bluuebunny/crossref_metadata_2025 huggingface dataset:
Metformin Has Direct Protective Effect on Human Cardiac Mitochondria
Authors: Emelyanova Larisa , Rudic Theodore , Ross Gracious R. , Rizvi Farhan , Kress David C. , Jahangir Arshad
Published: June 2021

Objective

Metformin (Metf), an oral antihyperglycemic agent, is widely used to treat type 2 diabetes mellitus. The primary action of the drug is to lower hepatic glucose synthesis and enhance peripheral glucose uptake via activation of the insulin receptor. It also exerts a cardioprotective effect independent of its glucose lowering action; however, mechanisms underlying cardioprotection in the human heart remain poorly defined. We hypothesize that the cardioprotective effect of Metf is associated with mild inhibition of mitochondrial oxidative phosphorylation (OXPHOS) and reduction of oxidative stress that protect mitochondria against permeability transition pore (PTP) opening.

Methods

Left atrial appendage tissue was collected from middle‐aged and elderly patients (n=21) undergoing open‐heart surgery. Mitochondria were isolated from fresh tissue using differential centrifugation and exposed to different concentrations of Metf (0–10 mM). The functional activity of mitochondrial OXPHOS complexes (CI‐V) was measured spectrophotometrically using specific OXPHOS inhibitors, electron donors, and acceptors. Superoxide production was measured by monitoring changes in fluorescence intensity of dihydroethidium (DHE) in the absence and presence of antimycin A (AA, 5 μM). The sensitivity of mitochondria toward PTP opening was assessed by exposing isolated mitochondria to 10 μM of Ca2+ pulses and monitoring abrupt mitochondrial Ca2+ release (Fluo‐5N fluorescence) and swelling of the matrix (decrease in absorbance). The study was approved by the Aurora Institutional Review Board and adhered to the Health Insurance Portability and Accountability Act and Aurora Health Care patient privacy and security guidelines. The study followed the principles of the Declaration of Helsinki.

Results

Metf had a dose‐dependent mild inhibitory effect on the activity of CI, CIV, and CV without affecting CII and CIII (Figure A). This was associated with reduction of superoxide production before and after exposure of the mitochondria to AA (Figure B). Metf in a dose‐dependent manner also attenuated Ca2+‐induced mPTP opening (Figure C) with delayed swelling of mitochondrial matrix (Figure D).

Conclusion

Metf improves cardioprotection by increasing tolerance to stress‐induced PTP opening due to its mild inhibitory effect on the OXPHOS and reduction of superoxide production. This can be a beneficial alternative to patients with impaired endogenous cardioprotective responses.

Support or Funding Information

N 570‐3657, Cardiac Research Award, Aurora Health Care

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding the Role of Polar Residues on the c‐Ring of E. coli F1Fo ATP Synthase
Authors: Founds Michael , Shrestha Rashmi , Steed Ryan
Published: June 2021

F1Fo ATP synthases catalyze the synthesis of adenosine triphosphate (ATP), the universal biological energy carrier, and are thus integral to life. The membrane‐embedded Fo sector utilizes the proton electrochemical gradient to generate rotation in the rotor subunits, which drives ATP synthesis in the F1 sector. In addition to ATP synthesis, F1Fo can also hydrolyze ATP to pump protons to create an electrochemical gradient. Recent structural models of ATP synthase from cryo‐electron microscopy have revealed the architecture of the rotor‐stator interface; however, the intricacies of the rotation mechanism are not fully understood. Previous research has identified specific residues of the c subunit of E. coli F1Fo, including Arg50 and Thr51 on the cytoplasmic end of transmembrane helix 2, that may be necessary for the H+ translocation mechanism. Mutation of Arg50 or Thr51 to Cys abolished ATP‐driven H+ pumping and blocked H+‐permeability. Interestingly, these mutants were able to grow in succinate minimal medium, suggesting that ATP synthesis activity was unaffected. Arg50 is part of a proposed charge interaction between subunits a and c, and the polar Thr51 may also participate in this interaction. Therefore, we studied the importance of positive charge and polarity at positions 50 and 51 using chemical and genetic modifications. Chemically appending positive charge onto Cys50 with methanethiosulfonate did not restore ATP‐driven H+ pumping activity. However, mutation of Arg50 to Lys or His supported activity. Mutation of Thr51 to Ser or Asp also supported H+ pumping activity. These studies provide insight into the role of polar residues on the surface of the c‐ring and their contribution to the H+‐driven rotational mechanism of F1Fo ATP synthase.

Support or Funding Information

Supported by the North Carolina GlaxoSmithKline Foundation and the University of North Carolina Asheville Undergraduate Research Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of Carnitine Palmitoyltransferase‐2 in skeletal muscle results in muscle remodeling and tissue‐specific sensitivity to insulin
Authors: Pereyra Andrea S , Ellis Jessica
Published: June 2021

Dysregulated mitochondrial fatty acid oxidation in skeletal muscle occurs with overfeeding and obesity and is implicated in the development of insulin resistance. To assess the effects of eliminating mitochondrial long‐chain fatty acid oxidation on insulin sensitivity we generated mice with a muscle‐specific deficiency in Carnitine Palmitoyltransferase‐2 (Cpt2Sk−/− mice), an irreplaceable enzyme required for mitochondrial oxidation of long‐chain fatty acids.

