Low-dose dexamethasone prevents endotoxaemia-induced muscle protein loss and impairment of carbohydrate oxidation in rat skeletal muscle

Abstract

We recently provided evidence suggesting a role for cytokine-mediated inhibition of Akt/Forkhead box O 1 (FOXO1) signalling in the induction of muscle atrophy and impairment of muscle carbohydrate oxidation during lipopolysaccharide (LPS)-induced endotoxaemia in rats. We hypothesized that a low-dose dexamethasone (Dex; anti-inflammatory agent) infusion during endotoxaemia would prevent the LPS-induced impairment of Akt/FOXO1 signalling, and therefore prevent the muscle atrophy and impairment of carbohydrate oxidation. Chronically instrumented Sprague-Dawley rats received a continuous intravenous infusion of LPS (15 microg kg(-1) h(-1)), Dex (12.5 microg kg(-1) h(-1)), Dex+LPS or saline for 24 h at 0.4 ml h(-1). LPS infusion caused haemodynamic changes consistent with a hyperdynamic circulation and induced increases in muscle tumour necrosis factor-alpha (TNF-alpha; 10-fold, P < 0.001), interleukin-6 (IL-6; 14-fold, P < 0.001) and metallothionein-1A (MT-1A; 187-fold, P < 0.001) mRNA expression. Dex co-administration abolished most of the haemodynamic effects of LPS and reduced the increase in muscle TNF-alpha, IL-6 and MT-1A by 51% (P < 0.01), 85% (P < 0.001) and 58% (P < 0.01), respectively. Dex infusion during endotoxaemia also prevented the LPS-induced 40% reduction in the muscle protein:DNA ratio and decrease in Akt phosphorylation, and partially prevented the reduction in FOXO1 phosphorylation. However, Dex did not prevent the LPS-mediated increase in muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) mRNA expression, but did significantly reduce the LPS-mediated increase in cathepsin-L mRNA expression and enzyme activity by 43% (P < 0.001) and 53% (P < 0.05), respectively. Furthermore, Dex suppressed LPS-induced pyruvate dehydrogenase kinase 4 (PDK4) mRNA upregulation by approximately 50% (P < 0.01), and prevented LPS-mediated muscle glycogen breakdown and lactate accumulation. Thus, low-dose Dex infusion during endotoxaemia prevented muscle atrophy and the impairment of carbohydrate oxidation, potentially through suppression of cytokine-mediated Akt/FOXO inhibition, and blunting of cathepsin-L-mediated lysosomal protein breakdown.

Figure 1. Changes in cardiovascular variables during 25 h infusion of saline (left hand panel) or LPS (right hand panel) in rats pre-treated with saline (filled symbols) or dexamethasone (Dex; open symbols)

Values are means and vertical bars represent s.e.m. *P < 0.05 vs. baseline (Friedman's test).

Figure 2. Alkaline-soluble protein to DNA ratio in extensor digitorum longus (EDL) muscle following 24 h of dexamethasone (Dex), lipopolysaccharide (LPS) or Dex plus LPS infusion

Alkaline-soluble protein (A) and DNA (B) concentration and protein:DNA ratio (C) in rat EDL muscle following 24 h Dex, LPS, Dex+LPS or saline infusion. Values represent means +s.e.m. Significantly different from saline group: *P < 0.05; **P < 0.01. Significantly different from Dex group: †P < 0.05; ††P < 0.01. Significantly different from LPS group: ‡P < 0.05; ‡‡P < 0.01.

Figure 3. mRNA expression of selected genes in rat extensor digitorum longus muscle, following 24 h of dexamethasone (Dex), lipopolysaccharide (LPS) or Dex plus LPS infusion

Relative mRNA expression of saline controls was set at 1. Values are means and vertical bars represent s.e.m. Significantly different from saline group: *P < 0.05; **P < 0.01; ***P < 0.001. Significantly different from Dex group: †P < 0.05; ††P < 0.01; †††P < 0.001. Significantly different from LPS group: ‡P < 0.05; ‡‡P < 0.01; ‡‡‡P < 0.001.

