bluuebunny/crossref_metadata_2025 huggingface dataset:
CysLT1R is essential for optimal TLR4 responses and associated macrophage polarization and sepsis in response to LPS
Authors: Teegala Lakshminarayan Reddy , Pokhrel Sabita , Sabu Kattuman Emma Elizabeth , Gudneppanavar Ravindra , Thodeti Charles , Paruchuri Sailaja
Published: May 2023

Background: Cysteinyl leukotrienes (cys-LTs; LTC4, LTD4 and LTE4) are arachidonic acid-derived inflammatory lipds, acting mainly via two G-protein-coupled receptors (GPCRs), cysteinyl leukotriene receptor 1 (CysLT1R) and cysteinyl leukotriene receptor 2 (CysLT2R). Cys-LTs are mainly recognized as potent modulators of asthma and allergy with their effects on mast cells. However, their role in other pathophysiological conditions are not well understood. Higher amounts of cys-LTs were reported in the lungs of mice subjected to sepsis than compared to sham mice, suggesting that CysLTs play critical role in sepsis via modulation of macrophage inflammatory responses. Objective: To investigate how cys-LT/CysLTR axis determines the inflammatory fate of macrophages in LPS/TLR4-induced sepsis. Methods: Bone marrow-derived stem cells (BMSC) from wild type (WT) and CysLT1R null ( Cysltr1-/-) mice were differentiated into macrophages and polarized to inflammatory phenotype using LPS via TLR4 pathway. Transcript and protein expression was analyzed by qPCR/RNA seq and western blotting/multiplex ELISA respectively. Results: LPS/TLR4 axis polarized WT macrophages to inflammatory phenotype by activating inflammatory intracellular signals (p38, CREB, and NFkB), upregulating surface expression of inflammatory markers (CD68, CD11b and LFA-1), enhancing phagocytosis and augmenting inflammatory cytokine secretion, in vitro. In contrast, LPS treated Cysltr1–/– macrophages exhibited attenuated TLR4 signaling observed with WT mice, suggesting that inhibition of CysLT1R prevents optimal TLR4 activation in response to LPS. Notably, reporter assays revealed a strong interaction between TLR4 and CysLT1R in HEK blue TLR4 cells overexpressing CysLT1R, further emphasizing a potential CysLT1R-TLR4 cross talk in driving TLR4-dependent inflammatory macrophage phenotype. Finally, Cysltr1–/– mice were protected from LPS-induced sepsis compared to WT mice with comcomitant reduction in the production of inflammatory cytokines. Conclusions: Our results uncover a potential crosstalk between two important innate immune receptors, TLR4-CysLT1R, in mediating macrophage inflammatory phenotype and sepsis in vivo. Understanding this interaction may offer novel potential therapeutic targets for macrophage-associated inflammatory disorders like sepsis.

This work was supported by National Institutes of Health (R01AI144115 and R01HL148585)

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.
Intestinal epithelial transcription factor HNF4A protects against microbiota dependent colitis and expansion of Akkermansia muciniphila
Authors: Rawls John , Kelly Cecelia , Jawahar Jayanth , Davey Lauren , Everitt Jeffrey , Galanko Joseph , Anderson Chelsea , Avendano Jonathan , McCann Jessica , Sartor Ryan , Valdivia Raphael
Published: May 2023

Development of the inflammatory bowel diseases (IBD) has been linked to altered homeostatic relationships between the intestine and the microbiota it harbors. However early preclinical stages of IBD remain poorly understood, especially with respect to host-microbiota interactions. The nuclear receptor transcription factor Hepatocyte nuclear factor 4 alpha (HNF4A) has important roles in intestinal epithelial cell (IEC) physiology, and our recent studies in zebrafish and mice revealed that HNF4A activity in IEC is suppressed by intestinal microbiota. Variants at HNF4A have been associated with human IBD, and IEC-specific knockout of Hnf4a in mice ( Hnf4a ΔIEC) leads to spontaneous colonic inflammation by 6-12 months. We hypothesized that intestinal pathology in Hnf4a ΔIEC mice may begin earlier in life and that microbiota may contribute to that process. Longitudinal analysis revealed that Hnf4a ΔIEC mice reared in specific pathogen-free (SPF) conditions develop fecal lipocalin 2 (LCN2) flares and episodic loose stools beginning as early as 4-5 weeks of age. Lifetime cumulative LCN2 levels correlated with histopathological features of colitis at 12 months of age. Antibiotic and gnotobiotic tests showed that these phenotypes in Hnf4a ΔIEC mice were dependent on the presence of microbiota. Fecal 16S rRNA gene sequencing in an SPF cohort of Hnf4a ΔIEC mice and controls at 12 months of age disclosed that genotype was a significant contributor to alterations in microbiota composition. Longitudinal analysis in a subset of inflamed Hnf4a ΔIEC and wild-type mice revealed that microbial community differences emerged at early life stages when fecal LCN2 flares first arose. These microbiota differences included a salient increase in the relative abundance of Akkermansia muciniphila in a subset of Hnf4a ΔIEC mice. We isolated an A. muciniphila strain from an aged, inflamed Hnf4a ΔIEC mouse, and its genome sequence revealed it to be a member of a new Akkermansia phylogroup with genetic features distinct from the known phylogroups. We conclude that HNF4A functions in IEC throughout the lifespan to protect against episodic flares of inflammation induced by microbiota while also shaping microbiota assembly. These results establish Hnf4a ΔIEC mice as a useful model for investigating preclinical pathology and host-microbiota interactions that precede overt IBD onset.

