DHT-treated Mice Research Articles

MicroRNA-21 modulates brown adipose tissue adipogenesis and thermogenesis in a mouse model of polycystic ovary syndrome

BackgroundPolycystic ovary syndrome (PCOS), the most common endocrine disorder in premenopausal women, is associated with increased obesity, hyperandrogenism, and altered brown adipose tissue (BAT) thermogenesis. MicroRNAs play critical functions in brown adipocyte differentiation and maintenance. We aim to study the role of microRNA-21 (miR-21) in altered energy homeostasis and BAT thermogenesis in a PCOS mouse model of peripubertal androgen exposure.MethodsThree-week-old miR-21 knockout (miR21KO) or wild-type (WT) female mice were treated with dihydrotestosterone (DHT) or vehicle for 90 days. Body composition was determined by EchoMRI. Energy expenditure (EE), oxygen consumption (VO2), carbon dioxide production (VCO2), and respiratory exchange ratio (RER) were measured by indirect calorimetry. Androgen receptor (AR), and markers of adipogenesis, de novo lipogenesis, angiogenesis, extracellular matrix remodeling, and thermogenesis were quantified by RT-qPCR and/or Western-blot.ResultsMiR-21 ablation attenuated DHT-mediated increase in body weight while having no effect on fat or BAT mass. MiR-21 ablation attenuated DHT-mediated BAT AR upregulation. MiR-21 ablation did not alter EE; however, miR21KO DHT-treated mice have reduced VO2, VCO2, and RER. MiR-21 ablation reversed DHT-mediated decrease in food intake and increase in sleep time. MiR-21 ablation decreased some adipogenesis (Adipoq, Pparγ, and Cebpβ) and extracellular matrix remodeling (Mmp-9 and Timp-1) markers expression in DHT-treated mice. MiR-21 ablation abolished DHT-mediated increases in thermogenesis markers Cpt1a and Cpt1b, while decreasing CIDE-A expression.ConclusionsOur findings suggest that BAT miR-21 may play a role in regulating DHT-mediated thermogenic dysfunction in PCOS. Modulation of BAT miR-21 levels could be a novel therapeutic approach for the treatment of PCOS-associated metabolic derangements.

Time-Dependent Progression of Metabolic Dysfunction in a Mouse Model of Polycystic Ovary Syndrome

Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women. PCOS is closely linked to metabolic disorders such as obesity, dyslipidemia, systemic insulin resistance (IR), adipose IR and white adipose tissue (WAT) dysfunction. Hyperandrogenism is a clinical hallmark of PCOS, and it plays a fundamental role in the development of metabolic perturbations associated with PCOS. Androgen excess is linked to metabolic dysfunction in multiple tissues, including WAT, in PCOS women. However, data on the disease onset and progression of WAT dysfunction and associated metabolic derangements in PCOS women are lacking. Therefore, we aim to test the hypothesis that the androgen dihydrotestosterone (DHT) induces time-dependent progression of metabolic dysfunction on both the molecular and functional levels in PCOS. This hypothesis will be tested using a well-characterized DHT-induced PCOS mouse model after 2, 4, 8 and 12 weeks of DHT administration. Methods: Three-week old female mice (C57BL/6N) were implanted with Silastic tubes filled with DHT (8 mg, s.c.) or vehicle (n=6/grp) for 2, 4, 8 and 12 weeks. Weekly body weight (BW, gravimetry), body composition (EchoMRI), retroperitoneal fat (RPF) mass (gravimetry) were assessed. Serum leptin, adiponectin and insulin were measured by ELISA, while cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and non-esterified free fatty acid (NEFA) were measured using a clinical chemistry analyzer. Adipose IR (insulin x NEFA) was calculated. RPF androgen receptor (AR), lipolytic marker adipose triglyceride lipase (ATGL), and adipogenesis marker peroxisome proliferator-activated receptor-γ (PPAR-γ) protein levels were assessed by Western blot. Results: DHT-treated mice showed significant (p<0.05) increases in BW (20.23±0.3 vs 17.63±0.1 g) and lean mass (17.77±0.5 vs 15.50±0.3 g) starting 2 weeks following DHT exposure where higher cholesterol (90.0±2.9 vs 70.0±5.8 mg/dL), insulin (0.51±0.08 vs 0.22±0.03 ng/mL) and adipo-IR (2.5-fold) as well as lower insulin-sensitizing adipokine adiponectin levels (4.9±0.2 vs 9.70±1.1 mg/mL) were also observed compared to vehicle. The increases in RPF mass (105.40±10.3 vs 32.78±1.6 mg) and total fat mass (2.43±0.5 vs 1.09±0.1 g) were only significant starting at weeks 4 and 8, respectively. DHT significantly increased NEFA (1.3- and 1.2-fold, 8- and 12-weeks post DHT, respectively) and leptin (1.5-fold, 12 weeks post DHT). Unexpectedly, DHT increased HDL-C (58.80±1.42 vs 41.40±1.2 mg/dL, 4 weeks post DHT), while decreasing triglycerides (76.17±5.4 vs 107.17±9.4 mg/mL) and LDL-C (6.5±0.8 vs 9.0±0.5 mg/mL) after 8 weeks post DHT. At the molecular level, DHT upregulated RPF AR expression (5-fold) and downregulated ATGL expression by 40% as early as 2 weeks post DHT, and downregulated PPARγ expression by 40% at 4 weeks post DHT indicating early onset of adipose tissue dysfunction. Conclusion and significance: Our findings suggest that some PCOS metabolic traits occur very early following the induction of hyperandrogenemia such as serum adiponectin, insulin and cholesterol, highlighting these markers as an early diagnosis potential biomarkers. The onset of WAT lipolysis and adipogenesis alterations as well as adipo-IR were also early events that may be drivers of disease progression. Therefore, actively screening and managing risk factors for metabolic derangements from early on in PCOS is critical for disease management. Supported by NIH-NIGMS P20GM121334 and NIH-NIMHD P50MD017338 grants and American Heart Association Predoctoral Fellowship 903804. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

OR35-02 Novel Androgen Receptor PROTAC Degrader ARV-110 Ameliorates Androgen Mediated Renal Injury in a Mouse Model of Polycystic Ovary Syndrome

