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Neonatal nicotine exposure affects adult rat hepatic pathways involved in endoplasmic reticulum stress and macroautophagy in a sex-dependent manner

Published online by Cambridge University Press:  01 December 2023

Luana Lopes Souza
Laboratory of Endocrine Physiology, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Camila Lüdke Rossetti
Laboratory of Endocrine Physiology, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Thamara Cherem Peixoto
Laboratory of Endocrine Physiology, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Alex Christian Manhães
Laboratory of Neurophysiology, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Egberto Gaspar de Moura
Laboratory of Endocrine Physiology, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Patrícia Cristina Lisboa*
Laboratory of Endocrine Physiology, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Corresponding author: P. C. Lisboa; Email:


Nonalcoholic fatty liver disease (NAFLD) involves changes in hepatic pathways, as lipogenesis, oxidative stress, endoplasmic reticulum (ER) stress, and macroautophagy. Maternal nicotine exposure exclusively during lactation leads to fatty liver (steatosis) only in the adult male offspring, not in females. Therefore, our hypothesis is that neonatal exposure to nicotine sex-dependently affects the signaling pathways involved in hepatic homeostasis of the offspring, explaining the hepatic lipid accumulation phenotype only in males. For this, between postnatal days 2 and 16, Wistar rat dams were implanted with osmotic minipumps, which released nicotine (NIC; 6 mg/Kg/day) or vehicle. The livers of offspring were evaluated at postnatal day 180. Only the male offspring that had been exposed to nicotine neonatally showed increased protein expression of markers of unfolded protein response (UPR), highlighting the presence of ER stress, as well as disruption of the activation of the macroautophagy repair pathway. These animals also had increased expression of diacylglycerol O-acyltransferase 1 and 4-hydroxynonenal, suggesting increased triglyceride esterification and oxidative stress. These parameters were not altered in the female offspring that had been neonatally exposed to nicotine, however they exhibited increased phospho adenosine monophosphate-activated protein kinase pAMPK expression, possibly as a protective mechanism. Thus, the disturbance in the hepatic homeostasis by UPR, macroautophagy, and oxidative stress modifications seem to be the molecular mechanisms underlying the liver steatosis in the adult male offspring of the nicotine-programming model. This highlights the importance of maternal smoking cessation during breastfeeding to decrease the risk of NAFLD development, especially in males.

Original Article
© The Author(s), 2023. Published by Cambridge University Press in association with The International Society for Developmental Origins of Health and Disease (DOHaD)

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Younossi, ZM, Koenig, AB, Abdelatif, D, Fazel, Y, Henry, L, Wymer, M. Global epidemiology of nonalcoholic fatty liver disease-meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016; 64(1), 7384.CrossRefGoogle ScholarPubMed
Lonardo, A, Nascimbeni, F, Ballestri, S, et al. Sex differences in nonalcoholic fatty liver disease: state of the art and identification of research gaps. Hepatology. 2019; 70(4), 14571469.CrossRefGoogle ScholarPubMed
Lazo, M, Hernaez, R, Eberhardt, MS, et al. Prevalence of nonalcoholic fatty liver disease in the United States: the third national health and nutrition examination survey, 1988-1994. Am J Epidemiol. 2013; 178(1), 3845.CrossRefGoogle ScholarPubMed
