Akt Thr308 Phosphorylation Research Articles

Dietary creatine promotes creatine reserves, protein deposition, and myofiber hyperplasia in muscle of juvenile largemouth bass (Micropterus salmoides)

Creatine can increase muscle mass, enhance performance, prevent disease-induced muscle atrophy, and improve overall cellular energy. This study investigated the effects of creatine as a feed additive on muscle growth and intrinsic molecular regulation mechanism in largemouth bass (Micropterus salmoides). Seven diets supplemented with different concentrations of creatine (0%, 0.1%, 0.25%, 0.5%, 1%, 2%, and 4%) were fed to juvenile largemouth bass (4.5–5.1 g) for eight weeks. Dietary creatine significantly increased body weight and body length, and reduced feed conversion ratio. High-performance liquid chromatography analysis revealed that dietary creatine addition notably increased creatine and phosphocreatine levels by 19.94% and 16.54% in the muscle of largemouth bass, respectively. Histological observation showed that dietary creatine significantly increased the total muscle fiber number and decreased the mean muscle fiber diameter. The Western blot results indicated that creatine ingestion significantly increased the protein level of MyoD1 in the muscle and promoted muscle fiber hyperplasia. In addition, dietary creatine also contributed to mTOR-S6K/4EBP1-mediated protein synthesis by maintaining AMPKα Thr172 dephosphorylation and AKT Thr308 phosphorylation. Moreover, the protein levels of the muscle-specific E3 ligases Atrogin1 and Murf1 responsible for ubiquitination degradation exhibited no significant change after feeding with creatine in largemouth bass. Overall, our data showed that creatine as a feed additive increased creatine and phosphocreatine contents, thus promoting muscle protein deposition and myofiber hyperplasia, eventually elevating growth performance and feed utilization efficiency in juvenile largemouth bass. The regression analysis of weight gain rate, specific growth rate, and feed conversion ratio determined 0.46%–0.54% creatine concentration range as optimal supplementation concentration for the culture of juvenile largemouth bass.

Investigating the pharmacological mechanism of Zhengyuan jiaonang for treating colorectal cancer via network pharmacology analysis and experimental verification

Ethnopharmacological relevanceZhengyuan jiaonang (ZYJN) is a traditional Chinese patent medicine (CPM) used in China for adjuvant cancer therapy, which has been proved to have anti-fatigue effects. Aim of studyThe study aims to investigate the antitumor effects of ZYJN and its underlying mechanisms using subcutaneous transplant CT26 model. Materials and methodsFingerprint analysis of ZYJN was performed using high performance liquid chromatography. The potential targets of ZYJN were predicted using bioinformatic analysis, which were further validated by Western Blot assay. Subcutaneous transplant CT26 model was used to evaluate the antitumor effects of ZYJN. The effects of ZYJN on the tumor immune microenvironment were investigated by flow cytometry. Transparent imaging was used to investigate the effects of ZYJN on fibrosis and angiogenesis. ResultsZYJN could inhibit colorectal cancer growth when administered alone or in combination with 5-FU. The combination of ZYJN and 5-FU could significantly increase the serum level of albumin (ALB) and decrease the serum level of aspartate aminotransferase (AST). In addition, the combination of ZYJN at 0.75 g/kg and 5-FU significantly decreased the serum level of vascular endothelial growth factors (VEGF) and inhibited the angiogenesis of CT26 cancer. The combination of ZYJN at 1.50 g/kg and 5-FU could promote the fibrosis process of CT26 cancer. Additionally, combination of ZYJN and 5-FU could significantly increase the percentage of tumor-infiltrating T cells and CD4+ T cells in the late stage of CT26 model, while ZYJN at 1.50 g/kg increased the percentage of NK cells as well as CD8+ T cells in the early stage of CT26 model. Western Blot analysis revealed that administration of ZYJN at 0.75 g/kg reduced the expression of PI3K-p110α, CDK1, CCNB1 and MMP-9, and inhibited the phosphorylation of Akt (Thr308). ConclusionsZYJN could inhibit the tumor growth of CT26 colorectal cancer by promoting tumor fibrosis, suppressing angiogenesis, migration, and invasion and modulating the tumor immune microenvironment. ZYJN enhanced the efficacy and reduced the toxicity of chemotherapy drugs in combination therapy. Our findings provide evidence for the clinical application of ZYJN in cancer treatment.

Dendrobium officinale polysaccharide decreases podocyte injury in diabetic nephropathy by regulating IRS-1/AKT signal and promoting mitophagy.

