bluuebunny/crossref_metadata_2025 huggingface dataset:
Signaling Mechanism of Cytochrome P450 1B1‐Dependent Angiotensin II‐Induced Activation of NADPH Oxidase in Vascular Smooth Muscle Cells
Authors: Song Chi Young , Jennings Brett L , Lin Jonathan , Fang Xiao R , Malik Kafait U
Published: June 2021

Previously, we have shown that angiotensin II (Ang II) activates NADPH oxidase and generates superoxide via release of arachidonic acid (AA) and its metabolism by cytochrome P450 (CYP1B1) in rat vascular smooth muscle cells (VSMCs). This study was conducted to determine the signaling mechanism involved in CYP1B1‐dependent Ang II‐ and AA‐induced NADPH oxidase activity in VSMCs isolated from aorta of rat, and wild type (Cyp1b1+/+) and CYP1B1 knockout (Cyp1b1−/−) mice. Ang II‐and AA‐induced increase in NADPH oxidase activity, measured using lucigenin as a chemiluminescent probe, was inhibited in rat VSMCs transduced with adenovirus (Ad) shRNA CYP1B1 but not its scrambled control shRNA, and in VSMCs from Cyp1b1−/− but not Cyp1b1+/+ mice. Ang II and AA increased ERK1/2, p38 MAPK, and c‐Src activities as determined by their phosphorylation by western blot analysis, in VSMCs from rat and Cyp1b1+/+ mice; increases in the activities of these signaling molecules were inhibited by Ad shRNA CYP1B1 in rat VSMCs, and in VSMCs from Cyp1b1−/− mice. Inhibitors of ERK1/2 (U0126), p38 MAPK (SB202190), and c‐Src (PP2), blocked Ang II‐ and AA induced increase in activities of these signaling molecules and NADPH oxidase, but not CYP1B1, measured using P450‐Glo™ Assay Kit, in rat VSMCs. These data suggest that Ang II stimulates NADPH oxidase activity via AA metabolism by CYP1B1 and activation of ERK1/2, p38 MAPK, and c‐Src, in VSMCs.
Restoration of endothelial function in Pparα−/− mice by Tempol
Authors: Silswal Neerupma , Wacker Michael J , Andresen Jon
Published: June 2021

Peroxisome proliferator‐activated receptor alpha (PPARα) is expressed throughout the vascular wall, and in the present study we investigated the vascular consequences of PPARα deficiency using Pparα−/− mice. By using isometric tension myography, concentration‐dependent relaxation to acetylcholine (ACh) was reduced by 20% in Pparα−/−compared to WT mice while no difference was observed in response to sodium nitroprusside (SNP). We also found that aortic expression of endothelial nitric oxide synthase (eNOS) protein was reduced by 25% in Pparα−/−mice indicating that the observed endothelial dysfunction may be due to reduced nitric oxide (NO) bioavailability. In support of this, using lucigenin‐enhanced chemiluminescence the aortic superoxide levels in Pparα−/−were double that of WT mice. To improve the endothelial function in Pparα−/−, we administered the antioxidant Tempol (1 mM) in the drinking water for 2 weeks which restored aortic relaxation to ACh, had no effect on SNP responses, and normalized superoxide levels. These results suggest that reduced eNOS expression in Pparα−/− mice, combined with elevated superoxide levels, promotes oxidative stress that impairs vascular function and Tempol likely restored endothelial function by enhancing NO bioavailability. This work was supported by AHA SDGs 0735053N (JA), 11SDG5330016 (MJW), 11POST7650044 (NS), and a NIH‐NIAMS grant RC2 AR058962 (JA & MJW).
PPARα Modulates Acrolein‐Induced Inflammatory Signaling in gp91phox Knock‐Out Mice
Authors: Marek Katarzyna , Chug Neha , Yousefipour Zivar , Newaz Mohammad
Published: June 2021

Objective of this study was to identify the effect of Peroxisome Proliferator Activated Receptor (PPAR) α induction on acrolein‐induced oxidative stress (OS) in gp91phox knockout mice. gp91phox knockout (KO) mice were treated with acrolein (Acr: 0.5μg/kg; i.p) with/without PPAR α ligand Clofibrate (clof: 250mg/kg; i.p). Age/weight matched wild type (WT) were used as control. KO mice had a higher (97%) 8‐Isoprostane in response to Acr than WT, and clof had no effect. Total Antioxidant Status (TAS) was significantly reduced and this was more prominent in WT (69%) than KO (53%). Xanthine Oxidase (XO) activity was higher in KO (0.53±0.05) than WT (0.35+0.02). Acr and clof increased XO activity in WT. Acr increased cycloxygenase (COX) in WT (4.58±0.3) and was unchanged in KO. Clof reduced COX in WT (54%) and KO mice (31%). NFκB activity was twice in Acr‐treated WT and stayed unchanged in KO. Clof blunted this increase in WT. Acr increased CD36 activity in KO (56%) which was attenuated by Clof. We propose that in absence of a NAD(P)H oxidase other oxygenase contributes in Acr‐induced OS and PPARα ligand clof is beneficial in attenuating inflammatory signaling.
Role of TRPC6 in High Glucose‐Induced Podocyte Apoptosis
Authors: Liu Bing‐Chen , Song Xiang , Yang Li‐Li , Eaton Douglas , Ma He‐Ping
Published: June 2021

It has been reported that the number of podocytes is significantly reduced in diabetic patients and animal models. However, the mechanism remains unclear. In the present study, we found that high glucose induced apoptosis in control podocytes which express TRPC6 channels, but not in TRPC6− podocytes in which TRPC6 was knocked down by its shRNA. This effect was reproduced by treatment of the podocytes with hydrogen peroxide (H2O2), one form of reactive oxygen species (ROS). Single‐channel data from cell‐attached patch‐clamp experiments showed that high glucose activated the TRPC6 channel, which was mimicked by exposure of podocytes to H2O2. Confocal microscopy data showed that high glucose elevated ROS in podocytes and H2O2 elevated intracellular Ca2+ via activation of TRPC6. These data together suggest that high glucose induces apoptosis in podocytes by inducing TRPC6 activity and subsequently increasing intracellular Ca2+ via elevation of ROS.
Superoxide and hydrogen peroxide stimulate renin release from juxtaglomerular cells
Authors: Mendez Mariela
Published: June 2021

