The main finding of the present study is the unexpected attenuation of all proteasome activities in skeletal muscle during the early hours of LPS-induced endotoxemia. The same pattern of regulation was also observed in cardiac tissue while only β5 activities were decreased in liver. As detailed in the methods, enzymatic activities were determined fluorometrically using specific substrates and inhibitors, following a validated protocol  that was previously used to show increase in proteasome activities following denervation-induced muscle atrophy .
The regulation of proteasome activity in skeletal muscle in response to LPS administration was investigated by previous in vivo studies. An increased β5 activity of rat soleus and extensor digitorum longus was observed 24 hours after LPS for doses ranging from 1 to 12 mg/kg [17, 20] and similar results were reported for the diaphragm of rat 48 hours after LPS administration . Intravenous administration of a low dose of endotoxin to human reduced protein degradation without changing proteasome β5 activity after 3 hours . Taken together, these results suggest that different rodent models (rat or mice) as well as the delay between LPS injection and muscle collection might explain the discrepancy between our results and those previously reported in the literature. Therefore, the choice of the delay between LPS injection and the animal sacrifice is critical. We choose a delay of 8 hours because this delay was known to drastically increase MuRF1 and MAFbx expression , which are considered to be master regulators of UPP-related proteolysis in skeletal muscle .
The mechanisms responsible for the decrease in proteasome activities in the early hours after LPS injection remain unclear. This decrease was not related to changes in proteasome 20S abundance, as the various 20S subunits, including β1, β5 and β5i were not affected. Nevertheless, these subunits must be assembled to fulfill their degradation function. Hence, it is possible that proteasome assembly was altered by acute LPS administration without showing any changes in protein subunit expression levels. Further research should be conducted to investigate that hypothesis. Alternatively, proteasome 20S activity is regulated through association with regulatory particles, the best described of them being the proteasome 19S. A reduction in proteasome activities has been proposed as a mechanism for sparing energy since less ATP would be consumed by proteasome 19S . The reduced level of RPT1 ATPase due to sepsis is an element in favor of that hypothesis, which is also supported by the close association between endotoxemia and a decreased ability to generate ATP through oxidative metabolism during acute phase of sepsis .
Proteasome assays measure the catalytic activity of the 20S (ATP-independent) or 26S forms (ATP-dependent) for a given substrate concentration. Our results suggest the presence of an inhibitory process, which represses all proteasome activities in response to acute LPS administration. Oxidative stress could be the inhibitory link between LPS and proteasome inhibition. Proteasomal degradation is known to be repressed in vitro through carbonylation of ATPase subunits of proteasome 19S . 19S ATPases – also known as Rpts - are located in the base of the 19S particle. Their functions are to bind substrates selectively, to open the gate formed by the α-ring of the 20S, to unfold substrates and to allow substrate translocation inside the proteolytic room of the 20S. For all these reasons, their association with the 20S proteasome stimulates proteasomal protein degradation . In this study, we showed that the level of protein carbonyls increased with LPS administration and was associated with a large decrease in RPT1 protein level. This coincides with the decrease in proteasome 20S activities. As carbonyls can be degraded independently of ATP directly by the 20S, this supports a role of oxidative stress in LPS-induced proteasome inhibition.
Transcripts coding for proteasome subunits PSMB1 and PSMC2 were increased 8 hours after LPS administration. This suggests that a feed-back mechanism occurs to restore/increase the level of proteasome 19S/20S subunits, which is consistent with the later increase in proteasome activity reported by other investigators [17, 18, 20].
Even though the catalytic activities of the proteasome were decreased, the total protein amount was likely lower after LPS administration . Indeed, the GAS/body weight ratio was decreased supporting the idea that atrophy was already occurring at the time of the sacrifice, i.e. 8 hours after LPS injection. The decrease in the assayed proteasome activities does not necessary implicate an in vivo repression of the overall UPP activity. A higher amount of substrate available for proteasome degradation could increase the rate of protein breakdown through the proteasome. Although the amount of protein available for 20S proteasome degradation is unknown, the lack of any change in the levels of steady state polyubiquitinated proteins after LPS injection argues against a substrate-dependent regulation of the 26S form. It is possible that although the proteasome activities are reduced by LPS, they remain high enough to ensure the removal of ubiquinated substrates available for degradation in vivo. Taken together, our results clearly show that UPP components are not necessarily regulated in the same way in response to a catabolic signal such as LPS and suggest that mechanisms of retro-feedback could occur to prevent any excessive protein breakdown.
Moreover, increased activity of other degradation pathways like autophagy might also explain the apparent discrepancy between the reduced proteasome activities and the decrease in muscle weight. Indeed, we observed changes in autophagy induction marker phospho-ULK1Ser757, autophagosome presence marker LC3bII and autophagosome formation marker LC3aII/LC3aI as well as autophagic flux marker p62, which are all consistent with a strong activation of protein degradation through the autophagy-lysosomal pathway in response to LPS injection, even if cathepsin L and cathepsin B activities were not affected.
A decrease in protein synthesis was observed by Lang et al. 4 hours after LPS administration and was associated to a massive dephosphorylation of 4E-BP1 . Our results also show that LPS induces a dephosphorylation of 4E-BP1 8 hours after LPS injection and reinforce the arguments for a negative protein balance.
