Mice
We enrolled 960 feminine DO mice in 12 waves, equivalent to delivery cohorts from generations 22 to 24 and 26 to twenty-eight with 80 first-parity and 80 second-parity animals from every technology born round 3 weeks aside. No a couple of mouse per litter was enrolled within the examine. The primary cohort entered the examine in March 2016 and the examine was totally populated in November 2017. This schedule was designed to make environment friendly use of our phenotyping capability and reduce the potential for seasonal confounding. The pattern measurement was decided to detect a ten% change in imply lifespan between intervention teams with allowance for some lack of animals on account of non-age-related occasions. We used feminine mice on account of considerations about male aggression. Mice had been assigned to housing teams of eight animals in large-format wean bins with optimistic stress air flow and incoming air temperature of 24.4 to 25.6 °C. Environmental enrichments had been offered together with nestlets, biotubes and gnawing blocks. Mice had been randomized by housing group to one in all 5 dietary interventions. Blinding was not attainable because of the completely different feeding necessities in every intervention group. Of the 960 mice that had been entered into the examine, 937 mice had been alive when interventions had been initiated at 6 months of age and solely these mice are included in our evaluation. The entire procedures used within the examine had been reviewed and accepted beneath Jackson Lab IACUC protocol 06005.
DR
DR was carried out by controlling the timing and quantity of meals offered to mice. Feeding schedules for DR had been began at 6 months of age. All mice had been fed a typical mouse chow weight loss plan (5K0G, LabDiet). The AL feeding group was supplied with limitless entry to meals and water. The IF mice had been offered limitless entry to meals and water. On Wednesday of every week at 15:00, IF mice had been positioned into clear cages and meals was withheld for the following 24 or 48 h for the 1D and 2D teams, respectively. CR mice had been supplied with limitless entry to water and measured quantities of meals each day at round 15:00, 2.75 g per mouse per day for 20% CR and a pair of.06 g per mouse per day for 40% CR. These quantities had been primarily based on AL consumption of three.43 g per mouse per day that we estimated primarily based on historic feeding knowledge from DO mice. For the 40% CR protocol, a gradual discount in meals consumption was carried out: the mice had been first subjected to twenty% CR for two weeks, then to 30% CR for an extra 2 weeks, earlier than transitioning to the total 40% CR. Within the 2D IF protocol, mice had been initially acclimatized to the 1D IF routine for two weeks. Mice had been co-housed with as much as eight mice per pen. Co-housing is customary observe for CR research39; competitors for meals was minimized by putting meals instantly into the underside of the cage, permitting particular person mice to ‘seize’ a pellet and isolate whereas they eat. CR mice had been supplied with a 3 day ration of meals on Friday afternoon, which resulted in weekly durations of feasting adopted by a interval of meals deprivation of roughly 1 day for the 20% CR mice and a pair of days for the 40% CR mice, corresponding to the IF fasting durations. The 15:00 feeding time carefully approximates the circadian alignment of feeding, beginning simply earlier than the start of the darkish cycle, which is the conventional energetic and feeding time of day for mice. This timing has been proven to maximise lifespan extension in mice subjected to 30% CR8. The feast–famine cycle induced by the Friday triple feeding has been utilized in different research of CR57,58,59 however direct evaluation of the well being impacts is missing.
Meals consumption (160 mouse unbiased cohort)
To acquire an correct estimate of meals consumption and modifications in physique weight in response to weekly fasting cycles, we arrange an unbiased cohort of 160 feminine DO mice. Mice had been positioned on the identical DR protocols as in the primary examine. Meals was weighed each day for a interval of 1 week when mice had been 30, 36 and 43 weeks of age. Meals consumption knowledge had been normalized to models of g per mouse per day and offered as each day and weekly averages throughout timepoints by weight loss plan. Physique composition was decided at 43 and 45 weeks of age utilizing non-imaging nuclear magnetic resonance (NMR) utilizing the Echo MRI instrument, a NMR machine with a 5-gauss magnet that’s tailored to small-animal research. NMR knowledge had been used to detect modifications in physique weight and composition earlier than and after fasting. Values of physique weight, lean mass, fats mass and adiposity (100% × fats mass/whole mass) pre-fasting had been co-plotted with the distinction between earlier than and after fasting (Friday to Monday for AL and CR; Tuesday to Thursday for 1D IF; Tuesday to Friday for 2D IF).
