Androgen Receptor Knockout Research Articles

Epidermal growth factor receptor contributes to indirect regulation of skeletal muscle mass by androgen.

Androgen is widely acknowledged to regulate skeletal muscle mass. However, the specific mechanism driving muscle atrophy resulting from androgen deficiency remains elusive. Systemic androgen receptor knockout (ARKO) mice exhibit reduction in both muscle strength and muscle mass while skeletal muscle fiber specific ARKO mice have decreased muscle strength without affecting skeletal muscle mass in the limbs. Therefore, androgens may indirectly regulate skeletal muscle mass through effects on non-myofibers. Considering this, our investigation focused on blood fluid factors that might play a role in the regulation of skeletal muscle mass under the influence of androgens. Using a male mouse model of sham, orchidectomy and DHT replacement, mass spectrometry for serum samples of each group identified epidermal growth factor receptor (EGFR) as a candidate protein involving the regulation of skeletal muscle mass affected by androgens. Egfr expression in both liver and epididymal white adipose tissue correlated with androgen levels. Furthermore, Egfr expression in these tissues was predominantly elevated in male compared to female mice. Interestingly, male mice exhibited significantly elevated serum EGFR concentrations compared to their female counterparts, suggesting a connection with androgen levels. Treatment of EGFR to C2C12 cells promoted phosphorylation of AKT and its downstream S6K, and enhanced the protein synthesis in vitro. Furthermore, the administration of EGFR to female mice revealed a potential role in promoting an increase in skeletal muscle mass. These findings collectively enhance our understanding of the complex interplay among androgens, EGFR, and the regulation of skeletal muscle mass.

Unveiling the critical role of androgen receptor signaling in avian sexual development

Gonadal hormone activities mediated by androgen and estrogen receptors, along with cell-autonomous mechanisms arising from the absence of sex-chromosome dosage compensation, are key factors in avian sexual development. In this study, we generate androgen receptor (AR) knockout chickens (AR−/−) to explore the role of androgen signaling in avian sexual development. Despite developing sex-typical gonads and gonadal hormone production, AR−/− males and females are infertile. While few somatic sex-specific traits persist (body size, spurs, and tail feathers), crucial sexual attributes such as comb, wattles and sexual behaviors remain underdeveloped in both sexes. Testosterone treatment of young AR−/− males fails to induce crow behavior, comb development, or regression of the bursa of Fabricius, which are testosterone-dependent phenotypes. These findings highlight the significance of androgen receptor mechanisms in fertility and sex-specific traits in chickens, challenging the concept of a default sex in birds and emphasizing the dominance of androgen signaling in avian sexual development.

Dynamic changes in Beclin-1, LC3B, and p62 in aldose reductase-knockout mice at different time points after ischemic stroke

Ischemic stroke is a brain injury caused by cerebral blood circulation disorders and is closely related to oxidative stress. Aldose reductase (AR) is a critical enzyme involved in oxidative stress. Autophagy has previously been found to play a key role in cerebral ischemia‒reperfusion injury. However, it is still unclear how autophagy molecules change after cerebral ischemia‒reperfusion injury in AR knockout mice (AR−/−). A transient middle cerebral artery occlusion (tMCAO) model was generated in AR−/− mice, and the neurological deficit scores of the mice were observed and recorded on Days 1, 3 and 5 after tMCAO. Neuronal damage in the ischemic penumbra was observed by TTC, HE, and Nissl staining. The expression of the autophagy-related molecules Beclin-1, LC3II/I, and P62 as well as that of molecules related to inflammation, oxidative stress, and neurological damage was detected by RT‒qPCR, western blotting, and immunofluorescence. Autophagosomes were observed using a transmission electron microscope. Cerebral ischemia‒reperfusion injury caused neurological deficits and ischemic infarction in tMCAO mice (P < 0.01). Beclin-1, Bcl2/Bax, SOD, GSH-px, P62, PSD95, and TOM20 levels decreased (P < 0.05), while IL-6, LC3II/I, and GFAP levels increased (P < 0.01) in the AR−/− tMCAO-1d group and the AR−/− tMCAO-3d group, compared to those in the sham group. Beclin-1, Bcl2/Bax, NOX4, GSH-px, P62, and PSD95 levels increased (P < 0.01), while IL-6, LC3II/I, and GFAP levels decreased (P < 0.01) in the AR−/− tMCAO-5d group compared to those in the AR−/− tMCAO-1d group. Autophagosome formation was observed in tMCAO mice. In summary, the changes in autophagy proteins in the brain tissue of the AR−/− mice after tMCAO were more obvious on Days 1 and 3 after tMCAO. The expression of Beclin-1 and P62 decreased, and the expression of LC3B increased after cerebral ischemia‒reperfusion injury in AR−/− mouse brain tissue.

Testosterone and Erythrocyte Lifespan.

