Vancomycin-Induced Modulation of Gram-Positive Gut Bacteria and Metabolites Remediates Insulin Resistance in iNOS Knockout Mice

Abstract

The role of oxidative and nitrosative stress has been implied in both physiology and pathophysiology of metabolic disorders. Inducible nitric oxide synthase (iNOS) has emerged as a crucial regulator of host metabolism and gut microbiota activity. The present study examines the role of the gut microbiome in determining host metabolic functions in the absence of iNOS. Insulin-resistant and dyslipidemic iNOS^-/- mice displayed reduced microbial diversity, with a higher relative abundance of Allobaculum and Bifidobacterium, gram-positive bacteria, and altered serum metabolites along with metabolic dysregulation. Vancomycin, which largely depletes gram-positive bacteria, reversed the insulin resistance (IR), dyslipidemia, and related metabolic anomalies in iNOS^-/- mice. Such improvements in metabolic markers were accompanied by alterations in the expression of genes involved in fatty acid synthesis in the liver and adipose tissue, lipid uptake in adipose tissue, and lipid efflux in the liver and intestine tissue. The rescue of IR in vancomycin-treated iNOS^-/- mice was accompanied with the changes in select serum metabolites such as 10-hydroxydecanoate, indole-3-ethanol, allantoin, hippurate, sebacic acid, aminoadipate, and ophthalmate, along with improvement in phosphatidylethanolamine to phosphatidylcholine (PE/PC) ratio. In the present study, we demonstrate that vancomycin-mediated depletion of gram-positive bacteria in iNOS^-/- mice reversed the metabolic perturbations, dyslipidemia, and insulin resistance.

Keywords: dyslipidemia; gut microbiota; iNOS−/− mice; insulin resistance; metabolome analysis; obesity.

Figure 1

Insulin-resistant iNOS^−/− mice display atypical gut microbiota with gram-positive bacteria dominance and altered serum metabolome. Gut microbiota analysis in wild-type (WT) and insulin-resistant iNOS^−/− mice. α-Diversity indices in stool samples: (A) observed, (B) Shannon, (C) Simpson, and (D) Chao1. (E) β-Diversity analysis via principal coordinate analysis (PCA) plot based on Bray–Curtis distance. Each dot represents an animal, projected onto the first (horizontal axis) and second (vertical axis) variables. (F) Differentially abundant microbiota at the phylum, family, and genus levels. Serum metabolomic analysis in chow-fed WT and iNOS^−/− mice in ESI (+) mode. (G) PCA score plot and (H) volcano plot of differential metabolites (p < 0.05) between WT and iNOS^−/− mice. Red in the volcano plot indicates significantly upregulated metabolites, green indicates the downregulated metabolites, and gray shows no significant difference. (I) Heatmap of differential metabolites found by metabolomics analysis in chow-fed WT and iNOS^−/− mice. Data are represented as mean ± SEM (n ≥ 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. WT. See also Figure S1 .

Figure 2

Vancomycin-induced modulation of gut microbiota rescues iNOS^−/− mice from systemic IR and dyslipidemia. Gut microbiota analysis in untreated and vancomycin-treated iNOS^−/− mice. α-Diversity indices in stool samples: (A) observed, (B) Shannon, (C) Simpson, and (D) Chao1. (E) PCA plot based on the Bray–Curtis distance. (F) Differentially abundant microbiota at the phylum, family, and genus levels. Systemic IR analysis in vancomycin-treated and untreated WT and iNOS^−/− mice. (G) Intraperitoneal glucose tolerance test (GTT), (H) area under the curve (AUC) calculated from IPGTT data, (I) intraperitoneal insulin tolerance test (ITT), (J) AUC calculated from ITT, (K) intraperitoneal pyruvate tolerance test (PTT), (L) AUC calculated from PTT, (M) fasting blood glucose levels, and (N) fasting serum insulin levels. Indices of insulin sensitivity: (O) HOMA-IR and (P) QUICKI. Serum lipids: (Q) total cholesterol (TC), (R) triglycerides (TG), (S) low-density lipoprotein (LDL), and (T) non-esterified free fatty acids (NEFA). Data are represented as mean ± SEM (n ≥ 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 between indicated groups. ^## p < 0.01, ^### p < 0.001 and ^#### p < 0.0001 vs. iNOS^−/−. See also Figures S2, S3, S5, S6 .

Figure 3

Vancomycin-induced alterations in serum metabolites in iNOS^−/− mice. Serum metabolomic analysis in untreated and vancomycin-treated iNOS^−/− mice in ESI (+) mode. (A) PCA score plot and (B) volcano plot of differential metabolites between iNOS^−/− mice with or without vancomycin treatment. Red in the volcano plot indicates significantly upregulated metabolites, green indicates the downregulated metabolites, and gray shows no significant difference. Heatmap of differential metabolites found by metabolomics analysis in iNOS^−/− mice with and without vancomycin treatment related to (C) nucleic acid metabolism; (D) vitamins, hormones, and bile acid metabolism; (E) carbohydrate metabolism; (F) miscellaneous/microbiota-derived metabolites; (G) amino acid metabolism; and (H) lipid metabolism. Data are represented as mean ± SEM (n ≥ 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. iNOS^−/−. See also Figures S4, S5 .

Figure 4

Improvement in the disrupted lipid and glucose homeostasis in the liver in iNOS^−/− mice following treatment with vancomycin. Hepatic lipid levels in WT and iNOS^−/− mice with and without vancomycin treatment: (A) TC, (B) TG, and (C) FFA. (D) Hepatic glycogen levels with or without insulin stimulation in WT and iNOS^−/− mice with and without vancomycin treatment. Hepatic mRNA expression analysis of genes involved in (E) gluconeogenesis, (F) lipid synthesis, (G) lipid oxidation, (H) lipid uptake, and (I) lipid efflux. Data are represented as mean ± SEM (n ≥ 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 between indicated groups. See also Figure S7 .

Figure 5

Improvement in the disrupted lipid and glucose homeostasis in adipose tissue and intestine in iNOS^−/− mice following treatment with vancomycin. Adipose tissue mRNA expression analysis of genes involved in (A) gluconeogenesis, (B) glucose homeostasis, (C) lipid synthesis, (D) lipid oxidation, and (E) lipid uptake. Intestinal tissue mRNA expression analysis of genes involved in (F) lipid synthesis, (G) lipid uptake, and (H) lipid efflux. Data are represented as mean ± SEM (n ≥ 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 between indicated groups. See also Figures S8, S9 .

Figure 6

Association of gut microbiota with metabolic parameters in iNOS^−/− mice following treatment with antibiotics. Heatmap of Pearson’s correlation coefficients between changes in different metabolic parameters and microbial taxa at the (A) phylum and (B) genus levels caused by gut microbiota modulation by vancomycin and Abx in WT and iNOS^−/− mice. *p < 0.05, **p < 0.01, ***p < 0.001 represent significant correlations between metabolic biomarker and bacterial taxa. Blue color represents negative and red positive correlations. See also Figure S11 .

Figure 7

Vancomycin-induced depletion of gram-positive gut bacteria and modulation of associated metabolites rescue the IR and dyslipidemia observed in iNOS^−/− mice. Red up or down arrows or dash represents increase, decrease, or no change in iNOS^−/− mice in comparison with WT. Violet up or down arrows or dash represents increase, decrease, or no change in vancomycin-treated iNOS^−/− mice respectively in comparison with untreated iNOS^−/−.