Developmental Programming: Impact of Gestational Steroid and Metabolic Milieus on Mediators of Insulin Sensitivity in Prenatal Testosterone–Treated Female Sheep

Muraly Puttabyatappa; Victoria Andriessen; Makeda Mesquitta; Lixia Zeng; Subramaniam Pennathur; Vasantha Padmanabhan

doi:10.1210/en.2017-00460

. 2017 Jun 26;158(9):2783–2798. doi: 10.1210/en.2017-00460

Developmental Programming: Impact of Gestational Steroid and Metabolic Milieus on Mediators of Insulin Sensitivity in Prenatal Testosterone–Treated Female Sheep

Muraly Puttabyatappa ¹, Victoria Andriessen ¹, Makeda Mesquitta ¹, Lixia Zeng ², Subramaniam Pennathur ^2,³, Vasantha Padmanabhan ^1,^3,^✉

PMCID: PMC5659659 PMID: 28911168

Abstract

Prenatal testosterone (T) excess in sheep leads to peripheral insulin resistance (IR), reduced adipocyte size, and tissue-specific changes, with liver and muscle but not adipose tissue being insulin resistant. To determine the basis for the tissue-specific differences in insulin sensitivity, we assessed changes in negative (inflammation, oxidative stress, and lipotoxicity) and positive mediators (adiponectin and antioxidants) of insulin sensitivity in the liver, muscle, and adipose tissues of control and prenatal T–treated sheep. Because T excess leads to maternal hyperinsulinemia, fetal hyperandrogenism, and functional hyperandrogenism and IR in their female offspring, prenatal and postnatal interventions with antiandrogen, flutamide, and the insulin sensitizer rosiglitazone were used to parse out the contribution of androgenic and metabolic pathways in programming and maintaining these defects. Results showed that (1) peripheral IR in prenatal T–treated female sheep is related to increases in triglycerides and 3-nitrotyrosine, which appear to override the increase in high-molecular-weight adiponectin; (2) liver IR is a function of the increase in oxidative stress (3-nitrotyrosine) and lipotoxicity; (3) muscle IR is related to lipotoxicity; and (4) the insulin-sensitive status of visceral adipose tissue appears to be a function of the increase in antioxidants that likely overrides the increase in proinflammatory cytokines, macrophages, and oxidative stress. Prenatal and postnatal intervention with either antiandrogen or insulin sensitizer had partial effects in preventing or ameliorating the prenatal T–induced changes in mediators of insulin sensitivity, suggesting that both pathways are critical for the programming and maintenance of the prenatal T–induced changes and point to potential involvement of estrogenic pathways.

Prenatally testosterone excess induces tissue-specific changes in negative and positive mediators of insulin sensitivity in a manner consistent with their insulin sensitivity status.

Peripheral insulin resistance (IR) is a common metabolic phenotype associated with obesity, type 2 diabetes mellitus, and polycystic ovary syndrome (PCOS) (1). Other metabolic dysfunctions observed are impaired glucose tolerance, dyslipidemia, and increased risk for cardiovascular disease (1, 2). Although the modern lifestyle, with its high-caloric diet and overnutrition together with a sedentary way of life, has contributed to the development of metabolic disorders (3), considerable evidence suggests that developmental insults (4), including exposure to excess native steroids during disease states (5) and environmental endocrine-disrupting chemicals with steroidogenic potential (6, 7) during the perinatal period, can also culminate in metabolic disorders. For instance, gestational exposure to excess testosterone (T) in rodents, sheep, and nonhuman primates leads to reproductive and metabolic defects similar to those seen in women with PCOS (8, 9). In the sheep model of gestational exposure to excess T, the main metabolic abnormalities observed are peripheral IR, dyslipidemia with increased palmitic acid in circulation, and reduced adipocyte size (10–12). In addition, tissue-specific studies of members of the insulin signaling pathway have suggested that liver and muscle are insulin resistant, whereas adipose tissue is insulin sensitive (13, 14). Similarly, rats exposed to excess prenatal dihydrotestosterone manifest hyperinsulinemia, with an associated increase in adiposity, dyslipidemia, and hepatic steatosis (15).

