**3.1 Hypoxia**

146 Dyslipidemia - From Prevention to Treatment

Fig. 4. Metabolically activated "brite adipocytes" as found in inguinal white adipose tissue

In collaboration with laboratory of dr. Kvatnansky we have recently observed that catecholamines, important regulators of lipolysis in adipose tissue, could be produced within adipocytes. Adipocytes isolated from mesenteric adipose tissue expressed genes encoding the catecholamine biosynthetic enzymes and produced catecholamines *de novo*. Administration of tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine, significantly reduced concentration of catecholamines in isolated adipocytes *in vitro* (Fig. 5.). We therefore hypothesized that the sympathetic innervation of adipose tissue is not the only source of catecholamines and that adipocyte-derived catecholamines could dynamically modulate metabolic or thermogenic properties of the white adipose tissue perhaps by

The prevalence of obesity and its consequent pathologies in modern society is of serious health concern. Although the expansion of adipose tissue mass during pathological obesity is in itself not a grave problem, rather it is the ensuing pathologies resultant of this state, including development of hypertension, type 2 diabetes and cardiac myopathies, that

of mice lacking the UCP1-thermogenesis after gradual exposure to cold.

enhancing "brite adipocyte" function (Vargovic et al. 2011).

impacts peoples lives and health services worldwide.

(**A**) histomorphology, (**B**) O2 consumption by oxymetry, (**C**) adipose tissue *ex vivo* palmitate oxidation, (**D**&**E**) evidence for AMPK activation (Ukropec et al. 2006).

**3. Adipocentric view on the pathophysiology of metabolic disease** 

Hypothesis that adipose tissue populated by large adipocytes contains the local microhypoxia suffering areas, which has a profound effect on the tissue metabolic and inflammatory phenotype, has been largely accepted. Hypoxia is one of the major triggers strongly inhibiting adipocyte differentiation. Tissue hypoxia in obesity is associated with the defects in the nutrient and oxygen supply into the tissue, related to a defective blood flow regulation which might be perpetuated by the increased fat cell size (Yun et al. 2002). This is happening in spite of the almost unlimited capacity of adipose tissue to expand in a nontransformed form, which is a very unique property of adipose tissue that cannot be seen in any other organ. To accomplish this adipose tissue requires potent mechanisms to remodel, acutely and chronically, as it can rapidly reach the diffusional limit of oxygen; molecular response to hypoxia is therefore an early determinant that limits healthy adipose tissue expansion. Proper expansion requires a highly coordinated response among many different cell types, including endothelial precursor cells, immune cells, and preadipocytes (Sun et al. 2011). It has also been demonstrated that mitochondrial oxidative apparatus is essential for the white fat adipocyte differentiation (De Pauw et al. 2009). Beside their key role in ATP

Adipose Tissue and Skeletal Muscle Plasticity in Obesity and Metabolic Disease 149

depends upon the expression of osteopontin, an extracellular matrix protein and proinflammatory cytokine which promotes the monocyte chemotaxis and cell motility. Mice exposed to a high-fat diet exhibited increased plasma osteopontin level, and elevated expression of osteopontin in macrophages recruited into adipose tissue. In addition, obese mice lacking osteopontin displayed improved insulin sensitivity in the absence of an effect on the diet-induced obesity, body composition or energy expenditure. These mice further demonstrated decreased macrophage infiltration into adipose tissue, which may reflect both impaired macrophage motility and attenuated monocyte recruitment by stromal vascular cells. Finally, obese osteopontin-deficient mice exhibited decreased markers of

Adipose tissue resident macrophages show significant heterogeneity in their properties and activation state, reflecting the local metabolic and immune microenvironment (Gordon & Taylor 2005). Different stimuli activate macrophages to express distinct patterns of chemokines, surface markers and enzymes that ultimately generate the diversity of macrophage function seen in inflammatory and non-inflammatory settings. It has recently been proposed that adipose tissue macrophages, which accumulate with obesity and are implicated in insulin resistance switch their phenotype from one of an alternatively activated (M2) to pro-inflammatory (M1) cells (Lumeng et al. 2007). Characteristic features of the IFN- induced pro-inflammatory (M1) macrophages include enhanced MHC class II expression, but distinctive up-regulation of i-NOS. Alternative activation of macrophages (M2) is strongly associated with extracellular parasitic infections, allergy, humoral immunity, and fibrosis. It is characterized by up-regulation of the endocytic lecithin-like receptors and arginase rather than i-NOS (Gordon 2007). Therefore, the alternatively activated (M2) macrophages seem to have high capacity for the tissue remodeling and

