Presence Of Wortmannin Research Articles

Activation of G Protein-Coupled Estrogen Receptor (GPER) Negatively Modulates Cardiac Excitation-Contraction Coupling (ECC) through the PI3K/NOS/NO Pathway.

The G-protein-coupled estrogen receptor (GPER) has been described to exert several cardioprotective effects. However, the exact mechanism involved in cardiac protection remains unclear. The aim of this study is to investigate the role of GPER activation on excitation-contraction coupling (ECC) and the possibility that such effect participates in cardioprotection. The cardiac myocytes of male Wistar rats were isolated with a digestive buffer and loaded with Fura-2-AM for the measurement of intracellular calcium transient (CaT). Sarcomere shortening (SS) and L-type calcium current (ICaL) were also registered. The confocal technique was used to measure nitric oxide (NO) production in cells loaded with DAF-FM-diacetate. Cardiac myocytes exposed to 17-β-estradiol (E2, 10 nM) or G-1 (1 μM) for fifteen minutes decreased CaT, SS, and ICaL. These effects were prevented using G-36 (antagonist of GPER, 1 μM), L-Name (NO synthase -NOS- inhibitor, 100 nM), or wortmannin (phosphoinositide-3-kinase -PI3K- inhibitor, 100 nM). Moreover, G1 increased NO production, and this effect was abolished in the presence of wortmannin. We concluded that the selective activation of GPER with E2 or G1 in the isolated cardiac myocytes of male rats induced a negative inotropic effect due to the reduction in ICaL and the decrease in CaT. Finally, the pathway that we proposed to be implicated in these effects is PI3K-NOS-NO.

The Marine Factor 3,5-Dihydroxy-4-methoxybenzyl Alcohol Represses Adipogenesis in Mouse 3T3-L1 Adipocytes In Vitro: Regulating Diverse Signaling Pathways

The augmentation of adipocytes in the adipose tissues brings disordered pathophysiological conditions, including type 2 diabetes, hyperlipidemia, hypertension, cardiovascular disease, and cancer. The phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) prevents oxidative stress as radical scavenging in cells. However, the role of the disorder as a pharmacologic factor has been poorly understood. This study elucidates the regulatory effects of DHMBA on adipogenesis in mouse 3T3-L1 adipocytes in vitro. The 3T3-L1 preadipocytes were cultured in DMEM containing 10% calf fetal serum in the presence of DHMBA. Culturing with DHMBA repressed the growth of 3T3-L1 preadipocytes cultured in a medium without differentiation factors. Interestingly, when 3T3-L1 preadipocytes were cultured in a medium including differentiation factors containing insulin, DHMBA did not affect the number of cells with the differentiation process of adipogenesis. Culturing with DHMBA (1, 10, or 100 μM) inhibited lipid accumulation in adipocytes and repressed adipogenesis in 3T3-L1 cells. The potent inhibitory effects of DHMBA on adipogenesis were seen at the later stage of culture. Adipogenesis was inhibited by the presence of wortmannin, PD98059, or Bay 11-7082, which are inhibitors of pathways related to insulin signaling pathway. Notably, the suppressive effects of DHMBA on adipogenesis were expressed by the presence of these inhibitors. DHMBA treatment declined the levels of PPARy and C/EBPα related to preadipocyte differentiation and PI3 kinase 100α, Akt, MAPK, phosphor-MAPK, and mTOR implicated in the insulin signaling pathway, leading to adipogenesis promotion. Thus, DHMBA may inhibit adipogenesis via regulating diverse signaling pathways, providing a new strategy for the therapy of obesity.

Surface morphology live-cell imaging reveals how macropinocytosis inhibitors affect membrane dynamics

Macropinocytosis is a unique endocytic pathway that involves dynamic membrane ruffling and is important in various cellular functions beyond nutrient uptake. Due to its impact, macropinocytosis has been the subject of various studies and macropinocytosis inhibitors have been widely used. However, inhibitors exhibit offtarget effects and could be less specific for macropinocytosis, thereby affecting various cellular mechanisms with effects on nanoscale membrane dynamics that remain elusive. In this study, we revealed how five different inhibitors affect membrane dynamics before and after treatment using scanning ion conductance microscopic imaging on living cells. Our results indicated that cytochalasins and sodium azide exhibited similar slowing effects on horizontal ruffle movements, even though their inhibitory mechanisms are different. In contrast, ruffle deformation and elongation were less strongly inhibited compared to the movements along cell membrane direction. These results suggested the involvement of mechanisms beyond actin polymerization. The ruffles disappeared and the cell surface smoothened upon the 5-(N-ethyl-N-isopropyl)-Amiloride treatment. In the presence of wortmannin, we observed active ruffles but no cup-like structures. These findings and scanning ion conductance microscopy live-cell imaging would potentially contribute to a better understanding of inhibitor effects.

