Female WT Research Articles

IL‐1 beta and TNF‐alpha play an essential role in modulating the risk of both periodontitis and Alzheimer’s disease

AbstractBackgroundPeriodontitis, a chronic systemic inflammatory disease, is a common health problem in the elderly. With increasing recognition that inflammation plays a key role in the pathophysiology of Alzheimer's disease (AD), our research team has been studying how systemic inflammation affects the brain immune responses. Aging is a major risk factor for both periodontitis and AD. As aging causes dysregulation of immune responses, we aimed to define the roles of cytokines in the pathogenesis of periodontitis and AD.MethodsTo induce experimental periodontitis, female WT mice were injected with heat‐killed P. gingivalis into their buccal mucosa three times per week every other week for a total of 5 weeks. Another group of mice were injected with IL‐1b and TNF‐a following the same timeline as above. Sickness Behavior and cognitive functions were assessed through open field and puzzle box test. Mandibular jaws were harvested for Micro‐CT scan. Different brain regions were harvested for biochemical analysis.ResultsMice injected with heat‐killed bacteria had worsened long‐term memory functions. It also significantly increased periodontal bone loss, which was accompanied by increased gene expression levels of IL‐1b and TNF‐a in the gums. Significant elevated levels of pro‐inflammatory cytokines in the liver as well as different brain regions were also observed. Immunostaining revealed increased levels of phospho‐tau in WT mice injected with heat‐killed bacteria, suggesting that elevated systemic cytokine levels caused by the injection of heat‐killed bacteria can lead to the appearance of AD pathology. Another group of mice injected with IL‐1b and TNF‐a displayed hyperactivity and worsened short‐term memory functions. Injections of cytokines also induced periodontal bone loss, which was accompanied by elevated levels of inflammatory cytokines in the liver as well as different brain regions.ConclusionTaken together, our study revealed that increased cytokine immune response in the gums led to periodontal bone loss, brain inflammation and modulated the cognitive functions of WT mice. This research demonstrated that cytokines play a crucial role in the pathogenesis of both periodontitis and AD.Acknowledgement: The study is supported by Health and Medical Research Fund (HMRF 04151216) to RCCC. RPHW is awarded by Hong Kong PhD Fellowship.

Abstract PO-110: Targeting cathepsin B in the pancreatic stellate cells stimulates CD8+ T cell dependent anti-tumor immune response

Abstract Background: Cathepsin B (CTSB) is a lysosomal cysteine protease which has been described to have a role in pancreatic cancer development. However, its significance in the tumor microenvironment has never been investigated. Pancreatic stellate cells (PSCs) are one of the main sources of Cancer Associated Fibroblasts (CAFs) in the pancreatic cancer microenvironment. Herein, we describe the effects of targeting CTSB in the pancreatic stellate cells (PSCs) in pancreatic cancer (PDAC). Methods: To specifically study the role of CTSB in PSCs, PSCs were isolated from C57BL/6J (WT) or CTSB -/- mice and were orthotopically injected with KPC cancer cells into the tail of pancreas of 6-week old female WT mice. Tumors were analyzed at endpoint using flow cytometry. To further study the dependence of effects of CTSB in PSCs on adaptive immune cells, WT or CTSB -/- PSCs were co-injected with KPC cells into Rag 1-/- mice or CD8+ T cell depleted mice (anti-CD8 mAb, 250ug/dose every 3 days). In vitro, WT or CTSB-/- PSCs were co-cultured with KPC across a transwell. CD8+ T cells, isolated from KPC tumor bearing mice, were exposed to conditioned media from this co-culture ex-vivo and their cytotoxicity was measured using calcein release assay. In another experiment, WT mice injected with KPC cancer cells were treated with vehicle control or the CTSB inhibitor, CA-074 methyl ester (10 mg/kg), through daily intraperitoneal injections. Results: Co-injections of WT PSC with KPC cells formed larger tumors as compared to KPC cancer cells alone, however, co-injection with CTSB-/- PSCs abrogated this tumor promoting effect. Flow cytometric analysis revealed increased infiltration of Ifn-γ secreting CD8+ T cells in the KPC+CB-/- PSC group as compared to KPC+WT PSC group. Tumor decreasing effect of CTSB-/- PSCs was lost when co-injections were performed in Rag 1-/- mice or CD8+ T cell depleted mice, indicating that this effect relies on a functional adaptive immune response, specifically CD8+ T cells. In vitro, co-culture of CTSB-/- PSCs with KPC led to reduced expression of inflammatory CAF (iCAF) markers like IL-6 and IL-11 when compared to co-culture of WT PSCs with KPC. CD8+ T cells exposed to conditioned media from CTSB-/- PSC co-culture showed increased cytotoxicity against KPC cells ex-vivo, as compared to those exposed to conditioned media from WT PSC co-culture. Finally, treatment with CA-074 was able to decrease orthotopic PDAC tumor growth with increased CD8+ T cell infiltration evident on flow cytometry. Conclusion: Targeting CTSB in the PSCs stimulates anti-tumor adaptive immune response against pancreatic cancer by inhibiting conversion of PSCs into the tumor-promoting iCAF phenotype. CTSB might emerge as a novel target for CAF-directed therapy in pancreatic cancer. Citation Format: Bharti Garg, Tejeshwar Jain, Utpreksha Vaish, Vikas Dudeja. Targeting cathepsin B in the pancreatic stellate cells stimulates CD8+ T cell dependent anti-tumor immune response [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-110.

