Dysfunction In Female Mice Research Articles

Downregulation of lysine-specific histone demethylase 1A (KDM1A/LSD1) in medial prefrontal cortex facilitates chronic stress–induced pain and emotional dysfunction in female mice

Chronic primary pain, characterized by overlapping symptoms of chronic pain, anxiety, and depression, is strongly associated with stress and is particularly prevalent among females. Recent research has convincingly linked epigenetic modifications in the medial prefrontal cortex (mPFC) to chronic pain and chronic stress. However, our understanding of the role of histone demethylation in the mPFC in chronic stress-induced pain remains limited. In this study, we investigated the function of lysine-specific histone demethylase 1A (KDM1A/LSD1) in the context of chronic overlapping pain comorbid with anxiety and depression in female mice. We employed a chronic variable stress model to induce pain hypersensitivity in the face and hindpaws, as well as anxiety-like and depression-like behaviors, in female mice. Our findings revealed that chronic stress led to a downregulation of KDM1A mRNA and protein expression in the mPFC. Notably, overexpressing KDM1A in the mPFC alleviated the pain hypersensitivity, anxiety-like behaviors, and depression-like behaviors in female mice, without affecting basal pain responses or inducing emotional distress. Conversely, conditional knockout of KDM1A in the mPFC exacerbated pain sensitivity and emotional distress specifically in females. In summary, this study highlights the crucial role of KDM1A in the mPFC in modulating chronic stress-induced overlapping pain, anxiety, and depression in females. Our findings suggest that KDM1A may serve as a potential therapeutic target for treating chronic stress-related overlap pain and associated negative emotional disorders.

Aberrant CD8+T cells drive reproductive dysfunction in female mice with elevated IFN-γ levels.

Interferon-gamma (IFN-γ) is pivotal in orchestrating immune responses during healthy pregnancy. However, its dysregulation, often due to autoimmunity, infections, or chronic inflammatory conditions, is implicated in adverse reproductive outcomes such as pregnancy failure or infertility. Additionally, the underlying immunological mechanisms remain elusive. Here, we explore the impact of systemic IFN-γ elevation on cytotoxic T cell responses in female reproduction utilizing a systemic lupus-prone mouse model with impaired IFN-γ degradation. Our findings reveal that heightened IFN-γ levels triggered the infiltration of CD8+T cells in the pituitary gland and female reproductive tract (FRT), resulting in prolactin deficiency and subsequent infertility. Furthermore, we demonstrate that chronic IFN-γ elevation increases effector memory CD8+T cells in the murine ovary and uterus. These insights broaden our understanding of the role of elevated IFN-γ in female reproductive dysfunction and suggest CD8+T cells as potential immunotherapeutic targets in female reproductive disorders associated with chronic systemic IFN-γ elevation.

Ezh2 emerges as an epigenetic checkpoint regulator during monocyte differentiation limiting cardiac dysfunction post-MI

Epigenetic regulation of histone H3K27 methylation has recently emerged as a key step during alternative immunoregulatory M2-like macrophage polarization; known to impact cardiac repair after Myocardial Infarction (MI). We hypothesized that EZH2, responsible for H3K27 methylation, could act as an epigenetic checkpoint regulator during this process. We demonstrate for the first time an ectopic EZH2, and putative, cytoplasmic inactive localization of the epigenetic enzyme, during monocyte differentiation into M2 macrophages in vitro as well as in immunomodulatory cardiac macrophages in vivo in the post-MI acute inflammatory phase. Moreover, we show that pharmacological EZH2 inhibition, with GSK-343, resolves H3K27 methylation of bivalent gene promoters, thus enhancing their expression to promote human monocyte repair functions. In line with this protective effect, GSK-343 treatment accelerated cardiac inflammatory resolution preventing infarct expansion and subsequent cardiac dysfunction in female mice post-MI in vivo. In conclusion, our study reveals that pharmacological epigenetic modulation of cardiac-infiltrating immune cells may hold promise to limit adverse cardiac remodeling after MI.

