Stimulated Akt Phosphorylation Research Articles

Effect of galectin-3 on synovial inflammation in knee osteoarthritis via stimulating phosphatidylinositol-3-kinase/Akt pathway

Galectin-3 (Gal-3), a glycan-binding protein responsible for inflammation, has been reportedly implicated in inflammatory arthritis. This study aimed to determine clinical and pathological effects of Gal-3 on inflammation in knee osteoarthritis (OA). Gal-3 mRNA and protein levels in synoviocytes, synovium, synovial fluid, and plasma of knee OA patients were determined using real-time polymerase chain reaction, immunohistochemistry, and enzyme-linked immunosorbent assay. Signaling mechanism underlying inflammatory effect of Gal-3 was further elucidated in human knee OA synoviocytes. Clinical study uncovered significant increases in plasma and synovial fluid Gal-3 levels in knee OA patients, particularly those with advanced-stage. In knee OA patients, plasma Gal-3 was significantly associated with radiographic severity and indicators of body composition, physical performance, and knee pain and disability. In the inflamed synovium of knee OA patients, further analysis depicted a marked up-regulation of Gal-3 mRNA expression, consistent with immunohistochemical analysis showing localization of Gal-3 protein in the lining and sublining layers of the inflamed synovium. An in vitro study unveiled that aberrant Gal-3 mRNA expression was regulated by tumor necrosis factor (TNF)-α in knee OA synoviocytes. Gal-3 significantly enhanced production of NO and IL-6, up-regulated mRNA expressions of IL-6, NF-κB, and MMP-13, and down-regulated mRNA expressions of ACAN and SOX-9 via stimulating Akt phosphorylation in knee OA synoviocytes. Gal-3 exerted an inflammatory action, which might emerge as a possible mediator of synovitis and cartilage degeneration in knee OA.

Kaempferol improves glucose uptake in skeletal muscle via an AMPK-dependent mechanism

Insulin resistance is a hallmark of type-2 diabetes (T2D) pathogenesis. Because skeletal muscle (SkM) is the major tissue for insulin-mediated glucose disposal, insulin resistance in SkM is considered a major risk factor for developing T2D. Thus, the identification of compounds that enhance the ability of SkM to take up glucose is a promising strategy for preventing T2D. Our previous work showed that kaempferol, a flavonol present in many foods, improves insulin sensitivity in obese mice, however, the mechanism underlying this beneficial action remains unclear. Here, we show that kaempferol directly stimulates glucose uptake and prevents lipotoxicity-impaired glucose uptake in primary human SkM. Kaempferol stimulates Akt phosphorylation in a time-dependent manner in human SkM cells. The effect of kaempferol on glucose uptake was blunted by inhibition of glucose transporter 4, phosphoinositide 3-kinase (PI3K), or AMPK. In addition, kaempferol induced AMPK phosphorylation, and inhibition of AMPK prevented kaempferol-stimulated Akt phosphorylation. In vivo, kaempferol administration induced rapid glucose disposal accompanied with increased Akt and AMPK phosphorylation in SkM tissue of the mice. Taken together, these findings suggest that kaempferol stimulates glucose uptake in SkM via an AMPK/Akt dependent mechanism, and it may be a viable therapeutic agent for insulin resistance.

Primaquine activates Keratin 7 to treat diabetes and its complications.

The global prevalence of type 2 diabetes mellitus (T2DM) raises the rates of its complications, such as diabetic nephropathy and cardiovascular diseases. To conquer the complications, new strategies to reverse the deterioration of T2DM are urgently needed. In this project, we aimed to examine the hypoglycemic effect of primaquine and explore its specific target. In vitro T2DM insulin resistance model was built in HepG2 cells to screen the potential anti-diabetic chemicals. On the other hand, the potential protein targets were explored by molecular docking. Accordingly, we chose C57BL/6N mice to establish T2DM model to verify the effect of the chemicals on anti-hyperglycemia and diabetic complications. By targeting the Keratin 7 (K7) to activate EGFR/Akt glucose metabolism signaling pathway, primaquine poses a potent hypoglycemic effect. The level of acetyl-CoA is enhanced markedly, supporting that primaquine upregulates the aerobic glycolysis. Moreover, primaquine ameliorates kidney function by reducing the secretion of urinary proteins and creatinine, especially for the urea nitrogen which is significantly decreased compared to no-treatment T2DM mice. Notably, primaquine restores the level of plasma low-density lipoprotein cholesterol (LDL-C) nearly to normal, minimizing the incidence of cardiovascular diseases. We find that primaquine may reverse the dysregulated metabolism to prevent diabetic complications by stimulating EGFR/Akt signaling axis, shedding new light on the therapy of T2DM. Insulin resistance is characterized by reduced p-Akt and glucose metabolism, dominated by anaerobic glycolysis. Primaquine activates the complex made of K7 and EGFR, further stimulating Akt phosphorylation. Then, p-Akt promotes the aerobic glucose metabolism and upregulates Ac-CoA to mobilize TCA cycle, improving insulin sensitivity. The online version contains supplementary material available at 10.1007/s40200-022-01135-8.

