Fig 1.
Glucagon secretion from mouse-isolated islets and InR1G cells.
Islets were isolated from C57B6 mice, and then batches of 20 islets were subjected to static incubation experiments for glucagon (A) and insulin (B) secretion after pretreatment with 7 mM (white) or 15 mM (black) glucose (n = 3–4, each group). InR1G cells were pretreated with 11.1 mM (white) or 25 mM (black) glucose. (C) InR1G cell glucagon secretion (top) and protein content (bottom) (n = 3–4, each group). (D) Number of the InR1G cells pre- (hatched) and post-treatment (n = 3–4, each group). Data are expressed as mean ± SEM; *P<0.05, between the indicated groups.
Fig 2.
Glucagon content and preproglucagon expression.
Cells were pretreated with 11.1 mM (white) or 25 mM (black) glucose. (A) Glucagon content following static incubation (n = 3–4, each group). (B) Preproglucagon expression (n = 6, each group). Data are expressed as mean ± SEM; *P<0.05, between the indicated groups.
Fig 3.
Akt and PDK1 phosphorylation status.
InR1G cells were exposed to regular (11.1 mM, white) or high (25 mM, black) glucose levels for 12 h. (A) Ser473 and Thr308 phospho-(p)Akt/Akt ratios were determined. (B) Following the stimulation of 11.1 mM or 25 mM glucose-treated cells with insulin (100 nM) for 0, 5, or 15 min, the ratio of Ser473 pAkt/Akt was determined. Representative images selected from 3 independent experiments are shown. (C) Phospho-(p)PDK1/PDK1 ratios were determined. Relative expression of pAkt and pPDK1 was determined using densitometry and normalized using total Akt and PDK1 levels. n = 3–4 in each group. Data are expressed as mean ± SEM; *P<0.05, between the indicated groups.
Fig 4.
ROS content and the effects of oxidative stress on InR1G cells.
(A) ROS content determination. Cells were exposed to regular (11.1 mM, white) or high (25 mM, black) glucose levels for 12 h; n = 7; data are expressed as mean ± SEM; *P<0.05, between the indicated groups. (B) JNK phosphorylation levels. InR1G cells were exposed to 11.1 mM glucose for 12 h, and stimulated using H2O2 (100 μM) for 5 or 15 min. (C) Akt phosphorylation and (D) glucagon secretion levels following the exposure of the cells to regular (11.1 mM) glucose levels in combination with (dotted)/without (white) 50 μM H2O2, or high (25 mM, black) glucose levels for 12 h, and the glucagon secretion was assessed after the stimulation with 25 mM glucose. Relative pAkt expression was determined using densitometry and normalized using total Akt levels; n = 4, each group. Data are expressed as mean ± SEM; *P<0.05, between the indicated groups.
Fig 5.
Time-dependent effects of different glucose concentrations on Akt and JNK phosphorylation levels.
InR1G cells were treated with regular (11.1 mM, white) or high (25 mM, black) glucose levels for 1, 2, 6, and 12 h. (A) JNK phosphorylation levels. (B) Akt phosphorylation levels. (C) InR1G cells were exposed to regular (11.1 mM) glucose concentration, in combination with (dotted) or without (white) 13.9 mM 2-DG, or high (25 mM, black) glucose concentration for 12 h (n = 3, each group). Relative pJNK and pAkt expressions were determined by densitometry, and they were normalized using total JNK and Akt levels. Representative images were selected from 3 independent experiments; n = 3 in each group; data are expressed as mean ± SEM; *P<0.05, between the indicated groups.
Fig 6.
Effects of JNK inhibition on InR1G glucagon secretion.
(A) Cells were cultured with DMSO or 10 μM SP600125 for 1 h, and stimulated using H2O2 (100 μM) for 15 min, and pJNK and JNK levels were determined. (B) JNK and pJNK levels in cells treated with regular (11.1 mM) or high (25 mM) glucose concentrations for 12 h, with the addition of DMSO or 10 μM SP600125. Relative pJNK expressions were determined by densitometry, and they were normalized using total JNK levels. (C) Glucagon secretion in cells treated with regular (11.1 mM, white) or high (25 mM, black) glucose concentrations, together with DMSO or 10 μM SP600125 for 12 h before the static incubation with the indicated glucose levels. (D) Cells were treated with DMSO or 10 μM SP600125, for 12 h before the static incubation with 7 mM glucose with (hatched)/without the addition of 10 mM L-Arg. Representative graphs/images selected from the results of 3 or 4 independent experiments are shown. n = 3–4 in each group; data are expressed as mean ± SEM; *P<0.05, between the indicated groups.
Fig 7.
Effects of DN-JNK expression on InR1G glucagon secretion.
Cells were infected with adenoviruses expressing GFP or DN-JNK 24 h prior to the 12-h treatment with 11.1 or 25 mM of glucose. (A) pJNK, JNK, pAkt, and Akt expression levels in these cells. Relative pJNK and pAkt expressions were determined by densitometry, and they were normalized using total JNK and Akt levels. (B) Glucagon secretion was assessed at 25 mM glucose stimulation in cells infected with adenoviruses carrying GFP- or DN-JNK expression vector. n = 4~6 in each group; data are expressed as mean ± SEM; *P<0.05, between the indicated groups.