Endogenous and exogenous androgens increase circulating erythrocytes and hemoglobin but their effects on erythrocyte lifespan is not known. To investigate androgen effects on immature and mature erythrocyte lifespan in humans and mice using novel non-radioactive minimally invasive methods. Human erythrocyte lifespan was estimated using alveolar carbon monoxide concentration and blood hemoglobin in Levitt's formula in hypogonadal or transgender men before and up to 18 weeks after commencing testosterone (T) treatment. Erythrocyte lifespan was estimated in androgen receptor (AR) knockout and wild-type mice after T or dihydrotestosterone (DHT) treatment of intact females or orchidectomized males using in vivo biotin labelling of erythrocyte surface epitopes for reticulocytes (Ter119+CD71+) and two markers of erythrocytes (CD45-, Ter119+CD71-) monitoring their blood disappearance rate by flow cytometry. Before treatment, hypogonadal and transgender men had marked reduction in erythrocyte lifespan compared with controls. T treatment increased erythrocyte lifespan at 6 weeks but returned to pre-treatment levels at 18 weeks while serum T and blood hemoglobin were increased by T treatment remaining elevated at 18-weeks. In mice T and DHT treatment had higher erythrocyte (but not reticulocyte) lifespan but neither orchidectomy nor AR inactivation significantly influenced erythrocyte or reticulocyte lifespan. We conclude that hypogonadal men have reduced erythrocyte lifespan and acute androgen-induced increase in circulating erythrocyte lifespan may contribute to the well-known erythropoietic effects of androgens, but longer-term effects require further investigation to determine how much they contribute to androgen-induced increases in circulating hemoglobin.

Glucoselysine, a unique advanced glycation end-product of the polyol pathway and its association with vascular complications in type 2 diabetes

Glucoselysine (GL) is a unique advanced glycation end-product derived from fructose. The main source of fructose in vivo is the polyol pathway, and an increase in its activity leads to diabetic complications. Here, we aimed to demonstrate that GL can serve as an indicator of the polyol pathway activity. Additionally, we propose a novel approach for detecting GL in peripheral blood samples using LC-MS/MS and evaluate its clinical usefulness. We successfully circumvent interference from fructoselysine, which shares the same molecular weight as GL, by performing ultrafiltration and hydrolysis without reduction, successfully generating adequate peaks for quantification in serum. Furthermore, using immortalized aldose reductase knockout mouse Schwann cells, we demonstrate that GL reflects the downstream activity of the polyol pathway and that GL produced intracellularly is released into the extracellular space. Clinical studies reveal that GL levels in patients with type 2 diabetes are significantly higher than those in healthy participants, while Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine (MG-H1) levels are significantly lower. Both GL and MG-H1 show higher values among patients with vascular complications; however, GL varies more markedly than MG-H1 as well as hemoglobin A1c, fasting plasma glucose, and estimated glomerular filtration rate. Furthermore, GL remains consistently stable under various existing drug treatments for type 2 diabetes, whereas MG-H1 is impacted. To the best of our knowledge, we provide important insights in predicting diabetic complications caused by enhanced polyol pathway activity via assessment of GL levels in peripheral blood samples from patients.

Investigating GABA Neuron-Specific Androgen Receptor Knockout in two Hyperandrogenic Models of PCOS.

Androgen excess is a hallmark feature of polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility. Clinical and preclinical evidence links developmental or chronic exposure to hyperandrogenism with programming and evoking the reproductive and metabolic traits of PCOS. While critical androgen targets remain to be determined, central GABAergic neurons are postulated to be involved. Here, we tested the hypothesis that androgen signaling in GABAergic neurons is critical in PCOS pathogenesis in 2 well-characterized hyperandrogenic mouse models of PCOS. Using cre-lox transgenics, GABA-specific androgen receptor knockout (GABARKO) mice were generated and exposed to either acute prenatal androgen excess (PNA) or chronic peripubertal androgen excess (PPA). Females were phenotyped for reproductive and metabolic features associated with each model and brains of PNA mice were assessed for elevated GABAergic input to gonadotropin-releasing hormone (GnRH) neurons. Reproductive and metabolic dysfunction induced by PPA, including acyclicity, absence of corpora lutea, obesity, adipocyte hypertrophy, and impaired glucose homeostasis, was not different between GABARKO and wild-type (WT) mice. In PNA mice, acyclicity remained in GABARKO mice while ovarian morphology and luteinizing hormone secretion was not significantly impacted by PNA or genotype. However, PNA predictably increased the density of putative GABAergic synapses to GnRH neurons in adult WT mice, and this PNA-induced plasticity was absent in GABARKO mice. Together, these findings suggest that while direct androgen signaling in GABA neurons is largely not required for the development of PCOS-like traits in androgenized models of PCOS, developmental programming of GnRH neuron innervation is dependent upon androgen signaling in GABA neurons.

