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Hyperandrogenic disorders, such as polycystic ovary syndrome, are often associated with metabolic disruptions such as insulin resistance and hyperinsulinemia. Studies in sheep, a precocial model of translational relevance, provide evidence that in utero exposure to excess testosterone during days 30–90 of gestation (the sexually dimorphic window where males naturally experience elevated androgens) programs insulin resistance and hyperinsulinemia in female offspring. Extending earlier findings that adverse effects of testosterone excess are evident in fetal day 90 pancreas, the end of testosterone treatment, the present study provides evidence that transcriptomic and phenotypic effects of in utero testosterone excess on female pancreas persist after cessation of treatment, suggesting lasting organizational changes, and induce a male-like phenotype in female pancreas. These findings demonstrate that the female pancreas is susceptible to programmed masculinization during the sexually dimorphic window of fetal development and shed light on underlying connections between hyperandrogenism and metabolic homeostasis. • Exposure to excess testosterone programs masculinization of female sheep pancreas. • Programming of beta-cell phenotype parallels transcriptomic masculinization. • Pancreatic alpha-cell phenotype is inconsistent with masculinization. • Fetal beta- and alpha-cell changes may underlie adverse adult pancreatic phenotype. [ABSTRACT FROM AUTHOR]

Inappropriate developmental exposure to steroids is linked to metabolic disorders. Prenatal testosterone excess or bisphenol A (BPA, an environmental estrogen mimic) leads to insulin resistance and adipocyte disruptions in female lambs. Adipocytes are key regulators of insulin sensitivity. Metabolic tissue-specific differences in insulin sensitivity coupled with adipose depot-specific changes in key mRNAs, were previously observed with prenatal steroid exposure. We hypothesized that depot-specific changes in the non-coding RNA (ncRNA) - regulators of gene expression would account for the direction of changes seen in mRNAs. Non-coding RNA (lncRNA, miRNA, snoRNA, snRNA) from various adipose depots of prenatal testosterone and BPA-treated animals were sequenced. Adipose depot-specific changes in the ncRNA that are consistent with the depot-specific mRNA expression in terms of directionality of changes and functional implications in insulin resistance, adipocyte differentiation and cardiac hypertrophy were found. Importantly, the adipose depot-specific ncRNA changes were model-specific and mutually exclusive, suggestive of different regulatory entry points in this regulation. [Display omitted] • Prenatal testosterone (T) excess programs adipose depot-specific changes in ncRNA. • Prenatal T induced ECAT miRNA changes is consistent with its cardioprotective role. • Prenatal T induced VAT ncRNA changes link to metabolism regulating coding genes. • Prenatal bisphenol A (BPA) programmed ncRNA changes was specific to VAT not SAT. • ncRNA changes in pre-T and BPA models indicate different mechanisms of action. [ABSTRACT FROM AUTHOR]

Prenatal testosterone (T)- treated female sheep manifest juvenile insulin resistance, post-pubertal increase in insulin sensitivity and return to insulin resistance during adulthood. Since compensatory hyperinsulinemia is associated with insulin resistance, altered pancreatic islet ontogeny may contribute towards metabolic defects. To test this, pregnant sheep were treated with or without T propionate from days 30–90 of gestation and pancreas collected from female fetuses at gestational day 90 and female offspring at 21 months-of-age. Uterine (maternal) and umbilical (fetal) arterial blood insulin/glucose ratios were determined at gestational day 90. The morphological and functional changes in pancreatic islet were assessed through detection of 1) islet hormones (insulin, glucagon) and apoptotic beta cells at fetal day 90 and 2) islet hormones (insulin, glucagon and somatostatin), and pancreatic lipid and collagen accumulation in adults. At gestational day 90, T-treatment led to maternal but not fetal hyperinsulinemia, decrease in pancreatic/fetal weight ratio and alpha cells, and a trend for increase in beta cell apoptosis in fetal pancreas. Adult prenatal T-treated female sheep manifested 1) significant increase in beta cell size and a tendency for increase in insulin and somatostatin stained area and proportion of beta cells in the islet; and 2) significant increase in pancreatic islet collagen and a tendency towards increased lipid accumulation. Gestational T-treatment induced changes in pancreatic islet endocrine cells during both fetal and adult ages track the trajectory of hyperinsulinemic status with the increase in adult pancreatic collagen accumulation indicative of impending beta cell failure with chronic insulin resistance. Image 1 • Gestational T excess induces maternal but not fetal hyperinsulinemia. • Gestational T excess reduced pancreatic/fetal weight ratio and fetal islet α cells. • Prenatal T-excess increased β cell size and collagen content. • Prenatal T excess tended to increase β cell number islet insulin and lipid content. • Prenatal T excess-induced islet disruptions contribute to hyperinsulinemia. [ABSTRACT FROM AUTHOR]

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. Image 1 • 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 FROM AUTHOR]

Prenatal testosterone (T)-treated female sheep exhibit an enhanced inflammatory and oxidative stress state in the visceral adipose tissue (VAT) but not in the subcutaneous (SAT), while surprisingly maintaining insulin sensitivity in both depots. In adult sheep, adipose tissue is predominantly composed of white adipocytes which favor lipid storage. Brown/beige adipocytes that make up the brown adipose tissue (BAT) favor lipid utilization due to thermogenic uncoupled protein 1 expression and are interspersed amidst white adipocytes, more so in epicardiac (ECAT) and perirenal (PRAT) depots. The impact of prenatal T-treatment on ECAT and PRAT depots are unknown. As BAT imparts a metabolically healthy phenotype, the depot-specific impact of prenatal T-treatment on inflammation, oxidative stress, differentiation and insulin sensitivity could be dictated by the distribution of brown adipocytes. This hypothesis was tested by assessing markers of oxidative stress, inflammation, adipocyte differentiation, fibrosis and thermogenesis in adipose depots from control and prenatal T (100 mg T propionate twice a week from days 30–90 of gestation) -treated female sheep at 21 months of age. Our results show prenatal T-treatment induces depot-specific changes in inflammation, oxidative stress state, collagen accumulation, and differentiation with changes being more pronounced in the VAT. Prenatal T-treatment also increased thermogenic gene expression in all depots indicative of increased browning with effects being more prominent in VAT and SAT. Considering that inflammatory and oxidative stress are also elevated, the increased brown adipocyte distribution is likely a compensatory response to maintain insulin sensitivity and function of organs in the proximity of respective depots. • Prenatal testosterone excess programs adipose depot-specific changes. • Depot-specific changes include negative/positive mediators of insulin sensitivity. • Prenatal testosterone excess increases thermogenic markers in all adipose depots. • Increase in thermogenesis may underlie preserved VAT and SAT insulin sensitivity. [ABSTRACT FROM AUTHOR]