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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Toxicol Appl Pharmacol. 2020 Nov 18;410:115337. doi: 10.1016/j.taap.2020.115337

Diets Enriched with Coconut, Fish, or Olive Oil Modify Peripheral Metabolic Effects of Ozone in Rats

Samantha J Snow 1,*, Andres R Henriquez 2, Jenifer I Fenton 3, Travis Goeden 3, Anna Fisher 1, Beena Vallanat 4, Michelle Angrish 1, Judy E Richards 1, Mette C Schladweiler 1, Wan-Yun Cheng 1, Charles E Wood 1,*, Haiyan Tong 1, Urmila P Kodavanti 1,**
PMCID: PMC8552221  NIHMSID: NIHMS1652926  PMID: 33217375

Abstract

Dietary factors may modulate metabolic effects of air pollutant exposures. We hypothesized that diets enriched with coconut oil (CO), fish oil (FO), or olive oil (OO) would alter ozone-induced metabolic responses. Male Wistar-Kyoto rats (1-month-old) were fed normal diet (ND), or CO-, FO-, or OO-enriched diets. After eight weeks, animals were exposed to air or 0.8 ppm ozone, 4h/day for 2 days. Relative to ND, CO- and OO-enriched diet increased body fat, serum triglycerides, cholesterols, and leptin, while all supplements increased liver lipid staining (OO>FO>CO). FO increased n-3, OO increased n-6/n-9, and all supplements increased saturated fatty-acids. Ozone increased total cholesterol, low-density lipoprotein, branched-chain amino acids (BCAA), induced hyperglycemia, glucose intolerance, and changed gene expression involved in energy metabolism in adipose and muscle tissue in rats fed ND. Ozone-induced glucose intolerance was exacerbated by OO-enriched diet. Ozone increased leptin in CO- and FO-enriched groups; however, BCAA increases were blunted by FO and OO. Ozone-induced inhibition of liver cholesterol biosynthesis genes in ND-fed rats was not evident in enriched dietary groups; however, genes involved in energy metabolism and glucose transport were increased in rats fed FO and OO-enriched diet. FO- and OO-enriched diets blunted ozone-induced inhibition of genes involved in adipose tissue glucose uptake and cholesterol synthesis, but exacerbated genes involved in adipose lipolysis. Ozone-induced decreases in muscle energy metabolism genes were similar in all dietary groups. In conclusion, CO-, FO-, and OO-enriched diets modified ozone-induced metabolic changes in a diet-specific manner, which could contribute to altered peripheral energy homeostasis.

Keywords: Ozone, fish-oil-enriched diet, olive oil-enriched diet, coconut-oil-enriched diet, metabolic homeostasis

INTRODUCTION

Epidemiological studies have identified a clear relationship between air pollutant exposure and various systemic metabolic conditions, including diabetes (Alderete et al., 2018; Puett et al., 2019), obesity (An et al., 2018), and cardiovascular disease (Rajagopalan et al., 2018). Metabolomic profiling has further identified distinct effects of air pollution on circulating metabolite biomarkers (Miller et al., 2016a; Lucht et al., 2018; Matthiessen et al., 2018; Rajkumar et al., 2019; van Veldhoven et al., 2019). Experimental evidence using animal models further supports a causal relationship between metabolic alterations and exposure to air pollutants, both acute (Bass et al., 2013; Miller et al., 2015) and long-term (Pardo et al., 2018; Qiu et al., 2017; Goettems-Fiorin et al., 2016; Miller et al., 2016b).

Ozone is a ubiquitous air pollutant with well-established adverse human health effects (U.S. EPA, 2020). The systemic metabolic effects of ozone are mediated primarily through neuroendocrine pathways. In previous work, we have shown that acute exposure to ozone is associated with the activation of sympathetic-adrenal-medullary (SAM) and hypothalamus-pituitary-adrenal (HPA) axes leading to release of epinephrine and corticosterone (Bass et al., 2013; Miller et al., 2015; 2016a; 2016c; Snow et al., 2018a) and concomitant inhibition of the release of thyroid stimulating hormone (TSH), luteinizing hormone (LH), and prolactin (PRL) (Henriquez et al., 2019). We have also shown that these neuroendocrine changes are coupled with metabolic alterations characterized by glucose intolerance, increased gluconeogenesis, suppression of insulin release from pancreas, adipose lipolysis, and muscle protein catabolism (Miller et al., 2015; 2016a, 2016b, 2016c). When the circulating stress hormones are depleted through adrenalectomy, these effects of acute ozone exposure on metabolic indicators and pulmonary injury/inflammation are diminished (Miller et al., 2016c; Henriquez et al., 2017; 2018), implying that a classical fight-or-flight stress response is induced, resulting in organ-specific metabolic and immune changes.

Given this evidence, and the broad impact of dietary factors on metabolism, it is conceivable that nutritional status, including dietary supplements, may alter metabolic responses to an air pollution exposure. A number of experimental animal and clinical studies have investigated how carbohydrate- or lipid-rich diets and popular dietary supplements can alter responsiveness to air pollutants (reviewed in Whyand et al., 2018). This evidence suggests that the specific types of supplements and/or nutrients may have differential interactions with the biological response induced by air pollutant stressors. For example, it has been shown in clinical studies that dietary intake of n-3 and n-9 monounsaturated fatty acids supplements resulted in reduction of particulate matter-induced acute vasoconstriction (Tong et al., 2012; 2015). We have recently shown that in a rat model, n-3 supplementation through a fish oil (FO)-enriched diet reduced ozone-induced vasoconstriction (Snow et al., 2018b). However, in the same study, we noted that this supplement was associated with impairment of lipid metabolism in the lung and accumulation of foamy macrophages. Dietary fatty acids are readily incorporated into cell membranes and other structural compartments. The changes in systemic, membrane, and cellular fatty acids can in turn alter receptor activities and cellular functions (Cholewski et al., 2018; Lankinen et al., 2018), as well as metabolic responses to air pollutants.

