Procedures

Procedure 1: Diet administration

Reagents and solutions

• Chow diet AIN-76A, 20.8% kcal protein, 67.7% kcal carbohydrate, 11.5% kcal fat (Research Diets, D100001, New Brunswick, NJ, USA)
• High-fat high-sucrose (HFHS) diet, 16.8% kcal protein, 51.4% kcal carbohydrate, 31.8% kcal fat (Research Diets, D12266B, New Brunswick, NJ, USA)
• High protein (HP) diet, 40% kcal protein, 40% kcal carbohydrate, 20% kcal fat (Research Diets, D12083101, New Brunswick, NJ, USA)

Steps

1. During the two week acclimation period, mice are maintained on a defined synthetic diet until 8-11 wks of age when they are placed on either a synthetic high-fat high-sucrose diet or a high protein diet (see Reagents and Solutions for details). Diets were administered for the remainder of the study (after 8 wks).

Procedure 2: Body composition and weight

Equipment, software, and supplies

• Body composition analyzer (EchoMRI, MRI-130, Houston, TX, USA)
• Scales

Steps

1. Body composition is assessed during the first week of the acclimation period to establish baseline phenotypes as well as after 8 wks of the diet challenge using the body composition analyzer. Body weight is calculated as lean + fat + free water + undetectable substances + systemic errors. Fat mass is all the fat molecules in the body expressed as equivalent weight of canola oil. Lean mass is muscle tissue mass equivalent of all the body parts containing water, excluding fat, bone minerals, and such substances which do not contribute to the NMR signal, such as hair, claws, etc. Note that free water mass is mostly from bladder.
2. Mice are weighed separately using scales to compute percentage fat and percentage lean.

Procedure 3: Metabolic rate and activity

Equipment, software, and supplies

• Indirect calorimetry cages, activity measured by beam breaks (x- y-, z-axes) (TSE SYSTEMS, Phenomaster, Chesterfield, MO, USA)
• Software (TSE SYSTEMS, LabMaster, Chesterfield, MO, USA)

Steps

1. Mice are placed into individual indirect calorimetry cages the week immediately following 8 wks of the experimental diet challenge for ~3 days and 2 nights.
2. Data are collected for the second day and second night, including oxygen consumption, carbon dioxide production, activity, and feed and water intake. Data are presented for day, night, and the average of day and night.
3. Heat production calculations are performed by the TSE software as described in Yam et al., 2021.

Procedure 4: Plasma clinical chemistries

Fasting (duration): 4h

Equipment, software, and supplies

• Heparinized capillary tubes
• EDTA tubes
• Centrifuge
• 1.5 mL Eppendorf tubes
• Clinical chemistry analyzer (Roche Diagnostics, Cobas Integra 400 Plus, Indianapolis, IN, USA)
• ELISA (ALPCO Diagnostics, Mouse Insulin, Salem, NH, USA)
• Spectrophotometric plate reader (BioTek Instruments Inc., BioTek Synergy 2, Winooski, VT, USA)
• Software (BioTek Instruments Inc., BioTek Gen5 software, Winooski, VT, USA)

Reagents and solutions

• Isoflurane

Steps

1. Blood samples are collected immediately before putting mice on experimental diets to establish baseline values. After 8 wks on experimental diets, mice are anesthetized via isoflurane inhalation and euthanized using cervical dislocation following a 4h fast. Blood samples are collected via retro-orbital bleed with heparinized capillary tubes into EDTA tubes, placed on ice and centrifuged at 6,000 rpm for 10 min at 4°C. Plasma is then transferred to Eppendorf tubes and stored at -80°C until analysis.
2. Clinical chemistry measures (except insulin) are quantified using a plasma chemistry analyzer (an internal control (human UTAK) is used to assess run variation).
3. Baseline and post-diet circulating insulin are measured using ultrasensitive mouse insulin ELISA per manufacturer's instructions except for the following adjustment: 15 µL of plasma sample dilutions are used in the assay and back calculations are performed to determine actual plasma concentrations. Insulin optical density is measured at 450 nm using a spectrophotometric plate reader; concentrations are determined from measured optical densities using manufacturer's software.

Procedure 5: Liquid chromatography - Mass spectrometry (LC-MS)

Fasting (duration): 4h

Equipment, software, and supplies

• LC-MS

Reagents and solutions

• Standards of non-deuterated analytes (Sigma-Aldrich, St. Louis, MO, USA)
• Methanol (Fisher Scientific, Waltham, MA, USA)

Steps

1. Analytes are quantified using liquid chromatography-mass spectrometry (LC-MS) methods described by Wang et al., 2014 with modifications detailed in Yam et al., 2021.
2. Standards ranging from 0 to 100 µM of non-deuterated analytes in methanol are run in order to establish analyte standard curves. Two-fold serial dilutions of a 100 µM stock solution in methanol is used to make 13 standards.

Procedure 6: Metabolic health assessment

Fasting (duration): 4h

Steps

1. Metabolic health scores are calculated for all mice at baseline and at 9 wks post-diet. Z-scores are calculated for metabolic risk factors including glucose, insulin, glucose:insulin ratio, cholesterol, triglyceride, and percent body fat. Z-scores are added together and then multiplied by -1 so that decreased health is reflected by a lower health score.