Akt-dependent Phosphorylation Research Articles

Breast cancer suppression by aplysin is associated with inhibition of PI3K/AKT/FOXO3a pathway.

Aplysin, a bromosesquiterpene isolated from Aplysia kurodai, was explored as a potential anti-breast cancer agent by us. However, the mechanisms underlying the anticarcinogenic effect of aplysin remain unclear. Here, aplysin was found to remarkably suppress tumor growth in vivo, inhibit cell proliferation and promote apoptosis in vitro. Additionally, we demonstrated that aplysin attained these effects in part by down-regulating PI3K/AKT/FOXO3a signaling pathway. Aplysin treatment inhibited the phosphorylation levels of AKT (Ser-473) and AKT-dependent phosphorylation of FOXO3a (Ser-253) in breast cancer cell lines and breast cancer tissues. The expression levels of FOXO3a-targeted genes were also destabilized by aplysin, cyclin D1 and Bcl-XL were declined; however, p21CIP1, p27KIP1, Bim, TRAIL and FasL were increased both in vivo and in vitro. Furthermore, activation of the PI3K/AKT signaling pathway by an activator and silencing of FOXO3a by shRNA protected the cells from aplysin mediated growth suppression and apoptosis. In summary, our findings revealed that aplysin could suppress breast cancer progression by inhibiting PI3K/AKT/FOXO3a pathway, thereby suggesting a potential role of aplysin as a chemoprevention drug for patients with breast cancer.

IGF-1 mediated Neurogenesis Involves a Novel RIT1/Akt/Sox2 Cascade

Insulin-like growth factor 1 (IGF-1) is known to have diverse effects on brain structure and function, including the promotion of stem cell proliferation and neurogenesis in the adult dentate gyrus. However, the intracellular pathways downstream of the IGF-1 receptor that contribute to these diverse physiological actions remain relatively uncharacterized. Here, we demonstrate that the Ras-related GTPase, RIT1, plays a critical role in IGF-1-dependent neurogenesis. Studies in hippocampal neuronal precursor cells (HNPCs) demonstrate that IGF-1 stimulates a RIT1-dependent increase in Sox2 levels, resulting in pro-neural gene expression and increased cellular proliferation. In this novel cascade, RIT1 stimulates Akt-dependent phosphorylation of Sox2 at T118, leading to its stabilization and transcriptional activation. When compared to wild-type HNPCs, RIT1−/− HNPCs show deficient IGF-1-dependent Akt signaling and neuronal differentiation, and accordingly, Sox2-dependent hippocampal neurogenesis is significantly blunted following IGF-1 infusion in knockout (RIT1−/−) mice. Consistent with a role for RIT1 function in the modulation of activity-dependent plasticity, exercise-mediated potentiation of hippocampal neurogenesis is also diminished in RIT1−/− mice. Taken together, these data identify the previously uncharacterized IGF1-RIT1-Akt-Sox2 signaling pathway as a key component of neurogenic niche sensing, contributing to the regulation of neural stem cell homeostasis.

RACK1 promotes lung cancer cell growth via an MCM7/RACK1/Akt signaling complex

MCM7, a member of the miniature chromosome maintenance (MCM) protein family, is crucial for the initiation of DNA replication and proliferation in eukaryotic cells. In this report, we demonstrate that RACK1 regulates cell growth and cell cycle progression in human non-small-cell lung cancer by mediating MCM7 phosphorylation through an MCM7/RACK1/Akt signaling complex. RACK1 functions as a central scaffold that brings Akt into physical proximity with MCM7. Overexpression of RACK1 increases interactions between Akt and MCM7 and promotes Akt-dependent MCM7 phosphorylation, which in turn increases MCM7 binding to chromatin and MCM complex formation. Together, these changes promote DNA replication and cell proliferation. Our findings reveal a novel signaling pathway that regulates growth in non-small cell lung cancer.