Cpt2 Sk−/− mice have lower basal glycaemia, hypo‐phosphorylation of Akt in liver and white adipose tissue with preserved Akt‐phosphorylation in muscle, and moderately increased sensitivity to glucose and insulin tolerance tests. Acute insulin stimulation increased Akt phosphorylation to similar levels in control and Cpt2 Sk−/− liver, adipose, and muscle. Thus, the induction in Akt phosphorylation from no‐insulin to insulin‐stimulated is heightened in Cpt2 Sk−/− liver and white adipose, but not in muscle.

Cpt2 Sk−/− muscles are free of oxidative stress and do not accumulate lipotoxic ceramides or triacylglycerol. However, Cpt2 Sk−/− muscles, compared to controls, accumulate up to 500‐fold more long‐chain acylcarnitines. Also, Cpt2 Sk−/− muscles undergo remodeling by increasing mitochondria, fiber‐type switching, and alteration in myocyte nuclei size, number, and distribution. Taken together these data suggest that loss of muscle Cpt2 causes adaptive muscle remodeling and improves insulin sensitivity of liver and adipose, but not muscle.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of programming on the hepatic epigenome by maternal low‐fat diet persist in adult offspring fed an obesogenic diet
Authors: Moody Laura , Shao Justin , Xu Guanying Bianca , Chen Hong , Pan Yuan‐Xiang
Published: June 2021

Obesity and metabolic disease present a risk to long‐term health outcomes. Epigenetic marks such as DNA methylation are established during early life and have effects on later life through developmental programming. In the present study, we examined whether maternal diet alters DNA methylation and whether such modifications persist after an obesogenic postnatal dietary challenge. During gestation and lactation, male Sprague Dawley rats were exposed to either a high‐fat diet (HF; n = 10) or low‐fat diet (LF; n = 10). After weaning, all animals were fed the HF diet until postnatal week 12. There were no differences observed in food intake or body weight between the two groups of offspring animals. Hepatic DNA methylation was quantified using both Methylated DNA Immunoprecipitation sequencing (MeDIP‐seq) and Methylation‐sensitive Restriction Enzyme sequencing (MRE‐seq). Overall, 1,419 differentially methylated regions (DMRs) were identified. DMRs tended to be located in CpG shores, as opposed to CpG islands, and were enriched for genes involved in metabolism and cancer. Gene expression was measured for 31 candidate genes in these pathways. Among these, Map3k5 and Igf1r were confirmed to be differentially expressed. When the functional relevance of intergenic DMRs was quantified using chromatin contact data, we discovered that conserved DMRs were topologically associated with metabolism genes, which was associated with differential expression of Adh5, Enox1, and Pik3c3. Overall, although maternal dietary fat was unable to reverse offspring weight gain in response to postnatal obesogenic diet, early life diet did program the hepatic methylome. Epigenetic alterations on the hepatic methylome occur primarily in metabolic and cancer pathways and are associated with altered gene expression. Physiological consequences of these epigenetic marks will require future investigations for a better understanding of epigenomic programming.

Support or Funding Information

This project was supported by UIUC Research Board grant #12192, the USDA Cooperative State Research, Education and Extension Service, Hatch project numbers # ILLU‐698‐923 and ILLU‐698‐369, ILLU‐698‐391, and the Data Purchase Program from the University of Illinois Library.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Silk Peptide from Bombyx mori Promotes Browning and Inhibits Fat Accumulation in Subcutaneous White Adipocytes
Authors: Lee Kippeum , Seo Young‐Jin , Chei Sungwoo , Song Ji‐Hyeon , Jin Heegu , Oh Jae‐Hoon , Oh Hyun‐Ji , Lee Boo‐Yong
Published: June 2021

Obesity is characterized as an excessive accumulation of fat in subcutaneous tissues. It is very influential for various diseases including cardiovascular disorders, type 2 diabetes. Adipose tissue is consisted of two types of cell, which are WATs and BATs. WAT principally stores lipid in the form of TGs, thereby acting as a repository of surplus energy in the body. On the other hands, BAT utilizes stored lipid for nonshivering thermogenesis, involving β‐oxidation and uncoupling of oxidative phosphorylation in mitochondria.

Silk peptide and extracts of silkworm have diverse pharmacological activities, inducing hyperglycemia, hyperlipidemia, and anti‐oxidant effect. As well as, silk peptide decreases lipid metabolism and body fat accumulation. However adipose browning mechanism as an one of anti‐obesity effect has not been elucidated.

In this research, silk peptides increases UCP1 expression and mitochondrial biogenesis in subcutaneous white adipose cells isolated from ICR mice. As well as, silk peptides induces BAT‐like differentiation of adipocytes and thermogenic activation, a process that also involves the upregulation of FAO. In conclusion, we suggest that silk peptides‐induced UCP1 activation in adipocytes have prospective therapeutic value for obesity suppression and associated metabolic disease.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protective Role of Lycium Barbarum Polysaccharides and C‐Phycocyanin against Ethanol‐Induced Gastric Ulcer
Authors: Lian Yu Zhi , Ni Po‐Yuan , Chao Jane C‐J
Published: June 2021

