bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of epicatechin on glucose transporters in cardiac tissue and peripheral nerves in type 2 diabetes (1086.5)
Authors: Gonzalez Nelson 
Published: June 2021

An abnormally high level of blood glucose is the key characteristic of type 2 diabetes (T2D). Studies have established that this results from decreased translocation of glucose transporters. Due to the consequences of T2D, i.e. cardiovascular disease and diabetic neuropathy, two glucose transporters of interest are the type 4 (GLUT4) and the glucose transporter type 3 (GLUT3). T2D has been linked to energy deficiency leading to cardiovascular disease. A key protein in an insulin independent pathway is 5' adenosine monophosphate‐activated protein kinase (AMPK), which is directly involved with the translocation of GLUT4. Thus, one of the main objectives of this study is to investigate the translocation of GLUT4 and relative AMPK expression in T2D. Moreover, studies have shown that hyperglycemia can cause peripheral nerve damage. GLUT3 translocation is independent of insulin signaling and can lead to an accumulation of glucose resulting in damage to the nerves. Peripheral nerve damage triggers the innate immune response which exacerbates the degeneration of peripheral nerves. In particular, the pro‐inflammatory cytokine tumor necrosis factor‐α (TNF‐α) has been observed to play a role. Therefore, another objective of this study is to investigate the expression of GLUT3 and TNF‐α in T2D to evaluate the progression of diabetic neuropathy. Using an in vivo high fat, streptozotocin (STZ) induced T2D model, we will inspect whether (‐)‐epicatechin (EPI), a naturally occurring flavonoid with demonstrated anti‐diabetic properties, alters the relative expression of the aforementioned proteins. Using samples collected from the heart and sciatic nerves of these animals, biochemical and immunohistological analysis will be performed to evaluate the expression and location of the proteins of interest. Ultimately, these results will further validate the anti‐diabetic properties of EPI and provide crucial data on its relatively elusive mechanism.Grant Funding Source: Supporter by Dr. Katrina Yamazaki and IMPACT LA
Fatal pulmonary arterial hypertension in female Fischer 344 rats (1089.20)
Authors: Crockett Edward , McLendon Jared , Alzoubi Abdallah , McMurtry Ivan , Stevens Troy 
Published: June 2021

Background: Male Fischer 344 (F344) rats exposed to Sugen‐5416, hypoxia, and then normoxia (Su/Hx/Nx) develop severe pulmonary arterial hypertension that is accompanied by death within 8 weeks. The mechanism responsible for death in these animals is incompletely understood, although acute heart failure was suspected. Here, we test the hypothesis that development of pulmonary arterial hypertension is accompanied by decreased right ventricular perfusion, which corresponds with decreased right ventricular function and decreased cardiac output. Methods: Six female F344 rats were injected with Sugen‐5416 (20 mg/kg) followed by three weeks of hypoxia (10% normobaric oxygen) and then five weeks of normoxia (21% oxygen at sea level). Survival was plotted as a Kaplan‐Meier curve. Echocardiography was utilized to monitor the development and severity of pulmonary arterial hypertension. Microsphere analysis was used to assess coronary and microvascular perfusion in the right ventricle. Histology was used to determine right ventricular matrix deposition, and right ventricular hypertrophy was estimated using the Fulton index (RV/[LV+S]). Preliminary Results: Five of six female F344 PAH rats died within 5‐weeks following SU/Hx/Nx exposure. Echocardiography showed right ventricular dilation and septal encroachment on the left ventricle, as well as decreased cardiac output. RV/[LV+S] confirmed significant hypertrophy of the right ventricular wall. Microsphere analysis indicated a substantial decrease in right ventricular coronary and microvascular perfusion. Histological trichrome staining displayed increased extracellular matrix deposition surrounding coronary arteries and cardiac myocytes. Conclusion: Decreased coronary and microvascular perfusion, increased fibrosis of the hypertrophied right ventricle and decreased cardiac output contribute to maladaptive cardiac remodeling and death in female F344 rats with severe pulmonary arterial hypertension.Grant Funding Source: Sponsred by NIH
Utilizing transgenic rats with eGFP‐tagged serotonergic (5‐HT) neurons to assess differential gene expression associated with age‐dependent changes in cellular CO2/H+ chemosensitivity (1092.7)
Authors: Puissant Madeleine M. , Mouradian Gary C. , Geurts Aron M. , Hodges Matthew R. 
Published: June 2021