Renin is the rate‐limiting enzyme in angiotensin formation, playing a pivotal role in blood pressure regulation. In the kidney, superoxide and hydrogen peroxide (H202) are involved in the development of hypertension by affecting nephron transport and vascular reactivity. However, it is not known whether superoxide and H202 are endogenously produced in juxtaglomerular (JG) cells, the source of these reactive oxygen species and whether they regulate renin release. We hypothesized that superoxide and H2O2 are endogenously produced in JG cells and that they stimulate renin release. Primary cultures of mouse JG cells were treated for 60 min with different inhibitors and renin released to the supernatant measured by radioimmunoassay. Decreasing endogenous superoxide with superoxide dismutase (300U/ml) inhibited renin release by 35±10% (p<0.03). By Western Blot we found that NOX4, one of the NADPH oxidase isoforms is present in JG lysates (n=3). Apocynin (120 μM), a general inhibitor of NADPH oxidases, decreased renin release by 28±7% (p<0.04). Since dismutation of superoxide results in H202 accumulation, we tested the effect of scavenging endogenous H202. Adding catalase (100U/ml) inhibited renin release by 37±10% (p<0.02). We concluded that endogenous superoxide and H202 stimulate renin release from mouse JG cells. These are the first data identifying endogenous sources of superoxide in JG cells. HFHS Award.
ASC plays a role in alveolar epithelial integrity
Authors: Fukumoto Jutaro , Fukumoto Itsuko , Phillips Oluwakemi , Cox Ruan , Parthasarathy Prasanna Tamarapu , Lgishetty Venu , Lockey Richard , Kolliputi Narasaiah
Published: June 2021

Apoptosis‐associated speck‐like protein containing a carboxy‐terminal CARD (ASC) is one of the major components of the NLRP3 inflammasome. We previously reported that inflammsome plays a role in macrophage activation under oxidant stress. In contrast to the widely studied inflammasome component NLRP3, the role of ASC under conditions of oxidant stress remains elusive. In this study, we tested the hypothesis that ASC deficiency in alveolar macrophages would lead to blunted inflammatory cytokine secretion and modified alveolar epithelial cell (A549) integrity. To test this, we used bone marrow‐derived macrophages (BMMs) harvested from wild type and ASC−/− mice. BMMs were subjected to hyperoxic conditions in the presence or absence of LPS. Supernatants from these cells were used to treat A549 cells and transepitheleal resistance (TER) was measured. Further, protein expression of E‐cadherin by A549 cells exposed to BMM‐derived supernatants was used as a measure of epithelial integrity. Our results reveal that A549 cells treated with ASC−/− BMM supernatant under hyperoxic conditions showed decreased TER in comparison to WT BMM supernatant treatment. The expression of E‐cadherin in these cells was also decreased in comparison to WT hyperoxic control. These results suggest that ASC may play a role in aiding alveolar macrophages maintaining epithelial integrity under oxidant stress.
Nitric oxide deficit elicits oxidative stress and augments T‐type calcium channel contribution to vascular tone
Authors: Hill Caryl Elizabeth , Kuo Ivana Y. , Ellis Anthie , Chaston Daniel J. , Shin Hee‐Sup , Hansen Pernille B. , Howitt Lauren
Published: June 2021

Nitric oxide (NO) bioavailability is reduced in cardiovascular disease however the impact on the mechanisms controlling vascular tone of pressurised arteries is unknown. Acute inhibition of NO with L‐NAME (10μM) significantly increased the T‐type (3μM NNC 55–0396) but not L‐type (1μM nifedipine) channel contribution to mouse cremaster arteriolar tone in vivo; an effect reversed by superfusion with the NO donor, SNP (100nM). Smooth muscle expression of Cav3.1 and Cav3.2 T‐type channels was also increased. In pressurised mesenteric arteries of Cav3.1ko and Cav3.2ko mice, acutely treated with L‐NAME, the contribution of T‐type channels was significantly less than in arteries from untreated mice. Chronic L‐NAME treatment (40mg/kg/day 2 weeks) increased vascular superoxide and the contribution of T‐type channels to cremaster tone. This was reversed by acute treatment with tempol (1mM), or inhibition of NADPH oxidase (500μM apocynin; 5μM DPI). Exogenous superoxide (xanthine oxidase, 3mU/ml) mimicked the effect of NO inhibition. We conclude that NO deficit significantly increases the contribution of Cav3.1and Cav3.2 T‐type calcium channels to vascular tone by increasing superoxide produced by NADPH oxidase.
HVCN1 contributes to superoxide production independent of intracellular pH regulation
Authors: O'Connor Paul Michael , Sun Jingping , Mazer Benjamin Aaron
Published: June 2021

