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PubMed1996

Expression of islet amyloid polypeptide in fetal and adult porcine and human pancreatic islet cells.

The synthesis and intracellular localization of the putative hormone islet amyloid polypeptide (IAPP) and its relation to insulin and glucagon during ontogenesis was investigated in fetal and adult porcine and human pancreatic islets. By means of ultrastructural immunogold immunocytochemistry, it was revealed that IAPP is produced by the hormonally pluripotent endocrine stem cells from the earliest time point studied. IAPP was colocalized with insulin and glucagon in the immature and nondifferentiated cell granules in both species. In adult man, highly intense IAPP immunoreactivity was found in beta-cell granules and, at lower intensity, in delta-cell granules. Some alpha-cells also contained a small amount of IAPP in their granules, and among these occasional granules displayed an intense immunoreactivity. In adult pig, IAPP was stored in quantity in beta-cell granules and in small amounts in granules of alpha- and delta-cells. It was difficult to determine the presence of IAPP in pancreatic polypeptide cells, because they were so seldom seen in this material. It is concluded that, in both man and pig, fetal pancreatic islet stem cells synthesize and store IAPP together with insulin and glucagon. The storage in different types of cells and granules was not as predictable as that of the classical islet hormones. The substance is more widely distributed within the pancreatic islet cell types than are any of the other islet hormones, which presumably has functional implications.

Aged↗

PubMed1995

Islet amyloid polypeptide in pancreatic tissue of children with persistent hyperinsulinemic hypoglycemia caused by primary islet hyperplasia and nesidioblastosis.

Islet amyloid is a recognized characteristic finding in insulinoma and secondary islet hyperplasia resulting from severe insulin resistance. Little information is available about the presence of islet amyloid in primary islet hyperplasia (nesidioblastosis) of childhood. Here we report that islet amyloid was not present in 12 children with primary islet hyperplasia associated with hyperinsulinemic hypoglycemia.

Amyloid↗

PubMed1999

Adenoviral gene transfer of the interleukin-1 receptor antagonist protein to human islets prevents IL-1beta-induced beta-cell impairment and activation of islet cell apoptosis in vitro.

The beta-cells in the pancreatic islets of Langerhans are the targets of autoreactive T-cells and are destroyed in type 1 diabetes. Macrophage-derived interleukin-1beta (IL-1beta) is important in eliciting beta-cell dysfunction and initiating beta-cell damage in response to microenvironmental changes within islets. In particular, IL-1beta can impair glucose-stimulated insulin production in beta-cells in vitro and can sensitize them to Fas (CD95)/FasL-triggered apoptosis. In this report, we have examined the ability to block the detrimental effects of IL-1beta by genetically modifying islets by adenoviral gene transfer to express the IL-1 receptor antagonist protein. We demonstrate that adenoviral gene delivery of the cDNA encoding the interleukin-1 receptor antagonist protein (IL-1Ra) to cultured islets results in protection of human islets in vitro against IL-1beta-induced nitric oxide formation, impairment in glucose-stimulated insulin production, and Fas-triggered apoptosis activation. Our results further support the hypothesis that IL-1beta antagonism in in situ may prevent intra-islet proinsulitic inflammatory events and may allow for an in vivo gene therapy strategy to prevent insulitis and the consequent pathogenesis of diabetes.

Adenoviridae↗

PubMed2000

The constitutive secretory pathway is a major route for islet amyloid polypeptide secretion in neonatal but not adult rat islet cells.