The underlying mechanisms leading to development of IR with prenatal exposure to excess androgens remain to be delineated. In other conditions of IR, such as that induced by obesity, chronic low-grade inflammation in adipose tissue, along with dyslipidemia, oxidative stress, and lipotoxicity in metabolic tissues, appear to contribute to the development of IR (16–18). In these scenarios, the development of chronic low-grade inflammation in adipose tissues appears to involve infiltration of macrophages, leading to activation of the immune system and to a consequent increase in proinflammatory cytokines (17, 19), which act in an autocrine and paracrine manner to interfere with insulin signaling (17) and to promote lipolysis, causing dyslipidemia (18). Increased accumulation of lipids also correlates with oxidative stress (20), which can interfere with insulin action in metabolic tissues (18). Additionally, an increase in circulating lipids can lead to ectopic accumulation of lipids in liver and muscle, causing lipotoxicity, which, coupled with the ability of lipids to negatively regulate glucose metabolism and insulin action (21, 22), can result in disruption of insulin signaling and development of IR (23).

Alternatively, decreased expression of mediators that promote tissue insulin sensitivity could also lead to development of IR. One such molecule that has gained prominence is the adipokine adiponectin (ADIPOQ). Adiponectin, secreted predominantly by adipose tissue [although other tissues, such as muscle, produce them (24)], exists in the circulation as a wide range of multimer complexes, with low (LMW), middle (MMW), and high molecular weight (HMW) forms being the major ones. They act through the adiponectin receptors ADIPOR1 and ADIPOR2, which are expressed in majority of the tissues (25) and signal through phosphorylation of 5′ AMP-activated protein kinase (AMPK) (26). Of the oligomeric forms of adiponectin, the HMW form has been demonstrated to be the most metabolically active (27). Adiponectins exert antidiabetic, antiatherogenic, and anti-inflammatory activities, with hypoadiponectinemia strongly associated with metabolic disorders (28–30).

Antioxidant enzymes, such as glutathione reductase (GSR) and superoxide dismutases (SODs), can also promote insulin signaling by neutralizing reactive oxygen or nitrogen species, which increase oxidative stress (31). Considering the multiple pathways that can affect insulin signaling, the primary goal of this study was to determine whether the insulin-sensitive status of the metabolic tissues in prenatal T–treated female sheep is reflective of changes in adiponectin, inflammatory/oxidative stress pathways, and lipotoxicity.

Because gestational testosterone excess induces maternal hyperinsulinemia and increases fetal testosterone and estradiol levels (32, 33), the developmental changes in mediators of tissue-specific insulin sensitivity could be programmed by androgenic, estrogenic, or insulin-dependent pathways. Additionally, because prenatal T treatment in female sheep culminates in functional hyperandrogenism and hyperinsulinemia in the female offspring (10), the prevailing steroidal and insulin status may regulate expression of mediators of insulin sensitivity in target tissues. Therefore, using an androgen antagonist and an insulin sensitizer as pharmacological interventions, the second goal of this study was to dissect out the organizational (prenatal) as well as the activational (postnatal) role of androgen and insulin in programming as well as the maintenance of the tissue-specific mediators of insulin sensitivity with focus on adiponectin, markers of macrophage infiltration, inflammation, oxidative stress, and lipotoxicity.

