With the epidemic of obesity, nonalcoholic fatty liver disease (NAFLD) has become the most common pediatric liver disease. The influence of a perinatal obesity-inducing diet (OID) on the development and progression of NAFLD in offspring is important but incompletely studied. Hence, we fed breeding pairs of C57BL/6J mice during gestation and lactation (perinatally) either chow or an OID rich in fat, fructose, and cholesterol (FFC). The offspring were weaned to either chow or an FFC diet, generating four groups: perinatal (p)Chow-Chow, pChow-FFC, pFFC-Chow, and pFFC-FFC. Mice were sacrificed at 10 weeks of age. We examined the whole-liver transcriptome by RNA sequencing (RNA-seq) and whole-liver genome methylation by reduced representation bisulfite sequencing (RRBS). Our results indicated that the pFFC-FFC mice had a significant increase in hepatic steatosis, injury, inflammation, and fibrosis, as assessed histologically and biochemically. We identified 189 genes that were differentially expressed and methylated in the pFFC-FFC mice versus the pChow-FFC mice. Gene set enrichment analysis identified hepatic fibrosis/hepatic stellate cell activation as the top canonical pathway, suggesting that the differential DNA methylation events in the mice exposed to the FFC diet perinatally were associated with a profibrogenic transcriptome. To verify that this finding was consistent with perinatal nutritional reprogramming of the methylome, we exposed pFFC-Chow mice to an FFC diet in adulthood. These mice developed significant hepatic steatosis, injury, inflammation, and more importantly fibrosis when compared to the appropriate controls. Conclusion: Perinatal exposure to an OID primes the immature liver for an accentuated fibrosing nonalcoholic steatohepatitis (NASH) phenotype, likely through nutritional reprogramming of the offspring methylome. These data have potential clinical implications for monitoring children of obese mothers and risk stratification of children with NAFLD.

Department of Molecular Pharmacology and Experimental Therapeutics Mayo Clinic Rochester MN

Division of Biomedical Statistics and Informatics Mayo Clinic Rochester MN

Division of Gastroenterology and Hepatology Mayo Clinic Rochester MN

Division of Pediatric Gastroenterology and Hepatology Mayo Clinic Rochester MN

Institute of Clinical Biochemistry and Diagnostics University Hospital Hradec Kralove Hradec Kralove Czech Republic

See more in PubMed

Ogden CL, Carroll MD, Lawman HG, Fryar CD, Kruszon‐Moran D, Kit BK, et al. Trends in obesity prevalence among children and adolescents in the United States, 1988–1994 through 2013–2014. JAMA 2016;315:2292‐2299. PubMed PMC

Welsh JA, Karpen S, Vos MB. Increasing prevalence of nonalcoholic fatty liver disease among United States adolescents, 1988–1994 to 2007–2010. J Pediatr 2013;162:e491. PubMed PMC

McPherson S, Hardy T, Henderson E, Burt AD, Day CP, Anstee QM. Evidence of NAFLD progression from steatosis to fibrosing‐steatohepatitis using paired biopsies: implications for prognosis and clinical management. J Hepatol 2015;62:1148‐1155. PubMed

Angulo P, Kleiner DE, Dam‐Larsen S, Adams LA, Bjornsson ES, Charatcharoenwitthaya P, et al. Liver fibrosis, but no other histologic features, is associated with long‐term outcomes of patients with nonalcoholic fatty liver disease. Gastroenterology 2015;149:e310. PubMed PMC

Doycheva I, Issa D, Watt KD, Lopez R, Rifai G, Alkhouri N. Nonalcoholic steatohepatitis is the most rapidly increasing indication for liver transplantation in young adults in the United States. J Clin Gastroenterol 2018;52:339‐346. PubMed

Cioffi CE, Welsh JA, Cleeton RL, Caltharp SA, Romero R, Wulkan ML, et al. Natural history of NAFLD diagnosed in childhood: a single‐center study. Children (Basel) 2017;4:E34. PubMed PMC