PI3K/Akt pathway mediates the positive inotropic effects of insulin in Langendorff-perfused rat hearts

Insulin exerts positive inotropic effects on cardiac muscle; however, the relationship between cardiac contractility and phosphoinositol-3-kinase/Akt (PI3K/Akt) activation remains unclear. We hypothesized that the positive inotropic effects of insulin are dose-dependent and mediated via the PI3K/Akt pathway in isolated normal rat hearts. The Institutional Animal Investigation Committee approved the use of hearts excised from rats under pentobarbital anesthesia. The hearts were perfused at a constant pressure using the Langendorff technique. After stabilization (baseline), the hearts were randomly divided into the following four insulin (Ins) groups: 1) Ins0 (0 IU/L), 2) Ins0.5 (0.5 IU/L), 3) Ins5 (5 IU/L), and 4) Ins50 (50 IU/L) (n = 8 in each group). To clarify the role of the PI3K/Akt pathway in insulin-dependent inotropic effects, we also treated the insulin groups with the PI3K inhibitor wortmannin (InsW): 5) InsW0 (0 IU/L), 6) InsW0.5 (0.5 IU/L), 7) InsW5 (5 IU/L), and 8) InsW50 (50 IU/L). Hearts were perfused with Krebs–Henseleit buffer solution with or without wortmannin for 10 min, followed by 20 min perfusion with the solution containing each concentration of insulin. The data were recorded as the maximum left ventricular derivative of pressure development (LV dP/dt max). Myocardial p-Akt levels were measured at 3 min, 5 min, and at the end of the perfusion. In the Ins groups, LV dP/dt max in Ins5 and Ins50 increased by 14% and 48%, respectively, 3 min after insulin perfusion compared with the baseline. Tachyphylaxis was observed after 10 min in the Ins5 and Ins50 treatment groups. Wortmannin partially inhibited the positive inotropic effect of insulin; although insulin enhanced p-Akt levels at all time points compared with the control group, this increase was suppressed in the presence of wortmannin. The positive inotropic effect of insulin is dose-dependent and consistent with Akt activation. This effect mediated by high doses of insulin on cardiac tissue was temporary and caused tachyphylaxis, potentially triggered by Akt overactivation, which leads beta 1 deactivation.

Rosmarinic Acid Ameliorates Pulmonary Ischemia/Reperfusion Injury by Activating the PI3K/Akt Signaling Pathway.

Pulmonary ischemia/reperfusion (IR) injury is the leading cause of acute lung injury, which is mainly attributed to reactive oxygen species (ROS) induced cell injuries and apoptosis. Since rosmarinic acid (RA) has been identified as an antioxidant natural ester, this natural compound might protect against pulmonary IR injury. In this study, the mice were given RA daily (50, 75, or 100 mg/kg) by gavage for 7 days before the pulmonary IR injury. We found that hypoxemia, pulmonary edema, and serum inflammation cytokines were aggravated in pulmonary IR injury. RA pretreatment (75 and 100 mg/kg) effectively reversed these parameters, while 50 mg/kg RA pretreatment was less pronounced. Our data also indicated RA pretreatment mitigated the upregulation of pro-oxidant NADPH oxidases (NOX2 and NOX4) and the downregulation of anti-oxidant superoxide dismutases (SOD1 and SOD2) upon IR injury. In vitro studies showed RA preserved the viability of anoxia/reoxygenation (AR)-treated A549 cells (a human lung epithelial cell line), and the results showed the protective effect of RA started at 5 μM concentration, reached its maximum at 15 μM, and gradually decreased at 20–25 μM. Besides, RA pretreatment (15 μM) greatly reduced the lactate dehydrogenase release levels subjected to AR treatment. Moreover, the results of our research revealed that RA eliminated ROS production and reduced alveolar epithelial cell apoptosis through activating the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway, which was supported by using wortmannin, because in the presence of wortmannin, the RA-mediated protection was blocked. Meanwhile, wortmannin also reversed the protective effects of RA in mice. Together, our results demonstrate the beneficial role of RA in pulmonary IR injury via PI3K/Akt-mediated anti-oxidation and anti-apoptosis, which could be a promising therapeutic intervention for pulmonary IR injury.