Stimulation of Akt Phosphorylation and Glucose Transport by Metalloporphyrins with Peroxynitrite Decomposition Catalytic Activity.

Iron porphyrin molecules such as hemin and iron(III) 4,4',4″,4‴-(porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid) (FeTBAP) have previously been shown to influence insulin signaling and glucose metabolism. We undertook this study to determine whether a catalytic action of iron porphyrin compounds would be related to their stimulation of insulin signaling and glucose uptake in C2C12 myotubes. FeTBAP did not display nitrite reductase activity or alter protein S-nitrosylation in myotubes, eliminating this as a candidate mode by which FeTBAP could act. FeTBAP displayed peroxynitrite decomposition catalytic activity in vitro. Additionally, in myotubes FeTBAP decreased protein nitration. The peroxynitrite decomposition catalyst Fe(III)5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato chloride (FeTPPS) also decreased protein nitration in myotubes, but the iron porphyrin Fe(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachlorideporphyrin pentachloride (FeTMPyP) did not. FeTBAP and FeTPPS, but not FeTMPyP, showed in vitro peroxidase activity. Further, FeTBAP and FeTPPs, but not FeTMPyP, increased Akt phosphorylation and stimulated glucose uptake in myotubes. These findings suggest that iron porphyrin compounds with both peroxynitrite decomposition activity and peroxidase activity can stimulate insulin signaling and glucose transport in skeletal muscle cells.

CaMKII and Kalirin, a Rac1-GEF, regulate Akt phosphorylation involved in contraction-induced glucose uptake in skeletal muscle cells

Rac1 plays an important role in contraction-stimulated muscle glucose uptake, but the mechanism is not fully elucidated. We previously identified Rac1-dependent activation of Akt played a partial role in contraction-stimulated GLUT4 translocation to the cell surface of C2C12 myotubes. Recognizing that contraction activates CaMKII in muscle and CaMKII is known to regulate Rac1 activity in other systems, here we investigated the relationship between CaMKII, Akt and contraction-stimulated glucose uptake. Expression of a constitutively-active mutant of CaMKIIδ stimulated Akt phosphorylation that was inhibited by Rac1 inhibitor II. C2C12 myotubes were contracted by electrical pulse stimulation (EPS). We observed the CaMKII inhibitor, KN-93 and CaMKIIδ siRNA-mediated knockdown, reduced EPS-induced Akt phosphorylation in C2C12 myotubes. ITX3, an inhibitor of the Rac-GTPase Kalirin and Kalirin siRNA-mediated knockdown reduced EPS-stimulated Akt phosphorylation in myotubes. In addition, the Akt inhibitor MK2206 partly reduced EPS-stimulated glucose uptake without simultaneously affecting CaMKII phosphorylation and Kalirin protein abundance. Our findings demonstrate EPS leads to Akt activation through a CaMKII-Kalirin-Rac1 signaling pathway and partly regulates contraction-stimulated glucose uptake in muscle cells.

A novel adipokine WISP1 attenuates lipopolysaccharide-induced cell injury in 3T3-L1 adipocytes by regulating the PI3K/Akt pathway

BackgroundTo study the effect of WISP1 on lipopolysaccharide (LPS)-induced cell injury in 3T3-L1 adipocytes. MethodLentivirus was transiently transfected into log phase 3T3-L1 adipocytes, which were then treated with LPS at a concentration of 10 μg/mL for 24 h. The cells were divided into the following groups: group A (control, untreated cells); group B (LPS-treated cells); group C (GFP), cells transfected with lentivirus-containing GFP; group D (GFP+LPS), group C treated with LPS;group E (WISP1OE), cells transfected with lentivirus, group F (shNC+LPS), cells transfected with lentivirus-containing nshRNA treated with LPS; group G (shWISP1 +LPS), cells transfected with lentivirus-containing shRNA treated with LPS; group H (WISP1OE+LPS), group E treated with LPS; group I (WISP1OE+LPS+LY294002), group E treated with LPS followed by LY294002 for 24 h. ResultsWISP1 overexpression notably ameliorated cell apoptosis, accompanied with the increased expression of bcl-2, the decreased expressions of bax and cleaved-caspase-3, and promoted the release of inflammatory factors, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in LPS-treated 3T3-L1 adipocytes. WISP1 knockdown exhibited the opposite results. In addition, WISP1 stimulated Akt phosphorylation and reduced nuclear translocation of Fork head box protein O3 (FoxO3a) in 3T3-L1 adipocytes treated by LPS. The inhibition of the PI3K/Akt signaling pathway diminished the protective effect of WISP1. ConclusionWISP1 prevents 3T3-L1 adipocytes from being injured by LPS by regulating the PI3K/Akt pathway.

