In this paper we evaluated the role of ambient air pollution on energy metabolism, glucose homeostasis, and inflammatory signaling pathways in a genetically predisposed model that develops severe Type II DM within 6–10 weeks. There are several important findings in this study that support an adjunctive role of air-pollution exposure in potentiating development of Type II DM. Firstly, PM2.5 exposure had effects on energy metabolism including reduction of O2 consumption, CO2 production, respiratory exchanging ratio and heat generation. Secondly, PM2.5 exposure exaggerated IR, visceral adiposity and peripheral inflammatory response. Thirdly, PM2.5 exposure resulted in lowered UCP1 expression in BAT together with increased expression of inflammatory genes. Finally, PM2.5 exposure resulted in activation of p38 and ERK but not JNK in the liver of KKay mice.
There is increasing evidence suggesting links between exposure to environmental toxins and susceptibility to Type II DM. Consistent with the study in a diet-induced obesity model[6, 8, 19], PM2.5 elevated blood glucose levels and impaired insulin signaling evidenced by reduced Akt phosphorylation in the genetic KKay mice. These results indicate the reproducible effect of air pollution in mediating adverse metabolic consequences in a genetically susceptible model of Type II DM. Thus it provides additional experimental evidence to support the association between PM2.5 and cardiometabolic disease.
One of the central findings of this study is the changes in metabolic indices demonstrated in carefully performed metabolic cage studies. Recent studies have suggested a compelling role for BAT in regulating metabolism and insulin resistance[20–22]. Brown adipocytes play an important role in dissipation of energy in the form of heat, a process called non-shivering thermogenesis. A reduction in thermogenic function of BAT has been linked to the development of IR and obesity[20–22]. UCP1, which is specifically expressed in BAT mitochondria, is largely responsible for the uncoupling of respiration from ATP synthesis resulting in dissipation of energy as heat, playing a pivotal role in thermogenesis. We have previously demonstrated abnormalities in BAT structure and function with chronic exposure to PM2.5. These changes include electron microscopic changes in BAT mitochondria as well as transcriptional reprogramming of BAT genes which include UCP1 expression.
The substantial down-regulation of UCP1 protein in BAT in the PM2.5 exposed mice may potentially account for decreased heat production and thereby explain reduction in O2 consumption and CO2 production. Interestingly, high fat diet treated mice showed substantial compensatory increase in the expression of UCP1 both at the protein levels and mRNA levels, likely representing an adaptive mechanism to excess caloric intake. Fromme et al. summarized 62 relevant studies of which 42 studies have previously demonstrated that high fat diet itself enhanced UCP1 expression. Thus our findings suggest a unique role of PM2.5 exposure in preventing the adaptive increase of UCP1. These findings are consistent with our previous studies showing that long term PM2.5 exposure reduced UCP1 expression in C57B/L6 mice. An important point in our study that deserves further explanation is the finding that in many end-points we observed a difference at 5 weeks but not at 8 weeks. It is possible that the severity of phenotype at 8 weeks in the KKay makes it very difficult to distinguish end-points at 8 weeks between groups.
White adipose tissue is a major source of energy for the human body. The increased epididymal (visceral) fat mass in mice exposed to concentrated PM2.5 demonstrated that PM2.5 inhalation induced visceral adiposity likely through adipocyte hypertrophy (indicating excess energy storage which was supported by decreased energy expenditure in PM2.5-exposed KKay mice) and enhanced inflammatory cell infiltration. Macrophages in the adipose tissue have been shown to increase from 10-15% to 45-60% of total cellular content during obesity and may independently have contributed to expansion of VAT mass. In addition, the adipose tissue is also a source of major adipocytokines such as adiponectin and leptin. The expression of adiponectin decreased with increase in the adiposity and reduction of adiponectin has been associated with insulin resistance, dyslipidemia, and atherosclerosis in humans. In line with this, circulating adiponectin levels were significantly reduced by PM2.5 inhalation as has been shown by us previously. Adiponectin plays an important role in mediating insulin-sensitization through binding to its receptors AdipoR1 and AdipoR2, leading to activation of adenosine monophosphate dependent kinas (AMPK) and presumably other yet-unknown signaling pathways. A trend towards a decrease in AMPK phosphorylation in liver of the KKay mice exposed to PM2.5 could potentially be attributable to reduced adiponectin release. Leptin is another major adipokine from adipose tissue. Contrary to the remarkably decreased circulating leptin levels in response to PM2.5 exposure in C57BL/6 mice fed on regular chow, we observed an increase in leptin levels in the PM2.5 inhaled KKay mice. The mechanisms by which leptin expression is regulated need further study. The net action of leptin is to inhibit appetite, stimulate thermogenesis, enhance fatty acid oxidation, decrease glucose, and thus reduce body weight and fat through central mechanisms. Recent studies have suggested that central leptin resistance may contribute to attenuation of the well known effects of leptin. Since the circulating leptin levels in our studies were increased in response to PM2.5 exposure, our results raise the possibility of leptin resistance as an additional component and will need further study. Consistent with our findings, Bremer et al. demonstrated the novel observation that adipose tissue in subjects with nascent metabolic syndrome have increased levels of leptin as well as decreased levels of adiponectin and omentin-1, concomitant with increased adipokines such as IL-1, IL-6, IL-8, PAI-1 and MCP1[28, 29]. Thus it can be assumed that adipose tissue dysregulation and aberrant adipokine secretion contribute towards the syndrome’s low-grade chronic proinflammatory state and IR accompanied by correction for increased adiposity in the current KKay mice.
