Protein Phosphorylation Levels Research Articles

Methylglyoxal reduces resistance exercise-induced protein synthesis and anabolic signaling in rat tibialis anterior muscle.

Resistance exercise provides significant benefits to skeletal muscle, including hypertrophy and metabolic enhancements, supporting overall health and disease management. However, skeletal muscle responsiveness to resistance exercise is significantly reduced in conditions such as aging and diabetes. Recent reports suggest that glycation stress contributes to muscle atrophy and impaired exercise-induced muscle adaptation; however, its role in the muscle response to resistance exercise remains unclear. Therefore, in this study, we investigated whether methylglyoxal (MGO), a key factor in glycation stress, affects the acute responsiveness of skeletal muscles to resistance exercise, focusing on protein synthesis and the key signaling molecules. This study included 12 8-week-old male Sprague-Dawley rats divided into two groups: one received 0.5% MGO-supplemented drinking water (MGO group) and the other received regular water (control group). After 10 weeks, the left tibialis anterior muscle of each rat was subjected to electrical stimulation (ES) to mimic resistance exercise, with the right muscle serving as a non-stimulated control. Muscle protein-synthesis rates were evaluated with SUnSET, and phosphorylation levels of key signaling molecules (p70S6K and S6rp) were quantified using western blotting. In the control group, stimulated muscles exhibited significantly increased muscle protein synthesis and phosphorylation levels of p70S6K and S6rp. In the MGO group, these increases were attenuated, indicating that MGO treatment suppresses the adaptive response to resistance exercise. MGO diminishes the skeletal muscle's adaptive response to ES-simulated resistance exercise, affecting both muscle protein synthesis and key signaling molecules. The potential influence of glycation stress on the effectiveness of resistance exercise or ES emphasizes the need for individualized interventions in conditions of elevated glycation stress, such as diabetes and aging.

Silibinin Induces Both Apoptosis and Necroptosis with Potential Anti-tumor Efficacy in Lung Cancer.

Lung cancer incidence is steadily on the rise, posing a growing threat to human health. The search for therapeutic drugs from natural active substance and elucidating their mechanism have been the focus of anti-tumor research. In our work, Silibinin (SiL) was chosen as a possible substance that could inhibit lung cancer. and its effects on inducing tumor cell death have been studied. CCK-8 analysis and morphological observation were used to assess the cytotoxic impacts of SiL on lung cancer cells in vitro. The alterations in mitochondrial membrane potential (MMP) and apoptosis rate of cells were detected by flow cytometry. The level of lactate dehydrogenase (LDH) release out of cells was measured. The expression changes of apoptosis or necroptosis-related proteins were detected using western blotting. Protein interactions among RIPK1, RIPK3 and MLKL were analyzed using the co-immunoprecipitation technique. In vivo, SiL was evaluated for its antitumor effects using LLC tumor-bearing mice with mouse lung cancer. With an increased dose of SiL, the proliferation ability of A549 cells was considerably inhibited, and the accompanying cell morphology changed. The results of flow cytometry showed that after SiL treatment, MMP levels decreased, and the proportion of cells undergoing apoptosis increased. The proteins associated with apoptosis were upregulated and activated. The amount of LDH released from the cells increased following SiL treatment, accompanied by augmented expression and phosphorylation levels of necroptosis-related proteins. The co-IP assay further confirmed necrosome formation induced by SiL. Furthermore, Necrosulfonamide (an MLKL inhibitor) increased the apoptotic rate of SiL-treated cells and aggravated the cytotoxic effect of SiL, indicating that necroptosis blockade could switch cell death to apoptosis and increase the inhibitory effect of SiL on A549 cells. In LLC-bearing mice, gastric administration of SiL significantly inhibited tumor growth. This study helped clarify the anti-tumor mechanism of SiL against lung cancer, elucidating its role in dual induction of apoptosis and necroptosis. In particular, necroptosis blockade could switch cell death to apoptosis and increase the inhibitory effect of SiL. Our work provided an experimental basis for the research on cell death induced by SiL and revealed its possible applications for improving the management of lung cancer.</p>.

