Myostatin(MSTN) 유전자 제거 상향 Wnt/β-catenin 신호통로 골대사에 영향을 줌으로써 제2형 당뇨병 생쥐 골다공증 개선 = Knockout of Mstn gene attenuates osteoporosis through Wnt/β-catenin signaling pathway in mice with type 2 diabetic mellitus

다국어 초록 (Multilingual Abstract)

Background:Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily and is a secreted protein highly produced by skeletal muscle that negatively regulates its growth and development. MSTN also plays a role in regulatingglucose and lipid metabolism.Previous studies have shown that people with type 2 diabetes mellitus(T2DM) and insulin resistance have elevated serum and skeletal muscle MSTN levels.The expression of MSTN in skeletal muscle of T2DM mice increased. After receiving MSTN antibody injection, the fasting and postprandial blood glucose levels improved. The available evidences show that MSTN can affect the body's glucose uptake and utilization and participate in the occurrence of insulin resistance. However, the molecular mechanism of MSTN involved in regulating T2DM insulin resistance is not clear.In this study, based on the MSTN knockout (KO) mice constructed by CRISPR/Cas9 gene editing, a high-fat diet combined with low-dose streptozotocin (STZ) was used to construct a T2DM animal model. The body composition, muscle strength, fatigue resistance, glucose regulation and histopathological changes in skeletal muscle were observed, and the expression levels of proteins on insulin signaling pathway were measured to explore the effect and mechanism of MSTN on bone metabolism and bone microstructure in mice with T2DM. Objective:To analyze effects ofMSTN on insulin resistance and insulinsignalingpathway in skeletal muscle of mice with T2DM.Methods:MSTN knockout heterozygous C57BL/6N mice were constructed by CRISPR/Cas9 gene editing techniques. We acquired finally male mice consisting of 12 wild type mice, 12 heterozygous mice and 12 homozygous mice through breeding and PCR. All animals were randomly assigned to 2 groups including 6 groups in totaland 6 in each group: WT group, MSTN(+/-) group, MSTN(-/-) group, WT+DM group, MSTN(+/-)+DM group and MSTN(-/-)+DM group.The first three groups were fed 6-week standard chow and the rest were fed 6-week high-fat diet for combined with small-dosestreptozocin (STZ) injection to structure models of T2DM. After measuring fasting plasma glucose (FPG) to evaluate animal models, all mice were measured body weight (BW), body length (BL), abdominal circumference (AC), gastrocnemius muscle weight (BMW), abdominal adipose weight (VAW) and calculated ratios (GMW/BW, VAW/BW) to evaluate the changes of body composition. All animals were measured gripping force and rotating time to exhaustion to evaluate muscle strengthand fatigue resistance. All groups were measured fasting serum insulin (FIns), tolerance test (GTT), insulin tolerance test (ITT) and calculated insulin sensitivity index (ISI), homeostasis model assessment-insulin resistance index (HOMA-IR) and area under curve (AUC)to evaluate directly and indirectlyinsulin sensitivity, insulin resistance and regulation of bloodsugar.HE staining analyzed the muscle fiber morphology and cross-sectional area in gastrocnemius muscles. Western blot (WB) quantified expressions of MSTN, InsR, IRS1, p-IRS1, PI3K, p-PI3K, Akt, p-Akt, GLUT4, GSK3β andp-GSK3βon the insulinsignaling pathway in gastrocnemius muscles. Measurement data among multiple groups were conducted using one-way analysis of variance (ANOVA)by SPSS software. Images of HE stainingandWBwereanalyzedsemiquantitativelybyImage Jsoftware.Graphs were produced usingGraphPad Prism software.