It had been recently proposed that PPARγ is required for maturation of alternatively activated macrophages (M2), which could also participate to its insulin sensitizing effect (Odegaard et al. 2007). Disruption of PPAR in myeloid cells impaired alternative macrophage activation and predisposed to the development of diet-induced obesity, insulin resistance, and glucose intolerance. This might be related to the concomitant downregulation of oxidative phosphorylation gene expression in skeletal muscle and liver (Odegaard et al. 2007). Phenotype of macrophages in the pathogenic-hypoxic adipose tissue might also be regulated by HIF-1 since the functional loss of HIF-1α resulted in a dramatic reduction of the intracellular ATP stores in macrophages to approximately 15-20%, most likely due to the inhibition of the HIF-1α regulated glycolytic energy generation (Cramer & Johnson 2003; Cramer et al. 2003). It could be hypothesized that resident alternatively activated macrophages have a beneficial role in regulating nutrient homeostasis and suggest that macrophage polarization towards the alternative state might be a useful strategy for

Fatty acid binding proteins (FABPs), which are common to adipocytes and macrophages, could also play an important role in metabolic and inflammatory disease, and might therefore represent desirable therapeutic targets for metabolic syndrome (Erbay et al. 2007). Macrophage-derived foam cells express the adipocyte fatty acid-binding protein (FABP) aP2 that plays an important role in regulating the development of insulin resistance in obesity. It has been shown that macrophages deficient in aP2 display alterations in the inflammatory cytokine production. Through its distinct actions in adipocytes and macrophages, aP2 links

treating type 2 diabetes, by modulating adipose tissue phenotype.

inflammation, both in adipose tissue and systemically (Nomiyama et al. 2007).

repair.

production, mitochondria constitute the primary source of reactive oxygen species (ROS), which have a great potential to influence the tissue plasticity.

ROS are not only considered a negative factors contributing to degenerative processes and ageing, but also a physiological signal molecules participating in the oxygen sensing mechanisms (Chandel & Budinger 2007). Mitochondrial ROS production influences the size of the white adipocytes, and ROS are in fact antiadipogenic signaling molecules triggering the hypoxia-dependent inhibition of adipocyte differentiation (Carriere et al. 2004). In addition, decreased oxygen availability stimulates the programming of cellular metabolism towards increased glycolysis and fatty acid and triacylglyceride synthesis (Halperin et al. 1969; Kinnula et al. 1978). Hypoxia-inducible factor (HIF), dimers composed of HIF1, HIF2 or HIF3 (collectively HIF) and HIF1/ARNT subunits, play a key role in the coordination of these metabolic adaptations (Trayhurn et al. 2008; Krishnan et al. 2009). HIF subunits are constitutively expressed but degraded under normoxia due to prolyl hydroxylase activity, which marks them for recognition by the von Hippel-Lindau (VHL) tumor suppressor protein pVHL, that acts as part of an E3 ubiquitin ligase complex to target HIF subunit for proteasomal degradation. Loss of pVHL function or hypoxia leads to accumulation of HIF, dimerization with HIF/ARNT and the activation of numerous hypoxia-inducible genes (Krek 2000; Semenza 2001). Previous work by Krek`s laboratory provided the seminal observation that hypoxia activated pVHL and HIF1 oxygen sensing system affects normal physiological function of heart and pancreatic beta cells by triggering the changes in the glucose and fatty acid metabolism (Zehetner et al. 2008; Krishnan et al. 2009). Interestingly, hypoxia present in atherosclerotic lesions contributes to the proinflammatory lipid-loaded foam cells formation, as it decreases expression of enzymes involved in -oxidation and increases expression of enzymes related to fatty acid synthesis and lipid droplet formation. The aforementioned processes possibly stimulate progression of the atherosclerotic plaque formation (Bostrom et al. 2007). Finally, tissue hypoxia largely modulates adipocytokine production and possibly contributes to the adipose tissue inflammation in obesity (Hosogai et al. 2007; Wang et al. 2007; Ukropec et al. 2008).

