bluuebunny/crossref_metadata_2025_split huggingface dataset:
Forkhead box transcription factors are required for expression of the glycemia‐protective enzyme angiotensin‐converting enzyme 2 in pancreatic β‐cells (1108.10)
Authors: Pedersen Kim , Lazartigues Eric 
Published: June 2021

Angiotensin‐converting enzyme 2 (ACE2) opposes Ang‐II signaling and improves β‐cell function in several diabetic animal models. We previously reported that diabetes progression reduces islet ACE2 expression and that HNF1α dose‐dependently increases ACE2 expression in pancreatic islets. Because HNF1α is not sufficient for the full ACE2 expression, we hypothesized that ACE2 expression in β‐cells is regulated by other factors binding to evolutionarily conserved motifs of the human ACE2 promoter. A putative motif for forkhead box transcription factors is located 144‐153 bases upstream of the translational start site. Mutation of the motif in luciferase reporters decreased ACE2 promoter activity in 832/13 insulinoma cells by 56% (p<0.001). Overexpression of FOXO1 slightly increased (< 3‐fold) promoter activity and led to FOXO1 binding to the motif as observed by EMSA. However, the endogenous complexes in 832/13 cells binding to the motif contained FOXA proteins with the most abundant of these complexes containing FOXA1/2. Furthermore, ENCODE data suggest that an additional FOXA1/2 binding site is located further upstream in the ACE2 promoter. We conclude that forkhead box transcription factors, in particular of the FOXA class, are required for expression of ACE2 in β‐cells. Depletion of nuclear FOXA2 occurring in islets of type 2 diabetics may thus contribute to reduced islet ACE2 levels.Grant Funding Source: Supported by NIH/NIDDK (DK084466)
Proteomic profiling of ubiquitinated proteins in human urinary exosomes (1109.1)
Authors: Huebner Alyssa , Cheng Lei , Fenton Robert , Pisitkun Trairak 
Published: June 2021

Urinary exosomes are small (20‐100 nm) membrane vesicles that contain various molecular constituents of every epithelial cell type facing the urinary space. Based on our previous proteomic studies of urinary exosomes, ubiquitin was identified throughout the molecular weight range of the analysis, suggesting the presence of multiple ubiquitinated proteins in urinary exosomes. Ubiquitination is the classical signal that marks plasma membrane proteins for incorporation into multivesicular bodies where exosomes are originated as internal vesicles. We postulated that ubiquitinated plasma membrane proteins are present in urinary exosomes, potentially providing non‐invasive clues for ubiquitin‐dependent signaling processes in renal epithelial cells. Immunoelectron microscopy revealed ubiquitin labeling on many, mostly inside, urinary exosomes. LC‐MS/MS analysis of urinary exosomes with and without enrichment by di‐glycine‐lysine antibody identified 747 ubiquitinated peptides corresponding to 593 proteins, out of 5007 total proteins. Ubiquitinated proteins were mainly associated with plasma membrane including transporters, channels, and receptors and comprising a number of novel ubiquitinated sites. All but one polyubiquitin chain linkages were identified, with K63‐linked chains being the most frequently observed suggesting non‐proteolytic roles of urinary exosomal ubiquitination.Grant Funding Source: Supported by the Lundbeck Foundation, AU Research Foundation, Danish Medical Research Foundation
Biphasic effect of PGF2a on the basolateral 40 pS K channel (a heterotetramer of Kir4.1 and Kir5.1) in the early distal convoluted tubule (DCT1) (1109.9)
Authors: Wang Lijun , Zhang Chengbiao , Su Xiaotong , Wang Wen‐Hui 
Published: June 2021