Pre-IVM with C-type natriuretic peptide promotes mitochondrial biogenesis of bovine oocytes via activation of CREB

The aim of this study was to evaluate the effects of C-type natriuretic peptide (CNP) treatment prior to in vitro maturation (IVM) on mitochondria biogenesis in bovine oocyte matured in vitro and explore the related causes. The results showed that treatment with CNP before IVM significantly improved mitochondrial content, elevated the expression of genes related to mitochondria biogenesis, and increased the protein levels of phosphorylation of cAMP-response element binding protein (p-CREB) in bovine oocytes following IVM. However, further studies revealed that treatment with CNP before IVM could not increased the protein levels of p-CREB in bovine oocytes when natriuretic peptide receptor 2 activities was inhibited using the relative specific inhibitor Gö6976. In addition, treatment with CNP before IVM could not improved mitochondrial content or elevated the expression of genes related to mitochondria biogenesis in bovine oocytes when CREB activities was abolished using the specific inhibitor 666–15. In summary, these results provide evidence that treatment of bovine oocytes with CNP before IVM promotes mitochondrial biogenesis in vitro, possibly by activating CREB.

HGF ameliorates cardiomyocyte apoptosis and inflammatory response in sepsis via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway

ObjectiveThis study aimed to analyze the impact of HGF on cardiomyocyte injury, apoptosis, and inflammatory response induced by lipopolysaccharide (LPS). MethodsEnzyme-linked immunosorbent assay (ELISA) was utilized to quantify the levels of HGF, interleukin (IL)-6, IL-10, creatine phosphokinase-isoenzyme-MB (CK-MB), and cardiac troponin I (cTnI) in the samples. qPCR and Western blotting (WB) were employed to assess the mRNA and protein expressions of HGF, IL-10, IL-6, PI3K, AKT, p-PI3K, and p-AKT. ResultsThe outcomes of the in vivo experiment revealed that serum levels of IL-6, IL-10, HGF and SOFA scores in the SC group were elevated in contrast to the non-SC group. The correlation analysis indicated a substantial and positive association among serum HGF, IL-6, and IL-10 levels and SOFA scores. Relative to IL-6, IL-10 levels, and SOFA scores, serum HGF demonstrated the highest diagnostic value for SC. Following LPS administration to stimulate H9c2 cells across various periods (0, 12, 24, 48, and 72 h), the levels of myocardial injury markers (CK-MB and cTnI) in the cell supernatants, intracellular inflammatory factors (mRNA and protein levels of IL-10 and IL-6), apoptosis and ROS levels, exhibited a gradual increase followed by a subsequent decline. Following the overexpression of HGF, there was an increase in cell viability, and a decrease in apoptosis, inflammation, oxidative stress injuries, and the protein phosphorylation expressions of PI3K and AKT. After knockdown of HGF expression, the activity of LPS-induced H9c2 cells was further reduced, leading to increased cell injury, apoptosis, inflammation, oxidative stress,and the expression levels of PI3K and Akt protein phosphorylation were further elevated. ConclusionHGF was associated with decreased LPS-induced H9c2 apoptosis and inflammation in H9c2 cells, alongside an improvement in cell viability, indicating potential cytoprotective effects. The mechanism underlying these impacts may be ascribed to the suppression of the PI3K/AKT signaling pathway.

Exploring allocryptopine as a neuroprotective agent against oxidative stress-induced neural apoptosis via Akt/GSK-3β/tau pathway modulation

Alzheimer’s disease (AD) is characterized by neuronal loss due to hyperphosphorylated proteins induced by oxidative stress. AD remains a formidable challenge in the medical field, as current treatments focusing on single biomarkers have yielded limited success. Hence, there’s a burgeoning interest in investigating novel compounds that can target mechanisms, offering alternative therapeutic approaches. The aim of this study is to investigate the effects of allocryptopine, an isoquinoline alkaloid, on mechanisms related to AD in order to develop alternative treatment strategies. In this study, the in vitro AD cell model was obtained by inducing nerve growth factor (NGF)-differentiated PC12 (dPC12) cells to oxidative stress with H2O2, and also the effect mechanism of different allocryptopine concentrations on the in vitro AD cell model was studied. The treatments’ antioxidative effects at the ROS level and their regulation of the cell cycle were assessed through flow cytometry, while their anti-apoptotic effects were evaluated using both flow cytometry and qRT-PCR. Additionally, the phosphorylation levels of Akt, GSK-3β, and tau proteins were analyzed via western blot, and the interactions between Akt, GSK-3β, CDK5 proteins, and allocryptopine were demonstrated through molecular docking. Our study’s conclusive results revealed that allocryptopine effectively suppressed intracellular ROS levels, while simultaneously enhancing the Akt/GSK-3β signaling pathway by increasing p-Akt and p-GSK-3β proteins. This mechanism played a critical role in inhibiting neural cell apoptosis and preventing tau hyperphosphorylation. Moreover, allocryptopine demonstrated its ability to regulate the G1/S cell cycle progression, leading to cell cycle arrest in the G1 phase, and facilitating cellular repair mechanisms, potentially contributing to the suppression of neural apoptosis. The in silico results of allocryptopine were shown to docking with the cyclin-dependent kinase 5 (CDK 5) playing a role in tau phosphorylation Akt and GSK-3β from target proteins. Therefore, the in silico study results supported the in vitro results. The results showed that allocryptopine can protect dPC12 cells from oxidative stress-induced apoptosis and hyperphosphorylation of the tau protein by regulating the Akt/GSK-3β signaling pathway. Based on these findings, it can be suggested that allocryptopine, with its ability to target biomarkers and its significant effects on AD-associated mechanisms, holds promise as a potential candidate for drug development in the treatment of AD. Further research and clinical trials are recommended in the future.

