**4.1 Intracellular hsp70**

248 Type 1 Diabetes – Complications, Pathogenesis, and Alternative Treatments

2011). The function of HSPs during stress goes beyond their intracellular localization and chaperone role as they have been detected outside cells activating signaling pathways. Extracellular HSPs are likely to act as indicators of the stress conditions, priming other cells, particularly of the immune system, to avoid the propagation of the insult (see De Maio, 2011 for review). As we shall present below, the delicate balance between the "danger signalling" extracellular HSPs and its intracellular counterparts may dictate pancreatic -cell response to cytokines and, eventually, the precipitation of diabetes. By regulating L-arginine consumption through iNOS, and, consequently, NO generation, intracellular HSP response

(or its deficiency) may unravel unpredicted facets of both type 1 and type 2 diabetes.

encoded by *HSPA1A* gene (Heck et al., 2011).

Heck, P.I. Homem de Bittencourt Jr., unpublished work).

Heat shock proteins (HSPs) are a set of highly conserved polypeptides in both eukaryotic and prokaryotic organisms. They are categorised in families according to their molecular sises and include HSP110, HSP100, HSP90, HSP70, HSP60 HSP30 and HSP10 subclasses. By far, the most studied (due to its evident high expression in mammalian cells under stress conditions) and conserved is the 70-kDa family (HSP70), which comprises a number of related proteins whose molecular weights range from 66 to 78 kDa. HSP70 isoforms are encoded by a multigene family consisting presently of, at least, 13 distinct genes in humans so far studied (Kampinga et al., 2009; Henderson, 2010). Human HSP70 is 73% identical to *Drosophila* HSP70 and 47% identical to *E. coli* DnaK (the *E. coli* orthologue of eukaryotic HSP70) while, surprisingly, the nucleotide sequences of the human and *Drosophila* genes are 72% identical and human and *E. coli* genes are 50% identical (Hunt & Morimoto, 1985). HSP70s function as molecular chaperones that facilitate protein transport, prevent protein aggregation during folding, and protect newly synthesised polypeptide chains against misfolding and protein denaturation (Henderson, 2010). While the constitutive form is expressed in a wide variety of cell types at basal levels (being only moderately inducible), the so-called inducible HSP70 forms (which are barely detectable under non-stressful conditions) could be promptly synthesised under a condition of "homeostatic stress", this being any "homeostasis threatening" condition, such as heat, glucose deprivation, lack of growth factors and so forth. Traditionally, research groups indistinctly use HSP70 as a unified term for both inducible (72 kDa, HSP72 encoded by the *HSPA1A* human gene) and constitutive (73 kDa, HSP73 or HSC70, for heat shock cognate protein, encoded by the human *HSPA8* gene whose product differs from *HSPA1A* protein by only 2 amino acids, Kampinga et al., 2009; Tavaria et al., 1996; Arya et al., 2007; Tavaria et al., 1995). However, HSP70 is the preferable form to be used only when one refers to the inducible HSP72 protein

Many different events can induce HSP expression, among them are environmental, pathological and physiological factors, such as heavy metal exposure, UV radiation, amino acid analogues, bacterial or viral infection, inflammation, cyclo-oxygenase inhibitors (including acetylsalicylic acid), oxidative stress, cytostatic drugs, growth factors, cell differentiation and tissue development, which strongly activate the main eukaryotic heat shock transcription factor, HSF-1, leading to HSP70 expression (Lindquist & Craig, 1988). Physical exercise, even at single low-intensity bouts (Silveira et al., 2007), is able to induce HSP70 expression in different cell types leading to augmented plasma HSP70 concentrations (see Heck et al., 2011 for review). In our hands, rats submitted to swimming sessions of as short as 20 min (2-4% body weight overload, a mild exercise) demonstrate increased HSP72 (mRNA and protein) in circulating monocytes and lymphocytes and in lymph node lymphocytes and peritoneal macrophages, which is paralleled by a rise in plasma HSP70 levels immediately after the exercise (C.M. Schöler, S.P. Scomazzon, P. Renck Nunes, T.G.

