findings with pol η / mice suggest port previous studies that have shown pol η bypasses oxidative that the vulnerability for metabolic abnormalities is increased by − − DNA lesions (13, 14) because pol η / mice exhibit an increased a loss of genome integrity with age. Our findings also suggest that amount of DNA lesions and elevated DNA damage responses. DNA damage-induced adipocyte senescence and metabolic ab- The increase in SA–β-gal activity; up-regulation of p53, p16, and normalities are not limited to genetic defects due to DNA p21; and increased SASP circulating and cellular levels of IL-6 damage repair or response enzymes. A high-fat or high-sugar − − and TNF-α that occur at a very early age in pol η / mice provide diet and environmental toxicants that have a direct or indirect evidence for a causative role of adipocyte senescence in obesity impact on genome integrity may also cause obesity and the and the development of insulin resistance. These results also metabolic syndrome by inducing DNA damage and adipocyte imply that senescence is the primary response to DNA damage in senescence. On the other hand, diets that are rich in antioxidant adipose tissue, which may explain the low incidence of malignant components may prevent oxidative DNA lesions and the devel- cancer in adipose tissue, because cellular senescence counteracts opment of senescent adipocytes, which may prevent the devel- tumorigenesis. Although increased inflammatory responses, in- opment of obesity and the metabolic syndrome in response to

cluding infiltration of macrophages into the adipose tissue and proinflammatory diets and environmental toxins. These findings, GENETICS increased levels of IL-6 or TNF-α, are commonly observed in together with other DNA repair enzyme-defective mouse models adipose tissue from obese subjects or mouse models (37), these (3, 4, 6–8), suggest that reduction of genome integrity, regardless observations may be a consequential phenomenon, because tis- of whether it is induced by defective DNA repair or exogenous sue samples were harvested from subjects or mice with an agents, contributes causatively to the development of the meta- established obesity phenotype. Deficiency of the DNA repair bolic syndrome. Given that most people become overweight or enzyme Neil1 or OGG1 also induces the metabolic syndrome moderately obese with age rather than severely obese (48), the and obesity in mice (3, 4), at least in part because of impaired mechanistic relationship between loss of genome integrity and mitochondrial function due to DNA damage (3, 4). Given the development of the metabolic syndrome with age needs to be essential role of mitochondria in energy and ROS production, it further studied. New insight may also be gained into the ap- will be worthwhile to investigate the mitochondrial function of parent link between obesity and cancer. these DNA repair/replication mice at different ages to understand its correlation with the development of the metabolic syndrome. Materials and Methods In summary, our findings suggest that pol η plays an important Animals. All animal experimental protocols were approved by the Institutional role in maintaining genome stability and preventing cellular se- Animal Care and Use Committee, Indiana University School of Medicine. WT C57BL/6 −− nescence in adipose tissue. This study extends the function of pol mice were purchased from The Jackson Laboratory. Pol η / mice were obtained tm1Rak η from its established role in suppressing tumorigenesis in sun- from the National Cancer Institute (B6;129-Polh /NCI) and backcrossed for 12 exposed skin to reducing the negative effects of endogenous generations with WT C57BL/6 mice. All experiments were conducted with male littermates maintained in the animal facility at 25 °C with a 12-h light/dark cycle. DNA damage on adipose tissue metabolism. It would be im- For body weight gain and body fat percentage measurements, mice were weighed portant to investigate the detailed mechanisms of how the de- weekly, and weight gain was calculated by subtracting the original weight at 8 wk ficiency of pol η increased DNA damage in adipose tissue and of age from the weight of the individual mice. Body fat content was analyzed the types of lesions that contribute to the observed metabolic using dual-energy X-ray absorptiometry scanning (PIXImus β mouse densitometer; abnormalities. It is not clear from the available literature whether Lunar Corp.). Treatments were started when mice were 8 wk of age, with a normal patients with XP-V are prone to the development of obesity and chow diet, high-fat diet (Harlan Laboratories, Inc.), or high-fructose diet (normal the metabolic syndrome. Given that