original article - diabetes€¦ · nuclear chop was not detected in lean nondiabetic and rare in...

Original Article

High Expression Rates of Human Islet AmyloidPolypeptide Induce Endoplasmic ReticulumStress–Mediated �-Cell Apoptosis, a Characteristicof Humans With Type 2 but Not Type 1 DiabetesChang-jiang Huang,

1Chia-yu Lin,

1Leena Haataja,

1Tatyana Gurlo,

1Alexandra E. Butler,

1

Robert A. Rizza,2

and Peter C. Butler1

OBJECTIVE—Endoplasmic reticulum (ER) stress–inducedapoptosis may be a common cause of cell attrition in diseasescharacterized by misfolding and oligomerisation of amyloido-genic proteins. The islet in type 2 diabetes is characterized byislet amyloid derived from islet amyloid polypeptide (IAPP) andincreased �-cell apoptosis. We questioned the following: 1)whether IAPP-induced �-cell apoptosis is mediated by ER stressand 2) whether �-cells in type 2 diabetes are characterized by ERstress.

RESEARCH DESIGN AND METHODS—The mechanism ofIAPP-induced apoptosis was investigated in INS-1 cells andhuman IAPP (HIP) transgenic rats. ER stress in humans wasinvestigated by �-cell C/EBP homologous protein (CHOP) ex-pression in 7 lean nondiabetic, 12 obese nondiabetic, and 14obese type 2 diabetic human pancreata obtained at autopsy. Toassure specificity for type 2 diabetes, we also examined pancre-ata from eight cases of type 1 diabetes.

RESULTS—IAPP induces �-cell apoptosis by ER stress in INS-1cells and HIP rats. Perinuclear CHOP was rare in lean nondia-betic (2.6 � 2.0%) and more frequent in obese nondiabetic(14.6 � 3.0%) and obese diabetic (18.5 � 3.6%) pancreata.Nuclear CHOP was not detected in lean nondiabetic and rare inobese nondiabetic (0.08 � 0.04%) but six times higher (P � 0.01)in obese diabetic (0.49 � 0.17%) pancreata. In type 1 diabeticpancreata, perinuclear CHOP was rare (2.5 � 2.3%) and nuclearCHOP not detected.

CONCLUSIONS—ER stress is a mechanism by which IAPPinduces �-cell apoptosis and is characteristic of �-cells in hu-

mans with type 2 diabetes but not type 1 diabetes. These findingsare consistent with a role of protein misfolding in �-cell apopto-sis in type 2 diabetes. Diabetes 56:2016–2027, 2007

Both type 1 and type 2 diabetes are characterizedby deficits in �-cell mass and increased �-cellapoptosis (1–6). The mechanism that initiates�-cell apoptosis in type 1 diabetes is believed to

be autoimmune-mediated cytokine production (5). Severalmechanisms have been proposed for increased �-cellapoptosis in type 2 diabetes, including oxygen free radi-cals (7), free fatty acid toxicity (8), interleukin-1� (9), andformation of islet amyloid polypeptide (IAPP) toxic oli-gomers (10–12).

Programmed cell death, or apoptosis, is important inmulticellular organisms to permit organ development andremodeling (13). In disease states, apoptosis permits se-lective removal of cells that are damaged, particularly inrelation to cell cycle, so that damage is not propagated(3,14). Apoptosis may be initiated by a wide variety ofcellular insults, which are currently thought to act throughat least three pathways that converge to accomplishirreversible destruction of the cell’s chromosomes. Thesethree major pathways have been designated as the extrin-sic and intrinsic pathways and endoplasmic reticulum(ER) stress pathway (15,16). The extrinsic pathway isclassically exemplified by cytokine-induced cell death,mediated through cell surface death receptors (17). Theintrinsic pathway is most often described as a response tomitochondrial disruption, for example, secondary to oxy-gen free radicals (18). ER stress–induced apoptosis isclassically ascribed to aggregates of misfolded protein thatare believed to compromise the ER membrane (15).

The human pancreatic �-cell is vulnerable to all threeforms of apoptosis. Cytokines are recognized as importantin the pathophysiology of type 1 diabetes (5) and havebeen proposed as potential mediators of glucose toxicityin type 2 diabetes (9). The �-cell bears a large burden ofprotein synthesis, protein folding and processing, andregulated protein secretion, with the primary client pro-teins being insulin and IAPP. Islet amyloid derived fromIAPP is a characteristic of the islet in type 2 diabetes (3).Both mice and rats with high expression rates of humanIAPP have been reported to develop diabetes because ofloss of �-cells through increased �-cell apoptosis (11,12).

From the 1Larry Hillblom Islet Research Center, University of California, LosAngeles, Los Angeles, California; and the 2Endocrine Research Unit, MayoMedical College, Rochester, Minnesota.

Address correspondence and reprint requests to Peter C. Butler, LarryHillblom Islet Research Center, UCLA, Los Angeles, CA 90024-2852. E-mail:[email protected].

Received for publication 12 February 2007 and accepted in revised form 10April 2007.

Published ahead of print at http://diabetes.diabetesjournals.org on 2 May2007. DOI: 10.2337/db07-0197.

