Protein kinaseB Akt Research Articles

Lipoic acid prevents mirtazapine-induced weight gain in mice without impairs its antidepressant-like action in a neuroendocrine model of depression

Mirtazapine (MIRT) is a multi-target antidepressant used in treatment of severe depression with promising efficacy, but also with important side effects, mainly sedation and weight gain. Thus, the present study aimed to test the effects of the neuroprotective antioxidant lipoic acid (ALA) in the reversal of weight and metabolic changes induced by MIRT in corticosterone-induced depression model in mice, as well as proposed mechanisms for their association antidepressant and pro-cognitive effects. To do these male Swiss mice received Tween 80 (control), corticosterone (CORT 20 mg / kg), MIRT (3 mg / kg) and ALA (100 or 200 mg / kg), alone or associated for 21 days. After this, the animals were subjected to behavioral tests for affective and cognitive domains. Daily weight changes, blood cholesterol fractions and corticosterone were measured. Also, hippocampus (HC) protein expression of the serotonin transporter (SERT), synaptophysin, protein kinase B-Akt (total and phosphorylated) and the cytokines IL-4 and IL-6 were investigated. CORT induced a marked depression-like behavior, memory deficits, metabolic changes (total cholesterol and LDL) and increased serum corticosterone. Also, CORT increased SERT expression in the HC. MIRT alone or combined with ALA sustained its antidepressant-like effect, as well as reversed CORT-induced impairment in spatial recognition memory. Additionally, the association MIRT+ALA200 reversed the weight gain induced by the former antidepressant, as well as reduced serum corticosterone levels and SERT expression in the HC. ALA alone induced significant weight loss and reduced total cholesterol and HDL fraction. Our findings provide promising evidence about the ALA potential to prevent metabolic and weight changes associated to MIRT, without impair its antidepressant and pro-cognition actions. Therefore, ALA+MIRT combination could represent a new therapeutic strategy for treating depression with less side effects.

Role of A-kinase anchoring proteins in cyclic-AMP-mediated Schwann cell proliferation

Proliferation of Schwann cells during peripheral nerve development is stimulated by the heregulin/neuregulin family of growth factors expressed by neurons. However, for neonatal rat Schwann cells growing in culture, heregulins produce only a weak mitogenic response. Supplementing heregulin with forskolin, an agent that elevates cyclic AMP levels, produces a dramatic increase in the proliferation of cultured Schwann cells. The mechanisms underlying this synergistic effect required for Schwann cell proliferation in vivo is not well established. Characterizing the A-kinase anchoring proteins (AKAPs) in Schwann cells might help identify substrates tethered to and phosphorylated by the cAMP-dependent protein kinase A (PKA). Using an RII overlay assay that detects AKAPs that are bound to the type II regulatory subunits of PKA, we identified AKAP150 in Schwann cells. Western blot analysis revealed that additional AKAPs, specifically AKAP95, and yotiao were also present. Disruption of PKA/AKAP interaction with Ht-31 peptide resulted in an increase in luciferase-conjugated cyclin D3 promoter activity. Transfection with sequence-specific AKAP siRNAs for AKAP150 and AKAP95 produced a marked reduction in cell proliferation. Immunoblot analysis revealed that knock down of AKAP95 protein caused a significant decrease in expression of the cell cycle regulatory proteins cyclin D2, cyclin D3 and the cell survival signal Akt/Protein Kinase B (Akt/PKB). Morphological characterization of Schwann cell AKAPs indicated the presence of nuclear (AKAP95), cytoplasm-associated (AKAP150) and perinuclear (yotiao) A-kinase anchoring proteins. These results indicate a role for AKAP95 and AKAP150 in the synergistic response of Schwann cells to treatment with heregulin and forskolin.

Obesity and chronic leptin resistance foster insulin resistance

Leptin is secreted from adipose tissue, maintains energy balance in our body, and regulates appetite via arcuate nucleus of the hypothalamus. It binds with its receptor (LepR) to kick-start multiple reaction cascades such as Janus kinase 2/signal transducer and activator of transcription 3, suppressor of cytokine signaling-3, insulin receptor substrate 1, phosphatidyl inositol 3-kinase, and protein kinase B-Akt. Insulin increases the uptake of fatty acids and enhances cellular glucose uptake and utilization. Insulin's metabolic effects are mediated by a number of tissue-specific pathways, some of which crosstalk leptin-mediated signaling. Studies showed that leptin resistance is instigated due to the excess release of leptin from adipocytes. It causes a lack of sensitivity toward leptin, for which the body fails to attain satiety and results in more food intake which in turn induces more obesity and aggravates further leptin resistance. Emphasizing on obesity, this review directs toward a possibility of chronic leptin resistance being responsible for insulin resistance. The above statement has been elicited by delineating the point of convergence between insulin and leptin signaling pathways.

