Sex-dependent Manner Research Articles

Prefrontal-medullary Circuitry Impacts Sex-specific Microvascular Function Following Chronic Stress

Vascular dysfunction is a prominent factor underlying cardiovascular disease, the leading cause of death worldwide. Additionally, chronic stress contributes to cardiovascular pathologies in a sex-dependent manner. However, the neurobiological mechanisms mediating the sex-specific impact of chronic stress on vascular function are not known. Our previous studies found that the prefrontal infralimbic cortex (IL)-rostral ventrolateral medulla (RVLM) circuit inhibits sympathetic and endocrine responses to stress; therefore, the current study tested the hypothesis that the IL-RVLM circuit is necessary to mitigate microvascular dysfunction following chronic stress. To this end, an intersectional genetic approach was used to induce Cre-dependent expression of tetanus toxin light chain and inhibit neurotransmitter release from RVLM-projecting IL neurons in male and female rats. Rats were then exposed to 2 weeks of chronic variable stress (CVS) or remained unstressed. Left ventricle structure was assessed by echocardiography and microvascular function was subsequently assessed by resistance arteriole pressure myography. Arteriole myogenic tone, endothelial-independent vasoconstriction, as well as endothelial-dependent vasodilation and vasoconstriction were determined at both normotensive (70 mmHg) and hypertensive (120 mmHg) pressures. Our results indicate that heart size and ventricular thickness were impacted in a sex-dependent manner. Circuit inhibition increased heart index in females regardless of stress. In males, circuit inhibition reduced ventricular wall thickness in diastole. Microvasculature also exhibited sex-dependent function. Following CVS, males had increased myogenic tone compared to females. Circuit inhibition also differentially impacted vasodilation in males and females. In chronically stressed females, circuit inhibition increased endothelial-dependent vasodilation at both normotensive and hypertensive pressures and normotensive endothelial-independent relaxation. These changes indicate that chronic stress leads to female microvascular dysfunction that is exacerbated by IL-RVLM inhibition. Chronically stressed males had no changes in normotensive vasodilation. However, with elevated pressure, circuit-inhibited CVS males had increased endothelial-dependent vasodilation. In contrast, unstressed males with circuit inhibition had decreased endothelial-dependent vasodilation at 120 mmHg, indicating that the male IL-RVLM circuit may be necessary for vascular function during acute hypertensive events. Broadly, these results support the hypothesis that the IL-RVLM circuit is necessary for appropriate vascular function and that circuit inhibition and chronic stress lead to sex-specific responses that may differentially impact the progression of cardiovascular disease. NIH R01 HL150559 (B.Myers) NIH F30 OD032120 (C. Dearing). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex Dependent Exacerbation of Salt Sensitive Hypertension and Cardiac Dysfunction Is Associated With Upregulation of miRNA Targeting ADAM17 and ACE2 in the Brain

Background: The DOCA-salt model has been widely used to study salt-sensitive hypertension and we previously reported that it is associated with ADAM17-mediated ACE2 shedding in the central nervous system. However, previously reported data are mainly from male mice, and it is unclear whether female mice exhibit the same phenotypes. Objective: We aimed to compare sex specific development of salt sensitive hypertension and cardiac dysfunction and investigate miRNA regulation associated with these changes. Methods: C57 mice (male and females, n = 8 -15) underwent uninephrectomy and telemetry implantation (DSI Inc.) for blood pressure (BP) conscious recording. After baseline recording, a DOCA pellet (50 mg, 21 days, Innovative Research of America) was implanted subcutaneously and drinking water was replaced by 1% NaCl solution to induce salt sensitive hypertension. Cerebral Spinal fluid (CSF) was collected at baseline and after 3 weeks of DOCA-salt treatment for exosome miRNA sequencing (Creative Biolab). In another set of mice, cardiac function was assessed by echocardiography (Visualsonics Vevo F2) and invasive left ventricular hemodynamics (Transonic). Mortality from each group was analyzed as well. Results: Baseline BP was not different between male and female mice. Following DOCA-salt treatment for 3 weeks, BP was increased in both sexes, with higher systolic BP observed in males compared to females (active phase: 173.0 ± 19.6 vs. 154.2 ± 19.1 mmHg, p<0.05). Although ejection fraction was not changed in either male or female mice, mitral peak early diastolic filling velocity and tissue doppler showed that E/E’ was increased in both sexes compared to baselines (Male: 33.4 ± 4 vs. 26.6 ± 1.9; Female: 30.3 ± 5.0 vs. 26.4 ± 4), with a greater increase in males (25.3% vs. 14.4%). In addition, hemodynamics revealed increased left ventricular end-diastolic pressure (11.1 ± 1.9 vs. 4.5 ± 0.5 mmHg, p<0.01) and relaxation time constant Tau (6.7 ± 1.0 vs. 4.9 ± 0.5 msec, p<0.01) in DOCA-salt treated males compared to non-treated controls, with no changes shown in female mice. These data suggest worsened cardiac diastolic function in males compared to females following DOCA-salt treatment. Moreover, a 20% mortality was observed in males while no death occured in females, confirming a more severe phenotype in males. Furthermore, miRNAseq analysis of CSF exosomes showed several upregulated miRNA in males but not in females following DOCA-salt treatment. Notably, miR206 associated with increased ADAM17 activity was increased by 8-fold in males but only 3-fold n females. Conclusion: DOCA-salt treatment promotes exacerbated hypertension, impaired cardiac function and mortality in a sex dependent manner, associated with upregulation of key miRNA targeting the brain renin-angiotensin system. The changes of ADAM17 and ACE2 targeted exosome miRNAs in the central nervous system might contribute to the sex difference in the cardiovascular dysfunction phenotypes in this model. This work was supported in part by a research grant from the National Institutes of Health (HL163588). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

