bluuebunny/crossref_metadata_2025 huggingface dataset:
Behavioral and Pharmacokinetic Properties of the Putatively‐Inert DREADD Ligand Clozapine‐N‐Oxide (CNO) in Rats and Mice
Authors: Porter Joseph H. , Manvich Daniel F. , Webster Kevin A. , Foster Stephanie L. , Farrell Martilias S. , Weinshenker David
Published: November 2023

Clozapine‐N‐oxide (CNO) is a major metabolite of the antipsychotic drug clozapine that has been utilized in many studies as an “inert” molecule to activate DREADDs (Designer Receptors Exclusively Activated by Designer Drugs). Recently, MacLaren et al (2016) demonstrated thatCNO produced reduced acoustic startle reflex and significantly attenuated d‐amphetamine‐induced hyperlocomotion in rats that lacked DREADD receptor expression. The present study examined the discriminative stimulus properties of CNO in both mice and rats trained to discriminate 1.25 mg/kg clozapine from vehicle in a two‐lever drug discrimination paradigm. After clozapine discrimination was established, substitution tests with CNO (1.0 – 40.0 mg/kg) revealed that CNO partially substituted for the clozapine stimulus in both species. Furthermore, pharmacokinetic studies using plasma samples from separate groups of rats and mice revealed that doses of CNO, which produced partial substitution for the clozapine stimulus, also resulted in the detection of clozapine and its secondary metabolite, N‐desmethylclozapine. Importantly, the emergent levels of clozapine were equivalent to those previously shown to exert behavioral effects. Together, these data demonstrate that CNO is a behaviorally active compound in both mice and rats lacking DREADD receptors and trained to discriminate clozapine. Our results, along with recent findings by others, emphasize the need for appropriate control groups in studies employing DREADDs.

Support or Funding Information

DA039991, DA038453
Effects of (‐)‐Epigallocatechin gallate (EGCG) on proliferation and viability of Jurkat T‐cells treated with ethanol
Authors: Snitsarev Vladislav
Published: November 2023

Adverse effects of ethyl alcohol (EtOH), the most abused substance, on the immune system are not fully understood. We hypothesized that Jurkat T‐lymphocytes (JTL) cell counts will decrease in the presence of alcohol, and that after 48h growing in increasing EtOH concentrations (0.001%‐50% v/v) would result in a simple dose‐response curve with defined IC50 for EtOH.

The Countess automated cell counter was used to measure the number of JTL in the Trypan Blue exclusive manner. The dose‐response relationship for EtOH alone obtained was far from the expected sigmoidal or biphasic shape. First, cell count was significantly increased at 0.01% EtOH compared to control. Second, cell count remained the same at 5%, 10%, and 20% EtOH with no proliferation. This complex shape of the dose response curve indicates potential multiple effects of EtOH on JTL with possibly different mechanisms. To define the components of the dose‐response, further studies will be performed and more data points obtained to allow curve fitting. Live cells were also found at 30% EtOH but were uncountable by the Countess's image analysis because the media turned cloudy at this and higher EtOH concentrations. No live cells were detected at 50% EtOH.

The EGCG concentration that can be achieved in plasma by drinking green tea is 10 μM. When 10 μM of EGCG was added 5 min before EtOH treatment, it abrogated the effects of 0.01% EtOH and shifted the IC50 for EtOH from 3% to 10%. At this point we conclude that protective effects of EGCG may be due to potential interactions with one or more pathways by which EtOH acts on Jurkat T‐cells.
N6‐Cyclohexyladenosine, an A1 Adenosine Receptor Agonist, as a Novel Thermolytic Adjunctive Pharmacotherapy for Targeted Temperature Management
Authors: Laughlin Bernard , Baily Isaac , Rice Sarah , Bararti Zeinab , Drew Kelly
Published: November 2023

Targeted temperature management (TTM) previously known as therapeutic hypothermia, the lowering of core body temperature (Tb) over a 24h period, is a lifesaving and neuroprotective standard of care intervention for cardiac arrest and neonatal hypoxic ischemic encephalopathy. Prior work shows that the A1 Adenosine receptor (A1AR) agonist N6‐Cyclohexyladenosine (CHA), inhibits shivering and nonshivering thermogenesis and lowers core body temperature. However, an optimal delivery protocol has not been developed. Here our purpose is to optimize a CHA delivery protocol to achieve and maintain a predictable and consistent target Tb of 32°C over a 24h period and also to quantify and characterize physiological responses to CHA assisted cooling at a temperature range and cooling interval consistent with current evidence based guidelines for targeted temperature management in cardiac arrest.

Sprague‐Dawley rats (n = 22) were instrumented with CTA‐F40 transmitters (DSI International) for continuous recording of Tb and ECG, arterial and venous cannulas and in some cases, brain cannula for monitoring brain temperature. Rats were administered CHA via continuous IV infusion or intraperitoneal injections to inhibit thermogenesis. In the first experiment ambient temperature was modulated to lower Tb with increasing doses of CHA to optimize cooling. In the second experiment cage surface temperature was modulated to control Tb with the CHA dose determined from the first experiment. Core body and brain temperatures, heart rate and rhythm, blood gas and electrolytes were monitored throughout the experiments

Results indicate a pronounced individual variation in response to CHA and the development of tolerance, necessitating a higher starting dose and use of dynamic conductive temperature modulation to prevent overcooling. We show that core body temperature predicts brain temperature during CHA induced cooling. CHA inhibits thermoregulatory shivering and core body temperature of 32°C can be safely maintained and modulated with surface cooling and continuous IV infusion of CHA over a 24h period. Acid‐base, electrolyte and blood gas analysis indicated acute hyperglycemia and a mild respiratory acidosis during the initiation of cooling. The hyperglycemia was transient and all rats were normoglycemic at the end of cooling and arterial pH normalized via metabolic compensation.

