Journal: The Journal of nutrition

Article Title: Suppression of High-Fat Diet-Induced Obesity by Platycodon Grandiflorus in Mice Is Linked to Changes in the Gut Microbiota.

doi: 10.1093/jn/nxaa159

Figure Lengend Snippet: FIGURE 2 Fatty acid catabolism and lipid anabolism in the adipose tissues of male C57BL/6J mice fed CON, HFD, or HFPG. (A) Map of genes involved in fat accumulation and fatty-acid metabolism affected by PG supplementation. (B) Western blot analysis for p-AMPK, p-ACC, and FASN in epididymal adipose tissues. (C) Fold change in densitometric concentrations of p-AMPK/AMPK, p-ACC/ACC, and FASN/ACTB relative to the CON group (n = 3). (D) Relative levels of mRNA expression in epididymal adipose tissues tested by qRT-PCR (n = 3). Values represent means ± SEMs, n = 7 if not specified. Labeled means without a common letter differ, P < 0.05. ACC, acetyl-CoA carboxylase; ACTB, actin, β; Adipoq, adiponectin; AMPK, AMP-activated protein kinase; ATGL, adipose triglyceride lipase; CEBPA, CCAAT/enhancer binding protein α; CON, control diet; FASN, fatty acid synthase; HFD, high-fat diet; HFPG, high-fat diet and PG supplementation; Lep, leptin; p-ACC, phosphorylated acetyl-CoA carboxylase; p-AMPK, phosphorylated AMP-activated protein kinase; PG, Platycodon grandiflorus; PPARG, peroxisome proliferator– activated receptor γ ; Srebf1, sterol regulatory element binding transcription factor 1; SREBP-1c, sterol regulatory element binding protein-1c; WAT, white adipose tissue.

Article Snippet: The primary antibodies against AMP-activated protein kinase α (AMPKα) (no. 5831), phosphorylated-AMPKα (p-AMPKα) (Thr172) (no. 2535), acetyl-CoA carboxylase (ACC) (no. 3676), phosphorylated-ACC (p-ACC) (Ser79) (no. 11818), fatty acid synthase (FASN) (no. 3180), and horseradish peroxidase–conjugated anti-rabbit secondary antibody (no. 7074) were obtained from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Labeling, Binding Assay, Control

Journal: Chinese Medical Journal

Article Title: Metformin improved oxidized low-density lipoprotein-impaired mitochondrial function and increased glucose uptake involving Akt-AS160 pathway in raw264.7 macrophages

doi: 10.1097/cm9.0000000000000333

Figure Lengend Snippet: Figure 3: Metformin improved lipid and glucose oxidation in Ox-LDL-loaded macrophages. (A) Effects of metformin on protein expression of enzymes mediating lipid synthesis and oxidation. (B) Metformin increased Ox-LDL-impaired glucose consumption. Glucose consumption was normalized by protein concentrations of cellular lysates. (C) Metformin decreased lactic acid production in Ox-LDL-loaded macrophages. Lactic acid levels were normalized by protein concentrations of cellular lysates. (D) Metformin down-regulated the ratio of lactic acid output by glucose input in the presence of Ox-LDL. ∗P < 0.05, †P < 0.01, xP < 0.001 vs. control; ‡P < 0.01, jjP < 0.001 vs. Ox-LDL. CPT-1b: Carnitine palmitoyltransferase 1b; NDUFS3: Nicotinamide adenine dinucleotide:ubiquinone oxidoreductase core sub-unit S3; Ox-LDL: Oxidized low-density lipoprotein; p-ACC: Acetyl-CoA carboxylase phosphorylation.

Article Snippet: After being blocked with 5% bovine serum albumin (BSA), blots were incubated with various primary antibodies including rabbit anti-Akt 1/2/3 (Thermo Fisher), rabbit anti-dynamin-related protein 1 (DRP-1), optic atrophy 1 (OPA1), mitofusin 2 (MFN2), Akt substrate of 160 kDa Cellular reactive oxygen species detection (AS160), nicotinamide adenine dinucleotide:ubiquinone oxidoreductase core sub-unit S3 (NDUFS3; Abcam, Cambridge, UK), rabbit anti-AMPK, P-AMPK (Thr172), acetyl-CoA carboxylase (ACC), ACC phosphorylation (pACC; Ser79), P-Akt (Ser473), TBC1 family domain member 1 (TBC1D1) phosphorylation (p-TBC1D1; Ser660) (CST, Beverly, MA, USA), anti-P-AS160 (T642) (Biorbyt, Cambridge, UK), anti-carnitine palmitoyltransferase (CPT)-1b (SAB, Nanjing, China), and rabbit anti-b-actin (Bio-world, St. Louis Park, MN, USA) at 4°C overnight.

