bluuebunny/crossref_metadata_2025_split huggingface dataset:
Plasma derived exosomes from diabetic mice cause accumulation of neutral lipids in C2C12 myotubes
Authors: Pitzer Christopher , Paez Hector , Ferrandi Peter , Mohamed Junaith , Alway Stephen 
Published: May 2023

Purpose: Skeletal muscle is the largest metabolically active organ in the body and a major site of glucose disposal. Ectopic neutral lipid accumulation in skeletal muscle is correlated with skeletal muscle insulin resistance. Exosomes are released by nearly every mammalian cell type and are found in almost all biofluids including the blood. Exosome biomolecular cargo is altered by multiple physiological stimuli. Exosomes found in the blood can readily enter skeletal muscle where their cargo regulates cellular metabolism. We hypothesized that exosomes derived from the serum of diabetic animals would alter expression of genes important for maintenance of lipid content in C2C12 myotubes, resulting in lipid accumulation. Methods: Exosomes were isolated from plasma of diabetic (Db/Db) and cage control mice using a commercially available exosome isolation reagent (exoQuick, System Biosciences Inc. Paolo Alto, CA) and resuspended in PBS. C2C12 myoblasts were grown to confluence and allowed to differentiate into myotubes for 5 days. Myotubes were treated with ARAC for 24 hours on day 3 of differentiation to remove proliferating myoblasts. Myotubes were incubated with plasma derived exosomes for 24 hours beginning on day 5 of differentiation. Oil Red O stain was performed to visualize lipid accumulation and then accumulated Oil Red O was eluted from washed myotubes. The eluent absorbance was quantified to determine intracellular lipid abundance. In a separate set of exosome exposed myotubes, qPCR was used to determine gene expression of genes important for lipid oxidation. Results: Oil Red O showed visible accumulation of lipids in myotubes exposed to exosomes from diabetic animals. Quantification of the Oil Red O elution showed a significant increase in neutral lipid presence in myotubes incubated with diabetic animal exosomes as compared to controls (32% increase, unpaired t-test P-value = 0.0004). qPCR showed no significant alterations in gene expression of FATP1, FATP4, ATGL, ACADM, or FASN. Conclusions: Together, these data show that exosomes from diabetic animals induce the accumulation of neutral lipids in C2C12 myotubes without altering expression of genes that maintain lipid content in these cells. This indicates a possibility that exosomes deliver neutral lipids to myotubes and that these lipids may be contained within exosomes and trafficked as cargo to the muscle. 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.
The circadian clock repressor Cry2 promotes adipogenesis by suppressing Wnt signaling via interaction with Per2
Authors: Li Weini , Xiong Xuekai , Kiperman Tali , Ma Ke 
Published: May 2023

The circadian clock is driven by a transcriptional-translational feedback loop that elicits ~24 hour rhythms in behavior and physiology. The cell-autonomous clock exerts temporal regulation in adipocyte development and clock disruption leads to the development of obesity. There is current intense effort to identify clock-targeting interventions for metabolic disease therapy. Cryptochrome 2 (Cry2) is a circadian clock repressor, and it binds with Period proteins as a heterodimer complex to inhibit CLOCK/Bmal1-activated clock transcription that constitutes the negative feedback arm of the clock circuit. Our previous studies demonstrated that clock activator Bmal1 inhibits adipogenesis via the Wnt signaling pathway. To date, the role of Cry2 in adipogenic regulation remains unknown. Here we show that Cry2 inhibits adipogenesis and identify a critical residue that mediates interaction with Per2 required for this regulation. Cry2 transcript and protein are markedly induced during adipogenic differentiation. Via site-directed mutagenesis, we identified that Cysteine 432 within the lid domain of Cry2 protein is required for interaction with Per2, and a C432A mutant abolished this interaction. As a result, Cry2 repression activity of CLOCK/Bmal1-mediated transcription was abolished by the C432A mutant, as shown by Per2-luc reporter luciferase assay. Cry2 C432 mutation also led to elevated ubiquitination that accelerated protein degradation, an additional mechanism contributing to its loss of function. In 3T3L1 preadipocyte, we found that ectopic expression of Cry2 enhanced adipogenesis, whereas its loss-of-function suppressed adipocyte differentiation. In line with these findings, a Cry-stabilizing compound KL001 markedly augmented adipogenic differentiation. As compared to wild-type Cry2 effect on promoting adipogenesis, Cry2 C432A mutant largely abolished differentiation consistent with loss of repressive function in clock modulation. Mechanistically, we show that Cry2 induces adipogenesis via negative regulation of Wnt signaling pathway leading to inhibition of CCAAT/enhancer binding protein α (C/EBPα) expression. Together, our findings reveal a novel function of Cry2 in promoting adipocyte development via a critical residue involved in interaction with Per2. Our study thus provides a mechanistic basis for clock-targeting interventions to inhibit Cry2 activity for the treatment of obesity and its associated complications This work is supported by NIDDK grants 1R01DK112794, 1R01DK130499 and AR-DMRI 2023 T2D Innovation Grant. 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.