High glucose (HG) caused oxidative stress and mitochondrial dysfunction, resulting in insulin resistance in podocytes, a key mechanism of diabetic nephropathy. Dendrobium officinale polysaccharide (DOP) was able to improve insulin resistance and antioxidant capability. The purpose of this study is to explore the mechanism by which DOP decreases the podocyte injury induced by HG. MPC5 cells were treated with HG, DOP, and IRS-1/2 inhibitor NT157. Afterwards, glucose consumption, generations of ROS and MDA were measured using the detection kits. Mitophagy was monitored using both MtphagTracyker and LysoTracker. The mitochondrial membrane potential was evaluated by JC-1 staining. DOP was also used in a mouse model of diabetes, with the measurements of urine albumin, blood creatinine and blood urea nitrogen. Treatment with DOP suppressed the HG-induced reduction of glucose consumption, the phosphorylation of IRS-1 (phospho Y632), AKT (phospho Ser473 and Thr308) and Nephrin. In addition, HG-induced augment of ROS and MDA, formation of γ-H2A.X foci and translocation of AKT to nucleus were inhibited by DOP. DOP enhanced mitophagy, which was associated with decreased mitochondrial membrane potential and ROS production. DOP conferred protective effect on podocyte in the diabetic mouse by reducing the albumin/creatinine ratio and blood urea nitrogen, and restoring Nephrin expression in podocytes. DOP alleviates HG-induced podocyte injuryby regulating IRS-1/AKT signal and promoting mitophagy.

190-OR: Lrtm1, a Novel Regulator of Insulin Signaling and Metabolism

Patients with obesity and metabolic syndrome, as well as individuals with pre-type 2 diabetes, exhibit hyperinsulinemia prior to development of hyperglycemia due to underlying skeletal muscle insulin resistance. To mimic the effect of chronic hyperstimulation of the insulin receptor (IR), we overexpressed the IR specifically in skeletal muscle (IRMOE). This led to skeletal muscle post-receptor desensitization, with increased IR and IRS-1(Tyr) phosphorylation but decreased phosphorylation of Akt Ser473 and Thr308. To identify the regulators involved in this post-receptor insulin resistance, we performed RNA sequencing of IRMOE muscle. Among the most upregulated genes was Lrtm1, a poorly characterized leucine rich repeat transmembrane molecule while highly expressed in the heart and skeletal muscle. Overexpression of Lrtm1 in C2C12 myoblasts led to increased phosphorylation of early insulin signaling molecules (the insulin receptor, IRS-1, and Shc), but reduced phosphorylation of the downstream molecules Akt (at both Ser473 and Thr308) and Erk Thr202/204 following insulin stimulation. Mice with knockout of Lrtm1 (Lrtm1KO) showed significant increases in percent lean mass and decreases in percent fat mass, as well as a slight reduction in overall body weight. In addition, Lrtm1KO mice were more active than controls in both metabolic cages and an open field behavioral tests. Lrtm1KO mice also exhibited improved glucose tolerance and insulin sensitivity, especially in males, even on normal chow diet. Most importantly, Lrtm1KO mice were protected from high fat diet induced obesity and metabolic syndrome, with the knockouts showing 18% less body weight gain, 30% lower fat mass, significantly improved glucose tolerance and improved insulin sensitivity. Thus, Lrtm1 is a novel regulator of post-receptor insulin signaling and glucose metabolism that may provide a new target for therapy insulin resistant prediabetes and type 2 diabetes. Disclosure G. Wang: Employee; Agilent. Y. Yu: None. W. Chen: None. W. Cai: None. X. Liu: None. C. Kahn: None.

COX5A Alleviates Doxorubicin-Induced Cardiotoxicity by Suppressing Oxidative Stress, Mitochondrial Dysfunction and Cardiomyocyte Apoptosis.

Doxorubicin (DOX) as a chemotherapeutic agent can cause mitochondrial dysfunction and heart failure. COX5A has been described as an important regulator of mitochondrial energy metabolism. We investigate the roles of COX5A in DOX-induced cardiomyopathy and explore the underlying mechanisms. C57BL/6J mice and H9c2 cardiomyoblasts were treated with DOX, and the COX5A expression was assessed. An adeno-associated virus serum type 9 (AAV9) and lenti-virus system were used to upregulate COX5A expression. Echocardiographic parameters, morphological and histological analyses, transmission electron microscope and immunofluorescence assays were used to assess cardiac and mitochondrial function. In a human study, we found that cardiac COX5A expression was dramatically decreased in patients with end-stage dilated cardiomyopathy (DCM) compared to the control group. COX5A was significantly downregulated following DOX stimulation in the heart of mice and H9c2 cells. Reduced cardiac function, decreased myocardium glucose uptake, mitochondrial morphology disturbance, reduced activity of mitochondrial cytochrome c oxidase (COX) and lowered ATP content were detected after DOX stimulation in mice, which could be significantly improved by overexpression of COX5A. Overexpression of COX5A effectively protected against DOX-induced oxidative stress, mitochondrial dysfunction and cardiomyocyte apoptosis in vivo and in vitro. Mechanistically, the phosphorylation of Akt (Thr308) and Akt (Ser473) were also decreased following DOX treatment, which could be reserved by the upregulation of COX5A. Furthermore, PI3K inhibitors abrogated the protection effects of COX5A against DOX-induced cardiotoxicity in H9c2 cells. Thus, we identified that PI3K/Akt signaling was responsible for the COX5A-mediated protective role in DOX-induced cardiomyopathy. These results demonstrated the protective effect of COX5A in mitochondrial dysfunction, oxidative stress, and cardiomyocyte apoptosis, providing a potential therapeutic target in DOX-induced cardiomyopathy.