Support or Funding Information

NIH R15 NS070779
Effect of Hydrogen Sulfide‐Releasing Compounds on Aqueous Humor Outflow Facility Under Different Perfusion Pressures
Authors: Robinson Jenaye , Mitchell Leah , Ngele Kalu , Opere Catherine , Ohia Sunny E , Njie‐Mbye Ya Fatou
Published: November 2023

Hydrogen sulfide (H2S)‐releasing compounds have been reported lower intraocular pressure in rabbits and increase aqueous humor outflow facility in a porcine ocular anterior segment model, ex vivo. The aim of the present study was to investigate the pharmacological actions of L‐cysteine, a substrate for the biosynthesis of H2S, on aqueous humor outflow facility using the porcine ocular anterior segment model maintained under normal and elevated ocular perfusion pressure.

Methods

Porcine ocular anterior segment explants were perfused with Dulbecco's Modified Eagle's Medium maintained at 37°C and gassed with 5% CO2 and 95% under a constant normal perfusion pressure of 7.35 mmHg or under an elevated perfusion pressure of 15 mmHg. Stabilized explants were exposed to L‐cysteine (1 nM – 1 μM) and outflow facility was monitored for 4 hours. Control experiments containing vehicle (0.1% saline) were run in parallel.

Results

Under normal pressure conditions, L‐cysteine elicited a concentration‐dependent increase in aqueous humor outflow facility that reached a maximal effect at 100 nM [(150.6 ± 17.2% of basal; (p < 0.04)], an effect that lasted for a duration of four hours. Interestingly, under conditions of elevated perfusion pressure, L‐cysteine (100 nM) also significantly increased outflow facility reaching a maximal effect of 173.0 ± 23.0% of basal (p < 0.01).

Conclusion

We conclude that L‐cysteine can increase aqueous humor outflow facility in the porcine anterior segment model in a manner that was independent of perfusion pressure. However, further studies are needed to determine the role of H2S in regulating aqueous humor outflow under these conditions.
Structure Activity Relationship of PEG‐Peptide Scavenger Receptor Inhibitors
Authors: Allen Rondine , Rice Kevin
Published: November 2023

The rapid capture and degradation of viral and non‐viral gene delivery nanoparticles by scavenger receptors (SR) on Kupffer cells and fenestrated endothelial cells in the liver results in a decreased half life of all nanoparticles in an in vivo system, as well as a lower percentage of the dose available for delivery to the target organ. Current attempts to achieve SR inhibition involves co‐administering high molecular weight, polyanionic inhibitors. Polyinosinic acid (Poly‐I), the most widely used and potent inhibitor, competes for SR binding and successfully inhibits the uptake and metabolism of viral gene delivery nanoparticles in the liver. However, Poly‐I also activates the immune system, resulting in toxicity in mice, making Poly‐I clinically unacceptable.

We have discovered peptide based SR inhibitors that block SR uptake of DNA nanoparticles and improves their metabolic half‐life by forming protein nanoparticles in the blood. These Poly (ethylene) glycol (PEG) polylysine peptides potently inhibit SR and allow DNA to transfect hepatocytes up to 12 hours after administration in mice.

Radio‐iodinated PEG‐peptides were used to study the pharmacokinetics and biodistribution to understand the structural properties which influence transfection competency and potency. We hypothesize that the in vivo potency and activity is influenced by the stability of protein nanoparticles formed in the blood. Peptides which are able to form stable nanoparticles are able to effectively block liver uptake of DNA nanoparticles by saturating SRs on Kupffer cells in the liver. The results of this study provide a framework for the design and synthesis of future PEG‐peptide scavenger receptor inhibitors, as well as an assay to quickly determine the activity of each inhibitor.

Support or Funding Information

The Iowa Biosciences Academy is supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R25GM058939, the University of Iowa (UI) Office of the Vice President for Research and the UI Chief Diversity Office. This work was also supported by the NIH Pharmacological Sciences Training Grant T32 GM067795 and NIH grant R01 GM087653.
Belinostat, A Clinically‐Used Histone Deacetylase Inhibitor, Inhibits Rifampicin Induced CYP3A4 and MDR1 Gene Expression
Authors: Pondugula Satyanarayana R , Abbott Kodye L , Chaudhury Chloe S , Schwartz Dean D
Published: November 2023

Pregnane X receptor (PXR), a ligand‐dependent nuclear receptor, is primarily involved in regulating the gene expression of enzymes and proteins involved in endobiotic and xenobiotic metabolism. In particular, PXR plays a crucial role in regulating the gene expression of drug‐metabolizing enzymes and drug‐efflux pumps, including cytochrome p450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). In cancer patients, upregulation of the levels of the PXR target genes, including CYP3A4 and MDR1, can lead to chemoresistance as well as adverse drug interactions. It is possible to inhibit the activity of PXR in order to overcome PXR‐mediated chemoresistance and adverse drug interactions. However, a selective and less‐toxic PXR antagonist has yet to be developed. We sought to determine whether belinostat, a clinically‐used histone deacetylase inhibitor, inhibits the PXR target gene expression in human liver cells (primary hepatocytes & hepatocells) and human intestinal cells (LS174T colon cancer cells). Rifampicin, an agonist of human PXR, was used to activate PXR. Cell viability and CYQUANT cell proliferation assays were performed to determine cytotoxicity and cell proliferation, respectively. Quantitative RT‐PCR assays were conducted to study gene expression of CYP3A4 and MDR1. CYP3A4 P450‐Glo and Rhodamine 123 intracellular accumulation assays were performed to determine the function of CYP3A4 and MDR1, respectively. Belinostat, at its non‐cytotoxic and therapeutically‐relevant concentrations, inhibited rifampicin induced gene expression of CYP3A4 and MDR1 in the liver/intestinal cells. Belinostat also attenuated the PXR agonist‐induced activity of CYP3A4 and MDR1 in the liver/intestinal cells. Furthermore, belinostat decreased rifampicin‐induced proliferation of LS174T human colon cancer cells, suggesting that belinostat suppresses PXR‐mediated cell proliferation of the cancer cells. These results are consistent with the conclusion that belinostat, at its non‐cytotoxic and therapeutically relevant concentrations, downregulates PXR target gene expression by inhibiting the transcriptional activity of PXR. Future studies will be directed to study the selectivity of belinostat to PXR, mechanisms of belinostat inhibition of PXR, and belinostat inhibition of PXR target gene expression in PXR mouse models.