Phenotypic Sexual Dimorphism in Response to High Fat Diet in C57BL/6J Mice Lacking Liver Androgen Receptor

Previous research has indicated that liver androgen receptors may play a role in modulating disease. This study aims to investigate the pathophysiology of high fat diet (HFD) induced dysglycemia in male and female liver androgen receptor knockout (LivARKO) mice. We performed metabolic tests on LivARKO female and male mice fed a high fat diet (HFD) or a control diet (CD) (from Research Diets Inc) during months 1 or 2 after starting the diet. Additionally, we performed western blot and qRT-PCR analysis on the livers of the mice to examine intermediates in the insulin signaling pathway. LivARKO-HFD female mice displayed no difference in glucose tolerance compared to female LivARKO-Control mice. Whereas in WT female mice, HFD impaired glucose tolerance (IGT). Our data suggests that starting at one-month LivARKO may be protecting female mice from HFD-induced metabolic dysfunction. LivARKO-HFD female mice displayed significantly worse insulin sensitivity at 15 minutes compared to LivARKO-Control (Con) female mice; but, strangely, LivARKO-HFD female mice had significantly better insulin sensitivity at 60 and 90 minutes compared to LivARKO-Con female mice. Despite protecting against IGT, LivARKO did not protect against HFD-induced hyperinsulinemia (HI) in female mice. In contrast to females, male LivARKO-HFD mice displayed impaired glucose tolerance (IGT) compared to male LivARKO-Con mice. Thus, LivARKO is not protective against HFD-induced glucose metabolic dysfunction in male mice. Lastly, LivARKO-HFD female mice maintained hepatic insulin sensitivity whereas LivARKO-HFD male mice displayed hepatic insulin resistance. These findings suggest that LivARKO delayed the onset of HFD-Induced dysglycemia in female mice. Funding: S.A. grant support from the National Science Foundation (NSF) Inclusion across the Nation of Communities of Learners of Underrepresented Discoverers in Engineering and Science (INCLUDES, HRD-1931045). S.A. grant from the National Institutes of Health (NIH) 1R01DK126892-01A1. 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.

Liver Androgen Receptor Knockout (LivARKO) Male Mice on 3-months of High Fat Diet (HFD) Did Not Experience Insulin Resistance at the Level of p-AKT in White Adipose Tissue

Introduction: Insulin resistance affects up to a third of the US adult population and polycystic ovary syndrome (PCOS) affects up to 10% of reproductive-age adult women. Andrisse et al 2021 (PMCID: PMC9097557) showed that deleting the liver androgen receptor (LivARKO) prevented female mice from developing hyperandrogenemia (HA)-induced insulin resistance. Here, we asked the question, does LivARKO prevent high fat diet (HFD) induced insulin resistance? Hypothesis: It was hypothesized that (unlike HA-LivARKO) HFD LivARKO male mice would display impaired insulin action (lowered insulin-stimulated p-AKT) in white adipose tissue in comparison to the Control diet fed LivARKO male mice, suggesting that AR does not play a significant role in regulating HFD-induced insulin resistance. Methods: Male LivARKO mice were placed on two diets: Control (Con, Research Diets Inc, RDI D12450J, Protein (20%), Carbs (70%, corn starch-50%, sucrose-maldextrin-20%, fructose-0%), Fat 10% (lard)) and High Fat (HFD, RDI D12492, Protein (20%), Carbs (20%, corn starch-0%, sucrose-maldextrin-20%, fructose-0%), Fat 60% (lard)). The mice were sacrificed after 3 months on the diet. Half of the mice were given a dose of 0.5 U/kg insulin before sacrificing to investigate the effects of the diets on insulin signaling. Western blots were used to determine protein expression in white adipose tissue (WAT). BCA assays were used to standardize the protein concentration in each sample. Insulin action can be measured molecularly by examining p-AKT Serine 473 (positive regulator, Santa Cruz sc-514032). If p-AKT S473 levels increase in the presence of insulin, this indicates that insulin is likely functioning properly in the sample. Results: Male LivARKO fed a control diet (LivARKO-Con) and administered with 0.5 U/kg insulin displayed significantly lowered p-AKT protein levels compared to male LivARKO-Con that did not get administered an i.p. injection of insulin (Basal), suggesting that male LivARKO-Con mice were experiencing insulin resistance. Additionally, male LivARKO fed a high fructose diet (LivARKO-HFrD) and administered with 0.5 U/kg insulin displayed slightly increased p-AKT protein levels compared to male LivARKO-HFrD-Basal mice. Conclusion: In conclusion, LivARKO male mice on 3-months of HFD were not experiencing insulin resistance at the level of p-AKT in WAT. We are not certain why insulin negatively regulated (or lowered) p-AKT in WAT of LivARKO-Con male mice. This could be an aspect of needing a higher n-value. Further studies in different energy storage tissues and higher n-values are warranted. We would like to thank the NSF for award # 1931045 and the NIH for award # 1R01DK126892-01A1 for supporting this work. 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.

