Ambient air pollution exaggerates adipose inflammation and insulin resistance in a mouse model of diet-induced obesity

Abstract

Background: There is a strong link between urbanization and type 2 diabetes mellitus. Although a multitude of mechanisms have been proposed, there are no studies evaluating the impact of ambient air pollutants and the propensity to develop type 2 diabetes mellitus. We hypothesized that exposure to ambient fine particulate matter (<2.5 mum; PM(2.5)) exaggerates diet-induced insulin resistance, adipose inflammation, and visceral adiposity.

Methods and results: Male C57BL/6 mice were fed high-fat chow for 10 weeks and randomly assigned to concentrated ambient PM(2.5) or filtered air (n=14 per group) for 24 weeks. PM(2.5)-exposed C57BL/6 mice exhibited marked whole-body insulin resistance, systemic inflammation, and an increase in visceral adiposity. PM(2.5) exposure induced signaling abnormalities characteristic of insulin resistance, including decreased Akt and endothelial nitric oxide synthase phosphorylation in the endothelium and increased protein kinase C expression. These abnormalilties were associated with abnormalities in vascular relaxation to insulin and acetylcholine. PM(2.5) increased adipose tissue macrophages (F4/80(+) cells) in visceral fat expressing higher levels of tumor necrosis factor-alpha/interleukin-6 and lower interleukin-10/N-acetyl-galactosamine specific lectin 1. To test the impact of PM(2.5) in eliciting direct monocyte infiltration into fat, we rendered FVBN mice expressing yellow fluorescent protein (YFP) under control of a monocyte-specific promoter (c-fms, c-fms(YFP)) diabetic over 10 weeks and then exposed these mice to PM(2.5) or saline intratracheally. PM(2.5) induced YFP cell accumulation in visceral fat and potentiated YFP cell adhesion in the microcirculation.

Conclusions: PM(2.5) exposure exaggerates insulin resistance and visceral inflammation/adiposity. These findings provide a new link between air pollution and type 2 diabetes mellitus.

Figure 1

Glucose tolerance test and vasomotor response in FA- or PM_2.5-exposed C57BL/6 mice fed HFC. A, Blood glucose levels by glucose tolerance test. B, Mean vasorelaxation of aortic rings in response to acetylcholine (Ach). C, Insulin-mediated vasorelaxation in aortic rings preconstricted with phenylephrine (10⁻⁷ mol/L). D, Maximum phenylephrine-induced vasoconstriction in aortic rings preincubated with the nitric oxide synthase inhibitor L-NMMA (10⁻⁴ mol/L). n=7. *P<0.05 vs FA.

Figure 2

PM_2.5 exposure impairs insulin signaling via the PI3-kinase/Akt pathway in FA- or PM_2.5-exposed C57BL/6 mice fed HFC. A, Immunoblotting and analysis of Akt (Ser⁴⁷³) phosphorylation in intact aortic tissues. B, Immunoblotting and analysis of Akt (Ser⁴⁷³) phosphorylation in denuded aortic tissues. C, Time course of Akt phosphorylation in response to insulin (500 mU/mL) in intact aortic rings. n=5. *P<0.05 vs FA.

Figure 3

Exposure to ambient PM2.5 increases circulatory adipokines and inflammatory biomarkers in C57BL/6 mice fed HFC. A, TNF-α and IL-6. B, E-selectin, ICAM-1, and plasminogen activator inhibitor-1 (PAI-1). C, Resistin and leptin. n=7. *P<0.05 vs FA.

Figure 4

PM_2.5 exposure increases visceral fat mass measured by magnetic resonance imaging in C57BL/6 mice fed HFC. A and B, T1-weighted spin echo images in the FA and PM_2.5 group, respectively. C, Analysis of fat mass distribution in the 2 groups. n=5. ◃ points to visceral fat tissue. Subcu indicates subcutaneous; Retro, retroperitoneal; and Mesen, mesenteric. *P<0.05 vs FA.

Figure 5

PM₂.5 exposure increases ATM infiltration and adipocyte size in C57BL/6 mice fed HFC. A, Immunohistochemistry for macrophage-specific marker F4/80 in sections of epididymal fat pads from FA- and PM_2.5-exposed mice. B, Quantification of ATMs in fat pads. F4/80⁺ nuclei were counted and expressed as a percentage of the total nuclei per ×400 power field. Four sections were evaluated from each mouse in each group. C, Adipocyte-size histograms show increased adipocyte hypertrophy in PM_2.5-exposed mice. Areas were calculated from 90 adipocytes from each of 5 mice in each group. D, Box plot of adipocyte size. The box represents the upper and lower quartiles. The whiskers or bars show the 5th or 95th percentiles. The line in the box represents the median. The + signs represent the mean. Raw numbers: FA: mean, 883±21 μm²; median, 839 μm²; PM_2.5: mean, 973±26 μm²; median, 905 μm². n=7. *P<0.05 vs FA.

Figure 6

Exposure to ambient PM_2.5 increases macrophage infiltration in adipose tissue and induces a shift in macrophage phenotype characteristics in C57BL/6 mice fed HFC. A, Immunofluorescence localization of ATMs (F4/80) in epididymal fat pads from mice exposed to FA or PM_2.5. Adipocytes identified by caveolin (Cav) staining and nuclei labeled with TOPRO3. Scale bar=100 μm. B, Real-time polymerase chain reaction measurement of macrophage M1/M2 gene expression. PM_2.5 treatment resulted in significant increases in the M1 phenotypic genes TNF-α and IL-6 in the F4/80⁺ cells of stromal vascular fraction (SVF). Nos2 indicates nitric oxide synthase-2; Itgax, integrin αX, CD11c; and Pparγ, peroxisome proliferator–activated receptor. n=7. *P<0.05 vs FA.

Figure 7

Glucose and insulin tolerance tests and YFP cell fluorescence staining of live adipose tissue in c-fms^YFP mice fed HFC. A, Blood glucose levels during glucose tolerance test. B, Blood insulin levels during glucose tolerance test. C, Unfixed live adipose tissue from HFC-fed transgenic mice that express yellow fluorescent protein (c-fms^YFP, yellow) was stained with Hoechst 33342 (blue) and isolectin (red) by confocal microscopy. Isolectin is an endothelium-specific marker. PM_2.5 treatment resulted in increased YFP cell infiltration into the adipose tissue compared with the saline control. n=4. *P<0.05, normal chow vs HFC.

Figure 8

PM_2.5 exposure increases YFP cell infiltration in mesenteric tissue and YFP cell adhesion in the cremasteric vasculature in c-fms^YFP mice fed HFC. A, B, Representative images of YFP cells in mesenteric tissue treated by either saline (A) or PM_2.5 (B) and the quantification of YFP cells (C). D, Representative image of YFP cells in cremasteric tissue treated by PM_2.5. The arrows depict YFP cells inside the vessel; closed arrowheads, YFP cells outside the vessel. E, Quantification of adherent YFP cells. The postcapillary venule boundaries are outlined in white lines. n=4. *P<0.05, saline vs PM_2.5.