Contents lists available at ScienceDirect Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/taap Ozone modifies the metabolic and endocrine response to glucose: Reproduction of effects with the stress hormone corticosterone Errol M. Thomsona,⁎, Shinjini Pilona, Josée Guénettea, Andrew Williamsa, Alison C. Hollowayb a Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9, Canada bDepartment of Obstetrics and Gynecology, McMaster University, Hamilton, Ontario L8N 3Z5, Canada A R T I C L E I N F O Keywords: Air pollution Ozone Glucose tolerance test Stress hormone Metabolic Endocrine A B S T R A C T Air pollution is associated with increased incidence of metabolic disease (e.g. metabolic syndrome, obesity, diabetes); however, underlying mechanisms are poorly understood. Air pollutants increase the release of stress hormones (human cortisol, rodent corticosterone), which could contribute to metabolic dysregulation. We as- sessed acute effects of ozone, and stress axis involvement, on glucose tolerance and on the metabolic (trigly- ceride), endocrine/energy regulation (insulin, glucagon, GLP-1, leptin, ghrelin, corticosterone), and in- flammatory/endothelial (TNF, IL-6, VEGF, PAI-1) response to exogenous glucose. Male Fischer-344 rats were exposed to clean air or 0.8 ppm ozone for 4 h in whole body chambers. Hypothalamic-pituitary-adrenal (HPA) axis involvement in ozone effects was tested through subcutaneous administration of the glucocorticoid synthesis inhibitor metyrapone (50mg/kg body weight), corticosterone (10 mg/kg body weight), or vehicle (40% propylene glycol) prior to exposure. A glucose tolerance test (2 g/kg body weight glucose) was conducted immediately after exposure, with blood samples collected at 0, 30, 60, 90, and 120min. Ozone exposure im- paired glucose tolerance, an effect accompanied by increased plasma triglycerides but no impairment of insulin release. Ozone diminished glucagon, GLP-1, and ghrelin responses to glucose, but did not significantly impact inflammatory/endothelial analytes. Metyrapone reduced corticosterone but increased glucose and triglycerides, complicating evaluation of the impact of glucocorticoid inhibition. However, administration of corticosterone reproduced the profile of ozone effects, supporting a role for the HPA axis. The results show that ozone-de- pendent changes in glucose tolerance are accompanied by altered metabolic and endocrine responses to glucose challenge that are reproduced by exogenous stress hormone. 1. Introduction A number of recent epidemiological studies have linked exposure to air pollution with metabolic disorders including insulin resistance, metabolic syndrome, and type 2 diabetes (Chen et al., 2013; Eze et al., 2015; Jiang et al., 2016a; Kramer et al., 2010; Thiering et al., 2013). Because of the ubiquitous exposure of the population, and the in- creasing global prevalence of metabolic disorders (Engin, 2017), im- pacts of air pollution on cardiometabolic diseases could represent a significant public health burden. Several plausible mechanisms have been advanced to explain how air pollutants may contribute to the pathogenesis of metabolic diseases (Liu et al., 2013; Rajagopalan and Brook, 2012; Thomson, 2014). Experimental models of acute exposure to ozone or particulate matter (Bass et al., 2013; Thomson et al., 2013; Vella et al., 2015) and chronic exposure to particulate matter (Sun et al., 2009) provide compelling evidence that pollutant exposure can acutely provoke systemic effects including impacts on metabolic homeostasis and in the longer term contribute to the acceleration of disease processes. However, there remains uncertainty regarding the nature and relative importance of underlying biological mechanisms, and how the various biological processes collectively contribute to the pathogenesis of metabolic disease. Although there is considerable evidence to support a central role for inflammatory processes in metabolic dysfunction (Liu et al., 2013), several recent studies suggest that air pollutants need not necessarily produce proinflammatory effects to provoke insulin resistance or dys- regulate glucose metabolism (Brook et al., 2013, 2016; Liu et al., 2017; Ying et al., 2016). We have previously shown that short-term exposure to ozone and particulate matter activates the hypothalamic-pituitary- adrenal (HPA) axis, resulting in increased circulating levels of the https://doi.org/10.1016/j.taap.2018.01.020 Received 1 November 2017; Received in revised form 16 January 2018; Accepted 28 January 2018 ⁎ Corresponding author at: Inhalation Toxicology Laboratory, Hazard Identification