NIH P01-DK094779

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.
Wet-Bulb Temperature or Heat Index: Which Better Predicts Fatal Heat in a Warming Climate?
Authors: Lu Yi-Chuan , Romps David
Published: May 2023

The wet-bulb temperature of 35 °C has been widely used as the threshold for human survivability in the field of climate science. However, recent experiments have shown that a person's core temperature starts to rise at a range of critical wet-bulb temperatures well below this threshold. It is shown here why this state of severe heat stress cannot be predicted using the wet-bulb temperature. Instead, it is shown that the recently extended heat index can explain nearly all of the variance in the observed critical combinations of temperature and humidity in those heat-stress experiments. For light and moderate exertion in an indoor setting, the heat index predicts that the critical wet-bulb temperature ranges from 20 to 32 °C, depending on the relative humidity, consistent with experimental results. For the same setting and exertion, the heat index predicts fatal wet-bulb temperatures ranging from 24 to 37 °C.

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.
Renoprotection conferred by empagliflozin in heart failure is associated with M2 macrophage polarization
Authors: Wisnivesky Aline , Bertacchi João Guilherme , Girardi Adriana
Published: May 2023

Background: SGLT2 inhibitors, primarily developed as antidiabetic agents, exert beneficial effects on the heart and kidneys beyond glucose control. We have previously demonstrated that the prevention of heart failure (HF) progression in rats by the SGLT2 inhibitor empagliflozin (EMPA) is associated with reduced proximal tubule NHE3 activity, restoration of euvolemia, and preservation of renal mass. The present study was undertaken to test the hypothesis that empagliflozin alleviates kidney injury in heart failure by eliciting anti-inflammatory responses. Methods: Male Wistar rats were subjected to myocardial infarction or sham operation. After four weeks, rats that developed HF and sham rats were treated with EMPA (10 mg/kg/day) or not for an additional four weeks, resulting in 4 experimental groups: [Sham (n=8) and Sham+EMPA (n=8), HF (n=10), HF+EMPA (n=9)]. The glomerular filtration rate (GFR) was estimated by creatinine clearance. Histological analysis was performed to evaluate renal congestion and tubular atrophy. Immunohistochemistry and quantitative RT-PCR were performed to assess inflammatory markers. Results are reported as the mean ± standard error of the mean. Comparisons among the means were conducted using a two-way variance analysis (ANOVA) followed by Tukey's post hoc test. P<0.05 was considered significant. Results: Untreated HF rats exhibited lower GFR and kidney weight to tibia length ratio than the Sham. EMPA treatment preserved GFR and renal mass and markedly attenuated HF-induced kidney congestion and tubular atrophy. There was a similar elevation in the number of pan-macrophage marker CD68-positive cells in the kidneys of untreated and EMPA-treated HF rats compared to Sham groups. However, EMPA-treated HF rats displayed downregulation in the kidney mRNA expression levels of M1 macrophage polarization markers (TNFα, iNOS, and CCR2) and upregulation of M2 macrophage polarization markers (CD206, Arg-1, and CD163) compared to untreated HF animals. Additionally, HF+EMPA rats displayed reduced renal infiltration of CD3+ T cells per field compared to untreated HF rats (21±3 vs. 12±1, P<0.01). Conclusion: These results suggest that empagliflozin alleviates renal injury in cardiorenal syndrome. This renoprotective effect may be attributable at least partially to the activation of anti-inflammatory pathways that modulate macrophage polarization.

FAPESP

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.
Endothelial expression of iron transporters is dependent on contact with smooth muscle
Authors: Dunaway Luke , Luse Melissa , Isakson Brant
Published: May 2023

Iron is essential for heme-protein function. In the microcirculation, alpha hemoglobin is an important heme protein in endothelium and is essential for nitric oxide sequestration or production. In the endothelium, its expression is dictated by cellular contact with smooth muscle cells. Thus, we sought to determine if there was a similar contact dependency for iron import into endothelium. We did this initially by using our vascular cell co-culture model of primary human coronary endothelial cells (ECs) and smooth muscle cells (SMCs) in three conditions: 1. cultured without SMCs (monolayer ECs), 2. co-cultured without direct contact with SMCs (no-contact ECs), and 3. co-cultured with SMCs such that there is cellular contact with ECs (contact ECs). We then collected the ECs from each condition and performed bulk RNA sequencing for an unbiased screen of iron transporters. We found transferrin receptor 1 (TFRC) and divalent metal transporter 1 (SLC11A2) expression to be significantly increased in contact ECs compared to monolayer ECs. TFRC was also found to be significantly increased relative to no-contact ECs. This is important because both of these transport iron into cells. To determine if the contact dependency for iron import transporters was valid in vivo, we used mice with a YFP reporter on the Cdh5 Cre such that all endothelial cells could be analyzed from a particular tissue. Single cell RNA sequencing from mesenteric and adipose tissue was segregated into lymphatic, capillary, veinous and arterial endothelium. Similar to the contact co-culture, we found Tfrc and Slc11a2 to be enriched in arterial endothelium (where contact with smooth muscle is prevalent) as compared to any other endothelial cell type. Based on our data, we propose endothelial cell contact regulates iron import, which may be vital to alpha globin function.

