Procedures

Procedure 1: Diet administration

Reagents and solutions

• Standard rodent chow (CD), 24% protein, 18% fat, 58% carbohydrates, 3.1kcal/g (Harlan, 2018, Indianapolis, IN, USA)
• High-fat diet (HFD), 18.3% protein, 60.3% fat, 21.4% carbohydrates, 5.1 kcal/g (Harlan, 06414, Indianapolis, IN, USA)

Steps

1. All mice are fed the chow diet until 8 weeks of age.
2. At 8 weeks of age half the mice are fed the high-fat diet and the rest are continued to be fed the chow diet.

Procedure 2: Body weight

Equipment, software, and supplies

• Balance scale

Steps

1. Body weights are measured at four time points during the study. The first measurement is recorded at 8 weeks; the second is recorded at 9 am on the morning of the day of the blood response to a test meal (at 20 weeks), the third is recorded after it; and the fourth before sacrifice (at 29 weeks).

Procedure 3: Blood glucose response to test meal test

Fasting (duration): Overday fast, 9 am to 7 pm

Equipment, software, and supplies

• Glucometer strips
• Glucometer
• EDTA-coated tubes
• Centrifuge

Reagents and solutions

• Ensure Plus, 1.5 Cal/mL; Proteins: 15% of total Cal, Carbohydrates: 57% of total Cal, Fat: 28% of total Cal (Abbott Laboratories, Abbott Park, IL, USA)

Steps

1. Mice at 20 weeks of age are fasted overday on the day of the test, in order to allow for bile accumulation in the gallbladder. Then at 7 pm mice are gavaged with a test meal (Ensure Plus at a dose of 10mL/Kg body weight). Blood is collected from the tail vein before gavage, and additionally at 30 and 60 minutes following gavage.
2. Blood glucose levels are measured directly from a drop of blood using Glucometer strips, prior to blood collection. Approximately 75 µL of blood is collected in EDTA-coated tubes, and kept on ice until centrifugation at 4500 rpm for 10 min at 4°C. Plasma is then extracted and stored at -80°C for bile acid quantification (see below).

Procedure 4: Feces collection

Equipment, software, and supplies

• Single housing cages, with grids and without bedding

Steps

1. Mice are housed in single cages with access to food and water ad libitum and their feces collected over a 24h time period.
2. Feces are stored at a temperature of -20°C for bile acid quantification analysis (see below), and this test is carried out when the mice are 24 weeks of age.

Procedure 5: Animal sacrifice and organ collection

Fasting (duration): overday fast, and then refed either CD or HFD 4h before sacrifice

Equipment, software, and supplies

• Surgical tools
• Isoflurane anesthetic machine
• Balance scale
• Ruler

Reagents and solutions

• Isoflurane anesthesia
• Ice
• Liquid nitrogen

Steps

1. In order to avoid multiple sacrifices at night, the light/dark cycle of the mice is inverted 2 weeks before sacrifice to ensure their adaptation.
2. Mice are refed at the beginning of the dark cycle. They are anesthetized by isoflurane and sacrifice is carried out at 29 weeks of age. Blood is collected by cardiac puncture and the mice are perfused with PBS. The liver (with the gallbladder removed), spleen, both kidneys, subcutaneous white adipose tissue, epididymal white adipose tissue, together with the gastrocnemius and soleus muscles, and cecum are all excised and weighed. In addition, the entire small intestine is excised starting from the pylorus to the entry of the cecum including the duodenum, ileum and jejunum. Mesenteric tissues are then removed and the entire small intestine is laid along a straight line on a flat surface and the length measured using a ruler.
3. All the excised organs are immediately frozen in liquid nitrogen and stored at -80°C. The liver is used for bile acid quantification (see below).

Procedure 6: Bile acid quantification

Definitions & Abbreviations: LC-HRMS # Liquid chromatography - high-resolution mass spectrometry analysis

Equipment, software, and supplies

• Mortar and pestle
• Liquid nitrogen
• Lysis tubes
• Tissue homogenizer (Bertin Instruments, Precellys Cryolys, Montigny-le-Bretonneux, France)
• Centrifuge
• Lyophilizer, for fecal samples
• Ceramic beads
• Solid phase extraction (SPE) plates
• Liquid chromatography - high-resolution mass spectrometry (LC-HRMS) system (Thermo Fisher Scientific, Vanquish Horizon UHPLC System, Waltham, MA, USA)
• TraceFinder 4.1 software (Thermo Fisher Scientific, Waltham, MA, USA)

Reagents and solutions

• Standard solutions for calibration and quality control
• Methanol (MeOH)
• 0.2% Formic acid
• Ammonium acetate

Steps

1. Bile acids (BAs) are profiled with the stable isotype dilution mass spectrometry assay at the Metabolomics Platform of the University of Lausanne. Standard solutions for calibration, quality controls (QCs) and samples are described by Sorrentino G. et al. (2020). The standards for calibration consisted of stock solutions of BAs and D5-labeled BAs (at 1 mg/mL) in methanol, that are then used for the preparation of standard mixtures with a final concentration from 2.5 to 10 μg/mL. Calibrators are prepared by serial dilutions with stripped serum (C0-C7). Internal standard (IS) mixture contained 13 deuterium-labeled BAs.
2. Samples consist of frozen plasma, liver (100 mg, larger lobe) and fecal powders. Liver and fecal samples are ground with liquid nitrogen using mortar and pestle, pre-weighed in the lysis tubes, and then homogenized in a tissue homogenizer with the addition of MeOH:H₂O (2:1) with 0.1% formic acid (1500 µL) and ceramic beads for 2 x 20 seconds at 10,000 rpm. Homogenized extracts are then centrifuged for 15 min at 21,000 g at a temperature of 4°C; the resulting aliquots (50 µL) of supernatant are premixed with 100 µL of the ice-cold internal standard solution (in 100% MeOH), and 600 µL of H₂O with 0.2% formic acid, and loaded onto solid-phase extraction (SPE) plates, for phospholipid removal as described by Sorrentino G. et al. (2020). Plasma aliquots (25 µL) are also prepared in a similar way and processed with SPE plates for phospholipid removal and BA pre-concentration.
3. BA's quantification is carried out with a LC-HRMS system (Vanquish Horizon ultra-high performance liquid chromatography (UHPLC) system coupled with a Q-Exactive Focus mass spectrometer interfaced with a HESI source, Thermo Fisher Scientific) operating in negative ionization mode. Chromatographic separation is carried out using an Acquity UPLC® HSS T3 1.8 µm 2.1 x 100 mm column. The mobile phase is composed of A = 5 mM Ammonium Acetate and 0.1% formic acid in H₂O, and B = 0.1% formic acid in ACN. Gradient elution is set as described in the below table. The flow rate is 350 µL/min, column temperature 30°C, and the injection volume is 20 µL . Mass spectrometry parameters are set to: full scan in narrow mass range m/z 370-522, mass resolving power = 70,000 FWHM and AGC target = 5e5.
4. Data is processed using Trace-Finder 4.1 software (Thermo Fisher Scientific). The linearity is evaluated for each BA. LOD is determined with signal-to-noise (S/N) ratio of greater than 3, LLOQ is determined with S/N ratio greater than 5 and less than 20% CV. In addition, to accurate m/z ratio, the retention time (RT in min) is used to distinguish between different isobaric species. Absolute concentrations are calculated using calibration curves and considering the response factor of corresponding IS. Absolute concentrations are calculated using calibration curves and considering the response factor of corresponding IS. BA's concentrations are reported to sample dry weight (liver and feces) or to µL of plasma.

Steps Images: