Exposure of human islets to cytokines can result in disproportionately elevated proinsulin release.

Infiltration of immunocytes into pancreatic islets precedes loss of beta cells in type 1 diabetes. It is conceivable that local release of cytokines affects the function of beta cells before their apoptosis. This study examines whether the elevated proinsulin levels that have been described in prediabetes can result from exposure of beta cells to cytokines. Human beta-cell preparations were cultured for 48 or 72 hours with or without IL-1beta, TNF-alpha, or IFN-gamma, alone or in combination. None of these conditions were cytotoxic, nor did they reduce insulin biosynthetic activity. Single cytokines did not alter medium or cellular content in insulin or proinsulin. Cytokine combinations, in particular IL-1beta plus IFN-gamma, disproportionately elevated medium proinsulin levels. This effect expresses an altered functional state of the beta cells characterized by preserved proinsulin synthesis, a slower hormone conversion, and an increased ratio of cellular proinsulin over insulin content. The delay in proinsulin conversion can be attributed to lower expression of PC1 and PC2 convertases. It is concluded that disproportionately elevated proinsulin levels in pre-type 1 diabetic patients might result from exposure of their beta cells to cytokines released from infiltrating immunocytes. This hormonal alteration expresses an altered functional state of the beta cells that can occur independently of beta-cell death.

Prolonged exposure of human beta-cells to high glucose increases their release of proinsulin during acute stimulation with glucose or arginine.

The disproportionate hyperproinsulinemia in type 2 diabetes has been attributed to either a primary beta-cell defect or a secondary dysregulation of beta cells under sustained hyperglycemia. This study examines the effect of a 10- to 13-day exposure to 20 mmol/L glucose on subsequent proinsulin and insulin release by human islets isolated from nondiabetic donors. Compared to control preparations kept at 6 mmol/L glucose, the high glucose cultured beta-cells released more proinsulin and less insulin during perifusion at 5, 10, or 20 mmol/L glucose. The lower amounts of secreted insulin resulted from a marked reduction in cellular insulin content (5-fold lower than in controls). The higher amount of secreted proinsulin is attributed to the sustained state of cellular activation that is known to occur after prolonged exposure to high glucose levels. This activated state of the beta-cell population is also held responsible for its higher secretory responsiveness to 5 mmol/L arginine at a submaximal (5 mmol/L) glucose concentration (8-fold higher proinsulin levels than in the control population). It results, together with the reduction in cellular insulin content, in 7- to 10-fold higher proinsulin over insulin ratios in the medium; at 5 mmol/L glucose, this extracellular ratio is similar to that in the cells. These data add direct support to the view that a disproportionate hyperproinsulinemia can result from a sustained activation of human beta-cells after prolonged exposure to elevated glucose levels.

Production of enzymatically active rat protein disulfide isomerase in Escherichia coli.

We report the development of a bacterial expression system allowing high-level synthesis of enzymatically active rat protein disulfide isomerase (rPDI). After expression of the rpdi gene under control of the inducible trc promoter (Ptrc), a significant amount of soluble, active rPDI was detected in the periplasmic contents, which were released from the cells by cold osmotic shock. However, the exported molecules were incompletely or improperly processed, while the major amount of synthesized rPDI was in fact detected in the soluble cellular fraction. Substitution of the autologous eukaryotic export signal with the nucleotide (nt) sequence encoding the signal peptide (sOmpA) of the bacterial outer membrane protein A, and expression of the sompA::rpdi fusion gene under control of both the lpp promoter (Plpp) and the lac promoter-operator (POlac), resulted in high-level production of rPDI. Furthermore, the latter was efficiently exported into the periplasmic compartment, from where it was recovered as a soluble, fully active form with the sOmpA precisely removed. The synthesis of a small 21-kDa peptide accompanying the production of rPDI was also observed. This rPDI-related peptide (rPDIf), which represented a C-terminal fragment of rPDI including the second active site, arose by internal translation initiation within rpdi. Replacement of the presumed internal start codon by CTC completely eliminated the aforementioned phenomenon and resulted in the production of a slightly mutated, enzymatically active enzyme (rPDIm).