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

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 Ca2+-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 Ca2+ and hyperactivation of calpain-2. Cleavage of α-spectrin, a marker of calpain hyperactivation, is increased in beta cells in T2DM. We conclude that overactivation of Ca2+-calpain pathways contributes to beta cell dysfunction and apoptosis in T2DM.

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 reticulum stress (ER stress) (12,13).
The proximal molecular events that link formation of toxic oligomers and induction of ER stress are unknown. One plausible explanation is that local membrane instability caused by toxic oligomers permits unregulated Ca 2ϩ surges from the ER or other intracellular Ca 2ϩ -enriched compartments. Toxic IAPP oligomers appear to form and act intracellularly (14) within the secretory pathway (15). Moreover, they escape the secretory pathway, apparently by disrupting intracellular membranes (15). Therefore, disturbance of the usually discrete cellular compartmentalization of Ca 2ϩ is a logical candidate to link membrane-permeant toxic oligomers and induction of cellular dysfunction and, under more extreme circumstances, cell death. There is as much as a 10,000-fold concentration difference between cytoplasmic Ca 2ϩ (100 nM) and ER lumen Ca 2ϩ (0.5-1.0 mM) so that even modest disruption of the ER membrane integrity might activate aberrant cytoplasmic Ca 2ϩ -activated signaling pathways. Also, because a high ER Ca 2ϩ concentration is required for appropriate ER function, ER membrane disturbance might be expected to exacerbate the ER dysfunction that permitted protein misfolding initially.
In support of the postulate that unregulated Ca 2ϩ release from the ER to the cytoplasm might be a mediator of beta cell dysfunction and apoptosis in T2DM, it is well recognized that cytoplasmic Ca 2ϩ overload is a ubiquitous cause of cell death in neurons, cardiomyocytes, and insulin-producing beta cells (16,17). Effectors or executors of calcium overload include protease calpains, kinases/phosphatases, calmodulin, and calcineurin (18). Sustained hyperactivation of calpain is provoked in many pathological processes, including ischemia, traumatic injury, and neurodegenerative disorders such as Alzheimer disease (17,19,20).
Calcium-dependent protease calpains belong to the cysteine protease family that has previously been implicated in the pathophysiology of several inflammatory disorders, including myocardial reperfusion injury, cerebral ischemia/reperfusion, circulatory shock, and T2DM (17). Insulin-producing beta cells express several calpains, including calpain-10, -calpain (or calpain-1), and m-calpain (or calpain-2). Polymorphisms in calpain-10 are associated with the risk of developing T2DM in some ethnic groups (21). The ubiquitous -calpains and m-calpains are activated by micromolar and millimolar levels of calcium, respectively. Calpains mediate a variety of physiological functions such as cytoskeleton remodeling, vesicle trafficking. and membrane fusion (22).
This study was designed to test the hypothesis that IAPPmediated ER stress-induced apoptosis is mediated in part through increased cytosolic Ca 2ϩ and activation of Ca 2ϩ -dependent calpain. We report that overexpression of human IAPP in INS 832/13 cells and isolated human islets led to increased cytoplasmic Ca 2ϩ , activation of Ca 2ϩ -sensitive calpain-2. and beta cell apoptosis. Inhibition of calpain by calpeptin attenuated the toxicity of human IAPP. Also, we detected calpain-cleaved fragments of the cytoskeleton protein ␣-spectrin, a surrogate indicator of Ca 2ϩ -initiated and calpain-mediated cytotoxicity, in beta cells of humans with T2DM.

Human and Rodent Prepro-IAPP Adenovirus
Adenovirus generation and transduction were performed according to the procedure described by Huang et al. (13). Briefly, to generate human and rat prepro-IAPP adenovirus, KpnI and XhoI or EcoRI and EcoRV restriction sites, respectively, were introduced in the front of ATG and after the stop codon. A 290-bp human prepro-IAPP PCR fragment was digested with KpnI and XhoI, and a 300-bp rat prepro-IAPP PCR fragment was digested with EcoRI and EcoRV. The fragments were inserted into pENTR2B and subsequently into pAd/ CMV/DEST adenovirus vector (Invitrogen). Recombinant adenoviruses expressing human and rat prepro-IAPP (hIAPP and rIAPP, respectively) were generated and purified according to the manufacturer's instructions (Clontech).