Based on our recent study that diets enriched with FO (high in n-3 fatty acids), olive oil (OO, high in monounsaturated fatty acids), or coconut oil (CO, high in saturated fatty acids) influenced acute ozone-induced vascular contractility and pulmonary response in rats (Snow et al., 2018b), we predicted that these dietary supplements might impact ozone-induced systemic metabolic alterations. The goal of this companion study was to examine how diets enriched with CO, FO vs OO will affect metabolic processes in liver, muscle, and adipose tissues at baseline and after acute ozone exposure. We hypothesized that since lipid-rich diets increase circulating triglycerides and cholesterols, they may dampen ozone-induced adipose lipolysis and metabolic alterations in liver and muscle tissue. We also postulated that ozone-induced changes in expression of genes involved in metabolic processes in liver, adipose tissue, and muscle tissue will be impacted by varied lipid-supplemented diets.

MATERIALS AND METHODS

Animals

Three-week old, male Wistar Kyoto (WKY) rats (Charles River Laboratories, Raleigh, NC) were acclimatized two/cage in polycarbonate cages with hardwood chip bedding in an AAALAC-approved animal facility (12h light/dark cycle, 23±1°C). All animal received Purine 5001 diet until the start of dietary regimen (Ralston Purina Laboratories, St. Louis, MO). Diet and water were provided ad libitum. Experimental protocols were approved by the U.S. Environmental Protection Agency’s Institutional Animal Care and Use Committee (IACUC).

Diets

Starting at 4 weeks of age (until the end of the study), animals were fed either a normal standard laboratory diet (ND; Purina 5001, Ralston Purina Laboratories, St. Louis, MO) or a diets enriched with 6% by weight coconut oil (CO; Teklad Custom Research Diets #TD.140728, Harlan Laboratories, Inc., Indianapolis, IN), fish oil (FO; Teklad Custom Research Diets #TD.140729, Harlan Laboratories, Inc.), or olive oil (OO; Teklad Custom Research Diets #TD.140727, Harlan Laboratories, Inc.) as has been reported in our previous study (Snow et al., 2018b). Detail about diet composition is provided in Supplementary Materials, Table 1. In brief, TD.140728 diet supplemented with CO had no eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) but had small amounts of monounsaturated fatty acids (n-6; ~6.2 g/kg diet). TD.140727 diet supplemented with OO contained no EPA or DHA and had 45.5 g oleic acid/kg diet (monounsaturated, n-9) and small amount of n-6 fatty acids (~11 g/kg diet). TD.140729 diet supplemented with menhaden FO contained EPA (10 g/kg diet) and DHA (~6.5 g/kg diet) and small amounts of oleic acid (n-9 fatty acid; 8.6 g/kg diet) and linoleic acid (n-6 fatty acid; 6.5 g/kg diet). Each diet was stored in refrigerated rooms and once taken out, was used in 2–3 days. Additional details regarding diet composition including specific fatty acid content have been described previously (Snow et al., 2018b). The group of rats fed ND were used to compare data to previous ozone exposure studies conducted in WKY rats at our animal facility (Miller et al., 2015; 2016b, 2016c; Henriquez et al., 2018). To assure adequate consumption and palatability of each diet, food consumption rates were determined daily for 5 days during week 1 and week 5 of the dietary regimen. Daily food consumption was calculated for each rat and averaged over 5 days for week 1 and 5.

Body Composition

Body weights were assessed prior to necropsy. Body composition was assessed between 7–8 weeks after beginning the dietary regimen prior to ozone exposure. A Bruker Minispec LF90 II TD-NMR body composition analyzer (Bruker Optics, Inc., Billerica, MA) was used to measure lean body mass (%), body fat (%), and body fluid (%) as previously described (Gordon et al., 2016).

Ozone Exposure

A silent arc discharge generator (OREC, Phoenix, AZ) was used to generate ozone from oxygen, which was transported to Rochester style “Hinners” chambers using mass flow controllers (Coastal Instruments Inc., Burgaw, NC). After the animals were on the diets for 8 weeks, they were exposed for 2 consecutive days to filtered air or 0.8 ppm ozone for 4h/day. Air temperature, relative humidity, and ozone concentration were continuously monitored throughout the exposures and previously reported (Snow et al., 2018b).

Glucose Tolerance Testing (GTT)

Immediately following the first day of ozone exposure, GTT was performed. Animals were fasted for approximately 6h prior to testing. After exposure, baseline glucose measurements (0 min time point) were taken using a Bayer Contour glucometer and strips via a tail prick with a sterile needle. Rats were then given an I.P. injection of glucose (2 g/kg/10 mL; 20% D-glucose solution in saline). Every 30 min for the next 120 min post glucose injection, blood glucose measurements were taken for a total of 5 readings. Area under the curve was measured using the trapezoidal method.

Necropsy and Sample Collection

Two cohorts of animals were used in this study with identical dietary and exposure conditions. Cohort 1 was used to obtain liver samples for Oil Red O staining (n=8/group); Cohort 2 was used for body composition, GTT, and for collection of blood and all other tissues (n=8/group). Within 2h following the second day of ozone exposure, animals were euthanized with an overdose of sodium pentobarbital (Fatal-Plus diluted 1:1 with saline; > 200 mg/kg; I.P. Vortech Pharmaceuticals, Ltd., Dearborn, MI). Liver samples from Cohort 1 were collected and processed for lipid staining. Briefly, a standardized portion from a liver lobe was collected from each animal, embedded in Histoprep media (Fisher Scientific, Fair Lawn, NJ), and transferred to dry ice for preparing a frozen tissue block and later sectioning. From Cohort 2, serum samples were collected from the abdominal aorta. In addition, liver tissue, left-side abdominal visceral adipose tissue, and gracilis muscle from the left leg were collected, flash frozen in liquid nitrogen, and stored at −80°C for RNA analysis.

