Journal: Cell Cycle

Article Title: Novel protective mechanism of reducing renal cell damage in diabetes: Activation AMPK by AICAR increased NRF2/OGG1 proteins and reduced oxidative DNA damage

doi: 10.1080/15384101.2016.1231259

Figure Lengend Snippet: AICAR upregulates AMPK and inhibits mTORC1 to increase protein expression of Nrf2 and OGG1 and decrease 8-oxodG in kidney of diabetic mice. Immunoblot analysis shows that AICAR treatment increased phosphorylation of AMPK at Thr172 and resulted in significant decrease in mTORC1 measured by (A) increase in phosphorylation of raptor at Ser92 and (C) decrease in phosphorylation of p70S6k at Thr389 in kidney cortex of diabetic mice compared to non-treated mice. (C) Activation of AMPK resulted in significant increase in transcription factor Nrf2 and OGG1 protein expression in treated mice compared to non-treated mice. Actin was used as a loading control. (B&D) Histograms represent means ± SE (n = 4). (E) 8-OxodG levels were measured according to the instruction manual of 8-oxodG kits showed significant decrease in DNA damage in kidney of treated mice compared to non-treated mice. Significant difference from non-treated mice is indicated by **P < 0.01. Kidney sections of db/db mice (F) treated or non-treated with AICAR were stained with 8-oxodG antibody followed by FITC-anti-rabbit IgG as secondary antibodies. Staining of nucleus with propidium Iodide (PI) (red) and 8-oxodG with FITC (green) were detected using a filter with excitation range 450–490 nm and excitation at 535 nm.

Article Snippet: Increase OGG1 protein expression in kidney of mice treated with AICAR resulted in significant decrease in accumulation of the oxidative DNA damage, 8-oxodG (pg/ml), quantitatively measured by 8-oxodG Stress MARQ kit ( ) compared to non-treated animals ( ).

Techniques: Expressing, Western Blot, Activation Assay, Staining

Journal: Cell Cycle

Article Title: Novel protective mechanism of reducing renal cell damage in diabetes: Activation AMPK by AICAR increased NRF2/OGG1 proteins and reduced oxidative DNA damage

doi: 10.1080/15384101.2016.1231259

Figure Lengend Snippet: AICAR activates AMPK and decreases mTORC1 expression resulting in increased protein/mRNA expression and promoter activity of OGG1 and decreased 8-oxodG in HG-treated renal proximal tubular cells. High glucose significantly decreased AMPK phosphorylation and activates mTORC1 measured by decrease (A) in phosphorylation of raptor at Ser92 and (B) an increase in phosphorylation of p70S6k at Thr389 in MCT cells. (A) Cells treated with AICAR before exposure to HG show a significantly increased in activation of AMPK, which decreases mTORC1 expression. (B) Enhance AMPK activity and inhibition of mTORC1 by AICAR leads to upregulation of Nrf2 and OGG1 protein expression in cell exposed to HG. (C) RT-PCR of OGG1 was performed in RNA isolated from MCT cells grown in NG or HG and treated with AICAR. PCR products were analyzed on an ethidium bromide-stained gel. GAPDG was used a loading control. (D) AMPK activity was measured as phosphorylation levels of AMPK at Thr172 in HG or/and AICAR treated MCT cell lysates by AMPK ELISA kit. (E) A reporter plasmid containing the OGG1 promoter driving expression of the luciferase and a control Renilla reporter gene were co-transfected into the cells using LipofectAMINE Plus Reagent™. AICAR pretreatment reversed the inhibitory effect of HG on OGG1 promoter activity as well as significantly increased OGG1 activity in cells grown in NG. (F) DNA was extracted from treated and non-treated cells and digested with nuclease P1. The detection of dG and 8-oxodG was performed using 8-oxodG kits. Authentic standards of 8-oxodG were analyzed simultaneously. Experiment represent means±SE (n = 6). Significant difference from cells grown in normal glucose is indicated by *P < 0.01, cells grown in NG and treated with AICAR by **P < 0.01 and cells exposed to HG and treated with AICAR compared to cells grown in HG by #P < 0.01.

Article Snippet: Increase OGG1 protein expression in kidney of mice treated with AICAR resulted in significant decrease in accumulation of the oxidative DNA damage, 8-oxodG (pg/ml), quantitatively measured by 8-oxodG Stress MARQ kit ( ) compared to non-treated animals ( ).