Islet amyloid polypeptide (IAPP or amylin) is a normal secretory product of the pancreatic beta-cell that is cosecreted with insulin and is the major constituent of islet amyloid deposits in individuals with type 2 diabetes or insulinomas. We have previously reported that glucose stimulates IAPP, but not insulin secretion, from neonatal rat beta-cells when regulated secretion is prevented by use of calcium-free media, suggesting that IAPP secretion occurs via a constitutive secretory pathway. To directly test this hypothesis, we examined the effects of 2 substances-brefeldin A (BFA) and cycloheximide (CHX)-that are predicted to selectively block constitutive secretion on the release of IAPP-like immunoreactivity (IAPP-LI) and immunoreactive insulin (IRI) from neonatal rat islet cell monolayer cultures. When regulated release was prevented by use of calcium-free media, glucose-stimulated IAPP-LI release was nearly abolished by blocking constitutive release with 10 microg/ml BFA (mean +/- SD: 8.7 +/- 7.7 vs. 29.3 +/- 14.3 pmol/l; n = 5; P < 0.05), an inhibitor of constitutive vesicle formation. Similarly, calcium-independent, glucose-stimulated IAPP-LI secretion was markedly suppressed when new protein synthesis was blocked by administration of 20 microg/ml CHX (4.6 +/- 2.1 vs. 29.5 +/- 14.0 pmol/l; n = 5; P < 0.005). Secretion of IRI was low in the absence of calcium, and neither BFA nor CHX had any further effect. When calcium was added to the incubation media to allow regulated secretion of both IRI and IAPP-LI, both BFA (47.7 micro 8.7 vs. 80.7 micro 10.3 pmol/l; P < 0.001) and CHX (37.3 +/- 5.8 vs. 73.3 +/- 6.2 pmol/l; n = 5; P < 0.0001) inhibited glucose-stimulated IAPP-LI secretion by approximately 40%, but again had no inhibitory effect on IRI secretion. These data indicate that approximately 40% of glucose-stimulated IAPP-LI release occurs via a constitutive secretory pathway in neonatal rat islet cells. By contrast, in adult rat islets, glucose-stimulated IAPP-LI release was almost abolished in the absence of calcium (86 +/- 3% inhibition; P < 0.05) and unaffected by addition of BFA (275 +/- 28 vs. 205 +/- 89 pmol/l; NS) or CHX (160 +/- 20 vs. 205 +/- 89 pmol/l; NS), suggesting that constitutive secretion of IAPP does not occur in mature beta-cells. Collectively, these data suggest that a significant proportion of glucose-stimulated IAPP secretion from neonatal, but not adult, rat islet cells occurs via a constitutive secretory pathway.

Aging↗

PubMed2002

Human pancreatic islets produce and secrete MCP-1/CCL2: relevance in human islet transplantation.

We investigated the capacity of human islets to produce monocyte chemoattractant protein-1 (MCP-1). Primary cultures of pancreatic islets expressed and secreted MCP-1, as determined by Northern blot, immunohistochemistry, in situ hybridization, and enzyme-linked immunosorbent assay. The produced MCP-1 was biologically active as it attracted monocytes in chemotaxis assay, and chemotactic activity was almost abrogated by a neutralizing anti-MCP-1 monoclonal antibody. Expression of MCP-1 was increased by primary inflammatory cytokines (interleukin-1 beta, tumor necrosis factor-alpha) and lipopolysaccharide at both the mRNA and protein levels but not by glucose. However, MCP-1 did not modulate insulin secretion. MCP-1 secreted by pancreatic islets plays a relevant role in the clinical outcome of islet transplant in patients with type 1 diabetes. In fact, low MCP-1 secretion resulted as the most relevant factor for long-lasting insulin independence. This finding opens new approaches in the management of human islet transplantation. Finally, the finding that MCP-1 appears constitutively present in normal human islet beta-cells (immunohistochemistry and in situ hybridization), in the absence of an inflammatory infiltrate, suggests that this chemokine could have functions other than monocyte recruitment and opens a new link between the endocrine and immune systems.

Adult↗

PubMed2002

Inhibition of thrombin abrogates the instant blood-mediated inflammatory reaction triggered by isolated human islets: possible application of the thrombin inhibitor melagatran in clinical islet transplantation.

