bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional factor‐X and related small molecule herbal inducer, ST32DB, against obesity and metabolic dysfunction through lipogenic transcription factor ChREBP and white adipocytes trans‐differentiation axis
Authors: Chen Hsi‐Hsien , Lin Heng
Published: November 2023

Altering the balance between lipogenesis/lipolysis or altered white fat remodeling is a potential strategy for treating obesity and metabolic syndrome. Here, we demonstrate that genetic deletion of Transcriptional factor‐X (TFX) decreased energy expenditure but increased body weight, insulin resistance and hepatic steatosis during high‐fat diet regimens, in addition, restored TFX expression by AAV‐mediated in TFX knockout mice were abrogate these metabolic changes. In vitro study in 3T3‐L1 adipocytes also shown that TFX‐overexpressing exhibited less lipid accumulation with diminished expression of adipogenesis, lypogenesis and inflammation‐related genes but increased loogenesis, β‐oxidation and brown/beige genes as compared through ChREBP and SCD1 dependent pathway. Finally, the beneficial metabolic change, thermogenesis elicited, reduced lipogenesis and increased lypolysis by a new finding TFX inducer selected by TFX stable clone in 3T3‐L1 adipocytes were emerged in normal mice but in TFX knock out mice when feeding high fat diet. In addition, consistent results between in vitro and in mice. Overall, our finding identify TFX and a new TFX single compound inducer from herbal as a regulator of lipogenesis/lipolysis balance and enhance energy expenditure and mediate adipocyte differentiation from white fat to brown fat through CHREBP and SCD1 pathway represents a possible therapeutic strategy go treat obesity and obesity induced metabolism syndrome.
Myometrial Response to Oxytocin Is Affected by Parity
Authors: Porta Maura , Sondgeroth Korie
Published: November 2023

Objective

The complex array of hormonal interactions that develop during pregnancy exert a profound impact in the reproductive system and in various other aspects of a woman's physiology. Some systems are permanently changed via epigenetic DNA modification, as it has been shown recently in the mammary gland (dos Santos, 2015). The aim of the present study was to investigate the hypothesis that pregnancy can affect some aspects of the uterine function, as well. Thus, we compared the response of non‐pregnant myometrium harvested from virgin and parous female rats to increasing concentrations of oxytocin, PGE2, and PGF2, which are endogenous stimulators of contractility in the uterus.

Methods

Virgin and parous female non‐pregnant CD Sprague Dawley rats (Charles River Laboratories) were euthanized at 18 weeks of age (to eliminate the confounding effect of age on contractility measurements) during proestrus (to eliminated variability due to hormonal fluctuations). The uterine horns were removed and one was flash frozen for later use (ELISA), while the other was dissected into six 3×10 mm strips. These were then suspended in a tissue bath containing Krebs solution (in mM, NaCl 117, KCl 4.7, NaHCO3 25, MgCl2 1.2, KH2PO4 1.2, CaCl2 2.5, glucose 11, pH= 7.4) at 37°C and bubbled with 95% O2 and 5% CO2. Separate dose responses curves to oxytocin, PGE2, and PGF2 were generated by incremental additions of each drug to the tissue bath (10E‐10 to 10E‐5 M in all cases). After the maximal dose of stimulator, 10E‐6 M of terbutaline (beta‐2 agonist; tocolytic) was added to the system.

Results

We found that indeed parous myometrium displays a significantly stronger response to oxytocin than its virgin counterpart (P<0.05). The EC50 of the drug for the receptor appears unchanged, but the maximal response to the drug is higher (P<0.05). Furthermore, the ability of a 10E‐6 M dose of terbutaline to inhibit activation of myometrial strips by maximal doses of oxytocin (10E‐5 M) also differs. Terbutaline inhibits 60±6% of oxytocin‐activated virgin myometrial contraction, vs. 39±5% of oxytocin‐activated parous myometrial contraction (P<0.005). Preliminary ELISA results suggest a ~30% higher expression of oxytocin receptor in non‐pregnant parous uterine tissue. No significant differences in the myometrial dose‐response relationship in virgin versus parous uterine tissue emerged in PGE2 and PGF2 treated strips.

Conclusions

Our results suggest that at least in the response to oxytocin, parity affects uterine contractility. This supports the general hypothesis that some (likely epigenetic) changes occur in the uterine smooth muscle during pregnancy that persist post‐parturition.

Support or Funding Information

This study was supported by Midwestern University.
Effects of Calorie Restriction and Fiber Type on Glucose Uptake in Single Fibers from Old Rats
Authors: Wang Haiyan , Arias Edward B. , Yu Carmen S. , Verkerke Anthony R.P. , Cartee Gregory D.
Published: November 2023

