Chemicals and reagents
Caffeoylquinic acid, bacitracin, neomycin, streptomycin, and vancomycin were purchased from Dalian Meilun Biotech Co. Ltd (Dalian, China). Sodium propionate was purchased from Aladdin Reagent (Shanghai, China). MCT inhibitor 7ACC1 was purchased from MedChem Express (New Jersey, USA). SCFA standards for LC–MS analysis including acetic, propionic, butyric, and lactic acids were obtained from Macklin Reagent (Shanghai, China). O-Benzylhydroxylamine hydroxylamine hydrochloride (O-BHA), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), and d3-AA were purchased from Sigma-Aldrich (Missouri, USA). CER (17:0) was purchased from Avanti Polar Lipids (Alabaster, USA). UCP1 antibody (GB112174) was purchased from Servicebio (Wuhan, China). MCT1 antibody (20,139–1-AP) and MCT4 antibody (22,787–1-AP) were purchased from Proteintech (Wuhan, China). α-Tubulin antibody (SC-5286) was purchased from Santa Cruz (CA, USA). HRP-conjugated anti-mouse IgG (7076S) and anti-rabbit IgG (7074S) were purchased from Cell Signaling Technology (MA, USA). A high-fat diet (HFD, 60 kcal%, D12492) and a matched low-fat diet (LFD, 10 kcal%, D12450J) were purchased from Research Diets Inc. (New Jersey, USA).
Animal studies
Six- to eight-week-old C57BL/6 J male mice were purchased from HuaFukang BioScience Company (Beijing, China). UCP1 knockout (UCP1 KO) mice and the control wild-type (WT) mice were kindly provided by Professor Jiqiu Wang at Shanghai Jiao Tong University School of Medicine. All the mice were housed in a pathogen-free facility under a 12-h dark–light cycle with ad libitum access to food and water.
For chronic treatment, sample size estimation was performed before experiments. Adult male C57BL/6 J mice were exposed to a HFD or LFD diet for 8 weeks. After 8 weeks of feeding, mice were randomized and assigned to the following treatment groups: HFD-V, HFD-CQA (150 mg/kg/day), and LFD, and then subjected to daily intragastric treatment for an additional 8 weeks. UCP1 KO mice and control wild-type mice were exposed to a HFD diet for 4 weeks, and then subjected to daily intragastric treatment for an additional 4 weeks. CQA was dissolved in saline, and the pH was adjusted to 5–6 using sodium hydroxide. During the treatment, the body weight and food/water intake were monitored.
For gut microbiota depletion treatment, mice were administered the antibiotics cocktail (bacitracin, neomycin, and streptomycin) in drinking water at 0.1% (w/v) of each compound along with saline or 150 mg/kg CQA daily.
For fecal transplantation, donor mice were treated as described for chronic treatment. After 2 weeks of treatment, feces were collected twice per week for the subsequent 8 weeks under a laminar flow hood under sterile conditions. Feces from donor mice of each group were pooled and resuspended in sterile ice-cold saline. The solution was vigorously vortexed for 30 s, before natural sedimentation for 1 min. The supernatant was collected within 10 min before oral gavage to prevent changes in bacterial composition. Recipient mice were fed a HFD for 7 weeks, followed by 5-day antibiotics cocktail treatment, and then transplanted twice per week with fresh fecal slurry by oral gavage for 8 weeks.
For selective antibiotics treatment, DIO mice were fed with HFD for 10 weeks, followed by giving the antibiotic (0.1% (w/v) bacitracin, or 0.05% (w/v) vancomycin) in drinking water, respectively, along with daily administration of saline or 150 mg/kg CQA for 4 weeks.
For simplified microbial community colonization, DIO mice were colonized with a simplified bacterial community (Community 1) consisting of Lachnospiraceae bacterium, Clostridium ramosum, Limosilactobacillus reuteri, Bacteroides acidifaciens, Escherichia coli, and Bifidobacterium animalis subsp. lactis. A second group of DIO mice was colonized with Community 2, which consists similar bacterial members except for L. reuteri. Bacterial strains of the microbial community were selected based on the following criteria: reported to be altered in obesity patients or to affect experimental obesity, showed high abundance in 16S rRNA amplicon sequencing data of DIO mice, and were able to form a stable community in rodents. DIO mice were fed with HFD for 9 weeks, followed by 5-day antibiotics cocktail treatment, before being colonized with 1.0 × 109 CFU/mice by oral gavage twice per week for 4 weeks. The composition of the bacterial consortia differed between Community 1 and 2 as shown in Fig. 5j. Along with colonization, the mice were daily administrated of saline or 150 mg/kg CQA for 4 weeks.
