**2. Pathophysiology of T2DM development: Role of inflammation, oxidative stress, and heat shock proteins**

The plasma glucose level at any given time is determined by the balance between the amount of glucose entering the bloodstream and the amount leaving it. The principal determinants are therefore the dietary intake; the rate of entry into the cells of muscle, adipose tissue, and other organs; and the glucostatic activity of the liver. Thus, there are biochemical abnormalities as fundamental defects to T2DM development as reduced entry of glucose into various peripheral tissues and increased release of glucose into the circulation from the liver. The extracellular glucose excess (hyperglycemia) represents for many cells challenge to maintenance of intracellular glucose level [13, 14].

In animals, hyperglycemia state can be produced by pancreatectomy, by toxins administration that in appropriate doses cause selective destruction of the β cells of the pancreatic islets (as streptozocin or alloxan), by administration of drugs that inhibit insulin secretion, or by administration of anti-insulin antibodies. Also, strains of mice, rats, hamsters, guinea pigs, miniature swine, and monkeys that have a high incidence of spontaneous diabetes mellitus have also been described. However, due to high prevalence of T2DM related to lifestyle, several experimental data obtained from high fat diet (HFD) animal models have been used with success to induce the disruption of insulin signaling in liver, skeletal muscle, or adipose tissue causing hyperinsulinemia and thus, the development of T2DM [15, 16].

days), increased the risk for acute myocardial infarction (by 2.5%), hospitalization or death from heart failure (2.1%), stroke (1.1%), and arrhythmia (1.5%). The risk increases for longterm exposure when people live in unhealthy urban area that exceeds PM2.5 levels, reaching more than 10% increase in cardiovascular mortality. Also, if people live in polluted area, a peak of PM2.5 levels increases 10–50 fold the risk for cardiovascular events [11]. Furthermore, elderly people and women are high susceptible profile to PM2.5 effects and for T2DM develop-

The pathophysiologic mechanisms evolved in susceptibility to cardiorespiratory PM2.5 effects in T2DM subjects, as well as the enhancing effect of PM2.5 exposure on development of T2DM, are discussed below. The number and the complexity of these mechanisms are positive correlated to the importance for life maintenance. In this chapter, we presented pathophysiologic mechanisms based on oxidative stress, inflammation, and heat shock response, with major contribution from experimental studies. These issues were selected considering as representative of the ability of an organism to respond physiologically (by adequate and quick ways) to the environmental challenges or internal changes in the metabolism as an essential characteristic that permits the life. As background of this discussion, there is the comprehension of the concept that homeostasis regulation of one variable is dependent on many cooperative or synergic mechanisms, that may be activated simultaneity or by steps, in terms of redox response, cell by cell signaling, and/or by molecular stress response. Since T2DM and PM2.5 may be considered as stress situations that can promote damage to organism, and also are conditions that require adaptation/protection responses in the stressed cells, the comprehension of multi-integrative physiologic response can provide mechanistic explanation of epidemiological data listed above. Whereas high-intensity challenges to organism can overload the defensive response mechanisms, chronic and moderate intensity challenges can induce internal "recalibration" of many systems to survive [12]. Then, in the light of this "integrative" and "evolutionary" perspective is important to consider the expressive and complex effects of PM2.5 exposure and T2DM on these variables discussed below: (a) the pro/anti-inflammatory balance; (b) the metabolic regulation (flux and consumption of energy sources); (c) the redox status (pro/antioxidant balance); and (d) heat

**2. Pathophysiology of T2DM development: Role of inflammation,** 

The plasma glucose level at any given time is determined by the balance between the amount of glucose entering the bloodstream and the amount leaving it. The principal determinants are therefore the dietary intake; the rate of entry into the cells of muscle, adipose tissue, and other organs; and the glucostatic activity of the liver. Thus, there are biochemical abnormalities as fundamental defects to T2DM development as reduced entry of glucose into various peripheral tissues and increased release of glucose into the circulation from the liver. The extracellular glucose excess (hyperglycemia) represents for many cells challenge to mainte-

**oxidative stress, and heat shock proteins**

nance of intracellular glucose level [13, 14].

ment, mainly in menopause [11].

