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Université de Montréal (R.R., J.D., N.H., J.-P.H.), Guy-Bernier Research Centre, Maisonneuve-Rosemont Hospital, Montréal, Québec, Canada H1T 2M4; and McGill University Health Center (L.R.), Montréal, Québec, Canada H3G 1A4
Address all correspondence and requests for reprints to: Jean-Pierre Hallé, M.D., Guy-Bernier Research Centre, Maisonneuve-Rosemont Hospital, 5415 boulevard de l’Assomption, Montréal, Québec, Canada H1T 2M4. E-mail: hallejp{at}videotron.ca.
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Abstract |
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Introduction |
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Islet cell mass is a function of balance between ß-cell apoptosis and necrosis, and islet neogenesis and ß-cell replication. Islet cells may be damaged during the isolation and encapsulation procedures, during in vitro culture, and after in vivo implantation (18, 19, 20, 21). Both apoptosis and necrosis occur during islet isolation and culture (18, 19, 20, 21). A recent publication suggests that necrosis predominates in vivo for islets encapsulated in large diameter (≥700 µm) microcapsules (16). We have proposed that islets require trophic support to maintain long-term viability and that this support is provided within the pancreas (22, 23). A prerequisite for successful islet transplantation is the reconstitution of the trophic support or, alternatively, the provision of relevant components (growth factors, extracellular matrix and duct cells including pluripotential precursor cells) necessary to increase islet cell mass (22, 23). We have recently shown that incubations of hamster islets with pancreatic duct cells or duct-conditioned medium prevents in vitro apoptosis and necrosis that occur after the islet isolation procedure (24). Duct-conditioned medium was shown to contain a small amount of nerve growth factor and much larger amounts of IGF-II, but no IGF-I (24). Incubations of islets with IGF-II alone have reproduced the effect of duct cells, and prevented apoptosis and necrosis (24). Hill and co-workers (25) had previously reported that a fall in the islet cell expression of IGF-II is temporally associated with the developmental apoptosis that occurs in neonatal rats. In addition, they have shown, in a transgenic mouse model in which IGF-II is overexpressed, that persistent circulating levels of IGF-II suppress neonatal islet cell apoptosis (26).
We hypothesized that pretransplant incubations of microencapsulated islets with IGF-II would have beneficial trophic effects on the maintenance of a functional islet cell mass. Potential mechanisms could include decreasing the susceptibility of microencapsulated islet cells to apoptosis and necrosis. The objective of the present study was to evaluate the effect of incubating microencapsulated islets with IGF-II on the in vitro and in vivo (post transplantation) survival of encapsulated islets, as well as to investigate the mechanisms of cell death involved. Our results indicate that in vitro incubations with IGF-II, during the pretransplantation period, prevent apoptosis and necrosis of encapsulated islet cells and allow normalization of blood glucose in streptozotocin (STZ)-induced diabetic mice, using a smaller number of encapsulated islets (i.e. <50%) than required in the absence of IGF-II.
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Materials and Methods |
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Islet isolation
Islets were isolated and purified from rat pancreases according to a previously described procedure (1). Briefly, pancreases were infused via the common bile duct with Hanks’ balanced salt solution, set apart, and minced on ice. Digestion was performed with type V collagenase (7.5 mg/ml; Sigma-Aldrich Ltd., Oakville, Ontario, Canada) for 5 min at 37 C. Islets were purified on a discontinuous Euroficoll gradient (Mediatech Inc., Herndon, VA), handpicked under an inverted light microscope, pooled and then separated to form four experimental groups: islets cultured without IGF-II and with 50, 100, and 500 ng/ml of IGF-II (ID Labs Inc., London, Ontario, Canada). Islets were cultured overnight in RPMI 1640 medium (Invitrogen Life Technologies, Inc., Burlington, Ontario, Canada) with 11 mM glucose supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Inc.), 1% penicillin-streptomycin-glutamine 100x solution (Invitrogen Life Technologies, Inc.) and with or without IGF-II in humidified air atmosphere containing 5% CO2. The IGF-II supplementation started the same day as islet isolation and lasted for the period of the in vitro study, and until the transplantation for the in vivo study.
Islet microencapsulation
In vitro experiments.