SerpinA3N in Leptin Receptor Neurons Regulates Metabolic and Glucose Homeostasis

Obesity and associated conditions such as type 2 diabetes are among the most concerning issues for health in the U.S. Leptin receptor signaling in the brain plays a pivotal role in the regulation of food intake and energy expenditure. High fat diet induces inflammation within the hypothalamus, leading to leptin insensitivity which contribute to obesity. Previous studies have shown that a novel gene SerpinA3N, a serine protease inhibitor, which is highly expressed in the arcuate nucleus of the hypothalamus, where the leptin receptor is also highly expressed, is upregulated in diet-induced obesity and by leptin stimulation. However, the role of SerpinA3N in the leptin receptor expressing cells in the control of body weight and glucose homeostasis remain unknown. RNAseq analysis showed that SerpinA3N mRNA levels were significantly upregulated in the hypothalamus of mice fed high fat high sucrose diet (HFHSD) for 12 weeks. Upregulation of hypothalamic SerpinA3N in HFHSD fed mice was confirmed by Western blot. This increase in hypothalamic SerpinA3N appears to be caused by excess of nutrient as we found that exposure to fatty acid significantly increased SerpeinA3N promoter activity in mouse hypothalamic N39 cells. Using immunofluorescence staining, we confirmed that SerpinA3N is expressed the leptin receptor positive cells of the hypothalamus. Next, we investigated whether loss of SerpinA3N in leptin receptor expressing neurons affects metabolic and glucose homeostasis. For this, we crossed mice harboring floxed alleles of the SerpinA3N gene (SerpinA3Nfl/fl) with mice expressing Cre recombinase in leptin receptor cells (LepRCre). We found that female, but not male, conditional knockout (CKO, LepRCre/SerpinA3Nfl/fl) mice have significantly lower body weight when fed normal chow or HFHSD. This lower body weight was due to decreased fat mass, measured by NMR. However, no alteration in food intake was observed between female CKO mice and controls, indicating that increased energy expenditure may account for the lower body weight in female CKO mice. Interestingly, CKO mice displayed exaggerated weight loss and anorectic response following leptin treatment (1 mg/g, twice daily, IP), indicating that loss of SerpinA3N enhances leptin sensitivity. Female CKO mice fed normal chow diet also exhibited enhanced insulin sensitivity compared to control animals. On the other hand, male CKO mice displayed significantly improved glucose handling, but not change in insulin sensitivity, only when fed HFHSD. Finally, insulin-induced phosphorylation of AKT was elevated in skeletal muscle, but not in liver and white adipose tissue, of CKO mice relative to controls. These results demonstrate that SerpinA3N in leptin receptor-expressing neurons regulate body weight and glucose homeostasis in a sex-dependent manner. NIH and VA. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Adrenal circadian clock protein BMAL1 regulates aldosterone and renal excretory function in a sex- and time-dependent manner

BMAL1 is a core circadian clock protein that is important for circadian rhythms of physiological function, as well as maintaining physiological homeostasis, including aspects of kidney function. Global Bmal1 knockout (KO) male mice exhibit loss of diurnal rhythm of renal sodium (Na) excretion in response to a normal or high salt diet. However, renal Na excretion rhythm remained intact in kidney-specific Bmal1 KO male mice. The adrenal gland synthesizes hormones, such as aldosterone, which influence renal electrolyte handling. The role of adrenal BMAL1 in the regulation of renal function remains unknown. The study objective was to test the hypothesis that adrenal BMAL1 is required for normal rhythms of aldosterone and renal Na handling. Male and female adrenal-specific aldosterone synthase Cre positive (AS) Bmal1 KO and littermate Cre negative, floxed control mice ( n=5-9/genotype) were acclimated to metabolic cages for 3 days then samples were collected for three days with mice on a normal salt diet followed by 7 days on a low salt diet to challenge the mice to stimulate aldosterone production. Twelve-hour food intake was measured, and urine samples were collected to measure night/day patterns in renal Na balance. Urinary aldosterone excretion was measured by ELISA. Both day and night urinary aldosterone excretion were increased following a low salt diet in control and KO male mice (ANOVA main effect of diet, p<0.05). Male AS Bmal1 KO mice displayed a significant increase in day urinary aldosterone excretion following a normal and low salt diet (ANOVA main effect of genotype, p=0.0230), with a trend for an increase in night urinary aldosterone excretion ( p=0.0571). Interestingly, these genotype effects were not seen in female AS Bmal1 KO mice. Urinary aldosterone was increased following a low salt diet in female mice, but only during the nighttime in control and KO mice ( p=0.0205). Sex differences were also observed in renal Na balance, where Na balance was more positive during the day in male, but not female, AS Bmal1 KO vs. control mice (ANOVA main effect of genotype, p=0.0282). Together, these data suggest adrenal BMAL1 is required for normal rhythms of aldosterone and renal Na balance in a sex-dependent manner. Future work will investigate whether the changes in aldosterone contribute to changes in renal Na balance. This work was supported by the American Heart Association (Postdoctoral FellowshipP0240441 to H.C.; Established Investigator Award 19EIA34660135 to M.G), theRobert and Mary Cade Professorship in Physiology (to M.G.) and the NIH(F32DK121424 to G.R.C.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Metabolic dysfunction is associated with alterations in gut microbiota in obese adolescents