Hypoglycemic activity of CDCA (chenodeoxycholic acid) and GW4064 in type I and type II diabetic mouse model systems

Farnesoid X receptor (FXR) is an orphan nuclear receptor that plays roles in diverse metabolic pathways including blood glucose. Previously we tested a signal transduction pathway of the phosphoinositide 3‐kinase (PI3K) pathway in in vitro model. In HepG2 cells and Caco‐2 cells, FXR in the presence of GW4064 (synthetic FXR agonist) and CDCA (chenodeoxycholic acid, physiological FXR agonist) enhanced activity of PI3K in FXR:PI3K immunocomplex that led to increase in glucose (GLC) uptake and glycogen synthase (GS) activity. However, positive effects on PI3K were reversed in the presence of wortmannin. We also confirmed that in vivo effects of FXR were comparable to those of insulin in mouse models. GW4064 reversed hyperglycemia in Db/Db mice in vivo. In addition, we found the comparable hypoglycemic effects of FXR in the C57BL/6 mice which were treated with streptozotocin that resulted in type I diabetic hyperglycemia. Time course and levels of antihyperglycemic effects of GW4064 were comparable to those of insulin. Accordingly, we report insulin‐mimetic action of FXR both in vitro and in vivomodel systems.

Class II Phosphatidylinositol-3-kinases Promotes CCR7 Recycling to the Cell Membrane in Human T-Cells

Abstract C-C Chemokine Receptor 7 (CCR7) and its ligands (CCL19 and CCL21) are important for immune cell migration/co-localization during the primary and subsequent immune responses. We have previously shown both in humans and in mice that signaling through CCR7/CCL19 promotes rapid receptor internalization via arrestin-3. Therefore, we questioned whether internalized CCR7/CCL19 recycles to the membrane in T-cells, and if so, what are the drivers for recycling. G protein-coupled receptors can activate phosphatidylinositol-3-kinase (PI3K) family members to regulate membrane remodeling events, which include receptor recycling to the cell surface. To better understand the role of PI3K in the recycling of CCR7, we used wortmannin at 5μM to block class I and class II PI3K’s and at 0.5μM to block class II PI3K during CCR7 recycling. We found that under both conditions, receptor recycling was reduced by 20% suggesting a role for PI3K family members in recycling of CCR7 to the cell membrane. Treatment of the cells with the class I δ specific inhibitor, GS-1101, however, did not affect recycling, suggesting that CCR7/CCL19 recycling to the cell membrane is dependent on class II PI3K. To determine the site within the cells where CCR7 was retained following inhibition of only class II PI3K, we used HEK-293 cells transfected with CCR7-eGFP and the trans-Golgi network (TGN) marker, TPST2-mCherry, ± 1μM wortmannin, which is required to inhibit class II PI3K in HEK-293 cells. We found that after receptor internalization with CCL19 and 30 minutes of recycling, CCR7-eGFP remained within the TGN in the presence of wortmannin, in contrast CCR7-eGFP, which recycled to the cell membrane in the absence of wortmannin, demonstrating a role for class II PI3K in CCR7 recycling. Supported by NIH-SC1GM111172 NIH-2U54MD007592 NSF-HRD-182675 NIH-5UL1GM118970

Ceramide modulates electrophysiological characteristics and oxidative stress of pulmonary vein cardiomyocytes.

Ceramide is involved in regulating metabolism and energy expenditure, and its abnormal myocardial accumulation may contribute to heart injury or lipotoxic cardiomyopathy. Whether ceramide can modulate the electrophysiology of pulmonary veins (PVs) remains unknown. We used conventional microelectrodes to measure the electrical activity of isolated rabbit PV tissue preparations before and after treatment with various concentrations of ceramide with or without H2 O2 (2mM), MitoQ, wortmannin or 740 YP. A whole-cell patch clamp and fluorescence imaging were used to record the ionic currents, calcium (Ca2+ ) transients, and intracellular reactive oxygen species (ROS) and sodium (Na+ ) in isolated single PV cardiomyocytes before and after ceramide (1μM) treatment. Ceramide (0.1, 0.3, 1 and 3μM) reduced the beating rate of PV tissues. Furthermore, ceramide (1μM) suppressed the 2mM H2 O2 -induced faster PV beating rate, triggered activities and burst firings, which were further reduced by MitoQ. In the presence of wortmannin, ceramide did not change the PV beating rate. The H2 O2 -induced faster PV beating rate could be counteracted by MitoQ or wortmannin with no additive effect from the ceramide. Ceramide inhibited pPI3K. Ceramide reduced Ca2+ transients, sarcoplasmic reticulum Ca2+ contents, L-type Ca2+ currents, Na+ currents, late Na+ currents, Na+ -hydrogen exchange currents, and intracellular ROS and Na+ in PV cardiomyocytes, but did not change Na+ -Ca2+ exchange currents. C2 ceramide may exert the distinctive electrophysiological effect of modulating PV activities, which may be affected by PI3K pathway-mediated oxidative stress, and might play a role in the pathogenesis of PV arrhythmogenesis.

