Recovery of Islet -Cell Function in Streptozotocin- Induced Diabetic M
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《糖尿病学杂志》
1 Section of Transplantation, Department of Surgery, The University of Chicago, Chicago, Illinois
2 Section of Endocrinology, Department of Medicine, The University of Chicago, Chicago, Illinois
[Ca2+]i, intracellular calcium contration; FISH, fluorescence in situ hybridization; FITC, fluoroscein isothiocyanate; IPGTT, intraperitoneal glucose tolerance test; STZ, streptozotocin
ABSTRACT
Limitations in islet -cell transplantation as a therapeutic option for type 1 diabetes have prompted renewed interest in islet regeneration as a source of new islets. In this study we tested whether severely diabetic adult C57BL/6 mice can regenerate -cells. Diabetes was induced in C57BL/6 mice with high-dose streptozotocin (160–170 mg/kg). In the absence of islet transplantation, all diabetic mice remained diabetic (blood glucose >400 mg/dl), and no spontaneous reversal of diabetes was observed. When syngeneic islets (200/mouse) were transplanted into these diabetic mice under a single kidney capsule, stable restoration of euglycemia for 120 days was achieved. Removal of the kidney bearing the transplanted islets at 120 days posttransplantation revealed significant restoration of endogenous -cell function. This restoration of islet function was associated with increased -cell mass, as well as -cell hypertrophy and proliferation. The restoration of islet cell function was facilitated by the presence of a spleen; however, the facilitation was not due to the direct differentiation of spleen-derived cells into -cells. This study supports the possibility of restoring -cell function in diabetic individuals and points to a role for the spleen in facilitating this process.
Autoimmune-mediated destruction of -cells is the predominant cause of type 1 diabetes. Current curative therapy for type 1 diabetes is based on replacement of -cells through pancreas or islet transplantation with concomitant immune suppression. The limited supply of allogeneic pancreata and complications arising from the need for continued immunosuppression have prompted renewed investigations into the natural ability of -cells to regenerate and restore glucose homeostasis. This line of investigation is prompted, in part, by observations that 11–40% of patients with longstanding type 1 diabetes have detectable C-peptide levels and residual -cells (1). However, it is unclear whether such patients have the ability to regenerate -cells to levels that can restore normal glycemia.
It is recognized that -cell mass must be actively regulated throughout life (2–5). The essential cellular and molecular bases for the regulation of -cell mass in adults are incompletely understood, but they are currently thought to include three major processes: replication of differentiated -cells, differentiation and neogenesis of -cells from precursor cells, and the inhibition of -cell apoptosis (6,7). Earlier studies in rodents favored the concept that adult -cell regeneration recapitulated development, with a prominent role for stem cells of pancreatic or extrapancreatic origin as the main mechanism for increasing -cell mass following injury and loss of -cells (8–11). Indeed, cells thought to be capable of developing into insulin-secreting -cells have been located in the pancreatic duct or elsewhere in the pancreas (12–15), liver (16–18), intestine (19; 20), bone marrow (20–22), or spleen (23). More recently, studies by Dor et al. (24) challenged the concept of differentiation/neogenesis and have suggested that proliferation of existing adult -cells is the predominant basis for adult islet regeneration.
An important issue arising from the observations of Dor et al. (24) is whether diabetic patients with significant loss of -cells have the ability to restore sufficient -cell mass to maintain euglycemia. We have developed a mouse model to test whether severely diabetic adult mice can regenerate -cells. In this model, diabetes (blood glucose >400 mg/dl) was induced in adult C57BL/6 mice within 1 week after treatment with high-dose streptozotocin (STZ; 160–170 mg/kg). In the absence of islet transplantation, all untreated diabetic mice remained diabetic (blood glucose >400 mg/dl) and no spontaneous reversal of diabetes was observed. Syngeneic islets (200/mouse) transplanted under a single kidney capsule resulted in the stable restoration of euglycemia for 120 days. Removal of the kidney bearing the transplanted islets at 120 days posttransplantation allowed for subsequent interrogation of endogenous -cell function. We observed significant restoration of -cell mass and function in this model. Using splenectomy and spleen cell transfer approaches, we also observed an important but indirect role for spleen cells in the restoration of endogenous -cell function.
RESEARCH DESIGN AND METHODS
C57BL/6 mice were used as donors and recipients of kidney subcapsular islet transplantation. The mice were purchased from NCI (Frederick, MD) or Jackson Laboratory (Bar Harbor, ME). GFP transgenic B/6 mice (backcrossed 10 generations) were used as donors of spleen cells (25). All mice were housed in pathogen-free conditions at The University of Chicago, following National Institutes of Health guidelines.
Recipients were made diabetic by a single intraperitoneal injection of STZ (160–170 mg/kg, Sigma Chemical, St. Louis, MO). Diabetic mice with nonfasted blood glucose values >400 mg/dl, measured by a blood glucose monitor (SureStep; Lifescan, Milpitas, CA), for more than 2 consecutive days were used as recipients of islet grafts.
Islet isolation and transplantation.
Islet isolation used methods previously reported (26) and involved intraductal collagenase digestion (Collagenase P, 0.3 mg/ml; Roche, Indianapolis, IN) and purification by Ficoll gradient centrifugation (Sigma, St. Louis, MO). Purified islets were transplanted under the kidney capsule immediately after purification. In some recipients, BrdU (Sigma) was administered at a dose of 100 mg/kg i.p., five times a week for 2 weeks.
Splenectomy and nephrectomy.
Splenectomy was performed on the day of islet transplantation. The abdominal cavity was opened with a mid-abdominal incision, the splenic artery and vein were ligated, and the spleen resected.
At 120 days postislet transplantation, nephrectomy of the islet-containing kidney was performed. The left renal artery and vein and the ureter were ligated, and the kidney was resected.
Preparation of spleen cells.
Spleen cells were isolated following procedures established by Kodama et al. (23). Briefly, donor mice were sacrificed by cervical dislocation and the spleen immediately removed and pressed through a Nylon cell strainer (BD Falcon) using a 1-ml syringe bottom (Becton Dickinson). The cells were washed and resuspended in a volume of 300 μl PBS (GIBCO). Mice received the equivalent of half a spleen per injection into the tail vein (4–6 x 107cells/spleen) twice a week for 6 weeks posttransplantation.
Intraperitoneal glucose tolerance test.
