Journal: Scientific Reports

Article Title: Selective targeting of PPARγ by the natural product chelerythrine with a unique binding mode and improved antidiabetic potency

doi: 10.1038/srep12222

Figure Lengend Snippet: ( a ) In vitro CDK5 assay on PPARγ LBD incubated with gradient concentration of rosiglitazone or chelerythrine. ( b ) Quantification of PPARγ phosphorylation relative to total PPARγ in vitro . ( c ) In vivo phosphorylation of PPARγ was analyzed in epididymal fat pads of mice with chelerythrine treatment as shown in . ( d ) Quantification of PPARγ phosphorylation relative to total PPARγ in vivo . IB, immunoblot; pPPARγ, phosphorylated PPARγ; pSer273, phosphorylated PPARγ at Ser273; Rosi, rosiglitazone; CHE, chelerythrine.

Article Snippet: For western blotting, a rabbit polyclonal phospho-specific antibody against PPARγ Ser273 was produced by AbMax Biotechnology Co., Ltd, China, with a synthetic phosphopeptide previously described .

Techniques: In Vitro, Incubation, Concentration Assay, Phospho-proteomics, In Vivo, Western Blot

Journal: Nature Communications

Article Title: A systems study reveals concurrent activation of AMPK and mTOR by amino acids

doi: 10.1038/ncomms13254

Figure Lengend Snippet: ( a ) Schematic representation of the mTOR-AMPK network. Amino acids (aa) activate mTORC1. Do they also activate other parts of the network? Nutrients (aa), growth factors and energy (AMP/ATP ratio) impinge on the mTOR (mammalian target of rapamycin) signalling cascade. Growth factors such as insulin activate the IR (insulin receptor). The IR binds IRS1 (insulin receptor substrate 1) which acts as an adaptor for PI3K (phosphoinositide 3-kinase). PI3K generates phosphatidylinositol (3,4,5)-trisphosphate, which binds and translocates PDK1 and Akt/PKB to the plasma membrane. Here, Akt is activated by PDK1 and subsequently inhibits the heteromeric TSC1–TSC2 (tuberous sclerosis protein) complex. TSC1–TSC2 serves as a GTPase-activating protein (GAP) for the small GTPase Rheb (Ras homologue enriched in brain) that activates mTORC1. mTORC1 phosphorylates p70-S6K (p70-S6-kinase), and regulates anabolic processes such as translation and catabolic processes including autophagy. A negative-feedback loop (NFL) from mTORC1 and p70-S6K to IR and IRS1 renders upstream insulin signalling refractory to the insulin input. AMP levels rise when energy demand enhances ATP conversion to ADP. AMP and the kinases LKB1 (liver kinase B1), and CaMKKβ (Ca 2+ / calmodulin-dependent protein kinase kinase β) activate AMPK (AMP-activated protein kinase), which inhibits mTORC1 by phosphorylating TSC2 and the mTORC1 component Raptor. Next to growth factors and the AMP/ATP ratio, aa activate mTORC1, and this is mediated by the Rag GTPases. ( b ) Simulated response of p70-S6K-pT389, Akt-pS473, Akt-pT308 and AMPK-pT172 to aa stimulation in a system with PI3K perturbation (PI3K activity 10 to 100%; experimental equivalent: PI3K inhibition with wortmannin). ( c ) Aa enhance AMPK-pT172 during PI3K inhibition with wortmannin. Shown are immunoblot results of aa-stimulated C2C12 cells in the presence or absence of 100 nM wortmannin. Data are representative of three experiments. ( d ) Quantitative representations of simulated (PI3K inhibition: residual activity 10% as shown in b ) and experimentally determined dynamics of p70-S6K-pT389, AMPK-pT172, Akt-pS473 and Akt-pT308 upon stimulation with aa with or without wortmannin. Shown are the mean and s.e.m., N =3. Statistical significance between control and treatment in experimental quantitations across the time course was detected by repeated measures analysis of variance (ANOVA). Exp Ctrl, experimental control condition (DMSO); Exp Inhib, experimental PI3K inhibition (wortmannin); Sim Ctrl, simulated control condition; Sim Inhib, simulated PI3K perturbation.

Article Snippet: The rabbit polyclonal antibody against p70-S6K (phospho T229, GTX25231-50) was obtained from Biozol, Eching, Germany and used at a final dilution of 1:1,000 in TBST buffer with 5% BSA.

