Protein Degradation In Myotubes Research Articles

Contribution of the autophagy-lysosomal and ubiquitin-proteasomal proteolytic systems to total proteolysis in rainbow trout (Oncorhynchus mykiss) myotubes.

The ubiquitin-proteasome system (UPS) is recognized as the major contributor to total proteolysis in mammalian skeletal muscle, responsible for 50% or more of total protein degradation in skeletal muscle, whereas the autophagic-lysosome system (ALS) plays a more minor role. While the relative contribution of these systems to muscle loss is well documented in mammals, little is known in fish species. The current study uses myotubes derived from rainbow trout myogenic precursor cells as an in vitro model of white muscle tissue. Cells were incubated in complete or serum-deprived media or media supplemented with insulin-like growth factor-1 (IGF-1) and exposed to selective proteolytic inhibitors to determine the relative contribution of the ALS and UPS to total protein degradation in myotubes in different culture conditions. Results indicate that the ALS is responsible for 30-34% and 50% of total protein degradation in myotubes in complete and serum-deprived media, respectively. The UPS appears to contribute much less to total protein degradation at almost 4% in cells in complete media to nearly 17% in serum-deprived cells. IGF-1 decreases activity of both systems, as it inhibited the upregulation of both proteolytic systems induced by serum deprivation. The combined inhibition of both the ALS and UPS reduced degradation by a maximum of 55% in serum-deprived cells, suggesting an important contribution of other proteolytic systems to total protein degradation. Collectively, these data identify the ALS as a potential target for strategies aimed at improving muscle protein retention and fillet yield through reductions in protein degradation.

Docosahexaenoic acid antagonizes palmitate‐induced protein degradation in myotubes

Muscle atrophy is a debilitating consequence of many chronic diseases, some of which are characterized by accumulation of lipids in skeletal muscle. High levels of saturated fatty acids (i.e. palmitic acid, PA) cause dysregulated protein metabolism in cultured myotubes, whereas the omega‐3 fatty acid docosahexaenoic acid (DHA) has beneficial metabolic effects. We hypothesized that DHA prevents PA‐induced muscle atrophy by counteracting its effects on protein degradation. To test our hypothesis, we studied C2C12 myotubes treated with 500uM PA and/or 100uM DHA. PA significantly increases the rate of protein degradation, and the response is reversed by co‐treatment with DHA. The protein kinase Akt is a key regulator of protein balance and inhibits the FoxO3 transcription factor. PA reduces the ratio of phospho‐to‐total Akt and increases the mRNA and total and nuclear protein levels of FoxO3. It also increases the levels of mRNA encoding three FoxO3 targets, the MuRF‐1 and atrogin‐1/MAFbx E3 ubiquitin ligases and the autophagy mediator Bnip3. DHA attenuates the effects of PA on Akt, FoxO3, and all three atrogenes mRNAs. These data indicate that PA induces protein degradation by inducing the ubiquitin‐proteasome and autophagic proteolytic systems and that DHA counters the catabolic effects of PA by improving Akt signaling.Support NIH DK95610, AHA GRNT 7660020

Growth hormone stimulates protein synthesis in bovine skeletal muscle cells without altering insulin-like growth factor-I mRNA expression1