Results:1. The levels of serum PINP and CTX in wt+dm group, MSTN + / - DM group and mstn-/- + DM group were higher than those in WT group, MSTN + / - group and mstn-/- (p<0.05). The levels of serum PINP and CTX in WT group, MSTN + / - group and mstn-/- group decreased successively (p<0.05); The levels of serum PINP and CTX in wt+dm group, mstn+/- +dm group and mstn-/- +dm group decreased successively (p<0.05).2. In WT group, MSTN + / - group and mstn-/- group, the trabeculae were continuous, the structure was intact, and the bone cortex was smooth. The number of trabeculae in mstn-/- group increased significantly; In wt+dm group and mstn+/- +dm group, the number of trabeculae decreased, trabeculae were broken, and the edge of bone cortex was rough, while in mstn-/- +dm group, the number of trabeculae was significantly increased and the continuity was significantly improved.3.ALP calcium cobalt staining showed that the dark brown precipitates in the cytoplasm of wt+dm group, MSTN + / - DM group and mstn-/- + DM group were more than WT group, MSTN + / - group and mstn-/- group, respectively. Dark brown precipitates in the cytoplasm of WT group, MSTN + / - group and mstn-/- group decreased in turn; Dark brown precipitates in the cytoplasm of wt+dm group, mstn+/- +dm group and mstn-/- +dm group decreased in turn.4. Trap staining showed that the purplish red precipitates in the cytoplasm of wt+dm group, MSTN + / - DM group and mstn-/- + DM group were more than WT group, MSTN + / - group and mstn-/- group, respectively. The purplish red precipitates in the cytoplasm of WT group, MSTN + / - group and mstn-/- group decreased in turn; The purplish red precipitates in the cytoplasm of wt+dm group, mstn+/- +dm group and mstn-/- +dm group decreased in turn. However, the change of osteoclast activity after MSTN gene knockout is more obvious than that of osteoblasts.5.Wt+dm group, mstn+/+dm group, mstn-//+dm group bone tissue Wnt β- The expression of catenin was lower than that of WT group, MSTN + / - group and mstn-/- group (p<0.05), GSK-3 β The expression was higher than that of WT group, MSTN + / - group and mstn-/- group (p<0.05). Wnt of bone tissue in mstn-/- group β- The expression of catenin was higher than that of WT group and MSTN + / - group, GSK-3 β The expression was lower than that of WT group and MSTN + / - group (p<0.05); Wnt of bone tissue in mstn-/-+dm group β- The expression of catenin was higher than that of wt+dm group, mstn+/+dm group and GSK-3 β The expression was lower than that in wt+dm group and mstn+/+dm group (p<0.05); Wt group and MSTN + / - group, wt+dm group and MSTN + / - DM group bone tissue Wnt, GSK-3 β, β- There was no significant difference in the expression of catenin (p>0.05).

Conclusion:1. Inhibition of Mstn gene expression can improve bone formation and bone resorption caused by T2DM, but mainly inhibit excessive bone resorption, and the degree of change is related to the expression of Mstn. 2. Inhibition of Mstn gene expression in T2DM mice can improve bone microstructure and increase bone strength, and the degree of change is related to the expression of Mstn. 3. Inhibition of Mstn gene expression in T2DM mice can up-regulate the Wnt/β-catenin signaling pathway in bone tissue, and the degree of change is related to the expression of Mstn.

국문 초록 (Abstract)