Abstract The gut microbiota (MB) has been implicated in adult metabolic dysfunction given evidence of dysbiosis in obesity and type 2 diabetes. However, few studies have investigated the relationship between metabolic disease, inflammation and the gut microbiota in children. We characterized the gut MB and immunometabolic profile of a cohort of obese, non-diabetic adolescents (MI4D) using high throughput fecal DNA sequencing with absolute bacterial quantitation. In the absence of antibiotics, we observed multi-log differences in fecal biomass correlated with stool water content and MB composition. Moreover, fecal bacterial biomass was associated with metabolic measures including serum triglycerides and insulin resistance in a sex-dependent manner. Mass cytometry demonstrated no significant associations of major immune cell subsets to fecal biomass or metabolic markers. In contrast, there were male-specific associations between serum markers of systemic inflammation and neutrophil activity with fecal biomass and waist circumference. Given the infiltration of visceral adipose tissue by myeloid cells in metabolic inflammation, our findings suggest that lower fecal biomass is associated with elevated systemic inflammation, especially in obese adolescent males. This study expands the understanding of sex-specific mechanism driving metabolic dysfunction in obese youth and suggests that gut MB alterations contribute to their metabolic and inflammatory dysregulation before diabetes diagnosis.

Sex-dependent impairment of kidney function in megalin knockout mice

Megalin ( Lrp2) is a multiligand receptor that drives endocytic flux in the kidney proximal tubule (PT) and is necessary for the recovery of albumin and other filtered proteins that escape the glomerular filtration barrier. We previously found that knocking out Lrp2 in the opossum kidney (OK) PT cell line leads to differential expression of genes involved in mitochondrial and metabolic function. Additionally, we observed large reductions in sodium glucose co-transporter 2 (Sglt2) transcript (>80%) and protein (~40%) expression levels. While Sglt2 transcript and protein levels in Lrp2 KO in mice were more modestly reduced, male and female KO mice fed regular chow or a Western diet (WD) had increased tolerance to a high glucose challenge compared with control mice. Surprisingly, male Lrp2 KO mice fed a WD developed substantial kidney injury, while female KO mice had significantly less injury. In metabolic studies, Lrp2 KO mice preferentially utilized fatty acid oxidation compared to control mice. We hypothesize that compromised endocytic flux in Lrp2 KO mice impairs mitochondrial and metabolic function to worsen kidney damage in a sex-dependent manner. Current studies are focused on a variety of approaches to test how reduced endocytic capacity impacts mitochondrial function and metabolism in Lrp2 KO cells and control OK cells. We will confirm our findings in age-matched male and female control and Lrp2 KO mice. These studies will help to determine the role of megalin expression and function on PT metabolism, function, and overall health. National Institutes of Health: T32DK061296, T32AG021885-20, RO1 DK125049, RO1 DK118726, S10 OD028596, U54 DK137329. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Is cerebral blood flow altered by anxiety and depression in a sex-dependent manner?

Background: It is well-known that premenopausal females have greater cerebral blood flow (CBF) than age-matched males. People with anxiety and/or depression (A/D) have altered CBF compared to those without A/D. For example, males with depression may have greater CBF, and females with depression may have lower CBF, than their healthy counterparts. In contrast, anxiety may be correlated with higher CBF in certain brain regions such as the amygdala and hippocampus in both sexes. However prior studies assessing the effect of sex and A/D on CBF did not control for the menstrual cycle, something which is known to influence CBF in females. Objective: To determine if A/D is linked to CBF alterations in young healthy adults, in a sex-specific pattern, while controlling for menstrual cycle and hormonal contraception (HC). Hypothesis: As most of our subjects had depression, we hypothesized that CBF would be higher in control (CON) females than CON males and this difference will be abolished in A/D subjects, due to a greater CBF in A/D males compared to CON males and a lower CBF in A/D females compared to CON females. Methods: Twenty females (4 A/D) and 21 males (8 A/D) had their CBF (ml/100gm/min) measured using magnetic resonance imaging (MRI, 3T) with arterial spin labelling (ASL). A/D was diagnosed by a physician. Subjects in all 4 groups were matched for age (mean ± SD; 21 ± 2 y). Females were studied during days 1-5 of the menstrual cycle and were not taking HC. CBF data were tested using 2-way mixed effects analyses (sex*group (i.e., CON vs. A/D)) in: grey matter (GM), white matter (WM), and 8 emotional/memory regions of the brain, including the accumbens, amygdala, caudate, anterior cingulate, hippocampus, entorhinal, parahippocampus, and precuneus. Results: A trend for significant sex*group interaction (all p ≥ 0.052) but not group main effects (all p ≥ 0.100) was observed. CBF in GM and WM as well as in the 8 emotional/memory regions was higher in females compared to males (main effect of sex, p ≤ 0.004). This sex effect appeared to be driven by the CON groups ( p < 0.0001 for all areas), as there were no CBF differences in any area between males and females in the A/D group (all p ≥ 0.089).Exploratory analyses showed significantly greater CBF in A/D males compared to CON males in GM as well as the accumbens, caudate, hippocampus, precuneus, and anterior cingulate gyrus (all p ≤ 0.049). No differences were observed for any outcome. Conclusion: These data suggest that while CON females have greater CBF than CON males at rest, this sex difference may be abolished in those with A/D. Albeit preliminary, these data are in line with previous findings whereby CBF appears to decrease more in females with depression and either remain unchanged or increase in males with depression. The higher CBF in certain regions in A/D males is interesting and in line with previous research showing increased CBF in the hippocampus with anxiety. As our sample size was small and included a mix of people with either anxiety or depression, future research should be conducted to assess the differential effects of these conditions in young adults. Support by NIH (HL150361). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Maternal Limosilactobacillus reuteri rescues social and emotional recognition behavior in an environmental mouse model of autism