Growth hormone is a major stimulator of skeletal muscle growth in animals, including cattle. In this study, we determined whether GH stimulates skeletal muscle growth in cattle by direct stimulation of proliferation or fusion of myoblasts, by direct stimulation of protein synthesis, or by direct inhibition of protein degradation in myotubes. We also determined whether these direct effects of GH are mediated by IGF-I produced by myoblasts or myotubes. Satellite cells were isolated from cattle skeletal muscle and were allowed to proliferate as myoblasts or induced to fuse into myotubes in culture. Growth hormone at 10 and 100 ng/mL increased protein synthesis in myotubes (P < 0.05), but had no effect on protein degradation in myotubes or proliferation of myoblasts (P > 0.05). Insulin-like growth factor-I at 50 and 500 ng/mL stimulated protein synthesis (P < 0.01), and this effect of IGF-I was much greater than that of GH (P < 0.05). Besides stimulating protein synthesis, IGF-I at 50 and 500 ng/mL also inhibited protein degradation in myotubes (P < 0.01), and IGF-I at 500 ng/mL stimulated proliferation of myoblasts (P < 0.05). Neither GH nor IGF-I had effects on fusion of myoblasts into myotubes (P > 0.1). These data indicate that GH and IGF-I have largely different direct effects on bovine muscle cells. Growth hormone at 10 and 100 ng/mL had no effect on IGF-I mRNA expression in either myoblasts or myotubes (P > 0.1). This lack of effect was not because the cultured myoblasts or myotubes were not responsive to GH; GH receptor mRNA was detectable in them and the expression of the cytokine-inducible SH2-containing protein (CISH) gene, a well-established GH target gene, was increased by GH in bovine myoblasts (P < 0.05). Overall, the data suggest that GH stimulates skeletal muscle growth in cattle in part through stimulation of protein synthesis in the muscle and that this stimulation is not mediated through increased IGF-I mRNA expression in the muscle.

Role of the dsRNA-dependent protein kinase (PKR) in the attenuation of protein loss from muscle by insulin and insulin-like growth factor-I (IGF-I)

Proteolysis-inducing factor (PIF), a tumour-produced cachectic factor, induced a dose-dependent decrease in protein synthesis in murine myotubes, together with an increase in phosphorylation of eucaryotic initiation factor 2 (eIF2) on the alpha-subunit. Both insulin (1 nM) and insulin-like growth factor I (IGF-I) (13.2 nM) attenuated the depression of protein synthesis by PIF and the increased phosphorylation of eIF2alpha, by inhibiting the activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) by induction of protein phosphatase 1. A low-molecular weight inhibitor of PKR also reversed the depression of protein synthesis by PIF to the same extent, as did insulin and IGF-I. Both insulin and IGF-I-stimulated protein synthesis in the presence of PIF, and this was attenuated by Salubrinal, an inhibitor of phospho eIF2alpha phosphatase, suggesting that at least part of this action was due to their ability to inhibit phosphorylation of eIF2alpha. Both insulin and IGF-I also attenuated the induction of protein degradation in myotubes induced by PIF, this effect was also attenuated by Salubrinal. These results suggest an alternative mechanism involving PKR to explain the effect of insulin and IGF-I on protein synthesis and degradation in skeletal muscle in the presence of catabolic factors.

Angiotensin II directly induces muscle protein catabolism through the ubiquitin–proteasome proteolytic pathway and may play a role in cancer cachexia

The ability of angiotensin I (Ang I) and II (Ang II) to induce directly protein degradation in skeletal muscle has been studied in murine myotubes. Angiotensin I stimulated protein degradation with a parabolic dose–response curve and with a maximal effect between 0.05 and 0.1 μM. The effect was attenuated by coincubation with the angiotensin-converting enzyme (ACE) inhibitor imidaprilat, suggesting that angiotensin I stimulated protein degradation through conversion to Ang II. Angiotensin II also stimulated protein breakdown with a similar dose–response curve, and with a maximal effect between 1 and 2.5 μM. Total protein degradation, induced by both Ang I and Ang II, was attenuated by the proteasome inhibitors lactacystin (5 μM) and MG132 (10 μM), suggesting that the effect was mediated through upregulation of the ubiquitin–proteasome proteolytic pathway. Both Ang I and Ang II stimulated an increased proteasome ‘chymotrypsin-like’ enzyme activity as well as an increase in protein expression of 20S proteasome α-subunits, the 19S subunits MSS1 and p42, at the same concentrations as those inducing protein degradation. The effect of Ang I was attenuated by imidaprilat, confirming that it arose from conversion to Ang II. These results suggest that Ang II stimulates protein degradation in myotubes through induction of the ubiquitin–proteasome pathway. Protein degradation induced by Ang II was inhibited by insulin-like growth factor and by the polyunsaturated fatty acid, eicosapentaenoic acid. These results suggest that Ang II has the potential to cause muscle atrophy through an increase in protein degradation. The highly lipophilic ACE inhibitor imidapril (Vitor™) (30 mg kg−1) attenuated the development of weight loss in mice bearing the MAC16 tumour, suggesting that Ang II may play a role in the development of cachexia in this model.

