Decreased Serine Phosphorylation Research Articles

Targeting CDK9 in KMT2A-Rearranged Infant Leukemia: Evidence for Activity and Drug Synergy with Enitociclib

Introduction: Translocations of the lysine methyltransferase 2A (KMT2A) gene on 11q23 are seen in most infant leukemias. The outcomes in cases with KMT2A rearrangements (KMT2Ar) are significantly worse due to higher disease relapse rates and the emergence of chemo-resistance. Studies have shown that P-TEFb (CDK9/Cyclin T1) is an important component of the KMT2Ar complex and drives the transcriptional elongation step, subsequently contributing to the development of leukemia (Mueller, Dorothee et al.,2009). Based on this rationale, we investigated the potential of the selective P-TEFb/CDK9 inhibitor enitociclib in infant leukemia. Enitociclib potently inhibits MYC and MCL-1 in various tumor models (Sher, Steven et al.,2023). In addition, menin has been shown to be an important protein in the activation of target genes by KMT2Ar fusions, and menin inhibitors are considered promising therapeutic agents against infant leukemias, with at least four menin inhibitors currently in clinical development. We show preclinical data to support the activity of enitociclib with respect to cellular cytotoxicity, apoptosis induction and drug synergy in infant leukemia cells. Methods: A panel of pediatric leukemia cell lines representing high-risk and treatment resistant KMT2Ar cells were used. Leukemia cells with wild-type KMT2A as well as normal CD34+ cells and PBMCs from healthy donors were used as controls. Leukemic cells were treated with enitociclib (0.5µM - 0.03µM) and cell viability was measured using alamar blue assay. Apoptotic cell death following enitociclib treatment was measured by Annexin V/Propidium Iodide flow cytometry. Target modulation analyses were carried out by western blotting. Bone marrow (BM) stroma and leukemia co-culture experiments were performed to identify the effect of enitociclib on BM-derived growth support provided to leukemic cells. Drug combination assays were carried out with the menin inhibitor MI-463 and combination indices were calculated using the Chao-Talalay method. Rat xenograft models bearing infant leukemia cells were used to study the efficacy and tolerability of enitociclib therapy. Results: Data from in vitro cytotoxicity assays showed high sensitivity of KMT2Ar infant leukemia cells to enitociclib with IC 50 values of 30nM for KMT2Ar cells and 406nM for KMT2A-WT cells. Annexin V/PI staining showed a dose-dependent increase in cells in the late apoptotic stage for KMT2Ar cells after treatment. Target validation assays in KMT2Ar cells showed inhibition of transcriptional elongation by decreased serine phosphorylation by approximately 75% of RNA POL II CTD starting as early as 6 hours after treatment with 0.1µM enitociclib. Additionally, in KMT2Ar cells, an 80%-85% reduction in the amount of MCL-1 and MYC protein levels was observed after treatment with 0.1µM enitociclib for 6 hours. Furthermore, our data show that enitociclib may help to overcome the BM derived stromal mediated survival advantage in KMT2Ar leukemic cells. Enitociclib also potentiated the anti-leukemic effect of chemotherapies for childhood leukemia with the most effective combination observed with doxorubicin (0.01µM IC 50 for doxorubicin alone, 0.002µM IC 50 when combined with enitociclib) and prednisolone (25µM IC 50 for prednisolone only, 0.01µM IC 50 in combination). Drug combination assays showed effective synergy between enitociclib and the KMT2A-menin inhibitor MI-463 in KMT2Ar infant leukemia cells with a combination index of less than 1. As a detectable biomarker of activity, HOXA9 protein levels were found to be robustly diminished with 60% decrease after 24-hour treatment with the drug combination. In vivo, enitociclib given once weekly for 3 weeks led to complete remission in 10 out of 12 rats bearing KMT2Ar xenografts. Discussion: Our data demonstrate the ability of enitociclib to induce effective cellular cytotoxicity in high-risk KMT2Ar leukemia cells. We further show that enitociclib potentiated the anti-leukemic effect of chemotherapies that constitute the backbone of childhood leukemia protocols. Importantly, we show synergy with menin inhibition that is currently in consideration to be included in new generation clinical trials, suggesting the effective use of this novel combination in future treatment protocols. We provide the first in vitro and animal data supporting enitociclib in future treatment approaches for infants with KMT2Ar leukemia.