AKT-dependent phosphorylation of the SAM domain induces oligomerization and activation of the scaffold protein CNK1

Scaffold proteins are hubs for the coordination of intracellular signaling networks. The scaffold protein CNK1 promotes several signal transduction pathway. Here we demonstrate that sterile motif alpha (SAM) domain-dependent oligomerization of CNK1 stimulates CNK1-mediated signaling in growth factor-stimulated cells. We identified Ser22 located within the SAM domain as AKT-dependent phosphorylation site triggering CNK1 oligomerization. Oligomeric CNK1 increased the affinity for active AKT indicating a positive AKT feedback mechanism. A CNK1 mutant lacking the SAM domain and the phosphorylation-defective mutant CNK1S22A antagonizes oligomerization and prevents CNK1-driven cell proliferation and matrix metalloproteinase 14 promoter activation. The phosphomimetic mutant CNK1S22D constitutively oligomerizes and stimulates CNK1 downstream signaling. Searching the COSMIC database revealed Ser22 as putative target for oncogenic activation of CNK1. Like the phosphomimetic mutant CNK1S22D, the oncogenic mutant CNK1S22F forms clusters in serum-starved cells comparable to clusters of CNK1 in growth factor-stimulated cells. CNK1 clusters induced by activating Ser22 mutants correlate with enhanced cell invasion and binding to and activation of ADP ribosylation factor 1 associated with tumor formation. Mutational analysis indicate that EGF-triggered phosphorylation of Thr8 within the SAM domain prevents AKT binding and antagonizes CNK1-mediated AKT signaling. Our findings reveal SAM domain-dependent oligomerization by AKT as switch for CNK1 activation.

Amyloid β attenuates metabotropic zinc sensing receptor, mZnR/GPR39, dependent Ca2+ , ERK1/2 and Clusterin signaling in neurons.

A hallmark of Alzheimer's disease is accumulation of amyloid beta (Aβ) deposits, which are associated with neuronal dysfunction, spine loss, and impaired Ca2+ homeostasis. Amyloid beta (Aβ) binds to and is aggregated by Zn2+ , a metal released from synaptic glutamatergic vesicles during neuronal activity. Synaptically released Zn2+ activates a metabotropic Gq-coupled Zn2+ -sensing receptor, mZnR/GPR39, and induces Ca2+ -signaling in post-synaptic neurons. We examined if Aβ, as a Zn2+ binding protein, regulates neuronal Zn2+ -signaling mediated by mZnR/GPR39 using SHSY-5Y cells and cortical neurons from GPR39 wild-type and knockout mice. Following acute or chronic treatment with Aβ neuronal Zn2+ -dependent Ca2+ release via mZnR/GPR39 is significantly reduced. This impairment is overcome when excess Zn2+ is applied, suggesting that impaired Ca2+ -signaling results from Aβ binding of Zn2+ . The Zn2+ -dependent mZnR/GPR39 activation triggers phosphorylation of extracellular regulated kinase and up-regulates expression of the chaperone protein clusterin (Clu). Importantly, neuronal Zn2+ -dependent extracellular regulated kinase1/2 phosphorylation and up-regulation of Clu are attenuated by silencing mZnR/GPR39 as well as by Aβ treatment. In contrast, Zn2+ -dependent AKT phosphorylation is not mediated by mZnR/GPR39 and is not attenuated by Aβ treatment. Thus, Zn2+ signaling via mZnR/GPR39 is distinctively disrupted by a critical pathological component of Alzheimer's disease. Synaptically released Zn2+ activates a Zn2+ -sensing receptor, mZnR/GPR39, and induces Ca2+ -signaling, followed by ERK1/2 MAPK activation and up-regulation of clusterin. Amyloid beta (Aβ) binds to Zn2+ thus forming oligomers that are a hallmark of Alzheimer's disease. We show that Aβ attenuates Zn2+ -dependent Ca2+ -responses, abolishes ERK1/2 activation and down-regulates clusterin expression. Thus, Zn2+ signaling via mZnR/GPR39 is disrupted by Aβ, a critical pathological component of Alzheimer's disease.

Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress.

Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2–3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin.