Support or Funding Information

The authors would like to thank Drs. Coleman and Tao for sharing their research facilities. This work was supported by the Auburn University Research Initiative in Cancer Grant, Animal Health and Disease Research Grant, and Auburn University Startup Funds to Pondugula SR.
Saikosaponins Induced Hepatotoxicity Through Disruption of Lipid Metabolism and Induction of Oxidative Stress in Mice: A Proteomic Analysis
Authors: Sun Rong , Li Xiaoyu , Yang Qian , Li Xiaojiaoyang , Liu Runping
Published: November 2023

INTRODUCTION

Radix Bupleuri (RB) has been popularly used for treating many liver diseases such as chronic hepatic inflammation and viral Hepatitis in China. Increasing clinical and experimental evidences indicate the potential hepatotoxicity of RB or prescriptions containing RB. Recently, Saikosaponins (SS) has been identified as major bioactive compounds isolated from RB, which may be also responsible for RB‐induced liver injury.

MATERIALS AND METHODS

Serum AST, ALT and LDH were determined to evaluate SS‐induced liver injury in mice. Serum and liver total triglyceride and cholesterol were used to indicate lipid metabolism homeostasis. Liver ROS, GSH, MDA and iNOS were used to examine the oxidative stress level after SS administration. A 8‐Plex iTRAQ Labeling Coupled with 2D LC ‐ MS/MS technique was applied to analyze the protein expression profiles in livers of mice administered with different doses of SS for different time periods. Gene ontology analysis and comparative toxigenomics were employed to elucidate potential mechanism involved.

RESULTS

SS dose and time dependently induced liver injury in mice, indicated by increased serum AST, ALT and LDH levels. According to proteomic analysis, 487 differentially expressed proteins were identified in mice administrated with different dose of SS for different time periods. Altered proteins were enriched in pathways such as lipid metabolism, protein metabolism, macro molecular transportation, cytoskeleton structure and response to stress. Cluster analysis demonstrated that SS rapidly induced lipid transportation and lipid metabolism related genes in less than 1 hour, followed by stress‐induced proteins, occurred from 4–8 hours and further regulated proteins involved in protein metabolism and energy metabolism (8–24 hours). Lipid metabolism dysfunction and oxidative stress were significantly induced by SS in liver.

CONCLUSION

These results identified hepatotoxicity of SS administration and suggested potential mechanism, including impaired lipid and protein metabolism and oxidative stress. The proteomic approach gave us new insights to understand toxicity of traditional Chinese medicine and provided experimental evidence for the clinical safe usage of RB.

Support or Funding Information

This work was supported by grants from the National Natural Science Foundation of China (Grant No. 81073148, 30672649, 81374059); the National Major Fundamental Research Program of China (Grant No. 2009CB522802); the Project supported by the Shangdong Committee of Science and Technology, China(Grant No. 2008GG2NS02021); the Key Project of Shandong Natural Science Foundation of China (Grant No. ZR2011HZ005). R. Sun is supported by “Traditional Chinese medicine pharmacology and toxicology expert”, the post of Taishan scholar.
Imbalanced Periaortic Adipose Tissue Brown‐to‐Inflammatory Phenotype is Related to Aortic Stiffness
Authors: Grunewald Zachary I , Winn Nathan C. , Gastecki Michelle L. , Woodford Makenzie L. , Ball James R. , Hansen Sarah A. , Sacks Harold S. , Vieira‐Potter Victoria J. , Padilla Jaume
Published: November 2023

Obesity‐mediated periaortic adipose tissue (AT) inflammation has been implicated in aortic stiffness, which is an established independent predictor for cardiovascular disease and mortality. However, whether increased browning of periaortic AT confers vascular protection in the setting of obesity remains unknown. Herein, we hypothesized that enhanced periaortic AT browning via interscapular brown AT lipectomy (iBATx) would mitigate obesity‐induced aortic stiffness. Accordingly, four groups of male mice (C57BL/6J) fed normal chow (NC) or Western diet (WD) for 18 weeks following sham or iBATx surgery were studied. NC‐fed leptin receptor deficient (db−/db−) adult male mice were also studied as another model of diet‐induced obesity. Aortic stiffness was assessed via pulse wave velocity and periaortic AT phenotype was determined via gene expression. Counter to our hypothesis, in the absence of periaortic AT inflammation, increased expression of browning genes in iBATx mice was associated with increased aortic stiffness (1.5‐fold vs. sham, P<0.05). This association was abrogated when increased expression of browning genes co‐existed with increased expression of inflammatory genes (i.e., WD‐fed sham and iBATx mice). Reciprocally, low expression of browning genes in db−/db− mice was also associated with increased aortic stiffness, relative to wild‐type controls (1.6‐fold, P<0.05). Importantly, when computing the ratio of browning‐to‐inflammatory gene expression (B/I ratio) across groups using a composite score (average of fold‐differences) of classical browning genes (UCP1, PGC1α, PRDM16, Cidea, FGF‐21) vs. inflammatory genes (IL‐1β, TNFα, MCP‐1, CD68), we found a U‐shaped polynomial relationship between the periaortic AT B/I ratio and aortic stiffness with an R2 of 0.97. That is, increased and decreased periaortic AT B/I ratios were associated with increased aortic stiffness. Of note, this polynomial relationship between AT B/I ratio and aortic stiffness was less apparent in visceral white (i.e., epididymal) AT (R2=0.36), supporting the notion that the phenotype of periaortic AT is particularly related to aortic stiffness, compared to other distant AT depots. In conclusion, these findings set forth the new hypothesis that an imbalanced periaortic AT browning‐to‐inflammatory phenotype may be an important determinant of aortic stiffness in mice.
Cardiovascular regulation in mice with attenuation or overexpression of the vesicular acetylcholine transporter (VAChT) gene: parasympathetic function in heart failure
Authors: Tezini Geisa C. S. V. , Martins Dias Daniel P. , Oliveira Mauro , Guatimosim Silvia , Prado Marco A. M. , Salgado Helio C.
Published: November 2023