Introduction: Polybrominated diphenyl ethers (PBDEs) are thyroid disrupting chemicals with adverse neurodevelopmental effects in children. Our group has previously shown that PBDEs produce autism (ASD)-like traits (Kozlova et al. 2022 Arch Tox; PMID: 34687351) in combination with reduced hypothalamic oxytocin expression in developmentally exposed female mice offspring. Limosilactobacillus reuteri (LR) is a beneficial probiotic that can increase oxytocin and thyroid hormones (THs) in other ASD mouse models. Therefore, we hypothesized that LR would ameliorate PBDE-induced deficits in social behavior, oxytocin and thyroid status. Methods: C57BL/6N dams (n=8/group) were dosed with a commercial mixture of PBDE congeners, DE-71, at 0.1 mg/kg/d (DE-71), or corn oil vehicle control (VEH/CON) during preconception, gestation and lactation). Dams were gavaged with L. reuteri (LR) ATCC-PTA-6475 at 107-108 CFU/mL (gift of Biogaia, Sweden). Cohort 1 pups (C1) received LR via dam until weaning (PND 21). Cohort 2 pups (C2) continued to receive gavage of LR post-weaning, daily, through 6 mo of age. Results: Fecal microbiome analysis confirmed colonization of LR in dams after 25 doses and their offspring at postnatal day 22 and 40. Beta diversity PCoA using unweighted Unifrac revealed differences in microbial communities in 6 month old female (p<.05-.01) and male offspring (apparent) between DE-71 and VEH/CON (C2). LR treatment normalized DE-71-induced abnormal social novelty preference (30 min retention) in adult male (p<.01) and females (p<.05) and long-term social recognition memory (24 h retention) in adult C2 females (p<.001). Using an emotional recognition task DE-71-exposed C2 male and female offspring were unable to distinguish between emotional state of stimulus mice but LR supplementation normalized the DE-71-induced deficits in C2 males (p<.01) and females (p<.05). On a marble burying test DE-71 exposed offspring showed abnormally elevated scores compared to VEH/CON, which were normalized by LR treatment only in C1 females (p<.05). In females, maternal LR treatment ameliorated DE-71-induced locomotor hyperactivity in C1. Since social behavior involves discrimination between social odors we also investigated the effects of DE-71 and LR treatment on olfactory discrimination ability. LR treatment ameliorated DE-71 effects compared to VEH/CON, i.e., reduced dishabituation from social odor 1 to 2 in males and female (p<.0001). Single molecule in situ hybridization performed on paraventricular hypothalamic nucleus (PVN) sections revealed upregulation of thyroid hormone transporter monocarboxylate transporter 8 (Mct8) and downregulation of iodothyronine deiodinase 3 (Dio3) on oxytocin-positive neurons in DE-71 vs VEH/CON in C2 females. Dio3 expression was normalized in DE-71+LR. Conclusions: These results indicate that early life exposure to PBDEs alters thyroid hormone system genes in PVN neurons that express oxytocin, suggesting that PBDEs may alter ASD-relevant behavior via brain thyroid signaling. In addition, maternal LR supplementation provides normalization in a sex-dependent manner. This work is supported by a Danone North America Gut Microbiome, Yogurt and Probiotics Fellowship Grant and a University of California President's Pre-Professoriate Fellowship to E.V.K and an NIH/NIGMS R35GM124724 to A.H. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deletion of Myostatin Significantly and Preferentially Improves Muscle Function in a Sex-Dependent Manner in Both an Obese and Aging Mouse Model

