Inhibition of Monoacylglycerol Lipase Activity Decreases Glucose-Stimulated Insulin Secretion in INS-1 (832/13) Cells and Rat Islets

Abstract

Lipid signals derived from lipolysis and membrane phospholipids play an important role in glucose-stimulated insulin secretion (GSIS), though the exact secondary signals remain unclear. Previous reports have documented a stimulatory role of exogenously added mono-acyl-glycerol (MAG) on insulin secretion from cultured β-cells and islets. In this report we have determined effects of increasing intracellular MAG in the β-cell by inhibiting mono-acyl-glycerol lipase (MGL) activity, which catalyzes the final step in triacylglycerol breakdown, namely the hydrolysis of MAG to glycerol and free fatty acid (FA). To determine the role of MGL in GSIS, we used three different pharmacological agents (JZL184, MJN110 and URB602). All three inhibited GSIS and depolarization-induced insulin secretion in INS-1 (832/13). JZL184 significantly inhibited both GSIS and depolarization-induced insulin secretion in rat islets. JZL184 significantly decreased lipolysis and increased both mono- and diacyglycerol species in INS-1 cells. Analysis of the kinetics of GSIS showed that inhibition was greater during the sustained phase of secretion. A similar pattern was observed in the response of Ca2+ to glucose and depolarization but to a lesser degree suggesting that altered Ca2+ handling alone could not explain the reduction in insulin secretion. In addition, a significant reduction in long chain-CoA (LC-CoA) was observed in INS-1 cells at both basal and stimulatory glucose following inhibition of MGL. Our data implicate an important role for MGL in insulin secretion.

Fig 1. Inhibition of MGL reduced GSIS in INS-1 cells and isolated rat islets.

(A) JZL184 dose-dependently reduced GSIS during static incubations in INS-1 cells (n = 3). (B) JZL184 (10 μM) reduced GSIS in INS-1 cells regardless of the temporal exposure (n = 3). (C) INS-1 cells demonstrated a partial recovery of GSIS following the removal of JZL184 (10 μM) for 2 hours (n = 3). (D) MJN110 dose-dependently reduced GSIS during static incubations in INS-1 cells (n = 4). (E) URB602 (50 μM) inhibited GSIS during static incubation in INS-1 cells (n = 3). (F) JZL184 dose-dependently reduced GSIS in isolated whole rat islets during a static incubation (n = 3). Error bars represent S.E. from independent experiments (*, p<0.05).

Fig 2. JZL184 reduced sustained insulin secretion during glucose stimulation in whole isolated rat islets.

Islets were perifused with basal glucose (3 mM) KRB buffer for approximately 10 minutes followed by a 30-minute perifusion with 15 mM glucose with or without 10 μM JZL184. At 40 minutes the islets were perifused with 15 mM glucose KRB with 0.4 mM DZ and 30 mM KCl with or without 10 μM JZL184. n = 3 independent experiments from different islet isolations. Error bars represent S.E. and are displayed every 4^th data point for clarity. All points after 23.1 minutes were significantly different (p<0.05).

Fig 3. JZL184 inhibited KCl-induced insulin secretion.

Insulin secretion from batch incubations of (A) INS-1 cells and (B) dissociated rat islets (n = 3 independent experiments for both A and B). Error bars represent S.E. (*, p<0.05).

Fig 4. Exposure to JZL184 reduced glycerol release and increased MAG species in INS-1 cells.

(A) Incubation with 10 μM JZL184 time-dependently reduced glycerol release at both basal and stimulatory glucose (n = 6). (B) Acute exposure to JZL184 for 30 minutes increased total MAG species at both basal and stimulatory glucose. (C) Quantitation of total MAGs from 4 experiments. Error bars represent S.E. from independent experiments (*, p<0.05).

Fig 5. Small decreases in Ca²⁺ influx following exposure to JZL184 in isolated rat islets and INS-1 cells.

(A.) Islets were brought from 3 mM glucose to 15 mM glucose with or without 10 μM JZL184 and cytosolic Ca²⁺ was documented using the probe fura-2 (n = 4). (B) INS-1 cells were brought from 2 mM glucose plus 0.25 mM DZ to 8 mM glucose with 0.25 mM DZ and 30 mM KCl with or without 10 μM JZL184 (n = 3). Ca²⁺ levels are expressed as the fura-2 ratio at 340/380 nm or the increment above basal in response to treatment (peak and sustained).

Fig 6. Significant reduction in total LC-CoA following acute treatment of INS-1 cells with JZL184.

Cells were exposed to JZL184 at both basal and stimulatory glucose and LC-CoA was measured as described in the methods (n = 3). Error bars represent S.E. from independent experiments (*, p<0.05).