[논문]Effects of alpha-linolenic acid and essential amino acids on the proliferation and differentiation of C2C12 myoblasts

Zhou, Dongjie; Li, Xiao-Han; Lee, Song‑Hee; Heo, Geun; Cui, Xiang-Shun

doi:10.12750/jarb.37.1.17

Effects of alpha-linolenic acid and essential amino acids on the proliferation and differentiation of C2C12 myoblasts 원문보기

Journal of animal reproduction and biotechnology = 한국동물생명공학회지, v.37 no.1, 2022년, pp.17 - 26

Zhou, Dongjie (Department of Animal Science, Chungbuk National University) , Li, Xiao-Han (Department of Animal Science, Chungbuk National University) , Lee, Song‑Hee (Department of Animal Science, Chungbuk National University) , Heo, Geun (Department of Animal Science, Chungbuk National University) , Cui, Xiang-Shun (Department of Animal Science, Chungbuk National University)

Abstract ▼ AI-Helper

Alpha-linolenic acid is an important polyunsaturated fatty acid that exhibits anticancer, anti-inflammatory, and antioxidative effects. In this study, we investigated the protective effects of alpha-linolenic acid on the cell proliferation and differentiation of C2C12 cells under essential amino acid-deficient conditions. Different concentrations of alpha-linolenic acid and essential amino acids were added to the growth and differentiation media. The concentrations of 10 µM of alpha-linolenic acid and 2% essential amino acid were chosen for subsequent experiments. Supplementation with alpha-linolenic acid and essential amino acids improved the proliferation and differentiation of C2C12 cells and significantly increased the mRNA levels of catalase, superoxide dismutase, B-cell lymphoma-2, and beclin-1 as well as the protein levels of PPARγ coactivator-1α compared to those in the controls. Moreover, supplementation with alpha-linolenic acid and essential amino acids reduced the levels of phosphorylated H2A.X variant histone, Bcl-2-associated X, p53, and light chain 3 during C2C12 cell proliferation, and increased the expression levels of myogenic factors 4 (myogenin) and 5 during C2C12 cell differentiation. Overall, we determined that alpha-linolenic acid and essential amino acids maintained the cell proliferation and differentiation of C2C12 cells via their anti-oxidative, anti-apoptotic, and anti-autophagic effects.

주제어

표/그림 (8)

