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J Sternesjo and S Sandler

Administration of the T-helper 1 (Th 1) cell promoting cytokine interleukin-12 (IL-12) accelerates the development of autoimmune diabetes in non-obese diabetic (NOD) mice. In this study we examined the effects of IL-12 on isolated islets from NMRI (Naval Medical Research Institute-established) mice, Sprague-Dawley (S-D) rats and NOD mice. NMRI and S-D islets were cultured in medium RPMI 1640 + 10% fetal calf serum and exposed for 48 h to recombinant mouse IL-12 (0, 0.1, 1 and 10 ng/ml). Islet glucose metabolism, as measured by glucose oxidation rate, was suppressed by about 25% in NMRI islets exposed to 10 ng/ml IL-12. In rat islets 0.1 ng/ml IL-12 induced a 20% decrease in glucose oxidation rate. Islets cultured with 10 ng/ml IL-12 showed a decrease in medium insulin accumulation both in mouse and rat. Glucose-stimulated insulin release was lowered in rat islets exposed to 10 ng/ml IL-12, but not affected in NMRI islets. In NMRI islets IL-12 did not influence nitric oxide production as measured by nitrite formation. In rat islets IL-12 induced a decrease in nitrite formation compared with control islets. Islets were isolated from female NOD mice (age 5, 12, 20 and 26 weeks) and examined either immediately or cultured for 7 days with 10 ng/ml IL-12 alone or in combination with 4 ng/ml of the T-cell stimulating cytokine interleukin-2 (IL-2). In the age groups > 5 weeks of age the glucose-stimulated insulin release was lower in freshly isolated compared with cultured control islets. IL-2 + IL-12 addition induced a small decrease in glucose-stimulated insulin release in islets from 12-week-old animals. With increasing age the DNA content in freshly isolated islets increased due to immune cell infiltration. The DNA content in cultured islets was decreased by 40-60% compared with freshly isolated islets in the age groups over 5 weeks. Islet insulin content was similar in both freshly isolated and cultured islets. None of the cytokines, either alone or in combination, affected islet DNA or insulin content. We conclude that IL-12 has minor suppressive effects in vitro on normal rodent islets. It is likely that the reported accelerated diabetes development of IL-12 administration to NOD mice in vivo is not mediated by a direct toxic effect to the islets. The suppressed insulin release in NOD mouse islets treated with IL-2 + IL-12 suggests, however, that the accelerating effect might partly be attributed to stimulation of immune cells present in the insulitic lesion.

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M Holstad and S Sandler

In earlier studies it has been shown that prolactin (PRL) is a stimulating factor for the immune system, and it has been suggested that PRL might antagonize immunosuppressive effects of glucocorticoids. PRL has been reported to affect the cytokine secretion pattern, by elevating cytokine gene expression in macrophages, after the onset of sepsis. It also promotes the antibody response in mice where it increases the production of interferon-gamma (IFN-gamma) and inhibits interleukin-1 (IL-1) production. Due to these properties, PRL might influence the development of autoimmune type 1 diabetes. The aim of the present study was to examine the effects of two drugs; PRL and bromocriptine (BC) in vivo on the development of hyperglycemia and pancreatic insulitis in mice treated with multiple doses of streptozotocin (STZ) (40 mg/kg body weight, i.p.). The dopaminergic agonist BC is known to inhibit PRL secretion. In another set of experiments, the direct effects of PRL on the function of pancreatic islets exposed to STZ in vitro were studied. Mice treated with STZ became gradually hyperglycemic, and concomitant treatment with PRL (4 mg/kg body weight) for 21 days significantly reduced the elevation in blood glucose levels from day 10 onwards (P<0.05). Morphologic examinations of the pancreas on day 21 of mice receiving STZ injections revealed a marked insulitis, but only moderate insulitis in the STZ treated animals given PRL. BC administration (10 mg/kg body weight) in combination with STZ did not significantly affect the elevation in blood glucose levels or the insulitis. PRL or BC administration alone did not change the serum glucose concentration. This study indicates that PRL may affect hyperglycemia in the early phase of autoimmune diabetes. We suggest that it might be due to counteraction of autoimmune immunologic mechanisms and/or enhancement of beta-cell regeneration.

