bluuebunny/crossref_metadata_2025 huggingface dataset:
Gcn5l1 Promotes Enhanced Cardiac Fatty Acid Oxidation through Acetylation of Mitochondrial Proteins
Authors: Thapa Dharendra , Zhang Manling , Guimarães Danielle A , Stoner Michael W , O'Doherty Robert M , Shiva Sruti , Scott Iain
Published: November 2023

Lysine acetylation is a reversible post‐translational modification, and is particularly important in the regulation of mitochondrial metabolic enzymes. Acetylation uses acetyl‐CoA derived from fuel substrate metabolism as a co‐factor, which links nutritional inputs to metabolic activity. The objective of this study was to investigate the role of mitochondrial acetylation in the heart. Specifically, we assessed how food intake controls cardiac mitochondrial acetylation and affects mitochondrial metabolism. Western blots, immunoprecipitations, qPCR and mitochondrial bioenergetic assessments were performed to achieve these objectives. We found that there was a significant increase in cardiac mitochondrial protein acetylation in mice fed with a long‐term high fat diet, and that this change correlated with an increase in the abundance of the mitochondrial acetyltransferase‐related protein Gcn5l1. Further, we show that the acetylation status of two mitochondrial fatty acid oxidation enzymes was significantly upregulated in high fat diet mice, and that this was linked to increase in their enzymatic activity. Finally, we demonstrate that the acetylation of mitochondrial fatty acid oxidation proteins is decreased following Gcn5l1 knockdown, and that reduced acetylation leads to diminished fatty acid oxidation in cultured H9C2 cells. Based on our findings, we conclude that lysine acetylation promotes fatty acid oxidation in the heart, and that this modification is regulated in part by the activity of Gcn5l1.

Support or Funding Information

NHLBI K22HL116728
Myocardin regulates mitochondrial calcium homeostasis and prevents permeability transition in cardiac myocytes.
Authors: Gordon Joseph , Mughal Wajihah , Field Jared , Hai Yan , Chapman Donald , Keijzer Richard , Rattan Sunil , Dixon Ian , Huang Jianhe , Parmacek Michael
Published: November 2023

Myocardin is a transcriptional co‐activator required for cardiovascular development and cardiomyocyte differentiation. Recent studies have shown that genetic inhibition of myocardin results in embryonic lethality with impaired cardiomyocyte proliferation and increased programmed cell death. Mitochondrial permeability transition, triggered by matrix calcium accumulation, has been implicated in regulated necrotic cell death, while permeability transition pore closure is involved with myocyte differentiation and mitochondrial maturation during development. We show that a genetic loss of myocardin function leads to endocardial necrosis, determined by HMGB1 staining, at embryonic day 9.5, concurrent with elevated expression of the death gene Nix. Mechanistically, we demonstrate that myocardin knockdown in primary ventricular myocytes reduces microRNA‐133a levels to allow Nix accumulation, leading to mitochondrial permeability transition, reduced mitochondrial respiration, and necrosis. Using organelle‐targeted calcium biosensors, we demonstrate that myocardin knockdown leads to sarcoplasmic reticulum (SR) calcium release and mitochondrial calcium accumulation, while mitochondrial permeability transition was prevented by pharmacological inhibition of the inositol triphosphate (IP3)‐activated calcium channel or the mitochondrial calcium uniporter. Gain of function studies confirm that myocardin can desensitize myocytes to permeability transition elicited by protein kinase‐A (PKA) activating agents by opposing SR calcium release. Furthermore, restoring microRNA‐133a function with mimicking oligonucleotides, or knockdown of Nix rescues mitochondrial calcium accumulation induced by myocardin knockdown. Molecular studies using Nix constructs targeted to the SR or mitochondria revealed that only SR‐targeted Nix leads to mitochondrial calcium accumulation, which could be attenuated by SR‐targeted Bcl‐2, but not mitochondrial‐targeted Bcl‐2. Finally, we observed reduced myocardin expression in vivo within the infarction border zone following coronary ligation in rodents, with corresponding reduced microRNA‐133a and elevated Nix expression. These findings identify a novel myocardin‐regulated genetic pathway that maintains cardiomyocyte calcium homeostasis and mitochondrial function during development, and is attenuated during ischemic heart disease.

Support or Funding Information

Supported by NSERC Canada, the Thorlakson Foundation, Research Manitoba, and the Children's Hospital Foundation of Manitoba.
Shear stress promotes mitochondrial homeostasis and attenuates TNFα‐induced mitochondrial dysfunction
Authors: Wu Li‐Hong , Ting Pei‐Ching , Wang Danny Ling
Published: November 2023

