Developmental programming: Prenatal testosterone-induced changes in epigenetic modulators and gene expression in metabolic tissues of female sheep☆
Graphical abstract
Introduction
Formation and maturation of organ systems during critical periods of development involves coordinated biological processes that are subject to modulation by endogenous and exogenous factors (Padmanabhan et al., 2016). Considering they play a critical role in the organ development and differentiation (Markey et al., 2003) and inappropriate exposure during the perinatal period are associated with inadvertent health outcomes (Padmanabhan et al., 2010a; Diamanti-Kandarakis and Dunaif, 2012; Yusuf et al., 2001; Diamanti-Kandarakis et al., 2009) steroids are receiving considerable attention as programming agents. Epidemiological and experimental studies that show inappropriate exposure to either native or environmental steroids during critical periods of development programs cardiometabolic disorders (Padmanabhan et al., 2016; Demissie et al., 2008; Eisner et al., 2000; Puttabyatappa and Padmanabhan, 2017) are consistent with the developmental origin of health and disease (DOHaD) hypothesis.
Specifically, gestational exposure to native steroid testosterone (T), for instance, compromises metabolic functions leading to peripheral insulin resistance and adipose tissue defects in rhesus macaques (Eisner et al., 2000; Bruns et al., 2007), sheep (Puttabyatappa and Padmanabhan, 2017), rat (Demissie et al., 2008; Lazic et al., 2011), and the mouse (Roland et al., 2010). The peripheral metabolic defects in prenatal T-treated sheep (Puttabyatappa and Padmanabhan, 2017), the model used in this study, has also been found to extend to metabolic organs with tissue-specific changes manifested as insulin resistance (Lu et al., 2016) and ectopic lipid accumulation in liver and muscle (Puttabyatappa et al., 2017a), along with hepatic oxidative stress (Puttabyatappa et al., 2017a) and metabolic disruptions (Hogg et al., 2011). Ectopic lipid accumulation is driven by genes that promote lipogenesis and lipid droplet formation. Lipogenesis are promoted by transcription factors belonging to the peroxisome proliferator-activated receptor (PPAR) and sterol regulatory element-binding transcription factor (SREBF) family, which show strong associations with development of hepatic and muscular lipotoxicity (Shimano and Sato, 2017; Silva and Peixoto, 2018). On the other hand, lipases namely hormone-sensitive lipase (HSL/LIPE) and hepatic lipase (LIPC), and lipid droplet surface protein, perilipin (PLIN) are essential for release of fatty acids and optimal storage of lipids. These genes have also been shown to be associated with pathological states. For example, upregulation of PLINs has been noted in liver of non-alcoholic fatty liver disease (NAFLD) patients as well as muscle of obese sedentary people (Carr and Ahima, 2016; Zacharewicz et al., 2018). Similarly, lipases are involved in the development of hepatic steatosis in mice with ablation of LIPC augmenting (Andres-Blasco et al., 2015) and overexpression of LIPE protecting from development of hepatic steatosis (Haemmerle et al., 2002). The contribution of these regulators in prenatal T-programmed increase in lipid accumulation in the liver and muscle are not known.
At the level of adipose tissue, the visceral adipose tissue (VAT) maintains insulin sensitivity (Lu et al., 2016) in spite of increases in inflammatory and oxidative stress status (Puttabyatappa et al., 2017a), reduction in adipocyte size (Lu et al., 2016; Veiga-Lopez et al., 2013), and disruptions in adipocyte differentiation markers (Puttabyatappa et al., 2017b). Interestingly, prenatal T-treatment induced adipose tissue-specific disruptions extended to subcutaneous (SAT), epicardiac (ECAT) and perirenal (PRAT) depots although they differed in grades of inflammatory and oxidative states and depot-specific expression of markers of adipocyte differentiation, thermogenesis, inflammation, oxidative stress and insulin signaling (Puttabyatappa et al., 2017a, 2019; Hogg et al., 2011; Nada et al., 2010). As a main function of adipose tissue is to store lipids, if depot-specific differences in expression of regulators of lipid metabolism (Kimmel and Sztalryd, 2016) contribute to the diversity in prenatal T-induced metabolic defects is of interest.
