Muscle protein degradation

In acute OPP the cardinal event that leads to muscle weakness is the inhibition of AChE at the neuromuscular junction. OP inhibition of neuronal and neuromuscular junction AChE are a consequence of the amount of blood borne OP reaching these sites after detoxification and removal by binding to plasma proteins such as albumin and carboxylesterases. OP inhibition of neuronal and neuromuscular junction AChE cannot be determined in humans as the tissues are not available for testing. In humans , OPP is determined by inhibition of plasma BuChE and erythrocyte AChE, which reflect blood levels of the pesticide. BuChE is a serine esterase that hydrolyses butyrylcholine with greater affinity than acetylcholine. The enzyme is highly susceptible to inhibition by organophosphates although the inhibition does not induce pathology. The easy accessibility of serum for testing in the poisoned patient make s BuChE it the preferred diagnostic marker of OPP. Erythrocyte AChE is considered to reflect the neuronal enzyme in kinetics of OP inhibition and reactivation although not necessarily the extent of inhibition. To understand muscle weakness pertaining to the course and outcome of OPP there is need to study muscle AChE.

Treatment of hyperlipidemia with clofibrate may result in development of a muscular syndrome. Our previous investigation (1979. J. Clin. Invest.64: 405.) showed that chronic administration of clofibrate to rats causes myotonia and decreases glucose and fatty acid oxidation and total protein of skeletal muscle. In the present experiments we have

Metabolic acidosis often leads to loss of body protein due mainly to accelerated protein breakdown in muscle. To identify which proteolytic pathway is activated, we measured protein degradation in incubated epitrochlearis muscles from acidotic (NH4Cl-treated) and pair-fed rats under conditions that block different proteolytic systems. Inhibiting lysosomal and calcium-activated proteases did not reduce the acidosis-induced increase in muscle proteolysis. However, when ATP production was also blocked, proteolysis fell to the same low level in muscles of acidotic and control rats. Acidosis, therefore, stimulates selectively an ATP-dependent, nonlysosomal, proteolytic process. We also examined whether the activated pathway involves ubiquitin and proteasomes (multicatalytic proteinases). Acidosis was associated with a 2.5- to 4-fold increase in ubiquitin mRNA in muscle. There was no increase in muscle heat shock protein 70 mRNA or in kidney ubiquitin mRNA, suggesting specificity of the response. Ubiquitin mRNA in muscle returned to control levels within 24 h after cessation of acidosis. mRNA for subunits of the proteasome (C2 and C3) in muscle were also increased 4-fold and 2.5-fold, respectively, with acidosis; mRNA for cathepsin B did not change. These results are consistent with, but do not prove that acidosis stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent, proteolytic pathway.

We found that insulinopenia induced by STZ causes substan- tial loss of body weight and the mass of muscle, liver, and adi- pose tissue. Others have provided evidence that the loss of muscle mass with insulinopenia is related to accelerated pro- tein degradation. For example, urinary 3-methylhistidine ex- cretion is increased in diabetic patients (4) and rats (3) and the rate of protein degradation is increased in the perfused hind- quarter muscles of insulinopenic rats (5). The latter experi- ments were performed in rats the first day after injection with 65 mg/kg STZ, but accelerated muscle proteolysis was not present at 3, 7, or 28 d after the same dose of STZ, nor was the proteolytic pathway stimulated by insulinopenia identified (32). When rats were given a higher dose of STZ (125 mg/kg), muscle protein degradation increased but it was concluded that this response was due in large part to decreased food in- take because control rats given the same amount of food also had increased muscle protein degradation (32). In the present studies, we controlled for this factor by using a pair-feeding protocol. Moreover, the food intake was sufficient to support growth in pair-fed, control rats (Fig. 1).

