Acetoacetate Accelerates Muscle Regeneration and Ameliorates Muscular Dystrophy in Mice

Abstract

Acetoacetate (AA) is a ketone body and acts as a fuel to supply energy for cellular activity of various tissues. Here, we uncovered a novel function of AA in promoting muscle cell proliferation. Notably, the functional role of AA in regulating muscle cell function is further evidenced by its capability to accelerate muscle regeneration in normal mice, and it ameliorates muscular dystrophy in mdx mice. Mechanistically, our data from multiparameter analyses consistently support the notion that AA plays a non-metabolic role in regulating muscle cell function. Finally, we show that AA exerts its function through activation of the MEK1-ERK1/2-cyclin D1 pathway, revealing a novel mechanism in which AA serves as a signaling metabolite in mediating muscle cell function. Our findings highlight the profound functions of a small metabolite as signaling molecule in mammalian cells.

Keywords: acetoacetate; cyclin D1; extracellular-signal-regulated kinase (ERK); muscle regeneration; muscular dystrophy.

FIGURE 1.

AA promotes C2C12 cell proliferation. a, C2C12 cells were treated with 5 mm AA, 3HB, or acetone for 24 h, and cell proliferation was measured by FACS analysis. b, proliferation of C2C12 cells treated with increasing amounts of AA, recombinant IGF1, or PBS for 24 h was analyzed by FACS and [³H]thymidine incorporation assay. c, proliferation of C2C12 cells treated with PBS or 5 mm AA for different times (24, 36, and 48 h) was analyzed by FACS and [³H]thymidine incorporation assay. d, proliferation of C2C12 cells stimulated by combined treatment with 5 mm AA and IGF1 (or myostatin) was assessed by FACS. Similar results were obtained in three separate experiments. Data are presented as means ± S.E. (error bars; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 2.

AA treatment stimulates satellite cell activation and proliferation during muscle regeneration. The data are representative of those obtained in experiments on TA muscles from five mice. a, H&E-stained serial cross-sections from mouse TA muscle treated with different dosages of AA at post-injury day 3. b, cross-sectional areas of regenerated myofibers in PBS control (Con) and 30 mm AA-treated TA muscle 7 days after CTX injury were measured by Image-Pro Plus based on H&E staining of CTX-injured TA muscle sections. c, representative image of sections from TA muscles, injected CTX and treated with AA for 3 days, immunostained for Pax7 (red), MyoD (green), and nuclei (DAPI; blue). Right panel shows merged image with DAPI. d, quantification of Pax7⁺ and MyoD⁺ cells. The histogram represents the percentage of cells retaining Pax7⁺ and MyoD⁺ in each subpopulation (≥2000 cells/condition). e, representative image of Pax7 and MyoD staining and quantification of Pax7⁺/MyoD⁺ cells 1 day post-injury with or without 30 mm AA treatment (≥300 cells each). f, quantification of Pax7⁺ and MyoD⁺ cells in sections of 30 mm AA-injected TA muscles 2, 3, and 5 days (d) after muscle damage. g, representative images of MyoD (red) and BrdU (green) immunostaining 3 days post-injury showing the percentage of BrdU⁺ cells in the MyoD⁺ cell population (≥300 cells each). h, double staining for Pax7 and MyoD in expanded satellite cells from single fibers isolated from gastrocnemius muscle and cultured for 48 h. i, 1st panel shows the expression of Pax7 and MyoD in TA muscles damaged for 3 days treated with different dosages of AA. The other two panels show the expression level of Pax7, MyoD, and embryonic MHC (eMHC) protein levels in 30 mm AA-treated TA muscles 2, 3, and 5 days post-injury. GAPDH protein served as a loading control. Scale bar, 50 μm. More than five pairs of mice were used for each experiment. Data are presented as mean ± S.E. (error bars; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 3.

AA treatment restores muscle function and morphology in mdx mice. a, H&E staining of TA muscles from mdx mice treated with AA or PBS control (Con) for 60 days. b, fiber cross-sectional fiber area (CSA) was calculated on the sections in a. C57BL/6 normal mice (B6) served as control. c, Evans blue dye uptake in EDL muscles. d, quantification of the percentage of Evans blue positive fiber area in AA-treated versus control mdx muscle. e, quantitation of serum creatine kinase (CK) levels in B6 and mdx mice treated with AA or PBS control. f, EDL muscles isolated from B6 and mdx mice treated with AA or PBS control were electrically stimulated in vitro to elicit tetanic contractions. g, peak twitch force was measured on EDL muscles isolated from B6 and mdx mice treated with AA or PBS control. h, treadmill test. Scale bar, 50 μm (a) and 100 μm (c). More than five mice were used for each group. Data are presented as mean ± S.E. (error bars; *, p < 0.05; **, p < 0.01).

FIGURE 4.

