OBJECTIVE—Treatment of diabetic patients by pancreatic islet transplantation often requires the use of islets from two to four donors to produce insulin independence in a single recipient. Following isolation and transplantation, islets are susceptible to apoptosis, which limits their function and probably long-term islet graft survival.

RESEARCH DESIGN AND METHODS—To address this issue, we examined the effect of the cell-permeable apoptosis inhibitor pentapeptide Val-Pro-Met-Leu-Lys, V5, on pancreatic islets in a mouse model.

RESULTS—V5 treatment upregulated expression of anti-apoptotic proteins Bcl-2 and XIAP (X-linked inhibitor of apoptosis protein) by more than 3- and 11-fold and downregulated expression of apoptosis-inducing proteins Bax, Bad, and nuclear factor-κB–p65 by 10, 30, and nearly 50%, respectively. Treatment improved the recovered islet mass following collagenase digestion and isolation by 44% and in vitro glucose-responsive insulin secretion nearly fourfold. Following transplantation in streptozotocin-induced diabetic mice, 150 V5-treated islet equivalents functioned as well as 450 control untreated islet equivalents in normalizing blood glucose.

CONCLUSIONS—These studies indicate that inhibition of apoptosis by V5 significantly improves islet function following isolation and improves islet graft function following transplantation. Use of this reagent in clinical islet transplantation could have a dramatic impact on the number of patients that might benefit from this therapy and could affect long-term graft survival.

Pancreatic islet transplantation holds great promise as a treatment for type 1 diabetes. Insulin independence has been accomplished using a glucocorticoid-free immunosuppression regimen but often requires transplantation of islets from two to four donors (13). Since there is a considerable shortage of pancreas donors suitable for islet isolation, relatively few diabetic patients have benefited from this form of therapy. More effective recovery of islets from donor pancreata would dramatically increase the number of patients that could be treated by islet transplantation and could improve long-term graft survival (46).

Following transplantation, islets undergo apoptosis and necrosis from transient local hypoxia, a lack of nutrient support (7,8), and hyperglycemia-induced toxicity (9,10). While use of fibroblast growth factor-2 (FGF-2) at the time of transplantation improves revascularization of islet grafts and facilitates their engraftment (11), this approach addresses only part of the problem. Collagenase digestion of the pancreas has been shown to induce apoptosis of isolated islets from anoikis and loss of cell-matrix interactions (12,13). In addition, islets express proinflammatory nuclear factor-κB (NF-κB)–dependent genes after isolation, amplifying apoptosis signaling and potentially inducing immunological rejection (1416). Optimization of the isolation process to reduce islet stress has failed to improve recovery, leaving uncontrolled apoptosis as the main cause of poor islet yield (3,13,17).

Investigators have attempted to prevent β-cell apoptosis by transferring antiapoptosis molecules (A20, Bcl-2, the Iκβ [inhibitor of κβ] repressor, and X-linked inhibitor of apoptosis protein [XIAP]) and growth factors (hepatocyte and vascular endothelial) into islet grafts (1827). In most cases, these genes have been delivered by recombinant adenovirus. This approach, however, is not without potential risk (28). Use of IGF-II (29), leptin (30), and 17 β-estradiol (31) has improved islet mass recovery and viability but has not affected the number of islets that are needed following transplantation to correct diabetes in mice. The caspase-3 inhibitor Z-DEVD-fmk (32) improves islet recovery and viability but inhibits only caspase-3. Whereas these methods have helped reduce apoptosis during islet isolation, few methods have been developed to reduce apoptosis after islet transplantation.

We examined the efficacy of the cell-permeable pentapeptide apoptosis inhibitor V5, which inhibits a wide range of caspases, on improving pancreatic islet recovery. This molecule binds Bax and prevents mitochondrial cytochrome c translocation (33), resulting in global inhibition of caspases through activation of NF-κB–dependent and BH1–4 genes (14,15,22). In this study, we demonstrate that culture of isolated islets with V5 improved islet recovery and their capacity for glucose-responsive insulin secretion. Use of FGF-2 and V5 decreased the number of islets needed to correct diabetes following transplantation threefold and allowed routine correction of glucose homeostasis in mice using islets from a single donor.

Peptide synthesis and preparation.

Synthesis of a V5 was carried out at Sigma Genosis (Ishikari, Japan). The purity of the material was 98.8%, and the total amount of the product was 48.9 mg. Dried peptide powders were stored at −80°C and dissolved in fresh pure water for the experiments.

Islet isolation and culture.

Male inbred Balb/C mice, 20 g and 10–12 weeks old, were used as pancreas donors. All the experiments performed were approved by the institutional ethical committee and were conducted according to its guidelines. Mouse islet isolation was performed with Hank's balanced salt solution (HBSS) (GibcoBRL, Grand island, NY), containing 2 mg/ml type-V collagenase (Sigma-Aldrich, St. Louis, MO), 2 mg/ml soybean trypsin inhibitor (Sigma-Aldrich), and 0.2% BSA (Sigma-Aldrich), using a modified Gotoh's method with Histopaque 1077-RPMI 1640 medium gradient (Sigma-Aldrich).

