Journal: Frontiers in Immunology

Article Title: Branched-chain ketoacids derived from cancer cells modulate macrophage polarization and metabolic reprogramming

doi: 10.3389/fimmu.2022.966158

Figure Lengend Snippet: Cancer cells consume BCAAs and secrete BCKAs. (A) , BCKAs (KIV, KIC and KMV) in the DMEM complete medium or conditioned media (CM) from indicated cells were detected by GC-MS. (B) , Representative mass spectra of KIV, KIC and KMV by GC-MS. (C) , BCAAs (leucine, isoleucine and valine) in the DMEM complete medium or conditioned media from indicated cells were detected by GC-MS. (D) , Diagram of workflow. Conditioned media from cancer cells and three BCKAs were added to BMDM or iBMDM, followed by multiomics analysis. Data in A and C show the mean ± SEM of n = 3 technical experiments.

Article Snippet: KIV (Cat: HY-W006057) and KMV (Cat: HY-113063) were purchased from MedChemExpress.

Techniques: Gas Chromatography-Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: Branched-chain ketoacids derived from cancer cells modulate macrophage polarization and metabolic reprogramming

doi: 10.3389/fimmu.2022.966158

Figure Lengend Snippet: BCKAs in cancer cell CM induce murine BMDM polarization. (A) , Relative proliferation rate of BMDM grown in DMEM complete medium or indicated cancer cell CM. (B) , Relative proliferation rate of BMDM grown in DMEM complete media or equivalent media containing 200 μM KIV or 200 μM KIC or 200 μM KMV (n = 3). (C) , mRNA expression of Arg1 , Mrc1 , Mgl1 and Mgl2 in BMDM was measured by qPCR after incubation with AE17 CM for 24 hours. (D) , ARG1 protein level was measured by western blot after incubation with AE17 CM for 24 hours. (E) , mRNA expression of Arg1 , Mrc1 , Mgl1 , Mgl2 , Vegf , Il1β , Il6 , Nos2 and Slc16a1 was measured in BMDM after stimulation with DMEM (Control), 200 μM KIV, 200 μM KIC and 200 μM KMV, respectively for 24 hours. (F) , Cytokines TGF-β, TNF-α, IL6 and growth factor VEGF in BMDMs after the indicated treatments were measured by ELISA kits. (G) , mRNA expression of Arg1 and Vegf was measured after pretreatment with the MCT1 inhibitor 250 nM AZD3965 for 1 hour, and stimulation with 200 μM KIV, 200 μM KIC and 200 μM KMV, respectively for 24 hours. Data show the mean ± SEM of n = 3 biological experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired two-tailed t test).

Article Snippet: KIV (Cat: HY-W006057) and KMV (Cat: HY-113063) were purchased from MedChemExpress.

Techniques: Expressing, Incubation, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Branched-chain ketoacids derived from cancer cells modulate macrophage polarization and metabolic reprogramming

doi: 10.3389/fimmu.2022.966158

Figure Lengend Snippet: BCKAs stimulation enhance TCA cycle and polyamine metabolism in macrophages. (A) , PCA analysis of total identified metabolites among DMEM, KIV, KMV and KIC treatment at two time points (2 h and 24 h) is shown. (B) , The hierarchal clustering heatmap of identified metabolites upon DMEM and three BCKAs treatment at two time points (2 h and 24 h) is shown. The rows indicate different metabolites, and the columns indicate different conditions. The log-transformed metabolite intensities (normalized to internal standards) were scaled by autoscaling (mean-centering/standard deviation). (C) , Schematic representation of the TCA cycle, the corresponding metabolite is shown. (D) , Schematic representation of the arginine metabolism at 24 h, the corresponding metabolites from (B) are shown. PCA (A) and hierarchical clustering (B) analysis were applied via online tool MetaboAnalyst. Data shown the mean ± SEM of n = 3 or 4 biological experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired two-tailed t test) (unpaired two-tailed t test).

Article Snippet: KIV (Cat: HY-W006057) and KMV (Cat: HY-113063) were purchased from MedChemExpress.

