bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelial nitric oxide synthase in the amphibian, Xenopus tropicalis
Authors: Donald John Alexander , Trajanovska Sofie
Published: June 2021

Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS, inducible NOS, and endothelial NOS (eNOS or NOS III). The presence of eNOS and endothelial NO signalling in fish and amphibian blood vessels has been controversial. This study utilised the genome of Xenopus (Silurana tropicalis) to clone eNOS and determine its tissue expression, and then establish if eNOS is involved in vascular regulation. The open reading frame of Xenopus eNOS (XteNOS) cDNA encoded an 1177 amino acid protein that showed closest structural identity to mammalian eNOS. XteNOS mRNA expression was highest in lung and skeletal muscle, with lower expression in the liver, gut, kidney, heart and brain. No discernable XteNOS mRNA expression was observed in the lateral and dorsal aortae. Western analysis of lung protein using an affinity‐purified anti‐XteNOS produced a single band at approximately 140 kDa, which is similar to mammalian eNOS. Immunohistochemistry showed XteNOS immunoreactivity in the collecting duct of the kidney and the lung parenchyma, but not in the endothelium of blood vessels of the kidney or mesentery. Myography using endothelium‐denuded lateral aorta of X. tropicalis found an acetylcholine‐induced NO‐mediated vasodilation that is most likely attributed to perivascular nitrergic nerves. Thus, Xenopus has an eNOS gene that is not expressed in the vascular endothelium.
Effects of Fecundity on Grasshopper Locomotory Performance
Authors: Barbara Matthew Thomas Santa , Kirkton Scott D.
Published: June 2021

Across taxa, evolutionary tradeoffs between fecundity and locomotory performance exist. Previous studies have suggested that the maximum jumping ability of gravid grasshoppers is reduced by their egg masses. In gravid American locusts (Schistocera americana), the egg mass is approximately twenty percent of the animals' body mass. To address the cost of added mass during jumping, weights equal to 20% of body mass were attached to female grasshoppers (n=8). The mean maximum jumping distances for the unweighted control group (100±7 cm) and 20% weighted group (105±10 cm) were not significantly different. To determine if males could also compensate for increased weight during jumping, weights equal to 20%, 40%, or 80% of body mass were attached to male grasshoppers (n=8). Maximum jump distance showed an inverse relationship with additional weight in male grasshoppers: unweighted control males jumped 123±9cm, 20% weight males jumped 97.0±4.9 cm, 40% weight males jumped 85.5±10.4 cm, and 80% weight males jumped 83.6±7.3 cm. Gravid females may be capable of compensating for their relatively larger mass by increasing jump energy production. For example, high‐speed video analysis of maximum jump performance showed that gravid females produced greater take‐off velocities (2.88±0.09 m/s) and accelerations (237±18 m/s2) during jumping than non‐gravid females (2.46±0.21 m/s) and (214±22 m/s2), respectively. Future experiments will involve attaching greater amounts of weights to females and analyzing high‐speed video to determine possible mechanisms gravid grasshoppers utilize to mitigate the effects of increased mass on jumping performance.
Differentially expressed microRNAs in dystrophin‐deficient muscle.
Authors: Gardan‐Salmon Delphine , Fritz Eric R. , Nettleton Dan , Reecy James M. , Selsby Joshua T.
Published: June 2021

Mutations in the dystrophin gene, resulting in the production of non‐functional protein products, are responsible for causing Duchenne muscular dystrophy. What remains less clear is the underlying mechanisms whereby dystrophin deficiency results in well recognized pathologies. MicroRNAs are a subclass of RNA that generally cause degradation of specific transcripts, resulting in reduced protein expression. To better understand downstream consequences of dystrophin deficiency, we analyzed microRNA expression in dystrophic skeletal muscle. Our hypothesis was that dystrophin deficiency would cause a change in expressed microRNAs compared to control muscle. Purified total RNA from six week old, male gastrocnemii (n=4 mdx; n=4 C57) were analyzed with miRCURY™ LNA arrays (Exiqon, Denmark). We identified 37 microRNAs that were differentially expressed (p<0.0001). Of these, 17 were down‐regulated and 20 were up‐regulated, including mir‐206, in mdx mice compared to controls. Evaluation of the genes these microRNAs are predicted to regulate revealed an array of processes that are potential candidates for miRNA regulation. Therefore, these results may help to identify novel pathways that contribute to disease pathology. Partially supported by CIAG.
Tyrosine phosphorylation regulates mechanical stretch‐induced activation of protein translation initiation in C2C12 myoblasts
Authors: Nakai Naoya , Kawano Fuminori , Oke Yoshihiko , Nomura Sachiko , Ohira Takashi , Fujita Ryo , Ohira Yoshinobu
Published: June 2021

It has been proposed that mechanically‐induced tension was the critical event for initiating muscle hypertrophy. However, the molecular mechanisms involved in this process are still under investigation. In the present study, effect of mechanical stretch on the protein translation initiation was studied in C2C12 myoblasts. C2C12 cells were plated on fibronectin‐coated silicone elastomer chamber in DMEM containing 10% fetal bovine serum. Cells were grown in CO2 incubator and subjected to 30 min of cyclic stretch (15% uniaxial stretch at a frequency of 1 Hz). Western blot analysis revealed that 30 min of cyclic stretch increased the phosphorylation of p70 S6 kinase (p70S6K), which is a key regulator for translation initiation. Pre‐treatment of cells with broad range tyrosine kinase inhibitor, genistein, completely blocked the stretch‐induced phosphorylation of p70S6K. These results suggest that tyrosine phosphorylation plays a critical role in the activation of protein translation initiation process in response to cyclic stretch. This work is supported by Grant‐in‐Aid for Scientific Research C (20500577 to N.N.) from the Japan Society for the Promotion of Science (JSPS).
Discrepancy between increased mTORC1 signaling and total muscle protein accretion after leucine stimulation
Authors: Haegens Astrid , Schols Annemie M , van Essen Anon LM , van Loon Luc JC , Langen Ramon C
Published: June 2021

An important goal of nutritional intervention in muscle wasting disorders is to stimulate protein synthesis. The branched chain amino acids (BCAA), and leucine in particular have been suggested to promote protein synthesis via activation of the mammalian target of rapamycin (mTOR) pathway. The endpoints determined so far were mostly cell signaling parameters leaving the question open whether the activation of mTORC1 by leucine leads to protein accretion? The aim of this study was to investigate, in C2C12 cells cultured in the presence of physiological AA levels, whether the stimulating effects of leucine on mTORC1 lead to total and muscle specific protein accretion. Myoblasts were differentiated in DMEM diluted with HBSS (supplemented with NaHCO3) to yield culture medium containing glucose (1g/L), FBS (1%) and physiological AA levels (10% of unmodified DMEM) in presence or absence of 5mM leucine.

