**6. Inflammatory mediators and suppression of β-cell function**

Since inflammation and β-cell death is common to both T1 and T2DM, it is reasonable to assume that shared inflammatory mediators may exist between the two conditions. It is these mediators that promote infiltration of immune cells, suppression of β-cell function, culminat‐ ing in reduced insulin exocytosis and increased β-cell apoptosis. These mediators can be loosely classified into four categories, cytokines, chemokines, ROS/RNS and other inflamma‐ tory products. However, it must be noted that the activity of these modulators can be heavily influenced by nutrient availability, such as in hyperglycaemia and dyslipidaemia conditions. Further to this, there is significant crossover between the molecules in these categories, and several can significantly impact on the others, indicating a complex role in both T1 and T2DM.

precise causes of inflammation in T1DM are a consequence of T-cell failure to respond to Treg, or whether defective or low Treg numbers are to blame for disease progression. Nonetheless, the interplay between these cell populations offers a potential therapeutic strategy for T1DM

Interestingly, an autoimmune element has also been reported in patients with T2DM, along with the accepted thesis of insulin-resistance [76,87]. Hyperglycaemia, dyslipidaemia and lowgrade inflammation (consisting of circulating inflammatory cytokines or adipokines released by adipocyte expansion), are considered important factors in the progression of T2DM and are generally present in obese individuals who are at risk of T2DM development [77]. These conditions lead to β-cell stress through a variety of processes that mainly include uncontrolled generation of ROS/RNS and cytokine-dependent initiation of death signals. Both processes combine to reduce β-cell function and decrease β-cell mass by inducing apoptotic cell death, leading to further hyperglycaemic and dyslipidaemic complications, and causing amplifica‐ tion of ROS/RNS generation, cytokine release and cytokine-mediated recruitment of the immune system (i.e. inflammation). These inflammatory factors are all detrimental for β-cell survival. As mentioned previously, IL-1β is elevated in the hyperglycaemic state, is increased in T1DM and is also expressed by β-cells in T2DM [77-79,88]. Moreover, concomitant downregulation of the receptor antagonist IL-1Ra was also observed in β-cells cultured in hyper‐ glycaemic conditions [76]. β-Cells are similar to immune cells and dysregulated expression of IL-1β in islets can cause auto-stimulation and subsequent release of IL-1β by other β-cells, via NFκB activation [76,88]. In addition, IL-1β can promote the local expression of other cytokines, for example IL-6 and IL-8. These cytokines aid in the recruitment of patrolling macrophages, which may subsequently become activated by high microenvironment levels of IL-1β and amplify IL-1β content in their own right [76]. In terms of islet inflammation, IL-1β expression and its effects on β-cell death appears to be a uniting factor, in both T1 and T2DM and is being

While inflammation is essential to maintain tissue homeostasis, it is also beneficial and allows repair of damaged organs. However, it is the presence of chronic, out of control and unchecked inflammatory factors that contribute to β-cell death and ensuing DM. Ultimately, increased local microenvironment cytokine production in islets is detrimental and understanding the mechanisms of cytokine release and regulation, and also suppression of β-cell function, will

Since inflammation and β-cell death is common to both T1 and T2DM, it is reasonable to assume that shared inflammatory mediators may exist between the two conditions. It is these mediators that promote infiltration of immune cells, suppression of β-cell function, culminat‐ ing in reduced insulin exocytosis and increased β-cell apoptosis. These mediators can be loosely classified into four categories, cytokines, chemokines, ROS/RNS and other inflamma‐ tory products. However, it must be noted that the activity of these modulators can be heavily

**6. Inflammatory mediators and suppression of β-cell function**

treatment [82].

140 Type 1 Diabetes

considered a possible therapeutic target [77,89].

allow the development of new treatment regimens.

T1DM is an autoimmune disease and it comes as no surprise that cytokine expression is elevated in these patients [79]. Interestingly, it is becoming more evident that cytokines also play a critical role in T2DM progression, and increased levels have been reported in T2DM patients [76,87]. The most obvious source of cytokine production is from islet invading immune cells, although other researchers have illustrated that islet β-cells could also express cytokines [76,79]. Cytokine and adipokine release also occurs from adipose tissue since it expands rapidly in obese patients. Here, hypoxia also plays a key part in cytokine release due to an inflamma‐ tory response to lack of vasculature in rapidly growing adipose tissue [90,91]. Recent evidence has suggested that adipocyte invading macrophages are a significant supplier of TNFα to the circulation in obese T2DM patients, and this could be a contributing-factor that modulates inflammation in disease progression [92,93]. It is likely that all sources contribute in some way or another to elevate cytokine levels, and consequently compound inflammation in DM patients.