Studies have shown that a parasympathetic hypoactivity has been identified as an independent risk factor in patients with myocardial infarction (Kleiger et al., 1987; La Rovere et al., 1998). Therefore, this study aims to evaluate the effects of the attenuation, or the overexpression, of the vesicular acetylcholine transporter (VAChT) gene on cardiovascular responses during the development of heart failure (HF). The study was conducted in mice knockdown homozygous for the VAChT gene (VAChT KDHOM, n=3) and transgenic subjects with overexpression of the VAChT gene (ChAT–ChR2–EYFP, n=4). Mice were implanted with probes for arterial pressure (AP) and heart rate (HR) recording by telemetry. Ten days after probe implantation the hemodynamics (AP and HR) and cardiac sympathovagal balance were assessed. Next, the mice were submitted to left coronary artery ligation for producing myocardial infarction (MI) and the development of HF. Data were collected 1 and 4 weeks after MI. Morphological analysis revealed similar infarct size among groups (32±4% in VAChT KDHOM vs. 28±2% in ChAT–ChR2–EYFP). VAChT KDHOM mice showed 1 week after MI increased mean AP (MAP) and HR; these parameters were back to normal after 4 weeks. ChAT–ChR2–EYFP mice presented 1 week after MI opposite results, with reduction of MAP and HR. MAP was back to normal after 4 weeks, while the HR remained at lower levels. Regarding the cardiac sympathovagal balance, there were no differences between groups. Overall, the results suggest that even though the extent of the MI was similar in both groups, the hemodynamic profile after this cardiac insult was opposite; VAChT KDHOM mice responded to MI with increased MAP and HR, whereas ChAT–ChR2–EYFP mice with reduction of both hemodynamic variables.

Support or Funding Information

FAPESP
Methotrexate Scavenges Xanthine Oxidase‐Derived Superoxide without Inhibiting Xanthine Oxidase Activity
Authors: Sarmiento Cleafes , Chiou Anderw , Duryee Michael J. , Tian Jun , Thiele Geoffrrey M. , Anderson Daniel R. , Mikuls Ted R. , Zimmerman Matthew C.
Published: November 2023

Background/Hypothesis

Rheumatoid arthritis (RA), an autoimmune disorder affecting 1.5 million Americans, is associated with chronic inflammation and elevated levels of reactive oxygen species (ROS) in joints resulting in painful swelling, bone erosion, and joint deformity. Methotrexate (MTX), a disease‐modifying anti‐rheumatic drug, has anti‐inflammatory, anti‐proliferative, and anti‐fibrogenic properties; however, the precise mechanism(s) by which MTX provides relief to RA patients is unclear. Although previous studies have suggested that MTX inhibits ROS production, these studies failed to determine if MTX truly inhibits production or increases scavenging of ROS. Herein, we tested the hypothesis that MTX directly scavenges a specific ROS, that is, superoxide.

Methods

In a cell‐free in vitro system, superoxide generated by hypoxanthine (HX, 50mM) and xanthine oxidase (XO, 10mU/mL) was measured using electron paramagnetic resonance (EPR) spectroscopy and the EPR spin probe 1‐hydroxy‐3‐methoxycarbonyl‐2,2,5,5‐tetramethylpyrrolidine (CMH, 200mM) in the absence or presence of MTX (2mM). In addition, we examined the ability of MTX to inhibit XO activity, by measuring uric acid levels produced by the HX + XO reaction ± MTX.

Results

The EPR spectra amplitude obtained from samples containing HX + XO were significantly elevated compared to control samples without HX and XO (3.69×106 ± 0.34×106 vs. 5.14×104 ± 0.69×104 EPR arbitrary units, p<0.05); thus, indicating increased levels of superoxide. MTX significantly attenuated levels of superoxide as determined by decreased EPR spectra amplitude (1.67×106 ± 0.24×106 EPR arbitrary units, p<0.05 vs. HX and XO alone). MTX did not inhibit XO activity as uric acid levels increased 8.77% ± 1.20% without MTX and 8.44% ± 1.00% with MTX.

Conclusions

Methotrexate, a first line of defense for RA patients, directly scavenges XO‐derived superoxide, but does not inhibit XO activity. Future studies are needed to determine if MTX's anti‐inflammatory, anti‐proliferative, and/or anti‐fibrogenic actions are due to its ability to directly scavenge superoxide.

