Histopathological Changes in Insulin,Glucagon and Somatostatin Cells in the Islets of NOD Mice During Cyclophosphamide-accelerated Diabetes: A Combined Immunohistochemical and Histochemical Study

Summary The cyclophosphamide model of accelerated diabetes in the NOD mouse is a useful model of insulin-dependent diabetes mellitus (IDDM). Knowledge on the progressive destruction of beta cells and the fate of other islet endocrine cell-types in this model is sparse. We employed immunohistochemistry and histochemistry, to study temporal changes in islet cell populations, insulitis and glucose transporter-2 expression during cyclophosphamide administration. Cyclophosphamide was administered to day 95 female NOD mice and the pancreas studied at days 0 ( = day 95), 4, 7, 11 and 14 after treatment and in age-matched control mice. At day 0, a majority of the endocrine cells were insulin-positive. Glucagon and somatostatin cells were mostly in the islet periphery and also internally. In the cyclophosphamide group, insulitis was moderate at day 0, declined at day 4 but increased progressively from day 7. The extent of insulitis in treated mice which were diabetes-free at day 14 was comparable to age-matched control mice. From day 11, the marked increase in insulitis correlated with a reciprocal decline in the extent of insulin immunostained islet area. At day 14, the mean insulin area per islet was markedly less in diabetic mice than in age-matched non-diabetic treated and controls. At diabetes, some islets showed co-expression of glucagon and insulin. Our studies suggest that the mean number of glucagon or somatostatin cells per islet does not vary during the study. Glucose transporter-2 immunolabelling was restricted to beta cells but declined in those adjacent to immune cells. We conclude that in the cyclophosphamide model, there is specific and augmented destruction of beta cells immediately prior to diabetes onset. We speculate that the selective loss of glucose transporter-2 shown in this study suggests the existence of a deleterious gradient close to the immune cell and beta cell surface boundary.     

Rotavirus infection accelerates type 1 diabetes in mice with established insulitis

Infection modulates type 1 diabetes, a common autoimmune disease characterized by the destruction of insulin-producing islet beta cells in the pancreas. Childhood rotavirus infections have been associated with exacerbations in islet autoimmunity. Nonobese diabetic (NOD) mice develop lymphocytic islet infiltration (insulitis) and then clinical diabetes, whereas NOD8.3 TCR mice, transgenic for a T-cell receptor (TCR) specific for an important islet autoantigen, show more rapid diabetes onset. Oral infection of infant NOD mice with the monkey rotavirus strain RRV delays diabetes development. Here, the effect of RRV infection on diabetes development once insulitis is established was determined. NOD and NOD8.3 TCR mice were inoculated with RRV aged > or = 12 and 5 weeks, respectively. Diabetes onset was significantly accelerated in both models (P < 0.024), although RRV infection was asymptomatic and confined to the intestine. The degree of diabetes acceleration was related to the serum antibody titer to RRV. RRV-infected NOD mice showed a possible trend toward increased insulitis development. Infected males showed increased CD8(+) T-cell proportions in islets. Levels of beta-cell major histocompatibility complex class I expression and islet tumor necrosis factor alpha mRNA were elevated in at least one model. NOD mouse exposure to mouse rotavirus in a natural experiment also accelerated diabetes. Thus, rotavirus infection after beta-cell autoimmunity is established affects insulitis and exacerbates diabetes. A possible mechanism involves increased exposure of beta cells to immune recognition and activation of autoreactive T cells by proinflammatory cytokines. The timing of infection relative to mouse age and degree of insulitis determines whether diabetes onset is delayed, unaltered, or accelerated.     

Fas and Fas ligand immunolocalization in pancreatic islets of NOD mice during spontaneous and cyclophosphamide-accelerated diabetes