74 Diabetes and Its Complications

shock response.

The HFD models help us to comprehension of the mechanisms described up to now for T2DM. As reviewed recently, it was proposed that the activation of transcription factor forkhead box protein O1 (FOXO1) in the liver and disruption of glucose-transporter translocation (GLUT4) to the surface membrane in skeletal muscle as the first steps of insulin resistance [17]. The resultant hyperglycemia and chronic hyperinsulinemia are hypothesized to disrupt insulin suppression of adipocyte lipolysis [17]. Additionaly, the active metabolism of adipose tissue may contribute to hyperinsulinemia since in HFD feeding, it occurs in the deregulation of hepatocyte gluconeogenesis (such as FOXO1), which causes increased hepatic glucose output, and deregulate the glucose transporter GLUT4 response to insulin in muscle, which results in decreased glucose uptake by muscle. In this case, the hypertrophy of adipose tissue can be interpreted as the first step of insulin resistance development that results in hyperglycemia and T2DM.

Persistent hyperglycemia causes tissue damage by different mechanism that involves oxidative stress. Increased uptake of glucose results in increased intracellular glucose concentration, that in turns, increased polyol pathway flux. This metabolic pathway uses dihydronicotinamide adenine dinucleotide phosphate (NADPH) that is required for maintaining the levels of the major intracellular nonenzymatic antioxidant defense, the glutathione. Nonenzymatic reaction of glucose and other glycation compounds formed advanced glycation products (AGEs) that modify intracellular proteins functions. Also, AGEs binding to specific receptors (RAGES) can induces reactive oxygen species (ROS) production. Finally, increased levels of AGEs and glucose (intracellularly and extracellularly) increased protein kinase C activation and hexosamine pathway flux. All these mechanisms listed above are involved in decrease nitric oxide (NO) production (vascular impaired function) and activation of factor nuclear kappa B (NF-kB), major pro-inflammatory transcript factor (for details, please see Giacco and Borwnlee, 2010).

Chronic hyperglycemia is strongly associated with enhanced oxidative stress with overproduction of ROS and nitrosative species (RNS), a reduction of the activity of antioxidant enzymes is known to cause endothelial dysfunction and insulin resistance [19]. Thus, oxidative stress constitutes as an important factor implicated not only in the T2DM development itself but also in the development of diabetic complications [18, 20]. T2DM is well known a cause of microangiopathies, observed at least by the three major diabetic complications, namely, diabetic retinopathy, nephropathy, and neuropathy. Also, T2DM constitutes a major risk factor for macroangiopathy, such as coronary artery disease and cerebrovascular disease. Thus, oxidative stress in T2DM is associated with a wide array of complications associated with decreased quality of life of affected patients, thus contributing to the staggering increase in health-care expenditure.

from 70 kDa HSP family, including inducible 72 kDa and constitutive 73 kDa forms. Also we use the prefix "e" or "i" to identify protein location, as extracellular (eHSP70) or intracellular

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Pro-inflammatory signaling, oxidative stress, and hyperglycemia in T2DM is related to decreased iHSP70 levels. In obesity, it observed a reduction in iHSP70 levels and an increase in JNK activation in skeletal muscle. This effect may be a result of heat shock factor (HSF-1) inhibition. The levels of iHSP70 are correlated with the level of insulin resistance and negatively correlated with fast glucose levels [27]. Heat therapy, that increases iHSP70 levels and decreases JNK activation in muscle, protects against hyperglycemia, hyperinsulinemia, glu-

Studies about cell stress response and oxidative stress using biopsies from T2DM patients showed that mRNA expression of HSP70 and heme-oxygenase-1 is reduced in this subjects. Furthermore, mRNA HSP70 levels were correlated with β-hidroxiacil-CoA dehydrogenase and citrate synthase enzymes activities, suggesting that insulin resistance is associated with poor heat shock and antioxidant defense of muscle [29]. In this way, mitochondrial dysfunction plays an essential role in T2DM development [30]. This organelle dysfunction may be a result of hyperglycemic state and/or oxidative state. The activation of key pathways that increases lipid oxidation and decreases lipid esterification reduces insulin resistance levels.

