bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of the Membrane Topology of the PORCN O‐Acyl Transferase
Authors: Fraley J Gabriel , Galli Lisa , Burrus Laura , Sanchez Luis
Published: November 2023

WNT signaling is critical for proper embryonic development and adult tissue homeostasis. WNT proteins require palmitoylation for optimal secretion and receptor binding. Porcupine (PORCN), a membrane bound O‐acyltransferase (MBOAT) family member is the protein that palmitoylates WNT. PORCN is localized to the Endoplasmic Reticulum (ER). Though PORCN is required for proper tissue patterning in developing embryos, its membrane topology is poorly understood. Determining the topology of PORCN may have useful applications in areas of embryonic development studies. Although numerous bioinformatics algorithms that predict membrane topology are available, no clear consensus for the topology of PORCN has emerged. Therefore, our goal is to experimentally determine the topology of PORCN.

To determine topology, we developed two strategies. The first strategy utilized differential solubilization and immunofluorescent imaging of single, and dual tagged PORCN variants. Results from single Myc‐tagged N‐and C‐terminus PORCN variants in transfected COS‐7 cells orient the N‐terminus in the ER lumen, and the C‐terminus in the cytosol. To further determine topology, we developed twelve dual tagged PORCN variants with Myc epitopes within predicted loops, and a C‐terminal 3X FLAG tag. The construction of dual tagged variants provided us with qualitative information, as well as quantitative values using Mander's coefficients of colocalization. Our studies show that five of the PORCN variant loops exhibit cytosolic orientation while one loop was oriented toward the lumen. As a control, we showed that the N‐ and C‐terminal tags remained lumenal and cytosolic, respectively. However, the remaining constructs provided conflicting results. Additionally, a PORCN antibody was generated against an immunogenic region that includes residues 282–303. Immunofluorescent imaging and colocalization quantitation orient this epitope towards the cytosol.

As a second strategy, we developed ten mutants which resulted in the introduction of ectopic consensus sites for the addition of N‐linked carbohydrates (NxT/NxS). These PORCN mutants were transfected into HEK‐293T cells and subjected to Western blot analysis to identify glycosylated variants. This analysis identified two glycosylated residues at A134N and D283N, which indicates localization of these two residues to the lumen of the ER. Intriguingly, these results do not agree with those from our differential solubilization studies. We are now carrying out additional controls to help us distinguish between these two results.

Support or Funding Information

NIH‐MARC T34‐GM008574
Multiple Modes of Inhibition of Human Cytochrome P450 2J2 by Dronedarone, Amiodarone and their Active Metabolites
Authors: Chan Eric Chun Yong , Karkhanis Aneesh , Lam Hui Yuan , Hong Yanjun , Venkatesan Gopalakrishnan , Chai Christina Li Lin , Koh Siew Kwan , Zhou Lei , Kojodjojo Pipin
Published: November 2023

Dronedarone, a multiple ion channel blocker is prescribed for the treatment of paroxysmal and persistent atrial fibrillation. While dronedarone does not precipitate toxicities like its predecessor amiodarone, its clinical use has been associated with idiosyncratic hepatic and cardiac adverse effects and drug‐drug interactions (DDIs). As dronedarone is a potent mechanism‐based inactivator of CYP3A4 and CYP3A5, a question arose if it exerts similar inhibitory effect on CYP2J2, a predominant cardiac CYP450 enzyme. In this study, we demonstrated that CYP2J2 is reversibly inhibited by dronedarone (Ki=0.0293 μM), amiodarone (Ki=4.782 μM) and their respective pharmacologically active metabolites namely N‐desbutyldronedarone (NDBD) (Ki=0.550 μM) and N‐desethylamiodarone (NDEA) (Ki=7.352 μM). Moreover, time‐, concentration‐ and NADPH‐dependent irreversible inactivation of CYP2J2 was investigated where inactivation kinetic parameters (KI, kinact) and partition ratio (r) of dronedarone (0.0498 μM, 0.0336 min−1, 3.3), amiodarone (0.213 μM, 0.0146 min−1, 20.7) and NDBD (0.479 μM, 0.0235 min−1, 21.7) were observed except for NDEA. The absence of the characteristic Soret's peak, lack of recovery of CYP2J2 activity upon dialysis, and biotransformation of dronedarone and NDBD to quinone‐oxime reactive metabolites further confirmed the irreversible inactivation of CYP2J2 by dronedarone and NDBD is via the covalent adduction of CYP2J2. Our novel findings illuminate the possible mechanisms of DDIs and cardiac adverse effects due to both reversible inhibition and irreversible inactivation of CYP2J2 by dronedarone, amiodarone and their active metabolites.

Support or Funding Information

Dean's Chair Professorship (Faculty of Science, National University of Singapore)
Discovery and development of tylophorine derived dibenzoquinolines‐33b compounds into therapeutic agents
Authors: Lee Shiow‐Ju , Li Chih‐Hung , Lee Yue‐Zhi , Yang Cheng‐Wei
Published: November 2023

We investigated the role of the tylophorine E ring on the biological activities through synthesis of a series of derivatives, bearing modifications at the E ring and N‐substitutions. All the derivatives were submitted for a variety of tests, anti‐cell growth against a panel of cancer cell lines, suppressing nitric oxide production inLPS/IFNgamma stimulated RAW264.7 cells, and anti‐viral replication in TGEV infected ST cells detected by inhibition of TGEV N and S protein expression. The uncyclized derivatives, dibenzoquinolines, do not have the enantimerism issue at C13a position. We have synthesized a series of novel tylophorine‐derived dibenzoquinolines and evaluated for their biological activities. The role of tylophorine E ring was explored unprecedentedly for the first time. Unlike other reported tylophorine derivatives, the potent tylophorine‐derived dibenzoquinolines appear to retain similar modes of action to those of tylophorine in terms of multi‐biological activities for anti‐inflammation, anti‐cancer cell proliferation, and anti‐coronavirus. The most potent compound dibenzoquinolines‐33b (DBQ‐33b) showed improved solubility, in vivo efficacies in a murine tumor xenograft model administrated orally and a murine paw edema model, good bioavailability, and no significant neurotoxicity tested by a rota‐rod test for motor coordination. More orally active leads derived from DBQ‐33b have been designed and synthesized and development of DBQ‐33b derived lead compounds into therapeutic agents is ongoing.