NIH HL088554 and University of Virginia Basic and Translational Research Training Grant T32 007284

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.
A myogenic program is enacted by cardiac protein CSRP3 in human and mouse kidney
Authors: Funahashi Yoshio , Nelson Jonathan , Burfeind Kevin , Munhall Adam , Groat Tahnee , Hutchens Michael
Published: May 2023

Introduction: Cardiac injury with renal injury (acute cardiorenal syndrome) can induce renal fibrosis, but mechanisms by which heart injury signals to kidney are poorly understood. The myocardiocyte differentiation factor cardiac LIM protein (CSRP3) is released into plasma after cardiac injury, and taken up by megalin (Lrp2) in renal proximal tubular epithelial cells in vivo. We evaluated the relationship between cardiac injury and renal fibrosis in a mouse model and in human cells by characterizing novel primary culture of human tubular epithelial cells and then testing the hypothesis that CSRP3 alters PTEC transcription of myogenesis, transport, and extracellular matrix genes in vitro and in vivo. Methods: In mice, cardiac arrest (CA) was induced with potassium chloride and resuscitated with epinephrine and chest compressions (CPR) as previously described. SnRNAseq was conducted on mouse kidney cells harvested 24h after CA/CPR and analyzed using Seurat. Research-designated human kidneys were obtained under IRB exemption for non-human subjects research and underwent dissection, digestion and FACS sorting for PTEC selection. hPTEC were passaged up to 4x before transporters were characterized by qPCR, immunoblot, and immunofluorescence. Recombinant human CSRP3 (Origene) was administered to hPTEC followed by RNASeq and qPCR. CSRP3 (5μg) was retroorbitally injected in mice and renal fibrosis was assessed 7 weeks later. Results: In mice, snRNAseq demonstrated that CA/CPR significantly upregulated muscle cell differentiation ontologies in proximal tubule cells, driven by regulation of 110/162 myogenesis genes. Following FACS sorting for CD10 and CD13, hPTEC were 95% pure PTEC and expressed RNA and protein for PT transporters Lrp2, Aqp1, Slc34a1, and Slc5a2. In hPTEC, CSRP3 induced myogenesis differentiation factor 1 (MyoD1)>30 fold, and 20 myogenesis pathway and extracellular matrix genes. 8/20 genes were coordinately regulated between mouse kidney after CA/CPR and hPTEC after CSRP3. CSRP3 injection caused tubulointerstitial fibrosis in mice (VαSMA/Vkidney (%): 1.05 (CSRP3) vs 0.68 (control), n=8/group, p<0.01). Conclusion: hPTEC cultured from research-designated donor kidneys express a PT phenotype. The myocardiocyte differentiation factor CSRP3 causes transcriptional change in hPTEC, resulting in myogenic differentiation which is highly similar to that observed in mouse kidney after CA/CPR with coordinate regulation of myogenesis genes. Intravenous administration of CSRP3 to mice causes tubular interstitial fibrosis. Together these data strongly suggest CSRP3 may drive renal fibrosis via the myogenesis pathway.

Merit grant I01BX004288-01

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.
Novel tools for neuronal activity, vessel diameter and red blood cell velocity imaging
Authors: Tiret Brice , Schuette Peter , Ollerenshaw Doug , Zitelli Kevin , Gulati Srishti , Adil Nosheen , Neufeld Shay , Nassi Jonathan
Published: May 2023

Healthy brain function requires a delicate balance between neural activity and local vascular dynamics. Perturbations to this balance, such as vessel diameter changes or fluctuations in red blood cell velocity, are thought to underlie a number of neurodegenerative and neurovascular diseases, including migraine, stroke, and epilepsy. Here, for the first time, we demonstrate high fidelity simultaneous acquisition of vascular and neural circuit dynamics in freely-moving animals using our newly developed nVue dual color miniscope. Our system enables simultaneous imaging of blood flow and neural activity in both superficial and deep-brain structures in awake, behaving rodents. By multiplexing two LEDs at up to 100hz, the nVue enables direct, real-time visualization of both hemodynamics and calcium activity in two channels. Additionally, we have augmented the Inscopix Data Processing Software package to capture time-varying estimates of vessel diameter and red blood cell (RBC) velocity in specific vessels. Vessel diameter is estimated as the full-width half maximum of a Lorentzian function fit to vessel cross-section, while RBC velocity is calculated based on time-lagged correlations between seed-pixels within individual regions of interest and all immediately neighboring pixels. Distances between correlation peaks are then plotted against time. Manual annotation of blood flow data was performed to establish a ground truth against which to validate the algorithm. The nVue blood flow imaging application offers a powerful, turn-key, multisystem workflow for simultaneously interrogating hemodynamics and neural circuit function in freely behaving animals which will profoundly expand the neurovascular coupling toolkit not only for basic and translational research but also for the development of novel preclinical therapeutics.

