bluuebunny/crossref_metadata_2025 huggingface dataset:
Establishment of Anoxia/cytokine-induced Injury and Regeneration in Colonoids
Authors: Miko Corina , Kim Jain , Jin Younggeon
Published: May 2024

Intestinal epithelial cells (IECs) play key roles in nutrient absorption and fluid secretion, providing a barrier to protect cells. IECs, derived from intestinal stem cells (ISCs), are crucial in maintaining intestinal homeostasis and regeneration, which is crucial in gastrointestinal diseases such as inflammatory bowel disease (IBD). To study epithelial injury/regeneration, primary cultured colonoids were utilized as a model system to mimic injury. They retain the composition and differentiation of the tissue, making it a suitable model for studying homeostasis and regeneration of ISCs in vitro. Anoxia and proinflammatory cytokines frequently coexist and exacerbate one another during intestinal injury. However, anoxic conditions have been mainly ignored in previous research. Thus, we developed a regeneration model using cytokines/anoxia injury to better recapitulate intestinal inflammation. Colonoids were treated with cytokines TNF-α and IFN-γ (5 ng/μL) in anoxic conditions for 24 hours to provoke an injury. After 24 hours of injury, the colonoids were exposed to normal oxygen and media conditions for regeneration. Regeneration was observed for 24, 48, and 72 hours and documented using photomicrographs. Morphological analysis detailed that injury was most severe 24 hours post-injury with the combined induction of cytokines and anoxia resulting in decreased colonoid area and number of buds compared to control groups. RT-PCR analysis detailed mRNA expression of stem cell markers ( Lgr5, Bmi1, and Hopx), Wnt ( Axin2, Myc2, and Ascl2), and YAP target genes ( Sca1 and Ctgf) were increased at peak injury and returned to pre-injury levels at 72 hours of recovery. Regeneration of the colonoids is consistent with in vivo observations, thus confirming the validity of the model. This versatile model can be used to study responses to injury and regeneration of intestinal epithelium and the function of ISCs with applications in intestinal disease therapeutics.

University of Maryland College of Agriculture and Natural Resources and the Maryland Agricultural Experiment Station Undergraduate Research Grant Grant #4307572 from the Maryland Stem Cell Research Fund.

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.
Muscle atrophy is coupled with ferroptosis in the skeletal muscle of Friedreich’s ataxia
Authors: Jee Eunbin , Medha Maisha , Kim Yuho , Kim Jonghan
Published: May 2024

Friedreich’s ataxia (FRDA) is a rare genetic disorder caused by an expansion of GAA repeats in the frataxin gene (FXN), resulting in frataxin deficiency. As frataxin is crucial for mitochondrial function by recruiting key iron components such as iron-sulfur clusters and hemes, FRDA is characterized by mitochondrial dysfunction along with abnormal iron metabolism. Recently, ferroptosis, an iron-dependent cell death, has been suggested to play an important role in the development of FRDA, yet if and how ferroptosis impacts FRDA-related muscle weakness has not been studied. Therefore, the objective of this study was to identify the role of ferroptosis in the skeletal muscle of FRDA using whole-body frataxin deficient mice (Fxnnull::YG8s(GAA)>800, #030395, The Jackson Laboratory). FRDA mice (male, 3-months old) had a significantly smaller ratio of muscle mass to body mass, compared to strain-, age-, and sex-matched C57BL/6J control mice, suggesting that this animal model develops muscle atrophy as early as 3 months old upon frataxin deficiency. Furthermore, the FRDA muscles showed elevated non-heme iron levels, which were accompanied by altered iron regulatory protein expressions, including ferritin and ferroportin. In addition, mitochondrial function was decreased in the skeletal muscles of FRDA mice, as evidenced by significantly decreased protein levels of oxidative phosphorylation complexes (OXPHOS I, II, and III; decrease by 31%, 26%, and 15% compared to control, respectively), as well as other mitochondrial capacity markers such as nuclear factor erythroid 2-related factor 1 (Nrf1) and heme oxygenase-1 ( p=0.06). Notably, ferroptosis markers such as Nrf2 and glutathione peroxidase 4 were significantly downregulated in the skeletal muscles of FRDA mice, while apoptotic markers (p53 and caspase 3) were not altered. Collectively, ferroptosis could promote FRDA-related muscle atrophy, which is associated with abnormal mitochondrial iron status and function. Our results also suggest that ferroptosis may represent a novel target for correcting or ameliorating the development and progression of muscle atrophy in FRDA patients. Future studies are warranted to determine the molecular mechanism of ferroptosis in FRDA-related mitochondrial dysfunction and to develop therapeutic strategies for muscle weakness in FRDA or other types of ataxias.

This study was supported in part by the NIH NS121394 and UMass Lowell Internal Seed Grant.

