**4. Exercise**

can be identified since the pro-inflammatory mediators JNK and NF-κB can be inhibited by the HSP70 expression, which is low in age, T2DM, obesity, and finally in sedentary subjects, suggesting that active muscle can be a non-pharmacological option to prevention or repair this

Older adults with diabetes are vulnerable to accelerated loss of lean body mass. Declines in lower extremity muscle mass in particular, can be associated with decreased muscle strength and lead to muscle weakness, poor lower-extremity performance, and mobility loss, all of which have been reported in persons with diabetes [28]. Aging also produces muscle changes such as decrease in type II fibers, decline in oxidative capacity of type IIa and I fibers, and forming a favorable development of metabolic alterations. During aging, changes in the morphological structure of the skeletal muscle and also in the number and function of mitochondria are observed in the decline in this tissue. Studies have consistently shown that PGC-1α, a transcription factor that promotes mitochondrial biogenesis, decline with aging and in many age-related chronic diseases suggesting that such decline may explain the progressive

"meta-inflammation" process that harm the human health.

mitochondrial dysfunction with aging [23].

92 Muscle Cell and Tissue

**Figure 3.** Aging alterations in the muscle related to insulin resistance.

As previously described, both the lifestyle and aging promote body changes, which may be directly related to skeletal muscle dysfunctions. Changes in composition and functional capacity of type 1 and type 2 fibers, which reduce the mass and muscle strength, occur due to molecular disorders affecting the muscle, whether caused by the high consumption of HFD and physical inactivity or by aging. Myosteatosis, reduction of PGC-1α development of a lowgrade inflammatory with increased expression of pro-inflammatory cytokines such as TNF-

Human lifestyle changed from daily strong physical activity patterns of hunter-gatherer societies (about 1,000–1,500 kcal/day with 3–4 h/day of moderate-to-vigorous physical activities) to digital industrialized society with dramatic reductions in daily activity levels. Physical inactivity is a "modern" human behavior that increases the risk of many adverse health conditions, including the development of diseases, such as T2DM and metabolic syndrome, decreasing the life expectancy. Although nowadays health professionals and population has knowledge about the necessity of exercise practice) to avoid many diseases, the definition of sedentarism still lacks a consensual definition [29, 30].

From ancient physicians, including Hippocrates and Plato, scientists believed in the benefits of physical activity for human and animal health. Classical epidemiological studies, as performed by Morris et al. [31], showed comparative evidences about the risk of sedentary routine to the cardiovascular system. However, in quantitative analyses, many protocols and descriptions were used in the sedentarism research field to classify an inactive person. "Active" definition may be based on total energy expenditure since an increase in daily energy expen‐ diture (aprox.150 kcal) can promote health benefits. Other definition is about accumulative activities during one week, since less than 30 minutes of moderate-intensity physical activity represent also a cardiovascular risk factor. Recently, it was estimated that physical inactivity causes 7% of the burden of disease from T2DM and causes 9% of premature mortality, or more than 5 million of deaths [30]. If the percentage of inactivity decreases at least 10% in the world's population, it is estimated that around more than one million deaths could be averted per year.

A recent meta-analysis has reported that exercise training is associated with a decline in glycosylated hemoglobin (HbA1c) levels. This effect is presented in aerobic, resistance, or combined aerobic and resistance training modes and also is compared to reductions achieved by commonly used oral antidiabetic medications. Three times a week of an exercise program with more than 30 minutes and at moderate intensity (above 50% VO2) is sufficient to reduce 10% in HbA1c levels mainly in insulin resistance subjects, according to the Diabetes Associa‐ tion in the past decades [32]. However, recent data suggest that structured exercise training of more than 150 minutes per week is associated with greater HbA1c declines than that of 150 minutes or less per week and that physical activity advice is also associated with lower HbA1c, but only when combined with dietary advice [33]. Current opinion appointed that the volume of exercise training (and not the type or intensity of exercise) is a major determinant of glycemic control in patients with T2DM. Reduction in HbA1c is associated with exercise frequency in supervised aerobic training, and with weekly volume of resistance exercise in supervised combined training promotes better results in the reduction of HbA1c [34]. Furthermore, a dose response can be estimated to exercise benefits with longer life expectancy for those who accumulate more than 450 minutes of exercise per week [35].