Division, Environmental Health Science and Research Bureau, Health Canada, 0802B Tunney's Pasture, Ottawa, Ontario K1A 0K9, Canada. E-mail addresses: errol.thomson@canada.ca (E.M. Thomson), shinjini.pilon@canada.ca (S. Pilon), josee.guenette@canada.ca (J. Guénette), andrew.williams@canada.ca (A. Williams), hollow@mcmaster.ca (A.C. Holloway). Toxicology and Applied Pharmacology 342 (2018) 31–38 Available online 31 January 2018 0041-008X/ Crown Copyright © 2018 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). T http://www.sciencedirect.com/science/journal/0041008X https://www.elsevier.com/locate/taap https://doi.org/10.1016/j.taap.2018.01.020 https://doi.org/10.1016/j.taap.2018.01.020 mailto:errol.thomson@canada.ca mailto:shinjini.pilon@canada.ca mailto:josee.guenette@canada.ca mailto:andrew.williams@canada.ca mailto:hollow@mcmaster.ca https://doi.org/10.1016/j.taap.2018.01.020 http://crossmark.crossref.org/dialog/?doi=10.1016/j.taap.2018.01.020&domain=pdf glucocorticoid corticosterone (Thomson et al., 2013). Pharmacological blockade of glucocorticoid synthesis prevented a subset of transcrip- tional responses in metabolic pathways across multiple organs, and effects of ozone exposure were reproduced by administration of corti- costerone, establishing stress hormone involvement in downstream ef- fects of ozone exposure (Thomson et al., 2016). Glucocorticoids are important regulators of glucose metabolism and insulin sensitivity, and chronic activation and dysregulation of the HPA axis can lead to me- tabolic dysfunction and disease (Bose et al., 2009; Chrousos and Kino, 2009). Evidence that ozone's ability to cause metabolic perturbations may be secondary to its effects on the HPA axis is therefore of interest in establishing plausible underlying mechanisms. While there has been considerable interest in determining whether air pollutants contribute to the development and progression of meta- bolic diseases, little is known about how exposure to air pollutants may modify the response to glucose of biological systems implicated in en- ergy homeostasis and inflammation. Abnormal responses of metabolic, endocrine, and inflammatory factors to exogenous glucose may serve as early indicators of pathogenic processes (Ahren, 2006; Corica et al., 2001; Esposito et al., 2002; Giugliano et al., 2008; Reynolds et al., 2003; Vossen et al., 2011); accordingly, examination of the effect of pollutants on responses of circulating factors to glucose may be useful in identifying effects and mechanisms that contribute to the develop- ment and progression of metabolic diseases. We have established an approach to monitor the responses of a variety of plasma factors during a glucose tolerance test (Pilon et al., 2017). In the present study, our objectives were to 1) determine whether exposure to ozone alters glu- cose tolerance in Fischer-344 rats, a common toxicological model for which we have previously characterised systemic effects of air pollu- tants (Thomson et al., 2013, 2016); 2) assess whether ozone inhalation modifies the metabolic, endocrine, and inflammatory response to exo- genous glucose; and 3) test whether glucocorticoids are involved in observed effects. We found that ozone reduced glucose tolerance in Fischer rats and modified metabolic and endocrine impacts of glucose, with exogenous corticosterone administration reproducing effects of ozone exposure. 2. Methods 2.1. Animals Specific pathogen-free male Fischer-344 rats (200–250 g) were ob- tained from Charles River (St. Constant, Québec, Canada). Animals were housed in individual plexiglass cages on wood-chip bedding under HEPA-filtered air and held to a 12 h dark/light cycle. Food and water were provided ad libitum until 2 h prior to inhalation exposures (6 h fast prior to glucose tolerance test). All experimental protocols were reviewed and approved by the Animal Care Committee of Health Canada. 2.2. Inhalation exposures Rats were trained in whole-body chambers for 2 h/day for 3 days prior to inhalation exposures for acclimatization. One hour prior to ozone exposure, rats were administered vehicle (40% propylene glycol in buffered saline), 50mg/kg body weight metyrapone, or 10mg/kg body weight corticosterone by subcutaneous injection as previously described (Thomson et al., 2016). Inhalation exposures were conducted using clean air or 0.8 ppm ozone for 4 h in whole body chambers (n=8/group with the exception of corticosterone group exposed to air where n=6; Table 1). A silent arc generator (Erwin Sander, Uetze, Germany) made ozone from medical-grade oxygen. A feedback loop (Guenette et al., 1997) maintained a steady ozone concentration of 0.8 ppm (average 0.791 ppm) by measuring the ozone concentration (TECO model 49; CD Nova-Tech, Markham, Ontario) in the centre of the chamber and adjusting the ozone bypass flow mixing with the main airstream (400 lpm HEPA-filtered air). A slightly negative pressure (~−0.5mm water) was maintained by continuously removing air from the chamber. 