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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0079
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Right arrow Diabetes and Insulin
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 12 6678-6686
Copyright © 2005 by The Endocrine Society

Activation of Peroxisome Proliferator-Activated Receptor-{gamma} by Rosiglitazone Protects Human Islet Cells against Human Islet Amyloid Polypeptide Toxicity by a Phosphatidylinositol 3'-Kinase-Dependent Pathway

Chia-Yu Lin, Tatyana Gurlo, Leena Haataja, Willa A. Hsueh and Peter C. Butler

Larry Hillblom Islet Research Center, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095-7073

Address all correspondence and requests for reprints to: Dr. Peter C. Butler, Larry Hillblom Islet Research Center, Division of Endocrinology, David Geffen School of Medicine at University of California Los Angeles, 900A Weyburn Place North, Los Angeles, California 90095-7073. E-mail: pbutler{at}mednet.ucla.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Type 2 diabetes mellitus (T2DM) is characterized by a deficit in ß-cell mass, increased ß-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). Human IAPP (h-IAPP) applied to ß-cells forms toxic oligomers that induce apoptosis. Thiazolidinediones, ligands of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}), can delay the onset of T2DM.

Objective: We questioned whether activation of endogenous PPAR-{gamma} in human islets by rosiglitazone (RSG) inhibits h-IAPP-induced islet cell death and, if so, by which mechanism.

Methods and Results: Vehicle or h-IAPP was applied to human islets with or without RSG (10 and 50 µM) for 48 h. A 2-fold increase in the number of terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling-positive nuclei was detected in h-IAPP-treated human islets (P < 0.001). RSG (10 and 50 µM) prevented h-IAPP-induced apoptosis in human islets (P < 0.001). Thioflavin T binding assays confirmed that this effect was not mediated by interference with h-IAPP oligomerization. Expression of dominant negative PPAR-{gamma} in human islets prevented the protective effect of RSG. RSG activation of PPAR-{gamma} resulted in downstream activation of the serine/threonine protein kinase Akt, an outcome that was inhibited by a specific phosphatidylinositol 3-kinase inhibitor, which ablated RSG protection against h-IAPP-induced islet cell apoptosis.

Conclusion: We conclude that in human islets, activation of PPAR-{gamma} inhibits h-IAPP-induced islet cell apoptosis, and this action is at least in part mediated through activation of the phosphatidylinositol 3'-kinase-Akt cascade. If this action is present in vivo, then thiazolidinediones have the potential to decrease ß-cell apoptosis in T2DM and reduce loss of ß-cell mass.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 2 DIABETES mellitus (T2DM) is increasing in epidemic proportions, and strategies for its prevention are under investigation in clinical trials. An important candidate for the prevention of T2DM is the thiazolidinedione (TZD) class of compounds. TZDs are high affinity ligands for peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}), a member of the nuclear hormone receptor superfamily of ligand-activated transcriptional factors (1). The first clinically available TZD, troglitazone, reduced the incidence of diabetes in women with a history of gestational diabetes at high risk of T2DM, an action that appeared to be related to decreased demands on pancreatic islets (2).

The islet in T2DM is characterized by a deficit in ß-cells resulting from increased ß-cell apoptosis (3). The mechanisms underlying the increased frequency of ß-cell apoptosis are unknown, although several have been proposed, including actions of toxic oligomers of islet amyloid polypeptide (IAPP) (4, 5, 6). IAPP is a 37-amino acid peptide coexpressed and secreted with insulin by ß-cells (7). Human IAPP (h-IAPP) has the propensity to form toxic oligomers that induce ß-cell apoptosis (8). Addition of h-IAPP to cells in culture results in the formation of IAPP oligomers that are cytotoxic (3, 4, 5, 6). Because PPAR-{gamma} ligands are reported to preserve islet cell mass in diabetes-prone obese rats (9), we hypothesized that TZDs might have a direct action on islets to decrease h-IAPP-induced apoptosis.