AA plays a non-metabolic role in ERK activation and muscle cell proliferation. a, cell proliferation was analyzed in Oxct1-knockdown C2C12 cells treated with 5 mm AA or PBS for 24 h. The efficiency of Oxct1 knockdown was examined by Western blotting. b, proliferation of HepG2 cells treated with PBS or AA (5 mm) was analyzed by FACS. c, SCOT catalytic activity was measured in the C2C12 cells treated with different dosages of AA (0, 1, 3, and 5 mm) for 24 h. d, cell proliferation was analyzed in C2C12 cells treated with 5 mm AA or succinate for 24 h. e, normalized Cyto-c, β-ATP synthase, and TFAM mRNA levels in C2C12 cells treated with 5 mm AA or 3HB for 6 or 24 h were determined by quantitative RT-PCR. f, normalized phosphofructokinase I (PFK1) and hexokinase II (HK2) mRNA levels in C2C12 cells treated with 5 mm AA or 3HB for 6 or 24 h were determined by quantitative RT-PCR. Lactate concentration in cell media was measured using a commercial kit. Similar results were obtained in three separate experiments. Data are presented as means ± S.E. (error bars; *, p < 0.05). NS, not significant.

FIGURE 5.

MEK-ERK1/2 activation is required for AA-induced C2C12 cell proliferation through up-regulation of cyclin D1 expression. a, C2C12 cells were treated with 5 mm AA for 24 h. Cell lysates were used for Western blot detection of cyclin D1, cyclin E, Cdk2, and Cdk4. β-Actin served as a loading control. PBS treatment served as control (Con). b, proteins in total lysates of C2C12 cells treated with different concentrations of AA (0.1, 0.5, 1, and 5 mm) and IGF1 (100 ng/ml) for 24 h were resolved by SDS-PAGE and analyzed by Western blotting for cyclin D1, Cdk2, and β-actin. c, efficiency of cyclin D1 knockdown in C2C12 cells was examined by Western blotting. d, cell proliferation was analyzed in cyclin D1-knockdown C2C12 cells treated with 5 mm AA or PBS for 24 h. e, expression levels of phospho (p)-MEK, total (t)-MEK, p-ERK, t-ERK, and β-actin proteins in C2C12 cells treated with 5 mm AA for different times (0.5, 1, 3, 6, and 12 h) were analyzed by Western blotting. f, C2C12 cells were pre-treated with the MEK inhibitor PD98059 (20 μm) for 1 h prior to AA (5 mm) treatment. The levels of cyclin D1, p-ERK, t-ERK, and β-actin were determined by Western blotting. g, proliferation of C2C12 cells treated with AA (5 mm) with or without PD98059 (20 μm) was analyzed by FACS. h, cyclin D1 expression was detected in the ERK1/2-knockdown C2C12 cells treated with 5 mm AA or PBS for 12 h. i, cell proliferation was analyzed in ERK1/2-knockdown C2C12 cells treated with 5 mm AA or PBS for 24 h. j, representative sections of AA-treated TA muscles, with and without PD98059 (20 μm) treatment, 2 days after injury immunostained for MyoD (green), and nuclei (DAPI; blue). Right panel shows merged image with DAPI. k, quantification of MyoD⁺ cells in sections described in j. l, expression of Pax7, cyclin D1, p-ERK, and t-ERK in damaged TA muscles treated with AA in the presence or absence of PD98059 was analyzed by Western blotting. Scale bar, 50 μm. Similar results were obtained in three separate experiments. Data are presented as means ± S.E. (error bars; *, p < 0.05). NS, not significant.

FIGURE 6.

AA activates the MEK-ERK1/2 pathway and promotes C2C12 cell proliferation independent of Ras/Raf. a, levels of Ras, t-Raf (c-Raf), and p-Raf (p-c-Raf) proteins in C2C12 cells in response to AA (5 mm) treatment were assessed by Western blotting at the indicated times. b, normalized level of Ras and p-Raf. Ras protein was normalized to β-actin, and p-Raf was normalized to total Raf. c, protein levels of Ras, t-Raf/p-Raf, t-MEK/p-MEK, and t-ERK1/2/p-ERK1/2 were determined by Western blot analysis in normal C2C12 and DN-Ras C2C12 cells treated with AA (5 mm) or PBS for 12 h. d, quantified levels of p-MEK and p-ERK relative to t-MEK and t-ERK, respectively, based on blots in c. e, protein levels of Ras, p-Raf, t-Raf, p-MEK, t-MEK, p-ERK, t-ERK, and β-actin in C2C12 cells treated with AA (5 mm) for 12 h in the presence or absence of the inhibitors FTA or GW5074 (GW) were determined by Western blotting. f, cell proliferation was analyzed in C2C12 cells or DN-Ras expressing C2C12 cells treated with 5 mm AA. g, cell proliferation was analyzed in C2C12 cells treated with AA (5 mm) in the presence or absence of FTA or GW5074. h, schematic model of AA function. Similar results were obtained in three separate experiments. Data are presented as means ± S.E. (error bars; *, p < 0.05; ***, p < 0.001).