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Citrus Polymethoxyflavones Attenuate Metabolic Syndrome by Regulating Gut Microbiome and Amino Acid Metabolism

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Citrus Polymethoxyflavones Attenuate Metabolic Syndrome by Regulating Gut Microbiome and Amino Acid Metabolism

Su-Ling Zeng et al. Sci Adv.

Abstract

Metabolic syndrome (MetS) is intricately linked to dysregulation of gut microbiota and host metabolomes. Here, we first find that a purified citrus polymethoxyflavone-rich extract (PMFE) potently ameliorates high-fat diet (HFD)-induced MetS, alleviates gut dysbiosis, and regulates branched-chain amino acid (BCAA) metabolism using 16S rDNA amplicon sequencing and metabolomic profiling. The metabolic protective effects of PMFE are gut microbiota dependent, as demonstrated by antibiotic treatment and fecal microbiome transplantation (FMT). The modulation of gut microbiota altered BCAA levels in the host serum and feces, which were significantly associated with metabolic features and actively responsive to therapeutic interventions with PMFE. Notably, PMFE greatly enriched the commensal bacterium Bacteroides ovatus, and gavage with B. ovatus reduced BCAA concentrations and alleviated MetS in HFD mice. PMFE may be used as a prebiotic agent to attenuate MetS, and target-specific microbial species may have unique therapeutic promise for metabolic diseases.