Commercial

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.
Effects of Chronic Intermittent Hypoxia on Cognitive Function in Rats
Authors: Cheung Emily , Escobar Joan , Schunke Kathryn , Kay Matthew , Mendelowitz David
Published: May 2023

Obstructive sleep apnea (OSA) affects approximately 2.2 million people worldwide and is characterized by frequent bouts of hypoxia during sleep, imbalance of the autonomic nervous system, and increased risk of cardiovascular diseases. Recent work has shown OSA is also strongly linked to cognitive impairments. In this study, we tested whether chronic intermittent hypoxia (CIH), an animal model of OSA, elicits anxiety and impairs learning and memory.Eight female adult Sprague Dawley rats were exposed to CIH (94% N2, 6% O2) for eight hours a day during their sleep cycle. Age matched controls were maintained under similar noise conditions, but at room air. After twenty-six weeks of CIH, learning skills were assessed. Using the Morris water maze protocol, animals were trained to swim to a visible platform in a circular pool. Animals underwent the training protocol for five consecutive days with six daily sessions. After training, animals were exposed to CIH for eight more weeks before tests were performed. During the testing session, spatial memory was tested by repeating the Morris water maze test with a hidden platform. In conjunction, anxiety levels were assessed using an open field maze test to quantify the duration of time an animal explores open regions on a platform.Animals exposed to CIH displayed significantly longer learning times (30.94 vs. 14.60 seconds, p < 0.0001, n=8) and lower levels of spatial recognition and memory (latency time 45.25 vs 27.33 seconds, p<0.05, n=8) than controls. Corresponding quantification of tau protein via ELISA demonstrated significant differences between the CIH and control groups (325.7 vs 206.1 pg/mL, p<0.05, n=8). Our results show, in an animal model of OSA, CIH impairs cognitive function. Future work will test if new treatment paradigms for OSA, such as oxytocin network activation, blunts decreases in cognitive abilities and increases in anxiety.

NIH 5R01HL147279

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.
Mild hypoxia as therapy for post-ischemic mitochondrial injury
Authors: Piao Lin , Fang Yong-Hu , Wu Rongxue , Hamanaka Robert , Mutlu Gökhan , Archer Stephen , Garcia Alfredo , Sharp Willard
Published: May 2023

Introduction: Mitochondrial injury occurs following cellular ischemia in the setting of myocardial infarction, stroke, and cardiac arrest (CA). Effective strategies for reducing injury are limited. Hypoxia has shown benefit in reversing mitochondrial mediated neurodegenerative disease but is little studied in the setting of post-ischemic injury and is regarded as potentially harmful. In this study, we investigated the effects of mild hypoxia on global post-ischemic injury following CA. We hypothesized that brief mild hypoxia following CA would improve mitochondrial function resulting in improved cellular metabolism and physiological recovery. Methods and Results: Anesthetized C57BL6 mice underwent brief asystolic CA (12 min) followed bycardiopulmonary resuscitation(CPR). One hour following successful CPR, mice were randomized to receive a brief episode (6 hours) of normoxia (21% O2) or hypoxia (10% O2) in an environmental chamber. Post intervention, the mice were returned to room air (21% O2). Mild hypoxia improved cardiac mitochondrial complex 1 respiration in the heart by 28% and ATP-related oxygen consumption by 49% (n=3, P<0.05 vs normoxia, respectively) compared to normoxia treated mice. Hypoxia was further associated with improved glucose oxidation in heart as evidenced by decreases in pyruvate dehydrogenase kinase 4 (PDK4) gene and protein expression (n=4, P<0.05 vs normoxia, respectively) as well as decreases in pyruvate dehydrogenase phosphorylation (n=4, P<0.05, respectively) in heart. Hypoxia decreased lactate dehydrogenase (LDH) expression and lactate concentration in heart compared to normoxia treated controls (n=4, P<0.05 vs normoxia, respectively). ROS production in heart by 20% (n=6, P<0.05) was also decreased by mild hypoxia when compared to normoxia post CA. These changes were associated with improved recovery of myocardial function, neurological recovery, and 10-day survival following CA (n=13, P<0.05 vs normoxia, respectively). Conclusion: Brief mild hypoxia treatment following global ischemic injury paradoxically improved recovery of myocardial mitochondrial respiration and metabolism and was associated with improved physiological recovery and survival. Post-ischemia hypoxia is a potential therapeutic strategy for mitochondrial ischemic injury and requires further study.

No financial disclosures.

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.
Interleukin-1 beta Increases Glycolysis Through AKT and HIF-1 alpha in Colorectal Cancer Cells
Authors: Kim Ji Yeon , Park Bohye , Riffey Olivia , Donohoe Dallas
Published: May 2023

Increased glucose utilization and glycolysis is an important component in colorectal cancer. Inflammation is a known promoter of cancer development and progression. It has been reported that increased production of pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNFα) promote glycolysis. Previously, in colorectal cancer cells, we have demonstrated that IL-1β increased glycolysis, while suppressing oxidative metabolism. However, the mechanism underlying how this pro-inflammatory cytokine increased glycolysis was unclear. We hypothesized that IL-1β promoted glycolysis by increasing AKT activation given the metabolic role of this protein in metabolism. AKT is downstream of the interleukin-1 receptor, which is the major receptor IL-1β signals through to elicit a cellular response. Here, we show that upregulation of glycolysis by IL-1β is mediated through the AKT and more specifically the downstream transcription factor hypoxia inducible factor-1 alpha (HIF-1α). Knockout of HIF-1α completely abolished the elevated glycolysis induced by IL-1β. In addition, IL-1β had profound effects on mitochondrial function through reducing basal respiration, spare respiratory capacity, and maximal respiration. These data suggest a mechanism by which IL-1β through AKT activation and HIF-1α, regulates glycolysis in colorectal cancer cells.

