apoptosis through induction of oxidative stress and bioenergetic decline marked by attenuation of intracellular ATP132,133 and NAD+ 134,135,136 levels, massive increase in NO,74 and elevation of cytoplasmic free Ca2+ .137 In certain settings, mitochondria dysfunction associated with these insults may also manifest in beta cell necrosis. NO is the major source of oxidative stress in beta cells, to which they are especially sensitive as a result of their low levels of antioxidant defense mechanisms.138,139,140 Although small amounts of NO are protective,141,142 supraphysiologic levels of this reactive oxygen species become cytotoxic during the course of T1D progression. Indeed, prevention of oxidative damage maintains beta cell viability in the presence of inflammatory cytokines in vitro133,143,144 and in experimental mod-

els of T1D in vivo.145,146,147,148,149,150,151,152,153

In addition to macrophage production of NO, beta cells synthesize their own NO on cytokine-induced upregulation of inducible nitric oxide synthase (iNOS).74 Accordingly, beta cells deficient in iNOS are more resistant to apoptosis induced by inflammatory cytokines,154 and inhibition of iNOS function in vivo either through genetic approaches or pharmacological inhibitors is protective in preclinical models of T1D.145,155 Multiple intracellular targets of NO compromise both insulin secretion and viability of beta cells. NO-induced DNA damage activates poly (ADP-ribose) polymerase (PARP), a nuclear enzyme that is activated in response to DNA strand breaks, which in the process of DNA repair, consumes NAD+ and thereby depletes beta cell ATP levels.156 Interestingly, toxins that cause T1D-like disease by destroying beta cell mass, such as streptozotocin and alloxan, are known to specifically deplete beta cells from their ATP and NAD+ reservoirs.157 PARP-deficient mice are resistant to T1D,158,159,160 and PARP inhibitors are being pursued in antidiabetes strategies.156 NO can also interfere with mitochondrial function at multiple levels, including inactivation of the mitochondrial TCA cycle enzyme aconitase, leading to diminished glucose oxidation and ATP synthesis161,162 and induction of mutations in mitochondrial genes such as components of respiratory chain complexes.163

3.3. Mechanisms of beta cell death in type 2 diabetes

Increase in beta cell mass and function normally compensates for insulin resistance. Although obesity is associated with insulin resistance, not all obese individuals are diabetic as a result of sufficient beta cell compensation both at the level of function (insulin secretion) and beta cell mass expansion.33,34,38 In lean or obese

individuals, insulin resistance progresses to T2D on failure of beta cell mass expansion that may in turn be further exasperated by genetic and/or environmental factors.164,165,166 Beta cell failure is believed to occur early during the course of disease progression.167 Thus T2D is marked by abnormalities in both insulin production and action. Although subject to some controversies in the past, the significance of beta cell demise in pathophysiology of T2D has increasingly gained support from analysis of human autopsies and rodent models of the disease.10,35,37 Indeed, assessment of a large number of pancreatic biopsies obtained from T2D subjects compared with lean and obese counterparts revealed 41% and 63% reduction in beta cell mass in lean and obese T2D individuals, respectively.35 Remarkably, comparison of beta cell apoptotic, replicative, and neogenic rates indicated significant increase in apoptosis as the underlying mechanism of beta cell loss. Pathways implicated in beta cell apoptosis include glucolipotoxicity, oxidative stress, inflammation, and ER stress. Glucolipotoxicity, inflammation, and possibly ER stress may be shared apoptotic mechanisms in both disease subtypes, with differential predominance.

3.3.1. Glucolipitoxicity

Although elevated lipids signal beta cell mass expansion as an adaptive response to insulin demand (lipoadaptation),168 chronic exposure to free fatty acids in the presence of elevated glucose levels leads to the progressive impairment of insulin secretory response169 and eventually culminates in apoptotic demise of beta cells.170,171,172,173,174 This paradigm of beta cell damage, also known as glucolipotoxicity, is believed to be associated with altered metabolism or “partitioning” of lipids to long-chain fatty acyl-CoA (LC-CoA) esters in lieu of their detoxification via mitochondrial oxidation.175 LCCoAs are the activated form of fatty acids that normally serve as substrates for carnitine palmitoyl transferase- 1 (CPT-1) and undergo beta oxidation in mitochondria. However, under hyperglycemic conditions, these activated fatty acid esters accumulate in the cytoplasm and exert cytotoxic effects. The following sections highlight the molecular mechanisms underlying the shift in the metabolic fate of fatty acids and associated beta cell damage in T2D.