The major tissue for insulin‐stimulated glucose disposal is skeletal muscle. Calorie restriction (CR; reducing calorie intake by ~40% below ad libitum) can increase glucose uptake by insulin‐stimulated muscle. Skeletal muscle is comprised of multiple, heterogeneous fiber types, and the efficacy of CR for enhancing insulin‐stimulated glucose uptake across all fiber types is uncertain. The conventional approach to evaluate possible fiber type differences has been to measure glucose uptake by multiple muscles or regions of muscles with differing fiber type profiles. However, tissue analyses cannot resolve fiber type differences in glucose uptake at the cellular level because: 1) whole muscles include multiple fiber types; 2) apparently no rodent muscle has been identified that is mostly comprised of type IIX fibers; 3) measurement of glucose uptake by fibers expressing multiple myosin heavy chain isoforms (i.e., hybrid fibers) requires single fiber analysis; and 4) various cell types (including neural, vascular, and adipose cells) contribute to glucose uptake determined in muscle tissue samples. The limitations of muscle tissue analysis motivated us to recently develop and validate the first method to determine both glucose uptake and fiber type in a single rat skeletal muscle fiber. We used this method in the current study with the primary aim to determine in 23–26 months‐old rats the effects of chronic CR (60% of ad libitum, AL, initiated at 14 weeks‐old) and fiber type on insulin‐stimulated glucose uptake by single fibers of diverse fiber types. Isolated epitrochlearis muscles from AL and CR rats were incubated with [3H]‐2‐deoxyglucose ± 1.8nMinsulin. Glucose uptake and fiber type (based on myosin heavy chain expression) were determined for single fibers dissected from the muscles. We also determined CR‐effects on the abundance of hexokinase II (a key enzyme that favors greater glucose uptake by catalyzing the conversion of glucose to glucose 6‐phosphate) in single fibers by immunoblotting. We found in single fibers from CR compared to AL rats: 1) significantly greater insulin‐stimulated glucose uptake by type I, IIA, IIB, IIBX and IIX fibers (P<0.01 for I, IIA and IIB; P<0.001 for IIBX and IIX); and 2) unaltered hexokinase II abundance in each fiber type. These observations revealed at the cellular level that long‐term CR by older rats enhanced insulin‐stimulated glucose uptake in rat epitrochlearis muscle regardless of fiber type and independent of elevated hexokinase II protein content.

Support or Funding Information

This work was supported by a grant from the National Institute on Aging at the National Institutes of Health (grant number AG10026) to GDC.
Prior Treatment with the AMPK Activator AICAR Induces Subsequently Enhanced Glucose Uptake in Isolated Skeletal Muscles from Old Rats
Authors: Oki Kentaro , Arias Edward B , Cartee Gregory D
Published: November 2023

Prior treatment of isolated skeletal muscles from young rats (≤4 months‐old) to the 5′ adenosine monophosphate‐activated protein kinase (AMPK) activator 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR) in the presence of serum can induce elevated insulin‐stimulated glucose uptake (GU) several hours after of removal of the muscles from AICAR and serum. Previous research did not determine if both AICAR and serum were required for the observed effect on insulin‐dependent GU, or if incubation with either was sufficient. Earlier research also has not evaluated the effects of prior AICAR incubation with and without serum on the GU of muscles from older rats. Accordingly, we examined the separate and combined effects of AICAR and serum on subsequent insulin‐independent and insulin‐dependent GU in skeletal muscles from aged rats (23–24 month old; n=20). Both epitrochlearis muscles from each rat were dissected and split into two separate muscle strips. Each muscle strip underwent a four‐step incubation. During step 1 (60 minutes), one muscle strip from each animal was incubated with: Krebs‐Henseleit Buffer (KHB) alone, KHB with AICAR, serum alone, or serum with AICAR. During step 2 (180 minutes), all muscle strips were incubated with KHB. During steps 3 (30 minutes) and 4 (20 minutes), all 4 strips from 10 rats were incubated without insulin, and all 4 strips from the remaining 10 rats were incubated with insulin. During step 4, muscle strips were incubated with radiolabeled 3‐O‐methyl‐D‐glucose and the same insulin concentration as the previous step. Repeated measures analysis of variance (RM ANOVA) in the basal (no insulin) group indicated a significant interaction (serum × AICAR) for insulin‐independent GU. In the insulin group, RM ANOVA revealed a significant main effect of AICAR on insulin‐dependent GU without a significant main effect of serum or significant serum × AICAR interaction. Our working hypothesis is that the elevated insulin‐independent GU after combined AICAR and serum incubation is secondary to subsequently elevated phosphorylation of TBC1D1 on Ser237 (an AMPK‐phosphosite), and the AICAR‐induced elevation of insulin‐dependent GU is secondary to subsequently elevated phosphorylation of TBC1D4 on Ser711 (an AMPK phosphosite) and Thr642 (an Akt‐phosphosite).

Support or Funding Information

NIA Training Grant AG 000114 and NIA R01 AG10026 (GDC)
Atorvastatin Reduces Adipose Tissue Browning And Prolong Survival In Cancer Cachexia Mice
Authors: Henriques Felipe Santos , Franco Felipe , Lopes Magno , Santos Kaltinaitis , Guilherme Adilson , Junior Miguel Luiz Batista
Published: November 2023

Background and aims

Cancer cachexia (CC) is a wasting syndrome characterized by systemic inflammation, body weight loss, atrophy of white adipose tissue (WAT) and skeletal muscle. Epidemiological data show that 60–80% of advanced cancer patients are affected by the syndrome, in which 22–40% of deaths are due to CC. During the development of cancer cachexia, a well‐characterized loss of both WAT and skeletal muscle occurs. White‐to‐brown adipose tissue remodeling (browning) in response to CC was demonstrated to take place early during CC stages, before skeletal muscle atrophy. WAT browning is associated with an increased expression of uncoupling protein 1 (UCP1), which leads to increased lipid mobilization and energy expenditure in CC mice. Recently, atorvastatin (ATOR) was proposed to exhibit anti‐cancer properties, including a reduction in adipose tissue loss; nevertheless, few studies have evaluated its effect on survival and WAT browning.

