Journal: Diabetes & Metabolism Journal

Article Title: Lactate-Induced Lipid Accumulation in Hepatocytes through GPR81 Activation

doi: 10.4093/dmj.2024.0531

Figure Lengend Snippet: Lactate-induced lipid accumulation in hepatocytes in vitro . Alpha mouse liver 12 (AML12) cells were treated with different doses of sodium L-lactate for 4 days. (A) Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (B) Intracellular lipid is stained with Oil Red O. The upper images representative gross morphology of Oil Red O staining of AML12 cells. The below representative pictures of cells were taken by a microscope at 200× original magnification. Scale bar: 100 μm. (C) Quantification of intracellular triglyceride (TG) content. Statistical significance compared with control is indicated by a P <0.05, b P <0.01, c P <0.0001.

Article Snippet: The alpha mouse liver 12 (AML12) mouse hepatocyte cell line was procured from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 supplemented with 1X insulin-transferrin-selenium (ITS) supplement, 40 ng/mL dexamethasone, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.25 g/L glutamine, and 10% fetal bovine serum at 37°C with 95% air and 5% CO2.

Techniques: In Vitro, MTT Assay, Staining, Microscopy, Control

Journal: Diabetes & Metabolism Journal

Article Title: Lactate-Induced Lipid Accumulation in Hepatocytes through GPR81 Activation

doi: 10.4093/dmj.2024.0531

Figure Lengend Snippet: G-protein-coupled receptor 81 (GPR81) may regulate monocarboxylate transporter 1 (MCT1) expression in hepatocytes in vitro . (A) Alpha mouse liver 12 (AML12) cells were treated with sodium L-lactate 20 and 40 mM for different time course. Western blot analysis was performed to assess the expression levels of GPR81, MCT1, and MCT4 in AML12 cells. (B) AML12 cells were treated with small interfering RNA (siRNA) 50 nM with or without lactate 20 mM for 3 days. Western blot analysis was conducted to evaluate the expression levels of GPR81 and MCT1 in AML12 cells. The intensities of the bands in the Western blot images were quantified using Image Lab software and are displayed in the corresponding plot alongside the representative blot images. The protein levels were normalized to β-actin expression. Statistical significance compared with control is indicated by a P <0.05, b P <0.01, c P <0.001, lactate 20 mM treated group vs. control; d P <0.05, e P <0.01, f P <0.001, lactate 40 mM treated group vs. control; statistical significance compared with si-scramble is indicated by g P <0.05, h P <0.01, i P <0.001, with siGPR81 indicated j P <0.01.

Article Snippet: The alpha mouse liver 12 (AML12) mouse hepatocyte cell line was procured from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 supplemented with 1X insulin-transferrin-selenium (ITS) supplement, 40 ng/mL dexamethasone, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.25 g/L glutamine, and 10% fetal bovine serum at 37°C with 95% air and 5% CO2.

Techniques: Expressing, In Vitro, Western Blot, Small Interfering RNA, Software, Control

Journal: Diabetes & Metabolism Journal

Article Title: Lactate-Induced Lipid Accumulation in Hepatocytes through GPR81 Activation

doi: 10.4093/dmj.2024.0531

Figure Lengend Snippet: G-protein-coupled receptor 81 (GPR81) played a major role in regulating lipid accumulation in lactate-treated alpha mouse liver 12 (AML12) cells. (A) AML12 cells were treated with L-lactate at 20 mM with or without AZD3965 at 100 nM for 4 days. Lipid accumulation was evaluated using Oil Red O staining. (B) AML12 cells were treated with sodium L-lactate 20 and 40 mM for 3 days. The isolation of plasma membrane (PM) and cytosol fractions was performed, and the expression of GPR81 and monocarboxylate transporter 1 (MCT1) in AML12 cells was assessed. Na/K ATPase served as a housekeeping marker for the PM, while tubulin served as a housekeeping marker for the cytosol. (C) Immunofluorescence of MCT1 (red) and nuclei (4ʹ,6-diamidino2-phenylindole [DAPI] blue). Scale bar: 20 μm. (D) AML12 cells were treated with small interfering RNA (siRNA) 50 nM with or without lactate 20 mM for 4 days. The accumulation of lipids in AML12 cells was visualized using Oil Red O staining. Statistical significance compared with control is indicated by a P <0.05, b P <0.01. Statistical significance compared with si-scramble is indicated by c P <0.05, d P <0.001, with siGPR81 indicated e P <0.001.

Article Snippet: The alpha mouse liver 12 (AML12) mouse hepatocyte cell line was procured from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 supplemented with 1X insulin-transferrin-selenium (ITS) supplement, 40 ng/mL dexamethasone, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.25 g/L glutamine, and 10% fetal bovine serum at 37°C with 95% air and 5% CO2.