Exploring Allocryptopine as a Neuroprotective Agent Against Oxidative Stress-Induced Neural Apoptosis via Akt/GSK-3β/Tau Pathway Modulation

Alzheimer’s disease (AD) is characterized by neuronal loss due to hyperphosphorylated proteins induced by oxidative stress. AD remains a formidable challenge in the medical field, as current treatments focusing on single biomarkers have yielded limited success. Hence, there’s a burgeoning interest in investigating novel compounds that can target mechanisms, offering alternative therapeutic approaches. The aim of this study is to investigate the effects of allocryptopine, an isoquinoline alkaloid, on mechanisms related to AD in order to develop alternative treatment strategies. In this study, the in vitro AD cell model was obtained by inducing nerve growth factor (NGF)-differentiated PC12 (dPC12) cells to oxidative stress with H2O2, and also the effect mechanism of different allocryptopine concentrations on the in vitro AD cell model was studied. The treatments’ antioxidative effects at the ROS level and their regulation of the cell cycle were assessed through flow cytometry, while their anti-apoptotic effects were evaluated using both flow cytometry and qRT-PCR. Additionally, the phosphorylation levels of Akt, GSK-3β, and tau proteins were analyzed via western blot, and the interactions between Akt, GSK-3β, CDK5 proteins, and allocryptopine were demonstrated through molecular docking. Our study’s conclusive results revealed that allocryptopine effectively suppressed intracellular ROS levels, while simultaneously enhancing the Akt/GSK-3β signaling pathway by increasing p-Akt and p-GSK-3β proteins. This mechanism played a critical role in inhibiting neural cell apoptosis and preventing tau hyperphosphorylation. Moreover, allocryptopine demonstrated its ability to regulate the G1/S cell cycle progression, leading to cell cycle arrest in the G1 phase, and facilitating cellular repair mechanisms, potentially contributing to the suppression of neural apoptosis. The in silico results of allocryptopine were shown to docking with the cyclin-dependent kinase-5 (CDK 5) playing a role in tau phosphorylation Akt and GSK-3β from target proteins. Therefore, the in silico study results supported the in vitro results. The results showed that allocryptopine can protect dPC12 cells from oxidative stress-induced apoptosis and hyperphosphorylation of the tau protein by regulating the Akt/GSK-3β signaling pathway. Based on these findings, it can be suggested that allocryptopine, with its ability to target biomarkers and its significant effects on AD-associated mechanisms, holds promise as a potential candidate for drug development in the treatment of AD. Further research and clinical trials are recommended in the future.

Hyperglycemia impairs EAAT2 glutamate transporter trafficking and glutamate clearance in islets of Langerhans: implications for type 2 diabetes pathogenesis and treatment.

Pancreatic endocrine cells employ a sophisticated system of paracrine and autocrine signals to synchronize their activities, including glutamate, which controls hormone release and β-cell viability by acting on glutamate receptors expressed by endocrine cells. We here investigate whether alteration of the excitatory amino acid transporter 2 (EAAT2), the major glutamate clearance system in the islet, may occur in type 2 diabetes mellitus and contribute to β-cell dysfunction. Increased EAAT2 intracellular localization was evident in islets of Langerhans from T2DM subjects as compared with healthy control subjects, despite similar expression levels. Chronic treatment of islets from healthy donors with high-glucose concentrations led to the transporter internalization in vesicular compartments and reduced [H3]-d-glutamate uptake (65 ± 5% inhibition), phenocopying the findings in T2DM pancreatic sections. The transporter relocalization was associated with decreased Akt phosphorylation protein levels, suggesting an involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the process. In line with this, PI3K inhibition by a 100-µM LY294002 treatment in human and clonal β-cells caused the transporter relocalization in intracellular compartments and significantly reduced the glutamate uptake compared to control conditions, suggesting that hyperglycemia changes the trafficking of the transporter to the plasma membrane. Upregulation of the glutamate transporter upon treatment with the antibiotic ceftriaxone rescued hyperglycemia-induced β-cells dysfunction and death. Our data underscore the significance of EAAT2 in regulating islet physiology and provide a rationale for potential therapeutic targeting of this transporter to preserve β-cell survival and function in diabetes.NEW & NOTEWORTHY The glutamate transporter SLC1A2/excitatory amino acid transporter 2 (EAAT2) is expressed on the plasma membrane of pancreatic β-cells and controls islet glutamate clearance and β-cells survival. We found that the EAAT2 membrane expression is lost in the islets of Langerhans from type 2 diabetes mellitus (T2DM) patients due to hyperglycemia-induced downregulation of the phosphoinositide 3-kinase/Akt pathway and modification of its intracellular trafficking. Pharmacological rescue of EAAT2 expression prevents β-cell dysfunction and death, suggesting EAAT2 as a new potential target of intervention in T2DM.

