**7. Pancreatic β-cell failure and death in T1DM**

Pancreatic β-cell failure can be defined as a reduction in insulin secretion or a failure to respond to plasma glucose (i.e. insulin-resistance). β-cell dysfunction in T1DM is characterised by an autoimmune-mediated destruction of β-cells leading to a decrease in pancreatic β-cell mass and reduced insulin secretion [109]. On the other hand, progression of T2DM is more variable and β-cell death occurs against the backdrop of insulin-resistance [109]. Here, the pathogenesis of T2DM usually involves a response to increased metabolic load by increased β-cell mass and enhanced insulin secretion [110]. A period of normoglycaemia ensues before a reduction in insulin secretion and β-cell function is observed. Finally, this phase is followed by a decrease in β-cell mass due to apoptotic cell death and is referred to as overt diabetes [110,111]. The shared feature associated with both T1 and T2DM is the failure of pancreatic β-cells resulting in reduced cell mass, dysfunction and ultimately apoptotic death. While there are common‐ alities associated with both types, the main mechanism of cell death associated with T1DM is immune-related.

At the time of diagnosis, T1DM patients present with a 70-80% reduction β-cell mass [109]. Insulitis and infiltration of leukocytes into islets is common in these patients. Several types of leukocytes are present including B-cells, macrophages and Natural Killer (NK) cells, but the principal invading cell type is the cytotoxic T-cell (CD4 and CD8) [81,82]. Immune cells promote β-cell death via several mechanisms, and these can be simplified to include phago‐ cytosis, production of cytokines and T-cell-induced initiation of death-receptor-mediated apoptosis. The intracellular generation of ROS/RNS and activation of caspase enzymes occurs inside target cells and ultimately seals the fate of these cells.

Generation of autoreactive effector T-cells is extremely important in the pathogenesis of T1DM, but the precise biochemical mechanisms behind release of self-antigen and development of autoreactive T-cells remains unknown. However, work in this field has identified a potential role for decreased expression of peripheral tissue antigens (PTA) in pancreatic draining lymph nodes, which possibly allows unchallenged escape of differentiated autoreactive T-cells [82,83,112]. These T-cells kill pancreatic β-cells through expression of Fas ligand and expres‐ sion of extracellular cytotoxic factors including cytokines, and lytic granules containing granzyme B and perforin [113]. In death-receptor-mediated apoptosis, Fas ligand or TNFα initiates death signals through association with FasR (Fas receptor) or TNFR (tumour necrosis factor receptor). An intracellular conformational change occurs that results in activation of caspase-8 [114,115]. This in turn serves to activate caspase-3 downstream promoting the apoptotic cascade [116].

T-cell-mediated release of granzyme B and perforin also leads to caspase activation in target cells. Here, perforin creates pores in the plasma membrane of the target cell, while granzyme 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 mitochondrialmediated death pathway.

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

Pancreatic β-cell failure can be defined as a reduction in insulin secretion or a failure to respond to plasma glucose (i.e. insulin-resistance). β-cell dysfunction in T1DM is characterised by an autoimmune-mediated destruction of β-cells leading to a decrease in pancreatic β-cell mass and reduced insulin secretion [109]. On the other hand, progression of T2DM is more variable and β-cell death occurs against the backdrop of insulin-resistance [109]. Here, the pathogenesis of T2DM usually involves a response to increased metabolic load by increased β-cell mass and enhanced insulin secretion [110]. A period of normoglycaemia ensues before a reduction in insulin secretion and β-cell function is observed. Finally, this phase is followed by a decrease in β-cell mass due to apoptotic cell death and is referred to as overt diabetes [110,111]. The shared feature associated with both T1 and T2DM is the failure of pancreatic β-cells resulting in reduced cell mass, dysfunction and ultimately apoptotic death. While there are common‐ alities associated with both types, the main mechanism of cell death associated with T1DM is

At the time of diagnosis, T1DM patients present with a 70-80% reduction β-cell mass [109]. Insulitis and infiltration of leukocytes into islets is common in these patients. Several types of leukocytes are present including B-cells, macrophages and Natural Killer (NK) cells, but the principal invading cell type is the cytotoxic T-cell (CD4 and CD8) [81,82]. Immune cells promote β-cell death via several mechanisms, and these can be simplified to include phago‐ cytosis, production of cytokines and T-cell-induced initiation of death-receptor-mediated apoptosis. The intracellular generation of ROS/RNS and activation of caspase enzymes occurs

Generation of autoreactive effector T-cells is extremely important in the pathogenesis of T1DM, but the precise biochemical mechanisms behind release of self-antigen and development of autoreactive T-cells remains unknown. However, work in this field has identified a potential role for decreased expression of peripheral tissue antigens (PTA) in pancreatic draining lymph nodes, which possibly allows unchallenged escape of differentiated autoreactive T-cells [82,83,112]. These T-cells kill pancreatic β-cells through expression of Fas ligand and expres‐ sion of extracellular cytotoxic factors including cytokines, and lytic granules containing granzyme B and perforin [113]. In death-receptor-mediated apoptosis, Fas ligand or TNFα initiates death signals through association with FasR (Fas receptor) or TNFR (tumour necrosis factor receptor). An intracellular conformational change occurs that results in activation of caspase-8 [114,115]. This in turn serves to activate caspase-3 downstream promoting the

T-cell-mediated release of granzyme B and perforin also leads to caspase activation in target cells. Here, perforin creates pores in the plasma membrane of the target cell, while granzyme

and T2DM, and examine the commonalities between both.

**7. Pancreatic β-cell failure and death in T1DM**

inside target cells and ultimately seals the fate of these cells.

immune-related.

144 Type 1 Diabetes

apoptotic cascade [116].

Conversely, macrophages induce β-cell death through production of ROS, cytokines and eventually phagocytosis. Macrophages express high levels of NOX, and use O2 - to kill invading 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 death by degradation of DNA and cytoskeletal proteins [124].

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

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 in a sub-population of beta cells in T2DM.
