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The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D). We determined by GC-MS the extent of asymmetric dimethylation (prADMA) and citrullination (prCit) of L-arginine residues in organ proteins (pr) of normoglycaemic control (ngCo, n = 6), acutely diabetic (acT1D, n = 6), chronically diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Pancreatic prCit and prADMA did not differ between the groups but were correlated (r = 0.728, P = 0.0003, n = 20). acT1D rats had lower prCit levels in spleen and kidney than ngCo rats. cuT1D rats had higher prADMA levels than chT1D rats only in the spleen. Combination therapy re-established normoglycaemia and increased prADMA in the spleen without altering pancreatic prADMA and prCit. Western blotting demonstrated the presence of different prADMA pattern, especially an ≈ 50-kDa prADMA in spleen and pancreas, and an ≈ 25-kDa prADMA in the pancreas only, with the kidney showing only a very faint and small prADMA. Besides the changes in the pancreas during different metabolic states, the spleen may play a stronger role for the recognition of metabolic changes in T1D than thought thus far.

SCOPUS: ar.j, info:eu-repo/semantics/published

Increased circulating levels of saturated fatty acids (FFAs) and glucose are considered to be major mediators of β-cell dysfunction and death in T2DM. Although it has been proposed that endoplasmic reticulum (ER) and oxidative stress play a crucial role in gluco/lipotoxicity, their interplay and relative contribution to β-cell dysfunction and apoptosis has not been fully elucidated. In addition it is still unclear how palmitate - the physiologically most abundant long-chain saturated FFA - elicits ER stress and which immediate signals commit β-cells to apoptosis. To study the underlying mechanisms of palmitate-mediated ER stress and β-cell toxicity, we exploited the observation that the recently described ER-resident GPx7 and GPx8 are not expressed in rat β-cells. Expression of GPx7 or GPx8 attenuated FFAs-mediated H2O2 generation, ER stress, and apoptosis induction. These results could be confirmed by a H2O2-specific inactivating ER catalase, indicating that accumulation of H2O2 in the ER lumen is critical in FFA-induced ER stress. Furthermore, neither the expression of GPx7 nor of GPx8 increased insulin content or facilitated disulfide bond formation in insulin-secreting INS-1E cells. Hence, reduction of H2O2 by ER-GPx isoforms is not rate-limiting in oxidative protein folding in rat β-cells. These data suggest that FFA-mediated ER stress is partially dependent on oxidative stress and selective expression of GPx7 or GPx8 improves the ER antioxidative capacity of rat β-cells without compromising insulin production and the oxidative protein folding machinery.

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.

Aims/hypothesis Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium (www.imidia.org) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). Methods Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (

International audience; We recently found that renal carbonic anhydrase (CA) is involved in the reabsorption of inorganic nitrite (NO2−), an abundant reservoir of nitric oxide (NO) in tissues and cells. Impaired NO synthesis in the endothelium and decreased NO bioavailability in the circulation are considered major contributors to the development and progression of renal and cardiovascular diseases in different conditions including diabetes. Isolated human and bovine erythrocytic CAII and CAIV can convert nitrite to nitrous acid (HONO) and its anhydride N2O3 which, in the presence of thiols (RSH), are further converted to S-nitrosothiols (RSNO) and NO. Thus, CA may be responsible both for the homeostasis of nitrite and for its bioactivation to RSNO/NO. We hypothesized that enhanced excretion of nitrite in the urine may contribute to NO-related dysfunctions in the renal and cardiovascular systems, and proposed the urinary nitrate-to-nitrite molar ratio, i.e., UNOxR, as a measure of renal CA-dependent excretion of nitrite. Based on results from clinical and experimental animal studies, here, we report on a first evaluation of UNOxR. We determined UNOxR values in preterm neonates, healthy children, and adults, in children suffering from type 1 diabetes mellitus (T1DM) or Duchenne muscular dystrophy (DMD), in elderly subjects suffering from chronic rheumatic diseases, type 2 diabetes mellitus (T2DM), coronary artery disease (CAD), or peripheral arterial occlusive disease (PAOD). We also determined UNOxR values in healthy young men who ingested isosorbide dinitrate (ISDN), pentaerythrityl tetranitrate (PETN), or inorganic nitrate. In addition, we tested the utility of UNOxR in two animal models, i.e., the LEW.1AR1-iddm rat, an animal model of human T1DM, and the APOE*3-Leiden.CETP mice, a model of human dyslipidemia. Mean UNOxR values were lower in adult patients with rheumatic diseases (187) and in T2DM patients of the DALI study (74) as compared to healthy elderly adults (660) and healthy young men (1500). The intra- and inter-variabilities of UNOxR were of the order of 50% in young and elderly healthy subjects. UNOxR values were lower in black compared to white boys (314 vs. 483, P = 0.007), which is in line with reported lower NO bioavailability in black ethnicity. Mean UNOxR values were lower in DMD (424) compared to healthy (730) children, but they were higher in T1DM children (1192). ISDN (3 × 30 mg) decreased stronger UNOxR compared to PETN (3 × 80 mg) after 1 day (P = 0.046) and after 5 days (P = 0.0016) of oral administration of therapeutically equivalent doses. In healthy young men who ingested NaNO3 (0.1 mmol/kg/d), UNOxR was higher than in those who ingested the same dose of NaCl (1709 vs. 369). In LEW.1AR1-iddm rats, mean UNOxR values were lower than in healthy rats (198 vs. 308) and comparable to those in APOE*3-Leiden.CETP mice (151).

Background/Aims: Elevated levels of non-esterified fatty acids (NEFAs) are under suspicion to mediate β-cell dysfunction and β-cell loss in type 2 diabetes, a phenomenon known as lipotoxicity. Whereas saturated fatty acids show a strong cytotoxic effect upon insulin-producing cells, unsaturated fatty acids are not toxic and can even prevent toxicity. Experimental evidence suggests that oxidative stress mediates lipotoxicity and there is evidence that the subcellular site of ROS formation is the peroxisome. However, the interaction between unsaturated and saturated NEFAs in this process is unclear. Methods: Toxicity of rat insulin-producing cells after NEFA incubation was measured by MTT and caspase assays. NEFA induced H2O2 formation was quantified by organelle specific expression of the H2O2 specific fluorescence sensor protein HyPer. Results: The saturated NEFA palmitic acid had a significant toxic effect on the viability of rat insulin-producing cells. Unsaturated NEFAs with carbon chain lengths >14 showed, irrespective of the number of double bonds, a pronounced protection against palmitic acid induced toxicity. Palmitic acid induced H2O2 formation in the peroxisomes of insulin-producing cells. Oleic acid incubation led to lipid droplet formation, but in contrast to palmitic acid induced neither an ER stress response nor peroxisomal H2O2 generation. Furthermore, oleic acid prevented palmitic acid induced H2O2 production in the peroxisomes. Conclusion: Thus unsaturated NEFAs prevent deleterious hydrogen peroxide generation during peroxisomal β-oxidation of long-chain saturated NEFAs in rat insulin-producing cells.

