2013 Progress Report: Long Term Metabolic Consequences of Exposures to Multipollutant Atmospheres in the Great Lakes Region

EPA Grant Number: R834797C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R834797
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Great Lakes Air Center for Integrative Environmental Research
Center Director: Harkema, Jack
Title: Long Term Metabolic Consequences of Exposures to Multipollutant Atmospheres in the Great Lakes Region
Investigators: Rajagopalan, Sanjay , Sun, Qinghua
Institution: The Ohio State University - Main Campus
EPA Project Officer: Ilacqua, Vito
Project Period: December 1, 2010 through November 30, 2015 (Extended to December 31, 2016)
Project Period Covered by this Report: August 1, 2012 through July 31,2013
RFA: Clean Air Research Centers (2009) RFA Text |  Recipients Lists
Research Category: Health Effects , Air

Objective:

We have recently demonstrated that short-term exposure to inhaled concentrated airborne particulate (CAP) matter <2.5μm (PM2.5) results in components of cardiometabolic syndrome (CMS) including development of hypertension and insulin resistance. In this project, we hypothesize that chronic inhalation of CAP in conjunction with gaseous components such as ozone from distinct multipollutant atmospheres synergistically interacts with diet and genetic susceptibility to influence development of CMS. Project 3 is an integral component of the overarching theme of this center that primary air pollutants, fine PM (PM2.5) and ozone (O3), cause cardiometabolic health effects that are dependent on the local atmospheric multipollutant milieu, predisposing factors, and the interactive toxicity of multipollutant coexposure. The experiments proposed are natural extensions of human research outlined in Project 1 and acute experiments in Project 2 and will focus on conducting chronic inhalation toxicology studies in diet fed and genetic models of obesity/diabetes. In Aim 1, simultaneous chronic exposure to multipollutant CAP from two locations in Columbus, OH representing near-roadside/traffic or remotely transported/aged emissions will be examined in combination with high fat chow (HFC). The impact of CAP on glucose/insulin homeostasis, adipokines, insulin signaling, adipose and pulmonary inflammation and an analysis of dose dependence and CAP components most likely associated with these effects will be evaluated in diet sensitive (C57BL/6) and genetic models of Type II diabetes susceptibility (KKA/y). In Aim 2, we will investigate the effect of co-exposure of multipollutant CAP with ozone on the temporal development of insulin resistance and adipose/lung inflammation using the KKA/y model. We will assess dose response relationship of multipollutant-O3 mixture on insulin resistance measures (HOMA-IR and IPGTT) and novel mediators of innate immune, pivotal in the development of metabolic derangement. Based on data from Aims 1 and 2, we will design experiments in Aim 3, which will help us assess chronic effects of multipollutant CAP in potentiating inflammatory monocyte activation and infiltration into tissue niches as a central mechanism for mediating adverse metabolic effects of CAP. Using state of the art multiple exposure systems available at OSU (OASIS-1 and OASIS-2) and MI in conjunction the resources available at the ECC including the use of several novel and novel high-time resolution exposure characterization methods, GLACIER offers an unprecedented opportunity to elucidate relevant mechanisms responsible for the effects of multipollutant CAP on the pathogenesis of insulin resistance and inflammation. The insights gleaned from the acute studies planned in Projects 1 and 2 in conjunction with chronic studies in Project 3, have significant public health ramifications and may eventually lead to policy changes to avert environmental exposure to PM2.5.

Progress Summary:

Conduct of Simultaneous Exposures in OASIS-1 and OASIS-2
During the last year we have accomplished significant progress towards beginning exposures in our near road-way site (OASIS-2) and comparing this site with OASIS-1 (regional site). We are now analyzing the data and are on our way towards preparation of a paper that will provide comparative information on the characterization of these two sites in Columbus. We are also in the process of determining if source apportionment can be performed to understand the differences in sources at these two sites. Our preliminary data indicates higher BC concentration at the OASIS-2 site in keeping with its proximity to the roadway.
 