Methods

Male C57BL/6 mice (6–8 week‐old) were subcutaneously inoculated with 300μl (3,5×105) of Lewis Lung Carcinoma (TB) and vehicle‐saline (control). Daily oral treatment with Atorvastatin (Citolar®, Pfizer) (100mg/kg per day) or vehicle was started 1 week before tumor cell injection. Samples of different AT depots were collected at day 27 stored at −80° C (5 to 8 animals per each group). Morphological and immunohistochemistry analyses were obtained by light microscopy, while the gene expression analysis was performed by qPCR. For survival studies, rats received daily oral ATOR or vehicle for up to 27 days. Tumor induction was successful in 100% of the animals.

Results

ATOR treatment reduced WAT browning cachexia‐induced in TB, showing down‐regulation of both gene and protein expression of UCP1 in SCAT (p< 0,01). Subcutaneous adipose tissue mass loss was attenuated in ATOR when compared with TB (25%, p<0.05). In addition, life span increased (29%, P<0.05) as well as treated animals survived for a significantly longer time than TB group. WAT demonstrated a decrease in TNFa, CD68 and CD3 positive cells in the ATOR when compared to with TB.

Conclusion

In the present study we demonstrated that ATOR treatment seems to be efficient in to attenuate classical cachexia marker such as adipose tissue loss followed by an attenuated browning effect induced by cachexia. In addition, ATOR treatment was efficient into increase both life span and survival end point.

Support or Funding Information

Financial Support: 15/19259–0
Examining the Impact of Different Exercise Protocols on PGC‐1α and FNDC5 mRNA Expression in Human Skeletal Muscle
Authors: Bonafiglia Jacob T. , Edgett Brittany A. , Scribbans Trisha D. , Little Jonathan P. , Gurd Brendon J.
Published: November 2023

A recent overexpression model demonstrated that peroxisome proliferator‐activated receptor coactivator 1‐alpha (PGC‐1α) regulates the expression of fibronectin type III domain containing protein 5 (FNDC5); a novel myokine with a potential role in stimulating brown‐fat‐like development in white adipose tissue. Although FNDC5 mRNA expression remains unaltered following an acute bout of endurance exercise (END) and high intensity interval training (HIIT), it is unclear whether FNDC5 mRNA expression increases following aerobic exercise protocols that demonstrate elevations in PGC‐1α mRNA expression. Therefore, the purpose of the present study was to test the hypothesis that aerobic exercise protocols that increase PGC‐1α mRNA expression will concomitantly increase FNDC5 mRNA expression in human skeletal muscle. Skeletal muscle biopsies were obtained from healthy men at rest (PRE) and three hours after one bout of moderate intensity interval exercise (MIIT; n = 10; 11 one‐minute intervals at 73% WR at VO2peak separated with one‐minute rest periods), HIIT (n = 10; 8 one‐minute intervals at 100% WR at VO2peak separated with one‐minute rest periods), or sprint interval training (SIT; n = 14; 8 20‐second intervals at 170% WR at VO2peak separated with 10 second rest periods). All samples and PGC‐1α mRNA expression data were obtained from previously published studies (1,2). PGC‐1α mRNA expression significantly (p < 0.05) increased following MIIT (+393% ± 345%), HIIT (+720% ± 392%), and SIT (+284% ± 140%). However, FNDC5 mRNA expression did not change following acute MIIT (−8% ± 29%, p = 0.3), HIIT (+16% ± 32%, p = 0.3), or SIT (−5% ± 18%, p = 0.3). Furthermore, changes in PGC‐1α and FNDC5 mRNA expression did not significantly correlate following any exercise protocol. This study demonstrates that although PGC‐1α mRNA expression increased following all exercise protocols, FNDC5 mRNA did not change. Therefore, these results suggest that interval exercise protocols that induce increases in PGC‐1α mRNA expression do not increase FNDC5 mRNA expression.

Support or Funding Information

This study was supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) to J.T.B., J. P. L., and B.J.G. B. A. E. and T. D. S. were supported by Ontario Graduate Scholarships. J. P. L. was also supported by a Canadian Institutes of Health Research (CIHR) New Investigator Award.
Muscle contractile activity regulates SDF‐1α/CXCL12 expression in skeletal muscle
Authors: Yamada Mami , Hokazono Chihiro , Tokizawa Ken , Marui Shuri , Iwata Masahiro , Suzuki Katsuhiko , Miura Shinji , Nagashima Kei , Okutsu Mitsuharu
Published: November 2023

Objective

Exercise training induces an increase in capillarity density in skeletal muscle. Chemokines, a family of 8‐ to 10‐kDa chemotactic cytokines, provide signals that regulate cell distribution, differentiation and migration through transmembrane receptors coupled to heterotrimeric GTP‐binding proteins. Among various chemokines, stromal cell‐derived factor‐1 (SDF‐1α/CXCL12) seems to be a key chemokine to control endothelial cell migration. The aim of this study was to examine whether muscle contractile activity regulates CXCL12 expression in skeletal muscle.

Methods

Male C57BL/6 mice were individually housed in cages equipped with a running wheel for 4 weeks. Oxidative soleus, plantaris and gastrocnemius with mixed fiber types, and glycolytic white vastus lateralis muscle were harvested at 24 hours after exercise training periods to measure mRNA expression and protein. Gastrocnemius muscle was harvested from muscle‐specific PGC‐1α transgenic mice (PGC‐1αTg) to measure CXCL12 protein. C2C12 myoblasts and myotubes were cultured for 1, 6 and 24 hours with or without mechanical stretch. Total RNA was isolated at 1 and 6 hours after stretch to measure CXCL12 mRNA expression. Protein was isolated at 24 hours after stretch to measure CXCL12 protein. Medium from cultured C2C12 myoblasts and myotubes were also collected at 24 hours after stretch to measure CXCL12 protein.

