bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Rhbg‐mediated NH3 and NH4+ transport by DIDS (1098.2)
Authors: Caner Tolga , Abdulnour‐Nakhoul Solange , Brown Karen , Hamm L. , Nakhoul Nazih
Published: June 2021

Rhbg is a non‐erythroid Rh glycoprotein expressed at the basolateral membrane of renal collecting duct. In previous studies, we demonstrated that Rhbg transported both NH4+ and NH3. As such, the Rh transporter is unique in being able to transport both the ionic and the gaseous components of ammonia. Other studies have shown that DIDS inhibited CO2 transport by AQP1. This suggested that DIDS might also inhibit transport of other gases such as NH3 by Rh proteins. This study was conducted to test this hypothesis. To do so we used ion‐selective microelectrodes and two‐electrode voltage clamp to measure changes in surface pH (pHs) and whole cell currents (I) induced by NH3/NH4+. All experiments were conducted on Xenopus oocytes expressing Rhbg. Our results indicate that in oocytes expressing Rhbg, exposure to 5mM NH4Cl caused a decrease in pHs by 0.85±0.12 and an inward current of ‐58±10 nA. The decrease in pHs is caused by NH3 influx whereas the inward current is due to electrogenic NH4+ influx. In the presence of DIDS, NH4Cl caused a significantly smaller decrease in pHs (0.57±0.07) and current (‐29±5 nA). Similarly, exposing Rhbg‐oocytes to 5mM MA/MA+ (a substitute to NH3/NH4+) caused a decrease in pHs of 0.27±0.04 and an inward current of ‐29±6 nA. In the presence of DIDS, the MA/MA+ induced changes in pHs (0.17±0.03) and ΔI (‐11±2 nA) were also inhibited. DIDS had no effect on NH3/NH4+ transport in H2O‐injected oocytes.

Our data indicate that 1) DIDS inhibits transport of NH3 & MA by Rhbg without affecting endogenous NH3 & MA transport. 2) DIDS also inhibits the electrogenic transport of NH4+ & MA+ by Rhbg. 3) This is the first report to show inhibition of both gas (NH3) and ionic (NH4+) transport by Rhbg.

Grant Funding Source: Supported by VA Merit grant
Maintained intracellular acidification reduces myogenic tone and vasomotion in mice cerebral arteries (1098.3)
Authors: Aalkjaer Christian , Thomsen Axel , Kim Sukhan , Aalbaek Filip , Boedtkjer Ebbe
Published: June 2021

The effect of smooth muscle (VSMC) and endothelial cell pH (pHi) on myogenic tone is unknown. Here the role of Na+,HCO3‐‐cotransporter NBCn1 for pHi in these cells, and the influence of low pHi on myogenic tone and vasomotion were determined in intact mouse middle cerebral arteries. Na+,HCO3‐‐cotransport contributed to extrusion of acid from wildtype arteries but not from arteries where NBCn1 was knocked out. pHi of VSMCs was approximately 0.3 units lower in arteries from NBCn1 knockout than wildtype mice. Substantial myogenic tone developed after inhibition of NO‐synthase with L‐NAME in wildtype arteries. Less tone developed in arteries from NBCn1 knockout mice. This myogenic tone was in both type of arteries abolished by the rho‐kinase inhibitor Y‐27632. The arteries displayed vasomotion, which was attenuated in arteries from NBCn1 knockout mice. No differences in membrane potential or [Ca2+]i were seen between VSMC in arteries from NBCn1 knockout and wildtype mice. We propose that NO production and rho‐kinase‐dependent VSMC Ca2+‐sensitivity are reduced at low pHi in mouse middle cerebral arteries, which likely impedes the ability of these arteries to adjust to changes in perfusion pressure.
Frontiers beyond the hydroxylation paradigm in the diverse chemistry of non‐heme Fe(IV)‐oxo (ferryl) enzyme intermediates (110.2)
Authors: Bollinger J.
Published: June 2021

Non‐heme‐iron (NH‐Fe) enzymes activate O2 for an impressive array of biomedically, agriculturally, and environmentally important oxidation reactions. Our past decade's work has characterized iron(IV)‐oxo (FeIV=O or ferryl) complexes in the reactions of several such NH‐Fe enzymes.1 In five of these enzymes, the ferryl complexes generate substrate radicals by abstracting hydrogen (H•) from aliphatic carbons,2‐6 leading to formation of new C‐O,2‐4 C‐Cl/Br,5,6 and C‐S bonds.1 Motivated by our success in rationalizing the divergent outcomes of the NH‐Fe α‐ketoglutarate‐dependent aliphatic hydroxylases and halogenases,7 we now seek both to exploit the ferryl manifold for novel, unnatural carbon‐functionalization reactions and to explain the structural and mechanistic bases for several other natural reaction types, including 1,3‐dehydrogenation of an alcohol to epoxide,8 stereo‐inversion of a chiral carbon, and desaturation and cleavage of C‐C bonds, that are likely to be initiated by ferryl complexes in other NH‐Fe enzymes. Recent successes in explaining and exploiting this potent and versatile enzymatic strategy will be highlighted.
Suppression of physical activity and non‐resting energy expenditure during calorie restriction: role in individual differences in adaptive thermogenesis (1101.8)
Authors: Mukherjee Sromona , Demuth Emily , Spriggs Addison , Britton Steven , Koch Lauren , Novak Colleen
Published: June 2021

Weight loss requires negative energy balance, which can induce adaptive thermogenesis‐the reduction of energy expenditure (EE) beyond that accounted for by the weight lost. Adaptive thermogenesis varies between individuals. Here, we measured EE and physical activity (PA) before and after 21 days of 50% calorie restriction (CR) in female rats with lean and obesity‐prone phenotypes‐rats strains selectively bred for high and low intrinsic aerobic capacity (HCR and LCR, respectively). Use of female rats minimized potentially confounding effects of group differences in body size. We found that CR decreased EE more than was predicted by loss of weight and lean mass, demonstrating adaptive thermogenesis. Both groups showed similar suppression in resting EE (HCR, 39%; LCR, 34%). The CR‐induced suppression in non‐resting EE, which includes activity EE, was significantly greater in HCR (37%) than in LCR (24%). CR also significantly suppressed PA in HCR but not LCR, but PA was still higher in HCR than in LCR even after CR. Weight loss did not differ between groups. These results suggest that individual differences in CR‐induced adaptive thermogenesis may be accounted for by variation in aerobic capacity. Moreover, it is likely activity EE, not resting or basal metabolism, accounts for individual variation in adaptive thermogenesis.

