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(A) Graph showing the mitochondrial OCR of C2C12 cells overexpressing pMIG GFP and pMIG RBM3-GFP, basal OCR and OCR after treatment with oligomycin (1 uM), FCCP (3 uM), antimycin and rotenone (1.5 uM), where the x-axis represents time in minutes and the y-axis represents oxygen consumption rate in pMol/min. (B) Bar graph measuring the basal respiration, maximum respiration (OCR after FCCP addition), spare respiratory capacity (basal respiration-maximum respiration) and ATP-linked respiration (basal respiration-respiration after oligomycin addition) of C2C12 cells overexpressing pMIG-GFP and pMIG-RBM3 GFP where the y-axis represents oxygen consumption rate in pMol/Min (n=2). (C) Heat map showing levels of TCA metabolites using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (D) Heat map showing levels of TCA metabolites using media (48 hrs.) from C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). (E) Graphical representation of levels of acetyl-CoA using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). mRNA expression levels of glycolytic genes (F) PKM1 , (G) PKM2 in C2C12 overexpressing pMIG-GFP control and pMIG-RBM3. (H) Western blot analysis of glycolytic protein levels (PKM1, PKM2) using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (I) mRNA expression levels of glycolytic genes PKM1 , PKM2 in mouse primary myoblasts overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). (J) Western blot analysis of <t>AMPK-beta</t> and 4E-BP1 using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (K) Western blot analysis of acetyl-CoA carboxylase (ACC) using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (L) Bar graph quantifying phosphorylated/total 4E-BP1, ACC and AMPK-beta respectively. *, **, *** represents p-value < 0.05, 0.01 and 0.001 respectively.

Total Ampk Beta, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "PROTEOMICS OF HYPOTHERMIC ADAPTATION REVEALS THAT RBM3 ENHANCES MITOCHONDRIAL METABOLISM AND MUSCLE STEM-CELL DIFFERENTIATION"

Article Title: PROTEOMICS OF HYPOTHERMIC ADAPTATION REVEALS THAT RBM3 ENHANCES MITOCHONDRIAL METABOLISM AND MUSCLE STEM-CELL DIFFERENTIATION

Journal: bioRxiv

doi: 10.1101/2023.05.05.539524

Figure Legend Snippet: (A) Graph showing the mitochondrial OCR of C2C12 cells overexpressing pMIG GFP and pMIG RBM3-GFP, basal OCR and OCR after treatment with oligomycin (1 uM), FCCP (3 uM), antimycin and rotenone (1.5 uM), where the x-axis represents time in minutes and the y-axis represents oxygen consumption rate in pMol/min. (B) Bar graph measuring the basal respiration, maximum respiration (OCR after FCCP addition), spare respiratory capacity (basal respiration-maximum respiration) and ATP-linked respiration (basal respiration-respiration after oligomycin addition) of C2C12 cells overexpressing pMIG-GFP and pMIG-RBM3 GFP where the y-axis represents oxygen consumption rate in pMol/Min (n=2). (C) Heat map showing levels of TCA metabolites using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (D) Heat map showing levels of TCA metabolites using media (48 hrs.) from C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). (E) Graphical representation of levels of acetyl-CoA using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). mRNA expression levels of glycolytic genes (F) PKM1 , (G) PKM2 in C2C12 overexpressing pMIG-GFP control and pMIG-RBM3. (H) Western blot analysis of glycolytic protein levels (PKM1, PKM2) using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (I) mRNA expression levels of glycolytic genes PKM1 , PKM2 in mouse primary myoblasts overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). (J) Western blot analysis of AMPK-beta and 4E-BP1 using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (K) Western blot analysis of acetyl-CoA carboxylase (ACC) using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (L) Bar graph quantifying phosphorylated/total 4E-BP1, ACC and AMPK-beta respectively. *, **, *** represents p-value < 0.05, 0.01 and 0.001 respectively.