The ventilatory response to hypercapnia in rodents increases throughout postnatal development, which parallels the increases in cellular CO2 sensitivity of serotonergic (5‐HT) neurons in primary cultures. Herein we begin to test the hypothesis that specific changes in gene expression in 5‐HT neurons during development contribute to the changes in cellular and/or organismal CO2/pH sensitivity. To begin to test this, acutely dissociated medullary raphe neurons obtained from adult (P56) transgenic rats (n=2) that express enhanced green fluorescent protein (eGFP) in cells expressing Pet‐1 (ePet‐1:eGFP rats) were sorted into pools (n= 5000 cells) using Fluorescent‐Assisted Cell Sorting (FACS). Total RNA was then extracted, and RNA quality and abundance measured before quantifying transcript abundance (Δ ct) with qRT‐PCR of 5‐HT neuron‐specific (slc6a4 (SERT); slc18a2 ( VMAT2); Tph2, and Trh) or non‐5‐HT specific raphe (Gad2 and Hprt) genes. We found high expression of 5‐HT neuron‐specific genes in the eGFP+ cell pools, including TRH (∆ct 6.94), TPH (∆ct 6.95), SERT (∆ct 3.18), and VMAT2 (∆ct 2.27), with little or no detectable expression of these genes in eGFP‐ cell pools. Expression levels of Gad2 and the housekeeping gene Hprt were comparable in both eGFP+ and eGFP‐ cells. These data indicate effective sorting of 5‐HT and non‐5‐HT raphe neurons in adult transgenic rats, and allow for further studies utilizing RNA sequencing to identify, measure and compare age‐related differences in 5‐HT neuron and non‐5‐HT neuron raphe transcriptomes.Grant Funding Source: NIH HL097033
The role of O‐linked‐N‐acetylglucosamine transferase in regulating autophagy (1094.7)
Authors: McLarty Jennifer , Paterson Andrew , Chatham John 
Published: June 2021

OGT catalyzes the addition of β‐N‐acetylglucosamine (O‐GlcNAc) to the hydroxyl group to serine/threonine residues of nuclear and cytoplasmic proteins, which regulates many biological processes critical for normal cell function. Autophagy is a highly conserved cellular self repair and survival mechanism also critical for normal cell function. We have previously reported that increases in cellular O‐GlcNAc levels interfere with autophagic signaling; therefore, we hypothesized that protein O‐GlcNAcylation may also regulate autophagy. O‐GlcNAc turnover was modulated in cultured human embryonic kidney 293 (HEK293) cells by overexpressing either OGT or a dominant negative OGT (dnOGT). Autophagy was induced either by nutrient deprivation (i.e., ND, serum and glucose free media) or rapamycin (RAP, 100 nM) for 3 hours, which was associated with an increase in overall O‐GlcNAc levels as well as O‐GlcNAcylation of Beclin‐1. Overexpressing OGT increased basal O‐GlcNAc levels and was associated with a decrease in the autophagic response in both ND and RAP treatment. Overexpression of dnOGT decreased overall basal O‐GlcNAcylation as well as attenuated the autophagic response in both ND and RAP groups. This study demonstrates that normal O‐GlcNAc cycling may play a critical role in the regulation of autophagy and provides novel mechanism by which changes in cellular O‐GlcNAcylation mediate cell survival responses.Grant Funding Source: Supported by NIH‐NHLBI grant RO1‐HL101192
cAMP/PKA‐mediated regulation of amino acid metabolism in murine T‐lymphoma cells (1095.16)
Authors: Wilderman Andrea , Guo Yurong , Taylor Susan , Insel Paul 
Published: June 2021