2.3. Glucose tolerance test Glucose tolerance tests were conducted immediately following ex- posure. Blood glucose was measured prior to glucose administration (50% dextrose, Vétoquinol N.-A.Inc., Lavaltrie, Québec, Canada, 2 g/kg body weight via intraperitoneal injection), and then 30, 60, 90, and 120min after administration using a OneTouch®Verio™IQ glucometer (LifeScan Canada Ltd., Burnaby, British Columbia, Canada). At each time point, approximately 200 μL of blood was collected from the tail vein with a heparinized needle and transferred to a BD plasma micro- tainer with plasma separator (BD, Mississauga, Ontario, Canada, cat# B365985). Tubes were centrifuged immediately and kept on ice until the end of the glucose tolerance test at which point plasma was ali- quoted for future analyses and stored at e80C. 2.4. Plasma analyses Insulin was analyzed using an ultra-sensitive rat enzyme-linked immunosorbent assay (ELISA) kit (Crystal Chem Inc., Downers Grove, IL, USA). Corticosterone was analyzed using the DetectX® ELISA kit (Arbor Assays, Ann Arbor, Michigan, USA). Triglycerides were analyzed using a colorimetric assay kit (Cayman Chemical Company, Ann Arbor, Michigan USA). Cytokine and metabolic hormone levels were analyzed by multiplexing glucagon, glucagon-like peptide (GLP)-1, ghrelin, leptin, plasminogen activator inhibitor (PAI)-1, tumour necrosis factor (TNF), interleukin (IL)-6, and vascular endothelial growth factor (VEGF) assays (Bio-Rad Laboratories (Canada) Ltd., Mississauga, Ontario, Canada) as previously described (Pilon et al., 2017). All ana- lyses were conducted in duplicate. 2.5. Indices of insulin resistance, sensitivity and β-cell function Surrogate indices of insulin resistance and β-cell function were calculated (Matthews et al., 1985) according to the following equations: − = × = ×HOMA IR G I /22.5 and HOMA%β (20 I )/(G –3.5)0 0 0 0 where G0 and I0 are fasting glucose (mmol/L) and insulin (μIU/mL) values, respectively. The Quantitative Insulin Sensitivity Check Index (QUICKI) (Katz et al., 2000) was calculated as follows: = +QUICKI 1/(logG logI )0 0 where G0 and I0 are fasting glucose (mg/dL) and insulin (μIU/mL) va- lues, respectively. The Matsuda Index (Matsuda and DeFronzo, 1999) was calculated according to: = × × × Matsuda 10, 000/sqrt(G I glucose mean (30–120 min) insulin mean (30–120 min) 0 0 0.5 2.6. Statistical analyses A generalized estimating equation (GEE) analysis was utilized to analyze the repeated measures data for each endpoint independently. Table 1 Experimental groups. Vehicle (n/group) Metyrapone (n/group) Corticosterone (n/group) Air 8 8 6 Ozone 8 8 N/A E.M. Thomson et al. Toxicology and Applied Pharmacology 342 (2018) 31–38 32 For these analyses, the GEE approach used the Gaussian distribution for the error while accounting for the clustering effect of the animal. The GEE model was fitted using the R statistical environment (R Core Team, 2016) by invoking the geepack library (Højsgaard et al., 2006; Yan, 2002; Yan and Fine, 2004), which implements the GEE approach for fitting marginal generalized linear models to clustered data (Liang and Zeger, 1986). The model in this analysis consisted of three fixed effects, namely OZONE, MET, and TIME, including the two and three interac- tion terms. Model diagnostics were conducted using the robustlmm li- brary (Koller, 2006). Observations that had weights < 0.3 were re- moved from the analysis. Post hoc comparisons were conducted using the doBy package (Højsgaard and Halekoh, 2016) as directed by sig- nificant main effects or interactions, with p-values adjusted using the Holm-Sidak approach (Holm, 1979). All other statistical analyses (e.g. one- and two-way ANOVAs) were performed using SigmaPlot 13.0 (Systa Software, Inc., San Jose, California, USA). Significant main ef- fects or factor interactions are described in text; for simplicity, only statistically significant differences arising from pairwise comparisons of air and ozone groups are indicated in Figs. 2-5. 3. Results 3.1. Corticosterone To confirm delivery of a biologically-effective dose of metyrapone, we assessed plasma corticosterone levels. As expected, corticosterone levels remained significantly lower in metyrapone-treated groups throughout the glucose tolerance test (MET× TIME, p < 0.001; Fig. 1). Corticosterone levels increased with time after glucose admin- istration, but the profile was not significantly impacted by ozone. 