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

<p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p><h3>List of scientific papers</h3><p>I. Ma Z, Portwood N, Foss A, Grill V, Bjorklund A (2005). Evidence that insulin secretion influences SNAP-25 through proteasomal activation. Biochem Biophys Res Commun. 329(3): 1118-26. <br><a href="https://doi.org/10.1016/j.bbrc.2005.02.086">https://doi.org/10.1016/j.bbrc.2005.02.086</a><br><br> </p><p>II. Yoshikawa H, Ma Z, Bjorklund A, Grill V (2004). Short-term intermittent exposure to diazoxide improves functional performance of beta-cells in a high-glucose environment. Am J Physiol Endocrinol Metab. 287(6): E1202-8. <br><a href="https://doi.org/10.1152/ajpendo.00255.2004">https://doi.org/10.1152/ajpendo.00255.2004</a><br><br> </p><p>III. Ma Z, Portwood N, Brodin D, Grill V, Björklund A (2006). Effects of diazoxide on gene expression in rat pancreatic islets are largely linked to elevated glucose and potentially serve to uphold beta cell sensitivity. Diabetes. [Accepted] <br><a href="https://doi.org/10.2337/db06-0322">https://doi.org/10.2337/db06-0322</a><br><br> </p><p>IV. Ma Z, Lavebratt C, Almgren M, Portwood N, Falkmer S, Björklund A (2006). Presence and functional importance of Kv1.1 channel in mouse islets: Evidence from mice with truncated Kv1.1. [Manuscript]</p>