Cell Lines
Rat insulinoma cell line INS 832/13 was kindly provided by Dr. C. Newgard (Durham, NC) (23). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 10 mM Hepes, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 g/ml streptomycin (Invitrogen), and 50 M 2-mercaptoethanol (Sigma) in a 37°C 5% CO 2 tissue culture incubator. Rat insulinoma cells (RIN) that express a low level of endogenous IAPP were used to establish the dose-response relationship between the m.o.i. of adenovirus-expressing hIAPP or rIAPP (Fig. 1, A and B) and protein expression levels. We chose to use 400 m.o.i. for all experiments with INS 832/13 cells. The cells were transduced with adenoviruses expressing human or rodent IAPP and then 48 or 72 h later were washed with PBS and either fixed for immunocytochemistry or lysed by boiling in Laemmli sample buffer for immunoblotting. Protein concentrations were determined using the DC protein assay (Bio-Rad). Calpeptin and BAPTA-AM were from Calbiochem.

Human Islets
Isolated human islets were obtained from the Islet Cell Resource Consortium. The islet purity was 90 -95% as assessed by dithizone staining. The donors, aged 35-55 years, were heart-beating cadaver organ donors, and none had a previous history of diabetes or metabolic disorders. Islet viability was assessed by the live/dead kit (Molecular Probes). Islets were cultured in a 6-well plate or 4-well chamber slide in RPMI 1640 medium (5.5 mM glucose) containing 100 units/ml penicillin, 100 g/ml streptomycin, and 10% fetal bovine serum (Invitrogen).
To ensure that islets were functional and viable during the course of the experiment, we performed functional evaluation (glucose-stimulated insulin secretion) of isolated islets. After 72 h in culture, 40 islets were preincubated for 1 h in complete RPMI 1640 medium containing 4.0 mM glucose. The medium was replaced with fresh medium containing either 4.0 mM glucose or 16.7 mM glucose for 5 min, and aliquots were collected for insulin measurements. Islets were lysed for insulin content measurement. Insulin concentration was measured by a human insulin enzyme-linked immunosorbent assay kit (Linco Inc., St. Charles, MO).

Cytosolic Ca 2؉ Measurement Using Reporter System
To measure the elevation of cytosolic Ca 2ϩ , we used the NFAT-SEAP reporter system (Clontech) based on the activation of calcineurin by the elevation of cytosolic Ca 2ϩ (24). Calcineurin is a calmodulin-dependent phosphatase (PP2B) that de-phosphorylates and activates the transcriptional factor nuclear factor of activated T-cells (24). INS 832/13 cells were plated in 4-well chamber permanox slides (Nunc, Rochester, NY) at 80,000 cells per well and cultured for 16 -24 h. Cells were transfected overnight with the NFAT-SEAP construct (1.0 g of plasmid DNA per well) using Lipofectamine (Invitrogen) and then transduced with adenoviruses expressing hIAPP versus rIAPP at 400 m.o.i. Medium was collected 6, 12, and 18 h after transduction, and the SEAP activity was measured according to the manufacturer's instructions (Calbiochem). Similar procedure was used for isolated human islets.