Results

CXCL12 mRNA and protein in soleus muscle was higher than white vastus lateralis muscle. Voluntary running resulted in increased CXCL12 mRNA and protein in gastrocnemius muscle. PGC‐1αTg mice expressed higher CXCL12 protein compared to wild‐type littermate. Mechanical stretch increased CXCL12 mRNA but not protein in C2C12 myotube. CXCL12 protein in medium from stretched C2C12 myotube increased by mechanical stretch. CXCL12 mRNA expression and protein in C2C12 myoblast has no effect to mechanical stretch.

Conclusions

Muscle contractile activity regulates CXCL12 expression in skeletal muscle, which may contribute to exercise‐induced angiogenesis in skeletal muscle.

Support or Funding Information

Grant‐in‐Aid for Challenging Exploratory Research (16K13019) and Grant‐in‐Aid for Scientific Research (B) (15H03080) to Okutsu M.
Voluntary Wheel Running in Mice Is Safe During Vaccinia Virus Infection and Does not Impair Vaccine Responses
Authors: Pence Brandt D. , Ryerson Melissa R. , Bravo‐Cruz Ariana G. , Woods Jeffrey A. , Shisler Joanna L.
Published: November 2023

Vaccinia virus (VACV) is a double‐stranded DNA virus of the poxvirus family. Since the eradication of smallpox, VACV is most commonly used as an experimental infection model, but VACV‐based vaccines (either live or attenuated) have received increased attention in previous years as vehicles for inoculation against other agents. Exercise has previously been shown to impair immune responses to viral infections. Although this has primarily been described in intense exercise, to our knowledge no study has examined voluntary wheel running (VWR) in mice, which impacts immune responses differently compared to forced exercise. Therefore, we examined the impact of VWR on infection and antibody responses to several strains of VACV. Male C57Bl/6J mice ran for 8 weeks on wheels or remained sedentary, then were infected with 10^5 PFU of pathogenic VACV strain Western Reserve (WR) intranasally or inoculated with 10^6 PFU of replication‐deficient VACV strain Modified Vaccinia Ankara (MVA) intraperitoneally. Mice receiving WR were followed for morbidity and mortality for 14 days, while mice receiving MVA had blood collected prior to inoculation and at week 1, 2, and 4 post‐inoculation for examination of antibody responses. VWR in mice did not alter mortality or body weight loss due to WR infection, although food intake was significantly reduced in sedentary mice (p=0.05), indicating a potential protective effect of VWR against morbidity. VWR also did not alter anti‐VACV IgG response, although mice with wheel access had non‐significantly increased IgG (p=0.22). In conclusion, VWR is safe prior to and during VACV infection and does not impair vaccine responses to the attenuated MVA strain. VWR may increase immune responses and reduce morbidity, although the present study was underpowered to determine this.

Support or Funding Information

Funded by a grant from the American College of Sports Medicine to BDP.
Heatstroke causes hypotension by decreasing both mechanical efficiency and arterial elastance in rat
Authors: Chang Ching‐Ping , Lin Mao‐Tsun , Cheng Bor‐Chih
Published: November 2023

We attempted to test the hypothesis that heatstroke causes arterial hypotension by modulating left ventricular performance in rats. Sprague‐Dawley male adult rats, under general anesthesia, were subjected to severe heat loss (43 °C for 70 min) to induce heatstroke. A 1.2F catheter‐tip pressure transducer was inserted into the left ventricle to record hemodynamic in the closed chest with pressure‐volume module data recording and analysis system. At the time point of heatstroke onset, compared with normothermic controls, rats with normal levels of cardiac HSP72 had significantly decreased end‐systolic pressure, decreased end‐diastolic pressure, decreased stroke volume, increased end‐systolic volume, decreased cardiac output, decreased ejection fraction, decreased stroke work, decreased arterial elastance, and increased time constant of fall in ventricular pressure by Glantz‐methods. With the overexpression of cardiac HSP72 by mild heat preconditioning (43°C for 30 min), a significant protection against heatstroke‐induced suppressed left ventricular performance was observed. Heatstroke causes hypotension by decreasing both cardiac mechanical efficiency and arterial elastance, which can be attenuated by the pre‐induction of cardiac HSP72.
Regulation of Erythropoietin Expression in Response to Exercise in Mouse and Human Skeletal Muscle
Authors: Edgett Brittany A. , Farquharson Laura , Romanova Nadya , Brunt Keith R. , Gurd Brendon J. , Simpson Jeremy A.
Published: November 2023

Erythropoietin (EPO) is the master regulator of erythropoiesis and also plays a role in cytoprotection, cell proliferation and heart contractility. In human skeletal muscle, EPO mRNA expression increases in response to acute exercise, however, whether glycolytic or oxidative muscle fibers differentially produce EPO is unknown. Thus, the purpose of this study was to examine EPO mRNA expression in oxidative and glycolytic muscles following acute exercise in mice. Further, we also examined whether training status had an effect on EPO expression. Experiment 1 involved young healthy men (age, 21.4 ± 2.8 years; VO2peak, 47.1 ± 11.8 mL min−1 kg−1) from a previously published study (Edgett et al. 2016) who performed a single bout of exercise at ~55% of peak aerobic work rate for 1 hour. Muscle biopsies were obtained from the vastus lateralis at rest and 3 hours post‐exercise. Experiment 2 involved 8 week‐old male C57/B6 mice that were either exercise‐trained by treadmill running (1 hour/day, 4 days/week) or remained sedentary (untrained) for 5 weeks. Following the intervention, all mice performed an exhaustive exercise bout that began at a speed of 12 m/min with a 20‐degree grade; speed was subsequently increased by 1 m/min at 2, 5, 10, 20, 30 and 40 min, or until mice reached exhaustion. Soleus (oxidative muscle) and extensor digitorum longus (EDL, glycolytic muscle) were harvested at 1, 2 and 4 hours post‐exercise. All samples were analyzed by real‐time qPCR for changes in EPO mRNA expression. In agreement with a previous report, EPO mRNA expression increased 14 ± 5% in human skeletal muscle 3 hours post‐exercise. In experiment 2, EPO expression increased 4.1 ± 2.8 and 7.4 ± 1.7 fold at 1 hour post‐exercise in the soleus muscle of untrained and trained mice, respectively, before returning towards baseline. In EDL, post‐exercise EPO expression increased 4.1 ± 1.0 fold by 2 hours and plateaued thereafter in only untrained mice; this response was blunted in trained mice. Here we show that EPO expression increased post‐exercise in both glycolytic and oxidative murine skeletal muscles; training had a fiber‐type‐specific differential effect in these muscles. Future studies should determine the physiological significance of skeletal muscle‐derived EPO.

