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The rising prevalence of type 1 diabetes (T1D) over the past decades has been linked to lifestyle changes, but the underlying mechanisms are largely unknown. Recent findings point to gut-associated mechanisms in the control of T1D pathogenesis. In nonobese diabetic (NOD) mice, a model of T1D, diabetes development accelerates after deletion of the Toll-like receptor 4 (TLR4). We hypothesized that altered intestinal functions contribute to metabolic alterations, which favor accelerated diabetes development in TLR4-deficient (TLR4−/−) NOD mice. In 70–90-day-old normoglycemic (prediabetic) female NOD TLR4+/+ and NOD TLR4−/− mice, gut morphology and microbiome composition were analyzed. Parameters of lipid metabolism, glucose homeostasis, and mitochondrial respiratory activity were measured in vivo and ex vivo. Compared with NOD TLR4+/+ mice, NOD TLR4−/− animals showed lower muscle mass of the small intestine, higher abundance of Bacteroidetes, and lower Firmicutes in the large intestine, along with lower levels of circulating short-chain fatty acids (SCFA). These changes are associated with higher body weight, hyperlipidemia, and severe insulin and glucose intolerance, all occurring before the onset of diabetes. These mice also exhibited insulin resistance–related abnormalities of energy metabolism, such as lower total respiratory exchange rates and higher hepatic oxidative capacity. Distinct alterations of gut morphology and microbiota composition associated with reduction of circulating SCFA may contribute to metabolic disorders promoting the progression of insulin-deficient diabetes/T1D development.

Background. Heat shock proteins (Hsp) act as intracellular chaperones and in addition are used as adjuvant in vaccines of peptides complexed with recombinant Hsp. By interacting with autologous peptides, Hsp may promote the induction of autoimmune reactivity. Objective. Here, we analysed whether the effect of Hsp on macrophages is modulated by insulin peptides known to interact with Hsp. Results. Combinations of the 70 kDa Hsp DnaK with peptide B11-23 from the core region of the proinsulin B-chain induced the release of the inflammatory mediators interleukin-6, tumor necrosis factor α, and interleukin-1β from cells of human and murine macrophage lines. In parallel, there was high-affinity binding of B11-23 to DnaK. DnaK mixed with peptides from other regions of the insulin molecule did not stimulate cytokine secretion. DnaK alone induced little cytokine production, and peptides alone induced none. Conclusion. The macrophage-stimulating potential of Hsp70 family proteins when combined with the proinsulin B-chain peptide B11-23 may contribute to the immunodominance of this peptide in the development of beta cell-directed autoimmunity in type 1 diabetes.

Measurement of ATP concentrations and synthesis in humans indicated abnormal hepatic energy metabolism in obesity, non-alcoholic fatty liver disease (NAFLD) and Type 2 diabetes. Further mechanistic studies on energy metabolism require the detailed phenotyping of specific mouse models. Thus, this study aimed to establish and evaluate a robust and fast single voxel

It is still an enigma why T cell autoreactivity in type 1 diabetes targets few beta cell antigens only. Among these, one primary autoantigen is pro(insulin). Autoimmune T cells preferentially recognise three epitopes on the proinsulin molecule, of which the peptide region B:11-23 is the dominant one. Interestingly, the three regions superimpose with binding sites of the chaperone hsp70, the region B:11-23 being the strongest binding one. Absence of an intact core region B:15-17 prevents autoimmune diabetes in NOD as well as binding of hsp70. A role of hsp70 in selecting autoimmune epitopes is supported by the ability of this and other chaperones to deliver bound peptides to MHC class I and II molecules for efficient antigen presentation. Binding of hsp70 to receptors on antigen presenting cells such as TLR4 results in costimulatory signals for T cell activation. Strongest effects are seen for the mixture of hsp70 with the peptide B:11-23. Thus, hsp70 may assist in proinsulin epitope selection and efficient presentation to autoreactive T cells. The concept of chaperone guided immune reactivity may also apply to other autoimmune diseases.

Objective The lack of effective treatments against diabetic sensorimotor polyneuropathy demands the search for new strategies to combat or prevent the condition. Because reduced magnesium and increased methylglyoxal levels have been implicated in the development of both type 2 diabetes and neuropathic pain, we aimed to assess the putative interplay of both molecules with diabetic sensorimotor polyneuropathy. Methods In a cross-sectional study, serum magnesium and plasma methylglyoxal levels were measured in recently diagnosed type 2 diabetes patients with (n = 51) and without (n = 184) diabetic sensorimotor polyneuropathy from the German Diabetes Study baseline cohort. Peripheral nerve function was assessed using nerve conduction velocity and quantitative sensory testing. Human neuroblastoma cells (SH-SY5Y) and mouse dorsal root ganglia cells were used to characterize the neurotoxic effect of methylglyoxal and/or neuroprotective effect of magnesium. Results Here, we demonstrate that serum magnesium concentration was reduced in recently diagnosed type 2 diabetes patients with diabetic sensorimotor polyneuropathy and inversely associated with plasma methylglyoxal concentration. Magnesium, methylglyoxal, and, importantly, their interaction were strongly interrelated with methylglyoxal-dependent nerve dysfunction and were predictive of changes in nerve function. Magnesium supplementation prevented methylglyoxal neurotoxicity in differentiated SH-SY5Y neuron-like cells due to reduction of intracellular methylglyoxal formation, while supplementation with the divalent cations zinc and manganese had no effect on methylglyoxal neurotoxicity. Furthermore, the downregulation of mitochondrial activity in mouse dorsal root ganglia cells and consequently the enrichment of triosephosphates, the primary source of methylglyoxal, resulted in neurite degeneration, which was completely prevented through magnesium supplementation. Conclusions These multifaceted findings reveal a novel putative pathophysiological pathway of hypomagnesemia-induced carbonyl stress leading to neuronal damage and merit further investigations not only for diabetic sensorimotor polyneuropathy but also other neurodegenerative diseases associated with magnesium deficiency and impaired energy metabolism., Highlights • Magnesium and methylglyoxal levels were inversely associated in individuals with type 2 diabetes and distal sensorimotor polyneuropathy. • Magnesium, methylglyoxal, and their interaction were associated with methylglyoxal-dependent nerve dysfunction. • Under experimental conditions, magnesium supplementation prevented methylglyoxal-mediated neurotoxicity. • Magnesium downregulates intracellular methylglyoxal production.