High Fructose Diet (HFrD) Induced Hepatic Insulin Resistance Is Not Present in Liver Androgen Receptor Knockout (LivARKO) Male Mice

Background: Hyperandrogenism (HA), a common symptom of polycystic ovary syndrome (PCOS), involves action of the androgen receptor (AR) and can induce insulin resistance among 35-80% of PCOS women (PMID 35432749: Amisi 2022). The liver plays an essential role in the metabolism of insulin and androgen signaling. HA has been shown to increase insulin resistance in women and female mice. Previous studies have suggested that knocking out liver AR can lead to improvements in metabolic and reproductive functions of PCOS (PMID 34547140: Andrisse 2021). Hypothesis: We hypothesized that liver androgen receptor knockout (LivARKO) mice on a high fructose diet would exhibit increased insulin resistance compared to controls, as measured by expression of phospho-Akt (p-Akt) and phospho-FoxO1 (p-foxo1), proteins involved in insulin signaling. Methods: LivARKO mice were divided into groups based on an assigned diet (Control or High Fructose; Con, Research Diets Inc, RDI D12450J, Protein (20%), Carbs (70%, corn starch-50%, sucrose-maldextrin-20%, fructose-0%), Fat 10% (lard)) and High Fructose (HFrD, RDI D02022704, Protein (20%), Carbs (70%, corn starch-9%, sucrose-maldextrin-1%, fructose-60%), Fat 10% (lard)). The mice were sacrificed and liver tissue samples were extracted for western blotting analysis examining p-Akt S473 and p-foxo1 S256 levels. Results: LivARKO male mice fed the control diet for one month displayed a significant increase in p-AKT expression when stimulated with insulin compared to basal controls. Similarly, LivARKO male mice fed the high fructose diet for one month displayed a significant increase in p-AKT expression when stimulated with insulin compared to basal mice on the same diet. After one month, LivARKO male mice on a control diet stimulated with insulin displayed significantly decreased p-Foxo1 expression compared to non-stimulated basal control. A similar trend was seen between basal and insulin-stimulated groups in the LivARKO-HFrD male mice, though this trend was not significant. Discussion: These findings suggested that the HFrD did not induce hepatic insulin resistance in LivARKO male mice. Here, we observed a similar pattern of increased insulin stimulated p-AKT to what is seen in WT mice on a chow diet (PMCID PMC3296881: Lu 2012); and this suggested that LivARKO did not disrupt insulin signaling in the liver of male mice when fed a control diet, which was similar to what was seen previously (DOI: 10.1002/hep.22252, Lin 2008, insulin increased PI3K activity in chow diet LivARKO male mice). Our data suggested that the HFrD induced hepatic insulin resistance seen in some WT models (such as PMCID PMC3131094: Nagai 2009) is not present in LivARKO male mice.This also suggested that unlike HFD, HFrD did not cause insulin resistance in LivARKO male mice. These results illuminate LivARKO as a potential therapeutic target for insulin resistance. This project was supported by the following grants to Dr. Stanley Andrisse. (1) NIH 1R01DK126892; (2) NIH 1T34GM142610; and (3) NSF 1931045. 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.

The Effect of High Fructose Diet on Insulin Signaling in Pituitary Tissue of Liver Androgen Receptor Knockout in Female Mice

Intro: Insulin resistance affects up to a third of the US adult population and polycystic ovary syndrome affects up to 10% of reproductive-age adult women. Hyperandrogenism in females can increase predisposition to insulin resistance. Andrisse et al 2021 (PMCID: PMC9097557) showed that deleting the liver androgen receptor (LivARKO) prevented female mice from developing hyperandrogenemia (HA)-induced insulin resistance. This study placed female LivARKO mice on a high fructose diet (HFrD) to determine if LivARKO mice demonstrate blunted insulin resistance on HFrD compared to control diet. Hypothesize: It was hypothesized that (unlike HA-LivARKO) HFrD LivARKO female mice would display impaired insulin action (lowered insulin-stimulated p-AKT) in PIT tissue in comparison to the Control diet fed LivARKO female mice, suggesting that liver AR does not play a significant role in regulating HFrD-induced insulin resistance in the pituitary. Methods: Female LivARKO mice were placed on two diets: Control (Con, Research Diets Inc, RDI D12450J) and High Fructose (HFrD, RDI D02022704). After 3 months on the diet, the mice were sacrificed. Half of the mice were given a dose of 0.5 U/kg insulin before sacrificing to investigate the effects of the diets on insulin signaling. Western blots were used to determine protein expression in tissue from the pituitary tissue (PIT). BCA assays were used to standardize the protein concentration in each sample. Insulin action can be measured molecularly by examining p-AKT Serine 473 (positive regulator, Santa Cruz sc-514032). If p-AKT S473 levels increase in the presence of insulin, this indicates that insulin is likely functioning properly in the sample. Results:LivARKO female mice on a control diet (Con) for 3-months administered with insulin displayed a 3-fold significant increase in p-AKT levels in the pituitary compared to LivARKO-Con female mice not injected with insulin (basal), suggesting that LivARKO-Con mice pituitaries were insulin sensitive. However, LivARKO female mice fed a high fructose diet (HFrD) for 3-months and then administered with insulin displayed significantly lowered p-AKT levels in the pituitary compared to basal LivARKO-HFrD female mice and compared to insulin LivARKO-Con, indicating that LivARKO-HFrD female mice were experiencing insulin resistance (lowered insulin sensitivity compared to LivARKO-Con). Interestingly, p-AKT was significantly increased by 2.5 fold in LivARKO-HFrD female mice compared to LivARKO-Con, suggesting that HFrD increased insulin sensitivity in the pituitary. Conclusion: In conclusion, these data suggest that LivARKO indirectly disrupted insulin-stimulated p-AKT in PIT possibly via altering metabolites in the blood. Additionally, HFrD is known to cause insulin resistance, however, it is not known to alter p-AKT levels. Thus, the change in p-AKT levels is presumably associated with the LivARKO. Further research is required on what components of the Control Diet are prompting this difference in insulin action and if it only takes place in the LivARKO mouse model. We would like to thank the National Science Foundation for award # NSF 1931045 and the National Institutes of Health 1R01DK126892-01A1. 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.

