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Objective: We hypothesize that in women experiencing obesity and PCOS, the women with higher levels of free androgen index will display more dysregulation in the insulin signaling pathway in adipose tissue. More specifically, we believe that visceral more than subcutaneous adipose tissue in the women in the study will display more dysregulation; and lastly that bariatric surgery will alleviate insulin signaling dysregulation. Methods: Here, we performed molecular analyses (Western blots and qPRC) exploring the insulin signaling pathway in visceral and subcutaneous (SubQ) adipose tissue from women with obesity and PCOS undergoing RYGB bariatric surgery; and SubQ WAT from women with obesity and PCOS at baseline and six-months after medical intervention with metformin (MTF) . Results: Subcutaneous (SubQ) white adipose tissue (WAT) displayed a trending (p=0.2) but non-significant increase in p-ACC compared to visceral WAT from women with PCOS and obesity pre-bariatric surgery. Six months of MTF increased p-ACC in SubQ WAT by 5-fold compared to zero months (baseline) in women with PCOS and obesity. IR displayed similar expression in the visceral compared to SubQ WAT in women with PCOS and obesity pre-bariatric surgery. IR expression increased by 20-fold at 6-months of MTF compared to baseline in SubQ WAT of women with PCOS and obesity. Phosphorylated forkhead box 01 (p-FOXO1) displayed similar expression in the visceral compared to SubQ WAT in women with PCOS and obesity. However, p-FOXO1 was decreased at 6-months of MTF compared to baseline in women with PCOS and obesity. p-AKT levels were similar in visceral compared to SubQ WAT in women with PCOS and obesity pre-bariatric surgery. However, p-AKT levels were significantly increased by 4-fold at 6-months of MTF compared to 0-months in women with PCOS and obesity. Conclusion: None of the insulin signaling intermediates measured were altered in visceral compared to SubQ in women with obesity and PCOS. Disclosure S. Andrisse: None.

Polycystic ovarian syndrome (PCOS) is a hormonal disorder with insulin resistance (IR) and hyperandrogenism (HA) as drivers of the condition. PCOS women have an increased risk of developing nonalcoholic fatty liver disease (NAFLD). Our lab explores NAFLD in PCOS through a mouse model with a liver androgen receptor (AR) knockout (LivARKO). The role of AR in high-fat diet (HFD) induced metabolic disease is not fully understood. We hypothesized that LivARKO would cause dysglycemia in males but protect from dysglycemia in females. To test the hypothesis, we collected data from mice fed a HFD or a caloric adjusted control diet (CD) over the course of two months. We performed glucose tolerance (GTT), insulin tolerance (ITT), and pyruvate tolerance (PTT) tests. At 1-month, HFD female LivARKO mice displayed no difference in glucose tolerance compared to CD LivARKO female mice, suggesting that at one month LivARKO is protecting female mice from HFD-induced metabolic dysfunction. HFD male LivARKO mice displayed impaired glucose tolerance (IGT) compared to CD LivARKO mice. Thus, LivARKO was not protective against HFD-induced glucose metabolic dysfunction in male mice. At 2-months, HFD showed IGT compared to Control diet in male and female LivARKO mice, suggesting that LivARKO slowed the progression to metabolic disease in female mice but did not fully stop it. HFD male and female LivARKO mice did not display significant changes in insulin tolerance at 1-month compared to CD mice. HFD male and female LivARKO mice showed impaired pyruvate tolerance starting at 1-month and continuing at 2-months compared to CD LivARKO mice. The impairment was significantly greater in male mice. In conclusion, we found that it took LivARKO female mice longer to show dysglycemia from HFD than male LivARKO mice. Thus, in females, LivARKO slowed the progression to metabolic disease, but did not fully stop it. The impaired PTT suggests the effects of HFD may alter hepatic gluconeogenic regulation. Disclosure T. R. Oliver: None. A. Osei-ntansah: None. S. Andrisse: None. T. Lofton: None. C. Falzarano: None. Funding National Science Foundation (HRD-1931045)

