Dietary Supplemental Glutamine Enhances the Percentage of Circulating Endothelial Progenitor Cells in Mice with High-Fat Diet-Induced Obesity Subjected to Hind Limb Ischemia

Abstract

This study investigated whether glutamine (GLN) pretreatment can enhance circulating endothelial progenitor cells (EPCs) and attenuate inflammatory reaction in high-fat diet-induced obese mice with limb ischemia. Mice were assigned to a normal control (NC), high-fat control (HC), limb ischemia (HI), and GLN limb ischemia (HG) groups. The NC group provided chow diet and treated as a negative control. Mice in the HC and HI groups were fed a high-fat diet which 60% energy provided by fat for 8 weeks. Mice in the HG group were fed the same diet for 4 weeks and then transferred to a high-fat diet with 25% of total protein nitrogen provided as GLN to replace part of the casein for the subsequent 4 weeks. After feeding 8 weeks, mice in the HC group were sham-operated, while the HI and HG groups underwent an operation to induce limb ischemia. All mice except the NC group were euthanized on either day 1 or 7 after the operation. The results showed that the 8 weeks' high-fat diet feeding resulted in obesity. The HG group had higher circulating EPCs on day 1 while muscle vascular endothelial growth factor, matrix metalloproteinase-9, and hypoxia-inducible factor-1 gene expressions were higher on day 7 postischemia than those of the HI group. The superoxide dismutase activity and reduced glutathione content in affected muscles were higher, whereas mRNA expressions of interleukin-6 and tumor necrosis factor-α were lower in the HG than those in the HI group. These findings suggest that obese mice pretreated with GLN-supplemented high-fat diet increased circulating EPC percentage, enhanced the antioxidant capacity, and attenuated inflammatory reactions in response to limb ischemia.

Figure 1

Percentage of blood total epithelial progenitor cells (EPCs). EPC populations were determined as the percentages of CD34⁺/CD133⁺/CD309⁺ cells among mononuclear cells. HC: high-fat sham-operated group; HI: high-fat ischemia group; HG: high-fat glutamine ischemia group. Values are presented as the means ± SEM. All data are representative of duplicate measurements on day 1 or 7 (n = 8 for the respective groups). Differences among groups were analyzed by a one-way ANOVA followed by Tukey's post hoc test. ⁺Significant differences from the HC group. ^#Significant differences from the HI group at the same time point (p < 0.05).

Figure 2

Plasma leptin and adiponectin concentrations in the sham-operated and ischemic groups. HC: high-fat sham-operated group; HI: high-fat ischemia group; HG: high-fat glutamine ischemia group. Values are expressed as the means ± SEM. All data are representative of duplicate measurements on day 1 or 7 (n = 8 for the respective groups). Differences among groups were analyzed by a one-way ANOVA followed by Tukey's post hoc test. ^∗Significant differences from the two other groups. ^#Significant differences from the HI group at the same time point (p < 0.05).

Figure 3

Superoxide dismutase (SOD) enzyme activity and reduced glutathione (GSH) content in ischemic muscle tissues. HC: high-fat sham-operated group; HI: high-fat ischemia group; HG: high-fat glutamine ischemia group. Values are expressed as the means ± SEM. All data are representative of duplicate measurements on day 1 or 7 (n = 8 for the respective groups). Differences among groups were analyzed by a one-way ANOVA followed by Tukey's post hoc test. ^∗Significant differences from the two other groups. ⁺Significant differences from the HC group. ^#Significant differences from the HI group at the same time point (p < 0.05).

Figure 4

Expression of cytokine genes in muscle tissues. TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; IL-6: interleukin-6; IL-10: interleukin-10; HC: high-fat sham-operated group; HI: high-fat ischemia group; HG: high-fat glutamine ischemia group. Values are expressed as the means ± SEM. All data are representative of duplicate measurements on day 1 or 7 (n = 8 for the respective groups). Differences among groups were analyzed by a one-way ANOVA followed by Tukey's post hoc test. ^∗Significant differences from the two other groups. ⁺Significant differences from the HC group. ^#Significant differences from the HI group at the same time point (p < 0.05).

Figure 5

Messenger RNA expression of endothelial progenitor cell (EPC) mobilization-related protein in muscle tissues after limb ischemia. HIF-1α: hypoxia-inducible factor-1α; VEGF: vascular endothelial growth factor; SDF-1: stromal cell-derived factor; MMP-9: matrix metalloproteinase-9; HC: high-fat sham-operated group; HI: high-fat ischemia group; HG: high-fat glutamine ischemia group. Values are expressed as the means ± SEM. All data are representative of duplicate measurements on day 1 or 7 (n = 8 for the respective groups). Differences among groups were analyzed by a one-way ANOVA followed by Tukey's post hoc test. ^∗Significant differences from the two other groups. ⁺Significant differences from the HC group. ^#Significant differences from the HI group at the same time point (p < 0.05).