Procedures

General Information for All Procedures: Protocol details can be found in Svenson et al., 2012.

Procedure 1: Diet administration

Equipment, software, and supplies

• Standard rodent chow (LabDiet (Scott Distributing), 5K52, Hudson, NH, USA)
• High fat high sucrose (HFHS), 45 kcal % fat diet, 40% carbohydrates and 15% protein (Envigo, TD.08811, Madison, WI, USA)

Steps

1. Mice are received at wean age (3 weeks of age), equal numbers of each sex mice are randomly assigned to either a standard rodent chow diet or a high fat, high sucrose (HFHS) diet; until approximately 6 mo of age (up to 30 weeks).

Procedure 2: Body weight

Equipment, software, and supplies

• Balance scale

Steps

1. Body weight of mice are recorded weekly from 3 to 30 weeks.

Procedure 3: Clinical chemistry

Fasting (duration): 4 h fast in the morning

Equipment, software, and supplies

• Topical anesthetic (tetracaine HCl)
• Heparin-coated microcapillary tubes
• Eppendorf tubes (1.5 mL)
• Clinical chemistry analyzer (Beckman Coulter, DxC 600Pro Synchron (A10400), Brea, CA, USA)

Steps

1. Whole blood from the retro-orbital sinus is collected via a heparin-coated microcapillary tube into 1.5-mL Eppendorf tubes, following the application of a topical anesthetic.
2. Clinical Chemistry at 8 weeks. Approximately 150 µL whole blood is added to a tube containing 2 µL of 10% sodium heparin, plasma separated by centrifugation at 10,000 rpm for 10 min at 4℃ and decanted into a clean Eppendorf tube. Total plasma bilirubin (TBIL), Calcium (Ca), Phosphorus (P), fructosamine (FRUC), glutamate dehydogenase (GLDH), glucose (GLU), blood urea nitrogen (BUN), HDL cholesterol (HDL), cholesterol (CHOL), non-esterified fatty acids (NEFA) and triglycerides (TG) are measured with a Clinical chemistry analyzer.
3. Clinical chemistry at 19 weeks. All the measures assessed at 8 weeks are repeated at 19 weeks, with the addition of plasma lipase (LIP) also being evaluated.

Procedure 4: Complete blood count

Equipment, software, and supplies

• Hematology analyzer (Siemens, Adiva 2120, Erangen, Bavaria, Germany)
• Tubes containing sodium EDTA

Steps

1. Blood (200 µL) is collected in a tube containing 2 µL of 10% sodium EDTA. Hematological parameters including white blood cell count (WBC), eosinophil count (EOS), lymphocyte count (LYM), monocyte count (MONO) neutrophil count (NEUT, red blood cell count (RBC), hematocrit (HCT) are assessed; in addition to RBC corpuscular hemoglobin (CHCM) and hemoglobin concentrations, along with platelet count (PLT) and volume (MPV).
2. The assessments are measured using a Siemens hematology analyzer at 10 weeks.
3. The measurements are repeated at 22 weeks.

Procedure 5: Urinalysis

Steps

1. Urinalysis to assess glucose, creatinine and microalbumin in urine are carried out at 11 and 20 weeks.

Procedure 6: Bone analysis and body composition by DXA

Definitions & Abbreviations: DXA # Dual-energy X-ray absorptiometry

Equipment, software, and supplies

• Bone densitometer (GE Healthcare, Lunar PIXImus, Chicago , IL, USA)
• Balance scale, with InCal calibration (Ohaus Corp., Navigator, Parsippany, NJ, USA)

Reagents and solutions

• Tribromoethanol anesthetic, (0.2 mL 2% solution/10 g body weight)

Steps

1. Mice are anesthetized intraperitoneally with tribromoethanol and body weight, length and area, lean, fat, total tissue mass, and bone mineral content, density and area are assessed.
2. The skull is omitted from assessment as it is bone dense. Mice are weighed on a balance scale to accommodate animal movement.
3. Mice are assessed by a bone densitometer at 12 and 21 weeks.

Procedure 7: Electrocardiogram (ECG)

Equipment, software, and supplies

• ECGenie, electrocardiogram (Mouse Specifics, Quincy, MA, USA)
• AnonyMouse software, v2.2 (Mouse Specifics, Quincy, MA, USA)

Steps

1. Unanesthetized mice are placed on a platform, containing a lead plate, raised 18 inches from the laboratory bench. The test begins when test animals contact the plate with any three paws.
2. Fast Fourier analysis is carried out from which interval durations are used to define heart rate, variability as well as other features of cardiac conductions. Tests are carried out at 13 weeks of age.

Procedure 8: Whole body composition by qNMR

Definitions & Abbreviations: qNMR # Quantitative Nuclear Magnetic Resonance

Equipment, software, and supplies

• Body composition analyzer, whole body composition for live animals (EchoMRI, Houston, TX, USA)

Steps

1. Whole body composition of fat and lean tissue mass, and free and total water together with body weight is assessed by a qNMR. Evaluation is carried out at 16 weeks of age.

Procedure 9: Plasma sample assessments

Fasting (duration): Non-fasted

Equipment, software, and supplies

• Electrochemiluminscent assay detection system (Meso Scale Discovery, Rockville, MD, USA)

Steps

1. Plasma calcium, phosphorus, albumin, creatinine, adiponectin, ghrelin, insulin and leptin are measured using the Meso Scale Discovery electrochemiluminescent assay detection system, at 17 weeks of age from non-fasted mice.

Procedure 10: Intraperitoneal glucose tolerance test (IPGTT)

Fasting (duration): Overnight fast (15 hours)

Equipment, software, and supplies

• Balance scale
• Abbott glucometer system

Steps

1. Mice are fasted overnight.
2. Mice are weighed and a small blood sample, taken from a tail tip incision, used to measure fasted glucose (GTT time 0, 0 minutes). A 20% glucose solution is injected intraperitoneally and tail tip blood samples are obtained at 15, 30, 60, 120 and 180 minutes after injection. The IPGTT is carried out at 23-24 weeks of age.

Procedure 11: Necropsy and body and organ weights

Fasting (duration): 4 h fast prior to necropsy

Equipment, software, and supplies

• Balance scale

Steps

1. Between 26-30 weeks of age mice are fasted and prepared for necropsy. Body weights are measured and the heart, kidneys and spleen are excised and weighed.