Impact of prior alcohol use on the subsequent development of cancer cachexia in male and female mice.

Alcohol is a carcinogen and its intake prior to developing cancer and throughout its duration exacerbates cancer cachexia in rodent models. However, the effects on cancer cachexia of stopping alcohol prior to tumor establishment are unknown. Male and female mice consumed either a nonalcohol control liquid diet (CON) or a 20% ethanol (kcal/day) liquid diet (EtOH) for 6 weeks. All mice then consumed a control diet and mice in the cancer groups were inoculated with C26 colon cancer cells. Gastrocnemius muscles were collected and analyzed after ~2 weeks. Skeletal muscle weight and male epididymal and female perigonadal fat mass were reduced more by the combination of cancer and prior EtOH than either exposure alone in both males and females. In males, protein synthesis was reduced by 30% following alcohol exposure, while no reductions were observed in female mice. AMPK Thr172 phosphorylation was increased in both male and female EtOH-Cancer groups, while Akt Thr308 phosphorylation was reduced only among males in EtOH-Cancer mice. Substrates in the mTORC1 pathway were reduced by cancer in both males and females, but prior alcohol intake only reduced phosphorylation of 4E-BP1 Ser65 and rpS6 Ser240/244 to a greater extent in male, but not female, mice. Autophagic and proteasomal signaling were largely unaffected by prior alcohol intake in cancer mice, despite a greater increase in Murf1 mRNA in both sexes. Prior alcohol consumption accelerates or worsens the onset of certain aspects of cancer cachexia in a sex-dependent manner, with males being more sensitive to these exposures, even with abstinence from alcohol prior to tumor initiation.

Insulin-resistant female rat skeletal muscles display diacylglycerol-mediated protein kinase C activation and inflammation without ceramide accumulation.

This study investigated the role of diacylglycerol (DAG)-mediated protein kinase C (PKC) activation, ceramide accumulation and inflammation in insulin-resistant female oxidative and glycolytic skeletal muscles induced by an obesogenic high-fat sucrose-enriched (HFS) diet. The HFS diet impaired insulin-stimulated AKTThr308 phosphorylation and glycogen synthesis, whereas rates of fatty acid oxidation and basal lactate production were significantly elevated in soleus (Sol), extensor digitorum longus (EDL) and epitrochlearis (Epit) muscles. Insulin resistance was accompanied by increases in triacylglycerol (TAG) and DAG contents in Sol and EDL, whereas in Epit muscles only TAG content and markers of inflammation were associated with HFS diet-induced insulin resistance. Analysis of membrane-bound/cytoplasmic PKC fractions revealed that the HFS diet promoted activation/translocation of PKCδ and θ isoforms in Sol, EDL and Epit muscles. However, none of these muscles displayed alterations in ceramide content in response to HFS feeding. This could be explained by a significant increase in Dgat2 mRNA expression in Sol, EDL and Epit muscles, which likely diverted most of the intramyocellular acyl-CoAs toward TAG synthesis instead of ceramides. Overall, this study helps elucidate the molecular mechanisms underlying insulin resistance caused by diet-induced obesity in female skeletal muscles with distinct fibre type compositions. KEY POINTS: Feeding female Wistar rats a high-fat sucrose-enriched diet (HFS) led to diacylglycerol (DAG)-induced PKC activation and insulin resistance in oxidative and glycolytic skeletal muscles. HFS diet-induced toll-like receptor 4 (Tlr4) expression did not lead to increased ceramide content in female skeletal muscles. In highly glycolytic female muscles, elevated TAG content and markers of inflammation underlay HFS diet-induced insulin resistance. The HFS diet suppressed glucose oxidation and increased lactate production in oxidative and glycolytic female muscles. Increased Dgat2 mRNA expression likely diverted most of the intramyocellular acyl-CoAs toward TAG synthesis and prevented ceramide formation in skeletal muscles of HFS-fed female rats.

MicroRNA‐17‐3p protects against excessive posthypoxic autophagy in H9C2 cardiomyocytes via PTEN–Akt–mTOR signaling pathway

The activity of phosphatase and tensin homolog (PTEN) can be inhibited by miR-17-3p, which results in attenuating myocardial ischemia/reperfusion injury (IRI), however, the mechanism behind this phenomenon is still elusive. Suppression of PTEN leads to augmented protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling strength and constrained autophagy activation, which might be the one mechanism for the ameliorated myocardial IRI. Thus, we tested the hypothesis that miR-17-3p attenuated hypoxia/reoxygenation (H/R)-mediated damage in cardiomyocytes by downregulating excessive autophagy via the PTEN-Akt-mTOR axis. The expression of miR-17-3p was remarkably increased after H/R treatment (6-h hypoxia followed by 6-h reoxygenation; H6/R6), which was concomitant with the increase of the release of lactic acid dehydrogenase (cell injury marker) and the enhancement LC3II/I ratio (autophagy markers) in H9C2 cardiomyocytes. Ectoexpression of miR-17-3p agomir led to remarkable augmentation of miR-17-3p expression and evidently attenuated H/R-mediated cell damage and excessive autophagy. Furthermore, an increase in miR-17-3p expression elicited constrained phosphorylation of PTEN (Ser380 ) while enhanced the phosphorylation of Akt (Thr308 , Ser473 ) and mTOR (Ser536 ) after H/R stimulation. In addition, pretreatment with LY-294002 (an Akt selective inhibitor) and rapamycin (an mTOR selective inhibitor) significantly abrogated the protective function of miR-17-3p on H/R-mediated cell damage and autophagy in H9C2 cardiomyocytes. Taken together, these observations indicated that the enhancement of the PTEN/Akt/mTOR axis and the consequent suppression of autophagy overactivation might represent an underlying mechanism by which miR-17-3p attenuated H/R-mediated damage in H9C2 cells.