Abstract CT085: Targeting androgen signaling in urothelial carcinoma

Abstract Background: Men are 3-4 times more likely to develop urothelial carcinoma (UC) than women, even after accounting for differences in smoking and occupational exposures. A potential cause for this difference may be androgen signaling. Testosterone receptors are present in up to 53% of UC in men and women. Androgen signaling promotes proliferation and chemotherapy resistance, while blocking androgen has been shown to sensitize UC to chemotherapy. When exposed to a chemical carcinogen, castrated male mice and androgen receptor (AR) knockout mice are less likely to develop UC. Additionally, men taking testosterone lowering agents for prostate cancer, who are concurrently treated for UC, have a 50% lower UC recurrence rate than men with normal testosterone signaling. In a study of 9 men and 1 woman with metastatic UC, all patients were treated with gemcitabine/cisplatin and concurrent enzalutamide. Patients’ testosterone levels were not medically lowered. Importantly, dihydrotestosterone, the native ligand for the AR, has higher affinity for the AR than enzalutamide. Interestingly, the only woman in the study (with presumably low dihydrotestosterone levels) had a complete response to therapy. No other complete responses were observed. The standard therapy for non-metastatic muscle invasive UC is chemotherapy followed by cystectomy. Currently this results in about a 35% pathologic complete response (pCR) rate. pCR is associated with improved overall survival. In our study (TASUC; NCT05839119), we add degarelix to standard neoadjuvant chemotherapy to treat men and women who have AR positive UC. Methods: The main eligibility criteria are: (1) Muscle-invasive urothelial carcinoma of the bladder (2) AR positive disease by nuclear staining on immunohistochemistry (3) pT2 - T4, N0 - N1, M0 (Stage II or IIIA disease) (4) Eligible for cisplatin-based chemotherapy (5) Eligible for cystectomy In this single-arm study, all patients receive standard of care gemcitabine/cisplatin neoadjuvant chemotherapy (4 cycles) with the addition of monthly degarelix during chemotherapy. The primary outcome is pCR rate at cystectomy. We are employing a Simon 2-stage minmax design. In stage I we will enroll 20 patients. If there are at least 7 pCRs (35%) we will enroll an additional 12 patients in stage II, for a total of 32 patients. This design will provide 80% statistical power to detect a change in pCR rate from 35% to 50% with a significance level of less than 0.20. If we observe at least 14 pCRs (of 32 patients) the study will be considered positive and we will plan a follow-up randomized, controlled phase II (or phase III) study. Exploratory outcomes include correlation of AR positivity with proliferation at diagnosis, identification of oncogenic androgen response elements from matched pre- and post- treatment tissue, and identification of gene signatures associated with pCR. We will also measure 2- and 5-year disease free survival. TASUC opened to accrual on October 2, 2023 and is actively recruiting. Citation Format: Mojisola Araoye, Anthony Mega, Galina G. Lagos, Benedito Carneiro, Sari Khaleel, Dragan Golijanin, SHELDON L. HOLDER. Targeting androgen signaling in urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT085.

High incidence of sebaceous gland inflammation in aldose reductase-deficient mice

Aldose reductase is a member of the 1B1 subfamily of aldo-keto reductase gene superfamily. The action of aldose reductase (AR) has been implicated in the pathogenesis of a variety of disease states, most notably complications of diabetes mellitus including neuropathy, retinopathy, nephropathy, and cataracts. To explore for mechanistic roles for AR in disease pathogenesis, we established mutant strains produced using Crispr-Cas9 to inactivate the AKR1B3 gene in C57BL6 mice. Phenotyping AR-knock out (ARKO) strains confirmed previous reports of reduced accumulation of tissue sorbitol levels. Lens epithelial cells in ARKO mice showed markedly reduced epithelial-to-mesenchymal transition following lens extraction in a surgical model of cataract and posterior capsule opacification. A previously unreported phenotype of preputial sebaceous gland swelling was observed frequently in male ARKO mice homozygous for the mutant AKR1B3 allele. This condition, which was shown to be accompanied by infiltration of proinflammatory CD3+ lymphocytes, was not observed in WT mice or mice heterozygous for the mutant allele. Despite this condition, reproductive fitness of the ARKO strain was indistinguishable from WT mice housed under identical conditions. These studies establish the utility of a new strain of AKR1B3-null mice created to support mechanistic studies of cataract and diabetic eye disease.