#### **3.2 Inflammation – Macrophage, adipocyte and preadipocyte plasticity**

Chronic low level of inflammation present in the "pathogenic" adipose tissue has been found to have adverse effects on the adipose tissue physiological functions contributing thus to the metabolic disease. It has been shown that increase in both body fat mass and adipocyte cell size are directly related to the number of macrophages found in the adipose tissue (Wellen & Hotamisligil 2005; Weisberg et al. 2006; Goossens 2008). A net proinflammatory response of the adipose tissue may result from adipose tissue secretion of proinflammatory factors; adipose tissue secretion of factors that stimulate other tissues to produce inflammatory factors; and decreased production of anti-inflammatory factors. Although the contribution of specific cell types to inflammation is uncertain, evidence is mounting that implicates adipose tissue macrophages as the significant contributor to inflammation in insulin resistant adipose tissue (Kanda et al. 2006; Neels & Olefsky 2006). There are controversial reports related to the importance of the CC chemokine ligand 2 (CCL2, monocyte chemoattractant protein-1) for the macrophage-recruitment and activation in obesity (Kamei et al. 2006; Inouye et al. 2007). Interestingly CCL2 has also been proposed to affect metabolism independently of its macrophage-recruiting capabilities (Inouye et al. 2007). There is also preliminary data indicating that the tissue infiltration by macrophages

production, mitochondria constitute the primary source of reactive oxygen species (ROS),

ROS are not only considered a negative factors contributing to degenerative processes and ageing, but also a physiological signal molecules participating in the oxygen sensing mechanisms (Chandel & Budinger 2007). Mitochondrial ROS production influences the size of the white adipocytes, and ROS are in fact antiadipogenic signaling molecules triggering the hypoxia-dependent inhibition of adipocyte differentiation (Carriere et al. 2004). In addition, decreased oxygen availability stimulates the programming of cellular metabolism towards increased glycolysis and fatty acid and triacylglyceride synthesis (Halperin et al. 1969; Kinnula et al. 1978). Hypoxia-inducible factor (HIF), dimers composed of HIF1, HIF2 or HIF3 (collectively HIF) and HIF1/ARNT subunits, play a key role in the coordination of these metabolic adaptations (Trayhurn et al. 2008; Krishnan et al. 2009). HIF subunits are constitutively expressed but degraded under normoxia due to prolyl hydroxylase activity, which marks them for recognition by the von Hippel-Lindau (VHL) tumor suppressor protein pVHL, that acts as part of an E3 ubiquitin ligase complex to target HIF subunit for proteasomal degradation. Loss of pVHL function or hypoxia leads to accumulation of HIF, dimerization with HIF/ARNT and the activation of numerous hypoxia-inducible genes (Krek 2000; Semenza 2001). Previous work by Krek`s laboratory provided the seminal observation that hypoxia activated pVHL and HIF1 oxygen sensing system affects normal physiological function of heart and pancreatic beta cells by triggering the changes in the glucose and fatty acid metabolism (Zehetner et al. 2008; Krishnan et al. 2009). Interestingly, hypoxia present in atherosclerotic lesions contributes to the proinflammatory lipid-loaded foam cells formation, as it decreases expression of enzymes involved in -oxidation and increases expression of enzymes related to fatty acid synthesis and lipid droplet formation. The aforementioned processes possibly stimulate progression of the atherosclerotic plaque formation (Bostrom et al. 2007). Finally, tissue hypoxia largely modulates adipocytokine production and possibly contributes to the adipose tissue

inflammation in obesity (Hosogai et al. 2007; Wang et al. 2007; Ukropec et al. 2008).