Serum/Plasma Analysis of Lipids and Hormones

Total cholesterol (TECO Diagnostics, Anaheim, CA), low-density lipoprotein (LDL) (Thermo Fisher Scientific, Inc., Middletown, VA), high-density lipoprotein (HDL), and triglyceride levels (TECO Diagnostics) were measured in serum using kits modified for the Konelab Arena 30 system (Thermo LabSystems, Espoo, Finland). The results of total cholesterol and triglycerides analysis are recently published (Snow et al., 2018b). Serum branched-chain amino acid levels (BCAA) were measured using an ELISA kit and protocol based on chemiluminescence detection (Abcam, Cambridge, MA). Insulin serum levels were detected using rat-specific chemiluminescence assay kit (Millipore, Billerica, MA) via manufacturer’s instructions. Serum samples collected from rats were analyzed for leptin using rat-specific electrochemiluminescence assays (Meso Scale Discovery, Gaithersburg, MD) via manufacturer’s instructions.

Fatty Acid (FA) Analysis

Serum samples from air control animals were analyzed for saturated, n-3, n-6, and n-9 fatty acids, and all samples from air and ozone exposure groups were analyzed for total fatty acids. For the creation of fatty acid methyl esters (FAMEs), a modified methylation protocol described by Jenkins (2010) was employed. A 100 μL aliquot of serum was added to a sample containing 20 μg of internal standard (methyl 12-tridecenoate, U-35M, Nu-Chek Prep, Elysian, MN) suspended in isooctane. Two mL of 0.5 N anhydrous potassium methoxide was added and samples were heated at 50˚C for 10 min and cooled to RT. Then 3 mL of 5% methanolic HCl was added and, samples were heated at 80˚C for 10 min. After cooling, 2 mL of water and 2 mL hexane were added, and the upper organic phase was removed and dried to obtain FAMEs. FAMEs were suspended in 1 mL isooctane, and 200 mL was transferred to gas chromatography vials. Samples were stored at –20°C until analysis.

The PerkinElmer (Waltham, MA) 680/600S gas chromatography-mass spectrometry (GC-MS) in the electron impact mode (70 eV) equipped with an Agilent Technologies (Santa Clara, CA) HP-88 column (100 m, 0.25 mm ID, 0.2 μM film thickness) was used for FAME quantification. Injection temperature was set at 250°C, and the GC temperature parameters were as follows: initial temperature at 80°C for 4 min; ramp 13°C/min to 175°C; hold 27 min; ramp 4°C/min to 215°C; hold 35 min with a helium flow of 1 mL/min (modified from Kramer, et al., 2008). The MS data were recorded in full scan mode (mass range of m/z 70–400 amu). MS transfer line and ion source temperature were set at 180°C.

A GC reference standard was created by combining Supelco 37 Component FAME Mix (Sigma-Aldrich, St. Louis, MO) with mead acid, docosatetraenoic acid, n-3 docosapentaenoic acid (DPA), n-6 DPA, and palmitelaidic acid (Cayman Chemicals, Ann Arbor, MI). Fatty acids were identified using the reference standard and confirmed by the EI mass fragmentation in the GC-MS. Quantification of FAMEs was conducted using internal standard and chromatographic peak area. Data were analyzed using MassLynx V4.1 SCN 714 (Waters Corporation, Milford, MA).

Histological Assessment of Liver Lipid Accumulation

Fresh frozen liver tissue sections (10 micrometers in thickness) were prepared from frozen Histoprep-blocks on a cryotome and mounted on slides. Sections were stained with Oil Red O and scanned at 40x magnification using the Aperio Digital Pathology Slide Scanner (Leica Biosystems, Buffalo Grove, IL). To quantify lipid, one Oil Red O-labeled liver slide was analyzed for each animal using the Aperio Positive Pixel Count macro. Available sections of adrenal gland were used as a positive control. Staining was quantified using positivity (number of positive pixels divided by total number of pixels) and total number of strong positive pixels per section of liver. Image analysis was performed blinded to treatment.

RNA Isolation and Targeted Illumina mRNA Sequencing

Approximately 20 mg of liver, gracilis leg muscle, or white abdominal adipose tissues from each animal were extracted for RNA isolation using RNeasy mini kits (Qiagen, Valencia, CA). RNeasy Fibrous Tissue Mini Kit was used for muscle RNA isolation and RNeasy Lipid Tissue Mini Kit was used for adipose tissue RNA isolation. RNA yield was determined using Qubit fluorometric quantitation (Thermo Fisher Scientific Inc., Waltham, MA). Custom Illumina® TruSeq® Targeted RNA Expression kits were ordered from Illumina Inc. (San Diego, CA, USA) based on the gene list selected by us that included genes involved in metabolic and pathological processes (Supplementary Materials, Table 2). Targeted RNAseq libraries were prepared from total RNA using the Illumina TruSeq Targeted RNA Expression Guide 15034665 C protocol. Intact total RNA (50 ng) was used per well for first strand cDNA synthesis and followed the guide for a 37 cycle PCR amplification. Resulting libraries were pooled based on tissue type and quantification of the pooled library was done using Agilent Technologies High sensitivity DNA assay chip and Qubit. On-board Clustering and sequencing of libraries was performed using Illumina MiSeq system and V3 kit. 50-Cycle sequencing was performed at a final library concentration of 10 pM and 5% Phix spike-in. TruSeq_CRT_Manifest_TC0040455-CRT.txt was used by Miseq reporter as the manifest file for secondary analysis. Clusters passing filter and percent aligned sequences (>85%) were used as primary quality metrics.

Data Analysis

GraphPad Prism v6.0 software (San Diego, CA) was used for statistical analysis of serum hormones, metabolites, body weight, and body composition data. A Two-Way ANOVA was performed using diet and exposure as independent factors followed by Holm-Sidak’s post-hoc test. Fatty acid data for air-exposed dietary groups were analyzed using One-Way ANOVA followed by Holm-Sidak’s post-hoc test. A p-value < 0.05 was considered statistically significant.

For each tissue, the gene expression data were normalized, median centered, and visualized in a heatmap. For each rat, the number of standard deviations from the median was calculated, averaged per group, and included in the heatmap (row z-score, n=4–6 rats per group). Hierarchical clustering was performed using average linkage clustering method and Euclidean distances for each tissue. The analysis was performed using the online tool provided by http://heatmapper.ca/. Significant differences (p<0.05) were calculated by multiple t-tests grouped analyses without assuming consistent standard deviation and correcting for multiple comparisons using Holm-Sidak method.

