Eva Strandell

Reversal of beta-cell suppression in vitro in pancreatic islets isolated from nonobese diabetic mice during the phase preceding insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is characterized by a progressive autoimmune destruction of the pancreatic beta-cells. One of the best-suited animal models for IDDM is the nonobese diabetic (NOD) mouse. In this investigation pancreatic islets were isolated from female NOD mice aged 5-7, 8-11, and 12-13 wk and examined immediately (day 0) or after 7 d of culture (day 7). The mice showed a progressive disturbance in glucose tolerance with age, and a correspondingly increased frequency of pancreatic insulitis. Islets isolated from the oldest mice often contained inflammatory cells on day 0, which resulted in an elevated islet DNA content. During culture these islets became depleted of infiltrating cells and the DNA content of the islets decreased on day 7. Islets of the eldest mice failed to respond with insulin secretion to high glucose, whereas a response was observed in the other groups. After culture all groups of islets showed a markedly improved insulin secretion. Islets from the 12-13-wk-old mice displayed a lower glucose oxidation rate at 16.7 mM glucose on day 0 compared with day 7. Islet (pro)insulin and total protein biosynthesis was essentially unaffected. In conclusion, islets obtained from 12-13-wk-old NOD mice exhibit an impaired glucose metabolism, which may explain the suppressed insulin secretion observed immediately after isolation. This inhibition of beta-cell function can be reversed in vitro. Thus, there may be a stage during development of IDDM when beta-cell destruction can be counteracted and beta-cell function restored, provided the immune aggression is arrested.

Functional Characteristics of Rat Pancreatic Islets Maintained in Culture After Exposure to Human Interleukin 1

Recent observations suggest a role for interleukin 1 β (IL-1) in the autoimmune β-cell destruction observed in type I (insulin-dependent) diabetes mellitus. We investigated the acute and long-term effects of IL-1 on pancreatic β-cell function in vitro. Rat pancreatic islets were isolated and kept in tissue culture for 5 days. The islets were subsequently transferred to media containing RPMI-1640 plus 1% human serum with or without human recombinant IL-1 β (300 pM) and cultured for another 48 h. The islets were examined either immediately after IL-1 exposure (day 0) or after an additional 6-day culture period without IL-1. On day 0, IL-1 was found to totally inhibit glucose-stimulated insulin release, partially inhibit glucose oxidation, and induce a decrease in islet DNA content. However, these islets were able to release insulin after stimulation with glucose plus theophylline, although the absolute rate of insulin secretion was lower than that of the control group. After 6 days in culture, the insulin-secretory response to glucose and the glucose oxidation rates of the IL-1-pretreated islets were completely restored, but there remained a reduced islet DNA content. We conclude that IL-1 is cytotoxic to islet β-cells. However, surviving β-cells are able to recover their functional capacity after a period of inhibited function.

Efficient Gene Transfer to Dispersed Human Pancreatic Islet Cells In Vitro Using Adenovirus-Polylysine/DNA Complexes or Polycationic Liposomes

The establishment of gene delivery systems that result in efficient transfection of the pancreatic β-cells may generate an important tool for the study of IDDM and may also represent one critical step toward a clinical application of gene transfer for the prevention or early treatment of the disease. Using the reporter gene vectors pCAT and pCMV β-gal, we have investigated the efficiency of transfection mediated by calcium phosphate precipitation, the monocationic liposome Lipofectin, the polycationic liposome Lipofectamine, and adenovirus-polylysine (AdpL) DNA complexes in human, mouse, rat, and fetal porcine islet cells. In all species studied, calcium phosphate–mediated transfection resulted in lower chloramphenicol acetyl transferase (CAT) activities than the other methods. Intact human, mouse, and rat islets were poorly transfected by Lipofectin, Lipofectamine, and AdpL. When dispersed by trypsin treatment, however, human, mouse, rat, and fetal pig islet cells were efficiently transfected by Lipofectamine. Moreover, transfection of dispersed human and mouse islet cells using AdpL also resulted in high CAT activities. The percentage of cells staining positively for β-galactosidase after transfection with Lipofectamine was 49% for mouse, 56% for rat, and 57% for dispersed human islet cells. Transfection of human islet cells using AdpL, however, yielded 70% βgal–positive cells. Fluorescence-activated cell sorting-purified rat islet α- and β-cells were transfected with similar efficiency using Lipofectamine. CAT expression in human islet cells transfected with either Lipofectamine or AdpL reached a peak value after 5–7 days, followed by a gradual decline. It is concluded that transfection with AdpL or Lipofectamine are both efficient means to achieve transient expression of gene constructs in human and mouse islet cells, while for rat and fetal porcine islet cells, Lipofectamine is the most efficient of the agents investigated in this study.