One way through which developmental exposures induce programmed changes in gene expression involves epigenetic modifications (Jimenez-Chillaron et al., 2012; Skinner et al., 2010). Epigenetic modifications involve DNA methylation (Chen and Riggs, 2005), histone modification (Turner, 1998) and expression of non-coding RNA (Moss, 2000). Methylation of DNA is carried out by members of DNA methyltransferases (DNMT), while epigenomic changes to chromatin involve posttranscriptional histone modification brought by enzymes that regulate the histone acetylation and/or methylation. Dysregulated expression of these enzymes are linked to various disease states (Copeland et al., 2010) and developmental exposures (Foulds et al., 2017; Lee et al., 2017; Smith and Ryckman, 2015). Additionally, as steroids are powerful programming agents (Skinner et al., 2010) and regulators of epigenetic machinery (Forger, 2018), it is intuitive to expect inappropriate exposure to excess steroids to induce epigenomic changes, reprogram gene expression, and contribute to tissue and adipose depot specific changes in insulin sensitivity.
The objective of this study is two-fold: 1) to address if prenatal T-treatment induced changes in lipases, lipid droplet related proteins, and the transcriptional factors involved in regulation of lipid metabolism are contributors to the dyslipidemia, adipose defects, and ectopic lipid accumulation seen in this model; and 2) to understand the contribution of tissue-specific changes in epigenetic mediators in development of the metabolic phenotype of prenatal T-treated sheep.
Section snippets
Animals and prenatal treatment
All animal procedures involved are performed as per the National Research Council's recommendations under the approved protocol of the University of Michigan Institutional Animal Care and Use Committee. Details on animal housing, breeding, general husbandry, nutrition provided and the prenatal treatments have been described previously (Manikkam et al., 2006). Prenatal T-treated animals were generated by intramuscular administration of 100 mg T propionate (1.2 mg/kg; Sigma-Aldrich St. Louis, MO)
Effect of prenatal T-treatment on expression of genes in metabolic tissues
Prenatal T-treatment induced a significant large magnitude increase in lipid droplet-associated proteins PLIN1 in the liver and muscle, and PLIN2 in the muscle (Fig. 1, left panel) and a trend for a large magnitude decrease in PLIN2 (d = 1.7) expression in liver. Among the adipose depots, prenatal T-treatment did not alter the expression of PLIN1 and 2 in VAT, SAT and ECAT but resulted in a non-significant large magnitude decrease in expression PLIN1 (d = 0.8) and trend (p = 0.08) for a large
Discussion
Supportive of our hypothesis, prenatal T excess induced a tissue and adipose depot-specific dysregulation in the expression of lipid metabolism, lipid storage and epigenetic regulatory genes. The observed changes in lipid metabolism and storage genes are consistent with the dyslipidemia and ectopic lipid accumulation observed in this model (Puttabyatappa et al., 2017a). On the other hand, the directionality of observed changes in tissue and adipose-depot specific changes in epigenetic modifying
CRediT authorship contribution statement
Xingzi Guo: Conceptualization, Methodology, Formal analysis, Writing - original draft. Muraly Puttabyatappa: Conceptualization, Methodology, Formal analysis, Writing - original draft. Steven E. Domino: Writing - review & editing, Funding acquisition. Vasantha Padmanabhan: Conceptualization, Writing - original draft, Project administration, Funding acquisition.
Acknowledgement
We thank Mr. Douglas Doop and Gary McCalla for their valuable assistance in breeding, lambing, and careful animal care; Dr. Almudena Veiga-Lopez, Dr. Bachir Abi Salloum, Mr. Evan Beckett, Mrs. Carol Herkimer and students supported through the Undergraduate Research Opportunity Program (University of Michigan) for the help provided with administration of treatments and tissue collection.
References (81)
- et al.
Polycystic ovarian syndrome (PCOS): long-term metabolic consequences
Metabolism
(2018) - et al.
Non-CpG methylation of the PGC-1alpha promoter through DNMT3B controls mitochondrial density
Cell Metabol.
(2009) - et al.
Pathophysiology of lipid droplet proteins in liver diseases
Exp. Cell Res.