A thrombotic/inflammatory reaction is elicited when isolated islets of Langerhans come in contact with ABO-compatible blood. The detrimental effects of this instant blood-mediated inflammatory reaction (IBMIR) provide a reasonable explanation for the observation that an unexpectedly high number of islets, from several donors, are needed to produce normoglycemia in transplant patients with type 1 diabetes. In this study, the hypothesis that a specific thrombin inhibitor, Melagatran, could reduce IBMIR in an in vitro model in which human islets are exposed to ABO-compatible blood was tested. The administration of Melagatran abrogated IBMIR dose-dependently. Islets exposed to blood, in the absence or presence of 0.4 micromol/l Melagatran, exhibited a loss of integrity and were found to be trapped in macroscopic clots containing platelets and CD11b(+) leukocytes. At concentrations from 1 to 10 micromol/l, Melagatran inhibited both coagulation and complement activation. Also, platelet and leukocyte activation and consumption were decreased. Islet morphology was maintained with almost no platelets adhering to the surface, and infiltration by CD11b(+) leukocytes was considerably reduced. In conclusion, Melagatran significantly reduced IBMIR in this model system. This protective effect indicates that thrombin plays a pivotal role in IBMIR and suggests that thrombin inhibition can improve the outcome of clinical islet transplantation.

ABO Blood-Group System↗

PubMed2005

Tissue factor produced by the endocrine cells of the islets of Langerhans is associated with a negative outcome of clinical islet transplantation.

There are strong indications that only a small fraction of grafts successfully engraft in clinical islet transplantation. One explanation may be the instant blood-mediated inflammatory reaction (IBMIR) elicited by tissue factor, which is produced by the endocrine cells. In the present study, we show that islets intended for islet transplantation produce tissue factor in both the transmembrane and the alternatively spliced form and that the membrane-bound form is released as microparticles often associated with both insulin and glucagon granules. A low-molecular mass factor VIIa (FVIIa) inhibitor that indirectly blocks both forms of tissue factor was shown in vitro to be a promising drug to eliminate the IBMIR. Thrombin-antithrombin complex (TAT) and FVIIa-antithrombin complex (FVIIa-AT) were measured in nine patients who together received 20 infusions of isolated human islets. Both the TAT and FVIIa-AT complexes increased rapidly within 15-60 min after infusion. When the initial TAT and FVIIa-AT levels were plotted against the increase in C-peptide concentration after 7 days, patients with an initially strong IBMIR showed no significant increase in insulin synthesis after 7 days. In conclusion, tissue factor present in both the islets and the culture medium and elicits IBMIR, which affects the function of the transplanted islets.

Adult↗

PubMed2000

Sertoli cell-induced reversal of adult rat pancreatic islet beta-cells into fetal-like status: potential implications for islet transplantation in type I diabetes mellitus.

BACKGROUND: Clinical success of pancreatic islet allograft (TX) for the therapy of diabetes mellitus is hampered by several pitfalls, primarily including the restricted availability of donor tissue and the immune- and/or non-immune-related TX's early loss, with the latter not necessarily being prevented by the host's general immunosuppression. Finally, adult islet beta-cells normally exhibit minimal proliferation capacity, which would not permit restoration of an eventually declining TX mass. METHODS: To address the limited beta-cell growth capacity, we have examined whether in vitro co-culturing adult rat islets (I) with prepubertal homologous Sertoli cells (SC) would stimulate I beta-cell expansion. SC-derived effects on the islets were studied in vitro, both morphologically (confocal laser microscopy) and functionally (glucose-stimulated insulin release). We have also preliminarily examined the in vivo impact of microencapsulated SC + I co-cultures on TX in diabetic mice. RESULTS: In vitro, we observed that SCs promoted significant beta-cell replication, as I beta-cell mitotic activity increased from 1% to greater than 8%, which coincided with the adult elements reversing into fetal-like status. This finding was coupled with significantly greater insulin release either in basal or in response to glucose, as compared with controls. CONCLUSIONS: Addition of SC to islets promotes reversal of the adult beta-cell elements into fetal-like conditions, thereby providing a new, potentially powerful tool that could significantly enhance the functional performance of islet TX in diabetic recipients.

Animals↗

PubMed1993

Transplantation of free and microencapsulated islets in rats: evidence for the requirement of an increased islet mass for transplantation into the peritoneal site.

