bluuebunny/crossref_metadata_2025_split huggingface dataset:
Potentiation Of TRPM5 Leads To Increased Taste Perception And Insulin Secretion
Authors: Philippaert Koenraad , Vennekens Rudi 
Published: November 2023

TRPM5 is a monovalent cation selective ion channel that is activated by raises in the intracellular Ca2+ concentration. This ion channel is functionally expressed in the type II taste receptor cells in the tongue, where it has an essential role in taste signal transduction. TRPM5‐expression is also present in the insulin secreting beta‐cells in the pancreas, where TRPM5 has a regulating role in glucose‐induced insulin secretion. There are currently no good pharmacological tools available to assess the potential of TRPM5 to modulate taste signalling or stimulate insulin secretion.We show a new class of natural compounds with TRPM5‐potentiating activity. We show potentiation of TRPM5‐mediated current in a heterologous expression system. We observe increased glucose‐induced calcium activity in isolated pancreatic islets of Langerhans when potentiating TRPM5. There is increased insulin secretion in the presence of our potentiator and WT but not TRPM5‐KO mice respond with an increased glucose tolerance during acute and long‐term treatment. Administration of a TRPM5‐potentiator to mice on a high fat diet leads to a healthier glycemic profile and delays or largely prevents the onset of diet‐induced type II diabetes.In a murine animal model we show stimulation of sweet bitter and umami taste perception. In a two bottle preference test with WT, TRPM5‐KO and sweet receptor‐KO mice, we show that specific potentiation of TRPM5 increases the preferences for sweet solution, without increasing the sugar load.Our work shows a novel class of compounds to potentiate TRPM5. Stimulating TRPM5 in taste leads to increased perception of nutrients and can lead do a decrease of the calorie intake. We further confirm TRPM5 as a valid pharmacological target in the battle against diabetes to potentiate the physiological profile of glucose induced insulin secretion.
Supplementation of whole flaxseed reduced hepatic steatosis in aged laying hens
Authors: Davis Jeremy E , Cain James , Small Carrie , Hales Dale B 
Published: November 2023

Flaxseed has been investigated for its many hepatoprotective properties. Although the essential fatty acid content is often suggested as the underlying mechanism, our recent work as indicated that whole flaxseed may be more protective against the progression of hepatic steatosis. The objective of this study was to determine the effect of feeding different flax‐based diets on the development of fatty liver in aged laying hens—a physiologically relevant model of human liver disease. Laying hens aged three years were fed standard hen diet (CON) or experimental diet supplemented with corn (CRN), fish (FISH), or flaxseed (FXO) oil for 46 weeks. To elucidate the components of flaxseed responsible for hepatoprotective properties, defatted flaxseed (DFM) and whole flaxseed (WFX) meal were also supplemented to experimental diets. At termination, plasma and tissues were collected and analyzed for metabolic and morphologic parameters. Our findings show that WFX supplementation was associated with markedly lower body and liver weight (p< 0.05). This corresponded with a 10–30% reduction in hepatic steatosis (p< 0.05), as well as 40–60% reduction in plasma AST concentration (p< 0.05). Taken together, these data suggest that dietary supplementation with whole flaxseed represents a potential therapy for attenuating the progression of fatty liver.Support or Funding InformationThis work was supported by the following grant NIH/NCCAM RO1AT005295.
Expression and Function of Androgen Receptor in Human Breast Cancer‐Associated Fibroblasts: May Androgens Shape Breast Tumor Microenvironment?
Authors: Lanzino Marilena , Campana Antonella , Giordano Cinzia , Barone Ines , Bonofiglio Daniela , Catalano Stefania , Andò Sebastiano 
Published: November 2023

