bluuebunny/crossref_metadata_2025 huggingface dataset:
Drosha‐dependent miRNA regulate aldosterone synthesis by increasing StAR and HSD3B2 expression
Authors: Oki Kenji , Gomez-Sanchez Elise P , Gomez-Sanchez Celso E
Published: June 2021

Drosha initiates the production of miRNAs from precursors. To study the functions of miRNA in adrenal steroid production, we knocked down Drosha expression in the HAC15 cell, a subclone of the H295R human adrenocortical cell, using lentiviral vectors to deliver shRNA of Drosha. Drosha protein levels in the infected HAC15 cells were reduced by 74.3% compared to empty vector infected control cells. Drosha knock‐down led to 36.3% and 46.4% suppression of aldosterone production under basal and after angiotensin II (A‐II) stimulated conditions, respectively. Basal and A‐II stimulated StAR and HSD3B2 mRNA expression levels in the Drosha knock‐down cells were also significantly suppressed; expression of other enzymes required for aldosterone synthesis were not altered. In conclusion, suppression of Drosha significantly diminished basal and A‐II stimulated StAR and HSD3B2 mRNA expression and aldosterone production, suggesting that at least one miRNA is essential for aldosterone production.

Support: VA Medical Research Funds, NIH HL27255 & HL75321
The effects of angiotensin II receptor antagonism on fear memory and immune cell modulation
Authors: Marvar Paul J , Geem Duke , Khoury Nayla , Ressler Kerry J
Published: June 2021

Pharmacological inhibition of the renin‐angiotensin system (RAS) is commonly used to treat hypertension and more recently been suggested as a treatment of emotional stress. In a clinical study we have recently shown a role for the RAS in the regulation of the stress response in patients with post‐traumatic stress disorder (PTSD). Psychological stress is also known to perturb the homeostasis of the immune system. The purpose of this study was to further examine the role of RAS in an animal model of PTSD and its impact on the immune system. We performed Pavlovian fear conditioning pairing auditory cues with footshocks to examine the effects of the angiotensin II receptor antagonist Losartan (1mg/kg and 10mg/kg) on fear memory extinction. 24 h after fear conditioning, Losartan was administered 1h prior to fear memory extinction training. While no effect of Losartan was observed on extinction training, there was a significant decrease in fear as measured by freezing behavior to the auditory cue when tested the following day (14.9% vs 27.3%; p<0.05). Immune cells of the spleen were quantified using flow cytometry and Losartan treated mice exhibited significantly fewer CD45+CD11b+ (macrophage– like) cells (p<0.05). These data suggest that the RAS may contribute to the retention of extinction fear memory and alter the effectiveness of the immune system to respond to a challenge.
Steroidhormones in the saliva of children after acute physical stress in school
Authors: Budde Henning , Windisch Claudia , Wegner Mirko , Arafat Ayman , Voelcker-Rehage Claudia
Published: June 2021

Acute bouts of physical activity can influence the secretory processes of the adrenal cortex releasing cortisol (C) in adolescents (Budde et al., 2010). After 12 minutes of exercise and an adequate workload of > 70% of the HFmax adolescents revealed increased salivary C levels (Budde et al., 2010).

The aim of our study was to assess to what extent these results are replicable in primary school students.

53 students of a 4th grade (9–10 years of age) were randomly assigned to an experimental (EG) and a control group (CG). Saliva collection took place after a normal school lesson (pre‐test) and 5 minutes after a 12‐min. control condition (movie watching) (CG, n = 21) or intensive exercise in a defined heart rate (HR) interval (EG, n = 32) (post‐test).

Repeated measures ANOVA showed a significant interaction between group (EG, CG) and test session (pre‐test, post‐test), F (1,49) = 5.00, p = .030, Î·2 = .093, pointing to a different response to physical exercise for the EG compared with the CG. However, post‐hoc t‐tests showed no significant increase of C concentration in the EG, but a significant decrease of C in the CG from pre‐ to post‐test.

Results, also for testosterone, are being discussed.
Effect of the peroxisome proliferator‐activated receptor gamma (PPARγ) agonist Rosiglitazone on the hypothalamic GT1‐7 cell line
Authors: Hwang Vicky , Fernandez Marina Olga , Webster Nicholas Julian
Published: June 2021

Polycystic ovary syndrome (PCOS) is a major cause of infertility in women and is linked to obesity and insulin resistance. Rosiglitazone, a PPARγ agonist, increases insulin sensitivity and improves PCOS and diabetes. PPARγ ligands have also been found to affect the hypothalamus‐pituitary‐gonadal axis, although their site and mechanism of action are not entirely known. The aim of this study was to determine the effects of rosiglitazone on insulin and kisspeptin stimulation of GT1‐7 hypothalamic GnRH neurons. Timecourse and dose response experiments were conducted using 24 hour pretreatment of rosiglitazone or vehicle, and with either insulin or kisspeptin stimulation. Akt and ERK phosphorylation were analyzed with Western Blot and GnRH mRNA expression was analyzed with RT‐QPCR. Preliminary results indicated that treatment with rosiglitazone increased insulin‐induced phosphorylation of Akt in GT1‐7 cells. Maximal stimulation occurred between 10 and 15 minutes, and an insulin concentration of 10 nM. Kisspeptin alone increased GnRH mRNA, with maximal stimulation at 1 nM, as reported by others. Furthermore, rosiglitazone appears to increase kisspeptin induced GnRH mRNA. In conclusion, rosiglitazone appears to have a direct role in regulating hypothalamic GT1‐7 neurons by acting as an insulin sensitizer and modulating the effects of kisspeptin.

