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. 2011 Dec;179(6):2855-65.
doi: 10.1016/j.ajpath.2011.08.014. Epub 2011 Oct 1.

Insulin resistance and metabolic hepatocarcinogenesis with parent-of-origin effects in A×B mice

Ian N Hines  1 , Hadley J Hartwell, Yan Feng, Elizabeth J Theve, Gregory A Hall, Sara Hashway, Jessica Connolly, Michelle Fecteau, James G Fox, Arlin B Rogers
Affiliations

Insulin resistance and metabolic hepatocarcinogenesis with parent-of-origin effects in A×B mice

Ian N Hines et al. Am J Pathol. 2011 Dec.

Abstract

Insulin resistance is a defining feature of metabolic syndrome and type 2 diabetes mellitus but also may occur independently of these conditions. Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of these disorders, increases the risk of hepatocellular carcinoma (HCC). However, mechanisms linking hyperinsulinemia to NAFLD and HCC require clarification. We describe a novel model of primary insulin resistance and HCC with strong parent-of-origin effects. Male AB6F1 (A/JCr dam × C57BL/6 sire) but not B6AF1 (B6 dam × A/J sire) mice developed spontaneous insulin resistance, NAFLD, and HCC without obesity or diabetes. A survey of mitochondrial, imprinted, and sex-linked traits revealed modest associations with X-linked genes. However, a diet-induced obesity study, including B6.A chromosome substitution-strain (consomic) mice, showed no segregation by sex chromosome. Thus, parent-of-origin effects were specified within the autosomal genome. Next, we interrogated mechanisms of insulin-associated hepatocarcinogenesis. Steatotic hepatocytes exhibited adipogenic transition characterized by vacuolar metaplasia and up-regulation of vimentin, adipsin, fatty acid translocase (CD36), peroxisome proliferator-activated receptor-γ, and related products. This profile was largely recapitulated in insulin-supplemented primary mouse hepatocyte cultures. Importantly, pyruvate kinase M2, a fetal anabolic enzyme implicated in the Warburg effect, was activated by insulin in vivo and in vitro. Thus, our study reveals parent-of-origin effects in heritable insulin resistance, implicating adipogenic transition with acquired anabolic metabolism in the progression from NAFLD to HCC.

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Figures

Figure 1
Figure 1
Clinical metabolic parameters in AB6F1 versus B6AF1 male mice. AB6F1 male mice developed early and progressive insulin resistance and hepatic steatosis (fatty liver). Although AB6F1 male mice also had moderately increased body mass compared with B6AF1 mice, neither morbid obesity nor hyperglycemia (not shown) was a component of the phenotype. *P < 0.05 compared with age-matched B6AF1 mice.
Figure 2
Figure 2
Histopathologic and genomic comparison of AB6F1 versus B6AF1 male mice. A: AB6F1 male mice exhibited more severe fatty liver lesions than B6AF1 male mice at all time points; lipid-filled vacuoles were verified by Oil Red O staining of frozen sections; immunohistochemical demonstration of PKM2 in AB6F1 neoplastic hepatocytes but not B6AF1 normal tissue. B: AB6F1 liver demonstrating focus of cellular alteration and dysplasia characterized by marked pleomorphism, intranuclear pseudoinclusions (arrow) and oval cell hyperplasia (arrowheads; left); HCC arising on a background of metabolic hepatopathy (right). C: Survey of nonhepatic, insulin resistance–associated histologic lesions in AB6F1 male mice, including WAT degeneration with macrophage proliferation forming F4/80+ crown-like rosettes, metaplasia of thoracic BAT to a WAT phenotype, focal skeletal muscle degeneration with fatty replacement, and pancreatic hyperplasia with occasional megaislets. D: Microarray heat map demonstrating hierarchal clustering of AB6F1 versus B6AF1 male mice at 9 months. E: Histologic comparison of macrovesicular steatosis with WAT (top) and microvesicular steatosis with BAT (bottom), consistent with transcriptional alterations characteristic of adipogenic transition. Original magnification: ×100 (A); ×400 (A, bottom); ×400 (B, left); ×40 (B, right); ×200 (C); ×400 (E).
Figure 3
Figure 3
Serum insulin and body weight comparison of A/J, B6.AX, B6.AY, B6AF1, and AB6F1 mice on a HF diet. Note dissociation between body weight and serum insulin levels in F1 mice compared with other groups. *P < 0.05, **P < 0.1.
Figure 4
Figure 4
Primary hepatocyte isolation and culture. A: Flow cytometry demonstrating less than 2% white blood cell contamination of enriched hepatocyte population; approximately 25% of hepatocytes labeled with anti-CD95/Fas antibody. B: 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide assay demonstrating strong trend (P = 0.07) for increased viability of insulin-supplemented cells versus dexamethasone alone after 4 days of culture. Photomicrographs demonstrate organization of hepatocytes into crude canaliculi, with increased clear (lipid) cytoplasmic vacuoles in insulin-supplemented cells (original magnification, ×1000). C: qRT-PCR of insulin-supplemented primary hepatocytes (dark gray columns), especially on day 4 of culture, demonstrating increased expression of adipocyte-associated genes, including vimentin, collagen 3α-1, fatty acid translocase (CD36), and PKM2, compared with nonsupplemented cells (light gray columns); these genes also were up-regulated in AB6F1 versus B6AF1 male mice at 15 months of age (black columns).
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