Skeletal muscle mass and function are essential components of health and homeostasis. In addition to its role in balance and locomotion, skeletal muscle is the body’s largest metabolic reservoir, absorbing significant amounts of glucose and amino acids from the blood stream. The loss of muscle mass is associated with impaired glucose tolerance, threatens vascular health and contributes to cardiovascular disease in obese and aging (sarcopenic) populations, along with driving overall mortality. The myokine myostatin (GDF-8) is a potent negative regulator of skeletal muscle growth that is upregulated in humans and animal models of obesity and aging. Loss of myostatin is known to increase the number of glycolytic muscle fibers and has been shown to correct indices that govern cardiometabolic health in obesity and aging, improving endothelial function and glucose homeostasis, and protecting against renal injury and hypertension. However, these studies have largely been conducted using male mice, leaving the question regarding the effectiveness of myostatin deletion on skeletal muscle function, independent of sex, largely unexplored. As such, our hypothesis is that the deletion of myostatin will improve skeletal muscle function in both male and female mice, regardless of the disease phenotype being obesity or aging. Young (3-5 mo.) lean and obese mice were used, along with aged (18-24 mo.) mice. The obese db/db model was utilized as it is a well characterized model of hyperphagia that results in obesity and subsequent metabolic disease, including significant whole-body adiposity and reduced muscle mass. On this background we have crossed mice harboring constitutive genetic deletion of myostatin to reverse muscle loss. Thus, myostatin deletion was crossed onto the relevant backgrounds in combination with aging or obesity and included both sexes. Muscle function was assessed in all groups using in vivo plantarflexion. Young male mice demonstrated an ~20% increase in force production compared to the females. Results show that aging and obesity inhibited skeletal muscle function in both sexes, with female mice showing a lessened impact of obesity or aging. Myostatin deletion improved muscle function in male mice, returning both obesity-derived weakness and aging-induced sarcopenia back to levels of young control. However, female mice without myostatin experienced a relative gain-of-function, with force production significantly exceeding that of the young control. The implication of these results is that weight loss may be more effective in protecting muscle function in diseases in the male population whereas myostatin inhibition, either pharmaceutically or with prescription exercise, may result in greater improvements in the female sex comparatively. This research gratefully acknowledges support from the NIA (K01 AG064121), OCAST (HR21-045-1) and the Niblack Research Scholars Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex-Dependent Effects of Angiotensin Type 2 Receptor Expressing Medial Prefrontal Cortex (mPFC) Interneurons in Fear Extinction Learning

Background: The renin-angiotensin system (RAS) has been identified as a potential therapeutic target for PTSD, though its mechanisms are not well understood. Brain angiotensin type 2 receptors (AT2Rs) are a subtype of angiotensin II receptors located in stress and anxiety-related regions, including the medial prefrontal cortex (mPFC). Their function and mechanism in the mPFC, however, remain unexplored. We therefore used a combination of neuroanatomical, chemogenetic, and behavioral methods to investigate mPFC-AT2R-expressing neuron involvement in fear learning. Methods: To characterize mPFC-AT2R-expressing neurons in the mPFC, AT2R-Cre/td-Tomato male and female brains were perfused and used for immunohistochemistry. Brain sections were stained with glutamatergic or interneuron markers, and density of AT2R+ cells and colocalization with each marker was quantified. To assess fear-related behaviors in AT2R-flox mice, we selectively deleted AT2R from mPFC neurons using an AAV-Cre or GFP virus. Mice then underwent Pavlovian auditory fear conditioning and anxiety-like and locomotor behavior testing using open field (OF) and elevated plus maze (EPM) tests. Results: IHC revealed that AT2R is expressed throughout the mPFC in males ( 208.6±48.8 cells/mm2) and females ( 139.0±59.8 cells/mm2) and has low co-expression on glutamatergic neurons ( TBR1 co-staining, males: 7.1%±1.8%; females: 13.2%±4.2%). Of the interneuron markers tested, AT2R is primarily expressed on somatostatin interneurons, and has higher colocalization in the mPFC of females than males ( males: 16.5%±1.2%, females: 31.8%±4.2%, p=0.02). Following fear conditioning and extinction, mPFC-AT2R deletion impaired extinction in female ( p=0.03) but not male ( p=0.76) mice. Locomotion in the OF was unaltered by mPFC-AT2R deletion in males or females ( total distance travelled; males: GFP 44.11m±5.92m, Cre 56.00m±10.29m, p=0.35; females: GFP 42.86m±4.85m, Cre 42.39m±4.97m, p=0.95), while AT2R-deleted females had increased exploratory behavior in the EPM ( open arm entries; males: GFP 12.38±1.02, Cre 13.22±1.54, p=0.66; females: GFP 12.50±0.96, Cre 20.00±2.81, p=0.02). Conclusion: These results lend support for mPFC-AT2R+ neurons as a novel somatostatin subgroup that influences fear extinction in a sex-dependent manner. This furthers underscores the role of mPFC in top-down regulation and a unique role for peptidergic (ie., angiotensin) mPFC regulation of fear and sex differences, and increases the understanding of circuitry and function of the brain RAS in disordered fear learning. This may lead to improved therapeutic treatments for PTSD. This research and PJM was supported by NIH1R01HL137103-01A1 and the American Heart Association 15CSA24340001. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differential Role for the Clock Gene, BMAL1, on Kidney Function in Male versus Female Rats