variation in the expression chow with 20% fructose in drinking water). Agents, including NAC (1 mg/mL; level of the XP-V gene affects the sensitivity to DNA-damaging wt/vol), Met (1.5 mg/mL; wt/vol), or sodium arsenite (2.5 mg/L; wt/vol), were ad- α – agents in various tissues (10, 38–40), an epidemiological study will ministered through the drinking water. PFT- (2.2 mg/kg; wt/body wt; Sigma Aldrich) was injected i.p. three times a week for 4 wk. For histological analysis, mice be necessary to examine the correlation between the pol η ex- η were euthanized with CO2, and tissues were harvested and fixed in 4% parafor- pression level and the metabolic syndrome. In addition to pol , maldehyde (PFA). Samples were subjected to H&E staining for visualization. The other translesion synthesis polymerases have been shown to han- cross-sectional area of fat tissue was used to measure the size of adipocytes uti- dle various types of DNA lesions (41), and it will be interesting to lizing ImageJ software (NIH). For Oil Red O staining, fresh liver tissue was immersed determine whether KO mice for other lesion bypass polymerases and frozen in optimal cutting temperature compound, followed by sectioning and are prone to obesity and metabolic abnormalities. The current staining with Oil Red O and Harris hematoxylin (49). For energy expenditure results also suggest that senescence in adipose tissue caused by measurement, mice were individually housed in metabolic chambers (Lab Master; chronic DNA damage plays a causative role in the development of TSE Systems). Oxygen consumption, respiratory exchange ratio, and food/drink metabolic abnormalities. Our data with mice suggest that adipo- intake were recorded and calculated as previously described (50, 51). cyte senescence plays an important role in the development of Blood Glucose and Glucose Tolerance Test. Mice were fasted overnight before obesity. Because senescent preadipocytes are present in severely a glucose tolerance test was performed. Blood glucose was measured using a obese subjects (25), the molecular mechanism responsible for se- glucometer (Contour; Bayer Healthcare) at 0, 30, 60, 90, and 120 min after i.p. nescence cells in adipose tissue requires additional study. Our injection of glucose (2 mg/g). The data were analyzed by one-way ANOVA model of adipose tissue senescence and obesity due to DNA using GraphPad Prism 5.0 software (GraphPad Software), and the area under damage provides an additional testable paradigm for other DNA the curve was calculated by the trapezoid method.

Chen et al. PNAS Early Edition | 7of9 Downloaded by guest on September 27, 2021 ELISA. Mice were fasted overnight, and plasma samples were collected from a pared with the corresponding control after normalization with actin or facial vein for ELISA analyses using ultrasensitive mouse leptin and insulin actinin (ImageJ software). Blots are representative of six independent ELISA kits (Crystal Chem, Inc.). The mouse IL-6 and TNF-α kits were purchased experiments. For immunofluorescence staining, cells were fixed with 4% from Biolegend, Inc., and the mouse adiponectin ELISA kit was obtained PFA, followed by incubation in permeabilization buffer (0.5% saponin,

from Life Technologies Invitrogen. The HOMA-IR was calculated from the 0.1% BSA, and 1% NaN3 in PBS). The cells were then stained with rabbit product of fasting insulin concentration (micro-international units per mil- anti-mouse phospho-γ-H2AX and DyLight488–anti-rabbit IgG (Jackson liliter) and plasma glucose (milligrams per deciliter) divided by 405 (52). Immune Research laboratory) before monitoring under a Leica florescence microscope (Leica DMI6000B) using Leica imaging LAS.AF software. The Isolation of Adipocytes and Macrophage Detection. Briefly, collected fat pads images represent results from four independent experiments. were minced and digested with type I collagenase before centrifugation using similar procedures as described (53). The pellet is the stromal vascular Single-Cell Gel Electrophoresis Assay (Comet Assay) and Immunohistochemistry. fraction (SVF), and the floating cells were collected as the mature adipocyte Adipocytes isolated from the adipose tissue of WT and pol η−/− mice (53) were fraction. The mature adipocyte fraction was collected and incubated with subjected to a single-cell gel electrophoresis assay (Trevigen, Inc.). The data, Alexa Fluor 488-conjugated anti-mouse F4/80 and R-PE–conjugated anti- which were analyzed by CometScore 1.5 software (Triek Corp.), are presented mouse CD11b (Molecular Probes, Life Technologies) before being analyzed as the percentage of DNA in the tail. For detection of 8-oxoG, the fixed adi- by flow cytometry (FACSCalibur; BD Biosciences). Cells stained with both pose tissue sections were deparaffinized and rehydrated before being in- + + F4/80 and CD11b were defined as the macrophage fraction. The amount cubatedwithmouseanti–8-oxoG antibody (Trevigen, Inc.), followed by + + of macrophages was calculated as the percentage of F4/80 /CD11b among incubation with biotin-conjugated anti-mouse IgG. The 3.3′-diaminobenzidine all live cells. (Thermo Scientific) reaction was used to visualize the bound secondary anti- body. The sections were counterstained with methyl green for the cell nucleus. Preparation of Protein Lysates and mRNA for Real-Time PCR. Adipose tissues Images were acquired using a Leica microscope (DM2500) and Leica imaging were homogenized with modified radioimmunoprecipitation assay buffer, LAS software. followed by centrifugation at 4 °C. The fat layer was removed, and cell ly- sates were subjected to Western blot analysis. Total RNA was isolated from SA–β-Gal Staining and Apoptosis Assessment. SA–β-gal activity in visceral − − mature adipose tissue fractions using TRIzol (Molecular Research Center, adipose tissues from WT and pol η / mice was determined using an SA–β-gal Inc.), and cDNA was prepared using a QuantiTect Reverse Transcription kit staining kit from Cell Signaling Technology. The percentage of cells positive and a QuantiFast SYBR Green PCR kit (both from Qiagen). Real-time PCR was for SA–β-gal was quantitated as described previously (28). performed using a StepOne Plus Real-Time PCR System (Applied Biosystems). The mouse primers were designed and validated using a QuantiTect Primer Cell Culture. Murine 3T3-L1 cells (American Type Culture Collection) were Assay System (Qiagen). Relative mRNA expression represents the target gene transfected with nontarget shRNA control or shRNA (Sigma–Aldrich) to mRNA level compared with the corresponding control mRNA level. generate 3T3L1-NTC and 3T3-sh-pol η cell lines. Embryos from WT and pol − − η / mice were harvested on day 13.5 for the generation of MEF cells (54). − − Western Blot Analysis and Immunofluorescence Staining. Whole-cell lysates The established pol η / MEF cells were transfected with either WT human (50 μg) were resolved by SDS/PAGE and transferred onto nitrocellulose pol η (hPol η WT) or the R61F mutant form of human pol η (hPol η R61F) (55). membranes (Bio-Rad) before being incubated with primary antibodies, The protein expression level of pol η was examined by Western blot analysis followed by addition of HRP-conjugated secondary antibody (Jackson with anti-human or anti-mouse pol η antibodies from Biorbyt. The harvested Immuno Research Lab). Specific proteins were detected using SuperSignal SVF cells from mice adipose tissue, 3T3L1 transfectants, and MEF cells were West Femto chemiluminescence substrates (Thermo Scientific) and docu- cultured and differentiated into adipocytes (56), and then examined for SA– mented with a Fujifilm LAS-4000 Image Analyzer (GE Life Science). The β-gal staining and Oil Red O staining. primary antibodies used for Western blotting were the following anti- mouse antibodies: anti-ATM, anti–phospho-γ-H2AX, and anti–Parp-1 an- Statistical Analyses. The staining images and blots represent results from four tibodies from Cell Signaling Technology; anti–phospho-ATM (Ser1981) and to six independent experiments. The graphical data, which are shown as the anti–Ki-67 antibodies from Millipore; anti-p53, anti–phospho-p53 (Ser15), mean ± SEM, were analyzed by one-way ANOVA for multiple comparison anti-p21, anti–phospho-p21, anti–PPAR-γ, anti-Bax, anti-Noxa, anti-Puma, tests or by the Student t test for individual pairwise comparisons. The anti-Actinin, and anti-Actin antibodies from Sigma–Aldrich; and anti- threshold for statistical significance was P < 0.05 vs. the corresponding Ink4a p16 from Abcam; anti–SREBP1-p68, anti–SREBP1-p125, anti–NF-κB- control values. Analyses were performed with statistical software (GraphPad – κ κ α – κ α p65, anti phospho-NF- B-p65, anti-I B ,andantiphospho-I B anti- Prism 5.0). bodies from Santa Cruz Biotechnology. The samples derived from the same experiment, and the blots were processed in parallel. Loading controls, ACKNOWLEDGMENTS. We thank Dr. Richard Honkanen (University of South positive and negative controls, and molecular markers were run on the Alabama) for suggestions and comments during the study. This study was same blot, and the loading controls were used for normalization. The supported, in part, by NIH Grant R01 CA 112446 (to K.-m.C.) and a Veterans quantitation of protein expression represents relative protein level com- Affairs Merit Award (to R.A.H.).

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