Mice and Semaglutide Treatment
Female C57BL/6 mice aged 20 months were obtained from the National Institute on Aging. After arrival, the mice were acclimatized to the facility for 1 week before baseline measurements and experimental treatments began. As described previously12, mice were group housed, consistent with standard practice for calorie-restriction studies. Animals were maintained under a 12 h–12 h light–dark cycle at 20–26 °C and 30–70% humidity.
Control and semaglutide-treated mice had ad libitum access to water and standard laboratory chow (LabDiet, Rodent Diet 5053). Calorie-restricted mice had unlimited access to water and received a measured amount of food each day. To minimize competition, food pellets were placed directly on the cage floor so that individual mice could access them. The degree of calorie restriction was based on the mean reduction in food intake produced by semaglutide and was not adjusted during the study.
Mice received daily subcutaneous injections of semaglutide at 10 nmol per kg or an equivalent volume of saline. In the lifespan study, 39 mice received saline and 40 mice received semaglutide throughout their lifespan. A separate cohort was treated for 3 months for physiological, molecular and cellular analyses, including 10 pairs for physiological assessments, five pairs for neural stem cell studies requiring tissue fixation and six pairs for other molecular and cellular analyses. For the longitudinal study, another cohort received saline, semaglutide or calorie restriction for 5 months, with 10 mice per group. Measurements were collected at baseline and after 2 or 4 months of treatment.
Subcutaneous injections were administered according to the standard operating procedure approved by the animal care committee at the University of California, Berkeley. Briefly, injections were performed daily with a 28-gauge needle in the dorsal subcutaneous region. A new needle was used for each mouse to reduce discomfort. Injection sites were systematically rotated across the dorsal surface to minimize local tissue irritation and the risk of chronic inflammation. No single site was used on consecutive days. Mice were monitored daily for erythema, swelling, ulceration and other tissue damage. No adverse effects were observed at any timepoint, and on-site veterinarians monitored animal health. All procedures complied with the requirements of the University of California, Berkeley animal care committee.
Mouse Lifespan Assessment
Mouse lifespan was assessed as described previously12. Mice were examined daily for survival and clinical signs of illness. Animals found dead were recorded during daily inspections. Severely moribund mice were euthanized and recorded as deaths. Severe morbidity was defined by the presence of any of the following: (1) tumour formation; (2) untreatable wounds; (3) untreatable skin ulceration or abscesses; (4) severe rectal prolapse; (5) inability to eat or drink, a condition interfering with eating or drinking, or lack of response to stimulation; (6) severe dehydration; (7) respiratory distress, agonal breathing or cyanosis; (8) paralysis or paresis; (9) uncontrollable haemorrhage; (10) progressive hypothermia that could not be relieved; or (11) a body condition score of <2 out of 5.
Consistent with established procedures12, both euthanized mice and mice found dead were classified as deaths in survival-curve analyses. No animals were removed from the study for reasons unrelated to age-associated decline or censored in the survival analysis.
Mouse Body Composition Analysis
Mouse body composition was measured using an EchoMRI-100V Body Composition Analyzer (EchoMRI). Fat mass and lean mass were recorded for each animal.
Open-Field Behavioural Test
As described previously33, mice were acclimatized to the testing room under normal lighting for 1 h before testing. Each mouse was placed in the centre of a 50 × 50 cm plastic chamber and allowed to explore freely for 15 min. Activity was recorded with a digital video camera and analysed using EthoVision XT14 (Noldus Information Technology). The central zone was defined as the area located from the wall at a distance equivalent to the length of the mouse.
Elevated Plus-Maze Test
As described previously33, mice were acclimatized to the behavioural testing room under red light for 1 h. The elevated plus maze consisted of two open arms and two closed arms. Mice were placed in the centre of the maze facing one of the closed arms and allowed to explore freely for 10 min. Activity was recorded with a digital video camera and analysed using EthoVision.