Phenotyping
We carried out three cycles of well being assessments of mice at early, center and late life. These assessments included a 7-day metabolic cage run at round 16, 62 and 114 weeks of age; blood assortment for movement cytometry evaluation at 24, 71 and 122 weeks; rotarod, physique composition, echocardiogram, acoustic startle, bladder operate, free wheel operating and a blood assortment for CBC evaluation at round 44, 96 and 144 weeks of age. Moreover, physique weights had been recorded weekly and guide frailty and grip energy assessments had been carried out at 6 month intervals. All assays had been carried out on the Jackson Laboratory in keeping with customary working procedures.
Physique weight
Mice had been weighed weekly all through their lives, leading to over 100,000 values longitudinally collected for the 937 mice. Physique weights had been analysed after native polynomial regression becoming inside mouse (that’s, loess smoothing).
Frailty, grip energy and physique temperature
We utilized a modified model of the clinically related FI38, which was calculated as the common of 31 traits which might be indicators of age-associated deficits and well being deterioration. Every trait was scored on a scale of 0, 0.5 or 1, the place 0 indicated the absence of the deficit; 0.5 indicated delicate deficit; and 1 indicated extreme deficit. Measurements had been taken on the baseline (5 months) and had been repeated roughly each 6 months. Easy averaging yielded a uncooked FI rating of between 0 and 1 for every mouse. Frailty scores had been adjusted by estimating batch, coat color and experimenter results as random elements that had been subtracted from uncooked frailty rating values earlier than statistical evaluation.
Physique composition
We carried out twin X-ray absorptiometry evaluation utilizing the LUNAR PIXImus II densitometer to gather bone density and physique composition (together with fats and non-fat lean tissue). Mice had been anaesthetized and individually positioned onto a disposable plastic tray that was then positioned onto the publicity platform of the PIXImus. The method to amass a single scan lasts roughly 4 min. Measurements had been taken at round 44, 96 and 144 weeks of age.
Immune cell profiling utilizing movement cytometry
Peripheral blood samples had been analysed by movement cytometry to find out the frequency of main circulating immune cell subsets. Evaluation was carried out earlier than the beginning of dietary interventions at 5 months, then at 16 and 24 months of age. These timepoints corresponded to 11 and 19 months of dietary intervention. Pink blood cells in PBL samples had been lysed and the samples had been washed in FACS buffer (Mitenyi, 130-091-222). Cells had been resuspended in 25 μl FACS buffer with 0.5% BSA (Miltenyi, 130-091-222 with 130-091-376). Antibodies together with Fc block (2.42, Leinco Applied sciences) had been added and incubated for 30 min at 4 °C. Labelled cells had been washed and DAPI was added earlier than evaluation on the LSRII (BD Bioscience) system. The antibody cocktail contained CD11c FITC, N418 (35-0114-U100, Tonbo Biosciences, 1:100); NKG2D (CD314) PE, CX5 (558403, BD Biosciences, 1:80); CD3e PE-CF594, 145-2C11 (562286, BD Biosciences, 1:40); CD19 BB700, 1D3 (566411, BD Biosciences, 1:40); CD62L PE-Cy7, MEL-14, (60-0621-U100, Tonbo Biosciences, 1:100); CD25 APC, PC61 (102012, BioLegend, 1:80); CD44 APC-Cy7, IM7 (25-0441-U100, Tonbo Biosciences, 1:40); Ly6G BV421, 1A8, (562737, BD Biosciences, 1:80); CD4 BV570, RM4-5 (100542, BioLegend, 1:40); CD11b BV650, M1/70 (563402, BD Biosciences, 1:160); CD45R/B220 BUV496 (RA3-6B2, 564662, BD Biosciences, 1:20); Fc Block, 2.4G2 (C247, Leinco Applied sciences, 1:100).
Owing to the outbred nature of those mice, movement cytometry markers had been restricted, and T cell subsets had been typically assigned as naive and non-naive by the presence of CD62L and CD44 (immune cell subtype designations are proven in Supplementary Desk 7). NKG2D-positive cells had been enumerated and will characterize reminiscence T cells that accumulate after immune responses60. Owing to limitations in movement cytometry markers that establish NK cells and their subsets within the mouse strains contributing to the outbred DO mouse line, NK cells had been outlined as non-T non-B lymphocytes expressing NKG2D. Inside this inhabitants, CD11c and CD11b had been used to typically outline maturation subsets. CD11b expression marks extra mature NK cells and CD11c is lowered on the least-mature NK subset61.