Aside the now classical molecular chaperone action, the most remarkable intracellular effect of HSP70s is the inhibition of NF-B activation, which has profound implications for immunity, inflammation, cell survival and apoptosis. Indeed, HSP70 blocks NF-B activation at different levels, by inhibiting the phosphorylation of the inhibitor of B (IBs), by directly binding to IB kinase- (IKK) thus inhibiting tumour necrosis factor- (TNF) induced apoptosis (Ran et al., 2004). In fact, the supposition that HSP70 might act intracellularly as a suppressor of NF-B pathways has been raised after a number of discoveries in which HSP70 was intentionally induced, such as the suppression of astroglial iNOS expression paralleled by decreased NF-B activation (Feinstein et al., 1996) and the protection of rat hepatocytes from TNF-induced apoptosis by treating cells with the NO-donor *S*-Nitroso-*N*-acetylpenicillamine (SNAP), which reacts with intracellular glutathione (GSH) molecules generating *S*-nitrosoglutathione (SNOG) that induces HSP70, and, consequently, HSP70 expression (Kim et al., 1997).

HSP70 confers protection against sepsis-related circulatory mortality via the inhibition of iNOS gene expression in the rostral ventrolateral medulla through the prevention of NF-B activation, inhibition of IB kinase activation and consequent inhibition of IB degradation (Chan et al., 2004). This is corroborated by the finding that HSP72 assembles with hepatocyte NF-B/IB complex in the cytosol thus impeding further transcription of NF-Bdepending *TNF-* and *NOS-2* genes that would worsen sepsis in rats (Chen et al., 2005). This may also be unequivocally demonstrated by treating cells or tissues with HSP70 antisense oligonucleotides that completely reverses the beneficial NF-B-inhibiting effect of heat shock and inducible HSP70 expression (see, for instance, Kim et al., 1997; Chan et al., 2004). Hence, HSP70 is anti-inflammatory *per se*, when intracellularly located, which also explains why cyclopentenone prostaglandins (cp-PGs) are powerful anti-inflammatory autacoids (Rossi et al., 2000; Homem de Bittencourt & Curi, 2001; Beere, 2004; Gutierrez et al., 2008).

Another striking effect of HSP70 is the inhibition of apoptosis, which occurs via many intracellular downstream pathways (e.g. JNK, NF-B and Akt) that are both directly and indirectly blocked by HSP70, besides the inhibition of Bcl-2 release from mitochondria (Beere, 2004). Therefore, intracellularly activated HSP70s are cytoprotective and antiinflammatory by avoiding protein denaturation and excessive NF-B activation which may be damaging to the cells.

It is strikingly noteworthy that L-glutamine attenuates TNF- release and enhances HSP72 expression in human peripheral blood mononuclear cells (Wischmeyer et al., 2003). In fact, L-glutamine induces HSP70 expression via *O*-glycosylation and phosphorylation of HSF-1 and Sp1 (Singleton, K.D. & Wischmeyer, P.E., 2008) in a process that is mediated, at least partially, by the increase in the flux through the hexosamine biosynthetic pathway (Hamiel et al., 2009). Also, it has been shown that a single dose of L-glutamine relieve renal ischaemia-reperfusion injury in rats in 24 h by a mechanism associated with enhanced HSP70 expression (Zhang et al., 2009).

#### **4.2 Extracellular hsp70**

HSP70s may also be found in the circulation and its presence is associated to oxidative stress. While healthy people usually have low plasma levels of HSP70, the association of increased blood concentrations of such proteins with illness and disease progression has been hypothesised. In this way, oxidative stress, inflammation, cardiovascular disorders and

A Novel L-Arginine/L-Glutamine Coupling Hypothesis: Implications for Type 1 Diabetes 251

the heart is not able to do the same, thus suggesting that the mechanisms of HSP70 protein synthesis are specifically driven in each tissue (Harris & Starnes, 2001; Skidmore et al., 2005; Morton et al., 2007; Staib et al., 2007) and that augmented temperature is insufficient to elicit HSP70 synthesis during exercise. Moreover, the susceptibility of tissues to be stressed by the environmental changes elicited by exercise varies enormously and other protective pathways may be activated in the heart, as we have shown for MRP/GS-X pump ATPases whose expression seems to prevent HSP70 expression in the cardiac muscle after exercise bouts (Krause et al., 2007). In spite of free radicals may be produced under normal conditions, a burst in reactive oxygen species does occur during exercise (Fisher-Wellman & Bloomer, 2009). Besides enzymatic and non-enzymatic antioxidant apparatus, studies in both animal models and humans implicate HSP70s as a complementary protection against oxidative damage (Smolka et al., 2000; Silmar et al., 2007; Hamilton et al., 2003), particularly because HSP70s may recover oxidatively denatured proteins. After an acute exercise session, skeletal muscle (Hernando & Manso, 1997), cardiac muscle (Locke et al., 1995) and other tissues, such as the liver (Gonzalez & Manso, 2004; Kregel & Moseley, 1996), have shown a state of oxidative stress, concomitantly to high concentrations of intracellular HSP70 (Salo et al., 1991). Even though oxidative stress is a strong factor to induce HSP70s in response to exercise, free radical production is not the only pathway involved in this process, since sexual hormones and adrenergic stimuli may modulate HSP70 response (Parro & Noble, 1999; Paroo et al., 2002a, 2002b; Paroo et al., 1999) and circulating monocytes from acutely exercised rats do not show appreciable changes in erythrocyte glutathione disulphide (GSSG) to glutathione (GSH) ratio (an index of intracellular redox status) and plasma thiobarbituric acid-reactive substances (TBARS), even in a state of high-