The second messenger cyclic AMP (cAMP), acting through PKA, influences gene and protein expression through CREB and other transcription factors. In order to define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we conducted quantitative proteomic studies of wild‐type (WT) and kin‐ (PKA‐null) S49 murine T lymphoma cells and compared the impact on protein expression of increases in cAMP by a cell‐permeable analog (8‐CPT‐cAMP, CPT). Numerous proteins involved in branched chain amino acid metabolism had markedly increased expression in WT S49 cells, but not kin‐ cells, treated with CPT for 16h. Expression of mRNA levels of these genes also increased in WT, but not kin‐, S49 cells in response to treatment with CPT. Consistent with this finding, incubation of WT S49 cells with CPT enhanced cell survival in growth media lacking glutamine, whereas kin‐ S49 cells show decreased viability in such media even in the presence of 8‐CPT‐cAMP. We conclude that cAMP acts via PKA to regulate expression of genes and proteins for branched chain amino acids, an action that may help protect cells from a decrease in viability in glutamine‐deficient media. Since not all of the cAMP/PKA‐regulated branched chain amino acid metabolism genes with increased expression have strong predicted binding of CREB, regulation of those genes by cAMP/PKA appears to occurs through as‐yet to be defined transcriptional pathways.Grant Funding Source: Supported by HHMI (S. S.T.), and DK54441 (S.S.T.), GM066232 (P.A.I.) and HL107200 (P.A.I.) from NIH
Anti‐aging gene klotho regulates cellular antioxidant capacity via binding to S‐fromylglutathione hydrolase through the N285 glycosylation site (1095.3)
Authors: Xu Yuechi , Dutcher Lincoln , Sun Zhongjie 
Published: June 2021

Background. Klotho is a recently‐identified aging‐suppressor gene. Mutation of klotho causes extensive premature aging phenotypes and shortens lifespan while overexpression of this gene extends lifespan by 20%. The secreted Klotho (Skl) circulates in the blood. However, the receptor of Skl is unknown. The purpose of this study is to identify the binding sites of Skl.Methods & Results. We apply proteomics method to identify the associated proteins of Skl. Mass spectrometry anal[[Unsupported Character ‐ Codename &amp;shy;]]ysis and coimmunoprecipitation identified a direct interaction between Skl and S‐formylglutathione hydrolase (FGH), which was confirmed by western blotting and immune fluorescent staining. Overexpression of Skl upregulated FGH protein levels in cells. In KL‐/‐ mice, FGH was decreased in kidneys and red blood cells. Skl decreased cellular O2‐ levels (DHE staining) and increased FGH activity (ratio of Glutathione/Glutathione disulfide). Knocked down FGH by siRNA abolished the Skl‐induced increase in anti‐oxidant activity, suggesting that the anti‐oxidant function of Skl is mediated by FGH. The point mutation experiment confirmed 3 N‐glycosylation sites Asp 161, 285 and 346 on Skl. The Asp 285 is the key site that determines Skl anti‐oxidant activity.Conclusion. Skl exerts anti‐oxidant activity by binding to FGH. The Asp 285 is the key site to the anti‐oxidant activity of Skl.Grant Funding Source: Supported by HL105302 and HL 102074.
c‐Jun N‐terminal kinase inhibits TNFα‐induced NF‐κB activation through endosomal Nox1 in vascular smooth muscle cells (1096.4)
Authors: Choi Hyehun , Lamb Fred 
Published: June 2021