RESULTS

Food Consumption, Body Weight, and Body Composition

The body weights of rats receiving CO, FO, and OO supplements were significantly higher than ND (FO>CO>OO) (Fig. 1A). The body composition analysis between 7–8 weeks into dietary regimen indicated slight but significant increases in body fat % (Fig. 1B) and small decreases in body fluid % (Fig. 1D) in rats fed CO and OO, but not FO-enriched diet. This pattern was associated with increases in the % of lean body mass in all three dietary groups including FO (Fig. 1C). As rats grew in weight over 5 weeks, the amount of each diet consumed was increased as expected (Fig. 1E). In general, the daily consumption of CO, FO, and OO supplemented diets was 2–3 grams less than the ND, indicating calorie intake adjustment. The palatability of each dietary supplement was similar across all oil-based diets (Fig. 1E).

Figure 1. Body weight, body composition, and food intake in WKY rats fed a normal or oil-based diet.

Figure 1.

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Body weight (A), body composition (i.e., body fat % (B), lean body mass % (C), body fluid % (D)), and daily food consumption (E). Body weights were obtained at the end of the experimental period. Body composition was assessed between 7–8 weeks after beginning the dietary regiment prior to ozone exposure. Food consumption rates were measured 1 and 5 weeks after animals were placed on either a normal diet (ND) or diet enriched with coconut oil (CO), fish oil (FO), or olive oil (OO). Data show mean ± SEM (n=8/group). † = p<0.05 significantly different than ND group. ‡ = p<0.05 significantly different than OO diet group. § = p<0.05 significantly different than FO diet group. * = p<0.05 significantly different than Week 1 within same diet.

The Impact of Diets on Liver Lipid Accumulation in Air- and Ozone-Exposed Rats

To assess the role of liver in lipid distribution after intake of lipid-enriched diets and ozone exposure, we stained fresh-frozen liver sections with Oil Red O to visualize lipids. Examination of the distribution of lipids in the liver indicated that increased lipid accumulation occurred by CO, FO, and OO in the periportal regions (OO>FO>CO), but not centrilobular regions in both air- and ozone-exposed rats, with acute ozone exposure having no major impact in any of the dietary groups (Fig. 2A). Quantification of lipid accumulation using image analysis indicated that OO resulted in maximum lipid accumulation relative to ND, CO, and FO (Fig. 2B).

Figure 2. Oil-based diets alter liver lipid levels.

Figure 2.

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Representative images of liver sections stained with Oil Red O to detect the presence of triglycerides and lipids following exposure to filtered air or 0.8 ppm ozone, 4h/day for 2 consecutive days in animals fed either a normal diet (ND) or diet enriched with coconut oil (CO), fish oil (FO), or olive oil (OO) (A). Positive staining in the images were determined using the Positive Pixel Count Macro (B). Data show mean ± SEM (n=8/group). † = p<0.05 significantly different than ND group for the same exposure.

Diet-Induced Changes in Circulating Saturated and Unsaturated Fatty Acids

To determine the influence of specific dietary fatty acid enrichment on levels of circulating lipid composition, we assessed serum levels of saturated, n-3, n-6, and n-9 fatty acids in air-exposed rats (Table 1). Saturated fatty acids were significantly increased in animals receiving CO-, FO-, and OO-enriched diets when compared to ND (OO>FO>CO). As expected, n-3 fatty acids in FO were 4–6 times the levels in ND, CO, and OO. Circulating n-6 fatty acids increased only in OO relative to other dietary groups. Levels of circulating n-9 fatty acids were lower than any other fatty acids determined in all dietary groups. Animals fed the OO diet had increased levels of n-9 relative to all other dietary groups (Table 1) as expected since the OO-supplemented diet is rich in n-9 fatty acids (Supplementary Materials, Table 1).

Table 1.

Diet-related changes in circulating lipids in air-exposed animals.

Lipid Type Normal Diet (ND) Coconut Oil (CO)- Enriched Diet Fish Oil (FO)- Enriched Diet Olive Oil (OO)- Enriched Diet
Saturated Fatty Acids (μg/mL) 723.6 ± 41.4 1255.3 ± 75.2 † 1654.1 ± 139.9 †‡ 2770.9 ± 118.9 †‡§
n-3 Fatty Acids (μg/mL) 272.4 ± 21.1 198.1 ± 14.1 1228.6 ± 140.8 †‡ 289.1 ± 8.9 §
n-6 Fatty Acids (μg/mL) 1180.1 ± 66.4 1427.9 ± 133.8 1285.1 ± 131 3183.5 ± 154.6 †‡§
n-9 Fatty Acids (μg/mL) 4 ± 0.7 14.2 ± 0.8 9 ± 1.2 43.2 ± 5.9 †‡§
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Data show mean ± SEM (n=8/group),

†

= p<0.05 significantly different than ND group.

‡

= p<0.05 significantly different than CO diet group.

§

= p<0.05 significantly different than FO diet group.

