L. A. Håkan Borg

A spectrophotometric method for determination of catalase activity in small tissue samples

A simple and rapid method for determination of catalase activity in small tissue samples is described. Using a new approach, we have exploited the peroxidatic function of catalase for the determination of enzyme activity. The method was based on the reaction of the enzyme with methanol in the presence of an optimal concentration of hydrogen peroxide. The formaldehyde produced was measured spectrophotometrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald) as a chromogen. With this method, a detection limit of 12.5 ng of purified catalase from bovine liver was possible, and it was successfully applied to microgram amounts of mouse liver and pancreatic islet homogenates. The catalase activity in liver was about 50 times higher than that in pancreatic islets.

Diabetes and Embryonic Malformations: Role of Substrate-Induced Free-Oxygen Radical Production for Dysmorphogenesis in Cultured Rat Embryos

The aim of this study was to evaluate the role of free-oxygen radicals in the embryonic maldevelopment of diabetic pregnancy. Rat embryos cultured in vitro during early organogenesis showed growth retardation and severe malformations after exposure to 50 mM glucose, 3 mM PYR, 10 mM HBT, or 3 mM KIC. Combinations of 25 mM glucose, 2.5 mM HBT, and 1 mM KIC also elicited embryonic growth retardation and malformations. The deleterious effects on embryonic development by all agents were alleviated by addition of SOD to the culture media, which yielded increased enzyme activity in the embryos and their membranes. The endogenous SOD activity also increased in embryos subjected to a high concentration of glucose or PYR in the culture medium. Addition of the mitochondrial PYR transport inhibitor CHC to the culture media blocked the dysmorphogenesis caused by glucose and PYR, but was without effect on the teratogenic actions of HBT and KIC. These findings implicate the embryonic mitochondria as a likely site for enhanced substrate-induced production of free-oxygen radicals mediating the teratogenic effect of a diabetic environment. In particular, the teratogenic process in diabetic pregnancy may depend on an increased production of free-oxygen radicals in immature embryonic mitochondria in response to a metabolic overload. This notion implies that every oxidative substrate entering the mitochondrial metabolism in excess may induce embryonic malformations and emphasizes the need for an extended metabolic surveillance of pregnant diabetic women. Consequently, optimal metabolic control should aim at normalizing the maternal serum concentrations of all possible oxidative substrates.

Pathogenesis of Diabetes-Induced Congenital Malformations

Abstract The increased rate of fetal malformation in diabetic pregnancy represents both a clinical problem and a research challenge. In recent years, experimental and clinical studies have given insight into the teratological mechanisms and generated suggestions for improved future treatment regimens. The teratological role of disturbances in the metabolism of inositol, prostaglandins, and reactive oxygen species has been particularly highlighted, and the beneficial effect of dietary addition of inositol, arachidonic acid and antioxidants has been elucidated in experimental work. Changes in gene expression and induction of apoptosis in embryos exposed to a diabetic environment have been investigated and assigned roles in the teratogenic processes. The diabetic environment appears to simultaneously induce alterations in several interrelated teratological pathways. The complex pathogenesis of diabetic embryopathy has started to unravel, and future research efforts will utilize both clinical intervention studies and experimental work that aim to characterize the human applicability and the cell biological components of the discovered teratological mechanisms.

MATERNAL ANTIOXIDANT TREATMENTS PREVENT DIABETES-INDUCED ALTERATIONS OF MITOCHONDRIAL MORPHOLOGY IN RAT EMBRYOS

Previous studies have suggested that production of reactive oxygen species by embryonic mitochondria may have a role in the induction of both high‐amplitude mitochondrial swelling and embryonic dysmorphogenesis in diabetic pregnancy. The present study analyzed the relationships between a putative metabolite‐induced production of free oxygen radicals, mitochondrial lipid peroxidation, and high‐amplitude mitochondrial swelling in embryos during organogenesis.

