bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitory effect of diosmetin on lipolysis and inflammation in adipocyte‐macrophage co‐culture model
Authors: Lee Hana , Sung Jeehye , Lee Junsoo
Published: June 2021

Obesity has become one of the most common metabolic syndromes and has come to pose a major worldwide threat to human health. Paracrine interaction between macrophages and adipocytes in obese tissue is thought to be a trigger of metabolic syndromes. Diosmetin is a flavone found in citrus fruits. Diosmetin has been shown to alleviate inflammatory cell infiltration and to repress the nuclear factor‐kappa B(NF‐κB) signaling pathway. However, not much is known about the anti‐inflammatory and anti‐lipolytic activities of diosmetin in co‐culture system of adipocytes and macrophages. The objective of this study was to evaluate the inhibitory effect of diosmetin on the inflammation and lipolysis in the interaction between adipocytes and macrophages. The secretion of inflammatory mediators such as nitric oxide, monocyte chemoattractant protein‐1, and tumor necrosis factor‐α increased in a co‐culture medium, however, diosmetin significantly decreased the inflammatory mediators. Diosmetin down‐regulated the expression of inducible nitric oxide synthase in co‐cultured macrophages and adipocytes. Diosmetin inhibited the phosphorylation of mitogen‐activated protein kinases and translation of NF‐κB. In addition, diosmetin suppressed the protein production of hormone sensitive lipase and fatty acid binding protein 4 and increased the mRNA expression of adiponectin in co‐cultured adipocytes. These results indicated that diosmetin inhibited inflammation and lipolysis in the crosstalk between adipocytes and macrophages and diosmetin‐containing foods could be used in dietary therapy for the prevention of obesity‐related metabolic diseases.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of oxidative stress and inflammatory cytokines in Type 2 Diabetes Mellitus patients with and without small intestinal bacterial overgrowth
Authors: Rana Satyavati , Malik Aastha , Morya Rajesh Kumar , Sharma Surendra Kumar , Bhadada Sanjay Kumar
Published: June 2021

Background

In type 2 diabetes mellitus (T2DM), insufficient insulin or resistance to insulin in body results in reduced uptake of glucose causing hyperglycemia. This may lead to oxidative stress and further may increase cytokine production. Inflammation may affect gut neural apparatus that may lead to gastrointestinal dysmotility which may amplify occurrence of small intestinal bacterial overgrowth in these patients.

Aim

To study role of oxidative stress, inflammatory cytokines, gut motility(orocecal transit time: OCTT) and small intestinal bacterial overgrowth (SIBO) in T2DM patients

Materials and Methods

150 T2DM patients and 150 age and sex matched healthy controls were enrolled. SIBO and OCTT were measured using noninvasive glucose and lactulose hydrogen breath tests respectively. Plasma levels of IL‐6, TNF‐α and IL‐10 were measured by ELISA. Oxidative stress (lipid per‐oxidation (LPO) and anti‐oxidant parameters such as reduced glutathione (GSH), Super‐oxide dismutase(SOD) and catalase were measured by standard methods.

Results

Out of 150 T2DM patients, 72 (48%) were males with Mean ± SEM age 56.7±0.74 years. IL‐6, TNF‐α and IL‐10 in T2DM patients were significantly higher(p<0.05) as compared to controls. LPO in T2DM patients was significantly increased(p<0.05) while GSH was significantly decreased(p<0.001). Moreover, SOD and Catalase were significantly higher as compared to controls. OCTT was delayed and SIBO was significantly higher in patients as compared to controls. Furthermore, SIBO +ve patients showed delayed OCTT and inflammatory cytokines were higher in SIBO +ve patients as compared to SIBO –ve patients. Also, LPO, SOD and Catalase were higher whereas GSH lower in SIBO +ve patients as compared to SIBO –ve patients

Conclusion

In T2DM patients, oxidative stress would have caused inflammation in gut wall. leading to delayed OCTT and SIBO.

Support or Funding Information

Department of Biotechnology, New Delhi, India vide no BT/PR8369/MED/30/996/2013

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crystallization and Study of Dihydrofolate Reductases (DHFR) in Wuchereria bancrofti and Brugia malayi.
Authors: Lange Keith Richard , Janson Cheryl , Goodey Nina
Published: June 2021

Lymphatic Filariasis, commonly regarded as Elephantiasis, is a vastly untreated parasitic disease primarily caused by the worms Wuchereria bancrofti and Brugia malayi which affects ~63 million people in 73 countries. Current drug treatments, such as diethylcarbamazine and ivermectin, are effective drug treatments when the host is initially infected; however, upon onset of limb swelling, these treatments fail to eradicate the parasites. Dihydrofolate Reductase (DHFR), a vital and ubiquitous enzyme that aids in DNA synthesis and folate metabolism, is a well‐established drug target and may result in a treatment option for many suffering from the disease. Selective inhibition of DHFR in both organisms may lead to progress in combating the disease and therefore aiding those who are infected. Currently, the lack of three‐dimensional understanding of both enzymes poses as an obstacle for selective inhibition. Crystallization and X‐Ray diffraction of these enzymes would elucidate their respective binding sites which will provide an understanding of how these enzymes function and thus, providing insight as to how to introduce inhibition. Once crystal structures of both enzymes are obtained with and without ligand bound, rational drug design can begin. Currently, both DHFR enzymes are being expressed and purified by Dr. Goodey's research lab and several inhibition studies have already been carried about; however, crystallization procedures have recently begun to ultimately provide a detailed model as to how the enzymes function within these organisms. In the future and as a result of this project, kinetic studies can then subsequently be carried out to test possible selective inhibitors.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Syndecan‐4 and PECAM‐1‐Dependent Cell Motility
Authors: Abraham Valsamma , Parambath Andrew , Murarka Monisha , Zoga Kristi , Saeed Danyall , Tran Thuongvu , Kumar Rahul , DeLisser Horace
Published: June 2021