Tumor necrosis factor‐α (TNFα), a pro‐inflammatory cytokine, can cause either vascular smooth muscle cell (VSMC) apoptosis (JNK‐dependent) or proliferation (NF‐κB‐dependent). We hypothesized that JNK modifies NADPH oxidase (Nox1)‐dependent NF‐κB stimulation by TNFα in VSMC. We quantified the impact of siRNA targeting JNK1 or 2 on TNFα dependent NF‐κB activation using an adenoviral reporter construct in cultured aortic VSMC from wildtype (WT) or Nox1 knockout (Nox1KO) mice. NF‐κB activation was potentiated 1.5 + 0.5 fold (n=6) by siRNA targeting JNK1 and 2 in WT cells, however, there was no effect on NF‐κB activation in Nox1KO cells (0.9 + 0.1 fold, n=4). NF‐κB activation was endocytosis‐dependent as it was blocked by dominant‐negative dynamin (Ad‐DynK44A, 61% reduction, n=4). JNK inhibition with SP600125 did not alter endocytosis of dextran‐conjugated Texas Red. Endosomal ROS production is required for NF‐κB activation by TNFα in VSMC. JNK regulates NF‐κB activation either at the level of Nox1 activation or at a step downstream of ROS. Reduction of NF‐κB activation by JNK may tip the balance of signaling to favor cell death over proliferation.Grant Funding Source: Supported by American Heart Association
Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying ouabain‐triggered Ca2+ oscillations (1097.10)
Authors: Fontana Jacopo , Kruusmägi Markus , Brismar Hjalmar , Aperia Anita 
Published: June 2021

Our group has studied the origin of Ca2+ oscillations triggered by the cardiotonic steroid ouabain, a highly specific ligand of Na+‐K+‐ATPase. It has been shown that these low frequency oscillations protect the cells from apoptosis by activating the NF κB/p65 survival subunit and inhibiting the intrinsic mitochondrial apoptotic pathway by reversing an imbalance between the apoptotic factor Bax and the antiapoptotic factor Bcl‐xL.The aim of this study is to reveal the identity and role of the intracellular compartments that are involved in ouabain‐triggered Ca2+ oscillations.Studies were first performed on COS7 cells which expressed genetic Ca2+ indicators targeted to mitochondria and cytosol. Following application of low doses of ouabain, we found that each cytosolic calcium wave was followed by a delayed mitochondrial calcium transient. The recovery of Ca2+ to normal levels was slower in mitochondria compared to cytosol, while the initial rate of increase was found to be similar in both compartments. Next, using an endoplasmic reticulum (ER)‐targeted genetic Ca2+ indicator, we observed rhythmic and simultaneous Ca2+ transients in both mitochondria and ER after ouabain application.We propose that Ca2+ shuttling between the ER and mitochondria may have a pivotal role in the propagation of ouabain triggered slow Ca2+ oscillations.Grant Funding Source: Swedish Research Council, Family Erling‐Persson Foundation, Märta and Gunnar V Philipson
MED28 regulates the development of epithelial‐mesenchymal transition and invasion in human lung cancer cells (1097.14)
Authors: Hsieh Nien‐Tsu , Li Chun‐I , Huang Chun‐Yin , Lee Ming‐Fen 
Published: June 2021