HVCN1 is a voltage‐gated ion channel, required for maximal superoxide (O2−) production by NADPH oxidase during the respiratory burst in macrophages (MØ). During activation of NADPH oxidase, H+ ions are produced resulting in cellular acidification. By extruding H+, HVCN1 maintains the respiratory burst by preventing feedback inhibition of the oxidase by low pH. Recent evidence from our laboratory indicates that HVCN1 may also contribute to superoxide formation by additional mechanisms. We hypothesized that ‘HVCN1 acts to enhance NADPH oxidase activity independent of its role in intracellular pH regulation’. Intracellular pH was clamped using nigericin (100μM)/K+(135μM) in isolated peritoneal MØ from the rat at pH 8.0, 7.5, 7.0 or 6.5. Maximal O2− production (L‐012 luminescence (AU)) in MØ in response to (PMA 100μM) was linearly related to intracellular pH (r2=0.97). Addition of Zn2+ (100μM), to specifically inhibit HVCN1, significantly reduced maximal O2− production by >; 50% at all pH levels (Table 1). As these reductions in O2− production could not be explained by changes in intracellular pH, we conclude that HVCN1 enhances O2− production in MØ by pH independent mechanisms.
Inspiratory Muscle Exercise Reduces Lipoperoxidation in Obese and Healthy Subjects: A Pilot Study
Authors: Callegaro Carine Cristina , Maldaner Loani , Donato Letícia , Spring Amanda , Porto Fernando , Chaves Leandro , Cattaneo Roberta
Published: June 2021

Introduction

High‐intensity inspiratory exercise seems to increase oxidative stress in healthy subjects. Although obesity is related to impaired inspiratory muscle function, the acute effects of the inspiratory exercise remain unclear in obese subjects.

Methods

We studied 10 obese (35 ± 3 kg/m2; 32 ± 6 years old) and 10 healthy subjects (23 ± 2 kg/m2; 24 ± 5 years old). Maximal inspiratory pressure (PImax) was assessed by a pressure transducer. Blood sample was collected to assess thiobarbituric acid reactive substances (T‐BARS) before and after the inspiratory exercise performed in different days to inspiratory exercise without load maintained for 30 min, inspiratory exercise at 30% PImax maintained for 30 min and inspiratory exercise at 60% PImax maintained until reach the fatigue.

Results

Inspiratory muscle strength was similar in obese and healthy subjects. Inspiratory exercise performed at 60% PImax reduced T‐BARS in obese (from 6 ± 2 nmol MDA/mL to 4 ± 2 nmol MDA/mL) and in healthy subjects (from 7 ± 4 nmol MDA/mL to 5 ± 3 nmol MDA/mL, ANOVA: Time= 0,003; Group= 0,52; Interaction= 0,98). As expected, inspiratory exercise performed without load and at 30% PImax did not change T‐BARS in obese and healthy subjects.

Conclusion

This study shows that acute inspiratory exercise may reduce lipoperoxidation in humans.
β‐adrenergic receptor agonists known to stimulate β‐arrestin‐dependent signaling inhibit CFTR activity, cell migration and airway epithelial restitution
Authors: Peitzman Elizabeth R. , O'Grady Scott M.
Published: June 2021

Previously, the β‐adrenergic receptor antagonist carvedilol was shown to function as an inverse agonist, capable of stabilizing a receptor conformation uncoupled from Gs, but able to stimulate β‐arrestin‐dependent signaling and induce desensitization. In this study, measurements of cell migration using impedance sensing technology showed that stimulation of normal human bronchial epithelial cells and Calu‐3 cells with epinephrine or salbutamol reduced cell migration and increased the time required for epithelial restitution. Pretreatment with CFTR inhibitor CFTRinh‐172 or CFTR silencing blocked the effect of epinephrine and salbutamol. Carvedilol also inhibited migration and restitution to the same extent as epinephrine and salbutamol. Moreover, short circuit current (Isc) measurements revealed that unlike β‐agonists which increased Isc, carvedilol decreased basal current and the peak current following 8‐cpt cAMP stimulation. Pretreatment with carvedilol also abolished the effect of 8‐cpt cAMP on Cl− secretion. The carvedilol effect was abolished by pretreatment with the β2‐adrenergic receptor antagonist ICI‐118551, which also rescued Clsecretion following 8‐cpt cAMP stimulation. Therefore, agonists that activate β‐arrestin signaling and facilitate β2 adrenergic receptor desensitization inhibit cell migration and wound repair by a mechanism involving CFTR inhibition.
Adenosine inhibits human T lymphocyte migration via inhibition of KCa3.1 channels
Authors: Chimote Ameet Ajit , Kucher Vladimir , Boiko Nina , Hajdu Peter , Kuras Zerrin , Yun Yeo‐Heung , Szilagyi Orsolya , Conforti Laura
Published: June 2021

Adenosine (Ado), a purine nucleoside, is present at high concentrations in tumors where it contributes to the failure of immune cells to fight cancer cells. The mechanisms mediating the immunosuppressive properties of Ado are not fully understood. The activity of T lymphocytes relies on ion channels. KCa3.1 and Kv1.3 channels control T cell effector functions such as cytokine release while KCa3.1, together with the Ca2+/Mg2+ channel TRPM7, controls T cell motility. We tested the hypothesis that Ado asserts its immunosuppressive function in human T lymphocytes via KCa3.1 inhibition. Ado selectively inhibited KCa3.1, but not Kv1.3 and TRPM7, in a dose‐dependent manner in activated T cells. The action of Ado was mediated by A2A adenosine receptors. KCa3.1 inhibition by Ado was reversed by SCH58261 (selective A2A receptor antagonist), but not by MRS1754 (A2B receptor antagonist). The effects of Ado were mimicked by the A2A receptor agonist CGS21680. Furthermore, adenyl cyclase and PKAI inhibition reversed Ado effect on KCa3.1. Ado and CGS21680 inhibited T cell migration. Comparable effects on migration were obtained by the selective KCa3.1 blocker TRAM37, but not Kv1.3 blockade. Our data suggest that Ado inhibits KCa3.1 in human T cells via A2A receptor and PKAI thereby resulting in decreased T cell motility. This mechanism is likely to contribute to decreased immune surveillance in solid tumors. (NIH 2R01CA095286)
Lysophosphatidic acid inhibits megakaryocyte differentiation in CD34+ hematopoietic stem cells
Authors: Lin Kuan‐Hung , Yao Chao‐Ling , Lee Hsinyu
Published: June 2021

