Allan E. Karlsen

The harmony of the spheres: inducible nitric oxide synthase and related genes in pancreatic beta cells

SummaryThe radical nitric oxide (NO) is a possible mediator of pancreatic beta-cell damage in insulin-dependent diabetes mellitus (IDDM). NO is produced by the enzyme nitric oxide synthase (NOS), in a reaction where arginine is the main substrate. There are different isoforms of NOS, but in the context of immune mediated beta-cell damage the inducible form of NOS (iNOS) is the most relevant. The beta-cell iNOS is similar and encoded by the same gene on chromosome 17 as the iNOS expressed in macrophages and other nucleated cells. iNOS activation depends on gene transcription and de novo enzyme synthesis, and NO seems to induce a negative feedback on iNOS expression. While iNOS mRNA is induced by interleukin-1Β (IL-1Β) alone in rodent insulin-producing cells, a combination of two (IL-1Β + interferon γ) (IFN-γ) or three (IL-1Β + IFNγ + tumour necrosis factor α) cytokines is required for iNOS activation in human pancreatic islets. The promoter region of the murine iNOS gene has at least 25 binding sites for different transcription factors, and the nuclear transcription factor κB is necessary for cytokine-induced iNOS transcription in both rodent and human pancreatic islets. The nature of other transcription factors relevant for iNOS regulation in these cells remains to be determined. Induction of iNOS is paralleled by induction of several other cytokine-dependent genes in beta cells, including argininosuccinate synthetase, cyclooxygenase and manganese superoxide dismutase. Some of these genes may contribute to beta-cell damage, while others are probably involved in beta-cell defence and/or repair. Regulation of iNOS and other related genes in beta cells is complex, and differs in several aspects from that observed in macrophages. There are also important differences in iNOS regulation between rodent and human pancreatic islets. A detailed knowledge of the molecular regulation of these genes in beta cells may be instrumental in the development of new approaches to prevent beta-cell destruction in early IDDM.

Detection of GAD65 Antibodies in Diabetes and Other Autoimmune Diseases Using a Simple Radioligand Assay

Autoantibodies to glutamic acid decarboxylase (GAD) are frequent at or before the onset of insulin-dependent diabetes mellitus (IDDM). We have developed a simple, reproducible, and quantitative immunoprecipitation radioligand assay using as antigen in vitro transcribed and translated [35S]methionine-labeled human islet GAD65. By using this assay, 77% (77 of 100) of serum samples from recent-onset IDDM patients were positive for GAD65 antibodies compared with 4% (4 of 100) of serum samples from healthy control subjects. In competition analysis with unlabeled purified recombinant human islet GAD65, binding to tracer was inhibited in 74% (74 of 100) of the GAD65-positive IDDM serum samples compared with 2% of the control samples. The levels of GAD antibodies expressed as an index value relative to a standard serum, analyzed with or without competition, were almost identical (r = 0.991). The intra- and interassay variations of a positive control serum sample were 2.9 and 7.6%, respectively (n = 4). The frequency of GAD antibodies was significantly higher with IDDM onset before the age of 30 (80%, 59 of 74) than after the age of 30 (48%, 10 of 21) (P < 0.01). The prevalence of islet cell antibodies showed a similar pattern relative to age at onset. Because simultaneous occurrences of multiple autoimmune phenomena are common, we analyzed sera from patients with other autoimmune diseases. The frequency of GAD antibodies in sera positive for DNA autoantibodies (8% [2 of 25] and 4% [1 of 25] in competition analysis) or rheuma factor autoantibodies [12% (4 of 35) and 3% (1 of 35) in competition analysis] was not different from that in control samples. In contrast, in sera positive for ribonucleoprotein antibodies the frequency of GAD antibodies was significantly increased (73% [51 of 70] and 10% [7 of 70] in competition analysis [P < 0.025]). In conclusion, even large numbers of serum samples can now be tested for GAD65 antibodies in a relatively short time, allowing screening of individuals without a family history of IDDM for the presence of this marker.