In a study with wild type and HSP70-knockout mice (HSP70-KO), it was demonstrated that HSP70 level is critical to maintenance of mitochondrial morphology and is a biomarker/sensor of mitochondrial stress levels and insulin signaling function in skeletal muscle. HSP70-KO mice showed impaired glucose homeostasis, insulin resistance, and increased adiposity levels. Also, muscles of HSP70 mice accumulated lipids probably as a result of reduction in fat acids oxidation, which in turns, promotes muscle inflammation. Moreover, muscle cells without HSP70 showed low levels of basal oxygen consumption and high levels of ROS mitochondrial production [31], whereas HSP72 overexpression mice are protected against insulin resistance by positive regulation of oxidative metabolism. Induction of HSP70 expression in skeletal muscle of these mice promoted an increase in mitochondrial number and oxidative

Intracellular HSP70 expression is associated with antiapoptotic and anti-inflammatory actions. Inhibition of NF-KB activation and translocation is a marked anti-inflammatory function of iHSP70 with great implications in immune system, inflammatory process and cell survival regulation [33]. Thus, HSP70 is well known by its molecular chaperon cytoprotective roles. However, this protein is also found in blood of health subjects [34] and a crescent number of studies have been demonstrated higher levels of HSP70 in blood in T2DM, T1DM, and gestational diabetes [35, 36]. The role of HSP70 in the extracellular space (eHSP70) involves immune regulatory actions, pro-inflammatory signaling, and alert/danger signal of cell dam-

In T2DM patients, it observed an increase in eHSP70 levels, and this increase is associated with diabetes duration, a biomarker of chronicity of the disease [36]. Also, chronic exposure of pancreatic β-cell *in vitro* to high levels of eHSP70 induces cell death and modifies cellular

(iHSP70) located.

cose intolerance, and insulin resistance [28].

capacity decreasing insulin resistance [32].

age [37].

Overweight and obesity is a risk factor for development of T2DM and is strongly related to chronic low grade inflammatory state. Adipose tissue metabolism is responsible for systemic oxidative stress and increase pro-inflammatory signaling, observed by increased plasma/serum cytokine levels. The development and the severity of the disease are related to immuno-inflammatory responses and thus, biomarkers. Inflammatory cells (monocytes/ macrophage and Th1 lymphocytes) are stimulated to express high amounts of the inducible form of nitric oxide synthase (iNOS, that is, encode by NOS-2 gene) by activation of transcriptors factor of inflammation, as NF-kB [21]. Studies in human obesity and insulin resistance (as well as in animal models) have revealed a clear association between the chronic activation of pro-inflammatory signaling pathways and decreased insulin sensitivity.

Elevated levels of TNF-α, IL-6, and IL-8 have all been reported in various diabetic and insulin-resistant states. As part of the chronic inflammatory process, locally secreted chemokines attract pro-inflammatory macrophages to the adipose tissue, where they form crown-like structures around large dead or dying adipocytes. These "infiltrated" macrophages release cytokines that further activate the inflammatory response in neighboring adipocytes, exacerbating inflammation and insulin resistance. In addition, overnutrition and obesity are often accompanied by elevations in tissue and circulating free fat acids (FFA) concentrations, and saturated FFAs can directly activate pro-inflammatory responses in vascular endothelial cells, adipocytes, and myeloid-derived cells. Excess of free fatty acids accumulate (FFA), resulting in lipotoxicity and an increase in potentially harmful intracellular lipid products activating the NF-κB pathway and inflammation. Adipose tissue macrophages (ATMs) infiltrate adipose tissue to clear these excess lipids and produce pro-inflammatory cytokines, such as IL-1β, TNF-α and IL-6, which further propagate inflammation [22].