Abstract Disclosure: J. Basnet: None. S. Rezq: None. A.M. Huffman: None. T.E. Asala: None. L.L. Yanes Cardozo: None. D.G. Romero: None. Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women. PCOS women often have increased central adiposity, insulin resistance, and renal injury. Hyperandrogenism is prevalent in PCOS women and is associated with renal injury and dysfunction. Androgens bind to the androgen receptor (AR) to impart downstream transcriptional activity. Targeting the AR may alleviate PCOS complications; however, the use of AR blockers is limited by their severe adverse effects in females; thus, safer alternative approaches are highly needed. The novel AR Proteolysis Targeting Chimera (PROTAC) degrader ARV-110 has shown high efficacy to prevent AR signaling in prostate cancer; however, its efficacy in PCOS is unknown. Using a well-established hyperandrogenemic mouse model of PCOS, we aim to test the hypothesis that decreasing AR protein levels with ARV-110 ameliorates androgen-mediated derangements in PCOS. Methods: Three-week old female mice (C57BL/6N) were implanted with Silastic tubes filled with the androgen dihydrotestosterone (DHT, 8mg, s.c., 90 days) or vehicle (n=6/grp). Four weeks post-DHT administration, mice were treated with ARV-110 (1 mg/kg/day, s.c.) for an additional 8 weeks. Body composition (EchoMRI) and oral glucose tolerance test (OGTT) were assessed. Kidney weight was measured by gravimetry. AR, extracellular matrix (ECM) remodeling marker MMP9, pro-fibrotic marker TGF-β, renal injury markers (urinary albumin to creatinine ration (UACR), KIM1, and NGAL) were assessed by Western-blot or ELISA. Results: DHT mice showed significant (p<0.05) increases in body weight (27.3±0.6 vs 23.9±0.26 g), fat mass (4.2±0.6 vs 2.1±0.1 g), lean mass (21.6±0.2 vs 19.8±0.2 g), as well as kidney weight (491.3±7.69 vs 303.1±5.5 mg) compared to controls. DHT increased fasting glucose (183.2±25.2 vs 141.5±9.9 mg/dL, p<0.05) and impaired OGTT (AUC:34557±6255 vs 23459±1896 mg.min/dL, p<0.05). ARV-110 treatment ameliorated all aforementioned DHT-mediated changes. At the molecular level, DHT-treated mice had higher renal AR (2.5-fold) and MMP9 (1.5-fold) protein expression. DHT upregulated renal TGF-β levels (2-fold, p<0.05), UACR (966.8±160.9 vs 381.5±41.2 ng albumin/mg creatinine), and urinary KIM1 (3.5-fold) and NGAL (6-fold). ARV-110 successfully decreased renal AR protein levels by 70% in DHT mice compared to vehicle-treated DHT. Excitingly, while ARV-110 did not modulate renal TGF-β levels, it completely abolished DHT-mediated increase in renal MMP9 while significantly lowering the levels of urinary renal injury markers UACR, KIM1 and NGAL. Conclusion and significance: Our findings suggest that DHT-mediated kidney hypertrophy, associated ECM remodeling and injury are ameliorated by ARV-110 in DHT-treated mice. AR PROTACs could be a novel therapeutic approach to treat renal dysfunction in PCOS. Supported by NIH P20GM121334. Presentation Date: Sunday, June 18, 2023

Differential Strain-dependent Ovarian and Metabolic Responses in a Mouse Model of PCOS.

Several mouse models have been developed to study polycystic ovarian syndrome (PCOS), a leading cause of infertility in women. Treatment of mice with DHT for 90 days causes ovarian and metabolic phenotypes similar to women with PCOS. We used this 90-day DHT treatment paradigm to investigate the variable incidence and heterogeneity in 2 inbred mouse strains, NOD/ShiLtJ and 129S1/SvlmJ. NOD mice naturally develop type 1 diabetes, and recent meta-analysis found increased androgen excess and PCOS in women with type 1 diabetes. The 129S1 mice are commonly used in genetic manipulations. Both NOD and 129S1 DHT-treated mice had early vaginal opening, increased anogenital distance, and altered estrus cycles compared with control animals. Additionally, both NOD and 129S1 mice had reduced numbers of corpora lutea after DHT exposure, whereas NOD mice had decreased numbers of preantral follicles and 129S1 mice had reduced numbers of small antral follicles. NOD mice had increased body weight, decreased white adipocyte size, and improved glucose sensitivity in response to DHT, whereas 129S1 mice had increased body weight and white adipocyte size. NOD mice had increased expression of Adiponectin, Cidea, Srebp1a, and Srebp1b and 129S1 mice had decreased Pparg in the white adipose tissues, whereas both NOD and 129S1 mice had increased expression of Glut4 and Prdm16, suggesting DHT may differentially affect glucose transport, thermogenesis, and lipid storage in white adipose tissue. DHT causes different ovarian and metabolic responses in NOD and 129S1 mice, suggesting that strain differences may allow further elucidation of genetic contributions to PCOS.

RF10 | PMON249 MicroRNA-21 Ablation Exacerbates Androgen-Mediated White Adipose Tissue Dysfunction and Impaired Insulin Signaling in a Mouse Model of Polycystic Ovary Syndrome

