**1. Introduction**

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

Zhang, W., Feng, D., Li, Y., Iida, K., McGrath, B. & Cavener, D.R. (2006). Perk Eif2ak3

Zinszner, H., Kuroda, M., Wang, X., Batchvarova, N., Lightfoot, R.T., Remotti, H., Stevens,

No.11, (Jun), pp.(3864-3874), 0270-7306

4131

pp.(982-995), 0890-9369

Alpha Kinase Is Required for the Development of the Skeletal System, Postnatal Growth, and the Function and Viability of the Pancreas. *Mol Cell Biol,* Vol. 22,

Control of Pancreatic Beta Cell Differentiation and Proliferation Is Required for Postnatal Glucose Homeostasis. *Cell Metab,* Vol. 4, No.6, (Dec), pp.(491-497), 1550-

J.L. & Ron, D. (1998). Chop Is Implicated in Programmed Cell Death in Response to Impaired Function of the Endoplasmic Reticulum. *Genes Dev,* Vol. 12, No.7, (Apr 1),

> L-Arginine is synthesised *in vivo* from L-glutamine, L-glutamate, or L-proline via the intestinal-renal axis (**Fig. 1A**) in humans and most other mammals (Wu et al., 2009). In humans, plasma L-glutamine is the precursor of 80% of plasma L-citrulline while plasma L-citrulline, in turn, is the precursor of 10% of plasma L-arginine (van de Poll et al., 2007). Although the intestine consumes L-glutamine at a high rates, dependent on L-glutamine supply (and production from the skeletal muscle), approximately 13% of L-glutamine taken up by the intestine is converted to L-citrulline, so that quantitatively, L-glutamine is the major precursor for intestinal release of L-citrulline (van de Poll et al., 2007), which can be further converted to L-arginine. These observations highlight the importance of L-arginine/L-glutamine metabolic coupling, especially as L-arginine is one of the most potent secretagogues of insulin from the pancreatic beta cells (Palmer et al., 1976), whereas L-arginine deficiency is associated with insulinopenia and failure to secrete insulin in response to glucose (Spinas et al., 1999). L-Arginine is essential for metabolism and function of multiple body organs, with decreased plasma and cellular levels of L-arginine reported in type 2 diabetic subjects (Pieper & Dondlinger, 1997).

> Since L-arginine is the precursor of nitric oxide (NO)\*, which serves as a key cell signalling molecule in pancreatic islet -cells, restriction in the availability of L-arginine is likely to

<sup>\*</sup> **Abbreviations used:** CAT, catalase; GSH, glutathione; GSSG, glutathione disulphide; GSPx, glutathione peroxidase; GSRd, glutathione disulphide reductase; HSP70, 70-kDa member of heat shock protein family; eHSP70, extracellular heat shock protein of 70 kDa; IFN-, interferon-; IB, a member of the inhibitors of nuclear factor B; IKK, inhibitor of B kinase; IL-1, interleukin-1; IL1-ra, IL-1 receptor antagonist; iNOS, inducible nitric oxide synthase; NF-B, a member of nuclear transcription factor B; NO, nitric oxide free radical ( N=O); PPAR-, peroxisome proliferator activated receptor-; RNS, reactive nitrogen species; ROS, reactive oxygen species; SNOG, *S-*nitrosoglutathione; SOD,

A Novel L-Arginine/L-Glutamine Coupling Hypothesis: Implications for Type 1 Diabetes 243

mechanical activity, where blood glutamine stores may be challenged. Metabolic acidosis, by increasing L-glutamine utilisation by the kidney, may also favour glutamine depletion unless enteral supplementation or enhanced physical activity takes place. This is also the case of psychological-stress motivated inflammatory reactions that may underlie by the activation of sympathetic-CRH-histamine system (**Fig. 3**), which ultimately leads to a Th1 centered immune response that augments glutamine utilisation. Therefore, L-glutamine imbalance, by virtue of deficiently supplying L-arginine to the pancreas, deviates -cell glutamate metabolism from the synthesis of GSH to that of NO, leading to oxidative stress, impairment of insulin release and insulitis. This ongoing inflammation feeds forward NO metabolism, which enhances L-glutamine consumption thus perpetuating this cyclic condition that leads to type 1 diabetes mellitus (T1DM) **(B).** Physical exercise, on the other hand, may improve L-glutamine supply from the skeletal muscle and

counteract Th1-mediated inflammation due to the production of type 2 cytokines, such as

production and other anti-inflammatory mediators. Arrow widths indicate the intensity of

IL-6. Immunomodulatory action of exercise may also involve heat shock protein

the metabolic flux through each pathway.