Long, MT, Pedley, A, Massaro, JM, et al. A simple clinical model predicts incident hepatic steatosis in a community-based cohort: the framingham heart study. Liver Int Off J Int Assoc Study Liver. 2018; 38(8), 14951503.Google Scholar
Ipsen, DH, Lykkesfeldt, J, Tveden-Nyborg, P. Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease. Cell Mol Life Sci. 2018; 75(18), 3313–3327.CrossRefGoogle ScholarPubMed
Lebeaupin, C, Vallee, D, Hazari, Y, Hetz, C, Chevet, E, Bailly-Maitre, B. Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease. J Hepatol. 2018; 69(4), 927947.CrossRefGoogle ScholarPubMed
Czaja, MJ. Function of autophagy in nonalcoholic fatty liver disease. Dig Dis Sci. 2016; 61(5), 13041313.CrossRefGoogle ScholarPubMed
Karkucinska-Wieckowska, A, Simoes, ICM, Kalinowski, P, et al. Mitochondria, oxidative stress and nonalcoholic fatty liver disease: a complex relationship. Eur J Clin Invest. 2022; 52(3), e13622.CrossRefGoogle ScholarPubMed
Arab, JP, Arrese, M, Trauner, M. Recent insights into the pathogenesis of nonalcoholic fatty liver disease. Annu Rev Pathol. 2018; 13(1), 321350.CrossRefGoogle ScholarPubMed
Hanson, MA, Gluckman, PD. Early developmental conditioning of later health and disease: physiology or pathophysiology? Physiol Rev. 2014; 94(4), 10271076.CrossRefGoogle ScholarPubMed
Souza, AFP, Woyames, J, Miranda, RA, et al. Maternal isocaloric high-fat diet induces liver mitochondria maladaptations and homeostatic disturbances intensifying mitochondria damage in response to fructose intake in adult male rat offspring. Mol Nutr Food Res. 2022; 66(8), e2100514.CrossRefGoogle ScholarPubMed
Kjaergaard, M, Nilsson, C, Rosendal, A, Nielsen, MO, Raun, K. Maternal chocolate and sucrose soft drink intake induces hepatic steatosis in rat offspring associated with altered lipid gene expression profile. Acta Physiol (Oxf). 2014; 210(1), 142153.CrossRefGoogle ScholarPubMed
Oliveira, LS, Souza, LL, Souza, AFP, et al. Perinatal maternal high-fat diet promotes alterations in hepatic lipid metabolism and resistance to the hypolipidemic effect of fish oil in adolescent rat offspring. Mol Nutr Food Res. 2016; 60(11), 24932504.CrossRefGoogle Scholar
Arciello, M, Gori, M, Maggio, R, et al. Environmental pollution: a tangible risk for NAFLD pathogenesis. Int J Mol Sci. 2013; 14(11), 2205222066.CrossRefGoogle ScholarPubMed
Ayonrinde, OT, Adams, LA, Mori, TA, et al. Sex differences between parental pregnancy characteristics and nonalcoholic fatty liver disease in adolescents. Hepatology. 2018; 67(1), 108122.CrossRefGoogle ScholarPubMed
Nighbor, TD, Doogan, NJ, Roberts, ME, et al. Smoking prevalence and trends among a U.S. national sample of women of reproductive age in rural versus urban settings. PLoS One. 2018; 13(11), e0207818.CrossRefGoogle Scholar
Hammoud, AO, Bujold, E, Sorokin, Y, Schild, C, Krapp, M, Baumann, P. Smoking in pregnancy revisited: findings from a large population-based study. Am J Obstet Gynecol. 2005; 192(6), 18531856.CrossRefGoogle ScholarPubMed
Colman, GJ, Joyce, T. Trends in smoking before, during, and after pregnancy in ten states. Am J Prev Med. 2003; 24(1), 2935.CrossRefGoogle ScholarPubMed
American Academy of Pediatrics. Transfer of drugs and other chemicals into human milk. Pediatrics. 2001; 108, 776789.CrossRefGoogle Scholar
Lange, S, Probst, C, Rehm, J, Popova, S. National, regional, and global prevalence of smoking during pregnancy in the general population: a systematic review and meta-analysis. Lancet Glob Heal. 2018; 6(7), e769–e776.Google ScholarPubMed