An intraperitoneal glucose tolerance test (IPGTT) was performed after the mice were fasted for 4 h. The blood was sampled from the tail vein before and 30, 60, 90, and 120 min after an intraperitoneal injection of 2g/kg body wt dextrose. Blood glucose levels were measured using a blood glucose meter (SureStep).
Pathologic examination.
The pancreas was harvested, embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA), and snap-frozen in liquid nitrogen. Islets were stained with anti-insulin polyclonal antibody (Zymed, South San Francisco, CA) and rabbit anti-glucagon polyclonal antibody (Dako, Carpinteria, CA). A standard avidin-biotin-peroxidase complex method was used to visualize the immunostaining.
To detect BrdU incorporation, cryosections were fixed in acetone and endogenous peroxidase was quenched with 0.3% H2O2. After antigen retrieval with 2x sodium chloride–sodium citrate, the sections were blocked with 2.4G2 monoclonal antibody and 5% goat serum and then serially incubated with mouse anti-BrdU (Sigma), biotinylated rat anti-mouse IgG1 (BD Pharmigen, San Diego, CA), and horseradish peroxidase–streptavidin (Zymed, South San Francisco, CA). The immunostaining was visualized by 3,3-diaminobenzidine (DAB) and counterstained with Mayer’s hematoxylin.
Fluorescence in situ hybridization (FISH) was used to detect male cells in the pancreas. Briefly, after tissue fixation with methanol/acetic acid (3:1) and denaturation with formamide, cryosections were serially incubated with fluoroscein isothiocyanate (FITC)-conjugated mouse Y chromosome probe (CONC mouse chromosome Y paint kit; Cambio, Cambridge, U.K.), rabbit anti-FITC antibody, and FITC-labeled goat anti-rabbit IgG. Finally, cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI-1).
Quantification of -cell mass.
Quantification of -cell mass was performed on normal mice, STZ-treated mice that were diabetic for 7 days, and in mice that remained normoglycemic for 60 days following the removal of islet grafts (restored group). Whole pancreata were removed from each mouse and weighed, and serially step-sectioned. Every 10th section was stained with anti-insulin polyclonal antibody (10 sections per mouse) and images of each section were captured on a Zeiss Axiovert 200M microscope. The -cell and total pancreas area were quantified with Image J (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/), and the relative ratio of -cell area compared with entire pancreas area determined. Finally the -cell mass was calculated by multiplying the relative ratio by the total weight of the pancreas.
Measurement of intracellular calcium concentration.
Dual-wavelength excitation spectrophotometry was used to measure intracellular calcium concentration ([Ca2+]i) as described previously (46). Briefly, isolated pancreatic islets were loaded with Fura-2 by a 25-min incubation at 37°C in Krebs-Ringer buffer containing 2 mmol/l glucose and 5 μmol/l Fura 2-AM (Molecular Probes, Eugene, OR). Fluorescence imaging was performed using a charged-coupled device (CCD) camera-based imaging system (Cascade camera from Photometrics, Tucson, AZ) and MetaFluor software (Universal Imaging, Downingtown, PA). During imaging experiments, islets were kept at 37°C and perifused with Krebs-Ringer buffer–based solutions containing glucose or other agonists as indicated, at a flow rate of 2.5 ml/min. [Ca2+]i was expressed as the ratio of fluorescence intensity at excitation wavelengths 340 and 380 nm (F340/F380).
Statistics.
All reported values are presented as means ± SEM and evaluated for statistical significance by ANOVA (SuperANOVA v. 1.11; Abacus Concepts, Berkeley, CA). A P value of <0.05 was considered to be statistically significant.
RESULTS
Restoration of -cell function in STZ-induced diabetic mice.
We have developed a mouse model of restoration of -cell function that combines the high-dose STZ diabetes model with syngeneic islet transplantation under a single kidney capsule (Fig. 1). A single injection of STZ (160–170 mg/kg body wt) induced diabetes in 70–80% of C57BL/6 mice, with 5% of mice succumbing to STZ toxicity (defined as early death before the development of diabetes). Approximately 70% of the STZ-injected mice had to be killed or died of diabetes by day 20 post–STZ treatment, and 100% mortality was observed by day 45 (Fig. 2A). Blood glucose levels of >400 mg/dl were observed within 1 week of STZ treatment (Fig. 2B and C), and no mice recovered spontaneously from STZ-induced diabetes.
In one group of diabetic STZ-treated mice (blood glucose levels >400 mg/dl for 2 consecutive days), syngeneic islets were transplanted under one kidney capsule. Normal glycemia (99 ± 2 mg/dl, n = 13) was restored by 1–2 days after transplantation and was maintained for the entire 120 days of observation. When the kidney bearing the transplanted islets was removed after 120 days, the mice were followed for another 45–100 days. The mean blood glucose postnephrectomy was 206 ± 7 mg/dl (Fig. 2B); this was significantly higher than for normal mice but significantly improved compared with before islet transplantation (P < 0.001). The modest hyperglycemia following the removal of transplanted islets was stable (Fig. 2C), and no mice died of diabetes up to 45–100 days postnephrectomy (Fig. 2A), consistent with stable endogenous -cell function. We experienced a <5% loss in mice as a result of the nephrectomy (i.e., death within 7 days of surgery with no evidence of diabetes); these mice were excluded from the survival data.
Our findings suggest that the regulation of blood glucose by the islet grafts facilitated the gradual, but incomplete, restoration of functional -cells in the pancreas of adult C57BL/6 mice treated with a single high dose of STZ. Further tests of these mice at 45 days postnephrectomy revealed abnormal IPGTT responses compared with normal C57BL/6 mice (n = 10; P < 0.001), even after 120 days of normoglycemia to allow for regeneration (Fig. 2D). We interpret these collective observations to mean that recovery, albeit partial, of endogenous -cell function had occurred in the STZ-induced diabetic mice during the period when normal glycemia was maintained by syngeneic islet transplantation.
Histology of regenerated -cells.
Examination of the cellular content of islets 7–14 days after STZ treatment using standard immunohistochemical approaches indicated a reduction in the proportion of insulin-positive -cells and an increase in glucagon-positive -cells compared with normal controls (Fig. 3). These observations are consistent with previous reports that high-dose STZ results in diabetes associated with -cell destruction (17,27). In mice with restored -cell function, the proportion of insulin-positive cells in the islets was increased.