Fig. 1. The l-arginine/l-glutamine coupling hypothesis of insulin-secreting -cells. (A) Pancreatic islet -cells utilise l-arginine for the biosynthesis of NO and l-glutamate for GSH generation during secretagogue-stimulated insulin secretion. l-Arginine is provided to the pancreas by the intestinal-renal axis from l-glutamine, while l-glutamate is furnished by the liver mainly from muscle-derived alanine. In the -cell, NH4+ may contribute to l-arginine biosynthesis, through the concerted action of carbamoyl phosphate synthetase I (CPS), ornithine transcarbamoylase (OTC), argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) that eventually produces l-arginine. Skeletal muscle-derived l-glutamine is also substrate for the maintenance of GSH metabolism in -cells, but rapidlyproliferating cells of the gut as well as immune cells compete with -cell for the utilisation of l-glutamine. Hence, any minimal reduction in the supply of l-arginine to the pancreas may shift l-glutamate metabolism towards the synthesis of NO instead of GSH, thus leading to oxidative stress, inhibition of insulin secretion and eventually -cell death. This is the case of undernourishment, cancer states, trauma, sepsis, major burns and low skeletal muscle

superoxide dismutase; TBARS, thiobarbituric acid-reactive substances; TNF-, tumor necrosis factor-; TNFR, TNF- receptor; UCP, uncoupling protein-2.

Fig. 1. The l-arginine/l-glutamine coupling hypothesis of insulin-secreting -cells. (A) Pancreatic islet -cells utilise l-arginine for the biosynthesis of NO and l-glutamate for GSH generation during secretagogue-stimulated insulin secretion. l-Arginine is provided to the pancreas by the intestinal-renal axis from l-glutamine, while l-glutamate is furnished by the liver mainly from muscle-derived alanine. In the -cell, NH4+ may contribute to l-arginine biosynthesis, through the concerted action of carbamoyl phosphate synthetase I (CPS),

argininosuccinate lyase (ASL) that eventually produces l-arginine. Skeletal muscle-derived l-glutamine is also substrate for the maintenance of GSH metabolism in -cells, but rapidlyproliferating cells of the gut as well as immune cells compete with -cell for the utilisation of l-glutamine. Hence, any minimal reduction in the supply of l-arginine to the pancreas may shift l-glutamate metabolism towards the synthesis of NO instead of GSH, thus leading to oxidative stress, inhibition of insulin secretion and eventually -cell death. This is the case of undernourishment, cancer states, trauma, sepsis, major burns and low skeletal muscle

superoxide dismutase; TBARS, thiobarbituric acid-reactive substances; TNF-, tumor necrosis factor-;

ornithine transcarbamoylase (OTC), argininosuccinate synthetase (ASS) and

TNFR, TNF- receptor; UCP, uncoupling protein-2.

mechanical activity, where blood glutamine stores may be challenged. Metabolic acidosis, by increasing L-glutamine utilisation by the kidney, may also favour glutamine depletion unless enteral supplementation or enhanced physical activity takes place. This is also the case of psychological-stress motivated inflammatory reactions that may underlie by the activation of sympathetic-CRH-histamine system (**Fig. 3**), which ultimately leads to a Th1 centered immune response that augments glutamine utilisation. Therefore, L-glutamine imbalance, by virtue of deficiently supplying L-arginine to the pancreas, deviates -cell glutamate metabolism from the synthesis of GSH to that of NO, leading to oxidative stress, impairment of insulin release and insulitis. This ongoing inflammation feeds forward NO metabolism, which enhances L-glutamine consumption thus perpetuating this cyclic condition that leads to type 1 diabetes mellitus (T1DM) **(B).** Physical exercise, on the other hand, may improve L-glutamine supply from the skeletal muscle and counteract Th1-mediated inflammation due to the production of type 2 cytokines, such as IL-6. Immunomodulatory action of exercise may also involve heat shock protein production and other anti-inflammatory mediators. Arrow widths indicate the intensity of the metabolic flux through each pathway.

A Novel L-Arginine/L-Glutamine Coupling Hypothesis: Implications for Type 1 Diabetes 245