BackgroundThe interaction between breast tumor epithelial and stromal cells is fundamental for tumor growth and progression. Indeed, while providing a scaffold for the breast, the stroma also regulates epithelial cell function through physical and hormonal paracrine exchanges, thus providing a favorable environment for proliferation and metastasis. There is extensive knowledge of androgen receptor (AR) signaling in breast epithelial cancer cells, but less regarding AR‐mediated action in stromal cells of breast tumor microenvironment.In the present report, we provide evidence of AR expression in breast cancer‐associated fibroblasts (CAFs) isolated from breast cancer patients. We also examined the effect of CAFs exposure to androgens on the migratory behavior of an estrogen‐responsive breast cancer cell line, MCF‐7.MethodsCAFs were isolated from biopsies of primary breast tumors (n=3); RT‐PCR, immunocytochemistry and immunoblotting analyses were used for evaluating AR expression; CAFs and MCF‐7 breast cancer cells were employed for co‐colture experiments; wound healing and transmigration assays were used for evaluating cell migration.ResultsCAFs do express the AR. RT‐PCR analysis demonstrated the presence of AR mRNA whose expression levels didn't appear to be influenced by administration of 10 nM of the nonmetabolizable synthetic androgen Mibolerone (MIB). Western Blot confirmed the presence, in CAFs lysates, of the AR protein as a ~110kDa protein which was up‐regulated in the presence of MIB, suggesting that AR protein levels might be controlled by post‐traslational mechanisms in CAFs. Immunocytochemical studies further demonstrated that AR is expressed in breast CAFs as a protein with both cytoplasm and nuclear distribution. Immunostaining was more pronounced under MIB treatment, especially in the nucleus.To explore the relationship between AR expression in the tumor stromal compartment and epithelial cancer cells behavior, co‐colture experiments were carried out by using CAFs and MCF‐7 cells. Interestingly, conditioned medium (CM) from MIB‐treated CAFs was less effective in stimulating MCF‐7 cell motility compared to CM from untreated ones, suggesting the intriguing hypothesis that androgens, via AR, may influence CAFs secretion of paracrine soluble factors involved in tumor cell motility sustainment.ConclusionsAndrogens, via AR, may drive stromal paracrine effects on epithelial breast cancer cells. Disclosing the effectors of androgen response in fibroblast and their impact on tumor microenvironment may provide an alternative perspective on breast cancer progression and therapeutic requirements of patients.Support or Funding InformationThis work was supported by Fondazione Italiana per la Ricerca sul Cancro (FIRC‐AIRC) grants: IG‐11595
Protective Effect of Resveratrol on Mitochondria and Oxidative Stress induced by Cisplatin in HK‐2 Cells
Authors: Valentovic Monica , Murphy Rachel , Lamyaithong Andre Benja , Stafford Reagan , Schnelle Anthony , Ball John G 
Published: November 2023

Cisplatin is used in treating many cancers including testicular, ovarian and cervical cancer. Impaired renal function is a major side effect of cisplatin and occurs in 33% of patients treated with cisplatin. Resveratrol (RES) is a polyphenolic compound that possesses anticancer activity. Part of the mechanism for reducing cisplatin toxicity by RES may be mediated by maintaining mitochondrial function and reducing oxidative stress. The purpose of this study was to evaluate the effect of cisplatin on mitochondrial function in a human proximal tubular kidney cell line (HK‐2). Specifically the levels of ATP and ADP were measured following exposure to cisplatin in the presence of RES or DMSO (vehicle). HK‐2 cells were pretreated for 1 h with 0–15 uM RES (vehicle control 10% DMSO). Renal cells were subsequently exposed to cisplatin at a final concentration of 0–30 uM for 24 or 48 h. Viability was assessed using MTT release and cell count with an n=6/treatment group and repeated 3 separate times. Cells pretreated with 10 uM RES were protected from cisplatin cytotoxicity at 24 and 48 h. Cisplatin caused a concentration dependent decline in MTT viability after 24 h exposure to 0, 7.5 and 15 uM cisplatin. RES pretreatment for 1 h with 10 or 15 uM RES increased HK‐2 viability relative to cells exposed only to RES Vehicle (DMSO). Additional studies examined whether RES stimulated cell growth as part of the mechanism for cellular protection. RES did not increase cell number as part of its protective mechanism. ATP levels were diminished by cisplatin causing an increase in the relative balance of ADP/ATP. RES (5 and 7.5 uM) did not alter ATP or ADP/ATP ratios. Additional studies examined Caspase cleavage, protein carbonylation and 4‐hydroxynonenal adduction (4‐HNE) following cisplatin exposure of HK‐2 cells pretreated with DMSO or RES. Western analysis detected an increase in protein carbonylation with 24 h cisplatin exposure which was reversed by RES. In summary, RES protects HK‐2 cells from cisplatin cytotoxicity and preserves mitochondrial ATP levels.Support or Funding Information(Supported by NIH Grants INBRE 3P20RR016477‐09S4; 5P20RR016477 and 8P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).
Molecular Mechanisms of All‐Trans‐Retinoic Acid Mediated Cytoprotection Against Renal Cell Injury
Authors: Sapiro Jessica M , Burrell Katherine L , Lau Serrine S 
Published: November 2023