For single-microbe treatment, DIO mice were fed with HFD for 7 weeks, followed by 5-day antibiotics cocktail treatment prior to assignment to the following four treatment groups: HFD-V (gavaged with saline), HFD-L. reuteri (108 CFU/mouse, suspended in saline), and HFD-L. lactis (108 CFU/mouse, suspended in saline). Each group was treated twice per week for 8 weeks.
For co-administration, DIO mice were fed with HFD for 4 weeks, followed by 5-days antibiotics cocktail-treatment prior to assignment to the following four treatment groups: HFD-V (gavaged with saline), HFD-CQA (50 mg/kg), HFD-L. reuteri (108 CFU/mouse), and HFD-L. reuteri + CQA. Each group was treated twice per week for 5 weeks.
For metabolite replenishment and inhibitor treatment, DIO mice were fed with HFD for 3 weeks, followed by 5-day antibiotics cocktail treatment prior to assignment to the following six treatment groups: HFD-V (gavaged with saline), HFD-RQ (108 CFU of L. reuteri plus 50 mg/kg CQA), HFD-PA (gavaged with 1.8% sodium propionate in drinking water, w/v) with or without 7ACC1 (0.3 mg/kg/d, i.p.) treatment for 4 weeks.
For adeno-associated virus (AAV) injections, DIO mice were fed with HFD for 10 weeks, followed by 2.0 × 1011 vg virus-packed mouse Mct1 (mMct1)-shRNA (AAV-shMct1) or control (AAV-shScramble) (Vector OBiO, Shanghai, China) injection at multiple sites on the inguinal fat pads. Three weeks after injection, antibiotics cocktail-treated mice were given L. reuteri (108 CFU/mouse) plus CQA (50 mg/kg) or saline twice per week for 3 weeks.
Glucose tolerance test
Glucose tolerance tests were performed after 16-h fasting. Blood samples were taken from the tail tip at 0, 15, 30, 60, 90, and 120 min after oral gavage of glucose (2 g/kg body weight), and blood glucose concentrations were measured with blood sugar test paper and glucometer (Sannuo Biosensors, China).
Insulin tolerance test
For the insulin tolerance test, insulin (0.75 U/kg body weight) was administered via an intraperitoneal injection after 4-h fasting. All of the ITT tests were performed at indicated times the same as GTT.
Body composition and indirect calorimetry
Body fat and lean mass were determined by 1H-NMR spectroscopy (Minispec LF90 II, Bruker, Karlsruhe, Germany) following the manufacturer’s protocol. Indirect calorimetry was performed on mice after 8-week CQA treatment using a 16-chamber indirect calorimeter (TSE PhenoMaster, TSE system, Germany) with one mouse per chamber as previously described [51]. Oxygen consumption (VO2) and carbon dioxide production (VCO2) were recorded, and heat production and RER (VCO2/VO2 ratio) were calculated. Mass-adjusted metabolic rate (MR) ratios were tested by global regression-based analysis-of-covariance (ANCOVA) using body mass as a covariate.
Core temperature measurement
Mice were exposed to acute cold stress (4 ± 1 °C) in the artificial climate chamber (Yanghui, Ningbo, China) for 4 h, and their rectal temperature was measured at the indicated time points with a temperature feedback system (RWD; Shenzhen, Guangdong, China).
Fasting blood glucose and fasting insulin assays
Fasting blood glucose was measured after 8-h fasting. Serum fasting insulin concentration was assessed in a 96-well microplate using an insulin mouse ELISA kit purchased from ThermoFisher (PA, USA). Homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated as follows:
$$\text{HOMA-IR} = {\text{FBG}} \times \text{FINS/22.5}$$
$$\text{(FBG, mmol/L and FINS, mIU/L)}$$
Biochemical analyses
For serum indices, levels of triglyceride, T-CHO, HDL-C, and LDL-C were measured according to the instruction of each assay kit (Jiancheng, Nanjing, China). For analysis of liver lipid content, 20 mg of the frozen liver was homogenized using a hybrid grinding machine (Biheng Bio-Technique Co. Ltd., Shanghai, China) in 10% (w/v) 50 mmol/L Tris with 1% Triton X-100. After centrifugation, supernatants were quantified using each assay kit (Jiancheng, Nanjing, China).