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.
Liraglutide protects against microvascular dysfunction and sepsis-mediated inflammation and organ injury
Authors: Aslaner David , Baer Brandon , Meegan Jamie , Bastarache Julie , Ware Lorraine
Published: May 2024

Introduction: Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection characterized by increased systemic inflammation and microvascular injury. Recently, commonly used diabetes and obesity medications, glucagon-like peptide-1 (GLP-1) receptor agonists, have demonstrated unexpected anti-inflammatory effects. We tested GLP-1 receptor agonist liraglutide in a clinically relevant two-hit murine model of polymicrobial abdominal sepsis and hyperoxia and microvascular endothelial injury in vitro, hypothesizing that GLP-1 receptor agonists exert protective effects during sepsis, limiting microvascular permeability, inflammation, organ injury and death. Methods: Sepsis was induced in male and female mice by intraperitoneal (IP) injection of cecal contents (CS;1.8 mg/g body weight) collected from euthanized donor mice. Mice were also exposed to hyperoxia (FiO2 90-96%) for 24 hours. Six and 18 hours after CS injection, mice were administered saline or liraglutide (0.1 mg/kg). At 12 hours, mice were given fluids and antibiotics. At 24 hours, mice were euthanized for plasma and bronchoalveolar lavage (BAL; right lung) collection. Plasma cytokines, organ injury markers, and BAL immune cells and protein were measured. The left lung was tied off and excised to measure wet-to-dry lung weight ratios. Additionally, for in vitro studies, primary human lung microvascular endothelial cells (HLMVECs) were treated with saline or liraglutide for 24 hours prior to saline (control) or LPS (100 ng/mL). HLMVEC barrier dysfunction was quantified using express permeability testing (XPerT) assay or Electric Cell-Substrate Impedance Sensing (ECIS) to measure transendothelial electrical resistance (TER). Results: In murine sepsis, illness severity scores and lung injury were improved in mice pretreated with liraglutide (N=12). Plasma blood urea nitrogen (BUN; P=0.0581), alanine transaminase (ALT; P=0.0311) and aspartate aminotransferase (AST; P=0.0263), and cytokines IL-1β (P=0.0078), IL-6 (P=0.0038), and TNF-α (P=0.0183) in plasma were reduced in mice treated with liraglutide. In HLMVECs, liraglutide (1.5 nM) significantly reduced LPS-induced barrier dysfunction measured by XPerT assay (P=0.0030) and ECIS (P=0.0075). Conclusion: Liraglutide reduces illness severity, inflammation, and organ injury in a two-hit model of sepsis-induced ALI. Liraglutide has direct effects in the microvascular endothelium, limiting LPS-mediated barrier dysfunction. These findings support a protective role for GLP-1 receptor agonism in sepsis, mediated through the microvasculature.

HL150783, AG075341, HL158906.

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.
The impact of repeat opioid use (ROU) on fentanyl-induced respiratory depression
Authors: Rai Karan G. , Nwakudu Chinwendu U. , Szujewski Caroline C. , Tryba Andrew K. , Garcia Alfredo J.
Published: May 2024

Objective: Repeat opioid use (ROU) is a primary driver for the ongoing opioid epidemic, and while a substantial understanding exists for the impact of ROU on behavioral neurophysiology, the consequences of ROU on respiratory-related physiology including opioid-induced respiratory depression (OIRD) remain poorly understood. Hypothesis: ROU leads to respiratory tolerance to fentanyl that is characterized by the blunting of respiratory depression and improvement of O2 consumption (VO2). Methods: We utilized a pre-clinical rodent model. Breathing was measured via whole body-plethysmography in freely behaving adult mice (P>90) prior to and immediately following fentanyl administration. A single dose of fentanyl (0.7 mg/kg, i.p.) was given for up to 6 consecutive days. In a subset of experiments, O2 consumption was measured simultaneously using whole-body plethysmography. Results: We identified two distinct respiratory phenotypes in response to OIRD following ROU. Type 1 phenotype (n=18) adapted over time where breathing during OIRD is greater on day 5 versus day 1. In contrast, the Type 2 phenotype (n=5) showed no such adaptation, with unchanged or worsened breathing by day 5. In a subset of animals (n=12) the relationship between breathing (i.e. minute ventilation (Ve)) and oxygen consumption (VO2) was examined before and following ROU. Under baseline conditions the Ve to VO2 relationship positively correlated on day 1 and 5. During OIRD on day 1 this correlation became negative, but began to normalize by day 5, resembling the baseline relationship. These phenomena also coincided with an increase in the ventilatory drive observed at the whole-animal level and in the isolated preBötzinger complex following ROU. Conclusions: ROU leads to two distinct respiratory phenotypes in response to OIRD, where the majority of subjects show a blunted respiratory depression. Additionally, ROU improves the relationship between breathing and oxygen consumption and enhances inspiratory drive in the presence of fentanyl. These findings suggest that fentanyl-induced hypoxia experienced by repeat opioid users vastly differ from naïve users. Such differences may contribute to the unpredictable occurrence of overdose amongst repeat users.

This work was supported by NIH: R01 HL163965 and DA057767.

This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Sex and tissue-specific downregulation of miR410-3p in a mouse model of cardiometabolic diseases
Authors: Alnoud Mohammed , Elgazzaz Mona , Martinez Alexa , Miao Kevin , lazartigues Eric
Published: May 2024