3.2. Glucose homeostasis Levels of all analytes, and derived variables from glucose and in- sulin data following acute exposure to air or ozone ± metyrapone, are presented in Table 2. Glucose tolerance was reduced after ozone ex- posure, with the duration of the effect dependent upon the presence of metyrapone (OZONE×MET interaction, p= 0.02; Fig. 2A, B). The ozone-exposed vehicle group exhibited an increase in the peak glucose response to challenge (Fig. 2A). Ozone increased the total glucose re- sponse to glucose challenge, as indicated by a significant main effect of ozone on the glucose area-under-the-curve. Metyrapone independently raised fasting glucose. Metyrapone treatment also impaired glucose homeostasis, with the metyrapone plus ozone group exhibiting a re- duced ability to clear the glucose load resulting in higher glucose levels at 120min (Fig. 2B) as well as an increased total glucose response (i.e. glucose area-under-the-curve) to the challenge. Insulin peaked at 30min post-glucose administration, and the profile of response was not significantly impacted by ozone or metyrapone (TIME main effect, p < 0.001; Fig. 2C, D). Ozone alone did not alter any indices of insulin secretion or effect, whereas metyrapone significantly reduced HOMAβ and insulin secretion. 3.3. Triglycerides The profile of triglyceride response to glucose was dependent upon ozone and metyrapone (OZONE×MET× TIME interaction, p= 0.01; Fig. 3). In the vehicle-treated group, triglyceride levels initially de- creased from pre-glucose administration levels, but were elevated in ozone-exposed animals 2 h after glucose administration compared to air-exposed animals (Fig. 3A). Triglyceride levels were generally higher in the metyrapone group, and tended to be higher in ozone-exposed group throughout the glucose tolerance test, although pairwise com- parisons were not significantly different (Fig. 3B). 3.4. Glucagon and GLP-1 The response of glucagon to glucose administration was reduced in ozone-exposed animals, and was not impacted by metyrapone treat- ment (OZONE× TIME interaction, p < 0.001; Fig. 4A, B). GLP-1 levels increased in air-exposed animals following glucose administration, an effect blunted by ozone independent of metyrapone (OZONE× TIME, p < 0.001; Fig. 4C, D). 3.5. Ghrelin and leptin Ghrelin levels were lower in ozone-exposed groups 60–90min after glucose challenge independent of metyrapone (OZONE× TIME inter- action, p= 0.002; Fig. 5A, B). Leptin levels increased with time fol- lowing glucose administration (TIME main effect, p < 0.001), and tended to be lower in metyrapone-treated animals (MET main effect, p= 0.047), but were not significantly impacted by ozone (Fig. 5C, D). 3.6. Cytokines and endothelial markers IL-6, PAI-1, and TNF were below the limit of detection, and levels were not increased by glucose administration, ozone, or metyrapone (data not shown). VEGF levels varied with time following glucose challenge (TIME main effect, p= 0.001), but were not significantly impacted by ozone or metyrapone (data not shown). 3.7. Effects of corticosterone administration To independently assess the effect of corticosterone on the response of plasma analytes to glucose, and to determine whether corticosterone could reproduce effects of ozone inhalation, corticosterone was ad- ministered to one group of animals prior to air exposure, and analyte responses to glucose were compared to responses in the ozone-exposed group (each expressed relative to the vehicle air control group). We included in these comparisons all analytes above the limit of detection, including those that were not significantly affected by ozone, as a common lack of response is also indicative of a similarity in profile. Complete baseline (pre-glucose challenge) data and responses to glu- cose administration for the corticosterone group are presented in Supplemental Table 1 and Supplemental Fig. 1 respectively. While there was no similarity in the profile of response to ozone and corti- costerone at baseline (r=0.23, p=0.55), acute responses to glucose at 0 30 60 90 120 0 200 400 600 800 1000 VEH Ozone VEH Air MET Ozone MET Air VEH Ozone MET Ozone MET Air VEH Air Time post-glucose (min) C or tic os te ro ne ( ng /m L) x 10 3 Fig. 1. Corticosterone response to glucose challenge following exposure to ozone and metyrapone. Rats were administered vehicle (VEH) or metyrapone (MET) and exposed for 