Acute hyperglycemia results in dynamic changes in the superoxide anion concentration of HeLa cells.

<p>(A) 5x10⁵ HeLa cells were exposed to glucose concentrations ranging from 4 mmol/L (control) to 20 mmol/L and sequentially imaged. Representative image is 15 minutes post treatment. (B) Quantification of coelenterazine chemiluminescence at each time point for each sample (n = 3). (C) 5x10⁵ RIN-5F cells were exposed to glucose concentrations ranging from 4 mmol/L (control) to 20 mmol/L and sequentially imaged. Representative image is 15 minutes post treatment. (D) Quantification of coelenterazine chemiluminescence at each time point for each sample (n = 3). (E) 5x10⁵ INS-1 cells were exposed to glucose concentrations ranging from 4 mmol/L (control) to 20 mmol/L and sequentially imaged. Representative image is 15 minutes post treatment. (F) Quantification of coelenterazine chemiluminescence at each time point for each sample (n = 3). (G) 5x10⁵ HeLa cells were exposed to glucose concentrations ranging from 13.2 mmol/L (control) to 27.8 mmol/L and sequentially imaged. Representative image is 120 minutes post treatment. (H) Quantification of coelenterazine chemiluminescence for HeLa, RIN-5F and INS-1 cells treated as described in G at 120 minutes post-treatment (n = 3). (Error bars represent the mean ± SEM, * denotes a significant difference from the 13.2 mmol/L HeLa cells of P < 0.015, + denotes a significant difference from the 13.2 mmol/L RIN-5F cells of P < 0.015, and # denotes a significant difference from the 13.2 mmol/L INS-1 cells of P < 0.015.) (I) Dihydroethidium assay of 5x10⁵ HeLa cells exposed to glucose concentrations of 4 mmol/L (control) or 20 mmol/L with the quantification of 2-hydroxyethidium fluorescence as each time point minus the pre-treatment (-5 minutes) fluorescence intensity for each sample (n = 3). (Error bars represent the mean ± SEM, * denotes a significant difference from the 4 mmol/L control of P < 0.04.) (J) Coelenterazine assay of 5x10⁵ HeLa cells exposed to glucose concentrations of 4 mmol/L (control) or 20 mmol/L with the quantification as the cumulative area under the curve at each time point minus the pre-treatment (-5 minutes) chemiluminescence for each sample (n = 3). (Error bars represent the mean ± SEM, * denotes a significant difference from the 4 mmol/L control of P < 0.004.)</p

HeLa cells produce coelenterazine chemiluminescence <i>in vitro</i>.

<p>(A) 5x10⁵ HeLa cells were imaged sequentially, at 5 min intervals for 1 hour. (B) Quantification of HeLa (black) or buffer (white) coelenterazine chemiluminescence at each time point (n = 3). (C) 5x10⁵ HeLa cells were imaged at 15 minutes following the addition of native coelenterazine ranging in concentration from 0 μmol/L to 100 μmol/L. (D) Quantification of HeLa (black) or buffer (white) coelenterazine chemiluminescence (n = 3). (E) A range of 500 to 1x10⁶ HeLa cells were imaged 15 minutes following the addition of 10 μmol/L of native coelenterazine. (F) A range of 500 to 1x10⁶ HeLa cells were imaged 1 minute following the addition of 1.07 mmol/L luciferin. (G) Correlation graph of coelenterazine chemiluminescent signal intensity (y-axis) versus the corresponding luciferase signal intensity (x-axis) for a given cell concentration. (Error bars represent the mean ± SEM.)</p

Superoxide anion concentration, as detected with coelenterazine, serves as an <i>in vivo</i> reporter of beta-cell mass.