The brain Renin-Angiotensin System (RAS) plays a pivotal role in controlling cardiometabolic homeostasis, fluid balance, and other functions including inflammation. Previous studies have shown that more than a thousand microRNA (miRNA) families regulate RAS genes expression, and their dysregulation has been associated with HTN, cardiac ischemia, and aging. Recently, we observed that hypercaloric diet (HCD) programming resulted in differentially expressed miRNA in the hypothalamus of only the male progeny, including miR-410-3p which is thought to target angiotensin-II type 1 receptors (AT1R). Here, we aimed to investigate whether miR-410-3p regulate AT1R expression in a mouse model of cardiometabolic disease (CMD) exhibiting hypertension, diabetes and obesity. C57BI/6J mice (3-week-old, both sexes, n=10) were fed a HCD (D12492. 60 kcal% Fat, Research Diets, USA) while weight and fasting blood glucose levels were measured weekly to ensure the acquisition of metabolic syndrome. At 12 weeks of age, the mice were infused subcutaneously (osmotic pump model 1004, Alzet) with Ang-II (600 ng/kg/min/4 weeks). Gene expression of miR-410-3p (QRT-PCR) was reduced in the hypothalamus of CMD male mice (0.5 ±0.1 vs.1.0 ±0.1, p<0.05) but increased in females (1.6 ±0.4 vs.0.7 ±0.2, p<0.001), while both sexes showed a reduction in the heart (Males: 0.2 ±0.0 vs.1.7 ±0.3; Females: 0.23 ±0.0 vs. 3.2 ±0.3, p<0.01). AT1R protein expression (Capillary Western) was increased in the hypothalamus of CMD male (1.5 ±0.1 vs.1.0 ±0.2, p<0.05) mice but not in females (1.0 ±0.1 vs.1.2 ±0.1, P>0.05) while both sexes exhibited elevated levels in the heart (Males: 2.0 ±0.1 vs. 1.0 ±0.1; Females: 2.3 ±0.1 vs. 1.3 ±0.2, p<0.05). Binding specificity (luciferase assay) was further assessed between miR-410-3p and the 3’UTR of AT1R. Binding was decreased in presence of miR410-3p mimic (0.9 ±0.0 vs. 2.1 ±0.0; p<0.001) and enhanced by a miR410-3p inhibitor (2.5 ±0.1 vs. 2.1 ±0.0; p<0.01), confirming that AT1R is a direct target of miR410-3p. Together, our data suggest that in CMD downregulation of miR410-3p is associated with the upregulation of AT1R both in the hypothalamus and heart, notably in males. This positions miR410-3p as a possible new therapeutic strategy to control RAS overactivity in CMD.

This work was supported in part by a research grant from the National Institutes of Health (HL163588).

This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
APOE Genotype Differentially Regulates Respiratory Motor Plasticity in the Active vs Rest phase
Authors: Marciante Alexandria , Mitchell Gordon
Published: May 2024

Acute intermittent hypoxia (AIH) elicits respiratory motor plasticity and is emerging as a promising strategy to improve breathing and other (non-respiratory) movements in clinical disorders that end life due to respiratory insuffciency ( e.g., spinal cord injury & ALS). AIH-induced phrenic long-term facilitation (pLTF) is the most commonly studied form of respiratory motor plasticity and presents as a persistent increase in phrenic nerve activity after AIH. Specific factors ( e.g. aging, inflammation, time of day) undermine AIH-induced pLTF. However, little information is available concerning the role of specific genes that regulate AIH-induced phrenic motor plasticity. Apolipoproteins (ApoE) are highly expressed in the central nervous system and function as lipid carriers. The APOE gene is tri-allelic ( E2, E3 and E4); ApoE2 and E3 proteins are associated with neuroprotective and neuro-neutral phenotypes, respectively. ApoE4 impairs synaptic plasticity and is implicated in neurodegeneration, particularly Alzheimer’s Disease. Indeed, we recently discovered that specific APOE alleles predict human expression of mAIH-induced plasticity in cortico-spinal pathways to the phrenic/diaphragm motor system. Initial experiments using human APOE4 knock-in rats demonstrate a causal effect in limiting phrenic motor plasticity (vs APOE3) during the active phase. However, the impact of APOE genotype on respiratory plasticity across the daily rest/active cycle is unknown. We tested the hypothesis that APOE4 undermines AIH-induced pLTF vsAPOE3 in a time-of-day-dependent manner. Anesthetized, vagotomized, paralyzed and ventilated adult (3-4 months) male Sprague-Dawley rats with knock-in humanized (h) APOE3 and h APOE4 were presented with AIH (15,1 min hypoxic episodes; arterial PO2 = 40-50mmHg). During the active phase, AIH-induced pLTF was significantly greater in h APOE3 (33±11%) vs h APOE4 rats (-26±7%; p<0.001; both n=6). In contrast, rest phase AIH-induced pLTF was similar in both h APOE3 and h APOE4 rats (121±12% and 122±5%, respectively; both n=7). These experiments demonstrate a causal relationship whereby APOE genotype and time-of-day interact to undermine AIH-induced pLTF in a manner that is unique to the active phase in rats. Since AIH has emerged as a promising neurotherapeutic to preserve/restore breathing in clinical disorders such as spinal cord injury and neuromuscular disease, understanding how genetic factors and time-of-day of AIH delivery impact phrenic motor plasticity will help identify individuals most/least likely to respond to treatment and reveal new targets for precision interventions of AIH.

NIH R01HL148030 (GSM), NIH R01HL149800 (GSM), NIH T32HL134621-5 (ABM).

