1.Aging parameters of the accelerated aging procedure through D-Galactose induction
Ronald Winardi Kartika ; Kris Herawan Timotius ; Veronika Maria Sidharta ; Tena Djuartina ; Cynthia Retna Sartika
Acta Medica Philippina 2024;58(Early Access 2024):1-6
Background and Objectives:
Intraperitoneal injection (i.p.) of D-galactose (D-gal) accelerates aging and develops aging models. A low dose of long-term use and a high dose of short-term use of D-gal can induce natural aging in mice, like brain, cardiac, liver, renal, and skin aging, and erectile dysfunction. Our research aims to determine whether a high dose of short-term use of D-gal. i.p. in rats can induce natural aging and affect the following parameters: body weight (BW), Superoxide Dismutase (SOD), Vascular endothelial growth factor (VEGF), C-reactive protein (CRP), and myostatin.
Methods:
A daily D-gal i.p. dose of 300 mg/ml/kg for seven days was carried out to induce aging parameters in the rats. After seven days, the body and gastrocnemius circumference of the rats were weighed, and biochemical analysis for SOD, VEGF, CRP, and myostatin in the blood plasma was done.
Results:
The data obtained were analyzed using nonparametric statistics Friedman test and Mann-Whitney test. After the seven day-intervention, both the control (NaCl 0.9% i.p.) and the high dose of short-term use of D-gal i.p. groups showed no significant difference in the body weight and gastrocnemius circumference. However, D-gal administration could increase the blood plasma level of SOD, VEGF, CRP, and myostatin.
Conclusion
We conclude that a high dose of short-term intraperitoneal D-galactose can be administrated to induce aging in rat models. The SOD, VEGF, CRP and myostatin can be used as aging parameters.
Aging
;
Galactose
;
Myostatin
;
Vascular Endothelial Growth Factor A
2.Decreasing Lean Body Mass with Age: Challenges and Opportunities for Novel Therapies.
Chrysoula BOUTARI ; Christos S MANTZOROS
Endocrinology and Metabolism 2017;32(4):422-425
No abstract available.
Body Composition
;
Body Mass Index
;
Myostatin
;
Adiposity
;
Obesity
;
Muscle, Skeletal
;
Muscular Diseases
3.Construction of a New T-Vector: Nickase (Nt.BspQI)-Generated T-Vector Bearing a Reddish-Orange Indicator Gene.
Ji Young CHOI ; Chulman JO ; Sangmee Ahn JO
Tissue Engineering and Regenerative Medicine 2016;13(1):66-69
T-vectors are widely used for cloning the polymerase chain reaction (PCR) products. However, the low conversion efficiency of a plasmid into the linear T-vector usually results in non-recombinants. Here, we designed a new plasmid pNBQ-T to easily select the recombinant colonies harboring PCR products. pNBQ-T plasmid, which contains a DsRed indicator gene between two Nt.BspQI restriction cassettes, each of which contains palindromic sequences susceptible to Nt.BspQI nickase (5′-GCTCTTCT^GAAGAGC-3′) at each end. Thus, this plasmid can be easily converted into T-vectors by a nickase (quadruple nicking), which results in two double strand breaks with 3′-thymidine overhangs. DsRed indicator gene, which is inserted between the restriction sites, helps identifying the PCR recombinants. Using this pNBQ-T plasmid the insertion efficiency of a PCR product was examined. We successfully identified white colony of the recombinants with the inserted myostatin promoter gene: the cloning efficiency was 93%. Therefore, this simple method utilizing pNBQ-T plasmid will serve as a useful and efficient technique for preparation of home-made T-vectors.
Clone Cells
;
Cloning, Organism
;
Deoxyribonuclease I*
;
Methods
;
Myostatin
;
Plasmids
;
Polymerase Chain Reaction
4.Propagation and phenotypic analysis of mutant rabbits with MSTN homozygous mutation.
Liqing SHANG ; Shaozheng SONG ; Ting ZHANG ; Kunning YAN ; Heqing CAI ; Yuguo YUAN ; Yong CHENG
Chinese Journal of Biotechnology 2022;38(5):1847-1858
Myostatin gene (MSTN) encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, MSTN-/- homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of MSTN gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. MSTN-/- primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology. The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits, whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain. Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits. The MSTN mutant rabbits with 14-19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed. Five primary rabbits with MSTN gene mutation were obtained, among which three were used for homozygous breeding. A total of 15 homozygous rabbits (5 types of mutants) were obtained (M2-a: 3; M2-b: 2; M3-a: 2; M7-a: 6; M7-b: 2). The body weight of MSTN-/- homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of MSTN+/+ wild-type rabbits of the same age ((2 718±120) g vs. (1 969±53) g, P < 0.01, a 38.0% increase). The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits ((3 512.2±439.2) μm2 vs. (1 274.8±327.3) μm2, P < 0.01), but also significantly higher than that of MSTN+/- hemizygous rabbits ((3 512.2±439.2) μm2 vs. (2 610.4±604.4) μm2, P < 0.05). In summary, five homozygous mutants rabbits of MSTN-/- gene were successfully bred, which showed a clear lean phenotype. The results showed that the primary breeds were non-chimeric mutant rabbits, and the mutant traits could be inherited from the offspring. MSTN-/- homozygous mutant rabbits of F2 generation could be obtained from F1 hemizygous rabbits by inbreeding or backcrossing. The progenies of the primary biallelic mutant rabbits were separated into two single-allelic mutants, both of which showed a "double-muscle" phenotype. Thus, this study has made progress in breeding high-quality livestock breeds with gene editing technology.
