Adiponectin protects against development of PNAS PLUS metabolic disturbances in a PCOS mouse model Anna Benricka,b,1, Belén Chanclóna, Peter Micallefa, Yanling Wua, Laila Hadia, John M. Sheltonc, Elisabet Stener-Victorina,d,2, and Ingrid Wernstedt Asterholma,2 aDepartment of Physiology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, 40530 Gothenburg, Sweden; bSchool of Health and Education, University of Skövde, 54128 Skövde, Sweden; cMolecular Pathology Core, University of Texas Southwestern Medical Center, Dallas, TX 75390-8573; and dDepartment of Physiology and Pharmacology, Karolinska Institute, 17177 Stockholm, Sweden Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved July 11, 2017 (received for review May 30, 2017) Adiponectin, together with adipocyte size, is the strongest factor (15). Furthermore, overexpression of adiponectin is associated associated with insulin resistance in women with polycystic ovary with improved whole-body insulin sensitivity and profound positive syndrome (PCOS). This study investigates the causal relationship effects within adipose tissue, such as increased mitochondrial between adiponectin levels and metabolic and reproductive functions density in adipocytes, smaller adipocyte size, and transcriptional in PCOS. Prepubertal mice overexpressing adiponectin from adipose up-regulation of factors involved in lipid storage through efficient tissue (APNtg), adiponectin knockouts (APNko), and their wild-type esterification of free fatty acids (16, 17). (WT) littermate mice were continuously exposed to placebo or Adipose tissue is now also recognized as an important factor in dihydrotestosterone (DHT) to induce PCOS-like traits. As expected, the complex equation by which nutritional status regulates female DHT exposure led to reproductive dysfunction, as judged by con- reproductive function (18). We have shown that adiponectin re- tinuous anestrus, smaller ovaries with a decreased number of corpus ceptors 1 and 2 are increased in ovaries in PCOS rat models (19, luteum, and an increased number of cystic/atretic follicles. A two-way 20), and accumulating evidence suggests a direct role for adipo- between-groups analysis showed that there was a significant main nectin in female reproductive tissues (18). Moreover, a lower effect for DHT exposure, but not for genotype, indicating adiponectin proportion of theca cells express adiponectin receptors in poly- does not influence follicle development. Adiponectin had, however, cystic ovaries compared with normal ovaries (21). In cultured some protective effects on ovarian function. Similar to in many women theca cells, adiponectin suppresses androstenedione production with PCOS, DHT exposure led to reduced adiponectin levels, larger and key enzymes in the androgen synthesis pathway (21), and adipocyte size, and reduced insulin sensitivity in WTs. APNtg mice placental cytotrophoblasts exposed to adiponectin modulate their remained metabolically healthy despite DHT exposure, while APNko- DHT mice were even more insulin resistant than their DHT-exposed steroidogenesis (22). However, adiponectin signaling is not es- littermate WTs. DHT exposure also reduced the mRNA expression of sential for normal mouse reproduction, as adiponectin knockout genes involved in metabolic pathways in gonadal adipose tissue of WT mice can produce viable offspring. and APNko, but this effect of DHT was not observed in APNtg mice. Based on these observations, we hypothesize that altered Moreover, APNtg-DHT mice displayed increased pancreatic mRNA adiponectin levels are causally involved in both the reproductive levels of insulin receptors, Pdx1 and Igf1R, suggesting adiponectin and metabolic disturbances associated with PCOS. To test this stimulates beta cell viability/hyperplasia in the context of PCOS. In hypothesis, we exposed adiponectin knockout (APNko) and conclusion, adiponectin improves metabolic health but has only mi- overexpressing transgenic (APNtg) mice along with their litter- nor effects on reproductive functions in this PCOS-like mouse model. mate wild-type (WT) controls to continuous dihydrotestosterone polycystic ovary syndrome | insulin resistance | adipose tissue Significance olycystic ovary syndrome (PCOS) is the most common en- Women with polycystic ovary syndrome (PCOS) have reduced Pdocrine and metabolic disorder occurring in females (1). fertility and increased risk of developing type 2 diabetes. Adi- PCOS is one of the leading causes of poor fertility and is associated ponectin, an adipocyte-derived hormone, together with adipo- with abdominal obesity, metabolic syndrome, and an increased risk cyte size, is the strongest factor associated with insulin resistance of developing type 2 diabetes (1, 2). Indeed, it has been demon- in PCOS. The potential causal relationship among adiponectin strated that 30–40%ofwomenwithPCOShaveimpairedglucose levels, infertility, and metabolic dysfunction in PCOS is, however, tolerance, and as many as 10% develop diabetes by the age of 40 y unknown. Exploiting mouse models, we found that adiponectin (3, 4). Despite the negative effect of PCOS on women’s health, is indeed involved in PCOS-related insulin resistance, and, albeit very little is known about its etiology, including the causal re- to a much smaller extent, is also involved in the development of lationship between the reproductive and metabolic features of reproductive dysfunction. Thus, increased levels of adiponectin PCOS. Adipose tissue dysfunction is implicated as a key feature, can prevent prepubertal androgen-induced metabolic dysfunc- and both lean and obese women with PCOS have aberrant adipose tion in dihydrotestosterone-exposed mice with PCOS-like traits. tissue morphology (5–7). Specifically, we have found that large Collectively, our findings support the notion that altered adipose tissue functionality is a key mediator of metabolic dysfunction in adipocyte size, low circulating adiponectin levels, and increased womenwithPCOS. waist circumference, but surprisingly, not androgen excess, are factors that are most strongly associated with insulin resistance in Author contributions: A.B. and E.S.-V. designed research; A.B., B.C., P.M., Y.W., L.H., women with PCOS (8). Others have shown that hyperandrogenism J.M.S., and I.W.A. performed research; A.B., B.C., P.M., Y.W., L.H., J.M.S., and I.W.A. is associated with intra-abdominal fat deposition (9). Adiponectin analyzed data; and A.B., E.S.-V., and I.W.A. wrote the paper. is an adipocyte-derived hormone exerting potent positive effects on The authors declare no conflict of interest. whole-body insulin sensitivity (10, 11), as well as on pancreatic This article is a PNAS Direct Submission. beta-cell survival and functionality (12–14). In line with the bene- 1To whom correspondence should be addressed. Email: [email protected]. ficial effects of adiponectin on metabolic regulation, adiponectin 2E.S.-V. and I.W.A. contributed equally to this work. PHYSIOLOGY knockout mice exposed to a high-fat diet display decreased hepatic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. insulin sensitivity and an impaired response to thiazolidinediones 1073/pnas.1708854114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1708854114 PNAS | Published online August 8, 2017 | E7187–E7196 Downloaded by guest on September 26, 2021 (DHT) exposure from puberty to adult life. DHT exposure has pattern was not different in APNtg-DHT compared with WT- been shown to be a reproducible mouse model of PCOS, effec- DHT mice, but APNko-DHT mice spent more time in diestrus tively mimicking many of the reproductive and metabolic disease and less time in proestrus compared with DHT-exposed WTs (P < features of patients with PCOS (23, 24). Using this approach, we 0.05). In agreement with the disrupted estrus cycle, all DHT- found that elevated levels of adiponectin protect against DHT- exposed mice, irrespective of genotype, showed no or very few induced insulin resistance and that this effect is associated with corpora lutea in the ovaries (Fig. 1D). reduced adipocyte size, decreased visceral fat, and a more Ovaries of DHT-exposed mice of all three genotypes weighed healthy adipose tissue, as reflected by restored adipose tissue and less than their placebo controls, and there was no effect of adi- improved pancreatic gene expression. In contrast, lack of adi- ponectin on this parameter (Fig. 1C). Uterus weight was not ponectin led to more severe metabolic consequences in response affected by either DHT or genotype (Fig. S1). Ovaries of DHT- to DHT/PCOS. Adiponectin had, however, only minor effects on exposed mice contained a similar number of antral follicles to reproductive functions in PCOS mice. placebo-treated control mice (Fig. 1E). The number of cystic and atretic follicles was increased in DHT-exposed WT and APNko Results mice (Fig. 1F); however, this difference was not found to be DHT Exposure Alters Sex Hormone Levels, but Not LH and FSH. To significant in APNtg mice (Fig. 1F). confirm that DHT exposure resulted in elevated serum DHT lev- els, sex hormone levels were determined. As expected, DHT levels Adiponectin Protects Against DHT-Induced Alterations in Cyp19a1 and Hsd3b, Which Are Genes Involved in Sex Hormone Steroidogenesis in were increased and testosterone levels decreased in DHT-exposed the Ovary. We measured the ovarian gene expression of four animals compared with in placebo (P) groups (Table 1). enzymes involved