Many activities of modern life, such as social visits with friends at night, long working hours, jet lag, and shift work cause circadian rhythm disturbances, which increase the risk of renal decline and eventual disease. Chronic kidney disease (CKD) is defined by decreases in glomerular filtration rate (GFR) which is higher during the daytime and lower at night in healthy individuals. Prior studies indicate that processes critical for maintaining blood pressure ( e.g. autoregulation) contribute to this time-of-day-dependence mediated by a molecular timekeeping mechanism known as the molecular circadian clock that resides in all tissues including the kidney. We hypothesized that loss of the molecular clock would impair normal kidney physiology through misalignment of functional rhythms with normal environmental patterns associated with day and night. We used a rat model of circadian disruption to interrogate our hypothesis. Male and female brain and muscle ARNT-like protein 1 knockout (BMAL1 KO) and littermate control (CON) rats aged 12-14 weeks underwent GFR measurements using FITC-labeled sinistrin clearance at two times of the day corresponding to the dark (active) and light (inactive) periods. Following GFR measurements, rats were placed in metabolic cages with food to record water intake and urine excretion over 12h active and inactive periods. Implantable telemeters were used for 24h measurement of systolic and diastolic blood pressure, locomotor activity and body temperature. Autoregulatory behavior was assessed using the isolated juxtamedullary nephron preparation to determine afferent arteriole diameter responses to changes in perfusion pressure. Diurnal measurements of GFR revealed that male BMAL1 KO rats had a significantly lower GFR in the active period as compared to CON males (1.87±0.37 vs 2.55±0.47 ml/min/100g; 2-way ANOVA, p<0.05; n=8-9). As a result, there was no diurnal variation in the KO males as seen in the CON males (Δ GFR 0.63±0.24 vs 0.04±0.21 ml/min/100g/12h CON vs KO respectively) (Sidak post-hoc, p<0.05; n=8-9) in parallel with a lack of diurnal variation in urine volume and Na excretion. Both male (n=8) and female (n=6-9) BMAL1 KO rats also displayed a significantly lower 24h mean arterial pressure (Males; 103±1 vs 107±1 mmHg, p<0.05; Females; 110±1 vs 100±2 mmHg, p<0.05) compared to CON with diurnal variation and autoregulatory reactivity preserved. Male BMAL1 KO rats consumed significantly more water compared to CON (n=5-9, 22.5±5.4 vs 14.7±4.0 mL/12h, p<0.05) concomitant with increased urine excretion (n=5-9, 13.3±6 vs 4.2±0.8 mL/12h, p<0.05) and reduced urine osmolality (n=5-7, 967±398 vs 2140±811 mmol/kg, p<0.05) regardless of time of day. Taken together these data suggest that BMAL1 is necessary for maintaining renal functional rhythms and urine concentrating ability in a sex-dependent manner. R01 DK134562 P01 HL158500. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2039: Limb Flexion Compresses The Anterior Tibial Artery And Changes Its Take-off Angle In A Sex-Dependent Manner

Introduction: Peripheral Arterial Disease (PAD) operations and reconstructions continue to fail at high rates, particularly below the knee. During limb flexion, distal superficial femoral and popliteal arteries experience significant deformations that are thought to contribute to disease development, but the biomechanics of the tibial arteries below the knee have not been characterized. Methods: Perfused human cadavers (n=15, 81±9 years old, range 60-93 years, 9F/6M) were imaged with their limbs in standing (180°), walking (110°), sitting (90°), and fetal/gardening (60°) positions using Computerized Tomography (CT). Three-dimensional geometries of the below-knee arteries were reconstructed in Mimics software and used to assess the take-off angle of the anterior tibial artery (AT) and the distance from the AT take-off to the nearest AT side branch. Results: The AT take-off angle relative to the tibioperoneal trunk increased with more acute limb flexion, from 70±19° in standing to 81±24° in walking, 83±24° in sitting, and 89±27° in fetal/gardening postures (p<0.01 for all postures). The AT take-off angle was larger in females than in males (24% in standing and 35% in walking, sitting, and gardening postures, p <0.01), but there were no differences between left and right limbs of either sex (p=0.66-0.99). The distance from the AT take-off to the nearest AT side branch decreased with more acute limb flexion (p< 0.01 for all postures). Conclusions: Limb flexion increases the AT take-off angle in a sex-dependent manner and compresses the AT, potentially contributing to the differences in hemodynamics and arterial mechanics and affecting the development and progression of tibial artery pathology. A better understanding of the below-knee artery biomechanics can provide valuable insights for surgical planning and may lead to improvements in the success rate and durability of PAD repairs.

Moderate Endurance Exercise Increases Arrhythmia Susceptibility and Modulates Cardiac Structure and Function in a Sexually Dimorphic Manner.

Although moderate endurance exercise has been reported to improve cardiovascular health, its effects on cardiac structure and function are not fully characterized, especially with respect to sexual dimorphism. We aimed to assess the effects of moderate endurance exercise on cardiac physiology in male versus female mice. C57BL/6J mice of both sexes were run on a treadmill for 6 weeks. ECG and echocardiography were performed every 2 weeks. After 6 weeks of exercise, mice were euthanized, and triple parametric optical mapping was performed on Langendorff perfused hearts to assess cardiac electrophysiology. Arrhythmia inducibility was tested by programmed electrical stimulation. Left ventricular tissue was fixed, and RNA sequencing was performed to determine exercise-induced transcriptional changes. Exercise-induced left ventricular dilatation was observed in female mice alone, as evidenced by increased left ventricular diameter and reduced left ventricular wall thickness. Increased cardiac output was also observed in female exercised mice but not males. Optical mapping revealed further sexual dimorphism in exercise-induced modulation of cardiac electrophysiology. In female mice, exercise prolonged action potential duration and reduced voltage-calcium influx delay. In male mice, exercise reduced the calcium decay constant, suggesting faster calcium reuptake. Exercise increased arrhythmia inducibility in both male and female mice; however, arrhythmia duration was increased only in females. Lastly, exercise-induced transcriptional changes were sex dependent: females and males exhibited the most significant changes in contractile versus metabolism-related genes, respectively. Our data suggest that moderate endurance exercise can significantly alter multiple aspects of cardiac physiology in a sex-dependent manner. Although some of these effects are beneficial, like improved cardiac mechanical function, others are potentially proarrhythmic.

