Functional Gut Microbiota Remodeling Contributes to the Caloric Restriction-Induced Metabolic Improvements

Abstract

Caloric restriction (CR) stimulates development of functional beige fat and extends healthy lifespan. Here we show that compositional and functional changes in the gut microbiota contribute to a number of CR-induced metabolic improvements and promote fat browning. Mechanistically, these effects are linked to a lower expression of the key bacterial enzymes necessary for the lipid A biosynthesis, a critical lipopolysaccharide (LPS) building component. The decreased LPS dictates the tone of the innate immune response during CR, leading to increased eosinophil infiltration and anti-inflammatory macrophage polarization in fat of the CR animals. Genetic and pharmacological suppression of the LPS-TLR4 pathway or transplantation with Tlr4-/- bone-marrow-derived hematopoietic cells increases beige fat development and ameliorates diet-induced fatty liver, while Tlr4-/- or microbiota-depleted mice are resistant to further CR-stimulated metabolic alterations. These data reveal signals critical for our understanding of the microbiota-fat signaling axis during CR and provide potential new anti-obesity therapeutics.

Keywords: TLR4; beige fat; browning; caloric restriction; fatty liver; gut microbiota; innate immunity; insulin sensitivity.

Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract

Figure 1

Short-Term Caloric Restriction Changes the Gut Microbiota Composition (A and B) Principal coordinate analysis of fecal (A) (Adonis test p = 0.519 at time 0, p = 0.001 at 3 weeks, and 0.013 at 6 weeks) and cecum (B) (Adonis test, p = 0.002) samples performed by computing weighted UniFrac. Each symbol represents a fecal (A) or cecum (B) sample from caloric-restricted (CR) or their ad libitum-fed (AL) control mice at the beginning of the experiment (T0), after 3 and 6 weeks (T3 and T6, respectively) (n = 8 per group). (C) Comparison of phylum-level proportional abundance of cecum and feces of AL and CR mice at the indicated time points. (D) Heatmap showing OTUs associated with a p value of Z score (scale shown in color bar) computed on the relative abundances of the selected OTUs across AL and CR samples. An idealized tree represents the taxonomic hierarchy of their phyla and families. Final leaves are labeled with the corresponding family name. (E) Relative abundance of different phyla in feces. Each graph represents the sum of the sequences annotated with the phylum relative to all analyzed sequences. Significance in (E) was calculated using non-paired two-tailed Student’s t test.∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

Figure 2

Gut Microbiota Remodeling during Caloric Restriction Provides Metabolic Benefits (A and B) Oral glucose tolerance test (OGTT) (A) and insulin tolerance test (ITT) (B) of AL- and CR-microbiota-transplanted GF mice (n = 6 per group). AUC, area under the curve. (C–E) Glucose infusion rate (C), 2-DG glucose uptake (D), and glucose disposal (E) in AL- and CR-microbiota-transplanted GF mice (n = 6 per group). (F) Quantification of the total ingSAT and pgVAT volume and their 3D reconstitution using the MicroPET-CT scans from mice as in (A). Scale bar, 2.5 mm. (G) Weight of indicated organs post mortem of AL- and GF-transplanted mice (n = 6 per group). (H) Representative H&E-stained histological sections from ingSAT, pgVAT, and iBAT. Scale bar, 200 μm. (I) Relative mRNA expression of browning markers in ingSAT of AL- and GF-transplanted mice (n = 8 per group). (J) Representative images of immunofluorescent staining for UCP1 in ingSAT of AL- and GF-transplanted mice. Scale bar, 200 μm. (K) Relative mRNA expression of type 2 cytokines Il4 and Il13 in ingSAT of mice as in (A). (L) Quantification of eosinophils in stromal vascular fractions (SVF) from mice as in (A). (M) Quantification of CD301 fluorescence intensity in CD11b+ F4/80+ macrophages from ingSAT SVF of AL- and CR-transplanted GF mice. (N) NOS2 mean fluorescence intensity (MFI) in macrophages from ingSAT SVF as in (A). Unless otherwise indicated, error bars show mean ± SD. Significance was calculated using non-paired two-tailed Student's t test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

