Protective Effects of Glutamine and Leucine Supplementation on Sepsis-Induced Skeletal Muscle Injuries

Abstract

This study investigated the effects of l-glutamine (Gln) and/or l-leucine (Leu) administration on sepsis-induced skeletal muscle injuries. C57BL/6J mice were subjected to cecal ligation and puncture to induce polymicrobial sepsis and then given an intraperitoneal injection of Gln, Leu, or Gln plus Leu beginning at 1 h after the operation with re-injections every 24 h. All mice were sacrificed on either day 1 or day 4 after the operation. Blood and muscles were collected for analysis of inflammation and oxidative damage-related biomolecules. Results indicated that both Gln and Leu supplementation alleviated sepsis-induced skeletal muscle damage by reducing monocyte infiltration, calpain activity, and mRNA expression levels of inflammatory cytokines and hypoxia-inducible factor-1α. Furthermore, septic mice treated with Gln had higher percentages of blood anti-inflammatory monocytes and muscle M2 macrophages, whereas Leu treatment enhanced the muscle expressions of mitochondrion-related genes. However, there were no synergistic effects when Gln and Leu were simultaneously administered. These findings suggest that both Gln and Leu had prominent abilities to attenuate inflammation and degradation of skeletal muscles in the early and/or late phases of sepsis. Moreover, Gln promoted the switch of leukocytes toward an anti-inflammatory phenotype, while Leu treatment maintained muscle bioenergetic function.

Keywords: calpain; hypoxia-inducible factor-1α; macrophage; mitochondria; monocyte.

Figure 1

Blood leukocyte distribution. The groups are described in the legend to Table 1. Leukocyte populations are presented as the percentages of neutrophils (Ly6G⁺), macrophages (F4/80⁺), inflammatory monocytes (Ly6C^highCCR2⁺), and anti-inflammatory monocytes (Ly6C^lowCX3CR1⁺) among blood leukocytes (CD45⁺). Student’s t-test was used to analyze differences between the C and S groups at the same time point. Differences among septic groups on the same day were analyzed by a one-way ANOVA with Tukey’s post-hoc test. * Significantly differs from the C group. † Significantly differs from the S group. # Significantly differs from the L group (p < 0.05).

Figure 2

Leukocyte populations in muscles. The groups are described in the legend to Table 1. Leukocyte populations are presented as the percentages of neutrophils (Ly6G⁺), monocytes (Ly6C^high), M1 macrophages (F4/80⁺CD206^-), and M2 macrophages (F4/80⁺CD206⁺) among leukocytes (CD45⁺). The M1/M2 macrophage ratio was also demonstrated. Student’s t-test was used to analyze differences between the C and S groups at the same time point. Differences among septic groups on the same day were analyzed by a one-way ANOVA with Tukey’s post-hoc test. * Significantly differs from the C group. † Significantly differs from the S group (p < 0.05).

Figure 3

Calpain activity and malondialdehyde (MDA) content in muscles. The groups are described in the legend to Table 1. Student’s t-test was used to analyze differences between the C and S groups at the same time point. Differences among septic groups on the same day were analyzed by a one-way ANOVA with Tukey’s post-hoc test. * Significantly differs from the C group. † Significantly differs from the S group (p < 0.05).

Figure 4

Muscle mRNA expressions of inflammatory genes. The groups are described in the legend to Table 1, and gene abbreviations are given in the footnotes to Table 2. Student’s t-test was used to analyze differences between the C and S groups at the same time point. Differences among septic groups on the same day were analyzed by a one-way ANOVA with Tukey’s post-hoc test. * Significantly differs from the C group. † Significantly differs from the S group (p < 0.05).

Figure 5

Muscle mRNA expressions of mitochondrion-related genes. The groups are described in the legend to Table 1, and gene abbreviations are given in the footnotes to Table 2. Student’s t-test was used to analyze differences between the C and S groups at the same time point. Differences among septic groups on the same day were analyzed by a one-way ANOVA with Tukey’s post-hoc test. * Significantly differs from the C group. † Significantly differs from the S group (p < 0.05).

Figure 6

H&E staining of the gastrocnemius (GA) muscle. Representative histological images on day 4 postoperatively (200× magnification) are provided. The groups are described in the legend to Table 1.

Figure 7

Schematic overview of the protective effects l-glutamine (Gln) and l-leucine (Leu) on sepsis-induced skeletal muscle injuries. HIF, hypoxia-inducible factor.