Animals and husbandry

BCAA longevity study

Three hundred and twelve male and female C57BL/6J mice were obtained from the Australian Resource Centre at 4 weeks old and housed at the Charles Perkins Centre at the University of Sydney under a 12 h light–dark cycle. Experiments using C57BL/6J mice were conducted under the approval of Sydney University’s Animal Ethics Committee (protocol no. 2014/752). Animals were housed four per cage in standard approved cages and were not exercised for the duration of the study (see Nature Research Reporting Summary for additional details of animals and study design). At 12 weeks of age, mice were allocated to one of four experimental ad libitum diet treatments. An additional 192 male and female mice were obtained and assigned to a 20% calorie-restricted BCAA100 or BCAA200 diet. Daily aliquots were estimated from the ad libitum intake of the BCAA100 mice. Food intake and body weight were measured fortnightly until 6 months of age, followed by monthly measurements thereafter. Mice were checked twice weekly; animals losing more than 15% body weight were killed. At 15 months of age, plasma and tissue were collected from one mouse from each cage. Animals were anaesthetized using a mixture of ketamine and xylazine (100 mg kg−1 ketamine and 16 mg kg−1 xylazine) and killed by exsanguination via cardiac bleed using a 23 G needle. Tissues collected were snap-frozen in liquid nitrogen or fixed for histology; they included liver, muscle, white and brown fat, spleen, pancreas, kidneys, heart and reproductive organs. Tissue collection was performed between 10:00 and 12:00 consistently, 4–6 h after the initiation of the light cycle and when animals had completed their normal overnight feeding period. The rationale behind this collection time was to provide a stable, postprandial sample several hours after the animals had completed their normal overnight feeding period. The collection time ensured that significant alterations to the normal diurnal pattern of feeding/fasting and active/sleeping were not introduced. This provides the best indication of their baseline response to the dietary interventions over 15 months of feeding, whereas overnight fasting introduces a significant fasting response in mice that would interfere with the normal hormonal and metabolite plasma profile due to innate nocturnal feeding patterns.

Short-term (6-week) dietary interventions

For short-term feeding experiments adding back Met, Thr or Trp to a BCAA200 diet (Fig. 2), 60 male C57BL/6J mice obtained from the Australian Resource Centre, housed 2 per cage at 12 weeks of age, were fed for 6 weeks on either a BCAA100, BCAA200, BCAA200 + Trp, BCAA200 + Thr or BCAA200 + Met diet (see Supplemental Tables 1 and 2 for a detailed breakdown). Food intake was measured every 2 d and body weight was recorded weekly. For studies investigating the effect of diet on serotonin (5-HT) and fluoxetine administration on food intake (Fig. 3), an additional 52, 6-week-old male C57BL/6J mice from the were purchased, housed 2 or 3 per cage and fed on either a BCAA200 or BCAA100 diet for 6 weeks. Electrophysiological experiments were conducted on five mice per diet. The remaining 42 mice were treated with either fluoxetine (n = 24 mice; 8 cages) or saline (n = 18 mice; 7 cages).

Exome-matching longevity study

C3B6F1 female hybrids were used for studies using exome-matched diets (Supplementary Table 4); female hybrids were generated by a cross between C3H female and C57BL/6J male mice. Parental strains were obtained from the Charles River Laboratories and experimental animals were bred in an in-house animal facility at the Max Planck Institute for Biology of Ageing. Four-week-old female mice were housed in groups of five, in individually ventilated cages under specific-pathogen-free conditions. Experiments were conducted under the approval of the State Office for Nature, Environment and Consumer Protection North Rhine-Westphalia (approval nos. 84-02.04.2012.A245 and 84-02.04.2017.A175).

Experimental diets

BCAA longevity study

All experimental diets, except for exome-matched diets, were custom-designed and manufactured in dry, pelleted form by Specialty Feeds. Diets were isocaloric (14.4 kJ g−1) and matched in the total calculated net metabolizable energy from protein (18%), carbohydrate (64%) and fat (18%) (Supplementary Table 1). BCAA200: twice the BCAA content of control diet AIN-93 G; BCAA100: standard content of BCAAs; BCAA50 and BCAA20: containing one-half and one-fifth of the standard content of BCAAs.

