PM2.5 concentration and compositional assessment
The exposure period covered seasons of winter (2016) and spring (2017). The PM2.5 levels in the exposure chamber and in the ambient air were correlated while the maximum values were 228.70 μg/m3 and 182.70 μg/m3, and minimum values 4.70 μg/m3 and 9.27 μg/m3 in the exposure chamber and in the ambient air, respectively (Fig. 1a). The regression coefficient between exposure chamber and ambient air concentrations was 0.365 (P < 0.001). Mean daily PM2.5 concentrations in the ambient air at the study site was 62.63 μg/m3 (SEM, 2.60 μg/m3), while mean daily concentrations of PM2.5 in the filtered chamber and exposure chamber were 0.09 μg/m3 (SEM, 0.02 μg/m3) and 62.74 μg/m3 (SEM, 2.96 μg/m3), respectively (Fig. 1b). The mean daily concentrations in the exposure chamber were very closed to the ones in the ambient air at the study site during the same time period (both were ranged between 60 and 70 μg/m3) and the maximum concentrations of 228.70 μg/m3 in the exposure chamber, although 46 μg/m3 higher than the ambient one (182.70 μg/m3), is frequently seen during peak hours in major cities in China. Elemental composition was demonstrated in Supplemental Material, Table S1.
Ambient PM2.5 exposure decreased insulin sensitivity, being more prominent in female than in male
Prior to assignment to exposure protocols, no significant difference between groups at baseline in body weight, fasting blood glucose within male or female mice was observed (Supplemental Fig. 1, A-D). After 24 weeks of PM2.5 exposure, we observed no effects of PM2.5 exposure on body weight (Fig. 2a, Supplemental Fig. 2A), blood glucose (Fig. 2b, Supplemental Fig. 2B), glucose tolerance (Fig. 2c), serum insulin levels (Fig. 2d, Supplemental Fig. 2C) or the homeostasis model assessment of the IR (HOMA-IR, Supplemental Fig. 2D) index (Fig. 2e) in each sex. Nevertheless, PM2.5-exposed mice displayed attenuation of whole-body insulin sensitivity in response to intraperitoneal insulin injection (Fig. 2h, Supplemental Fig. 2E), evidenced by higher blood glucose 30 min after insulin injection in male mice (Fig. 2f) and clear separation of insulin tolerance test (ITT) curves in FA and PM groups in female mice (Fig. 2g). Interestingly, insulin sensitivity was worse in PM2.5-exposed female mice than that in male mice (Fig. 2i). Taken together, these results suggest that PM2.5-induced dysregulation in whole body insulin sensitivity but not in control of post-prandial glycemic response. In addition, sex-dependence was observed for PM2.5-associated attenuation in insulin sensitivity, with female mice being more susceptible
Ambient PM2.5 exposure induced hepatic lipid deposition in female mice
To get a more comprehensive understanding on the effect of PM2.5 on organ metabolism, hepatic lipid deposition was examined with Oil Red O staining. As shown in Fig. 3, PM2.5-exposed mice displayed more intracytoplasmic lipids in female than male mice (Fig. 3a), but we observed no effects of PM2.5 on liver mass (Fig. 3b).
