[Effects of insulin on proteolysis of cultured rabbit skeletal muscular myotubes in vitro].
OBJECTIVE
To study the effects of insulin on the proteolysis of cultured rabbit skeletal muscular myotubes in vitro, and their possible mechanisms.


METHODS
Muscles of lower limbs of juvenile rabbits were isolated for tissue-block culture. After passage, myoblasts were formed and fused into myotubes. Then the protein in myotubes was radiolabelled with L-[ 3,5-3H] tyrosine. The myotubes were cultured in DMEM medium containing 100 nmol/L insulin (n = 24, group B) , 100 nmol/L dexamethasone (n = 24, group C) , 100 nmol/L insulin and 100 nmol/L dexamethasone (n = 24, group D) , no insulin or dexamethasone (n =24, group A), respectively. Twenty-four hours after culture, the L-[3,5-3H] tyrosine content in culture medium and cells were determined, and the degradation rates of protein were calculated. The mRNA expressions of ubiquitin and protease C2 subunit were determined by Northern blot.


RESULTS
The degradation rates of myotube protein in group A(0. 38+/-0.04) was obviously lower than that in group C (0.50+/-0.03, P <0.01), but it was obviously higher than that in group B(0. 35+/-0.03, P <0.05). Though the degradation rates of myotube protein in group D (0.41+/-0. 03) was evidently lower than that in group C ( P < 0.01) , it was still higher than that in group A( P < 0.05 ). The mRNA expressions of ubiquitin and protease C2 subunit in group A ( the scale: 2. 4 kb ubiquitin was 0. 82+/-0. 15, 1. 2 kb ubiquitin was 0. 60+/-0. 10, C2 subunit was 0. 75+/-0. 16) was obviously lower than that in group C ( the scale: 2.4 kb ubiquitin was 2. 15+/-0. 23, 1.2 kb ubiquitin was 1.50+/-0. 14,C2 subunit was 1.50+/-0. 13 , P <0. 01) , but it in group D was lower than that in group C (the scale: 2. 4 kb ubiquitin was 1. 25+/-0. 17, 1. 2 kb ubiquitin was 0. 85+/-0. 09, C2 subunit was 0. 90+/-0. 15, P <0. 01) , and it was similar to that in group B (the scale: 2.4 kb ubiquitin was 0. 85+/-0.07, 1.2 kb ubiquitin was 0. 65+/- 0. 12, C2 subunit was 0. 76 +/-0. 09, P > 0. 05).


CONCLUSIONS
The effects of insulin on the activity of ubiquitin-proteasome pathway and the proteolytic rate in normal myotubes were relatively weak. However, insulin can significantly inhibit the effects of dexamethasone on the gene expressions of ubiquitin system and the proteolytic rate in myotubes, but the mechanism needs further research.
Shen
Chuan-an
CA
Burn Institute, the First Affiliated Hospital to General Hospital of PLA, Beijing 100037, PR China.
Chai
Jia-ke
JK
Sheng
Zhi-yong
ZY
Yang
Hong-ming
HM
Yin
Hui-man
HM
Feng
Rui
R
D000818:Animals; D002478:Cells, Cultured; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism; D011817:Rabbits; D025801:Ubiquitin / Q000378:metabolism
D007328:Insulin; D009124:Muscle Proteins; D025801:Ubiquitin