Grant Funding Source: Supported by NIH R01NS055859 and NIH R15DK097644 to CMN
Instrument‐assisted soft tissue manipulation has no effect on the alpha7beta1 integrin pathway (1102.1)
Authors: Graham Zachary , Vardiman John , Siedlik Jake , Deckert Jake , Gallagher Philip
Published: June 2021

Instrument assisted soft tissue mobilization (IASTM) is a manual therapy technique used by clinicians to treat soft tissue injuries or aid in recovery. However, the mechanism of IASTM has yet to elucidated. A potential mechanism that IASTM may work is through mechanoreceptors of the sarcolemma. The alpha7beta1 integrin is a mechanoreceptor that relays membrane stretch signals to the cytoplasm through focal adhesion kinase (FAK), which can then relay the signal through cSrc and ERK1/2. In this study IASTM was administered using a standardized protocol on the gastrocnemius of 11 male subjects. Muscle biopsies were taken pre‐treatment and 24, 48, and 72 hours post‐treatment and analyzed via Western immunoblotting. We saw no change in the expression of the alpha7beta1 integrin or changes in expression of total or phosphorylated FAK, cSrc or ERK1/2 at any point of the study. We conclude that IASTM has no effect on the alpha7beta1 signaling pathway at 24‐72 hours post‐treatment.

Grant Funding Source: Supported by a General Research Grant from the University of Kansas awarded to J.P. Vardiman
The effects of KATP channels on skeletal muscle fatigue and recovery are dependent on muscle stimulus frequency and fibre type (1102.9)
Authors: Charter Mackenzie , Murrant Coral
Published: June 2021

ATP sensitive potassium (KATP) channels are hypothesized to protect against ATP depletion during maximal muscle contraction by decreasing force production, however their function in submaximal contractions and recovery is unknown. We sought to test whether KATP channels altered the force of contraction during fatigue and recovery of mouse slow (soleus; SOL) and fast (extensor digitorum longus; EDL) twitch muscle, in vitro, at maximal and submaximal stimulus frequencies. We fatigued SOL (60Hz, 60 contractions per minute (CPM) and 20Hz, 60CPM) and EDL (100Hz, 60CPM and 40Hz, 60CPM) for 5 minutes in the absence or presence of a KATP channel inhibitor (10‐5M glibenclamide; GLIB) or a KATP channel opener (10‐5M pinacidil; PIN) and then observed recovery (SOL: 60Hz 0.6 CPM and 20Hz, 0.6CPM; EDL:100Hz 0.6CPM and 40Hz 0.6 CPM). GLIB had no effect on fatigue or recovery of SOL or EDL at maximum stimulus frequencies but significantly attenuated force in SOL (12.2%) and EDL (6.9%) at submaximal frequencies. GLIB significantly enhanced force during recovery of SOL by 22.5% at submaximal stimulus frequencies. PIN did not affect fatigue of SOL or EDL at maximal stimulus frequencies or the recovery of SOL but significantly attenuated force of EDL during recovery by 8.9%. PIN had no effect on fatigue or recovery during submaximal contractions. Thus, the effect of KATP channels is dependent on both fibre type and stimulus frequency.

Grant Funding Source: Supported by NSERC, Canada
Aerobic training prevents the impaired vascular reactivity of fructose‐treated rats (1106.22)
Authors: Medeiros Renata , Gaique Thaiane , Bernardes Thais , Motta Nadia , Brito Fernanda , Bertoldi Julia , Santos Caroline , Oliveira Karen , Nóbrega Antonio
Published: June 2021

Fructose intake is associated with cardiometabolic disorders, but few studies investigated its role on vascular reactivity and the potential effects of exercise training. Thus, we assessed the effect of fructose intake on vascular reactivity in trained rats. Therefore male Wistar rats (n=7/group) received tap water (control group, C) or Fructose 10% (F group) ad libitum for two weeks. Then, rats underwent treadmill exercise for eight weeks (FT group). Caloric intake was higher in fructose groups compared with C group (P<0.01), although we found no change in body weight and in lipid profile, except the higher HDL in FT group (P<0.01). Training increased serum NO (C=296.5±42.8; F=325.8±51.6; FT=718.4±151.2; P<0.01). Regarding body composition, F increased body fat content compared with C and FT groups (P=0.03), whereas the FT group showed higher protein content (P<0.01). Finally, vascular vasoconstrictor reactivity was increased by fructose since F group required lower concentrations of phenylephrine to promote 50% of the maximum contractile effect when compared to C and FT groups (C=1.31‐6±2.4‐7; F=2.43‐7±6.5‐8; FT= 7.99‐7±1.5‐7 M; P<0,01). Vasodilator response to acetylcholine was also impaired in F group and was preserved by exercise training (C= 3.13‐7±1.9‐7; F=3.64‐6±9.6‐7; FT=3.13‐7±1.9‐7M; P<0.01). Thus, aerobic exercise prevented fructose‐impaired vascular reactivity in rats.