Ratiometric Measurement of Cytosolic Ca 2؉
INS 832/13 cells on coverslips were loaded with the fluorescent Ca 2ϩ indicator Fura-2 by incubation in media containing 5 M Fura-2/AM (Molecular Probes, Eugene, OR) for 60 min at 37°C (25). Coverslips were then mounted in an experimental chamber (RC-25F; Warner Instrument Corp.) that was perfused with culture medium at 1.5 ml/min. The perfusion medium was heated using an in line heater (TC-344B; Warner Instrument Corp.), which maintained bath temperature at 37°C. The chamber was placed on the stage of an inverted microscope (Zeiss TV 100; Carl Zeiss, Inc., Thornwood, NY) with attached digital imaging system (Attofluor; Atto Instruments, Rockville, MD) with electronically controlled excitation filter positions (software RatioVision). Cells were continuously perfused with media for 3-5 min before the start of the acquisition of data. Ratios of images (340 nm excitation/380 nm excitation, emission filter 520 nm) of 30 -50 cells per field were obtained at 30-s intervals. A region of interest was defined over each cell, and the average ratio intensity over the region was converted to [Ca 2ϩ ] i using a calibration curve constructed with a series of calibrated buffered calcium solutions (calcium calibration buffer kit number 2; Molecular Probes). For each cell, [Ca 2ϩ ] i values and their times of acquisition were stored on computer disk.

Calpain Activity Assay
The fluorometric calpain activity assay is based on the detection of the cleavage of the calpain substrate Ac-LLY-amino-4trifluoromethylcoumarin (calpain activity assay kit, Biovision, Sunnyvale, CA). Upon cleavage of this substrate in lysates, free amino-4-trifluoromethylcoumarin emits a yellow-green fluorescence (505 nm), which is detected by the CCD camera (UVP, Upland, CA). The cells were plated on a 6-well plate for overnight and transduced with hIAPP, rIAPP, adenoviruses, or none as a control. After 12 h, cells were washed with cold PBS and lysed with 100 l of lysis buffer on ice for 20 min. Protein content in the lysates was measured by DC-protein assay (Bio-Rad). Equal amount of protein lysates were subjected to calpain assay according to the manufacturer's instruction. Purified calpain (provided in the kit) was used as a positive control, and calpain inhibitor (benzyloxycarbonyl-LLY-fluoromethyl ketone) was included as a negative control (data not shown).

Caspase-3 Activity Assay
We employed a functional assay to detect the activation of procaspase-3 in human islets (Caspase-Glo 3/7 Assay, Promega, Madison, WI). Briefly, 100 l of reagents were used to measure the activity of about 100 islets at the end of the experiments. The values were normalized by the protein content. A specific inhibitor of caspase-3 (catalog no. 218832, Calbiochem) was used to verify the specificity of the assay. Calcium ATPase inhibitor, 0.1 M thapsigargin, was used as a positive control.

Human Pancreatic Tissue
Institutional Review Board approval was obtained from both the Mayo Clinic (Institutional Review Board number 1516-03) and UCLA (number 06-04-021-01). We obtained human pancreatic tissue at autopsy from seven obese humans with T2DM and seven age-and BMI-matched nondiabetic controls (Table  1). In addition, aliquots of nine surgically removed human pancreatic tissue specimens (pancreatectomy for tumor but nontumor-affected pancreas used for study) from T2DM (n ϭ 3) and control subjects (n ϭ 6) were studied with UCLA Institutional Review Board approval (Fig. 6C). The detection of IAPP toxic oligomers in these pancreas samples was reported previously (15).
To assess cell death, 48 or 72 h after transduction, culture medium was replaced with medium containing 50 g/ml pro- . Random areas (six per chamber) were imaged first in blue (DAPI) and then in red (PI or Cy3) channel. Immunofluorescent staining of human pancreatic sections was performed as described (13). A specific antibody against the calpain-cleaved ␣-spectrin (Santa Cruz Biotechnology) (26,27) was used as a surrogate for calpain hyperactivation. Sections were double-stained with cleaved spectrin and insulin antibodies.