Disruption of Glucocorticoid Action on CD11c+ Dendritic Cells Favors the Generation of CD4+ Regulatory T Cells and Improves Fetal Development in Mice.

A wealth of innate and adaptive immune cells and hormones are involved in mounting tolerance towards the fetus, a key aspect of successful reproduction. We could recently show that the specific cross talk between the pregnancy hormone progesterone and dendritic cells (DCs) is significantly engaged in the generation of CD4+ FoxP3+ regulatory T (Treg) cells while a disruption led to placental alterations and intra-uterine growth restriction. Apart from progesterone, also glucocorticoids affect immune cell functions. However, their functional relevance in the context of pregnancy still needs clarification. We developed a mouse line with a selective knockout of the glucocorticoid receptor (GR) on DCs, utilizing the cre/flox system. Reproductive outcome and maternal immune and endocrine adaptation of Balb/c-mated C57Bl/6 GRflox/floxCD11ccre/wt (mutant) females was assessed on gestation days (gd) 13.5 and 18.5. Balb/c-mated C57Bl/6 GRwt/wtCD11ccre/wt (wt) females served as controls. The number of implantation and fetal loss rate did not differ between groups. However, we identified a significant increase in fetal weight in fetuses from mutant dams. While the frequencies of CD11c+ cells remained largely similar, a decreased expression of co-stimulatory molecules was observed on DCs of mutant females on gd 13.5, along with higher frequencies of CD4+ and CD8+ Treg cells. Histomorphological and gene expression analysis revealed an increased placental volume and an improved functional placental capacity in mice lacking the GR on CD11c+ DCs. In summary, we here demonstrate that the disrupted communication between GCs and DCs favors a tolerant immune microenvironment and improves placental function and fetal development.

Amyloid-β Processing in Aged S100B Transgenic Mice Is Sex Dependent.

(1) Background: Calcium-binding protein S100B is involved in neuroregeneration but has also been associated with neurodegeneration. These contrasting effects may result from concentration or duration of exposure. We investigated the effect of long-term increased S100B levels on amyloid-β processing in one-year-old transgenic (tg) mice with 12 copies of the murine S100B gene with specific consideration of sex and specific brain regions. (2) Methods: S100B and amyloid-β 42 (Aβ42) were quantified in serum, cerebrospinal fluid (CSF), adipose tissue, and different brain regions by ELISA in wild-type (wt) and S100Btg mice (each n = 7 per group). Thioflavin T (ThT) and Aβ immunostaining were performed for visualization of Aβ deposition. (3) Results: S100B in serum, CSF, and brain was significantly increased in S100Btg mice of both sexes. Aβ42 was significantly increased in the hippocampus of male S100Btg mice (p = 0.0075), and the frontal cortex of female S100Btg mice (p = 0.0262). ThT and Aβ immunostaining demonstrated Aβ deposition in different brain regions in S100Btg mice of both sexes and female wt. (4) Conclusion: Our data validate this experimental model for studying the role of S100B in neurodegeneration and indicate that Aβ processing is sex-dependent and brain region-specific, which deserves further investigation of signaling pathways and behavioral responses.

Cigarette smoke and nicotine effects on behavior in HIV transgenic rats

HIV-related neurocognitive impairment can be worsened by cigarette smoking and be more severe in women. Therefore, we analyzed the effects of sex on behavioral function in HIV transgenic (Tg) rats that were exposed to either nicotine alone, to smoke from either nicotine-containing or nicotine-free cigarettes, or non-exposed. The animals were then assessed on the open field test for the total distance traveled and for the fraction of the total distance traveled and the total time spent in the center of the field, and the results then compared to WT rats subjected to the same exposures and testing. Higher total distances indicate greater locomotor activity and a higher center field measures imply a lower anxiety state. Total distances were overall higher for female and for Tg rats exposed to nicotine-free CS. Also, the total distance and both center field measures were overall higher for female rats in the control and nicotine-free CS-exposed groups. This was observed specifically for WT females as compared to WT males and, for the center field measures, for WT females as compared to Tg males. No genotype or sex-related differences were found for rats in the nicotine-free cigarette smoke (CS) and nicotine-containing CS exposed groups. Therefore, nicotine exposure did not impact genotype- and sex-related differences in motor responses and anxiety levels that were found in the control state. However, exposure to the non-nicotine components of CS resulted in locomotor activation in the presence of the HIV genes and was anxiogenic in WT and Tg male animals.