Chemerin Affects P4 and E2 Synthesis in the Porcine Endometrium during Early Pregnancy.

Chemerin, belonging to the adipokine family, exhibits pleiotropic activity. We hypothesised that the adipokine could be involved in the regulation of steroidogenesis in the porcine endometrium. Thus, the aim of this study was to determine the effect of chemerin on the key steroidogenic enzyme proteins’ abundance (Western blot), as well as on P4 and E2 secretion (radioimmunoassay) by the porcine endometrium during early pregnancy and the mid-luteal phase of the oestrous cycle. Moreover, we investigated the hormone impact on Erk and Akt signalling pathway activation (Western blot). Chemerin stimulated E2 production on days 10 to 11 of pregnancy. On days 10 to 11 and 15 to 16 of gestation, and on days 10 to 11 of the cycle, chemerin enhanced the expression of StAR and all steroidogenic enzyme proteins. On days 12 to 13 of pregnancy, chemerin decreased StAR and most of the steroidogenic enzyme proteins’ abundance, whereas the P450C17 abundance was increased. On days 27 to 28 of pregnancy, chemerin increased StAR and P450C17 protein contents and decreased 3βHSD protein amounts. It was noted that the adipokine inhibited Erk1/2 and stimulated Akt phosphorylation. The obtained results indicate that chemerin affected P4 and E2 synthesis through the Erk1/2 and Akt signalling pathways.

Anticancer Effects of Ursi Fel Extract and Its Active Compound, Ursodeoxycholic Acid, in FRO Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid cancer (ATC) is one of the most fatal human malignancies. Ursi Fel (UF) is the bile of a brown bear that has been traditionally used for heat clearance and toxin relief in Korean and Chinese medicines. In this study, we determined the anticancer effects of a UF extract and its active compound, ursodeoxycholic acid (UDCA), in FRO human ATC cells. FRO cells were treated with UF extract and UDCA at different concentrations for various durations. Cell viability was measured using an MTT assay. Cell apoptosis was investigated by flow cytometric analysis following Annexin V and propidium iodide (PI) staining, and Hoechst staining was used to observe nuclear fragmentation. The expression of pro-apoptotic (Bax, caspase-3, cytochrome c, and PARP), anti-apoptotic (Bcl-2), and angiogenetic (TGF-β, VEGF, N-cadherin, and sirtuin-1) proteins and the phosphorylation of Akt and mechanistic target of rapamycin (mTOR) were determined by western blot analysis. Treatment with UF extract at 10, 25, and 50 μg/mL and UDCA at 25, 50, and 100 μM/mL significantly inhibited the growth of FRO cells in a dose-dependent manner. Flow cytometry and Hoechst staining revealed an increase in the apoptosis of FRO cells mediated by UF extract and UDCA in a dose-dependent manner. UF extract (25 and 50 μg) and UDCA (50 and 100 μM) significantly increased the expression of Bax, caspase-3, cytochrome c, and PARP and inhibited the expression of Bcl-2, TGF-β, VEGF, N-cadherin, and sirtuin-1 in FRO cells. Furthermore, UF extract and UDCA treatment stimulated Akt phosphorylation and inhibited mTOR phosphorylation in these cells. These results indicate that UF extract and UDCA exert anticancer properties in FRO cells by inducing apoptosis and inhibiting angiogenesis via regulating the Akt/mTOR signaling pathway.