Circadian disruption is associated with the development of diabetes. Non-obese diabetic (NOD) mice show abnormal diurnal profiles in energy balance and locomotor activity suggesting circadian misalignment. Therefore, we analyzed cFos and mPER1 as markers for rhythmic neuronal activity within the suprachiasmatic nucleus (SCN) of wildtype (WT) and non-diabetic (nNOD) as well as acutely diabetic NOD (dNOD) mice. cFos levels show a day/night difference in both WT and nNOD but not in dNOD. mPER1 levels did not show a day/night difference in both nNOD and dNOD. This suggests that disruption of SCN rhythmicity in NOD mice precedes the actual onset of diabetes.

Obesity is frequently associated with excessive accumulation of lipids in ectopic tissue and presents a major risk factor for type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). Diacylglycerols (DAGs) and ceramides (CERs) were identified as key players in lipid-induced insulin resistance, typical for such diseases. Recent results suggest that the subcellular distribution of these lipids affects their lipotoxic properties. However, the subcellular dynamics of these lipids and the role of lipid droplets (LDs) as a potential storage site is not understood. Here, we developed a liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS)-method for the rapid and simultaneous quantification of DAG and CER species in tissue sample fractions. The assay is characterized by excellent recovery of analytes, limit of quantification, accuracy and precision. We established a fractionation protocol that allows the separation of subcellular tissue fractions. This method was subsequently tested to measure the concentration of DAGs and CERs in subcellular fractions of human muscle and several mouse tissues. In a mouse model of NAFLD, application of this method revealed a prominent role for LDs as repository for lipotoxic DAG and CER species. In conclusion, the new method proved as a valuable tool to analyse the subcellular dynamics of lipotoxins, related to the pathogenesis of insulin resistance, T2D and NAFLD.

Adipocytes represent an important cellular source of inflammatory mediators. However, the signals for the induction of proinflammatory adipocyte activities are largely unknown. Here, we demonstrate that heat shock protein (Hsp) 60, a potent stimulator of innate immunity, induces the release of the inflammatory mediators interleukin-6, CXCL1 and monocyte chemoattractant protein-1 in a time- and concentration-dependent manner from cells of the adipocyte line 3T3-L1 and from adipocytes of obese mice. These results identify Hsp60 as an important regulator of adipocyte functions which contribute to the development of inflammatory processes as observed in diabetes and diabetes-associated complications.

In the present study, we characterized regions of human heat shock protein (HSP) 60 responsible for binding to primary macrophages. Studies using 20-mer peptides of the HSP60 sequence to compete with HSP60-binding to macrophages from C57BL/6J mice showed that regions aa241–260, aa391–410 and aa461–480 are involved in surface-binding. HSP60 mutants, lacking the N-terminal 137, 243 or 359 amino acids, inhibited HSP60-binding to primary macrophages to different degrees, demonstrating that all three regions are required for optimal binding. Analysis of different pro- and eukaryotic HSP60 species indicated that phylogenetically separate HSP60 species use different binding sites on primary macrophages.

In the present study, we identified the human heat shock protein 60 (HSP60) epitope responsible for binding to macrophages. Studies using overlapping 15- and 20-mer peptides of the human HSP60 sequence to compete with binding of HSP60 to macrophages indicated that surface binding was accounted for by the region aa481–500. Deletion mutants of HSP60, lacking the N-terminal 137, 243 or 359 amino acids, strongly inhibited HSP60 binding to macrophages. Monoclonal antibodies addressing regions aa1–200, aa335–366 or aa383–447 did not block HSP60 binding. We conclude that a single C-terminal region, aa481–500, accounts for the binding of HSP60 to macrophages.

Heat shock protein 60 (hsp60) is a target antigen in autoimmune diabetes and injections of human hsp60 for tolerance induction were found to protect non-obese diabetic (NOD) mice, an animal model of human type 1 diabetes, from disease development. We tested whether innate immune cells of NOD mice exhibit an abnormal response to extracellular hsp60. Bone marrow derived macrophages (BMM) were grown from NOD, C57BL/6J, non-obese non-diabetic (NON) mice, and NOD-related congenic variants differing in the Idd-3, Idd-10/18, or major histocompatibility complex (MHC) region. Hsp60-stimulated BMM of NOD mice were found to produce high levels of interleukin (IL)-12(p70). The addition of IL-10 downregulated, whereas cyclooxygenase inhibitors elevated, IL-12(p70) production of activated BMM. BMM of NON, NON-NOD-H-2(g7) as well as of NOD-NON-H-2(nbl) mice produced significantly less IL-12(p70) than BMM of NOD mice, indicating that an interaction between the MHC haplotype and non-MHC genes of the NOD mouse is required for hyperresponsiveness to hsp60.

The cholera toxin B chain (CTB) has been reported to suppress T cell-dependent autoimmune diseases and to potentiate tolerance of the adaptive immune system. We have analyzed the effects of CTB on macrophages in vitro and have found that preincubation with CTB (10 μg/ml) suppresses the proinflammatory reaction to LPS challenge, as demonstrated by suppressed production of TNF-α, IL-6, IL-12(p70), and NO (p < 0.01) in cells of macrophage lines. Pre-exposure to CTB also suppresses LPS-induced TNF-α and IL-12(p70) formation in human PBMC. Both native and recombinant CTB exhibited suppressive activity, which was shared by intact cholera toxin. In cells of the human monocyte line Mono Mac 6, exposure to CTB failed to suppress the production of IL-10 in response to LPS. Control experiments excluded a role of possible contamination of CTB by endotoxin or intact cholera toxin. The suppression of TNF-α production occurred at the level of mRNA formation. Tolerance induction by CTB was dose and time dependent. The suppression of TNF-α and IL-6 production could be counteracted by the addition of Abs to IL-10 and TGF-β. IFN-γ also antagonized the actions of CTB on macrophages. In contrast to desensitization by low doses of LPS, tolerance induction by CTB occurred silently, i.e., in the absence of a measurable proinflammatory response. These findings identify immune-deviating properties of CTB at the level of innate immune cells and may be relevant to the use of CTB in modulating immune-mediated diseases.