Abstract P389: Free Fatty Acid Receptor 4 Is Necessary For An Adaptive Response Following Heart Failure Induced By Metabolic Syndrome In Mice

Non-resolving inflammation is central to the pathogenesis of heart failure (HF). Heart failure preserved ejection fraction (HFpEF) is a type of HF that is particularly associated with inflammation provoked by metabolic syndrome (MetS). The G-protein coupled receptor, free fatty acid receptor 4 (Ffar4), is a receptor for medium and long chain fatty acids (FA) that regulates metabolism and attenuates inflammation. Ffar4 is expressed in the human heart, and downregulated in heart failure. Furthermore, polymorphisms in Ffar4 have been associated with eccentric remodeling in a patient cohort. Previously, Ffar4 was shown to protect the heart from pathologic stress by attenuating oxidative stress in a mouse model of pressure overload. Here, we tested the hypothesis that Ffar4 would attenuate the development of heart failure using a mouse model of MetS-induced HFpEF. Metabolic syndrome was induced in mice by feeding a high-fat, high-sucrose diet (42% fat, 30% sucrose) to produce obesity and delivering the nitric oxide synthase inhibitor, L-NAME, in the drinking water to induce hypertension. The combined intervention (referred to as HFpEF diet) resulted in mice developing excessive adiposity, glucose intolerance (in males only), and mild hypertension. After 20 weeks on the HFpEF diet, both male and female WT mice, developed diastolic dysfunction (increased E/A and E/e’) and preserved ejection fraction (EF), consistent with clinical HFpEF. In Ffar4KO male mice HFpEF diet induced a greater degree of diastolic dysfunction compared to WT mice, despite equivalent metabolic parameters. Female Ffar4KO mice fed the HFpEF diet had a greater increase in weight gain and adiposity compared to WT female mice. Surprisingly, diastolic function was equivalent between WT and FFAR4KO female mice, suggesting a sex-based difference in FFAR4 cardioprotection. Our data show that Ffar4 attenuates HFpEF secondary to MetS.

Abstract MP61: Sex Difference In The Response Of Human Renal Proximal Tubule Cells To A Nephrotoxic Agent.

Our previous work indicated that the renal dopamine D2 receptor (D2R) has a significant role in regulating renal inflammation and injury, as well as in blood pressure control. In mice, D2R has protective effects in the kidney by limiting the inflammatory and fibrotic reaction; impaired D2R function results in renal inflammation and damage. Some common single nucleotide polymorphisms (SNPs; rs 6276 and 6277) in the human DRD2 gene are associated with decreased D 2 R expression and function and high blood pressure. To determine the effects of the presence of SNPs in the response to the nephrotoxic aristolochic acid (AA, 5μg/ml, 24 h), we studied immortalized human renal proximal tubule cells isolated from normal tissue of nephrectomies and genotyped for DRD2 SNPs and DRD2 wild-type (WT). We also determined whether this response is sex dependent. D2R protein was higher in male than in female WT (135±5 vs 100±4%; n=3/group; P<0.04) and lower in males with SNPs (43±2%, P<0.05) and females with SNPs (23±2%,P<0.05), compared with their respective WT counterparts. In both male groups (WT and SNPs), AA increased D2R protein by 80-100% but had no effect in WT females and increased ~50% in females with SNPs. The TNFα mRNA was higher in males with WT and SNPs which was increased by AA 9-10-fold in WT males and females but only 2-3-fold in those with SNPs. The TGFβ mRNA was similar in WT males and females and increased to the same extent in both groups with SNPs and was not affected by AA in all groups. Col1a1 mRNA was higher (30%) in WT males and females than those with SNPs; AA decreased Col1a1 mRNA in all groups. FN1 mRNA was higher (30-40%) in males and females with SNPs than WT; AA increased FN1 mRNA only in males and females with SNPs. The mRNA expression of the cell proliferation marker Ki-67 was higher in WT females than WT males (1.5-2-fold) and higher with SNPs than WT in both groups; AA increased Ki-67 mRNA in both groups and to a greater extent in males than in females. Taken together our data indicate that the presence of DRD2 SNPs affects the baseline expression of inflammatory and fibrotic factors and the response to AA is dependent on both sex and the presence of DRD2 SNPs. These data may have potential clinical translation since rs6276/6277 is commonly expressed (42%/23%) in the human population.

Accelerated developmental adipogenesis programs adipose tissue dysfunction and cardiometabolic risk in offspring born to dams with metabolic dysfunction.

This study determined if a perturbation in in utero adipogenesis leading to later life adipose tissue (AT) dysfunction underlies programming of cardiometabolic risk in offspring born to dams with metabolic dysfunction. Female mice heterozygous for the leptin receptor deficiency (Hetdb) had 2.4-fold higher prepregnancy fat mass and in late gestation had higher plasma insulin and triglycerides compared with wild-type (Wt) females (P < 0.05). To isolate the role of the intrauterine milieu, wild-type (Wt) offspring from each pregnancy were studied. Differentiation potential in isolated progenitors and cell size distribution analysis revealed accelerated adipogenesis in Wt pups born to Hetdb dams, accompanied by a higher accumulation of neonatal fat mass. In adulthood, whole body fat mass by NMR was higher in male (69%) and female (20%) Wt offspring born to Hetdb versus Wt pregnancies, along with adipocyte hypertrophy and hyperlipidemia (all P < 0.05). Lipidomic analyses by gas chromatography revealed an increased lipogenic index (16:0/18:2n6) after high-fat/fructose diet (HFFD). Postprandial insulin, ADIPO-IR, and ex vivo AT lipolytic responses to isoproterenol were all higher in Wt offspring born to Hetdb dams (P < 0.05). Intrauterine metabolic stimuli may direct a greater proportion of progenitors toward terminal differentiation, thereby predisposing to hypertrophy-induced adipocyte dysfunction.NEW & NOTEWORTHY This study reveals that accelerated adipogenesis during the perinatal window of adipose tissue development predisposes to later life hypertrophic adipocyte dysfunction, thereby compromising the buffering function of the subcutaneous depot.

