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The aim of this study was to perform a detailed analysis of cytokine toxicity in the new human EndoC-βH1 beta cell line. The expression profile of the antioxidative enzymes in the new human EndoC-βH1 beta cells was characterised and compared with that of primary beta cells in the human pancreas. The effects of proinflammatory cytokines on reactive oxygen species formation, insulin secretory responsiveness and apoptosis of EndoC-βH1 beta cells were determined. EndoC-βH1 beta cells were sensitive to the toxic action of proinflammatory cytokines. Glucose-dependent stimulation of insulin secretion and an increase in the ATP/ADP ratio was abolished by proinflammatory cytokines without induction of IL-1β expression. Cytokine-mediated caspase-3 activation was accompanied by reactive oxygen species formation and developed more slowly than in rodent beta cells. Cytokines transiently increased the expression of unfolded protein response genes, without inducing endoplasmic reticulum stress-marker genes. Cytokine-mediated NFκB activation was too weak to induce inducible nitric oxide synthase expression. The resultant lack of nitric oxide generation in EndoC-βH1 cells, in contrast to rodent beta cells, makes these cells dependent on exogenously generated nitric oxide, which is released from infiltrating immune cells in human type 1 diabetes, for full expression of proinflammatory cytokine toxicity. EndoC-βH1 beta cells are characterised by an imbalance between H2O2-generating and -inactivating enzymes, and react to cytokine exposure in a similar manner to primary human beta cells. They are a suitable beta cell surrogate for cytokine-toxicity studies.

Research on the pathogenesis of type 1 diabetes relies heavily on good animal models. The aim of this work was to study the translational value of animal models of type 1 diabetes to the human situation.We compared the four major animal models of spontaneous type 1 diabetes, namely the NOD mouse, BioBreeding (BB) rat, Komeda rat and LEW.1AR1-iddm rat, by examining the immunohistochemistry and in situ RT-PCR of immune cell infiltrate and cytokine pattern in pancreatic islets, and by comparing findings with human data.After type 1 diabetes manifestation CD8(+) T cells, CD68(+) macrophages and CD4(+) T cells were observed as the main immune cell types with declining frequency, in infiltrated islets of all diabetic pancreases. IL-1β and TNF-α were the main proinflammatory cytokines in the immune cell infiltrate in NOD mice, BB rats and LEW.1AR1-iddm rats, as well as in humans. The Komeda rat was the exception, with IFN-γ and TNF-α being the main cytokines. In addition, IL-17 and IL-6 and the anti-inflammatory cytokines IL-4, IL-10 and IL-13 were found in some infiltrating immune cells. Apoptotic as well as proliferating beta cells were observed in infiltrated islets. In healthy pancreases no proinflammatory cytokine expression was observed.With the exception of the Komeda rat, the animal models mirror very well the situation in humans with type 1 diabetes. Thus animal models of type 1 diabetes can provide meaningful information on the disease processes in the pancreas of patients with type 1 diabetes.

The LEW.1AR1-iddm rat is an animal model of spontaneous type 1 diabetes mellitus. This study analysed how adoptive transfer of selective T cell subpopulations affects the incidence of diabetes. CD4+ or CD8+ T cells were isolated from diabetic LEW.1AR1-iddm rats or diabetes-resistant LEW.1AR1 rats. Cells were selectively transferred into athymic LEW.1AR1-Whn rnu or prediabetic LEW.1AR1-iddm rats. The animals were monitored for blood glucose, islet infiltration and immune cell composition of pancreas-draining lymph nodes. After adoptive transfer of CD4+ T cells from diabetic LEW.1AR1-iddm rats into athymic LEW.1AR1-Whn rnu rats, 50% of the recipients developed diabetes. Transfer of CD8+ T cells failed to induce diabetes. Only 10% of the athymic recipients became diabetic after co-transfer of CD4+ and CD8+ T cells. Adoptive transfer of CD8+ T cells from LEW.1AR1 or diabetic LEW.1AR1-iddm rats into prediabetic LEW.1AR1-iddm rats significantly reduced the incidence of diabetes. In protected normoglycaemic animals regulatory CD8+/CD25+ and CD4+/CD25+ T cell subpopulations that were also FOXP3-positive accumulated in the pancreas-draining lymph nodes. In this lymphatic organ, gene expression of anti-inflammatory cytokines was significantly higher than in diabetic rats. Our results show that adoptive transfer of CD4+ but not CD8+ T cells from diabetic LEW.1AR1-iddm rats induced diabetes development. Importantly, CD8+ T cells from diabetic LEW.1AR1-iddm rats and diabetes-resistant LEW.1AR1 rats provided protection against beta cell destruction. The accumulation of regulatory T cells in the pancreas-draining lymph nodes from protected rats indicates that transferred CD8+ T cells may have beneficial effects in the control of beta cell autoimmunity.