Singh, PK, Singh, L, Wehrmeister, FC, et al. Prevalence of smoking and smokeless tobacco use during breastfeeding: a cross-sectional secondary data analysis based on 0.32 million sample women in 78 low-income and middle-income countries. EClinicalMedicine. 2022; 53, 101660.CrossRefGoogle ScholarPubMed
Lauria, L, Lamberti, A, Grandolfo, M. Smoking behaviour before, during, and after pregnancy: the effect of breastfeeding. Sci World J. 2012; 2012, 19.CrossRefGoogle ScholarPubMed
Slotkin, TA. If nicotine is a developmental neurotoxicant in animal studies, dare we recommend nicotine replacement therapy in pregnant women and adolescents? Neurotoxicol Teratol. 2008; 30(1), 119.CrossRefGoogle ScholarPubMed
Quezada, A, Vafai, K. Thermal effects on transport in the resting mammary glands. Int J Heat Mass Transf. 2015; 85, 987995.CrossRefGoogle Scholar
Miranda, RA, de Moura, EG, Soares, PN, et al. Thyroid redox imbalance in adult Wistar rats that were exposed to nicotine during breastfeeding. Sci Rep. 2020; 10(1), 15646.CrossRefGoogle ScholarPubMed
Oliveira, E, Moura, EG, Santos-Silva, AP, et al. Short- and long-term effects of maternal nicotine exposure during lactation on body adiposity, lipid profile, and thyroid function of rat offspring. J Endocrinol. 2009; 202(3), 397405.CrossRefGoogle Scholar
Pinheiro, CR, Oliveira, E, Trevenzoli, IH, et al. Developmental plasticity in adrenal function and leptin production primed by nicotine exposure during lactation: gender differences in rats. Horm Metab Res. 2011; 43(10), 693701.Google ScholarPubMed
Conceicao, EP, Peixoto-Silva, N, Pinheiro, CR, Oliveira, E, Moura, EG, Lisboa, PC. Maternal nicotine exposure leads to higher liver oxidative stress and steatosis in adult rat offspring. Food Chem Toxicol. 2015; 78, 5259.CrossRefGoogle ScholarPubMed
Bertasso, IM, Pietrobon, CB, Lopes, BP, et al. Programming of hepatic lipid metabolism in a rat model of postnatal nicotine exposure – sex-related differences. Environ Pollut. 2020; 258, 113781.CrossRefGoogle Scholar
Rossetti, CL, de Oliveira Costa, HM, Barthem, CS, da Silva, MH, de Carvalho, DP, da-Silva, WS. Sexual dimorphism of liver endoplasmic reticulum stress susceptibility in prepubertal rats and the effect of sex steroid supplementation. Exp Physiol. 2019; 104(5), 677690.CrossRefGoogle ScholarPubMed
Oliveira, LS, Caetano, B, Miranda, RA, et al. Differentiated hepatic response to fructose intake during adolescence reveals the increased susceptibility to non-alcoholic fatty liver disease of maternal high-fat diet male rat offspring. Mol Nutr Food Res. 2020; 64(3), e1900838.CrossRefGoogle ScholarPubMed
Gual, P, Gilgenkrantz, H, Lotersztajn, S. Autophagy in chronic liver diseases: the two faces of janus. Am J Physiol Cell Physiol. 2017; 312(3), C263C273.CrossRefGoogle ScholarPubMed
Allaire, M, Rautou, PE, Codogno, P, Lotersztajn, S. Autophagy in liver diseases: time for translation? J Hepatol. 2019; 70(5), 985998.CrossRefGoogle ScholarPubMed
Yamamoto, H, Zhang, S, Mizushima, N. Autophagy genes in biology and disease. Nat Rev Genet. 2023; 24(6), 382400.CrossRefGoogle ScholarPubMed
Feng, Y, He, D, Yao, Z, Klionsky, DJ. The machinery of macroautophagy. Cell Res. 2014; 24(1), 2441.CrossRefGoogle ScholarPubMed
Marques, RG, Morales, MM, Petroianu, A. Brazilian law for scientific use of animals. Acta Cir Bras. 2009; 24(1), 6974.CrossRefGoogle ScholarPubMed
Kilkenny, C, Browne, W, Cuthill, IC, Emerson, M, Altman, DG. Animal research: reporting in vivo experiments: the ARRIVE guidelines. J Gene Med. 2010; 12(7), 561563.CrossRefGoogle ScholarPubMed
