bluuebunny/crossref_metadata_2025 huggingface dataset:
Sorafenib induces autophagic cell death and apoptosis in hepatoma HepG2 cells via PERK/EIF2α signaling pathway
Authors: Liu Ze‐feng , Liu Qi‐Lin , Li Fu‐Jian , Yang Zhou‐Fan , Chen Li , Zhang Guo
Published: June 2021

Backgound & Aims

Previous studies have documented cellular apoptosis, autophagy and endoplasmic reticulum (ER) stress participated in the hepatocellular carcinoma (HCC). Sorafenib has been reported to regulate autophagy and apoptosis in hepatoma cells associated with different unfolded protein response (UPR). However, resistance problem dramatically limited the usage of sorafenib in HCC with uncertain mechanism. Therefore, this research is focused on whether and how sorafenib induces autophagy and apoptosis via specific PERK/EIF2a signaling pathway of ER stress in HepG2 cells.

Methods

Firstly, stable infected HepG2 cell lines of PERK knockdown and vehicle control cells were established with lentivirus‐mediated RNA interference, RT‐PCR and western blot were employed to identify the efficiency of PERK silencing. Trypen blue assay was then applied to investigate the effect of PERK knockdown, and sorafenib (10 μM) induced‐lethality in these established HepG2 cell lines in presence or absence of thapsigargin (Tg, 500 nM). Western blot was further performed to analyze the protein expression of PERK, p‐PERK, EIF2α, p‐EIF2α, CHOP, caspase3 and P62. Finally, the numbers of apoptotic cells in these cells were measured by flow cytometry.

Results

After PERK silencing, the expression of PERK mRNA and protein were significantly lower (P<0.05) in the established HepG2 cell lines v.s. control. In parallel, the expression of p‐PERK, EIF2α, p‐EIF2α, CHOP and cleaved caspase3 protein were significantly decreased (P<0.05) and apoptotic numbers were dramatically inhibited in PERK silencing cells (P<0.05). In basal condition, sorafenib was not only found to induce autophagic flux but also drive up‐regulation of PERK, p‐PERK, EIF2α, p‐EIF2α, CHOP in vehicle HepG2 cells. Interestingly, PERK overexpression further induced autophagy with decrement in P62 in HepG2 cells in response to sorafenib or sorafenib plus Tg (P<0.05). Meanwhile, trypan blue assay found that the sorafenib alone and sorafenib combined with Tg induced‐cell death was substantially decreased with about 10% and 11% in PERK silencing cells but increased with 30% in PERK overexpression cells, respectively (P<0.05).

Conclusion

PERK, one of the hallmarks of ER stress signaling, might cause procelldeath autophagy and thereby contribute to enhance sorafenib derived apoptosis in hepatoma HepG2 cells, which may provide a potential critical target for overcoming the resistance of sorafenib in HCC in the future.

Support or Funding Information

Supported by NSFC Grants 81360077, 81860654‐30630145, 81172260 and GXNSFC Grants 2015GXNSFDA139022 to Guo Zhang

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Antioxidant and Cytotoxic Properties of Tridax Procumbens Leaf Extract on Liver and Pancreatic Cancer Cell Lines
Authors: Samuel Ekundayo Stephen , Akinola Tolulope Joseph , Tosh David , Odunola Oyeronke Adunni
Published: June 2021

Human hepatocellular carcinoma and pancreatic ductal adenocarcinoma are two of the deadliest emerging forms of cancer worldwide. Hence, the increased search for phytochemicals with promising anticancer properties. In the present study, we assessed the phytochemistry, antioxidant activities and the role of Tridax procumbens leaf extract and fractions on HepG2 and Panc‐1 cells (models of hepatocellular and pancreatic ductal adenocarcinoma). HepG2 and Panc‐1 cell lines were maintained in supplemented Dulbecco's modified eagle's medium at 37°C in a humidified incubator with 5% CO2. The HepG2 and Panc‐1 cells were treated with 20, 50, 100 and 250 μg/ml of dimethyl sulfoxide (control), extract and fractions (n‐hexane, dichloromethane, ethyl acetate, aqueous) for 24 and 48hours. Phytochemistry (total flavonoid, total phenol) and antioxidant (2,2‐diphenyl‐1‐picrylhydrazyl hydrate, ferric reducing antioxidant power, nitric oxide radical, total antioxidant capacity) assays were performed on the test samples. The expression levels of catalase, alkaline phosphatase, glutathione S transferase‐pi and caspase 3 were monitored with immunofluorescence. Live/Dead and MTT assay were used to determine cytotoxicity of cultured cells. The dichloromethane and ethyl acetate fractions showed significant higher antioxidant activities as compared to other test samples. The dichloromethane fraction caused a significant level of cytotoxicity and induction in the activity of ALP, catalase and caspase 3 as compared to the control. Conversely, there was a significant cellular reduction and nuclear translocation of GST‐pi in dichloromethane fraction treated cell as compared to the control. The extract and fractions of Tridax procumbens have antioxidant activities. However, the dichloromethane fraction's activities depict a promising anticancer agent.