연구배경：제2형 당뇨병(T2DM)의 발병률은 해마다 증가하고 있으며 인류 건강에 큰 위협이 되고 있다. 제2형 당뇨병(T2DM) 발병 시 골대사가 비정상적으로 작동하고 골다공증 발생 위험이 높아진다. 그러나 구체적인 분자 메커니즘이 아직 밝혀지지 않았다. 마이오스타틴(myostatin, Mstn)는 전환성장인자 TGF-베타그룹(TGF-β family)에 속한 단백질로 골격근의 발생과 성장을 억제하는 분비형 단백질이다. Mstn는 근육의 이화작용을 시킬 수 있을 뿐만 아니라 골대사를 조절하는 역할도 하고 있는 것으로 나타났다.따라서 Mstn는 T2DM 골다공증의 발생과 관련될 수 있다고 추측할 수 있다. 본 연구에서는 Mstn 유전자 녹아웃 마우스를 연구 대상으로 고지방 식이와 저용량 스트렙토조토신으로 유도된 제2형 당뇨병(T2DM) 동물 모델을 이용하여 Mstn는 T2DM 골대사에 미치는 영향 및 분자 메커니즘을 탐구하고 당뇨병 골다공증의 치료에 새로운 방향과 대안을 모색한다.
연구목적：Myostatin(MSTN) 유전자 제거 제2형 당뇨병(type 2 diabetic mellitus, T2DM) 생쥐의 골 대사 및 골 미세구조에 미치는 영향과 메커니즘을 탐구한다.연구방법：수컷 생쥐 야생형(WT) 12마리, 이형 접합형(Mstn+/-) 12마리, 동형 접합형(Mstn-/-) 12마리를 각각 2개의 그룹 한 그룹당 6마리로 나누어, WT그룹, Mstn+/-그룹, Mstn-/-그룹, WT+DM그룹, Mstn+/-+DM그룹, Mstn-/-+DM그룹으로 분류한다. 앞에 3개의 그룹에게 일반 식단을 제공하고 뒤에 3개의 그룹에게 고지방 식이와 저용량 스트렙토조토신을 급여하여 T2DM 모델을 구축한다.생쥐 모델 6개 그룹을 만든 뒤 ELISA방법으로 혈청 P1NP(type I procollagen N-terminal propeptide, 골형성표지자), CTX(type I collagencross-linked C-telopeptide, 골흡수표지자) 수치를 측정하고, 마이크로-CT를 이용하여 왼쪽 경골의 골밀도(bone mineral density, BMD), 골 부피분율(bone volume/total volume, BV/TV), 골소주 개수(trabecular number, Tb.N), 골소주 두께(trabecular thickness, Tb.Th), 골소주 가격(trabecular separation, Tb.Sp), 구조적 모델 지수(structure model index, SMI)를 분석한다. 그리고, 알칼리성 인산분해효소 （alkaline phosphatase, ALP）ATPase 염색법으로 골조직 조골세포의 활성도를 확인하고, 타르타르산염 저항성 인산분해효소 (tartrate resistant acid phosphatase, TRAP) 염색법으로 골조직 파골세포의 활성도를 확인한다. 또한, 면역조직화학법을 통해 골조직 Wnt, 글리코겐 합성효소 키나아제 3β (glycogen synthase kinase-3β, GSK-3β), 베타 카테닌(β-catenin)의 발현을 측정한다.연구결과：WT그룹과 비교하면 WT+DM그룹의 혈청 PINP, CTX 수치, Tb.Sp, SMI 및 골조직 중의 조골세포와 파골세포의 활성, 그리고 GSK-3β의 발현은 모두 증가하고, BMD, BV/TV, Tb.N, Tb.Th 및 골조직 Wnt, β-catenin의 발현은 모두 감소한 것으로 나타났다.

WT그룹, Mstn+/-그룹과 비교하면 Mstn-/-그룹의 BMD, BV/TV, Tb.N, Tb.Th 및 골조직 Wnt, β-catenin의 발현은 모두 증가하고, 혈청 PINP, CTX 수치, Tb.Sp, SMI 및 골조직 중의 조골세포와 파골세포의 활성, GSK-3β의 발현은 모두 감소한 것으로 나타났다.

WT+DM그룹, Mstn+/-+DM그룹과 비교하면 Mstn-/-+DM그룹의 BMD, BV/TV, Tb.N, Tb.Th 및 골조직 Wnt, β-catenin의 발현은 모두 증가하고, 혈청 PINP, CTX 수치, Tb.Sp, SMI 및 골조직 중의 조골세포와 파골세포의 활성, GSK-3β의 발현은 모두 감소했다.

1. WT+DM그룹, Mstn+/-+DM그룹, Mstn-/-+DM그룹의 혈청 PINP, CTX수치는 각각 WT그룹, Mstn+/-그룹, Mstn-/-그룹보다 높은 것으로 확인됐다 (P<0.05). WT그룹, Mstn+/-그룹, Mstn-/-그룹의 혈청 PINP, CTX 수치는 순차적으로 감소하고 (P<0.05), WT+DM 그룹, Mstn+/-+DM그룹, Mstn-/-+DM그룹의 혈청 PINP, CTX 수치는 순차적으로 감소한 것으로 나타났다 (P<0.05).