Supported by NIH grant U54HD12303. VH was supported by Julia Brown Undergraduate Research Scholarship.
Blast brain injury elevates catecholamine biosynthesis in the rat adrenal medulla
Authors: Tümer Nihal , Svetlov Stanislav , Kirichenko Nataliya , Whidden Melissa , Erdos Benedek , Prima Victor , Sherman Alexandra , Kobeissy Firas , Yezierski Robert , Vierck Charles Charles , Wang Kevin
Published: June 2021

Traumatic Brain Injury (TBI) produces major health problems impacting the lives of both military and civilian personnel. TBI disrupts autonomic function but the nature of this disruption is unknown. Following brain injury, we assesed selective biochemical markers for autonomic function in adult male Sprague Dawley rats. Rats were subjected to head‐directed overpressure blast injury (OBI) of 358 kPa magnitude at the target. At the same time for sham controls, rats were anesthetized as the previous group but instead of OBI were exposed just to noise being placed at ~ 2 m distance from the shock tube nozzle. Sympathetic nervous system activation of the adrenal medullae (AM) was evaluated at 6 hours following blast injury by assessing the expression of catecholamine biosynthesizing enzymes, tyrosine hydroxylase (TH), dopamine‐β hydroxylase (DβH), along with Neuropeptide Y (NPY). TH and DβH expression increased by 20% and 25%, respectively, following OBI (P<0.05). NPY expression was also significantly elevated by 91% (P<0.01) following OBI. NPY is synthesized and co‐realesed with catecholamines in the AM. Collectively, the increased TH, DβH and NPY expression in the rat AM suggests that OBI results in increased sympathoexcitation. Such effects may be one of the important factors contributing to autonomic dysfunction following OBI.
CXCL12α induces proliferation and migration of endometrial epithelial cells expressing CXCR4
Authors: Ruiz Abigail , Monteiro Janice , Colon Mariano , Echevarria Martha , Morales Luisa , Bayona Manuel , Fazleabas Asgerally , Flores Idhaliz
Published: June 2021

Endometriosis defined as the presence of endometrial stroma and glands outside the uterus, is associated with chronic pain, inflammation, and increased levels of chemokines. We reported high CXCR4 mRNA levels in endometriotic lesions in a rat model and protein expression in human endometriosis tissues vs. control endometrium. CXCR4 is predominantly expressed by endometrial epithelial cells (EEC) and its ligand CXCL12 by endometrial stromal cells. The CXCR4‐CXCL12 axis plays roles in angiogenesis, invasion, and cell proliferation. We hypothesized that CXCL12α may induce proliferation and migration in EEC expressing CXCR4 via activation of the PI3K/Akt pathway. Cell proliferation and migration of EEC treated with CXCL12α was analyzed by BrdU proliferation and wound healing assays, respectively. We also studied CXCL12α‐mediated induction of Akt phosphorylation by immunoblotting. CXCL12α increased proliferation and migration of EEC vs. media (p<0.05). In addition CXCL12α induced rapid and transient Akt phosphorylation. These data suggest that the CXCR4‐CXCL12α axis can be activated in endometrial cells to promote cell proliferation and migration. These biological functions have been previously shown to be dysregulated in endometriosis, by still undefined mechanisms, leading to survival and growth of ectopic endometrium. R01‐ HD050509, S06‐GM08239, 1R25‐GM082406, R25GM096955‐01
Depression and anxiety behaviors are influenced by sex and estrous cycle stage
Authors: Alvarado John Kenneth , Rivera Johanna Stephanie , Jimenez Esther Angelica , Ramos-Ortolaza Dinah Lee , Torres-Reveron Annelyn
Published: June 2021

It is well‐documented that as compared to men, women experience higher levels of mood disorder and depression during the reproductive years. However, it has not been fully elucidated how variations in gonadal hormones are linked to depression and anxiety disorders. During eight weeks, adult females and males Sprague Dawley rats were exposed to a mild stressor consisting of housing isolation (HI) and were compared to paired‐housed controls. Vaginal smears were taken to determine the estrus cycle stage. At the end of the eight‐week period all animals were tested on the open field (OF) followed by the forced swim test (FST) for measuring anxiety and depression‐like behaviors. Myeloperoxidase activity in the colon of female rat showed a significant increase in HI females as compared to the paired housed females, suggesting underlying inflammatory processes produced by stress. However, the stressor (HI) only produced mild effects on the animal's behaviors. There were significant effects of sex and estrous cycle on OF and FST. During the OF the males presented reduced locomotor activity as compared to females. Females in proestrus showed less depressive behaviors as compared to males, confirming previously reported antidepressant effects of estrogen. These experiments demonstrate a significant role of gonadal hormones on the expression of depression‐like behaviors. NSU‐CFRDG 335513 & R25GM096955‐01.
Impact of SOD on Endothelial Cell Free Radical Distribution during eNOS Uncoupling
Authors: Kar Saptarshi , Kavdia Mahendra
Published: June 2021

Uncoupling of endothelial nitric oxide synthase (eNOS) results in a lower NO production and an increase in O2•− production from eNOS. The excess O2•− produced in the endothelial cells either reacts with the reduced NO available to from peroxynitrite (ONOO−) or gets dismutated by the enzyme superoxide dismutase (SOD) to form another oxidizing species H2O2. The excess H2O2 is scavenged by certain enzymes. It has been reported that oxidative stress reduces the activity of SOD and H2O2 scavenging enzymes causing excess build up of H2O2 and ONOO− in the endothelial cells. In this study, we quantify the contribution of eNOS uncoupling and SOD concentration on endothelial cell based H2O2 and ONOO− concentration for functional and dysfunctional endothelium. A single cell based computational model involving the biochemical pathways of eNOS and the reactions involving NO, O2•− and ONOO− was developed. The extent of endothelial functionality was defined by biopterin ratio ([BH4]/[TBP]). The results show that (1) both ONOO− and H2O2 concentration vary significantly with SOD concentration at all biopterin ratios and (2) for a given SOD concentration, H2O2 concentration increased by 3 orders of magnitude on transition from functional to chronic dysfunctional endothelium. The results show that eNOS uncoupling is a significant contributor to oxidative stress and the limitations of SOD to reduce it. Supported by NIH R01 HL084337.
PERIPHERAL PRAZOSIN COMBINED WITH NORADRENALINE INTO THE LATERAL PARABRACHIAL NUCLEUS FACILITATES CHOLINERGIC‐INDUCED SODIUM APPETITE
Authors: Gasparini Silvia , Gomide Joelma Maria Cardoso , Andrade-Franzé Glaucia Maria Fabricio , Andrade Carina Aparecida Fabricio , de Almeida Colombari Débora Simões , De Luca Laurival Antônio , De Paula Patrícia Maria , Moreira Thiago Santos , Menani José Vanderlei
Published: June 2021

Objectives

In the present study, we investigated the effects of the combination of noradrenaline into the LPBN and prazosin (α1‐adrenoceptor antagonist) intraperitoneally (ip) on water and 0.3 M NaCl intake in rats treated with ip injection of the cholinergic agonist pilocarpine.