Protective effects of intravitreal administration of mesenchymal stem cell-derived exosomes in an experimental model of optic nerve injury

Traumatic optic neuropathy results in the loss of retinal ganglion cells (RGCs), leading to unavoidable visual impairment. However, there is no effective therapy by far. Accumulated studies support the perception that mesenchymal stem cells (MSCs) secrete exosomes that serve as a protective paracrine factor. The study aimed to explore and evaluate the potential therapeutic effects of intravitreal transplantation of MSC-derived exosomes (MSC-exos) in an experimental model of optic nerve crush (ONC). Exosomes were isolated from rat MSCs and characterized by transmission electron microscope and western blotting. At the onset of ONC, a single intravitreal injection of exosomes or PBS was administered to the rats. At day 30, hematoxylin and eosin staining, immunohistochemistry, and βIII-tubulin staining were performed to evaluate the survival of RGCs. Moreover, TUNEL assay was used to examine the apoptosis of RGCs. Inflammation-relevant factors were identified via quantitative polymerase chain reaction. The expression levels of cell apoptosis-related molecules and key members of the PI3K/AKT signaling pathway were determined via western blot analysis. We found that MSC-exos exhibited typical characteristic morphologies (cup-shaped) and sizes (peak size of 93 nm). Furthermore, they exhibited substantial expression of the exosome markers CD63 and TSG101, but lacked the expression of the cellular marker GM130. Treatment with intravitreal MSC-exos notably promoted the survival of RGCs in ONC rats. The level of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-8, and MCP-1, were reduced, whereas those of the anti-inflammatory factor IL-10 were increased. Moreover, the apoptosis induced by ONC was decreased by the administration of MSC-exos via upregulation of the Bcl-2/Bax ratio and downregulation of caspase-3 activity. Furthermore, MSC-exos significantly stimulated AKT phosphorylation, whereas LY294002 restored the apoptosis-preventing effects of MSC-exos. The results of our results demonstrated that intravitreal administration of MSC-exos ameliorates ONC-induced injury in a rat model. These findings might aid in the development of effective exosome-based therapeutic strategies for the treatment of optic nerve degeneration.

LncRNA lnc-ISG20 promotes renal fibrosis in diabetic nephropathy by inducing AKT phosphorylation through miR-486-5p/NFAT5.

Long non‐coding RNA (lncRNA) lnc‐ISG20 has been found aberrantly up‐regulated in the glomerular in the patients with diabetic nephropathy (DN). We aimed to elucidate the function and regulatory mechanism of lncRNA lnc‐ISG20 on DN‐induced renal fibrosis. Expression patterns of lnc‐ISG20 in kidney tissues of DN patients were determined by RT‐qPCR. Mouse models of DN were constructed, while MCs were cultured under normal glucose (NG)/high glucose (HG) conditions. The expression patterns of fibrosis marker proteins collagen IV, fibronectin and TGF‐β1 were measured with Western blot assay. In addition, the relationship among lnc‐ISG20, miR‐486‐5p, NFAT5 and AKT were analysed using dual‐luciferase reporter assay and RNA immunoprecipitation. The effect of lnc‐ISG20 and miR‐486/NFAT5/p‐AKT axis on DN‐associated renal fibrosis was also verified by means of rescue experiments. The expression levels of lnc‐ISG20 were increased in DN patients, DN mouse kidney tissues and HG‐treated MCs. Lnc‐ISG20 silencing alleviated HG‐induced fibrosis in MCs and delayed renal fibrosis in DN mice. Mechanistically, miR‐486‐5p was found to be a downstream miRNA of lnc‐ISG20, while miR‐486‐5p inhibited the expression of NFAT5 by binding to its 3'UTR. NFAT5 overexpression aggravated HG‐induced fibrosis by stimulating AKT phosphorylation. However, NFAT5 silencing reversed the promotion of in vitro and in vivo fibrosis caused by lnc‐ISG20 overexpression. Our collective findings indicate that lnc‐ISG20 promotes the renal fibrosis process in DN by activating AKT through the miR‐486‐5p/NFAT5 axis. High‐expression levels of lnc‐ISG20 may be a useful indicator for DN.

The Role of Inositol Phosphate Multikinase (IPMK) in Time Restricted Feeding in Animal Model

