Thr Phosphorylation Research Articles

14-3-3 binding creates a memory of kinase action by stabilizing the modified state of phospholamban.

The cardiac membrane protein phospholamban (PLN) is targeted by protein kinase A (PKA) at Ser16 and by Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr17 β-Adrenergic stimulation and PKA-dependent phosphorylation of Ser16 acutely stimulate the sarcoplasmic reticulum calcium pump (SERCA) by relieving its inhibition by PLN. CaMKII-dependent phosphorylation may lead to longer-lasting SERCA stimulation and may sustain maladaptive Ca2+ handling. Here, we demonstrated that phosphorylation at either Ser16 or Thr17 converted PLN into a target for the phosphoadaptor protein 14-3-3 with different affinities. 14-3-3 proteins were localized within nanometers of PLN and endogenous 14-3-3 coimmunoprecipitated with pentameric PLN from cardiac membranes. Molecular dynamics simulations predicted different molecular contacts for peptides phosphorylated at Ser16 or Thr17 with the binding groove of 14-3-3, resulting in varied binding affinities. 14-3-3 binding protected either PLN phosphosite from dephosphorylation. β-Adrenergic stimulation of isolated adult cardiomyocytes resulted in the membrane recruitment of endogenous 14-3-3. The exogenous addition of 14-3-3 to β-adrenergic-stimulated cardiomyocytes led to prolonged SERCA activation, presumably because 14-3-3 protected PLN pentamers from dephosphorylation. Phosphorylation of Ser16 was disrupted by the cardiomyopathy-associated ∆Arg14 mutation, implying that phosphorylation of Thr17 by CaMKII may become crucial for 14-3-3 recruitment to ∆Arg14 PLN. Consistent with PLN acting as a dynamic hub in the control of Ca2+ handling, our results identify 14-3-3 binding to PLN as a contractility-augmenting mechanism.

The involvement of PI3K/Akt/mTOR/GSK3β signaling pathways in the antidepressant-like effect of AZD6765

AZD6765 (lanicemine) is a non-competitive NMDA receptor antagonist that induces a fast-acting antidepressant effect without presenting psychotomimetic effects. However, the mechanisms underlying its effects remain to be established. In this context, we demonstrated that a single administration of AZD6765 (1 mg/kg, i.p.) was able to induce an antidepressant-like effect in mice submitted to tail suspension test (TST), an effect reversed by LY294002 (a reversible PI3K inhibitor, 10 nmol/site, i.c.v.), wortmannin (an irreversible PI3K inhibitor, 0.1 μg/site, i.c.v.) and rapamycin (a selective mTOR inhibitor, 0.2 nmol/site, i.c.v.). In addition, the administration of sub-effective doses of AZD6765 (0.1 mg/kg, i.p.) in combination with lithium chloride (non-selective GSK-3β inhibitor, 10 mg/kg, p.o.) or AR-A014418 (selective GSK-3β inhibitor, (0.01 μg/site, i.c.v.) caused a synergistic antidepressant-like effect. These results suggest the involvement of PI3K/Akt/mTOR/GSK3β signaling in the AZD6765 antidepressant-like effect. In addition, western blotting analysis showed an increased immunocontent of synapsin in the prefrontal cortex and a tendency to an increased immunocontent of this protein in the hippocampus 30 min after AZD6765 administration, but no significant effect of AZD6765 was observed in P70S6K (Thr389) phosphorylation and GluA1 immunocontent. A single dose of AZD6765 (3 mg/kg, i.p.), similarly to ketamine (1 mg/kg, i.p.), decreased the latency to feed in the novelty suppressed feeding (NSF) test, a behavioral paradigm that evaluates depression/anxiety-related behavior. This effect was reversed by rapamycin administration, suggesting the activation of mTOR signaling in the effect of AZD in the NSF test. In addition, a single administration of AZD6765 (1 mg/kg, i.p.) or ketamine (1 mg/kg, i.p.) reversed the depressive-like behavior induced by chronic unpredictable stress (CUS). Altogether, the results provide evidence for the fast-acting antidepressant profile of AZD6765, by a mechanism likely dependent on PI3K/Akt/mTOR/GSK3β.