Techniques Used: Control, Expressing, Western Blot

Incubation:

Article Title: PROTEOMICS OF HYPOTHERMIC ADAPTATION REVEALS THAT RBM3 ENHANCES MITOCHONDRIAL METABOLISM AND MUSCLE STEM-CELL DIFFERENTIATION
Article Snippet: .. Primary antibodies MyHC (Invitrogen 14650382), MF-20 (DHSB AB_2147781) MYOG (Invitrogen MA5-11486), MyoD1 (Santa Cruz SC-377460), RBM3 (Invitrogen PA5-51976), beta-ACTIN (CST 4967S), beta-Tubulin (CST 2146), PKM1(CST D30G6), beta-Actin (CST 4967) PKM2 (CST D78A4), PDH (CST 3205), SDHA (CST 5839), phospho-4E-BP1 (CST 2855), total 4E-BP1 (CST 9452), phospho-AMPK-alpha (CST 2535), total-AMPK-alpha (CST 5831), phospho-AMPK-beta (CST 4186), total-AMPK-beta (CST 4150), phospho-ACC (CST 11818) and total ACC (CST 3676) were used in 1:1000 dilution and incubated at 4 0 C overnight. .. Secondary antibodies: anti-mouse IgG, HRP-linked (CST 7076) and anti-rabbit IgG, HRP-linked (CST 7074) were used in 1:5000 dilution and incubated at room temperature for 1hr.

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Journal: bioRxiv

Article Title: PROTEOMICS OF HYPOTHERMIC ADAPTATION REVEALS THAT RBM3 ENHANCES MITOCHONDRIAL METABOLISM AND MUSCLE STEM-CELL DIFFERENTIATION

doi: 10.1101/2023.05.05.539524

Figure Lengend Snippet: (A) Graph showing the mitochondrial OCR of C2C12 cells overexpressing pMIG GFP and pMIG RBM3-GFP, basal OCR and OCR after treatment with oligomycin (1 uM), FCCP (3 uM), antimycin and rotenone (1.5 uM), where the x-axis represents time in minutes and the y-axis represents oxygen consumption rate in pMol/min. (B) Bar graph measuring the basal respiration, maximum respiration (OCR after FCCP addition), spare respiratory capacity (basal respiration-maximum respiration) and ATP-linked respiration (basal respiration-respiration after oligomycin addition) of C2C12 cells overexpressing pMIG-GFP and pMIG-RBM3 GFP where the y-axis represents oxygen consumption rate in pMol/Min (n=2). (C) Heat map showing levels of TCA metabolites using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (D) Heat map showing levels of TCA metabolites using media (48 hrs.) from C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). (E) Graphical representation of levels of acetyl-CoA using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). mRNA expression levels of glycolytic genes (F) PKM1 , (G) PKM2 in C2C12 overexpressing pMIG-GFP control and pMIG-RBM3. (H) Western blot analysis of glycolytic protein levels (PKM1, PKM2) using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (I) mRNA expression levels of glycolytic genes PKM1 , PKM2 in mouse primary myoblasts overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). (J) Western blot analysis of AMPK-beta and 4E-BP1 using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (K) Western blot analysis of acetyl-CoA carboxylase (ACC) using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (L) Bar graph quantifying phosphorylated/total 4E-BP1, ACC and AMPK-beta respectively. *, **, *** represents p-value < 0.05, 0.01 and 0.001 respectively.

Article Snippet: Primary antibodies MyHC (Invitrogen 14650382), MF-20 (DHSB AB_2147781) MYOG (Invitrogen MA5-11486), MyoD1 (Santa Cruz SC-377460), RBM3 (Invitrogen PA5-51976), beta-ACTIN (CST 4967S), beta-Tubulin (CST 2146), PKM1(CST D30G6), beta-Actin (CST 4967) PKM2 (CST D78A4), PDH (CST 3205), SDHA (CST 5839), phospho-4E-BP1 (CST 2855), total 4E-BP1 (CST 9452), phospho-AMPK-alpha (CST 2535), total-AMPK-alpha (CST 5831), phospho-AMPK-beta (CST 4186), total-AMPK-beta (CST 4150), phospho-ACC (CST 11818) and total ACC (CST 3676) were used in 1:1000 dilution and incubated at 4 0 C overnight.

Techniques: Control, Expressing, Western Blot

Figure 4. Effects of 991 on AMPK activity and mitochondrial pyruvate uptake in WT versus AMPKβ1−/−mice.

