Tissue‐Specific Effects of Dietary Protein on Cellular Senescence Are Mediated by Branched‐Chain Amino Acids

All procedures were performed in conformance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) of the William S. Middleton Memorial Veterans Hospital and the University of Wisconsin‐Madison IACUC. Male C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) at 11 weeks of age. p16‐3MR reporter mice on the C57BL/6J background, in which the p16^INK4a promoter drives expression of the 3MR fusion protein, (Demaria et al. 2014) were received at 13 weeks of age from the Harris and Ricke labs at UW‐Madison, which were generously gifted to them by Dr. Judith Campisi. All mice were acclimated to the animal research facility for at least 1 week before entering studies. All animals were housed in static microisolator cages in a specific pathogen‐free mouse facility with a 12:12 h light–dark cycle, maintained at approximately 22°C.

Mice were fed amino acid‐defined diets with varying levels of protein and BCAAs (full diet compositions are provided in Table; Inotiv, Madison, WI, USA). Diets were started at either 12 weeks of age or 16 months of age in the C57BL/6J mice, or at 14 weeks of age in the p16‐3MR mice, and continued for at least 16 weeks. 8‐week‐old male C57BL/6J mice were placed on a high‐fat high‐sucrose western diet (WD; TD.160186) or a WD with restricted BCAAs (WD‐BR; TD.160188) for 45 weeks. S1

Glucose, insulin, and alanine tolerance tests were performed by fasting all mice for 4 h or overnight (~16 h) and then injecting either glucose (1 g/kg), insulin (0.75 U/kg) or pyruvate (2 g/kg) intraperitoneally (Bellantuono et al. 2020; Yu et al. 2019).

Blood glucose levels were determined at the indicated times using a Bayer Contour blood glucose meter (Bayer, Leverkusen, Germany) and test strips. Mouse body composition was determined using an EchoMRI Body Composition Analyzer. For the assay of multiple metabolic parameters (O₂, CO₂, food consumption, and activity tracking), mice were acclimatized to housing in a Columbus Instruments Oxymax/CLAMS‐HC metabolic chamber system for approximately 24 h, and data from a continuous 24‐h period was then recorded and analyzed.

In vivo luminescence assessment was performed by the Small Animal Imaging & Radiotherapy Facility (SAIRF). p16‐3MR reporter mice were injected intraperitoneally with 15 μg of RediJect Coelenterazine h Bioluminescent Substrate (Fischer Scientific; Catalog no. 50‐209‐9325). 15 min later, the mice were anesthetized with isoflurane, and luminescence was measured with a Revvity IVIS Spectrum in vivo imaging system (Revvity Health Sciences; 1 min medium binning).

Mice were euthanized in the fasted or fed state at the indicated age. Mice euthanized in the fed state were fasted overnight starting the day prior to sacrifice; in the morning, mice were refed for 3 h and then sacrificed 3 h later. Following blood collection via submandibular bleeding, mice were euthanized by cervical dislocation, and tissues were rapidly collected, weighed, and snap frozen in liquid nitrogen. A portion of the liver was fixed in 10% formalin for 4 h, transferred to sucrose for 24 h, and then embedded in Tissue‐Tek Optimal Cutting Temperature (OCT) compound, and then cryosectioned and stained for SA‐β‐gal activity and immunofluorescence staining for gamma‐H2AX. Images of the liver were taken using an EVOS microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA) at a magnification of 40X as previously described (Calubag et al. 2022; Cummings et al. 2018; Yu et al. 2021). For quantification, three independent fields were obtained for each tissue from each mouse and quantified using ImageJ (NIH, Bethesda, MD, USA).