We used the patch‐clamp technique to examine the effect of PGF2α on the basolateral 40 pS K channel in the DCT1. Application of PGF2α at concentrations lower than 500 nM stimulated the 40 pS K channel. The stimulatory effect of PGF2α was abolished by inhibiting src‐family protein kinase (SFK) and NADPH oxidase. Moreover, adding 100 μM H2O2 mimicked the effect of PGF2α and increased the 40 pS K channel activity in DCT1 and augmented whole cell K currents in HEK293 cells transfected with KCNJ10/16 which are known to form the heterotetramer in the basolateral membrane of DCT1. The inhibition of SFK also blocked the stimulatory effect of H2O2 on the basolateral 40 pS K channel in DCT1. The role of SFK in mediating the effect of H2O2 on the 40 pS K channel was further indicated by the observation that H2O2 failed to stimulate the K channels in cells transfected with KCNJ16+KCNJ10Y9F in which tyrosine phosphorylation site was mutated. This suggests that PGF2α and H2O2 stimulate the basolateral 40 pS K channel in DCT1 by SFK‐induced phosphorylation of KCNJ10. While PGF2α stimulates the K channels at low dose, PGF2α at high dose (>1 uM) inhibited the basolateral K channels. The inhibitory effect of PGF2α was abolished by inhibiting PKC. We conclude that PGF2α at low doses stimulates the basolateral 40 pS K channel by a SFK‐dependent pathway, while at high concentrations it inhibited the K channel in the DCT1 by a PKC‐dependent pathway.Grant Funding Source: NIH
The role of mitofusin 2 in saturated fatty acid induced ER stress in H4IIE liver cells (1116.4)
Authors: Moran G. , Wei Y. , Wang D. , Pagliassotti M.J. 
Published: June 2021

Genetic ablation of mitofusin 2 (Mfn2) in mice results in ER stress and insulin resistance in the liver. In liver cells, saturated fatty acids (SFA) induce ER stress and activate c‐Jun NH2 terminal kinase (JNK) which can mediate Mfn2 degradation. We hypothesized that SFA would reduce Mfn2 expression. We examined the effects of the unsaturated fatty acid oleate (O250µM) and the SFA palmitate (P250 µM) on Mfn2 mRNA, translation and protein expression in H4IIE liver cells (n=3). O250 µM and P250 µM had no significant effect on Mfn2 mRNA. P250 µM significantly increased Mfn2 translational efficiency based on polysome profiling, however no increase in Mfn2 protein expression was observed. JNK‐mediated degradation of Mfn2 involves serine phosphorylation (pSer) and ubiquitination (Ub), however P250 µM did not increase pSer or Ub of Mfn2. These data suggest that either the increase in translational efficiency was not sufficient to alter Mfn2 protein levels or that Mfn2 degradation occurs via mechanisms independent of pSer and Ub. We utilized si‐RNA mediated knockdown of Mfn2 to determine whether Mfn2 could protect against palmitate‐induced ER stress (n=3). Knockdown of Mfn2 had no significant effect on thapsigargin (Thap) or P250 µM ‐induced ER stress in H4IIE cells. These findings indicate that Mfn2 does not appear to play a protective role in Thap or P250 µM mediated ER stress in H4IIE cells.Grant Funding Source: NIH DK 072017
ErbB4 deletion compromises the murine small intestinal stem cell niche and sensitizes the epithelium to TNF‐induced apoptosis (1119.6)
Authors: Almohazey Dana , Bernard Jessica , Frey Mark 
Published: June 2021

The ErbB4 receptor tyrosine kinase is induced by inflammation in intestinal epithelial cells (IECs). Its ligand, NRG4, promotes survival signaling in colonocytes. However, the effects of ErbB4 loss on the small intestine or the intestinal stem cell (ISC) niche have not been defined. We asked whether ErbB4 deletion enhances tumor necrosis factor (TNF)‐induced apoptosis, and if it compromises the ISC niche. METHODS: Epithelial enteroid cultures from ileal crypts of ErbB4flox/flox (WT) and ErbB4flox/flox; Villin‐Cre (ErbB4KOIE) mice were given TNF (200 ng/ml) daily for 5 days, then expression of Paneth cell (Lyz1) and ISC (Lgr5) markers were tested by qPCR. WT and ErbB4KOIE mice were injected with 250 μg/kg TNF; after 24 h, ileums were fixed, sectioned, and apoptosis detected by In Situ Oligo Ligation (ISOL). RESULTS: ErbB4KOIE enteroids were dysmorphic, with 64% fewer buds/enteroid than WT, and had lower expression of Lyz1 (57% reduction, p<0.05) and Lgr5 (75% reduction, p<0.01). ErbB4‐null enteroids were highly sensitive to TNF, losing all crypt bud architecture after exposure. In vivo, ErbB4KOIE mice had increased TNF‐induced ileal epithelial apoptosis (1.8‐fold increase in ISOL+ cells, p=0.001) than WT. CONCLUSIONS: ErbB4 loss exacerbates TNF‐induced IEC death both in vivo and in vitro. This is associated with loss of PC and ISC markers, suggesting a role for ErbB4 in regulating the ISC niche.Grant Funding Source: Supported by NIH grant R01DK095004 and ACS grant 124678‐RSG‐13‐199‐01‐DDC
The brain renin‐angiotensin system suppresses digestive efficiency (1126.13)
Authors: Weidemann Benjamin , Littlejohn Nicole , Cole Renee , Grobe Justin 
Published: June 2021