According Fontana et al., obesity is strongly associated with plasma IL-6 levels so that has been calculated that one third of total circulating concentrations of IL-6 originate from adipose tissue. A feed-forward paracrine inflammatory cycle involves co-cultured adipocyte release of FFA and macrophage FFA-induced TNF-α production, which blocks insulin-stimulated glucose uptake in adipocytes, and leads to increased release of FFA. TNF-α induces insulin resistance through several mechanisms including inhibition of insulin receptor signaling and increases in FFA. In addition, macrophages secrete a chemotactic pro-inflammatory lectin Galectin-3 that directly decreases insulin signaling and promotes adipose tissue inflammation. Thus, the overexpressed pro-inflammatory cytokines in obesity are considered the link between obesity and inflammation [23–25] and also, obesity and the concomitant development of inflammation are major components of insulin resistance [26].

Additionally with pro-inflammatory signaling and oxidative stress, hyperglycemia in T2DM is also associated with modifications in the cell stress response ability, with markedly undesirable effects in the metabolism. Cell stress response may be studied observing heat shock proteins (HSP) amount, synthesis, and release from cells and tissues. Since HSP are classified by their molecular weight, in this chapter, we use the term "HSP70" to describe all proteins from 70 kDa HSP family, including inducible 72 kDa and constitutive 73 kDa forms. Also we use the prefix "e" or "i" to identify protein location, as extracellular (eHSP70) or intracellular (iHSP70) located.

Thus, oxidative stress in T2DM is associated with a wide array of complications associated with decreased quality of life of affected patients, thus contributing to the staggering increase

Overweight and obesity is a risk factor for development of T2DM and is strongly related to chronic low grade inflammatory state. Adipose tissue metabolism is responsible for systemic oxidative stress and increase pro-inflammatory signaling, observed by increased plasma/serum cytokine levels. The development and the severity of the disease are related to immuno-inflammatory responses and thus, biomarkers. Inflammatory cells (monocytes/ macrophage and Th1 lymphocytes) are stimulated to express high amounts of the inducible form of nitric oxide synthase (iNOS, that is, encode by NOS-2 gene) by activation of transcriptors factor of inflammation, as NF-kB [21]. Studies in human obesity and insulin resistance (as well as in animal models) have revealed a clear association between the chronic activation of

Elevated levels of TNF-α, IL-6, and IL-8 have all been reported in various diabetic and insulin-resistant states. As part of the chronic inflammatory process, locally secreted chemokines attract pro-inflammatory macrophages to the adipose tissue, where they form crown-like structures around large dead or dying adipocytes. These "infiltrated" macrophages release cytokines that further activate the inflammatory response in neighboring adipocytes, exacerbating inflammation and insulin resistance. In addition, overnutrition and obesity are often accompanied by elevations in tissue and circulating free fat acids (FFA) concentrations, and saturated FFAs can directly activate pro-inflammatory responses in vascular endothelial cells, adipocytes, and myeloid-derived cells. Excess of free fatty acids accumulate (FFA), resulting in lipotoxicity and an increase in potentially harmful intracellular lipid products activating the NF-κB pathway and inflammation. Adipose tissue macrophages (ATMs) infiltrate adipose tissue to clear these excess lipids and produce pro-inflammatory cytokines, such as IL-1β,

According Fontana et al., obesity is strongly associated with plasma IL-6 levels so that has been calculated that one third of total circulating concentrations of IL-6 originate from adipose tissue. A feed-forward paracrine inflammatory cycle involves co-cultured adipocyte release of FFA and macrophage FFA-induced TNF-α production, which blocks insulin-stimulated glucose uptake in adipocytes, and leads to increased release of FFA. TNF-α induces insulin resistance through several mechanisms including inhibition of insulin receptor signaling and increases in FFA. In addition, macrophages secrete a chemotactic pro-inflammatory lectin Galectin-3 that directly decreases insulin signaling and promotes adipose tissue inflammation. Thus, the overexpressed pro-inflammatory cytokines in obesity are considered the link between obesity and inflammation [23–25] and also, obesity and the concomitant develop-