AML12 cells were obtained from American Type Culture Collection (ATCC). AML12 cells were cultured in Gibco Dulbecco's modified Eagle's medium with F12 (DMEM/F12) without Amino Acids, Glucose, L‐Glutamine, Sodium Bicarbonate, HEPES, Sodium Pyruvate, Hypoxanthine, Thymidine, Phenol Red (Powder) (D9807‐10; US Biological Life Sciences, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (12306C; Life Technologies, Carlsbad, CA, USA) and 1% Penicillin/Streptomycin (15140122; Gibco, Billings, MT, USA). Amino acids and glucose were added to create custom DMEM lacking BCAAs. See Table S5 for amino acid and complete cell culture composition and catalog numbers. Senescence was induced in AML12 cells by treating them with 1 μM Antimycin A (A8674‐25MG; Sigma, St. Louis, MO, USA) for 14–21 days prior to collection (concentration chosen based on (Hytti et al. 2019; Stöckl et al. 2006; Wiley et al. 2016)).

RNA was extracted from liver or inguinal white adipose tissue (iWAT) using TRI Reagent according to the manufacturer's protocol (Sigma‐Aldrich). The concentration and purity of RNA were determined by absorbance at 260/280 nm using Nanodrop (Thermo Fisher Scientific). 1 μg of RNA was used to generate cDNA (Superscript III; Invitrogen, Carlsbad, CA, USA). Oligo dT primers and primers for real‐time PCR were obtained from Integrated DNA Technologies (IDT, Coralville, IA, USA); sequences are in Table. Reactions were run on a StepOne Plus machine (Applied Biosystems, Foster City, CA, USA) with Sybr Green PCR Master Mix (Invitrogen). If our technical replicates have a difference in Ct larger than 1, we excluded the sample entirely and did not do further analysis. Actin was used to normalize the results from gene‐specific reactions. S2

Animals used for Western blotting were sacrificed following an overnight fast and 3‐h refeed. Tissue samples from muscle were lysed in cold RIPA buffer supplemented with phosphatase inhibitor and protease inhibitor cocktail tablets (Thermo Fisher Scientific, Waltham, MA, USA) as previously described (Pak et al. 2021; Richardson et al. 2021) using a FastPrep 24 (M.P. Biomedicals, Santa Ana, CA, USA) with screw cap microcentrifuge tubes (822‐S) from (Dot Scientific, Burton, MI) and ceramic oxide bulk beads (10158–552) from VWR (Radnor, PA, USA). Protein lysates were then centrifuged at 13,300 rpm for 10 min, and the supernatant was collected. Protein concentration was determined by Bradford (Pierce Biotechnology, Waltham, MA, USA). 20 μg protein was separated by SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) on 16% resolving gels (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to PVDF membrane (EMD Millipore, Burlington, MA, USA). pT389‐S6K1 (9234), S6K1 (9202), pS240/244‐S6 (2215), S6 (2217), p‐Thr37/46 4E‐BP1 (2855), and 4E‐BP1 (9644) were purchased from Cell Signaling Technologies (CST, Danvers, MA, USA) and used at a dilution of 1:1000. Imaging was performed using a Bio‐Rad Chemidoc MP imaging station (Bio‐Rad, Hercules, CA, USA). Quantification was performed by densitometry using NIH ImageJ software.

Enhanced lysosomal biogenesis, a common feature of cellular senescence, can be detected by measuring β‐galactosidase (lysosomal hydrolase) activity at pH 6.0 (Lee et al. 2006). This senescence‐associated β‐galactosidase (SA‐β‐Gal) activity appears to be restricted to senescent cells at a low pH (Dimri et al. 1995; Zhao et al. 2017). SA‐β‐Gal activity is shared by almost all senescent cells, but particular care should be taken when detecting this in cultured cells because high confluency and contact inhibition can increase SA‐β‐gal activity (Dimri et al. 1995; Melk et al. 2004). Fresh tissue from mice was collected and fixed in 10% neutral buffered formalin (NBF) on ice for 3–4 h. Tissues were then transferred to 30% sucrose at 4°C for 24 h before being embedded in O.C.T. compound in a cryomold and stored at −80°C. Prior to cryosectioning, tissues were equilibrated at −20°C and then cryosectioned into 5–7 μm sections before being attached to Superfrost Plus slides. Fresh SA‐β‐gal staining solution at a pH of 6 was prepared as previously described (Yousefzadeh et al. 2020). Tissue slides were stained in SA‐β‐gal staining solution for 18–24 h at 37°C in a non‐CO2 incubator before being rinsed three times with PBS. To prevent crystal formation, a parafilm Coplin jar was used to prevent evaporative loss of staining solution. Stained sections were imaged using the EVOS microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA) at a magnification of 40X as previously described. The percent of SA‐β‐Gal‐positive area for each sample will be quantified using ImageJ.