Ad-hIAPP, adenovirus-expressing human islet amyloid polypeptide precur-sor protein; Ad-rIAPP, adenovirus-expressing rat islet amyloid polypeptideprecursor protein; CHOP, CEBP homologous protein; EGFP, enhanced greenfluorescent protein; ER, endoplasmic reticulum; GFP, green fluorescentprotein; HBSS, Hanks’ balanced salt solution; IAPP, islet amyloid polypeptide;IHC, immunohistochemistry; MOI, multiplicity of infection; preproIAPP, IAPPprecursor protein; siCHOP, small interfering CHOP; siRNA, small interferingRNA; TBS, Tris-buffered solution; TUNEL, transferase-mediated dUTP nick-end labeling; UPR, unfolded protein response.

© 2007 by the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked “advertisement” in accordance

with 18 U.S.C. Section 1734 solely to indicate this fact.

2016 DIABETES, VOL. 56, AUGUST 2007

There is increasing evidence that induction of apoptosis byamyloidogenic proteins is mediated by membrane-disrupt-ing oligomers that are distinct from amyloid fibrils (19).Moreover, the pathway initiating apoptosis in severalother diseases characterized by accumulation of unfoldedproteins has been reported to be ER stress (10,20).

In the present studies, we first sought to establishwhether the mechanism subserving human IAPP–induced�-cell death is ER stress. To this end, we used adenoviralexpression of human IAPP versus rodent IAPP in a �-cellline (INS-1 cell). To affirm these findings in primary �-cellsin an animal model, we studied islets from the human IAPP(HIP) rat, a transgenic rat model that develops isletpathology comparable with that in humans with type 2diabetes (12). To place these findings in the context ofhuman disease, we then examined pancreata from bothlean and obese humans with type 2 diabetes and fromnondiabetic control subjects. Recognizing that high glu-cose per se could theoretically induce ER stress by theactions of oxygen radicals inducing mitochondrial dys-function and subsequent deprivation of energy required tomaintain appropriate protein folding, we also examinedpancreata from humans with type 1 diabetes, where suffi-cient �-cells were still present to make a meaningfulevaluation.

RESEARCH DESIGN AND METHODS

Adenovirus generation and INS-1 cell line studies. The complementarycDNA encoding the full-length human and rat IAPP precursor protein (pre-proIAPP) was ligated into pEGFP-N2 vector (Clontech, Palo Alto, CA), and thesequences were verified. PreproIAPP–enhanced green fluorescent protein(EGFP) construct was digested with XhoI and NotI, ligated into pENTR2B(Invitrogen, Carlsbad, CA), and subsequently inserted into pAd/cytomegalovi-rus/DEST adenovirus vector (Invitrogen). Recombinant adenovirus-express-ing human and rat preproIAPP (Ad-hIAPP and Ad-rIAPP, respectively) fusedto EGFP were generated and purified according to the manufacturer’sinstructions (Invitrogen). PreproIAPP is 36 kDa, but after the signaling peptideis removed when IAPP reaches ER, the fusion protein is 34 kDa (unprocessedproIAPP EGFP). Cleavage by prohormone convertase PC1/3 at the COOH-terminus creates a 6-kDa NH2-terminal proIAPP and a 28-kDa processedCOOH-terminal proIAPP plus EGFP. Finally, prohormone convertase 2cleaved at the NH2-terminal creates a fully processed 4-kDa mature IAPP.Adenovirus-expressing green fluorescent protein (GFP) was kindly providedby Dr. Christopher Rhodes (University of Chicago, Chicago, IL).

INS-1 cells were grown in RPMI medium, supplemented with 10% FBS, 50�mol/l �-mercaptoethanol, 10 mmol/l HEPES, and 1 mmol/l sodium pyruvate.One day after plating, INS-1 cells were transduced at multiplicity of infection(MOI) � 100 with adenovirus-expressing GFP, Ad-rIAPP-EGFP, Ad-hIAPP-EGFP, or nontransduced control. For Western blotting experiments, cellswere washed with PBS and lysed in 2� Laemmli sample buffer aftertransduction. As a positive control to induce ER stress, cells were culturedovernight in the presence of 0.5 �g/ml Tunicamycin (Sigma, St. Louis, MO).Protein concentrations were determined using Bio-Rad protein assay reagents(Hercules, CA). Proteins (10 �g) were separated on a 4–12% Bis-Tris NuPAGEgel (Invitrogen) and transferred to polyvinylidine fluoride membranes (Bio-Rad). Membranes were blocked with 5% nonfat dry milk in Tris-bufferedsolution (TBS)/0.1% Tween-20 and incubated overnight at 4°C with anti-GFP(1:1,000; Zymed Laboratories, San Francisco, CA), anti–�-actin, anti–caspase-3 (1:1,000; Cell Signaling Technology, Beverly, MA), or anti–C/EBPhomologous protein (CHOP) (1:500; Santa Cruz Biotechnology, Santa Cruz,CA) antibodies. Membranes were washed with TBS/0.1% Tween-20 andincubated with horseradish peroxidase–conjugated secondary antibodies(1:3,000; Jackson Laboratories, Bar Harbor, ME) for 1 h. After washes,proteins were visualized using enhanced chemiluminescence (Bio-Rad).

