Body weight, bone densitometry, and body composition

Equipment

• Ohaus Portable Navigator Series Electronic Toploading Balance (Model NV-210)
• Lunar PIXImus II Mouse Densitometer (GE Medical Systems Model 51045; Madison, WI, USA), designated computer, and printer.

Preparation for Densitometry
Each mouse was weighed and anesthetized under approved protocols with Avertin (Tribromoethanol; 0.2 mL/gm body weight, injected i.p). Once anesthetized, each mouse was placed on the specimen sticky tray (body must be within blue line on tray). The tail is placed alongside the body, the front legs are extended to the side, and the neck and spine are gently straightened.

Controls: The PIXImus is calibrated with a "phantom mouse" according to manufacturer's protocol.

Running: Each mouse was tested individually by placing the sticky tray on the platform under the PIXImus beam path. The X-ray takes about 5 min for full scan. The region of interest (ROI) was adjusted manually for each mouse and includes the tail and excludes the head. The ROI is defined for quality control and analysis purposes.

Data: PIXImus data are from the body and tail; head excluded. BMD, BMC, total tissue mass, and % fat were measured. BMD and BMC adjusted for body mass and weights of lean and fat portions were derived and are available in the data set for analysis.

Safety: Gloves must be worn and safety procedures followed for anesthetizing animals. Radiation safety guidelines indicate that technicians must be 6 feet away from the PIXImus machine during scanning.

Glucose

insulin ELISA

Sample preparation: Whole blood was obtained via retro-orbital sinus through heparinized capillary tubes from non-fasted mice. Blood was transferred to 1.5 mL microfuge tubes containing 7.5 uL of Heparin 1000 U/mL (Sigma-Aldrich #H3393), and tubes were spun in a microcentrifuge at 14,000 rpm for 5 min. Plasma was transferred to a clean microfuge tube and remaining material discarded. Samples may be frozen for later analysis or used immediately.

Equipment and reagents: The commercially available ALPCO Diagnostics UltraSensitive Mouse Insulin ELISA Kit (# 008-10-1150-01, Windham, NH, USA) was used to measure the amount of insulin in mouse plasma. This is a solid phase two-site enzyme immunoassay based on a direct sandwich technique using two monoclonal antibodies with different specificities to antigenic determinants of mouse insulin. The second antibody is horse radish peroxidase (HRP)-conjugatged and is detected by reacting with a substrate, 3,3',5,5'- tetramethylbenzidine (TMB). Plates are read with the SpectraMax 190 Spectrophotometer using Softmax software (Molecular Devices, Sunnyvale, CA, USA) driven by a PC.

The kit contains:

• MicroPlates (twelve strips, eight wells each coated with mouse anti-insulin)
• Mammalian/Mouse Insulin control
• Insulin standards of 0, 0.025, 0.063, 0.25, 0.55, 1.40, 4.0, 7.5 ng/mL (lyophilized; reconstitute in 1 mL distilled water)
• Anti-Insulin HRP-Conjugate Stock (must be diluted to working concentration)
• Conjugate Stock Solution (for diluting Anti-Insulin Conjugate Stock)
• Washing Solution (must be diluted to working concentration)
• TMB Substrate (light sensitive; ready for use)
• Stop Solution (ready for use; contains sulfuric acid - handle with care)

Loading and Running: Load blanks, standards, controls, and samples in the microplate. Samples are run in duplicate or triplicate (see below).

All wells: 15 uL Insulin Zero Standard
Blanks: 10 uL Insulin Zero Standard
Standards: 10 uL Insulin Standard
Samples: 10 uL each sample
All wells: 50 uL Working Conjugate Buffer

Incubate the plate on a microplate rotator for 2 h at 18-28°C at 800-1100 rpm. Incubation can also be done by placing the plate overnight at 4°C. Once incubation is done, wash the plate 6 times with Wash Solution (manual or an automatic plate washer can also be used.)

Pipette 200 uL of TMB Substrate Solution into each well. Incubate for 30 min at 18-28°C. NOTE: TMB is light sensitive; it is very important that the plate is placed in a dark area for this incubation step. Stop the reaction by adding 50 uL of Stop Solution to each well.