Obesity is a major public health problem of the U.S. and is associated with diabetes, cardiovascular diseases and other diseases. Most research studies focus on excessive food consumption as the main cause of obesity. However, emerging data indicate that the timing of feeding can have significant effects on body weight and metabolism. Numerous studies in animals and small clinical studies in humans have shown that eating erratically over the 24 hour period or out of phase with the circadian rhythm predisposes toward weight gain, steatosis, dyslipidemia, insulin resistance and diabetes. Furthermore, studies indicate that restricting food intake to the active period synchronizes the circadian rhythm and metabolism, enhances weight loss and improves metabolic outcomes. Time restricted feeding (TRF) increases the amplitudes of clock gene expression and pathways mediating nutrient sensing and hepatic metabolism. However, the mechanisms mediating the effects of TRF are not fully understood. Here we characterized mice (10 week-old) fed a high-fat diet ad libitum (ALF) or from 7 pm to 7 am (TRF) for 2 weeks. The basal glucose production rate was similar between the two groups. Under hyperinsulinemic-euglycemic clamp, the glucose infusion rate (GIR) was significantly greater in TRF group compared to ALF group indicating an increase in insulin sensitivity. Using radioisotopic tracers, we demonstrated that the hepatic glucose production (HGP) was significantly reduced and the glucose disappearance rate was increased in TRF group compared to ALF group. Moreover, a biochemical analyses of liver tissues revealed that Inositol phosphate multikinase (IPMK) act as a key enzyme for inositol polyphosphate biosynthesis and play a role in insulin-, nutrient-, and energy-mediated metabolic signaling, was increased during TRF. Moreover, deletion of IPMK in hepatocytes decreased insulin stimulated AKT phosphorylation while increased lipid accumulation and gluconeogenesis. Importantly, hepatic deletion of IPMK attenuated the beneficial effects of TRF suggesting that IPMK in the liver may contributes to beneficial effects of TRF. Our findings provide the potential mechanism by which TRF confers the beneficial effects and may provide a novel therapeutic strategy for treating diabetes.

Transfer of MicroRNA-216a-5p From Exosomes Secreted by Human Urine-Derived Stem Cells Reduces Renal Ischemia/Reperfusion Injury.

Human urine-derived stem cells (USCs) protect rats against kidney ischemia/reperfusion (I/R) injury. Here we investigated the role of USCs exosomes (USCs-Exos) in protecting tubular endothelial cells and miRNA transfer in the kidney. Human USCs and USCs-Exos were isolated and verified by morphology and specific biomarkers. USC-Exos played a protective role in human proximal tubular epithelial cells (HK-2) exposed to hypoxia/reoxygenation (H/R). USCs-Exos were rich in miR-216a-5p, which targeted phosphatase and tensin homolog (PTEN) and regulated cell apoptosis through the Akt pathway. In HK-2 cells exposed to H/R, incubation with USC-Exos increased miR-216-5p, decreased PTEN levels, and stimulated Akt phosphorylation. Exposure of hypoxic HK-2 cells to USCs-Exos pretreated with anti-miR-216a-5p can prevent the increase of miR-216-5p and Akt phosphorylation levels, restore PTEN expression, and promote apoptosis. The dual-luciferase reported gene assay in HK-2 cells confirmed that miR-216a-5p targeted PTEN. In rats with I/R injury, intravenous infusion of USCs-Exos can effectively induce apoptosis suppression and functional protection, which is associated with decreased PTEN. Infusion of exosomes from anti-miR-216a-5p-transfected USCs weakened the protective effect in the I/R model. Therefore, USCs-Exos can reduce renal I/R injury by transferring miR-216a-5p targeting PTEN. Potentially, USCs-Exos rich in miR-216a-5p can serve as a promising therapeutic option for AKI.

Activation of protein kinase B by WNT4 as a regulator of uterine leiomyoma stem cell function

To investigate the functional interaction between the Wnt/β-catenin and protein kinase B (Akt) pathways in leiomyoma stem cells (LSC). Laboratory study. Research laboratory. Premenopausal women (n = 36; age range: 28 to 49 years) undergoing hysterectomy or myomectomy for leiomyoma. None. Gene expression, protein phosphorylation, and cell proliferation. Cells from human leiomyoma tissues were sorted by fluorescence-activated cell sorting (FACS) into three populations: LSC, intermediate cells (LIC), and differentiated cells (LDC) with the function of the Wnt/β-catenin and Akt signaling pathways in leiomyoma cells evaluated using real-time quantitative polymerase chain reaction and immunoblot analyses. The Wnt/β-catenin signaling pathway components were differentially expressed in each leiomyoma cell population. WNT4 was distinctly overexpressed in LIC, and its receptor FZD6 was primarily expressed in LSC. WNT4 stimulated Akt phosphorylation, activated β-catenin, and increased primary leiomyoma cell proliferation. These stimulatory effects were abolished by cotreatment with the Akt inhibitor, MK-2206. WNT4 up-regulated the expression of pro-proliferative genes, c-Myc and cyclin D1, specifically in LSC; this was also abrogated by Akt inhibition. Our data suggest that WNT4 regulates LSC proliferation via Akt-dependent β-catenin activation, representing a key step toward a better understanding of LSC regulation and potentially novel therapeutic targets.