Ligand interactions between PECAM‐1 and heparan sulfate proteoglycans have been implicated in the ability of PECAM‐1 to promote cell motility by stimulating Cdc42 activation and the extension of filopodia. However, the endothelial proteoglycan(s), involved in these activities are undefined. We therefore investigated the potential involvement of syndecans (SDCs), a family of four cell surface heparan sulfate proteoglycans, in these PECAM‐1‐dependent processes. For these studies, we employed HUVEC and REN cells (an EC surrogate) expressing mouse PECAM‐1 (REN‐MP). For both cell types, we observed that while syndecan‐1 (SDC1) and syndecan‐4 (SDC4) are surface expressed, SDC4, but not SDC1, co‐immunoprecipitated with PECAM‐1. This PECAM‐1/SDC4 interaction was disrupted by targeting PECAM‐1‐dependent, heparan/glycosaminoglycan (GAG) ligand binding. Further, antibody‐ and/or siRNA‐mediated inhibition of SDC4 in REN‐MP cells inhibited PECAM‐1‐dependent, heparan/GAG heterophilic binding, cell motility, filopodia extension and Cdc42 activation. The targeting of SDC4 did not suppress the concentration of PECAM‐1 in the tips of filopodia, a process previously shown to be mediated by PECAM‐1‐PECAM‐1 homophilic, but not heterophilic, ligand binding. These data suggest that cis binding interactions between PECAM‐1 and SDC4 in the membrane of endothelial cells are involved in PECAM‐1‐dependent cell motility during angiogenesis.

Support or Funding Information

Veterans Administration Merit Award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intravenous Lipid Emulsion Induces Endocytosis in Human Coronary Artery Endothelial Cells by a CD36/Caveolin‐1 Pathway
Authors: McVey Natalie , Richman Michael , Struve Janine , Baumgardt Shelley , Weihrauch Dorothee
Published: June 2021

Introduction

Intravenous lipid emulsion like Intralipid™ (ILP) are fatty emulsions commonly used in parenteral nutrition. Previous research has shown that they protect against myocardial ischemia/reperfusion injury (IR) in animal models through a mechanism involving activation of Akt/extracellular signal‐regulated kinase‐1/glycogen synthase kinase‐3β pathway ultimately leading to delayed mitochondrial permeability transition pore (mPTP) opening and the induction of a protective effect by ILP. They are also studied in connection with local anesthetic toxicity and mechanisms of scavenging anesthetics from the blood stream.

Hypothesis

The direct effect of ILP on endothelial cells has not been studied. Hence, the mechanisms by which ILP crosses the endothelium are not well understood especially the effects of ILP on human coronary artery endothelial cells (HCAEC). We hypothesize that uptake of ILP occurs through endocytosis in HCAECs by the formation of a CD36/caveolin‐1 cell membrane complex.

Methods

ILP in a 1% dilution was administered to HCAECs for 2 hours or control was left untreated. Western blots were conducted with anti‐CD36, caveolin‐1, dynamin‐2, src kinase‐1, eNOS, and phospho‐eNOS antibodies and equal loading of protein confirmed with beta‐actin. Additional cells were exposed to ILP, and then immunohistochemistry was performed, to visualize uptake of ILP into cells. This was accomplished by co‐labeling of lysosomal associated protein 3 CD63 and styryl peptide and lysosomal marker FM5‐95.

Results

The association of CD36 with caveolin‐1 is increased after stimulation with ILP. The tyrosine kinase Src kinase‐1 acts to phosphorylate dynamin‐2, a GTPase responsible for separating the vesicle from the plasma membrane. ILP stimulation induced phosphorylation of Src kinase 1 and increase in expression of dynamin2. ILP induced increased phosphorylation and Hsp90 association of eNOS, leading to increased synthesis of nitric oxide. Furthermore, the association and expression of lysosomal markers CD63 and FM5‐95 are increased after ILP treatment compared to no treatment.

Summary and Conclusion

Our data suggest that administration of ILP to endothelial cells induces an endosomal pathway by formation of CD36/caveolin association which leads to enhanced endosome formation seen by increased lysosomal markers. Furthermore, we can report that stimulation of endothelial cells with ILP results in an increase of eNOS activity and increased NO synthesis which might be important for the cardioprotective abilities.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of Lymphangiomas by Freund's Incomplete Adjuvant (FIA) in Fatty Acid Binding Protein (FABP)‐3, 5 and 7 Knock‐out Mice
Authors: Ezaki Taichi , Sagawa Hiromi , Tokuda Nobuko , Tokuda Kazuhiro , Yamashiro Chiemi , Kimura Kazuhiro , Owada Yuji
Published: June 2021

Introduction

In an FIA‐induced benign lymphangioma model (Mancardi et al., 1999), we have suggested a sequential transformation from peritoneal mesothelial cells to functional lymphatic vessels via fat storing tumor cells after the FIA stimulation (EB2009 & 2015). However, the actual mechanism of the tumorigenesis and cellular involvement have yet to be established. FABPs are known to be involved in the promotion of cellular uptake and transport of fatty acids and the targeting of them to specific metabolic pathways. The aim of this study is, therefore, to investigate the possible involvement of FABPs during the FIA‐induced tumor development using FABP KO mice.