 
Progress in Aim 1
We have completed Specific Aim 1 and as part of these experiments report on data on the effects of concentrated ambient PM2.5 when used alone in potentiation of glucose intolerance and IR in a genetic model of Type II DM. KKay mice, which are susceptible to Type II DM, were assigned to either concentrated ambient PM2.5 or filtered air (FA) for 4-8 weeks. The mice were exposed for 6 h/d, 5 d/wk, 5 -8 weeks in a mobile trailer exposure system (“Ohio Air Pollution Exposure System for Interrogation of Systemic Effects 1,” located at the near-roadway site located in Polaris (OASIS-2). The figure outlines the BC and PM2.5 concentrations at the site during 3 separate exposures and EC/OC concentrations for one of the exposures (Exposure 3).

 

PM2.5 and BC concentration to which mice were exposed at the study site. A, BC concentration in the ambient air in Columbus at OASIS-2 from Dec.2011-Nov.2012. Exposure 1-Exposure 3 labeled under the X-axis with exact date information. B, PM2.5 concentration in the filter air, ambient air and concentrated ambient air for Exposure 1. C, PM2.5 concentration in the filter air, ambient air and concentrated ambient air, and BC concentration in the ambient air and concentrated air for Exposure 2. D, PM2.5 concentration in the filter air, ambient air and concentrated ambient air, and BC concentration in the ambient air and concentrated air for Exposure 3. The values of PM2.5/BC concentration were shown above the according bar graphs in B-D. E, OC & EC concentration in the PM2.5 from Exposure 3. BC denotes black carbon; OC denotes organic carbon; EC denotes element carbon.
 
Demonstration of Time Course of Development of IR and Inflammation in KKay with PM2.5
As part of our original proposal we had proposed investigating the time course of development of the IR response to airpollution using a genetically pre-disposed model. We found that there were no changes in body weight or food intake during exposure. Circulating adiponectin was decreased at 5 weeks of exposure, accompanied by an increase in leptin levels. There were no further changes in adiponectin or leptin after 8 weeks exposure. An increase in fasting blood glucose was seen within 1-week of exposure to PM2.5, while insulin levels trended upwards, with the highest values after 6 weeks of exposure and significant differences between FA/ PM2.5 at the 3- and 8-week time-points. Corresponding HOMA-IR levels were significantly higher with concentrated ambient PM2.5 at the 1, 3- and 8-week time-points. Responses to intra-peritoneal glucose challenges were abnormal after 5 weeks of PM2.5 exposure, characterized by marked worsening of glycemic response to glucose challenge. This change in glucose tolerance persisted at 8 weeks (Figure 1H). Insulin tolerance was also impaired at 5 and 8 weeks.
 
Effect of PM2.5 exposure on energy homeostasis and BAT gene expression
We next investigated the effect of concentrated ambient PM2.5 exposure on whole body energy homeostasis. PM2.5 exposure reduced oxygen consumption, carbon dioxide production, respiratory exchange ratio and thermogenesis. In light of the significant change in thermogenesis, we investigated uncoupling protein 1(UCP1), a pivotal player in thermogenesis via uncoupling of oxidative phosphorylation. UCP1 protein but not mRNA expression was downregulated in interscapular brown adipose tissue (BAT) of PM2.5 exposed mice, consistent with decreased thermogenic activity. PPARγ-coactivator-1alpha (PGC1α) and PRD1-BF1-RIZ1 homologous domain-containing 16 (PRDM16) expression, transcriptional activators that play a key role in BAT development and acquisition of thermogenic phenotype were however no different. An increase in IL-6 and TNFα was also noted in BAT, indicating PM2.5 induced inflammation in brown adipose tissues. Taken together, alteration of UCP1 via post-transcriptional mechanisms and inflammation in BAT may account for the decreased thermogenesis and oxygen consumption in response to PM2.5 exposure.
 