The main cytokines that are responsible for inflammation in T1 and T2DM, include IL-1β, TNFα, INF-γ, IL-6 and IL-8. Central to the inflammatory role of each is stimulation of stressinduced kinases, IKKβ (inhibitor of nuclear factor kappaB kinase subunit β) and/or c-JNK (c-Jun-N-terminal kinase) [90]. Activation of IKKβ leads to translocation and activation of the NFκB. This factor targets transcription of genes associated with inflammation, and can cause subsequent up-regulation and release of IL-1β, TNFα, IL-6 and IL-8 [94,95]. Therefore, the aforementioned cytokines can initiate an auto-stimulatory or feed-forward inflammatory effect through NFκB-signalling in β-cells, resulting in amplification of inflammation. IL-1β and TNFα initiate NFκB-signalling directly via association with their relative receptors (IL-1R and TNFR) [96] and can also activate the apoptotic JNK pathway indirectly by intracellular interaction of TNF receptor associated factor (TRAF) with the cytoplasmic portion of IL-1R or TNFR [97]. NFκB can play either a pro-survival or pro-death role given the correct circum‐ stances [98]. Both NFκB and JNK are intrinsically connected, and NFκB can prevent JNKmediated cell death, the regulatory interactions of which have been reviewed expertly elsewhere [99]. An important component of NFκB activation and function is the presence/ absence of ROS/RNS. Therefore, ROS/RNS can influence NFκB-dependent cytokine expression and consequently immune response [98].

ROS/RNS can activate nuclear translocation of NFκB which promotes gene expression. However, ROS/RNS can also have an inhibitory effect when NFκB has already translocated to the nucleus [98]. The process by which ROS/RNS affects NFκB function is not entirely known but is believed to involve alteration of the NFκB catalytic site through interaction with cysteine residues, or by inhibiting specific kinase enzymes such as IκBα, that results in phosphorylation of NFκB [98]. Cellular ROS can be generated from Electron Transport Chain (ETC) respiratory complexes or from specific enzymes (e.g. NOX-mediated production for phagocytosis) [27,98]. Most notably, in hyperglycaemic/glucotoxic conditions (in T1 or T2DM), mitochondria are the major source of ROS/RNS primarily because of high oxidative phosphorylation and ATP production [100]. As a result of unavoidable oxidative chemistry and prolonged ETC activity, superoxide (O2 - ) anions can be formed and may "leak" from the mitochondria and elicit cellular damage [100]. Additionally, excess glucose can cause increased intracellular calcium, which may enhance mitochondrial O2 output, but also activate NOX-derived ROS via protein kinase C (PKC) [100]. High glucose can also induce NOX activity through NADPH production from the conversion of glucose-6-phosphate to pentose leading to increased O2 - [100]. Superoxide is a precursor reactive species and can be converted to other forms of strong oxidants including H2O2, and free radicals such as hydroxyl radicals and also peroxynitrite following reaction with NO [27,100]. These reactive species can cause DNA, lipid and protein damage and can also activate/regulate NFκB, who in turn can promote cytokine release and increased NO/O2 production by activation of iNOS and NOX expression [100,101]. Thus, ROS/RNS can exacer‐ bate the immunological response and lead to cell death in a cyclic fashion (Fig. 5).

Lipid- and adipose-derived factors are considered other inflammatory mediators. In T2DM patients, dyslipidaemia occurs along with hyperglycaemia and consequently vascular circulation and intracellular accumulation of lipids can have a profound effect on the inflam‐ matory response. Excess fatty acids can induced ROS generation through increased TCA metabolite production, increased NADH/NAD+ ratio and elevated intracellular Ca2+ [100]. They can also increase O2 - and NO production via activation of NOX and iNOS, respectively, all potentially activating the NFκB pathway [97,100,102]. Formation of ceramide from long chain fatty acids also contributes to precipitation of lipotoxicity in β-cells and results in ROS generation and apoptotic death [97,100]. Ceramide, synthesised by serine palmitoyltransferase from long chain fatty acids like palmitic acid [100], is capable of inhibiting the pro-survival PI3K pathway, activating caspase-9 [100]. Like other fatty acids, ceramide can associate with and activate TLR's, which may elicit an immune response [90].

Since adipose tissue expands in obese patients, increased adipose-derived factors have been detected in patient serum, including leptin, TNFα and IL-6. Leptin and adiponectin can play a role in the immune reaction in DM patients. Leptin, an appetite control endocrine factor, inhibits feeding by interaction with receptors in the hypothalamus and a subsequent stimu‐ lation of neurotransmitter release, for example norepinephrine [103]. It is considered a cytokine due to its homology in structure with IL-6, and its receptor-mediated effects [77,103,104]. It has been shown to induce β-cell death by up-regulating IL-1β, and has also been implicated in exacerbation of T1DM in animal models [77,105]. Conversely, adiponectin is considered an anti-inflammatory protein, and enhances IL-1Ra and IL-10 expression [90,106], leading to reduced IL-1β and enhanced suppression of T-cell mediated inflammation.