Grant Funding Source: CNPq, FAPERJ, CAPES, FINEP
Glucolipotoxicity‐induced p21 suppresses pro‐survival signaling in pancreatic β cells (1108.9)
Authors: Hernandez Angelina , Colvin E. , Fueger Patrick
Published: June 2021

Type 2 diabetes manifests from peripheral insulin resistance and a loss of functional β cell mass due to decreased β cell function, survival, or proliferation. The glucolipotoxic environment in the diabetic milieu activates a stress response in β cells, resulting in cell death and decreased survival through various mechanisms. One key player implicated in β cell dysfunction is FOXO‐1, a transcription factor phosphorylated and inhibited by the pro‐survival kinase Akt. Interestingly, palmitate in combination with high glucose induced expression of p21, a cell cycle inhibitor that activates the intrinsic apoptotic pathway and reduces Akt cell survival signaling. The molecular mechanism by which p21 activates apoptosis in β cells during metabolic stress is still unknown. We hypothesized that p21‐mediated suppression of Akt leads to FOXO‐1 activation, ultimately resulting in the expression of pro‐apoptotic factors. To investigate the molecular events that p21 acts through to increase death and reduce cell survival, we adenovirally overexpressed p21 in isolated islets and 832/13 cells. p21 overexpression increased phosphorylation of the stress‐activated protein kinase JNK. In contrast, FOXO‐1 phosphorylation was suppressed, indicating activation of FOXO‐1. We are actively investigating whether a constitutively‐active Akt will reverse this p21‐mediated phenomena of FOXO‐1 activation. Therefore, we propose that p21‐mediated inactivation of Akt leads to cell death via JNK and FOXO1 activation. Our findings suggest a molecular mechanism by which metabolic stress‐ or glucolipotoxicity‐induced p21 expression leads to β cell death‐a mechanism that could potentially be targeted to protect β cell mass.

Grant Funding Source: Supported by NIH grant DK078732 and William Townsend Porter Fellowship from APS
A long form of the alpha subunit of the human epithelial Na+ channel forms hyperactivated channels (1109.12)
Authors: Berman Jonathan , Brand Cristin , Awayda Mouhamed
Published: June 2021

The Alpha subunit of ENaC is critical to the ability to form a channel. Alpha‐like subunits have been described and have been shown to affect channel activity and/or regulation. A new isoform of the alpha subunit containing an extended N‐terminus resulting in a 728 a.a. subunit was recently identified. We examined the electrophysiological properties of channels formed by this isoform. Channels formed by α728βγ exhibited much higher activity than those formed by α669βγ in the Xenopus oocyte expression system. These differences were much larger under conditions which elevated the intracellular [Na+]. α728βγ also exhibited reduced current/voltage rectification. These differences occurred despite similar single channel conductances (~5.5 pS). The role of intracellular Na+ and Na+ self‐inhibition in these differences was examined. We find that α728βγ sustained much higher activity under elevated [Na+] than α669βγ and that intracellular and acute increase of [Na+] by injection of Na2SO4 led to further increase of conductance. α728βγ channels were further activated by extracellular cleavage by trypsin or subtilisin; with 2 notable differences: 1) fold activation was smaller than that observed with α669βγ, and 2) the large baseline activity of α728βγ channels was observed in the absence of detectable cleavage at the plasma membrane. These data indicate that α728 forms the structural basis of an ENaC with differing activity and regulation than α669.

Grant Funding Source: National Institute of Diabetes and Digestive and Kidney Diseases Grant DK‐55626
Glial cell line‐derived neurotrophic factor (GDNF) promotes barrier maturation and wound healing in intestinal epithelial cells in vitro (1111.1)
Authors: Meir Michael , Bergauer Lisa , Flemming Sven , Germer Christoph‐Thomas , Schlegel Nicolas
Published: June 2021

Recent data suggest that neurotrophic factors that derive from the enteric nervous system are involved in intestinal epithelial barrier regulation. In this context the glial cell line‐derived neurotrophic factor (GDNF) was shown to affect gut barrier properties in vivo directly or indirectly by yet undefined processes. Here we investigated whether GDNF directly affects intestinal epithelial barrier functions. Permeability (PE) was estimated by measurements of 4 kD FITC‐dextran flux across differentiated and across immature Caco2 monolayers. Recombinant human GDNF (100 ng/ml) was applied for 24h to test possible effects on epithelial barrier functions. GDNF application resulted in significant epithelial barrier stabilisation only in Caco monolayers with immature barrier functions. This effect was paralleled by a more linearized and augmented staining pattern of tight junction proteins claudin1 and occludin at the cell borders. Barrier maturation by GDNF correlated with augmented cAMP‐levels in immature Caco2 cells. Moreover, wound healing assays showed a significantly faster closure of the wounded areas following application of GDNF.

These data show that GDNF directly affects intestinal epithelial cells and promotes epithelial wound healing and barrier maturation. This effect may be mediated by augmented cAMP levels following GDNF application.

Grant Funding Source: Supported by DFG
MLCK regulation of claudin 3 expression contributes to IL1β mediated weakening of colonic epithelial tight junctions (1113.1)
Authors: Haines Ricci , Beard Richard , Yuan Sarah , Wu Mack
Published: June 2021

Interleukin 1β (IL1β) is an inflammatory cytokine well recognized for its role in autoinflammatory diseases including multiple sclerosis and ulcerative colitis, both associated with increased endothelial and epithelial barrier dysfunction respectively. Epithelial barrier integrity is maintained, in part, by expression of tight junction proteins such as occludin, zonula occludens, and claudin family of proteins. The major claudins involved in colonic epithelial barrier function are claudin 2, claudin 3, and claudin 4. In addition to structural components of tight junctions, kinases play a role in mediating signaling events that can affect gene expression. Recently, myosin light chain kinase (MLCK) has been shown to play a role in IL1β mediated abrogation of epithelial tight junctions, however the manner in which MLCK contributes to this dysfunction is not known. Our studies show that IL1β downregulated claudin 3, a sealing claudin, in young adult mouse epithelial (YAMC) and CACO 2 cells. In addition, we show that the MLCK inhibitor ML7 attenuated IL1β induced claudin 3 downregulation, and improved barrier function as measured by electric cell‐substrate impedance sensing (ECIS). Moreover, the role of MLCK mediated downregulation of claudin 3 was further characterized by immunocytochemistry. These results suggest that MLCK plays a critical gene regulatory role in addition to its known place in epithelial cell contractility in response to inflammation.

