AMP-dependent Protein Kinase Research Articles

Mitochondrial pyruvate carrier inhibition initiates metabolic crosstalk to stimulate branched chain amino acid catabolism

ObjectiveThe mitochondrial pyruvate carrier (MPC) has emerged as a therapeutic target for treating insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis (NASH). We evaluated whether MPC inhibitors (MPCi) might correct impairments in branched chain amino acid (BCAA) catabolism, which are predictive of developing diabetes and NASH. MethodsCirculating BCAA concentrations were measured in people with NASH and type 2 diabetes, who participated in a recent randomized, placebo-controlled Phase IIB clinical trial to test the efficacy and safety of the MPCi MSDC-0602K (EMMINENCE; NCT02784444). In this 52-week trial, patients were randomly assigned to placebo (n = 94) or 250 mg MSDC-0602K (n = 101). Human hepatoma cell lines and mouse primary hepatocytes were used to test the direct effects of various MPCi on BCAA catabolism in vitro. Lastly, we investigated how hepatocyte-specific deletion of MPC2 affects BCAA metabolism in the liver of obese mice and MSDC-0602K treatment of Zucker diabetic fatty (ZDF) rats. ResultsIn patients with NASH, MSDC-0602K treatment, which led to marked improvements in insulin sensitivity and diabetes, had decreased plasma concentrations of BCAAs compared to baseline while placebo had no effect. The rate-limiting enzyme in BCAA catabolism is the mitochondrial branched chain ketoacid dehydrogenase (BCKDH), which is deactivated by phosphorylation. In multiple human hepatoma cell lines, MPCi markedly reduced BCKDH phosphorylation and stimulated branched chain keto acid catabolism; an effect that required the BCKDH phosphatase PPM1K. Mechanistically, the effects of MPCi were linked to activation of the energy sensing AMP-dependent protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) kinase signaling cascades in vitro. BCKDH phosphorylation was reduced in liver of obese, hepatocyte-specific MPC2 knockout (LS-Mpc2−/−) mice compared to wild-type controls concomitant with activation of mTOR signaling in vivo. Finally, while MSDC-0602K treatment improved glucose homeostasis and increased the concentrations of some BCAA metabolites in ZDF rats, it did not lower plasma BCAA concentrations. ConclusionsThese data demonstrate novel cross talk between mitochondrial pyruvate and BCAA metabolism and suggest that MPC inhibition leads to lower plasma BCAA concentrations and BCKDH phosphorylation by activating the mTOR axis. However, the effects of MPCi on glucose homeostasis may be separable from its effects on BCAA concentrations.

Inhibition of phosphodiesterase 5A by tadalafil improves SIRT1 expression and activity in insulin-resistant podocytes

A decrease in intracellular levels of 3′,5′-cyclic guanosine monophosphate (cGMP) has been implicated in the progression of diabetic nephropathy. Hyperglycemia significantly inhibits cGMP-dependent pathway activity in the kidney, leading to glomerular damage and proteinuria. The enhancement of activity of this pathway that is associated with an elevation of cGMP levels may be achieved by inhibition of the cGMP specific phosphodiesterase 5A (PDE5A) using selective inhibitors, such as tadalafil. Hyperglycemia decreased the insulin responsiveness of podocytes and impaired podocyte function. These effects were associated with lower protein amounts and activity of the protein deacetylase sirtuin 1 (SIRT1) and a decrease in the phosphorylation of adenosine monophosphate-dependent protein kinase (AMPK). We found that PDE5A protein levels increased in hyperglycemia, and PDE5A downregulation improved the insulin responsiveness of podocytes with reestablished SIRT1 expression and activity. PDE5A inhibitors potentiate nitric oxide (NO)/cGMP signaling, and NO modulates the activity and expression of SIRT1. Therefore, we investigated the effects of tadalafil on SIRT1 and AMPK in the context of improving the insulin sensitivity in podocytes and podocyte function in hyperglycemia. Our study revealed that tadalafil restored SIRT1 expression and activity and activated AMPK by increasing its phosphorylation. Tadalafil also restored stimulating effect of insulin on glucose transport in podocytes with high glucose-induced insulin resistance. Additionally, tadalafil improved the function of podocytes that were exposed to high glucose concentrations. Our results display novel mechanisms involved in the pathogenesis of glomerulopathies in diabetes, which may contribute to the development of more effective treatment strategies for diabetic nephropathy.

