Steffen Helqvist

Heat shock protein induction in rat pancreatic islets by recombinant human interleukin 1β

SummaryInterleukin 1β, potentiated by tumour necrosis factor α, is cytotoxic to pancreatic Beta cells in vitro. We have hypothesized that interleukin 1β induces oxygen free radicals in Beta cells. Since cytotoxicity induced by free radicals and by heat may activate the same cellular repair mechanism (the heat shock response), the aim of this study was to investigate the pattern of protein synthesis in isolated islets after exposure to interleukin 1β (150 pg/ml, 24 h), tumour necrosis factor α (50 ng/ml, 24 h), heat shock (43°C, 30 min) and H2O2 (0.1 mmol/l, 20 min). By polyacrylamide gel electrophoresis, autoradiography, Western-blot analysis and partial peptide mapping of 35S-methionine labelled islets, interleukin 1β was found to induce a 73 kilodalton protein belonging to the heat shock protein family heat shock protein 70, a heat shock protein 90, and haem oxygenase. A minor induction of heat shock protein 73 and haem oxygenase was seen after H2O2. Interleukin 1β did not induce heat shock proteins in rat thyroid cells, rat mesangial cells or in human monocytes. Tumour necrosis factor α did not induce selective protein synthesis. Pre-exposure of islets to heat, tumour necrosis factor α, or H2O2 did not prevent the impairment of glucose-stimulated insulin release seen after 24 h of interleukin 1β exposure. The data are compatible with free radical induction by interleukin 1β. However, the heat shock response is not specific for oxidative injury, and previous studies have shown discrepant effects as to a protective effect of free radical scavengers against interleukin 1β-mediated beta-cytotoxicity. Thus, a role for free radicals in this context is not definitely proven.

Lack of Predictive Value of Islet Cell Antibodies, Insulin Antibodies, and HLA-DR Phenotype for Remission in Cyclosporin-Treated IDDM Patients

The effect of immunosuppression on the humoral immune response to islet autoantigens and exogenously administered insulin and the predictive value of islet cell cytoplasmic antibodies (ICAs), insulin antibodies (IAs), and HLA-DR phenotype for remission during immunosuppression were studied in a prospective randomized double-blind trial of cyclosporin administration in 98 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients. HLA-DR phenotype and glycosylated hemoglobin were determined at study entry, and insulin requirement, glucagon-stimulated C-peptide, ICAs, and IAs were measured at entry and after 1, 3, 6, 9, and 12 mo of follow-up. Cyclosporin therapy caused significant suppression of the prevalence and serum concentrations of ICAs and lAs. Cyclosporin-treated IDDM patients ICA+ at study entry had higher levels of stimulated C-peptide after 1 mo of study, but the increased (β-cell function was not associated with a higher frequency of insulin-free remission at 1 mo. ICA and IA status at entry did not predict cyclosporininduced remission as assessed by the prevalence of insulin-free remission or β-cell function at 3–12 mo of study, and significant decrements in the titers or total disappearance of ICAs were not associated with an increased prevalence or duration of non-insulinrequiring remission or higher stimulated C-peptide values. There was no correlation between the serum levels of ICAs and lAs at entry and β-cell function at 12 mo of follow-up. Although patients who were HLADR3/X seemed to respond better to cyclosporin therapy at 6 mo than patients who were HLA-DR3/4 or -DR4/X when analyzed cross-sectionally, a split-plot analysis of variance with time as the split-plot factor showed that the HLA-DR phenotype failed to identify patients with an overall better response to cyclosporin therapy. These data indicate that, although cyclosporin therapy inhibits the humoral immune response to islet components and exogenous insulin, significant decrements in ICA titers are not useful in monitoring efficacy of immunosuppression with cyclosporin, and the determination of ICA and IA status and immune response phenotype at diagnosis is of no predictive value for remission in selecting recent-onset IDDM patients for cyclosporin immunointervention.

Repetitive Exposure of Pancreatic Islets to Interleukin-1β. An In Vitro Model of Pre-diabetes?