Support or Funding Information

Commonwealth Scholarship Commission, Department for International Development, United Kingdom and University of Ibadan, Ibadan, Nigeria

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Study the Effects of Capsaicin on Triple Negative Breast Cancer Cells
Authors: Alasmari Moudi M. , Alshaeri Heba K. , Alashari Rawabi A. , Böhlke Mark , Maher Timothy , Pino‐Figueroa Alejandro
Published: June 2021

Triple negative breast cancer (TNBC) is one of the most aggressive types of breast cancer accounting for 12% of breast cancer cases. It is characterized by the lack of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER‐2), which limits treatment options and enhances its ability to metastasize with the risk of recurrence. Patients with TNBC are not responsive to conventional therapies. Capsaicin (CAP) is the most abundant capsaicinoid produced in chili pepper fruits and has been utilized for its analgesic and anti‐inflammatory effects. While several studies have demonstrated that capsaicin has anti‐carcinogenic properties in various types of human cancers, the underlying molecular mechanisms remain to be explored. The aim of this study was to investigate the effects of capsaicin in human TNBC, utilizing a BT‐20 cell model. Capsaicin demonstrated concentration‐ and time‐dependent in the viability of BT‐20 cells, as determined by the MTS assay. Capsaicin caused significant increases in cytochrome C release, caspase 3/7 activity and expression of cleaved poly‐(ADP‐ribose) polymerase (PARP), all of which are markers of apoptotic activation. These effects were accompanied by down‐regulation of cyclin D1 production, an indicator of cell cycle arrest at the G0/G1 phase. Further analyses of signaling mechanisms revealed that capsaicin significantly inhibited EGFR phosphorylation and the phosphorylation of its downstream signaling proteins AKT and MAPK, which may provide some explanation for its mechanism of action in TNBC. In conclusion, capsaicin has demonstrated inhibitory effects on cell growth and cell cycle progression on TNBC cells by enhancing apoptosis. Our data demonstrated a novel mechanism for capsaicin, modulating EGFR signaling by AKT/MAPK pathways in BT‐20 cells. These results provide useful information relevant to the development of a potential new therapy to treat TNBC with capsaicin.

Support or Funding Information

This work was funded by the College of Medicine, King Saud bin Abdulaziz University for Health Sciences (KSAU‐HS), Jeddah, Saudi Arabia.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prevention of Cardiac Dysfunction Development by a Methanocarba Adenosine Monophosphonate Derivative
Authors: Shen Jian‐Bing , Toti Kiran S , Chakraborty Saibal , Kumar T. Santhosh , Cronin Chunxia , Liang Bruce T , Jacobson Kenneth A
Published: June 2021

Accumulating evidence supports a therapeutic role of purinergic signaling in cardiac diseases. Previous studies showed that MRS2339, a charged methanocarba derivative of 2‐Cl‐adenosine monophosphate, is efficacious in animal models of heart failure when infused systemically. However, MRS2339 lacks oral bioavailability as a nucleoside 5’‐phosphate derivative and is subject to hydrolysis in vivo leading to the inactive nucleoside MRS1873. We now test the hypothesis that uncharged adenine nucleoside phosphonates, suitable as oral agents with a hydrolysis‐resistant phospho moiety, can prevent the development of cardiac dysfunction in a post‐infarction ischemic or pressure overloaded heart failure model. Of the diester‐masked uncharged phosphonates evaluated, MRS2978 but not MRS4074, was efficacious in preventing cardiac dysfunction with improved left ventricular (LV) fractional shortening (FS increased from 21±0.79% with vehicle (N=13) to 25.1±0.63% with MRS2978 (N=11), P<0.05) and with preservation of LV wall thickness when administered orally at onset of ischemic heart failure (within 24h after left anterior descending artery (LAD) ligation). MRS2925, the charged, unmasked analog of MRS2978, was able to prevent heart dysfunction only when it was infused subcutaneously; its oral gavage was not efficacious. In the established heart failure models caused either by LAD ligation or transverse aortic constriction (TAC), orally administrated MRS2978 (4 weeks after LAD ligation or 3 weeks after TAC), but not MRS2925, could partially restore cardiac dysfunction by increasing the FS in both ischemic (from 19.7±0.95% with vehicle (N=18) to 26.1±1.37% with MRS2978 (N=11), P<0.05) and pressure overload (FS from 17.5±0.76% with vehicle(N=9) to 23.7±1.25% with MRS2978 (N=10), P<0.05) heart failure models. MRS2978 was orally bioavailable (57–75%F) and highly stable in human, mouse and rat plasma, with 96–100% remaining after 2 h. MRS2925 could be formed from hydrolysis of MRS2978 in vivo, but as a minor metabolite (only 10%), suggesting that the effects of MRS2978 are not dependent on the MRS2925. Either MRS2978 or MRS2925, combining with 2me‐SATP, was capable of activating cardiac myocyte eNOS, consistent with myocyte eNOS activation as a mechanism for the salutary effect of these two phosphonates nucleotides. These data demonstrated the ability of oral therapy with MRS2978 to treat heart failure, both at the inception of heart failure for prevention and after the development of heart failure as a rescue.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resveratrol Protects Against Angiotensin II‐Induced Cellular Hypertrophy through Inhibition of CYP1B1/Mid‐Chain Hydroxyeicosatetraenoic Acid Mechanism
Authors: Shoieb Sherif M. , El‐Kadi Ayman O. S.
Published: June 2021

Objective

Cytochrome P450 1B1 (CYP1B1) and its associated cardiotoxic mid‐chain hydroxyeicosatetraenoic acid (HETEs) metabolites have been reported to directly contribute to the development of cardiac hypertrophy. Resveratrol (RESV) is naturally occurring and commercially available polyphenol that possess beneficial effects in wide array of cardiovascular diseases. Several reports have shown the capacity of RESV to treat cardiac hypertrophy, myocardial infarction and heart failure. However, the underlying mechanisms, responsible for RESV beneficial effects, are not fully elucidated. Since RESV is a well‐known CYP1B1 inhibitor, the purpose of this study is to investigate whether RESV protects against angiotensin II (Ang II)‐induced cellular hypertrophy through inhibition of CYP1B1/mid‐chain HETEs mechanism.

