Respective Wild-type Controls Research Articles

Abstract 1036: Mechanisms Of Foamy Monocyte Formation In Atherosclerosis

Diseases linked to atherosclerosis are the leading cause of death in the United States. Transmigration of monocytes across the endothelial layer and their differentiation into macrophages play an important role in the initiation and progression of atherosclerosis. These monocyte-derived macrophages internalize modified LDL in the arterial wall and drive the pathogenesis of atherosclerosis. Recent studies have shown that pharmacological depletion of foamy monocytes inhibits atherosclerosis development in mice, identifying monocyte uptake of plasma LDL as a therapeutic target in atherosclerosis. However, the precise mechanisms by which monocytes internalize plasma LDL remain unknown. The goal of our study was to investigate the role, and relative contribution, of macropinocytosis and scavenger receptors ( Cd36 and Sr-a ) to foamy monocyte formation. The presence of foamy monocytes was confirmed in hypercholesterolemic mice and humans via flow cytometry analysis of Nile Red fluorescence. High resolution scanning electron microscopy and flow cytometry analysis of dextran internalization confirmed macropinocytosis stimulation in THP-1 and primary murine monocytes. Stimulation of macropinocytosis in monocytes induced uptake of both native LDL and oxLDL, leading to foamy monocyte formation in vitro . Pharmacological (EIPA) and genetic inhibition of macropinocytosis ( Lysm - CreER T2+/- Nhe1 -/- ) inhibited foamy monocyte formation (58.9%) in hypercholesterolemic mice in vivo . RT-PCR confirmed expression of Cd36 and Sr-a in monocytes and deletion of Cd36 and Sr-a inhibited foamy monocyte formation in hypercholesterolemic mice (10.3%) compared to wild type controls. FACS and bulk RNA sequencing characterized monocyte subsets including classical, intermediate, and non-classical monocytes in hypercholesterolemic Cd36 -/- / Sr-a -/- and Lysm - CreER T2+/- Nhe1 -/- mice and their respective wild type controls. Mechanistic studies identified NADPH oxidase 2 (Nox2) and downstream redox regulation of actin-cytoskeleton as a major stimulator of monocyte macropinocytosis. These results provide novel insights into the mechanisms of foamy monocyte formation and potentially identify new therapeutic targets in the treatment of atherosclerosis.

Brown Adipose Tissue-Derived GDF15 Mediates Energy Homeostasis in a Sex-Dependent Manner

Growth differentiation factor 15 (GDF15) is a distant member of the transforming growth factor-β (TGFβ) superfamily that can be expressed in several tissues. Studies have shown that in response to prolonged high-fat feeding and cold stress GDF15 can be induced in brown adipocytes. Importantly, administration of recombinant GDF15 in rodents and non-human primates (Xiong, Y. et al. Long-acting MIC-1/GDF15 molecules to treat obesity: Evidence from mice to monkeys. Sci. Transl. Med. 9, eaan8732 (2017) reduces food intake and body mass. Indeed, we have recently shown that in a model of mitochondrial stress, brown adipose tissue (BAT)-derived GDF15 is required to maintain energy homeostasis and cold-induced thermogenesis in mice. Nonetheless, the role of BAT-derived GDF15 during physiological stress is not known. In the present study, we tested the hypothesis that BAT-derived GDF15 is required to regulate cold-induced thermogenesis and energy homeostasis in high-fat fed mice. To test this hypothesis, we generated mice selectively lacking GDF15 in thermogenic adipocytes (brown and beige) (GDF15 BKO). GDF15 BKO male and female mice had normal body weight, body composition and glucose homeostasis under baseline conditions. In response to 4 hours of cold exposure following fasting, the decline in core body temperature in GDF15 BKO female mice was similar to that observed in their wild type (WT) counterparts. Conversely, GDF15 BKO male mice became hypothermic. Surprisingly, after 12 weeks on a high-fat diet (HFD), GDF15 BKO female mice weighed significantly less and had reduced fat mass relative to their WT controls, while male mice had increased body weight and fat accumulation. Furthermore, resistance to DIO in female mice was associated with increased energy expenditure, while food intake and locomotor activity were unchanged. When ovariectomized, GDF15 BKO females experienced similar weight gain and fat accumulation as WT mice and no longer had increased energy expenditure. Of note, estrogen receptor alpha ( Esr1) expression in BAT was significantly repressed in GDF15 BKO male mice fed high-fat diet but was maintained in female mice relative to their respective WT controls. Taken together, our data suggest that GDF15 expression in BAT is dispensable for thermoregulation in female mice but is required to regulate core body temperature in males. Furthermore, while GDF15 BKO male mice are susceptible, female mice are resistant to DIO via mechanisms likely involving sex hormones and increased resting metabolic rates. NIH DK125405 to R.O.P.; Diabetes Research Training Program funded by the NIH (T32 DK112751) to JJ and by the NIH 1R25GM116686 to L.M.G.P. 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.