balance of protein turnover. During a prolonged period with no food consumption, protein synthesis decreases by 15 to 30%, resulting in a net catabolic period. That period of catabolism continues until adequate energy and amino acids are ingested to stimulate protein synthesis (Norton & Layman, 2006). Both exercise and nutrition have been found to strongly influence net muscle protein balance (Alvestrand et al., 1990; Morgan et al., 1971; Rannels et al., 1974; Tipton et al., 2009). Protein metabolism in skeletal muscle tissue is highly responsive to nutrient intake in healthy individuals and in addition to food ingestion, exercise can effectively alter protein turnover, as it stimulates both protein synthesis and protein breakdown (Biolo et al., 1995; Koopman et al., 2006). Protein gains occur only as a result of positive net muscle protein over a given time period. Positive net muscle protein balance results either from increased muscle protein synthesis and/or decreased muscle protein degradation. For hypertrophy of muscle to take place, muscle protein synthesis (MPS) must surpass muscle protein catabolism (Coburn et al., 2006). Consumption of amino acids through protein ingestion or intravenous injection is essential in accentuating post-exercise muscle protein synthesis. Contractile activity and availability of nutrients are strong modulators of exercise-induced adaptive responses (Moore et al., 2009; Wilkinson et al., 2008). Furthermore, lack of nutrient provision in timely proximity to high-intensity exercise bouts is possibly detrimental for maintenance or promotion of muscle mass (Coffey et al., 2011).

hindquarters. This injury increased muscle protein degradation (PD) from 140 +/- 5 to 225 +/- 5 nmol tyrosine/g per h, but did not alter protein synthesis. Muscle lactate release was increased greater than 70%, even though plasma catecholamines and muscle cyclic AMP were not increased. Insulin dose-response studies revealed that the burn decreased the responsiveness of muscle glycogen synthesis to insulin but did not alter its sensitivity to insulin. Rates of net glycolysis and glucose oxidation were increased and substrate cycling of fructose-6-phosphate was decreased at all levels of insulin. The burn-induced increase in protein and glucose catabolism was not mediated by adrenal hormones, since they

sis are not uncommon in CRF (3). Most studies in rats with ex- perimental CRF demonstrate enhanced net skeletal muscle protein degradation, i.e., the sum of both protein synthetic and degradative rates; both increased protein degradation and/or reduced protein synthesis have been described (4, 5). Re- ported causes for enhanced catabolism or reduced synthesis of skeletal muscle protein in CRF include acidemia, elevated glu- cocorticoids, and insulin resistance (for review see reference 6). No studies have examined whether there is resistance to the actions of insulin-like growth factor 1 (IGF-1) in skeletal muscle. IGF-1 is an anabolic hormone that has about 50% struc- tural homology with proinsulin (7). IGF-1 is synthesized in skeletal muscle as well as other tissues and acts on skeletal muscle primarily through the type 1 IGF receptor (IGF-1R). Like insulin, IGF-1 acts on skeletal muscle to stimulate cellular uptake of glucose and amino acids (8, 9) and enhance protein synthesis and suppress protein degradation (10). IGF-1 is nec- essary for skeletal muscle growth (11), and individuals with growth hormone receptor deficiency who also have low IGF-1 levels (i.e., Laron’s dwarfism) have markedly decreased stat- ure with essentially a proportionate reduction in skeletal mus- cle mass (12). IGF-1 may act in tissues in an autocrine or para- crine fashion, but may also function as an endocrine hormone (11). Several reports describe circulating inhibitors to IGF-1 (formerly referred to as somatomedin C) in patients with CRF (13, 14). Elevated levels of IGF binding proteins (IGFBPs), IGFBP fragments and small hemodialyzable compounds have been implicated as inhibitors of IGF-1 (14). Because of the po- tentially anabolic role of IGF-1 in skeletal muscle and other tissues, a number of investigators have begun to give re- combinant growth hormone or recombinant human IGF-1 (rhIGF-1) to patients with CRF (15, 16). The fact that there is no information on the actions of IGF-1 in skeletal muscle in CRF led us to investigate the effects of this hormone on pro- tein turnover in this organ. We first evaluated the dose- response relationship between rhIGF-1 and protein synthesis and degradation in skeletal muscle in CRF rats and sham- operated (SO), pair-fed rats. It was found that there was a sup- pressed skeletal muscle response to the anabolic effects of rhIGF-1 on both protein synthesis and degradation in CRF rats. Additional studies examined the potential mechanisms of this inhibition. The results indicate that a postreceptor defect Address correspondence to Joel D. Kopple, M.D., Division of Neph-

nonacidotic animals were given 3 micrograms/100 g of body weight dexamethasone twice a day, muscle protein degradation was increased if the muscles were simply incubated in acidified media. We conclude that chronic metabolic acidosis depresses nitrogen utilization and increases glucocorticoid production. The combination of increased glucocorticoids and acidosis stimulates muscle proteolysis but does not affect protein synthesis. These changes in […]