Leucine stimulated the mTORC1 targets 4E‐BP1 and S6K1, measured by western blot. Creatine kinase activity and total protein accretion were not increased in cells that had differentiated in the presence of leucine for 5 days. Myosin Heavy Chain fast and slow, measured by western blot, were however increased under these conditions. These findings suggest a differential response of muscle proteins in the presence of leucine.
IL‐6‐induced Cachexia and Muscle IGF‐1 Signaling
Authors: White James P , Sato Shuichi , Puppa Melissa J , Baynes John W , Welle Stephen L , Carson James A
Published: June 2021

Inflammatory cytokine IL‐6 is necessary for the development and progression of cancer cachexia in the ApcMin/+ mouse, a model of colorectal cancer and cachexia. The purpose of this study is to determine if circulating IL‐6 over‐expression (OE) regulates muscle protein synthesis and associated IGF‐1 signaling in wild type (Wt) and ApcMin/+ mice. Twelve week, weight stable, male ApcMin/+ and Wt mice were randomly assigned to IL‐6 OE or control groups. Circulating IL‐6 OE was induced by electroporation of an IL‐6 expressing vector into the quadriceps muscle while an empty vector served as the control. At the end of the 2 week OE period, mice were given an IP injection of deuterium‐labeled phenylalanine to measure myofibrillar protein synthesis rates. IL‐6 OE reduced ApcMin/+ mice body weight 14% over the 2 week time period. ApcMin/+ mice over‐expressing IL‐6 had a 16% decrease in gastrocnemius mass when compared to controls. Body and muscle weights in Wt mice were not affected by IL‐6 OE. ApcMin/+ mice had a 90% reduction in muscle IGF‐1 mRNA expression and an 80% reduction in the phosphorylation of eIF 4EBP‐1 protein. Myofibrillar protein synthesis was reduced 53% in the ApcMin/+ mouse over‐expressing IL‐6. There was no difference in protein synthesis in the Wt mice. IL‐6 OE can accelerate cachexia in the ApcMin/+ mouse, in part, through the repression of muscle IGF‐1 signaling and muscle protein synthesis. R01CA121249‐01A2
Elevated IL‐1RA levels blunt exercise‐induced increases in Hsp25 and Hsp72 in mouse muscle
Authors: Huey Kimberly , Meador Benjamin , Freund Gregory
Published: June 2021

Evidence using heat or transgenic up‐regulation of heat shock proteins (Hsp)72 and 25 suggest they may protect against obesity‐induced insulin resistance. We reported that running wheel training significantly increased Hsp72 and Hsp25 in active muscles of lean mice suggesting their contribution to improved glucose regulation with exercise. However, obesity is associated with higher levels of circulating IL‐1 receptor antagonist (IL‐1RA) that may impact Hsp up‐regulation. We tested the hypothesis that high IL‐1RA levels blunt exercise‐induced Hsp up‐regulation in muscle by blocking the actions of IL‐1. We compared Hsp72 and Hsp25 protein levels with Western blot in muscle of C57 mice (n=4/group) following 3 days of wheel running ± Kineret® (KIN), a recombinant IL‐1RA. Exercise increased Hsp25 and Hsp 72 protein from 0.08±0.02 to 0.16±0.03 and 0.10±0.04 to 0.19±0.03 arbitrary units in saline groups, respectively. No changes in either Hsp were seen with exercise and KIN. Average daily wheel running time (4.6±0.5 vs. 4.6±0.4 h) and distance (4.0±0.5 vs. 4.1±0.4 km) was not different between saline and KIN, respectively. These results suggest that high levels of circulating IL‐1RA may attenuate Hsp up‐regulation in active muscles. However, longer‐term training may reduce adiposity which subsequently may decrease circulating IL‐1RA and improve Hsp responses. Funded in part by NIH DK64862 and NS58525 to GGF
Hypoxic exercise training affects Ox‐LDL‐stimulated thrombin generation of monocyte‐derived microparticles
Authors: Chang Ya‐Lun , Wang John‐Shyan
Published: June 2021

Thrombin activity is critical to determining the severity of haemostatic reactions. Oxidative‐low density lipoprotein (ox‐LDL) promotes the shedding of procoagulant‐rich microparticles from monocytes, thereby accelerating the pathogenesis of atherothrombosis. This study explicates the manner in which hypoxic exercise training affects monocyte‐derived microparticles (MDMP) release and MDMP‐mediated thrombin generation (TG) stimulated by ox‐LDL. Fifteen sedentary healthy men cycled on an ergometer at about 60% of maximal oxygen consumption under 15%O2 condition for 30 minutes per day, 5 days per week for 5 weeks. The MDMP characteristics and dynamic TG parameters were measured by two‐color flow cytometry and calibrated, automatic thrombinography, respectively. Our results showed that acute 12%O2 exercise increase levels of ox‐LDL‐induced releases of total, tissue factor‐rich, phosphatidylserine‐exposed MDMP, which were accompanied by elevated thrombin peak height and increased TG rate in MDMP‐rich plasma. However, 15%O2 exercise training for 5 weeks suppressed the enhancement of coagulant‐related MDMP release and MDMP‐mediated dynamic TG rate under ox‐LDL stimulation following acute 12%O2 exercise. Therefore, our results suggest that long‐term moderate‐intensity exercise at 15%O2 environment ameliorates the promotion of ox‐LDL‐induced TG of MDMP caused by acute 12%O2 exercise, which may in turn decrease the risk of athero‐thrombosis at performing extremely hypoxic exertion.
Chronic hypoxia increases insulin‐stimulated glucose uptake in mouse skeletal muscle
Authors: Gamboa Jorge , Garcia‐Cazarin Mary , Andrade Francisco
Published: June 2021