One of the major etiologies of acute kidney injury is chemical‐induced nephrotoxicity. Pretreatment of LLC‐PK1 cells with all‐trans‐retinoic acid (ATRA, 25 μM, 24 hr) affords cytoprotection against p‐aminophenol (PAP), and 2‐(glutathion‐S‐yl)hydroquinone‐induced necrosis. In contrast co‐treatment of toxicant with ATRA does not afford cytoprotection, suggesting that ATRA modulates critical signaling pathways prior to toxicant exposure. Interestingly, ATRA did not alter PAP‐induced ROS generation and had a modest effect on Nrf2 induction suggesting other stress response proteins beyond ROS reduction contribute to ATRA protection. Therefore, additional protecting mechanisms were examined. During ATRA‐mediated pretreatment, the ER stress proteins Grp78 and p‐eIF2α increased 2‐fold in a time‐dependent manner (ATRA 24 hr and 4 hr, respectively). Furthermore, ATRA increased expression of the cellular stress kinases p‐ERK and p‐AKT with maximum levels at 30 min. p‐ERK decreased to basal levels after 4 hr, while p‐AKT remained elevated for 24 hr, coinciding with the time required for maximal ATRA cytoprotection. Moreover, induction of these stress kinases was observed at concentrations of ATRA (10 and 25 μM) required for cytoprotection. Interestingly, the p‐ERK inhibitor PD98059 reduced ATRA protection against PAP toxicity indicating a possible function of p‐ERK and its downstream target genes in the ATRA mechanism for protection. We further determined that ATRA led to a prompt increase in cyclin D1 levels (1–4 hr, 3‐fold). Collectively, UPR proteins (Grp78, p‐eIF2α), stress kinases (p‐AKT, p‐ERK) and mitogenic effects (cyclin D1) function interdependently to afford ATRA protection against renal cell damageSupport or Funding InformationES006694, T32ES007091, ES016578, ASPET‐SURF
Cytochrome P450 enzymes involved in the primary metabolism of the novel constitutive androstane receptor inhibitor, CINPA1
Authors: Cherian Milu T , Yang Lei , Chai Sergio C , Lin Wenwei , Chen Taosheng 
Published: November 2023

This study aims to identify the metabolic enzymes involved in the breakdown of CINPA1, a recently discovered small molecule inhibitor of the xenobiotic receptor CAR (constitutive androstane receptor).CAR and PXR are xenobiotic sensors that respond to drugs and endobiotics by modulating the expression of metabolic genes that enhance detoxification and elimination. Elevated levels of drug metabolizing Phase I, Phase II enzymes and efflux transporters resulting from CAR activation promote the elimination of chemotherapeutic agents leading to reduced therapeutic effectiveness. Multidrug resistance in tumors after chemotherapy could be associated with errant CAR activity, as shown in the case of neuroblastoma. CAR inhibitors used in combination with existing chemotherapeutics could be utilized to attenuate multidrug resistance and resensitize chemo‐resistant cancer cells. But such studies have been confounded so far as previously reported CAR inhibitors are also activators of pregnane X receptor (PXR). This renders them mechanistically counterproductive since CAR and PXR have overlapping functions. Through a directed screening approach we identified new CAR inhibitors from a subset of small molecules shortlisted as PXR inhibitors in a high‐throughput screen of 320,000 chemicals.The lead small molecule discovered was CINPA1, capable of reducing CAR‐mediated transcription with an IC50 of ~70 nM but without activating PXR. The mechanisms by which CINPA1 inhibits CAR function were also extensively examined and elucidated. CINPA1 association with the CAR‐LBD reduced recruitment of coactivators. Reduced CAR occupancy at the promoter regions of its target genes was observed in the presence of CINPA1. The pharmacokinetic properties of CINPA1 were determined and CINPA1 confirmed to be broken down into two main metabolites. In vitro enzyme assays analyzed by LC‐MS facilitated the identification of the cytochrome P450 enzymes responsible for metabolism of CINPA1 in human liver microsomes. CINPA1 is converted to the first metabolite by CYP3A4 and then further metabolized by CYP2D6. The metabolic profiling information for CINPA1 will help guide the in vivo PK/PD studies in mice. Ongoing structure activity relationship studies have also revealed some key features of this inhibitor and metabolites. Metabolite 1 is a mild CAR inhibitor while metabolite 2 has no effect on CAR function. Computational modeling using the CAR‐LBD structure shows that while CINPA1 and metabolite 1 can bind in the CAR ligand binding pocket, metabolite 2 might be unable to make the necessary molecular interactions required for binding.We describe the pharmacokinetic properties of CINPA1, a potent inhibitor of CAR that could be a novel molecular tool for understanding CAR function and could be utilized for resensitizing chemoresistant cancer cells as in the case of neuroblastoma.Support or Funding InformationNIH, ALSAC
Nitric Oxide Stimulates CYP51A1 Down Regulation
Authors: Byrd Aria , Lee Choon‐myung , Park Ji Won , Morgan Edward Thomas 
Published: November 2023