Role of NADP+-dependent isocitrate dehydrogenase from muscle tissue of Rana sylvatica in ROS defense during freeze-tolerance

The wood frog, Rana sylvatica, employs freeze tolerance as a winter survival strategy in seasonally cold environments. At subzero temperatures, up to 65–70% of total body water can freeze in extracellular spaces, halting vital functions (breathing, heartbeat) and causing ischemia that, in turn, can have numerous consequences including the generation of damaging reactive oxygen species (ROS). NADPH serves as a key donor of reductive power for most ROS detoxifying enzymes and can be generated by several metabolic pathways. One source of NADPH reducing power is the NADP-dependent isocitrate dehydrogenase (IDH) reaction. The present study evaluated the properties and regulation of IDH from skeletal muscle of R. sylvatica when frogs were exposed to stress conditions: freezing, dehydration or anoxia. Purified IDH exhibited higher affinity for isocitrate under all stress conditions as compared to controls, suggesting that the enzyme is primed to synthesize NADPH relative to the control state. Immunoblotting showed reduced serine and threonine phosphorylation of muscle IDH from frozen frogs and decreased serine phosphorylation on IDH from dehydrated frogs relative to control and anoxic states, demonstrating a reversible phosphorylation regulatory mechanism for IDH activity during freezing stress. Taken together, these results suggest activation and maintenance of IDH activity despite hypometabolic conditions. This initiation in activity of IDH during freezing may play a role in antioxidant defense by contributing to maintenance of the NADPH pool under stress conditions.

One-step purification and regulation of fructose 1,6-bisphosphatase from the liver of the freeze-tolerant wood frog, Rana sylvatica.

The wood frog (Rana sylvatica) undergoes numerous changes to its physiology and metabolic processes to survive the winter months, including adaptations that let them endure whole-body freezing. The regulation of key enzymes of central carbohydrate metabolism in the liver plays a crucial role in mediating the synthesis and maintenance of high concentrations of glucose as a cryoprotectant during freezing as well as glucose reconversion to glycogen after thawing. The present study characterized the regulation of fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) from wood frog liver during freezing, FBPase being a crucial enzyme regulating gluconeogenesis. Liver FBPase was purified to homogeneity from control and frozen wood frogs by a one-step chromatographic process. Kinetic and regulatory parameters of the enzyme were investigated and demonstrated a significant decrease in sensitivity to its substrate fructose-1,6-bisphosphatein the liver of frozen frogs, as compared with controls. Immunoblotting also revealed freeze-responsive changes in posttranslational modifications with a significant decrease in serine phosphorylation (by 53%) for FBPase from frozen frogs. Taken together, these results suggest that FBPase is suppressed, and gluconeogenesis is inhibited during freezing. This response acts as an important component of the metabolic survival strategy of the wood frog.

Phenolic compounds from coffee by-products modulate adipogenesis-related inflammation, mitochondrial dysfunction, and insulin resistance in adipocytes, via insulin/PI3K/AKT signaling pathways

The aim of this study was to evaluate the inhibitory potential of aqueous extracts from coffee silverskin (CSE) and husk (CHE) and their main phenolics on adipogenesis, obesity-related inflammation, mitochondrial dysfunction, and insulin resistance, in vitro. Coffee by-products extracts (31–500 μg mL−1) and pure phenolics (100 μmol L−1) reduced lipid accumulation and increased mitochondrial activity in 3T3-L1 adipocytes. Also reduced the expression of inducible nitric oxide synthase and cyclooxygenase-2 and diminished secretion of pro-inflammatory factors in LPS-stimulated RAW2643.7 macrophages. Cytokine release diminished (tumor necrosis factor α: 23–57%; monocyte chemoattractant protein 1: 42–60%; interleukin-6: 30–39%) and adiponectin increased (7–13- fold) in adipocytes treated with macrophage-conditioned media. ROS scavenging and activation of peroxisome proliferator-activated receptor γ coactivator 1-α pathway counteracted mitochondrial dysfunction. Increases in insulin receptor (1.4 to 4-fold), phosphoinositide 3-kinase (2 to 3-fold) and protein kinase B (1.3 to 3-fold) phosphorylation, in conjunction with a decrease in serine phosphorylation of insulin receptor substrate 1, evoked glucose transporter 4 translocation (8–15-fold) and glucose uptake (44–85%). CSE and CHE phenolics inhibited adipogenesis and elicited adipocytes browning. Suppressing macrophages-adipocytes interaction alleviated inflammation-triggered mitochondrial dysfunction and insulin resistance. CSE and CHE are beneficial in reducing adipogenesis and inflammation-related disorders.

