Reactive Oxygen Species Stimulate Insulin Secretion in Rat Pancreatic Islets: Studies Using Mono-Oleoyl-Glycerol

Chronic exposure (24–72 hrs) of pancreatic islets to elevated glucose and fatty acid leads to glucolipoxicity characterized by basal insulin hypersecretion and impaired glucose-stimulated insulin secretion (GSIS). Our aim was to determine the mechanism for basal hypersecretion of insulin. We used mono-oleoyl-glycerol (MOG) as a tool to rapidly increase lipids in isolated rat pancreatic ß-cells and in the clonal pancreatic ß-cell line INS-1 832/13. MOG (25–400 µM) stimulated basal insulin secretion from ß-cells in a concentration dependent manner without increasing intracellular Ca2+ or O2 consumption. Like GSIS, MOG increased NAD(P)H and reactive oxygen species (ROS). The mitochondrial reductant ß-hydroxybutyrate (ß-OHB) also increased the redox state and ROS production, while ROS scavengers abrogated secretion. Diazoxide (0.4 mM) did not prevent the stimulatory effect of MOG, confirming that the effect was independent of the KATP-dependent pathway of secretion. MOG was metabolized to glycerol and long-chain acyl-CoA (LC-CoA), whereas, acute oleate did not similarly increase LC-CoA. Inhibition of diacylglycerol kinase (DGK) did not mimic the effect of MOG on insulin secretion, indicating that MOG did not act primarily by inhibiting DGK. Inhibition of acyl-CoA synthetase (ACS) reduced the stimulatory effect of MOG on basal insulin secretion by 30% indicating a role for LC-CoA. These data suggest that basal insulin secretion is stimulated by increased ROS production, due to an increase in the mitochondrial redox state independent of the established components of GSIS

Reactive Oxygen Species Stimulate Insulin Secretion in Rat Pancreatic Islets: Studies Using Mono-Oleoyl-Glycerol

Chronic exposure (24–72 hrs) of pancreatic islets to elevated glucose and fatty acid leads to glucolipoxicity characterized by basal insulin hypersecretion and impaired glucose-stimulated insulin secretion (GSIS). Our aim was to determine the mechanism for basal hypersecretion of insulin. We used mono-oleoyl-glycerol (MOG) as a tool to rapidly increase lipids in isolated rat pancreatic ß-cells and in the clonal pancreatic ß-cell line INS-1 832/13. MOG (25–400 µM) stimulated basal insulin secretion from ß-cells in a concentration dependent manner without increasing intracellular Ca2+ or O2 consumption. Like GSIS, MOG increased NAD(P)H and reactive oxygen species (ROS). The mitochondrial reductant ß-hydroxybutyrate (ß-OHB) also increased the redox state and ROS production, while ROS scavengers abrogated secretion. Diazoxide (0.4 mM) did not prevent the stimulatory effect of MOG, confirming that the effect was independent of the KATP-dependent pathway of secretion. MOG was metabolized to glycerol and long-chain acyl-CoA (LC-CoA), whereas, acute oleate did not similarly increase LC-CoA. Inhibition of diacylglycerol kinase (DGK) did not mimic the effect of MOG on insulin secretion, indicating that MOG did not act primarily by inhibiting DGK. Inhibition of acyl-CoA synthetase (ACS) reduced the stimulatory effect of MOG on basal insulin secretion by 30% indicating a role for LC-CoA. These data suggest that basal insulin secretion is stimulated by increased ROS production, due to an increase in the mitochondrial redox state independent of the established components of GSIS

Scavenging ROS with NAC (A, B) or Resveratrol (C) blocked MOG, ß-OHB and glucose-stimulated insulin secretion. The effect of MOG to stimulate basal (2 mM glucose) insulin secretion was not enhanced by the prescence of ß-OHB (D).

<p>A. 3 mM NAC (black bars) inhibited 10 mM ß-OHB-, 100 µM MOG- and 8 mM glucose-stimulated insulin secretion from INS-1 cells. B. 10 mM ß-OHB-stimulated insulin secretion was inhibited by NAC in a concentration-dependent manner (0.3–3 mM). C. 100 µM resveratrol (black bars) inhibited 10 mM ß-OHB-, 100 µM MOG- and 8 mM glucose-stimulated insulin secretion from INS-1 cells. D. The increase in insulin secretion stimulated by addition of ß-OHB and MOG together was equal to the sum of their individual effects. A–D. n = 9–12 per condition from 3 separate experiments. * p<0.005 compared to 2G control. A, C. # p<0.005 compared to its own control. B. + p,0.005 compared to no NAC. D. # p<0.005 compared to ß-OHB or MOG added separately.</p

MOG and ß-OHB generated ROS in insulin secreting cells.