Integrating Network Pharmacology and Bioinformatics to Explore the Effects of Dangshen (Codonopsis pilosula) Against Hepatocellular Carcinoma: Validation Based on the Active Compound Luteolin.

This study aimed to explore the pharmacological mechanism of Dangshen (Codonopsis pilosula) against hepatocellular carcinoma (HCC) based on network pharmacology and bioinformatics, and to verify the anticancer effect of luteolin, the active ingredient of Codonopsis pilosula, on HCC cells. The effective compounds and potential targets of Codonopsis pilosula were established using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database. The genes related to HCC were obtained through the GeneCards database. The interactive genes were imported into the Visualization and Integrated Discovery database for Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal enrichment, and the hub genes were screened out. The Cancer Genome Atlas database was used to construct a prognosis model, and the prognosis and clinicopathological correlation were analyzed. In in vitro experiments, we verified the effects of luteolin, an active compound of Codonopsis pilosula, on the proliferation, cell cycle, apoptosis and migration of HCC cells. A total of 21 effective compounds of Codonopsis pilosula and 98 potential downstream target genes were screened through the TCMSP database, and 1406 HCC target genes were obtained through the GeneCards database. Finally, 53 interacting genes between the two databases were obtained, among which, the 10 key node genes were CASP3, TP53, MDM2, AKT1, ESR1, BCL2L1, MCL1, HSP90AA1, CASP9, and CCND1, involving 77 typical GO terms and 72 KEGG signals. The Kaplan-Meier survival curve of the model group showed that the overall survival of the low-risk group was significantly higher than that of the high-risk group. Luteolin significantly inhibited the proliferation and migration of HCC cells, induced apoptosis, and increased the G2/M phase ratio. Mechanistically, luteolin significantly inhibited the phosphorylation of MAPK-JNK and Akt (Thr308) and subsequently led to upregulation of ESR1. Pharmacological inhibition of ESR1 with fulvestrant enhanced cell viability and migration and attenuated apoptosis. Codonopsis pilosula has potential for clinical development due to its anti-HCC properties. Luteolin, the effective component of Codonopsis pilosula, plays anti-HCC role through AKT- or MAPK-JNK signaling mediated ESR1.

Finnish-specific AKT2 gene variant leads to impaired insulin signalling in myotubes.

Finnish-specific gene variant p.P50T/AKT2 (MAF=1.1%), is associated with insulin resistance and increased predisposition to type 2 diabetes. Here, we have investigated in vitro the impact of the gene variant on glucose metabolism and intracellular signalling in human primary skeletal muscle cells, that were established from 14 male p.P50T/AKT2 variant carriers and 14 controls. Insulin-stimulated glucose uptake and glucose incorporation into glycogen were detected with 2-[1,2-3H]-deoxy-D-glucose and D-[14C]-glucose, respectively, and the rate of glycolysis was measured with a Seahorse XFe96 analyzer. Insulin signalling was investigated with western blotting. Binding of variant and control AKT2-PH domains to phosphatidylinositol (3,4,5)-trisphosphate ((PI(3,4,5)P3)) was assayed using PIP Strips™ Membranes. Tyrosine (PTK) and serine-threonine (STK) kinase assay was performed using the PamGene® kinome profiling system. Insulin-stimulated glucose uptake and glycogen synthesis in myotubes in vitro were not significantly affected by the genotype. However, insulin-stimulated glycolytic rate was impaired in variant myotubes. Western blot analysis showed that insulin-stimulated phosphorylation of Akt-Thr308, AS160-Thr642 and GSK3β-Ser9 was reduced in variant myotubes compared to controls. Binding of variant AKT2-PH domain to PI(3,4,5)P3 was reduced as compared to control protein. PamGene® kinome profiling revealed multiple differentially phosphorylated kinase substrates, e.g. calmodulin, between the genotypes. Further in silico upstream kinase analysis predicted a large-scale impairment in activities of kinases participating for example in intracellular signal transduction, protein translation and cell cycle events. In conclusion, myotubes from p.P50T/AKT2 variant carriers show multiple signalling alterations which may contribute to predisposition to insulin resistance and T2D in the carriers of this signalling variant.

Interactive contribution of hyperinsulinemia, hyperglycemia, and mammalian target of rapamycin signaling to valvular interstitial cell differentiation and matrix remodeling.

