Dichloroacetate Prevents Sepsis Associated Encephalopathy by Inhibiting Microglia Pyroptosis through PDK4/NLRP3.

Dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, is often used to treat lactic acidosis and malignant tumors. Increasing studies have shown that DCA has neuroprotective effects. Here, we explored the role and mechanism of DCA in Sepsis associated encephalopathy (SAE). Single-cell analysis was used to determine the important role of PDK4 in SAE and identify the cell type. GO and GSEA analysis were used to determine the correlation between DCA and pyroptosis. Through LPS + ATP stimulation, a microglia pyroptosis model was established to observe the expression level of intracellular pyroptosis-related proteins under DCA intervention, and further detect the changes in intracellular ROS and JC-1. Additionally, a co-culture environment of microglia and neuron was simply constructed to evaluate the effect of DCA on activated microglia-mediated neuronal apoptosis. Finally, Novel object recognition test and the Morris water maze were used to explore the effect of DCA on cognitive function in mice from different groups after intervention. Based on the above experiments, this study concludes that DCA can improve the ratio of peripheral and central M1 macrophages, inhibit NLRP3-mediated pyroptosis through ROS and mitochondrial membrane potential (MMP). DCA can reduce neuron death caused by SAE and improve cognitive function in LPS mice. In SAE, DCA may be a potential candidate drug for the treatment of microglia-mediated neuroinflammation.

HIF-1α protects nucleus pulposus cells from oxidative stress-induced mitochondrial impairment through PDK-1.

The pathogenesis of intervertebral disc degeneration (IVDD) involves complex signaling networks and various effector molecules, and our understanding of the pathogenesis of IVDD is limited. Hypoxia inducible factor-1α (HIF-1α) is closely related to IVDD, and there is excessive oxidative stress concurrent with IVDD. In this study, we found that HIF-1α could protect nucleus pulposus cells from excessive oxidative stress by reversing the imbalance between oxidants and antioxidants and thus mitigating the oxidative stress-induced mitochondrial impairment. With further exploration, we found that pyruvate dehydrogenase kinase 1 (PDK-1) was involved in the protective effect of HIF-1α on nucleus pulposus cells under oxidative stress. We suggested that HIF-1α could preserve the mitochondrial integrity and activate glycolysis in nucleus pulposus cells via PDK-1, and the addition of DCA, a PDK-1 inhibitor, could blunt the protective effect of HIF-1α. In addition, the HIF-1α/PDK-1 regulatory axis was also confirmed in vivo through HIF-1α knockout mice model. Therefore, we propose that HIF-1α protects nucleus pulposus cells from excessive oxidative stress by maintaining the mitochondrial integrity and glycolysis via PDK-1, thus enriching the insight into the protective mechanism of HIF-1α against IVDD, and providing a novel therapeutic target for the treatment of IVDD.

The comparison of meat yield, quality, and flavor between small-tailed Han sheep and two crossbred sheep and the verification of related candidate genes.

Introduction: In Northeast China, Dorper and Australian White rams are commonly crossbred with small-tailed Han (STH) ewes to improve the offspring's meat yield and quality. However, the differences in traits and the flavor between the crossbred sheep and STH sheep remain unclear. In addition, the candidate genes potentially influencing the meat quality in the three sheep breeds require further verification. Methods: A total of 18 2-month-old healthy rams were raised over a period of 5 months, which included 6 STH, 6 Dorper and small-tailed Han crossbred (Do × STH), and 6 Australian white and small-tailed Han crossbred (Au × STH) offspring. The differences in slaughter, meat quality traits, fatty acid and amino acid composition in the muscular longissimus dorsi (MLD), and volatile compounds in the semitendinosus muscle were compared between the sheep breeds. The candidate genes related to intramuscular fat (IMF) content and fatty acids were validated. Results: The results of this study revealed that the crossbred sheep had higher body weight, carcass weight, bone weight, net meat weight, and IMF content than the STH sheep (p < 0.05). The Do × STH offspring had a higher pH value (24 h), moisture content, and cooking percentage; they also had redder and brighter meat color. The content of myristate, palmitic, and margaric acids in the crossbred sheep was higher than that in the STH sheep (p < 0.05). The Do × STH offspring had the highest saturated fatty acid content (p < 0.05). The Au × STH offspring had the highest protein content (p < 0.05). The arachidonic acid and amino acid (Asp, Ala, Ile, Leu, Lys, Thr, and essential amino acid) contents were higher in the STH sheep than in the crossbred sheep (p < 0.05). The odor activity value (OAV) analysis showed that most of the aldehydes in the Au × STH offspring had higher values. The PDK4 gene expression was positively associated with the IMF content and was negatively correlated with the linoleic acid content in the Do × STH sheep (p < 0.05). The TMEM273 gene expression was positively associated with linoleic and arachidonic acid contents and was negatively correlated with oleic and palmitic acid contents in the Do × STH sheep (p < 0.05). Discussion: The results showed the differences between the crossbred sheep and STH sheep and provided the candidate genes related to meat quality in sheep.