2. WT그룹, Mstn+/-그룹, Mstn-/-그룹은 골소주의 연속성, 구조가 완전하고 골피질이 매끄러우며, 그 중에 Mstn-/-그룹의 골소주 개수가 현저히 증가했다. WT+DM그룹, Mstn+/-+DM그룹은 골소주 개수가 감소하고 골소주가 파열되며 골피질의 가장자리가 거친 반면 Mstn-/-+DM그룹은 골소주 개수가 현저히 증가하고, 연속성도 확실히 많이 개선되었다.

3. 알칼리성 인산분해효소(ALP) ATPase 염색 결과에 따르면 WT+DM그룹, Mstn+/-+DM그룹, Mstn-/-+DM그룹은 WT그룹, Mstn+/-그룹, Mstn-/-그룹보다 세포질 내에 흑갈색의 침전물이 더 많은 것으로 나타났다. WT그룹, Mstn+/-그룹, Mstn-/-그룹은 세포질 내에 흑갈색의 침전물이 순차적으로 감소하고, WT+DM그룹, Mstn+/-+DM그룹, Mstn-/-+DM그룹은 세포질 내에 흑갈색의 침전물이 순차적으로 감소했다.

4. 타르타르산염 저항성 인산분해효소(TRAP) 염색 결과에 따르면 WT+DM그룹, Mstn+/-+DM그룹, Mstn-/-+DM그룹은 WT그룹, Mstn+/-그룹, Mstn-/-그룹보다 세포질 내에 자홍색의 침전물이 더 많은 것으로 나타났다. WT그룹, Mstn+/-그룹, Mstn-/-그룹은 세포질 내에 자홍색의 침전물이 순차적으로 감소하고, WT+DM그룹, Mstn+/-+DM그룹, Mstn-/-+DM그룹은 세포질 내에 자홍색의 침전물이 순차적으로 감소했다. 그러나 마이오스타틴 (Mstn) 녹아웃 후 파골세포의 활성은 조골세포의 활성보다 변화가 뚜렷하게 진행된다는 것을 보여주었다.

5. WT+DM그룹, Mstn+/-+DM그룹, Mstn-/-+DM그룹의 골조직 Wnt, β-catenin 발현은 각각 WT그룹, Mstn+/-그룹, Mstn-/-그룹보다 낮고 (P<0.05), GSK-3β 발현은 각각 WT그룹, Mstn+/-그룹 , Mstn-/-그룹 보다 높은 것으로 나타났다 (P<0.05). Mstn-/-그룹의 골조직 Wnt, β-catenin 발현은 WT그룹, Mstn+/-그룹보다 높고, GSK-3β 발현은 WT그룹, Mstn+/-그룹 보다 낮다 (P<0.05). Mstn-/-+DM그룹의 골조직 Wnt, β-catenin 발현은 WT+DM그룹, Mstn+/-+DM그룹보다 높고 (P<0.05), GSK-3β 발현은 WT+DM그룹, Mstn+/-+DM그룹보다 낮은 것으로 나타났다 (P<0.05). WT그룹과 Mstn+/-그룹, WT+DM그룹과 Mstn+/-+DM그룹의 골조직 Wnt, GSK-3β, β-catenin 발현 관련 비교 및 차이는 통계학적으로 의미가 없다 (P<0.05).

결론：1. 마이오스타틴(Mstn) 유전자의 발현을 억제하면 제2형 당뇨병(T2DM)으로 인한 골흡수를 억제하고, 골형성을 촉진시키는 효과를 볼 수 있다. 그 중에 과도한 골흡수를 억제하는 것을 주요 기능으로 하고, 변화 정도는 또한 마이오스타틴(Mstn) 발현량과 관련이 있는 것으로 밝혔다.