Abstract Background Polycystic ovary syndrome (PCOS) is characterized by oligo- or anovulation, polycystic ovaries, and hyperandrogenism. PCOS is associated with white adipose tissue (WAT) expansion and dysfunction, which has been linked to impaired insulin signaling in a number of metabolic diseases. MicroRNAs dysregulation in WAT is involved in the development of obesity and its associated complications. MicroRNA-21 (miR-21), the most abundant circulatory microRNA in women with PCOS, regulates androgen receptor (AR) expression in prostate cancer however, its role in WAT dysfunction in PCOS, and the associated systemic insulin resistance, is unknown. Using a well-established PCOS model of hyperandrogenemia we aim to test the hypothesis that miR-21 regulates AR expression to modulate androgen-mediated WAT dysfunction and the associated local and systemic insulin resistance. Methods Three-week-old miR-21 knockout (miR-21KO) or wild type (WT) female mice were treated with dihydrotestosterone (DHT, 8 mg/Silastic tube) or vehicle for 90 days (n=6/group). Body composition (EchoMRI) and oral glucose tolerance test (OGTT) were assessed. HOMA-IR index was calculated from fasting serum glucose and insulin levels. Retroperitoneal fat (RPF), a WAT depot that is linked to poor plasma glycemic indices and systemic inflammation, was collected to assess adipocyte size and perform molecular determinations. RPF miR-21 levels, as well as gene and protein expression of AR, and insulin signaling markers (IRS-1/2, insulin receptor-β, PI3K, AKT, Glut4, GSK-α, PTEN) were quantified by RT-qPCR and Western blot. Results In WT mice, DHT increased body weight (25.07 ± 0.52 vs 21.79 ± 0.47 g, p<0.05), fat mass (4.60 ± 0.46 vs 1.98 ± 0.12 g, p<0.05), RPF mass (29.68 ± 3.39 vs. 172.64 ± 32.75 g, p<0.05), RPF miR-21 (2.5-fold) and AR expression (2.1-fold). Adipocyte size analysis revealed a hypertrophic response to DHT, which was associated with impaired OGTT (186.10 ± 5.99 vs 250.70 ± 14.76 mg. min/dL, p<0.05) and downregulation of IRS-1/2, insulin receptor-β, and AKT, indicating impaired insulin signaling. MiR-21 ablation had no effect on DHT-mediated total fat or RPF mass increases, but it exacerbated DHT-mediated AR upregulation, RPF hypertrophy, and insulin resistance as measured by the HOMA-IR index. DHT-mediated reduction in GSK-α, which mediates obesity-induced inflammation, was abolished in miR-21KO mice. Glut4 protein was only downregulated in miR-21KO DHT-treated mice, despite DHT downregulating Glut4 mRNA in both strains. Furthermore, DHT only upregulated PTEN, a negative insulin signaling regulator and known target of miR-21 in miR-21KO mice. Conclusion and significance: These findings imply that WAT miR-21 plays a protective role in PCOS by alleviating androgen-mediated adipose tissue structural and molecular derangements, as well as the associated local and systemic insulin resistance via downregulating AR. Modulation of miR-21 levels in adipose tissue could be a novel therapeutic approach for the treatment of PCOS-related metabolic derangements. Presentation: Saturday, June 11, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

High‐Fat Diet Exacerbates Androgen‐Mediated Obesity and White Adipose Tissue Hypertrophy in a Mouse Model of Polycystic Ovary Syndrome

BackgroundPolycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in reproductive age women. PCOS is characterized by hyperandrogenemia, oligo‐ or anovulation and polycystic ovaries and is associated with obesity and white adipose tissue (WAT) dysregulation. The deleterious metabolic profile of women with PCOS is exacerbated by obesity. However, the molecular mechanisms that mediate WAT dysfunction in PCOS, as well as its modulation by the diet, are poorly understood. We aim to investigate the effect of an obesogenic diet on androgen‐mediated WAT hypertrophy by assessing the adipocyte size of the different WAT depots [mesenteric fat (MF), subcutaneous fat (SCF) and retroperitoneal fat (RPF)] using a well‐established model of PCOS in mice.MethodsThree‐week old female mice (C57BL/6J) were implanted with Silastic tubes filled with the androgen dihydrotestosterone (DHT, 8.0 mg, S.C.) or vehicle (empty tubes). Animals were fed high‐fat diet (HFD; 60% kcal fat) or low‐fat diet (LFD; 10% kcal fat, sucrose‐matched) for 90 days. Weekly body weights and body composition (EchoMRI) were assessed. Animals were sacrificed and multiple WAT depots (MF, SCF and RPF) were harvested for histological analysis. Formalin‐fixed paraffin‐embedded tissues from the three WAT depots were sectioned (5 µm thick) and stained with hematoxylin and eosin. The stained sections were imaged at 40X magnification. Adipocyte size (cross‐sectional area) was determined using ImageJ software with the Adiposoft plugin [~2,000 adipocytes/section, 3 sections/animal, n = 6/group]. GraphPad Prism 8.4.2 was used for statistical analysis, and P‐values of 0.05 were considered statistically significant.ResultsLFD‐fed DHT‐treated mice showed significant increases in body weight (28.00 ± 0.60 vs. 23.59 ± 0.41 g, p<0.05), lean mass (21.70 ± 0.30 vs. 20.04 ± 0.23 g, p<0.05), and fat mass (6.20 ± 0.50 vs. 3.42 ± 0.33 g, p<0.05) compared to their vehicle‐treated controls. Adipocyte size analysis showed that LFD‐fed DHT‐ treated mice have significantly larger adipocytes when assessed in MF (1512.0 ± 15.55 vs. 833.3 ± 6.50 µm2, p<0.05), SCF (1907.0 ± 22.04 vs. 1395.0 ± 12.43 µm2, p<0.05) and RPF (2284.0 ± 26.20 vs. 1396.0 ± 11.61 µm2, p<0.05), compared to their vehicle‐treated controls. Notably, HFD‐fed DHT‐treated mice showed greater MF and RPF adipocyte size compared to HFD‐fed vehicle‐treated mice, which was not observed in SCF. Interestingly, HFD exacerbated DHT‐mediated increases in body weight (1.54‐fold), lean mass (1.17‐fold), and fat mass (3.03‐fold) compared to LFD‐fed DHT‐treated mice. Additionally, the increase in adipocyte size in all fat depots in response to DHT was greater in mice fed HFD by 1.50‐fold compared to those fed LFD.Conclusion and significanceThe hypertrophic effect of androgens on the WAT is modulated by the diet. Increased adipocyte size is associated with insulin resistance in women with PCOS. Our results may help explain why obesity is associated with a worsened metabolic profile in women with PCOS at the cellular level. Weight loss with a low‐fat diet may be an effective approach to ameliorate WAT dysfunction and the subsequent metabolic dysregulation in PCOS.