Grant Funding Source: Supported by VA Merit Review 5101BX000799, the NIH grants HL96640, HL61507 and HL70752
Histology of liver oxidative stress in an obese mouse model of nonalcoholic fatty liver disease treated with Ω3 fatty acids, TNFα‐receptor blocker, or curcumin (1116.1)
Authors: Likhitsup Alisa , Alba Laura , Herndon Betty , Mansour Ahmad , Zia Hamid , Molteni Agostino
Published: June 2021

Nonalcoholic fatty liver disease (NAFLD) origins are unclear but progression involves inflammation and correlation with obesity and insulin resistance. Advanced glycation end products (AGEs) are oxidative stress markers that induce inflammation and release reactive oxygen species. AGEs play key roles in the development of vascular inflammation of many types. Tissue accumulation of AGEs is identified by receptor staining (RAGE) . This study evaluated dietary and drug intervention to interrupt RAGE staining in a model of NAFLD. OB/OB leptin knock‐out mice (10/group) received clinical levels of curcumin, Ω3 fatty acids, TNF‐α receptor blocker or control chow for 8 wk. Liver steatosis was scored at 400x. Liver was also stained with PAS for glycoproteins and RAGE antibody. The Image J program was used for quantification of RAGE stain percentage. By H&E, significant reduction of liver fat was seen in groups receiving curcumin or Ω‐3 fatty acids compared to controls. By RAGE stain, interventions reduced AGES from 38.1% (untreated) to 26.4% (TNF blocker) and 26.8% (Ω3). p value, 0.15. This pilot experiment suggests that AGEs contribute to the development of nonalcoholic fatty liver disease and/or the progression of NAFLD to nonalcoholic steatohepatitis. The progression, in our model, is interrupted by food additives or an anti‐inflammatory. Studies to expand this approach to interrupt NAFLD are planned.

Grant Funding Source: Funding: St Luke’s Hospital Foundation grant to LA
Weight loss alone does not fully resolve non‐alcoholic steatohepatitis markers in western diet fed Ldlr‐/‐mice: a pilot study (1116.5)
Authors: Lytle Kelli , Jump Donald
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is prevalent in >80% of obese adults and >60% of type 2 diabetic patients. NAFLD can progress to non‐alcoholic steatohepatitis (NASH) which is characterized by hepatosteatosis, inflammation, oxidative stress & fibrosis. Current clinical recommendations for NASH include weight loss coupled with therapies to improve insulin sensitivity and lower plasma lipids. There is, however, little evidence to support this therapeutic strategy. In a pilot study, we examined the effect of weight loss on NASH. Ldlr‐/‐ mice were fed a chow diet (CD) or Western diet (WD) for 22 weeks. A third group was fed the WD for 15 weeks, followed by 7 weeks on the CD (WD‐CD). WD‐fed mice became obese and developed a robust NASH phenotype. Body weight, plasma lipids, and multiple markers of inflammation (Mcp1, CD68, Tlr4, Tlr9, Cd14) and oxidative stress (Nox2, p22Phox, Hmox1) were not different in the CD and WD‐CD groups. Hepatic steatosis and fibrosis in the WD‐CD group was lower than that seen in the WD‐group, but significantly higher than that seen in the CD group. As such, weight loss alone does not fully resolve hepatic steatosis and fibrosis associated with WD‐induced NASH.
Evaluation of novel inflammation biomarkers GlycA and GlycB in overweight and obese adolescents with NAFLD (1116.9)
Authors: Jin Ran , Otvos James , Le Ngoc‐Anh , Vos Miriam
Published: June 2021

Objective: Cardiovascular disease (CVD) is a leading cause of increased mortality in patients with nonalcoholic fatty liver disease (NAFLD). One of the proposed roles of NAFLD in the pathogenesis of CVD is through the systemic release of inflammatory mediators. GlycA and GlycB are two novel inflammation biomarkers that are both associated with future CVD and diabetes. However, these markers have not been measured in pediatric NAFLD and their relationships to inflammation in NAFLD are unknown.

Methods: We examined 37 Hispanic overweight and obese adolescents who had presumed NAFLD (hepatic fat > 5% by MRI). GlycA and GlycB were measured by nuclear magnetic resonance (LipoScience, Raleigh, NC).

Results: In our cohort, both GlycA and GlycB were significantly correlated with hs‐CRP (r=0.744, p<0.001 and r=0.694, p<0.001). GlycA, but not GlycB, was also associated with BMI z‐score (r=0.334, p=0.043) and leptin (r=0.427, p=0.026). However, no significant correlations of GlycA and GlycB were observed with hepatic fat content and liver enzyme ALT.

Conclusion: Our preliminary data suggest that in pediatric NAFLD, GlycA and GlycB are associated with systemic inflammation and may have applications similar or complementary to hs‐CRP and other inflammatory biomarkers.

Grant Funding Source: Supported by NIH K23 DK080953 (Vos)
Gastric antral organoids are sustained by intrinsic Notch signaling (1119.1)
Authors: Gifford Gail , Demitrack Elise , Spence Jason , Simeone Diane , Samuelson Linda
Published: June 2021

The gastric epithelium undergoes constant turnover, with adult stem cells replenishing mature cell types to maintain homeostasis. The Notch signaling pathway is known to regulate gastric epithelial proliferation and differentiation, but little is known about mechanism. In this study we tested the hypothesis that Notch signaling is intrinsic to the epithelium and supports antral stem cell function. Mouse and human gastric organoids were used as a model to define epithelial Notch signaling. Organoids were established from antral glands and maintained in 3D culture in media containing EGF, Noggin, R‐spondin1 and Wnt3A. Gastric organoids were highly proliferative and contained differentiated gastric cell types, suggesting that they are sustained by antral stem cells. Notch pathway expression was determined by qRT‐PCR and function was tested using the Notch inhibitor DAPT. Pathway inhibition reduced cellular proliferation and organoid growth, and lowered the efficiency of organoid re‐establishment. Together these studies show that Notch signaling is intrinsically established by gastric epithelial cells in vitro to promote organoid proliferation and to maintain stem cell function.