To investigate the time course of apoptosis and CHOP protein expression,INS-1 cells were plated in a chamber slide. One day after plating, INS-1 cellswere transduced (MOI � 100) with Ad-rIAPP-EGFP or Ad-hIAPP-EGFP andthen cultured for 8, 16, 24, and 48 h. At the end of each indicated time, cellswere washed and fixed with 4% paraformaldehyde, followed by staining fortransferase-mediated dUTP nick-end labeling (TUNEL) (cell death detectionkit TMR Red; Roche Diagnostics, Mannheim, Germany) according to themanufacturer’s instructions. For CHOP staining, cells were permeabilized

with 0.2% Triton X-100 in PBS at room temperature for 10 min after fixationand incubated with anti-CHOP antibody (1:100; Santa Cruz Biotechnology)overnight, followed by secondary antibody conjugated to Cy3, diluted in 1:200(Jackson Laboratories). Fluorescent slides were viewed using a Leica DM6000microscope and images acquired using Openlab software (Improvision, Lex-ington, MA). Experiments were repeated four times for each time point.Small interfering RNA and transfection. CHOP protein levels werereduced by the small interfering RNA (siRNA) technique. The sequences of thesiRNA duplexes were chosen from CHOP coding region (Ddit3, NM_024134).The sense strand (regions 5–23) of siRNA was GAA UCU AAU ACG UCG AUCAdTdT (dT is deoxythymidine). The specific small interfering CHOP (siCHOP)and nonspecific control siRNA were synthesized by and purchased fromQIAGEN (Valencia, CA). Transfection of cultured INS-1 cells with siCHOP andnonspecific control siRNA was carried out by the chemical transfectantLipofectamine (Invitrogen) according to the manufacturer’s instructions.Transduction (MOI � 150) of the INS-1 cells by the Ad-hIAPP precededtransfection of siCHOP versus nonspecific control siRNA by 1 h. Cell lysateswere collected 48 h later. Immunoblotting was conducted as described above.CHOP and cleaved caspase-3 expression were quantified by measuring theoptical density of the desired bands in Western blots using Labwork software(UVP Bioimaging Systems, Upland, CA).Animal model and islet isolation. The generation of the HIP has previouslybeen described in detail (12). Briefly, the transgene is the fusion of the ratinsulin gene II promoter linked to the cDNA encoding for IAPP. Sprague-Dawley–expressing human IAPP was designed as HIP rats and without thehuman IAPP as Sprague-Dawley wild-type nontransgenic controls. Both werebred and housed at an animal housing facility at the University of California,Los Angeles. The University of California, Los Angeles institutional animalcare and use committee approved all surgical and experimental procedures inthese studies. Five-month-old rats were studied because, by this age, HIP ratshave increased �-cell apoptosis but do not yet have diabetes, avoiding theconfounding effects of glucose toxicity. To obtain rat islets, 5-month-old HIP(n � 12) and wild-type (n � 12) rats were killed by intraperitoneal injectionof pentobarbital (50 mg/kg). The bile duct was cannulated, and Hanks’balanced salt solution (HBSS) (Flow Labs, Irvine, Scotland) containing 7.5mmol/l calcium chloride, 20 mmol/l HEPES buffer, and 1 mg/ml collagenase(type II; Sigma, St. Louis, MO) was injected to uniformly distend the pancreas.The pancreas was then removed and incubated for 20 min in HBSS at 37°C,followed by transfer into HBSS containing 5 g/l BSA and 20 mmol/l HEPESbuffer at 4°C. The pancreas was dispersed by gentle shaking and washed fourtimes. After being handpicked three times, the islets from two to three animalswere pooled, washed with PBS, and immediately lysed in 2� Laemmli samplebuffer, aided by 10 strokes of a plastic micropestler. Antibodies used were asfollows: anti–caspase-12 (rat monoclonal, 1:1,000; Sigma), anti-CHOP (rabbitpolyclonal, 1:500; Santa Cruz Biotechnology), anti–�-actin (rabbit polyclonal,1:1,000), and anti–caspase-3 (rabbit polyclonal, 1:1,000; Cell Signaling). TheWestern blotting procedure of islet extracts was the same as that of INS-1cells, described above.Human subjects and pancreatic tissue. Institutional review board approvalwas obtained from both the Mayo Clinic (institutional review board no.1516-03) and the University of California, Los Angeles (no. 06-04-021-01). Weobtained human pancreatic tissue at autopsy from 7 lean and 12 obesenondiabetic humans and from 14 obese humans with type 2 diabetes. We alsoobtained pancreata at autopsy from three humans with long-standing type 1diabetes and four with relatively recent-onset type 1 diabetes who died ofdiabetic ketoacidosis (see Table 1). In addition, we obtained pancreas fromone 89-year-old man with recent-onset type 1 diabetes by surgery as previ-ously described (6). The three autopsy cases of long-standing type 1 diabeteswere selected as those with the largest number of �-cells per islet from a priorstudy of 42 cases of pancreas from patients with long-standing type 1 diabetes(1).

Potential autopsy cases were identified by retrospective analysis of theMayo Clinic autopsy database. To be included, cases were required to 1) havehad a full autopsy within 12 h of death; 2) have had a general medicalexamination, including at least one fasting blood glucose documented withinthe year before death (the exception being those with recent-onset type 1diabetes, who died at the first admission shortly after diagnosis); and 3)pancreatic tissue stored that was of adequate size and quality. Cases wereexcluded if 1) potential secondary causes of diabetes were present, 2)subjects had been exposed to chronic glucocorticoid treatment, or 3) pancre-atic tissue had undergone autolysis or showed evidence of pancreatitis. Thedefinitions of lean and obese were BMI �25 and �27 kg/m2, respectively.Pancreas processing and immunohistochemistry

Rat pancreas. To obtain pancreata for immunohistochemistry (IHC),5-month-old HIP and wild-type rats (n � 5 for each group) were perfused andfixed with 4% paraformaldehyde. The tissue was paraffin embedded and cut

