Foxtail Millet Improves Blood Glucose Metabolism in Diabetic Rats through PI3K/AKT and NF-κB Signaling Pathways Mediated by Gut Microbiota

Abstract

Foxtail millet (FM) is receiving ongoing increased attention due to its beneficial health effects, including the hypoglycemic effect. However, the underlying mechanisms of the hypoglycemic effect have been underexplored. In the present study, the hypoglycemic effect of FM supplementation was confirmed again in high-fat diet and streptozotocin-induced diabetic rats with significantly decreased fasting glucose (FG), glycated serum protein, and areas under the glucose tolerance test (p < 0.05). We employed 16S rRNA and liver RNA sequencing technologies to identify the target gut microbes and signaling pathways involved in the hypoglycemic effect of FM supplementation. The results showed that FM supplementation significantly increased the relative abundance of Lactobacillus and Ruminococcus_2, which were significantly negatively correlated with FG and 2-h glucose. FM supplementation significantly reversed the trends of gene expression in diabetic rats. Specifically, FM supplementation inhibited gluconeogenesis, stimulated glycolysis, and restored fatty acid synthesis through activation of the PI3K/AKT signaling pathway. FM also reduced inflammation through inhibition of the NF-κB signaling pathway. Spearman's correlation analysis indicated a complicated set of interdependencies among the gut microbiota, signaling pathways, and metabolic parameters. Collectively, the above results suggest that the hypoglycemic effect of FM was at least partially mediated by the increased relative abundance of Lactobacillus, activation of the PI3K/AKT signaling pathway, and inhibition of the NF-κB signaling pathway.

Keywords: NF-κB signaling pathway; PI3K/AKT signaling pathway; foxtail millet; glucose metabolism; gut microbiota.

Figure 1

Effect of foxtail millet supplementation on glucose metabolism (A–F), weight gain (G), food intake (H), and lipid metabolism (I–K) in HFD/STZ-induced diabetic rats. Data were represented as mean ± SD. NC, normal control group (n = 8); DC, diabetic control group (n = 8); FM, foxtail millet supplementation group (n = 8); GTT, glucose tolerance tests; HDL-C, high-density lipoprotein cholesterol. Differences between groups were compared using one-way ANOVA with Tukey’s multiple comparison post hoc test, * p < 0.05, ** p < 0.01.

Figure 2

Effect of foxtail millet supplementation on the structure of gut microbiota: (A) Venn diagram on the OTU level; (B) unsupervised principal component analysis (PCA) on the OTU level; (C) supervised partial least squares discriminant analysis (PLS-DA) on the OTU level. NC, normal control group (n = 5); DC, diabetic control group (n = 5); FM, foxtail millet supplementation group (n = 7).

Figure 3

Effect of foxtail millet supplementation on the composition of gut microbiota: (A) phylum-level taxonomic distributions; (B,C) mean proportions of 15 key genera in different groups; (D) LDA scores derived from LefSe analysis, LDA > 3.0. Data were represented as mean ± SD. NC, normal control group (n = 5); DC, diabetic control group (n = 5); FM, foxtail millet supplementation group (n = 7). Differences between three groups were compared using one-way ANOVA with Tukey–Kramer post hoc test; differences between two groups were compared using Student’s T-test, * p < 0.05, ** p < 0.01.

Figure 4

Effect of foxtail millet supplementation on liver transcriptomic profiles: (A–C) differentially expressed genes (DEGs) between different groups; (D) heatmap of 86 shared DEGs; (E) signaling pathways involved in upregulated DEGs; (F) signaling pathways involved in downregulated DEGs. NC, normal control group; DC, diabetic control group; FM, foxtail millet supplementation group.

Figure 5

The expression of mRNA and protein of core factors in PI3K/AKT signaling pathway and its downstream effectors: (A–D) core factors in PI3K/AKT signaling pathway; (E,F) key enzymes in glycolysis; (G–J) key enzymes in gluconeogenesis. Data were represented as mean ± SEM. NC, normal control group; DC, diabetic control group; FM, foxtail millet supplementation group; PI3K, phosphatidylinositol-3-kinase; AKT, protein kinase B; G6P, glucose-6-phosphatase; GK, glucose kinase; PEPCK, phosphoenolpyruvate carboxy. Differences between groups were compared using Student’s T-test, * p < 0.05, ** p < 0.01.

Figure 6

The expression of inflammatory cytokines (A,B), core factors in NF-κB signaling pathway (C–G), and nuclear translocation of p- NF-κB-p65 (H). Data were represented as mean ± SEM. NC, normal control group; DC, diabetic control group; FM, foxtail millet supplementation group; IΚB, κB kinase; IKK, inhibitor of κB kinase. Differences between groups were compared using Student’s T-test, * p < 0.05, ** p < 0.01.

Figure 7

Heatmap of Spearman’s correlation analysis between gut microbiota (top 15 at genus level) and metabolic parameters (A), liver gene expression and metabolic parameters (B), gut microbiota (top 10 at genus level), and liver gene expression (C). Significant correlations are marked by * p < 0.05; ** p < 0.01. IRS, insulin receptor substrate; PI3K, phosphatidylinositol-3-kinase; AKT, protein kinase B; GK, glucose kinase; G6P, glucose-6-phosphatase; PEPCK, phosphoenolpyruvate carboxy; WG, weight gain; TC, total triglycerides; FG, fasting glucose; HDL-C, high-density lipoprotein cholesterol; AUC, areas under the glucose tolerance test.

Figure 8

The mechanisms underlying the hypoglycemic effect of foxtail millet from the perspective of signaling pathways. Foxtail millet supplementation might improve the blood glucose metabolism by inhibiting gluconeogenesis, stimulating glycolysis, and repairing fatty acid synthesis through the insulin-mediated PI3K/AKT signaling pathway, as well as reducing inflammation through the NF-κB signaling pathway. Red background, significantly upregulated genes or proteins; grey background, significantly downregulated genes or proteins; arrow for promotion and —| for inhibition; IRS, insulin receptor substrate; PI3K, phosphatidylinositol-3-kinase; AKT, protein kinase B; GK, glucose kinase; PK, pyruvate kinase; G6P, glucose-6-phosphatase; FBP, fructose bisphosphatase; PEPCK, phosphoenolpyruvate carboxy; FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; SREBP1c, sterol regulatory element-binding protein-1c; IΚB, κB kinase; IKK, inhibitor of κB kinase.