Androgen Receptor Knockout Mice Research Articles

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.

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

Effect of AR gene-specific knockout on the process of radiation-induced pulmonary fibrosis and its mechanism.

Numerous studies have proved that epithelial-mesenchymal transition (EMT) of lung epithelial cells is one of the important causes of radiation-induced pulmonary fibrosis (RIPF). Aldose reductase (AR) is a monomer enzyme in the polyglycolic metabolic pathway and belongs to the aldo-keno reductase protein superfamily. Our previous studies have found that AR as one of the most significantly up-regulated genes was associated with the development of bleomycin-induced PF in rats. It is not clear whether aldose reductase is related to the regulation of radiation-induced EMT and mediates RIPF. AR-knockout mice, wild-type mice and lung epithelial cells were induced by radiation to establish a RIPF animal model and EMT system, to explore whether AR is mediation to RIPF through the EMT pathway. In vivo, AR deficiency significantly alleviated radiation-induced histopathological changes, reduced collagen deposition and inhibited collagen I, matrix metalloproteinase 2 (MMP2) and Twist1 expression. In addition, AR knockout up-regulated E-cadherin expression and up-regulated α-SMA and Vimentin expression. In vitro, AR, collagen I and MMP2 expression were increased in lung epithelial cells after radiation, which was accompanied by Twist1 expression up-regulation and EMT changes evidenced by decreased E-cadherin expression and increased α-SMA and Vimentin expression. Knockdown or inhibition of AR inhibited the expressions of Twist1, MMP2 and collagen I, and reduced cell migration and reversed radiation-induced EMT. These results indicated that aldose reductase may be related to radiation-induced lung epithelial cells EMT, and that inhibition of aldose reductase might be a promising treatment for RIPF.

Liver Androgen Receptor Knockout Delayed the Onset of High Fructose Diet-Induced Dysglycemia in Female Mice

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. The cellular molecular regulation of differing mechanisms of insulin resistance. 1R01DK126892-01A1 This is the full abstract presented at the American Physiology Summit 2023 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.

Novel mechanisms of androgen receptor-centered transcriptional regulatory network in regulating CD8 +T cell exhaustion and sex bias in cancer

Abstract Sex bias in cancers arising from nonreproductive organs is known but poorly understood. We recently reported on a T cell-intrinsic role of androgen receptor (AR) in driving CD8 +T cell exhaustion which underlies the poorer tumor control in males (Kwon et al., Sci Immunol, 2022). This study aims to understand how AR regulates CD8 +tumor-infiltrating lymphocytes (TILs) by identifying its genome-wide targets. We created a CD8 +T cell-specific AR knockout (KO) mouse model by crossing E8I-cre mice with Ar-floxed mice, which we challenged with syngeneic bladder tumor MB49. We then monitored tumor growth and performed spectral flow cytometry using a T cell exhaustion panel. Further, we used Cleavage Under Targets and Tagmentation – sequencing (CUT&amp;Tag-seq) to map the entire AR targets in CD8 +TILs. Loss of AR in CD8 +T cells significantly slowed the growth of MB49 in male but not female mice. CD8 +TILs from male CD8 AR KO mice showed reduced TOX +TCF1 −terminally exhausted subset (69%; P≤ 0.05) and TOX expression (72%; P = 0.0583) compared to controls. Similarly, TOX expression decreased by 43% in AR-deleted CD8 +T cells following chronic TCR stimulation in vitro (P ≤ 0.0001). Finally, using CUT&amp;Tag-seq, we found that AR binds directly to promoters of multiple key transcriptional regulators of T cell exhaustion, including Tcf7 and Tox. Through identifying the genome-wide targets of AR in CD8 +TILs, we characterized the cell-intrinsic mechanism by which AR regulates CD8 +T cell exhaustion. Our work suggests AR is a master transcriptional regulator that negatively impacts CD8 +T cell immunity, which may contribute to poorer survival in males and supports combining androgen deprivation therapy with immune checkpoint blockage for cancer treatment. Special Institutional Funding

OR25-1 Bilateral and Unilateral Malformations of Male Reproductive Tracts in Mice with Androgen Receptor Gene Deletion in the Mesenchyme