The renin‐angiotensin system (RAS) contributes to metabolic control through local actions within the brain and adipose. All components of the RAS are also expressed in the gastro‐intestinal (GI) tract, leading to the hypothesis that the RAS controls digestive efficiency. Mice with transgenic hyperactivity of the brain RAS (sRA) were generated through neuron‐specific expression of human renin, and expression of human angiotensinogen via its own promoter. sRA exhibit reduced body mass and elevated resting metabolic rate through elevated sympathetic nervous activity and a chronic suppression of the circulating RAS. Caloric densities of food/feces were assessed by bomb calorimetry. sRA had increased caloric intake (15.4±0.7 kcal/day, n=8) vs littermate control mice (13.0±0.7, n=7, P<0.01) as well as an increase in caloric loss to feces (6.3±0.8 vs 2.9±0.4 kcal/day, P<0.01). Thus, sRA had decreased digestive efficiency (59±5 vs 78±2 %, P<0.01) versus littermates on chow diet (Teklad 7013), but total daily caloric absorption (9.1±0.8 vs 10.0±0.5 kcal/day, P=0.36) was normal. Infusion of the angiotensin (Ang) II type 2 receptor agonist CGP‐42112a (50 ng/kg/min, s.c., 8 weeks) had no effect on efficiency or absorption (sRA 63±6 vs control 80±1 %; 9.3±0.8 vs 9.8±1.0 kcal/day). We conclude that the brain and circulating RAS help control digestive efficiency, and hypothesize a role for GI tract Ang II type 1 receptors.Grant Funding Source: Supported by NIH: HL098276, HL084207.
Brain endoplasmic reticulum stress mediates sympathetic and hemodynamic responses to blood‐borne TNF‐α in rats (1130.14)
Authors: Yu Yang , Zhang Zhi‐Hua , Wei Shun‐Guang , Felder Robert 
Published: June 2021

The pro‐inflammatory cytokine tumor necrosis factor (TNF)‐ α acts upon the CNS to augment sympathetic drive in normal and pathophysiological conditions, including heart failure. The underlying mechanisms are poorly understood. We hypothesized that TNF‐ α induces endoplasmic reticulum (ER) stress in the brain, upregulating inflammatory mechanisms that drive sympathetic activity. In anesthetized rats, a centrally directed intracarotid artery (ICA) injection of TNF‐α (0.5 µg/kg) increased (p<0.05, vs baseline) renal sympathetic nerve activity, mean blood pressure and heart rate. These responses were attenuated by continuous intracerebroventricular (ICV) infusion of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 5 µg/hr). ICA TNF‐α induced molecular markers of ER stress, reactive oxygen species (ROS), NF‐кB, cyclooxygenase‐2 (COX‐2) and phosphorylation of p44/42 and p38 mitogen‐activated protein kinase (MAPK) in the subfornical organ (SFO), a CNS site at which circulating proinflammatory cytokines act to elicit sympathetic responses. TUDCA significantly reduced TNF‐α induced indicators of ER stress, phosphorylation of p44/42 and p38 MAPK and NF‐кB and COX‐2 activity in SFO, but not ROS generation. The data suggest that ER stress induced in brain tissues by blood‐borne TNF‐α contributes to central inflammatory mechanisms driving sympathetic excitation, likely via MAPK and NF‐кB signaling.Grant Funding Source: supported by NIH Grants HL‐073986 and HL‐096671 and the Department of Veterans Affairs
Vagal nerve stimulation can elicit both activation and inhibition of brown adipose tissue sympathetic nerve activity (1131.6)
Authors: Morrison Shaun , Madden Christopher , Tupone Domenico 
Published: June 2021

