Inulin reduces visceral adipose tissue mass and improves glucose tolerance through altering gut metabolites

Animals and diet composition

All experimental procedures were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine (Permit number: 2020-50). Seven-week-old male C57BL/6J mice were purchased from Shimizu Laboratory Supplies (Kyoto, Japan) and acclimated under a controlled environment (12 h light/12 h dark cycle; temperature, 22–24 °C; and humidity, 30–60%) with free access to water in the animal facility of the Kyoto Prefectural University of Medicine. The mice were randomly divided into two groups at the age of 8 weeks and received either high-fat and high-sucrose based control diet (Control) or high-fat and high-sucrose based inulin diet (Inulin) for 12 weeks (n = 12 per group). The diets were formulated based on the high-fat/high-sucrose diet (D12327, Research Diets Inc., New Brunswick, NJ, USA) and modified to contain equivalent compositions of fat and sucrose in both Control and Inulin diets. The composition of experimental diets was 40% kcal fat, 32% kcal sucrose, 6% cellulose, and 7.5% kcal maltodextrin for the Control diet (D19100801, Research Diets, New Brunswick, NJ), and 40% kcal fat, 32% kcal sucrose, 6% cellulose, and 7.5% kcal inulin for the Inulin diet (D19100802, Research Diets, New Brunswick, NJ). Further details of both the diets are presented in Additional file 1: Table S1. The body weight was examined weekly for 12 weeks, and food intake was measured every day. The mice were carefully paired-fed to equalize the intake of total calories.

The mice at the age of 20 weeks were sacrificed after fasting for 3 h (n = 6 per group) or in the postprandial state (n = 6 per group) through administering 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol [17]. The feces samples were collected from individual mice through placing them into empty cages. The blood samples were collected from the portal vein in the postprandial state or cardiac cavity after fasting for 3 h, and then the plasma was separated through centrifugation (5000 rpm, 20 min, 4 °C). Epididymal and subcutaneous white adipose tissues were harvested and weighed. The content in the jejunum was collected. Adipose tissues, feces, and plasma samples were stored at − 80 °C until further use.

Indirect calorimetry

In vivo indirect open-circuit calorimetry analysis was performed when the mice were at the age of 18 weeks. The rates of oxygen consumption (VO2) and carbon dioxide production (VCO2) were assessed for 48 h during 12-h light/12-h dark cycles at the inlets and outlets of the sealed chambers with the O2/CO2 metabolism measurement system for the small animals (MM202R; Muromachi Kikai Co., Ltd., Tokyo, Japan). A constant air flow (0.6 L/min) was drawn through the chamber to maintain CO2 concentration below 0.5%. The system was controlled under a strict 12-h light/12-h dark cycle in atmospheric conditions of 22 °C and 30–60% humidity. Throughout the experiments, the mice had ad libitum access to food and water. Respiratory quotients (RQ) and energy expenditure [18] were calculated based on VO2 and VCO2 values.

Intraperitoneal glucose tolerance test (IPGTT)

In 20-week-old mice, the IPGTT was performed after fasting for 16 h. Blood glucose levels were assessed in the drops of blood collected at the following time points: 0, 30, 60, and 120 min after the intraperitoneal injection of glucose (2 g/kg body weight). The glucose concentration was measured using a glucometer (Glutest Neo Alpha; Sanwa Kagaku Kenkyusho, Nagoya, Japan). The area under the curve (AUC) of the IPGTT results was analyzed.

Histological examination

The samples of epididymal white adipose tissue (eWAT) as visceral adipose tissue and subcutaneous white adipose tissue (sWAT) were fixed with 4% paraformaldehyde, and embedded in paraffin. They were sectioned into 7-mm thick slices that were stained with hematoxylin and eosin for microscopic examination. The average cell area and lipid droplets per cell were measured using the ImageJ software according to the method described in the previous study [19]. After deparaffinization using xylene and ethanol, immunolocalization was performed in the paraffin sections of eWAT and sWAT. Primary antibodies (4 °C, 12 h) and secondary antibodies (room temperature, 1 h) were applied to the slides upon dilution with PBS. Primary antibody used was Rabbit anti-UCP1 (Abcam, 1:250), and secondary antibodies used were Alexa 488-conjugated (Invitrogen) and HRP conjugated (DAKO). Biotinylated IB4 (Vector Labs, 1:50) was used with Alexa 488-conjugated streptavidin. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Roche). Images were acquired using a BZ-X710 fluorescence microscope (Keyence, Osaka, Japan). For quantification, at least ten 10 × view fields per sample were analyzed. UCP1 expression in the white adipose tissues were quantified through measuring the cumulative pixel intensity in multiple fields of view using the ImageJ software.