Materials and Methods

Animals

All procedures involving animals in this study were approved by the Institutional Animal Care and Use Committee of the University of Michigan and are consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All studies were conducted using multiparous Suffolk sheep at the University of Michigan Sheep Research Facility (Ann Arbor, MI). Animal maintenance, breeding, prenatal treatments, and lambing were performed as described previously (34). In brief, about 2 to 3 weeks before and until the time of breeding, ewes were group-fed daily with 0.5 kg shelled corn and 1.0 to 1.5 kg alfalfa hay per ewe. Ewes were mated to raddled Suffolk rams of proven fertility and housed under a natural photoperiod in the pasture and group-fed with a daily maintenance diet of 1.25 kg alfalfa/brome mix hay per ewe.

After weaning at ∼8 weeks of age, female lambs were maintained outdoors and fed a pelleted diet (Shur-Gain; Nutreco Canada Inc., Guelph, Canada) comprised of 3.6 MCal/kg digestible energy and 18% crude protein.

Prenatal and postnatal treatments

Pregnant ewes were blocked by body score and weight and randomly assigned to four prenatal treatment groups: control (C), prenatal T–treated, prenatal T + androgen receptor antagonist (flutamide)–treated (TF), and prenatal T + insulin sensitizer (rosiglitazone)–treated (TR) (Fig. 1). Gestational T treatment consisted of twice weekly intramuscular administration of 100 mg T propionate (1.2 mg/kg) (Sigma-Aldrich, St. Louis, MO) suspended in 2 mL corn oil from day 30 to day 90 of gestation. To generate the TF group, capsules (Profill Capsule Filling System; Torpac, Fairfield, NJ) filled with flutamide (15 mg/kg) (Sigma-Aldrich) were administered daily at the dose shown to block the phenotypic virilization effects of both exogenous and endogenous androgens in male and prenatal T–treated female sheep (36). To generate the TR group, rosiglitazone (∼0.11 mg/kg) (Avandia; GlaxoSmithKline, Research Triangle Park, NC), at the dose shown previously to normalize the insulin to glucose ratio during gestation (33) and to restore insulin sensitivity (37) in prenatal T–treated sheep, was administered orally on a daily basis.

Figure 1. — Schematic showing the study design with the time and duration of different pre- and postnatal treatments used in this study. Modified from Padmanabhan *et al.* (35).

A subset of prenatal T–treated female sheep received postnatal treatments from 8 weeks of age (Fig. 1). They received flutamide (T+F; 15 mg/kg/d) or rosiglitazone (T+R; 0.11 mg/kg/d) by oral administration of packed capsules or tablets, respectively, which continued until the time of tissue harvest. The dosage was adjusted on the basis of weekly body weight measurements. The sample sizes for the prenatal treatment groups were C = 6, T = 5, TF = 8, and TR = 5. The numbers of animals in each postnatal treatment groups were T+F = 5 and T+R = 7. When multiple pregnancies were involved, only one offspring was used in any given treatment group. The effects of prenatal as well as postnatal interventions on estradiol-positive feedback, puberty, insulin sensitivity, and adipocyte size involving animals from this cohort have been previously published (11, 14, 35, 38).

Tissue collection

All animals were ovariectomized at the end of the second breeding season to prevent confounding effects from differing steroid background, and a 1-cm estradiol implant was placed subcutaneously as described previously (39) to maintain early follicular phase levels of estradiol. Twenty-eight days later, animals were implanted subcutaneously with two controlled internal drug-releasing implants containing progesterone (CIDR-G; InterAg, Hamilton, New Zealand). Progesterone implants were removed 14 days later, and four 30-mm estradiol implants were inserted subcutaneously. Tissues were harvested 18 hours later during the artificial follicular phase after euthanization by barbiturate overdose (Fatal Plus; Vortech Pharmaceuticals, Dearborn, MI). Blood samples were collected in a heparinized tube prior to barbiturate administration. All animals were fasted for 48 hours prior to tissue collection. Liver was obtained from the tip of the left lobe, skeletal muscle from the vastus lateralis, subcutaneous adipose tissue (SAT) from the sterna region, and visceral adipose tissue (VAT) from the mesenteric fat surrounding the ventral sac of the rumen. Tissues were flash-frozen and stored at −80°C until processed.