Long-term effects of glibenclamide on the insulin production, oxidative metabolism and quantitative ultrastructure of mouse pancreatic islets maintained in tissue culture at different glucose concentrations

SummaryIn order to evaluate long-term effects of sulphonylureas on pancreatic islet structure and function, isolated mouse islets were maintained in tissue culture for one week at various glucose concentrations, and in the absence or presence of glibenclamide. When the islets were cultured at 3.3 or 5.5 mmol/1, but not at 16.7 mmol/1 glucose, it was found that the drug stimulated insulin secretion into the culture medium during the initial 3 days of culture. During the remainder of the culture period no such enhancement of secretion was demonstrated. Insulin release due to glibenclamide apparently resulted in rapid depletion of intracellular insulin stores. The finding of an enlarged B-cell Golgi apparatus in the drug-treated islets was probably associated with granule discharge. The failure of glibenclamide to promote insulin secretion during the whole culture period could reflect the adverse effects of the drug on islet insulin biosynthesis as indicated by short-term experiments performed after culture. Similar experiments showed that the impaired insulin biosynthesis could not be restored by withdrawal of the drug from the culture medium for 3 days. Furthermore, the capacity for insulin release in response to an acute glucose challenge at the end of the culture period, was abolished by culture in the presence of glibenclamide. The drug effects on insulin biosynthesis and intracellular insulin stores, which were most pronounced at 5.5 mmol/1 glucose, possibly resulted from changes in B-cell metabolism as suggested by the diminished islet glucose-oxidation rate. The spatial characteristics of islet mitochondria indicated that these changes might involve an adaptation to substrates other than glucose. In conclusion, our findings suggest that sulphonylureas have an insulinotropic effect, which is however transient. Indeed, it rather seems as if long-term exposure of islet B-cells to sulphonylureasin vitro were accompanied by functional deficiency.

Antioxidant enzyme activity and mRNA expression in the islets of Langerhans from the BB/S rat model of type 1 diabetes and an insulin-producing cell line

It has been proposed that low activities of antioxidant enzymes in pancreatic beta cells may increase their susceptibility to autoimmune attack. We have therefore used the spontaneously diabetic BB/S rat model of type 1 diabetes to compare islet catalase and superoxide dismutase activities in diabetes-prone and diabetes-resistant animals. In parallel studies, we employed the RINm5F beta cell line as a model system (previously validated) to investigate whether regulation of antioxidant enzyme activity by inflammatory mediators (cytokines, nitric oxide) occurs at the gene or protein expression level. Diabetes-prone rat islets had high insulin content at the age used (58–65 days) but showed increased amounts of DNA damage when subjected to cytokine or hydrogen peroxide treatments. There was clear evidence of oxidative damage in freshly isolated rat islets from diabetes-prone animals and significantly lower catalase and superoxide dismutase activities than in islets from age-matched diabetes-resistant BB/S and control Wistar rats. The mRNA expression of antioxidant enzymes in islets from diabetes-prone and diabetes-resistant BB/S rats and in RINm5F cells, treated with a combination of cytokines or a nitric oxide donor, DETA-NO, was analysed semi-quantitatively by real time PCR. The mRNA expression of catalase was lower, whereas MnSOD expression was higher, in diabetes-prone compared to diabetes-resistant BB/S rat islets, suggesting regulation at the level of gene expression as well as of the activities of these enzymes in diabetes. The protein expression of catalase, CuZnSOD and MnSOD was assessed by Western blotting and found to be unchanged in DETA-NO treated cells. Protein expression of MnSOD was increased by cytokines in RINm5F cells whereas the expression of CuZnSOD was slightly decreased and the level of catalase protein was unchanged. We conclude that there are some changes, mostly upregulation, in protein expression but no decreases in the mRNA expression of catalase, CuZnSOD or MnSOD enzymes in beta cells treated with either cytokines or DETA-NO. The lower antioxidant enzyme activities observed in islets from diabetes-prone BB/S rats could be a factor in the development of disease and in susceptibility to DNA damage in vitro and could reflect islet alterations prior to immune attack or inherent differences in the islets of diabetes-prone animals, but are not likely to result from cytokine or nitric oxide exposure in vivo at that stage.

Amyloid deposition in transplanted human pancreatic islets : a conceivable cause of their long-term failure

Following the encouraging report of the Edmonton group, there was a rejuvenation of the islet transplantation field. After that, more pessimistic views spread when long-term results of the clinical outcome were published. A progressive loss of the β-cell function meant that almost all patients were back on insulin therapy after 5 years. More than 10 years ago, we demonstrated that amyloid deposits rapidly formed in human islets and in mouse islets transgenic for human IAPP when grafted into nude mice. It is, therefore, conceivable to consider amyloid formation as one potential candidate for the long-term failure. The present paper reviews attempts in our laboratories to elucidate the dynamics of and mechanisms behind the formation of amyloid in transplanted islets with special emphasis on the impact of long-term hyperglycemia.

