Altered proinsulin conversion in rat pancreatic islets exposed long-term to various glucose concentrations or interleukin-1beta.

In order to elucidate a possible relationship between beta-cell function and conversion of proinsulin to insulin, isolated rat pancreatic islets were maintained in tissue culture for 1 week at various glucose concentrations (5 x 6-56 mM). Studies were also conducted on islets cultured for 48 h with interleukin-1beta (IL-1beta). By pulse-chase labelling and immunoprecipitation, the relative contents of newly synthesized proinsulin and insulin were determined. ELISA was used to analyse insulin and proinsulin content in medium and within islets. Using real-time PCR, the mRNA levels of proinsulin converting enzymes (PC1 and PC2) were studied. Islets cultured at 56 mM glucose had an increased proportion of newly synthesized proinsulin when compared with islets cultured at 5 x 6 mM glucose after a 90-min chase periods, however, no difference was observed after culture at 11 and 28 mM glucose. ELISA measurements revealed that culture at increased glucose concentrations as well as islet exposure to IL-1beta increased proinsulin accumulation in the culture media. The mRNA expression of PC1 was increased after culture at 11 and 28 mM glucose. Treatment for 48 h with IL-1beta increased the proportion of proinsulin both at 45 and 90 min when compared with control islets. These islets also displayed a decreased mRNA level of PC1 as well as PC2. Calculations of the half-time for proinsulin demonstrated a significant prolongation after treatment with IL-1beta. We conclude that a sustained functional stimulation by glucose of islets is coupled to a decreased conversion of proinsulin which is also true for islets treated with IL-1beta. This may contribute to the elevated levels of proinsulin found both at the onset of type 1 diabetes as well as in type 2 diabetes.

Prolactin regulation of the expression of TNF-alpha, IFN-gamma and IL-10 by splenocytes in murine multiple low dose streptozotocin diabetes.

Previously, the hormone prolactin (PRL) has been found to protect against development of type 1 diabetes induced by multiple injections of streptozotocin (STZ) in mice. To further investigate this effect of PRL, C57BL/Ks mice were injected intraperitoneally with STZ (40 mg/kg body weight) or NaCl for 5 days and PRL (4 mg/kg body weight) or NaCl for 14 days. On day 15, splenocytes were isolated from the in vivo treated mice. Spleen cell preparations depleted in erythrocytes and macrophages were stained for cytoplasmic TNF-alpha, IFN-gamma and IL-10 and analyzed with flow cytometry. Isolated spleen cells were also cultured (RPMI 1640+10% fetal bovine serum) for 24 h. Thereafter, cytokine mRNA expression by the spleen cells was measured by real-time PCR and cytokine secretion determined by enzyme linked immunosorbent assay (ELISA). Freshly isolated spleen cell preparations from PRL and STZ+PRL treated animals seemed to have an increased frequency of IL-10 positive cells compared to controls. In cultured spleen cells isolated from STZ treated mice, IFN-gamma and IL-10 mRNA expression was up-regulated. PRL treatment down-regulated the mRNA expression of these cytokines and also TNF-alpha in the splenocytes obtained from animals treated with STZ. The accumulation of these cytokines in the cultures of the explanted splenocytes showed only minor differences between the experimental groups. Overall, the data seems to favor the view that PRL enhanced a Th2 response, which may reflect the preventive effect of PRL against development of multiple low dose STZ diabetes in mice.

Survival of an islet allograft deficient in iNOS after implantation into diabetic NOD mice.

Proinflammatory cytokines play a major role in rejection of pancreatic islet allografts and in type 1 diabetes (T1D). In rodent islets, exposure to IL-1beta alone or combined with IFN-gamma induces expression of inducible nitric oxide synthase (iNOS). Inhibition of iNOS or a deletion of the iNOS gene has been shown to be protective in animal models of T1D. In the present study we tested the hypothesis that transplantation of pancreatic islets deficient in iNOS (iNOS-/-) would permit increased graft survival. Pancreatic islets isolated from wild-type (wt) mice and iNOS-/- mice were allogeneically transplanted beneath the kidney capsule of spontaneously diabetic NOD mice. When blood glucose increased above 12.0 mM after preceding normalization of hyperglycemia, animals were sacrificed. Histological examinations of grafts were performed and graft gene expression was analyzed by real-time PCR. Transplantations of the two types of islets could reverse hyperglycemia and the grafts functioned for on average 1 week posttransplantation. Morphological examination of both types of islet grafts showed immune cell infiltration around and within the grafts. Remaining endocrine cells could be observed in wt and iNOS-/- islet grafts. In the removed grafts iNOS-/- islet tissue contained higher mRNA levels of insulin, proinsulin convertases (PC-1 and PC-2), and IL-1beta compared to transplanted wt islets. The assessments of insulin, PC-1 and PC-2 mRNAs of the grafts suggest that the iNOS-/- islets may be more resistant to destruction in the transplantation model used; however, this was not sufficient to prolong the period of normoglycemia posttransplantation.

Cytokines affect PDX-1 expression, insulin and proinsulin secretion from iNOS deficient murine islets.

In rodent islets, exposure to interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) induces expression of inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) formation, which may inhibit islet function. However, cytokines may also induce NO-independent islet suppression. The present aim was to investigate the effect of cytokine exposure to iNOS deficient (iNOS-/-) mouse islets on various islet functions. Islets from iNOS-/- and wt mice exposed to IL-1beta or (IL-1beta + IFN-gamma) for 2-20 h showed different kinetics of glucose-stimulated insulin secretion. In iNOS-/- islets, IL-1beta at high glucose induced a delayed and prolonged stimulation of insulin secretion, and this was followed by an increase in phospholipase D mRNA expression. After 6 and 24 h, proinsulin convertase 1 and 2 (PC1 and PC2) mRNA expression was suppressed and proinsulin secretion increased from wt islets. In iNOS-/- islets, PC1 expression was recovered after 24 h, and there was no difference in proinsulin secretion. PDX-1 mRNA expression was suppressed independent of NO-formation. We conclude that cytokines induce both NO-dependent and NO-independent functional inhibition of murine beta-cells.

Morbidity and immune response to natural schistosomiasis in baboons ( Papio anubis).

The morbidity and immunological response to naturally acquired Schistosoma mansoni infection in a population of wild baboons ( n=28) was investigated. Serum obtained from the baboons was assayed for adult worm (SWAP) and schistosome egg (SEA)-specific immunoglobulin (Ig)G and IgM antibodies. The animals were euthanised, perfused to recover adult schistosome worms and schistosome-related pathology was assessed. Nineteen animals (68%) had high serum levels of SWAP-specific IgG antibodies and 15 (54%) had high levels of SEA-specific IgG antibodies. Nine animals (32%) had high levels of SWAP-specific IgM antibodies and six (21%) had high levels of SEA-specific IgM antibodies. Mild schistosome-related pathology was noted in 18 animals (64%). However, adult schistosome worms were recovered from only three animals (10%). The results indicate a high exposure to schistosomiasis for free-ranging baboons inhabiting an endemic area, as evidenced by the high prevalence of parasite-specific humoral antibody response. However, this high exposure is associated with low worm recovery and mild pathology. In addition, parasite-specific IgM antibodies provided a good indicator of an active schistosome infection.