High altitude populations have lower blood glucose levels and decreased incidence of diabetes. Faster glucose uptake and increased insulin sensitivity are likely explanations for these findings: skeletal muscle is the largest glucose sink in the body and its adaptation to the hypoxia of altitude may influence glucose uptake and insulin sensitivity. This study tested the hypothesis that chronic normobaric hypoxia increases glucose uptake in mouse skeletal muscle. Adult male C57BL/6J mice were kept in normoxia (FIO2=21%, Control) or normobaric hypoxia (FIO2=10%, Hypoxia) for 4 weeks. Blood glucose and insulin concentrations were lower in the Hypoxia group (glucose: Control, 14.3 ± 0.65 vs. Hypoxia, 9.9 ± 0.83 mM, p<0.001; and insulin: Control, 1.2 ± 0.2 vs. Hypoxia, 0.7 ± 0.1 ng/ml, p<0.05). There was no difference in basal glucose uptake in soleus muscle (1.59±0.24 vs 1.71±0.15 umol/g/h; Control and Hypoxia respectively). However, insulin‐stimulated glucose uptake was 30% higher after 4 weeks of hypoxia (6.24±0.23 umol/g/h) compared to Control (4.87±0.37 umol/g/h, p<0.02). There was no significant difference in muscle glycogen content. While the content of glucose transporters 4 and 1 (GLUT4 and GLUT1), phosphoinositide 3‐kinase (PI3K), glycogen synthase kinase 3 (GSK3), and protein kinase B/AKT was not affected by chronic hypoxia, AKT phosphorylation following insulin stimulation in soleus muscle was significantly higher (25% greater in Hypoxia vs. Control, p<0.05). GSK3 phosphorylation was not different. These results demonstrate that increased insulin‐stimulated glucose uptake in skeletal muscles is the likely mechanism leading to lower blood glucose and insulin levels in mice adapted to chronic hypoxia. The molecular mechanisms mediating this adaptation may be useful for the treatment of insulin resistant states.
Effect of Acclimation to Intermittent Hypobaric Hypoxia on NO Homeostasis
Authors: Chang Jae Seung , Sim Yun Bok , Lee Jeong Hee , Nam Sang Seok , Sunoo Sub , Kong In Deok
Published: June 2021

Hypoxia is one of the major stimuli to produce nitric oxide (NO). Recent studies have shown that conversion of nitrogen dioxide (NO2) to NO is independent of nitric oxide synthase (NOS) activity, and exercise is responsible for NO production by its reduction of metabolic products NOx, nitrite (NO2−)/nitrate (NO3−). This study was to examine the effects of training on NO homeostasis after high altitude exposure.

Aerobic capacity (i.e., VO2max) and NO related molecules, cyclic guanosine monophosphate (cGMP), NOx or NO2, were measured by use of an fluorometric and enzyme immunoassay (EIA) analysis (Cayman Inc., USA) from bloods: Fourteen healthy exercisers comparatively corresponding to mountaineers during high‐intensity aerobic exercise who has climbed up 5,000 meters above the sea level, and volunteers who had 7 days before and after aerobic training on treadmill and bicycle ergometer based on 75% HR max and sleeping in hypobaric hypoxic conditions (3000m~5000m) for 4 days.

VO2/75%HRmax of acclimation at an altitude of 5,000 meters was significantly increased by 21.45% (p = 0.0016). In addition, cGMP concentration in post‐training group (3.35 ± 1.082 pmol/ml) was also increased compared to pre‐training one (1.54 ± 0.751 pmol/ml) (p < 0.001), whereas NOx or NO2 concentration was decreased significantly (i.e., NOx, 16.49 ± 6.357 vs. 11.32 ± 2.805 μM, p = 0.0027; NO2, 12.62 ± 8.321 vs. 7.39 ± 2.273 nM, p = 0.0395) These findings suggest that increase of cGMP via an NO production may ameliorate physical activity at high altitudes, which is associated with the changes of systemic concentration of NOx and NO2.
High Skin Temperature Accentuates Aerobic Performance Degradation when Hypohydrated.
Authors: Kenefick Robert W. , Cheuvront Samuel N. , Ely Brett R. , Palombo Laura J. , Sawka Michael N.
Published: June 2021

High skin temperature (Tsk) and hypohydration (HYPO) degrade aerobic performance, but the impact of Tsk on HYPO mediated performance degradation is unknown.

Hypothesis

graded elevations in Tsk would proportionally reduce performance when HYPO. Thirty‐two males (22 ± 4 yrs; VO2peak, 45 ± 8 ml·kg−1·min−1) were divided into 4 matched cohorts (n=8) and then were tested during compensable heat stress in one ambient temperature (Ta; 10, 20, 30, 40°C) when euhydrated (EU) and HYPO (−4% body mass). Volunteers completed a 30‐min exercise bout (cycle ergometer, 50% VO2peak) and 15 min self‐paced time trial. Within EU and HYPO, core temperatures were similar across Ta, but Tsk was elevated proportionally, ~4°C Tsk per 10°C Ta. Total work (TW) expressed as % change from EU (with 95% CI), was degraded incrementally with increased Ta by −2.9% (−5.8 to 0.07) at 10°C, −5.1% (−10.62 to 0.40) at 20°C, −12.1% (−18.79 to −5.5) at 30°C and −22.9% (−40.5 to −5.4) at 40°C. The % decrement in TW from EU (y) was predicted (r=0.60, P<0.05) from Tsk (x), y=−1.650x+39.86 where each 1° elevation in Tsk yields −1.65% decline in TW. The HYPO mediated degradation in aerobic exercise performance is amplified with increasing skin temperature.