C.-J. HUANG AND ASSOCIATES

DIABETES, VOL. 56, AUGUST 2007 2017

into 4-�m sections. The IHC procedure for rat pancreatic tissue was the sameas that described for the human pancreatic tissue.Human pancreas. At autopsy, pancreas was resected from the tail and, witha sample of spleen, fixed in formaldehyde and embedded in paraffin forsubsequent analysis. The surgical specimen was a distal pancreatectomy, asrecently described (6). Sections (4 �m) were cut from these paraffin blocksand mounted on Fisherbrand Ink Jet White IJL-6109-Plus-600621 chargedslides (Fisher category no. 12550109; Fisher Scientific, Pittsburgh, PA).Sections were stained by immunofluorescence for either insulin and CHOP(two slides per case) or glucagon and CHOP (one slide per case), usingmethods as previously described (6), totaling three slides per case. Pancreaticsections were deparaffinized in toluene, rehydrated in grades of alcohols,washed in H2O followed by antigen retrieval using antigen-unmasking buffer(Vector Laboratories, Burlingame, CA), permeabilized in 0.4% Triton X-100/TBS, and blocked with 0.2% Triton X-100 and 3% BSA/TBS. Primary antibodies

used were as follows: rabbit polyclonal anti-CHOP (Santa Cruz Biotechnol-ogy), guinea pig polyclonal anti-insulin (Zymed Laboratories), and mouseanti-porcine monoclonal glucagon (Sigma). The working dilution of theprimary antibodies was 1:100 of the antibody solution with 0.2% Tween-20 and3% BSA/TBS. Donkey-derived secondary antibodies conjugated to Cy3 andfluorescein isothiocyanate were diluted to 1:200 (Jackson ImmunoResearchLaboratories). All slides were mounted with Vectashield (Vector Laboratories)with DAPI and coverslipped. As a positive control for nuclear CHOP toidentify ER stress, INS-1 cells were treated with 2.5 �m thapsigargin (Sigma)versus DMSO for 16 h; then, the INS-1 cells were fixed in 4% paraformaldehydeat room temperature for 30 min and permeabilized with the 0.2% Triton X-100in PBS for 10 min. Then, the same anti-CHOP IHC procedure described abovewas used for detection of CHOP expression. Nuclear CHOP staining wasdetected in 73.2 � 5.9 vs. 0.8 � 0.1% (n � 4) of INS-1 cells treated withthapsigargin vs. DMSO, respectively. To assure specificity of primary antibod-

TABLE 1Clinical characteristics of human case subjects

Age(years) Sex

BMI(kg/m2)

FBG(mg/dl)

Duration ofdiabetes (years) Treatment

Obese type 2 diabetic53 Female 42 426 Unknown Insulin68 Female 38 116 1 Insulin65 Male 35 188 Unknown Oral62 Male 34 180 4 Insulin62 Female 45 121 7 Insulin76 Female 31 116 15 Insulin69 Female 36 187 22 Insulin64 Male 33 283 6 Oral38 Female 53 165 4 Oral49 Male 37 195 Unknown Diet74 Male 37 137 Unknown Diet75 Female 33 210 10 Oral63 Male 35 400 Unknown Insulin67 Male 30 121 22 Diet

Obese nondiabetic59 Male 35 97 — —63 Female 32 93 — —43 Female 44 90 — —44 Male 56 98 — —84 Male 45 95 — —75 Female 30 97 — —71 Female 29 86 — —58 Female 42 97 — —82 Male 30 106 — —50 Female 38 90 — —32 Male 38 99 — —77 Female 35 101 — —

Lean nondiabetic86 Male 25 107 — —85 Male 24 88 — —68 Male 25 78 — —56 Female 24 100 — —65 Male 22 95 — —81 Male 25 94 — —81 Male 23 97 — —

Recent-onset type 1diabetes

89* Male 18 349 0.25 Insulin12 Female 18 532 Days Insulin12 Male 15 300� 2 Insulin14 Female 14 530 2 weeks Insulin23 Female 19 517 1 Insulin

Long-standing type 1diabetes

42 Male 41 130 23 Insulin36 Female 19 422 32 Insulin14 Female 15 402 4 Insulin

*Surgical pancreas (all others obtained at autopsy). FBG, fasting blood glucose.

HUMAN IAPP INDUCES ER STRESS–INDUCED APOPTOSIS


ies, both a mouse monoclonal anti-CHOP (Abcam, CA) and a rabbit polyclonalanti-CHOP (Santa Cruz Biotechnology) were used, side by side, in theimmunofluorescence protocol. The same pattern and frequency of nuclearCHOP staining was observed with both primary antibodies in human pancre-atic tissues (Table 1) and INS-1 cells. We subsequently used the rabbitpolyclonal anti-CHOP antibody in the human and rat studies.