Image Analysis
To assess cell death, propidium iodide-positive cells were counted in each image and related to the total numbers of cells in the image (DAPI-labeled nuclei). To quantify the expression of calpain 2, the area labeled with anti-calpain 2-Cy3 was measured using Image-Pro Plus software (Media Cybernetics, Inc., Silver Spring, MD) and related to the total number of cells in the image (DAPI-labeled nuclei). For cleaved spectrin analysis, 10 -15 islets from each human pancreas section were imaged. Total beta cells and beta cells stained for cleaved spectrin were counted (Table 1).

Cryo-immunogold Labeling and Electron Microscopy
Sample Preparation-INS 832/13 cells were transduced with hIAPP or rIAPP for 30 h and then fixed with 4% paraformaldehyde ϩ 0.1% glutaraldehyde for 6 h, scraped, spun, and embedded and sectioned as described previously (28).
Staining Procedure-Sections were incubated with antibody against toxic oligomers (A11 (29)) diluted in 20 mM Tris, 150 mM NaCl, 1% bovine serum albumin at 4°C overnight, washed three times with the same buffer, and then incubated with secondary antibody for 45 min at room temperature. After washing, the sections were fixed with 0.8% glutaraldehyde, treated with 1% uranyl acetate in 1.3% methylcellulose, and air-dried. Samples were analyzed within 1-3 days after staining using a JEM 1200-EX transmission microscope (JEOL, Japan) equipped with a BioScan 600W digital camera (Gatan, Inc., Pleasanton, CA).

Confocal Time Lapse Microscopy
Time-lapse confocal microscopy was used as described previously (30). Briefly, INS 832/13 cells were transfected with plasmid DNA expressing NFAT-GFP for 12 h, and then the same cells were transduced with Ad-hIAPP at 400 m.o.i. Confocal time-lapse microscopy was conducted to follow individual cells in the presence with 500 ng/ml of PI for 2 h.

Statistical Analysis
All values were presented as the mean Ϯ S.E. Student's t test was performed to compare the differences between the rIAPP versus hIAPP adenovirus expressing cells or islets. Values of p Ͻ 0.05 were considered to be statistically significant.

Overexpression of Human IAPP in INS 832/13 Cells Reduces Cell Viability and Increases Cell Apoptosis
We previously reported that the overexpression of hIAPP with a green fluorescent protein tag induced ER stress in INS 832/13 cells (13). Because the presence of the green fluorescent protein tag may interfere or distort the toxic effects of hIAPP, we created an adenovirus expressing hIAPP without any tag. The nonamyloidogenic rIAPP was used as a control because it contains three proline residues in the 20 -29-amino acid region of IAPP rendering it soluble. The sequence of IAPP in the mouse and rat is identical, hence the term rodent. To establish a dose response for m.o.i. of adenoviral expression and toxicity, and to ensure comparable expression of experimental hIAPP and control rIAPP, RIN cells were transduced with an m.o.i. of 150 -500 with a resulting progressive increase in apoptosis quantified by cleavage of caspase-3 48 h after transduction (Fig. 1A). For subsequent experiments, we used an m.o.i. of 400 for hIAPP or rIAPP as a control for a comparable burden of protein expression (Fig. 1B). hIAPP expression caused increased apoptosis compared with control or rIAPP (Fig. 1D). Furthermore, toxic IAPP oligomers were detected in hIAPP-transduced INS 832/13 cells but rarely in cells transduced with rIAPP (Fig. 1C).

