G-protein-coupled receptor 40 agonist GW9508 potentiates glucose-stimulated insulin secretion through activation of protein kinase Cα and ε in INS-1 cells

Abstract

Objective: The mechanism by which G-protein-coupled receptor 40 (GPR40) signaling amplifies glucose-stimulated insulin secretion through activation of protein kinase C (PKC) is unknown. We examined whether a GPR40 agonist, GW9508, could stimulate conventional and novel isoforms of PKC at two glucose concentrations (3 mM and 20 mM) in INS-1D cells.

Methods: Using epifluorescence microscopy, we monitored relative changes in the cytosolic fluorescence intensity of Fura2 as a marker of change in intracellular Ca2+ ([Ca2+]i) and relative increases in green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) as a marker of PKC activation in response to GW9508 at 3 mM and 20 mM glucose. To assess the activation of the two PKC isoforms, relative increases in membrane fluorescence intensity of PKCα-GFP and PKCε-GFP were measured by total internal reflection fluorescence microscopy. Specific inhibitors of each PKC isotype were constructed and synthesized as peptide fusions with the third α-helix of the homeodomain of Antennapedia.

Results: At 3 mM glucose, GW9508 induced sustained MARCKS-GFP translocation to the cytosol, irrespective of changes in [Ca2+]i. At 20 mM glucose, GW9508 induced sustained MARCKS-GFP translocation but also transient translocation that followed sharp increases in [Ca2+]i. Although PKCα translocation was rarely observed, PKCε translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKCα and sustained translocation as well as transient translocation of PKCε. While the inhibitors (75 μM) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKCε inhibitor had a more potent effect.

Conclusion: GW9508 activated PKCε but not PKCα at a substimulatory concentration of glucose. Both PKC isotypes were activated at a stimulatory concentration of glucose and contributed to glucose-stimulated insulin secretion in insulin-producing cells.

Fig 1. GW9508 enhances insulin secretion only at 20 mM glucose in INS-1D cells.

INS-1D cells were incubated for 1 h with Krebs-Ringer buffer (KRB) containing 3 mM or 20 mM glucose in the presence or absence of GW9508, an agonist of G-protein coupled receptor 40 (GPR40). Data shown are mean ± standard error of the mean of three independent experiments with triplicate samples in each group. *p < 0.05 vs. absence of GW9508; #p < 0.05 vs. 3 mM glucose.

Fig 2. GW9508 induces sustained MARCKS-GFP translocation at 3 mM glucose.

Experiments were performed using epifluorescence microscopy. Simultaneous monitoring of green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP; filled circles) translocation (as a marker of PKC activation) and intracellular Ca²⁺ concentration ([Ca²⁺]_i) (open circles). The intensity of Fura2 for [Ca²⁺]_i measurement is represented as the 340/380 nm ratio. These values and the fluorescence intensity (F) of MARCKS-GFP were normalized to each initial value (F₀; ratio [Ca²⁺]_i and MARCKS F/F₀). INS-1D cells were perfused with extracellular solution containing 3 mM glucose at 1 mL per minute from 5 minutes before the start of the experiment to the end. To avoid light-induced cell damage, monitoring was paused from 7 to 16 minutes after starting the experiment. At 16 minutes, the distribution of MARCKS-GFP in cells was similar to that observed just after starting the experiment. Images were taken at the times indicated by the arrows. The bar represents 10 μm. The regions of interest in the cytosol are indicated by a white box. When MARCKS-GFP (green) was translocated from the plasma membrane to the cytosol, MARCKS F/F₀ in the cytosol increased. When the fluorescence intensity at 380 nm (red) declined, [Ca²⁺]_i, represented as the 340/380 nm ratio, increased (eight independent experiments, 81 cells).

Fig 3. GW9508 evokes transient translocation of MARCKS-GFP following Ca²⁺ oscillations at 20 mM glucose.

(A, B): Representative data showing the translocation of green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) and intracellular Ca²⁺ concentration [Ca²⁺]_i under stimulation by GW9508 in an extracellular solution containing (A) 3 mM glucose (the same data as in Fig 2 but with different scales; eight independent experiments, 81 cells) or (B) 20 mM glucose (four independent experiments, 80 cells). Biphasic translocation of MARCKS-GFP occurred after the second sharp increase in Ca²⁺ at 20 mM glucose. Scatterplots of the ratio of [Ca²⁺]_i-related increases in the relative fluorescence intensity of MARCKS-GFP in the cytosol versus the elevation in ratio [Ca²⁺]_i during GW9508 application at (C) 3 mM glucose (eight independent experiments, 56 cells) or (D) 20 mM glucose (four independent experiments, 63 cells). Cells that did not respond to GW9508 were excluded from the calculation. To count the cells that responded to GW9508, only cells that were almost unchanged in fluorescence of MARCKS-GFP before the application of GW9508 were chosen for study at 3 mM glucose. (E, F) To equalize the [Ca²⁺]_i elevation, only cells with an [Ca²⁺]_i ratio elevated by 1.5 were selected for analysis from C (all 56 cells) and D (47 cells) (dashed box in C and D).

Fig 4. Temporal profile of PKCα and PKCε translocation at 3 or 20 mM using total internal reflection fluorescence microscopy.

Representative data of the relative change in fluorescence intensity (F/F₀) of PKCα-GFP (A, B) or PKCε-GFP (C, D) at the plasma membrane. Images a-f were taken at the times indicated in each graph. Scale bars = 10 μm. When green fluorescent protein (GFP)-tagged PKCα or PKCε is translocated from the cytosol to the plasma membrane, F/F₀ increases. (A, B) Translocation of PKCα-GFP evoked by GW9508 at 20 mM glucose (B; 15 independent experiments, 165 cells) was higher than that at 3 mM glucose (A; 19 independent experiments, 283 cells), but lower than that induced by 40 mM KCL. (C, D) Translocation of PKCε-GFP induced by GW9508 at 20 mM glucose (D; 15 independent experiments, 57 cells) was biphasic, higher than that at 3 mM glucose (C; 18 independent experiments, 47 cells), and comparable with that induced by 40 mM KCL.

Fig 5. PKC inhibitors suppress GW9508-induced insulin secretion in INS-1D cells.

INS-1D cells were incubated for 1 h in Krebs-Ringer buffer containing 3 mM or 20 mM glucose with stimulation by GW9508 in the absence or presence of 75 μM antennapedia (antp), 75 μM antp-PKCα (antpα), 75 μM antp-PKCε (antpε), both antpα and antpε, 1 μM Gö 6976, or 1 μM bisindolylmaleimide I (BIS I). Data are shown as mean ± standard error of the mean of 12 independent experiments with triplicate samples in each group. *p < 0.05; **p < 0.01; #p < 0.05 vs. GW9508 at 20 mM glucose; ##p < 0.01 vs. GW9508 at 20 mM glucose.