Further morphometric analysis indicated that STZ treatment significantly reduced -cell mass, determined by anti-insulin staining, by 90% (Fig. 4A). After 120 days of normoglycemia, a statistically significant 3.7-fold increase in -cell mass was observed (P < 0.05). These observations are consistent with islet -cell regeneration. We acknowledge the possibility that numerous -cells could still be present after STZ treatment but remained undetected because of extensive degranulation, and that the -cells observed at 160 days post–STZ treatment are due to restoration of insulin granules in the -cells. However, this possibility is not supported by numerous findings that high-dose STZ is toxic to -cells (28–34) and by our additional studies in which the -cell mass was deduced by as the nonstaining portions of islets stained with a cocktail of anti-glucagon, anti-stomatostatin, and anti–pancreatic peptide antibodies. These latter studies confirmed a reduced islet -cell mass in STZ-induced diabetic mice and a 1.8-fold increase in -cell mass in mice after 120 days of normoglycemia (data not shown).
A predominant morphologic feature of the restored insulin-expressing islets was -cell hypertrophy (Figs. 4B–D). Cell hypertrophy was defined as statistically significant increases in the cell and nuclear sizes of restored islets (125 and 137%, respectively) compared with normal controls. Cell and nuclear sizes were defined as means of the maximal and minimal diameters of >300 islet cells or nuclei (from 4–8 islets randomly selected from the pancreas of experimental or control mice [n = 3 per group]).
To test whether -cell proliferation contributed to the restoration of -cell function, STZ-diabetic mice received BrdU (10x, i.p.) from days 0–14 postsyngeneic islet transplantation. The numbers of BrdU-positive cells observed in the islets was marginally (Fig. 4E and F) but statistically different, between the two groups (149 of 2,365 [6.2%] vs. 119 of 1,370 [8.7%] for nave versus restored groups, respectively, unpaired one-tail t test, P = 0.036). We conclude from these observations that the restoration of -cells was associated with cell hypertrophy and modest levels of -cell proliferation.
In vitro function of restored -cells.
We analyzed the function of the restored islets in vitro by harvesting the islets from the pancreata of the mice at 45–100 days postnephrectomy. The number of islets recovered from these mice was 3–17% of that recovered from normal C57BL/6 mice (5–20 per restored pancreas vs. 120–150 per normal pancreas; n = 8 per group). The isolated islets were maintained in culture for 48–72 h, and Ca2+ responses to glucose were imaged using Fura-2. Two examples of individual islet responses are shown (Fig. 5) and were compared with islets from a normal mouse. Similar results were obtained with 11 restored islets from a total of five mice. The results indicate that the glucose-induced calcium responses were perturbed in the restored islets, as compared with the normal ones, and suggest that this defect, as well as reduced islet numbers, accounted for the observed hyperglycemia and abnormal IPGTT responses in mice with restored -cell function.
Removal of spleen reduces the restoration of -cell function.
Recent interest in a possible role for the spleen in -cell restoration came from striking observations by Kodama et al. (23). We first investigated whether the presence of the spleen influenced the restoration of blood glucose regulation in our mouse model. Mice underwent splenectomy on the day of islet transplantation and were then subjected to the same protocol as described in Fig. 1 to allow for the restoration of islet -cells (n = 10). The blood glucose levels of splenectomized mice after nephrectomy were significantly higher (340 ± 12 mg/dl) than those of non-splenectomized mice (Fig. 6A and B; P < 0.001), suggesting a role for the spleen in facilitating the restoration of -cell function.
Spleen cells do not transdifferentiate into -cells.
The studies of Kodama et al. (23) suggested that spleen cells had the ability to differentiate directly into -cells, thereby contributing to -cell regeneration. We first tested whether infusion of spleen cells altered the rate of restoration of -cell function, following the protocol established by Kodama et al. Spleen cells from syngeneic C57BL/6 mice were injected intravenously (2–4 x 107) for 6 weeks (two injections per week), starting on day 1 postislet transplantation (splenectomized + STZ-treated recipients; n = 10). The random blood glucose concentrations in splenectomized mice inoculated with splenocytes, after nephrectomy, were comparable (218 ± 11 mg/dl) to those of non-splenectomized mice (Fig. 6B; P > 0.5) and significantly improved compared with the splenectomized mice without spleen cell infusion (P < 0.001). IPGTT responses were inferior in the splenectomized compared with the non-splenectomized (restored) groups (P < 0.001) and significantly improved in the splenectomized group receiving spleen cells (Fig. 6C; P < 0.001). These findings support the hypothesis that spleen cells can facilitate the restoration of -cell function in this model. However, we acknowledge that the very large number of spleen cells used in this study is irrelevant for clinical studies.
To test whether the spleen cell transfer facilitated the restoration of -cell function by directly converting into -cells, we transferred either syngeneic male spleen cells (n = 3) or spleen cells from syngeneic MIP-GFP mice (n = 6) into C57BL/6 recipients. At 45–100 days postnephrectomy, we looked for the presence of male (Y chromosome by FISH) or MIP-GFP+ -cells. We observed Y chromosome–positive male cells surrounding the restored islets, but the locations of the FISH-positive cells were consistent with peri-islet infiltration and not -cells (Fig. 7A and B). Indeed, significant numbers of CD4+, and some CD8+, cells were observed around the islets at 45–100 days postnephrectomy (Fig. 7C and D).
To test more directly the ability of spleen cells to differentiate into islet -cells, we used spleen cells from MIP-GFP+ mice and probed for the presence of MIP-GFP+ -cells in pancreas and other organs of the mice with restored -cell function by fluorescence microscopy or by immunofluorescence with anti-GFP staining. We were not able to detect GFP+ -cells in any of the restored islets or any other organ examined (Fig. 7E and F and data not shown). Our data do not support a conclusion that splenic stem cells are a source of -cells in our model, but suggest that transfused spleen cells detected in the vicinity of the restored islets were likely to have been infiltrating leukocytes.
DISCUSSION
Failure of pancreatic -cells to secrete insulin is the common physiologic defect in both type 1 and type 2 diabetes, and replacement of -cell mass with islet cell or pancreas transplantation is considered a viable therapeutic option. Because of the significant limitations of allogeneic islet cell and pancreas transplantation, there is renewed interest in the possibility that islet regeneration may provide new islets in diabetic patients. In a study of 2,432 patients who were 18 years of age at onset of diabetes, 15 and 33% of patients had stimulated C-peptide levels of >0.5 and 0.2–0.5 nmol/l, respectively, within the first 5 years of diagnosis (35). Postmortem studies demonstrated the presence of insulin-staining cells in new-onset diabetic patients (35–38). These observations, together with the recent report that preexisting pancreatic -cells in adult mice were capable of proliferation and generating new islets (24), have lent support to the notion that islet regeneration may represent a possible means of increasing islet -cell mass in type 1 and 2 diabetic patients. However, those studies did not specifically address the critical issue of whether severely diabetic adult individuals with depleted -cells can regenerate to levels that can maintain normal glycemia.