Two new competing pathways establish the threshold for cyclin-B–Cdk1 activation at the meiotic G2/M transition

Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K–Akt pathway through the Gβγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B–Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B–Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gβγ-dependent pathway enhances PI3K–Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gβγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries.

Peptidomimetic Targeting of Cavβ2 Overcomes Dysregulation of the L-Type Calcium Channel Density and Recovers Cardiac Function.

L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavβ2 chaperone regulates channel density at the plasma membrane. On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavβ2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavβ2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavβ2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavβ2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavβ2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavβ2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.

Insulin-Dependent Regulation of mTORC2-Akt-FoxO Suppresses TLR4 Signaling in Human Leukocytes: Relevance to Type 2 Diabetes

Leukocyte signaling in patients with systemic insulin resistance is largely unexplored. We recently discovered the presence of multiple Toll-like receptor 4 (TLR4) signaling intermediates in leukocytes from patients with type 2 diabetes or acute insulin resistance associated with cardiopulmonary bypass surgery. We extend this work to show that in addition to matrix metalloproteinase 9, hypoxia-inducible factor 1α, and cleaved AMPKα, patient leukocytes also express IRS-1 phosphorylated on Ser(312), Akt phosphorylated on Thr(308), and elevated TLR4 expression. Similar signaling intermediates were detected in leukocytes and neutrophils treated with lipopolysaccharide (LPS), a ligand of TLR4, in vitro. In contrast, insulin, but not LPS, induced mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt on Ser(473) and FoxO1/O3a on Thr(24/32) in leukocytes and neutrophils. Insulin suppressed LPS-induced responses in a dose- and time-dependent manner. AS1842856, a FoxO1 inhibitor, also suppressed TLR4 signaling. We propose that insulin is a homeostatic regulator of leukocyte responses to LPS/TLR4 and that the signaling intermediates expressed in leukocytes of patients with type 2 diabetes indicate TLR4 signaling dominance and deficient insulin signaling. The data suggest that insulin suppresses LPS/TLR4 signals in leukocytes through the mTORC2-Akt-FoxO signaling axis. Better understanding of leukocyte signaling in patients with type 2 diabetes may shed new light on disease causation and progression.

Smurf1 regulation of DAB2IP controls cell proliferation and migration.

Tumor cell proliferation, survival and migration are regulated by the deletion of ovarian carcinoma 2/disabled homolog 2 (DOC-2/DAB2) interacting protein (DAB2IP), a tumor suppressor that serves as a scaffold protein for H-Ras and TRAF2. Importantly, the oncogenic histone methyl-transferase EZH2 epigenetically down-regulates DAB2IP in a variety of tumors. Recently, we demonstrated that DAB2IP is negatively regulated by Akt-dependent phosphorylation and SCFFbw7-mediated degradation. Here, we further identify the oncoprotein Smurf1, an E3-ubiquitin ligase, as a novel negative regulator of DAB2IP. Smurf1-mediated cellular proliferation and migration are largely dependent on the presence of DAB2IP, suggesting that DAB2IP is a key effector molecule of Smurf1 oncogenic function. Additionally, we identify that similar to DAB2IP, Smurf1 is also a target of phosphorylation by both Akt1 and Akt2 kinases, which enhances Smurf1 abundance, leading to a reduction in DAB2IP. Given the role of DAB2IP in tumorigenesis and metastasis, our data identify Smurf1 as an upstream oncogenic factor that negatively regulates DAB2IP to govern aberrant cell growth and migration.

Roflumilast inhibits leukocyte–platelet interactions and prevents the prothrombotic functions of polymorphonuclear leukocytes and monocytes