Molecular-dynamics simulation methods for macromolecular crystallography.

It is investigated whether molecular-dynamics (MD) simulations can be used to enhance macromolecular crystallography (MX) studies. Historically, protein crystal structures have been described using a single set of atomic coordinates. Because conformational variation is important for protein function, researchers now often build models that contain multiple structures. Methods for building such models can fail, however, in regions where the crystallographic density is difficult to interpret, for example at the protein-solvent interface. To address this limitation, a set of MD-MX methods that combine MD simulations of protein crystals with conventional modeling and refinement tools have been developed. In an application to a cyclic adenosine monophosphate-dependent protein kinase at room temperature, the procedure improved the interpretation of ambiguous density, yielding an alternative water model and a revised protein model including multiple conformations. The revised model provides mechanistic insights into the catalytic and regulatory interactions of the enzyme. The same methods may be used in other MX studies to seek mechanistic insights.

OXPHOS deficiency induces mitochondrial DNA synthesis through non-canonical AMPK-dependent mRNA compartmentalization

Eukaryotic cells contain multiple copies of mitochondrial DNA (mtDNA) in discrete organelles or as tubular networks throughout the cytoplasm. The mtDNA copy number is dynamically regulated by mitochondrial biogenesis and mitophagy processes. However, the conditions regulating mtDNA replication, an essential component of biogenesis, are unknown. We observed that short-term (2 h) treatment of rat myoblasts with oligomycin, a specific inhibitor of the mitochondrial F1F0 ATP synthase, resulted in stimulation of mtDNA synthesis from the OH replication origin. This effect was abrogated by Compound C, an antagonist of the AMP-dependent protein kinase (AMPK), a universal intracellular energy sensor, and in AMPK-knockdown cells, indicating that mtDNA replication is regulated by AMPK under oxidative phosphorylation (OXPHOS)- deficient conditions. Using antibody decoration, enzymatically active AMPK, phosphorylated at T172 of the α1 subunit, was found to be located on the mitochondrial surface. Furthermore, oligomycin induced the compartmentalization of several mRNAs encoding OXPHOS components and mtDNA replication factors to mitochondria. Compartmentalization of mRNAs was inhibited by Compound C. We infer that AMPK is locally activated by inhibition of the F1F0 ATP synthase to stimulate association of mtDNA replication factor mRNAs, leading to stimulation of mtDNA synthesis. The findings have implications for the clonal expansion of OXPHOS-deficient mtDNA mutant mitochondria in human patients, with clinical consequences.

PGC-1β modulates catabolism and fiber atrophy in the fasting-response of specific skeletal muscle beds

ObjectiveSkeletal muscle is a pivotal organ for the coordination of systemic metabolism, constituting one of the largest storage site for glucose, lipids and amino acids. Tight temporal orchestration of protein breakdown in times of fasting has to be balanced with preservation of muscle mass and function. However, the molecular mechanisms that control the fasting response in muscle are poorly understood. MethodsWe now have identified a role for the peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) in the regulation of catabolic pathways in this context in muscle-specific loss-of-function mouse models. ResultsMuscle-specific knockouts for PGC-1β experience mitigated muscle atrophy in fasting, linked to reduced expression of myostatin, atrogenes, activation of AMP-dependent protein kinase (AMPK) and other energy deprivation signaling pathways. At least in part, the muscle fasting response is modulated by a negative effect of PGC-1β on the nuclear factor of activated T-cells 1 (NFATC1). ConclusionsCollectively, these data highlight the complex regulation of muscle metabolism and reveal a new role for muscle PGC-1β in the control of proteostasis in fasting.

GZ17-6.02 kills prostate cancer cells in vitro and in vivo.