Culture of mouse pancreatic islets in different glucose concentrations modifies B cell sensitivity to streptozotocin.

SummaryThere have previously been divergent data published regarding the effects of glucose on the diabetogenic effects of streptozotocin. In order to further explore this issue, two separate sets of experiments were performed. In the first, mouse pancreatic islets were maintained in culture for 3 days at different glucose concentrations (5.6,11.1 and 28 mmol/l) and then exposed to streptozotocin. After another 3 days in culture at 11.1 mmol/l glucose, the B cell function was evaluated by measurement of glucose-stimulated insulin release, the number of islets recovered after culture, and the islet DNA and insulin contents. In the second group of experiments islets were first maintained in culture at 11.1 mmol/l glucose, then treated with streptozotocin and subsequently cultured for 6 days at the different glucose concentrations given above. It was found that islets maintained in a medium containing 28 mmol/l glucose before or after streptozotocin exposure showed less signs of damage than islets cultured in 11.1 mmol/l glucose. A similar, but less pronounced, de creased sensitivity to streptozotocin was found in islets precultured in 5.6 mmol/l glucose, in comparison with those islets cultured in 11.1 mmol/l glucose. Culture at 5.6 mmol/l glucose just after streptozotocin treatment did not induce any improvement in islet survival or function. It is suggested that the increased damage induced by streptozotocin to islets precultured at 11.1 mmol/l glucose, in comparison with 5.6 mmol/l glucose, can be related to the fact that an increased metabolic activity of B cells render them more susceptible to the toxin. The improved preservation of islets cultured at 28 mmol/l glucose before or after streptozotocin treatment may reflect an additional effect of glucose, i. e. activation of defense mechanisms in the B cells against cytotoxins.

Metabolism and β-cell function of rat pancreatic islets exposed to human interleukin-1β in the presence of a high glucose concentration

It has been postulated that one of the factors causing immune-mediated pancreatic beta-cell destruction in insulin-dependent diabetes mellitus (IDDM) is interleukin-1 (IL-1). Rat pancreatic islets exposed to human recombinant IL-1 beta (rIL-1 beta) for 48 h in vitro exhibit a markedly reduced glucose-stimulated insulin secretion. Also, a deleterious effect of glucose on beta-cell function, especially under conditions of a reduced beta-cell mass, which may exist in the early phase of IDDM has been suggested. In this study the response of rat pancreatic islets in vitro to a combination of the cytokine and high glucose concentration have therefore been assessed. Thus, islets were cultured for 48 h at either 11.1 or 56 mM glucose with or without 25 U/ml rIL-1 beta. Exposure to the cytokine reduced the islet DNA content at both glucose concentrations by 20-25%. In short-term incubations in the absence of rIL-1 beta after the preceding culture with the cytokine, the glucose-stimulated insulin release was reduced by 70% in islets cultured at 11.1 mM glucose and by only 40% after culture at 56 mM glucose, when compared to the corresponding control islets. The utilization of D-[5-3H]glucose, i.e., the catabolism of glucose in the glycolytic pathway, was the same in all groups of islets. However, the D-[6-14C]glucose oxidation rate, i.e., the metabolism of glucose in the Krebs cycle, was reduced by about 65% in rIL-1 beta exposed islets kept at 11.1 mM glucose and 46% in islets cultured at 56 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1β induces the expression of HSP70, heme oxygenase and Mn-SOD in FACS-purified rat islet β-cells, but not in α-cells