Support or Funding Information

This study was supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) to B.A.E., K.R.B., B.J.G. and J.A.S.
A role for focal adhesion kinase in the contraction of murine gastric fundus smooth muscle evoked by cholinergic neurotransmission
Authors: Xie Yeming , Perrino Brian
Published: November 2023

Cholinergic neurotransmission‐induced smooth muscle contraction involves the regulation of myosin light chain (LC20) phosphorylation and dephosphorylation by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). C‐kinase potentiated protein phosphatase‐1 inhibitor protein of 17 kDa (CPI‐17), and myosin phosphatase target subunit 1 (MYPT1) are crucial for gastrointestinal smooth muscle contractile regulation via their inhibition of MLCP activity. Besides these mechanisms, integrin‐mediated cell‐matrix adhesions connect the cytoskeleton with the extracellular matrix to facilitate cohesion and to generate tension throughout the whole muscle in response cholinergic neurotransmission. Cell‐matrix adhesions and integrin signaling involve the dynamic recruitment and assembly of several proteins, primarily focal adhesion kinase (FAK), at the adhesive site. FAK is a tyrosine kinase whose phosphorylation state and activity is tightly linked with cell adhesion to the extracellular matrix through integrin receptors. FAK participates in transmitting the force generated by the activation of contractile proteins in smooth muscle cells (SMC), to the extracellular matrix filaments and throughout the smooth muscle tissue.

Here, we address the hypothesis that FAK activation is involved in the contraction of gastric fundus smooth muscle in response to cholinergic stimulation. FAK (not Pyk2) was identified as a major tyrosine‐phosphorylated protein in response to electric field stimulation (EFS). EFS in the presence of L‐NNA and MRS2500 contracted gastric fundus smooth muscle strips and significantly increased FAK phosphorylation at Tyr397. In addition, EFS increased the association of FAK with Beta‐1 integrin. The FAK inhibitor PF‐431396 inhibited the EFS‐stimulated contractions, the increase in FAK Tyr397 phosphorylation, and the association of FAK with Beta‐1 integrin. PF‐431396 also blocked the EFS‐induced increase in MYPT1 Thr853, CPI‐17 Thr38, and LC20 Ser19 phosphorylation. The muscarinic acetylcholine receptor antagonist atropine, but not the L‐type Ca2+ channel blocker nicardipine, prevented the EFS‐induced increase in FAK Tyr397 phosphorylation. However, FAK Tyr397 phosphorylation was increased during high K+‐induced contraction, and both were inhibited by PF‐431396. Our findings demonstrate that FAK Tyr397 phosphorylation and association with integrin receptors in SMC plays a role in the contractile response of gastric fundus smooth muscles to cholinergic transmission.

Support or Funding Information

This project was supported by a Takeda Innovation Center Grant, Takeda Pharmaceuticals, Takeda California, San Diego, CA 92121
S. aureus induction of Hif1α drives airway epithelial inflammatory signaling
Authors: Jurado Andrea , Wickersham Matthew , Lung Tania Wong Fok , Miller Alex , Parker Dane , Prince Alice
Published: November 2023

RATIONALE

Staphylococcus aureus is a major cause of healthcare associated pneumonia and leads to high morbidity and mortality rates. Immune cells depend on glycolysis, particularly through Hif1α signaling, to drive pro‐inflammatory defenses to clear bacterial infections. We postulated that airway epithelial cells similarly use Hif1α stabilization and glycolysis to defend against S. aureus infection.

METHODS

A proteomic analysis of bronchoalveolar lavage fluid (BALF) from S. aureus USA300 infected C57BL/6 mice was performed comparing infected and uninfected mice. The metabolic and immunological consequences of Hif1a signaling were demonstrated in vitro. Human bronchial epithelial (16HBE) cells were studied through qRT arrays, western blots and ELISAs.

RESULTS

S. aureus wild type USA300 (WT) were found to activate the canonical Hif1a and metabolic pathways that support glycolysis, as defined by the Ingenuity Pathway Analysis (IPA) of the secreted proteins in the BALF of infected mice. These results were verified in immunoblots demonstrating that S. aureus WT and toxin‐deficient agr mutants both stabilize Hif1α in 16HBE cells, despite significantly increased intracellular accumulation of the agr strain. Increased Hif1α corresponded with increased IL‐1b, IL‐8 and phosphofructokinase 1 (PFK1) transcript levels under infected conditions. The induction of HIF1a signaling, as a consequence of the metabolic stress caused by infection, was dependent on the glycolytic capacities of the bacteria, as a glycolytic mutant of S. aureus lacking pyruvate kinase (pyk) failed to induce Hif1α or secrete pro‐inflammatory cytokines IL‐1β and IL‐18. This was in contrast to WT and phosphoenolpyruvate kinase (pckA) deficient strains, with impaired gluconeogenesis.