Real-time reverse transcription-polymerase chain reaction

Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA), DNAse treated, and purified using the RNAeasy kit (Qiagen, Germantown, MD) as per the manufacturer’s guidelines. Total RNA was reverse transcribed into cDNA using the SuperScrit Vilo cDNA synthesis kit (Invitrogen) for use in the gene expression analysis. An SYBRgreen-based real-time reverse transcription polymerase chain reaction assay was used to determine the messenger RNA (mRNA) levels on a BioRad myiQ iCycler real-time polymerase chain reaction instrument. Oligonucleotide primers for the genes under study were designed using Primer Express software (Life Technologies, Carlsbad, CA), and the sequences for the primers used are shown in Supplemental Table 1^{(15.1KB, docx)}. The relative amount of each transcript was calculated using the ΔΔCT method and normalized to the endogenous reference gene ribosomal protein L19.

Immunoblotting

Plasma adiponectin was assessed using a previously published method (40) using the bovine antiadiponectin antibody kindly provided by Dr. Helga Sauerwein from University of Bonn, Germany. Briefly, 2.0 μL plasma was diluted 10-fold in phosphate-buffered saline and loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis gels. Plasma proteins were separated under reducing conditions to detect LMW forms and under nonreducing conditions to detect HMW forms and transferred to nitrocellulose membranes. Membranes were first stained with Ponceau to visualize the protein loading.

To determine tissue-specific AMPK levels, tissue samples were homogenized in radio-immunoprecipitation assay buffer (Pierce RIPA Buffer; Thermo Scientific, Rockford, IL) containing protease inhibitors (Complete Mini; Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitors (PhosSTOP; Roche Diagnostics) as described previously (14). Briefly, tissue homogenates were centrifuged at 10,000 g for 15 minutes at 4°C, and the supernatant containing whole-tissue protein extract was collected. Protein concentrations were determined using the Biorad DCA kit as per manufacturer’s recommendations. Equal amounts of protein (∼40 µg) were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane.

For determining plasma adiponectin and tissue AMPK levels, nitrocellulose membranes with transferred protein were incubated with primary antibody (Table 1) overnight at 4°C, washed, and probed with horseradish peroxidase–tagged secondary antibody raised against the species in which primary antibody was generated. Proteins were detected by electrochemiluminescent reaction using either Kodak X-OMAT film (Fisher Scientific) or the ProteinSimple FluorChem E system (San Jose, CA). The levels of each protein, as well as the corresponding loading control (glyceraldehyde 3-phosphate dehydrogenase), were determined in the same membrane after stripping and reblotting. Band densities were determined using ImageJ software.

Table 1.

Antibodies Used

Peptide/Protein Target	Name of Antibody	Manufacturer, Catalog Number, and/or Name of Individual Providing the Antibody	Species Raised in; Monoclonal or Polyclonal	Dilution Used	RRID
Bovine Adiponectin	Bovine antiadiponectin	Dr. Helga Sauerwein (University of Bonn, Germany)	Rabbit; polyclonal	WB 1:1K	AB_2650604
p-AMPK	Phospho-AMPKα (Thr172) (D79.5E)	Cell Signaling, 4182	Rabbit; monoclonal	WB 1:1K	AB_2169396
AMPK	AMPKα (D63G4)	Cell Signaling, 5832	Rabbit; monoclonal	WB 1:1K	AB_10624867
GAPDH	GAPDH (14C10)	Cell Signaling, 3683	Rabbit; monoclonal	WB 1:1K	AB_1642205

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Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RRID, research resource identifier; WB, Western blot.

