Modulate Autocrine Secretion of Insulin-like Growth Factor-1 and Insulin-like Growth Factor Binding Proteins from Differentiated Skeletal Muscle Cells*

Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/micrograms of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli.


Stretch-induced
Insulin-likegrowth factors(IGFs) I are potent mitogens involved in stimulating skeletal m'uscle growth (i-41 The:: increase amino acid uptake and proteinI synthesis, decrease prorein degradation, and stimulate the proliferation and differentiationof skeletal muscle cells (2,(5)(6)(7)(8)(9)(10). IGF's have been shown to be secreted from several mammalian skeletal muscle celllines (8,II,12).A number of studies have revealed that IGF-2 is released during myoblast proliferationwhile IGF-I effluxisobserved during skeletalmuscle differentiation (I, 8). Increases in IGF-I mRNA have been observed during muscle regeneration after injury (13)(14)(15), and during workinduced compensatory hypertrophy (16).It has been suggested * This work was supported by NASA Grant NAG2-414 and National Institutes of Health Grant ROI AR 39998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked _advertisement _ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The abbreviations used are: IGF, insulin-like growth factor; MM medium, muscle maintenance medium; PGM, program.
that the increased secretion of IGF-I during work-induced hypertrophy (16) may promote the accumulation of proteins in skeletalmuscle cellsby an autocrine mechanism but the level of IGF-I release from skeletalmuscle cellsundergoing hypertrophy isnot known. The mitogenic effectsof insulin-likegrowth factorsare regulated by their binding proteins ¢reviewed in Refs. [17][18][19][20].IGF binding proteins are released from cells which also secrete insulin-likegrowth factorsii, 18. 21,22 acetic acid-insoluble material during a 4-6-h incubation peraod as deserabed previously by Vandenburgh et al. t29). Incorporation is linear during this _ime period and excess nonradioactive phenylalanme was included in the medium (0.5 rn_) to allow rapid equilibration of the intracellular and extracellular amino acid pools (30). DNA was measured fluorometrically by the modified method of Labarca and Palgen !31).
After extensive rinsing in saline, the membrane was air-dried, and exposed to x-ray film for varyang times at -80°C with two intensifying screens. The amounts of labeled binding proteins were determined quantitatively by densitometric scanning of preflashed x-ray autoradiographs using a computerized image analysis system (JAVA and PEAK FIT, Jandel Scientific, Corte Madera, CA).
Statistical analyses of the data were performed by t tests for unpaired values using a statistical software program (SIGMASTAT, Jandel Scientific).

Stretch
Responses  (Fig. 5,4). Similarly, a 6-fold increase in the frequency of mechanical stimulation (5-rain rest periods, TRIAL52.PGM) showed no effect on the release of IGF-1 from the muscle cells (Fig. 5B).  (Fig. 6). IGF-1 release from stretched cells was signifi-   (Fig. 7). Noncollagen-embedded cells released lower amounts of IGF-1 than collagen-embedded cells in both cell types (Fig. 7, A versus B) (30), the production of IGF-1 by the myofibers in these cultures on a microgram cell protein basis constitutes the major part of total IGF-1 release. It is difficult, however, to determine the exact contribution of each celltype in the mixed cultures since the two celltypes appear to interactin regulating total IGF-I efflux in a complex manner when co-cultured (Fig.7), as found previously for the regulation of total protein degradation in the two celltypes (45). IGF-1 secretion in mixed cultures was less than in either cell type alone, indicating some form of feedback inhibition