Methods and results

Male Holtzman rats with bilateral stainless steel guide‐cannulas implanted into LPBN were used. Pilocarpine (1 mg/kg) injected ip induced water intake (2.9 ± 0.3 ml/180 min), without affecting 0.3 M NaCl intake (0.5 ± 0.3 ml/180 min). Bilateral injections of noradrenaline (80 nmol/0.2 μl) into the LPBN combined with ip prazosin (1 mg/kg) increased ip pilocarpine‐induced water intake (6.3 ±1.7 ml/180 min) and 0.3 M NaCl intake (14.7 ± 3.5 ml/180 min). LPBN noradrenaline injections combined with ip saline did not affect 0.3 M NaCl (0.8 ± 2.4 ml/180 min) and decreased water intake (0.8 ± 0.3 ml/180 min) induced by ip pilocarpine. Prazosin ip did not modify 0.3 M NaCl (4.8 ± 3.6 ml/180 min) or water intake (0.5 ± 0.3 ml/180 min) in rats treated with pilocarpine ip combined saline into the LPBN.

Conclusion

The results suggest that strong cholinergic‐induced sodium intake arises when the inhibitory mechanisms are deactivated with noradrenaline injected into the LPBN combined with the blockade of pressor responses with ip prazosin.
Mast Cell Corticotropin Releasing Factor Receptor Subtypes Play Opposing Roles in Mediating Stress‐Induced Colonic Barrier Dysfunction
Authors: Moeser Adam , Gibson Amelia
Published: June 2021

It has long been known that gastrointestinal disorders are profoundly influenced by life stress; however the mechanisms are poorly understood. Previous research showed that psychological stress causes disturbances in intestinal epithelial barrier function via corticotropin releasing factor (CRF)‐dependent activation of intestinal mast cells (MC). The precise pathway of MC activation by CRF in the stress response remains unclear. Here we definitively explore the role of MC CRF receptors 1 and 2 (CRF1 and CRF2) in stress‐induced intestinal barrier dysfunction. MC‐deficient mice (W‐sh/W‐sh) were repleted with bone marrow‐derived mast cells (BMMCs) from wild type (WT), CRF1KO, or CRF2 KO mice. Following the successful repletion of BMMCs, mice were subjected to 3 hours of restraint stress (RS). Transepithelial electrical resistance (TER) and paracellular flux of FITC‐dextran (FD4) were measured in Ussing chambers as indices of barrier function. There was a significant increase (p=0.003) in the rate of FD flux and reduction in TER in the colon of WT mice subjected to RS but not in W‐sh/W‐sh mice. Repletion of W‐sh/W‐sh mice with WT BMMCs restored stress‐induced changes in FD4 flux and TER (p=0.01). Repletion of W‐sh/W‐sh mice with CRF1 KO BMMCs ameliorated stress‐induced increases in FD4 flux. In contrast, repletion of W‐sh/W‐sh mice with CRF2 KO BMMCS exacerbated stress‐induced barrier dysfunction compared with WT BMMC controls (p<0.05). These data definitively show that MC CRF1 mediates stress‐induced intestinal barrier dysfunction while CRF2 may play a critical barrier protective role. These opposing roles of CRF receptors may help in identifying novel preventative and therapeutic targets to mitigate stress‐related intestinal disorders.
No evidence that copper is a transported substrate of the iron transporter DMT1
Authors: Shawki Ali , Niespodzany Eric J , Mackenzie Bryan
Published: June 2021

Divalent metal‐ion transporter 1 (DMT1) is essential for intestinal iron absorption and erythroid iron utilization. Mechanisms for the cellular uptake of copper are less clearly understood. We have previously demonstrated that DMT1 exhibits promiscuity towards a broad range of divalent metal ions that includes Cd2+, Co2+, Fe2+, Mn2+, Ni2+ and VO2+ but not copper; however, other investigators have claimed that DMT1 transports copper. Here, we further tested the hypothesis that copper is a transported substrate of DMT1 in vitro by measuring metal‐ion fluxes and currents in RNA‐injected Xenopus oocytes expressing DMT1. Expression of DMT1 in oocytes stimulated over 80‐fold the uptake of 2 μM 55Fe2+ at pH 6.0 (P < 0.001) but transport of 64Cu1+ or 64Cu2+ did not differ from control (P ≥ 0.71); however, Cu1+ inhibited DMT1‐mediated 55Fe2+ uptake in a competitive fashion (Ki ≈ 58 μM). In both control oocytes and oocytes expressing DMT1, Cu1+ evoked an inward current that markedly differed from typical DMT1‐dependent transport currents. Cu2+ did not evoke a current. Whereas DMT1 substrates (e.g. Fe2+ and Cd2+)—but not Cu1+—abolished the presteady‐state currents observed in the absence of metal ion, Cu2+ instead altered the time constant of the decay and did not abolish the transient currents. We conclude that Cu1+ and Cu2+ are capable of interacting with DMT1 but that neither is a transported substrate.
Exposure to tributyltin alters the secretion of tumor necrosis factor alpha from human lymphocytes
Authors: Hurt Kelsi , Whalen Margaret M.
Published: June 2021

Tributyltin (TBT) is an organotin compound that contaminates the environment and has been found in human blood samples. TBT‐exposed mammals show increased incidences of tumors. Human natural killer (NK) cells are the earliest defense against tumors and viral infections and secrete the cytokine tumor necrosis factor (TNF) alpha. TNF alpha is an important regulator of both the adaptive and innate immune responses. It promotes the inflammatory response and an association between malignant transformation and inflammation has been established. Previously, we have shown that TBT was able to interfere with the ability of NK cells to lyse tumor target cells. It important to determine if TBT can alter cytokine secretion by NK cells as this is also a critical function of these cells. The current study examines the consequences of 24 hour, 48 h and 6 day exposures to TBT on TNF alpha secretion by highly enriched human NK cells as well as mixture of T cells and NK cells. The results indicate that concentrations of TBT of 200 ‐ 2.5 nM caused significant alterations of TNF alpha secretion. Supported by NIH grant S06 GM008092‐35
Effects of Korean red ginseng on cyclophosphamide‐induced immunosuppression: comparative effects of 6‐year and 4‐year old Korean red ginseng
Authors: Hyun Sun Hee
Published: June 2021

