05-669 Sigma-AldrichAnti-phospho-Akt1/PKBα (Ser473) Antibody, clone 11E6

Sarcosine, an endogenous amino acid, is a competitive inhibitor of the type I glycine transporter and an N-methyl-d-aspartate receptor (NMDAR) coagonist. Recently, we found that sarcosine, an NMDAR enhancer, can improve depression-related behaviors in rodents and humans. This result differs from previous studies, which have reported antidepressant effects of NMDAR antagonists. The mechanisms underlying the therapeutic response of sarcosine remain unknown. This study examines the role of mammalian target of rapamycin (mTOR) signaling and α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR) activation, which are involved in the antidepressant-like effects of several glutamatergic system modulators. The effects of sarcosine in a forced swim test (FST) and the expression levels of phosphorylated mTOR signaling proteins were examined in the absence or presence of mTOR and AMPAR inhibitors. In addition, the influence of sarcosine on AMPAR trafficking was determined by analyzing the phosphorylation of AMPAR subunit GluR1 at the PKA site (often considered an indicator for GluR1 membrane insertion in neurons). A single injection of sarcosine exhibited antidepressant-like effects in rats in the FST and rapidly activated the mTOR signaling pathway, which were significantly blocked by mTOR inhibitor rapamycin or the AMPAR inhibitor 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) pretreatment. Moreover, NBQX pretreatment eliminated the ability of sarcosine to stimulate the phosphorylated mTOR signaling proteins. Furthermore, GluR1 phosphorylation at its PKA site was significantly increased after an acute in vivo sarcosine treatment. The results demonstrated that sarcosine exerts antidepressant-like effects by enhancing AMPAR-mTOR signaling pathway activity and facilitating AMPAR membrane insertion. Highlights-A single injection of sarcosine rapidly exerted antidepressant-like effects with a concomitant increase in the activation of the mammalian target of rapamycin mTOR signaling pathway.-The antidepressant-like effects of sarcosine occur through the activated AMPAR-mTOR signaling pathway.-Sarcosine could enhance AMPAR membrane insertion via an AMPAR throughput.

Different cancer cells exhibit altered sensitivity to metformin treatment. Recent studies suggest these findings may be due in part to the common cell culture practice of utilizing high glucose, and when glucose is lowered, metformin becomes increasingly cytotoxic to cancer cells. In low glucose conditions ranging from 0 to 5 mM, metformin was cytotoxic to breast cancer cell lines MCF7, MDAMB231 and SKBR3, and ovarian cancer cell lines OVCAR3, and PA-1. MDAMB231 and SKBR3 were previously shown to be resistant to metformin in normal high glucose medium. When glucose was increased to 10 mM or above, all of these cell lines become less responsive to metformin treatment. Metformin treatment significantly reduced ATP levels in cells incubated in media with low glucose (2.5 mM), high fructose (25 mM) or galactose (25 mM). Reductions in ATP levels were not observed with high glucose (25 mM). This was compensated by enhanced glycolysis through activation of AMPK when oxidative phosphorylation was inhibited by metformin. However, enhanced glycolysis was either diminished or abolished by replacing 25 mM glucose with 2.5 mM glucose, 25 mM fructose or 25 mM galactose. These findings suggest that lowering glucose potentiates metformin induced cell death by reducing metformin stimulated glycolysis. Additionally, under low glucose conditions metformin significantly decreased phosphorylation of AKT and various targets of mTOR, while phospho-AMPK was not significantly altered. Thus inhibition of mTOR signaling appears to be independent of AMPK activation. Further in vivo studies using the 4T1 breast cancer mouse model confirmed that metformin inhibition of tumor growth was enhanced when serum glucose levels were reduced via low carbohydrate ketogenic diets. The data support a model in which metformin treatment of cancer cells in low glucose medium leads to cell death by decreasing ATP production and inhibition of survival signaling pathways. The enhanced cytotoxicity of metformin against cancer cells was observed both in vitro and in vivo.