Decrease in insulin-containing secretory granules and mitochondrial gene expression in mouse pancreatic islets maintained in culture following streptozotocin exposure.

SummaryWe have previously described a preferential reduction in the secretory response to nutrient secretagogues in pancreatic mouse islets maintained in culture after in vitro exposure to streptozotocin (SZ). This reduction was associated with an impaired substrate metabolism at the mitochondrial level. To further clarify this issue, mouse pancreatic islets were exposed in vitro to 2.2 mM SZ for 30 min. At 4 h after SZ treatment ultrastructural changes were apparent in the endoplasmic reticulum and Golgi areas of the B-cells. However, 2 and 6 days following SZ exposure the B-cells appeared well preserved, except for a marked decrease in the number of insulin-containing secretory granules. A morphometric analysis of the B-cells 6 days after SZ exposure showed a normal B-cell size and a normal volume fraction of B-cell mitochondria. However, there was a decrease in total islet size and a 13% decrease in the volume fraction of B-cells in the islets. These mouse islets exhibited a decreased content of the mitochondrial DNA-encoded cytochrome b mRNA, as evaluated by dot-blot analysis. As a whole, the data obtained indicate that SZ treatment does not induce a decrease in the number of mitochondria or long-lasting ultrastructural damage to this organelle. However, there is a clear decrease in the cytochrome b mRNA, suggesting that SZ can induce damage to the mitochondrial DNA.

Intracellular degradation of insulin and crinophagy are maintained by nitric oxide and cyclo-oxygenase 2 activity in isolated pancreatic islets

Background information. Pancreatic β‐cells require an optimal insulin content to allow instantaneous secretion of insulin. This is maintained by insulin biosynthesis and intracellular degradation of insulin. Degradation may be effected by crinophagy, i.e. the fusion of secretory granules with lysosomes. IL‐1β (interleukin 1β) induces distinct changes of β‐cell lysosomes. To study the mechanisms for intracellular insulin degradation and crinophagy, isolated mouse pancreatic islets were exposed to IL‐1β and known pathways for IL‐1β actions were blocked. Intracellular insulin degradation was determined by following the fate of radioactively labelled insulin. Crinophagy was studied by ultrastructural analysis. The effects of blocking pathways for IL‐1β were monitored by measurements of nitrite and PGE2 (prostaglandin E2).

Steroid effects on intracellular degradation of insulin and crinophagy in isolated pancreatic islets

Under physiological conditions substantial amounts of hormone may be degraded within endocrine cells by a crinophagic process comprising fusions of secretory granules with lysosomes. Glucocorticoids may stabilise and progesterone destabilise lysosomal membranes. The effects of corticosterone and progesterone on intracellular degradation of insulin and crinophagy were determined in pancreatic beta-cells, and possible pathways mediating these effects were evaluated. Pancreatic islets were isolated from mice, intracellular degradation of insulin was measured by a pulse-chase method, and crinophagy was studied by electron microscopy. The islets were exposed to 3.3, 5.5 or 28 mM glucose with or without corticosterone, progesterone or the receptor ligands A-224817.0 and WAY-161358. Mifepristone was used to block steroid receptors and indomethacin to inhibit prostaglandin synthesis. Corticosterone caused a concentration-dependent decrease of insulin degradation at the lower glucose concentrations. Progesterone effected a concentration-dependent stimulation of insulin degradation. These results were paralleled with changes of the crinophagic activity in the beta-cells. Corticosterone decreased and progesterone increased islet production of prostaglandin E(2). Mifepristone abolished the steroid actions on insulin degradation and prostaglandin production. The effects of corticosterone were mimicked by the selective glucocorticoid receptor modulator A-224817.0, but in contrast to progesterone, the selective progesterone receptor agonist WAY-161358 had no effect on insulin degradation or prostaglandin production. Inhibition of cyclooxygenase blocked insulin degradation. The findings indicate that both corticosterone and progesterone could affect intracellular insulin degradation and crinophagy solely via the glucocorticoid receptor, and that prostaglandins may have a regulatory role in intracellular turnover of secretory material in pancreatic islet beta-cells.