In this study, 2 wk of hrGH treatment increased the rate of muscle protein degradation, assessed from 3-MH excretion, by an average of 16% across all the groups, with the largest stimu- lation, 2868%, in the control and HIV1 groups, and much smaller, statistically nonsignificant changes in the AIDS groups. The increase in protein degradation in the control and HIV 1 groups would tend to offset the stimulation in protein synthesis observed in these individuals (Table III), so that the accumula- tion of muscle protein would be less than that suggested by the enhancement of synthesis alone. Correspondingly, the cata- bolic effect on muscle protein synthesis in the AIDS groups (Table III) would also tend to be moderated by the fact that protein degradation was not enhanced by hrGH in these indi- viduals. Concurrent changes in protein synthesis and degrada- tion have been observed in other anabolic conditions such as those that accompany normal growth (50) or increased muscle mass brought about by resistance training (51) when both synthesis and degradation of muscle are stimulated, with the increase in protein synthesis exceeding the increase in degra- dation. Although a direct effect of hrGH on muscle protein degradation has not been reported, Fryburg et al. (52) have demonstrated an enhancement in muscle protein degradation by hrGH through an inhibition of insulin’s suppressive effect on muscle proteolysis.

The continual degradation and synthesis of protein, i.e., protein turnover, is crucial for homeostatic functions of normal cells [45]. Studies show that the ubiquitin- proteasome system plays a major role in the regulation of muscle protein degradation [29]. The abundance of the muscle-specific ubiquitin protein ligases (E3), atrogin- 1/MAFbx and MuRF1, is crucial for skeletal muscle degradation in catabolic states [46]. The target protein substrates of atrogin-1 include MyoD, a transcriptional regulator which controls muscle size [46]. MuRF1 pre- fers structural protein such as titin and myosin light chain-1 (MLC1) as target proteins [46]. Taken together, these ligases regulate the substrate targets that play an important role in skeletal muscle growth. In this study, we determined the abundance of these ubiquitin ligases. We found that only atrogin-1 was affected by age. Al- though these results are consistent with the recent results of Orellana et al. [47], the differential response of the two ligases seems inconsistent with their functions as major players of protein degradation. The finding that these ligases are differentially expressed in certain experimental conditions is not uncommon. Frost et al. [48] found that the sepsis-induced increase in atrogin-1 mRNA expres- sion, but not MuRF1, was completely blocked by IGF-I. Other studies show that the atrogin-1 mRNA expression, but not MuRF1, is increase by interleukin 6 (IL6) [49], and angiotensin II (ANG II) [50]. Conversely, the skeletal muscle MuRF1 mRNA expression, but not atrogin-1, is

As concerns the possible physiological mechanisms, the postprandial inhibition of forearm protein degradation might have been mediated by hyperinsulinemia, by hyperaminoaci- demia, or by other factors. Insulin was shown to inhibit either forearm or leg proteolysis in some studies (15, 16, 18) but not in others (17, 19, 46). These discrepancies could arise because the insulin inhibitory effect might become evident, provided that arterial amino acid concentrations are not concurrently decreased (15, 16, 18), as it occurs after a mixed meal. The mo- lecular mechanism(s) for the insulin effect are however still unclear, since insulin was shown to suppress mainly lysosomal proteolysis, which, in turn, is not the predominant pathway of protein degradation in muscle (47, 48). Hyperaminoacidemia was shown to inhibit muscle protein degradation in muscle, both in vitro (47, 48) and in vivo in man (26), although a recent study in pigs did not confirm this finding (49). The possible mechanisms for amino acid-induced suppression of proteolysis are discussed in detail (48). They may involve inhibition either of macroautophagy of nonmyofibrillar proteins, or of break- down of nonlysosomal myofibrillar proteins. The branched chain amino acid leucine may be important in the suppression of muscle proteolysis, either directly or through its transamina- tion product, a-ketoisocaproate [KIC] (48). Leucine (and other branched-chain amino acid) concentrations exhibited the largest increments after the meal (Table II). Interestingly, however, KIC concentrations did not increase (Table IV), con- sistently with other reports (3, 4, 8, 9, 11). On the whole, it is likely that both insulin and amino acids cooperated in the meal- induced inhibition of forearm muscle proteolysis observed in

