Fig 1.
Accumulation of C/EBPβ due to ER stress lowers AMPK activity.
A: MIN6 cells were treated with 1 μg/mL tunicamycin (Tm) for 24 h and analyzed with the indicated antibodies (left). Quantitation of the AMPK phosphorylation level is normalized to total AMPK (right). B: MIN6 cells were transfected with C/EBPβ expression vectors or empty vector. The cells were analyzed with the indicated antibodies (left and middle). Quantitation of the AMPK phosphorylation level is normalized to total AMPK (right). C: Isolated islets from wild-type (WT) and TG mice at 12 weeks of age were analyzed with the indicated antibodies (left). Quantitation of the AMPK phosphorylation level is normalized to total AMPK (right). D: Isolated islets from WT and TG mice at 12 weeks of age were analyzed for AMP and ATP levels by LC/MS. Data are means ± SE of 3 independent experiments. *P < 0.05, **P < 0.01. n.s., not significant.
Fig 2.
C/EBPβ expression is inhibited when AMPK is activated pharmacologically.
A, B: MIN6 cells were transfected with expression vectors for C/EBPβ. The cells were incubated with 100 μM AICAR (A) or 500 μM metformin (B) for 24 h and analyzed with the indicated antibodies (left). Quantitation of C/EBPβ expression is normalized to β-actin (right). C: MIN6 cells were transfected with expression vectors for c-Myc-tagged AMPK, CA-AMPK, DN-AMPK, and/or human influenza hemagglutinin epitope-tagged C/EBPβ, as indicated. These transfected cells were analyzed with the indicated antibody (left and right). Quantitation of C/EBPβ expression is normalized to β-actin (middle). D: MIN6 cells treated with scramble siRNA (control) and AMPK siRNA were lysed and subjected to immunoblot analysis with antibodies against AMPK, ACC, p-AMPK, p-ACC, and C/EBPβ. E: Isolated islets from WT and TG mice following 8 weeks of vildagliptin (vilda) treatment were analyzed with the indicated antibodies (left). Quantitation of the AMPK phosphorylation level is normalized to total AMPK (right). Data are means ± SE of 3 (C, D) or 4 (A, B, E) independent experiments. *P < 0.05, **P < 0.01.
Fig 3.
C/EBPβ phosphorylation is required for protein stabilization.
A: MIN6 cells were incubated with 2 μg/mL tunicamycin and/or 20 μM H7. B: MIN6 cells were transfected with expression vectors for C/EBPβ and then incubated with 20 μM H7 and 20 μM MG132. C: MIN6 cells were incubated with 5 μg/mL tunicamycin for the indicated periods. C/EBPβ expression and the phosphorylation of C/EBPβ at Thr-188 were analyzed by western blot analysis. D: MIN6 cells were transfected with expression vectors for C/EBPβ and then incubated with 20 μM U0126 and 20 μM SB203580 (left). Quantitation of C/EBPβ expression is normalized to β-actin (right) (n = 3). *P < 0.05, **P < 0.01. n.s., not significant.
Fig 4.
AMPK destabilizes C/EBPβ via T188 dephosphorylation.
A: MIN6 cells were co-transfected with expression vectors for C/EBPβ, T188A-mutant C/EBPβ, or T188E-mutant and mock or DN-AMPK (leftmost and second right). Quantitation of C/EBPβ expression is normalized to β-actin (second left and rightmost). B: MIN6 cells were co-transfected with expression vectors for C/EBPβ or T188A-mutant C/EBPβ, T188E-mutant and mock or CA-AMPK (leftmost and second right). Quantitation of C/EBPβ expression is normalized to β-actin (second left and rightmost). C, D: MIN6 cells were transfected with expression vectors for C/EBPβ or HA-tagged C/EBPβ. The cells were pretreated with 1 μg/mL CHX and incubated with 1 μg/mL CHX and 1 μM thapsigargin. C/EBPβ (C) or T188A-mutant C/EBPβ (D) expression was measured by western blot analysis (C; left and middle, D; left). Quantitation of C/EBPβ expression is normalized to β-actin (right). Data are means ± SE of 3 independent experiments. *P < 0.05, **P < 0.01. n.s., not significant.
Fig 5.
Effect of metformin on glucose tolerance in pancreatic beta cell-specific C/EBPβ TG mice.
A: Blood glucose levels and B: plasma insulin concentrations during the 8-week treatment period (n = 10–16 per group). C: Blood glucose levels and D: plasma insulin levels after an oral glucose load (1.5 mg/g body weight) following 8 weeks of metformin (met) treatment (n = 10–13 per group). *P < 0.05.
Fig 6.
Metformin has no significant impact on AMPK activity or pancreatic beta cell mass in pancreatic beta cell-specific C/EBPβ TG mice.
A: Western blot analysis of islets isolated from TG mice following 8 weeks of metformin (met) treatment for analysis with the indicated antibodies. B: Quantitation of the AMPK phosphorylation level is normalized to total AMPK in A (n = 4 per group). C: Quantitation of C/EBPβ expression in A (n = 3 per group). D: Immunofluorescence staining of pancreas sections for insulin (red) and glucagon (green) following 8 weeks of metformin administration. Magnification bar = 100 μm. E: Quantification of beta cell mass in WT and TG mice (n = 5–7 per group). *P < 0.05, **P < 0.01. n.s., not significant.
Fig 7.
Effect of the combined use of metformin and vildagliptin on glucose tolerance in pancreatic beta cell-specific C/EBPβ TG mice.
A: Blood glucose levels and B: plasma insulin concentrations during the 8-week treatment period (n = 11–15 per group). C: Blood glucose levels and D: plasma insulin levels after an oral glucose load (1.5 mg/g body weight) following 8 weeks of vildagliptin (vilda) and/or metformin (met) treatment (n = 5–11 per group). WT-(vilda), open triangles; WT-(vilda+met), open diamonds; TG-(vilda), black triangles; and TG-(vilda+met), black diamonds. *P < 0.05, **P < 0.01.
Fig 8.
Effect of combined use of metformin and vildagliptin on the islets in pancreatic beta cell-specific C/EBPβ TG mice.
A: Western blot analysis of islets isolated from TG mice following 8 weeks of vildagliptin (vilda) and/or metformin (met) treatment for analysis with the indicated antibodies. B: Quantitation of the AMPK phosphorylation level is normalized to total AMPK in A (n = 4 per group). C: Quantitation of C/EBPβ expression in A (n = 4 per group). D: Immunofluorescence staining of pancreas sections for insulin (red) and glucagon (green) following 8 weeks of vildagliptin and/or metformin administration. Magnification bar = 100 μm. E: Quantification of beta cell mass in WT and TG mice (n = 6–7 per group). *P < 0.05, **P < 0.01.