Abstract 2468: Phosphorylation of Thr55 mediates the tumor suppression activities of p53

Abstract P53 is a transcription factor that plays a central role in the regulation of cell cycle, apoptosis and senescence. In unstressed cells, p53 is kept at low level under the strict control of its negative regulators. Upon DNA damage or oncogenic stress, p53 is activated via post-translational modifications. The phosphorylation of serine and threonine, such as Ser15 and Ser46, play important roles in p53 regulation. However, data for Thr55 (T55) phosphorylation is limited and functions of T55 phosphorylation remain elusive. We have previously defined an intramolecular interaction in p53 which modulates DNA binding. The observation of altered intramolecular interactions in T55A and T55D mutants leads to the hypothesis that T55 phosphorylation might affect p53 functions. We generate a T55 phospho-specific antibody to investigate the regulation through this site. The results show T55 phosphorylation is induced by DNA damage compounds. We induce the expression of wild type and T55A mutant p53 in p53 null H1299 cells via the transduction of doxycycline-inducible lentiviral gene expression plasmids. T55A mutant p53 exhibits significantly weaker inhibition of cell viability compared with wild type p53 containing cell in the existence of DNA damaging compounds. Consistently, T55A mutant p53 induces lower mRNA expression level of several important apoptosis related genes and higher MDM2 level in comparison with wild type p53. Overall, our results reveal that phosphorylation of Thr55 is induced by several DNA damaging compounds and alters the expression of p53 target genes, leading to an enhanced tumor suppression. Citation Format: Fan He, Lihong Chen, Jiandong Chen. Phosphorylation of Thr55 mediates the tumor suppression activities of p53 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2468.

Abstract 293: Heat shock factor 1 directly suppresses AMP-activated protein kinase to promote tumorigenesis

Abstract Heat shock factor 1 (HSF1), a master transcriptional regulator of the evolutionarily conserved proteotoxic stress response, preserves proteomic stability upon environmental insults. HSF1 is also a potent pro-oncogenic factor, suggesting that proteomic stability enables oncogenesis. AMP-activated protein kinase (AMPK), a key cellular metabolic sensor and downstream effector of the tumor suppressor LKB1, activates catabolic pathways but suppresses anabolic pathways, including fatty acid and cholesterol biosynthesis, thereby maintaining energy homeostasis. We previously showed that upon activation by metabolic stressors, AMPK interacts with and phosphorylates HSF1 to inactivate it and cause proteomic instability, leading to tumor suppression. Intriguingly, we also discovered that HSF1 suppresses AMPK and controls body fat mass. This study is to investigate the mechanisms by which HSF1 reciprocally suppresses AMPK and the roles of this suppression in lipid metabolism and tumorigenesis. By utilizing recombinant proteins, HSF1 peptide libraries and transcription-deficient HSF1 mutants, our in vitro studies reveal that through physical interactions HSF1 imposes multilayer regulations on AMPK, including blocking AMP binding to γ subunits, impairing LKB1-mediated Thr172 phosphorylation, promoting Thr172 de-phosphorylation by PP2A, and impeding ATP binding to catalytic α subunits. Results from circular dichroism spectroscopy reveal that HSF1 induces global AMPK conformational changes. Biologically, Hsf1 deficiency suppresses lipogenesis and decreases lipid content via AMPK activation. Moreover, Hsf1-deficient cells and mice display reduced cholesterol levels. Interestingly, this defect leads to impaired cholesteroylation of sonic hedgehog (SHH). Consequently, the oncogenic SHH signaling is impaired in HSF1-deficient cells. In vivo, Hsf1 deficiency reduces body fat mass in mice, which can be markedly rescued by either pharmacological or genetic inhibition of AMPK. Importantly, the transcription-deficient HSF1 mutant, through AMPK suppression, enhances the lipid content and SHH cholesteroylation, and promotes the in vivo growth of xenografted human melanomas. In conclusion, HSF1 is a direct AMPK antagonist. This transcription-independent interaction of HSF1 with AMPK epitomizes a reciprocal kinase-substrate regulation whereby lipid metabolism and proteomic stability intertwine and promotes cancer anabolism and oncogenesis. Citation Format: Kuo-Hui Su, Siyuan Dai, Zijian Tang, Meng Xu, Chengkai Dai. Heat shock factor 1 directly suppresses AMP-activated protein kinase to promote tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 293.

The structural determinants of PH domain-mediated regulation of Akt revealed by segmental labeling.

Akt is a critical protein kinase that governs cancer cell growth and metabolism. Akt appears to be autoinhibited by an intramolecular interaction between its N-terminal pleckstrin homology (PH) domain and kinase domain, which is relieved by C-tail phosphorylation, but the precise molecular mechanisms remain elusive. Here, we use a combination of protein semisynthesis, NMR, and enzymological analysis to characterize structural features of the PH domain in its autoinhibited and activated states. We find that Akt autoinhibition depends on the length/flexibility of the PH-kinase linker. We identify a role for a dynamic short segment in the PH domain that appears to regulate autoinhibition and PDK1-catalyzed phosphorylation of Thr308 in the activation loop. We determine that Akt allosteric inhibitor MK2206 drives distinct PH domain structural changes compared to baseline autoinhibited Akt. These results highlight how the conformational plasticity of Akt governs the delicate control of its catalytic properties.