Short-term amino acid study

Diets where Thr, Trp or Met were added to a BCAA200 diet were matched for total energy content, BCAA content and macronutrient composition (Supplementary Table 2).

Exome-matching longevity study

Four-week-old C3B6F1 female hybrid mice were fed a 6% protein diet ad libitum for 3 months. At the age of 4 months, mice were assigned one of three experimental diets and fed ad libitum for 3 weeks, during which food intake was recorded twice per week. The group with the lowest food intake (23% protein) was taken as the baseline group to which other groups were pair-fed. Pair-feeding was done by measuring the food intake of mice fed the 23% protein diet with ad libitum access to food and adjusting the food intake of the rest of the mice accordingly. The food intake of control mice was measured twice a week and the average value per week was used to adjust the food intake for the rest of the mice for the following week. Food aliquots were prepared 1 week in advance and all mice were fed daily at 9:00. To minimize the differences in daily rhythms of feeding and activity between the control group and the rest of the groups, controls were also given daily aliquots of food that exceeded the amount normally eaten by this group.

To generate the theoretical requirement of mice for dietary amino acids, we used an in silico technique called exome matching23. Briefly, we used the complete set of protein-coding sequences in the mouse genome (Ensembl v.54, May 2009, downloaded 2 July 2009) to calculate the median proportional representation of each amino acid across all proteins. To determine the theoretically limiting amino acids, we divided the proportional representation of each dietary EAA (expressed in moles) by its proportional representation in the mouse exome and found the amino acid with the lowest value. Every other amino acid is then considered to be in excess. For each mole of cysteine (Cys) or Tyr that was considered undersupplied in the food, the availability of Met (for Cys) or Phe (for Tyr) was reduced by 1 mol. Non-essential amino acids are not considered because they can be generated de novo as long as the general supply of nitrogen from other amino acids is sufficient. The exome-matched diets used in the lifespan study were manufactured by ssniff and were isocaloric (16.6 kJ g−1; Supplementary Table 6). All amino acids were crystalline; 35–40% of BCAAs were added to the 6% protein diet (6% protein + BCAA) and all other amino acids were reduced to accommodate for BCAA supplementation.

Body composition

Body composition of C57BL/6J male and female mice was assessed using an EchoMRI 900 (EchoMRI) at 15 months of age. Additionally, dual-energy X-ray absorptiometry (DXA) scans were obtained using the UltraFocus 100 DXA (Faxitron) before tissue collection at 15 months. Animals were anaesthetized with 3% isoflurane, maintained at 1.5% and imaged. The region of interest was set to exclude the head and tail during scanning. Body composition of C3B6F1 female mice on exome-matched diets were measured with the minispec LF50H Body Analyzer (Bruker) at 12 months of age.

Glucose metabolism

Glucose tolerance tests (GTTs) were performed on C57BL/6J male and female mice at 15 months of age, by fasting mice for 4 h before testing. Basal blood samples were obtained by tail tip excision; blood glucose was measured using a clinical glucometer (Accu-Chek Performa; Roche Diagnostics). Glucose (2 g kg−1 lean mass) was then administered via oral gavage. Blood was collected at baseline, then 15, 30, 45, 60 and 90 min from the original tail wound and serial tail tip excision was not required. The incremental area under the curve (AUC) was calculated. The AUC indicates the time taken to clear a bolus dose of glucose from the bloodstream and return to basal levels.

Metabolic hormones

FGF21 was measured in plasma collected from 15-month-old C57BL/6J male and female mice using the mouse/rat FGF21 enzyme-linked immunosorbent assay (ELISA) Kit (BioVendor Laboratory Medicine) according to the manufacturer’s protocol. Mouse leptin, insulin and IGF1 were also measured by ELISA, according to the manufacturer’s instructions (Crystal Chem).

Plasma amino acids

Amino acids from C57BL/6J male and female 15-month-old mice were analysed at the Australian Proteome Analysis Facility, Macquarie University, using the AccQ-Tag Ultra Chemistry Kit (Waters).

Liver histology and triglyceride content

Paraffin-embedded liver tissue collected from 15-month-old C57BL/6J male and female mice was sectioned at 5 µm and stained with hematoxylin and eosin (H&E). The extent of steatosis and inflammation was assessed and scored (0 = 0% fat present, 1 = 1–33%, 2 = 34–66% and 3 ≥ 67%) by four independent observers blinded to the dietary treatment groups.

