Endogenous Pancreas Research Articles

CAR Tregs protect alloimmune islets against autoimmune destruction

Abstract Regulatory T cell (Treg) therapy is a promising approach to induce tolerance of transplanted tissues and/or self antigens that drive autoimmunity. Early clinical trials with polyclonal Tregs have proven feasible, safe and tolerable. The next wave of Treg therapies is genetically enhanced with antigen specificity. We have previously shown that Tregs engineered to express a chimeric antigen receptor specific for HLA-A2 (A2-CAR) delay rejection of HLA-A2+ grafts; however, it was unknown whether they would limit T cell responses targeting antigens other than HLA-A2 via bystander suppression. To investigate this possibility, we used a model of HLA-A2-mismatched islet transplantation and asked if A2-CAR Tregs could inhibit rejection mediated by diabetogenic T cells. HLA-A2+ islets (150–200) isolated from NOD-HLA-A2+ mice were transplanted into immunodeficient NSG mice. After 3.5 weeks, diabetogenic BDC2.5 CD4+ T cells were adoptively transferred with or without polyclonal or A2-CAR Tregs (1 CD4+:5 Tregs). Mice that received BDC2.5 CD4+ T cells without Tregs or polyclonal Tregs rapidly developed hyperglycemia. In contrast, mice that received A2-CAR Tregs were completely protected from hyperglycemia. Longitudinal monitoring of injected Tregs showed that A2-CAR Tregs were more numerous and activated relative to polyclonal Tregs. Removal of the HLA-A2+ islet graft from a protected A2-CAR Treg-treated mouse did not induce hyperglycemia, suggesting that the ability of BDC2.5 CD4+ T cells to mediate autoimmune attack of the endogenous (HLA-A2-) pancreas was suppressed. These data suggest that A2-CAR Tregs mediate bystander suppression in vivo and may have the ability to induce long-term tolerance to both allo- and autoantigens. Supported by a CIHR Canada Graduate Scholarship doctoral research award and a CIHR foundation grant (FDN-154304).

The development and evolution of insulin pumps: from early beginnings to future prospects.

Diabetes technology has proliferated extensively over the past few decades with vast ameliorations in glucose monitoring and in insulin delivery systems. From a treatment based on daily insulin injections, we have moved to increasingly advanced technologies. Despite such advancements which have allowed better glycemic control, decreased diabetes-related complications, and improved the quality of life among diabetic patients, it has left many individuals unsatisfied with the current rate of commercial artificial pancreas development, stemming the need for further research into novel technologies. Accordingly, the Juvenile Diabetes Research Foundation has marked three generations for the development of an artificial pancreas comprising historical landmarks and future prospects which aim to produce an advanced technological system that attempts to mimic the endogenous pancreas, eliminating the need for user input. This review presents a synopsis of the development and evolution of insulin pumps, starting with the earliest technologies available such as continuous subcutaneous insulin infusion and continuous glucose monitoring as separate components, to currently available integrated advanced closed-loop hybrid systems and possible future technologies. The aim of the review is to provide insight of the advantages and limitations of past and currently available insulin pumps with the hope of driving research into novel technologies that attempt to mimic endogenous pancreatic function as closely as possible.

The endocrine pancreas during exercise in people with and without type 1 diabetes: Beyond the beta-cell.

Although important for digestion and metabolism in repose, the healthy endocrine pancreas also plays a key role in facilitating energy transduction around physical exercise. During exercise, decrements in pancreatic β-cell mediated insulin release opposed by increments in α-cell glucagon secretion stand chief among the hierarchy of glucose-counterregulatory responses to decreasing plasma glucose levels. As a control hub for several major glucose regulatory hormones, the endogenous pancreas is therefore essential in ensuring glucose homeostasis. Type 1 diabetes (T1D) is pathophysiological condition characterised by a destruction of pancreatic β-cells resulting in pronounced aberrations in glucose control. Yet beyond the beta-cell perhaps less considered is the impact of T1D on all other pancreatic endocrine cell responses during exercise and whether they differ to those observed in healthy man. For physicians, understanding how the endocrine pancreas responds to exercise in people with and without T1D may serve as a useful model from which to identify whether there are clinically relevant adaptations that need consideration for glycaemic management. From a physiological perspective, delineating differences or indeed similarities in such responses may help inform appropriate exercise test interpretation and subsequent program prescription. With more complex advances in automated insulin delivery (AID) systems and emerging data on exercise algorithms, a timely update is warranted in our understanding of the endogenous endocrine pancreatic responses to physical exercise in people with and without T1D. By placing our focus here, we may be able to offer a nexus of better understanding between the clinical and engineering importance of AIDs requirements during physical exercise.