Journal: The Biochemical journal

Article Title: Inhibition of basal and glucagon-induced hepatic glucose production by 991 and other pharmacological AMPK activators.

doi: 10.1042/BCJ20220170

Figure Lengend Snippet: Figure 4. Effects of 991 on AMPK activity and mitochondrial pyruvate uptake in WT versus AMPKβ1−/−mice.

Article Snippet: Anti-total ACC (Merck Millipore, Catalogue No. 04-322), anti-P-Ser79-ACC (MerckMillipore, Catalogue No. 07-303), anti-GAPDH (Merck-Millipore, Catalogue No. MAB374), anti-total AMPKβ1 (R&D Systems, Catalogue No. AF2854), anti-P-Thr172-AMPK (Cell Signaling Technologies, Catalogue No. 2535), anti-total Raptor (Cell Signaling Technologies, Catalogue No. 2280), anti-P-Ser792Raptor (Cell Signaling Technologies, Catalogue No. 2083), anti-P-Thr56-eukaryotic elongation factor-2 (eEF2) (Cell Signaling Technologies, Catalogue No. 2331) and anti-total eEF2 (Cell Signaling Technologies, Catalogue No. 2332) antibodies were from the sources cited.

Techniques: Activity Assay

Figure 5. Effects of 991 on glucose production in hepatocytes from WT versus AMPKβ1−/−mice together with in vivo

Journal: The Biochemical journal

Article Title: Inhibition of basal and glucagon-induced hepatic glucose production by 991 and other pharmacological AMPK activators.

doi: 10.1042/BCJ20220170

Figure Lengend Snippet: Figure 5. Effects of 991 on glucose production in hepatocytes from WT versus AMPKβ1−/−mice together with in vivo

Techniques: In Vivo

Figure 6. Effects of AMPK activators on basal and glucagon-stimulated glucose production and PKA activity in hepatocytes from WT versus AMPKβ1−/−mice.

Journal: The Biochemical journal

Article Title: Inhibition of basal and glucagon-induced hepatic glucose production by 991 and other pharmacological AMPK activators.

doi: 10.1042/BCJ20220170

Figure Lengend Snippet: Figure 6. Effects of AMPK activators on basal and glucagon-stimulated glucose production and PKA activity in hepatocytes from WT versus AMPKβ1−/−mice.

Techniques: Activity Assay

DKI mice have reduced skeletal muscle AMPK α and β2 content but intact AMPK, ACC and TBC1D1 signaling in response to a maximal running test at a 5° incline versus WT. Skeletal muscles were collected from rested and maximally exercised male WT and DKI mice, and phosphorylation of AMPK and downstream substrates was assessed using Western blotting. (A) Representative immunoblots of p-T172 and total AMPK, p-S182 and total AMPK β, p-S79 and total acetyl-CoA carboxylase (ACC) and p-S660 and total TBC1 domain family member 1 (TBC1D1) with representative stain free image. Quantified relative (B) AMPK p-T172, (C) AMPK β p-S182, (D) ACC p-S79, and (E) TBC1D1 p-S660; (F) Total AMPK α; (G) Total AMPK β. Male mice, 17–20 weeks, n = 6–8. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Journal: Frontiers in Physiology

Article Title: Disrupting AMPK-Glycogen Binding in Mice Increases Carbohydrate Utilization and Reduces Exercise Capacity

doi: 10.3389/fphys.2022.859246

Figure Lengend Snippet: DKI mice have reduced skeletal muscle AMPK α and β2 content but intact AMPK, ACC and TBC1D1 signaling in response to a maximal running test at a 5° incline versus WT. Skeletal muscles were collected from rested and maximally exercised male WT and DKI mice, and phosphorylation of AMPK and downstream substrates was assessed using Western blotting. (A) Representative immunoblots of p-T172 and total AMPK, p-S182 and total AMPK β, p-S79 and total acetyl-CoA carboxylase (ACC) and p-S660 and total TBC1 domain family member 1 (TBC1D1) with representative stain free image. Quantified relative (B) AMPK p-T172, (C) AMPK β p-S182, (D) ACC p-S79, and (E) TBC1D1 p-S660; (F) Total AMPK α; (G) Total AMPK β. Male mice, 17–20 weeks, n = 6–8. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Antibodies against total AMPK α (2532), phospho-AMPK T172 (2531), total AMPK β (4150), phospho-AMPK β1 S182 (4186), total ACC (3662), phospho-ACC S79 (11,818), GLUT4 (2213), total TBC1D1 (66,433), phospho-TBC1D1 S660 (6928) and horseradish peroxidase-conjugated anti-rabbit (7074) and anti-mouse (7076) IgG secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States).