The duration of hypercaloric feeding seems to have an effect on protein metabolism. Long-term (5 month) high-fat feeding of C57BL/6 mice [11] or life-long gen- etic mutation of the leptin receptor ( db/db ) [12] leads to severe obesity, increased basal rate of muscle protein degradation, and loss of skeletal muscle mass. In both these models, blood glucose and insulin concentrations are profoundly elevated demonstrating that the mice are overtly diabetic, a condition known to induce muscle at- rophy [13]. Conversely, mice fed a high-fat diet for a shorter duration (2 months or less) have no apparent de- fects in basal muscle protein metabolism [14], but dem- onstrate whole-body insulin resistance and impaired muscle mitochondria function [15, 16]. High-fat fed mice also have an impaired ability to increase translation of muscle proteins during load-induced hypertrophy [17] and do not have the typical increased rate of muscle protein synthesis in response to a meal [14] demonstrat- ing that the ability to regulate protein synthesis in re- sponse to external cues is impaired. However, whether a short-term high fat diet affects the response of muscle to atrophy signals is unknown.

In addition to fi nding that autophagic protein degradation is the type of protein degradation most commonly triggered in response to knockdown of any individual kinase or phos- phatase, we have found that functional MPK-1 is very fre- quently required for the protein degradation that is triggered in response to knockdown of any individual kinase 20 or phosphatase (Figures 3 and 4). Thus, analysis of both the kinome and phosphatome suggests a central role of MPK-1 in modulating muscle protein degradation in re- sponse to phosphorylation events. This observation, like the observation of both increased and decreased phosphoryla- tion events being associated with increased autophagy, sug- gests that perhaps a central integrator of multiple favourable growth conditions exists. Our connectivity analysis of the kinome and phosphatome with respect to protein deg- radation suggests that LET-92 is a central node and that it ap- pears to be a modulator of muscle protein degradation with knockdown producing mpk-1-dependent autophagic degra- dation. These results, coupled with the fact that ERK is known to be expressed and active in human skeletal muscle, 34 raise the question of if Raf-MAPK is a central modulator of autoph- agic degradation, with a signi fi cant number of kinases and phosphatases providing modulatory signals for this central pathway. This also raises the question of if Raf-MAPK is not just a central player in controlling protein synthesis but also of autophagy, perhaps acting to either modulate or comple- ment a similar role of mTor. Thus, our results from C. elegans open the door to further mechanistic studies of the regula- tion of human muscle metabolism.

Several possible mechanisms may result in insulin reg- ulation of Ub-proteasome activity. Several animal experiments and clinical evidence suggest that in dia- betes, the PI3K-Akt pathway plays a key role in inhibit- ing the activity of the Ub system [29-31]. However, whether PI3K-Akt has the same effect under sepsis is not yet known. Our preliminary experiments showed that after administering LY294002, an inhibitor of the PI3K-Akt pathway, the inhibiting effect of insulin was clearly decreased. Insulin resistance is common in septic conditions, resulting in a relative lack of insulin in vivo [29,32,33]. Hu et al. [14] found that insulin deficiency activated the Ub-proteasome system, resulting in cardiac muscle protein catabolism in diabetes mellitus. They also found that insulin resistance accelerates muscle protein degradation by activation of the Ub-proteasome [34]. Other studies suggested administration of insulin significantly reduced Ub mRNA [14-16]. However in our study, the relationship between insulin resistance and activation of Ub system were not confirmed. Our preliminary results show that insulin significantly inhib- ited the release of inflammatory cytokines such as TNF- a , IL-1 and IL-6 in septic patients [35]. These inflamma- tory cytokines are key factors for activity of the Ub-pro- teasome system [36]. Thus, insulin may inhibit the activity of the Ub system by inhibiting inflammatory cytokines. However, the correlation between insulin, cytokines and the Ub system remains to be further investigated. Another possibility is that insulin may inhi- bit the proteasome through an associated protein, IDE. The catalytic properties of the proteasome can vary widely, depending on its association with regulatory pro- teins [37]. Previous studies showed that insulin inhibits the proteasome in vitro and in cultured cells. Removal of IDE from the extracts or introduction of a neutraliz- ing antibody into cells results in a loss of insulin regula- tion of the proteasome [38,39].