GZ17-6.02 is undergoing clinical evaluation in solid tumors and lymphoma. We defined the biology of GZ17-6.02 in prostate cancer cells and determined whether it interacted with the PARP1 inhibitor olaparib to enhance tumor cell killing. GZ17-6.02 interacted in a greater than additive fashion with olaparib to kill prostate cancer cells, regardless of androgen receptor expression or loss of PTEN function. Mechanistically, GZ17-6.02 initially caused peri-nuclear activation of ataxia-telangiectasia mutated (ATM) that was followed after several hours by activation of nuclear ATM, and which at this time point was associated with increased levels of DNA damage. Directly downstream of ATM, GZ17-6.02 and olaparib cooperated to activate the AMP-dependent protein kinase (AMPK) which then activated the kinase ULK1, resulting in autophagosome formation that was followed by autophagic flux. Knock down of ATM, AMPKα or the autophagy-regulatory proteins Beclin1 or ATG5 significantly reduced tumor cell killing. GZ17-6.02 and olaparib cooperated to activate protein kinase R which phosphorylated and inactivated eIF2α, i.e., enhanced endoplasmic reticulum (ER) stress signaling. Knock down of eIF2α also significantly reduced autophagosome formation and tumor cell killing. We conclude that GZ17-6.02 and olaparib interact to kill prostate cancer cells in vitro by increasing autophagy and by enhancing ER stress signaling. In vivo, GZ17-6.02 as a single agent profoundly reduced tumor growth and significantly prolonged animal survival. GZ17-6.02 interacted with olaparib to further suppress the growth of LNCaP tumors without ultimately enhancing animal survival. Our data support the consideration of GZ17-6.02 as a possible therapeutic agent in patients with AR+ prostate cancer.

Andrographolide Promotes Uptake of Glucose and GLUT4 Transport through the PKC Pathway in L6 Cells.

Glucose transporter 4 (GLUT4) is a membrane protein that regulates blood glucose balance and is closely related to type 2 diabetes. Andrographolide (AND) is a diterpene lactone extracted from herbal medicine Andrographis paniculata, which has a variety of biological activities. In this study, the antidiabetic effect of AND in L6 cells and its mechanism were investigated. The uptake of glucose of L6 cells was detected by a glucose assay kit. The expression of GLUT4 and phosphorylation of protein kinase B (PKB/Akt), AMP-dependent protein kinase (AMPK), and protein kinase C (PKC) were detected by Western blot. At the same time, the intracellular Ca2+ levels and GLUT4 translocation in myc-GLUT4-mOrange-L6 cells were detected by confocal laser scanning microscopy. The results showed that AND enhanced the uptake of glucose, GLUT4 expression and fusion with plasma membrane in L6 cells. Meanwhile, AND also significantly activated the phosphorylation of AMPK and PKC and increased the concentration of intracellular Ca2+. AND-induced GLUT4 expression was significantly inhibited by a PKC inhibitor (Gö6983). In addition, in the case of 0 mM extracellular Ca2+ and 0 mM extracellular Ca2+ + 10 μM BAPTA-AM (intracellular Ca2+ chelator), AND induced the translocation of GLUT4, and the uptake of glucose was significantly inhibited. Therefore, we concluded that AND promoted the expression of GLUT4 and its fusion with plasma membrane in L6 cells through PKC pathways in a Ca2+-dependent manner, thereby increasing the uptake of glucose.

Genome-Wide Association Analysis of Over 170,000 Individuals from the UK Biobank Identifies Seven Loci Associated with Dietary Approaches to Stop Hypertension (DASH) Diet.

Diet is a modifiable risk factor for common chronic diseases and mental health disorders, and its effects are under partial genetic control. To estimate the impact of diet on individual health, most epidemiological and genetic studies have focused on individual aspects of dietary intake. However, analysing individual food groups in isolation does not capture the complexity of the whole diet pattern. Dietary indices enable a holistic estimation of diet and account for the intercorrelations between food and nutrients. In this study we performed the first ever genome-wide association study (GWA) including 173,701 individuals from the UK Biobank to identify genetic variants associated with the Dietary Approaches to Stop Hypertension (DASH) diet. DASH was calculated using the 24 h-recall questionnaire collected by UK Biobank. The GWA was performed using a linear mixed model implemented in BOLT-LMM. We identified seven independent single-nucleotide polymorphisms (SNPs) associated with DASH. Significant genetic correlations were observed between DASH and several educational traits with a significant enrichment for genes involved in the AMP-dependent protein kinase (AMPK) activation that controls the appetite by regulating the signalling in the hypothalamus. The colocalization analysis implicates genes involved in body mass index (BMI)/obesity and neuroticism (ARPP21, RP11-62H7.2, MFHAS1, RHEBL1). The Mendelian randomisation analysis suggested that increased DASH score, which reflect a healthy diet style, is causal of lower glucose, and insulin levels. These findings further our knowledge of the pathways underlying the relationship between diet and health outcomes. They may have significant implications for global public health and provide future dietary recommendations for the prevention of common chronic diseases.