Opinions contained herein should not to be construed as official views of the Army or DoD.
Heat acclimation induces peripheral modifications in cutaneous vascular function in humans
Authors: Lorenzo Santiago , Sawka Michael N. , Minson Christopher T.
Published: June 2021

The aim of this study was to explore heat acclimation effects on cutaneous vascular responses and sweat rate to local acetylcholine (Ach) infusions. Ach (1, 10, and 100mM) was infused in 12 cyclists via microdialysis before and after a period of heat acclimation. Eight subjects served as controls to eliminate training effects. Skin blood flow was monitored via laser‐Doppler flowmetry (LDF) and cutaneous vascular conductance (CVC) was calculated as LDF/MAP. Sweat rate was measured by resistance hygrometry. Maximal forearm blood flow (FBF) was obtained by heating the contralateral forearm in a water spray device and measured by Doppler ultrasound. Heat acclimation increased CVC to 1, 10, and 100 mM Ach (43.5 ± 3.4 vs. 52.6 ± 2.6 %CVCmax; 67.7 ± 3.4 vs. 78.0 ± 3.0 %CVCmax; 81.0 ± 3.8 vs. 88.5 ± 1.1 %CVCmax; all P < 0.05). Maximal FBF remained unchanged after heat acclimation (290.9 ± 12.7 vs. 269.9 ± 23.6 ml min−1). Heat acclimation also increased sweat rate to Ach doses (0.13 ± 0.02 vs. 0.18 ± 0.02 mg cm−2 min−1; 0.21 ± 0.03 vs. 0.31 ± 0.03 mg cm−2 min−1; 0.45 ± 0.05 vs. 0.67 ± 0.06 mg cm−2 min−1; all P < 0.05). No differences were found in the control group in any of the variables. Local adaptations within the skin microcirculation and sweat gland apparatus play major roles in the increased thermoregulatory responses induced by heat acclimation. Sponsored by the Eugene and Clarissa Evonuk Memorial Graduate Fellowship
TRPV‐1 channels contribute to local and reflex cutaneous vasodilation in humans
Authors: Wong Brett J. , Fieger Sarah M. , Ade Carl J.
Published: June 2021

TRPV‐1 channels are putative channels in afferent sensory nerves. The purpose of this project was to determine the contribution of TRPV‐1 channels to cutaneous vasodilation elicited via local heating (LH) and whole body heating (WBH). In 2 separate protocols, subjects were equipped with 2 microdialysis fibers on the forearm: saline (control; CON) or 20 mM capsazepine (CPZ), a selective TRPV‐1 antagonist. Systemic arterial pressure (SAP) was measured and skin blood flow (SkBF) was monitored via laser‐Doppler flowmetry (LDF). In protocol 1, CPZ was infused for at least 1 hour after which local vasodilation was elicited in both sites via LH from 32°C to 40°C (0.5°C/5 sec). In protocol 2, CPZ was infused for at least 1 hour after which subjects underwent WBH via water‐perfused suits sufficient to raise oral temperature 1.0°C above baseline and elicit reflex vasodilation. At the end of each protocol, maximal SkBF was achieved via LH to 43°C and infusion of 28 mM nitroprusside. Cutaneous vascular conductance (CVC) was calculated (LDF/SAP) and normalized to maximal SkBF. In protocol 1, CPZ attenuated the initial peak to LH compared to CON (45±3 vs. 78±4 %CVCmax) as well as the secondary plateau phase (35±2 vs. 82±4 %CVCmax). In protocol 2, CPZ attenuated the SkBF response to WBH compared to CON (51±4 vs. 68±4 %CVCmax). These data indicate TRPV‐1 channels contribute to local vasodilation elicited via LH as well as reflex‐mediated vasodilation elicited via WBH.
Low salt intake during pregnancy and lactation causes changes in energy metabolism in adult female Wistar offspring
Authors: Jang Karen Lucasechi Lopes , Furukawa Luzia Naoko Shinohara , Dolnikoff Miriam Stermann , Heimann Joel Claudio
Published: June 2021

Objective

To study some factors related with the energy metabolism in adult offspring of dams fed low salt diet during pregnancy and lactation.

Methods

Female Wistar rats were fed low (LS 0.15%) or normal (NS 1.3% NaCl) salt diet since 8 weeks of age. At 12 weeks of age, they were mated. Offspring were fed NS from weaning and at 12 weeks of age, they were kept on 4oC or room temperature (RT) during 24 hours. Body weight (BW) and temperature (BT), blood glucose (GLU) plasma norepinephrine (NOR), and leptin (LEP) were measured. Gene expression (RT‐PCR) for uncoupling protein‐1 (UCP1), coenzyme Q1 (Q1) and Q2 (Q2) was evaluated in brown adipose tissue.

Results

(mean±SEM, n=5 to 8/group): No differences were observed in BW between adult offspring groups. UCP1, Q1 and Q2 were lower (p<0.05) in LS (UCP1 ‐ 1.5±0.1, Q1‐ 0.7±0.1, Q2 ‐ 1.0±0.1) than in NS (UCP1 ‐ 2.0±0.1, Q1 ‐ 1.1±0.1, Q2 ‐ 1.3±0.1) at RT but not at 4°C. At 4°C BT decreased (p<0.05) in NS but not in LS. NOR (pg/dL) was higher on 4°C (471±42) than on RT (206±28) in NS but not in LS. GLU did not change after exposure to 4°C in NS and LS. At 4°C, LEP decreased (p<0.05) in NS but not in LS.

Conclusions

Low salt diet during pregnancy and lactation modifies expression of some genes involved in energy metabolism in adult female offspring. Supported by FAPESP.
Thermoregulatory responses of cattle to controlled dehydration during heat challenge
Authors: Scharf Brad Allan , Evans Tim , Spiers Donald E.
Published: June 2021