Techniques: Western Blot, Staining

Lack of USF2 promotes autophagy and activates ERK1/2 and AKT (A, B) Western blot analyses and densitometric quantification of the protein levels of autophagy-related LC3, ATG5 as well as SDHB, USF2, and α-tubulin in control, ΔUSF2, and ΔUSF2+USF2 cells. The protein levels of the corresponding proteins in control cells were set to 100%. *significant difference control vs. ΔUSF2 and **ΔUSF2 cells vs ΔUSF2+USF2 cells, p ≤ 0.05. (C, D) Representative fluorescence images from control, ΔUSF2, and ΔUSF2+USF2 cells in the presence and absence of chloroquine (Clq; 50 μM for 6 h) probed with an antibody against LC3, stained with DAPI and quantified with the Operetta high-content imaging system. The LC3 levels in control cells were set to 100%. *significant difference control vs. ΔUSF2, ** Control chloroquine treated vs ΔUSF2 chloroquine treated cells, and # untreated vs chloroquine treated p ≤ 0.05. (E, F) Western blot analyses and densitometric quantification of the LC3II and p62 protein levels from control, ΔUSF2, and ΔUSF2+USF2 cells in the presence and absence of chloroquine (Clq; 50 μM for 6 h). The LC3II and p62 levels in control cells were set to 100%. *significant difference control vs. ΔUSF2, **Control chloroquine treated vs ΔUSF2 chloroquine treated cells, and # untreated vs chloroquine treated p ≤ 0.05. (G, H) Western blot analyses and densitometric quantification of the protein levels of pERK1/2, ERK1/2, pAKT, AKT, AMPKα, AMPKβ, USF2, V5-tag, and α-tubulin in control, ΔUSF2 and ΔUSF2+USF2 cells. The protein levels of the corresponding phospho (p) protein levels in the control cells were quantified and normalized to their total levels and the corresponding levels in control cells were set to 100%. *significant difference control vs. ΔUSF2, **ΔUSF2 cells vs ΔUSF2+USF2 cells, and # control vs ΔUSF2+USF2 cells, p ≤ 0.05.

Journal: Redox Biology

Article Title: Loss of USF2 promotes proliferation, migration and mitophagy in a redox-dependent manner

doi: 10.1016/j.redox.2020.101750

Figure Lengend Snippet: Lack of USF2 promotes autophagy and activates ERK1/2 and AKT (A, B) Western blot analyses and densitometric quantification of the protein levels of autophagy-related LC3, ATG5 as well as SDHB, USF2, and α-tubulin in control, ΔUSF2, and ΔUSF2+USF2 cells. The protein levels of the corresponding proteins in control cells were set to 100%. *significant difference control vs. ΔUSF2 and **ΔUSF2 cells vs ΔUSF2+USF2 cells, p ≤ 0.05. (C, D) Representative fluorescence images from control, ΔUSF2, and ΔUSF2+USF2 cells in the presence and absence of chloroquine (Clq; 50 μM for 6 h) probed with an antibody against LC3, stained with DAPI and quantified with the Operetta high-content imaging system. The LC3 levels in control cells were set to 100%. *significant difference control vs. ΔUSF2, ** Control chloroquine treated vs ΔUSF2 chloroquine treated cells, and # untreated vs chloroquine treated p ≤ 0.05. (E, F) Western blot analyses and densitometric quantification of the LC3II and p62 protein levels from control, ΔUSF2, and ΔUSF2+USF2 cells in the presence and absence of chloroquine (Clq; 50 μM for 6 h). The LC3II and p62 levels in control cells were set to 100%. *significant difference control vs. ΔUSF2, **Control chloroquine treated vs ΔUSF2 chloroquine treated cells, and # untreated vs chloroquine treated p ≤ 0.05. (G, H) Western blot analyses and densitometric quantification of the protein levels of pERK1/2, ERK1/2, pAKT, AKT, AMPKα, AMPKβ, USF2, V5-tag, and α-tubulin in control, ΔUSF2 and ΔUSF2+USF2 cells. The protein levels of the corresponding phospho (p) protein levels in the control cells were quantified and normalized to their total levels and the corresponding levels in control cells were set to 100%. *significant difference control vs. ΔUSF2, **ΔUSF2 cells vs ΔUSF2+USF2 cells, and # control vs ΔUSF2+USF2 cells, p ≤ 0.05.