This work was supported by USDA NIFA (2019-67017-29261).

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.
Alteration in S6K1-IRS1 signaling is uncoupled from whole body insulin sensitivity in animals treated with leucine or ketoisocaproic acid
Authors: Mann Gagandeep
Published: May 2023

Introduction: Plasma levels of branched-chain amino acids and their metabolites, branched-chain ketoacids are increased in insulin resistance, a condition that can lead to type 2 diabetes mellitus. Questions remain as to whether elevated levels of these metabolites cause, or are a consequence of insulin resistance. We previously showed that leucine and its metabolite ketoisocaproic acid (KIC) suppressed insulin-stimulated glucose uptake in L6 myotubess. Leucine and KICwith an increase in e S6K1 activation and phosphorylation of an inhibitory serine IRS-1 residue. The objective of this study was to analyze the effect of KIC gavage on skeletal muscle and liver insulin signaling and how this impacts whole-body insulin sensitivity. We hypothesized that KIC gavage will reduce whole-body insulin sensitivity and in response to the activation of the S6K1-IRS-1 pathway as shown in vitro. alter skeletal muscle and liver insulin signaling. Methods: Five-week-old male Sprague-Ddawley rats were starved for 24 hours. Rats were then gavaged with 0.75ml/100g of water, leucine (22.3g/L) or KIC (30g/L) twice, ten minutes apart, and then either sacrificed at different timepoints post-gavage (0.5-3h). A different set of rats were starved for 6 hours and then or injected with insulin an hour after the first gavageleucine or KIC gavage. Basal blood glucose levels and post insulin injection (10-120min after injection) blood glucose levels were measured., where blood glucose was measured pre (basal) and post (10-120min) insulin injection. Results: Leucine or KIC gavage had no effect on whole-body insulin sensitivity. This was consistent with no effect on ph-Akt (S473) phosphorylation. However, IRS-1 (S612, p<0.0001), S6K1 (Thr389, p<0.001) and S6 (S235/6, p<0.0001) phosphorylation were increased 30 minutes after leucine gavage in skeletal muscle and liver, but this was alleviated by the 60-minute mark. KIC gavage had no effect on the activation of these proteins. Conclusion: Despite increased S6K1 threonine phosphorylation and IRS-1 serine phosphorylation in skeletal muscle and liver in response to leucine gavage, there was no effect on whole-body insulin sensitivity. Unlike in studies with myotubes, KIC did not activate S6K1-IRS-1 signaling. Thus, although insulin resistant states exhibit increased plasma leucine and KIC levels, their supplementation does not reduce insulin sensitivity, suggesting that their increased levels are a consequence of insulin resistance and not causative. s. This suggests other mechanisms and/or tissues may play a role in regulating whole-body insulin sensitivity during leucine/KIC gavage.

NSerc, York University

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.
Drosophila Clc-c as a model of human CLC-5 and Dent Disease type 1
Authors: Reynolds Carmen , Gillen Christopher , Dow Julian , Romero Michael
Published: May 2023

Introduction: Dent Disease type 1 (DD1) results from X-linked, mutations in CLC-5 (2Cl-/H+ exchanger) and results in progressive renal failure by 20-40. DD1 is characterized by kidney Ca2+ mishandling, and subsequent hypercalcuria, calcium oxalate (CaOx) kidney stones, and kidney calcification as well as proteinuria. We identified Drosophila Clc-c as the CLC-5 homolog with conserved amino acids at DD1 mutation sites. We hypothesize that Clc-c shares functional similarities in Cl- transport, Ca2+ homeostasis and protein processing. Methods and Results: By voltage clamping Xenopus oocytes expressing Clc‑c and CLC-5, we find that both are electrogenic and have the same outward-rectifying current in Cl- solutions. Cl- transport (current at +80mV) was lowered by acidity (2.37±0.55 μA, pH 6.0, P=0.06), but not by alkaline solution (4.45±0.96 μA, pH 8.5, P= 0.7) when compared to the standard solution of pH 7.5 (4.97±1.12 μA). Similarly, Cl- transport was lowered by homologous DD1 mutations S393L (0.67±0.098 μA, P<0.0001), R494W (2.9±0.46 μA, P =0.006), and Q777x (1.13±0.28 μA, P =0.0001) vs. WT (7.4±1.2 μA) as observed previously for CLC-5 DD1 mutations: S244L, R345W, and Q629x (Tang et al, Physiol Rep 4[8], 2016). To determine cell membrane and possible intracellular localization of DD1 mutations, HA-tagged Clcc and mutations (identical placement as in CLC-5-HA, Tang 2016) were made. Likewise, transgenic Drosophila expressing Clc-c-GFP were made to localize Clc-c in cell and intracellular membranes of renal tubules (Malpighian tubules, MT) in vivo (Judd-Mole, U Monash, Dissertation, 2016). RNAi MT knockdowns of Clc-c (Clc-c-KD) were made and resulted in 50% decrease in Clc-c mRNA expression. CaOx crystals in anterior MT (7 days after eclosure) were present in all Clc-c-KD and were more abundant (15±4 crystals/fly) than w1118 controls (5±2 crystals/fly, P= 0.02). MT secretions showed that Clc-c KD flies contained higher [Ca2+] while all other cations (Na+, NH4+, K+, Mg2+), and volume were the same as w1118 . Similarly, urinary protein concentration (4-6 pooled flies per n) appears elevated in DD1 flies (1.41 ± 0.14 mg/mL, n=3) vs w1118 (1.16 ± 0.08 mg/mL, n=5). Conclusion: Drosophila Clc-c has similarities to human CLC-5 including impaired function of voltage-gated Cl- transport, homologous DD1 mutations, increased CaOx crystal formation, and elevated urinary [Ca2+] and protein. Our results indicate that the Drosophila Clc-c avatar may help to provide more direct insights to the cellular and organismal pathophysiology for future investigations of Cl- transport, Ca2+ homeostasis and urinary protein loss in DD1.