Effects of oridonin on sperm function and the PI3K/PDK1/AKT signaling pathway: Implications for reproductive toxicity.

Oridonin, a natural terpenoid isolated from the leaves of Isodon rubescens (Hemsley) H.Hara, is widely used in oriental medicine for its anticancer properties across various cancer types. Despite its prevalent use, the toxic effects of oridonin on male reproduction, particularly its impact on sperm functions and the mechanisms involved, are not well understood. This study aimed to explore the effects and underlying mechanisms of oridonin on sperm functions. We initially treated Duroc boar spermatozoa with varying concentrations of oridonin (0, 5, 50, 75, 100, and 150 µM) and incubated them to induce capacitation. We then assessed cell viability and several sperm functions, including sperm motility and motion kinematics, capacitation status, and ATP levels. We also analyzed the expression levels of proteins associated with the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide-dependent kinase-1 (PDK1)/protein kinase B (AKT) signaling pathway and phosphotyrosine proteins. Our results indicate that oridonin adversely affects most sperm functions in a dose-dependent manner. We observed significant decreases in AKT, p-AKT (Thr308), phosphatase and tensin homolog (PTEN), p-PDK1, and p-PI3K levels following oridonin treatment, alongside an abnormal increase in phosphotyrosine proteins. These findings suggest that oridonin may disrupt normal levels of tyrosine-phosphorylated proteins by inhibiting the PI3K/PDK1/AKT signaling pathway, which is crucial for cell proliferation, metabolism, and apoptosis, thus potentially harming sperm functions. Consequently, we recommend considering the reproductive toxicity of oridonin when using it as a therapeutic agent.

Post-translational modifications in prion diseases.

More than 650 reversible and irreversible post-translational modifications (PTMs) of proteins have been listed so far. Canonical PTMs of proteins consist of the covalent addition of functional or chemical groups on target backbone amino-acids or the cleavage of the protein itself, giving rise to modified proteins with specific properties in terms of stability, solubility, cell distribution, activity, or interactions with other biomolecules. PTMs of protein contribute to cell homeostatic processes, enabling basal cell functions, allowing the cell to respond and adapt to variations of its environment, and globally maintaining the constancy of the milieu interieur (the body's inner environment) to sustain human health. Abnormal protein PTMs are, however, associated with several disease states, such as cancers, metabolic disorders, or neurodegenerative diseases. Abnormal PTMs alter the functional properties of the protein or even cause a loss of protein function. One example of dramatic PTMs concerns the cellular prion protein (PrPC), a GPI-anchored signaling molecule at the plasma membrane, whose irreversible post-translational conformational conversion (PTCC) into pathogenic prions (PrPSc) provokes neurodegeneration. PrPC PTCC into PrPSc is an additional type of PTM that affects the tridimensional structure and physiological function of PrPC and generates a protein conformer with neurotoxic properties. PrPC PTCC into PrPSc in neurons is the first step of a deleterious sequence of events at the root of a group of neurodegenerative disorders affecting both humans (Creutzfeldt-Jakob diseases for the most representative diseases) and animals (scrapie in sheep, bovine spongiform encephalopathy in cow, and chronic wasting disease in elk and deer). There are currently no therapies to block PrPC PTCC into PrPSc and stop neurodegeneration in prion diseases. Here, we review known PrPC PTMs that influence PrPC conversion into PrPSc. We summarized how PrPC PTCC into PrPSc impacts the PrPC interactome at the plasma membrane and the downstream intracellular controlled protein effectors, whose abnormal activation or trafficking caused by altered PTMs promotes neurodegeneration. We discussed these effectors as candidate drug targets for prion diseases and possibly other neurodegenerative diseases.