CONCLUSIONS

The induction of epithelial glycolysis contributes to the metabolic and inflammatory response to S. aureus pulmonary infection. Both the organisms and the host use glycolysis to meet their increased metabolic requirements, promoting competition for glucose and stimulating Hif1a signaling. Our data highlights that metabolic stress is a driving factor in the host response to S. aureus in the lung, suggesting that manipulating metabolism might be a novel approach in the prevention and treatment of bacterial pneumonia.

Support or Funding Information

FUNDING: NIH R35 HL135300
Rupture of Cancer Cells Under Microcirculatory Conditions
Authors: Kamyabi Nabiollah , Vanapalli Siva
Published: November 2023

Survival of circulating tumor cells (CTCs) in blood circulation is essential for cancer metastasis. However, fluid forces in microcirculation have been shown to cause cell rupture, lending to the notion that such mechanical trauma of CTCs is responsible for metastatic inefficiency. Literature evidence for tumor cell death due to mechanical damage remains ambiguous and moreover the physical mechanisms of cell rupture are poorly understood. Here, we develop a microfluidic capillary model of microcirculation that contains an array of flow bifurcations and investigate rupture of tumor cells. We find that when a cancer cell arrives at a narrow bifurcation, it is temporarily arrested, followed by strong cellular deformation causing a cytosol‐filled membrane‐bound vesicle to be pinched‐off. The vesicle size was found to increase with the size of tumor cell suggesting that the cytosol content is larger for bigger cells. To identify the essential features of the mechanism underlying the rupture process, we used a library of drugs that (i) either enhance or inhibit the polymerization of actin and microtubules (ii) inhibit myosin II and decrease cortical tension, and (iii) condense and decondense nuclear chromatin. Results from these studies show that the cytoskeletal elements and membrane play an important role in regulating the time‐scale for rupture of tumor cells, but not the nucleus. The vesicle size was not sensitive to any of the drug treatments indicating again that the cytosol content determines the vesicle volume. Collectively, the drug studies support the poroelastic model of a cell, where under mechanical stress, the cytosol flows through the elastic cytoskeleton and fills the membrane that is detached from the cortex. Experiments with different cancer cell lines showed that highly metastatic cells take longer to rupture suggesting that their cytoskeleton and membrane properties are different than weakly metastatic cell lines. Finally, we find that less than 5% of the fragmented tumor cells undergo apoptosis, suggesting that CTCs can survive the impact of fluid forces in microcirculation and that mechanical trauma is not a significant cause for metastatic inefficiency.

Support or Funding Information

Cancer Prevention and Research Institute of Texas (Grant No. RP 140298)
KLF15 is modulated by leucine in skeletal muscle submitted to immobilization in rats
Authors: Zuanazzi Flávia Aparecida Graça , Bento Mirella , Silvestre João Guilherme , Moriscot Anselmo Sigari
Published: November 2023

Leucine is a branched chain amino acid known not only as a component of proteins, but also as a pharmacological agent, which promotes important anti‐catabolic effects, mitigating muscle atrophy. Previous studies from our laboratory demonstrated that daily administration of leucine was able to attenuate the weight loss in soleus muscle and cross‐sectional area after 7 days unilateral immobilization of the left hind limb. This protective effect of leucine was correlated with reduced gene expression of key components of the Ub‐proteasome proteolytic system atrogin‐1 and MuRF1. The understanding of regulatory factors involved in the anticatabolic effects of leucine is of great interest. Accordingly, the Krüppel like factor 15 (KLF15) is a recently discovered transcriptional factor that is involved in skeletal muscle atrophy at least in part by upregulating atrogin‐1 and MuRF1 gene expression. In this study, our hypothesis contemplates that KLF15 is involved in the anti‐catabolic effects of leucine.

The protocols used in this study submitted and approved by Institute of Biomedical Sciences/University of São Paulo – Ethical Committee for Animal Research (#99/2015). Male Wistar rats (n=6 per group), 2 months old, weighing 260g, were divided into four groups: control (untreated animal), leucine supplemented only, immobilized (left hind limb immobilization), immobilized and leucine supplemented. Leucine was orally administered (via gavage) once a day in a dose of 2.7 g/kg body mass per days for 7 days. Monolateral hind limb immobilization was performed in the left hind limb by applying a cast with total plantar flexion and knee extension. Rats were killed after 7 days and soleus muscle removed. Protein and mRNA expression were analyzed by Western Blotting and RT‐PCR, respectively. Intracellular localization of KLF15 was determined by immunofluorescent analysis.

Immobilization for 7 days resulted in an increased in protein (50%) and mRNA (100%) levels of KLF15 in soleus muscle from rats, nonetheless not altered by leucine supplementation. Interestingly, immobilization for 7 days enhanced nuclear localization of KLF15, which was reduced by leucine supplementation in soleus muscle from immobilized group.

These results suggest that the anti‐catabolic effects of leucine involve blocking KLF15 action.