Oxidative stress has long been recognised to play an important role in the development of type 1 diabetes and its subsequent complications (Wierusz-Wysock et al., 1997) which are aggravated due to the low activities of oxygen free radical scavenging enzymes in islet cells, especially mitochondrial manganese-type superoxide dismutase (Mn-SOD; Asayama et al., 1986), glutathione peroxidase (GSPx; Malaisse et al., 1982; Mathews & Leiter, 1999) and glutathione disulphide (GSSG) reductase (GSRd; Mathews & Leiter, 1999). Also, the expression of mRNA encoding for several antioxidant enzymes, such as Mn-SOD, cytoplasmic copper-zinc type SOD (Cu/Zn-SOD), GSPx, and catalase (CAT), has been reported to be lower in islets of Langerhans compared with other mouse tissues (Lenzen et al., 1996). Additionally, the administration of antioxidants (nicotinamide, SOD, -tocopherol, probucol and the 21-aminosteroid lazaroids), as well as oxygen free radical scavengers, have been used in vitro to protect islets from the cytotoxic effects of some proinflammatory cytokines (IL-1, TNF and INF), concurrently providing *in vivo* protection against the development of the autoimmune diabetes process (Nomikos et al., 1986). Conversely, studies on MnSOD and CAT transgenics have shown that protection of islets from oxidative stress does not alter cytokine toxicity (Chen et al., 2005), which indicates that, although related to each other, oxidative stress and cytokine-induced islet toxicity may use

An additional complication to this scenario is the fact that -cells express mitochondrial uncoupling protein 2 (UCP2) which dissipates the coupling between electron transport from ATP formation favouring O2- generation. Since O2- anion is a powerful activator of UCP2, a positive feedback mechanism exists in that O2- generation enhances its own formation. This is particularly critical under prolonged hyperglycaemia, where UCP2 activity may be extremely high thus further depressing insulin secretion by -cells (Newsholme et al., 2007). This situation is probably associated with the development of type 2 diabetes. Furthermore, the high-glucose, high fatty-acid environment created by either insulin-deficiency or insulinresistance favours the expression of NAD(P)H oxidase with consequently enhanced ROS

Type 1 diabetic patients exhibit major defects in antioxidant protection compared with healthy, non-diabetic controls. A significant reduction in total antioxidant status in both plasma and serum samples from these patients is typically observed (Maxwell et al., 1997). Diabetic children show significant reduction in GSH and GSPx in erythrocytes, as well as in plasma -tocopherol and -carotene levels (Dominguez et al., 1998). Incubation of rat (Rabinovitch et al., 1992) and human (Rabinovitch et al., 1996) islet cells with a cytotoxic combination of cytokines (IL-1, TNF and IFN) has been reported as an inducing factor for lipid peroxidation (also known as lipoperoxidation). When individually administered, however, the same cytokines have been shown to inhibit insulin release without any increase in lipid peroxidation or cytodestructive effects in rat islets (Sumoski et al., 1989). Taken together, these findings suggest that cytokine-induced inhibition of insulin release may not be oxygen free radical-mediated, whereas the cytodestructive effects of cytokines on -cells do appear to involve free radical-mediated events that induce the formation of toxic derivatives within the islets of Langerhans (Suarez-Pinz et al., 1996). This strongly suggests that type 1 cytokines interfere in -cell metabolism at some point that is intimately related to insulin secretion. But where does reside this extreme sensitivity of -cells to cytokine signals? The expression of iNOS, necessary for the synthesis of NO during insulin

production and -cell death (Morgan et al., 2007, Newsholme et al., 2009b).

specific and diverse pathways to induce -cell death.

secretion, may provide an explanation.

contribute to derangements in the secretion and action of insulin (Newsholme et al., 2009a). Hypertension associated with diabetes is related with a decrease in levels of L-arginine (Spinas, 1999), as are inflammatory conditions characterised by release of L-arginase by activated macrophages (Murphy & Newsholme, 1998). While excessive NO production can trigger oxidative/nitrosative stress and is undoubtedly a key mechanism that results in cell death (Newsholme et al., 2009a; Spinas, 1999; Michalska et al., 2010), good evidence now suggests that lesser amounts of cellular NO, produced by the NF-B-regulated inducible nitric oxide synthase (iNOS, EC 1.14.13.39), encoded by the *NOS-2* gene, serves as an important coupling factor in insulin secreting cells (Newsholme et al., 2009a; Spinas, 1999; Michalska et al., 2010). Recent data from the authors' laboratories has demonstrated that L-arginine is an important stimulator of -cell glucose consumption and intermediary metabolism (M.S. Krause, N.H. McClenaghan, P.R. Flatt, P.I. Homem de Bittencourt Jr., C. Murphy & P. Newsholme, unpublished results). Such actions lead to increased insulin secretion, enhanced antioxidant and protective responses with greater functional integrity when challenged with pro-inflammatory cytokines. Given that insulin-secreting cells have very low expression levels of antioxidant enzymes, such as catalase (CAT) and glutathione peroxidase (GSPx), -cells are particularly prone to chemical stress in the diabetogenic or inflammatory environment typical of type 1 and possibly type 2 diabetes (Newsholme et al., 2009a; Spinas, 1999). In fact, the pathogenesis of type 2 diabetes is now recognised to involve both innate and adaptive immunity, since type 2 diabetes is associated with low-grade systemic inflammation, infiltration of adipose tissue and pancreatic islets with CD8+ T lymphocytes that precede invasion by inflammatory macrophages and activation of these cells resulting in pro-inflammatory cytokine secretion (Mandrup-Poulsen, 2010).