Enhancement of cGMP-dependent pathway activity ameliorates hyperglycemia-induced decrease in SIRT1-AMPK activity in podocytes: Impact on glucose uptake and podocyte function

Hyperglycemia significantly decreases 3′,5′-cyclic guanosine monophosphate (cGMP)-dependent pathway activity in the kidney. A well-characterized downstream signaling effector of cGMP is cGMP-dependent protein kinase G (PKG), exerting a wide range of downstream effects, including vasodilation and vascular smooth muscle cells relaxation. In podocytes that are exposed to high glucose concentrations, crosstalk between the protein deacetylase sirtuin 1 (SIRT1) and adenosine monophosphate-dependent protein kinase (AMPK) decreased, attenuating insulin responsiveness and impairing podocyte function. The present study examined the effect of enhancing cGMP-dependent pathway activity on SIRT1-AMPK crosstalk in podocytes under hyperglycemic conditions. We found that enhancing cGMP-dependent pathway activity using a cGMP analog was associated with increases in SIRT1 protein levels and activity, with a concomitant increase in the degree of AMPK phosphorylation. The beneficial effects of enhancing cGMP-dependent pathway activity on SIRT1-AMPK crosstalk also included improvements in podocyte function. Based on our findings, we postulate an important role for SIRT1-AMPK crosstalk in the regulation of albumin permeability in hyperglycemia that is strongly associated with activity of the cGMP-dependent pathway.

Irisin inhibits PCSK9 expression through activating AMPK-SREBP2 pathway

AimsPrevious studies found that irisin attenuated the vascular wall inflammation caused by Oxidized low-density lipoprotein (ox-LDL), and recent experiments have shown that proprotein convertase subtilisin/kexin type 9 (PCSK9) can act on various cells in the vascular wall to induce inflammatory responses. But, the relationship between irisin and PCSK9 has not been reported. The aim of this study was to investigate the effect of irisin on PSCK9 in endothelial cells and hepatocytes under the induction of ox-LDL. MethodsExperiments were performed using human umbilical vein endothelial cells and Hep G2, and cells were treated with irisin and (or) ox-LDL for evaluating expression of PCSK9 and downstream inflammatory proteins, while the expression levels of AMP-dependent protein kinase (AMPK) and sterol-regulatory element binding protein 2 (SREBP2) were also examined. Then Compound C was used to inhibit AMPK activation and SiAMPK for silencing of AMPK mRNA, and the above assays were also performed to deeply validate the role of the AMPK-SREBP2 pathway. ResultsIrisin treatment significantly downregulated the expression of PCSK9 and inflammation-related proteins induced by ox-LDL, also restored the content of p-AMPK and reduced the SREBP2 content. After the use of Compound C or SiAMPK, the content of p-AMPK was obviously decreased, and the positive effect of irisin was greatly weakened. ConclusionsThis study demonstrates that irisin suppresses PCSK9 expression through the AMPK-SREBP2 pathway and ameliorates ox-LDL-induced endothelial cells inflammation.

Cyclocarya paliurus triterpenoids suppress hepatic gluconeogenesis via AMPK-mediated cAMP/PKA/CREB pathway