The purpose of this study is to investigate the effects of exercise on cancer progression, metastasis, and underlying mechanisms in an orthotopic model of murine prostate cancer. C57BL/6 male mice (6-8 wk of age) were orthotopically injected with transgenic adenocarcinoma of mouse prostate C-1 cells (5 × 10(5)) and randomly assigned to exercise (n = 28) or a non-intervention control (n = 31) groups. The exercise group was given voluntary access to a wheel 24 h/day for the duration of the study. Four mice per group were serially killed on days 14, 31, and 36; the remaining 38 mice (exercise, n = 18; control, n = 20) were killed on day 53. Before death, MRI was performed to assess tumor blood perfusion. Primary tumor growth rate was comparable between groups, but expression of prometastatic genes was significantly modulated in exercising animals with a shift toward reduced metastasis. Exercise was associated with increased activity of protein kinases within the MEK/MAPK and PI3K/mTOR signaling cascades with subsequent increased intratumoral protein levels of HIF-1α and VEGF. This was associated with improved tumor vascularization. Multiplex ELISAs revealed distinct reductions in plasma concentrations of several angiogenic cytokines in the exercise group, which was associated with increased expression of angiogenic and metabolic genes in the skeletal muscle. Exercise-induced stabilization of HIF-1α and subsequent upregulation of VEGF was associated with "productive" tumor vascularization with a shift toward suppressed metastasis in an orthotopic model of prostate cancer.

Activated protein C (APC) has a protective efficacy against ischemia-reperfusion (I/R) injury in several organs. The objective of this study was to investigate effect of APC in myocardium with possible mechanism.We used regional and global myocardial I/R injury models of rats. They consisted of I/R injuries (1) by ligation of left coronary artery, or (2) using Langendorff apparatus. Langendorff was used to focus the mechanism of APC excluding coagulation cascade in a working heart. Each experiment had an APC group (n=10) and a control group with normal saline (n=10). Injections of these solutions into rats were performed 30 minutes before the planned-I/R injury. Cardiac performance after the procedure was evaluated by echocardiography or indices with Langendorff apparatus. Coronary flow (CF) was measured in the global I/R injury model. Western blotting was performed to detect the change of AKT1 signal in myocardium after global I/R injury.LV FUNCTION IMPROVED SIGNIFICANTLY IN THE APC GROUP: %EF at 2 weeks after procedure, 70.8%± 4.5% vs. 56.5%± 0.7%; APC vs. control; pless than 0.01. Percent LV development pressure (LVDP) also improved in the APC group significantly, 88.8%± 45.3% vs. 28.1%± 15.4%; APC vs. control; pless than 0.01. In APC group, %CF improved significantly, 88.5%± 15.8% vs. 65.0%± 13.4%; APC vs. control; pless than 0.01. It was enhanced significantly when acetylcholine was administered; % CF: 103.5%± 9.9% vs. 87.0%± 12.1%; APC vs. control; pless than 0.05. Western blotting revealed that APC significantly induced activation of phosphorylated AKT1 in myocardium (pless than 0.05).APC has a novel effect to protect myocardium and cardiac performance against I/R injury through improvement of endothelial function and activation of AKT1.