Effect of insulin on natriuretic peptide gene expression in porcine heart

Gut hormones affect cardiac function and contractility. In this study, we examined whether insulin affects the cardiac atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) gene expression and release of proANP-derived peptides in pigs. Anaesthetized pigs were included in an experimental study comparing the effect of hyperinsulinemia in 15 pigs submitted to two different protocols versus 11 control pigs receiving saline infusion. Phosphorylation of Akt on Thr308 was determined by western blotting with a pAkt-Thr308 antibody. The mRNA contents of ANP and BNP were determined with real-time PCR; plasma and cardiac tissue proANP was measured with an immunoluminometric assay targeted against the mid-region of the propeptide and a processing-independent assay. Insulin stimulation increased phosphorylation of Akt Thr308 in both left atrium and left ventricle of porcine hearts (p < 0.005). No change was observed in ANP and BNP mRNA contents in the right or left atrium. BNP mRNA contents in the left ventricle, however, decreased 3-fold (p = 0.02) compared to control animals, whereas the BNP mRNA content in the right ventricle as well as ANP mRNA content in the right and left ventricle did not change following hyperinsulinemia. Moreover, the peptide contents did not change in the four cardiac chambers. Finally, proANP concentrations in plasma did not change during the insulin infusion compared to the control animals. These results suggest that insulin does not have direct effect on atrial natriuretic peptide expression but may have a role in the left ventricle.

1739-P: Insulin Rapidly Increases Skeletal Muscle Mitochondrial ADP Sensitivity, Mitigating HFD-Induced Mitochondrial Dysfunction

Introduction: Causality of the association between impairments in mitochondrial function and insulin resistance remains debatable. We have previously demonstrated that mitochondrial ADP sensitivity is impaired in high-fat diet (HFD)-induced insulin resistance, which contributes to bioenergetic imbalance and higher reactive oxygen species (ROS) production. However, insulin regulates several metabolic processes within skeletal muscle, and we tested the hypothesis that insulin could rapidly improve mitochondrial ADP sensitivity, a phenomenon possibly lost in HFD-induced insulin resistance. Methods: Male and female C57Bl/6 mice were fed a control diet (10% fat) or HFD (60% fat) for 8 weeks. At the end of treatment, insulin was injected intraperitoneally (3 U/kg) twice (0 and 30 minutes). At 60 minutes red gastrocnemius muscle was excised to evaluate mitochondrial bioenergetics, ADP sensitivity, and ROS production. Results: In control mice, insulin increased respiration by ∼2-fold in the presence of submaximal (100-1000 μM ADP) but not maximal ADP-supported respiration. As a result, insulin decreased the apparent ADP Km from 700 to 380 μM ADP. Despite these respiratory changes, insulin had no effect on mitochondrial maximal succinate-supported ROS emission or the ability of ADP to suppress ROS emission. HFD consumption impaired whole-body glucose intolerance, skeletal muscle insulin resistance (decreased insulin-stimulated Akt Thr-308 and Ser-473 phosphorylation) and impaired mitochondrial submaximal ADP-supported respiration by ∼50% (ADP Km ∼1100 μM). However, despite the reduction in insulin signaling following HFD, insulin increased submaximal ADP-supported respiration by ∼2-fold, decreased the apparent ADP Km to ∼400 μM and improved the ability of ADP to suppress mitochondrial ROS emission. Conclusion: Insulin rapidly improves mitochondrial ADP sensitivity, mitigating the apparent HFD-induced mitochondrial dysfunction. Disclosure H. Brunetta: None. H.L. Petrick: None. E.A. Nunes: None. G. Holloway: None. Funding Natural Sciences and Engineering Research Council of Canada (400360); Coordination for the Improvement of Higher Education Personnel

CaMKII is activated in opioid induced conditioned place preference, but αCaMKII Thr286 autophosphorylation is not necessary for its establishment