Few studies have quantified the impact of dehydration on bovine thermal status during heat stress. Angus steers (n=8) in trial 1 were dehydrated and rehydrated over 6 days at thermoneutrality (TN; 19–21°C), with 4 month recovery. Trial 2 consisted of 5 days of cyclic heat stress (HS; 26–36°C) followed by water restriction and rehydration as in trial 1. Measurements included respiration rate (RR) and rectal temperature (Tre) taken 6 times daily, with body weight (BW), feed intake (FI), and sweat rate (SR) determined at selected times. Dehydration‐induced reductions in FI (70–75%), BW (4–9%), and RR (~15bpm) were similar in magnitude regardless of ambient temperature. FI and BW quickly returned to baselines after 1 day rehydration, while RR took 4 days. SR approached 100 g/m2h during HS, declined to 30 g/m2h with dehydration, and did not recover until after 3 days rehydration. Tre exhibited no significant change with dehydration, due to reduction in FI that counterbalanced decreases in RR and SR. Rehydration decreased Tre (~0.5°C), with slow recovery over 4 days. Surprisingly, dehydration of cattle did not result in a change in thermal status during heat stress. USDA Agreement No. 58‐6227‐3‐016
Effects of Skin Temperature on Hypohydration Mediated Hemodynamic Responses to Submaximal Exercise.
Authors: Kenefick Robert W. , Ely Brett R. , Cheuvront Samuel N. , Palombo Laura J. , Sawka Michael N.
Published: June 2021

This study delineated the impact of skin temperature elevation on hypohydration (−4% body mass) mediated hemodynamic responses to submax exercise. Thirty‐two males (22 ± 4 yrs, VO2peak, 45 ± 8 ml·kg−1·min−1) were divided into 4 matched cohorts (n=8). Each cohort then completed euhydrated (EU) and hypohydrated (HYPO) trials in one ambient condition (Ta; 10, 20, 30 or 40°C) consisting of 30‐min of cycle ergometry (50% VO2peak). Cardiac output (Qc), blood pressure (MAP), core (Tre) and skin (Tsk) temperature measures were taken at rest and 20 min of exercise. Tre values were similar within EU and HYPO across Ta, however Tsk increased proportionally ~4°C Tsk per 10°C Ta. EU plasma volume changes were 0, −5, −7 and −11% and −4, −6, −10 and −15% when HYPO at 10, 20, 30 or 40oC, respectfully. The core‐to‐skin temperature gradient was not different between EU and HYPO at 10, 20, or 30°C and was smaller as Ta increased. However, at 40°C the core‐to‐skin gradient was wider (p<0.05) in HYPO. The % change in Qc (rest to exercise) was lower with HYPO (p<0.05) in 30 and 40°C. Exercise MAP decreased (p<0.05) at 40o when HYPO. Combined effects of hypovolemia and lowering of core‐to‐skin gradient resulted in an inability to maintain Qc and blood pressure during sub‐max exercise and are likely associated with performance degradation.

Opinions contained herein should not to be construed as official views of the Army or DoD.
Central efferent pathways mediating shivering thermogenesis
Authors: Nakamura Kazuhiro , Morrison Shaun F.
Published: June 2021

Shivering is a remarkable thermogenic response when body temperature needs to be defended from environmental cold challenges as well as during the development of fever. The preoptic area (POA) receives both environmental thermal information from the skin and pyrogenic signals from the immune system activated by infection and the POA sends descending signals that induce shivering in skeletal muscles. However, the efferent pathway from the POA that is responsible for shivering is unknown. In this study, we examined the involvement of several brain regions in the cooling‐evoked or febrile shivering response in isoflurane‐anesthetized rats. Cooling the trunk skin or nanoinjecting prostaglandin E2 (PGE2) into the POA evoked an increase in the EMG level recorded from the nuchal muscle. Both cooling‐ and PGE2‐evoked EMG increases were blocked by bilateral injections of muscimol, a GABAA receptor agonist, into the dorsomedial hypothalamic nucleus (DMH). In addition, injection of muscimol or 8‐OH‐DPAT, a 5‐HT1A receptor agonist, into the rostral raphe pallidus nucleus (rRPa) also blocked the EMG increase. These results suggest that the DMH and rRPa are essential synaptic integration sites in the efferent neuronal pathway from the POA that mediates the shivering responses to skin cooling and to pyrogens and that this pathway can be modulated by serotonergic inputs to the medullary raphe. Supported by NIH and MEXT Japan.
Effects of ganoderma lucidum spores powder on expression of NCAM‐1 and NCAM‐L1 in hippocampus of rats with epilepsy
Authors: Wang Shu‐Qiu , Kang Yu‐Ming , Qi Zong‐Chun , Ma Xiao‐Ru , Liang Yan‐Feng , Li Dian‐Gang , Zhang Bo
Published: June 2021

To investigate the effects of ganoderma lucidum spores powder on expression of neural cell adhesion molecule (NCAM‐1) and NCAM‐L1 of nerve cells in hippocampus of rats with epilepsy emblazed by pentylenetetrazol (PTZ) and study the mechanism of epilepsy and improvement mechanism by ganoderma lucidum spores powder. Thirty male Wister rats were divided into three groups randomly: ganoderma lucidum spores powder, epilepsy model and control groups. The expression of NCAM‐1 and NCAM‐L1 can be detected in each group. The results showed that, compared with control group the NCAM‐1 positive cells and the expression of NCAM‐1 and NCAM‐L1 were increased obviously in epilepsy model group, while the expression of NCAM‐1 and NCAM‐L1 ganoderma spores powder group decreased obviously compared with epilepsy model group.

Conclusion

Both of NCAM‐1 and NCAM‐L1 play a role in the starting and developing of epilepsy caused by PTZ; Ganoderma lucidum spores powder could inhibit the growth of axons of neurons and transmembrane signal transduction in the nervous system, and reduce cell‐cell adhesion by down‐regulating expression of NCAM‐1 and NCAM‐L1, while synaptic plasticity of the nervous system was reduced and have anti‐epileptic effect. Ganoderma lucidum spores powder is an assistant medicine to cure epilepsy, and it may become a clinical drug to protect brain against epilepsy.
Mechanisms of neuropeptide AF‐induced anorexia in chickens
Authors: Newmyer Brandon , Siegel Paul , Cline Mark
Published: June 2021