S100A8 Protects Human Primary Alveolar Type II Cells against Injury and Emphysema.

Pulmonary emphysema is characterized by alveolar wall destruction, and cigarette smoking is the main risk factor in this disease development. S100A8 is a member of the S100 protein family, with an oxidative stress-related and antiinflammatory role. The mechanisms of human alveolar type II (ATII) cell injury contributing to emphysema pathophysiology are not completely understood. We wanted to determine whether S100A8 can protect ATII cells against injury induced by cigarette smoke and this disease development. We used freshly isolated ATII cells from nonsmoking and smoking organ donors, as well as patients with emphysema to determine S100A8 function. S100A8 protein and mRNA levels were low in individuals with this disease and correlated with its severity as determined by using lung tissue from areas with mild and severe emphysema obtained from the same patient. Its expression negatively correlated with high oxidative stress as observed by 4-hydroxynonenal levels. We also detected decreased serine phosphorylation within S100A8 by PKAα in this disease. This correlated with increased S100A8 ubiquitination by SYVN1. Moreover, we cultured ATII cells isolated from nonsmokers followed by treatment with cigarette smoke extract. We found that this exposure upregulated S100A8 expression. We also confirmed the cytoprotective role of S100A8 against cell injury using gain- and loss-of-function approaches in vitro. S100A8 knockdown sensitized cells to apoptosis induced by cigarette smoke. In contrast, S100A8 overexpression rescued cell injury. Our results suggest that S100A8 protects ATII cells against injury and cigarette smoke-induced emphysema. Targeting S100A8 may provide a potential therapeutic strategy for this disease.

Rare ginsenosides ameliorate lipid overload-induced myocardial insulin resistance via modulating metabolic flexibility

BackgroundRare ginsenosides are found in ginseng and notoginseng, two medicinal plants widely used in China for treatment of cardiovascular diseases and type 2 diabetes. However, their pharmacological studies regarding myocardial fuel metabolism and insulin signaling are not clear. PurposeTo explore the effect of a rare ginsenoside-standardized extract (RGSE), derived from steamed notoginseng, on cardiac fuel metabolism and insulin signaling. Study designWe used palmitic acid (PA) to treat H9c2 cells in vitro and high fat diet (HFD) to mice to induce insulin resistance in vivo. MethodsIn vitro, differentiated H9c2 cells were pretreated with RGSE, metformin, mildronate or dichloroacetate (DCA) and stimulated with PA. In vivo, mice were fed with HFD and received RGSE, metformin or DCA for 6 weeks. Protein expression was determined by Western blotting. Mitochondrial membrane potential (Δψm), glucose uptake and reactive oxygen species (ROS) production were measured by fluorescence labeling. Other assessments including oxygen consumption rate (OCR) were also performed. ResultsRGSE prevented PA-induced decrease in pyruvate dehydrogenase (PDH) activity and increase in carnitine palmitoyltransferase 1 (CPT1) expression, and ameliorated insulin-mediated glucose uptake and utilization in H9c2 cells. Metformin and mildronate exhibited similar effects. In vivo, RGSE counteracted HFD-induced increase in myocardial expression of p-PDH and CPT1 and ameliorated cardiac insulin signaling. Metformin and DCA also showed beneficial effects. Further study showed that RGSE decreased OCR and mitochondrial complex I activity in PA-treated H9c2 cells, reduced ROS production and relieved mitochondrial oxidative stress, thus decreased serine phosphorylation in IRS-1. ConclusionRGSE ameliorated myocardial insulin sensitivity under conditions of lipid overload, which was tightly associated with the decrease in mitochondrial oxidative stress via modulating glucose and fatty acid oxidation.

