Hepatic transcriptional profile reveals the role of diet and genetic backgrounds on metabolic traits in female progenitor strains of the Collaborative Cross

Abstract

Mice have provided critical mechanistic understandings of clinical traits underlying metabolic syndrome (MetSyn) and susceptibility to MetSyn in mice is known to vary among inbred strains. We investigated the diet- and strain-dependent effects on metabolic traits in the eight Collaborative Cross (CC) founder strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). Liver transcriptomics analysis showed that both atherogenic diet and host genetics have profound effects on the liver transcriptome, which may be related to differences in metabolic traits observed between strains. We found strain differences in circulating trimethylamine N-oxide (TMAO) concentration and liver triglyceride content, both of which are traits associated with metabolic diseases. Using a network approach, we identified a module of transcripts associated with TMAO and liver triglyceride content, which was enriched in functional pathways. Interrogation of the module related to metabolic traits identified NADPH oxidase 4 (Nox4), a gene for a key enzyme in the production of reactive oxygen species, which showed a strong association with plasma TMAO and liver triglyceride. Interestingly, Nox4 was identified as the highest expressed in the C57BL/6J and NZO/HILtJ strains and the lowest expressed in the CAST/EiJ strain. Based on these results, we suggest that there may be genetic variation in the contribution of Nox4 to the regulation of plasma TMAO and liver triglyceride content. In summary, we show that liver transcriptomic analysis identified diet- or strain-specific pathways for metabolic traits in the Collaborative Cross (CC) founder strains.

Keywords: Collaborative Cross; diet; genetic backgrounds; metabolic syndrome; transcriptomics.

Figure 1.

Effect of diet on liver gene expression in female eight Collaborative Cross (CC) founder strains mice. We identified the effect of an atherogenic diet on global liver gene expression in eight CC founder strains (n = 48). Principal component (PC) analysis (A) and Volcano plot (B) between high-fat and cholic acid (HFCA) diet and AIN-93M diet in liver gene expression in eight CC founder strains. B: horizontal dotted lines indicate adj. P < 0.05, vertical dotted gray lines indicate a twofold difference. Venn diagram to identify overlapping gene ontology (GO) Biological Process 2018 terms (C) and Kyoto Encyclopedia of Genes and Genomes (KEGG) 2019 Mouse pathways (D) between upregulated genes in HFCA diet, upregulated genes in AIN-93M diet, or nondiet responsive genes in enrichment analysis. Top 10 GO terms and KEGG pathways of upregulated genes in HFCA diet (E) and AIN-93M diet (F) identified in enrichment analysis. Pathways were ordered from top to bottom by significance (highest to lowest) and colored by gene richness.

Figure 2.

Effect of genetic background on hepatic gene expression in the eight Collaborative Cross (CC) founder strains in female mice. Principal component analysis from AIN-93M diet or HFCA diet (A) and hierarchical clustering (B) determined that the major source of variation in gene expression was due to genetic variation among the eight strains. Venn diagrams to identify overlapping upregulated genes between A/J strain and B6 strain (C) and between CAST strain and B6 strain (D) in each diet. Volcano plots between high-fat and cholic acid (HFCA) diet and AIN-93M diet in liver gene expression in A/J strain (E) and CAST strain (F). Horizontal dotted lines indicate adj. P < 0.05, vertical dotted gray lines indicate a twofold difference. Top 10 GO terms of upregulated genes in HFCA diet and AIN-93M diet identified in enrichment analysis in A/J strain (G) and CAST strain (H). Pathways were ordered from top to bottom by significance (highest to lowest) and colored by gene richness. n = 6 mice for each founder, three for AIN-93M diet and three for HFCA diet-fed mice. A/J (yellow), B6 (gray), C57BL/6J; 129 (pink), 129S1/SvlmJ; NOD (blue), NOD/ShiLtJ; NZO (light blue), NZO/HILtJ; CAST (green), CAST/EiJ; PWJ (red), PWK/PhJ; WSB (purple), WSB/EiJ.