Chronic low level of inflammation present in the "pathogenic" adipose tissue has been found to have adverse effects on the adipose tissue physiological functions contributing thus to the metabolic disease. It has been shown that increase in both body fat mass and adipocyte cell size are directly related to the number of macrophages found in the adipose tissue (Wellen & Hotamisligil 2005; Weisberg et al. 2006; Goossens 2008). A net proinflammatory response of the adipose tissue may result from adipose tissue secretion of proinflammatory factors; adipose tissue secretion of factors that stimulate other tissues to produce inflammatory factors; and decreased production of anti-inflammatory factors. Although the contribution of specific cell types to inflammation is uncertain, evidence is mounting that implicates adipose tissue macrophages as the significant contributor to inflammation in insulin resistant adipose tissue (Kanda et al. 2006; Neels & Olefsky 2006). There are controversial reports related to the importance of the CC chemokine ligand 2 (CCL2, monocyte chemoattractant protein-1) for the macrophage-recruitment and activation in obesity (Kamei et al. 2006; Inouye et al. 2007). Interestingly CCL2 has also been proposed to affect metabolism independently of its macrophage-recruiting capabilities (Inouye et al. 2007). There is also preliminary data indicating that the tissue infiltration by macrophages

**3.2 Inflammation – Macrophage, adipocyte and preadipocyte plasticity** 

which have a great potential to influence the tissue plasticity.

depends upon the expression of osteopontin, an extracellular matrix protein and proinflammatory cytokine which promotes the monocyte chemotaxis and cell motility. Mice exposed to a high-fat diet exhibited increased plasma osteopontin level, and elevated expression of osteopontin in macrophages recruited into adipose tissue. In addition, obese mice lacking osteopontin displayed improved insulin sensitivity in the absence of an effect on the diet-induced obesity, body composition or energy expenditure. These mice further demonstrated decreased macrophage infiltration into adipose tissue, which may reflect both impaired macrophage motility and attenuated monocyte recruitment by stromal vascular cells. Finally, obese osteopontin-deficient mice exhibited decreased markers of inflammation, both in adipose tissue and systemically (Nomiyama et al. 2007).

Adipose tissue resident macrophages show significant heterogeneity in their properties and activation state, reflecting the local metabolic and immune microenvironment (Gordon & Taylor 2005). Different stimuli activate macrophages to express distinct patterns of chemokines, surface markers and enzymes that ultimately generate the diversity of macrophage function seen in inflammatory and non-inflammatory settings. It has recently been proposed that adipose tissue macrophages, which accumulate with obesity and are implicated in insulin resistance switch their phenotype from one of an alternatively activated (M2) to pro-inflammatory (M1) cells (Lumeng et al. 2007). Characteristic features of the IFN- induced pro-inflammatory (M1) macrophages include enhanced MHC class II expression, but distinctive up-regulation of i-NOS. Alternative activation of macrophages (M2) is strongly associated with extracellular parasitic infections, allergy, humoral immunity, and fibrosis. It is characterized by up-regulation of the endocytic lecithin-like receptors and arginase rather than i-NOS (Gordon 2007). Therefore, the alternatively activated (M2) macrophages seem to have high capacity for the tissue remodeling and repair.

It had been recently proposed that PPARγ is required for maturation of alternatively activated macrophages (M2), which could also participate to its insulin sensitizing effect (Odegaard et al. 2007). Disruption of PPAR in myeloid cells impaired alternative macrophage activation and predisposed to the development of diet-induced obesity, insulin resistance, and glucose intolerance. This might be related to the concomitant downregulation of oxidative phosphorylation gene expression in skeletal muscle and liver (Odegaard et al. 2007). Phenotype of macrophages in the pathogenic-hypoxic adipose tissue might also be regulated by HIF-1 since the functional loss of HIF-1α resulted in a dramatic reduction of the intracellular ATP stores in macrophages to approximately 15-20%, most likely due to the inhibition of the HIF-1α regulated glycolytic energy generation (Cramer & Johnson 2003; Cramer et al. 2003). It could be hypothesized that resident alternatively activated macrophages have a beneficial role in regulating nutrient homeostasis and suggest that macrophage polarization towards the alternative state might be a useful strategy for treating type 2 diabetes, by modulating adipose tissue phenotype.

Fatty acid binding proteins (FABPs), which are common to adipocytes and macrophages, could also play an important role in metabolic and inflammatory disease, and might therefore represent desirable therapeutic targets for metabolic syndrome (Erbay et al. 2007). Macrophage-derived foam cells express the adipocyte fatty acid-binding protein (FABP) aP2 that plays an important role in regulating the development of insulin resistance in obesity. It has been shown that macrophages deficient in aP2 display alterations in the inflammatory cytokine production. Through its distinct actions in adipocytes and macrophages, aP2 links

Adipose Tissue and Skeletal Muscle Plasticity in Obesity and Metabolic Disease 151

is likely (Hong et al. 2010; Ukropec et al. 2010; Howard & Lee 2011; Lee, D. H. et al. 2011;

Fig. 6. The prevalence of prediabetes increases with increased circulating levels of PCBs (sum 15 congeners of polychlorinated biphenyls); DDE (2,2'-bis(4-chlorophenyl)-1,1 dichloroethylene); DDT (2,2'-bis(4-chlorophenyl)-1,1,1-trichloro-ethane); HCB

of orders for all 5 pollutants. Odds ratios were adjusted for age, gender and BMI.