Techniques: Expressing, Activity Assay, Activation Assay, Inhibition, Reverse Transcription Polymerase Chain Reaction, Isolation, Staining, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Luciferase, Transfection

Journal: Cell Cycle

Article Title: Novel protective mechanism of reducing renal cell damage in diabetes: Activation AMPK by AICAR increased NRF2/OGG1 proteins and reduced oxidative DNA damage

doi: 10.1080/15384101.2016.1231259

Figure Lengend Snippet: Downregulation of Nrf2 resulted in significant decrease in OGG1 protein expression and lead to sharp decrease in OGG1 promoter activity in renal roximal tubular cells treated with HG. (A) Cells transfected with siRNA against Nrf2 showed a significant decrease in OGG1 protein compared to cells transfected with control (nonspecific siRNA) and grown under both conditions of NG and HG. In addition, cells transfected with siRNA against Nrf2 and treated with AICAR showed no changes in protein expression of Nrf2 and OGG1. (B) Cells co-transfected with siRNA against Nrf2 and reporter plasmid construct carried OGG1 promoter showed a significant decrease in OGG1 promoter activity in cells grown under low and high concentrations. Cells transfected with siRNA against Nrf2 and treated with AICAR showed significant decrease in OGG1 promoter activity under both NG and HG conditions. Experiment represent means ± SE (n = 6). Significant difference from cells grown in NG compared to cells in HG is indicated by *P < 0.01, cells grown in NG and transfected with siRNA of Nrf2 by **P < 0.01, cells transfected with siRNA of Nrf2 and exposed to HG by #P < 0.01, cells grown in NG and transfected with siRNA of Nrf2+treated with AICAR by §P < 0.01, and cells transfected with siRNA of Nrf2 and exposed to HG+treated with AICAR by ¶P < 0.01.

Article Snippet: Increase OGG1 protein expression in kidney of mice treated with AICAR resulted in significant decrease in accumulation of the oxidative DNA damage, 8-oxodG (pg/ml), quantitatively measured by 8-oxodG Stress MARQ kit ( ) compared to non-treated animals ( ).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Construct

Journal: Cell Cycle

Article Title: Novel protective mechanism of reducing renal cell damage in diabetes: Activation AMPK by AICAR increased NRF2/OGG1 proteins and reduced oxidative DNA damage

doi: 10.1080/15384101.2016.1231259

Figure Lengend Snippet: High glucose treatment significantly reduces binding of Nrf2 to the OGG1 promoter element and AICAR treatment reversed the effect of HG in renal proximal tubular cells. (A) EMSA analysis of a DNA probe corresponding to the putative Nrf2 binding site in the OGG1 promoter. Labeled probes were incubated with nuclear extracts isolated from MCT cells grown in NG or HG and treated or non-treated with AICAR (2 mM). (B) Treatment of MCT cells with HG significantly reduced binding of Nrf2 to OGG1 promoter compared to cells grown in NG. While AICAR treatment showed significant increased in binding of Nrf2 to OGG1 promoter activity in cells grown in NG and cells exposed to HG. (C) The specificity of binding of the DNA/protein complex to Nrf2 was demonstrated by adding an Nf2 antibody (1 and 2ug) to the reaction mixture. Including the Nrf2 antibody in the reaction results in marked reduction of the specific DNA/protein complex.

Article Snippet: Increase OGG1 protein expression in kidney of mice treated with AICAR resulted in significant decrease in accumulation of the oxidative DNA damage, 8-oxodG (pg/ml), quantitatively measured by 8-oxodG Stress MARQ kit ( ) compared to non-treated animals ( ).

Techniques: Binding Assay, Labeling, Incubation, Isolation, Activity Assay

Journal: Cell Cycle

Article Title: Novel protective mechanism of reducing renal cell damage in diabetes: Activation AMPK by AICAR increased NRF2/OGG1 proteins and reduced oxidative DNA damage

doi: 10.1080/15384101.2016.1231259

Figure Lengend Snippet: Proposed model for the role of AICAR in preventing kidney damage in diabetes. AICAR activates AMPK and inhibits binding of raptor to mTORC1 to decrease mTOR and increase OGG1 activity. Inactivation of AMPK results in downregulation Nrf2 and leads to decrease the functional activity of OGG1. On the other hand, inactivation of Akt and inhibition of mTORC2 results in upregulation of OGG1 activity to reduce accumulation of oxidative DNA damage in renal cells. The consequences steps of upregulation and downregulation of major signals that activate the DNA repair pathways and prevent cell damage lead to improved kidney parameters in diabetic patients under the therapeutic effect of AMPK activator.