(2016) - et al.
Targeting epigenetic enzymes for drug discovery
Curr. Opin. Chem. Biol.
(2010) - et al.
Peroxisome proliferator-activated receptor alpha-isoform deficiency leads to progressive dyslipidemia with sexually dimorphic obesity and steatosis
J. Biol. Chem.
(1998) - et al.
A quantitative Western Blot method for protein measurement
J. Biol. Stand.
(1985) - et al.
Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis
J. Biol. Chem.
(2002) - et al.
The role of nutrition on epigenetic modifications and their implications on health
Biochimie
(2012) - et al.
Epigenetics in non-alcoholic fatty liver disease
Mol. Aspect. Med.
(2017) Non-coding RNA's: lightning strikes twice
Curr. Biol.
(2000)
DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development
Cell
Sheep models of polycystic ovary syndrome phenotype
Mol. Cell. Endocrinol.
Hepatic epigenetic phenotype predetermines individual susceptibility to hepatic steatosis in mice fed a lipogenic methyl-deficient diet
J. Hepatol.
Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation
Cell Metabol.
Epigenetic transgenerational actions of environmental factors in disease etiology
Trends Endocrinol. Metabol.
Developmental programming: prenatal and postnatal contribution of androgens and insulin in the reprogramming of estradiol positive feedback disruptions in prenatal testosterone-treated sheep
Endocrinology
Scientists rise up against statistical significance
Nature
Preadipocytes from obese humans with type 2 diabetes are epigenetically reprogrammed at genes controlling adipose tissue function
Int. J. Obes.
Hepatic lipase deficiency produces glucose intolerance, inflammation and hepatic steatosis
J. Endocrinol.
Epigenetic regulation of PLIN 1 in obese women and its relation to lipolysis
Sci. Rep.
Prenatal androgen excess negatively impacts body fat distribution in a nonhuman primate model of polycystic ovary syndrome
Int. J. Obes.
Maintenance and regulation of DNA methylation patterns in mammals
Biochem. Cell. Biol.
Models of 'obesity' in large animals and birds
Front. Horm. Res.
A power primer
Psychol. Bull.
Genetic and epigenetic regulation in nonalcoholic fatty liver disease (NAFLD)
Int. J. Mol. Sci.
Transient prenatal androgen exposure produces metabolic syndrome in adult female rats
Am. J. Physiol. Endocrinol. Metab.
Insulin resistance and the polycystic ovary syndrome revisited: an update on mechanisms and implications
Endocr. Rev.
Endocrine-disrupting chemicals: an Endocrine Society scientific statement
Endocr. Rev.
A grape seed procyanidin extract inhibits HDAC activity leading to increased Pparalpha phosphorylation and target-gene expression
Mol. Nutr. Food Res.
Timing of prenatal androgen excess determines differential impairment in insulin secretion and action in adult female rhesus monkeys
J. Clin. Endocrinol. Metab.
Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice
J. Clin. Invest.
Past, present and future of epigenetics in brain sexual differentiation
J. Neuroendocrinol.
Endocrine-disrupting chemicals and fatty liver disease
Nat. Rev. Endocrinol.
Importance of variations in behavioural and feedback actions of oestradiol to the control of seasonal breeding in the Ewe
J. Endocrinol.
The effect of estrogen on the lipoprotein lipase activity of rat adipose tissue
J. Clin. Invest.
The in utero programming effect of increased maternal androgens and a direct fetal intervention on liver and metabolic function in adult sheep
PloS One
Metabolic syndrome, dyslipidemia and regulation of lipoprotein metabolism
Curr. Diabetes Rev.
Hypermethylation of hepatic Gck promoter in ageing rats contributes to diabetogenic potential
Diabetologia
Systems genetics reveals the functional context of PCOS loci and identifies genetic and molecular mechanisms of disease heterogeneity
PLoS Genet.
Increased expression of DNA methyltransferase 3a in obese adipose tissue: studies with transgenic mice
Obesity
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This work was supported by National Institutes of Health grant P01 HD44232. Dr. Guo is recipient of international exchange funding from XiangYa Famous Doctor Central South University.