Diabetes and its major key determinants insulin resistance and hyperglycemia are known risk factors for calcific aortic valve disease (CAVD). The processes leading to molecular and structural alterations of the aortic valve are yet not fully understood. In previous studies, we could show that valvular interstitial cells (VIC) display canonical elements of classical insulin signaling and develop insulin resistance upon hyperinsulinemia and hyperglycemia accompanied by impaired glucose metabolism. Analyses of cultured VIC and aortic valve tissue revealed extracellular matrix remodeling and degenerative processes. Since PI3K signaling through mammalian target of rapamycin (mTOR) is involved in fibrotic processes of the heart, we aim at further functional investigation of this particular Akt-downstream signaling pathway in the context of diabetes-induced CAVD. Primary cultures of VIC were treated with hyperinsulinemia and hyperglycemia. Phosphorylation of mTOR(Ser2448) was determined by Western blot analysis after acute insulin stimulus. Inhibition of mTOR phosphorylation was performed by rapamycin. Phosphorylation of mTOR complex 1 (MTORC1) downstream substrates 4E-BP1(Thr37/46) and P70S6K(Thr389), and MTORC2 downstream substrate Akt(Ser473) as well as the PDK1-dependent phosphorylation of Akt(Thr308) was investigated. Markers for extracellular matrix remodeling, cell differentiation and degenerative changes were analyzed by Western blot analysis, semi-quantitative real-time PCR and colorimetric assays. Hyperinsulinemia and hyperglycemia lead to alterations of VIC activation, differentiation and matrix remodeling as well as to an abrogation of mTOR phosphorylation. Inhibition of mTOR signaling by rapamycin leads to a general downregulation of matrix molecules, but to an upregulation of α-smooth muscle actin expression and alkaline phosphatase activity. Comparison of expression patterns upon diabetic conditions and rapamycin treatment reveal a possible regulation of particular matrix components and key degeneration markers by MTORC1 downstream signaling. The present findings broaden the understanding of mitogenic signaling pathways in VIC triggered by hyperinsulinemia and hyperglycemia, supporting the quest for developing strategies of prevention and tailored treatment of CAVD in diabetic patients.

CircSEMA4B inhibits the progression of breast cancer by encoding a novel protein SEMA4B-211aa and regulating AKT phosphorylation

PI3K/AKT signaling pathway plays an important role in regulating the tumorigenesis, recurrence, and metastasis of breast cancer (BC). In this study, we discovered a circRNA with protein-coding potential, which we named circSEMA4B. CircSEMA4B could encode a novel protein, SEMA4B-211aa. Both circSEMA4B and SEMA4B-211aa were remarkably downregulated in BC tissues and cell lines. Low expression of circSEMA4B was positively associated with TNM stage, tumor size, lymph node metastasis, and distant metastasis of BC patients. The functional investigation showed that circSEMA4B and SEMA4B-211aa could significantly inhibit the proliferation and migration of BC in vivo and in vitro. Of note, SEMA4B-211aa inhibited the generation of PIP3 by binding to p85, thereby inhibiting the phosphorylation of AKT (Thr308). CircSEMA4B inhibited the phosphorylation of AKT (Ser473) through miR-330-3p/PDCD4 axis. Taken together, circSEMA4B is a novel negative regulator of PI3K/AKT signaling pathway, providing novel mechanistic insights into the underlying mechanisms of BC.

Hepatic mTORC2 Signaling Facilitates Acute Glucagon Receptor Enhancement of Insulin-Stimulated Glucose Homeostasis in Mice.

Long-term glucagon receptor (GCGR) agonism is associated with hyperglycemia and glucose intolerance, while acute GCGR agonism enhances whole-body insulin sensitivity and hepatic AKTSer473 phosphorylation. These divergent effects establish a critical gap in knowledge surrounding GCGR action. mTOR complex 2 (mTORC2) is composed of seven proteins, including RICTOR, which dictates substrate binding and allows for targeting of AKTSer473. We used a liver-specific Rictor knockout mouse (RictorΔLiver) to investigate whether mTORC2 is necessary for insulin receptor (INSR) and GCGR cross talk. RictorΔLiver mice were characterized by impaired AKT signaling and glucose intolerance. Intriguingly, RictorΔLiver mice were also resistant to GCGR-stimulated hyperglycemia. Consistent with our prior report, GCGR agonism increased glucose infusion rate and suppressed hepatic glucose production during hyperinsulinemic-euglycemic clamp of control animals. However, these benefits to insulin sensitivity were ablated in RictorΔLiver mice. We observed diminished AKTSer473 and GSK3α/βSer21/9 phosphorylation in RictorΔLiver mice, whereas phosphorylation of AKTThr308 was unaltered in livers from clamped mice. These signaling effects were replicated in primary hepatocytes isolated from RictorΔLiver and littermate control mice, confirming cell-autonomous cross talk between GCGR and INSR pathways. In summary, our study reveals the necessity of RICTOR, and thus mTORC2, in GCGR-mediated enhancement of liver and whole-body insulin action.