Techniques: Staining, Isolation, Clinical Proteomics, Membrane, Expressing, Marker, Immunofluorescence, Small Interfering RNA, Control

Journal: Diabetes & Metabolism Journal

Article Title: Lactate-Induced Lipid Accumulation in Hepatocytes through GPR81 Activation

doi: 10.4093/dmj.2024.0531

Figure Lengend Snippet: Lactate-induced G-protein-coupled receptor 81 (GPR81) activation promotes hepatocyte lipogenesis and fatty acid storage in vitro . (A) Alpha mouse liver 12 (AML12) cells were treated with sodium L-lactate 20 and 40 mM for 3 days. (B) AML12 cells were treated with small interfering RNA (siRNA) 50 nM with or without lactate 20 mM for 3 days. Cell lysates were then analyzed via Western blot to determine protein levels. Representative images of immunoblots of lipogenesis markers. β-Actin is a loading control. SREBP1c, sterol regulatory element-binding protein 1c; ACC, acetyl-CoA carboxylase; SCD1, stearoyl-CoA desaturase-1; FABP4, fatty acid binding protein 4; PPARα, peroxisome proliferator-activated receptor alpha; CPT1, carnitine palmitoyltransferase I; NS, not significant. Statistical significance compared with control is indicated by a P <0.05, b P <0.01, c P <0.0001. Statistical significance compared with si-scramble is indicated by d P <0.05, e P <0.01, with siGPR81 indicated f P <0.05, g P <0.01.

Article Snippet: The alpha mouse liver 12 (AML12) mouse hepatocyte cell line was procured from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 supplemented with 1X insulin-transferrin-selenium (ITS) supplement, 40 ng/mL dexamethasone, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.25 g/L glutamine, and 10% fetal bovine serum at 37°C with 95% air and 5% CO2.

Techniques: Activation Assay, In Vitro, Small Interfering RNA, Western Blot, Control, Binding Assay

Journal: Diabetes & Metabolism Journal

Article Title: Lactate-Induced Lipid Accumulation in Hepatocytes through GPR81 Activation

doi: 10.4093/dmj.2024.0531

Figure Lengend Snippet: Lactate-mediated G-protein-coupled receptor 81 (GPR81) activation regulates 5’ adenosine monophosphate-activated protein kinase (AMPK) in alpha mouse liver 12 (AML12) cells. (A) AML12 cells were treated with sodium L-lactate 20 and 40 mM for 3 days. (B) AML12 cells were treated with small interfering RNA (siRNA) 50 nM with or without lactate 20 mM for 4 days. Cell lysates were analyzed by Western blot to measure the protein levels of phosphorylation AMPK (p-AMPK) and AMPK. (C) AML12 cells were treated with lactate for 2 days and then co-treated with or without 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) 100 µM for next 2 days. Lipid accumulation was evaluated using Oil Red O staining. (D) AML12 cells were treated with lactate for 2 days and then co-treated with or without AICAR 100 µM for next 1 day. Representative images of immunoblots of mature form of sterol regulatory element-binding protein 1c (SREBP1c), CD36, and fatty acid binding protein 4 (FABP4). β-Actin or tubulin is a loading control. NS, not significant. Statistical significance compared with control is indicated by a P <0.05, b P <0.01, c P <0.0001. Statistical significance compared with si-scramble is indicated by d P <0.01, with siGPR81 indicated e P <0.01. Statistical significance compared with lactate 20 mM is indicated by f P <0.05, g P <0.01, h P <0.001.

Article Snippet: The alpha mouse liver 12 (AML12) mouse hepatocyte cell line was procured from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 supplemented with 1X insulin-transferrin-selenium (ITS) supplement, 40 ng/mL dexamethasone, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.25 g/L glutamine, and 10% fetal bovine serum at 37°C with 95% air and 5% CO2.

Techniques: Activation Assay, Small Interfering RNA, Western Blot, Phospho-proteomics, Staining, Binding Assay, Control

Journal: Diabetes & Metabolism Journal

Article Title: Lactate-Induced Lipid Accumulation in Hepatocytes through GPR81 Activation

doi: 10.4093/dmj.2024.0531

Figure Lengend Snippet: Lactate- induced glycolysis in alpha mouse liver 12 (AML12) cells and hepatic lipid accumulation zebrafish. (A) Seahorse analysis of extracellular acidification rate (ECAR), (B) non-glycolytic acidification, (C) glycolysis, (D) glycolytic capacity, and (E) glycolytic reserve were assessed in AML12 cells treated with sodium L-lactate 20 and 40 mM for 3 days. (F) To detect the hepatic response to lactate, we utilized selective fluorescent staining (Nile red) for intracellular lipid droplets in transgenic (Tg) (fabp10a: cyan fluorescent protein [CFP]) zebrafish larvae treated with or without lactate 10 mM. Figures are magnified as ×200. Quantitative analysis of the area of lipid droplet in liver based on Nile Red staining. (G) Lactate-induced lipid accumulation mostly in the liver not in muscle or adipose tissue in zebrafish model Nile red staining for intracellular lipid droplets in Tg (fabp10a: CFP) zebrafish larvae treated with or without lactate 10 mM. Scale bar indicated 100 µm. OD, optical density; 2-DG, 2-deoxy-d-glucose; DMSO, dimethyl sulfoxide; DA, dorsal aorta; L, liver; SB, swim bladder; SIA, supra-intestinal artery; VTA, vertebral artery. Statistical significance compared with control is indicated by a P <0.05, b P <0.01, c P <0.001, d P <0.0001.

Article Snippet: The alpha mouse liver 12 (AML12) mouse hepatocyte cell line was procured from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 supplemented with 1X insulin-transferrin-selenium (ITS) supplement, 40 ng/mL dexamethasone, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.25 g/L glutamine, and 10% fetal bovine serum at 37°C with 95% air and 5% CO2.

Techniques: Staining, Transgenic Assay, Control

Journal: Advanced Science

Article Title: Depletion of Hepatic SREBP2 Protects Against Hypercholesterolemia and Atherosclerosis through the ANGPTL3‐LPL Axis

doi: 10.1002/advs.202412677

Figure Lengend Snippet: Depletion of hepatic SREBP2 promotes Lipoprotein lipase activity in the absence of LDLR. A,B) Plasma lipoprotein lipase contents (A) and lipoprotein lipase activity (B) measured in heparinized C57BL/6J or Ldlr −/− male mice injected with AAV8‐scramble control or AAV8‐ Srebf2 ‐shRNA viruses under chow diet feeding ( n = 8 mice per group, by two‐way ANOVA). C,D) Plasma lipoprotein lipase contents (C) and lipoprotein lipase activity (D) measured in heparinized Ldlr −/− male mice injected with AAV8‐scramble control or AAV8‐ Srebf2 ‐shRNA viruses under 5‐week chow diet feeding followed by 3‐week western diet feeding ( n = 6–8 mice per group, by student's t test). E) Relative expression of the genes involved in de novo lipogenesis, cholesterol synthesis and LPL regulation measured by RNA‐seq using different CRISPR/Cas9‐mediated genetically modified AML12 mouse hepatocyte cell line ( n = 3 replicates for each cell line). F,G) qPCR expression analysis of genes modulating LPL activity in the liver of C57BL/6J (F) and Ldlr −/− (G) mice injected with AAV8‐scramble control or AAV8‐ Srebf2 ‐shRNA viruses under 5‐week chow followed by 5‐week western diet feeding ( n = 5–6 mice per group, by student's t test). H) qPCR expression analysis of ANGPTL3 , ANGPTL4 , and ANGPTL8 in SREBF2 knockout and control Huh7 human hepatoma cells ( n = 3 replicates for each cell line, representative of 3 independent experiments, by student's t test). I) qPCR expression analysis of ANGPTL3 , ANGPTL4 , and ANGPTL8 in SREBF2 knockout or control Huh7 human hepatoma cells lacking LDLR ( n = 3 replicates for each cell line, representative of 3 independent experiments, by student's t test). J–M) Western blot (J and L) and quantification (K and M) of ANGPTL3 (FL stands for full length) and ANGPTL8 in heparinized plasma of 4h‐fasted male C57BL6J mice (J and K, n = 7 mice per group, by t student's t test) and mice lacking LDLR (L and M, n = 6–8 mice per group, by student's t test) injected with AAV8‐scramble control or AAV8‐ Srebf2 ‐shRNA viruses fed chow. N,O) Western Blot of cellular (N) and secreted (O) ANGPTL3 protein in SREBF2 knockout and control LDLR +/+ and LDLR −/− Huh7 human hepatoma cells (representative of 3 independent experiments). P,Q) Western blot and relative quantification of ANGPTL3 in heparinized plasma of 4 h fasted male C57BL/6J mice and mice lacking LDLR injected with AAV8‐scramble control or AAV8‐ Srebf2 ‐shRNA viruses fed chow (3 representative mice of each group consisting of 6–8 mice, by one‐way ANOVA). * p < 0.05, ** p < 0.01, and *** p < 0.001; Error bars indicate mean ± SD.

Article Snippet: The AML12 (alpha mouse liver 12) cell line was established from hepatocytes obtained from a male mouse (CD1 strain, line MT42) transgenic for human TGF alpha (ATCC, ATCC CRL‐2254).

Techniques: Activity Assay, Clinical Proteomics, Injection, Control, shRNA, Western Blot, Expressing, RNA Sequencing, CRISPR, Genetically Modified, Knock-Out, Quantitative Proteomics