The high-dose STZ-induced diabetes model has been extensively used to demonstrate the function of transplanted islets (28–30). Those studies were based on the assumption that mice treated with high-dose STZ are unable to recover endogenous -cell function. Indeed, there have been few reports of functional -cell regeneration in this high-dose model, despite reports of limited increases in the numbers of insulin-positive cells shortly after high-dose STZ treatment (13,39,40). In this study we report that STZ-induced diabetic adult C57BL/6 mice, with blood glucose levels of >400 mg/dl, have modest abilities to recover -cell function and mass in vivo. Thus, these observations confirm and extend observations of a modest two- to threefold increase in pancreatic -cells in high-dose STZ-induced diabetic adult rats (41).
The percent of -cells in the islets and the islet size were significantly reduced following STZ treatment, as compared with those of normal mice, but increased in STZ-diabetic mice after 120 days of normal glycemia. We demonstrated that the recovery of -cell function was the result of increased -cell mass due to -cell hypertrophy. A modest but statistically increased level of -cell proliferation was observed following BrdU labeling in the islets of restored compared with normal controls. Our observations, albeit at lower rates of BrdU uptake, are therefore consistent with enhanced -cell proliferation reported in other models of -cell regeneration (42) (43).
The recovery of -cell function was only observed in mice in which normal blood glucose levels were maintained by the transplanted syngeneic islets, as none of the diabetic mice recovered from diabetes without islet transplantation. These observations are consistent with the notion that recovery is dependent on glucose control, although the possibility exists that the presence of islets may contribute to the recovery of endogenous -cell function, independent of effects on glucose control.
Our in vivo and in vitro studies also underscore the limited nature of -cell regeneration in this STZ model. Random blood glucose levels remained elevated even after 120 days of normal glycemia, and the IPGTT responses remained impaired. The restored islets also had abnormal abilities to flux [Ca2+]i in response to high glucose challenge. The partial recovery of -cell function could be due to permanent destruction of -cell–to–-cell contacts, alterations in the architecture of the restored islet, or scarring, leading to defective -cell sensing. Whether these features are specific to the STZ-induced diabetes model or reflect a more generalized limitation of regenerated -cells will have to be elucidated in other more physiologic models of -cell destruction.
Our observations of limited restoration of -cells in the STZ model are in contrast to the more robust levels of regeneration reported for autoimmune diabetic NOD mice (23,44,45), and they suggest that the rate of -cell regeneration may be regulated by multiple factors. In the RIP-LCMV model of autoimmune diabetes, von Herrath et al. (46) reported that the ability to resist the development of diabetes differed drastically between the 129 and the C57BL/6 strain, and they speculated that this difference was the result of differences in the abilities of these two strains of mice to regenerate -cells. It has also been reported that partial pancreactectomy enhanced resistance to STZ-induced diabetes (11) and that neonatal rats are able to spontaneously recover from STZ-induced diabetes (47). Thus, it appears that multiple factors, including the genetic background, the age of the diabetic animal, and the type of injury/inflammation causing -cell destruction, affect the rate of -cell regeneration in rodents. It is likely that these same factors will shape the ability of diabetic human patients to regenerate -cells. We speculate that -cell regeneration in adult humans is likely to be a slow process, and the significant question of how effectively diabetic patients with depleted -cell mass can regenerate remains to be answered.
Understanding how -cell regeneration is normally regulated could lead to the identification of new approaches for enhancing -cell regeneration. To this end we tested the effect of the spleen in the restoration of -cell function in this model. A role for the spleen in -cell regeneration was suggested by clinical data showing that the incidence of diabetes was significantly higher in patients undergoing partial pancreatectomy and splenectomy than in those undergoing pancreatectomy alone (48). Further evidence for a role of the spleen in -cell regeneration came from the NOD mouse model of autoimmune diabetes (23). Kodama et al. (23) reported that stem cells residing in the spleen of NOD mice were capable of differentiating into -cells, thereby restoring -cell mass and curing diabetes. We here demonstrate that the removal of the spleen resulted in significantly reduced restoration of -cell function in this model, consistent with the clinical data. We also confirmed that the administration of high doses of spleen cells partially restored the ability to recover -cell function in the splenectomized mice. We observed a peri-islet localization of the donor spleen cells, but, contrary to the observations by Kodama et al. (25), we were unable to demonstrate a direct contribution of spleen-derived cells to the regenerated islets. These data are, however, consistent with recent reports that spleen cells do not differentiate into islet -cells (49–51). It is currently unclear what role the spleen plays in this model of restoration of -cell function, but it may resemble the indirect effects of bone marrow–derived stem cells in initiating pancreatic -cell regeneration as described by Hess et al. (52). Indeed, we are currently testing whether the spleen contributes to the inflammatory process in the pancreas (and thereby stimulates recovery of the STZ damaged islet), as well as defining the cell types, namely T, B, or other cells, involved in this effect. Finally, we are testing whether an infusion of a more physiologic number of spleen cells can enhance the recovery of -cell function.
In conclusion, there is recent interest in utilizing the natural ability of -cells to regenerate as an innovative approach for the treatment of type 1 diabetes. We have developed a new mouse model, whereby we demonstrate a partial restoration of -cell mass and function in adult mice made diabetic with STZ. While the end goal is to completely eliminate the need for exogenous insulin, the benefits of partial recovery of -cell function in severe diabetic patients cannot be ignored, as patients with residual -cell function require less insulin, have better and easier glycemic control, and fewer end-organ complications compared with patients with no residual function (35). We also observed a significant requirement for the spleen and spleen cells in islet cell regeneration, and we demonstrate that this is not due to direct differentiation of the spleen cells into -cells. This study supports the possibility of regeneration as a means for generating new -cells even in severely diabetic individuals with significantly reduced -cell mass. The rate of normal -cell regeneration is slow, and it is critical that strategies are identified to enhance -cell regeneration in severely diabetic individuals.
ACKNOWLEDGMENTS
This work was supported by grant support from the JDRF, National Institutes of Health(DK073529), Chester Foundation, and Lyman Family Fund. We thank Dr. Denise Faustman for her advice on the spleen cell preparations, Dr. Ian Boussy for his assistance throughout the study and Christine Ding for technical assistance.