Next, high-coverage quantitative lipidomics analysis was conducted on liver samples collected from both FA and PM2.5 exposed mice. A total of 421 lipids species from 24 subclasses were quantified. Although we observed no significant difference in either total hepatic diacylglycerols (DAG) or individual lipid species (Fig. 3, Supplemental Fig. 3A), a significant increase in levels of triacylglycerols (TAGs) was observed in PM exposed female mice but not in male mice (Fig. 3e, Supplemental Fig. 3B). Then, individual lipid species were examined. PM2.5 exposure induced significant increase in 15 of the 105 TAG species in male mice (Fig. 3f, Supplemental Material, Table S2). They were three saturated fatty acids (SFAs) of palmitic acid (16:0)-containing TAG, two docosahexaenoic acid (22:6)-containing TAG, two monounsaturated fatty acids (MUFAs) (16:1, 18:1)-containing TAG, and eight polyunsaturated fatty acids (PUFAs) (18:2, 18:3, 20:4, 22:5)-containing TAG (Supplemental Material, Table S2). PM2.5 exposure induced significant increase in 27 of the 105 TAG species in female mice (Fig. 3g, Supplemental Material, Table S2). They were four SFA of palmitic acid (16:0)-containing TAG, five DHA (22:6)-containing TAG, four MUFA (16:1, 18:1)-containing TAG, and 14 PUFA (16:2, 18:2, 18:3, 20:4, 22:5)-containing TAG (Supplemental Material, Table S2). Interestingly, consistent with the total level of TAG which showing higher level in female mice than male mice in response to PM2.5 exposure, 64 of the 105 TAG species increased in female mice compared to male mice (Fig. 3h, Supplemental Material, Table S2). The sex difference in response to PM2.5 exposure was further emphasized by principal component analysis (PCA), which discriminated the data obtained in ovals and circles (Fig. 3i). In addition, we also found a significant increase in levels of hepatic free cholesterols in PM2.5 exposed female mice but not in male mice (Fig. 3j, Supplemental Fig. 3C). There was no significant difference in levels of total cholesterol ester (CE) or CE species (Supplemental Fig. 3D, G, E and H) in response to PM2.5 exposure in male mice, whereas 2 of 18 CE species (CE-16:1, CE-20:3) elevated in PM2.5 exposed female mice compared to FA exposed female mice (Supplemental Fig. 3F and I). These observations cumulatively suggest a PM2.5-induced lipid accumulation in the liver, in particular in female mice.
Ambient PM2.5 exposure changes the profile of fatty acids in the liver
Next, levels of free fatty acid (FFA) were examined to confirm PM2.5-induced lipid metabolic dysregulation in female mice. Appreciable accumulation in total FFA was detected in the livers of PM2.5 exposed female but not male animals as shown by Fig. 4a-c, Supplemental Fig. 4A-C. A closer look into individual FFAs revealed that unsaturated fatty acid of 16:1 increased in female mice exposed to PM2.5, albeit not reaching significant difference. Further, FFA levels of n-3 family or n-6 family (22:6, 22:5, 22:4, 20:5, 20:4, 18:3) but not n-9 family (20:3,18:1) displayed significant or a trend toward increase (Fig. 4c, Supplemental Fig. 4C). These observations suggest an enhancement of FFA levels in female mice induced by PM2.5 exposure.
To investigate the mechanism that serves to retain fatty acid in the liver, we examined molecules of fatty acid export, fatty acid uptake and TAG hydrolysis. Expression of ApoB, the molecules involved in TAG and fatty acid export, increased in PM2.5-exposed female mice at both mRNA and protein levels. However, no significant difference was observed with Apo E (Fig. 4d-h). Interestingly, PM2.5 exposure upregulated expression of ApoB at protein levels, but not mRNA levels in male mice (Fig. 4d, f and g, Supplemental Fig. 4D). Expression of microsomal triglyceride transort protein (MTTP), which produces beta-lipoprotein including ApoB, increased in response to PM2.5 exposure at both mRNA level and protein levels in female mice (Fig. 4e, f and h, Supplemental Fig. 4E). No significant difference in levels of MTTP expression was observed in male mice (Fig. 4d, f and g, Supplemental Fig. 4D). None of fatty acid-binding protein 1 (FABP1), FABP2, FABP5 or CD36, were altered at transcriptional levels in the liver of PM2.5-exposed mice, independent of sex factors (Fig. 4d and e). In addition, examination of molecules for lipolysis demonstrated that hepatic expression of both adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) increased at both mRNA levels and protein levels in female, but not male, mice in response to PM2.5 exposure (Fig. 4d-h, Supplemental Fig. 4D-E). These results indicate that PM2.5 exposure enhanced hydrolysis of TAG and production of ApoB in the liver of female mice.