The Epithelial Sodium Channel is a Zinc Sensitive Renal Sodium Reabsorption Pathway that Mediates Zinc Deficiency-induced Hypertension
Authors: McMichael Kelia , Wenegieme Tara-Yesomi , Waite Aston , Ume Adaku , Adams Danielle , Williams Clintoria 
Published: May 2023

Background: Zinc deficiency (ZnD) is comorbid with many chronic diseases including kidney disease and diabetes. Individuals in these vulnerable populations have a higher prevalence of hypertension. Recently, we reported that ZnD induces hypertension by promoting renal Na+ reabsorption. However, the renal Na+ reabsorption mechanisms involved continue to be identified. The renal epithelial Na+ channel (ENaC) plays an essential role in regulating whole-body Na+ and water balance and is involved in hypertension. We hypothesize that ENaC is a Zn2+-sensitive channel that mediates ZnD-induced hypertension. Experimental Design: To establish the role of ENaC in ZnD-induced hypertension, WT C57Bl/6 mice were switched from a Zn2+ adequate to a Zn2+ deficient diet for 10 weeks. During weeks 9 and 10, mice were treated with benzamil, an ENaC inhibitor. Systolic blood pressure (BP) was monitored by tail-cuff plethysmography; Renal ENaC expression was examined by immunohistochemistry and Western blot. To authenticate the Zn2+ sensitivity of ENaC, mouse distal convoluted tubule cells (mDCT) were treated with a Zn2+ chelator (TPEN) or vehicle (DMSO) for 24 hours. A subset of TPEN-treated cellular monolayers were supplemented with Zn2+ for 24 hours to replete intracellular Zn2+. Cellular ENaC expression was examined by Western blot analysis. Results: B lood pressure: Compared to zinc adequate mice (101.4 mm Hg ± 4.225), zinc deficient mice had increased BP (154.6 mm Hg ± 2.422). However, benzamil treatment lowered BP (115.0 mm Hg ± 4.626) of zinc deficient mice. Immunohistochemistry: Next, Zn2+ effects on ENaC expression was examined. In zinc deficient mice, αENaC protein abundance significantly increased compared with zinc adequate mice. Western blot analysis: To directly investigate Zn2+ effects on ENaC expression, in vitro studies were performed using mDCT cells. Western blot analysis showed that TPEN-induced ZnD stimulated both αENaC and βENaC protein expression. Summary: Collectively, our findings reveal that (1) ENaC is a Zn2+ sensitive renal Na+ reabsorption pathway and (2) ENaC mediates ZnD-induced hypertension. Significance: Understanding the renal mechanisms by which Zn2+ regulates blood pressure may have an important therapeutic impact on hypertension. Funding Source: AHA-16SDG27080009 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.
