The obesity epidemic in the U.S. has led to extensive research into potential contributing dietary factors, especially fat and fructose. Recently, increased consumption of soybean oil, which is rich in polyunsaturated fatty acids (PUFAs), has been proposed to play a causal role in the epidemic. Here, we designed a series of four isocaloric diets (HFD, SO-HFD, F-HFD, F-SO-HFD) to investigate the effects of saturated versus unsaturated fat, as well as fructose, on obesity and diabetes. C57/BL6 male mice fed a diet moderately high in fat from coconut oil and soybean oil (SO-HFD, 40% kcal total fat) showed statistically significant increases in weight gain, adiposity, diabetes, glucose intolerance and insulin resistance compared to mice on a diet consisting primarily of coconut oil (HFD). They also had fatty livers with hepatocyte ballooning and very large lipid droplets as well as shorter colonic crypt length. While the high fructose diet (F-HFD) did not cause as much obesity or diabetes as SO-HFD, it did cause rectal prolapse and a very fatty liver, but no balloon injury. The coconut oil diet (with or without fructose) increased spleen weight while fructose in the presence of soybean oil increased kidney weight. Metabolomics analysis of the liver showed an increased accumulation of PUFAs and their metabolites as well as γ-tocopherol, but a decrease in cholesterol in SO-HFD. Liver transcriptomics analysis revealed a global dysregulation of cytochrome P450 (Cyp) genes in SO-HFD versus HFD livers, most notably in the Cyp3a and Cyp2c families. Other genes involved in obesity (e.g., Cidec, Cd36), diabetes (Igfbp1), inflammation (Cd63), mitochondrial function (Pdk4) and cancer (H19) were also upregulated by the soybean oil diet. Taken together, our results indicate that in mice a diet high in soybean oil is more detrimental to metabolic health than a diet high in fructose or coconut oil.

Funding: This work was supported by the National Institute of Environmental Health Sciences T32 5T32-ES018827 to PD ( http://www.niehs.nih.gov/ ), the National Institutes of Health R01DK053892 to FMS ( http://nih.gov/ ), and University of California Riverside Collaborative Seed grant to FMS ( http://www.research.ucr.edu/ ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2015 Deol et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

In this study, we examined the effect of both soybean oil and fructose on the development of obesity and its associated co-morbidities in C57/BL6 male mice, in the context of a diet moderately high in total fat. We formulated diets with 40% kcal fat from coconut oil (HFD) or coconut oil plus soybean oil (SO-HFD) and then supplemented those with fructose (F-HFD and F-SO-HFD). The total amount of fat is comparable to current American consumption [ 35 ] as is the soybean oil [ 16 , 36 ] and fructose [ 37 ]. Mice on the high soybean oil diet (SO-HFD) showed increased weight gain, adiposity, fatty liver with balloon injury, diabetes and IR, compared to mice on the HFD. Fructose in the diet (F-HFD and F-SO-HFD) had less severe metabolic effects than soybean oil but caused rectal prolapse and seemed to synergize with soybean oil to increase kidney weight. The SO-HFD produced distinct changes in the liver transcriptome from the HFD, most notable of which was a global dysregulation of cytochrome P450 (Cyp) genes as well as genes involved in obesity, diabetes, lipid metabolism and cancer. Metabolomic analyses of the HFD and SO-HFD livers revealed a greater accumulation of PUFAs and their metabolites but also increased antioxidant capacity and decreased cholesterol in SO-HFD. Taken together, these results indicate that while there may be some health benefits to a diet rich in soybean oil, and while fructose induced some negative effects in the gut and kidney, overall soybean oil induced more obesity, diabetes, IR and liver injury than either fructose or saturated fat from coconut oil in mice.

Another component of the American diet that has increased substantially in the last four decades is fructose, primarily in the form of high fructose corn syrup (HFCS) in processed foods and sodas [ 26 – 28 ]. Fructose consumption has increased from ~37 g/day in 1977 [ 29 ] to ~49 g/day in 2004, with the highest intake among teenage males (75 g/day) [ 30 ]. Despite extensive investigation of the effects of HFCS, especially in human studies (reviewed in [ 28 , 31 ]), there is still a debate over the role of fructose in the development of obesity and the Metabolic Syndrome: while some studies link the increase in obesity to an increased use of HFCS in the American diet [ 32 ], others cite a lack of definitive clinical evidence on the topic [ 33 ]. Aside from one recent study that examined the effects of soybean oil and fructose on butyrate production in rat intestine [ 34 ], we found no reports in the literature that compared the metabolic effects of HFCS to those of soybean oil, nor any that examined these two components together.