Barnes Maze Spatial Memory Test
As described previously35, the Barnes maze consisted of a 92 cm-diameter circular acrylic platform with 20 evenly spaced holes, each 5 cm in diameter. One hole contained a target escape box. Testing comprised three phases: habituation on day 1, training on days 2–3 and a probe trial on day 5. Mice were acclimatized to the behavioural testing room for 1 h before each session.
On day 1, each mouse was placed beneath a clear beaker at the centre of the maze for 30 s while noise was presented using a 65 dB, 1 Hz metronome. The mouse was then guided towards the target hole and given 3 min to enter the escape box. If it failed, the mouse was placed directly into the box for 1 min with the noise turned off.
On days 2–3, mice were placed beneath an opaque beaker for 10 s before release. After the beaker was removed, the noise was initiated and the mouse was given 2 min to locate the escape box. If unsuccessful, the mouse was guided to the target hole using a clear beaker and given an additional 3 min to enter the box. If the mouse still did not enter, it was placed directly into the box for 1 min with the noise turned off. On day 5, a single 2 min probe trial was conducted with the noise turned on and the escape box removed to assess spatial memory. All trials were video-recorded, and day 5 videos were analysed using EthoVision.
Accelerating Rotarod Motor Coordination Test
As described previously22, mice were placed on a rotating rod 3 cm in diameter and elevated 44.5 cm above the floor (Rotamex-5, Columbus Instruments). During training on days 1–3, mice completed habituation trials at a constant speed of 4 rpm for 60–300 s. On test day 4, mice were acclimatized to the testing room for 30 min. The accelerated rotarod test consisted of three trials in which the speed increased from 4 to 40 rpm over 300 s. Trials were separated by 1 h. Infrared sensors automatically recorded the latency to fall, and the mean fall latency was calculated for each mouse.
Inverted Screen Strength Test
As described previously35, mice were acclimatized to the testing room for 30 min. Each mouse was placed in the centre of a wire cage lid, which was then inverted and suspended 40–50 cm above a padded surface. Timing began when the mouse assumed a fully inverted position. The latency to fall was recorded. Each mouse completed three trials separated by 1 h, and the mean fall latency was calculated.
Treadmill Exhaustion and Exercise Capacity Test
As described previously24, mice were tested on an Exer-6M Open Treadmill (Columbus Instruments). Animals were food deprived for 2 h before training on days 1 and 2 and before testing on day 4.
On day 1, mice were habituated on the stationary treadmill for 30 s and then ran for 10 min with the following stepwise speed increases: 5 m min−1 from 0–2.5 min, 6 m min−1 from 2.5–5 min and 8 m min−1 from 5–10 min. On day 2, the protocol was repeated at 5 m min−1 from 0–2.5 min, 7 m min−1 from 2.5–5 min and 10 m min−1 from 5–10 min.
On day 4, mice were habituated for 30 s and began running at 12 m min−1 for 40 min. The treadmill speed was then increased by 1 m min−1 every 10 min. Exhaustion was defined as an inability to resume running for at least 20 s despite gentle prodding. Running time and distance were recorded.
Glucose Tolerance Test
As described previously25, mice were fasted for 14 h with free access to water. Baseline blood glucose was measured from tail-vein blood using a CONTOUR NEXT glucometer. Mice then received an intraperitoneal injection of d-glucose at 2 g per kg body weight, followed by additional blood glucose measurements.
Metabolic Cage Measurements
Metabolic parameters were measured using the Oxymax Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments).
Liver RNA Sequencing and Bioinformatics Analysis
Total RNA was extracted from liver samples using the RNeasy Mini Kit (Qiagen). Poly(A)-enriched RNA-sequencing libraries were prepared by Novogene and sequenced on the NovaSeq X Plus platform (Illumina). Bioinformatics analysis was performed using the Galaxy public server (https://usegalaxy.org).