The number of gamma‐H2AX foci increases in damaged and senescent cells (Bernadotte et al. 2016), making it a useful marker for DNA damage. We used the same tissues prepared for SA‐β‐Gal staining, and prior to cryosectioning, tissues were equilibrated at −20°C and then cryosectioned into 5–7 μm sections before being attached to Superfrost Plus slides. After blocking the slides with 1X PBS + 5% normal serum +0.3% Triton X‐100 for 1 h, slides were incubated with the primary antibody (p‐H2AX, Ser139, 9718, CST; 1:800 dilution) at 4°C overnight. Slides were further incubated with secondary antibody (Alexafluor 488 Conjugate, 4412: 1:200 dilution) at room temperature for 2 h. After rinsing slides with PBS three times, slides were mounted with Fluoro‐Gel II with DAPI (Electron Microscopy Scientific, Cat#17985–50). Stained sections were imaged using the EVOS microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA) at a magnification of 40X as previously described. The percent of gamma‐H2AX‐positive area for each sample was quantified using ImageJ.

Blood plasma for FGF21 was obtained at Week 15 in the fasted state and from blood collected at the time of euthanasia in the refed state. Blood FGF21 levels were assayed by a mouse/rat FGF‐21 Quantikine ELISA kit (MF2100) from R&D Systems (Minneapolis, MN, USA).

Data are presented as the mean ± SEM unless otherwise specified. Statistical analyses were performed using one‐way or two‐way ANOVA followed by Tukey–Kramer post hoc test, as specified in the figure legends. Outliers were excluded using the Robust Regression Outlier Test (ROUT) in Graphpad Prism (v10), Q = 1%, and are indicated by an asterik(*) and blue colored font in the Source Data. Other statistical details are available in the figure legends. Energy expenditure differences were detected using analysis of covariance (ANCOVA). ANCOVA analysis assumes a linear relationship between the variables and their covariates. If the slope is equal between groups, then the regression lines are parallel, and elevation is then tested to determine any differences (i.e., if slopes are statistically significantly different, elevation will not be determined). In all figures, n represents the number of biologically independent animals. Sample sizes were chosen based on our previously published experimental results with the effects of dietary interventions (Cummings et al. 2018; Fontana et al. 2016; Yu et al. 2021, 2019, 2018). Data distribution was assumed to be normal, but this was not formally tested.

All studies were performed on animals or on tissues collected from animals. Young animals of each sex and strain were randomized into groups of equivalent weight, housed 2–3 animals per cage, before the beginning of the in vivo studies. Aged animals were randomized into groups of equivalent weight, housed 2–3 animals per cage, when possible.

We began our study by designing a series of diets in which the total amount of calories derived from amino acids (AAs) was either 7% (LP, Low Protein), 21% (CP, Control Protein), or 36% (HP, High Protein). These diets were isocaloric. Dietary fat was held constant (19%) and the levels of carbohydrates adjusted in order to maintain equivalent caloric density. We further designed CP and HP diets in which the levels of all three BCAAs were reduced to the level of BCAAs found in the LP diet (CP‐BR and HP‐BR, respectively); in these diets, the reduction in BCAAs was balanced by a proportional increase in non‐essential AAs, keeping the percentage of calories derived from AAs constant. The full composition of these diets is summarized in Table. S1