Synthesis, Self-Assembly and In Vitro Cellular Uptake Kinetics of Nanosized Drug Carriers Based on Aggregates of Amphiphilic Oligomers of N-Vinyl-2-pyrrolidone.

Development of nanocarrier-based drug delivery systems is a major breakthrough in pharmacology, promising targeted delivery and reduction in drug toxicity. On the cellular level, encapsulation of a drug substantially affects the endocytic processes due to nanocarrier–membrane interaction. In this study we synthesized and characterized nanocarriers assembled from amphiphilic oligomers of N-vinyl-2-pyrrolidone with a terminal thiooctadecyl group (PVP-OD). It was found that the dissolution free energy of PVP-OD depends linearly on the molecular mass of its hydrophilic part up to = 2 × 104, leading to an exponential dependence of critical aggregation concentration (CAC) on the molar mass. A model hydrophobic compound (DiI dye) was loaded into the nanocarriers and exhibited slow release into the aqueous phase on a scale of 18 h. Cellular uptake of the loaded nanocarriers and that of free DiI were compared in vitro using glioblastoma (U87) and fibroblast (CRL2429) cells. While the uptake of both DiI/PVP-OD nanocarriers and free DiI was inhibited by dynasore, indicating a dynamin-dependent endocytic pathway as a major mechanism, a decrease in the uptake rate of free DiI was observed in the presence of wortmannin. This suggests that while macropinocytosis plays a role in the uptake of low-molecular components, this pathway might be circumvented by incorporation of DiI into nanocarriers.

The calcimimetic R-568 attenuates subarachnoid hemorrhage-induced vasospasm through PI3K/Akt/eNOS signaling pathway in the rat model

Cerebral vasospasm (CVS) causes mortality and morbidity in patients after subarachnoid hemorrhage (SAH). The mechanism and adequate treatment of CVS are still elusive. R-568 is a calcimimetic agent known to exert a vasodilating effect. However, there is no report on its vasodilator effect against SAH-induced vasospasm. In the present study, we investigated the therapeutic effect of R-568 on the SAH-induced CVS model in rats. Seventy-two adult male Sprague-Dawley rats were divided into 8 groups: sham surgery; SAH only; SAH + Vehicle, SAH + R-568; SAH + R-568 + Wortmannin (the PI3K inhibitor); SAH + Wortmannin; SAH + R-568 + Calhex-231 (a calcilytic agent); SAH + Calhex-231. SAH was induced by blood (0.3 mL) given by intracisternal injection. R-568 (20 µM) was administered intracisternal immediately prior to experimental SAH. Basilar arteries (BAs) were obtained to evaluate PI3K/Akt/eNOS pathway (immunoblotting) and morphological changes 48 h after SAH. Perimeters of BAs were decreased by 24.1% in the SAH group compared to the control group and the wall thickness was increased by 75.3%. With R-568 treatment, those percentages were 9.6% and 29.6%, respectively, indicating that vasospasm was considerably improved when compared with the SAH group (P < 0.001 in both). While p-PI3K/PI3K and p-Akt/Akt ratio and eNOS protein expression were markedly decreased in the SAH rats, treatment with R-568 resulted in a significant increase in these levels. The beneficial effects of R-568 were partially blocked in the presence of Calhex-231 and completely blocked in the presence of Wortmannin. Herein, we found that treatment with R-568 would attenuate SAH-induced CVS through the PI3K/Akt/eNOS pathway and demonstrate therapeutic promise in CVS treatment following SAH.

Everolimus: A potential therapeutic agent targeting PI3K/Akt pathway in brain insulin system dysfunction and associated neurobehavioral deficits.