Activation of PPAR-{gamma} improves insulin signaling in human adipocytes, as evidenced by inducing the expression of p85{alpha} regulatory subunit of phosphatidylinositol 3'-kinase (PI3 kinase) (10, 11) and by increasing insulin-induced PI3 kinase and Akt activities (10, 11). The level of phosphorylated Akt is increased in skeletal muscle and adipose tissue of humans after TZD treatment (12, 13, 14), and administration of rosiglitazone (RSG) to diabetes-prone obese Zucker rats results in amplification of insulin-stimulated Akt phosphorylation (15). Overexpression of Akt in mouse ß-cells increases ß-cell mass and survival and improves glucose tolerance and resistance to experimental diabetes (16). An important function of activated PI3 kinase is inhibition of apoptosis, and Akt is a good candidate for mediating PI3 kinase-dependent cell survival responses. Akt is a serine-threonine kinase that is activated by PI3 kinase in response to insulin as well as to various growth factors. It is well established that Akt mediates some of the metabolic actions of insulin in peripheral tissues. Activation of Akt also results in the phosphorylation of various downstream protein targets affecting cell cycle and proliferation and intracellular apoptotic pathways (17).

In the present study we addressed the following questions. First, does the PPAR-{gamma} ligand RSG inhibit h-IAPP toxicity in human islets? Second, does this action require PPAR-{gamma} activation, or is it a PPAR-{gamma}-independent action? Third, if RSG is protective against h-IAPP-induced apoptosis, is this action mediated through the PI3 kinase pathway?


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Design

Human islets were cultured in the presence of 40 µM h-IAPP vs. vehicle for 48 h before paraformaldehyde fixation and measurement of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. Having established that h-IAPP applied by this protocol does increase islet cell apoptosis, we repeated these experiments in the presence of 10 and 50 µM RSG to investigate whether RSG protects islet cells against h-IAPP-induced toxicity. These studies demonstrated that RSG is indeed protective against h-IAPP-induced toxicity, and we thereafter examined the potential mechanisms by which this protective effect might be mediated.

To establish whether RSG prevents h-IAPP toxicity by inhibiting aggregation of h-IAPP, we used a thioflavin T (TFT) binding assay to detect h-IAPP oligomerization in the presence and absence of RSG. To establish whether RSG actions are mediated through the PPAR-{gamma} activation, we transduced human islets with dominant negative (dn) PPAR-{gamma} adenovirus to abolish PPAR-{gamma}-mediated activation. Finally, to establish that the protect action of RSG is mediated through activation of PI3 kinase, we performed h-IAPP cytotoxicity experiments in the presence and absence of RSG and a specific PI3 kinase inhibitor, LY294002, at 50 µM.

Preparation of h-IAPP, RSG, and LY294002

Lyophilized h-IAPP (from Dr. C. G. Glabe, Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, CA) was reconstituted at 0.5% acetic acid and vortexed to prepare a 1-mM stock solution, which was then applied to culture medium to generate a final concentration of 40 µM. Vehicle was 0.5% acetic acid added to culture medium in the same manner, but without h-IAPP. The acetic acid concentration in culture medium (vehicle equal to h-IAPP) was less than 0.02%. RSG (from GlaxoSmithKline, Research Triangle Park, NC) was dissolved in dimethylsulfoxide (DMSO) to make 100 mM stock and diluted in culture medium to obtain final concentrations of 10 and 50 µM. LY294002 (Cell Signaling Technology, Inc., Beverly, CA) was also prepared in DMSO to make 10 mM stock and diluted in culture medium to a final concentration of 50 µM. The final concentration of DMSO in culture medium was less than 0.2%.

Human islet culture

Human pancreatic islets were isolated from the whole pancreas of heart-beating organ donors (10 men and one woman; 45.1 ± 2.7 yr old; body mass index, 28.7 ± 1.3 kg/m2) mostly by the Diabetes Institute for Immunology and Transplantation, University of Minnesota (Minneapolis, MN; Bernhard J. Hering), as well as University of Miami and University of Illinois at Chicago. Donors were nondiabetic. After isolation, islets were maintained in RPMI 1640 culture medium (5.5 mM glucose) supplemented with 10% fetal bovine serum at 37 C in humidified air containing 5% CO2. Experiments were performed 2–3 d after shipment to Los Angeles. In each of the five independent experiments, replicate aliquots of approximately 30 islets/chamber slide were studied under the different experimental conditions described. Chamber slides were previously coated with a human tumor bladder-9 matrix as previously described (6, 18).