Role of the JAK/STAT3 Pathway in Obesity‐Induced Renal Injury in Polycystic Ovary Syndrome

BackgroundPolycystic ovary syndrome (PCOS), the most common endocrine disorder in reproductive‐age women, is characterized by oligo‐ or anovulation, hyperandrogenism, and polycystic ovaries. Both animal models and women with PCOS exhibit inflammation, obesity, and renal damage. The molecular mechanisms that mediate renal injury and dysfunction in PCOS, as well as its modulation by the diet are poorly understood. The JAK/STAT3 pathway, which is activated by interleukin‐6 (IL‐6) and leptin receptors, is involved in the pathophysiology of different models of renal injury. Despite its chronic activation in extra‐renal tissues in PCOS and obesity models, the role of the JAK/STAT3 pathway in PCOS‐mediated renal damage remains unknown. We aim to test the hypothesis that an obesogenic diet, via increased leptin and IL‐6, activates the JAK/STAT3 signaling pathway to exacerbate androgen‐induced renal injury.MethodsThree‐week‐old peripubertal female mice were implanted with Silastic tubes filled with the non‐aromatizable androgen dihydrotestosterone (DHT, 8 mg) or vehicle (empty tubes). Animals were fed either a high‐fat diet (HFD; 60% kcal fat) or a low‐fat diet (LFD; 10% kcal fat, sucrose‐matched) for 90 days. Fat mass (EchoMRI), serum leptin (ELISA), kidney weight (gravimetry), and urinary albumin to creatinine ratio (UACR, clinical chemistry analyzer) were measured. Markers of renal inflammation [IL‐6, tumor necrosis factor‐α (TNF‐α)], oxidative stress [NADPH oxidase2 (Nox2)], and fibrosis [collagen I, collagen III, transforming growth factor‐β (TGF‐β), and fibronectin] were quantified by RT‐qPCR. Western blot analysis of active STAT3 [phosphorylated‐STAT3 (Tyr705)] and total STAT3 proteins expression in the kidneys was also performed.ResultsIn LFD‐fed mice, DHT increased fat mass (20.28 ± 1.10 vs. 6.78 ± 0.43 g, p<0.05), serum leptin (1114.13 ± 133.68 vs. 299.27± 25.32 pg/mL, p<0.05), kidney weight (33.79 ± 1.35 vs. 26.52 ± 1.08 g, p<0.05) and UACR (1,227 ± 420 vs. 404 ± 169 μg albumin/g creatinine, p<0.05). DHT also increased the expression of renal inflammatory markers (TNF‐α and IL‐6). Notably, while DHT upregulated the expression of the fibrotic marker TGF‐β in LFD‐fed mice, it only induced the expression of Collagen I and Nox2 in the HFD‐fed mice. Furthermore, HFD exacerbated DHT‐mediated increases in adiposity and circulating leptin and IL‐6, as well as renal inflammation, hypertrophy, and UACR. The disturbance in the aforementioned parameters was associated with increased renal STAT3 phosphorylation in HFD‐fed DHT‐treated mice compared to LFD‐ or HFD‐fed controls (2.00‐ and 2.86‐fold, respectively, p<0.05).Conclusion and significanceOur findings suggest that diet‐induced obesity plays a significant role in the renal outcomes in PCOS, and that increased STAT3 activation may explain the exacerbated renal injury in HFD‐fed DHT‐treated female mice. JAK/STAT3 pathway modulation could be novel therapeutic approach for diet‐induced renal injury in PCOS. The implications of inhibiting specific STAT3 pathway components on the exacerbated renal impairment caused by obesity in our model of PCOS are currently being investigated.

MicroRNA‐21 Genetic Ablation Exacerbates Insulin Signaling Dysregulation in Hyperandrogenemic Female Mice

IntroductionPolycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in reproductive‐age women and is characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. PCOS is associated with obesity and insulin resistance, both being risk factors associated with cardiovascular disease. Micro‐RNAs are small, non‐coding RNAs capable of altering gene expression by binding to specific mRNA targets. MicroRNA‐21(miR21) is cell‐specifically expressed and dynamically regulated in tissues that are implicated in cardiometabolic dysfunction in PCOS including the liver, skeletal muscle, and the heart. We hypothesize that miR‐21 genetic ablation exacerbates androgen‐induced dysregulation of insulin signaling to modify insulin resistance and cardiac damage in a mouse model of PCOS.MethodsThree‐week‐old peripubertal miR‐21 knockout (miR21KO) or wild‐type (WT) C57BL/6N female mice were treated s.c. with dihydrotestosterone (DHT, 8 mg) or vehicle for 90 days. Fasting glucose was determined by an enzymatic method and both fasting insulin and advanced glycation end products (AGES) were measured by ELISA. microRNA, mRNA, and protein levels were quantified by RT‐qPCR and Western‐blot. Data were analyzed by t‐test or two‐way ANOVA. All differences are considered significant p<0.05.ResultsIn WT animals DHT treatment increased miR21 expression in the soleus muscle (2.15±0.47‐fold, p<0.05), with a trend to increase in the tibialis anterior muscle (1.25‐fold), and the liver (1.41‐fold) compared with their vehicle counterparts. DHT‐treated miR21KO females showed exacerbated insulin resistance, having increased HOMA‐IR compared with both vehicle miR21KO and WT groups (6.50 ± 3.64 vs. 3.32±1.83 and 3.16±1.31, p<0.05) calculated using fasting insulin and glucose. In the soleus, miR21KO DHT‐treated mice showed an increase in Irs2 (1.45‐fold, p<0.05) compared with vehicle WT, whereas no increase was found in DHT‐treated WT animals. DHT‐treated miR21KO animals showed significantly increased Akt2 (1.40‐fold) and the glucose transporter Slc2a2 (1.24‐fold) mRNA expression in the liver. Additionally, liver protein expression of total mTOR was increased only in miR21KO DHT‐treated animals (2.40, p<0.05) compared to vehicle WT animals. Lastly, miR21KO increased left ventricular advanced glycation end products (AGEs), having significantly higher AGEs compared to all three treatment groups (3.10±0.52μg /mg protein, p<0.05).ConclusionsOur results suggest that miR21 upregulation in insulin sensitive tissues is a compensatory mechanism to counteract androgen‐inducted dysregulation of insulin signaling pathways and downstream cardiac damage. The differences observed in androgen‐treated mice highlight the potential importance of miR21 in the crosstalk between several insulin signaling networks. Notably, given the worsened DHT‐induced insulin resistance and increased left ventricular AGEs in miR21KO animals, a miR‐21 mimic may be a potential novel therapeutic option for women with PCOS.