The slowly progressing loss of glucose tolerance over years before clinical onset of Type 1 (insulin-dependent) diabetes mellitus may be due to repetitive immunological attacks on the pancreatic beta-cell mass. Accordingly, we studied the effects of repetitive exposure of isolated rat pancreatic islets to the beta-cytotoxic immune-mediator interleukin-1 beta. Islets were exposed thrice to 60 U/ml of recombinant interleukin-1 beta for 24 hr. The islets were allowed to recover for 6 d between the interleukin-1 beta exposure periods. After each of the three interleukin-1 beta exposure periods, islet capacity to release insulin was decreased to 12, 6 and 3% of control, respectively, and islet insulin content decreased to 75, 56 and 21%, respectively. After the two recovery culture periods, the capacity for insulin release reversed to 75 and 30% of control, respectively. An increase in islet insulin content was only seen after the first recovery culture. During repetitive as well as long-term (6 d) interleukin-1 beta exposure of islets, medium accumulation of glucagon was either increased or unaffected. In analogy, beta-cells exposed to interleukin-1 beta for 6 d showed ultrastructural signs of degeneration and cytolysis, whereas alpha-cells were intact. In conclusion, interleukin-1 beta injury to beta-cells was partially reversible, but successive episodes of islet interleukin-1 beta exposure were increasingly detrimental; alpha-cell function and structure did not show susceptibility to damage by interleukin-1 beta. These findings may contribute to our understanding of islet cell behaviour before and during onset of Type 1 diabetes.

On the Pathogenesis of Insulin-Dependent Diabetes Mellitus — A Discussion of Three Recently Proposed Models

Only in 1974 was it unequivocally shown that insulin-dependent (IDDM) and noninsulin-dependent (NIDDM) diabetes mellitus are two distinct disease entities [1]. In their paper Nerup et al. presented the first attempt to formulate a fully comprehensive model to explain the etiology and pathogenesis of IDDM: Histocompatibility complexes like HL-A contain, in addition to genes controlling serologically detectable antigens, so-called immune-response genes which control the development of cell-mediated immunity to certain antigens. One or more immune-response genes associated with HL-A8 and/or W15 might be responsible for an altered T-lymphocyte response. The genetically determined host response could fail to eliminate an infecting virus (Coxsackie B4 and others) which in turn might destroy the pancreatic beta-cells or trigger an autoimmune reaction against the infected organ.

Interleukin 1β Increases the Cytosolic Free Sodium Concentration in Isolated Rat Islets of Langerhans

Interleukin l (1L‐I) exerts both stimulatory and inhibitor) (cytotoxic) effects on insulin producing βcells in isolated pancreatic islets. Since alteration in ion fluxes is crucial for endocrine cell activation and is a denominator of cell death, and since IL‐1 was recently shown to increase the total sodium content in a murine pre‐B‐lymphocyte cell line, we investigated the effect of recombinant human IL‐lβ (rhIL‐1β) on the cytosolic tree sodium concentration (Na +) in rat islets, furthermore, long‐term rhIL‐1βeffects on islet cell function were studied during exposure of islets to amiloride. a blocker of the plasma membrane Na+/H+ exchange. One hour of islet exposure to 60 U/ml of rhIL‐lβ caused a threefold increase in I Na + . in islet cells, and this effect was abolished by deplelion of extracellular sodium. Blockade of Na+/H+ exchange with amiloride abolished the inhibitory effect of rhIL‐1β Son insulin release. In conclusion. rhIL‐lβwas found to increase sodium influx in pancreatic islet cells. This might underlie the widespread effects of rhIL‐lβ on β‐cell function and morphology, possibly related to IL‐l‐mediated toxic free radical formation.

Intra-Peritoneal Administration of Interleukin-1β Induces Impaired Insulin Release from the Perfused Rat Pancreas

Previous studies have demonstrated a stimulatory effect of interleukin-1 beta (IL-1 beta) on insulin and glucagon release from the perfused rat pancreas, accompanied by selective lysis of 20% of beta-cells as assessed by electronmicroscopy. However, we have not observed an inhibitory action of IL-1 beta on insulin release from the perfused pancreas as shown for isolated islets. To test whether periodical exposure of the endocrine pancreas to circulating IL-1 beta in vivo affects insulin release from the intact perfused pancreas, rats were treated with daily intraperitoneal injections of 4 micrograms IL-1 beta/kg or saline for 5 days. On day 5 the pancreata were isolated 2 h after the last injection and perfused from 0 to 72 min with 11 mmol/l D-glucose and from 72 to 84 min with 20 mmol/l D-glucose. Saline or IL-1 beta was added from 12 to 72 min. In pancreata from animals pre-treated with IL-1 beta glucose-stimulated as well as IL-1 beta potentiated glucose-stimulated insulin release was almost completely abolished. Furthermore, a decline in insulin release was observed at 11 mmol/l D-glucose, in contrast to an increase in insulin release in controls. The total extractable insulin content in pancreata from IL-1 beta pre-treated rats was higher than in pancreata from saline-treated controls. In contrast to the inhibitory effect of in vivo administration of IL-1 beta on beta-cell function glucagon secretion was stimulated. These observations suggest that circulating IL-1 beta is an important modulator of alpha- and beta-cell secretory function in vivo and that IL-1 beta should be considered a contributory pathogenetic factor in the development of insulin-dependent (type 1) diabetes mellitus.