The type II insulin-like growth factor (IGF) receptor has low affinity for IGF-I analogs: pleiotypic actions of IGFs on myoblasts are apparently mediated by the type I receptor.

We have characterized binding and several actions of the somatomedins [insulin-like growth factors I and II (IGF-I and IGF-II)] on L6 myoblasts. Both IGF-I and IGF-II are potent stimulators of amino acid uptake, cell proliferation, and differentiation; they also suppress protein degradation in these cells. In all measurements, the relative potencies are IGF-I greater than IGF-II greater than insulin. Two recombinant DNA-produced analogs of IGF-I, (Thr59)IGF-I and (N-Met)IGF-I, were as active as native IGF-I in all four assays. However, when 125I-labeled hormones were used for studies of binding to IGF receptors, there was a striking difference between the native and recombinant IGF-I molecules. Both were bound significantly by the type I receptor (a 350K molecule that is dissociated upon sulfhydryl reduction), but the recombinant analogs exhibited little cross-reactivity with the type II receptor (a 220K molecule that is not dissociated by reduction). Thus, the equal activity of native IGF-I and its recombinant DNA-produced analogs coupled with the higher potency of IGF-I (compared to IGF-II) suggest that the type II receptor plays little or no role in the four actions of the somatomedins studied in L6 myoblasts.

Transforming growth factor-beta. A very potent inhibitor of myoblast differentiation, identical to the differentiation inhibitor secreted by Buffalo rat liver cells.

Transforming growth factor-beta (TGF-beta) is now known to have a number of actions in addition to the induction of phenotypic transformation in fibroblastic cells. In this paper, we characterize its inhibition of differentiation in rat myoblasts of Yaffe's L6 strain and demonstrate its identity or very close similarity to the differentiation inhibitor (DI) secreted by Buffalo rat liver cells cultured in serum-free medium. At concentrations as low as 60 pg/ml, TGF-beta gave detectable inhibition of differentiation measured as myoblast fusion and creatine kinase elevation; maximal inhibition was observed at and above 0.5 ng/ml (20 pM). The inhibition persisted as long as fresh TGF-beta was added at 48-h intervals, but it was reversed upon removal of the factor. By itself or in the presence of mitogens, TGF-beta had no mitogenic activity in the L6 cells. Concentration dependencies of human TGF-beta and the rat DI were closely parallel in three assays: inhibition of myoblast differentiation, stimulation of normal rat kidney cell growth in soft agar, and competition for displacement of labeled TGF-beta from binding sites on A549 human lung carcinoma cells. We conclude that most if not all of the DI activity found in medium conditioned by Buffalo rat liver cells can be attributed to the presence of TGF-beta or a very similar molecule. These observations also offer a potentially useful approach to study the control of myogenesis; the process(es) can be blocked in cloned L6 myoblasts by incubation with very small quantities of a pure protein in fully defined serum-free medium.

Biphasic concentration dependency of stimulation of myoblast differentiation by somatomedins.

It is widely believed that mitogens inhibit in vitro differentiation of myoblasts to form postmitotic myotubes, but we and others have shown that the mitogenic hormones insulin and the insulin-like growth factors (IGFs) stimulate myoblast differentiation. We now report the results of concentration-dependency studies that resolve this disagreement. We found that the IGFs give a biphasic dose-response curve; at low concentrations, there is progressive stimulation of L6 myoblast differentiation; at higher concentrations, there is a progressive decrease. Similar results were obtained with IGF-II and insulin. When differentiation was maximally stimulated (by 1,280 ng/ml insulin), adding rat IGF-II gave decreases in differentiation similar to those reported for other mitogens. Two trivial explanations have been eliminated: stimulation of differentiation (at low concentrations) is not due to enhanced survival or growth of the cells, and inhibition (at higher concentrations) is not a toxic effect. In L6 cells, epidermal growth factor and fibroblast growth factor had no effect on proliferation or differentiation. We conclude that the effects of medium components on myoblast differentiation cannot be generalized to indicate inhibition by all mitogens; depending on the cell lines and concentrations used, certain mitogens may either stimulate or inhibit differentiation.

Stimulation and inhibition of myoblast differentiation by hormones.

The growth and differentiation of L6 myoblasts are subject to control by two proteins secreted by cells of the Buffalo rat liver line. The first of these, rat insulinlike growth factor-II (formerly designated multiplication stimulating activity) is a potent stimulator of myoblast proliferation and differentiation, as well as associated processes such as amino acid uptake and incorporation into protein, RNA synthesis, and thymidine incorporation into DNA. In addition, this hormone causes a significant decrease in the rate of protein degradation. All of these actions seem to be attributable to a single molecular species, although their time courses and sensitivity to the hormone differ substantially. The second protein, the differentiation inhibitor (DI), is a nonmitogenic inhibitor of all tested aspects of myoblast differentiation, including fusion and the elevation of creatine kinase. Indirect immunofluorescence experiments demonstrated that DI also blocks accumulation of myosin heavy chain and myomesin. Upon removal of DI after 72 h incubation, all of these effects were reversed and normal myotubes containing the usual complement of muscle-specific proteins were formed. Thus, this system makes it possible to achieve specific stimulation or inhibition of muscle cell differentiation by addition of purified proteins to cloned cells in serum-free medium.