Androgen receptor actions on AgRP neurons are not a major cause of reproductive and metabolic impairments in peripubertally androgenized mice.

Excess levels of circulating androgens during prenatal or peripubertal development are an important cause of polycystic ovary syndrome (PCOS), with the brain being a key target. Approximately half of the women diagnosed with PCOS also experience metabolic syndrome; common features including obesity, insulin resistance and hyperinsulinemia. Although a large amount of clinical and preclinical evidence has confirmed this relationship between androgens and the reproductive and metabolic features of PCOS, the mechanisms by which androgens cause this dysregulation are unknown. Neuron-specific androgen receptor knockout alleviates some PCOS-like features in a peripubertal dihydrotestosterone (DHT) mouse model, but the specific neuronal populations mediating these effects are undefined. A candidate population is the agouti-related peptide (AgRP)-expressing neurons, which are important for both reproductive and metabolic function. We used a well-characterised peripubertal androgenized mouse model and Cre-loxP transgenics to investigate whether deleting androgen receptors specifically from AgRP neurons can alleviate the induced reproductive and metabolic dysregulation. Androgen receptors were co-expressed in 66% of AgRP neurons in control mice, but only in <2% of AgRP neurons in knockout mice. The number of AgRP neurons was not altered by the treatments. Only 20% of androgen receptor knockout mice showed rescue of DHT-induced androgen-induced anovulation and acyclicity. Furthermore, androgen receptor knockout did not rescue metabolic dysfunction (body weight, adiposity or glucose and insulin tolerance). While we cannot rule out developmental compensation in our model, these results suggest peripubertal androgen excess does not markedly influence Agrp expression and does not dysregulate reproductive and metabolic function through direct actions of androgens onto AgRP neurons.

Liver Androgen Receptor Knockout Improved High-fat Diet Induced Glucose Dysregulation in Female Mice But Not Male Mice.

Previous research has indicated that liver androgen receptors may play a role in modulating disease. This study aims to investigate the pathophysiology of high-fat diet (HFD) induced dysglycemia in male and female liver androgen receptor knockout (LivARKO) mice. We performed metabolic tests on LivARKO female and male mice fed a HFD or a control diet (from Research Diets Inc.) during months 1 or 2 after starting the diet. Additionally, we performed Western blot and quantitative real-time PCR analysis on the livers of the mice to examine intermediates in the insulin signaling pathway. LivARKO-HFD female mice displayed no difference in glucose tolerance compared to female LivARKO-Control (Con) mice, whereas in wild-type female mice, HFD impaired glucose tolerance (IGT). Our data suggests that starting at 1 month, LivARKO may be protecting female mice from HFD-induced metabolic dysfunction. LivARKO-HFD female mice displayed significantly worse insulin sensitivity at 15 minutes compared to LivARKO-Con female mice, but, strangely, LivARKO-HFD female mice had significantly better insulin sensitivity at 60 and 90 minutes compared to LivARKO-Con female mice. Despite protecting against IGT, LivARKO did not protect against HFD-induced hyperinsulinemia in female mice. In contrast to females, male LivARKO-HFD mice displayed impaired glucose tolerance compared to male LivARKO-Con mice. Thus, LivARKO is not protective against HFD-induced glucose metabolic dysfunction in male mice. Lastly, LivARKO-HFD female mice maintained hepatic insulin sensitivity whereas LivARKO-HFD male mice displayed hepatic insulin resistance. These findings suggest that LivARKO delayed the onset of HFD-induced dysglycemia in female mice.

Proteomic analysis shows decreased type I fibers and ectopic fat accumulation in skeletal muscle from women with PCOS.