Grant Funding Source: Supported by NIH grants T32GM008322‐21/22 (GBG), T32DK094775‐02 (GBG), P01‐DK062041 (LCS)
Activation of Gα12 regulates ectodomain shedding of cadherins in renal epithelial cells (1124.1)
Authors: Xu Jen , Wu Yong , Li Suyang , Hsieh Yu‐Chung , Kong Tianqing , Lu Tzongshi
Published: June 2021

Disruption of cell‐cell and cell‐matrix adhesions in kidney epithelial cells is involved in the progression of autosomal dominant polycystic kidney disease (ADPKD). We previously showed that polycystin‐1 (PC1) inhibits activation of the G‐protein, which results in downregulation of focal adhesions. The aim of this study was to investigate whether adheren junction proteins are involved in the progression of renal cyst formation in ADPKD after Gα12 activation. Using mass spectral analysis, we first showed expression of E‐, N‐ and K‐cadherins are altered in kidney epithelial cells after Gα12 activation. Gα12 activation or Pkd1 knockout increases ectodomain shedding of E‐cadherin and N‐cadherin. In addition, the early form of N‐cadherin is increased and the late form is decreased after Gα12 activation. In renal cystic fluid collected from Pkd1 knockout mice and ADPKD patients, shed fragments from both E‐cadherin and N‐cadherin are increased but no change in K‐cadherin, which indicates a specific change of E‐cadherin and N‐cadherin by Gα12 activation. Immunostaining of kidney tissue showed that N‐cadherin is localized in nucleus after Gα12 activation, consistent with WNT/β‐catenin signaling pathway. In conclusion, our data shows that Gα12 regulates shedding of E‐cadherin and N‐cadherin and affects the cell‐cell adhesion and cell polarity of kidney epithelial cells in the development of kidney cysts in ADPKD.
Inhibition of a hyperpolarization‐activated inward current (Ih) facilitates cold‐activation of locus coeruleus neurons of bullfrogs, Lithobates catesbeianus (1128.2)
Authors: Santin Joseph , Watters Kayla , Hartzler Lynn
Published: June 2021

Mechanisms that determine neuronal excitability under variable temperatures are unclear. In bullfrogs, cooling increases action potential (AP) firing in locus coeruleus (LC) neurons. We used whole‐cell patch clamp electrophysiology in brainstem slices to test whether inhibition of a hyperpolarization‐activated current (Ih) promotes increases in LC neuron excitability during cooling from 20°C to 10°C. Cooling reduced maximum Ih amplitude from ‐211.5±55.0pA to ‐69.5±7.8pA (P<0.05) and shifted the voltage at half maximal activation (V50) from ‐69.9±11.4mV to ‐92.9±5.8mV (P<0.05). Application of 2mM CsCl inhibited Ih (‐142.8±53.3pA vs. ‐3.3±14.3pA; P<0.05), and may potentiate AP firing increases induced by cooling (increase in AP firing; control: 0.9±0.5Hz vs. Cs+: 2.2±1.6Hz; P=0.2; n=3). Elevating intracellular cyclic AMP to 100μM increased the proportion of activated Ih near resting membrane potential (Vm ) at 20°C and 10°C (V50 =‐63.7±9.5mV and ‐71.3±10.2mV); yet, activation of Ih suppressed the firing increase elicited by cooling (0.2±0.7Hz decrease vs. increased firing in controls; P=0.06; n=5). Our data suggest inhibition of Ih enhances LC excitability during cooling and implicate Ih as a temperature‐sensitive mechanism that modulates neuronal excitability during changes in brain temperature. Given extensive involvement of LC neurons in physiological functions, understanding the regulation of LC neuron excitability during temperature changes has broad ramifications.

Grant Funding Source: Supported by NSF IOS 1257338
Sociopsychological stress activates hypothalamo‐medullary neurons that drive brown adipose tissue thermogenesis and hyperthermia (1130.6)
Authors: Kataoka Naoya , Nakamura Kazuhiro
Published: June 2021

Psychological stress induces hyperthermia in many mammalian species. We have shown that psychological stress induces heat production in brown adipose tissue (BAT) through activation of sympathetic premotor neurons in the rostral medullary raphe (rMR) (Lkhagvasuren et al., 2011). The present study investigated the central circuit mechanism that drives stress signaling to the BAT‐controlling premotor neurons. Rats exposed to social defeat stress, a sociopsychological stress model, exhibited increases in BAT and abdominal temperature. This stress response was eliminated by a nanoinjection of AP5 and CNQX, glutamate receptor antagonists, into the rMR. This stress activated neurons in the dorsomedial hypothalamus (DMH) that directly project to the rMR and inhibition of neurons in the DMH with muscimol injections eliminated the stress‐induced BAT thermogenesis and hyperthermia. Supporting functional contribution of the DMH‐rMR monosynaptic pathway in the stress‐driven BAT thermogenesis, in vivo optogenetic stimulation of DMH‐derived nerve endings in the rMR elicited BAT thermogenesis, which was blocked by antagonizing glutamate receptors in the rMR. These results indicate that the DMH‐rMR glutamatergic monosynaptic pathway likely mediates stress signaling to drive BAT thermogenesis, contributing to psychological stress‐induced hyperthermia.
Integrative bioinformatical analysis of clear cell renal cell carcinoma (1138.10)
Authors: Butz Henriett , Nofech Mozes Roy , Rotondo Fabio , Kovacs Kalman , Patocs Attila , Szabo Peter , Yousef George
Published: June 2021

Background. Clear cell renal cell carcinoma (ccRCC) is the most common adult neoplasm of the kidney. Better understanding of the molecular pathogenesis may lead to new therapeutic options.

Aim. Our aim was to assemble transcriptomic, proteomic and miRNA data using an integrative approach.

Methods. Available mRNA, miRNA and protein data were collected from 593 ccRCC and 389 normal kidneys. We performed pathway analysis. TCGA database and immunohistochenmitry were used for validation. Functional analyses were undertaken on cell line model.