Radioprotective effect of polyvinylpyrrolidone modified selenium nanoparticles and its antioxidation mechanism in vitro and in vivo.

Polyvinylpyrrolidone (PVP) is a commonly used biomedical polymer material with good water solubility, biocompatibility, low immunogenicity, and low toxicity. The aim of this study is to investigate the antioxidant mechanism and clinical potential of PVP modified selenium nanoparticles (PVP-Se NPs) as a new radioprotective agent. A laser particle size analyzer and transmission electron microscope were used to characterize PVP-Se nanoparticles prepared by chemical reduction. Human umbilical vein endothelial cells (HUVECs) were used to evaluate the radiation protective effects of PVP-Se NPs. SD rats were employed as an in vivo model to identify the most effective concentration of PVP-Se NPs and assess their potential radioprotective properties. Western blot (WB) was used to detect the expression of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling proteins in human umbilical vein endothelial cells (HUVECs) and rat liver and kidney tissues. PVP-Se NPs could reduce the oxidative stress injury and inflammatory response caused by X-ray irradiation in HUVECs and rats, and inhibit cell apoptosis by modulating NF-κB and MAPK signaling pathways. PVP-Se NPs could increase HUVECs viability, reduce apoptosis, inhibit inflammatory factors IL-1β, IL-6 and TNF-α, improve the survival rate of rats, promote antioxidant enzyme activities in cells and rats, reduce malondialdehyde concentration in serum, and reduce the expression of inflammatory factors such as IL-1β, IL-6 and TNF-α in cell supernatant and liver and kidney tissues. PVP-Se NPs could significantly reduce the phosphorylation levels of NF-κB and MAPK pathway-associated proteins in HUVECs and rat liver and kidney tissues (p < 0.05). PVP-Se NPs can protect against radiation-induced oxidative damage by modulating NF-kB and MAPK pathways, providing a theoretical basis and experimental data for their use as an effective radioprotective agent.

Cinnamaldehyde inhibits the NLRP3 inflammasome by preserving mitochondrial integrity and augmenting autophagy in Shigella sonnei-infected macrophages

BackgroundWorldwide, more than 125 million people are infected with Shigella each year and develop shigellosis. In our previous study, we provided evidence that Shigella sonnei infection triggers activation of the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome in macrophages. NLRP3 inflammasome is responsible for regulating the release of the proinflammatory cytokines interleukin (IL)-1β and IL-18 through the protease caspase-1. Researchers and biotech companies have shown great interest in developing inhibitors of the NLRP3 inflammasome, recognizing it as a promising therapeutic target for several diseases. The leaves of Cinnamomum osmophloeum kaneh, an indigenous tree species in Taiwan, are rich in cinnamaldehyde (CA), a compound present in significant amounts. Our aim is to investigate how CA affects the activation of the NLRP3 inflammasome in S. sonnei-infected macrophages.MethodsMacrophages were infected with S. sonnei, with or without CA. ELISA and Western blotting were employed to detect protein expression or phosphorylation levels. Flow cytometry was utilized to assess H2O2 production and mitochondrial damage. Fluorescent microscopy was used to detect cathepsin B activity and mitochondrial ROS production. Additionally, colony-forming units were employed to measure macrophage phagocytosis and bactericidal activity.ResultsCA inhibited the NLRP3 inflammasome in S. sonnei-infected macrophages by suppressing caspase-1 activation and reducing IL-1β and IL-18 expression. CA also inhibited pyroptosis by decreasing caspase-11 and Gasdermin D activation. Mechanistically, CA reduced lysosomal damage and enhanced autophagy, while leaving mitochondrial damage, mitogen-activated protein kinase phosphorylation, and NF-κB activation unaffected. Furthermore, CA significantly boosted phagocytosis and the bactericidal activity of macrophages against S. sonnei, while reducing secretion of IL-6 and tumour necrosis factor following infection.ConclusionCA shows promise as a nutraceutical for mitigating S. sonnei infection by diminishing inflammation and enhancing phagocytosis and the bactericidal activity of macrophages against S. sonnei.

