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: Antibodies for GPR81 (MBS821939, My BioSource, San Diego, CA, USA), MCT1 (sc-365501, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MCT4 (sc-376140, Santa Cruz Biotechnology), β-actin (sc-47778, Santa Cruz Biotechnology), phosphorylation 5’ adenosine monophosphate-activated protein kinase (p-AMPK) (Thr172 #2535, Cell Signaling Technology, Danvers, MA, USA), AMPK (#2532, Cell Signaling Technology), peroxisome proliferator-activated receptor alpha (PPARα) (sc-398394 Santa Cruz Biotechnology), sterol regulatory element-binding protein 1 (SREBP1) (NB100-2215, Novus Biologicals, Centennial, CO, USA), stearoyl-CoA desaturase-1 (SCD1) (#2794, Cell Signaling Technology), acetyl-CoA carboxylase (ACC) (#3676, Cell Signaling Technology), and α-tubulin (#12351, Cell Signaling Technology) were used in this study.

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: Antibodies for GPR81 (MBS821939, My BioSource, San Diego, CA, USA), MCT1 (sc-365501, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MCT4 (sc-376140, Santa Cruz Biotechnology), β-actin (sc-47778, Santa Cruz Biotechnology), phosphorylation 5’ adenosine monophosphate-activated protein kinase (p-AMPK) (Thr172 #2535, Cell Signaling Technology, Danvers, MA, USA), AMPK (#2532, Cell Signaling Technology), peroxisome proliferator-activated receptor alpha (PPARα) (sc-398394 Santa Cruz Biotechnology), sterol regulatory element-binding protein 1 (SREBP1) (NB100-2215, Novus Biologicals, Centennial, CO, USA), stearoyl-CoA desaturase-1 (SCD1) (#2794, Cell Signaling Technology), acetyl-CoA carboxylase (ACC) (#3676, Cell Signaling Technology), and α-tubulin (#12351, Cell Signaling Technology) were used in this study.

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: Antibodies for GPR81 (MBS821939, My BioSource, San Diego, CA, USA), MCT1 (sc-365501, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MCT4 (sc-376140, Santa Cruz Biotechnology), β-actin (sc-47778, Santa Cruz Biotechnology), phosphorylation 5’ adenosine monophosphate-activated protein kinase (p-AMPK) (Thr172 #2535, Cell Signaling Technology, Danvers, MA, USA), AMPK (#2532, Cell Signaling Technology), peroxisome proliferator-activated receptor alpha (PPARα) (sc-398394 Santa Cruz Biotechnology), sterol regulatory element-binding protein 1 (SREBP1) (NB100-2215, Novus Biologicals, Centennial, CO, USA), stearoyl-CoA desaturase-1 (SCD1) (#2794, Cell Signaling Technology), acetyl-CoA carboxylase (ACC) (#3676, Cell Signaling Technology), and α-tubulin (#12351, Cell Signaling Technology) were used in this study.

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: Antibodies for GPR81 (MBS821939, My BioSource, San Diego, CA, USA), MCT1 (sc-365501, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MCT4 (sc-376140, Santa Cruz Biotechnology), β-actin (sc-47778, Santa Cruz Biotechnology), phosphorylation 5’ adenosine monophosphate-activated protein kinase (p-AMPK) (Thr172 #2535, Cell Signaling Technology, Danvers, MA, USA), AMPK (#2532, Cell Signaling Technology), peroxisome proliferator-activated receptor alpha (PPARα) (sc-398394 Santa Cruz Biotechnology), sterol regulatory element-binding protein 1 (SREBP1) (NB100-2215, Novus Biologicals, Centennial, CO, USA), stearoyl-CoA desaturase-1 (SCD1) (#2794, Cell Signaling Technology), acetyl-CoA carboxylase (ACC) (#3676, Cell Signaling Technology), and α-tubulin (#12351, Cell Signaling Technology) were used in this study.

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