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1.
In this study, we addressed the direct effect of leucine on insulin signaling. In investigating the associated mechanisms, we found that leucine itself does not activate the classical Akt- or ERK1/2 MAP kinase-dependent signaling pathways but can facilitate the insulin-induced phosphorylations of Akt473 and ERK1/2 in a time- and dose-dependent manner in cultured hepatocytes. The leucine-facilitated insulin-induced phosphorylation of Akt at residue 473 was not affected by knocking down the key component of mTORC1 or -2 complexes but was blocked by inhibition of c-Src (PP2), PI3K (LY294002), Gαi protein (pertussis toxin or siRNA against Gαi1 gene, or β-arrestin 2 (siRNA)). Similarly, the leucine-facilitated insulin activation of ERK1/2 was also blunted by pertussis toxin. We further show that leucine facilitated the insulin-mediated suppression of glucose production and expression of key gluconeogenic genes in a Gαi1 protein-dependent manner in cultured primary hepatocytes. Together, these results show that leucine can directly facilitate insulin signaling through a Gαi protein-dependent intracellular signaling pathway. This is the first evidence showing that macronutrients like amino acid leucine can facilitate insulin signaling through G proteins directly.  相似文献   

2.
We examined mechanisms by which L-4F reduces obesity and diabetes in obese (ob) diabetic mice. We hypothesized that L-4F reduces adiposity via increased pAMPK, pAKT, HO-1, and increased insulin receptor phosphorylation in ob mice. Obese and lean mice were divided into five groups: lean, lean-L-4F-treated, ob, ob-L-4F-treated, and ob-L-4F-LY294002. Food intake, insulin, glucose adipocyte stem cells, pAMPK, pAKT, CB1, and insulin receptor phosphorylation were determined. Subcutaneous (SAT) and visceral adipose tissue (VAT) were determined by MRI and hepatic lipid content by magnetic resonance spectroscopy. SAT and VAT volumes decreased in ob-L-4F-treated animals compared with control. L-4F treatment decreased hepatic lipid content and increased the numbers of small adipocytes (P < 0.05) and phosphorylation of insulin receptors. L-4F decreased CB1 in SAT and VAT and increased pAKT and pAMPK in endothelium. L-4F-mediated improvement in endothelium was prevented by LY294002. Inhibition of pAKT and pAMPK by LY294002 was associated with an increase in glucose levels. Upregulation of HO-1 by L-4F produced adipose remodeling and increased the number of small differentiated adipocytes. The anti-obesity effects of L-4F are manifested by a decrease in visceral fat content with reciprocal increases in adiponectin, pAMPK, pAKT, and phosphorylation of insulin receptors with improved insulin sensitivity.  相似文献   

3.
IRS1 and IRS2 are key substrates of the insulin receptor tyrosine kinase. Mass spectrometry reveals more than 50 phosphorylated IRS1 serine and threonine residues (Ser(P)/Thr(P) residues) in IRS1 from insulin-stimulated cells or human tissues. We investigated a subset of IRS1 Ser(P)/Thr(P) residues using a newly developed panel of 25 phospho-specific monoclonal antibodies (αpS/TmAbIrs1). CHO cells overexpressing the human insulin receptor and rat IRS1 were stimulated with insulin in the absence or presence of inhibitors of the PI3K → Akt → mechanistic target of rapamycin (mTOR) → S6 kinase or MEK pathways. Nearly all IRS1 Ser(P)/Thr(P) residues were stimulated by insulin and significantly suppressed by PI3K inhibition; fewer were suppressed by Akt or mTOR inhibition, and none were suppressed by MEK inhibition. Insulin-stimulated Irs1 tyrosine phosphorylation (Tyr(P)Irs1) was enhanced by inhibition of the PI3K → Akt → mTOR pathway and correlated with decreased Ser(P)-302Irs1, Ser(P)-307Irs1, Ser(P)-318Irs1, Ser(P)-325Irs1, and Ser(P)-346Irs1. Metabolic stress modeled by anisomycin, thapsigargin, or tunicamycin increased many of the same Ser(P)/Thr(P) residues as insulin, some of which (Ser(P)-302Irs1, Ser(P)-307Irs1, and four others) correlated significantly with impaired insulin-stimulated Tyr(P)Irs1. Thus, IRS1 Ser(P)/Thr(P) is an integrated response to insulin stimulation and metabolic stress, which associates with reduced Tyr(P)Irs1 in CHOIR/IRS1 cells.  相似文献   