Multi-omic analysis of the cardiac cellulome defines a vascular contribution to cardiac diastolic dysfunction in obese female mice

Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Clinical data further support that CMD associates with development of heart failure with preserved ejection fraction and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the impact of smooth muscle cell (SMC)-specific MR deletion on obesity-associated coronary and cardiac diastolic dysfunction in female mice. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Global MR blockade with spironolactone prevented coronary and cardiac dysfunction in obese females and specific deletion of SMC-MR was sufficient to prevent obesity-associated coronary and cardiac diastolic dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in non-myocyte populations including B/T cells, macrophages, and endothelium as well as increased coronary VCAM-1 protein expression, independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling in obesity-associated coronary and cardiac dysfunction, thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity.

Lack of STAT1 co-operative DNA binding protects against adverse cardiac remodelling in acute myocardial infarction.

In this study, we addressed the functional significance of co-operative DNA binding of the cytokine-driven transcription factor STAT1 (signal transducer and activator of transcription 1) in an experimental murine model of acute myocardial infarction (MI). STAT1 knock-in mice expressing a phenylalanine-to-alanine substitution at position 77 in the STAT1 amino-terminal domain were examined for the early clinical effects produced by ligation of the left anterior descending coronary artery (LAD), an established model for MI. The F77A mutation has been previously reported to disrupt amino-terminal interactions between adjacent STAT1 dimers resulting in impaired tetramerization and defective co-operative binding on DNA, while leaving other protein functions unaffected. Our results demonstrate that a loss of STAT1 tetramer stabilization improves survival of adult male mice and ameliorates left ventricular dysfunction in female mice, as determined echocardiographically by an increased ejection fraction and a reduced left intra-ventricular diameter. We found that the ratio of STAT3 to STAT1 protein level was higher in the infarcted tissue in knock-in mice as compared to wild-type (WT) mice, which was accompanied by an enhanced infiltration of immune cells in the infarcted area, as determined by histology. Additionally, RNA sequencing of the infarcted tissue 24 h after LAD ligation revealed an upregulation of inflammatory genes in the knock-in mice, as compared to their WT littermates. Concomitantly, genes involved in oxidative phosphorylation and other metabolic pathways showed a significantly more pronounced downregulation in the infarcted tissue from STAT1F77A/F77A mice than in WT animals. Based on these results, we propose that dysfunctional STAT1 signalling owing to a lack of oligomerisation results in a compensatory increase in STAT3 expression and promotes early infiltration of immune cells in the infarcted area, which has beneficial effects on left ventricular remodelling in early MI following LAD ligation.

Neuronal SIRT3 Deletion Predisposes to Female-Specific Alterations in Cellular Metabolism, Memory, and Network Excitability.

Mitochondrial dysfunction is an early event in the pathogenesis of neurologic disorders and aging. Sirtuin 3 (SIRT3) regulates mitochondrial function in response to the cellular environment through the reversible deacetylation of proteins involved in metabolism and reactive oxygen species detoxification. As the primary mitochondrial deacetylase, germline, or peripheral tissue-specific deletion of SIRT3 produces mitochondrial hyperacetylation and the accelerated development of age-related diseases. Given the unique metabolic demands of neurons, the role of SIRT3 in the brain is only beginning to emerge. Using mass spectrometry-based acetylomics, high-resolution respirometry, video-EEG, and cognition testing, we report targeted deletion of SIRT3 from select neurons in the cortex and hippocampus produces altered neuronal excitability and metabolic dysfunction in female mice. Targeted deletion of SIRT3 from neuronal helix-loop-helix 1 (NEX)-expressing neurons resulted in mitochondrial hyperacetylation, female-specific superoxide dismutase-2 (SOD2) modification, increased steady-state superoxide levels, metabolic reprogramming, altered neuronal excitability, and working spatial memory deficits. Inducible neuronal deletion of SIRT3 likewise produced female-specific deficits in spatial working memory. Together, the data demonstrate that deletion of SIRT3 from forebrain neurons selectively predisposes female mice to deficits in mitochondrial and cognitive function.SIGNIFICANCE STATEMENT Mitochondrial SIRT3 is an enzyme shown to regulate energy metabolism and antioxidant function, by direct deacetylation of proteins. In this study, we show that neuronal SIRT3 deficiency renders female mice selectively vulnerable to impairment in redox and metabolic function, spatial memory, and neuronal excitability. The observed sex-specific effects on cognition and neuronal excitability in female SIRT3-deficient mice suggest that mitochondrial dysfunction may be one factor underlying comorbid neuronal diseases, such as Alzheimer's disease and epilepsy. Furthermore, the data suggest that SIRT3 dysfunction may predispose females to age-related metabolic and cognitive impairment.