표 Table 1. Primer sequences for RT-PCR (F: forward, R: reverse)
그림 Fig. 1. Effects of α-linolenic acid (ALA) and essential amino acids (EAAs) on C2C12 cell proliferation. The control growth medium (GM) contained 0.5% EAA. (A) Schematic diagram of the proliferation experiment. (B) Images of morphological changes were obtained using phase contrast photomicrography after 48 h of treatment. Scale bars represent 100 μm. (C) Cell viability of C2C12 cells was measured using the cell counting kit (CCK)-8 assay. Cells were treated with ALA (0, 5, 10, and 25 μM) or EAA (1 and 2%) for 48 h. (D, E) Expression levels of proliferating cell nuclear antigen (PCNA) were determined using western blotting and immunofluorescence assays. Scale bars represent 20 μm. (F) mRNA expression levels of PCNA and Ki-67 were determined using reverse transcriptionquantitative polymerase chain reaction (RT-qPCR). *p < 0.05, **p < 0.01, ***p < 0.005 vs. the control group.
그림 Fig. 2. Effects of ALA and EAAs on antioxidative factors. (A) Micrographs show the PPARγ coactivator (PGC)-1α (green) and Hoechst 33258 nuclear fluorochrome (blue). (B) Bar graph shows mean fluorescence intensity (MFI) analysis (MFI ± standard error of the mean (SEM) from n = 250-300; **** p < 0.001 vs. control). (C) Micrographs show sirtuin 1 (SIRT1) (green) and Hoechst 33258 nuclear fluorochrome (blue). (D) mRNA expression levels of catalase (CAT) and superoxide dismutase (SOD) were determined using RT-qPCR. Scale bars represent 20 μm. *p < 0.05, ***p < 0.005 vs. the control group.
그림 Fig. 3. Effects of ALA and EAAs on the DNA damage of C2C12 cells during proliferation. (A) Immunostaining images of the DNA damage marker, phosphorylated H2A.X variant histone (p-H2AX) (green), and the nuclear marker, Hoechst 33258 (blue), in C2C12 cells. All experiments were performed thrice and the representative result is shown. Scale bars represent 20 μm. (B) Quantification of colocalization of p-H2AX with Hoechst 33258 from random fields. Bar graph represents the mean ± SEM values from three independent experiments.
그림 Fig. 4. Effects of ALA and EAAs on the apoptosis and autophagy of C2C12 cells. (A, B) Terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL) assay and apoptosis index after treatment for 48 h. Scale bars represent 20 μm. (C) mRNA expression levels of B-cell lymphoma (Bcl)-2, Bcl-2-associated X (Bax), and p53 were determined using RT-qPCR. (D) mRNA expression levels of beclin-1 were determined using RT-qPCR. (E) Representative bands of light chain 3 (LC-3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained via western blotting.
그림 Fig. 5. Effects of ALA and EAAs on the differentiation of C2C12 cells. (A) Schematic diagram of the differentiation experiment. (B) Images of morphological changes were obtained using phase contrast photomicrography after 6 d of treatment. Scale bars represent 100 μm. (C, D) Cell lengths and widths of C2C12 cells.
그림 Fig. 6. Regulation of ALA and EAAs on the differentiation markers. (A) mRNA expression levels of myogenin (MYOG/MYF4) and MYF5 were determined using RT-qPCR. (B, C) Expression levels of PCNA were determined using western blotting and immunofluorescence assays. Scale bars represent 20 μm.
그림 Fig. 7. Scheme of the apoptosis pathway modulated by ALA and EAAs in C2C12 cells. Bax, Bcl-2 associated X; Bcl-2, B-cell lymphoma- 2; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha.

참고문헌 (23)

Cho HJ and Kim TM. 2021. CDDO-Me alleviates oxidative stress in human mesenchymal stem cells. J. Anim. Reprod. Biotechnol. 36:285-291.

원문보기 상세보기
Daval JL, Pourie G, Grojean S, Lievre V, Strazielle C, Blaise S, Vert P. 2004. Neonatal hypoxia triggers transient apoptosis followed by neurogenesis in the rat CA1 hippocampus. Pediatr. Res. 55:561-567.
Dickinson JM, Gundermann DM, Walker DK, Reidy PT, Borack MS, Drummond MJ, Arora M, Volpi E, Rasmussen BB. 2014. Leucine-enriched amino acid ingestion after resistance exercise prolongs myofibrillar protein synthesis and amino acid transporter expression in older men. J. Nutr. 144:1694-1702.

상세보기
Dickinson JM, Volpi E, Rasmussen BB. 2013. Exercise and nutrition to target protein synthesis impairments in aging skeletal muscle. Exerc. Sport Sci. Rev. 41:216-223.

상세보기
Fry CS, Drummond MJ, Glynn EL, Dickinson JM, Gundermann DM, Timmerman KL, Walker DK, Dhanani S, Volpi E, Rasmussen BB. 2011. Aging impairs contraction-induced human skeletal muscle mTORC1 signaling and protein synthesis. Skelet. Muscle 1:11.

상세보기
Guo K and Walsh K. 1997. Inhibition of myogenesis by multiple cyclin-Cdk complexes. Coordinate regulation of myogenesis and cell cycle activity at the level of E2F. J. Biol. Chem. 272:791-797.
Hussain G, Schmitt F, Loeffler JP, Gonzalez de Aguilar JL. 2013. Fatting the brain: a brief of recent research. Front. Cell. Neurosci. 7:144.

상세보기
Lee HJ, Jeon RH, Park BJ, Jang SJ, Lee SL, Rho GJ, Kim SJ, Lee WJ. 2019. Differentiation inductions altered telomere length and telomerase activity in human dental pulpderived mesenchymal stem cell. J. Anim. Reprod. Biotechnol. 34:93-99.

