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. 2020 Mar 10;15(3):e0229896.
doi: 10.1371/journal.pone.0229896. eCollection 2020.

Gender differences in diet-induced steatotic disease in Cyp2b-null mice

Melissa M Heintz  1   2 , Rebecca McRee  2 , Ramiya Kumar  2 , William S Baldwin  1   2
Affiliations

Gender differences in diet-induced steatotic disease in Cyp2b-null mice

Melissa M Heintz et al. PLoS One. .

Abstract

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease; however, progression to nonalcoholic steatohepatitis (NASH) is associated with most adverse outcomes. CYP2B metabolizes multiple xeno- and endobiotics, and male Cyp2b-null mice are diet-induced obese (DIO) with increased NAFLD. However, the DIO study was not performed long enough to assess progression to NASH. Therefore, to assess the role of Cyp2b in fatty liver disease progression from NAFLD to NASH, we treated wildtype (WT) and Cyp2b-null mice with a normal diet (ND) or choline-deficient, L-amino acid-defined high fat diet (CDAHFD) for 8 weeks and determined metabolic and molecular changes. CDAHFD-fed WT female mice gained more weight and had greater liver and white adipose tissue mass than their Cyp2b-null counterparts; males experienced diet-induced weight loss regardless of genotype. Serum biomarkers of liver injury increased in both CDAHFD-fed female and male mice; however CDAHFD-fed Cyp2b-null females exhibited significantly lower serum ALT, AST, and ASP concentrations compared to WT mice, indicating Cyp2b-null females were protected from liver injury. In both genders, hierarchical clustering of RNA-seq data demonstrates several gene ontologies responded differently in CDAHFD-fed Cyp2b-null mice compared to WT mice (lipid metabolism > fibrosis > inflammation). Oil Red O staining and direct triglycerides measurements confirmed that CDAHFD-fed Cyp2b-null females were protected from NAFLD. CDAHFD-fed Cyp2b-null mice showed equivocal changes in fibrosis with transcriptomic and serum markers suggesting less inflammation due to glucocorticoid-mediated repression of immune responses. In contrast to females, CDAHFD-fed Cyp2b-null males had higher triglyceride levels. Results indicate that female Cyp2b-null mice are protected from NAFLD while male Cyp2b-null mice are more susceptible to NAFLD, with few significant changes in NASH development. This study confirms that increased NAFLD development does not necessarily lead to progressive NASH. Furthermore, it indicates a role for Cyp2b in fatty liver disease that differs based on gender.

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Conflict of interest statement