It is well accepted that PI3k/Akt signaling pathway is a potential therapeutic window which regulates metabolism and energy homeostasis within the brain, and is an important mediator of normal neuronal physiological functions. Dysregulation of this pathway results in impaired insulin signaling, learning and memory and neuronal survival. Elucidating the role of everolimus in intracerebroventricular (ICV) streptozotocin induced Insulin/IGF-1 dependent PI3K/Akt/mTOR pathway dysregulation and associated neurobehavioral deficits. Rats were administered with streptozotocin (3mg/kg) intracerebroventricular, followed by administration of everolimus (1mg/kg) orally for 21days. After that, Morris water maze and passive avoidance tests were performed for assessment of memory. Animals were sacrificed to evaluate brain insulin pathway dysfunction, neurotrophic, apoptotic, inflammatory, and biochemical markers in rat brain. To elucidate the mechanism of action of everolimus, PI3K inhibitor, wortmannin was administered in the presence of everolimus in one group. Streptozotocin administration resulted in a significant decrease of brain insulin, insulin growth factor-1 levels, and alterations in behavioral, neurotrophic (BDNF), inflammatory (TNF-α), apoptotic (NF-κB, Bcl2 and Bax) and biochemical (AChE and ChAT assay) parameters in comparison to sham group rats. Everolimus significantly mitigated the deleterious behavioral, biochemical, and molecular changes in rats having central insulin dysfunction. However, the protective effect of everolimus was completely abolished when it was administered in the presence of wortmannin. Findings from the study reveal that mTOR inhibitors can be an important treatment strategy for neurobehavioral deficits occurring due to central insulin pathway dysfunction. Protective effect of drugs is via modulation of PI3K/Akt pathway.

Deteriorating insulin resistance due to WL15 peptide from cysteine and glycine-rich protein 2 in high glucose-induced rat skeletal muscle L6 cells.

This study investigates the antioxidant and antidiabetic activity of the WL15 peptide derived from Channa striatus on regulating the antioxidant property in the rat skeletal muscle cell line (L6) and enhancing glucose uptake via glucose metabolism. Increased oxidative stress plays a major role in the development of diabetes and its complications. Strategies are needed to mitigate the oxidative stress that can reduce these pathogenic processes. Our results showed that with treatment with WL15 peptide, the reactive oxygen species significantly decreased in L6 myotubes in a dose-dependent manner, and increased antioxidant enzymes help to prevent the formation of lipid peroxidation in L6 myotubes. The cytotoxicity of WL15 is evaluated in the L6 cells and found to be non-cytotoxic at the tested concentration. Also, for the analysis of glucose uptake activity in L6 cells, the 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxy- d -glucose assay was performed in the presence of wortmannin and genistein inhibitors. WL15 demonstrated antidiabetic activities through a dose-dependent increase in glucose uptake (64%) and glycogen storage (7.8 mM). The optimal concentration for the maximum activity was found to be 50 µM. In addition, studies of gene expression in L6 myotubes demonstrated upregulation of antioxidant genes and genes involved in the pathway of insulin signaling. In cell-based assays, WL15 peptide decreased intracellular reactive oxygen species levels and demonstrated insulin mimic activity by enhancing the primary genes involved in the insulin signaling pathway by increased glucose uptake indicatingthat glucose transporter type 4 (GLUT4) is regulated from the intracellular pool to the plasma membrane.

Role of protein tyrosine phosphatase 1B inhibitor in central insulin resistance and associated cognitive deficits

BackgroundProtein tyrosine phosphatase 1B (PTP1B) inhibitors are potential candidates for the treatment of peripheral insulin resistance and diabetes mellitus. Similar to peripheral action within the brain also, PTP1B activation impairs insulin signaling pathways. Activation of PTP1B in brain also accentuates neuroinflammation, oxidative stress and decreases neurotrophic factors in various brain dysfunctions including cognitive decline. ObjectivesThe main objective of our study was to elucidate the role of alendronate, a potent PTP1B inhibitor (blood brain barrier crossing bisphosphonate) in central insulin resistance and associated memory deficits. MethodologyTo induce central insulin resistance, streptozotocin (3 mg/kg) intracerebroventricular (ICV) was administered in two alternate days (1st and 3rd). After 21 days, memory was assessed via using the passive avoidance and Morris water maze paradigm. At the end of behavioral studies, animals were sacrificed to assess a variety of biochemical and molecular parameters in the hippocampus and cerebral cortex region of the brain. Treatment drug alendronate (3 mg/kg/day, p.o) and standard drug donepezil (3 mg/kg/i.p.) were administered from the 3rd day of STZ administration till the end of the study. Inhibition of PTP1B activates phosphoinsotide-3 kinase (PI3 K) (down-stream regulator of insulin signaling pathway).Thus, to illuminate the mechanism of action of alendronate, PI3 K inhibitor, wortmannin was administered in presence of alendronate in one group. ResultsAdministration of alendronate to ICV streprozotocin treated rats resulted in modulation of the insulin signaling pathway and associated behavioral, biochemical and molecular changes in central insulin resistance. However, the protective effect of alendronate was entirely vanished when it was administered in the presence of wortmannin. ConclusionAlendronate can be an important treatment strategy in central insulin signaling pathway dysfunction and associated cognitive deficits. Protective effect of alendronate is via modulation of PI3-K/Akt signaling pathway.