Materials & Methods

Lymphangiomas were induced in C57BL/6 (B6) mice and FABP3, 5 and 7 KO mice by an intraperitoneal injection of FIA (0.2 ml emulsion). For histological analyses, tissue cryosections were made and immunostained with various antibodies against FABP3, 4, 5, 7, and various lymphatic markers, such as podoplanin (PDPN), LYVE‐1 and LA102.

Results & Conclusions

After FIA injection, peritoneal mesothelial cells became tall in height losing their polarity and gradually formed stratified cell masses on the peritoneal surface in B6 mice. The tumors became honeycomb‐like lymphangiomas consisted of ring‐like fat storing cells with various sizes. Some fat storing cells fused with each other and gradually formed either tubular structures like lymphatic vessels or huge follicles. These cells were strongly positive for PDPN, F4/80 and FABP5, but negative for FABP4. The follicular structures mainly consisted of FABP7+ cells. Interestingly, only the functional lymphatic vessel‐like structures expressed LYVE‐1 and LA102. In FABP‐KO mice, there was no significant difference in the incidence of tumors as compared with normal B6 mice. However, tumors developed more vigorously in FABP7 KO mice than other KO strains. Among FABP KO mice, relatively smaller fat storing cells were seen in FABP3 KO mice, whereas follicular type structures were more commonly found in FABP5 KO mice. In contrast, the tumors in FABP7 KO mice showed less fat storing cells with more inflammatory and fibrous components. In conclusion, a close relationship between the extrinsic FIA metabolism and the lymphatic system may be suggested in this tumor model, although the actual role and biological significance of each FABP in the tumorigenesis remain to be further clarified.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of selective induction of Korean red ginseng extract on cytochrome P450 2D and 3A
Authors: Jeon Ju Mi , Cho Pil Joung , Jo Jung Jae , Shrestha Riya , Lee Sangkyu
Published: June 2021

Ginseng (Panax ginseng Meyer) has been used as a traditional herbal drug in Asian countries. In this study, herb‐drug interaction (HDI) of Korean red ginseng (KRG) extract was investigated using in vitro and in vivo models. Male 5‐week‐old C54BL/6N mice were administered orally with KRG (0, 0.5, 1, and 2 g/kg) once a day for 2 and 4 weeks. The activities of 5 CYP enzymes were determined by monitoring the concentration of probes in blood by using cocktail assay. CYP2D‐catalyzed dextromethorphan O‐demethylation was found to be significantly induced after repeated administration of KRG. The AUCratio of probe for CYP2D decreased with the elevation of the dose and duration of KRG administration. Contrarily, AUCratio of probe for CYP3A increased on increasing the dose and duration of KRG administration. Similarly, CYP3A‐catalyzed midazolam 1′‐hydroxylation also increased in dose‐dependent manner in vitro. Herein, we suggested the possibility of KRG‐induced drug interaction upon continuous administration for 2 – 4 weeks.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Amelioration of N‐acetyl‐1,4‐benzoquinone imine‐Induced Hepatotoxicity in Rats by Aqueous Leaf Extract of Annona Muricata (Soursop)
Authors: Nosiri Chidi Ijeoma , Anyanwu Chukwuma , Okwara Elizabeth Nnenna
Published: June 2021

The leaf of A.muricata is traditionally used to boost the liver and kidney. Acetaminophen (ACT), an analgesic and over the counter drug is usually abused. Overdose of it could lead to hepatic oxidative stress where the intermediate N‐acetyl‐1,4‐benzoquinoneimine which is rapidly detoxified by Glutathione S‐transferase becomes too much for this enzyme to handle. This mechanism is important in the development of acetaminophen toxicity. 20 albino rats randomly divided into 4 groups of 5 rats were used for the study. (Group 1:normal saline, group 2: ACT alone (400 mg/kg p.o), group 3: ACT and plant extract (400mg/kg p.o) and group 4: ACT and extract 800 mg/kg p.o. For the results of ALT (Alanine amino transferase) and AST (Aspartate amino transferase) levels, group 2 showed a significant increase compared to the control and treated groups. ALT for the control group was 17.00±1.00, group 2 value: 35.00±1.00, group 3 value: 31.00±1.00 and group 4 values: 21.00±1. For AST, the control value gave 26.00±1.00, group 2 value: 45.00±2.50, group 3 value: 38.00±1.00 and group 4 value: 29.00±1. For ALP (Alkaline phosphatase), the control value: 65.16±1.44, group 2 value: 70.05±0.81, the group 3 value 67.79±0.58 while group 4: 64.37±2. In all, the treated groups dose dependently ameliorated the hepatotoxicity shown by the levels of the liver enzymes. The high dose of (NAPQI) may have reacted with hepatic glutathione (GSH) leading to low concentration of total hepatic GSH that generated the production of free radicals which destroyed the liver cells causing the liver enzymes to leak into the blood. The histopathological effect also showed moderate to severe portal inflammation in group 2 while groups 1,3 and 4 showed normal hepatocytes. These findings suggest that annona muricata leaf extract may have both tissue protective and antioxidant properties.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Photoaffinity‐Mediated Identification of a Third Citalopram Analog Binding Site on the Serotonin Transporter
Authors: Tomlinson Michael J. , Kumar Vivek , Newman Amy H. , Lever John R. , Henry L. Keith , Vaughan Roxanne A.
Published: June 2021