TUNEL assay

The TUNEL technique detects the free 3-hydroxy strand breaks resulting from DNA degradation. Human islet cultures in chamber slides were fixed and permeabilized, followed by TUNEL staining, which was performed according to the manufacturer’s instructions (In Situ Cell Death Detection Kit, AP, Roche, Indianapolis, IN). Stained islet images were then taken by mounting chamber slides onto the motorized stage (H107, ProScan, Prior Scientific, Cambridge, UK) of an inverted microscope (Inverted System Microscope IX 70, Olympus, Melville, NY) with an analog camera (3-CCD camera, Optronics International, Chelmsford, MA), connected to a Hewlett-Packard computer (Palo Alto, CA). Islets pictures were taken using a x10 objective. Apoptotic cells were counted in each islet, and islet area was measured using image process software, Image-Pro Plus (Media Cybernetics, Silver Spring, MD). The number of TUNEL-positive apoptotic cells per islet was normalized to islet area. The results were expressed as a percentage of the corresponding control value.

TFT binding assay

The same stock h-IAPP and vehicle solutions used in the islet studies was used to prepare vehicle only vs. 40 µM h-IAPP in 10 mM phosphate buffer. RSG (50 µM) was similarly prepared. The samples of 40 µM h-IAPP and vehicle were incubated at 37 C with or without 50 µM RSG for 48 h. Aliquots of each preparation were collected at 0, 16, 24, and 48 h for analysis of TFT binding. A 20-µl sample from each preparation was added to a 1-cm path length cuvette containing 480 µl 5 µM thioflavin prepared in 50 mM glycine buffer. Fluorescence was measured for 5 min in each sample using a spectrofluorometer with excitation and emission wavelengths of 450 and 482 nm, respectively. The final concentrations of h-IAPP and RSG used in these TFT binding assays were the same as those used in islet experiments.

Recombinant adenovirus vector with dnPPAR-{gamma}

The dominant negative PPAR-{gamma}1 was constructed by mutating the full-length human PPAR-{gamma} expression vector obtained from Alex Elbrecht (Merck Research Laboratories, Rahway, NJ) (19). The Leu468 and Glu471 in PPAR-{gamma} were site-mutated into Ala to create dnPPAR-{gamma}. The dnPPAR-{gamma} DNA fragment was cloned into the type 5 adenoviral vector (Adeno-X Expression System, BD Clontech, Palo Alto, CA). Recombinant adenovirus was generated as described previously (20), expressing full-length dnPPAR-{gamma}. The recombinant adenovirus was expanded in HEK293 cells (American Type Culture Collection, Manassas, VA) and purified using a BD Adeno-X Virus Purification Kit (BD Clontech). Adenovirus vector with green fluorescence protein expression was used as a virus infection control.

Western blotting

Islets were cultured in suspension in culture medium containing 5.5 mM glucose, supplemented with 10% fetal bovine serum. On the day of the experiments, medium was changed, and groups of islets were treated with vehicle or the specific PI3 kinase inhibitor, LY294002 (50 µM), for 1 h before addition of vehicle, h-IAPP (40 µM), and/or RSG (10 and 50 µM) for 48 h. The preparations of h-IAPP, RSG, and LY294002 were the same as for the toxicity experiments described above. At the end of the incubation, islets were washed in PBS and collected in Laemmli sample buffer. Lysates were boiled, then frozen at –80 C until assayed. Protein content was analyzed by a DC protein assay from Bio-Rad Laboratories (Hercules, CA). Equal amounts of protein samples were loaded onto 4–15% SDS-PAGE gels. Proteins were then electrically transferred to a polyvinylidene fluoride membrane. After transfer, membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBS-T) at room temperature for 1 h. Primary antibody incubation was performed overnight at 4 C. Rabbit polyclonal antiphosphorylated serine 473 residue of Akt antibody, anti-Akt, PI3 kinase p85, nuclear factor-{kappa}B (NF{kappa}B) p65, ß-actin (Cell Signaling Technology, Inc.) were used at 1:1000 dilutions, PPAR-{gamma} and insulin receptor-ß (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used at 1:1000 and 1:200 dilutions, respectively, and antiinsulin receptor substrate-2 (anti-IRS2; Upstate Biotechnology, Inc., Lake Placid, NY) at a 1:1000 dilution, followed by incubation with horseradish-peroxidase-linked antirabbit IgG (Zymed Laboratories, Inc., South San Francisco, CA). After washing in TBS-T, the blots were developed using enhanced chemiluminescence (Bio-Rad Laboratories) and detected by an Autochemi system (UVP, Inc., Upland, CA). Membranes were stripped in stripping buffer (Pierce Chemical Co., Rockford, IL) and reprobed with primary antibody. Data were normalized to ß-actin.