Autoimmunity as an Endogenous Source of Heterologous Alloreactivity.

In autoimmune Type 1 diabetic (T1D) recipients, the disease can recur in pancreatic islet transplants. However, because autoimmune T cells recognize autoantigens in the context of self MHC antigens, it is not clear how autoreactive (islet-specific) T cells may recognize MHC-unrelated islet allografts. In this study, determined the specificity of T cells infiltrating islet allografts in the non-obese diabetic (NOD) mouse model of T1D. We grafted 400-500 syngeneic (NOD, H-2g7) or allogeneic (C3H, H-2k) islets beneath the kidney capsule of spontaneously diabetic female NOD mice. Curiously, although the autoimmune repertoire is self (H-2g7) MHC-restricted, C3H allografts were more rapidly rejected in NOD mice (n = 6; MST 7days) than were syngeneic (NOD) islet grafts (n = 19; MST 11 days), p < .001. We tested the hypothesis that within the activated autoimmune T cell repertoire was a sub-population of T cell receptors (TCR) that cross-reacted with allogeneic MHC. To determine whether islet-autoreactive T cells infiltrate C3H islet allografts, we used 454 high-throughput TCR sequencing to compare the TCR repertoires expressed in the NOD recipient's residual endogenous pancreatic islets to those expressed by islet allograft-infiltrating T cells. A portion of TCRs were shared between the endogenous pancreatic islets and the C3H allograft. We next determined the specificity of TCRs used in high frequency in rejected C3H islet allografts. To this end, TCRs expressed in high frequency in the allograft (>1%) were cloned and tested for islet autoreactivity and/or alloreactivity following TCR cDNA transfection into IL-2 producing hybridoma cells. Of note, three TCRs studied that were the most frequent in the graft were also detected in the endogenous pancreas. As expected, all three TCRs were islet reactive. Surprisingly, all three of these TCR also were directly alloreactive to C3H spleen cells in the absence of either islet antigens or NOD APCs. Two of the three TCRs were MHC class I reactive (anti-H-2Dk), and the other was MHC class II-reactive (anti-I-Ak). These results suggest that dual auto/allo-reactive T cells are selectively expanded in islet allografts. As such, we propose that autoimmunity is an unusual source of endogenous heterologous alloreactivity that may target an allograft via simultaneous auto- and allo-reactive specificities.

Autoimmunity as an endogenous source of heterologous alloimmunity (TRAN3P.868)

Abstract Curiously, although autoimmunity is restricted to self MHC molecules, spontaneously diabetic NOD mice reject MHC-unrelated islet allografts more rapidly than syngeneic (NOD) grafts. We hypothesized that the autoimmune (islet-reactive) T cell repertoire harbors a subset of TCRs that also cross-react to allogeneic MHC molecules. To test this concept, we performed high throughput TCR sequencing of T cells infiltrating MHC-mismatched (C3H, H-2k) islet allografts on the day of rejection in diabetic female NOD mice. TCRs expressed in high frequency in the allograft (&amp;gt;1%) were cloned and tested for islet autoreactivity and/or alloreactivity following TCR cDNA transfection into IL-2 producing hybridoma cells. Of note, three TCRs studied that were the most frequent in the graft were also detected in the endogenous pancreas. All three transfectants expressing these TCRs were islet reactive. Importantly, all three of these TCR also were directly alloreactive to C3H spleen cells. Two of the three TCRs were MHC class I reactive (anti-H-2Dk), and the other was MHC class II-reactive (anti-I-Ak). These results suggest that dual auto/allo-reactive T cells are selectively expanded in islet allografts. As such, we propose that autoimmunity is an unusual source of endogenous heterologous alloreactivity that may accelerate allograft rejection by simultaneous auto- and allo-reactivity.