The recommendation for decreased saturated fat consumption, as well as other factors, led to a dramatic, >1000% increase in the consumption of soybean oil in the U.S. from 0.01 to 11.6 kg/yr/capita between 1909–1999 [ 16 ]. Approximately 40 million tons of soybean oil were produced worldwide in 2007, which is about one half of all the edible vegetable oil and one-third of all fats and seed oils produced [ 17 ]. Soybean oil is heavily used in processed foods, margarines, salad dressings and snack foods, and is the oil of choice in many restaurants and fast food establishments [ 18 ]. While there has been extensive investigation of the role of various other dietary components in obesity, especially SFAs, soybean oil has received relatively little attention. Soybean oil is high in PUFAs whereas most experimental animal studies employ diets high in saturated fats, typically lard. While several studies have looked at the effects of soybean oil on obesity and diabetes, the experimental designs were quite variable as were the results [ 19 – 25 ]. There has also not been any global transcriptomics or metabolomics analysis of the effects of soybean oil on the liver, a primary metabolic organ.

Saturated fatty acids (SFAs) were deemed unhealthy due to studies in the 1950s and 1960s that showed a positive correlation between dietary SFAs and the risk for cardiovascular disease [ 6 , 7 ]. As a result, nutritional guidelines were developed that encouraged people to reduce their intake of saturated fat, typically found in meat and dairy products, and increase their intake of polyunsaturated fatty acids (PUFAs) found in plant oils [ 8 , 9 ]. These guidelines are still in effect today [ 10 ]. In recent years, however, there has been a shift in the dialogue surrounding which dietary fats are the most harmful, with some studies suggesting a reconsideration of nutritional guidelines [ 11 , 12 ]. In particular, there is a growing body of evidence that suggests that saturated fat from sources such as coconut and palm oil, which are rich in medium chain triglycerides (MCTs), may actually be beneficial for the prevention and treatment of the Metabolic Syndrome [ 13 – 15 ].

There has been an alarming increase in obesity and its associated co-morbidities—diabetes and heart disease—in the U.S. during the last four decades. Recent estimates suggest that 36% of the U.S. population is currently obese and by 2030 this will increase to ~50% [ 1 , 2 ]. Furthermore, obesity is no longer a problem of developed countries but has become a major global health issue [ 3 ] with an estimated 3.4 million deaths worldwide being attributed to it annually [ 4 ]. Often associated with obesity are diabetes, insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD), which along with heart disease and hypertension, are referred to as the Metabolic Syndrome [ 5 ]. There are many contributing factors to obesity, including genetics, lifestyle, environmental factors and microbiota, but diet is still one of the most relevant, both in terms of the number of calories that are consumed as well as the source of those calories.

Data are presented as means ± standard error of means. One-way ANOVA with Tukey’s post-hoc analysis (GraphPad Prism version 6 for Mac, GraphPad Software, La Jolla, CA USA) was used to test for differences between groups. Welch’s two-sample t-test and an estimate of the false discovery rate (q-value) were used for analyzing the metabolomics data. Student’s t-test was used when comparing two conditions. Statistical significance for all data was set at P ≤ 0.05 and approaching significance at 0.05 < P < 0.10.