ESSENTIALS: Thrombosis is a major comorbidity in patients with chronic obstructive pulmonary disease (COPD). Roflumilast is a selective phosphodiesterase type-4 (PDE4) inhibitor approved for treatment of severe COPD. PDE4 blockade by roflumilast inhibits prothrombotic functions of neutrophils and monocytes. PDE4 inhibitors may reduce thrombotic risk in COPD as well as in other vascular diseases. Roflumilast, an oral selective phosphodiesterase type 4 inhibitor, is approved for the treatment of severe chronic obstructive pulmonary disease (COPD). A recent meta-analysis of trials on COPD revealed that treatment with roflumilast was associated with a significant reduction in the rate of major cardiovascular events. The mechanisms of this effect remain unknown. We tested the hypothesis that roflumilast N-oxide (RNO), the active metabolite of roflumilast, curbs the molecular mechanisms required for leukocyte-platelet (PLT) interactions and prevents the prothrombotic functions of polymorphonuclear leukocytes (PMNs) and monocytes (MNs). Using well-characterized in vitro models, we analysed the effects of RNO on: (i) PMN adhesiveness; (ii) the release of neutrophil extracellular traps (NETs); and (iii) tissue factor expression in MNs. Key biochemical events underlying the inhibitory effects of RNO were defined. In PMNs, RNO prevented phosphoinositide 3-kinase (PI3K)-dependent phosphorylation of Akt on Ser473, and Src family kinase (SFK)-mediated Pyk2 phosphorylation on Tyr579-580, while inducing protein kinase A-mediated phosphorylation of C-terminal Src kinase, the major negative regulator of SFKs. Modulation of these signaling pathways by RNO resulted in a significant impairment of PMN adhesion to activated PLTs or human umbilical vein endothelial cells, mainly mediated by inhibition of the adhesive function of Mac-1. Moreover RNO curbed SFK/PI3K-mediated NET release by PMNs adherent on fibrinogen-coated surfaces. In MNs interacting with activated PLTs, RNO curbed PI3K-mediated expression of tissue factor. The efficacy of RNO was significantly potentiated by formoterol, a long acting β-adrenergic receptor agonist. This study reveals novel antithrombotic activities by which roflumilast may exert protective effects against cardiovascular comorbodities in COPD.

Hepatic FOXO1 Target Genes Are Co-regulated by Thyroid Hormone via RICTOR Protein Deacetylation and MTORC2-AKT Protein Inhibition

MTORC2-AKT is a key regulator of carbohydrate metabolism and insulin signaling due to its effects on FOXO1 phosphorylation. Interestingly, both FOXO1 and thyroid hormone (TH) have similar effects on carbohydrate and energy metabolism as well as overlapping transcriptional regulation of many target genes. Currently, little is known about the regulation of MTORC2-AKT or FOXO1 by TH. Accordingly, we performed hepatic transcriptome profiling in mice after FOXO1 knockdown in the absence or presence of TH, and we compared these results with hepatic FOXO1 and THRB1 (TRβ1) ChIP-Seq data. We identified a subset of TH-stimulated FOXO1 target genes that required co-regulation by FOXO1 and TH. TH activation of FOXO1 was directly linked to an increase in SIRT1-MTORC2 interaction and RICTOR deacetylation. This, in turn, led to decreased AKT and FOXO1 phosphorylation. Moreover, TH increased FOXO1 nuclear localization, DNA binding, and target gene transcription by reducing AKT-dependent FOXO1 phosphorylation in a THRB1-dependent manner. These events were associated with TH-mediated oxidative phosphorylation and NAD(+) production and suggested that downstream metabolic effects by TH can post-translationally activate other transcription factors. Our results showed that RICTOR/MTORC2-AKT can integrate convergent hormonal and metabolic signals to provide coordinated and sensitive regulation of hepatic FOXO1-target gene expression.

Abstract B40: AKT-ACLY signaling preferentially maintains histone acetylation over lipid synthesis during nutrient limitation