During insulin-dependent diabetes mellitus, immune cells which infiltrate pancreatic islets mediate beta cell destruction over a prolonged asymptomatic prediabetic period. The molecular mechanisms of beta cell death in vivo remain unresolved. At least two major molecular processes of destruction have been proposed. One involves the Fas–FasL (Fas–Fas ligand) system and the other, the perforin pathway. Here, dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of Fas and FasL in the NOD mouse, during spontaneous diabetes (days 21, 40 and 90) and following acceleration of diabetes with cyclophosphamide (days 0, 4, 7, 11 and 14 after cyclophosphamide administration). The expression of the proteins was correlated with advancing disease. FasL was expressed constitutively in most beta cells but not in glucagon or somatostatin cells or islet inflammatory cells and paralleled the loss of insulin immunolabelling with advancing disease. It was also expressed in beta cells of non-diabetes prone CD-1 and C57BL/6 mice from a young age (day 21). Strong immunolabelling for Fas was first observed in extra-islet macrophages and those close to the islet in NOD and non-diabetes-prone mice. During spontaneous and cyclophosphamide diabetes, it was observed in a higher proportion of islet infiltrating macrophages than CD4 and CD8 T cells, concomitant with advancing insulitis. In cyclophosphamide-treated mice, the proportion of Fas-positive intra-islet CD4 and CD8 T cells at day 14 (with and without diabetes) was considerably higher than at days 0, 4, 7 and 11. At days 11 and 14, a proportion of Fas-positive intra-islet macrophages co-expressed interleukin-1 and inducible nitric oxide synthase. Fas was not detectable in beta cells and other islet endocrine cells during spontaneous and cyclophosphamide induced diabetes. Our results show constitutive expression of FasL in beta cells in the NOD mouse and predominant expression of Fas in intra-islet macrophages and to a lesser extent in T cells prior to diabetes onset. Interleukin-1 in intra-islet macrophages may induce Fas and inducible nitric oxide synthase expression in an autocrine and paracrine manner and mediate beta cell destruction or even death of some macrophages and T cells. However, other mechanisms of beta cell destruction during spontaneous and cyclophosphamide-accelerated diabetes and independent of Fas–FasL, require examination.     

Activation of natural killer T cells by alpha-galactosylceramide treatment prevents the onset and recurrence of autoimmune Type 1 diabetes

Type 1 diabetes (T1D) in non-obese diabetic (NOD) mice may be favored by immune dysregulation leading to the hyporesponsiveness of regulatory T cells and activation of effector T-helper type 1 (Th1) cells. The immunoregulatory activity of natural killer T (NKT) cells is well documented, and both interleukin (IL)-4 and IL-10 secreted by NKT cells have important roles in mediating this activity. NKT cells are less frequent and display deficient IL-4 responses in both NOD mice and individuals at risk for T1D (ref. 8), and this deficiency may lead to T1D (refs. 1,6-9). Thus, given that NKT cells respond to the alpha-galactosylceramide (alpha-GalCer) glycolipid in a CD1d-restricted manner by secretion of Th2 cytokines, we reasoned that activation of NKT cells by alpha-GalCer might prevent the onset and/or recurrence of T1D. Here we show that alpha-GalCer treatment, even when initiated after the onset of insulitis, protects female NOD mice from T1D and prolongs the survival of pancreatic islets transplanted into newly diabetic NOD mice. In addition, when administered after the onset of insulitis, alpha-GalCer and IL-7 displayed synergistic effects, possibly via the ability of IL-7 to render NKT cells fully responsive to alpha-GalCer. Protection from T1D by alpha-GalCer was associated with the suppression of both T- and B-cell autoimmunity to islet beta cells and with a polarized Th2-like response in spleen and pancreas of these mice. These findings raise the possibility that alpha-GalCer treatment might be used therapeutically to prevent the onset and recurrence of human T1D.     

An Immunohistochemical Study of Macrophage Influx and the Co-localization of Inducible Nitric Oxide Synthase in the Pancreas of Non-obese Diabetic (NOD) Mice During Disease Acceleration with Cyclophosphamide

