**4. Heat shock protein pathways**

Heat shock proteins (HSPs) have been found to play a fundamental role in the recovery from multiple stress conditions and to offer protection from subsequent insults (De Maio,

A Novel L-Arginine/L-Glutamine Coupling Hypothesis: Implications for Type 1 Diabetes 249

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,

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-B-

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

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

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

and *NOS-2* genes that would worsen sepsis in rats (Chen et al., 2005). This

**4.1 Intracellular hsp70** 

depending *TNF-*

be damaging to the cells.

**4.2 Extracellular hsp70** 

HSP70 expression (Zhang et al., 2009).

and, consequently, HSP70 expression (Kim et al., 1997).

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.

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

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 Heck, P.I. Homem de Bittencourt Jr., unpublished work).