4 h to air or ozone. Plasma corticosterone levels were measured in samples collected before glucose administration and at 30, 60, 90, and 120min (n= 5–8/point). †MET vs. VEH at all timepoints, p < 0.001; ‡30, 60, 90, 120 vs. 0 min, p < 0.001 for both VEH and MET groups. E.M. Thomson et al. Toxicology and Applied Pharmacology 342 (2018) 31–38 33 30 (r=0.64, p=0.066; Fig. 6A), 60 (r=0.94, p < 0.001; Fig. 6B), and 90min (r=0.63, p= 0.069; Fig. 6C) were similar in the ozone and corticosterone groups compared to vehicle air controls. Restriction of the comparison to statistically significant ozone effects improved the regression (r=0.97, p=0.001; Fig. 6D), confirming that exogenous corticosterone administration effectively reproduced the profile of re- sponses to ozone. In contrast, although metyrapone also reduced glu- cose tolerance, there was no similarity to ozone in the profile of analyte response to glucose challenge (data not shown). 4. Discussion Metabolic disorders are characterised by dysfunctional metabolic, endocrine, and inflammatory processes that collectively contribute to disease processes, including changes in the way the body responds to carbohydrate and lipid intake (Parhofer, 2015; Tomkin and Owens, 2017). Air pollutants are associated with metabolic disease, impaired glucose tolerance, and insulin resistance (Liu et al., 2013), but the effect of air pollutants on the metabolic and endocrine response to glucose has not, to our knowledge, been examined. As responses of relevant meta- bolic, endocrine, and inflammatory factors to glucose challenge have been used as early indicators of metabolic dysfunction (Ahren, 2006; Corica et al., 2001; Esposito et al., 2002; Giugliano et al., 2008; Reynolds et al., 2003; Vossen et al., 2011), we reasoned that challen- ging pollutant-exposed animals with glucose could reveal biological effects of pollutant exposure not evident from simply measuring post- Table 2 Metabolic indices for Fischer rats (n= 8/group) administered vehicle or metyrapone and exposed for 4 h to air or ozone (mean ± SEM). Vehicle Air Vehicle Ozone 50 MET Air 50 MET Ozone P valuea Fasting glucose (mmol/L) 7.0 ± 0.1 6.7 ± 0.2 7.9 ± 0.2 7.9 ± 0.2 MET, p < 0.001 Fasting insulin (pmol/L) 312 ± 32 339 ± 18 309 ± 26 281 ± 22 NS Corticosterone (ng/mL) 430 ± 77 494 ± 111 225 ± 81 262 ± 36 MET, p < 0.001 Triglycerides (mg/dL) 92.1 ± 19.7 107.5 ± 37.7 140.6 ± 41.8 169.8 ± 35.3 MET, p < 0.001 Ghrelin (pg/mL) 17,147 ± 7509 13,438 ± 2146 19,066 ± 11,412 21,436 ± 5506 NS GLP-1 (pg/mL) 173 ± 57 151 ± 25 132 ± 22 139 ± 14 NS Glucagon (pg/mL) 347 ± 61 307 ± 69 275 ± 35 329 ± 37 MET x Ozone p= 0.041 Leptin (pg/mL) 3355 ± 1118 3315 ± 513 3153 ± 1922 2763 ± 434 NS VEGF (pg/mL) 27.6 ± 4.0 25.8 ± 3.3 25.2 ± 8.1 28.7 ± 3.4 NS HOMA-IR 16.3 ± 1.9 16.9 ± 1.2 18.2 ± 1.7 16.8 ± 1.7 NS HOMA-ß 293 ± 26 362 ± 35 239 ± 20 206 ± 11 MET, p < 0.001 QUICKI 0.264 ± 0.004 0.261 ± 0.002 0.260 ± 0.004 0.262 ± 0.003 NS Matsuda Index 3.4 ± 0.3 3.1 ± 0.2 3.0 ± 0.3 2.8 ± 0.3 NS AUC Glucose 33.0 ± 0.8 33.7 ± 0.8 42.2 ± 2.3 48.9 ± 2.1 MET, p < 0.001 Ozone, p=0.037 AUC Insulin 12.0 ± 1.8 12.1 ± 1.6 9.9 ± 1.1 9.4 ± 0.7 NS Insulin Secretion (AUCins/AUCglu) 0.36 ± 0.05 0.35 ± 0.04 0.25 ± 0.04 0.19 ± 0.02 MET, p < 0.001 NS, not significant; MET, metyrapone; AUC, area-under-the-curve. a Two-way ANOVA assessment of factors OZONE and MET. 0 30 60 90 120 )L/o m m( esocul G 6 8 10 12 14 16 18 MET Air MET Ozone 0 30 60 90 120 6 8 10 12 14 16 18 * * * * 0 30 60 90 120 1 2 3 4 5 6 7 8 9 VEH Air VEH Ozone 0 30 60 90 120 )L m/gn( nilusnI 1 2 3 4 5 6 7 8 9 MET Air MET Ozone VEH Air VEH Ozone VEH Air VEH Ozone MET Air MET Ozone MET Air MET Ozone Time post-glucose (min) Time post-glucose (min)Time post-glucose (min) Time post-glucose (min) G lu co se (m m ol /L ) G lu co se (m m ol /L ) L m/gn( nilusnI ) In su lin ( ng /m L) Fig. 2. Effects of ozone and metyrapone on glucose and insulin levels during the glucose tolerance test. Glucose (n=8/point) and insulin (n=5–8/point) levels were measured in vehicle (A, C) or metyrapone (B, D)-treated animals exposed to air or ozone. *p < 0.05, air vs ozone. E.M. Thomson et al. Toxicology and Applied Pharmacology 342 (2018) 31–38 34 exposure levels of these analytes, and provide insight into mechanisms underlying metabolic health impacts. In the present study we show that acute exposure to ozone reduced glucose tolerance in male Fischer-344 rats, consistent with effects seen in other rodent models (Bass et al., 2013; Miller et al., 2016; Vella et al., 2015). The ozone-induced re- duction in glucose tolerance was accompanied by an increased trigly- ceride