4.
Increased mammalian target of rapamycin complex 1 (mTORC1) activity has been suggested to play important roles in development of insulin resistance in obesity. mTORC1 hyperactivity also increases endoplasmic reticulum (ER) stress, which in turn contributes to development of insulin resistance and glucose intolerance. Increased IRS1 phosphorylation at Ser307 in vitro is correlated with mTORC1- and ER stress-induced insulin resistance. This phosphorylation site correlates strongly with impaired insulin receptor signaling in diabetic mice and humans. In contrast, evidence from knock-in mice suggests that phosphorylation of IRS1 at Ser307 is actually required to maintain insulin sensitivity. To study the involvement of IRS1Ser307 phosphorylation in mTORC1-mediated glucose intolerance and insulin sensitivity in vivo, we investigated the effects of liver specific TSC1 depletion in IRS1Ser307Ala mice and controls. Our results demonstrate that blockade of IRS1Ser307 phosphorylation in vivo does not prevent mTORC1-mediated glucose intolerance and insulin resistance.  相似文献   

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7.
The number and quality of endothelial progenitor cells (EPCs) are damaged to varying degrees in patients at risk for developing atherosclerosis. The improvement of the quantity and functions of EPCs can enhance repair of injured endothelial monolayer resulting in inhibiting atherosclerosis. The purpose of this study was to investigate the effect of pinocembrin (PIN), a major flavonoid in propolis on the differentiation and biological functions of EPCs and the potential mechanisms of these effects. Flow cytometry analysis revealed that PIN treatment increased the number of CD34+, CD133+, FLK-1+, CD133+/FLK-1+ and CD34+/FLK-1+ mononuclear cells (MNCs) in the peripheral blood of apoE−/− mice compared to untreated control mice. In vitro PIN treatment significantly increased the number of CD34+, CD133+, FLK-1+ and CD133+/FLK-1+ MNCs derived from SD bone marrow compared to untreated controls by 42.1, 84.6, 165.9 and 23.1 %, respectively. Additionally, PIN can improve biological functions of EPCs, such as proliferation, migration, adhesion, and in vitro tube formation and NO release. All of these improvements were inhibited by LY294002, while L-NAME only inhibited the PIN-induced increase in EPC proliferation and adhesion. We conclude that PIN can both promote the differentiation of EPCs in vitro and ex vivo and improve the biological functions of EPCs. The PI3K-eNOS-NO signaling pathway may be involved in the PIN-induced increase in the proliferation and adhesion of EPCs.  相似文献   

8.
Ginsenoside Rb1 shows a strong antioxidant effect and has potential activation effects on Akt. The aim of the present study was to investigate the protective effect of Rb1 on age-related ovarian granulosa cell injury. Ovarian granulosa cells(GCs) were obtained from 50 young women(≤30 years) and 50 aged women(≥38 years) at an IVF center. Young and aged ICR mice were administered with or without Rb1(10 mg kg-1, i.p.) for 2 weeks. The protective effects of Rb1 were investigated and the r...  相似文献   

9.
Thioridazine has been known as an antipsychotic agent, but it also has anticancer activity. However, the effect of thioridazine on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitization has not yet been studied. Here, we investigated the ability of thioridazine to sensitize TRAIL-mediated apoptosis. Combined treatment with thioridazine and TRAIL markedly induced apoptosis in various human carcinoma cells, including renal carcinoma (Caki, ACHN, and A498), breast carcinoma (MDA-MB231), and glioma (U251MG) cells, but not in normal mouse kidney cells (TMCK-1) and human normal mesangial cells. We found that thioridazine downregulated c-FLIP(L) and Mcl-1 expression at the post-translational level via an increase in proteasome activity. The overexpression of c-FLIP(L) and Mcl-1 overcame thioridazine plus TRAIL-induced apoptosis. We further observed that thioridazine inhibited the Akt signaling pathway. In contrast, although other phosphatidylinositol-3-kinase/Akt inhibitors (LY294002 and wortmannin) sensitized TRAIL-mediated apoptosis, c-FLIP(L) and Mcl-1 expressions were not altered. Furthermore, thioridazine increased the production of reactive oxygen species (ROS) in Caki cells, and ROS scavengers (N-acetylcysteine, glutathione ethyl ester, and trolox) inhibited thioridazine plus TRAIL-induced apoptosis, as well as Akt inhibition and the downregulation of c-FLIP(L) and Mcl-1. Collectively, our study demonstrates that thioridazine enhances TRAIL-mediated apoptosis via the ROS-mediated inhibition of Akt signaling and the downregulation of c-FLIP(L) and Mcl-1 at the post-translational level.  相似文献   