Gastric ulcer is a major complication of gastrointestinal disease. This study investigated the gastroprotective effect of Lycium barbarum polysaccharids (LBPs) and C‐phycoyanin (C‐PC) on ethanol‐induced gastric ulcer in rats. Male Sprague‐Dawley rats were divided into 5 groups: normal, ulcer, ulcer treated with 100 mg/kg BW LBPs, ulcer treated with 50 mg/kg BW C‐PC, and ulcer treated with a mixture of 50 mg/kg BW LBPs and 25 mg/kg BW C‐PC. The treatments were given daily by gavage from week 1 to 5, and gastric ulcer was induced by 50% ethanol via gavage every other day from week 2 to 5. Ulcer area, serum hydroxyproline ‐ a potential biomarker for gastric ulcer, serum lipid peroxide ‐ malondialdehyde (MDA), gastric inflammatory markers ‐ interleukin‐1β (IL‐1β), myeloperoxidase (MPO) activity, and intercellular adhesion molecule‐1 (ICAM‐1), and gastric cytoprotective factors ‐ prostaglandin E2 (PGE2) and heat shock protein 70 (HSP70) were measured. The ulcer group had significantly increased ulcer area, serum MDA, gastric IL‐1β, MPO activity, and ICAM‐1, but decreased serum hydroxyproline and gastric PGE2 levels. Pretreatment with LBPs or C‐PC significantly attenuated the severity of ethanol‐induced gastric mucosal damage, decreased serum MDA, gastric IL‐1β, MPO activity, and ICAM‐1, and increased serum hydroxyproline and gastric HSP70 levels. Pretreatment with a mixture of LBPs and C‐PC had similar effects with pretreatment with LBPs or C‐PC, however, a mixture of LBPs and C‐PC significantly decreased gastric ICAM‐1 levels compared with the ulcer and LBPs‐treated groups, but did not change serum hydroxyproline, gastric MPO activity, and PGE2 levels. Therefore, LBPs and/or C‐PC exert gastroprotective effects on ethanol‐induced gastric ulcer through suppression of lipid peroxidation and gastric inflammation and improvement of cytoprotective mechanisms.

Support or Funding Information

The study is supported by the Ministry of Science and Technology, Taiwan (grant no. MOST105‐2320‐B‐038‐036‐MY3).

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 Catalytic Activity of the PAH1‐encoded Phosphatidate Phosphatase is Required for Lipid Biosynthesis in the Oleaginous Yarrowia lipolytica
Authors: Carmon Taylor , Ukey Rahul , Fakas Stylianos
Published: June 2021

The PAH1 gene encodes phosphatidate phosphatase (PAP) which catalyzes the conversion of phosphatidate (PA) to diacylglycerol used for the synthesis of triacylglycerol (TAG). Previous research in our lab showed that the lack of Pah1 (i.e. pah1Δ) in Y. lipolytica causes a significant decrease in cellular lipid content and TAG levels, while the phospholipid levels are increased. However, it is unclear whether this phenotype is due to the loss of the catalytic activity of Pah1 or due to some other function of the protein (e.g. transcriptional regulation). The objective of this work was to confirm that the loss of the catalytic activity of Pah1 is responsible for the phenotypes presented by pah1Δ cells.

Yarrowia lipolytica pah1Δ cells expressing either wild‐type Pah1 fused with Green fluorescent protein (GFP) or a catalytically inactive Pah1 (i.e., D350E) fused with GFP were grown in media that favor lipid biosynthesis. PAP activity was measured following the release of water‐soluble 32Pi from chloroform‐soluble [32P] PA, while lipid profiles were analyzed by thin layer chromatography. The expression of the GFP fusions was confirmed by immunoblot analysis using antibodies directed against GFP. The results showed that the pah1Δ strain expressing an inactive Pah1 presented a decrease in TAG levels as well as overall lipid content and an increase in phospholipids compared to the pah1Δ strain expressing the wild‐type Pah1. These results showed that the catalytic activity of Pah1 is required for TAG biosynthesis in Y. lipolytica.

Support or Funding Information

This work was supported, in whole or in part, by National Institutes of Health Grant R15GM124602‐01 from the United States Public Health Service. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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 acyltransferase Gpc1 impacts PC molecular species and phenotypic outcomes
Authors: Nikiforov Alexiy , Skinner Brelan , Godshalk Maeve , Patton‐Vogt Jana
Published: June 2021

Phospholipids are major components of cellular membranes and their biochemical properties, such as fatty acid composition, help define the biophysical properties of membranes. Phosphatidylcholine (PC) is the most abundant phospholipid in cellular membranes and is synthesized primarily by two well defined metabolic pathways in Saccharomyces cerevisiae. Our laboratory recently characterized a novel glycerophosphocholine (GPC) acyltransferase, encoded by GPC1, that provides a third route for PC biosynthesis and is involved in the newly defined PC deacylation‐reacylation remodeling pathway (PC‐DRP). PC‐DRP begins with GPC, the product of complete PC deacylation by phospholipases of the B type. Gpc1 acylates GPC to form lysophosphatidylcholine (LysoPC). LysoPC can subsequently be converted to PC by Ale1. Previous results have shown that Gpc1 prefers the addition of saturated fatty acids during PC remodeling, which results in more saturated PC species and has the potential to influence the biophysical properties of the membrane. To better understand how GPC1 is regulated and to determine its physiological role in the cell, we assessed the effect of GPC1 dosage and temperature alterations on growth, expression, and PC molecular species. Preliminary data indicates that while increasing the temperature from 30°C to 37°C slows growth in a wild type strain, the gpc1Δ mutant is much less effected. As expected, overexpression of GPC1 further negatively impacts growth at 37°C. GPC1 message abundance, as measured by qRT‐PCR, is decreased at 37°C as compared to 30°C in a wild type strain. Other members of PC‐DRP, including ALE1 and PLB1 also display decreased expression upon elevated temperature. Mass spectrometry‐based analysis of the PC molecular species under these conditions are ongoing. Other stressors expected to impact membrane events, including inositol availability, are being examined. These experiments will provide insight into the physiological role of Gpc1.

