Ginsenoside Rb1 Improves Metabolic Disorder in High-Fat Diet-Induced Obese Mice Associated With Modulation of Gut Microbiota

Abstract

Gut microbiota plays an important role in metabolic homeostasis. Previous studies demonstrated that ginsenoside Rb1 might improve obesity-induced metabolic disorders through regulating glucose and lipid metabolism in the liver and adipose tissues. Due to low bioavailability and enrichment in the intestinal tract of Rb1, we hypothesized that modulation of the gut microbiota might account for its pharmacological effects as well. Here, we show that oral administration of Rb1 significantly decreased serum LDL-c, TG, insulin, and insulin resistance index (HOMA-IR) in mice with a high-fat diet (HFD). Dynamic profiling of the gut microbiota showed that this metabolic improvement was accompanied by restoring of relative abundance of some key bacterial genera. In addition, the free fatty acids profiles in feces were significantly different between the HFD-fed mice with or without Rb1. The content of eight long-chain fatty acids (LCFAs) was significantly increased in mice with Rb1, which was positively correlated with the increase of Akkermansia and Parasuttereller, and negatively correlated with the decrease of Oscillibacter and Intestinimonas. Among these eight increased LCFAs, eicosapentaenoic acid (EPA), octadecenoic acids, and myristic acid were positively correlated with metabolic improvement. Furthermore, the colonic expression of the free fatty acid receptors 4 (Ffar4) gene was significantly upregulated after Rb1 treatment, in response to a notable increase of LCFA in feces. These findings suggested that Rb1 likely modulated the gut microbiota and intestinal free fatty acids profiles, which should be beneficial for the improvement of metabolic disorders in HFD-fed mice. This study provides a novel mechanism of Rb1 for the treatment of metabolic disorders induced by obesity, which may provide a therapeutic avenue for the development of new nutraceutical-based remedies for treating metabolic diseases, such as hyperlipidemia, insulin resistance, and type 2 diabetes.

Keywords: fecal metabolome; free fatty acid receptor; ginsenoside Rb1; gut microbiota; lipidomics; long-chain fatty acids; metabolic disorder.

FIGURE 1

Rb1 treatment improved lipid metabolism and insulin resistance in high-fat diet (HFD)-fed mice. (A) Mice were fed with a high-fat diet for 12 weeks and then subjected to Rb1 treatment (120 mg/kg) by gavage for 4 weeks. Blood samples of day 24 were used to test serum metabolic parameters. Tissues were collected on day 28. (B) Low-density lipoprotein cholesterol (LDL-c), (C) triglyceride (TG), (D) total cholesterol (TC), (E) fasting blood glucose (FBG), (F) fasting insulin was tested in serum and (G) homeostasis model assessment of insulin resistance (HOMA-IR) index was assessed. (H) An Intraperitoneal glucose tolerance test was performed on day 16. (I) Liver TG, (J) liver TC, and (K) epididymal fat were assessed. (L) Body weight and (M) food intake data were recorded twice a week. All data were presented as the mean ± SEM (n = 5–7). A one-way analysis of variance followed by Tukey’s post hoc test was applied to compare data among groups. *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant.

FIGURE 2

Rb1 treatment improved lipid metabolism in HFD-fed rats. (A) Rats were provided with a high-fat diet for 2 weeks and then subjected to treatment with Rb1 (60 mg/kg) by gavage for 4 weeks. Blood samples of day 28 were used to test serum metabolic parameters, including (B) low-density lipoprotein cholesterol (LDL-c), (C) triglyceride (TG), (D) total cholesterol (TC), (E) high-density lipoprotein cholesterol (HDL-c), (F) glycosylated hemoglobin (GHb), and (G) blood glucose (Glu). (H) Body weight and (I) food intake were also recorded. (J) Oil red O (ORO) staining and hematoxylin and eosin (H&E) staining of liver tissues. (K) H&E staining of epididymal fat tissues. All data were expressed as the mean ± SEM (n = 8). Significant difference among groups were evaluated using one-way analysis of variance followed by Duncan’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant.

FIGURE 3

Rb1 treatment altered the composition of gut microbiota in HFD-fed mice. Fecal samples of mice were collected before Rb1 treatment (day 0) and after Rb1 treatment (day 9, day 20, and day 27). Bacterial 16S rRNA (V3-V4 region) sequencing was conducted for these fecal samples. The alpha diversity was estimated by the (A) Shannon Index and (B) Pielou evenness index. The beta diversity was assessed by (C) principal coordinates analysis (PCoA). Relative abundance of taxa (D) at the phylum level and (E) at the genus level is shown. All data were expressed as the mean ± SEM (n = 5–7). Significant difference among groups were evaluated using two-way analysis of variance followed by Tukey’s multiple comparison test. *P < 0.05; ** P < 0.01; ***P < 0.001.

FIGURE 4

Key bacterial alterations in response to Rb1 treatment in HFD-fed mice and functional pathway prediction. Key genera significantly altered by Rb1 treatment were selected by Linear discriminate analysis effect size (LEfSe) analysis. Functional pathway prediction was performed by the tax4fun software. (A) Heatmap presents the relative abundance of key genera significantly altered by Rb1 treatment (LDA scores > 3). (B) Heatmap presents the abundance of differential pathways in day 0, day 9, day 20, and day 27. Pathways with significant differences between Rb1 and HFD are shown in the top 20 (pathways with the same P-value are shown all). The abundance of pathways was normalized by Z-score method. Kruskal-Wallis rank-sum test was used to calculate the significant difference between groups (P < 0.05).

FIGURE 5

Rb1 modified fecal free fatty acids profiles in HFD-fed mice. The content of free fatty acids in fecal samples collected after Rb1 treatment for 24 days was determined by LC-MS/MS. (A) Heatmap of fecal free fatty acids in mice of the HFD and Rb1 group is shown. (B) Fold change of differential fecal free fatty acids of the Rb1 group compared to the HFD group is displayed. All data were expressed as the mean ± SEM (n = 5). P-values were evaluated using a t-test analysis. *P < 0.05; **P < 0.01. LCFA, long-chain fatty acid, MCFA: medium-chain fatty acid; SCFA, short-chain fatty acid.

FIGURE 6

Rb1 treatment adjusted colonic gene expression associated with free fatty acids receptors and the intestinal barrier function in HFD-fed mice. Relative mRNA expression in colon was determined by real-time PCR. (A–E) Free fatty acids receptor-related genes, (F–I) intestinal barrier function-related genes, (J–L) oxidate stress-related genes are shown. Relative expression levels were normalized to those of GAPDH. All data were expressed as the mean ± SEM (n = 5–7). P-values were determined using a t-test analysis. *P < 0.05; **P < 0.01.

FIGURE 7

Correlation analysis and potential mechanism of improvement of metabolic disorder via Rb1 treatment. Spearman rank correlation between gut microbiota against the corresponding host metabolic parameters, fecal free fatty acids profiles or colonic gene expression for samples of day 20 (A) or day 27 (B) were individually calculated. The correlation analysis inferred potential mechanism of Rb1-treatment improved metabolic disorder is shown in panel (C). *P < 0.05, **P < 0.01, ***P < 0.001.