Hemodynamic blood flow‐induced laminar shear flow exerts atheroprotective responses in endothelial cells (ECs) have been intensively studied. Our earlier studies indicated that laminar shear flow attenuated cytokine‐induced inflammatory responses in ECs. However, the effects of laminar shear flow on mitochondrial homeostasis and functions in ECs and during the inflammation remain unclear. We examined the dynamic changes of mitochondrial phenotype and its regulatory proteins associated with mitochondrial functions in ECs under physiological shear flow. Cultured human ECs were subjected to a constant shear stress (12 dynes/cm2) with a parallel‐plate flow chamber system. Mitochondrial biogenesis, dynamics and function were analyzed from ECs after shear treatment. Shear‐treated ECs increased mitochondrial biogenesis as indicated by the elevated levels of mitochondrial transcription factor (TFAM), transcriptional coactivator (PGC1‐α), nuclear respiratory factor (NRF1) and mitochondrial proteins (SDHA and COX4). Shear flow triggered a time‐dependent increment of mitochondrial tubular formation (fusion). This phenomenon was coupled with an increased expression of fusion proteins (MFN2, OPA1) but decreased fission protein (FIS1). Laminar shear flow also increased the long form of OPA‐1 that promotes fusion. Consistently, an increase of phosphorylation at S637 but decrease at S616 on Drp1 and the decrease of mitochondria‐associated Drp1 were observed in ECs after flow treatment. Laminar flow increased the expression of mitochondrial antioxidant enzymes MnSOD2, Thioredoxin (TRX2), Peroxiredoxins (PRX3 and PRX5). As a consequence, ECs exposed to laminar shear flow improve mitochondrial function as revealed by a decreased mitochondrial reactive oxygen species levels (mROS), and an increase of mitochondrial membrane potential and ATP production. Furthermore, ECs exposed to TNFα decreased OPA1 and MFN2 expression and increased mitochondrial fission as revealed by the mitochondrial fragmentation. Consequently, TNFα treatment resulted in a decreased mitochondrial function in ECs. However, ECs under shear flow attenuated this TNFα‐induced decrease of OPA1 and mitochondrial dysfunction. ECs treated with TNFα decreased OPA1 and MFN2 expression, whiles under shear condition, these effects were attenuated. Our results suggest that steady laminar flow promotes endothelial function and helps to prevent the inflammation‐induced mitochondrial dysfunction in ECs. Our study further supports that laminar shear flow exerts atheroprotective role to ECs.
Elevated Pax3+ cell number potentially compensates for Pax7+ cell depletion in diaphragm muscle of running mice
Authors: Ghazala Lina S , Confides Amy L , Murach Kevin L , Ho Angel L , Jackson Janna L , McLoon Linda , Dupont‐Versteegden Esther E
Published: November 2023

Satellite cell depletion in hind limb muscles has minimal effect on hypertrophy, sarcopenia, or the ability to adapt to aerobic exercise. However, whether the ability of the constantly active diaphragm muscle to adapt to a stressor such as exercise is decreased with a loss of satellite cells is unknown. We hypothesized that satellite cell depletion would negatively affect diaphragm muscle, particularly with running and in aged mice. We used the Pax7‐DTA mouse model to deplete over 90% of Pax7 positive cells in the diaphragm muscle upon treatment with tamoxifen which induces CreER‐mediated expression of DTA. Female mice were treated with vehicle or tamoxifen at 4 months of age and were either given running wheels at 6 (young) or 22 (aged) months of age or remained sedentary for 8 weeks after which they were euthanized. Satellite cell depletion was associated with 25–28% decreased running activity in young and aged mice. Satellite cell depletion had no effect on fiber type distribution or cross sectional area of fibers in both young and aged mice regardless of running activity. Also, minimal effect was observed on diaphragm function measured using ultrasound analysis. There was no significant difference in myonuclear abundance among treatment and activity groups suggesting that there may be another stem cell type present in diaphragm muscle of satellite cell depleted mice which could compensate for the loss of Pax7+ cells. We used fluorescent in situ hybridization to detect Pax3+ cells and found an increased abundance of these cells in young and aged diaphragm muscle in satellite depleted mice. Additionally, only very few Pitx2 positive cells were detected in young and aged diaphragm muscle and no difference was observed with satellite cell depletion. These results indicate that satellite cell depletion does not negatively affect diaphragm muscle in young and aged mice even under running conditions and we conclude that the increased presence of Pax3+ cells in satellite cell depleted diaphragm muscle potentially compensates for the loss of Pax7+ cells.

Support or Funding Information

NIA AG043721 and APS Undergraduate Summer Research Fellowship.
Satellite cell activation by voluntary wheel running is associated with improved recovery from muscle disuse in mice
Authors: Brooks Matthew J. , Mohamed Junaith S. , Alway Stephen E
Published: November 2023

The reloading of atrophied muscles from weightlessness or disuse results in injury and prolonged recovery. Satellite cells, or muscle stem cells, have a key role in facilitating injury‐induced muscle repair but have not been shown to be required in muscle remodeling following atrophy. However, it has been less clear if low‐impact exercise, such as voluntary wheel running, could provide a sufficient stimulus to enhance muscle recovery from atrophy through satellite cell activation. This study aimed to determine if voluntary wheel running improved recovery from muscle disuse following hindlimb suspension unloading (HSU), and if the recovery was associated with exercise‐induced satellite cell activity. Young adult male and female C57BL/6 background mice (n=6/group) were subjected to either 14 days of normal weight bearing, 14 days of HSU, 14 days of HSU + 14 days of re‐ambulation, 14 days of voluntary wheel running, or 14 days of HSU + 14 days of voluntary wheel running. Mice were given 5‐bromo‐2′‐deoxyuridine (BrdU) in their drinking water during the final 14 days of the experiment to measure satellite cell proliferation in gastrocnemius muscle fibers. HSU significantly reduced in vivo maximal force and decreased the rate of fatigue in the plantarflexor muscles. Voluntary wheel running during reloading after HSU improved resistance to fatigue, which correlated with significant increases in both muscle fiber cross sectional area (CSA) and an increased percentage of oxidative Type IIA muscle fibers in the gastrocnemius muscle as compared to the mice recovering without exercise. BrdU positive nuclei that had proliferated and were located inside the muscle sarcolemma were identified by immunohistochemical labeling and quantified in tissue cross sections. Following HSU, wheel running mice had a significantly greater percentage of BrdU positive nuclei that were inside gastrocnemius muscle fibers. Western blot analysis showed HSU mice with wheel running had a higher MyoD to Pax7 ratio as compared to mice that did not exercise after HSU. Mice in the wheel running group had improved fatigue resistance, an increased oxidative fiber phenotype, and increased fiber CSA as compared to non‐exercised animals after HSU. These results indicate that voluntary wheel running increased satellite cell activity during the recovery phase following HSU and this was associated with improved recovery from muscle disuse. While satellite cells did not exhibit increased activity in passive recovery (non‐wheel running), manipulating their activation through low‐impact exercise appears to enhance morphological recovery and improve muscle function.
Early Rehabilitation and Volumetric Muscle Loss Injury Adapts the Genetic Response of the Remaining Muscle Mass
Authors: Greising Sarah M , Corona Benjamin T , Warren Gordon L , Call Jarrod A
Published: November 2023