The Role of Collagen VIII in the Aging Mouse Kidney.

The gradual loss of kidney function due to increasing age is accompanied by structural changes such as fibrosis of the tissue. The underlying molecular mechanisms are complex, but not yet fully understood. Non-fibrillar collagen type VIII (COL8) could be a potential factor in the fibrosis processes of the aging kidney. A pathophysiological significance of COL8 has already been demonstrated in the context of diabetic kidney disease, with studies showing that it directly influences both the development and progression of renal fibrosis occurring. The aim of this study was to investigate whether COL8 impacts age-related micro-anatomical and functional changes in a mouse model. The kidneys of wild-type (Col8-wt) and COL8-knockout (Col8-ko) mice of different age and sex were characterized with regard to the expression of molecular fibrosis markers, the development of nephrosclerosis and renal function. The age-dependent regulation of COL8 mRNA expression in the wild-type revealed sex-dependent effects that were not observed with collagen IV (COL4). Histochemical staining and protein analysis of profibrotic cytokines TGF-β1 (transforming growth factor) and CTGF (connective tissue growth factor) in mouse kidneys showed significant age effects as well as interactions of the factors age, sex and Col8 genotype. There were also significant age and Col8 genotype effects in the renal function data analyzed by urinary cystatin C. In summary, the present study shows, for the first time, that COL8 is regulated in an age- and sex-dependent manner in the mouse kidney and that the expression of COL8 influences the severity of age-induced renal fibrosis and function.

Do high-protein diets have the potential to reduce gut barrier function in a sex-dependent manner?

PurposeImpaired gut barrier function is associated with systemic inflammation and many chronic diseases. Undigested dietary proteins are fermented in the colon by the gut microbiota which produces nitrogenous metabolites shown to reduce barrier function in vitro. With growing evidence of sex-based differences in gut microbiotas, we determined whether there were sex by dietary protein interactions which could differentially impact barrier function via microbiota modification.MethodsFermentation systems were inoculated with faeces from healthy males (n = 5) and females (n = 5) and supplemented with 0.9 g of non-hydrolysed proteins sourced from whey, fish, milk, soya, egg, pea, or mycoprotein. Microbial populations were quantified using fluorescence in situ hybridisation with flow cytometry. Metabolite concentrations were analysed using gas chromatography, solid phase microextraction coupled with gas chromatography-mass spectrometry and ELISA.ResultsIncreased protein availability resulted in increased proteolytic Bacteroides spp (p < 0.01) and Clostridium coccoides (p < 0.01), along with increased phenol (p < 0.01), p-cresol (p < 0.01), indole (p = 0.018) and ammonia (p < 0.01), varying by protein type. Counts of Clostridium cluster IX (p = 0.03) and concentration of p-cresol (p = 0.025) increased in males, while females produced more ammonia (p = 0.02), irrespective of protein type. Further, we observed significant sex-protein interactions affecting bacterial populations and metabolites (p < 0.005).ConclusionsOur findings suggest that protein fermentation by the gut microbiota in vitro is influenced by both protein source and the donor’s sex. Should these results be confirmed through human studies, they could have major implications for developing dietary recommendations tailored by sex to prevent chronic illnesses.

Deletion of mouse lysyl oxidase in megakaryocytes affects bone properties in a sex-dependent manner

AbstractLysyl oxidase (LOX) is a facilitator of extracellular matrix cross-linking. Using newly developed megakaryocyte-specific LOX knockout mice, we show that LOX expressed in these scarce bone marrow cells affects bone volume and collagen architecture in a sex-dependent manner.

Metformin Induces MeCP2 in the Hippocampus of Male Mice with Sex-Specific and Brain-Region-Dependent Molecular Impact.

Rett Syndrome (RTT) is a progressive X-linked neurodevelopmental disorder with no cure. RTT patients show disease-associated symptoms within 18 months of age that include developmental regression, progressive loss of useful hand movements, and breathing difficulties, along with neurological impairments, seizures, tremor, and mental disability. Rett Syndrome is also associated with metabolic abnormalities, and the anti-diabetic drug metformin is suggested to be a potential drug of choice with low or no side-effects. Previously, we showed that in vitro exposure of metformin in a human brain cell line induces MECP2E1 transcripts, the dominant isoform of the MECP2 gene in the brain, mutations in which causes RTT. Here, we report the molecular impact of metformin in mice. Protein analysis of specific brain regions in the male and female mice by immunoblotting indicated that metformin induces MeCP2 in the hippocampus, in a sex-dependent manner. Additional experiments confirm that the regulatory role of metformin on the MeCP2 target "BDNF" is brain region-dependent and sex-specific. Measurement of the ribosomal protein S6 (in both phosphorylated and unphosphorylated forms) confirms the sex-dependent role of metformin in the liver. Our results can help foster a better understanding of the molecular impact of metformin in different brain regions of male and female adult mice, while providing some insight towards its potential in therapeutic strategies for the treatment of Rett Syndrome.

Sex-dependent responses to high concentration of binge ethanol in spleen of adolescent F344 rats.