More recently, however, it has been demonstrated the presence of HSP70s in the circulation in response to exercise (Walsh et al., 2001). Since exercise is able to induce high concentrations of HSP70s in both muscle and plasma, the most obvious hypothesis was, primarily, that skeletal muscle should be the releaser of HSP70 during exercise. However, further studies have revealed that this is not the case, at all. Postural muscles express high levels of HSP70s under basal conditions, which has led to the belief in a preventive role for these proteins against muscle damage through the stabilization of ionic channels (Tupling et al., 2007), as well as myotube development (Kayani et al., 2008). HSP70s were also believed to be an important way to preserve low twitch (oxidative) muscle phenotype after frequent activation, as in physical training (Kelly et al., 1996; Murlasits et al., 2006). Preservation of intracellular muscular function during different exercises, venous-arterial HSP70 differences in different territories (Febbraio et al., 2002a), and the lack of evidence supporting the proposition that the muscle could be the major source of circulatory eHSP70 precluded the 'muscle hypothesis' and suggested that other tissues/cells should be responsible for the increase of eHSP70 in the circulation. Once HSP70 protein release from the muscle to the extracellular fluid could eventually happen by lysis process, and considering that the lysis of muscle fibre occurs only under severe cellular stress condition, the presence of eHSP70 during moderate exercise, as we normally employ, was found to be unfeasible. Though it had been shown that both the intensity and duration of exercise have effects in plasma eHSP70 (Fehrenbach et al., 2005) and muscle (Milne & Noble, 2002) HSP70 immunocontents, this rise in circulating levels of eHSP70 precedes, however, any gene or protein expression

profile synthesis of hydrogen peroxide (Silveira et al., 2007).

pulmonary fibrosis have been directly correlated with HSP70 concentration in the bloodstream (Ogawa et al., 2008). On the other hand, L-glutamine supplementation, which rises circulating HSP70 levels in critically ill patients, is associated with lower hospital treatment period (Ziegler et al., 2005). Therefore, these studies may suggest that elevation of HSP70 levels could be an important immunoinflammatory response against physiological disorders or disease.

Inasmuch as HSP70s exist in the extracellular space, molecular interactions with cell surface receptors may occur and signalling pathways could be triggered in many cell types, whereas there are a variety of receptors to HSP70 binding, amplifying the possible targets to these extracellular molecules (Calderwood et al., 2007a, 2007b). However, the function of circulating HSP70 is incompletely understood yet. HSP70s are released towards the extracellular space by special mechanisms that include pumping across cell membranes through the highly conserved ABC cassette transport proteins. Recent studies have demonstrated that exosomes provide the major pathway for the vesicular secretory release of HSP70s and that heat stress strikingly enhances the amount of HSP70 secreted per vesicle, but does not influence the efficiency of stress-induced rate of HSP70 release and the number of exosomes neither (Sun et al., 2005; Lancaster & Febbraio, 2005; Multhoff, 2007). A similar profile was observed in our hands (T.G. Heck; P. Renck Nunes; S.P. Scomazzon & P.I. Homem de Bittencourt Jr., manuscript in preparation), in which lymph node lymphocytes from exercised rats submitted to a further (other than the exercise bouts) challenge (heat shock) presented an HSP70 accumulation into the culture medium that is dependent on previous exercise load. Apparently, systemic extracellular HSP70 (eHSP70) could arise from many tissues and different cell types and this may involve distinct mechanisms of release (including necrosis) and a large variety of inducing factors (Mambula et al., 2007). Finally, HSP72 is clearly the major component of the secreted eHSP70 found in the circulation, although recent evidence suggests that other forms may also be released into the blood, as recently pointed out by De Maio (2011). eHSP70 has been shown to bind to type 2 and 4 tolllike receptors (TLR2 and TLR4) on the surface of antigen-presenting cells (APCs) similarly to lipopolysaccharides (LPS), inducing the production of the pro-inflammatory cytokines IL-1 and TNF-, as well as NO (a product with prominent anti-microbial activity), in an NF-Bdependent fashion (Ao et al., 2009; Asea, 2003; Asea, 2008).