Glucose
On the movement cytometry blood collections at 16, 62 and 114 weeks, mice had been fasted for 4 h and glucose was measured utilizing the OneTouch Ultra2 glucose meter from LifeScan together with OneTouch Extremely take a look at strips. At every of the CBC blood collections at 24, 71 and 122 weeks, non-fasted glucose was measured utilizing the glucose meter.
Echocardiogram
Ultrasonography was carried out utilizing the VisualSonics (VSI) Vevo 770/2100 high-frequency ultrasound system with 30 and 40 MHz probes. Echocardiography makes use of pulsed Doppler sonography, utilized by way of the ultrasound probe, to measure blood movement charges and volumes.
Metabolic monitoring cages
Mice had been individually housed for 7 days in a metabolic cage (Promethion Mannequin from Sable Techniques Worldwide) and the exercise, feeding and respiration had been tracked. Feeding protocols for dietary intervention had been maintained. Metabolic cage knowledge had been used to evaluate metabolism, power expenditure and exercise of mice in Y1, Y2 and Y3. Animal-level knowledge had been cleaned to take away outliers and instrument failure and summarized as cumulative (meals, wheel distance) or median throughout 5 min intervals (respiratory quotient, power expenditure). The imply and s.d. had been computed at 4 h and 1 h intervals and had been plotted as imply ± 2 s.e.m. throughout timepoint intervals. Animal-level summaries had been computed as the common throughout 7 days of the each day (24 h), gentle section (12 h) or darkish section (12 h) median values. Furthermore, we computed ‘change’ traits (for instance, delta respiratory quotient and delta power expenditure) because the distinction between the fifth to ninety fifth percentiles of all 1 h summaries throughout all the 7 day run.
CBC evaluation
Blood samples had been run on the Siemens ADVIA 2120 haematology analyzer with mouse-specific software program as described beforehand62.
Acoustic startle
Startle response was measured in rodents utilizing automated startle chambers, wherein a mouse was positioned in a transparent, acrylic tube connected to a extremely delicate platform that’s calibrated to trace their startle reflex whereas being uncovered to a collection of stimuli at various decibels and occasions. Mice had been initially uncovered to white noise from an overhead speaker, which transitions to a collection of randomized, computer-generated stimuli ranging in quantity from 70 to 120 decibels at 40 ms in period and an interval of 9–22 s. The take a look at runs for about 30 min.
Rotarod
We used the Ugo–Basile rotarod, which has 5 lanes evenly spaced alongside a motorized horizontal rotating rod, permitting for as much as 5 mice to be examined concurrently. Under every lane is a platform outfitted with a visit plate that information the latency for every mouse to fall. Initially of the session, mice had been positioned onto the rod, which started rotating at 4 rpm, slowly rising to a most of 40 rpm, over 300 s. Mice got three consecutive trials. We reported the imply latency (time to fall) and the slope of latencies throughout trials, in addition to the variety of trials with no falls and variety of trials with fast falls. In case a mouse didn’t cooperate with the take a look at, trials had been recorded as lacking.
Voiding assay
Cages had been ready by reducing a chunk of cosmos blotting paper, 360 gsm, to straightforward duplex cage dimensions. Shavings had been faraway from a clear cage, and the paper was taped to the underside of the cage. Meals was offered throughout this take a look at; nonetheless, water was eliminated to stop attainable leaking onto the blotting paper. Mice had been individually housed in a ready cage for 4 h. On the finish of the trial, the mice had been returned to their unique housing models, and papers had been eliminated and dried for two–4 h, earlier than being individually bagged. Papers had been shipped to Beth Israel Deaconess Medical Heart, the place they had been scanned with ultraviolet gentle to picture and quantify the void spots.
Residence cage wheel operating
Free-wheel-running knowledge had been collected at round 44, 96 and 144 weeks of age. Mice had been individually housed for no less than 36 h in a particular cage suited to deal with the Med Affiliate low profile operating wheel with a wi-fi transmitter. The meals hopper was eliminated to permit for seamless motion of the wheel, and meals was positioned onto the cage ground. The 15.5-cm-diameter plastic wheel sits at an angle on an digital base, which tracks the revolutions. The battery-powered base permits for steady monitoring of knowledge, which is then wirelessly transmitted, in 30 s intervals, to an area laptop.