Results. Metabolic processes were the most significant signalling pathways, and we validated Aryl‐Hydrocarbon Receptor (AHR) signalling to be involved in tumorigenesis. Using network analysis, we identified GRHL2 as diagnostic marker and drug target. KIAA0101 was found to enhance tumor cell migration and invasion. We also demonstrated that KIAA0101 overexpression correlates with poor prognosis and may represent a diagnostic marker. We identified miR‐139‐5p as the miRNA mostly affecting the network targeting ZEB1, TCF4, ETS1 and CCND2.

Conclusions. Using integrative bioinformatical analyses we identified the most characteristic pathways and molecules. Some of them may be applicable as biomarkers or new, potential drug targets.
Effect of Capsosiphon fulvescnes extract and pheophorbide A on glucose metabolism and oxidative stress in liver of streptozotocin‐induced diabetic rats (1139.10)
Authors: Kim Se‐Wook , Oh Ji‐sun , Hong Chung‐Oui , Nam Mi‐Hyun , Yang Sung‐Yong , Jung Ha‐Young , Lee Kwang‐Won
Published: June 2021

We carried out to evaluate the effect of Capsosiphon fulvescnes extract (CFE) and Pheophorbide A (PhA) on blood glucose level and glucose tolerance. Male Sprague‐Dawley rats (160‐200 g), 9 weeks old, were administered with 40 mg/kg B.W. of streptozotocin (STZ) intraperitoneally to induce experimental diabetes. When we repeatedly administrated CFE and PhA for 9 weeks, administrations of CFE and PhA significantly improved glucose tolerance of STZ‐induced rats. Hyperglycemic effect of CFE was observed in an oral glucose tolerance test (OGTT) in which, a dose of CFE (4, 20 mg/kg B.W.) and PhA (0.2 mg/kg B.W.). An OGTT of rats treated low dose of CFE (CL), high dose of CFE (CH), and PhA decreased than it of STZ‐induced rats. Group treated CFE and PhA significantly reduced the serum glucose level, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total cholesterol and total triglyceride compared with the levels of STZ‐ induced rats group (diabetic control group). To confirm the hepatic protective effect, reduced glutathione (GSH), oxidized glutathione (GSSG), malondialdehyde (MDA) and antioxidant enzyme activities such as superoxide dismutase (SOD) and catalase (CAT) levels were measured in liver cytosol. Groups treated CFE and PhA increased the levels of GSH, SOD and CAT than that of STZ‐induced group significantly. And the levels of MDA and GSSG decreased compared with the levels of STZ‐induced group. Therefore, we suggested that PhA, activity compound of CFE, has hyperglycemic effects and ameliorate diabetic state.
Angiotensin II stimulation alters vasomotor response to adenosine agonist in mesenteric artery: ole for A1 and A2B receptors (1140.4)
Authors: Yadav Vishal , Nayeem Mohammed , Tilley Stephen , Mustafa S. Jamal
Published: June 2021

Angiotensin II (Ang II) is one of the major mediators that lead to the development of hypertension. However the signaling pathway that leads to hypertension are not well understood. Ang II activates several intermediate molecules which may alter localized signaling events to further regulate vascular tone that may contribute to the development of hypertension. Adenosine is the important metabolite which controls local blood flow through activation of four subtypes of adenosine receptors (ARs). Previously, we have reported that A1AR is the major determinant of contractions while A2B produces relaxation in mouse mesenteric arteries (MAs). In this study, we sought to investigate whether acute exposure to Ang II alters vasomotor responses to non‐selective AR agonist, NECA, in MAs. We used DMT myograph system for muscle tension measurement in isolated resistant MAs. We have found that acute exposure (15 min) to Ang II (10 nM) altered dose‐dependent vasodilatation to NECA producing significantly higher contractions (~37%) in MAs. Notably, these higher contractions were absent in MAs from A1 KO mice. Interestingly, we have also found that NECA induced relaxation in MAs from A1 KO mice were inhibited (~10%) after exposure to Ang II. Importantly, in MAs from WT mice, we have found that pre‐treatment with HET0016 (10 µM, 20HETE inhibitor) significantly inhibited Ang II and NECA‐induced contractions. In addition, in MAs from A2B KO mice, NECA produced contractions which were significantly higher after Ang II stimulation. These contractions were considerably inhibited by HET0016 and completely blocked by DPCPX (1 µM, A1 antagonist). Moreover, Ang II contractions were higher in MAs from A2B KO mice (p<0.05). Taken together, our data indicate that acute Ang II stimulation enhances A1 and reduces A2B‐dependent signaling in MAs. Thus, these altered A1 and A2B dependent signaling pathway may contribute to the development of hypertension.

Grant Funding Source: Supported by HL 094447, HL027339, HL114559 and AI 096139
Effect of alcohol on clopidogrel metabolism: Identification of a new alcohol‐dependent metabolic pathway (1141.14)
Authors: Hu Zheyi , Laizure S. , Herring Vanessa , Parker Robert
Published: June 2021

The majority of orally administrated clopidogrel, a platelet aggregation inhibitor, is hydrolyzed by human carboxyesterase‐1 (CES1) to the inactive clopidogrel acid metabolite with the remainder metabolized by CYP450 enzymes into the inactive intermediate, 2‐oxo‐clopidogrel, that is further oxidized to the active thiol metabolite. The objective of this study was to determine the effect of alcohol, a known CES1 inhibitor, on the metabolism of clopidogrel. Clopidogrel metabolism was studied in human recombinant CES1, human liver S9 fractions (HLS9), and in plasma‐carboxylesterase knockout mice. Liquid chromatography/triple quadrupole mass spectrometry (LC‐MS/MS) was utilized for the quantification of clopidogrel and its metabolites. Alcohol‐derived metabolites were identified by product ion scan using LC‐MS/MS. The addition of alcohol resulted in the formation of the transesterified clopidogrel metabolite, ethyl‐clopidogrel in CES1 and HLS9 incubations. Similar to clopidogrel, ethyl‐clopidogrel was metabolized to ethyl‐2‐oxo‐clopidogrel followed by the subsequent oxidation to thiol metabolites in HLS9. Co‐administration of alcohol (3 g/kg) and clopidogrel (25 mg/kg) to plasma‐carboxylesterase knockout mice resulted in a reduced systemic exposure to clopidogrel acid (control AUC0‐8h 39 µg·h/mL versus alcohol AUC0‐8h 20 µg·h/mL) and the formation of transesterified metabolites. Alcohol significantly inhibits the hydrolysis of clopidogrel to clopidogrel acid, and produces transesterified metabolites of clopidogrel, 2‐oxo‐clopidogrel, and the active metabolite.