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E Strandell and S Sandler

Abstract

The aim of the present study was to examine if the islet donor gender influences the β-cell sensitivity to possible mediators of β-cell destruction in insulin-dependent diabetes mellitus (IDDM). We have currently addressed this issue by comparing the action of the cytokine interleukin-1β (IL-1β) and the alkylating agent streptozotocin (STZ), on isolated pancreatic islets derived from prediabetic female and male nonobese diabetic (NOD) mice. In this mouse strain the females have a much higher incidence of spontaneous IDDM than the males. Pancreatic islets were isolated from female and male mice of the same litter, cultured for 6 days and thereafter either 5–50 U/ml IL-1β were added for 48 h or 1·8 mm STZ for 30 min. The cytokine exposure caused a strong increase in the medium insulin accumulation in the cultures of islets obtained from both males and females. The insulin and DNA contents were similar when comparing female and male islets before and after cytokine exposure. The male pancreatic islets exhibited a higher rate of islet glucose oxidation than islets obtained from females after IL-1β addition. Non-cytokine treated islets from males showed a higher insulin content compared with corresponding female islets. After IL-1β addition male and female islets had a similar inhibition of insulin secretion following stimulation by glucose. On the other hand, islets of both genders were equally sensitive to the inhibitory action of STZ, when studied 6 days after STZ incubation, as assessed by glucose oxidation and insulin release experiments. In conclusion the data of the present study did not demonstrate a clear gender difference in the islet β-cell sensitivity to immune mediated suppression by IL-1β or alkylation damage in IDDM-prone NOD mice. However, it cannot be excluded that in vivo, under the influence of sex hormones, a decisive alteration in β-cell sensitivity to damage might develop.

Journal of Endocrinology (1997) 153, 81–86

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D. L. Eizirik and S. Sandler

ABSTRACT

The aim of this study was to clarify whether prolonged in-vitro exposure of either normal or damaged β cells to a high glucose environment can be toxic to these cells. For this purpose NMRI mice were injected intravenously with a diabetogenic dose of streptozotocin (SZ; 160 mg/kg) or vehicle alone (controls). Their islets were isolated 15 min after the injection and subsequently maintained in culture for 21 days in the presence of 11·1 or 28 mmol glucose/l. After this period, during acute glucose stimulation, the control islets showed a marked increase in their insulin release in response to a high glucose stimulus. In the SZ-exposed islets there was a decrease in DNA and insulin contents, and a deficient insulin secretory response to glucose. However, in the SZ-damaged islets as well as in the control islets, culture with 28 mmol glucose/l compared with 11·1 mmol glucose/l did not impair islet retrieval after culture, islet DNA content or glucose-induced insulin release. Thus, the degree of damage was similar in the SZ-treated islets cultured at the two concentrations of glucose. These results suggest that glucose is not toxic to normal or damaged mouse pancreatic islets over a prolonged period in tissue culture.

Journal of Endocrinology (1989) 123, 47–51

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C. Svensson, S. Sandler and C. Hellerström

ABSTRACT

Previous studies have shown that 4 weeks after syngeneic transplantation of a suboptimal number of islets into either C57BL/6J (BL/6J) or C57BL/KsJ (BL/KsJ) diabetic mice there is an impaired insulin secretion by the perfused grafts. After normalization of the blood glucose level with a second islet graft, the BL/6J strain showed restored insulin secretion whilst that of the BL/KsJ strain remained impaired. The aim of the present work was to study the effects of glucose on the in-vitro function of islet β-cells from these two mouse strains, with different sensitivities of their β-cells to glucose in vivo. Isolated pancreatic islets from each strain were kept for 1 week in tissue culture at 5·6, 11, 28 or 56 mmol glucose/l and were subsequently analysed with regard to insulin release, (pro)-insulin and total protein biosynthesis, insulin, DNA and insulin mRNA contents and glucose metabolism. Islets from both strains cultured at 28 or 56 mmol glucose/l showed an increased accumulation of insulin in the culture medium and an enhanced glucose-stimulated insulin release compared with corresponding control islets cultured at 11 mmol glucose/l. After culture at either 5·6 or 56 mmol/l, rates of (pro)insulin biosynthesis were decreased in BL/KsJ islets in short-term incubations at 17 mmol glucose/l, whereas islets cultured at 56 mmol glucose/l showed a marked increase at 1·7 mmol glucose/l. In BL/6J islets, the (pro)insulin biosynthesis rates were similar to those of the BL/KsJ islets with one exception, namely that no decrease was observed at 56 mmol glucose/l. Islets of both strains showed a decreased insulin content after culture with 56 mmol glucose/l. Insulin mRNA content was increased in islets cultured in 28 or 56 mmol glucose/l from both mouse strains. Glucose metabolism showed no differences in the rates of glucose oxidation, however, in islets cultured in 56 mmol glucose/l the utilization of glucose was increased in both BL/6J and BL/KsJ animals. There were no differences in DNA content in islets cultured at different glucose concentrations, suggesting no enhancement of cell death.