BackgroundAbnormal enhancement of hepatic gluconeogenesis is a vital mechanism of the pathogenesis of Type 2 diabetes mellitus (T2DM); thus, its suppression may present an efficient therapeutic strategy for T2DM. Cyclocarya paliurus (CP), a plant species native to China, has been reported to have anti-hyperglycemia activity. Our previous studies have revealed that Cyclocarya paliurus triterpenic acids (CPT) exert the favorable glucose-lowering activity, but the regulatory effect of CPT on hepatic gluconeogenesis is still unclarified. PurposeThis study aimed to investigate the potential role and mechanism of CPT in gluconeogenesis. Study designIn this study, the ameliorative effect and underlying mechanism of CPT on gluconeogenesis were investigated: high-fat diet and streptozotocin-induced T2DM mice and glucagon-challenged mouse primary hepatocytes. MethodsT2DM model mice with or without oral administration of CPT for 4 weeks were monitored for body weight, glucose and lipid metabolism. Hematoxylin and eosin staining was used to observe liver lipid deposition. Real-time PCR assays were performed to examine the mRNA expression of glucose-6-phosphate (G6Pase), and phosphoenolpyruvate carboxykinase (PEPCK), two key enzymes involved in liver gluconeogenesis. Western blotting was used to determine AMP-dependent protein kinase (AMPK) expression and induction of the glucagon signaling pathway. The possible mechanism of CPT on liver gluconeogenesis was further explored in glucagon-induced mouse primary hepatocytes. ResultsIn vivo and in vitro experiments revealed that CPT treatment significantly reduced fasting blood glucose, total cholesterol and triglyceride levels, and improved insulin resistance. Furthermore, CPT could obviously decreased the mRNA and protein expression of G6Pase and PEPCK, the cyclic AMP content, the phosphorylation level of protein kinase A and cyclic AMP response element-binding protein. But CPT promoted the phosphorylation of AMP-dependent protein kinase (AMPK) and activation of phosphodiesterase 4B. Mechanistically, intervention with Compound C (an AMPK inhibitor) partially blocked the suppressive effect of CPT on hepatic gluconeogenesis. ConclusionThese findings suggested that CPT may inhibit hepatic gluconeogenesis against T2DM by activating AMPK.

Impact of Redox Modification on PKA Substrate Selection

Protein kinases regulate multiple signaling pathways involved in health and disease. For instance, cyclic adenosine monophosphate‐dependent protein kinase (PKA) controls a diverse set of cellular functions, including gene expression, cell cycle progression, metabolism, and apoptosis. Dysregulation of PKA has been linked to many diseases, including diabetes, cancer, and neurodegenerative disorders. PKA is a tetramer consisting of two regulatory and two catalytic subunits. Cyclic AMP binding to the regulatory subunits leads to dissociation of the catalytic subunits, unleashing them to phosphorylate serine and threonine residues on their target substrates. Previous studies suggest that the alpha isoform of the PKA catalytic subunit (PKA Cα) is oxidized on C199 within the P+1 substrate binding region. We hypothesized that, by modifying the substrate selectivity of PKA Cα, oxidation may regulate which pathway is activated in response to a given signal. Specifically, we investigated the impact of redox modification on the substrate selection of PKA Cα. To this end, we used fluorescence polarization (FP) spectroscopy to assess the ability of oxidized and reduced forms of PKA Cα to bind a variety of model peptide ligands (e.g., PKI, Kemptide, and CREBtide). The binding of each ligand was monitored under equilibrium binding conditions and the apparent equilibrium binding parameters (i.e., KD,app) were determined for each kinase‐ligand pair. These studies suggest that H2O2‐dependent oxidation of PKA Cα alters its interactions with some ligands while having little effect on others. To gain further insights into the factors driving the differential changes in the interaction between PKA Cα and its ligands, we are currently using surface plasmon resonance (SPR) to measure changes in the apparent rate constants, kon,app and koff,app, as well as KD,app. Together, these studies offer important insights into the mechanisms of crosstalk between redox and phosphorylation‐dependent signaling at the level of kinase substrate selection and have implications for kinase regulation during normal and pathological states. Interestingly, the site of oxidation is highly conserved among AGC kinase family members. Therefore, in the future, we will use a multipronged approach to explore the impact of redox modification on the substrate selection of other AGC family members, such as PKA Cβ and AKT1, in neuronal signaling and the etiology of various diseases.

Markers of Hypoxia and Metabolism Correlate With Cell Differentiation in Retina and Lens Development.

