Fig. 6: Mitochondrial substrates elevate ATP and insulin secretion but do not suppress AMPK signalling. | Nature Communications

Fig. 6: Mitochondrial substrates elevate ATP and insulin secretion but do not suppress AMPK signalling.

From: Altered glycolysis triggers impaired mitochondrial metabolism and mTORC1 activation in diabetic β-cells

Fig. 6

a, b Insulin secretion (a, n = 3 biologically independent experiments) and ATP/ADP ratio (b, n = 5 biologically independent experiments) in LG-cells stimulated with 20 mM glucose (G), methyl pyruvate (Pyr), leucine (Leu) or monomethylsuccinate (MMS) for 1 h. c Representative Western blot of lysates from LG-cells acutely exposed to 2 mM glucose or 20 mM of the indicated mitochondrial substrates for 1 h. Uncropped blots in source data. d, e Quantitative densitometry analysis of p-AMPK/AMPK (d) and p-S6/S6 (e) in LG-cells stimulated with 20 mM glucose (G), methyl pyruvate (Pyr), leucine (leu) or monomethysuccinate (MMS) for 1 h (n = 3 biologically independent experiments). f Representative Western blot of lysates from cells transfected with scrambled siRNA (Scr) or Pfkl and Pfkm siRNA (Pfkl + m KD) and cultured at low (LG) or high (HG) glucose for 48 h. Cells were subsequently stimulated with 2 mM or 20 mM glucose for 1 h. Phosphorylated (p) and total AMPK and S6. Uncropped blots in source data. g, h Quantitative densitometry analysis of p-AMPK/AMPK (b, n = 4 biologically independent experiments) and p-S6/S6 (c, n = 4 biologically independent experiments). LG-cells (black), HG-cells (red), LG-cells with Pfkl+m KD (blue), HG-cells with Pfkl+m KD (orange). i Representative Western blot of lysates from LG- and HG-cells cultured for 48 h in the presence of 0.05% DMSO (veh) or 5 µM of the PFKFB3 inhibitor, PFK-15, and then stimulated with 2 mM or 20 mM glucose for 1 h. Phosphorylated (p) and total AMPK and S6. Uncropped blots in source data. j, k Quantitative densitometry analysis of p-AMPK/AMPK (b, n = 3 biologically independent experiments) and p-S6/S6 (c, n = 4 biologically independent experiments). LG-cells (black), HG-cells (red), LG-cells + PFK-15 (blue), HG-cells + PFK-15 (orange). Veh, vehicle (0.05% DMSO). Panels show individual data points and mean ± sem. *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA with Bonferroni post hoc test (a, b, d, e) and two-tailed unpaired Student’s t test (g, h, j, k). Source data are provided as a Source Data file.

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