Recently we demonstrated an anorectic effect of neuropeptide AF (NPAF). The present study was designed to further elucidate mechanisms mediating NPAF‐induced anorexia and to test in hypo‐ and hyperphagia animals. In Exp 1–3, blockade of the mu and kappa but not delta subtypes of opioid receptors and co‐injection of NPAF did not have an additive anorexic effect, suggesting NPAF is associated with mu and kappa opioid receptors. In Exp 4–5, NPAF injection into both divisions of the paraventricular nucleus (PVN) did not affect food or water intake. In Exp 6, NPAF was centrally injected and food intake was quantified in lines of chicks that have undergone long‐term divergent selection for low (LWS) or high (HWS) body weight and are models of hypo‐ and hyperphagic respectively. LWS chicks responded 120 min sooner and their threshold was 4 orders lower than noted in HWS chicks. In Exp 7, several behaviors were quantified, and feeding and exploratory pecks, time spent standing and sitting were all reduced, but not differentially between lines. However, total distance moved was reduced in LWS‐ but not HWS‐NPAF‐treated chicks. Also, LWS‐ slept more while HWS‐NPAF‐treated chicks slept less. These data support that NPAF‐induced anorexia is mediated through opioid receptors, that the PVN is not primary activated in NPAF‐induced anorexia, and hypo‐ and hyperphagic individuals respond differently to NPAF.
Loss of Interleukin‐6 Blunts Glucagon Secretion During Hypoglycemia
Authors: Chukwuma Valentine U. , Morris R. Tyler , Mulligan Kimberly X. , McGuinness Owen P.
Published: June 2021

We have shown that the glucagon response to lipopolysaccharide was blunted in interleukin 6 knock‐out (IL6 KO) mice. In this study, we investigated the effects of a non‐inflammatory stimulus on the glucagon response in IL6 KO mice. Chronically catheterized conscious male mice (C57BL6/j; 8–10 weeks old) were subjected to a 5‐hr fast, after which hypoglycemic clamps were performed. Insulin (10mU/kg/min) and glucose were infused to maintain arterial blood glucose between 50–70 mg/dl. Initial glucagon response was blunted in the IL6 KO mice compared to the wild‐type (WT) (213±74 vs. 867±294, pg/ml). Epinephrine was significantly higher in the IL6 KO mice compared to WT (1049±298 vs. 308±123, pg/ml). There was no significant difference in the insulin concentration, corticosteroids, and norepinephrine response. Perifused islets were also stimulated in vitro using hypoglycemia, epinephrine and arginine. There was no significant difference in the glucagon response between the islets from WT and IL6 KO mice. In summary, IL6 KO mice have an impaired glucagon response when subjected to a non‐ inflammatory stimulus in vivo, but the blunted response is not observed in islets in vitro. Since the epinephrine response to hypoglycemia was not blunted, IL6 may play a role in augmenting pancreatic autonomic tone during hypoglycemia. Funding: DK043748, DK078188 and DK059637
Inhibition of soluble epoxide hydrolase reduces food intake and increases metabolic rate in mice fed high fat‐high fructose diet
Authors: do Carmo Jussara M , Silva Alexandre A , Morgan Jarrett , Hall John E
Published: June 2021

This study evaluated the responses to soluble epoxide hydrolase (s‐EH) inhibition on food intake, body weigh and metabolic parameters in mice fed a high fat‐high fructose diet (HFD) for 10 weeks. After 5 weeks of HFD, mice were divided into two groups: 1) s‐EH inhibitor (AR1153, 200 mg/kg/day), and 2) vehicle (0.3 ml) were administered orally. Food intake, body weight, oxygen consumption (VO2), carbon dioxide production (CO2), respiratory quotient (RQ), and motor activity were measured weekly. HFD increased body weight (37¡Ó1 vs 26¡Ó1 g), glucose (316¡Ó8 vs 188¡Ó27 mg/dl), insulin (62.1¡Ó8.1 vs 15.5¡Ó5.0 ƒÝU/ml), and leptin levels (39.4¡Ó3.6 vs 7.5¡Ó0.1 ng/ml) and reduced VO2, CO2 and motor activity. s‐EH inhibition for 5 weeks decreased caloric intake by ~32% and increased VO2 by ~17% compared to week 5 of HFD leading to significant weight loss. No significant changes in VO2 or body weight were observed in vehicle‐treated group. AR1153 or vehicle caused similar reductions on glucose and insulin levels, whereas leptin levels and visceral fat were reduced only in AR1153‐treated group. Uncoupling protein 1 (UCP1) content in brown adipose tissue was also elevated by ~50% during s‐EH inhibition compared to vehicle treatment. These results suggest that s‐EH inhibition promotes weight loss by reducing appetite and increasing metabolic rate, and that increased UCP1 content may have contributed to the increase in energy expenditure
Effects of chronic fructose feeding on leptin sensitivity and obesity in mice
Authors: Tillman Erik James , Swoap Steven John
Published: June 2021

Chronic 60% fructose feeding decreases the sensitivity to leptin in rats as assessed by decreases in food intake and body weight after administration of exogenous leptin. We hypothesized that a 60% fructose diet would also lead to leptin resistance in mice, as assessed by 1) a lack of response to exogenous leptin, 2) elevated circulating leptin, and 3) a lack of entry into torpor upon fasting. Male Hsd:NSA(CF‐1) mice were fed either a 60% fructose diet or an isocaloric fructose‐free diet and food intake and body weight were monitored. After 4 weeks of the diet, mice were injected intraperitoneally with leptin (5 mg/kg) and consequent changes in food intake and body weight were monitored. After 8 more weeks on the diet, plasma leptin levels were measured, and then mice were fasted at 20°C for 24 hours. The two feeding groups had similar caloric intake and gained the same amount of weight over the 12 week feeding period. Additionally, high fructose feeding did not attenuate the decrease in either food intake or body weight in response to exogenous leptin. Further, mice on the high fructose diet showed no difference in circulating leptin from mice on the fructose‐free diet (22.9±6.3 and 24.4±3.2 ng/ml, respectively). Finally, neither feeding group entered torpor when fasted at 20°C. These observations indicate the failure of a 60% fructose diet to induce leptin resistance in Hsd:NSA(CF‐1) mice over 12 weeks.
Long‐chain n‐3 polyunsaturated fatty acid supplementation improves the muscle protein anabolic response to hyperaminoacidemia‐hyperinsulinemia in healthy older adults
Authors: Smith Gordon Ian , Reeds Dominic N. , Mohammed B. Selma , Mittendorfer Bettina
Published: June 2021