Adaptive changes of human islets to an obesogenic environment in the mouse

Aims/hypothesisIn this study, we used an immunodeficient mouse model to explore, in vivo, the longitudinal adaptation of human islets to an obesogenic environment.MethodsNon-diabetic Rag2 –/– mice (n = 61) were transplanted with human islets (400 islet equivalents [IEQ]) from six pancreases: four non-diabetic and two with overt metabolic dysfunction (older, high HbAlc or history of diabetes). Animals were fed for 12 weeks with a control or high-fat diet (HFD), and followed for weight, serum triacylglycerol, fasting blood glucose and human C-peptide. After the mice were killed, human grafts and the endogenous pancreas were analysed for endocrine volume, distribution of beta and alpha cells, and proliferation.ResultsTransplanted mice on an HFD gained significantly more weight (p < 0.001) and had higher fasting glycaemia (2–12 weeks; p = 0.0002) and consistently higher fasting human C-peptide levels (2–12 weeks; p = 0.04) compared with those on the control diet. Histology demonstrated doubling of human islet graft volume at 12 weeks in animals on the HFD and increased beta cell volume (p < 0.001), but no change in alpha cell volume. Human islet function (hyperbolic product HOMA2%BS) at 12 weeks was four times lower in HFD animals (p < 0.001 vs controls) because of insufficient beta cell adaptation to decreased (70%) sensitivity (HOMA%S). Human islets obtained from donors with metabolic dysfunction failed to adapt to the HFD.Conclusions/interpretationThis longitudinal study provides direct evidence that human islets adapt both endocrine and beta cell mass, function and gene expression to obesity in vivo. The present model will facilitate the identification of mechanisms by which human islets adapt to obesity in vivo and the cell type(s) responsible, and factors predisposing human beta cells to decompensation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-012-2775-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Rosiglitazone Treatment Does Not Decrease Amyloid Deposition in Transplanted Islets From Transgenic Mice Expressing Human Islet Amyloid Polypeptide

In human islet transplantation, insulin independence decreases over time. We previously showed that amyloid deposition following transplantation of islets from human islet amyloid polypeptide (hIAPP) transgenic mice resulted in ß-cell loss and that rosiglitazone treatment decreased islet amyloid deposition and preserved ß-cell area in the endogenous pancreas of hIAPP transgenic mice. Thus, we sought to determine if rosiglitazone treatment decreases islet amyloid deposition and the associated ß-cell loss after islet transplantation. Streptozocin-diabetic mice were transplanted with 100 islets from hIAPP transgenic (T) mice or nontransgenic (NT) littermates under the kidney capsule and received either rosiglitazone (R) in drinking water or plain drinking water (C). The resultant groups (NTC [n = 11], NTR [n = 9], TC [n = 14], and TR [n = 10]) were followed for 12 weeks after which the graft was removed and processed for histology. Amyloid was detected in nearly all T islet grafts (TC = 13/14, TR = 10/10) but not in NT grafts. Rosiglitazone did not alter amyloid deposition (% graft area occupied by amyloid; TC: 2.15 ± 0.7, TR: 1.72 ± 0.66; P = .86). % ß-cell/graft area was decreased in the TC grafts compared to NTC (56.2 ± 3.1 vs 73.8 ± 1.4; P < .0001) but was not different between TC and TR groups (56.2 ± 3.1 vs 61.0 ± 2.9; P = .34). Plasma glucose levels before and after transplantation did not differ between NTC and TC groups and rosiglitazone did not affect plasma glucose levels post–islet transplantation. Rosiglitazone did not decrease amyloid deposition in hIAPP transgenic islet grafts. Therefore, rosiglitazone treatment of recipients of amyloid forming islets may not improve transplantation outcomes.