In this study, the protective effects of Korean red ginseng (KRG) against cyclophosphamide (CY)‐induced immunosuppression were investigated in animal models. Male BALB/c mice were divided into 6 groups: Normal and CY‐control groups were fed normal diet and the four test groups received modified diets containing 5 or 10% of 4‐year old or 6‐year old KRG powder for 4 weeks. In the subtype and NK cell activity assay, CY was given intraperitoneally in a single dose of 50 mg/kg three days before sacrifice. For the T‐dependent antibody response, CY was given intraperitoneally in a single dose of 50 mg/kg five days before sacrifice and mice were immunized with sheep red blood cells (SRBCs) intraperitoneally in the next day.

CY, as reported, showed suppressed effects on body weights, organ weights, splenic lymphocyte subpopulation (total T cells, CD4+ T cells, CD8+ T cells, macrophages and B cells), antibody‐forming cells to SRBC and NK cell activity in these animals. The administration of KRG powder elevated the depressed relative weights of thymus and spleen and increased the number of splenic lymphocyte subpopulation, anti‐SRBC producing cells that were remarkably decreased by the treatment of CY. 6‐year old KRG powder showed more potent effect than 4‐year old KRG in enhancing immune function in the CY‐immunosuppressed mice. These results suggest that 6‐year old KRG is more potent in stimulating immunity in immunosupressed condition than 4‐year old KRG.
Pharmacological characterization of NOP receptor agonists as abuse‐free and constipation‐free analgesics in monkeys
Authors: Wladischkin Kathryn A , Dysko Robert C , Collins Gregory T , Ko Yi-An , Winger Gail , Ko Mei-Chuan
Published: June 2021

The nociceptin/orphanin FQ peptide (NOP) receptor, the fourth opioid receptor subtype, mediates distinctive actions in monkeys that suggest a possibility that NOP agonists may result in strong analgesia in the absence of side effects that are found in mu opioid receptor (MOP) agonists in primates. The aim of this study was to investigate and compare the behavioral effects of NOP agonists, Ro 64‐6198 and SCH221510, in side‐by‐side comparisons with MOP agonists, morphine and alfentanil, in rhesus monkeys. Both NOP and MOP agonists dose‐dependently produced antinociceptive effects against a noxious stimulus. Antagonist studies demonstrated that NOP and MOP receptors independently mediate antinociceptive actions. However, at antinociceptive doses, MOP agonists, but not NOP agonists, produced respiratory depression. NOP agonists did not produce reinforcing effects comparable with those of MOP agonists under the self‐administration procedure. In addition, at equianalgesic doses, injections of 3 mg/kg of morphine, but not 0.01 mg/kg of SCH221510, significantly delayed the gastrointestinal transit rate of monkeys, indicating that NOP agonists lack of constipating effects of MOP agonists. These results suggest that activation of NOP receptors produces antinociception with reduced abuse liability and fewer side effects in primates (Supported by USPHS grant AR‐059193).
Oral administration of L‐leucine reduces nitric oxide synthesis by endothelial cells of rats
Authors: Tekwe Carmen D. , Lei Jian , Yao Kang , Li Xilong , Rezaei Reza , Dahanayaka Sudath , Meininger Cynthia J. , Carroll Raymond J. , Bazer Fuller W. , Wu Guoyao
Published: June 2021

High concentrations of branched‐chain amino acids in plasma are commonly associated with endothelial dysfunction in obese humans and animals. This study tested the hypothesis that elevated levels of leucine in plasma can reduce nitric oxide (NO) synthesis by endothelial cells (EC). Beginning at 19 weeks of age, male Sprague‐Dawley rats were fed a semi‐purified diet containing a normal fat content and received drinking water containing either 1% L‐leucine or 0.68% L‐alanine (isonitrogenous control) (n=8/group). Twenty‐four‐hour whole‐body energy expenditure of rats was determined every 4 weeks using Oxymas (an open circuit calorimeter, Columbus Instruments, Ohio). At 31 weeks of age, rats were euthanized to obtain tissues. Oral administration of leucine increased (P < 0.01) concentrations of leucine in plasma by 35%, but did not affect (P > 0.05) water consumption (25 mL/kg BW per day), food intake, body weight, skeletal muscle weight, white adipose tissue weight, or whole‐body heat production. Leucine administration enhanced (P < 0.05) glutamine:fructose‐6‐phosphate transaminase activity and decreased (P < 0.05) NO synthesis in EC and whole‐body insulin sensitivity. These novel results indicate that elevated concentrations of leucine promote glucosamine synthesis and reduce NO production by EC, contributing to increased risk for cardiovascular disease. (Supported by AHA and NIH grants).
Activation of Protease‐activated receptor −3 signals independently to phosphorylate endothelial nitric oxide synthase in human endothelial cells
Authors: Tillery Lakeisha C. , Epperson Tenille , Mantey Jane , Motley Evangeline
Published: June 2021

Protease‐activated receptor‐3 (PAR‐3) is widely recognized as a non‐signaling elusive member of a G protein‐coupled receptor family known to regulate the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway via phosphorylation of its regulatory sites. Failure of the eNOS/NO pathway leads to endothelial dysfunction, a precursor to cellular induced hypertension. Using human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) as a model for human endothelium, we demonstrated that PAR‐3 is highly co‐expressed with PAR‐1 and ‐2 and can be activated using a synthetic peptide, SFNGGP‐NH2. We hypothesize that PAR‐3 can signal independently of PAR‐1 and ‐2 to regulate eNOS. Analysis using eNOS specific antibodies showed that 50uM of SFNGGP lead to the phosphorylation of the positive site of eNOS at ‐Ser1177 and the negative site ‐Thr495 in a time‐dependent manner in both cell lines. Human endothelial cells pretreated with 10uM of SCH79797, a selective PAR‐1 inhibitor, and then stimulated with SFNGGP; we observed an increase in phosphorylation at both eNOS regulatory sites. Using siRNA, transient knock‐down of PAR‐1 confirms PAR‐3 independent signaling potential. Our data suggests that PAR‐3 is able to regulate eNOS/NO signaling via site specific phosphorylation that is independent of PAR‐1 activation. Cardiovascular disease is the leading cause of death among Americans, where hypertension can be a silent aggressor. Studying the signaling pathways responsible for eNOS‐induced NO production will lead to a better understanding of endothelial dysfunction in cardiovascular diseases.