Methods

Human ventricular cardiomyocytes RL‐14 and rat cardiomyoblast H9c2 cells were treated with vehicle or 10 μM Ang II in the absence and presence of 2, 10 or 50 μM RESV for 24 h. Thereafter, the level of mid‐chain HETEs was determined using liquid chromatography–mass spectrometry (LC/MS). Hypertrophic markers and CYP gene expression and protein levels were measured using real‐time PCR and Western blot analysis, respectively.

Results

Our results demonstrated that RESV, at concentrations of 10 and 50 μM, was able to protect against Ang‐II‐induced cellular hypertrophy as evidenced by a substantial inhibition of hypertrophic markers, β‐myosin heavy chain (MHC)/α‐MHC and atrial natriuretic peptide (ANP). Ang II significantly induced the protein expression of CYP1B1 and increased the metabolite formation rate of its associated mid‐chain HETEs namely 5‐, 8‐, 9‐, 12‐ and 15‐HETE in both cell lines. Interestingly, the protective effect of RESV, at concentrations 10 and 50 μM, was associated with a significant decrease of CYP1B1 protein expression and mid‐chain HETEs to nearly control levels.

Conclusion

Our results provided the first evidence that RESV protects against Ang II‐induced cellular hypertrophy, at least in part, through CYP1B1/mid‐chain HETEs‐dependent mechanism.

Support or Funding Information

This work was supported by a grant from the Canadian Institutes of Health Research [Grant 106665] to A.O.S.E. S.M.S. is the recipient of Antoine Noujaim Graduate Scholarship in Pharmaceutical Sciences.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
20‐HETE Antagonism Reduces Left Ventricular Remodeling Post‐Myocardial Infarction
Authors: D'Addario Catherine , Joseph Gregory , Soler Amanda , Brechoire Ines , Hutcheson Brenda , Falck John R , Proctor Spencer , Schwartzman Michal , Rocic Petra
Published: June 2021

20‐Hydroxyeicosatetraenoic acid (20‐HETE) is a cytochrome p450‐derived eicosanoid that stimulates endothelial dysfunction and inflammation via binding to its receptor, GPR75 (GPCR‐Gαq/11). Increased 20‐HETE in animals and humans is associated with hypertension, stroke, myocardial infarction (MI), metabolic syndrome (MetS), and increased reactive oxygen species (ROS). However, investigated effectiveness of 20‐HETE antagonists on ROS generation and MI size has been limited by short‐term follow up and administration of inhibitors prior to onset of MI only in healthy animals. Here, we evaluated the effect of a 20‐HETE antagonist, 20‐SOLA, administered at onset of reperfusion on post MI remodeling 48 hours and 8 weeks after reperfusion in normal (SD) and MetS (JCR:LA‐cp) rats. 20‐HETE was elevated in response to ischemia and more so during reperfusion; this elevation was greater in MetS (ischemia: 2‐fold SD and JCR; reperfusion: 3‐fold SD, 4‐fold JCR). MI size was markedly greater in JCR vs. SD rate (50% vs. 25% of LV). Treatment with 20‐SOLA significantly decreased MI size in both SD (~50%) and JCR (~65%) rats. Equivalent results were obtained in animals treated with GPR75‐shRNA‐Lnv at onset of reperfusions. 20‐SOLA improved coronary blood flow (1.00 ± 0.01 to 1.84 ± 0.03 mg/ml/g in SD to 0.99 ± 0.03 to 1.75±0.01 mg/ml/g in JCR). Mechanistically, smaller MI size in 20‐SOLA‐treated animals correlated with decreased ROS production in JCR rats (90%). Furthermore, survival and left ventricular (LV) function were preserved 8 weeks post MI in 20‐SOLA‐treated animals (ejection fraction (EF) = 80.5% (SD, 100% survival) and 82.5% (JCR, 100% survival)). This correlated with decreased ROS, preserved myocyte morphology, and preserved intact collagen. Differential activation of MMPs in SD vs. JCR rats vs. 20‐SOLA‐treated animals underlie the observed morphological, structural and functional changes. ROS production was decreased in JCR rats treated with a NOX inhibitor (~60%); NOX inhibition also markedly reduced MI size at 48h (60%) and preserved LV function at 8 weeks (EF = 74%, 85% survival) vs. non‐treated rats demonstrating an important role for NOX‐derived ROS in determining MI size and long‐term post‐MI remodeling. In the aggregate, these results indicated that targeting 20‐HETE actions may be an important consideration in prevention of detrimental LV remodeling and mortality post MI. Funding: HL093052.

Support or Funding Information

Funding: HL093052

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Bauhinia variegata Leaf Extract in Diabetic Cardiomyopathy
Authors: Kulkarni Yogesh Anant , Laddha Ankit Pravin
Published: June 2021