Effects of Different Na+ Concentrations on cAMP-Dependent Protein Kinase Activity in Postmortem Meat.

cAMP-dependent protein kinase (PKA) activity regulates protein phosphorylation, with Na+ playing a crucial role in PKA activity. The aim of this study was to investigate the effects of different Na+ concentrations on PKA activity and protein phosphorylation level in postmortem muscle. The study consisted of two experiments: (1) NaCl of 0, 20, 100, 200 and 400 mM was added to a muscle homogenate incubation model to analyze the effect of Na+ concentration on PKA activity, and (2) the same concentrations were added to pure PKA in vitro incubation models at 4 °C to verify the effect of Na+ on PKA activity. The PKA activity of the muscle homogenate model increased with storage time in groups with different Na+ concentrations. High concentrations of Na+ inhibited sarcoplasmic protein phosphorylation. The PKA activity at 24 h of storage and the sarcoplasmic protein phosphorylation level at 12 h of storage in the group with 200 mM Na+ was lower than that of the other groups. After 1 h incubation, the PKA activity of samples in the 200 mM Na+ group was inhibited and lower than that in the other Na+ groups in the in vitro incubation model. These results suggest that the Na+ concentration at 200 mM could better inhibit PKA activity. This study provided valuable insights for enhancing curing efficiency and improving meat quality.

Wogonin Inhibits Colorectal Cancer Proliferation and Epithelial Mesenchymal Transformation by Suppressing Phosphorylation in the AKT Pathway.

Colorectal cancer is the third leading cause of cancer-related death worldwide. Hence, there is a need to identify new therapeutic agents to improve the current repertoire of therapeutic drugs. Wogonin, a flavonoid from the herbal medicine Scutellaria baicalensis, has unique antitumor activity. Our study aimed to further explore the inhibitory effects of wogonin on colorectal cancer and its specific mechanism. The results showed that wogonin significantly inhibited the proliferation of colorectal cancer cells as well as their ability to invade and metastasize. We detected phosphorylation of tumor-associated signaling pathways using a phosphorylated protein microarray and found that wogonin intervention significantly inhibited the phosphorylation level of the AKT protein in colorectal cancer cells. Through in vitro and in vivo experiments, it was confirmed that wogonin exerted its antitumor effects against colorectal cancer by inhibiting phosphorylation in the AKT pathway. Our discovery of wogonin as an inhibitor of AKT phosphorylation provides new opportunities for the pharmacological treatment of colorectal cancer.

Magnolol ameliorates fumonisin B1-induced oxidative damage and lipid metabolism dysfunction in astrocyte-like C6 cells

The neurotoxicity of fumonisin B1 (FB1), a commonly detected mycotoxin in crops and the environment, has attracted considerable attention in recent years. However, no effective method for eliminating FB1 completely exists due to the thermal stability and water solubility of this mycotoxin. Magnolol (MAG) is a neolignane with antioxidative and neuroprotective effects. It has been applied in neurotoxicity treatment. However, the application of MAG to attenuate FB1-induced toxicity has not been reported. This study explored the protective mechanism of MAG against FB1-induced damage in C6 cells through antioxidant and lipid metabolism modulation. Results showed that exposure to 15 μM FB1 caused oxidative stress by changing the levels of malondialdehyde, reactive oxygen species, total superoxide dismutase, catalase, and total glutathione. These changes were reversed by MAG addition, especially at the concentration of 80 μM. The protective effects of MAG were further confirmed by the reduction in the phosphorylation levels of proteins in the MAPK signaling pathway. Lipidomics analysis identified 263 lipids, which belong to 24 lipid classes. Among all of the identified lipids, triglycerides (TGs), diglycerides (DGs), phosphatidylcholines (PCs), wax monoesters (WEs), Cers, and phosphatidylethanolamines (PEs) were major categories. Moreover, nine categories of lipids showed the opposite change trend in the FB1 exposure and MAG 80 groups. A further investigation of the 34 co-occurring differential lipids with remarkable changes (P value < 0.05 and VIP value > 1) in the control, FB1 exposure, and MAG 80 groups was performed. Therein, nine lipids (PCs, LPCs, and SM) were screened out as potential biomarkers to reveal the cytoprotective effects of MAG. This work is the first to investigate the rescue mechanism of MAG in FB1-induced cytotoxicity. The obtained results may expand the application of MAG to alleviate the toxicity of mycotoxins.

