Dose-dependent effects of vitamin D on transdifferentiation of skeletal muscle cells to adipose cells - PubMed (original) (raw)

Dose-dependent effects of vitamin D on transdifferentiation of skeletal muscle cells to adipose cells

Kevin J P Ryan et al. J Endocrinol. 2013.

Abstract

Fat infiltration within muscle is one of a number of features of vitamin D deficiency, which leads to a decline in muscle functionality. The origin of this fat is unclear, but one possibility is that it forms from myogenic precursor cells present in the muscle, which transdifferentiate into mature adipocytes. The current study examined the effect of the active form of vitamin D₃, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), on the capacity of the C2C12 muscle cell line to differentiate towards the myogenic and adipogenic lineages. Cells were cultured in myogenic or adipogenic differentiation media containing increasing concentrations (0, 10⁻¹³, 10⁻¹¹, 10⁻⁹, 10⁻⁷ or 10⁻⁵ M) of 1,25(OH)₂D₃ for up to 6 days and markers of muscle and fat development measured. Mature myofibres were formed in both adipogenic and myogenic media, but fat droplets were only observed in adipogenic media. Relative to controls, low physiological concentrations (10⁻¹³ and 10⁻¹¹ M) of 1,25(OH)₂D3 increased fat droplet accumulation, whereas high physiological (10⁻⁹ M) and supraphysiological concentrations (≥10⁻⁷ M) inhibited fat accumulation. This increased accumulation of fat with low physiological concentrations (10⁻¹³ and 10⁻¹¹ M) was associated with a sequential up-regulation of PPARγ2 (PPARG) and FABP4 mRNA, indicating formation of adipocytes, whereas higher concentrations (≥10⁻⁹ M) reduced all these effects, and the highest concentration (10⁻⁵ M) appeared to have toxic effects. This is the first study to demonstrate dose-dependent effects of 1,25(OH)₂D₃ on the transdifferentiation of muscle cells into adipose cells. Low physiological concentrations (possibly mimicking a deficient state) induced adipogenesis, whereas higher (physiological and supraphysiological) concentrations attenuated this effect.

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Figures

Figure 1

Figure 1

Dose-dependent effects of 1,25(OH)2D3 on accumulation of lipid droplets in C2C12 cells cultured in myogenic (A, B, C, D, E and F) or adipogenic (G, H, I, J, K and L) media. Representative images of C2C12 cells stained with Oil Red-O (as indicated by white arrows) and counterstained with haematoxylin to indicate lipid droplet and nuclei/myofibre structures respectively. Cells were cultured for 6 days in myogenic (A, B, C, D, E and F) or adipogenic (G, H, I, J, K and L) media with vehicle (A and G) or increasing concentrations of 1,25(OH)2D3 (10−13 M (B and H), 10−11 M (C and I), 10−9 M (D and J), 10−7 M (E and K) or 10−5 M (F and L)).

Figure 2

Figure 2

Dose-dependent effects of 1,25(OH)2D3 on percentage area of lipid accumulation in C2C12 cells cultured in adipogenic media for 6 days. C2C12 cells were stained with Oil Red-O and counterstained with haematoxylin, before the percentage area of the red stain within a field of view (FOV) was quantified by image analysis to give a representative percentage area per well (_n_=4 wells, five FOV/well). One-way ANOVA indicated a significant effect of 1,25(OH)2D3 (P<0.001). Significant differences compared to DMSO control cells were determined by post-hoc Bonferroni's test (**P<0.01 and ***P<0.001).

Figure 3

Figure 3

Dose-dependent effects of 1,25(OH)2D3 on expression of white adipocyte marker genes. Expression of white adipocyte marker genes was determined by quantitative RT-PCR analysis. Levels of (A) Ppar γ 2, (B) Fabp4, (C) Adipoq/adiponectin and (D) Ppar γ 1 mRNAs were quantified in C2C12 cells cultured in the absence or presence of 10−13, 10−11, 10−9, 10−7 or 10−5 M 1,25(OH)2D3 for 2, 4 or 6 days in adipogenic differentiation media. Expression at day 0 (before differentiation media and 1,25(OH)2D3 was added) is also included and is indicated by a bar (in some instances, this was very low). Significant two-way interactions between day of differentiation and 1,25(OH)2D3 concentration were observed for PPARγ2, FABP4 and AdipoQ (P<0.001 for all). For PPARγ1, there was a significant effect of stage of differentiation (_P_=0.002) and a significant effect of 1,25(OH)2D3 concentration (_P_=0.005), but no interaction.

