Volume 120, Issue 5, Pages 1183-1192 (April 2001) Nonalcoholic steatohepatitis: Association of insulin resistance and mitochondrial abnormalities  Arun J. Sanyal, Carol Campbell–Sargent, Faridoddin Mirshahi, William B. Rizzo, Melissa J. Contos, Richard K. Sterling, Velimir A. Luketic, Mitchell L. Shiffman, John N. Clore  Gastroenterology  Volume 120, Issue 5, Pages 1183-1192 (April 2001) DOI: 10.1053/gast.2001.23256 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Two-step hyperinsulinemic euglycemic clamping was performed in 3 groups of subjects: normal controls (■), fatty liver alone (2), and NASH (●). At each step (10 and 40 mU · m−2 · min−1 insulin infusion) of the clamping, there was a step-wise significant (normal vs. either fatty liver or NASH) decrease from normal controls to fatty liver and then NASH in the glucose infusion rates required to maintain euglycemia, indicating the presence of insulin resistance in those with fatty liver and NASH. At high-dose insulin infusion, patients with NASH required a significantly lower glucose infusion rate than those with fatty liver or normal controls. Mean (±SEM) data from a total of 6 subjects in each group are shown. Gastroenterology 2001 120, 1183-1192DOI: (10.1053/gast.2001.23256) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of insulin infusion (40 mU · m−2 · min−1) on serum (FFA and glycerol) in normal controls (■) and subjects with fatty liver alone (▨) or NASH (●). Insulin infusion produced a decrease from baseline values (% change) in both FFA and glycerol in all groups. There was a step-wise decrease in percent change from normal controls to fatty liver alone to NASH. This difference reached significance for normal versus either fatty liver or NASH. Mean (± SEM) data from a total of 6 subjects in each group are shown. Gastroenterology 2001 120, 1183-1192DOI: (10.1053/gast.2001.23256) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Transmission electron micrograph of hepatocytes from (A) fatty liver and (B) NASH. The mitochondria from those with fatty liver had a normal morphology with well-defined cristae. In contrast, the mitochondria in those with NASH (B) were swollen and rounded and had a markedly abnormal morphology with loss of cristae, appearance of multilamellar membranes, and stacks of intramitochondrial paracrystalline inclusion bodies. Gastroenterology 2001 120, 1183-1192DOI: (10.1053/gast.2001.23256) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Prevalence of intramitochondrial, paracrystalline inclusion bodies in subjects with NASH, subjects with fatty liver alone, normal controls, and subjects with hepatitis C. NASH was associated with a high prevalence of such inclusions compared with those in fatty liver subjects who did not have such inclusions. A small minority of normal subjects and those with hepatitis C had such defects. These data indicate that NASH is associated with intramitochondrial paracrystalline inclusion bodies. Gastroenterology 2001 120, 1183-1192DOI: (10.1053/gast.2001.23256) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Serum (β-OH butyrate) at baseline and during the 2 steps of the hyperinsulinemic euglycemic clamp are shown. Under baseline fasting conditions, subjects with NASH (●) had a significantly (P < 0.0001) higher β-OH butyrate than those with fatty liver (▨) or normal controls (■). Those with fatty liver had values intermediate between those with NASH and normal controls. This pattern was maintained during both steps of the clamping and indicate increased mitochondrial fatty acid β oxidation in subjects with NASH. Mean (± SEM) data from 6 subjects in each group are shown. Gastroenterology 2001 120, 1183-1192DOI: (10.1053/gast.2001.23256) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Evidence of lipid peroxidation in hepatocytes from (B) normal, (C) fatty liver, and (D) NASH subjects. Formalin-fixed, paraffin-embedded sections (5 μm thick) were incubated with antinitrotyrosine antibody followed by anti-rabbit antibody, streptavidin-enzyme conjugate, and 3,3-diaminobenzidine. The nuclei were counterstained with hematoxylin. In sections in which (A) nonimmune sera were used (negative control) and (B) in normal liver, there was no staining for 3-NT. In contrast, both (C) fatty liver and (D) NASH were associated with substantial staining for 3-NT. Gastroenterology 2001 120, 1183-1192DOI: (10.1053/gast.2001.23256) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Quantitation of differences in peroxynitrite concentrations in normal liver, fatty liver, and NASH as assessed by immunohistochemical staining. DAB-positive pixels were consistently in the 100–150-pixel intensity range in the blue channel of the RGB histogram, whereas background staining was in the >200-pixel intensity range. The percents of pixels in the 100–150 range in the blue channel in normal livers, fatty livers stained with nonimmune sera (negative controls), fatty liver, and NASH livers are shown. There was a significant increase in percent of positive pixels in fatty liver as well as NASH compared with normal controls (small circle, P < 0.0001, ANOVA). The percent of positive pixels was also significantly higher in NASH livers than in fatty liver (star). Gastroenterology 2001 120, 1183-1192DOI: (10.1053/gast.2001.23256) Copyright © 2001 American Gastroenterological Association Terms and Conditions