Islets were handpicked up under a microscope and cultured with RPMI-1640 (GibcoBRL) at 37°C and 5% CO2 for in vitro analyses. Freshly isolated islets were used immediately for transplantation experiments. Islet viability was evaluated using a Live & Dead detection kit (Molecular Probes, Eugene, OR) in accordance with the manufacturer's instructions. Purity of islets was assessed by dithizone staining, and islet equivalents' yield was determined using a phase-contrast microscopy with a squared calibrated grid. One islet equivalent was equal to a spherical islet of 150 μm in diameter. ATP content of islets, directly after isolation and following 24 h of culture, was measured at SRL (Tokyo, Japan) using 500 islet-equivalent aliquots per experiment in three separate studies.

Measurement of mitochondrial activity of islets.

Islets of 50 islet equivalents were cultured for 24 h with or without 100 μmol/l V5 in each well of six-well plates (BD Biosciences, San Jose, CA), and mitochondrial dehydrogenases activity of islets were comparatively measured using 0.5 mg/ml of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT reagent; Sigma). Freshly isolated islets were used as a positive control (34). Three independent experiments were performed.

Detection of apoptosis of islets by annexin-V expression.

Expression of annexin-V was measured in islets treated with or without 100 μmol/l V5 at 24 h of culture. Islets were washed twice with HBSS and dispersed to a single cell by gentle pipeting in trypsin-EDTA (Sigma-Aldrich). Single islet cells were washed twice with HBSS containing 10% newborn calf serum (Sigma-Aldrich) and labeled with an annexin V–enhanced green fluorescent protein apoptosis detection kit (MBL, Nagoya, Japan), according to the manufacturer's instructions, and analyzed by a MoFlo cell sorter (Dako-Cytomation, Tokyo, Japan). Apoptotic cells were identified by the fluorescence of enhanced green fluorescent protein. Freshly isolated islets were used as a control (3). Three independent experiments were performed.

Power blot analysis for apoptosis-associated molecules.

Islets (1,000 islet equivalents) were cultured with RPMI-1640-S with or without V5 (100 μmol/l) for 24 h. Then, islets were washed with HBSS twice, rinsed with ice-cold lysis buffer of PBS containing 1.0% Triton X-100 (Sigma-Aldrich), sonicated for 30 s, and placed on ice for 10 min. Lysates were centrifuged at 15,000 rpm for 10 min at 4°C to exclude cellular debris. Protein concentrates were collected and analyzed for 50 apoptosis-associated molecules according to the manufacture's protocol (Clonotech, Tokyo, Japan). Three independent experiments were performed.

Measurement of insulin secretion, insulin content, and stimulation index of islets.

Islets (10 islet equivalents/well of six-well plates) were cultured in RPMI-1640-S supplemented with or without V5 (100 μmol/l) in both standard and ultralow attachment plates (BD Bioscience, Tokyo, Japan). Islets were treated for 20 min in 2 ml of Krebs-Ringer balanced buffer (KRBB) (containing 143.0 mmol/l Na, 5.8 mmol/l K, 2.5 mmol/l Ca, 1.2 mmol/l Mg2, 124.1 mmol/l Cl, 1.2 mmol/l PO-4, 1.2 mmol/l SO-4, 25 mmol/l HCO-3, 10 mmol/l HEPES, 0.2% BSA, and 3.3 or 25 mmol/l glucose) at pH 7.4 for RPMI-1640-S equilibration. Insulin secretion of the islets was measured under static incubation using a Mercodia mouse insulin enzyme-linked immunosorbent assay kit (Uppsala, Sweden) at 0, 24, 72, 120, and 168 h, as previously reported (35,36). Briefly, islets were first incubated at 37°C and 5% CO2 for 2 h in KRBB with 3.3 mmol/l glucose, then in KRBB with 25 mmol/l glucose for 2 h, and finally in KRBB with 3.3 mmol/l glucose. Amount of insulin content of islets was measured at the end of static incubation. Three independent experiments were performed.

Transplantation experiments.

Female inbred Balb/C mice, 20 g and 10 weeks old, received a single intraperitoneal injection of 220 mg streptozotocin per kg body wt. Mice with blood glucose levels >360 mg/dl on a minimum of two consecutive measurements were selected as recipients (34). Gelatinized microspheres for islet transplantation (30–50 μm in size) containing both FGF-2 (100 ng) and V5 (100 μmol/l) or FGF-2 (100 ng) only were prepared through glutaraldehyde cross-linking of an aqueous gelatin solution, as previously reported (37). For transplantation, freshly isolated islets were suspended in 10 μl RMPI-1640-S medium, embedded with the gelatinized microspheres, and then transplanted under the kidney capsule of diabetic mice.

