Journal: Journal of Nutrition and Metabolism

Article Title: Undaria pinnatifida (Wakame) Intake Ameliorates High-Fat Diet-Induced Glucose Intolerance via Promoting GLUT4 Expression and Membrane Translocation in Muscle

doi: 10.1155/2023/9774157

Figure Lengend Snippet: Effect of wakame components on AKT and AMPK phosphorylation and GLUT4 levels in C2C12 cells. Differentiated C2C12 cells were treated with PBS, DMSO, metformin (1 mM), fucoidan (1 mg/mL), ethanol extract (50 μ g/mL), hexane extract (50 μ g/mL), fucoxanthinol (1 μ g/mL), or wakame peptide (100 μ g/mL) for 24 h before lysis. The lysates were analyzed with Western blot using (a) GLUT4, (b) P-AKT (s473) and T-AKT, and (c) P-AMPK (T172) and T-AMPK antibodies and quantified. The level of GLUT4 was normalized by that of GAPDH. The intensity of P-AKT and P-AMPK bands was normalized by that of their respective total protein bands. The data are presented as mean ± SD ( n = 4). The data were analyzed with one-way ANOVA combined with the Tukey test. ∗ p < 0.05, compared to PBS; # p < 0.05, compared to DMSO.

Article Snippet: The phosphor-specific antibodies, P-AKT (S473, 4051S; Cell Signaling Technology) and P-AMPK (T172, 2531S; Cell Signaling Technology), were used at 1 : 1000 dilution.

Techniques: Lysis, Western Blot

Journal: Journal of Nutrition and Metabolism

Article Title: Undaria pinnatifida (Wakame) Intake Ameliorates High-Fat Diet-Induced Glucose Intolerance via Promoting GLUT4 Expression and Membrane Translocation in Muscle

doi: 10.1155/2023/9774157

Figure Lengend Snippet: Wakame's effect on level and phosphorylation of insulin signaling molecules and GLUT4 in skeletal muscle. The skeletal muscle cells were collected via dissection from the ND, ND + W, HFD, and HFD + W mice. The cell lysates were analyzed by Western blotting with (a) GLUT4, (b) P-AKT (s473) and T-AKT, and (c) P-AMPK (T172) and T-AMPK antibodies. The level of GLUT4 was normalized to that of GAPDH. The level of P-AKT and T-AMPK was normalized to that of their respective total protein. The data were presented as mean ± SD ( n = 4-5) and analyzed with one-way ANOVA combined with the Tukey test. ∗ p < 0.05.

Article Snippet: The phosphor-specific antibodies, P-AKT (S473, 4051S; Cell Signaling Technology) and P-AMPK (T172, 2531S; Cell Signaling Technology), were used at 1 : 1000 dilution.

Techniques: Dissection, Western Blot

Journal: iScience

Article Title: Perturbation of placental protein glycosylation by endoplasmic reticulum stress promotes maladaptation of maternal hepatic glucose metabolism

doi: 10.1016/j.isci.2022.105911

Figure Lengend Snippet:

Article Snippet: Phospho-AMPKα (Thr172) , Cell Signaling Technology , Cat#2531; RRID: AB_330330.

Techniques: Recombinant, Plasmid Preparation, Avidin-Biotin Assay, Lysis, Isolation, Bradford Protein Assay, Labeling, SYBR Green Assay, Mutagenesis, Software

Journal: Antioxidants

Article Title: Cranberry Proanthocyanidins as a Therapeutic Strategy to Curb Metabolic Syndrome and Fatty Liver-Associated Disorders