Lysophosphatidic acid (LPA) is a membrane‐derived lysophosphoipid which exerts its functions through binding to various LPA receptors (LPARs), which belong to family of G‐protein coupled receptors (GPCRs). In our previous studies, we have demonstrated that LPA induces erythropoiesis in the cord blood‐derived human CD34+ hematopoietic stem cells (hHSC) by activating LPAR3 with addition of erythropoietin under serum‐free culture. In this study, we attempt to investigate the role of LPA in megakaryopoiesis. Our data demonstrated that under TPO/SCF induced MK differentiation condition, megakaryocyte (MK) increased LPAR expression during differentiation period, and LPA treatment can significantly decrease megakaryopoiesis, which is detected by protein expression of CD41a and CD61. Simultaneously treatment with LPA and Ki16245, which is an antagonist of LPAR1 and LPAR3, rescued the inhibition of MK differentiation by LPA. We further demonstrated that specific agonist for LPAR3 block the megakaryopoiesis. On the other hand, the specific agonist of LPAR2 didn¡¦t have significant effects on megakaryocyte differentiation. These results indicate that LPA plays an important role in hHSC differentiation, and LPAR3 may be a critical regulator for both erythropoiesis and megakaryopoiesis.
Osteogenic differentiation of human mesenchymal stem cells induced by nanostructures
Authors: CHOI SEON YOUNG , RYU PAN DONG , JOO SANG‐WOO , LEE SO YEONG
Published: June 2021

In our previous study, chitosan‐conjugated gold nanoparticles altered expression of miRNAs related to bone and cartilage formation. Several studies suggest that many types of nanoparticles induce differentiation of human mesenchymal stem cells (hMSCs) into determined lineage. We hypothesized that various nanostructures promote osteogenic differentiation of hMSCs. In order to examine promoted osteogenic differentiation of hMSCs, PEGylated graphene oxide, gold nanorods and chitosan‐conjugated gold nanoparticles were added to an osteogenic induction medium for 7 and 14 days. For the study, alizarin red staining, alamar blue assay and quantitative real time PCR were performed. Our results demonstrate that nanostructures promote the deposit of calcium content in hMSCs at non‐toxic concentrations. Also, the expression of marker genes related to the early stage of osteogenic differentiation was increased. These results indicate that nanostructures may be used as a promoting reagent for bone formation. This study received support from the National Research Foundation of Korea (2012R1A2A2A01047151).
Impact of Augmenting Intracellular ATP on the Inducible Release of ATP from Banked Erythrocytes
Authors: Kirby Brett S , Zhu Hongmei , Riccio Daniel A , McMahon Timothy J
Published: June 2021

Prolonged banking of human erythrocytes (RBCs) coincides with adverse clinical outcomes following blood transfusion. Healthy RBCs release ATP that is vasoactive and anti‐adhesive, but hypoxia‐induced release of ATP from banked RBCs is markedly impaired. We tested the hypothesis that stored RBCs (35–42 days in blood bank) demonstrate impairments in ATP release that are common among disparate stimuli, and that release can be improved with an intracellular ATP ([ATP] i) boosting solution (Rejuvesol). ATP release from human RBCs (fresh or stored) was measured after mechanical deformation (5μm filtration) or direct G‐protein activation (mastoparan7; Mas7). Average basal ATP release was 146±19 and 107±14 nM from fresh and stored RBCs, respectively. Deformation‐induced release from stored RBCs was blunted compared to fresh (Δ34 vs 180%), but similar from Mas7 (Δ78 vs 95%). [ATP] i was lower in stored vs fresh RBCs, but elevated by Rejuvesol (2.8 vs 4.8 vs 6.8 umol/g Hb). Augmenting [ATP] i increased ATP release upon deformation by 2‐fold, but had no impact on Mas7‐induced release. These data suggest a specific impairment in inducible ATP release from stored human RBCs that is upstream of G‐protein coupled receptor activation, and that augmenting [ATP] i may improve ATP release in response to deformation. Improved ATP export may in turn support better clinical outcomes following RBC transfusion.

Support NIH HL113943
Differences in the association between stress, depression and cardiovascular disease risk factors in children and adults
Authors: Stanley Shyla C , D'Audiffret Alexandre C , Pagano Nunzio , Frisbee Stephanie J , Frisbee Jefferson C
Published: June 2021

Links between stress, depression and CVD risk factors in adults and children are unclear, although these likely involve consequences to stress‐sensitive systems (e.g., immune, endocrine) impacting CVD outcomes. To interrogate this, 245 participants in a community study completed Centers for Epidemiologic Studies Depression screener (CES‐D) and the Reeder Stress Index (RSI), had anthropometric measurements taken ((BMI, MAP, % body adiposity (%BA), lipids, etc.), and provided plasma samples for biomarker analyses. Univariate analyses suggested that correlations between inflammation, stress and depression differed widely between adults and children. CVD RF correlating with stress in children included %BA, HDL, glucose and BMI, while adult stress correlated with %BA, TG, and glucose. Correlation between inflammation and stress varied between ages: childhood stress correlated with EGF, IFN‐γ, IL‐1β, IL‐6 and IL‐8; adult stress correlated with GLP‐1 and IL‐1β. Multiple regression analyses suggest 29.5% of variance in adult RSI was predicted by tPAI‐1, IL‐1β, MAP and gender, while CES‐D score was driven by RSI (35.2% of variance). In children, CES‐D score and RSI were similarly predicted by a common cluster of variables (%BA, IFN‐γ, IL‐1β). The differences in stress and depression predictors between adults and children suggest distinct mechanisms for adapting to external stressors across age groups.
Role of the epithelial Na+ channels (ENaC) in development of ARPKD
Authors: Pavlov Tengis S , Levchenko Vladislav , Ilatovskaya Daria V , Palygin Oleg , Staruschenko Alexander
Published: June 2021

