Fig 2: Examination of genes related to BCAA and fatty acid metabolism in PDAC tissue. A–F: Data mining in GSE62165 dataset. (A) The expression level of SLC carriers (SLC7A5, SLC1A5, SLC3A2, SLC43A1 and SLC43A2) between PDAC and normal tissue. (B) GSEA analysis revealed enriched MYC and NOTCH signaling pathway in PDAC tissue. (C) The expression level of MYC and NOTCH (NOTCH1-4) between PDAC and normal tissue. (D) BCAA catabolism related genes BCAT1, BCAT2, BCKDHA, BCKDHB, BCKDK, ACADS and ACADSB were found significantly down-regulated in PDAC group. (E) The expression of lipogenesis related enzymes including FAS, ACACA and ACLY were significantly up-regulated in PDAC tissue. (F) CPT1A, the rate-limiting enzyme for fatty acid oxidation was notably upregulated in PDAC tissue. G–J: Data mining in GSE102238 dataset. (G) The expression level of SLC carriers (SLC7A5, SLC1A5, SLC3A2, SLC43A1 and SLC43A2) between PDAC and adjacent normal tissue. (H) NOTCH3 and NOTCH4 were upregulated in PDAC tissue while MYC was down-regulated. (I) Gene expression of BCAT1, BCAT2, BCKDHA, BCKDHB and ACADSB were decreased in PDAC tissue. (J) FAS and ACACA were increased in PDAC tissue.

Fig 3: Immunoblots of selected BCAA metabolic enzymes. (A, B) Immunoblots of Bcat2, Dbt (A) and Bckdha (B) in liver tissue samples from control mice and tumor tissue samples from model mice. Images of full-length immunoblots are shown in Supplementary Fig. 7. (C–E) Quantification ratios of Bcat2 (C), Dbt (D) and Bckdha (E) in the above tissues with Gapdh as a loading control (C = control, T = treatment, n = 4). *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 4: Regulation of BCAA degradation and ubiquitin‐dependent proteolysis in hypophysectomized rats. Overview and number of differentially expressed phosphoproteins (DEPPs) among WT, PR and rhGH groups (A); Heat map of DEPPs between the rhGH and PR groups (B). The unit of the colour gradient was shown by Z‐score. The gene set enrichment of DEPPs by KEGG between the rhGH and PR groups (C). Differences in the key enzymes related to BCAA degradation in liver tissues (D), including western blotting of BCKDHA and p‐BCKDHA (E); Western blotting of BCAT1, BCAT2 and MuRF1 in skeletal muscle among the different groups (F). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Fig 5: Bcat1 activation remodels BCAA catabolism in Müller cells during diabetic retinopathy (DR). (A) Relative BCAA levels in the vitreous humor were detected from DR patients (n = 26) and non-DR patients (n = 22). (B) Schematic overview of the reaction catalyzed by Bcats. (C) Protein expression of Bcat1 and Bcat2 in the retinas of WT and db/db mice were determined by western blotting. (D) Enzymatic activity of Bcats from WT and db/db mice was quantified and normalized to total protein as indicated. (E) A UMAP plot showing 10 distinct clusters of retina cells (left). Cell types are color coded. The distribution of cells expressing Bcat1 or Bcat2 is shown in panels in the middle and right, respectively. The circled area indicates Müller cells. (F) Representative immunostaining images of Bcat1 (green), Bcat2 (green), Gs (red), and Tom20 (red) of mouse retinas. Scale bar: 50 µm. (G) rMC-1 cells were incubated in either normal glucose (5.5-mM) with mannitol (24.5-mM) as an osmotic control or in high glucose (30-mM) for the indicated conditions. After transfecting siRNA against Bcat1 (#2) or scrambled control (scr), relative levels of α-KG were detected and normalized to cell numbers. (H) siRNA against Bcat1 (#2) or scr were transfected into rMC-1 cells. Cells were treated with normal glucose or high glucose as indicated. Relative levels of Leu, Ile, Val, and Glu in rMC-1 cells were determined and normalized to cell numbers. All data are shown as mean ± SD from at least three independent experiments. *P < 0.05, **P < 0.01; n.s., no significant change.