For elderly individuals, combined training (resistance and aerobic) performed twice a week promotes similar muscular adaptation (strength and thickness) and some similar cardiovas‐ cular adaptations when compared to three times per week, suggesting that it is applicable as an exercise prescription for aged people to improve the adherence to an health life style. [36]. Regular exercise can block the aging-associated increase in sympathetic nervous system activity to peripheral tissues (probably an adaptation to improve energy consumption). Chronically augmented sympathetic stimulation promotes reductions in the peripheral blood flow and vascular conductance and thus can also contribute to the metabolic dysfunction, by increasing glucose intolerance and insulin resistance. Each exercise session possibly substitutes the necessity of an increase in the sympathetic activity induced by aging and then stimulates thermogenesis to prevent increasing adiposity [37]. In this way, daily physical activity is able to promote weight loss without caloric restriction and also can reduce obesity, particularly abdominal obesity, and insulin resistance in men. Exercise programs without weight loss reduce abdominal fat and prevent further weight gain, preventing insulin resistance [38].

The epidemiologic and clinical trials results listed above exist because exercise represents a physical stress that challenges human body homeostasis. In response to this stressor, autonomic nervous system and the hypothalamic-pituitary-adrenal axis are known to react and to participate in the maintenance of homeostasis. It is well known that exercise induces several changes on both immune system [39, 40] and endocrine system. Cytokines may be modulated by the secretion of hormones from the hypopituitary-hypothalamus axis and in an integration cross-talk, hypopituitary-hypothalamus axis is also modulated by immuno‐ logic status by secretion of cytokines, showing an important neuroendocrine-immune loop in the human body [41, 42]. Skeletal-muscle fibers also can produce several hundred secreted factors, including proteins, growth factors, cytokines, and metallopeptidases, with such secretory capacity increasing during muscle contractions or after exercise training. Muscle-derived molecules exerting either paracrine or endocrine effects are termed "myokines" and are strong candidates to make up a substantial fraction of the exercise "polypill" effect to the whole body [37].

These signaling actors can be influenced by multiple factors including mode, duration, and intensity of exercise. Changes are proportional to exercise intensity and duration of exercise, although the effect of intensity is more marked, as showed by impaired immune functions and high secretion of hormones after an acute bout of vigorous exercise [39]. Some adaptations from regular training appears to be related to modified circulating hormones, as cortisol, insulin and glucagon, and also by alterations in the pro/anti-inflammatory cytokine balance (IL-2/IL-10 ratio and more th1/th2 lymphocytes cytokines ratio).

Thus, chronic moderate exercise improves immune functions [43] by the induction of an antiinflammatory environment with each bout of exercise promoted. The IL-6 is expressed in high amounts in active muscle and is the first cytokine released into the circulation during exercise, followed by increased anti-inflammatory cytokines as IL-10 [44, 45]. Also, each exercise session promotes an increase in ROS production that result in improved antioxidant defense in the muscle, plasma, and other tissues [37]. In parallel, some studies demonstrated that exercise is a physiological stimulus that promotes an increase in the muscle HSP70 expression and eHSP70 concentration (plasma), influenced by the intensity [46] and duration [47]. Increased intracellular HSP70 expression in leukocytes can be associated to less TNF-α plasma concen‐ tration after an exercise session [48]. Thus, regular exercise can shift the whole body from a pro-oxidant and pro-inflammatory state to an equilibrium induced by molecular, redox, hormonal, and "immune-metabolic" adaptations.

control in patients with T2DM. Reduction in HbA1c is associated with exercise frequency in supervised aerobic training, and with weekly volume of resistance exercise in supervised combined training promotes better results in the reduction of HbA1c [34]. Furthermore, a dose response can be estimated to exercise benefits with longer life expectancy for those who