Cytochrome P450 proteins (P450) are a family of enzymes found in virtually all living organisms. These proteins are responsible for the metabolism of xenobiotics as well as clearance and biosynthesis of endogenous compounds in the body. Cholesterol is an endogenous sterol that is vital for maintaining cell membrane fluidity and serving as a precursor for the production of important biological compounds such as vitamin D, steroid hormones and bile acids. The production of cholesterol is reliant on CYP51A1, which is the only P450 that converts lanosterol to 4, 4‐dimethylcholesta‐8(9), 14, 24‐trien‐3β‐ol via 14α‐demethylation. CYP51A1 is also believed to be the oldest recognizable P450, as it is the only P450 with a conserved function across animal, fungal and plant kingdoms. Previous studies have shown that nitric oxide (NO) formed in hepatocytes in response to inflammatory stimuli can cause down regulation of a number of drug metabolizing P450s. The objective of this study is to determine if CYP51A1 is down regulated by NO. Huh7 cell lines were transduced with lentiviral vectors containing pLX304‐CYP51A1V5 to generate v5‐tagged CYP51A1 expressing cell lines. HeLa cell lines were transduced with lentivirus containing doxycycline inducible pLIX304‐hNOS2, in which human nitric oxide synthase2 (hNOS2) is regulated through a tetracycline promoter region, and additionally transduced with lentiviral vectors containing pLX304‐CYP51A1V5. Huh7 cell lines were treated with NO donors (the NONOates DPTA and NOC‐18, and S‐nitrosoglutathione, GSNO) and then CYP51A1 protein expression was determined by Western Blot Analysis. Results showed that NOC‐18 down regulated 51A1 in both a time and dose dependent manner. Treatment with DPTA (6 h) and NOC‐18 (18 h) were more effective than GSNO in down regulation of 51A1. The HeLa cell lines treated with doxycycline produced amounts of NO comparable to those observed in human hepatocytes, which was blocked by the competitive hNOS2 inhibitor L‐NG‐nitroarginine methyl ester (L‐NAME). Doxycycline treatment for 12 or 18 hours down regulated 51A1 protein expression to 25–30% of control values. L‐NAME treatment blocked the down‐regulation of CYP51A1 in doxycycline‐treated cells. We conclude that NO stimulates the down regulation of CYP51A1 in both HeLa and Huh7 cell lines. Future studies will investigate whether this down regulation is achieved through proteosomal degradation subsequent to ubiquitin tagging, as has been seen in the down regulation of drug metabolizing P450s like CYP2B1.Support or Funding InformationThis research is supported by the National Institutes of Health grant R01GM069971
Synergistic Interaction of Histone Deacetylase Inhibitors with Interferon‐β in Inducing Apoptosis of Human Neuroblastoma Cells
Authors: Dedoni Simona , Olianas Maria C. , Onali Pierluigi 
Published: November 2023