We randomized 12‐week‐old C57BL/6J male mice to these diets, following them longitudinally for 16 weeks (Figure 1A). As we anticipated based on our previous studies, we found that mice consuming diets low in BCAAs (i.e., LP, CP‐BR, HP‐BR) gained significantly less weight and fat mass than CP‐ and HP‐fed mice, with an overall reduction in adiposity (Figure S1A–H). Importantly, this weight reduction was not due to decreased calorie intake; mice consuming the LP, CP‐BR, and HP‐BR diets consumed more calories than CP and HP‐fed mice (Figure S1I–L). Due to this increase, animals consuming the CP‐BR and HP‐BR diets had an overall increase in calories derived from AAs, even while BCAAs were effectively restricted (Figure S1M–P).

We have previously observed that the paradoxical decreased weight gain of BCAA‐restricted mice is associated with increased energy expenditure (Cummings et al. 2018; Fontana et al. 2016). Using metabolic chambers, we determined energy expenditure via indirect calorimetry while also assessing food consumption, activity, and fuel source utilization. As we anticipated, mice with reduced levels of BCAAs had increased energy expenditure relative to the respective BCAA‐replete diet (Figure S2A–E). We also noted a tendency for increased spontaneous activity in animals with lower levels of BCAAs that reached statistical significance in some cases (Figure S2F).

Increased energy expenditure in LP‐fed mice is mediated by FGF21, which is induced by PR and stimulates the beiging of inguinal white adipose tissue (iWAT) and the activation of brown adipose tissue (BAT) by stimulating sympathetic nerve activity (Hill et al. 2017; Laeger et al. 2014; Owen et al. 2014). We observed that BCAA restriction increased plasma FGF21 in the context of the CP (21% protein) diet, but not the HP diet (36% protein diet) (Figure S2G,H). We observed that thermogenic genes were upregulated in the inguinal white adipose tissue (iWAT) in response to an LP diet, but not in response to BCAA restriction; conversely, in BAT, we observed a BCAA‐restriction induction of several thermogenic genes (Figure S2I,J). Mice consuming diets low in BCAAs have a higher respiratory exchange ratio (RER), suggesting an increase in carbohydrate/protein substrate utilization (Figure S2K–M). This data is consistent with BCAA restriction increasing energy expenditure via FGF21‐induced activation of thermogenesis.

We also assessed glucose homeostasis by performing glucose, insulin, and alanine tolerance tests. Mice consuming LP and low BCAA diets had improved glucose and pyruvate tolerance and lower fasting blood glucose relative to mice consuming CP and HP diets; however, insulin sensitivity was improved only in LP‐fed mice (Figure). S3A–G

As described above, we hypothesized that the metabolic benefits of low BCAA diets are in part through altered senescence. We therefore examined the mRNA expression of common senescence and SASP genes. We observed that BCAA restriction reduced the expression of the senescence marker p16, while the expression of p21 was not significantly altered (Figure 1B,C). We also performed hepatic SA‐β‐Gal staining and livers from mice fed diets with low levels of BCAAs (LP, CP‐BR, and HP‐BR) had lower SA‐β‐Gal‐positive staining than livers from CP and HP‐fed mice (Figure 1D,E). BCAA restriction also had strong effects on the expression of multiple SASP genes, reducing the expression of Il1α, Il6, Ccl2, and Cxcl1, while not affecting the expression of several other SASP genes (Figure 1F,G; Figure S4A–D).

The effects of BCAA restriction were generally stronger in the context of the CP diet, while BCAA restriction in the context of the HP diet either had no effect or did not reach statistical significance. To better understand the roles of BCAAs and protein in the regulation of these senescence and SASP genes, we analyzed the mRNA expression using a Multiple Linear Regression (MLR) approach with gene expression as the dependent variable and the level of either dietary protein or BCAAs as the independent variables. We found that dietary BCAAs substantially contributed to the expression of most of the senescence and SASP genes we examined, while dietary protein level did not (Figure 1H).