Gastrin Attenuates Renal Ischemia/Reperfusion Injury by a PI3K/Akt/Bad-Mediated Anti-apoptosis Signaling.

Ischemic/reperfusion (I/R) injury is the primary cause of acute kidney injury (AKI). Gastrin, a gastrointestinal hormone, is involved in the regulation of kidney function of sodium excretion. However, whether gastrin has an effect on kidney I/R injury is unknown. Here we show that cholecystokinin B receptor (CCKBR), the gastrin receptor, was significantly up-regulated in I/R-injured mouse kidneys. While pre-administration of gastrin ameliorated I/R-induced renal pathological damage, as reflected by the levels of serum creatinine and blood urea nitrogen, hematoxylin and eosin staining and periodic acid-Schiff staining. The protective effect could be ascribed to the reduced apoptosis for gastrin reduced tubular cell apoptosis both in vivo and in vitro. In vitro studies also showed gastrin preserved the viability of hypoxia/reoxygenation (H/R)-treated human kidney 2 (HK-2) cells and reduced the lactate dehydrogenase release, which were blocked by CI-988, a specific CCKBR antagonist. Mechanistically, the PI3K/Akt/Bad pathway participates in the pathological process, because gastrin treatment increased phosphorylation of PI3K, Akt and Bad. While in the presence of wortmannin (1 μM), a PI3K inhibitor, the gastrin-induced phosphorylation of Akt after H/R treatment was blocked. Additionally, wortmannin and Akt inhibitor VIII blocked the protective effect of gastrin on viability of HK-2 cells subjected to H/R treatment. These studies reveals that gastrin attenuates kidney I/R injury via a PI3K/Akt/Bad-mediated anti-apoptosis signaling. Thus, gastrin can be considered as a promising drug candidate to prevent AKI.

Molecular process of glucose uptake and glycogen storage due to hamamelitannin via insulin signalling cascade in glucose metabolism.

Understanding the mechanism by which the exogenous biomolecule modulates the GLUT-4 signalling cascade along with the information on glucose metabolism is essential for finding solutions to increasing cases of diabetes and metabolic disease. This study aimed at investigating the effect of hamamelitannin on glycogen synthesis in an insulin resistance model using L6 myotubes. Glucose uptake was determined using 2-deoxy-D-[1-3H] glucose and glycogen synthesis were also estimated in L6 myotubes. The expression levels of key genes and proteins involved in the insulin-signaling pathway were determined using real-time PCR and western blot techniques. The cells treated with various concentrations of hamamelitannin (20µM to 100µM) for 24h showed that, the exposure of hamamelitannin was not cytotoxic to L6 myotubes. Further the 2-deoxy-D-[1-3H] glucose uptake assay was carried out in the presence of wortmannin and Genistein inhibitor for studying the GLUT-4 dependent cell surface recruitment. Hamamelitannin exhibited anti-diabetic activity by displaying a significant increase in glucose uptake (125.1%) and glycogen storage (8.7mM) in a dose-dependent manner. The optimum concentration evincing maximum activity was found to be 100µm. In addition, the expression of key genes and proteins involved in the insulin signaling pathway was studied to be upregulated by hamamelitannin treatment. Western blot analysis confirmed the translocation of GLUT-4 protein from an intracellular pool to the plasma membrane. Therefore, it can be conceived that hamamelitannin exhibited an insulinomimetic effect by enhancing the glucose uptake and its further conversion into glycogen by regulating glucose metabolism.

In vitro insulin treatment reverses changes elicited by nutrients in cellular metabolic processes that regulate food intake in fish.