Techniques: Clinical Proteomics, Membrane, Activity Assay, Inhibition, Western Blot, Control

Journal: Nature Communications

Article Title: A systems study reveals concurrent activation of AMPK and mTOR by amino acids

doi: 10.1038/ncomms13254

Figure Lengend Snippet: ( a ) Simulated time courses corresponding to the perturbation as shown in , but using the model including the new p70-S6K extended module. The simulated time courses for p70-S6K-pT389 and pT229 also incorporate the corresponding experimental data that was used for parameter estimation . The simulated time courses for Akt and AMPK represent predictions using this new model. ( b ) Equivalent quantitative representations as shown in , but using the model including the new p70-S6K extended module. Simulated quantifications are based on data from a of this figure. Experimental data are the same as shown in and reported here to compare it with the new model. Statistical significance between control and treatment in experimental quantitations across the time course was detected by repeated measures analysis of variance (ANOVA). Exp Ctrl, experimental control condition (DMSO); Exp Inhib, experimental PI3K inhibition (wortmannin); Sim Ctrl, simulated control condition; Sim Inhib, simulated PI3K perturbation. Shown are the mean and s.e.m., N =3. ( c ) The allosteric Akt inhibitor MK-2206 reduces mTORC1 activation by aa. Aa-enhanced p70-S6K-pT389 is reduced upon Akt inhibition with MK-2206, whereas AMPK-T172 remains phosphorylated. Shown are immunoblot results of aa-stimulated C2C12 cells in the presence or absence of 1 μM MK-2206. Data are representative of three experiments. ( d ) IRS1 knockdown does not inhibit mTOR signalling upon aa stimulation. Aa readdition enhances p70-S6K-pT389 and pT229 in control C2C12 myocytes transfected with non-targeting siRNA (siControl) or with IRS1 knockdown (siIRS1). Shown are immunoblot results of aa-stimulated C2C12 cells with and without IRS1 knockdown. Data are representative of three experiments.

Article Snippet: The rabbit polyclonal antibody against p70-S6K (phospho T229, GTX25231-50) was obtained from Biozol, Eching, Germany and used at a final dilution of 1:1,000 in TBST buffer with 5% BSA.

Techniques: Control, Inhibition, Activation Assay, Western Blot, Knockdown, Transfection

Journal: Nature Communications

Article Title: A systems study reveals concurrent activation of AMPK and mTOR by amino acids

doi: 10.1038/ncomms13254

Figure Lengend Snippet: ( a ) Simulated response of p70-S6K-pT389, Akt-pS473, Akt-pT308 and AMPK-pT172 to aa induction in a system with mTORC2 inhibition (mTORC2 activity 10 to 100%; experimental equivalent: mSin1 knockdown). ( b ) Aa induce p70-S6K-pT389 and AMPK-pT172, when mTORC2 is inhibited by mSin1 knockdown (shSin1). Representative immunoblot results of aa-stimulated C2C12 cells in the absence or presence of shSin1 (4 days). Data are representative of three experiments. ( c ) Quantitative representations of simulated (mTORC2 inhibition: reduction to 40%, as shown in a ) and experimentally determined dynamics of p70-S6K-pT389, AMPK-pT172, Akt-pS473 and Akt-pT308 upon stimulation with aa with mTORC2 inhibition using shSin1. Data are the mean and s.e.m., N =3. Statistical significance between control and treatment in experimental quantitations across the time course was detected by repeated measures analysis of variance (ANOVA). Exp Ctrl, experimental control condition (shControl); Exp Inhib, experimental mTORC2 inhibition (shSin1); Sim Ctrl, simulated control condition; Sim Inhib, simulated mTORC2 perturbation.

Article Snippet: The rabbit polyclonal antibody against p70-S6K (phospho T229, GTX25231-50) was obtained from Biozol, Eching, Germany and used at a final dilution of 1:1,000 in TBST buffer with 5% BSA.

Techniques: Inhibition, Activity Assay, Knockdown, Western Blot, Control

Journal: Nature Communications

Article Title: A systems study reveals concurrent activation of AMPK and mTOR by amino acids

