Changes In Akt Phosphorylation Research Articles

Dual inhibition of MEK and PI3Kβ/δ-a potential therapeutic strategy in PTEN-wild-type docetaxel-resistant metastatic prostate cancer.

Background: Docetaxel remains the standard treatment for metastatic castration-resistant prostate cancer (mCRPC). However, resistance frequently emerges as a result of hyperactivation of the PI3K/AKT and the MEK/ERK pathways. Therefore, the inhibition of these pathways presents a potential therapeutic approach. In this study, we evaluated the efficacy of simultaneous inhibition of the PI3K/AKT and MEK/ERK pathways in docetaxel-resistant mCRPC, both in vitro and in vivo. Methods: Docetaxel-sensitive and docetaxel-resistant mCRPC cells were treated with selumetinib (MEK1/2 inhibitor), AZD8186 (PI3Kβ/δ inhibitor) and capivasertib (pan-AKT inhibitor) alone and in combination. Efficacy and toxicity of selumetinib+AZD8186 were tested in docetaxel-resistant xenograft mice. CRISPR-Cas9 generated a PTEN-knockdown docetaxel-resistant cell model. Changes in phosphorylation of AKT, ERK and downstream targets were analyzed by Western blot. Antiapoptotic adaptations after treatments were detected by dynamic BH3 profiling. Results: PI3K/AKT and MEK/ERK pathways were hyperactivated in PTEN-wild-type (wt) docetaxel-resistant cells. Selumetinib+AZD8186 decreased cell proliferation and increased apoptosis in PTEN-wt docetaxel-resistant cells. This observation was further confirmed in vivo, where docetaxel-resistant xenograft mice treated with selumetinib+AZD8186 exhibited reduced tumor growth without additional toxicity. Conclusion: Our findings on the activity of selumetinib+AZD8186 in PTEN-wt cells and in docetaxel-resistant xenograft mice provide an excellent rationale for a novel therapeutic strategy for PTEN-wt mCRPC patients resistant to docetaxel, in whom, unlike PTEN-loss patients, a clinical benefit of treatment with single-agent PI3K and AKT inhibitors has not been demonstrated. A phase I-II trial of this promising combination is warranted.

Shear stress facilitates Akt activation in human pulmonary microvascular endothelial cells

Introduction: Pulmonary arterial obstruction due to acute thromboembolism causes local reduction or cessation of blood flow. Under normal conditions, pulmonary blood flow exerts shear stress on endothelial cells, known to play an important role in maintaining vascular homeostasis. We hypothesized that pulmonary microvascular endothelial cells exposed to abrupt reductions in shear stress would demonstrate attenuated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and phosphorylation of downstream effectors Akt and endothelial nitric oxide synthase (eNOS). Methods: We tested our hypothesis in primary cultures of human lung microvascular endothelial cells (hLMVECs). We cultured early passage (p≤8) hLMVECs from 5 unique donor lots in standard 6-well culture plates and exposed cells to shear stress using an orbital shaker. Using a fixed depth (volume) and viscosity of tissue culture media, we adjusted the magnitude of shear stress exerted on hLMVECs by changing the angular velocity of orbital rotation. We acclimatized hLMVECs to physiologically-relevant shear stress (~12 dyn/cm 2 ) for 24 hours, then exposed cells to low shear stress (~3 dyn/cm 2 ) or no shear stress (0 dyn/cm 2 ) for 30 minutes. Control cells were maintained at 12 dyn/cm 2 of shear stress. In some experiments, PI3K activity was inhibited throughout the acclimatization and shear stress manipulation period by including LY294002 (10 μM) or vehicle (DMSO, 0.1% v/v) in culture media. We prepared whole cell lysates and used western blot to evaluate the expression and phosphorylation of Akt (ser473) and eNOS (ser1177). Results: We found that phosphorylation of Akt, but not Akt expression, decreased significantly (p<0.05) when shear stress was acutely reduced (~3 dyn/cm 2 ) or removed (0 dyn/cm 2 ). Inhibition of PI3K activity with LY294002 further diminished shear-mediated changes in Akt phosphorylation in all 3 groups. Conversely, eNOS phosphorylation was not modified by acute reductions in shear stress, but eNOS expression was markedly attenuated by PI3K inhibition under all conditions. Conclusions: Our results indicate that phosphorylation of Akt, but not its downstream target eNOS, is rapidly attenuated by abrupt reductions in shear stress in hLMVECs. However, shear stress appears to regulate eNOS expression in a PI3K-dependent manner. Taken together, these findings suggest that acute disruption of flow, as seen in pulmonary embolism, may disrupt human lung microvascular endothelial cell homeostasis and nitric oxide bioavailability via multiple PI3K-dependent signaling pathways. This work is supported by HL126514 and HL159906. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 103: Regulation Of Cardiac PTEN/Akt Signaling By Moderate Intra-Ischemic Therapeutic Hypothermia