Muscle atrophy is a major health matter. Development of therapeutic strategies aiming to counteract muscle loss is crucial. When physical exercise is infeasible and when nutritional strategies are ineffective, the use of pharmacological agents is the only way to prevent muscle atrophy. As UPP is one of the two main pathways responsible for the degradation of the bulk of the proteins in skeletal muscle, proteasome inhibitor administration seems an interesting approach to prevent muscle wasting, especially knowing that a proteasome inhibitor has been approved for hematological malignancy therapy. Additionally, administration of proteasome inhibitors in rodents reduced plasma cytokine increase and prolonged survival in septic shock, which makes them attractive therapeutic agents [13, 14]. Epoxomicin was chosen because of its specific inhibition on proteasome activity and its high affinity for the β5 subunit , which is believed to be the rate limiting step for proteasomal degradation . Our results showed that in vivo administration of epoxomicin was effective for inhibiting 20S and 26S proteasome β5 activities in the hepatic and cardiac muscle cells whereas this inhibition was much weaker and not significant in the skeletal muscle cell. The unexpected inhibition of proteasome activity due to acute LPS administration made it unlikely that proteasome inhibition would have an effect on muscle atrophy under the conditions investigated. A lower sensitivity to proteasome inhibitors inherent to skeletal muscle cells could be due to tissue specific reactivity. Protein degradation was reported to be less sensitive to proteasome inhibitors in isolated skeletal muscles than in cultured cells possibly due to slower up-take or a faster degradation in skeletal muscle than in other tissues . Alternatively, epoxomicin was administered by intraperitoneal injection, a commonly used route for small laboratory animals. Drugs administered intraperitoneally are primarily absorbed through the portal circulation. Therefore, they must pass through the liver before reaching other organs . The liver plays numerous important physiological roles, including detoxification. It is therefore conceivable that epoxomicin was partly removed before reaching the inferior vena cava and being distributed to other tissues. The fact that proteasome activity was strongly inhibited in the liver and to a lesser extent in the heart is an element in favor of that hypothesis. Intravenous and subcutaneous injections are two approved routes of administration of the proteasome inhibitor bortezomib in humans and inhibit 20S proteasome activity to the same extent . Future animal studies could use one of these two routes to be able to correlate the results with human studies.
The results of the present study confirm that the expressions of muscle specific ligases MuRF1 and MAFbx are repressed by proteasome inhibitors. In a previous study, we showed that the administration of another less specific proteasome inhibitor MG132 reduced muscle atrophy caused by a 6-day hindlimb suspension . This was associated with a repression of the increase of MuRF1 and MAFbx. Another study reported similar results in response to a 7-day hindlimb immobilization protocol . Among signaling pathways regulating muscle ligase mRNA expression, NF-κB is a transcription factor potentially repressed by proteasome inhibitor. Under atrophy signal, the inhibitory protein of NF-κB, IκBα becomes phosphorylated and so is marked for ubiquitination and subsequent proteasomal degradation. Therefore, proteasome inhibitors could prevent IκBα degradation and thereby NF-κB activation as well as subsequent increase in ligase mRNA. In our previous study, IκBα was unchanged at the end of the 6-day hindlimb suspension protocol. However, this does not preclude an activation of NF-κB as IκBα may have been degraded at the onset of the unloading and returned to a basal level at the time of sample collection, while MuRF1 and MAFbx remained elevated. Caron et al. also showed a repression of the increase in MuRF1 and MAFbx in immobilized animals treated with MG-132 which was associated to a reduced increase of the Tnf-α, interleukin-6 and interleukin-1 cytokines, which are known to be regulated by NF-κB . The activation of the NF-κB pathway assessed in the present study by a decreased IκBα level and an increased Tnf-α in response to LPS, was not altered by epoxomicin administration. This is consistent with the fact that proteasome β5 activity was not inhibited in skeletal muscle.
Circulating pro-inflammatory cytokines – especially TNF-α - can regulate MuRF1 and MAFbx through p38 activation. Therefore, another explanation for a decrease in muscle ligase mRNA expression could be an anti-inflammatory effect of epoxomicin. However, the lack of change in Tnf-α and in p38 phosphorylation state after epoxomicin injection argues against this hypothesis.
Among signaling pathways regulating catabolism, the phosphoinositide-3-kinase (PI3K)/Akt/FoxO3 pathway coordinately regulates UPP and autophagy . Studies dealing with LPS provide controversial results on the activation of this pathway. While LPS increases Akt phosphorylation state in C2C12 cell culture , LPS administration in vivo seems to repress Akt [21, 39]. Here we show a drastic dephosphorylation of both Akt and FoxO3 with LPS administration, which was not reversed by epoxomicin treatment.
Muscle ligase expression can also be regulated by oxidative stress . In the present study, protein oxidation was rescued in LPS animals following epoxomicin administration suggesting indirect anti-oxidant properties for epoxomicin, which could explain the lower increase in MuRF1 and MAFbx.
The main limitation of this study is the lack of a group receiving epoxomicin only. Based on available literature we hypothesized an increase in proteasome activity after LPS injection. The number of groups (3) was chosen with the goal of studying the protective effect of epoxomicin. Due to the LPS-induced decrease in proteasome activities, it is difficult to interpret if epoxomicin was ineffective in muscle because of tissue insensitivity or if epoxomicin is not useful at this time-point because proteasome activities are lowered. To investigate the various components of UPP and the regulatory signaling pathways, we chose an early time-point for sample collection. Therefore, the experimental design used in this study does not allow an investigation of any potential interaction between LPS and epoxomicin.