Table 1.
Composition of diets used in this study.
Table 2.
Fatty acid profile of oils used in diets in this study.
Fig 1.
Soybean oil induces greater weight gain and adiposity than fructose.
A-C) Average weekly body weights of male C57/BL6 mice started on the indicated diets at weaning. All diets are isocaloric with 40 kcal% total fat except Viv chow, which has 13.5 kcal% fat. HFD, high fat diet largely from coconut oil; SO-HFD, high soybean oil diet; SO-F-HFD, high soybean oil and high fructose diet; F-HFD, high fructose diet. N = 6–12. * Significantly higher than all others; ** HFD significantly higher than Viv. Significance is defined as P ≤ 0.05 by Student’s T-test. D) Average weight of different types of white adipose tissue. Diets are color coded as in A-C. * Significantly lower than all others; ** HFD significantly lower than SO-HFD; *** SO-HFD significantly higher than all others; N = 6–12 per diet. Significance is defined as P ≤ 0.05 by ANOVA with Tukey’s post-hoc analysis.
Fig 2.
Fructose enhances liver/body weight ratio and kidney weight; soybean oil is protective in spleen and kidney.
Average weight of organs harvested from C57/BL6 mice on the indicated diets as in Fig 1 for 35 weeks. A) Left, total liver weight. * Significantly lower than SO-HFD and F-HFD. Right, liver as percent of body weight. B) Spleen weights. * Significantly lower than all others except SO-HFD; C) Kidney weights (both combined). * Significantly higher than Viv and SO-HFD;. N = 6–12 per diet. Significance is defined as P ≤ 0.05 by ANOVA with Tukey’s post-hoc analysis.
Fig 3.
Soybean oil and fructose affect intestinal morphology.
Length of small intestine (A) and colon (B) of C57/BL6 male mice on the indicated diets for 35 weeks. N = 6–12 per diet. * Significantly higher than all others. ** Significantly higher than SO-HFD and HFD. Average crypt length in the proximal (C) and distal (D) colon. N = 3–4 mice per group (up to 25 crypts measured/mouse). * Significantly higher than all others. ** Significantly higher than SO-HFD. Significance is defined as P ≤ 0.05 by ANOVA with Tukey’s post-hoc analysis. E) Left, incidence of rectal prolapse at 35 weeks, n = 12 per group. Right, representative image of rectal prolapse (arrow) in a mouse on F-HFD for 35 weeks.
Fig 4.
Soybean oil induces diabetes and insulin resistance (IR).
A) Left panel: GTT assay of male mice on the indicated diets for 20 weeks. Right panel: Area under the curve (AUC) for GTT. * SO-HFD significantly higher than HFD, F-HFD and Viv. ** F-SO-HFD significantly higher than Viv and HFD. B) Left panel: ITT assay of mice on the indicated diets for 33 weeks. Right panel: Area under the curve (AUC) for ITT. * SO-HFD significantly higher than others. Significance is defined as P ≤ 0.05 by ANOVA with Tukey’s post-hoc analysis. N = 6–12 per group.
Fig 5.
Soybean oil causes fatty liver and hepatic balloon injury.
Representative Oil Red O staining for fatty liver in male mice on the various diets for 35 weeks (A-E) or 16 weeks (F). (E,F) Arrows indicate ballooning injury in mice on SO-HFD. Scale bar (100 microns) is shown in (F). Livers from 4–9 mice per group were examined: see S2 Fig for images from additional mice.
Fig 6.
Soybean oil causes a distinct dysregulation of hepatic gene expression from coconut oil.
A) Principle components analysis (PCA) of RNA-seq from livers of male mice fed Viv, HFD or SO-HFD for 35 weeks showing the three biological replicates clustering together for each diet except for one HFD outlier. B) Heat map showing differential gene expression between SO-HFD, HFD and Viv livers. C) Venn diagrams showing genes dysregulated in HFD or SO-HFD versus Viv. D) Increased Cidea mRNA expression confirmed by qPCR in the livers of mice fed SO-HFD compared to mice fed Viv or HFD for 16 or 35 weeks. * SO-HFD (35 weeks) is significantly higher than all others. Significance is defined as P ≤ 0.05 using a Student’s t-test. Three livers were assayed in triplicate for all conditions except Viv, for which two livers were analyzed. See S1 Dataset for raw cycle count (Cq) values obtained by qPCR for Cidea and cyclophilin A.
Fig 7.
Gene ontology analysis of liver genes dysregulated by soybean oil.
Functional annotation clustering of genes dysregulated ≥ 1.5-fold (log2) in SO-HFD versus HFD male mouse livers in RNA-seq. Top, upregulated genes; Bottom, downregulated genes.
Fig 8.
Select liver genes dysregulated in SO-HFD related to metabolic disease and cancer.
Absolute expression levels from RNA-seq data in FPKM of dysregulated genes in the livers of Viv, HFD and SO-HFD fed male mice at 35 weeks. Shown are six representative genes for each category out of 13 obesity, 14 diabetes, 27 inflammation and 27 mitochondrial dysfunction and 31 cancer related genes identified by comparison with Pubmed Gene lists. Pro- and anti-cancer genes were curated manually. Some genes may belong to more than one category. SO-HFD values are significantly different (q-value ≤ 0.05) from both Viv and HFD for all genes except for Hyou1, Idh2, Lars2, Sc25a30, Cish, Dkk4, Socs3 and Wif1 where SO-HFD is significantly different only from HFD.
Fig 9.
Dysregulation of Cyp genes in HFD and SO-HFD livers.
A) Dysregulated (≥1.5-fold log2) Cyp genes in liver RNA-seq of male mice at 35 weeks. B) Absolute expression in FPKM of various Cyp genes as in Fig 8. SO-HFD is significantly different (q-value ≤ 0.05) from both Viv and HFD for all genes except Cyp4a14 (SO-HFD not different from Viv) and Cyp17a1 (SO-HFD not different from HFD).
Fig 10.
PUFA metabolites are enriched in SO-HFD versus HFD at 35 weeks.
A) Cytoscape visualization of PUFA pathway enrichment (overall 8.4-fold) in SO-HFD versus HFD at 35 weeks. Circles indicate fold enrichment for SO-HFD versus HFD at 35 weeks (bold): red or pink, significantly (P ≤ 0.05) or trending significantly (P ≤ 0.1) up; blue or purple, significantly or trending significantly down; black, no change. Size of circles depicts relative fold change. Bar graphs below each metabolite show relative change for other comparisons as noted. B) Table showing fold change in medium chain and long chain fatty acids for the indicated comparisons. Significance for all data was set at P ≤ 0.05 and approaching significance at 0.05 < P < 0.10 as determined by Welch’s two-sample t-test.
Fig 11.
Hepatic metabolite levels change with diet and over time.
Box-plots of liver metabolomics data from male mice fed the indicated diets for 16 or 35 weeks showing changes in levels of A) Linoleic acid, B) Arachidonic acid, C) 12-HETE, D) 13 HODE +9 HODE, E) Glutathione, oxidized, F) γ-Tocopherol, G) α-Tocopherol, H) 3-Hydroxybutyrate, I) Lactate, and J) Cholesterol. At 16 weeks SO-HFD is significantly different from HFD for all metabolites except γ-tocopherol, lactate and cholesterol. At 35 weeks there is no statistical difference between SO-HFD and HFD except for linoleic acid, γ-tocopherol and lactate; cholesterol is approaching significance. Significance, P ≤ 0.05; approaching significance, 0.05 < P < 0.10 by Welch’s two sample t-test. (See S4 Dataset for additional box plots.)