The authors have declared that no competing interests exist

Figures

Fig 1
Fig 1. Changes in body weight and organ weight over the 8 weeks of diet-induced NASH treatment.
Body and select organ weights of female (A) and male (B) WT and Cyp2b-null mice were monitored during the 8 weeks of treatment. (C) Immunoblots of PCNA in female and male mice with β-actin as the reference gene. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD as the post-hoc test (n = 9; n = 2 for immunoblots). An ‘a’ indicates ND-fed WT different than CDAHFD-fed WT, ‘b’ indicates ND-fed Cyp2b-null different than CDAHFD-fed Cyp2b-null, ‘c’ indicates ND-fed WT different than ND-fed Cyp2b-null, ‘d’ indicates CDAHFD-fed WT different than CDAHFD-fed Cyp2b-null.
Fig 2
Fig 2. Gender-dependent differences in serum glucose and glucose tolerance in response to CDAHFD.
Fasting blood glucose levels were measured during weeks 4 (A) and 6 (B) in female and male mice. During week 6 glucose tolerance tests were performed on all treatment groups (C) and glucose measured at 20, 40, 60, 90, and 120 minutes after the glucose challenge. Results are also represented as area under the curve (D). Data are presented as mean serum glucose ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD as the post-hoc test (n = 9). An ‘a’ indicates ND-fed WT different than CDAHFD-fed WT, ‘b’ indicates ND-fed Cyp2b-null different than CDAHFD-fed Cyp2b-null, ‘c’ indicates ND-fed WT different than ND-fed Cyp2b-null. No asterisk indicates a p-value < 0.05, * indicates a p-value < 0.01, and ** indicates a p-value < 0.0001.
Fig 3
Fig 3. Biomarkers of liver tissue damage in ND and CDAHFD-fed WT and Cyp2b-null mice.
Serum ALT, AST, ALP, and CK concentrations were measured, and histopathological changes were evaluated by H&E staining of liver tissues from female (A) and male (B) mice. Images were taken at 100x (0.2 mm) and 400x (0.05 mm) magnification. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD as the post-hoc test (n = 5). An ‘a’ indicates ND-fed WT different than CDAHFD-fed WT, ‘b’ indicates ND-fed Cyp2b-null different than CDAHFD-fed Cyp2b-null, ‘c’ indicates ND-fed WT different than ND-fed Cyp2b-null, ‘d’ indicates CDAHFD-fed WT different than CDAHFD-fed Cyp2b-null. No asterisk indicates a p-value < 0.05, * indicates a p-value < 0.01, and ** indicates a p-value < 0.0001.
Fig 4
Fig 4. RNAseq demonstrates changes in gene expression in livers of CDAHFD-fed Cyp2b-null mice.
Heat maps showing log2-transformed, Z-score scaled RNA-seq expression of significant differentially expressed genes (log2FC > 1.0) between CDAHFD-fed Cyp2b-null and CDAHFD-fed WT groups in female (A) and male (B) mice. Yellow and blue color intensity indicate gene up- or down-regulation, respectively. Dendrogram clustering on the y-axis groups genes by expression profile across samples. GO term enrichment analysis summary using Revigo [43] for up-regulated GO terms in CDAHFD-fed Cyp2b-null female (C) and male (D) mice compared to CDAHFD-fed WT mice. Each scatterplot contains enriched GO terms from the biological process class that remain after term redundancy is reduced and are displayed in a two-dimensional space where semantically similar GO terms are positioned closer together within the plot. Each circle represents an enriched GO term; the cooler the color of a term, the greater signficance (p < 0.05) of that term with measured changes in gene expression. Circle size indicates the frequency of the GO term in the underlying GO database, i.e. circles of more general terms are larger.
Fig 5
Fig 5. Measured liver fibrosis and inflammatory markers in CDAHFD-treated Cyp2b-null female mice.
Changes in the expression of fibrosis, inflammation and stress response-associated genes were investigated and grouped by respective biomarkers and/or KEGG pathways (A). LogFC values with an asterisk indicates a significant difference (p < 0.05) between two groups, e.g. CDAHFD-fed versus ND-fed WT mice, and no asterisk denotes significance by one-way ANOVA across all treatment groups. Histopathological changes were evaluated by Masson’s trichrome staining of female liver tissues (B). Images were taken at 100x (0.2 mm) and 400x (0.05 mm) magnification. Liver hydroxyproline (C), as well as serum C-reactive protein (D) and corticosterone (E) were measured in all treatment groups. Graph data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD as the post-hoc test (n = 5). An ‘a’ indicates ND-fed WT different than CDAHFD-fed WT, ‘b’ indicates ND-fed Cyp2b-null different than CDAHFD-fed Cyp2b-null, ‘c’ indicates ND-fed WT different than ND-fed Cyp2b-null, ‘d’ indicates CDAHFD-fed WT different than CDAHFD-fed Cyp2b-null. No asterisk indicates a p-value < 0.05, * indicates a p-value < 0.01, and ** indicates a p-value < 0.0001.