Abstract The cytokine IL-1β has previously been demonstrated to induce the expression of the stress genes iNOS, hsp70, heme oxygenase and Mn-SOD in rat pancreatic islets in vitro. The aim of this study was to determine whether the IL-1β-induced effects are specific for the insulin producing β-cell, or whether other islet cells, such as the glucagon-producing α-cell, respond to IL-1β addition. Purified rat α- and β-cell suspensions were obtained by fluorescence-activated cell sorting and incubated with or without IL-1β (25 U/ml) for 24 h. The α- and β-cell contents of hsp70, heme oxygenase and Mn-SOD and medium nitrite levels were determined. It was found that IL-1β exposure induced the production of nitric oxide in β-cells, but not in α-cells. Moreover, the expression of hsp70, heme oxygenase and Mn-SOD was also induced in β-cells, but not in α-cells. There were no detectable levels of hsp70 in α-cells. It is concluded that the stress gene response following IL-1β exposure in markedly different in α- and β-cells. This finding may be of importance for the understanding of the autoimmune destruction of β-cells in insulin-dependent diabetes mellitus.

Functional characteristics of cultured mouse pancreatic islets following exposure to different streptozotocin concentrations

The present study was undertaken to investigate the acute and long-term effects of streptozotocin (SZ) on pancreatic islet function and survival in vitro. Isolated mouse pancreatic islets, that had been cultured overnight, were exposed to SZ (0.55-4.4 mM) or critic acid buffer in the case of the control group. The islets were examined either immediately after SZ exposure or after one week in culture. There was a marked loss of islets treated with 2.2 and 4.4 mM SZ during the culture; however, the DNA content of the remaining islets was unaffected. The islet insulin content was reduced 7 days after treatment with 2.2 and 4.4 mM SZ. At 4.4 mM the glucagon and somatostatin content of the islet was also decreased but not to the same degree as the insulin content. SZ-induced inhibition of glucose-stimulated insulin release and (pro)insulin biosynthesis was more pronounced on day 7 as compared to day 0. A similar pattern of inhibitory action of SZ was observed on islet glucose oxidation rates. Islet ATP contents were depressed on day 7 in islets exposed 4.4 mM SZ, but were otherwise similar to the control group. Islet NAD + NADH contents were decreased by 50% after exposure to 2.2 mM SZ, compared to the control islets on day 0. This decrease in NAD + NADH contents was to a large extent restored during the one-week culture. The present study shows that islets failed to completely repair the acute damage caused by SZ, and that the impairment of the islet glucose-stimulated insulin release induced by SZ seemed to progress in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of mRNA contents of IL-1b, and nitric oxide synthase in pancreatic islets isolated from female and male nonobese diabetic mice.

SummaryInterleukin-1Β (IL-1Β) has been suggested to mediate beta-cell destruction in insulin-dependent diabetes mellitus (IDDM) by inducing nitric oxide production. In this study, we assessed the levels of IL-1Β and the inducible form of nitric oxide synthase (iNOS), using a semi-quantitative polymerase chain reaction assay, and performed determinations of nitrite accumulation and IL-1Β bioactivity, on pancreatic islets isolated from 5- and 16-week-old female and male nonobese diabetic (NOD) mice and from nondiabetes prone NMRI mice. NOD mouse islets contained notable amounts of IL-1Β mRNA. At 5 weeks of age, but not at 16 weeks, the values were higher in islets isolated from NOD females compared to males. The IL-1Β bioactivity showed differences roughly reflecting the mRNA levels in the NOD mouse islets. In the NMRI mouse islets the IL-1Β bioactivity was very low. The expression of iNOS mRNA increased in both male and female islets between 5 and 16 weeks of age. Immunocytochemistry of pancreatic sections indicated the presence of macrophages especially in the peri-insular area of the NOD mice which suggests that IL-1Β was produced by macrophages. The levels of IL-1Β activity and mRNA in freshly isolated islets from NOD 5-week-old females did not correlate to the iNOS mRNA content or to the nitrite production. However, after incubation with IL-1Β in vitro, both NOD and NMRI islets responded with a marked increase in nitric oxide production. It is concluded that the presence of IL-1Β in isolated NOD mouse islets, via an induction of iNOS expression and nitric oxide production, cannot explain the gender difference in diabetes incidence in NOD mice.