Taken together, the above findings suggest that the body must attain a precise equilibrium between pro-inflammatory eHSP70 and anti-inflammatory intracellular HSP70 production in order to avoid chronic non-resolved inflammations, such as those observed in sepsis and during the onset of type 1 diabetes. However, why such a balance is not achieved in these illnesses is a matter of intense study.

#### **4.3 Heat shock proteins and exercise**

As recently reviewed (Heck et al., 2011), physical exercise and its inherent physiological alterations induce HSP70 expression in many tissues and cell types, not only in the muscle cells. The breakdown of cell homeostasis produced by modifications in temperature, pH, ion concentrations, oxygen partial pressure, glycogen/glucose availability, and ATP depletion are among the factors that activate HSP70 synthesis during exercise (Noble et al., 2008). Rise in core and muscle temperature during exercise seems an obvious way to induce HSP70. However, while skeletal muscle sustains HSP70 expression in the absence of heat stimulus,

pulmonary fibrosis have been directly correlated with HSP70 concentration in the bloodstream (Ogawa et al., 2008). On the other hand, L-glutamine supplementation, which rises circulating HSP70 levels in critically ill patients, is associated with lower hospital treatment period (Ziegler et al., 2005). Therefore, these studies may suggest that elevation of HSP70 levels could be an important immunoinflammatory response against physiological

Inasmuch as HSP70s exist in the extracellular space, molecular interactions with cell surface receptors may occur and signalling pathways could be triggered in many cell types, whereas there are a variety of receptors to HSP70 binding, amplifying the possible targets to these extracellular molecules (Calderwood et al., 2007a, 2007b). However, the function of circulating HSP70 is incompletely understood yet. HSP70s are released towards the extracellular space by special mechanisms that include pumping across cell membranes through the highly conserved ABC cassette transport proteins. Recent studies have demonstrated that exosomes provide the major pathway for the vesicular secretory release of HSP70s and that heat stress strikingly enhances the amount of HSP70 secreted per vesicle, but does not influence the efficiency of stress-induced rate of HSP70 release and the number of exosomes neither (Sun et al., 2005; Lancaster & Febbraio, 2005; Multhoff, 2007). A similar profile was observed in our hands (T.G. Heck; P. Renck Nunes; S.P. Scomazzon & P.I. Homem de Bittencourt Jr., manuscript in preparation), in which lymph node lymphocytes from exercised rats submitted to a further (other than the exercise bouts) challenge (heat shock) presented an HSP70 accumulation into the culture medium that is dependent on previous exercise load. Apparently, systemic extracellular HSP70 (eHSP70) could arise from many tissues and different cell types and this may involve distinct mechanisms of release (including necrosis) and a large variety of inducing factors (Mambula et al., 2007). Finally, HSP72 is clearly the major component of the secreted eHSP70 found in the circulation, although recent evidence suggests that other forms may also be released into the blood, as recently pointed out by De Maio (2011). eHSP70 has been shown to bind to type 2 and 4 tolllike receptors (TLR2 and TLR4) on the surface of antigen-presenting cells (APCs) similarly to lipopolysaccharides (LPS), inducing the production of the pro-inflammatory cytokines IL-1 and TNF-, as well as NO (a product with prominent anti-microbial activity), in an NF-B-

Taken together, the above findings suggest that the body must attain a precise equilibrium between pro-inflammatory eHSP70 and anti-inflammatory intracellular HSP70 production in order to avoid chronic non-resolved inflammations, such as those observed in sepsis and during the onset of type 1 diabetes. However, why such a balance is not achieved in these

As recently reviewed (Heck et al., 2011), physical exercise and its inherent physiological alterations induce HSP70 expression in many tissues and cell types, not only in the muscle cells. The breakdown of cell homeostasis produced by modifications in temperature, pH, ion concentrations, oxygen partial pressure, glycogen/glucose availability, and ATP depletion are among the factors that activate HSP70 synthesis during exercise (Noble et al., 2008). Rise in core and muscle temperature during exercise seems an obvious way to induce HSP70. However, while skeletal muscle sustains HSP70 expression in the absence of heat stimulus,

dependent fashion (Ao et al., 2009; Asea, 2003; Asea, 2008).

illnesses is a matter of intense study.