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.
Signaling of GPR17 in Hepatic and Renal Tissues
Authors: Spellman Michael , Samimi Safa , Kurtz Ryan , Shepard Blythe
Published: May 2024

G protein-coupled receptors (GPCRs) are the largest class of proteins in the human body and are considered to be excellent targets for drug discovery. However, the vast majority of GPCRs are understudied. Using a large-scale TaqMan array screen on both male and female C57BL6 murine livers, we identified GPR17 as one of the most highly expressed orphan receptors within the liver (ΔCt = 19.9, normalized to GAPDH). GPR17 expression in the liver was confirmed using reverse transcriptase polymerase chain reaction (RT-PCR) and was also identified in murine whole kidney from both males and females indicating that GPR17 RNA expression is not sex-dependent. While GPR17 has known roles in oligodendrocyte maturation and myelination in the central nervous system, its functions within the liver and kidney are undetermined. To investigate its physiological functions, we started by uncovering its localization within the liver and kidney and identifying its ligand profile. A series of RNAScope in-situ hybridization assays were performed to localize GPR17 within hepatic tissue. Gpr17 mRNA was found within hepatocytes indicating that its highest expression is in the parenchymal cells of the liver. To identify possible activators for GPR17, we cloned the coding region into both flag-tagged PME18s and the Presto-Tango vectors. Live-cell surface labeling of HEK293T expressing the GPR17 constructs confirmed robust surface expression on the plasma membrane facilitating ligand screening. GPR17 downstream signaling has yet to be studied. Thus, we used the PRESTO-TANGO assay, an exogenous chemical ligand screening assay that relies on a luminescence readout to indicate -arrestin recruitment and receptor activation. A large, non-biased chemical screen of over 500 physiological compounds was tested for activation of GPR17. To date, we identified seven exogenous activators of GPR17: 3-[methyl(1,2,3,4- tetrahydronaphthalen-2-yl)amino]pyrazine-2-carbonitrile, 4-(4-fluorophenyl)-5-methyl-N-(2-methyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)thiophene-2-carboxamide,({1-[(2-chlorophenyl)methyl]-3,5-dimethyl-1H-pyrazol-4-yl}methyl)(methyl)[(1-methyl-1H-imidazol-2-yl)methyl]amine,N,N-dimethyl-6-[(5-phenyl-1,3-oxazol-2-yl)methyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-4-amine,4-phenyl-1-[6-(pyrrolidin-1-yl)-9H-purin-9-yl]butan-2-ol, 2-(3-fluorophenyl)-3-methyl-1-{[1,2,4]triazolo[4,3-a]pyridine-6-carbonyl}pyrrolidine, and 2,6,6-trimethyl-N-({2-methyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrimidin-6-yl}methyl)-4,5,6,7-tetrahydro-1-benzofuran-4-amine. Ongoing studies are aimed at narrowing down the in vivo activators of GPR17 to ultimately shed light on the physiological functions that this receptor plays in the kidney and liver.

NIH/NCATS.

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.
Proteomic and Transcriptomic Analysis of Human Colonoids Exposed to Bacterial Serine Protease, EspP
Authors: Thompson Myranda , Appell Lidia , Atanga Roger , In Julie
Published: May 2024

Gut regeneration mechanisms remain an important, yet elusive, facet of intestinal injury research. The intestinal epithelium must be intact for critical gut functions to remain in place, yet injury via microbe interactions or environmental toxicants is common. EspP is a potent serine protease that functions as a cytotoxin and is secreted by enterohemorrhagic Escherichia coli. This bacterial toxin has been recently shown to cause significant colonic epithelial damage in human colonoids. Previous proteomics and single cell RNA-seq analysis of human colonoids exposed to EspP found that there is a marked expansion of enteroendocrine cells (EECs) at acute exposures. There is also an increase in vascular endothelial growth factor (VEGF)A and B, but the reasons for this remains unknown. Therefore, our goals are to understand the effects of altered secretory lineage differentiation upon EspP exposure during EHEC infection and characterize angiogenesis as a key factor in disease progression. We hypothesize that EspP induced EEC differentiation leads to increased enterohormone section, such as 5-HT, which ultimately influences the mucosal immune response and indirectly leads to increased VEGFA and VEGFB expression. Together, these factors promote systemic disease progression through epithelial-endothelial interactions. Human colonoids derived from healthy adult colonic biopsies (UNM IRB approved study 18-626 and 18-171) were established and shown to physiologically model the colonic epithelia. Colonoids were acutely exposed to EspP. Control and EspP-exposed colonoids (n=3 unique donors) were dissociated into single cells and processed for droplet-based single cell sequencing (scRNA-seq) or tandem mass spectrometry. The scRNA-seq data was analyzed using the Seurat pipeline and integrated with the proteomics dataset. scRNA-seq results identified a 4-fold expansion of EECs, with increases in EEC hormones 5-HT and PYY as well as a 2-fold expansion of VEGF expression. These transcriptomic changes seen were validated in colonoids (>5 biologically unique human donors) via RNA FISH and immunofluorescence. Our results demonstrate that acute EspP exposure directly induces transcriptomic and cellular changes in intestinal epithelia independent of microbiota, stroma, and immune cells. As EEC expansion is seen broadly in disease states from colorectal cancer to celiac disease, determining the role of enterohormones upon these changes, as well as the function of EECs during acute injury, will fill a gap in knowledge relevant to a wider problem of intestinal dysfunction.

This work was supported by grants from the National Institutes of Health: K01DK106323, R56ES034400, P42ES025589; and AGA SURF.