Support or Funding Information

This work was supported by a grant (MOST 104‐2325‐B‐400‐006‐) from the National Research Program for Biopharmaceuticals, Ministry of Science and Technology, Taiwan ROC.
In Vitro Invastigation Effects of 4‐hydroxyacetophenone on Rat Torasic Aorta's Vasomotor Activity
Authors: GELEN Volkan , ÇELEBİ Fikret
Published: November 2023

Aim

Z. clinopodioides Lam. is also known as a “Field Mint” Seven compounds which have vasodilator activity have been isolated from Z. clinopodioides Lam. and two of them are phenolics compounds and these are acetovanillone, 4‐HAP, four of them are flavonoids and these are acacetin, apigenin, chrysin, thymonin one derivat of cinnamic acid and ethyl 4‐coumarate. In this study, it was aimed to define vasodilator activiy mechanisms of fenolic compound 4‐HAP on isole rat torasic aorta.

Material and Method

In this study 40 male adult Sprague Dawley type of rat were used. Prepared rings were laid out into the 20 ml organ bath with Kreb solution. Rings were stretched by 1g and they were left to 1 hour incubation period. In the end of the incubation period, FE, KCI, nifedipine, L‐NAME, 4‐HAP, SQ22.536, ODQ, ACh, SKF96365, Propranolol, Atropin, TEA, Gibenclamide, 4‐aminopyridine and U73122 were implemented to bath with a protocol.

Results

Mechanisms of this relaxed effect was assigned by using antagonists. It was observed that Vasorelaksan effect of 4‐HAP on endothelilal aorta straight muscle contractions which had been inductioned by FE under the existance of L‐NAME was considerably inhibited.

Conclusion

It was stated that 4‐HAP relaxed FE and KCI contractions and owing to this activity endothelilal tissues on L‐NAME existance notably decreased because of NO networkway. It is firmly believed that as relaxed effect of 4‐HAP declines remarkably under the existance of 4‐aminopyridine and nifedipine on unendothelilal aorta rings, activity could be on K+ chanal and L‐type Ca+2 chanal.

Support or Funding Information

Supported of ÖYP
Physcion Induces Vasorelaxation In Rat Aorta through Endothelium‐dependent and Endothelium‐independent Mechanisms
Authors: Zhou Zi Qi , Sun Sha Sha , Zhou Guang Hai , Kang Dae Gill , Lee Ho Sub , Yang Sun Nye , Cho Kyung Woo , Jin Song Nan , Wen Jin Fu
Published: November 2023

Background

Physcion, an active anthraquinone constituent isolated from the rhubarb, a traditional Chinese herbal medicine which is widely used in clinical treatment, has cardiovascular protective properties. However, the effects of physcion on the vascular function and its pharmacological mechanisms of action have not been studied. The aim of the present study was to evaluate the vascular effects of physcion and its action mechanisms in rats.

Methods and Results

Aortic rings from Sprague‐Dawley rats were mounted in organ baths, and the effects of physcion on vascular function were studied by means of isometric tension recording. Physcion induced a concentration‐dependent relaxation of pre‐contracted aortic rings in endothelium‐intact (+E) and endothelium‐denuded (−E) vessels. The nitric oxide synthase inhibitor L‐NAME, as well as the soluble guanylate cyclase inhibitor ODQ, attenuated the relaxation in +E but not in −E vessels. Inhibition of cyclooxygenases with indomethacin had no effects on the physcion‐induced vasorelaxation in +E vessels. A modulator of the store‐operated Ca2+ entry (SOCE), 2‐aminoethyl diphenylborinate markedly attenuated the physcion‐induced vasorelaxation in +E vessels. In −E preparations, K+ channel blockers, such as tetraethylammonium, large‐conductance calcium‐activated K+ (KCa) channel blocker iberiotoxin, and KATP channel blocker glibenclamide significantly attenuated the relaxation elicited by physcion.

Conclusions

These data demonstrate that physcion induces vasorelaxation through activation of endothelium‐dependent and ‐independent signaling pathways: endothelium‐dependent NO‐cGMP signaling and endothelium‐independent KCa channel and KATP channel signaling.

Support or Funding Information

Acknowledgments: This work was supported by research grants from the National Natural Science Foundation of China (Nos. 81270920, 31571175), the Natural Science Foundation of Shandong Province (No. ZR2015HM037).
Mechanisms of Dietary Fructose and Copper in the Development and Progression of NAFLD Spectrum Disorders
Authors: Morrell Austin , Burkhead Jason
Published: November 2023

Dietary induced oxidative stress appears to be factor which affects the development and progression of non‐alcoholic fatty liver disease (NAFLD) spectrum disorders, however no clear mechanism(s) for this have been identified. Preliminary studies by the Burkhead lab and others suggest that exposure to high oxidative stresses through the diet, lowers hepatic copper import even in copper deeffiecnt conditions. This downregulation of copper import in combination with high fructose consumption could have a synergist effect increasing the levels of oxidative stress (and therefore cellular damage) experienced by hepatocytes while lowering their ability to respond to it via superoxide dismutase. Using cell as well as animal models we aim to identify the mechanisms that allow oxidative stress, particularly that caused by diet, to regulate hepatic copper import and the systematic effects of this, as they related to NAFLD. These results could help explain the rising prevalence of NAFLD/NASH as well as identifying one potential mechanism that aids the progression of NAFLD to NASH.

