An ancestral role for the mitochondrial pyruvate carrier in glucose-stimulated insulin secretion

Abstract

Objective: Transport of pyruvate into the mitochondrial matrix by the Mitochondrial Pyruvate Carrier (MPC) is an important and rate-limiting step in its metabolism. In pancreatic β-cells, mitochondrial pyruvate metabolism is thought to be important for glucose sensing and glucose-stimulated insulin secretion.

Methods: To evaluate the role that the MPC plays in maintaining systemic glucose homeostasis, we used genetically-engineered Drosophila and mice with loss of MPC activity in insulin-producing cells.

Results: In both species, MPC deficiency results in elevated blood sugar concentrations and glucose intolerance accompanied by impaired glucose-stimulated insulin secretion. In mouse islets, β-cell MPC-deficiency resulted in decreased respiration with glucose, ATP-sensitive potassium (KATP) channel hyperactivity, and impaired insulin release. Moreover, treatment of pancreas-specific MPC knockout mice with glibenclamide, a sulfonylurea KATP channel inhibitor, improved defects in islet insulin secretion and abnormalities in glucose homeostasis in vivo. Finally, using a recently-developed biosensor for MPC activity, we show that the MPC is rapidly stimulated by glucose treatment in INS-1 insulinoma cells suggesting that glucose sensing is coupled to mitochondrial pyruvate carrier activity.

Conclusions: Altogether, these studies suggest that the MPC plays an important and ancestral role in insulin-secreting cells in mediating glucose sensing, regulating insulin secretion, and controlling systemic glycemia.

Keywords: DILP2, Drosophila insulin-like peptide 2; Diabetes; Drosophila; GSIS, glucose-stimulated insulin secretion; GTT, glucose tolerance test; IMM, inner mitochondrial membrane; IPCs, Insulin-producing Cells; ITT, insulin tolerance test; Insulin; MPC1 and MPC2, Mitochondrial Pyruvate Carrier 1 and 2; Mitochondria; OCR, oxygen consumption rates; Pdx1, pancreatic and duodenal homeobox 1; Pyruvate; RESPYR, REporter Sensitive to PYRuvate; Stimulus-coupled secretion; β-Cell.

Figure 1

Drosophila MPC1 mutants are hyperglycemic and sensitive to dietary sugar. A: dMPC1 mutants die more rapidly as dietary sugar is increased. The percentage of surviving adult control (left panel) or dMPC1 mutants (right panel) fed a low (2% sucrose, 10% yeast; purple lines) or high (18% sucrose, 10% yeast; light blue lines) sugar diet was assayed every 2–5 days. N > 115 male flies per genotype under each feeding condition. Mean ± SEM is shown. B–E: Control (blue) or dMPC1 mutants (red) were aged 8–12 days on the low or high sugar diet and whole animal metabolite levels were measured to determine the abundance of B: trehalose C: glucose, D: glycogen, or E: triglycerides (TAG), all normalized to protein. N = 5–6 samples per genotype. Mean ± SEM is shown. *p < 0.05, **p < 0.01, and ***p < 0.001. F: Hemolymph glucose concentrations were determined in control and dMPC1 mutants aged 10 days on the high sugar media. N = 4 per genotype. **p < 0.01 G: Control and dMPC1 mutants were aged 5–9 days on standard laboratory media and fasted overnight for 16 h. After fasting, whole-animal glucose levels were determined and normalized to protein. N = 5 per genotype. **p < 0.01.