In the CNS, serotonin (5HT) controls anxiety and mood, and disorders caused by imbalanced 5HT include Major Depressive Disorder and Autism Spectrum Disorder. The serotonin transporter (SERT) is a major regulator of synaptic 5HT levels and is the target for antidepressants such as escitalopram (S‐CIT) and fluoxetine, which bind to and inhibit the transporter to elevate free transmitter. The efficacy of these drugs is moderate and many patients experience inadequate relief, indicating the need for better understanding of mechanisms or development of novel therapeutics. Antidepressant effects on SERT are complex, as many induce high‐affinity actions at the S1 orthosteric site, as well as allosteric modulation of S1 function through low‐affinity binding to a distinct S2 domain. To examine S‐CIT mechanisms we developed two novel photoaffinity ligands, [125I]VK 03‐83 and [125I]VK 03‐51, that possess identical S‐CIT pharmacophores but have cross‐linking moieties appended to different positions on the citalopram core. Our findings show that both ligands bind to SERT, inhibit 5HT transport, and allosterically regulate S1 function, but display distinct pharmacological characteristics that indicate differential binding mechanisms. [125I]VK 03‐83 labeling of SERT is fully inhibited by 5HT and typical SERT inhibitors, with an S‐CIT Ki of ~1 nM, consistent with binding to S1. In contrast, although [125I]VK 03‐51 labeling is fully displaced by the homologous ligand, it is inhibited only ~30% by S‐CIT or other standard SERT inhibitors, and shows no displacement by 10 μM 5HT, indicative of binding to multiple sites. The S‐CIT‐displaceable portion of [125I]VK 03‐51 labeling occurs with a Ki of ~1 nM, consistent with binding at S1, whereas the fraction of labeling that is not sensitive to ≥10 μM concentrations of S‐CIT, other inhibitors, or 5HT likely does not occur at either S1 or S2, which should both be fully occupied by these conditions. These findings suggest that [125I]VK 03‐51 interacts with SERT at a third site that has not been previously detected by reversible binding or co‐crystallization approaches, but is revealed by covalent cross‐linking. This site may function in transport inhibition or allosteric regulation, and suggests a domain for exploration of transporter mechanisms or medication development.

Support or Funding Information

NIH DA027845 and UNDSMHS Seed Grant (LKH and RAV), NIDA Intramural Research Program (AHN)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ACETAZOLAMIDE INCREASES LOCOMOTION, EXPLORATORY BEHAVIOR, AND WEIGHT LOSS FOLLOWING SOCIAL STRESS: A TREATMENT FOR EMOTIONAL EATING?
Authors: Student Jeff , Woodman Ryan , Lockette Warren
Published: June 2021

Background

Amphetamines and sympathomimetics (e.g. phenylpropanolamine and ephedrine) are effective, centrally acting drugs that induce weight loss through their potent anorexic and locomotor properties. We first reported that amphetamines and these sympathomimetic agents antagonize catecholamine‐dependent, alpha‐2 adrenergic receptor‐dependent signal transduction mediated by chloride/bicarbonate transport.

Hypothesis

Other drugs that target and inhibit cellular bicarbonate/chloride antiport would similarly demonstrate anorectic properties, induce locomotion, and diminish weight gain ‐‐especially in disorders such as depression or social isolation where there is an increase in emotional eating.

Methods

Male and female inbred mice were housed in groups or they underwent the stress of living in social isolation or in solitary metabolic chambers for five days. Mice consumed either normal rodent chow or a rodent food supplemented with high fat, high fructose corn syrup; water was provided ad libitum. To inhibit chloride/bicarbonate transport, acetazolamide (ACT, 3 mM) or vehicle was added to the drinking water. There were no noticeable adverse effects in mice receiving ACT. Following a period of habituation, rodents underwent evaluations of exploratory locomotion and learning with the object recognition test. Food intake and weights were also tracked; for all groups, n ≥ 9.

Results

Compared to the control, unstressed, group housed rodents, living in social isolation reduced spontaneous exploration time in male mice (values expressed as means in sec ± SEM) from 23 ± 4 to 14 ± 3 (p<0.001), and in female mice, from 47 ± 6 to 23 ± 3 (p<0.001). Drinking water supplemented with ACT had no effect on exploration time in group housed mice, but ACT completely restored the diminished exploration time found in mice stressed by isolation to control, grouped mice values. The ratio of time spent exploring new objects compared to familiar items (DR ratio) was also reduced following social isolation from 2.6 ± 0.5 to 1.2 ± 0.2 (p<0.001). ACT completely normalized the DR ratio of the stressed mice. The body weight of female mice fed high fat, high fructose corn syrup diets for 12 weeks and given ACT was significantly reduced when compared to mice drinking only water (weight in g ± SEM), 23.7 + 0.8 v. 21.0 + 0.5, p=0.02. This difference in weight was not due to changes in body water volume, but instead, due to the significant decrease in food consumption and greater locomotor activity recorded in mice fed the ACT.