The dependence of BAT thermogenesis on the availability of metabolic substrates suggests that metabolic signals from the viscera could influence the sympathetic outflow driving BAT thermogenesis. Viscerosensory afferents in the proximal end of the sectioned left cervical vagus nerve were activated with electrical stimulation in anesthetized, ventilated rats while recording the SNA to the contralateral interscapular BAT pad. Paired‐pulse VNS delivered once every 5 sec evoked excitatory evoked potentials in BAT SNA with a peak latency of ~ 180 ms, followed by a ~2 s period of quiescent BAT SNA. Within a few seconds of stimulus onset, continuous, single‐pulse VNS at 2 Hz produced a complete inhibition of cold‐evoked BAT SNA that was sustained for at least 2 hrs of maintained VNS. Similarly, activation of neurons in the intermediate nucleus of the solitary tract (iNTS), at the level of the area postrema, with nanoinjections (60 pmol) of bicuculline also inhibits the VNS‐evoked excitation of BAT SNA. Inhibition of neurons in the rostral ventromedial medullary raphe pallidus region also eliminates the VNS‐evoked excitation of BAT SNA. These data support the existence of populations of vagal afferents whose stimulation can (1) increase BAT SNA via activation of BAT sympathetic premotor neurons or (2) inhibit BAT SNA via activation of BAT sympathoinhibitory neurons in the iNTS (see Am. J. Physiol. Regul. Integr. Comp. Physiol., 299:R277‐R290, 2010. PMC2904145).Grant Funding Source: Supported by NIH NS40987
The role of activation of chemokine receptor 2 in TRPV1‐mediated renal protection during salt‐sensitive hypertension (1136.2)
Authors: Wang Youping , Zhu Mingjun , Liu Weihong , Wang Xiaoxiao , Cui Lin , Shen Si , Xu Hui , Wang Donna 
Published: June 2021

To determine whether deletion of TRPV1 exaggerates salt‐sensitive hypertension‐induced renal injury via activation of chemokine receptor 2 (CCR2), wild type (WT) and TRPV1‐null mutant (TRPV1‐/‐) mice were subjected to uninephrectomy and DOCA‐salt treatment for 4 weeks with or without selective CCR2 antagonist, RS504393. DOCA‐salt treatment increased systolic blood pressure (SBP), urinary excretion of albumin and 8‐isoprostane, and decreased creatinine clearance compared with the control. DOCA‐salt treatment also caused obvious glomerulosclerosis, tubulointerstitial injury and monocyte/macrophage infiltration compared with the control. The changes in the parameters were greater in TRPV1‐/‐ mice compared with WT mice with the exception of SBP. Blockade of the CCR2 with RS504393 (4 mg/kg) had no effect on SBP. However, treatment with RS504393 prevented renal functional and morphological damage, and reduced the increase in monocyte/macrophage infiltration in DOCA‐salt hypertensive WT and TRPV1‐/‐ mice with the greater in the latter. Our results suggest that TRPV1 attenuates salt‐sensitive hypertension‐induced renal damage possibly via suppression of CCR2. (This work was supported by grants from the National Natural Science Foundation of China (No. 81170243) and Henan Provincial Science and Technology Innovative Program for Outstanding Scholarship (No. 124200510007).Grant Funding Source: NNSF of China (No. 81170243), Henan Provincial STlPOS(No.124200510007).
Identification of protein kinases that phosphorylate the urea channel protein UT‐A1 (1137.11)
Authors: Hwang Gloria , Chou Chung‐Lin , Hoffert Jason , Knepper Mark 
Published: June 2021