A Low-Oxygenated Subpopulation of Pancreatic Islets Constitutes a Functional Reserve of Endocrine Cells

OBJECTIVEThe blood perfusion of pancreatic islets is highly variable and tightly regulated by the blood glucose concentration. Thus, oxygen levels are considered crucial for islet metabolism and function. Although islet oxygenation has been extensively studied in vitro, little is known about it in vivo. The current study aimed to investigate the oxygenation of the endocrine pancreas in vivo.RESEARCH DESIGN AND METHODSThe reductive metabolism of 2-nitroimidazoles, such as pimonidazole, has previously been extensively used in studies of oxygen metabolism both in vitro and in vivo. At tissue oxygen levels <10 mmHg, pimonidazole accumulates intracellularly and may thereafter be detected by means of immunohistochemistry. Islet oxygenation was investigated in normal, 60% partially pancreatectomized, as well as whole-pancreas–transplanted rats. Moreover, leucine-dependent protein biosynthesis was performed using autoradiography to correlate islet oxygenation with metabolic activity.RESULTSIn vivo, 20–25% of all islets in normal rats showed low oxygenation (pO2 <10 mmHg). Changes in the islet mass, by means of whole-pancreas transplantation, doubled the fraction of low-oxygenated islets in the endogenous pancreas of transplanted animals, whereas this fraction almost completely disappeared after a 60% partial pancreatectomy. Moreover, oxygenation was related to metabolism, since well-oxygenated islets in vivo had 50% higher leucine-dependent protein biosynthesis, which includes (pro)insulin biosynthesis.CONCLUSIONSThe current study suggests a novel subpopulation of dormant low-oxygenated islets, which seems to constitute a functional reserve of endocrine cells. This study establishes a novel perspective on the use of the endocrine pancreas in glucose homeostasis.

Co-transplantation of mesenchymal stem cells maintains islet organisation and morphology in mice

Recent studies have shown that mesenchymal stem cells (MSCs) secrete several factors that improve survival and function of transplanted islets. Implantation of islets beneath the kidney capsule results in morphological changes, due to interactions of the graft with the host, thus impairing islet function. We co-transplanted MSCs with islets to determine their effects on the remodelling process and studied graft function in a mouse model of minimal islet mass. Islets were syngeneically transplanted, either alone or with kidney-derived MSCs, underneath the kidney capsule of streptozotocin-induced diabetic C57Bl/6 mice. Blood glucose levels were monitored and intraperitoneal glucose tolerance tests carried out. Hormone contents of grafts and pancreas were assessed by radioimmunoassay. Graft morphology and vascularisation were evaluated by immunohistochemistry. MSCs improved the capacity of islet grafts to reverse hyperglycaemia, with 92% of mice co-transplanted with MSCs reverting to normoglycaemia, compared with 42% of those transplanted with islets alone. Average blood glucose concentrations were lower throughout the 1 month monitoring period in MSC co-transplanted mice. MSCs did not alter graft hormone content. Islets co-transplanted with MSCs maintained a morphology that more closely resembled that of islets in the endogenous pancreas, both in terms of size, and of endocrine and endothelial cell distribution. Vascular engraftment was superior in MSC co-transplanted mice, as shown by increased endothelial cell numbers within the endocrine tissue. Co-transplantation of islets with MSCs had a profound impact on the remodelling process, maintaining islet organisation and improving islet revascularisation. MSCs also improved the capacity of islets to reverse hyperglycaemia.

Hyaluronidase Treatment of Graft Pancreatitis in Rats: Marked Effects on the Blood Perfusion of the Transplanted Pancreas

Hyaluronan is known to accumulate in tissues during inflammatory diseases associated with graft implantation and rejection of organ allografts. The aim was to evaluate whether hyaluronidase treatment affected hyaluronan content and blood perfusion in graft pancreatitis. Syngeneic rat pancreatic-duodenal transplantations were performed. Two days later blood flow measurements were made with a microsphere technique in both grafted and endogenous pancreas in animals treated with daily injections of vehicle or hyaluronidase (20.000 U/kg). Non-transplanted rats served as controls. Also, samples for analysis of hyaluronan and water content were taken. The hyaluronan content of the pancreatic graft was increased after transplantation. Hyaluronidase treatment markedly reduced total pancreatic and islet blood flow in both grafted and endogenous pancreas, whereas duodenum blood flow was unaffected. No blood flow effects were seen in non-transplanted control rats. Hyaluronan content was increased in the grafted pancreas, but hyaluronidase treatment decreased it to levels comparable to those of the endogenous gland. There were no differences in hyaluronan content in the endogenous pancreases of transplanted and non-transplanted rats. Graft pancreatitis after rat pancreas transplantation is associated with an increased hyaluronan content, which can be reduced by treatment with hyaluronidase. Hyaluronidase treatment of the graft recipients effected a 50% reduction in total pancreatic and islet blood flow in the graft, as well as in the endogenous pancreas. The functional importance of this is at present unknown.