NIH‐NIGMS‐SCORE and Porter Physiology Developmental Fellowship
Differences in resting metabolic rate and physical activity patterns in lean and overweight/obese pregnant women
Authors: Ruebel Meghan Lynne , Shankar Kartik , Badger Thomas , Andres Aline
Published: June 2021

Energy requirements vary during pregnancy due to changes in physical activity (PA) and maternal fat stores. This study measured resting metabolic rate (RMR) and PA patterns in healthy lean and overweight/obese (OW) pregnant women. RMR was measured using indirect calorimetry (MOXUS), activity patterns were estimated by omnidirectional accelerometers (Actical®) and body composition was measured using air displacement plethysmography (BodPod®). Nineteen pregnant women including 12 lean (BMI 18.5–24.9 kg/m2) and 7 OW (BMI 25–35 kg/m2) were assessed at 6–10, 12 and 24 weeks (wk) of gestation. Absolute RMR differed significantly between lean and OW women (1438 ± 49 vs. 1753 ± 62 kcal/day, p<0.05) at 6–10 wk. Moreover, when adjusted for weight, RMR (kcal/kg/d) differed (p<0.05) throughout pregnancy for lean and OW women (23 ± 1 vs. 21 ± 1, 24 ± 1 vs. 20 ± 1, 22 ± 1 vs. 19 ± 1 at 6–10, 12 or 24 wk, respectively). RMR was also positively correlated with fat mass and fat free mass in both groups (r=0.77, and r= 0.52, respectively, p<0.05). Although not significant, lean women spent ~50 more minutes in moderate to vigorous and ~150 less minutes in sedentary time compared to the OW over the 24 wk. In conclusion, OW women had lower RMR (kcal/kg/d) and decreased PA compared to lean women, suggesting a greater risk of excessive gestational weight gain and post partum weight retention. USDA‐ARS CRIS# 6251‐51000‐005‐03S.

Grant Funding Source : USDA‐ARS CRIS# 6251‐51000‐005‐03S
Effect of molsidomine and tempol on ischemic tolerance of chronically hypoxic rat hearts
Authors: Mandikova Petra , Micova Petra , Kozichova Kristyna , Dabrowska Ludmila , Kolar Frantisek , Neckar Jan
Published: June 2021

Chronic hypoxia (CH) is associated with increased formation of reactive oxygen species (ROS), pulmonary hypertension (PH) and improved myocardial tolerance to ischemia/reperfusion injury. The aim was to find out whether nitric oxide donor (molsidomine; MOL) and superoxide dismutase mimetic (tempol; TMP) attenuate i) development of PH and ii) increased ischemic tolerance of CH hearts. Adult male Wistar rats were adapted to continuous normobaric CH (10 % O2, 4 wks). Subgroups of the animals were treated with MOL or TMP given either acutely before ischemia or chronically during adaptation. Right ventricular systolic pressure (RVSP) was increased in CH rats by 70 % compared to the normoxic group. Chronic TMP significantly attenuated the increase in RVSP while no effect was observed in chronic MOL‐treated group. Adaptation to CH significantly decreased infarct size induced by coronary artery occlusion (43.4 ± 2.9 % of the area at risk) compared to normoxic controls (57.5 ± 2.7 %). Chronic TMP abolished the protection induced by CH (55.3 ± 2.6 %). Acute TMP had any effect on infarct size in neither group. Chronic MOL reduced infarct size only in the normoxic group (50.7 ± 2.3 %) while acute MOL treatment protected both normoxic and CH groups by 40 – 45 %. Our results confirm that ROS play a role in the development of PH and cardioprotection induced by CH. Supported by grants IAAX01110901 and GAUK 411911.
Exercise Training Improves Coronary Microvascular Arteriolar Function in Familial Hypercholesterolemia Porcine Model via Nrf2
Authors: Lee Sewon , Park Yoonjung , Zuidema Mozow Y. , Laughlin M. Harold , Bowles Douglas K. , Baines Christopher , Hannink Mark , Hill Michael A. , Dellsperger Kevin C. , Zhang Cuihua
Published: June 2021

We tested the hypothesis that exercise in Rapacz‐Familial Hypercholesterolemic (FH, a model of established atherosclerosis) swine may improve endothelial‐dependent vasodilation in coronary arterioles via NF‐E2 related factor (Nrf2), a nuclear transcription factor that regulates antioxidant gene expression. Male FH swine were divided into exercise (EX; n=7) and sedentary (SED; n=6) groups. We assessed endothelium‐dependent and ‐independent dilation from isolated and pressurized coronary arterioles from FH swine. Although SNP‐induced dilation was not different between SED and EX, both adenosine‐ and flow‐mediated dilation were significantly higher in EX compared to SED. TEMPOL, a superoxide dismutase (SOD) mimetic, improved the adenosine‐mediated vasodilation in FH SED coronary arterioles (n=7). Extraluminal incubation of coronary arterioles with a Nrf2 inhibitor, all‐trans retinoic acid (ATRA; 1μM, 60 min) reduced endothelial‐dependent vasodilation in both SED and EX FH swine (n=7). In addition, EX increased Nrf2 mRNA expression in the heart homogenates. In conclusion, our results indicate that EX of FH pigs may exert a beneficial effect in coronary arterioles by activating Nrf2, resulting in attenuating oxidative stress and increasing the bioavailability of nitric oxide (NO). Supported by HL077566, HL085119, HL52490, AHA, SDG and 10POST3870022.
Role of BKCa in endothelium‐dependent dilation of soleus feed arteries
Authors: Rawicki Nathaniel L. , Gray Eric J. , Carter Matthew R. , Jasperse Jeffrey L.
Published: June 2021

