Glutathionylation state of uncoupling protein-2 and the control of glucose-stimulated insulin secretion

Abstract

The role of reactive oxygen species (ROS) in glucose-stimulated insulin release remains controversial because ROS have been shown to both amplify and impede insulin release. In regard to preventing insulin release, ROS activates uncoupling protein-2 (UCP2), a mitochondrial inner membrane protein that negatively regulates glucose-stimulated insulin secretion (GSIS) by uncoupling oxidative phosphorylation. With our recent discovery that the UCP2-mediated proton leak is modulated by reversible glutathionylation, a process responsive to small changes in ROS levels, we resolved to determine whether glutathionylation is required for UCP2 regulation of GSIS. Using Min6 cells and pancreatic islets, we demonstrate that induction of glutathionylation not only deactivates UCP2-mediated proton leak but also enhances GSIS. Conversely, an increase in mitochondrial matrix ROS was found to deglutathionylate and activate UCP2 leak and impede GSIS. Glucose metabolism also decreased the total amount of cellular glutathionylated proteins and increased the cellular glutathione redox ratio (GSH/GSSG). Intriguingly, the provision of extracellular ROS (H(2)O(2), 10 μM) amplified GSIS and also activated UCP2. Collectively, our findings indicate that the glutathionylation status of UCP2 contributes to the regulation of GSIS, and different cellular sites and inducers of ROS can have opposing effects on GSIS, perhaps explaining some of the controversy surrounding the role of ROS in GSIS.

FIGURE 1.

Effect of different doses of H₂O₂ on insulin release and ROS levels in Min6 cells exposed to 25 or 1 mm glucose. A, insulin release. Cells were washed once with KRB, starved for 1 h, and then exposed to KRB containing either 25 or 1 mm glucose with H₂O₂ for 1 h. Following the incubation, the assay medium was collected and tested for insulin content. Data were normalized to total cell protein content/well. B, ROS levels. Cells were washed once with KRB, starved for 1 h in the presence of DCFHDA, and then exposed to KRB containing 25 or 1 mm glucose with H₂O₂ for 1 h. The assay medium was then removed, and DCFHDA fluorescence was measured. Data were normalized to total cell protein content/well and background fluorescence. n = 4, mean ± S.E., one-way ANOVA with Fisher's protected least significant difference post hoc test. A.U., absorption units.

FIGURE 2.

Impact of diamide on Min6 cell viability and physiology. Min6 cells were washed with KRB, starved for 1 h with KRB containing 1 mm glucose, and then incubated for 1 h in KRB containing 20 mm glucose and varying amounts of diamide (0–1000 mm). Exposure to H₂O₂ (Per; 5 mm) served as the control. A, ROS levels determined with 20 μm DCFHDA. For ROS measurements, cells were loaded with DCFHDA prior to exposure to 25 mm glucose and diamide. ROS levels were then measured fluorometrically and normalized to protein and background fluorescence. B, assessment of the reductive cellular environment. Cells were exposed simultaneously to MTT and diamide/glucose and then tested for the amount of reduced tetrazolium. Values were normalized to background cellular absorption. C, measurement of cell death using propidium iodide (PI). Following exposure to diamide/glucose, cells were treated for 10 min with 1 μg/ml PI diluted in PBS. Amount of cell death was then tested fluorometrically. Values were normalized to background fluorescence and amount of protein. n = 6, mean ± S.E., one-way ANOVA with Fisher's protected least significant difference post hoc test. D, impact of diamide on insulin release. For insulin release determinations, Min6 cells were washed once with KRB, starved for 1 h, and then treated for 1 h with KRB containing 25 mm glucose and different amounts of diamide. The supernatant was then collected and tested for insulin content. n = 4, mean ± S.D. one-way ANOVA with Fisher's protected least significant difference post hoc test. E, effect of BioGEE on insulin release. Min6 cells were washed once with KRB, starved for 1 h, and then treated for 1 h with KRB containing 25 mm glucose and BioGEE (1 mm). n = 4, mean ± S.E., Student's t test. A.U., absorption units.

