Maternal diet alters long-term innate immune cell memory in fetal and juvenile hematopoietic stem and progenitor cells in nonhuman primate offspring.

Maternal overnutrition increases inflammatory and metabolic disease risk in postnatal offspring. This constitutes a major public health concern due to increasing prevalence of these diseases, yet mechanisms remain unclear. Here, using nonhuman primate models, we show that maternal Western-style diet (mWSD) exposure is associated with persistent pro-inflammatory phenotypes at the transcriptional, metabolic, and functional levels in bone marrow-derived macrophages (BMDMs) from 3-year-old juvenile offspring and in hematopoietic stem and progenitor cells (HSPCs) from fetal and juvenile bone marrow and fetal liver. mWSD exposure is also associated with increased oleic acid in fetal and juvenile bone marrow and fetal liver. Assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling of HSPCs and BMDMs from mWSD-exposed juveniles supports a model in which HSPCs transmit pro-inflammatory memory to myeloid cells beginning in utero. These findings show that maternal diet alters long-term immune cell developmental programming in HSPCs with proposed consequences for chronic diseases featuring altered immune/inflammatory activation across the lifespan.

The combined impact of testosterone and Western-style diet on endometriosis severity and progression in rhesus macaques†.

Polycystic ovary syndrome (PCOS) is associated with irregular menstrual cycles, hyperandrogenemia, and obesity. It is currently accepted that women with PCOS are also at risk for endometriosis, but the effect of androgen and obesity on endometriosis has been underexplored. The goal of this study was to determine how testosterone (T) and an obesogenic diet impact the progression of endometriosis in a nonhuman primate (NHP) model. Female rhesus macaques were treated with T (serum levels approximately 1.35 ng/ml), Western-style diet (WSD; 36% of calories from fat compared to 16% in standard monkey chow) or the combination (T + WSD) at the time of menarche as part of a longitudinal study for ~7 years. Severity of endometriosis was determined based on American Society for Reproductive Medicine (ASRM) revised criteria, and staged 1-4. Stages 1 and 2 were associated with extent of abdominal adhesions, while stages 3 and 4 were associated with presence of chocolate cysts. The combined treatment of T + WSD resulted in earlier onset of endometriosis and more severe types associated with large chocolate cysts compared to all other treatments. There was a strong correlation between glucose clearance, homeostatic model assessment for insulin resistance (HOMA-IR), and total percentage of body fat with presence of cysts, indicating possible indirect contribution of hyperandrogenemia via metabolic dysfunction. An RNA-seq analysis of omental adipose tissue revealed significant impacts on a number of inflammatory signaling pathways. The interactions between obesity, hyperandrogenemia, and abdominal inflammation deserve additional investigation in NHP model species.

Androgen-mediated Perturbation of the Hepatic Circadian System Through Epigenetic Modulation Promotes NAFLD in PCOS Mice.

In women, excess androgen causes polycystic ovary syndrome (PCOS), a common fertility disorder with comorbid metabolic dysfunctions including diabetes, obesity, and nonalcoholic fatty liver disease. Using a PCOS mouse model, this study shows that chronic high androgen levels cause hepatic steatosis while hepatocyte-specific androgen receptor (AR)-knockout rescues this phenotype. Moreover, through RNA-sequencing and metabolomic studies, we have identified key metabolic genes and pathways affected by hyperandrogenism. Our studies reveal that a large number of metabolic genes are directly regulated by androgens through AR binding to androgen response element sequences on the promoter region of these genes. Interestingly, a number of circadian genes are also differentially regulated by androgens. In vivo and in vitro studies using a circadian reporter [Period2::Luciferase (Per2::LUC)] mouse model demonstrate that androgens can directly disrupt the hepatic timing system, which is a key regulator of liver metabolism. Consequently, studies show that androgens decrease H3K27me3, a gene silencing mark on the promoter of core clock genes, by inhibiting the expression of histone methyltransferase, Ezh2, while inducing the expression of the histone demethylase, JMJD3, which is responsible for adding and removing the H3K27me3 mark, respectively. Finally, we report that under hyperandrogenic conditions, some of the same circadian/metabolic genes that are upregulated in the mouse liver are also elevated in nonhuman primate livers. In summary, these studies not only provide an overall understanding of how hyperandrogenism associated with PCOS affects liver gene expression and metabolism but also offer insight into the underlying mechanisms leading to hepatic steatosis in PCOS.