doi: 10.1038/ncomms13254

Figure Lengend Snippet: ( a ) Different aa mixes enhance AMPK-T172 and p70-S6K-T389 phosphorylation. C2C12 cells were starved in HBSS (16 h), with subsequent readdition of HBSS+aa mix 1, HBSS+aa mix 2 or HBSS only. N =3. aa mix 1: Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, OH-Pro, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val; aa mix 2: Arg, Cys, Glu, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, Val. ( b ) AMPK-pT172 induction by aa is inhibited by the CaMKKβ inhibitor STO-609. Aa-stimulated C2C12 cells were analysed with or without 10 μg ml −1 STO-609. N =3. ( c ) Simulated response of p70-S6K-pT389, Akt-pS473, Akt-pT308 and AMPK-pT172 to aa stimulation and AMPK perturbation (AMPK activity 10 to 100%; experimental equivalent: STO-609). ( d ) Aa readdition enhances p70-S6K-pT389, Akt-pT308 and Akt-pS473 in the presence of STO-609. Aa-stimulated C2C12 cells were analysed with or without 10 μg ml −1 STO-609. N =4. ( e ) Quantitative representations of simulated (AMPK inhibition 50%, corresponds to c ) and measured dynamics of p70-S6K-pT389, AMPK-pT172, Akt-pS473 and Akt-pT308 upon aa, with or without STO-609. Shown are the mean and s.e.m., N =4. Statistical significance between control and treatment in experimental quantitations across the time course was detected by repeated measures analysis of variance (ANOVA). Exp Ctrl, experimental control condition (DMSO); Exp Inhib, experimental AMPK perturbation (STO-609); Sim Ctrl, simulated control condition; Sim Inhib, simulated AMPK perturbation. ( f ) Aa readdition enhances p70-S6K-pT389, Akt-pT308 and mTOR-pS2481 in cells with AMPK knockdown (siAMPKα1+2). Aa-stimulated HeLa cells were analysed with or without siAMPKα1+2 (2 days). N =3. ( g ) Aa enhance ULK1-pS757 (mTORC1 substrate site), and ULK1-pS317 (AMPK substrate site) remains phosphorylated. STO-609 reduces ULK1-pS317 but not ULK1-pS757. Aa-stimulated C2C12 cells were analysed with or without 10 μg ml −1 STO-609. N =3. ( h ) ULK1 can be phosphorylated at the AMPK substrate site serine 317, when the mTORC1 substrate site (ULK1-S758 in human, corresponding to S757 in mouse) is mutated to glutamate (ULK1-S758E). ULK1/2 double-knockout (DKO) mouse embryonic fibroblasts (MEFs) were left untransfected, or stably transfected with human wild-type ULK1 (ULK1-WT), or ULK1-S758E. N =6. ( i ) Relative quantitation of data shown in h . Signals for ULK1-pS317 and ULK1-pS758 in ULK1-WT and ULK1-S758E were quantified and normalized to ULK1 total levels. Values for ULK1-WT, starvation condition were set to 1. Mean values and s.e.m., N =6. ( j ) STO-609 reduces LC3-II autophagy marker levels in aa-stimulated cells. Aa-stimulated C2C12 cells were analysed with or without 10 μg ml −1 STO-609. All cells were pre-treated with Bafilomycin A 1 (BafA) for 60 min. N =3.

Article Snippet: The rabbit polyclonal antibody against p70-S6K (phospho T229, GTX25231-50) was obtained from Biozol, Eching, Germany and used at a final dilution of 1:1,000 in TBST buffer with 5% BSA.

Techniques: Phospho-proteomics, Activity Assay, Inhibition, Control, Knockdown, Double Knockout, Stable Transfection, Transfection, Quantitation Assay, Marker

Journal: Nature Communications

Article Title: A systems study reveals concurrent activation of AMPK and mTOR by amino acids

doi: 10.1038/ncomms13254

Figure Lengend Snippet: ( a ) Simulated response of p70-S6K-pT389, Akt-pS473, Akt-pT308 and AMPK-pT172 to aa stimulation in a system with mTORC1 perturbation (mTORC1 activity 10 to 100%; experimental equivalent: mTORC1 inhibition by Raptor knockdown). ( b ) Aa readdition enhances Akt-pT308, Akt-pS473 and AMPK-pT172 during mTORC1 inhibition by Raptor knockdown (shRaptor). Representative immunoblot results of aa-stimulated C2C12 cells with and without shRaptor (4 days). Data are representative of four experiments. ( c ) Quantitative representations of simulated (mTORC1 inhibition: reduction to 25%, as shown in a ) and experimentally determined dynamics of p70-S6K-pT389, AMPK-pT172, Akt-pS473 and Akt-pT308 upon stimulation with aa with mTORC1 inhibition using shRaptor. Data are the mean and s.e.m., N =4. Statistical significance between control and treatment in experimental quantitations across the time course was detected by repeated measures analysis of variance (ANOVA). Exp Ctrl, experimental control condition (shControl); Exp Inhib, experimental mTORC1 inhibition (shRaptor); Sim Ctrl, simulated control condition; Sim Inhib, simulated mTORC1 perturbation. ( d ) Schematic representation of the mTOR-AMPK network with four aa inputs (known input green, new inputs magenta). Aa activate (1) mTORC1, (2) PI3K/PDK1, directly activating Akt and p70-S6K, (3) mTORC2 and (4) AMPK via CaMKKβ. Of note, AMPK activation by aa does not inhibit mTORC1, but activates ULK1. mTORC1 inhibits ULK1 in response to aa. Via these events, AMPK and mTORC1 balance ULK1 activity and autophagy under nutrient sufficiency.

Article Snippet: The rabbit polyclonal antibody against p70-S6K (phospho T229, GTX25231-50) was obtained from Biozol, Eching, Germany and used at a final dilution of 1:1,000 in TBST buffer with 5% BSA.

Techniques: Activity Assay, Inhibition, Knockdown, Western Blot, Control, Activation Assay