Article Snippet: Western blot analyses were performed using standard protocols with antibodies against LC3B (#2775S; 1:1000; Cell Signaling), ATG5 (sc-133158; 1:1000; Santa Cruz Biotechnology), SDHB (sc:13315; 1:1000; Santa Cruz Biotechnology), p62/SQSTM1 (#5114; 1:1000; Cell Signaling), pERK1/2 (#9101; 1:1000; Cell Signaling), total ERK1/2 (#9107; 1:1000; Cell Signaling), pAMPKα/β and total AMPKα/β (AMPK and ACC Antibody Sampler Kit #9957; 1:1000; Cell Signaling), USF-2 (N-18; 1:500; Santa Cruz Biotechnology) as well as monoclonal antibodies against V5-tag (#R960-25; 1:5000; Invitrogen), and α-tubulin (clone B-5-1-2; #T5168, 1:10000; Sigma-Aldrich).

Techniques: Western Blot, Fluorescence, Staining, Imaging

Primary mouse hepatocytes were incubated for 1 h with DMSO as vehicle, 10 μM 991, 500 μM phenformin, 2 mM AICAR, 100 μM A769662, 10 nM glucagon or with concentrations of 991 and glucagon as indicated for measurements of intracellular adenine nucleotide concentrations ( a ), AMPK activity by immunoprecipitation using anti-AMPKα1 and anti-AMPKα2 antibodies ( b , d ) and total PDE activity ( d , e ). In e , the 100% value for PDE activity in the DMSO-treated control condition was 6.4±0.7 μU per mg of protein. In c , mouse hepatocytes were incubated for 20 min with 10 μM 991 or DMSO as vehicle before incubation with 10 nM glucagon for ELISA measurements of cAMP concentrations at the indicated times. In f , mouse hepatocytes were incubated for 20 min with 10 μM 991 or DMSO as vehicle and 5 mM pan-PDE inhibitor IBMX before incubation with the indicated concentrations of glucagon for 15 min and measurement of cAMP (ELISA method). Values are means±s.e.m. for n =3 ( a – d ), n =5 ( e ) or n =4 ( f ) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05) compared with control incubations with DMSO.

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: Primary mouse hepatocytes were incubated for 1 h with DMSO as vehicle, 10 μM 991, 500 μM phenformin, 2 mM AICAR, 100 μM A769662, 10 nM glucagon or with concentrations of 991 and glucagon as indicated for measurements of intracellular adenine nucleotide concentrations ( a ), AMPK activity by immunoprecipitation using anti-AMPKα1 and anti-AMPKα2 antibodies ( b , d ) and total PDE activity ( d , e ). In e , the 100% value for PDE activity in the DMSO-treated control condition was 6.4±0.7 μU per mg of protein. In c , mouse hepatocytes were incubated for 20 min with 10 μM 991 or DMSO as vehicle before incubation with 10 nM glucagon for ELISA measurements of cAMP concentrations at the indicated times. In f , mouse hepatocytes were incubated for 20 min with 10 μM 991 or DMSO as vehicle and 5 mM pan-PDE inhibitor IBMX before incubation with the indicated concentrations of glucagon for 15 min and measurement of cAMP (ELISA method). Values are means±s.e.m. for n =3 ( a – d ), n =5 ( e ) or n =4 ( f ) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05) compared with control incubations with DMSO.