Hyperglycemia is marked by exaggerated glucose flux through mitochondria and diversion of glucose-derived carbons from the TCA cycle (cataplerosis) to the cytoplasm in the form of intermediates such as citrate.176,177 Citrate accumulation leads to inhibition of beta oxidation by giving rise to malonyl CoA, an inhibitor of

CPT-1178,179 (Figure 19-2). In addition to malonyl CoA production, chronic elevation of glucose metabolism in this setting negatively regulates fatty acid oxidation by decreasing the cellular adenosine monophosphate (AMP)/ATP ratio and dampening AMP-activated protein kinase (AMPK), a cellular energy sensor that activates key enzymes necessary for mitochondrial metabolism of fatty acids.180 Inhibition of fatty acid oxidation in turn leads to their accumulation in the cytosol and redirects their metabolism to esterification and ceramide production.41,174 The importance of this shift in lipid partitioning or channeling in lipotoxicity-induced death is corroborated by the protective effect of pharmacologic or genetic maneuvers that activate AMPK and CPT-1 or inhibit LC-CoA synthesis.170,181,182,183

Several mechanisms have been implicated in death induced by glucolipotoxicity, including the effect of

fatty acids on the anionic phospholipid cardiolipin (CL), increased ceramide synthesis, and ROS production. Interestingly, these cytotoxic effects are only associated with saturated fatty acids.170,172 Monounsaturated fatty acids are metabolized to triglycerides and do not produce ceramide.171,184,185 Because cardiolipin is necessary for the attachment of cytochrome c to the inner mitochondrial membrane186 and the assembly of higher order complexes of respiratory chains,187 elevated fatty acids interfere with mitochondrial function and further facilitate cytochrome c egress from mitochondria in response to apoptotic stimuli.188,189 Consequently, impairment of CL synthesis in the presence of elevated fatty acids sensitizes beta cells to apoptosis.185,190,191

Long-chain fatty acyl CoAs also serve as precursors for de novo synthesis of the lipid messenger ceramide.171,192 Apoptosis associated with glucolipotoxicity can be

blocked by inhibition of ceramide synthesis or overexpression of BCL-2.41,174,181 Notably, direct association of CPT-1 and BCL-2 was recently reported.193 Whether the protective role of BCL-2 in glucolipotoxicity may be linked to its potential capacity to regulate fatty acid metabolism through CPT-1 is an intriguing thesis that remains to be experimentally tested. The proapoptotic effects of ceramide have been attributed to multiple downstream targets, including BAX conformational change,194,195 BID cleavage,196 downregulation of PI3 kinase activity,197,198 BAD dephosphorylation,198,199 and transcriptional induction of BNIP3.200 However, the requirement of these proapoptotic BCL-2 proteins in glucolipotoxicity-induced apoptosis awaits loss-of- function studies. Interestingly, a recent report indicated that necrosis is an alternate form of death under glucolipotoxic conditions if execution of apoptosis is blocked by caspase inhibition.170

The proapoptotic consequence of malonyl CoA and LC-CoA elevation in the context of glucolipotoxicity is intriguing because these same signals are transiently elevated under normal physiologic conditions of stimulatory nutrient/fuel concentration, serving as metabolic coupling factors and amplifying signals, respectively, to couple glucose metabolism and insulin exocytosis.201,202 Thus nutrient overload as in glucolipotoxicity appears to render a metabolic signaling pathway, which is normally operative during physiologic control of insulin secretion, into a proapoptotic signal.