Unveiling the PDK4-centered rituximab-resistant mechanism in DLBCL: the potential of the "Smart" exosome nanoparticle therapy.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) represents a prevalent malignant tumor, with approximately 40% of patients encountering treatment challenges or relapse attributed to rituximab resistance, primarily due to diminished or absent CD20 expression. Our prior research identified PDK4 as a key driver of rituximab resistance through its negative regulation of CD20 expression. Further investigation into PDK4's resistance mechanism and the development of advanced exosome nanoparticle complexes may unveil novel resistance targets and pave the way for innovative, effective treatment modalities for DLBCL. METHODS: We utilized a DLBCL-resistant cell line with high PDK4 expression (SU-DHL-2/R). We infected it with short hairpin RNA (shRNA) lentivirus for RNA sequencing, aiming to identify significantly downregulated mRNA in resistant cells. Techniques including immunofluorescence, immunohistochemistry, and Western blotting were employed to determine PDK4's localization and expression in resistant cells and its regulatory role in phosphorylation of Histone deacetylase 8 (HDAC8). Furthermore, we engineered advanced exosome nanoparticle complexes, aCD20@ExoCTX/siPDK4, through cellular, genetic, and chemical engineering methods. These nanoparticles underwent characterization via Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM), and their cellular uptake was assessed through flow cytometry. We evaluated the nanoparticles' effects on apoptosis in DLBCL-resistant cells and immune cells using CCK-8 assays and flow cytometry. Additionally, their capacity to counteract resistance and exert anti-tumor effects was tested in a resistant DLBCL mouse model. RESULTS: We found that PDK4 initiates HDAC8 activation by phosphorylating the Ser-39 site, suppressing CD20 protein expression through deacetylation. The aCD20@ExoCTX/siPDK4 nanoparticles served as effective intracellular delivery mechanisms for gene therapy and monoclonal antibodies, simultaneously inducing apoptosis in resistant DLBCL cells and triggering immunogenic cell death in tumor cells. This dual action effectively reversed the immunosuppressive tumor microenvironment, showcasing a synergistic therapeutic effect in a subcutaneous mouse tumor resistance model. CONCLUSIONS: This study demonstrates that PDK4 contributes to rituximab resistance in DLBCL by modulating CD20 expression via HDAC8 phosphorylation. The designed exosome nanoparticles effectively overcome this resistance by targeting the PDK4/HDAC8/CD20 pathway, representing a promising approach for drug delivery and treating patients with Rituximab-resistant DLBCL.

Molecular insights into the regulatory landscape of PKC-related kinase-2 (PRK2/PKN2) using targeted small compounds.

The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

Inhibition of the ubiquitin-proteasome system reduces the abundance of pyruvate dehydrogenase kinase 1 in cultured myotubes.

Pyruvate dehydrogenase kinase (PDK), which phosphorylates the pyruvate dehydrogenase complex, regulates glucose metabolism in skeletal muscle. PDK1, an isozyme whose expression is controlled by hypoxia-inducible factor-1α (HIF-1α), is thought to play a role in muscle adaptation to hypoxia. While transcriptional upregulation of PDK1 by HIF-1α is well characterised, mechanisms controlling proteolysis of PDK1 in skeletal muscle have not been thoroughly investigated. Proteasome inhibitor MG132 paradoxically reduced the abundance of PDK1 in human cancer cells and rat L6 myotubes, suggesting that MG132 might direct PDK1 towards autophagic degradation. The objectives of our current study were to determine (1) whether MG132 suppresses PDK1 levels in primary human myotubes, (2) whether chloroquine, an inhibitor of autophagy, prevents MG132-induced suppression of PDK1 in L6 myotubes, and (3) whether PYR-41, an inhibitor of ubiquitination, suppresses PDK1 in L6 myotubes. Using qPCR and/or immunoblotting, we found that despite markedly upregulating HIF-1α protein, MG132 did not alter the PDK1 expression in cultured primary human myotubes, while it suppressed both PDK1 mRNA and protein in L6 myotubes. The PDK1 levels in L6 myotubes were suppressed also during co-treatment with chloroquine and MG132. PYR-41 markedly increased the abundance of HIF-1α in primary human and L6 myotubes, while reducing the abundance of PDK1. In L6 myotubes treated with PYR-41, chloroquine increased the abundance of the epidermal growth factor receptor, but did not prevent the suppression of PDK1. Collectively, our results suggest that cultured myotubes degrade PDK1 via a pathway that cannot be inhibited by MG132, PYR-41, and/or chloroquine.