NF{kappa}B activation

Human islets were cultured in suspension with h-IAPP and/or RSG for 2 d. At the end of experiment, islets were washed with PBS and fixed in 4% paraformaldehyde. After which, islets were resuspended in Histogel (Richard-Allan Scientific, Kalamazoo, MI) followed by rapid centrifugation and paraffin embedding. For immunostaining, sections were deparaffinized in toluene, rehydrated in grades of alcohol, and 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-100/3% BSA/TBS. Primary antibodies mouse anti-NF{kappa}B p65 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and guinea pig anti-insulin (Zymed Laboratories) were diluted in 1:100 in the antibody solution with 0.2% Tween 20/3% BSA/TBS. Donkey-derived secondary antibodies conjugated to Cy3 and fluorescein isothiocyanate were diluted 1:200 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). All slides were mounted with Vectashield (Vector Laboratories). Fluorescent slides were viewed using a Leica DM6000 microscope (Deerfield, IL) and images acquired using Openlab (Improvision, Inc., Lexington, MA) software.

Glucose-stimulated insulin secretion

Approximately 35 human islets/dish were cultured in suspension for 48 h with h-IAPP and/or RSG, then transferred to Krebs-Ringer bicarbonate buffer, 0.2% human serum albumin, and 20 mM HEPES containing 4 mM glucose for 1 h, followed by 1 h with 16 mM glucose. Krebs-Ringer bicarbonate buffer was collected at the end of the basal glucose and glucose-stimulated periods for measurement of insulin secretion as previously described (21, 22).

Statistical analysis

Data are presented as the mean ± SEM and were analyzed by two-tailed t test or ANOVA for multiple group comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RSG protects human islets from h-IAPP toxicity

Apoptosis was increased by approximately 2.2-fold (P < 0.001) in islets exposed to h-IAPP (Fig. 1Go). Islets treated with RSG (10 and 50 µM) and h-IAPP had a markedly lower frequency of apoptosis than islets treated with h-IAPP alone (P < 0.001), implying that RSG protected islets from the cytotoxic actions of h-IAPP. RSG alone had no effect on apoptosis compared with control islets.



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  		FIG. 1. RSG inhibited h-IAPP-induced toxicity. Top, TUNEL staining of human islets treated with vehicle control (A), h-IAPP (B), and h-IAPP and RSG (C) for 48 h. Bottom, Bar graph of TUNEL-positive cell expressed as a percentage of the control. Treatment with h-IAPP significantly increased apoptosis compared with control values (P < 0.001). RSG prevented h-IAPP toxicity (P < 0.001). A specific PI3 kinase inhibitor, LY294002, decreased the protective effect of RSG (P < 0.01).

 
Lack of effect of RSG on h-IAPP oligomerization

As expected, an aqueous solution of h-IAPP showed TFT binding that was maximal by approximately 16 h, indicating IAPP oligomer formation (Fig. 2Go). Neither control solutions nor solutions containing RSG alone showed TFT binding. RSG and h-IAPP, at concentrations comparable to those used in the islet cytotoxicity studies and over the same time course, showed comparable TFT binding to h-IAPP alone. We conclude that RSG inhibition of h-IAPP-induced apoptosis is not accomplished by inhibition of h-IAPP oligomerization.



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  		FIG. 2. RSG did not interfere with amyloidogenesis of h-IAPP in the TFT binding assay. Both h-IAPP and h-IAPP plus RSG showed increased fluorescence signals, indicating IAPP oligomer formation, whereas there were only background signals in control and RSG groups.

 
RSG activation of Akt

The ratio of phosphorylated Akt/Akt expression levels in human islets was increased by RSG alone (50 µM, 1.6 ± 0.2-fold; 10 µM, 1.5 ± 0.1-fold; P < 0.05 vs. control) or h-IAPP plus RSG (50 µM, 1.5 ± 0.2-fold; 10 µM, 1.3 ± 0.1-fold; P < 0.05 vs. control; Fig. 3Go). However, h-IAPP treatment alone did not change the ratio of phosphorylated Akt/Akt. Pretreatment of islets with a PI3 kinase-specific inhibitor, LY294002, significantly inhibited RSG-induced Akt phosphorylation in the presence of h-IAPP (50 µM RSG, 0.9 ± 0.1-fold; 10 µM RSG, 0.6 ± 0.1-fold; P < 0.05). Interestingly, PI3 kinase inhibitor also abolished the protective effect of RSG from h-IAPP toxicity (P < 0.01; Fig. 1Go). There was a 1.8-fold increase in cell death compared with the control (P < 0.001), suggesting that activation of the PI3 kinase-Akt pathway is one of mechanisms by which RSG protects human islets from h-IAPP toxicity.