Support or Funding Information

NIH R15 GM104876 (to JPV)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pannexin 1 and Pannexin 3 regulate body fat accumulation in mouse models of dietinduced obesity
Authors: Penuela Silvia , Lee Vanessa R , Barr Kevin , Kelly John J , Johnston Danielle , Brown Cody FC , Robb Kevin P , Sayedyahossein Samar , Huang Kenneth , Gros Robert , Flynn Lauren
Published: June 2021

Pannexins (Panx) are channel‐forming glycoproteins important in paracrine signaling and cellular development. Panx1 and Panx3 are expressed in adipocytes and adipose stromal cells. We discovered that mice globally lacking Panx1 (KO) have significantly greater total fat mass and reduced lean mass compared to wild type (WT) mice under a normal diet, while Panx3 KO mice have reduced total fat mass and weigh less than WT controls. Despite having higher fat content, Panx1 KO mice on a high fat diet exhibited no differences in weight gain but showed an increase in glucose and insulin levels compared to WT. However, metabolic cage data revealed that these Panx1 KO mice display significantly increased activity levels, higher ambulatory activity, and reduced sleep duration relative to their WT littermates on a high‐fat diet. To uncover the cellular mechanism responsible for the increased fat content in the Panx1 KO, we isolated primary cultures of adipose‐derived stromal cells (ASCs) from WT and KO fat pads. In WT ASCs we observed that Panx1 protein levels increase upon induction into an adipogenic lineage. ASCs isolated from Panx1 KO mice proliferate less but demonstrate enhanced adipogenic differentiation with increased intracellular lipid accumulation, glycerol‐3‐phosphate dehydrogenase (GPDH) enzyme activity, and adipokine secretion, as compared to WT ASCs. This was consistent with the increased adipocyte size (hypertrophy) and decreased adipocyte numbers observed in subcutaneous fat of the Panx1 KO mice compared to WT. Panx3 is also expressed in adipose stromal cells and Panx3 protein levels significantly increase in differentiated adipocytes. However, the effect of the Panx3 KO seems to be the opposite of the Panx1 KO, with decreased weight under a high fat diet and lower fat mass (even under a normal chow diet). We propose that Panx1 and Panx3 may play opposing roles in adipose stromal cells during the early stages of adipogenic proliferation and differentiation, regulating fat accumulation in vivo.

Support or Funding Information

Funded by the Canadian Institutes of Health Research (CIHR) grants to SP and LF

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Proteins Released from the Egg Cell Surface During Fertilization
Authors: Wiseman Emily , Carroll David
Published: June 2021

Multiple events occur at the interface between the sperm and egg membrane during fertilization, including sperm‐egg binding, species‐specific discrimination, membrane fusion, and egg activation. Each of these events is believed to rely upon discrete cell surface molecules. To date, only a handful of egg membrane proteins have been identified in any species. The process of fertilization as a whole is not well understood on a molecular level, from the initial sperm and egg binding, to the activation of early development. An excellent model organism to address these problems is the Pacific bat star, Patiria miniata, because they produce a large quantity of synchronized oocytes that are large, nearly transparent, and rigid enough to hold their shape despite removal of extracellular layers. Furthermore, we are learning that their genome is similar to humans with less complexity. Recent work suggests that proteases are necessary for successful fertilization, raising the possibility that fragments of egg membrane proteins could be released from the surface during fertilization. This could provide a mechanism for the identification of fertilization‐relevant membrane proteins. Using biotinylation and western blotting, we show that peptides ranging from 50kD to 250kD are being cleaved from the egg surface and released into the sea water within three minutes of introducing sperm. These peptides have been collected by affinity interaction against streptavidin conjugated mag‐sepharose beads and their identities are being determined by tandem mass spectrometry. Since a proteome has not yet been created for P. miniata, a recently developed transcriptome of the mature unfertilized egg (NCBI accession number: PRJNA398668) will be used to identify the peptides revealed by tandem mass spectrometry. Furthermore, this transcriptome is also being examined computationally with the purpose of identifying and grouping transcripts that may be integral to fertilization, as a whole, and with a specific focus on the egg cell surface. Understanding how fertilization takes place at the molecular level in starfish could eventually lead to understanding infertility issues, as well as offer new possible non‐hormonal prophylactics for use in humans.

Support or Funding Information

NIH R15

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
GlycoSense™: A Simple Alternative to Existing Methods for Glycosylation Detection and Monitoring
Authors: Woods Robert J. , Saunders Matthew J. , Yang Loretta
Published: June 2021

Lectenz® Bio has developed an innovative technology called GlycoSense™ that detects binding between glycans and glycan‐specific reagents that are conjugated to spectrally‐unique beads. By combining the individual bead‐based reagents into a multiplex array, a profile of key glycan features can be obtained in less than a minute on a basic cytometer. We illustrate the performance of the method using glycoprotein standards and demonstrate its utility by monitoring changes in glycosylation patterns upon treatment with glycoenzymes. The GlycoSense™ approach addresses the unmet need for a rapid and robust tool to analyze protein glycosylation. In addition to established carbohydrate‐detection reagents such as antibodies and lectins, this approach can employ the engineered proteins (known as Lectenz®), developed by Lectenz® Bio.

