Lactobacillus reuteri ZJ617 attenuates metabolic syndrome via microbiota-derived spermidine

0
Lactobacillus reuteri ZJ617 attenuates metabolic syndrome via microbiota-derived spermidine

Ethics declarations

All animal experiments were performed in accordance with the “Regulation for the Use of Experimental Animals” of Zhejiang Province, China, and approved by the Animal Care and Use Committee of Zhejiang University (Ethics Code Permit Number: ZJU20170529; ZJU20240770). Fecal samples from healthy volunteers with obesity (BMI ≥28) was performed with the approval of the Ethical Committee of The Second Affiliated Hospital of Zhejiang University (Ethics Code Permit Number: 2024-1195). All participants provided written informed consent for sample collection and subsequent experiments.

Animals and L. reuteri ZJ617 administration

Four-week-old male, specific pathogen-free (SPF) C57BL/6J mice were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Before the onset of the experiment, they were acclimatized to the environment (23 ± 1 °C, 12-h light-dark cycle, 50% ± 5 humidity) with free and unlimited access to food and drinking water for one week. Then mice were randomly assigned into three groups (3 cages/group): LFD (Low-fat diet, containing 70% kcal from carbohydrate and 10% kcal from fat (~5.5% kcal from soybean Oil and ~4.5% from kcal Lard); PD450J), HFD (High-fat diet, containing 20% kcal from carbohydrate and 60% kcal from fat (~5.5% kcal from soybean Oil and ~54.5% kcal from Lard); PD6001) and HFD supplement with L. reuteri ZJ617. All diets were from Changzhou SYSE Bio-Tec. Co., Ltd. (Changzhou, China), and the diet composition is given in Supplementary Table 1. The mice were allowed free access to food and were maintained for 14 weeks.

L. reuteri ZJ617 had previously been isolated from piglet small intestines and stored in 20% glycerol at −80 °C until usage. The strain was anaerobically cultured for 18 h in sterilized De Man Rogosa and Sharpe (MRS) medium at 37 °C. Cultures were collected in the log phase and diluted to 109 colony-forming units/mL in drinking water. There was no significant difference in the number of alive L. reuteri ZJ617, after placement in water for 24 h. The drinking water was refreshed every day. The abundance of Lactobacillus was calculated using plate colony-counting.

Fecal microbiota transplantation

The procedure of fecal microbiota transplantation was done according to a previous study and modified47. Feces were collected from the donor LFD, HFD, and HFD + ZJ617 groups, respectively, at the end of week 14, and the pooled sample in each group was used for the following experiment. The fecal pellets were diluted with sterile PBS (100 mg/mL). The mixtures were centrifuged at 1000×g, 4 °C for 5 min. The supernatant was transferred into a new tube and centrifuged for 5 min at 15,000×g to precipitate the bacteria. Then, the bacterial pellets were resuspended in 600 μL sterile PBS, and an equal volume of 40% sterile glycerol was added. The resuspended bacteria were stored at -80 °C for later use with transplantation.

Four-week-old male C57BL/6J mice were acclimatized for 1 week, then their intestinal microbiota was first depleted by an antibiotic mixture ((vancomycin (0.5 g/L), neomycin sulfate (1 g/L), metronidazole (1 g/L), and ampicillin (1 g/L)) in drinking water for 1 week. Then, the 100 μL of bacteria suspension was transplanted to the mice that had been pre-treated with antibiotics via oral gavage for 14 weeks (three times per week). All mice were fed the same diet, HFD, for 14 weeks.

Histological analysis

After euthanasia, the liver and adipose tissues of mice were preserved in 4% paraformaldehyde (4% PFA). The fixed paraffin-embedded sample sections were stained with hematoxylin and eosin (H&E) to assess liver steatosis and adipocyte size, respectively, followed by microscopical examination. All processes were performed according to the manufacturer’s instructions. Adipocyte sizes were quantified by Cellpose 2.0 v and Image J 1.52 v. The slices were collected and evaluated in a blinded manner.

Transmission electron microscope

Adipose tissues were preserved in 2.5% glutaraldehyde for more than 4 h and then were post-fixed with 1% OsO4. The double-fixed Spurr resin-embedded sample sections were stained by uranyl acetate and alkaline lead citrate for 5 min, respectively, and then mitochondria were observed with a TEM (Hitachi Model H-7650). All processes were performed according to the instructions of the Bio-ultrastructure analysis Laboratory of Analysis center of Agrobiology and environmental sciences, Zhejiang University. The slices were collected and evaluated in a blinded manner.

Glucose homeostasis

For oral glucose tolerance testing (OGTT), all mice were placed in clean cages and provided with water but without food. After 6 h of fasting, the mice were administrated an oral glucose (2 g/kg, Sigma-Aldrich, 158968). Blood glucose levels were measured before and after oral glucose administration at 15, 30, 60, 90, and 120 min and determined with a glucose meter (Accu-Chek Performa, Roche) on blood samples collected from the tip of the tail vein during week 12.