Adipocytes release immune mediators that contribute to diabetes-associated inflammatory processes. As the stress protein heat shock protein 60 (Hsp60) induces proinflammatory adipocyte activities, we hypothesized that adipocytes of diabetes-predisposed mice exhibit an increased proinflammatory reactivity to Hsp60. Preadipocytes and mature adipocytes from nonobese diabetic (NOD), New Zealand obese (NZO), and C57BL/6J mice were analyzed for Hsp60 binding, Hsp60-activated signaling pathways, and Hsp60-induced release of the chemokine CXCL-1 (KC), interleukin 6 (IL-6), and macrophage chemoattractant protein-1 (MCP-1). Hsp60 showed specific binding to (pre-)adipocytes of NOD, NZO, and C57BL/6J mice. Hsp60 binding involved conserved binding structure(s) and Hsp60 epitopes and was strongest to NZO mouse-derived mature adipocytes. Hsp60 exposure induced KC, IL-6, and MCP-1 release from (pre-)adipocytes of all mouse strains with a pronounced increase of IL-6 release from NZO mouse-derived adipocytes. Compared to NOD and C57BL/6J mouse derived cells, Hsp60-induced formation of IL-6, KC, and MCP-1 from NZO mouse-derived (pre-)adipocytes strongly depended on NFκB-activation. Increased Hsp60 binding and Hsp60-induced IL-6 release by mature adipocytes of NZO mice suggest that enhanced adipocyte reactivity to the stress signal Hsp60 contributes to inflammatory processes underlying diabetes associated with obesity and insulin resistance.

Activation of the nuclear enzyme poly(ADP-ribose)polymerase (PARP) is a critical step in beta-cell death in response to exposure with free radicals or other DNA damaging agents. Nicotinamide, a B vitamin, exerts its beta-cell protective action primarily via its ability to block excessive PARP activity. We show here that the isoquinolinone derivative PD128763, a specific PARP inhibitor, provides protection from cell death in islet cells exposed in vitro to nitric oxide or oxygen radical generating compounds or to the beta-cell toxin streptozotocin, at concentrations 100 times less than required for nicotinamide. Furthermore, while the protective action of nicotinamide is rapidly lost after washing of islet cells, the effects of PD128763 are more long lasting. Both compounds had little capacity to rescue damaged islet cells from subsequent lysis. We conclude that the isoquinolinone derivative PD128763 is superior to nicotinamide in enhancing the resistance of beta-cells towards inflammatory attacks.

Human type 1 diabetes results from the selective destruction of insulin-producing pancreatic beta cells during islet inflammation. Cytokines and reactive radicals released during this process contribute to beta-cell death. Here we show that mice with a disrupted gene coding for poly (ADP-ribose) polymerase (PARP-/- mice) are completely resistant to the development of diabetes induced by the beta-cell toxin streptozocin. The mice remained normoglycemic and maintained normal levels of total pancreatic insulin content and normal islet ultrastructure. Cultivated PARP-/- islet cells resisted streptozocin-induced lysis and maintained intracellular NAD+ levels. Our results identify NAD+ depletion caused by PARP activation as the dominant metabolic event in islet-cell destruction, and provide information for the development of strategies to prevent the progression or manifestation of the disease in individuals at risk of developing type 1 diabetes.

Superantigens have been implicated in the pathogenesis of type I diabetes and other immune-mediated diseases. We therefore tested the hypothesis of an abnormal reactivity of the immune system toward bacterial superantigens during the prediabetic phase. For this purpose, splenocytes from NOD (H-2g7) mice were exposed to two well-characterized superantigens: Staphylococcal aureus enterotoxin-B (SEB) and toxic shock syndrome toxin-1 (TSST-1). Cells from BALB/c (H-2d) and C57BL/6 (H-2b) mice as well as those from NON (H-2non) and NOR (H-2g7) mice were used as controls. After 72 h of co-culture with the superantigens or the mitogen concanavalin A (Con A), proliferative response and mitochondrial activity were determined. In the culture supernatants, the cytokines gamma-interferon (IFN-gamma) and interleukin 10 (IL-10) were measured. Striking similarities between NOD cells and major histocompatiblity complex (MHC)-identical NOR cells could be observed with regard to a low proliferative and mitochondrial response to SEB, accompanied by a normal response to TSST-1 and Con A, respectively. In addition, only NOD and NOR spleen cells were low producers of the T-helper 1 (Th1) cytokine IFN-gamma in response to SEB. Conversely, abnormally high IFN-gamma levels were induced by TSST-1 in NOD and NOR spleen cells. The cytokine response to Con A was also biased toward IFN-gamma in both NOD and NOR. Since IFN-gamma and IL-10 are crucial disease-promoting or -protecting mediators in prediabetic NOD mice, superantigens may affect pathogenesis by acting on the Th1/Th2 cytokine balance. The low responder status toward SEB in NOD spleen cells may be of pathogenetic relevance in view of recent findings that the insulin B-chain also interacts with the SEB binding site on MHC class II molecules. In conclusion, we show here that immune cells from mice with a diabetes-associated MHC type respond differently to common environmental superantigens than do immune cells from control strains.

In islet cells isolated from normal outbred Wistar rats, the known high vulnerability of islet cells toward oxygen radicals or nitric oxide can be abolished by inducing a stress response, such as by heat shock. We show here that islet cells from diabetes-prone BB rats are unable to mount such a protective response. Islet cells from diabetes-prone BB rats without recognizable insulitis were heat stressed. Subsequently, cells were exposed to nitric oxide, to oxygen radicals, or to the β-cell toxin streptozotocin. While prior heat shock substantially increased the survival of toxin-treated Wistar rat islet cells, no protective stress response was noted for islet cells from diabetes-prone BB rats. Islet cells from diabetes-resistant BB rats were protected by heat stress to the same extent as Wistar rats. A survey of four additional major histocompatibility complex (MHC)-disparate rat strains confirmed the existence of a low and high responder type to stress. Parallel analysis of heat shock protein (hsp)70 induction by Western blot showed a low and high hsp70 response phenotype. A high hsp70 response coincided with a protective stress response. The presence (or absence) of a protective stress response correlated with the preservation (or loss) of intracellular NAD+ in toxin-treated islet cells. The lack of a protective stress response in islet cells from diabetes-prone BB rats, but not in diabetes-resistant BB rats, may promote β-cell lysis and autoantigen release, and hence could be important for initiation or propagation of the disease process.