Diabetic mice were divided into the following four groups: 1) n = 21, transplantation with 150 islet equivalents obtained from one mouse prepared with FGF-2 only; 2) n = 21, transplantation with 150 islet equivalents obtained from one mouse prepared with both FGF-2 and V5; 3) n = 21, transplantation with 450 islet equivalents obtained from three mice prepared with FGF-2 only; and 4) n = 5, animals received no islet transplants. Normal healthy mice were used as a positive control (group 5; n = 5).

In vivo evaluation after islet transplantation in diabetic mice.

Blood glucose levels were monitored at regular intervals for 27 weeks. Normoglycemia was defined to be <126 mg/dl in at least two consecutive measurements. An intraperitoneal glucose tolerance test was performed at 24 weeks. Mice were fasted overnight and then glucose (1g/kg body wt) was injected intraperitoneally, as previously described (34). Nephrectomy was performed at 26 weeks in the mice of groups 1, 2, and 3, and total insulin content of the samples was measured per microgram graft (34).

Histological studies of kidneys bearing islet grafts.

Kidneys bearing islet grafts were removed at 3 days and at 26 weeks after transplantation (n = 3 from groups 1, 2, and 3), fixed in 10% formalin for 24 h, and embedded in paraffin for hematoxylin-eosin staining, insulin staining, and transferase-mediated dUTP nick-end labeling (TUNEL) staining. Serial-matched paraffin sections were used for these stainings. Polyclonal anti-insulin guinea pig primary antibodies (Dakocytomation) were applied. Then, secondary antibody phycoethrin-labeled anti–guinea pig (Amersham Biosciences) was added. Green fluorescent nuclear counterstaining was used for all the samples. Immunofluorescent stained slides were observed under a confocal laser-scanning microscope (LSM510; Carl Zeiss) (34). An in situ cell death detection tetramethylrhodamine red kit (Roche, Mannheim, Germany) was used for TUNEL staining.

PCR analysis.

For detection of inflammatory molecules, total RNA was extracted from kidneys bearing islet grafts 2 days after transplantation using RNA Trizol (Invitrogen), as previously reported (38). RT-PCR was performed at 22°C for 10 min and then at 42°C for 20 min using 1.0 μg RNA per reaction to ensure that the amount of cDNA amplified was proportional to the mRNA present in the original samples. The following specific primers were used: interleukin (IL)-1β (NM_008361), 5′-caggcaggcagtatcactca-3′ forward and 5′-agctcatatgggtccgacag-3′ reverse; tumor necrosis factor (TNF)-α (NM_01369), 5′-agtccgggcaggtctacttt-3′ forward and 5′-ggtcactgtcccagcatctt-3′ reverse; Bcl-2 (NM_009741), 5′-aggagcaggtgcctacaaga-3′ forward and 5′-gcattttcccaccactgtct-3′ reverse; and GAPDH (NM_008084) 5′-acccagaagactgtggatgg-3′ forward and 5′-cacattgggggtaggaacac-3′ reverse.

Immunoelectron microscopic examination of kidneys bearing islet grafts.

At 26 weeks, kidneys bearing islet grafts were harvested (n = 3 each from groups 1, 2, and 3) and fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 mol/l phosphate buffer at pH 7.4. Samples were embedded in LR White (London Resin, London, U.K.). Ultrathin sections on nickel grids were incubated with 6 mol/l urea in 0.1 mol/l glycine-HCl buffer (pH 3.5) for 5 min to etch the surface of sections. The grids were incubated with polyclonal anti-insulin–guinea pig primary antibodies (DakoCytomation) at 4°C overnight. After washing, the grids were incubated for 1.5 h with 10 nm colloidal gold-conjugated goat anti–guinea pig IgG (British Biocell International). The grids were washed, postfixed with 2% glutaraldehyde, rinsed with ddH2O, and dried. The sections were stained with 2% uranyl acetate for 15 min and 3% lead citrate for 1 min and observed with a Hitachi H-7100 transmission electron microscope (35).

Statistical analysis.

Results were expressed as means ± SE. For comparisons between two groups, the paired or unpaired Student's t test (two tailed) was used. For multiple comparisons, the one-way ANOVA was used. Kaplan-Meier method was used to calculate the survival data. A P value <0.05 was considered significant when determined by the Mann-Whitney U test.

Effect of treatment with V5 on islet viability and function, as well as parameters associated with apoptosis in vitro.