Anti-metastasis by reducing cellular migration and invasion and by deregulating the expression of matrix metalloproteinases (MMPs) is a therapeutic approach for cancer treatment. The objective of this study focused on the effects of the novel compound 6-pyrrolidinyl-2-(2-hydroxyphenyl)-4-quinazolinone (MJ-29) regarding anti-metastatic actions on human oral squamous cell carcinoma CAL 27 cells and on the verification of the underlying related molecular mechanisms of this event. MJ-29 concentration- and time-dependently caused a suppression of cell adhesive ability utilizing cell adhesion assay; it also inhibited the migration and invasion of CAL 27 cells using scratch wound closure and transwell invasion assays in a concentration-dependent response. Importantly, we confirmed that the applied concentration range of MJ-29 exhibited no dramatic influence of cytotoxicity on CAL 27 cells using the thiazolyl blue tetrazolium bromide assay. MJ-29 also attenuated the enzymatic activity of MMP-2 and MMP-9. Furthermore, we found that activation of their upstream protein kinases, by MJ-29, potentially exerted an inhibitory effect on the phosphorylated protein levels of extracellular regulated protein kinase 1/2, p38 and c-Jun N-terminal kinase 1/2, as well as serine/threonine kinase AKT by MJ-29 in CAL 27 cells. The expression of RAS and focal adhesion kinase was also down-regulated in MJ-29-treated CAL 27 cells. Collectively, these findings provide further evidence for the molecular signaling basis of the effects of MJ-29 on suppression of migration and invasion which might be useful as a therapeutic strategy to treat human oral cancer.

In contemporary reinforcement learning models, reward prediction error (RPE), the difference between the expected and actual reward, is thought to guide action value learning through the firing activity of dopaminergic neurons. Given the importance of dopamine in reward learning and the involvement of Akt1 in dopamine-dependent behaviors, the aim of this study was to investigate whether Akt1 deficiency modulates reward learning and the magnitude of RPE using Akt1 mutant mice as a model. In comparison to wild-type littermate controls, the expression of Akt1 proteins in mouse brains occurred in a gene-dosage-dependent manner and Akt1 heterozygous (HET) mice exhibited impaired striatal Akt1 activity under methamphetamine challenge. No genotypic difference was found in the basal levels of dopamine and its metabolites. In a series of reward-related learning tasks, HET mice displayed a relatively efficient method of updating reward information from the environment during the acquisition phase of the two natural reward tasks and in the reverse section of the dynamic foraging T-maze but not in methamphetamine-induced or aversive-related reward learning. The implementation of a standard reinforcement learning model and the Bayesian hierarchical parameter estimation show that HET mice have higher RPE magnitudes and that their action values are updated more rapidly among all three test sections in T-maze. These results indicate that Akt1 deficiency modulates natural reward learning and RPE. This study showed a promising avenue for investigating RPE in mutant mice and provided evidence for the potential link from genetic deficiency, to neurobiological abnormalities, to impairment in higher-order cognitive functioning.

To elucidate the molecular mechanisms behind physical inactivity-induced insulin resistance in skeletal muscle, 12 young, healthy male subjects completed 7 days of bed rest with vastus lateralis muscle biopsies obtained before and after. In six of the subjects, muscle biopsies were taken from both legs before and after a 3-h hyperinsulinemic euglycemic clamp performed 3 h after a 45-min, one-legged exercise. Blood samples were obtained from one femoral artery and both femoral veins before and during the clamp. Glucose infusion rate and leg glucose extraction during the clamp were lower after than before bed rest. This bed rest-induced insulin resistance occurred together with reduced muscle GLUT4, hexokinase II, protein kinase B/Akt1, and Akt2 protein level, and a tendency for reduced 3-hydroxyacyl-CoA dehydrogenase activity. The ability of insulin to phosphorylate Akt and activate glycogen synthase (GS) was reduced with normal GS site 3 but abnormal GS site 2+2a phosphorylation after bed rest. Exercise enhanced insulin-stimulated leg glucose extraction both before and after bed rest, which was accompanied by higher GS activity in the prior-exercised leg than the rested leg. The present findings demonstrate that physical inactivity-induced insulin resistance in muscle is associated with lower content/activity of key proteins in glucose transport/phosphorylation and storage.