Autosomal recessive polycystic kidney disease (ARPKD) is characterized by the development of renal cysts of tubular epithelial cell origin. Dysfunction and aberrant regulation of the epithelial Na+ channel (ENaC) lead to a spectrum of diseases ranging from hyper‐and hypotension associated with improper renal Na+ conservation and wasting, respectively, to cystic fibrosis. Members of the epidermal growth factor (EGF) family have direct actions on Na+ reabsorption in the distal nephron. It has also been reported that EGF receptor (EGFR) is overexpressed and mislocalized to the apical membranes in the cystic epithelia. Here we tested whether ENaC plays a role in the cyst development in PCK rats, model of ARPKD. 4 and 12 weeks treatment with ENaC inhibitor benzamil caused more severe cyst formation in PCK rats. Patch clamp studies of ENaC in freshly isolated cysts and immunohistochemical analysis confirm that ENaC expression and activity are modulated in the freshly isolated cysts of PCK compared to benzamil‐treated and noncystic collecting duct. We conclude that abnormal EGF signaling leads to inappropriately low ENaC activity which contributes to cyst formation in PCK rats. Thus, the disruption of the EGF‐EGFR axis reported in PKD is linked to suppression of normal Na+ reabsorption and further progress of this kidney disease. Supported by the AHA 10POST4140109; ADA 1–10‐BS‐168; NIH HL108880.
Inhibition of NFκB activity in renal collecting duct cells abolishes transepithelial voltage difference
Authors: Svenningsen Per , Jensen Boye L
Published: June 2021

The renal collecting ducts are of major importance for regulation whole body sodium homeostasis and blood pressure. NFκB is a transcription factor which has been shown to influence transepithelial sodium transport in the renal collecting duct. The aim of the present study was to test the effect of NFκ inhibition on transepithelial voltage (TEV) and resistance (TER) in the mouse cortical collecting duct cell line M‐1. M‐1 cells were grown for 8 days with vehicle or 100 nM NFκB inhibitor on Snapwell supports, and TEV and TEV were measured daily. NFκB inhibition reduced the TEV significantly, which on day 8 reached 17.4 ± 1.5 mV in vehicle treated cells versus 0 ± 0 mV in inhibitor treated cells. In contrast, NFκB inhibition increased TER, which on day 8 reached 2118 ± 411 Ω in vehicle treated cells versus 3066 ± 208 Ω in inhibitor treated cells. It was observed that treatment of the cells with NFκB inhibition led to acidification of the apical and basolateral culture medium; thus, the pH in vehicle treated cells were 6.89±0.01 and 6.99±0.01, respectively, whereas pH of the inhibitor treated cells were 6.82±0.02 and 6.91±0.01, respectively. Thus, the current data suggest that NFκB activity is essential for development of a functional renal collecting duct.
Intestinal epithelial endocytosis of commensal bacteria is dependent on IFNγ‐induced terminal web myosin phosphorylation and brush border fanning
Authors: Wu Li‐Ling , Kuo Wei‐Ting , Peng Wei‐Hao , Lu Kuo‐Shyan , Ni Yen‐Hsuan , Yu Linda Chia‐Hui
Published: June 2021

Bacterial dissemination is commonly seen in intestinal obstruction (IO). Paracellular influx was mediated by ROCK signaling for phosphorylated myosin light chain (pMLC) and tight junctional disruption. Myosin phosphorylation in terminal web (TW) is associated with brush border (BB) fanning. The mechanism of bacterial endocytosis in enterocytes remains unclear.

Aim

To investigate the role of IFNγ in TW pMLC for bacterial endocytosis.

Methods

Mouse intestines were obstructed by loop ligation in which inhibitors were luminally administered. Next, mice were injected i.p. with anti‐IFNγ. Bacterial endocytosis in isolated enterocytes was measured by gentamycin resistance assay and fluorescence in situ hybridization. Epithelial Ca2+/ATP‐dependent TW contraction and BB fanning were visualized using TEM.

Results

Bacterial endocytosis was observed without paracellular permeability change at 3–6 h of IO. Enterocytic pMLC localized to TW was associated with BB fanning, which may be inhibited by ML‐7 but not Y27632. IO‐induced bacterial endocytosis through intermicrovillus space was reduced by ML‐7 or anti‐IFNγ. Caco‐2 cells treated with IFNγ showed MLCK‐dependent TW contraction and E. coli endocytosis and translocation.

Conclusions

IFNγ‐induced epithelial MLCK‐dependent TW contraction and BB fanning is involved in bacterial endocytosis. Funding support by National Science Council and NTU.
Rab22 is involved in regulation of the epithelial sodium channel (ENaC)
Authors: Liu Xiaoning , Bertuccio Claudia , Devor Daniel , Butterworth Michael
Published: June 2021

We previously demonstrated a role for small GTPases in the regulation of the epithelial sodium channel (ENaC) in epithelial cells of the distal kidney nephron. To further map the trafficking itinerary of ENaC in mouse cortical collecting duct (mCCD) cells, we investigated a number of other GTPases for their ability to alter ENaC function. Overexpression of wt‐Rab22 in mCCD cells resulted in a 45 +/−5% increase in ENaC‐mediated sodium transport over control transfect cells. A Rab22 dominant‐negative mutant, S19N reduced ENaC activity, while a GTPase deficient mutation Q64L enhanced ENaC activity 72 +/− 7% over control levels. To determine the location of a Rab22‐positive compartment we carried out indirect immunofluorescent labeling of both Rab11 and Rab22 in mCCD cells. Both Rabs localized to puncta (vesicles) near the membrane, and ENaC could be localized to both compartments, however there was a clear and non‐overlapping localization for each Rab. Finally, we tested the impact of the Rab22 mutants on cAMP activation of ENaC. There was a significantly greater response to cAMP stimulation in cells overexpressing the Q64L mutation compared to control or wt‐Rab22 transfected cells. These data introduce a new protein regulator in the trafficking itinerary of ENaC at a location distinct from the previously reported GTPases. The role of Rab22 and mechanism of ENaC regulation is still under investigation.
Heterogeneous nuclear ribonucleoprotein A2/B1 is a novel aldosterone target gene in the rat distal colon epithelium
Authors: de la Rosa Diego Alvarez , Hernandez‐Diaz Ivan , Morales Silvia , Hernandez Guadalberto , Salido Eduardo , Canessa Cecilia M , Giraldez Teresa
Published: June 2021