Cyclophosphamide has been used to accelerate and synchronize diabetes in non-obese diabetic (NOD) mice. It was injected to 70-day-old female NOD mice and its effect on the progression of insulitis studied at days 0, 4, 7, 11 and at onset of diabetes. Pancreatic sections were also examined for the influx of CD4 and CD8 T cells and macrophages following immunofluorescence staining. The kinetics of macrophage immunoreactive cells in the exocrine and intra-islet areas were also investigated. Light and confocal microscopy were employed to examine the expression and co-localization of inducible nitric oxide synthase following dual- and triple-label immunofluorescence histochemistry. After cyclophosphamide administration, the severity of insulitis remained similar from days 0 to 4 but began to rise at day 7 and markedly by day 11 and at onset of diabetes. At these two later stages, the insulitis scores were close to 100% while in age-matched control groups the insulitis scores were considerably lower. Immunohistochemical staining showed increasing numbers of CD4 and CD8 T cell subsets and macrophages within the islets and in exocrine, sinusoidal and peri-vascular regions. At onset of diabetes, several islets contained prominent clusters of macrophage immunoreactive cells. Macrophage influx into the islets increased sharply from day 7 (mean number per islet: 119±54 SEM), peaked at day 11 (mean number per islet: 228±42), and then declined at onset of diabetes (mean number per islet: 148±49). Several cells with immunolabelling for inducible nitric oxide synthase were detectable from day 7 onwards until the onset of diabetes. Dual- and triple-label immunohistochemistry showed that a significant proportion of macrophages and only a few beta cells contained the enzyme. Macrophages positive for the enzyme were located as clusters or occasionally contiguously, in the peri-islet and intra-islet areas but rarely in the exocrine region. Islets with minimal distribution of macrophages in the peri-islet areas were not positive for inducible nitric oxide synthase. Beta cells positive for the enzyme were observed in islets with significant macrophage infiltration in locations close to macrophages. The present results show that cyclophosphamide administration to female NOD mice results in a rapid influx of CD4 and CD8 cells and macrophages. The marked up-regulation of inducible nitric oxide synthase in a selective proportion of macrophages, within the islets, immediately preceding and during the onset of diabetes suggests that nitric oxide released by islet macrophages may be an important molecular mediator of beta cell destruction in this accelerated model of insulin-dependent diabetes mellitus.     

Cortisone-induced islet cell hyperplasia in hamsters

Pancreatic islet cell hyperplasia was studied in hamsters during one to eight weeks of cortisone treatment. Measurement of serum glucose and insulin; pancreatic insulin, glucagon, somatostatin, pancreatic polypeptide as well as islet tissue morphometry were performed. Serum glucose was highest at week 2, followed by mild to moderate hyperglycemia. Serum insulin was increasingly higher from week 1 to week 8. Pancreatic insulin was maximal at week 5 then declined through week 8 in the presence of beta cell neurosis in markedly hyperplastic islets. Pancreatic concentration of somatostatin and pancreatic polypeptide moderately increased more than the control levels; however, compared with the controls, glucagon was reduced by cortisone treatment. Effect of cortisone in the four types of islet cells is discussed, particularly on beta cell hyperplasia, which appears to be a response to decreased insulin binding to the target organs with no changes in receptor concentration.     

Immunohistochemical Demonstration of Leptin in Pancreatic Islets of Non-Obese Diabetic and CD-1 mice: Co-localization in Glucagon Cells and its Attenuation at the Onset of Diabetes

Leptin is a 16 kD polypeptide hormone produced predominantly by white adipose tissue and exerts profound effects on food intake and energy balance. More recent studies have shown extra sites of leptin production in human and rodent tissues and have ascribed additional roles for the hormone, e.g., in immune and reproductive functions. A role for the hormone has also been implicated in insulin-dependent diabetes mellitus in the non-obese diabetic (NOD) mouse. However, whether leptin originates from islet cells of the mouse is not known. Here dual-label immunohistochemistry was employed to examine leptin expression in islet cells, and its distribution and cellular sources in pancreatic sections of female NOD/Ak and CD-1 mice of various ages. For comparison, leptin immunolabelling was examined in adult pancreatic sections from male NOD/Ak CD-1, Balb/c and FVB/N mice and female severe combined immunodeficient CB. 17 mice. Pancreatic tissues from adult female guinea pig, sheep and cattle and neonatal pigs were also studied. Our results show that in the day 1 NOD and CD-1 mice, leptin immunolabelling was observed in selective glucagon cells within the developing islets while at days 15 and 22, it became more intense and co-incident. This pattern of staining was maintained at days 40, 90, 150 and 250. In the female NOD mouse, leptin was absent in intra-islet immune cells. Its expression was variable in islets from male NOD and CD-1 mice. In spontaneously diabetic female NOD mice and following acceleration of diabetes with cyclophosphamide, despite the persistence of strong immunolabelling for glucagon in the re-distributed alpha cells, leptin expression was either absent, diminished or present in only a proportion of alpha cells. The reduction in leptin labelling was often associated with diabetic islets which had insulitis in association with only a small number of residual beta cells. Leptin expression was absent in guinea pig, ovine, bovine and neonatal porcine islet cells, despite the expression of intensely labelled glucagon cells. The present results demonstrate leptin co-localization in glucagon cells of the mouse islet. Its expression diminishes in the presence of inadequate insulin. Leptin produced within the mouse islet may have bi-directional influences on leptin and insulin regulation and may play local functions in islet development and metabolism.     