Activation of calcium/calmodulin-dependent protein kinase II (CaMKII), particularly its α isoform, is known to be important for neuronal processes central for learning and memory and has also been implicated in the maladaptive learning involved in drug addiction.Thr286 autophosphorylation of αCaMKII has been shown to be indispensable for establishment of cocaine-induced CPP (Easton et al., 2014). To study the contribution of CaMKII in opioid induced conditioned learning, we examined how establishment of conditioned place preference (CPP) induced by 10 or 30 μmol/kg morphine or its active metabolite morphine-6-glucuronide (M6G) affects the levels and Thr286 autophosphorylation of the α- and β-isoforms of CaMKII, as well as β-actin levels, in dorsal and ventral striatum and in hippocampus of mice. An acute and a sub-chronic treatment were used as controls. Whereas an acute single administration of morphine or M6G caused increases in CaMKII levels and phosphorylation at Thr286 and β-actin in striatal areas, CPP induced by these opioids was accompanied primarily by an increase in the protein levels of both CaMKII isoforms and β-actin in dorsal striatum and hippocampus. Decreases in CaMKII Thr286 phosphorylation were observed in dorsal striatum after the sub-chronic pharmacological treatment. Despite the changes observed in αCaMKII activity in wild type mice, morphine-induced CPP was not affected in αCaMKIIT286A autophosphorylation-deficient mice. These results indicate that opioid-induced CPP is accompanied by activation of α- and βCaMKII in striatum and hippocampus, but, in opposition to what has been observed with cocaine, αCaMKII autophosphorylation is not essential for establishment of opioid-induced CPP.

OR14-07 Association Between Placental Glucose Uptake and Protein O-Glcnacylation and Birth Outcomes in Obese Non-Diabetic Mothers

Increased transport of nutrients such as glucose, across the placenta, has been linked to abnormal fetal growth and pregnancy complications in obese non-diabetic mothers (1); however, the underlying mechanisms are poorly understood. We hypothesized that in placenta, the metabolic and nutrient sensor O-GlcNAc transferase (OGT), highly sensitive to glucose flux through the hexosamine biosynthetic pathway (HBP), responds to maternal obesogenic environment by increasing O-GlcNAc post-translational modification of nucleocytoplasmic proteins in the placenta altering fetal growth trajectories. Tissue biopsies were isolated from placentas collected at term from 26 non-diabetic mothers alongside routine biochemistry and anthropometric data (maternal fasting glucose, glycated hemoglobin (HbA1c), early pregnancy body weight (BMI) and birth weight). OGT and glucose transporter 1 (GLUT1) protein expression as well as tissue levels of O-GlcNAcylation were determined by immunoblotting using specific antibodies. The BeWo choriocarcinoma cell line was also used as an in vitro model of trophoblast to test the effect of high glucose and GLUT1 silencing on the OGT activity by immunoblotting. Maternal BMI was positively correlated to birth weight centile (BWC) (p=0.0130, R2=0.231), maternal fasting glucose (p=0.0177, R2=0.221) and HbA1c levels (p= 0.0156, R2=0.229) as well as placental OGT protein expression (p=0.0294, R2=0.183). The latter was positively associated to the levels of protein O-GlcNAcylation (p=0.0023, R2=0.326). Interestingly, GLUT1 protein levels were positively correlated to BWC (p=0.0056, R2=0.279) and strongly correlated to protein O-GlcNAcylation (p<0.0001, R2=0.507), suggesting an increase in the placental flux of glucose. In agreement with findings in placenta biopsies, in BeWo cells total protein O-GlcNAcylation levels were altered by cell exposure to different glucose levels (5 mM vs 15 mM, p<0.01). This was prevented by downregulating OGT or GLUT1 expression (p<0.001) using gene silencing. In addition, OGT protein levels were negatively associated to AMP-activated protein kinase (AMPK) activation (p=0.0005, R2=0.402) in placenta biopsies identifying a novel cross-talk between two placental nutrient sensors, OGT and AMPK, previously shown in other tissues (2). Accordingly, the silencing of OGT promoted the activation of AMPK (p<0.01) and its downstream target acetyl-CoA carboxylase (ACC) (p<0.01) in BeWo cells, as demonstrated by increased phosphorylation of residues Thr172 and Ser79 for AMPK and ACC respectively. Such obesity-associated cross talk between metabolic and nutrient sensors might disrupt multiple cellular pathways involved in fetal development and growth.

Increased AMP deaminase activity decreases ATP content and slows protein degradation in cultured skeletal muscle