Modulation of brain insulin signaling in Alzheimer’s disease: New insight on the protective role of green coffee bean extract

Objective: Alzheimer's disease (AD), a neurodegenerative disorder, involves brain insulin signaling cascades and insulin resistance (IR). Because of limited treatment options, new treatment strategies are mandatory. Green coffee bean extract (GCBE) was reported to attenuate IR and improve brain energy metabolism. We aimed to investigate the possible use of GCBE as a prophylactic strategy to delay the onset of AD or combined with pioglitazone (PIO) as a strategy to retard the progression of AD.Methods: Rats received 10% fructose in drinking water for 18 weeks to induce AD. GCBE-prophylactic group received GCBE for 22 weeks started 4 weeks prior to fructose administration. The PIO group treated with PIO for 6 weeks started on week 12 of fructose administration. The GCBE+PIO group received GCBE for 22 weeks started 4 weeks prior to fructose administration and treated with PIO for the last 6 weeks of fructose administration.Results: Pretreatment with GCBE, either alone or combined with PIO, alleviated IR-induced AD changes. GCBE improved cognition, decreased serine phosphorylation of insulin receptor substrate, increased phosphoinositol-3 kinase (PI3K) activity and protein kinase B (Akt) gene expression, decreased glycogen synthase kinase-3β (GS3Kβ) gene expression and Tau hyperphosphorylation.Discussion: GCBE exerted neuroprotective effects against IR-induced AD mediated by alleviating IR and modulating brain insulin signaling cascade.

A heteromeric molecular complex regulates the migration of lung alveolar epithelial cells during wound healing

Alveolar type II epithelial cells (ATII) are instrumental in early wound healing in response to lung injury, restoring epithelial integrity through spreading and migration. We previously reported in separate studies that focal adhesion kinase-1 (FAK) and the chemokine receptor CXCR4 promote epithelial repair mechanisms. However, potential interactions between these two pathways were not previously considered. In the present study, we found that wounding of rat ATII cells promoted increased association between FAK and CXCR4. In addition, protein phosphatase-5 (PP5) increased its association with this heteromeric complex, while apoptosis signal regulating kinase-1 (ASK1) dissociated from the complex. Cell migration following wounding was decreased when PP5 expression was decreased using shRNA, but migration was increased in ATII cells isolated from ASK1 knockout mice. Interactions between FAK and CXCR4 were increased upon depletion of ASK1 using shRNA in MLE-12 cells, but unaffected when PP5 was depleted. Furthermore, we found that wounded rat ATII cells exhibited decreased ASK1 phosphorylation at Serine-966, decreased serine phosphorylation of FAK, and decreased association of phosphorylated ASK1 with FAK. These changes in phosphorylation were dependent upon expression of PP5. These results demonstrate a unique molecular complex comprising CXCR4, FAK, ASK1, and PP5 in ATII cells during wound healing.

Clove and Its Active Compound Attenuate Free Fatty Acid-Mediated Insulin Resistance in Skeletal Muscle Cells and in Mice

Several reports indicate anti-hyperglycemic effects of Syzygium aromaticum. In the present study, we report for the first time that clove extract (SAM) and its compound nigricin (NGC) decreases free fatty acid-mediated insulin resistance in mouse myoblasts. In addition, NGC was able to diminish insulin resistance in a diabetic mouse model. We observed that SAM and its compound NGC exhibited significant antioxidant activity in murine skeletal muscle cells. They also modulated stress signaling by reducing p38 MAP kinase phosphorylation. NGC and SAM treatments enhanced proximal insulin signaling by decreasing serine phosphorylation of insulin receptor substrate-1 (IRS-1) and increasing its tyrosine phosphorylation. SAM and NGC treatments also modified distal insulin signaling by enhancing protein kinase B (PKB) and glycogen synthase kinase-3-beta (GSK-3 beta) phosphorylation in muscle cells. Glucose uptake was enhanced in muscle cells after treatment with SAM and NGC. We observed increased glucose tolerance, glucose-stimulated insulin secretion, decreased insulin resistance, and increased beta cell function in diabetic mice treated with NGC. The results of our study demonstrate that clove extract and its active agent NGC can be potential therapeutic agents for alleviating insulin resistance.

