Protection Against Insulin Resistance in Obesity

SCREENING PROCEDURES Participants will undergo a battery of initial screening tests
including: a detailed health history and physical examination, a physical activity
questionnaire, and a body composition assessment. All women will undergo a urine pregnancy
test.

GENERAL STUDY DESIGN To avoid potential confounding hormonal effects, all women will be
studied in the early follicular phase of their menstrual cycle. Subjects will be advised of
what they should be eating in the 3 days leading up to their hospital visit to maintain their
weight. Subjects will also complete a three day food journal before their hospital visit. The
day prior to the study trial admission, subjects will need to eat pre-prepared standardized
meals for breakfast, lunch, dinner and an evening snack that the study team has given them.

Subjects will be admitted to the Michigan Clinical Research Unit (MCRU) in the morning after
a 12-hour, overnight fast. After arrival, subjects will rest quietly in their room for about
30 minutes. The study team will then measure subjects resting metabolic rate for about 20
minutes. After this test, an intravenous (IV) catheter will be placed for infusions and a
second IV will be placed in vein of the opposite arm for blood sampling.

After the IV placement, the study team will begin a tracer-labeled glucose, or "sugar,"
infusion. The study team will then remove a small sample of muscle from the subjects thigh.
This muscle biopsy procedure involves numbing a nickel-sized portion of the skin of the thigh
with a local anesthetic, making a small incision (1/4 inch), and removing a small piece of
muscle (approximately the size of 2-3 grains of rice). The incision will then be closed with
a piece of sterile tape. The study team will also obtain a small sample of fat tissue from
the area just underneath the skin near the belly button. This procedure involves numbing a
small region of skin near the belly button with a local anesthetic, using a needle a small
amount of fat tissue is removed from underneath the skin. During the procedure subjects will
feel some pinching and pulling, but it should not be very painful. The injury resulting from
the muscle and fat tissue biopsies is rather minor. Subjects will likely have some bruising
and mild soreness, but it should not prevent them from performing their general daily
activities.

Approximately 2.5 hours after admission, the study team will begin the tracer-labeled fat
infusion. The tracer-labeled fats the study team are infusing occur naturally in the body. By
injecting a slightly greater amount of this substance than what is already in the body the
study team will be able to measure the rate of fat breakdown and fat burning. After about 50
minutes of the infusion, three blood samples will be obtained in 5 min intervals.

After, the study team will begin a hyperinsulinemic-euglycemic clamp procedure (a procedure
to measure the body's sensitivity to insulin). During this procedure the study team will
infuse insulin and glucose through the IV and will collect blood samples every 5 minutes for
the duration of the procedure to monitor the subjects blood glucose and blood insulin
concentrations. This procedure is designed to last 2 hours. The study team will change the
infusion rate of glucose accordingly to ensure that the subjects blood sugar is maintained at
a healthy/normal level. The study team will also infuse a small amount of potassium to make
sure potassium concentrations remain at healthy/normal levels. About 30 minutes after
starting this procedure to measure the body's sensitivity to insulin, the study team will
collect another sample of muscle from the thigh and fat tissue from the area just underneath
the skin near the belly button. After completing the hyperinsulinemic-euglycemic clamp
procedure, subjects will be provided a meal, and the study team will continue to monitor the
subjects blood glucose concentration until stable.

When the subjects vital signs are stable, the study team will also measure percent body fat
using a Dual Energy X-ray Absorptiometry (DEXA) and intra-abdominal fat by Magnetic resonance
imaging (MRI). When the DEXA scan and MRI tests have been completed, subjects will be
discharged from the hospital. Depending on scheduling, however, the DEXA scan and MRI tests
may be performed on a separate occasion from the study trial admission.

Inclusion Criteria:

- Body mass index [BMI]: 30-40 kg/m2

- Age: 18-45 years

- All women must be pre-menopausal

- Non-exerciser: no regularly planned exercise/physical activity

Exclusion Criteria:

- Weight instability ≥ ±3kg ≥ 6 months

- Medications known to affect lipid and/or glucose metabolism

- Pregnancy or actively breast feeding