Chemokines can also be secreted from adipose tissue and are elevated in the adipose tissue of obese mice and humans [90,107]. CC-chemokine ligand-2 (CCL-2) functions to recruit mono‐ cytes toadipose tissue resultingindifferentiationintoactivatedmacrophages [108].Others such as CCL-3, CCL-6, CCL-7, CCL-8 and CCL-9, have also been reported to be elevated in high-fat fed mice, suggesting they may play a role in immune cell recruitment and inflammation [90].

**Figure 5.** Illustration depicting the potential convergence points of the immunological NFκB and the metabolic ROS/ER stress pathways in pancreatic β-cells. Islet inflammation is characterised by the presence of leukocyte infil‐ trates that mediate the destruction of β-cells by release of cytokines, generation of ROS (NO, cytokine-NFκB-depend‐ ent]) and by activating the granzyme b- and death-receptor-mediated death pathways. Also shown is the effect of excess glucose on ROS production and ER stress that ultimately activates caspase enzymes via mitochondrial- and ERmediated death pathways. ROS/RNS can also activate and regulate the NFκB stress pathway, which may lead to ex‐

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pression of cytokines that promotes immune cell infiltration exacerbating β-cell death.

Several mechanisms and modulators may contribute to the inflammatory response observed in T1 and T2DM. Cytokines, ROS and NFκB-signalling appear to be critical in mediating immune cell infiltration and further cytokine production. The balance between β-cell survival

The Impact of Inflammation on Pancreatic β-Cell Metabolism, Function and Failure in T1DM and T2DM… http://dx.doi.org/10.5772/55349 143

production [100]. As a result of unavoidable oxidative chemistry and prolonged ETC activity,

damage [100]. Additionally, excess glucose can cause increased intracellular calcium, which

C (PKC) [100]. High glucose can also induce NOX activity through NADPH production from

is a precursor reactive species and can be converted to other forms of strong oxidants including H2O2, and free radicals such as hydroxyl radicals and also peroxynitrite following reaction with NO [27,100]. These reactive species can cause DNA, lipid and protein damage and can also activate/regulate NFκB, who in turn can promote cytokine release and increased NO/O2

production by activation of iNOS and NOX expression [100,101]. Thus, ROS/RNS can exacer‐

Lipid- and adipose-derived factors are considered other inflammatory mediators. In T2DM patients, dyslipidaemia occurs along with hyperglycaemia and consequently vascular circulation and intracellular accumulation of lipids can have a profound effect on the inflam‐ matory response. Excess fatty acids can induced ROS generation through increased TCA metabolite production, increased NADH/NAD+ ratio and elevated intracellular Ca2+ [100].

all potentially activating the NFκB pathway [97,100,102]. Formation of ceramide from long chain fatty acids also contributes to precipitation of lipotoxicity in β-cells and results in ROS generation and apoptotic death [97,100]. Ceramide, synthesised by serine palmitoyltransferase from long chain fatty acids like palmitic acid [100], is capable of inhibiting the pro-survival PI3K pathway, activating caspase-9 [100]. Like other fatty acids, ceramide can associate with

Since adipose tissue expands in obese patients, increased adipose-derived factors have been detected in patient serum, including leptin, TNFα and IL-6. Leptin and adiponectin can play a role in the immune reaction in DM patients. Leptin, an appetite control endocrine factor, inhibits feeding by interaction with receptors in the hypothalamus and a subsequent stimu‐ lation of neurotransmitter release, for example norepinephrine [103]. It is considered a cytokine due to its homology in structure with IL-6, and its receptor-mediated effects [77,103,104]. It has been shown to induce β-cell death by up-regulating IL-1β, and has also been implicated in exacerbation of T1DM in animal models [77,105]. Conversely, adiponectin is considered an anti-inflammatory protein, and enhances IL-1Ra and IL-10 expression [90,106], leading to

Chemokines can also be secreted from adipose tissue and are elevated in the adipose tissue of obese mice and humans [90,107]. CC-chemokine ligand-2 (CCL-2) functions to recruit mono‐ cytes toadipose tissue resultingindifferentiationintoactivatedmacrophages [108].Others such as CCL-3, CCL-6, CCL-7, CCL-8 and CCL-9, have also been reported to be elevated in high-fat fed mice, suggesting they may play a role in immune cell recruitment and inflammation [90]. Several mechanisms and modulators may contribute to the inflammatory response observed in T1 and T2DM. Cytokines, ROS and NFκB-signalling appear to be critical in mediating immune cell infiltration and further cytokine production. The balance between β-cell survival

bate the immunological response and lead to cell death in a cyclic fashion (Fig. 5).