**4. Skeletal muscle in metabolic health and disease** 

et al. 2008).

mood scale (**B**) (Boecker et al. 2008).

(hexachlorobenzene) and b-HCH (beta-hexachlorocyclohexane); POLL5 represents the sum

Skeletal muscle represents a large mass of tissue, and its primary function is to use energy, though quite inefficiently, to enable us the 3D life, voluntary positioning and moving our bodies in a surrounding space. This makes an active muscle to be the most effective energy burner. In addition to obvious metabolic consequences, regular exercise activates central reward mechanisms and makes us happy (Figure 7.) (Sher 1998; Boecker

A B

Fig. 7. Correlation of opidergic ligand 6-O-(2-[18F]fluoroethyl)-6-O-desmethyldiprenorphine binding in right orbitofrontal cortex (OFC) with the visual analog mood scale (for euphoria) in runners (**A**) and effect of exercise on the individual's mood expressed in the visual analog

Lee, D. H. et al. 2011; Lee, H. K. 2011).

together the features of the metabolic syndrome including insulin resistance, obesity, inflammation, and atherosclerosis (Linton & Fazio 2003).

#### **3.3 Phospholipid membrane composition**

In the last decades, free radical processes delineated an interdisciplinary field linking chemistry to biology and medicine. Free radical mechanisms became of importance as molecular basis of physiological and pathological conditions. Lipids, in particular unsaturated fatty acids, are susceptible to free radical attack. The reactivity of the double bond toward free radicals is well known; in particular the reversible addition of radical species to this functionality determines the cis-trans double bond isomerization. Since the prevalent geometry displayed by unsaturated fatty acids in eukaryotes is cis, the occurrence of the cis-trans isomerization by free radicals corresponds to the loss of an important structural information linked to biological activity (Ferreri & Chatgilialoglu 2009). Formation of trans isomers of unsaturated fatty acid in biological membranes can have important meaning and consequences connected to radical stress associated with nutritional overload and mitochondrial defects. It might, together with changes in membrane lipid composition (Pietilainen et al. 2011), substantially modulate lipid membrane biophysical characteristics such as thickness, fluidity, protein lateral diffusion capacity, permeability to small molecules in expanding adipocytes and contribute thus to the development of metabolic disease in obesity.

#### **3.4 Pollutants and metabolic health**

Physical inactivity and unhealthy diet are well recognized environmental influences largely increasing the risk for metabolic disease development. Recent advances in detecting the presence of various persistent organic pollutants in the surrounding world as well as within our bodies, prompted us to evaluate its possible role in pathogenesis of different endocrine and metabolic pathological states (Langer et al. 2003; Langer et al. 2007; Langer 2010; Langer et al. 2010; Ukropec et al. 2010; Langer et al. 2011).

A heavily polluted area of Eastern Slovakia was targeted by the PCBrisk cross-sectional survey to search for possible links between environmental pollution and both prediabetes and diabetes. Associations of serum levels of five persistent organic pollutants (POPs), namely polychlorinated biphenyls (PCBs), 2,2'-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE), 2,2'-bis(4-chlorophenyl)-1,1,1-trichloro-ethane (p,p'-DDT), hexachlorobenzene (HCB) and beta-hexachlorocyclohexane (-HCH), with prediabetes and diabetes were investigated in 2,047 adults. Prevalence of prediabetes and diabetes increased in a dosedependent manner, with individuals in upper quintiles of individual POPs showing striking increases in prevalence of prediabetes (Fig. 6.) Interestingly, unlike PCBs, DDT and DDE, increased levels of HCB and -HCH seemed not to be associated with increased prevalence of diabetes (Ukropec et al. 2010). Cumulative effect of all five persistent organic pollutants (sum of orders) more than tripled the prevalence of prediabetes while that of diabetes was increased more than six times as compared to the referent quintile composed of individuals with lowest levels of pollutants in serum. We as well as the others have clearly shown that increasing serum concentrations of individual persistent organic pollutants considerably increased prevalence of prediabetes and diabetes in a dose-dependent manner. Interaction of industrial and agricultural pollutants in increasing prevalence of prediabetes or diabetes

together the features of the metabolic syndrome including insulin resistance, obesity,

