Alterations in Adipose Tissue and Adipokines in Heterozygous APE1/Ref-1 Deficient Mice

Abstract

Backgruound: The role of apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) in adipose tissue remains poorly understood. This study investigates adipose tissue dysfunction in heterozygous APE1/Ref-1 deficiency (APE1/Ref-1+/-) mice, focusing on changes in adipocyte physiology, oxidative stress, adipokine regulation, and adipose tissue distribution.

Methods: APE1/Ref-1 mRNA and protein levels in white adipose tissue (WAT) were measured in APE1/Ref-1+/- mice, compared to their wild-type (APE1/Ref-1+/+) controls. Oxidative stress was assessed by evaluating reactive oxygen species (ROS) levels. Histological and immunohistochemical analyses were conducted to observe adipocyte size and macrophage infiltration of WAT. Adipokine expression was measured, and micro-magnetic resonance imaging (MRI) was used to quantify abdominal fat volumes.

Results: APE1/Ref-1+/- mice exhibited significant reductions in APE1/Ref-1 mRNA and protein levels in WAT and liver tissue. These mice also showed elevated ROS levels, suggesting a regulatory role for APE1/Ref-1 in oxidative stress in WAT and liver. Histological and immunohistochemical analyses revealed hypertrophic adipocytes and macrophage infiltration in WAT, while Oil Red O staining demonstrated enhanced ectopic fat deposition in the liver of APE1/Ref-1+/- mice. These mice also displayed altered adipokine expression, with decreased adiponectin and increased leptin levels in the WAT, along with corresponding alterations in plasma levels. Despite no significant changes in overall body weight, microMRI assessments demonstrated a significant increase in visceral and subcutaneous abdominal fat volumes in APE1/Ref-1+/- mice.

Conclusion: APE1/Ref-1 is crucial in adipokine regulation and mitigating oxidative stress. These findings suggest its involvement in adipose tissue dysfunction, highlighting its potential impact on abdominal fat distribution and its implications for obesity and oxidative stress-related conditions.

Keywords: APE1/Ref-1; Abdominal fat distribution; Adipokine expression; Adipose tissue dysfunction; Oxidative stress.

Fig. 1.

Generation of heterozygous apurinic/apyrimidinic endonuclase1/redox factor-1 (APE1/Ref-1) deficiency mice. (A) Targeting schema of the APE1/Ref-1 gene deletion with loxP sites flanking exons 2 and 3. Knockout of the APE1/Ref-1 gene was generated via the repair of double-strand breaks using non-homologous end joining. (B) Polymerase chain reaction genotyping for detection of the APE1/Ref-1 in genomic DNA from heterozygous (APE1/Ref-1^+/-) mice, wild-type (APE1/Ref-1^+/+) mice. The presence of a 1,460 bp band indicates the wild-type allele, while the presence of a 568 bp band confirms the detection of the deleted allele. (C) The body weight of 20-week-old male heterozygous (APE1/Ref-1^+/-) mice and wild-type (APE1/Ref-1^+/+) mice (n=10). There is no significant (NS) change in body weight observed between both groups. (D) Reduced APE1/Ref-1 mRNA expression in the white adipose tissue (WAT) of APE1/Ref-1^+/- mice (n=5). mRNA levels were analyzed with quantitative reverse transcription-polymerase chain reaction as described in the ‘Methods’ section. (E) Western blotting results of APE1/Ref-1 protein expression in WAT. Data presented are representative of three independent experiments. (F) Quantitative analysis for Western blotting of APE1/Ref-1 protein expression in WAT of APE1/Ref-1^+/- and APE1/Ref-1^+/+ mice (n=9). (G) Western blotting results of APE1/Ref-1 protein expression in liver. (H) Quantitative analysis to measure APE1/Ref-1 protein expression in liver of APE1/Ref-1^+/- and APE1/Ref-1^+/+ mice (n=6). The data represent the mean±standard error of the mean. Statistical significance is indicated as NS. EcoRI and BamHI means restriction endonuclease enzyme sites. ^aP<0.001 vs. APE1/Ref-1^+/+ mice.

Fig. 2.

Abnormal oxidative stress state in the white adipose tissue (WAT) of heterozygous apurinic/apyrimidinic endonuclase1/redox factor- 1 (APE1/Ref-1)-deficient mice. (A) Increased total reactive oxygen species (ROS) production in WAT of APE1/Ref-1^+/- mice (n=5–6). (B) Increased superoxide production in APE1/Ref-1^+/- compared to APE1/Ref-1^+/+ mice in WAT (n=5–6). (C) Increased total ROS production in APE1/Ref-1^+/- compared to APE1/Ref-1^+/+ mice (n=5). Total ROS was measured with dichlorofluorescein assay. (D) Increased superoxide production in the liver of APE1/Ref-1^+/- mice (n=6). Superoxide production was measured with lucigenin-enhanced chemiluminescence. The data represent the mean±standard error of the mean. RFU, relative fluorescence unit; RLU, relative luminescence unit. ^aP<0.05 vs. APE1/Ref-1^+/+ mice.

Fig. 3.