Techniques: Transformation Assay, Standard Deviation, Two Tailed Test

Journal: Molecular Biology Reports

Article Title: Branched-chain amino acids and their metabolites decrease human and rat hepatic stellate cell activation

doi: 10.1007/s11033-024-10027-4

Figure Lengend Snippet: Effect of BCAAs and BCKAs on activation markers in rHSCs cultured with BCAAs from day 1 to 7. Panel A shows the protein expression of Collagen type 1 and α-SMA, given as the integrated density of the protein band divided by the integrated density of α-tubulin (used as a housekeeping protein), and then normalized to the control sample, in samples from rHSCs at day 7 after treatment with BCAAs and BCKAs as indicated. Panel B corresponds to the gene expression levels of Col1a1 and Acta2, as delta-delta Ct with the housekeeping gene 36B4 in rHSCs at day 7. Panel C illustrates the cell proliferation in rHSCs treated with leucine, valine, isoleucine, KIC, KIV, KMV (blue lines), without any treatment (black line), or the control without cells (orange line). Panel D shows a representative immunofluorescence staining of Collagen type 1 (green) and α-SMA (red) under the indicated conditions. α-SMA alpha-smooth muscle actin, BCAAs branched-chain amino acids, BCKAs Branched-chain Keto Acids, Ile Isoleucine, KIC α-Ketoisocaproate, KIV α-Ketoisovalerate, KMV α-Keto-β-methylvalerate, Leu Leucine, NT Non-treatment, Val Valine. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Figure created with Biorender.com

Article Snippet: Regarding the experiments with BCKAs, both quiescent and activated rHSCs, as well as hHSCs from cirrhotic and non-cirrhotic livers, were treated with KIC (H60076.03, Thermo Fisher Scientific, Waltham, MA, USA), KMV (189,720,010, Thermo Fisher Scientific), KIV (198,978, Sigma-Aldrich), or their combination in a 1:1:1 ratio (BCKAs) at a concentration of 2 mM in culture medium.

Techniques: Activation Assay, Cell Culture, Expressing, Control, Immunofluorescence, Staining

Journal: Molecular Biology Reports

Article Title: Branched-chain amino acids and their metabolites decrease human and rat hepatic stellate cell activation

doi: 10.1007/s11033-024-10027-4

Figure Lengend Snippet: Effects of BCAAs and BCKAs on activation markers in rHSCs cultured with BCAAs from day 7 to 11. Panel A shows the protein expression of Collagen type 1 and α-SMA, given as the integrated density of the protein band divided by the integrated density of α-tubulin (used as a housekeeping protein), and then normalized to the control sample, in samples from rHSCs at day 11 after 4 days of treatment with BCAAs and BCKAs as indicated. Panel B shows the gene expression levels of Col1a1 and Acta2 , indicated as delta-delta Ct with the housekeeping gene 36b4 , under the indicated treatment conditions in rHSCs at day 11. Panel C indicates a representative immunofluorescence staining of Collagen type 1 (green) and α-SMA (red) under the indicated conditions. α-SMA alpha-smooth muscle actin, BCAAs Branched-chain Amino Acids, BCKAs Branched-chain Keto Acids, Ile Isoleucine, KIC α-Ketoisocaproate, KIV α-Ketoisovalerate, KMV α-Keto-β-methylvalerate, Leu Leucine, NT non-treatment, Val Valine. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Figure created with Biorender.com

Article Snippet: Regarding the experiments with BCKAs, both quiescent and activated rHSCs, as well as hHSCs from cirrhotic and non-cirrhotic livers, were treated with KIC (H60076.03, Thermo Fisher Scientific, Waltham, MA, USA), KMV (189,720,010, Thermo Fisher Scientific), KIV (198,978, Sigma-Aldrich), or their combination in a 1:1:1 ratio (BCKAs) at a concentration of 2 mM in culture medium.

Techniques: Activation Assay, Cell Culture, Expressing, Control, Immunofluorescence, Staining

Journal: Molecular Biology Reports

Article Title: Branched-chain amino acids and their metabolites decrease human and rat hepatic stellate cell activation

doi: 10.1007/s11033-024-10027-4

Figure Lengend Snippet: Protein expression of BCAT and BCKDH in human HSCs from cirrhotic and non-cirrhotic livers. Panel A illustrates the protein expression of BCAT and BCKDH in human HSCs isolated from cirrhotic livers (blue bars), treated with BCAAs or BCKAs as indicated. Panel B shows the protein expression of BCAT and BCKDH in human HSCs from non-cirrhotic livers (orange bars), also treated with BCAAs or BCKAs as indicated. BCAAs branched-chain amino acids, BCAT branched-chain amino acid transaminase, BCKAs branched-chain keto acids, BCKDH branched-chain keto acid dehydrogenase, Ile isoleucine, KIC α-ketoisocaproate, KIV α-ketoisovalerate, KMV α-keto-β-methylvalerate, Leu leucine, Val valine. * p < 0.05; ** p < 0.01; *** p < 0.001. Figure created with Biorender.com

Article Snippet: Regarding the experiments with BCKAs, both quiescent and activated rHSCs, as well as hHSCs from cirrhotic and non-cirrhotic livers, were treated with KIC (H60076.03, Thermo Fisher Scientific, Waltham, MA, USA), KMV (189,720,010, Thermo Fisher Scientific), KIV (198,978, Sigma-Aldrich), or their combination in a 1:1:1 ratio (BCKAs) at a concentration of 2 mM in culture medium.