Cytosolic Calcium Is Increased in INS 832/13 Cells Overexpressing Human IAPP
Having established that the new hIAPP adenovirus without a tag induced apoptosis in INS 832/13 cells, we sought to determine what caused apoptosis in INS 832/13 cells overexpressing hIAPP. Because hIAPP oligomers may compromise cellular membrane integrity (7, 31, 32), we designed several experiments to establish if overexpression of hIAPP induces an increase in cytosolic Ca 2ϩ . First, we established that hIAPP protein expression was detected 4 -6 h after viral infection (data not shown), and the cytosolic Ca 2ϩ was elevated 12 h ( Fig. 2A, reporter assay) and 6 h (Fig. 2B, ratiometric assay) after adenoviral transduction. We observed an increased level of cytosolic Ca 2ϩ in cells overexpressing hIAPP and a more modest increase in cells overexpressing rIAPP compared with nontransduced control cells (Fig. 2A). The minor effect of rIAPP was not detected by the ratiometric assay, which measured the cytosolic Ca 2ϩ in less than a second, but was detectable by the reporter assay, which measured the accumulative effects of cytosolic Ca 2ϩ elevation over 12 h. The increased Ca 2ϩ levels in rIAPP-expressing cells may come from two sources as follows: adenoviral transfection itself can increase Ca 2ϩ levels (33) or the increased number of peptides entering the ER can permit some passive leakage of Ca 2ϩ through the ER translocon pore (34). We employed confocal time-lapse video micrography to show that hIAPP-induced abnormal elevation of cytosolic Ca 2ϩ ions is followed by cell death (Fig. 2C, right panel). In Fig. 2C, left panel, one cell in the lower portion of the image is positive for cytosolic NFAT-GFP at the beginning of imaging (8 h post-transduction). Two hours later, in the same cell, we observed an abnormally high level of dephosphorylated NFAT-GFP (Fig. 2C, green color) due to Ca 2ϩ -activated calcineurin. This dephosphorylated NFAT-GFP translocated to the nucleus (Fig. 2C, middle panel). Twenty minutes later, PI was incorporated to the nucleus of this cell indicating cell death (Fig. 2C, right panel). As further evidence of the role of elevated Ca 2ϩ in mediating hIAPP, induced apoptosis was obtained using Ca 2ϩ chelator. When the intracellular calcium chelator BAPTA-AM was added 4 h after viral transduction, apoptosis (caspase-3 activation) was reduced at a dose of 0.25 M (Fig.  2D). We do not see significant protective effect at higher concentrations of BAPTA-AM. We believe that at higher concentrations BAPTA-AM penetrates the ER lumen and depletes ER Ca 2ϩ leading to loss of function of ER chaperones, compromising protein folding and thus compromising its protective effects (35).

Calcium-dependent Calpain Activity Is Increased in INS 832-13 Cells Overexpressing Human IAPP
We further asked what was the consequence of increased cytosolic Ca 2ϩ induced by hIAPP. Among many cellular events initiated by increased cytosolic Ca 2ϩ , calpain, a neutral Ca 2ϩ -dependent protease, was a prime suspect. Previous study has shown that overexpression of Alzheimer amyloid precursor protein in cultured neurons induced activation of calpain (19). The expression of hIAPP in INS 823/13 cells induced increased calpain protease activity (Fig. 3A). This functional assay of calpain protease activity does not specify which calpain is activated, so a calpain-2-specific antibody was then used to address this question.
The antibody used to detect calpain-2 recognizes both the 80-kDa inactive and 58-kDa active forms of calpain-2 (catalog no. 208755, Calbiochem). Inactive calpain-2 is associated with the ER membrane at the cytosolic side (36), but following activation, calpain-2 is released from this site and rapidly degraded. Rapid degradation of activated calpain-2 is presumably to prevent this potent protease from degrading proteins distant from the intended local site of activation (37). Consistent with this, treatment with thapsigargin (ER Ca 2ϩ -ATPase inhibitor) induced an almost complete loss of calpain-2 immunoreactivity on the ER surface (Fig. 3B, top  right panel). Overexpression of hIAPP also caused a loss of calpain-2 immunoreactivity (Fig. 3B, lower right panel) compared with cells overexpressing rIAPP (Fig. 3B, lower left panel). To ensure the localization of calpain-2 on the ER of beta cells, we performed ER fractionation and performed immunoblots using the same calpain-2-specific antibody. The result of enriched ER protein immunoblotting confirmed the observation from immunocytochemistry that treatment with thapsigargin led to the loss of inactive calpain in the ER-enriched fraction (data not shown). Immunoblotting showed that overexpression of hIAPP decreased total calpain content (Fig. 3C), presumably as a consequence of rapid degradation following Ca 2ϩ activation (see above, Fig. 3B) (37). Furthermore, the observed hIAPP-induced elevated cytosolic Ca 2ϩ , increased calpain activity, and the decrease in total calpain protein content by immunoblot are consistent with Etoposide-induced cytosolic Ca 2ϩ elevation, increased calpain activity, and decreased total calpain protein content (38).