<p>(A) Images of a representative nonprogressive mouse at 10, 12, 14 and 16 weeks of age. (B) Images of a representative progressive mouse at 10, 12, 14 and 16 weeks of age. The progressive mouse was normoglycemic at the time of the images. (C) The signal intensities for the two groups of mice, nonprogressive (white) (n = 8) and progressive (black) (n = 7), for a given age normalized to the first day of imaging for the corresponding mouse and expressed as the fold change in signal intensity. (D) The signal intensities for the nonprogressive mice during the last week of the study normalized to the first day of imaging for the corresponding mouse and expressed as the fold change in signal intensity. The signal intensities for progressive mice for the last day of the study during which their fasting blood glucose levels were not exceeding 5.6, 6.9 or 8.3 mmol/L normalized to the first day of imaging for the corresponding mouse and expressed as the fold change in signal intensity. (E) The mean of the fasting blood glucose levels that were less than 8.3 mmol/L for nonprogressive and progressive mice. (Error bars represent the mean ± SEM, and * denotes a significant difference between the groups of P < 0.039.)</p

Coelenterazine administration <i>in vivo</i> results in chemiluminescent detection of superoxide anion in a dose-dependent manner that is of pancreatic origin.

<p>(A) Healthy mice were administered 0, 1, 2.5, 5, 7.5 and 10 mg/kg coelenterazine and imaged for chemiluminescence. (B) Quantification of the chemiluminescent signal for each coelenterazine dose (n = 1). (C) The fold change in the abdominal chemiluminescent signal from before to 20 hours following treatment with 600 units of PEG-SOD (SOD) or vehicle control (CON) (n = 3). (D) Tissues collected from a mouse euthanized 3 minutes post-administration of 5 mg/kg native coelenterazine. (E) Tissues from the mouse in D fluorescently imaged with an excitation of 430 nm and emission of 520 nm. (F) Isolated tissues treated with 20 μg of native coelenteraine <i>ex vivo</i>. (G) Quantification of the chemiluminescent signal from each well (n = 3). (Error bars represent the mean ± SEM, and * denotes a significant difference of P < 0.02.) (H) Whole mouse pancreas was excised and incubated in 20 μmol/L coelenterazine, fixed and stained with anti-insulin antibody followed by an Alexa Fluor 750 secondary. (Scale bar represents 400 μm.)</p

Treatment with mitochondrial shuttle inhibitors, GKA50, Ranolazine and glucose results in significant decreases in superoxide concentration as detected by coelenterazine chemiluminescence.

<p>(A) Bold, red font indicates methods of perturbation. The physiological response of cells was assessed by addition of glucose, GKA50, an activator of glycolysis, AOAC, an inhibitor of the malate-aspartate shuttle, 4-OHCA, an inhibitor of the pyruvate and lactate shuttle, ranolazine, an inhibitor of beta-oxidation and an activator of pyruvate dehydrogenase. (B) HeLa cells were treated with vehicle control, 250 μmol/L of 4-OHCA, 100 μmol/L AOAC, a combination of 4-OHCA and AOAC, 1 μmol/L GKA50 or 1 μmol/L Ranolazine and the final glucose concentrations adjusted from 4 mmol/L to 4 mmol/L, 11.7 mmol/L or 30 mmol/L and sequentially imaged. Representative image is 95 minutes post treatment. (C) Quantification of coelenterazine chemiluminescence 95 minutes post-treatment (n = 3). (Error bars represent the mean ± SEM, * denotes a significant difference from the 4 mmol/L control of P < 0.05, # denotes a significant difference from the 11.7 mmol/L control of P < 0.015, and + denotes a significant difference from the 30 mmol/L control of P < 0.015.)</p

Coelenterazine chemiluminescence is dependent upon superoxide anion concentrations <i>in vitro</i>.

<p>(A) HeLa cells were treated with range of 0 units/mL to 300 units/mL of PEG-SOD and imaged sequentially. The image was taken at 95 minutes post PEG-SOD addition. (B) Quantification of HeLa (black) or buffer (white) coelenterazine chemiluminescence for each concentration 95 minutes post PEG-SOD addition (n = 3). (C) HeLa cells were treated with range of 0 units/mL to 600 units/mL of PEG-CAT and imaged sequentially. Image is 95 minutes post PEG-CAT addition. (D) Quantification of HeLa (black) or buffer (white) coelenterazine chemiluminescence for each concentration 95 minutes post PEG-CAT addition (n = 3). (E) HeLa cells were treated with range of 0 μmol/L to 500 μmol/L of uric acid and imaged sequentially. Image is 95 minutes post uric acid addition. (F) Quantification of HeLa (black) or buffer (white) coelenterazine chemiluminescence for each concentration 95 minutes post uric acid addition (n = 3). (Error bars represent the mean ± SEM, and * denotes significant differences of P < 0.02.)</p