response and by a blunting of glucagon, GLP-1, and ghrelin re- sponses to glucose. Pretreatment with the glucocorticoid synthesis inhibitor metyrapone reduced corticosterone levels but also increased glucose and triglyceride levels, complicating determination of gluco- corticoid involvement in observed effects. However, exogenous corti- costerone reproduced the profile of effects of ozone. The results show that short-term exposure to ozone, in addition to impacting glucose tolerance, modifies the metabolic and endocrine response to glucose, and that the profile of response is similar to that generated by gluco- corticoid administration. Acute ozone inhalation resulted in differential responses of endo- crine and metabolic factors to a glucose challenge when compared to air exposure. Importantly, there is evidence that such effects may be relevant to disease processes. For example, postprandial hyperlipidemia is a feature of obesity and metabolic syndrome and predictor of cardi- ovascular risk (Lopez-Miranda et al., 2007; Ridker, 2008). In the cur- rent study, ozone administration resulted in higher triglyceride re- sponses to the glucose challenge; this has been proposed to be an early indicator of metabolic dysfunction (Vossen et al., 2011). Similarly, some studies suggest that glucagon suppression by glucose is aug- mented in the presence of insulin resistance and its risk factors (Ahren, 2006). The mechanisms underlying the altered responsiveness of 0 30 60 90 120 40 60 80 100 120 140 160 180 200 VEH Air VEH Ozone * 0 30 60 90 120 40 60 80 100 120 140 160 180 200 MET Air MET Ozone VEH Air VEH Ozone MET Air MET Ozone Time post-glucose (min) Time post-glucose (min) )L m/gn( sed irecylgir T T rig ly ce rid es ( ng /m L) Fig. 3. Effects of ozone and metyrapone on triglyceride response to glucose. Triglyceride levels were measured in vehicle (A) or metyrapone (B)-treated animals exposed to air or ozone (n=6–8/point). *p < 0.05 air vs ozone. 0 30 60 90 120 0 200 400 600 800 1000 1200 1400 1600 1800 2000 0 30 60 90 120 0 200 400 600 800 1000 0 30 60 90 120 0 200 400 600 800 1000 0 30 60 90 120 0 200 400 600 800 1000 1200 1400 1600 1800 2000 A C D B * * ** * * VEH Air VEH Ozone MET Air MET Ozone 8 9 10 11 VEH Air VEH Ozone MET Air MET Ozone Time post-glucose (min) Time post-glucose (min)Time post-glucose (min) Time post-glucose (min) ( nogacul G pg /m L) G lu ca go n (p g/ m L) G LP -1 ( pg /m L) G LP -1 ( pg /m L) Fig. 4. Effects of ozone and metyrapone on glucagon and glucagon-like peptide (GLP)-1 responses to glucose. Glucagon and GLP-1 levels were assessed in vehicle (A, C) or metyrapone (B, D)-treated animals exposed to air or ozone (n= 3–7/point). *p < 0.05, air vs ozone. E.M. Thomson et al. Toxicology and Applied Pharmacology 342 (2018) 31–38 35 glucagon and GLP-1 to glucose in ozone-exposed animals have not been fully elucidated, but it is tempting to speculate that it may relate to activation of AMP-activated protein kinase (AMPK) signaling, which has been described in the lungs following ozone exposure (Hulo et al., 2011). Activation of AMPK has been shown to reduce GLP-1 secretion (Jiang et al., 2016b) and antagonise hepatic glucagon signaling (Johanns et al., 2016), both of which could have profound effects on glucose homeostasis. However, whether AMPK activity is altered sys- temically following acute exposure to ozone remains to be determined before perturbation in glucose homeostasis can be causally ascribed to this pathway. The perturbations in glucose homeostasis seen in this study may also be related to ozone-induced activation of the HPA axis. Glucocorticoids are well known for their involvement in regulating glucose uptake and metabolism, and glucocorticoid levels and altered glucocorticoid sen- sitivity are associated with changes in glucose tolerance and other metabolic parameters (Majer-Lobodzinska and Adamiec-Mroczek, 2017; Rafacho et al., 2014). We have previously shown in this Fischer rat model that ozone and particulate matter stimulate the HPA axis, increasing levels of the stress hormones adrenocorticotrophic hormone and corticosterone (Thomson et al., 2013). Furthermore, transcriptional responses to ozone in a number of organs were blocked by metyrapone treatment and reproduced by corticosterone (Thomson et al., 2016), highlighting the role of this stress hormone in mediating pulmonary and extrapulmonary effects of ozone exposure. It should be noted that stress associated with the additional handling inherent to preparation and conduct of glucose tolerance tests could also acutely impact stress hormone levels, as suggested by the