Figure 2

Sugars and sugar alcohols accumulate in dMPC1 mutants in response to dietary sugar. Control (blue) or dMPC1 mutants (dMPC1^–)(red) were aged 8–12 days on the low or high sugar diets and whole animal metabolite levels were measured by GC/MS. The abundance of pyruvate, ribose, inositol, erythrose, sorbitol, mannitol, threitol, and xylitol or ribitol (which cannot be distinguished in our analysis) is shown. N = 3 biological replicates per condition. Mean ± SEM is shown. P values comparing mutants to controls under either condition were calculated by Student's t test. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3

Drosophila MPC1 mutants are glucose intolerant and have reduced glucose-stimulated insulin secretion. A: Oral-glucose tolerance test performed on adult control (blue) and dMPC1 mutants (red) that were aged 5–9 days on standard laboratory media, fasted overnight, fed 10% glucose for 2 h, and then re-fasted for either 2 or 4 h. Data represents free glucose levels from whole animal homogenates normalized to protein concentration. N = 5 biological replicates per genotype at each timepoint. Mean ± SEM is shown. *p < 0.05 and ***p < 0.001. B: Control and dMPC1 mutants were aged 8–12 days on the high sugar diet and phosphorylated AKT (P-AKT), total AKT, and tubulin protein levels were determined by western blot analysis. C: The insulin-producing cells (IPCs) embedded in the adult brain were stained with antibodies directed against either DILP2 (left, red) or MPC1 (green, center) in either control or dMPC1 mutants. dMPC1 staining is evident in the IPCs of control, but not mutant, flies. D: dMPC1 mutants display defects in GSIS. Control and dMPC1 mutants carrying DILP2-HF in a dilp2¹ mutant background were aged 5–9 days on standard laboratory media, fasted overnight, and fed glucose for 30 min. Hemolymph was collected to measure circulating DILP2-HF under fasted and fed conditions. Y axis depicts the ratio of secreted DILP2-HF to total DILP2-HF. (fasted n = 4, fed n = 5). **p ≤ 0.01. NS = not significant. E: IPC-specific dMPC1 RNAi results in reduced GSIS. Levels of circulating DILP2-HF were assayed in controls (blue) or animals with IPC-specific RNAi against dMPC1 (red) using a dilp2-GAL4 driver. *p < 0.05, **p ≤ 0.01.

Figure 4

Mice with pancreas-specific MPC2 deletion have reduced GSIS despite normal insulin content. A: Immunohistochemical staining of islets showed no difference in immunodetectable insulin, glucagon, or β and α cell number. B: Histologic assessment of islet number and individual cross-sectional area detected no difference between WT and PdxCreMpc2−/− mice. C: Mice were fasted for 16 h and injected i.p. with 1.5 g/kg glucose. PdxCreMpc2−/− mice displayed significantly elevated blood glucose concentrations after the overnight fast and after bolus glucose injection. D: Mice were fasted for 4 h and injected i.p. with 0.5 U/kg insulin. PDXCreMpc2−/− mice displayed elevated basal blood glucose levels but similar insulin response curves. E: Plasma insulin concentrations were reduced in PdxCreMpc2−/− mice 15 min after bolus glucose injection. F: Loss of Mpc2 in beta cells impairs GSIS by isolated islets. Isolated islets from WT and PdxCreMpc2−/− mice were incubated with the indicated concentrations of glucose and insulin concentration of the medium determined. Insulin secretion was corrected in PdxCreMpc2−/− islets with either 1 μM glibenclamide treatment or stimulation with 30 mM KCl + 1 mM glucose. 10 mM glutamine + 23 mM glucose-stimulated insulin secretion was also normal. N = 10–12 mice per genotype with 10 pancreas sections analyzed per mouse in A and B. N = 10–13 mice per genotype in C and D. N = 5–6 mice per genotype in E. N = 2–3 mice per genotype and two technical replicates per mouse in F. Mean ± SEM is shown. P values comparing KOs to controls were calculated using a Student's t test. *p < 0.05, **p < 0.01, and ***p < 0.001, ^Ϯp < 0.05 compared to non-glib treatment.