Image analysis using an epifluorescent microscope and a confocal laser

scanning microscope. Fluorescent slides (three slides per case) were viewedusing a Leica DM6000 microscope (Leica Microsystems, Wetzlar, Germany)connected to an Apple computer, and images were acquired using Openlabsoftware (Improvision).Humans. The number of islets with at least 20 �-cells per islet section on

FIG. 1. Human IAPP–induced CHOP expression precedes apoptosis in human IAPP–expressing INS-1 cells. A: INS-1 cells were transduced withadenovirus-expressing GFP, Ad-rIAPP EGFP, Ad-hIAPP EGFP, or nothing. Cell lysates were prepared after 24 h. Equal amounts of proteinsamples were separated by SDS-PAGE and transferred. Membranes were probed with anti-GFP (A), anti-CHOP (B), and anti-actin antibodies.Unprocessed IAPP EGFP is 34 kDa (upper band), whereas processed COOH-terminal IAPP plus EGFP is 28 kDa (lower band). After 8, 16, 24,or 48 h, cells were fixed and immunostained for TUNEL (apoptosis) (C) or CHOP (D). Values are TUNEL- or CHOP-positive cells per 100 cells(means � SEM) with three experiments. *P < 0.05 relative to INS-1 cells expressing GFP, rIAPP, or nontransduced, by ANOVA. E: Representativemicrophotograph shows the colocalization of CHOP (panel A) (blue) and TUNEL (panel B) (red) of INS cells (panel C) (EGFP positive)transduced with Ad-hIAPP EGFP (MOI � 100) for 36 h. The bar scale is 10 �m. F: The representative Western blot shows that CHOP siRNAreduced CHOP levels by 81% and decreased caspase-3 cleavage by 68% as measured by optical density and normalized with actin.



FIG. 2. Caspase-12 is highly ex-pressed and activated in �-cellsof HIP rats. A: Representativemicrophotographs of rat isletsshow that caspase-12 (red)–posi-tive cells were insulin-staining(green) �-cells by fluorescenceIHC of paraffin-embedded pan-creatic sections. For quantifica-tion, please see Table 2. B:Representative Western blotsconfirm the presence of activatedcaspase-12 (four cleaved bands)in the islets from HIP rats butonly a small amount of pro-caspase-12 (51 kDa) presence inislets from wild-type rats. Threeindependent Western blots hadsimilar results. C: Western blotshows the cleaved caspase-3 (17kDa) in HIP rats but a muchlower level in wild-type rats(four animals per group withsimilar results). D: TUNELassays show positive TUNEL nu-clei (panels D–F) in HIP rat is-lets but rarely in wild-typecontrols (panels A–C). Activatedcaspase-12 can cleave pro-caspase-3, as reported by Hitomiet al. (ref. 43) in 2004. Bar scalerepresents 10 �m.



each slide varied from case to case, ranging from 71 to 157 in lean and obesecontrols and obese cases of type 2 diabetes. For detailed evaluation of CHOPexpression, 15 islets per case (with a minimum of 20 �-cells per islet in planeof section) were selected at random in these cases. In the recent-onset type 1diabetes case, there were 8–110 islets per section; four sections were studied,with detailed morphometic analysis of CHOP expression performed in 100islets. As expected, insulin-positive �-cells were much less frequent in thethree cases of long-standing type 1 diabetes. In these cases, 1–8 islets with�145 �-cells per plane of section were present in each slide (28 isletsevaluated). The nuclear localization of CHOP was confirmed by use of a Leicaspinning-disc laser confocal microscope (DMIRE2; Leica, Dearfield, IL) with adigital camera (Hamamatsu, Japan), and images were acquired using Volocitysoftware (McBain Instruments, Chatsworth, CA). The series (z) sections wereacquired with 0.4-�m step size. The images (512 � 512 pixels) were saved as

TIF files, and the contrast levels of the images were adjusted with AdobePhotoshop (Adobe, Mountain View, CA). All morphometric analysis wasevaluated independently in a blinded manner by two observers.HIP rats. Ten islets per tissue section were photographed and analyzed fromeach pancreas. The total number of insulin-positive cells that were CHOPpositive or caspase-12 positive were counted and expressed as a fraction ofthe �-cells. The detailed procedure was the same as that for humans.Statistical analysis. To test specific hypotheses posed, we used ANOVA, andpost hoc testing was performed when a significant difference existed. A P

value �0.05 was considered statistically significant.

RESULTS

Apoptosis induced by human IAPP was mediated byCHOP in INS-1 cells. To establish the mechanism ofendogenously expressed human IAPP-induced apoptosis,we generated adenoviruses expressing human and ratproIAPP EGFP. Human and rat IAPP were expressed atsimilar levels in INS cells when transduced at MOI � 100(Fig. 1A). Expression of CHOP, a mediator of ER stress(21), was increased in response to transduction withAd-hIAPP EGFP but not Ad-rIAPP EGFP (Fig. 1B). Con-sistent with the actions of CHOP to mediate ER stress–induced apoptosis, increased CHOP expression andnuclear translocation preceded DNA fragmentation(TUNEL) in human IAPP–expressing cells (Fig. 1C vs. D).

FIG. 3. ER stress marker GADD153/CHOP is induced in thepancreatic �-cells of HIP rats. A: IHC staining demonstratesthe colocalization of CHOP (arrow) with insulin-positive�-cells. The arrow and inset indicate the nuclear CHOP inHIP but not wild-type rats. The arrow head shows a perinu-clear cytosolic CHOP. For quantification, see Table 2. B:Western blot analysis (three independent immunoblotting)confirms abundant CHOP (29 kD) in islets from HIP rats andonly a very low expression level in wild-type rats. Bar scalerepresents 10 �m. M, molecular weight.

TABLE 2Percentage of �-cells that expressed ER stress markers inpancreatic islets from HIP and wild-type rats at 5 month of age

Marker HIP rats (%) Wild-type rats (%) P

Caspase-12 58 � 8.0 7.3 � 3.0 0.05CHOP 48 � 9.8 4.9 � 1.4 0.05Nuclear CHOP 0.5 � 0.2 0.0 � 0.0 0.05

Data are means � SEM (n � 3) from the quantifications of caspase-12–positive �-cells in Fig. 2 and CHOP–positive �-cells in Fig. 3. Pvalues were obtained by t test.