Microencapsulation of islets of Langerhans may avoid the necessity of a permanent immunosuppressive drug therapy and opens up new perspectives for xenotransplantation in the treatment of insulin dependent diabetes. In a mouse model we recently showed long-term normoglycemia after microencapsulated xenotransplantation. Since the acceptance of mice to any kind of foreign material is quite high we assume that the rat model better reflects the situation of higher mammalians or even humans. Due to the volume of the transplanted material (i.e. islets+alginate-capsule) only the peritoneal cavity can be used up to now. The quantity of islets necessary to normalize the non-fasting blood glucose level was much higher than expected and free transplants needed even a higher amount of islets than encapsulated ones (3000 encapsulated vs. 2 x 3000 non-encapsulated). Transplantation beneath the kidney capsule was successful with only 1200-1500 islets per rat proving the metabolic potency of the islets. Implantation of empty capsules did not alter the diabetic state. We conclude that the alginate matrix may act as a "spacer" creating a distance between the consuments of a lacking substrate esp. oxygen in an unfavourable environment and perhaps protect it from unspecific mediators released during the postoperative period. Our findings underline the necessity for smaller capsules that would enable us to use other transplantation sites.

Animals↗

PubMed1997

Expression of islet amyloid polypeptide in fetal and adult porcine and human pancreatic islet cells.

Adult↗

PubMed2000

A modified method of islet isolation preserves the ability of pancreatic islets to increase protein O-glycosylation in response to glucose and streptozotocin.

An important link has recently been shown in vivo between beta-cell O-linked protein glycosylation and beta-cell apoptosis, with hyperglycemia having been demonstrated to reversibly increase beta-cell O-linked protein glycosylation by providing substrate for the glucosamine pathway. In contrast, the same study showed that the administration of streptozotocin to rats prior to the induction of hyperglycemia results in irreversible increases in O-glycosylation and subsequent beta-cell apoptosis. In light of these data, we investigated beta-cell O-glycosylation in vitro by exposing isolated rat islets to high glucose, glucosamine, or streptozotocin and analyzing the pattern of O-glycosylated proteins present. All three compounds acutely increased O-glycosylation of a predominate 135-kDa protein (p135); however, their ability to stimulate p135 O-glycosylation was only consistently observed when islets were isolated in the presence of high glucose and 1 mM L-glutamine. Islets isolated in low glucose and no added L-glutamine demonstrated no consistent increase in p135 O-glycosylation in response to glucose, glucosamine, or streptozotocin. These data suggest that during islet isolation, beta-cell enzymes responsible for regulating p135 O-glycosylation may be adversely affected by the absence of high glucose and glutamine, which together are necessary for O-linked N-acetylglucosamine synthesis. We propose that the combination of high glucose and glutamine during islet isolation generates UDP-N-acetylglucosamine and O-linked N-acetylglucosamine, thus providing substrate protection for these enzymes and preserving the ability of isolated islets to O-glycosylate p135.

Animals↗

PubMed1996

Complement-fixing islet cell antibodies in type-1 diabetes can trigger the assembly of the terminal complement complex on human islet cells and are potentially cytotoxic.

Forty-one sera of patients with IDDM (insulin-dependent diabetes mellitus) containing complement-fixing islet cell antibodies were analyzed for their ability to activate TCC (terminal complement complex). Eighteen sera were found to promote deposition of TCC on human islets of pancreatic cryostat sections with a nonhomogeneous pattern of distribution corresponding to that of insulin. Activation of TCC by IDDM serum and binding of this complex to islet cells was confirmed using purified islets. Flow cytometric analysis of islet cell treated with a TCC+ IDDM serum showed IgG binding to the cell surface. The same serum had a cytotoxic effect on islet cells in the presence of human C. These results obtained with a homologous system of C activation by IDDM sera suggest that TCC may contribute, at least in part, to the pancreatic beta cell damage.

Autoantibodies↗

PubMed1997

Nitric oxide-induced expression of C-reactive protein in islet cells as a very early marker for islet stress in the rat pancreas.