Support or Funding Information

NIH U01 CA221213, R43 GM123863

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanical Stress and Alteration of Cellular Morphology Influences O‐GlcNAc Modification
Authors: Nagy Tamas , Cser Annamária , Kolarovszki Béla , Miseta Attila , Frank Dorottya
Published: June 2021

Protein O‐linked N‐acetylglucosamine modification (O‐GlcNAc) is the result of a post‐translational modification that involves the reversible transfer of an N‐acetyl‐glucosamine molecule to Ser/Thr OH‐group of intracellular proteins. O‐GlcNAc modification is increasingly recognized as a key regulatory element, capable to modify the functionality and the life‐cycle of hundreds of proteins. O‐GlcNAc's participation in stress adaptation processes was evidenced after several environmental challenges such as heat shock, hypoxia or oxidative stress. However, intracellular O‐GlcNAc modification was not yet studied under mechanical stress, despite the fact that physical stress is a constant challenge for many cells in our body.

In our present work, we used human periodontal ligament (PDL) cells isolated from teeth extracted for orthodontic indications. PDL cells were cultivated under standard cell culture conditions and exposed to various amount of compression force for 12 hours.

We have analyzed cell survival by flow‐cytometry and protein O‐GlcNAc by Western‐blot. Since cytoskeletal filaments are essential to the structural integrity of the cells both in normal condition and under mechanical stress, we also studied the intracellular distribution of O‐GlcNAc in relation to actin, alpha‐tubulin, vimentin and the cellular shape. We have found that O‐GlcNAc elevated under moderate mechanical load (1.5 – 3 g/cm2) but did not changed significantly under higher load ( ≥ 7 g/cm2). Intracellular distribution of O‐GlcNAc in non‐loaded cell demonstrated an accumulation at several membrane protrusion sites which showed the most overlap with tubulin and no overlap at all with vimentin. Under mechanical stress, the cells retracted these protrusions and O‐GlcNAc distribution became more centralized as well.

In conclusion, we found that O‐GlcNAc regulation responds to mechanical challenges and our data suggest that O‐GlcNAc is coupled to cellular morphology.

Support or Funding Information

This work was funded by the Research Fund of the University of Pécs, Faculty of Medicine (grant No.: AOK‐KA 2/2015 (06.30)) and the EU founded Hungarian project GINOP 2.3.2. –15 (grant No.: 2.3.2. –15‐2016‐00050).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acute Exposure to E‐Cigarette Vapor Causes Changes in Apoptotic Pathways in the Lung
Authors: Heyob Kathryn M , Sugar Sophia S , Johnson Brooke , Mieth Saya , Pavlek Leeann R , Adelstein Jeremy , Schwieterman Neill A , Wold Loren E , Rogers Lynette
Published: June 2021

Background

Electronic cigarette usage is increasing at an alarming rate primarily because of reports implicating their use as safer than traditional cigarettes. However, little is known about the acute effects on lung injury and inflammation. These studies tested the hypothesis that acute exposure to e‐cigarette vapor would result in lung inflammation and injury.

Methods

C57Bl/6 mice were exposed to e‐cigarette vapor consisting of 10 mg/mL nicotine in a 50/50 mixture of propylene glycol/vegetable glycerin (PG/VG) or PG/VG alone for 6 hr/day, 5 days/week for either 1 or 2 weeks. Lungs were excised and snap‐frozen for analysis. Lung homogenates were analyzed by western blot for pAKT, pSTAT3, PECAM, BCL‐2, and BAX. RNA was isolated for measurement of MMP9, KC, and VEGF by RT‐PCR.

Results

pAKT protein expression was decreased (0.54 vs 1.0 relative density, p=0.02) and pSTAT3 was increased (1.7 vs 1.0 relative density, p=0.02) in the e‐cigarette exposed mice vs PG/VG exposed mice after 1 week of exposure. At 2 weeks, pSTAT3 remained increased (1.9 vs 1.0 relative density, p=0.01) and both BCL‐2 (2.5 vs 1.0 relative density, p=0.02) and BAX (1.7 vs 1.0 relative density, p<0.05) were increased in the exposed mice compared to mice exposed to PG/VG alone. No differences were observed in PECAM, MMP9, KC, or VEGF.

Conclusions

Acute exposure to e‐cigarette vapor caused changes in signaling molecules associated with apoptosis. While we did not observe differences in the inflammatory pathways tested, additional inflammatory and apoptotic markers may yield insights into the acute effects of e‐cigarette vapor exposure on the lung.

Support or Funding Information

NIH NHLBI R01 HL139348 02

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Involvement of Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166) in Pulmonary Fibrosis
Authors: Kim Mi Na , Hong Jung Yeon , Kwak Eun Ji , Kim Eun Gyul , Lee Yong Ju , Kim Kyung Won , Sohn Myung Hyun
Published: June 2021

Rationale

Idiopathic pulmonary fibrosis is a chronic, progressive lung disease characterized by fibroproliferative matrix molecule accumulation, collagen deposition and apoptosis, eventually resulting in pulmonary dysfunction and death. Activated leukocyte cell‐adhesion molecule (ALCAM; CD166) is a cell adhesion molecule that has been implicated in adhesive and migratory attribution including leukocyte homing and trafficking and the cancer metastasis. We investigated the involvement of ALCAM on development of bleomycin‐induced pulmonary fibrosis in murine model.