AMP-activated protein kinase (AMPK), an evolutionarily conserved serine-threonine kinase that senses cellular energy status, is activated by stress and neurohumoral stimuli. We investigated the mechanisms by which adrenergic signaling alters AMPK activation in vivo. Brown adipose tissue (BAT) is highly enriched in sympathetic innervation, which is critical for regulation of energy homeostasis. We performed unilateral denervation of BAT in wild type (WT) mice to abolish neural input. Six days post-denervation, UCP-1 protein levels and AMPK α2 protein and activity were reduced by 45%. In β(1,2,3)-adrenergic receptor knock-out mice, unilateral denervation led to a 25-45% decrease in AMPK activity, protein expression, and Thr(172) phosphorylation. In contrast, acute α- or β-adrenergic blockade in WT mice resulted in increased AMPK α Thr(172) phosphorylation and AMPK α1 and α2 activity in BAT. But short term blockade of α-adrenergic signaling in β(1,2,3)-adrenergic receptor knock-out mice resulted in decreased AMPK activity in BAT, which strongly correlated with enhanced phosphorylation of AMPK on Ser(485/491), a site associated with inhibition of AMPK activity. Both PKA and AKT inhibitors attenuated AMPK Ser(485/491) phosphorylation resulting from α-adrenergic blockade and prevented decreases in AMPK activity. In vitro mechanistic studies in BAT explants showed that the effects of α-adrenergic blockade appeared to be secondary to inhibition of oxygen consumption. In conclusion, adrenergic pathways regulate AMPK activity in vivo acutely via alterations in Thr(172) phosphorylation and chronically through changes in the α catalytic subunit protein levels. Furthermore, AMPK α Ser(485/491) phosphorylation may be a novel mechanism to inhibit AMPK activity in vivo and alter its biological effects.

FGF21 is a novel metabolic regulator involved in the control of glucose homeostasis, insulin sensitivity and ketogenesis. The liver has been considered the main site of production and release of FGF21 into the blood. Here we show that, after thermogenic activation, brown adipose tissue (BAT) becomes a source of systemic FGF21. This is due to a powerful cAMP-mediated pathway of regulation of FGF21 gene transcription. Norepinephrine, acting via β-adrenergic, cAMP-mediated, mechanisms and subsequent activation of protein kinase-A and p38 MAP kinase, induces FGF21 gene transcription and also FGF21 release in brown adipocytes. ATF2 binding to the FGF21 gene promoter mediates cAMP-dependent induction of FGF21 gene transcription. FGF21 release by brown fat in vivo was directly assessed by analyzing arterio-venous differences in FGF21 concentration across interscapular brown fat, in combination with blood flow to BAT and assessment of FGF21 half-life. This analysis demonstrates that exposure of rats to cold induced a marked release of FGF21 by brown fat in vivo, in association with a reduction in systemic FGF21 half-life. The present findings lead to the recognition of a novel pathway of regulation the FGF21 gene and an endocrine role of brown fat, as a source of FGF21 that may be specially relevant in conditions of activation of thermogenic activity.

Lung cancer is the leading cause of death from cancer in the world. Gefitinib is known to its inhibition of EGFR tyrosine kinase and worldwide used for antitumor in non-small cell lung cancer (NSCLC). Here, we show that Gefitinib reduces p-Akt levels, concomitant with elevation of p21 levels and suppression of cdk2/4 and cyclinE/D1 activities which result in impaired cell cycle progression through G1 arrest only in NSCLC cells in which it inhibits growth. We find that Gefitinib-induced p21 protein stability, rather than increased RNA accumulation, was responsible for the elevated p21 levels. More, treatment of beta-elemene, a natural plant drug extracted from Curcuma wenyujin, restored sensitivity to Gefitinib via the mechanism modulated the elevation of p21 levels in the cells which are acquired resistance to Gefitinib. These data suggest that administration of Gefitinib in combination with beta-elemene may offer great opportunities for NSCLC which are acquired resistance to Gefitinib. The p21 effect on the cells to response to Gefitinib was further confirmed by p21 over-expression and knockdown studies pointing to a requirement of p21 for the cells sensitive to Gefitinib. Thus, we propose that p21 is required for Gefitinib-sensitive NSCLC cells.Copyright © 2011 Elsevier Masson SAS. All rights reserved.