Journal: bioRxiv

Article Title: Branched-chain amino acid metabolism is a crucial modulator of cellular senescence

doi: 10.1101/2024.09.10.612139

Figure Lengend Snippet: (A) Schematic of BCAA metabolism. BCAT, BCAA aminotransferase; BCKDH, branched-chain α-ketoacid dehydrogenase; BCKDK, BCKDH kinase; α-KG, α-ketoglutarate; α-KIC, α-ketoisocaproic acid; α-KMV, α-ketomethylvaleric acid; α-KIV, α-ketoisovaleric acid; IV-CoA, isovaleryl-coenzyme A (CoA); MV-CoA, α-methylbutanoyl-CoA; IB-CoA, isobutyryl-CoA; BCKA, branched-chain α-ketoacid; BC-CoA, branched-chain acyl-CoA; BC-carnitine, branched-chain acyl-carnitine. (B, C) Suppression of BCAT2 expression in Dox-induced tetTRF2ΔB cells. IMR90-tetTRF2ΔB or IMR90-tetOne cell clones were incubated with Dox for the indicated times, and then subjected to immunoblotting (B) or RT-qPCR (C). β-Actin was used as a loading control (B). Data are normalized to β-actin, and the relative ratios to IMR90-tetOne clone #6 without Dox are presented (C). Bars represent the mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. ** p < 0.01 and *** p < 0.001, by two-way ANOVA with Dunnett’s test. (D) Decreased BCKA to BCAA ratio in Dox-treated tetTRF2ΔB cells. IMR90-tetTRF2ΔB and IMR90-tetOne cell clones were incubated with Dox for the indicated times, and then subjected to metabolome analysis. Ratios of keto-valine to valine (α-KIV/Val), and the combined ratio of keto-leucine and keto-isoleucine to the sum of leucine and isoleucine (α-KIC+α-KMV/Leu+Ile) are presented. Bars represent the mean relative ratios to IMR90-tetOne clone #6 from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. ** p < 0.01 and *** p < 0.001 by two-way ANOVA with Dunnett’s test. (E) Reduced KLF15 expression in Dox-treated tetTRF2ΔB cells. IMR90-tetTRF2ΔB and IMR90-tetOne cell clones were incubated with Dox for 9 days, and then subjected to RT-qPCR. Data are normalized to β-actin, and the ratios relative to tetOne clone #6 are presented. Bars represent the mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. *** p < 0.001, by one-way ANOVA with Dunnett’s test. (F, G) Regulation of BCAT2 expression by KLF15. (F) IMR90-hTERT cells were lentivirally introduced with two independent shRNAs for KLF15 (sh KLF15 #1, #2) and an shRNA control (shCon), and then subjected to RT-qPCR to detect BCAT2. (G) IMR90-tetTRF2ΔB cells infected lentivirally with the KLF15 expression vector (pLVX-KLF15) or empty vector (pLVX-empty) were incubated with Dox for 9 days, and then subjected to RT-qPCR to detect BCAT2. Data are normalized to β-actin, and the ratios relative to shCon (F) and to pLVX-empty without Dox (G) are presented. Bars represent the mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. ** p < 0.01 and *** p < 0.001, by one-way ANOVA with Dunnett’s test (F) and two-way ANOVA with Tukey’s test (G). NS, not significant.

Article Snippet: The lentiviral human BCAT2 expression plasmid using a pLVXneo backbone was designed and ordered from VectorBuilder (Chicago, IL, USA).

Techniques: Expressing, Clone Assay, Incubation, Western Blot, Quantitative RT-PCR, Control, shRNA, Infection, Plasmid Preparation

Journal: bioRxiv

Article Title: Branched-chain amino acid metabolism is a crucial modulator of cellular senescence