Pancreatic islet yield following isolation was 152.5 ± 3.46 islet equivalents per mouse. Islet viability immediately after isolation, and 12 and 24 h later in control culture, was 99.1 ± 0.7%, 81.0 ± 1.2%, and 72.0 ± 0.9%, respectively, whereas viability at the same time points after culture in the presence of 100 μmol/l V5 was 98.9 ± 0.6%, 95.4 ± 0.8%, and 93.5 ± 0.6%. To assess the effect of V5 on islets recovered under clinically relevant donor recovery conditions, we also evaluated the effect of V5 on mouse islets isolated in association with 12 h of cold ischemia and 30 min of warm ischemia. We found that V5 was effective in protecting mouse islets under these conditions (Table 1). Since apoptosis is often reflected in mitochondrial function, we assessed mitochondrial dehydrogenase activity 24 h after isolation. As shown in Fig. 1A, mitochondrial dehydrogenase activity was significantly greater in V5-treated islets (96.4 ± 1.5%) than that in control untreated islets (54.0 ± 5.7%). We also measured the expression of annexin-V, an early marker of apoptosis, 24 h after isolation. V5-treated islets expressed significantly less annexin-V (14.0%) than control untreated islets (58.5%) (Fig. 1B–D). A power blot was then performed to identify proteins significantly (more than twofold) altered in isolated islets after treatment for 24 h in V5. Power blot analysis screened over 50 proteins associated with apoptosis (Fig. 1E). Western blot analysis showed a 0.11-fold reduction in the expression of the pro-apoptotic protein Bax, a 0.34-fold reduction in Bad, and a 0.46-fold reduction in NF-κB–p65. Treatment of islets with V5 also generated an 11.76-fold upregulation in XIAP and a 3.31-fold increase in Bcl-2 expression (Fig. 1E and F).

To further investigate the effect of V5 on recovered islets, we analyzed glucose-responsive insulin secretion, or insulin secretion index, immediately after isolation and 24, 72, 120, and 168 h later. Since islets adherent to culture plates lose function quickly, analysis was performed on islets under both adherent and nonadherent conditions. V5-treated islets had a 2.7- to 3.7-fold higher insulin secretion index than control untreated islets at all time points under both adherent and nonadherent conditions (Fig. 2A and B), and V5-treated islets maintained their insulin content (128 ± 1 at 24 h and 76 ± 6 at 168 h under adherent culture conditions; 128 ± 3 at 24 h and 101 ± 5 at 168 h under nonadherent culture conditions) significantly better than untreated control islets (116 ± 5 at 24 h and 9 ± 2 at 168 h under adherent culture conditions; 117 ± 4 at 24 h and 80 ± 4 at 168 h under nonadherent culture conditions) (Fig. 2C and D), respectively.

Effect of V5 on recovered islets after transplantation.

To investigate the effect of V5 treatment on recovered islets after transplantation, we transplanted V5-treated and control untreated islets into streptozotocin-induced diabetic mice. Transplantation procedures induce a meaningful grade of apoptosis, dramatically reducing the islet engraftment capacities and its survival by induction of the inflammatory reactions. We examined the potential prevention of apoptosis in vivo by microspheres containing V5 and FGF-2 within the islets grafts, 3 days after transplantation, by insulin staining and TUNEL assay. We found that the number of insulin-positive cells was significantly maintained by V5 treatment (150 control untreated islet equivalents: 51 ± 9 cells/high power field vs. 150 V5-treated islet equivalents: 202 ± 23 cells/high power field; Fig. 3A–F and J). Notably, a significantly larger number of TUNEL-positive cells were observed in control untreated islet grafts than found in V5-treated islets (Fig. 3G–I and K). V5 treatment significantly reduced inflammatory molecule IL-1β and TNF-α gene expression and enhanced Bcl-2 gene expression in islet grafts (Fig. 3).

In consistency with this, following transplantation of 150 V5-treated islet equivalents (recovered from one donor), normoglycemia was achieved in all diabetic recipients within 12 days and 100% 6-month survival obtained (Fig. 4A and B). In contrast, transplantation of 150 control untreated islet equivalents (recovered from one donor) failed to tightly control blood glucose levels, and 60% of diabetic recipients died 6 months after transplantation. Transplantation of 450 control untreated islet equivalents (recovered from three donors) corrected hyperglycemia to the same degree as transplantation of 150 V5-treated islet equivalents and produced a similar degree of blood glucose control to that after transplantation of 150 V5-treated islet equivalents following glucose challenge (Fig. 4C). Removal of kidneys bearing islet grafts 26 weeks after transplantation produced hyperglycemia in all transplanted mice, indicating that the islet grafts were responsible for correction of diabetes. Histological analysis of V5-treated islet grafts showed a comparatively equal amount of both total insulin content (16.1 ± 0.4 μg/graft), compared with those (16.7 ± 1.4 μg/graft) of V5-untreated 450 islet equivalents (Fig. 4D), and size of the functional islet grafts (Fig. 5B, C, E, and F). Insulin immunoelectron microscopy 26 weeks after transplantation showed β- cells with numerous secretory granules and well-preserved ultrastructure organelles following V5-treatment (Fig. 5H and J), whereas control untreated islet grafts showed fewer β-cells, each containing fewer insulin secretory granules and organelles (Fig. 5G and J).