Ambient PM2.5 exposure increased the Plasmalogens in the liver
Plasmalogens are subclass of glycerophospholipid that contains vinyl ether and play multiple roles in cellular function by acting as components of the cell plasma membrane. Most plasmalogens have choline (PC-plasmalogens, PCp) in the polar head group and are enriched with n-3 or n-6 PUFAs, such as DHA (22:6 n-3) or arachidonic acid (20:4 n-6), in the sn-2 position. An investigation into the hepatic plasmalogens revealed increases in the levels of PCp in PM2.5 exposed female mice compared to their FA controls, while no such change was found in male mice exposed to PM2.5 (Fig. 5a, Supplemental Fig. 5A). Interestingly, detected PCp containing long chain and 0, 1, 2 or 5 double bonds (PC34:2p, PC34:1p, PC34:0p, PC36:2p, PC36:1p, PC36:0p, PC38:2p, PC38:1p, PC40:2p, PC40:1p, PC40:5p), PCp containing 4 double bonds (PC40:4p, PC38:4p, but not PC36:4p) in their structures significantly increased with PM2.5 exposure, whereas those with 3 double bonds (PC36:3p, PC38:3p, PC40:3p), were not altered (Fig. 5b, Supplemental Fig. 5B).
We next investigated potential reasons for increased plasmalogen levels. As shown in Fig. 5b-c, PM2.5 exposure showed no effect on hepatic mRNA expression of the rate-limiting enzymes in plasmalogen biosynthesis [glyceronephosphate O-acyltransferase (Gnpat) or alkylglycerone phosphate synthase (Agps)] or fatty acid desaturase (Fads1 and Fads2) which is associated with a decrease in DHA in hepatic lipids during plasmalogen synthesis either in male mice or female mice. Plasmalogens play a pivotal role in fatty acid metabolism in the liver and achieved this function by peroxisome proliferator-activated receptor alpha (PPARα). We found distinct increase in expression of PPARα and peroxisome proliferator-activated receptor gamma coactivator 1 beta (PGC1β) in liver from PM2.5-exposed female mice, whereas there was no significant difference in response to PM2.5 exposure in male mice (Fig. 5b and c). Carnitine palmitoyl transferase-1 (Cpt1α), acyl-CoA oxidase-1 (Acox1), very-long-chain acyl-CoA dehydrogenase (Vlcad), and D-bifunctional protein (Dbp1) are molecules implicated in mitochondrial and peroxisomal FAO. Consistent with changes in PPARα and PGC1β, Cpt1α and Acox1 were increased in expression at both mRNA levels (Fig. 5d) and protein levels (Fig. 5e and g, Supplemental Fig. 5C-D) in PM2.5-exposed female mice, while only Cpt1α increased at mRNA level in male mice (Fig. 5c).
Ambient PM2.5 exposure inhibited HPA Axis
To explore the mechanism of PM2.5-induced metabolic dysfunction and female susceptibility, plasma hormones in relation to PM2.5 exposure were examined. Lower levels of steroids were observed in both male and female mice after PM2.5 exposure (Fig. 6a and b, Supplemental Fig. 6A-B). Level of corticosterone decreased in male mice whereas cortisol decreased in female mice (Fig. 6a and b, Supplemental Fig. 6A-B). Next, mRNA levels of corticotropin-releasing hormone (CRH) in hypothalamus decreased in male mice (Fig. 6c) and adrenocorticotropic hormore (ACTH) in pituitary decreased in female mice (Fig. 6d) in response to PM2.5 challenge. In addition, circulating sex hormones were also examined with mice exposed to PM2.5. As shown in Supplemental Fig. 6C-F, PM2.5 significantly increased testosterone levels in the plasma in male mice (Supplemental Fig. 6C, E). There was no significant alteration in other hormones observed (Supplemental Fig. 6C-F).