<p>A. Both 200 µM MOG (squares) and 10 mM ß-OHB (triangles) increased ROS in INS-1 cells compared to basal (2 mM glucose) alone (circles). B. Area under curve of panel A. 200 µM MOG increased ROS generation in dissociated rat islets (C) compared to 3 mM glucose controls as measured with DCF fluorescence. D. 200 µM MOG (squares) and 10 mM ß-OHB (triangles) increased ROS generation in dissociated islet cells expressing the cytosolic HyPer protein compared to the basal (2 mM glucose) control as measured by changes in fluorescence ratio. E. Area under curve of D. A, B. n = 16 from 4 separate passages of cells. C. Results are typical of three separate experiments. D, E. Average of 14–33 cells from 3 separate animals. (A–C). ROS monitored for 1 hr in cells loaded with CM-H₂DCFA using a Tecan fluorescence platereader. (D–E). HyPer fluorescence ratio in dissociated islet cells monitored using a Zeiss 710 LIVE fluorescence microscope at 20× magnification. B and E, * p<0.005% compared to basal.</p

The DG kinase inhibitor R59949, failed to mimic the effects of MOG to stimulate insulin secretion from INS-1 ß-cells at 2 mM glucose.

<p>0.2 mM MOG (hatched bar) increased basal (2 mM glucose) insulin secretion from INS-1 cells 2.5-fold while 12 mM glucose resulted in a 3.5-fold increase (first black bar). The effect of the DG kinase inhibitor R59949 to stimulate basal insulin secretion (white bars) was small and inconsistent over the concentration range tested (0.25–10 µM) compared to the MOG-stimulated increase in basal release (hatched bar). R59949 increased glucose-stimulated insulin release 2-fold at low concentration (0.25 µM) with higher concentrations (1–10 µM) having no stimulatory effect compared to the control (black bars). N = 9 from 3 separate experiments. *p<0.05; **p<0.005 compared to control basal value. #p<0.005 compared to high glucose control.</p

MOG was metabolized by INS-1 ß-cells leading to an increase in glycerol (B), LC-CoA (C) and the LC-CoA/CoA ratio (D). Inhibition of LC-CoA synthetase with triacsin C reduced the stimulatory effect of MOG on basal (2 mM glucose) insulin secretion (E).

<p>A. 200 µM MOG increased basal (2 mM glucose) insulin secretion from INS-1 cells in a time-dependent manner. Glycerol release (B) and LC-CoA formation (C) were increased over time from the same cells. D. 200 µM MOG increased LC-CoA: free CoASH ratio to a greater extent than 200 µM oleic acid bound (4∶1 molar ratio) to BSA (FA) compared to the 2 mM glucose control after 1 hr incubation. E. Triacsin C (96 µM) inhibited insulin secretion stimulated by 100 µM MOG by 30% without affecting basal (2 mM glucose) insulin secretion from INS-1 cells. Triacsin C was included in both the preincubation and test conditions. A and B p<0.005 for all. C and E. *p<0.05 n = 9 to 12 from 3–4 different passages of cells. D. *p<0.05 n = 4 from single experiment.</p

MOG mimicked long-term exposure to glucose and FA by stimulating basal insulin secretion from pancreatic islet cells.

<p>A. Long-term exposure to 11 mM glucose and either 0.16 mM or 0.3 mM oleate complexed to FBS (FA) increased basal (2 mM glucose, white bars) and decreased glucose-stimulated insulin release (12 mM glucose, shaded bars) from INS-1 832/13 cells. MOG stimulated basal insulin secretion from dissociated rat islets in a concentration-dependent manner (25–400 µM) (B). C. Perifusion of rat islets demonstrated the pattern of MOG-stimulated basal insulin secretion. Arrow indicates addition of 200 µM MOG to 3 mM glucose. A and B. n = 12 from 3 separate experiments. C. Representative of 3 separate experiments. A and B, *p<0.005 compared to control.</p

The effect of MOG to stimulate basal insulin secretion was independent of changes in cytosolic Ca²⁺ and oxygen consumption.

<p>A. 200 µM MOG did not increase cytosolic Ca²⁺ in dissociated rat islet cells. Arrows indicate addition of 11 mM glucose (black trace) and 200 µM MOG (gray trace). B. 200 µM MOG (white squares) did not affect basal O₂ consumption in INS-1 cells. Incubation with and without MOG started 30 min prior to O₂ consumption measurements. Arrows indicate addition of 5 µM oligomycin (O) to inhibit respiration and 100 µM dinitrophenol (DNP) to stimulate maximal respiration. C. 200 µM MOG did not affect glucose-stimulated O₂ consumption in INS-1 cells. Arrow indicates addition of 2 mM glucose (black diamonds) and 12 mM glucose alone (white squares) or with 200 µM MOG (white triangles). A. Average signal from more than 10 cells per condition. Representative of 3 separate measurements. B and C. n = 4 from single experiment repeated three times.</p