Abstract Metabolic reprogramming by oncogenes is a mechanism to support biosynthesis and growth in cancer cells. Activation of the PI3K/AKT pathway increases glucose consumption and glycolysis, leading to higher acetyl-CoA production. Extra-mitochondrial acetyl-CoA is utilized in lipid synthesis and protein acetylation. We previously showed that tumor cell histone acetylation is regulated in a glucose-dependent manner, through ATP-citrate-lyase (ACLY) conversion of glucose-derived citrate into acetyl-CoA. In low glucose conditions, acetyl-CoA and histone acetylation levels decrease. However, AKT-dependent phosphorylation of ACLY at serine 455 promotes acetyl-CoA production to allow maintenance of global levels of histone acetylation under glucose limitation. It is unclear whether partitioning of acetyl-CoA between lipid synthesis and histone acetylation in the setting of nutrient limitation impacts cell growth or survival. In this study, we examined whether acetyl-CoA utilization for lipid synthesis is impacted by AKT-ACLY signaling during nutrient limitation. Using 13C-glucose labeling and mass spectrometry, we determined that de novo lipid synthesis is suppressed to a much greater extent than histone acetylation in low glucose conditions. Moreover, phosphorylation of ACLY does not sustain fatty acid synthesis under glucose limitation, in contrast to its effects on histone acetylation. These data indicate that the PI3K-AKT-ACLY axis preferentially maintains histone acetylation over lipid synthesis during nutrient limitation. This regulation is likely due to AMPK-dependent phosphorylation of acetyl-CoA carboxylase (p-ACC), blocking acetyl-CoA commitment to fatty acid synthesis. This study reveals that cancer cells employ robust mechanisms to maintain histone acetylation in nutrient limited environments. Citation Format: Joyce V. Lee, Zuo-Fei Yuan, Shichong Liu, Benjamin A. Garcia, Kathryn E. Wellen. AKT-ACLY signaling preferentially maintains histone acetylation over lipid synthesis during nutrient limitation. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B40.

Fluid shear triggers microvilli formation via mechanosensitive activation of TRPV6.

Microvilli are cellular membrane protrusions present on differentiated epithelial cells, which can sense and interact with the surrounding fluid environment. Biochemical and genetic approaches have identified a set of factors involved in microvilli formation; however, the underlying extrinsic regulatory mechanism of microvilli formation remains largely unknown. Here we demonstrate that fluid shear stress (FSS), an external mechanical cue, serves as a trigger for microvilli formation in human placental trophoblastic cells. We further reveal that the transient receptor potential, vanilloid family type-6 (TRPV6) calcium ion channel plays a critical role in flow-induced Ca2+ influx and microvilli formation. TRPV6 regulates phosphorylation of Ezrin via a Ca2+-dependent phosphorylation of Akt; this molecular event is necessary for microvillar localization of Ezrin in response to FSS. Our findings provide molecular insight into the microvilli-mediated mechanoresponsive cellular functions, such as epithelial absorption, signal perception and mechanotransduction.

Akt-dependent Girdin phosphorylation regulates repair processes after acute myocardial infarction

Myocardial infarction is a leading cause of death, and cardiac rupture following myocardial infarction leads to extremely poor prognostic feature. A large body of evidence suggests that Akt is involved in several cardiac diseases. We previously reported that Akt-mediated Girdin phosphorylation is essential for angiogenesis and neointima formation. The role of Girdin expression and phosphorylation in myocardial infarction, however, is not understood. Therefore, we employed Girdin-deficient mice and Girdin S1416A knock-in (GirdinSA/SA) mice, replacing the Akt phosphorylation site with alanine, to address this question. We found that Girdin was expressed and phosphorylated in cardiac fibroblasts in vitro and that its phosphorylation was crucial for the proliferation and migration of cardiac fibroblasts. In vivo, Girdin was localized in non-cardiomyocyte interstitial cells and phosphorylated in α-smooth muscle actin-positive cells, which are likely to be cardiac myofibroblasts. In an acute myocardial infarction model, GirdinSA/SA suppressed the accumulation and proliferation of cardiac myofibroblasts in the infarcted area. Furthermore, lower collagen deposition in GirdinSA/SA mice impaired cardiac repair and resulted in increased mortality attributed to cardiac rupture. These findings suggest an important role of Girdin phosphorylation at serine 1416 in cardiac repair after acute myocardial infarction and provide insights into the complex mechanism of cardiac rupture through the Akt/Girdin-mediated regulation of cardiac myofibroblasts.