Sevoflurane preconditioning inhibits cardiomyocyte injury induced by oxygen‑glucose deprivation by modulating TXNIP.

The thioredoxin interaction protein (TXNIP) has been reported to be closely related to cell oxidative stress, apoptosis and inflammation. TXNIP is involved in the regulation of oxidative stress in lung and renal injury. However, it is unclear as to whether it participates in the protective effects of sevoflurane preconditioning in cardiomyocyte injury caused by oxidative stress in ischemia. In the present study, H9c2 cardiomyocytes were cultured with 0, 1.5, 2, 3.5, 5 or 6% sevoflurane for 3h, followed by exposure to oxygen and glucose deprivation. The results demonstrated that oxygen and glucose deprivation induced an increase in TXNIP expression, lactate dehydrogenase (LDH) release, caspase‑3 activity, reactive oxygen species and malondialdehyde production. Preconditioning of the H9c2 cells with 3.5% sevoflurane suppressed TXNIP expression, LDH leakage, caspase‑3 activity, reactive oxygen species and malondialdehyde production, and it promoted cell viability. TXNIP overexpression reversed the effects of 3.5% sevoflurane preconditioning on caspase‑3 activity, reactive oxygen production and cell viability. Furthermore, TXNIP modulated p27 expression via PKB (protein kinase B/AKT) phosphorylation following preconditioning with 3.5% sevoflurane, and oxygen and glucose deprivation. On the whole these findings indicated that sevoflurane preconditioning protected the H9c2 cells against injury induced by oxygen and glucose deprivation by modulating TXNIP, AKT activation and p27 signaling.

Serine/Threonine Kinase 3-Phosphoinositide-Dependent Protein Kinase-1 (PDK1) as a Key Regulator of Cell Migration and Cancer Dissemination.

Dissecting the cellular signaling that governs the motility of eukaryotic cells is one of the fundamental tasks of modern cell biology, not only because of the large number of physiological processes in which cell migration is crucial, but even more so because of the pathological ones, in particular tumor invasion and metastasis. Cell migration requires the coordination of at least four major processes: polarization of intracellular signaling, regulation of the actin cytoskeleton and membrane extension, focal adhesion and integrin signaling and contractile forces generation and rear retraction. Among the molecular components involved in the regulation of locomotion, the phosphatidylinositol-3-kinase (PI3K) pathway has been shown to exert fundamental role. A pivotal node of such pathway is represented by the serine/threonine kinase 3-phosphoinositide-dependent protein kinase-1 (PDPK1 or PDK1). PDK1, and the majority of its substrates, belong to the AGC family of kinases (related to cAMP-dependent protein kinase 1, cyclic Guanosine monophosphate-dependent protein kinase and protein kinase C), and control a plethora of cellular processes, downstream either to PI3K or to other pathways, such as RAS GTPase-MAPK (mitogen-activated protein kinase). Interestingly, PDK1 has been demonstrated to be crucial for the regulation of each step of cell migration, by activating several proteins such as protein kinase B/Akt (PKB/Akt), myotonic dystrophy-related CDC42-binding kinases alpha (MRCKα), Rho associated coiled-coil containing protein kinase 1 (ROCK1), phospholipase C gamma 1 (PLCγ1) and β3 integrin. Moreover, PDK1 regulates cancer cell invasion as well, thus representing a possible target to prevent cancer metastasis in human patients. The aim of this review is to summarize the various mechanisms by which PDK1 controls the cell migration process, from cell polarization to actin cytoskeleton and focal adhesion regulation, and finally, to discuss the evidence supporting a role for PDK1 in cancer cell invasion and dissemination.

SiO2@antisense molecules covered by nepetalactone, extracted from Nepeta gloeocephala, inhibits ILK phosphorylation and downstream PKB/AKT signaling in HeLa cells.