Liver triglyceride content was measured by homogenizing 30 mg of frozen tissue from C57BL/6J male and female 15-month-old mice in a 1:2 ratio of methanol and chloroform using a bead-based tissue lyser (TissueLyserLT, Qiagen). Lipids were extracted overnight on a roller and dried down using a nitrogen apparatus and a heating block at 37–45 °C. The dried sample was then resuspended in absolute ethanol (RNA grade), quantified by GPO-PAP method (catalogue no. 11730711 216, Roche/Hitachi) with absorbance measured using an Infinite M1000 PRO plate reader (Tecan) at 490 nm.

Blood lipids and biochemistry

Blood cholesterol, triglycerides and liver function tests (ALT and AST) analyses were performed on plasma collected from C57BL/6J male and female 15-month-old mice at the Diagnostic Pathology Unit, Concord Hospital, NSW Health using a cobas 8000, c702 Photometric Modular Analyzer (Hitachi).

Metabolic phenotyping

To determine the whole-animal metabolic rate, substrate use and activity, 12–16 C57BL/6J male and female mice per diet were housed individually and assessed by indirect calorimetry in a Promethion high-definition continuous respirometry system for mice (Sable Systems International) at 15 months of age. Oxygen consumption (VO 2 ) and carbon dioxide production (VCO 2 ) were measured over 48 h, following an 8 h acclimation period, and maintained at approximately 22 °C under a 12:12 h light–dark cycle. Energy expenditure is shown a kcal h−1 corrected for lean mass. Mice were not given access to a running wheel.

Electrophysiology

Following 6 weeks on either a BCAA200 or BCAA100 diet, coronal dorsal raphe brain slices (280 µM) from 12-week-old C57BL/6J male mice were cut with the VT-1200 S Vibratome (Leica Biosystems). Slices were initially incubated in recovery solution containing 93 mM N-methyl-d-glucamine chloride, 2.5 mM KCl, 1.2 mM NaH 2 PO 4 , 30 mM NaHCO 3 , 20 mM HEPES, 25 mM d-glucose, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 10 mM MgCl 2 , 0.5 mM CaCl 2 , pH 7.3, 300–310 mOsm l−1 heated at 34 °C and saturated with carbogen for 10 min and then stored in physiological saline (artificial cerebrospinal fluid (ACSF)) for at least 1 h either with or without the addition of Trp (50 µM). Slices were transferred to a recording chamber and superfused continuously at 2.5 ml min−1 with 34 °C ACSF containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 × 2H 2 O, 1 mM MgCl 2 , 2 mM CaCl 2 , 25 mM NaHCO 3 and 11 mM d-glucose saturated with carbogen. Dorsal raphe nuclei were visualized with an Olympus BX51 microscope using Dodt gradient contrast optics. Whole-cell patch-clamp recordings were made from the dorsal raphe nuclei using an internal solution containing 95 mM potassium gluconate, 30 mM KCl, 10 mM HEPES, 2 mM EGTA, 15 mM NaCl, 1 mM MgCl 2 , 2 mM MgATP and 0.3 mM NaGTP. Neurons were voltage-clamped at −60 mV and liquid junction potentials of −12 mV were not corrected. Electrically evoked IPSCs were elicited via bipolar tungsten stimulating electrodes (10 stimuli delivered at 166 Hz, 100 V, 100 µs). Membrane currents were recorded with a Multiclamp 700B Amplifier (Molecular Devices), digitized and then acquired and analysed with the Axograph Acquisition software (Axon Instruments). All recordings were made in the presence of 100 µM picrotoxin (Sigma-Aldrich), 50 µM DL-2-amino-5-phosphonopentanoic acid (Abcam), 500 nM prazosin (Sigma-Aldrich), 10 µM cyanquixaline (Abcam) and 1 µM CGP 55845 (Tocris). Stock solutions of drugs were made in distilled water except for NAN-190 (Sigma-Aldrich), which was dissolved in dimethylsulfoxide, then diluted to their final concentration in ACSF and applied by superfusion.