Luck, W, Nau, H. Nicotine and cotinine concentrations in serum and urine of infants exposed via passive smoking or milk from smoking mothers. J Pediatr. 1985; 107(5), 816820.CrossRefGoogle ScholarPubMed
Hukkanen, J, Jacob, P 3rd, Benowitz, NL. Metabolism and disposition kinetics of nicotine. Pharmacol Rev. 2005; 57(1), 79115.CrossRefGoogle ScholarPubMed
Oliveira, E, Pinheiro, CR, Santos-Silva, AP, et al. Nicotine exposure affects mother’s and pup’s nutritional, biochemical, and hormonal profiles during lactation in rats. J Endocrinol. 2010; 205(2), 159170.CrossRefGoogle ScholarPubMed
Miranda, RA, Rodrigues, VST, Peixoto, TC, Manhães, AC, de Moura, EG, Lisboa, PC. Nicotine exposure during breastfeeding alters the expression of endocannabinoid system biomarkers in female but not in male offspring at adulthood. J Dev Orig Health Dis. Published online February. 2023; 14(3), 111.Google Scholar
Corthell, JT. Immunoblotting (Western blot). in: basic molecular protocols in neuroscience: tips, tricks, and pitfalls . Elsevier. 2014, 5575.Google Scholar
Lee, JS, Zheng, Z, Mendez, R, Ha, SW, Xie, Y, Zhang, K. Pharmacologic ER stress induces non-alcoholic steatohepatitis in an animal model. Toxicol Lett. 2012; 211(1), 2938.CrossRefGoogle ScholarPubMed
Donnelly, N, Gorman, AM, Gupta, S, Samali, A. The eIF2α kinases: their structures and functions. Cell Mol Life Sci. 2013; 70(19), 34933511.CrossRefGoogle ScholarPubMed
Oyadomari, S, Harding, HP, Zhang, Y, Oyadomari, M, Ron, D. Dephosphorylation of translation initiation factor 2alpha enhances glucose tolerance and attenuates hepatosteatosis in mice. Cell Metab. 2008; 7(6), 520532.CrossRefGoogle ScholarPubMed
Marciniak, SJ, Ron, D. Endoplasmic reticulum stress signaling in disease. Physiol Rev. 2006; 86(4), 11331149.CrossRefGoogle ScholarPubMed
Thapaliya, S, Wree, A, Povero, D, et al. Caspase 3 inactivation protects against hepatic cell death and ameliorates fibrogenesis in a diet-induced NASH model. Dig Dis Sci. 2014; 59(6), 11971206.CrossRefGoogle Scholar
Kelly, DM, Nettleship, JE, Akhtar, S, et al. Testosterone suppresses the expression of regulatory enzymes of fatty acid synthesis and protects against hepatic steatosis in cholesterol-fed androgen deficient mice. Life Sci. 2014; 109(2), 95103.CrossRefGoogle ScholarPubMed
Zhang, Y, Cao, C, Du, S, et al. Estrogen regulates endoplasmic reticulum stress-mediated apoptosis by ERK-p65 pathway to promote endometrial angiogenesis. Reprod Sci. 2021; 28(4), 12161226.CrossRefGoogle ScholarPubMed
Cui, W, Sathyanarayan, A, Lopresti, M, Aghajan, M, Chen, C, Mashek, DG. Lipophagy-derived fatty acids undergo extracellular efflux via lysosomal exocytosis. Autophagy. 2021; 17(3), 690–705.CrossRefGoogle ScholarPubMed
Bhatt-Wessel, B, Jordan, TW, Miller, JH, Peng, L. Role of DGAT enzymes in triacylglycerol metabolism. Arch Biochem Biophys. 2018; 655, 111.CrossRefGoogle ScholarPubMed
Maiers, JL, Malhi, H. Endoplasmic reticulum stress in metabolic liver diseases and hepatic fibrosis. Semin Liver Dis. 2019; 39(2), 235248.Google ScholarPubMed
Singh, R, Kaushik, S, Wang, Y, et al. Autophagy regulates lipid metabolism. Nature. 2009; 458(7242), 1131–1135.CrossRefGoogle ScholarPubMed
Klionsky, DJ, Abdelmohsen, K, Abe, A, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy, 2016; 12(2), 443.CrossRefGoogle ScholarPubMed
Kimura, S, Fujita, N, Noda, T, Yoshimori, T. Monitoring autophagy in mammalian cultured cells through the dynamics of LC3. Methods Enzymol. 2009; 452, 112.CrossRefGoogle ScholarPubMed
Fernandes, MS, Pedroza, AA, de Andrade Silva, SC, et al. Undernutrition during development modulates endoplasmic reticulum stress genes in the hippocampus of juvenile rats: involvement of oxidative stress. Brain Res. 2022; 1797, 148098.CrossRefGoogle ScholarPubMed