and activate TLR's, which may elicit an immune response [90].

reduced IL-1β and enhanced suppression of T-cell mediated inflammation.

the conversion of glucose-6-phosphate to pentose leading to increased O2

) anions can be formed and may "leak" from the mitochondria and elicit cellular

output, but also activate NOX-derived ROS via protein kinase




superoxide (O2

142 Type 1 Diabetes


may enhance mitochondrial O2

They can also increase O2

**Figure 5.** Illustration depicting the potential convergence points of the immunological NFκB and the metabolic ROS/ER stress pathways in pancreatic β-cells. Islet inflammation is characterised by the presence of leukocyte infil‐ trates that mediate the destruction of β-cells by release of cytokines, generation of ROS (NO, cytokine-NFκB-depend‐ ent]) and by activating the granzyme b- and death-receptor-mediated death pathways. Also shown is the effect of excess glucose on ROS production and ER stress that ultimately activates caspase enzymes via mitochondrial- and ERmediated death pathways. ROS/RNS can also activate and regulate the NFκB stress pathway, which may lead to ex‐ pression of cytokines that promotes immune cell infiltration exacerbating β-cell death.

and death is dependent on the interactions of these mediators, but also on the glycaemic and lipidaemic environment. We will now discuss the precise mechanisms of β-cell death in T1 and T2DM, and examine the commonalities between both.

B is released into the cytosol and activates caspase-3 [113,117]. Interestingly, in order to yield activation of caspase-3, both caspase-8 from the death-receptor pathway outlined above, and granzyme B converge and initiate the mitochondrial-mediated death pathway via cleavage of BID [a member of the B-cell lymphoma-2 (Bcl-2) family of proteins], to truncated BID (tBID). In this process, cytosolic tBID translocates to the mitochondrial membrane and activates other Bcl-2-related proteins, such as BAX. Release of cytochrome *c* is then stimulated, which acts as the trigger for mitochondrial-mediated activation of caspase -9 and -3 [118,119,120]. Therefore, both the death-receptor and granzyme B-mediated death pathways activate the mitochondrial-

The Impact of Inflammation on Pancreatic β-Cell Metabolism, Function and Failure in T1DM and T2DM…

Conversely, macrophages induce β-cell death through production of ROS, cytokines and

organisms or possibly damaged β-cells. Expression of high amounts of ROS/RNS or reduced antioxidant defences, results in mitochondrial dysfunction, which can culminate in mitochon‐ drial-mediated apoptosis. Briefly, this involves major structural changes caused by ROS/RNSmediated lipid/protein oxidation on both the inner and outer mitochondrial membranes, thus increasing the membrane permeability to proteins [121]. This is regulated by the interaction of pro- and anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-XL, BAX, BAK, BIM, BID and BAD) [122]. The release of cytochrome *c* to the cytosol and its association with apoptosis protease activation factor-1 (Apaf-1) and pro-caspase-9, forms a heptameric wheel-like caspaseactivating complex, known as the apoptosome, which subsequently leads to activation of caspase-9 and effector caspase-3, further down-stream [123]. Caspase activation promotes cell

In addition, immune cells release cytokines (e.g. TNFα, INF-γ and IL-1β) that also promote up-regulation of ROS/RNS via activation of NFκB (e.g. NO), who in turn can be regulated by ROS [100,101]. Induction of NO expression can cause activation of tumour suppressor protein (p53) leading to inhibition of cell cycle and death [109]. Cytokines can also inflict cell death via stimulation of the JNK pathway [97]. Here, IL-1β and TNFα activate mitochondrial translo‐ cation of JNK, who is a regulator of Bcl-2 proteins. JNK phosphorylates BIM, which results in the release of BAX-dependent cytochrome *c* and initiation of mitochondrial-mediated apop‐ tosis [125,126]. Additionally, release of INF-γ by T-cells, can also phosphorylate BIM in β-cells,

A variety of biochemical signalling pathways are available by which autoimmune cells utilise to initiate β-cell destruction. Consequently, due to a complete lack of insulin secretion and subsequent diminished glucose-uptake by muscle and adipose tissue, hyperglycaemia ensues in T1DM patients. High levels of blood glucose leads to further complications including, glucotoxicity, lipotoxicity and glucolipotoxicity and these are key players in exacerbation of the disease, and can lead to the clinical complications of T2DM [108]. Therefore, the precise way in which these factors affect β-cell turnover and survival will be discussed in the next section. Nonetheless, β-cell death in T1DM is based on classical immune-related death processes, but also relies on involvement of ROS and mitochondrial mediated which may occur


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eventually phagocytosis. Macrophages express high levels of NOX, and use O2

death by degradation of DNA and cytoskeletal proteins [124].

promoting cell death in a similar manner [77,86].

in a sub-population of beta cells in T2DM.

mediated death pathway.