Novel humanin analogs confer neuroprotection and myoprotection to neuronal and myoblast cell cultures exposed to ischemia-like and doxorubicin-induced cell death insults

Humanin (HN) is a 24-amino acid mitochondrial-derived peptide, best known for its ability to protect neurons from damage caused by ischemic stroke and neurodegenerative insults and cardiomyocytes from myocardial infarction or doxorubicin (Dox)-induced cardiotoxicity. This study examines the neuroprotective and myoprotective effects of HN novel synthetic analogs HUJInin and c(D-Ser14-HN), prepared by solid-phase peptide synthesis. The cellular models employed were oxygen-glucose-deprivation (OGD) followed by reoxygenation (R)-induced neurotoxicity in PC12 and SH-SY5Y neuronal cell cultures and Dox-induced cardiotoxicity in H9c2 and C2C12 myoblast cell cultures, respectively. Necrotic and apoptotic cell death was measured by LDH release and caspase-3 activity. Erk 1/2 and AKT phosphorylations were examined by western blotting. Mitochondrial calcium and mitochondrial membrane potential were measured using the fluorescent dye tetramethylrhodamine-methyl ester. It was found that HUJInin and c(D-Ser14-HN) conferred significant dose-dependent neuroprotection, a phenomenon related to attenuation of OGD insult-induced Erk 1/2 phosphorylation, stimulation of AKT phosphorylation and improvement of mitochondrial functions. These peptides also conferred myoprotective effect towards Dox-induced apo-necrotic cell death insults. HUJInin and c(D-Ser14-HN) synthetic analogs may provide new lead compounds for the development of a potential candidate drug for stroke treatment and/or Dox-induced cardiotoxicity therapy in cancer patients.

Taurine attenuates gossypol-induced apoptosis of C2C12 mouse myoblasts via the GPR87-AMPK/AKT signaling.

Gossypol, a toxic polyphenol extracted from cotton seeds, is hazardous to human and animal health. Taurine is considered as an essential or semi-essential amino acid and has diverse cytoprotective effects. This study was aimed to investigate the protective effect and molecular mechanism of taurine against apoptosis of C2C12 mouse myoblasts induced by gossypol. C2C12 mouse myoblasts were exposed to gossypol (0, 1nM, 10nM, 100nM, 1μM, and 10μM). Cell numbers were rapidly decreased with increasing concentrations of gossypol. Gossypol significantly induced apoptosis, decreased Bcl2 expression, and increased the protein levels of Bax and the cleaved caspase 3. Taurine (0.24mM) treatment largely rescued the cell number decreased by gossypol, attenuated gossypol-induced cell apoptosis. GPR87 knockdown abolished the inhibition by taurine of cell apoptosis. Furthermore, GPR87 overexpression attenuated cell apoptosis induced by gossypol. Both taurine treatment and GPR87 overexpression stimulated AKT phosphorylation but inhibited AMPK phosphorylation, whereas gossypol had the opposite effects. Taurine treatment promoted GPR87 expression and subcellular localization and partially rescued the inhibition of gossypol on this expression. In summary, these data reveal that taurine attenuates gossypol-induced apoptosis of C2C12 mouse myoblasts via the GPR87-AMPK/AKT signaling.

Abstract 1430: CaMKK2 regulates EGF-dependent activation of oncogenic Akt in ovarian cancer cells