Animals
;
CRISPR-Cas Systems/genetics*
;
Gene Editing
;
Muscle, Skeletal/metabolism*
;
Mutation
;
Myostatin/metabolism*
;
Phenotype
;
Rabbits
5.Follistatin N terminus differentially regulates muscle size and fat in vivo.
Hui ZHENG ; Chunping QIAO ; Ruhang TANG ; Jianbin LI ; Karen BULAKLAK ; Zhenhua HUANG ; Chunxia ZHAO ; Yi DAI ; Juan LI ; Xiao XIAO
Experimental & Molecular Medicine 2017;49(9):e377-
Delivery of follistatin (FST) represents a promising strategy for both muscular dystrophies and diabetes, as FST is a robust antagonist of myostatin and activin, which are critical regulators of skeletal muscle and adipose tissues. FST is a multi-domain protein, and deciphering the function of different domains will facilitate novel designs for FST-based therapy. Our study aims to investigate the role of the N-terminal domain (ND) of FST in regulating muscle and fat mass in vivo. Different FST constructs were created and packaged into the adeno-associated viral vector (AAV). Overexpression of wild-type FST in normal mice greatly increased muscle mass while decreasing fat accumulation, whereas overexpression of an N terminus mutant or N terminus-deleted FST had no effect on muscle mass but moderately decreased fat mass. In contrast, FST-I-I containing the complete N terminus and double domain I without domain II and III had no effect on fat but increased skeletal muscle mass. The effects of different constructs on differentiated C2C12 myotubes were consistent with the in vivo finding. We hypothesized that ND was critical for myostatin blockade, mediating the increase in muscle mass, and was less pivotal for activin binding, which accounts for the decrease in the fat tissue. An in vitro TGF-beta1-responsive reporter assay revealed that FST-I-I and N terminus-mutated or -deleted FST showed differential responses to blockade of activin and myostatin. Our study provided direct in vivo evidence for a role of the ND of FST, shedding light on future potential molecular designs for FST-based gene therapy.
Activins
;
Animals
;
Follistatin*
;
Genetic Therapy
;
In Vitro Techniques
;
Mice
;
Muscle Fibers, Skeletal
;
Muscle, Skeletal
;
Muscular Dystrophies
;
Myostatin
;
Negotiating
6.Increased circulating myostatin in patients with type 2 diabetes mellitus.
Fang WANG ; Yonggan LIAO ; Xuefeng LI ; Chunlin REN ; Changming CHENG ; Yongsheng REN
Journal of Huazhong University of Science and Technology (Medical Sciences) 2012;32(4):534-539
The changes of plasma myostatin levels in patients with type 2 diabetes mellitus (T2D) and their clinical correlation were investigated. We recruited 43 T2D patients and 20 age-matched healthy subjects. Plasma myostatin, lipid and glucose, and serum insulin were determined. T2D patients showed significantly higher fasting plasma glucose (FPG), serum insulin and triglyceride levels, and lower high-density lipoprotein levels than normal control subjects (P<0.01). Mean plasma myostatin level in T2D patients and health controls was (66.5±17.8) and (46.2±13.8) ng/mL, respectively. An unpaired t test showed that the increase of myostatin in the T2D patients was significant (P<0.001). In both healthy control and T2D groups, the female subjects showed higher myostatin levels than the male subjects. In the T2D patients, plasma level of myostatin was negatively correlated with body mass index (BMI, r=-0.42, P<0.01) and FPG (r=-0.51, P[Symbol: see text]0.01), but positively correlated with insulin resistance index (HOMA-IR, r=0.48, P<0.01). Up-regulation of plasma myostatin in the T2D patients and its correlation with BMI, FPG and blood insulin sensitivity suggests that plasma myostatin may be implicated in the pathogenesis of T2D and thus presented as a therapeutic target for treating the disease. Furthermore, circulating myostatin levels may be used as a biomarker for the disease.
Blood Glucose
;
Diabetes Mellitus, Type 2
;
blood
;
Female
;
Humans
;
Insulin
;
blood
;
Lipids
;
blood
;
Male
;
Middle Aged
;
Myostatin
;
blood
7.Relation between serum myostatin with BMI and PaO₂/PaCO₂ in patients with chronic obstructive pulmonary disease.