For elderly individuals, combined training (resistance and aerobic) performed twice a week promotes similar muscular adaptation (strength and thickness) and some similar cardiovas‐ cular adaptations when compared to three times per week, suggesting that it is applicable as an exercise prescription for aged people to improve the adherence to an health life style. [36]. Regular exercise can block the aging-associated increase in sympathetic nervous system activity to peripheral tissues (probably an adaptation to improve energy consumption). Chronically augmented sympathetic stimulation promotes reductions in the peripheral blood flow and vascular conductance and thus can also contribute to the metabolic dysfunction, by increasing glucose intolerance and insulin resistance. Each exercise session possibly substitutes the necessity of an increase in the sympathetic activity induced by aging and then stimulates thermogenesis to prevent increasing adiposity [37]. In this way, daily physical activity is able to promote weight loss without caloric restriction and also can reduce obesity, particularly abdominal obesity, and insulin resistance in men. Exercise programs without weight loss reduce abdominal fat and prevent further weight gain, preventing insulin resistance [38]. The epidemiologic and clinical trials results listed above exist because exercise represents a physical stress that challenges human body homeostasis. In response to this stressor, autonomic nervous system and the hypothalamic-pituitary-adrenal axis are known to react and to participate in the maintenance of homeostasis. It is well known that exercise induces several changes on both immune system [39, 40] and endocrine system. Cytokines may be modulated by the secretion of hormones from the hypopituitary-hypothalamus axis and in an integration cross-talk, hypopituitary-hypothalamus axis is also modulated by immuno‐ logic status by secretion of cytokines, showing an important neuroendocrine-immune loop in the human body [41, 42]. Skeletal-muscle fibers also can produce several hundred secreted factors, including proteins, growth factors, cytokines, and metallopeptidases, with such secretory capacity increasing during muscle contractions or after exercise training. Muscle-derived molecules exerting either paracrine or endocrine effects are termed "myokines" and are strong candidates to make up a substantial fraction of the exercise

These signaling actors can be influenced by multiple factors including mode, duration, and intensity of exercise. Changes are proportional to exercise intensity and duration of exercise, although the effect of intensity is more marked, as showed by impaired immune functions and high secretion of hormones after an acute bout of vigorous exercise [39]. Some adaptations from regular training appears to be related to modified circulating hormones, as cortisol, insulin and glucagon, and also by alterations in the pro/anti-inflammatory cytokine balance

Thus, chronic moderate exercise improves immune functions [43] by the induction of an antiinflammatory environment with each bout of exercise promoted. The IL-6 is expressed in high

accumulate more than 450 minutes of exercise per week [35].

94 Muscle Cell and Tissue

"polypill" effect to the whole body [37].

(IL-2/IL-10 ratio and more th1/th2 lymphocytes cytokines ratio).

Associated to the progression of cardiovascular disease was observed higher levels of inflam‐ matory markers such as IL-6 and CRP that are related to reductions in nitric oxide (NO) concentrations caused by reduced eNOS activity [49]. These extracellular pro-inflammatory signaling is increased in obese and type two diabetic people. Curiously, higher eHSP70 content in the bloodstream is also observed in these individuals, connecting immune-inflammatory events that promote a pro-atherogenic profile. eHSP70 levels were associated with multiple biomarkers of the acute-phase reaction, inflammation, and endothelial-cell activation, indi‐ cating the presence of a complex stress response that involves immune-metabolic signaling by eHSP70, by interaction of eHSP70 with cell surface receptors in immune cells. The release of these proteins promotes the bind to Toll-like receptor 4 (TLR4)/CD14 receptors, resulting in endothelial cells expressing adhesion molecules in smooth muscle cells leading to prolifera‐ tion, and in macrophages inducing a range of proinflammatory cytokines [50].