Conclusion

Drugs that inhibit chloride/bicarbonate transport could offer a safe, new approach to achieving behavioral weight loss in obese individuals with emotional eating.

Support or Funding Information

University of Missouri

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activated Protein C‐mediated Crosstalk Between PAR1 and S1PR1 in Endothelial Barrier Stabilization
Authors: Molinar Olivia , Chen Buxin , Grimsey Neil J. , Lin Huilan , Coronel Luisa Julieta , Trejo JoAnn
Published: June 2021

Vascular leakage is a hallmark of sepsis, a life‐threatening condition induced by systemic microbial infection. Mortality rates for severe sepsis are high, due to the lack of available effective treatments. The aim of this study is to define the key signaling pathways that promote endothelial barrier stabilization to identify new cellular targets that can be manipulated to reduce vascular leakage. Activated protein C (APC) is an anti‐coagulant protease that was previously shown to promote endothelial barrier protection through protease‐activated receptor‐1 (PAR1), a GPCR. Activation of PAR1 by APC stimulates Rac‐1 activity, which enhances endothelial barrier stability through modulation of the actin cytoskeleton and adherens junction integrity. In addition, PAR1 appears to require the sphingosine 1‐phosphate receptor‐1 (S1PR1) to promote endothelial barrier stabilization, but how signaling by the two receptors integrate to promote endothelial barrier stability is unknown. We previously demonstrated that APC/PAR1‐mediated endothelial barrier protection requires compartmentalization in caveolae and is dependent on β‐arrestin‐2 and dishevelled‐2 (Dvl‐2) scaffolds. However, it is unknown whether β‐arrestin‐2 and Dvl‐2 are required for PAR1 and S1PR1 signaling crosstalk via APC. Here, we report that PAR1 and S1PR1 co‐localize in endothelial cells and co‐segregate in caveolin‐1 enriched fractions, consistent with PAR1 and S1PR1 signaling from caveolae. We found that APC treatment of endothelial cells results in SK1 translocation to membrane fractions and activation. In addition, we show that loss of β‐arrestin‐2 expression causes a significant loss in APC‐induced SK1 activation. Thus, we hypothesize that endothelial barrier stabilization induced by APC/PAR1 and S1PR1 is integrated by β‐arrestin‐2 and SK1.

Support or Funding Information

Olivia Molinar is an Institutional Research and Academic Career Developmental Award (IRACDA) Postdoctoral Fellow and is supported by NIGMS/NIH award K12GM068524. This work is funded by two “GPCR CYTOPROTECTIVE SIGNALING MECHANISMS” R01 grants: 3R01GM116597‐01S1 and 5R01GM116597‐02.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Impact of Altered Subunit Expression on Sweet Taste Receptor Surface Trafficking and Signaling
Authors: Tan Susan , Healey Robert , Thordarson Pall , Finch Angela
Published: June 2021

The sweet taste receptor is a heterodimeric G protein‐coupled receptor (GPCR), consisting of the subunits Tas1R2 (T1R2) and Tas1R3 (T1R3). Expression of this receptor has been demonstrated in various tissues throughout the body, including the pancreas, small intestine and adipose tissue. This receptor is thought to play a role in maintaining the body's energy homeostasis, and may potentially be a target in the treatment of metabolic disorders such as diabetes and obesity. Some studies show that the two sweet taste receptor subunits are expressed in unequal quantities in pancreatic β cells and adipocytes, however the impact of this unequal expression on receptor function is not fully understood. This study aims to determine the impact of varying the degree of expression of the two subunits on receptor surface trafficking and downstream signaling pathways. To address this aim, sweet taste receptor‐expressing stable cell lines were generated in AD293 cells by sequential selection of vectors, and in HEK293 TRex cells using bicistronic vectors and the FlpIn system. Receptor expression was characterized through real‐time PCR and biotinylation pull‐down experiments, while receptor signaling was determined in live cells using a genetically encoded Fluorescence Resonance Energy Transfer (FRET) sensor and a Bioluminescence Resonance Energy Transfer (BRET) sensor. Our results show that expression of both receptor subunits is predominantly intracellular, and that co‐expression of both subunits does not improve surface expression. Co‐expression of both subunits was, however, found to be necessary for signaling, as cell lines expressing only one subunit did not show functional responses to aspartame. The magnitude of these functional responses was found to be greatest when subunit expression was closest to 1:1, suggesting that the sweet taste receptor is most efficacious as a 1:1 heterodimer. Cell lines expressing both subunits were found to signal through Gi, as well as undergo extracellular signal‐regulated kinase (ERK) phosphorylation, demonstrating the ability of this receptor to couple to multiple signaling pathways. While some studies using mouse sweet taste receptors have reported Gs signaling at high concentrations of sweeteners, this could not be replicated in this study using human receptors, suggesting that signaling pathways may be species‐specific. Overall, this study shows that co‐expression of both sweet taste receptor subunits is necessary for signaling but not for surface trafficking and that this receptor is most efficacious when both subunits are expressed at equal levels.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the Effects of E‐Cigarette Vapor on Oral Epithelial Cells in a Cultured Cell Model
Authors: Duggar Marygrace , Swanson Hollie , Hill‐Odom Miriam
Published: June 2021