Support or Funding Information

This research was made possible by grants from the National Center for Research Resources (5P20RR016469) and the National Institute for General Medical Science (NIGMS) (8P20GM103427), a component of the National Institutes of Health (NIH) and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
Lateral Hypothalamic Regulation of Energy Homeostasis and Sympathetic Outflow: The Role of Leptin and Melanocortin Signaling
Authors: Cruz Eva Nilda Rodriguez , Morgan Donald A , Davis Kevin Carroll , Saito Kenji , Toth Brandon A , Martinez Eduardo , Jiang Jingwei , Rahmouni Kamal , Cui Huxing
Published: November 2023

Hypothalamus plays a fundamental role in both body weight homeostasis and sympathetic regulation, but the underlying neural basis of this association remains incompletely understood. Brain leptin and melanocortin systems have emerged as critical regulators of body weight homeostasis, sympathetic nerve activity (SNA) and blood pressure. However, the neural circuits by which leptin and melanocortin regulate SNA and blood pressure have not been fully defined. We have previously identified a unique subpopulation of GABAergic neurons in the lateral hypothalamic area (LHA), a historically recognized “feeding center” in the brain, that coexpress both leptin receptor (LepR) and melanocortin 4 receptor (MC4R), indicating that this unique subset of LHA inhibitory neurons are direct targets of endogenous leptin and melanocortin. Because of a well‐established role of LHA in both feeding and sympathetic regulation, we hypothesized that LHA MC4R signaling pathway may plays a key role in sympathetic overactivity and elevated blood pressure associated with obesity. Here we show that in vivo Cre/loxP system‐mediated re‐expression of endogenous MC4Rs in the LHA of severely obese MC4R‐null mice restores the blunted responses of MC4R agonist MTII‐mediated increase in sympathetic outflow to various beds, including brown adipose tissue, muscle, and kidney, and elevates blood pressure in obese normotensive MC4R‐TB mice without significantly affecting food intake and body weight. Finally, optogenetic and/or chemogenetic stimulation of LHA LepR‐positive neurons (some of which are also MC4R‐positive) suppresses food intake and deceases blood pressure and renal SNA in conscious mice. In conclusion, our findings identify a novel brain circuit by which leptin and melanocortin signaling regulate sympathetic traffics and blood pressure, which may contribute to the development of obesity‐associated hypertension.

Support or Funding Information

NIH/NHLBI

R01 HL127673‐01A1
Human Antigen R (HuR) Regulates Structure and Function of Brown Adipose Tissue
Authors: Yamamoto Kaila , Lanzillotta Lindsey , Anthony Sarah R , Jones Shannon , Yoshida George , Owens Albert Phillip , Tranter Michael
Published: November 2023

Human antigen R (HuR) is an RNA binding protein widely expressed throughout the body, including in both white (WAT) and brown (BAT) adipose tissue. Recent work from our lab has shown that adipocyte‐specific HuR knockout (adipo‐HuR−/−) mice gain less weight following a high fat diet (HFD) than the wild type (WT) controls. In this study, we focused on the role of HuR deletion on the function of BAT, which mediates non‐shivering thermogenesis.

Our results show that, when subjected to a cold stress (4°C), HFD‐fed adipo‐HuR−/− mice are less cold tolerant compared to their WT counterparts. However, when the cold challenge was repeated with chow fed mice, there was no difference between the HuR knockout and wildtype mice in their ability to thermoregulate, suggesting that a change induced by high fat feeding rendered the adipo‐HuR−/− mice less cold tolerant. Histological analysis revealed that, while the BAT of the chow fed adipo‐HuR−/− mice appeared normal, following HFD, the BAT from adipo‐HuR−/− mice appears less dense with a cell size that more closely resembles WAT. Since uncoupling protein‐1 (UCP1) plays a key role in BAT mitochondrial uncoupling and non‐shivering thermogenic regulation, we assessed the ability of HuR to directly mediate UCP1 mRNA expression. Interestingly, analysis of the UCP1 mRNA sequence predicts at least four distinct HuR binding sites in the UCP1 3′UTR, and our data shows that UCP1 mRNA expression is decreased in BAT from adipo‐HuR−/− mice.

In conclusion, our results indicate that HuR regulates both the structure and function of BAT, at least in part through direct modulation of UCP1 mRNA expression.

Support or Funding Information

This work was supported by a University of Cincinnati Heart, Lung, and Vascular Institute Near Horizons Grant (MT, APO).
Attenuated Sweating Responses in Tattooed Skin During a Passive Whole‐body Heat Stress
Authors: Pizzey Faith K , Parupia Iqra M , Allen Dustin R , Huang Mu , Luetkemeier Maurie J , Davis Scott L
Published: November 2023

Tattoos have increased in popularity in the last 10–15 years among individuals between the ages of 18–40, with a high prevalence in athletes and members of the military. The tattooing process involves repeated needle insertions (50 to 3,000 times per minute) to permanently deposit ink at a depth of 3–5 mm into the dermal layer, potentially damaging and impairing thermoregulatory mechanisms (i.e., eccrine sweat glands and cutaneous vasculature). The aim of the present study was to test the hypothesis that reflex increases in sweat rate (SR) and cutaneous vasodilation are blunted in tattooed skin (TAT) compared to adjacent healthy skin (CON) during a passive whole‐body heat stress (WBH). Four individuals (2M, 2F) with a substantial area of tattooed skin participated in the study. Skin blood flow (laser‐Doppler flowmetry; LDF) and SR (capacitance hygrometry) were continuously measured during normothermic baseline (34 °C water perfusing a tube‐lined suit) and WBH (increased core temperature 1.0 °C via 48 °C water perfusing the suit). LDF and SR were assessed in areas not exposed to direct heating. Cutaneous vascular conductance (%CVCmax) was calculated from the ratio of LDF to mean arterial pressure and normalized to maximal responses obtained by local heating at 42 °C following WBH. Results indicated that the laser‐Doppler measurement technique is clearly affected by tattooing not allowing for an accurate measurement of skin blood flow in TAT. The accumulated sweating responses throughout the duration of WBH (area under the curve) were attenuated in TAT relative to CON (TAT: 19.06 ± 13.20 vs CON: 24.34 ± 15.61 mg/cm2, p=0.04). The attenuated thermoregulatory reflex increases in SR in TAT could be due to the accumulation of ink within the dermal layer, damage to the sweat gland from repeated needle insertions, or some combination therein. Further research is warranted to assess whether attenuated sweating responses in tattooed skin translate to increased risk of heat‐related injury especially when tattooing covers a higher percentage of body surface area.
Effects of Lactobacillus rhamnosus on uptake and lipid accumulation in Caco‐2 cells
Authors: Ipinmoroti Ayodeji O , Onyango E M
Published: November 2023