In this study, the anticancer property of SiO2@antisense molecules (SiO2@AMs) and SiO2@AM covered by nepetalactone (SiO2@AM/CN), extracted from Nepeta gloeocephala, was investigated. Here integrin-linked kinase (ILK) phosphorylation and protein kinase B/AKT (PKB/AKT) signaling was studied when HeLa cells were exposed to SiO2@AM and SiO2@AM/CN. First, N. gloeocephala was identified at the Iranian National Herbarium. Then, its essential oil (EO) was obtained by the hydrodistillation method. In the next step, 4aα,7α,7aα-nepetalactone was extracted from the EO, based on the spectroscopic data. To obtain SiO2@AM/CN, 1 ml of SiO2@AM was mixed with extracted nepetalactone and then strongly shaken for 30 min. Finally, serial concentrations (100, 50, 25 and 12.5 μg ml-1) of SiO2@AM and SiO2@AM/CN were prepared and then exposed to HeLa cells (2 × 105 cells per ml) for 24 h at 37 °C. After incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell-cycle analysis, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and western blots were carried out. To find ILK phosphorylation and PKB/AKT signaling, the expression of threonine-173 (Thr-173), serine-246 (Ser-246), total ILK, AKT-Ser473, AKT-Thr308 and total AKT was investigated. HeLa cells that were treated with SiO2@AM/CN had G2/M arrest. Based on the TUNEL assay, many apoptotic cells have been shown when they were exposed to SiO2@AM/CN. Importantly, SiO2@AM/CN decreased ILK phosphorylation at Thr-173 and Ser-246 without affecting total ILK levels. Moreover, SiO2@AM/CN decreased AKT-Ser473 and AKT-Thr308 phosphorylation without affecting total PKB/AKT protein.

The dipeptidyl peptidase IV inhibitor vildagliptin suppresses development of neuropathy in diabetic rodents: effects on peripheral sensory nerve function, structure and molecular changes.

Incretin-related therapy was found to be beneficial for experimental diabetic neuropathy, but its mechanism is obscure. The purpose of this study is to explore the mechanism through which dipeptidyl peptidase IV inhibitor, vildagliptin (VG), influences neuropathy in diabetic rodents. To this end, non-obese type 2 diabetic Goto-Kakizaki rats (GK) and streptozotocin (STZ)-induced diabetic mice were treated with VG orally. Neuropathy was evaluated by nerve conduction velocity (NCV) in both GK and STZ-diabetic mice, whereas calcitonin-gene-related peptide expressions, neuronal cell size of dorsal root ganglion (DRG) and intraepidermal nerve fiber density were examined in GK. DRG from GK and STZ-diabetic mice served for the analyses of GLP-1 and insulin signaling. As results, VG treatment improved glucose intolerance and increased serum insulin and GLP-1 in GK accompanied by the amelioration of delayed NCV and neuronal atrophy, reduced calcitonin-gene-related peptide expressions and intraepidermal nerve fiber density. Diet restriction alone did not significantly influence these measures. Impaired GLP-1 signals such as cAMP response element binding protein, protein kinase B/Akt (PKB/Akt) and S6RP in DRG of GK were restored in VG-treated group, but the effect was equivocal in diet-treated GK. Concurrently, decreased phosphorylation of insulin receptor substrate 2 in GK was corrected by VG treatment. Consistent with the effect on GK, VG treatment improved NCV in diabetic mice without influence on hyperglycemia. DRG of VG-treated diabetic mice were characterized by correction of GLP-1 signals and insulin receptor substrate 2 phosphorylation without effects on insulin receptor β expression. The results suggest close association of neuropathy development with impaired signaling of insulin and GLP-1 in diabetic rodents. Diabetic neurons are resistant to insulin and such insulin resistance may contribute to development of neuropathy. DPP-IV inhibitor, vildagliptin, corrected insulin resistance and improved neuropathy irrespective of blood glucose via augmented action of GLP-1.

Abstract 1951: Identification of novel molecular targets for treatment of castration-resistant prostate cancer