Figure 4. Cytosolic Akt protein expression in rat extensor digitorum longus (EDL) muscle

A, representative Western blots of phosphorylated Akt protein. B, representative blot of total Akt protein. C, density of Akt protein bands, measured by Western blotting, in EDL muscle from rats administered dexamethasone (Dex), lipopolysaccharide (LPS), Dex+LPS or saline for 24 h. D, ratio of phosphorylated to total cytosolic Akt protein in EDL muscle of Dex-, LPS-, Dex+LPS- or saline-treated rats. Significantly different from saline group: *P < 0.05. Significantly different from Dex group: †P < 0.05; ††P < 0.01. Significantly different from LPS group: ‡P < 0.05.

Figure 5. Forkhead box O (FOXO) 1 protein expression in rat extensor digitorum longus (EDL) muscle

A and B, representative Western blots of cytosolic phosphorylated and total FOXO1 protein. C, density of cytosolic FOXO1 protein bands in EDL muscle from rats administered dexamethasone (Dex), lipopolysaccharide (LPS), Dex+LPS or saline for 24 h. D, ratio of phosphorylated to total cytosolic FOXO1 protein in EDL muscle of Dex, LPS, Dex+LPS or saline-treated rats. Significantly different from control: *P < 0.05; **P < 0.01.

Figure 6. Pyruvate dehydrogenase kinase 4 (PDK4) protein expression, and glycogen and lactate concentrations in rat extensor digitorum longus (EDL) muscle

A, representative blot of PDK4 protein. B, density of PDK4 protein bands, measured by Western blotting, in EDL muscle of rats administered with dexamethasone (Dex), lipopolysaccharide (LPS), Dex+LPS or saline for 24 h. C and D, glycogen (C) and lactate (D) content in EDL muscle from rats administered with dexamethasone (Dex), lipopolysaccharide (LPS), Dex+LPS or saline for 24 h. Values are means and vertical bars represent s.e.m. Significantly different from saline control: *P < 0.05; **P < 0.01. Significantly different from Dex group: †P < 0.05; ††P < 0.01; †††P < 0.001. Significantly different from LPS group: ‡P < 0.05; ‡‡P < 0.01.

Figure 7. Proteasome and cathepsin-L activity in rat extensor digitorum longus (EDL) muscle, following 24 h of lipopolysaccharide (LPS) or dexamethasone (Dex) + LPS infusion

Values are means and vertical bars represent s.e.m. Significantly different from saline control group: *P < 0.05. Significantly different from LPS group: ‡P < 0.05.

Figure 8. Proposed mechanism of dexamethasone (Dex)-mediated prevention of muscle atrophy and impairment of carbohydrate oxidation during lipopolysaccharide (LPS)-induced endotoxaemia

A, during LPS-induced endotoxaemia, cytokine-mediated impairment of Akt/Forkhead box O (FOXO) signalling is thought to be important in both the induction of muscle atrophy, via FOXO-mediated upregulation of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1), leading to an increase in ubiquitin–proteasome pathway (UPP)-dependent protein degradation, and the impairment of carbohydrate oxidation, through upregulation of pyruvate dehydrogenase kinase 4 (PDK4) and subsequent pyruvate dehydrogenase complex (PDC) inhibition. An increase in lysosome-mediated proteolysis is also thought to contribute to muscle protein loss, via cytokine-induced cathepsin-L upregulation. B, low-dose Dex infusion during endotoxaemia provides protection against muscle atrophy and the impairment of carbohydrate oxidation, most likely due to the blunting of cytokines e.g. tumour necrosis factor-α (TNF-α). Dex treatment prevents LPS-induced inhibition (i.e. reduced phosphorylation) of Akt and activation (i.e. reduced phosphorylation) of FOXO. Furthermore, LPS-induced upregulation of PDK4, and muscle glycogen breakdown and lactate accumulation, are suppressed by administration of Dex during endotoxaemia. In contrast, LPS-induced MAFbx and MuRF1 upregulation are not suppressed by Dex, and may be upregulated via an unknown, Akt/FOXO-independent mechanism. However, the LPS-induced increase in cathepsin-L mRNA expression is blunted by Dex. and arrows indicate upregulation/increased activity and downregulation/decreased activity, respectively, and arrow thickness represents the magnitude of upregulation/increased activity and downregulation/decreased activity.