Diabetes mellitus is a chronic metabolic disorder associated with development of micro and macro vascular diabetic complications. According to World Health Organization cardiovascular disease causes up to 65% of deaths among diabetic population. The present work was designed to study effect of Bauhinia variegata, a medicinal plant described in Ayurveda for its medicinal benefits in diabetic cardiomyopathy. Alcoholic extract of Bauhinia variegata leaf (AlcE) was prepared by Soxhlet extraction technique and was standardized using HPTLC. Male Sprague Dawley rats were injected with streptozotocin (55 mg/kg, i.p.) for induction of diabetes. After 42 days of diabetes induction, the animals were grouped and treated with alcoholic extract at dose of 250, 500 and 1000 mg/kg by oral route for next 28 days. At the end of treatment, plasma and heart tissue were collected for evaluation of various parameters. AlcE at 1000 mg/kg significantly reduced plasma glucose as compared to diabetic control animals. AlcE at dose of 500 and 1000 mg/kg significantly (P<0.001) reduced elevated level of creatine kinase‐MB, lactate dehydrogenase and aspartate transaminase in plasma as compared to diabetic animals. AlcE treatment prevented cardiac tissue from oxidative damage, it significantly reduced the elevated level of malonyl dialdehyde at dose of 500 and 1000 mg/kg whereas it significantly increased the level of antioxidant enzymes at dose of 1000 mg/kg (P<0.001). Present study showed that Bauhinia variegata leaf extract has significant effect in management of diabetic cardiomyopathy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Baicalein Neutralizes Hypercholesterolemia‐Induced Aggravation of Cardiac Oxidative Injury
Authors: Al‐Rejaie Salim Salih
Published: June 2021

Hypercholesterolemia is a global well‐known contributing factor in cardiovascular diseases through provoking cardiac oxidative damage and depleting energy production. Baicalein (BL) is a bioactive polyphenolic flavonoid that has multiple cardiovascular beneficial effects. This study aims to investigate the potential cardio‐protective properties of BL against cardiac oxidative injury in high cholesterol diet (HCD) fed rats. HCD in pellet form was prepared by adding 1% cholesterol + 0.5% cholic acid with normal cholesterol rat chow powder. Eighteen animals were fed with HCD for 10 weeks. At the 6th week, twelve animals started BL treatment in two doses (25 and 50 mg/kg/day, orally). Histologically, BL protected the myocardial morphology against HCD associated multi‐focal vacuolar degeneration. BL markedly reduced serum total cholesterol (TC), triglycerides (TG), creatine kinase‐B (CK‐B), lactate dehydrogenase (LDH) and creatine kinase‐MB (CK‐MB) levels in hypercholesteremic rats. In cardiac tissue homogenate, the levels of lipid‐peroxidation biomarker, thiobarbituric acid reactive substances (TBARS) were significantly prohibited, while glutathione (GSH) levels were increased markedly by BL in HCD fed rats. In addition, hypercholesterolemia‐induced inhibition of cardiac enzymatic activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) was considerably attenuated by BL treatment. Overall, the current findings elucidated that BL exhibits a promising therapeutic value for cardiac metabolic changes linked with hypercholesterolemia via enhancement of the redox balance.

Support or Funding Information

Deanship scientific research at King Saud University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Water‐pipe Smoke Negatively Distress Cardiovascular Biomarkers in Mice
Authors: Rababah Abeer , Bsoul Ragad , Alkhatatbeh Mohammad , Alzoubi Karem
Published: June 2021

Tobacco use is one of the main risk factors to cardiovascular diseases (CVDs) and atherosclerosis in particular. It was mis‐considered that water‐pipe smoking (WPS) is less harmful than cigarette smoking due to its flavor. Studies had shown that more than 100 million people smoke waterpipe daily. WPS contains several toxic materials such as: nicotine, carcinogens, tar, carbon monoxide and heavy metals. Thus, WPS is considered to be as one of the toxic environmental factors that should be investigated intensively. The aim of this study is to investigate the effect of WPS on several cardiovascular biological markers that may cause atherosclerosis in mice. The study also conducted to study the temporal effects of WPS on the atherosclerotic biomarkers upon short (2 weeks) and long‐term (8 weeks) exposures. Mice were exposed to WPS and heart homogenates were analyzed to elucidate the effects of WPS on matrix metalloproteinase (MMPs), endothelin‐1 (ET‐1) and, myeloperoxidase (MPO). Following protein estimation, enzyme‐linked immunosorbent assays were done to measure the levels of MMPs (isoforms 1, 3, and 9), MPO, and ET‐1 protein. Our data showed that acute exposure to WPS significantly enhances the levels of MMP‐3, MMP‐9, and MPO expressions (p<0.05) compared to their corresponding control. However, the body was capable to compromise the levels for such parameters following continuous exposure for 8 weeks (p>0.05). Additionally, we showed that the level of ET‐1 was significantly higher upon chronic exposure to WPS compared to both control and acute exposure groups (p<0.05). In conclusion, WPS has a significant negative effect on cardiac health and the enhancement of the atherosclerotic biomarkers (MMP‐3 and 9, MPO, and ET‐1) might represent an early scavenger of compensatory efforts to maintain cardiac function after WP exposure.

Support or Funding Information

This project was supported by a grant from the Deanship of Research at Jordan University of Science and Technology (grant number 2015/249 to AR).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deficiency of Sirtuin 3 Accentuates Angiotensin II‐Induced Arterial/Myocardial Stiffness and Hypertension
Authors: Chen Jian‐Xiong , Zeng Heng
Published: June 2021