Abstract One of the major limitations of current therapies for treatment of prostate cancer is the acquisition of castration-resistance by tumor cells. Castration-resistant prostate cancer (CRPC) proceeds through multiple signaling pathways that utilize the PI3K/AKT axis to direct anti-apoptotic and pro-survival responses in CRPC cells. We performed in-depth genomic and phenotypic analysis which revealed that two transcription factors, Cyclic AMP Response Element Binding protein (CREB) and Forkhead Box O3 (FOXO3a), were inversely expressed in CRPC cells. Specifically, an upregulation of CREB in stage-specific manner in CRPC tumor cells was suggestive of its proliferative role in tumor growth. Conversely, downregulation of FOXO3a expression suggested inhibition of this transcription factor's pro-apoptotic function, which may also facilitate tumor growth. In pre-clinical models of CRPC, both CREB and FOXO3a transcriptionally regulated the function of prostate tumor suppressor, Par-4 (prostate apoptotic response gene-4). Promoter bashing studies identified consensus regulatory target binding sites of CREB and FOXO3a on Par-4 promoter. The sequential deletion of three CREB binding sites in the Par-4 promoter relieved CREB-mediated suppression of Par-4, whereas sequential deletion of FOXO3a binding sites in Par-4 promoter abrogated Par-4 promoter activity. We further dissected upstream events of CREB and FOXO3a, which are regulated by protein kinase-B AKT using a small-molecule AKT inhibitor (SMAI) drug. Overexpression of AKT in CRPC (DU-145/AKT, C4-2B/AKT and PC-3/AKT) cells induced pro-survival machinery by activating CREB and inhibiting pro-apoptotic function of FOXO3a and resulting in resistance of cells to drug. The in vivo efficacy of our SMAI drug was evaluated in DU-145/AKT and C4-2B/AKT tumor xenografts in nude mouse model. AKT overexpressing tumors exhibited aggressive growth rates and exhibited high vascularisation and angiogenesis as compared to vector-transfected tumors. Daily oral administration of SMAI drug significantly inhibited tumor growth in both DU-145 and C4-2B AKT-overexpressing tumors. IHC analysis of these tumor tissues revealed reduced FOXO3a expression and increased pCREB expression following treatment with SMAI drug in AKT versus control tumors. The cooperative effects of CREB and FOXO3a in AKT-mediated signaling suggests that the development of small molecule inhibitors targeting AKT/CREB function while simultaneously inducing FOXO3a/Par-4 activation in CRPC tumors, represents an attractive target for drug development. Citation Format: Jim Moselhy, Ram V. Roy, Suman Suman, Trinath P. Das, Murali Ankem, Chendil Damodaran. Identification of novel molecular targets for treatment of castration-resistant prostate 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 1951. doi:10.1158/1538-7445.AM2015-1951

Amplified lipid rafts of malignant cells constitute a target for inhibition of aberrantly active NFAT and melanoma tumor growth by the aminobisphosphonate zoledronic acid.

Nuclear factors of activated T cells (NFAT) are critical modulators of cancer cell growth and survival. However, the mechanisms of their oncogenic dysregulation and strategies for targeting in tumors remain elusive. Here, we report coupling of anti- apoptotic NFAT (NFAT2) activation to cholesterol-enriched lipid raft microdomains of malignant melanoma cells and interruption of this pathway by the aminobisphosphonate zoledronic acid (Zol). The pathway was indicated by capability of Zol to promote apoptosis and to retard in vivo outgrowth of tumorigenic melanoma cell variants through inhibition of permanently active NFAT2. NFAT2 inhibition resulted from disintegration of cholesterol-enriched rafts due to reduction of cellular cholesterol by Zol. Mechanistically, raft disruption abolished raft-localized robust store-operated Ca(2+) (SOC) entry, blocking constitutive activation of protein kinase B/Akt (PKB) and thereby reactivating the NFAT repressor glycogen synthase kinase 3β (GSK3β). Pro-apoptotic inactivation of NFAT2 also followed reactivation of GSK3β by direct inhibition of PKB or SOC, whereas GSK3β blockade prevented Zol-induced NFAT2 inhibition and cell death. The rescuing effect of GSK3β blockade was reproduced by recovery of entire SOC/PKB/GSK3β cascade after reconstitution of rafts by cholesterol replenishment of Zol-treated tumorigenic cells. Remarkably, these malignant cells displayed higher cholesterol and lipid raft content than non-tumorigenic cells, which expressed weak SOC, PKB and NFAT2 activities and resisted raft-ablating action of Zol. Together, the results underscore the functional relevance of amplified melanoma rafts for tumor-promoting NFAT2 signaling and reveal these distinctive microdomains as a target for in vitro and in vivo demise of tumorigenic cells through NFAT2 inhibition by the clinical agent Zol.

Activation of spinal phosphatidylinositol 3-kinase/protein kinase B mediates pain behavior induced by plantar incision in mice

The etiology of postoperative pain may be different from antigen-induced inflammatory pain and neuropathic pain. However, central neural plasticity plays a key role in incision pain. It is also known that phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (PKB/Akt) are widely expressed in laminae I–IV of the spinal horn and play a critical role in spinal central sensitization. In the present study, we explored the role of PI3K and Akt in incision pain behaviors. Plantar incision induced a time-dependent activation of spinal PI3K-p110γ and Akt, while activated Akt and PI3K-p110γ were localized in spinal neurons or microglias, but not in astrocytes. Pre-treatment with PI3K inhibitors, wortmannin or LY294002 prevented the activation of Akt brought on by plantar incision in a dose-dependent manner. In addition, inhibition of spinal PI3K signaling pathway prevented pain behaviors (dose-dependent) and spinal Fos protein expression caused by plantar incision. These data demonstrated that PI3K signaling mediated pain behaviors caused by plantar incision in mice.