원문보기 상세보기
Leick L, Lyngby SS, Wojtaszewski JF, Pilegaard H. 2010. PGC1alpha is required for training-induced prevention of age-associated decline in mitochondrial enzymes in mouse skeletal muscle. Exp. Gerontol. 45:336-342. (Correcction published 2010, Exp. Gerontol., 45, p. 988)
McKinsey TA, Zhang CL, Olson EN. 2001. Control of muscle development by dueling HATs and HDACs. Curr. Opin. Genet. Dev. 11:497-504.
Park MH, Park JE, Kim MS, Lee KY, Yun JI, Choi JH, Lee E, Lee ST. 2014. Effects of suspension culture on proliferation and undifferentiation of spermatogonial stem cells derived from porcine neonatal testis. Reprod. Dev. Biol. 38:85-91.

원문보기 상세보기
Porrello A, Cerone MA, Coen S, Gurtner A, Fontemaggi G, Cimino L, Piaggio G, Sacchi A, Soddu S. 2000. p53 regulates myogenesis by triggering the differentiation activity of pRb. J. Cell Biol. 151:1295-1304.

상세보기
Qu A, Jiang C, Xu M, Zhang Y, Zhu Y, Xu Q, Zhang C, Wang X. 2009. PGC-1alpha attenuates neointimal formation via inhibition of vascular smooth muscle cell migration in the injured rat carotid artery. Am. J. Physiol. Cell Physiol. 297:C645-C653.
Rescan PY. 2001. Regulation and functions of myogenic regulatory factors in lower vertebrates. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 130:1-12.
Roqueta-Rivera M, Abbott TL, Sivaguru M, Hess RA, Nakamura MT. 2011. Deficiency in the omega-3 fatty acid pathway results in failure of acrosome biogenesis in mice. Biol. Reprod. 85:721-732.
Roy S, Rawat AK, Sammi SR, Devi U, Singh M, Gautam S, Yadav RK, Rawat JK, Singh L, Ansari MN, Saeedan AS, Pandey R, Kumar D, Kaithwas G. 2017. Alpha-linolenic acid stabilizes HIF-1 α and downregulates FASN to promote mitochondrial apoptosis for mammary gland chemoprevention. Oncotarget 8:70049-70071.
Shen L, Sun B, Sheng J, Yu S, Li Y, Xu H, Su J, Sun L. 2018. PGC1α promotes cisplatin resistance in human ovarian carcinoma cells through upregulation of mitochondrial biogenesis. Int. J. Oncol. 53:404-416.
Skapek SX, Rhee J, Kim PS, Novitch BG, Lassar AB. 1996. Cyclin-mediated inhibition of muscle gene expression via a mechanism that is independent of pRB hyperphosphorylation. Mol. Cell. Biol. 16:7043-7053.

상세보기
Valente LJ, Gray DH, Michalak EM, Pinon-Hofbauer J, Egle A, Scott CL, Janic A, Strasser A. 2013. p53 efficiently suppresses tumor development in the complete absence of its cell-cycle inhibitory and proapoptotic effectors p21, Puma, and Noxa. Cell Rep. 3:1339-1345.

상세보기
Walker DK, Dickinson JM, Timmerman KL, Drummond MJ, Reidy PT, Fry CS, Gundermann DM, Rasmussen BB. 2011. Exercise, amino acids, and aging in the control of human muscle protein synthesis. Med. Sci. Sports Exerc. 43:2249-2258.

상세보기
Wang X, Breeze A, Kulka M. 2015. N-3 polyunsaturated fatty acids inhibit IFN-γ-induced IL-18 binding protein production by prostate cancer cells. Cancer Immunol. Immunother. 64:249-258.
Wathes DC, Abayasekara DR, Aitken RJ. 2007. Polyunsaturated fatty acids in male and female reproduction. Biol. Reprod. 77:190-201.
Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270:1326-1331.

상세보기

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증