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. Author manuscript; available in PMC: 2019 Aug 28.
Published in final edited form as: Cell Rep. 2019 Jul 16;28(3):759–772.e10. doi: 10.1016/j.celrep.2019.06.058

Figure 3. The yeast GTP/GDP Exchanger Increases mtGTP Permeability and Insulin Secretion.

Figure 3.

(A–C) Expression of yeast GGC1, a GTP/GDP exchanger, exchanges cytosolic GTP down its concentration gradient for matrix GDP. mtGTP is also generated by endogenous SCS-GTP (red circle). PEPCK-M uses mtGTP to metabolize anaplerotic OAA into cataplerotic PEP (A). mRNA of GFP (B) and GGC1 (C) from cells transiently transfected with GGC1-GFP versus empty vector (control).

(D) Western blot of anti-GFP and anti-voltage-dependent anion channel (VDAC) as loading control in whole-cell lysates, mitochondrial (mito), and cytosolic (cyto) fractions of INS-1 cells transfected with GGC1-GFP (GGC1+) or empty vector (control).

(E) NADH oxidation via GDH in isolated mitochondria as a function of GTP concentration.

(F) Insulin secretion from INS-1 cells overexpressing control (empty vector), mtGFP, and GGC1 after 45 min of exposure to basal 3 mM (gray bars) or stimulatory 15 mM (black bars) glucose.

(G) Mitochondria from cells overexpressing hSCS-ATP (blue circles) have decreased mtGTP, making GGC1 the principal source of matrix GTP.

(H–J) mRNA of (H) GFP, (I) GGC1, and (J) hSCS-ATP from cells transiently transfected with GGC1-GFP versus empty vector (control).

(K) Insulin secretion from constitutive hSCS-ATP cells (solid bars) and hSCS-ATP cells transfected with empty vector (blue striped) or GGC1-GFP vector (red striped) after 45 min exposure to basal 2.5 mM or stimulatory 16.7 mM glucose.

The data are means ± SEMs (for B, C, and H–J: n = 2 per group; for E: n = 7; for F: n = 6 per group; for K: basal glucose hSCS-ATP n = 6, control hSCS- ATP n = 25, GGC1 hSCS-ATP n = 6, stimulatory glucose hSCS-ATP n = 6, control hSCS- ATP n = 23, GGC1 hSCS-ATP n = 5; technical replicates for all panels). Statistical significance is *p < 0.05, ***p < 0.001, and ****p < 0.0001 by unpaired two-tailed t test (B, C, and E), two-way ANOVA with Tukey’s multiple comparisons test (F and K), one-way ANOVA with Tukey’s multiple comparisons test (H and J), and one-way ANOVA with Holm-Sidak’s multiple comparisons test (I).