High-fat loading abolishes sex differences in blood pressure (BP) responses to high-salt in Dahl rats
Authors: Godley Hannah , Dasinger John Henry , Cherian-Shaw Mary , Zamaro Aleksandra , Spires Denisha , Ilatovskaya Daria , Mattson David , Abias-Battad Justine , Sullivan Jennifer 
Published: May 2023

There are numerous reports that male Dahl rats have greater increases in BP with a high-salt (HS) diet than females, yet women have been suggested to be more salt-sensitive than men. Since the typical Western diet consists of both HS and high-fat (HF), the goal of the present study was to test the hypothesis that HF loading and a combined HF/HS would abolish the protection observed in female Dahl rats vs. males. To test our hypothesis, 6 week old male and female Dahl rats were implanted with telemetry devices to continuously measure mean arterial BP; n=6. Rats were allowed to recover for one week followed by one week of baseline BP recording. Rats were then placed on a HF diet (60% kcal from lard; Bio-Serv) for 2 weeks followed by a HF/HS (4% salt; Bio-Serv) for an additional 2 weeks. Rats were placed in metabolic cages prior to starting HS and after 2 weeks to collect a 24 hour urine sample to measure urinary protein and albumin excretion. Glomerular filtration rate (GFR) was measured at the end of the study conscious freely moving animals using FITC-inulin elimination method. Baseline BP values were comparable between males (115±5 mmHg) and females (117±3 mmHg; p=0.67). BP increased during HF diet treatment, yet BP values remained similar in males and females (133±7 vs. 134±3 mmHg, respectively; p=0.93). Interestingly, females had a greater increase in BP on HF/HS vs. males that reached significance by the end of the study (146±7 vs. 160±6 mmHg, respectively; p=0.03). Despite this, urinary protein (185±23 vs. 94±19 mg/day; p=0.001) and albumin (142±22 vs. 56±13 mg/day; p=0.006) excretion were greater in males than in females. However, GFR was comparable between males and females (0.55 ± 0.03 vs. 0.45 ± 0.04 ml/min/100 g of body weight; p=0.07). In conclusion, female Dahl rats are not protected from HS diet when also challenged with HF, supporting our hypothesis that HF loading eliminates cardiovascular protection in females vs. males. However, males exhibited greater renal injury. 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.
Inhibiting Cyclooxygenase Activity Attenuates the Exaggerated Exercise Pressor Reflex in Male UCD-Type 2 Diabetes Mellitus Rats
Authors: Samora Milena , Huo Yu , Graham James , Stanhope Kimber , Havel Peter , Harrison Michelle , Stone Audrey 
Published: May 2023

It is well established that prostaglandins, a product of cyclooxygenase (COX) enzymes contribute to evoking the exercise pressor reflex. Previous studies suggest that COX expression is upregulated in the peripheral nerves of those with type 2 diabetes (T2D), likely due to low grade inflammation associated with the disease. The purpose of this study was to determine if COX activity in the exercising skeletal muscle exaggerates the exercise pressor reflex in T2D. We hypothesized that inhibiting COX activity in the hindlimb would reduce the exaggerated pressor response to muscle contraction in T2D rats. To do this, we evoked the exercise pressor reflex by statically contracting the skeletal muscle for 30s in unanesthetized, decerebrate rats. Experiments were performed on male University of California Davis (UCD)-T2D (n=8; body weight: 526 ± 42g) and Sprague Dawley rats (n=3; body weight: 473 ± 10g). Changes (Δ) in peak mean arterial pressure (MAP) and heart rate (HR) responses to exercise were compared before and after locally injecting indomethacin (1 mg/kg in 0.2 mL of 100 mM sodium carbonate), a COX 1 and 2 inhibitor, or its vehicle (0.2 mL of 100 mM sodium carbonate) into the arterial supply of the ipsilateral (contracting) and contralateral (non-contracting) hindlimb. Data are presented as mean ± SD. We found that local injection of indomethacin into the contracting hindlimb reduced the exaggerated peak pressor (ΔMAP: 30 ± 14 mmHg to 18 ± 8 mmHg; P<0.01) but not cardioaccelerator (ΔHR: 21 ± 8 bpm to 19 ± 11 bpm; P=0.59) responses in T2D rats, but did not reduce these responses in healthy rats (ΔMAP 14 ± 3 mmHg to 14 ± 6 mmHg; P>0.99; ΔHR: 10 ± 5 bpm to 13 ± 7 bpm; P=0.67). Conversely, local injection of indomethacin into the non-contracting hindlimb (n=4), as well as injection of the vehicle in the contracting hindlimb (n=4), did not affect the pressor response to muscle contraction in T2D rats. Developed tensions during muscle contraction were similar for all comparisons. In summary, our findings suggest that COX activity contributes to the exaggerated pressor response to muscle contraction in T2D rats. This project was supported by NIH R01 HL144723. This project was supported by NIH R01 HL144723. 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.