Identification and evolution of PDK-1-like involving lamprey innate immunity.

3-phosphoinositide-dependent protein kinase-1 (PDK-1) is a key kinase regulating the activity of the PI3K/AKT pathway and a major regulator of the AGC protein kinase family. It is essential in the physiological activities of cells, embryonic development, individual development and immune response. In this study, we have identified for the first time an analogue of PDK-1 in the most primitive vertebrate, lamprey, and named it PDK-1-like. The protein sequence similarity of lamprey PDK-1-like to human, mouse, chicken, African xenopus and zebrafish PDK-1 were 64.4 %, 64.5 %, 65.0 %, 61.3 % and 63.2 %, respectively. The phylogenetic tree showed that PDK-1-like of lamprey were located at the base of the vertebrate branch, in line with the trend of biological evolution. Meanwhile, homology analysis showed that PDK-1 proteins across species shared a conserved kinase structural domain and a Pleckstrin Homology (PH) domain. Genomic synteny analysis revealed that the large-scale duplication blocks were not found in lamprey genome and neighbor genes of lamprey PDK-1-like presented dramatic differences compared with jawed vertebrates. More importantly, qPCR analysis showed that PDK-1-like was widely expressed in lamprey. Its mRNA expression levels varied in response to different pathogenic stimuli, and its expression was generally up-regulated under Polyinosinic-Polycytidylic acid (Poly(I:C)) stimulation. Pearson's correlation analysis showed that PDK-1-like was involved in co-expressed with MyD88-independent TLR-3 pathway during the immune response of lamprey, instead of MyD88-dependent TLR-3 pathway. In summary, our composite results offer valuable clues to the origin and evolution of PDK-1, and imply that PDK-1 s are among the most ancestral immune regulators in vertebrates.

Cordycepin Modulates Microglial M2 Polarization Coupled with Mitochondrial Metabolic Reprogramming by Targeting HKII and PDK2.

The microenvironment mediated by the microglia (MG) M1/M2 phenotypic switch plays a decisive role in the neuronal fate and cognitive function of Alzheimer's disease (AD). However, the impact of metabolic reprogramming on microglial polarization and its underlying mechanism remains elusive. This study reveals that cordycepin improved cognitive function and memory in APP/PS1 mice, as well as attenuated neuronal damage by triggering MG-M2 polarization and metabolic reprogramming characterized by increased OXPHOS and glycolysis, rather than directly protecting neurons. Simultaneously, cordycepin partially alleviates mitochondrial damage in microglia induced by inhibitors of OXPHOS and glycolysis, further promoting MG-M2 transformation and increasing neuronal survival. Through confirmation of cordycepin distribution in the microglial mitochondria via mitochondrial isolation followed by HPLC-MS/MS techniques, HKII and PDK2 are further identified as potential targets of cordycepin. By investigating the effects of HKII and PDK2 inhibitors, the mechanism through which cordycepin targeted HKII to elevate ECAR levels in the glycolysis pathway while targeting PDK2 to enhance OCR levels in PDH-mediated OXPHOS pathway, thereby inducing MG-M2 polarization, promoting neuronal survival and exerting an anti-AD role is elucidated.

Elucidation of the anti-gastric cancer mechanism of Guiqi Baizhu Formula by integrative approach of chemical bioinformatics.

