bcat inhibitor  (MedChemExpress)

Journal: Investigative Ophthalmology & Visual Science

Article Title: BCAT1 Activation Reprograms Branched-Chain Amino Acid Metabolism and Epigenetically Promotes Inflammation in Diabetic Retinopathy

doi: 10.1167/iovs.66.6.59

Figure Lengend Snippet: Bcat1 is inhibited by Plk4-mediated phosphorylation. ( A ) Flag–BCAT1 was overexpressed in rMC-1 cells. Cells were incubated either in normal glucose (5.5-mM) with mannitol (24.5-mM) as an osmotic control or in high glucose (30-mM). Flag–BCAT1 was immunoprecipitated for the enzymatic activity assay. Immunopurified protein was further subjected to western blotting to determine serine/threonine phosphorylation (p-S/T), tyrosine phosphorylation (p-Y), lysine acetylation (ace-K), and lysine methylation (me-K). Bcat1 activity was normalized to immunopurified Flag protein. ( B ) Volcano plot showing differentially expressed protein kinases between WT and db/db retinas. ( C ) Flag–BCAT1 was co-expressed with HA-tagged BMPR1B, MASTL, or PLK4 in rMC-1 cells. Flag–BCAT1 was immunoprecipitated. Its enzymatic activity was analyzed and normalized to Flag protein. p-S/T of Bcat1 and p-Bcat1 T333 was determined. ( D ) rMC-1 cells overexpressing Flag–BCAT1 was cultured either in normal glucose (5.5-mM) with mannitol (24.5-mM) as an osmotic control or in high glucose (30-mM). Flag–BCAT1 was immunoprecipitated. Plk4 protein and Bcat1 T333 phosphorylation levels were determined by western blotting. ( E ) Plk4 and Bcat1 protein expression and p-Bcat1 T333 in the retinas of WT and db/db mice were determined by western blotting. ( F ) The correlation between Bcat enzymatic activity and p-Bcat1 T333 levels from mouse retinas is shown. Pearson correlation was determined. ( G ) WT and db/db mice were treated with or without BAY-069. Retina vessels were stained with Evans Blue to visualize vascular leakage. White arrows indicate the vascular leakage. Scale bar : 1 mm. ( H ) The working model shows that Bcat1 activity is increased in diabetic retinopathy, which is negatively regulated by Plk4. Increased Bcat1 activity promotes BCAA catabolism, leading to a reduction in α-KG levels. α-KG reduction results in higher H3K4me3 levels and overexpression of Il6 , Tnf-α , and Gfap , accelerating diabetic retinopathy. All data are shown as mean ± SD from at least three independent experiments. * P < 0.05, ** P < 0.01; n.s., no significant change.

Article Snippet: Then, 1 μL of Bcat inhibitor (1 mmol/L, BAY-069, #HY-148242; MedChemExpress, Monmouth Junction, NJ, USA), 1 μL of Bcat1-specific inhibitor (100 μmol/L or 200 μmol/L, ERG240, #HY-W193545A; MedChemExpress), or PBS (mock) was intravitreally injected under general anesthesia with ketamine (80 mg/kg) and xylazine (4 mg/kg).

Techniques: Phospho-proteomics, Incubation, Control, Immunoprecipitation, Enzyme Activity Assay, Western Blot, Methylation, Activity Assay, Cell Culture, Expressing, Staining, Over Expression

Journal: Nature Communications

Article Title: Branched-chain α-ketoacids are preferentially reaminated and activate protein synthesis in the heart

doi: 10.1038/s41467-021-21962-2

Figure Lengend Snippet: a Simplified schematic figure diagraming the potential metabolic fates of [U- 13 C]KIV, as well as the effects of LY3351337 to inhibit BCAT activity, and BT2 to inhibit BDK. Label incorporation from first (red circles) and second (blue circles) passes through the TCA cycle are shown in dashed boxes. Measured metabolites in the TCA cycle are highlighted with a gray background and black dashed border. b The fractional percent labeling with 13 C is shown for the indicated metabolites; ( c ) The absolute amounts of 13 C-labeled valine, 3-HIB, citrate, and succinate are shown. Data in b–d are from hearts isolated from Wistar rats and perfused with [U- 13 C]KIV (100 μM) in the absence (Veh; n = 5; gray) or presence of the BDK inhibitor, BT2 ( n = 4; red) or the BCAT inhibitor, LY3351337 ( n = 6; blue). d Rate of reamination (nmol/min) of [U- 13 C]KIV to valine and rate of formation of 13 C-labeled 3-HIB from [U- 13 C]KIV in isolated hearts following treatment with LY3351337 or BT2. Data represent mean ± SEM. Statistical differences indicated by Tukey’s HSD post-hoc test following one-way ANOVA: * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Thirty minutes after a single injection of BCAT inhibitor, LY3351337 (10 mg/kg i.p., Eli Lilly Co.), or vehicle (DMSO, Sigma), male Wistar rats received an i.p. injection of [U- 13 C]KIV (100 mg/kg, Cambridge Isotopes), followed by blood sampling from the tail vein at 2, 5, 10, 15, 30, and 60 min.

Techniques: Activity Assay, Labeling, Isolation

Journal: Nature Communications

Article Title: Branched-chain α-ketoacids are preferentially reaminated and activate protein synthesis in the heart

doi: 10.1038/s41467-021-21962-2

Figure Lengend Snippet: Thirty minutes after a single injection of BCAT inhibitor, LY3351337 (10 mg/kg i.p.; n = 5; blue), or Veh (DMSO; n = 5; gray), Wistar rats received an i.p. injection of [U- 13 C]KIV (100 mg/kg), followed by blood sampling from the tail vein at 2, 5, 10, 15, 30, and 60 min. Fractional percent labeling with 13 C of ( a ) plasma KIV and ( b ) plasma valine. N = 5–6 per group. A separate cohort of Wistar rats was treated with LY3351337 (10 mg/kg i.p.) or vehicle and received an i.p. injection of [U- 13 C]KIV (100 mg/kg) 30 min later. Tissues were harvested 30 min after the [U- 13 C]KIV injection. c Fractional percent labeling with 13 C of valine in plasma and the indicated tissues in the absence (Veh; n = 8) or presence of LY3351337 ( n = 10). The dashed line indicates the plasma enrichment level. Data represent mean ± SEM. Statistical differences indicated by a two-way paired Student’s t -test: * P < 0.05, ** P < 0.005.

Article Snippet: Thirty minutes after a single injection of BCAT inhibitor, LY3351337 (10 mg/kg i.p., Eli Lilly Co.), or vehicle (DMSO, Sigma), male Wistar rats received an i.p. injection of [U- 13 C]KIV (100 mg/kg, Cambridge Isotopes), followed by blood sampling from the tail vein at 2, 5, 10, 15, 30, and 60 min.

Techniques: Injection, Sampling, Labeling, Clinical Proteomics