In this chapter, we discuss how the continued supply of L-arginine, physiologically provided by the metabolism of L-glutamine via the intestinal-renal axis and from active skeletal muscle (which will be enhanced during exercise) is essential for -cell functional integrity and indeed for -cell defence, which will be required to avoid/attenuate islet inflammation associated with the pathogenic mechanisms underlying type 1 and type 2 diabetes (**Fig. 1B**). L-arginine is therefore preserved for essential NO generation and stimulation of glucose metabolism, critical for insulin secretion. Additionally, the role of skeletal muscle (during exercise) on these metabolic processes is discussed.

#### **2. Oxidative metabolism and oxidative stress in -cells and type 1 diabetes**

The intense aerobic metabolism, intrinsic to pancreatic -cells, exposes these cells to the deleterious effects of high-turnover oxygen-based reactions. In fact, during secretagoguestimulated insulin secretion, -cells are associated with accelerated mitochondrial flux of electrons and, consequently, elevated tendency towards reactive oxygen species (ROS) production (Newsholme et al., 2007). However and notably, -cells present a very low level of expression of antioxidant enzymes such as CAT and GSPx compared with other tissues and this reduced antioxidant activity is associated with significant increases in lipid hydroperoxides, conjugated dienes and protein carbonyls, which are markers for oxidative stress (Santini et al., 1997), so that -cells are intrinsically prone to oxidative stress.

Moreover, a growing body of evidence indicates that, in the pre-diabetic condition, antioxidant status may be impaired (Rocie et al., 1997). Hence, the low antioxidant defence in certain individuals (even if transiently) may predispose to an enhanced oxidative stress and the eventual -cell death that categorises the onset of type 1 and type 2 diabetes.

contribute to derangements in the secretion and action of insulin (Newsholme et al., 2009a). Hypertension associated with diabetes is related with a decrease in levels of L-arginine (Spinas, 1999), as are inflammatory conditions characterised by release of L-arginase by activated macrophages (Murphy & Newsholme, 1998). While excessive NO production can trigger oxidative/nitrosative stress and is undoubtedly a key mechanism that results in cell death (Newsholme et al., 2009a; Spinas, 1999; Michalska et al., 2010), good evidence now suggests that lesser amounts of cellular NO, produced by the NF-B-regulated inducible nitric oxide synthase (iNOS, EC 1.14.13.39), encoded by the *NOS-2* gene, serves as an important coupling factor in insulin secreting cells (Newsholme et al., 2009a; Spinas, 1999; Michalska et al., 2010). Recent data from the authors' laboratories has demonstrated that L-arginine is an important stimulator of -cell glucose consumption and intermediary metabolism (M.S. Krause, N.H. McClenaghan, P.R. Flatt, P.I. Homem de Bittencourt Jr., C. Murphy & P. Newsholme, unpublished results). Such actions lead to increased insulin secretion, enhanced antioxidant and protective responses with greater functional integrity when challenged with pro-inflammatory cytokines. Given that insulin-secreting cells have very low expression levels of antioxidant enzymes, such as catalase (CAT) and glutathione peroxidase (GSPx), -cells are particularly prone to chemical stress in the diabetogenic or inflammatory environment typical of type 1 and possibly type 2 diabetes (Newsholme et al., 2009a; Spinas, 1999). In fact, the pathogenesis of type 2 diabetes is now recognised to involve both innate and adaptive immunity, since type 2 diabetes is associated with low-grade systemic inflammation, infiltration of adipose tissue and pancreatic islets with CD8+ T lymphocytes that precede invasion by inflammatory macrophages and activation of these

cells resulting in pro-inflammatory cytokine secretion (Mandrup-Poulsen, 2010).

skeletal muscle (during exercise) on these metabolic processes is discussed.

In this chapter, we discuss how the continued supply of L-arginine, physiologically provided by the metabolism of L-glutamine via the intestinal-renal axis and from active skeletal muscle (which will be enhanced during exercise) is essential for -cell functional integrity and indeed for -cell defence, which will be required to avoid/attenuate islet inflammation associated with the pathogenic mechanisms underlying type 1 and type 2 diabetes (**Fig. 1B**). L-arginine is therefore preserved for essential NO generation and stimulation of glucose metabolism, critical for insulin secretion. Additionally, the role of

**2. Oxidative metabolism and oxidative stress in -cells and type 1 diabetes** 

stress (Santini et al., 1997), so that -cells are intrinsically prone to oxidative stress.

and the eventual -cell death that categorises the onset of type 1 and type 2 diabetes.