Article Snippet: Increase OGG1 protein expression in kidney of mice treated with AICAR resulted in significant decrease in accumulation of the oxidative DNA damage, 8-oxodG (pg/ml), quantitatively measured by 8-oxodG Stress MARQ kit ( ) compared to non-treated animals ( ).

Techniques: Binding Assay, Activity Assay, Functional Assay, Inhibition

Journal: Physiological Reports

Article Title: Nitric oxide activates AMPK by modulating PDE3A in human pulmonary artery smooth muscle cells

doi: 10.14814/phy2.14559

Figure Lengend Snippet: siRNA silencing of PDE3A (siPDE3A) blunted NO‐induced AMPK activation. Human PASMC were transfected with siPDE3A, siPDE3B, or scramble siRNA. After 48 hr, cells were treated with DETA NONOate (250 μM) for 24 hr. Protein was harvested, analyzed by Western blot, and expression quantified by densitometry. (a) Representative Western blots for each PDE3A, PDE3B, β‐actin, and p‐ and total AMPK. Data are shown as fold changes ± SEM relative to scramble siRNA control for (b) PDE3A/β‐actin after siPDE3A or siPDE3B transfection; n = 5–6 for each group, * p < .025 different from scramble siRNA (two‐way ANOVA). (c) PDE3B/β‐actin after siPDE3A or siPDE3B transfection; n = 6 for each group, * p < .005 different from scramble siRNA (two‐way ANOVA), and (d) p/T‐AMPK after siPDE3A or siPDE3B transfection; n = 5–6 for each group, * p < .02 different from scramble siRNA, control; # p < .001 different from scramble siRNA, DETA NONOate; & p < .003 different from siPDE3A, DETA NONOate (two‐way ANOVA)

Article Snippet: AICAR (5‐Aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside), an AMPK agonist, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Transfection, Western Blot, Expressing, Control

Journal: Physiological Reports

Article Title: Nitric oxide activates AMPK by modulating PDE3A in human pulmonary artery smooth muscle cells

doi: 10.14814/phy2.14559

Figure Lengend Snippet: sGC stimulator increased PDE3A protein expression and AMPK phosphorylation, whereas sGC inhibitor prevented the NO‐induced increase in PDE3A protein expression and AMPK phosphorylation in hPASMC. Human PASMC were treated with the NO donor DETA NONOate (250 μM) or sGC stimulator, BAY 41‐2272 (10 μM). Protein was harvested, analyzed by Western blot, and expression quantified by densitometry. Representative Western blots with data shown below as fold changes ± SEM relative to vehicle control for (a) PDE3A and β‐actin; n = 6–11 for each group, * p < .04 different from control (one‐way ANOVA) and (b) p‐AMPK and total AMPK; n = 6–9 for each group, * p < .03 different from control (one‐way ANOVA)l. Alternatively, hPASMC were pretreated for 4 hr with the sGC inhibitor, ODQ (10 μM), then incubated with DETA NONOate (250 μM) for 48 hr. Representative Western blots with data below shown as fold changes ± SEM relative to vehicle control for (c) PDE3A and β‐actin; n = 8–11 for each group, * p < .001 different from control, # p < .001 different from DETA NONOate (one‐way ANOVA) and (d) p‐AMPK and total AMPK; n = 8–9 for each group, * p < .001 different from control, # p < .001 different from DETA NONOate (one‐way ANOVA)

Article Snippet: AICAR (5‐Aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside), an AMPK agonist, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Incubation

Journal: Physiological Reports

Article Title: Nitric oxide activates AMPK by modulating PDE3A in human pulmonary artery smooth muscle cells

doi: 10.14814/phy2.14559

Figure Lengend Snippet: Treatment with NO, AMPK agonist, and PDE3 inhibitor decreased hPASMC proliferation. Human PASMC were treated with DETA NONOate (250 μM), the AMPK agonist, AICAR (5 μM), the PDE3 inhibitor, milrinone (10 μM), the combination of DETA NONOate + milrinone, or vehicle and incubated for 48 hr. MTT proliferation assay was performed. Data shown are means ± SEM . n = 6–10, * p < .001 different from control, # p < .003 different from AICAR (one‐way ANOVA)

Article Snippet: AICAR (5‐Aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside), an AMPK agonist, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Proliferation Assay, Control