RNA extraction and quantification
Total RNA was isolated from tissues with TRIzol reagent (Invitrogen, California, USA). cDNA was synthesized with PrimeScript Reverse Transcriptase (Takara Bio, Japan) according to the manufacturer’s instructions. Real-time PCR was performed using SYBR Premix Ex Taq (Takara Bio, Japan) on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad, CA, USA). The results were analyzed by the ΔCt method and normalized to reference genes. The sequence of primers is shown in Supplemental table 1.
Protein extraction and western blotting
Total protein was extracted using RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) and phosphorylase inhibitor. Protein concentration was determined using the BCA Protein Assay Kit. Protein from the indicated samples was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane. The blots were incubated with the respective primary antibody. After washing, anti-rabbit IgG was used as the secondary antibody. The blots were rewashed and developed by chemiluminescence (Immobilon Western Chemiluminescent HRP substrate, Millipore, MA, USA).
Histology and microscopy
H&E staining or Oil red O staining was performed on formalin-fixed paraffin-embedded or OCT-embedded sections using a standard protocol, respectively. Brightfield images were scanned with the NanoZoomer 2.0-HT slide scanner (Hamamatsu, Japan) at different magnifications.
Immunohistochemical staining was performed on formalin-fixed paraffin-embedded sections using a standard protocol. Briefly, slides were dewaxed with xylene 3 times for 15 min each, hydrated through an alcohol gradient, and soaked in DPBS for 5 min. Antigen was recovered by pre-heating in the EDTA buffer (pH 8.0). Endogenous peroxidase activity was blocked with 3% H2O2 in PBS for 25 min. Non-specific binding sites were blocked using 3% bovine serum albumin in PBS. The slides were incubated overnight at 4 °C with rabbit polyclonal anti-UCP1 primary antibody diluted at 1:200 in PBS. According to the manufacturer’s instructions, the slides were rinsed in distilled water, followed by treatment with a secondary antibody (HRP labeled). Immuno-visualization was performed with 3,3’-diaminobenzidine (DAB) as substrate and counterstained with hematoxylin. Slide digital images were scanned with the NanoZoomer 2.0-HT slide scanner (Hamamatsu, Japan) at different magnifications.
DNA isolation and 16S rRNA amplicon sequencing
Cecum contents or fecal samples of mice were collected and immediately frozen at − 80 °C. Metagenomic DNA was extracted using a fecal DNA isolation kit (QIAGEN, Dusseldorf, Germany). The concentration and quality of the DNA were assessed using a NanoDrop instrument and agarose gel electrophoresis, respectively. Sterile water was served as the negative control.
The universal primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′) were used to amplify the V3-V4 regions of the 16S rRNA gene. All PCRs were performed with 15 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs, USA), 0.2 μmol/L of forwards and reverse primers, and approximately 10 ng of template DNA. Thermal cycling consisted of initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and elongation at 72 °C for 30 s. Finally, the samples were incubated at 72 °C for 5 min. Sequencing libraries were generated using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer’s recommendations, and sequenced on the NovaSeq PE250 platform (Illumina, USA).
16S rRNA gene sequencing analysis was performed using QIIME (Quantitative Insights Into Microbial Ecology, http://qiime.org/). Briefly, sequence analysis was performed by Uparse software (v7.0.1001) for operational taxonomic units (OTUs) production. Each representative sequence was then assigned taxonomy against SILVA 132 database with 97% identity. The Mothur method and the SSUrRNA database were used to assign taxonomic categories to all OTUs at a confidence threshold of 0.8. Analyses for alpha diversity indices were calculated with QIIME and displayed with R programming language (v2.15.3). Cluster analysis was performed by principal component analysis (PCA) using the stats package (v3.5.0) and ggplot2 package (v3.2.0) in R programming language. Bacterial taxonomic profiling is displayed in a bar plot at the phylum level and in a heatmap at the species level using stats package (v3.6.3) and pheatmap package (v1.0.12). A linear discriminant analysis (LDA) column chart was generated to detect differentially abundant taxa across groups using the OmicStudio tools (https://www.omicstudio.cn/tool/).
Bacterial strains and culture conditions
Limosilactobacillus reuteri subsp. reuteri (ATCC 23272) and Lactococcus lactis subsp. lactis (ATCC 19435) were grown at 37 °C under anaerobic conditions (anaerobic gas mixture, 80% N2, 10% CO2, and 10% H2) in MRS (Hope Bio-Technology Co., Ltd., China) broth. Lachnospiraceae bacterium (BNCC 354474), Clostridium ramosum (ATCC 25582), and Bacteroides acidifaciens (BNCC 353574) were grown at 37 °C under anaerobic conditions in trypticase soy broth with defibrinated sheep blood (ATCC® Medium #260). Escherichia coli (ATCC 25922) was aerobically grown at 37 °C in Luria-Bertani medium. Bifidobacterium animalis subsp. lactis (JCM 10602) was grown at 37 °C under anaerobic conditions in BBL medium (Hope Bio-Technology Co., Ltd., China). Microbial growth was monitored by measuring the optical density (OD600) and the growth curve was plotted. Common antibiotics, including neomycin, streptomycin, bacitracin, and vancomycin, were added separately for screening the susceptibility of L. reuteri. The feces from DIO mice were homogenized and cultured at 37 °C under anaerobic conditions in GAM (Gifu Anaerobic Medium, Hope Bio-Technology Co., Ltd. China) broth.