Adipocyte hypertrophy in white adipose tissue (WAT) and hepatic fat accumulation in heterozygous apurinic/apyrimidinic endonuclase1/ redox factor-1 (APE1/Ref-1)-deficient mice. (A) Low power (4×) and high power (10×) image of hematoxylin and eosin (H&E) staining in WAT from the APE1/Ref-1^+/+ mice and APE1/Ref-1^+/- mice. Note that APE1/Ref-1^+/- mice exhibit larger adipocytes compared to APE1/Ref-1^+/+ mice. (B) Mean adipose diameters in WAT of APE1/Ref-1^+/- mice and APE1/Ref-1^+/+ mice. Mean adipose diameters in WAT of APE1/Ref-1^+/- mice are significantly larger than those of APE1/Ref-1^+/+ mice, indicating adipose hypertrophy (n=3). (C) Immunohistochemical detection of the macrophage-specific antigen F4/80 in WAT of APE1/Ref-1^+/- mice. (D) Quantitative analysis of F4/80 expression in WAT of APE1/Ref-1^+/- mice. Macrophage infiltrations are increased in adipose tissue of APE1/Ref-1^+/- mice (n=3). (E) Low power (4×) and high power (40×) image H&E staining in liver tissue from APE1/Ref-1^+/+ mice and APE1/Ref-1^+/- mice. Fat vacuoles are observed in the liver of APE1/Ref-1^+/- mice, not in APE1/Ref-1^+/+ mice. (F) Quantitative analysis of hepatic steatosis in non-adipose tissue of APE1/Ref- 1+/- mice. Hepatic steatosis was analyzed by histopathological scoring as described in the ‘Methods’ section (n=3). (G) Oil Red O staining for lipid detection in frozen liver tissue sections of APE1/Ref-1^+/+ and APE1/Ref-1^+/- mice. (H) Quantification of the percentage of increased triglyceride content in liver tissue of APE1/Ref-1^+/- mice compared to APE1/Ref-1^+/+ mice. The data represent the mean±standard error of the mean (n=3–5). ^aP<0.05 vs. APE1/Ref-1^+/+ mice.

Fig. 4.

Decreased adiponectin in heterozygous apurinic/apyrimidinic endonuclase1/redox factor-1 (APE1/Ref-1)-deficient mice. (A) Decreased mRNA expression of adiponectin in the white adipose tissue (WAT) of APE1/Ref-1^+/- mice (n=5). mRNA levels were analyzed with quantitative reverse transcription-polymerase chain reaction as described in the ‘Methods’ section. (B) Representative Western blot analysis of adiponectin protein expression in WAT from three individual mice. (C) Quantitative analysis of adiponectin protein expression in WAT normalized to β-actin showing reduced expression in APE1/Ref-1^+/- mice, compared to APE1/Ref-1^+/+ mice (n=9). (D) Immunohistochemistry staining of adiponectin showing reduced expression in WAT of APE1/Ref-1^+/- mice. (E) Reduced plasma adiponectin levels in APE1/ Ref-1+/- mice compared to APE1/Ref-1^+/+ mice (n=6). The data represent the mean±standard error of the mean. ^aP<0.05; ^bP<0.01; ^cP<0.001 vs. APE1/Ref-1^+/+ mice.

Fig. 5.

Elevated leptin expression in heterozygous apurinic/apyrimidinic endonuclase1/redox factor-1 (APE1/Ref-1)-deficient mice. (A) Increased leptin mRNA expression in the white adipose tissue (WAT) of APE1/Ref-1^+/- mice than in APE1/Ref-1^+/+ mice (n=5). mRNA levels were analyzed with quantitative reverse transcription-polymerase chain reaction as described in the ‘Methods’ section. (B) Representative Western blotting results of leptin protein expression in WAT from three individual mice. (C) Quantitative analysis of leptin protein expression in WAT normalized to β-actin showing increased expression in APE1/Ref-1^+/- mice, compared to APE1/Ref-1^+/+ mice (n=6). (D) Immunohistochemistry staining of leptin showing increased expression in the WAT of APE1/Ref-1^+/- mice. (E) Increased leptin levels of plasma in APE1/Ref-1^+/- mice compared to APE1/Ref-1^+/+ mice (n=4). The data represent the mean±standard error of the mean. ^aP<0.001 vs. APE1/Ref-1^+/+ mice.

Fig. 6.

Quantification of increased abdominal fat area in heterozygous apurinic/apyrimidinic endonuclase1/redox factor-1 (APE1/Ref-1) deficiency mice using micro-magnetic resonance imaging (MRI). (A) Representative microMRI images of coronal sections to determine abdominal localization. The gray box indicates the abdominal analysis area yielding the results in panels C-F. The white line indicates the position of the transverse sectional image that is shown in panel B. (B) Representative transverse sectional image of the abdomen (visceral fat [V], subcutaneous fat [S]). (C) Results of increased visceral fat volume in APE1/Ref-1^+/- mice compared to APE1/Ref-1^+/+ mice (n=5–6). (D) Thicker abdominal subcutaneous fat volume in APE1/Ref-1^+/- mice (n=5–6). (E) APE1/Ref-1^+/- mice with greater total abdominal fat, which is the sum of visceral and subcutaneous fat (n=5–6). (F) Average abdominal cross-sectional area between the two groups without difference (n=5–6). Data are presented as mean±standard error of the mean. NS, no significant. ^aP<0.05 vs. APE1/Ref-1^+/+.