**4.3 Heat shock proteins and exercise** 

disorders or disease.

the heart is not able to do the same, thus suggesting that the mechanisms of HSP70 protein synthesis are specifically driven in each tissue (Harris & Starnes, 2001; Skidmore et al., 2005; Morton et al., 2007; Staib et al., 2007) and that augmented temperature is insufficient to elicit HSP70 synthesis during exercise. Moreover, the susceptibility of tissues to be stressed by the environmental changes elicited by exercise varies enormously and other protective pathways may be activated in the heart, as we have shown for MRP/GS-X pump ATPases whose expression seems to prevent HSP70 expression in the cardiac muscle after exercise bouts (Krause et al., 2007). In spite of free radicals may be produced under normal conditions, a burst in reactive oxygen species does occur during exercise (Fisher-Wellman & Bloomer, 2009). Besides enzymatic and non-enzymatic antioxidant apparatus, studies in both animal models and humans implicate HSP70s as a complementary protection against oxidative damage (Smolka et al., 2000; Silmar et al., 2007; Hamilton et al., 2003), particularly because HSP70s may recover oxidatively denatured proteins. After an acute exercise session, skeletal muscle (Hernando & Manso, 1997), cardiac muscle (Locke et al., 1995) and other tissues, such as the liver (Gonzalez & Manso, 2004; Kregel & Moseley, 1996), have shown a state of oxidative stress, concomitantly to high concentrations of intracellular HSP70 (Salo et al., 1991). Even though oxidative stress is a strong factor to induce HSP70s in response to exercise, free radical production is not the only pathway involved in this process, since sexual hormones and adrenergic stimuli may modulate HSP70 response (Parro & Noble, 1999; Paroo et al., 2002a, 2002b; Paroo et al., 1999) and circulating monocytes from acutely exercised rats do not show appreciable changes in erythrocyte glutathione disulphide (GSSG) to glutathione (GSH) ratio (an index of intracellular redox status) and plasma thiobarbituric acid-reactive substances (TBARS), even in a state of high-

profile synthesis of hydrogen peroxide (Silveira et al., 2007). More recently, however, it has been demonstrated the presence of HSP70s in the circulation in response to exercise (Walsh et al., 2001). Since exercise is able to induce high concentrations of HSP70s in both muscle and plasma, the most obvious hypothesis was, primarily, that skeletal muscle should be the releaser of HSP70 during exercise. However, further studies have revealed that this is not the case, at all. Postural muscles express high levels of HSP70s under basal conditions, which has led to the belief in a preventive role for these proteins against muscle damage through the stabilization of ionic channels (Tupling et al., 2007), as well as myotube development (Kayani et al., 2008). HSP70s were also believed to be an important way to preserve low twitch (oxidative) muscle phenotype after frequent activation, as in physical training (Kelly et al., 1996; Murlasits et al., 2006). Preservation of intracellular muscular function during different exercises, venous-arterial HSP70 differences in different territories (Febbraio et al., 2002a), and the lack of evidence supporting the proposition that the muscle could be the major source of circulatory eHSP70 precluded the 'muscle hypothesis' and suggested that other tissues/cells should be responsible for the increase of eHSP70 in the circulation. Once HSP70 protein release from the muscle to the extracellular fluid could eventually happen by lysis process, and considering that the lysis of muscle fibre occurs only under severe cellular stress condition, the presence of eHSP70 during moderate exercise, as we normally employ, was found to be unfeasible. Though it had been shown that both the intensity and duration of exercise have effects in plasma eHSP70 (Fehrenbach et al., 2005) and muscle (Milne & Noble, 2002) HSP70 immunocontents, this rise in circulating levels of eHSP70 precedes, however, any gene or protein expression