Figure 3

CR Gut Microbiota Transplantation Is Functional at Thermoneutrality (A and B) OGTT (A) and normalized ITT (B) of thermoneutrality housed AL- and CR-transplanted GF mice (n = 6 per group). (C) Representative H&E-stained histological sections from ingSAT of mice as in (A). Scale bar, 200 μm. (D) Relative mRNA expression of browning markers in ingSAT (q) and pgVAT (r) of mice as in (A) (n = 8 per group). (E and F) OGTT (E) and ITT (F) of mice as in AL and CR GF mice (n = 9–10 per group). (G) Relative mRNA expression of browning markers in ingSAT of mice as in (E) (n = 5–6 per group). (H) Representative H&E-stained histological sections from ingSAT of mice as in (E). Scale bar, 200 μm. (I and J) OGTT (I) and ITT (J) of mice as in AL and CR Abx-treated mice (n = 9–10 per group). (K) Relative mRNA expression of browning markers in ingSAT of mice as in (E) (n = 5–6 per group). (L) Representative H&E-stained histological sections from ingSAT of mice as in (E). Scale bar, 200 μm. Error bars show mean ± SD. Significance was calculated using non-paired two-tailed Student's t test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

Figure 4

LPS Reconstitution Reverts the Metabolic Improvements Caused by CR (A) PCoA analysis based on Bray-Curtis similarity matrix constructed using the square root-transformed relative abundance of SEED functional roles in AL and CR mice in feces (p = 0.0095) samples (p = 0.027). (B) SEED level 1 subsystems significantly different (Wilcoxon rank-sum test, p Lbp and Myd88 in liver of AL and CR mice (n = 4 per group). (F) Relative mRNA expression of browning markers in ingSAT of sham-operated and LPS-infused AL and CR mice (n = 4–6 per group). (G) Representative H&E histological sections and immunofluorescent staining for UCP1 from ingSAT of sham-operated and LPS-infused AL and CR mice. Scale bars, 100 μm. (H and I) OGTT (H) and ITT (I) of sham-operated and LPS-infused AL and CR mice (n = 6 per group). (J and K) Relative mRNA expression of browning markers in ingSAT (J) and pgVAT (K) of sham-operated AL and CR mice, and LPS-infused AL LPSlow and CR LPSlow mice (n = 5 per group). (L) Infrared temperature changes relative to initial values in eye, ventral, or dorsal regions, or eye during 3 hr of cold exposure of mice as in (J) and (K) (n = 5 per group). Unless otherwise indicated, error bars show mean ± SD and significance was calculated using non-paired two-tailed Student's t test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

Figure 5

TLR4 Suppression Leads to Beige Fat Development (A) Body weight gain of HCD mice injected daily with either 10% DMSO (HCD Vehicle) or CLI-095 (HCD CLI-095) for 5 weeks (n = 10). (B and C) OGTT (B) and normalized ITT (C) of mice as in (A) (n = 10). (D) Infrared temperature changes relative to initial values in eye, ventral, or dorsal regions, or eye during 3 hr of cold exposure of HCD mice injected daily with either 10% DMSO (HCD Vehicle) or CLI-095 (HCD CLI-095) for 5 weeks (n = 5 per group). (E) Weight of indicated organs post mortem of mice as in (A) (n = 10 per group). (F–H) Transversal [18F]FDG PET-CT images (F), standardized uptake values (SUV) of radiolabeled tracer [18F]FDG in ingSAT (G), and quantification of the total ingSAT and pgVAT volume (H) using MicroPET-CT on mice as in (A) (n = 5). (I) Representative H&E-stained histological sections from ingSAT of HCD vehicle and CLI-095 mice. Scale bar, 200 μm. (J) Relative mRNA expression of browning markers in ingSAT of HCD Vehicle and CLI-095 mice (n = 5 per group). (K) OGTT in 8-week-old wild-type (WT) and Tlr4 KO mice (n = 5–6 per group). (L) Fat/lean mass ratio in WT and Tlr4 KO mice at the indicated ages (n = 3–12 per group). (M) ITT in 8-week-old WT and Tlr4 KO mice (n = 5–6 per group). (N and O) Relative mRNA expression of browning markers in ingSAT (N) and pgVAT (O) of WT and Tlr4−/− mice (n = 6 per group). (P) Representative images of immunofluorescent staining of UCP1 in ingSAT of WT and Tlr4 KO mice. Scale bar, 200 μm. (Q) Oxygen consumption rate (OCR) in ingSAT biopsies from WT and Tlr4 KO mice detected with Seahorse analyzer. Iso, isoproterenol (n = 4 mice per group assayed in quadruplicates). (R) Relative mRNA expression of browning markers in ingSAT of AL and CR Tlr4 KO mice (n = 5 per group). (S and T) Quantification of CD11b+Siglec F+ eosinophils (S) and CD301 mean fluorescence intensity (MFI; T) in CD11b+ F4/80+ macrophages in ingSAT SVF of WT and Tlr4 KO mice (n = 5 per group). Unless otherwise indicated, error bars show mean ± SD. Significance was calculated using non-paired two-tailed Student's t test.∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