FOOTNOTES
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.
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2 Section of Endocrinology, Department of Medicine, The University of Chicago, Chicago, Illinois
[Ca2+]i, intracellular calcium contration; FISH, fluorescence in situ hybridization; FITC, fluoroscein isothiocyanate; IPGTT, intraperitoneal glucose tolerance test; STZ, streptozotocin
ABSTRACT
Limitations in islet -cell transplantation as a therapeutic option for type 1 diabetes have prompted renewed interest in islet regeneration as a source of new islets. In this study we tested whether severely diabetic adult C57BL/6 mice can regenerate -cells. Diabetes was induced in C57BL/6 mice with high-dose streptozotocin (160–170 mg/kg). In the absence of islet transplantation, all diabetic mice remained diabetic (blood glucose >400 mg/dl), and no spontaneous reversal of diabetes was observed. When syngeneic islets (200/mouse) were transplanted into these diabetic mice under a single kidney capsule, stable restoration of euglycemia for 120 days was achieved. Removal of the kidney bearing the transplanted islets at 120 days posttransplantation revealed significant restoration of endogenous -cell function. This restoration of islet function was associated with increased -cell mass, as well as -cell hypertrophy and proliferation. The restoration of islet cell function was facilitated by the presence of a spleen; however, the facilitation was not due to the direct differentiation of spleen-derived cells into -cells. This study supports the possibility of restoring -cell function in diabetic individuals and points to a role for the spleen in facilitating this process.
Autoimmune-mediated destruction of -cells is the predominant cause of type 1 diabetes. Current curative therapy for type 1 diabetes is based on replacement of -cells through pancreas or islet transplantation with concomitant immune suppression. The limited supply of allogeneic pancreata and complications arising from the need for continued immunosuppression have prompted renewed investigations into the natural ability of -cells to regenerate and restore glucose homeostasis. This line of investigation is prompted, in part, by observations that 11–40% of patients with longstanding type 1 diabetes have detectable C-peptide levels and residual -cells (1). However, it is unclear whether such patients have the ability to regenerate -cells to levels that can restore normal glycemia.
It is recognized that -cell mass must be actively regulated throughout life (2–5). The essential cellular and molecular bases for the regulation of -cell mass in adults are incompletely understood, but they are currently thought to include three major processes: replication of differentiated -cells, differentiation and neogenesis of -cells from precursor cells, and the inhibition of -cell apoptosis (6,7). Earlier studies in rodents favored the concept that adult -cell regeneration recapitulated development, with a prominent role for stem cells of pancreatic or extrapancreatic origin as the main mechanism for increasing -cell mass following injury and loss of -cells (8–11). Indeed, cells thought to be capable of developing into insulin-secreting -cells have been located in the pancreatic duct or elsewhere in the pancreas (12–15), liver (16–18), intestine (19; 20), bone marrow (20–22), or spleen (23). More recently, studies by Dor et al. (24) challenged the concept of differentiation/neogenesis and have suggested that proliferation of existing adult -cells is the predominant basis for adult islet regeneration.
An important issue arising from the observations of Dor et al. (24) is whether diabetic patients with significant loss of -cells have the ability to restore sufficient -cell mass to maintain euglycemia. We have developed a mouse model to test whether severely diabetic adult mice can regenerate -cells. In this model, diabetes (blood glucose >400 mg/dl) was induced in adult C57BL/6 mice within 1 week after treatment with high-dose streptozotocin (STZ; 160–170 mg/kg). In the absence of islet transplantation, all untreated diabetic mice remained diabetic (blood glucose >400 mg/dl) and no spontaneous reversal of diabetes was observed. Syngeneic islets (200/mouse) transplanted under a single kidney capsule resulted in the stable restoration of euglycemia for 120 days. Removal of the kidney bearing the transplanted islets at 120 days posttransplantation allowed for subsequent interrogation of endogenous -cell function. We observed significant restoration of -cell mass and function in this model. Using splenectomy and spleen cell transfer approaches, we also observed an important but indirect role for spleen cells in the restoration of endogenous -cell function.
RESEARCH DESIGN AND METHODS
C57BL/6 mice were used as donors and recipients of kidney subcapsular islet transplantation. The mice were purchased from NCI (Frederick, MD) or Jackson Laboratory (Bar Harbor, ME). GFP transgenic B/6 mice (backcrossed 10 generations) were used as donors of spleen cells (25). All mice were housed in pathogen-free conditions at The University of Chicago, following National Institutes of Health guidelines.
Recipients were made diabetic by a single intraperitoneal injection of STZ (160–170 mg/kg, Sigma Chemical, St. Louis, MO). Diabetic mice with nonfasted blood glucose values >400 mg/dl, measured by a blood glucose monitor (SureStep; Lifescan, Milpitas, CA), for more than 2 consecutive days were used as recipients of islet grafts.
Islet isolation and transplantation.
Islet isolation used methods previously reported (26) and involved intraductal collagenase digestion (Collagenase P, 0.3 mg/ml; Roche, Indianapolis, IN) and purification by Ficoll gradient centrifugation (Sigma, St. Louis, MO). Purified islets were transplanted under the kidney capsule immediately after purification. In some recipients, BrdU (Sigma) was administered at a dose of 100 mg/kg i.p., five times a week for 2 weeks.
Splenectomy and nephrectomy.
Splenectomy was performed on the day of islet transplantation. The abdominal cavity was opened with a mid-abdominal incision, the splenic artery and vein were ligated, and the spleen resected.
At 120 days postislet transplantation, nephrectomy of the islet-containing kidney was performed. The left renal artery and vein and the ureter were ligated, and the kidney was resected.
Preparation of spleen cells.
Spleen cells were isolated following procedures established by Kodama et al. (23). Briefly, donor mice were sacrificed by cervical dislocation and the spleen immediately removed and pressed through a Nylon cell strainer (BD Falcon) using a 1-ml syringe bottom (Becton Dickinson). The cells were washed and resuspended in a volume of 300 μl PBS (GIBCO). Mice received the equivalent of half a spleen per injection into the tail vein (4–6 x 107cells/spleen) twice a week for 6 weeks posttransplantation.
Intraperitoneal glucose tolerance test.
An intraperitoneal glucose tolerance test (IPGTT) was performed after the mice were fasted for 4 h. The blood was sampled from the tail vein before and 30, 60, 90, and 120 min after an intraperitoneal injection of 2g/kg body wt dextrose. Blood glucose levels were measured using a blood glucose meter (SureStep).