doi: 10.1101/2024.09.10.612139

Figure Lengend Snippet: (A) Immunoblot analysis of BCAT2 knockdown efficiency. IMR90-hTERT cells were lentivirally introduced with either of two independent shRNAs for BCAT2 (sh BCAT2 #1, #2) or the shRNA control (shCon), and then subjected to immunoblotting to detect the BCAT2 protein. β-actin was used as a loading control. (B) Inhibition of cell proliferation by BCAT2 knockdown. IMR90-hTERT cells lentivirally introduced with shCon or sh BCAT2 were incubated for the indicated times, and cumulative population doublings (PDs) were calculated. Complementation of BCAT2 was performed by lentiviral infection of BCAT2 cDNA expression vector (pLVX-BCAT2) before the introduction of sh BCAT2 #1 targeting the 3′-UTR (untranslated region) of BCAT2 mRNA (sh BCAT2 #1 + BCAT2). Data are presented as mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. ** p < 0.01 and *** p < 0.001, by two-way ANOVA with Tukey’s test. (C, D) Increase of SA-β Gal activity by BCAT2 knockdown. (C) Representative images of SA-β Gal staining of BCAT2 -knockdown cells. IMR90-hTERT cells lentivirally introduced with shCon or sh BCAT2 were incubated for 9 days after puromycin selection for shRNA-introduced cells, and then subjected to the SA-β Gal staining assay. Images on the right are magnified images of the boxed regions. Scale bars, 100 µm. (D) Quantification of the SA-β Gal-positive cells from the experiment shown in (C). Data are presented as mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. * p < 0.05, ** p < 0.01, and *** p < 0.001, by two-way ANOVA with Dunnett’s test. (E, F) Loss of lamin B1 in BCAT2 -knockdown cells. (E) Representative images of lamin B1 immunofluorescence (IF) of BCAT2 -knockdown cells. IMR90-hTERT cells were incubated for 9 days after shRNA introduction, and then subjected to IF to detect lamin B1. Scale bars, 50 µm. (F) Quantification of lamin B1 fluorescence intensity at the nuclear periphery, from the experiment shown in (E). *** p < 0.001; versus shCon, by one-way ANOVA with Dunnett’s test. (G, H) Increased expression of CDKN s and SASP-related genes in BCAT2 -knockdown cells. Total RNAs were isolated from IMR90-hTERT cells 9 days after shRNA introduction, and analyzed by RT-qPCR to detect CDKN2A ( p16 ), CDKN1A ( p21 ), IL6 , IL8 , CXCL1 , and CXCL2 mRNA. Data are normalized to β-actin, and the relative ratios to shCon are presented. Bars represent the mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. *** p < 0.001, by one-way ANOVA with Dunnett’s test. (I–L) Inhibition of cellular senescence by BCAT2 overexpression. (I) Immunoblot analysis of BCAT2 overexpression. IMR90-tetTRF2ΔB #2 cells were infected lentivirally with pLVX-BCAT2 or empty vector (pLVX-empty), and then subjected to immunoblotting to detect the BCAT2 protein. β-Actin was used as a loading control. (J) Recovery of cell growth arrest by BCAT2 overexpression in Dox-treated tetTRF2ΔB cells. IMR90-tetTRF2ΔB #2 cells infected lentivirally with pLVX-BCAT2 or pLVX-empty were incubated with Dox for the indicated times, and cumulative PDs were calculated. Data are presented as mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. *** p < 0.001, by two-way ANOVA with Tukey’s test. NS, not significant. (K) Representative images of SA-β Gal staining of BCAT2-overexpressing Dox-treated tetTRF2ΔB cells. IMR90-tetTRF2ΔB #2 cells infected lentivirally with pLVX-BCAT2 or pLVX-empty were incubated with Dox for 9 days, and then subjected to the SA-β Gal staining assay. Images at the bottom are magnified images of the boxed regions. Scale bars, 100 µm. (L) Quantification of SA-β Gal-positive cells in the experiment shown in (K). Data are presented as mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. *** p < 0.001, by two-way ANOVA with Tukey’s test. NS, not significant. (M) Abrogation of growth arrest by BC-carnitine supplementation in Dox-treated tetTRF2ΔB cells. IMR90-tetTRF2ΔB #2 cells were induced by Dox in a medium containing 30 µM BC-carnitines for the indicated times, and cumulative PDs were calculated. Data are presented as mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. *** p < 0.001, by two-way ANOVA with Dunnett’s test.

Article Snippet: The lentiviral human BCAT2 expression plasmid using a pLVXneo backbone was designed and ordered from VectorBuilder (Chicago, IL, USA).