The development of the epithelial tumors from epithelial‐mesenchymal transition (EMT) to metastasis includes degradation and remodeling of the extracellular matrix and acquisition of a motile and invasive phenotype. MED28, facilitating transcriptional activation as a Mediator subunit, is also found highly expressed in epithelial tumors. Recently, we have reported that MED28 enhanced migration and invasion in human MCF7 and MDA‐MB‐231 breast cancer cells. The aim of this current study is to explore the role of MED28 in EMT and invasion in human non‐small cell lung cancer (NSCLC). Suppression of MED28 reduced the expression of vimentin, a mesenchymal marker, in human A549 NSCLC cells. In addition, TNFα induced EMT, whereas MED28 knockdown alleviated the morphological alterations. MED28 modulated TNFα‐induced Matrigel invasion and NFκB functional activity as assessed by heterologous promoter/reporter assay. The increased expression of several TNFα‐enhanced proteins, including MMP9, vimentin, p‐p65, and pIκB, was suppressed upon MED28 knockdown. Out data indicate a role of MED28 in the development of EMT and invasion in human NSCLC cells.Grant Funding Source: Supported by NSC101‐2320‐B‐309‐001 and NSC102‐2320‐B‐309‐001‐MY3 to M‐F Lee
Myocardial infarction rapidly induces diaphragm muscle weakness (1102.11)
Authors: Bowen T Scott , Mangner Norman , Werner Sarah , Glaser Stephanie , Schrepper Andrea , Doenst Torsten , Linke Axel , Schuler Gerhard , Adams Volker 
Published: June 2021

Chronic heart failure (CHF) induced by myocardial infarction (MI) results in diaphragm muscle weakness, with increased inflammation and oxidative stress directly implicated. It remains unknown, however, if diaphragm muscle function is impaired immediately post‐MI and whether this is associated with increased markers of inflammation and oxidative stress.Methods: Ligation of the left coronary artery to induce MI (n=21; confirmed by echocardiography) or sham operation (n=22) was performed on 8 wk old C57BL/6 mice. Three days later, in vitro isometric force of diaphragm muscle fibre bundles was assessed.Results: Specific force was depressed (p<0.05) between frequencies of 80‐300 Hz post‐MI, as was maximal tetanic force (25±1 vs. 21±1 N/cm²). MI had no influence on local mRNA expression of TNF‐α and IL‐6, but both were increased (p<0.05) at the systemic level. Compared to sham, MI increased (p<0.05) mRNA expression and enzyme activity of xanthine oxidase (65% and 19%, respectively) and NADPH oxidase (140% and 44%, respectively), however antioxidant enzymes remained unchanged.Conclusion: Diaphragmatic contractile dysfunction is rapidly induced following MI, and this is associated with increased systemic inflammation and markers of local oxidant production. These findings suggest diaphragm muscle weakness is early‐onset in CHF, potentially mediated by inflammation and oxidative stress.Grant Funding Source: Supported by the Alexander von Humboldt Foundation
Mechanisms of skeletal muscle deformity in hyperhomocysteinemia (1102.24)
Authors: Veeranki Sudhakar , Givvimani Srikanth , Sathnur Pushpakumar , Metreveli Naira , Tyagi Suresh 
Published: June 2021

Hyperhomocysteinemia (HHcy) is associated with elderly frailty, skeletal muscle malfunction and injury, reduced vascular integrity and function and mortality. Although HHcy was implicated in impairment of vasculogenesis after hind limb ischemia in murine models, the underlying mechanisms are unclear. We hypothesize that HHcy compromises perfusion, collateral formation and vasculogenesis through diminishing post‐ischemic vasculogenic responses in the muscle fibers. To test this hypothesis, we created femoral artery ligation in wild type (WT) and CBS+/‐ mice (a model for HHcy) and assessed tissue perfusion, collateral formation and skeletal muscle function using Laser Doppler perfusion imaging, barium angiography and fatigue tests. In addition, we evaluated post‐ischemic levels of VEGF, and levels of its muscle specific regulators: HIF‐1α and PGC‐1α in the skeletal muscle fibers. The observations indicated that dysregulation of VEGF, HIF‐1 alpha and PGC‐1 alpha levels in the CBS+/‐ mice ischemic skeletal muscles. Moreover, there was enhanced FOXO dependent atrogene transcription in the muscles and reduced muscle function after ischemia in the CBS+/‐ mice. These results suggest that HHcy adversely affects muscle specific ischemic responses and contribute to muscle frailty.Grant Funding Source: 1RO1HL108621
Dysregulation of dysferlin during 7 days mechanical unloading is mediated by ROS in rat soleus muscle (1102.27)
Authors: Lee Yang , Lawler Matthew , Botchlett Rachel , Hord Jeff , Lawler John 
Published: June 2021