Fluoxetine administration

Following 6 weeks on a BCAA200 diet, 12-week-old male C57BL/6J mice were gavaged with either fluoxetine (20 mg kg−1; Selleck Chemicals) or saline for 4 d. Food intake was recorded daily.

Gene expression

Total RNA from the liver and hypothalamus of 15-month-old male and female C57BL/6J mice were extracted using the TRIzol method (Sigma-Aldrich) and quantified spectrophotometrically using a NanoDrop (2000c, Thermo Fisher Scientific). For RNA-seq, hypothalamic RNA was sequenced by the Australian Genome Research Facility using an Illumina HiSeq 2500 System. The sequence reads were analysed according to Australian Genome Research Facility quality control measures. The cleaned sequence reads were then aligned against the Mus musculus genome (GRCm38). The TopHat aligner (v.2.0.14) was used to map the reads to the genomic sequences. The transcripts were assembled with StringTie v.1.2.4, using the read alignment with gencode M14. The fragments per kilobase of transcript per million mapped reads (FPKM) were generated using StringTie based on the formula FPKM = 106 C/(NL/103). C is the number of reads uniquely aligned to a gene, N is the total number of reads that is uniquely aligned to all genes and L is the number of bases of a gene. Heat maps were generated to model the relationship of BCAA intake with gene expression as measured by standardized z-scores. The rows are organized by hierarchical clustering using agglomerative clustering with complete linkage and Euclidean distance metric.

For real-time PCR, complementary DNA was synthesized (iScript; Bio-Rad Laboratories) and mRNA expression analysed using the SYBR Green methodology (LightCycler 480; Roche Molecular Systems). Primer pairs were designed using the Roche Universal Probe Library and BLASTed against the National Center for Biotechnology Information mouse genomic sequence database. Reactions were performed in triplicate and target gene expression normalized with eukaryotic elongation factor 2 as the endogenous control. The fold change was calculated based on a pooled sample. Primers were 300 nM in concentration and sequences for each gene are described: Npy-forward CGACACTACATCAATCTCATC, Npy-reverse AAGTTTCATTTCCCATCACC; Agrp-forward CTTCTTCAATGCCTTTTGC, Agrp-reverse TTTTTAAACCGTCCCATCC; Lepr-forward TGCTGAATTATACGTGATCG, Lepr-reverse AGACGTAGGATGAATAGATGG; Socs3-forward ATTTCGCTTCGGGACTAGC, Socs3-reverse AACTTGCTGTGGGTGACCAT; Pepck-forward CCAACGTGGCCGAGACTAGCG, Pepck-reverse GGCACATGGTTCCGCGTCCT; Eef2-forward TGTCAGTCATCGCCCATGTG, Eef2-reverse CATCCTTGCGAGTGTCAGTGA; Fgfr1c-forward TCCTCTTCTGGGTGTGC, Fgfr1c-reverse CTCCACTTCCACAGGGACTC; Klb-forward GAGGATGATCAGATCCGAAAGT, Klb-reverse AGCCTTTGATTTTGACCTTGTC.

Protein quantification

For all western blots, protein concentration was determined with a bicinchoninic acid assay (Sigma-Aldrich) and lysates loaded and standardized to contain 25 µg of protein. Liver, white adipose tissue and BAT from 15-month-old C57BL/6J male and female mice were resolved in either a 4–12% Bis-Tris gel (Bio-Rad Laboratories) or 3–7% Tris-Acetate Gel (Bio-Rad Laboratories). Following electrophoresis, proteins were transferred to a nitrocellulose membrane, blocked with 5% BSA in Tris-buffered saline with Tween 20 and incubated with various primary antibodies (all Cell Signaling Technology): mTOR, catalogue no. 2972; phospho-mTOR (Ser2448), catalogue no. 2971; UCP1 (D9D6X), catalogue no. 14670; ACLY (D1X6P), catalogue no. 13390; SCD1 (M38), catalogue no. 2438; Fatty Acid Synthase (C20G5), catalogue no. 3180; Acetyl-CoA Carboxylase, catalogue no. 3662; β-actin (13E5), catalogue no. 4970; cyclophilin A, catalogue no. 2175 (see Nature Research Reporting Summary) overnight at 4 °C. Bands were imaged following incubation for 1 h at room temperature in anti-rabbit horseradish peroxidase-linked secondary antibody (catalogue no. 7074; Cell Signaling Technology) on a ChemiDoc Imaging System (Bio-Rad Laboratories) using the ImageLab software v.5.1 to quantify relative protein expression.