Livers from mice that had been fed Viv, HFD or SO-HFD for 16 weeks or 35 weeks were harvested, rinsed briefly in PBS, and immediately frozen in liquid nitrogen. Frozen liver samples (6–8 per treatment group) were shipped to Metabolon Inc (Durham, NC) where they were extracted and prepared for analysis using a previously described standard solvent extraction method [ 51 ]. The extracted samples were split into equal parts for analysis on the GC/MS and LC/MS/MS platforms. Also included were several technical replicate samples created from a homogeneous pool containing a small amount of all study samples. The analysis yielded a dataset comprising a total of 398 compounds of known identity (referred to as biochemicals). Metabolic pathways were visualized using the Cytoscape plugin in the Metabolync Portal ( https://portal.metabolon.com )

Quantitative real-time PCR (qRT-PCR) was performed using the Bio-Rad-CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) to verify the relative expression of Cidea in the livers of mice fed Viv, HFD or SO-HFD for 16 or 35 weeks. Total RNA was extracted as described above and RT-PCR was performed using the QuantiTect Reverse Transcription Kit (Qiagen) for cDNA synthesis, followed by PCR using the QuantiFast SYBR Green PCR Kit (Qiagen). Thermal cycling conditions were 5 min at 95°C followed by 35 cycles of 10 s at 95°C and 30 s at 60°C. Cidea primers (forward: 5’-ATCACAACTGGCCTGGTTACG-3’; reverse: 5’-TACTACCCGGTGTCCATTTCT-3’) were gifts from Dr. Joseph Baur at University of Pennsylvania, Philadelphia. The relative expression level of Cidea was determined by normalizing to cyclophilin A (Ppia) expression as described previously [ 50 ].

Paired-end sequencing reads were aligned to the mouse reference genome (GRCm38/mm10 assembly) with Tophat v1.2 [ 45 ] and processed by Cufflinks [ 46 ] to assemble transcripts and measure their relative abundances in FPKM units (fragments per kilobase of exon per million fragments mapped). Assembled transcripts from experimental samples were compared with the RefSeq refFlat annotated transcriptome downloaded from the UCSC Genome Browser and examined for differential expression using the Cuffcompare and Cuffdiff utilities included in the Cufflinks package. Cuffdiff was run with FPKM upper-quartile normalization and a false discovery rate (FDR, q-value) threshold of 5%. Dysregulated genes (≥ 1.5 log2 fold change) in SO-HFD versus HFD livers were uploaded to DAVID ( http://david.abcc.ncifcrf.gov ) for functional annotation clustering [ 47 ]. Lists of various disease-associated mouse genes were generated using Pubmed Genes (searched for obesity, diabetes, inflammation, cancer) and MitoCarta (mitochondrial genes) [ 48 ] and cross-referenced with genes significantly altered between HFD and SO-HFD. Venn diagrams were created using the online tool VENNY [ 49 ]. All RNA-seq data have been submitted to GEO, accession number GSE68360.

Total RNA was isolated from liver samples using a miRNeasy kit (Qiagen, Inc., Valencia, CA) and evaluated for purity and concentration by NanoDrop (Wilmington, DE) and Agilent 2100 Bioanalyzer (Santa Clara, CA). Poly(A) + RNA (4 μg) with an RNA Integrity Number (RIN) of 7.6 or higher was used to construct sequencing libraries with the TruSeq Long RNA Sample Prep Kit (Illumina, San Diego, CA). Libraries were validated by Bioanalyzer, pooled in equimolar amounts, and sequenced on an Illumina HiSeq 2000 at the UCR Genomics Core to generate 50 base, paired-end reads. Three biological replicates from livers (large lobe) of mice fed Viv, HFD or SO-HFD for 35 weeks were sequenced twice (nine samples per lane) yielding in >70 million reads per sample, more than twice the recommended number for mammalian tissues [ 44 ].

Tissues were collected and snap frozen in liquid nitrogen before storage at -80°C or fixed in 10% neutral-buffered formalin for 24 h before storing in 30% sucrose solution at 4°C. Liver tissue was put into RNAlater (Ambion-Life Technologies, Carlsbad, CA) for 24 h before storage at -80°C. Mesenteric, peri-renal, gonadal and flank subcutaneous adipose tissues were excised and weighed. Frozen liver tissues were sectioned at 5 μm on a Microm cryostat (Thermo Scientific, Waltham, MA) set to −19°C and then air-dried. Rehydrated liver sections were placed in 100% propylene glycol for 2 min, and stained in 0.5% Oil Red O (Sigma-Aldrich, St. Louis, MO) solution in propylene glycol for 10 min at 60°C. Slides were transferred to an 85% propylene glycol solution for 2 min and rinsed twice with distilled water. The slides were counterstained with Mayer’s hematoxylin for 40 sec, rinsed in running tap water for 3 min followed by 30 sec in distilled water and mounted with glycerin jelly. Images were captured at 40X (Zeiss Axioplan).