Inhibition of Calpain Activity by Calpeptin Reduces Toxicity of hIAPP in INS 832/13 Cells
If calpain mediates hIAPP-induced apoptosis, then inhibition of calpain would be expected to reduce apoptosis. We chose the specific calpain inhibitor calpeptin, a fragment of the endogenous calpain inhibitor calpastatin, to test this hypothesis. A dose-response study was undertaken in INS 832/13 to identify a nontoxic dose (data not shown). Based on those findings, we chose to use calpeptin at 1.25 M for 66 h. At that dose, caspase-3 cleavage and PI incorporation were decreased by 30 and 50%, respectively, in calpeptin-treated hIAPP-expressing cells compared with hIAPP-expressing cells without treatment (Fig. 4, A and B).

Calcium Elevation and Calpain Activation Are Observed in Human Islets Overexpressing Human IAPP
After establishing that overexpression of hIAPP induced increased cytosolic Ca 2ϩ , activation of calpain, and apoptosis in INS 832/13 cells, we extended these findings to isolated human islets. Expression of hIAPP increased cytosolic Ca 2ϩ levels, monitored by the same Ca 2ϩ reporter system (Fig. 5A). Activation of calpain by hIAPP was also confirmed in human islets assayed by calpain activity (Fig. 5B). Inhibition of calpain by calpeptin in human islets again led to attenuation of caspase-3 activation (Fig. 5C).

Cleaved Spectrin Was Detected in Beta Cells in Humans with T2DM
Having demonstrated that high expression levels of human IAPP induce increased cytosolic Ca 2ϩ and hyperactivation of calpain, we posed the following question. Is there evidence of chronically increased cytosolic Ca 2ϩ and hyperactivation of calpain in beta cells of humans with T2DM? Living beta cells from humans with T2DM are rarely available. Therefore, to address this, we analyzed the consequences of sustained elevated cytosolic Ca 2ϩ and hyperactivation of calpain in sections of fixed paraffin-embedded human tissue available from T2DM.
The cytoskeletal protein ␣-spectrin (also called ␣-fodrin), when subjected to calpain cleavage, generates a specific ␣-spectrin fragment that can be detected as a measurement of calpain activity (39,40). Using a specific antibody against the calpainspecific (26), cleaved fragment of ␣-spectrin, we detected increased cleaved fragments (Fig. 6, A and B) in beta cells from humans with T2DM (n ϭ 7), compared with age-and BMImatched nondiabetic subjects (n ϭ 7). Exocrine cells also express ␣-spectrin, but the cleaved spectrin in T2DM was rarely detected in exocrine cells, ensuring that the detected calpain hyperactivation in beta cells was not an artifact of tissue collection or preservation. To substantiate this observation, we  examined the relationship between the frequency of cleaved spectrin and the frequency of intracellular toxic IAPP oligomers detected with a specific anti-toxic oligomer antibody (A11) in pancreatic beta cells of nine human pancreas samples obtained at surgery as reported previously (15). We observed a positive correlation (r 2 ϭ 0.8, p Ͻ 0.01) between the frequency of beta cells with cleaved spectrin and the oligomer-specific immunoreactivity (Fig. 6C). Using immunoblotting, we also detected cleaved spectrin in isolated human islets (Fig. 6D) and in INS 832/13 cells overexpressing hIAPP (Fig. 6E). In the same preparation, inhibition of calpain activation by calpeptin reduced the cleavage of spectrin induced by overexpressing hIAPP (Fig. 6D).