In the present study we investigated the effect of metabolic activation on the susceptibility of isolated rat pancreatic islet cells to the alkylating beta-cell toxin streptozocin (SZ), reactive oxygen intermediates (ROI), and nitric oxide (NO). The latter two represent physiologically occurring mediators involved in the autoimmune destruction of islet cells. ROI were generated by the enzyme xanthine oxidase, and NO was released from sodium nitroprusside. During 18 h of culture at a physiological glucose concentration (5 mmol/L), 75% of the islet cells were lysed by SZ, 81% by ROI, and 74% by NO, as determined by the trypan blue exclusion assay. Increasing concentrations of glucose or the nonnutrient stimulators theophylline and glibenclamide dose-dependently reduced SZ- and ROI-mediated islet cell lysis. In the presence of 29 mmol/L glucose, 5.5 mmol/L theophylline, or 10 micrograms/mL glibenclamide, SZ-induced lysis was reduced to 15%, 22%, or 15%, and ROI-induced lysis was reduced to 20%, 34%, or 15%, respectively. In contrast, stimulation by glucose, theophylline, or glibenclamide did not improve resistance against NO. The protection against SZ and ROI was associated with preserved mitochondrial activity, as determined by the ability of the islet cells to convert the tetrazolium salt 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide into its formazan. Elevation of the glucose concentration from 5.5 to 29 mmol/L increased the residual mitochondrial activity from 45% to 80% in SZ-exposed islet cells and from 21% to 78% in ROI-exposed cells. Conversely, the lack of protection against NO correlated with no preservation of mitochondrial activity in the presence of high concentrations of glucose, theophylline, or glibenclamide. In conclusion, our results show that metabolic stress does not render islet cells more susceptible to inflammatory insults in vitro. Rather, an increased mitochondrial energy supply improves the resistance against SZ and ROI, whereas the toxicity of NO was independent of islet cell activity.

A single dose of cyclophosphamide (250 mg/kg) is known to synchronize and accelerate development of diabetes in non-obese diabetic mice. We have reported previously that cyclophosphamide treatment of 10-week-old female non-obese diabetic mice induces a shift from T-helper type 2 to T-helper type 1 activity in islet lesions. We now show that this shift in regulatory T-cell function is preceded by the expression of interleukin-12 in the islets as well as in the spleen. In the spleen macrophages were identified as the interleukin-12 expressing cell type. At the same time there was little induction of tumour necrosis factor alpha gene expression by macrophages. Since interleukin-12 is well known to drive T-helper cell type 1 responses we assume that interleukin-12 released by macrophages mediates the accelerating effect of cyclophosphamide on islet inflammation in non-obese diabetic mice. mRNA expression of the p40 chain of interleukin-12 in response to cyclophosphamide was not seen in macrophages of Balb/c mice and thus represents an immune abnormality of non-obese diabetic mice favouring T-helper type 1 reactions.

Background/objectiveToll-like receptors (TLR) mediate the recognition of microbial constituents and stress-induced endogenous ligands by the immune system. They may also be involved in the maintenance or break down of tolerance against autologous antigens. The aim of our investigation was to study the consequence of TLR4 deficiency on the development of insulin-deficient diabetes in the NOD mouse.MethodsThe TLR4 defect of the C57BL/10ScN mouse was backcrossed onto the NOD background and the effect of TLR4 deficiency on diabetes development was analysed by in vivo and in vitro studies.ResultsCompared to animals with wildtype TLR4 expression (TLR4(+/+)), female NOD mice carrying a homozygous TLR4 defect (TLR4(-/-)), showed significant acceleration of diabetes development, with a younger age at diabetes onset (TLR4 (+/+) 177±22 d, TLR(-/-): 118±21 d; pConclusionsOur findings demonstrate that TLR4 deficiency results in an acceleration of diabetes development and immune cell infiltration of islets in NOD mice. We conclude that TLR4 is involved in the progression of the insulitis process thereby controlling the development of insulin-deficient diabetes in NOD mice.

Earlier preclinical and clinical trials indicate that fusidic acid, a triterpenoid compound originally described as an antimicrobial drug may protect islet beta cells from destruction in type I (insulin-dependent) diabetes mellitus. Since nitric oxide appears to be an important mediator of inflammatory islet cell death we analyzed whether fusidic acid interferes with nitric oxide production or action. We report here that fusidic acid dose-dependently inhibits lysis of isolated islet cells by activated macrophages, a process mediated by nitric oxide. In the presence of 100 microM fusidic acid macrophage-mediated islet cell lysis was reduced from 52.5 to 1.7% (P0.001). Fusidic acid only slightly affected macrophage function and did not inhibit the release of nitric oxide. We therefore tested whether fusidic acid suppresses nitric oxide toxicity in target cells. Isolated islet cells were exposed to the nitric oxide donor nitroprusside which led to DNA strand breaks and plasma membrane lysis. DNA strand breaks were reduced from 54.6 to 34.9% (P0.001) in the presence of 100 microM fusidic acid and cell lysis was reduced from 60.1 to 27.5% with 100 microM (P0.001). In the presence of 500 microM fusidic acid DNA strand breaks and cell lysis were reduced further to 27.1 and 10.7%, respectively (P0.001). No protection by fusidic acid was observed when cells were exposed to oxygen radicals or the alkylating beta cell toxin streptozotocin. The suppression of nitric oxide toxicity by fusidic acid was not due to its known inhibitory action on protein biosynthesis and thus represents a hitherto unknown activity of this drug.