A Novel L-Arginine/L-Glutamine Coupling Hypothesis: Implications for Type 1 Diabetes 253

in turn, allow iNOS expression (needed to NO-assisted insulin secretion) but not at exaggerated ratios that culminate with -cell death and failure in insulin secretion. In fact, physical exercise, which may also present an anti-inflammatory effect by virtue of its ability to induce the expression of HSP70, is inversely associated with L-arginine utilisation by -cell iNOS (Atalay et al., 2004). Furthermore, a dramatic scenario does exist in that the susceptibility to oxidative damage to -cells in type 1 diabetes is associated to the impairment of HSP70-induced cytoprotection, while endurance training may offset some of the adverse effects of diabetes by upregulating tissue HSP70 expression (Atalay et al., 2004). Indeed, in many, if not all, severe inflammatory manifestations of acute nature, such as sepsis or insulitis, the stage of HSP70-based "resolution of inflammation" is simply not seen at all. For instance, in the serum of septic patients with highly oxidative profile (whose prognosis is death), it is observed 30-fold increase in serum HSP70 (eHSP70) compared with control subjects (Gelain et al., 2011), whereas the amount of intracellular HSP70 expressed in the cells of such subjects is, as a rule, lower that that expected. Corroborating this proposition, the expression of HSP70 by pancreatic islets from diabetes-prone BB rats has been found to be lower than that in diabeticresistant LEW rats of same age and, in the diabetes-prone BB rats, HSP70 expression has shown to be much lower in young as compared to adult animals (Wachlin et al., 2002). Since intracellular HSP70 functions as a potent anti-inflammatory cellular tool due to the impairment over NF-B downstream pathways, a deficient HSP70 may threaten -cell survival

Results from our group have also shown that, besides a reduction in peripheral insulin resistance, heat shock treatment (which also enhances HSP70 export towards the plasma) may impair insulin action under hypoglycaemic conditions in the rat model (M.S. Ludwig.; V.C. Mingueti; P. Renck Nunes; T.G. Heck; R.B. Bazotte & Homem de Bittencourt, P.I. Jr., manuscript in preparation) so that HSP70 balance seems to be crucial for glucose-insulin homeostasis. Now, we are currently evaluating the possibility that exercise may stimulate Th2 based immune response and protect -cells from pro-inflammatory cytokine pathways through HSP70 induction, which, ultimately, may prevent type 1 diabetes. Since **a)**  L-glutamine is a major precursor of L-arginine, which is capital for -cell survival, **b)**  L-arginine-dependent moderate NO synthesis induces HSP70 and **c)** physical exercise is able of directly inducing HSP70 and of enhancing L-glutamine production by the skeletal muscle, both exercise and/or L-glutamine supplementation are argued as preventive agents against the installation of type 1 diabetes by re-establishing the HSP70 equilibrium between the intra and extracellular spaces, as previously hypothesised (Krause & Homem de Bittencourt, 2008).

From the above discussion, it seems clear that the development of diabetes is not simply a question of cytokine imbalance culminating in a redox disruption and consequent oxidative stress that disrupts or kills -cells. This, in fact, raises another question: is beta cell susceptibility to stress solely a question of compromised antioxidant defence? If this were the case, it would appear preposterous that such a sophisticated cell remains prone to endogenously-generated NO-mediated self-destruction. The intricate metabolism of

In -cells, pro-inflammatory cytokines induce the production of NO, synthesised from L-arginine, via a reaction catalysed by iNOS, whose functionality depends on NF-B-driven gene transcription and *de novo* enzyme synthesis. iNOS also utilises NADPH and O2 as co-

**5. Participation of L-arginine/L-glutamine coupling in diabetes** 

L-arginine in -cells may unravel some important points in this regard.

(see Hooper & Hooper, 2005, for review).

of HSP70 in skeletal muscle (Febbraio et al., 2002b), which is another strong argument against the 'muscle hypothesis'. As stated above, other tissues synthesise HSP70s during physiological challenges to the homeostasis, as in an acute physical exercise bout. In this way, after treadmill exercise protocol, the rat liver has been found to enhance the expression of HSP70s (Gonzalez & Manso, 2004). Moreover, and finally, in a human study featuring leg and hepatosplanchnic venous-arterial eHSP70 difference in response to exercise it was unequivocally demonstrated that the contracting muscle does not contribute to eHSP70 circulating levels, while hepatosplanchnic viscera release eHSP70 from undetectable levels at rest to 5.2 pg/min after 120 min of exercise (Febbraio et al., 2002a). Additional studies have shown that oral glucose administration may exclusively reduce HSP70 release from the liver without any effect on muscle glycogen content or intracellular expression of HSP70 (Febbraio et al., 2004). Taken together, these results suggest that other cells may release eHSP70 during exercise, as verified during an experiment that analysed cerebral venous-arterial HSP70 difference (Lancaster et al., 2004). Although the liver seems to participate in this process, the nature of eHSP70-releasing cell(s) during exercise remains to be established.