On the pathogenesis of IDDM.

SummaryA model of the pathogenesis of insulin-dependent diabetes mellitus, i.e. the initial phase of beta-cell destruction, is proposed: in a cascade-like fashion efficient antigen presentation, unbalanced cytokine, secretion and poor beta-cell defence result in beta-cell destruction by toxic free radicals (O2− and nitric oxide) produced by the beta cells themselves. This entire process is under polygenetic control.

Suppressor of cytokine signaling 3 (SOCS-3) protects β-cells against interleukin-1β- and interferon-γ-mediated toxicity

Suppressor of cytokine signaling 3 (SOCS-3) is a negative feedback regulator of IFN-γ signaling, shown up-regulated in mouse bone marrow cells by the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IFN-γ. IL-1β and IFN-γ alone, or potentiated by TNF-α, are cytotoxic to the insulin producing pancreatic β-cells and β-cell lines in vitro and suggested to contribute to the specific β-cell destruction in Type-1 diabetes mellitus (T1DM). Using a doxycycline-inducible SOCS-3 expression system in the rat β-cell line INS-1, we demonstrate that the toxic effect of both IL-1β or IFN-γ at concentrations that reduced the viability by 50% over 3 days, was fully preventable when SOCS-3 expression was turned on in the cells. At cytokine concentrations or combinations more toxic to the cells, SOCS-3 overexpression yielded a partial protection. Whereas SOCS-3-mediated inhibition of IFN-γ signaling is described in other cell systems, SOCS-3 mediated inhibition of IL-1β signaling has not previously been described. In addition we show that SOCS-3 prevention of IL-1β-induced toxicity is accompanied by inhibited transcription of the inducible nitric oxide synthase (iNOS) by 80%, resulting in 60% decreased formation of the toxic nitric oxide (NO). Analysis of isolated native rat islets exposed to IL-1β revealed a naturally occurring but delayed up-regulated SOCS-3 transcription. Influencing SOCS-3 expression thus represents an approach for affecting cytokine-induced signal transduction at a proximal step in the signal cascade, potentially useful in future therapies aimed at reducing the destructive potential of β-cell cytotoxic cytokines in T1DM, as well as other cytokine-dependent diseases.

Differential Expression of Glutamic Acid Decarboxylase in Rat and Human Islets

The GABA synthesizing enzyme GAD is a prominent islet cell autoantigen in type I diabetes. The two forms of GAD (GAD64 and GAD67) are encoded by different genes in both rats and humans. By in situ hybridization analysis of rat and human pancreases, expression of both genes was detected in rat islets, whereas only GAD64 mRNA was detected in human islets. Immunocytochemical analysis of rat and human pancreatic sections or isolated islets with antibodies to GAD64 and GAD67 in combination with antibodies to insulin, glucagon, or SRIF confirmed that a GAD64 and GAD67 expression were β-cell specific in rat islets. In contrast, only GAD64 was detected in human islets and was, in addition to β-cells, also surprisingly localized to some α-cells, δ-cells, and PP-cells. In long-term (4 wk) monolayer cultures of newborn rat islet cells, GAD64 expression remained β-cell specific as observed in vivo, whereas GAD67 was localized not only to the β-cells but also in the α-cells and δ-cells. A small but distinct fraction of GAD positive cells in these monolayer cultures did not accumulate GABA immunoreactivity, which may indicate cellular heterogeneity with respect to GABA catabolism or GAD enzyme activity. In a rat insulinoma cell line (NHI-6F) producing both glucagon and insulin depending on the culture conditions, GAD64 expression was detected only in cultures in which the insulin producing phenotype dominated. In conclusion, these data demonstrate that the two GAD isoforms are differentially expressed in rat and human islets but also that the expression differs according to culture conditions. These findings emphasize the need to consider both the species and culture conditions of islets.

