Effects of Ca2+, microRNAs, and rosuvastatin on insulin-secreting beta cell function

Type 2 diabetes (T2D) is a condition of high blood glucose levels due to insulin resistance and defective insulin secretion. Impaired insulin secretion plays a major role in the pathophysiology of T2D, it is mainly attributed to beta cell function i.e. failure to secrete insulin or reduced beta cell mass. The exocytotic process is crucial for beta cell function and its dysregulation leads to impaired insulin secretion. Therefore, understanding the central mechanisms involved in the regulation of exocytosis is essential to recognize possible targets for therapeutic intervention and treatment of T2D. In this thesis I have investigated the role of Ca2+, miRNAs and rosuvastatin in the regulation of ion channels, exocytosis and insulin secretion of beta cells. For this purpose, pancreatic rat INS-1 832/13 beta cells, rodent animal models, and islets from human cadaver donors has been used. Whole-cell patch clamp was used to study exocytosis measured as changes in cell membrane capacitance. In beta cells, biphasic exocytotic pattern was previously mainly attributed to insulin granule pool depletion. In paper I, we used the pulse length protocol and mixed-effect modelling; the latter takes care of cellular heterogeneity, to study exocytosis as a function of Ca2+ influx (measured as Q). The data suggests that pool depletion plays a minor role in observed biphasic exocytotic pattern in INS-1 832/13 cells; instead exocytosis is mostly determined by the kinetics of Ca2+ current inactivation. In paper II and III, we have investigated the effects of miRNA modulation on insulin secretion and exocytosis. First we investigated miRNA-regulation of voltage-gated Na+ channels since their role in beta cell function is not yet clear. Down-regulation of miR-375 differentially affected Na+ channel inactivation properties in INS-1 832/13 cells and miR-375 knock-out mice beta cells, suggesting species differences. As steady-state inactivation determines the number of channels available for generation of action potential, this study is a proof of principle that mir-375 could be important in regulating electrical activity in human beta cells. Next, miRNA-regulation of the exocytotic process was investigated. Overexpression of miR-335 reduced exocytosis and thereby insulin secretion through decreased expression of the exocytotic proteins STXBP1, SNAP25 and SYT11. In this paper I also made the novel observation that SYT11 increase basal insulin secretion and decrease rapid exocytosis, two phenomenons associated with T2D. The work on miR-335 emphasizes the importance of miRNAs in the regulation of the exocytotic process. In paper IV and V the effects of the cholesterol-lowering drug rosuvastatin was investigated. Rosuvastatin treatment dose dependently affected Ca2+ influx, exocytosis, basal and glucose-induced insulin secretion in INS-1 832/13 cells. Interestingly, most of this effect was though mevalonate pathway and not from the cholesterol lowering ability of rosuvastatin. In vivo rosuvastatin had an overall positive effect on glucose homeostasis in mice but negative effects on beta cell function such as disturbed Ca2+-signalling. In conclusion, the data in my thesis demonstrate the need for investigations of the mechanisms behind defective insulin secretion and exocytosis in order to understand and treat T2D

Regulation of Pancreatic Beta Cell Stimulus-Secretion Coupling by microRNAs.

Increased blood glucose after a meal is countered by the subsequent increased release of the hypoglycemic hormone insulin from the pancreatic beta cells. The cascade of molecular events encompassing the initial sensing and transport of glucose into the beta cell, culminating with the exocytosis of the insulin large dense core granules (LDCVs) is termed "stimulus-secretion coupling." Impairment in any of the relevant processes leads to insufficient insulin release, which contributes to the development of type 2 diabetes (T2D). The fate of the beta cell, when exposed to environmental triggers of the disease, is determined by the possibility to adapt to the new situation by regulation of gene expression. As established factors of post-transcriptional regulation, microRNAs (miRNAs) are well-recognized mediators of beta cell plasticity and adaptation. Here, we put focus on the importance of comprehending the transcriptional regulation of miRNAs, and how miRNAs are implicated in stimulus-secretion coupling, specifically those influencing the late stages of insulin secretion. We suggest that efficient beta cell adaptation requires an optimal balance between transcriptional regulation of miRNAs themselves, and miRNA-dependent gene regulation. The increased knowledge of the beta cell transcriptional network inclusive of non-coding RNAs such as miRNAs is essential in identifying novel targets for the treatment of T2D