Hypertension is prevalent disease worldwide and is considered as the key factor for the development of heart failure with preserved ejection fraction (HFpEF). Arterial stiffness is the center feature of hypertension and has significant impact upon disease etiology and outcomes. Sirtuin 3 (SIRT3) emerged as a protein of particular interest to the aging and cardiovascular disease in humans. Our previous study has shown that deletion of SIRT3 resulted in impairment of coronary flow reserve (CFR) and diastolic dysfunction in mice. Little is known about role of SIRT3 in angiotensin II‐induced arterial stiffness and hypertension. In present study, wild type (WT) mice and SIRT3KO mice were infusion with Ang II (1000 ng/kg/min) for 4 weeks, blood pressure, pulse‐wave velocity (PWV), CFR and diastolic function (IVRT and MPI) were measured before and after Ang II treatment. Aorta and heart were collected and fibrosis were measured by Masson staining and fibroblast specific protein‐1 (FSP‐1) immunostaining. Our results revealed that Ang II infusion led to a significant increase in PWV, whereas knockout of SIRT3 further enhanced Ang II‐induced elevation of PWV. This was accompanied by increases in systolic, diastolic pressure and MAP in SIRT3KO mice + Ang II as compared to WT mice + Ang II. Knockout of SIRT3 further reduced CFR and increased IVRT and MPI in mice. Histologic and immunostaining studies revealed that Ang II‐induced aortic thickness and perivascular fibrosis were significantly increased in SIRT3KO mice compared to WT mice. Similarly, there were significant increase in Ang II‐induced fibrosis area and higher number of FSP‐1+ in the heart of SIRT3KO mice than WT mice. In addition, the expression of PDGF‐β was higher in the aorta and heart of SIRT3KO mice than WT mice. Double staining PDGF‐β with FSP‐1 showed an increased number of PDGF‐β+/FSP‐1+. Using NG2 pericyte tracing reporter NG2DsRedBAC mice, we further confirmed that Ang‐II‐induced NG2 pericyte recruitment was significantly increased in the SIRT3 KO mice, suggesting deletion of SIRT3 promotes Ang‐II‐induced pericyte recruitment which may differentiate into fibrosis and contribute to increased arterial and myocardial stiffness. Our study demonstrated that deletion of SIRT3 exacerbated Ang II‐induced arterial stiffness, hypertension and diastolic dysfunction. Deletion of SIRT3 increased arteries and myocardial stiffness via enhancing pericyte recruitment and pericytes‐to‐fibroblast transition.

Support or Funding Information

This work was supported by R01HL102042‐06

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Yerba Mate (Ilex paraguariensis) and Dimethyl Fumarate Can Improve Metabolic Syndrome Condition Induced by High Fat Diet in Mice
Authors: Valenca Samuel Santos , Valenca Helber Maia , Lanzetti Manuella
Published: June 2021

Introduction

Metabolic syndrome (MS) is a combination of clinical signs and symptoms that occur together and directly contribute to the risk of developing cardiovascular disease and diabetes. Some studies suggest that the prevalence of MS in the US is 32% of the population, while in Brazil this level reaches 23%. The criteria for the classification of MS are diverse, but according to the WHO, the presence of diabetes or reduced glucose tolerance or insulin resistance, associated with at least two of the following conditions: elevation of blood pressure, dyslipidemia and obesity are considered. MS confers a two‐fold increased risk for developing cardiovascular disease, two to four times the risk of stroke, three to four times the risk of acute myocardial infarction. Our hypothesis is that toasted yerba mate and dimethyl fumarate have a therapeutic effect on the metabolic syndrome in mice.

Methods

Metabolic syndrome was induced by hyperlipidic diet (60% of total calorie) in C57BL / 6 mice for 12 weeks, and treatment was performed concomitantly with yerba mate (2 mg / g) and dimethyl fumarate (0.24 mg / g) diluted in water, to which the animals had free access. The groups were separated as follows: control group (standard diet and water), HFD group (hyperlipidic diet and water), HFD + Mate group (hyperlipidic diet and mat herb tea) and HFD + DMF group (hyperlipidic diet and dimethyl fumarate).

Results

It was observed that the HFD + Mate group obtained an increase in body mass in the same way as the HFD group, also increasing triglyceride levels, but maintained normal HDL levels. The HFD + DMF group, however, remained with body mass equal to the control group, maintaining normal levels of HDL and triglycerides. Both the HFD + Mate group and the HFD + DMF group remained at optimal glycemic levels, including glucose tolerance and insulin resistance tests. Histological analysis of the liver showed hepatic steatosis in the HFD group, but not in the HFD + Mate and HFD + DMF groups.

Conclusion

DMF was effective to prevent metabolic syndrome, once the hallmarks glucose, body weight, triglycerides and HDL cholesterol were kept similar to control group. DMF also protected against hepatic steatosis. About mate tea, the effect was more modest than DMF, once only HDL cholesterol and glucose were protected, as well as hepatic steatosis was avoided. These data suggest DMF can modulate satiation preventing the syndrome metabolic development.

Support or Funding Information

Brazilian Research Agencies ‐ CAPES, CNPq and FAPERJ

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pre‐ and Postconditioning Effects of Forced Treadmill Running on Gastric Injury Induced by Indomethacin in Rats
Authors: Filaretova Ludmila , Komkova Olga , Punin Yuriy , Yarushkina Natalia
Published: June 2021

The preconditioning effect of a mild stressor can reduce ulcerogenic effect of a severe stressor on the gastric mucosa. Our previous findings suggest that glucocorticoids produced in response to stress contribute to the gastroprotective effect of stress preconditioning and promote a healing of gastric injury. In this study, we tested if forced treadmill running can attenuate gastric injury induced by indomethacin (IM) in rats. IM administration (35 mg/kg, sc) in fasting (24 h) rats caused formation of gastric erosions (4 h after injection) that then gradually healed over the next 48 hours. Rats were subjected forced treadmill running (30 min) 1 h before IM administration (preconditioning effect) or 4 h after IM administration when gastric erosions have been already developed (postconditioning effect). Gastric mucosa was examined for injury 4 h (formation of gastric erosions) and 24 h (healing of gastric erosions) after IM administration. Treadmill was set up horizontally (15 m/min), run was stimulated by shocks 0.2 mA. Control animals were at rest and they were also injected by IM. Corticosterone plasma levels and somatic pain sensitivity (tail flick test) were measured. Forced treadmill running resulted in an increase of tail flick latencies (analgesic effect) which was accompanied by an increase in plasma corticosterone levels. The onset of forced treadmill running 1 h before IM administration increased the plasma corticosterone levels, normalized somatic pain sensitivity and attenuated IM‐induced gastric injury 4 h after IM administration. The onset of forced treadmill running after formation of gastric injury resulted in a decrease of gastric injury 24 h after IM injection comparing with control rats. Thus, forced treadmill running can attenuate gastric injury formation induced by indomethacin (IM) and promote a healing of IM‐induced injury in rats.