Visfatin-Induced Smooth Muscle Cell Stiffness Syndrome during Lysosomal Acid Ceramidase Deficiency
Authors: Li Guangbi , Zhang Fan , Huang Dandan , Zou Yao , Li Pin-Lan 
Published: May 2023

Recent studies have reported that intrinsic stiffness of smooth muscle cells (SMCs) plays an important role in contributing to arterial wall stiffness, which led to the concept of smooth muscle cell stiffness syndrome (SMCSS). However, the molecular mechanisms regulating the intrinsic stiffness of SMCs are still poorly understood. The present study tested whether lysosomal acid ceramidase (AC) determines intrinsic stiffness of mouse aortic SMCs via regulation of actin polymerization and integrin recycling. Atom force microscopy was used to study mechanical properties of SMCs. As an injurious adipokine, visfatin was found to reduce SMC height, but elevate cortical stiffness and focal adhesion in SMCs, indicating the augmentation of intrinsic stiffness. Moreover, visfatin-induced elevation of cortical stiffness was associated with increased actin polymerization in peripheral areas of SMCs. These pathological changes were much more remarkable in SMCs from smooth muscle-specific Asah1 gene (gene code of AC) knockout (Asah1fl/fl/SMcre) mice compared to SMCs from WT/WT mice. Super-resolution microscopy showed that multivesicular bodies (MVBs) as integrin degrading organelles were much less interacted or fused with lysosomes in SMCs from Asah1fl/fl/SMcre mice than WT/WT mice upon visfatin stimulation. In contrast, MVB-α5 integrin encounters increased more in SMCs from Asah1fl/fl/SMcre mice compared to SMCs from control littermates, suggesting that AC deficiency increases α5 integrin accumulation in MVB, which may enhance recycling endosome moving to cell membrane. Given the vital role of lysosomal transient receptor potential mucolipin 1 (TRPML1) channel in the regulation of lysosome trafficking and its interaction with MVB, we tested whether this lysosomal channel is disturbed by visfatin. By GCaMP3 Ca2+ imaging and whole-lysosome patch clamp recording, we found that ML-SA5 (TRPML1 channel agonist) induced remarkable lysosomal Ca2+ release in WT/WT SMCs, which was attenuated by visfatin. In SMCs lacking Asah1 gene, however, ML-SA5 induced much less Ca2+ release from lysosomes, which was almost abolished by visfatin. Lysosome trafficking analysis showed that ML-SA5 activation of lysosomal TRPML1 channels markedly increased lysosome movement in SMCs, which was significantly inhibited by visfatin and Asah1 gene deletion. Based on these results, we concluded that lysosomal AC deficiency leads to dysregulation of actin polymerization and integrin recycling in SMCs, which may be a novel molecular mechanism of SMCSS and ultimate arterial wall stiffening during obesity. This study was supported by NIH grants HL057244 and HL075316. 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.