Read at 450 nm and 640 nm on the plate spectrophotometer. The plate should be kept in the dark and read within 30 min of adding the Stop Solution. Discard the plate in a hazardous waste bin.

Investigator Notes:

• Thaw reagents the day before the test
• Standards: add 1 mL of distilled water to each; store at -20°C
• Washing Solution: mix 40 mL of Washing Solution Concentrate to 800 mL distilled water
• Working Conjugate Buffer: for every strip, 50 uL Anti-Insulin Conjugate stock plus 500 uL Conjugate Buffer is needed.
• Wear Gloves (Stop Solution contains sulfuric acid)
• Quality Control: Mouse insulin controls are tested each time for quality assurance. Data for all controls, standards, and unknowns are kept on file.

The homeostasis model assessment (HOMA) index was calculated for mice fed a high-fat diet. HOMA is a surrogate measure of in vivo insulin sensitivity using the following formula:

leptin ELISA

Sample preparation: Whole blood was obtained via retro-orbital sinus through heparinized capillary tubes from non-fasted mice. Blood was transferred to 1.5 mL microfuge tubes containing 7.5 uL of Heparin 1000 U/mL (Sigma-Aldrich #H3393), and tubes were spun in a microcentrifuge at 14,000 rpm for 5 min. Plasma was transferred to a clean microfuge tube and remaining material discarded. Samples may be frozen for later analysis or used immediately.

Equipment and reagents: The Mouse Leptin ELISA Kit (Crystal Chem, Inc., #90030; Downers Grove, IL, USA) was used to measure the amount of leptin in mouse plasma. Plates were washed using a Skan Washer 300 (Model 12010) and read with the SpectraMax 190 Spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) using Softmax software driven by a PC.

Components & preparation:

• Thaw Mouse Leptin ELISA Kit Microplate
• Mouse Leptin Stock: reconstitute with 100 uL Sample Diluent and dissolve completely (=25,600 pg/mL)
• Mouse Leptin Standards: perform doubling dilutions with Mouse Leptin Stock and Sample Diluent to give 0 (diluent only), 200, 400, 800, 1600, 3200, 6400, and 12,800 pg/mL mouse leptin
• Washing Buffer Solution: dilute Washing Buffer stock solution to give 1 L and mix well
• Anti-Guinea Pig IgG Enzyme Conjugate: Dilute one bottle of Anti-Guinea Pig IgG Enzyme Conjugate Stock by adding one bottle of Enzyme Conjugate Diluent; mix until clear
• Enzyme substrate

Loading and running: Samples are run in duplicate or triplicate (see below).

Pretreatment: wash wells twice with 300 uL Washing Buffer.

First Reaction

• All wells: 45 uL Sample Diluent
• All wells: 50 uL of Guinea Pig Anti-Mouse Leptin
• Standards: 5 uL of each standard
• Samples: 5 uL of each sample

Cover plate and incubate overnight (16-20 h) at 4°C. Wash wells 5 times with 300 uL of Washing Buffer.

Second Reaction

• All wells: 100 uL of Anti-Guinea Pig IgG Enzyme Conjugate (working concentration)

Wash wells 7 times with 300 uL of Washing Buffer.

Add 100 uL of Enzyme Substrate Solution into each well. Incubate for 30 min at 18-28°C. NOTE: enzyme is light sensitive; it is very important that the plate is placed in a dark area for this incubation step. Stop the reaction by adding 100 uL of Stop Solution to each well. Wear gloves (Stop Solution is sulfuric acid).

Read at 450 nm and 630 nm on the plate spectrophotometer. The plate should be kept in the dark and read within 30 min of adding the Stop Solution. Discard the plate in a hazardous waste bin.

Data: samples were run in duplicate or triplicate. Leptin concentrations were determined by comparing to the absorbance measurements of the standards (standard curve). Averages were calculated for each animal and are available for analysis. (The number of replicates and standard deviation between replicates are available for each animal in the project data set; these values are not used for analysis).

Safety: This test must be conducted wearing safety gloves as mouse plasma and sulfuric acid are handled. Caution must be practiced, and all materials must be dispensed into hazardous waste bins.