Lipidomics
Serum lipidomics was performed based on an established protocol [52]. Briefly, a 50 μL aliquot of serum was extracted with 200 μL of ice-cold chloroform: methanol (2:1, v/v) solution containing CER (17:0) as an internal standard. The samples were vibrated and incubated for 20 min at 37 °C. The organic and aqueous phases were separated by centrifugation at 14,000 rpm for 15 min. The lower organic phase was transferred to another clean tube and evaporated to dryness at room temperature under vacuum. The residue was dissolved in 50 μL of chloroform: methanol (1:1, v/v) and then diluted with isopropanol: acetonitrile: H2O (2:1:1, v/v/v) before LC/MS analysis. Lipidomics analysis was performed on a Waters UPLC-ESI-Q/TOF MS (Waters Corp., MA) equipped with an electrospray ionization source. Separation was achieved on an Acquity UPLC CSH C18 column (100 × 2.1 mm, 1.7 μm, Waters Corp.). The mobile phase was a water/acetonitrile solution containing 10 mmol/L ammonium formate (2:3, A phase) and acetonitrile/isopropanol containing 10 mmol/L ammonium formate (1:9, B phase). The column temperature was maintained at 55 °C and the flow rate was 0.4 mL/min. Mass spectrometry data were acquired in negative ESI mode at a range of m/z 100–1500.
Short-chain fatty acids analysis
The method of SCFAs determination was modified based on an established protocol [53]. Briefly, for feces or cecum contents, 10 mg samples were homogenized in 0.2 mL of extraction solution (200 μmol/L d3-AA as an internal standard in 50% aqueous methanol, 1:20, w:v) for 5 min, followed by centrifugation at 14,000 rpm, 4 °C for 10 min. An aliquot of 80 μL of supernatant was incubated with 10 μL of 0.1 mol/L O-BHA in methanol and 10 μL of 0.25 mol/L EDC in methanol at 25 °C for 1 h. After the incubation, the slurry exact was diluted by twofold in 50% aqueous methanol. An aliquot of 200 μL of diluted sample was extracted by 600 μL of chloroform with 10 min of vigorous shaking. After the centrifugation, 100 μL of the lower organic layer was transferred and evaporated to the dryness by a MiniVac system (Gene Company Limited, USA). The residue was reconstituted in 100 μL of 50% aqueous methanol, briefly vortexed, and centrifuged. Then, 5 μL of the sample was injected for LC–MS/MS. For microbial supernatant extraction, 20 μL of supernatant was precipitated by adding 60 μL of extraction solution. The resulting mixture was shaken briefly and centrifuged at 14, 000 rpm for 10 min at 4 °C. Then, 60 μL of supernatant was mixed with 20 μL of water prior to the derivatization and reconstitution described above.
A Thermo Scientific™ Vanquish™ Flex UHPLC system (Thermo Fisher Scientific, Germering, Germany) was coupled with an Orbitrap Elite™ mass spectrometer (LC–MS/MS). Chromatographic separation was performed on an Acquity UPLC® BEH C18, 50 × 2.1 mm, with a particle size of 1.7 μm (Waters, USA). Here, 0.1% formic acid in water with 10 mmol/L ammonium formate (phase A) and 0.1% FA in methanol: isopropanol (9:1 v/v, phase B) served as mobile phases with a flow rate of 0.4 mL/min. The separation was achieved by gradient elution. Mass spectrometry data were acquired in positive ESI modes at a range of m/z 100–1000.
Statistical analysis
The data obtained are presented as the mean ± SD and displayed using GraphPad Prism 8.0 program (GraphPad Software, San Diego, Canada). When comparing two groups, statistical significance was determined using a two-tailed Student’s t-test. When more than two groups were investigated, a one-way analysis of variance with Tukey’s correction was applied for comparisons between different groups. The QIIME platform (v1.9.1) and R language packages were used for microbiota analysis. P values < 0.05 were considered statistically significant.