Abstract Congenital unilateral defects of male reproductive tracts have been documented in men, suggesting the potential left-right asymmetry in male reproductive tract development. However, classic textbooks of endocrinology have been teaching us that the development of the paired male reproductive tract is governed by the universal action of the androgen receptor (Ar). During sexual differentiation, testis-derived androgens activate the Ar to promote the stabilization of the bilateral Wolffian ducts, the progenitor for the male reproductive tract. The Wolffian ducts then differentiate into the paired male reproductive tract organs, which include the epididymes, vas deferentes, and seminal vesicles. The Ar is expressed both in the epithelium and mesenchyme of the male reproductive tract during fetal development. Landmark tissue recombinant studies and the observation of normal morphology in the epithelium-specific Ar knockout mouse all support the notion that Ar action in the mesenchyme dictates the morphogenesis of the male reproductive tract. However, no genetic study has been performed to investigate the consequence of the loss of mesenchymal Ar on male reproductive tract development. To test the functional significance of the mesenchymal Ar, we designed a mesenchyme-specific Ar knockout mouse model (ARcKO) to ablate Ar specifically in the mesenchyme prior to the initiation of sexual differentiation. We performed immunohistochemistry of AR to confirm that Ar expression was absent in the mesenchyme while epithelial Ar remained intact in this ARcKO model. Based on the widely accepted notion that mesenchymal Ar regulates epithelial morphogenesis of the male reproductive tract, we expected to see abnormal patterning of the male reproductive tract. Indeed, the epididymis lost its characteristic coiling and became cystic. However, 40% of the 23 collected ARcKO males displayed these abnormalities on both horns; 43% and 17% displayed these abnormal phenotype solely on the left and right horn, respectively. This surprising observation might result from asynchronous activities of Cre on the left and right horns. However, when the Cre was crossed with a reporter (tdTomato), we observed comparable reporter expression on both horns at the onset of sexual differentiation of reproductive tracts. These observations suggest that the developmental programs in the morphogenesis of the left and right male reproductive tracts are asymmetrical and there could be a compensation mechanism for the loss of mesenchymal AR. We are currently in the process of comparing the left and the right horns in Ar knockout male mice to determine any biased signaling. Taken together, our study provides a unique model for not only studying congenital defects of male reproductive tracts but also for investigating the potential asymmetry in the masculinization program. Presentation: Monday, June 13, 2022 11:00 a.m. - 11:15 a.m.

Androgen Actions in Adipose Tissue, But Not Skeletal Muscle, are Involved in the Development of Metabolic PCOS Features in a Mouse Model

Background: Polycystic ovary syndrome (PCOS) is a common disorder characterised by endocrine, reproductive and metabolic dysfunction. Hyperandrogenism is a key diagnostic trait and evidence supports a role for androgen receptor (AR) mediated actions in PCOS pathogenesis. Aberrant AR signalling in adipose tissue and muscle have been proposed as being involved in PCOS pathogenesis, but their importance and the precise AR signalling mechanisms involved remain undefined. Aim: To investigate the role of AR signalling in white adipose tissue (WAT), brown adipose tissue (BAT) and skeletal muscle in the development of PCOS. Method: WT, global androgen receptor knockout (ARKO) and skeletal muscle-specific (SkMARKO) mice were exposed to DHT to induce PCOS. ARKO (AR−/−) WAT/BAT was transplanted into WT (AR+/+) DHT-induced PCOS females, and WT WAT/BAT was transplanted into ARKO DHT-induced PCOS females. After 12 weeks of DHT exposure all mice were assessed for the presence of reproductive and metabolic PCOS traits. Results: DHT exposure in WT females induced key reproductive and metabolic PCOS traits. SkMARKO mice treated with DHT displayed a similar phenotype to DHT-treated WT females, with full development of reproductive and metabolic PCOS traits. Transplantation of ARKO WAT/BAT into DHT-treated WT females prevented the development of some metabolic PCOS features, as mice exhibited a significantly lower body weight (P&lt;0.05), decreased visceral adiposity (P&lt;0.05), and smaller adipocyte size (P&lt;0.05) compared to DHT-treated WT females with sham surgery. In contrast, reproductive PCOS traits were not ameliorated by WAT or BAT transplantation. Furthermore, DHT exposure in ARKO female mice transplanted with WT WAT/BAT did not lead to the development of PCOS traits. Conclusion: WAT and BAT, but not skeletal muscle, are key sites of AR-mediated actions involved in the development of metabolic PCOS traits. These data support targeting adipocyte AR-driven pathways in future research aimed at developing novel therapeutic interventions for PCOS.

