FIGURE 6.

PEPCK-M is required for PEP synthesis and insulin secretion. A, two different siRNAs were used to silence PEPCK-M expression in INS-1 832/13 cells. -Fold change in PEPCK-M mRNA levels after 36 h of transfection with a control siRNA (non-silencing siRNA), siRNA1, or siRNA2 against PEPCK-M normalized to GAPDH. Western blot analysis of PEPCK-M after knockdown using siRNA in INS-1 832/13 cells is shown below. B, PEPCK activity measurement in INS-1 832/13 cells after silencing of PEPCK-M with siRNA1 compared with non-treated cells. C, time course of M+1 labeled PEP by PEPCK-M. INS-1 832/13 cells were preincubated in DMEM base without glucose for 60 min and then 15 mm glucose with 5 mm [3-¹³C]pyruvate. Shown are the concentrations of the M+1 isotopologue of PEP metabolized by PEPCK-M in non-transfected and siRNA1-transfected cells at the indicated times. D, glucose-stimulated insulin secretion in INS-1 832/13 cells that were non-transfected or transfected with a control siRNA, siRNA1, or siRNA2 against PEPCK-M. Cells were preincubated in KRBH with 3 mm glucose for 90 min and then with either 3 or 15 mm glucose for 90 min. Error bars are S.E. and significance by t test and ANOVA; *, p < 0.05; **, p < 0.01; ***, p < 0.001.