2. 제2형 당뇨병(T2DM) 생쥐의 마이오스타틴(Mstn) 유전자 발현을 억제하면 골 미세구조를 개선하고 골강도를 증가시킬 수 있으며 변화 정도는 또한 마이오스타틴(Mstn) 발현량과 관련되어 있다.
3. 제2형 당뇨병(T2DM) 생쥐에게 마이오스타틴(Mstn) 유전자 발현을 억제하면 골조직 Wnt/β-catenin 신호 전달 경로를 상향 조절할 수 있다. 또한, 변화 정도는 마이오스타틴 (Mstn) 발현량과 관련이 있는 것으로 나타났다.

목차 (Table of Contents)

• Ⅰ. 서 론 1
• 1. 머리말 1
• 2. 연구배경 2
• 3. 연구목적 4
• 4. 연구 의의 4
• Ⅱ. 재료 및 방법 5
• 1. 재료 5
• 1.1. 실험동물 5
• 1.2. 실험 기자재 및 시약 5
• 2. 방법 8
• 2.1. 동물모델 구축 및 평가 8
• 2.2. 생쥐 체중, 신장, 복부둘레, 그립력, 지구력 운동 수행 능력 측정 9
• 2.3. 골 미세구조 지표 측정 10
• 2.4. 골조직 조골세포의 활성도 관찰 10
• 2.5. 골조직 파골세포의 활성도 관찰 11
• 2.6. 골조직 Wnt, GSK-3β 및 β-catenin의 발현 측정 11
• 2.7. 통계학적 분석 12
• 3. 결 과 12
• 3.1. 모델링 후 생쥐의 체중, 신장, 복부둘레, 체성분 비교 12
• 3.2. 모델링 후 생쥐의 근력, 항피로 능력 비교 15
• 3.3. 모델링 후 생쥐의 혈청 PINP 및 CTX 수치 비교 16
• 3.4. 모델링 후 생쥐의 골 미세구조 비교 17
• 3.5. 모델링 후 생쥐의 골조직 조골세포의 활성도 비교 18
• 3.6. 모델링 후 생쥐 골조직의 파골세포 활성도 비교 19
• 3.7. 모델링 후 생쥐 골조직 Wnt, GSK-3β, β-catenin 발현 비교 20
• Ⅲ. 토 론 23
• 1. MSTN은 체질량 및 체성분에 미치는 영향 23
• 2. MSTN은 근력, 항피로 능력에 미치는 영향 24
• 3. MSTN은 골소주에 미치는 영향 26
• 4. MSTN은 골전환 표지자 및 골대사에 미치는 영향 27
• 5. MSTN은 Wnt/β-catenin 신호 전달 경로에 의한 골대사에 미치는 영향 28
• Ⅳ. 총론 : MSTN 개요 31
• 1. MSTN의 합성 및 조절 32
• 2. MSTN의 생물학적 기능 33
• 3. MSTN과 골격근 33
• 4. MSTN과 심근 34
• 5. MSTN과 지방 36
• 6. MSTN과 인슐린 저항성 37
• 7. MSTN과 골격 38
• 8. MSTN과 노화 39
• 9. MSTN과 운동, 질병의 관계 40
• 9.1 MSTN과 운동 40
• 9.2 MSTN과 근감소증 42
• 9.3 MSTN과 심혈관질환 43
• 9.4 MSTN과 비만 44
• 9.5 MSTN과 당뇨병 44
• 9.6 MSTN과 골다공증 45
• 10. 결론 및 전망 46
• 참고문헌 47
• Abstract 67

참고문헌 (Reference)

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36 Juvvuna PK, Subramanian S, Sriram S, Sharma M., Salerno MS, McFarlane C, Kambadur R, "Myostatin induces DNA damage in skeletal muscle of streptozotocin-induced type 1 diabetic mice", 289(9):5784-98. doi: 10.1074/jbc. M113.483115. Epub 2014 Jan 14. Retraction in: J Biol Chem. 2015 Nov 6;290(45):27012. PMID: 24425880; PMCID: PMC3937650., 2014