doi: 10.3390/antiox12010090

Figure Lengend Snippet: PAC modulates hepatic lipid metabolism. Protein expression of pivotal biomarkers influencing lipid metabolism and biogenesis was determined in liver by Western blot as described in Materials and Methods. ( A ) ACC, ( B ) p-ACC, ( D ) FAS, ( E ) SREBP1c, ( F ) CPT1A, ( G ) PPARα, ( H ) PGC1α, ( I ) AMPKα and ( J ) p-AMPKα protein mass was determined. ( A ) ACC and ( B ) p-ACC as well as ( I ) AMPKα and ( J ) p-AMPKα were blotted on separate gels and normalized using their respective β-actins; the ( C ) p-ACC/ACC and ( K ) p-AMPKα/AMPKα ratios were then calculated. Data are expressed as the mean ± SEM for n = 4–8 mice/group with a representative gel per experiment. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HFHS mice. Each set of experiments, including the proteins of interest and their β-actin (as a reference protein and a loading housekeeping control) was run on the same gel. ACC: acetyl-CoA carboxylase; p-ACC: Phosphorylated-acetyl-CoA carboxylase; FAS: Fatty acid synthase; SREBP1c: Sterol regulatory element binding protein-1c; CPT1A: Carnitine palmitoyl transferase 1 isoform A; PPARα: Peroxisome proliferator activated receptor alpha; PGC1α: Peroxisome proliferator activated receptor gamma coactivator 1 alpha; AMPKα: AMP-activated protein kinaseα; p-AMPKα: Phosphorylated-AMP-activated protein kinaseα.

Article Snippet: Fat-free milk was used for the initial blocking of non-specific sites, followed by overnight incubation at 4 °C with the following primary antibodies at 1/1000 dilution unless otherwise specified: Sterol regulatory element binding protein-1c (SREBP1c), Peroxisome proliferator-activated receptor alpha (PPARα) from Cayman Chemical); Fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), AMP-activated protein kinaseα (AMPKα) and its phosphorylated form Phosphorylated AMPKα Thr172 (p-AMPKα), Carnitine palmitoyl transferase 1 isoform A (CPT1A), Inhibitor of kappa B (IκB; 1/500) from Cell signalling; Peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1α), Nuclear factor erythroid-2-related factor 2 (NRF2) from Abcam; Superoxide dismutase 2 (SOD2), Carbohydrate response element binding protein (ChREBP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Invitrogen; Glutathione peroxidase 1 (GPx), Cyclooxygenase-2 (COX-2) from Novus Biologicals; Nuclear factor kappa B (NF-κB; 1/250), Glucose 6-phosphatase (G6Pase), Phosphoenolpyruvate carboxykinase (PEPCK) from Santa Cruz Biotechnology; Phosphorylated acetyl-CoA carboxylase Ser79 (p-ACC; 1/500, Millipore); Tumor necrosis factor-alpha (TNFα; ThermoFisher Scientific, Waltham, MA, USA) and β-actin (1/250,000; Sigma-Aldrich, Burlington, MA, USA).

Techniques: Expressing, Western Blot, Binding Assay

Journal: Frontiers in Endocrinology

Article Title: Canagliflozin promotes osteoblastic MC3T3-E1 differentiation via AMPK/RUNX2 and improves bone microarchitecture in type 2 diabetic mice

doi: 10.3389/fendo.2022.1081039

Figure Lengend Snippet: Molecular docking analysis. Phosphorylation AMPKα (Thr172) structure download from PDB database binding with canagliflozin, dapagliflozin, and empagliflozin respectively, the image on the right is enlarged of the left.

Article Snippet: The primary antibodies were RUNX2 (Abcam, USA, ab192256), OCN (Santa Cruz Biotechnology Cat# sc-365797, RRID : AB_10859392), and p-AMPKα (Thr172) (Cell Signaling Technology, USA, CST2535).

Techniques: Binding Assay

Journal: Frontiers in Endocrinology

Article Title: Canagliflozin promotes osteoblastic MC3T3-E1 differentiation via AMPK/RUNX2 and improves bone microarchitecture in type 2 diabetic mice

