No abstract

In proliferating C2C12 myoblasts, serum and physiological concentrations of insulin and IGF-I stimulated protein synthesis and RNA accretion. After fusion, the multinucleated myotubes remained responsive to serum but not to insulin or IGF-I, even though both insulin and type-I IGF receptor mRNAs increased in abundance. Protein synthetic responses to insulin and IGF-I in myoblasts were not inhibited by dexamethasone, ibuprofen or Ro-31-8220, thus phospholipase A2, cyclo-oxygenase and protein kinase C did not appear to be involved in the signalling mechanisms. Neither apparently were polyphosphoinositide-specific phospholipase C or phospholipase D since neither hormone increased inositol phosphate, phosphatidic acid, choline or phosphatidylbutanol production. Only the phosphatidylinositol-3-kinase inhibitor, wortmannin, and the 70 kDa S6-kinase inhibitor, rapamycin, wholly or partially blocked the effects of insulin and IGF-I on protein synthesis. 2-deoxyglucose uptake remained responsive to insulin and IGF-I after fusion and was also inhibited by wortmannin. The results suggest that the loss of responsiveness after fusion is not due to loss of receptors, but to the uncoupling of a post-receptor pathway, occurring after the divergence of the glucose transport and protein synthesis signalling systems, and that, if wortmannin acts at a single site, this is prior to that point of divergence.

No abstract

The rates of [3H]N(tau)-methylhistidine (3-MH) accumulation in the medium, following pulse labelling of cells for 48 h with [3H]methionine, were used to measure myofibrillar protein degradation. In fused C2C12 myotubes, incubation for 24 or 48 h after the labelling period gave rates of myofibrillar degradation of 38 and 42%/day. In a leucine free medium, these rates were similar; 40 and 47%/day, respectively. Using identical conditions +/- leucine, but in the absence of [3H]-methionine, rates of protein accretion and synthesis over 24-48 h were measured. From these data, rates of total protein degradation were calculated by difference and were similar to myofibrillar degradation rates. We have used the same pulse labelling protocol to assess whether the method is applicable to non-muscle cell lines based on the knowledge that 3T3 fibroblasts contain actin in the cytoskeleton. 3-MH was detected both in protein and upon its release into the medium. Actin degradation measured over a 48 h period gave a value half that obtained for total degradation, but the results suggest that the release of 3-MH by fibroblasts in vivo could be appreciable. The development of this methodology should provide a useful tool to investigate signalling mechanisms regulating actin degradation in a variety of cell types.

The role of cyclic AMP as a second messenger in the stimulation of protein synthesis and the potential involvement of mitogen activated protein (MAP) kinase in this response was studied in L6 myoblasts. Dibutyryl-cAMP (dbt-cAMP) increased protein synthesis at 90 min and 6 h in a concentration-dependent manner. The responses at 90 min were probably mediated by increased translation as they were not blocked by actinomycin D; effects at 6 h were accompanied by increases in RNA content implying a transcriptional component. 100 nM 12-0-tetradecanoylphorbol-13-acetate (TPA), 1 nM Insulin (90 min incubations) and 100 nM vasopressin (6 h incubation) also increased protein synthesis and these responses were additive with those of 500 micron dbt-cAMP. Responses to forskolin were similar to dbt-cAMP whilst 1,9-dideoxyforskolin had no effect. Cell extracts immunoblotted with MAP kinase antibody showed bands corresponding to approx. 42, 44, 54 and 83 kDa. 500 micron dbt-cAMP elicited an increase in activity of both the 42 and 44 kDa bands when assayed by the 'in gel' method and a similar response was also observed with forskolin. TPA and vasopressin also stimulated the activity of these two isoforms, but had no significant additive or inhibitory effects when added in combination with 500 micron dbt-cAMP. In contrast, although 1 nM insulin alone had no effect, a synergistic response in terms of MAP kinase activation was observed in the presence of dbt-cAMP. The data demonstrate that cAMP stimulates protein synthesis in L6 cells and suggest a role for MAP kinase in this event.