10.
Insulin stimulates phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinases (ERK) in various mammalian cells. To study the role of PI3K in insulin stimulation of ERK, we employed PI3K inhibitor LY294002 and mouse embryonic R? fibroblasts lacking IGF-1 receptors. In these R? cells, PI3K inhibition by LY294002 enhanced insulin stimulation of ERK phosphorylation whereas LY294002 inhibited insulin stimulation of Akt phosphorylation. The enhanced insulin stimulation of ERK phosphorylation was accompanied by increased IRS-1 tyrosine phosphorylation. Insulin stimulation of insulin receptor tyrosine phosphorylation was not altered. PI3K inhibition increased IRS-1–Grb2 complex formation and ras activity following insulin treatment of cells. Increased insulin stimulation of ERK by PI3K inhibition was mediated by the MEK/ERK pathway, but did not involve inhibitory Ser259 phosphorylation of raf that was reported to be mediated by Akt. In summary, PI3K inhibition in R? cells enhanced insulin stimulation of ERK phosphorylation by mechanisms involving enhancement of IRS-1 tyrosine phosphorylation, IRS-1–Grb2 complex formation and the ras/MEK/ERK pathway.  相似文献   

11.
Previous report showed that epidermal growth factor (EGF) promotes tumor progression. Several studies demonstrated that growth factors can induce heme oxygenase (HO)-1 expression, protect against cellular injury and cancer cell proliferation. In this study, we investigated the involvement of the c-Src, NADPH oxidase, reactive oxygen species (ROS), PI3K/Akt, and NF-κB signaling pathways in EGF-induced HO-1 expression in human HT-29 colon cancer cells. Treatment of HT-29 cells with EGF caused HO-1 to be expressed in concentration- and time-dependent manners. Treatment of HT-29 cells with AG1478 (an EGF receptor (EGFR) inhibitor), small interfering RNA of EGFR (EGFR siRNA), a dominant negative mutant of c-Src (c-Src DN), DPI (an NADPH oxidase inhibitor), glutathione (an ROS inhibitor), LY294002 (a PI3K inhibitor), and an Akt DN inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF caused an increase in c-Src phosphorylation at Tyr406 in a time-dependent manner. Treatment of HT-29 cells with EGF induced an increase in p47phox translocation from the cytosol to membranes. The EGF-induced ROS production was inhibited by DPI. Stimulation of cells with EGF resulted in an increase in Akt phosphorylation at Ser473, which was inhibited by c-Src DN, DPI, and LY 294002. Moreover, treatment of HT-29 cells with a dominant negative mutant of IκB (IκBαM) inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF induced p65 translocation from the cytosol to nuclei. Treatment of HT-29 cells with EGF induced an increase in κB-luciferase activity, which was inhibited by a c-Src DN, LY 294002, and an Akt DN. Furthermore, EGF-induced colon cancer cell proliferation was inhibited by Sn(IV)protoporphyrin-IX (snPP, an HO-1 inhibitor). Taken together, these results suggest that the c-Src, NADPH oxidase, PI3K, and Akt signaling pathways play important roles in EGF-induced NF-κB activation and HO-1 expression in HT-29 cells. Moreover, overexpression of HO-1 mediates EGF-induced colon cancer cell proliferation.  相似文献   

12.

Background

Transplantation with bone marrow-derived mesenchymal stem cells (BMSCs) improves the survival of neurons and axonal outgrowth after stroke remains undetermined. Here, we investigated whether PI3K/AKT signaling pathway is involved in these therapeutic effects of BMSCs.

Methodology/Principal Findings

(1) BMSCs and cortical neurons were derived from Sprague-Dawley rats. The injured neurons were induced by Oxygen–Glucose Deprivation (OGD), and then were respectively co-cultured for 48 hours with BMSCs at different densities (5×103, 5×105/ml) in transwell co-culture system. The average length of axon and expression of GAP-43 were examined to assess the effect of BMSCs on axonal outgrowth after the damage of neurons induced by OGD. (2) The injured neurons were cultured with a conditioned medium (CM) of BMSCs cultured for 24 hours in neurobasal medium. During the process, we further identified whether PI3K/AKT signaling pathway is involved through the adjunction of LY294002 (a specific phosphatidylinositide-3-kinase (PI3K) inhibitor). Two hours later, the expression of pAKT (phosphorylated AKT) and AKT were analyzed by Western blotting. The length of axons, the expression of GAP-43 and the survival of neurons were measured at 48 hours.

Results

Both BMSCs and CM from BMSCs inreased the axonal length and GAP-43 expression in OGD-injured cortical neurons. There was no difference between the effects of BMSCs of 5×105/ml and of 5×103/ml on axonal outgrowth. Expression of pAKT enhanced significantly at 2 hours and the neuron survival increased at 48 hours after the injured neurons cultured with the CM, respectively. These effects of CM were prevented by inhibitor LY294002.

