Effect of Diabetes Mellitus on Cholesterol Metabolism

Hypothesis:

1. Cholesterol absorption inhibitors like ezetimibe are more effective in lowering
cholesterol in subjects with type 1 diabetes mellitus . The primary outcome measures
are LDL cholesterol and cholesterol tracer absorption.

2. Cholesterol synthesis inhibitors like statins are more effective in lowering
cholesterol in subjects with type 2 diabetes mellitus. The primary outcomes are LDL
cholesterol and 24 Hour urinary mevalonic acid levels.

3. Response to statin is related to basal cholesterol synthesis rates. The primary
outcomes are LDL cholesterol and 24 Hour urinary mevalonic acid levels.

4. Response to ezetimibe is related to basal absorption rates. The primary outcome
measures are LDL cholesterol, phytosterol levels and cholesterol tracer absorption.

Specific Aims:

1. Measure baseline sterol absorption using plant sterol levels and and synthesis by using
24 h excretion of urinary mevalonic acid levels in type 1 and type 2 subjects

2. Measure changes in lipid parameters, cholesterol synthesis and absorption markers in
type 1 and type 2 subjects before and after 6 week therapy with simvastatin and 6 week
therapy with ezetimibe after a 4 week washout period

3. Start collecting blood for future genetic analysis for polymorphisms in cholesterol
absorption genes

Specific Methods:

VISIT 1: Evaluation of all the subjects that are willing to participate in the study would
be carried out at the GCRC until required sample size is recruited and will include the
following:

1. Sign informed consent

2. Complete a health questionnaire that has a standardized format to collect basic
information regarding past and current medical history

3. Physical exam that includes measurement of blood pressure, height, weight, waist and
hip circumference will be performed.

4. Blood (30ml) would be drawn for baseline chemistry including creatinine, ALT, CPK,
fasting lipid panel, Apo-B, Apo A1, glycated hemoglobin (HbA1c) and serum pregnancy
test in women of child bearing age. Urine will be collected for urine analysis and
presence of protein in the urine

5. Evaluation of inclusion/exclusion criteria Subjects fulfilling the inclusion and
exclusion criteria would be recruited into the study

Following specific procedures will be followed:

At the start of the study, subjects who are already on any lipid lowering medication will be
asked to stop the medications 4 weeks prior to obtaining the baseline labs. No changes will
be made to their diet, exercise pattern or in treatment for the DM. This will be done by a
telephone conversation.

VISIT 2 (Day 1): Subjects will report fasting to GCRC at 8 AM. Subjects will briefly meet
with bionutritionist for 24 hour dietary recall and instructions to keep a food diary on day
2 and 3. Blood sample (50ml) will be collected for fasting glucose, insulin, lipid panel,
Apo B, Apo A-1, CPK, Hb A1c, plasma and serum frozen for sterol analyses (by GC) and WBC
separated for DNA extraction.

The subject will be asked to consume with a cholesterol tracer (Cholesterol D5). Subjects
will be asked to consume the 8 ounces in entirety. They will be asked not to eat/drink
anything (except water) until the lunch time. If 8 Ounces are inadequate for breakfast,
subjects will be offered another 8 Oz serving of Carnation® Instant Breakfast in the same or
different flavor. If the subject is lactose intolerant, Lactaid tablets will be offered to
offset the effects of milk. If subjects are unable to consume milk (with or without
Lactaid®) or allergic to soy bean oil, they will be ineligible to participate in the study.
Subjects will be sent home with a urine jug to collect 24-hour urine on day 3.

VISIT 3 (Day 4): Subject will bring the urine jug and food diary back for analysis and that
same day will have blood (20ml) collected for evaluation of tracers. Food diary will be
reviewed by the bionutritionist. Urine pregnancy test will be performed in women of child
bearing age. Subjects will be started on either simvastatin or ezetimibe (we will alternate
the subjects so that half the sample will initially be treated with simvastatin and half
will be started on ezetimibe). The dose of Simvastatin (Merck) is 40 mg orally at nighttime
for 6 weeks and the dose of ezetimibe (Schering-Plough) is 10 mg taken orally once a day.
Subjects will be instructed on low-fat diet (therapeutic life style changes diet)
recommended by American Heart Association by the bionutritionist.