Calcium Current Inactivation Rather than Pool Depletion Explains Reduced Exocytotic Rate with Prolonged Stimulation in Insulin-Secreting INS-1 832/13 Cells

Impairment in beta-cell exocytosis is associated with reduced insulin secretion and diabetes. Here we aimed to investigate the dynamics of Ca2+-dependent insulin exocytosis with respect to pool depletion and Ca2+-current inactivation. We studied exocytosis, measured as increase in membrane capacitance (ΔCm), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis. The observed linear relationship between ΔCm and Q suggests that Ca2+-channel inactivation rather than granule pool restrictions is responsible for the decline in exocytosis observed at longer depolarizations. INS-1 832/13 cells possess an immediately releasable pool (IRP) of ∼10 granules and most exocytosis of granules occurs from a large pool. The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected. These findings suggest that most insulin release occurs away from Ca2+-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation

Modulation of microRNA-375 expression alters voltage-gated Na(+) channel properties and exocytosis in insulin-secreting cells.

MiR-375 has been implicated in insulin secretion and exocytosis through incompletely understood mechanisms. Here we aimed to investigate the role of miR-375 in the regulation of voltage-gated Na(+) channel properties and glucose-stimulated insulin secretion in insulin-secreting cells

Mixed-effects analysis of the pulse-length data from the second pulse, following a 50 ms prepulse, in the control and EGTA groups.

<p>Capacitance data (ΔCm) plotted against Ca²⁺ influx measured as charge (Q) from individual cells with single-cell fits indicated by solid lines, while group fits (fixed-effects) are given by the dashed lines. Ctrl (panel 1–14); EGTA (panel 15–32).</p

Analysis of the double-pulse data.

<p>The exocytotic response (ΔC_m) is plotted against Ca²⁺-influx (charge; Q). Capacitance increases and Ca²⁺ influxes evoked by the first 50 ms-depolarization (Pulse 1) are shown as open symbols with fixed-effects fits indicated by dashed lines, while data evoked by the second 50-ms depolarization (Pulse 2) are plotted as filled symbols with their fixed-effects fits given by the solid lines. The colors and symbols indicate the groups (Black circles and lines: CTRL; Blue triangles and lines: EGTA). The graph contains data from n = 14 and n = 18 experiments from the control and EGTA group, respectively.</p

Illustration of the protocol where a 50 ms prepulse is followed by depolarizations of varying length (50, 100, 200, 400, and 800 ms).

<p>The applied changes in membrane potential due to the depolarizations (V; top) evoke Ca²⁺-currents (ICa; middle) and increases in membrane capacitance (ΔCm; bottom). The panels show the data from a single cell in response to 3 (50 ms prepulse + 50, 200 or 800 ms depolarization) of the 5 double-pulses applied to the cell. We analyzed the data from the depolarizations of varying length following the prepulse.</p

Current recovery <i>Q</i>_{3,50 ms}/<i>Q</i>₁ (see text) for the different durations of the second pulse and 200 ms (protocol I, squares) or 10 s (protocol II, crosses) resting period.

<p>Current recovery <i>Q</i>_{3,50 ms}/<i>Q</i>₁ (see text) for the different durations of the second pulse and 200 ms (protocol I, squares) or 10 s (protocol II, crosses) resting period.</p

No difference in Ca²⁺ current sensitivity between protocol I and protocol II.

<p>Capacitance increases are plotted vs. Ca²⁺ influx <i>Q</i> for the third 500-ms depolarizations following a 50 ms prepulse, a second pulse of either 200 ms (squares), 400 ms (circles) or 800 ms (triangles), and a resting period of either 200 ms (protocol I, black) or 10 s (protocol II, gray). The line indicates the fixed-effects fit from the linear mixed-effects model, which fitted the entire data set (both protocols).</p