Ambient NO2 exposure affects hepatic glycolipid metabolism in mice with a sex-dependent property

NO 2 is one of the major air pollutants. Exposure to NO 2 is associated with various metabolic diseases, such as non-alcoholic fatty liver disease (NAFLD), diabetes, cardiovascular disease, etc. Abnormal hepatic glycolipid metabolism is a key feature of metabolic diseases. However, few studies provide evidence for the effects of NO 2 inhalation on hepatic glycolipid metabolism. In this study, female and male C57BL/6 J mice were exposed to NO 2 (2.5 ppm, 5 h/day) for 4 weeks using a systemic inhalation exposure system. The results indicated that NO 2 exposure increased the levels of hepatic enzymes in serum and led to liver dysfunction in female mice, but not male ones. Especially, female mice developed glycolipid metabolic disorders, including increased blood glucose, decreased hepatic glycogen and glucose tolerance, and increased lipid level in liver and blood. Metabolomic analysis clarified 33 differential metabolites of glycolipids, which were mainly enriched through several pathways, including synthesis and degradation of ketone bodies, fat digestion and absorption, galactose metabolism, fatty acid degradation, and glycerophospholipid metabolism. The mRNA and protein expression implicated that the exposure disrupted glucose metabolism by reducing hepatic glycogen synthesis and increasing gluconeogenesis, and caused lipid deposition by increasing lipogenesis and uptake, thereby increasing lipid oxidation and secretion. The findings highlight the risk of metabolic diseases upon exposure to NO 2 in the air. • NO 2 exposure caused female-specific hepatic glycolipid metabolism disorders. • NO 2 inhalation altered glycogen synthesis and gluconeogenesis. • NO 2 inhalation affected lipogenesis and uptake, and induced lipid deposition.

Galectin-3 protects auditory function in female mice

Sex differences in the development of sensorineural hearing loss have been recognized in various inner ear disorders, but the molecular basis for such differences is poorly understood. Autosomal genes have been shown to cause sex differences in disease susceptibility, but many genes exerting sex-dependent effects on auditory function remain to be identified. Galectin-3 (Gal-3), a protein encoded by the autosomal gene Lgals3, is a member of the β-galactoside-binding protein family, and has been linked to multiple biological processes, including immune responses, apoptosis, and cell adhesion. Here, we investigated auditory function and hair cell integrity in Gal-3 knockout (KO, Lgals3−/−) and wild-type (WT, Lgals3+/+) mice from age 1 to 6 months. KO mice show a more rapid age-related increase in ABR thresholds compared to WT mice. Noticeably, the threshold deterioration in female KO mice is significantly greater than in the male KO and WT mice. The ABR threshold elevation manifests over a broad frequency range in female KO mice, whereas the threshold elevations are confined to high frequencies in the male KO and WT mice. Moreover, DPOAE input/output functions reveal a similar pattern of auditory dysfunction, with the female KO mice displaying a significantly greater reduction in DPOAE amplitudes than male KO mice and WT mice of both sexes. Finally, age-related outer hair cell loss is greater for female KO mice compared to male KO mice and WT mice of both sexes. Together, these results indicate that Gal-3 deficiency exacerbates age-related cochlear degeneration and auditory dysfunction in female mice. Our study identifies Gal-3 as a sex-dependent molecule for maintaining female cochlear integrity.

Deficiency of ataxia-telangiectasia mutated kinase attenuates Western-type diet-induced cardiac dysfunction in female mice.

Chronic consumption of Western‐type diet (WD) induces cardiac structural and functional abnormalities. Previously, we have shown that WD consumption in male ATM (ataxia‐telangiectasia mutated kinase) deficient mice associates with accelerated body weight (BW) gain, cardiac systolic dysfunction with increased preload, and exacerbation of hypertrophy, apoptosis, and inflammation. This study investigated the role of ATM deficiency in WD‐induced changes in functional and biochemical parameters of the heart in female mice. Six‐week‐old wild‐type (WT) and ATM heterozygous knockout (hKO) female mice were placed on WD or NC (normal chow) for 14 weeks. BW gain, fat accumulation, and cardiac functional and biochemical parameters were measured 14 weeks post‐WD. WD‐induced subcutaneous and total fat contents normalized to body weight were higher in WT‐WD versus hKO‐WD. Heart function measured using echocardiography revealed decreased percent fractional shortening and ejection fraction, and increased LV end systolic diameter and volume in WT‐WD versus WT‐NC. These functional parameters remained unchanged in hKO‐WD versus hKO‐NC. Myocardial fibrosis, myocyte hypertrophy, and apoptosis were higher in WT‐WD versus WT‐NC. However, apoptosis was significantly lower and hypertrophy was significantly higher in hKO‐WD versus WT‐WD. MMP‐9 and Bax expression, and Akt activation were higher in WT‐WD versus WT‐NC. PARP‐1 (full‐length) expression and mTOR activation were lower in WT‐WD versus hKO‐WD. Thus, ATM deficiency in female mice attenuates fat weight gain, preserves heart function, and associates with decreased cardiac cell apoptosis in response to WD.