Proteins of the hnRNP A/B subfamily have been shown to regulate all aspects of mRNA metabolism and are associated with the modulation of cell proliferation. hnRNP A2/B1 binds the mRNA of the γ subunit of the epithelial Na+ channel (ENaC). ENaC is expressed in the apical surface of tight epithelia such as the renal distal tubule and collecting duct or distal colon. It is the rate‐limiting step of Na+ reabsorption and is essential in maintaining extracellular volume homeostasis. Aldosterone enhances ENaC activity in all its epithelial targets, but the molecular mechanisms underlying the effect are tissue‐specific. We studied whether physiological variations in aldosterone levels modulate hnRNP A2/B1 expression in target tissues in the rat. The results demonstrate that chronic or acute increases in aldosterone circulating levels potently enhance hnRNP A2/B1 mRNA and protein expression in the rat distal colon, but not in kidney or other aldosterone target tissues. This effect is mediated by the mineralocorticoid receptor. The genomic region upstream of hnRNP A2/B1 contains aldosterone‐responsive elements involved in the control of gene expression. Our data suggest that hnRNP A2/B1 may be part of tissue‐specific post‐transcriptional mechanisms involved in aldosterone‐mediated control of ENaC. Supported by grants BFU2010–16625 and CSD‐2008–00005 from Ministerio de Ciencia y Competitividad, Spain.
Effect of Hypovitaminosis D on Muscle Function and Physical Performance in Saudi Women‐Cross sectional
Authors: Hussein Khulood Sami
Published: June 2021

INTRODUCTION AND AIMS

Vitamin D deficiency is associated with reduced muscle strength. Data on the effect of poor vitamin D status on physical performance in Saudi women are lacking. The aim of this study was to investigate the correlation between vitamin D level and measures of physical performance.

METHODS

A total of 223 postmenopausal [>;51 years] healthy women were randomly recruited. Fasting blood samples were collected for assessment of 25(OH)D status. Muscle function was assessed by the time taken to perform the following tests: get up and go (GUG); 8 feet walk (8FW); five‐times sit to stand (5‐STS). SPSS (version 16) was used for data analysis.

RESULTS & DISCUSSION

39% of women were < 25nmol/L, 77% < 50 nmol/L, and 95% < 75 nmol/L. A significant negative correlation was found between 25(OH)D and GUG (P<0.05). After adjusting for key confounding factors including BMI the correlation disappeared. Linear regression analysis was used to examine the association between vitamin D level and measures of physical performance adjusting for potential confounders. Vitamin D level was not among the predictors of physical performance measures in this study group.

CONCLUSION

These data suggest that low vitamin D status is not associated with poor physical performance and may be a reflection of muscle adaptation to prolonged, life‐cycle of hypovitaminosis D.
Stereological analysis of Akt in muscles of young and old rats following a single bout of stretch‐shortening contractions
Authors: Rader Erik P , Ensey James , Baker Brent A
Published: June 2021

Careful design of resistance training programs is critical for offsetting age‐related sarcopenia. To address this, we implemented a non‐injurious stretch‐shortening contraction (SSC) protocol to muscles of rats. Following 4.5 weeks of resistance‐type training, muscles of young rats increased force by ~30% and mass by ~16%, while old rats sustained force deficits of ~30% with no change in muscle mass; no overt signs of histopathology were noted. Age‐related maladaptation to training may be due to aberrant Akt signaling – signaling influential in cellular survival/growth.

Purpose

To characterize volume density of Akt positive muscle fibers following an acute bout of non‐injurious SSCs with aging.

Methods

Dorsiflexor muscles of 12wk. and 30mo. old Fischer 344xBN Hybrid rats were exposed to SSCs in vivo. At 6h, 3d, and 5d, tibialis anterior muscles were removed for stereological analysis of transverse sections labeled for pan‐Akt (N =3 per group).

Results

For control muscles and muscles at 6h, pan‐Akt volume density represented ~40% of total tissue volume and was independent of age. At 3d and 5d post SSC, pan‐Akt levels were unaltered for old rats while values for young rats were decreased relative to values for old (p<0.05).

Conclusion

While the training‐induced decrease in Akt at young age needs further study, the lack of change in Akt levels at old age is consistent with an overall blunted response to training.
Old age of male C57/BL6 mice coincides with development of sarcopenia and anabolic resistance
Authors: van Dijk Miriam , Nagel Jolanda , Dijk Francina J , Walrand Stephane , van Norren Klaske , Luiking Yvette
Published: June 2021

Ageing is associated with sarcopenia, a progressive decline of skeletal muscle mass, muscle quality and muscle function. Although muscle parameters have been studied in mice of various ages/strains, no single study characterized these parameters in mice of various ages of the same strain. Therefore, male 10 and 24 mo old C57BL6/6J mice (n=45/group) were studied for the parameters of sarcopenia and the presence of anabolic resistance as part of its pathophysiology, with the aim of characterizing sarcopenia in old mice. Statistics were performed by ANOVA with post hoc LSD. Muscle mass of the hind limb was lower in 24mo (426±10mg) compared to 10mo (481±8mg) old mice (P<0.05). A significant decrease in physical daily activity (−62.8%), muscle grip strength (−11.1%) and ex vivo muscle contractile function (−22.4% maximal force production) was observed in 24mo compared to 10mo old mice (P<0.05). The muscle anabolic response to a single standard meal was tested and showed increased muscle protein synthesis in young, but not in old mice (P<0.05) indicative of anabolic resistance in the old mice. This study shows clear characteristics of sarcopenia and anabolic resistance in 24mo old C57/BL6 male mice. These observations offer potential to explore the effects of interventions targeting sarcopenia in the future using this mouse model.
Muscle‐specific deletion of p38α/β MAPK improves glucose tolerance and reduces body fat but impairs exercise capacity
Authors: Greene Nicholas Perry , Lira Vitor A , Chain Kristopher H , Zhang Mei , Hargett Stefan R , Keller Susanna R , Yan Zhen
Published: June 2021