Figure 6

Hematopoietic Tlr4 Deficiency Promotes Browning (A) OGTT of WT (left) and Tlr4 KO (right) mice lethally irradiated and subsequently transplanted with WT and KO bone marrow (n = 5–9 per group). (B) Infrared eye temperature readings relative to initial values during 12-hr cold-exposed WT (left) and KO (right) BMT mice (n = 5–9 per group). Error bars show mean ± SEM. (C) Representative H&E-stained histological sections from ingSAT and pgVAT of BMT mice (n = 5–9 per group). Scale bars, 200 μm. (D) Weight of fat depots post mortem in BMT mice (n = 5–9 per group). (E and F) Relative mRNA expression of browning markers in ingSAT (E) and pgVAT (F) in BMT mice (n = 5–9 per group). Unless otherwise indicated, error bars show mean ± SD. Significance was calculated using non-paired two-tailed Student's t test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

Figure 7

iAP Induces Browning and Improves Metabolic Health During HCD (A and B) Colon mucin layer thickness quantification (A) and representative Alcian blue-stained (arrows) sections (B) of AL and CR mice (n = 6 per group). Scale bar, 200 μm. (C–G) Relative abundance of LpxA (C), LpxL (D), KdtA (E), LpxB (F), and LpxK (G) involved in lipid A biosynthesis detected by metagenomics analysis as level 4 functions in fecal samples from AL and CR mice (n = 8 per group). (H and I) Endotoxin levels in serum (H) and feces (I) of AL and CR mice determined by limulus amebocyte lysate (LAL) colorimetric assay (n = 6–11 per group). (J and K) Endotoxin levels in serum (J) and feces (K) of HCD-fed control and iAP mice determined by LAL colorimetric assay (n = 6–8 per group). (L and M) OGTT (L) and ITT (M) of HCD control and iAP mice (n = 12 per group). (N and O) Glucose infusion rate (N) and 2-[14C]DG uptake (O) in indicated tissues during hyperinsulinemic-euglycemic clamp. Error bars show mean ± SEM (n = 8). (P) Representative H&E-stained histological sections from livers of the indicated mice. Scale bars, 100 μm. (Q and R) Liver triglyceride content in HCD vehicle and CLI-095 (Q) of control and iAP (R) mice. (S) Representative H&E-stained histological sections from livers of the indicated BMT mice. Scale bars, 100 μm. (T) Liver triglyceride content in HCD BMT mice (e). (U) Weight of indicated organs post mortem of HCD control and iAP mice (n = 12). (V) Representative H&E-stained histological sections from ingSAT of HCD control and iAP mice. Scale bar, 200 μm. (W and X) Relative mRNA expression of browning markers (W) and type 2 cytokines (X) in ingSAT of HCD control and iAP mice (n = 6). Unless otherwise indicated, error bars show mean ± SD. Significance was calculated using non-paired two-tailed Student's t test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; n.s., not significant.