Aspects on the modulation of potassium channels in insulin-producing beta-cells

This thesis attempts to further clarify mechanisms behind the negative effects of overstimulation and the beneficial effect of β-cell rest. For this purpose diazoxide - opener of K+-ATP channels, which reversibly inhibits glucose-induced insulin secretion, was used as a probe. Additionally, another type of potassium channel namely voltage dependent potassium channels (Kv-channels) specifically Kv1.1 has been tested for functional effects. Long-term (24 h) exposure of SD rat islets to elevated glucose (27 mmol/l) in vitro decreases glucoseinduced insulin response. The decrease is prevented by the co-culture with diazoxide. These effects were associated with reciprocal changes in certain exocytotic proteins (SNAP-25, syntaxin). Proteasomal inhibitors (MG132, ALLN and epoxomicin) but not a lysosomal inbitor (NH4Cl) blocked the inhibitory effects of diazoxide (tested for SNAP-25). This blocking effect was accompanied by a similar effect on glucose induced insulin secretion. The effects of short-term intermittent vs. continuous exposure to diazoxide in a high glucose environment appeared to have the same the benefit on K+-ATP dependent insulin secretion but not on K+-ATP independent insulin secretion. Intermittent and continuous diazoxide alike increased post-culture ATP-to-ADP ratios, failed to affect glucose oxidation, but decreased oleate oxidation. Continuous, but not intermittent, diazoxide decreased significantly mRNA for UCP-2. A 2 h exposure to 20 mmol/ll KCl or 10 µmol/l cycloheximide abrogated the postculture effects of intermittent, but not of continuous, diazoxide. Intermittent diazoxide islets levels of the SNARE protein SNAP-25, and KCl antagonized this effect. All in all, intermittent diazoxide exposure is sufficient to induce important functional changes in βcells. The overall effects by diazoxide on gene expression at high and low glucose were assessed by microarry. 114 genes were up-regulated (signal log2 ratio ≥0.5) and 173 genes down-regulated (signal log2 ratio ≤ -0.5) by diazoxide. 86% of diazoxide's effects (up and down regulation) were observed only after co-culture with 27 mmol/1 glucose. Up-regulation was to 3 1 % and down-regulation to 79 % contrary to effects of glucose per se. Diazoxide down-regulated genes of fatty acid oxidation and upregulated synthesis, whereas glucose per se had no effect. Irrespective of glucose concentration diazoxide up regulated certain genes which support β-cell functionality (nkx6.1 and pdx 1) and downregulated UCP-2, a potentially desensitizing gene. All in all, diazoxide effects were markedly glucose dependent and included genes known to be crucial for normal insulin secretion. The presence and functionality of Kv1.1 channels was assessed in BALB/cByJ mice and Kv1.1 truncated mceph/mceph mice islets. Gene expression (mRNA) was demonstrated in wild type and -as a smaller molecule- in mceph/mceph. Incremental glucose-induced insulin release was lower in BALB/cByJ than in mceph/mceph. Reciprocally, blocking Kvl.l by dendrotoxin-k increased secretion in BALB/cByJ but not in meeph/mceph mouse islets. These results strongly indicate the presence and functionality of Kv1.1 channels at least in mouse β-cells