The p38 MAPK belong to the stress‐activated kinase family, and p38γ MAPK is vital to exercise‐induced PGC‐1α expression, mitochondrial biogenesis and angiogenesis in skeletal muscle. However, the roles of p38α and p38β in skeletal muscle remain unclear likely due to redundanct function. To determine the function of these isoforms in skeletal muscle, we generated muscle‐specific p38α/β double knockout mice (DMKO). We then subjected these mice to 12 weeks of 45% high‐fat diet (HFD). DMKO mice showed improved glucose tolerance compared with wild‐type (WT) littermates on normal chow and HFD. DMKO mice showed reductions in total body weight, and muscle and epididymal fat mass compared with WT mice, accompanied by increased energy expenditure at equivalent caloric consumption. DMKO mice showed reduced exercise capacity as assessed by treadmill running. These findings demonstrate protective effects of p38α/β MAPK deletion in skeletal muscle regarding the development of insulin resistance and gains in body weight and fat mass, associated with increased energy expenditure. However, p38α/β MAPK deletion impairs exercise capacity suggesting a critical role in muscle function that requires further examination.
Fiber type specific PGC‐1α content and its relation to capillarization and mitochondrial content
Authors: Gouspillou Gilles , Sgarioto Nicolas , Norris Brandon , Hepple Russell Thomas
Published: June 2021

PGC‐1α is a central protein in skeletal muscle physiology. In addition of being the master regulator of mitochondrial biology, this coactivator of transcription has been implicated in the regulation of angiogenesis and has been shown to drive the formation of slow‐twitch fibers. The objective of the present study was, for the first time, to determine the relationship between PGC‐1α content, fiber type, capillarization, and mitochondrial content in rat skeletal muscle. To this end, serial sections of plantaris muscle were used to determine (i) fiber type (immunolabeling for MHC I, IIa, IIx, and IIb), (ii) mitochondrial content (succinate dehydrogenase stain), (iii) capillarization (Lead ATPase) and (iv) PGC‐1α content (immunolabeling). Surprisingly, we found that type IIa fibers have the highest PGC‐1α content, whereas no significant differences were observed between other fiber types. Associated with their higher PGC‐1α content, Type IIa fibers also displayed the highest mitochondrial content, followed by type I>;IIx>;IIb. However, type I displayed the highest number of capillaries per fiber perimeter, followed by type IIa>;IIx>;IIb. Although type IIa fibers show the higest PGC‐1α and mitochondrial content, our results suggest that neither basal mitochondrial content nor capillarization are directly related to differences in PGC‐1α content between fiber types in rat skeletal muscle.
Satellite Cell Depletion Negatively Impacts Voluntary Wheel Running Performance in Mice
Authors: Jackson Janna Renee , Fry Christopher S. , Kirby Tyler J. , Lee Jonah D. , Dean Clyde S. , Werker Justin H. , McCarthy John J. , Dupont‐Versteegden Esther E. , Peterson Charlotte A.
Published: June 2021

Satellite cells have long been thought to be responsible for muscle plasticity. Recent studies in the field using genetically‐modified mouse models that allow for conditional satellite cell ablation have challenged this dogma. Although these studies confirmed that satellite cells are required for muscle regeneration, they surprisingly show that they are not required for muscle growth. While the role that muscle stem cells play in muscle growth and regeneration are being defined, their role in muscle response to aerobic exercise remains unexplored. Therefore the purpose of the current study is to assess the involvement of satellite cells in response to voluntary wheel running. Female Pax7‐DTA mice were satellite cell depleted following tamoxifen administration. Mice were either ambulatory, or were wheel run for 8 weeks. Satellite cell depleted animals ran ~38% less km/day and 23% slower than non‐depleted animals. Succinate dehydrogenase was significantly elevated in plantaris muscles with running, but staining intensity tended to be attenuated in satellite cell‐depleted muscle. Myosin heavy chain isoforms were significantly altered with running, independent of satellite cell ablation. Similarly, fiber vascularization, quantified using the endothelial marker CD31, was elevated with running, but was unaffected by satellite cell depletion. In conclusion, the presence of satellite cells appears to be beneficial to voluntary running performance. The metabolic processes that may be altered in the absence of satellite cells that contribute to decreased endurance are currently under investigation. Funding: NIAMS R01AR060701
Body‐on‐a‐chip BOAC: A tool to elucidate clinical observations that involve modulations of the “methylation pathway” in association with the expression of adipokines
Authors: Ariza‐Nieto Magnolia , Alley Joshua B , Samy Sanjay A. , Fitzgerald Laura , Shuler Michael L
Published: June 2021