37 Chandra M, Santana LF, Rodgers BD, Nelson OL, Murry CE, Mamidi R, Interlichia JP, Garikipati DK, "Myostatin represses physiological hypertrophy of the heart and excitation-contraction coupling", 587(Pt 20):4873-86. doi: 10.1113/jphysiol.2009.172544. Epub 2009 Sep 7. PMID: 19736304; PMCID: PMC2770153., 2009

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39 Brown M, Yao X, Wang Y, Raw CE, Phillips CL, Pfeiffer FM, Oestreich AK, Gentry BA, Carleton SM, "Myostatin deficiency partially rescues the bone phenotype of osteogenesis imperfecta model mice", 27(1):161-70. doi: 10.1007/s00198-015-3226-7. Epub 2015 Jul 16. PMID: 26179666; PMCID: PMC8018583., 2016

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41 Ge X, Sathiakumar D, McFarlane C., Lua BJ, Lee M, Kukreti H, "Myostatin signals through miR-34a to regulate Fndc5 expression and browning of white adipocytes", 41(1):137-148. doi: 10.1038/ijo.2016.110. Epub 2016 Jun 14. PMID: 27297797; PMCID: PMC5220162., 2017

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43 Alvares LE, Mantovani CS, Grade CVC, "Myostatin gene promoter: structure, conservation and importance as a target for muscle modulation", 10:32. doi: 10.1186/s40104-019-0338-5. PMID: 31044074; PMCID: PMC6477727, 2019

44 Du H, Zhang X, Zhang D, Yang X, Tang L, Sun L., Han Y, Gao X, "Combination of Weight-Bearing Training and Anti-MSTN Polyclonal Antibody Improve Bone Quality In Rats", 26(6):516-524. doi: 10.1123/ijsnem.2015-0337. Epub 2016 Aug 24. PMID: 27098383., 2016

45 Abudukadier A, Shulman GI, Petersen MC, Petersen KF, Moreira GV, Jurczak MJ, Haqq CM, Friedman G, Camporez JP, "Anti-myostatin antibody increases muscle mass and strength and improves insulin sensitivity in old mice", 113(8):2212-7. doi: 10.1073/pnas.1525795113. Epub 2016 Feb 8. PMID: 26858428; PMCID: PMC4776508., 2016

46 Bhasin S, Yarasheski KE, Sinha-Hikim I, Pak-Loduca J, Gonzalez-Cadavid NF, "Serum myostatin-immunoreactive protein is increased in 60-92 year old women and men with muscle wasting", 6(5):343-8. PMID: 12474026, 2002

47 Hafer-Macko C., Ryan AS, Prior S, Li G, Ivey FM, "Skeletal muscle hypertrophy and muscle myostatin reduction after resistive training in stroke survivors", 42(2):416-20. doi: 10.1161/STROKEAHA.110.602441. Epub 2010 Dec 16. PMID: 21164115; PMCID: PMC3026882., 2011

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52 De Bock K, Russell AP, Léger B, Hespel P, Derave W, "Human sarcopenia reveals an increase in SOCS-3 and myostatin and a reduced efficiency of Akt phosphorylation", 11(1):163-175B. doi: 10.1089/rej.2007.0588. PMID: 18240972, 2008

53 Bae KH, Park SG, Lee SC, Ko Y, Kim WK, Choi HR, "Myostatin inhibits brown adipocyte differentiation via regulation of Smad3-mediated β-catenin stabilization", 44(2):327-34. doi: 10.1016/j. biocel.2011.11.004. Epub 2011 Nov 11. PMID: 22094186., 2012

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55 Bowser M, Wenger K, Hamrick M., Fulzele S, Elkasrawy M, "Myostatin (GDF-8) inhibits chondrogenesis and chondrocyte proliferation in vitro by suppressing Sox-9 expression", 29(6):253-62. doi: 10.3109/08977194.2011.599324, 2011

56 Brimioulle S, Ray L, Naeije R, McEntee K., Mathieu M, Mahmoudabady M, Jespers P, Hadad I, Dewachter L, "Activin-A, transforming growth factor-beta, and myostatin signaling pathway in experimental dilated cardiomyopathy", 14(8):703-9. doi: 10.1016/j. cardfail.2008.05.003. Epub 2008 Jun 11. PMID: 18926443., 2008