To measure glucose tolerance (GTT), mice were fasted overnight (~18 h) according to the standard operating procedures outlined by the NIH Mouse Metabolic Phenotyping Center Consortium [ 43 ] and glucose (2 g/kg body weight) was administered by intraperitoneal (IP) injection of a 20% glucose solution in 0.9% saline. Tail blood glucose was measured at 0 (pre-injection), 15, 30, 60 and 120 min after injection using OneTouch Ultra Glucose Meter and OneTouch Ultra Test Strips (LifeScan Inc, Milpitas, CA). To measure insulin sensitivity (ITT), mice were fasted for 4.5 h and then injected IP with 0.75 U/kg of Humulin R (Eli Lilly and Company, Indianapolis, IN). Tail blood glucose was measured at 0, 15, 30, 60 and 90 min as for the GTT.

The basic high fat diet (HFD) had 40 kcal% total fat with 36 kcal% from coconut oil and 4 kcal% from soybean oil. A small amount of soybean oil was added to all HFDs to provide the essential fatty acids linoleic acid (LA, C18:2) and α-linolenic acid (C18:3) ( Table 2 ) [ 39 – 41 ]. In the high soybean oil diet (SO-HFD) a portion of the coconut oil was replaced with soybean oil to give a final concentration of 21 kcal% fat calories from coconut oil and 19 kcal% from soybean oil, of which 10 kcal% were from LA. (Soybean oil is ~55% LA [ 42 ]). The high fructose diets (F-HFD and F-SO-HFD) had 25.9 kcal% of energy from added fructose and the same fatty acid composition as HFD and SO-HFD, respectively. The total amounts of carbohydrates and protein were constant across all the diets. Regular vivarium (Viv) chow (Purina Test Diet 5001, Newco Distributors, Rancho Cucamonga, CA) was used as a low fat control. Diets were provided in pellet form, twice weekly for up to 16 or 35 weeks; the amount of food consumed was monitored on a per cage basis.

Four isocaloric diets with 4.87 kcal/gm (5.56 kcal total) ( Table 1 ) were formulated in conjunction with Research Diets, Inc. (New Brunswick, NJ). The diets are based on the Surwit diet, which is widely used in diet-induced obesity studies and formulated with elements from the AIN-93 diet. The 5% fiber from cellulose in the AIN diet is replaced with cornstarch [ 38 ].

Care and treatment of animals was in accordance with guidelines from and approved by the University of California Riverside Institutional Animal Care and Use Committee (AUP#20110015). All mice had ad libitum access to food and water (other than the indicated fasting times). At the end of the study, mice were euthanized by carbon dioxide inhalation, in accordance with stated NIH guidelines.

Male C57/BL6 mice were weaned at three weeks of age and assigned randomly to one of the five diets used in the study. The animals were maintained on a 12:12 h light-dark cycle in a non-specific pathogen free vivarium at the University of California, Riverside (UCR). Twelve mice were put on each diet with three to four animals per cage. Individual mouse weights were recorded once a week.