Moreover, nuclear CHOP was colocalized with TUNEL inAd-hIAPP EGFP cells (Fig. 1E). Furthermore, after siRNAwas used to knockdown CHOP expression by 81%, Ad-hIAPP EGFP–induced cleavage of caspase-3 was reducedby 68% (Fig. 1F).Human IAPP–induced expression of caspase-12 andCHOP in HIP rats. To affirm that human IAPP–inducedER stress occurs in primary �-cells in vivo, we examinedthe HIP rat, a well-characterized model of type 2 diabetes,characterized by islet pathology comparable with that inhumans with type 2 diabetes (12). The HIP rat is transgenicfor human IAPP, expressing high levels of human IAPP.Activated caspase-12, a marker of ER stress in rodents(22,23), was much more abundant in HIP than in wild-typeislets (Fig. 2B and Table 2). Caspase-12 was present in aperinuclear pattern and colocalized with insulin-positive�-cells but not other islet cell types (Fig. 2A and Table 2).Functional caspase-12 protein is not expressed in humantissues, owing to mutations leading to nine alternativesplicing sites (24), so caspase-12 was not examined inhuman tissue. The IHC caspase-12 findings in the HIP ratwere confirmed by Western blot analysis (Fig. 2B). Fur-thermore, islet lysates from HIP rats had a high expressionlevel of cleaved caspase-3 compared with that in isletlysates from wild-type rats (Fig. 2C). Specificity forcaspase-12 in IHC and Western blots was confirmed byusing two different anti–caspase-12 antibodies, one mono-clonal (22) and one polyclonal (21). TUNEL assaysshowed positive nuclear staining in HIP rats (Fig. 2D,panels D–F), which was rarely seen in wild-type rats (Fig.2D, panels A–C).

CHOP expression was increased in �-cells of HIP versuswild-type rats, predominantly in a perinuclear pattern (Fig.3A and Table 2). In HIP rats, CHOP expression wasoccasionally nuclear (Fig. 3A, inset and arrow), with afrequency of 0.5 � 0.2% of �-cell nuclei vs. none inwild-type rats (Table 2). While the frequency of �-cell

apoptosis was increased in the HIP versus wild-type rat,only a minority of �-cells are at any time apoptotic (Fig.2D) in the HIP rat at a frequency consistent with nuclearCHOP staining observed here. This finding of occasionalnuclear CHOP is also consistent with prior reports of ERstress–induced apoptosis in tissue rather than isolatedcells in culture (25). Western blot analysis of proteinlysates from isolated islets confirmed increased CHOPprotein expression in HIP rats (Fig. 3B).CHOP expression in human pancreasType 1 diabetes. Perinuclear �-cell CHOP expression wasrarely detected in cases of type 1 diabetes or in leannondiabetic humans (Figs. 4 and 5). This was true both inthe recent-onset case of type 1 diabetes (Fig. 4A–C) and inthe cases of long-standing type 1 diabetes (Fig. 4D–F). Thefrequency of occasional CHOP-positive cells in the nonen-docrine pancreas was comparable in all groups, includingthe cases of type 1 diabetes (Fig. 4).Impact of obesity. Perinuclear cytoplasmic CHOP ex-pression was rarely detected in �-cells of lean nondiabetichumans (Fig. 5A–C) but more frequently in obese nondia-betic humans (Fig. 5D–F) (2.6 � 2.0 vs. 14.6 � 3.0%, P �0.05 [see Fig. 6]). Nuclear CHOP was not detected in�-cells from lean nondiabetic cases and was only veryrarely detected in obese nondiabetic cases.Type 2 diabetes. The frequency of perinuclear cytoplas-mic CHOP expression was nonsignificantly increased inobese cases with type 2 diabetes compared with obesenondiabetic controls (18.5 � 3.6 vs. 14.6 � 3.0%, respec-tively; P � 0.2) (Fig. 6). Within the islet, this perinuclearcytoplasmic CHOP expression was confined to �-cells andspecifically not detected in -cells (data not shown). �-Cellnuclear CHOP was detected six times more frequently(0.49 � 0.17 vs. 0.08 � 0.04%, P � 0.05) in type 2 diabeticcases compared with obese nondiabetic controls (Fig.7A–E) (by high-power light microscopy and by laserconfocal microscopy) (Figs. 6 and 7F).

FIG. 4. CHOP is not expressed in type 1 diabetic human pancreas. A–C: Representative microphotograph of pancreatic islet from a human withrecent-onset type 1 diabetes, with T-cell infiltrate marked by an arrow head. D–F: Representative micrographs of a human with long-standing type1 diabetes. In contrast with type 2 diabetes, CHOP immunostaining was rarely seen in islets of recent-onset or long-standing type 1 diabetes andno more frequently than in nondiabetic controls. Occasional CHOP-positive cells (B and C, white arrows) were present in parenchymal tissue ata frequency comparable with that in nondiabetic pancreas. Scale bar � 10 �m.



DISCUSSION

In the present study, we report that the �-cell ER stresscharacterized by increased expression and nuclear trans-location of CHOP is a characteristic of type 2 but not type1 diabetes in humans. Moreover, we report that highexpression levels of human IAPP induces ER stress–mediated �-cell apoptosis that can be overcome by inhibi-tion of CHOP expression. These data imply that differentmechanisms initiate �-cell apoptosis in type 1 and type 2diabetes and suggest that toxic oligomers of IAPP mayplay a role in ER stress–induced apoptosis in type 2diabetes.