Neonatal Tolerization With Glutamic Acid Decarboxylase But Not With Bovine Serum Albumin Delays the Onset of Diabetes in NOD Mice

To test the role of glutamic acid decarboxylase (GAD65) or bovine serum albumin (BSA) autoimmunity in the pathogenesis of diabetes, GAD65 or BSA was injected intraperitoneally into neonatal female NOD mice (100 micrograms/mouse of each protein). Treatment with GAD65, but not with BSA, significantly delayed the onset of diabetes compared with control mice (P < 0.05). At 18 weeks, 6 of 10 control mice compared with 0 of 10 GAD65-treated mice (P = 0.005) and 7 of 14 BSA-treated mice had developed diabetes. However, after 79 weeks, 6 of 10 of the GAD65-treated mice were diabetic compared with 9 of 10 of the control mice and 12 of 14 of the BSA-treated mice. In GAD65-treated mice without diabetes, insulitis was markedly reduced compared with control or BSA-treated mice (P < 10−4). To further elucidate why GAD becomes an autoantigen, the expression in NOD mice islets was studied. Quantitative immunohistochemistry revealed that islet cell expression of GAD was increased in 5-week-old NOD mice compared with BALB/c mice (P = 0.02). With the occurrence of insulitis (9–15 weeks), the GAD expression was further increased relative to 5-week-old NOD mice (P < 0.02). In conclusion, GAD, but not BSA, autoimmunity is important for the development of diabetes in NOD mice. Furthermore, concordant with the appearance of insulitis, the GAD expression increased in NOD mouse islets, which could possibly potentiate the β-cell-directed autoimmunity.

Inhibition of glucose stimulated insulin secretion by neuropeptide Y is mediated via the Y1 receptor and inhibition of adenylyl cyclase in RIN 5AH rat insulinoma cells

Summary Neuropeptide Y (NPY) has been shown to inhibit insulin secretion from the islets of Langerhans. We show that insulin secretion in the insulinoma cell line RIN 5AH is inhibited by NPY. 125I-Peptide YY (PYY) saturation and competition-binding studies using NPY fragments and analogues on membranes prepared from this cell line show the presence of a single class of NPY receptor with a Y1 receptor subtype-like profile. Inhibition of insulin secretion in this cell line by NPY fragments and analogues also shows a Y1 receptor-like profile. Both receptor binding and inhibition of insulin secretion showed the same orders of potency with NPY > [Pro34]-NPY > NPY 3–36 > > NPY 13–36. The Y1 receptor antagonist, BIBP 3226, blocks NPY inhibition of insulin secretion from, and inhibits 125I-PYY binding to, RIN 5AH cells. Northern blot analysis using a Y1-receptor specific probe shows that NPY Y1 receptors are expressed by RIN 5AH cells. Y5 receptors are not expressed in this cell line. Neuropeptide Y inhibition of insulin secretion is blocked by incubation with pertussis toxin, implying that the effect is via a G-protein (Gi or Go) coupled receptor. Neuropeptide Y inhibits the activation of adenylyl cyclase by isoprenaline in RIN 5AH cell lysates, and the stimulation of cAMP by glucagon-like peptide-1 (7–36) amide (GLP-1). It also blocks insulin secretion stimulated by GLP-1, but not by dibutyryl cyclic AMP. Hence, we suggest that NPY inhibits insulin secretion from RIN 5AH cells via a Y1 receptor linked through Gi to the inhibition of adenylyl cyclase. [Diabetologia (1998) 41: 1482–1491]

Unraveling the Pathogenesis of Type 1 Diabetes with Proteomics: Present And Future Directions