doi: 10.3389/fendo.2022.1081039

Figure Lengend Snippet: Canagliflozin promotes differentiation of osteoblastic MC3T3-E1 partially through the AMPK/RUNX2 pathway under HG. (A, B) Expression of t-AMPK, phosphorylation AMPKα (Thr172), and RUNX2 detected under HG (with or without canagliflozin and compound C) on the 3rd day during differentiation and quantified. (C, D) Expression of phosphorylation AMPKα (Thr172) and RUNX2 examined under HG (with or without canagliflozin and AICAR) on the 3rd day during differentiation and quantified. (E, F) ALP staining under HG (with or without canagliflozin, compound C, and AICAR) on the 7th day during differentiation and quantified. (G, H) Alizarin Red S under HG (with or without canagliflozin, compound C, and AICAR) on the 14th day during differentiation the number of calcium nodules was calculated under the microscope. (I, J) Alizarin Red S under HG (with or without canagliflozin, compound C, and AICAR) on the 21st day during differentiation and the number of calcium nodules was calculated under the microscope. (K) Expression of phosphorylation AMPKα (Thr172) under HG (with or without canagliflozin, compound C, and AICAR) (on the 3rd day during differentiation) and captured the picture by fluorescence microscopy. (L) Expression of RUNX2 under HG (with or without canagliflozin, compound C, and AICAR) (on the 3rd day during differentiation) and captured the picture by fluorescence microscopy. (The concentration of canagliflozin was 5 μM, compound C was 1 μM, AICAR was 10 μM). * P < 0.05, ** P < 0.01, *** P < 0.001 compared with HG group. Data were analyzed using one-way ANOVA for three independent experiments.

Article Snippet: The primary antibodies were RUNX2 (Abcam, USA, ab192256), OCN (Santa Cruz Biotechnology Cat# sc-365797, RRID : AB_10859392), and p-AMPKα (Thr172) (Cell Signaling Technology, USA, CST2535).

Techniques: Expressing, Staining, Microscopy, Fluorescence, Concentration Assay

Journal: Frontiers in Endocrinology

Article Title: Canagliflozin promotes osteoblastic MC3T3-E1 differentiation via AMPK/RUNX2 and improves bone microarchitecture in type 2 diabetic mice

doi: 10.3389/fendo.2022.1081039

Figure Lengend Snippet: Canagliflozin can improve bone microarchitecture in Type 2 Diabetic Mouse Models. (A) HE staining on femoral tissue in DM and DM+Cana groups, the image below is the magnified of the picture above, the bar is in the bottom left corner of the picture. (B) Toluidine blue staining on femoral tissue in DM and DM+Cana groups, the bar is in the bottom left corner of the picture. (C, D) Expression of phosphorylation AMPKα (Thr172) in DM and DM+Cana groups and quantified. (E, F) Expression of RUNX2 in DM and DM+Cana groups and quantified. (G) The concentration of OCN in plasma determined by OCN Elisa Kit. * P < 0.05; ** P < 0.01; *** P < 0.001; Data were analyzed using t test.

Article Snippet: The primary antibodies were RUNX2 (Abcam, USA, ab192256), OCN (Santa Cruz Biotechnology Cat# sc-365797, RRID : AB_10859392), and p-AMPKα (Thr172) (Cell Signaling Technology, USA, CST2535).

Techniques: Staining, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: LKB1/AMPK and PKA Control ABCB11 Trafficking and Polarization in Hepatocytes

doi: 10.1371/journal.pone.0091921

Figure Lengend Snippet: Western blot analyses were performed with total cell lysates of cultured hepatocytes on day 6 using antibodies specific to LKB1 ( A ), AMPK ( B ), and phospho-Thr172 AMPK (phAMPK) ( C ). Immunoblots were quantified by densitometry; data were expressed as relative values to the expression level of untreated control cells. ( D ) Relative phosphorylation of AMPK was determined by dividing the phAMPK value by the total AMPK expression level. Pretreatments: TC – 100 µM taurocholate, AICAR - 500 µM AICAR, cAMP - 200 µM 8-Br-cAMP. Means ± S.E.M. of three experiments are shown. LKB1 expression is absent in the hepatocytes of the liver-specific LKB1 −/−, whereas AMPK expression is 64% of the control level. Both absolute and relative level of phosphorylated AMPK is greatly reduced in the LKB1-decifient hepatocytes.

Article Snippet: Rabbit anti-LKB1, anti-AMPK, anti-phospho-Thr172 AMPK antibodies were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Cell Culture, Expressing