Pathologic examination.
The pancreas was harvested, embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA), and snap-frozen in liquid nitrogen. Islets were stained with anti-insulin polyclonal antibody (Zymed, South San Francisco, CA) and rabbit anti-glucagon polyclonal antibody (Dako, Carpinteria, CA). A standard avidin-biotin-peroxidase complex method was used to visualize the immunostaining.
To detect BrdU incorporation, cryosections were fixed in acetone and endogenous peroxidase was quenched with 0.3% H2O2. After antigen retrieval with 2x sodium chloride–sodium citrate, the sections were blocked with 2.4G2 monoclonal antibody and 5% goat serum and then serially incubated with mouse anti-BrdU (Sigma), biotinylated rat anti-mouse IgG1 (BD Pharmigen, San Diego, CA), and horseradish peroxidase–streptavidin (Zymed, South San Francisco, CA). The immunostaining was visualized by 3,3-diaminobenzidine (DAB) and counterstained with Mayer’s hematoxylin.
Fluorescence in situ hybridization (FISH) was used to detect male cells in the pancreas. Briefly, after tissue fixation with methanol/acetic acid (3:1) and denaturation with formamide, cryosections were serially incubated with fluoroscein isothiocyanate (FITC)-conjugated mouse Y chromosome probe (CONC mouse chromosome Y paint kit; Cambio, Cambridge, U.K.), rabbit anti-FITC antibody, and FITC-labeled goat anti-rabbit IgG. Finally, cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI-1).
Quantification of -cell mass.
Quantification of -cell mass was performed on normal mice, STZ-treated mice that were diabetic for 7 days, and in mice that remained normoglycemic for 60 days following the removal of islet grafts (restored group). Whole pancreata were removed from each mouse and weighed, and serially step-sectioned. Every 10th section was stained with anti-insulin polyclonal antibody (10 sections per mouse) and images of each section were captured on a Zeiss Axiovert 200M microscope. The -cell and total pancreas area were quantified with Image J (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/), and the relative ratio of -cell area compared with entire pancreas area determined. Finally the -cell mass was calculated by multiplying the relative ratio by the total weight of the pancreas.
Measurement of intracellular calcium concentration.
Dual-wavelength excitation spectrophotometry was used to measure intracellular calcium concentration ([Ca2+]i) as described previously (46). Briefly, isolated pancreatic islets were loaded with Fura-2 by a 25-min incubation at 37°C in Krebs-Ringer buffer containing 2 mmol/l glucose and 5 μmol/l Fura 2-AM (Molecular Probes, Eugene, OR). Fluorescence imaging was performed using a charged-coupled device (CCD) camera-based imaging system (Cascade camera from Photometrics, Tucson, AZ) and MetaFluor software (Universal Imaging, Downingtown, PA). During imaging experiments, islets were kept at 37°C and perifused with Krebs-Ringer buffer–based solutions containing glucose or other agonists as indicated, at a flow rate of 2.5 ml/min. [Ca2+]i was expressed as the ratio of fluorescence intensity at excitation wavelengths 340 and 380 nm (F340/F380).
Statistics.
All reported values are presented as means ± SEM and evaluated for statistical significance by ANOVA (SuperANOVA v. 1.11; Abacus Concepts, Berkeley, CA). A P value of <0.05 was considered to be statistically significant.
RESULTS
Restoration of -cell function in STZ-induced diabetic mice.
We have developed a mouse model of restoration of -cell function that combines the high-dose STZ diabetes model with syngeneic islet transplantation under a single kidney capsule (Fig. 1). A single injection of STZ (160–170 mg/kg body wt) induced diabetes in 70–80% of C57BL/6 mice, with 5% of mice succumbing to STZ toxicity (defined as early death before the development of diabetes). Approximately 70% of the STZ-injected mice had to be killed or died of diabetes by day 20 post–STZ treatment, and 100% mortality was observed by day 45 (Fig. 2A). Blood glucose levels of >400 mg/dl were observed within 1 week of STZ treatment (Fig. 2B and C), and no mice recovered spontaneously from STZ-induced diabetes.
In one group of diabetic STZ-treated mice (blood glucose levels >400 mg/dl for 2 consecutive days), syngeneic islets were transplanted under one kidney capsule. Normal glycemia (99 ± 2 mg/dl, n = 13) was restored by 1–2 days after transplantation and was maintained for the entire 120 days of observation. When the kidney bearing the transplanted islets was removed after 120 days, the mice were followed for another 45–100 days. The mean blood glucose postnephrectomy was 206 ± 7 mg/dl (Fig. 2B); this was significantly higher than for normal mice but significantly improved compared with before islet transplantation (P < 0.001). The modest hyperglycemia following the removal of transplanted islets was stable (Fig. 2C), and no mice died of diabetes up to 45–100 days postnephrectomy (Fig. 2A), consistent with stable endogenous -cell function. We experienced a <5% loss in mice as a result of the nephrectomy (i.e., death within 7 days of surgery with no evidence of diabetes); these mice were excluded from the survival data.
Our findings suggest that the regulation of blood glucose by the islet grafts facilitated the gradual, but incomplete, restoration of functional -cells in the pancreas of adult C57BL/6 mice treated with a single high dose of STZ. Further tests of these mice at 45 days postnephrectomy revealed abnormal IPGTT responses compared with normal C57BL/6 mice (n = 10; P < 0.001), even after 120 days of normoglycemia to allow for regeneration (Fig. 2D). We interpret these collective observations to mean that recovery, albeit partial, of endogenous -cell function had occurred in the STZ-induced diabetic mice during the period when normal glycemia was maintained by syngeneic islet transplantation.
Histology of regenerated -cells.
Examination of the cellular content of islets 7–14 days after STZ treatment using standard immunohistochemical approaches indicated a reduction in the proportion of insulin-positive -cells and an increase in glucagon-positive -cells compared with normal controls (Fig. 3). These observations are consistent with previous reports that high-dose STZ results in diabetes associated with -cell destruction (17,27). In mice with restored -cell function, the proportion of insulin-positive cells in the islets was increased.