Increased Insulin Resistance in 10‐month compared to 5‐month Old Male Hepatic Androgen Receptor Knockout Mice

Insulin resistance is an important component of type 2 diabetes and metabolic syndrome. The liver plays an essential component in the metabolism of insulin and androgen signaling. Sex differences exist in type 2 diabetes with men having a higher prevelance than women. In our study we placed male liver androgen receptor knock‐out mice (Liv‐ARKO) on chow diets to determine if Liv‐ARKO had an effect on insulin resistance on younger age (5 month) compared to older age (10 month).In this study, male Liv‐ARKO mice were kept on chow diets for either 5 months or 10 months and then sacrificed. Some mice were given a dose of 0.5 U/kg insulin before sacrificing to investigate the effects of age on insulin signaling. Western blots were used to determine protein expression in tissue from the liver, skeletal muscle, white adipose tissue. BCA assays were used to standardize the protein concentration in each sample.Upon activation of the insulin signaling pathway, the insulin receptor substrate (IRS) proteins, initiates activation of the phosphatidylinositol 3‐kinase (PI3k) pathways, resulting in stimulation of protein kinases such as Akt. As seen in a previous study1 approximately 5 month old (22 week old) hepatic‐AR‐knockout chow mice showed that when stimulated with insulin there was an increase in PI3k activity. This study did not take it a step further to show what was going on with p‐AKT.It was hypothesized that p‐AKT is lowered in both 5 and 10 month old mice compared to mice not given insulin. Additionally, after 10 months, total AKT was increased. Insulin stimulated p‐Foxo was lowered at 5 months but at 10 months p‐Foxo was increased.Our results suggest that Liver Androgen Receptor is causing decreased insulin action at the level of p‐AKT in both young (5 month) and old (10 month) mice. If insulin is not stimulating p‐AKT, the body may try to make more total AKT to try to compensate; hence the increase in total AKT seen in the 10 month mice. Further investigation should also be performed on the gender differences in these results.

Increased Insulin Resistance in Male Hepatic Androgen Receptor Knockout Mice Fed High Fructose Diet

IntroductionInsulin resistance is a common hepatic dysfunction amongst individuals suffering from diabetes or obesity and affects 10‐25% of the general population1. The intracellular signaling pathway is reliant on every step being executed correctly. It is evident that excess dietary fats and sugars will result in obesity and likely diabetes. This cellular machinery can be altered when there is a build‐up of lipids which impact molecular insulin signaling. Our objective is to determine if chronic intake of fructose by male liver androgen receptor knockout (Liv‐ARKO) mice will show cascade alteration resulting in increased resistance to insulin when compared to male Liv‐ARKO mice on a control diet. We hypothesized that LivARKO mice on HFrD would show impaired insulin action when compared to control and chow diet mice.MethodsWe placed male Liv‐ARKO mice on high fructose (HFrD), Control (Research Diets Inc), and Chow diets. We performed insulin tolerance tests (ITT), glucose tolerance tests (GTT), and pyruvate tolerance tests (PTT) after 1‐2 months of being on diets. The mice were later sacrificed, and their tissues were extracted for further tests. To assess differences in insulin action that may arise from increased fructose some mice were given a dose of 0.5 U/kg insulin before being sacrificed to further investigate insulin signaling proteins. BCA protein assays were done to ensure that protein concentration in each sample was standardized. Western blots were performed using tissue from liver to measure molecular insulin action, via p‐AKT presence.ResultsMale mice on control diet for 2‐months showed improved glucose tolerance and enhanced pyruvate tolerance when compared to male mice on control diet for 1 month. Mice on 2‐month control diet showed impaired insulin sensitivity compared to mice on the same diet for 1 month.Male LivARKO mice displayed no insulin stimulated p‐AKT when on a chow diet. Levels of p‐AKT were the same in basal and insulin male LivARKO mice fed a chow diet. Male LivARKO mice fed a control diet displayed a 4‐fold increase in levels of p‐AKT when stimulated by insulin compared to mice not given insulin (basal). Moreover, male LivARKO mice fed a HFrD displayed no insulin stimulated p‐AKT. Foxo1 levels were not altered by insulin stimulation in male LivARKO mice fed a chow, control, or HFrD. However, it should be noted that control and HFrD both increased Foxo1 levels compared to chow diet.ConclusionOur results suggest that hepatic insulin action or the ability for insulin to initiate glucose uptake in liver is decreased in male LivARKO mice on HFrD. Further investigation of Research Diets Inc. data is necessary to determine why the control diet and HFrD lead to the results discovered.