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  		FIG. 3. Increased Akt phosphorylation after RSG treatment in the presence or absence of h-IAPP compared with control values after 48 h (P < 0.05; upper panel, 50 µM RSG; lower panel, 10 µM RSG). The PI3 kinase inhibitor, LY294002, blocked the activation of Akt expression. Treatment with h-IAPP alone did not change Akt phosphorylation.

 
Insulin signaling upstream of Akt

IRS2 protein expression was increased by 10 µM RSG treatment in human islets (P < 0.05; Fig. 4Go). Protein expression of insulin receptor-ß (data not shown) and PI3 kinase p85 did not change significantly with RSG treatment. Treatment with h-IAPP did not affect insulin receptor-ß, but enhanced IRS2 and PI3 kinase p85 protein expression (P < 0.05). RSG and h-IAPP together had an additive effect on insulin receptor-ß, IRS2, and PI3 kinase p85 expression levels (P < 0.05), whereas the presence of LY294002 in addition to RSG and h-IAPP caused an even greater level of IRS2 expression (P < 0.01), presumably due to feedback-induced up-regulation of insulin signaling.



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  		FIG. 4. RSG enhanced the protein expression of IRS2 in the presence and absence of h-IAPP (P < 0.05). PI3 kinase p85 expression was increased in the presence of RSG and h-IAPP (P < 0.05), but not RSG alone. The specific PI3 kinase blocker, LY294002, caused a feedback up-regulation of IRS2 (P < 0.01). Treatment with h-IAPP also increased IRS2 and PI3 kinase p85 levels (P < 0.05).

 
NF{kappa}B activation

Treatment with h-IAPP increased NF{kappa}B p65 protein expression in islet extracts (P < 0.05). However, this was not modified in the presence of RSG or RSG plus LY294002 (Fig. 5Go). To evaluate the proportion of these NF{kappa}B that underwent nuclear translocation, we performed immunocytochemistry of human islets for NF{kappa}B. Under all conditions studied, only approximately 1–3% of ß-cells were positive for NF{kappa}B nuclear translocation. h-IAPP treatment did not increase NF{kappa}B nuclear translocation, and RSG did not alter it.



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  		FIG. 5. Treatment with h-IAPP increased the NF{kappa}B p65 protein level (P < 0.05), which was not modified by addition of RSG (top). Double immunostaining for insulin (green) and NF{kappa}B p65 (red) in human islets after treatment with h-IAPP and/or RSG for 2 d is shown. The arrows indicate ß-cell nuclei stained for activated NF{kappa}B.

 
Lack of protective effect of RSG on h-IAPP toxicity in human islets after dnPPAR-{gamma} misexpression

To examine the role of PPAR-{gamma} in mediating the inhibitory actions of RSG on h-IAPP toxicity in human islets, a mutant dnPPAR-{gamma} (L468A, E471A PPAR-{gamma}) was generated, and human islets were transduced with adenovirus carrying dnPPAR-{gamma}. Both endogenous PPAR-{gamma} and dnPPAR-{gamma} were detected by Western blot in human islets (Fig. 6Go). Misexpression of dnPPAR-{gamma} in human islets resulted in loss of the protective effect of RSG on h-IAPP-induced apoptosis (P < 0.05; Fig. 7Go, top), suggesting the requirement for PPAR-{gamma} activation and downstream gene transactivation for the protective effect of RSG. Also, the level of phosphorylated Akt was not increased after treatment with RSG plus h-IAPP in the presence of dnPPAR-{gamma} misexpression compared with that in the dnPPAR-{gamma} misexpression control (data not shown). There was a slight, but not significant, increase in apoptosis after infection with adeno-green fluorescence protein and dnPPAR-{gamma} compared with that in control noninfected islets. There was no difference between adenovirus transduction and dnPPAR-{gamma} misexpression, indicating that dnPPAR-{gamma} per se did not alter basal apoptosis (Fig. 7Go, bottom).



