Insulin Resistance in Non-alcoholic Fatty Liver Disease

The purpose of this study is to determine whether nonalcoholic fatty liver disease (NAFLD) is associated with altered peripheral and hepatic insulin sensitivity and to investigate potential mechanisms underlying insulin resistance in NAFLD by determining associations between hepatic and peripheral insulin sensitivity, hepatic steatosis, dyslipidemia, inflammatory cytokines, glucose metabolism, beta-cell function and body fat distribution.

NAFLD and nonalcoholic steatohepatitis (NASH) are common liver disorders that are strongly associated with obesity, type 2 diabetes and dyslipidemia. The underlying pathophysiology of fatty infiltration of the liver is thought to be related to insulin resistance, which is an almost universal finding in patients with NAFLD. It is also possible that fat infiltration and inflammation in the liver may impair insulin sensitivity, either locally in the liver, or peripherally via the actions of inflammatory cytokines. We hypothesize that insulin resistance is a major causal factor leading to fat deposition in the liver and NAFLD, and thus interventions aimed at improving insulin sensitivity will result in a reduction of hepatic inflammation and steatosis. Specific Aim 1: To determine in a cross-sectional study whether NAFLD is associated with altered peripheral and hepatic insulin sensitivity and to study their relationships with hepatic steatosis, dyslipidemia, inflammatory cytokines, glucose metabolism, -cell function and body fat distribution. Specific Aim 2: To determine in a 6 month placebo-controlled double-blinded treatment study if treatment with rosiglitazone, an insulin sensitizer, or fenofibrate, a triglyceride lowering agent, will improve both hepatic as well as peripheral insulin sensitivity and thereby improve hepatic steatosis and inflammation in subjects with NAFLD. The results of the proposed study will have important implications for our understanding of the mechanisms underlying insulin resistance and abnormalities in lipid and glucose metabolism in subjects with NAFLD and for the design of future studies aimed at the prevention and treatment of this condition.

Liver fat was estimated by non-contrast CT scan measuring the density ratio between the liver and spleen by Hounsfield units (liver/spleen ratio), which has been previously correlated with liver fat quantification by magnetic resonance spectroscopy.Ten separate measurements equally distributed throughout the liver and spleen were obtained and the Hounsfield units averaged. In subjects with more than one slice through the liver and spleen, the values for all slices were averaged.

Change in the Liver Spleen Ratio by CT Scan From Baseline to 6 Months as a Measure of Fat in the Liver

A two-step stable isotope labeled, hyperinsulinemic-euglycemic clamp procedure was performed with a low dose insulin infusion (20 mU/m2/min) for 3 hours followed by a primed high dose insulin infusion (160 mU/m2/min x 5 minutes then 80 mU/m2/min) for two hours. D20 was infused and adjusted to maintain the blood glucose at 90 mg/dl. Samples for glucose, insulin and 6,6 2d glucose were drawn every 15 minutes during the final half hour of the basal, low dose and high dose insulin periods. Whole body insulin sensitivity was calculated as the rate of glucose disposal (Rd)/lean body mass during the high dose insulin infusion.

Unenhanced CT scan images were obtained on a General Electric Discovery HD750 CT scanner. Intra-abdominal (IAF) areas were measured at the top of the iliac crest and quantified using the Tomovision program (SliceOMatic V4.3) by one trained technologist.

Hepatic insulin sensitivity was determined as the percent suppression of endogenous glucose production (EGP) at the end of the low dose insulin clamp.

Inclusion Criteria: Age 18-80 years old Controls: otherwise healthy Case subjects: NAFLD on liver biopsy within the past 3 years or presumed NAFLD with otherwise unexplained elevated ALT and fatty liver by CT or ultrasound Able to comply with taking 3 pills a day for 6 months and follow-up safety visits Exclusion Criteria: Controls: history or evidence of hepatic steatosis Cases: Cirrhosis on liver biopsy or by clinical exam or fibrosis score Causes of liver dysfunction other than NASH Use of medications associated with hepatic steatosis: glucocorticoids estrogens tamoxifen amiodarone accutane sertraline Use of medications that cause insulin resistance: niacin glucocorticoids anti-HIV drugs or atypical antipsychotics Use of lipid-lowering medications except stable dose statin Use of anti-NASH drugs such as: ursodeoxycholic acid betaine milk thistle Use of coumadin Use of nitrates Significant alcohol consumption: Average >20 grams/day In subjects with diabetes a HbA1c >7.5% or use of insulin metformin rosiglitazone or pioglitazone Liver transaminases: Cases: ALT >5x upper limit of normal Controls: ALT or AST above the normal range Iron saturation >50% Creatinine >1.5 mg/dl for men and >1.4 mg/dl for women Hematocrit <33% Pregnancy or lactation Significant weight loss within the past 6 months for controls, or since the liver biopsy for case subjects, history of significant coronary artery disease or congestive heart failure Retinopathy

Utzschneider KM, Largajolli A, Bertoldo A, Marcovina S, Nelson JE, Yeh MM, Kowdley KV, Kahn SE. Serum ferritin is associated with non-alcoholic fatty liver disease and decreased Beta-cell function in non-diabetic men and women. J Diabetes Complications. 2014 Mar-Apr;28(2):177-84. doi: 10.1016/j.jdiacomp.2013.11.007. Epub 2013 Nov 26.

Kratz M, Marcovina S, Nelson JE, Yeh MM, Kowdley KV, Callahan HS, Song X, Di C, Utzschneider KM. Dairy fat intake is associated with glucose tolerance, hepatic and systemic insulin sensitivity, and liver fat but not beta-cell function in humans. Am J Clin Nutr. 2014 Jun;99(6):1385-96. doi: 10.3945/ajcn.113.075457. Epub 2014 Apr 16.

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