Early autoantibody responses in prediabetes are IgG1 dominated and suggest antigen-specific regulation.

The islet autoimmunity of preclinical type 1 diabetes remains poorly characterized in humans. In this paper, the IgG subclass response to the islet autoantigens insulin, glutamic acid decarboxylase, and IA-2 was studied sequentially from birth to diabetes onset or current follow-up in 26 autoantibody positive offspring of parents with diabetes. Islet autoantibody appearance was characterized by an early IgG1 peak response to one or more Ags, most commonly to insulin, at a median age of 2.2 yr (interquartile range, 2-2.9 yr). In five offspring, an acute fulminant beta-cell destruction and diabetes onset occurred during this initial Ab response. In the remainder, early Ab levels declined markedly, and Ab peaks against other beta cell Ags arose sequentially over several years suggesting regulation and spreading of autoimmunity. Second peak Ab responses to the same Ag were observed in only two offspring, both developing diabetes at this time. Two others developed diabetes with declining Ab levels. Abs of IgG1 subclass dominated against each Ag, and other subclasses, were usually only detected during peak IgG1 responses. The IgG4 response to insulin was exceptional, being dominant over IgG1 in four offspring and in five others appeared and/or persisted after IgG1 levels declined. These Th2-associated IgG4 responses were not correlated with protection from diabetes. The presence of IgG1-restricted responses to DA2 were associated with diabetes development. These findings suggest that type 1 diabetes has an early acute destructive phase of beta cell autoimmunity, which may be regulated and which spreads chronically until diabetes onset.     

Cross-priming of diabetogenic T cells dissociated from CTL-induced shedding of beta cell autoantigens

Cross-presentation of self Ags by APCs is key to the initiation of organ-specific autoimmunity. As MHC class I molecules are essential for the initiation of diabetes in nonobese diabetic (NOD) mice, we sought to determine whether the initial insult that allows cross-presentation of beta cell autoantigens in diabetes is caused by cognate interactions between naive CD8(+) T cells and beta cells. Naive splenic CD8(+) T cells from transgenic NOD mice expressing a diabetogenic TCR killed peptide-pulsed targets in the absence of APCs. To ascertain the role of CD8(+) T cell-induced beta cell lysis in the initiation of diabetes, we expressed a rat insulin promoter (RIP)-driven adenovirus E19 transgene in NOD mice. RIP-E19 expression inhibited MHC class I transport exclusively in beta cells and rendered these cells resistant to lysis by CD8(+) (but not CD4(+)) T cells, both in vitro and in vivo. Surprisingly, RIP-E19 expression impaired the accumulation of CD8(+) T cells in islets and delayed the onset of islet inflammation, without affecting the timing or magnitude of T cell cross-priming in the pancreatic lymph nodes, which is the earliest known event in diabetogenesis. These results suggest that access of beta cell autoantigens to the cross-presentation pathway in diabetes is T cell independent, and reveal a previously unrecognized function of MHC class I molecules on target cells in autoimmunity: local retention of disease-initiating clonotypes.     

IFN beta accelerates autoimmune type 1 diabetes in nonobese diabetic mice and breaks the tolerance to beta cells in nondiabetes-prone mice

Genetic and environmental factors are decisive in the etiology of type 1 diabetes. Viruses have been proposed as a triggering environmental event and some evidences have been reported: type I IFNs exist in the pancreata of diabetic patients and transgenic mice expressing these cytokines in beta cells develop diabetes. To determine the role of IFNbeta in diabetes, we studied transgenic mice expressing human IFNbeta in the beta cells. Autoimmune features were found: MHC class I islet hyperexpression, T and B cells infiltrating the islets and transfer of the disease by lymphocytes. Moreover, the expression of beta(2)-microglobulin, preproinsulin, and glucagon in the thymus was not altered by IFNbeta, thus suggesting that the disease is caused by a local effect of IFNbeta, strong enough to break the peripheral tolerance to beta cells. This is the first report of the generation of NOD (a model of spontaneous autoimmune diabetes) and nonobese-resistant (its homologous resistant) transgenic mice expressing a type I IFN in the islets: transgenic NOD and nonobese-resistant mice developed accelerated autoimmune diabetes with a high incidence of the disease. These results indicate that the antiviral cytokine IFNbeta breaks peripheral tolerance to beta cells, influences the insulitis progression and contributes to autoimmunity in diabetes and nondiabetes- prone mice.     