Introduction: Previous studies demonstrate a critical role for Akt in mediating cardioprotection by therapeutic hypothermia. However, the mechanism of hypothermia-induced Akt activation remains poorly understood. Akt activation is determined in part by PTEN activity and degradation. Hypothesis: We hypothesized that moderate intra-ischemic cooling activates Akt in the ischemic-reperfused heart due to enhanced PTEN degradation. Methods: Rat hearts were isolated and perfused media containing 5 mM glucose. LV function was monitored continuously by an intraventricular balloon. Rate-pressure product (RPP=heart rate x LV developed pressure) was monitored as an index of cardiac function. Hearts in the normothermic control group (I/R) were subjected to 20 min. of global, no-flow ischemia followed by reperfusion at 37 °C. In the hypothermia intervention group (I/R+TH), hearts were cooled to 30 °C at the start of ischemia and then rewarmed 10 min. after reperfusion. Coronary perfusate was collected from the pulmonary artery at preischemic baseline and 10 min. after reperfusion. Ventricular tissue was harvested at 0 or 30 min. after reperfusion for western blotting. Results: RPP was significantly higher in the I/R+TH group (n=7) compared to the I/R group (n=8) at 30 min. post-reperfusion (24,725 ± 1,112 mmHg*beats/min. vs. 18,237 ± 2,223 mmHg*beats/min., p<0.05). Coronary perfusate taurine concentration was significantly higher in the I/R group compared to the I/R+TH group at 10 min. post-reperfusion (19 ± 4 μM vs. 5 ± 1 μM, p<0.01). Akt serine473 phosphorylation was significantly higher in the I/R+TH group compared to the I/R group at 0 min. post-reperfusion (p<0.01), but significantly lower at 30 min. post-reperfusion (p<0.01). Additionally, TH enhanced total PTEN expression at 0 min. (p<0.05) and 30 min. (p<0.01) post-reperfusion without affecting the ratio of phospho-PTEN/PTEN. Conclusion: Intra-ischemic cooling was associated with temporal changes in Akt phosphorylation despite enhanced PTEN expression. Additionally, modulation of cardiac taurine release by hypothermia should be carefully considered in future studies assessing taurine as a biomarker for cardiac arrest and ischemic heart pathologies.

Reduction in Insulin Mediated ERK Phosphorylation by Palmitate in Liver Cells Is Independent of Fatty Acid Induced ER Stress.

Saturated free fatty acids (FFAs) such as palmitate in the circulation are known to cause endoplasmic reticulum (ER) stress and insulin resistance in peripheral tissues. In addition to protein kinase B (AKT) signaling, extracellular signal-regulated kinase (ERK) has been implicated in the development of insulin resistance. However, there are conflicting data regarding role of ERK signaling in ER stress-induced insulin resistance. In this study, we investigated the effects of ER stress on insulin resistance and ERK phosphorylation in Huh-7 cells and evaluated how oleate prevents palmitate-mediated ER stress. Treatment with insulin resulted in an increase of 38–45% in the uptake of glucose in control cells compared to non-insulin-treated control cells, along with an increase in the phosphorylation of AKT and ERK. We found that treatment with palmitate increased the expression of ER stress genes, including the splicing of X box binding protein 1 (XBP1) mRNA. At the same time, we observed a decrease in insulin-mediated uptake of glucose and ERK phosphorylation in Huh-7 cells, without any change in AKT phosphorylation. Supplementation of oleate along with palmitate mitigated the palmitate-induced ER stress but did not affect insulin-mediated glucose uptake or ERK phosphorylation. The findings of this study suggest that palmitate reduces insulin-mediated ERK phosphorylation in liver cells and this effect is independent of fatty-acid-induced ER stress.

Toward Development of a Diabetic Synovium Culture Model.

Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation and inflammation of synovium, the specialized connective tissue that envelops the diarthrodial joint. Type 2 diabetes mellitus (DM) is often found in OA patients, with nearly double the incidence of arthritis reported in patients with diabetes (52%) than those without it (27%). The correlation between OA and DM has been attributed to similar risk factors, namely increasing age and joint loading due to obesity. However, a potential causative link is not well understood due to comorbidities involved with treating diabetic patients, such as high infection rates and poor healing response caused by hyperglycemia and insulin resistance. The purpose of this study was to investigate the effect of hyperglycemic and insulin culture conditions on synovium properties. It was hypothesized that modeling hyperglycemia-induced insulin resistance in synovium would provide novel insights of OA pathogenesis in DM patients. To simulate DM in the synovial joint, healthy synovium was preconditioned in either euglycemic (EG) or hyperglycemic (HG) glucose concentrations with insulin in order to induce the biological response of the diseased phenotype. Synovium biochemical composition was evaluated to determine ECM remodeling under hyperglycemic culture conditions. Concurrent changes in AKT phosphorylation, a signaling pathway implicated in insulin resistance, were measured along with gene expression data for insulin receptors, glucose transporters, and specific glycolysis markers involved in glucose regulation. Since fluid shear stress arising during joint articulation is a relevant upstream stimulus for fibroblast-like synoviocytes (FLS), the predominant cell type in synovium, FLS mechanotransduction was evaluated via intracellular calcium ([Ca2+]i). Incidence and length of primary cilia, a critical effector of cell mechanosensing, were measured as potential mechanisms to support differences in [Ca2+]i responses. Hyperglycemic culture conditions decreased collagen and GAG content compared to EG groups, while insulin recovered ECM constituents. FLS mechanosensitivity was significantly greater in EG and insulin conditions compared to HG and non-insulin treated groups. Hyperglycemic treatment led to decreased incidence and length of primary cilia and decreased AKT phosphorylation, providing possible links to the mechanosensing response and suggesting a potential correlation between glycemic culture conditions, diabetic insulin resistance, and OA development.

How does prolonged moderate intensity walking affect gait in children with cerebral palsy?

The mitogen-activated kinase phosphatase-3 (MKP-3) has gained great importance in the scientific community by acting as a regulator of the cell cycle through dephosphorylation of FoxO1, an important transcription factor involved in the insulin intracellular signaling cascade. When dephosphorylated and translocated to the nuclei, FoxO1 can promote the transcription of orexigenic neuropeptides (NPY/AgRP) in the hypothalamus, whereas insulin signaling is responsible for the disruption of this process. However, it is not understood if the hypothalamic activation of MKP-3 affects FoxO1 phosphorylation, and we hypothesized that MKP-3 overexpression reduces the capacity of the insulin signal to phosphorylate FoxO1. In the present study, we overexpressed the DUSP6 gene through an injection of adenovirus directly into the hypothalamic third ventricle of Swiss mice. The colocalization of the adenovirus was confirmed by the immunofluorescence assay. Then, MKP-3 overexpression resulted in a significant reduction of hypothalamic FoxO1 phosphorylation after insulin stimulation. This effect was independent of changes in Akt phosphorylation. Thus, the role of MKP-3 in the hypothalamus is closely associated with FoxO1 dephosphorylation and may provide a potential therapeutic target against hypothalamic disorders related to obesity and unbalanced food intake control.

α-Synuclein Regulates Peripheral Insulin Secretion and Glucose Transport.

AimPopulation based studies indicate a positive association between type 2 diabetes (T2D) and Parkinson’s disease (PD) where there is an increased risk of developing PD in patients with T2D. PD is characterized by the abnormal accumulation of intraneuronal aggregated α-synuclein (α-syn) in Lewy bodies, which negatively impact neuronal viability. α-syn is also expressed in both pancreatic islets and skeletal muscle, key players in glucose regulation. Therefore, we examined the functional role of α-syn in these tissues.MethodsUsing mice lacking, overexpressing or transiently injected with α-syn, effects on glucose and insulin tolerance and insulin secretion were determined, with further characterization of the effects on GLUT4 translocation using GLUT4myc myotubes.ResultsMice genetically ablated for α-syn became glucose intolerant and insulin resistant with hyperinsulinemia and reduced glucose-stimulated insulin secretion (GSIS). Mice overexpressing human α-syn are more insulin senstive and glucose tolerant compared to controls with increased GSIS. Injection of purified α-syn monomers also led to improved glucose tolerance and insulin sensitivity with hightened GSIS. α-syn monomer treatments increased surface GLUT4 levels in myotubes but without any significant change in Akt phosphorylation. The increase in cell surface GLUT4 was largely due to a large reduction in GLUT4 endocytosis, however, with a compensatory reduction in GLUT4 exocytosis.ConclusionCumulatively, this data suggests that α-syn modulates both pancreatic beta cell function and glucose transport in peripheral tissues, thereby playing a pivitol role in the maintenance of normal glucose homeostasis.