In searches for marker molecules specifically expressed in nitric oxide-treated islet cells as a means to recognize early events in islet destruction, we now establish the presence of neo-C-reactive protein (neoCRP) in rat islet cells as early as 2 hr after treatment. We detected this altered molecular form of the acute-phase-reactant C-reactive protein (CRP) using immunocytochemistry with an anti-neoCRP-specific monoclonal antibody as well as reverse transcription-polymerase chain reaction with CRP-specific primers and in situ hybridization to demonstrate the presence of CRP-specific mRNA. After induction of a generalized inflammatory reaction in rats with heat-inactivated Corynebacterium parvum in vivo, neoCRP expression in islets is also found and within the pancreas restricted to pancreatic islet cells only. Our findings suggest an early heat-shock-like expression of this molecule in response to local nitrite oxide production or to exogeneously added nitric oxide in islet cells.

Animals↗

PubMed1995

Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats.

We investigated the growth of islet beta and alpha cells in adult rats which had undergone partial pancreatic duct ligation. Whereas the non-ligated head portion of the pancreas remained unaffected in terms of histology and cell population dynamics, the ligated tail part of the pancreas showed pronounced changes in histology and cell growth. These changes included replacement of exocrine acini by ductal complexes and significant growth of islet cells. Using immunocytochemistry and morphometry, we found that the beta-cell population had nearly doubled within 1 week and that a smaller, but also significant growth of the alpha-cell population had occurred. In addition, small islets and islet-cell clusters were more numerous in the pancreatic tail, indicating islet neogenesis. The bromodeoxyuridine (BrdU) pulse labelling index of beta and alpha cells increased five fold and threefold, respectively, in the tail. However, the observed beta-cell labelling index remained below 1% which was largely insufficient to explain the increased number of beta cells. This indicates that recruitment from a proliferating stem-cell compartment was the main source for the beta-cell hyperplasia. A tenfold-elevated BrdU labelling index (18%) was observed in the duct-cell compartment which was identified by specific immunostaining for cytokeratin 20. Transitional cytodifferentiation forms between duct cells expressing cytokeratin 20 and beta cells expressing insulin, or alpha cells expressing glucagon, were demonstrated by double immunostaining. Pancreatic duct ligation also induced the expression of the beta-cell-specific glucose transporter type 2 (GLUT-2) in duct cells, indicating their metaplastic state. We concluded that in this adult rat model, the proliferation and differentiation of exocrine duct cells represents the major mechanism of endocrine beta-cell neogenesis. Our study thus demonstrates that in normal adult rats islet-cell neogenesis can be reactivated by stimulation of pancreatic duct cells.

Animals↗

PubMed1990

Maintenance of embedded pig pancreatic pseudo-islets in a collagen gel matrix: study of the effect of hydrocortisone, a collagenase inhibitor, and nicotinamide on collagenolysis and the morphogenesis of pancreatic islet-cells in collagen gel matrix.

We describe a method for maintaining neonatal pig pancreatic isletlike cell clusters (as pseudo-islets) embedded in a collagen gel matrix for long periods. The pseudo-islets were formed from single cells of pig pancreas maintained in a suspension culture and then embedded in pepsin-solubilized type I collagen. When the pseudo-islets were cultured in the collagen matrix, the amount of collagen in the culture decreased gradually during the culture period as soluble hydroxyproline-containing material accumulated in the medium. A low concentration of collagen (0.16%) degraded the collagen gels more rapidly than did high concentrations of collagen (0.64%). The degradation of collagen depended both on the number of pseudo-islets embedded in the gel matrix and on the culture conditions used to maintain them. With added nicotinamide, the accumulation of hydroxyproline decreased in the medium and the structure of the gel matrix was well maintained. Hydrocortisone or a specific inhibitor of collagenase did not decrease the solubilization of embedded pseudo-islet cultures and did not help to maintain their structure. These observations indicate the possible utility of long-term maintenance of pseudo-islets in collagen gel matrix in the presence of nicotinamide.

Animals↗

PubMed1986

Time-course of altered islet phospholipids and of calcium binding and ionophoretic properties of islet lipids following glucose stimulation.