DISCUSSION
We tested the hypothesis that human IAPP-induced apoptosis is mediated through disturbance of intracellular Ca 2ϩ homeostasis. We report the following: 1) that overexpression of hIAPP induced the formation of toxic IAPP oligomers and increased cytosolic Ca 2ϩ concentration and hyperactivation of calpain-2 protease in insulin-secreting beta cells and/or isolated human islets; 2) that inhibition of calpain attenuated the toxicity of human IAPP in INS 832/13 cells and isolated human islets; and 3) that calpain-cleaved products of ␣-spectrin were present in isolated human islets and INS 832/13 cells overexpressing hIAPP and were identified in pancreatic beta cells from humans with T2DM. These studies imply that chronically and aberrantly activated Ca 2ϩ /calpain pathways contribute to the cytotoxicity of aggregated human IAPP. Moreover, these data suggest that dysfunction and increased beta cell apoptosis in T2DM are mediated, at least partially, through chronic overactive Ca 2ϩ /calpain pathways. These findings support and extend the IAPP toxic oligomer hypothesis in T2DM.
Aberrantly increased cytoplasmic Ca 2ϩ has been shown to mediate cellular dysfunction and cell death in neurodegenerative diseases, also characterized by abnormal intracellular aggregates of amyloidogenic proteins (17,18,41). We report that the overexpression of hIAPP in insulin-producing cells or islets leads to an early increase in cytoplasmic Ca 2ϩ . This finding was obtained by using a gene reporter system, in which the Ca 2ϩsensitive transcriptional factor nuclear factor of activated T-cells was linked to the secreted alkaline phosphatase to measure the accumulated effects of Ca 2ϩ elevation. This finding was substantiated by the ratiometric measurement of intracellular Ca 2ϩ using fura-2-AM (Fig. 2B), a standard protocol in the field (25).
The mechanism by which hIAPP aggregates cause inappropriately increased cytoplasmic Ca 2ϩ is unknown but is most likely mediated by the property of hIAPP oligomers to induce FIGURE 6. Cleavage of calpain substrate spectrin was detected in beta cells of paraffin-embedded pancreatic tissues from humans with T2DM. A, immunofluorescent images of pancreatic sections stained with anti-cleaved spectrin (red) and anti-insulin antibodies (green). Nuclei were visualized by DAPI (blue). Beta cell with cleaved (Cl.) spectrin was indicated by an arrow (inset shows beta cell with cleaved spectrin in the periphery). B, quantification of beta cells positive for cleaved spectrin in tissue sections from age-and BMI-matched obese nondiabetic (OND, n ϭ 7) and from obese diabetic subjects (OD, n ϭ 7). C, there was a positive correlation between frequency of beta cells with cleaved spectrin and frequency of beta cells positive for toxic oligomers detected by immunofluorescent labeling with oligomer-specific antibody A11 in surgically obtained human pancreas. These tissue samples were previously used to report the increased frequency of toxic IAPP oligomers in beta cells of individuals with T2DM compared with nondiabetic controls (15 nonselective membrane leakage (7). In support of this, it has been shown that application of ␤-amyloid aggregates to cells caused increased cytoplasmic Ca 2ϩ concentrations and subsequently reduced ER Ca 2ϩ stores (42). It has been reported that exogenous hIAPP can activate transient receptor potential channels (43). Cytosolic Ca 2ϩ elevation induced by hIAPP overexpression may arise as a result of hIAPP induced leakage into the cytosol from the secretory pathway or mitochondria because membrane-permeant IAPP toxic oligomers are identified at each of these sites (14,15). Also, oligomeric ␤-amyloidinduced Ca 2ϩ release can be blocked by the inhibition of the ER calcium release channels (42). Furthermore, increased cytoplasmic Ca 2ϩ can be associated with the malfunction of both ER ryanodine and inositol 1,4.5-triphosphate receptors, which are substrates of activated calpains (44,45). Because toxic hIAPP oligomers have been detected in the secretory pathway (15), they might also be secreted and act on the plasma membrane, a site that also leads to cytotoxicity (7,10,32,46,47) and elevated cytoplasmic calcium levels (18,42).