VISIT 4 (Day 46 ± 7 days): Subjects will report fasting to GCRC at 8 AM. The subject will
consume Carnation® Instant Breakfast with a cholesterol tracer (Cholesterol D5). Subjects
will be asked to maintain same dietary restriction as visit 2. Subjects will be sent home
with a urine jug to collect 24-hour urine on day 3. Subjects will start food diary on day 47
and 48.

VISIT 5 (Day 49 ± 7 days): Subject will bring the urine jug and food diary back for analysis
and that same day will have blood (40ml) collected for evaluation of tracers, lipid panel,
Apo A1, Apo B 100, ALT, CPK, plasma and serum stored for sterol analyses (by GC). Same day,
weight, waist and hip circumference will be measured again. Food diary will be reviewed by
bionutritionist. Subjects will be asked to bring back pill bottles also for pill count.
Simvastatin or ezetimibe will be stopped for 4 weeks.

VISIT 6 (Day 77 ± 7 days): Subjects will be asked to come fasting to GCRC at 8 AM. 15ml of
blood will be collected to measure lipid panel, apo B100 and Apo A1, Hb A1C, and ALT levels.
Urine pregnancy test will be performed in women of child bearing age. Subjects will be
started on 10 mg of ezetimibe or 40 mg of simvastatin as mentioned above for next 6 weeks.

VISIT 7 (Day 119 ± 7 days): Subjects will report fasting to GCRC at 8 AM. The subject will
consume a Carnation® Instant Breakfast with cholesterol tracer (Cholesterol D5). Subjects
will be asked to maintain same dietary restriction as visit 2. Subjects will be sent home
with a urine jug to collect 24-hour urine on day 3.

Subjects will start food diary on day 120 and 121

VISIT 8 (Day 122 ± 7 days): Subject will bring the urine jug and food diary back for
analysis and that same day will have blood (40 ml) collected for evaluation of tracers,
lipid panel, Apo B100, Apo A1, Hb A1c, ALT, CPK, plasma and serum stored for sterol analyses
(by GC). Weight, waist and hip circumference will be measured again. Food diary will be
reviewed by bionutritionist. Ezetimibe or simvastatin therapy will be stopped. Subjects will
be asked to resume their usual cholesterol lowering medications.

Ending the Study:

Study will be ended when all the required participants are enrolled. We will also consider
stopping the study when accumulated data suggests that risks exceed benefits of the study or
if preliminary data suggests there is a clear advantage of treating a particular group with
a certain agent.

Inclusion Criteria:

- Type 1 DM:

- Age > 18 years

- Subjects diagnosed with type 1 DM (diagnosed based upon history of ketoacidosis,
proven insulin dependence, absent C-peptide and or positive autoantibody profile
(such as anti-GAD etc.)

- Stable A1C < 8.5%

- BMI < 31

- Type 2 DM:

- Age > 18 years

- Subjects diagnosed with type II DM (diagnosed as adult onset, not-insulin dependent
and not on insulin)

- Stable A1C < 8.5%

- BMI < 31

Exclusion Criteria:

- History of active, unstable cardiovascular disease (including MI, CHF, Stroke,
Angina, CABG, stenting/PTCA, peripheral vascular disease, intermittent claudication)

- Pregnancy, nursing or likely to get pregnant during the course of the study (not on
oral contraceptives and premenopausal)

- Chronic Kidney Disease (creatinine > 2.0)

- Liver function test abnormalities, not previously worked up (AST or ALT >4x upper
limit of normal)

- Active substance abuse including alcohol

- History of severe Hypertriglyceridemia (untreated TG > 500) and on therapy

- Use of agents that interfere with cholesterol absorption (such as fiber, resins etc.)
which can not be discontinued for the duration of the study

- Actively enrolled in a weight loss program or following a special diet ( e.g.: Atkins
diet)

- History of malignancy <5y

- History of Rhabdomyolysis and Myopathy

- Use of on-going oral corticosteroids

- History of HIV infection

- Use of following drugs/compounds: cyclosporine, itraconazole, ketoconazole,
erythromycin, clarithromycin, HIV protease inhibitors, nefazodone, gemfibrozil,
niacin, amiodarone, verapamil or large quantities of grape fruit juice (> 1 quart per
day)

- Proteinuria: more than or equal to 300mg/24 hours calculated from random urine
specimen.

- BMI >31

- Anyone with hypersensitivity to either one of the study medications

- Allergy to Soy bean products

- Unable to consume milk products with or without Lactaid®