Oxidative stress measures

As markers of oxidative stress, levels of protein-bound oxidized tyrosine moieties [3-nitrotyrosine (NY); o, o'-dityrosine (DY); and 3-chlorotyrosine (ClY)] were quantified by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry as described previously (41) in plasma and in tissue extracts from VAT, SAT, muscle, and liver. Briefly, the plasma and tissue proteins were precipitated with ice-cold trichloroacetic acid (10% vol/vol) and then delipidated with the mixture containing water, methanol, and water-washed diethyl ether (1:3:7; vol/vol/vol). Tissues were homogenized before precipitation of proteins and delipidation. Known amounts of isotopically labeled internal standards ¹³C₆-Y ¹³C₆-NY, ¹³C₆-ClY, and ¹³C₁₂-o, o'-DY were added, and precipitated proteins were hydrolyzed at 110°C for 24 hours in 4 M methanesulfonic acid solution saturated with 1% benzoic acid. Samples were then subjected to solid-phase extraction, and the amount of oxidized amino acids was determined by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry. Multiple reactions were monitored by integrating peak areas of the labeled standards and the analytes. The levels of the oxidized amino acids in each sample were then normalized to the amino acid tyrosine content in the respective samples and expressed as the ratio of the oxidized product over the total tyrosine. Intra-assay coefficients of variation for NY, DY, and ClY were 8.47%, 8.16%, and 12.98%, respectively.

Assessment of ectopic lipid accumulation

Lipid accumulation in liver and muscle were assessed by Oil Red O staining of tissue cryosections as well as hematoxylin and eosin (H&E) staining of paraffin-embedded tissue sections. In addition, tissue triglyceride content in both liver and muscle was measured using a commercially available assay kit. For Oil Red O staining, frozen sections mounted in optimum cutting temperature medium were sectioned at 5 μm on a Leica CM3050S cryostat (Leica Microsystems, Germany), and the cryosections were fixed in neutral buffered formalin (20 minutes) and washed twice with tap water (5 minutes) and 60% isopropanol (5 minutes) prior to staining with Oil Red O (Sigma-Aldrich) for 15 minutes. Slides were washed in 60% isopropanol (5 minutes), counterstained with Mayer’s hematoxylin, and cover slipped with an aqueous mounting medium (Fisher Scientific). For H&E staining, paraffin-embedded tissue sections were stained following a standard protocol using Gill’s hematoxylin (42). For both Oil Red O and H&E staining, three slides containing sections chosen 50 μm apart were used. Five regions corresponding to the four corners and center of each section were imaged for determining the Oil Red O–positive area. The ratio of Oil Red O–positive stain area to the total area of the imaged section was calculated using the Image Pro-Plus 3.0.1 system (Media Cybernetics, Rockville, MD) using a well-validated densitometrical method (43) as described previously (44). The distribution of lipid deposits did not allow quantitative estimation to be performed on the muscle sections.

To assess the tissue triglyceride content, 100 mg of the tissue was homogenized in 1 mL of phosphate-buffered saline, and tissue lipids were extracted using the Bligh and Dyer method (45) with slight modifications. Briefly, the tissue homogenate was placed into a borosilicate glass tube containing 3.75 ml of 1:2 (v/v) chloroform/methanol, 1.25 mL chloroform, and 1.25 mL distilled water, vortexed, and centrifuged for 5 minutes at room temperature. The bottom phase was carefully recovered, air dried overnight, and reconstituted in 200 µL isopropyl alcohol for assessment of triglyceride content using the Wako L-Type TG M kit (Wako Diagnostics, Mountain View, CA) as per the manufacturer’s recommendations. Each sample was assayed in duplicate, and the intra- and interassay coefficients of variance were 3.32 ± 0.57% and 5.2 ± 0.73%, respectively. Plasma triglyceride content was assessed using a colorimetric assay kit (Cayman Chemicals, Ann Arbor, MI) as per the manufacturer’s recommendation. The intra- and interassay coefficients of variance were 2.14% and 6.71%, respectively.