Conclusions/Significance

BMSCs promote axonal outgrowth and the survival of neurons against the damage from OGD in vitro by the paracrine effects through PI3K/AKT signaling pathway.  相似文献   

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14.
Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca2+ ([Ca2+]i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca2+]i are linked critically by the ATP-driven plasma membrane Ca2+-ATPase (PMCA) important for maintaining low resting [Ca2+]i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca2+]i was measured by fura-2 imaging. An in situ [Ca2+]i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca2+ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca2+ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca2+ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca2+ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis.  相似文献   

15.

Objective

Tetrameric α2-macroglobulin (α2M), a plasma panproteinase inhibitor, is activated upon interaction with a proteinase, and undergoes a major conformational change exposing a receptor recognition site in each of its subunits. Activated α2M (α2M*) binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α2M* in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells.

Methods

Employing immunoprecipitation techniques and Western blotting as well as kinase assays, activation of the mTORC1 and mTORC2 complexes, as well as down stream targets were studied. RNAi was also employed to silence expression of Raptor, Rictor, or GRP78 in parallel studies.

Results

Stimulation of cells with α2M* promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, AktT308, and AktS473 in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α2M* treatment of cells elevated mTORC1 kinase activity as determined by kinase assays of mTOR or Raptor immunoprecipitates. mTORC1 activity was sensitive to LY294002 and rapamycin or transfection of cells with GRP78 dsRNA. Down regulation of Raptor expression by RNAi significantly reduced α2M*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α2M*-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of AktS473 phosphorylation and levels of p-AktS473 in mTOR and Rictor immunoprecipitates. mTORC2 activity was sensitive to LY294002 and transfection of cells with GRP78 dsRNA, but insensitive to rapamycin. Down regulation of Rictor expression by RNAi significantly reduces α2M*-induced phosphorylation of AktS473 phosphorylation in Rictor immunoprecipitates.

Conclusion

Binding of α2M* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein synthesis in the prostate cancer cells.  相似文献   

16.
AimsThe expression of brain-derived neurotrophic factor (BDNF) may be a downstream target of a variety of antidepressant treatments, and selective serotonin reuptake inhibitors (SSRIs) are used clinically for the treatment of depression. BDNF binds to and activates tyrosine kinases receptor (TrkB) to exert its effects. TrkB, after activation by ligands, stimulates phosphoinositide 3-kinase (PI3K). The downstream target of PI3K is Akt-1, a serine-threonine kinase. BDNF has signaling through the PLC-?IP3/Ca2+ pathway. Furthermore, the PLC-?γ/IP3/Ca2+ pathway is regulated by the sigma-1 receptors. Here, we examined whether fluvoxamine (FLV) activated Akt-1 and increased phosphorylation of Akt-1 via sigma-1 receptor in PC12 cells.Main methodsWe examined the effect of the SSRI, FLV and BDNF on the phosphorylation levels of serine-threonine kinase Akt-1 in PC12 cells using immunoblotting techniques.Key findingsTreatment with 10 μM and 100 μM FLV of PC12 cells stimulated a 2.4- and 3.8-fold maximal increase in Ser473-phosphorylated Akt-1 levels at 40 min, respectively. Treatment with 50 ng/ml BDNF also stimulated Ser473 -phosphorylated Akt-1 by 2.6-fold with a maximal increase at 5 min. In addition, the phosphorylation induced by FLV and BDNF was blocked by LY294002, a selective inhibitor of PI3K. The sigma-1 receptor agonists dehydroepiandrosterone (DHEA)-sulfate also stimulated a 2.1-fold increase in the level of Ser473-phosphorylated Akt-1.SignificanceThis study demonstrates that fluvoxamine treatment rapidly increased phosphorylation of Akt-1. And BDNF activated Akt-1 phosphorylation by the TrkB/PI3K/Akt-1 pathway. We conclude that the phosphorylation of Akt-1, downstream of PI3K, was the key to their antidepressant effects.  相似文献   

17.
The homeostasis of protein metabolism is maintained and regulated by the rates of protein biosynthesis and degradation in living systems. Alterations of protein degradation may regulate protein biosynthesis through a feedback mechanism. Whether a change in protein biosynthesis modulates protein degradation has not been reported. In this study, we found that inhibition of protein biosynthesis induced phosphorylation/activation of AKT and led to phosphorylation of AKT target substrates, including FoxO1, GSK3α/β, p70S6K, AS160, and the E3 ubiquitin ligase MDM2. Phosphorylation of ribosomal protein S6 was also modulated by inhibition of protein biosynthesis. The AKT phosphorylation/activation was mediated mainly through the PI3K pathway because it was blocked by the PI3K inhibitor LY294002. The activated AKT phosphorylated MDM2 at Ser166 and promoted degradation of the tumor suppressor p53. These findings suggest that inhibition of protein biosynthesis can alter degradation of some proteins through activation of AKT. This study reveals a novel regulation of protein degradation and calls for caution in blocking protein biosynthesis to study the half-life of proteins.  相似文献   