Methods

Bleomycin‐induced pulmonary fibrosis model was established with wild type and ALCAM knockout mice. Pulmonary fibrosis was also induced in TGF‐ β transgenic mice that conditionally overexpress TGF‐β upon doxycycline administration. Levels of ALCAM were measured in lung tissue and bronchial alveolar lavage fluid (BALF) of wild type mice and TGF‐ β transgenic mice. Histopathologic investigation, collagen deposition were evaluated.

Result

The protein and mRNA levels of ALCAM were decreased in the BALF and lung tissue of bleomycin‐treated wild type mice. Reduced ALCAM level was observed in BALF of doxycycline administered TGF‐β transgenic mice relative to those of control mice. ALCAM knockout mice showed aggravated lung fibrosis response compared with wild type mice upon bleomycin treatment.

Conclusion

ALCAM contributes to bleomycin‐induced pulmonary fibrosis associated with TGF‐β response.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of a Low Fat Diet versus a High Fat Diet on Lymphoid Cell Infiltration in a Mouse Model of Oral Cancer
Authors: Pitstick Lenore , Green Jacalyn M , Goral Joanna , Barakat Ameir , Meyer Alice , Pytynia Matthew , Ciancio Mae , Jham Bruno
Published: June 2021

Introduction

Risk factors for oral cancer include tobacco use, male sex, and possibly dietary fat. Exposure to 4‐nitroquinoline 1‐oxide (4NQO) in the drinking water causes oral cancer in mice and models the effects of tobacco use. This study was undertaken to investigate the effects of a high saturated fat (HF) or low fat (LF) diet and sex on presence of lymphoid cells in cancerous tongues. We hypothesized that inflammation, characterized by the increased presence of lymphoid cells, would accompany development of tumor lesions and that sex and diet would affect the levels of inflammation.

Methods

Male and female C57Bl/6 mice (36 each gender), 5 weeks old, were divided into a low fat (10kcal% fat; LF) or HF (60 kcal% fat) diet. Within each dietary group, mice were randomly assigned to one of 3 water treatment groups for 17 weeks: water alone (control); propylene glycol in water (1.25%; PG‐H2O); or 4NQO in PG‐H2O (50 mg/ml; 4NQO). After 17 weeks, all mice were given water alone for 6 more weeks. Tongues harvested from the euthanized animals were fixed in formalin and processed for histological examination. T cells (CD3+ cells), macrophages (CD68+ cells), and neutrophils (Ly6+ cells) were detected by immunohistochemistry, counted, and normalized to tissue area. Analysis was done using GraphPad Prism 7.0. All animal work was in compliance with the regulations of IACUC at Midwestern University, Downers Grove, IL.

Results

Oral cancer developed in all mice exposed to 4NQO and was absent in all controls. All three lymphoid cell types were significantly higher (p<0.05) in the 4NQO mice versus the control mice: CD3+ (11.2 vs 0.9 cells/mm2), CD68+ (3.6 vs 1.5 cells/mm2), and Ly6+ (9.4 vs 0.38 cells/mm2). Among the 4NQO treated mice, there were no significant differences between males and females, or between HF and LF mice for T cells or macrophages. 4NQO treated males had a significantly higher numbers of neutrophils than females (12 vs 6.6 cells/mm2). LF diet increased the levels of neutrophils in both males and females (15 and 7.7 cells/mm2 respectively) when compared to the HF diet in both males and females (9.3 and 5.3 cells/mm2 respectively); however, this trend was not statistically significant. We speculate that the higher carbohydrate content of the LF diet may support increased anaerobic glycolytic metabolism via the Warburg effect.

Conclusion

Development of 4NQO‐induced oral cancer was accompanied by inflammatory responses as manifested by the presence of T cells, macrophages, and neutrophils. Neutrophils were the only cell type affected by sex and possibly by the content of dietary fat.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Immunological and Bacteriological Assessment of Women with Breast Cancer in Benin, Nigeria
Authors: Okojie Rachel Obhade , Ojo Matilda Adesuwa
Published: June 2021

Neutrophils and lymphocytes are important cells involved in cellular immune response. Pre‐treatment neutrophil‐lymphocyte ratio (NLR) is a biomarker which can be used to predict disease progression, mortality and morbidity in breast cancer patients. Fungating breast lesions found in advanced stages of breast cancer patients are usually infected with opportunistic mixed bacteria flora and treatment with systemic antibiotics is often geared at alleviating symptoms and optimizing the quality of life of the patients.

A total of 60 participants (30 patients and 30 apparently healthy women as controls) were involved in this study. Full blood count analysis was done using Sysmex KX‐21N Automated Hematology Analyzer. Swab samples were also collected from the breast wounds. These were cultured and isolated using appropriate media and standard microbiological techniques. Antibiotic susceptibility test was determined using commonly used antibiotics.

It was observed that there is a preponderance of breast cancer within the study area. Majority of the subjects presented at the advanced stages (stage 3 and 4) and they had high values of neutrophil‐lymphocyte ratio. The mean value of the neutrophil‐lymphocyte ratio (NLR) obtained for the women with breast cancer (3.50±2.7 x103/μl) was found to be higher than the ratio of the control group (2.1±0.56 x103/μl). Using 2.3 as the cut‐off marker for NLR, 36.7 % of the subjects had NLR less than 2.3 while 63.3 % were higher or equal to 2.3; which were found to be statistically significant (p < 0.05). It showed that majority (40%) of the cancer subjects presented at stage 4 and out of which 30% had fugitive breast lesions. The predominant bacteria isolated from the lesions were Escherichia coli (35.7 %), Proteus mirabilis (28.6 %), Pseudomonas aeruginosa (21.4 %) and Staphylococcus aureus (14.3 %). All the bacterial isolates showed high susceptibility to pefloxacin and ceftriaxone (78.50 %) respectively and exhibited very low susceptibility to amoxicillin (7 %) and ampiclox (0 %).