Islets from the four experimental groups were microencapsulated in purified Keltone LV sodium alginate (ISP Alginates Inc., San Diego, CA) under endotoxin-free conditions. The sodium alginate was purified according to a previously described method (27). Islets were mixed with 1.8% sodium alginate at a final concentration of 5000 islets/ml. Islets were entrapped in small alginate beads by extrusion of the alginate solution through a 25-gauge needle using an electrostatic droplet generator (28). The alginate beads fell into a 100-mM calcium lactate solution, which cross-linked the alginate to form gel beads. The microcapsule membrane was formed by successively soaking the alginate beads in 0.05% poly-L-lysine (Sigma-Aldrich) for 5 min and 0.18% alginate for 5 min. This procedure was performed at room temperature. Microencapsulated islets (microcapsule diameter: 300–350 µm) used for in vitro experiments were cultured for an additional 6 d in serum-free RPMI 1640 medium with 5.5 mM glucose supplemented with 1% penicillin-streptomycin-glutamine 100x solution and with or without IGF-II at 37 C in humidified air atmosphere containing 5% CO2. This prolonged time in serum-free culture medium (used only for in vitro experiments) was designed to evaluate the potential protective effect of IGF-II under conditions where islets have minimal nutrient supply. Islets are transplanted within 24 h after encapsulation. The culture medium was changed every second day as previously described (24).
In vivo experiments.
Poly-L-lysine was recently shown to impair microcapsule biocompatibility (17, 29, 30). For in vivo experiments, the encapsulation protocol was modified to exclude poly-L-lysine coating to allow the study of islet survival without the confounding effect of biocompatibility issues. In brief, islets were microencapsulated in purified LVG sodium alginate (FMC Biopolymer, Philadelphia, PA) cross-linked with 10 mM barium solution under endotoxin-free conditions. Microencapsulated islets used for transplantation experiments were cultured overnight in UltraCULTURE medium (BioWhittaker, Inc., Walkersville, MD) supplemented with 1% penicillin-streptomycin-glutamine 100x solution with or without 500 ng/ml IGF-II at 37 C in humidified air atmosphere containing 5% CO2. This protocol was used to evaluate the additional benefits provided by IGF-II over the results obtained in optimal conditions.
Microencapsulated islet morphology
Inverted light microscopy.
Aliquots of 150 islets per experimental condition were studied under an inverted microscope. The number of encapsulated islets with central necrosis was evaluated and expressed as the percentage of the total islet number as previously described (24). Islet diameter (micrometers) and the diameter (micrometers) of the area of necrosis were determined using a calibrated scale mounted in the eyepiece of the microscope.
Histology.
At the end of the incubation period, samples of encapsulated islets from each experimental group were entrapped in 2% low-melting agarose (Invitrogen Life Technologies, Inc.), fixed in 10% buffered formalin and embedded in paraffin. Serial sections (4 µm thick and 150 µm apart) were cut from each block and processed for routine light microscopy (staining with heamatoxylin and eosin) or for immunocytochemistry.
Microencapsulated islet cell viability and function
Fluorescence microscopy.
Fluorescence microscopy was used, as previously described (31, 32), to evaluate islet cell viability at the end of the incubation period. In brief, aliquots of 150 microencapsulated islets were stained with propidium iodide (0.5 µg/ml; Sigma-Aldrich), and acridine orange (2.4 ng/ml; Sigma-Aldrich) and the number of microencapsulated islets showing more than 50% of viable cells were counted and expressed as a percentage of the total.
Sodium 3'-[1-(phenyl-amino-carbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)-benzene sulfonic acid hydrate (XTT) assay.
Islet viability was also quantitatively assessed, at the end of the incubation period, using the XTT assay (Roche Diagnostics, Laval, Québec, Canada). An aliquot of 150–500 islet equivalents (one islet equivalent equals one 150 µm-diameter islet) was incubated in an RPMI 1640 medium with XTT salt in a 96-well plate for 24 h at 37 C in humidified air atmosphere containing 5% CO2. XTT is metabolized by mitochondria of viable cells to soluble formazan, which can be detected with an ELISA plate reader (absorbance wavelength: 450 nm; reference wavelength: 620 nm). The formazan production was normalized for 150 islet equivalents.
Apoptosis detection assay.