In the last decades, free radical processes delineated an interdisciplinary field linking chemistry to biology and medicine. Free radical mechanisms became of importance as molecular basis of physiological and pathological conditions. Lipids, in particular unsaturated fatty acids, are susceptible to free radical attack. The reactivity of the double bond toward free radicals is well known; in particular the reversible addition of radical species to this functionality determines the cis-trans double bond isomerization. Since the prevalent geometry displayed by unsaturated fatty acids in eukaryotes is cis, the occurrence of the cis-trans isomerization by free radicals corresponds to the loss of an important structural information linked to biological activity (Ferreri & Chatgilialoglu 2009). Formation of trans isomers of unsaturated fatty acid in biological membranes can have important meaning and consequences connected to radical stress associated with nutritional overload and mitochondrial defects. It might, together with changes in membrane lipid composition (Pietilainen et al. 2011), substantially modulate lipid membrane biophysical characteristics such as thickness, fluidity, protein lateral diffusion capacity, permeability to small molecules in expanding adipocytes and contribute thus to the development of

Physical inactivity and unhealthy diet are well recognized environmental influences largely increasing the risk for metabolic disease development. Recent advances in detecting the presence of various persistent organic pollutants in the surrounding world as well as within our bodies, prompted us to evaluate its possible role in pathogenesis of different endocrine and metabolic pathological states (Langer et al. 2003; Langer et al. 2007; Langer 2010; Langer

A heavily polluted area of Eastern Slovakia was targeted by the PCBrisk cross-sectional survey to search for possible links between environmental pollution and both prediabetes and diabetes. Associations of serum levels of five persistent organic pollutants (POPs), namely polychlorinated biphenyls (PCBs), 2,2'-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE), 2,2'-bis(4-chlorophenyl)-1,1,1-trichloro-ethane (p,p'-DDT), hexachlorobenzene (HCB) and beta-hexachlorocyclohexane (-HCH), with prediabetes and diabetes were investigated in 2,047 adults. Prevalence of prediabetes and diabetes increased in a dosedependent manner, with individuals in upper quintiles of individual POPs showing striking increases in prevalence of prediabetes (Fig. 6.) Interestingly, unlike PCBs, DDT and DDE, increased levels of HCB and -HCH seemed not to be associated with increased prevalence of diabetes (Ukropec et al. 2010). Cumulative effect of all five persistent organic pollutants (sum of orders) more than tripled the prevalence of prediabetes while that of diabetes was increased more than six times as compared to the referent quintile composed of individuals with lowest levels of pollutants in serum. We as well as the others have clearly shown that increasing serum concentrations of individual persistent organic pollutants considerably increased prevalence of prediabetes and diabetes in a dose-dependent manner. Interaction of industrial and agricultural pollutants in increasing prevalence of prediabetes or diabetes

inflammation, and atherosclerosis (Linton & Fazio 2003).

**3.3 Phospholipid membrane composition** 

metabolic disease in obesity.

**3.4 Pollutants and metabolic health** 

et al. 2010; Ukropec et al. 2010; Langer et al. 2011).

is likely (Hong et al. 2010; Ukropec et al. 2010; Howard & Lee 2011; Lee, D. H. et al. 2011; Lee, D. H. et al. 2011; Lee, H. K. 2011).

Fig. 6. The prevalence of prediabetes increases with increased circulating levels of PCBs (sum 15 congeners of polychlorinated biphenyls); DDE (2,2'-bis(4-chlorophenyl)-1,1 dichloroethylene); DDT (2,2'-bis(4-chlorophenyl)-1,1,1-trichloro-ethane); HCB (hexachlorobenzene) and b-HCH (beta-hexachlorocyclohexane); POLL5 represents the sum of orders for all 5 pollutants. Odds ratios were adjusted for age, gender and BMI.