Results

Soybean oil induces more weight gain and adiposity than fructose In order to compare the effects of a diet enriched in soybean oil to one consisting primarily of saturated fat from coconut oil, and to examine the effects of fructose, we designed four isocaloric diets: high fat diet (HFD, 40 kcal% fat, primarily from coconut oil), soybean oil-enriched HFD (SO-HFD), fructose-enriched HFD (F-HFD) and fructose-enriched SO-HFD (F-SO-HFD) (Table 1). The total fat content in these diets is similar to the current American diet (37–39 kcal%) as is the amount of soybean oil, based on LA composition (~10 kcal%) (Table 1) [16,36,52]. Coconut oil, which consists mainly of saturated fats of chain length 12 to 18 (Table 2), was used as the primary source of fat as it is naturally low in LA and other PUFAs, whereas diets made from lard (which is typically used in rodent studies) can have variable amounts of PUFAs depending on what the animals have been fed [24,53,54]. Therefore, the use of coconut oil allowed us to study the metabolic effects of soybean oil in a saturated fat background, without affecting the final PUFA concentrations. The amount of fructose in our diets (25 kcal%) is comparable to human consumption and that used in rodent studies [55–58]. Male C57/BL6 mice at weaning were put on one of the four HFDs or a standard low fat, high fiber vivarium chow (Viv). Food intake did not differ significantly between the four HFDs (S1 Fig). The Viv chow-fed mice consumed more grams of food than the HFD-fed mice: this is to be expected since the chow is high in fiber and low in calories (3.36 kcal/gm) compared to the HFD (4.87 kcal/gm). Mice fed SO-HFD gained more weight and at a faster rate than mice fed HFD (Fig 1A left). Addition of fructose also increased body weight above HFD (Fig 1A right), although not as much as soybean oil (Fig 1B left). SO-HFD mice gained slightly more weight than F-SO-HFD mice, although this difference was significant only between weeks 8 and 16 (Fig 1B right). Weights of mice on the high fructose diets, irrespective of soybean oil content, did not differ significantly from each other (Fig 1C). PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pone.0132672.g001 The amount of mesenteric and subcutaneous white adipose tissue (WAT) was significantly greater in SO-HFD than HFD mice; the amount of peri-renal fat was trending in the same direction. Fructose-fed mice (F-HFD and F-SO-HFD) had similar amounts of mesenteric fat and peri-renal fat as SO-HFD mice but lower amounts of subcutaneous WAT. SO-HFD mice had the lowest amount of gonadal WAT compared to the other three HFDs although the difference was not statistically significant (Fig 1D). The most notable difference was in the subcutaneous fat where SO-HFD was markedly greater than the other three HFDs (Fig 1D).

Fructose increases liver/body weight ratio and synergizes with soybean oil to increase kidney weight; coconut oil increases spleen weight Liver size is well known to adjust to body size. Mice fed F-HFD or SO-HFD had larger livers (by weight) at the time of harvest compared to the Viv chow fed mice (Fig 2A left). When normalized to body weight, F-HFD had the greatest liver-to-body weight ratio, while F-SO-HFD was the lowest, although the differences were not statistically significant (Fig 2A right). PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pone.0132672.g002 Spleen weight was examined as a potential sign of infection, liver disease and inflammation. All four HFDs had elevated spleen weights compared to Viv, although the difference between SO-HFD and Viv was not statistically significant (Fig 2B). Kidneys showed a profile similar to spleen, although with larger differences between the diets: F-SO-HFD had the largest kidney weight while SO-HFD was similar to Viv (Fig 2C). These results suggest that the diet high in saturated fat from coconut oil may have a negative effect on the immune system and that soybean oil may counter those effects. In contrast, in the kidney there may be a synergistic effect between fructose and soybean oil.

Intestinal length is shortened by all four HFDs Since the intestines play a major role in nutrient absorption [59], we examined their overall morphology. Mice on all four HFDs had significantly shorter small intestines compared to Viv-fed mice (Fig 3A and 3B). Colon length was also shorter in these mice although the difference was significant only between Viv versus SO-HFD or HFD. These effects are most likely due to the reduced amount of fiber in the HFD diets compared to the fiber-rich Viv chow as fiber is known to increase intestinal mass and crypt cell production [60]. Colonic inflammation and gross changes in colon morphology were not observed in the colons of any of the diet-treated mice (data not shown), although all four HFDs had decreased crypt length in the distal colon (Fig 3C). In contrast, in the proximal colon SO-HFD had a significantly shorter crypt length than Viv; crypt length was also shorter than in HFD although that difference was not significant (Fig 3D). It is known that crypt length and response of the colonic epithelial cells to external stimuli can vary along the length of the rodent intestine [61,62]. The most notable effect on the gut was that fructose dramatically increased the incidence of rectal prolapse to 30% in F-SO-HFD and 44% in F-HFD (Fig 3E). Taken together, these results indicate that soybean oil may protect against the effects of a diet high in saturated fat (and/or low in fiber) in the distal but not the proximal colon, and that fructose severely impacts the rectum. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pone.0132672.g003