Severe ischemia reperfusion injury (IRI) results in rapid complement activation, acute kidney injury and progressive renal fibrosis. Little is known about the roles of the C5aR1 and C5aR2 complement receptors in IRI. In this study C5aR1–/– and C5aR2–/– mice were compared to the wild type in a renal IRI model leading to renal fibrosis. C5a receptor expression, kidney morphology, inflammation, and fibrosis were measured in different mouse strains one, seven and 21 days after IRI. Renal perfusion was evaluated by functional magnetic resonance imaging. Protein abundance and phosphorylation were assessed with high content antibody microarrays and Western blotting. C5aR1 and C5aR2 were increased in damaged tubuli and even more in infiltrating leukocytes after IRI in kidneys of wild-type mice. C5aR1–/– and C5aR2–/– animals developed less IRI-induced inflammation and showed better renal perfusion than wild-type mice following IRI. C5aR2–/– mice, in particular, had enhanced tubular and capillary regeneration with less renal fibrosis. Anti-inflammatory IL-10 and the survival/growth kinase AKT levels were especially high in kidneys of C5aR2–/– mice following IRI. LPS caused bone marrow–derived macrophages from C5aR2–/– mice to release IL-10 and to express the stress response enzyme heme oxygenase-1. Thus, C5aR1 and C5aR2 have overlapping actions in which the kidneys of C5aR2–/– mice regenerate better than those in C5aR1–/– mice following IRI. This is mediated, at least in part, by differential production of IL-10, heme oxygenase-1 and AKT.

Interleukin (IL)-1β, the sole proinflammatory cytokine released from pancreas-infiltrating macrophages, inhibits glucose-stimulated insulin secretion (GSIS), causing hyperglycemia in Cohen diabetes-sensitive (CDs) rats fed a diabetogenic-diet (CDs-HSD). Because IL-1β blockade is a potential therapeutic target in diabetes, we examined whether treating CDs rats with IL-1β antibody (IL-1βAb; 0.5 mg/kg body weight) could counteract the inhibition of GSIS and hyperglycemia. We found that daily IL-1βAb injections had a beneficial effect on glucose tolerance and insulin secretion in CDs-HSD rats. In the oral glucose tolerance test, IL-1βAb–treated CDs-HSD rats showed lower blood glucose concentrations (P < 0.001) and higher GSIS (P < 0.05) compared with nontreated CDs-HSD rats. IL-1βAb treatment also protected the exocrine pancreas; the number of infiltrating macrophages decreased by 70% (P < 0.01) and IL-1β expression decreased by 85% (P < 0.01). In parallel, a 50% reduction (P < 0.01) in the rate of apoptosis and in fat infiltration (P < 0.05) was noted in the exocrine parenchyma of IL-1βAb–treated CDs-HSD rats compared with nontreated CDs-HSD rats. Altogether, these data demonstrate that blocking IL-1β action by IL-1βAb counteracted β-cell dysfunction and glucose intolerance, supporting the notion that prevention of pancreas infiltration by macrophages producing IL-1β is of crucial importance for the preservation of β-cell function and prevention of diabetes.

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.

β-Cell mitochondrial dysfunction as well as proinflammatory cytokines have been suggested to contribute to reduced glucose-stimulated insulin secretion (GSIS) in type 2 diabetes. We recently demonstrated that Cohen diabetic sensitive (CDs) rats fed a high-sucrose, low-copper diet (HSD) developed hyperglycemia and reduced GSIS in association with peri-islet infiltration of fat and interleukin (IL)-1β-expressing macrophages, whereas CD resistant (CDr) rats remained normoglycemic on HSD. We examined: 1) the correlation between copper concentration in the HSD and progression, prevention, and reversion of hyperglycemia in CDs rats, 2) the relationship between activity of the copper-dependent, respiratory-chain enzyme cytochrome c oxidase (COX), infiltration of fat, IL-1β-expressing macrophages, and defective GSIS in hyperglycemic CDs rats. CDs and CDr rats were fed HSD or copper-supplemented HSD before and during hyperglycemia development. Blood glucose and insulin concentrations were measured during glucose tolerance tests. Macrophage infiltration and IL-1β expression were evaluated in pancreatic sections by electron-microscopy and immunostaining. COX activity was measured in pancreatic sections and isolated islets. In CDs rats fed HSD, GSIS and islet COX activity decreased, while blood glucose and infiltration of fat and IL-1β-expressing macrophages increased with time on HSD ( P < 0.01 vs. CDr-HSD rats, all parameters, respectively). CDs rats maintained on copper-supplemented HSD did not develop hyperglycemia, and in hyperglycemic CDs rats, copper supplementation restored GSIS and COX activity, reversed hyperglycemia and infiltration of fat and IL-1β-expressing macrophages ( P < 0.01 vs. hyperglycemic CDs-HSD rats, all parameters, respectively). We provide novel evidence for a critical role of low dietary copper in diminished GSIS of susceptible CDs rats involving the combined consequence of reduced islet COX activity and pancreatic low-grade inflammation.

The prevention of diabetes by the immunomodulatory agent FTY720 (fingolimod) was studied in the LEW.1AR1-iddm (IDDM) rat, an animal model of human type 1 diabetes. Immune cell subtypes and cytokine profiles in pancreatic islets, secondary lymphoid tissue, and serum were analyzed for signs of immune cell activation. Animals were treated with FTY720 (1 mg/kg body weight) for 40 d starting on d 50 of life. Changes in gene and protein expression of cytokines, CD8 markers, monocyte chemoattractant protein-1, inducible NO synthase, and caspase 3 were evaluated. Treatment with FTY720 prevented diabetes manifestation and islet infiltration around d 60 of life, the usual time of spontaneous diabetes development. On d 120, 30 d after the end of FTY720 therapy, diabetes prevention persisted. However, six of 12 treated animals showed increased gene expression of IL-1β, TNF-α, and CD8 markers in pancreas-draining lymph nodes, indicating immune cell activation. In parallel, serum concentrations of these proinflammatory cytokines were increased. These six animals also showed macrophage infiltration without proinflammatory cytokine expression in a small minority (2–3%) of islets. Interestingly, regulatory T lymphocytes were significantly increased in the efferent vessels of the pancreas-draining lymph nodes only in animals without signs of immune cell activation but not in the rats with immune cell activation. This provides an indication for a lack of protective capacity in the animals with activated immune cells. Thus, FTY720 treatment prevented the manifestation of diabetes by promoting the retention of activated immune cells in the lymph nodes, thereby avoiding islet infiltration and β-cell destruction by proinflammatory cytokines.