Grant Funding Source: Supported by National Institutes of Health National Institute of General Medical Sciences
Overfeeding of saturated fat and fructose induces alterations of hepatic fatty acid composition in rats (1142.10)
Authors: Tricoli de Souza Verônica , Priviero Fernanda , Claudino Mario , Rocha Thalita , Messias Marcia , Carvalho Patricia
Published: June 2021

Fatty acids (FAs) are essential components of lipids and exhibits important biological functions. The analysis of FAs profiles in tissues has become increasingly important as end points in understanding the roles of FAs in physiological or pathological processes. This study aimed to describe the changes in fatty acid composition of the liver lipids caused by excessive consumption of high saturated fat diet associated with fructose in Wistar rats. Rats were fed ad libitum with chow (3 Kcal/g; 2.3% soybean oil) or high‐fat diet (5.5 Kcal/g; 30% lard and 4% soybean oil) associated with fructose (100mg/mL) for 12 weeks. The lipids of liver homogenates were extracted with chloroform‐MeOH and the fatty acids methyl esters were analyzed by gas‐chromatography. Fatty acid composition of the liver was found to shift from a pattern dominated by saturated (SFA, C16:0), monounsaturated (MUFA, 18:1 n‐9) and polyunsaturated FA (PUFA, C18:2 n‐6). Oleic acid (C18:1 n‐9) increased (approximately 36%, p=0.0001) whereas PUFA (C20:4 n‐6 and C22:6 n‐3) and SFA (18:0) decreased in the liver of animals fed with high saturated fat and fructose. This difference in the fatty acid profile in the liver is reflected in the increased MUFA/SFA ratio. The data indicated that the overfeeding of high saturated fat and fructose diets alter the hepatic fatty acid composition and the oleic acid is greatly accumulated in the liver.

Grant Funding Source: FAPESP ‐ 2012/11050‐6
Establishment and evaluation of physical and psychological stress‐induced PTSD animal model (1144.7)
Authors: Yu Bin , Zhang Xueqiong , Cui Xiangyu , Cui Suying , Zhang Yonghe
Published: June 2021

It is well known that not only direct recipients but also the witnesses of traumatic events could suffer PTSD. However, there are no recognized animal models to mimic psychological stress induced PTSD. So we tried to establish psychological stress induced PTSD by communication box. Physical stress (foot‐shock, FS) was generated by receiving foot‐shock directly which mimicked the direct recipients of trauma, while psychological stress (non‐foot shock, PS) was generated by exposure to emotional responses without direct physical stress, for example, the visual and auditory stimuli that arose from foot shock stress rats, to mimic the witnesses of trauma. Serum corticosterone levels were increased three times 30 minutes after stress in both FS and PS rats, this result suggested that the modeling process was intense threat for both FS and PS rats. 21 days after trauma, we evaluated whether rats presented PTSD symptoms, like anxiety, high vigilance, avoiding trauma‐related cues and strong memory of trauma, by using elevated plus maze/ open field test, acoustic startle response test, ball (trauma related cue) burying test, and freezing time (re‐exposed to communication box) test. The results showed that about 40% FS rats and 30% PS rats could simulate PTSD clinical symptoms, and this animal model could be helpful to explore PTSD mechanism specifically and comprehensively.

Grant Funding Source: This study was supported by the NNSFC (No.81173031,81202511 and 81302746).
The effect of S‐equol in attenuation of cell injury under oxidative stress in human umbilical vein endothelial cells (1146.1)
Authors: Zhang Ting , Mi Mantian , Liang Xinyu , Shi Linying
Published: June 2021

Objective To study the protective effects of S‐equol on oxidative stress‐induced cell injury in human umbilical vein endothelial cells (HUVECs).

Method HUVECs were used in this study. To establish the oxidative stress‐induced cell model, effect of H2O2 on cell viability was detected by CCK‐8 assay. The same method was used to detect the effect of S‐equol on cell apoptosis using flow cytometry and TUNEL assay. Western blot assay was used to detect the effect of S‐equol on caspase‐3 and Bcl‐2 protein expression. SOD, CAT and GSH levels were determined using SOD, CAT and GSH kits.

Results The IC50 of H2O2 was 650μM. S‐equol (1 nM, 10 nM, 100 nM) significantly dose‐dependently inhibited H2O2‐induced decrease of cell viability in HUVECs. S‐equol (100 nM) significantly inhibited cell apoptosis in e HUVECs under oxidative stress, and reduce the number of TUNEL labeling‐positive cells. S‐equol (100 nM) up‐regulated Bcl‐2 protein expression level and significantly inhibited caspase‐3 protein expression increased by H2O2. S‐equol dose‐dependently up‐regulated SOD, CAT and GSH levels, and inhibited H2O2‐induced decrease in SOD, CAT and GSH levels.

Conclusion Low concentration (physiological concentration) of S‐equol attenuated oxidative stress‐induced cell injury in HUVECs, which relates to the increase in SOD, CAT and GSH level.