Recent studies have provided novel insights of co-development of the neural and vascular elements of the retina. Knowledge of these relationships are crucial to understand the impact of therapeutic measures in Retinopathy of Prematurity (ROP). ROP is imposed by therapeutic oxygen upon immature retinal blood vessels and neural cells causing delayed development and vascular regression. However, the impact of hyperoxia on developing retinal neurons is less understood because some aspects of normal development remain unknown. The metabolic changes during differentiation of retinal progenitor cells to functional neurons is one such aspect. We correlated immunomarkers of hypoxia with markers of metabolic change in developing retinal neurons during the early postnatal period in mice. The same marker proteins were studied in secondary lens fiber differentiation at postnatal day-3 (P3). Nuclear localization of the oxygen-sensitive subunits of hypoxia inducible factor, HIF-1α and HIF-2α was correlated with increasing mitochondrial content in differentiating neurons. Nuclear HIF was also correlated with AMP-dependent protein kinase (AMPK), and the AMPK phosphorylation target PPAR-gamma coactivator-1alpha (PGC-1α), the principal regulator of mitochondrial biogenesis. Expression of AMPK, PGC1α and HIF-2α in secondary fiber differentiation was visible in each profile of the lens equator. Strong nuclear localization for all markers was present at the onset of secondary fiber differentiation, and reflected changes in size, mitochondrial content, and metabolism. We speculate that the 'physiological hypoxia' that drives retinal vascular development is cell-specific and reliant upon neuronal differentiation and mitochondrial biogenesis. We suggest that the onset of differentiation increases energy consumption that is detected by AMPK. In turn AMPK increases mitochondrial biogenesis via PGC-1α. Mitochondrial oxygen consumption may then create intracellular hypoxia that activates HIF. This progression is congruent with the expression of these markers in secondary lens fiber differentiation and nuclear localization of HIF-2α. Nuclear localization of HIF-1α and HIF-2α in the postnatal retina is less defined than in the lens as it may involve the remnant of HIF expression from the embryonic period that is sustained and increased by intracellular hypoxia caused by increasing mitochondrial oxygen consumption. This the first report of the involvement of HIF-2α, AMPK and PGC-1α in lens development.

Calcium/calmodulin-dependent protein kinase kinase 2 mediates pleiotropic effects of epidermal growth factor in cancer cells

AimsEngagement of epidermal growth factor (EGF) with its receptor (EGFR) produces a broad range of cancer phenotypes. The overriding aim of this study was to understand EGFR signaling and its regulation by the Ca2+/calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) in cancer cells. ResultsIn ovarian cancer cells and other cancer cell types, EGF-induced activation of oncogenic Akt is mediated by both the canonical PI3K-PDK1 pathway and by CaMKK2. Akt activation induced by EGF occurs by both calcium-dependent and calcium-independent mechanisms. In contrast to the canonical pathway, CaMKK2 neither binds to, nor is regulated by phosphoinositides but is activated by Ca2+/CaM. Akt activation at its primary activation site, T308 occurs by direct phosphorylation by CaMKK2, but activation at its secondary site (S473), is through an indirect mechanism requiring mTORC2. In cells in which another CaMKK2 target, 5′AMP-dependent protein kinase (AMPK) was deleted, Akt activation and calcium-dependency of activation were still observed. CaMKK2 accumulates in the nucleus in response to EGF and regulates transcription of phosphofructokinase platelet (PFKP) a glycolytic regulator. CaMKK2 is required for optimal PFK activity. CaMKK2 regulates transcription of plasminogen activator, urokinase (PLAU) a metastasis regulator. The EGFR inhibitor gefitinib synergizes with CaMKK2 inhibition in the regulation of cell survival and increases the dose-reduction index. CRISPR/Cas9 knockout of CaMKK2 leads to compensatory PTEN downregulation and upregulation of Akt activation. ConclusionsCaMKK2-mediation of EGFR action may enable cancer cells to use intracellular calcium elevation as a signal for growth and survival.

SIRT3-AMPK signaling pathway as a protective target in endothelial dysfunction of early sepsis

Extensive vascular endothelial dysfunction usually occurs in sepsis, resulting in high mortality. The purpose of this study was therefore to investigate the role of AMP-dependent protein kinase (AMPK) in the aortic endothelial dysfunction of early sepsis in mice, and the relationship between AMPK and Sirtuin3 (SIRT3). Cecal ligation and puncture (CLP) surgery was performed to establish a mouse sepsis model, and human umbilical vein endothelial cells (HUVECs) were treated with lipopolysaccharide (LPS) to mimic a sepsis model in vitro. We suppressed and increased the activities of AMPK with Dorsomorphin (CC) and Acadesine (AICAR), respectively. 3-TYP (SIRT3 inhibitor) and Honokiol (SIRT3 agonist) were used to alter SIRT3 activity. Then, the inflammatory and endothelial function parameters of the vascular tissue and survival rate were determined. In vivo, the expression of Ser1177 phosphorylation of endothelial nitric oxide synthase (p-eNOS), endothelium-dependent relaxation function, and survival decreased (P < 0.05), while NF-κB and NLRP3 pathways were activated in CLP-induced early sepsis (P < 0.05). Moreover, activation of AMPK significantly reversed the reduction of p-eNOS expression (P < 0.05), prevented endothelial dysfunction (P < 0.05), deactivated NF-κB and NLRP3 pathways (P < 0.05), and improved survival (P < 0.05) in septic mice. However, AMPK inhibition led to opposite effects (P < 0.05). In addition, changing the activity of AMPK had little effect on SIRT3 expression (P > 0.05), while the expression of p-AMPK varied with the inhibition or activation of SIRT3 (P < 0.05), which was further demonstrated using in vitro experiments. Together, the results showed that the SIRT3-AMPK signaling pathway played an important role in inhibiting vascular inflammation and endothelial dysfunction during early sepsis.