Gastric cancer (GC) has posed a great threat to the lives of people around the world. To date, safer and more cost-effective therapy for GC is lacking. Traditional Chinese medicine (TCM) may provide some new options for this. Guiqi Baizhu Formula (GQBZF), a classic TCM formula, has been extensively used to treat GC, while its bioactive components and therapeutic mechanisms remain unclear. In this study, we evaluated the underlying mechanisms of GQBZF in treating GC by integrative approach of chemical bioinformatics. GQBZF lyophilized powder (0.0625 mg/mL, 0.125 mg/mL) significantly attenuated the expression of p-IGF1R, PI3K, p-PDK1, p-VEGFR2 to inhibit the proliferation, migration and induce apoptosis of gastric cancer cells, which was consistent with the network pharmacology. Additionally, atractylenolide Ⅰ, quercetin, glycyrol, physcione and aloe-emodin, emodin, kaempferol, licoflavone A were found to be the key compounds of GQBZF regulating IGF1R and VEGFR2, respectively. And among which, glycyrol and emodin were determined as key active compounds against GC by farther vitro experiments and LC/MS. Meanwhile, we also found that glycyrol inhibited MKN-45 cells proliferation and enhanced apoptosis, which might be related to the inhibition of IGF1R/PI3K/PDK1, and emodin could significantly attenuate the MKN-45 cells migration, which might be related to the inhibition of VEGFR2-related signaling pathway. These results were verified again by molecular dynamics simulation and binding interaction pattern. In summary, this study suggested that GQBZF and its key active components (glycyrol and emodin) can suppress IGF1R/PI3K/PDK1 and VEGFR2-related signaling pathway, thereby inhibiting tumor cell proliferation and migration and inducing apoptosis. These findings provided an important strategy for developing new agents and facilitated clinical use of GQBZF against GC.

Gynostemma Pentaphyllum ameliorates CCl4-induced liver injury via PDK1/Bcl-2 pathway with comprehensive analysis of network pharmacology and transcriptomics.

BACKGROUND: Gynostemma pentaphyllum (Thunb.) Makino, commonly known as "southern ginseng", contains high amounts of ginsenoside derivatives and exhibits similar biological activities with Panax ginseng (C. A. MEY) (ginseng), which is usually used as a low-cost alternative to ginseng. G. pentaphyllum has therapeutic effects on liver diseases. However, the mechanisms underlying its hepatoprotective action have not been fully elucidated. METHODS: The protective effects of the ethanolic extract of G. pentaphyllum (GPE) were evaluated using an experimental carbon tetrachloride (CCl4)-induced liver disease model. Potential targets of GPE were predicted using the "Drug-Disease" bioinformatic analysis. Furthermore, comprehensive network pharmacology and transcriptomic approaches were employed to investigate the underlying mechanisms of GPE in the treatment of liver disease. RESULTS: The pathological examinations showed that GPE significantly alleviated hepatocyte necrosis and liver injury. GPE significantly downregulated Bax and cleaved-PARP expression and upregulated Bcl-2 expression during CCl4-induced hepatocyte apoptosis. We compared the effects of four typical compounds in GPE -a ginsenoside (Rb3) shared by both GPE and ginseng and three unique gypenosides in GPE. Notably, Gypenoside A (GPA), a unique saponin in GPE, markedly reduced hepatocyte apoptosis. In contrast, ginsenoside Rb3 had a weaker effect. Network pharmacology and transcriptomic analyses suggested that this anti-apoptotic effect was achieved by upregulating the PI3K/Akt signaling pathway mediated by PDK1. CONCLUSIONS: These results suggested that G. pentaphyllum had a promising hepatoprotective effect, with its mechanism primarily involving the upregulation of the PDK1/Bcl-2 signaling pathway by GPA, thereby preventing cell apoptosis.

12-O-deacetyl-phomoxanthone A inhibits ovarian tumor growth and metastasis by downregulating PDK4.