Dihydrotanshinone I inhibits ovarian tumor growth by activating oxidative stress through Keap1-mediated Nrf2 ubiquitination degradation

Dihydrotanshinone I (DHT), a bioactive compound in Salvia miltiorrhiza, was reported to exhibit cytotoxicity against various malignancies. However, the underlying mechanism on ovarian cancer remains unclear. Here, DHT inhibited cell viability of ovarian cancer HO8910PM, SKOV3, A2780 and ES2 cells. It showed moderate inhibitory effect on ovarian epithelial IOSE80 cells and lower toxicity than chemotherapy drugs. DHT induced apoptosis and G2 cell cycle arrest accompanied by reduced expression of Bcl-2, Caspase-3, and increased Bax. Meanwhile, DHT increased ROS accumulation, decreased mitochondrial membrane potential and activated oxidative stress in HO8910PM and ES2 cells. Mechanistically, DHT inhibited Nrf2 and p62 expression, Nrf2 target genes and enzymes, and Nrf2 nuclear translocation, while increased the expression of Nrf2 inhibitor Keap1. NAC, a ROS scavenger, rescued DHT-induced proliferation inhibition, ROS generation and Nrf2 inhibition. DHT alleviated tBHQ-induced Nrf2 expression and increased its mRNA level. However, the proteasome inhibitor MG132 blocked DHT-induced Nrf2 inhibition, suggesting a post-translational regulation manner. DHT enhanced Nrf2 binding with Keap1, leading to potentiated Nrf2 ubiquitination degradation. Furthermore, Nrf2 and p62 overexpression blocked DHT-induced Nrf2 and p62 inhibition. Consistent with the in vitro results, DHT significantly delayed tumor growth in HO8910PM and ES2 xenograft nude mice, decreased tumor marker HE4 and CA125 levels, reversed the abnormally expressed proteins including Ki67, Nrf2, p62, Keap1, Bcl-2, CyclinB1, Cdc-2, and antioxidant enzymes SOD, CAT in vivo. Serum from DHT-treated mice also inhibited cell growth in vitro. Taken together, DHT exhibits anti-ovarian tumor effect by activating oxidative stress through ubiquitination-mediated Nrf2 degradation. Our findings implicate a potential application of DHT for ovarian cancer therapy.

Inhibition of Col6a5 Improve Lipid Metabolism Disorder in Dihydrotestosterone-Induced Hyperandrogenic Mice.

Hyperandrogenism is a key pathological feature of polycystic ovarian syndrome (PCOS). Excess androgen can lead to PCOS-like cell hypertrophy in the ovaries and adipose tissue of rodents. Here, we established a dihydrotestosterone (DHT)-induced hyperandrogenic mouse model to analyze the differences in gene expression and signaling pathways of the ovaries and gonad fat pads of mice treated with or without DHT by RNA microarray analysis. From the results, we focused on the overlapping differentially expressed gene—Col6a5—and the major differentially enriched signaling pathway—lipid metabolism. We employed DHT-induced mouse ovarian stromal cell, adipogenic 3T3-L1 cell and hepatic cell line NCTC1469 models to investigate whether androgens directly mediate lipid accumulation and hypertrophy. We found that DHT increased lipid droplet accumulation in ovarian stromal cells and adipogenic 3T3-L1 cells but not NCTC1469 cells. DHT significantly altered stromal cell cholesterol metabolism and steroidogenesis, as indicated by changes in cholesterol levels and the expression of related genes, but these effects were not observed in 3T3-L1 cells. Moreover, Col6a5 expression was significantly increased in ovaries and gonadal fat pads of DHT-treated mice, and Col6a5 inhibition alleviated DHT-induced excess lipid accumulation and hypertrophy of ovarian stromal cells and adipogenic 3T3-L1 cells, even improved lipid metabolism in overnourished NCTC1469 cells. Our results indicate that Col6a5 plays important roles in the pathogenesis of DHT-induced lipid metabolism disorder and the hypertrophy of ovarian stromal cells and adipocytes.

Androgens and Diet Regulation of SARS‐CoV‐2 Viral Entry Proteins: Implications for COVID‐19 Cardiorenal Outcomes Severity in Polycystic Ovary Syndrome