β cell regeneration

Replacing the 350 million β cells destroyed in people with type 1 diabetes has traditionally been sought by transplanting human pancreatic tissue obtained after death. Alternative sources of exogenous surrogate β cells being examined include stem cells, fetal pancreas and cells derived from the liver, with pluripotent stem cells offering the most hope for a large supply of usable tissue. Whilst induced pluripotent stem cells offer the benefit of being autologous, genetic manipulation required for their formation creates safety concerns for future therapeutic use. Encapsulation of pancreatic progenitors derived from embryonic stem cells seems to offer a solution allowing acceptance of the histoincompatible cells, and prevention not only of teratomas but also recurrence of autoimmune rejection. Regenerating β cells from non-β cells in the endogenous pancreas is being attempted using several agents, with most promise ones arising from the administration of gastrin and an incretin. The peptide INGAP, or its humanized version, HIP, also may be beneficial. At present, more benefit has been achieved in rodents, but clinical trials are underway. The main source of new β cells in humans is from pancreatic progenitors, whereas in rodents it is from pre-existing β cells.

Hemodynamic effect of iopromide in pancreas-duodenum transplanted rats

Radiological contrast media (CM) have been suggested to be able to impair pancreatic microcirculation, especially in acute pancreatitis. To evaluate the effects of the low-osmolar CM iopromide on total pancreatic and especially islet blood perfusion after whole pancreas transplantation. Rats receiving a pancreas-duodenum transplantation 2 days earlier, i.e., with graft pancreatitis, were injected with iopromide. Blood perfusion measurements were then made with a microsphere technique. The graft blood perfusion was decreased in control rats when compared to the endogenous pancreas. Administration of iopromide increased both total pancreatic and islet blood perfusion in the grafted pancreas, but not in the endogenous gland. No effects on blood perfusion to either the native or transplanted duodenum were seen after iopromide administration. Iopromide increases the blood perfusion of a whole pancreas transplant 2 days after implantation, i.e., when graft pancreatitis is present. The consequences of this CM-induced hyperperfusion for graft pancreatic function remain to be established.Key words: Intravascular contrast media; islet blood perfusion; graft pancreatitis;pancreas transplantation; pancreatic blood perfusion

SYSTEMIC TGF-BETA1 GENE THERAPY INDUCES CD4+CD25+ FOXP3+ REGULATORY T CELLS, RESTORES SELF-TOLERANCE AND FACILITATES ISLET REGENERATION IN OVERTLY DIABETIC NOD MICE