E‐cigarette usage is growing, especially among adolescents and young adults. This trend is especially concerning due to the lack of regulations governing the e‐cigarette industry and lack of scientific data on the effects of e‐cigarettes on human health. Using a cultured oral keratinocyte model, we hypothesized that exposure to e‐cigarette vapor would adversely affect the growth of cells that reside in the oral cavity. To test this hypothesis, we cultured the cells with condensate prepared from the vapors of grape flavored e‐cigarette fluids, both with and without nicotine. After three days of treatment, cell numbers were determined using a colorimetric assay. Results indicate that the e‐cigarette condensate adversely affects cell growth, regardless of the absence or presence of nicotine. To understand the underlying mechanisms, changes in gene expression in the cells treated with condensate were evaluated using real‐time qPCR after 24, 48, and 72 hours of treatment. A time‐dependent increase in the levels of CYP1A1 and IL8, gene targets of the aryl hydrocarbon receptor, were observed. Results obtained from similar experiments using the isolated flavoring agent, methyl anthranilate, indicate that formation of a heat‐induced byproduct of this agent may be involved in the observed changes in cell growth. Future experiments will focus on determining the mechanisms by e‐cigarette condensates impact cell growth and identifying the specific chemical byproduct mediating these effects.

Support or Funding Information

Funded in part by a grant from the American Society of Pharmacology and Experimental Therapeutics for Summer Undergraduate Research.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
HRH1: A Novel GPCR Drug Target in Pancreatic Cancer
Authors: Rodriguez Alyssa , Sriram Krishna , Moyung Kevin , Insel Paul
Published: June 2021

Patients with pancreatic cancer, in particular pancreatic ductal adenocarcinoma (PDAC), have a ~6% chance of surviving 5 years after diagnosis. Approved drugs only add weeks or months to patient lifespan. PDAC is difficult to treat, in part because the tumor often has poor vascularization and dense extracellular matrix, which yield a hypoxic tumor microenvironment. Finding new drugs for PDAC is thus an unmet medical need. The G protein‐coupled receptor (GPCR) superfamily is the most frequently targeted class of proteins for FDA‐approved drugs. We hypothesized that one or more GPCR may be a novel therapeutic target for PDAC.

Bioinformatic analysis of PDAC tumors in TCGA [The Cancer Genome Atlas] revealed prominent (~32‐fold) overexpression of H1 histamine receptor (HRH1) compared to normal pancreatic tissue [GTex database]. KRAS and p53 mutations occur, respectively, in ~70% and ~60% of PDAC tumors whereas increased HRH1 expression of HRH1 in 100% of tumor samples in TCGA. HRH1 is an attractive target for repurposing since many FDA approved antagonists are directed at this receptor. We thus sought to validate HRH1 expression in PDAC cells.

Using qPCR, we found high HRH1 expression and low expression of HRH2‐4 in BxPC, AsPC‐1, and CAPAN‐2 (PDAC) cell lines and normal pancreatic ductal epithelial cells. Addition of histamine increased Ca++ in ASPC‐1 and CAPAN‐2 cells; this increase in Ca++ was blocked by multiple FDA‐approved HRH1 antihistamines. Histamine increased PDAC cell proliferation in cells grown in hypoxia but not normoxia. High expression of HRH1 in PDAC cells may thus confer a growth advantage for PDAC cells in hypoxic conditions. These preliminary data suggest that FDA‐approved HRH1 antihistamines are candidates for repurposing as novel therapeutics for the treatment of pancreatic cancer.

Support or Funding Information

Supported by HHMI and an ASPET David Lehr Award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
GPR68, a proton sensing GPCR, mediates interaction of pancreatic cancer associated fibroblasts and cancer cells
Authors: Wiley Shu , Sriram Krishna , Liang Wenjing , Chang Sarah , McCann Thalia , Nishihara Hiroshi , French Randall , Lowy Andrew , Insel Paul
Published: June 2021

Pancreatic ductal adenocarcinoma (PDAC) has a unique tumor microenvironment that is characterized by a dense fibrotic stroma (desmoplasia) that is generated by pancreatic cancer associated fibroblasts (PCAFs) derived from pancreatic stellate cells (PSCs) and pancreatic fibroblasts (PFs). Using an unbiased GPCRomic array approach we identified GPR68, a proton sensing GPCR, has much higher expression in PCAFs compared to both PFs and PSCs. GPR68 also had increased expression in PDAC tumors compared to normal pancreas and its expression was increased in PSCs by co‐culture with PDAC cells or incubation with TNFα. GPR68 activation in PCAFs enhanced expression of interleukin‐6 (IL‐6) via a cAMP/PKA/CREB signaling pathway and GPR68 knockdown with siRNA diminished IL‐6 production by PCAFs and an enhancement in proliferation of PDAC cells by PCAF conditioned media. Use of the allosteric modulator compound ogerin enhanced pH‐dependent increase in cAMP. Furthermore, a pilot screening of 96 GPCR specific compounds tested with GPR68 overexpressing HEK cells identified five compounds that decrease intracellular cAMP at pH6.4, indicating that they may be inhibitors of GPR68. We conclude that PDAC cells induce expression of GPR68 in PCAF, resulting in increased IL‐6 production by PCAFs and enhanced PDAC cell proliferation. This PCAF‐expressed GPR68 thus contributes to PDAC cell‐PCAF interaction and may be a novel therapeutic target for pancreatic cancer, a type of cancer for which new therapies are an important unmet medical need.