Support or Funding Information

Institutional Development Award (IDeA)
Suppression of the gut microbiome with broad‐spectrum antibiotic cocktail reverses age‐related arterial dysfunction in mice
Authors: Richey James , Cuevas Lauren , Neilson Andrew , Smithson Andrew , Gilley Andrew , Seals Douglas , Gioscia‐Ryan Rachel
Published: November 2023

Aging is a primary risk factor for cardiovascular diseases (CVD), driven largely by the development of arterial dysfunction, characterized by impaired endothelial function and stiffening of the large elastic arteries secondary to oxidative stress. Increasing evidence suggests a role for the gut microbiome in modulating host physiology, as dysregulation of the abundance and type of gut microbes can have systemic consequences, including promotion of oxidative stress and production of adverse gut microbe‐dependent metabolites such as trimethylamine n‐oxide (TMAO). TMAO is causally linked to atherosclerosis and independently predictive of CVD risk. However, the potential role of the gut microbiome in modulating arterial function with primary aging is unknown. We assessed endothelial function (carotid artery endothelium‐dependent dilation [EDD] to acetylcholine), arterial stiffness (aortic pulse‐wave velocity [aPWV]), circulating levels of TMAO, and aortic markers of oxidative stress (via electron paramagnetic resonance [EPR] and Western blot) in young (YC; 6 mo; n=5), old (OC; ~27 mo; n=5) and old antibiotic (Abx)‐treated mice (OA; n=4; 3‐wk broad‐spectrum Abx cocktail in drinking water to suppress endogenous gut microbes). Plasma TMAO was dramatically reduced with Abx treatment (OC: 14.7 ±5.3 vs. OA: 0.8 ±0.2 μM, p <0.05), indicating gut microbe suppression. Abx treatment reversed the age‐related impairment in peak EDD (OC: 69.2 ±6.4, p<0.05 vs. YC: 90.4 ±6.9 and OA: 83.5 ±3.9 %) and the age‐associated elevation in aPWV (OC: 487 ±30, p <0.05 vs. YC: 373 ±14 and OA: 360 ±47 cm/s). Improved arterial function with Abx was accompanied by reduced arterial oxidative stress, as indicated by attenuation of the age‐related increase in aortic superoxide production (EPR; OC: 9859 ±6086, p<.05 vs. YC: 2983 ±1711 and p=0.11 vs. OA: 6057 ±1806 AU) and augmentation of antioxidant enzyme extracellular superoxide dismutase expression (OC: 2.7 ±0.11, p =0.06 vs OA: 3.3 ±.32 AU). Our results identify the gut microbiome as an important mediator of age‐related arterial dysfunction and oxidative stress, and suggest that therapeutic strategies targeting the gut microbiome may hold promise for preserving arterial function with aging, and reducing risk of CVD.

Support or Funding Information

Supported by: NIH F31AG047784, R01HL107120, R01AG013038
Oxidized Low‐Density Lipoproteins Enhance Endothelial Barrier Function
Authors: Zhang Chongxu , Crystal Crystal , Meliton Lucille , Dudek Steven , Levitan Irena
Published: November 2023

Oxidized modifications of low‐density lipoprotein (OxLDL) are known to contribute to the formation of atherosclerotic plaques and early endothelial dysfunction. Earlier studies showed that exposure to oxLDL results in the disruption of the endothelial barrier suggesting that it may contribute to the initiation of the disease. An important constraint of these studies, however, was that oxLDL was generated by copper oxidation, an artificial process that does not occur in vivo and that may generate oxLDL species with chemical and biological properties different from those in vivo. In this study, therefore, we compared two types of oxLDL: LDL oxidized by copper sulfate (Cu‐oxLDL) or LDL oxidized by lipoxygenase (LPO‐oxLDL), a physiological process that is responsible for oxLDL generation in vivo. Surprisingly, we found that both, Cu‐oxLDL and LPO‐oxLDL transiently enhance the barrier function of Human Aortic Endothelial cells (HAECs). Specifically, endothelial permeability of HAECs was first measured using Electric Cell‐substrate Impedance Sensing (ECIS), a well‐established method to assess endothelial barrier function. Our results show that exposure to 100 μg/ml LPO‐oxLDL resulted in 40% increase in the resistance of HAECs monolayer, an effect that peaked at 2 hours and dissipated after 20 hours. Exposure to Cu‐oxLDL had a similar but smaller effect: the resistance of HAECs monolayer increased by 10% after 2 h and dissipated after 20 hours. No further change was observed at longer exposures. To verify these observations, we used an alternative method, by measuring the leak of a fluorescent FITC‐dextran (40 kDa) in the trans‐well system. Exposure to both Cu‐oxLDL and LPO‐oxLDL resulted in a ~50% decrease of HAECs permeability to FITC‐dextran. We further tested if ROS play a role in the oxLDL‐induced enhancement of HAECs barrier function using MnTMPyP, a cell‐permeable ROS scavenger. Our data show that MnTMPyP attenuated the effects of both Cu‐oxLDL and LPO‐oxLDL on HAECs permeability. In conclusion, our data suggest that oxLDL may have a protective effect on endothelial barrier function and that this effect is mediated by ROS. Further studies are needed to evaluate the role of this process to vascular permeability in vivo.

