siRNA screen for mitochondrial regulators. (A) Schematic diagram of the screen. (B) Distribution of MitoTracker Green versus MitoTracker Red CMXRos signals (left plot) and MitoTracker Green versus the Red-to-Green ratio (right plot) in the primary screen. (C) Assessment of mitochondrial content by MitoTracker Green signal and mitochondrial DNA quantitation after siRNA depletion of Sin3A and Ccnd1. (*) P < 0.05 by unpaired t-test. (D) Assessment of mitochondrial membrane potential by the Red-to-Green ratio and TMRE signal after siRNA depletion of Sin3A and Ccnd1. (*) P < 0.05. (E) Network extension of the WNT pathway. The network was constructed by anchoring on canonical WNT pathway components (diamonds) using protein interaction data, incorporating interactions with human orthologs of screen hits (red) via up to one bridging component (blue). The interactions shown fulfilled a Benjamini and Hochberg (BH)-adjusted P-value of <0.05 for the given network size.

Increased Wnt signals activate mitochondrial biogenesis and OXPHOS gene expression. Cells received either 0.1% BSA in PBS (control) or the recombinant Wnt3A protein at the doses indicated. (*) P < 0.05 by unpaired t-test; (**) P < 0.01. (A) Mitochondrial proliferation in C2C12 cells following Wnt3A treatment as assessed by mitochondrial DNA quantitation. (B) Electron micrographs of C2C12 cells treated with control or 50 ng/mL Wnt3A protein for 3 d. (C) Wnt3A-treated cells show a significant increase in mitochondrial volume density (P < 0.05), as quantified by using a grid superimposed on electron micrographs (N = 15 each). The number of points falling within mitochondria was expressed as a percentage of the number of points falling within cytoplasm. A small increase in cristae surface density was also noted (data not shown). Error bars represent SEM. (D,E) Increased cellular oxygen consumption results from Wnt3A treatment. Drugs were added at the indicated times. (F) Activation of mitochondrial OXPHOS gene expression from Wnt3A treatment. (G,H) Mitochondrial proliferation and increased OXPHOS gene expression in MEFs following 3 d of Wnt3A exposure. (I) Treatment with the Wnt3A antagonist Dkk-1 (5 μg/mL) for 1 wk reduces mitochondrial DNA in MEFs. (J) Stable expression of dominant-negative TCF4 (DN TCF4) cDNA reduces mitochondrial DNA and abrogates the Wnt3A effect on mitochondrial proliferation. C2C12 cells were transduced with retroviruses carrying an empty vector or the dominant-negative TCF4 cDNA, and were selected for puromycin resistance. (*) P < 0.05 for control versus Wnt3A; (†) P < 0.05 for vector versus dominant-negative TCF4 (DN TCF4). (K,L) Dominant-negative TCF4 cDNA expression reduces oxygen consumption. DNP and oligomycin were injected at the indicated times to measure the maximal respiratory capacity and uncoupled respiration, respectively.

Wnt-driven effects on mitochondria lead to increased oxidative stress. (*) P < 0.05 by unpaired t-test; (**) P < 0.01. (A,B) Wnt3A treatment increases mitochondrial superoxide and hydrogen peroxide levels. This is observed after 3 d of Wnt3A treatment (shown), as well as following long-term exposure. (C) Continuous exposure of MEFs to WNT3A results in increased oxidative DNA damage, as measured by 8-oxoguanine levels, which is prevented by concominant NAC treatment. NAC also blocks the Wnt3A-induced increase in the SAβ-gal staining (D) and decrease in BrdU incorporation of cells given a 1-h pulse of BrdU (E). In C–E, MEFs were examined after 2 wk of Wnt3A treatment in 3% oxygen. (F) Wnt3A treatment induces mitochondrial proliferation after 3 d of treatment with or without NAC present.

Wnt-driven mitochondrial biogenesis involves Myc and IRS-1. (A) Depletion of Myc by shRNA reduces Wnt-driven mitochondrial proliferation. Cells transduced with viruses expressing Myc shRNAs or controls were treated with Wnt3A for 3 d and assayed for MitoTracker Green signal. (B) IRS-1 mRNA is rapidly induced in MEFs by Wnt3A treatment, while the Myc mRNA induction shows delayed kinetics. Similar induction was seen in C2C12 cells (not shown). (C) The IRS-1 protein is induced within 12 h following Wnt3A treatment. (D) Depletion of IRS-1 by shRNA reduces Wnt-driven mitochondrial proliferation. Cells expressing IRS-1 shRNAs or controls were treated with Wnt3A for 3 d and were analyzed as in A. (E) Stable expression of Wnt3A induces IRS-1 in C2C12 cells, while expressing Dkk-1 has the opposite effect. Cells were transduced with viruses expressing the Wnt3A cDNA or the Dkk-1 cDNA, selected, and examined. (F) Stable expression of dominant-negative TCF4 cDNA reduces the IRS-1 protein levels and abrogates Wnt3A-mediated induction of IRS-1. Cells were treated with recombinant Wnt3A protein for 3 d. (G) TCF4 binds the IRS-1 promoter, as determined by ChIP and quantitative PCR. C2C12 cells were treated with Wnt3A protein for 1 d and subjected to chemical cross-linking, and the DNA was sheared and immunoprecipitated with rabbit IgG or the TCF4 antibody. (H) The IRS-1 protein accumulates in the chromatin fraction following Wnt3A treatment in C2C12 cells. (TOT) Total lysate; (CYT) cytoplasmic proteins; (SN) soluble nuclear proteins; (CHR) chromatin fraction. Histone H2A and Ras are chromatin and cytosolic markers, respectively. (I) IRS-1 occupies the Myc promoter in a Wnt-dependent fashion. C2C12 cells were treated for 1 d with vehicle or Wnt3A protein, cross-linked, and processed for ChIP with rabbit IgG or the IRS-1 antibody. (J) Reciprocal coimmunoprecipitation studies of TCF4 and IRS-1 in Wnt3A-treated C2C12 cells. (K) Depletion of IRS-1 impairs Wnt3A-induced chromatin accumulation of β-catenin.

Wnt regulates insulin signaling in muscle cells in culture. (A) Stable expression of Wnt3A cDNA enhances insulin-stimulated Akt activation. C2C12 cells virally transduced to express the Wnt3A cDNA were incubated in serum-free medium for 3 h, treated with different doses of insulin, and examined for the phosphorylation of Akt at Ser473. (B) Treatment with recombinant Wnt3A protein augments insulin-stimulated Akt activation. C2C12 cells were treated with control or 50 ng/mL Wnt3A protein for 1 d, incubated in serum-free medium, and stimulated with insulin. (C) Treatment with Wnt3A protein enhances insulin-stimulated glycogen synthesis in C2C12 myocytes. Cells were treated with control or Wnt3A protein for 1 d, stimulated with varying doses of insulin, incubated in medium containing ¹⁴C-glucose, and lysed, and the glycogen was extracted for measurement of incorporated ¹⁴C radioactivity. (*) P < 0.05 by unpaired t-test. (D) A model of Wnt3A-mediated regulation of mitochondria and insulin signaling.