Support or Funding Information

This work was supported by National Institutes of Health (NIH), National Cancer Institute (NCI) Therapeutic Training Grant 5T32CA121938, NIH/NCI Research Grants R21 CA189477, R01 CA155620, and Padres Pedal the Cause PTC2017, and by an American Society for Pharmacology and Experimental Therapeutics (ASPET)‐David Lehr Research Award.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of G protein‐coupled receptors in pancreatic cancer cells
Authors: Liu Dongling , McCann Thalia , French Randall , Wiley Shu , Lowy Andy , Insel Paul
Published: June 2021

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer and has an overall 5‐year survival rate ~7%. PDAC has poor response to chemotherapy and radiotherapy; thus new treatments for PDAC are an important, unmet medical need. G protein‐coupled receptor (GPCR) regulate many biological functions, including in cancer cells, but GPCR expression and the role of GPCRs in PDAC cells is unclear. We generated two primary PDAC cell lines (79E and 34E) and an immortalized normal pancreatic ductal epithelial cell line (HPDE6). Hypothesizing that GPCRs may regulate PDAC cells, we used Taqman GPCR array to assess GPCR expression in four PDAC cell lines, 79E, 34E, Miapaca, and AsPC. We identified 68 commonly expressed GPCRs in all four PDAC cell lines, among which 22 GPCRs were significantly up‐regulated (at least 2‐fold) in PDAC cells compared to normal control cell HPDE6; 13 GPCRs were significantly down‐regulated and 3 GPCRs were uniquely expressed in PDACs. We further tested the 13 most up‐regulated GPCRs using real time qPCR and confirmed that LPHN1, CHRM4, FZD4, HRH1 and CD97 have much higher expression in PDACs compared to HPED6, suggesting that these GPCRs might contribute to the malignant phenotype and perhaps could be novel treatment targets for pancreatic cancer, in particular for PDAC.

Support or Funding Information

This work is supported by NIH/NCI Research Grants R21 CA189477, R01 CA155620, and Padres Pedal the Cause PTC2017.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TARGETING THE METABOLIC SIGNALING NETWORK FOR IMMUNE CELL LONGEVITY AND FUNCTION IN ORAL CANCER THERAPY
Authors: Allevato Michael
Published: June 2021

Oral squamous cell carcinoma (OSCC), which includes cancer of the oral cavity and oropharynx, is a significant health issue. Despite aggressive multimodality therapies, OSCC has a poor five year survival rate (60%), which results in ~300,000 deaths each year worldwide1. OSCC is associated with human papillomavirus (HPV) infection, and abusive intake of tobacco and alcohol 2–3. OSCC evades the immune recognition and anti‐tumor immune response, by exploiting the immune system's regulatory immune suppressive mechanisms 2–3. A key mechanism of tumor immune suppression has been depicted as T‐cell exhaustion 4–7. In this case, T‐cell's effector function is restrained due to the activation of checkpoints, such as PD‐1 by its ligand PD‐L1, expressed on cancers cells, T‐cells, natural killer cells and macrophages 4–7. Immune checkpoint inhibitors have demonstrated revolutionary restoration of T‐cell mediated anti‐tumor immunity in OSCC 4–7. Despite this success, the overall response rate of these immune therapies in OSCC is only ~20 percent 4–7. New therapeutic options to prevent and treat OSCC are urgently needed. The unrestrained growth of cancer cells and the effector function of immune cells are often supported by the same metabolic pathway, namely aerobic glycolysis8. We believe that by taking advantage of the unique metabolic requirement of tumor cells and the cancer‐fighting immune cells, we can discover new insights into how such a relationship regulates anti‐tumor immunity. Our aim is to elucidate the role of immune and cancer cell metabolic properties in regulating the efficacy of the anti‐tumor response. Targeting both the metabolic signaling pathways of the OSCC tumor cells and tumor infiltrating immune cells has the potential to achieve cancer remission by both halting OSCC progression and enhancing the longevity of the anti‐tumor immune response.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Provoked Cardiovascular and Autonomic Effects of Endotoxemia in Ovariectomized Rats Are Distinctly Affected by Estrogen and Progesterone Supplementation
Authors: El‐Mas Mahmoud M. , El‐Lakany Mohammed A. , Fouda Mohamed A. , El‐Gowelli Hanan M.
Published: June 2021

