Effects Of AMPK Activation On Proteolysis In Skeletal Muscle Of Scald Rats And Its Signaling Mechanisms

Objective:The protein metabolism in skeletal muscle post severe burn is characterized by hyperproteolysis and negative nitrogen balance. Our previous studies have deduced that ubiquitin-proteasome played a key role in skeletal muscle proteolysis. Especially, muscle atrophy F-box (MAFbx) and muscle-specific RING finger protein 1 (MuRF1) are key enzymes in regulating muscle proteolysis. Furthermore, the enhanced proteolysis can be inhibited by administration of intensive insulin therapy. However, the changes and molecular mechanisms in expression of skeletal muscle MAFbx and MuRF1 post-burn are still obscure.Adenosine triphosphate (ATP) supplies energy to ubiquitin-proteasome pathway of proteolysis. Energy status may affect proteolysis in skeletal muscles. It is well known that AMP-activated protein kinase (AMPK), the'Energy Gauge', regulates production of ATP. Recently, several studies revealed the inhibitory effects of AMPK activation on protein synthesis. However, little has been studied on the effects of energy status and AMPK activity on skeletal muscle proteolysis and its mechanisms. In order to further elucidate the mechanisms of proteolysis in skeletal muscles post-burn, and to guide clinical treatment, this project was aimed to explore:(1) changes of energy status, AMPK and proteolysis in skeletal muscles of scalded rats, (2) changes of energy status, AMPK and proteolysis in L6 myotubes treated with serum from scalded rats, (3) molecular mechanisms of interaction among AMPK, proteolysis and insulin signaling pathway in L6 myotubes.Methods:1. Animal experiments:One hundred male Wistar rats (180-220g) were randomly divided into two groups:(1) control group (n=50), (2) scald group (n=50). In scald group, rats were inflicted on with 94℃water for 12s to create full-thickness burns, 30% total body surface area (TBSA). The rats were anesthetized 6h, Id,3d,5d, or 7d after injury and sacrificed for collecting muscles:tibialis anterior, extensor digitorum longus (EDL), and soleus. Rats and muscles were weighed. Then, protein levels and activity of AMPK were determined by Western blotting. Quantitative real time PCR (Q-PCR) was used to measure mRNA levels of AMPKa, serine/threonine kinase 11 (LKB1), MAFbx and MuRF1. Furthermore, adenosine monophosphate (AMP)/ATP ratio and energy charge (EC) were measured by high performance liquid chromatography (HPLC).2. Cell culture experiment A:Forty male Wistar rats were randomly divided into sham scald group (n=20) and scald group (n=20). Scald animal model was established according to aforementioned method 1. Rats were sacrificed to harvest serum 12h post-injury. Furthermore, after pre-incubation with DMEM plus 10% FBS for 12h, the differentiated L6 myotubes were cultured with DMEM containing 10% sham scald rat serum (10%SS) or 10% scald rat serum (10% BS) for different durations of time (0, 10min, 1h,6h,24h). The levels of protein, mRNA and nucleotides were measured according to method 1.3. Cell culture experiment B:The L6 myotubes were pre-incubated with DMEM for 12h, followed by replacement with DMEM plus different doses of AMPK activator (AICAR,5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside) or insulin for various time durations. In some groups, PI3K inhibitor (wortmannin), protein kinases B (PKB, Akt) inhibitor (Akt Inhibitor IV) or AMPK inhibitor (Compound C) was applied as pretreatment for 1h. Protein levels and activity of AMPK, acetyl-CoA carboxylase (ACC), Akt, and glycogen synthase kinase (GSK) 3-βwere determined by Western blotting, mRNA levels of AMPKa, LKB1, MAFbx and MuRF1 by Q-PCR, nucleotides levels by HPLC.Results:1. Scald reduced body weight of rats, especially on 3d post-injury (p<0.05). Meanwhile, muscle mass of EDL, and EDL to body weight ratio were decreased from Id through 3d in scald group compared with that in control group (p<0.01). Further, compared with control group, the mRNA level of MAFbx in tibialis anterior was-increased 6h post-injury (p<0.01), the mRNA level of MuRF1 increased both at 6h and on 1d post-injury (p<0.01), followed by a decrease from 5d to 7d in scald group (p<0.01 or p<0.05). Compared with control group, phosphorylation level of tibialis anterior AMPKa (Thr172) in scald group was increased at 6h (p<0.05), then was decreased to the lowest level on 3d (p<0.01), and finally was increased on 7d (p<0.05). But there were no differences in protein levels of AMPKa between the two groups (p>0.05). The mRNA level of AMPKa2 subtype decreased on 1d in scald group (p<0.01). The gene expression of LKB1 in scald group was upregulated at 6h (p<0.01), then reduced significantly 3d and 5d (p<0.01), followed by an increase on 7d. Interestingly, tibialis anterior AMP/ATP ratio was higher in scald group, except on 3d, than that in control group (p<0.01), while changes of EC was contrary to changes of AMP/ATP ratio (p<0.01 or p<0.05).2. Scald rat serum caused upregulation of MAFbx and MuRF1 mRNA expression in L6 myotubes at 24h (p<0.05). Compared with 10%SS group, the protein level of AMPKa was decreased in 10%BS group (30min, 1h and 6h, p<0.01), while pAMPKa level was increased in 10%BS group (1h and 6h,p<0.05; 24h,p<0.01). In 10%BS group, the mRNA level of AMPKα1 were reduced at 24h (p<0.01), and the mRNA level of AMPKa2 were reduced at 1h,6h and 24h (p<0.05), and the mRNA level of LKB1 was increased at 24h (p<0.01), as compared to that in 10%SS group. But the AMP/ATP ratio and EC showed no significant differences between the two groups (p>0.05).3. In L6 myotubes, AMPK activator, AICAR, downregulated mRNA expression of MAFbx and MuRF1 in a dose dependent pattern. Howerver, pretreatment with Compound C inhibited the upregulation of MuRF1 level caused by AICAR (p<0.01). Furthermore, AICAR enhanced both pAkt (Ser473) and pGSK3-β(Ser9) levels, relating to activating PI3K. On the other hand, insulin reduced both MAFbx and MuRF1 mRNA levels dose-dependently. This inhibitory effect could be blocked by administration of AICAR (p<0.01). Concomitantly, insulin inhibited AMPKa through activation of Akt, and downregulated mRNA level of LKB1 dose-dependently. Moreover, insulin had no effects on AMP/ATP ratio and EC (p>0.05).Conclusion:A lower energy status activates AMPK, which then enhancing expression of ubiquitin E3 ligases and skeletal muscle proteolysis in scalded rats. There are tight connections between AMPK and insulin signaling pathway. AMPK phosphorylates Akt, while insulin inhibits AMPK, which might be one of signaling pathways counting for downregulating proteolysis. Thus, enhanced proteolysis in skeletal muscle could be caused by activated AMPK after burns. It could be implied that targeting AMPK would be a new strategy to inhibit skeletal muscle proteolysis after burns.