Maternal Obesity and Western-Style Diet Impair Fetal and Juvenile Offspring Skeletal Muscle Insulin-Stimulated Glucose Transport in Nonhuman Primates.

Infants born to mothers with obesity have a greater risk for childhood obesity and metabolic diseases; however, the underlying biological mechanisms remain poorly understood. We used a Japanese macaque model to investigate whether maternal obesity combined with a Western-style diet (WSD) impairs offspring muscle insulin action. Adult females were fed a control or WSD prior to and during pregnancy through lactation, and offspring subsequently weaned to a control or WSD. Muscle glucose uptake and signaling were measured ex vivo in fetal (n = 5-8/group) and juvenile (n = 8/group) offspring. In vivo signaling was evaluated after an insulin bolus just prior to weaning (n = 4-5/group). Maternal WSD reduced insulin-stimulated glucose uptake and impaired insulin signaling at the level of Akt phosphorylation in fetal muscle. In juvenile offspring, insulin-stimulated glucose uptake was similarly reduced by both maternal and postweaning WSD and corresponded to modest reductions in insulin-stimulated Akt phosphorylation relative to controls. We conclude that maternal WSD leads to a persistent decrease in offspring muscle insulin-stimulated glucose uptake even in the absence of increased offspring adiposity or markers of systemic insulin resistance. Switching offspring to a healthy diet did not reverse the effects of maternal WSD on muscle insulin action, suggesting earlier interventions may be warranted.

Maternal High-Fat Diet Consumption and Chronic Hyperandrogenemia Are Associated With Placental Dysfunction in Female Rhesus Macaques.

The risk of adverse perinatal outcomes with maternal polycystic ovary syndrome may differ among hyperandrogenic and nonhyperandrogenic phenotypes and is likely modulated by maternal obesity and diet. The relative contribution of maternal hyperandrogenism and nutritional status to placental dysfunction is unknown. Female rhesus macaques (N = 39) were assigned at puberty to one of four treatment groups: subcutaneous cholesterol implants and a standard chow diet (controls); testosterone (T) implants and a normal diet; cholesterol implants and a high-fat, Western-style diet (WSD); and testosterone implants in combination with a high-fat diet. After 3.5 years of treatment, contrast-enhanced and Doppler ultrasound analyses of placental blood flow were performed for a representative subset of animals from each treatment group during pregnancy, and placental architecture assessed with stereological analysis. Placental growth factors, cellular nutrient sensors, and angiogenic markers were measured with ELISA and Western blotting. WSD consumption was associated with a 30% increase in placental flux rate relative to that in animals receiving a normal diet. T and WSD treatments were each independently associated with increased villous volume, and T also was associated with an ∼ 40% decrease fetal capillary volume on stereological analysis. T treatment was associated with significantly increased mTOR and SOCS3 expression. WSD consumption was associated with decreased GLUT1 expression and microvillous membrane localization. Hyperandrogenemic and nonhyperandrogenemic phenotypes are associated with altered placental angiogenesis, nutrient sensing, and glucose transport. WSD and T appear to have distinct effects on vascular impedance and capillary angiogenesis.

Chronic hyperandrogenemia in the presence and absence of a western-style diet impairs ovarian and uterine structure/function in young adult rhesus monkeys.