Chronic renal failure (CRF) is associated with metabolic acidosis and abnormal muscle protein metabolism. As we have shown that acidosis by itself stimulates muscle protein degradation by a glucocorticoid-dependent mechanism, we assessed the contribution of acidosis to changes in muscle protein turnover in CRF. A stable model of uremia was achieved in partially nephrectomized rats (plasma urea nitrogen, 100-120 mg/dl, blood bicarbonate less than 21 meq/liter). CRF rats excreted 22% more nitrogen than pair-fed controls (P less than 0.005), so muscle protein synthesis and degradation were measured in perfused hindquarters. CRF rats had a 90% increase in net protein degradation (P less than 0.001); this was corrected by dietary bicarbonate. Correction of acidosis did not reduce the elevated corticosterone excretion rate of CRF rats, nor did it improve a second defect in muscle protein turnover, a 34% lower rate of insulin-stimulated protein synthesis. Thus, abnormal nitrogen production in CRF is due to accelerated muscle proteolysis caused by acidosis and an acidosis-independent inhibition of insulin-stimulated muscle protein synthesis.

It is not surprising to find that many, if not all, viruses have evolved strategies to impede host IFN responses (15). Evolu- tion of enhanced IFN resistance can lead to highly infectious viruses and/or persistent infections (4, 11, 13, 14, 27, 47). Re- cently, the IFN antagonist strategies used by some negative- stranded RNA viruses have been determined to act directly on the ISGF3 STAT protein subunits. The paramyxovirus simian virus 5 (SV5) was found to evade IFN responses by specifically targeting the STAT1 protein for proteolytic degradation. This destruction of STAT1 was found to be mediated by expression of a single virus-encoded protein called V (11, 12, 54). Human parainfluenza virus 2 (HPIV2) blocks IFN signaling by prefer- entially inducing degradation of STAT2 and not STAT1 (40, 55). In common with SV5, the expression of the HPIV2 V protein from a cDNA clone is sufficient to abolish IFN-respon- sive transcription as a result of STAT2 destabilization (40). These two paramyxovirus V proteins have ⬃50% amino acid sequence identity in their ⬃220-amino-acid length, yet they specifically recognize and catalyze the destruction of only one of the two IFN-responsive STAT proteins.

Considering that C-EGFP protein expressed at a low level and decreased over time, we assume that C-EGFP protein might be degraded after expression. Since degradation of intracellular proteins are generally mediated by 26S prote- asome and autophagolysosome, we added inhibitors, MG132 and 3-MA, of them to the cell medium, respect- ively, after plasmid transfection. MG132 is commonly used in experiments about degradation of proteins by 26S proteasome. Results showed that MG132 increased pro- tein level of cleaved C-EGFP in PK-15 and 3-MA had no effect on protein level of cleaved C-EGFP (Fig. 2a). To examine whether the 87 residues at N-terminal were af- fected by 26S proteasome or not, plasmid pEGFP-C1-C encoding EGFP tagged C protein at N-terminal, EGFP-C (Fig. 2b), was constructed and transfected in PK-15 cells. EGFP-C protein could be detected and MG132 up- regulated its level (Fig. 2b), showing that the 87 residues at N-terminal may be less affected by 26S proteasome than the 12 residues at C-terminal. Plasmids pEGFP-N1-C and pEGFP-N1 were transfected in PK-15 cells, respectively. Western blot results showed that cleaved form of C-EGFP protein expressed at a relatively low level compared with EGFP protein (Fig. 2c). Transcription levels of EGFP and C were analysed by qRT-PCR and the result showed that MG132 down-regulated average mRNA level of both EGFP (Fig. 2c-d) compared with the untreated group, though the impact of MG132 on mRNA level of C was not significant. Although MG132 has opposite functions on mRNA and protein level of C protein, the conclusion that MG132 up-regulated protein level of C protein could also be obtained. Similar results were observed in 3D4/2 cells, that MG132 up-regulated the level of C-EGFP