Further morphometric analysis indicated that STZ treatment significantly reduced -cell mass, determined by anti-insulin staining, by 90% (Fig. 4A). After 120 days of normoglycemia, a statistically significant 3.7-fold increase in -cell mass was observed (P < 0.05). These observations are consistent with islet -cell regeneration. We acknowledge the possibility that numerous -cells could still be present after STZ treatment but remained undetected because of extensive degranulation, and that the -cells observed at 160 days post–STZ treatment are due to restoration of insulin granules in the -cells. However, this possibility is not supported by numerous findings that high-dose STZ is toxic to -cells (28–34) and by our additional studies in which the -cell mass was deduced by as the nonstaining portions of islets stained with a cocktail of anti-glucagon, anti-stomatostatin, and anti–pancreatic peptide antibodies. These latter studies confirmed a reduced islet -cell mass in STZ-induced diabetic mice and a 1.8-fold increase in -cell mass in mice after 120 days of normoglycemia (data not shown).
A predominant morphologic feature of the restored insulin-expressing islets was -cell hypertrophy (Figs. 4B–D). Cell hypertrophy was defined as statistically significant increases in the cell and nuclear sizes of restored islets (125 and 137%, respectively) compared with normal controls. Cell and nuclear sizes were defined as means of the maximal and minimal diameters of >300 islet cells or nuclei (from 4–8 islets randomly selected from the pancreas of experimental or control mice [n = 3 per group]).
To test whether -cell proliferation contributed to the restoration of -cell function, STZ-diabetic mice received BrdU (10x, i.p.) from days 0–14 postsyngeneic islet transplantation. The numbers of BrdU-positive cells observed in the islets was marginally (Fig. 4E and F) but statistically different, between the two groups (149 of 2,365 [6.2%] vs. 119 of 1,370 [8.7%] for nave versus restored groups, respectively, unpaired one-tail t test, P = 0.036). We conclude from these observations that the restoration of -cells was associated with cell hypertrophy and modest levels of -cell proliferation.
In vitro function of restored -cells.
We analyzed the function of the restored islets in vitro by harvesting the islets from the pancreata of the mice at 45–100 days postnephrectomy. The number of islets recovered from these mice was 3–17% of that recovered from normal C57BL/6 mice (5–20 per restored pancreas vs. 120–150 per normal pancreas; n = 8 per group). The isolated islets were maintained in culture for 48–72 h, and Ca2+ responses to glucose were imaged using Fura-2. Two examples of individual islet responses are shown (Fig. 5) and were compared with islets from a normal mouse. Similar results were obtained with 11 restored islets from a total of five mice. The results indicate that the glucose-induced calcium responses were perturbed in the restored islets, as compared with the normal ones, and suggest that this defect, as well as reduced islet numbers, accounted for the observed hyperglycemia and abnormal IPGTT responses in mice with restored -cell function.
Removal of spleen reduces the restoration of -cell function.
Recent interest in a possible role for the spleen in -cell restoration came from striking observations by Kodama et al. (23). We first investigated whether the presence of the spleen influenced the restoration of blood glucose regulation in our mouse model. Mice underwent splenectomy on the day of islet transplantation and were then subjected to the same protocol as described in Fig. 1 to allow for the restoration of islet -cells (n = 10). The blood glucose levels of splenectomized mice after nephrectomy were significantly higher (340 ± 12 mg/dl) than those of non-splenectomized mice (Fig. 6A and B; P < 0.001), suggesting a role for the spleen in facilitating the restoration of -cell function.
Spleen cells do not transdifferentiate into -cells.
The studies of Kodama et al. (23) suggested that spleen cells had the ability to differentiate directly into -cells, thereby contributing to -cell regeneration. We first tested whether infusion of spleen cells altered the rate of restoration of -cell function, following the protocol established by Kodama et al. Spleen cells from syngeneic C57BL/6 mice were injected intravenously (2–4 x 107) for 6 weeks (two injections per week), starting on day 1 postislet transplantation (splenectomized + STZ-treated recipients; n = 10). The random blood glucose concentrations in splenectomized mice inoculated with splenocytes, after nephrectomy, were comparable (218 ± 11 mg/dl) to those of non-splenectomized mice (Fig. 6B; P > 0.5) and significantly improved compared with the splenectomized mice without spleen cell infusion (P < 0.001). IPGTT responses were inferior in the splenectomized compared with the non-splenectomized (restored) groups (P < 0.001) and significantly improved in the splenectomized group receiving spleen cells (Fig. 6C; P < 0.001). These findings support the hypothesis that spleen cells can facilitate the restoration of -cell function in this model. However, we acknowledge that the very large number of spleen cells used in this study is irrelevant for clinical studies.
To test whether the spleen cell transfer facilitated the restoration of -cell function by directly converting into -cells, we transferred either syngeneic male spleen cells (n = 3) or spleen cells from syngeneic MIP-GFP mice (n = 6) into C57BL/6 recipients. At 45–100 days postnephrectomy, we looked for the presence of male (Y chromosome by FISH) or MIP-GFP+ -cells. We observed Y chromosome–positive male cells surrounding the restored islets, but the locations of the FISH-positive cells were consistent with peri-islet infiltration and not -cells (Fig. 7A and B). Indeed, significant numbers of CD4+, and some CD8+, cells were observed around the islets at 45–100 days postnephrectomy (Fig. 7C and D).
To test more directly the ability of spleen cells to differentiate into islet -cells, we used spleen cells from MIP-GFP+ mice and probed for the presence of MIP-GFP+ -cells in pancreas and other organs of the mice with restored -cell function by fluorescence microscopy or by immunofluorescence with anti-GFP staining. We were not able to detect GFP+ -cells in any of the restored islets or any other organ examined (Fig. 7E and F and data not shown). Our data do not support a conclusion that splenic stem cells are a source of -cells in our model, but suggest that transfused spleen cells detected in the vicinity of the restored islets were likely to have been infiltrating leukocytes.
DISCUSSION
Failure of pancreatic -cells to secrete insulin is the common physiologic defect in both type 1 and type 2 diabetes, and replacement of -cell mass with islet cell or pancreas transplantation is considered a viable therapeutic option. Because of the significant limitations of allogeneic islet cell and pancreas transplantation, there is renewed interest in the possibility that islet regeneration may provide new islets in diabetic patients. In a study of 2,432 patients who were 18 years of age at onset of diabetes, 15 and 33% of patients had stimulated C-peptide levels of >0.5 and 0.2–0.5 nmol/l, respectively, within the first 5 years of diagnosis (35). Postmortem studies demonstrated the presence of insulin-staining cells in new-onset diabetic patients (35–38). These observations, together with the recent report that preexisting pancreatic -cells in adult mice were capable of proliferation and generating new islets (24), have lent support to the notion that islet regeneration may represent a possible means of increasing islet -cell mass in type 1 and 2 diabetic patients. However, those studies did not specifically address the critical issue of whether severely diabetic adult individuals with depleted -cells can regenerate to levels that can maintain normal glycemia.