Feldstein AE, Charatcharoenwitthaya P, Treeprasertsuk S, Benson JT, Enders FB, Angulo P. The natural history of non‐alcoholic fatty liver disease in children: a follow‐up study for up to 20 years. Gut 2009;58:1538‐1544. PubMed PMC

Poston L, Caleyachetty R, Cnattingius S, Corvalan C, Uauy R, Herring S, et al. Preconceptional and maternal obesity: epidemiology and health consequences. Lancet Diabetes Endocrinol 2016;4:1025‐1036. PubMed

Stewart MS, Heerwagen MJ, Friedman JE. Developmental programming of pediatric non‐alcoholic fatty liver disease: redefining the "first‐hit". Clin Obstet Gynecol 2013;56:577‐590. PubMed PMC

Wesolowski SR, Kasmi KC, Jonscher KR, Friedman JE. Developmental origins of NAFLD: a womb with a clue. Nat Rev Gastroenterol Hepatol 2017;14:81‐96. PubMed PMC

Heijmans BT, Tobi EW, Stein AD, Putter H, Blauw GJ, Susser ES, et al. Persistent epigenetic differences associated with prenatal exposure to famine in humans. Proc Natl Acad Sci U S A 2008;105:17046‐17049. PubMed PMC

Zeybel M, Mann DA, Mann J. Epigenetic modifications as new targets for liver disease therapies. J Hepatol 2013;59:1349‐1353. PubMed

Page A, Mann DA, Mann J. The mechanisms of HSC activation and epigenetic regulation of HSCs phenotypes. Curr Pathobiol Rep 2014;2:163‐170. PubMed PMC

Gabory A, Attig L, Junien C. Developmental programming and epigenetics. Am J Clin Nutr 2011;94:1943S‐1952S. PubMed

Hardy T, Zeybel M, Day CP, Dipper C, Masson S, McPherson S, et al. Plasma DNA methylation: a potential biomarker for stratification of liver fibrosis in non‐alcoholic fatty liver disease. Gut 2017;66:1321‐1328. PubMed PMC

Charlton M, Krishnan A, Viker K, Sanderson S, Cazanave S, McConico A, et al. Fast food diet mouse: novel small animal model of NASH with ballooning, progressive fibrosis, and high physiological fidelity to the human condition. Am J Physiol Gastrointest Liver Physiol 2011;301:G825‐G834. PubMed PMC

Ibrahim SH, Hirsova P, Malhi H, Gores GJ. Animal models of nonalcoholic steatohepatitis: eat, delete, and inflame. Dig Dis Sci 2016;61:1325‐1336. PubMed PMC

Johnson F, Wardle J. Variety, palatability, and obesity. Adv Nutr 2014;5:851‐859. PubMed PMC

Krenkel O, Tacke F. Liver macrophages in tissue homeostasis and disease. Nat Rev Immunol 2017;17:306‐321. PubMed

Idrissova L, Malhi H, Werneburg NW, LeBrasseur NK, Bronk SF, Fingas C, et al. TRAIL receptor deletion in mice suppresses the inflammation of nutrient excess. J Hepatol 2015;62:1156‐1163. PubMed PMC

Khazen W, M'Bika JP, Tomkiewicz C, Benelli C, Chany C, Achour A, et al. Expression of macrophage‐selective markers in human and rodent adipocytes. FEBS Lett 2005;579:5631‐5634. PubMed

McDonald B, Kubes P. Innate immune cell trafficking and function during sterile inflammation of the liver. Gastroenterology 2016;151:1087‐1095. PubMed

Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Nonalcoholic Steatohepatitis Clinical Research Network. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:1313‐1321. PubMed

Zatloukal K, French SW, Stumptner C, Strnad P, Harada M, Toivola DM, et al. From Mallory to Mallory‐Denk bodies: what, how and why? Exp Cell Res 2007;313:2033‐2049. PubMed

Lackner C, Gogg‐Kamerer M, Zatloukal K, Stumptner C, Brunt EM, Denk H. Ballooned hepatocytes in steatohepatitis: the value of keratin immunohistochemistry for diagnosis. J Hepatol 2008;48:821‐828. PubMed

Holcik M, Korneluk RG. XIAP, the guardian angel. Nat Rev Mol Cell Biol 2001;2:550‐556. PubMed