High Fat Diet and High Fructose Diet‐fed Male and Female Liv‐ARKO Mice Displayed Impair Glucose‐Stimulated Insulin Secretion

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. The liver plays an essential component in the metabolism of insulin and androgen signaling. Hyperandrogenism in females can also increase predisposition to insulin resistance. In our study, we placed female and male liver androgen receptor knock‐out mice (Liv‐ARKO) on high fructose diets (HFrD) and high fat diets (HFD) to determine if Liv‐ARKO mice demonstrated altered glucose‐stimulated insulin secretion on special diets compared to a control diet.In this study, female and male Liv‐ARKO mice were placed on three distinct diets: Control (Research Diets Inc), High Fat (HFD), and High Fructose (HFrD). After one month and two months on these diets, Glucose‐Stimulated Insulin Secretion tests were performed: each group was injected with a 20% glucose solution at 2g of glucose/kg body mass and blood samples were taken via tail milking at 0 minutes, 15 minutes, and 30 minutes. Blood samples were immediately centrifuged and serum supernatant was extracted for further analysis via Sigma Insulin Assay.In the current study, after two months, female mice on HFrD demonstrated lower basal insulin levels compared to female mice fed a control diet. Interestingly, mice on a HFD showed insulin levels that were lower than the basal levels of both control mice and HFrD mice. Furthermore, compared to the control mice, the HFD mice demonstrated lower insulin levels at both basal level and 30 minutes post‐glucose administration.It was hypothesized that HFD and HFrD‐fed Liv‐ARKO mice would impair glucose‐stimulated insulin secretion, which our current study supports. These data suggest that diets high in fructose and fat both blunt insulin secretion following a meal, and to a greater extent in high fat diets. This dysfunction may ultimately play a role in the development of insulin resistance. More research is necessary in order to further investigate the effects of high fat and high fructose diets on glucose‐stimulated insulin secretion and consequent development of insulin resistance.

Study of Insulin Resistance with Female Liver Androgen Receptor Knock‐Out Mice (Liv‐AKRO) on a High Fructose Diet

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. The liver plays an essential component in the metabolism of insulin and androgen signaling. Hyperandrogenism in females can also increase predisposition to insulin resistance. In our study, we placed female liver androgen receptor knock‐out mice (Liv‐ARKO) on high fructose diets (HFrD) to determine if Liv‐ARKO mice demonstrated blunted insulin resistance on HFrD compared to control diet.In this study, female Liv‐ARKO mice were placed on three distinct diets: Control (Research Diets Inc) (CD), chow, and High Fructose (HFrD). The mice were sacrificed after 1 or 2 months. Some 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 liver, skeletal muscle, white adipose tissue, ovary, and pituitary glands. BCA assays were used to standardize the protein concentration in each sample. Insulin action can be measured molecularly by examining p‐AKT. If p‐AKT levels increase in the presence of insulin, this indicates that insulin is likely functioning properly in the sample.As seen in previous studies, insulin increased p‐AKT in livers of Liv‐ARKO mice on a chow diet. In contrast, insulin did not increase p‐AKT in livers of Liv‐ARKO mice on the CD for 2 months in comparison to those not given insulin (basal). Furthermore, Liv‐ARKO mice on a HFrD for 2 months displayed an even further decrease in p‐AKT in the liver compared to Liv‐ARKO mice on a CD stimulated with insulin. Liv‐ARKO mice on the CD for 2 months showed enhanced glucose tolerance (EGT) compared to those on the CD for only 1 month. The CD increased glucose tolerance at the whole body level (GTT) and the molecular level (pAKT). Glucose tolerance also increased at 2 months in comparison to 1 month in Liv‐ARKO mice on the CD. This effect is the same and equally as significant for male and female Liv‐ARKO mice. After 2 months on the CD, Liv‐ARKO mice showed impaired insulin sensitivity (IIS) compared to 1 month on the diet. This IIS is significantly greater in female Liv‐ARKO mice compared to male.It was hypothesized that HFrD‐fed Liv‐ARKO would impair insulin action compared to the CD and chow fed mice. The Liv‐ARKO mouse model did not rescue HFrD insulin resistance. The results confirmed this hypothesis and further determined that the CD increased insulin action in comparison to the chow diet. The data suggests that the CD is reducing insulin action in the Liv‐ARKO mice. There is insulin action in Liv‐ARKO mice on the CD as pAKT is present, although at lower levels than in the basal mice. However, compared to the control, the HFrD prohibits the ability of insulin to increase pAKT, indicating complications in the early stages of the insulin signaling pathway. Further research is required into what components of the CD are prompting this difference in insulin action and if it only takes place in the Liv‐ARKO mouse model. Further investigation should be performed on the gender discrepancy in these results.