Volumetric muscle loss (VML) injuries occur due to orthopaedic trauma or the surgical removal of skeletal muscle and result in debilitating long‐term functional deficits. Current treatment strategies do not promote significant restoration of function; it is possible that limitations to traditional rehabilitation may be related to the attenuated potential of the remaining muscle to adapt following injury. We examined early rehabilitation interventions involving range of motion, muscle stimulation, and pharmacologic targeting to augment mitochondrial capacity (via guanidionpropionic acid) in isolation or combination to understand the capability of the remaining muscle mass to adapt during the acute phase of healing after VML injury. Adult male mice underwent an ~20% multi‐muscle VML injury to the posterior compartment (gastrocnemius, soleus, and plantaris muscle) and were randomized to rehabilitation twice per week beginning 72 hours post‐injury or no treatment. During each 30 minute rehabilitation session, passive muscle torque, a measure of muscle stiffness, was recorded. At 3, 7, and 14 days post‐injury muscles from the injured and contralateral limb were harvested and used to quantify the fibrotic, myogenic, inflammatory, and mitochondrial gene expression. The response to VML injury and associated early rehabilitation appeared to be transient over days post‐injury. VML injury without rehabilitation was associated with a large initial increase in inflammatory gene expression at 3 days, which later decreased at 7 days and then increased again at 14 days post‐ injury. The myogenic gene response was initially increased at 3 days but then declined at both 7 and 14 days. Combined rehabilitation increased the acute myogenic response and suppressed the inflammatory response post‐injury. Under normal circumstances, mitochondrial biogenesis accompanies myogenic muscle recovery from injury and is likely to meet the energy demands of muscle repair. Mitochondrial genes were downregulated across all measured time points post‐ injury when left untreated; suggesting mitochondrial dysfunction following injury, with a combined rehabilitation approach this downregulation appears to be blunted. Collectively, early rehabilitation appears to mitigate the acute maladaptive response to VML injury. Additional work is needed to determine if long‐term functional gains can be accomplished by implementing early rehabilitation.

Support or Funding Information

Studies funded by the Alliance for Regenerative Rehabilitation Research and Training Grant supported by the NICHD, NINDS, and NIBIB.
Exercise Mitigates Bone Loss in Type 2 Diabetes Mellitus Mice
Authors: Zhai Yuankun , Behera Jyotirmaya , Bala Jyoti , George Akash , Tyagi Suresh , Tyagi Neetu
Published: November 2023

AIM

The aims of the present study were to investigate the effectiveness of exercise intervention on the bone loss in leptin receptor‐deficiency (db/db) type 2 diabetes mellitus mice, and examined the influence of homocysteine (Hcy) and hydrogen sulfide (H2S) level to clarify its molecular mechanisms.

METHODS

Forty mice were used in our study, include twenty db/db control mice and twenty db/db mice. All these mice were divided into four groups, db/db control (CT), db/db control plus exercise (CT+EX), db/db mice (DB), db/db plus exercise (DB+EX), every group have 10 mice. CT+EX and DB+EX were present in the treadmill training for 8 weeks (7 m/min the first week, 10 m/min the second week, and 11 m/min third week until the end. Run for 330 m total with a 5 minute break every 110 m, 6 times/week). After sacrificed, alkaline phosphatase (ALP), tartrate‐resistant acid phosphatase 5b (TRACP‐5b), and gasotransmitter [hydrogen sulfide (H2S) and nitric oxide (NO)] in serum were assayed, bone quality, including bone mineral density (BMD), micro structure, biomechanical parameters were measured by DXA, micro CT and three bending test separately. Total mRNA and protein were isolated, and factors in Hcy/H2S cycle were detected by western‐blot and RT‐PCR.

RESULTS

Physical exercise induced lose bodyweight, especial in DB+EX (P < 0.05), also decreased the blood glucose (P < 0.01), but showed no effects on db/db control mice. Exercise increases the bone quality index (BMD, trabecular microstructure, break force) both in db/db control mice and db/db mice, but there also shown a more obvious difference in db/db mice than db/db control mice. We also found exercise decrease the Hcy level, but increase H2S level in serum in db/db mice. Exercise regulate the factors involved in homocysteine metabolism to balance the level of Hcy and H2S, such increased MTHFR and CBS, but decreased SAHH.

CONCLUSION

The exercise intervention could decrease bodyweight and blood glucose in db/db mice, proved moderate exercise is helpful for diabetes treatment. Meanwhile, exercise also rebalance the Hcy and H2S level in db/db mice, and then prohibit bone loss. All these results represents that moderate exercise improve bone quality in type 2 diabetes patients who also with secondary osteoporosis.

Support or Funding Information

Financial support from National Institute of health grant AR‐067667 and HL‐107640 are greatly acknowledged.
Transcriptional regulator PGC1α regulates amino acid metabolism activated by exercise in skeletal muscles
Authors: Hatazawa Yukino , Miura Shinji , Kamei Yasutomi
Published: November 2023

Skeletal muscles play important roles not only in exercise and nutrition metabolism, but also in human health. Peroxisome proliferator‐activated receptor (PPAR) γ coactivator 1α (PGC1α) is a coactivator of various nuclear receptors and other transcription factors, whose expression increases in the skeletal muscles during exercise. We have previously made transgenic mice overexpressing PGC1α in skeletal muscles (PGC1α‐Tg mice). PGC1α Tg mice showed increased red fiber and capacity for longer treadmill running. In this study, we analyzed metabolic changes of skeletal muscles in PGC1α‐Tg mice by global gene expression (microarray) and metabolomic analyses.