Protective effects of 2,4-dinitrophenol in okadaic acid-induced cellular model of Alzheimer's disease

Alzheimer's disease (AD) research started several decades ago and despite the many efforts employed to develop new treatments or approaches to slow and/or revert disease progression, AD treatment remains an unsolved issue. Knowing that mitochondria loss of function is a central hub for many AD-associated pathophysiological processes, there has been renewed interest in exploring mitochondria as targets for intervention. In this perspective, the present study was aimed to investigate the possible beneficial effects of 2,4 dinitrophenol (DNP), a mitochondrial uncoupler agent, in an in vitro model of AD. Retinoic acid-induced differentiated SH-SY5Y cells were incubated with okadaic acid (OA), a neurotoxin often used as an AD experimental model, and/or with DNP. OA caused a decrease in neuronal cells viability, induced multiple mitochondrial anomalies including increased levels of reactive oxygen species, decreased bioenergetics and mitochondria content markers, and an altered mitochondria morphology. OA-treated cells also presented increased lipid peroxidation levels, and overactivation of tau related kinases (GSK3β, ERK1/2 and AMPK) alongside with a significant augment in tau protein phosphorylation levels. Interestingly, DNP co-treatment ameliorated and rescued OA-induced detrimental effects not only on mitochondria but also but also reinstated signaling pathways homeostasis and ameliorated tau pathology. Overall, our results show for the first time that DNP has the potential to preserve mitochondria homeostasis under a toxic insult, like OA exposure, as well as to reestablish cellular signaling homeostasis. These observations foster the idea that DNP, as a mitochondrial modulator, might represent a new avenue for treatment of AD.

Role of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1a) in regulating mitochondrial energy effciency in skeletal muscle

Previous studies have shown that exercise training improves mitochondrial energy effciency in skeletal muscle, but the molecular mechanism is unclear. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1a) is a transcriptional co-activator that coordinately regulates exercise-induced mitochondrial biogenesis and quality control. This study aimed to test the hypothesis that PGC-1a is involved in the improvement of mitochondrial energy effciency in skeletal muscle by using a skeletal muscle-specific PGC-1a overexpression mouse model. Ten to twelve-week-old skeletal muscle-specific PGC-1a transgenic mice (MCK-PGC-1a) and their wildtype littermates (WT) were used. Protein levels of oxidative phosphorylation (OXPHOS) subunits in skeletal muscle were determined by Western blot analysis. The rate of O2 consumption ( JO2) and ATP production ( JATP) in isolated mitochondria from skeletal muscle were evaluated using high-resolution respirometry and fluorometry, respectively, and mitochondrial energy effciency (ATP produced per O2 consumed, or P/O) was calculated. Overexpression of PGC-1a in skeletal muscle did not affect body weights and muscle weights. Consistent with previous studies, MCK-PGC-1a mice had significantly higher OXPHOS protein content than WT mice. On the other hand, JO2, JATP, and P/O in isolated muscle mitochondria were not different between genotypes. Our results suggest that exercise training-induced improvement in muscle mitochondrial energy effciency is not mediated by PGC-1a. This research is supported by NIH grant DK107397 to K.F. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acute Peripheral Irisin Administration Increases Hippocampal Mass in Young, Healthy Rat Cohort

Introduction: Skeletal muscle-brain crosstalk is highlighted as an important contributor to the beneficial relationship between exercise and the brain. Irisin is a key crosstalk myokine due to the ability of irisin to cross the blood-brain barrier and mediate effects of exercise in the brain. Importantly, the influence of irisin in the brain is often demonstrated through chronic administration or supraphysiological dosing of irisin. The aim of the present study is to determine if an acute, non-supraphysiological dose of exogenous irisin is suffcient to induce previously established effects in the hippocampi of young, healthy rats. Methods: Twenty-four 2–3-month-old male and female F344 rats were divided into two groups of 12 (50% male/female) which either received an irisin (100 μg/kg) or vehicle intraperitoneal injection. Forty-eight hours following irisin injections, brains were sectioned and weighed. Levels of total and phosphorylated hippocampal cyclic-AMP Response Element Binding Protein (CREB), a downstream target of irisin signaling, were quantified with Jess Simple Western automated western blot analysis. Values are mean ± standard deviation. Results: Irisin administration resulted in greater hippocampi mass (relative to brain mass, %) compared to vehicle treated animals (3.65±0.24 vs 3.28±0.32%, p&lt;0.01). Further, pCREB/CREB in the hippocampus of irisin animals (0.58±0.13) was 24.6% greater than vehicle (0.47±0.09, p&lt;0.05). Irisin administration did not influence body mass or whole brain mass relative to body mass. Conclusions: The present study demonstrates that a non-supraphysiological dose of irisin is suffcient to increase hippocampal mass and CREB phosphorylation 48 hours post-injection. This work supports irisin as an important, physiologically relevant mediator of the beneficial relationship between exercise, skeletal muscle, and the brain. NIH P20GM113109. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelin-1 stimulated human preadipocyte growth through the AMPK, ERK, cJUN, PKC, and STAT3 pathways