O251* Aims: Type 1 diabetes results from autoaggressive T cell mediated destruction of insulin producing beta cells of the pancreas. Recent data support the idea that restoration of self-tolerance may facilitate islet cell regeneration/recovery. In view of the potent immunoregulatory activity of transforming growth factor beta1 (TGF-β1), we explored the hypothesis that systemic TGF-β1 therapy restores self-tolerance, facilitates islet regeneration and cures autoimmune diabetes. Methods: We designed and constructed a replication deficient adenovirus (Ad) vector encoding the active form of human TGF-β1 and tested the hypothesis that systemic TGF-β1 gene therapy restores self-tolerance and facilitates islet regeneration in the NOD model. Overtly diabetic NOD mice received intravenous injection of Ad.AH.TGF-β1 at a dose of 5x1010 particle units and seven to fourteen days after the injection, the mice were transplanted with 500-600 syngeneic islets under the kidney capsule. Results: Hyperglycemia occurred by day 17 in all untreated NOD mice whereas 50% of syngeneic grafts functioned until day 60 in the NOD mice treated with Ad.AH.TGF-β1. Analysis of retrieved islet grafts showed well-preserved islets with a peri-islet infiltrate primarily of CD4+ T cells and expression of CD25 and Foxp3. Staining for IFN-γ was negative whereas TGF-β1 and IL-10, and PD-L1 and PD-L2 all showed heightened expression. Having demonstrated that Ad.AH.TGF-β1 protects syngeneic islet grafts, we investigated whether systemic TGF-β1 gene therapy is associated with islet regeneration in the native pancreas. Thus, we removed the long-term functioning syngeneic islet grafts and examined whether the mice remained normoglycemic; we also removed the endogenous pancreas and examined the status of native islets. Normoglycemia was observed in 66% of the mice despite removal of the syngeneic islet grafts suggesting recovery of endogenous islet function upon treatment with Ad.AH.TGF-β1. Examination of the native pancreas revealed scattered islets, among pancreatic exocrine tissues, with intact islet structures (panel A). There were few intra-islet cellular infiltrates. However, similar to what was found in the syngeneic islet graft, there were well-demarcated lymphocytic infiltrates on the periphery of the islets without invasion of the islets themselves (Panel A. Peri-insulitis but not insulitis). Moreover, the intact islets stained strongly for insulin (Panel B) providing definitive evidence for functional restoration of endogenous beta cells.FigureConclusions: Our data demonstrate that systemic TGF-beta1 gene restores self-tolerance and facilitates islet regeneration in previously diabetic NOD mice. Our studies support the idea that in-vivo expansion of CD4+CD25+ Foxp3+ regulatory T cells is an effective strategy for constraining established islet directed autoimmunity and curing type 1 diabetes.

Recovery of the endogenous beta cell function in the NOD model of autoimmune diabetes.

In light of accumulating evidence that the endocrine pancreas has regenerative properties and that hematopoietic chimerism can abrogate destruction of beta cells in autoimmune diabetes, we addressed the question of whether recovery of physiologically adequate endogenous insulin regulation could be achieved in the nonobese diabetic (NOD) mice rendered allogeneic chimerae. Allogeneic bone marrow (BM) was transplanted into NOD mice at the preclinical and overtly clinical stages of the disease using lethal and nonlethal doses of radiation for recipient conditioning. Islets of Langerhans, syngeneic to the BM donors, were transplanted under kidney capsules of the overtly diabetic animals to sustain euglycemia for the time span required for recovery of the endogenous pancreas. Nephrectomies of the graft-bearing organs were performed 14 weeks later to confirm the restoration of endogenous insulin regulation. Reparative processes in the pancreata were assessed histologically and immunohistochemically. The level of chimerism in NOD recipients was evaluated by flow cytometric analysis. We have shown that as low as 1% of initial allogeneic chimerism can reverse the diabetogenic processes in islets of Langerhans in prediabetic NOD mice, and that restoration of endogenous beta cell function to physiologically sufficient levels is achievable even if the allogeneic BM transplantation is performed after the clinical onset of diabetes. If the same pattern of islet regeneration were shown in humans, induction of an autoimmunity-free status by establishment of a low level of chimerism, or other alternative means, might become a new therapy for type 1 diabetes.

Evidence for a circulating islet cell growth factor in insulin-resistant states.

Insulin resistance is a feature of many common disorders including obesity and type 2 diabetes mellitus. In these disorders, the beta-cells compensate for the insulin resistance for long periods of time with an increase in secretory capacity, an increase in beta-cell mass, or both. To determine whether the beta-cell response might relate to a circulating growth factor, we have transplanted normal islets under the kidney capsule of normoglycemic insulin-resistant mice with two different models of insulin resistance: lean mice that have a double heterozygous deletion of the insulin receptor and insulin receptor substrate-1 (DH) or the obese, hyperglycemic ob/ob mice. In the grafts transplanted into both hosts, there was a marked increase in beta-cell mitotic activity and islet mass that was comparable with that observed in the endogenous pancreas. By contrast, islets of the DH mouse transplanted into normal mice showed reduced mitotic index. These data suggest the insulin resistance is associated with a circulating islet cell growth factor that is independent of glucose and obesity.

