Received: 25 September 2020 | Revised: 26 November 2020 | Accepted: 7 December 2020 DOI: 10.1096/fj.202002212R RESEARCH ARTICLE Maternal adiponectin prevents visceral adiposity and adipocyte hypertrophy in prenatal androgenized female mice Yanling Wu1 | Belén Chanclón1 | Peter Micallef1 | qElisabet Stener-Victorin2 | Ingrid Wernstedt Asterholm1 | Anna Benrick1,3 1Department of Physiology, Institute of Neuroscience and Physiology, Sahlgrenska Abstract Academy, University of Gothenburg, Hyperandrogenism is the main characteristic of polycystic ovary syndrome, which Gothenburg, Sweden affects placental function and fetal growth, and leads to reproductive and metabolic 2 Department of Physiology and dysfunction in female offspring. Adiponectin acts on the placenta and may exert Pharmacology, Karolinska Institute, Stockholm, Sweden endocrine effects on the developing fetus. This study aims to investigate if mater- 3School of Health Sciences, University of nal and/or fetal adiponectin can prevent metabolic and reproductive dysfunction in Skövde, Skövde, Sweden prenatal androgenized (PNA) female offspring. Adiponectin transgenic (APNtg) and wild-type dams received dihydrotestosterone/vehicle injections between gesta- Correspondence Anna Benrick, Department of Physiology, tional days 16.5-18.5 to induce PNA offspring, which were followed for 4 months. University of Gothenburg, Institute of Offspring from APNtg dams were smaller than offspring from wild-type dams, in- Neuroscience and Physiology, Box 423, 405 30 Gothenburg, Sweden. dependent of genotype. Insulin sensitivity was higher in wild-type mice from APNtg Email: [email protected] dams compared to wild-types from wild-type dams, and insulin sensitivity correlated with fat mass and adipocyte size. PNA increased visceral fat% and adipocyte size in Funding information Novo Nordisk Fonden (NNF), Grant/ wild-type offspring from wild-type dams, while wild-type and APNtg offspring from Award Number: NNF19OC0056601; APNtg dams were protected against this effect. APNtg mice had smaller adipocytes Diabetesfonden (Stiftelsen Svenska than wild-types and this morphology was associated with an increased expression Diabetesförbundets Forskningsfond), Grant/Award Number: DIA2019-419; of genes regulating adipogenesis (Ppard, Pparg, Cebpa, and Cebpb) and metabo- Magnus Bergvalls Stiftelse (Magnus lism (Chrebp and Lpl). Anogenital distance was increased in all PNA-exposed wild- Bergvall Foundation), Grant/Award Number: 2017-02069; Adlerbertska type offspring, but there was no increase in PNA APNtg offspring, suggesting that Stiftelserna (Adlerbertska Foundations), adiponectin overexpression protects against this effect. In conclusion, elevated adi- Grant/Award Number: GU2019/86; ponectin levels in utero improve insulin sensitivity, reduce body weight and fat mass Stiftelsen Handlanden Hjalmar Svenssons (Hjalmar Svensson Foundation), Grant/ gain in the adult offspring and protect against PNA-induced visceral adiposity. In Award Number: HJSV2019056; Kungl. conclusion, these data suggest that PNA offspring benefit from prenatal adiponectin Vetenskaps- och Vitterhets-Samhället i supplementation. Göteborg (KVVS), Grant/Award Number: 2018-217; Vetenskapsrådet (VR), Grant/ Award Number: 2017-00792, 2018-02435, KEYWORDS 2013-7107 and 2020-02435 adiponectin, adiposity, imprinting, PCOS, PNA Abbreviations: AGD, anogenital distance; APNtg, adiponectin transgenic; DEXA, dual-energy x-ray absorptiometry; DHT, dihydrotestosterone; GD, gestational day; ITT, insulin tolerance test; PCOS, polycystic ovary syndrome; PNA, prenatal androgenized; wt, wild-type. Anna Benrick and Ingrid Wernstedt Asterholm jointly supervised this work. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology. The FASEB Journal. 2021;35:e21299. wileyonlinelibrary.com/journal/fsb2 | 1 of 13 2 of 13 | WU ET AL. 1 | INTRODUCTION chronically androgen exposed PCOS model.19 Interestingly, female adiponectin transgenic mice of this model have lower The prevalence of polycystic ovary syndrome (PCOS) ranges testosterone and higher estradiol levels compared to controls, from 4% to 21% depending on the diagnostic criteria used, supporting a direct effect of adiponectin on sex hormone ste- and the syndrome causes reproductive and metabolic disease roidogenesis in the ovaries.19,20 This is also supported by in in women of reproductive age.1 Both clinical and preclinical vitro studies.21,22 Prenatally androgenized (PNA) mice are evidence pinpoint excessive androgen secretion as the under- another well-described PCOS-like mouse model in which lying cause