Measurement of metabolites in the content of jejunum, feces, and portal vein serum

Amino acids, organic acids, and SCFAs in the content of jejunum, feces, and portal vein serum were analyzed using gas chromatography-mass spectrometry (GC/MS) on an Agilent 7890B/7000D System (Agilent Technologies, Santa Clara, CA, USA). The feces (20 mg) and serum (50 µL) samples were added in 500 μL of acetonitrile and 500 μL of diluted water and grinded in a ball mill at 4000 rpm for 2 min. Then, the samples were shaken at 1000 rpm for 30 min at 37 °C, and centrifuged at 14,000 rpm for 3 min at room temperature. The supernatant (500 uL) was separated and added in 500 μL of acetonitrile, and further shaken at 1000 rpm for 3 min at 37 °C. Then, the samples were centrifuged at room temperature for 3 min at 14,000 rpm, and adjusted for pH to 8 using 0.1 mol/L NaOH.

The concentrations of amino acids, organic acids, and SCFAs were then determined through GC/MS using the following on-line solid phase extraction (SPE) method. In the SPE-GC system SGI-M100 (AiSTI SCIENCE, Wakayama, Japan), SPE and injection into the GC/MS system are automatically performed after the sample has been added to the vial and set on the autosampler tray. Flash-SPE ACXs (AiSTI SCIENCE) were used for the solid stratification. For measuring the level of amino acids and organic acids, 50 µL aliquots of each of the aforementioned sample extracts were loaded onto the solid phase and washed with acetonitrile and water (1:1). Then, the samples were dehydrated with acetonitrile and impregnated with 4 μL of 0.5% methoxyamine-pyridine solution. Then, N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was supplied to the solid phase to perform methoxylation and trimethylsilylation while derivatization, and eluted with hexane. The final product was injected through PTV injector, LVI-S250 (AiSTI SCIENCE), and the temperature was maintained at 220 °C for 0.5 min, increased gradually 50 °C/min to 290 °C, and then held there for 16 min. The samples were loaded onto a capillary column, Vf-5 ms (30 m × 0.25 mm [inner diameter] × 0.25 μm [membrane thickness]; Agilent Technologies). The column temperature was maintained at 80 °C for 3 min, then increased gradually by 25 °C/min to 190 °C, by 3 °C/min to 220 °C and by 15 °C/min to 310 °C, which was held there for 4.6 min. The sample was injected in the split mode at a split ratio of 50:1. As for measuring SCFA, 50 μL aliquots of each of the aforementioned sample extracts were loaded onto the solid phase and washed with acetonitrile and water (1:1). Then, the samples were dehydrated with acetone and impregnated with 4 μL of N-tert-butyldimethylsilyl-N- methyltrifluoroacetamide (MTBSTFA)-toluene solution (1:3) and eluted with hexane after derivatization on the solid phase. The final product was injected through the PTV injector, LVI-S250, and the temperature was maintained at 150 °C for 0.5 min, increased gradually 25 °C/min to 290 °C, and then held there for 16 min. The samples were loaded onto a capillary column, Vf-5 ms (30 m × 0.25 mm [inner diameter] × 0.25 μm [membrane thickness]; Agilent Technologies). The column temperature was maintained at 60 °C for 3 min, increased gradually by 10 °C/min to 100 °C and 20 °C/min to 310 °C, and then held there for 7 min. The sample was injected in the split mode at a split ratio of 20:1. Each amino acid, organic acid, and SCFA were detected in the scan mode (m/z; 70–470). All results were normalized to the peak height of norleucine, adipic acid, and tetradeuteroacetic acid of 0.01 mM for each amino acid, organic acid and SCFA, respectively.

Measurement of lipidome in eWAT

The composition of fatty acids in murine adipose tissue was assessed using GC/MS, Agilent 7890B/7000D (Agilent Technologies, Santa Clara, CA). Briefly, the adipose tissue sample (15 mg) was methylated using the fatty acid methylation kit (Nacalai Tesque, Kyoto, Japan). The samples were loaded onto a capillary column, Vf-5 ms (30 m × 0.25 mm [inner diameter] × 0.25 μm [membrane thickness]; Agilent Technologies). The column temperature was maintained at 80 °C for 3 min, then increased gradually by 25 °C/min to 190 °C, by 3 °C/min to 220 °C and by 15 °C/min to 310 °C, which was held there for 4.6 min. The sample was injected in the split mode with a split ratio of 5:1. Each fatty acid methyl ester was detected in the selected ion monitoring mode. All results were normalized to the peak height of the C17:0 internal standard.

Statistical analysis

The data were analyzed using JMP version 13.0 software (SAS, Cary, NC). Statistical significance of differences between groups were determined using unpaired Student’s t test and two-way repeated-measures analysis of variance was performed to evaluate the body weight change. Analysis of covariance (ANCOVA) was used to evaluate energy expenditure [20]. A p < 0.05 were considered statistically significant. Figures were generated using the GraphPad Prism software Version 8.0 (San Diego, CA, USA).

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