Microarray data indicated that branched chain amino acid (BCAA) metabolism was activated in skeletal muscles of PGC1α‐Tg mice. Moreover, we observed that gene and protein expression of BCAA metabolism‐related enzymes were increased in PGC1α‐Tg mice (Hatazawa et al. PLOS ONE. 2014). Then, we examined low molecular weight compounds, especially water‐soluble metabolites, in skeletal muscles of PGC1α‐Tg mice by metabolomic analyses. Metabolic products of the TCA cycle increased in PGC1α‐Tg mice. Levels of β‐alanine and related metabolites, which may serve as a substrate for the TCA cycle, were markedly decreased in PGC1α‐Tg mice. Moreover, our metabolomics data showed the activation of the purine nucleotide pathway, malate–aspartate shuttle, and creatine metabolism, which are known to be active during exercise. Expression of these genes related with these pathways was increased in PGC1α‐Tg mice. Thus, the data suggests that PGC1α provide an energy source during exercise by using amino acids, including BCAA, as a substrate for the TCA cycle and activating the cycle. Meanwhile, PGC1α overexpression in skeletal muscles increased the muscle content of gamma‐aminobutyric acid (GABA), gamma‐aminoisobutyric acid (BAIBA) and serotonin, suggesting that these amino acid‐related molecules might act as secretory bioactive molecules (myokines) that have effects on other organs. Namely, this may be a new mechanism that explains the interaction of multiple organ networks in lifestyle diseases affected by exercise (Hatazawa et al. PLOS ONE. 2015).
Pyruvate Dehydrogenase Activity in Red and Superficial White Gastrocnemius Muscles of the Rat during Contraction
Authors: Kasper Jonathan D , Lewis Matthew T , Gudziak Gregory L , Meyer Ronald A , Wiseman Robert W
Published: November 2023

The increase in glucose oxidation during exercise is dependent upon the mitochondrial enzyme pyruvate dehydrogenase (PDH) which is allosterically regulated by reversible phosphorylation through a family of PDH kinases and phosphatases. However, the relationship between PDH activation and muscle fiber type during exercise has not been well described because fiber specific muscles have not been reported, and, because voluntary exercise involves motor unit recruitment which further complicates interpretation. To determine whether PDH activation differs in red versus superficial white muscle in vivo, gastrocnemius muscle contractile activity was controlled by electrical pacing via surgically implanted electrodes on the sciatic nerve of anesthetized male Wistar rats. Muscle force production of the triceps surae muscle group was recorded from the Achilles tendon and the knee pinned in place with a tungsten rod through the head of the femur. Gastrocnemius muscle samples were collected from the superficial white (predominantly Type IIb fibers) and deep red (predominantly Type IIb fibers) portions and freeze clamped at rest and during 5 min of twitch stimulation at 0.5Hz and 1.0Hz intensities. PDH activity was assayed in vitro using a radioisotopic tracer assay with [2‐14C]‐pyruvate as the substrate. Total PDH activity in the deep red gastrocnemius was almost two‐fold greater than that of the superficial white (1.9±0.1 vs 1.1±0.1 U/gWW, n=15). Fractional PDH activity (% total activity) was significantly increased in muscle samples collected from superficial white relative to deep red gastrocnemius at rest (15.0±2.0 vs 9.5±0.7%), and during 0.5Hz (60.9±8.1 vs 37.4±3.9%), and 1.0Hz stimulation frequencies (68.3±2.8 vs 40.5±2.8%) (n=4–5 for all groups). However, the active fraction of PDH activity was not different when comparing superficial white to deep red gastrocnemius at rest (0.12±0.02 vs 0.15±0.02 U/gWW) or during stimulation at 0.5Hz (0.67±0.08 vs 0.71±0.04 U/gWW), or 1.0Hz intensities (3±0.08 vs 0.82±0.02 U/gWW). These data suggest that the active fraction of PDH is dependent on the contraction intensity and therefore muscle fiber type differences do not appear to play a role in the number of PDH active sites during contractile activity. Therefore future designs of therapeutic exercise to optimize glucose disposal should be chosen based on the maximal intensity that can be maintained without fatigue to maximize the working muscle mass and PDH activity.

Support or Funding Information

Supported by NIH DK095210
Effects of combined practical cooling prior to and during exercise on thermoregulatory responses
Authors: Tokizawa Ken , Son Su‐young , Oka Tatsuo , Yasuda Akinori
Published: November 2023

Pre‐cooling is a popular strategy for alleviating heat strain. Because whole body water immersion that commonly used to pre‐cool in experimental studies is not always possible in all field settings, we have examined effects of fanning and hands and feet cooling on heat strain while wearing protective clothing. The practical pre‐cooling reduced core temperature elevation during exercise by nearly half compared with no‐cooling trial. In the present study, we evaluated effects of combined applications of the practical pre‐cooling and wearing a tube‐lined perfusion vest using field‐portable cooler during exercise on heat strain.

Eight males engaged in 60 min of walking (3 km/h) in a hot environment (33°C, 60%RH) while wearing protective clothing under three separate conditions: no cooling (NC), wearing a tube‐lined perfusion vest during walking (VEST), and pre‐cooling (fanning to the face, hands and feet cooling, and wearing the vest) for 30 min before walking and wearing the vest during walking (PC+VEST).