In an animal model of type I diabetes, the non-obese diabetic (NOD) mouse, the influence of the antioxidant lipoic acid (LA) on the development of diabetes was investigated. Acceleration of diabetes development with cyclophosphamide (CY) resulted in 60% diabetic animals with severely infiltrated islets within 1–3 weeks. Daily administration of lipoic acid for 20 or 30 days around cyclophosphamide treatment suppressed the incidence of diabetes to 30% (P

Despite extensive studies on streptozotocin, alloxan and nitric oxide toxicity in pancreatic islets the mechanism of oxygen radical induced islet cell death has not been determined. The present study shows at the level of single cells that following exposure to oxygen radicals generated from xanthine oxidase DNA strand breaks occur in cell nuclei within 5-60 min and precede cell death by several hours. Similar kinetics were seen when treating islet cells with the alkylating agent streptozotocin. Immunofluorescence studies demonstrated the endogenous formation of ADP-ribose polymers in nearly all islet cell nuclei within minutes of treatment with xanthine oxidase, indicating activation of the enzyme poly(ADP-ribose) polymerase (PARP). Concomitantly, cellular NAD+ depletion was noted. Nicotinamide largely prevented NAD+ depletion and in parallel resulted in islet cell survival. These findings identify islet cell nuclear DNA as a primary target of oxygen radical toxicity and suggest related pathways of oxygen radical, nitric oxide and streptozotocin toxicity.

The modulation of tumor necrosis factor alpha-mediated cytotoxicity by gangliosides was analyzed. When cells of the TNF alpha-sensitive fibrosarcoma cell line L929 were incubated for 16 h with recombinant TNF alpha (156 or 312 pg/ml), they were lysed to 65.2% or 78.0%, respectively. The presence of a bovine brain ganglioside mixture (BBG, Cronassial) inhibited the TNF alpha-mediated lysis in a dose-dependent manner (IC50 approximately 1 mg/ml). Maximum inhibition was achieved with 2 mg/ml BBG (82.6% inhibition for 156 pg/ml TNF alpha and 88.5% inhibition for 312 pg/ml TNF alpha). In situ nick translation revealed that BBG (2 mg/ml) significantly reduces the number of cells with TNF alpha-induced DNA-strand breaks from 71.2% to 6.6% (P < 0.0001), indicating a protection against TNF alpha-mediated apoptosis. We conclude that BBG prevents the cytotoxic action of TNF alpha on tumor cells. The inhibitory effect may be due to an interference of gangliosides with intracellular signal transduction pathways, resulting in an inhibition of the activation of DNA-cleaving endonucleases.

NO has been identified recently as the prime islet-toxic product of inflammatory macrophages. The adverse effects of IL-1 on isolated islets also have been reported to involve NO. We now show that exposure of an islet cell suspension to the NO donor nitroprusside or to activated macrophages leads to DNA strand breaks. Macrophages did not induce DNA damage in the presence of the NO synthase inhibitor NG-methyl-L-arginine. DNA strand breaks were demonstrated at the level of single cells by a modified nick-translation procedure and confirmed by analysis of DNA fragmentation by gel electrophoresis. DNA strand breaks occurred within 1 h and preceded islet cell lysis. DNA damage could not be prevented by inhibitors of endogenous endonucleases. We conclude that islet cell DNA is an early target of NO action.

Macrophages from autoimmune diabetes prone BB rats were found to produce radical oxygen intermediates (ROI) at an enhanced rate when compared to diabetes resistant BB or normal Wistar rats. The release of ROI was determined by chemiluminescence using in parallel luminol and lucigenin as detector molecules. In diabetes prone BB rats the spontaneous release of ROI was upregulated in macrophages from different compartments, i.e. peritoneum and spleen. Also, maximal output of ROI after activation of macrophages either in vivo by injection of Corynebacterium parvum or in vitro by LPS and IFN was highest for cells from diabetes prone BB rats. This macrophage abnormality was seen in animals prior to recognizable islet inflammation and also was present at the level of macrophages grown in vitro from precursor cells of diabetes prone BB rats. Hypersecretion of oxygen radicals may contribute to Beta cell loss and diabetes development in BB rats.

Adipocytes play important roles in lipid metabolism but also in the control of inflammatory processes. Based on our previous findings of heat shock protein (Hsp) 60-induced activation of preadipocytes we investigated whether the capacity of heat shock protein 60 (Hsp60) to interact with adipocytes and to stimulate their proinflammatory activity is determined by the differentiation state of the cells. Hsp60 bound to adipocytes and stimulated the release of inflammatory mediators independent of their differentiation state. Hsp60-adipocyte interactions revealed basic characteristics of a receptor-mediated process. Our findings characterize Hsp60 binding and Hsp60-induced release of proinflammatory mediators as fundamental properties of adipocytes independent of their differentiation state.

SUMMARY Multiple low doses of streptozotocin arc known to induce immune-mediated insulin deficient diabetes and depression of immune reactivity. We show here that immune depression by streptozotocin is not general but that some parts of the immune system are stimulated. Spleen cells from streptozotocin-treated mice showed enhanced cytotoxicity against syngeneic islet cells and various tumour cells including insulinoma cells. Several cell types served as effector cells, including macrophages, asialo GM1+ and Lyt-2+ lymphocytes. The increased cytotoxic activity towards islet cells was mostly due to macrophages and to non-asialo GM1+ and non-Lyt-2+ lymphocytes. A higher activation state of macrophages in low dose streptozotocin-treated mice was demonstrated by measurements of superoxide anion release. We conclude that multiple low doses of streptozotocin stimulate ‘natural cytotoxicity’, i.e. the non-MHC restricted cytotoxic activity of macrophages, T cells and natural killer lymphocytes.

IL-1 and TNF alpha are assumed to be major mediators of islet cell destruction during the pathogenesis of type 1 diabetes. Here we show by neutralization of the two cytokines with excess antibody that IL-1 and TNF alpha do not contribute to the cytotoxic activity of activated macrophages towards isolated islet cells. However, islet cells can be protected from lysis by depleting the culture medium of L-arginine or by adding the antagonist NG-monomethyl-L-arginine, both of which inhibit the generation of nitric oxide by activated macrophages. These results indicate a role of nitric oxide or its equivalent, the endothelium-derived relaxing factor in the development of type 1 diabetes. This is the first report showing that nitric oxide may damage normal cells and thus may be a hitherto unrecognized pathogenetic factor in tissue inflammation and autoimmune disence.