A number of studies have highlighted the innate immune mechanisms as the critical role that is responsible for the pathophysiological abnormalities, including IR. In line with this, our results demonstrated that although PM2.5 inhalation did not enhance inflammotary cytokines production in blood, it did result in increased circulating inflammatory monocytes (CD11b+/7/4hi/Gr-1low) accompanied by a corresponding reduction in bone marrow. We have previously demonstrated this same finding in our earlier studies and shown an important role for TLR4 in mobilization of inflammatory subsets of monocytes in response to PM2.5 exposure. This subset is believed to mediate pro-inflammatory effects and a decrease in this population has been associated with favorable end-points including regression of atherosclerotic lesions and macrophage accumulation. It is well known that monocytes originate from progenitors in the bone marrow and traffic via the bloodstream to peripheral tissues. As a homeostatic response to diverse triggers, circulating monocytes leave the bloodstream and migrate into tissues where, they differentiate into macrophage or dendritic cell populations following conditioning by local growth factors such as pro-inflammatory cytokines and microbial products. F4/80+/CD11c+ is a widely used marker to label “classically activated” macrophages that have been demonstrated to play a pathophysiological role in high-fat diet-induced obesity[26, 32–34]. Our findings in this study extend our previous observations where we have shown increased macrophage infiltration into VAT in response to concentrated PM2.5 exposure, in excess of the effects of high-fat diet alone. Furthermore, the macrophages in VAT in mice exposed to PM2.5 (72.7 μg/m3, 128 days) demonstrated an “M1” profile with increased cytokines such as TNFα and IL-6, and reduced expression of IL-10 and N-acetyl-galactosamine specific lectin 1. Using a mouse model with yellow fluorescent protein (YFP)-expressing monocytes (c-fmsYFP), we further confirmed enhanced monocyte adhesion in microcirculation of VAT and accumulation in visceral fat. The increased monocyte/macrophage infiltration in VAT in response to PM2.5 exposure further appears to be dependent on CCR2 (116.9 ± 34.2 μg/m3, ~17 weeks), as it was abolished by genetic ablation of CCR2. Thus, the increased population of F4/80+/CD11c+ in eWAT suggest mechanisms similar to those involved in diet mediated aggravation of the VAT infiltration via CCR2-dependent pathways. Except for the increase in excess oxidative and nitrosative stress in BAT after long term PM2.5 exposure, we demonstrated in the current study that PM2.5-mediated up-regulation of pro-inflammatory genes in BAT and provided additional explanation for the inhibited energy metabolism. Together with the redirection of Th1/Th2 balance towards a Th1 polarized state in response to PM2.5 exposure (Figure 5B), these results suggest the increased inflammation in visceral WAT, BAT and Th1 polarization in spleen indicated recruitment of monocytes into tissues, therefore, contributed to the pathogenesis of inflammatory diseases such as IR or diabetes.
p38 MAPK belongs to a family of evolutionarily conserved serine-threonine MAPKs that link extracellular signals to intracellular machinery regulating a plethora of cellular processes. Together with JNK, they are activated by environmental or genotoxic stress and described as stress-activated protein kinases[35–37]. Different from the observation with PM2.5-exposed C57BL/6 mice fed on regular chaw by Zheng et al., we found activation of p38 and ERK but not JNK in response to PM2.5 in the KKay mice. These differences likely represent differences in strains and genetic susceptibility, in addition to the complex interactions of diet and environmental signals. In keeping a line with our study, Jiao et al. have suggested that the increased hepatic ERK activity may contribute to increased liver glycogen content and decreased energy expenditure in obesity and may play a central role in hepatic glucose and lipid metabolism[39, 40]. However, whether increased MAPKs activity is causal or a homeostatic consequence remains to be determined as others have suggested that increases in p38 activity may regulate Xbp1 nuclear translocation and activity and thus may represent a compensatory mechanism to maintain homeostatic response. Therefore, the significance of these findings and precise role of p38 warrants further studies.
In summary, our results suggest that particulate air pollution exposure resulted in dysregulated metabolism and influenced IR likely through complex pathways involving the liver, visceral and brown adipose tissue in a genetic KKay diabetes model. These findings suggest an important role for PM2.5 in modulating susceptibility to Type II DM and may have important implications for public health at a global scale.