We examined the effects of BCAA restriction on senescence and the SASP in other contexts to determine the general applicability of these findings. We found that there was an overall beneficial effect of BCAA restriction on senescence and SASP gene expression in the livers of aged C57BL/6J male mice, as shown by the significant overall effect of diet, with restriction of BCAAs significantly reducing expression of p16 (Figure 1I). We further found that BCAA restriction had an overall beneficial effect on senescence and SASP gene expression in the livers of mice fed a high‐fat, high‐sucrose Western diet, reducing expression of p16, p21, and Il1α (Figure 1J).

We repeated this study in p16‐3MR mice. Overall, the effects of protein and BCAAs on body weight, food intake, and glucose regulation in p16‐3MR males were similar to what we observed in C57BL/6J males (Figure). S5A–O

At 20–22 weeks of age, we analyzed p16 expression in vivo using a bioluminescence assay. Surprisingly, we found no significant difference between mice fed any of the diets on whole‐body total luminescence, which represents p16 expression (Figure 2A and Figure S5P). We then plotted the total p16 luminescence against the calories of either protein or BCAAs consumed and performed a simple linear regression; a slope p‐value of less than 0.05 suggests that there is a linear relation between the variables. While there was no relationship between p16 luminescence and the calories of protein consumed, we found a statistically significant relationship between the calories of BCAAs consumed and p16 luminescence. Surprisingly though, this relationship was negative, with p16 luminescence decreasing as BCAA consumption increased (Figure 2B,C).

We therefore decided to examine the adipose depots, specifically, the iWAT and epididymal white adipose tissue (eWAT) since they are likely to show bioluminescence in the abdominal area, where we detected strong p16 activity, and diets have been shown to have a robust effect on adipose tissue senescence in other studies (Ogrodnik et al. 2021; Ogrodnik et al. 2019; Allyson K. Palmer et al. 2019; Wang et al. 2022). In contrast to our original hypothesis that BCAA restriction would reduce senescence, in our original C57BL/6J mice we found a strong increase in the expression of multiple senescence and SASP genes in BCAA‐restricted mice, with the expression of p16, p21, Il1α, Il1β, and Il6 increasing in BCAA‐restricted iWAT, and Il1α increasing in BCAA‐restricted eWAT (Figure 2D–I). This interpretation was further supported by MLR analysis, which found a strong contribution of BCAA levels—but not protein level—to the expression of these senescence and SASP genes (Figure 2J). Broadly, we find that opposed to what we observed in the liver, BCAA restriction increases senescence in adipose tissue, increasing expression of most senescence and SASP genes in iWAT, increasing expression of Il1α expression in eWAT, and increasing some senescence genes in BAT as well (Tables S3 and S4).

The differential effect of BCAAs on senescence in the liver and iWAT was quite surprising. We hypothesized that this might be due in part to the differential effect of FGF21, a hormone induced by protein restriction and BCAA restriction that has been shown to regulate senescence (Li et al. 2019; Lu et al. 2021). FGF21 has differential effects on mTOR protein kinase signaling in liver and adipose tissue (Minard et al. 2016), and mTOR signaling is a key regulator of aging, metabolism, and components of the senescence and SASP (Calubag et al. 2024). We examined Fgf21 expression in the liver and three adipose depots; Fgf21 expression was higher in the liver and iWAT of LP‐fed mice than in all other groups, with CP‐BR‐fed mice having the next highest expression; there were minimal effects of BCAAs on Fgf21 expression in eWAT or BAT (Figure S7A–D). Therefore, we then looked at mTORC1 signaling in the liver and iWAT. There were minimal effects of diet on the phosphorylation of the mTORC1 substrates S6K1 T389 or 4E‐BP1 T37/S46, or of the downstream readout S6 S240/S244 in either of the tissues (Figure S7E–J).