PI3K-Akt Signaling in the Basolateral Amygdala Facilitates Traumatic Stress Enhancements in Fear Memory.

BackgroundA core symptom of posttraumatic stress disorder is persistent fear memory, which can be defined as fear memory that is resistant to updating, inhibition, or extinction. posttraumatic stress disorder emerges after traumatic stress exposure, but neurobiological mechanisms via which traumatic stress leads to persistent fear memory are not well defined. Akt signaling within the amygdala (Amy) is enhanced with traumatic stress, and phosphatidylinositol kinase 3 (PI3K) activation of Akt within the basolateral Amy (BLA) has been implicated as critical to fear memory formation. These findings raise the possibility that traumatic stress enhances PI3K→Akt signaling in the BLA, which leads to persistent fear memory.MethodsTo test this hypothesis, rats were exposed to traumatic stress using the single prolonged stress model, and changes in Akt phosphorylation were assayed in the Amy at 0 and 30 minutes after fear conditioning (FC). In a separate experiment, we inhibited PI3K→Akt signaling in the BLA prior to FC and observed the effect this had on acquisition, expression, and extinction of FC in stressed and control rats.ResultsEnhanced Akt phosphorylation in the Amy at both time points was observed in stressed rats, but not in control rats. PI3K→Akt inhibition in the BLA had no effect on freezing in control rats but decreased freezing during extinction training and testing in stressed rats.ConclusionThese findings suggest that PI3K→Akt signaling in the BLA could be a mechanism via which traumatic stress leads to fear memory that is resistant to extinction.

The Potential Targets and Mechanisms of a Carbazole and Pyrazole Containing Anticancer Compound.

Characterization of a small anticancer compound. The development of small molecules as new anti-cancer therapeutics is necessary to improve anti-tumor efficacy and reduce toxicities, especially for the treatment of brain tumors, where only small molecules can effectively cross the brain-blood barrier. Several novel hits were previously selected by concurrently screening colon and glioma cancer cell lines with a sensorconjugated reporter system. Here, we focused on one of them. Elucidating the potential target(s) of a novel anticancer compound. Computer-assisted structural and motif analysis (least absolute shrinkage and selection operator or LASSO score) was used to assess compound's targets, then direct kinase activity assays were used for the confirmation; Western blot of phosphorylated kinases, as well as FACS and caspase 3/7 activity assays, were used to decipher the action mechanisms. Finally, the expression profiling of proteins involved in various G-protein pathways by real-time PCR was performed. The small chemical, (4E)-4-[2-(9-ethyl-9H-carbazol-3-yl)hydrazin-1-ylidene]-3-methyl- 4,5-dihydro-1H-pyrazol-5-one, with a formula C18H17N5O and MW of 319.36, designated as VUGX01, was predicted to be a ligand/inhibitor to receptor tyrosine kinases (RTKs) by computer analysis (least absolute shrinkage and selection operator or LASSO score). However, direct analysis with recombinant kinases showed that it is not an effective inhibitor to the popular receptor kinases at 1μM concentration. This compound can activate caspases in some tumor cell lines but has minimal effects on the cell cycle. Drug treatments lead to the changes in phosphorylation of AKT and c- RAF, as well as the expression level of MAP2K, suggesting this compound may interact with Gprotein coupled receptors (GPCRs). The expression profiling of 82 proteins involved in various Gprotein pathways by real-time PCR showed that the treatment up-regulates the expression of several proteins, including angiotensinogen, angiotensin II receptor, and IP3-kinase catalytic subunit gamma. VUGX01 can effectively block proliferation and induce apoptosis of certain types of cancer cells, even it is predicted by high LASSO score, but it is not an effective RTKs inhibitor, it may inhibit cell growth through acting as a novel ligand to one or several GPCRs.

Effects of inosine monophosphate and exercise training on neuronal nitric oxide synthase in the mouse brain

