Developmental programming: Prenatal testosterone-induced changes in epigenetic modulators and gene expression in metabolic tissues of female sheep

https://doi.org/10.1016/j.mce.2020.110913Get rights and content

Highlights

  • Prenatal T induces tissue-specific changes in genes involved in lipid metabolism.

  • Prenatal T induces tissue-specific changes in genes involved in lipid storage.

  • Prenatal T induces tissue-specific changes in DNA and histone modifying enzymes.

  • Epigenetic enzyme changes may underlie tissue-specific metabolic changes.

Abstract

Prenatal testosterone (T)-treated female sheep manifest peripheral insulin resistance and tissue-specific changes in insulin sensitivity with liver and muscle manifesting insulin resistance accompanied by inflammatory, oxidative and lipotoxic state. In contrast, visceral (VAT) and subcutaneous (SAT) adipose tissues are insulin sensitive in spite of VAT manifesting changes in inflammatory and oxidative state. We hypothesized that prenatal T-induced changes in tissue-specific insulin resistance arise from disrupted lipid storage and metabolism gene expression driven by changes in DNA and histone modifying enzymes. Changes in gene expression were assessed in liver, muscle and 4 adipose (VAT, SAT, epicardiac [ECAT] and perirenal [PRAT]) depots collected from control and prenatal T-treated female sheep. Prenatal T-treatment increased lipid droplet and metabolism genes PPARA and PLIN1 in liver, SREBF and PLIN1 in muscle and showed a trend for decrease in PLIN2 in PRAT. Among epigenetic modifying enzymes, prenatal T-treatment increased expression of 1) DNMT1 in liver and DNMT3A in VAT, PRAT, muscle and liver; 2) HDAC1 in ECAT, HDAC2 in muscle with decrease in HDAC3 in VAT; 3) EP300 in VAT and ECAT; and 4) KDM1A in VAT with increases in liver histone acetylation. Increased lipid storage and metabolism genes in liver and muscle are consistent with lipotoxicity in these tissues with increased histone acetylation likely contributing to increased liver PPARA. These findings are suggestive that metabolic defects in prenatal T-treated sheep may arise from changes in key genes mediated, in part, by tissue-specific changes in epigenetic-modifying enzymes.

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.

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      Prenatal T-treatment changes gene expression in VAT, including increased expression of proinflammatory cytokines, antioxidant and thermogenic genes, markers of macrophage infiltration, oxidative stress, and collagen accumulation genes (Puttabyatappa et al., 2017a, 2020), as well as reduced adipogenesis, adipocyte differentiation marker expression (Puttabyatappa et al., 2017b), and adipocyte size (Cardoso et al., 2016). Our earlier findings using RT-PCR found these changes were accompanied by changes in epigenetic enzymes, specifically increased expression of DNA methyltransferases (DNMT3A), histone acetyltransferases (EP300) and histone demethylase (KDM1A) (Guo et al., 2020). In the present study, prenatal T-treatment increased expression of DNMT3A and EP300 (Supplemental Table 5), validating our previous finding.

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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.

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