18.
19.
Ligation of cell surface-associated GRP78 by activated α(2) -macroglobulin triggers pro-proliferative cellular responses. In part, this results from activation of adenylyl cyclase leading to an increase in cAMP. We have previously employed the cAMP analog 8-CPT-2Me-cAMP to probe these responses. Here we show in 1-LN prostate cancer cells that 8-CPT-2Me-cAMP causes a dose-dependent increase in Epac1, p-Akt(T308) , p-Akt(S473) , but not p-CREB. By contrast, the PKA activator 6-Benz-cAMP caused a dose-dependent increase in p-CREB, but not Epac1. We measured mTORC2-dependent Akt phosphorylation at S473 in immunoprecipitates of mTOR or Rictor from 1-LN cells. 8-CPT-2Me-cAMP caused a two-threefold increase in p-Akt(S473) and Akt(S473) kinase activity in Rictor immunoprecipitates. By contrast, there was only a negligible effect on p-Akt(T308) in Rictor immunoprecipitates. Silencing Rictor gene expression by RNAi significantly suppressed 8-CPT-2Me-cAMP-induced phosphorylation of Akt at Ser(473) . These studies represent the first report that Epac1 mediates mTORC2-dependent phosphorylation of Akt(S473) . Pretreatment of these cells with the PI 3-Kinase inhibitor LY294002 significantly suppressed 8-CPT-2Me-cAMP-dependent p-Akt(S473) and p-Akt(S473) kinase activities, and both effects were rapamycin insensitive. This treatment caused a two to threefold increase in S6 Kinase and 4EBP1 phosphorylation, indices of mTORC1 activation. Pretreatment of the cells with LY294002 and rapamycin significantly suppressed 8-CPT-2Me-cAMP-induced phosphorylation of S6 Kinase and 4EBP1. We further demonstrate that in 8-CPT-2Me-cAMP-treated cells, Epac1 co-immunoprecipitates with AKAP, Raptor, Rictor, PDE3B, and PDE4D suggesting thereby that during Epac1-induced activation of mTORC1 and mTORC2, Epac1 may have an additional function as a "scaffold" protein.  相似文献   

20.
In cultured bovine adrenal chromaffin cells expressing Nav1.7 sodium channel isoform, veratridine increased Ser473-phosphorylation of Akt and Ser9-phosphorylation of glycogen synthase kinase-3β by 217 and 195%, while decreasing Ser396-phosphorylation of tau by 36% in a concentration (EC50 = 2.1 μM)- and time (t1/2 = 2.7 min)-dependent manner. These effects of veratridine were abolished by tetrodotoxin or extracellular Ca2+ removal. Veratridine (10 μM for 5 min) increased translocation of Ca2+-dependent conventional protein kinase C-α from cytoplasm to membranes by 47%; it was abolished by tetrodotoxin, extracellular Ca2+ removal, or Gö6976 (an inhibitor of protein kinase C-α), and partially attenuated by LY294002 (an inhibitor of phosphatidylinositol 3-kinase). LY294002 (but not Gö6976) abrogated veratridine-induced Akt phosphorylation. In contrast, either LY294002 or Gö6976 alone attenuated veratridine-induced glycogen synthase kinase-3β phosphorylation by 65 or 42%; however, LY294002 plus Gö6976 completely blocked it. Veratridine (10 μM for 5 min)-induced decrease of tau phosphorylation was partially attenuated by LY294002 or Gö6976, but completely blocked by LY294002 plus Gö6976; okadaic acid or cyclosporin A (inhibitors of protein phosphatases 1, 2A, and 2B) failed to alter tau phosphorylation. These results suggest that Na+ influx via Nav1.7 sodium channel and the subsequent Ca2+ influx via voltage-dependent calcium channel activated (1) Ca2+/protein kinase C-α pathway, as well as (2) Ca2+/phosphatidylinositol 3-kinase/Akt and (3) Ca2+/phosphatidylinositol 3-kinase/protein kinase C-α pathways; these parallel pathways converged on inhibitory phosphorylation of glycogen synthase kinase-3β, decreasing tau phosphorylation.  相似文献   

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