Techniques: Expressing, Isolation

Journal: Molecular Biology Reports

Article Title: Branched-chain amino acids and their metabolites decrease human and rat hepatic stellate cell activation

doi: 10.1007/s11033-024-10027-4

Figure Lengend Snippet: Effect of BCAAs and BCKAs on activation markers in hHSCs from cirrhotic tissue, cultured with BCAAs and BCKAs from day 1 to 7. Panel A shows the protein expression of Collagen type 1 and α-SMA, given as the integrated density of the protein band divided by the integrated density of α-tubulin (used as a housekeeping protein), and then normalized to the control sample when treating the cells with BCAAs and BCKAs as indicated. Panel B shows the gene expression levels of COL1A1 and ACTA2 , indicated as delta-delta Ct with the housekeeping gene 18S , under the indicated treatment conditions. Panel C indicates the cell proliferation in hHSCs treated with leucine, valine, or isoleucine (continued blue lines), KIC, KIV, or KMV (intermittent blue lines), without any treatment (black line), or the controls without cells (orange line). Panel D shows the immunofluorescence staining of Collagen type 1 (green) and α-SMA (red) under the indicated conditions. α-SMA alpha-smooth muscle actin, BCAAs branched-chain amino acids, BCKAs branched-chain keto acids, Ile isoleucine; KIC α-ketoisocaproate, KIV α-ketoisovalerate, KMV α-keto-β-methylvalerate, Leu leucine, NT non-treatment, Val valine. * p < 0.05; ** p < 0.01; *** p < 0.001. Figure created with Biorender.com

Article Snippet: Regarding the experiments with BCKAs, both quiescent and activated rHSCs, as well as hHSCs from cirrhotic and non-cirrhotic livers, were treated with KIC (H60076.03, Thermo Fisher Scientific, Waltham, MA, USA), KMV (189,720,010, Thermo Fisher Scientific), KIV (198,978, Sigma-Aldrich), or their combination in a 1:1:1 ratio (BCKAs) at a concentration of 2 mM in culture medium.

Techniques: Activation Assay, Cell Culture, Expressing, Control, Immunofluorescence, Staining

Journal: Molecular Biology Reports

Article Title: Branched-chain amino acids and their metabolites decrease human and rat hepatic stellate cell activation

doi: 10.1007/s11033-024-10027-4

Figure Lengend Snippet: Effect of BCAAs and BCKAs on activation markers in hHSCs from non-cirrhotic tissue, cultured with BCAAs and BCKAs from day 7 to 11. Panel A shows the protein expression of Collagen type 1 and α-SMA, given as the integrated density of the protein band divided by the integrated density of α-tubulin (used as a housekeeping protein), and then normalized to the control sample, when treating the cells with BCAAs and BCKAs as indicated. Panel B shows the gene expression levels of COL1A1 and ACTA2 , indicated as delta-delta Ct with the housekeeping gene 18S , under the indicated treatment conditions. Panel C indicates the immunofluorescence staining of Collagen type 1 (green) and α-SMA (red) under the indicated conditions. α-SMA alpha-smooth muscle actin, BCAAs branched-chain amino acids, BCKAs branched-chain keto acids, Ile isoleucine, KIC α-ketoisocaproate, KIV α-ketoisovalerate, KMV α-keto-β-methylvalerate, Leu leucine, NT non-treatment, Val valine. * p < 0.05; ** p < 0.01. Figure created with Biorender.com

Article Snippet: Regarding the experiments with BCKAs, both quiescent and activated rHSCs, as well as hHSCs from cirrhotic and non-cirrhotic livers, were treated with KIC (H60076.03, Thermo Fisher Scientific, Waltham, MA, USA), KMV (189,720,010, Thermo Fisher Scientific), KIV (198,978, Sigma-Aldrich), or their combination in a 1:1:1 ratio (BCKAs) at a concentration of 2 mM in culture medium.

Techniques: Activation Assay, Cell Culture, Expressing, Control, Immunofluorescence, Staining