FIGURE 3.

ROS and glutathionylation activate and deactivate, respectively, proton leak in a UCP2-dependent manner in Min6 cells. Min6 cells were transduced with short hairpin control (shCtl) or UCP2 (shUCP2) lentiviral particles and then tested for the effect of H₂O₂ (0 or 10 μm) and diamide (100 μm) on bioenergetics using the Seahorse XF24 analyzer. A, immunoblot detection of UCP2 in Min6 cells transduced with either shCtl or shUCP2. Succinate dehydrogenase (SDH) served as the loading control. B, summary of the method for determining the impact of diamide on Min6 cell bioenergetics using the XF24 analyzer. Following the injection of diamide, resting respiration was tested; this was then followed by the injection of glucose (G, 25 mm), oligomycin (O, 0.13 μg/ml), and antimycin A (A, 2 μm). All values were expressed as a percentage of resting respiration. n = 4, mean ± S.E. C, effect of diamide on absolute oligomycin-induced state 4 respiration rates in shCtl and shUCP2 Min6 cells. n = 4, mean ± S.E., Student's t test. D, effect of H₂O₂ on absolute oligomycin-induced state 4 respiration rates in shCtl and shUCP2 Min6 cells. Determinations were performed as described in B. n = 4, mean ± S.E., Student's t test. E, summary of the effect of reversible glutathionylation on proton leak through UCP2.

FIGURE 4.

UCP2 is required for the diamide-mediated regulation of insulin release. A, UCP2 knockdown increases the ATP/ADP ratio in Min6 cells. Min6 cells transduced with either short hairpin control (shCtl) or UCP2 (shUCP2) lentiviral particles were starved and then incubated for 1 h in KRB containing 25 or 1 mm glucose. ATP and ADP were detected as described under “Materials and Methods.” n = 3, mean ± S.E., Student's t test. B, UCP2 knockdown abolishes the diamide-mediated increase in insulin release. Min6 cells transduced with either shCtl or shUCP2 were starved and then incubated for 1 h in KRB containing 25 or 1 mm glucose with either diamide (100 μm) or H₂O₂ (10 μm). Media were then collected and tested for insulin release. Data were normalized to total cellular protein/well. n = 4, mean ± S.E., one-way ANOVA with Fisher's protected least significant difference post hoc test. * and ** denotes p ≤ 0.05 and 0.01, respectively, when compared with the 25 mm glucose control. † denotes p ≤ 0.05 when compared with the 1 mm glucose control. C, diamide modulates glucose-stimulated insulin release in a UCP2-dependent manner. Islets from control (RIPCre) and pancreas-specific UCP2 knock-out (UCP2BKO) mice were treated with diamide (0 and 10 μm) and then tested for insulin release as described under “Materials and Methods.” Insulin release was measured following a 1-h exposure to high glucose (16.7 mm) or low glucose (2.8 mm) conditions. n = 4, mean ± S.E., Student's t test. ** denotes p ≤ 0.01 when compared with 0 μm diamide control.

FIGURE 5.