Folic Acid Preconditioning Alleviated Radiation-Induced Ovarian Dysfunction in Female Mice.

Radiological therapy/examination is the primary source of artificial radiation exposure in humans. While its application has contributed to major advances in disease diagnosis and treatment, ionizing radiation exposure is associated with ovarian damage. The use of natural products, either alone or as an adjunct, has become increasingly common for reducing the side effects of radiological therapy during disease treatment. Herein, we explored the protective effect of folic acid (FA), a widely used B vitamin, against radiation-induced ovarian injury and its mechanism of action. Female mice with normal ovarian function were randomly divided into control, FA, radiation, and radiation + FA groups. The intervention strategy included daily intragastric administration of FA (5 mg/kg) for 3 weeks prior to radiation exposure. Mice in the radiation and radiation + FA groups received a single dose of 5 Gy X-ray irradiation. Changes in the estrous cycle were then recorded, and ovarian tissues were collected. Pathophysiological changes as well as reproductive and endocrine-related indexes were determined via H&E staining, immunohistochemistry, Western blot, and ELISA. The reproductive performance and emotional symptoms of animals were also monitored. Our results indicated that FA intervention effectively alleviated ovarian damage, leading to more regular estrous cycles, lesser impairment of follicular morphology and endocrine status, as well as greater germ cell preservation. Reduced levels of oxidative stress, inflammation, and enhanced DNA repair were associated these changes. FA pre-administration improved the reproductive performance, leading to higher pregnancy rates and greater litter sizes. Further, the anxiety levels of animals were significantly reduced. Our results indicate that FA pre-administration significantly alleviates radiation-induced ovarian damage in rodents, highlighting its potential as a protective strategy against radiation exposure in the female population.

Phenotypic differences based on lateralization of intrahippocampal kainic acid injection in female mice

Clinical evidence indicates that patients with temporal lobe epilepsy (TLE) often show differential outcomes of comorbid conditions in relation to the lateralization of the seizure focus. A particularly strong relationship exists between the side of seizure focus and the propensity for distinct reproductive endocrine comorbidities in women with TLE. Therefore, here we evaluated whether targeting of left or right dorsal hippocampus for intrahippocampal kainic acid (IHKA) injection, a model of TLE, produces different outcomes in hippocampal granule cell dispersion, body weight gain, and multiple measures of reproductive endocrine dysfunction in female mice. One, two, and four months after IHKA or saline injection, in vivo measurements of estrous cycles and weight were followed by ex vivo examination of hippocampal dentate granule cell dispersion, circulating ovarian hormone and corticosterone levels, ovarian morphology, and pituitary gene expression. IHKA mice with right-targeted injection (IHKA-R) showed greater granule cell dispersion and pituitary Fshb expression compared to mice with left-targeted injection (IHKA-L). By contrast, pituitary expression of Lhb and Gnrhr were higher in IHKA-L mice compared to IHKA-R, but these values were not different from respective saline-injected controls. IHKA-L mice also showed an increased rate of weight gain compared to IHKA-R mice. Increases in estrous cycle length, however, were similar in both IHKA-L and IHKA-R mice. These findings indicate that although major reproductive endocrine dysfunction phenotypes present similarly after targeting left or right dorsal hippocampus for IHKA injection, distinct underlying mechanisms based on lateralization of epileptogenic insult may contribute to produce similar emergent reproductive endocrine outcomes.