Rectal temperature increased by 0.8 ± 0.1°C during walking in NC. The increase in VEST was lower than in NC (0.6 ± 0.1°C, p < 0.05), and further reduction was observed in PC+VEST (0.2 ± 0.1°C, p < 0.05). Sweat rate in the chest and percentage reduction in body weight were also inhibited in VEST compared with NC (p < 0.05, respectively). In PC+VEST, the inhibitions were greater than in VEST (p < 0.05, respectively). Thermal sensation and pleasantness by ratings of visual analogue scale were lower in VEST and PC+VEST than in NC (p < 0.05), whereas no differences in the perceptions were observed between VEST and PC+VEST.

These results suggest that combined applications of the pre‐cooling and wearing a tube‐lined perfusion vest during exercise can additively inhibit core temperature elevation and dehydration in the heat.
Determination of seasonal differences of clock gene expressions in obese men using saliva sample
Authors: Sato Maki , Hayashi Hisaki , Ikemoto Tatsunori , Ushita Takahiro , Kanikowska Dominika , Sato Motohiko
Published: November 2023

Obesity is a significant health problem in the world during the past several decades. Obese people have an increased incidence for developing cardiovascular, renal, and hormonal diseases and sleep disorders. Disruption of circadian rhythms is also associated with a risk of obesity. Bmal1 and Rev‐erb‐α (Nr1d1) of clock genes were reported to be involved in adipocyte differentiation and lipogenesis. We have previously reported that the seasonal differences of melatonin secretion during sleep, which showed that melatonin concentration in obese subjects were decreased compared with non‐obese subjects in winter season. In the present study, we investigated that the clock gene expressions in obese and non‐obese men in summer and winter. We hypothesized that the clock gene expressions were decreased in obese men in winter. Non‐obese men and obese men participated in summer and winter. Total RNA was isolated from saliva sample of 2 mL. We measured the clock gene expressions of Bmal1 and Rev‐erb‐α by real‐time PCR. Bmal1 was significantly decreased in obese than in non‐obese men, and Rev‐erb‐α tended to be decreased in obese than in non‐obese men. Seasonal differences of clock gene expressions in obese and non‐obese men will be discussed.

Support or Funding Information

This work was supported by JSPS KAKENHI Grant Number JP15K16529.
LPS‐Induced TLR4 Activation During Prolonged Running and Cycling Events in Hot and Humid Environments
Authors: Hosokawa Yuri , Zinn Jacob R , Wright Skylar , Leduc Madeline , Howard Emily , Bivona Joseph J , Casa Douglas J , Stearns Rebecca L , Huggins Robert A , Adams William M , Jardine John , Davis Robert J , Armstrong Lawrence E , Vingren Jakob , Luk Hui Ying , Levitt Danielle , Ganio Matthew S , McDermott Brendon P , Williamson Keith , McKenzie Amy L , Munoz Colleen X , Kunces Laura J , Lee Elaine C
Published: November 2023

Lipopolysaccharide (LPS) is a gram‐negative bacterial marker that might be released into circulation during exercise‐heat stress. LPS induces an innate immune response by binding LPS‐binding protein (LBP) and its cognate receptor, TLR4 (toll‐like receptor 4), expressed on CD16+CD14+ circulating immune cells. LPS‐induced TLR4 activation results in robust immune responses which drive endotoxemia, sepsis, and putatively, exertional heatstroke pathophysiology.

PURPOSE

We aimed to test the hypothesis that competing in prolonged cycling (50–100 mile event) or high‐intensity running (7 mile race) events in hot and humid environments (heat index>80°F) result in LPS‐induced TLR4 activation and changes in cellular gene expression.

METHODS

We recruited 64 subjects participating in the Hotter n' Hell Hundred (HHH) ultraendurance cycling event (Wichita Falls, TX) and 36 subjects racing in the Falmouth (FAL) Road Race (Falmouth, MA). We sampled venous blood pre (PRE) and immediately post (IP) each event, and also 30 minutes after IP (30IP) in FAL. We isolated circulating PBMCs (peripheral blood mononuclear cells) which were stained for surface markers CD14, CD16, and TLR4 using fluor‐conjugated antibodies. We used multi‐color digital flow cytometry on‐site to analyze cell populations. We also isolated anticoagulant‐treated plasma samples from whole blood for analysis of circulating LPS (Hycult Biotech, ELISA) and LBP (R&D ELISA) concentrations. At both events, we measured environmental and physiological variables. We statistically analyzed data using repeated measures ANOVA (LSD post hoc, α‐level:p≤0.05) for differences (PRE vs. IP, PRE vs. 30IP).

RESULTS

Circulating LPS increased in both HHH riders and FAL racers by 25% (p<0.05) and 39% (p<0.04), respectively. By 30IP, FAL racers' circulating LPS levels had returned to PRE values (p>0.19). In both races, circulating LBP remained unchanged (p>0.33). CD14+CD16+ cells expressing TLR4 in circulation were significantly increased at IP (p<0.05).

CONCLUSION

We conclude that circulating LPS is increased post‐exercise heat stress and analysis of downstream activation is required to determine role of LPS in exercise‐heat stress.

Support or Funding Information

We would like to thank Connecticut Institute for Clinical and Translational Science and McNair Scholars Program for funding the research and thank Hotter'N Hell Hundred and Falmouth Road Race for their support.
Metabolic monitoring and cage activity in mice exposed to 7‐months of electronic cigarette vapor
Authors: Pitzer Christopher , Breit Matthew , Hoskinson Hannah , Fabrega Mireia , Bryner Randy , Olfert I.M.
Published: November 2023

Introduction

Nicotine is a known central nervous system stimulant that increases heart rate and metabolism. E‐cigarettes (E‐cig) are a new and highly effective nicotine delivery device, that include a vast array of flavorings and chemical by‐products that are likely to have deleterious consequences. Currently there is very little known about the long‐term effects of E‐cig vapor exposure, particularly in the context of whole body metabolism. We hypothesize that metabolic cage activity will be increased in mice chronically exposed to E‐cig vapor.