Optimization of carbon and nitrogen partitioning in DP202216 maize hybrids

ContextThe photoassimilates and nitrogen (N) allocation in grain crops can be studied via the harvest index (HI) and the N harvest index (NHI). These two variables should be considered in breeding programs seeking more effective use of resources in maize (Zea mays L.) and other grain crops. Molecular biotechnology has emerged as a complement to improve not only maize grain yield but also resource utilization efficiency. A recent study reported a beneficial impact of increasing and extending the expression of a maize MADS-box transcription factor (zmm28) on the NHI. ObjectiveThis research aimed to further identify enhancements of C-N partitioning (HI, NHI) in DP202216 maize hybrids recognizing the main physiological and morphological traits associated to the C-N dynamics via in-season intra-canopy measurements. MethodsTwo DP202216 elite hybrids were evaluated with their respective wild-type (WT) controls during two field growing seasons under two N regimes. The N nutrition index (NNI), as crop N status, was quantified to avoid misinterpretations of the results. ResultsThe DP202216 trait showed, on average, a ∼6% increase in the HI with a concomitant positive impact of ∼9% in the NHI across hybrids, N regimes, and seasons. Those increases were consistent at different crop NNI. After accounting for the NNI, it was shown that the increases in both HI and NHI were supported by the accumulation of more N in leaves (mainly in both lower and middle sections of the canopy vertical profile) and more water-soluble carbohydrates (WSC) in stems before flowering, combined with more aggressive post-anthesis remobilization. ConclusionIn this study, it was shown an increase in the efficient use of crop resources in DP202216 hybrids with respect to their isogenic WT controls at similar NNI via increments in C and N partitioning (HI and NHI). ImplicationsThis physiological and morphological understanding of C- and N-related traits addressed the challenge of optimizing the effective use of resources in maize breeding programs. To better understand traits by management interactions with yield and yield stability, DP202216 hybrid evaluations across a broad range of commercially relevant N scenarios would be valuable.

Characterization of metabolic phenotypes and distinctive genes in mice with low-weight gain.

Being overweight exacerbates various metabolic diseases, necessitating the identification of target molecules for obesity control. In the current study, we investigated common physiological features related to metabolism in mice with low weight gain: (1) G protein-coupled receptor, family C, group 5, member B-knockout; (2) gastric inhibitory polypeptide receptor-knockout; and (3) Iroquois-related homeobox3-knockout. Moreover, we explored genes involved in metabolism by analyzing differentially expressed genes (DEGs) between low-weight gain mice and the respective wild-type control mice. The common characteristics of the low-weight gain mice were low inguinal white adipose tissue (iWAT) and liver weight despite similar food intake along with lower blood leptin levels and high energy expenditure. The DEGs of iWAT, epididymal (gonadal) WAT, brown adipose tissue, muscle, liver, hypothalamus, and hippocampus common to these low-weight gain mice were designated as candidate genes associated with metabolism. One such gene tetraspanin 7 (Tspan7) from the iWAT was validated using knockout and overexpressing mouse models. Mice with low Tspan7 expression gained more weight, while those with high Tspan7 expression gained less weight, confirming the involvement of the Tspan7 gene in weight regulation. Collectively, these findings suggest that the candidate gene list generated in this study contains potential target molecules for obesity regulation. Further validation and additional data from low-weight gain mice will aid in understanding the molecular mechanisms associated with obesity.

THU051 GH Resistance And Adipose Tissue: Multispecies Measurement Of Morphology And Transcriptome

Abstract Disclosure: J.A. Young: None. A. Hinrichs: None. S.R. Bell: None. M. Soneson: None. D.K. Geitgey: None. E. Wolf: None. E.O. List: None. J.J. Kopchick: None. D.E. Berryman: None. Laron Syndrome (LS) is a condition caused by mutations in the growth hormone (GH) receptor gene that renders the individual GH resistant. LS was first described in humans in the 1960’s, and since then, animal models of LS have been developed, including pigs (GHR-KO pigs). One of the notable characteristics of LS, in both humans and animals, is obesity without some of its comorbidities, such as increased cancer and diabetes. The aim of this study was to examine the subcutaneous and visceral adipose tissue of GH resistant pigs and a human with LS using histology and transcriptomics and make cross-species and cross-depot comparisons where possible. Adipocyte sizing analysis showed that GH resistance resulted in a trend towards increased cell sizes in human subcutaneous (SubQ) and visceral (Visc) depots, and a significant increase in pig Visc. Interestingly, the human adipocyte size histogram was similar to that of the pig across genotypes and depots. When examining the adipose transcriptome, PCA analysis showed a strong species clustering, but not a strong depot or genotype clustering. Differentially expressed gene (DEG) analysis comparing GHRKO pigs to their respective wild type controls showed that IGF1 expression was decreased in both depots, as expected, and 38 other genes were also significantly altered in GHR-KO pigs compared to controls, in both depots. Enrichment analysis showed a decrease in an extracellular matrix term in the GHR-KO SubQ compared to controls, while terms associated with fatty acid and carbohydrate metabolism were increased in GHR-KO SubQ. When comparing GHR-KO visceral to WT, terms associated with IGF signaling and longevity regulation had decreased expression, while immune activation and cell motility were increased. Pathway analysis generally agreed with these results, but also showed a decrease in multiple signaling pathways including AMPK and PI3K-Akt in both depots. Paradoxically, the JAK-STAT signaling pathway was increased in GHR-KO Visc compared to controls. Depot differences were also explored using this dataset. Within each genotype, the gene expression of the SubQ and Visc depots resulted in no significant DEGs between depots in the WT pigs, while GHRKO pigs had 24 DEGs between SubQ and Visc. Enrichment analysis and pathway analysis showed no significant results in either depot, presumably due to the low number of DEGs. In conclusion, GHR-KO pigs had expected changes in adipocyte size and expression of metabolic and extracellular matrix genes, but other unexpected changes were also present, including changes to cell motility-related genes. There were few differences between depots within each genotype, indicating that depots are equally affected by GH. Further studies are required to understand the functional changes to adipose tissue caused by LS and the effectiveness of pigs as an LS model. Presentation: Thursday, June 15, 2023

