Calcium-activated Calpain-2 Is a Mediator of Beta Cell Dysfunction and Apoptosis in Type 2 Diabetes

Abstract

The islet in type 2 diabetes (T2DM) and the brain in neurodegenerative diseases share progressive cell dysfunction, increased apoptosis, and accumulation of locally expressed amyloidogenic proteins (islet amyloid polypeptide (IAPP) in T2DM). Excessive activation of the Ca 2؉ -sensitive protease calpain-2 has been implicated as a mediator of oligomer-induced cell death and dysfunction in neurodegenerative diseases. To establish if human IAPP toxicity is mediated by a comparable mechanism, we overexpressed human IAPP in rat insulinoma cells and freshly isolated human islets. Pancreas was also obtained at autopsy from humans with T2DM and nondiabetic controls. We report that overexpression of human IAPP leads to the formation of toxic oligomers and increases beta cell apoptosis mediated by increased cytosolic Ca 2؉ and hyperactivation of calpain-2. Cleavage of ␣-spectrin, a marker of calpain hyperactivation, is increased in beta cells in T2DM. We conclude that overactivation of Ca 2؉ -calpain pathways contributes to beta cell dysfunction and apoptosis in T2DM. Hyperglycemia in type 2 diabetes mellitus (T2DM)3 is due to impaired insulin secretion in the setting of relative insulin resistance (1). The islets of Langerhans in T2DM are characterized by a deficit in beta cells, increased beta cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP), a 37-amino acid highly conserved peptide co-expressed and secreted with insulin by pancreatic beta cells (2, 3).The pathology of the islet in T2DM and brain in neurodegenerative diseases such as Alzheimer disease share several parallels. In both, the loss of functional tissue is associated with deposition of a locally expressed protein with the potential to form amyloid fibrils (Alzheimer beta protein in Alzheimer disease and IAPP in T2DM) (2, 4). In both T2DM and Alzheimer disease, there has been a debate as to whether the amyloid deposits contribute to cell loss (the so-called amyloid hypothesis) or are secondary to the processes leading to cell loss. Evidence against a direct role of amyloid deposits on cell loss is the poor correlation between the extent of amyloid deposits and the severity of disease in both human and animal models (3,5,6). Moreover, preformed amyloid fibrils are not cytotoxic when applied to cells (7).However, several lines of evidence are supportive of a role of cytotoxicity by amyloidogenic proteins. These include genetic predisposition in occasional families with mutations leading to increased amyloidogenicity of the amyloid protein (8) and reproduction of the disease phenotype in rodent models transgenic for the relevant human amyloidogenic protein (9). There is an increasing appreciation that the cytotoxic forms of amyloidogenic proteins are small nonfibrillar oligomers that may form in membranes and cause nonselective membrane permeability (7, 10, 11), the toxic oligomer hypothesis. Moreover, misfolding and aggregation of amyloidogenic proteins into toxic oligomers induce apoptosis through the mechanism of endoplasmic retic...

Human-IAPP disrupts the autophagy/lysosomal pathway in pancreatic β-cells: protective role of p62-positive cytoplasmic inclusions

“…To further investigate the role of p62 accumulation in b-cells of h-IAPP transgenic rodents, we used the INS 832/13 (rat insulinoma cell line) cells transduced with adenoviruses expressing h-IAPP or the non-amyloidogenic and non-toxic r-IAPP for comparable load of protein. 20 Cells transduced with h-IAPP exhibited a B5-fold increase in LC3-II levels after 36 and 48 h (Po0.01), suggesting either an induction of autophagy or a decrease in autophagosome clearance (Figure 4), comparable to that reported above in vivo. In cells transduced with r-IAPP, LC3-II levels were also increased at 48 h (3.2 ± 0.3-fold, Po0.001), but to a much lesser extent than in cells transduced with h-IAPP (Figure 4).…”

“…The complementary cDNA encoding the full-length human and rat preproIAPP were used as templates to generate the human and rat IAPP adenoviruses, as described previously. 20 Recombinant adenovirus expressing human and rat preproIAPP (h-IAPP and r-IAPP, respectively) were produced and purified according to the manufacturer's instructions (Clontech, Palo Alto, CA, USA).…”

Autophagy defends pancreatic β cells from human islet amyloid polypeptide-induced toxicity

“…To test the hypothesis that p62 is required for IAPP degradation by autophagy, we evaluated the interaction between IAPP and p62 in INS 832/13 cells transduced with adenoviruses expressing h-IAPP or the nonamyloidogenic and nontoxic r-IAPP (used as a control for comparable burden of protein expression [Supplemental Figure 6A] as previously published; refs. [17][18][19]. In all 3 conditions (control and r-IAPP-and h-IAPP-transduced cells), IAPP immunoprecipitated with p62 ( Figure 2A).…”

A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome

“…1A). Previous studies have shown that calpain 2 activation is regulated on the ER membrane and it plays a role in ER stress-induced apoptosis and β cell death (20,(22)(23)(24), which prompted us to study the role of WFS2 in calpain 2 activation.…”