Methods

C57BL/6 female mice were randomly assigned to 3R4F reference cigarette (N=13), cappuccino flavored E‐vapor (18 mg/ml nicotine, N=12), or filtered air (N=13) exposed groups. Animals were exposed 4 h/day, 5 d/w for 7 months. Whole body metabolic gas exchange and cage activity was measured after the 7‐month exposure using a home‐cage chronic laboratory animal monitoring system (Oxymax CLAMS, Columbus Instruments). Food and water were administered ad libitum.

Results

Oxygen consumption (VO2) at night was significantly increased in E‐cig and 3R4F by 21% and 17%, respectively (p<0.05) compared to air exposed mice. However, VO2 was not significantly different between groups during the day. Carbon dioxide production (VCO2) at night was also increased in E‐cig and 3R4F by 19% (for both groups, p<0.05) compared to air, and also not significantly different during the day. Respiratory exchange ratio (RER) was not different between any of the groups at night or day, however indirect calorimetric assessment of heat production (kcal/hr) was significantly greater in E‐cig mice by 16% and 18% compared to 3R4F and air, respectively (p<0.05), at night. Heat, during the day, also tended to be greater in E‐cig mice by 12% and 11% compared to 3R4F and air, respectively (p=0.068). Spontaneous cage activity at night tended to be greater in E‐cig mice (p=0.06) compared to 3R4F and air groups, but no difference in cage activity occurred during the day.

Conclusion

Exposure to E‐cigs increased VO2, VCO2 and heat production, which corresponded to greater cage activity, in measurements made at night (the animal's period of greatest cage activity), but not during the day (the period of least cage activity). Similar to E‐cigs, 3R4F reference cigarettes also increased VO2 and VCO2, but not heat production, at night. These data suggest chronic E‐cig exposure can increase basal metabolic and cage activity in mice.

Support or Funding Information

NIH P20GM103434 (West Virginia IDeA Network for Biomedical Research Excellence) and the Marshall‐WVU Health Initiative Award
ORM: A Central Regulator of Sphingolipid Homeostasis and Composition in Arabidopsis
Authors: Cahoon Edgar
Published: November 2023

The sphingolipid metabolic network in Arabidopsis is tightly regulated to ensure sufficient biosynthetic flux to support cell viability and growth but to also rapidly respond to perturbations such as those induced by bacterial pathogenesis that trigger production of the bioactive sphingolipid metabolites. These metabolites include long‐chain bases and ceramides that signal programmed cell death (PCD) for plant defense. Regulation of sphingolipid biosynthesis is mediated primarily through reversible physical interactions, of serine palmitoyltransferase (SPT), the initial enzyme in long‐chain base biosynthesis, with ORM proteins (orosomucoid‐like proteins). Consistent with a role of ORMs as repressors of SPT activity, overexpression of the two Arabidopsis ORM proteins AtORM1 and AtORM2 strongly reduced the accumulation of free long‐chain bases in response to treatment with the mycotoxin fumonisin B1 (FB1), a ceramide synthase inhibitor. These plants also displayed increased resistance to the PCD‐inducing cytotoxicity of FB1. Conversely, RNAi suppression of AtORM1/2 resulted in large increases in long‐chain bases in response to FB1 treatment and increased sensitivity to FB1. AtORM1 and 2 overexpressing plants also had decreased activity of Class I ceramide synthase that generates ceramides with C16 fatty acids and dihydroxy long‐chain bases and increased activity of Class II ceramide synthases that produce ceramides with very‐long chain fatty acids and trihydroxy long‐chain bases. The reverse effect on ceramide synthase activities was observed in AtORM RNAi lines. These findings suggest an extended role of ORMs as regulators of the production of structurally and functionally distinct ceramides for glycosphingolipid synthesis to support plant growth and long‐chain base homeostasis.
Genetic Lineage Tracing Analysis of Endothelial Cells in Lung Vascular Injury Identifies Two Distinct Sources of Endothelial Regeneration
Authors: Liu Menglin , Gong Haixia , Zhang Lianghui , Xiong Shiqin , Rehman Jalees , Malik Asrar B
Published: November 2023

Loss of pulmonary vascular endothelial cells (ECs) is a key pathogenic feature of acute lung injury (ALI). This compromises endothelial barrier function and leads to severe pulmonary edema. The process responsible for regeneration of lung endothelium is not well understood. Regeneration may be due to proliferation of resident ECs and also non‐ECs may be mobilized to repair the barrier. Here, we used an mT/mG (membrane Tomato/membrane GFP) double fluorescent transgenic mouse crossed with a tamoxifen (tam)‐inducible EC Cre mouse model. Tam induced the labeling of ECs with GFP whereas all other cells remained labeled with TomatoRed, allowing us to trace EC fate following endothelial injury. We found that lung vascular injury induced by endotoxin (LPS) produced ~50% loss of ECs which peaked at 24h post‐LPS. EC regeneration occurred rapidly and peaked within 5 days, thus mirroring restoration of lung vascular barrier function. Thereafter, tam was re‐injected to assess whether non‐ECs transdifferentiating to ECs could also account for the EC regeneration. The 2nd tamoxifen labeling showed that half of ECs were derived from a non‐EC lineage. We identified a subpopulation of cells co‐expressing the endothelial surface marker CD31 and fibroblast‐specific protein 1 (FSP1) peaking at 1day post‐LPS and remained elevated during recovery, suggesting this population may contribute to replenishment of the endothelium. Inhibition of TGF‐β signaling promoted EC population recovery. Thus, there appear to be 2 mechanisms of EC recovery: a fibroblast subpopulation which transitions to ECs and acts in concert with proliferation of the native endothelium.