Recently, the purine nucleoside inosine has been demonstrated to have several neuroprotective effects. Similarly, exercise training has well-known beneficial effects on mental health and cognitive function. Neuronal nitric oxide synthase (nNOS) is a key neuronal messenger in several brain regions, and the downregulation of nNOS has been shown to improve brain function. However, whether inosine and exercise training have combined effects on nNOS pathway-related proteins in the brain remains unknown. We found, for the first time, that inosine monophosphate (IMP), which is a precursor of inosine, decreases nNOS levels in the ventral hippocampus (vHp) and the cerebellum (Ce), but not in the dorsal hippocampus (dHp). In the vHp, the phosphorylation of cAMP response element-binding protein (CREB) was also upregulated, which negatively correlated with nNOS protein levels. In the cerebral cortex (Cx), no significant activation of the nNOS pathway was observed. In the dHp, vHp, Cx, and Ce, no interactions between the effects of IMP and exercise on nNOS protein and CREB phosphorylation levels were observed. The phosphorylation of nNOS was regulated by the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Although IMP induced minor changes in Akt phosphorylation, nNOS phosphorylation was unchanged by either IMP or exercise. In conclusion, in the vHp, which is associated with emotional behavior, IMP decreased nNOS levels and activated CREB, suggesting that IMP can elicit anxiolytic effects.

Bombyxin/Akt signaling in relation to the embryonic diapause process of the silkworm, Bombyx mori

Our previous study showed that phosphorylation of glycogen synthase kinase (GSK)-3β is related to the embryonic diapause process in Bombyx. However, the upstream signaling pathway was not clearly understood. In the present study, we examined bombyxin/Akt signaling in relation to the embryonic diapause process of B. mori. Results showed that GSK-3β phosphorylation stimulated by dechorionation was blocked by LY294002, a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, indicating involvement of PI3K in GSK-3β phosphorylation in dechorionated eggs. Direct determination of Akt phosphorylation showed that dechorionation stimulated Akt phosphorylation. The Akt phosphorylation was blocked by LY294002. Temporal changes in Akt phosphorylation showed that different changing patterns exist between diapause and developing eggs. Relatively higher phosphorylation levels of Akt were detected between days 3 and 5 after oviposition in non-diapause eggs compared to those at the same stages in diapause eggs. Upon treatment with HCl, which prevents diapause initiation, Akt phosphorylation levels exhibited a later and much broader peak compared to diapause eggs. Examination of expression levels of the bombyxin-Z1 gene showed that in diapause eggs, a major peak occurred 1 day after oviposition, and its level then sharply decreased on day 2. However, in both non-diapause and HCl-treated eggs, a major broad peak was detected between days 1 and 4 after oviposition. These temporal changes in bombyxin-Z1 gene expression levels during embryonic stages coincided with changes in Akt phosphorylation, indicating that bombyxin-Z1 is likely an upstream signaling component for Akt phosphorylation. Taken together, our results indicated that PI3K/Akt is an upstream signaling pathway for GSK-3β phosphorylation and is associated with the diapause process of B. mori eggs. To our knowledge, this is the first study to demonstrate the potential correlation between bombyxin/Akt signaling and the embryonic diapause process.

Influences of Hypoxia Exercise on Whole-Body Insulin Sensitivity and Oxidative Metabolism in Older Individuals.

Aging is a primary risk factor for most chronic diseases, including type 2 diabetes. Both exercise and hypoxia regulate pathways that ameliorate age-associated metabolic muscle dysfunction. We hypothesized that the combination of hypoxia and exercise would be more effective in improving glucose metabolism than normoxia exercise. We randomized 29 older sedentary individuals (62 ± 6 years; 14 women, 15 men) to bicycle exercise under normobaric hypoxia (fraction of inspired oxygen = 15%) or normoxia (fraction of inspired oxygen = 21%). Participants trained thrice weekly for 30 to 40 minutes over 8 weeks at a heart rate corresponding to 60% to 70% of peak oxygen update. Insulin sensitivity measured by hyperinsulinemic-euglycemic glucose clamp and muscle protein expression before and after hyperinsulinemic-euglycemic glucose clamp. Heart rate and perceived exertion during training were similar between groups, with lower oxygen saturation when exercising under hypoxia (88.7 ± 1.5 vs 96.2 ± 1.2%, P < 0.01). Glucose infusion rate after 8 weeks increased in both the hypoxia (5.7 ± 1.1 to 6.7 ± 1.3 mg/min/kg; P < 0.01) and the normoxia group (6.2 ± 2.1 to 6.8 ± 2.1 mg/min/kg; P = 0.04), with a mean difference between groups of -0.44 mg/min/kg; 95% CI, -1.22 to 0.34; (P = 0.25). Markers of mitochondrial content and oxidative capacity in skeletal muscle were similar after training in both groups. Changes in Akt phosphorylation and glucose transporter 4 under fasting and insulin-stimulated conditions were not different between groups over time. Eight weeks of hypoxia endurance training led to similar changes in insulin sensitivity and markers of oxidative metabolism compared with normoxia training. Normobaric hypoxia exercise did not enhance metabolic effects in sedentary older women and men beyond exercise alone.

