Metabolomic and multi-tissue transcriptomic analysis reveals complex phenotypic interactions of eight-week social isolation and acute ozone exposure in rats
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Socially isolated individuals and communities often face psychosocial stressors in combination with exposure to high levels of air pollutants. Social isolation (SI) has been considered a risk factor for neuropsychiatric conditions and is also linked to increased metabolic disorders, and the SI model in rats is a well-validated model of depressive- and anxiety-like behavior. Based on our research demonstrating the mechanistic similarities on how air pollutants and psychosocial stressors through the neuroendocrine system might exacerbate chronic immune and metabolic alterations, we hypothesized that rats under long-term SI will have exacerbated metabolic phenotype through dysregulated neuroendocrine activity and that acute ozone inhalation will exacerbate metabolic response in SI relative to pair-housed rats. Male, 4-week-old Wistar-Kyoto rats were either 1) pair-housed (2/cage) with environmental enrichment and frequent handling as no stress (NS) – control group; or 2) socially isolated (SI) by single-housing, with no environmental enrichment provided and avoiding frequent handling for 8 weeks. Animals were then exposed to filtered air or ozone (0.8 ppm) as a challenge stressor for 4h followed immediately by necropsy. The body weight gain of NS and SI were similar during the course of the study, indicating similar food consumption. However, in air-exposed SI animals, circulating cholesterol assessed using clinical assays was increased along with small increases in circulating cytokines, and this effect of SI was exacerbated by acute ozone exposure. Serum metabolomic analysis revealed only a modest effect on circulating metabolites of air-exposed SI relative to NS animals, except for higher levels of several circulating sphingomyelins in SI rats. These increases in sphingomyelins were associated with the absence of higher levels of cholesterol when assessed through metabolomics, suggesting that these sphingomyelins are likely bound to cholesterol and thus some cholesterol signal may be lost during the mass spectrometry ionization process. A single ozone exposure caused remarkable changes in circulating metabolites – including higher levels of a variety of free fatty acids, glycerols, branched chain amino acids, and ketone metabolites – typical of an acute stress-mediated metabolic response we have previously demonstrated in rats and humans. These ozone effects also included increases in sphingomyelins in both NS and SI groups. Because sphingomyelins are widely distributed in all cell membranes and enriched in myelinated neurons, and because liver is the master regulator of metabolic processes, we examined transcriptional changes in the stress responsive region of the brain – hypothalamus – and also in the liver, to determine their potential contribution to circulating sphingomyelins in SI animals. There were no significant transcriptional changes attributable to SI either in the hypothalamus or in the livers of air-exposed animals. Acute ozone exposure, on the other hand, caused 940 hypothalamic genes to be changed in NS and 1192 in SI, and 2398 liver genes in NS and 3055 genes in SI, with no significant interaction between SI and ozone in either tissue. Thus, these data indicate that other complex mechanisms are likely responsible for exacerbation of systemic metabolic and inflammation phenotypes together with increases in circulating sphingomyelins in SI by acute ozone exposure. These subtle effects of subchronic SI modifying the metabolomic response to acute ozone exposure indicate the importance of understanding how prior stressors modify the subsequent stress responses. (This abstract does not reflect the US EPA policy).