Diet-Induced Changes in Circulating Lipids in Air- and Ozone-Exposed Animals

Circulating lipid composition can be changed based on specific lipid-enriched diets. While ozone alters circulating lipid distribution in rats and humans as a result of activation of SAM and HPA axes (Miller et al., 2015, Miller et al., 2016a; 2016b; 2016c), we determined the influence of CO-, FO-, and OO-enriched diets on ozone-induced changes in circulating lipids. Total cholesterol was significantly increased by CO and OO, but not FO-enriched diets relative to ND in air-exposed rats. Ozone exposure lead to increases in total cholesterol among animals receiving ND, CO, and OO diets, but not in animals receiving FO (Fig 3A). FO and OO decreased circulating LDL levels relative to ND in air-exposed rats. Ozone-exposure increased LDL levels in animals receiving ND and OO supplemented diet (Fig 3B). Circulating HDL levels were decreased in animals receiving FO and OO relative to those on ND and CO-enriched diets. No ozone effects were noted on HDL levels in any dietary group (Fig. 3C). In air-exposed animals, circulating triglycerides were markedly increased in animals receiving CO and OO, but not in animals receiving FO supplemented diet relative to ND. No major changes occurred in circulating triglycerides due to ozone exposure (Fig. 3D). The data for total cholesterol and triglycerides were recently published in a table form (Snow et al., 2018b). We have previously demonstrated that circulating free and total fatty acids levels are increased after a single ozone exposure as a result of adipose lipolysis (Miller et al., 2015, 2016a). Here we show that circulating total fatty acids increased significantly after a 2-day ozone exposure but only in rats fed CO-enriched diet (Fig. 2E). The lack of effect in ND could be due to 2-day exposure protocol as oppose to as single day exposure. Given that a single ozone exposure is associated with increases in circulating BCAA as a result of SAM and HPA activation, we also wanted to determine if any of these diets influenced this ozone-induced stress response. BCAA levels were not significantly changed by any of the dietary interventions in air-exposed rats; however, the levels were significantly increased by ozone exposure in animals receiving ND and CO but not FO or OO supplemented diets (Fig 3F).

Figure 3. Serum lipids and BCAA levels are altered by ozone exposure and oil-based diets.

Figure 3.

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Serum lipids including total cholesterol (A), low-density lipoprotein (LDL) cholesterol (B), high-density lipoprotein (HDL) cholesterol (C), triglycerides (D), total fatty acids (E) and branched-chain amino acids (BCAA) (F) were measured following exposure to filtered air or 0.8 ppm ozone, 4h/day for 2 consecutive days in animals fed either a normal diet (ND) or diet enriched with coconut oil (CO), fish oil (FO), or olive oil (OO). Data show mean ± SEM (n=8/group). * = p<0.05 significantly different than filtered air group within same diet. † = p<0.05 significantly different than ND group for the same exposure. ‡ = p<0.05 significantly different than CO diet group for the same exposure. § = p<0.05 significantly different than FO diet group for the same exposure.

The Impact of Diets on Ozone-Induced Hyperglycemia and Glucose Intolerance

Ozone exposure induces glucose intolerance in several of our prior studies (Bass et al., 2013, Miller et al., 2015, 2016b; 2016c). To determine if CO, FO or OO dietary supplements influence the effect of ozone-induced glucose intolerance, we performed GTT immediately after the first day of ozone exposure in rats. The dietary supplements in air-exposed rats did not induce hyperglycemia or glucose intolerance, although the air-exposed animals on OO tended to have lower glucose clearance after injection (Fig. 4A). Ozone, on the other hand, caused hyperglycemia in all four dietary groups and induced glucose intolerance (Fig. 4A). CO and FO did not affect this response to ozone; however, animals on OO had significantly exacerbated glucose intolerance (Fig. 4A). Baseline ozone-induced hyperglycemia was not affected by any dietary intervention (Fig. 4B); however, computation of area under the curve clearly indicated exacerbation of ozone response in animals on OO-enriched diet relative to all other dietary groups (Fig. 4C).

Figure 4. Glucose intolerance and hyperglycemia following exposure to ozone in animals fed a normal or oil-based diet.

Figure 4.

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GTT (A) was conducted following exposure to filtered air or 0.8 ppm ozone, 4h/day for 1 day in animals fed either a normal diet (ND) or diet enriched with coconut oil (CO), fish oil (FO), or olive oil (OO). Ozone-induced hyperglycemia (B) was measured at baseline prior to glucose injection. Area under the curve (C) was calculated for each group. Data show mean ± SEM (n=8/group). * = p<0.05 significantly different than filtered air group within same diet. † = p<0.05 significantly different than ND group for the same exposure. ‡ = p<0.05 significantly different than CO diet group for the same exposure. § = p<0.05 significantly different than FO diet group for the same exposure.

Diet- and Ozone-Induced Changes in Circulating Metabolic Hormones

Circulating leptin and insulin were measured to determine how FO- and OO-enriched diets influence metabolic status of rats and how ozone-induced metabolic response is modified by these diets. All three diets relative to ND resulted in increases in circulating leptin in air-exposed rats. Ozone exposure for 2 consecutive days was associated with further increases in circulating leptin in animals receiving CO and FO, but not in ND or OO-supplemented dietary group (Fig 5A). Circulating insulin was not significantly affected by any of the three dietary supplements in air-exposed rats. Ozone exposure, when considering individual dietary supplement, did not significantly decrease insulin levels; however, when the data for all diets were combined, insulin was significantly depleted (p=0.02) (Fig. 5B).

Figure 5. Tissue-specific metabolic markers are altered by ozone exposure and oil-based diets.

Figure 5.

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Leptin (A) and insulin (B) were measured following exposure to filtered air or 0.8 ppm ozone, 4h/day for 2 consecutive days in animals fed either a normal diet (ND) or diet enriched with coconut oil (CO), fish oil (FO), or olive oil (OO). Data show mean ± SEM (n=8/group). * = p<0.05 significantly different than filtered air group within same diet. † = p<0.05 significantly different than ND group for the same exposure.

Interactive Effects of Diet and Ozone on Genes Involved in Metabolic Processes

Since acute ozone exposure is associated with rapid systemic lipid alterations in rodents and humans (Miller et al., 2015; 2016a), we wanted to determine the expression of selected genes involved in lipid and glucose metabolic processes, insulin signaling, and inflammation in liver, adipose tissue, and muscle tissue using custom-designed arrays for 48-genes (Supplementary Materials, Table 2). Expression changes due to diet and ozone for each tissue are described below.