Effects of low-dose oxygen ions on cardiac function and structure in female C57BL/6J mice

Purpose: Astronauts in space vehicles beyond low-Earth orbit will be exposed to high charge and energy (HZE) ions, and there is concern about potential adverse effects on the cardiovascular system. Thus far, most animal studies that assess cardiac effects of HZE particles have included only males. This study assessed the effects of oxygen ions (16O) as a representative ion of the intravehicular radiation environment on the heart of female mice. Materials and methods: Female C57BL/6 J mice at 6 months of age were exposed to 16O (600 MeV/n) at 0.25−0.26 Gy/min to a total dose of 0, 0.1, or 0.25 Gy. Cardiac function and abdominal aorta blood velocity were measured with ultrasonography at 3, 5, 7, and 9 months after irradiation. At 2 weeks, 3 months, and 9 months, cardiac tissue was collected to assess collagen deposition and markers of immune cells. Results: Ultrasonography revealed increased left ventricle mass, diastolic volume and diameter but there was no change in the abdominal aorta. There was no indication of cardiac fibrosis however, a 75 kDa peptide of left ventricular collagen type III and α-smooth muscle cell actin were increased suggesting some remodeling had occurred. Left ventricular protein levels of the T-cell marker CD2 was significantly increased at all time points, while the neutrophil marker myeloperoxidase was decreased at 2 weeks and 9 months. Conclusions: These results taken together suggest 16O ion exposure did not result in cardiac fibrosis or cardiac dysfunction in female mice. However, it does appear mild cardiac remodeling occurs in response to HZE radiation.

The second-generation antipsychotic drug aripiprazole modulates the serotonergic system in pancreatic islets and induces beta cell dysfunction in female mice

Aims/hypothesisSecond-generation antipsychotic (SGA) drugs have been associated with the development of type 2 diabetes and the metabolic syndrome in patients with schizophrenia. In this study, we aimed to investigate the effects of two different SGA drugs, olanzapine and aripiprazole, on metabolic state and islet function and plasticity.MethodsWe analysed the functional adaptation of beta cells in 12-week-old B6;129 female mice fed an olanzapine- or aripiprazole-supplemented diet (5.5–6.0 mg kg−1 day−1) for 6 months. Glucose and insulin tolerance tests, in vivo glucose-stimulated insulin secretion and indirect calorimetry were performed at the end of the study. The effects of SGAs on beta cell plasticity and islet serotonin levels were assessed by transcriptomic analysis and immunofluorescence. Insulin secretion was assessed by static incubations and Ca2+ fluxes by imaging techniques.ResultsTreatment of female mice with olanzapine or aripiprazole for 6 months induced weight gain (p<0.01 and p<0.05, respectively), glucose intolerance (p<0.01) and impaired insulin secretion (p<0.05) vs mice fed a control chow diet. Aripiprazole, but not olanzapine, induced serotonin production in beta cells vs controls, likely by increasing tryptophan hydroxylase 1 (TPH1) expression, and inhibited Ca2+ flux. Of note, aripiprazole increased beta cell size (p<0.05) and mass (p<0.01) vs mice fed a control chow diet, along with activation of mechanistic target of rapamycin complex 1 (mTORC1)/S6 signalling, without preventing beta cell dysfunction.Conclusions/interpretationBoth SGAs induced weight gain and beta cell dysfunction, leading to glucose intolerance; however, aripiprazole had a more potent effect in terms of metabolic alterations, which was likely a result of its ability to modulate the serotonergic system. The deleterious metabolic effects of SGAs on islet function should be considered while treating patients as these drugs may increase the risk for development of the metabolic syndrome and diabetes.Graphical abstract

1148-P: Liver Androgen Receptor Knockout Delays the Onset of Diet-Induced Metabolic Dysfunction in Female Mice Compared with Male Mice

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)

6:2 Cl-PFESA has the potential to cause liver damage and induce lipid metabolism disorders in female mice through the action of PPAR-γ