Understanding the role of Cysteinyl leukotriene receptor signaling in cancer progression and metastasis
Authors: Sabu Kattuman Emma Elizabeth , Teegala Lakshminarayan Reddy , AV Thanusha , Thodeti Charles , Paruchuri Sailaja 
Published: May 2023

Background: Cysteinyl leukotrienes (cys-LTs) are pro-inflammatory mediators which enhance inflammation through cysteinyl leukotriene receptor 1 (CysLT1R) and cysteinyl leukotriene receptor 2 (CysLT2R). Although cys-LTs and their receptors are mainly implicated in asthma and inflammation, recent association of inflammation with cancer has led to enhanced interest in understanding these players in cancer progression and metastasis. However, the exact molecular mechanisms underlying key processes in cancer- proliferation, migration and invasion ( in vitro), tumor progression and metastasis (in vivo) by these mediators remain unexplored. Further, the contribution of these receptors in tumor vs stromal microenvironment, and their interactions is unknown. A thorough investigation of these receptor signaling can reveal their therapeutic potential in cancer progression and metastasis. Objective: To study how cysteinyl leukotriene receptors contribute to melanoma and breast cancer initiation, progression, and metastasis Methods: Expression and functional analysis of CysLTR was determined by transcript (qPCR), western blotting (protein) and by calcium flux assays. Cancer cell survival and proliferation were analyzed by XTT and BrDU assays. Activation of intracellular signaling molecules was determined by western blotting using phospho-specific antibodies. Migration and invasion were analyzed using trans-well assays. Tumors were induced in WT, Cysltr1-/- and Cysltr2-/- mice and their progression and metastasis were evaluated. Results: Breast cancer (EO771) and melanoma (Luc-B16F10-GFP) cells express high levels of CysLT2R compared to CysLT1R. Further, stimulation of tumor cells with cys-LTs caused phosphorylation of Erk, p38, JNK, Akt, and CREB independent of epidermal growth factor receptor (EGFR). Notably, treatment of tumor cells with CysLTR antagonists significantly reduced migratory capability of the cells suggesting the vital role played by CysLTRs in tumor cell migration. Finally, we observed a significant reduction in the tumor volume in mice deficient in CysLTR ( Cysltr1-/- and Cysltr2-/-) mice compared to the WT mice, indicating a role for CysLTR in mediating tumor growth in vivo. Conclusion: Cysteinyl leukotrienes play an important role in promoting tumor proliferation, and metastasis via CysLTR and therefore, understanding their signaling and targeting them can offer potential targets for cancer therapy. 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.
Activation of select airway afferent subpopulations evokes cardiopulmonary reflexes
Authors: Darcey Teresa , Taylor-Clark Thomas 
Published: May 2023

Activation of airway sensory nerves causes respiratory and autonomic reflexes. The majority of the sensory nerves are only sensitive to noxious stimuli, such as inflammation, infection, irritants and pollutants. Activation of these nociceptive sensory nerves evokes protective mechanisms such as apnea, cough and bradycardia. However, these reflexes may also contribute to disease morbidity when excessively or inappropriately activated. Airway nociceptive sensory nerves, which are largely projected from the vagal ganglia (nodose and jugular ganglion), are heterogeneous with respect to gene expression and neuroanatomy, and our objective is to characterize the reflexes evoked by activation of specific afferent subsets. To selectively activate vagal afferent subpopulations in vivo, mice were exposed to nebulized selective stimuli such as capsaicin (transient receptor potential (TRP) vanilloid 1 (V1) agonist), allyl isothiocyanate (AITC, TRP ankyrin 1 (A1) agonist), adenosine (nodose-selective agonist) and clozapine-N-oxide (CNO, selective agonist for the designer receptors exclusively activated by designer drugs (DREADD) stimulatory receptor hM3Dq). hM3Dq expression was selectively expressed in sensory subpopulations under the control of Cre recombinase in TRPV1-cre (all nociceptors), Tac1-cre (peptidergic/jugular-originating nerves) and P2X2-cre (nodose-originating nerves). ECGs were recorded via radiotelemetry following implantation of biopotential sensing modules, and respiration was measured via whole body plethysmography. Dose response experiments have been performed for capsaicin, AITC and CNO to determine the minimum dose required to evoke the cardiopulmonary reflex, all of which produce significant bradypnea and bradycardia. Capsaicin and AITC produced comparable levels of bradypnea, but AITC evoked a stronger bradycardia than capsaicin. Bradypnea also appeared at lower doses for both capsaicin and AITC compared to the higher doses required to evoke bradycardia. Adenosine administration produced minimal bradypnea and bradycardia compared to CNO administration on the P2X2+ nodose population which produced a larger bradypnea and bradycardia. CNO-driven activation of the Tac1+ jugular population also evoked significant bradypnea and bradycardia. Our data indicate that stimulation of nociceptive afferents (TRPV1+ and/or TRPA1+) selectively evokes bradypnea and bradycardia in freely moving mice. However, our data also suggests that functionally-distinct nociceptive subpopulations may differentially regulate cardiopulmonary reflexes. NIH R01: HL152219; NIH T32: HL160529 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.