Endothelium‐dependent dilation is mediated by nitric oxide (NO), prostacyclin (PGI2), and one or more endothelium‐derived relaxing factors (EDHF). We tested two hypotheses: 1) that large Ca2+ activated K+ (BKCa) channels contribute to EDHF‐mediated dilation in rat soleus feed arteries (SFA), and 2) that BKCa activity is increased by short term exercise training (rats ran 30 m/min, 10% incline, 1 hr/day, 5 days/wk for 4 wks). SFA (n=31) were isolated, cannulated, and pressurized at 90 cmH2O. Following a 1‐hr equilibration period, an acetylcholine dose‐response curve was performed, followed by curves using iberiotoxin (IBTX, 5μM) to inhibit BKCa, L‐NNA (300μM) and indomethacin (INDO, 50 μM) to inhibit production of NO and PGI2 respectively, and combined BKCa, NO, and PGI2 inhibition. There were no differences between sedentary and short‐term trained rats, so all data from both groups is presented together. Maximal dilation to ACh was reduced by IBTX (Con 83.7± 3.1 vs. IBTX 61.2 ± 6.3 % dilation). L‐NNA and INDO decreased ACh‐mediated dilation, but adding IBTX did not further reduce ACh dilation (Con 83.7± 2.3 vs. L‐NNA+ INDO 30.9 ± 6.1 vs. IBTX+ L‐NNA+ INDO 20.1± 5.5 % dilation). These data indicate that BKCa contribute to ACh‐mediated dilation, but are not altered by short term exercise training. Adding IBTX to L‐NNA and Indo did not further reduce ACh dilation, indicating that NO and BKCa share a common signaling pathway.
β‐Adrenergic Receptor (β‐AR)‐Stimulated Cardiac Myocyte Apoptosis and Myocardial Remodeling are Modulated by Exogenous Ubiquitin
Authors: Daniels Christopher R. , Foster Cerrone R. , Yakoob Sana , Singh Mahipal , Singh Krishna
Published: June 2021

β‐AR stimulation increases levels of extracellular ubiquitin (UB), and UB inhibits β‐AR‐stimulated cardiac myocyte apoptosis. We hypothesized that exogenous UB plays a protective role in chronic β‐AR‐stimulated myocyte apoptosis and myocardial remodeling. Mice were infused with vehicle (sham), UB (1μg/g/h), isoproterenol (ISO; 400μg/kg/h) or UB+ISO using mini‐osmotic pumps. Left ventricular (LV) structural and functional remodeling was studied 7 days after infusion. ISO‐infusion similarly increased heart weight:body weight ratio in both ISO groups. M‐mode echocardiography indicated increased percent fractional shortening and ejection fraction in ISO, not UB+ISO, group. Isovolumic contraction and relaxation times, as measured using Doppler echocardiography, were significantly lower in ISO vs UB+ISO. The increase in cardiac myocyte apoptosis and fibrosis was significantly greater in ISO vs UB+ISO. Akt activity was higher, while GSK‐3β activity was lower in UB+ISO vs ISO. MMP‐2 expression and activity was higher in UB+ISO, while increased MMP‐9 expression was only observed in UB+ISO hearts. In vitro, UB treatment enhanced MMP‐2 expression and activity and increased MMP‐9 expression in adult cardiac fibroblasts. Exogenous UB plays a crucial role in β‐AR‐stimulated myocardial remodeling with effects on LV function, fibrosis and myocyte apoptosis. Supported by HL‐091405, HL‐092459 and BX000640
Combined Effect of Flow‐Mediated Shear Stress and Resveratrol on Sirt1/PGC‐1α Pathway in Vascular Endothelial Cells.
Authors: Kim Ji Seok , Kim Boa , Park Joon-Young
Published: June 2021

The concept of enhancing structural integrity of mitochondria has emerged as a novel protective mechanism against CVD. Flow‐mediated laminar shear stress (LSS) and resveratrol (RSV) have vasoprotective effects, in part, by promoting mitochondrial biogenesis through Sirt1/PGC‐1α pathway.

Objective

The purpose of this study was to test a hypothesis that combined treatment of the two effective stimulators would have synergistic effect on the activation of Sirt1/PGC‐1α pathway in endothelial cells (ECs).

Methods

HUVECs were exposed to either high LSS (h‐flow, exercise‐mimic, 20 dyne/cm2), RSV (20 μM), or h‐flow + RSV conditions for 12 h. For the h‐flow conditions, cells were further exposed to low LSS (l‐flow, postexercise recovery‐mimic, 5 dyne/cm2) for 24 h.

Results

RSV treatment increased Sirt1 and PGC‐1α expression levels in a dose‐and time‐dependent fashion. We observed that the Sirt1 protein expression level was further increased under h‐flow + RSV condition (P < .05). Moreover, h‐flow resulted in 10‐fold increase in PGC‐1α expression level during l‐flow (P < .05). However, there was no additive effect of RSV on PGC‐1α expression level at any shear conditions.

Conclusion

The current preliminary data suggests that, although the combined RSV and h‐flow treatment has a synergistic effect on Sirt1 expression, h‐flow may override the effect of RSV with respect to the mitochondrial biogenesis in ECs.
Effects of physical training on neuronal hypothalamic activation induced by exercise
Authors: Santiago Henrique Pereira , Leite Laura Hora Rios , de Andrade Lima Paulo Marcelo , Rodovalho Gisele Vieira , Szawka Raphael Escorsim , Coimbra Cândido Celso
Published: June 2021

We assessed whether the effects of physical training on heat balance are followed by hypothalamic activation induced by exercise. After physical training (8 weeks), trained (T) and untrained (UT) Wistar rats were submitted to graded running until fatigue while body (Tb) and tail (Ttail) temperatures were recorded. Workload (W), body heating rate (BHR), heat storage (HS) and Tb threshold for tail vasodilation (TTbV) were determined. Afterwards, c‐Fos immunohistochemistry was conducted in paraventricular nucleus (PVN), medial preoptic nucleus (MPO) and median preoptic nucleus (MnPO). T animals had a 110% increase in W (p < 0.01), however, animals from both groups had similar increases in Tb and Ttail during exercise. A reduction of 0.6°C in TTbV was shown by T animals (p < 0.01), being possibly responsible for the lower BHR (0.019 ± 0.002 °C.min‐1, T vs 0.030 ± 0.005 °C, UT, p < 0.05) and ratio between HS and W (18.18 ± 1.65 cal/Kgm, T vs 31.38 ± 5.35 cal/Kgm, UT, p < 0.05). Physical training increased by 112%, 90% and 65% neuronal activation in the PVN, MPO and MnPO after running, respectively (p < 0.01), which was directly associated with W (PVN: r = 0.97; MPO: r = 0.86; MnPO: r = 0.77, p < 0.05). The data indicate that physical training enhances neuronal activation induced by exercise in these nuclei, related with improvement of exercise performance and probably with heat loss enhancement.