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.
Pathophysiology of Carnitine Palmitoyltransferase 2 (CPT2) Deficiency in the Dahl Salt-sensitive R at
Authors: Smith Brody , Dissanayake Lashodya , Zietara Adrian , Levchenko Vladislav , Upadhyay Gunjan , Palygin Oleg , Halade Ganesh
Published: May 2024

Carnitine palmitoyltransferase 2 (CPT2) is an essential enzyme of the carnitine shuttle, which facilitates fatty acid -oxidation in mitochondria. This study aims to characterize the various effects of CPT2 deficiency on the Dahl salt-sensitive (SS) rat metabolome and its concomitant influence on renal homeostasis. CRISPR/Cas9 was used to insert a single base pair into the SS Cpt2 gene, causing a frameshift mutation found to be lethal in homozygotes (SSCpt2−/−). Western blotting and qPCR analysis of renal cortex tissue confirmed a reduced expression of CPT2 in heterozygous (SSCpt2+/-) rats compared to wild-type (WT) littermates; therefore, male and female heterozygotes were used for all experiments. Lipid metabolomics of renal cortex samples collected from SSCpt2+/- animals at 8 weeks old on a baseline 0.4% NaCl diet revealed a significant upregulation in inflammatory lipids such as 11-HETE (p<0.01) and 12-HHT (p<0.05) when compared to WT littermates. Urine analysis from the SSCpt2+/- group demonstrated a severe reduction in acetoacetic acid production (p<0.001), indicating ketogenesis is likely impacted by the loss of CPT2 in -oxidation. Additionally, TCA cycle metabolites fumaric acid (p<0.05) and oxaloacetic acid (p<0.01) are significantly downregulated in urine from SSCpt2+/- animals. Radio telemeters were implanted into the rats at 8 weeks of age to continuously record mean arterial pressure (MAP) and monitor the development of SS hypertension over the course of several dietary treatment protocols. Three weeks of either a 4% NaCl high-salt (HS) diet or a HS + ketogenic (HSK) diet with equivalent caloric content (4:1 caloric ratio, fat: carbohydrate) were administered to the animals. No difference in MAP elevation occurred between groups after the HS challenge alone, but SSCpt2+/- animals on the HSK diet had a significantly attenuated development of hypertension (p=0.03). Further metabolic analysis after the HSK diet illustrated that both groups had similar increases in long-chain fatty acids, liver enzymes, and cholesterol levels with a decrease in short-chain fatty acids. These experiments show that the partial loss of CPT2 in the Dahl SS rat leads to dysregulation across multiple metabolic pathways. Moreover, a ketogenic diet appears to protect heterozygous animals from the development of salt-sensitive hypertension.

Funding: NIH R35 HL135749 and VA I01 BX004024.

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.
Obesity to Type 2 Diabetes Mellitus: Importance of Longitudinal Characterization of the Progressive Phases as Identified in Rhesus Monkeys Maintained Under Constant Diet and Environmental Conditions
Authors: Hansen Barbara , Pattnaik Aishaya , Alvoir Joshua , Volante Vernon
Published: May 2024

The complex physiological mechanisms that underlie the links between obesity and T2DM involve “adiposity-induced” alterations in beta cell function —not yet mechanistically understood. Clearly the changes in beta cell function are greatly increased during the progression of obesity, --not reduced!! AND multiorgan insulin resistance whose mechanisms are unknown (although likely reduced by weight loss). Rhesus monkeys have shown these and have provided an amazing look at the Phases that lead to T2DM. We have previously summarized, in a table of 49 variables, the many ways that the nonhuman primate rhesus monkey offers exciting opportunities for insight into these extraordinary conditions compared to the human with T2DM. The Importance of longitudinal characterization, with a number of measurements that are regularly and periodically carried out to assess longitudinally some of the pathophysiological processes that develop spontaneously and naturally in these monkeys as they progress from normal, lean, young animals to obese animals with or without various physiological disturbances undergo the aging process. Usually these disturbances develop at varying rates in different monkeys and include hypertension, hyperlipidemia, hyperinsulinemia, hyperglycemia, impaired glucose tolerance, insulin resistance, peripheral neuropathy, impaired renal clearance, and cardiovascular disease—in short, the entire syndrome that becomes manifest in aged and diabetic humans. The longitudinal studies depend specifically on regular assessment of each animal and determination of the rate at which that animal is or is not progressing in the development of pathophysiological disturbances. Identification of phases allows one to estimate where a given monkey is in regard to the progression to overt diabetes. Application to humans will greatly increase our predictive ability to understand the mechanisms underlying both obesity and T2DM.

NIA NO1AG3 1012.

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.
No relation between increases of spleen volume and reductions in circulating pro-inflammatory microparticles following fourteen days of apnea and hypoxia exposures
Authors: Keeler Jason , Hite M. Jo , Hess Hayden , Tourula Erica , Chapman Robert , Johnson Blair , Ilardo Melissa
Published: May 2024