Figure 3.

Effect of diet and genetic background on hepatic co-expression gene modules in the eight Collaborative Cross (CC) founder strains in female mice. Differences of principal component (PC)1 of module eigengenes (ME) including turquoise (A), brown (B), green (C), red (D), magenta (E), and tan (F) module ME by diet and genetic background were plotted. G: number and proportion of diet or nondiet responsive genes in six modules. The x-axis is the number of genes and the y-axis is the color of each module. Length of the bar corresponding to each module is the number of module genes, and portions corresponding to purple, gray, and light blue colors in each bar are the number of genes upregulated by the high-fat and cholic acid (HFCA) diet (purple), nondiet responsive genes (gray), and genes upregulated by the AIN-93M diet (light blue). Proportion of each color is written in white letters in the bar. ***P < 0.001, **P < 0.01, *P < 0.05. n = 6 mice for each founder, three for AIN-93M diet (light blue color) and three for HFCA diet (purple color)-fed mice. A/J (yellow), B6 (gray), C57BL/6J; 129 (pink), 129S1/SvlmJ; NOD (blue), NOD/ShiLtJ; NZO (light blue), NZO/HILtJ; CAST (green), CAST/EiJ; PWJ (red), PWK/PhJ; WSB (purple), WSB/EiJ.

Figure 4.

Diet-dependent differences in key metabolic traits in eight Collaborative Cross (CC) founder strains. Liver weight and triglyceride (TG) and plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, and betaine were higher in high-fat and cholic acid (HFCA) diet-fed mice (purple color), while oxygen consumption per body weight was higher in AIN-93M diet-fed mice (light blue color). Plasma trimethylamine N-oxide (TMAO) was higher in HFCA diet-fed mice, but not significant. A/J (yellow), B6 (gray), C57BL/6J; 129 (pink), 129S1/SvlmJ; NOD (blue), NOD/ShiLtJ; NZO (light blue), NZO/HILtJ; CAST (green), CAST/EiJ; PWJ (red), PWK/PhJ; WSB (purple), WSB/EiJ. ****P <0.001, **P < 0.01, *P < 0.05. Data were means ± S.E., n ≥ 4 mice/diet/strain.

Figure 5.

Association of hepatic co-expression gene modules with metabolic traits in the eight Collaborative Cross (CC) founder strains in female mice. A: Spearman correlation between liver gene modules and metabolic traits in all mice. Module names were shown along the right axis, and top-enriched gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms in the legend. B: Spearman correlation between the top 30 high-fat and cholic acid (HFCA)-specific differential expression genes (DEGs) identified in the magenta module and metabolic traits in all mice. The P values were adjusted using the Benjamini–Hochberg (BH) false discovery rate (FDR) procedure. ***P < 0.001, **P < 0.01, *P < 0.05, .P < 0.10.

Figure 6.

Association of hepatic gene modules with metabolic traits points to Nox4-associated plasma trimethylamine N-oxide (TMAO) and creatinine production. Effect of diet or genetic background on plasma TMAO (A) and liver triacylglycerol (TG) (B), and hepatic expression of Fmo3 (C) and Nox4 (D). Spearman correlation between plasma TMAO and Fmo3 (E) or Nox4 (F) abundance. Spearman correlation between liver TG and Fmo3 (G) or Nox4 (H) abundance. ***P < 0.001, **P < 0.01, *P < 0.05, n = 6 mice for each founder, three for AIN-93M diet (light blue color) and three for high-fat and cholic acid (HFCA) diet-fed mice (purple color). A/J (yellow), B6 (gray), C57BL/6J; 129 (pink), 129S1/SvlmJ; NOD (blue), NOD/ShiLtJ; NZO (light blue), NZO/HILtJ; CAST (green), CAST/EiJ; PWJ (red), PWK/PhJ; WSB (purple), WSB/EiJ.