Emodin, Physcion, and Crude Extract of Rhamnus sphaerosperma var. pubescens Induce Mixed Cell Death, Increase in Oxidative Stress, DNA Damage, and Inhibition of AKT in Cervical and Oral Squamous Carcinoma Cell Lines.

There have been few studies on the pharmacological properties of Rhamnus sphaerosperma var. pubescens, a native Brazilian species popularly known as “fruto-de-pombo.” The aim of this study was to investigate the scavenging capacity of emodin, physcion, and the ethanolic crude extract of Rhamnus sphaerosperma var. pubescens against reactive oxygen and nitrogen species, as well as their role and plausible mechanisms in prompting cell death and changes in AKT phosphorylation after cervical (SiHa and C33A) and oral (HSC-3) squamous cell carcinoma treatments. Emodin was shown to be the best scavenger of NO• and O2•−, while all samples were equally effective in HOCl/OCl− capture. Emodin, physcion, and the ethanolic extract all exhibited cytotoxic effects on SiHa, C33A, HSC-3, and HaCaT (immortalized human keratinocytes, nontumorigenic cell line), involving mixed cell death (apoptosis and necrosis) independent of the caspase activation pathway. Emodin, physcion, and the ethanolic extract increased intracellular oxidative stress and DNA damage. Emodin decreased the activation of AKT in all tumor cells, physcion in HSC-3 and HaCaT cells, and the ethanolic extract in C33A and HaCaT cells, respectively. The induction of cancer cell death by emodin, physcion, and the ethanolic crude extract of Rhamnus sphaerosperma var. pubescens was related to an increase in intracellular oxidative stress and DNA damage and a decrease in AKT activation. These molecules are therefore emerging as interesting candidates for further study as novel options to treat cervical and oral carcinomas.

Overexpression of Mitogen-activated protein kinase phosphatase-3 (MKP-3) reduces FoxO1 phosphorylation in mice hypothalamus.

The mitogen-activated kinase phosphatase-3 (MKP-3) has gained great importance in the scientific community by acting as a regulator of the cell cycle through dephosphorylation of FoxO1, an important transcription factor involved in the insulin intracellular signaling cascade. When dephosphorylated and translocated to the nuclei, FoxO1 can promote the transcription of orexigenic neuropeptides (NPY/AgRP) in the hypothalamus, whereas insulin signaling is responsible for the disruption of this process. However, it is not understood if the hypothalamic activation of MKP-3 affects FoxO1 phosphorylation, and we hypothesized that MKP-3 overexpression reduces the capacity of the insulin signal to phosphorylate FoxO1. In the present study, we overexpressed the DUSP6 gene through an injection of adenovirus directly into the hypothalamic third ventricle of Swiss mice. The colocalization of the adenovirus was confirmed by the immunofluorescence assay. Then, MKP-3 overexpression resulted in a significant reduction of hypothalamic FoxO1 phosphorylation after insulin stimulation. This effect was independent of changes in Akt phosphorylation. Thus, the role of MKP-3 in the hypothalamus is closely associated with FoxO1 dephosphorylation and may provide a potential therapeutic target against hypothalamic disorders related to obesity and unbalanced food intake control.

PI3-kinase cascade has a differential role in acquisition and extinction of conditioned fear memory in juvenile and adult rats.

The basolateral amygdala (BLA), medial prefrontal cortex (mPFC) circuit, plays a crucial role in acquisition and extinction of fear memory. Extinction of aversive memories is mediated, at least in part, by the phosphoinositide-3 kinase (PI3K)/Akt pathway in adult rats. There is recent interest in the neural mechanisms that mediate fear and extinction in juvenile animals and whether these mechanisms are distinctive from those in adult animals. In the present study, we examined (1) changes in phosphorylation of Akt in the BLA and mPFC after fear conditioning and extinction in juvenile and adult rats and (2) the effect of BLA and mPFC localized inhibition of the PI3K following acquisition and extinction of contextual fear memory. Our results show that Akt phosphorylation is increased following acquisition of contextual fear learning in the BLA but not in the mPFC in adult and juvenile rats. Extinction learning was not associated with changes in Akt phosphorylation. Although there were no differences in the pattern of phosphorylation of Akt either in adult or juvenile rats, microinjection of the PI3K inhibitor, LY294002, into the BLA or mPFC elicited differential effects on fear memory acquisition and extinction, depending on the site and timing of the microinjection, as well as on the age of the animal. These results suggest that PI3K/Akt has a differential role in formation, retrieval, and extinction of contextual fear memory in juvenile and adult animals, and point to developmental differences between adult and juvenile rats in mechanisms of extinction.