The contribution of ATP-sensitive K(+) channel (K(ATP) channel)-dependent and -independent signaling to the insulinotropic characteristics of imidazolines was explored using perifused mouse islets and beta-cells. Up to a concentration of 100 muM efaroxan had no insulinotropic effect in the presence of a basal glucose concentration, but enhanced the effect of a stimulatory concentration of glucose or nonglucidic nutrients (ketoisocaproate plus glutamine). The secretion by a non-nutrient (40 mM KCl) was not enhanced. At 500 microM, efaroxan stimulated insulin secretion when glucose was basal. Likewise, at 0.1 to 10 microM RX871024 [2-(imidazolin-2-yl)-1-phenylindole] showed a purely enhancing effect, but at 100 microM it elicited a strong KCl-like secretory response in the presence of basal glucose. At 0.1 and 1 microM RX871024 did not significantly depolarize the beta-cell membrane. However, at a purely enhancing drug concentration (10 microM RX871024 or 100 microM efaroxan) K(ATP) channel activity was strongly reduced, the membrane was depolarized, and the cytosolic Ca(2+) concentration was elevated in the presence of basal glucose. Insulin secretion by sulfonylurea receptor (SUR)1 knockout (KO) islets, which have no functional K(ATP) channels, was not increased by efaroxan (100 or 500 microM) or by 10 microM RX871024 but was increased by 100 microM RX871024. The imidazolines phentolamine and alinidine (100 microM) were also ineffective on SUR1 KO islets. It is concluded that a significant K(ATP) channel block is compatible with a purely enhancing effect of the imidazolines on nutrient-induced insulin secretion. Only RX871024 has an additional, nondepolarizing effect, which at a high drug concentration is able to elicit a K(ATP) channel-independent secretion.

Thyroid hormones reduce glucose tolerance in humans and animals. This effect is related to a decrease of glucose-induced insulin secretion following a reduction of pancreatic beta cell mass due to beta cell loss. The aim of this study was to analyze in vitro the mechanisms underlying the effects of triiodothyronine (T 3 ) on the cell viability and cell cycle caused by changes of cell death or proliferation rate of insulin-producing INS-1 cells. 72-h Exposure of INS-1 cells to increasing T 3 concentrations up to 500 μM resulted in a significant viability reduction. This T 3 toxicity was caused by an increased apoptotic cell death rate, which was accompanied by a decreased proliferation rate. Inhibitory effects of T 3 on glucose-induced insulin secretion were already seen after 24 h of incubation, indicating that the deleterious effects of T 3 were time-dependent, changing from specific cellular dysfunctions to a severe and extended disturbance of the cellular survival program. Only T 3 concentrations higher than 250 μM were able to decrease cell viability and proliferation rate, to increase the rate of apoptosis and to reduce glucose-induced insulin secretion. These micromolar T 3 concentrations were significantly higher than the concentration range of T 3 receptor binding, indicating that other non-receptor-mediated mechanisms beyond the receptor level must be responsible for the observed toxic effects of T 3 in vitro.

In this manuscript we describe two closely related inbred guinea pig strains, maintained at the Central Animal Facility of the Hannover Medical School, which develop a Type 2 diabetes mellitus-like syndrome. Both strains are characterised by a high incidence of hyperglycaemia, early onset and manifestation of hyperglycaemia (3 months of age), non-inflammatory chronic vacuolary degeneration of s-cells in the pancreas, and an opacity of the optic lens. The development of diabetes was monitored by determination of blood glucose concentration and light microscopy of various organs. The measurement of serum insulin revealed a severe decrease. Tentative treatment was done by implantation of a sustained release insulin implant, but showed no impact on the blood glucose concentration within an observation period of 8 weeks. Further functional and genetic studies are needed to verify whether these two strains of guinea pigs make valuable animal models for studying the interacting factors leading to Type 2 diabetes mellitus.

Type 1 and type 2 diabetes are characterized by progressive beta-cell failure. Apoptosis is probably the main form of beta-cell death in both forms of the disease. It has been suggested that the mechanisms leading to nutrient- and cytokine-induced beta-cell death in type 2 and type 1 diabetes, respectively, share the activation of a final common pathway involving interleukin (IL)-1beta, nuclear factor (NF)-kappaB, and Fas. We review herein the similarities and differences between the mechanisms of beta-cell death in type 1 and type 2 diabetes. In the insulitis lesion in type 1 diabetes, invading immune cells produce cytokines, such as IL-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. IL-1beta and/or TNF-alpha plus IFN-gamma induce beta-cell apoptosis via the activation of beta-cell gene networks under the control of the transcription factors NF-kappaB and STAT-1. NF-kappaB activation leads to production of nitric oxide (NO) and chemokines and depletion of endoplasmic reticulum (ER) calcium. The execution of beta-cell death occurs through activation of mitogen-activated protein kinases, via triggering of ER stress and by the release of mitochondrial death signals. Chronic exposure to elevated levels of glucose and free fatty acids (FFAs) causes beta-cell dysfunction and may induce beta-cell apoptosis in type 2 diabetes. Exposure to high glucose has dual effects, triggering initially "glucose hypersensitization" and later apoptosis, via different mechanisms. High glucose, however, does not induce or activate IL-1beta, NF-kappaB, or inducible nitric oxide synthase in rat or human beta-cells in vitro or in vivo in Psammomys obesus. FFAs may cause beta-cell apoptosis via ER stress, which is NF-kappaB and NO independent. Thus, cytokines and nutrients trigger beta-cell death by fundamentally different mechanisms, namely an NF-kappaB-dependent mechanism that culminates in caspase-3 activation for cytokines and an NF-kappaB-independent mechanism for nutrients. This argues against a unifying hypothesis for the mechanisms of beta-cell death in type 1 and type 2 diabetes and suggests that different approaches will be required to prevent beta-cell death in type 1 and type 2 diabetes.

The IDDM (LEW.1AR1/Ztm-iddm) rat is a type 1 diabetic animal model characterized by a rapid apoptotic pancreatic beta-cell destruction. Here we have analyzed the time course of islet infiltration, changes in the cytokine expression pattern, and beta-cell apoptosis in the transition from the pre-diabetic to the diabetic state. Transition from normoglycemia to hyperglycemia occurred when beta-cell loss exceeded 60-70%. At the early stages of islet infiltration, macrophages were the predominant immune cell type in the peripherally infiltrated islets. Progression of beta-cell loss was closely linked to a severe infiltration of the whole islet by CD8+ T-cells. With progressive islet infiltration, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were expressed in immune cells but not in beta-cells. This proinflammatory cytokine expression pattern coincided with the expression of inducible nitric oxide synthase (iNOS) and procaspase 3 in beta-cells and a peak apoptosis rate of 6.7%. Islet infiltration declined after manifestation of clinical diabetes, yielding end-stage islets devoid of beta-cells and immune cells without any sign of cytokine expression. The observed coincidence of IL-1beta and TNF-alpha expression in the immune cells and the induction of iNOS and procaspase 3 mRNA expression in the beta-cells depicts a sequence of pathological changes leading to apoptotic beta-cell death in the IDDM rat. This chain of events provides a mechanistic explanation for the development of the diabetic syndrome in this animal model of human type 1 diabetes.

