Control or βIRKO cells were treated with 100 nM of either insulin or IGF-I for 15 min and fractionated mitochondria and cytosol were examined. Protein levels were assessed by densitometry. Controls were set to an arbitrary evel of 1. In Figures A though G, the upper panels show representative Western blots and lower panels show mean values after quantitation of data. In H a representative Western blot is shown. Figures A, B, C and D relate to alterations in isolated mitochondrial fractions and Figures E, F, and G relate to alterations in cytosolic fractions. A, Reduced total BAD_S, * p<0.05 βIRKO vs control cells. B, Mitochondrial BADs phosphorylation at Ser-112, * p≤0.05, basal vs insulin stimulation, n = 3. ND, non-detectable. C, Mitochondrial BADs phosphorylation at Ser-136, * p≤0.05, basal vs insulin stimulation, n = 3. D, Mitochondrial BADs phosphorylation at Ser-112 and Ser-136 under basal or IGF-I stimulated conditions, * p≤0.05 vs. Control basal; # p<0.05, βIRKO±IGF-I; n = 3. Open bar: controls; Shaded bar: βIRKO. E, Total cytosolic BAD_S, * p<0.05. F and G, Ser-112 phosphorylation pattern of cytosolic BAD_S upon insulin (F) or IGF-I (G) treatments. * p<0.05 control basal vs βIRKO basal; # p<0.05, control±insulin/IGF-I. H, Total BAD in whole cell lysates. Open bar: controls; Shaded bar: βIRKO.

A–B, Reduced mitochondrial membrane potential response to glucose stimulation in βIRKO β-cells. A, dispersed islet β-cells from control or βIRKO mice were stained by TMRE and analyzed by confocal microscopy. B, Fluorescence intensity in images was quantitated by NIH ImageJ. n = 3–5; # p<0.05, 5.5 mM vs 18 mM glucose. Open bar: controls; shaded bar: βIRKO β-cells. C–D, Reduced mitochondrial glucokinase activity in βIRKO β-cells. Glucokinase activity in freshly purified mitochondria from either control or βIRKO cells is plotted as absorbance of glucose-6-phosphate dehydrogenase-driven increase in NADPH fluorescence (C). The activity is illustrated by the rates determined by measuring optical density changes over linear periods, between 5 and 20 minutes (D). Mean of three independent experiments is shown. The activities of controls were set to an arbitrary level of 1. * p<0.05. E–G, Altered mitochondrial number and size. Quantitative analyses of mitochondrial ultrastructrue in islet β-cells from control or βIRKO mice. Mitochondrial number (E), size (F), and volume density (G) were quantitated as described in Methods, * p<0.05, n = 5. H, Representative transmission electron microscopy (TEM) micrographs of islet β-cells from control or βIRKO mice. Arrowheads point mitochondria showing increased size in βIRKOs. n = 28–30 cells from 3–5 mice. Magnification bar: 2 µM

A, Upper panel, Protein profile after expression of insulin receptors in βIRKO cells was immunoblotted by antibodies as indicated. p-Akt of controls were set to an arbitrary level of 1, * p<0.05, n = 3. Lower panel shows quantitation of p-Akt to total Akt. B, Upper panel, P70S6K and phosphor-P70S6K were assessed in cytosolic fractions from control, βIRKO or βIRKO cells re-expressing insulin receptors (βIRKO+hIRB), p-P70S6K of controls were set to an arbitrary level of 1, * p<0.05, n = 3. Lower panel shows quantitation of p-P70S6K to total p70S6K. C, Upper panel, microcystin pull down assays to demonstrate the five protein components of BAD/GK complex in mitochondria isolated from control, βIRKO or βIRKO cells re-expressing insulin receptors (βIRKO+hIRB), * p<0.05. Lower panel, each component was normalized to PP1 which was set to an arbitrary level of 1 for each cell type. D, Mitochondrial fractions isolated from control, βIRKO or βIRKO cells re-expressing insulin receptors (βIRKO+hIRB) were used to evaluate phosphorylation of JNK1, p70S6K, BAD_S. Arrow indicates total JNK1.

Upper panels: Western blotting of five components in the BAD/GK complex with the indicated antibodies. Lower panels: Mean data after quantitation. Each component was normalized to PP1 and control samples were set to an arbitrary level of 1. A, control (Con) and βIRKO β-cell lines. B, control and βIRKO islet cells. C, islet cells from controls or patients with type 2 diabetes. Representative of 3–5 independent experiments. * p<0.05; ** p<0.001 versus Control. Open bar: controls; Shaded bar: βIRKO or type 2 diabetes β-cells. WAVE1, A protein kinase A scaffold; GK, glucokinase; PP1, protein phosphotase 1; PKA, protein kinase A; BAD_S, BCL2-antagonist of cell death. D, Western blotting of five components in BAD/GK complex in cytosolic and mitochondrial compartments fractionated from control (Con) or βIRKO (KO) β-cell lines. Equal amount of proteins was loaded in each lane. Using total amount of protein loaded for the cytosol and mitochondria ∼20% of total cellular GK is associated with mitochondria. L and S denote the large and small isoforms of BAD respectively.

Control or βIRKO cells were treated with 100 nM of either insulin or IGF-I for 15 min and fractionated cytosol and mitochondria were used for analyses. Controls were set to an arbitrary level of 1. In each Figure, the upper panel shows the representative Western blot and lower panel shows the mean value after quantitation of data. A, Responses in mitochondrial p70S6K to insulin treatment. * p<0.05; # p≤0.05. B, Responses in cytosolic p70S6K to insulin treatment. * p = 0.06; # p = 0.078; C and D, Responses in mitochondrial and cytosolic p70S6K to IGF-I treatment in control and βIRKO cells. * p<0.05; # p<0.05. E and F, Responses in mitochondrial and cytosolic JNK1 to IGF-I treatment in control and βIRKO cells. # p<0.05. Open bar: controls; Shaded bar: βIRKO.

A, Basal and 25 mM glucose stimulated oxygen consumption rates (OCR) in control, βIRKO or βIRKO cells re-expressing insulin receptors (βIRKO+hIRB) cells, * p<0.05, n = 6. B, OCR response to 60 µM DNP (2,4-Dinitrophenol) in control, βIRKO or βIRKO+hIRB cells. OCRs were normalized by cell numbers in all assays, * p<0.05, n = 22. C, Re-expression of insulin receptors recovered the reduction of ATP production in βIRKO cells in response to 25 mM glucose stimulation. After starvation, control, βIRKO or βIRKO+hIRB cells were treated with 2 mM and 25 mM glucose in KRB buffer for 30 min, ATP was extracted and measured, ** p<0.001, n = 3. D and E, Re-expression of insulin receptors reduced the PGC1α and UCP2 expression to control levels. Expression levels of PGC1α (D) and UCP2 (E) were assessed in control, βIRKO or βIRKO+hIRB cells using real-time PCR. All cells grown in normal culture conditions; mRNA was extracted and cDNA was synthesized for real-time PCR, * p<0.05, n = 3.

Impairment of insulin signaling alters BAD/GK complex and reduces mitochondrial function by inhibiting mitochondrial GK activity. The compensatory increase in IGF-I signaling can impact phosphorylation of mitochondrial BAD to influence glucokinase activity and the mitochondrial threshold to cell death cues; it can also impact mitochondrial p70S6K to phosphorylate BADs. Mitochondrial dysfunction due to impaired insulin signaling can activate mitochondrial JNK1, which can phosphorylate mitochondrial substrate, e.g. BADS, to influence mitochondrial function and sensitivity to stress-induced cell death.