SFRR-Europe Annual Award Lecture: Redox Biology and Metabolism in Brain Aging and Alzheimer׳s Disease

The gradual decline in energy metabolism during brain aging and some neurodegenerative disorders results in a hypometabolic state, which is a function of deficits in (a) substrate supply, (b) mitochondrial catalysis and energy transduction, and b) cytosolic metabolic, signaling, and transcriptional pathways. Mitochondria play a central role for they integrate several signaling processes and generate molecules such as H2O2 that coordinate cytosolic signaling and transcriptional pathways through thiol/disulfide exchange mechanisms. The hypometabolic state inherent in brain aging and a mouse model of Alzheimer׳s disease was examined in a model that integrates mitochondrial function, insulin signaling, and JNK signaling as well as the effects of nutraceutical interventions –likely acting through thiol/disulfide exchange mechanisms– in the regulation of these parameters. Brain aging proceeds with a decrease of glucose uptake (dynamic microPET imaging), which was associated with a decrease in the expression of the insulin-sensitive neuronal GLUT3/4 and microvascular endothelium GLUT1 (55 kD) but not astrocytic GLUT1 (45 kD). Brain aging was associated with an imbalance between the PI3K/Akt pathway of insulin signaling and JNK signaling and a down-regulation of the PGC1α-mediated transcriptional pathway of mitochondrial biogenesis, thus impairing on several aspects of energy homeostasis. Of note, these effects were observed in cortex- and hippocampal preparations but they are not necessarily cell specific, for astrocytes respond in a different manner: astrocytes showed an age-dependent increase in mitochondrial oxidative metabolism (respiring either on glucose or glucose plus pyruvate) and mitochondrial biogenesis. These metabolic changes were associated with an age-dependent increase in H2O2 generation (largely ascribed to NOX2) and NFκB signaling as well as augmented responses with age to inflammatory cytokines. Further, these inflammatory cytokines, IL-1β and TNFα stimulated mitochondrial oxidative metabolism and mitochondrial biogenesis in astrocytes. Impaired glucose uptake (accumulating into energy deficits) and synaptic plasticity are the major characteristics of the triple transgenic mouse model of Alzheimer׳s disease. These effects were accompanied with diminished membrane translocation of GLUT4 and GLUT3, increase in tyrosine phosphorylation of the IRS (inactivation) and decrease in serine phosphorylation (activation). The former effect coincided with increased activation of JNK1 and diminished activation of Akt. The JNK/Akt imbalance affected mitochondrial energy metabolism. The functional outcome of this hypometabolic state was translated into a substantially diminished long-term potentiation. The hypometabolic state in both models, brain aging and the mouse model of Alzheimer׳s disease, was rescued by treatment with lipoic acid, likely through a thiol/disulfide mechanism, that resulted largely in activation of the insulin receptor substrate and higher translocation to the membrane of the insulin-sensitive glucose transporters. In this manner, lipoic acid treatment appears to increase substrate supply; its effectiveness resulted in an increase of synaptic plasticity measured as long-term potentiation.

Abstract 668: Phosphoproteomic characterization of selumetinib action in KRAS mutant lung cancer

Abstract In the field of lung cancer therapeutics, KRAS-mutant lung cancer is a significant problem due to limited therapeutic options and poor prognosis. Pathway inhibition of the RAS-driven RAF-MEK-ERK pathway using small-molecule kinase inhibitors has been a key focus for treating cancers driven by oncogenic RAS; however, significant clinical responses are lacking. Our initial experiments assessed the efficacy of the clinical MEK inhibitor AZD6244 (selumetinib) in a panel of non-small cell lung cancer cell lines harboring oncogenic KRAS mutations. None of the cell lines showed pronounced apoptosis nor did we observe significant reduction in cell viability in nano-molar range (<1 μM). Given that cancer cells can rapidly rewire signaling cascades to adapt to loss of survival signaling, we hypothesized that a phosphoproteomics approach would provide further insight into adaptive signaling alteration associated with AZD6244. To this aim, we assessed global phosphoproteome change after AZD6244 treatment (1 μM, 24hr) in A427 and A549 lung adenocarcinoma cell lines harboring oncogenic KRAS mutations. We identified 7,730 unique phosphopeptides (corresponding to 3,137 unique phosphoproteins) after removing low-confidence phosphopeptides. We defined phosphopeptide significantly altered by AZD6244 as one that has had quantity changed by two-fold, corresponding to one standard deviation of fold change within the data. This resulted in 882 increased and 831 decreased phosphopeptides after MEK inhibition. As expected, AZD6244 decreased ERK phosphorylation and induced feedback increase of MEK and B- and C-RAF phosphorylation. In addition, we observed that AZD6244 increased tyrosine phosphorylation (Y1172) and decreased serine phosphorylation (S1045) of EGFR, which are involved in kinase activation and receptor trafficking, respectively. We also found reduced inhibitory serine phosphorylation of MET (S985), suggesting MEK inhibition could lead to MET activation by relieving inhibitory serine phosphorylation. These observations raise the possibility that MEK inhibition leads to feedback activation of RTKs, as well as its downstream activation (e.g., wild-type RAS). Notably, AZD6244 increased phosphorylation of KSR-1, a scaffolding protein required for assembly of MAPK signaling complex as well as phosphorylation of GEF-H1, a recently reportedly novel regulator of KSR-1 required for proper activation of RAS-driven MAPK activation. Collectively, our phosphoproteomics results indicate altered phosphorylation of a series of signaling proteins in MAPK signaling cascade, presumably contributing feedback activation of RAF, MEK and ERK reactivation following MEK inhibition. We are currently characterizing the importance of GEF-H1 and KSR-1 in lung cancer cell growth and survival and their role in adaptive signaling change associated with MEK inhibition. Updated work will be presented. Citation Format: Jae-Young Kim, Eric A. Welsh, Bin Fang, Fumi Kinose, John M. Koomen, Eric B. Haura. Phosphoproteomic characterization of selumetinib action in KRAS mutant lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 668. doi:10.1158/1538-7445.AM2015-668