Type 1 diabetes results from the loss of insulin-producing pancreatic β-cells by β-cell–specific autoimmune responses. Pancreatic islet transplantation is one possible method for the cure of diabetes; however, the shortage of human donor pancreata limits the widespread application of this procedure (1,5,6). Because a relatively large β-cell mass from two to four donor pancreata is needed to achieve normoglycemia in the recipient, it is crucial to develop treatments that would reduce loss of transplanted islets due to apoptosis and maximize use of the limited amount of donor tissue. Isolation of human islets is very stressful on the cells as it disrupts cell-cell and cell-matrix interactions and results in islet apoptosis (9,12,13,17,32,38,39). Alterations in islet fine structure can be seen shortly after isolation and culture in vitro. Prevention of apoptosis has been a target to maintain islet mass for transplantation; for example, overexpression of XIAP (26) or A20 (23) in mouse islets by adenoviral vector–mediated delivery prevented early posttransplant apoptosis and reduced the islet cell mass needed to achieve normoglycemia. Although ex vivo gene transfer procedures using viral vectors are attractive, adenovirally transduced islets should be cultured to eliminate the risk of viral gene transfer to recipients. Thus, it would be beneficial to develop a simple, efficient method to protect islets from apoptosis and reduce the number of islets required for transplantation.

Bax is a member of the Bcl-2 family of proteins and plays a key role in the induction of apoptosis. In response to apoptotic stimuli, Bax translocates from the cytosol to mitochondria and causes release of apoptogenic factors (40). Inhibition of Bax would be extremely useful in islet culture immediately after isolation procedure. Ku70 plays an important role in DNA double-strand break repair in the nucleus (41). Ku70 binds Bax in the cytosol and inhibits its translocation into mitochondria (33). The Bax-binding domain of human Ku70 consists of residues 578–583, and a pentapeptide (i.e.,V5) contained within these residues is cell permeable and suppressed Bax-mediated apoptotic cell death in several types of human cells, including hepatoma Hep3B cells and myeloid 32D (EpoR wt) cells (14). We previously found that V5 treatment of monkey hepatocyte cultures improved differentiated function and prolonged cell survival (42). In this study, we investigated the effect of V5 on islet viability and functionality during islet isolation and transplantation into diabetic mice.

First, we examined the effect of V5 on apoptosis of islets in vitro. We found that fluorescein isothyocyanate–labeled V5 (100 μmol/l) was uniformly taken up by islet cells within 3 h when added into the culture, and no cytotoxic effects were observed with a dose ≤500 μmol/l (data not shown). Treatment of islets isolated from Balb/c mice with V5 peptide significantly increased viability and inhibited apoptosis.

The mitochondrial metabolite, succinate, is a key metabolic mediator of glucose-stimulated preproinsulin gene transcription and translation (43). Therefore, we examined mitochondrial function in V5-treated islets and found that mitochondrial function was increased by ∼42% compared with untreated islets. Preservation of mitochondrial function in β-cells is critical for preserving their capacity to produce, store, and secrete insulin. V5 treatment significantly enhanced ATP levels in both 12-h cold preserved and 30-min warm preserved islets (Table 1) and markedly reduced apoptosis. Consistent with these findings, glucose-responsive insulin secretion was also increased by 2.7- to 3.7-fold in V5-treated islets. Islets treated with V5 maintained the insulin content of freshly isolated islets, even after 1 week of culture. Since islet culture seems to be an important step in the islet transplantation, the use of V5 might constitute an important tool for maintaining more viable islets with enhanced insulin secretion over the conventional cultures, floating better than adherent culture with matrices. Second, we examined the expression of proteins involved in the regulation of apoptosis in V5-treated islets. We found that the expression of pro-apoptotic molecules Bax, Bad, and NF-κB was markedly reduced, and the expression of anti-apoptotic molecules XIAP and Bcl-2 was upregulated. XIAP has previously been shown to improve β-cell growth, survival, and metabolic function during stress (35,44,45) and affects Akt/protein kinase B phosphorylation (36,45), modulating Bad, caspase-9, Bcl-2, cyclic AMP–response element-binding protein, and insulin receptor substrate-1 downstream. These changes may help to protect V5-treated islets from apoptosis and increase graft survival. The beneficial effects of XIAP have been reported. Overexpression of XIAP markedly enhanced β-cell survival and functional recovery of islets in hypoxia- and cytokine-induced injury in vitro (27). Overexpression of XIAP in human islets reversed the negative effects of immunosuppressive drugs on insulin secretion and cell viability (46). Recently, it was reported that XIAP overexpression in human islets prevented posttransplant apoptosis and reduced the islet mass required to treat diabetes (26).