Liver:

There were changes in the liver gene expression due to enriched diets relative to ND in air-exposed rats (Fig. 6A). CO significantly increased acetyl-CoA carboxylase alpha (Acaca) involved in fatty acid metabolism, but these increases did not reach significance in FO or OO. FO-enriched diet decreased expression of 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (Hmgcs1), and sterol regulatory element binding transcription factor 2 (Srebf2) involved in cholesterol biosynthesis in air-exposed rats. OO inhibited only Hmgcs1 in air-exposed rats. Ozone exposure in ND group increased carnitine palmitoyltransferase 2 (Cpt2) and peroxisome proliferator activated receptor gamma coactivator 1 alpha (Ppargc1a), but decreased nuclear receptor subfamily 1 group H member 3 (Nr1h3) and Hmgcs1, suggesting increased glucose and fatty acid metabolism while inhibiting cholesterol synthesis. In rats receiving CO-enriched diet, increase in Ppargc1a was associated with increases in acetyl-CoA carboxylase beta (Acacb) and Acaca, but without inhibition of Hmgcs1 and Nr1h3, suggesting that cholesterol synthesis inhibition effect of ozone was likely dampened in CO-enriched dietary group (Fig. 6A). Ozone-induced changes were not significant for any genes in the livers of FO and OO animals when compared to diet-matched air groups, which may further suggest dampening of ozone effects on cholesterol metabolism.

Figure 6. Heat maps showing diet- and ozone-induced changes in genes involved in metabolic processes in liver, adipose, and muscle tissues.

Figure 6.

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Liver (A), abdominal adipose (B), and muscle tissues (C) were collected following exposure to filtered air 0.8 ppm ozone, 4h/day for 2 consecutive days in animals fed either a normal diet (ND) or diet enriched with coconut oil (CO), fish oil (FO), or olive oil (OO). Gene expression values (average row Z-score from normalized counts; n=4–6/group) were used for hierarchical clustering. Increasing red intensity refers to increasing gene expression and increasing blue intensity refers to decreasing gene expression. * = p<0.05 significantly different than filtered air group within same diet. † = p<0.05 significantly different than ND group for the same exposure.

Adipose tissue:

Few diet-related changes in expression of genes were apparent in adipose tissue of air-exposed rats. CO did not significantly affect adipose tissue mRNA expression in air-exposed rats, however, FO increased Acaca and decreased insulin receptor substrate 1 (Irs1) and insulin receptor substrate 2 (Irs2) genes, which are involved in glucose uptake and metabolism, and CO decreased carnitine O-acetyltransferase (Crat) involved in faty acid transport and beta oxidation, in air-exposed rats relative to the ND air group (Fig. 6B). Some of the ozone-induced changes were common for all diets in adipose tissue, but some were diet-specific. Ozone exposure tended to increase adipose tissue expression of pyruvate dehydrogenase kinase 4 (Pdk4) and plasminogen activator inhibitor 1 (PAI-1 or Serpine1) and significantly decreased Crat, Ppargc1a, AKT serine/threonine kinase 1 (Akt1), AKT serine/threonine kinase 2 (Akt2), Irs1, and solute carrier family 2 member 4 (Slc2a4 or GLUT4) in animals fed ND (Fig. 6B). These genes impacted by ozone in ND are involved in insulin signaling, glucose uptake, and metabolism. This ozone-induced effect was also evident in the CO group; however, this response was somewhat dampened in the FO and OO groups. Ozone exposure led to increases in the expression of Pdk4, lipase E, hormone sensitive type (Lipe), and Serpine1 in animals fed FO- and OO-enriched diets, which may suggest increased oxidative stress and lipolysis (Fig. 6B).

Skeletal Muscle:

There were no significant gene expression changes because of the enriched diet in muscle tissue of air-exposed rats. However, ozone exposure had a significant impact on many genes in muscle regardless of diet. Marked increases in Pdk4, Serpine1, and phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1) after ozone exposure were noted in all dietary groups (Fig. 6C), suggesting oxidative stress-mediated changes. Ozone exposure resulted in inhibition of many genes across all diets. In the ND group, significant decreases were noted for Akt2 and Irs1. Following ozone exposure, decreased expression of glycogen synthase kinase 3 beta (Gsk3b), Crat, Hmgcs1, Srebf2, Akt2, Irs1 and mammalian target of rapamycin kinase (Mtor) were found in animals on CO enriched diet; C-C motif chemokine receptor 2 (Ccr2), Akt2, and Irs1 were significantly decreased in the FO group; and inhibition of Irs1 and Mtor were significant in animals fed OO (Fig. 6C). These changes are consistent with the inhibition of energy metabolism and insulin signaling in all dietary groups including ND.

DISCUSSION

Ozone induces a wide array of metabolic changes in multiple organs through the activation of SAM and HPA axes (Snow et al., 2018a; Miller et al., 2016c). Here, we postulated that metabolic effects induced by ozone will be modified in animals receiving CO (high in saturated fatty acids), FO (high in n-3 fatty acids), or OO (high in n-9, mono- and other polyunsaturated fatty acids) enriched diets in a diet-specific manner. As expected, CO-, FO- and OO-enriched diets increased circulating saturated, n-3, n-6, and/or n-9 fatty acids in air-exposed rats (Table 1). While all dietary supplements increased % lean body mass and liver lipid accumulation, only CO- and OO-enriched diets increased overall body fat %, and circulating cholesterol as well as triglycerides relative to ND (Table 2). In rats fed ND, ozone exposure led to hyperglycemia, glucose intolerance, cholesterolemia, BCAA increase, and inhibition of liver genes involved in cholesterol biosynthesis. Ozone exposure in ND also lead to inhibition of adipose tissue and muscle genes regulating insulin signaling and energy metabolism. FO but not CO dampened ozone-induced increases in cholesterol and BCAA, and adipose tissue gene expression regulating insulin signaling and energy metabolism. Relative to ND, OO also dampened ozone-induced BCAA increases and adipose tissue genes involved in glucose uptake and metabolism while exacerbating ozone-induced glucose intolerance and increased liver Akt expression, suggesting its differential impact on glucose handling (Table 2). Ozone-induced inhibition of genes involved in muscle tissue energy metabolism and increases in genes involved in oxidative stress were generally similar across all dietary groups. Overall, FO and OO dietary supplements blunted acute ozone-induced changes in BCAA, cholesterol, fatty acids, and liver genes involved in cholesterol biosynthesis as well as those in adipose tissue insulin signaling/energy metabolism (Fig. 6). These findings suggest that dietary polyunsaturated fatty acids may offer protection against some metabolic effects induced by ozone in a diet-specific manner.