Abstract P3105: Stress Exacerbates Ischemia/reperfusion Injury In The Female Heart Through The Glucocorticoid Receptor Signaling In Cardiomyocytes

It is estimated that over 30,000 women younger than 55-years-old die of acute myocardial infarction (AMI) in the United States annually. Clinical data suggest that exposure to mental stress exacerbates the outcomes of an AMI in women. However, a knowledge gap exists regarding the molecular mechanisms whereby stress contributes to AMI in women and how they differ in men. The present project aims to define the mechanisms underlying the link between stress, glucocorticoids (stress hormones), the glucocorticoid receptor (GR) signaling, and the worse outcomes of AMI in females than in their male counterparts. We challenged male and female transgenic mice lacking GR in cardiomyocytes (cardioGRKO) and their respective wild-type controls in a murine model of "mental" stress followed by ischemia/reperfusion (AMI) injury. Our data show that stress exacerbated the response to AMI injury in control females compared to their male counterparts. Females without cardiomyocyte GR were protected and presented significantly reduced infarct areas compared to cardioGRKO males and control males and females with intact GR signaling. These results demonstrated that stress directly affected the heart and that these effects are sexually dimorphic. The severity of the infarct and cardiomyocyte death was further exacerbated during the highest estrogen activity phase (proestrus) of the female cycle. Our in vitro studies additionally supported these data, showing that glucocorticoids block estrogen's pro-survival effects on cardiomyocytes in hypoxia/re-oxygenation conditions in a mechanism dependent upon GR inhibition of estrogen regulation of antioxidant pathways, including decreased levels of the master regulator of the antioxidant response, nuclear factor-erythroid factor 2-related factor 2 (Nrf2), and increased superoxide and lipid peroxides production. Our in vivo models displayed similar findings. Our study is the first to test the impact of mental stress on activating glucocorticoid signaling in the heart in the setting of AMI. This study is relevant to human health as a proof of concept that exposure to high stress levels aggravates AMI in premenopausal hearts by cardiomyocyte GR-regulated mechanisms that may be targeted to develop therapies tailored to women.

Resistin-like Molecule α and Pulmonary Vascular Remodeling: A Multi-Strain Murine Model of Antigen and Urban Ambient Particulate Matter Co-Exposure.

Pulmonary hypertension (PH) has a high mortality and few treatment options. Adaptive immune mediators of PH in mice challenged with antigen/particulate matter (antigen/PM) has been the focus of our prior work. We identified key roles of type-2- and type-17 responses in C57BL/6 mice. Here, we focused on type-2-response-related cytokines, specifically resistin-like molecule (RELM)α, a critical mediator of hypoxia-induced PH. Because of strain differences in the immune responses to type 2 stimuli, we compared C57BL/6J and BALB/c mice. A model of intraperitoneal antigen sensitization with subsequent, intranasal challenges with antigen/PM (ovalbumin and urban ambient PM2.5) or saline was used in C57BL/6 and BALB/c wild-type or RELMα-/- mice. Vascular remodeling was assessed with histology; right ventricular (RV) pressure, RV weights and cytokines were quantified. Upon challenge with antigen/PM, both C57BL/6 and BALB/c mice developed pulmonary vascular remodeling; these changes were much more prominent in the C57BL/6 strain. Compared to wild-type mice, RELMα-/- had significantly reduced pulmonary vascular remodeling in BALB/c, but not in C57BL/6 mice. RV weights, RV IL-33 and RV IL-33-receptor were significantly increased in BALB/c wild-type mice, but not in BALB/c-RELMα-/- or in C57BL/6-wild-type or C57BL/6-RELMα-/- mice in response to antigen/PM2.5. RV systolic pressures (RVSP) were higher in BALB/c compared to C57BL/6J mice, and RELMα-/- mice were not different from their respective wild-type controls. The RELMα-/- animals demonstrated significantly decreased expression of RELMβ and RELMγ, which makes these mice comparable to a situation where human RELMβ levels would be significantly modified, as only humans have this single RELM molecule. In BALB/c mice, RELMα was a key contributor to pulmonary vascular remodeling, increase in RV weight and RV cytokine responses induced by exposure to antigen/PM2.5, highlighting the significance of the genetic background for the biological role of RELMα.