Effects of D-serine treatment on outer retinal function

The role of the N-Methyl-D-Aspartate Receptor (NMDAR) in the outer retina is unclear despite expression of the NMDAR-complex and its subunits in the outer retina. The flash-electroretinogram (fERG) offers a non-invasive measurement of the retinal field potentials of the outer retina that can serve to clarify NMDAR contribution to early retinal processing. The role of the NMDAR in retinal function was assessed using a genetic mouse model for NMDAR hypofunction (SR-/-), where the absence of the enzyme serine racemase (SR) results in an 85% reduction of retinal D-serine. NMDAR hypo- and hyperfunction in the retina results in alterations in the components of the fERG. The fERG was examined after application of exogenous D-serine to the eye in order to determine whether pre- and post-topical delivery of D-serine would alter the fERG in SR-/- mice and their littermate WT controls. Amplitude and implicit time of the low-frequency components, the a- and b-wave, were conducted. Reduced NMDAR function resulted in a statistically significantly delayed a-wave and reduced b-wave in SR-/- animals. The effect of NMDAR deprivation was more prominent in male SR-/- mice. A hyperfunction of the NMDAR, through exogenous topical delivery of 5mM D-serine, in WT mice caused a significantly delayed a-wave implicit time and reduced b-wave amplitude. These changes were not observed in female WT mice. There were temporal delays in the a-wave and amplitude and a decrease in the b-wave amplitude and implicit time in both hypo- and NMDAR hyperfunctional male mice. These results suggest that NMDAR and D-serine are involved in the retinal field potentials of the outer retina that interact based on the animal's sex. This implicates the involvement of gonadal hormones and D-serine in retinal functional integrity.

Ghrelin receptor signaling is not required for glucocorticoid-induced obesity in female mice.

Chronic exposure to high circulating glucocorticoid or ghrelin concentrations increases food intake, weight gain and adiposity, suggesting that ghrelin could contribute to the metabolic effects of chronic glucocorticoids. In male mice, however, blocking ghrelin receptor (GHSR) signaling increased the weight gain and adiposity induced by chronic corticosterone (CORT), rather than attenuating them. In the current study, we investigated the role of GHSR signaling in the metabolic effects of chronic exposure to high circulating CORT in female mice. To do this, female WT and GHSR KO mice were treated with either CORT in a 1% ethanol (EtOH) solution or 1% EtOH alone in their drinking water for 32 days (n = 5-8/group). Body weight, food, and water intake as well as vaginal cyclicity were assessed daily. As expected, CORT treatment-induced significant increases in body weight, food intake, adiposity and also impaired glucose tolerance. In contrast to results observed in male mice, WT and GHSR KO female mice did not differ on any of these parameters. Neither plasma levels of ghrelin, LEAP-2, the endogenous GHSR antagonist produced by the liver, nor their ratio were altered by chronic glucocorticoid exposure. In addition, CORT treatment disrupted vaginal cyclicity, produced a reduction in sucrose consumption and increased locomotor activity regardless of genotype. Chronic CORT also decreased exploration in WT but not GHSR KO mice. Collectively, these data suggest that most metabolic, endocrine, reproductive and behavioral effects of chronic CORT exposure are independent of GHSR signaling in female mice.

Fine-Tuning the PI3K/Akt Signaling Pathway Intensity by Sex and Genotype-Load: Sex-Dependent Homozygotic Threshold for Somatic Growth but Feminization of Anxious Phenotype in Middle-Aged PDK1 K465E Knock-In and Heterozygous Mice.

According to the Research Domain Criteria (RDoC), phenotypic differences among disorders may be explained by variations in the nature and degree of neural circuitry disruptions and/or dysfunctions modulated by several biological and environmental factors. We recently demonstrated the in vivo behavioral translation of tweaking the PI3K/Akt signaling, an essential pathway for regulating cellular processes and physiology, and its modulation through aging. Here we describe, for the first time, the in vivo behavioral impact of the sex and genetic-load tweaking this pathway. The anxiety-like phenotypes of 61 mature (11–14-month-old) male and female PDK1 K465E knock-in, heterozygous, and WT mice were studied. Forced (open-field) anxiogenic environmental conditions were sensitive to detect sex and genetic-load differences at middle age. Despite similar neophobia and horizontal activity among the six groups, females exhibited faster ethograms than males, with increased thigmotaxis, increased wall and bizarre rearing. Genotype-load unveiled increased anxiety in males, resembling female performances. The performance of mutants in naturalistic conditions (marble test) was normal. Homozygotic-load was needed for reduced somatic growth only in males. Factor interactions indicated the complex interplay in the elicitation of different negative valence system’s items and the fine-tuning of PI3K/Akt signaling pathway intensity by genotype-load and sex.

Phlpp1 is induced by estrogen in osteoclasts and its loss in Ctsk-expressing cells does not protect against ovariectomy-induced bone loss.