Obesity is an epidemic in the Unites States that puts individuals at increased risk for cancer, cardiovascular disease and type 2 diabetes mellitus. The link between obesity and cancer may be related to decreased levels of adiponectin, an antiangiogenic, antiatherogenic and antioncogenic biomarker. The anti‐inflammatory and insulin sensitizing properties of adiponectin may be explained by its ability to inhibit endothelial progenitor cells (EPC's) and hematopoietic progenitor cells (HPC's). Our hypothesis is that by nutritionally limiting the levels of homocysteine, epigenetic alternations in the levels of NPY will lead to increased levels of adiponectin. The goal of this research is to observe the changes in circulating levels of adiponectin, homocysteine and NPY in vivo and an attempt is being made to replicate the observations in vitro using the well defined “body‐on‐a‐chip” BOAC systems. Liver, omental fat and mononuclear cells are used to genotype and phenotype each donor. Genetically characterized bone marrow and/or adipose derived mesenchymal stem cells are isolated, expanded and differentiated. The BOAC is a microfabricated device based on a physiologically based‐pharmacokinetic (PBPK) model and combined with cell cultures is being used to study changes in the epigenome. The genotyped BOAC is a new tool to study variability in nutrient bioavailability, modulations of the methylation pathway, and adipokine expression and signaling. Funding Cornell Center on the Microenvironment and Metastasis (CMM) outreach pilot grant and Guthrie Foundation Investigator‐Initiated Research Grant.
Dietary sodium suppresses high‐fat diet weight gain in C57Bl/6J mice
Authors: Pearson Nicole A. , Littlejohn Nicole K. , Lutter Michael L. , Grobe Justin L.
Published: June 2021

To examine the synergistic effects of dietary sodium and fat on weight gain, male C57Bl/6J mice (9 wk) were placed (n=5 each) on a 45% (kcal) high‐fat diet (HFD, Bio‐Serv) with 0.25, 0.5, 1, 2, or 4% sodium, or standard chow (Teklad 7013). While baseline body masses were indistinguishable, the rate of weight gain was increased with HFD, and this was reduced with increasing sodium content (chow = 0.52±0.02, vs HFD+0.25% = 1.88±0.17, HFD+4% = 0.70±0.10 g/week). NMR at 4 weeks revealed adipose mass gains that paralleled body mass gains, with increased sodium preventing gains in a dose‐dependent fashion. These effects were maintained and confirmed by tissue masses at 10 weeks of treatment. Further, it was determined that the adipose gains were made primarily in the inguinal and perigenital fat, with no changes in interscapular fat. Caloric intake at ten weeks of treatment was similar across groups (11.4 to 15.9 kcal/day), while sodium intake correlated tightly with diet content (0.284 to 5.942 mEq/day), suggesting that the mice regulated food intake based on caloric content rather than sodium content. These data support the hypothesis that dietary sodium is a positive regulator of metabolic rate. Ongoing studies are focused on determining whether these effects are mediated by suppression of circulating renin, and whether new lower (renin‐inducing) USDA sodium intake guidelines will have unintended metabolic consequences.
Impaired skeletal muscle metabolism in obesity prone rats on a high fat diet
Authors: Allerton Timothy Daniel , Bench Eli , Primeaux Stefany
Published: June 2021

CD36 and Glut4 are major transporters of fatty acids and glucose and PPARγ is an important modulator of insulin sensitivity and lipid sensing in skeletal muscle, especially when consuming a high fat diet (HFD). Dysregulation of these transporters and modulators in skeletal muscle may influence the susceptibility to obesity. The purpose of the current experiment was to determine the effects of a HFD on factors associated with glucose and lipid storage and metabolism in the skeletal muscle of obesity‐prone and obesity‐resistant rats. Osborne‐Mendel (OM) and S5B/Pl (S5B) rats were fed a HFD or a low fat diet for 2 weeks. The gastrocnemius muscle was removed and tissue was sliced and stained for oxidative enzymes or protein was isolated for determination of protein expression of CD36, Glut4, and PPARγ using Western Blot. OM rats had a higher proportion of oxidative muscle fibers compared to S5B rats. Additionally, OM rats had a greater expression of CD36 compared to S5B rats, which was independent of diet. The consumption of a HFD reduced Glut4 levels in the skeletal muscle of OM rats compared to S5B rats and OM rats expressed less PPARγ than S5B rats, regardless of diet. Overall, these differences suggest that OM rats consuming a HFD have impaired expression of the main glucose transporter and a key modulator of lipid detection, PPARγ, in skeletal muscle, which may contribute to their susceptibility to obesity.
Multivalent GLP‐1/Yhb for targeted therapy and imaging of pancreatic β‐cells
Authors: Ananthakrishnan Kameswari , Penrod Leah V. , Hart Nathaniel J. , Weber Craig S. , Anderson Miranda , Vagner Josef , Limesand Sean W. , Lynch Ronald M.
Published: June 2021

Glucagon like peptide‐1 (GLP‐1) has potential for treating diabetes due to its effects on β‐cell proliferation and function. Described is a GLP‐1 analog that targets β‐cells specifically, which may increase its therapeutic efficacy and make it useful for monitoring β‐cell mass. GLP‐1 was coupled with a flexible linker to Yohimbine (Yhb) (α2 Adrenergic Receptor antagonist). GLP‐1/Yhb binds to cells expressing both receptors at low nM concentrations and is rapidly internalized. In cells where either GLP‐1R or α2AR were knocked down, binding of GLP‐1/Yhb was severely impaired (≤ half of control cells) indicating both receptors are required for high affinity binding. Intravenous injection of 250nM GLP‐1/Yhb‐Cy5 showed a high β‐cell:acinar ratio of ~1.8:1 in pancreatic sections while binding of 111In‐labeled GLP‐1/Yhb in isolated rat islets was significantly higher than acinar cells. Only islet binding was blocked by unlabeled GLP‐1/Yhb indicating specific binding. Following tail vein injection of 111In‐GLP‐1/Yhb, >; 90% of ligand was cleared from circulation within 30 mins. At this time, specific binding in pancreas relative to a range of other tissues was observed because pretreatment with cold GLP‐1/Yhb blocked only islet binding. In all, these data show that GLP‐1/Yhb binds selectively to β‐cells, demonstrating its potential for targeted β‐cell imaging and therapy.

Support: Juvenile Diabetes Research Fnd