This research assessed the direct effects of insulin on nutrient-sensing mechanisms in the brain of rainbow trout (Oncorhynchus mykiss) using an in vitro approach. Cultured hypothalamus and hindbrain were exposed to 1 µmoll-1 insulin for 3 h, and signals involved in appetite regulation and nutrient-sensing mechanisms were measured. Additionally, the involvement of the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in the actions of insulin was studied by using the inhibitor wortmannin. Treatment with insulin alone did not elicit many changes in the appetite regulators and nutrient-sensing-related genes and enzymes tested in the hypothalamus and hindbrain. However, we found that, when insulin and nutrients were added together, insulin reversed most of the effects exerted by nutrients alone, suggesting that insulin changes responsiveness to nutrients at the central level. Effects reversed by insulin included expression levels of genes related to the sensing of both glucose (slc2a2, slc5a1, gck, pck1, pklr, g6pcb, gys1, tas1r3 and nr1h3 in the hindbrain, and slc2a2, pklr and pck1 in the hypothalamus) and fatty acid (cd36 in the hindbrain, and cd36 and acly in the hypothalamus). Nutrient-induced changes in the activity of Acly and Cpt-1 in the hindbrain and of Pepck, Acly, Fas and Hoad in the hypothalamus were also reversed by insulin. Most of the insulin effects disappeared in the presence of wortmannin, suggesting the PI3K/Akt pathway is a mediator of the effects of insulin reported here. This study adds new information to our knowledge of the mechanisms regulating nutrient sensing in fish.

Polyphyllin VII induces apoptotic cell death via inhibition of the PI3K/Akt and NF‑κB pathways in A549 human lung cancer cells.

Polyphyllin VII is an active compound isolated from Paris polyphylla, which is termed Chonglou in China. The present study was designed to investigate the underlying mechanisms of the antitumor effect of Polyphyllin VII in lung cancer cells. The cytotoxic effect of Polyphyllin VII in human lung cancer A549 cells was analyzed; the results revealed an IC50 value of 0.41±0.10 µM at 24 h. The associated mechanisms were investigated by phase-contrast microscopy, fluorescence microscopy, flow cytometry and western blot analysis. Exposure of A549 cells to Polyphyllin VII resulted in apoptosis. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, and wortmannin, an inhibitor of PI3K, both decreased the proportion of viable A549 cells in the presence of Polyphyllin VII. The ratio of apoptotic cells increased in the presence of wortmannin and PDTC. Western blot analysis revealed that PI3K, phosphorylated (p)-PI3K, Akt, p-Akt, NF-κB and p-NF-κB were downregulated following treatment with Polyphyllin VII. Increased caspase-3 activity, increased poly-(ADP-ribose) polymerase cleavage and a downregulation of inhibitor of caspase-activated DNase were observed following treatment with Polyphyllin VII, and these effects were enhanced by either wortmannin or PDTC. The present results revealed that Polyphyllin VII was able to induce apoptotic cell death in A549 human lung cancer cells via inhibition of the PI3K/Akt and NF-κB pathways.

Neuroprotective effect of zolpidem against glutamate-induced toxicity is mediated via the PI3K/Akt pathway and inhibited by PK11195

Excitotoxicity is a pathological process in which neuronal dysfunction and death are induced by excessive glutamate stimulation, the major fast excitatory neurotransmitter in the mammalian brain. Excitotoxicity-induced neurodegeneration is a contributing factor in ischemia-induced brain damage, traumatic brain injury, and various neurodegenerative diseases. It is triggered by calcium overload due to prolonged over-activation of ionotropic N-methyl-d-aspartate (NMDA) receptors. Enhanced Ca2+ release results in neuronal vulnerability through several intertwined mechanisms, including activation of proteolytic enzymes, increased production of reactive oxygen species (ROS), mitochondrial dysfunction and modulation of intracellular signalling pathways. We investigated the neuroprotective effect of hypnotic zolpidem, a drug that exerts its central effects at the GABAA receptor complex, against glutamate-induced toxicity in P19 neurons. Zolpidem prevented death of P19 neurons exposed to glutamate, and abolished the glutamate-induced increase in ROS production, p53 and Bax expression, and caspase-3/7 activity. Zolpidem effects were mediated by marked over-activation of Akt kinase. The pro-survival effect, as well as the pAkt induction, were prevented in the presence of wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3K) that functions upstream of Akt. The beneficial effect of zolpidem on neuronal survival was not prevented by flumazenil, a GABAA receptor antagonist. PK11195, a drug that modulates the mitochondrial translocator protein 18 kDa (TSPO) and F0F1-ATPase, prevented the beneficial effect of zolpidem, indicating that the mechanism of zolpidem action involves preservation of mitochondrial function and integrity. Zolpidem effects were further mediated by prevention of glutamate-induced increase in the expression of the NR2B subunit of NMDA receptor. The obtained results suggest the promising therapeutic potential of zolpidem against excitotoxic insults and highlight the importance of mitochondria and the Akt pathway as valuable targets for therapeutic interventions in glutamate-mediated neuropathological conditions.