Novel experimental strategies to prevent the development of type 1 diabetes mellitus.

Abstract Type 1 diabetes is an autoimmune disease leading to extensive destruction of the pancreatic β-cells. Our research focusses on the role of β-cells during the course of the disease, aiming at finding novel strategies to enhance β-cell resistance against the cytotoxic damage inflicted by the immune system. Special attention has been paid to the possibility that cytokines released by the immune cells infiltrating the pancreatic islets can directly suppress and kill β-cells. Certain cytokines (interleukin-1β, tumor necrosis factor-α and interferon-γ) either alone or in combination, are able to activate signal transduction pathways in β-cells leading to transcription factor activation and de novo gene expression. In this context, it has been found that induction of inducible nitric oxide synthase mediates an elevated production of nitric oxide, which impairs mitochondrial function and causes DNA damage eventually leading to apoptosis and necrosis. However, other induced proteins SUCH AS heat shock protein 70 and superoxide dismutase may reflect a defense reaction elicited in the β-cells by the cytokines. Our strategy is to further seek for proteins involved in both destruction and protection of β-cells. Based on this knowledge, we plan to apply gene therapeutic approaches to increase expression of protective genes in β-cells. If this is feasible we will then evaluate the function and survival of such modified β-cells in animal models of type 1 diabetes such as the NOD mouse. The long-term goal for this research line is to find novel approaches to influence β-cell resistance in humans at risk of developing type 1 diabetes.

Prolonged exposure of pancreatic islets isolated from “pre-diabetic” non-obese diabetic mice to a high glucose concentration does not impair Beta-cell function

SummaryIn the early stages of Type 1 (insulin-dependent) diabetes mellitus patients present a deficient insulin response to glucose. The reasons for this defective response are unknown, but it has been suggested that it reflects a deleterious effect of excessive glucose stimulation on a reduced Beta-cell mass. Female non-obese diabetic (NOD) mice from our colony, at the age of 12–13 weeks, have a normal basal glycaemia but an impaired intravenous glucose tolerance test, insulitis and a defective insulin response to glucose. In order to characterize the potential effect of glucose on the Beta cells at that “pre-diabetic” stage, pancreatic islets were isolated from 12–13 week old female NOD mice. Immediately after isolation (day 0) the NOD islets displayed a defective insulin response to an acute stimulation with 16.7 mmol/l glucose. After seven days in culture at both 11 and 28 mmol/l glucose these islets showed an increased insulin release in response to an acute glucose stimulation. This increase was more pronounced in the islets cultured at 28 mmol/l glucose. Experiments performed in parallel, using islets obtained from a non-diabetes prone strain of mice (Naval Medical Research Institute, NMRI) showed that these islets had a similar insulin release in response to glucose both on day 0 and after seven days in culture at 11 mmol/l glucose. The insulin mRNA levels of NOD islets did not change over one week in culture at 11 or 28 mmol/l glucose, but culture at the high glucose concentration induced a decrease in the islet insulin content. The present data show that culture at high glucose concentrations does not impair the function of islets isolated from NOD mice. These observations make excessive glucose stimulation, as a single factor, an unlikely explanation for the defective insulin release observed in NOD islets in the “prediabetic” period.