Probiotic microorganisms have been shown to influence the digestion and absorption of various nutrients including lipids. Accumulation of triglyceride (TAG) in enterocytes of the small intestine follows two specific absorption pathways: the monoacylglycerol acyltransferase pathway (MGAT) and an alternative glycerol acyltransferase pathway (GPAT). Triglycerides are the main constituents of body fat in humans and other animals derived from the esterification of glycerol and free fatty acids. In this study, we investigated the effects of Lactobacillus rhamnosus MRS6AN, an isolate from “amabere amaruranu” a Kenyan traditional cultured milk, on triglyceride accumulation and expression of GPAT3 in Caco2 cell enterocyte model. In the present study, we examined the effects of L. rhamnosus MRS6AN on lipid absorption mediated by glycerol‐3‐phosphate acyltransferase. Caco‐2 cells were treated overnight with various bacterial cell extracts (cytoplasmic fraction, CF; filtered spent broth, FSB; heat inactivated bacteria, HIB) or with live bacteria (LB). Treated Caco‐2 cells were further divided into 3 treatment groups: Treatment 1 cells were directly overlaid with a lipid mixture without washing, Treatment 2 cells were washed in PBS then overlaid with the lipid mixture, while treatment 3 cells were washed with PBS, overlaid with lipid mixture and fresh treatments. The cells were subjected to incorporated lipid content assay through triglyceride accumulation analysis and Oil‐Red staining to determine the lipid content. Lipid content was measured using absorbance (OD 540 nm). Compared to the controls, TAG accumulation in cells treated with LB or its FSB was reduced by 0.143 and 0.097, respectively, in Treatment 1 cells; 0.151 and 0.136, respectively, in Treatment 2 cells; and 0.147 and 0.087 in Treatment 3 cells. HIB had no effect on lipid accumulation in Treatment 1 and 3 cells, but a slight reduction in Treatment 2 cells by 0.065. CF reduced TAG in Treatment 2 and 3 cells by 0.08 and 0.093 respectively. Data from this study suggest that L. rhamnosus MRS6AN strain may reduce lipid uptake and subsequent TAG accumulation in enterocytes.

Support or Funding Information

East Tennessee State University
Diaphragm neuromuscular transmission dysfunction in chronic heart failure
Authors: Kelley Rachel Catherine , Coblentz Philip David , Ferreira Leonardo Franklin
Published: November 2023

Chronic heart failure (CHF) causes loss of inspiratory muscle pressure (~force) and endurance (inspiratory dysfunction), which contributes to dyspnea and exercise intolerance. Inspiratory dysfunction in patients may arise from phrenic nerve and diaphragm muscle abnormalities. Diaphragm contractile function is preserved in animals with mild CHF, yet patients with mild CHF have inspiratory dysfunction. Therefore, we hypothesized that CHF causes neuromuscular abnormalities that impair diaphragm function.

Methods

Male Sprague‐Dawley (SD) rats underwent ligation of the coronary artery to induce CHF or sham surgery. Approximately twelve weeks post‐surgery, we dissected costal diaphragm bundles for tests of maximal force and fatigue characteristics using field (direct) muscle or phrenic nerve stimulation. After stimulations at maximal frequency (200 Hz), we conducted an isometric fatigue protocol (30 Hz, 500 ms trains, 0.5 train/second). In bundles stimulated through the phrenic nerve, we superimposed direct muscle stimulation every 15 s during the fatigue protocol.

Results

Animals in the CHF groups demonstrated mild‐to‐moderate disease as evidenced by transmural infarct area <35% of left ventricle plus septum and normal right ventricle morphology. Phrenic nerve stimulation caused greater fatigue in diaphragm from animals with CHF (fatigue index at 90th contraction: 70 ± 1.4 % and 55 ± 3.1 %, n=3, p<0.05). CHF also exacerbated phrenic‐to‐diaphragm neuromuscular transmission failure compared to Sham (42 ± 1.1 % and 27 ± 1.4 %, p<0.05). Diaphragm fatigue characteristics were similar in Sham and CHF when contraction was stimulated via direct muscle stimulation. Maximal tetanic specific force (N/cm2) was also similar in Sham and CHF animals with either direct (Sham 29.4 ± 2.4, CHF 25.9 ± 2.6) or phrenic stimulation (Sham 23.5 ± 0.7, CHF 21.9 ± 1.4).

Conclusions

Our data suggest that mild/moderate CHF causes neuromuscular transmission failure in the diaphragm. Neuromuscular transmission failure occurred in the absence of diaphragm weakness, which is evident in severe CHF, and likely contributes to inspiratory dysfunction seen in patients.