F32 DK128987, T32 DK007013

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.
Sarcolipin ablation decreases whole body VO2 in young female nice
Authors: Brahmbhatt Aditya , Chambers Paige , Hashemi Ashkan , Tupling Arthur
Published: May 2023

The sarco(endo)plasmic reticulum calcium (Ca2+) ATPase (SERCA) pump is a major regulator of cytosolic Ca2+ in striated muscle. Sarcolipin (SLN) reduces SERCA-mediated Ca2+ pumping efficiency and is a key regulator of muscle metabolism and contractile function. With SERCA activity comprising approximately 12% of whole-body metabolic rate, the inefficient SERCA Ca2+ cycling promoted by SLN would be predicted to increase metabolic rate. Although SLN is involved in adaptive diet-induced thermogenesis and overexpression of SLN increases metabolic rate and protects against diet-induced obesity, we found previously that SLN ablation had no effect on whole body metabolic rate or body composition in male mice that were fed a standard chow diet. However, unpublished work from our lab indicates that SLN expression in mouse soleus is significantly higher in females compared with males. Therefore, the purpose of this study was to investigate the effects of SLN ablation on body composition and metabolic rate of female mice. Adult (4-6mo) female wild type (WT) and SLN knockout (KO) mice (n=4 per group) were used to collect anthropometric measures (adiposity and body weight) and were housed in a comprehensive lab animal monitoring system (CLAMS) over a 48-hour period to assess daily whole body VO2 and total cage activity. With SLN ablation, total whole body VO2 (ml/kg/hr) was lower (p<0.05) in the KO females (3534 ± 95.1) compared to WT females (5080 ± 98.8), with no differences (p>0.05) found for total cage activity (counts) (WT, 14935.42 ± 1799.6 vs. KO, 11860.83 ± 679.48), body weight (grams) (WT, 20.97 ± 0.76 vs. KO, 22.18 ± 0.80), or adiposity (arbitrary units) (WT, 5.18 ± 1.71 vs. KO, 2.12 ± 0.36). These results showing that high levels of endogenous muscle SLN expression in female mice influence whole-body metabolism even under chow-fed conditions, demonstrate a potential important sex difference in whole body physiology and metabolism.

Natural Sciences and Engineering Research Council of Canada (NSERC) [grant number 311922-05 (to A.R.T.)]

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.
Exercise-induced upregulation of APOE expression is attenuated by dietary supplementation with resveratrol
Authors: Allard Joanne , Graham-Hyatt Shantol , Mulgrave Verona , Alsayegh Abdulrahman
Published: May 2023

The ε4 variant of the Apolipoprotein E gene (APOE4) is the most prevalent genetic risk factor for Alzheimer’s disease (AD). Approximately 25% of people carry at least one copy of the variant which increases risk of AD two-to-three-fold relative to those who carry the ε3 variant. Low ApoE levels are typically reported in Alzheimer’s patients and animal studies have reveal lower levels of ApoE in ε4 carriers relative to ε3 carriers. Therefore, reduced ApoE levels has been associated with increased risk for dementia. Physical exercise and active lifestyles are well-accepted therapeutic strategies for the prevention or delay of Alzheimer’s disease and other dementias, Additionally, there is growing evidence that the consumption of antioxidant nutritional supplements may also be therapeutic and slow progression of AD. But the effect of physical activity or antioxidant intake on APOE expression is not well-studied.The objective of this study is to determine the impact of exercise and resveratrol intake on APOE expression. We hypothesized that both resveratrol intake and exercise would increase APOE expression in brain with an additive effect when combined. Targeted replacement mice, homozygous for either APOE3 or APOE4 were randomized into four groups based on activity and resveratrol dietary supplementation: sedentary standard, sedentary resveratrol, exercise standard, and exercise resveratrol. Exercised groups were subjected to daily treadmill running for 8 weeks. Resveratrol treated groups were fed a chow supplemented with resveratrol. Results showed that exercise induced a significant increase in ApoE protein in brain cortex of both APOE3 and APOE4 mice. Resveratrol treatment alone had no significant effect on brain APOE expression, but in combination with exercise, attenuated the exercise-induced increase. While biochemical variations between APOE3 and APOE4 mice likely contribute to the differential risk for AD, the exercise-induced increase in ApoE levels in both genotypes supports a role for APOE in the therapeutic mechanisms of exercise. The timing of antioxidant supplementation in conjunction with exercise interventions should be critically considered to ensure best therapeutic efficacy of exercise.