BackgroundThe susceptibility and the severity of coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are associated with hyperandrogenism, obesity, and pre‐existing pulmonary, metabolic, renal or cardiac conditions. Hyperandrogenemia and ovarian dysfunction characterize polycystic ovary syndrome (PCOS), the most common endocrine disorder in premenopausal women. Women with PCOS have higher prevalence of obesity and higher incidence of cardiovascular diseases and renal injury, making them a population at risk for severe COVID‐19 outcomes.MethodsThree‐week‐old female mice were implanted with Silastic tubes filled with the nonaromatizable androgen dihydrotestosterone (DHT, 8 mg) or vehicle (empty tubes). Animals were maintained on high‐fat diet (HFD; 60% kcal fat) or low‐fat diet (LFD; 10% kcal fat, sucrose‐matched) for 90 days. Fat mass (EchoMRI), kidney and left ventricle (LV) weights as well as the cardiac and renal expression of the inflammatory markers interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), and interleukin‐1β (IL‐1β) were assessed. Moreover, circulatory, urinary, renal and cardiac levels of SARS‐CoV‐2 host cell receptor angiotensin converting enzyme 2 (ACE2) were quantified. Additionally, renal and cardiac mRNA and protein expression of the multiple cellular proteases required for viral entry (TMPRSS2, TMPRSS4, furin, cathepsin L, and ADAM17) were assessed.ResultsHFD exacerbated DHT‐mediated increases in fat mass (20.28 ± 1.10 vs. 6.78 ± 0.43 g, p<0.05), kidney weight (33.79 ± 1.35 vs. 26.52 ± 1.08 g, p<0.05), LV hypertrophy (7.48 ± 0.21 vs. 6.41 ± 0.28 g, p<0.05) and renal and cardiac expression of the inflammatory marker IL‐6 (1.5 and 2.5‐fold, respectively). PCOS mice on both diets showed higher urinary ACE2 (LFD: 18‐fold, HFD: 10‐fold) and altered renal mRNA and/or protein expression of all the measured targets. DHT upregulated renal TMPRSS2 (1.19 ± 0.04 vs. 0.98 ± 0.03 AU, p<0.05) and cathepsin L (2.64 ± 0.25 vs. 0.62 ± 0.25 AU, p<0.05) proteins in HFD‐fed DHT‐treated mice compared to LFD‐fed DHT‐treated mice. In the heart, LFD‐fed DHT‐treated mice showed no change in ACE2 and the cellular proteases on either the mRNA or protein levels except for ADAM17 protein that was significantly upregulated (1.63‐fold) compared to the vehicle‐treated group. On the other hand, while HFD‐fed DHT‐treated mice showed similar increase in cardiac ADAM17 protein (1.64‐fold vs. LFD‐fed vehicle‐treated), they showed higher mRNA expression of ACE2 (1.83‐fold) and furin (1.32‐fold) compared to the HFD‐fed vehicle‐treated mice.Conclusion and significanceOur findings highlight women with PCOS as a population with a high risk of COVID‐19 associated cardiac and renal complications. Further, our study suggests dietary control recommendations for optimal management of women with PCOS that can ultimately attenuate COVID‐19 cardiorenal outcomes.

SARS‐CoV‐2 Viral Entry Proteins in Hyperandrogenemic Female Mice: Implications for Women with Polycystic Ovary Syndrome (PCOS) and COVID‐19

BackgroundSARS‐CoV‐2, the causative agent of COVID‐19, infects host cells using the angiotensin I converting enzyme 2 (ACE2) as its receptor after priming by host proteases, including the transmembrane serine protease TMPRSS2. Androgen action in target tissues may partially mediate entry of SAR‐CoV‐2 and male patients have been shown to suffer increased severity and mortality compared with females. Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in reproductive‐age women and is characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. PCOS is associated with obesity and cardiometabolic dysfunctions, both being risk factors associated with severe COVID‐19 pathology. The purpose of this study is to investigate the role of hyperandrogenism in a female mouse model of PCOS and to characterize the differences in host machinery required for SARS‐CoV‐2 viral entry in multiple tissues that are affected by COVID‐19 in humans.MethodsFemale mice were treated with dihydrotestosterone (DHT, 8 mg) s.c. for 90 days. Body composition was measured by EchoMRI. Fasting glucose was determined by an enzymatic method and the oral glucose tolerance test was performed with an oral glucose load (2g glucose per kg lean mass weight). mRNA and protein levels of ACE2, Tmprss2, Cathepsin L, Furin, Tmprss4, and Adam17 were quantified by RT‐qPCR, Western‐blot, and ELISA in tissues, serum, and urine. All differences are considered significant p<0.05.ResultsDHT treatment increased body weight, fat and lean mass. DHT treated females showed altered glucose homeostasis, having increased fasting glucose (201.10 ± 11.11 vs. 152.80 ± 9.23 mg/dL, p<0.05) and an increased area under the curve (209.2 ± 11.0 vs. 160.8 ± 3.5 mg.min/dL, p<0.05) by OGTT. The small intestine was shown to have the greatest relative Ace2 mRNA expression compared with the other tissues. Ace2 mRNA was upregulated in DHT‐treated animals in the lung (1.24‐fold), cecum (4.4‐fold), left ventricle (1.54‐fold), and kidney (2.37‐fold), and was downregulated in DHT‐treated animals in the brain (0.68‐fold) and colon (0.37‐fold). ACE2 protein expression was increased in the small intestine (1.98‐fold), left ventricle (1.30‐fold), and kidney (1.32‐fold). The SARS‐CoV‐2 priming proteases Tmprss2, Cathepsin L, and Furin mRNA were upregulated by DHT in the kidney, and protein TMPRSS2 was increased in the small intestine (1.38‐fold). ACE2 sheddase Adam17 mRNA was upregulated in the kidney by DHT, which corresponded with increased urinary ACE2 in DHT‐treated mice (14.13±3.33 vs. 0.55±0.19 pg/ng creatinine, p<0.05).ConclusionsOur results highlight the potential for increased cardiac, renal, and gastrointestinal dysfunction in PCOS women with COVID‐19. The differences observed in androgen‐treated mice highlight that several mechanisms involved in SARS‐CoV‐2entry may be altered in women with PCOS and translate to the reduced protection of specific target organs known to be susceptible to COVID‐19 injury.

Pathogenesis of Reproductive and Metabolic PCOS Traits in a Mouse Model.

Polycystic ovary syndrome (PCOS) is a common and heterogeneous disorder; however, the etiology and pathogenesis of PCOS are poorly understood and current management is symptom-based. Defining the pathogenesis of PCOS traits is important for developing early PCOS detection markers and new treatment strategies. Hyperandrogenism is a defining characteristic of PCOS, and studies support a role for androgen-driven actions in the development of PCOS. Therefore, we aimed to determine the temporal pattern of development of PCOS features in a well-characterized dihydrotestosterone (DHT)-induced PCOS mouse model after 2, 4, and 8 weeks of DHT exposure. Following 2 weeks of treatment, DHT induced the key PCOS reproductive features of acyclicity, anovulation, and multifollicular ovaries as well as a decrease in large antral follicle health. DHT-treated mice displayed the metabolic PCOS characteristics of increased body weight and exhibited increased visceral adiposity after 8 weeks of DHT treatment. DHT treatment also led to an increase in circulating cholesterol after 2 weeks of exposure and had an overall effect on fasting glucose levels, but not triglycerides, aspartate transaminase (AST) and alanine transaminase (ALT) levels, or hepatic steatosis. These data reveal that in this experimental PCOS mouse model, acyclicity, anovulation, and increased body weight are early features of a developing PCOS phenotype whereas adiposity, impaired glucose tolerance, dyslipidemia, and hepatic steatosis are later developing features of PCOS. These findings provide insights into the likely sequence of PCOS trait development and support the addition of body weight criteria to the early diagnosis of PCOS.