Table 2.

Summary of ozone effects on on circulating metabolites and tissue gene expression in rats fed ND or CO-, FO-, or OO-enriched diets.

Metabolic process Biological indicators Ozone effect: ND Ozone effect: CO Ozone effect: FO Ozone effect: OO
Hyperglycemia Baseline glucose ↑ ↑ ↑ ↑
Glucose intolerance Glucose tolerance test ↑ ↑ ↑ ↑↑
Cholesterolemia Total cholesterol ↑ ↑ -- ↑
LDL cholesterol ↑ -- -- ↑
HDL cholesterol -- -- -- ↑
Stress response BCAA ↑ ↑ -- --
Total fatty acids -- ↑ -- --
Leptinemia Serum leptin -- ↑ ↑ --
Liver: cholesterol biosynthesis Hmgcs1, Ppargc1a, and Nr1h3 expression ↓ -- -- --
Liver: fatty acid synthesis, oxidation Acaca and Acacb expression -- ↑ -- --
Liver: energy homeostasis and fat metabolism Crat expression -- -- ↑ --
Liver: insulin-mediated glucose transport Akt1 expression -- -- -- ↑
Adipose tissue: oxidative stress senescence, inflammation Serpine-1, Tlr4, and Retn expression ↑↓ ↑↓ ↑↓ ↑↓
Adipose tissue: triglycerides hydrolysis/metabolism Pdk4 and Lipe expression -- -- ↑ ↑
Adipose tissue: glucose uptake/metabolism Irs1, Slc2a4, Akt1, Akt2, and Pdk4 expression ↓ ↓ -- --
Adipose tissue: cholesterol biosynthesis Crat, Srebf2, Acaca, and Ppargc1a expression ↓ ↓ -- --
Muscle: atrophy, oxidative stress, senescence Pdk4, Serpine-1, and Ccr2 expression ↑ ↑ ↑ ↑
Muscle: energy metabolism Akt2, Gsk3b, Mtor, and Irs1 expression ↓ ↓ ↓ ↓
Muscle: lipid biosynthesis Crat, Srebf2, and Hmgcs1 expression -- ↓ -- --
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ND = normal diet; CO = coconut oil; FO = fish oil; OO = olive oil; LDL = low-density lipoprotein; HDL = high-density lipoprotein; BCAA = branched-chain amino acids. Biological processes were predicted based on the functions of a given metabolite or tissue gene expression change. Predicted increases and decreases are indicated by an “↑” or “↓” arrows, respectively, and no significant impact is indicated by “—“. Note that diet effects in air-exposed rats are not emphasized in this table.

Increased intake of n-6 fatty acids and saturated and unsaturated fats has been associated with increased body fat and circulating lipids in animal models (Domínguez-Vías et al., 2017). However, human studies have shown lipid-lowering effects of extra virgin olive oils rich in monounsaturated fatty acids (Elias et al., 2017). Diets enriched in n-3 fatty acids are associated with reduction in body fat and circulating cholesterol and triglycerides (Dias et al., 2017; Liu et al., 2013). The beneficial effects of n-3 containing fatty acids in chronic conditions, including non-alcoholic fatty liver diseases, have been reported (Hodson et al., 2019; Spooner and Jump, 2019). We show here that dietary supplements produced desired effects in our animal model, in that FO increased n-3, OO increased n-6 and n-9 fatty acids, while all supplements increased saturated fatty acids relative to ND (Table 1). Our dietary regimen reproduced the classical effects of n-3 fatty acid-enriched FO such as reduction in circulating triglycerides and cholesterols (Poudyal et al., 2012; Yepuri et al., 2011; Su and Jones, 1993); however, CO and OO resulted in higher fat mass and increases in circulating triglycerides and cholesterol relative to ND group (Fig. 3). The expression changes induced by FO in livers of air-exposed animals showing inhibition of genes involved in cholesterol biosynthesis (Fig. 6) are similar to those previously reported (Garay-Lugo et al., 2016) and may be linked to its cholesterol lowering effect. These findings led us to test the influence of distinct dietary conditions on homeostatic metabolic response to a challenge stressor, ozone.

A single ozone exposure has been associated with a wide-array of metabolic effects in rats and humans mediated via the activation of a stress response (Snow et al., 2018a). Here we show that the acute 2-day ozone-induced changes, such as hyperglycemia, glucose intolerance (Fig. 4), increases in BCAA and circulating cholesterols (Fig. 3) together with inhibition of liver genes involved in cholesterol biosynthesis in rats fed ND (Fig. 6), are consistent with stress-mediated homeostatic metabolic alterations (Miller et al., 2015). However, ozone effects, such as increases in serum fatty acid and leptin in rats fed ND were dampened with a 2-day exposure protocol.

Exposure to air pollutants has been linked to steatosis-like changes in the liver (Yin et al., 2019; Li et al., 2017). Acute ozone exposure has also been shown to induce changes in liver metabolic processes reflective of inhibition of lipid synthesis, increased gluconeogenesis, altered mitochondrial function, and activation of acute phase response (Bass et al., 2013; Miller et al., 2015). Although, acute ozone exposure did not increase pathology of the liver, it did increase the expression of Cpt2 and Ppargca1 and inhibited Hmgca1 and Nr1h3 in livers of ND-fed rats (Fig. 6), which is suggestive of an inhibition of lipid and sterol metabolic processes through increased glucocorticoid activity (Attia et al., 2010). These ozone-induced changes were insignificant in FO and OO animals despite OO exacerbating glucose intolerance. This suggests that ozone effects on lipid metabolism are likely dampened by the increased accumulation of n-3 and n-6 fatty acids in the liver. Targeted experiments will be needed to understand the mechanisms.