Differences in gene expression in human monocytes and macrophages from healthy and asthmatic donors in response to IL-4

Abstract The lungs from asthmatics are in a state of Th2-driven inflammation with a shift in macrophage polarization toward an M2 profile induced by IL-4 and IL-13. The number of M2 macrophages in the lung correlates with asthma severity. Women have more lung M2 cells than men and are more likely to have asthma than men. We hypothesized that sex differences affect macrophage polarization in humans. First, we determined that M2-related gene expression was different between healthy and asthmatic donors in response to IL-4 and IL-13. Monocytes from asthmatics expressed more CCL17 in response to IL-4, while CCL22 and MMP12 expression was similar. We found that CD200R was highly expressed in response to IL-4 in monocyte derived-macrophages (MDM) from asthmatics compared with those of healthy donors. Furthermore, this increase was greater in MDM from women compared to men. Moreover, MMP12 and CCL22 expression were not different between the sexes. Interestingly, some genes (TGM2) were only up-regulated in MDM from asthmatics. Second, we analyzed the effect of male and female sex steroid hormones on the IL-4 signaling pathway. In the presence of IL-4, we found that both estrogen (E2) and dihydrotestosterone(active metabolite of testosterone) can induce changes in phosphorylation of Akt but not in STAT6 in MDM. Similar effects on signaling were results were obtained in U937, a human monocytic cell line. The stronger M2 response in female cells could be due to increased expression of estrogen receptors. We found that monocytes from women displayed greater expression of ERa isoforms. These results indicate a potential role for both intrinsic sex differences and E2 as inducers of M2 polarization in macrophages in asthma, leading to worse lung inflammation in women.

Crosstalk Between MAPK/ERK and PI3K/AKT Signal Pathways During Brain Ischemia/Reperfusion.

The epidermal growth factor receptor (EGFR) is linked to the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Raf/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK1/2) signaling pathways. During brain ischemia/reperfusion, EGFR could be transactivated, which stimulates these intracellular signaling cascades that either protect cells or potentiate cell injury. In the present study, we investigated the activation of EGFR, PI3K/AKT, and Raf/MAPK/ERK1/2 during ischemia or reperfusion of the brain using the middle cerebral artery occlusion model. We found that EGFR was phosphorylated and transactivated during both ischemia and reperfusion periods. During ischemia, the activity of PI3K/AKT pathway was significantly increased, as judged from the strong phosphorylation of AKT; this activation was suppressed by the inhibitors of EGFR and Zn-dependent metalloproteinase. Ischemia, however, did not induce ERK1/2 phosphorylation, which was dependent on reperfusion. Coimmunoprecipitation of Son of sevenless 1 (SOS1) with EGFR showed increased association between the receptor and SOS1 in ischemia, indicating the inhibitory node downstream of SOS1. The inhibitory phosphorylation site of Raf-1 at Ser259, but not its stimulatory phosphorylation site at Ser338, was phosphorylated during ischemia. Furthermore, ischemia prompted the interaction between Raf-1 and AKT, while both the inhibitors of PI3K and AKT not only abolished AKT phosphorylation but also restored ERK1/2 phosphorylation. All these findings suggest that Raf/MAPK/ERK1/2 signal pathway is inhibited by AKT via direct phosphorylation and inhibition at Raf-1 node during ischemia. During reperfusion, we observed a significant increase of ERK1/2 phosphorylation but no change in AKT phosphorylation. Inhibitors of reactive oxygen species and phosphatase and tensin homolog restored AKT phosphorylation but abolished ERK1/2 phosphorylation, suggesting that the reactive oxygen species-dependent increase in phosphatase and tensin homolog activity in reperfusion period relieves ERK1/2 from inhibition of AKT.

