Insulin Resistance in Nonalcoholic Fatty Liver Disease: A Case Control Study

Purpose of the study:

To date, no case control study evaluating insulin resistance (IR) in nonalcoholic fatty liver
disease (NALFD) has been published. We hypothesize that IR is characteristic of NAFLD as
compared to age, gender, non-diabetic BMI-matched control subjects, both healthy and those
with non-cirrhotic, non-steatotic liver disease. The following aims test this central
hypothesis:

Specific Aim 1: Measure IR and insulin clearance using intravenous glucose tolerance testing
and Bergman's Minimal Modeling in patients with NAFLD as compared to matched controls
(healthy and those with non-steatotic hepatitis). This aim tests the postulate that IR is
necessary for the development of hepatic steatosis as compared to healthy (non-steatotic
livers). It will also test the postulate that IR is not present as a confounding factor
(impaired hepatic clearance) in NAFLD as compared to those with non-steatotic forms of
hepatitis.

Specific Aim 2: Determine if IR is associated with altered parameters of lipid metabolism as
compared to matched controls. This aim addresses whether IR (if present) is associated with
abnormal lipid parameters in NAFLD or can be observed irrespective of the presence of hepatic
steatosis.

Specific Aim 3: Measure the differential effects of IR and lipid metabolism on peripheral
mononuclear cell (PBMC) inflammatory response and the associated hepatocyte mitochondrial
ultrastructure and measures of oxidative stress. Since increased oxidative stress and
bioenergetic failure have been associated with hepatic steatosis, this aim will address
whether IR and/or steatosis results in impaired inflammatory response and increased oxidative
stress as compared to controls. Changes in innate immune response will be determined by
measuring inflammatory indices thought to correlate with obesity, IR, and/or chronic
hepatitis: 1) interleukins (IL-2, IL-4, IL-6, IL-10, IL-12, IL-18) 2) C-reactive protein, 3)
TNF-a and TGF-B 4) IFN-A & IFN-g, and 5) adiponectin. Changes in oxidative stress will be
determined by measuring key indices of oxidative stress and damage. These include, a) reduced
and oxidized glutathione (GSH and GSSG); oxidation/reduction status), b) malondialdehyde
(MDA;lipid peroxidation), c) nitrotyrosine (NO damage), and d) 8OHdG and 8OHG (DNA damage).

Background and significance:

Obesity, diabetes, hypertriglyceridemia, hypertension, and coronary heart disease constitute
a phenotype common to individuals with either the IR syndrome or NAFLD1-12. Furthermore, the
hepatic steatosis, fibrosis, and cirrhosis characteristic of NAFLD are also frequent
histologic findings in patients with IR 3-7. Together, these data beg the question whether
NAFLD and IR are causally associated. Support for this notion derives from recent
observations that IR may be a strong, independent predictor of NAFLD, even in the absence of
glucose intolerance 13. Moreover, since NAFLD has been reported to occur in lean subjects
(BMI greater than 25) with normoglycemia 14, 15, it is reasonable to postulate that NAFLD may
be an early manifestation and a consequence of IR. Increased oxidative stress is an important
pathogenic mechanism of obesity-associated metabolic syndrome 16. Fat accumulation correlated
with systemic oxidative stress in humans and mice. However, increase oxidative stress as an
instigator of the metabolic syndrome in patients with NAFLD as compared to controls has not
yet been investigated. Furthermore, impaired cytokine medicated inflammatory response has
been shown to correlate with body mass index across the broad range of obesity and may
mediate hepatic steatosis and/or lead to mitochondrial dysfunction in hepatocytes 17, 18.
Discerning whether NAFLD is attributable to increased oxidative stress and/or abnormalities
in innate immune response would be imperative in identifying potentially useful therapeutic
targets for obesity-associated liver disease.

Inclusion Criteria:

- Biochemical and liver histologic features to confirm the presence of a healthy liver,
fatty liver, and/or non-fatty liver with inflammation due to another etiology besides
NAFLD.

Exclusion Criteria:

- > 20 grams of alcohol/day

- Impaired oral glucose tolerance test

- Clinical or histologic evidence of cirrhosis (stage 5-6 fibrosis) or portal
hypertension.

- Chronic hepatitis C infection

- Known diabetes mellitus or need for insulin-sensitizing agents and/or insulin therapy.

- Pregnancy