The elevation of eHSP70 levels could be an important integrative response, from/to immuno‐ logic/metabolic center of homeostasis control in response against physiological disorder or disease [14, 17, 51, 52]. Whereby healthy people have low plasmatic levels of eHSP70, the association of these proteins with illness, disease progression, and mortality were hypothe‐ sized, as well as longevity and health parameter status were attributed to this protein [53]. Interestingly, in the last years, from Walsh et al. (2001) [54] to present [55-57], some studies investigated eHSP70 concentration in response to exercise. However, eHSP70 released from immune cells during exercise can be an immune signal from the periphery to the central nervous system (e.g. hypothalamus) leading to the "fatigue sensation/behavior". In other words, after eHSP70 concentrations had reached a critical level (not known yet), higher exercise loads or duration would be dangerous to the whole body [51]. Then, eHSP70 can be a signal to impose fatigue sensation and shutdown of exercise, avoid a pro-inflammatory state to maintaining homeostatic, metabolic, and hemodynamic equilibrium.

In the bloodstream, eHSP70s and cytokines might participate in the physiologic responses of physical exercise, as chemical messengers released during an effort. Increased levels of eHSP70 in the plasma during exercise may participate in the fatigue sensation, also acting as a danger signal from the immune system [51]. On the other hand, intracellular HSP70 (iHSP70) synthesis is necessary for homeostasis maintaining in the muscle and other tissues. In the intracellular millie, these proteins have molecular chaperone action of such proteins, limiting protein aggregation, facilitating protein refolding, and maintaining structural function of proteins. iHSP70 have further been demonstrated to provide cytoprotection by anti-apoptotic mecha‐ nisms, inhibiting gene expression and regulating cell cycle progression [58]. Intracellularly activated HSP70 are anti-inflammatory by avoiding protein denaturation and excessive NFkB activation that may be damaging to the cells. Thus, iHSP70 act as a suppressor of NF-kB pathways (inhibiting TNF-α expression), an important anti-inflammatory role of HSP70 family proteins [51].

The chaperone function of iHSP70 is more than microscopic measurements of laboratory research field. Muscle disuse result in muscular atrophy that is represented by the decrease in muscle mass, fiber cross sectional area, and total myofibrillar protein content. In this situation, contractile protein breakdown exceeds protein synthesis. Moreover, in atrophied muscle occurs an increase in the proportion of fibers containing the fast myosin heavy chain by the transformation from the slow myosin heavy chain (MyHC-I/β) to the fast myosin heavy chain (MyHC-IId/x). As early as 18 hours and as late as 18 days after muscle disuse, it is possible to measure a decrease in iHSP70 in the soleus muscle [59]. Interestingly, previous heat treatment is a strategy to induce iHSP70 expression in the muscle and this molecular adaptation results in the maintenance of muscle mass during a 7-day period of immobilization [60]. In this way, iHSP70 expression appears to have no full protective effect on muscle mass, fiber cross sectional area, and total myofibrillar protein content, but prevents the decrease of MyHC-I/β and the increase of MyHC-IId/x induced during the atrophy process [61]. These evidences suggest that HSP70 can inhibit a key signaling pathway for atrophy in muscle cells preventing the muscular atrophy.

Heat treatment also has been tested in humans. Short wave diathermy therapy is a clinical strategy that means to increase deep heating of tissues with higher water content. This strategy may promote a 58% increase of HSP70 expression in *vastus lateralis* [62]. It is possible that previous heat treatments cannot reduce markers of muscle damage but is able to reduce muscular pain, preserve strength, and improve range of motion following exccentric contrac‐ tions. Curiously, there is a gender difference in heat shock response in both basal and induced by exercise iHSP70 levels, with men showing low pre-exercise levels and an attenuated iHSP70 response. The gender difference may be explained by the effects of estrogen modulation in heat shock response [62].

If the disused muscle is in trouble, the reuse of the musculature may represent many stages of soreness. After immobilization, the reload process to the muscle implies in newest molecular adaptations. If less required muscle is submitted to a challenge, the iHSP70 expression increased greatly (~200%) in the first two weeks of reload process and the return to basal levels (above disuse levels) early as eight weeks [59, 63]. This effect is accompanied by an increase in the percentage of slow type I MHC fibers (MyHC-I/β). Although many factors appear to be related to the down- and upregulation of iHSP70, the expression of this protein is closely related to the morphological and functional changes of muscle cells.