Journal: Nature Communications

Article Title: Native chromatome profiling reveals hundreds of metabolic enzymes in the nucleus across tissues

doi: 10.1038/s41467-026-69217-2

Figure Lengend Snippet: A Chromatin-associated proteins differentially abundant across cancer lineages relative to healthy counterparts, defined as the 1% most extreme z-values per tissue. B ACBD5 and ATP5MJ relative protein abundance in chromatin samples, based on lineage, with abundance in healthy counterpart represented as a red point, while statistically significant samples are highlighted in blue (depleted) and brown (enriched). Significantly changing proteins were determined by taking the top 1% of most differentially abundant proteins between respective cancer and normal sample (two-sided). C Immunofluorescence images of ACBD5 (top) and ATP5MJ (bottom) (green) within Tubulin-stained cells (red) in prostate cancer (PC-3) and skin cancer (A-431) cell lines. Images were obtained from the HPA dataset. Scale bar, 20 μm. D Nuclear related GO-terms enriched in ProHD interactors of metabolic enzymes, with enzymes functionally associated to one significant term depicted. E Hierarchical clustering of spearman correlations based on normalized protein abundances across chromatin samples with positive relationships indicating covarying proteins across samples. F Network of statistically significant biological processes correlated with “one-carbon by folate” pathway enzymes, based on normalized chromatin-bound protein intensities. Proteins positively correlated with “one-carbon by folate” enzymes are represented as weighted edges; edge thickness corresponds to correlation strength. G Relative protein abundance per sample for GART-POLD1 and SHMT2-MCM7 protein correlation pairs across tissue types. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were Vinculin (E1E9V) XP ® (Cell Signaling Technology #13901; 1:1000), NDUFS3 (abcam #ab177471, 1:1000), SDHA (Thermo Fisher Scientific; #PA5-66693; 1:1000), UQCRB (Thermo Fisher Scientific; #PA5-106324; 1:1000), COX4 (Thermo Fisher Scientific; #PA5-19471; 1:1000), COX5A (Thermo Fisher Scientific; #PA5-27432; 1:1000), ATP5A1 (abcam; #ab14748; 1:1000), FDX1 (Thermo Fisher Scientific #PA5-59653, 1:1000), Histone H3 (1B1B2) (Cell Signaling Technology #14269, 1:10000), MTHFD1 (ProteinTech; #10794-1-AP; 1:1000), ATIC (ProteinTech; #11099-1-AP; 1:1000), GART (ProteinTech; #13659-1-AP; 1:1000), SHMT2 (ProteinTech; #10726-1-AP; 1:1000).

Techniques: Quantitative Proteomics, Immunofluorescence, Staining

Journal: Diabetologia

Article Title: Biguanides and thiazolidinediones inhibit stimulated lipolysis in human adipocytes through activation of AMP-activated protein kinase.

doi: 10.1007/s00125-009-1639-6

Figure Lengend Snippet: Fig. 3 Effects of AICAR, biguanides and thiazolidinediones on protein kinase A downstream targets in isolated human adipocytes. Human adipocytes were preincubated for 1 h in the presence of 500 μmol/l AICAR, 100 μmol/l phenformin (Phen), 250 μmol/l piogli- tazone (Pio), 250 μmol/l rosiglitazone (Rosi) or 4 h in the presence of 2 mmol/l metformin (Met) and then incubated for another hour with 1 μmol/l isoprenaline. Total adipocytes extracts were prepared and analysed by western blotting for phosphorylation of CREB (Ser133), for total CREB content and for phosphorylation of PKA substrates. These blots are representative of two independent experiments

Article Snippet: AICAR (500 μmol/l) (Cell Signaling Technology, Boston, MA, USA), phenformin (Sigma, St Louis, MO, USA) (100 μmol/l), pioglitazone (250 μmol/l) and rosiglitazone (250 μmol/l) (Alexis Biochemicals, Lausen, Switzerland) were added for 1 h, whereas metformin (2 mmol/l) (Alexis Biochemicals) was added for 4 h. When used, compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrrazolo[1,5-a]pyrimidine; Calbiochem, Darmstadt, Germany) (50 μmol/l) and triacsin C (Alexis Biochemicals, Lausen, Switzerland) (10 μmol/l) were added to the adipocyte medium 2 and 3 h, respectively, before addition of the effectors.