Prior studies demonstrated that deletion of the protein phosphatase Phlpp1 in Ctsk-Cre expressing cells enhances bone mass, characterized by diminished osteoclast activity and increased coupling to bone formation. Due to non-specific expression of Ctsk-Cre, the definitive mechanism for this observation was unclear. To further define the role of bone resorbing osteoclasts, we performed ovariectomy (Ovx) and Sham surgeries on Phlpp1 cKOCtsk and WT mice. Micro-CT analyses confirmed enhanced bone mass of Phlpp1 cKOCtsk Sham females. In contrast, Ovx induced bone loss in both groups, with no difference between Phlpp1 cKOCtsk and WT mice. Histomorphometry demonstrated that Ovx mice lacked differences in osteoclasts per bone surface, suggesting that estradiol (E2) is required for Phlpp1 deficiency to have an effect. We performed high throughput unbiased transcriptional profiling of Phlpp1 cKOCtsk osteoclasts and identified 290 differentially expressed genes. By cross-referencing these differentially expressed genes with all estrogen response element (ERE) containing genes, we identified IGFBP4 as potential estrogen-dependent target of Phlpp1. E2 induced PHLPP1 expression, but reduced IGFBP4 levels. Moreover, genetic deletion or chemical inhibition of Phlpp1 was correlated with IGFBP4 levels. We then assessed IGFBP4 expression by osteoclasts in vivo within intact 12-week-old females. Modest IGFBP4 immunohistochemical staining of TRAP+ osteoclasts within WT females was observed. In contrast, TRAP+ bone lining cells within intact Phlpp1 cKOCtsk females robustly expressed IGFBP4, but levels were diminished within TRAP+ bone lining cells following Ovx. These results demonstrate that effects of Phlpp1 conditional deficiency are lost following Ovx, potentially due to estrogen-dependent regulation of IGFBP4.

Understanding How Sex Influences the Impact of IL-10 on Bone Microarchitecture and Bone Metabolism Over Time

Abstract Objectives Dietary interventions with pre- and probiotics favorably affect the gut-bone axis, mediated in part by the anti-inflammatory cytokine, interleukin (IL)-10. This study sought to understand how IL-10’s impact on bone metabolism and microarchitecture differs with sex and time. Methods Six-week-old B6.129P2-Il10tm1Cgn/J (KO) and C57BL/6 (WT) mice were assigned in a 2 × 2 × 2 factorial design with strain (WT &amp; KO), sex, and time (3 &amp; 6 m) as factors. Mice were fed AIN-93G diet for 3 m followed by AIN-93 M for the study duration. Dual-energy x-ray absorptiometry was used to assess bone mineral content (BMC) and density (BMD). Micro-computed tomography was used to assess femur and lumbar vertebrae trabecular and cortical bone. Serum procollagen 1 intact N-terminal propeptide (P1NP) and C-terminal telopeptide of type I collagen (CTX-1), bone formation and resorption markers respectively, were assessed by ELISA. Data were analyzed using ANOVA; p &amp;lt; 0.05 was considered significant. Results Reductions in BMC and BMD (P &amp;lt; 0.05) in KO vs WT and at 3 vs 6 m were observed; a sex effect was found with reductions in BMC in KO females compared to KO males. Femoral trabecular bone volume (BV/TV) was lower (P &amp;lt; 0.05) in KO vs WT, females vs males, and at 6 vs 3 m. Trabecular thickness (TbTh) decreased (P &amp;lt; 0.05) in KO vs WT and increased from 3 to 6 m, while decreases in trabecular number (TbN) were greater (P &amp;lt; 0.05) in KO mice, females, and at 6 m compared to counterparts. Cortical area and thickness were decreased (P &amp;lt; 0.05) in KO vs WT and in females vs males, which was greater at 6 m, while cortical bone porosity was higher in KO vs WT and increased at 6 mo. Vertebral trabecular BV/TV was lower (P &amp;lt; 0.05) in KO vs WT at 3 and 6 m, with KO females showing reduced BV/TV (P &amp;lt; 0.05) from 3 to 6 m. Reduced TbTh and TbN were observed in KO vs WT, and females had increased (P &amp;lt; 0.05) TbTh and trabecular separation and reduced TbN. P1NP showed a time effect (P &amp;lt; 0.05) with reductions in WT females and males at 6 m compared to 3 m KO females (P &amp;lt; 0.05). CTX-1 shows a sex effect (P &amp;lt; 0.05) and a trending strain effect (P = 0.059), with elevated serum CTX-1 in 3 m KO males compared to WT and KO females at 6 m (P &amp;lt; 0.05). Conclusions While IL-10 plays an important role in maintaining both trabecular and cortical bone, it may have a more protective effect on the cortical bone of female mice over time. Funding Sources Oklahoma Agricultural Experiment Station.