Article Snippet: Anti-total ACC (Merck Millipore, Catalogue No. 04-322), anti-P-Ser79-ACC (Merck-Millipore, Catalogue No. 07-303), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Merck-Millipore, Catalogue No. MAB374), anti-total GP (Sigma, Catalogue No. HPA000962), anti-total AMPK β1(R&D Systems, Catalogue No. AF2854) and anti-total AMPK β2 (R&D Systems, Catalogue No. MAB3808), anti-PThr172-AMPK (T172) (Cell Signaling Technologies, Catalogue No. 2535, anti-P-AMPK-substrate (Cell Signaling Technologies, Catalogue No. 5759), anti-P-PKA-substrate (Cell Signaling Technologies, Catalogue No. 9624), anti-total Raptor (Cell Signaling Technologies, Catalogue No. 2280) and anti-P-Ser792-Raptor (Cell Signaling Technologies, Catalogue No. 2083), anti-total CREB (Cell Signaling Technologies, Catalogue No. 9197), anti-phospho-Ser133-CREB (Cell Signaling Technologies, Catalogue No. 9198) and anti-total PDE4B (Origene, Catalogue No. TA503471) antibodies were from the sources cited.

Techniques: Incubation, Activity Assay, Immunoprecipitation, Control, Enzyme-linked Immunosorbent Assay

Primary hepatocytes from wild-type mice or mice bearing a liver-specific deletion of the two AMPK catalytic subunits (AMPK α 1 −/− α LS−/− ) were treated as described in the legends to and . The cells were collected and lysed for immunoblotting levels of phosphorylated ACC and AMPK versus total proteins along with GAPDH as a loading control ( a ). Extracts were also prepared for the measurement of cAMP concentrations by ELISA ( b ), for PKA assay ( c ) and for PDE assay ( d ). Values are means±s.e.m. for n =3 ( b – d ) separate experiments, and in a representative immunoblots are shown. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05) compared with control incubations with DMSO or between the indicated conditions.

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: Primary hepatocytes from wild-type mice or mice bearing a liver-specific deletion of the two AMPK catalytic subunits (AMPK α 1 −/− α LS−/− ) were treated as described in the legends to and . The cells were collected and lysed for immunoblotting levels of phosphorylated ACC and AMPK versus total proteins along with GAPDH as a loading control ( a ). Extracts were also prepared for the measurement of cAMP concentrations by ELISA ( b ), for PKA assay ( c ) and for PDE assay ( d ). Values are means±s.e.m. for n =3 ( b – d ) separate experiments, and in a representative immunoblots are shown. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05) compared with control incubations with DMSO or between the indicated conditions.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Protein Kinase A Assay

PDE4B was cloned from mouse hepatocyte cDNA. The recombinant protein was overexpressed in E. coli and purified. PDE protein was phosphorylated for 1 h with purified recombinant activated AMPK and/or purified PKA catalytic subunits and [γ- 32 P] ATP, and analysed by SDS–PAGE followed by Coomassie blue staining and phosphorimaging for quantification ( a , c ). In b , PDE was phosphorylated for 1 h with recombinant activated AMPK and [γ- 32 P]. Phosphorylation sites were identified by LC–MS/MS after trypsin digestion and radioactive peak separation by high-performance liquid chromatography (HPLC). The phosphorylation sites that were identified are underlined in the right hand panel. In d and e , recombinant PDE was phosphorylated as above but with non-radioactive ATP for PDE assay as indicated. In d , separate determinations of V max and K M were made by linear regression of double reciprocal (Lineweaver Burk) plots. In e , the basal PDE activities of the wild-type (WT), S118A, S125A and S304A mutant proteins were 1.97±0.25, 0.14±0.01, 1.59±0.15 and 0.32±0.09 mU per mg of protein, respectively. Values are means±s.e.m. for n =3 ( c – e ) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05) compared with control incubations or between the indicated conditions.

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: PDE4B was cloned from mouse hepatocyte cDNA. The recombinant protein was overexpressed in E. coli and purified. PDE protein was phosphorylated for 1 h with purified recombinant activated AMPK and/or purified PKA catalytic subunits and [γ- 32 P] ATP, and analysed by SDS–PAGE followed by Coomassie blue staining and phosphorimaging for quantification ( a , c ). In b , PDE was phosphorylated for 1 h with recombinant activated AMPK and [γ- 32 P]. Phosphorylation sites were identified by LC–MS/MS after trypsin digestion and radioactive peak separation by high-performance liquid chromatography (HPLC). The phosphorylation sites that were identified are underlined in the right hand panel. In d and e , recombinant PDE was phosphorylated as above but with non-radioactive ATP for PDE assay as indicated. In d , separate determinations of V max and K M were made by linear regression of double reciprocal (Lineweaver Burk) plots. In e , the basal PDE activities of the wild-type (WT), S118A, S125A and S304A mutant proteins were 1.97±0.25, 0.14±0.01, 1.59±0.15 and 0.32±0.09 mU per mg of protein, respectively. Values are means±s.e.m. for n =3 ( c – e ) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05) compared with control incubations or between the indicated conditions.