Additionally with pro-inflammatory signaling and oxidative stress, hyperglycemia in T2DM is also associated with modifications in the cell stress response ability, with markedly undesirable effects in the metabolism. Cell stress response may be studied observing heat shock proteins (HSP) amount, synthesis, and release from cells and tissues. Since HSP are classified by their molecular weight, in this chapter, we use the term "HSP70" to describe all proteins

pro-inflammatory signaling pathways and decreased insulin sensitivity.

TNF-α and IL-6, which further propagate inflammation [22].

ment of inflammation are major components of insulin resistance [26].

in health-care expenditure.

76 Diabetes and Its Complications

Pro-inflammatory signaling, oxidative stress, and hyperglycemia in T2DM is related to decreased iHSP70 levels. In obesity, it observed a reduction in iHSP70 levels and an increase in JNK activation in skeletal muscle. This effect may be a result of heat shock factor (HSF-1) inhibition. The levels of iHSP70 are correlated with the level of insulin resistance and negatively correlated with fast glucose levels [27]. Heat therapy, that increases iHSP70 levels and decreases JNK activation in muscle, protects against hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance [28].

Studies about cell stress response and oxidative stress using biopsies from T2DM patients showed that mRNA expression of HSP70 and heme-oxygenase-1 is reduced in this subjects. Furthermore, mRNA HSP70 levels were correlated with β-hidroxiacil-CoA dehydrogenase and citrate synthase enzymes activities, suggesting that insulin resistance is associated with poor heat shock and antioxidant defense of muscle [29]. In this way, mitochondrial dysfunction plays an essential role in T2DM development [30]. This organelle dysfunction may be a result of hyperglycemic state and/or oxidative state. The activation of key pathways that increases lipid oxidation and decreases lipid esterification reduces insulin resistance levels.

In a study with wild type and HSP70-knockout mice (HSP70-KO), it was demonstrated that HSP70 level is critical to maintenance of mitochondrial morphology and is a biomarker/sensor of mitochondrial stress levels and insulin signaling function in skeletal muscle. HSP70-KO mice showed impaired glucose homeostasis, insulin resistance, and increased adiposity levels. Also, muscles of HSP70 mice accumulated lipids probably as a result of reduction in fat acids oxidation, which in turns, promotes muscle inflammation. Moreover, muscle cells without HSP70 showed low levels of basal oxygen consumption and high levels of ROS mitochondrial production [31], whereas HSP72 overexpression mice are protected against insulin resistance by positive regulation of oxidative metabolism. Induction of HSP70 expression in skeletal muscle of these mice promoted an increase in mitochondrial number and oxidative capacity decreasing insulin resistance [32].

Intracellular HSP70 expression is associated with antiapoptotic and anti-inflammatory actions. Inhibition of NF-KB activation and translocation is a marked anti-inflammatory function of iHSP70 with great implications in immune system, inflammatory process and cell survival regulation [33]. Thus, HSP70 is well known by its molecular chaperon cytoprotective roles. However, this protein is also found in blood of health subjects [34] and a crescent number of studies have been demonstrated higher levels of HSP70 in blood in T2DM, T1DM, and gestational diabetes [35, 36]. The role of HSP70 in the extracellular space (eHSP70) involves immune regulatory actions, pro-inflammatory signaling, and alert/danger signal of cell damage [37].

In T2DM patients, it observed an increase in eHSP70 levels, and this increase is associated with diabetes duration, a biomarker of chronicity of the disease [36]. Also, chronic exposure of pancreatic β-cell *in vitro* to high levels of eHSP70 induces cell death and modifies cellular bioenergetics profile. Since T2DM and T1DM patients exhibit higher eHSP70 levels, the perpetuation of this pro-inflammatory signal may induce loss of cell integrity and consequently β-cell dysfunction [38].