This research was supported by NIH grants R25AG047843 and 2U54MD007597-31

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.
Role of preeclampsia on insulin-induced vasorelaxation
Authors: Rodríguez Reyes Betzabel , Bobadilla Rosa , Tufiño Martínez Cecilia , Madrigal Aguilar Damian , Mungia Venegas Julio
Published: May 2023

The objective of this project was to evaluate whether PE alters the vascular response to insulin. The physiological insulin resistance characteristic of pregnancy is increased in preeclamptic (PE) conditions. With the hypothesis that PE diminishes insulin vascular effects we used a conventional isolated aorta rings preparation to study insulin-induced relaxation on phenylephrin (Phe) pre-contracted vessels and Phe induced contraction in the presence or absence of insulin. Vessels were obtained from non-pregnant rats (NP) with subrenal aortic coarctation (SRAC), pregnant (P) and pregnant rats with SRAC (PE). Experiments were performed in thoracic or abdominal aorta rings with or without endothelium. The results show that PE increases the contractile response to Phe in the thoracic and abdominal aorta compared to P (PE vs P p<0.01). Incubation with insulin did not modify the thoracic or abdominal aorta Phe induced contraction of any of the three groups with and without endothelium.On the other hand, insulin induced relaxation was more evident in vessels with endothelium and was decreased both in the thoracic and the abdominal aorta of PE group (PE vs P p<0.01; PE vs NP-CARS p<0.01).In conclusion, PE decreases endothelium-dependent insulin-induced relaxation suggesting PE deteriorates insulin-PI3K-NO pathway.

Funding: Consejo Nacional de Ciencia y Tecnología(CONACyT)

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.
Centrally derived gasotransmitter modulation of hypoglossal motoneuron activity
Authors: Browe Brigitte , Prabhakar Nanduri , Garcia Alfredo
Published: May 2023

Airway obstruction during inspiration is a hallmark trait of sleep apnea (SA). In mice, genetic elimination of the CO producing enzyme, heme oxygenase-2 (HO-2), leads to a SA phenotype predominately associated with recurrent airway obstruction. This phenotype is mitigated by either inhibition or genetic ablation of the H2S producing enzyme, cystathionine g-lysase (CSE). While these observations indicate that mutual interactions between HO-2/CO and CSE/H2S are important for regulating airway clearance, the extent of contribution from these interaction in the central nervous system to airway tone are unclear. Here, we determine how HO-2/CO and CSE/H2S impact inspiratory output from the hypoglossal nucleus (XIIn) in the isolated rhythmic brainstem slice preparation. We hypothesize that these enzymes and their respective gasotransmitter products locally interact in the inspiratory circuit to modulate the input-output (I/O) relationship between preBötC and XIIn. Electrophysiological studies conducted in brainstem slices from mice (P5-12) showed that HO-2 dysregulation, using either in wildtype slices exposed to Cr (III) Mesoporphyrin IX chloride or in slices from HO-2 null mice, increased transmission failure from preBötC to XIIn and altered the I/O relationship between the rhythm generating network and XIIn. HO-2 dysregulation also increased subnetwork activity in the preBötC that was associated with a decrease in amplitude regularity. While many transmission failures to the XIIn were associated with preBötC subnetwork activity, simultaneous recordings from the preBötC, premotor area, and the XIIn revealed that preBötC subnetwork activity was reliably transmitted to the premotor field but not to the XIIn. Dual extracellular and intracellular recordings from the preBötC and individual XIIn neurons under disinhibited conditions revealed that HO-2 dysregulation reduced inspiratory drive currents received by XIIn neurons, decreased the intrinsic excitability of XIIn neurons while also reducing the amount of preBötC subnetwork activity. Additionally, targeted inhibition of CSE/H2S activity using L-PAG or by genetic elimination of CSE in HO-2 null mice mitigated these effects of HO-2 dysregulation. Thus, the mutual interactions HO-2/CSE and CSE/H2S in the medullary brainstem appear to modulate XIIn output by: (1) causing a E/I imbalance that promotes preBötC subnetwork activity; (2) suppressing inspiratory drive received in XIIn; and (3) suppressing intrinsic excitability of XIIn neurons. These findings may be key to understanding the action by which central gasotransmitters actions contribute to upper airway muscle regulation.

This work was supported by NIH: P01-HL-44454; R01NS107421.