Impaired mitochondrial quality control in skeletal muscles from C57 and D2 mdx model of Duchenne Muscular Dystrophy

Mitochondrial dysfunction is an early pathological feature of Duchenne muscular dystrophy (DMD) and precedes myopathy. Balanced mitochondrial quality control (e.g., fission, fusion, biogenesis, autophagy, and mitophagy) is essential to maintain the overall mitochondrial health and skeletal muscle function. DBA/2J mdx (D2- mdx) has emerged as a more relevant mouse model of DMD as it more closely mirrors early stage of DMD pathology in humans than the C57BL/10 mdx (C57 -mdx) model. The purpose of this study was to determine whether the expression of proteins responsible for mitochondrial quality control were dysregulated in skeletal muscles from these two mouse models of DMD. We hypothesized that the expression of protein markers of mitochondrial fission and mitophagy would be higher, while markers of mitochondrial fusion would be lower in both models of DMD with greater alterations in D2- mdx mice. 8-10-week-old male C57 -mdx and D2- mdx mice, and their respective wildtype (WT) mice were used in this study. Muscle strength was measured using hang wire test. Gastrocnemius muscles were collected for immunoblot analysis to assess mitochondrial quality control proteins. Hang wire impulse, an indicator of muscle strength, was significantly lower in both models of DMD in comparison to their WT controls (main effect of mdx, P = 0.01). Regarding mitochondrial fission, while there was no difference in protein expression and phosphorylation (Ser 616) of Dynamin-Related Protein 1 (Drp1) between any groups, mitochondrial fission 1 protein (Fis1) was markedly higher in skeletal muscles from both models of DMD when compared to WT controls (main effect of mdx, P=0.0001). Greater expression of Fis1 was observed in D2- mdx than C57- mdx when compared to their respective WT controls (~5.7-fold vs. 3.8-fold). Furthermore, mitochondrial fusion marker Mitofusion 1 (Mfn1) was significantly lower in both models of DMD (main effect of mdx, P=0.029), however, it appears to be mainly driven by the reduction in D2-mdx (mdx x strain interaction, P=0.019). No difference of Mfn1 expression was found in C57-mdx mice. Finally, expression of protein markers of mitochondrial biogenesis (PGC1 a) and autophagy (LC3B) were higher in both models of DMD when compared to their respective WT controls (main effect of mdx, P=0.0001 and P=0.001, respectively). In conclusion, these data suggest that there is dysregulated mitochondrial dynamics towards pro-fission state in skeletal muscle from mdx mice with an exacerbated effect in D2- mdx model, which may contribute to the muscle weakness phenotype associated with DMD This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Genetic predisposition to Alzheimer's disease alters inflammasome activity after traumatic brain injury

Traumatic Brain Injury (TBI) is a major cause of death and disability in the US and a recognized risk factor for the development of Alzheimer's disease (AD). The relationship between these conditions is not completely understood, but the conditions may share additive or synergistic pathological hallmarks that may serve as novel therapeutic targets. Heightened inflammasome signaling plays a critical role in the pathogenesis of central nervous system injury (CNS) and the release of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck from neurons and activated microglia contribute significantly to TBI and AD pathology. This study investigated whether inflammasome signaling after TBI was augmented in AD and whether this signaling pathway impacted biochemical and neuropathological outcomes and overall cognitive function. Five-month-old, 3xTg mice and respective wild type controls were randomized and underwent moderate controlled cortical impact (CCI) injury or served as sham/uninjured controls. Animals were sacrificed at 1 hour, 1 day, or 1 week after TBI to assess acute pathology or at 12 weeks after assessing cognitive function. The ipsilateral cerebral cortex was processed for inflammasome protein expression by immunoblotting. Mice were evaluated for behavior by open field (3 days), novel object recognition (2 weeks), and Morris water maze (6 weeks) testing after TBI. There was a statistically significant increase in the expression of inflammasome signaling proteins Caspase-1, Caspase-8, ASC, and interleukin (IL)-1β after TBI in both wild type and 3xTg animals. At 1-day post injury, significant increases in ASC and IL-1β protein expression were measured in AD TBI mice compared to WT TBI. Behavioral testing showed that injured AD mice had altered cognitive function when compared to injured WT mice. Elevated Aβ was seen in the ipsilateral cortex and hippocampus of sham and injured AD when compared to respective groups at 12 weeks post injury. Moreover, treatment of injured AD mice with IC100, an anti-ASC monoclonal antibody, inhibited the inflammasome, as evidenced by IL-1β reduction in the injured cortex at 1-week post injury. These findings show that the inflammasome response is heightened in mice genetically predisposed to AD and suggests that AD may exacerbate TBI pathology. Thus, dampening inflammasome signaling may offer a novel approach for the treatment of AD and TBI.