Figure 4

Figure 4

Dose-dependent effects of 1,25(OH)2D3 on (A) CK activity (IU/well), (B) protein (μg/well) and (C) DNA content (μg/well) in C2C12 cells cultured for 4 days in myogenic media (MM) or adipogenic media (AM) with increasing concentrations (vehicle control, 10−13, 10−11, 10−9, 10−7 and 10−5 M) of 1,25(OH)2D3. Significant two-way interactions between media type and 1,25(OH)2D3 concentration were observed for CK activity (P<0.001), protein (P<0.01) and DNA (P<0.001) content.

Figure 5

Figure 5

Dose-dependent effects of 1,25(OH)2D3 on expression of skeletal muscle marker genes. Expression of skeletal muscle marker genes was determined by quantitative RT-PCR analysis. Levels of (A) CK, (B) myogenin, (C) MyoD and (D) Myf5 mRNAs were quantified in C2C12 cells cultured in the absence or presence of 10−13, 10−11, 10−9, 10−7 or 10−5 M 1,25(OH)2D3 for 2, 4 or 6 days in adipogenic differentiation media. Expression at day 0 (before differentiation media and 1,25(OH)2D3 was added) is also included for reference and is indicated by a bar. Significant two-way interactions between day of differentiation and 1,25(OH)2D3 concentration were observed for CK, myogenin, Myf5 (P<0.001 for all) and MyoD (P<0.05) mRNA transcripts.

Figure 6

Figure 6

Dose-dependent effects of 1,25(OH)2D3 on expression of brown adipocyte marker genes. Expression of brown adipocyte marker genes was determined by quantitative RT-PCR analysis. Levels of (A) Elovl3, (B) Cidea, (C) C/ebp β and (D) Pgc1 α mRNAs were quantified in C2C12 cells cultured in the absence or presence of 10−13, 10−11, 10−9, 10−7 or 10−5 M 1,25(OH)2D3 for 2, 4 or 6 days in adipogenic differentiation media. Expression at day 0 (before differentiation media and 1,25(OH)2D3 was added) is also included for reference and is indicated by a bar. Significant effects of day of differentiation were observed for Elovl3, Cidea and C/ebpβ (P<0.001 for all three), but not PGC1α (_P_>0.05). There were no significant effects of 1,25(OH)2D3 concentration on any of the brown adipocyte marker genes.

Figure 7

Figure 7

Dose-dependent effects of 1,25(OH)2D3 on expression of VDR, CYP27B1 (1α-hydroxylase) and CYP24A1 (24-hydroxylase) enzymes. Expression of VitD-related genes was determined by quantitative RT-PCR analysis. Levels of (A) Vdr, (B) Cyp27b1 and (C) Cyp24a1 mRNAs were quantified in C2C12 cells cultured in the absence or presence of 10−13, 10−11, 10−9, 10−7 or 10−5 M 1,25(OH)2D3 for 2, 4 or 6 days in adipogenic differentiation media. Expression at day 0 (before differentiation media and 1,25(OH)2D3 was added) is also included for reference and is indicated by a bar. A significant two-way interaction (P<0.001) between day of differentiation and 1,25(OH)2D3 concentration was observed for VDR only. There was a significant effect of day of differentiation (_P_=0.001) on expression of Cyp27b1 mRNA, but no effect of 1,25(OH)2D3 concentration. By contrast, there was a significant effect (P<0.001) of 1,25(OH)2D3 concentration on Cyp24a1 mRNA, but no effect of day of differentiation.

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References

    1. Aguiari P, Leo S, Zavan B, Vindigni V, Rimessi A, Bianchi K, Franzin C, Cortivo R, Rossato M, Vettor R, et al. High glucose induces adipogenic differentiation of muscle-derived stem cells. PNAS. 2008;105:1226–1231. doi: 10.1073/pnas.0711402105. - DOI - PMC - PubMed
    1. Asakura A, Komaki M, Rudnicki M. Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation. Differentiation. 2001;68:245–253. doi: 10.1046/j.1432-0436.2001.680412.x. - DOI - PubMed
    1. Bischoff HA, Stahelin HB, Dick W, Akos R, Knecht M, Salis C, Nebiker M, Theiler R, Pfeifer M, Begerow B, et al. Effects of vitamin D and calcium supplementation on falls: a randomized controlled trial. Journal of Bone and Mineral Research. 2003;18:343–351. doi: 10.1359/jbmr.2003.18.2.343. - DOI - PubMed
    1. Blumberg JM, Tzameli I, Astapova I, Lam FS, Flier JS, Hollenberg AN. Complex role of the vitamin D receptor and its ligand in adipogenesis in 3T3-L1 cells. Journal of Biological Chemistry. 2006;281:11205–11213. doi: 10.1074/jbc.M510343200. - DOI - PubMed
    1. Brown DM, Parr T, Brameld JM. Myosin heavy chain mRNA isoforms are expressed in two distinct cohorts during C2C12 myogenesis. Journal of Muscle Research and Cell Motility. 2012;32:383–390. doi: 10.1007/s10974-011-9267-4. - DOI - PubMed

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