Techniques: Western Blot, Knockdown, shRNA, Control, Inhibition, Incubation, Infection, Expressing, Plasmid Preparation, Activity Assay, Staining, Selection, Immunofluorescence, Fluorescence, Isolation, Quantitative RT-PCR, Over Expression

Journal: bioRxiv

Article Title: Branched-chain amino acid metabolism is a crucial modulator of cellular senescence

doi: 10.1101/2024.09.10.612139

Figure Lengend Snippet: (A,B) Increased BCAAs and decreased BCKA to BCAA ratios in BCAT2 -knockdown cells. Metabolites were extracted from IMR90-hTERT cells 3 days after lentiviral shRNA introduction, and then subjected to metabolome analysis. Bars represent the mean relative ratios to shCon from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. *** p < 0.001; versus shCon, by one-way ANOVA with Dunnett’s test. (C) Rapid inhibition of proliferation in BCAT2 -knockdown RPE1-hTERT cells. RPE1-hTERT cells lentivirally introduced with shCon or sh BCAT2 were incubated for the indicated times, and cumulative PDs were calculated. Data are presented as mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. *** p < 0.001, by two-way ANOVA with Dunnett’s test. (D) Inhibition of cell proliferation by a BCAT2 inhibitor. IMR90-hTERT cells were incubated with the BCAT2 inhibitor gabapentin (Gbp) at the indicated concentrations for the indicated times, and cumulative PDs were calculated. Data are presented as mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. * p < 0.05, *** p < 0.001, by two-way ANOVA with Tukey’s test. (E) Cell proliferation analysis in the presence of an excess amount of BCAA. IMR90-hTERT cells were incubated with the indicated concentrations of BCAAs for the indicated times, and cumulative PDs were calculated. Data are presented as mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. (F) Mitigated cell growth arrest by BCAA supplementation in Dox-treated tetTRF2ΔB cells. IMR90-tetTRF2ΔB #2 cells were induced by Dox in a medium containing the indicated concentrations of BCAAs for the indicated times, and cumulative PDs were calculated. Data are presented as mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. * p < 0.05, ** p < 0.01, *** p < 0.001, by two-way ANOVA with Tukey’s test.

Article Snippet: The lentiviral human BCAT2 expression plasmid using a pLVXneo backbone was designed and ordered from VectorBuilder (Chicago, IL, USA).

Techniques: Knockdown, shRNA, Inhibition, Incubation

Journal: bioRxiv

Article Title: Branched-chain amino acid metabolism is a crucial modulator of cellular senescence

doi: 10.1101/2024.09.10.612139

Figure Lengend Snippet: (A,B) Decreased intracellular glutamate and glutathione levels in BCAT2 -knockdown cells. Metabolites were extracted from IMR90-hTERT cells 3 days after lentiviral shRNA introduction, and then subjected to metabolome analysis. Bars represent the mean relative ratios to shCon from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. *** p < 0.001, by one-way ANOVA with Dunnett’s test. NS, not significant. (C, D) Increased intracellular ROS in BCAT2 -knockdown cells. (C) Representative images of intracellular ROS. IMR90-hTERT cells lentivirally introduced with shCon or shBCAT2 were incubated for 3 days with 5 mM GSH, 30 µM BC-carnitines, or both, and then subjected to the intracellular ROS detection assay using dihydroethidium (DHE). Oxidized DHE fluorescence images are presented. (D) Quantification of intracellular ROS in the experiment in (C). Red dots indicate mean values. Error bars indicate the standard error (SE). *** p < 0.001, by two-way ANOVA with Tukey’s test. (E) Abrogation of growth arrest by BC-carnitine supplementation and GSH in BCAT2 -knockdown cells. IMR90-hTERT cells introduced with shCon or shBCAT2 were incubated with 5 mM GSH, 30 µM BC-carnitines, or both for the indicated times, and cumulative PDs were calculated. Data are presented as mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. *** p < 0.001, by two-way ANOVA with Tukey’s test.

Article Snippet: The lentiviral human BCAT2 expression plasmid using a pLVXneo backbone was designed and ordered from VectorBuilder (Chicago, IL, USA).

Techniques: Knockdown, shRNA, Incubation, Detection Assay, Fluorescence

Journal: bioRxiv

Article Title: Branched-chain amino acid metabolism is a crucial modulator of cellular senescence

doi: 10.1101/2024.09.10.612139

Figure Lengend Snippet: (A) Proliferation of BCAT2 -knockdown cells incubated with NAC. IMR90-hTERT cells introduced with shCon or sh BCAT2 were incubated with or without 5 mM NAC, and cumulative PDs were calculated. Data are presented as mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. NS, not significant, by two-way ANOVA with Tukey’s test. (B, C) Intracellular cysteine and glycine levels in BCAT2 -knockdown cells. Metabolites were extracted from IMR90-hTERT cells 3 days after lentiviral shRNA introduction, and then measured by LC-MS. Metabolite levels were normalized to the total amount of all metabolites. The relative ratios to shCon are presented. Bars represent mean values from three independent experiments, and the dots represent the values from each experiment. Error bars indicate SD. NS, not significant, by one-way ANOVA with Dunnett’s test.