These promising in vitro results suggested that V5 treatment might preserve the islet mass in grafts and thus reduce the number of islets needed to obtain insulin independence. Local hypoxia and lack of nutrients can cause apoptosis in islet transplants (8). Early vascularization of islet grafts can overcome these problems and facilitate islet engraftment. We have previously found that use of gelatinized microspheres containing slow-release, cross-linked FGF-2 that persists for ∼2 weeks produced rapid islet revascularization at the site of implantation (37). In those mouse islet transplantation studies, islet graft function was improved by improved vascularization. However, we also encountered considerable transplantation-associated apoptosis that resulted in loss of islet mass (11). Therefore, in this study, we transplanted islets embedded in microspheres containing FGF-2, to enhance vascularization, and V5, to reduce apoptosis, and found that apoptosis was decreased and the number of insulin-positive cells was increased in grafts containing islets treated with V5. When we embedded 150 islet equivalents within FGF-2–conjugated microspheres along with V5, diabetes was remitted within 12 days in streptozotocin-induced diabetic mice, similar to the results seen in mice transplanted with 450 islet equivalents with FGF-2 only, suggesting that normoglycemia could be achieved with islets from a single donor if V5 is provided. Interestingly, V5 treatment significantly depressed inflammatory molecule IL-1β and TNF-α gene expression in islet grafts. We found that V5 treatment similarly affected allogenic islets following transplantation in preliminary studies (supplementary Fig. 1 [ available in an online appendix at http://dx.doi.org/10.2337/db06-1679]). We next plan to explore methods to protect transplanted islets from autoimmune attack and early recurrence of diabetes following allogeneic islet transplantation.

In conclusion, we have shown that treatment of islets with V5 increases islet viability, enhances islet function, and prevents apoptosis. Transplantation of islets along with FGF-2 and V5 allowed a smaller islet mass (single-donor pancreas) to be used for transplantation; normoglycemia was achieved and insulin content and islet function were preserved posttransplantation. Timed release of V5, perhaps by gelatinization, may result in long-term prevention of apoptosis and improve outcomes in human islet transplantation.

FIG. 1.

Mitochondrial function, annexin-V expression, and power blot assay of the islets treated with V5. A: Comparative measurements of mitochondrial function of the islets at 24 h by an MTT assay. V5-treated islets showed significantly better function than untreated islets [P < 0.01 for V5 (+): 96.4 ± 1.5%] and V5 (−): 54.0 ± 5.7%). The value for islets immediately after isolation was 100%. BD: Detection of annexin-V expression. V5-untreated islets revealed the high expression of annexin-V (58.5%) 24 h after isolation. The significantly lower expression of annexin-V (14.0%) was observed in the V5-treated islets at 24 h, which was relatively comparable with that of normal islets immediately after isolation (8.7%). E: List of the molecules assessed by a power blot study. F: The differential expression of the molecules in V5-treated or -untreated islets at 24 h. The marked reduction of the expression of pro-apoptotic proteins of Bax, Bad, and NF-κB p65 and the upregulated expression of XIAP and Bcl-2 were observed in V5-treated islets. The data are representative of three independent experiments.

FIG. 1.

Mitochondrial function, annexin-V expression, and power blot assay of the islets treated with V5. A: Comparative measurements of mitochondrial function of the islets at 24 h by an MTT assay. V5-treated islets showed significantly better function than untreated islets [P < 0.01 for V5 (+): 96.4 ± 1.5%] and V5 (−): 54.0 ± 5.7%). The value for islets immediately after isolation was 100%. BD: Detection of annexin-V expression. V5-untreated islets revealed the high expression of annexin-V (58.5%) 24 h after isolation. The significantly lower expression of annexin-V (14.0%) was observed in the V5-treated islets at 24 h, which was relatively comparable with that of normal islets immediately after isolation (8.7%). E: List of the molecules assessed by a power blot study. F: The differential expression of the molecules in V5-treated or -untreated islets at 24 h. The marked reduction of the expression of pro-apoptotic proteins of Bax, Bad, and NF-κB p65 and the upregulated expression of XIAP and Bcl-2 were observed in V5-treated islets. The data are representative of three independent experiments.

FIG. 2.

Insulin secretion index and insulin content of islets. A: V5-treated islets showed significantly better insulin secretion index than V5-untreated islets at days 1, 3, 5, and 7. C: The insulin content was significantly higher in V5-treated islets at days 1, 3, 5, and 7 compared with V5-untreated islets. These data are representative of three independent experiments. E: Treatment of V5 allowed significantly favorable maintenance of islet mass at days 1, 3, 5, and 7. BF: These findings, observed in adherent culture, were also confirmed in islets in floating culture using ultralow attachment dishes. IEQ, islet equivalent.

FIG. 2.

Insulin secretion index and insulin content of islets. A: V5-treated islets showed significantly better insulin secretion index than V5-untreated islets at days 1, 3, 5, and 7. C: The insulin content was significantly higher in V5-treated islets at days 1, 3, 5, and 7 compared with V5-untreated islets. These data are representative of three independent experiments. E: Treatment of V5 allowed significantly favorable maintenance of islet mass at days 1, 3, 5, and 7. BF: These findings, observed in adherent culture, were also confirmed in islets in floating culture using ultralow attachment dishes. IEQ, islet equivalent.

FIG. 3.