AIMS/HYPOTHESIS: The LEW.1AR1-iddm rat, an animal model of human type 1 diabetes, arose through a spontaneous mutation within the inbred strain LEW.1AR1. A susceptibility locus (Iddm8) on rat chromosome 1 (RNO1) has been identified previously, which is accompanied by autoimmune diabetes and the additional phenotype of a variable CD3(+) T cell frequency. METHODS: In the present study we characterised the Iddm8 region on RNO1 in backcross strains using the genetically divergent Brown Norway (BN) and Paris (PAR) rats. Candidate genes of the Iddm8 region were sequenced for mutation analysis. RESULTS: The Iddm8 region could be subdivided by single nucleotide polymorphism (SNP) analyses. In the first region, a mutation in exon 44 of the Dock8 gene was identified resulting in an amino acid exchange in the protein from glutamine to glutamate. This exchange is unique for the LEW.1AR1-iddm rat. In the second region, a SNP was detected in exon 11 of the Vwa2 gene with an exchange from arginine to tryptophan. This SNP is also present in other rat strains. CONCLUSIONS/INTERPRETATION: The Dock8 mutation gave rise to a new type 1 diabetes rat model with very close similarity to type 1 diabetes in humans, providing a deepened insight into the impact of genes involved in diabetes development.

It has recently been proposed that IL-1beta may be responsible for beta cell death in type 2 diabetes mellitus. Major support for this assumption was derived from experiments in the gerbil Psammomys obesus (sand rat), a model for nutritionally induced non-insulin-dependent type 2 diabetes. Using gerbil-specific primers for the analysis of gene expression, we investigated the validity of this hypothesis.Gene expression was analysed by real-time RT-PCR of isolated and laser-microdissected islets and by in situ RT-PCR, both in beta cells and in immune cells, as well as in lymph nodes and spleen.We were unable to detect Il-1beta and the IL-1beta-inducible enzyme inducible nitric oxide synthase (iNos) by in situ RT-PCR, either in the pancreatic beta cells, or in the small number of non-activated immune cells of healthy and diabetic Psammomys obesus after 1 and 3 weeks on a high-energy diet. Very low levels of Il-1beta and iNos mRNA were detectable in collagenase-isolated and laser-microdissected islets of normoglycaemic gerbils by real-time RT-PCR without any increase of these mRNAs in islets from diabetic animals. These results were confirmed by electron microscopy with immunogold staining for IL-1beta and insulin.The diabetic syndrome induced in Psammomys obesus by high-energy diet is a classical type 2 diabetes model, which does not show any evidence of an involvement of the proinflammatory cytokine IL-1beta or of activated immune cells in its pathogenesis. This is clearly at variance with the situation in type 1 diabetes.

Thyroid hormones reduce glucose tolerance in animals and humans. This effect is accompanied by a reduction in the beta-cell volume of the pancreas.We studied the underlying mechanism using terminal UTP nick end labelling (TUNEL) and caspase-3 to analyse apoptosis and BrdU labelling of beta-cell proliferation.The reason for the reduction of the beta-cell volume of the pancreas after thyroxine treatment is apparently an increased rate of beta-cell apoptosis by an increase of TUNEL and caspase-3 positive rat beta cells. In parallel, thyroxine treatment increased the rate of apoptosis in rat pancreatic ductal cells which are considered to contribute to the pool of stem cells from which insulin-producing beta cells originate. The effects of thyroid hormone treatment are reversible through an increase of the beta-cell replication rate when thyroxine is withdrawn as documented by an increase of the BrdU labelling index.An increased rate of beta-cell death due to apoptosis causes a decrease of insulin content and glucose-induced insulin secretion from the pancreas in hyperthyroidism. The resulting reduction of beta cells in the pancreas can provide an explanation for the decrease of glucose tolerance in hyperthyroidism.