The time-course of alteration in islet cell phospholipid content following D-glucose exposure in islet cells and in islet cell membranes was related to the ability of lipids extracted from both cultured pancreatic islet cells and from plasma membranes isolated from the islet cells to translocate calcium in two model membrane systems. The first model system (bulk-phase system) detected lipid species with the ability to bind calcium, irrespective of their ability to enhance calcium transport across cell membranes. The second system (multilamellar membrane system) detected lipid species with the ability to both bind calcium and to enhance calcium transport across cell membranes (true ionophores). Pre-exposure to high D-glucose concentration led to a rapid (within 1 min) fall in membrane phosphoinositides. This was partially blocked by mannoheptulose. A concurrent fall in calcium binding activity of lipids from the plasma membrane was observed. In the whole islet cell fraction, D-glucose induced a marked increase in Ca2+ ionophoretic activity. Unlike the fall in membrane polyphosphoinositides and membrane Ca2+ binding activity, these changes were dependent on the presence of added extracellular calcium. L-Glucose was without effect on membrane phosphoinositide content. It is concluded that altered membrane and intracellular phospholipids may contribute to the increased availability of intracellular Ca2+ following D-glucose stimulation by virtue of their Ca2+ binding and ionophoretic properties.

Animals↗

PubMed1993

Islet allograft, islet xenograft, and skin allograft survival in CD8+ T lymphocyte-deficient mice.

Despite extensive study, the immunologic mechanisms mediating allograft rejection have not been completely defined. In the current study, we evaluated the T cell subsets important in islet allograft, skin allograft, and islet xenograft rejection using a genetically engineered line of mice deficient in beta 2-microglobulin expression. Because these mice lack cell surface MHC class I expression, they are deficient in T cells of the CD8 subset (class I-restricted cytotoxic T cells). Pancreatic islet allografts transplanted to CD8+ T cell-deficient recipients showed prolonged survival compared with controls. No prolongation was observed in the survival of pancreatic islet xenografts or in the survival of skin allografts transplanted to the CD8+ T cell--deficient hosts. We conclude that CD8+ T cells play a prominent role in islet allograft, but not islet xenograft or skin allograft, rejection in mice.

Animals↗

PubMed1994

Evidence that intrathymic islet transplantation does not prevent diabetes or subsequent islet graft destruction in RT6-depleted, diabetes-resistant BioBreeding/Worcester rats.

Pancreatic islet allografts transplanted intrathymically are accepted and restore normoglycemia in streptozotocin-diabetic rats given one injection of antilymphocyte serum. Intrathymic allografts similarly restore normoglycemia in diabetes-prone (DP) Bio-Breeding (BB) rats that have developed spontaneous autoimmune diabetes. Intrathymic islets also reduce the frequency of subsequent diabetes when transplanted prophylactically into young DP rats. These findings suggest that intrathymic transplantation can prevent not only allograft rejection, but also the appearance and recurrence of autoimmune tissue destruction. To explore these hypotheses further, we attempted both to confirm previous studies and to extend them to another model of autoimmune diabetes, the RT6-depleted diabetes-resistant (DR) BB rat. Fewer than 1% of DR-BB rats develop spontaneous diabetes, but most become hyperglycemic after in vivo immune elimination of RT6+ T cells. Using the protocols described in the literature, we observed the following: (1) Consistent with previous reports, intrathymic islet allografts survived indefinitely in streptozotocin-diabetic, antilymphocyte serum-treated, non-BB recipient rats. (2) Consistent with previous reports, intrathymic islet grafts produced long-term normoglycemia in diabetic DP-BB rats and also reduced the frequency of spontaneous diabetes in young animals transplanted prophylactically. (3) In contrast, intrathymic islets (iso- and allografts) neither prevented nor reversed diabetes in RT6-depleted DR rats. We hypothesize that intrathymic islet grafts survive in DP-BB rats because they are lymphopenic and immunocompromised, whereas immunocompetent diabetic DR rats successfully recapitulate the autoimmune disease process. Although intrathymic allograft transplantation is postulated to induce a state of tissue-specific tolerance, our results indicate that this tolerant state may not extend to autoimmune destruction of either isografts or allografts.

ADP Ribose Transferases↗