The female gender is protected against immunological and cardiovascular complications of sepsis. This study investigated whether gonadal hormone depletion by bilateral ovariectomy (OVX) could uncover the cardiovascular and autonomic effects of endotoxemia and whether these effects can be reversed by hormone replacement therapies. Changes in blood pressure (BP), heart rate variability (HRV), and left ventricular (LV) function caused by i.v. administration of lipopolysaccharide (LPS, 10 mg/kg) were evaluated in conscious female rats with different hormonal states. While LPS caused no specific cardiovascular effects in intact female rats, LPS‐treated OVX rats exhibited significant decreases in blood pressure (BP) and increases in spectral low‐frequency/high‐frequency ratio (LF/HF) of HRV, denoting enhanced cardiac sympathetic dominance. Moreover, LPS increased the rate of rise in LV pressure (dP/dtmax, LV contractility) without affecting the isovolumic relaxation constant (Tau, a measure of diastolic function). LPS hypotension was totally eliminated in OVX rats pretreated with estrogen but not medroxyprogesterone acetate (MPA). Prior selective activation of estrogen receptor‐α (4,4′,4″‐(4‐Propyl‐[1H]‐pyrazole‐1,3,5‐triyl)trisphenol, PPT) or estrogen receptor‐β (Diarylpropionitrile, DPN) partially counteracted LPS hypotension. Each of the abovementioned drug treatments (estrogen, MPA, PPT, or DPN) abrogated the LPS‐evoked exaggeration in cardiac sympathetic control (LF/HF ratio). By contrast, the elevated dP/dtmax in LPS‐treated rats was largely compromised by PPT only. The data suggest differential modulatory roles for gonadal hormones (estrogen and progesterone) and estrogen receptors (α and β) on endotoxic cardiovascular and autonomic manifestations in female rats.

Support or Funding Information

Supported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Doxorubicin Temporally Modulates Cyclooxygenase‐2 Levels in Male and Female Human Vascular Smooth Muscle Cells
Authors: Tat Trinh , Bartel Robyn , So Mei , Sweazea Karen , Gonzales Rayna J.
Published: June 2021

Doxorubicin (Dox) is a chemotherapeutic agent that is highly effective at reducing recurrence and mortality in breast cancer patients. However, this anti‐cancer drug elicits dose‐ and time‐dependent cardiovascular toxicity. The pathogenesis of Dox is multifactorial, one of which involves the development and progression of inflammation mediated by activation of the TLR4/NFκB pathway. In a recent collaborative study, we demonstrated that intermittent bouts of Dox administered to mimic chemotherapeutic administration clinically attenuated protein levels of cyclooxygenase‐2 (COX‐2), a downstream proinflammatory mediator of the TLR4/NFκB pathway, in cerebral and peripheral vasculatures isolated from female ovariectomized rats. These findings were surprising and not in agreement with previously published data. Therefore, to address whether this Dox‐induced attenuated inflammatory response is time and dose dependent, we exposed cultured primary human female and male vascular smooth muscle cells to vehicle (saline) or Dox (0.3, 1.0, and 5.0 μM) for 6 and 24 hr. Following Dox treatment, cells were isolated, homogenized, and whole cell lysates analyzed for COX‐2 protein levels via immunoblotting. We also assessed levels of the upstream transcription factor, NFκBp65 as well as its activation. We observed that Dox dose dependently decreased levels of COX‐2 without altering NFκBp65 whole cell lysate protein levels at the 6 and 24 hr time points. However, NFκBp65 activity (6 hr time point), assessed indirectly using nuclear/cytosolic fractionation, was increased. Dox had no effect on cell morphology or density determined by light microscopy. Live versus dead cell percentages, determined using trypan blue, were also not affected by Dox suggesting that the anticancer drug did not alter cell death at 6 and 24 hr time points. In conclusion, these studies demonstrate a possible novel action for the anticancer agent eliciting both a dose‐ and time‐dependent effect on selective downstream mediators of inflammation such as COX‐2 in the female and male vasculature. Future studies are planned to evaluate the impact of acute versus chronic Dox treatment on COX‐2 at the transcriptional or translational levels to determine a mode of molecular regulation and to assess if sex differences exist.

Support or Funding Information

University of Arizona Sarver Heart Center Medical Student Award (MS) and the Valley Research Partnership P1 Grant (RJG).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial modulation of calcium pulsars through oxidative stress
Authors: Berlatie Marianne , Toussaint Fanny , Blanchette Alexandre , Ledoux Jonathan
Published: June 2021

Intracellular Ca2+ levels are central to endothelial mechanisms regulating smooth muscle contractility. Growing body of evidence suggests an important role for spatially‐restricted Ca2+ signals such as Ca2+ pulsars in endothelial‐dependent modulation of vascular tone. However, little information is available on mechanisms modulating these spontaneous Ca2+ dynamics. Recent work showed that clustered mitochondria near myoendothelial projections (MEP) regulate Ca2+ pulsars. Mitochondrial ATP production through oxidative respiration also generates reactive oxygen species (ROS). Interestingly, preliminary data suggests that ROS stimulate Ca2+ pulsars. Therefore, according to mitochondria production of ROS and organelle's clustering near MEP, it was hypothesized that mitochondria are modulators of local calcium dynamics through mitochondrial oxidative stress (mROS) in native endothelium. Intracellular Ca2+ dynamics were monitored in situ in Fluo‐4‐loaded endothelium of mesenteric arteries from mice with high‐speed confocal microscopy. Exposure to MitoTEMPO (10 μM), a mitochondria‐targeted antioxidant, decreased spontaneous Ca2+ pulsar frequency (2‐fold decrease). On the other hand, rotenone, a Complex 1 inhibitor, was used to stimulate mROS production and evaluate its impact on Ca2+ pulsars. mROS appears to stimulate Ca2+ dynamics since application of rotenone (10 μM) increased Ca2+ pulsars frequency. These results suggest that oxidative stress from mitochondrial origin (mROS) can modulate Ca2+ pulsars in native endothelium and might have a significant impact on vascular tone in pathophysiological conditions.

Support or Funding Information

Canadian Institutes of Health Research and Montreal Heart Institute Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.