BackgroundProtein degradation is an energy-dependent process, requiring ATP at multiple steps. However, reports conflict as to the relationship between intracellular energetics and the rate of proteasome-mediated protein degradation. MethodsTo determine whether the concentration of the adenine nucleotide pool (ATP + ADP + AMP) affects protein degradation in muscle cells, we overexpressed an AMP degrading enzyme, AMP deaminase 3 (AMPD3), via adenovirus in C2C12 myotubes. ResultsOverexpression of AMPD3 resulted in a dose- and time-dependent reduction of total adenine nucleotides (ATP, ADP and AMP) without increasing the ADP/ATP or AMP/ATP ratios. In agreement, the reduction of total adenine nucleotide concentration did not result in increased Thr172 phosphorylation of AMP-activated protein kinase (AMPK), a common indicator of intracellular energetic state. Furthermore, LC3 protein accumulation and ULK1 (Ser 555) phosphorylation were not induced. However, overall protein degradation and ubiquitin-dependent proteolysis were slowed by overexpression of AMPD3, despite unchanged content of several proteasome subunit proteins and proteasome activity in vitro under standard conditions. ConclusionsAltogether, these findings indicate that a physiologically relevant decrease in ATP content, without a concomitant increase in ADP or AMP, is sufficient to decrease the rate of protein degradation and activity of the ubiquitin-proteasome system in muscle cells. This suggests that adenine nucleotide degrading enzymes, such as AMPD3, may be a viable target to control muscle protein degradation and perhaps muscle mass.

Guanosine potentiates the antidepressant-like effect of subthreshold doses of ketamine: Possible role of pro-synaptogenic signaling pathway

Background Augmentation therapies may be effective strategies to potentiate the ketamine's actions with lower potential for knock-on effects. Thus, this study investigated the ability of combined administration of guanosine plus ketamine to elicit an antidepressant-like effect associated with mTOR pathway modulation. The ability of this combined administration to exert an antidepressant-like effect in a model of depression was also evaluated.Methods Mice were administered with subthreshold doses of ketamine (0.1 mg/kg, i.p.) and guanosine (0.01 mg/kg, p.o.) and submitted to the tail suspension test, and immunoblotting analyses (p-mTOR, p-p70S6K, PSD-95, GluA1, and synapsin) in the hippocampus and prefrontal cortex. The antidepressant-like effect of ketamine plus guanosine in mice subjected to administration of corticosterone (20 mg/kg, p.o., 21 days) was also evaluated.Results Ketamine plus guanosine treatment elicited an antidepressant-like effect, which was associated with increased mTOR (Ser2448) and p70S6K (Thr389) phosphorylation in the hippocampus, but not in the prefrontal cortex. Furthermore, increased PSD-95 and GluA1 immunocontent were observed in the prefrontal cortex, but not in the hippocampus of ketamine plus guanosine-treated mice. Reinforcing the notion that guanosine may potentiate the ketamine's behavioral response, a single administration of subthreshold doses of ketamine plus guanosine counteracted the corticosterone-induced depressive-like behavior.Conclusions Our results indicate that guanosine potentiates the antidepressant-like effect of subthreshold doses of ketamine, an effect likely associated with the stimulation of synaptogenic pathway in the hippocampus and prefrontal cortex, although with a different profile. The augmentation effect of ketamine by guanosine could have therapeutic relevance for patients with treatment-resistant depression.

GSK3 Activity is Decreased in The Hippocampus of Male Mice Supplemented with Low‐Dose Lithium in Drinking Water

Sporadic Alzheimer’s disease (AD) is a neurodegenerative disease characterized by neurofibrillary tangles composed of hyperphosphorylated tau proteins and amyloid‐beta (Aβ) plaques. Together, these two histopathological detriments in the brain lead to progressive cognitive decline. Glycogen synthase kinase‐3 β (GSK3β), a kinase found abundantly in the brain, has been implicated in the development of these AD pathologies. GSK3β remains constituently active until phosphorylated at its inhibitory Ser9 residue. When active, GSK3β is capable of inducing tau hyperphosphorylation and can phosphorylate amyloid‐precursor protein (APP), influencing greater levels of Aβ peptide production. Lithium (Li), a GSK3β inhibitor, has been suggested as an AD prophylaxis. However, Li has also been associated with several dose‐dependent adverse side effects. The purpose of this study was thus to examine the effects of a prophylactic low‐dose Li supplementation on brain GSK3β signaling and activity. C57BL/6J male mice were given 10mg/kg/day Li or no Li in their drinking water for 6 weeks (n=12/group) and the prefrontal cortex (PFC) and hippocampal (HIP) tissues were collected for GSK3 activity assay and western blot analyses. Li mice had increased serum Li concentrations (p&lt;0.0001) and decreased hippocampal GSK3 activity (p=0.06) as compared to control mice. pAkt Thr308 phosphorylation, a kinase immediately upstream of GSK3β capable of inducing inhibitory pGSK3β Ser9 phosphorylation, was increased in the PFC of Li mice compared to control mice (p&lt;0.05). However, no differences in pGSK3β Ser9 or Tyr216, an activating residue, were observed between control and Li mice in both the PFC and HIP. Changes in GSK3 activity without changes in inhibitory Ser9 phosphorylation may suggest that a longer supplementation is needed to affect significant changes in the brain. Furthermore, these results demonstrate that the phosphorylation status of GSK3β may not always be a direct translation of its activity. This highlights the importance of measuring GSK3β activity alongside of measures of upstream kinases (i.e. PI3K/Akt signaling) and downstream targets to obtain a better indication of GSK3β’s overall activity within the brain. Overall, this initial work provides evidence that Li can inhibit hippocampal GSK3 activity and alter the phosphorylation status of upstream kinases at a low‐dose. Future work will examine Li’s potential prophylactic effects in a diseased model which better mimics AD (i.e. aged and metabolically‐distressed mice). This is important to further elucidate the efficacy of low‐dose Li in the prevention of AD pathologies.Support or Funding InformationThis work is supported by the Alzheimer Society of Brant, Haldimand Norfolk, Hamilton Halton and NSERC.