Pathways of protein degradation in L6 myotubes.

To assess the importance of different proteolytic pathways in anabolic and catabolic states, L6 myotubes were labeled with 3H-phenylalanine for 24 hr, then exposed for another 24 hr to control medium, alone or with one of the following: insulin (1 microgram/ml), insulin-like growth factor-I (IGF-I, 100 ng/ml), clenbuterol (100 microM), or dexamethasone (1 microM). Release of acid-soluble radioactivity and 3-methylhistidine were used to determine rates of total and myofibrillar protein degradation, respectively. Within each hormone treatment, cells were also exposed to none, one, or both of the following protease inhibitors: chloroquine (25 microM); E-64d (100 microM). Chloroquine inhibits lysosomal proteases, and E-64d is a cell-penetrating inhibitor of cysteine proteases (including the calpains). Total protein degradation was reduced by insulin (-33%) and IGF-I (-40%), increased by clenbuterol (+20%), and was unaffected by dexamethasone. Myofibrillar protein degradation was reduced by insulin (-35%), IGF-I (-55%), and clenbuterol (-22%), and was unaffected by dexamethasone. Therefore, total (mainly sarcoplasmic) and myofibrillar protein degradation were regulated independently of one another. The influence of the inhibitors was not consistent across hormone treatments. Chloroquine inhibited total but not myofibrillar proteolysis, indicating that lysosomes may be rate limiting in the former but not the latter. In contrast, E-64d had no effect on total protein degradation, but increased myofibrillar proteolysis. Interactions between anabolic and catabolic hormones and these two protease inhibitors indicate that both lysosomal and nonlysosomal proteolytic pathways are involved in regulation of total protein degradation in myotubes. In contrast, lysosomes are probably not involved in regulating the rate of myofibrillar protein degradation, the initial step likely being due to calpain activity.

Effects of dexamethasone and anabolic agents on proliferation and protein synthesis and degradation in C2C12 myogenic cells.

The objective of this experiment was to determine the dose response of dexamethasone (DEX) on C2C12 myogenic cells and to examine the effects of the anabolic compounds estradiol (E), testosterone (T), and dihydrotestosterone (D) alone and in combination with DEX on proliferation and protein turnover in cultured C2C12 myogenic cells. In the first study, cells were treated with seven concentrations (0, 25, 50, 75, 100, 150, or 200 nM) of DEX in medium with or without 5% horse serum (HS) for the determination of protein synthesis and degradation, and six concentrations (0, 50, 100, 150, 200, or 250 nM) of DEX in medium with 5% fetal bovine serum for cell proliferation measurements. Proliferation of myoblasts decreased (P < .05) with DEX. As DEX concentration increased, protein degradation in myotubes increased (P < .05) up to 100 nM, then declined. Protein synthesis decreased linearly (P < .01) as DEX concentration increased. The presence of HS in the medium decreased (P < .01) protein degradation by 32% as compared with no HS and increased (P < .05) protein synthesis. In the second study, cells were treated with E, T, or D at four concentrations (0, 100, 500, or 1,000 nM) in medium containing 0 or 100 nM DEX. Cells were assayed for protein synthesis or protein degradation. Synthesis decreased (P < .01) and degradation increased (P < .01) with DEX. No differences (P > .05) were found between E, T, or D hormone treatments or concentrations. To measure proliferation, myoblasts were treated 1 d after plating with the same anabolic hormone treatments in medium containing 0 to 100 nM DEX. Cells were grown to confluence and assayed for proliferation. Proliferation decreased (P < .01) in the presence of DEX in each treatment compared with controls. Cells treated with E had significantly lower (P < .05) proliferation rates than cells treated with T and D. The presence of concentrations of DEX at 100 nM inhibited proliferation and protein synthesis and increased protein degradation. Anabolic agents at pharmacological doses do not inhibit the DEX effects on C2C12 myogenic cells, nor do they directly affect proliferation or protein turnover.