218-LB: Age, Sex, and Fat Mass Affects Erythropoietin Metabolic Response in Mice

Erythropoietin (EPO) is required for erythrocyte production. However, EPO receptor (EpoR) extends beyond erythroid tissue and ΔEpoR mice with EpoR restricted to hematopoietic tissue show an increase in fat mass. At 3 months of age, ΔEpoR mice show no difference in body weight compared with wild type (WT, C57BL/6) mice. By 6 months of age, male body weights of ΔEpoR mice are greater than WT mice, and at 8+ months body weights of male and female ΔEpoR mice are greater than their WT counterparts. To assess EPO activity on glucose homeostasis and fat mass, mice were fed a high fat diet (HFD, 60% kCal fat) and treated with EPO (3000U/kg 3X per week) or saline for 3 weeks at age 3, 6, and 8+ months. EPO increased hematocrit in all mice. EPO treatment in male WT mice reduces weight and fat mass at 6 and 8+ months of age, when their fat mass is 35% or greater with saline treatment. Female WT mice treated with EPO do not show significantly less weight or fat mass at any age. EPO treatment does not affect weight or fat mass in ΔEpoR mice. With HFD feeding and EPO treatment in 3 month old mice, body weights are not different between WT and ΔEpoR mice although fat mass is significantly higher in ΔEpoR mice. At 6 months, body weight and fat mass in males are greater in ΔEpoR than WT, while ΔEpoR females show no difference in body weight but significantly more fat mass compared with WT females. In the older mice (8+ months old), both sexes of ΔEpoR mice are significantly heavier and fatter than WT mice. EPO treatment improves glucose tolerance in female ΔEpoR mice at 3 and 6 months but not at 8+months, whereas male ΔEpoR mice only showed improvement at 3 months, not at 6 and 8+months. In male and female WT mice, EPO improves glucose metabolism at 3 and 6 months, and to a lesser extent at 8+ months with greater improvement in mice with &amp;lt;35% fat mass. These data show that EPO regulates metabolism in a sex and age dependent manner mediated via EpoR expression in non-hematopoietic tissue and is influenced by level of fat mass. Disclosure H. Rogers: None. C. T. Noguchi: None.

Mice With Targeted Deletion of ARC Kisspeptin Exhibit Immature Gametogenesis and Impaired Fertility

Hypothalamic kisspeptin is primarily synthesized in two discrete nuclei - the anteroventral periventricular (AVPV) and the arcuate (ARC) nuclei. We have previously developed a selective, conditional ARC kisspeptin knock-out (KO) mouse line, namely the Pdyn-Cre/Kissfl/fl KO mice, that exhibited normal puberty onset in both sexes, but impaired estrous cyclicity and LH pulsatility in Pdyn-Cre/Kissfl/fl KO females. To examine the end-organ effect of the lack of ARC kisspeptin, we examined gametogenesis, gonad morphology, and fertility. Hematoxylin and eosin (H&E) staining of serial-sectioned whole ovaries demonstrated that Pdyn-Cre/Kissfl/fl KO female mice lacked corpora lutea - their ovarian folliculogenesis did not progress beyond antral follicle development, suggesting an ovulatory defect in Pdyn-Cre/Kissfl/fl KO females. 75% of the Pdyn-Cre/Kissfl/fl KO male mice had testes exhibiting a striking decrease in mature sperm in the seminiferous tubules. The remaining 25% showed evidence of mature sperm. Further evidence of a hypogonadal phenotype of the Pdyn-Cre/Kissfl/fl KO mice included the significantly low weight and small size of the ovaries, uteri, and testes when compared to control littermates. In a controlled, continuous mating paradigm with proven WT males, 2-4-month-old Pdyn-Cre/Kissfl/fl KO female mice failed to become pregnant or produce any pups, whereas age-matched WT females exhibited normal pregnancies to term. Thus, Pdyn-Cre/Kissfl/fl KO females have complete infertility. Ongoing studies of male fertility data suggest that Pdyn-Cre/Kissfl/fl KO males are subfertile, in accordance with their variable spermatogenesis phenotype - some KO males sired pups when paired with proven, WT females, whereas other KO males are infertile. Future experiments include assessing the capability of Pdyn-Cre/Kissfl/fl KO mice to respond to chronic, exogenous kisspeptin and GnRH administration to rescue abnormal LH pulsatility and estrous cyclicity in females, as well as the impaired fertility in both sexes.

The Effects of Diet Induced Obesity (DIO) on Skeletal Muscle Transcription in MuRF1 KO Mice

Background: As obesity and Type II Diabetes rise globally, it is important to understand the similarities and differences in the response of metabolic tissues between males and females. We wanted to evaluate the impact of prolonged diet induced obesity (DIO) on the skeletal muscle transcriptome of our MuRF1 KO (KO) mice. Methods: RNA was isolated from the gastrocnemius muscle of male and female WT and KO mice that were fed either standard chow (Envigo 2918) or a 45% HFD (Research Diets D12451) for 22 weeks (n = 4). RNA was enriched for mRNA prior to library preparation. RNA sequencing was performed using 150 bp paired-end reads (~ 31.6 M reads per sample). Differentially expressed genes (DEGs) were identified using DESeq2 with an FDR set to 5%. Results: At baseline (chow diet), both male and female KO mice had DEGs compared to their WT counterparts (male, 1174; female, 105). Most DEGs were found to be unique by sex (male, 1151; female, 82), though 23 genes were found to be changed in common. After obesity was induced by 22 weeks of 45% HFD feeding, KO animals showed a greater transcriptional response than their WT counterparts. Males had 1821 DEGs (v. 179 in WT) while females had 4425 DEGs (v. 2090 in WT). In males, 78 genes were changed in common between WT and KO in response to DIO, with 76 of those genes changing in the same direction (Slc282a and Gm15427 did not). In females, 1445 genes were changed in common between WT and KO, with all but 2 genes (Pla2g7 and Zfp385b) changing in the same direction. In both male and female KO animals, oxidative phosphorylation and ribosomal pathways were most significant, though the direction of change in the DEGs was opposite. Conclusion: In skeletal muscle, sex highly influences the genes and pathways changed in response to DIO. Even among common pathways identified, the response between males and females differed. Loss of MuRF1 results in common and unique transcript changes in and between males and females under normal conditions and in DIO.