Exercise-induced expression of iHSP70 in the skeletal muscle has a major role in restoring muscle metabolic functionality as it provides cytoprotection to damaged cells. An iHSP70 inducing ability has been shown as a result in different protocols of exercise, including eccentric, concentric not damaging, and aerobic or resistive, all of which are capable to induce intramuscular HSP70 expression [64, 65]. Although time and intensity of the physical effort are determinant factors to increase of intramuscular iHSP70, its rise may be detectable just 2 h after the onset of an acute exercise session, when HSPA1A mRNA expression peaks [54]. Moreover, exercise-induced iHSP70 presents a time and intensity dependence [46].

millie, these proteins have molecular chaperone action of such proteins, limiting protein aggregation, facilitating protein refolding, and maintaining structural function of proteins. iHSP70 have further been demonstrated to provide cytoprotection by anti-apoptotic mecha‐ nisms, inhibiting gene expression and regulating cell cycle progression [58]. Intracellularly activated HSP70 are anti-inflammatory by avoiding protein denaturation and excessive NFkB activation that may be damaging to the cells. Thus, iHSP70 act as a suppressor of NF-kB pathways (inhibiting TNF-α expression), an important anti-inflammatory role of HSP70 family

The chaperone function of iHSP70 is more than microscopic measurements of laboratory research field. Muscle disuse result in muscular atrophy that is represented by the decrease in muscle mass, fiber cross sectional area, and total myofibrillar protein content. In this situation, contractile protein breakdown exceeds protein synthesis. Moreover, in atrophied muscle occurs an increase in the proportion of fibers containing the fast myosin heavy chain by the transformation from the slow myosin heavy chain (MyHC-I/β) to the fast myosin heavy chain (MyHC-IId/x). As early as 18 hours and as late as 18 days after muscle disuse, it is possible to measure a decrease in iHSP70 in the soleus muscle [59]. Interestingly, previous heat treatment is a strategy to induce iHSP70 expression in the muscle and this molecular adaptation results in the maintenance of muscle mass during a 7-day period of immobilization [60]. In this way, iHSP70 expression appears to have no full protective effect on muscle mass, fiber cross sectional area, and total myofibrillar protein content, but prevents the decrease of MyHC-I/β and the increase of MyHC-IId/x induced during the atrophy process [61]. These evidences suggest that HSP70 can inhibit a key signaling pathway for atrophy in muscle cells preventing

Heat treatment also has been tested in humans. Short wave diathermy therapy is a clinical strategy that means to increase deep heating of tissues with higher water content. This strategy may promote a 58% increase of HSP70 expression in *vastus lateralis* [62]. It is possible that previous heat treatments cannot reduce markers of muscle damage but is able to reduce muscular pain, preserve strength, and improve range of motion following exccentric contrac‐ tions. Curiously, there is a gender difference in heat shock response in both basal and induced by exercise iHSP70 levels, with men showing low pre-exercise levels and an attenuated iHSP70 response. The gender difference may be explained by the effects of estrogen modulation in

If the disused muscle is in trouble, the reuse of the musculature may represent many stages of soreness. After immobilization, the reload process to the muscle implies in newest molecular adaptations. If less required muscle is submitted to a challenge, the iHSP70 expression increased greatly (~200%) in the first two weeks of reload process and the return to basal levels (above disuse levels) early as eight weeks [59, 63]. This effect is accompanied by an increase in the percentage of slow type I MHC fibers (MyHC-I/β). Although many factors appear to be related to the down- and upregulation of iHSP70, the expression of this protein is closely

related to the morphological and functional changes of muscle cells.

proteins [51].

96 Muscle Cell and Tissue

the muscular atrophy.

heat shock response [62].

Since iHSP70 family members promote the facilitation of protein transport into the mitochon‐ dria, allowing and improving structural integrity of the organelle during fast energy flow, iHSP70 content has been correlated with an increase in the oxidative capacity of muscle cells. Several studies have demonstrated the relationship between high iHSP70 levels in the skeletal muscle and increased activity of mitochondrial enzymes after a short training period [66]. On the other hand, decreased mitochondrial function is known to be associated with the accu‐ mulation of intramyocyte triglycerides (and its byproducts), insulin resistance and diabetes. For this additional reason, exercise-induced iHSP70 expression can lead to improvements in metabolite oxidation and, consequently, insulin sensitivity.