Article Snippet: The lentiviral human BCAT2 expression plasmid using a pLVXneo backbone was designed and ordered from VectorBuilder (Chicago, IL, USA).

Techniques: Knockdown, Incubation, shRNA, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Branched-chain amino acid metabolism is a crucial modulator of cellular senescence

doi: 10.1101/2024.09.10.612139

Figure Lengend Snippet: (A) Linear regression analysis between plasma BCAAs and mouse age. Pearson’s correlation coefficients and p -values are presented at the top of the plots. The blue lines represent regression lines. The shaded areas show the 95% confidence intervals (CIs). (B, C) Linear regression analysis of Bcat2 mRNA expression in mouse tissues. (B) Bcat2 read counts from the Tabula Muris Senis Bulk dataset were normalized by calculating counts per million (CPM) and subjected to linear regression analysis. The blue lines represent regression lines. The shaded areas show the 95% CIs. (C) Pearson’s coefficients and statistical values of the data in (B). Tissues indicated in magenta have a significantly negative correlation with age. (D, E) Deceased Bcat2 expression and BCKA production in adipose tissue of aged mice. Inguinal subcutaneous adipose tissues were collected from the indicated months-old normal mice. Data were normalized to β-actin (D) or the total amount of all metabolites (E), and the ratios relative to 3 months-old mice are presented. Data are presented as mean values, and the dots represent the values from each mouse (n = 5 (D), n = 10 (E)). Error bars indicate SE. * p < 0.05, by the unpaired Student t -test. (F) Proposed working model of cellular senescence regulation by BCAT2, and graphical summary of BCAT2 -KO in mice and age-dependent alterations in BCAA metabolism. BCAT2 mitigates cellular senescence through two distinct mechanisms: the catabolism of BCAAs and the maintenance of intracellular glutathione levels via the production of glutamate (left). The disruption of BCAT2 in mice results in the accumulation of senescent cells and subsequent tissue atrophy, particularly in skeletal muscle and adipose tissue. Additionally, aging downregulates BCAT2 expression, leading to alterations in BCAA catabolism in adipose tissue (right). The figure was created using BioRender ( https://biorender.com ).

Article Snippet: The lentiviral human BCAT2 expression plasmid using a pLVXneo backbone was designed and ordered from VectorBuilder (Chicago, IL, USA).

Techniques: Clinical Proteomics, Expressing, Disruption

Journal: bioRxiv

Article Title: Branched-chain amino acid metabolism is a crucial modulator of cellular senescence

doi: 10.1101/2024.09.10.612139

Figure Lengend Snippet: (A) Schematic showing the Bcat2 -KO strategy using CRISPR-Cas9. Two sgRNAs were designed upstream of exon 4 and downstream of exon 6 (indicated by scissors). F1, R1, and R2, primers for PCR genotyping; bp, base pairs; WT, wild type. (B, C) PCR and immunoblot analysis of the indicated mice, to verify the deletion of the endogenous Bcat2 allele (B) and protein (C), respectively. (D) Survival plots of Bcat2 -KO mice. (E) Changes in body weight of Bcat2 -KO mice. Error bars indicate SE. * p < 0.05, ** p < 0.01, and *** p < 0.001, versus Bcat2 +/+ mice, by two-way ANOVA with Dunnett’s test. (F) Normal lower limb reflexes in Bcat2 -KO mice. Representative pictures of the hindlimb clasping test of 10-week-old control and Bcat2 -KO mice to assess signs of neurological deficits.

Article Snippet: The lentiviral human BCAT2 expression plasmid using a pLVXneo backbone was designed and ordered from VectorBuilder (Chicago, IL, USA).