γ-Aminobutyric A Receptor (GABAAR) Regulates Aquaporin 4 Expression in the Subependymal Zone: RELEVANCE TO NEURAL PRECURSORS AND WATER EXCHANGE

Activation of γ-aminobutyric A receptors (GABA(A)Rs) in the subependymal zone (SEZ) induces hyperpolarization and osmotic swelling in precursors, thereby promoting surface expression of the epidermal growth factor receptor (EGFR) and cell cycle entry. However, the mechanisms underlying the GABAergic modulation of cell swelling are unclear. Here, we show that GABA(A)Rs colocalize with the water channel aquaporin (AQP) 4 in prominin-1 immunopositive (P(+)) precursors in the postnatal SEZ, which include neural stem cells. GABA(A)R signaling promotes AQP4 expression by decreasing serine phosphorylation associated with the water channel. The modulation of AQP4 expression by GABA(A)R signaling is key to its effect on cell swelling and EGFR expression. In addition, GABA(A)R function also affects the ability of neural precursors to swell in response to an osmotic challenge in vitro and in vivo. Thus, the regulation of AQP4 by GABA(A)Rs is involved in controlling activation of neural stem cells and water exchange dynamics in the SEZ.

Astaxanthin prevents loss of insulin signaling and improves glucose metabolism in liver of insulin resistant mice

This study investigates the effects of astaxanthin (ASX) on insulin signaling and glucose metabolism in the liver of mice fed a high fat and high fructose diet (HFFD). Adult male Mus musculus mice of body mass 25-30 g were fed either normal chow or the HFFD. After 15 days, mice in each group were subdivided among 2 smaller groups and treated with ASX (2 mg·(kg body mass)⁻¹) in olive oil for 45 days. At the end of 60 days, HFFD-fed mice displayed insulin resistance while ASX-treated HFFD animals showed marked improvement in insulin sensitivity parameters. ASX treatment normalized the activities of hexokinase, pyruvate kinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, glycogen phosphorylase, and increased glycogen reserves in the liver. Liver tissue from ASX-treated HFFD-fed animals showed increased tyrosine phosphorylation and decreased serine phosphorylation of insulin receptor substrates (IRS)-1 and -2. ASX increased IRS 1/2 and phosphatidylinositol 3-kinase (PI3K) association and serine phosphorylation of Akt. In addition, ASX decreased HFFD-induced serine kinases (c-jun N-terminal kinase-1 and extracellular signal-regulated kinase-1). The results suggest that ASX treatment promotes the IRS-PI3K-Akt pathway of insulin signaling by decreasing serine phosphorylation of IRS proteins, and improves glucose metabolism by modulating metabolic enzymes.

Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake

The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.

