Cyp2b-null male mice are susceptible to diet-induced obesity and perturbations in lipid homeostasis

Abstract

Obesity is an endemic problem in the United States and elsewhere, and data indicate that in addition to overconsumption, exposure to specific chemicals enhances obesity. CYP2B metabolizes multiple endo- and xenobiotics, and recent data suggests that repression of Cyp2b activity increases dyslipidemia and age-onset obesity, especially in males. To investigate the role played by Cyp2b in lipid homeostasis and obesity, we treated wildtype and Cyp2b-null mice with a normal (ND) or 60% high-fat diet (HFD) for 10 weeks and determined metabolic and molecular changes. Male HFD-fed Cyp2b-null mice weigh 15% more than HFD-fed wildtype mice, primarily due to an increase in white adipose tissue (WAT); however, Cyp2b-null female mice did not demonstrate greater body mass or WAT. Serum parameters indicate increased ketosis, leptin and cholesterol in HFD-fed Cyp2b-null male mice compared to HFD-fed wildtype mice. Liver triglycerides and liver:serum triglyceride ratios were higher than their similarly treated wildtype counterparts in Cyp2b-null male mice, indicating a role for Cyp2b in fatty acid metabolism regardless of diet. Furthermore, RNAseq demonstrates that hepatic gene expression in ND-fed Cyp2b-null male mice is similar to HFD-fed WT male mice, suggestive of fatty liver disease progression and a role for Cyp2b in lipid homeostasis. Females did not show as demonstrative changes in liver health, and significantly fewer changes in gene expression, as well as gene expression associated with liver disease. Overall our data indicates that the repression or inhibition of CYP2B may exacerbate metabolic disorders and cause obesity by perturbing fatty acid metabolism, especially in males.

Keywords: Cyp2b; Non-alcoholic fatty liver disease (NAFLD); Obesity; P450; RNAseq; Triglycerides.

Fig. 1:. Changes in body weight during the 10-weeks of dietary treatments.

Body weight of A) male and B) female, WT, and Cyp2b-null mice were monitored during the 10-week feeding study. Male but not female Cyp2b-null mice show increased weight during HFD treatments. Data are represented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD as the post-hoc test (n= 8-9). An ‘a’ indicates WT-ND are different than Cyp2b-null-ND, ‘b’ indicates WT-ND are different than WT-HFD, ‘c’ indicates Cyp2b-null-ND are different than Cyp2b-null-HFD, ‘d’ indicates WT-HFD are different than Cyp2b-null-HFD.

Fig. 2:. Glucose tolerance tests do not show significant differences between WT and Cyp2b-null mice:

Glucose tolerance tests were performed on weeks 5 (A) and 8 (B) as described in the Materials and Methods. Results are shown for males and females over the time course of the assay and as area under the curve (AUC). 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= 8-9). ** Indicates a p-value ≤ 0.01. An ‘a’ indicates WT-ND are different than Cyp2b-null-ND, ‘b’ indicates WT-ND are different than WT-HFD, ‘c’ indicates Cyp2b-null-ND are different than Cyp2b-null-HFD, ‘d’ indicates WT-HFD are different than Cyp2b-null-HFD.

Fig. 3:. Liver triglyceride concentrations are significantly increased in Cyp2b-null mice.

Liver triglycerides were extracted and determined as described in the Materials and Methods using commercial kits. Data are presented as mean ± SEM for males (A) and females (B). Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD as the post-hoc test (n= 8-9). * indicates a p-value ≤0.05, *** indicates p-value ≤0.001 and **** indicates p-value ≤ 0.0001. An ‘a’ indicates WT-ND are different than Cyp2b-null-ND, ‘b’ indicates WT-ND are different than WT-HFD, ‘c’ indicates Cyp2b-null-ND are different than Cyp2b-null-HFD, ‘d’ indicates WT-HFD different than Cyp2b-null-HFD. H&E staining was performed in male (C) and female (D) mice with increases in vacuolization in HFD-fed males. Oil Red O staining was also performed in male (E) and female (F) mice and indicate weak liver lipid staining in all ND treatments and greater triglyceride staining in HFD-fed Cyp2b-null males relative to their HFD-fed WT counterparts.

Fig. 4:. Liver triglyceride:serum triglyceride (A), liver triglyceride:serum free fatty acid (B), and serum free fatty acidrserum b-hydroxybutyrate (C) ratios in ND and HFD-fed mice.

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= 8-9). * indicates a p-value ≤0.05, *** indicates p-value ≤0.001 and **** indicates p-value ≤ 0.0001. An ‘a’ indicates WT-ND are different than Cyp2b-null-ND, ‘b’ indicates WT-ND are different than WT-HFD, ‘c’ indicates Cyp2b-null-ND are different than Cyp2b-null-HFD, ‘d’ indicates WT-HFD different than Cyp2b-null-HFD.

Fig. 5:. Serum adiponectin and leptin concentrations in WT and Cyp2b-null mice.

Commercial kits were used to determine (A) adiponectin and (B) leptin concentrations in serum samples. Data are presented as mean ± SEM. Statistical significance was determined by oneway ANOVA followed by Fisher’s LSD as the post-hoc test (n= 8-9). * Indicates a p-value ≤ 0.05, ** indicates a p-value ≤0.01, *** indicates a p-value ≤0.001 and **** indicates p-value ≤ 0.0001. An ‘a’ indicates WT-ND are different than Cyp2b-null-ND, ‘b’ indicates WT-ND are different than WT-HFD, ‘c’ indicates Cyp2b-null-ND are different than Cyp2b-null-HFD, ‘d’ indicates WT-HFD are different than Cyp2b-null-HFD.

Fig. 6:. Cyp2b-null male mice fed a ND display a similar gene expression profile to WT mice fed a HFD.

RNAseq was performed on liver samples from WT and Cyp2b-null mice fed either a ND or HFD. (A) Heat map showing log2-transformed, Z-score scaled RNA-Seq expression of 500 genes with the highest variance between treatment groups. Red and blue color intensity indicate gene up- or down-regulation, respectively. Dendrogram clustering on the x-axis indicates sample similarity, whereas dendrogram clustering on the y-axis groups genes by expression profile across samples. (B) Venn diagrams of shared up- and down-regulated differentially expressed genes (p<0.05) between Cyp2b-null ND-fed and WT HFD-fed mice compared to WT ND mice. (C) GO term enrichment analysis summary using Revigo [58] for significantly up-regulated genes in Cyp2b-null ND mice compared to WT ND mice. The 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 more significantly (p < 0.05) associated that term is with the group of genes being studied. Circle size indicates the frequency of the GO term in the underlying GO database, i.e. circles of more general terms are larger.

Fig. 7:. Cyp2b-null female mice demonstrate relatively fewer gene expression changes.

RNAseq was performed on liver samples from WT and Cyp2b-null mice fed either a ND or HFD. (A) Heat map showing log2-transformed, Z-score scaled RNA-Seq expression of 500 genes with the highest variance between treatment groups. Red and blue color intensity indicate gene up- or down-regulation, respectively. Dendrogram clustering on the x-axis indicates sample similarity, whereas dendrogram clustering on the y-axis groups genes by expression profile across samples. (B) Venn diagrams of shared up- and down-regulated differentially expressed genes (p<0.05) between Cyp2b-null ND-fed and WT HFD-fed mice compared to WT ND mice.