Support or Funding Information

Funding support from NIH R01 HL130318
Neurons of the arcuate nucleus in mice express angiotensinogen
Authors: Sapouckey Sarah A , Claflin Kristin E , Gibson‐Corley Katherine N , Grobe Justin L
Published: November 2023

Recent studies from our laboratory have demonstrated that angiotensin II (ANG) type 1A receptors (AT1A) specifically localize to the subset of neurons which express both the leptin receptor (LepR) and agouti‐related peptide (AgRP). These are critically involved in the control of thermogenic adipose sympathetic nerve activity (SNA) and thereby resting metabolic rate (RMR).This mechanism appears to involve AT1A‐mediated suppression of gamma‐aminobutyric acid (GABA) synthesis and packaging in AgRP neurons of the arcuate nucleus (ARC). It remains unclear, however, how LepR signaling results in AT1A activation within AgRP neurons. We hypothesize that activation of LepR in POMC and/or AgRP neurons results in de novo synthesis and local release of angiotensinogen (AGT) within the ARC, and consequently increased autocrine or paracrine ANG signaling within the ARC. To test this hypothesis, we examined the localization of AGT mRNA expression within the ARC of wildtype C57BL/6J mice by fluorescent in situ hybridization methods (RNAscope) and confocal fluorescent microscopy. Consistent with our working hypothesis, cells within the ARC expressing AGT mRNA colocalize with both AgRP and POMC. Cells within the ARC expressing insulin II (INS2) also expressed AGT. Finally, as expected, AGT was also expressed in astrocytes, colocalizing with glial fibrillary acidic protein (GFAP). We and others have previously demonstrated that LepR activation increases phosphorylation of the signal transduction and activator of transcription‐3 (STAT3) transcription factor, and that p‐STAT3 appears to stimulate AGT expression in other cell types including cardiac myocytes and hepatocytes. We therefore hypothesize that the phosphorylation of STAT3 by LepR activation is mechanistically involved in the regulation of AGT expression by leptin in POMC and AgRP neurons. Ongoing studies are aimed at (i) examining AGT expression control by pSTAT3 in POMC and AgRP neurons of the ARC, and (ii) using Cre‐lox recombination methods to selectively disrupt the AGT gene in LepR, POMC and AgRP neurons to study the functional significance of AGT in these cells in the control of SNA and RMR, and thereby energy homeostasis.
Transduction of Sympathetic Nerve Activity to Blood Pressure in Type 2 Diabetes Patients
Authors: Young Ben E , Vranish Jennifer R , Holwerda Seth W , Newby Chase E , Keller David M , Hart Emma C , Fadel Paul J
Published: November 2023

The technique of microneurography has been used extensively to obtain direct recordings of muscle sympathetic nerve activity (MSNA) in both healthy and diseased populations. Much less commonly studied, however, is the transduction of MSNA into changes in arterial blood pressure. Recently, several methodologies have been developed to begin to better understand sympathetic transduction. Herein, we compared two methodologies and their ability to detect group differences in sympathetic transduction between Type 2 Diabetes (T2D) patients (N=15; age=47±2.0 years; 9 women) and age‐and BMI‐matched controls (CON: N=11; age=47±2.4 years; 8 women). T2D patients were of interest since previous work has reported enhanced α‐adrenergic sensitivity in this group. We tested the hypothesis that T2D patients would exhibit an enhanced sympathetic transduction to spontaneous changes in resting MSNA. MSNA, heart rate (ECG), and beat‐to‐beat arterial blood pressure (finger photoplethysmography) were continuously recorded during a 10‐minute resting baseline. Transduction of MSNA was assessed using two distinct methodologies. First, signal averaging was used to characterize the peak change in diastolic blood pressure over the subsequent 10 cardiac cycles following MSNA bursts. Normalized MSNA burst heights were divided into quartiles, and the associated peak diastolic blood pressures were used to derive a slope. Second, diastolic blood pressures were associated with normalized MSNA burst areas at a lag of 6–8 cardiac cycles, and binned into 1%·sec bins. Linear regression of these binned areas provided a slope. Resting MSNA, and diastolic blood pressure (CON: 78±2 mmHg, T2D: 81±3, p=0.28) were not different between groups. There was no difference in transduction slopes between groups using burst height quartiles (CON: 0.73±0.11 mmHg/AU, T2D: 0.53±0.09 mmHg/AU, p=0.18). Indeed, signal averaging of MSNA bursts produced a similar peak increase in diastolic blood pressure in CON (1.7±0.3 mmHg) and T2D patients (1.9±0.3 mmHg; p=0.68). Likewise, transduction slopes derived from binned MSNA areas were also not different between groups (CON: 0.29±0.03 mmHg/%·sec, T2D: 0.23±0.03 mmHg/%·sec, p=0.28). These preliminary findings suggest that, despite enhanced α‐adrenergic sensitivity in T2D, resting sympathetic transduction to arterial blood pressure is not different between T2D patients and healthy CON subjects. This finding was independent of the transduction analysis methodology employed.

Support or Funding Information

Supported by AHA Grant In Aid 20160072.
Progression of experimental autoimmune encephalomyelitis is associated with up‐regulation of major sodium transporters in the mouse kidney cortex under a normal salt diet
Authors: Zhou Xiaoming , Packialakshmi Balamurugan , Xiao Yao , Nurmukhambetova Saule , Lees Jason R
Published: November 2023