The present study indicates that, irrespective of genetic background, murine β-cells can adapt to very high glucose concentrations in vitro without any obvious signs of so-called glucotoxicity. Previously observed signs of glucotoxicity in vivo in BL/KsJ islets appear not to be related only to glucose but rather to an additional factor in the diabetic environment.

Journal of Endocrinology (1993) 136, 289–296

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Z Li, FA Karlsson and S Sandler

The aim of this study was to investigate the alpha cell population during the development of type 1 diabetes following multiple low-dose streptozotocin administration in mice. For this purpose C57BL/Ks male mice were injected with streptozotocin (40 mg/kg body weight for 5 days). Development of hyperglycemia was monitored over 28 days and a morphometric analysis of islet endocrine cells was performed. A reduction of islet cell area was observed after two injections of streptozotocin. The subsequent decrease of the area throughout the study period averaged 35%. Insulin-positive beta cells gradually disappeared from the identified islets. Hyperglycemia was present from day 7 onwards and in parallel with hyperglycemia, insulitis developed. An analysis of the alpha cell number per islet area revealed a 2- to 3-fold increase in this cell population, with the highest value on day 21. Confocal microscopy analysis of the ICA 512 protein tyrosine phosphatase revealed strong expression in the alpha cells at day 21, suggesting high secretory activity in the diabetic state. It is concluded that multiple low-dose streptozotocin treatment of C57BL/Ks male mice causes the disappearance of a fraction of the islets of Langerhans. In the remaining islet tissue an expansion of alpha cells occurs, reflecting a loss of intraislet beta cells as well as a regeneration of alpha cells.

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N Welsh, B Margulis, K Bendtzen and S Sandler

Abstract

The aim of the present investigation was to evaluate the putative involvement of oxygen free radicals in interleukin-1β (IL-1β)-induced suppression of islet glucose oxidation. Isolated adult rat pancreatic islets were exposed for 1 h to liposomally encapsulated superoxide dismutase (SOD; 10 mg/ml), catalase (CAT; 10 mg/ml) and glutathione peroxidase (GPX; 5 mg/ml), after which IL-1β (25 U/ml) or hydrogen peroxide (H2O2; 0·1 mm) was added, and the incubation was continued overnight. The following day, samples were taken from the incubation media for nitrite determinations, and islet glucose oxidation rates were measured. The CAT activity increased fourfold after addition of CAT-containing liposomes. It was found that IL-1β induced a marked increase in islet nitrite production, as an index of nitric oxide formation, and that this was paralleled by a decrease in islet glucose oxidation rates. H2O2-treated islets exhibited a modest decrease in glucose oxidation rates and a minor increase in the release of nitrite to the media. Treatment of islets with liposomes containing the antioxidant enzymes SOD, CAT and GPX, either alone or in combination, did not decrease the effect of IL-1β. However, the H2O2-induced decrease in glucose oxidation rates was counteracted by the combination of the antioxidants. It was concluded that, provided the intracellular delivery of the antioxidant enzymes to the islet cells was effective, oxygen free radicals probably do not play a decisive role in IL-1β suppression of islet glucose metabolism.

Journal of Endocrinology (1994) 143, 151–156