lack of significant effect of ozone on corticosterone measured here. Given that there is a lag between release of corticosterone into blood and receptor-mediated effects on target tissues, conclusions regarding the involvement of corticosterone in downstream effects cannot be definitively made on the basis of comparisons at a given point in time. Indeed, we have shown that adrenal production of corticosterone was suppressed 5 h after admin- istration of exogenous corticosterone, despite plasma corticosterone having returned to vehicle control levels by that time, confirming de- livery of a biologically-effective dose (Thomson et al., 2016). In the present study, the independent effect of metyrapone on plasma glucose impeded unambiguous assessment of how blockade of glucocorticoids synthesis altered effects of ozone on responses to glucose. Dose-de- pendent effects of metyrapone on plasma glucose that appear to be independent of effects on corticosterone have been described in other models (Rotllant et al., 2002). It is possible that the reduction in glucose tolerance observed in metyrapone-treated ozone-exposed animals may be a function of increased release of glucose in response to metyrapone coupled with less efficient clearance due to effects of ozone, in- dependent of any effects mediated by metyrapone-dependent reduction in corticosterone synthesis. Adrenalectomy has been shown to alleviate ozone-induced reductions in glucose tolerance in Wistar rats (Miller et al., 2016), supporting involvement of adrenal-derived factors in mediating effects of ozone on glucose tolerance. It is noteworthy that in the present study exogenous corticosterone produced an overall profile of response to glucose that was similar to that observed during the glucose tolerance test following ozone exposure, consistent with a possible role for glucocorticoids in mediating impacts of ozone on the response of plasma metabolic and endocrine factors to glucose. Because particulate matter, like ozone, activates the HPA axis and provokes systemic changes in gene expression (Thomson et al., 2013), these ef- fects may not be specific to ozone, but may instead represent a more general response to pollutant stressors. 0 30 60 90 120 0 5 10 15 20 25 30 35 40 VEH Air VEH Ozone 0 30 60 90 120 0 5 10 15 20 25 30 35 40 MET Air MET Ozone A C D B * * * 0 30 60 90 120 0 2 4 6 8 10 12 14 16 18 VEH Air VEH Ozone 0 30 60 90 120 0 2 4 6 8 10 12 14 16 18 MET Air MET Ozone VEH Air VEH Ozone MET Air MET Ozone VEH Air VEH Ozone MET Air MET Ozone Time post-glucose (min) Time post-glucose (min)Time post-glucose (min) Time post-glucose (min) ( nilerh G pg 0 1x )L m / 3 G hr el in ( pg /m L) x 10 3 ( ni tpeL pg 01x ) L m/ 3 Le pt in ( pg /m L) x 10 3 Fig. 5. Effects of ozone and metyrapone on ghrelin and leptin responses to glucose. Ghrelin and leptin levels were measured in vehicle (A, C) or metyrapone (B, D)-treated animals exposed to air or ozone (n= 4–8/point). *p < 0.05, air vs ozone. E.M. Thomson et al. Toxicology and Applied Pharmacology 342 (2018) 31–38 36 Several aspects of this work should be considered when interpreting results. One of the strengths of the present study is the repeated mea- surement of plasma endpoints throughout the glucose tolerance test that enabled characterisation of the temporal response of analytes to glucose and any modification by ozone. As a result, we were able to capture both early and later effects of ozone exposure on the response to glucose. Multiple measures were analyzed simultaneously, providing insight into impacts of ozone on the response to glucose that extend beyond assessment of glucose tolerance. The incretin effect is bypassed by intraperitoneal administration of glucose, and we have shown pre- viously that responses of a number of factors measured in this study differ depending upon route of glucose administration (Pilon et al., 2017). Although repeated handling and training sessions in the whole- body exposure chambers were part of the acclimatization process, it is clear that both the exposure and the subsequent glucose tolerance test can impose stress that could impact analyte levels. For example, glu- cagon can be elevated in response to glucose in psychological and metabolic stress states (Jones et al., 2012), and both corticosterone and ghrelin increase acutely under stress conditions (Schellekens et al., 2012), which may explain their rise during the glucose tolerance test in air-exposed animals. Thus, while experimental conditions were care- fully controlled to eliminate potential bias from nuisance variables (e.g. exposure chambers, handling, noise), effects