#### **4.4 HSP70 and glucose/insulin status**

Intracellular HSP70 expression produces a clear anti-inflammatory effect by knocking down the expression of pro-inflammatory NF-B-dependent pathways. However, the activation of HSP70 pathways produces a much more delicate effect. Accordingly, in obese insulinresistant mice, chronic heat shock treatment has been shown to dramatically reduce insulin resistance by HSP72-specific prevention of c-Jun N-terminal Kinase (JNK) phsophorylation, an effect which is also observed in high-fat fed HSP72+/+ transgenic mice (Chung et al., 2008). Also, elevated expression of HSP70 has also been found in circulating mononuclear cells from type 2 diabetic patients (Yabunaka et al., 1995), which, as discussed above, is a immunoinflammatory disease as well. On the other hand, in rat islets, L-glutamine, which is an activator of HSF-1, was shown to attenuate ischaemic injury through the induction of HSP70 (Jang et al., 2008). Moreover, the well known inhibitory effect of IL-1 and TNF- (alone or combined) on insulin secretion may be completely prevented by a 1-h heat shock (42°C) pre-treatment of both human and rat islets (Scarim et al., 1998). These authors have also shown that the protective effects of heat shock on islet metabolic function are associated with the inhibition of IL-1- and TNF-stimulated NF-B nuclear localization and the consequent iNOS expression. Conversely, NO was found to be one of the triggers of HSP70 expression in human islets (Scarim et al., 1998), which is similar to that previously encountered by Kim et al. (1997), who described a protective effect of NO (via the formation of SNOG that induces HSP70) in rat hepatocytes against TNF-induced apoptosis. Moreover, J-type cyclopentenone prostaglandins (cp-PGs), which are the most powerful anti-inflammatory substances ever known (see Gutierrez et al., 2008 for review) and natural ligands of peroxisome-proliferator activated receptor- (PPAR-; Forman et al., 1995; Kliewer et al., 1995), are the strongest inducers of HSP70 expression and consequent NF-B blockade, a pattern that is shared with synthetic antidiabetic thiazolidinediones (TZDs), such as rosiglitazone, pioglitazone, troglitazone, and ciglitazone (see Zingarelli & Cook, 2005, for review).

The above observations point out again to the importance of poised L-arginine-dependent NO production by -cells in order to achieve an optimum of HSP70 expression, which may,

of HSP70 in skeletal muscle (Febbraio et al., 2002b), which is another strong argument against the 'muscle hypothesis'. As stated above, other tissues synthesise HSP70s during physiological challenges to the homeostasis, as in an acute physical exercise bout. In this way, after treadmill exercise protocol, the rat liver has been found to enhance the expression of HSP70s (Gonzalez & Manso, 2004). Moreover, and finally, in a human study featuring leg and hepatosplanchnic venous-arterial eHSP70 difference in response to exercise it was unequivocally demonstrated that the contracting muscle does not contribute to eHSP70 circulating levels, while hepatosplanchnic viscera release eHSP70 from undetectable levels at rest to 5.2 pg/min after 120 min of exercise (Febbraio et al., 2002a). Additional studies have shown that oral glucose administration may exclusively reduce HSP70 release from the liver without any effect on muscle glycogen content or intracellular expression of HSP70 (Febbraio et al., 2004). Taken together, these results suggest that other cells may release eHSP70 during exercise, as verified during an experiment that analysed cerebral venous-arterial HSP70 difference (Lancaster et al., 2004). Although the liver seems to participate in this process, the nature of eHSP70-releasing cell(s) during exercise remains