Figures

Fig. 1
Fig. 1. Citrus PMFE shows potential metabolic protective effects via inhibition of the mTOR/P70S6K/SREBP pathway.
(A) Typical high-performance liquid chromatography (HPLC) chromatograms of citrus peel extract (ECP) and citrus PMFE. mAU, milli-absorbance unit. (B) Chemical structures of the four major PMFs. (C) PMFE inhibits SREBP luciferase activity in HL-7702/SRE-Luc reporter cells in a dose- and time-dependent manner. (D) PMFE reduces mature SREBP-1/2 levels in a dose-dependent manner. Representative Western blotting analysis of SREBP-1/2 is shown. Expression of SREBP (mature) protein was normalized to β-actin as the loading control, and the relative ratio to the vehicle group was labeled below (estimated using the ImageJ software). (E) PMFE inhibits de novo synthesis of cholesterol and fatty acid by decreasing SREBP target genes. HL-7702 cells were treated with dimethyl sulfoxide (DMSO) or indicated concentrations of PMFE for 18 hours. (F) HL-7702 cells were stained with nile red to assess lipids content. (G) PMFE inhibits the activity of mTOR and its downstream kinase P70S6K. Relative intensities of the bands were taken as a ratio of the phosphoprotein over total protein (normalized to internal controls). All experiments were repeated three times. Error bars represent SD. Significant differences compared with DMSO group are indicated by *P < 0.05, **P < 0.01, and ***P < 0.001 (assessed by Student’s t test).
Fig. 2
Fig. 2. Citrus PMFE exhibits robust metabolic protection in HFD-fed mice.
Mice were randomly divided into four groups (n = 8). Chow-fed mice were treated daily with 0.5% CMCNa suspension (Chow). HFD-fed mice were orally administrated 0.5% CMCNa suspension (HFD), PMFE (PMFE; 120 mg/kg per day), or lovastatin (Lov; 30 mg/kg per day). (A) Body weight of the chow- and HFD-fed mice treated daily with solvent (0.5% CMCNa), PMFE, or lovastatin for 8 weeks. (B) Average daily food intake for the above four groups of mice. (C) Epididymal fat. (D) Liver weight. (E) Liver lipid content was assessed using oil red O staining (scale bar, 100 μm). (F) Effect of PMFE on percentage of initial blood glucose level during insulin tolerance test (ITT). Right: Area under the curve (AUC). (G) Representative pictures of hematoxylin and eosin (H&E)–stained white adipose tissue (scale bar, 100 μm). (H) Effect of PMFE on glucose tolerance measured by oral glucose tolerance test (OGTT). Right: AUC. The PMFE-gavaged mice had significantly lower serum glucose levels compared to HFD mice [two-way analysis of variance (ANOVA)]. (I) Total TC, TG, LDL, and HDL levels in blood. Error bars are expressed as means ± SD. Statistical significance was determined by one-way or two-way ANOVA with Tukey tests for multiple-group comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 3
Fig. 3. Citrus PMFE alleviates HFD-induced gut dysbiosis.
Microbiota composition of chow-fed mice and HFD mice treated with PMFE (30, 60, and 120 mg/kg per day) was analyzed by 16S rDNA pyrosequencing (n = 8 for each group). (A) Weighted UniFrac PCoA analysis of gut microbiota based on the OTU data of chow, HFD, and PMFE groups. (B) Bacterial taxonomic profiling at the phylum level of intestinal bacteria from different mouse groups. (C) Firmicutes-to-Bacteroidetes ratio in the indicated groups. (D) Representative H&E pictures of intestine (scale bars, 250 μm). (E) Heatmap of the 50 OTUs in Chow group altered by HFD responding to PMFE treatment. The color of the spots in the left panel represents the relative abundance of the OTU in each group. In the middle panel, white circles represent less abundant OTUs in Chow and PMFE compared with HFD; black diamonds represent more abundant OTUs in Chow and PMFE compared with HFD. The phylum, family, and genus names of the OTUs are shown on the right panel. The analyses were conducted using R software version 3.3.1.
Fig. 4
Fig. 4. Citrus PMFE alters MetS-associated BCAA levels in HFD mice.
Chow-fed mice were treated daily with solvent (0.5% CMCNa) (Chow). HFD-fed mice were orally administered solvent (0.5% CMCNa) (HFD) or PMFE (120 mg/kg per day). (A) Principal components analysis (PCA) score plots for discriminating the fecal metabolome from Chow, HFD, and PMFE groups. (B) Heatmaps of the differential metabolites that were altered by HFD feeding compared with PMFE-fed mice. Asterisks represent metabolites whose abundance in chow-fed mice was altered by HFD and then regulated by PMFE. The differences of abundance distributions among metabolites between two groups were measured by the Mann-Whitney U test with Benjamini-Hochberg false discovery rate correction. Adjusted P values less than 0.05 were considered statistically significant. (C) Disturbed metabolic pathways in the Chow versus HFD and HFD versus PMFE groups. (D) Comparison of circulating levels of valine, leucine, isoleucine, serine, and phenylalanine in feces by GC-MS in the indicated groups. (E) Heatmap analysis of the Pearson correlation of fecal amino acids and metabolic syndrome–related indexes. Red represents positive correlation, and blue indicates negative correlation. Error bars are expressed as means ± SD. Statistical significance was determined by one-way ANOVA with Tukey tests for multiple-group comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 5
Fig. 5. Fecal transplants of citrus PMFE exhibit metabolic protection in HFD mice.
Mice were randomly divided into four groups (n = 8). HFD-fed mice were orally administered solvent (0.5% CMCNa) (HFD) or PMFE (120 mg/kg per day). Horizontal fecal transfer from solvent (0.5% CMCNa)–treated HFD mice is referred to as HFD receivers (HFD→HFD). Horizontal fecal transfer from PMFE-treated mice is referred to as PMFE receivers (PMFE→HFD). (A) Study design of fecal transplant experiment. (B) Body weight of the above four groups of mice. (C) Epididymal fat. (D) Liver weight. (E) Insulin tolerance measured by ITT. Right: AUC. (F) Total TC, TG, LDL, and HDL levels in blood. (G) Relative abundance of isoleucine, leucine, valine, phenylalanine, serine, and tyrosine in feces by GC-MS in the indicated groups. Error bars are expressed as means ± SD. Statistical significance was determined by one-way or two-way ANOVA with Tukey tests for multiple-group comparisons.
Fig. 6
Fig. 6. Citrus PMFE–mediated enrichment of B. ovatus attenuates MetS in HFD mice.
Chow-fed mice were treated daily with solvent (0.5% CMCNa) (Chow). HFD-fed mice were orally administered with high-dose PMFE (120 mg/kg per day, HFD and PMFE), B. ovatus (BO live), B. ovatus and high-dose PMFE (BO and PMFE), or killed B. ovatus (BO killed). (A) Study design of in vitro batch culture fermentation and in vivo verification experiment. (B) Relative abundance of Bacteroides species after incubation with PMFE (presented in percent initial levels). (C) Changes of the amino acid concentrations at 0, 12, 24, and 48 hours after incubation with different concentrations of PMFE. (D) Relative abundance of B. ovatus in the indicated groups measured by qPCR. (E) Body weight of the above four groups of mice. (F) Total TC, TG, LDL, and HDL levels in blood. (G) Relative abundance of isoleucine, leucine, valine, serine, tyrosine, and phenylalanine in feces by GC-MS in the indicated groups. Error bars are expressed as means ± SD. Statistical significance was determined by one-way or two-way ANOVA with Tukey tests for multiple-group comparisons.

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