The adaptive mechanisms that protect the ER fromaccumulation of unfolded proteins are often collectivelyreferred to as the unfolded protein response (UPR) andhave been studied for the most part in cell culture (25–29).The initial UPR depends on the property of three sensoryproteins—PERK, Ire1, and ATF6—to detect the presenceof unfolded proteins in the ER. This activates a sequenceof events that globally decreases translation of major ERclient proteins, increases transcription and translation ofER chaperone proteins (e.g., BiP), and increases expres-sion of proteins involved in clearance of misfolded ERproteins. The importance of these protective mechanisms

FIG. 5. Expression of perinuclear CHOP and insulin in human pancreas. Representative microphotographs of pancreatic islets from leannondiabetic (LN) (A–C), obese nondiabetic (OB) (D–F), and obese type 2 diabetic (G–I) subjects obtained at autopsy and stained for insulin (A,D, and G, green), CHOP (B, E, and H, red), and nucleus (DAPI, blue). Perinuclear CHOP expression is increased in �-cells of obese nondiabeticcompared with lean nondiabetic cases but is most abundant in type 2 diabetic subjects. See Fig. 6 for the quantification. Scale bar � 10 �m. Thearrows show perinuclear staining.



for pancreatic �-cells is illustrated by the increased �-cellapoptosis initiated by ER stress in PERK/ mice (30).Degenerative disease states characterized by ER stress aretypically characterized by formation of protein oligomers(20). Once protein oligomers form, it appears that cellstransition from the protective UPR to programmed celldeath or apoptosis through ER stress. One of the mostwell-characterized mediators of ER stress–induced apo-ptosis is the transcriptional regulator CHOP. For example,ER aggregates of mutant insulin in the Akita mouse induceER stress–mediated �-cell apoptosis mediated by CHOP(31).

We report that in nondiabetic humans, obesity wascharacterized by increased perinuclear CHOP expression,although this was not accompanied by an increase in the

frequency of nuclear CHOP translocation. Another recentreport has shown increased cytoplasmic CHOP detectedby IHC in humans with type 2 diabetes, but nuclear CHOPwas not reported (32). The �-cell workload has beenshown to predict vulnerability to apoptosis from severalproapoptotic stimuli, and �-cell rest has been shown todelay or prevent onset of diabetes (33), perhaps mediatedin part by differential expression of CHOP. The perinuclearexpression of CHOP was increased to an even greaterextent in obese individuals with type 2 diabetes, but thiswas now also accompanied by an increase in the fre-quency of �-cells with CHOP nuclear translocation. Takentogether, these data imply that obesity increases transcrip-tion of CHOP but that nuclear translocation of CHOP isprovoked by a factor (or factors) present in type 2 diabetesbut not in obesity. Based on these data, the transcriptionalregulation of CHOP expression and the factors(s) that leadto its nuclear translocation are apparently distinct. Furtherstudies are required to establish what factors mediate thetranslocation of cytoplasmic to nuclear CHOP and toestablish the relative roles of cytoplasmic CHOP versusthose of nuclear CHOP in promoting ER stress–inducedapoptosis. Most studies of ER stress–induced apoptosishave been carried out in cell lines where the ER stress–induced apoptosis appears to be related to nuclear CHOP(as we indeed observe in the INS-1 cell studies presentedhere). If, as we postulate, nuclear translocated CHOP isrequired to mediate ER stress–induced apoptosis, then inthe setting of increased CHOP expression in obesity, thefactor(s) that trigger nuclear translocation of CHOP wouldpresumably be amplified.

Several mechanisms have been proposed as initiatorsof �-cell apoptosis in type 2 diabetes, including expo-sure to high glucose or fatty acids (7,8). Exposure ofhuman �-cells to high glucose may initiate apoptosisthrough generation of oxygen free radicals or inductionof expression of interleukin-1� (7,9). However, theabsence of ER stress noted here in �-cells from humanswith either long-standing or recent-onset type 1 diabetesin cases with documented increased �-cell apoptosis(1,6) argues against the primacy of glucose and/orcytokine toxicity as the underlying mechanisms sub-serving ER stress in type 2 diabetes. Also, althoughmarked obesity appears to increase the �-cell expres-sion of CHOP, neither �-cell apoptosis (3) nor nucleartranslocation of CHOP was increased in islets of pa-tients with morbid obesity without type 2 diabetes,implying that increased free fatty acids alone are un-likely to be responsible for increased ER stress or �-cellapoptosis in type 2 diabetes, although exposure of a�-cell to toxic free fatty acids in culture does initiate ERstress (32). In reality, the pathways that induce apopto-sis are closely interrelated. For example, any damage tothe mitochondria (through the so-called intrinsic path-way of apoptosis) will likely lead to failure of the ER tosufficiently fold proteins and potentially provoke the ERstress pathway. The ER stress pathway has been shownto induce activation of proinflammatory cytokines thatmay induce the so-called extrinsic pathways throughdeath receptors (17,34). The ER and mitochondria ex-change membranes, and thus formation of membranepermeant oligomers, such as those that form intracellu-larly in human IAPP transgenic mice (19), may lead todisruption of mitochondria and induce the intrinsicpathway of ER stress.