IL-6 Responsiveness of CD4+ and CD8+ T Cells after Allogeneic Stem Cell Transplantation Differs between Patients and Is Associated with Previous Acute Graft versus Host Disease and Pretransplant Antithymocyte Globulin Therapy.

Graft-versus-host disease (GVHD), one of the most common and serious complications after allogeneic stem cell transplantation, is mediated by allocative T cells. IL-6 mediates both pro- and anti-inflammatory effects and modulates T cell response through classical signaling and trans-signaling. We investigated the effects on the mTOR and JAK/STAT pathways after various types of IL-6 signaling for circulating T cells were derived from 31 allotransplant recipients 90 days post-transplant. Cells were stimulated with IL-6 alone, hyper-IL-6 (trans-signaling), IL-6+IL-6 receptor (IL-6R; classical + trans-signaling) and IL-6+IL-6R+soluble gp130-Fc (classical signaling), and flow cytometry was used to investigate the effects on phosphorylation of AKT (Thr308), mTOR (Ser2442), STAT3 (Ser727) and STAT3 (Tyr705). CD3+CD4+ and CD3+C8+ T cells responded to classical and trans IL-6 stimulation with increased STAT3 (Tyr705) phosphorylation; these responses were generally stronger for CD3+CD4+ cells. STAT3 (Tyr705) responses were stronger for patients with previous acute GVHD; CD3+CD4+ cells from GVHD patients showed an additional STAT3 (Ser727) response, whereas patients without acute GVHD showed additional mTOR (Ser2448) responses. Furthermore, treatment with antithymocyte globulin as a part of GVHD prophylaxis was associated with generally weaker STAT3 (Tyr705) responses and altered STAT3 (Ser727) responsiveness of CD3+CD4+ cells together with increased mTOR (Ser2448) responses for the CD3+CD8+ cells. Thus, early post-transplant CD3+CD4+ and CD3+ CD8+ T cell subsets differ in their IL-6 responsiveness; this responsiveness is modulated by antithymocyte globulin and differs between patients with and without previous acute GVHD. These observations suggest that allotransplant recipients will be heterogeneous with regard to the effects of post-transplant IL-6 targeting.

Abstract PD3-01: Inducible nitric oxide synthase activates PI3K/Akt signaling via PTEN S-nitrosylation in triple-negative breast cancer

Abstract Our previous findings have shown that increased inducible nitric oxide synthase (iNOS) expression is a poor prognostic indicator and associated with worse overall survival in TNBC patients. Growing evidence has also suggested that hyperactivation of the phosphoinositide-3-kinase (PI3K) survival signaling pathway is one of the most common oncogenic aberrations in TNBC. Nitric oxide (NO) is a unique molecule in its ability to target multiple oncogenic pathways in a spatial and temporal manner, such as PI3K, extracellular signal-regulated kinase (ERK), β-catenin pathway, transforming growth factor beta (TGFβ) signaling, and hypoxia-inducible factor (HIF). This study investigates the role of NO in PI3K-Akt pathway activation in human TNBC cells, in conditions with extracellular NO donor exposure and increased intracellular iNOS (NOS2) expression. TNBC cell lines BT549, HCC1937, Hs578T, SUM159, MDA-MB-231, and BT-20 were exposed to the NO donor DETA NONOate at increasing concentrations (0-500 µM) for 12 hours. Significant elevation in phosphorylation of Akt (Ser473 and Thr308) in response to increase NO donor concentration was found only in cell lines with wild-type PTEN and PIK3R1, such as SUM159, MDA-MB-231, and BT20. To evaluate whether NO-induced activation of Akt was PI3K-dependent, SUM159 (wild-type PTEN) and HCC1937 (PTEN mutant) cells were also pre-treated with PI3K inhibitor alpelisib before NO exposure. PI3K inhibition was only able to eliminate basal phospho-Akt and prevent NO-induced Akt activation in the SUM159 cell line. Furthermore, compared to vehicle control treated cells, we found that NO donor-induced activation enhanced phosphorylation in 15/18 Akt signaling proteins in SUM159 cells (PTEN intact) and 3/18 Akt signaling proteins in HCC1937 cells (PTEN loss). In Hs578T cells in which NOS2 was either overexpressed or knocked down via lentiviral transduction, we found that enhanced expression of NOS2 was associated with increased p-Akt expression and knock-down of NOS2 led to reduced p-Akt. This result suggests that iNOS, as opposed to other NOS isoforms, is more associated with modulating PI3K signaling in TNBC.We also analyzed TNBC data from The Cancer Genome Atlas and found that patients with positive iNOS mRNA expression had significantly higher phospho-Akt (Ser473 and Thr308) [p≤0.0001] expression compared to patients with no iNOS expression. When we stratified this patient cohort based on both iNOS and PTEN expression, we only found significantly higher phospho-Akt (Ser473 and Thr308) [p≤0.01] expression in patients with positive iNOS and PTEN expression, supporting the hypothesis that NO may impair PTEN’s function as a PI3K antagonist. Ongoing studies are directed to investigate mechanisms of iNOS-PTEN physical interactions, iNOS-dependent post-translational modifications to PTEN, and whether iNOS and PTEN are dual prognostic markers in TNBC patients. Citation Format: Tejaswini P. Reddy, Liliana Guzman-Rojas, Roberto R. Rosato, Wei Qian, Hong Zhao, Jenny C. Chang. Inducible nitric oxide synthase activates PI3K/Akt signaling via PTEN S-nitrosylation in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-01.