We describe a new Type I (insulin-dependent) diabetes mellitus rat model (LEW.1AR1/Ztm-iddm) which arose through a spontaneous mutation in a congenic Lewis rat strain with a defined MHC haplotype (RT1.Aa B/Du Cu).The development of diabetes was characterised using biochemical, immunological and morphological methods.Diabetes appeared in the rats with an incidence of 20 % without major sex preference at 58+/-2 days. The disease was characterised by hyperglycaemia, glycosuria, ketonuria and polyuria. In peripheral blood, the proportion of T lymphocytes was in the normal range expressing the RT6.1 differentiation antigen. Islets were heavily infiltrated with B and T lymphocytes, macrophages and NK cells with beta cells rapidly destroyed through apoptosis in areas of insulitis.This Type I diabetic rat develops a spontaneous insulin-dependent autoimmune diabetes through beta cell apoptosis. It could prove to be a valuable new animal model for clarifying the mechanisms involved in the development of autoimmune diabetes.

To the editor: We read with interest the paper by Ren et al. [1] published in Diabetologia. In this study the liver of diabetic rats was transduced by a lentiviral vector system with the furincleavable insulin analogue B10. An instantaneous long-term correction of blood glucose concentrations was achieved within 2–3 days after virus application. At the end of the experiments, 500 days after virus application, an induced transdifferentiation of the transduced hepatocytes into cells immunohistochemically positive for insulin, glucagon and somatostatin was documented. Sixty days after virus injection, the treated rats showed a physiological insulin response and blood glucose concentrations after IVGTTs. The lentivirally transduced livers were positive in RT-PCR analyses for several beta cell typical transcription factors, but not for insulin or insulin proconvertase 2 as a sign of a partial transdifferentiation of hepatocytes into beta cells. Interestingly, the transduction by an empty lentiviral vector led to an induction of the beta cell transcription factors pancreatic and duodenal homeobox 1 (Pdx1) and neurogenic differentiation 1 (Neurod1). The lentiviral vector system used encodes for additional HIV accessory genes that might contribute to the induction of transdifferentiation. We performed similar experiments. In our own ongoing experiments in which streptozotocin (STZ)-diabetic rats were transduced by the latest lentiviral vector system (third generation, which provides the highest biosafety [2, 3]) via portal vein injection, no signs of transdifferentiation occurred (Fig. 1). In contrast to Ren et al. [1], in our experiments we used furin-cleavable insulin and not the furin-cleavable insulin analogue B10. In this insulin analogue the histidine in position 10 of the B chain of the insulin molecule is replaced by aspartic acid [4]. The mutation of this insulin analogue has been shown to induce mitogenic effects [5–7], and according to the authors’ [1] conclusion, could also be responsible for the observed liver transdifferentiation. Using the furin-cleavable insulin for the lentiviral transductions we detected a decrease in blood glucose concentration of STZ-diabetic rats from 22.3±3.4 to 15.6±2.1 mmol/l after 3 days and to 15.0±2.8 mmol/l after 30 days. About 20% of the hepatocytes of the transduced liver was positive for insulin (Fig. 1). Thus we can conclude that it is possible to improve blood glucose homeostasis through lentiviral overexpression of the insulin gene in the liver without concomitant transdifferentiation of the liver cells, purely by establishing insulin production in the liver, thereby replacing the loss of insulin supply from the endocrine pancreas to the organism. Use of the native insulin for expression in the liver prevents potential undesirable mitogenic effects. Diabetologia (2008) 51:694–695 DOI 10.1007/s00125-008-0931-1