Histological study of islets transplanted into the subrenal capsule in diabetic mice at 3 days. AC (hematoxylin and eosin staining): A few clusters of islets were found in the graft of V5-untreated 150 islet equivalents (group 1: obtained from one donor), but much larger clusters were detected in that of V5-treated 150 islet equivalents (group 2: obtained from one donor). Amounts of islets were found in the graft of V5-untreated 450 islet equivalents (group 3: obtained from three donors), which served as a positive control. DF (insulin staining): A few insulin-expressing cells, indicated by a red signal, were observed in the group 1 graft; in contrast, the group 2 graft showed significantly more insulin-positive cells, in which insulin intensity was stronger than in the cells in the group 3 graft. The observation was confirmed by counting the cell numbers of 10 different sections of each sample (n = 3) (J). The number of insulin-positive cells was 51.3 ± 8.7 for group 1, 202.1 ± 23.4 for group 2, and 212.5 ± 51.1 for group 3. GI (TUNEL staining): Significantly higher ratios of TUNEL-positive cells, indicated by a red signal, were observed in the grafts of groups 1 and 3 compared with the group 2 graft. K: The number of TUNEL-positive cells was counted in 10 different sections of each sample (n = 3), and it was 91.2 ± 15.7 cells for group 1, 8.1 ± 3.1 for group 2, and 211.7 ± 37.6 for group 3. Scale bars = 200 μm. L: The number of double- (TUNEL- and insulin-) positive cells was counted in 10 different sections of each sample (n = 3), and it was 8.3 ± 2.0 cells for group 1, 5.1 ± 2.0 for group 2, and 22.9 ± 5.8 for group 3. Scale bars = 200 μm. M: V5 treatment significantly depressed inflammatory molecule IL-1β and TNF-α gene expression and enhanced Bcl-2 expression in islet grafts 2 days after transplantation.

FIG. 3.

Histological study of islets transplanted into the subrenal capsule in diabetic mice at 3 days. AC (hematoxylin and eosin staining): A few clusters of islets were found in the graft of V5-untreated 150 islet equivalents (group 1: obtained from one donor), but much larger clusters were detected in that of V5-treated 150 islet equivalents (group 2: obtained from one donor). Amounts of islets were found in the graft of V5-untreated 450 islet equivalents (group 3: obtained from three donors), which served as a positive control. DF (insulin staining): A few insulin-expressing cells, indicated by a red signal, were observed in the group 1 graft; in contrast, the group 2 graft showed significantly more insulin-positive cells, in which insulin intensity was stronger than in the cells in the group 3 graft. The observation was confirmed by counting the cell numbers of 10 different sections of each sample (n = 3) (J). The number of insulin-positive cells was 51.3 ± 8.7 for group 1, 202.1 ± 23.4 for group 2, and 212.5 ± 51.1 for group 3. GI (TUNEL staining): Significantly higher ratios of TUNEL-positive cells, indicated by a red signal, were observed in the grafts of groups 1 and 3 compared with the group 2 graft. K: The number of TUNEL-positive cells was counted in 10 different sections of each sample (n = 3), and it was 91.2 ± 15.7 cells for group 1, 8.1 ± 3.1 for group 2, and 211.7 ± 37.6 for group 3. Scale bars = 200 μm. L: The number of double- (TUNEL- and insulin-) positive cells was counted in 10 different sections of each sample (n = 3), and it was 8.3 ± 2.0 cells for group 1, 5.1 ± 2.0 for group 2, and 22.9 ± 5.8 for group 3. Scale bars = 200 μm. M: V5 treatment significantly depressed inflammatory molecule IL-1β and TNF-α gene expression and enhanced Bcl-2 expression in islet grafts 2 days after transplantation.

FIG. 4.

Blood glucose levels, survival rate, and glucose challenge tests after islet transplantation and insulin content of islet grafts. A: Transplantation of V5-treated 150 islet equivalents (group 2) perfectly controlled the blood glucose levels of diabetic mice for 6 months; levels were comparable with those observed in group 3. B: All mice in group 2 survived for 6 months. C: Intraperitoneal glucose tolerance test performed at 24 weeks. The mice with group 2 showed a normal profile of blood glucose levels after glucose challenge. D: Insulin content of islet grafts. Significantly higher insulin content was observed in the group 2 graft (16.1 ± 0.4 mg/graft), which was comparable with that of the group 3 graft (16.7 ± 1.4), in comparison with the group 1 graft (6.2 ± 0.2). IEQ, islet equivalent.

FIG. 4.

Blood glucose levels, survival rate, and glucose challenge tests after islet transplantation and insulin content of islet grafts. A: Transplantation of V5-treated 150 islet equivalents (group 2) perfectly controlled the blood glucose levels of diabetic mice for 6 months; levels were comparable with those observed in group 3. B: All mice in group 2 survived for 6 months. C: Intraperitoneal glucose tolerance test performed at 24 weeks. The mice with group 2 showed a normal profile of blood glucose levels after glucose challenge. D: Insulin content of islet grafts. Significantly higher insulin content was observed in the group 2 graft (16.1 ± 0.4 mg/graft), which was comparable with that of the group 3 graft (16.7 ± 1.4), in comparison with the group 1 graft (6.2 ± 0.2). IEQ, islet equivalent.