Soybean oil induces diabetes, glucose intolerance and insulin resistance (IR) Since there is a considerable debate in the literature about whether HFCS in sodas or processed foods contributes to diabetes [33,63], we examined glucose tolerance and insulin sensitivity by GTT and ITT. To our surprise, we found that at 20 weeks the F-HFD did not cause diabetes (fasting blood glucose level > 200 mg/dL) whereas the SO-HFD did (Fig 4A). Furthermore, the F-HFD mice were just barely less tolerant to glucose than the Viv mice while the SO-HFD were extremely intolerant. Interestingly, the addition of fructose to SO-HFD (F-SO-HFD) actually slightly ameliorated the glucose intolerance of SO-HFD. Notably, the diet consisting primarily of coconut oil (HFD) did not show any diabetes or glucose intolerance at 20 weeks. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pone.0132672.g004 Even more striking were the results of the insulin tolerance test. At 33 weeks, the SO-HFD mice were the most insulin resistant and much more so than F-SO-HFD mice, which were indistinguishable from F-HFD and HFD (Fig 4B). All told, these results indicate that a moderately high fat diet of coconut oil, either in the presence or absence of fructose, does not induce significant diabetic symptoms (elevated fasting blood glucose and glucose intolerance) while isocaloric diets with soybean oil (either with or without fructose) do. Counter intuitively, our results also suggest that the addition of fructose to the diet may even protect against the IR caused by soybean oil.

Soybean oil causes fatty liver and hepatocyte ballooning Since the liver is a major metabolic organ involved in lipid metabolism, we stained the mouse livers with Oil Red O (ORO) and observed fatty livers in both the soybean oil and fructose-fed mice although there were important differences between the two diets (Fig 5). While fructose caused excessive but typically fairly uniform fat deposition (Fig 5C and 5D), as has been observed previously [64,65], the SO-HFD livers had very large lipid droplets that were consistently accompanied by severe hepatocyte ballooning, suggesting potential liver damage (Fig 5E). The macrovesicular steatosis and balloon injury was also observed in livers of mice that had been on SO-HFD for just 16 weeks although the effects were not as large as at 35 weeks (Fig 5F). Despite the fatty livers and extensive tissue injury (in the case of SO-HFD mice), there was minimal liver fibrosis in all five diets (data not shown). PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pone.0132672.g005

Soybean oil causes a dysregulation of hepatic Cyp gene expression The single most highly represented family of dysregulated genes (≥1.5-fold log2) was that of the cytochrome P450 (Cyp) genes (30 genes total) (Fig 9A). The most prevalent subfamilies were Cyp2c (nine genes total) and Cyp3a (six genes), followed by Cyp2b (three genes), and Cyp4a (three genes), all of which are in the LA or arachidonic acid (AA) metabolism pathways and most of which also metabolize steroids (Fig 9B). At first pass, this is consistent with soybean oil being highly enriched for LA and AA being a metabolite of LA. However, paradoxically, genes that encode P450 enzymes and use LA as a substrate, such as Cyp2c54, were downregulated even more in SO-HFD than HFD. Interestingly, Cyp3a family members (Cyp3a16, Cyp3a41a, Cyp3a41b, Cyp3a44), which play an important role in drug metabolism, are significantly downregulated in HFD livers but somewhat less downregulated in SO-HFD (Fig 9B and S3 Dataset). In contrast, a number of other Cyps (e.g., Cyp2a22, 2b9, 2b13, 2c38, 4a14, 46a1) are uniquely upregulated in SO-HFD. It is possible that downregulation of the Cyp3a family triggers a compensatory upregulation of these other Cyp genes, as was shown previously in Cyp3a-/- mouse studies [81]. Cyp17a1, an important enzyme in the steroidogenic pathway, is increased by both SO-HFD and HFD, while two other Cyp genes involved in cholesterol and bile acid metabolism (Cyp46a1 and Cyp7b1) show opposite effects in terms of activation in SO-HFD or HFD livers. Importantly, the expression of Cyp7b1, which is involved in the conversion of cholesterol to bile acids, is greatly reduced in SO-HFD but not HFD. This could result in lower levels of bile acids, which are known to play anti-obesogenic roles [82,83]. (See S3 Dataset for a complete list of significantly dysergulated Cyp genes.) PPT PowerPoint slide

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larger image TIFF original image Download: 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). https://doi.org/10.1371/journal.pone.0132672.g009