INTRODUCTION: Indigenous breath-hold diving populations have comparatively large spleen volumes, suggesting a functional benefit of a larger spleen in the context of diving. The spleen plays a role in clearing blood-borne microparticles (MPs). Pro-inflammatory MPs are increased with diving and have been associated with the etiology of decompression sickness. Thus, a potential benefit of a larger spleen may be enhanced splenic clearance of MPs. Repeated apneas and hypoxia exposure increases spleen volume in people without breath-hold diving ancestry. PURPOSE: Test the hypothesis that repeated apnea and hypoxia exposures reduce blood-borne pro-inflammatory MPs and the reduction is correlated with changes in spleen size. Methods: 20 healthy adults (10 women) completed 10 maximal apneas and 3 h of exposure to normobaric hypoxia (FiO2=0.14) for 14 days within a 16-day period. On Days 1 and 14, pre-exposure spleen volume was measured via ultrasound using the Pilström equation and venous blood was collected for blood-borne pro-inflammatory MPs analysis via flow cytometry. MPs data were log transformed for analyses. Data are presented as mean ± SD. Results: Average apnea duration increased from Day 1 (76±31 s) to Day 14 (91±30 s, p=0.004, dz=0.48). Pre-exposure spleen volume increased from Day 1 (169±36 ml) to Day 14 (182±39 ml, p=0.002, dz=0.33). Total MPs decreased from Day 1 (1407±814 #/μl) to Day 14 (1092±561#/μl, p=0.047, dz=0.45). MPs expressing CD66b+ (neutrophil origin; 194±89 vs. 156±72 #/μl, p=0.014, dz=0.47) and CD146 (endothelial origin; 337±197 vs. 250±116 #/μl, p=0.037, dz=0.53) were lower on Day 14, but MPs expressing CD41a (platelet origin) did not differ between Days 1 (82±78 #/μl) and 14 (55±38 #/μl, p=0.166, dz=0.45). Changes in MP counts from Day 1 to Day 14 were not correlated with changes in spleen volume for Total MPs (r=0.02, p=0.93) nor MPs expressing CD66b+ (r=0.30, p=0.23), CD146 (r=-0.04, p=0.86), or CD41a (r=0.31, p=0.20). CONCLUSION: Repeated apnea and hypoxia exposures increase spleen volume and reduce blood-borne pro-inflammatory MPs. However, there was no relation between exposure-induced changes in spleen size and MP counts. Thus, spleen size may not directly impact MP clearance. Rather, alterations in the MP phagocytic properties found within the spleen or another systemic change likely contribute to the observed reductions in MPs. Further work is needed to understand the role of splenic function in these observations.

Support: Offce of Naval Research (N00014-20-1-2593).

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.
Strategies for Inducing and Validating Zinc Deficiency and Zinc Repletion
Authors: Wenegieme Tara-Yesomi , McMichael Kelia , Elased Dalia , Hasrat Khanzada , Rockwood Jananie , Ume Adaku , Marshall Andrea
Published: May 2024

Background: According to the World Health Organization, zinc deficiency is a growing global health problem, impacting 31% of the world’s population. Zinc deficiency is a common comorbidity with many chronic diseases, from skin to kidney diseases. Given the increasing interest in zinc’s role in disease onset and progression, there is a dire need for reproducible approaches to manipulate zinc homeostasis. Here, we provide proven strategies for inducing and validating zinc deficiency and zinc repletion in a mouse model. Experimental Design: After acclimating to a ZnA (zinc adequate) diet, mice are randomly assigned to either a ZnA or ZnD (zinc deficient) diet. To replete zinc levels (ZnR), a subset of ZnD mice is returned to the ZnA diet. Animal growth rates and plasma zinc levels are assessed to validate zinc status. Results: Mice placed on the ZnD-diet have reduced body weights (26.48g ± 0.78) compared to mice maintained on the ZnA-diet (28.12g ± 0.13). However, zinc repletion promotes weight gain (27.87g ± 1.01) resulting in body weights comparable to mice maintained on the ZnA-diet. Furthermore, mice maintained on the ZnA-diet have plasma zinc levels of 3.19 μM/g Body Weight. While plasma zinc levels are 40% lower in mice place on the ZnD-diet (1.29 μM/g Body Weight). In contrast, plasma zinc levels (2.44 μM/g Body Weight) are increased in ZnR mice. Conclusion: These findings validate that the described dietary approach is an effective strategy for inducing zinc deficiency and zinc repletion. Significance: This cost-effcient approach will enhance the scientific rigor and reproducibility of studies examining the impact of zinc deficiency, and repletion on disease onset and progression.

Funding Source: R21 DK119879, R01 DK-133698.

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.
Chemogenetic phrenic motoneuron activation enables increased tidal volume
Authors: Benevides Ethan , Thakre Prajwal , Rana Sabhya , Oweiss Karim , Polotsky Vsevolod , Fuller David
Published: May 2024

Reduced output of the phrenic neuromuscular system and the resultant respiratory insuffciency occurs in many neuromuscular disorders. We hypothesized that expressing an excitatory DREADD (designer receptors exclusively activated by designer drugs) in phrenic motoneurons would enable DREADD-ligand induced increases in inspiratory tidal volume in awake, freely behaving rats. ChAT-Cre rats (n= 9; female n= 3) underwent bilateral injections of a Cre-dependent viral construct, AAV9-hSyn-DIO-hM3D(Gq)-mCherry (titer: 2.07x1012 vg/mL) into the mid-cervical (C4) ventral horn (1 μl injected per side). The combination of focal AAV injection and Cre-dependent expression was expected to drive hM3D(Gq) expression primarily in phrenic motoneurons, which are ChAT-positive and located in the C3-C5 ventral horn. Whole-body plethysmography was used to measure breathing frequency, tidal volume, and minute ventilation before and after intravenous delivery of a DREADD ligand, JHU37160 (J60), or saline (sham). Delivery of the ligand produced an increase in inspiratory tidal volume compared to saline infusion (two-way RM ANOVA, p= 0.037). Respiratory rate was similar between the two conditions (p= 0.582). Two weeks after plethysmography recordings, rats were urethane anesthetized and phrenic nerve electrical activity was directly recorded using suction electrodes. This procedure was done to directly assess the impact of intravenous delivery of the DREADD ligand on phrenic motoneuron output. The J60 ligand caused a rapid, sustained, and bilateral increase in phrenic nerve efferent burst amplitude (one-way RM ANOVA, p< 0.001), whereas saline injection had no impact. The increase in phrenic burst amplitude lasted up to 100 minutes, at which point the experiment was stopped. We conclude that expression of an excitatory DREADD in phrenic motoneurons enables DREADD ligand induced increases in tidal volume in the awake, unrestrained rat. The phrenic nerve recordings verify that this response very likely reflects activation of phrenic motoneurons.