Long-Term Androgen-Induced Nonalcoholic Fatty Liver Disease in a Polycystic Ovary Syndrome Mouse Model is Related to Mitochondrial Dysfunction

Abstract Objective: Metabolic disorders are markedly common in women with polycystic ovary syndrome (PCOS), and nonalcoholic fatty liver disease (NAFLD) is observed in 30%-55% of all PCOS patients. Many studies have reported that autophagy and apoptosis, which are closely related to mitochondrial function, play important roles in the development of NAFLD. Therefore, in this study, we aimed to explore the role of mitochondrial dysfunction caused by liver apoptosis and autophagy imbalance in the development of NAFLD in a PCOS mouse model. Methods: We used a dihydrotestosterone (DHT)-induced PCOS model to mimic the pathological process of hyperandrogenism. Hematoxylin and eosin and Oil Red O staining assays were used to observe the pathological changes in the liver. Western blotting and quantitative real-time polymerase chain reaction were used to perform mitochondrion-related assays. Results: Hepatic steatosis and different degrees of inflammation were observed in the DHT-treated mice. The expression of molecules involved in the respiratory chain and aerobic respiration process was altered. The levels of the key molecules associated with apoptosis and autophagy were abnormal. Conclusions: Androgens may play a role in the process of hepatic steatosis development by affecting mitochondrial function and subsequently inducing apoptosis and autophagy. Such phenomena might be involved in the pathogenesis of NAFLD in women with PCOS.

1766-P: Androgen Action in Hypothalamic Arcuate Nucleus Neurons Increases Hepatic Glucose Production in Female Mice

Androgen excess predisposes females to type 2 diabetes. Using mouse models, we reported that androgen excess causes insulin resistance via activation of the androgen receptor (AR) in the brain. Neurons of the arcuate nucleus of the hypothalamus (ARC) regulate hepatic glucose production (HGP). Thus, we hypothesized that in female mice, androgen excess in neurons of the ARC causes peripheral insulin resistance by increasing HGP. To test this, we injected AaV-Cre-GFP or AaV-GFP into the ARC of ARlox/lox female mice to generate ARC-specific AR knockout (ARC-ARKO) and control mice, respectively. When exposed to a Western diet, control female mice chronically treated with dihydrotestosterone (DHT) developed insulin resistance and fasting hyperglycemia compared to vehicle-treated control mice. In contrast, DHT-treated ARC-ARKO mice remained insulin sensitive and normoglycemic compared to vehicle-treated ARC-ARKO mice. During a hyperinsulinemic-euglycemic clamp, insulin’s ability to suppress HGP was blunted in DHT-treated control mice. In contrast, insulin was still able to suppress HGP in DHT-treated ARC-ARKO females. Additionally, during the clamp, DHT-treated control mice showed no alteration in hepatic activation of AKT, a marker of hepatocyte insulin sensitivity, but exhibited reduced activation of hepatic STAT3, a marker of hypothalamic insulin sensitivity. In contrast, in DHT-treated ARC-ARKO mice activation of hepatic STAT3 was increased. Together these results suggest that in female mice exposed to a Western diet, androgen excess causes insulin resistance in ARC neurons via action at the AR leading to impairments in the brain-IL6-STAT3 pathway which results in unsuppressed HGP. Disclosure J. Morford: None. F. Mauvais-Jarvis: None. Funding National Institutes of Health (R01DK074970, R01107444); U.S. Department of Veterans Affairs (BX003725)

VCAM1 Is Induced in Ovarian Theca and Stromal Cells in a Mouse Model of Androgen Excess.

Ovarian theca androgen production is regulated by the pituitary LH and intrafollicular factors. Enhanced androgen biosynthesis by theca cells contributes to polycystic ovary syndrome (PCOS) in women, but the ovarian consequences of elevated androgens are not completely understood. Our study documents the molecular events that are altered in the theca and stromal cells of mice exposed to high androgen levels, using the nonaromatizable androgen DHT. Changes in ovarian morphology and function were observed not only in follicles, but also in the stromal compartment. Genome-wide microarray analyses revealed marked changes in the ovarian transcriptome of DHT-treated females within 1 week. Particularly striking was the increased expression of vascular cell adhesion molecule 1 (Vcam1) specifically in the NR2F2/COUPTF-II lineage theca cells, not granulosa cells, of growing follicles and throughout the stroma of the androgen-treated mice. This response was mediated by androgen receptors (ARs) present in theca and stromal cells. Human theca-derived cultures expressed both ARs and NR2F2 that were nuclear. VCAM1 mRNA and protein were higher in PCOS-derived theca cells compared with control theca and reduced markedly by the AR antagonist flutamide. In the DHT-treated mice, VCAM1 was transiently induced by equine chorionic gonadotropin, when androgen and estrogen biosynthesis peak in preovulatory follicles, and was potently suppressed by a superovulatory dose of human chorionic gonadotropin. High levels of VCAM1 in the theca and interstitial cells of DHT-treated mice and in adult Leydig cells indicate that there may be novel functions for VCAM1 in reproductive tissues, including the gonads.

Crocetin attenuates DHT-induced polycystic ovary syndrome in mice via revising kisspeptin neurons

Polycystic ovarian syndrome (PCOS), the most common endocrine disorder of women in reproductive age, is typical with hyperandrogenism and disturbance of the hypothalamus–pituitary–ovary (HPO) axis, i.e. abnormal expression of hypothalamic gonadotropin-releasing hormone (GnRH) followed by the elevated ratio of serum luteinizing hormone (LH) level to follicle-stimulating hormone (FSH) level. This derangement might have a close relationship with hypothalamic kisspeptin expression that is thought to be a key regulator of GnRH. Crocetin, one of the main components of Saffron clinically used as traditional medicine in gynecology diseases, was evaluated for its therapeutic effects on PCOS induced by prenatally exposure to dihydrotestosterone (DHT) in mice. Herein, we found that DHT-treated mice showed a similar phenotype to human PCOS such as heavier ovary, prolonged diestrus, multiple enlarged follicles with fewer corpus luteum, and higher LH and testosterone levels. Kisspeptin expression was lower in anteroventral periventricular nucleus (AVPV) but higher in arcuate nucleus (ARC). Treatment of crocetin prevented the prolongation of diestrus and reduction in corpora luteum, recover the levels of GnRH, FSH, LH, progesterone (P4), estradiol (E2) and testosterone (T), and increase the kisspeptin level in AVPV but reduce that in ARC. The present study provides in vivo evidence that crocetin improved the PCOS in mice via increasing AVPV-kisspeptin and reducing ARC-kisspeptin expression.

