Dynamic Post-Prandial Metabolism in Patients With Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is the most common hepatic disorder in the Western
world, and is a leading cause of liver-related morbidity and mortality. Excessive caloric
intake and specific nutritional components have been implicated in its pathogenesis, but the
complex metabolic pathways linking nutrient consumption with hepatic fat accumulation have
not yet been fully elucidated. Analysis of pathophysiology and the search for biomarkers in
NAFLD has largely focused on fasting samples; the dynamic response to a caloric load has not
been interrogated, beyond glucose tolerance testing. Metabolomics is a novel high-throughput
technique, allowing for simultaneous detection of hundreds to thousands of metabolites, and
the evaluation of their interconnectivity and causal relationships. We propose a pilot study
to investigate the metabolic response of patients with NAFLD to a standardized food
challenge, by applying a metabolomics-based dynamic approach. In this study we will recruit
up to 50 patients with NAFLD and compare them to 12 controls with metabolic syndrome without
NAFLD, and 12 healthy controls. Following an overnight fast, subjects will be situated in a
metabolic chamber for continuous measurement of metabolic rates, and will be given a liquid
mixed meal containing approximately 30% of daily caloric requirement over a period of 15
minutes. The energy expenditure (EE) and respiratory quotient (RQ), reflecting preference for
carbohydrate or fat oxidation, will be measured at 1-minute resolution. Blood will be drawn
at baseline, and at 30 min, 1, 2, 4 and 24 hours after ingestion. Plasma will be separated
and stored in -80 (Infinite)C. Following completion of recruitment, stored samples will be
analyzed at the NCI Laboratory of Metabolism by LC/MS and GC/MS, metabolites of interest
identified, and individual metabolites quantitated and normalized to their pre-prandial
level. Study results will hopefully identify novel metabolic markers of disease, and uncover
altered signaling pathways unique to NAFLD that are activated primarily under fed state
conditions.

- INCLUSION CRITERIA:

For the entire cohort:

- Male or female aged > 18 years

- Ability to provide informed consent

For group 1 subjects (NAFLD)

- Biopsy-proven NAFLD within 2 years prior to screening, OR

- The presence of at least two of the following criteria:

- Suggestion of liver fat by an imaging study (ultrasound, CT scan, MRI or MR
spectroscopy) performed in the 6 months prior to screening

- Elevated aminotransferase levels (ALT > 31 U/L for men or > 19 U/L for women, or AST >
30 U/L) on at least two occasions in the 6 months preceding enrollment.

- Presence of the metabolic syndrome, defined according to the modified AHA/NCEP
criteria as the presence of at least three of:

i. Abdominal obesity, defined as waist circumference > 102 cm for men or > 88 cm for
women

ii. Elevated triglycerides (> 150 mg/dL) or the use of medication to lower triglycerides

iii. Reduced HDL cholesterol (< 40 mg/DL for men or < 50 mg/dL for women)

iv. Elevated blood pressure (> 135/80 mmHg) or use of medication for hypertension

v. Elevated fasting glucose levels (> 100 mg/dL) or use of anti-diabetic medication

For the purpose of inclusion, the presence of overt diabetes mellitus type 2 will be
considered equivalent to the presence of the metabolic syndrome, even if the other criteria
are absent.

For group 2 subjects (non-NAFLD metabolic syndrome):

- Evidence of metabolic syndrome

- Normal transaminases (ALT less than or equal to 31 U/L for men or less than or equal
to 19 U/L for women, or AST less than or equal to 30 U/L) at screening

- Absence of liver fat by imaging or liver biopsy within 6 months of screening

For group 3 subjects (healthy volunteers):

- No history of known liver disease

- Not on any regular systemic medications (with the exception of oral contraceptives)

- BMI <= 25 kg/m2

- Non diabetic

- Normal transaminases and fasting glucose (<95 mg/dL)

EXCLUSION CRITERIA:

1. Concomitant liver disease such as autoimmune hepatitis, primary biliary cirrhosis,
primary sclerosing cholangitis, Wilson s disease, alpha-1 antitrypsin deficiency

2. Chronic infection with hepatitis C virus (HCV) or hepatitis B virus (HBV). Patients
who were treated successfully for HCV and achieved sustained virological response can
be eligible for enrollment > 18 months after treatment cessation. Patients receiving
antiviral therapy are ineligible.

3. Estimated average alcohol consumption > 30 g/d for men or > 20 g/d for women in the 6
months prior to enrollment, or binge-drinking behavior .

4. Gain or loss of > 10% body weight within the 6 months prior to enrollment.

5. Decompensated liver cirrhosis, defined as a past or present occurrence of a
decompensation event (variceal bleeding, ascites, spontaneous bacterial peritonitis,
encephalopathy or hepatocellular carcinoma or by albumin < 3 g/dl, PT > 3 seconds
above the upper limit of the norm, platelet count < 70,000 or total bilirubin > 2
mg/dL (in the absence of Gilbert s syndrome).

6. Pregnancy or lactation

7. Treatment with medications known to cause fatty liver disease such as atypical
neuroleptics, tetracycline, methotrexate or tamoxifen

8. Disorders interfering with substrate absorption such as gastric bypass surgery,
malabsorption disorders, use of orlistat or bile acid sequestrants, or extensive small
bowel resection.

9. Diabetic patients requiring insulin treatment

10. Lactose intolerance or allergy to Ensure or one or more of its components

11. Hyper/hypothyroidism

12. Inability to remain sedentary for 4 hours, or to remain for 26-30 hours in the
metabolic chamber

13. Inability to obtain vascular access for the required blood samples