Anti–tumor necrosis factor-α (TNF-α) therapy (5 mg/kg body weight), alone or combined with the T-cell–specific antibody anti–T-cell receptor (TCR) (0.5 mg/kg body weight), was performed over 5 days immediately after disease manifestation to reverse the diabetic metabolic state in the LEW.1AR1-iddm rat, an animal model of human type 1 diabetes. Only combination therapy starting at blood glucose concentrations below 15 mmol/L restored normoglycemia and normalized C-peptide. Increased β-cell proliferation and reduced apoptosis led to a restoration of β-cell mass along with an immune cell infiltration–free pancreas 60 days after the end of therapy. This combination of two antibodies, anti-TCR/CD3, as a cornerstone compound in anti–T-cell therapy, and anti–TNF-α, as the most prominent and effective therapeutic antibody in suppressing TNF-α action in many autoimmune diseases, was able to reverse the diabetic metabolic state. With increasing blood glucose concentrations during the disease progression, however, the proapoptotic pressure on the residual β-cell mass increased, ultimately reaching a point where the reservoir of the surviving β-cells was insufficient to allow a restoration of normal β-cell mass through regeneration. The present results may open a therapeutic window for reversal of diabetic hyperglycemia in patients, worthwhile of being tested in clinical trials.

The therapeutic capacity of an antibody directed against the T cell receptor (anti-TCR) of the TCR/CD3 complex alone or in combination with fingolimod (FTY720) to reverse the diabetic metabolic state through suppression of autoimmunity and stimulation of β cell regeneration was analyzed in the LEW.1AR1-iddm (IDDM) rat, an animal model of human type 1 diabetes. Animals were treated with anti-TCR (0.5 mg/kg body weight for 5 days) monotherapy or in combination with fingolimod (1 mg/kg body weight for 40 days). Metabolic changes and β cell morphology were analyzed before, immediately after, and 60 days after end of therapy. Both therapies were started early after disease manifestation and led to normoglycemia in parallel with an increase of the C-peptide concentration. Combination therapy increased the β cell mass reaching a range of normoglycemic controls, decreased the apoptosis rate fivefold, and increased the proliferation rate threefold. Additionally, at 60 days after therapy, islets were virtually free of T cells, macrophages, and cytokine expression. In contrast, after anti-TCR monotherapy, β cell mass remained low with an activated immune cell infiltrate. A concomitant fivefold increased β cell apoptosis rate resulted in a complete loss of β cells. Only combination therapy yielded sustained normoglycemia with full reversal of islet infiltration and restoration of pancreatic β cell mass.Combination therapy of anti-TCR and fingolimod was effective in the reversal of T1D. Combination therapy increased the pancreatic β cell mass to normoglycemic control levels. Combination therapy leads to a full reversal of pancreatic islet infiltration. Anti-TCR monotherapy did not abolish islet infiltration. Combination therapy was successful only immediately after diabetes manifestation.

In the well-established, high-dose streptozotocin diabetic rat model, it is unknown whether normoglycemia after pancreas or islet transplantation may induce the expression of the glucose recognition structures and stimulate the replication of the few surviving pancreatic beta cells. Therefore, the endocrine pancreatic tissue was examined immunocytochemically in streptozotocin-treated major histocompatibility complex congenic Lewis rats at 10 and 100 days after transplantation of whole pancreata or isolated islets implanted under the kidney capsule. In the diabetic state the pancreatic beta cells displayed a weak immunostaining for insulin and glucokinase together with a lack of GLUT2 glucose transporter immunoreactivity in the plasma membrane. Ten days after transplantation, the surviving beta cells had regained their normal immunostaining for insulin and for the glucose recognition structures glucokinase and the A single high dose of streptozotocin causes severe experimental insulin-dependent diabetes mellitus in adult rats due to a selective destruction of the pancreatic beta cells in the islets of Langerhans. At doses between 50 and 60 mg/kg of body weight, only very few beta cells survive in the pancreas (1-3). The diabetic state is irreversible and insulin-dependent, thus representing an experimental animal model for type I diabetes (2). Because of the prevailing hyperglycemia, even the few residual beta cells in the pancreas do not function properly and therefore cannot contribute even to a basal supply of insulin to the organism (4). Pancreatic beta cells can function properly in a diabetic organism apparently only after restitution of normoglycemia (5). GLUT2 glucose transporter. One hundred days after transplantation, both of whole pancreas or isolated islets, the number of surviving beta cells in islets of the pancreata of the recipient animals had increased by two- to threefold. The regenerated beta cells were surrounded by a rim of other endocrine cells. The increase in the number of beta cells was not accompanied by signs of neogenesis from ductal structures in the pancreata. The authors' observations support the concept that strict long-term maintenance of normoglycemia through adequate supply of insulin from endocrine grafts is the ideal prerequisite for beta-cell recovery and restitution of the glucose recognition structures, as well as replication of beta cells in pancreata with end-stage diabetic beta-cell destruction after high-dose streptozotocin treatment.

The common marmoset New World monkey (Callithrix jacchus), is a primate model with great potential for scientific research, including research on diabetes. However, in opposite to Rhesus and Java monkeys (Macaca mulatta and Macaca fascicularis) little is known about the marmosets islet microarchitecture, glucose transporter and pancreatic marker gene expression. In this work we analyze differences and similarities in size, shape, cellular composition and intra-islet topography between the common marmoset and the human endocrine pancreas. Different sized, circular and a-circular shaped islets of the common marmoset and human display α-cells in the whole islet organ leading to a ribbon-like islet type. The number of islets was significantly higher in the common marmoset compared with humans. However, the area of insulin-producing cells was significantly higher in the human pancreas. Intra-islet distribution pattern of δ- and β-cells was similar in both species. The morphology of the exocrine pancreas regarding acinar and ductal cells was quite similar as confirmed by ultrastructural analysis. Additionally the ultrastructure of secretory granules from α-, δ- and β-cells of human and non-human primate pancreas showed the same characteristics. Molecular analysis showed the presence of endocrine pancreatic marker genes like PMCA2, NCX1, SUR1, KIR6.2, MAFA, NGN3 and PDX1 also expressed in the human. For the first time we could show presence of Glut 5 and 9 transporters in addition to the low abundance transporter Glut2 and the highly expressed Glut1 glucose transporter. We propose that Callithrix jacchus displays a new animal model for diabetes research and regenerative medicine.

Chromogranin A (CGA) is a secretory protein present in the adrenal medulla and in a variety of endocrine organs. This protein may serve as precursor for pancreastatin (PST) and for other biologically active peptides. Recently, chromostatin (CST), a CGA derivative, has been identified that possesses high biological activity. The cellular distribution of CST in various endocrine organs is completely unknown. Using immunohistochemistry on plastic sections, we investigated the occurrence and cellular distribution of CST, PST, and CGA in human endocrine pancreas of healthy and diseased states and in the adrenal medulla. In the normal and diabetic pancreas, CST immunoreactivity was localized exclusively in beta cells, which were mostly unreactive for PST and CGA. Both latter peptides were confined mainly to glucagon (alpha) cells. Insulinoma cells displayed strong insulin, PST, and CGA immunoreactivities, but they were faintly immunoreactive for CST or unreactive. Adrenal chromaffin cells exhibited strong immunoreactivity for CGA but lacked CST and PST immunoreactivities. Based on the peculiar distributive pattern of CST, PST, and CGA, we suggest that CGA is differentially processed in chromaffin and islet tissues and in insulinoma cells. The unique cellular localization of CST in the endocrine pancreas of normal and pathological conditions may indicate that CST is involved in beta-cell function.