An increase in cytosolic Ca 2ϩ leads to activation of calpain, which is implicated in a number of pathological disorders, including brain ischemia, injury, and neurodegeneration (17). Inappropriately high calpain activation has been reported in the affected hippocampus in Alzheimer disease (16). Our data indicate that increased cytosolic Ca 2ϩ concentrations preceded the hyperactivation of calpain. Furthermore, we showed that inhibition of calpain attenuated hIAPP-induced cytotoxicity, which is consistent with a recent report showing that the inhibition of calpain improves memory and synaptic transmission in a mouse model of Alzheimer disease with overexpression of human ␤-amyloid protein (48).
We also report that beta cells from humans with T2DM have cleaved ␣-spectrin, which is an indicator of a compromised cytoskeleton and cellular membrane. Scaffolding protein spectrin (␣II) is an actin-binding protein and is expressed in most cells, including pancreatic endocrine and exocrine cells. Spectrin functions as a membrane stabilizer by forming trimers, tetramers, and higher polymers (49). ␣II spectrin has a -calpain cleavage site based on the secondary and tertiary conformational features surrounding the cleavage site, rather than the linear sequence (49). Activated calpain cleaves spectrin and generates 145-and 150-kDa fragments, recognized by a specific antibody (50). It has been reported that calpain-cleaved fragments of ␣-spectrin are a hallmark of hyperactivation of the calcium-calpain system (51), and injection of 6-hydroxydopamine into rodent brains resulted in calpain activation and the cleavage of ␣-spectrin. When treated with calpastatin, the cleavage of ␣-spectrin was greatly reduced (52).
Therefore, the presence of cleaved spectrin in beta cells from humans with T2DM, but rarely in age-and BMI-matched nondiabetic subjects, is consistent with chronic overactivation of Ca 2ϩ -calpain pathways. Moreover, we report a positive correlation between the frequency of beta cells with cleaved spectrin (Fig. 6C) and the frequency of toxic oligomer-containing beta cells (15), consistent with the hypothesis that formation of intracellular toxic IAPP oligomers disrupt intracellular membrane fidelity with the adverse consequences of inappropriate activation of Ca 2ϩ -sensitive pathways. These observations were further substantiated by the demonstration of the presence of cleaved spectrin in isolated human islets and INS 832/13 cells overexpressing hIAPP and reduction of spectrin cleavage by concurrent use of a calpain inhibitor (Fig. 6, D  and E).
There are other lines of evidence to support Ca 2ϩ /calpain activation findings. Overexpression of calmodulin in mice results in insulin secretion defects, loss of beta cells, and diabetes (53,54). Also, abnormal elevation of cytosolic Ca 2ϩ induces ER stress and beta cell apoptosis by palmitate (55). Finally, ER stress is associated with Ca 2ϩ efflux from the ER (56), and the ER stress marker, nuclear CHOP, was detected in beta cells from humans with T2DM but only rarely in beta cells from obese nondiabetic subjects (13).
In conclusion, we report that the overexpression of human islet amyloid protein hIAPP leads to the formation of toxic oligomers, an elevation of cytosolic Ca 2ϩ , and hyperactivation of the Ca 2ϩ -dependent protease calpain. Furthermore, inhibition of calpain activity attenuates the beta cell apoptosis associated with chronic hyperactivation of Ca 2ϩ /calpain. Identification of calpain-cleaved fragments of ␣-spectrin in beta cells from humans with T2DM but not in control subjects indicates that the Ca 2ϩ -dependent protease calpain may play a key role in the pathophysiology of T2DM. These findings suggest that sustained hyperactivity of the Ca 2ϩ /calpain signaling pathway may be the molecular basis for oligomeric protein conformational disorders such as T2DM and Alzheimer disease (16,48).