Support or Funding Information

The study was supported by grant of Russian Science Foundation (RSF) N 19‐15‐00430.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In Vitro Studies on the Mechanism of CFTR Inhibition by a Fungus‐derived Compound and Its Potential Anti‐diarrheal Application
Authors: Akrimajirachoote Nattaphong , Satitsri Saravut , Rukachaisirikul Vatcharin , Muanprasat Chatchai
Published: June 2021

Secretory diarrheas caused by enterotoxin‐producing bacteria are a major global health problem. Overactivation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels play an important role in the pathogenesis of secretory diarrheas. Preliminary results in our laboratory found that the fungus‐derived N453 inhibited CFTR‐mediated Cl− secretion with a half maximal inhibitory concentration (IC50) of ̴ 2 μM in intestinal epithelial (T84) cells. This study aimed to explore its underlying mechanisms and to demonstrate its anti‐diarrheal application. As analyzed by apical Cl− current measurement, pretreatment with phosphodiesterase and multi‐drug resistance protein 4 inhibitors did not affect the inhibitory effect of N453 on cAMP‐induced Cl− secretion T84 cells. In addition, N453 inhibited Cl− secretion induced by CPT‐cAMP (non‐hydrolyzable cAMP). These results suggest that CFTR inhibition by N453 does not involve alteration of intracellular cAMP levels. Moreover, N453 inhibited CFTR‐mediated Cl− transport induced by genistein (direct CFTR activator), suggesting that N453 might directly inhibit CFTR activity. Pretreatment with inhibitors of AMP‐activated protein kinase (AMPK) and protein phosphatase (PP), negative regulators of CFTR, did not affect the inhibitory effect of N453 on cAMP‐induced Cl− secretion, suggesting that CFTR inhibition by N453 is not resulted from activation of these negative regulators. Interestingly, N453 inhibited Cl− secretion induced by cholera and heat‐stable enterotoxins. Collectively, N453 derived from fungi acts as CFTR inhibitors, which may be beneficial for the treatment of secretory diarrheas.

Support or Funding Information

This work was supported by the NSTDA Chair Professor grant (the Fourth Grant) of the Crown Property Bureau Foundation, Faculty of Science Mahidol University, Faculty of Graduate Studies Mahidol University and Kasetsart Veterinary Development Funds.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A truncation mutation in human SLC12A2 leads to abnormal goblet cell mucus secretion
Authors: Koumangoye Rainelli , Delpire Eric
Published: June 2021

The basolateral Na+‐K+‐2Cl− cotransporter (NKCC1) plays a critical role in maintaining the intracellular concentrations of Na+, K+, and Cl− ions, and cell volume. The cotransporter also plays a key role in mediating epithelial fluid secretion. In previous studies, we have described the first known human SLC12A2 (NKCC1) mutation (Cold Spring Harbor Mol. Case Stud. 2(6): a001289, 2016). The now 16‐year‐old patient carries a carboxyl‐terminal truncation mutation (NKCC1‐DFX) in the transporter that leads to complex clinical features including complete gastrointestinal and bladder failure, endocrine insufficiencies, small intestinal dysmotility, seizure‐like episodes, and multi‐organ failure. The 11 bp deletion leads to truncation of the cytosolic COOH‐terminal domain of the cotransporter, making it non‐functional. In polarized epithelial MDCK cells and a mouse model that recapitulates the patient mutation we demonstrated that expression of the truncated Na‐K‐2Cl cotransporter causes mistargeting of the mutant and wild‐type cotransporter to the apical membrane of Cl− secreting epithelia and affects proper lumen formation in MDCK cysts (Am. J. Physiol. Cell Physiol. 315: C258–C276, 2018). Recently due to chronic infections, the patient's colon was surgically resected and lately, the patient has been battling chronic yeast infections in the small intestine. To investigate whether NKCC1‐DFX affect the integrity and function of the gut epithelia, we used MDCK cells expressing the mutant transporter and a mouse model recapitulating the patient mutation. We show that expression of the NKCC1‐DFX mutant impairs tight junction integrity, as indicated by a decrease in trans‐epithelial electrical resistance and inability of MDCK‐I cells expressing NKCC1‐DFX to re‐establish barrier function after a calcium‐switch challenge. Compared to wild‐type mice, NKCC1‐DFX have increased intestinal transit time and displayed an increase in unhealthy mitochondria characterize by dense inclusion bodies. Relative to wild‐type mice, NKCC1‐DFX mice showed decreased expression of Claudin‐2, a tight junction protein involved in paracellular Na+ and water transport. NKCC1‐DFX expression causes defective goblet cells mucus granules exocytosis leading to secretion of intact granules in the lumen of the large intestine. In addition, NKCC1‐DFX colon submucosal glands secrete mucus that remained attached to the epithelium. Taken together, these data indicate that NKCC1‐DFX likely impairs gut barrier function by affecting epithelia tight junction integrity and mucus secretion.