Type 1 diabetes (T1D) is the result of selective destruction of the insulin-producing β-cells in the pancreatic islets of Langerhans. T1D is due to a complex interplay between the β-cell, the immune system, and the environment in genetically susceptible individuals. The initiating mechanism(s) behind the development of T1D are largely unknown, and no genes or proteins are specific for most T1D cases. Different pro-apoptotic cytokines, IL-1 β in particular, are present in the islets during β-cell destruction and are able to modulate β-cell function and induce β-cell death. In β-cells exposed to IL-1 β, a race between destructive and protective events are initiated and in susceptible individuals the deleterious events prevail. Proteins are involved in most cellular processes, and it is thus expected that their cumulative expression profile reflects the specific activity of cells. Proteomics may be useful in describing the protein expression profile and thus the diabetic phenotype. Relatively few studies using proteomics technologies to investigate the T1D pathogenesis have been published to date despite the defined target organ, the β-cell. Proteomics has been applied in studies of differentiating β-cells, cytokine exposed islets, dietary manipulated islets, and in transplanted islets. Although that the studies have revealed a complex and detailed picture of the protein expression profiles many functional implications remain to be answered. In conclusion, a rather detailed picture of protein expression in β-cell lines, islets, and transplanted islets both in vitro and in vivo have been described. The data indicate that the β-cell is an active participant in its own destruction during diabetes development. No single protein alone seems to be responsible for the development of diabetes. Rather the cumulative pattern of changes seems to be what favors a transition from dynamic stability in the unperturbed β-cell to dynamic instability and eventually to β-cell destruction.

Two-Dimensional Gel Electrophoresis of Rat Islet Proteins: Interleukin 1β-Induced Changes in Protein Expression are Reduced by L-Arginine Depletion and Nicotinamide

Interleukin (IL)-1 β-mediated damage to β-cells in isolated islets of Langerhans depends upon de novo synthesis of proteins that have not been fully identified. Further, IL-1 β-induced and tumor necrosis factor alpha-induced islet damage partly depends on the intracellular production of the nitric oxide (NO) radical. IL-1 β has also been reported to induce the synthesis of cellular defense proteins, e.g., heme-oxygenase and heat shock proteins 70 and 90. Nicotinamide, while in itself inactive, inhibited IL-1 β-induced NO production in a time- and dose-dependent manner. To enable the identification of IL-1 β-induced proteins with possible protective and deleterious effects, we characterized the effects of IL-1 β, nicotinamide, and NO synthesis inhibition by L-arginine depletion on rat islet protein expression detected by high-resolution two-dimensional gel electrophoresis. More than 1,600 proteins were reproducibly detected in control rat islets. Incubation with IL-1 β–, nicotinamide-, or L-arginine-depleted control medium upregulated 29, 3, and 1 protein, respectively, and downregulated 4, 0, and 1 protein, respectively. Addition of nicotinamide and L-arginine depletion reduced the upregulation of 16 and 20 IL-1 β-induced proteins, respectively. The identity of these proteins is under study. The demonstrated changes in protein expression caused by IL-1 β ± nicotinamide and L-arginine depletion may form the basis for identification of proteins with possible protective and deleterious roles in the initial β-cell destruction in insulin-dependent diabetes mellitus.

Cloning and Expression of Cytokine-Inducible Nitric Oxide Synthase cDNA From Rat Islets of Langerhans

An inducible nitric oxide (NO) synthase isoform (iNOS) is specifically induced in the β-cells of interleuldn (IL)-1β–exposed rat islets, suggesting a role for NO in the pathogenesis of type I diabetes. The aim of this study was to clone and characterize iNOS cDNA from cytokineexposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN-5AH β-cells cultured in the absence of cytokines. Addition of IL-1α alone or in combination with tumor necrosis factor-α induced a concentration- and time-dependent expression of the iNOS gene and associated NO production (measured as nitrite) from both islets and RIN cells. iNOS transcripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence analysis revealed a near 100% identity to the recently published iNOS cDNA cloned from cytokineexposed rat hepatocytes and smooth muscle cells. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inhibited by addition of the Larginine analogs, Nω-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as a series of spots with the expected molecular mass of 131 kDa and pi values in the range of 6.8 to 7.0. In conclusion, the IL-1β-induced iNOS cloned and expressed from rat islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types.