Baicalein Preconditioning Cardioprotection Involves Pro-Oxidant Signaling and Activation of Pyruvate Dehydrogenase.

Preconditioning has a powerful protective potential against myocardial ischemia-reperfusion injury (I/R). Our prior work demonstrated that baicalein, a flavonoid derived from the root of Scatellaria baicalensis Georgi (also known as Huangqin), confers this preconditioning protection. This study further explored the mechanisms of baicalein preconditioning (BC-PC) in mouse cardiomyocytes. Cells were treated with baicalein (10 μM) for a brief period of time (10 min) prior to simulated ischemia 90 min/reperfusion for 180 min. Baicalein triggered an induction of a small amount of mitochondrial reactive oxygen species (ROS) prior to the initiation of ischemia, assessed by 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (6-carboxy-H2DCFDA). It also significantly increased cell viability measured by propidium iodide (PI) and lactate dehydrogenase and preserved mitochondrial membrane potential assessed by TMRM fluorescence intensity. Myxothiazol, a mitochondrial electron transport chain complex III inhibitor, partially blocked ROS generation induced by BC-PC and reduced cell viability. BC-PC increased phosphorylation of Akt (Thr308 and Ser473) and eNOS Ser1177, and nitric oxide (NO) production measured using 4,5-diaminofluorescein diacetate (DAF-2 DA, 1 μM). Akt inhibitor API-2 abolished Akt phosphorylation and reduced DAF-2 production and cell viability. In addition, BC-PC decreased phosphorylation of pyruvate dehydrogenase (PDH) reflecting upregulated PDH activity, and increased ATP production at 30 min during reperfusion. Taken together, baicalein preconditioning-induced cardioprotection involves pro-oxidant generation, activates survival signaling Akt/eNOS/NO, and improves metabolic recovery after I/R injury. Our work provides new perspectives on the effect of baicalein on cardiac preconditioning against I/R injury.

Astrocyte-secreted TIMP-1 binds to CD63 and differentially phosphorylates Akt in protecting neurons and promoting cognitive recovery in 5xFAD mice.

Astrocytes respond dramatically to pathological alterations in the brain including Alzheimer's disease (AD) through a highly heterogeneous process called astrocyte reactivity. Astrocyte reactivity is characterized by changes in morphological, transcriptional and biochemical levels. The reactive astrocytes secrete a plethora of cytokines that regulate neuronal health through disease stages. Recently, we showed that oligomeric Aβ1-42 (Aβ) treatment to primary astrocytes induced an early reactivity and detected tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as the major candidate in the astrocyte secretome that promoted neuronal viability against Aβ toxicity. Moreover, in Aβ-injected rats, TIMP-1 recovered cognitive functions (Brain Behav Immun. 2020 87:804-819). However, the mechanisms underlying TIMP-1's neuroprotective role remained to be revealed which we aim to achieve through our present study. Primary cortical neurons were co-treated with Aβ and recombinant TIMP-1, and Akt phosphorylation at different sites and downstream protein levels were checked by western blotting and reconfirmed by immunofluorescence studies. Co-localization and immunoprecipitation studies were performed to determine TIMP-1's binding partner/s. Subsequently, TIMP-1 was stereotactically injected in 6-month old 5xFAD mice and cognitive behaviors were assessed through locomotion, passive avoidance, fear conditioning and elevated plus maze tests. Immunohistochemical analyses and western blotting were employed to analyze pathway inductions in vivo. In neurons, TIMP-1 activated Akt by phosphorylating at two different residues: S473 and Thr308. Akt S473 phosphorylation ameliorated impaired autophagy flux and downregulated Aβ-induced pro-apoptotic protein levels by inhibiting FoxO3a in cultured neurons and in cortex and hippocampus of 5xFAD mouse. Subsequently, CD63 was detected as the binding partner of TIMP-1 on the neuronal surface. TIMP-1 treatment recovered the cognitive deficits in 5xFAD mice. Synaptic proteins, SNAP-25 and PSD-95, expressions were increased following TIMP-1 treatment in 5xFAD. This was consistent with Akt thr308 phosphorylation and downstream GSK3β phosphorylation-mediated inhibition, implicated in improving synaptic plasticity. Data show that TIMP-1 regulates differential phosphorylation of Akt that is potentially central to regulating autophagy, apoptotic pathways and synaptic plasticity in models of AD resulting in cognitive recovery. We highlight that TIMP-1 mediates its neuroprotective role by binding to CD63 and propose it as a primary candidate in astrocyte-secreted cytokine-mediated AD therapy.