A TUNEL assay was used, as previously described (24), to identify apoptotic cells. Briefly, at the end of the incubation period, samples of microencapsulated islets were prepared for histology (see above) and the In Situ Cell Death Detection Kit (Roche Diagnostics) was used for labeling apoptotic cells. The sections were developed by immunocytochemistry using anti-fluorescein antibody conjugate with alkaline phosphatase conjugate and Fast Red substrate (Roche Diagnostics). To reduce nonspecific labeling, the sections were treated with Universal Protein Blocker (DAKO Diagnostics Inc., Mississauga, Ontario, Canada). The sections were counterstained with heamatoxylin. Approximately 800 cells from ten islets were counted in each group using image analysis software, and the apoptotic index was calculated (24). To identify which islet cells were apoptotic, the TUNEL assay and immunocytochemistry using antiinsulin antibody (DAKO Diagnostics Inc.) were performed on consecutive sections. The sections were immunostained using antiinsulin antibody (dilution 1:100) and streptavidin-biotin-peroxidase complex. To reduce nonspecific labeling, the sections were treated with Universal Protein Blocker. Insulin was detected using diaminobenzidine substrate (DAKO Diagnostics Inc.) to obtain a dark brown reaction product.
Perifusion (glucose stimulation of insulin secretion).
Aliquots of 150 islet equivalents were tested at the end of the incubation period using a perifusion procedure (Acusyst-S Cell Culture System; Forma Scientific, Marietta, OH). Islets were perifused at the rate of 1 ml/min for 1 h at 37 C with RPMI 1640 medium, which was gassed with 95% air-5% carbon dioxide, and maintained at pH 7.4. The RPMI 1640 medium contained 10% fetal bovine serum, 1% penicillin-streptomycin-glutamine 100x solution, and 3.3 mM basal glucose. Basal samples were collected at 15-min intervals for 60 min before the glucose concentration in the perifusate was raised to 16.5 mM, and effluent samples were also collected at 15-min intervals for 75 min. The medium was then supplemented with 10 mM theophylline (Sigma-Aldrich), and effluent samples were also collected at 15-min intervals for another 75 min. The islets were finally perifused with the basal glucose perifusate for 75 min with sample collections at 15-min intervals. All samples collected on ice were stored at frozen until RIA for insulin (Cederlane Laboratories Ltd., Hornby, Ontario, Canada). Data were presented as mean insulin-release stimulation index (SI). SIs were obtained by calculating the ratio of high-glucose-stimulated insulin release (16.5 mM glucose) on the basal insulin release (3.3 mM glucose).
Microencapsulated islet transplantation
Recipients.
Diabetes in mice was induced by ip injection of STZ (185 mg/kg body weight in sodium citrate buffer, pH 4.5; Sigma-Aldrich). Blood glucose of all mice rose above 20 mM within 2 d post injection of STZ. Daily sc ultralente insulin injections were used to maintain mice alive (Humulin U, 2 U/d·mouse, Eli Lilly, Toronto, Ontario, Canada). Diabetic mice (blood glucose > 20 mM for a successive 2 d) were used as recipients for microencapsulated islet transplantations 14 d after injection of STZ. Control groups included diabetic mice transplanted with nonencapsulated islets, and nontransplanted mice.
Transplantation.
After islet isolation, the islet preparation was carefully and uniformly suspended, and split in two equal aliquots, which were randomly assigned to the two experimental groups (incubations with and without IGF-II). After 24 h in culture with or without IGF-II, islets were microencapsulated, and incubated another 24 h, with or without IGF-II. Just before transplantation, islet-containing microcapsules were carefully and uniformly suspended, and aliquots of each group were counted, by a blinded observer, to determine the number of islet equivalents per volume using a calibrated scale mounted in the eyepiece of the microscope. Using the concentration of islets per volume, the islet preparation was separated to form the transplantation aliquots (150 and 300 islet equivalents). Microencapsulated or nonencapsulated (additional controls) rat islet aliquots were transplanted via a 16-gauge catheter into the peritoneal cavity of diabetic mice anesthetized with sodium pentobarbital (Somnotol, 50 mg/kg body weight; MTC Pharmaceutical, Cambridge, Ontario, Canada). The last insulin injection was given at the time of transplantation to minimize the hyperglycemic stress to which islets would be submitted during the first hours post transplantation.
