Aims/hypothesis. Insulin-producing beta cells are destroyed by oxidative and nitrosative stress during the pathogenesis of Type I (insulin-dependent) diabetes mellitus. These cells are more sensitive than others due to their deficiency of well known antioxidant enzymes like superoxide dismutase, glutathione peroxidase and catalase. However the peroxiredoxins discovered in the past decade form a large family of highly conserved thioredoxin-dependent peroxide reductases, which are present in most tissues. We investigated whether peroxiredoxins I and II are present in pancreatic beta cells and if they are inducible by oxidative and nitrosative stress. Methods. To detect these enzymes in insulin-producing beta cells we used semiquantitative RT-PCR, western blots and immunohistochemistry. The expression of peroxiredoxins I and II was analysed after treatment with cytokines, hydrogen peroxide, alloxan or streptozotocin in the rat insulinoma cells INS-1 using RT-PCR and western blots. Results. We show that peroxiredoxins I and II are present in the cytoplasm of pancreatic islet cells as well as in insulinoma cell lines βTC6-F7 and INS-1. Peroxiredoxins I and II were up-regulated by all stress agents used. Conclusion/interpretation. Beta cells, undersupplied with well characterized antioxidant enzymes, possess an additional antioxidant system which is inducible by oxidative as well as nitrosative stress. [Diabetologia (2002) 45:867-876]

Various pancreatic β-cell stressors including cytokines and saturated fatty acids are known to induce oxidative stress, which results in metabolic disturbances and a reduction in insulin secretion. However, the key mechanisms underlying dysfunction are unknown. We investigated the effects of prolonged exposure (24 h) to pro-inflammatory cytokines, H(2)O(2) or PA (palmitic acid) on β-cell insulin secretion, ATP, the NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase) component p47phox and iNOS (inducible nitric oxide synthase) levels using primary mouse islets or clonal rat BRIN-BD11 β-cells. Addition of a pro-inflammatory cytokine mixture [IL-1β (interleukin-1β), TNF-α (tumour necrosis factor-α) and IFN-γ (interferon-γ)] or H(2)O(2) (at sub-lethal concentrations) inhibited chronic (24 h) levels of insulin release by at least 50% (from islets and BRIN-BD11 cells), while addition of the saturated fatty acid palmitate inhibited acute (20 min) stimulated levels of insulin release from mouse islets. H(2)O(2) decreased ATP levels in the cell line, but elevated p47phox and iNOS levels as did cytokine addition. Similar effects were observed in mouse islets with respect to elevation of p47phox and iNOS levels. Addition of antioxidants SOD (superoxide dismutase), Cat (catalase) and NAC (N-acetylcysteine) attenuated H(2)O(2) or the saturated fatty acid palmitate-dependent effects, but not cytokine-induced dysfunction. However, specific chemical inhibitors of NADPH oxidase and/or iNOS appear to significantly attenuate the effects of cytokines, H(2)O(2) or fatty acids in islets. While pro-inflammatory cytokines are known to increase p47phox and iNOS levels in β-cells, we now report that H(2)O(2) can increase levels of the latter two proteins, suggesting a key role for positive-feedback redox sensitive regulation of β-cell dysfunction.

Type 1 diabetes mellitus is characterized by a progressive autoimmune destruction of insulin-producing b cells. Macrophages and T lymphocytes release cytokines, which induce the synthesis of oxygen and nitrogen radicals in the pancreatic islets. The resulting cellular and mitochondrial damage promotes b cell death. b cells are very sensitive to the autoimmune free radical-dependent attack due to their low content of antioxidant enzymes such as glutathione peroxidase and catalase. A focal point of b cell protection should be the control of the mitochondrial redox status, which will result in the preservation of metabolic stimulus-secretion coupling. For this reason, there is a considerable interest in the mitochondrial peroxiredoxin III (PRX III), a thioredoxindependent peroxide reductase, which was shown to be able to protect against both oxidative and nitrosative stress.Using the Tet-On-system, we generated stably transfected rat insulinoma cells over-or under-expressing PRX III in a doxycyclin-dependent manner to analyze the effect of increased or decreased amounts of cellular PRX III, following treatment with several stressors. We provide evidence that PRX III protects pancreatic b cells from cell stress induced by accumulation of hydrogen peroxide, or the induction of inducible nitric oxide synthase or caspase-9 and -3 by pro-inflammatory cytokines or streptozotocin. Basal insulin secretion was markedly decreased in cells expressing lower levels of PRX III. We suggest PRX III may be a suitable target for promoting deceleration or even prevention of stress-associated apoptosis in pancreatic b cells and the manifestation of insulin-dependent diabetes mellitus.