Administration of long chain n‐3 polyunsaturated fatty acids (LCn‐3PUFA) has been reported to increase whole‐body protein synthesis in burned rats and growing steers. The effects of LCn‐3PUFA on human protein metabolism are unknown. To fill this gap, we measured the skeletal muscle protein fractional synthetic rate (FSR) during basal, postabsorptive conditions and during intravenous infusion of insulin and amino acids in 11 healthy, 65–80 y old adults (BMI: 26 ± 1 kg/m2; means ± SEM) before and after 8 weeks of supplementation with either 4 g·d−1 Lovaza™ (GlaxoSmithKline) containing 3.36 g of the ethylesters of eicosapentanoic acid [20:5n‐3] and docosahexaenoic acid [22:6n‐3]; n = 6) or corn oil (n = 5). Corn oil supplementation had no effect on either the basal muscle protein FSR (P = 0.62) or the anabolic response to insulin and amino acid infusion (P = 0.58). LCn‐3PUFA supplementation also had no effect on the basal rate of muscle protein synthesis (0.055 ± 0.005 vs. 0.054 ± 0.011 %·h−1, before and after supplementation, respectively; P= 0.95) but augmented the hyperaminoacidemia hyperinsulinemia‐induced increase in the rate of muscle protein synthesis (from 0.010 ± 0.006 %/h to 0.030 ± 0.004 %·h−1; P= 0.04). We conclude that LCn‐3PUFA have anabolic properties in older adults and may be useful for the prevention and treatment of sarcopenia.

Supported by grants from the NIH, ASN, AFAR, GSK, and the Longer Life Foundation.
Angiotensin II Increases JNK and IRS‐1ser307 Activity via NAD(P)H and Inhibits Insulin Signaling in Rats with Insulin Resistance Induced by Low Salt Diet
Authors: Coelho Michella Soares , Negri Heloisa B , Ferreira Daniele N , Oliveira Ivone B , Hirata Aparecida E , Casarini Dulce E , Prada Patricia O , Dolnikoff Miriam S , Saad Mario JA , Heimann Joel C
Published: June 2021

Low salt diet (LS) increases angiotensin II (AII), which may enhance JNK and IRS1ser307 phosphorylation (PP) decreasing insulin signaling (INS‐S). AII stimulate ROS production through activation of NAD(P)H oxidase (NAD). However, whether AII, oxidative stress (OX) and inflammatory signaling (IS) are involved in INS resistance induced by LS is not established.

Objective

Evaluate if losartan (LOS) improves INS resistance by decreasing OX, JNK and IRS1ser307 PP.

Methods

Rats were fed a LS (0.15% NaCl) or normal salt diet (NS: 1.3%) since weaning. Groups: NS, NS+LOS, LS and LS+LOS. NAD activity (subunits p47 and gp91PHOX), tissue AII, blood glucose (G), plasma INS, HOMA, INS‐S and IS, AII type 1 (AT1) receptor gene and protein expression were evaluated.

Results

(P<0.05, n=8): INS level, HOMA index, muscle AII and NAD subunits PP were greater in LS (p47: 10,066±975, gp91: 740±106) than in NS (p47: 5,783±801, gp91: 416±70 AU). G and NAD activity were lower in LS+LOS than in LS. INS receptor and Akt PP were greater and JNK (58±3) and IRS1ser307 PP (47±3 AU) were lower in the muscle of LS+LOS than in LS. IkB‐α PP was lower in LS (50±3) than in NS (100±3 AU) suggesting an activation of IS in LS. AT1 did not differ among groups.

Conclusions

INS resistance induced by LS is associated with alterations in regulation of INS‐S, activated IS (JNK and IkB‐α), and higher tissue AII and NAD activity. In LS, LOS improves INS‐S by decreasing OX, JNK and IRS1ser307 PP, indicating that LOS may has anti‐inflammatory properties. AII may be a pathway that connects IS, OX and INS resistance in LS.

Funded by FAPESP
Cholera Toxin Enhances ENaC‐Mediated Sodium Absorption Across Cultured Human Mammary Epithelial Cells
Authors: Wang Qian , Wang Lin‐Hua , Schultz Bruce D.
Published: June 2021

Mechanisms that control ion transport across human mammary gland and contribute to the unique low sodium concentration in human milk have not been fully described. When grown on permeable supports, MCF10A cells, an epithelial cell line derived from human mammary gland, exhibited amiloride‐sensitive ion transport, suggesting activity of the epithelial Na+ channel, ENaC. When cultured in the presence of cholera toxin, MCF10A cells exhibited significantly greater amiloride sensitive short circuit current (Isc) at all time points tested (2 h to 7 d). Maximal amiloride‐sensitive Isc was observed with 1 d exposure to cholera toxin (4.76 ± 0.73 μA cm−2) when compared to vehicle‐treated monolayers (1.19 ± 0.27 μA cm−2). Effects of cholera toxin on Isc were neither mimicked by forskolin nor inhibited by H‐89 (10 μM), a protein kinase A (PKA) inhibitor. RT‐PCR analyses showed that cholera toxin increased the copy number for mRNA coding for each of the three ENaC subunits by 2 to 4 fold. Taken together, these results suggest that human mammary epithelial cells express ENaC, a mechanism that can remove Na+ from milk. Furthermore, the results suggest a novel mechanism to regulate ENaC expression that involves cholera toxin, but that is independent of cAMP and PKA. [NIH (P20‐RR017686) and KS Ag Exp Stn support]
FTY720 S‐ene‐phosphonate is a novel pan‐antagonist of the S1P receptors that inhibits lymphocyte egress
Authors: Tigyi Gabor , Valentine William J , Kiss Gyongyi N , Liu Jianxiong , Lu Xuequan , Sun Chaode , Bittman Robert
Published: June 2021