Human heat shock protein 60 (HSP60) has been shown to bind to the surface of innate immune cells and to elicit a proinflammatory response. In this study we demonstrate that the macrophage stimulatory property of recombinant human HSP60 is tightly linked to the HSP60 molecule and is lost after protease treatment. However, inhibition of macrophage stimulation was reached by the LPS-binding peptide magainin II amide. Indeed, HSP60 specifically bound [3H]LPS. [3H]LPS binding to HSP60 was saturable and competable by the unlabeled ligand. To identify the epitope region of the HSP60 molecule responsible for specific LPS binding, we analyzed the effect of several anti-HSP60 mAbs on HSP60-induced production of inflammatory mediators from macrophages. We identified only one mAb, clone 4B9/89, which blocked the macrophage stimulatory activity of the chaperone. The epitope specificity of this mAb points to the region aa 335–366 of HSP60. Clone 4B9/89 also strongly inhibited [3H]LPS binding to HSP60. A more detailed analysis was performed by screening with selected overlapping 20-mer peptides of the HSP60 sequence, covering the region aa 331–380. Only one peptide blocked LPS binding to HSP60, thereby restricting the potential LPS-binding region to aa 351–370 of HSP60. Finally, analysis of selected 15-mer peptides and a 13-mer peptide of the HSP60 sequence revealed that most of the LPS-binding region was accounted for by aa 354–365 of HSP60, with the motif LKGK being critical for binding. Our studies identified a defined region of HSP60 involved in LPS binding, thereby implicating a physiological role of human HSP60 as LPS-binding protein.

Previous studies have shown that human heat shock protein (hsp) 60 elicits a strong proinflammatory response in cells of the innate immune system with CD14, Toll-like receptor (TLR) 2, and TLR4 as mediators of signaling, but probably not of binding. In the present study, we directly demonstrate binding of hsp60 to the macrophage surface and find the binding receptor for hsp60 different from the previously described common receptor for several other heat shock proteins, including hsp70, hsp90, and gp96. Fluorescence-labeled human hsp60 bound to cell surfaces of the murine macrophage lines J774 A.1 and RAW264.7 and to mouse bone marrow-derived macrophages. By flow cytometry, we could demonstrate for the first time that hsp60 binding to macrophages occurred at submicromolar concentrations, is saturable, and can be competed by unlabeled hsp60, but not by unrelated proteins, thus confirming the classic characteristics of specific ligand-receptor interactions. Binding of hsp60 at 4°C was followed by endocytosis at 37°C. Hsp60 binding to macrophages could not be competed by excess hsp70, hsp90, or gp96, all of which share the α2-macroglobulin receptor as binding site. Hsp60 binding occurred in the absence of surface TLR4. However, no cytokine response was induced by hsp60 in TLR4-deficient macrophages. We conclude that hsp60 binds to a stereo-specific receptor on macrophages, and that different surface molecules are engaged in binding and signal transduction. Furthermore, the binding site for hsp60 is separate from the common receptor for hsp70, hsp90, and gp96, which suggests an independent role of hsp60 as danger Ag and in immunoregulation.

Heat shock protein (hsp) 70 protects cells against stress by means of its ability to chaperone denatured proteins and to modulate stress-activated signaling pathways. Because inflammatory processes are often accompanied by hsp expression and because stress and cytokines share several signaling pathways, we investigated the possibility that hsp70 might modulate the cellular response to cytokines. We found that stable cell clones overexpressing hsp70, or cells shortly after transfection with hsp70, produced 2 times more nitric oxide and inducible nitric-oxide synthase (iNOS) protein and mRNA in response to cytokines than control cells expressing undetectable amounts of hsp70. Since mitogen-activated protein kinases participate in the activation of iNOS by cytokines, we investigated whether hsp70 affected the activation of these signaling pathways. hsp70 overexpression led to a specific enhancement of the activation of the p38 pathway by cytokines, producing little or no effect on the activation of extracellular signal-regulated kinase or Jun N-terminal kinase. Blocking p38 activity with SB203580 totally abolished the enhancing effect of hsp70 on cytokine-induced endogenous iNOS mRNA accumulation or transcription of an iNOS promoter-driven luciferase gene, while having little effect on the cytokine response observed in control cells. We conclude that the p38 pathway acts as an enhancing factor in the activation of iNOS by cytokines and that hsp70 can modulate the cellular response to cytokines by acting on signaling elements upstream of p38.

Human beta cells exhibit increased resistance against nitric oxide (NO) radicals as compared with rodent islet cells. Here we tested whether endogenous heat shock protein 70 (hsp70) accounts for the resistance of human cells. Stable transfection of the human beta cell line CM with an antisense hsp70 mRNA-expressing plasmid (ashsp70) caused selective suppression (>95%) of spontaneously expressed hsp70 but not of hsc70 or GRP75 protein. ashsp70 transfection abolished the resistance of CM cells to the NO donors (Z)-1- (2-(2-aminoethyl)-N-(2-ammonioethyl)amino)diazen-1-ium -1,2-diolate and sodium nitroprusside and increased the proportions of necrotic cells 3-5-fold (p < 0.05) and of apoptotic cells about 2-fold (p < 0.01). Re-induction of hsp70 expression by heat shock re-established resistance to NO toxicity. hsp70 did not exert its protective effect at the level of membrane lipid integrity because radical induced lipid peroxidation appeared independent of hsp70 expression. However, after NO exposure only hsp70-deficient cells showed significantly decreased mitochondrial activity, by 40-80% (p < 0.01). These results suggest a key role of hsp70 in the natural resistance of human beta cells against NO induced injury, by preserving mitochondrial function. These findings provide important implications for the development of beta cell protective strategies in type 1 diabetes and islet transplantation.

Human heat shock protein 60 (hsp60) elicits a potent proinflammatory response in cells of the innate immune system and therefore has been proposed as a danger signal of stressed or damaged cells. We report here that macrophages of C3H/HeJ mice, carrying a mutant Toll-like-receptor (Tlr) 4 are nonresponsive to hsp60. Both the induction of TNF-α and NO formation were found dependent on a functional Tlr4 whereas stimulation of macrophages by CpG DNA was Tlr4 independent. We conclude that Tlr4 mediates hsp60 signaling. This is the first report of a putative endogenous ligand of the Tlr4 complex.