Techniques: Clone Assay, Recombinant, Purification, SDS Page, Staining, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, High Performance Liquid Chromatography, Mutagenesis, Control

In a , wild-type (WT) or mutant recombinant mouse liver PDE4B was incubated for 1 h with non-radioactive ATP in the presence (+) or absence (−) of recombinant activated AMPK. Proteins (0.1 μg) were seperated by SDS–PAGE for immunoblotting with the indicated antibodies. In b and c , mouse hepatocytes from either WT ( b ) or both WT and AMPK α 1 −/− α 2 LS−/− mice ( c ) were serum-starved overnight and incubated for 1 h with the indicated concentrations of 991 or phenformin. The cells were collected and lysed for immunoblotting with the indicated antibodies, except for PDE4B, which was immunoprecipitated as described in the Methods section, before immunoblotting. In c , phosphorylation levels of AMPK and its targets ACC, Raptor and PDE4B were quantified by densitometry and expressed relative to the corresponding total protein levels or GAPDH before normalization as indicated. Representative immunoblots are shown and for blot quantification in c , the values are means±s.e.m. for n =3 (p-ACC, p-Raptor and p-AMPK) or n =4 (p-PDE4B) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05).

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: In a , wild-type (WT) or mutant recombinant mouse liver PDE4B was incubated for 1 h with non-radioactive ATP in the presence (+) or absence (−) of recombinant activated AMPK. Proteins (0.1 μg) were seperated by SDS–PAGE for immunoblotting with the indicated antibodies. In b and c , mouse hepatocytes from either WT ( b ) or both WT and AMPK α 1 −/− α 2 LS−/− mice ( c ) were serum-starved overnight and incubated for 1 h with the indicated concentrations of 991 or phenformin. The cells were collected and lysed for immunoblotting with the indicated antibodies, except for PDE4B, which was immunoprecipitated as described in the Methods section, before immunoblotting. In c , phosphorylation levels of AMPK and its targets ACC, Raptor and PDE4B were quantified by densitometry and expressed relative to the corresponding total protein levels or GAPDH before normalization as indicated. Representative immunoblots are shown and for blot quantification in c , the values are means±s.e.m. for n =3 (p-ACC, p-Raptor and p-AMPK) or n =4 (p-PDE4B) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05).

Techniques: Mutagenesis, Recombinant, Incubation, SDS Page, Western Blot, Immunoprecipitation, Phospho-proteomics

Unlike biguanides, treatment with 991 activates AMPK without increasing cellular AMP levels. Both biguanides and 991 activate the major PDE 4B isoenzyme in hepatocytes in an AMPK-dependent manner. Metformin and phenformin activate hepatic AMPK either directly or via a rise in AMP, which could compete with ATP to inhibit adenylate cyclase. Phosphorylation-induced activation of PDE4B by AMPK reduces glucagon-stimulated cAMP accumulation. As a consequence, PKA activation by glucagon and downstream signalling are decreased in hepatocytes incubated with 991, the effect being AMPK-dependent.

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: Unlike biguanides, treatment with 991 activates AMPK without increasing cellular AMP levels. Both biguanides and 991 activate the major PDE 4B isoenzyme in hepatocytes in an AMPK-dependent manner. Metformin and phenformin activate hepatic AMPK either directly or via a rise in AMP, which could compete with ATP to inhibit adenylate cyclase. Phosphorylation-induced activation of PDE4B by AMPK reduces glucagon-stimulated cAMP accumulation. As a consequence, PKA activation by glucagon and downstream signalling are decreased in hepatocytes incubated with 991, the effect being AMPK-dependent.

Techniques: Phospho-proteomics, Activation Assay, Incubation

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total ampk beta (Cell Signaling Technology Inc)

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