FTY720 phosphate, an analogue of sphingosine‐1‐phosphate (S1P) is in a phase‐III clinical study for the treatment of multiple sclerosis. FTY720P causes S1P1 receptor internalization rendering the lymphocytes unresponsive to S1P. Here we describe the first enantioselective synthesis of chiral isosteric phosphonate analogues of FTY720 and the pharmacological characterization of two enantiomers, FTY720 S‐ene phosphonate and FTY720 R‐ene phosphonate. Pharmacological analysis of the enantiomers revealed that R‐ene was a full agonist of S1P1 (EC50 38±5 nM). In contrast, S‐ene was a full antagonist of S1P1,3,4 (Ki 208 nM, 15 nM, and 1190 nM, respectively) and a partial antagonist of S1P2 and S1P4. Both enantiomers dose‐dependently inhibited lysophospholipase D (IC50 182 nM for S‐ene and 169 nM for R‐ene). When injected into mice, S‐ene caused transient peripheral lymphopenia, which is consistent with its antagonist activity at S1P1. FTY720 S‐ene phosphonate also activated ERK1/2 in a pertussis toxin‐sensitive mechanism, suggesting that it also activated a yet unidentified GPCR. FTY720 S‐ene phosphonate exerted an antiapoptotic effect in IEC‐6 intestinal epithelial cells challenged with camptothecin. S‐ene is the first pan‐antagonist of S1P receptors and offers utility in probing S1P responses in vitro and in vivo. Supported by NIH Grants HL083187, AI80405 and CA92160.
Analysis of antibody responses to schistosome glycan antigens during the course of Schistosoma mansoni infection in mice
Authors: Dotson Dominique R. , Jenkins LaToria , Perez Ivy , Dorabawila Nelum , Mandalasi Msano , Nyame A. Kwame
Published: June 2021

Schistosomes are parasitic helminths of the genus Schistosoma that cause the vascular disease Schistosomiasis. Previous studies show that the major antibody responses to schistosome infections are directed against glycans, making these molecules potential targets for the development of vaccines. We are developing monoclonal antibodies to these antigens from spleens of infected mice. Twelve mice were infected with 25–30 cercariae and the antibody responses were monitored over the period of 14 weeks. IgM responses start at week 4 post infection, peak at week 8, and remained high during the course of the study. IgG responses start at week 6, peak at week 11, and remained high during the course of the study. Interestingly, the IgG responses to antigens were sensitive to periodate oxidation only up to week 10 post infection and became progressively resistant to periodate oxidation thereafter. This was confirmed by analyzing infected mice sera against three defined Schistosome glycan antigens. Taken together, IgM responses to glycan antigens persist throughout the course of infection while IgG responses switch from anti‐glycan responses to non‐glycan responses in the later stages of infection. Thus, there is a narrow window of time for harvesting spleens from infected mice for generation of IgG monoclonal antibodies to glycan antigens. Supported by grants T34GM008411 and 5 S06 GM078091‐04.
Hepatitis C virus (HCV) associated steatosis and increased gluconeogenic gene expression in Huh8 cells: essential role of NS5A and C/EBPâ
Authors: Choudhury Mahua , Qadri Ishtiaq , Rahman Mizanoor , Knotts Trina a , Janssen Rachel c , Iwahashi Mieko , Puljak Livia , Simon Francis r , Kilic Gordan , Fitz Gregory j , Friedman Jacob E
Published: June 2021

Chronic infection with hepatitis C virus (HCV) is associated with altered lipid metabolism, type II diabetes and steatohepatitis, however the cellular mechanisms are not well understood. PEPCK was dramatically up‐regulated in Huh.8 cells (stably infected HCV replicon) involving the activation of the CRE in the PEPCK promoter and responded robustly to NS5A expression/deletion. HCV induced an increase in C/EBPβ, Foxo1, and PGC1α gene at transcription level and activation of phosphorylated CREB at Ser133. Dominant negative CREB or C/EBPβ RNAi transduction in Huh.8 cells significantly reduced PEPCK, without affecting Foxo1 or PGC1a genes. Cellular lipids were greatly increased in Huh.8 cells, while a deletion in NS5A cause reduction but was without effect on insulin resistance. Huh8 cells demonstrated insulin resistance at the level of the decreased insulin receptor β and 473‐Ser‐Akt; and increased 307‐Ser‐IRS‐1 in response to insulin; accompanied by increased SREBP‐1c and SCD‐1. C/EBPβ RNAi reduced the expression of SREBP‐1c, PPARγ, DGAT1, LXRα but was unable to reduce the accumulation of triglycerides. We conclude that NS5A and C/EBPβ are partially responsible for the NASH like features in Huh8 cells. Collectively, these data suggests cellular mechanisms other than NS5A in the mechanism(s) for HCV‐induced insulin resistance and alteration in lipid metabolism. Support R01‐DK59767, ALF
Histone Deacetylase inhibitors modulate mitochondrial biogenesis in skeletal muscle
Authors: Galmozzi Andrea , Mitro Nico , Gers Elise , Gilardi Federica , Mai Antonello , Saez Enrique , Crestani Maurizio , De Fabiani Emma
Published: June 2021

The control of metabolic pathways occurs at the transcriptional level also through epigenetic mechanisms. Since it has been demonstrated that Histone Deacetylases (HDACs) are involved in muscle fiber type determination, our aim was to investigate the metabolic effects following the biochemical inhibition of HDACs in skeletal muscle and the underlying mechanisms.

In C2C12 myotubes we observed that HDAC inhibitors (HDACi) upregulate the mRNA of PGC‐1alpha and its target genes. Additionally, transcriptome analysis in C2C12 treated with HDACi revealed an increase in OXPHOS genes and in genes coding for fatty acids catabolism enzymes, most likely linked to PGC‐1alpha‐dependent activation of gene transcription. Consistent with the increase of PGC‐1alpha, FACS analysis revealed increased density and activity of mitochondria in C2C12 myoblasts. As well, staining of myotubes treated with HDACi showed an increase in mitochondrial density and activity. Using Class specific HDAC inhibitors we observed that Class I HDACs are the most involved in the promotion of these effects.

These results underscore a central role of HDACs, and most probably of Class I HDACs, in modulating metabolic adaptation in cells and suggest HDACi as valuable tools to study molecular mechanisms implicated in energy metabolism and possibly in metabolic diseases.

(Funded by grants: EC FP6 LSHM‐CT‐2006‐037498 SOUTH, PRIN 2008 ZTN724)