ApoE is important for the production of infectious HCV particles. Thus, the observation that ApoE was degraded by HCV-induced autophagy was curious. To address this question, we studied the possible role of autophagy in the interaction between ApoE and the HCV E2 envelope protein. It had previously been demon- strated that ApoE colocalized and interacted with HCV E2 in the perinuclear region (30, 42). We were able to confirm that previous finding and further demonstrated an important role of autophagy in the transport of ApoE to the perinuclear region, as impairing autophagy by suppressing the expression of ATG7 dispersed ApoE from the perinuclear region and reduced its colocalization with E2 (Fig. 5). This suppression of ATG7 expression did not reduce the HCV RNA level or significantly affect mature HCV particles in cells, but it reduced the HCV titer released from cells (Fig. 6). In contrast, the suppression of the late stage of autophagy using BafA1 increased the level of ApoE in the perinuclear region and enhanced its colocaliza- tion with HCV E2 (Fig. 7A). BafA1 also increased both intracellular and extracellular HCV titers (Fig. 7C and D). As BafA1 only slightly increased the amount of HCV RNA released from HCV-infected cells (Fig. 7E), it apparently primarily increased the infectivity of HCV particles. These results indicate that the efficient transport of ApoE to the perinuclear region to interact with E2 required autophagy, which then enhanced the production of infectious HCV particles. A model of the role of autophagy in the trafficking of ApoE is illustrated in Fig. 8. As shown in the figure, ApoE is localized to the ER in hepatocytes and can be transported by autophago- somes to lysosomes for degradation. Upon HCV infection, the association of ApoE with autophagosomes is enhanced by HCV-induced autophagy. This leads to its enhanced transport to the perinuclear region, which may be the ER or Golgi compartment, as previously suggested (30, 46), to interact with the HCV E2 protein and promotes the production of infectious viral particles. As autophagosomes induced by HCV also fuse with lysosomes in the later stage of HCV infection, part of ApoE is also degraded by autophagy during this trafficking process. In this model, the suppression of ATG7 expression inhibits the formation of autophagosomes, resulting in the retention of ApoE in the ER. This prevents ApoE from being delivered to the HCV assembly site and reduces the infectivity of released HCV. In contrast, the suppression of maturation of autophagosomes with BafA1 prevents ApoE from being degraded by autophagy, resulting in its enhanced transport to the HCV assembly site and the increase of the infectivity of both intracellular and extracellular HCV particles.

The branched chain amino acids (BCAAs) are leucine, valine and isoleucine. A multi-million dollar industry of nutritional supplements has grown around the concept that dietary supplements of BCAAs alone produce an anabolic response in humans driven by a stimulation of muscle protein synthesis. In this brief review the theoretical and empirical bases for that claim are discussed. Theoretically, the maximal stimulation of muscle protein synthesis in the post-absorptive state in response to BCAAs alone is the difference between muscle protein breakdown and muscle protein synthesis (about 30% greater than synthesis), because the other EAAs required for synthesis of new protein can only be derived from muscle protein breakdown. Realistically, a maximal increase in muscle protein synthesis of 30% is an over-estimate because the obligatory oxidation of EAAs can never be completely suppressed. An extensive search of the literature has revealed no studies in human subjects in which the response of muscle protein synthesis to orally-ingested BCAAs alone was quantified, and only two studies in which the effect of intravenously infused BCAAs alone was assessed. Both of these intravenous infusion studies found that BCAAs decreased muscle protein synthesis as well as protein breakdown, meaning a decrease in muscle protein turnover. The catabolic state in which the rate of muscle protein breakdown exceeded the rate of muscle protein synthesis persisted during BCAA infusion. We conclude that the claim that consumption of dietary BCAAs stimulates muscle protein synthesis or produces an anabolic response in human subjects is unwarranted.

sho r t e r and wid e r m y o t u b u l e s in his DMD m u s c l e t i s s u e c u l t u r e . N u m b e r s of n u c le i in the m y o t u b u l e s in the c u l t u r e d DMD m u s c l e s were fou n d to be i n c r e a s e d (Geiger and G a r vi n, 195 7 ) w h e r e a s another r e p o r t ( Morgan e ^ a 1 . 1973) s h o w e d a d ec r e a s e . Both M o r g a n ej^ a_l. (1973) and V a s s i l o p oui o s , Emery and G or d o n (1977) show e d larger n u c l e i in t he DMD c u l t u r e d muscle.