Support or Funding Information

Supported by Postdoctoral fellowship PNPD‐CAPES e FAPESP 2015/04090‐0.
The Mechanism of Exercise Preventing Skeletal Muscle Atrophy
Authors: Theilen Nicholas , Kunkel George , Jeremic Nevena , Tyagi Suresh
Published: November 2023

Background

Skeletal muscle atrophy is the consequence of protein degradation exceeding protein synthesis. This arises for a multitude of reasons including the unloading of muscle during microgravity, post‐surgery bedrest, immobilization of a limb after injury, and overall disuse of the musculature. Mitochondrial dysfunction is associated with skeletal muscle atrophy and contributes to the induction of protein degradation and cell apoptosis with the loss of organelle function. The development of therapies to enhance mitochondrial function prior to skeletal muscle atrophy settings to diminish protein degradation is scarce.

Objective

We are investigating whether an exercise treatment prior to an atrophic setting will diminish the skeletal muscle atrophy incurred during hindlimb suspension (HLS) in mice.

Methods

Male C57BL/6 (wild‐type) mice between 11–14 weeks of age were assigned to control, HLS only (HLS‐only), exercise only (Ex‐only), and exercise prior to HLS (Ex+HLS), groups. The exercise protocol consisted of 14 progressive exercise sessions over an 18‐day period and the HLS protocol lasted 7 consecutive days. At the end of treatments mice were first anesthetized and blood flow imaging of the hindlimb was done using laser Doppler technology. Mice were then sacrificed and the soleus and gastrocnemius muscle were excised and analyzed for muscle weight. Immunohistochemistry staining was done to detect fiber cross‐sectional area. Future measurements will assess concentrations of mitochondrial function proteins in the excised muscle tissue through western blotting techniques.

Results

HLS‐only resulted in reductions in soleus and gastrocnemius muscle weight of 33.6% and 25.4%, respectively, while exercising prior to HLS led to a decrease in soleus and gastrocnemius weight of 5.1% and 10.8%, respectively, compared to controls. A standardization measurement of soleus muscle weight (mg) to bodyweight (g) ratio decreased 32.7% (.218±.018) in HLS‐only while decreasing 8.1% (.298±.004) in Ex+HLS. Immunostaining revealed greater cross‐sectional area, continuity, and uniformity in Ex+HLS soleus and gastrocnemius muscle fibers compared to HLS‐only. Laser Doppler imaging suggests blood flow in the lower hindlimbs is greater in Ex+HLS mice compared to the HLS only group.

Conclusions

Data indicates the time before an atrophic setting, particularly caused by muscle unloading and disuse, is a useful period to intervene progressive exercise training to prevent skeletal muscle atrophy which we may be associated with enhanced mitochondrial function.

Support or Funding Information

NIH Grant HL74185 to SCT
Anti‐inflammatory Cytokine Receptor Prevents Muscle Atrophy
Authors: Kwon Young‐Won , Son Young Hoon , Park Sun‐Dong , Lee Ju‐Hee
Published: November 2023

Skeletal muscle plays a leading role in the metabolism and concurrently functions as a repository of protein and amino acids. In many serious diseases such as cancer cachexia and sepsis, excessive secretion of cytokines exacerbates skeletal muscle wasting and metabolic imbalance. Excessively increased cytokines due to disease and tissue damage are regulated by anti‐inflammatory cytokines. However, the precise mechanisms of anti‐inflammatory cytokine in skeletal muscle atrophy have not been fully elucidated. Interleukin‐10 (IL‐10) is an anti‐inflammatory cytokine that improved insulin resistance in high fat diet and aging. The physiological effects of IL‐10 in skeletal muscle were known, but the study of IL‐10 receptor (IL‐10R) was unknown. Our hypothesis was that IL‐10R regulates muscle atrophy in vitro model. In our study, we found that IL‐10 mRNA levels were significantly elevated in various muscle atrophy conditions including the treatment of H2O2, dexamethasone, and cancer cell conditioned media. This is the regulation for suppressing the pro‐inflammatory cytokine, but the mechanism of IL‐10R is unidentified. In order to confirm the directly role of IL‐10R in skeletal muscle, we used the IL‐10R siRNA in C2C12 myotubes. Knockdown of IL‐10R increased the protein level of Atrogin‐1 by Foxo dependent activation, but MuRF1 level by NF‐κB dependent activation remained unchanged. Increased Atrogin‐1 is associated with the reduction of Akt signaling. Declining Akt activity leads to decrease in overall protein synthesis. Using puromycin incorporation, we proved that suppression of IL‐10R reduced the protein synthesis in myotubes. As a result, inhibition of IL‐10R implies a deficit of protein synthesis due to reducing Akt activation. According to our data, the inhibition of IL‐10R is caused by activation of E3 ligase via inhibition of insulin signaling. Our findings suggest that activation of IL‐10R may exert the potential therapeutic effect of skeletal muscle atrophy.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (No.2015R1D1A1A01060340).
Effect of glycosylation of cyclooxygenase‐2 (COX‐2) on homodimerization
Authors: Vann‐Victorino Daniel DeWayne Colbert , Cunanan Jelaine , Chen Mindy , Chan Rena , Hall Randall W. , Sevigny Mary B.
Published: November 2023

