Effects of combined hormone replacement therapy or its effective agents on the IGF-1 pathway in skeletal muscle.
OBJECTIVE
To investigate the effects of combined hormone replacement therapy (HRT) and its effective agents on the IGF-1 signaling pathway.


METHODS
To examine the effects of HRT on skeletal muscle in vivo, we utilized pre- and post-intervention samples from a randomized double blinded trial with 50-57-year-old women. The intervention included the year-long use of either HRT preparation (2 mg 17β-estradiol, E₂; 1mg norethisterone acetate, NETA, n=10) or placebo (CO, n=9). Microarray technology and quantitative PCR (qPCR) were used to study the expression of insulin-like growth factor I (IGF-1) and its splice variants as well as IGF-1 receptor, Akt1, mTOR, FOXO1, FOXO3, atrogin, estrogen receptors and androgen receptor in muscle samples. Serum concentrations of IGF-1, E(2) and testosterone were measured. C2C12 myotubes were fed with E₂ or NETA followed by analyzing the expression of essentially the same gene transcripts as in human samples by qPCR and phosphorylation of Akt and mTOR by Western blotting.


RESULTS
The gene expression of IGF-1 and its splice variant, IGF-1Ec (also known as the mechano growth factor or MGF), mTOR, FOXO3, and AR was up-regulated among the HRT users compared to the CO (P<0.05), while Akt1 was down-regulated (P<0.05). The change in the level of IGF-1Ec transcript correlated positively with muscle size at post-intervention (r=0.5, P<0.05). In C2C12 myotubes, no statistically significant effects of either E₂ or NETA at the level of gene transcripts studied were identified. The amount of phosphorylated Akt appeared to respond to NETA, albeit the response was not statistically significant. Phosphorylation of mTOR did not respond to either of the treatments.


CONCLUSIONS
Year-long postmenopausal HRT was found to affect the expression of the genes along the IGF-1 signaling cascade reflecting the higher muscle mass compared to the CO women. By using cell culture model we were, however, unable to confirm the possible differential role of E₂ and NETA. It appears that the synchronous presence of both effective agents of the HRT or the presence of yet unidentified microenvironmental factors providing proper paracrine signals naturally existing in the intact muscle tissue is critical for appropriate signaling via sex steroid-IGF-1 axis to occur.
Pöllänen
Eija
E
Gerontology Research Centre, University of Jyväskylä, P.O. Box 35, FIN-40014 University of Jyväskylä, Jyväskylä, Finland.
Ronkainen
Paula H A
PH
Horttanainen
Mia
M
Takala
Timo
T
Puolakka
Jukka
J
Suominen
Harri
H
Sipilä
Sarianna
S
Kovanen
Vuokko
V
D004958:Estradiol / Q000097:blood / Q000627:therapeutic use*; D015914:Estrogen Replacement Therapy; D005260:Female; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D008875:Middle Aged; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000187:drug effects; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics; D018482:Muscle, Skeletal / Q000737:chemistry / Q000187:drug effects*; D009640:Norethindrone / Q000031:analogs & derivatives* / Q000627:therapeutic use; D000077563:Norethindrone Acetate; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000096:biosynthesis / Q000235:genetics; D016032:Randomized Controlled Trials as Topic; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis / Q000235:genetics; D011944:Receptors, Androgen / Q000096:biosynthesis / Q000235:genetics; D011960:Receptors, Estrogen / Q000096:biosynthesis / Q000235:genetics; D044843:SKP Cullin F-Box Protein Ligases / Q000096:biosynthesis / Q000235:genetics; D058570:TOR Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics; D013739:Testosterone / Q000097:blood; D014158:Transcription, Genetic / Q000187:drug effects
C085126:FOXO1 protein, human; C499517:FOXO3 protein, human; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D009124:Muscle Proteins; D020033:Protein Isoforms; D011944:Receptors, Androgen; D011960:Receptors, Estrogen; D013739:Testosterone; D004958:Estradiol; D007334:Insulin-Like Growth Factor I; D000077563:Norethindrone Acetate; C440907:FBXO32 protein, human; D044843:SKP Cullin F-Box Protein Ligases; C546842:MTOR protein, human; D017526:Receptor, IGF Type 1; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D009640:Norethindrone