Polycystic ovary syndrome's (PCOS) main feature is hyperandrogenism, which is linked to a higher risk of metabolic disorders. Gene expression analyses in adipose tissue and skeletal muscle reveal dysregulated metabolic pathways in women with PCOS, but these differences do not necessarily lead to changes in protein levels and biological function. To advance our understanding of the molecular alterations in PCOS, we performed global proteomic and phosphorylation site analysis using tandem mass spectrometry, and analyzed gene expression and methylation. Adipose tissue and skeletal muscle were collected at baseline from 10 women with and without PCOS, and in women with PCOS after 5 weeks of treatment with electrical stimulation. Perilipin-1, a protein that typically coats the surface of lipid droplets in adipocytes, was increased whereas proteins involved in muscle contraction and type I muscle fiber function were downregulated in PCOS muscle. Proteins in the thick and thin filaments had many altered phosphorylation sites, indicating differences in protein activity and function. A mouse model was used to corroborate that androgen exposure leads to a shift in muscle fiber type in controls but not in skeletal muscle-specific androgen receptor knockout mice. The upregulated proteins in muscle post treatment were enriched in pathways involved in extracellular matrix organization and wound healing, which may reflect a protective adaptation to repeated contractions and tissue damage due to needling. A similar, albeit less pronounced, upregulation in extracellular matrix organization pathways was also seen in adipose tissue. Our results suggest that hyperandrogenic women with PCOS have higher levels of extra-myocellular lipids and fewer oxidative insulin-sensitive type I muscle fibers. These could be key factors leading to insulin resistance in PCOS muscle while electric stimulation-induced tissue remodeling may be protective. Swedish Research Council (2020-02485, 2022-00550, 2020-01463), Novo Nordisk Foundation (NNF22OC0072904), and IngaBritt and Arne Lundberg Foundation. Clinical trial number NTC01457209.

Proteomic analysis shows decreased type I fibers and ectopic fat accumulation in skeletal muscle from women with PCOS

Background:Polycystic ovary syndrome’s (PCOS) main feature is hyperandrogenism, which is linked to a higher risk of metabolic disorders. Gene expression analyses in adipose tissue and skeletal muscle reveal dysregulated metabolic pathways in women with PCOS, but these differences do not necessarily lead to changes in protein levels and biological function.Methods:To advance our understanding of the molecular alterations in PCOS, we performed global proteomic and phosphorylation site analysis using tandem mass spectrometry, and analyzed gene expression and methylation. Adipose tissue and skeletal muscle were collected at baseline from 10 women with and without PCOS, and in women with PCOS after 5 weeks of treatment with electrical stimulation.Results:Perilipin-1, a protein that typically coats the surface of lipid droplets in adipocytes, was increased whereas proteins involved in muscle contraction and type I muscle fiber function were downregulated in PCOS muscle. Proteins in the thick and thin filaments had many altered phosphorylation sites, indicating differences in protein activity and function. A mouse model was used to corroborate that androgen exposure leads to a shift in muscle fiber type in controls but not in skeletal muscle-specific androgen receptor knockout mice. The upregulated proteins in muscle post treatment were enriched in pathways involved in extracellular matrix organization and wound healing, which may reflect a protective adaptation to repeated contractions and tissue damage due to needling. A similar, albeit less pronounced, upregulation in extracellular matrix organization pathways was also seen in adipose tissue.Conclusions:Our results suggest that hyperandrogenic women with PCOS have higher levels of extra-myocellular lipids and fewer oxidative insulin-sensitive type I muscle fibers. These could be key factors leading to insulin resistance in PCOS muscle while electric stimulation-induced tissue remodeling may be protective.Funding:Swedish Research Council (2020-02485, 2022-00550, 2020-01463), Novo Nordisk Foundation (NNF22OC0072904), and IngaBritt and Arne Lundberg Foundation. Clinical trial number NTC01457209.

Reproductive Profile of Neuronal Androgen Receptor Knockout Female Mice With a Low Dose of DHT.

Hyperandrogenism and polycystic ovarian syndrome result from the imbalance or increase of androgen levels in females. Androgen receptor (AR) mediates the effects of androgens, and this study examines whether neuronal AR plays a role in reproduction under normal and increased androgen conditions in female mice. The neuron-specific AR knockout (KO) mouse (SynARKO) was generated from a female mouse (synapsin promoter driven Cre) and a male mouse (Ar fl/y). Puberty onset and the levels of reproductive hormones such as LH, FSH, testosterone, and estradiol were comparable between the control and the SynARKO mice. There were no differences in cyclicity and fertility between the control and SynARKO mice, with similar impairment in both groups on DHT treatment. Neuronal AR KO, as in this SynARKO mouse model, did not alleviate the infertility associated with DHT treatment. These studies suggest that neuronal AR KO neither altered reproductive function under physiological androgen levels, nor restored fertility under hyperandrogenic conditions.

Crucial Roles of the Mesenchymal Androgen Receptor in Wolffian Duct Development.