Oxidative stress is a common factor in both conditions, T2DM and PM2,5. Furthermore, the oxidative stress induced by PM2,5 also represents a pathogenic stimulus for pancreatic β-cell

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The redox unbalance promoted by exposure to air pollution can stimulates an inflammatory processes, contributing to installation of a metabolic disorder. The role of inflammation in the toxicity mediated by PM2,5 is associated to the increase in alveolar immunological response (increased phagocytic cell count) and to pro-inflammatory cytokines production by these cells in the alveolar surface [49], accompanied by increased lung oxidative damage [50–52], which

Postulated mechanisms of action include oxidative stress and low-grade inflammation, endothelial dysfunction, visceral adipose tissue inflammation, endoplasmic reticulum stress, and mitochondrial dysfunction [5, 53]. Thus, both acute and chronic PM2,5 exposures are associated to inflammatory and oxidative markers, as well as in T1DM and T2DM, but it is not clear the real effects of diabetes plus air pollution combination. However, the pathophysiology involved in this case increases the global risk of death by increasing the susceptibility to air

Cell stress response, observed by alteration in HSPs levels in different organs, is a defensive and cytoprotective response in both conditions, exposure to pollutants and metabolic diseases. However, there are few pieces of evidence about PM2,5 exposure concomitant to T2DM

The HSPs naturally are very sensitive elements to any chemical attack to the cells and are extensive used as biomarker of environmental exposures. In this sense, the iHSP70 expression during cellular challenges indicates that these proteins can be candidate to monitoring air pollution aggression to the health organism [54]. One study showed increase in the iHSP70 in the lung and heart one day after course particle exposure [55], and the authors discussed the plausibility of oxidative stress and/or cytokines in HSPs-induced expression as cellular defense at molecular levels, inhibiting pro-inflammatory pathways. In this way, low doses (12.5 μg/ml) of PM2,5 can increase eHSP70 in human bronquial epithelial culture [56]. Thus, the strong correlation among oxidative stress and inflammation induced by PM2,5 inhalation promotes both increase in the iHSP70 and eHSP70 content, reinforcing the purpose of use these proteins as an important biomarker of homeostatic equilibrium in environmental chal-

Simulating urbanized conditions (consumption of high fat diet and exposure to PM2.5) [16] showed that subchronic exposure to PM2.5, even at low doses (5 μg-day, intranasal administration), potentiates metabolic dysfunction in HFD-fed mice, which are T2DM-susceptible. The effects of PM2.5 in T2DM mice presented a positive correlation between adiposity, increased body weight and glucose intolerance, and increased glucose and triacylglycerol plasma levels. Also, in this study, pancreas exhibited lower iHSP70 expression, accompanied by 3.7-fold increase in the plasma to pancreas [eHSP72]/[iHSP70] ratio (H-index). This study represents an experimental evidence that the combination of two relevant challenges to the organism, from different origins (environmental and dietary factors), promotes alterations in

dysfunction [47], since it is responsible for debility on the antioxidant defenses [48].

generally evolute to systemic oxidative stress, a risk for diabetes complications.

pollution damage [10].

lenges [16, 57, 58].

development that explores heat shock response [16].

In obese T2DM subjects, the eHSP70 level is higher than nonobese T2DM suggesting that adiposity, mainly visceral adiposity and its complications, may contribute to increasing eHSP70 levels. Actually, iHSP70 may be considered a cytoprotective proteins by anti-inflammatory functions associated with normal insulin sensitivity. On the other hand, increased levels of eHSP70 chronically may be a result of chronic low-grade inflammatory state of visceral obesity [39]. Therefore, the unbalance between eHSP70 and iHSP70 levels (eHSP70/iHSP70 ratio), known as H-index, can reveal the full context of inflammatory process and insulin resistance state [16, 40].