Techniques: Expressing, Control, Phospho-proteomics

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Melanocortin-induced PKA activation inhibits AMPK activity via ERK-1/2 and LKB-1 in hypothalamic GT1-7 cells.

doi: 10.1210/me.2011-1218

Figure Lengend Snippet: FIG. 3. -MSH-induced dephosphorylation of AMPK (Thr172) or of ACC (Ser79) in GT1-7 cells monitored by whole-cell ELISA. Approximately 25,000 GT1-7 cells were seeded into white 96-well dishes. After 20 h of serum starvation, cells were stimulated with 1 M -MSH for 10 min and phosphorylation of AMPK at Thr172 (A), total AMPK levels (B), or phosphorylation of ACC at Ser79 (C) monitored by specific antibodies in whole cells. Cell-bound, HRP-conjugated secondary antibody was detected by automatic injection of SuperSignal West Femto HRP substrate in a FLUOstar Omega plate reader at time point 1 sec (as indicated by the arrow) and total luminescence recorded for additional 8 sec. As a control, luminescence signals were measured in samples without any first antibody. A–C, Raw data (eight replicates) of one representative experiment are shown as the mean SEM. D, Data of three independent experiments were compiled, control values subtracted and normalized by setting RLU values of unstimulated cells (basal) at time point 9 sec as 100%. -MSH-induced dephosphorylation was than calculated by subtracting values of stimulated cells from 100%. Hashed signs (##, P 0.01) indicate a significant difference to unstimulated cells (basal).

Article Snippet: Primary antibodies phospho (p)-ERK-1/2 or total (t)-ERK-2 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and t-AMPK- , p-AMPKThr172, p-ACC-Ser79, or LKB-1, were from Cell Signaling (Danvers, MA).

Techniques: De-Phosphorylation Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Injection, Control

Journal: Biomolecules

Article Title: Linking Dysregulated AMPK Signaling and ER Stress in Ethanol-Induced Liver Injury in Hepatic Alcohol Dehydrogenase Deficient Deer Mice

doi: 10.3390/biom9100560

Figure Lengend Snippet: EtOH-induced altered expression for AMPKα signaling in the livers of ADH − and ADH + deer mice fed 1%, 2% or 3.5% EtOH daily for 2 months. Protein phosphorylation/expression for p-AMPKα/AMPKα ( A ), p-ACC1/ACC1 ( B ), CPT1A ( C ), FAS ( D ) and CaMKKβ ( E ). Western blots along with respective bar diagrams show relative intensities of p-AMPK/AMPK, p-ACC1/ACC1, CPT1A, FAS and p-CaMKKβ/CaMKKβ. Intensities were normalized to β-actin (loading control). Values are expressed as means ± SEMs ( n = 4). * p -value ≤ 0.05.

Article Snippet: The primary antibodies for AMPKα (62 kDa; catalogue number 5831), phospho (p)-AMPKα (Thr 172) (62 kDa; catalogue number 2535), Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ; 60, 50 kDa; catalogue number 4436), p-CaMKKβ (Thr286) (60, 50 kDa; catalogue number 12716), liver kinase B1 (LKB1; 54 kDa; catalogue number 3050), p-LKB1 (Ser 428) (54 kDa; catalogue number 53482), acetyl CoA carboxylase 1 (ACC1; 265 kDa; catalogue number 4190), p-ACC1 (Ser 79) (280 kDa; catalogue number3661), fatty acid synthase (FAS; 273 kDa; catalogue number 3189), glucose regulated protein 78 (GRP78; 78 kDa; catalogue number 3117), eukaryotic translation initiation factor 2α (eIF2α; 38 kDa; catalogue number 5324), p-eIF2α (Ser 51; 38 kDa; catalogue number 3398), caspase-3 (17, 19, 35 kDa; catalogue number 4436), caspase-8 (10, 57 kDa; catalogue number 4790) and β-actin (45 kDa; catalogue number 4970) were purchased from Cell Signaling Technology (Danvers, MA).

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

Journal: Phytomedicine Plus

Article Title: Berberine is as effective as the anti-obesity drug Orlistat in ameliorating betel-nut induced dyslipidemia and oxidative stress in mice

doi: 10.1016/j.phyplu.2021.100098

Figure Lengend Snippet: Fig. 6. Berberine and Orlistat restored phosphorylation of ACC at Ser-79 in AEBN-treated mice; a- histograms showing ratio of protein intensity of pACC (Ser-79) to total ACC in the liver of male and female mice; b- representative western blots immuno-probed for pACC (Ser-79) and total ACC with blots immuno-probed for β-actin serving as loading controls in the liver of male and female mice respectively. Data are expressed as mean ± SD of three indepen dent experiments. The p-values have been determined using one way ANOVA followed by Tukey’s Multiple Comparison Test; n = 30 mice per treatment group. Western blots are representative of 3 independent experiments.

Article Snippet: Polyvinyl Difluoride (PVDF) membrane was obtained from (Sigma Aldrich, St. Louis USA); antibodies against total and phosphorylated AMPK (Thr-172) (# 2603S and #2535S respectively) total and phosphorylated ACC (Ser-79) (# 3676S and #11818S respectively), FASN (#3180) and Beta- actin (# 4970S) were obtained from Cell Signaling Technology (Massachusetts, United States).

Techniques: Phospho-proteomics, Western Blot, Comparison