Branched-Chain Amino Acid Accumulation Fuels the Senescence-Associated Secretory Phenotype

Abstract

The essential branched-chain amino acids (BCAAs) leucine, isoleucine, and valine play critical roles in protein synthesis and energy metabolism. Despite their widespread use as nutritional supplements, BCAAs' full effects on mammalian physiology remain uncertain due to the complexities of BCAA metabolic regulation. Here a novel mechanism linking intrinsic alterations in BCAA metabolism is identified to cellular senescence and the senescence-associated secretory phenotype (SASP), both of which contribute to organismal aging and inflammation-related diseases. Altered BCAA metabolism driving the SASP is mediated by robust activation of the BCAA transporters Solute Carrier Family 6 Members 14 and 15 as well as downregulation of the catabolic enzyme BCAA transaminase 1 during onset of cellular senescence, leading to highly elevated intracellular BCAA levels in senescent cells. This, in turn, activates the mammalian target of rapamycin complex 1 (mTORC1) to establish the full SASP program. Transgenic Drosophila models further indicate that orthologous BCAA regulators are involved in the induction of cellular senescence and age-related phenotypes in flies, suggesting evolutionary conservation of this metabolic pathway during aging. Finally, experimentally blocking BCAA accumulation attenuates the inflammatory response in a mouse senescence model, highlighting the therapeutic potential of modulating BCAA metabolism for the treatment of age-related and inflammatory diseases.

Keywords: BCAA; SASP; age-related inflammation; mTORC1; senescence.

Figure 1

Altered BCAA metabolism in senescent cells. a) Altered amino acid metabolic pathways in senescent NHBE cells. b) Top differentially expressed genes in amino acid‐related metabolic pathways between senescent NHBE cells and proliferative cells. c) Immunoblots and d) RT‐qPCR showing increased SLC6A14 and decreased BCAT1 in senescent NHBE cells. Cells were induced to senescence by treatment with the EGFR inhibitor erlotinib (1 × 10^‐6 m). e,f) Increased SLC6A15 and reduced BCAT1 in oncogene‐induced senescence. IMR90 cells with Tet‐on HRasV12 were treated with doxycycline (1 µg mL^‐1) for 9 d. g,h) Increased SLC6A15 and reduced BCAT1 in DNA damage‐induced senescence. IMR90 cells were treated with 100 × 10^‐6 m etoposide for 24 h to induce DNA damage and then cultured for another 8 d. i,j) Increased SLC6A15 and reduced BCAT1 in replicative senescence. Early (passage doubling: 38) and late passage (passage doubling: 78) IMR90 cells were collected for immunoblotting and RT‐qPCR. d,f,h,j) n = 3, mean ± SD, two‐tailed Student's t‐test. *P < 0.05, **P < 0.05, ***P < 0.001. SLC6A14: solute carrier family 6 member 14; CLIC3: chloride intracellular channel 3; ASS1: argininosuccinate synthetase 1; OCA2: oculocutaneous albinism II; KMO: kynurenine 3‐monooxygenase; BCAT1: branched chain amino acid transaminase 1; CHAC1: ChaC glutathione specific gamma‐glutamylcyclotransferase 1; DDAH1: dimethylarginine dimethylaminohydrolase 1; PFAS: phosphoribosylformyl‐glycinamidine synthase; SLC39A8: solute carrier family 39 member 8; SLC6A15: solute carrier family 6 member 15; CDKN1A(p21): cyclin dependent kinase inhibitor 1A; CDKN2A(p16): cyclin dependent kinase inhibitor 2A; ACTB: actin beta; HRasV12: HRas proto‐oncogene with G12V mutation.

Figure 2

SLC6A15 and BCAT1 regulate the SASP. a,c) Immunoblots showing that a) SLC6A15 knockdown or c) BCAT1 overexpression impairs expression of IL6 and IL8 in oncogene‐induced senescence. IMR90 cells with Tet‐on HRasV12 were treated with doxycycline (1 µg mL^‐1) for 9 d to induce senescence. Cells were infected with lentivirus to express ShN, SLC6A15 shRNA, GFP, or BCAT1, as indicated. b) RT‐qPCR analysis showing that SLC6A15 knockdown inhibits IL6 and IL8 expression in oncogene‐induced senescence. n = 3, mean ± SD, one‐way ANOVA test with Dunnett's multiple comparisons, *P < 0.05, ****P < 0.0001. d) SLC6A15 knockdown or e) BCAT1 overexpression impairs the expression of IL6 and IL8 in DNA damage‐induced senescence. IMR90 cells were treated with 100 × 10^‐6 m etoposide for 24 h and cultured for 8 d before collecting samples. Cells were infected with lentivirus to express ShN, SLC6A15 shRNA, GFP, or BCAT1, as indicated. Eto.: Etoposide; IL6: interleukin 6; IL8: interleukin 8.