Trans-cortical vessels in the mouse temporal bulla bone are a means to recruit myeloid cells in chronic otitis media and limit peripheral leukogram changes.

Chronic otitis media, inflammation of the middle ear, is a sequel to acute otitis media in ∼8% of children. Chronic otitis media with effusion is the most common cause of childhood deafness and is characterised by effusion of white blood cells into the auditory bulla cavity. Skull flat bones have trans-cortical vessels which are responsible for the majority of blood flow in and out of the bone. In experimental models of stroke and aseptic meningitis there is preferential recruitment of myeloid cells (neutrophils and monocytes) from the marrow in skull flat bones. We report trans-cortical vessels in the mouse temporal bone connect to the bulla mucosal vasculature and potentially represent a means to recruit myeloid cells directly into the inflamed bulla. The mutant mouse strains Junbo (Mecom Jbo/+ ) and Jeff (Fbxo11 Jf/+ ) develop chronic otitis spontaneously; Mecom Jbo/+ mice have highly cellular neutrophil (90%) rich bulla exudates whereas Fbxo11 Jf/+ mice have low cellularity serous effusions (5% neutrophils) indicating differing demand for neutrophil recruitment. However we found peripheral leukograms of Mecom Jbo/+ and Fbxo11 Jf/+ mice are similar to their respective wild-type littermate controls with healthy bullae and infer preferential mobilization of myeloid cells from temporal bulla bone marrow may mitigate the need for a systemic inflammatory reaction. The cytokines, chemokines and haematopoietic factors found in the inflamed bulla represent candidate signalling molecules for myeloid cell mobilization from temporal bone marrow. The density of white blood cells in the bulla cavity is positively correlated with extent of mucosal thickening in Mecom Jbo/+ , Fbxo11 Jf/+ , and Eda Ta mice and is accompanied by changes in epithelial populations and bone remodelling. In Mecom Jbo/+ mice there was a positive correlation between bulla cavity WBC numbers and total bacterial load. The degree of inflammation varies between contralateral bullae and between mutant mice of different ages suggesting inflammation may wax and wane and may be re-initiated by a new wave of bacterial infection. Clearance of white blood cells and inflammatory stimuli from the bulla cavity is impaired and this may create a pro-inflammatory feedback loop which further exacerbates otitis media and delays its resolution.

Kynurenine metabolism is altered in mdx mice: a potential muscle to brain connection.

What is the central question in this study? Promoting muscle health with regular aerobic exercise can improve mental health through a kynurenine metabolic pathway: do conditions of muscle disease such as muscular dystrophy negatively influence this pathway? What is the main finding and its importance? The DBA/2J mdx model of Duchenne muscular dystrophy exhibits altered kynurenine metabolism with less kynurenic acid and peroxisome proliferator-activated receptor-γ coactivator 1-α and higher levels of tumour necrosis factor α mRNA- results associated with anxiety-like behaviour. Regular exercise can direct muscle kynurenine (KYN) metabolism toward the neuroprotective branch of the kynurenine pathway thereby limiting the accumulation of neurotoxic metabolites in the brain and contributing to mental resilience. However, the effect of muscle disease on KYN metabolism has not yet been investigated. Previous work has highlighted anxiety-like behaviours in approximately 25% of patients with Duchenne muscular dystrophy (DMD), possibly due to altered KYN metabolism. Here, we characterized KYN metabolism in mdx mouse models of DMD. Young (8-10week old) DBA/2J (D2) mdx mice, but not age-matched C57BL/10 (C57) mdx mice, had lower levels of circulating kynurenic acid (KYNA) and lower KYNA:KYN ratio compared with their respective wild-type (WT) controls. While both C57 and D2 mdx mice displayed signs of anxiety-like behaviour, spending more time in the corners of the arena during a novel object recognition test, this effect was more prominent in D2 mdx mice. Correlational analysis detected a significant negative association between KYNA:KYN levels and time spent in corners in D2 mice, but not C57 mice. In extensor digitorum longus muscles from D2 mdx mice, but not C57 mdx mice, we found lowered protein levels of peroxisome proliferator-activated receptor-γ coactivator 1-α and kynurenine amino transferase-1 enzyme when compared with WT. Furthermore, D2 mdx quadriceps muscles had the highest level of tumour necrosis factor α expression, which is suggestive of enhanced inflammation. Thus, our pilot work shows that KYN metabolism is altered in D2 mdx mice, with a potential contribution from altered muscle health.

Sex-specific Regulation of Prolactin Secretion by Pituitary Bradykinin Receptors.