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 fat diet (HFD) induced dysglycemia and hepatic insulin resistance in male and female liver androgen receptor knockout (LivARKO) mice. We performed insulin tolerance tests (ITT), glucose tolerance tests (GTT), and pyruvate tolerance tests (PTT) on LivARKO male and 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. Two-months on the CD showed significantly enhanced glucose tolerance (EGT) compared to 1-month on CD in both male and female LivARKO mice. This EGT phenotype was the same and equally as significant in males vs. females. Two-months on CD showed impaired insulin sensitivity (IIS) compared to 1-month on CD in male and female LivARKO mice. This IIS was significantly greater in female mice. Two-months on the CD displayed enhanced pyruvate tolerance compared to 1-month on the CD in male and female LivARKO mice. Two-months compared to 1-month on HFD displayed no significant difference in glucose tolerance in male and female LivARKO mice. Two-months on HFD showed impaired insulin sensitivity compared to 1-month in female but not male LivARKO mice. Pyruvate tolerance was not changed from 1- to 2-months on HFD in male or female LivARKO mice. In the CD mice, we see EGT but IIS. This suggests that insulin action is decreased. The enhanced pyruvate tolerance in CD mice suggests that the control diet lowers gluconeogenesis. Why the control diet is altering glucose metabolism from 1-2 months is still a question to be investigated. In conclusion, we show that 1- vs. 2-months on HFD showed no change in glucose metabolism in male and a slight change in female LivARKO mice. Disclosure A. Osei-ntansah: None. T. R. Oliver: None. C. Falzarano: None. T. Lofton: None. S. Andrisse: None. Funding National Science Foundation (HRD-1931045); Robert Wood Johnson Foundation

Hyperandrogenemia (HA) and insulin resistance are hallmarks of polycystic ovary syndrome (PCOS). These hallmarks are also integral elements of nonalcoholic liver disease (NALFD). Administering low dose dihydrotestosterone (DHT) induced a lean female mouse model with a PCOS-like phenotype. The molecular mechanism of HA-induced NAFLD has not been determined. We hypothesized that low dose DHT would interrupt hepatic lipid metabolism leading to NAFLD. We extracted white adipose tissue (WAT), liver, and skeletal muscle from control and low dose DHT female mice; and performed Western blot and real-time quantitative PCR (qRT-PCR) analysis of lipogenic intermediates. Low-dose DHT lowered the active form of cytosolic SREBP1 in the liver and WAT compared to controls. Additionally, low dose DHT increased inactive SREBP1 in the liver. However, DHT did not alter the levels of the active and inactive forms of SREBP2 in the liver and WAT. DHT increased SCAP protein expression and SCAP-SREBP1 binding, which helps activate SREBP. Regarding lipogenic enzymes, FAS mRNA and protein expression was increased in liver of DHT mice. p-ACC levels were unaltered in liver but decreased in WAT. Other lipid metabolism pathways were examined in liver and WAT but no changes were observed. Taken together, our findings support the hypothesis that cytosolic SREBP1 decreased due to its translocation to the nucleus, where it upregulated lipogenic mRNA expression. We showed that low-dose DHT increased SCAP-SREBP-1 association promoting the activation and translocation of SREBP1 from the cytosol to the nucleus to influence lipogenic gene expression leading to increased lipogenesis contributing to NAFLD. Disclosure S. Andrisse: None. Funding Robert Wood Johnson Foundation (74250); Eunice Kennedy Shriver National Institute of Child Health and Human Development (K23HD095075)

Hyperandrogenemia (HA) and insulin resistance (IR) are hallmarks of polycystic ovary syndrome (PCOS), a common endocrine disorder that affects 1 in 10 women. These hallmarks are also integral elements of nonalcoholic liver disease (NALFD), a disorder that is common in women with PCOS. Administering low dose dihydrotestosterone (DHT) induced a lean female mouse model with a PCOS-like phenotype, displaying IR and NAFLD. The molecular mechanism of HA-induced NAFLD has not been determined. We hypothesized that low dose DHT would interrupt hepatic lipid metabolism leading to NAFLD. To investigate the role of androgens on the master regulator of lipogenesis, sterol regulatory element-binding protein 1 (SREBP-1), we extracted white adipose tissue (WAT), liver, and skeletal muscle from wild type, control and low dose DHT female mice; and performed Western blot and real-time quantitative PCR (qRT-PCR) analysis of lipogenic intermediates of the tissue homogenates. Low-dose DHT lowered the active form of cytosolic SREBP1 in the liver and WAT compared to controls. Additionally, low-dose DHT lowered inactive SREBP1 in the liver. However, the condition did not alter the levels of the active and inactive forms of SREBP2 in the liver and WAT, though the active form was lowered in skeletal muscle. Further, p-ACC levels were unaltered in liver and WAT. FAS levels were unchanged in WAT and skeletal muscle. Taken together, our findings support the hypothesis that cytosolic SREBP1 decreased due to its translocation to the nucleus, where it regulates lipogenic protein levels. We speculate that low-dose DHT promotes the translocation of SREBP-1 from the cytosol to the nucleus to influence lipogenic gene expression leading to increased lipogenesis contributing to NAFLD. Disclosure S. Andrisse: None.