Glucolipotoxic conditions induce β-cell iron import, cytosolic ROS formation and apoptosis

in Journal of Molecular Endocrinology
Correspondence should be addressed to J B Hansen: jakobha@sund.ku.dk
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Type 2 diabetes (T2D) arises when the pancreatic beta-cell fails to compensate for increased insulin needs due to insulin resistance. Glucolipotoxicity (GLT) has been proposed to induce beta-cell dysfunction in T2D by formation of reactive oxygen species (ROS). Here, we examined if modeling glucolipotoxic conditions by high glucose-high free fatty acid (FFA) exposure (GLT) regulates beta-cell iron transport, by increasing the cytosolic labile iron pool (LIP). In isolated mouse islets, the GLT-induced increase in the LIP catalyzed cytosolic ROS formation and induced apoptosis. We show that GLT-induced ROS production is regulated by an increased LIP associated with elevated expression of genes regulating iron import. Using pharmacological and transgenic approaches, we show that iron reduction and decreased iron import protects from GLT-induced ROS production, prevents impairment of the mitochondrial membrane potential (MMP) and inhibits apoptosis. This study identifies a novel pathway underlying GLT-induced apoptosis involving increased iron import, generation of hydroxyl radicals from hydrogen peroxide through the Fenton reaction in the cytosolic compartment associated with dissipation of the MMP and beta-cell apoptosis.

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  • Figure S1 (A) Quantification of the LIP from isolated islets exposed to palmitate (400 µM) or vehicle (BSA and ethanol) for 24 hours using calcein assay. Means +SEM of three independent experiments, *p<0.05, Student’s t-test. (B) ROS production in MIN6 cells exposed to palmitate (400 µM) or vehicle (BSA and ethanol) for 24 hours measured by H2DCF fluorescence. Means +SEM of five independent experiments, *p<0.05, Student’s t-test. (C) GSIS in MIN6 cells exposed to palmitate (400 µM) or vehicle (BSA and ethanol) for 24 hours. Means +SEM of six independent experiments, *p<0.05, Student’s t-test. (D) Apoptosis measurement using DNA-histone complex detection by ELISA in MIN6 cells exposed to palmitate (400 µM) or vehicle (BSA and ethanol) for 24 hours. Means +SEM of five independent experiments, **p<0.01, Student’s t-test. (E-I) Expression of ferritin heavy chain (Fth), ferritin light chain (Ftl) ferroportin (Fpn) pancreatic duodenum homeobox (Pdx) 1, and insulin (Ins) 1 mRNA in MIN6 cells exposed to palmitate (400 µM) or vehicle (BSA and ethanol) for up to 24 hours. Means +SEM of six independent experiments, *p<0.05, Student’s t-test.
  • Figure S2 (A) Stimulatory index (fold of 3 mM glucose, right) of mouse islets exposed to various concentrations of DFO for 48 hours. Means +SEM of five independent experiments. (B) Quantification of accumulated insulin secreted into the media from isolated islets during treatment with DFO (0.1mM) for 48 hours. Means +SEM of three independent experiments. (C) Total islet insulin content of islets after treatment with DFO (0.1 mM) for 48 hours. Means +SEM of three independent experiments. (D) Quantification of LIP in MIN6 cells exposed to palmitate (400 µM) with increasing concentrations of DFO. Data shown as fold of vehicle control with means +SEM of 3-4 independent experiments, *p<0.05, Student’s t-test.
  • Figure S3 (A) DMT1 KO verification by RT-PCR in isolated Cre-Flox+ and Cre+Flox+ islets. Means +SEM of n=2 independent experiments. (B) GSIS and KCl induced insulin secretion from isolated Cre-Flox+ and Cre+Flox+ islets normalized to secretion at 3 mM glucose. Means +SEM of three independent experiments.
  • Figure S4 (A-C) Measurements of IL-6, TNF-α and MCP-1 secretion to the culture medium from islets exposed to GLT (400 µM palmitate and 30 mM glucose) or vehicle (BSA and ethanol) in the presence or absence of DFO (0.1 mM) for 48 hours, or (D-F) in GLT or vehicle exposed Cre-Flox+ and Cre+Flox+ islets. Means +SEM of 2-4 independent experiments, *p<0.05, Student’s t-test.

 

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    The beta-cell-labile iron pool is increased by glucolipotoxicity (GLT). (A) Quantification of the labile iron pool (LIP) from isolated islets of mice fed a high-fat diet (HFD) or chow for 7 weeks was measured by calcein assay. Means + s.e.m. of n = 4–5, *P < 0.05, Student’s t-test. (B) Quantification of the LIP from isolated mouse islets exposed to GLT (400 µM palmitate and 30 mM glucose) or vehicle (BSA and ethanol) for up to 72 h using calcein assay. Means + s.e.m. of 4–7 independent experiments, *P < 0.05, Student’s t-test. (C) Quantification of the LIP in human islets exposed to a combination of palmitate (200 µM) and oleate (200 µM) for 24 h using calcein assay. Means + s.e.m. of n = 4, *P < 0.05, Student’s t-test. (D) Quantification of the LIP in human islets exposed to a combination of IL-1β (150 pg/mL) and IFN-γ (5 ng/mL) for 24 h using calcein assay. Means + s.e.m. of n = 3, *P < 0.05, Student’s t-test. (E) Quantification of the LIP in MIN6 cells exposed to with palmitate (400 µM) or vehicle (BSA and ethanol) for up to 72 h using calcein assay. Means + s.e.m. of 3–4 independent experiments, *P < 0.05, Student’s t-test. (F and G) Transferrin (Trfr) and divalent metal transporter 1 (Dmt1) mRNA in MIN6 cells exposed to palmitate (400 µM) or vehicle (BSA and ethanol) for up to 24 h. Means + s.e.m. of six independent experiments, *P < 0.05, ****P < 0.0001, Student’s t-test.