Inhibition of the AMP-Activated Protein Kinase-α2 Accentuates Agonist-Induced Vascular Smooth Muscle Contraction and High Blood Pressure in Mice

The aim of the present study was to determine the effects and molecular mechanisms by which AMP-activated protein kinase (AMPK) regulates smooth muscle contraction and blood pressure in mice. In cultured human vascular smooth muscle cells, we observed that activation of AMPK by 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibited agonist-induced phosphorylation of myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1). Conversely, AMPK inhibition with pharmacological or genetic means potentiated agonist-induced the phosphorylation of MLC and MYPT1, whereas it inhibited both Ras homolog gene family member A and Rho-associated kinase activity. In addition, AMPK activation or Rho-associated kinase inhibition with Y27632 abolished agonist-induced phosphorylation of MLC and MYPT1. Gene silencing of p190-guanosine triphosphatase-activating protein abolished the effects of AMPK activation on MLC, MYPT1, and Ras homolog gene family member A in human smooth muscle cells. Ex vivo analyses revealed that agonist-induced contractions of the mesenteric artery and aortas were stronger in both AMPKα1(-/-) and AMPKα2(-/-) knockout mice than in wild-type mice. Inhibition of Rho-associated kinase with Y27632 normalized agonist-induced contractions of AMPKα1(-/-) and AMPKα2(-/-) vessels. AMPKα2(-/-) mice had higher blood pressure along with decreased serine phosphorylation of p190-guanosine triphosphatase-activating protein. Finally, inhibition of the Ras homolog gene family member A/Rho-associated kinase pathway with Y27632, which suppressed MYPT1 and MLC phosphorylation, lowered blood pressure in AMPKα2(-/-) mice. In conclusion, AMPK decreases vascular smooth muscle cell contractility by inhibiting p190-GTP-activating protein-dependent Ras homolog gene family member A activation, indicating that AMPK may be a new therapeutic target in lowering high blood pressure.

Acidic extracellular pH induces p120-catenin-mediated disruption of adherens junctions via the Src kinase-PKCδ pathway

An acidic microenvironment induces disruption of adherens junctions (AJs) of hepatoma cells. This study investigated the impact of an acidic extracellular pH (pHe) on p120-catenin (p120-ctn) serine phosphorylation. pH 6.6 treatment increased intracellular calcium levels, activated protein kinase C (PKC)α and PKCδ, and decreased serine phosphorylation of p120-ctn. Further knockdown of PKCα and δ by small interference RNA (siRNA) prevented the pH 6.6-induced downregulation of p120-ctn at AJ and the serine dephosphorylation of p120-ctn. Moreover, PP2 pretreatment and siRNA of c-Src abrogated the pH 6.6-induced PKCδ activation. Together, the c-Src-PKCδ cascade and PKCα regulate the acidic pHe-induced AJ disruption.

Glucose and fructose as functional modulators of overall dog, but not boar sperm function

The main aim of the present work was to test the effects of glucose and fructose on the phosphorylation levels of proteins linked to the control of overall sperm function in two species with very different metabolic characteristics, dog and boar. Incubation of dog spermatozoa with 10mM glucose increased serine phosphorylation of proteins related to cell cycle and signal transduction including cyclins B and E, Cdk2, Cdk6, Cdc6, PYK2, c-kit, Raf-1, TRK and several protein phosphatases. Incubation of dog spermatozoa with 10mM fructose decreased serine phosphorylation levels of cyclins B and D3, Cdk1/Cdc2, Cdk2, Cdk6, Akt, PI3 kinase, ERK-1 and protein kinase C. Incubation of boar spermatozoa with glucose or fructose did not modify any of the phosphorylation patterns studied. Given that one important difference between dog and boar spermatozoa is the presence of glucokinase (GK) in dog but not in boar, GK-transfected COS7 cells were incubated with either 10mM glucose or 10mM fructose. Incubation of GK-transfected cells with fructose decreased serine phosphorylation of cyclin A, ERK-2 and Hsp-70. In contrast, incubation of control COS7 cells with fructose increased serine phosphorylation of Cdk6, Cdk1/Cdc2, protein kinase C and Hsp-70. Incubation with glucose did not induce any significant effect. Our results indicate that monosaccharides act as signalling compounds in dog spermatozoa after ejaculation through changes in the phosphorylation levels of specific proteins. One of the factors that may be related to the action of sugars is the equilibrium of the total sperm hexokinase activity, in which the presence or absence of GK appears to be relevant.