Support or Funding Information

This work was supported in part by the National Institutes of Health: RO1HL118068, RO1HL090152 and T32HL007829.
Insulin Resistance Is Inversely Associated with the Intestinal Expression of Key Genes Involved in Chylomicron Assembly and Secretion
Authors: Drouin‐Chartier Jean‐Philippe , Tremblay André J , Lemelin Valéry , Lépine Marie‐Claude , Guay Valérie , Lamarche Benoît , Couture Patrick
Published: November 2023

Background

Insulin resistance is a chronic metabolic disorder associated with an overproduction of atherogenic intestinal triglyceride‐rich lipoproteins (TRLs). However, the mechanisms underlying this increased secretion of intestinal TRLs remain to be fully understood. The objective of the present study was to evaluate the association between the homeostasis model assessment of insulin‐resistance (HOMA‐IR) index and the intestinal expression of key genes involved in chylomicron assembly and secretion in a large sample of non‐diabetic men.

Methods

Duodenal biopsies were obtained by gastroduodenoscopy in 105 non‐diabetic men. Gene expression was measured using quantitative PCR in duodenal biopsy samples. Anthropometric parameters and fasting plasma glucose and insulin concentrations were measured in all participants.

Results

Participants' mean age (±SD) was 37.7 ± 11.1 years (range: 21.0 to 65.0 years). Mean BMI was 31.8 ± 4.3 kg/m2 (range: 20.4 to 43.8 kg/m2) and mean HOMA‐IR index was 4.10 ± 1.64 (range: 1.02 to 10.02).

The HOMA‐IR index was inversely associated with the duodenal expression of microsomal triglyceride transfer protein (MTP) (r=‐0.23; P=0.02), acetyl‐coenzyme A synthetase 1 (ACS1) (r=−0.22; P=0.03), diacylglycerol O‐acyltransferase 1 (DGAT1) (r=−0.25; P=0.01) and DGAT2 (r=−0.24; P=0.02). These associations were independent of body mass index (BMI), waist circumference and age. There was no significant association between the HOMA‐IR index and the duodenal expression of apolipoprotein (apo) B (r=−0.15; P=0.15) and SAR1 gene homolog B (SAR1B) (r=−0.20; P=0.07).

Duodenal expression of MTP was positively associated with the expression of apo B (r=0.60; P<0.0001), ACS1 (r=0.57; P<0.0001), DGAT1 (r=0.72; P<0.0001), DGAT2 (r=0.77; P<0.0001) and SAR1B (SAR1B) (r=0.66; P<0.0001).

Using multiple linear regression models, the HOMA‐IR index was the only factor significantly associated with variations in the duodenal expression of MTP, DGAT1 and DGAT2, whereas BMI and the HOMA‐IR index both contributed significantly to the variance of ACS1 expression.

Conclusions

This study demonstrates that insulin‐resistance is inversely associated with expression of the key genes involved in chylomicron assembly in the duodenum, independent of variations in BMI, waist circumference and age. The downregulation of the expression of these genes may represent an adaptive response to the chronic insulin‐resistance to decrease chylomicron secretion.
ELOVL3, 6 and 7 Specifically Elongate Saturated Branched Chain Fatty Acids and Compete with Normal Fatty Acids
Authors: Wang Zhen , Park Hui Gyu , Wang Dong Hao , Goykhman Yuliya , Lawrence Peter , Kothapalli Kumar S.D. , Brenna J. Thomas
Published: November 2023

Introduction

Branched chain fatty acids (BCFA) are saturated fatty acids (SFA) terminating with either an isopropyl or isobutyl group. They are common components in human diets, and are synthesized de novo in human skin sebaceous and meibomian glands as well as vernix caseosa. They are known to have structure specific activities but little known of the pathways for their endogenous interconversion. Multiple human ELOVLx (elongase of very long chain fatty acids) genes are responsible for interconversion of saturated fatty acids. Our objectives are to perform the first investigation of the activity of ELOVL on BCFA using anteiso‐15:0 and iso‐18:0 as examples, to characterize the substrate specificity to BCFA, and to investigate competition between BCFA and normal (n‐) SFA (n‐16:0 and n‐18:0) for the ELOVL‐mediated elongation.

Methods

Human breast cancer cell line MCF7 was used as the experiment model. MCF7 cells were grown in MEM‐α media with 10% FBS in a humidified environment at 37°C with 5% CO2. The open reading frame of ELOVL transcripts (ELOVL1, ELOVL3, ELOVL6 and ELOVL7) were cloned into a pcDNA3.1 expression vector. MCF7 cells were transfected with specific ELOVL transgene vector along with empty vector (control) for 24h using Polyplus jetPRIME transfection reagent. Then albumin‐bound fatty acids, anteiso‐15:0, iso‐18:0, n‐16:0 and n‐18:0 were dosed into cells at 0–100 μM and allowed to incubate for additional 24h. Cells were washed twice with 1 × PBS, harvested by trypsinization, fatty acid methyl esters were analyzed by GC‐FID and GC‐MS.

Results

ELOVL6 chiefly elongated anteiso‐15:0 à anteiso‐17:0; ELOVL3 predominantly elongated iso‐18:0 à iso‐20:0; ELOVL7 showed moderate elongase activity toward both anteiso‐15:0 and iso‐18:0. n‐16:0 competed with anteiso‐15:0 for ELOVL6 mediated elongation, and n‐18:0 competed with iso‐18:0 for ELOVL3 mediated elongation. These latter results verified the characterized elongation activities.