Chronic Stimulation of Arcuate Kiss1 Neurons Decreases Bone Mass in Female Mice

Loss of peripheral estrogen in postmenopausal women is often associated with decreased physical activity and loss of bone mass, leading to an increased risk of metabolic diseases, osteoporosis, and skeletal fragility. While it is well-established that loss of peripheral estrogen signaling results in bone loss, we previously found that eliminating central estrogen signaling paradoxically results in an unexpected massive increase in bone mass only in female mice. Specifically, deletion of estrogen receptor alpha (ERα) signaling in kisspeptin 1 (Kiss1) expressing neurons of the arcuate nucleus (ARCKiss1) increases bone mass at the expense of reproduction in female mice. Currently, the mechanisms and the neurocircuits that modulate these unexpected responses are unknown. Here, to begin addressing these questions, we asked if changing the neuronal output of ARCKiss1 neurons using chemogenetic manipulation of ARCKiss1 neurons might also alter bone mass and locomotion in female mice. To do this, we delivered stimulatory (AAV2-hM3Dq-mCherry) designer receptors exclusively activated by designer drugs (DREADDs) to the ARC of wild type and Kiss1-Cre+ (Kiss1-CrehM3q-DREADDs) female mice and asked if chronic activation of ARCKiss1 neurons might alter bone mass as analyzed by standard ex-vivo µCT imaging. Clozapine N-oxide (CNO) was delivered for 22 days (0.1 mg/mL). We also leveraged the ANY-Maze system to assess home cage activity over an extensive 96-hour period. Acute activation of ARCKiss1 tended to decrease home cage activity by nearly 40% in Kiss1-CrehM3q-DREADDs mice during the dark period compared to WT females. Interestingly, chronic activation of ARCKiss1 neurons significantly lowered trabecular bone volume by nearly 30%. Current studies are underway to ask if inhibiting ARCKiss1 neurons results in increased bone mass. Our findings collectively suggest that the neuronal activity of ARCKiss1 neurons is sufficient to shift energy allocation away from locomotion and bone-building to maximize reproductive capacity. We speculate that the widely used SERM in breast cancer treatment, Tamoxifen, might exert its bone sparing effect by silencing ARCKiss1 neurons.

Establishing a Role for Sarcospan as an Obesity‐Susceptibility Gene in Mice

This study verifies genome-wide association studies that connect variants in the SSPN locus, encoding the protein sarcospan, with increased waist circumference and heightened susceptibility to cardiovascular disease. We hypothesized that deletion of SSPN would protect SSPN-null mice from diet-induced obesity. To test this SSPN-null and WT (C57BL/6J) male and female mice were subjected to high fat diet (HFD) (60%) and compared to equivalent controls. After 16 weeks HFD, mice were assessed for changes in anthropometric indices, histology, glucose handling, blood chemistry, cardiac function and tissues for ectopic lipid deposition. Our in vivo findings suggest that SSPN plays a role in adiposity and specific variants in the gene locus may increase/decrease susceptibility to central adiposity thereby heightening risk of cardiometabolic disease. SSPN-null male mice were partially protected from weight gain, liver steatosis and had slightly improved glucose handling compared to WT males after HFD. After HFD, female SSPN-null mice had only a small increase in body weight compared to WT females. EchoMRI was used to measure % Fat per body weight. WT female mice had significantly higher % Fat after HFD compared to WT controls, while % Fat in female SSPN-null mice was largely similar after the HFD regimen. Blood chemistry measurements suggested liver damage in HFD WT mice since alanine aminotransferase (ALT) levels (U/L) were significantly elevated with levels higher in males than females. SSPN-null mice of both genders and diets however exhibited similar ALT levels. Cardiac function remained largely preserved in all groups of mice after HFD, therefore extended studies may be needed. In conclusion, deletion of the SSPN gene protects both females and males from diet-induced obesity and future studies will ascertain how it affects cardiovascular health.

Basal renal phenotype and response to induction of aristolochic acid nephropathy in mice lacking the neutral amino acid transporter B0AT1 (SLC6A19)