For insulin tolerance testing (ITT), all mice were placed in clean cages and provided with water but no food. After five hours of fasting, the mice were injected with insulin (0.75 U/kg, Sigma-Aldrich, I2643), and blood glucose levels were measured before and after injection at 15, 30, 60, 90, and 120 min after injection during week 13.

Biochemical analysis

Blood samples were centrifuged at 3000 × g for 15 min at 4 °C to produce serum samples. The levels of serum AST, ALT, TG, TC, and LDL-C were determined using the corresponding assay kits (C010-2-1, C009-2-1, A110-1-1, A111-1-1, and A113-1-1, respectively; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Serum insulin levels were measured using ELISA kits (H203-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Liver tissues were homogenized with ethanol and centrifugated at 2500×g for 10 min at 4 °C, and the supernatant was used to determine TG. All indexes were used a multifunctional microplate reader (TECAN, Switzerland), according to the manufacturer’s protocols.

Real-time qPCR

Total RNA was extracted using a SteadyPure Universal RNA Extraction kit (Accurate Biotechnology, AG21017, China) according to the manufacturer’s protocols. RNA concentrations were equalized and converted to cDNA using a kit (Accurate Biotechnology, AG11728, China). Gene expression was measured by qPCR detection (Bio-Rad, CFX96, USA) systems using SYBR Green (Accurate Biotechnology, AG11701, China). Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control. The primer is given in Supplementary Table 2.

Western blot

White adipose tissue and cells were lysed with RIPA buffer containing PMSF, proteinase inhibitor, and phosphatase inhibitors (Beyotime Technology, Shanghai, China) in an ice bath followed by centrifugation at ~13,000 × g for 10 min. The supernatant was collected and denatured in a sample buffer and resolved with SDS-PAGE. Samples were transferred to PVDF membranes with a pore size of 0.45 μM. Individual proteins were detected using 1:1000 dilution of primary antibodies, as follows: ADRB3 (1:1000, Abcam, ab94506, polyclonal antibody), UCP1 (1:1000, Abcam, ab234430, polyclonal antibody) and β-actin (1:1000, Cell Signaling Technology, 4970, polyclonal antibody). Proteins were visualized on film with horseradish peroxidase–conjugated secondary antibodies diluted 1:5000.

16S rDNA amplicon sequencing

The V3-V4 region of the bacteria 16S ribosomal RNA genes were amplified using primers 341 F 5′-CCTACGGGRSGCAGCAG)-3′ and 806 R 5′-GGACTACVVGGGTATCTAATC-3′. PCRs were performed, and the library was constructed using Qubit. The purified amplicons were pooled in equimolar amounts, and paired-end sequenced on an Illumina MiSeq PE250 platform (Study1)/NovaSeq PE250 platform (Study2) (Illumina, San Diego, USA) according to the manufacturer’s protocols. ASVs were selected via standard clustering with 100% similarity using Deblur. Each representative tag was assigned to a taxon using the RDP Classifier. The QIIME2 was implemented for ASVs profiling.

PICRUSt2 for prediction of metagenome functions

PICRUSt2 was utilized to predict metagenome functions48. Gene banks such as Kyoto Encyclopedia of Genes and Genomes (KEGG Orthologous, KO), Enzyme Commission numbers (EC no.), and Clusters of Orthologous Groups (COG) were used to support functional gene and pathway profiles. All predicted pathways and differently expressed genes were obtained using a Wilcoxon test.

Metabolomic profiling of intestinal contents

For metabolomic analysis, intestinal contents were collected and immediately snap-frozen in liquid nitrogen. The metabolites were analyzed by gas chromatography time-of-flight mass spectrometer (GC-TOF-MS), according to our previous study49. The pre-weighted intestinal contents were extracted and homogenized with a pre-cold mixture (methanol/chloroform, v: v = 3:1). The centrifuged and speed-vacuum-dried metabolites were solubilized in 40 μL of methoxyamination hydrochloride (20 mg/mL in pyridine) and then incubated at 80 °C for 30 min, then derivatized by 60 μL of BSTFA regent (1% TMCS, v/v) at 80 °C for 1.5 h. The cooled samples were mixed with fatty acid methyl esters (FAMEs; 5 μL in chloroform) and injected into the GC-TOF-MS system for detection and analysis.

For the data of the metabolites of intestinal contents, the abundance differences were obtained using a Wilcoxon test. OPLS-DA was conducted to identify the discrimination of variables. Differential metabolites were defined as those with variable importance in the projection (VIP) >1.0 obtained from OPLS-DA and P < 0.05.