AIMS: The xanthone dimer 12-O-deacetyl-phomoxanthone A (12-ODPXA) was extracted from the secondary metabolites of the endophytic fungus Diaporthe goulteri. The 12-ODPXA compound exhibited anticancer properties in murine lymphoma; however, the anti-ovarian cancer (OC) mechanism has not yet been explored. Therefore, the present study evaluated whether 12-ODPXA reduces OC cell proliferation, metastasis, and invasion by downregulating pyruvate dehydrogenase kinase (PDK)4 expression. METHODS: Cell counting kit-8, colony formation, flow cytometry, wound healing, and transwell assays were performed to examine the effects of 12-ODPXA on OC cell proliferation, apoptosis, migration, and invasion. Transcriptome analysis was used to predict the changes in gene expression. Protein expression was determined using western blotting. Glucose, lactate, and adenosine triphosphate (ATP) test kits were used to measure glucose consumption and lactate and ATP production, respectively. Zebrafish xenograft models were constructed to elucidate the anti-OC effects of 12-ODPXA. RESULTS: The 12-ODPXA compound inhibited OC cell proliferation, migration, invasion, and glycolysis while inducing cell apoptosis via downregulation of PDK4. In vivo experiments showed that 12-ODPXA suppressed tumor growth and migration in zebrafish. CONCLUSION: Our data demonstrate that 12-ODPXA inhibits ovarian tumor growth and metastasis by downregulating PDK4, revealing the underlying mechanisms of action of 12-ODPXA in OC.

The effect of PDK1 in maintaining immune cell development and function.

3-phosphoinositide-dependent protein kinase 1 (PDK1) exhibits a substantial influence on immune cell development by establishing a vital connection between PI3K and downstream mTOR signaling cascades. However, it remains unclear whether PDK1 signaling affects the homeostasis and functionality of immune cells. To explore the impact of PDK1 on different immune cells within immune organs, transgenic mouse strains with lymphocyte-specific PDK1 knockout (PDK1fl/fl CD2-Cre) were generated. Unlike wild-type (WT) mice, lymphocyte-specific PDK1 knockout (KO) mice exhibited thymic atrophy, elevated percentages of CD8+ T cells and neutrophils, and reduced proportions of γδ T cells, B cells, and NK cells in the spleen. Functional analysis revealed elevated release of IFN-γ and IL-17A by T cells in PDK1 KO mice, contrasting with diminished levels observed in γδ T cells and Treg cells. Furthermore, the activation, cytotoxicity, and migratory potential of γδ T cells in PDK1 KO mice are heightened, indicating a potential association with the regulation of the mTOR signaling pathway. To conclude, the findings of this research demonstrated that specific knockout of PDK1 in lymphocytes hindered T cell development in the thymus and exhibited a substantial influence on immune cell homeostasis in the spleen and lymph nodes.

Nirmatrelvir has detrimental effects on sperm function by altering the PI3K/PDK1/AKT signaling pathway.

Nirmatrelvir (NMV) is a recently developed selective inhibitor of the main protease of Sars-Cov-2 that reduces the severity of infection. Despite its widespread use and various side effects, NMV's effect on male fertility is still unclear. This study was thus established to investigate how NMV affects male fertility. For experiments, Duroc spermatozoa were incubated with various concentrations of NMV (0, 0.1, 1, 10, 50, and 100 µM). Then, sperm motility, motion kinematics, capacitation status, intracellular ATP level, and cell viability were evaluated. In addition, the expression levels of phospho-PKA substrates, tyrosine-phosphorylated proteins, and PI3K/PDK1/AKT signaling pathway-related proteins were measured by western blotting. Our results showed that sperm motility, motion kinematics, proportion of capacitated spermatozoa, and intracellular ATP level were significantly decreased by NMV in a dose-dependent manner. Moreover, PKA activation was significantly suppressed by NMV, and expression levels of PI3K, phospho-PDK1, AKT, and phospho-AKT (Thr308 and Ser473) were significantly increased in a dose-dependent manner. Combining these findings, it is suggested that NMV has detrimental effects on sperm function by inducing abnormal changes in the PI3K/PDK1/AKT signaling pathway, resulting in PKA deactivation. Therefore, there is a need to pay particular attention to its male reproductive toxicity when NMV is administered.