FTY720P Increases, at 2 hrs, the Activity of the Na+/K+ ATPase in Caco‐2 cells via PKC and PI3K

Fingolimod phosphate (FTY720P), an analogue of sphingosine‐1‐phosphate (S1P), was shown previously to modulate, in a time‐dependent manner, the activity of hepatic Na+/K+ ATPase with an inhibitory effect appearing at 15 min and a stimulatory one at 2hrs. Because in the colon, the ATPase plays a pivotal role in the absorption of sodium and water, a time response study was conducted in human epithelial colorectal adenocarcinoma (Caco‐2) cells to examine if FTY720P regulates the activity of the ATPase in a similar fashion. An inhibitory effect was already demonstrated at 15 min. This work investigates the effect of FTY720P at 2 hrs and attempts to determine the major signaling molecules involved. The activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate released in presence and absence of ouabain, a specific inhibitor of the enzyme. FTY720P exerted a significant and prominent stimulatory effect on the ATPase at 2 hours. This effect disappeared completely in presence of JTE‐013, a blocker of S1P receptor 2 (S1PR2), as well as in presence of each of calphostin and wortmannin, respective inhibitors of protein kinase C (PKC) and phosphoinositide 3‐kinase (PI3K). Moreover, Phorbol‐12‐Myristate‐13‐Acetate (PMA), an activator of PKC, simulated the stimulatory effect of FTY720P on Na+/K+ ATPase further confirming the involvement of PKC. BAPT/AM, a chelator of calcium ions, cancelled the effect of FTY720P on the ATPase, indicating thus an involvement of calcium ions in the signaling pathway. Since only conventional PKC isomers require the presence of calcium ions for their activation, FTY720P probably acts via a conventional PKC isomer. The stimulatory effect of PMA disappeared in presence of wortmannin, suggesting that PKC is upstream of PI3K. It was concluded that at 2 hours, FTY720‐P binds to S1PR2 leading to the activation of conventional PKC which, in turn, activates directly or indirectly PI3K, resulting in Na+/K+ ATPase stimulation.Support or Funding InformationUniversity Research BoardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of PI3K in myocardial ischaemic preconditioning: mapping pro-survival cascades at the trigger phase and at reperfusion.

The Reperfusion Injury Salvage Kinase (RISK) pathway is considered the main pro‐survival kinase cascade mediating the ischaemic preconditioning (IPC) cardioprotective effect. To assess the role of PI3K‐Akt, its negative regulator PTEN and other pro‐survival proteins such as ERK and STAT3 in the context of IPC, C57BL/6 mouse hearts were retrogradely perfused in a Langendorff system and subjected to 4 cycles of 5 min. ischaemia and 5 min. reperfusion prior to 35 min. of global ischaemia and 120 min. of reperfusion. Wortmannin, a PI3K inhibitor, was administered either at the stabilization period or during reperfusion. Infarct size was assessed using triphenyl tetrazolium staining, and phosphorylation levels of Akt, PTEN, ERK, GSK3β and STAT3 were evaluated using Western blot analyses. IPC reduced infarct size in hearts subjected to lethal ischaemia and reperfusion, but this effect was lost in the presence of Wortmannin, whether it was present only during preconditioning or only during early reperfusion. IPC increased the levels of Akt phosphorylation during both phases and this effect was fully abrogated by PI3K, whilst its downstream GSK3β was phosphorylated only during the trigger phase after IPC. Both PTEN and STAT3 were phosphorylated during both phases after IPC, but this was PI3K independent. IPC increases ERK phosphorylation during both phases, being only PI3K‐dependent during the IPC phase. In conclusion, PI3K‐Akt plays a major role in IPC‐induced cardioprotection. However, PTEN, ERK and STAT3 are also phosphorylated by IPC through a PI3K‐independent pathway, suggesting that cardioprotection is mediated through more than one cell signalling cascade.