The most clearly established causes of ER stress–

FIG. 6. Expression of CHOP in human pancreatic �-cells. Percentage of�-cells with CHOP expression (A: cytoplasmic CHOP; B: nuclearCHOP) detected by immunofluorescence in lean nondiabetic (LN) (n �7), obese nondiabetic (OB) (n � 12), obese type 2 diabetic (T2DM)(n � 14), and type 1 diabetic (T1DM) (n � 7) cases obtained atautopsy. See Figs. 4, 5, and 7 for the images of perinuclear and nuclearCHOP staining, respectively. *P < 0.05 compared with lean nondia-betic; **P < 0.01 compared with lean nondiabetic; and #P < 0.05compared with obese nondiabetic subjects.



FIG. 7. Expression of perinu-clear and nuclear CHOP inhuman pancreatic �-cells.Pancreatic tissue from anobese case with type 2 diabe-tes was stained for insulin(A, green), CHOP (B, red),and nuclei blue (C, blue) withDAPI. D: Merged image (40�).E: High-power (63�) image ofpancreatic �-cells. F: Confocalimage of �-cells from anotherobese case with type 2 diabe-tes, stained for insulin (green)and CHOP (red). White arrowsindicate CHOP-positive nucleiin �-cells. See Fig. 6 for quan-tification. Scale bar � 10 �m.



induced apoptosis involve protein mutations leading toprotein misfolding, for example, the missense mutationin insulin 2 in the Akita mouse (35) and peripheralneuropathy as a result of mutations in the P0 protein(36). High expression rates of human IAPP in mice leadsto the formation of toxic intracellular IAPP oligomers(37), implying misfolding and/or intracellular traffickingof human IAPP beyond a critical threshold of expres-sion. The islet in type 2 diabetes is characterized by isletamyloid derived from IAPP (2,3), implying that proteinmisfolding is characteristic of this disease, but, of note,islet amyloid is not a feature of type 1 diabetes (2).Proof of principal of a potential role of IAPP misfoldingplaying a role in type 2 diabetes in humans is the rareS20G mutation in IAPP that increases its oligomeric andcytotoxic properties (38) and is associated with in-creased risk for type 2 diabetes (39). This is analogousto point mutations in A�P (Alzheimer’s � protein)1– 42leading to hereditary forms of Alzheimer’s disease (40).However, in most cases of type 2 diabetes, as in mostcases of Alzheimer’s disease, there are no mutations ineither the IAPP transcript or promoter region (41) or,indeed, in the transcript or promoter region of A�P1– 42,respectively (42). Also, only species with a primarysequence of IAPP with the propensity to form mem-brane-permeant toxic oligomers (humans, cats, andmonkeys) spontaneously develop type 2 diabetes (10).However, it is not yet established why IAPP formsaggregates in humans who develop type 2 diabetes orwhether these aggregates underpin the increased �-cellapoptosis in this disease. Moreover, while �-cell apopto-sis is increased in type 2 diabetes, this only involves arelative minority of �-cells at any time, so loss of �-cellmass is gradual.

In contrast, a high proportion (the majority) of cellsexposed to inducers of ER stress in culture have nuclearCHOP staining and undergo apoptosis within 24 h, con-firmed by us in the positive control studies with INS-1 cellsexposed to thapsigargin (an ER stress inducer), as pre-sented here (25). If the frequency of ER stress–inducedapoptosis in vivo approached the proportion of cellsimpacted in studies of ER stress induced in culture, theconsequences would be short-lived and devastating. For-tunately, the loss of �-cell function (and presumably �-cellmass) in type 2 diabetes is a much more gradual process(11), and the frequency of nuclear CHOP observed here iscomparable with that of �-cell apoptosis (3,4), as previ-ously reported in type 2 diabetes. These findings empha-size the important protective mechanisms present in vivoto prevent ER stress–induced apoptosis in most humans(for example, obese individuals who do not develop type 2diabetes).

In summary, we report that increased expression ofhuman IAPP in INS-1 cells and human IAPP transgenic ratsleads to ER stress–induced apoptosis. We note that thisER stress–induced activation of caspase-3 is overcome byknockdown of CHOP. We also report that obesity inhumans is characterized by increased perinuclear cyto-plasmic CHOP expression, although by very minimal nu-clear translocation of CHOP. In contrast, in type 2diabetes, increased cytoplasmic CHOP is accompanied bynuclear translocation of CHOP. These observations haveled us to a novel hypothesis as to how obesity mightpredispose to �-cell ER stress. If, as we postulate, nuclearCHOP mediates �-cell apoptosis, then if the unknownsignal that causes nuclear CHOP translocation were trig-

gered, this signal would be amplified in the context ofobesity. Future studies will be required to test that hypoth-esis. Meanwhile, the present data support the postulatethat in patients with obesity and type 2 diabetes, ER stressis likely an important mechanism leading to increased�-cell apoptosis. In this context, the islet in type 2 diabetesis further characterized as revealing features of an un-folded protein disease, in common with most neurodegen-erative diseases.

ACKNOWLEDGMENTS

These studies were funded by the National Institutes ofHealth (grants DK 59579 [to P.C.B.] and DK29953 [toR.A.R.]) and by the Larry Hillblom Foundation.

We are grateful to Aleksey Matveyenko and HeatherGerber for their excellent technical support, Anil Bhushanand Kathrin Maedler for helpful suggestions, and BonnieLui for excellent editorial assistance.

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original article - diabetes€¦ · nuclear chop was not detected in lean nondiabetic and rare in...

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