Pyridoxine Improves Platelet Nitric Oxide Synthase Dysfunction Induced by Advanced Glycation End Products in vitro

Advanced glycation end products (AGEs) increase platelet aggregation and suppress vascular nitric oxide (NO) synthase (NOS) activity, and these effects may contribute to the atherothrombotic disease seen in diabetes. The aims of this study were to determine in vitro whether pyridoxine can abrogate the impairment in platelet NOS activity caused by AGEs, and to determine the mechanism by which it does this. Platelet aggregation was measured by Born aggregometry. Intraplatelet cyclic guanosine-3',5'-monophosphate (cGMP, an index of bioactive NO) was measured by radioimmunoassay. Serine-1177-specific phosphorylation of NOS type 3 (NOS-3) and phosphorylation of protein kinase Akt were determined in platelets by Western blotting. Phosphatidylinositol 3-kinase (PI3K) activity in platelets was ascertained by homogeneous time-resolved fluorescence (HTRF) assay. We found that AGE-modified albumin (AGEs) 200 mg/L increased platelet aggregability and decreased intraplatelet cGMP; these effects were largely attenuated by pyridoxine. Western blotting studies revealed that AGEs decreased NOS-3 phosphorylation on serine-1177, increased NOS-3 O-glycosylation, and decreased serine phosphorylation of protein kinase Akt; all of these changes were abrogated by pyridoxine. Direct measurement of PI3K activity in platelets demonstrated that all of the above effects could be attributed to a suppression by AGEs of PI3K activity, which was prevented by co-incubation with pyridoxine. We conclude that pyridoxine is effective in ameliorating the dysfunction of platelet NO signaling in response to AGEs, through improving PI3K activity, and hence downstream Akt phosphorylation and in turn serine-1177 phosphorylation of NOS-3.

Consumption of barley β-glucan ameliorates fatty liver and insulin resistance in mice fed a high-fat diet

Consumption of a diet high in barley beta-glucan (BG) has been shown to prevent insulin resistance. To investigate the mechanism for the effects of barley BG, three groups of male 7-wk-old C57BL/6J mice were fed high-fat diets containing 0, 2, or 4% of barley BG for 12 wk. The 2% BG and 4% BG groups had significantly lower body weights compared with the 0% BG group. The 4% BG group demonstrated improved glucose tolerance and lower levels of insulin-resistance index and glucose-dependent insulinotropic polypeptide. Consumption of the BG diet decreased hepatic lipid content. Mice on the BG diet also demonstrated decreased fatty acid synthase and increased cholesterol 7alpha-hydroxylase gene expression levels. The BG diet promoted hepatic insulin signaling by decreasing serine phosphorylation of insulin receptor substrate 1 and activating Akt, and it decreased mRNA levels of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. In summary, consumption of BG reduced weight gain, decreased hepatic lipid accumulation, and improved insulin sensitivity in mice fed a high-fat diet. Insulin signaling enhanced due to the expression changes of glucose and lipid metabolism genes by BG consumption. Consumption of barley BG could be an effective strategy for preventing obesity, insulin resistance, and the metabolic syndrome.

SIRT1 Exerts Anti-Inflammatory Effects and Improves Insulin Sensitivity in Adipocytes

SIRT1 is a prominent member of a family of NAD(+)-dependent enzymes and affects a variety of cellular functions ranging from gene silencing, regulation of the cell cycle and apoptosis, to energy homeostasis. In mature adipocytes, SIRT1 triggers lipolysis and loss of fat content. However, the potential effects of SIRT1 on insulin signaling pathways are poorly understood. To assess this, we used RNA interference to knock down SIRT1 in 3T3-L1 adipocytes. SIRT1 depletion inhibited insulin-stimulated glucose uptake and GLUT4 translocation. This was accompanied by increased phosphorylation of JNK and serine phosphorylation of insulin receptor substrate 1 (IRS-1), along with inhibition of insulin signaling steps, such as tyrosine phosphorylation of IRS-1, and phosphorylation of Akt and ERK. In contrast, treatment of cells with specific small molecule SIRT1 activators led to an increase in glucose uptake and insulin signaling as well as a decrease in serine phosphorylation of IRS-1. Moreover, gene expression profiles showed that SIRT1 expression was inversely related to inflammatory gene expression. Finally, we show that treatment of 3T3-L1 adipocytes with a SIRT1 activator attenuated tumor necrosis factor alpha-induced insulin resistance. Taken together, these data indicate that SIRT1 is a positive regulator of insulin signaling at least partially through the anti-inflammatory actions in 3T3-L1 adipocytes.