Effect of mevalonate and squalene on insulin secretion from rosuvastatin-treated INS-1 832/13 cells.

<p>(A) Insulin secretion at basal (2.8 mM) concentrations of glucose from cells treated with 20 μM rosuvastatin (Ros), 50 μM mevalonate (MVA), and 100 μM squalene (Sq) for 48 h as indicated in the figure. (B) Same as in (A) but with stimulatory (16.7 mM) concentrations of glucose instead. (C) Same as in (A) but the cells are stimulated with 50 mM K⁺ as well. Data are given as mean ± SEM from 3 experiments with 3 technical replicates in each experiment. * p≤ 0.05.</p

Effects of 48 h of rosuvastatin treatment on insulin secretion in INS-1 832/13 cells.

<p>(A) Insulin secretion at 2.8 mM glucose measured in the presence of rosuvastatin (Ros) at concentrations ranging from 20 nM-20 μM as indicated in the figure. Statistical significance is calculated compared to the control (DMSO). (B) Same as in (A) but insulin secretion is measured at 16.7 mM glucose instead. (C) Insulin secretion at 2.8 mM glucose from cells treated with 20μM rosuvastatin, 200 μM diazoxide (Dzx) or a combination of the two. (D) Insulin secretion at 2.8 mM glucose and 50 mM K⁺ with and without 20μM rosuvastatin. (E) Insulin secretion at 16.7 mM glucose from cells treated with 100 nM GLP-1 with and without 20 μM rosuvastatin. Data are given as mean ± SEM from 3 experiments with 3 technical replicates in each experiment. * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001.</p

Effects of 24–48 h of rosuvastatin treatment on exocytosis in INS-1 832/13 cells.

<p>(A) Example traces of depolarization-induced exocytosis measured as changes in cell membrane capacitance, from rosuvastatin-treated cells (20 μM; black trace) and control cells (grey trace). Exocytosis was evoked by a train of ten 500 ms depolarizing pulses from -70 mV to 0 mV. (B) Summary of the total capacitance change during the train in control cells (white bars) and rosuvastatin-treated cells (Ros; black bars). The concentration of rosuvastatin in these experiments ranged from 20 nM-20 μM as marked in the figure. (C) A graph describing the exocytotic response to all 10 pulses (∑_all) to the first 2 pulses (∑_1–2) or to the latter 8 pulses (∑_3–10) in cells incubated with 20 μM rosuvastatin (black bars) and their controls (white bars). (D) Calcium sensitivity of cells incubated with 20 μM rosuvastatin (Ros; black bars) or their controls (white bars). Calcium sensitivity is calculated by dividing the exocytotic response to the first pulse with the calcium charge measured during the same pulse. Data are given as mean ± SEM of 21–31 cells. * p≤ 0.05; ** p≤ 0.01.</p

Electrophysiological characterization of voltage-gated ion channels in rosuvastatin treated INS-1 832/13.

<p>Cells were treated with 20 μM rosuvastatin for 24-48h. (A) Example traces of currents evoked by a depolarization to 0 mV in a single rosuvastatin-treated (Ros; black trace) and control (grey trace) cell. I_sus and I_p measured in (B) and (D) are marked. (B) Sustained current (I_sus)-voltage (V) relationship (C) charge (Q)-voltage (V) relationship. Charge is measured as the area enclosed by the curve in (A). (D) peak current (I_p)-voltage (V) relationship in INS-1 832/13 cells treated with 20 μM rosuvastatin (Ros; black dots) or control cells (Control; white squares). Data are given as mean ± SEM of 28–38 cells.</p

Example traces from intracellular Ca²⁺ measurements in WT and OPN^-/- islets.

<p>Representative examples of Ca²⁺ traces obtained from an islet from a WT (A) or OPN^-/- (B) mouse. Marked on the traces are the measurements mentioned in the text. C₀ is the area of the Ca²⁺ dip that initially occurs upon glucose-stimulation. C₁ is the amplitude of the first peak. 16.7 G is 16.7 mM glucose and 2.8 G is 2.8 mM glucose. The baseline is marked with a dashed line. Histogram of the cytoplasmic Ca²⁺ concentration (measured as area under the curve but above the baseline; C), C₀ (D), C₁ (E), and the frequency of ocillations during the sustained phase (F) in islets from WT (white bars) and OPN^-/- (black bars) mice. Data are obtained from 16–21 islets from 3 mice per condition. ** p≤ 0.01; *** p≤ 0.001.</p

Pancreatic slices from OPN^-/- and WT mice have similar histology.

<p>Representative images from (A) eosin and hematoxylin stainings and (B) insulin stainings on pancreatic slices from WT (left) and OPN^-/- (right) mice. Scale bar 100 μm.</p

Altered cell-cell connections and insulin granule distribution in beta cells from OPN^-/- mice.

<p>(A) Ultrastructural images from a section of a WT (left) and OPN^-/- (right) islet. Scale bar 5 μM. (B) Ultrastructural images from a section of an OPN^-/- islet. The atypical structure on the beta cell is marked with a black arrow. Scale bar 2μM. (C, D, and E) Histogram of the calculated volume density (C), surface density (D), and distribution of the insulin containing large dense-core vesicles (LDCVs; E) in pancreatic beta cells from WT and OPN^-/- mice. Data are given as mean ± SEM from 24–26 cells taken from 3 animals per condition. ** p≤ 0.01; *** p≤ 0.001.</p

Expression pattern of proteins associated with ER and Ca²⁺ handling in WT and OPN^-/- islets.

<p>Expression pattern of <i>Mig 6</i> (A), <i>SEl1L</i> (B), <i>Atp2A2</i> (C) and <i>ATP 2A3</i> (D) in islets from WT and OPN^-/- mice as marked in the figure. Data are described using the median with interquartile range for 6 biological replicates with 3 technical replicates in each experiment. * p≤ 0.05.</p

Deletion of OPN has modest effects on metabolism.

<p>(A) Body weight in WT and OPN^-/- mice measured at 12 weeks. (B) Non-fasted blood glucose levels from WT and OPN^-/- mice measured at 12 weeks. (C) Insulin content in isolated islets from WT and OPN^-/- mice. (D) Glucose-induced insulin secretion at 11.1 mM glucose from isolated WT and OPN^-/- islets described as fold increase over basal insulin secretion at 2.8 mM glucose with and without the addition of 200 ng/ml OPN and/or 100 nM GIP. (E) Same experiment as in (D) but displaying the non-processed data as ng/islet/h. Data are given as mean ± SEM from 28–30 animals (A and B) or from 4 biological experiments with 3 technical replicates in each experiment (C, D, and E). * p≤ 0.05.</p