Support or Funding Information

Supported by NIH grant GM118944.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcineurin Inhibition Impairs the Function of Neuronal Potassium‐Chloride Cotransporter 2
Authors: Zessin Michael , Strauss Ulf , Deisz Rudolf , Borschewski Aljona , Schindler Maximilian , Endlich Nicole , Loyola Sebastian , Pleinis John , Rodan Aylin , Giesecke Torsten , McCormick James , Mrowka Ralf , Mutig Kerim
Published: June 2021

Calcineurin inhibitors (CnI) such as Cyclosporin A (CsA) are instrumental for immunosuppression after organ transplantation but may cause serious neurologic side effects, including seizures.

Neuronal excitability depends on intact Cl− homeostasis. Generation of hyperpolarizing synaptic responses to GABA and glycine requires a Cl− gradient across the cell membrane with a low [Cl−]i, mainly established by K+‐Cl− cotransporter 2 (KCC2) and to a lesser extent Na+‐K+‐Clcotransporter 1 (NKCC1); both are cation‐coupled chloride cotransporters (CCCs). Calcineurin has been implicated in the regulation of CCCs, whereas impaired CCC function is a well‐known condition in human pharmacoresistant epilepsy. Therefore, we hypothesized that CsA affects KCC2 or NKCC1 functions, thus causing neuronal hyperexcitability.

In ex vivo intracellular recordings with sharp microelectrodes, Wistar rat pyramidal neurons in neocortical slices responded to CsA (5 μM for 1h) with a less negative GABAA reversal potential (+7.2 mV) and prolonged Cl− extrusion time after iontophoretic Cl− loading (+3.9 s). 2‐photon fluorescence lifetime imaging in presence of Cl− sensitive dye MQAE showed an increased [Cl−]i in layer V neurons (+6.9 mM), suggesting reduced KCC2 activity. Co‐immunoprecipitation studies in rodent brain tissue suggested interactions of calcineurin Aβ (CnAβ) with KCC2 and NKCC1.

In vivo, CsA administration to rats (5–25 mg/kg i.p.) enhanced levels of inhibitory tyrosine KCC2 phosphorylation at short term (4h; +172%) and reduced levels of activating S940‐KCC2 phosphorylation in the long term (14d; −61%). Reduced phospho‐S940‐KCC2 levels were observed in further models of calcineurin inhibition such as genetic CnAβ‐deficiency in mice (−78%) or CsA treatment of zebrafish larvae (10 μM in water: −54% after 24h). In contrast, NKCC1 and its activating kinase SPAK were stimulated upon calcineurin inhibition in rodents. Similar data were obtained in Drosophila melanogaster.

Our results provide evidence that CnI attenuate KCC2 function but may stimulate NKCC1 leading to elevated [Cl−]i. These effects may increase neuronal excitability and contribute to CnI neurological adverse effects. The benefits of KCC2 activators or SPAK inhibitors for enhancing chloride extrusion in patients experiencing CnI neurotoxicity deserve further characterization.

Support or Funding Information

This study was supported by Deutsche Forschergemeinschaft.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Adipogenesis by α1 Na+/K+‐ATPase via Its Conserved Caveolin Binding Motif
Authors: Huang Minqi , Cai Liquan , Pierre Sandrine , Xie Zijian
Published: June 2021

In addition to its role as an ion pump, Na/K‐ATPase (NKA) α1 isoform forms a signal receptor complex with the non‐receptor tyrosine kinase Src and the scaffolding protein caveolin‐1. Stimulation of this receptor leads to an activation of Src/ERK cascade and an increase in generation of reactive oxygen species (ROS). Pharmacological inhibition of the NKA‐mediated signal transduction has been shown to inhibit adipogenesis and high fat diet‐induced obesity. However, there is no genetic evidence supporting a role of α1 NKA in the regulation of adipogenesis. To address this issue in a model with relevance to human phtsiology, we employed CRISPR/Cas9 system to mutate a conserved caveolin‐binding motif (F97A and F100A) in the gene coding for α1 NKA (ATP1A1), and measured the effects of this loss‐of‐function mutation on pathways relevant to adipogenesis during the differentiation of human induced pluripotent stem cells (iPSC) to adipocytes. We found that the CBM mutant human iPSC are capable of differentiation into adipocytes. However, Oil Red O staining indicated a significant reduction in lipid accumulation during adipocyte maturation in the mutant cells. This observation was confirmed by electron microscopy imaging and by changes in the expression of adipocyte marker genes. Mechanistically, the loss of CBM in ATP1A1 resulted in alteration in mitochondrial structure and function observed by electron microscopy imaging, mitochondrial ROS generation, membrane potential, biogenesis, and marker genes expression by RT‐qPCR. This was further validated by metabolic profiling by Seahorse Extracellular Flux Analysis and RNA sequencing followed by KEGG enrichment analysis of metabolic pathways. Taken together, these results indicate an important role of NKA‐mediated signal transduction in adipogenesis through a regulation of mitochondrial structure and function. Thus, we suggest that NKA‐mediated signal transduction may serve as a new target for developing therapeutic avenues in the management of obesity and metabolic syndromes.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Na+/K+‐ATPase α1 and Cardiac Resilience
Authors: Marck Pauline V. , Xu Yunhui , King Cierra , Perdoncin Maddie , Yan Yanling , Blanco Gustavo , Cai Liquan , Lingrel Jerry , Xie Zijian , Pierre Sandrine V.
Published: June 2021

Systemic levels of endogenous cardiotonic steroids (CTS) vary in response to physiological and pathophysiological stresses such as exercise, pregnancy, hypertension, and heart failure. Ample experimental evidence has shown that exposure to CTS modulates cardiac Na/K‐ATPase (NKA) α1 ion‐pumping and non‐ion‐pumping functions, which profoundly affects cardiac contractility and structure. However, the physiological role of CTS as a group of hormones with distinct but related modulatory effects on cardiovascular equipoise and dysfunction remains poorly understood. We used 2 genetically engineered mouse models to test the hypothesis that the endogenous CTS/NKA α1 interaction confers cardiac resilience during a sustained hemodynamic challenge.

