Volume 128, Issue 5, Pages 1381-1390 (May 2005) Mice Heterozygous for a Defect in Mitochondrial Trifunctional Protein Develop Hepatic Steatosis and Insulin Resistance  Jamal A. Ibdah, Peter Perlegas, Yiwen Zhao, Jerry Angdisen, Hermina Borgerink, Melanie K. Shadoan, Janice D. Wagner, Dietrich Matern, Piero Rinaldo, J. Mark Cline  Gastroenterology  Volume 128, Issue 5, Pages 1381-1390 (May 2005) DOI: 10.1053/j.gastro.2005.02.001 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Histopathologic analyses. Representative liver sections obtained from MTPa+/+ (A and C) and MTPa+/− (B and D) littermates and stained with H&E (A and B) or oil red O (C and D). (Original magnification, ×20.) Gastroenterology 2005 128, 1381-1390DOI: (10.1053/j.gastro.2005.02.001) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Representative electron micrograph of hepatocytes from MTPa+/+ (A) and MTPa+/− (B) at ×11,700 magnification. The mitochondria from the MTPa+/− were swollen with hypodense matrix and disrupted cristae. Gastroenterology 2005 128, 1381-1390DOI: (10.1053/j.gastro.2005.02.001) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 (A) Glucose tolerance test (GTT). Serial glucose measurements at the indicated time points were obtained from 9 MTPa+/+ (○) and 9 MTPa+/− (•) littermates using tail vein blood and a Fast Take Glucometer System after administering a glucose load of 1.5 g/kg body weight by oral gavage. Error bars represent SEM. (B) Insulin tolerance test (ITT). Serial glucose measurements at the indicated time points were obtained from 12 MTPa+/+ (○) and 9 MTPa+/− (•) littermates using tail vein blood and a Fast Take Glucometer System after administering an insulin challenge of 0.75 U/kg body weight by intraperitoneal injection. Error bars represent SEM. Gastroenterology 2005 128, 1381-1390DOI: (10.1053/j.gastro.2005.02.001) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Indices of hepatic steatosis, insulin sensitivity, and MTP α-subunit expression in MTPa+/− mice at various ages. (A) Hepatic steatosis score; (B) hepatic triglyceride level; (C) fasting serum insulin level; (D) insulin AUC after glucose challenge; (E) Kitt; (F) hepatic MTP α-subunit expression. Error bars represent SEM. The number of mice used in various experiments were as follows: for hepatic steatosis score and serum insulin level studies, n = 7 for all age groups except for 14–18-month age group (n = 12); for hepatic triglyceride measurements, n = 6 for all age groups; for insulin AUC and Kitt studies, n = 7 except for 2–4-month age group (n = 8 for insulin AUC and 6 for Kitt) and for 14–18-month age group (n = 6); for the MTP expression studies, n = 3 for all age groups. Comparison of means among the multiple groups was performed using ANOVA. For all variables, there were no significant differences in mean values among the 2–4-, 5–6-, and 7–8-month MTPa+/− mice (P > .05). Mean values in the 9–10- and 14–18-month MTPa+/− mice were significantly different from those in younger MTPa+/− mice groups (2–4-, 5–6-, and 7–8-month-old mice). The asterisks above the boxes for the 9–10- and 14–18-month MTPa+/− mice indicate the level of significance (*P < .05, **P < .01, ***P < .001) compared with the 7–8-month MTPa+/− mice. Age-associated changes in the MTPa+/+ mice were not statistically significant for hepatic steatosis score, hepatic triglyceride level, serum insulin level, insulin AUC, and Kitt as determined by ANOVA (P > .1, data not shown). Hepatic MTP α-subunit expression in 14–18-month MTPa+/+ mice was significantly different from that in the 2–4-month MTPa+/+ mice (P = .036, data not shown). Gastroenterology 2005 128, 1381-1390DOI: (10.1053/j.gastro.2005.02.001) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Hepatic cytochrome P-450 2E1 expression. Representative Western blot analyses performed after immunoprecipitation, using protein isolated from livers obtained from 4 MTPa+/+ and 4 MTPa+/− littermates. Anti-rat cytochrome P-450 2E1 raised in goat and anti-mouse albumin raised in rabbit were used in the immunoprecipitation and Western blot analyses. Densitometry measurements of bands were performed as described in the Materials and Methods section. The first MTPa+/+ band in each blot was given an arbitrary density of 1, and relative densities of the remaining bands were determined. Densitometry results from the Cyt P450-2E1 were divided by those from albumin, and the ratio is presented in the Figure as “Relative Density.” Gastroenterology 2005 128, 1381-1390DOI: (10.1053/j.gastro.2005.02.001) Copyright © 2005 American Gastroenterological Association Terms and Conditions