Vasopressin regulates the urea channel protein UT‐A1 by phosphorylation at Ser84 and Ser486. What kinases are responsible for these phosphorylation events remains an open question. To identify protein kinases that interact with UT‐A1 in IMCD, we employed crosslinking with BSOCOES, UT‐A1 immunoprecipitation, SDS‐PAGE separation, and quantitative mass spectrometry of trypsin digests of gel pieces. The peptides passing stringent spectral quality thresholds were quantified (label‐free) to identify those with high signal:noise (UTA1 antibody/preimmune IgG>4). This identified four protein kinases bound to the UT‐A1 (Phosphorylase Kinase [PhK], Calmodulin‐Dependent Kinase 2δ□[CAMK2], CDC42‐Binding Protein Kinase β□[CDC42BPK] and mTOR). In vitro phosphorylation assays were carried out by incubating these kinases or Protein Kinase A (purified, active recombinant) with synthetic peptides containing the known phosphorylation sites in UT‐A1. Phosphorylation was determined by immunoblotting using phospho‐specific antibodies. Four of the five tested kinases phosphorylated Ser486 (PKA > CDC42BPK > PhK > CAMK2). Kinetic studies of Ser486 phosphorylation (1 min‐24 hr) also showed PKA > CDC42BPK > PhK. In contrast, only CDC42BPK phosphorylated Ser84‐UT‐A1. mTOR did not phosphorylate either site. These results provide important data for modeling vasopressin signaling pathways.Grant Funding Source: Supported by NHLBI
The use of bioinformatics to identify potentially damaging BK channel and palmitoylation enzyme polymorphisms as novel research targets (1138.14)
Authors: Adeyileka Bernadette 
Published: June 2021

Large conductance Ca2+ and voltage‐gated potassium (BK) channels, coded for by the KCNMA1 gene, are found ubiquitously in humans and couple excitation‐contraction and stimulus‐secretion. BK channel dysfunction leads to pathophysiology affecting multiple body systems and processes (2). The BK channel undergoes palmitoylation, a post‐translational modification (PTM) that alters the trafficking, targeting and function of the protein. Palmitoylation is undertaken by a group of 23 palmitoyl‐acyl‐tranferase (PAT) enzymes coded for by the zDHHC genes. BK channel palmitoylation via PAT enzymes is crucial for BK channel function and thus has also been investigated as a novel therapeutic target. This study uses a bioinformatics approach to screen the genomic and proteomic data available for human KCNMA1 and zDHHC23, to identify and prioritise isoforms for future laboratory and clinical investigation. NCBI, Ensembl and Uniprot databases were utilised to identify: (a) splice variant information; (b) the number of non‐synonymous single‐nucleotide polymorphisms (nsSNPs); (c) nsSNP co‐location with key PTM and functional sites; (d) the damaging probability of prioritized nsSNP. Screening of KCNMA1 and zDHHC23 gene canonical proteins produced 154 and 63 unique nsSNPs respectively, with prioritisation of 13 KCNMA1 and 3 zDHHC23 nsSNP as amino acid (AA) co‐ordinates matched those of PTM residues or functionally important sites. nsSNP within 3 AA of key sites were recorded, with 64 (KCNMA1) and 22 (zDHHC23) nsSNP identified. The damaging probability of each prioritised nsSNP was assessed using SIFT and PolyPhen predictive software, with COSM196758, rs202024249, COSM920646, COSM332559 producing the highest scores. Prioritised SNPs will be investigated for functional impact in laboratory based assays. nsSNPs found to alter BK channel or PAT function will then be examined with respect to their distribution and incidence within appropriate populations and disease groups.Grant Funding Source: Supported by RGU IHWR
Characterization of UDP‐glucuronosyltransferase 2B expression and regulation by nuclear signaling pathways in HepaRG cells (1141.6)
Authors: Barrett Kathleen , Duniec‐Dmuchowski Zofia , Kocarek Thomas , Runge‐Morris Melissa 
Published: June 2021