Xiufang WEN ; Pian LIU ; Haiqiao WU ; Xiangdong ZHOU
Journal of Central South University(Medical Sciences) 2014;39(8):807-810
OBJECTIVE:
To determine the relation between serum myostatin with body mass index (BMI) and PaO₂/PaCO₂ in men with chronic obstructive pulmonary disease (COPD).
METHODS:
A cohort of outpatients with stable COPD was evaluated. We evaluated the myostatin, PaO₂/PaCO₂ and BMI, and the patients were stratified by BMI. The plasma level of myostatin and PaO₂/PaCO₂ was measured by high sensitivity ELISA or blood gas analysis.
RESULTS:
PaCO₂ and myostatin increased significantly compared with those in the control group (P<0.05), but PaO₂ decreased significantly. There was positive correlation between myostatin and PaCO₂ (P<0.05), and negative correlation between myostatin and BMI/FEV1/pred value/PaO₂ (P<0.05).
CONCLUSION
Patients with higher myostatin levels had a lower BMI, lower PaO₂ and higher PaCO₂, with poor pathogenetic condition and prognosis. Myostatin may be a potential treatment target in patients with chronic obstructive pulmonary disease.
Blood Gas Analysis
;
Body Mass Index
;
Humans
;
Male
;
Monitoring, Physiologic
;
Myostatin
;
blood
;
Prognosis
;
Pulmonary Disease, Chronic Obstructive
;
blood
8.Genes and their functional mechanisms in the pathogenesis of muscular dystrophy.
Acta Academiae Medicinae Sinicae 2005;27(3):394-400
Muscular dystrophy (MD), a group of inherited disorders characterized by progressive skeletal muscle wasting and weakness, can be classified into several groups according to Mendelian inheritance patterns and clinical features. Many genes related to MD have been identified and cloned by genetic linkage analysis and positional cloning strategy. Our understanding of the molecular mechanisms giving rise to muscular dystrophy have made a progress by the functional analysis of proteins encoded by candidate genes for MD. This article reviews genes and their functional mechanisms in the pathogenesis of muscular dystrophy.
Calpain
;
genetics
;
Dystrophin
;
genetics
;
Humans
;
Lamin Type A
;
genetics
;
Muscle Proteins
;
genetics
;
Muscular Dystrophies
;
etiology
;
genetics
;
Myostatin
;
Transforming Growth Factor beta
;
genetics
;
Tripartite Motif Proteins
;
Ubiquitin-Protein Ligases
;
genetics
9.Regulation of myostatin promoter activity by myocyte enhancer factor 2.
Jia LI ; Jie DENG ; Junlin ZHANG ; De CHENG ; Huayan WANG
Chinese Journal of Biotechnology 2012;28(8):918-926
Myostatin (Mstn) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals. The transcriptional regulation of Mstn is controlled by multiple genes including MEF2, which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying. In this study, we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells. Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs. Using a serial deletion strategy, we tested the activity of several promoter fragments by luciferase assay. Overexpression of MEF2C, but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds, in both C2C12 myoblasts and myotubes. When we transfected exogenous MEF2C, Mstn mRNA level was also upregulated in C2C12 cells, but the protein level was only significantly increased in myotubes. Thus, we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine. Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.
Animals
;
Cells, Cultured
;
Gene Expression Regulation
;
MEF2 Transcription Factors
;
Mice
;
Muscle, Skeletal
;
metabolism
;
Myoblasts
;
cytology
;
Myogenic Regulatory Factors
;
genetics
;
physiology
;
Myostatin
;
genetics
;
physiology
;
Promoter Regions, Genetic
;
Swine
10.Site-directed mutagenesis of MSTN gene by gene targeting in Qinchuan cattle.
Yonggang LIU ; Song HUA ; Jie LAN ; Yongli SONG ; Yulong HE ; Fusheng QUAN ; Yong ZHANG
Chinese Journal of Biotechnology 2010;26(3):410-416
Myostatin, a member of the transforming growth factor beta (TGF-beta) family, is a negative regulator for muscle growth. Loss of the function of this gene is associated with the phenotype described as "double muscling", an extreme form of muscle development characterized by a large increase in muscle mass. Two replacement vectors, pA2T-Mstn4.0 and pA2T-Mstn3.2, were constructed, linearized, and transfected into the bovine fetal fibroblasts through electroporation. 170 drug-resistant cell colonies were obtained in cell culture medium containing 600 microg/mL G418 and 50 nmol/L GCV. Targeted homologous integration occurred in colony No. 58 as identified by PCR, and the targeted colony was further confirmed by sequencing and Southern blotting. This suggested that one allele of myostatin was successfully mutagenized in bovine fetal fibroblasts.
Animals
;
Base Sequence
;
Cattle
;
China
;
Electroporation
;
Fetus
;
Fibroblasts
;
cytology
;
metabolism
;
Gene Targeting
;
Molecular Sequence Data
;
Mutagenesis, Site-Directed
;
veterinary
;
Myostatin
;
genetics