Glucose metabolic processes were differentially influenced by diets and ozone. Air-exposed animals receiving OO enriched diets tended to develop glucose intolerance and when exposed to ozone, this glucose intolerance was exacerbated (Fig. 4). Dietary intake of OO has been reported to induce glucose intolerance (Krygsman et al., 2010; Jelinek et al., 2013; Deol et al., 2015) and changes in liver gene expression (Deol et al., 2015) with some of the similar changes noted in this study. Increased Akt1 gene expression in the livers of animals receiving OO-enriched diet may indicate decreased Akt phosphorylation and the inhibition of glucose uptake leading to exacerbated ozone-induced glucose intolerance. Impaired Akt signaling has been linked to glucose intolerance (Pereira-da-Silva et al., 2005). This implies that under the dietary conditions used in this study, OO-enriched diet can contribute to impaired glucose metabolism in the liver and differentially impact ozone-induced changes in metabolic processes.

Since dietary fats are stored in adipose tissue and the fatty acids are readily incorporated in cellular components, it was conceivable that CO, FO, and OO would induce changes in abdominal adipose tissue mRNA expression for metabolic genes. While CO did not significantly influence adipose tissue gene expression relative to ND, FO was associated with inhibition of Irs1 and Irs2 and increases in Acaca in air-exposed rats, suggesting its potential role in reducing glucose uptake in adipose tissue that can feed to fatty acids synthesis. Ozone-induced activation of Serpine1 and Pdk4 in all groups and Lipe in FO and OO groups are markers of increased adipose lipolysis and inflammation. Repeated ozone exposure for 2 weeks in mice has been shown to induce inflammatory gene expression in adipose tissue (Zhong et al., 2016). While ozone-induced inhibition of adipose tissue Crat, Ppargc1a, Akt1, Akt2, Irs1, and Glut4 (Fig. 6B) might indicate inhibition of insulin signaling and glucose metabolism in ND and CO groups, the lack thereof in rats fed FO- and OO-enriched diets might indicate protection.

It has been widely reported that immediately following ozone or particulate pollutant exposures, rodents exhibit adaptive hypothermic response (Gordon et al., 2014; Watkinson et al., 1997), likely linked to diminished activity of the muscle tissue, which represents the largest tissue mass in the body. The metabolic changes after pollutant exposure in muscle are poorly studied, but likely depend on various factors, including the availability of nutrient substrates and the type of a stressor response. Impaired muscle function has been widely studied in smokers who develop chronic obstructive pulmonary disease (Krüger et al., 2015; Kneppers et al., 2019). Although dietary supplements did not significantly influence expression of muscle tissue genes in our selected markers, ozone-induced changes in expression are likely reflective of the metabolic deregulation in association with increased circulating glucocorticoids and adrenaline (Schakman et al., 2013; Snow et al., 2018a). Ozone-induced activation of Pdk4 (Attia et al., 2010; Pettersen et al., 2019) and Pik3r1 (Kuo et al., 2017) and inhibition of Fabp4 (Jiang et al., 2013) and Irs1 (Hu et al., 2019) could be due to increased glucocorticoid effects on muscle cells, with diet having little if any influence on these genes (Fig. 6). Thus, ozone-induced muscle gene expression changes are likely due to endocrine hormone changes such as glucocorticoids, adrenaline, and those related to thyroid function examined in several of our prior studies (Miller et al., 2016b; Henriquez et al., 2019). Moreover, the inhibition of Akt2, Gsk3b, Mtor, and Irs1 expression in ozone-exposed rats may indicate inhibition of energy production consistent with the hypothermic response after ozone exposure.

In the current study, only male WKY rats were examined based on the sensitivity of these rats to inhaled pollutants (Shen et al., 2017), however, females could also be susceptible to metabolic alterations induced by diet and ozone. Acute ozone exposure, used as a challenge stressor, occurred at high concentration (0.8 ppm) that will activate neuroendocrine response in a consistent manner. However, this concentration is not likely to be observed in the ambient environment. Nevertheless, humans inhaling 0.2–0.3 ppm ozone during intermittent exercise have been estimated to receive a similar dose as resting rodents inhaling ozone at 0.8–1 ppm (Hatch et al., 2013). Finally, the use of whole genome mRNA sequencing in multiple organs could provide a more complete systems view of the wide array of metabolic processes impacted by diet and/or ozone exposure.

In conclusion, we show that fatty acid-enriched diets led to increases in circulating fatty acids reflective of dietary composition of saturated and unsaturated (i.e., n-3, n-6, and n-9) fatty acids. CO- and OO- but not FO-enriched diets increased body fat %, circulating triglycerides, and cholesterol while all diets increased total fatty acids relative to ND (OO>FO>CO) in air-exposed rats. Serum BCAA, assessed to determine the intensity of the ozone-induced stress response, was increased only in ND and CO animals after exposure, suggesting dampening of this response by FO and OO. However, ozone-induced hyperglycemia and glucose intolerance were exacerbated in animals receiving OO-enriched diet. Overall, ozone-induced changes in liver and adipose tissue gene expression involved in glucose and energy metabolism and lipid biosynthesis were less remarkable in animals fed FO- and OO-enriched diets, whereas ozone-induced inhibition of muscle tissue genes linked to energy metabolism were similar across all dietary groups. Thus, diets enriched with n-3, n-6, and n-9 fatty acids could alleviate some of the metabolic effects of ozone, although these effects may depend on the type of fatty acid enrichment, metabolic process, and tissue being examined.

Supplementary Material

Sup1

ACKNOWLEDGEMENTS

The authors would like to thank Drs. M. Ian Gilmour and Mike Madden of the US EPA and Dr. Jonathan Shannahan of the Purdue University (West Lafayette, IN) for their critical review of the manuscript. We thank Dr. Mark Higuchi and Mr. Malek Khan of the EPA for their help in conducting ozone exposures, and Mr. Alan Tennant for assistance with slide scanning.

FUNDING INFORMATION

This work was supported in part by US EPA funds and the Fulbright (CONICYT) and EPA-UNC Cooperative Trainee Agreement (CR-83515201) to A.H.

Footnotes

DISCLOSURES

The research described in this article has been reviewed by the Center for Public Health and Environmental Assessment, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and the policies of the Agency, nor does mention of trade names of commercial products constitute endorsement or recommendation for use.

Declaration

Authors declare no competing financial interests.

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