Sex differences in the control of prolactin secretion are well documented. Sex-related differences in intrapituitary factors regulating lactotroph function have recently attracted attention. Sex differences in prolactinoma development are well documented in clinic, prolactinomas being more frequent in women but more aggressive in men, for poorly understood reasons. Kallikrein, the enzyme releasing kinins has been found in the pituitary, but there is no information on pituitary kinin receptors and their function. In the present work, we characterized pituitary bradykinin receptors (BRs) at the messenger RNA and protein levels in 2 mouse models of prolactinoma, Drd2 receptor gene inactivation and hCGβ gene overexpression, in both males and females, wild type or genomically altered. BR B2 (B2R) accounted for 97% or more of total pituitary BRs in both models, regardless of genotype, and was present in lactotrophs, somatotrophs, and gonadotrophs. Male pituitaries displayed higher level of B2R than females, regardless of genotype. Pituitary B2R gene expression was downregulated by estrogen in both males and females but only in females by dopamine. Activation of B1R or B2R by selective pharmacological agonists induced prolactin release in male pituitaries but inhibited prolactin secretion in female pituitaries. Increased B2R content was observed in pituitaries of mutated animals developing prolactinomas, compared to their respective wild-type controls. The present study documents a novel sex-related difference in the control of prolactin secretion and suggests that kinins are involved, through B2R activation, in lactotroph function and prolactinoma development.

148-OR: Partial Knockout of Skeletal Muscle Dynamin-Related Protein 1 Improves Insulin Sensitivity in Diet-Induced Insulin Resistant Mice, but Not in Normal Control Mice

Dynamin-related protein 1 (Drp1) is the key regulator of mitochondrial fission. We and others have reported excessive Drp1 activity in skeletal muscle from both animals and humans with insulin resistance, which is strongly correlated to impaired skeletal muscle insulin sensitivity. To examine whether Drp1 directly regulates skeletal muscle insulin sensitivity and whole-body glucose homeostasis, we generated tamoxifen-inducible skeletal muscle-specific heterozygous Drp1 partial knockout mice (mDrp1+/-) . Male mDrp1+/- and wildtype (WT) mice were fed with either a high-fat diet (HFD) or low-fat diet (LFD) for four weeks, received tamoxifen injections for five consecutive days, and remained on their respective diet for another four weeks. Improvements in whole-body glucose tolerance and insulin sensitivity were observed in HFD-fed mDrp1+/- mice (P<0.05) , but not in LFD-fed mDrp1+/- mice, when compared to their respective WT controls. Further, HFD-induced impairment in skeletal muscle insulin-stimulated Akt Ser473 phosphorylation (P<0.05) was mitigated in HFD-fed mDrp1+/- mice. In contrast, insulin-stimulated Akt Ser473 phosphorylation was reduced in LFD-fed mDrp1+/- mice compared to LFD-fed WT mice (P<0.05) . mDrp1+/- mice exhibited more fused and interconnected mitochondrial networks in skeletal muscle than WT mice. The examination of mitochondrial function revealed no change in mitochondrial respiration but reductions in mitochondrial Complex I and II and pyruvate dehydrogenase derived H2O2 production in HFD-fed mDrp1+/- mice compared to HFD-fed WT mice (P<0.05) . In conclusion, our data suggest that partial knockout of skeletal muscle Drp1 improves skeletal muscle insulin sensitivity and whole-body glucose homeostasis in diet-induced insulin-resistant mice but not in normal control mice. These improvements are, at least in part, due to reduced mitochondrial-derived H2O2 production. Disclosure B.A.Kugler: None. N.Lin: None. P.D.Nguyen: None. A.Ali: None. A.Sesay: None. B.Kalemba: None. H.Sesaki: None. K.Zou: None. Funding National Institutes of Health (R15DK131512) Diabetes Action Research and Education Foundation Grant (#505)

CADASIL mutations sensitize the brain to ischemia via spreading depolarizations and abnormal extracellular potassium homeostasis.

Cerebral autosomal dominant arteriopathy, subcortical infarcts, and leukoencephalopathy (CADASIL) is the most common monogenic form of small vessel disease characterized by migraine with aura, leukoaraiosis, strokes, and dementia. CADASIL mutations cause cerebrovascular dysfunction in both animal models and humans. Here, we showed that 2 different human CADASIL mutations (Notch3 R90C or R169C) worsen ischemic stroke outcomes in transgenic mice; this was explained by the higher blood flow threshold to maintain tissue viability compared with that in wild type (WT) mice. Both mutants developed larger infarcts and worse neurological deficits compared with WT mice, regardless of age or sex after filament middle cerebral artery occlusion. However, full-field laser speckle flowmetry during distal middle cerebral artery occlusion showed comparable perfusion deficits in mutants and their respective WT controls. Circle of Willis anatomy and pial collateralization also did not differ among the genotypes. In contrast, mutants had a higher cerebral blood flow threshold, below which infarction ensued, suggesting increased sensitivity of brain tissue to ischemia. Electrophysiological recordings revealed a 1.5- to 2-fold higher frequency of peri-infarct spreading depolarizations in CADASIL mutants. Higher extracellular K+ elevations during spreading depolarizations in the mutants implicated a defect in extracellular K+ clearance. Altogether, these data reveal a mechanism of enhanced vulnerability to ischemic injury linked to abnormal extracellular ion homeostasis and susceptibility to ischemic depolarizations in CADASIL.

