Ozone induces glucose intolerance and systemic metabolic effects in young and aged Brown Norway rats

Abstract

Air pollutants have been associated with increased diabetes in humans. We hypothesized that ozone would impair glucose homeostasis by altering insulin signaling and/or endoplasmic reticular (ER) stress in young and aged rats. One, 4, 12, and 24 month old Brown Norway (BN) rats were exposed to air or ozone, 0.25 or 1.0 ppm, 6 h/day for 2 days (acute) or 2 d/week for 13 weeks (subchronic). Additionally, 4 month old rats were exposed to air or 1.0 ppm ozone, 6 h/day for 1 or 2 days (time-course). Glucose tolerance tests (GTT) were performed immediately after exposure. Serum and tissue biomarkers were analyzed 18 h after final ozone for acute and subchronic studies, and immediately after each day of exposure in the time-course study. Age-related glucose intolerance and increases in metabolic biomarkers were apparent at baseline. Acute ozone caused hyperglycemia and glucose intolerance in rats of all ages. Ozone-induced glucose intolerance was reduced in rats exposed for 13 weeks. Acute, but not subchronic ozone increased α2-macroglobulin, adiponectin and osteopontin. Time-course analysis indicated glucose intolerance at days 1 and 2 (2>1), and a recovery 18 h post ozone. Leptin increased day 1 and epinephrine at all times after ozone. Ozone tended to decrease phosphorylated insulin receptor substrate-1 in liver and adipose tissues. ER stress appeared to be the consequence of ozone induced acute metabolic impairment since transcriptional markers of ER stress increased only after 2 days of ozone. In conclusion, acute ozone exposure induces marked systemic metabolic impairments in BN rats of all ages, likely through sympathetic stimulation.

Keywords: Aging; Air pollution; Epinephrine; Metabolic syndrome; Ozone; Serum biomarkers.

Fig. 1

Schematics of the study design for acute, subchronic and time-course experiments. Timing of glucose tolerance test (GTT), O₃ exposures, and necropsy/serum and tissue collection are shown for each sub-study. Post-exposure GTTs were done immediately following exposure whereas necropsy and tissue and serum collection were done either 18 h after exposure (acute, subchronic, and group 3 time-course study) or immediately following exposure (group 1 and 2 time-course study).

Fig. 2

Glucose tolerance test (GTT) comparison of 1, 4, 12, and 24 month old BN rats prior to the start of O₃ exposure. Inset graph shows fasting blood glucose levels for all ages. Each value is the mean blood glucose measurement ± S.E. of 18 to 21 rats. Mean values of blood glucose at each timepoint are compared for age-factor, relative to 4 month old rats († = p < 0.05 †† = p < 0.01). 0 ppm indicates FA exposure.

Fig. 3

Glucose tolerance test (GTT) comparison of BN rats after acute or subchronic exposure to FA or O₃, performed immediately after O₃ exposure. Each value is the mean blood glucose measurement ±S.E. of 4 to 10 rats. The individual plots show acute GTT results; a) 1 month b) 4 month c) 12 month d) 24 month; and subchronic GTT results, e) 4 month f) 12 month g) 24 month. Mean values of blood glucose at each timepoint are compared for age-factor, relative to 4 month old rats († = p < 0.05 ††=p < 0.01). Significant exposure effects are indicated relative to FA exposed group (* = p < 0.05 ** = p < 0.01). 0 ppm indicates FA exposure.

Fig. 4

Glucose tolerance test (GTT) comparison of BN rats after exposure to FA or O₃ in the time-course study. Each value is the mean blood glucose measurement ± S.E. of 4 to 10 rats. Mean values of blood glucose at each timepoint are compared for significant exposure effects relative to FA exposed group (* = p < 0.05 ** = p < 0.01). 0 ppm indicates FA exposure.

Fig. 5

Comparison of serum biomarkers in BN rats exposed to FA or O₃ in the time-course study. Serum was collected during necropsy immediately after 1 day or 2 days O₃ exposure, or 18 h after 2 days O₃ exposure and analyzed as described in methods. Control values may vary due to diurnal variation and the injection of glucose in glucose tolerance tests for animals in the 2 day 18 h timepoint group. Abbreviations: interleukin 6 (IL-6), α₁-acid glycoprotein (AGP), α₂-macroglobulin (A2M). Each value is the mean ± S.E. of 6 rats. Mean values of markers are compared for significant exposure effects relative to FA exposed group (* = p < 0.05 ** = p < 0.01). 0 ppm indicates FA exposure.

Fig. 6

Comparison of serum epinephrine in BN rats exposed to FA or O₃ in the time course study. Serum was collected during necropsy immediately after 1 day or 2 days O₃ exposure, or 18 h after 2 days O₃ exposure and analyzed for epinephrine. Control values may vary due to diurnal variation and the injection of glucose during glucose tolerance testing for animals in the 2 day 18 h timepoint group. Each value is the mean ± S.E. of 4–6 rats. Mean values of markers are compared for significant exposure effects relative to FA exposed group (* = p < 0.05). 0 ppm indicates FA exposure.