Objectives To compare the effects of glucocorticoids and thyroid hormones on the regulation of the beta cell mass in the pancreas, the rats were treated and analyzed for cell cycle changes in islet and duct cells as a source for beta cell neogenesis. Methods Different rat pancreases were morphometrically analyzed after immunohistochemical staining for markers of proliferation and apoptosis. Results Hydrocortisone increased the beta cell mass of rat pancreases through an increase of proliferation. This effect was counteracted by an increase of apoptosis. In contrast, thyroxine decreased the beta cell mass through an increase of apoptosis. This effect was counteracted by an increased rate of proliferation. Combined treatment with both hormones nullified the antagonistic effects on proliferation, apoptosis, and beta cell mass, thereby contributing to the maintenance of a stable total beta cell volume of the pancreas. Conclusions Hydrocortisone and thyroxine induced analogous changes in pancreatic duct cells, which represent a crucial pool for new beta cells through neogenesis. This may explain the positive effects of glucocorticoids in the immunosuppressive therapy regimen after whole pancreas transplantation upon long-term insulin independence, which is not achievable with isolated islets because of the loss of duct cells during the islet process before transplantation.

Background: Embryonic stem (ES) cells have the potential to produce unlimited numbers of surrogate insulin-producing cells for cell replacement therapy of type 1 diabetes mellitus. The impact of the in vivo environment on mouse ES cell differentiation towards insulin-producing cells was analysed morphologically after implantation. Methods: ES cells differentiated in vitro into insulin-producing cells according to the Lumelsky protocol or a new four-stage differentiation protocol were analysed morphologically before and after implantation for gene expression by in situ reverse transcription polymerase chain reaction and protein expression by immunohistochemistry and ultrastructural analysis. Results: In comparison with nestin positive ES cells developed according to the reference protocol, the number of ES cells differentiated with the four-stage protocol increased under in vivo conditions upon morphological analysis. The cells exhibited, in comparison to the in vitro situation, increased gene and protein expression of Pdx1, insulin, islet amyloid polypeptide (IAPP), the GLUT2 glucose transporter and glucokinase, which are functional markers for glucose-induced insulin secretion of pancreatic beta cells. Renal sub-capsular implantation of ES cells with a higher degree of differentiation achieved by in vitro differentiation with a four-stage protocol enabled further significant maturation for the beta-cell-specific markers, insulin and the costored IAPP as well as the glucose recognition structures. In contrast, further in vivo differentiation was not achieved with cells differentiated in vitro by the reference protocol. Conclusions: A sufficient degree of in vitro differentiation is an essential prerequisite for further substantial maturation in a beta-cell-specific way in vivo, supported by cell-cell contacts and vascularisation. Fil: Naujok, Ortwin. Hannover Medical School; Alemania Fil: Francini, Flavio. Hannover Medical School; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Centro de Endocrinología Experimental y Aplicada (i); Argentina Fil: Picton, Sally. Aston University; Reino Unido Fil: Bailey, Clifford J.. Aston University; Reino Unido Fil: Lenzen, Sigurd. Hannover Medical School; Alemania Fil: Jörns, Anne. Hannover Medical School; Alemania

Background Treatment of diabetes type 2 using chronic pharmacological inhibition of dipeptidyl peptidase 4 (DP4) still requires an in-depth analysis of models for chronic DP4 deficiency, because adverse reactions induced by some DP4 inhibitors have been described. Methods In the present study, a novel congenic rat model of DP4 deficiency on a "DP4-high" DA rat genetic background was generated (DA.F344-Dpp4(m)/ SvH rats) and comprehensively phenotyped. Results Similar to chronic pharmacological inhibition of DP4, DP4 deficient rats exhibited a phenotype involving reduced diet-induced body weight gain and improved glucose tolerance associated with increased levels of glucagon-like peptide-1 (GLP-1) and bound leptin as well as decreased aminotransferases and triglycerides. Additionally, DA.F344-Dpp4(m)/SvH rats showed anxiolytic-like and reduced stress-like responses, a phenomenon presently not targeted by DP4 inhibitors. However, several immune alterations, such as differential leukocyte subset composition at baseline, blunted natural killer cell and T-cell functions, and altered cytokine levels were observed. Conclusions While this animal model confirms a critical role of DP4 in GLP-1-dependent glucose regulation, genetically induced chronic DP4 deficiency apparently also affects stress-regulatory and immuneregulatory systems, indicating that the use of chronic DP4 inhibitors might have the potential to interfere with central nervous system and immune functions in vivo.

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.

Objective Psammomys obesus gerbil exhibits PKCe over-expression on high-energy (HE) diet. Muscle insulin receptor (IR) signalling and tyrosine kinase activity are inhibited eliciting insulin resistance. We aimed at preventing diabetes by inhibiting PKCe-induced serine phosphorylation of IRS-1 with novel PKCe abrogating peptides. Research design PKCe abrogating peptides were copied from catalytic domain of PKC molecule (PCT patent IL2006/000755). Psammomys fed a diabetogenic HE diet received i.p. peptides KCe-12 and KCe-16 (18 mg/kg) on days 0, 7 and 14 controls received peptide solvent. Results Food consumption and animal weight remained unchanged. On day 16, non-fasting blood glucose levels returned to normal (90 ± 5 versus 347 ± 16 mg/dL in untreated controls). Hyperinsulinemia fell from 584 ± 55 to 180 ± 22 mU/L. Western blot analysis showed that the increased phosphoserine636, 639 content on IRS-1 in gastrocnemius muscle of diabetic animals was reduced three fold, the PKB/AKT activity increased two fold and muscle GLUT4 tended to increase, compared with controls. Likewise, administration of KCe-12 prior to placing the HE diet prevented the onset of diabetes. KCe-12 treatment did not reduce muscle PKCe level. Damage and loss of insulin in pancreatic beta cells on HE diet were prevented by KCe-12, as shown in micrographs of islet hematoxylin-eosin staining and insulin immunostaining. The preserved secretory function enabled Psammomys to normalize glucose homeostasis. Conclusions KCe-16 and KCe-12 peptides derived from PKCe substrate-binding region prevented the nutritional diabetes and protected muscle IRS-1 from PKCe-induced serine phosphorylation, abrogating the insulin-signalling impediment in the Psammomys model of type 2 diabetes. Anti-diabetic peptides may lead to novel modalities preventing human overnutrition-induced insulin resistance and diabetes. Copyright © 2008 John Wiley & Sons, Ltd.