Multiple sclerosis is an auto‐immune disease. Experimental autoimmune encephalomyelitis (EAE) is a popular animal model to study the pathophysiology of the disease. Recent repeated demonstrations of the exacerbation of EAE by high salt diets suggest a critical role of Na in the progression of EAE. This discovery has prompted us to study whether EAE stimulates renal absorption of Na even under a normal NaCl diet. The renal cortex is the primary site to absorb Na. In the kidney cortex, Na enters the epithelial cells mainly through the apical Na‐H exchanger NHE3, NaCl co‐transporter NCC and Na channel ENaC and then exits the cells exclusively through the basolateral Na,K‐ATPase. Here we report that as EAE progressed from mild to severe stage, there were parallel increases in the protein abundance of NHE3 and αENaC but not in the protein levels of βENaC, γENaC or NCC. Severe EAE also correlated with augmentation of the Na,K‐ATPase activity with an affiliated elevation of its β1‐subunit protein. These effects cannot be explained alone by EAE‐associated dehydration or anorexia. To determine how NHE3, αENaC and Na,K‐ATPase are up‐regulated during severe EAE, we examined their up‐stream stimulators. We have identified that the protein levels of the kinase SGK1 and scaffold NHERF2 and phosphorylation of ERK1/2 were increased during severe EAE, whereas the protein level of SGK2 or NHERF1 was not. We conclude that severe EAE is associated with up‐regulation of NHE3, αENaC and Na,K‐ATPase, which is most likely contributed by increased expression of SGK1 and NHERF2 and activation of ERK1/2. These data suggest that severe EAE increases Na absorption by the renal cortex.

Support or Funding Information

National Multiple Sclerosis Society PP3448
Human and Murine Precision‐Cut Kidney Slices as a Tool to Study the Inflammatory Pathogenesis of Renal Disease
Authors: Mutsaers Henricus A.M. , Stribos Elisabeth G.D. , Nørregaard Rikke , Olinga Peter
Published: November 2023

Renal disease is a major global health problem and the 9th leading cause of death in the United States. Inflammation is one of the driving forces in acute and chronic kidney diseases, and cytokines act as both mediators and initiators of injury. Currently, there is a lack of good translational models to study the inflammatory basis of kidney disorders, hindering the search for therapeutic targets. The objective of our study was to investigate the inflammatory process in an in vitro model that reflects the cellular diversity as well as architecture of the kidney, namely precision‐cut kidney slices (PCKS).

PCKS were successfully prepared from both human and murine kidneys, and cultured up to 96 hours. Viability of PCKS was assessed by the ATP content of the slices. Furthermore, gene expression of several inflammatory markers was determined by quantitative PCR.

Human and murine PCKS remained viable during culture up to 72h. During culture of human PCKS we observed a marked increase in the gene levels of interleukin (IL)‐6, IL‐8, and IL‐1β, peaking at 3–24h. Afterwards, gene expression decreased but remained elevated as compared to control slices (0h). Conversely, expression of COX‐2 increased at 3h and remained high. Interestingly, a similar expression profile was observed in murine PCKS. During culture, gene levels of IL‐6 and CXCL1 spiked at 3h, after which gene expression decreased, while both IL‐1β and COX‐2 greatly increased at 3h and plateaued. Noteworthy, long‐term incubation (72–96h) induced fibrogenesis in PCKS, possibly driven by ILs. The impact of COX‐2 inhibitors on the inflammatory and fibrotic response in PCKS is currently being evaluated.

Thus, PCKS can be used to study the inflammatory pathogenesis of renal disease and identify therapeutic targets. Moreover, the model is useful to reveal species differences in disease development and progression.

Support or Funding Information

NA
ngPalmPISC: A Sensitive Method for Proteome‐wide Characterization of S‐acylation
Authors: Zhou Bo , Yan Yiwu , Freeman Michael R , Yang Wei
Published: November 2023

Protein S‐acylation (palmitoylation) reversibly modulates protein localization, and thus regulates protein activity, stability, and complex formation. Accumulating evidence suggests that S‐acylation plays a key role in the development and progression of various diseases, such as cancer and neurodegenerative diseases. Previously, we established an S‐acylproteomics method termed Palmitoyl‐protein Identification and Site Characterization (PalmPISC), and identified 67 known and 331 novel candidate S‐acylated proteins from human prostate cancer DU145 cells (Yang et al., Mol Cell Proteomics, 2010, 9: 54–70). In this study, we developed a next‐generation PalmPISC (ngPalmPISC) method that enables proteome‐wide characterization of S‐acylated proteins. Using ngPalmPISC, we identified 2,063 high‐confidence and 528 medium‐confidence candidate S‐acylated proteins from human prostate cancer LNCaP cells. Of these, 1,174 (56.9%) high‐confidence and 242 (45.8%) medium‐confidence candidates are contained in the SwissPalm database, corroborating the effectiveness of our ngPalmPISC method in identifying S‐acylated proteins. In addition, 1,191 and 457 cysteine residues were identified as candidate S‐acylation sites with high and medium confidence, respectively. To our knowledge, this represents the first proteome‐wide characterization of human S‐acylated proteins, with a proteomic coverage of almost an order of magnitude greater than any other single S‐acylproteomics study. Gene ontology analysis of the high‐confidence candidates indicates that S‐acylated proteins are highly significantly enriched in subcellular organelles including mitochondria, endoplasmic reticulum, Golgi apparatus, nucleolus, ribosome, and peroxisome, as well as in biological processes like rRNA processing, translation, and fatty acid beta‐oxidation. Interestingly, many candidate prostate cancer biomarkers, such as prostate‐specific antigen, prostate‐specific membrane antigen, and alpha‐methylacyl‐CoA racemase, are S‐acylated in LNCaP cells. This suggests that S‐acylation may play an important role in prostate cancer progression, and that the S‐acylproteome is a rich source for prostate cancer biomarker discovery. In summary, our ngPalmPISC method opens up a new avenue for proteome‐wide analysis of S‐acylated proteins in cells and clinical specimens, and its application reveals that S‐acylation is more prevalent than we previously thought.

Support or Funding Information

DoD PCRP W81XWH‐15‐1‐0167

Steve Spielberg Cancer Discovery Foundation (CSR205927)