Granzyme B is dispensable in the development of diabetes in non-obese diabetic mice

Pancreatic beta cell destruction in type 1 diabetes is mediated by cytotoxic CD8(+) T lymphoctyes (CTL). Granzyme B is an effector molecule used by CTL to kill target cells. We previously showed that granzyme B-deficient allogeneic CTL inefficiently killed pancreatic islets in vitro. We generated granzyme B-deficient non-obese diabetic (NOD) mice to test whether granzyme B is an important effector molecule in spontaneous type 1 diabetes. Granzyme B-deficient islet antigen-specific CD8(+) T cells had impaired homing into islets of young mice. Insulitis was reduced in granzyme B-deficient mice at 70 days of age (insulitis score 0.043±0.019 in granzyme B-deficient versus 0.139±0.034 in wild-type NOD mice p<0.05), but was similar to wild-type at 100 and 150 days of age. We observed a reduced frequency of CD3(+)CD8(+) T cells in the islets and peripheral lymphoid tissues of granzyme B-deficient mice (p<0.005 and p<0.0001 respectively), but there was no difference in cell proportions in the thymus. Antigen-specific CTL developed normally in granzyme B-deficient mice, and were able to kill NOD islet target cells as efficiently as wild-type CTL in vitro. The incidence of spontaneous diabetes in granzyme B-deficient mice was the same as wild-type NOD mice. We observed a delayed onset of diabetes in granzyme B-deficient CD8-dependent NOD8.3 mice (median onset 102.5 days in granzyme B-deficient versus 57.50 days in wild-type NOD8.3 mice), which may be due to the delayed onset of insulitis or inefficient priming at an earlier age in this accelerated model of diabetes. Our data indicate that granzyme B is dispensable for beta cell destruction in type 1 diabetes, but is required for efficient early activation of CTL.     

TRPV1+ sensory neurons control beta cell stress and islet inflammation in autoimmune diabetes

In type 1 diabetes, T cell-mediated death of pancreatic beta cells produces insulin deficiency. However, what attracts or restricts broadly autoreactive lymphocyte pools to the pancreas remains unclear. We report that TRPV1(+) pancreatic sensory neurons control islet inflammation and insulin resistance. Eliminating these neurons in diabetes-prone NOD mice prevents insulitis and diabetes, despite systemic persistence of pathogenic T cell pools. Insulin resistance and beta cell stress of prediabetic NOD mice are prevented when TRPV1(+) neurons are eliminated. TRPV1(NOD), localized to the Idd4.1 diabetes-risk locus, is a hypofunctional mutant, mediating depressed neurogenic inflammation. Delivering the neuropeptide substance P by intra-arterial injection into the NOD pancreas reverses abnormal insulin resistance, insulitis, and diabetes for weeks. Concordantly, insulin sensitivity is enhanced in trpv1(-/-) mice, whereas insulitis/diabetes-resistant NODxB6Idd4-congenic mice, carrying wild-type TRPV1, show restored TRPV1 function and insulin sensitivity. Our data uncover a fundamental role for insulin-responsive TRPV1(+) sensory neurons in beta cell function and diabetes pathoetiology.     

Pancreatic IL-4 expression results in islet-reactive Th2 cells that inhibit diabetogenic lymphocytes in the nonobese diabetic mouse.

When immunological tolerance breaks down, autoimmune destruction of insulin-producing beta cells in the pancreas can cause insulin-dependent diabetes mellitus. We previously showed that transgenic nonobese diabetic (NOD) mice expressing IL-4 in the pancreas (NOD-IL-4 mice) were protected from insulitis and diabetes. Here we have characterized the avoidance of pathological autoimmunity in these mice. The absence of disease did not result from a lack of T cell priming, because T cells responding to dominant islet Ags were present. These islet Ag-specific T cells displayed a Th2 phenotype, indicating that Th2 responses could account for the observed tolerance. Interestingly, islet Ag-specific Th1 T cells were present and found to be functional, because neutralization of the Th2 effector cytokines IL-4 and IL-10 resulted in diabetes. Histological examination revealed that NOD-IL-4 splenocytes inhibited diabetogenic T cells in cotransfer experiments by limiting insulitis and delaying diabetes. Neutralization of IL-4 in this system abrogated the ability of NOD-IL-4 splenocytes to delay the onset of diabetes. These results indicate that IL-4 expressed in the islets does not prevent the generation of pathogenic islet responses but induces islet Ag-specific Th2 T cells that block the action of diabetogenic T cells in the pancreas.     