The intense aerobic metabolism, intrinsic to pancreatic -cells, exposes these cells to the deleterious effects of high-turnover oxygen-based reactions. In fact, during secretagoguestimulated insulin secretion, -cells are associated with accelerated mitochondrial flux of electrons and, consequently, elevated tendency towards reactive oxygen species (ROS) production (Newsholme et al., 2007). However and notably, -cells present a very low level of expression of antioxidant enzymes such as CAT and GSPx compared with other tissues and this reduced antioxidant activity is associated with significant increases in lipid hydroperoxides, conjugated dienes and protein carbonyls, which are markers for oxidative

Moreover, a growing body of evidence indicates that, in the pre-diabetic condition, antioxidant status may be impaired (Rocie et al., 1997). Hence, the low antioxidant defence in certain individuals (even if transiently) may predispose to an enhanced oxidative stress Oxidative stress has long been recognised to play an important role in the development of type 1 diabetes and its subsequent complications (Wierusz-Wysock et al., 1997) which are aggravated due to the low activities of oxygen free radical scavenging enzymes in islet cells, especially mitochondrial manganese-type superoxide dismutase (Mn-SOD; Asayama et al., 1986), glutathione peroxidase (GSPx; Malaisse et al., 1982; Mathews & Leiter, 1999) and glutathione disulphide (GSSG) reductase (GSRd; Mathews & Leiter, 1999). Also, the expression of mRNA encoding for several antioxidant enzymes, such as Mn-SOD, cytoplasmic copper-zinc type SOD (Cu/Zn-SOD), GSPx, and catalase (CAT), has been reported to be lower in islets of Langerhans compared with other mouse tissues (Lenzen et al., 1996). Additionally, the administration of antioxidants (nicotinamide, SOD, -tocopherol, probucol and the 21-aminosteroid lazaroids), as well as oxygen free radical scavengers, have been used in vitro to protect islets from the cytotoxic effects of some proinflammatory cytokines (IL-1, TNF and INF), concurrently providing *in vivo* protection against the development of the autoimmune diabetes process (Nomikos et al., 1986). Conversely, studies on MnSOD and CAT transgenics have shown that protection of islets from oxidative stress does not alter cytokine toxicity (Chen et al., 2005), which indicates that, although related to each other, oxidative stress and cytokine-induced islet toxicity may use specific and diverse pathways to induce -cell death.

An additional complication to this scenario is the fact that -cells express mitochondrial uncoupling protein 2 (UCP2) which dissipates the coupling between electron transport from ATP formation favouring O2- generation. Since O2- anion is a powerful activator of UCP2, a positive feedback mechanism exists in that O2- generation enhances its own formation. This is particularly critical under prolonged hyperglycaemia, where UCP2 activity may be extremely high thus further depressing insulin secretion by -cells (Newsholme et al., 2007). This situation is probably associated with the development of type 2 diabetes. Furthermore, the high-glucose, high fatty-acid environment created by either insulin-deficiency or insulinresistance favours the expression of NAD(P)H oxidase with consequently enhanced ROS production and -cell death (Morgan et al., 2007, Newsholme et al., 2009b).

Type 1 diabetic patients exhibit major defects in antioxidant protection compared with healthy, non-diabetic controls. A significant reduction in total antioxidant status in both plasma and serum samples from these patients is typically observed (Maxwell et al., 1997). Diabetic children show significant reduction in GSH and GSPx in erythrocytes, as well as in plasma -tocopherol and -carotene levels (Dominguez et al., 1998). Incubation of rat (Rabinovitch et al., 1992) and human (Rabinovitch et al., 1996) islet cells with a cytotoxic combination of cytokines (IL-1, TNF and IFN) has been reported as an inducing factor for lipid peroxidation (also known as lipoperoxidation). When individually administered, however, the same cytokines have been shown to inhibit insulin release without any increase in lipid peroxidation or cytodestructive effects in rat islets (Sumoski et al., 1989). Taken together, these findings suggest that cytokine-induced inhibition of insulin release may not be oxygen free radical-mediated, whereas the cytodestructive effects of cytokines on -cells do appear to involve free radical-mediated events that induce the formation of toxic derivatives within the islets of Langerhans (Suarez-Pinz et al., 1996). This strongly suggests that type 1 cytokines interfere in -cell metabolism at some point that is intimately related to insulin secretion. But where does reside this extreme sensitivity of -cells to cytokine signals? The expression of iNOS, necessary for the synthesis of NO during insulin secretion, may provide an explanation.