Funding: R01HD052682-14 (DDF), R01HL153140-04 (DDF), University of Florida Graduate Fellowship (ESB).

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.
Comprehensive Assessment of the Impact of Alcohol Use on Cognitive Deficits in People with HIV
Authors: Fitzpatrick-Schmidt Taylor , Oral Evrim , Welsh David , Molina Patricia , Ferguson Tekeda , Edwards Scott
Published: May 2024

Human immunodeficiency virus (HIV) infection profoundly impacts the nervous system, leading to neurological deficits such as HIV-associated neurocognitive disorder (HAND). HAND represents the most common neurological comorbidity among people with HIV (PWH), and alcohol use may exacerbate the cognitive deficits seen in this condition, especially in underserved populations. This study aimed to investigate the relationship between alcohol use patterns and cognitive performance in an underserved cohort of PWH, with the hypothesis that alcohol misuse would exacerbate cognitive deficits in this population. Data collected from participants (n=259; 66.7% male; mean age 52 ± 10 years) enrolled in the New Orleans Alcohol Use in HIV (NOAH) study were utilized for cross-sectional analysis. Cognitive function was assessed using the Montreal Cognitive Assessment (MoCA), and alcohol use was comprehensively measured by four metrics: Alcohol Use Disorders Identification Test (AUDIT), 30-day timeline followback (TLFB), lifetime drinking history, and phosphatidylethanol (PEth) levels. The average MoCA score among participants was 20.7 ± 4.5, with 86.5% demonstrating cognitive impairment (MoCA < 26). Notably, individuals with total MoCA scores below 18 (classified as moderately or severely cognitively impaired) had a higher frequency of recent severe alcohol misuse (as assessed by PEth levels) and greater lifetime alcohol consumption. Participants at increased risk for AUD (as assessed by the AUDIT) also had worse global cognition and memory task performance, particularly those aged 50 and older. Further analysis of MoCA sub-score data indicated that participants with increased AUD risk had significant impairments in the cognitive domains of language, delayed recall, and memory. These findings highlight the importance of considering alcohol use and AUD risk as important factors that may exacerbate cognitive decline in vulnerable populations, including older patients and PWH. Future analyses will compare the relationship between cognition and alcohol in these HIV-positive patients with a cohort of HIV-negative patients to better understand the impact of concurrent HIV and alcohol misuse on cognitive function.

This research was supported by grant #F30AA030941, #T32AA007577, and #P60AA009803 from the NIAAA at the National Institutes of Health.

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.
Characterizing MetALD in a Nonhuman Primate Model using High Fat and Sucrose Diet and Chronic Binge Alcohol Administration
Authors: Gallegos Eden , Simon Liz , Molina Patricia
Published: May 2024

Mechanisms leading to metabolic and alcohol associated liver disease (MetALD) are not clearly delineated. This study examined the differential regulation of markers of hepatic mitochondrial function in nonhuman primates fed a high fat/high sucrose (HFSD) and administered chronic binge alcohol (CBA) or vehicle as compared to control-fed animals. We hypothesized that markers of hepatic dysregulation due to HFSD will be further altered by the combination of CBA/HFSD. Eight rhesus macaques were fed HFSD (27% sucrose and 42% fat, by kcal) with n=4 receiving vehicle and n=4 receiving 13-14g/kg/wk of ethanol through a gastric catheter. Rhesus macaques fed standard primate chow were used as reference controls (n=4-6). Liver RNA was isolated for determining expression of genes regulating mitochondrial biogenesis and function using RT-qPCR. Results were analyzed using one-way ANOVA and post-hoc pairwise multiple comparisons were performed using Tukey’s method, with alpha set to 0.05. Expression of inflammatory genes was significantly altered across the groups (p<0.05). HFSD increased tumor necrosis factor alpha (p=0.01), interleukin 1-beta (p=0.04), cluster of differentiation 38 (p=0.01), and transforming growth factor beta (p=0.01) expression compared to control. Both HFSD and CBA/HFSD increased interleukin-10 compared to control (p=0.02, p=0.03). Gene expression of mitochondrial function and oxidative stress regulators were significantly altered across the groups (p<0.05). CBA/HFSD decreased expression of peroxisome proliferator-activated receptor-gamma coactivator-1 beta (p=0.04) and nuclear respiratory factor 2 (p=0.04) compared to control. Both HFSD and CBA/HFSD decreased peroxisome proliferator-activated receptor alpha expression compared to control (p=0.01, 0.01). HFSD decreased superoxide dismutase 2 (SOD2) expression compared to control (p=0.01) and increased uncoupling protein 2 (p=0.01). SOD2 expression was increased only in CBA/HFSD compared to HFSD (p=0.01). These results reveal marked HFSD-mediated increases in markers of inflammation and CBA/HFSD-mediated changes in markers of mitochondrial function regulation and oxidative stress regulation. These findings suggest a greater impact of calorie-dense diets than CBA on hepatic inflammation and a combined impact of CBA and HFSD on mitochondrial regulation. Ongoing studies will examine oxidative phosphorylation complex protein activities and expression to elucidate and integrate HFSD or CBA mediated changes in hepatic bioenergetic capacity.