Abstract Background-The canonical PI3K/Akt pathway is hyperactive in a variety of cancers including ovarian cancer (OVCa). Using baculovirus-expressed, FPLC-purified enzymes, we previously demonstrated that Calcium/Calmodulin-dependent CaM Kinase Kinase 2 (CaMKK2) activates the oncogene Akt by direct phosphorylation of its primary activation site T308. In OVCAR-3 and other OVCa cells, RNAi-mediated knockdown of CaMKK2 decreased basal Akt phosphorylation at T308 in a Phosphoinositide-Dependent Kinase-1 (PDK1)-independent manner. CaMKK2 knockdown also decreased Akt phosphorylation at its secondary activation site, S473 by an apparently indirect mechanism. Results- In this study we show that CaMKK2 mediates downstream effects of epidermal growth factor (EGF) on the Akt cascade in OVCa cells. Treatment of OVCAR3 cells with EGF produced a prominent enhancement of p-Akt T308 which was reduced ~2-fold, by either CaMKK2 or PDK1 knockdowns. Knockdown of CaMKK2 but not PDK1 reduced p-Akt S473 after EGF treatment. Similar results were obtained with Insulin-like growth factor 1 (IGF1). To discern how CaMKK2 is engaged by EGF action we examined whether, in vitro, CaMKK2 phosphorylation of WT Akt or a synthetic peptide encompassing T308 is stimulated by Calcium/CaM and/or Phosphatidylinositol (3,4,5)-trisphosphate (PIP3). CaMKK2 activity was enhanced by Calcium/CaM but not by soluble PIP3. CaMKK2 did not bind to a dot blot of various phosphoinositides, although strong binding to PIP3 was observed with PDK1. Alternatively, Akt regulation by CaMKK2 may occur via a Calcium/CaM-dependent pathway. Increased p-Akt T308 and S473 upon treatment of cells with the calcium-ionophore, ionomycin, was significantly reduced by CaMKK2 knockdown. EGF stimulation of Akt phosphorylation at both T308 and S473 was significantly reduced by the intracellular calcium chelator, BAPTA-AM and by the CaM inhibitor, W7. We also probed the mechanism by which optimal cellular Akt phosphorylation at S473 requires CaMKK2. mTOR complex 2 (mTORC2) and DNA-dependent protein kinase (DNA-PKcs) are considered candidate kinases for p-Akt S473. CaMKK2 knockdown regulated p-Akt S473 in M059J cells, a DNA-PKcs null cell line similarly to that observed with OVCAR3 cells, suggesting that mTORC2 may be regulated by CaMKK2. Using an MS/MS Protein-protein interaction (PPI) screen we observed CaMMK2 interaction with multiple ribosomal proteins an observation consistent with suggested mTORC2 ribosomal regulation of Akt S473 phosphorylation. Conclusions-This study documents Akt activating phosphorylation by CaMKK2 in response to EGF in a Calcium/CaM-dependent, PI3K/PIP3 independent, manner. Besides directly phosphorylating Akt at T308, CaMKK2 regulates p-Akt S473 potentially through the regulation of mTORC2 activity. Our results highlight the importance of CaMKK2 activated by growth factor signals as a parallel system to the oncogenic PI3K/PDK1 pathway. Citation Format: Shuhang Dai, Angela M. Gocher, Arthur M. Edelman. CaMKK2 regulates EGF-dependent activation of oncogenic Akt in ovarian cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1430.

The green tea polyphenol epigallocatechin-3-gallate attenuates age-associated muscle loss via regulation of miR-486-5p and myostatin

(−)-Epigallocatechin-3-gallate (EGCG), the most abundant catechin component in green tea, has been reported to attenuate age-associated insulin resistance, lipogenesis and loss of muscle mass through restoring Akt activity in skeletal muscle in our previous and present studies. Accumulated data has suggested that polyphenols regulate signaling pathways involved in aging process such as inflammation and oxidative stress via modulation of miRNA expression. Here we found that miRNA-486-5p was significantly decreased in both aged senescence accelerated mouse-prone 8 (SAMP8) mice and late passage C2C12 cells. Thus, we further investigated the regulatory effect of EGCG on miRNA-486-5p expression in age-regulated muscle loss. SAMP8 mice were fed with chow diet containing without or with 0.32% EGCG from aged 32 weeks for 8 weeks. Early passage (<12 passages) and late passage (>30 passages) of C2C12 cells were treated without or with EGCG at concentrations of 50 μM for 24h. Our data showed that EGCG supplementation increased miRNA-486-5p expression in both aged SAMP8 mice and late passage C2C12 cells. EGCG stimulated AKT phosphorylation and inhibited FoxO1a-mediated MuRF1 and Atrogin-1 transcription via up-regulating the expression of miR-486 in skeletal muscle of 40-wk-old SAMP8 mice as well as late passage C2C12 cells. In addition, myostatin expression was increased in late passage C2C12 cells and anti-myostatin treatment upregulated the expression of miR-486-5p. Our results identify a unique mechanism of a dietary constituent of green tea and suggest that use of EGCG or compounds derived from it attenuates age-associated muscle loss via myostatin/miRNAs/ubiquitin-proteasome signaling.

Imoxin prevents dexamethasone-induced promotion of muscle-specific E3 ubiquitin ligases and stimulates anabolic signaling in C2C12 myotubes