Supported by: CNPq, FAPEMIG and CAPES.
Central NO‐signaling is important to PVN activation, improving heat dissipation and exercise performance
Authors: de Andrade Lima Paulo Marcelo , Santiago Henrique Pereira , Coimbra Candido Celso
Published: June 2021

To investigate whether central NO‐signaling is involved in activation of PVN induced by treadmill running, L‐NAME (n=9) or SALINE (n=9) were injected into the lateral cerebral ventricule of Wistar rats immediately before running until fatigue (18m.min, 5% inclination). Intraperitoneal temperature and tail temperature were measured during exercise. Animals were subjected to transcardiac perfusion 90 minutes after the end of the exercise session. Brains were removed for c‐Fos immunohistochemical analysis. Heat storage rate (HSR) and maximal variation of tail temperature (MVTt) were calculated. Running exercise increased 656% c‐Fos expression in the PVN (p<0.001). Rats injected with L‐NAME showed a reduction of 53% in c‐Fos expression in the PVN (p<0.01), and a reduction of 42% in running performance (p<0.01), with lower MVTt (3.2 ± 0.4 L‐NAME vs. 4.4 ± 0.3 ºC SAL, p<0.05), and consequentely, higher HSR (15.2 ± 2.2 L‐NAME vs. 10.7 ± 2.0 cal/min SAL, p<0.01) when compared with SAL‐group. c‐Fos expression was directly associated with running performance (r=0.917, p<0.01), with MVTt in the SAL‐group (r=0.978, p<0.05), and inversely associated with HSR (r=0.739, p<0.05). Central inhibition of brain nitrergic system decreases the exercise‐induced c‐Fos expression in the PVN, leading to decreased heat dissipation capacity, which in turn, increased HSR, and decreased physical performance. CNPq, Capes and Fapemig.
Exercise‐induced cardioprotection is dependent on enhanced glutathione reductase activity
Authors: Frasier Chad R , Sloan Ruben C , Hayes Brian , Brown David A
Published: June 2021

Oxidation of myocardial glutathione beyond a critical level collapses mitochondrial energetics and leads to cardiac ischemia‐reperfusion injury. We recently found that exercise‐induced cardioprotection was characterized by improved glutathione replenishment during varying oxidative insults. Since we observed increased glutathione reductase (GR) activity following exercise, we conducted this study to determine if inhibiting GR activity abolished exercise preconditioning. Female rats were either sedentary (Sed) or exercised for 10 consecutive days (Ex). Isolated hearts were exposed to 25 min ischemia and 2 h of reperfusion (I/R). Ex reduced infarct size (41±3% and 52±2% of risk zone for Ex and Sed respectively; P<0.05), protected against arrhythmia (arrhythmia scores were 3.0±0 and 3.7±0.3 for Ex and Sed respectively; P<0.05) and preserved coronary flow during reperfusion (10±1 and 7±1 mL/min/g for Ex and Sed respectively; P<0.05). Perfusion with the GR inhibitor BCNU (150 μM) during I/R abolished the exercise‐induced protection against infarction (57±1% in Ex+BCNU; P<0.05), arrhythmia (arrhythmia score 4.8±0.2 in Ex+BCNU; P<0.05) and coronary reactivity (5±1 mL/min/g in Ex+BCNU; P<0.05). Ex did not protect when animals received anti‐oxidant treatment, suggesting that increased GR activity is triggered by ROS. These data support our hypothesis that maintaining the reduced glutathione pool protects the Ex heart against I/R injury.
Presence of VDR and CYP27B1 in mouse C2C12 cells and skeletal muscle reveal the action of 25(OH)D3 on suppression of myoblast proliferation
Authors: Srikuea Ratchakrit , Zhang Xiping , McCarthy John J. , Esser Karyn A. , Park-Sarge Ok-Kyong
Published: June 2021

1α,25(OH)2D3, an active form of vitamin D3 was demonstrated to regulate myoblast proliferation via its nuclear vitamin D receptor (VDR). Local production of 1α,25(OH)2D3 was reported in the cells that express VDR and CYP27B1, the latter is used to convert 25(OH)D3 to 1α,25(OH)2D3. However, whether skeletal muscle expresses CYP27B1 and 25(OH)D3 can be used as a precursor for the local synthesis of 1α,25(OH)2D3 in skeletal muscle and regulate muscle cell functions are currently unknown. In this study, we investigated 1) VDR and CYP27B1 mRNA transcripts in C2C12 cells (mouse muscle cell line) and skeletal muscle tissue 2) the expression of VDR and CYP27B1 protein in C2C12 cells 3) the effect of 25(OH)D3 on C2C12 cell proliferation. Here, we demonstrated that C2C12 cells and skeletal muscle expressed VDR and CYP27B1 mRNA transcripts. Both genes were significantly up‐regulated at the mRNA level after skeletal muscle was overloaded. Nuclear VDR and mitochondrial CYP27B1 were expressed in C2C12 myoblasts but not myotubes. 25(OH)D3 treatment increased nuclear VDR expression and inhibited C2C12 myoblast proliferation. This effect was abolished by CYP27B1 knockdown, suggesting CYP27B1 is required for 25(OH)D3 action in myoblasts. Taken together, the presence of VDR and CYP27B1 in C2C12 cells and skeletal muscle revealed the possible contribution of 25(OH)D3 on the regulation of skeletal muscle function, in part on controlling of muscle cell proliferation.