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    Glucolipotoxicity (GLT) induces iron-dependent cytosolic ROS production, apoptosis and secretory dysfunction. (A) Quantification of the labile iron pool (LIP) from isolated islets treated with different concentrations of DFO using calcein assay. Means + s.e.m. of 4–6 independent experiments. (B) Apoptosis measurement using DNA–histone complex detection by ELISA in isolated islets exposed to GLT (400 µM palmitate and 30 mM glucose) or vehicle (BSA and ethanol) with or without DFO (0.1 mM) for 48 h. Means + s.e.m. of four independent experiments, *P < 0.05 Control GLT vs DFO GLT, #P < 0.05 Control Vehicle vs Control GLT, two-way ANOVA. Cytosolic (C) and mitochondrial (D) thiol oxidation in islets exposed to medium containing 11 mM glucose (G11), G11 supplemented with 400 µM palmitate (G11_PA), 30 mM glucose (G30) or G30 with 400 µM palmitate (G30_PA) with or without DFO (0.1 mM) for 48 h using cyto-roGFP1 and mito-roGFP1 probes, respectively. Means + s.e.m. of 3–5 independent experiments, **P < 0.01 G30_PA vs G30_PA DFO, ###P<0.001 G11 vs G30_PA, Student’s t-test. (E) GSIS from isolated islets exposed to FFA (200 µM palmitate and 200 µM oleate) or vehicle (BSA and ethanol) with or without DFO (0.1 mM) for 48 h. Means + s.e.m. of four independent experiments, **P < 0.01, Student’s t-test.

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    Iron is important for beta-cell mitochondrial function. (A) Quantification of the labile iron pool (LIP) from isolated islets of inducible beta-cell specific WT (CreFlox+) or KO (Cre+Flox+) mice. Means + s.e.m. of n = 5–6 independent experiments, ***P < 0.001, Student’s t-test. (B) ROS production in isolated islets of CreFlox+ and Cre+Flox+ islets measured by H2DCF fluorescence. Means + s.e.m. of n = 9, *P < 0.05, Student’s t-test. (C) MMP measurements using Rhod123 in isolated CreFlox+ and Cre+Flox+ islets. Left, means + s.e.m. fluorescence traces from islets analyzed, n = 8–9. Right, means + s.e.m. AUC of fluorescence traces normalized to time of n = 5 independent experiments, *P < 0.05, Student’s t-test.

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    Beta-cell-specific DMT1 KO reduces GLT-mediated apoptosis ROS production and mitochondrial damage. (A) Apoptosis measurement using DNA-histone complex detection by ELISA in isolated CreFlox+ and Cre+Flox+ islets exposed to GLT (400 µM palmitate and 30 mM glucose) or vehicle (BSA and ethanol). Means + s.e.m. of four independent experiments, **P < 0.01, Student’s t-test. (B) ROS production in isolated islets of CreFlox+ and Cre+Flox+ islets exposed to GLT (400 µM palmitate and 30 mM glucose) or vehicle (BSA and ethanol) measured by H2DCF fluorescence. Means + s.e.m. of three independent experiments, **P < 0.01 and **P < 0.01 Vehicle CreFlox+ vs GLT CreFlox+, ##P < 0.01 GLT CreFlox+ vs GLT Cre+Flox+, Student’s t-test. (C) Insulin secretion measured by perifusion in isolated CreFlox+ and Cre+Flox+ islets exposed to GLT (400 µM palmitate and 30 mM glucose) or vehicle (BSA and ethanol). Left panel, perifusion traces. Middle panel, stimulatory index of islet perifusion experiment. Right panel, total islet insulin after perifusion. Graphs show means + s.e.m. of four independent experiments, **P < 0.01, Student’s t-test. (D) MMP measurements using Rhod123 in isolated CreFlox+ islets exposed to GLT (400 µM palmitate and 30 mM glucose) or vehicle (BSA and ethanol). Left panel, means + s.e.m. of fluorescence traces from islets analyzed, n = 5–7. Right panel, means + s.e.m. AUC of fluorescence traces normalized to time of n = 4–7 independent experiments, *P < 0.05, **P < 0.01, Student’s t-test. (E) MMP measurements using Rhod123 in isolated Cre+Flox+ islet exposed to GLT (400 µM palmitate and 30 mM glucose) or vehicle (BSA and ethanol). Left panel, means + s.e.m. of fluorescence traces from islets analyzed, n = 4–5. Right, means + s.e.m. AUC of fluorescence traces normalized to time of n = 4–5 independent experiments, *P < 0.05, **P < 0.01, Student’s t-test.

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