The development of type 1 diabetes in animal models is T cell and macrophage dependent. Islet inflammation begins as peripheral benign Th2 type insulitis and progresses to destructive Th1 type insulitis, which is driven by the innate immune system via secretion of IL-12 and IL-18. We now report that daily application of IL-18 to diabetes-prone female nonobese diabetic mice, starting at 10 wk of age, suppresses diabetes development (p < 0.001, 65% in sham-treated animals vs 33% in IL-18-treated animals by 140 days of age). In IL-18-treated animals, we detected significantly lower intraislet infiltration (p < 0.05) and concomitantly an impaired progression from Th2 insulitis to Th1-dependent insulitis, as evidenced from IFN-γ and IL-10 mRNA levels in tissue. The deficient progression was probably due to lesser mRNA expression of the Th1 driving cytokines IL-12 and IL-18 by the innate immune system (p < 0.05). Furthermore, the mRNA expression of inducible NO synthase, a marker of destructive insulitis, was also not up-regulated in the IL-18-treated group. IL-18 did not exert its effect at the levels of islet cells. Cultivation of islets with IL-18 affected NO production or mitochondrial activity and did not protect from the toxicity mediated by IL-1β, TNF-α, and IFN-γ. In conclusion, we show for the first time that administration of IL-18, a mediator of the innate immune system, suppresses autoimmune diabetes in nonobese diabetic mice by targeting the Th1/Th2 balance of inflammatory immune reactivity in the pancreas.

When cultures of pancreatic islet cells are exposed to the nitric oxide donor sodium nitroprusside, to enzymatically generated reactive oxygen intermediates or to streptozotocin cell lysis occurs after 4-12 h. We report here that a heat shock at 43 degrees C for 90 min reduces cell lysis from nitric oxide (0.45 mM sodium nitroprusside) by 70%, from reactive oxygen intermediates (12 mU xanthine oxidase and 0.05 mM hypoxanthine) by 80% and from streptozotocin (1.5 mM) by 90%. Heat shock induced resistance was observed immediately after termination of the 90 min culture at 43 degrees C and correlated with enhanced expression of hsp70. The occurrence of DNA strand breaks, a major early consequence of nitric oxide, reactive oxygen intermediates, or streptozotocin action, was not suppressed by heat shock treatment. However, the depletion of NAD+, the major cause of radical induced islet cell death, was suppressed after heat shock (P < 0.01). We conclude that pancreatic islet cells can rapidly activate defence mechanisms against nitric oxide, reactive oxygen intermediates and streptozotocin by culture at 43 degrees C. Islet cell survival is due to the prevention of lethal NAD+ depletion during DNA repair, probably by slowing down poly(ADP-ribose)polymerase activation.

Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) is an early response of cells exposed to DNA-damaging compounds such as nitric oxide (NO) or reactive oxygen intermediates (ROI). Excessive poly-(ADP-ribose) formation by PARP has been assumed to deplete cellular NAD+ pools and to induce the death of several cell types, including the loss of insulin-producing islet cells in type I diabetes. In the present study we used cells from mice with a disrupted and thus inactivated PARP gene to provide direct evidence for a causal relationship between PARP activation, NAD+ depletion, and cell death. We found that mutant islet cells do not show NAD+ depletion after exposure to DNA-damaging radicals and are more resistant to the toxicity of both NO and ROI. These findings directly prove that PARP activation is responsible for most of the loss of NAD+ following such treatment. The ADP-ribosylation inhibitor 3-aminobenzamide partially protected islet cells with intact PARP gene but not mutant cells from lysis following either NO or ROI treatment. Hence the protective action of 3-aminobenzamide must be due to inhibition of PARP and does not result from its other pharmacological properties such as oxygen radical scavenging. Finally, by the use of mutant cells an alternative pathway of cell death was discovered which does not require PARP activation and NAD+ depletion. In conclusion, the data prove the causal relationship of PARP activation and subsequent islet cell death and demonstrate the existence of an alternative pathway of cell death independent of PARP activation and NAD+ depletion.

In activated macrophages the inducible form of the enzyme nitric oxide (NO) synthase generates high amounts of the toxic mediator NO. After 20 h of treatment with LPS rat peritoneal macrophages release 12-16 nmol NO2-/10(5) cells which is detectable in the culture supernatant by the Griess reaction as a measure of NO formation. The addition of aminoguanidine (1 mM), a preferential inhibitor of the inducible NO-synthase, completely abolished NO2-accumulation. Incubation with indomethacin or acetyl-salicylic acid, preferential inhibitors of the cyclooxygenase pathway of the arachidonic acid metabolism, did not influence NO2- levels. Nordihydro-guaiaretic acid (50 microM), a preferential inhibitor of the lipoxygenase pathway, caused strong reduction of NO2- accumulation to 1.9 +/- 0.3 nmol/200 microliter. Simultaneous inhibition of cyclo- and lipoxygenase by BW755c resulted in an intermediate effect (7.3 +/- 1.1 nmol/200 microliter NO2-). These results show that the induction of NO production in activated macrophages is regulated by products of the lipoxygenase-pathway of the arachidonic acid metabolism.

SUMMARY Islet cells cocultured with activated macrophages are lysed within 15 h in vitro. We showed previously that nitric oxide generated by macrophages is a major mediator of islet cell death. We have now probed several pathways to interfere with the chain of events leading to islet cell death. Scavenging of extracellular oxygen radicals by superoxide dismutase and catalase did not improve islet cell survival. Scavenging of extra- and intracellular oxygen radicals by two potent substances, citiolone and dimethyl-thiourea, also did not reduce islet cell lysis, while a lipid-soluble scavenger, probucol, provided partial protection. These findings argue against a synergistic action of nitric oxide and oxygen radicals in islet cell toxicity. The inhibition of poly(ADP-ribose)polymerase by 3-aminobenzamide significantly improved islet cell survival. Selective inhibitors of cyclooxygenase, such as indomethacin or acetylsalicylic acid, did not improve islet cell survival. Full protection was seen in the presence of NDGA, an inhibitor of lipoxygenase, and partial suppression was caused by BW755c, an inhibitor of both lipoxygenase and cyclooxygenase. We conclude that inflammatory islet cell death caused by activated macrophages involves the activation of arachidonic acid metabolism and of poly(ADP-ribose)polymerase, but that scavenging of oxygen free radicals provides little protection from lysis.