4-Octyl Itaconate Prevents Free Fatty Acid-Induced Lipid Metabolism Disorder through Activating Nrf2-AMPK Signaling Pathway in Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD), characterized with oxidative stress and hepatic steatosis, is a serious threat to human health. As a specific activator of nuclear factor E2-related factor 2 (Nrf2), the 4-octyl itaconate (4-OI) has the beneficial effects in antioxidant and anti-inflammation; however, whether 4-OI can alleviate hepatic steatosis and its mechanism is still unknown. The present study was aimed at investigating the protective effects of 4-OI on free fat acid- (FFA-) induced lipid metabolism disorder and its potential molecular mechanism in hepatocytes. The results showed that 4-OI treatment markedly alleviated FFA-induced oxidative stress and excessive lipid accumulation in hepatocytes. Mechanistically, 4-OI significantly suppressed the overproduction of reactive oxygen species (ROS) through activation of Nrf2; the downregulation of ROS level induced a downregulation of AMP-dependent protein kinase (AMPK) phosphorylation level which finally ameliorated excessive lipid accumulation in FFA-stimulated hepatocytes. In general, our data demonstrated that 4-OI relieves the oxidative stress and lipid metabolism disorder in FFA-stimulated hepatocytes; and these beneficial effects were achieved by activating the Nrf2-AMPK signaling pathway. These data not only expand the new biological function of 4-OI but also provide a theoretical basis for 4-OI to protect against lipid metabolism disorders and related diseases, such as NAFLD.

Roles of Phosphorylation of N-Methyl-D-Aspartate Receptor in Chronic Pain.

Phosphorylation of N-methyl-D-aspartate receptor (NMDAR) is widely regarded as a vital modification of synaptic function. Various protein kinases are responsible for direct phosphorylation of NMDAR, such as cyclic adenosine monophosphate-dependent protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, Src family protein tyrosine kinases, cyclin-dependent kinase 5, and casein kinase II. The detailed function of these kinases on distinct subunits of NMDAR has been reported previously and contributes to phosphorylation at sites predominately within the C-terminal of NMDAR. Phosphorylation underlies both structural and functional changes observed in chronic pain, and studies have demonstrated that inhibitors of kinases are significantly effective in alleviating pain behavior in different chronic pain models. In addition, the exploration of drugs that aim to disrupt the interaction between kinases and NMDAR is promising in clinical research. Based on research regarding the modulation of NMDAR in chronic pain models, this review provides an overview of the phosphorylation of NMDAR-related mechanisms underlying chronic pain to elucidate molecular and pharmacologic references for chronic pain management.

Mulberry polyphenol extracts attenuated senescence through inhibition of Ras/ERK via promoting Ras degradation in VSMC.