Effect of glucose energization on glutathione redox in Min6 cells. A, Min6 cells were starved for 1 h and then incubated in 25 or 1 mm glucose in the absence or presence of 10 μm H₂O₂ +1 mm BioGEE. Cells were then lysed in RIPA containing 25 mm N-ethylmaleimide, and BioGEE-tagged proteins were enriched using streptavidin beads. UCP2 was detected by immunoblot. B, HPLC analysis of GSH/GSSG ratio and total GSH associated with proteins in Min6 cells exposed to 25 or 1 mm glucose. Cells were starved, incubated in KRB containing 25 or 1 mm glucose, and then lysed with 0.1% (v/v) trifluoroacetic acid/methanol solution (90:10). Supernatant was then injected into the HPLC. For GSH associated with protein (total glutathione associated with proteome), protein was treated with KOH and then the resulting supernatant was injected into the HPLC. GSH and GSSG retention times were confirmed and quantified by injecting standard solutions. n = 3, mean ± S.E., Student's t test. C, immunodetection of BioGEE-modified proteins in Min6 cell extract. Cells were treated with BioGEE and lysed, and the amount of BioGEE-tagged protein was detected with avidin-HRP. D, UCP2 knockdown increases cellular ROS levels following H₂O₂ challenge. Min6 cells transduced with either short hairpin control (shCtl) or UCP2 (shUCP2) lentiviral particles were loaded with DCFHDA (20 μm) during cell starvation, washed with KRB, and then incubated for 1 h in 25 or 1 mm glucose with H₂O₂ (0–100 μm). Cells were then measured for DCFHDA fluorescence. Data were normalized to total cell protein/well and background fluorescence. n = 4, mean ± S.E., Student's t test. E, time course analysis of ROS production in mitochondria (MitoSOX) or total cell (DCFHDA) following exposure to 25 or 1 mm glucose. Amount of ROS was then detected following various incubation times. Data were normalized to total protein levels. n = 4, mean ± S.E., one-way ANOVA with Fisher's protected least significant difference post hoc test. A.U., absorption units.

FIGURE 6.

Matrix ROS activates leak through UCP2 that impedes GSIS. To simulate production of superoxide in the matrix without inhibiting the respiratory complexes, Min6 cells were incubated for 18 h with paraquat (PQ). A, PQ-mediated increases in matrix ROS are dose-dependent. Cells were pre-loaded with either MitoSOX (20 μm) or TMRE (10 nm) and energized for 1 h with 25 mm glucose. Fluorescent signals were then detected and compared to determine whether PQ generates superoxide in the matrix and whether PQ uptake has an effect on mitochondrial membrane potential. Data were normalized to total protein per well. B, impact of PQ on Min6 mitochondrial bioenergetics. Following an assessment of resting respiration, respiration rates in cells treated to PQ (0–250 μm) were tested following exposure to glucose (G; 25 mm), oligomycin (O; 0.13 μg/ml), FCCP (F; 2 μm), and antimycin A (A; 2 μm). Data were normalized to total protein per well. n = 4, mean ± S.E. C, PQ accumulates in mitochondria. Following exposure of Min6 cells to 0 or 50 μm PQ, mitochondria were isolated, lysed, and treated with dithionite. PQ was detected by UV-visible scan from 500 to 700 nm. D, PQ activates UCP2 proton leak. Min6 cells transduced with either short hairpin control (shCtl) or UCP2 (shUCP2) lentiviral particles and treated with or without PQ (50 μm) were sequentially treated with glucose (G; 25 mm), oligomycin (O; 0.13 μg/ml), FCCP (F; 2 μm), and antimycin A (A; 2 μm). Impact of PQ on UCP2-dependent proton leak is summarized to the right of the bioenergetic data. All data were normalized to total protein per well. n = 4, mean ± S.D. Student's t test. E, PQ impedes GSIS. Min6 cells were starved and then treated with different amounts of glucose (1–20 mm) for 1 h. Insulin levels in the incubation medium were normalized to total protein amounts/well. n = 4, mean ± S.E. Student's t test.

FIGURE 7.

Matrix ROS, glutathionylation of UCP2, and the control of GSIS. Following glucose catabolism, reducing equivalents enter complex I and complex II of the electron transport chain. The liberated electrons are transferred through complexes III and IV to the terminal electron acceptor O₂. The energetically favorable transfer of electrons to O₂ is coupled to the genesis of PMF, which is then used to drive complex V (ATP synthase). ATP is exported into the cytosol from the mitochondria increasing the ATP/ADP ratio, which induces insulin release. Over time with progressive glucose metabolism and the decrease in available ADP, PMF increases, as does matrix ROS generation from complexes I and III. The increase in matrix ROS leads to the deglutathionylation of UCP2 and the activation of proton leak. Proton leak decreases PMF and ATP/ADP, thus hindering further insulin release. SSG, protein-glutathione adduct; IMS, intermembrane space.