Supported by F30AA030910, P60AA009803, and T32AA007577.

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.
Empagliflozin, an SGLT2i, relaxes coronary smooth muscle at pharmacological concentrations
Authors: Essajee Sal , Warne Cooper , Tucker Selina , Tune Johnathan , Dick Gregory
Published: May 2024

Morbidity and mortality due to heart failure with preserved ejection fraction (HFpEF) is increasing and there are no universally recognized strategies for treatment. Sodium-glucose co-transporter type 2 inhibitors (SGLT2i), such as Empagliflozin, have been shown to reduce myocardial stiffness and improve coronary microvascular performance, but mechanisms are unclear. Previous studies in rabbit aorta and rat mesentery suggest Empagliflozin relaxes smooth muscle through activation of K+ channels. This study investigated the effects of therapeutic and pharmacological concentrations of Empagliflozin (10-300 μM) on the isometric tension of left circumflex (CFX) coronary arteries from Yorkshire swine. We tested the hypothesis that Empagliflozin would relax coronary smooth muscle via a concentration-dependent manner, involving K+ channels. CFX arteries were dissected, cleaned of adventitia, cut into 3 mm rings (n = 16), and mounted in organ baths containing Krebs-Henseleit buffer (37 °C) for isometric tension studies. Each CFX segment was adjusted to optimal preload (~3 g passive tension) as determined by <10 % change in active tension in response to 60 mM KCl. In order to determine whether Empagliflozin caused relaxation and, if so, by what mechanism, rings were contracted with either 30 mM K+ or the thromboxane mimetic U46619 (1 μM). Once tension plateaued, Empagliflozin (10-300 μM) was added in increasing concentrations. Therapeutic concentrations of Empagliflozin (10-30 μM), elicited no significant relaxation. Pharmacologic concentrations of Empagliflozin (≥100 μM) caused significant relaxation from ~15 ± 11% to ~45 ± 10% in response to 100 μM vs. 300 μM Empagliflozin, respectively. Relaxation was not different between rings contracted with K+ or U46619, suggesting membrane hyperpolarization via K+ channels is not involved. Our results represent the first study of the effects of Empagliflozin on coronary smooth muscle reactivity. Data support the idea that relaxation of coronary smooth muscle by Empagliflozin occurs at supra-pharmacologic concentrations in vitro, but K+ channel activation appears to be an unlikely mechanism.

This project was supported by National Institutes of Health grant R01 HL158723.

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.
Immune Cell Profile in Young Adults who Regularly Use E-cigarettes and the Role of Nicotine
Authors: Simovic Tijana , Matheson Chloe , Rodgers Abigail , Baig Fauzan , Breland Alison , Cobb O Caroline , Nana-Sinkam Patrick
Published: May 2024

Background: Cigarette smoking results in altered immune cell function and associated systemic inflammation, as observed in chronic obstructive pulmonary disease (COPD). Electronic (e-) cigarettes are novel, popular tobacco products frequently advertised as a safer alternative to traditional cigarettes; however, the effects of their regular use on immune cell function are currently unknown. Thus, the purpose of this study was to investigate the impact of e-cigarette usage and the role of nicotine on the immune cell profile of young, regular users. Methods: Twenty-five young, apparently healthy, regular users of e-cigarettes (EU, age: 23±3 y.) and twenty-two non-users (NU, age: 23±4 y.) participated in this study. A fasted venous blood sample was obtained from all participants for a complete blood count and nicotine concentrations. EU were randomized to either nicotine or nicotine-free products for fourteen days and completed a second assessment to investigate changes in the immune cell profile based on either increased (+C; n=11) or decreased (-C; n=9) nicotine usage accessed via cotinine levels. For comparison, the same assessments were completed in a group of 13 patients with a diagnosis of COPD (67±9 y., GOLD Stage II-IV). Results: Regular users of e-cigarettes exhibit significantly higher eosinophils (absolute, p=0.008, percentage, p=0.042) and platelets ( p=0.018) when compared to non-users but similar ( p≥0.431) to patients with COPD. White blood cell, neutrophil, monocyte, and lymphocyte counts were similar between the EU and NU. After fourteen days, despite significant changes in nicotine (Δ+C=+2±4 vs. Δ-C=-3±4 ng/mL, p=0.014) and cotinine (Δ+C=+28±27 vs. Δ-C=-141±120 ng/mL, p=0.001) between +C and -C groups, no differences in the immune cell profiles were observed. To note, no association between immune cells and nicotine levels at baseline or follow-up (nicotine: r≤0.352; p≥0.122; cotinine: r≤0.310; p≥0.126). Conclusions: Results of the study suggests that regular users of e-cigarettes present with altered immune cell profile in eosinophils and platelets when compared to non-users. The elevated immune cell markers were comparable to observed values in patients with COPD and have been associated with adverse effects on pulmonary and cardiovascular health. In addition, the elevated immune cell profile was not related to changes in nicotine, inferring that other e-liquid components may have an effect on innate immunity. Future studies are warranted to investigate the long-term effects of e-cigarette use and their components on immune cell profile.

Supported in part by a Rapid Response Project NIDA/FDA (PRM).

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.