6:2 Cl-PFESA is a polyfluoroalkyl ether with high environmental persistence that has been confirmed to have significant adverse effects on animals. In this study, 6-week-old female C57BL/6 mice were exposed to 0, 1, 3 and 10 μg/L 6:2 Cl-PFESA for 10 weeks to estimate the hepatotoxicity of 6:2 Cl-PFESA and explore its underlying molecular mechanism. The results indicated that 6:2 Cl-PFESA preferentially bioaccumulated in the liver and induced hepatic cytoplasmic vacuolation and hepatomegaly in mice. In addition, serum metabolic profiling showed that 6:2 Cl-PFESA exposure caused an abnormal increase in amino acids and an abnormal decrease in acyl-carnitine, which interfered with fatty acid transport and increased the risk of metabolic diseases. Further experiments showed that 6:2 Cl-PFESA formed more hydrogen bonds with PPAR-γ than PFOS, Rosi and GW9662, and the binding affinity of 6:2 Cl-PFESA toward PPAR-γ was the highest among the ligands. 6:2 Cl-PFESA promoted the differentiation of 3T3-L1 cells by increasing PPAR-γ expression. Therefore, our results showed that 6:2 Cl-PFESA has the potential to induce liver damage and dysfunction in female mice, and this effect was achieved through PPAR-γ. This study is the first to reveal the hepatic toxicity of 6:2 Cl-PFESA in female mammals and provides new insights for subsequent in-depth research.

Liver Androgen Receptor Knockout Delays the Onset of Diet Induced Metabolic Dysfunction in Female Mice Compared to Male Mice

Polycystic ovarian syndrome (PCOS) is a hormonal disorder with insulin resistance (IR) and hyperandrogenism (HA) as drivers of the condition. Women diagnosed with PCOS have an increased risk of developing non-alcoholic fatty liver disease (NAFLD). Our lab explores the condition of 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 male and female 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. In contrast to females, at 1-month of diet, 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. Further molecular analysis is warranted.

Mineralocorticoid Receptor in Myeloid Cells Mediates Angiotensin II-Induced Vascular Dysfunction in Female Mice

Enhanced mineralocorticoid receptor (MR) signaling is critical to the development of endothelial dysfunction and arterial stiffening. However, there is a lack of knowledge about the role of MR-induced adipose tissue inflammation in the genesis of vascular dysfunction in women. In this study, we hypothesize that MR activation in myeloid cells contributes to angiotensin II (Ang II)-induced aortic stiffening and endothelial dysfunction in females via increased pro-inflammatory (M1) macrophage polarization. Female mice lacking MR in myeloid cells (MyMRKO) were infused with Ang II (500 ng/kg/min) for 4 weeks. This was followed by determinations of aortic stiffness and vasomotor responses, as well as measurements of markers of inflammation and macrophage infiltration/polarization in different adipose tissue compartments. MyMRKO mice were protected against Ang II-induced aortic endothelial stiffening, as assessed via atomic force microscopy in aortic explants, and vasorelaxation dysfunction, as measured by aortic wire myography. In alignment, MyMRKO mice were protected against Ang II-induced macrophage infiltration and M1 polarization in visceral adipose tissue (VAT) and thoracic perivascular adipose tissue (tPVAT). Collectively, this study demonstrates a critical role of MR activation in myeloid cells in the pathogenesis of vascular dysfunction in females associated with pro-inflammatory macrophage polarization in VAT and tPVAT. Our data have potential clinical implications for the prevention and management of cardiovascular disease in women, who are disproportionally at higher risk for poor outcomes.

Abstract P60: Xanthine Oxidase Mediates Cerebrovascular Dysfunction in Chronically Stressed Female Mice

Chronic stress is a risk factor for many cerebrovascular diseases including stroke and vascular dementia. Our lab has preciously shown that chronic stress in male mice and rats reduces cerebrovascular function, mediated by an increase in oxidative stress, potentially via the xanthine oxidase pathway. However, the role of xanthine oxidase on cerebrovascular function in female mice has yet to be explored. Here, we examined the effect of chronic stress on middle cerebral artery function and xanthine oxidase expression in female mice. At 18 weeks of age, mice underwent the unpredictable chronic mild stress (UCMS; 5days/week for 7hrs/day) paradigm to elicit a chronic stress phenotype. On week 26, mice were euthanized and the middle cerebral arteries (MCA) were removed and positioned in a pressurized myobath. To test for vessel health, the MCA was exposed to increasing concentrations of acetylcholine (Ach; 10-9M to 10-4M) in the presence or absence of febuxostat (10uM), a xanthine oxidase inhibitor as well as increasing doses of phenylephrine (PE; 10-9M to 10-4M) and sodium nitroprusside (SNP; 10-9M to 10-4M). Coronal sections of brain tissue were then stained for the xanthine oxidase enzyme, imaged using a Nikon Eclipse 800 and fluorescence intensity was analyzed via ImageJ. MCA dilation to Ach was impaired in female mice that underwent UCMS compared to control mice (9.5±1um vs 20±1um; p&lt;0.05). Incubating the MCA with febuxostat had no effect on control mice but improved MCA dilation in the UCMS mice (9.5±1um to 17±0.5um; p&lt;0.05). No differences were found between control vs UCMS groups in response to PE or SNP. To support this, preliminary data (n=3-4/group) shows a trend of increased fluorescence intensity of xanthine oxidase expression within the somatosensory cortex of the mice exposed to UCMS compared to controls (41,666±2,923AU vs 31,091±3,759AU, respectively; p=0.059). Together, these data suggest increased xanthine oxidase activity plays a role in cerebrovascular dysfunction in female mice.