May the Force move you: TSC‐ing the mechanical activation of mTOR

That fact that lifting heavy loads causes muscles to grow bigger and stronger has been recognized for over 2000 years. However, it is only in the last 15 years that the necessary molecular tools have been developed to begin to understand how the mechanical signal of load across the muscle is converted into a chemical signal that results in a change in muscle phenotype. In this issue of The Journal of Physiology, that understanding takes another significant step forward courtesy of insight revealed by Jacobs and colleagues (2013). Up until now, it was clear that the mechanistic target of rapamycin (mTOR) was activated in a load-dependent manner (Baar & Esser, 1999), and that the increase in mTOR activity was required for the load-induced increase in muscle protein synthesis and hypertrophy (Goodman et al. 2011). This makes sense in that mTOR (1) can increase protein synthesis and limit protein breakdown, (2) can be activated by growth signals (activity, feeding, and hormones), and (3) is inhibited by stress (fasting, unfolded proteins, alcohol consumption, aging, etc.). But the issue of how mTOR was activated by activity in muscle remained unclear. In most cells, mTOR is activated when a growth factor binds to a receptor tyrosine kinase and activates phosphoinositide 3-kinase and protein kinase B/Akt (PKB). Once activated, PKB phosphorylates and inactivates tuberin (TSC2), a GTPase activating protein that turns off the Ras-homologue enriched in brain (Rheb). Since Rheb is the direct activator of mTOR, inactivating TSC2 (turning the inhibitor off) is the switch that activates mTOR, promoting protein synthesis and cell growth. The canonical growth factor pathway can activate mTOR. However, in muscle, the activation of mTOR by loading occurs in a growth factor-independent manner (Philp et al. 2011). That is, muscle loading enhances mTOR activity in the absence of classical growth factor-activated signals. This suggests the existence of a mechanoreceptor, a protein that can directly ‘feel the force’ and transduce it into a chemical signal. Jacobs and her colleagues show in this issue of The Journal of Physiology that, like growth factors, mechanotransduction targets TSC2. Using immunoprecipitation and λ phosphatase, the authors showed that TSC2 was phosphorylated in response to resistance exercise. Unlike growth factors, the phosphorylation of TSC2 did not occur at the previously described Thr1462 site. Instead, Jacobs showed that TSC2 was phosphorylated at other Ser/Thr residues within an RxRxx motif. The next interesting observation was that the phosphorylation of this motif was associated with a move of TSC2 from the membrane fraction to the cytoplasm. The highlight of the work was their use of frequency scatterplots to co-localize TSC2 and LAMP2, a marker of the lysosome. Using ultra-thin sections they showed that resistance exercise causes a 90% decrease in the association of TSC2 and LAMP2, suggesting that TSC2 had been moved away from the lysosome. This is important because the lysosome is where the mTOR activator Rheb is located. Together, these data suggest that a mechanoreceptor senses loading, induced by eccentric contractions in this case, and turns on an RxRxx-targeted kinase that phosphorylates TSC2 and moves it off the lysosome and away from its target Rheb, allowing mTOR activation (Fig. 1). Figure 1 A schematic of the activation of mTOR following resistance exercise Not only does TSC2 move away from the lysosome, but Jacobs also showed that mTOR moves to the lysosome 1 h after resistance exercise. However, whether the movement of mTOR to the lysosome is dependent on the mechanical load or amino acids is not entirely clear. We do know that within the first 90 min following resistance exercise there is a load-dependent increase in the amino acid leucine in the active muscle (MacKenzie et al. 2009). Since leucine causes the translocation of mTOR to the lysosome through the leucyl tRNA synthase–Rag–Ragulator pathway (Sancak et al. 2010), this suggests that mTOR is moving to the lysosome as a result of the increase in amino acids and not a direct mechanical signal. This observation also explains why taking extra amino acids immediately following resistance exercise can increase protein synthesis and muscle growth, probably by amplifying the movement of mTOR to the lysosome. As with any good research paper, the findings by Jacobs and colleagues raise further questions. What load-induced kinase targets RxRxx motifs in TSC2? How is such a kinase activated by load? Conversely, in the absence of load, what are the mechanisms that tether TSC2 to the lysosome? Can TSC2 be ‘encouraged’ to dissociate from the lysosome, perhaps to a therapeutic end? Answers to these questions, would finally give us a complete understanding of how muscle feels the force. Jacobs and colleagues have given us a good start.