Techniques: CRISPR, Western Blot, Control

Journal: bioRxiv

Article Title: Branched-chain amino acid metabolism is a crucial modulator of cellular senescence

doi: 10.1101/2024.09.10.612139

Figure Lengend Snippet: (A–C) Skeletal muscle atrophy in Bcat2 -knockout (KO) mice. (A) Representative images showing skeletal muscles from age-matched control and Bcat2 -KO mice (13 weeks old). Quad: quadriceps muscle, Gas: gastrocnemius muscle, Sol: soleus muscle. Scale bar, 5 mm. (B) Representative hematoxylin and eosin (H&E) staining images of the gastrocnemius muscle from age-matched control and Bcat2 -KO mice (11 weeks old). Images at the bottom are magnified images of the boxed regions. Scale bar, 50 µm. (C) Quantification of myofiber size of the gastrocnemius muscle. Red dots indicate mean values. Error bars indicate SE. *** p < 0.001, by the unpaired Student t -test. (D, E) Decreased lamin B1 expression in the myocytes of Bcat2 -KO mice. (D) Representative images of lamin B1 immunofluorescence in the gastrocnemius muscle of age-matched control and Bcat2 -KO mice (11 weeks old). Magnified images of the boxed areas are also presented. Scale bar, 20 µm. BF, bright field. (E) Quantification of lamin B1 intensity in the experiment shown in (D). Red dots indicate mean values. Error bars indicate SE. *** p < 0.001, by the unpaired Student t -test. (F, G) Increased expression of CDKN and SASP-related genes in skeletal muscle of Bcat2 -KO mice. Total RNAs were isolated from the gastrocnemius muscle of age-matched control and Bcat2 -KO mice (10–13 weeks old). Data are normalized to β-actin, and the heatmaps of z-scores (F) and the relative ratio to Bcat2 +/+ mice (G) are presented. Data are presented as mean values, and the dots represent the values from each mouse (n = 5). Error bars indicate SE. * p < 0.05, ** p < 0.01, comparing between Bcat2 +/+ and Bcat2 −/− using the Wilcoxon rank sum test. (H–M) Atrophy and increased SA-β Gal activity in adipose tissues of Bcat2 -KO mice. (H) Representative images show adipose tissues from age-matched control and Bcat2 -KO mice (11 weeks old). Gonadal: gonadal adipose tissue, Inguinal: inguinal subcutaneous adipose tissue. Scale bar, 5 mm. (I) Representative histological images of SA-β Gal (blue staining) and H&E staining of gonadal adipose tissues from age-matched control and Bcat2 -KO mice (11 weeks old). The bottom images are magnified images of the boxed areas. Scale bar, 50 µm. (J, K) Representative macroscopic images of SA-β Gal staining of gonadal adipose tissue (J) and inguinal subcutaneous adipose tissue (K) from age-matched control and Bcat2 -KO mice (11 weeks old). (L) Quantification of adipocyte size of the gonadal adipose tissues. Red dots indicate mean values. Error bars indicate SE. *** p < 0.001, by the unpaired Student t -test. (M) Quantification of SA-β Gal positive adipocytes in the experiment shown in (I). Bars represent the mean values, and the dots represent the values from each mouse (n = 4, 10–11 weeks old). Error bars indicate SE. *** p < 0.001, by the unpaired Student t -test.

Article Snippet: The lentiviral human BCAT2 expression plasmid using a pLVXneo backbone was designed and ordered from VectorBuilder (Chicago, IL, USA).

Techniques: Knock-Out, Muscles, Control, Staining, Expressing, Immunofluorescence, Isolation, Activity Assay

Journal: bioRxiv

Article Title: Branched-chain amino acid metabolism is a crucial modulator of cellular senescence

doi: 10.1101/2024.09.10.612139

Figure Lengend Snippet: (A–C) Increased expression levels of SASP-related genes in Bcat2 -KO mice. Total RNAs were isolated from gastrocnemius muscles of 10 to 13-week-old mice (A) and gonadal adipose tissues of 10 to 11-week-old mice (B, C), shown in and . Data are normalized to β-actin, and the relative ratios to the levels of Bcat2 +/+ mice (A, C), and heatmaps of the z-scores (B) are presented. Data are presented as mean values, and the dots represent the values from each mouse (n = 5). Error bars indicate SE. * p < 0.05, ** p < 0.01, comparing between Bcat2 +/+ and Bcat2 −/− using the Wilcoxon rank sum test. (D) Increased SA-β Gal activity in adipocytes from Bcat2 -KO mice. Representative histological images of SA β-Gal (blue) staining with eosin counterstaining (pink) of gonadal adipose tissues from age-matched control and Bcat2 -KO mice (11 weeks old). The bottom images are magnified images of the boxed regions. Scale bar, 50 µm.

Article Snippet: The lentiviral human BCAT2 expression plasmid using a pLVXneo backbone was designed and ordered from VectorBuilder (Chicago, IL, USA).

Techniques: Expressing, Isolation, Muscles, Activity Assay, Staining, Control