57 Buchanan SM, Wattrus S, Walker RG, Wagers AJ, Thompson TB, Rubin LL, Poggioli T, Oh J, Lee RT, Katsimpardi L, Heidecker B, Ganz P, Fong YW, "Biochemistry and Biology of GDF11 and Myostatin: Similarities, Differences, and Questions for Future Investigation", 118(7):1125-41, 2016

58 Brachat S, Sheppard K, Rivet H, Morvan F, Minetti GC, Lach-Trifilieff E, Koelbing C, Ibebunjo C, Hatakeyama S, Hartmann S, Glass DJ, Feige JN, "An antibody blocking activin type II receptors induces strong skeletal muscle hypertrophy and protects from atrophy", 34(4):606-18. doi: 10.1128/MCB.01307-13. Epub 2013 Dec 2. PMID: 24298022; PMCID: PMC3911487., 2014

59 Bjørklund Holven K, Raschke S, Jensen J, Eckel J, Eckardt K, "Identification and validation of novel contraction-regulated myokines released from primary human skeletal muscle cells", 8(4):e62008. doi: 10.1371/journal. pone.0062008. PMID: 23637948; PMCID: PMC3634789., 2013

60 Bauman WA, Liu XH, Cardozo CP, "Myostatin inhibits glucose uptake via suppression of insulin-dependent and -independent signaling pathways in myoblasts", 6(17):e13837. doi: 10.14814/phy2.13837. PMID: 30252210; PMCID: PMC6121119, 2018

61 Bi Y, Zheng X., Zhang L, Xiao H, Wang Z, Wang Y, Ren H, Liu X, Li L, Laible G, Hua Z, Hua W, Dong F, "Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP", 6:31729. doi: 10.1038/srep31729. PMID: 27530319; PMCID: PMC4987667., 2016

62 Adams V, Schur R, Schuler G., Matsumoto Y, Linke A, Lenk K, Erbs S, "Impact of exercise training on myostatin expression in the myocardium and skeletal muscle in a chronic heart failure model", 11(4):342-8. doi: 10.1093/eurjhf/hfp020. Epub 2009 Feb 13. PMID: 19218333., 2009

63 Arounleut P, Liang LF, Bialek P, "Amyostatininhibitor(propeptide-Fc)increasesmusclemassandmusclefibersizei nagedmicebutdoesnotincreasebonedensityorbonestrength", (9):898-904, 2013

64 Bakhurin KI, Shallal-Ayzin MV, Mendias CL, Gumucio JP, Faulkner JA, Davis CS, "Haploinsufficiency of myostatin protects against aging-related declines in muscle function and enhances the longevity of mice", 14(4):704-6. doi: 10.1111/acel.12339. Epub 2015 Mar 24. PMID: 25808276; PMCID: PMC4531085., 2015

65 Brown R, Xu T, Welle S., Han B, Burgess K, "Effect of myostatin depletion on weight gain, hyperglycemia, and hepatic steatosis during five months of high-fat feeding in mice", 6(2):e17090. doi: 10.1371/journal. pone.0017090. PMID: 21390326; PMCID: PMC3044753., 2011

66 Bonala S, Zhang C, Sharma M, McFarlane C, Masuda S, Lokireddy S, Kambadur R., Gluckman PD, Ge X, "Myostatin-deficient mice exhibit reduced insulin resistance through activating the AMP-activated protein kinase signalling pathway", 54(6):1491-501. doi: 10.1007/s00125-011-2079-7. Epub 2011 Feb 24. Erratum in: Diabetologia. 2015 Mar;58(3):643. PMID: 21347623., 2011

67 Li S, Zhang Z, Zhang H, Wu F, Sheng Z, Ou Y, Ma Y, Liao E., Li X, "Serum myostatin in central south Chinese postmenopausal women: Relationship with body composition, lipids and bone mineral density", 41(3):223-8. doi: 10.3109/07435800.2015.1044609. Epub 2016 May 4. PMID: 27144806., 2016