Li R, Grimm SA, Mav D, Gu H, Djukovic D, Shah R, et al. Transcriptome and DNA methylome analysis in a mouse model of diet‐induced obesity predicts increased risk of colorectal cancer. Cell Rep 2018;22:624‐637. PubMed PMC

Thiam AR, Beller M. The why, when and how of lipid droplet diversity. J Cell Sci 2017;130:315‐324. PubMed

Gong J, Sun Z, Wu L, Xu W, Schieber N, Xu D, et al. Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites. J Cell Biol 2011;195:953‐963. PubMed PMC

Tomita K, Kohli R, MacLaurin BL, Hirsova P, Guo Q, Sanchez LHG, et al. Mixed‐lineage kinase 3 pharmacological inhibition attenuates murine nonalcoholic steatohepatitis. JCI Insight 2017;2:94488. PubMed PMC

Krishnan A, Abdullah TS, Mounajjed T, Hartono S, McConico A, White T, et al. A longitudinal study of whole body, tissue, and cellular physiology in a mouse model of fibrosing NASH with high fidelity to the human condition. Am J Physiol Gastrointest Liver Physiol 2017;312:G666‐G680. PubMed PMC

Hardy T, Mann DA. Epigenetics in liver disease: from biology to therapeutics. Gut 2016;65:1895‐1905. PubMed PMC

Nano J, Ghanbari M, Wang W, de Vries PS, Dhana K, Muka T, et al. BIOS consortium. Epigenome‐wide association study identifies methylation sites associated with liver enzymes and hepatic steatosis. Gastroenterology 2017;153:e1092. PubMed

Gluckman PD, Hanson MA, Cooper C, Thornburg KL. Effect of in utero and early‐life conditions on adult health and disease. N Engl J Med 2008;359:61‐73. PubMed PMC

Brumbaugh DE, Friedman JE. Developmental origins of nonalcoholic fatty liver disease. Pediatr Res 2014;75:140‐147. PubMed PMC

Li J, Huang J, Li JS, Chen H, Huang K, Zheng L. Accumulation of endoplasmic reticulum stress and lipogenesis in the liver through generational effects of high fat diets. J Hepatol 2012;56:900‐907. PubMed

Mischke M, Pruis MG, Boekschoten MV, Groen AK, Fitri AR, van de Heijning BJ, et al. Maternal Western‐style high fat diet induces sex‐specific physiological and molecular changes in two‐week‐old mouse offspring. PLoS One 2013;8:e78623. PubMed PMC

Wankhade UD, Zhong Y, Kang P, Alfaro M, Chintapalli SV, Thakali KM, et al. Enhanced offspring predisposition to steatohepatitis with maternal high‐fat diet is associated with epigenetic and microbiome alterations. PLoS One 2017;12:e0175675. PubMed PMC

Bruce KD, Cagampang FR, Argenton M, Zhang J, Ethirajan PL, Burdge GC, et al. Maternal high‐fat feeding primes steatohepatitis in adult mice offspring, involving mitochondrial dysfunction and altered lipogenesis gene expression. Hepatology 2009;50:1796‐1808. PubMed

Sarosiek KA, Fraser C, Muthalagu N, Bhola PD, Chang W, McBrayer SK, et al. Developmental regulation of mitochondrial apoptosis by c‐Myc governs age‐ and tissue‐specific sensitivity to cancer therapeutics. Cancer Cell 2017;31:142‐156. PubMed PMC

Zeng H, Liu J, Jackson MI, Zhao FQ, Yan L, Combs GF Jr. Fatty liver accompanies an increase in lactobacillus species in the hind gut of C57BL/6 mice fed a high‐fat diet. J Nutr 2013;143:627‐631. PubMed PMC

Ma J, Prince AL, Bader D, Hu M, Ganu R, Baquero K, et al. High‐fat maternal diet during pregnancy persistently alters the offspring microbiome in a primate model. Nat Commun 2014;5:3889. PubMed PMC

Hepatocyte apoptosis is tumor promoting in murine nonalcoholic steatohepatitis