Endothelin-1 mediates natriuresis but not polyuria during vitamin D-induced acute hypercalcaemia.

Hypercalcaemia can occur under various pathological conditions, such as primary hyperparathyroidism, malignancy or granulomatosis, and it induces natriuresis and polyuria in various species via an unknown mechanism. A previous study demonstrated that hypercalcaemia induced by vitamin D in rats increased endothelin (ET)-1 expression in the distal nephron, which suggests the involvement of the ET system in hypercalcaemia-induced effects. In the present study, we demonstrate that, during vitamin D-induced hypercalcaemia, the activation of ET system by increased ET-1 is responsible for natriuresis but not for polyuria. Vitamin D-treated hypercalcaemic mice showed a blunted response to amiloride, suggesting that epithelial sodium channel function is inhibited. We have identified an original pathway that specifically mediates the effects of vitamin D-induced hypercalcaemia on sodium handling in the distal nephron without affecting water handling. Acute hypercalcaemia increases urinary sodium and water excretion; however, the underlying molecular mechanism remains unclear. Because vitamin D-induced hypercalcaemia increases the renal expression of endothelin (ET)-1, we hypothesized that ET-1 mediates the effects of hypercalcaemia on renal sodium and water handling. Hypercalcaemia was induced in 8-week-old, parathyroid hormone-supplemented, male mice by oral administration of dihydrotachysterol (DHT) for 3days. DHT-treated mice became hypercalcaemic and displayed increased urinary water and sodium excretion compared to controls. mRNA levels of ET-1 and the transcription factors CCAAT-enhancer binding protein β and δ were specifically increased in the distal convoluted tubule and downstream segments in DHT-treated mice. To examine the role of the ET system in hypercalcaemia-induced natriuresis and polyuria, mice were treated with the ET-1 receptor antagonist macitentan, with or without DHT. Mice treated with both macitentan and DHT displayed hypercalcaemia and polyuria similar to that in mice treated with DHT alone; however, no increase in urinary sodium excretion was observed. To identify the affected sodium transport mechanism, we assessed the response to various diuretics in control and DHT-treated hypercalcaemic mice. Amiloride, an inhibitor of the epithelial sodium channel (ENaC), increased sodium excretion to a lesser extent in DHT-treated mice compared to control mice. Mice treated with either macitentan+DHT or macitentan alone had a similar response to amiloride. In summary, vitamin D-induced hypercalcaemia increases the renal production of ET-1 and decreases ENaC activity, which is probably responsible for the rise in urinary sodium excretion but not for polyuria.

Reproductive and Metabolic Phenotype of a Mouse Model of PCOS

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women in their reproductive age, is characterized by both reproductive and metabolic features. Recent studies in human, nonhuman primates, and sheep suggest that hyperandrogenism plays an important role in the development of PCOS. We investigated whether chronic dihydrotestosterone (DHT) exposure in mice reproduces both features of PCOS. Such a model would allow us to study the mechanism of association between the reproductive and metabolic features in transgenic mice. In this study, prepubertal female mice received a 90 d continuous release pellet containing the nonaromatizable androgen DHT or vehicle. At the end of the treatment period, DHT-treated mice were in continuous anestrous, their ovaries contained an increased number of atretic follicles, with the majority of atretic antral follicles having a cyst-like structure. Chronic DHT-exposed mice had significantly higher body weights (21%) than vehicle-treated mice. In addition, fat depots of DHT-treated mice displayed an increased number of enlarged adipocytes (P < 0.003). Leptin levels were elevated (P < 0.013), adiponectin levels were diminished (P < 0.001), and DHT-treated mice were glucose intolerant (P < 0.001). In conclusion, a mouse model of PCOS has been developed showing reproductive and metabolic characteristics associated with PCOS in women.

Glucosensing by GnRH Neurons: Inhibition by Androgens and Involvement of AMP-Activated Protein Kinase

GnRH neurons integrate steroidal and metabolic cues to regulate fertility centrally. Central glucoprivation reduces LH secretion, which is governed by GnRH release, suggesting GnRH neuron activity is modulated by glucose availability. Here we tested whether GnRH neurons can sense changes in extracellular glucose, and whether glucosensing is altered by the steroids dihydrotestosterone (DHT) and/or estradiol (E). Extracellular recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice ± DHT and/or E implants. Firing rate was reduced by a switch from 4.5 to 0.2 mm glucose in cells from OVX, OVX+E, and OVX+DHT+E mice, but not OVX+DHT mice. This suggests that androgens reduce the sensitivity of GnRH neurons to changes in extracellular glucose, but E mitigates this effect. Next we investigated potential mechanisms. In the presence of the ATP-sensitive potassium channel antagonist tolbutamide, glucosensing persisted. In contrast, glucosensing was attenuated in the presence of compound C, an antagonist of AMP-activated protein kinase (AMPK), suggesting a role for AMPK in glucosensing. The AMPK activator N1-(b-D-ribofuranosyl)-5-aminoimidazole-4-carboxamide (AICAR) mimicked the effect of low glucose and was less effective in cells from DHT-treated mice. The effect of DHT to diminish responses to low glucose and AICAR was abolished by blockade of fast synaptic transmission. Both AICAR and low glucose activated a current with a reversal potential near -50 mV, suggesting a nonspecific cation current. These studies indicate that glucosensing is one mechanism by which GnRH neurons sense fuel availability and point to a novel role for AMPK in the central regulation of fertility.