Prostaglandins are a group of fatty acid cell signals made from arachidonic acid. The prostaglandins have many roles in the body including inflammation response, contraction and relaxation of smooth muscle cells, vasodilation, and platelet aggregation. Cyclooxygenase‐2 (COX‐2) catalyzes two steps in the prostaglandin pathway, wherein arachidonic acid is converted into prostaglandin G2 which is then converted into prostaglandin H2. Though COX‐2 is best known for its role in the inflammatory response, overexpression of COX‐2 plays a role in various diseases such as arthritis and certain cancers. COX‐2 exists as two glycoforms—72 and 74kDa— due to a variable glycosylation site at Asn580. Our lab has previously reported that this variable glycosylation affects the turnover rate of the COX‐2 protein and reduces the efficacy of several COX‐2 inhibitors. In this current study, we wish to determine the effect of this glycosylation on COX‐2 dimerization, since COX‐2 activity depends on dimerization. The molecular dynamics computer program NAMD was used to determine the difference in dimerization free energies between the 74kDa/74kDa COX‐2, the 72kDa/74kDa COX‐2, and the 72kDa/72kDa COX‐2 dimers to elucidate the influence of variable glycosylation on dimerization. The results indicate that the Gibbs free energy of dimerization for the 72kDa/74kDa dimer is more negative than the corresponding Gibbs free energy of dimerization for the 74kDa/74kDa dimer. These data indicate that dimerization between the 72kDa and the 74kDa COX‐2 monomers is more energetically favorable than dimerization between two 74kDa monomers. Further computational studies analyzing the DG of the 72kDa/72kDa dimer are currently being carried out, and future studies will include immunoprecipitation experiments to confirm these computer simulation results.
Impact of Metaboreflex Activation on Cerebrovascular Reactivity Index in Healthy Young Men
Authors: Maia‐Lopes Paulo Marcelo , Teixeira André L , Antonino Diego , Souza Mayara C , Ricardo Djalma R , Ramos Plinio S , Vianna Lauro C
Published: November 2023

Background

The control of cerebral perfusion during exercise appears to be highly complex and remains incompletely understood. During isometric exercise, activation of metabolically sensitive skeletal muscle afferents (muscle metaboreflex) markedly increases mean arterial pressure (MAP), and MAP is sustained when the ischemia is maintained following the cessation of exercise (post‐exercise muscle ischemia, PEI). Stimulation of muscle metaboreflex has been implicated in the brain blood flow responses to exercise, but this remains unclear.

Aim

To determine the influence of muscle metaboreflex activation on pressure reactivity index (PRx) and intracranial pressure (ICP) in healthy young men.

Methods

Fifteen healthy young men (24 ± 5 years, mean ± SD) randomly performed two protocols. In the experimental protocol the muscle metaboreflex was activated during 3‐min period via PEI, whereas during control protocol the muscle metaboreflex was not activated. Measurements were made at baseline and following isometric handgrip exercise (IHG) with or without PEI for experimental and control protocols, respectively. Participants completed a single bout of IHG at 40% of maximum voluntary contraction for 90 seconds. Heart rate (HR) and arterial BP was monitored on beat‐to‐beat basis using finger photoplethysmography (Human NIBP, AD instruments). In addition, ICP was monitored noninvasively via BrainCare Research 1.5 (BrainCare Health Technology, São Carlos, Brazil). Briefly, small deformations in the surface of the cranium caused by beat‐to‐beat ICP oscillations was captured by strain gauge type sensor (SR‐4) who was placed in contact to the surface of head and connected to personal monitor. PRx is determined by calculating the correlation coefficient between 40 consecutive, time averaged data points of ICP and MAP.

Results

Baseline physiological variables were similar between protocols (P>0.05). During PEI, however, MAP significantly increased from rest (Δ28±3 mmHg, P<0.01) whereas mean ICP, HR (69 ± 3 bpm, P>0.05), and PRx (0.19 ± 0.08, P>0.05) were unchanged. During control protocol, MAP (Δ2±1 mmHg, P>0.05) returned to baseline values following IHG exercise and mean ICP, HR and PRx were unchanged from rest.

Conclusion

Despite the robust and sustained elevation in systemic blood pressure during selective activation of metabolically sensitive skeletal muscle afferent fibers, the PRx is preserved, indicating that the cerebrovascular is normally reactive.

Support or Funding Information

CAPES, FAPDF, CNPq.
Cholinergic signaling in the forebrain regulates peripheral inflammation
Authors: Pavlov Valentin A. , Lehner Kurt R. , Chavan Sangeeta S. , Prado Vania F. , Prado Marco A.M. , Tracey Kevin J.
Published: November 2023

The brain regulates physiological functions integral to survival. Relatively little is known about the brain neuronal regulation of peripheral immune function. Brain cholinergic signals have been implicated in controlling peripheral inflammation, but specific insight is lacking. Here, utilizing neuron‐and receptor‐specific approaches, we studied the role of forebrain cholinergic signaling and the M1 muscarinic acetylcholine receptor (M1 mAChR) in the regulation of peripheral inflammation. Forebrain‐specific Cre‐loxP‐based ablation of the vesicular acetylcholine transporter (required for synaptic acetylcholine release) and vagotomy abolished the suppression of serum TNF by the centrally‐acting cholinergic drug galantamine in murine endotoxemia. Basal forebrain cholinergic neurons innervate areas with abundant M1 mAChR localization. Enhancement of acetylcholine action on the brain M1 mAChR with the selective positive allosteric modulator benzyl quinolone carboxylic acid (BQCA) suppressed serum TNF levels in endotoxemia. Peripheral administration of the centrally‐acting BQCA also suppressed TNF levels and improved survival in endotoxemia, effects abolished in M1 mAChR KO mice. Optogenetic stimulation of basal forebrain cholinergic neurons in the medial septum reduced serum TNF in endotoxemic mice as compared to sham stimulation. These findings provide novel insights into brain regulation of peripheral inflammation and are of interest for developing new brain‐based treatments for inflammatory conditions.

Support or Funding Information

This work was supported by the following grants from the National Institute of General Medical Sciences, NIH: RO1GM089807 (to VAP and KJT) and RO1GM057226 (to KJT).