Immune Depletion in Combination with Allogeneic Islets Permanently Restores Tolerance to Self-Antigens in Diabetic NOD Mice

The destruction of beta cells in type 1 diabetes (T1D) results in loss of insulin production and glucose homeostasis. Treatment of non-obese diabetic (NOD) mice with immune-depleting/modulating agents (e.g., anti-CD3, murine anti-thymocyte-globulin (mATG)) can lead to diabetes reversal. However, for preclinical studies with these and other agents seeking to reverse disease at onset, the necessity for exogenous insulin administration is debated. Spontaneously diabetic NOD mice were treated with a short-course of mATG and insulin provided as drug therapy or by way of allogeneic islet implants. Herein we demonstrate that exogenous insulin administration is required to achieve disease reversal with mATG in NOD mice. Unexpectedly, we also observed that provision of insulin by way of allogeneic islet implantation in combination with mATG leads to a pronounced reversal of diabetes as well as restoration of tolerance to self-islets. Expansion/induction of regulatory cells was observed in NOD mice stably cured with mATG and allogeneic islets. These data suggest that transient provision of allogeneic insulin-producing islets might provide a temporary window for immune depletion to be more effective and instilling stable tolerance to endogenous beta cells. These findings support the use of a never before explored approach for preserving beta cell function in patients with recent onset T1D.     

Multiple roles for tumor necrosis factor-alpha and lymphotoxin alpha/beta in immunity and autoimmunity

Tumor necrosis factor (TNF)-alpha and lymphotoxin (LT) alpha/beta play multiple roles in the development and function of the immune system. This article focuses on three important aspects of the effects of these cytokines on the immune response and on autoimmunity. In several experimental systems (Jurkat T cells, murine T-cell hybridomas), TNF-alpha appears to cause a downregulation of signaling through the TCR, revealed by changes in calcium flux, activation of p21, p23 and ZAP70, and a decrease in nuclear activation of NF-kappaB. Previous and present results suggest that TNF-alpha interferes in some manner with signaling through the TCR, at a locus yet to be delineated. Transgenic expression of LTbetaR-Fc in nonobese diabetic (NOD) transgenic mice results in prevention of type 1 diabetes in NOD mice as long as the level of expression of the fusion protein (under the control of the cytomegalovirus promoter) remains above a level of 2-3 microg/ml. Once the expression levels of the fusion protein have dropped below this critical level, the diabetic process resumes and the animals become diabetic at 40-50 weeks of age, whereas nontransgenic littermates develop diabetes by 25-30 weeks of age. The paradoxical effects of neonatal TNF-alpha administration in NOD mice in increasing incidence of and hastening onset of type 1 diabetes, while neonatal anti-TNF administration completely prevents all signs of islet cell autoimmunity, are due partly to the low levels of CD4+CD25+ T cells in NOD mice. These low levels are reduced by a further 50% on neonatal administration of nontoxic levels of TNF-alpha. In contrast, neonatal administration of anti-TNF-alpha results in a dramatic increase in the levels of CD4+CD25+ regulatory T cells, to levels beyond those seen in wild-type untreated NOD mice. TNF-alpha and LTalpha/beta thus have pleomorphic regulatory effects on the development and expression of autoimmunity.     

Insulin secretion and islet endocrine cell content at onset and during the early stages of diabetes in the BB rat: effect of the level of glycemic control.