Androgen conspires with the CD8+ T cell exhaustion program and contributes to sex bias in cancer.

Sex bias exists in the development and progression of nonreproductive organ cancers, but the underlying mechanisms are enigmatic. Studies so far have focused largely on sexual dimorphisms in cancer biology and socioeconomic factors. Here, we establish a role for CD8+ T cell-dependent antitumor immunity in mediating sex differences in tumor aggressiveness, which is driven by the gonadal androgen but not sex chromosomes. A male bias exists in the frequency of intratumoral antigen-experienced Tcf7/TCF1+ progenitor exhausted CD8+ T cells that are devoid of effector activity as a consequence of intrinsic androgen receptor (AR) function. Mechanistically, we identify a novel sex-specific regulon in progenitor exhausted CD8+ T cells and a pertinent contribution from AR as a direct transcriptional transactivator of Tcf7/TCF1. The T cell-intrinsic function of AR in promoting CD8+ T cell exhaustion in vivo was established using multiple approaches including loss-of-function studies with CD8-specific Ar knockout mice. Moreover, ablation of the androgen-AR axis rewires the tumor microenvironment to favor effector T cell differentiation and potentiates the efficacy of anti-PD-1 immune checkpoint blockade. Collectively, our findings highlight androgen-mediated promotion of CD8+ T cell dysfunction in cancer and imply broader opportunities for therapeutic development from understanding sex disparities in health and disease.

5-HT3A receptors maintain hippocampal LTP in a CB1 and GABAA receptor- dependent manner for spatial memory.

As the only ionotropic receptor in the 5-HT receptor family, the 5-HT3 receptor (5-HT3 R) is involved in psychiatric disorders and its modulators have potential therapeutic effects for cognitive impairment in these disorders. However, it remains unclear how 5-HT3 Rs shape synaptic plasticity for memory function. Extracellular as well as whole-cell electrophysiological recordings were used to monitor hippocampal LTP and synaptic transmission in hippocampal slices in 5-HT3 AR knockout or 5-HT3 AR-GFP mice. Immunocytochemistry, qRT-PCR and western blotting were used to measure receptor expression. We also assessed hippocampal dependent cognition and memory, using the Morris water maze (MWM) and novel object recognition. We found that 5-HT3 R dysfunction impaired hippocampal LTP in Schaffer collateral (SC)-CA1 pathway in hippocampal slices, by facilitating GABAergic inputs in pyramidal cells. This effect was dependent on 5-HT3 Rs on axon terminals. It resulted from reduced expression and function of the cannabinoid receptor 1 (CB1 R) co-localized with 5-HT3 Rs on axon terminals, and then led to diminishment of tonic inhibition of GABA release by CB1 Rs. Inhibition of CB1 Rs mimicked the facilitation of GABAergic transmission by 5-HT3 R disruption. Consequently, mice with hippocampal 5-HT3 R disruption exhibited impaired spatial memory in MWM tasks. These results suggest that 5-HT3 Rs are crucial in enabling hippocampal synaptic plasticity via a novel CB1 R-GABAA -dependent pathway to regulate spatial memory.