BACKGROUND Ischemia/reperfusion (I/R) injury after pancreas transplantation might result in graft pancreatitis. The role of heme oxygenase-1 (HO-1) in pancreas transplantation and prevention of graft pancreatitis is unknown. METHOD We studied the impact of HO-1 induction with cobalt protoporphyrin (CoPP) in experimental pancreas transplantation with moderate (6 hr) and prolonged (20 hr) cold ischemic time (CIT). Donor animals received CoPP 5 mg/kg intraperitoneal at 48 hr or intraperitoneal saline injections in the corresponding control groups before procurement. Harvested grafts were perfused with HTK solution and stored at 4 degrees C. RESULTS After prolonged CIT, graft survival was 100% with CoPP pretreatment in contrast to only 37.5% without pretreatment. CoPP-pretreated grafts demonstrated an unimpaired endocrine graft function at moderate and prolonged CIT. Serum lipase activity as a sign of exocrine preservation was significantly lower. In addition, morphological architecture was well preserved. CoPP pretreatment markedly increased HO-1 gene expression in donor pancreas (130-fold increase) by means of quantitative reverse transcriptase -polymerase chain reaction. Immunohistochemical examinations showed that the increase of HO-1 on the protein level was related to HO-1-positive donor macrophages in the pancreas grafts. HO-1 overexpression was accompanied by significant decrease of proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-2, IL-6, interferon-y, and by significant increase of the anti-inflammatory cytokine IL-10 and less expression of adhesion molecules such as e- and p-selectins. CONCLUSIONS HO-1 is highly inducible in the allograft rat pancreas and associated with a survival benefit and good graft function after transplantation. This study contributes to the beneficial potentials of HO-1 for the prevention of graft pancreatitis.

OBJECTIVE—The Cohen diabetes–sensitive rat develops postprandial hyperglycemia when fed a high-sucrose, copper-poor diet, whereas the Cohen diabetes–resistant rat maintains normoglycemia. The pathophysiological basis of diabetes was studied in the Cohen diabetic rat centering on the interplay between the exocrine and endocrine compartments of the pancreas. RESEARCH DESIGN AND METHODS—Studies used male Cohen diabetes–sensitive and Cohen diabetes–resistant rats fed 1-month high-sucrose, copper-poor diet. Serum insulin and glucose levels were measured during glucose and insulin tolerance tests. The pancreas was evaluated for weight, insulin content, macrophage, and fat infiltration. Glucose-stimulated insulin secretion (GSIS) was determined in isolated perfused pancreas and in islets. RESULTS—Hyperglycemic Cohen diabetes–sensitive rats exhibited reduced pancreatic weight with lipid deposits and interleukin-1β–positive macrophage infiltration in the exocrine pancreas. Islet morphology was preserved, and total pancreatic insulin content did not differ from that of Cohen diabetes–resistant rats. Lipids did not accumulate in skeletal muscle, nor was insulin resistance observed in hyperglycemic Cohen diabetes–sensitive rats. Intravenous glucose-tolerance test revealed markedly elevated glucose levels associated with diminished insulin output. Insulin release was induced in vivo by the non-nutrient secretagogues arginine and tolbutamide, suggesting a selective unresponsiveness to glucose. Decreased GSIS was observed in the isolated perfused pancreas of the hyperglycemic Cohen diabetes–sensitive rat, whereas islets isolated from these rats exhibited glucose-dependent insulin secretion and proinsulin biosynthesis. CONCLUSIONS—The association of the in vivo insulin secretory defect with lipid accumulation and activated macrophage infiltration in the exocrine pancreas suggests that changes in the islet microenvironment are the culprit in the insulin secretory malfunction observed in vivo.

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.

Insulin-secreting MIN6 cells show greatly enhanced secretory responsiveness to nutrients when grown as islet-like structures (pseudoislets). Since beta-cells use different mechanisms to respond to nutrient and non-nutrient stimuli, we have now investigated the role of homotypic beta-cell interactions in secretory responses to pharmacological or receptor-operated non-nutrient stimuli in MIN6 pseudoislets. In addition to an enhanced secretory responsiveness to glucose, insulin secretion from MIN6 pseudoislets was also enhanced by non-nutrients, including carbachol, tolbutamide, PMA, and forskolin. The improved secretory responsiveness was dependent on the cells being configured as pseudoislets and was lost on dispersal of the pseudoislets into single cells and regained on the re-formation of pseudoislet structures. These observations emphasise the importance of islet anatomy on secretory responsiveness, and demonstrate that homotypic beta-cell interactions play an important role in generating physiologically appropriate insulin secretory responses to both nutrient and non-nutrient stimuli.

The importance of the cellular immune system for the development of T1DM in the LEW.1AR1-iddm rat was investigated by use of polyinosinic-polycytidylic acid (Poly I:C) and by adoptive transfer of concanavalin A (Con A) activated lymphocytes from diabetic LEW.1AR1-iddm rats and the coisogenic LEW.AR1 background strain. Poly I:C treatment induced diabetes, characterized morphologically by a diffuse infiltration of the pancreas, in up to 20% of the animals of the coisogenic LEW.1AR1 background strain. It did not increase the diabetes incidence of 30% of the LEW.1AR1-iddm strain. In contrast Poly I:C treatment induced diabetes in up to 80% of the animals of the Mhc congenic LEW.1WR1 strain. Adoptive transfer of lymphocytes activated by the T-cell mitogen Con A from diabetic donors doubled the incidence of diabetes, characterized morphologically by a focal insulitis, in diabetes prone LEW.1AR1-iddm recipients. In contrast, animals of the LEW.1AR1 background strain did not develop diabetes after adoptive transfer. Moreover, adoptive transfer of Con A activated lymphocytes from LEW.1AR1 rats to LEW.1AR1-iddm rats with 30 or 60% diabetes incidence, significantly decreased the incidence of diabetes in LEW.1AR1-iddm rats with 60% diabetes incidence. The results show that autoreactive lymphocytes induce beta cell destruction in the LEW.1AR1-iddm rat, while the LEW.AR1 background strain apparently contains regulatory potential, which is able to counteract the autoimmune response.

Background Psammomys obesus is a desert gerbil developing hyperglycaemia, hyperinsulinaemia and insulin resistance when placed for 2 weeks on a high-energy (HE) diet. The mechanism underlying the antidiabetic effect of rosiglitazone (RG) treatment (20 mg/kg per day for 2 weeks) was studied. Methods The antidiabetogenic effect of RG treatment on serum insulin and metabolic parameters in serum and target tissues of insulin action was investigated in vivo and compared with the pancreatic beta cell protective effects of RG. Results Almost all RG-treated animals remained normoglycaemic compared to controls, but, at the same time, they were hyperinsulinaemic. RG had no effect on serum free fatty acid and serum and muscle triglyceride concentrations and did not appreciably affect body weight and fat depots. RG prevented a HE diet-induced reduction of GLUT 4 glucose transporter content in epididymal adipose tissue, but not in gastrocnemius muscle. The normoglycaemic effect was not associated with a suppression of liver PEPCK activity. Muscle PKCe expression, known to be elevated in diabetic Psammomys and to inhibit insulin signalling, was only marginally decreased. However, RG treatment prevented the marked decrease in insulin immunostaining as well as the vacuolization of the beta cells and accelerated beta cell proliferation. Conclusions These data indicate that the skeletal muscle is not the primary target of RG action, whereas the preservation of the insulin secretory capacity and the prevention of degenerative beta cell vacuolization in spite of persisting insulin resistance appear to be the basis for the anti-hyperglycaemic effect of RG in Psammomys. Copyright © 2005 John Wiley & Sons, Ltd.