Grant Funding Source: Supported by the National Natural Science Foundation of China (No. 81102129)
Study on mechanisms of action of liuwei dihuang decoction with proteomic and metabonomic techniques (1147.3)
Authors: Jiang Ning , Zhou Wenxia , Zhang Yongxiang , Yan Xianzhong , Zhang Qi
Published: June 2021

Objective: Liuwei Dihuang (LW) decoction are classical traditional Chinese medicinal prescription for anti‐aging. Senescence‐accelerated mouse (SAM) is a good model to study mechanisms of aging. To better understand the mechanisms of actions of LW on antiaging, systematic techniques, including proteomic and metabonomic methods, were employed in this study. Method: SAM‐prone/8 (SAMP8) were orally administered with LW for consecutive 30 days. Two dimensional electrophoresis (2‐DE) based proteomic technique and 1H nuclear‐magnetic‐resonance‐spectroscopy (NMR) based metabonomic technique were employed to investigate the proteins and metabolites that LW acted on. Results: The expressions of serum proteins in SAMP8 before and after LW treatment were quite different. There were 21 differentially expressed proteins after LW treatment, which were related to the functions of transcriptional control, cytoskeleton, cell signaling, etc.. Principle components analysis of 1HNMR spectra showed clear separation between serum samples of SAMP8 before and after LW treatment. Metabolites that LW affected were glucose, lactate, high density lipoprotein, etc. related to glucose, lipids, lipoproteins and amino acids metabolism. Conclusion: These findings suggested abnormal serum protein expressions and metabolism disequilibrium in SAMP8 caused by aging and the mechanisms of actions of the LW may lie in restoring of the normal condition of specific proteins and metabolism equilibrium in SAMP8.

Grant Funding Source: Supported by grants from the National Natural Science Foundation of China 30701073, 90709012
Interleukin‐6 modulates transforming growth factor‐β receptor II function and receptor compartmentalization in epidermal keratinocytes (1148.11)
Authors: Chastain Lerin , Cottrell Mackenzie , Ihnat Mike , Gallucci Randle
Published: June 2021

IL‐6 and TGF‐ β mediate a broad range of biological responses including growth, differentiation, and migration. We have previously shown that IL‐6 modulates Tgf‐β1 expression in fibroblasts, however; what role IL‐6 plays concerning TGFβ‐RII expression and function in skin cells is unknown. Therefore, the aim of this study was to investigate the mechanism by which IL‐6 might modulate TGFβ‐RII in skin.

Skin from WT and IL‐6 over‐expressing mice and IL‐6 treated keratinocyte cultures were analyzed for TGF‐βRII expression via immunohistology and western blot. TGF‐βRII function and activity was assessed by cell migration, proliferation, and Smad2/3 phosphorylation. TGF‐βRII membrane localization was determined by co‐immunoprecipitation. IL‐6 overexpression and IL‐6 treatment increased TGF‐βRII expression in epidermis and isolated keratinocytes respectively. IL‐6 treatment of keratinocytes augmented TGF‐β1 function as demonstrated through increased migration and decreased proliferation. TGF‐ βRII is down regulated when located within caveolin rich lipid rafts and co‐IP showed with IL‐6 treatment, association between TGF‐ βRII and caveolin increased resulting in decreased Smad2/3 phosphorylation. These results suggest that IL‐6 regulates TGF‐ β1 function by modulating TGF‐ βRII expression and increasing trafficking of the receptor to lipid raft pools.

Grant Funding Source: NIH/NIGMS R01GM67745
Atorvastatin inhibits induction of chemokine receptor CXCR7 to reduce macrophage migration (1148.12)
Authors: Liu Yiwei , Ma Wanshu , Crocker Laura , Shen Jianzhong
Published: June 2021

We recently reported that the new SDF‐1 receptor CXCR7 is induced during monocyte‐to‐macrophage differentiation in the context of atherosclerosis. This study aimed to determine whether CXCR7 induction during macrophage differentiation is inhibited by statins. Here we show that atorvastatin inhibited CXCR7 mRNA and protein expression in THP‐1 macrophages, without affecting CXCR4 expression. Suppression of CXCR7 expression was reversed by supplementation with mevalonate. Inhibition of squalene synthase, the enzyme committed to cholesterol biosynthesis, partially decreased CXCR7 induction. However, the geranylgeranyl transferase inhibitor, GGTI‐286, the farnesyl transferase inhibitor, FTI‐276, and the Rho kinase inhibitor, Y‐27632, all failed to mimic the effect of atorvastatin, suggesting that the protein prenylation pathways are not critical. Interestingly, the effect of atorvastatin was not fully mimicked by other statins. Furthermore, activation of CXCR7 by SDF‐1, TC14012, or I‐TAC all prompted macrophage migration, which was suppressed by atorvastatin treatment, but not by the CXCR4 antagonist. We conclude that atorvastatin modulates macrophage migration by down‐regulating CXCR7 expression, suggesting a new CXCR7‐dependent mechanism of atorvastatin to benefit atherosclerosis treatment independent of its lipid lowering effect.

Grant Funding Source: American Heart Association ─ National SDG grant 12SDG8850011
Selective estrogen receptor modulators resveratrol, genistein, tamoxifen, pterostilbene and clomiphene downregulates the expression of minute‐double minute2 (MDM2) and vascular endothelial growth factor (VEGF) genes in SNB‐19 glioblastoma cells (1148.9)
Authors: Bhatt Vrushank , Ratna Warren
Published: June 2021

Glioblastoma multiforme is the most common primary neoplasm of the brain. Our studies using SNB‐19 glioma cells have shown that selective estrogen receptor modulators (SERMs) at pharmacological concentrations modulate the expression of minute double minute (mdm2) and vascular endothelial growth factor (VEGF) genes. The SNB‐19 glioblastoma cells were treated with increasing concentrations of SERMs such as resveratrol, genistein, tamoxifen, pterostilbene and clomiphene, and their effects were studied using RNA and protein analyses. The cell viability studies on cells exposed to the above SERMs showed a concentration dependent cell growth arrest and cell death by apoptosis. Reverse‐Transcription Polymerase Chain Reaction (RT‐PCR) studies indicated decreased levels of transcription of mdm2 and VEGF mRNA. Western blot analysis showed a corresponding decrease in the expression of the VEGF protein. The above results show the down‐regulation of the mdm2 and VEGF expression and the induction of cell death in SNB‐19 glioma cells due to exposure to the above SERMs, indicating their potential use in causing the regression of glioblastomas.

Grant Funding Source: Supported by science research funds from Long Island University, Brooklyn, NY