Amanda Thom

Stimulation of myofibrillar protein degradation and expression of mRNA encoding the ubiquitin‐proteasome system in C2C12 myotubes by dexamethasone: effect of the proteasome inhibitor MG‐132

Addition of the synthetic glucocorticoid, dexamethasone (Dex) to serum‐deprived C2C12 myotubes elicited time‐ and concentration‐dependent changes in Nτ‐methylhistidine (3‐MH), a marker of myofibrillar protein degradation. Within 24 h, 100 nM Dex significantly decreased the cell content of 3‐MH and increased release into the medium. Both of these responses had increased in magnitude by 48 h and then declined toward basal values by 72 h. The increase in the release of 3‐MH closely paralleled its loss from the cell protein. Furthermore, Dex also decreased the 3‐MH:total cell protein ratio, suggesting that myofibrillar proteins were being preferentially degraded. Incubation of myotubes with the peptide aldehyde, MG‐132, an inhibitor of proteolysis by the (ATP)‐ubiquitin (Ub)‐dependent proteasome, prevented both the basal release of 3‐MH (>95%) and the increased release of 3‐MH into the medium in response to Dex (>95%). Northern hybridization studies demonstrated that Dex also elicited similar time‐ and concentration‐dependent increases in the expression of mRNA encoding two components (14 kDa E2 Ub‐conjugating enzyme and Ub) of the ATP‐Ub‐dependent pathway. The data demonstrate that Dex stimulates preferential hydrolysis of myofibrillar proteins in C2C12 myotubes and suggests that the ATP‐Ub‐dependent pathway is involved in this response. J. Cell. Physiol. 181:455–461, 1999.

Insulin and insulin-like growth factor-I responsiveness and signalling mechanisms in C2C12 satellite cells: effect of differentiation and fusion.

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.

Measurement of protein degradation by release of labelled 3-methylhistidine from skeletal muscle and non-muscle cells

The rates of [3H]Nτ‐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.

Growth and metabolism of fetal and maternal muscles of adolescent sheep on adequate or high feed intakes: possible role of protein kinase C-α in fetal muscle growth

From days 4-104 of pregnancy, adolescent sheep, weighing 43.7 (SE 0.87) kg were offered a complete diet at two different intakes (approximately 5 or 15 kg/week) designed to meet slightly, or well above, maternal maintenance requirements. The fetal and maternal muscles were taken on day 104 of pregnancy and analysed for total DNA, RNA and protein. Ewes offered a high intake to promote rapid maternal weight gain, weighed more (76.5 (SE 4.5) v 50.0 (SE 1.7) kg) and had muscles with a greater fresh weight, whilst their fetuses had smaller muscles, than those fed at a lower intake. Plantaris muscle of the ewes fed at the high intake contained more RNA and protein; again the opposite situation was found in the fetal muscle. On the higher maternal intakes, the DNA, RNA and protein contents of the fetal plantaris muscle were less than in fetuses of ewes fed at the lower intake. To investigate the possible mechanisms involved in this decrease in fetal muscle mass, cytosolic and membrane-associated muscle proteins were subjected to Western immunoblotting with antibodies to nine isoforms of protein kinase C (PKC), a family of enzymes known to play an important role in cell growth. Five PKC isoforms (alpha, epsilon, theta, mu, zeta) were identified in fetal muscle. One of these, PKC-alpha was located predominantly in the cytosolic compartment in the smaller fetuses of the ewes fed at a high plane of nutrition, but was present to a greater extent in the membranes of the more rapidly growing fetuses of the ewes fed at the lower intake. This was the only isoform to demonstrate nutritionally related changes in it subcellular compartmentation suggesting that it may mediate some aspects of the change in fetal growth rate.

Evidence that Protein Kinase C and Mitogen Activated Protein Kinase are not Involved in the Mechanism by which Insulin Stimulates Translation in L6 Myoblasts

Insulin stimulated a concentration-dependent increase in protein synthesis in L6 myoblasts which was significant at 1 nM. This response was not prevented by the transcription inhibitor, actinomycin D. The protein kinase C (PKC) inhibitor, Ro-31-8220, and downregulation of PKC by prolonged incubation of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), had no effect on the ability of insulin to stimulate protein synthesis whilst completely blocking the response to TPA. In contrast, insulin failed to enhance protein synthesis significantly in the presence of either ibuprofen, a selective cyclooxygenase inhibitor or rapamycin, an inhibitor of the 70 kDa S6 kinase. When cell extracts were prepared and assayed for total myelin basic protein kinase activity, a stimulatory effect of insulin was not observed until the concentration approached 100-fold (i.e. 100 nM) that required to elicit increases in protein synthesis. Upon fractionation on a Mono-Q column, 100 nM insulin increased the activity of 3 peaks which phosphorylated myelin basic protein. Two of these peaks were identified as the 42 and 44 kDa forms of Mitogen Activated Protein (MAP) kinase by immunoblotting. In contrast, 1 nM insulin had no effect on the activity of these peaks. The data suggest that physiologically relevant concentrations of insulin do not stimulate translation in L6 cells through either PKC or the 42/44 kDa isoforms of MAP kinase and that this response is, at least in part, mediated through the activation of the 70 kDa S6 kinase by cyclooxygenase metabolites.

Translocation of protein kinase C isoforms in rat muscle in response to fasting and refeeding

Weanling rats were offered food ad libitum, or fasted for 18 h, or fasted and refed for times ranging from 5 to 30 min. Five protein kinase C (PKC) isoforms (alpha, epsilon, zeta, theta and mu) were detected in the hindlimb muscles by Western immunoblotting. PKC forms epsilon and theta were abundant in plantaris, but not in soleus muscle, and no difference in localization was detected between fed rats and those fasted for 18 h. PKC forms alpha and mu were affected by fasting and refeeding. PKC-mu was found only in the cytosolic fraction of the plantaris muscle of the fasted animal, but in the fully-fed animals it was also associated with the membrane fraction. The pattern of localization observed in the fully-fed state was restored in the fasted rats by 20 min refeeding. In contrast, PKC-alpha was not detected in the cytosolic fraction of the plantaris in fasted animals but rapidly reappeared there on refeeding, being restored to 20% and 80% of the fed value within 5 and 30 min of refeeding respectively. The timing of these changes was correlated with the increase in serum insulin concentration, which was significantly elevated above the fasted value by 5 min and at subsequent times. These data suggest a possible role for PKC isoforms alpha and mu in the metabolic changes that occur in skeletal muscle on transition between the fasted and the fed state.