Follow up of diabetic animals.
Recipient mice had free access to water and food (mouse colony chow 5018; Agribrands, Purina, Ontario, Canada). Blood glucose levels in samples obtained from mouse-tail veins were measured (Glucometer Elite, Bayer Inc., Toronto, Ontario, Canada) every day during the first week post transplantation and then at least once a week for the remainder of the study. Mice were considered normoglycemic when glucose levels were less than 11 mM on a consecutive 2 d. Mice were killed after 7 d if still diabetic or 7 d after reoccurrence of diabetes. In order not to introduce a bias supporting our hypothesis, mice (1 mouse in each IGF-II treated group) that died while normoglycemic were considered diabetic at the time of death.
Statistical analysis
All in vitro experiments were performed five times, and the results are expressed as mean ± SEM. The differences between experimental groups were analyzed by unpaired Student’s t test with P < 0.05 considered significant. The predetermined primary end-point of the in vivo experiments was the number of mice achieving normoglycemia after transplantation. The differences between experimental groups within a category were analyzed by 
2 test with P < 0.05 considered significant.
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Results |
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). Seven percent of the encapsulated islet population showed signs of very dark tissue located in the center of the islets with sharply demarcating borders from the surrounding islet tissue (Fig. 1, A and B; Table 1). This central area of necrosis was confirmed by routine histology showing signs of very dark tissue located in the center of the islets with sharply demarcating borders (data not shown). IGF-II culture supplementation, at concentrations of 100 and 500 ng/ml, significantly reduced the percentage of encapsulated islets with central necrosis in a dose-dependent manner: 46% (P < 0.01) and 69% (P < 0.001) reduction, respectively (Fig. 1, C and D; Table 1). The presence of necrosis was associated with islet size (Table 1), being more prevalent in islets larger than 200 µm.
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). The number of dead cells was reduced when islets were cultured with IGF-II (Fig. 2B). IGF-II supplementation significantly improved the percentage of viable encapsulated islets in a dose-dependent manner (Fig. 3A). A 51% and 83% increase in the percentage of viable encapsulated islets was observed when the cultured medium was supplemented with 100 and 500 ng/ml IGF-II, respectively (Fig. 3A; P = 0.052 and P < 0.01, respectively).
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). A 44% and 51% increase in formazan production was observed in encapsulated islet cultured with 100 and 500 ng/ml IGF-II respectively (Fig. 3B; P < 0.05 and P < 0.01, respectively).
Apoptosis studies.
The TUNEL assay was used to evaluate islet cell apoptosis on d 6 post encapsulation. Without IGF-II supplementation, a large number of islet cells showed evidence of apoptosis (Fig. 4A). Most TUNEL-positive cells were located in the inner 80% of the islet, suggesting that these were primarily ß-cells. Labeling of islets for insulin confirmed that most apoptotic cells were ß-cells (data not shown), although, as expected, some TUNEL-positive cells did not show immunoreactivity for insulin. IGF-II supplementation significantly reduced the number of TUNEL-positive cells (Fig. 4B) and the apoptotic index in a dose-dependent manner (Fig. 5). A 20, 29, and 33% reduction in the apoptotic index was observed in encapsulated islets cultured with 50, 100, and 500 ng/ml IGF-II, respectively (Fig. 5; P < 0.05, P < 0.005 and P < 0.001, respectively).
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In vivo studies
Islets of the same size were used for transplant experiments because both experimental groups (incubations with and without IGF-II) had similar ratio of islets on islet equivalents (1.25 ± 0.09 and 1.18 ± 0.06 respectively). None (0/12) of the STZ-induced diabetic mice transplanted ip with 300 nonencapsulated rat equivalent islets became normoglycemic (data not shown). One (1/12) of the STZ-induced diabetic mice transplanted with 300 nonencapsulated islet equivalents preincubated with IGF-II 500 ng/ml became normoglycemic (data not shown).
