Surface and intracellular Fas expression associated with cytokine-induced apoptosis in rodent islet and insulinoma cells.

During the process of insulitis in the pathogenesis of type I (insulin-dependent) diabetes mellitus, proinflammatory cytokines induce expression of the death receptor Fas on the surface of pancreatic beta-cells and thereby contribute to the enhanced susceptibility of beta-cells for apoptosis. The aim of this study was to compare cell-surface and intracellular Fas expression associated with cytokine-induced apoptosis in commonly used beta-cell models such as isolated islets and insulinoma lines derived from mouse and rat. The cell line NIT-1 responded to the interleukin (IL)-1beta+interferon (IFN)-gamma stimulus with translocation of Fas to the cell surface. Likewise, islet cells from non-obese diabetic (NOD) mice and BB/OK rats expressed increasing amounts of the Fas receptor on their surfaces after exposure to IL-1beta in combination with IFN-gamma and tumour necrosis factor-alpha. Moreover, islets obtained from BB/OK rats at an age near the onset of diabetes had an increased surface expression of Fas compared with young rats. In contrast, western blot analysis of cell lysates from cytokine-exposed islets and insulinoma cells revealed total Fas expression levels comparable to those of untreated controls. In conclusion, islets from BB/OK rats and NOD mice, in addition to NIT-1 insulinoma cells, responded to cytokine exposure with surface expression of the Fas receptor, whereas in cell lysates the levels of expression of Fas were found to be independent of cytokine exposure. Taken together, the findings indicate that cytokine-treated beta-cells might possess two pools of Fas protein, one of which is inducible by cytokines and accounts for surface Fas expression, whereas the other is constitutively expressed in cytoplasmic compartments. The underlying mechanisms, including possible interactions between these two sources of cellular Fas expression, need to be investigated in future studies.

Impact of metabolic activity of beta cells on cytokine-induced damage and recovery of rat pancreatic islets.

The influence of beta cell activity on cytokine-induced functional and structural impairments as well as the ability of those damaged cells to recover were investigated. Rat islets cultured for 4 days in the presence of 5, 10, and 30 mmol/l glucose were exposed to interferon-gamma (IFN, 500 U/ml) and tumor necrosis factor-alpha (TNF, 250 U/ml) for the last 24 h. After cytokine removal islets were allowed to recover spontaneously in culture medium containing 10 mmol/l glucose for a further 7 days. Cytokines significantly inhibited insulin release into culture medium, insulin storage, glucose-stimulated insulin secretion, protein, and DNA synthesis. In the presence of cytokines there was a six- to eightfold increase in nitrite production by the islets. The functional impairments were more pronounced in metabolically stimulated beta cells. In addition, cytokines caused membrane alterations as indicated by increased spontaneous chromium-51 release. The cytokines specifically induced the synthesis of two proteins (72 and 88 kDa, respectively). By immunoblotting, the 72-kDa protein was identified as heat shock protein. After a 1-week recovery period, insulin storage and stimulated insulin secretion of cytokine-treated islets were still significantly diminished. However, protein and DNA synthesis of cytokine-exposed islets returned to pre-exposure levels. In conclusion, high beta cell activity increases islet susceptibility to TNF+IFN. Cytokine-induced, long-lasting, inhibitory effects are primarily directed to beta-cell-specific functions, while general vital cell functions clearly recover after cytokine removal. The induction of certain proteins and the increased protein synthesis and replication rate after cytokine removal might reflect activated repair processes.

Human amniotic fluid obtained from diabetic women. A potent stimulator of islet cell replication.

Recently, human amniotic fluid (HAF) from healthy women was found to stimulate growth and function of pancreatic B-cells. Here, the effect of HAF and serum from healthy probands (HS) was compared with that from probands with gestational (GD), noninsulin-dependent (NIDDM), or insulin-dependent diabetes (IDDM) on islet function and replication. Rat islets were cultured in the presence of either HAF or HS for 7 d. Insulin content and basal insulin release were not different after exposure of the islets to HAF or HS from healthy or diabetic women. In contrast to HS, HAF provoked the islets to deliver significantly more insulin during culture. Additionally, the same islets exhibited a more intense response to a glucose challenge. The degree of HAF-induced insulin release was not influenced by the type of diabetes. HAF and HS from GD and NIDDM women did not influence the islet DNA synthesis in comparison to HAF and HS from healthy pregnant women. However, HAF but not HS from IDDM pregnant women, elicited a significant increase in islet replication. Most effective in stimulating islet cell replication were HAFs from IDDM pregnant women belonging to the White D-type. It was shown that the relatively high concentration of insulin in the HAFs was not directly responsible for the observed increase of the islet DNA synthesis. HAF from women with long-term diabetes is supposed to contain factor(s) that might directly or indirectly enhance islet replication.

[Protein biosynthesis following heat shock in Bacillus subtilis]. / Proteinbiosynthesen nach Hitzeschock in Bacillus subtilis.

Heat-activated spores of Bacillus subtilis synthesize during the early outgrowth special proteins which are absent from vegetative cells (Hecker 1983). Some of these outgrowth-specific proteins may be synthesized in response to heat activation of spores. In vegetative cells of B. subtilis synthesis of several proteins is markedly induced when temperature is shifted up from 30 to 44 degrees C. None of these putative heat shock proteins is synthesized during early outgrowth of heat-activated spores at 30 degrees C.

[Activation of protein biosynthesis in outgrowing spores of Bacillus subtilis]. / Aktivierung von Proteinbiosynthesen in auswachsenden Sporen von Bacillus subtilis.

The programme of protein synthesis as an indicator for the control of gene expression was examined during outgrowth of Bacillus subtilis spores. At various stages of outgrowth cells of Bacillus subtilis were labelled with 35S-L-methionine. Extracted proteins were separated on two-dimensional gels according to O'Farrell (1975). Three groups of proteins were synthesized during outgrowth: 1. During all stages of outgrowth a great number of "vegetative genes" is expressed. The programme of protein synthesis of the outgrowing cell is very similar to that of a vegetative cell. 2. Only a few proteins--probably the products of outgrowth-genes--are synthesized especially in outgrowing spores and turned off at different stages of outgrowth. 3. The synthesis of a minor group of vegetative proteins is triggered during different stages of outgrowth. In contrast to earlier assumptions (comp. Torriani and Levinthal 1967, Hansen et al. 1970, Galizzi et al. 1976) we suggest that only a small portion of the genome is activated during outgrowth as a dependent sequence. These results are discussed on the basis of earlier concepts about the regulation of outgrowth as a developmentally regulated gene expression programme.