DPYD polymorphisms c.496A>G, c.2194G>A and c.85T>C and risk of severe adverse drug reactions in patients treated with fluoropyrimidine-based protocols.

Cancer patients with reduced dihydropyrimidine dehydrogenase (DPD) activity are at increased risk of severe fluoropyrimidine (FP)-related adverse events (AE). Guidelines recommend FP dosing adjusted to genotype-predicted DPD activity based on four DPYD variants (rs3918290, rs55886062, rs67376798 and rs56038477). We evaluated the relationship between three further DPYD polymorphisms: c.496A>G (rs2297595), *6 c.2194G>A (rs1801160) and *9A c.85T>C (rs1801265) and the risk of severe AEs. Consecutive FP-treated adult patients were genotyped for "standard" and tested DPYD variants, and for UGT1A1*28 if irinotecan was included, and were monitored for the occurrence of grade ≥3 (National Cancer Institute Common Terminology Criteria) vs. grade 0-2 AEs. For each of the tested polymorphisms, variant allele carriers were matched to respective wild type controls (optimal full matching combined with exact matching, in respect to: age, sex, type of cancer, type of FP, DPYD activity score, use of irinotecan/UGT1A1, adjuvant therapy, radiotherapy, biological therapy and genotype on the remaining two tested polymorphisms). Of the 503 included patients (82.3% colorectal cancer), 283 (56.3%) developed grade ≥3 AEs, mostly diarrhoea and neutropenia. Odds of grade ≥3 AEs were higher in c.496A>G variant carriers (n = 127) than in controls (n = 376) [OR = 5.20 (95% CI 1.88-14.3), Bayesian OR = 5.24 (95% CrI 3.06-9.12)]. Odds tended to be higher in c.2194G>A variant carriers (n = 58) than in controls (n = 432) [OR = 1.88 (0.95-3.73), Bayesian OR = 1.90 (1.03-3.56)]. c.85T>C did not appear associated with grade ≥3 AEs (206 variant carriers vs. 284 controls). DPYD c.496A>G and possibly c.2194G>A variants might need to be considered for inclusion in the DPYD genotyping panel.

Characterization of Alzheimer's disease-like neuropathology in Duchenne's muscular dystrophy using the DBA/2J mdx mouse model.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by a mutation in the dystrophin gene. In addition to muscle pathology, some patients with DMD will exhibit cognitive impairments with severity being linked to age and type of genetic mutation. Likewise, some studies have shown that mdx mice display impairments in spatial memory compared with wild‐type (WT) controls, while others have not observed any such effect. Most studies have utilized the traditional C57BL/10 (C57) mdx mouse, which exhibits a mild disease phenotype. Recently, the DBA/2J (D2) mdx mouse has emerged as a more severe and perhaps clinically relevant DMD model; however, studies examining cognitive function in these mice are limited. Thus, in this study we examined cognitive function in age‐matched C57 and D2 mdx mice along with their respective WT controls. Our findings show that 8‐ to 12‐week‐old C57 mdx mice did not display any differences in exploration time when challenged with a novel object recognition test. Conversely, age‐matched D2 mdx mice spent less time exploring objects in total as a well as less time exploring the novel object, suggestive of impaired recognition memory. Biochemical analyses of the D2 mdx brain revealed higher soluble amyloid precursor protein β (APPβ) and APP in the prefrontal cortex of mdx mice compared with WT, and lower soluble APPα in the hippocampus, suggestive of a shift towards amyloidogenesis and a similar pathogenesis to Alzheimer's disease. Furthermore, our study demonstrates the utility of the D2 mdx model in studying cognitive impairment.

Metabolomic and transcriptomic profiling of adult mice and larval zebrafish leptin mutants reveal a common pattern of changes in metabolites and signaling pathways

BackgroundLeptin plays a critical role in the regulation of metabolic homeostasis. However, the molecular mechanism and cross talks between leptin and metabolic pathways leading to metabolic homeostasis across different species are not clear. This study aims to explore the effects of leptin in mice and zebrafish larvae by integration of metabolomics and transcriptomics. Different metabolomic approaches including mass spectrometry, nuclear magnetic resonance (NMR) and high-resolution magic-angle-spinning NMR spectrometry were used to investigate the metabolic changes caused by leptin deficiency in mutant ob/ob adult mice and lepb−/− zebrafish larvae. For transcriptome studies, deep RNA sequencing was used.ResultsThirteen metabolites were identified as common biomarkers discriminating ob/ob mice and lepb−/− zebrafish larvae from their respective wild type controls: alanine, citrulline, ethanolamine, glutamine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, putrescine, serine and threonine. Moreover, we also observed that glucose and lipid levels were increased in lepb−/− zebrafish larvae compared to the lepb+/+ group. Deep sequencing showed that many genes involved in proteolysis and arachidonic acid metabolism were dysregulated in ob/ob mice heads and lepb mutant zebrafish larvae compared to their wild type controls, respectively.ConclusionsLeptin deficiency leads to highly similar metabolic alterations in metabolites in both mice and zebrafish larvae. These metabolic changes show similar features as observed during progression of tuberculosis in human patients, mice and zebrafish larvae. In addition, by studying the transcriptome, we found similar changes in gene regulation related to proteolysis and arachidonic acid metabolism in these two different in vivo models.