Insulino-mimetic and anti-diabetic effects of zinc

While it has long been known that zinc (Zn) is crucial for the proper growth and maintenance of normal biological functions, Zn has also been shown to exert insulin-mimetic and anti-diabetic effects. These insulin-like properties have been demonstrated in isolated cells, tissues, and different animal models of type 1 and type 2 diabetes. Zn treatment has been found to improve carbohydrate and lipid metabolism in rodent models of diabetes. In isolated cells, it enhances glucose transport, glycogen and lipid synthesis, and inhibits gluconeogenesis and lipolysis. The molecular mechanism responsible for the insulin-like effects of Zn compounds involves the activation of several key components of the insulin signaling pathways, which include the extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3-K)/protein kinase B/Akt (PKB/Akt) pathways. However, the precise molecular mechanisms by which Zn triggers the activation of these pathways remain to be clarified. In this review, we provide a brief history of zinc, and an overview of its insulin-mimetic and anti-diabetic effects, as well as the potential mechanisms by which zinc exerts these effects.

Lipid rafts couple store-operated Ca 2+ entry to constitutive activation of PKB/Akt in a Ca 2+ /calmodulin-, Src- and PP2A-mediated pathway and promote melanoma tumor growth

Central role of constitutively active protein kinase B/Akt (PKB) in melanoma drives the search for new targets to abolish its deranged signaling. PKB activation is promoted by cholesterol-enriched lipid rafts and is Ca(2+)-dependent, but the pathway linking rafts and Ca(2+) to deregulation of this enzyme remains poorly understood. Here employing B16BL6 melanoma model, we show that ablation of rafts with methyl-β-cyclodextrin (MβCD) inactivated PKB by inhibiting Src kinase and reactivating the negative PKB modulator, PP2A phosphatase. Blockade of PP2A with okadaic acid rescued PKB, indicating that raft ablation reactivated PP2A through inhibiting Src. Indeed, direct Src blockade with the Src kinase inhibitor-1 or the dominant-negative Src-mutant was sufficient for PP2A reactivation and downregulation of PKB, whereas reconstitution of rafts in MβCD-treated cells restored PKB, PP2A and Src activities to their basal levels. This pathway was also interrupted by inhibition of the Ca(2+) sensor calmodulin, either by its antagonist N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide or the Ca(2+)-insensitive calmodulin-mutant or the intracellular Ca(2+)-chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N#N#-tetraacetic acid tetra-(acetocymethyl)-ester or by diminishing the store-operated Ca(2+) entry with 2-aminoethoxydiphenyl borate or small hairpin RNA against Stim1. Ablation of rafts prevented Stim1-mediated store-operated Ca(2+) entry, aborted Ca(2+) stimulation of raft-residing calmodulin and disrupted its Ca(2+)-dependent binding to Src, abolishing Src activity and entire Src/PP2A/PKB cascade. Most importantly, blockade of this cascade in the tumor site by raft-ablating MβCD, administered to melanoma-bearing mice, robustly retarded tumor growth and extended animal survival. Together, our data suggest that lipid rafts couple store-operated Ca(2+) entry to sustained activation of major tumor-promoting signaling elements in melanoma cells and underscore the potential of raft-targeting agents as effective anticancer drugs.

Cellular Mechanism of Zinc–Hinokitiol Complexes in Diabetes Mellitus

Abstract Zinc complexes exhibit high insulin-like and antidiabetic activities in animals with type 2 diabetes mellitus (DM). However, molecular mechanisms underlying these activities have not yet been determined. In this study, we investigated activation of the insulin signaling pathway by Zn in 3T3-L1 adipocytes using di(hinokitiolato)zinc complex (hinokitiol: 2-hydroxy-4-isopropylcyclohepta-2,4,6-trienone, [Zn(hnk)2]), which exhibits antidiabetic effect in animals with type 2 DM. Our results show that [Zn(hnk)2] strongly induced Akt/protein kinase B (Akt/PKB) phosphorylation, and optimal phosphorylation was achieved at a concentration of 50 µM. The [Zn(hnk)2]-induced Akt/PKB phosphorylation was almost completely inhibited by wortmannin, whereas [Zn(hnk)2]-induced phosphorylation of glycogen synthase kinase-3β (GSK3β) was partially inhibited by wortmannin. Further, we examined cellular Zn uptake by [Zn(hnk)2] stimulation and evaluated cellular glucose uptake at the same time point. The intracellular Zn concentration incubated with [Zn(hnk)2] was approximately 4.7-fold higher than that in the control cells. Moreover, the glucose uptake of [Zn(hnk)2]-treated adipocytes was 3.7-fold higher than that of the control adipocytes. These results suggest that [Zn(hnk)2] was able to translocate glucose transporter 4 (GLUT4) protein to the plasma membrane. Thus, we propose that [Zn(hnk)2] produces the antidiabetic effect by inducing insulin signaling pathways and glucose uptake.