Systemic Inflammation in Response to Concentrated Ambient PM2.5 Exposure
Another essential question that we sought to address in our proposed investigations in Aim 1 was to elucidate the extent of systemic inflammatory response with concentrated ambient PM2.5. We investigated a population of inflammatory monocytes that we and others have shown to be relevant in response to diverse triggers including high-fat feeding and high cholesterol chow. We found that PM2.5 exposure increased CD11b+Ly6Glow7/4hi cells in the peripheral circulation with a corresponding trend towards a reduction in this population in the bone marrow. Although there were no differences in splenic CD11b+Ly6Glow7/4hi cells (Figure 3A), IFN production from splenocytes of PM2.5 exposed mice was significantly higher compared to that of FA mice with a corresponding decrease in IL-4 release. These results suggest a redirection of Th1/Th2 balance towards a Th1 polarized state in response to PM2.5 exposure. Type 2 DM in humans and animal models is associated with increased levels of recruitment and/or activation of innate immune cells in visceral adipose depots. It has been shown that PM2.5 exposure results in an increase in adipose tissue macrophages with a shift to a pro-inflammatory phenotype characterized by an increase in F4/80 macrophages in the visceral adipose and a pro-inflammatory “M1 phenotype” typified by TNF-α, IL-6 and a decrease in IL-10, MgI1 gene expression. We observed an increase in F4/80+/CD11c+ cells in VAT in response to PM2.5 exposure. Together with data demonstrating that PM2.5-mediated monocyte infiltration into VAT is CCR2-/- dependent (unpublished data), these results suggest mechanisms similar to those involved in diet mediated aggravation of the VAT infiltration by monocytes via CCR2 dependent pathways.
 
Hypothalamic Inflammation in Response to Concentrated Ambient PM2.5 in KKay Mice
This is a new observation that we have made as part of our investigations that may provide an integrated understanding of the diverse effects of PM2.5. CNS inflammation has been shown to be relevant to the pathogenesis of Type II DM and the development of peripheral inflammation in response to factors such as high-fat feeding in both animal models as well as studies in humans.
 
We examined mRNA encoding inflammatory mediators, including cytokines (IL-6, TNFα), Suppressor-of-cytokine signaling (Socs3), components of the NFκB pathway, and microglia/macrophage (MAC1) in hypothalamic centers. In mice exposed to PM2.5, TNFα and IL-6 expression was elevated after 5-weeks of exposure to PM2.5 as compared to FA. Longer duration (8 week) exposure to PM2.5 elevated TNFα and IL6 expression, as well as significantly increased IKK expression. These results suggest that even short-term exposure of a few weeks is sufficient to induce increases in cytokine expression in the medial basal hypothalamus. SOCS-3 mRNA levels were unaltered in the hypothalamus. SOCS proteins were originally identified as anti-inflammatory, negative regulators of cytokine receptor signaling and attenuate leptin receptor signaling contributing to central leptin resistance. Mice deficient in SOCS-3 or with brainspecific SOCS-3 deletion are more sensitive to the anorectic effects of leptin and are resistant to DIO. The lack of an elevation in SOCS-3 expression with PM2.5 in the face of hyperleptinemia could potentially drive CNS inflammation while allowing continued leptin signaling and may account for maintenance of body weight.
 
Metabolic Responses to intracerebroventricular Administration of TNF-a Blockade in Response to PM2.5 Exposure
Based on the increased hypothalamic TNF expression in PM2.5 mice and observations that increased hypothalamic inflammation appears to occur early following initiation of a high fat diet and may contribute to changes in peripheral inflammation including brown adipose tissue (BAT) dysfunction, we hypothesized that TNFα antagonism would restore peripheral glucose intolerance and altered thermogenesis following PM2.5 exposure. Mice at age of 5 weeks old were continuously administered infliximab (0.2ug/day) or artificial CSF (aCSF) at a rate of 0.11uL/hr through a minipump connected to a cannula directed at the lateral ventricle (+0.2 posterior and -0.95 lateral to Bregma, extending 2.75 mm below the skull). ICV infliximab did not influence peripheral glycemia or insulin tolerance in response to PM2.5 exposure; neither did body temperature differ between groups. However, infliximab treatment significantly impaired energy homeostasis, as evidenced by further decreases in O2 consumption, CO2 production, respiratory exchanging ratio, and heat generation in inflixmab treated mice compared to artificial CSF controls. This is contrary to reports in which the centrally TNFα administration reduced the expression of thermogenic proteins in brown adipose tissue and skeletal muscle, effects which were blunted in TNFα receptor knockout mice. It has been suggested that, depending on its local concentrations, TNFα can exert a dual functions in the hypothalamus, being catabolic at high concentrations and anabolic at low concentration. Consistent with these paradoxical effects on energy metabolism, TNFα levels in obese animals are higher than controls but significantly lower than tumor-bearing rats, accompanied by inhibition of feeding/anorexia in tumorbearing and increase in feeding/obesity in obese animals respectively.
 