High level of plasma endothelin (ET)-1 was noticed in obesity, and it was found to regulate adipogenesis and the endocrine activity of fat cells. However, relatively little is known about the ET-1 signaling pathway in preadipocyte growth. Our previous reports have shown that ET-1 and ET-3 stimulated murine 3T3-L1 preadipocyte growth through different kinase signaling cascades with ET type A receptor (ETAR) but not ETBR dependency. In this study, the hypothesis arose that ET-1 may regulate human visceral white preadipocyte growth through the AMPK, ERK, cJUN, PKC, and STAT3 pathways with ET receptor-specific dependency. As indicated by an increased number of cells and greater conversion of 3-4,5-dimethylthiazol-2-yl-2,5 diphenyl tetrazolium bromide (MTT) to formazan crystals by living cells, the stimulation of human preadipocyte growth by ET-1 depended on concentration. Neither ET-2 nor ET-3 stimulated human preadipocyte growth. When signaling pathways were examined, ET-1 signaling stimulated phosphorylation of AMPK, ERK, cJUN, PKC, and STAT3, proteins but not AKT, JNK, or p38 MAPK. Treatment with an ETAR antagonist, such as BQ610, or an ETBR antagonist, such as BQ788, blocked the ET-1-induced increase in cell growth and phosphorylated levels of AMPK, PKC, and STAT3 proteins. However, treatment with BQ788 but not BQ610 blocked the ET-1-increased levels of ERK and c-JUN protein phosphorylation. In addition, pretreatment with specific inhibitors of AMPK (compound C), ERK1/2 (U0126), JNK (SP600125), JAK2/STAT3 (AG490), or PKC (Ro318220) prevented the ET-1-induced increase in cell growth and reduced the ET-1-stimulated phosphorylation of AMPK, ERK1/2, c-JUN, STAT3, and PKC. However, neither the p38 MAPK antagonist nor the ceramide synthase inhibitor altered the effect of ET-1. The results indicate that the target molecules of ETAR and ETBR signaling pathways transducing ET-1 growth signals in human visceral preadipocytes can be similar (e.g., AMPK, JAK2/STAT3, and PKC) or different (e.g., ERK1/2 and JNK/c-JUN). This work was supported in part by the National Science and Technology Council, Taiwan, Grant NSTC 112-2320-B-008-001 to YHK and the Taoyuan Armed Forces General Hospital, Taiwan, Grant AFTGH to PJH, CCK, LJS, and YHK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BDTX-189, a novel tyrosine kinase inhibitor, inhibits cell activity via ERK and AKT pathways in the EGFR C797S triple mutant cells

The tertiary mutation C797S in the structural domain of the EGFR kinase is a common cause of resistance to third-generation EGFR tyrosine kinase inhibitors (TKIs). In this study, we used a potent, selective and irreversible inhibitor, BDTX-189, to target EGFR C797S triple mutant cells for cell activity. The study constructed the H1975–C797S (EGFR L858R/T790 M/C797S) cell line using the Lentiviral infection/Cas9 method and investigated its potential as a fourth-generation tyrosine kinase inhibitor via chemosensitivity approach. The results demonstrated its ability to induce cytotoxic effects, and inhibit EGFR L858R/T790 M/C797S cell growth and proliferation in a dose-dependent manner. Meanwhile, BDTX-189 reduces the protein phosphorylation levels of EGFR, ERK, and AKT, promoting apoptosis. Furthermore, BDTX-189 not only inhibits common EGFR triple mutations but also effectively inhibits EGFR L858R mutation and EGFR L858R/T790 M mutation. These findings support the cytotoxic effect of BDTX-189 and its inhibitory effect on cell division and proliferation with the EGFR C797S triple mutation.