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  		FIG. 6. Endogenous PPAR-{gamma} expression (left) and misexpression of dnPPAR-{gamma} (right) in human islets.

 


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  		FIG. 7. Top, RSG did not prevent h-IAPP toxicity in dnPPAR-{gamma}-misexpressed human islets (P < 0.05). Bottom, Basal rate of apoptosis was not affected by dnPPAR-{gamma} misexpression per se.

 
RSG preserves glucose-stimulated insulin release in the presence of h-IAPP toxicity

Static incubation of human islets at 16 vs. 4 mM glucose for 1 h resulted in a 4-fold increase in insulin secretion by control islets. When islets were previously exposed to h-IAPP, the increment in insulin secretion by islets exposed to 16 vs. 4 mM glucose was absent. However, the increment was partially restored (P < 0.05) in islets cultured with h-IAPP and RSG (Fig. 8Go).



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  		FIG. 8. Left, Basal and glucose-stimulated insulin concentration. Right, Fold increase in insulin secretion with glucose stimulation. Glucose (16 mM) stimulated insulin secretion by untreated islets and islets treated with both h-IAPP and RSG or with RSG only compared with baseline (4 mM glucose; P < 0.05). However, glucose-stimulated insulin secretion from islets exposed to h-IAPP alone was markedly attenuated (and did not reach significance) compared with baseline.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we report that activation of PPAR-{gamma} decreases h-IAPP-induced apoptosis in human islets. This action appears to be at least in part dependent on activation of the PI3 kinase-Akt pathway. These results may have implications for the prevention of T2DM. T2DM is characterized by ß-cell dysfunction and a deficit in ß-cell mass. This deficiency appears to precede the development of T2DM and stems from increased loss of ß-cells due to apoptosis (3). Therefore, strategies to prevent T2DM might reasonably include efforts to prevent ß-cell apoptosis. In humans, several studies have suggested that PPAR-{gamma} ligands preserve or improve ß-cell function in subjects at risk of T2DM (2, 23). Because TZDs lower glucose and free fatty acid levels, it is difficult to dissect out the direct benefits of TZDs, because these factors also promote islet cell injury as well as increased ß-cell demand due to worsening peripheral insulin resistance (24, 25, 26). To address the potential direct effects of PPAR-{gamma} on a specific cause of islet cell apoptosis, we investigated whether RSG could prevent h-IAPP-induced apoptosis in human islets in culture.

IAPP-toxic oligomers have been implicated in increased ß-cell apoptosis in T2DM (5, 8, 27). The islet in T2DM, like the brain in several neurodegenerative diseases, is characterized by formation of amyloid deposits from locally expressed proteins and loss of tissue. Little is known about why these proteins with the propensity to form toxic oligomers do so in the setting of disease (such as Alzheimer’s ß-protein in Alzheimer’s disease, synuclein in Parkinson’s disease, and IAPP in T2DM), although the structures of the resulting oligomers formed in the various diseases appear to be remarkably similar (8). We suggested that h-IAPP oligomers might act by disruption of cell membranes based on electron micrograph appearances of disrupted cell membranes adjacent to h-IAPP oligomers in human insulinoma cells (28). Subsequently, we were able to show that h-IAPP oligomers do cause nonselective ion channels and may also fully disrupt membranes (5). h-IAPP oligomers added to human islets caused numerous h-IAPP oligomer-induced vesicles adjacent to the cell membrane, and this correlated with apoptosis (5). In contrast, addition of h-IAPP-derived amyloid fibrils to human islets had no action on cell membranes and did not induce apoptosis. Recently, it has become apparent that this membrane-disruptive property of the oligomers of amyloidogenic proteins (but not amyloid fibrils) is a common mechanism by which these toxic oligomers induce apoptosis (8, 29, 30). Also, it has recently been shown that the membrane-disruptive action of these oligomers leads to an influx of ionized calcium (or leakage of ionized calcium from endoplasmic reticulum stores if intracellular membranes are disrupted) (31). Using a thioflavin binding assay to detect h-IAPP oligomerization, we found that RSG had no effect on h-IAPP oligomerization. Therefore, RSG prevented apoptosis through direct actions on islet cells.