Thus, exercise training or regular physical activity tend to reduce the stressful impact of each exercise session and improve glucose uptake and storage, antioxidant capacity, associated with the increase in the iHSP70 content in muscle [67, 68]. In fact, increased iHSP70 expression has been demonstrated to participate in cell signaling that prevents insulin resistance [67]. Since iHSP70 family members promote the facilitation of protein transport into the cytoplasm environment and help the cell to maintain the structural integrity of the organelles and proteins involved in cross-bridge contraction, iHSP70 can help prevent the loss of muscle mass, the decrease in the type II fiber content, and the loss of metabolic capacity as oxidative metabolism and thus improving glucose usage by the active muscle [66], which is important both in obese and aging conditions.

Exercise also promotes several redox related adaptations. The increase in ROS levels and adenosine monophosphate (AMP), signaling the activation of the enzyme AMPK to stimulate the catabolism of substrates in producing new ATP [69]. In this way, AMPK in skeletal muscles contributes to the maintenance of normoglycemia by two mechanisms—most glucose uptake occurs by increasing the translocation of GLUT4 and by reducing peripheral insulin resistance. In addition, this enzyme acts on hypothalamic functions by modulating the events related to hunger and satiety. Thus, plays a key role in the regulation of cellular metabolism and in the maintenance of energy homeostasis [70].

During a moderate aerobic exercise session the skeletal muscle is able to uptake glucose without insulin signaling and increase fatty acid mobilization to use as an energy source. Belonging to the family of enzymes activated by cellular stress caused primarily by ATP depletion, AMPK is responsible for the regeneration of ATP levels via oxidation of fatty acids and glucose [71]. When the AMPK is activated, it is capable of inhibiting both enzymes, the 3 hydroxy-3-methylglutaryl CoA reductase (HMGCoA reductase) and ACC. In the liver, the inactivation of the malonyl CoA ACC concentrations is insufficient to inhibit CPT1. Thus, there is a predominance of the β-oxidation of fatty acids synthesis, and the production of energy outweighs the expense [72, 73]. Considering intervention strategies for T2DM, exercise improves muscular oxidative capacity and, consequently, insulin sensitivity, up regulating the expression of PGC-1α in healthy subjects. In many rodent models, the increase of the OXPHOS is coordinated by PGC-1α, which increases the transcriptional activity of PPARγ, reducing the effects that promote insulin resistance.

The predominant oxidative metabolism in type I fibers, which are modified by the action of aerobic training at moderate or high intensity, may be a key point to glucose metabolism regulation. There is an increase in the number of mitochondria and GLUT4 vesicles, thus promoting the acceleration of oxidative metabolism with increased ROS production accom‐ panied by increased antioxidant defense capacity. Increase in antioxidant enzyme activity observed in superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, and also in glutathione (GSH) content, represent oxidative stress preventive adaptations induced by exercise training. The redox protection has direct impact in the maintenance of insulin sensitivity in the muscle [74, 75]. These changes may be triggered or stimulated by increased HSP70 expression [76].

Regular exercise promotes the acute increase of blood flow and shear stress and, in turn, improves the NO bioavailability, hence increasing the endothelium-dependent vasodilatation. This NO effect occurs in parallel to the decrease in pro-inflammatory biomarkers after exercise training. This improvement in NO and decrease in pro-inflammatory markers could represent one of the most important mechanisms of cardioprotection induced by regular exercise that prevent comorbidities associated to T2DM. However, the exercise intensity seems to be a crucial variable to future studies. iHSP70 expression depends on exercise intensity (level of physical challenge, measured by workload or time), while many other adaptations could not be influenced by exercise training intensity. We believe that exercise can induce both intracel‐ lular and extracellular HSP70, but promotes equilibrium in HSP70 signaling to the whole body in T2DM [77, 78].