Abstract 2669: Chaetoglobosin K, an Akt pathway inhibitor, prevents proliferation and migration of prostate carcinoma cells

Abstract Chaetoglobosin K (ChK) is an indolylcytochalasin isolated from the fungus Diplodia macrospora that has shown anti-tumorigenic effects in multiple human lung and ovarian carcinoma cell lines. We have previously reported that ChK decreases Akt activation and modulates key downstream Akt/mTOR pathway effectors in tumorigenic cells. The down-regulation of Akt activation by ChK is independent of class I PI3K enzyme inhibition or the upstream mediators PDK1, PTEN, and mTORC2. The purpose of these studies was to investigate the effect of ChK on the proliferation and migration of prostate carcinoma cells of various metastatic potential and PTEN status. The activation of Akt was monitored to assess the ability of ChK to inhibit Akt in prostate carcinoma cells. In these studies, MTT assays were used to measure the number of viable PC3, DU145, and LNCaP cells treated with ChK. Scratch wound and transwell migration assays were utilized to examine the effect of ChK on cell migration. To assess Akt activation, western blot analysis was performed to measure changes in Akt phosphorylation at the ser473 and thr308 sites after cells were treated with ChK or vehicle. ChK was found to inhibit the number of viable PC3, DU145, and LNCaP cells with IC50 values in the 3-5μM range. ChK inhibited migration of PC3 cells into the wound space at 1μM by approximately 60% after 72 hours as compared to vehicle treated controls. ChK inhibited transwell migration of PC3 cells by greater than 90% compared to controls after 5 hours. Additionally, ChK treatment lowered Akt phosphorylation at key activation sites. In summary, ChK inhibits the proliferation, migration, and Akt activation of prostate carcinoma cells in vitro. Further mechanistic studies of ChK will substantiate its therapeutic potential in the treatment of prostate and other cancers. Citation Format: Amna Ali, Silvia Caggia, Diane F. Matesic, Shafiq A. Khan. Chaetoglobosin K, an Akt pathway inhibitor, prevents proliferation and migration of prostate carcinoma cells. [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 2669. doi:10.1158/1538-7445.AM2015-2669

Enhanced insulin signaling in human skeletal muscle and adipose tissue following gastric bypass surgery.

Roux-en-Y gastric bypass (RYGB) leads to increased peripheral insulin sensitivity. The aim of this study was to investigate the effect of RYGB on expression and regulation of proteins involved in regulation of peripheral glucose metabolism. Skeletal muscle and adipose tissue biopsies from glucose-tolerant and type 2 diabetic subjects at fasting and during a hyperinsulinemic-euglycemic clamp before as well as 1 wk and 3 and 12 mo after RYGB were analyzed for relevant insulin effector proteins/signaling components. Improvement in peripheral insulin sensitivity mainly occurred at 12 mo postsurgery when major weight loss was evident and occurred concomitantly with alterations in plasma adiponectin and in protein expression/signaling in peripheral tissues. In skeletal muscle, protein expression of GLUT4, phosphorylated levels of TBC1D4, as well as insulin-induced changes in phosphorylation of Akt and glycogen synthase activity were enhanced 12 mo postsurgery. In adipose tissue, protein expression of GLUT4, Akt2, TBC1D4, and acetyl-CoA carboxylase (ACC), phosphorylated levels of AMP-activated protein kinase and ACC, as well as insulin-induced changes in phosphorylation of Akt and TBC1D4, were enhanced 12 mo postsurgery. Adipose tissue from glucose-tolerant subjects was the most responsive to RYGB compared with type 2 diabetic patients, whereas changes in skeletal muscle were largely similar in these two groups. In conclusion, an improved molecular insulin-sensitive phenotype of skeletal muscle and adipose tissue appears to contribute to the improved whole body insulin action following a substantial weight loss after RYGB.

Regulation of Peripheral Nerve Myelin Maintenance by Gene Repression through Polycomb Repressive Complex 2.

Myelination of peripheral nerves by Schwann cells requires coordinate regulation of gene repression as well as gene activation. Several chromatin remodeling pathways critical for peripheral nerve myelination have been identified, but the functions of histone methylation in the peripheral nerve have not been elucidated. To determine the role of histone H3 Lys27 methylation, we have generated mice with a Schwann cell-specific knock-out of Eed, which is an essential subunit of the polycomb repressive complex 2 (PRC2) that catalyzes methylation of histone H3 Lys27. Analysis of this mutant revealed no significant effects on early postnatal development of myelin. However, its loss eventually causes progressive hypermyelination of small-diameter axons and apparent fragmentation of Remak bundles. These data identify the PRC2 complex as an epigenomic modulator of mature myelin thickness, which is associated with changes in Akt phosphorylation. Interestingly, we found that Eed inactivation causes derepression of several genes, e.g., Sonic hedgehog (Shh) and Insulin-like growth factor-binding protein 2 (Igfbp2), that become activated after nerve injury, but without activation of a primary regulator of the injury program, c-Jun. Analysis of the activated genes in cultured Schwann cells showed that Igfbp2 regulates Akt activation. Our results identify an epigenomic pathway required for establishing thickness of mature myelin and repressing genes that respond to nerve injury.