In vitro gastrointestinal tract simulation

In vitro fecal microbiota fermentation was conducted with a gastrointestinal tract simulation system as described previously with some modification50,51. The feces were collected from six adult volunteers with obesity (BMI ≥28, aged 20–25) who had not taken any antibiotics and probiotics in the past 6 months. The fecal slurry (1:9; w/v) was prepared by diluting the homogenized fecal sample in sterile PBS medium, which was added into the colon simulation bioreactor with nutrient medium (1:9; v/v). After 24 h of inoculation and culture, fresh nutrient medium was added into the bioreactor and waste was drained at a certain rate (Stage 1) to maintain the normal growth of microorganisms. L. reuteri ZJ617 (109 CFU) and S-adenosylmethionine (0.05% w/v; equal to 0.8 g/day for an adult according to previous study52) was added to the reactor. The fermentation lasted for 6 days (Stage 2). During the entire fermentation process, the culture was automatically supplemented with 0.5 mol/L NaOH solution and diluted with HCl to adjust the pH to 6.8. The speed of the stirring rotor was set at 80 r/min. The temperature was kept at 37 °C modulated by the heating and cooling system. Anaerobic conditions were generated by flushing the headspace of all reactions and medium vessels with N2 for 30 min and three times per day. Each sample was collected from three independent fermentation waste extraction at the beginning and end of stage 2 for spermidine-producing bacteria or spermidine determination.

Bacteroides
uniformis

Bacteroides uniformis were obtained from our previous study53. Briefly, the experiment consisted of three groups: medium only (Control), medium supplemented with 10 μM S-adenosylmethionine (SAM), and medium supplemented with SAM and B. uniformis (B. uniformis + SAM). All groups were placed in a vinyl anaerobic chamber (Coy Drive 14,500, USA) and anaerobically cultured at 37 °C for 48 h. At the end of the experiment, all samples were harvested for spermidine determination.

Concentration of spermidine

The concentration of spermidine in intestinal contents, serum, adipose tissue, and bacteria fermentation supernatant was analyzed by UHPLC-MS/MS or HPLC, as previously described and modified54. The weighted intestinal contents, serum and adipose tissues and the bacteria fermentation supernatant were extracted and homogenized with ice-cold perchloric acid solution (0.4 mol/L), the bacteria were disrupted by sonication. All samples were derivatized after pH was adjusted to 9.0. Dansyl chloride acetone solution (10 mg/mL, Aladdin, D133513) was applied for derivatization. Nitrogen-dried metabolites were re-solubilized in HPLC-grade acetonitrile (Sigma-Aldrich, 34851) and injected into the HPLC system while obtaining spermidine ion traces.

Spermidine supplementation and inhibitor treatment

Four-week-old male C57BL/6J mice were acclimatized for one week. HFD-fed mice were supplemented with spermidine (20 mg/kg) in drinking water for 14 weeks, The dosage was determined based on a previous study35. The drinking water was refreshed every day. The concentration of spermidine in drinking water was adjusted as w/v based on the changes in body weight and water consumption. For the spermidine synthesis inhibitor, HFD-fed mice were supplemented with L. reuteri ZJ617 and oral gavage with diminazene aceturate (2.5 mg/kg) daily for 14 weeks. The mice were allowed free access to food and were maintained for 14 weeks.

Metabolic rate measurements

The mouse metabolic rate was assessed after 4 weeks of treatment with L.reuteri ZJ617 or 14 weeks of treatment with spermidine, using the TSE PhenoMaster System (TSE PhenoMaster, Germany) following the manufacturer’s instructions. Before the metabolic rate was monitored, mice were individually caged for 24 h to acclimate to the system.

Cell culture and staining

The 3T3-L1 preadipocytes (SCSP-5038) were purchased from the National Collection of Authenticated Cell Cultures, grown in high-glucose DMEM (Gibco) supplemented with 10% calf serum (Sigma-Aldrich, B7447) and 1% penicillin-streptomycin at 37 °C in an incubator containing 5% CO2, followed by feeding with fresh medium every 2 days to reach confluence. For beige adipocyte differentiation, the cells were induced with an induction medium containing DMEM, 10% FBS, 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, 4.5 μg/mL insulin, and 1 μM rosiglitazone with or without spermidine or PBS for 48 h, and further growth medium was supplemented with insulin with or without spermidine or PBS every 2 days for 6 days. The cultured 3T3-L1 adipocytes were stained using an Oil Red O staining Kit (Solarbio, G1262, China) according to the manufacturer’s protocols. The images of the ORO staining were recorded with a microscope and quantified by Image J 1.52v. The images were collected and evaluated in a blinded manner.

Statistics and reproducibility

For assessing differences between the two groups, a two-tailed t-test was performed. For differences among more than two groups, a one-way analysis variance (ANOVA) was performed, followed by Dunnett tests (Study1 and Study2) or Šídák tests (Study3). All data are shown as means ± SD. Statistical analyses were performed using GraphPad Prism 9.0v (USA). P < 0.05 was considered statistically significant. Results from representative experiments (such as micrographs) were obtained from at least three independent fields of view with similar results.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

link

Leave a Reply

Your email address will not be published. Required fields are marked *