The Photorhabdus Virulence Cassettes RRSP-Like Effector Interacts With Cyclin-Dependent Kinase 1 and Causes Mitotic Defects in Mammalian Cells.

The “Photorhabdus virulence cassettes” (PVCs) secreted by Photorhabdus are defined as “extracellular contractile injection systems” (eCISs) and can deliver effectors to eukaryotic hosts for cytotoxicity. Previously, we demonstrated the cryogenic electron microscopy (cryo-EM) structure and assembly process of an intact PVC particle from Photorhabdus asymbiotica. In this work, we characterized the biological functions of a PVC effector, which is defined as a homologous protein of Ras/Rap1-specific endopeptidase domain (RRSP) in the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin from Vibrio vulnificus. In this work, we found that the RRSP homologous protein (RRSPPa) was associated with inhibition of cell proliferation and increased cell apoptosis and death of HeLa cells. Furthermore, we discovered that RRSPPa disturbed mitotic progression, including the induction of cell cycle alteration, retardation of cell abscission time, and regression of the cleavage furrow. In addition, we revealed that RRSPPa could target the cyclin-dependent kinase 1 (CDK1) protein and block activation of CDK1 through inhibition of Thr161 phosphorylation, which partially explained the crucial role of this effector in cell mitosis.

Low shear stress damages endothelial function through STAT1 in endothelial cells (ECs).

Low shear stress (LSS) occurs in areas where atherosclerosis is prevalent. Many studies have revealed that signal transducer and activator of transcription 1 (STAT1) plays a significant role in cardiovascular disease. Nonetheless, the mechanism underlying the connection between STAT1 and LSS is not fully understood. The purpose of this study was to investigate the link between LSS and STAT1 in endothelial cells (ECs). Monolayer endothelial cells were stimulated or not stimulated by LSS. Protein expression and phosphorylation levels were determined by western blotting. Immunofluorescence was used to compare the protein expression differences in bifurcated and non-bifurcated human coronary arteries. Endothelial function was assessed by using a dihydroethidium assay, real-time PCR, western blotting and nitric oxide (NO)-sensitive fluorophore. Results showed that STAT1 played a key role in LSS-induced endothelium damage. Firstly, LSS activated STAT1, as evidenced by LSS-induced STAT1 (Tyr701) phosphorylation in ECs in vitro and the increased intimal STAT1 expression at bifurcation of human coronary arteries. Secondly, LSS-induced STAT1 phosphorylation was positively regulated by inhibitor of nuclear factor kappa-B kinase ε (IKKε). Additionally, LSS-promoted inflammatory factor expression was markedly reversed by silencing STAT1 (siSTAT1). LSS also increased reactive oxygen species (ROS) level and decreased endogenous NO release: however, siSTAT1 reversed these adverse effects through upregulating the antioxidant gene heme oxygenase-1(HO-1) and downregulating endothelial nitric oxide synthase (eNOS) Thr495 phosphorylation. According to our results, LSS-mediated EC injury may be associated with the activation of STAT1. Strategies designed to reduce STAT1 expression or inhibit STAT1 activation may be effective approaches for reducing the incidence of atherosclerosis.