To gain insight into what types of damage might be signaling liver and iWAT cells to undergo senescence, we looked at staining and several genes related to DNA damage. First, we confirmed that there was increased DNA damage using gamma‐H2AX immunofluorescence staining in the liver of the CP‐fed mice compared to the low BCAA diets (Figure 3A,B). We then found that a BCAA‐restricted diet affected the expression of genes related to mitochondrial dysfunction and mitochondrial biogenesis, without altering the expression of genes related to antioxidant function and oncogenesis in both the liver and adipose tissue (Figure 3C; Figure S6A). Specifically, we found that the livers of CP‐BR‐fed mice had lower Cycl1, Sdha, and Atp5ai expression as well as increased Pgc1a expression compared to CP‐fed mice, suggesting they had improved mitochondrial function (Figure 3C). In contrast, we found the opposite gene expression changes in Cyc1 and Sdha in the iWAT of CP‐BR‐fed mice, suggesting they had impaired mitochondrial function (Figure S6A).

This suggested to us that BCAAs, specifically at 21% protein, may mediate mitochondrial function/dysfunction, leading to the tissue‐specific differences in senescence. To confirm that mitochondrial dysfunction related to dietary BCAA may be mediating the effects on senescence, we turned to cell culture. We cultured mouse AML12 hepatocytes in media containing the normal level of BCAAs (100% BCAA) or restricted BCAAs (0% additive BCAA; this media still contains some BCAAs from serum). We treated these cells with antimycin A for 14–20 days to induce mitochondrial dysfunction‐related senescence, and then collected the cells to assess senescence (Figure 3D). As we expected, AML12 cells treated with antimycin A senesced, with increased expression of p16 and p21 (Figure 3E). Culture of cells in the BCAA‐restricted media (0% added BCAA) prevented antimycin βA‐induced senescence. This difference in senescence was associated with antimycin A‐induced mitochondrial dysfunction, which occurred in antimycin A‐treated cells cultured in 100% BCAA, and cells cultured in 0% BCAA media were protected from (Figure 3F). These results support a model in which BCAA restriction protects liver cells from senescence via cell‐autonomous effects on mitochondrial dysfunction‐related senescence induction.

To further explore the tissue‐specific effects of BCAAs on senescence, we explored the possibility of lipolysis playing a role. It was recently shown that DNA damage leads to altered mitochondrial structure and dynamics, which downregulates fatty acid oxidation (the first step of which is lipolysis) and induces cellular senescence (Yamauchi et al. 2024). In contrast, the induction of systemic lipolysis in Drosophila has been shown to reduce senescence in the ovary and gut (Shang et al. 2022). Therefore, we looked at gene expression of lipolysis in both the liver and iWAT and found that at least at 36% protein, lipolytic gene expression was increased in the liver and decreased in the iWAT by BCAA restriction (Figure S7K,L). This suggests that the increased lipolysis in the liver and reduced lipolysis in the iWAT in response to BCAA restriction may also be playing a role in the induction of senescence, particularly in the 36% protein context.

The metabolic benefits of the CP‐BR diet are due primarily to the restriction of isoleucine, with a lesser contribution from the restriction of valine (Yu et al. 2021). To determine if the effects of CP‐BR on cellular senescence are due to a single BCAA, we placed 12‐week‐old C57BL/6J male mice on the CP and CP‐BR diets, as well as on leucine‐restricted (Leu‐R), isoleucine‐restricted (Ile‐R) or valine‐restricted (Val‐R) diets. All of these diets are isocaloric with identical levels and sources of fats and carbohydrates and are isonitrogenous through the addition of non‐essential amino acids in restricted diets; the full diet composition is provided in Table S1.