No abstract

Geeignete Haftgruppen in der chemoselektiven Afinitatschromatographie sollten eine gunstige Gleichgewichtslage und eine sehr schnelle Gleichgewichtseinstellung mit den Substraten zeigen. Aldehyde und Ketone unterschiedlicher Struktur (1 -16), von denen man 3-7 und 10 erstmalig herstellte, wurden auf ihre Eignung zur Haftung von Monoalkoholen uber eine kovalente Acetalbildung untersucht. Als besonders gunstig erwiesen sich cyclische Halbacetale, die bei saurer Katalyse mit Monoalkoholen eine schnelle und reversible Vollacetalbildung zeigten. Von diesen waren 1,3-Dihydro-l -isobenzofuranol (1 a), 1,3-Dihydro-7-methoxy-I-isobenzofuranol(5a) und 2H-Chromen-2-01(10) mit einer sehr schnellen Kinetik besonders geeignet. Acetale verschiedener Alkohole wurden ebenfalls dargestellt und deren Hydrolysereaktion untersucht. Zur Darstellung von entsprechenden Polymeren wurde das polymerisierbare Derivat 20 synthetisiert. On the Chemistry of Binding Sites, VI') On the Suitability of Various Aldehydes and Ketones as Binding Sites for MonoalcoholsSuitable binding sites in the chemoselective affinity chromatography should posses a favourable state and a very fast adjustment of the equilibrium with substrates. Aldehydes and ketones of various structure (1-16), from which 3-7 and 10 were prepared for the first time, were investigated for their suitability to bind monoalcohols via a covalent acetal bond.Especially advantageous were cyclic hemiacetals which showed on acidic catalysis with monoalcohols a fast and reversible formation of acetals. Out of these 1,3-dihydro-l-isobenzofuranol (la), 1,3-dihydro-7-methoxy-l-isobenzofuranol (5a), and 2H-chromen-2-01 (10) with very fast kinetics seemed to be especially suitable. Acetals of different alcohols were prepared as well and their hydrolytic behaviour was studied. For the preparation of corresponding polymers the polymerizable derivative 20 was synthesized.Beim Aufbau von Enzymmodellen2) wie auch zur chemoselektiven Afinitatschromatographie 's3p4) werden selektive Haftgruppen zur schnellen und reversiblen Bindung von Substraten benotigt. Meist werden hierfur elektrostatische oder hydrophobe Wechselwirkungen ausgenutzt 3), Wegen der haufig recht langsamen Gleichgewichtseinstellung werden kovalente Wechselwirkungen seltener einge~e t z t~.~) .Besonders bewahrt hat sich hier die Boronsauregruppe zur Bindung von Diolen',''.

The safety of monotertiarybutylhydroquinone as an oil‐soluble food grade antioxidant was evaluated in acute studies with rats and dogs, in subacute feedings in rats, in rat reproductive efficiency and placental transfer studies, and in subacute feedings with monotertiarybutylhydroquinone heated in vegetable oil. In lifetime feedings in rats and 2 year feedings in dogs, wt gain, feed consumption, behavior, mortality, hemograms, clinical chemistries, gross, microscopic, and electron microscopy were evaluated. There were no toxic or untoward effects. Monotertiarybutylhydroquinone was handled similarly by rats, dogs, and humans. Rats eliminated single oral 0.1–0.4 g/kg doses mostly in the urine in 3–4 days as the 4‐0‐sulfate (57–80%) and the 4‐0‐glucuronide (4%) and with 4–12% unchanged. 2,3,5,6‐14C‐Monotertiarybutylhydroquinone was eliminated similarly with <0.1% as14CO2 and <0.2% remaining in the animal after 4 days. Oral 0.1 g/kg doses to dogs gave a somewhat higher glucuronide contribution. The elimination pattern was little altered in long term feedings. Humans eliminated 0.002 g/kg single doses (high fat vehicle) almost completely in the urine in 2–3 days, with <0.1% unchanged, 73–88% as the 4‐0‐sulfate, and 15–22% as the 0‐glucuronide. Monotertiarybutylhydroquinone residues in most tissues of long term animals were below lower detection limits or negligible. Monotertiarybutylhydroquinone did not induce liver microsomal mixed function oxidases in short and long term rat and dog feedings. The feeding studies and comparative biochemical studies showed monotertiarybutylhydroquinone to be safe for its intended use.

No abstract

The lysosomal hydrolase beta-glucuronidase (beta-gluc) can be used for the bioactivation of non-toxic glucuronide prodrugs of anticancer agents. The enzyme is present at high levels in many tumours and hence may lead to an enhanced drug targeting by tumour-selective release of the active anticancer drug. Individual expression and regulation of this enzyme is one factor modulating the bioactivation of glucuronide prodrugs. Nevertheless, in contrast to murine beta-gluc, which is inducible by androgens, the human enzyme has been regarded as an unregulated housekeeping gene due to a lacking TATA box and high G+C contents within the putative promotor sequence. Despite these facts, we were able to demonstrate downregulation of human beta-gluc expression by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin in the human hepatoma cell line HepG2. However, cis-acting elements responsible for this regulation have not yet been identified. We therefore characterised the 5'-untranslated region of the human beta-gluc gene using transient transfection assays with promotor-luciferase constructs in HepG2 cells and cloned fragments between 3,770 bp and 107 bp. A23187 reduced the beta-gluc promotor activity. This effect disappeared using fragments smaller than 356 bp. Using site-directed in vitro mutagenesis and gel-electrophoretic-mobility shift assays, we found evidence of an involvement of transcription factor activating protein-2 (AP-2) binding sites on the regulation of human beta-glucuronidase by A23187. Our studies provide a basis for the understanding of the transcriptional regulation of the human beta-glucuronidase gene and could be useful for the optimisation of glucuronide prodrug therapy.