Long non-coding RNA NEAT1 promotes aerobic glycolysis and progression of cervical cancer through WNT/ß-catenin/PDK1 axis.

BACKGROUND: Cervical cancer is one of the most common gynecological cancers. Accumulated evidence shows that long non-coding RNAs (lncRNAs) play essential roles in cervical cancer occurrence and progression, but their specific functions and mechanisms remain to be further explored. METHODS: The RT-qPCR assay was used to detect the expression of NEAT1 in cervical cancer tissues and cell lines. CCK-8, colony formation, flow cytometry, western blotting, and Transwell assays were used to evaluate the impact of NEAT1 on the malignant behavior of cervical cancer cells. Glucose consumption, lactate production, ATP levels, ROS levels, MMP levels, and the mRNA expressions of glycolysis-related genes and tricarboxylic acid cycle-related genes were detected to analyze the effect of NEAT1 on metabolism reprograming in cervical cancer cells. The expressions of PDK1, ß-catenin and downstream molecules of the WNT/ß-catenin signaling pathway in cervical cancer cells and tissues were detected by western blotting, RT-qPCR, immunofluorescence and immunohistochemistry assays. RESULTS: This study investigated the role and possible molecular mechanism of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in cervical cancer. Our results showed that NEAT1 was highly expressed in cervical cancer tissues and cell lines. Downregulation of NEAT1 inhibited the proliferation, migration, invasion and glycolysis of cervical cancer cells, while overexpression of NEAT1 led to the opposite effects. Mechanistically, NEAT1 upregulated pyruvate dehydrogenase kinase (PDK1) through the WNT/ß-catenin signaling pathway, which enhanced glycolysis and then facilitated cervical cancer metastasis. Furthermore, NEAT1 maintained the protein stability of ß-catenin but did not affect its mRNA level. We also excluded the direct binding of NEAT1 to the ß-catenin protein via RNA pull-down assay. The suppressive impact of NEAT1 knockdown on cell proliferation, invasion, and migration was rescued by ß-catenin overexpression. The WNT inhibitor iCRT3 attenuated the carcinogenic effect induced by NEAT1 overexpression. CONCLUSION: In summary, these findings indicated that NEAT1 may contribute to the progression of cervical cancer by activating the WNT/ß-catenin/PDK1 signaling axis.

Bawei Chenxiang Wan ameliorates right ventricular hypertrophy in rats with high altitude heart disease by SIRT3-HIF1α-PDK/PDH signaling pathway improving fatty acid and glucose metabolism.

BACKGROUND: Bawei Chenxiang Wan (BCW) is among the most effective and widely used therapies for coronary heart disease and angina pectoris in Tibet. However, whether it confers protection through a right-ventricle (RV) myocardial metabolic mechanism is unknown. METHODS: Male Sprague-Dawley rats were orally administrated with BCW, which was injected concurrently with a bolus of Sugen5416, and subjected to hypoxia exposure (SuHx; 5000 m altitude) for 4 weeks. Right ventricular hypertrophy (RVH) in high-altitude heart disease (HAHD) was assessed using Fulton's index (FI; ratio of RV to left ventricle + septum weights) and heart-weight-to-body-weight ratio (HW/BW). The effect of therapeutic administration of BCW on the RVH hemodynamics was assessed through catheterization (mean right ventricular pressure and mean pulmonary artery pressure (mRVP and mPAP, respectively)). Tissue samples were used to perform histological staining, and confirmatory analyses of mRNA and protein levels were conducted to detect alterations in the mechanisms of RVH in HAHD. The protective mechanism of BCW was further verified via cell culture. RESULTS: BCW considerably reduced SuHx-associated RVH, as indicated by macro morphology, HW/BW ratio, FI, mPAP, mRVP, hypertrophy markers, heart function, pathological structure, and myocardial enzymes. Moreover, BCW can alleviate the disorder of glucose and fatty acid metabolism through upregulation of carnitine palmitoyltransferase1ɑ, citrate synthase, and acetyl-CoA and downregulation of glucose transport-4, phosphofructokinase, and pyruvate, which resulted in the reduced levels of free fatty acid and lactic acid and increased aerobic oxidation. This process may be mediated via the regulation of sirtuin 3 (SIRT3)-hypoxia-inducible factor 1α (HIF1α)-pyruvate dehydrogenase kinase (PDK)/pyruvate dehydrogenase (PDH) signaling pathway. Subsequently, the inhibition of SIRT3 expression by 3-TYP (a selective inhibitor of SIRT3) can reverse substantially the anti-RVH effect of BCW in HAHD, as indicated by hypertrophy marker and serum myocardial enzyme levels. CONCLUSIONS: BCW prevented SuHx-induced RVH in HAHD via the SIRT3-HIF1ɑ-PDK/PDH signaling pathway to alleviate the disturbance in fatty acid and glucose metabolism. Therefore, BCW can be used as an alternative drug for the treatment of RVH in HAHD.