It is pertinent to increase public awareness of breast cancer, to encourage women to be involved in routine medical examination and to educate women with breast cancer to present early to the hospitals for adequate treatment, as these will help to manage the progression of the disease and also reduce mortality and morbidity. Appropriate use of antibiotics is also very important.

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 BTB domain protein‐Cul3 complex is a novel regulator of Collagen expression in Breast Cancer
Authors: kim kyungho , Kim Youn‐Jae
Published: June 2021

CUL3‐RING ubiquitin ligases are associated with diverse cellular processes through over two hundred BTB‐domain proteins. RHOBTB3, a BTB‐domain protein, harbors capable of assembling Cullin3‐ dependent ubiquitin ligase complexes and recent studies have been suggested to play an essential role in transport vesicle docking at the Golgi complex. Collagen type I alpha 1(Col1A1) reportedly associated with tumor interstitial macromolecular transport and cancer cell dissemination. However, the precise function and mechanism of Col1A1 remain unclear how Col1A1 is regulated in human breast cancer growth and metastasis. To address this question, we analysed RHOBTB3 and Col1A1 gene expression in breast cancer using the public database. We exemplify each three representative studies showing RHOBTB3 and Col1A1 are generally upregulated in breast cancer. To determine the effects of RHOBTB3 and Col1A1 expression on metastasis and involvement of RHOBTB3 and Col1A1, we performed knock‐down of RHOBTB3 in breast cancer cells. The knock‐down of RHOBTB3 significantly decreased expression of COL1A1 mRNA and protein and inhibition of cell growth and invasion in breast cancer. Collectively, these findings demonstrated RHOBTB3 is an important factor for regulation of the COL1A1 synthesis and inhibition of cell growth and invasion in breast cancer cells. Thus, RHOBTB3 is oncogenic and a potential oncotarget for breast cancer therapy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Overexpression of Elabela Transgene Ameliorates UUO‐Induced Renal Fibrosis through Suppressing TGF‐β in Sparague‐Dawley Rats
Authors: Wu Chunying , Zhu Yeyan , Li Haonan , Wang Lei , Zhu Qing
Published: June 2021

Elabela (ELA) is a newly discovered endogenous ligand of APJ receptor, which is widely distributed in various tissues and organs including kidney. Accumulated evidences demonstrate that ELA plays a critical role in the regulation of renal physiology and pathophysiology. Our preliminary data showed that ELA expression was significantly inhibited in the kidney of unilateral ureteral obstruction (UUO) rats. However it remains unknown whether ELA has renoprotective effects in UUO model. The present study tested the hypothesis that overexpression of ELA transgenes protect against renal fibrosis in UUO rats. Male Sprague‐ Dawley rats (200–250g body weight) were subjected to UUO surgery and plasmids expressing ELA transgenes or control vectors were transfected into the obstructed kidneys via renal arteries. After 7 days, the rats were scarified and the kidneys were collected for analysis. The ELA mRNA levels were decreased by 70% in UUO rats compared to that of the control rats, which were significantly increased by 2‐fold in the UUO+ELA plasmids‐treated rats, suggesting successful overexpression of ELA in the kidney via transfection of ELA overexpression plasmid. Siris red staining results showed that collagen accumulation was increased in the UUO rats, which was dramatically suppressed in UUO+ELA plasmids‐treated animals. The mRNA levels of Collagen I, Fibronectin and α‐SMA were all elevated in the UUO group and were notably attenuated in the UUO+ELA plasmids‐treated group. Furthermore we examined the expression of TGF‐β, a central mediator of renal fibrosis and the results showed that the mRNA and protein levels of TGF‐β were upregulated in UUO rats but greatly decreased in UUO+ELA plasmids‐treated rats. These results suggest that the impaired activation of ELA in the kidney may represent a novel mechanism for renal fibrosis under pathological condition and that correction of this impairment may serve as a novel therapeutic approach for the treatment of chronic renal fibrotic diseases.

Support or Funding Information

This work was supported by grants from the National Natural Science Foundation of China: No. 81600322 and No. 81770707

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sustained Delivery of Dihydrotestosterone Impact on the Pathophysiology of Isolated Endothelial and Smooth Muscle Cells
Authors: Benghuzzi Hamed a , Tucci Michelle a
Published: June 2021