Once hyperglycemia develops in diabetes, high glucose per se is probably an important cause of increased ß-cell apoptosis (32). In a previous study with the TZD pioglitazone, it was shown that human islets in culture at a glucose concentration of 600 mg/dl (33.3 mM) had increased ß-cell apoptosis that was overcome by either pioglitazone or salicylate (33). This is consistent with the intriguing findings by the same group that glucose-induced ß-cell apoptosis is mediated through production of IL-1ß, which, in turn, is proapoptotic through activation of NF{kappa}B, a pathway that is also repressed by PPAR-{gamma} activation and salicylate (33, 34, 35). However, we found activated NF{kappa}B p65 very occasionally and comparably in both h-IAPP- and h-IAPP/RSG-treated islets, suggesting that h-IAPP-induced apoptosis is not mediated primarily by NF{kappa}B activation, and RSG does not protect islet cells from h-IAPP by inhibition of NF{kappa}B activation.

Like many other members of the nuclear receptor family, PPAR-{gamma} is a transcription factor. Upon ligand binding, the receptor undergoes a conformational change and forms a heterodimer with retinoid X receptor-{alpha}, binding to a specific DNA sequence, PPAR response element, and regulating many target genes involved in lipid and glucose metabolism. Ligand binding to PPAR-{gamma} results in the release of corepressors bound to the receptor and the recruitment of coactivators to initiate transcription of target genes (36, 37, 38). Although PPAR-{gamma} ligands change the conformation of the receptor, they have antiinflammatory and other effects that may not be receptor mediated (39, 40, 41). The dnPPAR-{gamma} mutant used here has been shown to inhibit the transcriptional activity of wild-type PPAR-{gamma} by impairing coactivator recruitment and corepressor release, while retaining ligand binding (20). Because this dnPPAR-{gamma} mutant prevented the inhibitory action of RSG on h-IAPP toxicity in human islets, this antiapoptotic effect of RSG is PPAR-{gamma} dependent. Moreover, although partially PPAR-{gamma}-deficient islets from high-fat-fed mice had impaired glucose-stimulated insulin secretion, this defect was reversed by pioglitazone treatment (42). In contrast when PPAR-{gamma} was absent in mouse islets, troglitazone failed to restore insulin secretion (43). Taken together, these studies suggest the importance of the action of PPAR-{gamma} in the ß-cell and also support the PPAR-{gamma}-dependent effect of RSG found in this study.

The PI3 kinase-Akt pathway plays an important role in cell survival vs. apoptosis, and several studies have suggested that PI3 kinase-Akt signaling mediates the survival of isolated islets or ß-cells (44, 45, 46). Glucose promotes pancreatic islet ß-cell survival through the PI3 kinase-Akt signaling pathway, an action believed to be accomplished through insulin release upon glucose stimulation. Moreover, promotion of ß-cell survival has been attributed to the PI3 kinase-Akt signaling pathway of paracrine insulin action in the ß-cell (47, 48, 49). Indeed, a ß-cell-specific insulin receptor knockout leads to impaired glucose-induced insulin secretion and glucose intolerance, and IRS2-deficient mice develop diabetes as a result of a reduction in ß-cell mass with subsequent increased ß-cell apoptosis (50, 51). Pancreatic regeneration is associated with an increase in IRS2 and activated Akt in proliferating duct cells (52). Overexpression of active Akt in ß-cells of transgenic mice leads to expansion of ß-cell mass due to an increase in both cell size and number (16). Additionally, RSG has been shown to prevent fatty acid-induced inhibition of glucose-stimulated insulin secretion in human islets (53), which may be mediated through the IRS2-associated PI3 kinase pathway (54). However, it has also been shown that the presence of activated PPAR-{gamma} stimulates fatty acid oxidation, but decreases glucose oxidation, consequently contributing to the defective glucose-stimulated insulin secretion in rat pancreatic islets (55). Treatment with RSG in the present study enhanced IRS2 protein and Akt activation. Taken together, these findings support a central role of the PI3 kinase-Akt signaling pathway of insulin signaling for the survival of ß-cells.