When, using a minimal mass model, 300 encapsulated islet equivalents not incubated with IGF-II were transplanted into diabetic mice, normoglycemia was induced in 92% (11/12) of recipients (Fig. 6A). However, progressive recurrence of hyperglycemia was observed, and only 67% (8/12) and 25% (3/12) remained normoglycemic at 45 and 143 d, respectively (Fig. 6A). In contrast, when 300 encapsulated islet equivalents incubated with IGF-II were used, 100% (12/12) of recipients became normoglycemic, and 100% (12/12) and 75% (6/8) remained normoglycemic at 45 and 143 d, respectively (Fig. 6A). From d 143, both groups began to gradually converge and became statistically indistinguishable; so at the end of the study, the percentages of normoglycemic mice were 25% (2/8) in the IGF-II treated group and 0% (0/11) in the control group (Fig. 6A). The differences were statistically significant from d 45–143 (Fig. 6A; P < 0.05). The average blood glucose levels remained normal (< 7 mM) until the end of the study (213 d) in the IGF-II-treated group whereas it progressively increased up to 14.7 ± 1.0 mM at 213 d without IGF-II incubations (Fig. 6B). The differences between the two groups were statistically significant from d 21–143 (Fig. 6B; P < 0.05). Again, after 143 d the two groups began to converge and became statistically indistinguishable (Fig. 6B).
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). The average blood glucose remained over 12 mM throughout the study (Fig. 6B). In contrast, after the transplantation of 150 islet equivalents preincubated with IGF-II, 100% (11/11) of the recipients became normoglycemic and from d 21–101 more than 80% (8/10) remained so (Fig. 6A). At d 143, 50% (5/10) of the recipients were normoglycemic (Fig. 6A). The average blood glucose remained within normal limits until d 171, slightly higher than in the group transplanted with 300 IGF-II preincubated islet equivalents but significantly lower than with 150 (or even 300) non-IGF-II preincubated islet equivalents (Fig. 6B). The differences between treatment groups in both parameters (% normoglycemic mice and average blood glucose) were important and significant throughout the study (Figs. 6, A and B; P < 0.05). |
Discussion |
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The results of fresh islet examinations, histological studies, fluorescence microscopy studies with propidium iodide and acridine orange staining, XTT assays, and apoptosis studies all indicated that IGF-II supplementation significantly improves in vitro encapsulated rat islet cell viability in a dose-dependent manner. This confirms and expands the findings of our previous in vitro studies with nonencapsulated hamster islets (24). The latter study showed that the pancreatic ductal epithelium, duct-conditioned medium, or IGF-II promotes islet cell survival (24).
There is an experimental advantage to using encapsulated (as opposed to free) islets for in vitro viability studies, particularly if islets have to be cultured for long periods. Nonviable free islets disintegrate and are washed-out during culture medium changes, which are done every second day. As a result, the actual number of dead cells may be underestimated. Encapsulated islets remain within microcapsules, even when most islet cells are dead. In the present study, islets incubated with or without different concentrations of IGF-II were all encapsulated, allowing for more reliable comparisons between treatment groups.
A dual staining fluorescence method has advantages over the simple trypan blue exclusion technique for viability studies on multicellular three-dimensional structures like islets (31, 32). The former method allows the identification of both living and dead cells, whereas the latter identifies only living cells, which may be misleading if peripheral cells are alive while central cells are dead. In addition, with the former method, dead cells and living cells may be localized within the islets. Results were expressed as a percentage of viable islets defined as an islet with more than 50% of viable cells, because, in culture, islets with more than 50% of dead cells usually do not recover and all cells eventually die. IGF-II supplementation had a dose-dependent protective effect on the viability of islets undergoing the potentially damaging isolation and encapsulation procedures and during culture for 6 d in minimally nutritive conditions (Figs. 1, 2, and 3A). These results were confirmed by the XTT assay showing that IGF-II supplementation had a dose-dependent positive effect on encapsulated islet cell survival (Fig. 3B).