FIG. 5.

Histological study of islets transplanted into the subrenal capsule in diabetic mice at 6 months. AC (HE staining): Quite a few clusters of islets were found in the group 1 graft, but islet clusters were properly maintained in the group 2 graft, which was comparable with those in group 3 islets. Scale bars = 200 μm. DF (insulin staining): Significantly more insulin-positive cells were observed in the group 2 graft than in the group 1 graft, which were comparable with the group 3 graft. Scale bars = 200 μm. GJ (transmission electron microscopy–immunogold staining): Numerous insulin secretory granules and well-preserved ultrastructural organelles were observed in the group 2 graft (12.0 ± 1.2 granules/field) when the grafts of group 1 (3.4 ± 0.09) and group 3 (12.0 ± 1.5) were considered. Such insulin granules were present in the apical zone of the β-cells in the group 2 graft. Black arrows indicate the insulin secretory granules labeled by immunogold, and white arrows show the glucagons granules (J). Scale bars = 300 nm. These data are representative of three different samples.

FIG. 5.

Histological study of islets transplanted into the subrenal capsule in diabetic mice at 6 months. AC (HE staining): Quite a few clusters of islets were found in the group 1 graft, but islet clusters were properly maintained in the group 2 graft, which was comparable with those in group 3 islets. Scale bars = 200 μm. DF (insulin staining): Significantly more insulin-positive cells were observed in the group 2 graft than in the group 1 graft, which were comparable with the group 3 graft. Scale bars = 200 μm. GJ (transmission electron microscopy–immunogold staining): Numerous insulin secretory granules and well-preserved ultrastructural organelles were observed in the group 2 graft (12.0 ± 1.2 granules/field) when the grafts of group 1 (3.4 ± 0.09) and group 3 (12.0 ± 1.5) were considered. Such insulin granules were present in the apical zone of the β-cells in the group 2 graft. Black arrows indicate the insulin secretory granules labeled by immunogold, and white arrows show the glucagons granules (J). Scale bars = 300 nm. These data are representative of three different samples.

TABLE 1

Effect of V5 on mouse islets isolated in association with 12 h of cold ischemia and 30 min of warm ischemia

ExperimentsV5Islet yieldIslet viability (%)
ATP (pg/islet equivalent)
0 h12 h24 h0 h24 h
Cold ischemia 110.8 ± 9.4 93.3 ± 2.1 92.1 ± 2.3* 88.9 ± 3.8* 39.4 ± 8.2 129.3 ± 11.2* 
 − 108.3 ± 10.8 92.4 ± 3.7 71.4 ± 6.4* 57.3 ± 5.6* 40.2 ± 7.9 68.4 ± 8.4* 
Warm ischemia 83.5 ± 7.3 58.9 ± 10.6 46.0 ± 8.2§ 39.2 ± 7.9* 19.8 ± 3.5 45.9 ± 8.5* 
 − 82.3 ± 8.6 60.3 ± 9.2 37.8 ± 4.5 14.8 ± 4.7* 21.6 ± 2.8 16.1 ± 5.8* 
ExperimentsV5Islet yieldIslet viability (%)
ATP (pg/islet equivalent)
0 h12 h24 h0 h24 h
Cold ischemia 110.8 ± 9.4 93.3 ± 2.1 92.1 ± 2.3* 88.9 ± 3.8* 39.4 ± 8.2 129.3 ± 11.2* 
 − 108.3 ± 10.8 92.4 ± 3.7 71.4 ± 6.4* 57.3 ± 5.6* 40.2 ± 7.9 68.4 ± 8.4* 
Warm ischemia 83.5 ± 7.3 58.9 ± 10.6 46.0 ± 8.2§ 39.2 ± 7.9* 19.8 ± 3.5 45.9 ± 8.5* 
 − 82.3 ± 8.6 60.3 ± 9.2 37.8 ± 4.5 14.8 ± 4.7* 21.6 ± 2.8 16.1 ± 5.8* 

Data are means ± SE.

*

P < 0.01 for V5(+) vs. V5(−);

P < 0.05 for V5(+) vs. V5(−).

Published ahead of print at http://diabetes.diabetesjournals.org on 7 February 2007. DOI: 10.2337/db06-1679.

Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db06-1679.

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.

The work presented in this paper was supported in part by the Ministry of Education, Science, and Culture and the Ministry of Economy and Industry, Japan; by Life Science Project of 21st Century, Japan; by a Grant-in-Aid for Scientific Research (B) of the Japan Society for the Promotion of Science (to N.K.); by the American Diabetes Association; by National Institutes of Health Grant 1R21DK60192; and by Canadian Institutes of Health Research Grant 89687 (to H.S.J.).

We thank Dr. Ann Kyle for editorial assistance and Tae Yamanishi for her valuable technical assistance, providing with the histological assessment.

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