Prolonged stimulation of insulin secretion by depolarization and Ca2+ influx regularly leads to a reversible state of decreased secretory responsiveness to nutrient and nonnutrient stimuli. This state is termed “desensitization.” The onset of desensitization may occur within 1 h of exposure to depolarizing stimuli. Desensitization by exposure to sulfonylureas, imidazolines, or quinine produces a marked cross-desensitization against other ATP-sensitive K+ channel (KATP channel)-blocking secretagogues. However, desensitized β-cells do not necessarily show changes in KATP channel activity or Ca2+ handling. Care has to be taken to distinguish desensitization-induced changes in signaling from effects due to the persisting presence of secretagogues. The desensitization by depolarizing secretagogues is mostly accompanied by a reduced content of immunoreactive insulin and a marked reduction of secretory granules in the β-cells. In vitro recovery from a desensitization by the imidazoline efaroxan was nearly complete after 4 h. At this time point the depletion of the granule content was partially reversed. Apparently, recovery from desensitization affects the whole lifespan of a granule from biogenesis to exocytosis. There is, however, no direct relation between the β-cell granule content and the secretory responsiveness. Even though a prolonged exposure of isolated islets to depolarizing secretagogues is often associated with the occurrence of ultrastructural damage to β-cells, we could not find a cogent link between depolarization and Ca2+ influx and apoptotic or necrotic β-cell death.

We studied the histo- and immunopathology of the endocrine and exocrine pancreas and a number of other organs in a new insulin-dependent diabetes mellitus (IDDM) rat model (LEW.1AR1/Ztm- iddm rat). The pancreas of the acutely diabetic animals showed an inflammatory infiltrate, involving all islets and ducts. The islet infiltrate was composed mainly of ED1-positive macrophages and T lymphocytes, comprising a large number of CD8(+) lymphocytes and a few CD4(+) lymphocytes. In addition, the islets displayed apoptotic cells, characterized by condensation and fragmentation of nuclear chromatin. These cells were identified as beta cells by insulin immunostaining. Other endocrine and exocrine glands, including adrenals and thyroid, as well as salivary and submandibular glands, were unaffected. Organs from the digestive tract or systemic circulatory system, including small intestine, liver, heart, and lung also showed no involvement. The kidney was intact in acutely diabetic rats. However, 6 months after diabetes manifestation, pathological changes compatible with a diabetic nephropathy had developed, affecting both the glomerula and the proximal tubular segments. It was concluded that the autoimmune process in this new IDDM rat model is restricted to the endocrine pancreas and leads to apoptotic beta cell destruction.

A prolonged exposure of isolated pancreatic islets to insulin secretagogues, the imidazolines phentolamine, alinidine and idazoxan (100microM each), the sulfonylurea tolbutamide (500microM), or the alkaloid quinine (100microM) resulted in morphological damage of 4-18% of beta-cells compared to less than 2% in controls. Thus, the question arose whether K(ATP) channel-blocking insulin secretagogues are beta-cell toxic as has already been suggested for sulfonylureas. The concentration- and time-dependency of the secretagogue-associated toxicity was documented by viability assays in insulin-secreting HIT T15 cells. Treatment for 24h with idazoxan reduced MTT conversion by 50% at 100microM and by 98% at 1000microM. Phentolamine and quinine reduced viability comparably at 1000microM, but were less toxic at 100microM. On the other hand, the imidazoline alinidine and the sulfonylurea tolbutamide were only moderately toxic (less than 40% viability loss at 1000microM). The imidazoline efaroxan appeared even to be non-toxic. Apoptotic DNA fragmentation and DEVD-caspase activation was observed at 100microM of idazoxan and phentolamine, whereas at 1000microM signs of necrosis predominated. Alinidine, tolbutamide and quinine treatment did not increase markers of apoptotic cell death. Blocking Ca(2+) influx by D600 did not diminish secretagogue-associated toxicity. Electron microscopy confirmed the validity of these observations for beta-cells in intact mouse islets. In summary, beta-cell toxicity of the tested insulin secretagogues varied widely and did not depend on a prolonged Ca(2+) influx via L-type Ca(2+) channels. Thus, secretagogue-mediated closure of K(ATP) channels is apparently not per se beta-cell toxic.

The Psammomys obesus (sand rat) is a well-established model of nutritionally induced non-insulin-dependent type-2 diabetes. When fed a high-energy (HE) diet, the diabetes-prone animals develop hyperinsulinaemia and hyperglycaemia. Within 1 week, all animals become hyperinsulinaemic. However, a loss of immunostaining for insulin as well as for the GLUT2 glucose transporter in the plasma membrane and the glucokinase in the cytoplasm of the pancreatic beta cells became evident only when the animals subsequently developed hyperglycaemia. After 1 week of HE diet feeding, the pancreatic beta-cell volume was reduced by one-third in hyperglycaemic Psammomys. Insulin immunostaining as well as GLUT2 glucose transporter immunostaining in the plasma membrane and glucokinase immunostaining in the cytoplasm were reduced by more than 50%. After 3 weeks of HE diet feeding, all changes observed after 1 week were even more pronounced, with reductions in the range of 70–95%. The reduction of the total beta-cell volume of the pancreas due to beta-cell death and the diminution of insulin content of the remaining beta cells in the islets during the HE diet feeding was accompanied by a parallel fall of the pancreas insulin content. For all changes observed, there was a significant correlation with the increase of the blood glucose concentration (r>0.9) but not with the increase of the plasma insulin concentration (r>0.2). Thus, increasing glycaemia appears to be the factor responsible for the deterioration of the pancreatic beta-cell function and the resulting loss of the insulin secretory capacity in Psammomys. The final result of this development is an irreversible diabetic state due to the feeding of the HE diet.

Objective: Epidemiological evidence shows that chronic coffee consumption in humans is correlated with a lower incidence of type 2 diabetes mellitus. For the experimental exploration of the underlying mechanisms, this effect needs to be replicated in an animal model of type 2 diabetes with a short lifespan. Design: Male C57BL/6 mice consumed regular coffee or water ad libitum and the development of obesity and diabetes caused by high-fat diet (55% lipids, HFD) was observed from week 10 on for 35 weeks in comparison with mice feeding on a defined normal diet (9% lipids, ND). Results: The massive weight gain in HFD mice was dose-dependently retarded (P=0.034), the moderate weight gain in ND mice was abolished (P

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.