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.
Gastric Bypass reprograms hepatic clock machinery and improves glucose flux independent of weight
Authors: Mokadem Mohamad , Ye Yuanchao , Abu El Haija Marwa , Herz Hussein , Linden Benjamin , Chu Yi
Published: May 2023

Background: The molecular clock machinery regulates several homeostatic rhythms, including but not limited to glucose homeostasis. We previously demonstrated that Roux-en Y Gastric Bypass (RYGB) has a weight-independent effect on glucose metabolism, and transiently reduces food intake. In this study we investigate the effects of RYGB on diurnal eating behavior as well as its effects on the molecular clock, and its requirement for the metabolic effects of this bariatric procedure in obese mice. Results: We find that RYGB reverses the high fat diet-induced disruption in diurnal eating pattern during the early post-surgery phase of food reduction (Figure1). “Dark-cycle” pair-feeding experiments improved glucose tolerance to the level of bypass-operated animals during the physiologic “fasting” phase (Zeitgeber ZT2), but not the “feeding” phase (ZT14)(Figure 2). Using a clock gene reporter mouse model ( mPer2 luciferase), we reveal that RYGB induces a liver-specific phase shift in peripheral clock oscillation with no changes to the central clock activity within the suprachiasmatic nucleus (SCN) (Figure 3). In addition, we show that weight loss effects are attenuated in obese ClockΔ19 mutant mice post-RYGB (Figure 4) that also fails to improve glucose metabolism after surgery (Figure 5), specifically early hepatic glucose production (Figure 6). Conclusion: RYGB reprograms the peripheral clock within the liver early after surgery to alter diurnal eating behavior and regulate hepatic glucose flux.

US Veterans Merit Award (#I01 BX004774)

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.
Time dependent activation of molecular signatures is associated with Trimethylamine N oxide induced endothelial dysfunction in human microvascular endothelial cells
Authors: Shanmugham Meyammai
Published: May 2023

Cardiovascular diseases (CVD) are the principal cause of death worldwide. Endothelial dysfunction is recognized as a crucial initiating contributing factor for CVD. Trimethylamine–N–oxide (TMAO), a gut microbiome-derived metabolite is involved in CVD progression. However, the underlying time-dependent molecular mechanisms resulting in the pathogenesis of TMAO in endothelial cells remain unclear. To investigate the time-dependent molecular signatures of TMAO-induced endothelial dysfunction in human microvascular endothelial cells (HMEC–1), we performed transcriptomics and metabolomics analysis on TMAO (50 μM) treated cells for 24H or 48H. Differentially expressed genes were used for overrepresentation analysis of pathways to determine the various molecular signatures involved. These were validated with quantitative real–time PCR, cell viability (prestoblue), and reactive oxygen species generation (DCFDA). TMAO treatment for 24H and 48H induced endothelial dysfunction, demonstrated by lowered cell viability, and elevated oxidative stress. Interestingly, time–dependent modifications in the molecular signatures were unique between time points. Indicatively, there were fewer GO biological processes and KEGG pathways suppressed, after 24H TMAO treatment. They were involved in cellular response and developmental processes. Compared to 48H treatment, a greater number of molecular signatures were activated and suppressed for GO biological processes and KEGG pathways. Upregulated DEG was enriched in pathways like oxidative stress and the production of inflammatory phenotypes, while suppressed pathways were associated with the structural organization of the ECM, endothelial cell proliferation, and collagen metabolism. This study indicates that TMAO–induced endothelial dysfunction is regulated through the mitigation of molecular gene signatures involved in oxidative stress and inflammation, activating endothelial cell remodelling.

Funding source: SUTD Start-up Research Grant, and SUTD Kickstarter Initiative, Singapore

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.
Differences and the impact of G protein-coupled estrogen receptor deletion in the vascular and renal response to aldosterone
Authors: Leite Ana Paula , Ogola Ben , Visniauskas Bruna , Kilanowski-Doroh Isabella , Horton Alec , Diaz Zaidmara , Lindsey Sarah
Published: May 2023

We recently showed that the G protein-coupled estrogen receptor (GPER) protects female mice from arterial stiffening, an independent predictor of cardiovascular mortality. Previous work indicates that GPER may directly bind aldosterone and/or dimerize with the mineralocorticoid receptor. Therefore, we hypothesized that GPER may promote aldosterone-induced renal and vascular effects, particularly in male animals where less estrogen is available to activate GPER. Male (M) and female (F) wildtype (wt) and global GPER knockout (ko) mice were used between 6-7 months of age. Aldosterone was delivered to all mice subcutaneously via osmotic mini pumps at a dose of 600 μg/kg/day and drinking water was replaced with saline for 28 days. At the end of the study, tail-cuff systolic blood pressure (SBP) and metabolic cage data were collected. Data were analyzed by 2-way ANOVA followed by Sidak’s test and correlation analysis. In basal conditions, GPER ko increased systolic SBP in males but not females. After aldosterone-salt treatment, SBP was similar in Mwt versus Mko but was significantly higher in Fko versus Fwt (Pinteraction=0.002; 148 ± 12 vs. 108 ± 2 mmHg; P<0.001). Similarly, proteinuria was similar in males but greater in Fko than Fwt (Pinteraction=0.01; 6.0 ± 1.5 vs. 2.4 ± 0.4 mg/day; P=0.04), indicating greater kidney injury. In summary, GPER deletion in females revealed enhanced blood pressure and renal damage in response to an aldosterone-salt challenge. These findings do not support a role for GPER in promoting the impact of aldosterone on vascular and renal damage in either sex. In contrast, GPER protected female mice from the negative impact of aldosterone-salt on blood pressure and kidney function. More studies are needed to understand the interactions of GPER and aldosterone in the kidney of both males and females.

This work was supported by the National Institutes of Health (HL133619 and AG071746 to SL, HL155841 to BO) and the American Heart Association (BV and IK)

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.