Abrogating PDK4 activates autophagy-dependent ferroptosis in breast cancer via ASK1/JNK pathway.

BACKGROUND: Targeting ferroptosis mediated by autophagy presents a novel therapeutic approach to breast cancer, a mortal neoplasm on the global scale. Pyruvate dehydrogenase kinase isozyme 4 (PDK4) has been denoted as a determinant of breast cancer metabolism. The target of this study was to untangle the functional mechanism of PDK4 in ferroptosis dependent on autophagy in breast cancer. METHODS: RT-qPCR and western blotting examined PDK4 mRNA and protein levels in breast cancer cells. Immunofluorescence staining appraised light chain 3 (LC3) expression. Fe (2 +) assay estimated total iron level. Relevant assay kits and C11-BODIPY (591/581) staining evaluated lipid peroxidation level. DCFH-DA staining assayed intracellular reactive oxygen species (ROS) content. Western blotting analyzed the protein levels of autophagy, ferroptosis and apoptosis-signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) pathway-associated proteins. RESULTS: PDK4 was highly expressed in breast cancer cells. Knockdown of PDK4 induced the autophagy of breast cancer cells and 3-methyladenine (3-MA), an autophagy inhibitor, countervailed the promoting role of PDK4 interference in ferroptosis in breast cancer cells. Furthermore, PDK4 knockdown activated ASK1/JNK pathway and ASK1 inhibitor (GS-4997) partially abrogated the impacts of PDK4 absence on the autophagy and ferroptosis in breast cancer cells. CONCLUSION: To sum up, deficiency of PDK4 activated ASK1/JNK pathway to stimulate autophagy-dependent ferroptosis in breast cancer.

Stevioside Ameliorates Palmitic Acid-Induced Abnormal Glucose Uptake via the PDK4/AMPK/TBC1D1 Pathway in C2C12 Myotubes.

BACKGROUND: Stevioside (SV) with minimal calories is widely used as a natural sweetener in beverages due to its high sweetness and safety. However, the effects of SV on glucose uptake and the pyruvate dehydrogenase kinase isoenzyme (PDK4) as an important protein in the regulation of glucose metabolism, remain largely unexplored. In this study, we used C2C12 skeletal muscle cells that was induced by palmitic acid (PA) to assess the effects and mechanisms of SV on glucose uptake and PDK4. METHODS: The glucose uptake of C2C12 cells was determined by 2-NBDG; expression of the Pdk4 gene was measured by quantitative real-time PCR; and expression of the proteins PDK4, p-AMPK, TBC1D1 and GLUT4 was assessed by Western blotting. RESULTS: In PA-induced C2C12 myotubes, SV could significantly promote cellular glucose uptake by decreasing PDK4 levels and increasing p-AMPK and TBC1D1 levels. SV could promote the translocation of GLUT4 from the cytoplasm to the cell membrane in cells. Moreover, in Pdk4-overexpressing C2C12 myotubes, SV decreased the level of PDK4 and increased the levels of p-AMPK and TBC1D1. CONCLUSION: SV was found to ameliorate PA-induced abnormal glucose uptake via the PDK4/AMPK/TBC1D1 pathway in C2C12 myotubes. Although these results warranted further investigation for validation, they may provide some evidence of SV as a safe natural sweetener for its use in sugar-free beverages to prevent and control T2DM.