Unacylated ghrelin stimulates fatty acid oxidation to protect skeletal muscle against palmitate-induced impairment of insulin action in lean but not high-fat fed rats

BackgroundGhrelin is a gut hormone that spikes in circulation before mealtime. Recent findings suggest that both ghrelin isoforms stimulate skeletal muscle fatty acid oxidation, lending to the possibility that it may regulate skeletal muscle’s handling of meal-derived substrates. It was hypothesized in the current study that ghrelin may preserve muscle insulin response during conditions of elevated saturated fatty acid (palmitate) availability by promoting its oxidation. Methods and resultsSoleus muscle strips were isolated from male rats to determine the direct effects of ghrelin isoforms on fatty acid oxidation, glucose uptake and insulin signaling. We demonstrate that unacylated ghrelin (UnAG) is the more potent stimulator of skeletal muscle fatty acid oxidation. Both isoforms of ghrelin generally protected muscle from impaired insulin-mediated phosphorylation of AKT Ser473 and Thr308, as well as downstream phosphorylation of AS160 Ser588 during high palmitate exposure. However, only UnAG was able to preserve insulin-stimulated glucose uptake during exposure to high palmitate concentrations. The use of etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase (CPT-1) abolished this protection, strongly suggesting that UnAG’s stimulation of fatty acid oxidation may be essential to this protection. To our knowledge, we are also the first to investigate the impact of a chronic high-fat diet on ghrelin’s actions in muscle. Following 6 wks of a high-fat diet, UnAG was unable to preserve insulin-stimulated signaling or glucose transport during an acute high palmitate exposure. UnAG was also unable to further stimulate 5′ AMP-activated protein kinase (AMPK) or fatty acid oxidation during high palmitate exposure. Corticotropin-releasing hormone receptor-2 (CRF-2R) content was significantly decreased in muscle from high-fat fed animals, which may partially account for the loss of UnAG’s effects. ConclusionsUnAG is able to protect muscle from acute lipid exposure, likely due to its ability to stimulation fatty acid oxidation. This effect is lost in high-fat fed animals, implying a resistance to ghrelin at the level of the muscle. The underlying mechanisms accounting for ghrelin resistance in high fat-fed animals remain to be discovered.

Vitamin B6 inhibits macrophage activation to prevent lipopolysaccharide-induced acute pneumonia in mice.

Macrophage activation participates in the pathogenesis of pulmonary inflammation. As a coenzyme, vitamin B6 (VitB6) is mainly involved in the metabolism of amino acids, nucleic acids, glycogen and lipids. We have previously reported that activation of AMP‐activated protein kinase (AMPK) produces anti‐inflammatory effects both in vitro and in vivo. Whether VitB6 via AMPK activation prevents pulmonary inflammation remains unknown. The model of acute pneumonia was induced by injecting mice with lipopolysaccharide (LPS). The inflammation was determined by measuring the levels of interleukin‐1 beta (IL‐1β), IL‐6 and tumour necrosis factor alpha (TNF‐α) using real time PCR, ELISA and immunohistochemistry. Exposure of cultured primary macrophages to VitB6 increased AMP‐activated protein kinase (AMPK) Thr172 phosphorylation in a time/dose‐dependent manner, which was inhibited by compound C. VitB6 downregulated the inflammatory gene expressions including IL‐1β, IL‐6 and TNF‐α in macrophages challenged with LPS. These effects of VitB6 were mirrored by AMPK activator 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR). However, VitB6 was unable to inhibit LPS‐induced macrophage activation if AMPK was in deficient through siRNA‐mediated approaches. Further, the anti‐inflammatory effects produced by VitB6 or AICAR in LPS‐treated macrophages were abolished in DOK3 gene knockout (DOK3 −/−) macrophages, but were enhanced in macrophages if DOK3 was overexpressed. In vivo studies indicated that administration of VitB6 remarkably inhibited LPS‐induced both systemic inflammation and acute pneumonia in wild‐type mice, but not in DOK3 −/− mice. VitB6 prevents LPS‐induced acute pulmonary inflammation in mice via the inhibition of macrophage activation.

Quercetin and Isoquercitrin Inhibiting Hepatic Gluconeogenesis Through LKB1-AMPKα Pathway.

To observe the impact of quercetin and isoquercitrin on gluconeogenesis in hepatocytes. Mouse primary hepatocytes were cultured with lactic acid and pyruvic acid. After treatment with quercetin and isoquercitrin for 24 hours, the glucose concentration in the culture supernatant was determined. RT-PCR was used to detect the mRNAs of PEPCK, G6Pase, LKB1, and AMPKα. Protein levels of LKB1, AMPKα, and Thr172 phosphorylation were evaluated by Western blot. The glucose concentration in the gluconeogenesis group (GN) was significantly higher than in the control group (C), but the glucose concentrations in the high level quercetin(group 80Q) and high level isoquercitrin (group 80I) were significantly lower than in the group GN, P<0.01. In the group 80Q, and group 80I, the mRNA levels of PEPCK and LKB1were significantly lower than in the group GN (P<0.01), and the G6Pase mRNA were significantly lower than in the group GN (P<0.05). The protein levels of LKB1 and the phosphorylation of AMPKα Thr172 in the group 80Q, group 40I, and group 80I were higher than in the group GN. The effects of quercetin and isoquercitrin on LKB1 and AMPKα were similar to those of metformin. Quercetin and isoquercitrin inhibit gluconeogenesis in hepatocytes, which may be related to the LKB1 upregulation and phosphorylation of AMPKα.