Sex steroid hormones appear to be a risk factor for the development of atherosclerosis. Proliferation of vascular smooth muscle cells (SMC) was documented to be a mediator in atherosclerotic plaque formation. The objective of this study was to investigate the effect of sustained delivery of dihydrotestosterone (DHT) on the growth of cultured aortic endothelial cells (EC) and SMC incubated individually, or in combination. The SMC were explanted from the media and intima of the abdominal and thoracic aorta of adult male rabbits, and EC were prepared by trypsinization of the vessel. Cells were seeded and grown undisturbed for up to 6 weeks in culture. A total of four equal (n=4 wells) groups was used in Phase I. Groups 1–3 were plated with EC, SMC, and EC+SMC, respectively. Group IV served as control. Half of the wells (n=4 per group) in each group were exposed to tricalcium phosphates capsules loaded with 40 mg DHT (release profile: 3–5 ng/ml/day) and second half was exposed to sham capsules. Resultes revealed that sustained release of DHT to EC+SMC cultures induced greater cellular proliferation than in control wells, as determined by cell counts after 96 hours of culture. In contrast, the number of cells/well of a single cell type, grown in the presence of DHT, did not differ from the control wells. In the second phase of this investigation, various capsules releasing different concentrations of DHT were used. The release rate of 10 ng/ml/day of DHT regressed the proliferation rate by approximately 40% compared to other lower doses (2.4, or 6 ng/ml/day). Results of this investigation suggest that: (1) sustained delivery of a physiological dose of DHT to combined cultures of EC+SMC significantly increased cellular growth, while the exposure of DHT to EC or SMC grown individually had no effect on the rate of cellular proliferation, and (2) the effect of sustained delivery of DHT on vascular smooth muscle cells was found to be dose dependent.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Methamphetamine and cocaine administration increases CRF‐R2 and decreases S100‐beta astrocytic immunoreactivity in the VTA
Authors: Trzeciak Marta , Sharpe Amanda L. , Douglas Phillip , Beckstead Michael J.
Published: June 2021

Dopamine neurons in the ventral tegmental area (VTA) play a crucial role in the reinforcing properties of abused drugs including methamphetamine and cocaine. However, there is evidence suggesting the involvement of non‐dopaminergic transmitters, including glutamate and corticotropin‐releasing factor (CRF), on psychostimulant effects in the VTA. Astrocytes can express a large variety of receptors for neurotransmitters such as glutamate which could indirectly influence neurotransmission in the VTA. Additionally, corticotropin‐releasing factor (CRF) receptors have been reported on astrocytes in the cerebellum, although no one has yet identified them on astrocytes in the VTA. CRF in the VTA plays a role in stress‐induced relapse and drug‐seeking behavior, but the localization of its effects are not completely understood. Although tissue from mouse VTA shows a high level of CRF1 receptor transcript and lower levels of CRF2 receptor transcript, both receptors play a functional role in regulating VTA synaptic activity.

Here, we aimed to use immunofluorescence to identify the effect of methamphetamine and cocaine administration on astrocytic glutamate and CRF receptors. We treated adult male mice with i.p. injections of methamphetamine (3 mg/kg), cocaine (10 mg/kg), or saline (vehicle) for 12 days. Brains were then removed and sectioned (40 μm thickness) in the coronal plane. The tissue was then processed for immunofluorescence using tyrosine hydroxylase as a dopaminergic cell marker and S100‐beta as a marker for astrocytes. We examined the level of glutamate and glutamate/aspartate transporters (GLT‐1 and GLAST) and CRF receptors. Images were captured using fluorescent microscopy and processed using ImageJ software. The results showed a ~30% decrease in GLT‐1 and GLAST immunofluorescence in brains of mice treated with cocaine or methamphetamine compared to saline. In addition, we observed increased labelling of CRF2 receptors in drug‐treated groups, a decrease of S100beta, and an increase of co‐staining of these markers. Our results suggest that administration of either methamphetamine or cocaine increases immunoreactivity for CRF2 receptors in the VTA, an effect that is most pronounced in S100‐ beta positive cells.

Support or Funding Information

This work was supported by National Institute on Aging grant R01 AG052606.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex Differences in High Fat Diet‐Induced Enhanced Sensitivity to Methamphetamine and Dopamine D1 Receptor Agonist SKF 82958
Authors: Hardin Ethan , Ramos Jeremiah , Serafine Katherine
Published: June 2021

Eating a diet high in fat can lead to obesity and insulin resistance. Further, eating high fat laboratory chow enhances sensitivity of rats to the behavioral effects of drugs acting on dopamine systems (e.g., methamphetamine). For example, male rats eating high fat chow are more sensitive to the locomotor stimulating effects of methamphetamine than rats eating standard chow. The locomotor‐stimulating effects of methamphetamine are due, in part, to increased action of dopamine at dopamine D1 receptors; however, it is not known if diet enhances sensitivity of rats to the behavioral effects of dopamine D1 receptor agonists (e.g., SKF 82958). To test the hypothesis that eating a high fat diet enhances sensitivity rats to locomotion induced by dopaminergic drugs, male and female Sprague‐Dawley rats ate either standard (17% kcal from fat) or high fat (60% kcal from fat) chow and were tested once per week with SKF 82958 (0.01–3.2 mg/kg) or methamphetamine (0.1–3.2 mg/kg) using a cumulative dosing procedure for 6 weeks. Male and female rats eating high fat chow were more sensitive to the locomotor‐stimulating effects methamphetamine than control rats eating standard chow. Female rats eating high fat chow, but not males, were also more sensitive to the locomotor stimulating effects of SKF 82958 than female rats eating standard chow. Taken together with previous research, these results suggest that among females, eating high fat chow might impact sensitivity to methamphetamine via dopamine D1 receptor specific changes, while among males, high fat diet‐induced enhanced sensitivity to methamphetamine might be due to dopamine D2 receptor specific changes. Future experiments will examine differences in dopamine receptor expression among male and female rats eating different diets.

Support or Funding Information

UTEP COURI

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