Support or Funding Information

This study is supported by NIH grants HL‐073965 and HL‐083298 to I.L. and HL‐079356 to K.W.
Sympathoadrenal activity, blood pressure and depression in Mexican children 10–14 years old with type 2 diabetes mellitus risk factors
Authors: Fernandez Ana Cecilia , Flores Constantino , Esquivel Aurora , Martinez Beatriz Elina , Fernando Shane , Pina Alexandra Soto , Ramos Roxana Elina Valdés
Published: November 2023

Increased physical or emotional responses to stress may result in increased cardiovascular disease and Type 2 Diabetes Mellitus (T2DM) risk factors. The consequent raise in blood pressure may be a direct effect of catecholamines by sympathetic stimulation, but its relation to T2DM risk is not well known. Our aim was to evaluate sympathoadrenal activity and blood pressure in children with low and high T2DM risk. This was a cross‐sectional descriptive study with children from public schools with at least one risk factor: family history (FH) of T2DM; FH of hypertension; presence of Acantosis Nigricans; fasting capillary glucose 100–126 mg/dL; or BMI ≥85th percentile. Data collection included anthropometric and blood pressure measurements, blood samples and Infant Depression Inventory (CDI‐2). Data were analyzed by sex using descriptive statistics and linear regression model. The sample consisted of 116 children (52 girls and 64 boys). FH of T2DM was higher among girls compared with boys (78.84% vs. 65.62%, p < 0.05). Presence of depression and mean CDI‐2 scores were higher among girls (26.92% and 56.35±12.81) compared with boys (13.11% and 53.56±11.42) (p < 0.05). Mean systolic blood pressure was higher among boys compared with girls (104.81±11.45 mm Hg vs. 103.18±13.86 mm Hg; p < 0.001) and mean diastolic blood pressure was higher for girls (67.64 ± 10.47 mm Hg vs. 65.96 ± 12.65 mm Hg; p < 0.001). Concentrations of plasma total and fractionated catecholamines were lower in girls compared with boys: 511.63±168.91 pg/ml vs. 652.42±174.56 pg/ml for total catecholamines; 67.98±49.49 pg/ml vs. 104.54±77.24 pg/ml for epinephrine; 435.65±143.44 pg/ml vs. 535.54±144.96 pg/ml for norepinephrine; and 18.00±19.78 pg/ml vs. 19.20±11.59 pg/ml for dopamine, respectively. In summary, girls have a lower sympathetic activity, lower systolic blood pressure and higher depression scores compared with boys, and may be associated to their hormonal condition.

Support or Funding Information

Fondo Semilla Universidad Autonoma del Estado de Mexico and University of North Texas Health Science Center
Loss of local Ca2+ signaling networks in the endothelium in diet induced obesity
Authors: Hong Kwangseok , Cope Eric L , Marziano Corina , Sonkusare Swapnil K
Published: November 2023

Endothelial cells (ECs) play a crucial role in regulating vascular function. Weakening of local Ca2+ signaling networks in the ECs leads to the loss of endothelial vasodilation in hypertension (Sonkusare et al., Science Signaling, 2014). There is increasing evidence that obesity is also associated with a loss of endothelial function. The objective of this study was to determine the impact of diet‐induced obesity (DIO) on the local Ca2+ signaling networks that regulate endothelial vasodilation. We recently showed that elementary Ca2+ influx events through endothelial TRPV4 (transient receptor potential vanilloid 4) channels (TRPV4 Ca2+ sparklets) dilate mesenteric arteries (MAs) via Ca2+‐sensitive, intermediate conductance K+ (IK) channels (Sonkusare et al., Science, 2012). Moreover, muscarinic receptor agonist carbachol (CCh) dilates MAs via activation of TRPV4 sparklets at myoendothelial projections (MEPs), an interaction that requires MEP‐localized A kinase anchoring protein 150 (AKAP150). Diameter studies were performed in 3rd order mouse MAs constricted by intravascular pressure (80 mm Hg) and Ca2+ events were recorded in the slit‐open, pinned down MAs using a spinning disk confocal imaging system. After 8–10 weeks of high‐fat diet feeding, the vasodilations to CCh (0.3–10 μM) and GSK1016790A (TRPV4 channel agonist, 3–30 nM) were attenuated in the DIO mice. The activity of TRPV4 sparklets, recorded as the fluorescence integral of all the sparklet sites per field of view (~14 ECs), was 4‐fold lower in the DIO mice compared to the mice fed with a normal diet. Whereas 90% of MEPs showed identifiable AKAP150 staining in the control mice, only 32% MEPs showed AKAP150 staining in the DIO mice. These results support the concept that a loss of AKAP150 from the MEPs and resulting TRPV4 channel dysfunction may be responsible for impaired endothelial vasodilation in the DIO mice.