The protein tyrosine phosphatase PTP-PEST mediates hypoxia-induced endothelial autophagy and angiogenesis via AMPK activation.

Global and endothelial loss of PTP-PEST (also known as PTPN12) is associated with impaired cardiovascular development and embryonic lethality. Although hypoxia is implicated in vascular remodelling and angiogenesis, its effect on PTP-PEST remains unexplored. Here we report that hypoxia (1% oxygen) increases protein levels and catalytic activity of PTP-PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry revealed that α subunits of AMPK (α1 and α2, encoded by PRKAA1 and PRKAA2, respectively) interact with PTP-PEST under normoxia but not in hypoxia. Co-immunoprecipitation experiments confirmed this observation and determined that AMPK α subunits interact with the catalytic domain of PTP-PEST. Knockdown of PTP-PEST abrogated hypoxia-mediated tyrosine dephosphorylation and activation of AMPK (Thr172 phosphorylation). Absence of PTP-PEST also blocked hypoxia-induced autophagy (LC3 degradation and puncta formation), which was rescued by the AMPK activator metformin (500 µM). Because endothelial autophagy is a prerequisite for angiogenesis, knockdown of PTP-PEST also attenuated endothelial cell migration and capillary tube formation, with autophagy inducer rapamycin (200 nM) rescuing angiogenesis. In conclusion, this work identifies for the first time that PTP-PEST is a regulator of hypoxia-induced AMPK activation and endothelial autophagy to promote angiogenesis.

MTORC1 directly inhibits AMPK to promote cell proliferation under nutrient stress

Central to cellular metabolism and cell proliferation are highly conserved signalling pathways controlled by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK)1,2, dysregulation of which are implicated in pathogenesis of major human diseases such as cancer and type 2 diabetes. AMPK pathways leading to reduced cell proliferation are well established and, in part, act through inhibition of TOR complex-1 (TORC1) activity. Here we demonstrate reciprocal regulation, specifically that TORC1 directly down-regulates AMPK signalling by phosphorylating the evolutionarily conserved residue Ser367 in the fission yeast AMPK catalytic subunit Ssp2, and AMPK α1Ser347/α2Ser345 in the mammalian homologs, which is associated with reduced phosphorylation of activation loop Thr172. Genetic or pharmacological inhibition of TORC1 signalling led to AMPK activation in the absence of increased AMP:ATP ratios; under nutrient stress conditions this was associated with growth limitation in both yeast and human cell cultures. Our findings reveal fundamental, bi-directional regulation between two major metabolic signalling networks and uncover new opportunity for cancer treatment strategies aimed at suppressing cell proliferation in the nutrient-poor tumor microenvironment.

Effect of acute transcranial magnetic stimulation on intracellular signalling in human skeletal muscle.

To investigate the potential of an acute bout of transcranial electrical stimulation to induce anabolic signalling. Experimental intervention on healthy subjects. Ten healthy subjects, 5 women and 5 men (mean age (standard deviation (SD) 32 years (SD 4)). The quadriceps muscle was stimulated at a frequency of 10 Hz for 10 s, followed by 20 s of rest, repeated 40 times over 20 min. Electromyography and force data were collected for all transcranial electrical stimulation sequences. Muscle biopsies were obtained from the vastus lateralis muscle before and 1 and 3 h after stimulation. One bout of transcranial electrical stimulation decreased phosphorylation of AKT at Thr308 (1 h: -29%, 3 h: -38%; p < 0.05) and mTOR phosphorylation at Ser2448 (1 h: -10%; ns, 3 h: -21%; p < 0.05), both in the anabolic pathway. Phosphorylation of AMPK, ACC and ULK1 were not affected. c-MYC gene expression was unchanged following transcranial electrical stimulation, but rDNA transcription decreased (1 h: -28%, 3 h: -19%; p < 0.05). PGC1α-ex1b mRNA increased (1 h: 2.3-fold, 3 h: 2.6-fold; p < 0.05), which also correlated with vastus lateralis electromyography activity, while other PGC-1α variants were unchanged. Acute transcranial electrical stimulation of skeletal muscle in weight-bearing healthy individuals did not induce anabolic signalling, and some signs of impaired muscle anabolism were detected, suggesting limited potential in preventing muscle wasting.