Central IKK2 Inhibition Prevents PM2.5-Induced Disruption of Glucose/Energy Balance and Peripheral Inflammation
To determine whether increased hypothalamic IKK- NF-κB pathway contributes to PM2.5’s effects, we administered a pharmacological inhibitor of IKK2 (IMD-0354) ICV and concomitantly exposed mice at age of 7 weeks old to PM2.5 or FA for 4 weeks (3rd Exposure). IKK-2 inhibition had no effect on food intake and body weight in either FA or PM2.5 mice. In contrast, PM2.5-induced changes in glucose and insulin were normalized with IMD-0354 treatment. This effect was PM2.5 dependent, as IMD-0354 did not further improve these parameters in the FA group. Taken together, these results suggest that central IKK2 inhibition prevents PM2.5-induced abnormalities in glucose/insulin homeostasis. As expected, ICV IMD-0354 normalized energy metabolism, reflected by restored O2 consumption, CO2 production, respiratory exchanging ratio and heat generation, compared with PM2.5-exposed mice administered vehicle. Moreover, inhibition of central inflammation improved energy homeostasis including oxygen consumption and heat generation, in the FA group as well. Interruption of IKK-2 signaling was sufficient to restore weight gain in these experiments. We did not see weight gain in response to PM2.5 despite reduction in BAT thermogenesis and increased peripheral inflammation. While these changes may be specific to the model (KKay mice) and the short duration of exposure, they also suggest complex adaptations in satiation/adiposity signals that may warrant further study.
 
Central IKK2 Inhibition Ameliorates PM2.5-induced Peripheral Inflammation
We next investigated whether central IKK inhibition ameliorated peripheral inflammation induced by PM2.5 exposure. There was a clear trend (P = 0.0518) for an increase in circulating CD11b+Ly6Glow7/4hi cells with concentrated ambient PM2.5 exposure that was nearly normalized by IMD-0354 treatment. F4/80+/CD11c+ cells in the epididymal adipose was markedly higher in response to concentrated ambient PM2.5 exposure, which is consistent with the prior exposure period and this increase was attenuated by IMD-0354. These results indicate that peripheral inflammation in blood and adipose tissue in response to PM2.5 is dependent to a considerable degree on CNS inflammation.
 
Central IKK2 Inhibition Suppresses PM2.5-induced Hypothalamic Inflammation
IMD-0354 reduced IL-6 and IKK expression, both of which were upregulated by PM2.5 exposure, but had no effect on TNFα and IκB expression. We next investigated the presence of reactive gliosis, characterized by the recruitment, activation, and proliferation of glial cells, such as astrocytes, NG2 cells, and microglia, in the in the arcuate nucleus of the hypothalamus. Using anti-Iba1 (ionized calcium binding adaptor molecule 1), a microglia-specific cytoplasmic marker, we found a 50% increase in microglial number in the arcuate nucleus of mice exposed to PM2.5 compared to FA. Additionally, microglia from PM2.5 mice were larger with a more activated morphology. Central IMD-0354 infusion restored both the numbers and size of microglia in response to PM2.5. The effect of PM2.5 on astrocytes was assessed with GFAP immunostaining. Astrocytes are abundant throughout the CNS and were apparent in the arcuate nucleus of all groups. The intensity of GFAP staining was elevated by exposure to PM2.5 with the increase in GFAP staining among PM2.5 mice prevented by treatment with the IKK inhibitor. While cell boundaries of astrocytes in the arcuate nucleus of the KKay mice exposed to FA was relatively preserved, the boundaries of astrocytes exposed to PM2.5 coalesced into a dense network reminiscent of a syncytium (Figure 10), a finding pathognomonic of reactive gliosis.
 
Summary
During this project cycle we have generated the following new findings and contributed to further understanding of the link between PM2.5 and inflammation:
  1. Began exposures in a near roadway site and performed characterization of the exposure milieu.
  2. Concentrated ambient PM2.5 exaggerated Type II DM development in a genetic model with effects evident within 5 weeks including decreased thermogenesis, increased peripheral inflammation.
  3. Concentrated ambient PM2.5 exposure caused rapid effects (in KKay) of hypothalamic inflammation.
  4. Centrally inhibition of IKKβ but not TNFα blockade showed improvement in insulin resistance and metabolism.