Figure 5

Constitutive or Tamoxifen-inducible deletion of Mpc2 in pancreatic β-cells results in defective glucose-stimulated insulin secretion. A: Mice were fasted for 16 h and injected i.p. with 1.5 g/kg glucose. RipCreMpc2−/− mice display elevated blood glucose concentrations following bolus glucose injection. B: Plasma insulin concentration of RipCreMPC2−/− mice is reduced 15 min after bolus glucose injection C: Schematic of Tamoxifen-inducible Mpc2 deletion in PdxCre^ERMpc2−/− mice. Tamoxifen dose was 50 mg/kg i.p. for 5 consecutive days. D: Mice were fasted for 16 h and injected i.p. with 1.5 g/kg glucose. PdxCre^ERMpc2−/− mice displayed elevated blood glucose concentration during the GTT. Glucose AUC was also significantly elevated. E: Plasma insulin concentrations of PdxCre^ERMpc2−/− mice were reduced 15 min after i.p. injection of 1.5 g/kg glucose following a 6 h fast. F: Isolated islets from PdxCre^ERMpc2−/− mice contain normal insulin content, but defective glucose-stimulated insulin secretion. Insulin secretion was corrected in PdxCre^ERMpc2−/− islets with either 1 μM Glibenclamide treatment or stimulation with 30 mM KCl + 1 mM glucose, suggesting the defect prior to the membrane depolarization. N = 5–7 mice per group in A, B, and E. N = 11–12 mice per genotype in D, and N = 2–3 mice per genotype and two technical replicates per mouse in F. Mean ± SEM is shown. P values comparing KOs to controls were calculated using a Student's t test. *p < 0.05, **p < 0.01, and ***p < 0.001, ^Ϯp < 0.05 compared to non-glib treatment.

Figure 6

Defects in MPC2 KO islet K_ATPchannel activity and GSIS are correctable with sulfonylurea treatment. A: Isolated islets were stimulated with glucose and oxygen consumption rates (OCR) were determined. PdxCreMpc2−/− islets display reduced OCR in response to 20 mM glucose stimulation, but maintain similar responsiveness to oligomycin, FCCP, and Antimycin A + Rotenone treatment. B: PdxCreMpc2−/− islets display increased K_ATP channel activity in response to glucose stimulation. ⁸⁶Rb⁺ efflux was measured in cultured islets following stimulation with glucose (7 mM or 16.7 mM) or glucose plus glibenclamide (16.7 mM + glib). C: Blood glucose concentrations during the GTT performed using fl/fl and PdxCreMpc2−/− mice. Mice were fasted for 16 h and injected i.p. with 1.5 g/kg glucose and co-injected with vehicle or the K_ATP channel inhibitor glibenclamide (0.1 mg/kg). D: Glibenclamide also corrected plasma insulin concentrations 15 min after glucose injection in PdxCreMpc2−/− mice. N = 4–5 mice per genotype in A, N = 5 experimental replicates using islets from 2 to 3 mice per genotype in B, N = 13–14 mice per GTT group in C, and N = 6 per insulin secretion group in D. *p < 0.05, ^Ϯp < 0.05 compared to vehicle treatment.

Figure 7

Glucose stimulation increases MPC activity in INS-1 cells. A: Schematic of the BRET-based RESPYR biosensor used to measure real time activity of the mitochondrial pyruvate carrier. RLuc8 is c-terminally fused to MPC2 and Venus is c-terminally fused to MPC1. BRET signal increases above basal levels as carrier transport activity increases. B: BRET kinetics of INS-1 cells expressing RESPYR. Cells were stimulated after 5 min with PBS (black squares), 1 mM glucose (gray circles), 7 mM glucose (blue rectangles), 16.7 mM glucose (green diamonds) or 22 mM glucose (red circles) or 1 μM glibenclamide (purple triangles) in PBS. Data were analyzed by repeated measures ANOVA in Prism. Post Hoc analysis was performed using Tukey's multiple comparison tests. Data represents mean ± SEM of six independent experiments with 4–5 technical replicates per experiment.*p < 0.05 compared to PBS, 1 mM glucose and 1 μM glib treatments, ^Ϯp < 0.05 compared to all other treatments.