BMI-related progression of atypical PKC-dependent aberrations in insulin signaling through IRS-1, Akt, FoxO1 and PGC-1α in livers of obese and type 2 diabetic humans

Information on insulin resistance in human liver is limited. In mouse diet-induced obesity (DIO), hepatic insulin resistance initially involves: lipid+insulin-induced activation of atypical protein kinase C (aPKC); elevated Akt activity/activation but selective impairment of compartmentalized Akt-dependent FoxO1 phosphorylation; and increases in gluconeogenic and lipogenic enzymes. In advanced stages, e.g., in hepatocytes of type 2 diabetes (T2D) humans, insulin activation of insulin receptor substrate-1(IRS-1) and Akt fails, further increasing FoxO1-dependent gluconeogenic/lipogenic enzyme expression. Increases in hepatic PGC-1α also figure prominently, but uncertainly, in this scheme. Here, we examined signaling factors in liver samples harvested from human transplant donors with increasing BMI, 20→25→30→35→40→45.We found, relative to lean (BMI=20–25) humans, obese (BMI>30) humans had all abnormalities seen in early mouse DIO, but, surprisingly, at all elevated BMI levels, had decreased insulin receptor-1 (IRS-1) levels, decreased Akt activity, and increased expression/abundance of aPKC-ι and PGC-1α. Moreover, with increasing BMI, there were: progressive increases in aPKC activity and PKC-ι expression/abundance; progressive decreases in IRS-1 levels, Akt activity and FoxO1 phosphorylation; progressive increases in expression/abundance of PGC-1α; and progressive increases in gluconeogenic and lipogenic enzymes. Remarkably, all abnormalities reached T2D levels at higher BMI levels. Most importantly, both “early” and advanced abnormalities were largely reversed by 24-hour treatment of T2D hepatocytes with aPKC inhibitor. We conclude: hepatic insulin resistance in human obesity is: advanced; BMI-correlated; and sequentially involves increased aPKC-activating ceramide; increased aPKC levels and activity; decreases in IRS-1 levels, Akt activity, and FoxO1 phosphorylation; and increases in expression/abundance of PGC-1α and gluconeogenic and lipogenic genes.

Abstract 266: Screening and Validation of Small Molecule Inhibitors of Serum and Glucocorticoid Regulated Kinase-1 as Novel Therapy for Cardiac Arrhythmia Disorders

Alterations in sodium flux (INa) play an important role in the pathogenesis of cardiac arrhythmias and may also contribute to the development of cardiomyopathies. Recent data demonstrates a critical role for the serum and glucocorticoid regulated kinase-1 (SGK1) by modulation of INa in the heart, by regulating the voltage-gated sodium channel NaV1.5. To better understand and pharmacologically probe the significance of SGK1 in cardiac dysrhythmias, we have used computer aided drug discovery (CADD) to identify small molecule inhibitors of SGK1. Expression of a constitutively active form of SGK1 (SGK1-CA) increased INa (1.7 fold, p <0.005) in a stable line of HEK cells expressing NaV1.5. Conversely, expression of a dominant negative form (SGK-DN) decreased NaV1.5 channel activity (2.8 fold, p <0.005). We examined the effects of SGK1 inhibition in a LQT model, by quantifying the ability of SGK1 inhibition to rescue the 2:1 AV block phenotype of the potassium channel zebrafish mutant, breakdance (bkd). Morpholino injection or expression of SGK1-DN significantly rescued the 2:1 AV block phenotype as compared to controls (p < 0.05). Using CADD partnered with iterative empirical screens we identified several hit chemical scaffolds. Our lead compound inhibits the phosphorylation of the SGK1 target gene, GSK3-β in a dose dependent manner in cardiomyocytes (CMs) expressing SGK1-CA at the lowest effective concentration of 0.5μM. There was no significant inhibition of AKT dependent phosphorylation of GSK3-β up to a concentration of 50μM, demonstrating specificity of the inhibitor for SGK1. Incubation of bkd zebrafish mutants with the inhibitor rescued the 2:1 AV block in a dose dependent manner (60% rescue with 45μM, p < 0.05). Acute application of the inhibitor dramatically inhibited INa with either expression of SGK1-CA (90.8% reduction, p <0.05 ) or with RFP only (77.5% reduction p < 0.005). The half-time of inhibition was 200s with resulting current densities that were not statistically different than those observed with genetic inhibition by expression of SGK1-DN. We conclude SGK1 activity regulates INa and speculate that structure activity relationship (SAR) derivatives of our lead compound might have a role in treatment of human cardiac arrhythmias.