The high-dose STZ-induced diabetes model has been extensively used to demonstrate the function of transplanted islets (28–30). Those studies were based on the assumption that mice treated with high-dose STZ are unable to recover endogenous -cell function. Indeed, there have been few reports of functional -cell regeneration in this high-dose model, despite reports of limited increases in the numbers of insulin-positive cells shortly after high-dose STZ treatment (13,39,40). In this study we report that STZ-induced diabetic adult C57BL/6 mice, with blood glucose levels of >400 mg/dl, have modest abilities to recover -cell function and mass in vivo. Thus, these observations confirm and extend observations of a modest two- to threefold increase in pancreatic -cells in high-dose STZ-induced diabetic adult rats (41).
The percent of -cells in the islets and the islet size were significantly reduced following STZ treatment, as compared with those of normal mice, but increased in STZ-diabetic mice after 120 days of normal glycemia. We demonstrated that the recovery of -cell function was the result of increased -cell mass due to -cell hypertrophy. A modest but statistically increased level of -cell proliferation was observed following BrdU labeling in the islets of restored compared with normal controls. Our observations, albeit at lower rates of BrdU uptake, are therefore consistent with enhanced -cell proliferation reported in other models of -cell regeneration (42) (43).
The recovery of -cell function was only observed in mice in which normal blood glucose levels were maintained by the transplanted syngeneic islets, as none of the diabetic mice recovered from diabetes without islet transplantation. These observations are consistent with the notion that recovery is dependent on glucose control, although the possibility exists that the presence of islets may contribute to the recovery of endogenous -cell function, independent of effects on glucose control.
Our in vivo and in vitro studies also underscore the limited nature of -cell regeneration in this STZ model. Random blood glucose levels remained elevated even after 120 days of normal glycemia, and the IPGTT responses remained impaired. The restored islets also had abnormal abilities to flux [Ca2+]i in response to high glucose challenge. The partial recovery of -cell function could be due to permanent destruction of -cell–to–-cell contacts, alterations in the architecture of the restored islet, or scarring, leading to defective -cell sensing. Whether these features are specific to the STZ-induced diabetes model or reflect a more generalized limitation of regenerated -cells will have to be elucidated in other more physiologic models of -cell destruction.
Our observations of limited restoration of -cells in the STZ model are in contrast to the more robust levels of regeneration reported for autoimmune diabetic NOD mice (23,44,45), and they suggest that the rate of -cell regeneration may be regulated by multiple factors. In the RIP-LCMV model of autoimmune diabetes, von Herrath et al. (46) reported that the ability to resist the development of diabetes differed drastically between the 129 and the C57BL/6 strain, and they speculated that this difference was the result of differences in the abilities of these two strains of mice to regenerate -cells. It has also been reported that partial pancreactectomy enhanced resistance to STZ-induced diabetes (11) and that neonatal rats are able to spontaneously recover from STZ-induced diabetes (47). Thus, it appears that multiple factors, including the genetic background, the age of the diabetic animal, and the type of injury/inflammation causing -cell destruction, affect the rate of -cell regeneration in rodents. It is likely that these same factors will shape the ability of diabetic human patients to regenerate -cells. We speculate that -cell regeneration in adult humans is likely to be a slow process, and the significant question of how effectively diabetic patients with depleted -cell mass can regenerate remains to be answered.
Understanding how -cell regeneration is normally regulated could lead to the identification of new approaches for enhancing -cell regeneration. To this end we tested the effect of the spleen in the restoration of -cell function in this model. A role for the spleen in -cell regeneration was suggested by clinical data showing that the incidence of diabetes was significantly higher in patients undergoing partial pancreatectomy and splenectomy than in those undergoing pancreatectomy alone (48). Further evidence for a role of the spleen in -cell regeneration came from the NOD mouse model of autoimmune diabetes (23). Kodama et al. (23) reported that stem cells residing in the spleen of NOD mice were capable of differentiating into -cells, thereby restoring -cell mass and curing diabetes. We here demonstrate that the removal of the spleen resulted in significantly reduced restoration of -cell function in this model, consistent with the clinical data. We also confirmed that the administration of high doses of spleen cells partially restored the ability to recover -cell function in the splenectomized mice. We observed a peri-islet localization of the donor spleen cells, but, contrary to the observations by Kodama et al. (25), we were unable to demonstrate a direct contribution of spleen-derived cells to the regenerated islets. These data are, however, consistent with recent reports that spleen cells do not differentiate into islet -cells (49–51). It is currently unclear what role the spleen plays in this model of restoration of -cell function, but it may resemble the indirect effects of bone marrow–derived stem cells in initiating pancreatic -cell regeneration as described by Hess et al. (52). Indeed, we are currently testing whether the spleen contributes to the inflammatory process in the pancreas (and thereby stimulates recovery of the STZ damaged islet), as well as defining the cell types, namely T, B, or other cells, involved in this effect. Finally, we are testing whether an infusion of a more physiologic number of spleen cells can enhance the recovery of -cell function.
In conclusion, there is recent interest in utilizing the natural ability of -cells to regenerate as an innovative approach for the treatment of type 1 diabetes. We have developed a new mouse model, whereby we demonstrate a partial restoration of -cell mass and function in adult mice made diabetic with STZ. While the end goal is to completely eliminate the need for exogenous insulin, the benefits of partial recovery of -cell function in severe diabetic patients cannot be ignored, as patients with residual -cell function require less insulin, have better and easier glycemic control, and fewer end-organ complications compared with patients with no residual function (35). We also observed a significant requirement for the spleen and spleen cells in islet cell regeneration, and we demonstrate that this is not due to direct differentiation of the spleen cells into -cells. This study supports the possibility of regeneration as a means for generating new -cells even in severely diabetic individuals with significantly reduced -cell mass. The rate of normal -cell regeneration is slow, and it is critical that strategies are identified to enhance -cell regeneration in severely diabetic individuals.
ACKNOWLEDGMENTS
This work was supported by grant support from the JDRF, National Institutes of Health(DK073529), Chester Foundation, and Lyman Family Fund. We thank Dr. Denise Faustman for her advice on the spleen cell preparations, Dr. Ian Boussy for his assistance throughout the study and Christine Ding for technical assistance.
FOOTNOTES
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.
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