STUDY QUESTION: Does chronic hyperandrogenemia beginning at menarche, in the absence and presence of a western-style diet (WSD), alter ovarian and uterine structure-function in young adult rhesus monkeys? SUMMARY ANSWER: Phenotypic alterations in ovarian and uterine structure/function were induced by exogenous testosterone (T), and compounded in the presence of a WSD (T+WSD). WHAT IS KNOWN ALREADY: Hyperandrogenemia is a well-established component of PCOS and is observed in adolescent girls, indicating a potential pubertal onset of disease symptoms. Obesity is often associated with hyperandrogenemia and it is hypothesized that metabolic dysfunction exacerbates PCOS symptoms. STUDY DESIGN, SIZE, DURATION: Macaque females (n = 40) near the onset of menarche (~2.5 years of age) were assigned to a 2 by 2 factorial cohort design. Effects on reproductive characteristics were evaluated after 3 years of treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Rhesus macaques (Macaca mulatta) were fed either a normal balanced diet (n = 20) or a WSD (n = 20). Additionally, implants containing cholesterol (n = 20) or T (n = 20) were implanted subcutaneously to elevate serum T approximately 5-fold. This resulted in treatment groups of controls (C), T, WSD and T+WSD (n = 10/group). Vaginal swabbing was performed daily to detect menses. After 3 years of treatment, daily serum samples from one menstrual cycle were assayed for hormone levels. Ovarian structure was evaluated in the early follicular phase by 3D/4D ultrasound. Uterine endometrial size and ovarian/luteal vascular function was also evaluated in subgroups (n = 6/group) in the late follicular and mid-luteal phases by 3D/4D ultrasound and contrast-enhanced ultrasound, respectively. Expression of steroid hormone receptors and markers of decidualization and endometrial receptivity were assessed in endometrial biopsies at mid-luteal phase. MAIN RESULTS AND THE ROLE OF CHANCE: Approximately 90% of menstrual cycles appeared ovulatory with no differences in frequency or duration between groups. Serum estradiol (E2) levels during the early follicular phase were greatest in the T alone group, but reduced in T+WSD (P < 0.02). Serum LH was elevated in the T group (P < 0.04); however, there were no differences among groups in FSH levels (P > 0.13). Ovarian size at menses tended to be greater in the WSD groups (P < 0.07) and antral follicles ≥1 mm were more numerous in the T+WSD group (P < 0.05). Also, females in T and T+WSD groups displayed polycystic ovarian morphology (PCOM) at greater frequency than C or WSD groups (P < 0.01). Progesterone (P4) levels during the luteal phase were reduced in the T+WSD group compared to C and T groups (P < 0.05). Blood volume (BV) and vascular flow (VF) within the corpus luteum was reduced in all treatment groups compared to C (P < 0.01, P = 0.03), with the WSD alone group displaying the slowest BV and VF (P < 0.05). C and WSD groups displayed endometrial glands at mid-luteal phase with low estrogen receptor 1 (ESR1) and progesterone receptor (PGR) mRNA and immunohistochemical staining in the functionalis zone, but appreciable PGR in the stroma. In contrast, T and T+WSD treatment resulted in glands with less secretory morphology, high ESR1 expression in the glandular epithelium and low PGR in the stroma. Endometrial levels of TIMP3 and MMP26 mRNA and immunostaining were also decreased in the T and T+WSD groups, whereas AR expression was unchanged. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Females are young adults, so effects could change as they reach prime reproductive age. The T level generated for hyperandrogenemia may be somewhat greater than the 3-4-fold increase observed in adolescent girls, but markedly less than those observed in male monkeys or adolescent boys. WIDER IMPLICATIONS OF THE FINDINGS: Alterations to ovarian and uterine structure-function observed in T and, in particular, T+WSD-treated female macaques are consistent with some of the features observed in women diagnosed with polycystic ovary syndrome (PCOS), and suggest impaired fertility. STUDY FUNDING/COMPETING INTEREST(S): Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH) under Award Number P50HD071836 (to RLS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Additional funding was provided by Office of the Director, NIH under Award Number P51OD011092 (Support for National Primate Research Center). Authors declare no competing interests.