Statistical analysis

Heterogeneity of variance was tested with Bartlett χ² test, and when required data were log-transformed. Changes in gene expression, markers of oxidative stress, and triglyceride content were analyzed using one-way analysis of variance (ANOVA) followed by Tukey post hoc test using Prism 6 software (GraphPad, La Jolla, CA), and the threshold for significance was set as P ≤ 0.05. The prenatal and postnatal treatment effects were analyzed separately. Cohen effect size analysis (35, 46, 47) was also used to determine the magnitude of differences between the control and prenatal T–treated and the intervention groups vs the prenatal T–treated group. A computed Cohen d value of ≥0.5 indicates medium large effect size differences, and a value of ≥0.8 and above indicates large effect size differences (46, 47).

Results

Effect of prenatal interventions

Adiponectin

Effect size analysis found medium-sized effects of prenatal T treatment in decreasing mRNA expression of ADIPOQ in VAT and large-sized effects in increasing expression in SAT, liver, and muscle (Fig. 2). Prenatal T treatment increased mRNA expression of ADIPOR1 in VAT. Effect size analysis also revealed large effects in increasing the ADIPOR1 mRNA expression in VAT as well as muscle and a medium-sized effect in ADIPOR2 expression in liver and muscle. Prenatal intervention with androgen antagonist or insulin sensitizer did not alter prenatal T treatment–induced changes.

Figure 2. — mRNA expression of *ADIPOQ* (top panels), *ADIPOR1* (middle panels), and *ADIPOR2* (bottom panels) in the VAT, SAT, liver, and muscle tissues of C, T, TF, and TR groups. Data are presented as the mean ± standard error of the mean of the fold change in mRNA levels. Differing letters above the histogram indicate significant changes by ANOVA. Numerical superscripts are Cohen d values comparing (1) C vs T (within the circle) and (2) TF and TR vs T (within the square box).

Prenatal T treatment increased HMW forms of ADIPOQ in the plasma. Effect size analysis, in addition to revealing large effects of prenatal T treatment in increasing HMW forms, revealed a medium-sized effect in increasing LMW forms. Effect size analysis showed large effects of insulin sensitizer in preventing prenatal T–induced increase in both LMW and HMW forms and androgen antagonist in preventing the increase in HMW forms (Fig. 3).

Figure 3. — Plasma levels of LMW and HMW forms of adiponectin in C, T, TF, and TR groups. Top panels show representative blots under reducing conditions (left, LMW) and nonreducing conditions (right, HMW forms) along with Ponceau staining for protein loading. Bottom panels show mean ± standard error of the mean of density ratios between LMW (bottom left) and HMW (bottom right) forms of adiponectin with the 64-kDa protein from the Ponceau-stained membrane. Differing letters above the histogram indicate significant changes by ANOVA. Numerical superscripts are Cohen d values comparing (1) C vs T (within the circle) and (2) TF and TR vs T (within the square box).

At the tissue level, prenatal T excess increased phosphorylation of AMPK, a molecule activated by adiponectin (48) in the liver (Supplemental Fig. 1^{(1.1MB, TIF)}). In addition, effect size analysis showed large effects in prenatal T treatment–induced increases in AMPK phosphorylation not only in the liver but also in the muscle and showed a decrease in pAMPK in VAT. Both prenatal interventions prevented the effects of prenatal T excess in liver but not in muscle and VAT (Supplemental Fig. 1^{(1.1MB, TIF)}).

Proinflammatory cytokines

Prenatal T treatment increased mRNA expression of cytokines, tumor necrosis factor alpha (TNF), and chemokine (C-C) ligand 2 (CCL2) and trended (P = 0.06) to increase interleukin 1 beta (IL1B) in VAT (Fig. 4). Effect size analysis also revealed large effects of prenatal T treatment in inducing increases in all cytokines not only in VAT but also in liver and TNF and CCL2 only in SAT and muscle. Prenatal T treatment also increased the expression of macrophage marker CD68 in VAT, in addition to having a medium-sized effect in increasing CD68 in SAT, liver, and muscle.

