Arsenite and methylarsonite inhibit mitochondrial metabolism and glucose-stimulated insulin secretion in INS-1 832/13 β cells

Abstract

Growing evidence suggests that exposure to environmental contaminants contributes to the current diabetes epidemic. Inorganic arsenic (iAs), a drinking water and food contaminant, is one of the most widespread environmental diabetogens according to epidemiological studies. Several schemes have been proposed to explain the diabetogenic effects of iAs exposure; however, the exact mechanism remains unknown. We have shown that in vitro exposure to low concentrations of arsenite (iAs^III) or its trivalent methylated metabolites, methylarsonite (MAs^III) and dimethylarsinite (DMAs^III), inhibits glucose-stimulated insulin secretion (GSIS) from isolated pancreatic islets, with little effect on insulin transcription or total insulin content. The goal of this study was to determine if exposure to trivalent arsenicals impairs mitochondrial metabolism, which plays a key role in the regulation of GSIS in β cells. We used a Seahorse extracellular flux analyzer to measure oxygen consumption rate (OCR), a proxy for mitochondrial metabolism, in cultured INS-1 832/13 β cells exposed to iAs^III, MAs^III, or DMAs^III and stimulated with either glucose or pyruvate, a final product of glycolysis and a substrate for the Krebs cycle. We found that 24-h exposure to 2 μM iAs^III or 0.375-0.5 μM MAs^III inhibited OCR in both glucose- and pyruvate-stimulated β cells in a manner that closely paralleled GSIS inhibition. In contrast, 24-h exposure to DMAs^III (up to 2 µM) had no effects on either OCR or GSIS. These results suggest that iAs^III and MAs^III may impair GSIS in β cells by inhibiting mitochondrial metabolism, and that at least one target of these arsenicals is pyruvate decarboxylation or downstream reactions.

Keywords: Arsenic; Diabetes; Insulin secretion; Mitochondrial respiration; β cells.

Fig. 1

Oxygen consumption and GSIS in INS-1 832/13 cells during acute exposures to iAs^III, MAs^III, and DMAs^III. OCR was measured in INS-1 832/13 cells stimulated with 16.7 mM glucose (marked with vertical dotted line) and following injection of arsenicals (marked with vertical solid line): iAs^III (a), MAs^III (b) or DMAs^III (c). OCR was monitored at 6-min intervals. GSIS was measured in INS-1 832/13 cells cultured in 16.7 mM glucose after acute (2, 4, 6 h) exposure to arsenicals (d). Each time point was normalized to the high glucose control. Mean ± SD is shown for four technical replicates (OCR), and mean + SD is shown for three technical replicates (GSIS). *p < 0.05 and ⁺p < 0.01 for comparisons of treated versus untreated (0 μM) cells

Fig. 2

Oxygen consumption in INS-1 832/13 cells after 24-h exposures to iAs^III. OCR was measured in INS-1 832/13 cells stimulated with 16.7 mM glucose (a, b) or 10 mM pyruvate (c, d) following 24-h exposure to iAs^III. a, c Additions of 16.7 mM glucose or 10 mM pyruvate are marked by dotted lines. b, d OCR values recorded at low glucose (2.5 mM), high glucose (16.7 mM), and pyruvate (10 mM) were averaged to quantify the overall effects of the exposure. Mean + SEM is shown for four biological replicates. *p < 0.05, ⁺p < 0.01 for comparisons of treated versus untreated (0 μM) cells

Fig. 3

Oxygen consumption in INS-1 832/13 cells after 24-h exposures to MAs^III. OCR was measured in INS-1 832/13 cells stimulated with 16.7 mM glucose (a, b) or 10 mM pyruvate (c, d) following 24-h exposure to MAs^III. a, c Additions of 16.7 mM glucose or 10 mM pyruvate are marked by dotted lines. b, d: OCR values recorded at low glucose (2.5 mM), high glucose (16.7 mM), and pyruvate (10 mM) were averaged to quantify the overall effects of the exposure. Mean + SEM is shown for four biological replicates. *p < 0.05, ⁺p < 0.01 for comparison of treated versus untreated (0 μM) cells

Fig. 4

Mitochondrial respiration parameters of INS-1 832/13 cells after 24-h exposure to iAs^III or MAs^III. OCR was measured in INS-1 832/13 cells following 24-h exposure to iAs^III or MAs^III. Maximal respiration (a), spare respiratory capacity (b), non-mitochondrial respiration (c), and proton leak (d) were measured following sequential addition of: 16.7 mM glucose, oligomycin, FCCP, and a Rotenone/Antimycin A mix. Mean + SD is shown for six to seven technical replicates with two biological replicates. *p < 0.05, ⁺p < 0.01 for comparison of treated versus control cells

Fig. 5

Glucose stimulated insulin secretion in INS-1 832/13 cells after 24-h exposures to iAs^III, MAs^III, and DMAs^III. INS-1 832/13 cells were exposed to iAs^III (a), MAs^III (b), or DMAs^III (c) for 24 h prior to GSIS. Insulin secretion was measured after stimulation with 2.5 mM glucose (clear bars) followed by 16.7 mM glucose (gray bars). Mean + SD is shown for three biological replicates. *p < 0.05 for comparison of treated versus untreated (0 μM) cells

Fig. 6

Viability of INS-1 832/13 cells after 24-h exposure to iAs^III, MAs^III and DMAs^III: The MTT assay INS-1 832/13 cells were exposed for 24 h to iAs^III (a), MAs^III (b), or DMAs^III (c) and cell viability was measured by the MTT assay. Mean + SEM is shown for three biological replicates. ⁺p < 0.01 for comparisons of treated versus untreated (0 μM) cells

Fig. 7

Viability of INS-1 832/13 cells after 24-h exposures to iAs^III, MAs^III and DMAs^III: CellTox™ Green. INS-1 832/13 cells were exposed for up to 24 h to iAs^III (a), MAs^III (b), or DMAs^III (c). Cell viability was measured using CellTox™ Green at 0.5, 1, 1.5, and 24 h. Values were normalized to the control at each time point. Mean + SD is shown for four technical replicates. *p < 0.05, ⁺p < 0.01 for comparisons of treated versus untreated (control) cells