Deletion of P21-activated kinase-1 induces age-dependent increased visceral adiposity and cardiac dysfunction in female mice.

It is known that there is an age-related progression in diastolic dysfunction, especially prevalent in postmenopausal women, who develop heart failure with preserved ejection fraction (HFpEF, EF > 50%). Mechanisms and therapies are poorly understood, but there are strong correlations between obesity and HFpEF. We have tested the hypothesis that P21-activated kinase-1 (PAK1) preserves cardiac function and adipose tissue homeostasis during aging in female mice. Previous demonstrations in male mice by our lab that PAK1 activity confers cardio-protection against different stresses formed the rationale for this hypothesis. Our studies compared young (3-6months) and middle-aged (12-15months) female and male PAK1 knock-out mice (PAK1-/-) and wild-type (WT) equivalent. Female WT mice exhibited increased cardiac PAK1 abundance during aging. By echocardiography, compared to young WT female mice, middle-aged WT female mice showed enlargement of the left atrium as well as thickening of posterior wall and increased left ventricular mass; however, all contraction and relaxation parameters were preserved during aging. Compared to WT controls, middle-aged PAK1-/- female mice demonstrated worsening of cardiac function involving a greater enlargement of the left atrium, ventricular hypertrophy, and diastolic dysfunction. Moreover, with aging PAK1-/- female mice, unlike male PAK1-/- mice, exhibited increased adiposity with increased accumulation of visceral adipose tissue. Our data provide evidence for the significance of PAK1 signaling as an element in the preservation of cardiac function and adipose tissue homeostasis in females during aging.

A Sex-Specific Role of Endothelial Sirtuin 3 on Blood Pressure and Diastolic Dysfunction in Female Mice.

Background: Heart failure with preserved ejection fraction (HFpEF) is characterized by a diastolic dysfunction and is highly prevalent in aged women. Our study showed that ablation of endothelial Sirtuin 3 (SIRT3) led to diastolic dysfunction in male mice. However, the sex-specific role of endothelial SIRT3 deficiency on blood pressure and diastolic function in female mice remains to be investigated. Methods and Results: In this study, we demonstrate that the ablation of endothelial SIRT3 in females elevated blood pressure as compared with control female mice. Diastolic function measurement also showed that the isovolumic relaxation time (IVRT) and myocardial performance index (MPI) were significantly increased, whereas the E’ velocity/A’ velocity (E’/A’) ratio was reduced in the endothelial-specific SIRT3 knockout (SIRT3 ECKO) female mice. To further investigate the regulatory role of endothelial SIRT3 on blood pressure and diastolic dysfunction in metabolic stress, SIRT3 ECKO female mice were fed a normal diet and high-fat diet (HFD) for 20 weeks. The knockout of endothelial SIRT3 resulted in an increased blood pressure in female mice fed with an HFD. Intriguingly, SIRT3 ECKO female mice + HFD exhibited impaired coronary flow reserve (CFR) and more severe diastolic dysfunction as evidenced by an elevated IVRT as compared with control female mice + HFD. In addition, female SIRT3 ECKO mice had higher blood pressure and diastolic dysfunction as compared to male SIRT3 ECKO mice. Moreover, female SIRT3 ECKO mice + HFD had an impaired CFR and diastolic dysfunction as compared to male SIRT3 ECKO mice + HFD. Conclusions: These results implicate a sex-specific role of endothelial SIRT3 in regulating blood pressure and diastolic function in mice. Deficiency of endothelial SIRT3 may be responsible for a diastolic dysfunction in aged female.