Iron and Insulin Resistance in Overweight and Obese Humans

Screening:

If a potential subject meets the eligibility criteria after completing the general
pre-screening questionnaire, they will be contacted to set up an in-person screening visit
where the comprehensive written informed consent will be obtained. This visit may occur
either at the Michigan Clinical Research Unit (MCRU) or at the Substrate Metabolism
Laboratory (SML). Subjects will complete health history, food frequency, and physical
activity questionnaires. Subjects that meet eligibility requirements and provide written
informed consent will have a finger-stick performed for the assessment of hemoglobin
concentration. Women with hemoglobin < 13.0 g/dL and men with hemoglobin < 14.0 g/dL will be
withdrawn from the study.

Percent body fat will be measured using Dual Energy X-ray Absorptiometry [DEXA]. Women will
provide a urine sample for a urine pregnancy test prior to the DEXA scan; if a subject has a
positive pregnancy test prior to the DEXA scan, they will be withdrawn. The DEXA body
composition assessment test may be performed at the screening visit or scheduled anytime
between the screening visit and the baseline trial.

Baseline Trial:

Subjects will be provided general instructions on what to eat in the 3 days leading up to
their baseline trial and be required to abstain from planned exercise in the 3 days leading
up to the baseline and post-blood donation study trials. They will record their dietary
intake during the 3 days leading up to the baseline trial and replicate it during the 3 days
prior to the post-blood donation study trial. They will be required to fast the night before
the trials starting at 9:00PM.

Subjects will arrive at MCRU at ~07:00AM. After measurement of body weight and resting blood
pressure, intravenous catheters (IV) will be placed in each arm (one for blood sampling and
the other for infusion).

At ~7:50 AM blood samples (in 5 minute intervals) will be collected. A ~8:00 AM a
tracer-labeled glycerol infusion will begin. The tracer-labeled glycerol infusion that is
infused occurs naturally in the body. By injecting a slightly greater amount of this
substance than is already in the body, the rate of fat breakdown and fat burning is able to
be measured. One of the subject's hands will be placed within a "hot-hand box" to enable the
study team to collect arterialized venous blood samples from the intravenous catheter. At
~08:30AM, a small sample of abdominal fat tissue will be collected. Between ~09:50 and 10:00,
three blood samples will be collected to determine basal lipolysis. At ~10:00, we will start
the hyperinsulinemic, euglycemic clamp procedure to measure the body's sensitivity to
insulin. During this procedure insulin and glucose will be infused through the IV in the
forearm and blood samples will be collected periodically to monitor the subjects blood sugar.
The infusion rate of glucose will be changed accordingly to ensure that the subjects blood
sugar is maintained at a healthy/normal level. This procedure will take about two hours.
Following completion, The insulin will be discontinued and the subject will be provided a
lunch and the glucose will be gradually tapered off over the next hour. The study team will
ensure the subjects blood glucose levels are stable prior to discharge.

Blood Donation:

Subjects will donate blood within ~1 month after their baseline trial. Subjects will report
to the Substrate Metabolism Laboratory between 24 and 72 hours following blood donation. A
finger-stick will be performed for the measurement of hemoglobin concentration.

Post-Blood Donation Trial:

The exact same procedures as performed in the Baseline Trial will be repeated 4 weeks after
the subject's blood donation.

Diet/Physical Activity/Weight Maintenance:

Subjects will be required to maintain their normal diet and physical activity habits during
the study. Following the baseline trial, subjects will be provided with a scale and asked to
weigh themselves at home on a daily basis. If body weight changes ±2kg from their initial
weight, subjects will consult with our research dietitian for strategies to maintain weight
during the period between study trials.

Subject Inclusion Criteria

- Age: 18-40

- (Body Mass Index ≥27 kg/m2>27)

- Women must have regularly occurring menses and must be premenopausal

- Meet general blood donation eligibility requirements

Subject Exclusion Criteria

- Evidence/history of cardiovascular or metabolic disease

- Medications known to affect lipid or glucose metabolism, or inflammation

- Blood donation in the last 12 months

- Iron supplementation in the last 12 months (multivitamin with ≤100 %DV ok)

- Diagnosed anemia in the last 24 months

- Weight instability ≥ ±3kg in the last 6 months

- Women must not be pregnant or actively lactating