Lifespan
Analysis employees frequently evaluated mice for prespecified scientific symptomology: palpable hypothermia, responsiveness to stimuli, capability to eat or drink, dermatitis, tumours, stomach distention, mobility, eye situations (equivalent to corneal ulcers), malocclusion, trauma and wounds of aggression. If mice met the factors for remark in any of those classes, veterinary employees had been contacted. If the scientific group decided a mouse to be palpably hypothermic and unresponsive, unable to eat or drink, and/or met protocol standards for extreme dermatitis, tumours and/or struggle wounds, pre-emptive euthanasia was carried out to stop struggling; in any other case, the veterinary employees offered therapy. Each mice euthanized or discovered lifeless had been represented as deaths within the survival curves. Mice euthanized on account of accidents unrelated to imminent loss of life had been handled as censored (we recorded a complete of 13 censoring occasions).
Knowledge preparation and evaluation
Cloud-based analysis administration software program (Climb by Rockstep) was used to trace animals, schedule testing and supply a steady repository for major knowledge assortment. Knowledge had been frequently reviewed by a statistical analyst in the course of the examine for anomalies. Preliminary knowledge high quality management included figuring out and resolving gear miscalibration, mislabelled animals and technically unimaginable values. If we couldn’t manually appropriate these utilizing laboratory information, they had been eliminated. Quantitative assays together with physique weight and temperature had been explored for outliers. Quantitative traits aside from physique weights had been corrected for batch results. To quantify batch results, we match a totally random-effects linear combined mannequin conditioning on weight loss plan, physique weight at take a look at and age. We adjusted trait values by subtracting the batch mannequin coefficients. Lifespan knowledge had been recorded in days however are offered in months (30.4 days monthly) for ease of interpretation. Statistical significance for very small P values is reported as P −16 in the primary textual content; non-truncated P values are offered within the Supplementary Info. All analyses had been carried out utilizing R v.4.2.2 and RStudio v.2022.12.0+353. Knowledge evaluation scripts can be found (Knowledge availability).
Survival evaluation
We carried out survival evaluation to check lifespan outcomes for the 5 examine teams. We plotted Kaplan–Meier survival curves and examined the equality of survival distributions throughout weight loss plan teams utilizing log rank assessments utilizing an general take a look at (4 d.f.) and all pairwise comparisons between diets. P values are reported with no a number of testing adjustment, and we thought-about a comparability to be vital if P 30. Mortality doubling occasions had been estimated from a Gompertz log-linear hazard mannequin with 95% CI and proportion change relative to the AL group (flexsurv R package deal v.2.2.2).
Longitudinal trait evaluation
For traits collected yearly or biannually, we had been capable of discover hypothesized direct and oblique relationships with weight loss plan, physique weight and age. We used generalized additive combined fashions (GAMMs) with a gaussian/identification hyperlink to analyse these results by becoming a collection of nonlinear relationships between trait response and covariates. GAMMs for a mix of fastened and random results (formulae under) on trait response pre-adjusted for batch had been match utilizing the gam() operate of the mgcv package deal in R, with the Newton optimizer and default management parameters. Age was rescaled to proportion of life lived (PLL = age at take a look at/lifespan). The PLL scale removes artifacts on account of survivorship bias throughout teams with completely different lifespans. All steady variables aside from PLL had been rank regular scores reworked (RZ) previous to mannequin becoming.
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(1)
RZ(T) ~ D + s(BW) + s(PLL) + (1|ID)
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(2)
RZ(T) ~ D + s(PLL) + (1|ID)
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(3)
RZ(T) ~ D + s(BW) + (1|ID)
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(4)
RZ(T) ~ s(BW) + s(PLL) + (1|ID)
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(5)
RZ(T) ~ D + s(BW) + s(PLL|weight loss plan) + (1|ID)
the place T is trait, D is dietary project, BW is physique weight on the date closest to T’s assortment date, PLL is the proportion of life lived as of T assortment date and s() is the smoothing parameter. Every mouse had a number of datapoints throughout the T assortment date. This clustering was accounted for with a random intercept for ID, specified as (1|ID) above. We carried out speculation assessments associated to the GAMM suits to discover trait sensitivity to physique weight (mannequin 1 (M1) versus M2), PLL, (M1 versus M3), weight loss plan (M1 versus M4) and diet-by-trait interplay (M1 versus M5). Utilizing the fashions specified above and a conservative false-discovery charge (FDR 
The whole outcomes of the longitudinal trait evaluation are offered in Supplementary Desk 6.