of ozone and subsequent response to exogenous glucose should not be assumed to be occurring in a “stress-free” context. Clearly, ozone acts through both glucocorticoid- dependent and independent pathways (Thomson et al., 2016), and there is evidence for involvement of other processes, including oxida- tive stress (Vella et al., 2015), in adverse metabolic effects. Given the interrelated nature of metabolic, inflammatory, and stress pathways, continued characterisation of their collective responses to pollutant exposure in a variety of models should yield a better understanding of pollutant effects that underlie disease processes. Although levels of several cytokines and endothelial factors were not altered in response to glucose or ozone in the present study, the magnitude and duration of responses to glucose can indicate underlying disease processes (Esposito et al., 2002), and so effects on such endpoints warrant further in- vestigation in other models, particularly following chronic exposure where systemic inflammation has been reported (Sun et al., 2009). In summary, we found that ozone alters the metabolic and endo- crine response to glucose challenge. Interestingly, the profile of re- sponse was similar to that produced following glucose challenge of animals administered corticosterone. Altered metabolic and endocrine responses to glucose are predictive of metabolic diseases, suggesting that these ozone-induced changes in response to glucose could be re- levant to disease processes. Further studies are warranted to investigate the consequences of long-term exposure to air pollutants on the re- sponse to glucose administration. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.taap.2018.01.020. Acknowledgments We wish to acknowledge the technical assistance of Kevin Curtin, Alain Filiatreault, Marjolaine Godbout-Cheliak, Karine Chamberland, Michelle Lalande, Scott Smith, Cina Aghazadeh Sanaei, and Bruce Martin. Thanks to Dr. Azam Tayabali and Dr. Dalibor Breznan for 60 min -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.430 min -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 A B VEGF Trig Leptin Glucagon GLP-1 Ghrelin Glucose InsulinCort GLP-1 Ghrelin Glucagon Leptin Insulin Trig VEGF Glucose Cort r=0.636 r=0.938 C -0.8 -0.6 -0.4 -0.2 0.0 0.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Trig Glucose Ghrelin 90 Glucagon Ghrelin 60 GLP-1 r=0.973 D 90 min -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 r=0.630 Trig Insulin Cort Leptin VEGF GlucoseGlucagon GLP-1 Ghrelin Corticosterone vs Air (log2FC) ) C F2gol( ri A sv enoz O Corticosterone vs Air (log2FC) Corticosterone vs Air (log2FC) Corticosterone vs Air (log2FC) ) C F2gol( ri A sv enoz O O zo ne v s A ir (lo g2 F C ) O zo ne v s A ir (lo g2 F C ) Significant Effects Fig. 6. Comparison of responses to ozone and exogenous corticosterone. Corticosterone was administered to a group of animals (n= 6), and their mean response to glucose was compared to the mean response of the ozone-exposed group (n= 8) relative to the vehicle air group across all analytes at 30min (A), 60min (B), and 90min (C). The comparison to corticosterone was also performed on the subset of analytes and time points for which there was a statistically significant difference between air and ozone groups (D). Ghrelin 60 and 90 refers to measurements taken at 60 and 90min respectively; GLP, glucagon-like peptide; Trig, triglycerides; VEGF, vascular endothelial growth factor; FC, fold-change. E.M. Thomson et al. Toxicology and Applied Pharmacology 342 (2018) 31–38 37 https://doi.org/10.1016/j.taap.2018.01.020 https://doi.org/10.1016/j.taap.2018.01.020 helpful comments in reviewing the manuscript. This work was sup- ported by Health Canada (Clean Air Regulatory Agenda, project number 810504). References Ahren, B., 2006. 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http://refhub.elsevier.com/S0041-008X(18)30027-9/rf0260 http://refhub.elsevier.com/S0041-008X(18)30027-9/rf0260 http://refhub.elsevier.com/S0041-008X(18)30027-9/rf0260 http://refhub.elsevier.com/S0041-008X(18)30027-9/rf0260 Ozone modifies the metabolic and endocrine response to glucose: Reproduction of effects with the stress hormone corticosterone Introduction Methods Animals Inhalation exposures Glucose tolerance test Plasma analyses Indices of insulin resistance, sensitivity and β-cell function Statistical analyses Results Corticosterone Glucose homeostasis Triglycerides Glucagon and GLP-1 Ghrelin and leptin Cytokines and endothelial markers Effects of corticosterone administration Discussion Acknowledgments References