Indeed, given our observation that a specific PI3 kinase inhibitor prevented the antiapoptotic effect of RSG, it is likely that the PI3 kinase-Akt pathway plays a role in RSG-induced protection of islet cells from h-IAPP-induced apoptosis. Consistent with this, islets cultured with RSG increased activated Akt, which was abolished by a specific PI3 kinase blocker. However, LY294002, at 50 µM, did not completely block the effect of RSG on h-IAPP toxicity, but did completely deplete phospho-Akt levels, suggesting that RSG may activate other protective mechanisms. We observed an increase in IRS2 protein expression in response to PI3 kinase inhibition. This is consistent with the reported PI3 kinase-dependent feedback inhibition of IRS2 mRNA expression observed previously in primary liver cells, as well as breast and kidney cancer cell lines (56, 57, 58).

Although the present study design allows direct evaluation of the actions of RSG in protection of human islet cells, several limitations should be noted. First, there is never any assurance that actions observed in cultured islets ex vivo would also be present in vivo. It is necessary to culture human islets in vitro with a relatively high concentration of the drug of interest. In vivo each cell is exposed to vasculature, but in vitro the islet cells are present in an aggregate of approximately 2000, with no vasculature. Second, it is not yet established beyond a doubt whether h-IAPP plays an important role in the high frequency of ß-cell apoptosis present in T2DM. Furthermore, it is unknown by what mechanism h-IAPP induces ß-cell apoptosis in humans (if it does) and whether this is comparable to that present when h-IAPP oligomers are added to islets in culture. In a recent in vivo study, Hull and colleagues (59) reported that RSG treatment of mice transgenic for h-IAPP decreased the extent of islet amyloid derived from h-IAPP. That study is difficult to interpret. First, this h-IAPP-transgenic model apparently does not develop diabetes. Second, islet amyloid is not the form of h-IAPP that induces ß-cell death in h-IAPP-transgenic rodents (4, 27, 60). This is actually supported by the study by Hull et al. (59), because the h-IAPP transgenic mice developed islet amyloid, but not diabetes, and RSG treatment reduced islet amyloid, but did not alter blood glucose concentrations. Third, no data were provided about the effects of RSG on ß-cell apoptosis, presumably because ß-cell apoptosis is not increased in this nondiabetic h-IAPP-transgenic murine model. It is therefore difficult to interpret the relevance of the reported effects of RSG on islet amyloid in that particular murine model on any direct or indirect actions that RSG may have on islets in humans.

In conclusion, we report that activation of PPAR-{gamma} by RSG decreases h-IAPP-induced apoptosis in human islets to potentially preserve ß-cell mass. This effect is at least partly mediated through the effects of PPAR-{gamma} activation on the PI3 kinase-Akt pathway. These data are consistent with the prevention of T2DM in high-risk women with troglitazone (2, 23). With current technology, it is impossible to determine whether PPAR-{gamma} ligands preserve ß-cell mass in vivo in humans. Nevertheless, these studies support the inclusion of TZDs in additional prospective clinical studies of people at risk of T2DM aimed at prevention or delay of diabetes.


   Acknowledgments
 
We appreciate the helpful comments of our colleagues at the Larry Hillblom Islet Research Center, University of California Los Angeles (Drs. Anil Bhushan, Alexandra Butler, and Kathrin Maedler) and Dr. Christopher Rhodes (Pacific Northern Research Institute, Seattle, Washington). We thank Ryan Galasso for performing the human insulin ELISA assay. We also thank Dr. B. J. Hering (University of Minnesota Diabetes Institute for Immunology and Transplantation) for human islets, and Dr. A Hayek (Whittier Institute, University of California-San Diego) for HTB-9 cells, as well as Drs. Ralf Langen and Sajith Jayasinghe (Biochemistry and Molecular Biology, Zilkha Neurogenetic Institute, University of Southern California) for assistance with the thioflavin T assay.


   Footnotes
 
This work was supported by National Institutes of Health Grant DK-61539 and the Larry Hillblom Foundation.

First Published Online October 4, 2005

Abbreviations: DMSO, Dimethylsulfoxide; dn, dominant negative; h-IAPP, human IAPP; IAPP, islet amyloid polypeptide; IRS, insulin receptor substrate; NF{kappa}B, nuclear factor-{kappa}B; PI3 kinase, phosphatidylinositide 3'-kinase; PPAR-{gamma}, peroxisome proliferator-activated receptor-{gamma}; RSG, rosiglitazone; TBS, Tris-buffered saline; TBS-T, TBS with 0.1% Tween 20; T2DM, type 2 diabetes mellitus; TFT, thioflavin T; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling; TZD, thiazolidinedione.

Received January 13, 2005.

Accepted September 26, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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