Support or Funding Information

Funded by NIH award HL121484 to SKS.
Fibrocyte markers are increased in the pudendal artery of spontaneously hypertensive rats (SHR)
Authors: Ogbi Safia , Webb R. Clinton
Published: November 2023

Erectile dysfunction is associated with the presence of hypertension and its incidence increases with the severity and duration of blood pressure elevation. In hypertension, vascular dysfunction and corpus cavernosum dysfunction are characterized by similar hallmark alterations, such as decreased relaxation and augmented contractile responses, vascular inflammation and remodeling. Fibrocytes are bone marrow derived mesenchymal progenitor cells that have recently been linked with fibrosis and vascular remodeling and are increased in hypertension. We hypothesized that fibrocytes contribute to pudendal artery remodeling in the spontaneously hypertensive rat (SHR). Pudendal arteries were isolated from male SHR and normotensive Wistar‐Kyoto control rats (WKY). Co‐staining for pro‐collagen and CD45, markers of fibrocytes, were observed in pudendal arteries using confocal microscopy. Protein expression of pro‐collagen and CD45 was also quantified by western blotting. Our results indicate that protein expression of fibrocyte markers, as well as co‐staining, were increased in the pudendal artery of SHR compared to WKY. These data suggest that fibrocyte‐induced vascular dysfunction in the pudendal artery could be associated with ED in hypertension. Therefore, fibrocytes can be a putative target for novel therapeutic approaches of this condition.

Support or Funding Information

American Heart Association
Cannabinoid Receptor Type‐1 Regulates Inflammation and Insulin Resistance in Metabolic Endotoxemia
Authors: Grunewald Zachary Ian
Published: November 2023

Background

Circulating endotoxin, or lipopolysaccharide (LPS), is a suggested trigger of obesity‐associated inflammation and insulin resistance. In rodents, chronic low‐level administration of LPS induces weight gain, inflammation and insulin resistance, inducing metabolic endotoxemia. Additionally, obesity is associated with over‐activation of the endocannabinoid system, promoting insulin resistance. Interestingly, LPS has been shown to regulate endocannabinoid synthesis. We hypothesized that metabolic endotoxemia promotes inflammation and insulin resistance via a cannabinoid receptor type‐1 (CB1R) dependent pathway.

Methods

Male Wistar rats fed a chow diet were implanted with mini‐osmotic pumps delivering a low dose of LPS (n=20, 12.5 μg/kg/hr) or saline (n=10) continuously for 6 weeks (42 days). 10 rats receiving LPS were injected with a CB1R antagonist (SR141716, ip 3μg/g) daily. Rats were pair‐fed to the level of the LPS + CB1R antagonist group (CB1Rx) to control for differences in body weight. Glucose tolerance (OGTT, 2g/kg) and insulin sensitivity (ITT, 0.75u/kg) were measured after 5 weeks of treatment. Insulin was given 1 hour prior to sacrifice (n=5/group). RT‐PCR, ELISA assays, and western blot was used to determine experimental outcomes.

Results

After 6 weeks, pair‐feeding was successful to control for body weight and energy intake differences. LPS administration was sufficient to alter insulin sensitivity. However, alterations to both glucose and insulin sensitivity were further exacerbated in the CB1Rx group. Circulating LPS binding protein was elevated in the CB1Rx group only. Serum adiponectin was reduced in both groups receiving LPS infusion compared to control. Liver, visceral and periaortic adipose tissue, and muscle exhibited a pro‐inflammatory phenotype in LPS and LPS + CB1Rx treated animals. Liver total AKT/PKB was reduced in the CB1Rx group only. p‐AKT (T308) was reduced in the LPS treated group and further reduced in the CB1Rx group. Insulin degrading enzyme was partially attenuated with CB1Rx treatment compared to LPS treatment alone.

Conclusions

Our findings oppose our hypothesis that LPS induces inflammation and insulin resistance via a CB1R dependent pathway. Rather, inhibition of CB1R further exacerbates the inflammatory and insulin resistant state induced by chronic low‐level LPS administration. Moreover, our findings suggest that CB1R may play a role in regulating the pro‐inflammatory and insulin resistant phenotype associated with obesity. Thus, these findings suggest that CB1R inhibition may not be a beneficial strategy towards the treatment of obesity‐associated inflammation and insulin resistance.

Support or Funding Information

Funding for this project came from 1) UGA Junior Faculty Research Grant for Claire de La Serre, 2) Kappa Omicron Nu Eileen C. Maddex Master's Fellowship for Zachary I. Grunewald
Periodic Blue Light Exposure Activates Microglial Kinases and Alters Gene Expression
Authors: Cheng Kevin P , Williams Justin C , Watters Jyoti J.
Published: November 2023

The potential side effects of blue light used in either optogenetic and/or in vivo imaging applications in the CNS are poorly understood. Previously we reported that periodically delivered blue light (450nm, 25–100 mJ/cm2 in a single 1 second pulse per minute for 6 hours) altered the basal expression levels of a subset of genes in primary murine microglia that do not express exogenous light‐responsive proteins. However, the mechanism whereby blue light induces these changes, or whether they are temporary and/or reversible, remains unknown. Here we demonstrate that blue light‐induced changes in microglial gene expression are evident up to at least 18 hours following the cessation of light exposure, suggesting that constant light delivery is not necessary to observe long‐lasting effects. To further understand the mechanism underlying these gene expression changes, we tested the ability of blue light to activate the AMP‐activated protein kinase (AMPK) and Protein Kinase B (AKT) pathways, two signaling cascades known to exert strong downstream transcriptional effects on inflammatory and cell survival related genes. Preliminary data suggest that both pathways may play a role in the effects of blue light in microglia. These results demonstrate that long‐term optogenetic and in vivo imaging applications may have previously unappreciated off‐target effects as they may alter the basal gene expression profiles and kinase activity of all light‐exposed cells, not only of those optogenetically targeted.