Figure 3

BCAA accumulation drives the SASP by activating mTORC1 signaling. BCAA metabolite assay showing that intracellular BCAA levels increase in a) replicative, b) oncogene‐induced, and c) DNA damage‐induced senescence. Relative BCAA levels were quantified by normalizing to cell number and then to the proliferative control for each condition. d) SLC6A14 or SLC6A15 overexpression increases intracellular BCAA levels in HEK293T cells. e) BCAT1 knockdown in HEK293T cells increases intracellular BCAA levels. a–e) n = 3, mean ± SD, a) two‐tailed Student's t‐test or b–e) one‐way ANOVA test with Dunnett's multiple comparisons. *P < 0.05, ***P < 0.001, ****P < 0.0001, ns, not significant. f) SLC6A15 knockdown impairs BCAA accumulation in senescent IMR90 cells. g) BCAT1 rescue impairs BCAA accumulation in senescent IMR90 cells. f,g) n = 3, mean ± SD, two‐way ANOVA test with Tukey's multiple comparisons. **P < 0.01, ****P < 0.0001. h) SLC6A15 knockdown or i) BCAT1 overexpression inhibits mTORC1 activation. j,k,m) mTORC1 activation via DEPDC5 knockdown rescues SASP production in j,m) SLC6A15‐depleted or k) BCAT‐overexpressing senescent cells. m) n = 3, mean ± SD, two‐way ANOVA test with Tukey's multiple comparisons. The symbol “#” indicates statistical significance (P < 0.05) compared to the HRasV12‐ShN‐ShN group. l) Schematic of BCAA‐dependent SASP induction. Increase amino acid transport through SLC6A14 or SLC6A15 together with reduced BCAT1‐mediated catabolism leads to BCAA accumulation in senescent cells. BCAA buildup then activates mTORC1 signaling to promote SASP factor production. RS: replicative senescence; p4EBP(T37/46): eukaryotic translation initiation factor 4E‐binding protein 1 phosphorylated at threonine 37 and/or threonine 46; 4EBP: eukaryotic translation initiation factor 4E‐binding protein 1; pS6RP(S240/244): S6 ribosomal protein phosphorylated at serine 240 and serine 244; S6RP: S6 ribosomal protein; DEPDC5: DEP domain–containing 5; IL1A: interleukin 1 alpha; IL1B: interleukin 1 beta; CXCL1: chemokine (C‐X‐C motif) ligand 1; CXCL2: chemokine (C‐X‐C motif) ligand 2.

Figure 4

BCAA metabolism regulates cellular senescence and age‐related phenotypes in Drosophila. a) Overexpression of dSlc6a15‐a or b) dSlc6a15‐b in transgenic fruit flies using the actin‐Gal4/UAS system. n = 3, mean ± SD, two‐tailed Student's t‐test. *P < 0.05, **P < 0.01. c) dSlc6a15‐a or dSlc6a15‐b overexpression shortens fly lifespans and d) impairs climbing ability in 4‐week‐old female flies. c) n = 70–75, log‐rank test, ***P < 0.001. d) n = 30–40, One‐way ANOVA, *P < 0.05, **** P < 0.0001. e–g) dSlc6a15‐a or dSlc6a15‐b overexpression induces expression of e) Drosophila dacapo (dp21), f) Upd2, and g) multiple antimicrobial peptides. n = 3, mean ± SD. SE: short exposure; LE: long exposure; Upd2: Unpaired 2; Attc: Attacin‐C; CecA1: Cecropin A1; CecA2: Cecropin A2; CecB: Cecropin B; Mtk: Metchnikowin; DptA: Diptericin A; DptB: Diptericin B; Dro: Drosocin.

Figure 5

BCAA inhibition abrogates SASP factor production and senescent cell immune clearance in mice. Schematic illustrations of a) sleeping‐beauty transposon plasmids and b) experimental design. c) RT‐qPCR analysis showing that BCAT1 overexpression and SLC6A15 knockdown reduce SASP factor expression and immune cell markers on day 6. Representative images and quantification of liver tissue immunohistochemical staining for NRasV12 (brown) and CD45 (red) expression on d) days 6 and e) 12 in GFP‐ShN control and BCAT1‐15KD groups, as indicated. c–e) n = 5, mean ± SD, two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. Scale bar = 100 µm. IR: inverted repeat; NRasV12: NRas proto‐oncogene with G12V mutation; EF1: elongation factor‐1 promoter; PGK: phosphoglycerate kinase promoter; U6: U6 promoter; Il6: interleukin 6; Il1a: interleukin 1 alpha; Il1b: interleukin 1 beta; Tnf: tumor necrosis factor; Ptprc: protein tyrosine phosphatase receptor type c; CD45: protein tyrosine phosphatase receptor type C.