Inhibition of glycogen synthase kinase 3[beta] activity with lithium in vitro attenuates sepsis-induced changes in muscle protein turnover

Abstract

Loss of lean body mass is a characteristic feature of the septic response, and the mechanisms responsible for this decrease and means of prevention have not been fully elucidated. The present study tested the hypothesis that in vitro treatment of skeletal muscle with lithium chloride (LiCl), a glycogen synthase kinase (GSK) 3 inhibitor, would reverse both the sepsis-induced increase in muscle protein degradation and inhibition of protein synthesis. Sepsis decreased GSK-3[beta] phosphorylation and increased GSK-3[beta] activity, under basal conditions. Sepsis increased muscle protein degradation, with a concomitant increase in atrogin 1 and MuRF1 mRNA and 26S proteosome activity. Incubation of septic muscle with LiCl completely reversed the increased GSK-3[beta] activity and decreased proteolysis to basal nonseptic values, but only partially reduced proteosome activity and did not diminish atrogene expression. Lithium chloride also did not ameliorate the sepsis-induced increase in LC3-II, a marker for activated autophagy. In contrast, LiCl increased protein synthesis only in nonseptic control muscle. The inability of septic muscle to respond to LiCl was independent of its ability to reverse the sepsis-induced increase in eukaryotic initiation factor (eIF) 2B[varepsilon] phosphorylation, decreased eIF2B activity, or the reduced phosphorylation of FOXO3, but instead was more closely associated with the continued suppression of mTOR (mammalian target of rapamycin) kinase activity (e.g., reduced phosphorylation of 4E-BP1 and S6). These data suggest that in vitro lithium treatment, which inhibited GSK-3[beta] activity, (a) effectively reversed the sepsis-induced increase in proteolysis, but only in part by a reduction in the ubiquitin-proteosome pathway and not by a reduction in autophagy; and (b) was ineffective at reversing the sepsis-induced decrease in muscle protein synthesis. This lithium-resistant state seems mediated at the level of mTOR and not eIF2/eIF2B. Hence, use of GSK-3[beta] inhibitors in the treatment of sepsis may not be expected to fully correct the imbalance in muscle protein turnover.

Figure 1

Weight gain and muscle weight of nonseptic and septic animals. Panel A: On day 0 rats were implanted with either a sterile (nonseptic) or infected (septic) pellet. Rats were weighed daily and the differences in weight from day 0 are plotted. Panel B: Wet weight of the epitrochlearis was determined immediately prior to incubation. Panel C: Muscle protein content was determined at the end of the 2-h incubation period. Values shown are means ± SEM for nonseptic (n = 42) and septic (n = 45) rats. *P <0.001 versus time-matched nonseptic value.

Figure 2

Effect of lithium chloride (LiCl) on sepsis-induced changes in muscle protein synthesis and degradation. Epitrochlearis muscles from nonseptic and septic rats were excised on day 3 post-sepsis and incubated in vitro in the absence or presence of 10 mM LiCl. Panel A: rates of protein synthesis; Panel B, rates of protein degradation (net tyrosine release). Values are means ± SEM for 9–10 muscles in each group. Means with different letters (a, b, c) are significantly different (P < 0.05).

Figure 3

Effect of LiCl on Akt and GSK-3β phosphorylation as well as GSK-3 activity in muscle from nonseptic and septic rats. The phosphorylation state of Akt (Ser473) and GSK-3β (Ser9) were determined in muscle homogenates using phosphospecific antibodies. The blots were then stripped and re-probed with an antibody recognizing the respective total protein. Panel A, quantitation of all Western blot data for phosphorylated GSK-3 normalized to total GSK protein. The final 2 lanes for each Western blot are positive controls from control muscles treated with IGF-I (100 ng/ml). Panel B, GSK-3β activity was quantified in homogenates as described in Methods. Values are means ± SEM for 8–9 muscles in each group. Means with different letters (a, b, c) are significantly different (P < 0.05).

Figure 4

Lithium ameliorates the sepsis-induced increase in eIF2Bε Ser535-phosphorylation and decreased eIF2B activity in muscle. To determine the relative phosphorylation state of eIF2Bε, equal amounts of protein from homogenates of epitrochlearis from nonseptic and septic rats were immunoblotted with an anti-eIF2B antibody, specific for the phosphorylated form of eIF2Bε. The blots were then stripped of antibody and re-probed with an antibody recognizing total eIF2Bε. Panel A, bar graph indicates the amount of eIF2Bε in the phosphorylated form divided by the total eIF2Bε. Panel B, eIF2B activity was determined as in Methods. Results represent means ± SEM for 8–13 muscles in each group. Means with different letters (a, b) are significantly different (P < 0.05).

Figure 5

Effect of LiCl on 4E-BP1 and S6 phosphorylation in muscle of nonseptic and septic rats. Results represent means ± SEM for 8–13 muscles in each group. Total 4E-BP1 was determined and the γ-isoform (most heavy phosphorylated), β-isoform, and α-isoform (least phosphorylated) identified. α-Tubulin is also shown as a loading control. Panel A, quantitation of 4E-BP1 phosphorylation. Panel B, phosphorylated and total ribosomal protein S6 were determined and the bar graph indicates the phosphorylated form normalized to total S6. Means with different letters (a, b) are significantly different (P < 0.05).

Figure 6

Effect of LiCl on atrogene expression and proteasome activity in muscle from nonseptic and septic rats. The mRNA content in muscle for atrogin-1 (panel A) and MuRF1 (panel B) was quantitated by ribonuclease protection assay (RPA) and data were normalized to L32 which was unaffected by either sepsis and/or lithium treatment. The 20S and 26S proteasome activity was assessed as in Methods. Panel C, 26S proteasome activity data are presented, but the sepsis- and LiCl-induced changes in 20S proteasome activity were comparable (data not shown). Values are means ± SEM for 8–9 muscles in each group. Means with different letters (a, b, c) are significantly different (P < 0.05).

Figure 7

Effect of LiCl on the autophagic/lysosomal pathway as assessed by the LC3-II/LC3-I ratio in muscle from nonseptic and septic rats. LC3 was determined by Western blot analysis and the two isoforms so indicated (inset). Bar graph, represents quantitation of all data normalized to α-tubulin. Values are means ± SEM for 8–9 muscles in each group. Means with different letters (a, b) are significantly different (P < 0.05).

Figure 8

Effect of LiCl on FOXO3 phosphorylation in muscle from nonseptic and septic rats. Total and Thr32-phosphorylated FOXO3 were determined by Western blot analysis. Bar graph, represents quantitation of all data normalized to total FOXO3 protein. Values are means ± SEM for 8–9 muscles in each group. Means with different letters (a, b) are significantly different (P < 0.05).