This work was supported by NIH R01AR45617 to KAE and PGY funds to OKPS
Regulation of skeletal muscle oxidative phenotype by classical NF‐κB signalling
Authors: Remels Alexander , Gosker Harry , Schols Annemie , Langen Ramon
Published: June 2021

Molecular mechanisms underlying loss of muscle oxidative phenotype (OXPHEN: intrinsic features determining fatigue resistance and capacity for substrate oxidation) in diseases associated with chronic systemic inflammation are unclear. In C2C12 myotubes, TNF‐α reduced protein and mRNA levels of myosin heavy chain (MyHC) slow and oxidative phosphorylation (OXPHOS) complex (I‐III) subunits. These effects of TNF‐α were blocked when classical NF‐κB signalling was genetically abrogated (C2C12‐IκBα‐SR). Accordingly, TNF‐α‐induced reductions in mRNA levels of the key OXPHEN regulators PGC‐ 1α and mitochondrial transcription factor A (Tfam) were attenuated in C2C12‐IκBα‐SR myotubes. Also, TNF‐α reduced mitochondrial fission (MTP18) and fusion (mfn‐2) mRNA expression in a classical NF‐κB‐dependent manner. In analogy, IL‐ 1β or over‐expression of P65/RelA or a constitutive active form of IKK‐β (key constituents of classical NF‐κB signalling) significantly decreased Tfam promoter trans‐activation as well as nuclear respiratory factor 1 (NRF‐1) transcriptional activity. This data shows that classical NF‐κB activation is both sufficient and required for TNF‐α‐induced impairment of skeletal muscle OXPHEN, which is relevant for several pathological conditions characterized by systemic inflammation and muscle dysfunction.

This study was supported by the Netherlands Asthma Foundation (3.2.09.068).
Cachexia and loss of skeletal muscle mass in a murine model of pulmonary hypertension
Authors: Ahn Bumsoo , Frye Gregory S. , Ferreira Leonardo F.
Published: June 2021

Pulmonary hypertension (PH) causes skeletal muscle atrophy and weakness. Monocrotaline (MCT) has been used to induce PH. In rats, MCT‐induced PH rapidly evolves to heart failure and the animals also become anorexic. Mice are more resistant to MCT than rats and have a delayed transition to heart failure from PH. However, the effects of MCT‐induced PH on mouse skeletal muscles are unclear. We examined the effects of MCT on body weight, food intake and soleus muscle mass in mice. Eight‐week old C57BL/6J mice received weekly subcutaneous injections of MCT (n = 5; 600 mg/kg) or saline (n = 4; control) for 8 weeks. Body weight was measured weekly and food intake was measured 2–3 times a week. After 8 weeks, MCT body weight was 7.5 % lower than control (P < 0.05). Food intake was similar in both groups (P > 0.05), which shows that mice did not become anorexic. Soleus weight in MCT was 20.5 % lower than control (P < 0.05). Our data show that the murine model of pulmonary hypertension mimics the cachexia and loss of skeletal muscle mass reported in patients. These effects were independent of changes in food intake. Thus, mice can be used to examine the mechanisms of skeletal muscle atrophy and weakness induced by PH without the confounding effect of anorexia.
Doxorubicin impairs skeletal muscle mitochondrial respiratory capacity in skeletal muscle
Authors: Gilliam Laura A. A. , Fisher-Wellman Kelsey H. , Lin Chien-Te , Maples Jill M. , Neufer P. Darrell
Published: June 2021

Debilitating muscle weakness and fatigue are common side effects of chemotherapy in cancer patients, limiting recovery and increasing morbidity. Our previous work shows that doxorubicin, a common chemotherapy drug, elevates reactive oxygen species (ROS) and decreases contractile force in skeletal muscle. As mitochondria represent a primary source of oxidant generation in muscle, we hypothesized that doxorubicin may compromise mitochondrial respiratory control and increase ROS production. 72 hrs following a single doxorubicin injection (20 mg/kg), maximal ADP‐stimulated O2 consumption was decreased during respiration supported by palmitoylcarnitine (PC) (−46 ± 26 %, p<0.05) and pyruvate/malate (−43 ± 21 %, p<0.05, n=6/group) in permeabilized fiber bundles (PmFB) from red gastrocnemius muscle. PmFB from doxorubicin treated animals also display increased rates of mitochondrial H2O2 emission with PC (23 ± 2 %) and 3‐phosphoglycerate (49 ± 2 %, p=0.1). Our data indicate that doxorubicin impairs both maximal State 3, and fatty‐acid‐supported respiration. In addition, doxorubicin promotes mitochondrial ROS generation, a potential underlying cause of doxorubicin‐induced muscle dysfunction.

Supported by DK073488
Genioglossus motor unit properties in a rat model of sleep apnoea
Authors: O'Halloran Ken D. , Bradford Aidan , Jones James FX , Edge Deirdre
Published: June 2021

Intermittent hypoxia elicits plasticity in sensory and motor pathways involved in the control of breathing with potentially adaptive and maladaptive consequences for respiratory homeostasis. Chronic intermittent hypoxia (CIH) is a major feature of sleep‐disordered breathing. We sought to test the hypothesis that CIH alters pharyngeal dilator muscle motor unit properties.

Adult male Wistar rats were exposed to 20 cycles of normoxia and hypoxia (5% O2 at nadir; SaO2 ~ 80%) per hour, 8 hours a day for 7 days (CIH, N=5) or sham treatment (N=5). Following gas treatments, rats were anaesthetized (urethane 1.5g/kg i.p.) and genioglossus (GG) muscle single motor unit potentials were recorded during quiet basal breathing.

During basal breathing, the amplitude and duration of GG motor units was significantly different in sham vs. CIH‐treated rats (313±32μV vs. 430±46μV (amplitude) and 5.0±0.4ms vs. 3.4±0.2ms (duration); mean±SEM, Student's t test, p=0.0415 and 0.0013 respectively). The most common firing frequency of individual units determined from auto correlograms was significantly decreased (53±6Hz vs. 37±3Hz, sham vs. CIH, p=0.0318).

Our results indicate that CIH causes remodelling in the central respiratory motor network with potential consequences for the physiological control of upper airway patency.