Apoptosis largely predominated over necrosis. This is in contrast with the findings of De Vos (16), who reported that necrosis has been the principal mechanism of cell death in islets encapsulated in more than 700-µm diameter microcapsules. This discrepancy may be explained by microcapsule size. Considering the three-dimensional configuration of microcapsules, the more than 700-µm diameter microcapsules used by De Vos are 10-fold larger in volume than the 300–350 µm diameter microcapsules used in the present study. The use of smaller microcapsules considerably enhances the access of islets to nutrients and oxygen (33). It is noteworthy that, in the present study (Table 1), as well as in the De Vos study (16), central necrosis was located predominantly in larger islets, suggesting that deprivation of nutritional factors causes central necrosis (16). In spite of the damages induced by the encapsulation procedure, after 6 d in culture, the percentage of islets with necrosis was lower (7%) in the current study than that reported by Ilieva et al. (>15%) in nonencapsulated hamster islets (24). A better in vitro survival of encapsulated than nonencapsulated islet cells has been reported by others (9, 34), and previously observed in our laboratory (unpublished data). This may be explained by the formation of a favorable microenvironment in microcapsules. Another potential explanation is a difference between hamster and rat islets. Nevertheless, IGF-II incubations significantly decreased in a dose-dependent manner both the apoptosis and necrosis that occur following the islet isolation and encapsulation procedures and cell culture.
Encapsulated islets had an appropriate response to glucose stimulation with a biphasic insulin secretion that promptly returned to the basal rate on reduction of glucose to the basal level. The signaling pathways of insulin secretion appears to be preserved, at least in part, because in all conditions tested, when the secretagogue theophylline was introduced in the system, encapsulated islets increased their insulin secretion. The SI showed that IGF-II supplementation had a positive effect on encapsulated islet cell function. However, perifusion studies are known not to be predictive of the success of in vivo transplantation (35). Therefore, the definitive test for assessing islet function is the result of transplantation into diabetic animals.
The potential clinical significance of the results presented above was confirmed by transplantation studies in diabetic mice. For these in vivo studies, we chose to reduce the ex vivo incubation period to a minimum before transplantation, because prolonged culture time reduces islet function. In addition, this corresponds to clinical practice. Therefore, the total IGF-II exposure time was 48 h, approximately one half before and one half after the encapsulation procedure. UltraCULTURE medium was also used to avoid any reduction in encapsulated islet cell viability and to reduce exposure to xenoproteins. The combination of these conditions resulted in the transplantation of viable and functional encapsulated grafts. Because all islet preparations, including controls, were cultured in the same optimal conditions, except for the absence or the presence of 500 ng/ml IGF-II, the positive results represent the benefits provided by IGF-II incubations, in addition to the usual optimal procedures.
The most frequently used numbers of encapsulated islets for transplantation into diabetic mice have been 1000 or 500 (10, 11, 17, 36, 37, 38, 39, 40), which corresponds approximately to 40,000 and 20,000 islet equivalents/kg body weight respectively. In the present study, a minimal mass model, i.e. 300 and 150 islet equivalents, was used to be able to detect differences over a relatively short period, and to evaluate if the use of IGF-II could allow successful transplantations using a reduced islet cell mass. Without IGF-II incubations, the transplantation 150 encapsulated islet equivalents induced normoglycemia in only 50% of recipients, all of which, except one, presented a recurrence of diabetes within 2 months. Increasing the number of these islet equivalents to 300 allowed normalization of blood glucose in a higher proportion of recipients (92%), and the recurrence of diabetes was delayed but still observed in a progressive number of recipients up to 100% at the end of the study. With IGF-II incubations, the transplantation of 300 islet equivalents induced normoglycemia in all recipients and maintained normoglycemia up to d 143 in 75% of the recipients. The transplantation of 150 encapsulated islet equivalents induced normoglycemia in all recipients with only two recurrences up to 101 d post transplantation and 50% remaining euglycaemic at d 143. From d 73–101, the percentage of normoglycemic mice was significantly higher in the group treated with 150 IGF-II incubated islet equivalents than in the one treated with 300 non-IGF-II-incubated islet equivalents. The average blood glucose levels inversely paralleled these results. Therefore, IGF-II incubations decreased by more than a half the number of donors/pancreases required to inducing normoglycemia in diabetic mice.
The prolonged effect of IGF-II on islet survival could be explained either by a better preservation of the initial islet cell mass or by a proliferative or a functional component that allows 150 islet equivalents treated with IGF-II to perform better than 300 untreated islet equivalents. We believe that the former explanation is more likely for two reasons: 1) the four curves (of both parameters, percentage of normoglycemic mice and average blood glucose) are relatively parallel, suggesting that the speed of cell death/proliferation is similar; and 2) it is unlikely that in vitro 48-h incubations have a continuous in vivo proliferative effect many weeks after transplantation. However, further studies are required to better understand the mechanisms of such an important effect.