A Novel L-Arginine/L-Glutamine Coupling Hypothesis: Implications for Type 1 Diabetes 247

enzymic activities by the suppressors of cytokine signalling (SOCS) family. Upregulation of either SOCS-1 or SOCS-3 protects -cells against cytokine-induced cell death in vitro and in vivo (Karlsen et al., 2001; Flodstrom et al., 2003). SOCS-3 also protects insulin-producing cells against IL-1–mediated apoptosis via NF-B inhibition (Karlsen et al., 2004). Evidence indicates that the fate of -cells, after cytokine exposure, depends on the duration and

Besides its activation by cytokines, NF-B is also a potential target for reactive oxygen/nitrogen species (ROS/RNS). It is noteworthy that NF-B was the first redoxsensitive eukaryotic transcription factor shown to respond directly to oxidative stress in many types of cells (Dröge, 2002), while its activation leads to the expression of at least a hundred of inducible proteins directly involved in inflammation, such as cyclooxygenase-2 (COX-2), iNOS, TNF and IL-1 (Moynagh, 2005). Therefore, NF-B is, at the same time, both a target and an inducer of inflammation and inflammation-induced oxidative stress. In resting (unstimulated) cells, NF-B dimeric complexes are predominantly found in the cytosol where they are associated with members of the inhibitory IB family (Moynagh, 2005), so that NF-B gene products are entirely inducible proteins whose activation is dictated by specific stimuli that activate IB kinase (IKK) complexes. These stimuli include high intracellular GSSG levels and oxidative stress *per se* (Dröge, 2002). IKKs, in turn, phosphorylate IB proteins directing them to proteasome-mediated degradation, which sets

NF-B activation is responsible for both initiation and amplification of immune and inflammatory responses in all cells. Actually, NF-B activation is *sine qua non* for the control of immune and inflammatory responses (Baldwin, 1996; Nakamura et al., 1997; Winyard et al., 1997), and since inflammatory factors, such as pro-inflammatory cytokines, chemokines, adhesion molecules, colony-stimulating factors and inflammatory enzymes, are NF-Bdependent gene products, dysregulation or aberrant activation of NF-B could initiate inappropriate autoimmune and inflammatory responses. Conversely, inhibition of NF-B activation has been argued as a potential therapeutic approach in several immune and inflammatory-related diseases (Chen et al., 1999). This is why cyclopentenone prostaglandins (cp-PGs), which are powerful inhibitors of NF-B activation (Rossi et al., 2000), are now considered to be the physiological mediators of the "**resolution of inflammation**" (Piva et al., 2005), whereas cp-PG-based pharmacological approaches, *e.g.* LipoCardium technology, which is a liposome contained cp-PG-based formulation specifically directed towards atherosclerotic lesions in arterial walls (Homem de Bittencourt et al., 2007; Gutierrez et al., 2008) have proved to be powerful anti-atherosclerotic strategies

Finally, considering that all the known forms of inflammation finish with the formation of naturally-occurring anti-inflammatory agents (e.g. cp-PGs, IL-10), an important question remains as to how does -cell not resolve inflammation by triggering such responses? A fault in the expression of the anti-inflammatory heat shock proteins may give a clue to this

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,

severity of perturbation of key -cell gene networks.

NF-B dimers free to bind to DNA in the nucleus.

(Piva et al., 2005; Ianaro et al., 2003; Homem de Bittencourt Jr., 2007).

question.

**4. Heat shock protein pathways** 

NO has incontestably been shown to be a physiological regulator of insulin secretion in -cells, in an elegant experimental protocol designed by Prof. Anne Marie Salapatek's group in Canada and reported in a seminal paper (Smukler et al., 2002). They have also reported that endogenous NO production can be stimulated by glucose, and that this stimulation can be blocked by NOS inhibition, whereas scavenging of NO specifically blocks insulin release stimulated by physiological intracellular concentrations of NO-donors (2 mM), but has no effect on the release stimulated by elevated K+. It has also been reported that NO donation did not elicit a -cell intracellular Ca2+ ([Ca2+]i) response alone, but was able to potentiate a glucose-induced [Ca2+]i response. Since NO is a strong heme-reactant, it partially inhibits the mitochondrial respiratory chain by binding to cytochrome *c* and/or cytochrome oxidase. As a consequence, the mitochondrial membrane potential decreases and Ca2+ leaves the mitochondria. This is followed by restoration of the mitochondrial membrane potential and Ca2+ reuptake by mitochondria (Spinas, 1999). Therefore, overproduction of NO related to inflammatory stimuli may be related to cellular dysfunction but **not** normal levels of NO. As previously argued (Smukler et al., 2002), the precise level of NO is crucial in determining its resultant effect, with low levels being involved in physiological signalling and higher levels becoming cytotoxic (Moncada et al., 1991; Beck et al., 1999). Hence, the supraphysiological elevation of L-arginine, or the application of exogenous NO donors under the condition of already elevated NO, may result in excessive NO production, yielding cytotoxic effects (Smukler et al., 2002).