Insulin secretion and glucose tolerance after islet transplantation in rats with noninsulin-dependent diabetes induced by neonatal streptozotocin

The present study was designed to identify in a model of noninsulin-dependent diabetes induced by neonatal streptozotocin (n0-STZ), the long-term consequences of an islet graft upon 1) glucose handling of the recipient and, 2) glucose response of the residual β cells in the recipient pancreas. We have examined, 4 and 8 wk after islet implantation under the kidney capsule of syngeneic diabetic n0-STZ rats, their tolerance to glucose administered in vivo, together with their insulin release in response to glucose in vivo (oral glucose tolerance test) as well as in vitro (perfused pancreas). The results in the islet-grafted n0-STZ rats, were compared to those obtained in nongrafted nondiabetic rats and nongrafted n0-STZ rats. Our study shows that transplanting a limited number (900) of adult islets under the kidney capsule reverses to normal, many parameters of the noninsulin-dependent diabetic state in the n0-STZ rat model: these include body weight, basal plasma glucose in both the nonfasted and postabsorptive states, and basal plasma insulin in the postabsorptive state. Furthermore, tolerance to oral glucose administration was greatly improved in the transplanted rats and it was correlated with restoration of a manifest glucose-induced insulin secretion in vivo as evaluated (ΔI) during an oral glucose tolerance test. Our data clearly show that the insulin response to glucose from the endogenous pancreas of n0-STZ diabetic rat was not really improved by long-term (8 wk) basal normoglycemia. More precisely, we were able to detect a slight but significant improvement of the early phase of insulin release in vitro in response to glucose; however, the overall insulin response remained 15 times lower than the normal one with no reapparance of the late phase of insulin release. After cessation of glucose stimulation in vivo, off-response of insulin, which is also a landmark of the impaired insulin release by the β cells of n0-STZ rats, was still detectable in the perfused pancreas of the transplanted n0-STZ rats. Finally, because the reactivity to glucose of the endogenous residual β cells was not regained, the insulin released in vivo during the oral glucose test in the graft-bearing n0-STZ rats can be attributed mainly to functioning of the grafted islets population.

Insulin secretion and glucose tolerance after islet transplantation in rats with noninsulin-dependent diabetes induced by neonatal streptozotocin.

The present study was designed to identify in a model of noninsulin-dependent diabetes induced by neonatal streptozotocin (n0-STZ), the long-term consequences of an islet graft upon 1) glucose handling of the recipient and, 2) glucose response of the residual beta cells in the recipient pancreas. We have examined, 4 and 8 wk after islet implantation under the kidney capsule of syngeneic diabetic n0-STZ rats, their tolerance to glucose administered in vivo, together with their insulin release in response to glucose in vivo (oral glucose tolerance test) as well as in vitro (perfused pancreas). The results in the islet-grafted n0-STZ rats, were compared to those obtained in nongrafted nondiabetic rats and nongrafted n0-STZ rats. Our study shows that transplanting a limited number (900) of adult islets under the kidney capsule reverses to normal, many parameters of the noninsulin-dependent diabetic state in the n0-STZ rat model: these include body weight, basal plasma glucose in both the nonfasted and postabsorptive states, and basal plasma insulin in the postabsorptive state. Furthermore, tolerance to oral glucose administration was greatly improved in the transplanted rats and it was correlated with restoration of a manifest glucose-induced insulin secretion in vivo as evaluated (delta 1) during an oral glucose tolerance test. Our data clearly show that the insulin response to glucose from the endogenous pancreas of n0-STZ diabetic rat was not really improved by long-term (8 wk) basal normoglycemia. More precisely, we were able to detect a slight but significant improvement of the early phase of insulin release in vitro in response to glucose; however, the overall insulin response remained 15 times lower than the normal one with no reappearance of the late phase of insulin release. After cessation of glucose stimulation in vivo, off-response of insulin, which is also a landmark of the impaired insulin release by the beta cells of n0-STZ rats, was still detectable in the perfused pancreas of the transplanted n0-STZ rats. Finally, because the reactivity to glucose of the endogenous residual beta cells was not regained, the insulin released in vivo during the oral glucose test in the graft-bearing n0-STZ rats can be attributed mainly to functioning of the grafted islets population.

Potent inhibitory effects of transplantable rat glucagonomas and insulinomas on the respective endogenous islet cells are associated with pancreatic apoptosis.