Wild type mice expressing the relatively CTS‐insensitive NKA α1 and the CTS‐sensitive NKA α2 (α1r/rα2s/s), mice expressing NKA α1 mutated to CTS‐sensitivity (α1s/sα2s/s), and mice with cardiomyocyte‐specific NKA α1 knockout (α1−/−α2s/s) were treated with a pressor dose of Angiotensin II (Ang II) of 1.5 mg.kg−1. d−1 for 14 days via osmotic minipumps to induce adverse cardiac remodeling.

All mice were born with the expected Mendelian frequency and survived to adulthood. In the α1s/sα2s/s mouse, total cardiac expression of NKA α1 and α2 assessed by western blotting and ouabain‐sensitive ATPase activity were unchanged, and the expected increased affinity for the CTS ouabain was detected. NKA expression and activity in the α1−/−α2s/s heart was consistent with a nearly complete ablation of NKA α1, partially compensated by an upregulation of NKA α2. Ang II or vehicle treatment was administered to 3 month‐old males (n=6–8/group), and cardiac remodeling was assessed by gravimetry, histology, and qPCR. In contrast to wild‐type α1r/rα2s/s, the Ang II treatment did not induce any significant cardiac hypertrophy or fibrosis in α1s/sα2s/s hearts. Conversely, the Ang II treatment was lethal in α1−/−α2s/s mice, with a 70% mortality rate at 8 days. A shorter (7 days) Ang II treatment revealed massive cardiac fibrosis (50% increase compared to α1r/rα2s/s). Taken together, these results support a physiological role of endogenous CTS in cardiac resilience through an interaction with the signaling cardiac NKA α1 isoform.

Support or Funding Information

N/A

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylations in the Termini of Mitochondrial Calcium Uniporter Regulate Mitochondrial Calcium Uptake
Authors: O‐Uchi Jin , Cao Jessica L , Adaniya Stephanie M , Jhun Bong Sook , Sheu Shey Shing
Published: June 2021

Mitochondrial Ca2+ (mtCa2+) uptake via the mtCa2+ uniporter (MCU) mediates cell survival and death. Our group previously showed that activation and mitochondrial translocation of proline‐rich tyrosine kinase 2 (Pyk2), tyrosine phosphorylation (P‐Tyr) of MCU, and enhancement of mtCa2+ uptake concomitantly occur in cardiomyocytes under Gq protein–coupled receptor (GqPCR) stimulation, which activates apoptotic myocyte death. However, it is still unclear how Pyk2 access to MCU structure, phosphorylate MCU, and modulate its channel function as well as mitochondrial Ca2+ uptake profile. Here, we determined Pyk2‐specific P‐Tyr site(s) in MCU and investigated the detailed molecular mechanism underling the regulation of mtCa2+ uptake via P‐Tyr of MCU. Through computational prediction programs for phosphorylation sites, we identified three Tyr residues (Y158, Y289, and Y317) in human MCU as candidates for general PTK phosphorylation sites, which are highly conserved across all eukaryotic species. P‐Tyr levels at these candidate sites were biochemically determined before and after the stimulation of a GqPCR in HEK293T cells stably overexpressing wild type (WT)‐ or dephosphomimetic mutants of MCU (MCU‐YFs). Only two Tyr sites (Y517F at the N‐ and Y317 C‐termini of MCU) exhibited increased P‐Tyr levels in response to GqPCR stimulation. Overexpression of MCU‐Y317F failed to increase mtCa2+ uptake in response to cytosolic Ca2+ elevation, although the overexpression of WT‐ and the other two MCU‐YFs significantly augmented mtCa2+ uptake compared to non‐transfected cells. Furthermore, we also found that MCU‐Y317F‐flag exhibits less association with WT‐MCU‐GFP compared to those observed in WT‐MCU‐flag, indicating reduced subunit oligomerization by this point mutation. Finally, we demonstrated that increased in P‐Tyr of MCU levels occurs in the ventricle tissues from transgenic (TG) mice with cardiac‐specific overexpression of a constitutively active Gαq protein (Gαq‐Q209L TG mice) compared to those from WT, concomitant with higher Pyk2 activation and apoptotic signaling. In summary, we conclude that MCU contains Pyk2‐specific phosphorylation site(s) and Pyk2‐dependent P‐Tyr of MCU at these site(s) increases mitochondrial Ca2+ uptake possibly via enhanced subunit oligomerization.

Support or Funding Information

J.O.‐U. was supported by NIH/NHLBI R01HL136757, AHA 16SDG27260248, Rhode Island Foundation #20164376 Medical Research Grant, and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award. J.L.C. and S.M.A were supported by Undergraduate Teaching & Research Award (UTRA) from Brown University. B.S.J was supported by 18CDA34110091. S.S.S was supported by NIH/NHLBI R01HL093671 and R01HL122124.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.