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. 2009 Nov 24;4(11):e7983.
doi: 10.1371/journal.pone.0007983.

Insulin signaling regulates mitochondrial function in pancreatic beta-cells

Siming Liu  1 , Terumasa Okada, Anke Assmann, Jamie Soto, Chong Wee Liew, Heiko Bugger, Orian S Shirihai, E Dale Abel, Rohit N Kulkarni
Affiliations

Insulin signaling regulates mitochondrial function in pancreatic beta-cells

Siming Liu et al. PLoS One. .

Abstract

Insulin/IGF-I signaling regulates the metabolism of most mammalian tissues including pancreatic islets. To dissect the mechanisms linking insulin signaling with mitochondrial function, we first identified a mitochondria-tethering complex in beta-cells that included glucokinase (GK), and the pro-apoptotic protein, BAD(S). Mitochondria isolated from beta-cells derived from beta-cell specific insulin receptor knockout (betaIRKO) mice exhibited reduced BAD(S), GK and protein kinase A in the complex, and attenuated function. Similar alterations were evident in islets from patients with type 2 diabetes. Decreased mitochondrial GK activity in betaIRKOs could be explained, in part, by reduced expression and altered phosphorylation of BAD(S). The elevated phosphorylation of p70S6K and JNK1 was likely due to compensatory increase in IGF-1 receptor expression. Re-expression of insulin receptors in betaIRKO cells partially restored the stoichiometry of the complex and mitochondrial function. These data indicate that insulin signaling regulates mitochondrial function and have implications for beta-cell dysfunction in type 2 diabetes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Presence of BAD/GK complex in mitochondria.
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; BADS, 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.
Figure 2
Figure 2. Altered expression of BADS and phospho-BADS in mitochondria and cytosol.
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 BADS, * 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 BADS, * p<0.05. F and G, Ser-112 phosphorylation pattern of cytosolic BADS 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.
Figure 3
Figure 3. Altered phospho-p70S6K and phospho-JNK1 activation in mitochondria.
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.
Figure 4
Figure 4. Altered mitochondrial function in βIRKO β-cells.
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
Figure 5
Figure 5. Re-expression of insulin receptors improved altered mitochondrial function in βIRKO β-cells.
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.
Figure 6
Figure 6. Normalization of signaling complexes after re-expression of insulin receptor B isoforms in βIRKO β-cells.
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, BADS. Arrow indicates total JNK1.
Figure 7
Figure 7. Schematic showing the impact of insulin/IGF-I signaling on β-cell mitochondrial metabolism and function.
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.
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