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. 2018 Nov-Dec;32(6):1427-1431.
doi: 10.21873/invivo.11395.

Geranylgeraniol-induced Myogenic Differentiation of C2C12 Cells

Takuma Matsubara  1 , Mariko Urata  1 , Tsuyoshi Nakajima  1 , Mari Fukuzaki  1 , Ryo Masuda  1 , Yoshiyuki Yoshimoto  1 , William N Addison  2 , Chihiro Nakatomi  1 , Kazmasa Morikawa  3 , Min Zhang  4 , Katsura Saeki  5 , Yukiko Takahashi  6 , Atsuko Nakamichi  6 , Shoichiro Kokabu  7
Affiliations

Geranylgeraniol-induced Myogenic Differentiation of C2C12 Cells

Takuma Matsubara et al. In Vivo. 2018 Nov-Dec.

Abstract

Background: Geranylgeraniol (GGOH) is a C20 isoprenoid found in fruits, vegetables, and grains, including rice. As a food substance, GGOH is categorized as 'Generally Recognized as Safe'. GGOH is an intermediate product in the mevalonate pathway and acts as a precursor to geranylgeranyl pyrophosphate.

Materials and methods: C2C12 mouse myoblasts derived from muscle satellite cells were used. Quantitative reverse-transcriptase polymerase chain reaction, western blotting analysis, and immunocytochemical analysis were performed to respectively assess mRNA expression, protein levels, and the number of myofibers.

Results: GGOH reduced the expression levels of skeletal muscle atrophy-related ubiquitin ligases in myofibers derived from C2C12 cells. GGOH induced myogenic differentiation of C2C12 cells via geranylgeranylation. GGOH did not adversely affect the proliferation of C2C12 cells.

Conclusion: GGOH induces myoblast differentiation in C2C12 cells.

Keywords: C2C12 cells; Sarcopenia; geranylgeranylation; myogenesis; statin.

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Figures

Figure 1
Figure 1. Geranylgeraniol (GGOH) reduces the expression levels ofskeletal muscle atrophy-related ubiquitin ligases in myofibers derived fromC2C12 cells. C2C12 cells were cultured with 2% horse serum for 5 daysand then treated with or without (CtrI) 50 μM GGOH for another 3 days.Total RNA was isolated, then F-box protein 32 (Fbxo32) (A) and tripartitemotif containing 63 (Trim63) (B) mRNA levels were analyzed usingquantitative polymerase chain reaction. All data are expressed as themean±SD (n=3). *Significantly different at p&0.01 versus vehicle-treatedcells. Similar results were obtained by three independent experiments.
Figure 2
Figure 2. Geranylgeraniol (GGOH) induces myogenic differentiation of C2C12 cells via geranylgeranylation. C2C12 cells were treated with orwithout (CtrI) 50 μM GGOH for 2 days. Myogenic differentiation (Myod) (A), myogenin (Myog) (B), creatine kinase, M-type (Ckm) (C), and insulinlikegrowth factor-2 (Igf2) (D) mRNA levels were analyzed using quantitative polymerase chain reaction. All data are expressed as the mean±SD(n=3).*Significantly different at p&0.01 versus vehicle-treated cells. Cells were treated with 0, 5, 10, 50, or 100 μM GGOH for 3 (F) or 5 (G) days.The protein levels of MYOG (F) and myosin heavy chain (MYHC) (G) were assessed by western blotting analysis. Immunocytochemical analysiswas performed using antibody to MYHC on day 5. Scale bar represents 10 μm (H). Cells were treated with or without 50 μM GGOH in the presenceor absence of 100 μM GGTI-298 for 3 days. The protein levels of MYOG were determined by western blotting analysis (I). Similar results wereobtained by three independent experiments (F-I). Myogenic differentiation of C2C12 cells was induced by the treatment with Dulbecco’s modifiedEagle’s medium supplemented with 2% horse serum (Myo-Media) (A-F)
Figure 3
Figure 3. Geranylgeraniol (GGOH) does not affect the proliferation ofC2C12 cells. C2C12 cells were cultured in the presence of 0, 5, 10, 50,or 100 μM GGOH for the indicated times. The proliferation of cells wasassessed using a Cell Counting Kit-8. All data are expressed as themean±SD (n=3).
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