Over the course of 17 weeks, we tracked physiological parameters in all groups of mice (Figure 4A). As anticipated, CP‐BR‐, Ile‐R‐, and Val‐R‐fed mice consumed more food than CP and Leu‐R‐fed mice, yet these groups showed attenuated weight and fat mass gain (Figure 4B–F and Figure S8A–D) as a result of increased energy expenditure (Figure S8F–H). These phenotypes were associated with strong induction of FGF21 in the blood of CP‐BR, Ile‐R, and Val‐R‐fed mice and increased expression of the thermogenic genes Bmp8b and Elovl3 in the BAT of CP‐BR‐ and Val‐R‐fed mice (Figure S8I,J), and with improved blood glucose control in the same animals as well as Ile‐R‐fed mice (Figures S8L–R).

Finally, we assessed hepatic senescence. Although CP‐BR did reduce SA‐β‐Gal positivity and senescence gene expression, Ile‐R and Val‐R did not, despite their improvements in metabolic health (Figure 4G–L). Interestingly, restriction of individual BCAAs seemed to increase, not decrease, hepatic SASP expression (Figure 4K,L). This suggests that while Ile‐R and Val‐R may mediate the metabolic and physiological effects seen in CP‐BR diets, they do not mediate the effects of BCAA restriction on hepatic senescence and the SASP at this age.

To examine the role of sex in the response of senescence to dietary protein, we placed 14‐week‐old, female, p16‐3MR mice on the five diets (LP, CP, CP‐BR, HP, HP‐BR) for 28 weeks (Figure 5A). After 28 weeks on diet, we found largely no effect of diet in females (Figure 5B,C). In agreement with this, BCAA‐restricted females do not consume more food, but their consumption of protein and BCAAs was similar to that of the males (Figure 5D–F; Figures S9A–C and S1M–P). While females fed a low BCAA diet tended to have improved glucose tolerance (Figure S9D,E), BCAAs largely had no effect on fasting blood glucose, insulin sensitivity, nor suppression of hepatic gluconeogenesis (Figure S9F,J).

We next examined senescence in the livers of female p16‐3MR mice. We found no significant difference in SA‐β‐Gal activity between any of the groups, though HP‐fed females trended toward higher hepatic SA‐β‐Gal activity than LP‐, CP‐, and CP‐BR‐fed mice (p = 0.0696, p = 0.0980, and p = 0.0613, respectively) (Figure 5G,H), suggesting that higher protein consumption may induce senescence in females. While LP, CP‐BR, and HP‐BR‐fed females did have reduced expression of p16 compared to CP‐fed females, this was the only significant difference identified; using the MLR approach we used previously, we found that neither dietary BCAAs nor dietary protein contributed to the expression of any of the senescence and SASP genes we examined (Figure 5I,J). While females are thought to be more prone to senescence (Ng and Hazrati 2022; Yousefzadeh et al. 2020), it may not be the case in young animals, such as in this study, as we see higher SA‐β‐Gal‐positive staining in the liver of the males compared to females (Figures 1D,E and 5G,H). It may also be that in young mice, female‐specific senescence accumulation is tissue‐specific, and we would have seen senescence in other tissues (Jin et al. 2023). As LP and CP‐BR diets reduced p16 expression in the liver and we saw trends in reduced SA‐β‐Gal‐positive staining (Figure 5H,I), we decided to look at CS senescence and SASP gene expression in the livers of C57BL/6J female mice fed a CP‐BR diet for ~7 months (from 16 months of age until they were 23 months of age), using mRNA banked from a previous study (Richardson et al. 2021). We found that there was an overall significant effect of diet on senescence and SASP gene expression (Figure 5L), suggesting that a reduction of dietary BCAAs can reduce hepatic senescence in middle‐aged/aged C57BL/6J females.

We also assessed p16 bioluminescence; we observed no significant differences in p16 expression as quantified by total p16 luminescence in the females (Figure 5M,N). While there was a trending positive relationship between luminescence and BCAAs consumed (p = 0.085), we did not see significant effects of BCAAs when performing a MLR analysis in the iWAT (Figure 5O,P and Figure S9K,L).

Overall, we found a sex‐specific effect of BCAAs on metabolic health and senescence, with female mice largely not benefiting from BCAA restriction with respect to the effects on weight, body composition, and hepatic cellular senescence.