Determination of the Effect of Extreme Dietary Carbohydrate Restriction on Hepatic Glucose Production

All subjects will be evaluated by the Research Nurse at the Clinical Translational Research
Center (CTRC) Outpatient Clinic to obtain informed consent, a brief medical history, weight,
body mass index, waist/hip circumference and vital signs. Additionally, blood will be drawn
at this visit to screen for renal dysfunction (Chem 10), liver disease (liver function tests
(LFT's)), thyroid dysfunction (TSH), diabetes mellitus (HbA1C), and chronic inflammatory
states (erythrocyte sedimentation rate). Subjects recruited to the study will be placed on a
low-carbohydrate diet (<20 g/d without caloric restriction) designed either to promote weight
loss or maintain weight stability for fourteen days prior to the protocol or they will
consume a typical "Western" diet designed either to promote weight loss (restricted by 1300
kcal/d in the final fourteen days) or maintain weight stability for twenty-one days. Subjects
recruited to the study will be lean, overweight, or obese and also may carry a diagnosis of
NAFLD. Target accrual for total enrollment for this project will be set to 80 subjects to
accommodate attrition and screen failures. The details of the low-carbohydrate diet have been
discussed in consultation with Linda Brinkley, nutrition staff of the CTRC. Subjects chosen
to undergo carbohydrate-restriction will have a teaching session with a CTRC dietician.
Subjects will then initiate dietary carbohydrate-restriction on their own and keep a daily
dietary record for seven days. At the end of seven days, the dietary record will be analyzed.
The CTRC will then prepare all meals for the final seven days of the diet in accordance with
daily caloric intake calculated from the dietary record. Calorie-restricted participants will
keep a dietary record for one week while eating a typical "Western" diet. The dietician will
analyze this record and meals restricted by 1300 kcal/d will be prepared by the CTRC for the
final fourteen days of the diet. Alternatively, some subjects eating a typical "Western" diet
may consume prepared meals with a fixed content of carbohydrate, fat, and protein that is
unrelated to their pre-study dietary intake. In this scenario, the dietary carbohydrate
content may be varied between 30 and 60% to provide information on differences in hepatic
metabolism under conditions of differing, but clinically sustainable, carbohydrate intake.
All participants will be admitted to the CTRC the night prior to the study and begin an
overnight fast after dinner. Between 18:00 and 09:00, subjects will receive two stable
isotope tracers orally: [U-13C]propionate at 08:00 (three 400 mg doses given over 1 hour) and
70% 2H2O at 22:00, 02:00, and 06:00. During the study subjects will also be given 0.5% 2H2O
ad libitum. Two 500 mg acetaminophen tablets will also be given at 08:00. Between 08:00 and
09:00 subjects will undergo measurement of their respiratory quotient (RQ) using the CTRC
indirect calorimeter (Delta Trac II).. Subjects will then have an intravenous catheter placed
and an infusion of tracer amounts of [3,4-13C2]glucose, [1,2-13C]β-hydroxybutyrate and
[3,4-13C]acetoacetate will be initiated. At 1 ½ and 2 hours after initiation of the infusion,
a 50 cc blood draw will be performed. Voided urine will be collected every hour after
ingestion of acetaminophen until the protocol concludes. The subject will then be given a
meal and discharged from the CTRC, marking the completion of the protocol. The collected
blood and urine will be transported by the research coordinator to the Advanced Imaging
Research Center lab for analysis by MRS. Another blood draw (approximately 10 cc) will be
done in conjunction with the final MRI in order to measure changes in the lipid profile after
the diet intervention. The visits in their entirety will be as follows:

1 screening visit: 20 minutes

1. dietician visit: 20 minutes

2. MRS visits: 45 minutes each

1 (optional) overnight visit: 20 hours