Figure 4.

Insulin Regulates Mitochondrial Respiration via PI3K/AKT in hESCs

(A) Western blot analysis of p-AKT (Ser473) and total AKT in H1 hESCs after 1 h of incubation in E8 or Mito Stress Test assay medium supplemented with vehicle ± insulin, or BEZ235 (BEZ, 2.5 μM) ± insulin. GAPDH was used as loading control.

(B) OCR of H1 hESCs after 1 h of pre-incubation with vehicle ± insulin or BEZ235 ± insulin, measured with Mito Stress Test at baseline and in response to Oligo, FCCP, and Rote/AA (n = 3 independent replicates).

(C) Kinetic OCR response to vehicle ± insulin or BEZ235 ± insulin as indicated.

(D) OCR of H1 hESCs following 1 h of pre-incubation with vehicle ± insulin or AKTi VIII (10 μM) ± insulin, measured with Mito Stress Test (n = 3 independent replicates).

(E) Kinetic OCR response to vehicle ± insulin or AKTi VIII (10 μM) ± insulin as indicated.

(F and G) OCR of AKT1-overexpressing (F) and AKT2-overexpressing (G) H1 cell lines following 1 h of pre-incubation with or without insulin, measured with Mito Stress Test. The data were normalized to the cell numbers. The cell numbers were determined using a Celigo image cytometer (n = 3 independent replicates).

(H) Western blot analyses of p-AKT (Ser473) and total AKT in H1 hESCs, and AKT1- and AKT2-overexpressing H1 cell lines after 2 h of incubation in E8 or Mito Stress Test assay medium. GAPDH was used as loading control.

(I) Western blot analyses of p-AKT (Ser473) and AKT in whole-cell lysate and the mitochondrial fraction from H1 hESCs after 2 h of incubation in Mito Stress Test assay medium with or without insulin. COX IV and GAPDH were used as loading controls.

(J) Immunostaining of p-AKT (Ser473) in H1 hESCs (green) after 2 h of incubation in Mito Stress Test assay medium supplemented with insulin. Mitochondria were stained with MitoTracker (red) and nuclei with Hoechst (blue). Vehicle, DMSO. Scale bar, 5 μm.

Data are presented as means ± SD.