Conclusion

Here for the first time we showed the substrate specificity of human elongase to BCFA systematically, and verified it by establishing competition between BCFA and n‐SFA.

Support or Funding Information

Internal Fund
Exercise Effects on Fecal pH During A Period of Excess Dietary Fat and Sugar Consumption
Authors: Castillo Luisa F , Tucker Wesley J , Angadi Siddhartha S , Gaesser Glenn A , Whisner Corrie Marie
Published: November 2023

Background

Short chain fatty acids (SCFA), a product of microbial fermentation, have been observed in higher quantities in obese individuals compared to lean counterparts. Despite links between excess caloric consumption and adiposity, data are limited on whether fecal pH correlates with changes in body fat during periods of excess fat and sugar consumption and how exercise influences these relationships.

Purpose

The goal of this study was to assess whether changes in fecal pH (a proxy for SCFA production) correlated with changes in measures of adiposity during a fat‐sugar supplemented diet and exercise intervention.

Methods

Twelve otherwise healthy overweight/obese males (aged 18–50 years; BMI = 29.6±3.6 kg/m2; percent body fat = 31.9±5.1%) consumed 12 donuts/wk for 4 weeks which contributed an excess energy intake of 14,554 kcal. Participants were randomized to 3 groups: moderate‐intensity continuous training (MICT), high‐intensity interval training (HIIT), and no exercise (Control). Body mass index (BMI) and percent body fat (% fat) were assessed pre‐ and post‐intervention by Seca stadiometer‐scale and DEXA respectively. Baseline and 4‐week fecal samples were obtained from participants and fecal pH measured using a digital pH meter. Spearman correlations were used to assess correlations between changes in fecal pH, BMI and % fat. The Wilcoxon rank sum test was used to evaluate differences in fecal pH between groups.

Results

Mean fecal pH at baseline was 6.52±0.33. Median (IQR) changes in pH for control, HIIT and MICT groups (0.22 (−0.33, 0.39), 0.22 (−0.80, 0.55), −0.33 (−0.70, −0.08), respectively) did not differ significantly (p=0.375). Although mean % fat did not change significantly in any group, the change in fecal pH across all groups during the intervention was positively correlated with the change in % fat (r=0.69, p=0.013). No correlation was observed between changes in fecal pH and BMI (r=−0.44, p=0.155).

Conclusions

Findings from this study suggest that an increase in fecal pH was associated with an increase in percent body fat. These positive changes in adiposity during a period of increased fat and sugar intake suggest that further work is needed to understand the role of gut microbes and SCFA concentrations on body composition over time. Further, these findings suggest a need to understand how different exercise modalities mediate associations between gut SCFA production and changes in body fat. Finally, fecal pH as a proxy for SCFA production in the colon may be a risk marker for weight change during periods of fat‐sugar overfeeding.
Ten Weeks of Protein Consumption at the RDA Results in a Loss of Appendicular Lean Mass in Healthy Older Men, a Randomized Controlled Trial
Authors: Mitchell Cameron J , Milan Amber M , Mitchell Sarah M , Zeng Nina , Ramzan Farha , Sharma Pankaja , Knowles Scott , Roy Nicole , Sjödin Anders , Wagner Karl‐Heinz , Cameron‐Smith David
Published: November 2023

The age related loss of skeletal muscle mass and function, can lead to loss of independence and is detrimental to metabolic health. The current recommended daily allowance (RDA) for protein intake is 0.8 g/kg/day; both epidemiological and mechanistic evidence suggest that protein intake at this level may not be adequate to maintain skeletal muscle mass in older adults especially during negative energy balance. However, there is limited evidence from randomized controlled trials.

30 healthy older men (BMI: 28.3 ± 0.8 kg/m2, body fat: 27.8 ± 1.4 %, fasting glucose: 5.7 ± 0.1 mmol/L; Mean ± SEM) were randomized to consume protein either at the RDA or at twice the RDA ([2xRDA], 1.6g/kg/day) for ten weeks. Both diets intended to maintain body mass and conformed to U.S. Department of Agriculture guidelines. All meals were provided to the participants. Lean mass and fat mass were measured using Dual‐energy X‐ray absorptiometry, physical function was assessed by using the short physical performance battery (SPPB) and isometric dynamometry. Compliance was ensured with diet records and analysis of urinary nitrogen excretion.

Appendicular lean mass did not change in 2xRDA (25.0 ± 10.3 kg to 25.2 ± 9.6 kg, P=0.592) but decreased in RDA (24.4 ± 9.9 kg to 23.8 ± 9.3 kg, P=0.005). Whole body lean mass was unchanged in RDA (56.6 ± 21.2 kg to 56.1 ± 20.1 kg, P=0.149), while it increased in 2xRDA (55.7 ± 21.2kg to 57.2 ± 20.2 kg, P<0.001), however, this changes was mostly accounted for by an increase in trunk lean mass (+1.4 ± 0.3 kg, P<0.001). Both groups decreased total body fat mass and body fat percentage similarly. SPPB scores were unchanged in both groups but there was a trend towards an interaction (P=0.077) in isometric knee extension torque such that strength increased in 2xRDA (147.1 ± 17.7 Nm to 154.6 ± 13.4 Nm) and decreased in RDA (158.8 ± 14.2 Nm to 150.1 ± 13.0 Nm).

When older men consume a controlled diet containing protein at currently recommended levels, an observable loss of appendicular lean mass can occur in as little as ten weeks. Higher protein diets may be required to support the maintenance of muscle mass in older adults.

Trial registry: ACTRN12616000310460

Support or Funding Information

Ministry of Business, Innovation and Employment (New Zealand) FRIENZ European Study Tour Research Scheme