3.3.2. Endoplasmic reticulum stress

The ER in beta cells has evolved to efficiently handle synthesis, folding, processing, and export of large amounts of newly synthesized insulin, endowing beta cells with a high secretory capacity. Unfolded protein response (UPR) is an adaptive quality-control mechanism executed by the ER that ensures proper refolding of misfolded proteins or degradation of those that are not correctly folded or processed. Beta cells are especially sensitive to UPR because they heavily rely on ER and Ca2+ for proper protein folding and secretion of insulin granules.203,204 In beta cells, UPR can be induced by toxic oligomers of islet amyloid polypeptide (IAPP, also known as amylin), glucolipotoxicity, oxidative stress, cytokines, hypoxia, and reduced protein glycosylation.205 Prolonged UPR transforms into a proapoptotic stress response (ER stress) when the homeostatic folding of newly synthesized proteins is not achieved. ER stress can also be associated with genetic/environmental factors and aberrations in Ca2+ homeostasis that compromise the proper function of ER.205 Insulin resistance can

lead to ER stress in the beta cell as a state in which the demand for insulin secretion and the risk of ER overload simultaneously increase. Furthermore, because beta cell mass is progressively lost in both type 1 and type 2 diabetes, the remaining beta cells become prone to ER stress because of ever-increasing insulin demand. The following sections focus on the inducers of ER stress in diabetic beta cells, especially the pathophysiology of IAPP and the underlying apoptotic mechanisms.

IAPP is a 37–amino acid peptide processed and cosecreted with insulin206 that may carry physiologic roles in control of food intake207 and paracrine inhibition of insulin secretion by beta cells.208,209 IAPP has a high capacity to form insoluble toxic oligomers210 and constitutes a major inducer of ER stress in beta cells. During progression of T2D, with the increase in insulin demand, beta cells synthesize more IAPP to cosecrete with insulin. However, the increase in IAPP expression is much higher than insulin in this case,211 and the capacity of the ER to properly execute protein folding is eventually saturated. Consequently, toxic oligomers of IAPP accumulate leading to beta cell degeneration.212,213,214 Remarkably, IAPP oligomers exhibit similar three-dimensional structure to that of amyloid Aβ peptide in Alzheimer’s disease, α-synclein in Parkinson’s disease, polyglutamine in Huntington’s disease, and prions, despite amino acid sequence differences.215,216 Indeed, an antibody raised against Aβ oligomers recognizes IAPP oligomers.215 Thus IAPP-associated beta cell loss in T2D may share common pathophysiology with neuronal loss induced by amyloidogenic proteins in neurodegenerative disorders; notably, the contribution of ER stress-induced apoptosis to disease progression.53,217,218 Transgenic expression of human IAPP (hIAPP) in mice or rats is associated with elevated beta cell apoptosis, decreased beta cell mass, and hyperglycemia in a gene dosage-dependent manner.212,219,220 Furthermore, IAPP oligomers221,222 and ER stress markers can be selectively detected in pancreatic biopsies from T2D individuals compared with control

samples.223,224,225

The sensors and effector pathways in charge of executing UPR and ER-stress associated apoptosis have been recently reviewed.205,226 Briefly, three protein sensors, PERK (protein kinase-like ER kinase), ATF6 (activating transcription factor 6), and IRE1 (inositol requiring 1) are triggered in response to unfolded proteins and activate an adaptive program that reduces production of new client proteins for the ER folding machinery, helps refold misfolded proteins, and degrades protein aggregates. UPR initiates with PERK, which, on activation, phosphorylates the translation initiation factor eIF2α, leading to inhibition of general protein