#### **3. Nuclear factor B-dependent L-arginine metabolism in -cells**

Pancreatic -cells have to constantly express NF-B-regulated iNOS in order to achieve appropriate amounts of NO produced from L-arginine. However, inflammatory cytokines, such as IL-1 and TNF-, activate NF-B in rodent and human islet cells (Eizirik & Mandrup-Poulsen, 2001). Contrarily, prevention of NF-B activation protects pancreatic -cells against cytokine-induced apoptosis (Giannoukakis et al., 2000; Heimberg et al., 2001). It is impressive that about 70 NF-B–dependent genes have been currently identified in cells, including genes encoding for various inflammatory cytokines and iNOS (Darville & Eizirik, 1998). Remarkably, the expression of *ca.* 50% of the -cell genes that may be modified after cytokine exposure is secondary to iNOS-mediated NO formation (Kutlu et al., 2003). It is of note that treatment of human, as well as rodent -cells with purified IL-1 alone is not sufficient to induce apoptosis, but if IL-1 is combined with interferon- (IFN), -cells undergo apoptosis after few days in culture (Eizirik & Mandrup-Pouls, 2001). This suggests that an intracellular IFN signal must synergise with IL-1 signalling pathways in order to trigger -cell apoptosis. IFN binds to cell surface receptors and activates the Janus tyrosine kinases JAK1 and JAK2. These kinases phosphorylate and activate their downstream transcription factor STAT-1 (for signal transducers and activators of transcription), which dimerises and translocates to the nucleus where binding to -activated sites on target genes occurs (Eizirik & Mandrup-Pouls, 2001). STAT-1 mediates the potentiating effect of IFN on IL-1-induced iNOS expression (Darville & Eizirik, 1998). Because excessive activation of JAK/STAT signalling may lead to cell death, STAT transcriptional activity is regulated by multiple negative feedback mechanisms. These include dephosphorylation of JAK and cytokine receptors by cytoplasmic protein-tyrosine phosphatases SHPs (for Src homology 2 domain phosphatases), and inhibition of JAK

NO has incontestably been shown to be a physiological regulator of insulin secretion in -cells, in an elegant experimental protocol designed by Prof. Anne Marie Salapatek's group in Canada and reported in a seminal paper (Smukler et al., 2002). They have also reported that endogenous NO production can be stimulated by glucose, and that this stimulation can be blocked by NOS inhibition, whereas scavenging of NO specifically blocks insulin release stimulated by physiological intracellular concentrations of NO-donors (2 mM), but has no effect on the release stimulated by elevated K+. It has also been reported that NO donation did not elicit a -cell intracellular Ca2+ ([Ca2+]i) response alone, but was able to potentiate a glucose-induced [Ca2+]i response. Since NO is a strong heme-reactant, it partially inhibits the mitochondrial respiratory chain by binding to cytochrome *c* and/or cytochrome oxidase. As a consequence, the mitochondrial membrane potential decreases and Ca2+ leaves the mitochondria. This is followed by restoration of the mitochondrial membrane potential and Ca2+ reuptake by mitochondria (Spinas, 1999). Therefore, overproduction of NO related to inflammatory stimuli may be related to cellular dysfunction but **not** normal levels of NO. As previously argued (Smukler et al., 2002), the precise level of NO is crucial in determining its resultant effect, with low levels being involved in physiological signalling and higher levels becoming cytotoxic (Moncada et al., 1991; Beck et al., 1999). Hence, the supraphysiological elevation of L-arginine, or the application of exogenous NO donors under the condition of already elevated NO, may result in excessive NO production, yielding cytotoxic effects