Figure 4. — Proinflammatory cytokines (mRNA expression of *IL1B*, *TNF*, and *CCL2*) and macrophage marker (*CD68*) expression in C, T, TF, and TR groups (mean ± standard error of the mean). Differing letters above the histogram indicate significant changes by ANOVA. Numerical superscripts are Cohen d values comparing (1) C vs T (within the circle) and (2) TF and TR vs T (within the square box). ^#Indicates a significant difference between C and T groups as determined by Student t test.

Prenatal intervention with an androgen antagonist did not prevent the prenatal T–induced increase in IL1B, TNF, and CD68 but blocked the increase in CCL2 expression in VAT. In contrast, prenatal intervention with insulin sensitizer blocked the prenatal T–induced increase in TNF and partially prevented the increase in CD68 expression in the VAT. Neither intervention prevented the impact of prenatal T on any proinflammatory cytokines evident through effect size analyses in SAT, liver, and muscle. However, flutamide intervention partially prevented the effects of prenatal T treatment on CD68 in muscle, whereas rosiglitazone intervention fully prevented its effects on CD68 in SAT.

Oxidative stress markers

Exposure to gestational T excess increased plasma concentrations of NY but not DY (Fig. 5). Effect size analyses also found a large-magnitude difference between control and prenatal T–treated female sheep in plasma NY levels (d = 2.2) and only a marginal difference in plasma DY levels (d = 0.7). Androgen antagonist intervention but not insulin sensitizer treatment partially prevented the increase in plasma NY (TF not different from C or T; effect size Cohen d = 1.3 T vs TF).

Figure 5. — Plasma and tissue oxidized tyrosine concentrations in C, T, TF, and TR groups (mean ± standard error of the mean). The concentrations of NY and DY are expressed as the ratio of oxidized tyrosine to total tyrosine in plasma (top panels) and NY (middle panel) and DY (bottom panel) in the VAT, SAT, liver, and muscle. Differing letters above the histogram indicate significant changes by ANOVA. Numerical superscripts are Cohen d values comparing (1) C vs T (within the circle) and (2) TF and TR vs T (within the square box).

Concentrations of NY were also significantly increased in VAT and liver. Effect size analyses found large size differences in prenatal T–induced increases in NY in VAT and liver as well as in SAT and muscle. Effect size analyses also found large size increases in DY concentrations in VAT of prenatal T–treated animals. In both SAT and muscle, effect size analysis also showed marginal increases in NY and not DY content. ClY was below detection limit in the plasma and in all tissues examined. Both interventions failed to prevent prenatal T–induced increases in NY level in all tissues.

Antioxidant gene expression

Prenatal T treatment increased mRNA expression of GSR and SOD1 and tended to increase (P = 0.06) SOD2 in VAT (Fig. 6). Prenatal T treatment also increased SOD1 in muscle. Effect size analysis also found large-magnitude differences between control and prenatal T–treated female sheep in all three antioxidants in VAT and SOD1 in muscle. In addition, effect size analyses found large-magnitude increases in GSR and SOD1 in muscle, GSR in SAT, and SOD1 in liver. Neither intervention prevented prenatal T–induced increases in GSR, SOD1, and SOD2 in any of the tissues studied. The interventions magnified the amplitude of differences with levels being higher in the intervention groups compared with prenatal T–treated groups.

Figure 6. — Antioxidant [*GSR* (top panels), *SOD1* (middle panels), and *SOD2* (bottom panels)] expression in the VAT, SAT, liver, and muscle tissues of C, T, TF, and TR groups (mean ± standard error of the mean). Differing letters above the histogram indicate significant changes by ANOVA. Numerical superscripts are Cohen d values comparing (1) C vs T (within the circle) and (2) TF and TR vs T (within the square box). ^#Indicates significant difference between C and T groups as determined by Student t test.