We previously reported that binge ethanol induces atrophy of the spleen, a key immune organ, in adolescent male F344 rats. Because there are significant sex effects in immune function, we investigated whether binge ethanol exerts sex-dependent effects on the spleen, including producing splenic atrophy. We gave F344 rats ethanol (4.8 g/kg/day; 52% w/v; i.g.) on postnatal days [PND] 36 ~ 38 and sacrificed them on PND 39 for spleen collection. We performed immunophenotyping analysis of splenic cells and examined the expression of 158 genes related to alcohol metabolism, epigenetic modification, and immune regulation in the spleens of adolescent (PND 39) male and female rats. Following a 3-day ethanol exposure, a loss of body weight, and absolute and relative spleen weight, was seen only in male adolescent rats. Ethanol altered the relative proportions of lymphocyte subtypes in both sexes with different patterns. We also found that 3-day ethanol exposure induced sex-dependent gene expression changes in spleen. Among the 158 genes studied, the expression of only three genes was significantly increased in female rats. However, the expression of 30 genes was significantly increased/decreased in male rats. Female rats had greater expression of alcohol metabolizing enzyme genes in the spleen under physiological conditions and when stimulated by binge ethanol. The genes are involved in epigenetic modification were differentially expressed in a sex-dependent manner. We found that male adolescent rats were more sensitive to binge ethanol than female rats. Differential expression of the genes related to alcohol metabolism and epigenetic modification (of DNA methyltransferase and histone deacetylases) between the sexes could account for the observed sex-dependent responses to binge ethanol in adolescent rats.

Nanoplastics aggravated TDCIPP-induced transgenerational developmental neurotoxicity in zebrafish depending on the involvement of the dopamine signaling pathway

Plastics pose a hazard to the environment. Although plastics have toxicity, microplastics (MPs) and nanoplastics (NPs) are capable of interacting with the rest pollutants in the environment, so they serve as the carriers and interact with organic pollutants to modulate their toxicity, thus resulting in unpredictable ecological risks. PS-NPs and TDCIPP were used expose from 2 h post-fertilization (hpf) to 150 days post-fertilization (dpf) to determine the bioaccumulation of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and its potential effects on neurodevelopment in F1 zebrafish (Danio rerio) offspring under the action of polystyrene nano plastics (PS-NPs). The exposure groups were assigned to TDCIPP (0, 0.4, 2 or 10 µg/L) alone group and the PS-NPs (100 µg/L) and TDCIPP co-exposed group. F1 embryos were collected and grown in clean water to 5 dpf post-fertilization. PS-NPs facilitated the bioaccumulation of TDCIPP in the gut, gill, head，gonad and liver of zebrafish in a sex-dependent manner and promoted the transfer of TDCIPP to their offspring, thus contributing to PS-NPs aggravated the inhibition of offspring development and neurobehavior of TDCIPP-induced. In comparison with TDCIPP exposure alone, the combination could notably down-regulate the levels of the dopamine neurotransmitter, whereas the levels of serotonin or acetylcholine were not notably different. This result was achieved probably because PS-NPs interfered with the TDCIPP neurotoxic response of zebrafish F1 offspring not through the serotonin or acetylcholine neurotransmitter pathway. The increased transfer of TDCIPP to the offspring under the action of PS-NPs increased TDCIPP-induced transgenerational developmental neurotoxicity, which was proven by a further up-regulation/down-regulation the key gene and protein expression related to dopamine synthesis, transport, and metabolism in F1 larvae, in contrast to TDCIPP exposure alone. The above findings suggested that dopaminergic signaling involvement could be conducive to the transgenerational neurodevelopmental toxicity of F1 larval upon parental early co-exposure to PS-NPs and TDCIPP.

Perinatal exposure to atazanavir-based antiretroviral regimens in a mouse model leads to differential long-term motor and cognitive deficits dependent on the NRTI backbone.

Combination antiretroviral therapy (ART) use in pregnancy has been pivotal in improving maternal health and reducing perinatal HIV transmission. However, children born HIV-exposed uninfected fall behind their unexposed peers in several areas including neurodevelopment. The contribution of in utero ART exposure to these deficits is not clear. Here we present our findings of neurocognitive outcomes in adult mice exposed in utero to ART. Dams were treated with a combination of ritonavir-boosted atazanavir with either abacavir plus lamivudine (ABC/3TC + ATV/r) or tenofovir disoproxil fumarate plus emtricitabine (TDF/FTC + ATV/r), or water as a control, administered daily from day of plug detection to birth. Offspring underwent a battery of behavioral tests that investigated motor performance and cognition starting at 6-weeks of age and ending at 8 months. Changes in brain structure were assessed using magnetic resonance imaging and immunohistochemistry. Expression of genes involved in neural circuitry and synaptic transmission were assessed in the hippocampus, a region strongly associated with memory formation, using qPCR. Pups exposed to TDF/FTC + ATV/r showed increased motor activity and exploratory drive, and deficits in hippocampal-dependent working memory and social interaction, while pups exposed to ABC/3TC + ATV/r showed increased grooming, and deficits in working memory and social interaction. Significant volumetric reductions in the brain were seen only in the ABC/3TC + ATV/r group and were associated with reduced neuronal counts in the hippocampus. Altered neurotransmitter receptor mRNA expression as well as changes in expression of the neurotrophic factor BDNF and its receptors were observed in both ART-exposed groups in a sex-dependent manner. In our model, in utero ART exposure had long-term effects on brain development and cognitive and motor outcomes in adulthood. Our data show that neurological outcomes can be influenced by the type of nucleoside reverse transcriptase inhibitor backbone of the regimen and not just the base drug, and display sex differences.