Background B0AT1 (SLC6A19) mediates the reabsorption of neutral amino acids, including branched-chain amino acids (BCAA), in the intestine and kidney (expressed in early and mid-proximal tubule - S1/S2). Dietary restriction of BCAA has been proposed to improve metabolic health by inhibiting mTOR and inducing autophagy. Here we characterized basal kidney function and determined whether B0AT1 KO protects the kidney from aristolochic acid-induced nephropathy (AAN), a model of proximal tubular injury. Methods In male B0AT1 KO and littermate wild-type mice (WT) mice, we measured fractional renal amino acid excretion and performed renal FITC-sinistrin clearance studies under terminal anesthesia to determine renal creatinine handling. A set of female mice was injected with aristolochic acid (3 mg/kg i.p.) or vehicle every 3 days for 3 weeks, and kidneys were harvested 3 weeks later for RTqPCR analyses (n=7-16 mice per group). Results RTqPCR and Western blotting confirmed absence of renal B0AT1 mRNA in KO, associated with increased fractional renal excretion of neutral amino acids, including BCAA (to 20-40%), whereas plasma levels of BCAA were not changed. In vehicle-treated mice, plasma creatinine was slightly higher in KO vs WT (0.080±0.002 vs 0.074±0.002 mg/dl; *P<0.05). Clearance studies revealed a trend for lesser fractional excretion of creatinine in KO vs WT (P=0.057), suggesting lesser creatinine secretion contributed to higher plasma levels. Urinary albumin/creatinine ratio (UACR) and urinary glucose/creatinine ratio (UGCR) were tripled and doubled in female KO vs WT (180±18 vs 62±5 ng/μg*; 1.35±0.26 vs 0.70±0.06 mg/mg*), respectively, suggesting a modest impairment in proximal tubular albumin and glucose reabsorption. The renal expression of the alternative neutral amino acid transporter B0AT3 (SLC6A18), expressed in S1-S3, was reduced in KO (to 32±5% of WT*). This may be due to KO model generation, as B0AT1 and B0AT3 are co-expressed side-by-side on the same chromosome. Whole kidney CCR2 and KIM-1 mRNA expression were modestly higher in KO vs WT, which may reflect enhanced amino acid and glucose transport in the vulnerable S3 segment in the outer medulla. In the absence of robust effects on plasma creatinine, AAN significantly increased UACR vs vehicle in WT (+164±39 vs +52±14 ng/μg*) but not in KO (-18±30 vs +57±49 ng/μg) when measured after completion of injections. Moreover, increases in CCR2 and TIMP1 expression in response to AAN vs vehicle observed in WT (+206±28%; +203±45%) were attenuated in KO (+54±20%*; +81±39%*). Conclusions Genetic deletion of B0AT1 in male and female mice induces distinct effects on kidney transport beyond amino acids. Follow up studies with higher doses of AA are needed to more fully evaluate the nephroprotective potential of B0AT1 inhibition.

The 5xFAD Mouse Model of Alzheimer's Disease Displays Early Stunting of Neuronal Development in the Dentate Gyrus: a Potential Novel Treatment Window for Dementias Associated with Amyloidosis?

Approximately 5.8 million people over the age of 65 are living with Alzheimer's Disease (AD), and the number of individuals afflicted with AD is projected to double by 2050. Hallmarks of AD are intraneural plaques of amyloid-beta and neurofibrillary tangles with concomitant and progressive decline in neuro-cognitive function. Currently, there is no cure for AD, which stems partly from the sporadic nature of the disease and “after-the-fact” treatments which have failed to remediate disease progression. The goal of our lab is to elucidate early markers of AD onset prior to overt disease occurrence. To this end, we are using the 5XFAD transgenic mouse model which displays an aggressive form of amyloidosis and associated dementias, which develop because of insertion of the five most common genetic mutations associated with aberrant amyloid processing found in AD. Typically, intraneural amyloid plaques develop at 2-4 months of age in 5xFAD mice and cognitive deficits are observed at 4 to 6 months of age. The objective of our current study was to determine characteristics of hippocampal neuronal arborization in 5xFAD mice and non-transgenic (WT) littermates at at 15 days, 1-month, and 3-months of age, which is thought to be a pre-symptomatic period. Mice were euthanized in strict accordance with AALAC guidelines and approved protocols. Isolated brains from male and female 5xFAD and WT mice were fixed and Golgi stained (Rapid Stain Golgi kit, FDNeurotechnologies). Fixed tissue was sectioned by a cryostat (100 um), and slides were imaged using a Leica DM6000 at 40X. Images were then reassembled in Z-stacks and analyzed with FIJI. A Sholl analysis was performed on the hippocampal subregions of the dentate gyrus (DG), cortical area 1 (CA1), and cortical area 3 (CA3) where dendritic length was the primary endpoint. Results showed that there was no shortening at 15 days and 3-months in 5XFAD versus WT, but there was a shortening of neurites in the 5XFAD versus WT at 1-month of age, mainly within the DG (p = 0.04969; N = 6). At 1-month, the dentate gyrus neurons were 108.2μm (p = 0.04969; N = 6), or 5% shorter, with observed simplification of arborization. The other hippocampal subregions did not display significant alterations in neuronal development. These results show that at an extreme early age, 5xFAD mice already show a lack of neuronal development that occurs prior to intraneuronal amyloid plaque appearance, especially in a hippocampal subregion known to be vulnerable to AD-related neuronal loss. This subregion is especially vulnerable due to the fact that amyloid beta accumulates in this region initially, which could lead to the noted shortening in neuronal length. The DG receives input from the entorhinal cortex and is relayed to CA3 via mossy fibers, which is imperative for long-term potentiation and spatial memory. This suggests that the so-called pre-symptomatic period will have to be re-evaluated. Furthermore, our data suggest that a potential window for treatments have been found which may aid in developing novel therapeutic regimens to slow or prevent progression of AD symptomology associated with amyloidosis.