Techniques: Isolation, Incubation, Western Blot, Phospho-proteomics

Journal: Diabetologia

Article Title: Biguanides and thiazolidinediones inhibit stimulated lipolysis in human adipocytes through activation of AMP-activated protein kinase.

doi: 10.1007/s00125-009-1639-6

Figure Lengend Snippet: Fig. 5 Effects of AICAR, biguanides and thiazolidinediones on AMPK Thr172 and ACC Ser80 phosphorylation in the presence of compound C. Human adipocytes were preincubated in the absence or presence of compound C (50 μmol/l) for 2 h and then treated for 1 h with 500 μmol/l AICAR, 100 μmol/l phenformin (Phen), 250 μmol/l pioglitazone (Pio), 250 μmol/l rosiglitazone (Rosi) (a) or 4 h with 2 mmol/l metformin (Met) (b). Total adipocyte extracts were prepared and analysed by western blotting for phosphorylation of AMPK Thr172 and ACC Ser80 and for total ACC and AMPKα1 content. These blots are representative of at least three independent experiments

Article Snippet: AICAR (500 μmol/l) (Cell Signaling Technology, Boston, MA, USA), phenformin (Sigma, St Louis, MO, USA) (100 μmol/l), pioglitazone (250 μmol/l) and rosiglitazone (250 μmol/l) (Alexis Biochemicals, Lausen, Switzerland) were added for 1 h, whereas metformin (2 mmol/l) (Alexis Biochemicals) was added for 4 h. When used, compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrrazolo[1,5-a]pyrimidine; Calbiochem, Darmstadt, Germany) (50 μmol/l) and triacsin C (Alexis Biochemicals, Lausen, Switzerland) (10 μmol/l) were added to the adipocyte medium 2 and 3 h, respectively, before addition of the effectors.

Techniques: Phospho-proteomics, Western Blot

Journal: Diabetologia

Article Title: Biguanides and thiazolidinediones inhibit stimulated lipolysis in human adipocytes through activation of AMP-activated protein kinase.

doi: 10.1007/s00125-009-1639-6

Figure Lengend Snippet: Fig. 4 Effects of AICAR, biguanides and thiazolidinediones on AMPK Thr172 phosphorylation in the presence of isoprenaline or ANP. Human adipocytes were preincubated for 1 h in the presence of 500 μmol/l AICAR, 100 μmol/l phenformin (Phen), 250 μmol/l pioglitazone (Pio), 250 μmol/l rosiglitazone (Rosi) or 4 h in the presence of 2 mmol/l metformin (Met)

Article Snippet: AICAR (500 μmol/l) (Cell Signaling Technology, Boston, MA, USA), phenformin (Sigma, St Louis, MO, USA) (100 μmol/l), pioglitazone (250 μmol/l) and rosiglitazone (250 μmol/l) (Alexis Biochemicals, Lausen, Switzerland) were added for 1 h, whereas metformin (2 mmol/l) (Alexis Biochemicals) was added for 4 h. When used, compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrrazolo[1,5-a]pyrimidine; Calbiochem, Darmstadt, Germany) (50 μmol/l) and triacsin C (Alexis Biochemicals, Lausen, Switzerland) (10 μmol/l) were added to the adipocyte medium 2 and 3 h, respectively, before addition of the effectors.

Techniques: Phospho-proteomics

Journal: Diabetologia

Article Title: Biguanides and thiazolidinediones inhibit stimulated lipolysis in human adipocytes through activation of AMP-activated protein kinase.

doi: 10.1007/s00125-009-1639-6

Figure Lengend Snippet: Fig. 8 Effects of AICAR, biguanides and thiazolidinediones on HSL phosphorylation and translocation in the presence of ANP. Human adipocytes were preincubated for 1 h in the presence of 500 μmol/l AICAR, 100 μmol/l phenformin (Phen), 250 μmol/l pioglitazone (Pio), 250 μmol/l rosiglitazone (Rosi) or 4 h with 2 mmol/l metformin (Met) then 100 nmol/l ANP was added for 1 h. Adipocyte lysates were prepared and analysed by western blotting for phosphorylation of HSL Ser554 and for total HSL and ATGL content (a, b). These blots are representative of two independent experiments. Human adipocytes were preincubated for

Article Snippet: AICAR (500 μmol/l) (Cell Signaling Technology, Boston, MA, USA), phenformin (Sigma, St Louis, MO, USA) (100 μmol/l), pioglitazone (250 μmol/l) and rosiglitazone (250 μmol/l) (Alexis Biochemicals, Lausen, Switzerland) were added for 1 h, whereas metformin (2 mmol/l) (Alexis Biochemicals) was added for 4 h. When used, compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrrazolo[1,5-a]pyrimidine; Calbiochem, Darmstadt, Germany) (50 μmol/l) and triacsin C (Alexis Biochemicals, Lausen, Switzerland) (10 μmol/l) were added to the adipocyte medium 2 and 3 h, respectively, before addition of the effectors.

Techniques: Phospho-proteomics, Translocation Assay, Western Blot