Successful human nonencapsulated islet transplantations have required two donor pancreases for most recipients, which represents an average total transplanted graft of 12,000 islet equivalents/kg body weight (2, 3). A much larger number of encapsulated islets than nonencapsulated islets have been required to normalize blood glucose. For example, 15,000–50,000 encapsulated islet equivalents/kg body weight have been required in mice (10, 11, 17), rats (8, 9, 16), dogs (13, 14), and monkeys (12). Using IGF-II supplementation (and 300 µm microcapsules), it was possible, for the first time, to reverse hyperglycemia in diabetic mice by transplanting as few as 150 encapsulated islets or 6000 islet-equivalents/kg of body weight. It is noteworthy that the lowest number of nonencapsulated islets, transplanted in a well-vascularized site (in contrast to the peritoneum that is used for encapsulated islets), which has been reported to induce normoglycemia in diabetic mice has been 150 (41) (as previously mentioned, most studies have used 500 or 1000 islets). With 150 islet equivalents, a recurrence of hyperglycemia occurred in some of the recipients. However, with 300 islet equivalents (12,000 islet equivalents/kg body weight), which is equivalent to the number of islets/kg used in clinical transplantation, normoglycemia was maintained up to 4 months. In addition, methods could be developed to provide prolonged in vivo supply of IGF-II, such as transfection of the IGF-II gene into islets or coencapsulation of IGF-II producing cells.
IGF-II is a member of the IGF family, which includes IGF-I, insulin, IGF, insulin receptors, and IGF binding proteins (42). IGF biological effects are mediated primarily by the type I IGF receptor (IGF-I receptor), which is a tyrosine kinase (43, 44, 45). The key signaling pathways so far described to explain enhance proliferation and inhibition of apoptosis through type I IGF receptor are the RAS/RAF/MAPK pathway and the phosphatidylinositol 3'-kinase pathway (42, 46, 47). We aimed at reproducing conditions that we previously found in the pancreas. We found no IGF-I in duct cell secretions, whereas IGF-II was the principal secretion product of duct cells (24). Therefore, we selected to study IGF-II first. However, it is possible that IGF-I would be as potent or even more potent in our system because it is likely that the antiapoptotic effects we observed are mediated via the type I IGF receptor in ß-cells. Indeed, IGF-I has been shown to protect against anoikis (48) and apoptosis (49) and even enhance graft survival in the fetal pancreas transplantation model (50, 51, 52). However, further studies are required to better understand the mechanisms of IGF effects on ß-cell survival, to see whether IGF-II has novel effects, distinct from those of IGF-I, and to determine if IGFs could act synergistically.
In conclusion, the present study demonstrated that a total of 48 h in vitro incubation with IGF-II, in the pretransplantation period, decreases islet cell apoptosis and necrosis in a dose-dependent manner. These IGF-II incubations allowed the normalization of blood glucose in diabetic mice, using less than half the number of encapsulated islets required in the absence of IGF-II. Because apoptosis is more prevalent in dog and human than in rodent islets and some immunosuppressive drugs induce apoptosis, the effect of IGF-II could be even more important for clinical transplantation. Thus, IGF-II supplementation of culture medium may decrease the number of pancreases that have to be used to induce normoglycemia in diabetic patients. In addition, because IGF-II has been reported to be the most abundant growth factor released by pancreatic duct cells (24), the present study provides additional support to the concept that the microenvironment provided by the exocrine pancreas plays an important role in islet survival, and that partial or total reestablishment of this microenvironment is a good strategy to promote islet cell survival.
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Acknowledgments |
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Footnotes |
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Abbreviations: SI, Stimulation index; STZ, streptozotocin; XTT, sodium 3'-[1-(phenyl-amino-carbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)-benzene sulfonic acid hydrate.
Received December 23, 2002.
Accepted for publication March 10, 2003.
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, P < 0.05 vs. 50 ng/ml IGF-II. B, Quantitative assessment of islet function using the XTT assay, 6 d post encapsulation. Experiments were performed on 5 different islet preparations (n = 5). Data are presented as mean ± SEM. *, P < 0.05 vs. 0 ng/ml IGF-II. 
, P < 0.0005 vs. 0 ng/ml IGF-II. ||, P < 0.05 vs. 50 ng/ml IGF-II.