The type I interferon (IFN) family of cytokines, comprising IFN‐α, IFN‐β, IFN‐ω and IFN‐κ, displays antiproliferative activity and has been used with some success in the treatment of different human neoplasias. We have previously observed that IFN‐β causes cell death of human SH‐SY5Y neuroblastoma (NB) cells by inducing apoptosis through the intrinsic mitochondrial pathway (Dedoni et al. J. Neurochem. 115,1421‐1433, 2010). The histone deacetylase (HDAC) inhibitor valproic acid has been reported to enhance the antiproliferative effects of type I IFNs, but little is known on whether the two agents synergize in inducing NB cell apoptosis. In the present study we show that prolonged exposure (24 h) of SH‐SY5Y cells to therapeutic concentrations of valproic acid (0.6 and 1 mM) had no effect on cell viability but, when combined with IFN‐β (30 ng/ml), significantly enhanced the apoptotic cell death induced by the cytokine. The synergistic interaction was associated with enhanced expression of apoptotic markers, including caspase activation, DNA fragmentation, and cleavage of the DNA‐repairing enzyme poly‐(ADP ribose) polymerase (PARP) and the pro‐survival transcription factor β‐catenin. Valproic acid enhanced the IFN‐β‐stimulated Tyr‐phosphorylation of STAT1, which is required by the cytokine to trigger apoptosis. Sodium butyrate (1 mM) and trichostatin (100 and 300 nM), two additional HDAC inhibitors, mimicked the valproic acid potentiation of IFN‐β‐induced apoptosis, whereas valpromide (1 mM), which did not enhance histone acetylation, was without effect. Differently from SH‐SY5Y cells, MYCN‐amplified human LAN‐1 NB cells displayed no sensitivity to the pro‐apoptotic activity of IFN‐β, but underwent apoptosis when treated with either valproic acid or sodium butyrate. Co‐treatment of LAN‐1 cells with IFN‐β and HDAC inhibitors, but not valpromide, resulted in a significant increase in the expression of the apoptotic markers. These data demonstrate a synergistic interaction of IFN‐β and HDAC inhibitors in promoting the apoptotic death of NB cells and support the use of this combination for the treatment of human NB.Support or Funding InformationThe study was supported by Regione Autonoma della Sardegna, Italy, L.R. n. 7/2007‐CRP10810/2012.
Diminazene Aceturate Attenuates Obesity and Promotes a Balance of the Renin‐Angiotensin System in Fat and Muscle Tissue in Young and Aged Rats on High Fat Diet
Authors: Sakarya Yasemin , Bruce Erin , Morgan Drake , Tümer Nihal , Carter Christy S. , Scarpace Philip J. 
Published: November 2023

Obesity is a complex disorder, and increases the risk for a number of chronic diseases, including diabetes and cardiovascular diseases. Renin‐angiotensin system (RAS) is considered the major regulator of cardiovascular homeostasis. Recent studies suggest stimulation of the vasoprotective ACE2/Ang (1–7)/Mas axis of the RAS leads to significant reductions in body weight in obese animal models. We have shown that Diminazene Aceturate, a novel ACE2 activator, significantly reduced body weight of both young and aged rats fed 60% high fat diet. Notably we observed a significant loss of fat mass with a preservation of lean mass. We hypothesize that maintaining balance between the ACE/AngII/AT1R axis and vasoprotective components in muscle and white adipose tissues will prevent much of the associated pathophysiology of the obesity.Male Fisher 344 × Brown Norway rats, ages 4 (n=12) and 23 months (n=17) were fed 60% high fat diet for one week, whereupon, animals were further divided, and given either 15mg/kg/day DIZE s.c. (Young DIZE, n=6; Old DIZE, n=9) or vehicle (Young Control, n=6; Old Control, n=8). Body weight and food intake were measured throughout the experiment. Time Domain‐Nuclear Magnetic Resonance (TD‐NMR) was used to assess body composition 1 week and 3 weeks after the start of DIZE treatment. With the aim of elucidating the effect of DIZE on the relationship between RAS components, AT1R, AT2R, MasR, ACE, ACE2 mRNA were measured in retroperitoneal white adipose tissue (RTWAT), and tibialis anterior (TA) muscle, using qPCR.Relative quantitation of RTWAT mRNA expression showed DIZE treatment significantly increased AT2R (5 fold) and MasR (1 fold) in old animals when compared to non‐treated controls. AT1R expression was significantly increased in the muscle of old rats treated with DIZE; however, the AT1R/AT2R ratio was not different from control. Furthermore, the ACE/ACE2 ratio was decreased in the muscle tissue in both young and old rats. Our data suggests DIZE may be a useful tool in maintaining the balance between the two opposing arms of the RAS, and will be an effective treatment to ameliorate obesity and related disorders.Support or Funding InformationHypertension Center T32 Post Doctoral training grant, North Florida/South Georgia Veterans Health System (Research/GRECC), and National Institute's of Health Grant NIH DK 091710