Muscle atrophy is the loss of skeletal muscle mass during several pathological conditions such as long-term fasting, aging, cancer, diabetes, sepsis and immune disorders. Glucocorticoids are known to trigger skeletal muscle atrophy. Dexamethasone (DEX), a synthetic glucocorticoid, induces skeletal muscle atrophy by suppression of protein synthesis and promotion of protein degradation. The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) plays a significant role in mediating lipopolysaccharide-induced inflammation. However, pathological roles of PKR in muscle atrophy are not fully understood. The current study aimed to investigate the effect of imoxin, a PKR inhibitor, on DEX-induced muscle atrophy in C2C12 myotubes. Myotubes were incubated with imoxin at different concentrations with or without 5 μM DEX for 24 h. In the current study, imoxin treatment significantly reduced protein levels of MuRF1 and MAFbx induced by DEX by 88 ± 2% and MAFbx by 99 ± 0%, respectively. Moreover, 5 μM imoxin treatment reduced protein ubiquitination by 42 ± 4% and protein content of nuclear FoxO3α (77 ± 4%) in presence of DEX. Furthermore, 5 μM imoxin treatment stimulated Akt phosphorylation (195 ± 5%), mTOR phosphorylation (171 ± 21 %) and p70S6K1 phosphorylation (314 ± 31 %) under DEX-treated condition even though DEX treatment did not suppressed Akt/mTOR/p70S6K1 axis. These findings suggest that imoxin may protect against DEX-induced skeletal muscle atrophy by alleviating muscle specific E3 ubiquitin ligases and imoxin alone may promote protein synthesis via Akt/mTOR/S6K1 axis in muscle cells.

Imoxin Prevents Dexamethasone‐Induced Promotion of Muscle‐Specific E3 Ubiquitin Ligases and Stimulates Anabolic Signaling in C2C12 Myotubes

Muscle atrophy is the loss of skeletal muscle mass during several pathological conditions such as long‐term fasting, aging, cancer, diabetes, sepsis and immune disorders. Dexamethasone (DEX), a synthetic glucocorticoid, induces skeletal muscle atrophy by suppression of protein synthesis and promotion of protein degradation. Specifically, DEX increases the levels of muscle specific ubiquitin E3 ligases including MAFbx and MuRF1. The double‐stranded RNA (dsRNA)‐activated protein kinase R (PKR) plays a significant role in mediating inflammation and glucose homeostasis. However, pathological roles of PKR in muscle atrophy are not fully understood. The current study aimed to investigate the effect of imoxin, a PKR inhibitor, on DEX‐induced muscle atrophy in C2C12 myotubes. Myotubes were incubated with imoxin at different concentrations with or without 5 μM DEX for 24 h. Western blotting was performed to measure proteins involved in muscle protein synthesis as well as degradation. DEX significantly augmented the protein levels of MuRF1 and MAFbx (275 ± 5 % and 694 ± 36 %, respectively) compared with serum free control (CON). However, 5 μM imoxin treatment significantly reduced protein levels of MuRF1 by 88 ± 2 % and MAFbx by 99 ± 0 % compared with DEX, respectively. In addition, DEX suppressed FoxO4 phosphorylation (33 ± 9 %) compared to CON, but 5 μM imoxin treatment promoted FoxO4 phosphorylation (735 ± 44 %) compared to DEX. Additionally, DEX stimulated protein ubiquitination compared with control (124 ± 1 %) but 5 μM imoxin treatment reduced protein ubiquitination by 42 ± 4 % compared to DEX. At the same time, 5 μM imoxin treatment stimulated Akt phosphorylation (195 ± 5 %), mTOR phosphorylation (171 ± 21%) and p70S6K1 phosphorylation (314 ± 31 %) under DEX‐treated condition even though DEX treatment did not suppressed Akt/mTOR/p70S6K1 axis. These findings suggest that imoxin protects against DEX‐induced skeletal muscle atrophy by alleviating muscle specific E3 ubiquitin ligases and promoting protein synthesis via Akt/mTOR/S6K1 axis in muscle cells.Support or Funding InformationNone

IGFBP5 increases cell invasion and inhibits cell proliferation by EMT and Akt signaling pathway in Glioblastoma multiforme cells

BackgroundRecurrence of Glioblastoma multiforme (GBM) seems to be the rule despite combination therapies. Cell invasion and cell proliferation are major reasons for recurrence of GBM. And insulin-like growth factor binding protein 5 (IGFBP5) is the most conserved of the IGFBPs and is frequently dysregulated in cancers and metastatic tissues.ResultsBy studying the human glioma tissues, we find that IGFBP5 expression associate to the histopathological classification and highly expressed in GBM. Using IGFBP5 mutants we demonstrate that knockdown of IGFBP5 inhibited cell invasion, whereas promoting cell proliferation in GBM cells. Mechanistically, we observed that promoting GBM cell proliferation by inhibiting IGFBP5 was associated with stimulating Akt (Protein kinase B) phosphorylation. However, IGFBP5 promote GBM cell invasion was related to the epithelial-to-mesenchymal transition (EMT). Furthermore, the Chinese Glioma Genome Altas (CGGA) database show that IGFBP5 is significantly increased in recurrent glioma and it predicted worse survival.ConclusionsThe obtained results indicate that IGFBP5 has two sides in GBM—inhibiting cell proliferation but promoting cell invasion.