Correlation analysis of phosphorylation of myofibrillar protein and muscle quality of tilapia during storage in ice

In this study, the correlation between protein phosphorylation and deterioration in the quality of tilapia during storage in ice was examined by assessing changes in texture, water-holding capacity (WHC), and biochemical characteristics of myofibrillar protein throughout 7 days of storage. The hardness significantly decreased from 471.50 to 252.17 g, whereas cooking and drip losses significantly increased from 26.5% to 32.6% and 2.9% to 9.1%, respectively (P < 0.05). Myofibril fragmentation increased, while myofibrillar protein sulfhydryl content and Ca2+-ATPase activity decreased from 119.33 to 89.29 μmol/g prot and 0.85 to 0.46 μmolPi/mg prot/h, respectively (P < 0.05). Correlation analysis revealed that the myofibrillar protein phosphorylation level was positively correlated with hardness and Ca2+-ATPase activity but negatively correlated with WHC. Myofibrillar protein phosphorylation affects muscle contraction by influencing the dissociation of actomyosin, thereby regulating hardness and WHC. This study provides novel insights for the establishment of quality control strategies for tilapia storage based on protein phosphorylation.

Listeria-vectored cervical cancer vaccine candidate strains reduce MDSCs via the JAK-STAT signaling pathway.

Immunosuppressive status is prevalent in cancer patients and increases the complexity of tumor immunotherapy. It has been found that Listeria-vectored tumor vaccines had the potential ability of two-side regulatory effect on the immune response during immunotherapy. The results show that the combined immunotherapy with the LM∆E6E7 and LI∆E6E7, the two cervical cancer vaccine candidate strains constructed by our lab, improves the antitumor immune response and inhibits the suppressive immune response in tumor-bearing mice in vivo, confirming the two-sided regulatory ability of the immune response caused by Listeria-vectored tumor vaccines. The immunotherapy reduces the expression level of myeloid-derived suppressor cells (MDSCs)-inducing factors and then inhibits the phosphorylation level of STAT3 protein, the regulatory factor of MDSCs differentiation, to reduce the MDSCs formation ability. Moreover, vaccines reduce the expression of functional molecules associated with MDSCs may by inhibiting the phosphorylation level of the JAK1-STAT1 and JAK2-STAT3 pathways in tumor tissues to attenuate the immunosuppressive function of MDSCs. Immunotherapy with Listeria-vectored cervical cancer vaccines significantly reduces the level and function of MDSCs in vivo, which is the key point to the destruction of immunosuppression. The study for the first to elucidate the mechanism of breaking the immunosuppression.

Ba-Qi-Rougan formula alleviates hepatic fibrosis by suppressing hepatic stellate cell activation via the MSMP/CCR2/PI3K pathway

Ethnopharmacological relevanceThe Ba-Qi-Rougan formula (BQRGF) is a traditional and effective compound prescription from Traditional Chinese Medicine (TCM) utilized in treating hepatic fibrosis (HF). Aim of the studyWe aimed to evaluate the therapeutic efficacy of BQRGF on HF and explore the underlying mechanisms of action. Materials and methodsUPLC-Q-TOF-MS technology was employed to identify the material basis of BQRGF. Mice with carbon tetrachloride (CCl4)-induced HF received BQRGF at three doses (3.87, 7.74, and 15.48 g/kg per day). We examined serum and liver biochemical indicators and liver histology to assess the therapeutic impact. Primary mouse cells were isolated and utilized for experimental analysis. MSMP expression levels were examined in vitro and in vivo experimental models, including human and mouse tissue. Furthermore, lentivirus and small interfering RNA (siRNA) transfections were employed to manipulate microseminoprotein (MSMP) expression in LO2 cells (human normal liver cells). These manipulated LO2 cells were then co-cultured with LX2 human hepatic stellate cells (HSCs). Through the modulation of MSMP expression in co-cultured cells, administering recombinant MSMP (rMSMP) with or without BQRGF-medicated serum, and using specific pathway inhibitors or agonists in LX2 cells, we elucidated the underlying mechanisms. ResultsA total of 48 compounds were identified from BQRGF, with 12 compounds being absorbed into the bloodstream and 9 compounds being absorbed into the liver. Four weeks of BQRGF treatment in the HF mouse model led to significant improvements in biochemical and molecular assays and histopathology, particularly in the medium and high-dose groups. These improvements included a reduction in the level of liver injury and fibrosis-related factors. MSMP levels were elevated in human and mouse fibrotic liver tissues, and this increase was mitigated in HF mice treated with BQRGF. Moreover, primary cells and co-culture studies revealed that BQRGF reduced MSMP expression, decreased the expression of the hepatic stellate cell (HSC) activation markers, and suppressed critical phosphorylated protein levels in the CCR2/PI3K/AKT pathway. These findings were further validated using CCR2/PI3K/AKT signaling inhibitors and agonists in MSMP-activated LX2 cells. ConclusionsCollectively, our results suggest that BQRGF combats HF by diminishing MSMP levels and inhibiting MSMP-induced HSC activation through the CCR2/PI3K/AKT pathway.