Support or Funding Information

This work was supported by a DARPA HIST grant.
Theobromine, the primary methylxanthine found in Theobroma cacao, can pass through the blood‐brain barrier in mice
Authors: Sugimoto Naotoshi , Katakura Masanori , Matsuzaki Kentaro , Ohno‐Shosaku Takako , Yachie Akihiro , Shido Osamu
Published: November 2023

Theobromine, a caffeine derivative, is the primary methylxanthine produced by Theobroma cacao. We previously showed that theobromine has anti‐tumor effect and inhibits the cell growth of a glioma cell line derived from human malignant glioma [1]. Glioma is a cancer of brain. So, for exerting the anti‐tumor effect, theobromine must pass though the blood‐brain barrier.

Here, we investigate whether theobromine could pass though the blood‐brain barrier in mice. All animal experiments were performed in accordance with the Guidelines for Animal Experimentation of Shimane University Faculty of Medicine, which were compiled from the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science. Mice were divided into two groups. First group mice had a normal diet, and second group mice had a diet supplemented with 0.05% of theobromine for 30 days. We show that theobromine is detected in cortex as well as plasma in mice that had the diet with theobromine. These results suggest that theobromine can pass through the blood‐brain barrier in mice.

Support or Funding Information

This work was supported in part by the Grants‐in‐Aid for Science and Culture of Japan (25282021).
Chondrocyte Metabolic Flexibility and Early‐Stage Osteoarthritis in Mice Fed a High‐Fat Diet
Authors: Donovan Elise , Hudson Joanna , Kinter Mike , Griffin Timothy
Published: November 2023

Introduction

Obesity is a primary risk factor for osteoarthritis (OA). However, how chondrocyte metabolism changes in response to a high‐fat diet (HFD) and the mechanisms by which obesity initiates and promotes pro‐catabolic changes in chondrocytes that lead to OA are not fully understood. The objective of this study was to better understand the initiating cellular factors associated with OA pathogenesis in high‐fat diet (HFD)‐induced obese mice by examining changes in the expression of chondrocyte metabolic pathway components.

Methods

Male C57BL/6J mice (n = 10–12 per diet per time point) were fed either a HFD (60% kcal fat) or a control diet (Ctr; 10% kcal fat) beginning at 6 weeks of age. Knee OA histopathology was examined using a modified Mankin OA scoring system following 20, 32, and 46 weeks of HFD feeding. In the 32‐week cohort, chondrocyte intracellular lipid accumulation was measured by oil red O staining. In an additional cohort of animals at 20 weeks (n = 3–5), mRNA and protein were isolated from knee articular cartilage to examine chondrocyte transcriptional and protein expression changes.

Results

HFD for 20 weeks increased osteophyte formation (p = 0.03) and tidemark duplication (p = 0.03) but did not increase the modified Mankin OA score, which was only significantly increased after 46 weeks on the HFD (p = 0.02). Because early osteoarthritic changes were observed by 20 weeks of HFD feeding, we examined cartilage transcriptional changes at this time point. None of the extracellular matrix genes (Aggrecan, Col10, Col2a) or matrix degrading proteases (Mmp9, Mmp13, Adamts5) were differentially expressed in HFD animals compared to Ctr at this time point, consistent with the histology. Cpt1a gene (1.4 fold increase, p = 0.03) and protein (1.87 fold increase, p = 0.04) expression were increased in the HFD group, suggesting increased transport of fatty acids into the mitochondria for oxidation. Of the 16 glycolysis related proteins examined, 62% were decreased in HFD animals (outside the reference gene CV range of 0.94 – 1.06), and of the 15 β‐oxidation proteins examined, 63% were increased in HFD animals. After 32 weeks of HFD, the percentage of chondrocytes containing lipid droplets remained unchanged.

Conclusion

HFD did not alter lipid droplet accumulation but did increase Cpt1a gene and protein expression, and there was a general trend for an increase in β‐oxidation proteins and a decrease in glucose metabolism proteins. These data suggest that in response to HFD chondrocyte lipid uptake and flux are increased while glucose utilization is likely decreased; these metabolic changes preceded HFD induced cartilage degradation.

Support or Funding Information

Supported by the NIH and Arthritis Foundation.
Carbohydrate Mouth Rinsing Enhances Late‐Exercise Sprint Performance in Fatigued Cyclists
Authors: Roberson Paul , D'Lugos Andrew , Vining Caitlin , Pataky Mark , Baur Daniel , Saunders Michael , Luden Nicholas
Published: November 2023

PURPOSE

There is good evidence that mouth rinsing with carbohydrate (CHO) solutions can enhance endurance performance (≥30 min). The impact of CHO on sprint performance has been less consistent, suggesting that CHO may confer benefits in conditions of ‘metabolic strain’. To test this hypothesis, the current study examined the impact of late exercise mouth rinsing on sprint performance. Secondarily, we investigated the effects of a protein mouth rinse (PRO) on performance.

METHODS

Eight trained male cyclists participated in 3 trials consisting of 120 min of fixed load cycling (55% Wmax) followed by a 30‐km computer simulated time trial, during which only water was provided. Following 15 min of passive recovery, 10 min of fixed load cycling (3‐min at 35% Wmax, 7‐min at 55% Wmax) was performed. This was immediately followed by a 2‐km time trial. Subjects rinsed for 5 sec with 25mL of CHO, PRO, or PLA at min 5:00 and 14:30 of passive recovery, and min 8:00 of the 10‐min fixed load. Magnitude‐based inferential statistics were used to analyze the mouth rinse effects on 10‐min fixed load heart rate, glucose, lactate, RPE, and 2‐km time trial performance.

RESULTS

CHO ‘likely’ enhanced performance vs. PLA (4.8%). Further, CHO ‘likely’ elevated heart rate (3.8%) and glucose (5.8%) during Sub vs. PLA. All other comparisons were ‘unclear’.

