Chronic High Glucose and Pyruvate Levels Differentially Affect Mitochondrial Bioenergetics and Fuel-stimulated Insulin Secretion from Clonal INS-1 832/13 Cells

Abstract

Background: Elevated glucose may cause ␤-cell dysfunction in type 2 diabetes, i.e., glucotoxicity. Results: Elevated glucose, but not pyruvate, perturbed insulin secretion and content, plasma and mitochondrial membrane potentials, and proton leak, while increasing glycolytic metabolites in ␤-cells. Conclusion: Early metabolism of glucose exerts a toxic effect on clonal insulin-producing cells. Significance: Unraveling these mechanisms may provide protection of ␤-cells in diabetes.

Chronic Exposure to Excess Nutrients Left-shifts the Concentration Dependence of Glucose-stimulated Insulin Secretion in Pancreatic β-Cells

“…The mitochondrial leak, defined as proton flowback across the inner mitochondrial membrane not coupled to ATP synthesis (oligomycin-insensitive), was increased in INS-1 cells cultured at high glucose, which has been observed previously (Fig. 7F) (25). The increased leak accounted for approximately half of the elevated basal respiration in 11G cells.…”

“…Most studies in this area have focused on the reduced GSIS observed following exposure of ␤-cells to these conditions (22)(23)(24)(25). Whether the reduction in GSIS develops as a result of a defect in the pathway(s) leading to insulin secretion or a depletion of insulin stores is controversial.…”

Metabolomics applied to the pancreatic islet

“…Multiple contributors to β-cell dysfunction have been proposed, including oxidative stress [69,70], ER stress [70], altered glucose metabolism [71] or impaired exocytosis mechanisms [72–75]. Several GC–MS-based metabolomics studies have been reported for insulinoma cell models of glucotoxicity, three of which were combined with 13 C-tracer and NMR experiments [21,22,36,64,76]. Despite similarities in methods used, significant variation in metabolite coverage remains.…”

“…However, the elevated concentrations of these intermediates are rapidly lowered in response to lowering of the extracellular glucose concentration. Chronic exposure of β-cells to high levels of pyruvate does not have the same toxic effects as hyperglycemia, suggesting that unique metabolic fates of glucose rather than “bioenergetic overload” induced by provision of a bolus of mitochondrial substrate is driving glucotoxicity [76]. Similar to glucotoxicity, inhibition of 6PGDH inhibits insulin gene expression, GSIS and causes PPP intermediates to accumulate.…”

Inhibition of the malate–aspartate shuttle in mouse pancreatic islets abolishes glucagon secretion without affecting insulin secretion

“…On the day of analysis, the cells were incubated in 400 μl of buffer P (135 mM NaCl, 3.6 mM KCl, 1.5 mM CaCl 2 , 0.5 mM MgSO 4 , 0.5 mM Na 2 HPO 4 , 10 mM HEPES and 5 mM NaHCO 3 , pH 7.4) containing 2.8 mM glucose for 90 min at which 0.2 μM Fluo-4 acetoxymethyl ester (Invitrogen, Life Technologies), 0.25 mM sulfinpyrazone (a multi-specific inhibitor of organic anion transporters) and BSA (0.1 %) were added and the incubation was continued for a further 30 min. A vial from a FLIPR ® membrane potential assay kit, explorer format component A, containing a proprietary plasma membrane potential ( ψ p ) indicator (PMPI; Molecular Devices) was reconstituted in 10 ml of water and the buffer P containing Fluo-4 was removed and replaced with 400 μl of fresh buffer P containing 4 μl of PMPI immediately prior to imaging as described previously [38,39]. Excitation was performed at 488 nm (Fluo-4) and 514 nm (PMPI) and emission was recorded with a 530 nm long-pass filter [39] on a Zeiss LSM510 inverted confocal fluorescence microscope.…”