Angiopoietin-1 Protects the Endothelial Cells Against Advanced Glycation End Product Injury by Strengthening Cell Junctions and Inhibiting Cell Apoptosis

Endothelial dysfunction is a major characteristic of diabetic vasculopathy. Protection of the vascular endothelium is an essential aspect of preventing and treating diabetic vascular complications. Although Angiopoietin-1 (Ang-1) is an important endothelial-specific protective factor, whether Ang-1 protects vascular cells undergoing advanced glycation end product (AGE) injury has not been investigated. The aim of the present study was to determine the potential effects of Ang-1 on endothelial cells after exposure to AGE. We show here that Ang-1 prevented AGE-induced vascular leakage by enhancing the adherens junctions between endothelial cells, and this process was mediated by the phosphorylation and membrane localization of VE-cadherin. Furthermore, Ang-1 also protected endothelial cells from AGE-induced death by regulating phosphatidylinositol 3-kinase (PI3K)/Akt-dependent Bad phosphorylation. Our findings suggest that the novel protective mechanisms of Ang-1 on endothelium are achieved by strengthening endothelial cell junctions and reducing endothelial cell death after AGE injury.

Role of Akt and Ca2 + on cell permeabilization via connexin43 hemichannels induced by metabolic inhibition

Connexin hemichannels are regulated under physiological and pathological conditions. Metabolic inhibition, a model of ischemia, promotes surface hemichannel activation associated, in part, with increased surface hemichannel levels, but little is known about its underlying mechanism. Here, we investigated the role of Akt on the connexin43 hemichannel's response induced by metabolic inhibition. In HeLa cells stably transfected with rat connexin43 fused to EGFP (HeLa43 cells), metabolic inhibition induced a transient Akt activation necessary to increase the amount of surface connexin43. The increase in levels of surface connexin43 was also found to depend on an intracellular Ca2+ signal increase that was partially mediated by Akt activation. However, the metabolic inhibition-induced Akt activation was not significantly affected by intracellular Ca2+ chelation. The Akt-dependent increase in connexin43 hemichannel activity in HeLa43 cells also occurred after oxygen-glucose deprivation, another ischemia-like condition, and in cultured cortical astrocytes (endogenous connexin43 expression system) under metabolic inhibition. Since opening of hemichannels has been shown to accelerate cell death, inhibition of Akt-dependent phosphorylation of connexin43 hemichannels could reduce cell death induced by ischemia/reperfusion.

Akt-Girdin signaling in cancer-associated fibroblasts contributes to tumor progression.

PI3K-Akt signaling is critical for the development, progression, and metastasis of malignant tumors, but its role in the tumor microenvironment has been relatively little studied. Here, we report that the Akt substrate Girdin, an actin-binding protein that regulates cell migration, is expressed and activated by Akt phosphorylation in cancer-associated fibroblasts (CAF) and blood vessels within the tumor microenvironment. Lewis lung tumors grafted into mice defective in Akt-mediated Girdin phosphorylation (SA transgenic mice) exhibited a decrease in both CAF infiltration and tumor growth, compared with wild-type (WT) host control animals. Contrasting with the findings of other studies, we found that Akt-dependent phosphorylation of Girdin was not a rate-limiting step in the growth of endothelial cells. In addition, Lewis lung tumors displayed limited outgrowth when cotransplanted with CAF derived from tumor-bearing SA transgenic mice, compared with CAF derived from tumor-bearing WT mice. Collectively, our results revealed a role for Akt-mediated Girdin phosphorylation in CAF during tumor progression, highlighting the need to inhibit Akt function in both tumor cells and cells that comprise the tumor microenvironment.