CONCLUSION

These data clearly demonstrate that sprint performance is enhanced by CHO, but not PRO, under challenging metabolic conditions. The elevated heart rate and blood glucose levels following the rinse protocol suggest that the performance gain with CHO may have been mediated though an enhanced excitatory response.

Support or Funding Information

Supported by a Faculty Research Grant from James Madison University College of Health and Behavior Studies
Effects of Branched‐Chain Amino Acid Supplementation on Exercise Performance and Recovery in Highly Endurance‐Trained Athletes
Authors: Jafari Hanieeh , Ross J. Barrett , Emhoff Chi‐An W.
Published: November 2023

Branched‐chain amino acid (BCAA) supplementation has recently entered the spotlight as a potentially key nutritional component to skeletal muscle recovery during periods of high training volume in endurance athletes. The purpose of this study was to examine the effects of BCAA supplementation on anaerobic and aerobic performance, substrate utilization during constant‐load exercise to exhaustion, and plasma creatine kinase and lactate dehydrogenase levels during post‐exercise recovery. Nine well‐trained cyclists (5 male, 4 female) underwent a morning session and an afternoon session of exercise testing under BCAA or Placebo conditions. Morning tests included two 15‐second Wingates to determine peak and mean anaerobic power output, followed by an incremental test to exhaustion to determine peak oxygen consumption (VO2peak). Afternoon tests included two 15‐second Wingates, followed by a constant‐load exercise to exhaustion at 75% VO2peak, during which only water was provided. During the latter test, pulmonary gas exchange, blood lactate and blood glucose were periodically measured to monitor substrate utilization. Pre‐ and post‐exercise blood tests were conducted to quantify plasma creatine kinase and lactate dehydrogenase levels as biomarkers for muscle damage. Post‐exercise ratings of perceived soreness were also collected. Subjects were then instructed to continue training for at least 2000 MET‐min/week for the next four weeks while supplementing with either a BCAA (5 g amino acids + 56 g carbohydrates) or Placebo (56 g carbohydrates) powdered drink during each workout. At the completion of the four weeks, subjects repeated two 15‐second Wingates and an incremental test to exhaustion to determine any differences in anaerobic and aerobic power due to BCAA or Placebo supplementation. There were no differences in VO2peak pre‐ and post‐training between BCAA and placebo conditions. Peak anaerobic power was slightly improved when athletes supplemented their training with the BCAA drink. The total energy expenditure achieved during the constant‐load exercise test was not different between conditions. Post‐exercise plasma creatine kinase levels were significantly reduced with BCAA compared with Placebo, while there were no differences in plasma lactate dehydrogenase levels. Post‐exercise leg muscle soreness was slightly lower with BCAA supplementation, although not significant. In conclusion, BCAA supplementation with endurance training may augment adaptations in muscle anaerobic power and post‐exercise recovery in well‐trained athletes.

Support or Funding Information

This research was supported by the Saint Mary's College Faculty Development Fund and a gift from GU Energy Labs, Berkeley, CA.
NSAIDs Inhibit Cell Migration by Disrupting the Cellular Adhesion Signaling Cascade
Authors: Silver Kristopher , Lillich James
Published: November 2023

Treatment with non‐steroidal anti‐inflammatory drugs (NSAIDs) is associated with a wide range of negative side effects including gastrointestinal toxicity. Various mechanisms have been proposed to account for these effects, including promotion of ulceration and erosion by inhibition of cell migration and wound healing. Our lab has previously demonstrated that NSAIDs inhibit expression and activity of calpains resulting in stunted and uncoordinated cell migration, but the downstream effects of suppression of calpain activity/expression are currently unknown. Accordingly, we used a combination of immunoblotting and immunofluorescence in both migrating and non‐migrating IEC‐6 cells to identify total and phosphorylated forms of adhesion signaling proteins, such as vinculin, PAX, FAK, and talin, which are affected by NSAID treatment. Whereas vinculin was unaffected by NSAID treatment in either resting or migrating cells, there was significance suppression of expression of phosphorylated PAX and talin in both cell populations following treatment with indomethacin or NS‐398. Additionally, there was also a significant decrease in expression of phosphorylated FAK with indomethacin, but a significant increase in phosphorylated FAK with NS‐398 treatment. These data suggest that not only do NSAIDs affect calpain activity, as previously reported, but NSAIDs also significantly disrupt the adhesion signaling cascade required for coordinated cell migration. Further, our results also provide a mechanism to explain the apparent retention of rear cellular adhesions observed in NSAID‐treated migrating cells and provide a broad foundation for understanding the precise molecular mechanisms through which NSAIDs disrupt cell migration and wound healing.

Support or Funding Information

This work was supported by a grant from the National Institutes of Diabetes and Digestive and Kidney Disease (1R15DK091791)
Oxygen Kinetics and Muscle Twitch Tension in the Rat Extensor Digitorum Longus Muscle After Creatine Kinase Inhibition
Authors: Batten Spencer M , Dobard Sarah E , Buell Joshua H , Cammack Scott A , Sexton William L
Published: November 2023