 

Future Activities:

Before embarking on simultaneous exposures of PM2.5 + Ozone it was important for us to determine the extent of effects attributable to PM2.5 alone. We will perform the experiments in Aim 3, which assess chronic effects of multipollutant CAP in potentiating inflammatory monocyte activation and infiltration into tissue niches as a central mechanism for mediating adverse metabolic effects of CAP.


Journal Articles on this Report : 6 Displayed | Download in RIS Format

Other subproject views: All 46 publications 35 publications in selected types All 35 journal articles
Other center views: All 147 publications 71 publications in selected types All 71 journal articles
Type Citation Sub Project Document Sources
Journal Article Blazek A, Rutsky J, Osei K, Maiseyeu A, Rajagopalan S. Exercise-mediated changes in high-density lipoprotein: impact on form and function. American Heart Journal 2013;166(3):392-400. R834797 (2013)
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  • Journal Article Brook RD, Bard RL, Kaplan MJ, Yalavarthi S, Morishita M, Dvonch JT, Wang L, Yang H-Y, Spino C, Mukherjee B, Oral EA, Sun Q, Brook JR, Harkema J, Rajagopalan S. The effect of acute exposure to coarse particulate matter air pollution in a rural location on circulating endothelial progenitor cells: results from a randomized controlled study. Inhalation Toxicology 2013;25(10):587-592. R834797 (2013)
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  • Journal Article Liu C, Ying Z, Harkema J, Sun Q, Rajagopalan S. Epidemiological and experimental links between air pollution and type 2 diabetes. Toxicologic Pathology 2013;41(2):361-373. R834797 (2013)
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  • Journal Article Sun L, Liu C, Xu X, Ying Z, Maiseyeu A, Wang A, Allen K, Lewandowski RP, Bramble LA, Morishita M, Wagner JG, Dvonch JT, Sun Z, Yan X, Brook RD, Rajagopalan S, Harkema JR, Sun Q, Fan Z. Ambient fine particulate matter and ozone exposures induce inflammation in epicardial and perirenal adipose tissues in rats fed a high fructose diet. Particle and Fibre Toxicology 2013;10:43. R834797 (2013)
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  • Journal Article Wagner JG, Allen K, Yang HY, Nan B, Morishita M, Mukherjee B, Dvonch JT, Spino C, Fink GD, Rajagopalan S, Sun Q, Brook RD, Harkema JR. Cardiovascular depression in rats exposed to inhaled particulate matter and ozone: effects of diet-induced metabolic syndrome. Environmental Health Perspectives 2014;122(1):27-33. R834797 (2013)
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  • Journal Article Ying Z, Xu X, Bai Y, Zhong J, Chen M, Liang Y, Zhao J, Liu D, Morishita M, Sun Q, Spino C, Brook RD, Harkema JR, Rajagopalan S. Long-term exposure to concentrated ambient PM2.5 increases mouse blood pressure through abnormal activation of sympathetic nervous system:a role for hypothalamic inflammation. Environmental Health Perspectives 2014;122(1):79-86. R834797 (2013)
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  • Supplemental Keywords:

    Ozone (O3) PM2.5, Type II Diabetes Mellitus (DM), Insulin Resistance (IR), Scientific Discipline, Air, ENVIRONMENTAL MANAGEMENT, air toxics, Health Risk Assessment, Biochemistry, Biology, Risk Assessment, ambient air quality, particulate matter, aerosol particles, susceptible populations, acute cardiovascualr effects, human exposure, physiology, cardiopulmonary, cardiotoxicity

    Relevant Websites:

    GLACIER: Great Lakes Air Center For Integrated Environmental Research Exit

    Progress and Final Reports:

    Original Abstract
  • 2011 Progress Report
  • 2012 Progress Report
  • 2014 Progress Report
  • 2015 Progress Report
  • 2016 Progress Report
  • Final Report

  • Main Center Abstract and Reports:

    R834797    Great Lakes Air Center for Integrative Environmental Research

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R834797C001 Cardiometabolic Effects of Exposure to Differing Mixtures and Concentrations of PM2.5 in Obese and Lean Adults
    R834797C002 Cardiometabolic, Autonomic, and Airway Toxicity of Acute Exposures to PM2.5 from Multipollutant Atmospheres in the Great Lakes Region
    R834797C003 Long Term Metabolic Consequences of Exposures to Multipollutant Atmospheres in the Great Lakes Region