Soeda, J, Mouralidarane, A, Cordero, P, et al. Maternal obesity alters endoplasmic reticulum homeostasis in offspring pancreas. J Physiol Biochem. 2016; 72(2), 281291.CrossRefGoogle ScholarPubMed
Oliveira, LS, Caetano, B, Miranda, RA, et al. Differentiated hepatic response to fructose intake during adolescence reveals the increased susceptibility to non-alcoholic fatty liver disease of maternal high-fat diet male rat offspring. Mol Nutr Food Res. 2020; 64(3), e1900838.CrossRefGoogle ScholarPubMed
Soeda, J, Cordero, P, Li, J, et al. Hepatic rhythmicity of endoplasmic reticulum stress is disrupted in perinatal and adult mice models of high-fat diet-induced obesity. Int J Food Sci Nutr. 2017; 68(4), 455466.CrossRefGoogle ScholarPubMed
Melo, AM, Benatti, RO, Ignacio-Souza, LM, et al. Hypothalamic endoplasmic reticulum stress and insulin resistance in offspring of mice dams fed high-fat diet during pregnancy and lactation. Metabolis. 2014; 63(5), 682692.CrossRefGoogle ScholarPubMed
Smith, BK, Marcinko, K, Desjardins, EM, Lally, JS, Ford, RJ, Steinberg, GR. Treatment of nonalcoholic fatty liver disease: role of AMPK. Am J Physiol Endocrinol Metab. 2016; 311(4), E730E740.CrossRefGoogle ScholarPubMed
Garcia, D, Hellberg, K, Chaix, A, et al. Genetic liver-specific AMPK activation protects against diet-induced obesity and NAFLD. Cell Rep. 2019; 26(1), 192208.e6.CrossRefGoogle ScholarPubMed
Jung, TW, Choi, KM. Pharmacological modulators of endoplasmic reticulum stress in metabolic diseases. Int J Mol Sci. 2016; 17(2), 192.CrossRefGoogle ScholarPubMed
Bae-Gartz, I, Kasper, P, Großmann, N, et al. Maternal exercise conveys protection against NAFLD in the offspring via hepatic metabolic programming. Sci Rep. 2020; 10(1), 15424.CrossRefGoogle ScholarPubMed
Soares, PN, Rodrigues, VST, Peixoto, TC, et al. Cigarette smoke during breastfeeding in rats changes glucocorticoid and vitamin D status in obese adult offspring. Int J Mol Sci. 2018; 19(10), 3084.Google Scholar
Friedman, TC, Sinha-Hikim, I, Parveen, M, et al. Additive effects of nicotine and high-fat diet on hepatic steatosis in male mice. Endocrinology. 2012; 153(12), 58095820.CrossRefGoogle ScholarPubMed
Wong, MK, Nicholson, CJ, Holloway, AC, Hardy, DB. Maternal nicotine exposure leads to impaired disulfide bond formation and augmented endoplasmic reticulum stress in the rat placenta. PLoS One. 2015; 10(3), e0122295.CrossRefGoogle ScholarPubMed
Seoane-Collazo, P, Martinez de Morentin, PB, Ferno, J, Dieguez, C, Nogueiras, R, Lopez, M. Nicotine improves obesity and hepatic steatosis and ER stress in diet-induced obese male rats. Endocrinology. 2014; 155(5), 16791689.CrossRefGoogle ScholarPubMed
de Oliveira, E, Moura, EG, Santos-Silva, AP, et al. Neonatal nicotine exposure causes insulin and leptin resistance and inhibits hypothalamic leptin signaling in adult rat offspring. J Endocrinol. 2010; 206(1), 5563.CrossRefGoogle ScholarPubMed
Peixoto, TC, Moura, EG, Soares, PN, et al. Nicotine exposure during breastfeeding reduces sympathetic activity in brown adipose tissue and increases in white adipose tissue in adult rats: sex-related differences. Food Chem Toxicol. 2020; 140, 111328.CrossRefGoogle ScholarPubMed
Flister, KFT, Pinto, BAS, França, LM, et al. Long-term exposure to high-sucrose diet down-regulates hepatic endoplasmic reticulum-stress adaptive pathways and potentiates de novo lipogenesis in weaned male mice. J Nutr Biochem. 2018; 62, 155166.CrossRefGoogle ScholarPubMed
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