Metabolomics

Targeted LC–QQQ–MS analysis was performed to detect a different set of water-soluble metabolites in the positive and negative modes using an liquid chromatography mass spectrometry (LC–MS) system comprising a 1260 Infinity II LC System (Agilent) coupled to a QTRAP 5500 Mass Spectrometer (AB Sciex). Samples for analysis were from C57BL/6J 15-month-old male and female mice. For the hydrophilic interaction LC (HILIC) analysis, plasma and cortex samples (10 µl) were prepared via protein precipitation with the addition of 9 volumes of 74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid containing stable isotope-labelled internal standards (l-valine-d 8 (Sigma-Aldrich) and l-phenylalanine-d 8 (Cambridge Isotope Laboratories)). For the amide analysis, plasma samples (30 µl) were prepared via protein precipitation with the addition of 70 µl of 75:25 v/v of acetonitrile/methanol containing stable isotope-labelled internal standards (Thymine-d 4 (Sigma-Aldrich) and l-phenylalanine-d 8 (Cambridge Isotope Laboratories)). The samples were centrifuged (20 min, 14,000 r.p.m., 4 °C) and the supernatants (10 µl) were injected directly onto a 2.1 × 150 mm, 3 µm Atlantis Silica HILIC Column (Waters) and a 4.6 × 100 mm, 3.5 μm XBridge Amide (Waters), for the HILIC and amide analyses, respectively. Eighty metabolites from the 84 metabolites optimized for positive mode detection were detectable in the plasma extracts. Of 110 metabolites optimized for negative mode, 73 were detectable in the mice plasma and were included in the final multiple reaction monitoring method. Raw data files (Analyst Software v.1.6.2; AB Sciex) were imported into the MultiQuant v.3.0 analysis software for multiple reaction monitoring Q1/Q3 peak integration; data were normalized relative to the pooled plasma samples that were analysed in the sample queue after every 10 study samples.

Immunohistochemistry and islet analysis

Paraffin-embedded pancreatic tissue from 15-month-old C57BL/6J male and female mice was sectioned and then stained as follows: sections were deparaffinized and rehydrated through a xylene-ethanol series and washed twice in PBS containing 0.1% BSA and 0.01% sodium azide (wash buffer). Slides were incubated with blocking buffer (DAKO) for 1 h at room temperature, then incubated with guinea pig anti-insulin (catalogue no. A0564; DAKO) and monoclonal anti-glucagon antibody produced in mouse (catalogue no. G2654; Sigma-Aldrich) overnight at 4 °C in a humidified chamber (see Nature Research Reporting Summary for antibody details). The following day, slides were washed twice in wash buffer, incubated in goat anti-guinea pig immunoglobulin G (IgG) and donkey anti-mouse IgG secondary antibodies (Alexa Fluor 488 and 594, respectively; Themo Fisher Scientific), washed twice again and then mounted in ProLong Diamond Antifade Mountant with 4,6-diamidino-2-phenylindole (Thermo Fisher Scientific). Slides were imaged with Leica DM6000 widefield and SP8 confocal microscopes.

Images were analysed with Fiji Image J (v.1.52b). The total pancreas area was calculated by tile-stitching entire pancreas sections in each slide. Fluorescence levels for each stain (insulin and glucagon) were thresholded by using control samples to exclude/eliminate any background fluorescence or noise. The threshold values were then applied to all sections, and intensity values were calculated by using the remaining normalized fluorescence readings. The total insulin- and glucagon-positive area were calculated; the total islet area was measured as a sum of total insulin and glucagon staining. At least ten islets per pancreas were averaged to obtain the pancreas measurement of each individual mouse.