translation and selective increase in ATF-4 translation (Figure 19-3a). The transcription factor ATF-4 in turn increases expression of select chaperones and antioxidant defense genes. Another UPR sensor, IRE1, is activated by dimerization and transphosphorylation, leading to stimulation of its inherent endoribonuclease activity and processing of mRNA encoding the transcription factor XBP-1 (X-box binding protein-1). XBP- 1, together with ATF-6, regulates transcription of additional genes required for UPR, including chaperones, ER-associated degradation (ERAD) components, and autophagy genes (Figure 19-3a). Increased ERAD components and autophagy help clear unfolded protein, protein aggregates, and damaged organelles.227 Accumulation of unfolded proteins also leads to the release of ER Ca2+, which activates a signal transduction program mediated by Ca2+/calmodulin-dependent kinase kinase-β, leading to enhanced autophagy228,229 (Figure 19-3a). If the integrated outcome of these signaling pathways does not resolve the ER load of unfolded and aggregated proteins, then these same sensors can engage the intrinsic pathway of apoptosis.230 Consistent with the notion that UPR is primarily an adaptive mechanism evolved to preserve cellular survival, ablation of Perk in beta cells is associated with significantly higher sensitivity to apoptosis on stress associated with unfolded proteins and nutrient overload.231,232,233 Consistent with these findings, interference with eIF1α phosphorylation downstream of PERK is also associated with loss of beta cell mass.234 Importantly, polymorphisms in several genes associated with UPR and ER stress, such as PERK,235,236,237,238 ATF 6,239,240 and IAPP,241 are associated with diabetes in humans.

Apoptotic pathways downstream of ER stress are under active investigation and involve both transcriptional and post-translational mechanisms (Figure 19-3b). p53 and C/EBP homologous protein (CHOP)/ growth arrest and DNA damage induced gene-153 (GADD153), a transcription factor induced by ATF4, initiate an ER stress-associated transcription program that is marked by changes in expression levels of several BCL-2 family members, including downregulation of BCL-2242 and upregulation of BIM,243 NOXA, and p53-upregulated modulator of apoptosis (PUMA).244,245 Furthermore, CHOP has been implicated in increased expression of death receptors such as FAS and DR5246,247 and attenuation of AKT survival pathway through augmented expression of its inhibitor TRB3.248 Loss of CHOP protects beta cells from ER stress induced by NO or IAPP and associated diabetes.223,249,250 Downstream of IRE1, TRAF-2 modulates the apoptotic response to ER stress by multiple mechanisms, such as

activation of ER-linked caspases251 (Figure 19-3b). Alternatively, TRAF-2 is recruited to IRE1252 and mediates c-Jun N-terminal kinase (JNK) activation through apoptosis signal-regulating kinase (ASK-1).253 JNK-1 phosphorylation of BCL-2 inhibits its survival function.254 Beyond the transcriptional and post-translational mechanisms that impinge on BCL-2 family members on ER stress-induced apoptosis, select members of this family can functionally interact with the ER by regulating Ca2+ homeostasis255 and IRE1 activation.256

4. BETA CELL APOPTOSIS AND ISLET

TRANSPLANTATION THERAPY

Because loss of functional beta cell mass is central to the etiology of diabetes, beta cell transplantation is being actively pursued as a possible therapeutic approach.257,258 The success of transplantation therapy, however, has been limited because of an insufficient source of insulin-producing tissue available for transplantation and the loss of islet viability during isolation or expansion and immediately after transplantation. These therapeutic challenges have spurred active search for strategies to enhance beta cell viability and improve “engraftment” of transplanted islets.

Islet viability during isolation or expansion and shortly after transplantation is compromised by hypoxia as a result of loss of the normal vascularized islet microenvironment. On revascularization, islets undergo further oxidative stress.259,260 In addition to this metabolic stress, islets are subject to immunemediated damage. In response to tissue trauma during surgery and ischemic reperfusion, donor islets produce chemokines that activate an innate immune response in the host marked by release of inflammatory cytokines such as TNF-α, IL-1β, and IFN-γ,261 compromising the viability of donor islets.262 Furthermore, only a small percentage of transplanted islets that survive under these conditions display physiologic insulin secretory characteristics.263,264,265

Multiple strategies have been explored to attenuate apoptosis during islet transplantation.266 Expression of antioxidant enzymes such as glutathione peroxidase and superoxide dismutase alleviate oxidative damage associated with hypoxia and reoxygenation.267 Inhibition of signaling downstream of inflammatory cytokines by IL- 1 receptor antagonists268,269,270 or inhibition of the JAKSTAT pathway through SOCS proteins78,131,271 protects islet grafts. Studies in preclinical models of islet transplantation using both rodent and human islets have also shown that combined inhibition of the extrinsic and intrinsic pathway by blocking effector caspases through