In vitro models of pancreatic islet cell inflammation are the lysis of isolated islet cells by activated macrophages or by oxygen radicals released by the endothelial enzyme xanthine oxidase. Dihydrolipoic acid protected islet cells in both systems by different modes of action. Macrophage cytotoxicity towards islet cells, which is nitric-oxide-mediated, was suppressed by 2 h of preincubation of macrophages with lipoic acid. Similarly, 2 h of preincubation sufficed to protect islet cells against enzymatically produced oxygen radicals. Dihydrolipoic acid was found by chemiluminescence assay to scavenge directly such radicals. In macrophages dihydrolipoic acid suppressed the production of nitrite as a measure of nitric oxide release. These results suggest that dihydrolipoic acid is an anti-inflammatory agent which at the same time interferes with nitric oxide release from inflammatory macrophages and protects target cells from oxygen radical attack.

The endothelium-associated enzyme xanthine oxidase is known to generate reactive oxygen intermediates which may damage the surrounding tissue. We investigated whether reactive oxygen intermediates released by xanthine oxidase exert a toxic effect on isolated rat islet cells. The xanthine oxidase (25 mU/ml)/hypoxanthine (0.5 mmol/1) system released reactive oxygen intermediates in vitro as detected by luminol in a chemiluminescence analysing system. The addition of nicotinamide inhibited the release of reactive oxygen intermediates in a dose-dependent manner (50 % inhibition at 20 mmol/1). Exposure of islet cells to enzyme generated reactive oxygen intermediates caused lysis of 39% of the cells within 15 h. Monitoring the mitochondrial function of islet cells by the conversion of tetrazolium bromide to its formazan product revealed a significant reduction of the respiratory activity down to 51 % of that of the controls by 30 min after the initiation of the xanthine oxidase reaction. Mitochondrial dysfunction preceded plasma membrane damage. The addition of nicotinamide, a radical scavenger and inhibitor of the DNA repair enzyme poly(ADP-ribose) synthetase protected the islet cells from lysis and partially preserved their mitochondrial activity in the presence of reactive oxygen intermediates. We conclude that activation of the endothelial enzyme xanthine oxidase, known to be induced by mediators of immune cells or by episodes of ischaemia and reperfusion causes islet cell damage with subsequent cell death in early phases of pancreatic islet cell destruction.

Previous studies have indicated that nitric oxide is involved in the lysis of pancreatic islet cells by inflammatory macrophages. Here we show that the incubation of islet cells with chemical NO-donors leads to cell lysis in a concentration and time dependent way. Islet cell death could be prevented by nicotinamide and 3-aminobenzamide, which are known to inhibit ADP- ribosylation, while several scavengers of oxygen radicals, N-acetylcysteine, dihydrolipoic acid, dimethylthiourea and citiolone, provided no protection.

A number of observations indicate an essential role of macrophage activity in the development of hyperglycemia in animal models of Type I diabetes. Administration of macrophage-toxic silica particles prevents spontaneous diabetes development in BB rats or NOD mice. The same result was noted in the low-dose streptozotocin-induced diabetes model in mice. Macrophages appear to be the first immune cells infiltrating islets during early insulitis. Macrophages in inflamed islets of BB rats bear the ED1 marker, whereas resident islet macrophages are ED2-positive. In vitro, ED1-positive macrophages were found to lyse pancreatic islet cells to a similar degree to various tumor cells but not normal thyrocytes. Macrophage-mediated lysis of islet cells was inhibited in the presence of 10-100 mM nicotinamide.

Low-dose streptozotocin treatment in C57B1/6J mice causes development of hyperglycemia within two weeks. Diabetes development is due to the specific loss of beta cells from pancreatic islets which can be blocked by immunosuppressive treatment. The role of vascular permeability in pancreatic islet destruction was studied by administration of methysergide or pargyline in addition to low-dose streptozotocin. Both drugs impair serotonin-enhanced vascular permeability. Administration of methysergide or pargyline during the first 11 days following streptozotocin treatment caused substantial suppression of diabetes development. These observations suggest a role of enhanced vascular permeability in immune-mediated beta cell destruction.

In a study of seven different hsp60 species, we found that all mammalian and microbial proteins shared the property of eliciting an inflammatory response in mouse macrophages. In all cases, TNFalpha production was induced by 0.1 microM concentrations of hsp60. However, the different hsp60 preparations did not compete for the same binding site. The binding of fluorescence-labeled human hsp60 was inhibited by excess unlabeled human, rat or mouse hsp60, but not hamster, Escherichia coli, Chlamydia pneumoniae or Mycobacterium bovis hsp60. We conclude that phylogenetically separate hsp60 species interact with innate immune cells via different recognition pathways.

We show here that preincubation of pancreatic islet cells with alpha-tocopherol significantly improves their resistance to toxic doses of nitric oxide (NO). No protection was afforded by other antioxidants such as vitamin C or glutathione-monoethyl ester. The pathway of NO induced islet cell death involves DNA damage and excessive activation of poly(ADP-ribose)polymerase leading to irreversible depletion of intracellular NAD+. alpha-Tocopherol was found to interfere at early steps of this pathway, by preventing the occurrence of DNA strand breaks. This indicates that alpha-tocopherol directly interacts with NO or its reactive intermediates. We conclude that alpha-tocopherol is not only part of the cellular defence system against oxygen radicals but also protects eukaryotic cells from NO toxicity.

Heat stress is known to render rat islet cells resistant against the toxic effects of nitric oxide, reactive oxygen intermediates and the islet cell toxin streptozotocin. We report here for the first time that protection against nitric oxide is mediated by the major heat shock protein, hsp70, even in the absence of heat stress. The human hsp70 gene was stably transfected into the rat insulinoma cell line RINm5F. Constitutive expression of hsp70 caused protection from NO-induced cell lysis which was of the same extent as seen after heat stressing cells. Our results identify hsp70 as a defence molecule against nitric oxide.