Functional heterogeneity among pancreatic beta cells is a characteristic feature of the islets of Langerhans. Under physiological conditions, beta cells in the pancreas of fed rats exhibited heterogeneous immunohistochemical staining for insulin and glucokinase. Intracellular beta cell glucokinase staining was either faint or dense. In the pericapillary space beta cell glucokinase immunoreactivity had a polar orientation, with the highest density in cytoplasmic regions close to the blood vessels. Starvation resulted in a loss of heterogeneity with homogeneous insulin staining in all beta cells of the islets, and this was accompanied by a loss of heterogeneous glucokinase staining. The intracellular polarity of glucokinase staining in contact to blood vessels also disappeared after starvation. Refeeding resulted in the reappearance of intercellular heterogeneity. In dependence on the functional demand, the endocrine pancreas recruited insulin from beta cells according to a well-defined hierarchy, with an initial preferential mobilization of medullary beta cells. In the course of this process intracellular polarity of glucokinase staining reappeared in areas of the beta cell with functional contact to the GLUT2 glucose transporter in the plasma membrane. This can be regarded as the morphological correlate of an activation of the glucose signal recognition apparatus. Interestingly, the study also provides evidence that the changes in glucokinase distribution apparently preceded those in insulin distribution, which is in keeping with the central role of glucokinase as the glucose sensor of the pancreatic beta cell.

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

The in vitro toxicity of the diabetogenic agent alloxan as documented by the induction of beta cell necrosis was studied in isolated ob/ob mouse pancreatic islets. The effect of alloxan has been compared with that of a number of N-alkyl alloxan derivatives and with that of the structurally related compound, ninhydrin. Alloxan and its derivatives were selectively toxic to pancreatic beta cells, with other endocrine cells and exocrine parenchymal cells being well preserved, even at high concentration. In contrast, ninhydrin was selectively toxic to pancreatic beta cells only at comparatively low concentration, destroying all islet cell types at high concentrations. The ultrastructural changes induced by all the test compounds in pancreatic beta cells in vitro were very similar to those observed during the development of alloxan diabetes in vivo. The relative toxicity of the various compounds to pancreatic beta cells in vitro was not, however, related to their ability to cause diabetes in vivo. Indeed, the non-diabetogenic substances ninhydrin, N-butylalloxan and N-isobutylalloxan were very much more toxic to isolated islets than the diabetogenic compounds alloxan and N-methylalloxan. These results suggest that the differences in diabetogenicity among alloxan derivatives are not due to intrinsic differences in the susceptibility of the pancreatic beta cells to their toxicity, but may reflect differences in distribution or metabolism. High concentrations of glucose protected islets against the harmful effects of alloxan and its derivatives, but not those of ninhydrin. Low levels of glucose, and non-carbohydrate nutrients, afforded little protection, indicating that the effect of glucose is not due to the production of reducing equivalents within the cell, 3-O-Methylglucose, which protects against alloan diabetes in vivo, did not protect against alloxan toxicity in vitro. Since 3-O-methylglucose is known to prevent uptake of alloxan by pancreatic beta cells, it appears that uptake of alloxan by the cell is not a prerequisite for the induction of beta cell necrosis.

The diabetic Chinese hamster is a well-established animal model for NIDDM with a defective glucose-induced insulin secretory response. In the pancreas of nondiabetic hamsters, the GLUT2 glucose transporter was localized in the plasma membrane of insulin-positive beta cells. At variance with the rat, immunoreactivity was also detected in the cytoplasm. Other islet cell types were not GLUT2 positive. GLUT2 immunoreactivity was already significantly reduced in beta cells from mildly diabetic animals in spite of a normal insulin immunoreactivity. In severely diabetic animals the majority of the beta cells had lost GLUT2 immunostaining. This observation was confirmed in a Western blot analysis of the GLUT2 protein in isolated pancreatic islets. Only beta cells that were densely immunostained for insulin were still GLUT2 positive. However, around 40% of the beta cells devoid of GLUT2 immunoreactivity were still insulin immunoreactive. Thus, the loss of GLUT2 immunoreactivity, which is an important component of the glucose recognition apparatus of the pancreatic beta cell, is an early indicator of beta cell dysfunction before the development of degenerative lesions or the loss of insulin immunoreactivity. GLUT2 loss may be important in the deterioration of glucose-induced insulin secretion in the diabetic Chinese hamster.

It has already been shown that insulin cells studied under experimental conditions exhibit differences in insulin immunoreactivity and insulin release. The aim of this study, therefore, was to investigate whether insulin cells themselves exhibit morphological abnormalities after transplantation. Insulin cells in rat pancreas isografts with preserved or suppressed exocrine secretion were studied immunocytochemically and ultrastructurally and compared with those of unoperated rats. In isografts with preserved exocrine secretion, cortical insulin cells connected to the exocrine parenchyma or to glucagon or somatostatin cells expressed mostly dense immunoreactivities for insulin and amylin. In addition, medullary insulin cells connected only to other insulin cells displayed faint immunoreactivities for both constituents as found in unoperated animals. After duct ligation, however, pancreatic ducts and elongated capillaries extended into the islets. Corresponding to the stages of islet fragmentation, the heterogeneity among insulin cells underwent changes and was finally abolished. Ultrastructurally, differences in the number of secretory granules paralleled the heterogeneity in insulin immunoreactivity. It is interesting to note that the heterogeneity among insulin cells is preserved after transplantation, indicating that this phenomenon might be of physiological relevance. The heterogeneity may implicate differences in insulin storage and release as found in insulin cells under normal conditions.

The preservation of pancreatic B-cells is essential to ensure normal endocrine function of whole pancreas transplants. At present, however, it is unknown whether or not pancreatic B-cells in allografts display intercellular variations in insulin immunoreactivity and ultrastructure. It is also not clear whether the GLUT2 glucose transporter immunoreactivity is maintained in the plasma membrane after transplantation, as in B-cells under normal conditions. Therefore, pancreatic tissue of isografts and allografts, with or without signs of chronic rejection, was examined in streptozotocin-diabetic rats 10 to 100 days after transplantation by means of immunocytochemistry and electron microscopy. In isografts with preserved exocrine secretion pancreatic B-cells of larger islets exhibited normal heterogeneities in insulin immunoreactivity and in the number of secretory granules, whereas in isografts with suppressed exocrine secretion the heterogeneity was altered or abolished depending on the stage of islet dissociation. Differences in insulin immunoreactivity and ultrastructure were also observed in B-cells of tolerated allografts or those with signs of chronic rejection. In contrast to the localization of the GLUT2 glucose transporter under physiological conditions, GLUT2 glucose transporter immunoreactivity was not restricted to the pancreatic B-cell membrane but was also detected in the cytoplasm after transplantation. The cytoplasmic GLUT2 glucose-transporter localization developed in a time-dependent manner. It was evident 10 days after transplantation in isografts and was detectable still 100 days after transplantation in tolerated as well as in chronically rejected allografts. Interestingly, B-cells in the allografts with a dense insulin immunoreactivity exhibited GLUT2 glucose transporter expression in both localizations, whereas B-cells with a faint insulin immunoreactivity exhibited GLUT2 glucose transporter only in the plasma membrane. This may indicate a relation between the functional state of the pancreatic B-cells and the distribution of the GLUT2 glucose transporter in the cell. The heterogeneity between B-cells in pancreatic isografts and allografts implicates differences in the functional state of the B-cells with respect to insulin storage and release.