Statistical analyses

Data are presented as the mean ± s.e.m. and significance was reached when P < 0.05 using R (v.3.4.1) and Prism (v.7.02; GraphPad Software). Comparisons between dietary treatments on various responses were analysed with analysis of variance (ANOVA); non-parametric data were analysed with a Kruskal–Wallis test. Sex was added as a cofactor and responses showing a significant diet × sex interaction are shown in Supplementary Fig. 3 and Supplementary Table 5. Comparisons of metabolic responses between ad libitum-fed and calorie-restricted animals were performed using a two-sided unpaired t-test. Details of the statistical tests for each graph are described in each figure legend. See the Nature Research Reporting Summary for data analysis and software specifications.

Survival data for C57BL/6J male and female mice and C3B6F1 female mice were analysed with the ‘survival’ package in R, using Cox’s proportional hazards models (CPHMs) implemented using the ‘coxph’ function58. CPHMs were used because they allowed us to explore the interactions between diet and sex effects on survival, as well as, where required, time-dependent effects in expanded CPHMs. Including the additive or interactive effects of sex alongside diet did not improve model fits based on the Akaike information criterion (AIC; BCAA data, Fig. 1n: AIC diet = 1,935.9, AIC diet + sex = 1,935.7, AIC diet × sex = 1,936; calorie restriction data, Fig. 7a: AIC diet = 2,075.6, AIC diet + sex = 2,075.6, AIC diet × sex = 2,076.2), suggesting diet alone was the best predictor of survival in the experiments.

For BCAA data from C57BL/6J male and female mice (Fig. 1n), we explored an expanded, time-dependent CPHM (survival curves cross around week 60); however, this model detected no significant interactions between time (pre- versus post-week 60) and diet (CPHM BCAA × time estimated log hazard ratio (lnHR) = 0.95, s.e.m. = 0.94, P = 0.31). CPHMs detected significant differences in survival between animals on BCAA200 and all other diets (BCAA20 versus BCAA200 estimated lnHR = −0.58, s.e.m. = 0.21, P < 0.005; BCAA50 versus BCAA200 estimated lnHR = −0.48, s.e.m. = 0.20, P < 0.05; BCAA100 versus BCAA200 estimated lnHR = −0.49, s.e.m. = 0.19, P < 0.005). Ten BCAA20 animals were killed before 60 weeks of age because of weight loss (n = 5) or seizures (n = 5) and were excluded from the survival analysis. For data comparing ad libitum-fed and calorie-restricted mice (Fig. 7a), significant differences were detected (CPHM: calorie-restricted versus ad libitum-fed P < 0.001; 100 versus 200 ad libitum-fed P = 0.01; 100 versus 200 calorie-restricted P = 0.68). For the exome-matched feeding experiments using C3B6F1 female mice, we found that CPHMs detected significant differences in survival on 23% protein diets relative to 6% protein diets (estimated lnHR = 0.68, s.e.m. = 0.21, P < 0.005), but not between 6% protein diets and 6% protein diets + BCAAs (estimated lnHR = −0.24, s.e.m. = 0.23, P = 0.30).

Metabolites from C57BL/6J male and female 15-month-old mice were correlated with BCAA intake; those with a Pearson correlation coefficient r > 0.1 were analysed in MetaboAnalyst v.4 using the Kyoto Encyclopedia of Genes and Genomes (KEGG; Mus musculus) pathway library (v.4.0; https://www.metaboanalyst.ca/faces/home.xhtml)59,60,61. Both positively and negatively correlated metabolite sets were analysed using the integrated pathway analysis. Significant Holm-adjusted P values are plotted in Fig. 3.

For the hypothalamic data from male and female C57BL/6J 15-month-old mice analysed using RNA-seq, the correlation coefficients of BCAA intake and the FPKM value of each gene were plotted as heat maps and show genes with a significant P. For the volcano plots, the FPKM data were filtered with averaged FPKM for each gene >0.1. The Pearson correlation coefficient and corresponding P for each gene were calculated. Correlation coefficients with less than 0.5th percentile or greater than 99.5th percentile were considered as significant and marked with red dashed lines. FPKM data were correlated with BCAA intake; genes with moderate-to-strong correlation (r > 0.3) were analysed in the Database for Annotation, Visualization and Integrated Discovery (v.6.8; https://david.ncifcrf.gov/home.jsp)29,62. Both positively and negatively correlated gene sets were analysed using the Functional Annotation Tool and pathways assessed using the KEGG (Mus musculus) pathway library and shown in Supplementary Table 4.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.