Although it is agreed that autoimmune destruction of pancreatic islets in diabetic BB rats is rapid, reports of endocrine cell content of islets from BB diabetic rats at the time of onset of diabetes vary considerably. Because of the rapid onset of the disease (hours) and the attendant changes in islet morphology and insulin secretion, it was the aim of this study to compare islet beta-cell numbers to other islet endocrine cells as close to the time of onset of hyperglycemia as possible (within 12 h). As it has been reported that hyperglycemia renders the beta cell insensitive to glucose, the early effects of different levels of insulin therapy (well-controlled vs. poorly controlled glycemia) on islet morphology and insulin secretion were examined. When measured within 12 h of onset, insulin content of BB diabetic islets, measured by morphometric analysis or pancreatic extraction, was 60% of insulin content of control islets. Despite significant amounts of insulin remaining in the pancreas, 1-day diabetic rats exhibited fasting hyperglycemia and were glucose intolerant. The insulin response from the isolated perfused pancreas to glucose and the glucose-dependent insulinotropic hormone, gastric inhibitory polypeptide (GIP), was reduced by 95%. Islet content of other endocrine peptides, glucagon, somatostatin, and pancreatic polypeptide, was normal at onset and at 2 weeks post onset. A group of diabetic animals, maintained in a hyperglycemic state for 7 days with low doses of insulin, were compared with a group kept normoglycemic by appropriate insulin therapy. No insulin could be detected in islets of poorly controlled diabetics, while well-controlled animals had 30% of the normal islet insulin content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phe-met-arg-phe-amide (FMRF-NH2) inhibits insulin and somatostatin secretion and anti-FMRF-NH2 sera detects pancreatic polypeptide cells in the rat islet

FMRF-NH2-like immunoreactivity was localized in the pancreatic polypeptide containing cells of the rat islet. FMRF-NH2 was investigated with regard to its effect on insulin, somatostatin and glucagon secretion from the isolated perfused rat pancreas. FMRF-NH2 (1 microM) significantly inhibited glucose stimulated (300 mg/dl) insulin release (p less than 0.005) and somatostatin release (p less than 0.01) from the isolated perfused pancreas. FMRF-NH2 (1 and 10 microM) was without effect on glucagon secretion, either in low glucose (50 mg/dl), high glucose (300 mg/dl), or during arginine stimulation (5 mM). These findings indicate that these FMRF-NH2 antisera recognize a substance in the pancreatic polypeptide cells of the islet which may be capable of modulating islet beta and D cell activity.     

Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD) mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.     

Induction of glutamic acid decarboxylase 65-specific Th2 cells and suppression of autoimmune diabetes at late stages of disease is epitope dependent.

Peptide-based immunotherapy is one strategy by which to selectively suppress the T cell-mediated destruction of beta cells and treat insulin-dependent diabetes mellitus (IDDM). Here, we investigated whether a panel of T cell epitopes derived from the beta cell autoantigen glutamic acid decarboxylase 65 (GAD65) differ in their capacity to induce Th2 cell function in nonobese diabetic (NOD) mice and in turn prevent overt IDDM at different preclinical stages of disease development. The panel consists of GAD65-specific peptides spanning aa 217-236 (p217), 247-265 (p247), 290-309 (p290), and 524-543 (p524). Our studies revealed that all of the peptides effectively prevented insulitis and diabetes when administered to NOD mice before the onset of insulitis. In contrast, only a mixture of p217 and p290 prevented progression of insulitis and overt IDDM in NOD mice exhibiting extensive beta cell autoimmunity. Immunization with the GAD65-specific peptides did not block IDDM development in NOD mice deficient in IL-4 expression. These findings demonstrate that GAD65-specific peptide immunotherapy effectively suppresses progression to overt IDDM, requires the production of IL-4, and is dependent on the epitope targeted and the extent of preexisting beta cell autoimmunity in the recipient.     

The Non-Immune RIP-kb Mouse is a Useful Host for Islet Transplantation,as the Diabetes is Spontaneous,Mild and Predictable

Chemically-induced diabetic mice and spontaneously diabetic NOD mice have been valuable as recipients for experimental islet transplantation. However, their maintenance often requires parenteral insulin. Diabetogenic chemicals can be cytotoxic to the host’s immune system and to other organs some of which are often used as the transplant site. Procurement of diabetic cohorts in the NOD mouse is problematic due to variability in the age of disease onset. We show that RIP-K^b mice, which spontaneously develop non-immune diabetes due to over-expression of the H-2K^b heavy chain in beta cells, offer many advantages as islet transplant recipients. Diabetes is predictable with a relatively narrow range of onset (4 wk) and blood glucose levels (23.0± 4.0 mmol/l for 39 males at 6 weeks of age). The diabetes is mild enough so that most diabetic mice can be maintained to 40 weeks of age without parenteral insulin. This consistency of diabetes avails that outcomes of intervention can be interpreted with confidence.