**3. Nuclear factor B-dependent L-arginine metabolism in -cells** 

Pancreatic -cells have to constantly express NF-B-regulated iNOS in order to achieve appropriate amounts of NO produced from L-arginine. However, inflammatory cytokines, such as IL-1 and TNF-, activate NF-B in rodent and human islet cells (Eizirik & Mandrup-Poulsen, 2001). Contrarily, prevention of NF-B activation protects pancreatic -cells against cytokine-induced apoptosis (Giannoukakis et al., 2000; Heimberg et al., 2001). It is impressive that about 70 NF-B–dependent genes have been currently identified in cells, including genes encoding for various inflammatory cytokines and iNOS (Darville & Eizirik, 1998). Remarkably, the expression of *ca.* 50% of the -cell genes that may be modified after cytokine exposure is secondary to iNOS-mediated NO formation (Kutlu et al., 2003). It is of note that treatment of human, as well as rodent -cells with purified IL-1 alone is not sufficient to induce apoptosis, but if IL-1 is combined with interferon- (IFN), -cells undergo apoptosis after few days in culture (Eizirik & Mandrup-Pouls, 2001). This suggests that an intracellular IFN signal must synergise with IL-1 signalling pathways in order to trigger -cell apoptosis. IFN binds to cell surface receptors and activates the Janus tyrosine kinases JAK1 and JAK2. These kinases phosphorylate and activate their downstream transcription factor STAT-1 (for signal transducers and activators of transcription), which dimerises and translocates to the nucleus where binding to -activated sites on target genes occurs (Eizirik & Mandrup-Pouls, 2001). STAT-1 mediates the potentiating effect of IFN on IL-1-induced iNOS expression (Darville & Eizirik, 1998). Because excessive activation of JAK/STAT signalling may lead to cell death, STAT transcriptional activity is regulated by multiple negative feedback mechanisms. These include dephosphorylation of JAK and cytokine receptors by cytoplasmic protein-tyrosine phosphatases SHPs (for Src homology 2 domain phosphatases), and inhibition of JAK

(Smukler et al., 2002).

enzymic activities by the suppressors of cytokine signalling (SOCS) family. Upregulation of either SOCS-1 or SOCS-3 protects -cells against cytokine-induced cell death in vitro and in vivo (Karlsen et al., 2001; Flodstrom et al., 2003). SOCS-3 also protects insulin-producing cells against IL-1–mediated apoptosis via NF-B inhibition (Karlsen et al., 2004). Evidence indicates that the fate of -cells, after cytokine exposure, depends on the duration and severity of perturbation of key -cell gene networks.

Besides its activation by cytokines, NF-B is also a potential target for reactive oxygen/nitrogen species (ROS/RNS). It is noteworthy that NF-B was the first redoxsensitive eukaryotic transcription factor shown to respond directly to oxidative stress in many types of cells (Dröge, 2002), while its activation leads to the expression of at least a hundred of inducible proteins directly involved in inflammation, such as cyclooxygenase-2 (COX-2), iNOS, TNF and IL-1 (Moynagh, 2005). Therefore, NF-B is, at the same time, both a target and an inducer of inflammation and inflammation-induced oxidative stress. In resting (unstimulated) cells, NF-B dimeric complexes are predominantly found in the cytosol where they are associated with members of the inhibitory IB family (Moynagh, 2005), so that NF-B gene products are entirely inducible proteins whose activation is dictated by specific stimuli that activate IB kinase (IKK) complexes. These stimuli include high intracellular GSSG levels and oxidative stress *per se* (Dröge, 2002). IKKs, in turn, phosphorylate IB proteins directing them to proteasome-mediated degradation, which sets NF-B dimers free to bind to DNA in the nucleus.

NF-B activation is responsible for both initiation and amplification of immune and inflammatory responses in all cells. Actually, NF-B activation is *sine qua non* for the control of immune and inflammatory responses (Baldwin, 1996; Nakamura et al., 1997; Winyard et al., 1997), and since inflammatory factors, such as pro-inflammatory cytokines, chemokines, adhesion molecules, colony-stimulating factors and inflammatory enzymes, are NF-Bdependent gene products, dysregulation or aberrant activation of NF-B could initiate inappropriate autoimmune and inflammatory responses. Conversely, inhibition of NF-B activation has been argued as a potential therapeutic approach in several immune and inflammatory-related diseases (Chen et al., 1999). This is why cyclopentenone prostaglandins (cp-PGs), which are powerful inhibitors of NF-B activation (Rossi et al., 2000), are now considered to be the physiological mediators of the "**resolution of inflammation**" (Piva et al., 2005), whereas cp-PG-based pharmacological approaches, *e.g.* LipoCardium technology, which is a liposome contained cp-PG-based formulation specifically directed towards atherosclerotic lesions in arterial walls (Homem de Bittencourt et al., 2007; Gutierrez et al., 2008) have proved to be powerful anti-atherosclerotic strategies (Piva et al., 2005; Ianaro et al., 2003; Homem de Bittencourt Jr., 2007).

Finally, considering that all the known forms of inflammation finish with the formation of naturally-occurring anti-inflammatory agents (e.g. cp-PGs, IL-10), an important question remains as to how does -cell not resolve inflammation by triggering such responses? A fault in the expression of the anti-inflammatory heat shock proteins may give a clue to this question.