Trait associations with lifespan
To establish traits which might be related to lifespan, we carried out regression evaluation on lifespan with traits at every timepoint after adjusting for results of weight loss plan and physique weight. We match linear fashions:
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(1)
Lifespan ~ weight loss plan + BW6 + BWtake a look at,
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(2)
Lifespan ~ weight loss plan + BW6 + BWtake a look at + trait
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(3)
Lifespan ~ weight loss plan + BW6 + BWtake a look at + trait + weight loss plan:trait
the place BW6 is the final preintervention physique weight, and BWtake a look at is the physique weight at time of testing. For physique composition and change-in-body weight traits, we didn’t embody the physique weight phrases. All steady variables had been rank regular scores (RZ) reworked earlier than mannequin becoming. We carried out chance ratio assessments for the weight loss plan and physique weight adjusted affiliation (M2 versus M1) and for the weight loss plan × trait interplay (M3 versus M2). We utilized a FDR adjustment to every take a look at throughout traits and timepoints (one-step Benjamini–Hochberg technique). Traits had been categorized and vital (FDR 
We estimated the general energy of affiliation between trait and lifespan because the regression coefficient of the trait time period in M2. Because the traits are standardized, the regression coefficients are equal to diet- and body-weight-adjusted partial correlations (designated as r) and will be in contrast throughout traits. Furthermore, we stratified the information to estimate diet-specific body-weight-adjusted partial correlations which might be proven within the scatter plot panels (Fig. 2f).
The whole outcomes of lifespan affiliation evaluation are offered in Supplementary Desk 7.
Community modelling
We carried out a multivariate community evaluation to decompose the results of DR on lifespan throughout measured traits. An empirical covariance matrix was estimated utilizing the nonparanormal SKEPTIC estimator derived from the pairwise Kendall–Tau correlation utilizing pairwise-complete knowledge. The estimated covariance matrix was projected to the closest optimistic particular matrix by truncating eigenvectors with adverse eigenvalues. A sparse low-rank Gaussian graphical mannequin was fitted to this covariance estimate utilizing the ggLASSO Python package deal with the parameters lambda = 0.1, mu = 100. The outcome was normalized to acquire an inferred partial correlation matrix, which we use as our phenotype community for downstream evaluation.
To cluster the phenotypes, we constructed a weighted okay-nearest neighbours graph utilizing absolutely the partial correlation from above as similarity weights (that’s, we retained the okay-largest weights per node) utilizing okay = 10. The ensuing okay-nearest neighbours graph was clustered utilizing spectral clustering to acquire 20 clusters, which had been labelled by hand. Food plan and lifespan had been damaged out into their very own univariate clusters and the physique weight cluster was break up into lean tissue mass and adiposity (fats mass/whole mass) to account for physique composition results identified from the literature. This resulted in a summarized illustration of 23 clusters (weight loss plan, lifespan and 21 teams of traits, one per cluster plus FTM).
We obtained a covariance decomposition by recomputing a sparse low-rank community (as above) on the lowered illustration of 23 options. To acquire the relative significance of various results of DR on lifespan, we carried out covariance path decomposition48 of the covariance between weight loss plan and lifespan utilizing this lowered illustration graphical mannequin. Absolutely the values of path scores had been used to rank the relative significance of every path and normalized to sum to 1 to estimate the fraction of the covariance between DR and lifespan defined. To acquire community visualizations, the place of nodes and orientation of edges had been decided by computing max-flow by way of the trail community. We outline the trail community because the graph fashioned by taking the partial correlation community however reweighting edges to be the sum of all (absolute) path scores of paths that comprise that edge.
QTL mapping
Genetic mapping evaluation of all continuous-valued traits together with lifespan was carried out utilizing the R package deal qtl263. Additive covariates for the genome-scan fashions had been weight loss plan and physique weight variables as indicated in Supplementary Desk 5. Founder haplotype results had been estimated treating 8-state genotypes as random results. Trait heritability was estimated utilizing the R/qtl2 operate est.herit() to suit a linear combined mannequin together with an additive kinship matrix. Confidence intervals had been obtained by parametric bootstrap. Significance thresholds for QTL mapping had been estimated from 1,000 permutations of the trait and covariate knowledge; vital QTL exceed the genome-wide multiple-test adjusted 0.05 threshold (7.5) and suggestive QTL exceed the unadjusted 0.05 threshold (6.0)64.
Reporting abstract
Additional data on analysis design is accessible within the Nature Portfolio Reporting Abstract linked to this text.