Uncoupling protein 3 expression levels influence insulin sensitivity, fatty acid oxidation, and related signaling pathways

Controversy exists on whether uncoupling protein 3 (UCP3) positively or negatively influences insulin sensitivity in vivo, and the underlying signaling pathways have been scarcely studied. We studied how a progressive reduction in UCP3 expression (using UCP3 +/+, UCP3 +/-, and UCP3 -/- mice) modulates insulin sensitivity and related metabolic parameters. In order to further validate our observations, we also studied animals in which insulin resistance was induced by administration of a high-fat diet (HFD). In UCP3 +/- and UCP3 -/- mice, gastrocnemius muscle Akt/protein kinase B (Akt/PKB) (serine 473) and AMP-activated protein kinase (AMPK) (threonine 171) phosphorylation, and glucose transporter 4 (GLUT4) membrane levels were reduced compared to UCP3 +/+ mice. The HOMA-IR index (insulin resistance parameter) was increased both in the UCP3 +/- and UCP3 -/- mice. In these mice, insulin administration normalized Akt/PKB phosphorylation between genotypes while AMPK phosphorylation was further reduced, and sarcolemmal GLUT4 levels were induced but did not reach control levels. Furthermore, non-insulin-stimulated muscle fatty acid oxidation and the expression of several involved genes both in muscle and in liver were reduced. HFD administration induced insulin resistance in UCP3 +/+ mice and the aforementioned parameters resulted similar to those of chow-fed UCP3 +/- and UCP3 -/- mice. In conclusion, high-fat-diet-induced insulin resistance in wild-type mice mimics that of chow-fed UCP3 +/- and UCP3 -/- mice showing that progressive reduction of UCP3 levels results in insulin resistance. This is accompanied by decreased fatty acid oxidation and a less intense Akt/PKB and AMPK signaling.

Flavonoids as Protein Kinase Inhibitors for Cancer Chemoprevention: Direct Binding and Molecular Modeling

Protein kinases play crucial roles in the regulation of multiple cell signaling pathways and cellular functions. Deregulation of protein kinase function has been implicated in carcinogenesis. The inhibition of protein kinases has emerged as an important target for cancer chemoprevention and therapy. Accumulated data revealed that flavonoids exert chemopreventive effects through acting at protein kinase signaling pathways, more than as conventional hydrogen-donating antioxidants. Recent studies show that flavonoids can bind directly to some protein kinases, including Akt/protein kinase B (Akt/PKB), Fyn, Janus kinase 1 (JAK1), mitogen-activated protein kinase kinase 1 (MEK1), phosphoinositide 3-kinase (PI3K), mitogen-activated protein (MAP) kinase kinase 4 (MKK4), Raf1, and zeta chain-associated 70-kDa protein (ZAP-70) kinase, and then alter their phosphorylation state to regulate multiple cell signaling pathways in carcinogenesis processes. In this review, we report recent results on the interactions of flavonoids and protein kinases, especially their direct binding and molecular modeling. The data suggest that flavonoids act as protein kinase inhibitors for cancer chemoprevention that were thought previously as conventional hydrogen-donating antioxidant. Moreover, the molecular modeling data show some hints for creating natural compound-based protein kinase inhibitors for cancer chemoprevention and therapy.

Coupling of mitochondria to store-operated Ca2+-signaling sustains constitutive activation of protein kinase B/Akt and augments survival of malignant melanoma cells

Mitochondria are emerging as a major hub for cellular Ca2+-signaling, though their contribution to Ca2+-driven growth- and survival-promoting events in cancer is poorly understood. Here employing flow cytometry to monitor mitochondrial and cytosolic Ca2+, we assessed trans-mitochondrial Ca2+-transport and store-operated Ca2+-influx (store-operated channels (SOC)) in malignant vs. non-malignant B16BL6 melanoma clones. Remarkably, mitochondrial Ca2+-fluxes measured in whole cells or in isolated mitochondria were accelerated in the malignant clones compared to their non-malignant counterpart clones. This coincided with enhanced SOC-mediated Ca2+-influx and high levels of constitutively active protein kinase B/Akt (PKB). Interruption of trans-mitochondrial Ca2+-transport in the malignant cells with an antagonist of the mitochondrial Na+/Ca2+ exchanger, CGP-37157, abolsihed SOC-mediated Ca2+-influx, inactivated PKB, retarded cell growth and increased vulnerability to apoptosis. Similarly, direct SOC blockade by silencing Stim1 inhibited PKB, indicating that the crosstalk between SOC and mitochondria is essential to preserve PKB in constitutively active state. Finally, the retraction of mitochondria from sub-plasmalemmal micro-domains triggered by Fis1 over-expression inhibited SOC-coupled trans-mitochondrial Ca2+-flux, Ca2+-entry via SOC and PKB activity. Taken together, our data show that in the malignant melanoma cells, the functional and spatial relationship of up-regulated mitochondrial Ca2+-transport to the SOC sustains the robust Ca2+-responses and down-stream signaling critical for apoptosis-resistance and proliferation.