Cholecalciferol ameliorates insulin signalling and insulin regulation of enzymes involved in glucose metabolism in the rat heart

Context The evidence on potential cross-talk of vitamin D and insulin in the regulation of cardiac metabolism is very scanty. Objective Cholecalciferol was administered to male Wistar rats for six weeks to study its effects on cardiac glucose metabolism regulation. Materials and methods An expression, phosphorylation and/or subcellular localisation of insulin signalling molecules, glucose transport and metabolism key proteins were studied. Results Circulating non-esterified fatty acids (NEFA) level was lower after cholecalciferol administration. Cholecalciferol decreased cardiac insulin receptor substrate 1 Ser307 phosphorylation, while insulin-stimulated Akt Thr308 phosphorylation was increased. Cardiac 6-phosphofructo-2-kinase protein, hexokinase 2 mRNA level and insulin-stimulated glycogen synthase kinase 3β Ser9 phosphorylation were also increased. Finally, FOXO1 transcription factor cytosolic level was reduced. Conclusion Vitamin D-related improvement of insulin signalling and insulin regulation of glucose metabolism in the rat heart is accompanied by the decrease of blood NEFA level and dysregulation of cardiac FOXO1 signalling.

Abstract MP40: Breakpoint Cluster Region Protein Function As A Kinase Mediates The Atheroprotective Actions Of Hdl In Vascular Endothelium

Introduction: HDL has direct atheroprotective actions on endothelial cells (EC) that include the attenuation of vascular inflammation and promotion of endothelial monolayer integrity. These processes are mediated by scavenger receptor class B, type 1 (SR-BI) and its adaptor protein PDZK1. We have identified the breakpoint cluster region protein (BCR) as a PDZK1 interacting protein in EC. Best known as a gene in the BCR-ABL complex associated with the Philadelphia chromosome, BCR structure predicts both adaptor protein and kinase functions. Hypothesis: Via interaction with PDZK1 and kinase activity, BCR mediates the atheroprotective actions of HDL on EC. Methods: Newly-developed kinase assays that employ time-resolved Forster resonance energy transfer were used to detect kinase activity and identify kinase substrates. In vivo studies were done in ApoAI -/- versus ApoAI -/- ;BCR -/- mice administered control AAV or AAV-ApoAI. Leukocyte-EC interaction and carotid artery reendothelialization were evaluated by intravital microscopy and thermal injury, respectively. Atherosclerosis was invoked using AAV8-PCSK9. BCR expression was compared in human atherosclerotic and normal arteries in two independent publicly available patient cohorts. Results: In cultured EC, HDL activation of eNOS, blunting of EC-monocyte adhesion, and promotion of EC migration all required BCR, and reconstitution studies with BCR mutants revealed requirements for both BCR-PDZK1 interaction and kinase activity. In response to HDL, BCR directly phosphorylated Akt-Ser473 and it provoked Akt-Thr308 phosphorylation by PDK1. ApoAI restoration in vivo in ApoAI -/- mice prompted SR-BI-PDZK1-BCR protein complex formation and BCR phosphorylation of Akt-Ser473 in mouse aorta. It also blunted leukocyte-endothelial cell adhesion and enhanced carotid artery reendothelialization in BCR +/+ , but not in BCR -/- mice. Furthermore, the atheroprotective effect of ApoAI restoration was demonstrable in BCR +/+ , but not in BCR -/- mice. In both human cohorts BCR expression was decreased in atherosclerotic versus normal arteries. Conclusion: BCR function as a novel direct and indirect kinase of Akt mediates the atheroprotective actions of HDL in vascular endothelium.

Phosphorylation of Akt at Thr308 regulates p-eNOS Ser1177 during physiological conditions.

Endothelial nitric oxide synthase (eNOS)‐derived nitric oxide (NO) plays a crucial role in maintaining vascular homeostasis. As a hallmark of eNOS activation, phosphorylation of eNOS at Ser1177 induced by activated protein kinase B (PKB/Akt) is pivotal for NO production. The complete activation of Akt requires its phosphorylation of both Thr308 and Ser473. However, which site plays the main role in regulating phosphorylation of eNOS Ser1177 is still controversial. The purpose of the present study is to explore the specific regulatory mechanism of phosphorylated Akt in eNOS activation. Inhibition of Akt Thr308 phosphorylation by a specific inhibitor or by siRNA in vitro led to a decrease in eNOS phosphorylation at Ser1177 and to lower NO concentration in the cell culture medium of HUVECs. However, inhibiting p‐Akt Ser473 had no effect on eNOS phosphorylation at Ser1177. Next, we administered mice with inhibitors to downregulate p‐Akt Ser473 or Thr308 activity. Along with the inhibition of p‐Akt Thr308, vascular p‐eNOS Ser1177 protein was simultaneously downregulated in parallel with a decrease in plasma NO concentration. Additionally, we cultured HUVECs at various temperature conditions (37, 22, and 4 °C). The results showed that p‐Akt Ser473 was gradually decreased in line with the reduction in temperature, accompanied by increased levels of p‐Akt Thr308 and p‐eNOS Ser1177. Taken together, our study indicates that the phosphorylation of Akt at Thr308, but not at Ser473, plays a more significant role in regulating p‐eNOS Ser1177 levels under physiological conditions.