Ageing is one of the major risk factors of human diseases, including cancer, diabetes, and cardiovascular disease. Mulberry exhibits a wide range of functions, such as anti-oxidant, anti-inflammation, and anti-diabetes. In this study, we investigated the role of mulberry polyphenol extract (MPE) in K-Ras-induced senescence of smooth muscle cells. Forced expression of K-Ras enhanced senescence of smooth muscle A7r5 cells as shown by the elevation of β-galactosidase activity. Treatment with MPE significantly repressed the Ras, phosphorylated ERK, and β-galactosidase level. MPE triggered the association of cyclins with their corresponding cyclin-dependent protein kinases and hyperphosphorylated retinoblastoma (RB). MPE also down-regulated the levels of K-Ras-induced CDK inhibitors. MPE enhanced the phosphorylated AMP-dependent protein kinase (AMPK) and inducible nitric oxide synthase (iNOS) level in the presence of K-Ras. Pretreatment with either L-NAME or AMPK inhibitor reversed the effects of MPE. In addition, L-NAME and AMPK inhibitor repressed the MPE-induced total and phosphorylated 3-hydroxy-3-methylglutaryl coenzyme A (HMG-Co A) level. MPE repressed K-Ras-induced G0/G1 arrest, whereas L-NAME and AMPK inhibitor blocked the effects of MPE. Our results indicated that MPE recovered the K-Ras-induced senescence of vascular smooth muscle cells through iNOS and AMPK-dependent pathway. Our findings suggested that MPE may prevent ageing-induced atherosclerosis.

Isoglycyrrhizin protects mouse lungs against acute respiratory distress syndrome via regulation of AMPK/Nrf2/ARE pathway

Purpose: To study the effect of isoglycyrrhizin on LPS-mediated acute respiratory distress syndrome (ARDS) in a mouse model, as well as the associated mechanism of action.Methods: Ninety (90) wild-type C57BL/6 male mice were randomly assigned to 3 groups, viz, control, ARDS and isoglycyrrhizin groups. Pathological lesions in mice lungs were determined using H&amp;E staining. The mRNA and protein expressions of inducible nitric oxide synthase (iNOS), heme oxygenase (HO-1), cyclooxygenase-2 (COX-2), AMP- dependent protein kinase (AMPK), serine/threonine proteinkinase (Akt), glycogen synthase kinase 3 (GSK3), nucleotide-binding domain-like receptor protein 3 (NLRP3), and Nrf2 were assayed using quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting, respectively.Results: The levels of mRNA and protein expressions of INO) and COX-2 were significantly upregulated in ARDS, when compared to control, but were markedly down-regulated by isoglycyrrhizin (p &lt; 0.05). Similarly, exposure of ARDS mice to isoglycyrrhizin led to upregulations of mRNA and proteinlevels of Nrf2, NQO1, HO-1, GCLM, GCLC, p-GSK3, GSK3, p-AMPK, AMPK, p-Akt and AKT (p &lt; 0.05). Moreover, isoglycyrrhizin significantly downregulated p-IκB and Nucl-p65 with respect to protein and mRNA levels, but upregulated IκBα expression. Histopathological examination revealed that pretreatment of ARDS mice with isoglycyrrhizin significantly reduced the number of infiltrating inflammatory cells, edema and ARDS score (p &lt; 0.05).Conclusion: Isoglycyrrhizin protects mouse lungs against ARDS via regulation of AMPK/Nrf2/ARE pathway. Thus, this compound has potential for use in the treatment of ARDS.

Increased oxidative stress contributes to enhance brain amyloidogenesis and blunts energy metabolism in sucrose-fed rat: effect of AMPK activation

Metabolic disturbances are linked to neurodegenerative diseases such as Alzheimer disease (AD). However, the cellular mechanisms underlying this connection are unclear. We evaluated the role of oxidative stress (OS), during early metabolic syndrome (MetS), on amyloidogenic processes in a MetS rat model induced by sucrose. MetS caused OS damage as indicated by serum and hypothalamus lipid peroxidation and elevated serum catalase activity. Tissue catalase and superoxide dismutase activity were unchanged by MetS, but gene expression of nuclear factor erythroid-derived 2-like 2 (NFE2L2), which up-regulates expression of antioxidant enzymes, was higher. Expression of amyloid-β cleaving enzyme 1 (BACE-1) and amyloid precursor protein (APP), key proteins in the amyloidogenesis pathway, were slightly increased by sucrose-intake in the hippocampus and hypothalamus. Activation and expression of protein kinase B (PKB) and AMP-dependent protein kinase (AMPK), pivotal proteins in metabolism and energy signaling, were similarly affected in the hippocampus and hypothalamus of MetS rats. Brain creatine kinase activity decreased in brain tissues from rats with MetS, mainly due to irreversible oxidation. Chronic metformin administration partially reversed oxidative damage in sucrose-fed animals, together with increased AMPK activation; probably by modulating BACE-1 and NFE2L2. AMPK activation may be considered as a preventive therapy for early MetS and associated neurodegenerative diseases.