PPARs: Nuclear Receptors Controlled by, and Controlling, Nutrient Handling through Nuclear and Cytosolic Signaling.

Peroxisome proliferator-activated receptors (PPARs), which are known to regulate lipid homeostasis, are tightly controlled by nutrient availability, and they control nutrient handling. In this paper, we focus on how nutrients control the expression and action of PPARs and how cellular signaling events regulate the action of PPARs in metabolically active tissues (e.g., liver, skeletal muscle, heart, and white adipose tissue). We address the structure and function of the PPARs, and their interaction with other nuclear receptors, including PPAR cross-talk. We further discuss the roles played by different kinase pathways, including the extracellular signal-regulated kinases/mitogen-activated protein kinase (ERK MAPK), AMP-activated protein kinase (AMPK), Akt/protein kinase B (Akt/PKB), and the NAD+-regulated protein deacetylase SIRT1, serving to control the activity of the PPARs themselves as well as that of a key nutrient-related PPAR coactivator, PPARγ coactivator-1α (PGC-1α). We also highlight how currently applied nutrigenomic strategies will increase our understanding on how nutrients regulate metabolic homeostasis through PPAR signaling.

Important roles of Akt PKB signaling in the aging process

The increased costs associated with an ever-growing aged population are expected to pose a significant burden on health care resources. From a biological standpoint, aging is an accelerated deteriorative process in tissue structure and function that is associated with higher morbidity and mortality. The Akt / protein kinase B (PKB) is a family of serine / threonine protein kinases, which play prominent roles in a diverse number of processes including cell survival, cell growth, gene expression, apoptosis, protein synthesis, energy metabolism and oncogenesis. It is likely that age-related changes in tissue structure and function are related to alterations in Akt expression and Akt-dependent signaling. Here we review the role that Akt may play in the aging process and attempt, where possible, to highlight how these data may lead to new directions of inquiry and clinical relevance to the aged.

Sevoflurane Postconditioning Protects Chronically-Infarcted Rat Hearts against Ischemia-Reperfusion Injury by Activation of Pro-survival Kinases and Inhibition of Mitochondrial Permeability Transition Pore Opening upon Reperfusion

We evaluated the cardioprotection against myocardial ischemia-reperfusion injury induced by sevoflurane postconditioning (SpostC) in chronically-infarcted rat hearts, and investigated the roles of phosphoinositide 3-kinase (PI3K)-protein kinase B/Akt (PKB/Akt), mitogen-activated extracellular regulated kinase 1/2 (MEK 1/2)-extracellular regulated kinase 1/2 (ERK 1/2), and mitochondrial permeability transition pore (mPTP). Left anterior descending (LAD) coronary artery was ligated to induce myocardial infarction in rats. Six weeks later, chronically-infarcted hearts were isolated and subjected to 30 min of global ischemia, followed by 1 h of reperfusion with Krebs-Henseleit (K-H) buffer. SpostC was administered by perfusing the hearts with K-H buffer saturated with 3% sevoflurane during the first 15 min of reperfusion. To evaluate the role of PI3K-PKB/Akt and MEK 1/2-ERK 1/2 in SpostC, PI3K inhibitor LY294002 (15 microM) and MEK 1/2 inhibitor PD98059 (20 microM) were administered alone or together with sevoflurane during the first 15 min of reperfusion. We found that exposure of 3% sevoflurane during early reperfusion significantly improved functional recovery (improved left ventricular developed pressure (LVDP), +/-dp/dt, CF, HR and reduced left ventricular end-diastolic pressure (LVEDP)), decreased myocardial infarct size and reduced LDH and CK-MB release, when compared with unprotected hearts. However, these protective effects were abolished in the presence of either LY294002 or PD98059, which was accompanied by the prevention of PKB/Akt and ERK 1/2 phosphorylation, and reduction of myocardial nicotinamide adenine dinucleotide (NAD+) content. These findings suggest that sevoflurane postconditioning protects chronically-infarcted rat hearts against ischemia-reperfusion injury by inhibiting mPTP opening via recruitment of PKB/Akt and ERK 1/2.