HepaRG cells are bipotent human liver progenitor cells that can be differentiated into hepatocyte‐like cells that retain important liver functions. We evaluated the expression and regulation of the UDP‐glucuronosyltransferase (UGT) 2B subfamily of conjugating enzymes (UGT2B4, 2B7, 2B10, 2B11, 2B15, 2B17, and 2B28) in HepaRG cells. During proliferative culture conditions, expression of all UGT2Bs increased with cell confluency. After 2% DMSO‐induced differentiation, expression of UGT2B7and 2B17 initially decreased, but levels of all UGT2B mRNAs increased with continued differentiation. Treatment of differentiated HepaRG cells with the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) agonist, phenobarbital, increased expression of all UGT2B genes except UGT2B17. Rifampicin (PXR) increased expression of all UGT2B genes except UGT2B7. CITCO (CAR) increased UGT2B15 and decreased UGT2B17. GW3965 (liver X receptor) decreased all UGT2Bs except UGT2B11, while GW4064 (farnesoid X receptor) increased UGT2B10, 2B11, and 2B15 but decreased UGT2B4, 2B7 and 2B17. Treatment with ciprofibrate (peroxisome proliferator‐activated receptor α) increased UGT2B4, 2B7, and 2B15 but decreased UGT2B10 and 2B17. Treatment with 2,3,7,8‐tetracholorodibenzo‐p‐dioxin (Ah receptor) increased UGT2B4, 2B10, 2B11, 2B15, and 2B28. In conclusion, UGT2B genes are expressed in differentiating HepaRG cells and are differentially regulated by nuclear signaling pathways.Grant Funding Source: Supported by NIH grant ES005823
Prophylactic effect of calcium channel blockers against oxaliplatin‐induced acute peripheral neuropathy (1143.8)
Authors: Kawashiri Takehiro , Tsutsumi Kuniaki , Ushio Soichiro , Egashira Nobuaki , Masuda Satohiro 
Published: June 2021

Although oxaliplatin (L‐OHP) is an important anticancer agent against colorectal cancer, severe acute and chronic peripheral neuropathies are frequently occurred. We peviously reported that oxalate derived from L‐OHP is involved in acute cold hyperalgesia. In the present study, the mechanism of L‐OHP‐induced acute neuropathy was examined with the effects of calcium channel blockers in the rat model and primary cultured dorsal root ganglia (DRG).Administration of L‐OHP or oxalate to rats induced cold hyperalgesia, and increased in transient receptor potential melastatin 8 (TRPM8) mRNA levels in DRG. In addition, the treatment of L‐OHP or oxalate induced Ca²⁺ influx and nuclear factor of activated T‐cell (NFAT) nuclear translocation as well as up‐regulation of the TRPM8 mRNA levels in cultured DRG. These oxalate‐induced changes were inhibited by nifedipine, diltiazem and mexiletine. Comparable to the in vitro results, administration of these calcium channel blockers prevented the L‐OHP‐induced cold hyperalgesia in rats.These results suggest that the L type calcium channels/NFAT/TRPM8 pathway is a downstream mediator for L‐OHP‐induced cold hyperalgesia, and that calcium channel blockers may have prophylactic potential against L‐OHP‐induced acute neuropathy.
Vasopressin instead of epinephrine enhances efficacy of CPR without causing tachycardia (1150.5)
Authors: Cherry Brandon , Nguyen Anh , Williams Arthur , Scott Gary , Hollrah Roger , Ryou Myoung‐Gwi , Hoxha Besim , Olivencia‐Yurvati Albert , Mallet Robert 
Published: June 2021

Survival from cardiac arrest is highly dependent on the arterial pressure generated by cardiopulmonary resuscitation (CPR). To increase efficacy of precordial compressions, the potent vasoconstrictor epinephrine (EPI) is administered. However, EPI also elicits a robust, β1‐adrenoceptor‐mediated tachycardia following defibrillation, depleting the myocardium of the energy reserves it requires for recovery. We proposed that the adrenoceptor‐independent vasoconstrictor vasopressin (AVP) increases arterial pressure as effectively as EPI without producing tachycardia. After 6 min pacing‐induced cardiac arrest, domestic swine (25‐35 kg; 10 boars, 11 sows) received precordial compressions (100/min) for 4 min, and either EPI (0.1 mg; n=5) or AVP (10 U; n=16) was injected iv at 1 min CPR. EPI and AVP similarly increased mean arterial pressure from 31±3 to 66±4 mmHg vs. 34±3 to 59±3 mmHg at 4 min CPR. Countershock energy to achieve cardioversion was 16±4 J/kg in EPI‐ vs. 12±2 J/kg in AVP‐treated pigs. Post‐arrest tachycardia was less intense in AVP‐ (133±11 bpm) than EPI‐treated (174±14 bpm) pigs (P<0.05). Thus, AVP augments CPR as effectively as EPI but avoids tachycardia. Support: NINDS R01NS076975, NIA T32AG020494Grant Funding Source: Supported by: NINDS R01NS076975, NIA T32AG020494