of PCOS,2 and we have previously shown that the mice are exposed to androgens in utero, during late ges- daughters of women with PCOS are more likely to develop tation.23 PNA mice develop PCOS-like symptoms including the condition.3 Women with PCOS are also hyperandrogenic polycystic ovaries, irregular cycles, increased adipocyte size, during pregnancy,4-6 and have an increased risk of compli- and impaired glucose tolerance.24-26 Anogenital distance is cations during pregnancy and/or delivery, for example, pre- increased in PNA offspring and can be used as an sign of eclampsia and gestational diabetes.7 PCOS is associated with prenatal androgenization in utero in mice, and correlates with placental dysfunction5 that likely impacts fetal development androgen-dependent physiological effects in adulthood.27 and programing, and women with PCOS often deliver babies Adiponectin receptors are expressed in the placenta and adi- with a large or small birth weight for their gestational age.8 ponectin supplementation to pregnant dams during the rapid Birth weight is a sensitive marker of the overall impact of the fetal growth phase decreases glucose transport to the fetus intrauterine environment on fetal development and reduced or by inhibiting mTOR and insulin signaling in the placenta.8,28 excessive birth weight indicates an increased risk of the baby Lack of adiponectin, on the contrary, increases fetal growth developing obesity, diabetes, cardiovascular disease, and/or in mice.29 As adiponectin affects placental nutrient transport PCOS in adult life 9. The mechanisms underlying PCOS are during pregnancy, this allows for speculation that adiponectin poorly understood. However, it has been proposed that the may exert endocrine effects on the developing fetus during disease may be caused by an elevated exposure of circulat- pregnancy, and protect against the effects of prenatal andro- ing androgens during pregnancy, causing an unfavorable in- gen exposure, on the offspring. This study aims to investigate trauterine environment that can impair embryo development if maternal and/or fetal adiponectin can prevent metabolic and tissue patterning.10 and reproductive dysfunction in PNA female offspring. Adiponectin is a hormone that is secreted from adipose tissue that acts to improve insulin sensitivity and whole-body metabolism. Women with PCOS have decreased circulating 2 | MATERIAL AND METHODS adiponectin which, together with increased adipocyte size, represent the strongest contributing factors to the high-risk 2.1 | Animals PCOS women of developing insulin resistance.11 Jin and colleagues suggest that maternal adiponectin levels affect Wild-type (wt) and adiponectin transgenic (APNtg) mice, neonatal inflammation through effects on the milk compo- on a C57BL/6J background,30 were maintained under stand- sition,12 but its role during pregnancy, and in reproductive ard housing conditions at the animal facility, Sahlgrenska function, is underexplored. Adiponectin receptors are widely Academy, University of Gothenburg. They had ad libitum ac- distributed throughout the hypothalamic-pituitary-ovarian cess to food and water under controlled conditions (12-hour axis and reproductive organs, including the placenta and light/dark cycle environment). All experiments were per- endometrium, enabling adiponectin to affect metabolism in formed with permission from the Animal Ethics Committee these tissues,13-15 and potentially affect steroidogenesis.16 of the University of Gothenburg, and were performed in ac- Adiponectin is decreased before and throughout pregnancies cordance with EU guidelines for the care and use of labora- in women with PCOS, and women with PCOS who develop tory animals (2010/63/EU). gestational diabetes have lower adiponectin levels during pregnancy than euglycemic women with PCOS17 suggest- ing that low adiponectin levels lead to an impaired capacity 2.2 | Study design to handle metabolic changes during pregnancy. This is sup- ported by animal studies showing that pregnant adiponectin APNtg (n = 14) and wt (n = 11) female mice were mated with knockout mice are hyperlipidemic, with abnormal glucose wt males. Females in estrus phase, as determined by vaginal tolerance in late pregnancy.18 smears,31 were mated with a wt male overnight. Pregnancy We have shown that adiponectin overexpression pre- was confirmed by the presence of a post-copulation plug the vents the development of metabolic disorders in prepuber- following morning, and was this day was defined as gesta- tally androgenized PCOS-like mice.19 However, only minor tional day