68 Du H, Zhang D, Yang X, Wang Z, Tang L, Sun L., Han Y, Gao X, "Inhibiting myostatin signaling prevents femoral trabecular bone loss and microarchitecture deterioration in diet-induced obese rats", 241(3):308-16. doi: 10.1177/1535370215606814, 2016

69 Akashi H, Wang C, Sweeney HL, Schulze PC, Najjar M, Kennel PJ, Ji R, George I., Castillero E, "Cardiac myostatin upregulation occurs immediately after myocardial ischemia and is involved in skeletal muscle activation of atrophy", 457(1):106-11. doi: 10.1016/j. bbrc.2014.12.057. Epub 2014 Dec 18. PMID: 25528587; PMCID: PMC4967536., 2015

70 허칭화, "쑨밍샤오, 웨옌펀, 등.베이징지역 중장년 2형 당뇨병 환자 근소 증 유병률 연구 및 영향 요인 분석[J]", 중화당뇨병잡지, ,11(5):328-333, 2019

71 Ashby M, Thomas M, Smith H, Sharma M, Plummer E, McFarlane C, Ling N, Kambadur R., Hennebry A, "Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-kappaB-independent, FoxO1-dependent mechanism", 209(2):501-14. doi: 10.1002/jcp.20757. PMID: 16883577., 2006

72 Artaza JN, Tsao J, Reisz-Porszasz S, Kloner RA, Gonzalez-Cadavid NF, Dow JS, Bhasin S, "Alterations in myostatin expression are associated with changes in cardiac left ventricular mass but not ejection fraction in the mouse", 194(1):63-76. doi: 10.1677/JOE-07-0072. PMID: 17592022., 2007

73 Borup R, Vaag A., Torp-Pedersen C, Storgaard H, Spohr C, Palsgaard J, Jensen M, Friedrichsen M, Dominguez H, De Meyts P, Brøns C, "Gene expression in skeletal muscle biopsies from people with type 2 diabetes and relatives: differential regulation of insulin signaling pathways", 4(8):e6575. doi: 10.1371/journal. pone.0006575. PMID: 19668377; PMCID: PMC2719801., 2009

74 Bernardo BL, Wachtmann TS, Opsahl AC, LeBrasseur NK, Kuhn M, Judkins KM, Hadcock JR, Freeman TB, Cosgrove PG, "Postnatal PPARdelta activation and myostatin inhibition exert distinct yet complimentary effects on the metabolic profile of obese insulin-resistant mice", 5(6):e11307. doi: 10.1371/journal. pone.0011307. PMID: 20593012; PMCID: PMC2892469., 2010

75 유솔남, "PPARα흥분제 2형 당뇨병의 다른 발전 단계에 대한 실험 동물 모델 인슐린β세포기능의 영향 및 그 기능에 대한 연구[D]", 북경협화 의학원, 2010

76 Clark BC, Consitt LA, "The Vicious Cycle of Myostatin Signaling in Sarcopenic Obesity: Myostatin Role in Skeletal Muscle Growth, Insulin Signaling and Implications for Clinical Trials", 7(1):21-27. doi: 10.14283/jfa.2017.33. PMID: 29412438; PMCID: PMC6909929., 2018

77 Ngo S, Zarfeshani A, Sheppard AM, "Leucine alters hepatic glucose/lipid homeostasis via the myostatin-AMP-activated protein kinase pathway - potential implications for nonalcoholic fatty liver disease", 6(1):27. doi: 10.1186/1868-7083-6-27. PMID: 25859286; PMCID: PMC4391119., 2014

78 Geng S., Ju F, Han HS, "In vivo and in vitro effects of PTH1-34 on osteogenic and adipogenic differentiation of human bone marrow-derived mesenchymal stem cells through regulating microRNA-155", 119(4):3220-3235. doi: 10.1002/jcb.26478. Epub 2018 Jan 2. PMID: 29091308., 2018