Intracellular HSP70 expression produces a clear anti-inflammatory effect by knocking down the expression of pro-inflammatory NF-B-dependent pathways. However, the activation of HSP70 pathways produces a much more delicate effect. Accordingly, in obese insulinresistant mice, chronic heat shock treatment has been shown to dramatically reduce insulin resistance by HSP72-specific prevention of c-Jun N-terminal Kinase (JNK) phsophorylation, an effect which is also observed in high-fat fed HSP72+/+ transgenic mice (Chung et al., 2008). Also, elevated expression of HSP70 has also been found in circulating mononuclear cells from type 2 diabetic patients (Yabunaka et al., 1995), which, as discussed above, is a immunoinflammatory disease as well. On the other hand, in rat islets, L-glutamine, which is an activator of HSF-1, was shown to attenuate ischaemic injury through the induction of HSP70 (Jang et al., 2008). Moreover, the well known inhibitory effect of IL-1 and TNF- (alone or combined) on insulin secretion may be completely prevented by a 1-h heat shock (42°C) pre-treatment of both human and rat islets (Scarim et al., 1998). These authors have also shown that the protective effects of heat shock on islet metabolic function are associated with the inhibition of IL-1- and TNF-stimulated NF-B nuclear localization and the consequent iNOS expression. Conversely, NO was found to be one of the triggers of HSP70 expression in human islets (Scarim et al., 1998), which is similar to that previously encountered by Kim et al. (1997), who described a protective effect of NO (via the formation of SNOG that induces HSP70) in rat hepatocytes against TNF-induced apoptosis. Moreover, J-type cyclopentenone prostaglandins (cp-PGs), which are the most powerful anti-inflammatory substances ever known (see Gutierrez et al., 2008 for review) and natural ligands of peroxisome-proliferator activated receptor- (PPAR-; Forman et al., 1995; Kliewer et al., 1995), are the strongest inducers of HSP70 expression and consequent NF-B blockade, a pattern that is shared with synthetic antidiabetic thiazolidinediones (TZDs), such as rosiglitazone, pioglitazone, troglitazone, and ciglitazone (see Zingarelli & Cook,

The above observations point out again to the importance of poised L-arginine-dependent NO production by -cells in order to achieve an optimum of HSP70 expression, which may,

to be established.

2005, for review).

**4.4 HSP70 and glucose/insulin status** 

in turn, allow iNOS expression (needed to NO-assisted insulin secretion) but not at exaggerated ratios that culminate with -cell death and failure in insulin secretion. In fact, physical exercise, which may also present an anti-inflammatory effect by virtue of its ability to induce the expression of HSP70, is inversely associated with L-arginine utilisation by -cell iNOS (Atalay et al., 2004). Furthermore, a dramatic scenario does exist in that the susceptibility to oxidative damage to -cells in type 1 diabetes is associated to the impairment of HSP70-induced cytoprotection, while endurance training may offset some of the adverse effects of diabetes by upregulating tissue HSP70 expression (Atalay et al., 2004). Indeed, in many, if not all, severe inflammatory manifestations of acute nature, such as sepsis or insulitis, the stage of HSP70-based "resolution of inflammation" is simply not seen at all. For instance, in the serum of septic patients with highly oxidative profile (whose prognosis is death), it is observed 30-fold increase in serum HSP70 (eHSP70) compared with control subjects (Gelain et al., 2011), whereas the amount of intracellular HSP70 expressed in the cells of such subjects is, as a rule, lower that that expected. Corroborating this proposition, the expression of HSP70 by pancreatic islets from diabetes-prone BB rats has been found to be lower than that in diabeticresistant LEW rats of same age and, in the diabetes-prone BB rats, HSP70 expression has shown to be much lower in young as compared to adult animals (Wachlin et al., 2002). Since intracellular HSP70 functions as a potent anti-inflammatory cellular tool due to the impairment over NF-B downstream pathways, a deficient HSP70 may threaten -cell survival (see Hooper & Hooper, 2005, for review).

Results from our group have also shown that, besides a reduction in peripheral insulin resistance, heat shock treatment (which also enhances HSP70 export towards the plasma) may impair insulin action under hypoglycaemic conditions in the rat model (M.S. Ludwig.; V.C. Mingueti; P. Renck Nunes; T.G. Heck; R.B. Bazotte & Homem de Bittencourt, P.I. Jr., manuscript in preparation) so that HSP70 balance seems to be crucial for glucose-insulin homeostasis. Now, we are currently evaluating the possibility that exercise may stimulate Th2 based immune response and protect -cells from pro-inflammatory cytokine pathways through HSP70 induction, which, ultimately, may prevent type 1 diabetes. Since **a)**  L-glutamine is a major precursor of L-arginine, which is capital for -cell survival, **b)**  L-arginine-dependent moderate NO synthesis induces HSP70 and **c)** physical exercise is able of directly inducing HSP70 and of enhancing L-glutamine production by the skeletal muscle, both exercise and/or L-glutamine supplementation are argued as preventive agents against the installation of type 1 diabetes by re-establishing the HSP70 equilibrium between the intra and extracellular spaces, as previously hypothesised (Krause & Homem de Bittencourt, 2008).