Effects of transplantable rat insulinomas (IN) and glucagonomas (GLU) on the endogenous pancreas were analyzed using morphometry, immunocytochemistry, in situ hybridization, and staining for apoptotic cells. Hyperinsulinemia (IN-rats) and hyper-GLP-1/glucagonemia (GLU-rats) were both associated with marked islet atrophy (67 and 76% of control average planimetrical islet area, respectively). Selective islet B cell inhibition of proinsulin (I and II) genes as well as of expression of the insulin gene transcription factor, IPF1/STF1, was found in IN-rats. Moreover, these islets were characterized by significant B cells apoptosis in the absence of infiltrating lymphocytes. In GLU-rats selective islet A cell inhibition was observed at the level of glucagon mRNA. These islets contained small, highly condensed but clearly active B cells with prominent IPF1/STF1-positive nuclei, surrounded by densely packed glucagon-negative cells with reduced cytoplasm. Furthermore, an active apoptotic process was found exclusively in the exocrine pancreas of GLU-rats. Thus, in IN-rats, islet B cell mass reduction is distinguished by non-immune-mediated programmed cell death, while GLU-rats exhibit A cell mass reduction by cytoplasmic retraction and selective exocrine apoptosis.

The amelioration of hyperglycemia in streptozotocin-induced diabetic rats after the intraportal transplantation of an insufficient number of islets by nicotinamide treatment.

The purpose of the present study was to determine whether or not hyperglycemia in streptozotocin (STZ)-induced diabetic rats after the intraportal transplantation of an insufficient number of isogenic islets can be ameliorated by nicotinamide treatment. WKA/Qdj (RT 1u) rats were used both as donors and recipients. Islets were isolated by the collagenase technique. A total of 350 islets was transplanted into the liver via the portal vein of the STZ-induced diabetic rats. Either nicotinamide (NA, 0.5 g/kg) or a vehicle (saline) was administered ip once a day for 60 days after transplantation. All the diabetic rats without islet transplantation remained hyperglycemic irrespective of the NA treatment. All the recipients (n = 12) bearing the islet grafts and treated with saline remained hyperglycemic (> 400 mg/dl) at 60 days after transplantation. In marked contrast, all the recipients (n = 18) with islet grafts and treated with NA became normoglycemic (< 200 mg/dl) at 16.2 +/- 7.1 days (mean +/- SD) after transplantation. Morphologically, islets were easily found in the liver of the recipients. Aldehyde-fuchsin stain revealed that the beta cells in the islet grafts of the NA treated recipients were well granulated, whereas those treated with saline were degranulated. The insulin content of the liver bearing the grafts treated with either NA or saline was 116.3 +/- 26.0 micrograms/liver (n = 4) or 5.7 +/- 2.2 micrograms (n = 4), respectively, while that of 350 donor islets was 29.4 +/- 2.5 micrograms (n = 5). The insulin content of the pancreas in the NA- or saline-treated recipients was 27.3 +/- 10.6 micrograms/pancreas (n = 4) or 2.7 +/- 1.2 micrograms (n = 4), respectively, while those of the pancreas from the diabetic rats without transplantation was 1.9 +/- 0.7 micrograms (n = 4) or 1.6 +/- 0.8 micrograms (n = 5), respectively. These findings clearly demonstrate that the hyperglycemia in the STZ-diabetic recipients after transplantation of an insufficient number of islets can be ameliorated while, in addition, the islet mass in the liver as well as the endogenous pancreas both increase in size with nicotinamide treatment.

Prostaglandin D2, F2 alpha, E2, and E1 in early phase of experimental acute necrohemorrhagic pancreatitis in rats.

Changes in endogenous pancreas production of prostaglandins D2, F2 alpha, E2, and E1 in early stages of acute necrotizing pancreatitis induced by intraductal administration of 3.5% sodium taurocholate have been determined by radioimmunoassay of chromatographically purified tissue extracts. For this purpose 18 male Wistar rats were randomized in three groups: control, pancreatitis, and pancreatitis plus indomethacin. Pancreas tissue samples were obtained 5 min after pancreatitis induction. In the pancreatitis-induced group, prostaglandins D2, F2 alpha, and E2 show significantly increased tissue levels relative to the controls whereas prostaglandin E1 remains unmodified. These results suggest a role for series 2 prostaglandins in the earlier stages of pancreatitis.