Fig 6
Fig 6. Measured liver fibrosis and inflammatory markers in CDAHFD-treated Cyp2b-null male mice.
Changes in the expression of fibrosis, inflammation and stress response-associated genes were investigated and grouped by respective biomarkers and/or KEGG pathways (A). LogFC values with an asterisk indicates a significant difference (p < 0.05) between two groups, e.g. CDAHFD-fed versus ND-fed WT mice, and no asterisk denotes significance by one-way ANOVA across all treatment groups. Histopathological changes were evaluated by H&E, and Masson’s trichrome staining of male liver tissues (B). Images were taken at 100x (0.2 mm) and 400x (0.05 mm) magnification. Liver hydroxyproline (C), as well as serum C-reactive protein (D) and corticosterone (E) were measured in all treatment groups. Graph data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD as the post-hoc test (n = 5). An ‘a’ indicates ND-fed WT different than CDAHFD-fed WT, ‘b’ indicates ND-fed Cyp2b-null different than CDAHFD-fed Cyp2b-null, ‘c’ indicates ND-fed WT different than ND-fed Cyp2b-null, ‘d’ indicates CDAHFD-fed WT different than CDAHFD-fed Cyp2b-null. No asterisk indicates a p-value < 0.05, * indicates a p-value < 0.01, and ** indicates a p-value < 0.0001.
Fig 7
Fig 7. Steatosis and markers of steatosis in CDAHFD-fed WT and CDAHFD-fed Cyp2b-null female mice.
Changes in the expression of nonalcoholic fatty liver disease-related genes were investigated and grouped by respective biomarkers and/or KEGG pathways (A). LogFC values with an asterisk indicates a significant difference (p < 0.05) between two groups, e.g. CDAHFD-fed versus ND-fed WT mice, and no asterisk denotes significance by one-way ANOVA across all treatment groups. Fatty liver histopathological changes were evaluated by Oil red O staining in female mice (B). Images were taken at 100x (0.2 mm) and 400x (0.05 mm) magnification. Total liver triglycerides (C) were measured in female mice to confirm Oil Red O staining results. Liver lipid droplets were also quantified by total area (D) using ImageJ Fiji from Oil Red O slides (400x). Serum levels of β-hydroxybutyrate (E), leptin (F), and adiponectin (G) were also determined. Graphed data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD as the post-hoc test (n = 5). An ‘a’ indicates ND-fed WT different than CDAHFD-fed WT, ‘b’ indicates ND-fed Cyp2b-null different than CDAHFD-fed Cyp2b-null, ‘c’ indicates ND-fed WT different than ND-fed Cyp2b-null, ‘d’ indicates CDAHFD-fed WT different than CDAHFD-fed Cyp2b-null. No asterisk indicates a p-value < 0.05, * indicates a p-value < 0.01, and ** indicates a p-value < 0.0001.
Fig 8
Fig 8. Steatosis and markers of steatosis in CDAHFD-fed WT and CDAHFD-fed Cyp2b-null male mice.
Changes in the expression of nonalcoholic fatty liver disease-related genes were investigated and grouped by respective biomarkers and/or KEGG pathways (A). LogFC values with an asterisk indicates a significant difference (p < 0.05) between two groups, e.g. CDAHFD-fed versus ND-fed WT mice, and no asterisk denotes significance by one-way ANOVA across all treatment groups. Fatty liver histopathological changes were evaluated by Oil red O staining in female mice (B). Images were taken at 100x (0.2 mm) and 400x (0.05 mm) magnification. Total liver triglycerides (C) were measured in male mice to confirm Oil Red O staining results. Liver lipid droplets were also quantified by total area (D) using ImageJ Fiji from Oil Red O slides (400x). Serum levels of β-hydroxybutyrate (E), leptin (F), and adiponectin (G) were also determined. Graph data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD as the post-hoc test (n = 5). An ‘a’ indicates ND-fed WT different than CDAHFD-fed WT, ‘b’ indicates ND-fed Cyp2b-null different than CDAHFD-fed Cyp2b-null, ‘c’ indicates ND-fed WT different than ND-fed Cyp2b-null, ‘d’ indicates CDAHFD-fed WT different than CDAHFD-fed Cyp2b-null. No asterisk indicates a p-value < 0.05, * indicates a p-value < 0.01, and ** indicates a p-value < 0.0001.
Fig 9
Fig 9. qPCR confirmation of RNAseq analysis.
Changes in the expression of genes in females (A) and males (B) involved in fibrosis, inflammation, insulin signaling, fatty liver, and proliferation by qPCR confirmation. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD as the post-hoc test (n = 5). An ‘a’ indicates ND-fed WT different than CDAHFD-fed WT, ‘b’ indicates ND-fed Cyp2b-null different than CDAHFD-fed Cyp2b-null, ‘c’ indicates ND-fed WT different than ND-fed Cyp2b-null, ‘d’ indicates CDAHFD-fed WT different than CDAHFD-fed Cyp2b-null. No asterisk indicates a p-value < 0.05, * indicates a p-value < 0.01, and ** indicates a p-value < 0.0001.
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