Plasma triglycerides

Plasma triglyceride content was significantly elevated by prenatal T treatment, with neither intervention preventing this increase (Supplemental Fig. 3^{(559.7KB, TIF)}).

Ectopic lipid accumulation

Prenatal T treatment significantly increased the liver triglyceride content, and effect size analysis also showed large effects of prenatal T treatment in increasing liver lipid content, as evident from Oil Red O staining and a clear area in H&E-stained sections (Fig. 7). Although effect size analysis also revealed large effects of prenatal T treatment in increasing muscle triglyceride content, the lipid content in muscle after Oil Red O staining while appearing to be increased was not quantifiable (Supplemental Fig. 4^{(24.5KB, doc)}). Neither intervention affected the liver triglyceride content. However, effect size analysis found that androgen antagonist prevented the large effects of prenatal T treatment in liver lipid content (Oil Red O staining). Effect size analyses also found that androgen antagonist treatment prevented the increase in muscle triglyceride content.

Figure 7. — Ectopic lipid accumulation in liver from C, T, TF, and TR groups. Photomicrographs of histochemical analysis by H&E staining of paraffin-embedded and Oil Red O staining in cryosections from liver is shown in the right panels (bar = 25 μm). On the left are shown mean ± standard error of the mean of the percent staining of Oil Red O in liver (top left) and triglyceride content in liver (left middle) and muscle (left bottom). Differing letters above the histogram indicate significant changes by ANOVA. Numerical superscripts are Cohen d values comparing (1) C vs T (within the circle) and (2) TF and TR vs T (within the square box).

Effect of postnatal treatments

Assessment of the effect of postnatal intervention with androgen receptor antagonist or insulin sensitizer was restricted to variables affected by prenatal T treatment. These included adiponectin, NY and triglyceride content in plasma, ADIPOR1, proinflammatory cytokines, NY content and antioxidants in the VAT, pAMPK, NY and lipid content in liver, and pAMPK and lipid content in muscle.

Adiponectin

Neither postnatal intervention had an effect in reversing the prenatal T treatment–induced increase in HMW adiponectin forms or ADIPOR1 in VAT or pAMPK in the liver (Fig. 8; Supplemental Fig. 2^{(991.2KB, TIF)}).

Figure 8. — Plasma levels of LMW and HMW forms of adiponectin and mRNA expression of ADIPOR1 in VAT in C, T, T+F, and T+R groups. Top panels show representative blots under reducing conditions (left: LMW) and nonreducing conditions (right: HMW forms) along with Ponceau staining to depict protein loading. Bottom panels show mean ± standard error of the mean of density ratios between LMW (bottom left) and HMW (bottom middle) of adiponectin with the 64-kDa protein from the Ponceau-stained membrane. Bottom right shows mRNA expression levels of ADIPOR1. Differing letters above the histogram indicate significant changes by ANOVA. Numerical superscripts are Cohen d values comparing (1) C vs T (within the circle) and (2) T+F and T+R vs T (within the square box). ^#Indicates significant difference between C and T groups as determined by Student t test.

Proinflammatory cytokines

Postnatal insulin sensitizer but not antiandrogen treatment reversed the trend in increased expression of IL1B induced by prenatal T treatment (Fig. 9). Effect size analysis revealed that antiandrogen and insulin sensitizer treatment also had large- and medium-sized effects, respectively, in reversing the prenatal T–induced increase in TNF. Prenatal T treatment induced increases in CCL2 and CD68 expression in the VAT were not reversed by either treatment.

Figure 9. — Proinflammatory cytokines (mRNA expression of *IL1B*, *TNF*, and *CCL2*) and macrophage marker (*CD68)* expression in C, T, T+F, and T+R groups (mean ± standard error of the mean). Differing letters above the histogram indicate significant changes by ANOVA. Numerical superscripts are Cohen d values comparing (1) C vs T (within the circle) and (2) T+F and T+R vs T (within the square box). ^#Indicates significant difference between C and T groups as determined by Student t test.