Creatine kinase (CK) catalyzes the reversible reaction in which high‐energy phosphates are shuttled between phosphocreatine and ADP. CK plays a role in the initial production and maintenance of muscle force during contractions before the onset of glycolytic and oxidative metabolism. The onset of oxidative metabolism leads to changes in interstitial PO2 (PiO2) reflecting the dynamic balance between oxygen delivery (QO2) and oxygen consumption (VO2). The purpose of this study was to investigate the role of CK in QO2 to VO2 matching as reflected in changes in PiO2 at the onset of contractions in the rat extensor digitorum longus muscle (EDL). Female Sprague‐Dawley rats (n=9, 291 ± 5 g) were anesthetized (sodium pentobarbital, 50 mg/kg ip). The trachea was cannulated to maintain a patent airway, and the carotid artery was cannulated to monitor mean arterial pressure and heart rate. The EDL was isolated and connected to a force transducer coupled with a muscle tension analyzer. In each EDL, PiO2 (measured by phosphorescence quenching) was determined at rest and during a 150 s contraction bout (1 Hz, 6 V) where PiO2 and twitch tension were simultaneously measured during the transition to steady‐state contractions. After a 10 min recovery period the rats received an intravenous bolus of IA (20 mg/kg in saline). After a 15 min equilibration period, the same contraction protocol was repeated. Thus, each rat served as its own control. This protocol was validated in preliminary experiments wherein PiO2 and twitch tension data were not different during a repeated stimulation. Mean arterial pressure (120 ± 5, 128 ± 4 mmHg) and heart rate (363 ± 19, 349 ± 9 bpm) were similar before and after IA. There was no difference in resting PiO2 between the control stim and post‐IA (28 ± 3, 29 ± 2 mmHg). At the onset off contractions, PiO2 fell more rapidly after IA as reflected by a faster time constant (16 ± 2, 12 ± 1 s; P<0.05) and mean response time (20 ± 2, 16 ± 2 s; P<0.05). After IA treatment twitch tension (N/g) and the rate of tension development (dT/dt) were both lower (P<0.05) throughout the 150 s contraction bout. However, muscle fatigue was similar between both contraction bouts (3 ± 1, 3 ± 1%; P>0.05). The results of this study reveal that inhibition of creatine kinase with IA in fast‐twitch rat skeletal muscle depressed developed twitch tension and dT/dt. IA treatment also sped the fall in PiO2 reflecting a dramatic imbalance between QO2 and VO2 during the transition to steady state contractions, which may have contributed to the decline in contractile performance.

Support or Funding Information

Supported by a grant from the Graduate Program Committee, KCOM – ATSU.
Crystal Structure of Human Tetraspanin CD81 Reveals a Conserved Intramembrane Binding Cavity
Authors: Zimmerman Brandon , McMillan Brian , Seegar Tom , Kruse Andrew , Blacklow Stephen
Published: November 2023

Tetraspanins comprise a diverse family of four‐pass transmembrane proteins that play critical roles in cell activation, adhesion, migration and proliferation. CD81/Tspan28, the most widely studied member of the family, forms a complex with CD19, CD21 and CD225 to regulate B cell receptor function, and also acts as a receptor or co‐receptor for pathogens such as Hepatitis C virus and Plasmodium species. Despite the fundamental importance of tetraspanin proteins in a broad range of physiologic and pathophysiologic processes, structural information about a full‐length tetraspanin has remained elusive. Here we present the first crystal structure of a full‐length tetraspanin, human CD81. The transmembrane segments of CD81 pack as two largely separated pairs of helices, not as the four‐helix bundle predicted previously. The structure is capped by the large extracellular loop 2 (EC2) at the outer membrane leaflet, and the two pairs of helices converge at the inner leaflet to form a shape resembling an inverted teepee. The transmembrane region contains a large pocket suggestive of a ligand‐binding site, offering a potential mechanism for binding lipids or other molecules. In support of this idea, we observe electron density consistent with a bound cholesterol molecule in the pocket, and we detect specific binding of cholesterol to this site in vitro. The discovery of the hydrophobic pocket in the core of the transmembrane domain presents a novel therapeutic avenue for targeting CD81 and other tetraspanins in pathogenic infections or malignancies.

Support or Funding Information

Brandon Zimmerman is supported by a CIHR postdoctoral fellowship. NIH grant NCI 5ROI CA092433 to SCB and DP5 OD021345 to ACK.
Novel gene expression changes dependent on 2 mitochondrial enzymes in a rescued model of Parkinson's disease
Authors: Theisen Christopher Shane , Kogel William , Tiege Angela , Kaston Essa , Perry Kelley
Published: November 2023

Parkinson's disease (PD) is a progressive disorder of the central nervous system for which there is no cure. The pathogenesis of PD involves insufficient formation of dopamine as a result of dopaminergic cell death in the midbrain. The cause of demise of these cells is poorly understood, but mitochondrial dysfunction has been implicated as a central pathway in the neurodegenerative process. We have observed cellular protection in a model of PD by increasing the levels of 2 important mitochondrial enzymes, NAMPT and SIRT3. Our current model of Parkinson's disease employs the use of immortalized dopamine progenitor neurons and zebrafish larvae. In this study, we examined the effects of overexpressing Nampt in dopaminergic neurons as well as neurons stably expressing lentiviral mediated shRNA against Nampt and the essential mitochondrial sirtuin gene, Sirt3. After establishing stable populations of dopaminergic neurons with either increased or decreased levels NAMPT or SIRT3 we have identified various genes that may be involved in the pathways of neuroprotection in dopaminergic neurons susceptible to cell death in the progression of PD. We compared the expression levels of major genes involved in neurotoxicity. A sub cohort of genes has been identified as over expressers when NAMPT and SIRT3 have reduced expression. There are 15 genes that have significant changes in gene expression consistent with our hypothesis. 7 genes have dramatic changes reciprocal to NAMPT over vs under expression. Our results indicate that NAMPT and SIRT3 alter similar pathways. These mitochondrial enzymes are also being examined in a zebrafish model of PD that shows to have rescued motor function when the genes are overexpressed.

Support or Funding Information

KCUMB Division of Research