Wolffian duct (WD) maintenance and differentiation is predominantly driven by the androgen action, which is mediated by the androgen receptor (AR). It is well established that the mesenchyme indicates the fate and differentiation of epithelial cells. However, in vivo developmental requirement of mesenchymal AR in WD development is still undefined. By designing a mesenchyme-specific Ar knockout (ARcKO), we discovered that the loss of mesenchymal Ar led to the bilateral or unilateral degeneration of caudal WDs and cystic formation at the cranial WDs. Ex vivo culture of ARcKO WDs invariably resulted in bilateral defects, suggesting that some factor(s) originating from surrounding tissues in vivo might promote WD survival and growth even in the absence of mesenchymal Ar. Mechanistically, we found cell proliferation was significantly reduced in both epithelial and mesenchymal compartments; but cell apoptosis was not affected. Transcriptomic analysis by RNA sequencing of E14.5 mesonephroi revealed 131 differentially expressed genes. Multiple downregulated genes (Top2a, Wnt9b, Lama2, and Lamc2) were associated with morphological and cellular changes in ARcKO male embryos (ie, reduced cell proliferation and decreased number of epithelial cells). Mesenchymal differentiation into smooth muscle cells that are critical for morphogenesis was also impaired in ARcKO male embryos. Taken together, our results demonstrate the crucial roles of the mesenchymal AR in WD maintenance and morphogenesis in mice.

SAT050 Liver Androgen Receptor Knockout Delayed The Onset Of High Fructose Diet-induced Dysglycemia In Female Mice

Abstract Disclosure: C. Falzarano: None. T. Oliver: None. A. Osei-Ntansah: None. T. Lofton: None. S. Andrisse: None. Introduction: Previous research has indicated that a lipid-independent pathogenic mechanism plays a role in androgen-induced hepatic insulin resistance where liver androgen receptors may play a role in modulating disease. Consequently, this study aims to investigate the pathophysiology of high fructose diet (HFrD) induced dysglycemia in male and female liver androgen receptor knockout (LivARKO) mice. Andrisse et al 2021 showed that deleting the liver androgen receptor prevented female mice from developing hyperandrogenemia (HA)-induced insulin resistance. From this, we asked the question, does LivARKO prevent HFrD induced dysglycemia? Hypothesis: It was hypothesized that HFrD LivARKO female mice would display impaired whole body glucose metabolism in comparison to the Control diet fed LivARKO female mice, suggesting that AR does not play a significant role in regulating HFrD-induced dysglycemia. Methods: We performed insulin tolerance tests (ITT), glucose tolerance tests (GTT), pyruvate tolerance tests (PTT), and glucose stimulated insulin secretion (GSIS) tests on LivARKO female mice fed a high fat diet (HFD) or a control diet (CD) (from Research Diets Inc) during months 1 or 2 after starting the diet. Results: After 1-month, LivARKO female mice fed a Control diet had impaired glucose tolerance compared to LivARKO-HFrD female mice; however, after 2-months, the LivARKO-HFrD female mice displayed impaired glucose tolerance above that of the Control diet LivARKO female mice. LivARKO female mice fed a high fructose diet (HFrD) did not display increased fasting insulin levels compared to LivARKO female mice on a control diet. HFrD LivARKO mice did not display any increase in GSIS at 15-min or 30-min. Conclusion: These findings suggest that LivARKO delayed the onset of HFrD-Induced dysglycemia in female mice. Further research may consist of molecular data to elucidate the mechanistic pathways involved in these whole body phenotypes observed. Presentation: Saturday, June 17, 2023

SAT362 Low-dose DHT’s Effects On Gluconeogenesis And Energy Metabolism Are Ameliorated By Central Nervous System Knockout Of The Androgen Receptor

Abstract Disclosure: V. Ubba: None. S. joseph: None. R. Akbar: None. M. Dsilva: None. S. Wu: None. Polycystic ovarian syndrome (PCOS) is a leading cause of infertility in women of reproductive age. Mostly, the level of androgens are deviated from their physiological values in females, resulting in hyperandrogenemia. Androgen manifests its function in the cells via the Androgen Receptor (AR), making it a prime factor for studying PCOS pathophysiology. We generated central nervous system (CNS) specific AR knockout mouse (SynARKO), using synapsin promoter driven cre, to examine its role under normal and hyperandrogenemic conditions in female mice. Assessment of reproductive parameters under physiological levels of androgens, revealed no differences in the cyclicity and fertility of SynARKO mice when compared to Control (Con-veh) mice. These parameters were altered under hyperandrogenemia (induced using DHT) in Control mice (Con-DHT) mice and were not rescued in the SynARKO- DHT mice, suggesting CNS AR is dispensable to reproductive features in females. Evaluation of metabolic functions by Glucose tolerance test, we observed alleviated glucose intolerance in SynARKO-DHT (4 months). Energy expenditure and food intake were increased in Con-DHT mice compared to Con-veh, while showing no further differences in SynARKO-DHT mice compared to SynARKO-veh. At the molecular level, DHT treatment showed increased phosphorylated Akt in hypothalamus of control mice. Interestingly, it was restored in synARKO upon DHT treatment. Further, the inflammation marker, phosphorylated NF-kB p-65, was increased in the hypothalamus of Con-DHT mice. Additionally, IBA, a marker of macrophage activation was also increased upon DHT treatment in control mice. Taken together, our results suggest that while the reproductive functions are not mediated by the CNS AR, metabolic effects caused by hyperandrogenemia, is regulated by the central nervous system AR. Presentation Date: Saturday, June 17, 2023