Abstract P737: Human Homologs Of Differentially Expressed Plasma MicroRNAs In Preclinical Murine Models Target Fundamental Mechanisms Of Cerebral Cavernous Malformation

Introduction: There is a current need for sensitive and specific biomarkers of Cerebral Cavernous Malformation (CCM) that can be readily translated from preclinical to human models to accurately diagnose and monitor disease states and response to novel therapeutics. MiRNAs are small non-coding RNAs that influence gene expression and whose levels can be affected by disease states. We hypothesize that there are human homologs of differentially expressed (DE) miRNA in the plasma of CCM murine models that can be identified in CCM patients. We further hypothesize that these miRNAs have gene targets within previously published CCM transcriptomes, mechanistically linking them to CCM disease. Methods: Plasma miRNAs from homozygous and heterozygous Ccm1 and Ccm3 mice, as well as their respective wild type controls were sequenced and analyzed. Putative gene targets of DE miRNAs [p<0.05, false discovery rate (FDR) corrected] were queried in previously published mouse CCM transcriptomes. The human homologs of the DE miRNAs in the plasma of Ccm1 and Ccm3 mouse models were identified and assessed in the plasma of healthy controls (n=13), CCM1 (n=11), and CCM3 (n=11) patients using RT-qPCR. Results: 5 miRNAs in homozygous and 10 in heterozygous for Ccm1 , while 45 in homozygous and 2 in heterozygous for Ccm3 were DE in the plasma of mouse models (p<0.05, FDR corrected), had gene targets within CCM mouse transcriptomes, and have a human homolog. Preliminary results show mmu-miR-375-3p as DE in both Ccm1 +/- and Ccm3 -/- mice. RT-qPCR assays show that plasma relative quantification values of the human homolog hsa-miR-375-3p were higher in CCM3 than in CCM1 patients ( p <0.001) and in healthy controls ( p <0.05). Conclusion: DE plasma miRNAs identified in mouse models with human homologs and putative gene targets mechanistically implicated in CCM disease may be used as candidate biomarkers for specific clinical contexts.

Inhibiting PHGDH with NCT-503 reroutes glucose-derived carbons into the TCA cycle, independently of its on-target effect

The small-molecule inhibitor of phosphoglycerate dehydrogenase, NCT-503, reduces incorporation of glucose-derived carbons into serine in vitro. Here we describe an off-target effect of NCT-503 in neuroblastoma cell lines expressing divergent phosphoglycerate dehydrogenase (PHGDH) levels and single-cell clones with CRISPR-Cas9-directed PHGDH knockout or their respective wildtype controls. NCT-503 treatment strongly reduced synthesis of glucose-derived citrate in all cell models investigated compared to the inactive drug control and independent of PHGDH expression level. Incorporation of glucose-derived carbons entering the TCA cycle via pyruvate carboxylase was enhanced by NCT-503 treatment. The activity of citrate synthase was not altered by NCT-503 treatment. We also detected no change in the thermal stabilisation of citrate synthase in cellular thermal shift assays from NCT-503-treated cells. Thus, the direct cause of the observed off-target effect remains enigmatic. Our findings highlight off-target potential within a metabolic assessment of carbon usage in cells treated with the small-molecule inhibitor, NCT-503.

Helix-Loop-Helix Factor Id3 (Inhibitor of Differentiation 3): A Novel Regulator of Hyaluronan-Mediated Adipose Tissue Inflammation.

The aim of this study was to unravel mechanisms whereby deficiency of the transcription factor Id3 (inhibitor of differentiation 3) leads to metabolic dysfunction in visceral obesity. We investigated the impact of loss of Id3 on hyaluronic acid (HA) production by the 3 HAS isoenzymes (HA synthases; -1, -2, and -3) and on obesity-induced adipose tissue (AT) accumulation of proinflammatory B cells. Approach and Results: Male Id3-/- mice and respective wild-type littermate controls were fed a 60% high-fat diet for 4 weeks. An increase in inflammatory B2 cells was detected in Id3-/- epididymal AT. HA accumulated in epididymal AT of high-fat diet-fed Id3-/- mice and circulating levels of HA were elevated. Has2 mRNA expression was increased in epididymal AT of Id3-/- mice. Luciferase promoter assays showed that Id3 suppressed Has2 promoter activity, while loss of Id3 stimulated Has2 promoter activity. Functionally, HA strongly promoted B2 cell adhesion in the AT and on cultured vascular smooth muscle cells of Id3-/- mice, an effect sensitive to hyaluronidase. Our data demonstrate that loss of Id3 increases Has2 expression in the epididymal AT, thereby promoting HA accumulation. In turn, elevated HA content promotes HA-dependent binding of B2 cells and an increase in the B2 cells in the AT, which contributes to AT inflammation.