Dysferlin plays an important role in membrane repair of damaged sarcolemma, is bound to neuronal nitric oxide synthase‐mu (nNOSµ) during disuse, and can be regulated by reactive oxygen species (ROS). Previously, we showed that oxidative stress increased caveolin‐3 which contributed to nNOSµ dislocation from the sarcolemma and thus atrophy. However, the underlying mechanisms that contribute to nNOS translocation and muscle atrophy remain unknown. We hypothesized that oxidative stress would affect dysferlin localization during mechanical unloading, and thus nNOSµ translocation. F344 rats were divided (n=9/group) into control (CON), hindlimb unloaded for 7days (7DHU), and 7D HU+ EUK (EUK) groups. The superoxide dismutase/catalase mimetic EUK‐134 was injected daily (3 mg/kg/d) beginning 24 hours prior to HU. Upregulated ROS sources included sarcolemma (Nox2) and mitochondria (Nox4) during 7DHU were downregulated by EUK‐134. Dystrophin and beta‐sarcoglycan are not altered during 7 days mechanical unloading period. However, nNOS was translocated from sarcolemma to sarcoplasm in 7DHU group. Dysferlin was also downregulated and disrupted from sarcolemma in 7DHU groups, which might exacerbate perturbation of the membrane environment. EUK‐134 partially prevented translocation or nNOS and dysregulation of dysferlin, suggesting changes in these proteins were redox dependent. Our data indicated that oxidative stress is involved in unloading‐induced nNOSµ translocation that is coupled with dysferlin in the rat soleus.Grant Funding Source: Supported by NASA Space Biology grant (NNX12AR62G)
Quantitative phosphoproteomics studies reveal different signaling cascades in cultured distal convoluted tubular cells upon vasopressin or angiotensin II stimulation (1109.10)
Authors: Cheng Lei , Pistikun Trairak , Fenton Robert 
Published: June 2021

The thiazide‐sensitive NaCl cotransporter (NCC) plays an important role in sodium reabsorption in the kidney distal convoluted tubules (DCT). Both angiotensin II (ANGII) and vasopressin (AVP) are hormones known to exert effects in this segment and induce phosphorylation of NCC, which regulates NCC function. However, the signaling pathway from AVP to NCC and possible interplay between AVP and ANGII signaling cascades remain unknown. Here we performed large scale quantitative phosphoproteomics of cultured mpkDCT cells to map global changes in protein phosphorylation events upon treatment with ANG II or the V2‐selective analog dDAVP (30min). Experiments were performed using the stable isotope labeling by amino acids in cell culture (SILAC) quantification approach. LC‐ESI‐MS/MS identified 21616 quantifiable phosphopeptides and 12848 different phosphorylation sites, of which 7149 were confidently assigned . Kinase prediction of the regulated phosphorylation sites using NetworKIN indicated that ANGII stimulation increased activity of the kinase families CDK, MAPKs or PAK, whereas dDAVP stimulation increased the activity of the kinases PKA, DMPK, PIM2, PAK, or CLK. Motif analysis indicated that RRXSXXX (PKA substrate motif) was a preferential target for upregulated phosphorylation sites upon dDAVP stimulation, whereas for downregulated phosphorylation sites XXXSPXX (CDK substrate motif) was more prominent. cAMP assays showed that dDAVP induced an increase in cAMP levels in mpkDCT cells, supporting the PKA signaling cascades identified from proteomic analysis. Our results suggest that highly distinct signaling cascades are activated in cultured mpkDCT cells upon acute treatment by either dDAVP or ANG II, analysis of which will likely identify novel mechanisms for NCC regulation.Grant Funding Source: Supported by Lundbeck Foundation and the Danish Medical Research Foudation