Low-quality reads and adaptor sequences were removed before alignment to the mouse mm10 genome using HISAT2 (Galaxy v.2.2.1). Gene-level raw read counts were generated with HTSeq (Galaxy v.2.0.5). Differential gene expression was analysed using DESeq2 (Galaxy v.2.11.40.8), and volcano plots were produced with the Volcano Plot tool (Galaxy v.0.0.7). Pathway enrichment analysis was performed using GSEAPy (v.1.0.6). Transcription-factor enrichment analysis was conducted with TRRUST using differentially expressed genes with Padj < 0.1. Overlapping differentially expressed genes induced by semaglutide, ageing42 or calorie restriction42 were analysed for Gene Ontology enrichment using the enrichGO function in clusterProfiler (v.4.16.0).
Reverse-Transcription Quantitative PCR
As described previously40, total RNA was extracted using TRIzol reagent (Invitrogen). Complementary DNA was synthesized with qScript cDNA SuperMix (Quanta Biosciences). Gene expression was measured by quantitative PCR using the Eva qPCR SuperMix kit (BioChain Institute) on an ABI StepOnePlus system. Expression values were normalized to actin. Primer sequences are provided in Supplementary Table 2.
NAD+ and NAD+/NADH Measurement
Tissues were homogenized in PBS/bicarbonate/0.5% DTAB buffer. NAD+ and NADH concentrations were quantified using NAD/NADH-Glo (Promega). Acid-treated samples were used to measure NAD+, whereas base-treated samples were used to measure NADH. Luminescence was detected using a SpectraMax i3 plate reader (Molecular Devices).
ATP Quantification
ATP concentrations were measured using an ATP Assay Kit (Sigma-Aldrich). Protein concentration was determined with the BCA Protein Assay (Thermo Fisher Scientific), and ATP levels were normalized to total protein content.
Plasma IGF1 Measurement
As described previously41, plasma samples were pretreated with an acid–ethanol extraction solution to release IGF1 from binding proteins. Plasma IGF1 concentrations were measured using a mouse IGF1 ELISA Kit (Invitrogen).
Western Blot Analysis
As described previously41, tissues were homogenized in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using a BCA Protein Assay Kit. Equal amounts of protein were separated on 12% SDS–PAGE gels and transferred to nitrocellulose membranes (Bio-Rad).
Membranes were blocked with 5% BSA for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: phospho-AKT (Ser473) antibody (CST, 9271, 1:1,000), AKT antibody (CST, 9272, 1:1,000), HSP90 antibody (CST, 4877, 1:1,000), p-eIF2α (Ser52) polyclonal antibody (Invitrogen, 44-728G, 1:1,000), eIF2α antibody (CST, 9722, 1:1,000), GRP78 antibody (Santa Cruz, 166490, 1:1,000) and β-actin antibody (Santa Cruz, 47778, 1:2,000).
On the following day, membranes were incubated with horseradish-peroxidase-conjugated secondary antibodies (BioLegend, 406401 and 405306, 1:4,000) for 2 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence substrate (PerkinElmer, NEL103001EA) and an iBright CL1500 Imaging System (Invitrogen). Band intensity was quantified using Fiji/ImageJ (v.1.54p).
Immunostaining and Neural Stem Cell Analysis
Cryosections were fixed, permeabilized, blocked and incubated overnight at 4 °C with the following primary antibodies: anti-CD68 (BioLegend, 137001, 1:200), anti-IL-6 (CST, 12912S, 1:200), PE-CD11b (BioLegend, 101208, 1:200), FITC-CCR2 (BioLegend, 150607, 1:100) and anti-γ-H2AX (CST, 2577, 1:200). Sections stained for CD68, IL-6 and γ-H2AX were then incubated with secondary antibodies (Thermo Fisher Scientific, SA5-10018 (1:500), A-11036 (1:500) and A32731 (1:2,000)) for 2 h at room temperature. DAPI was used to stain nuclei. Images were acquired with a Zeiss LSM 880 confocal microscope. Five randomly selected fields per mouse were analysed using Fiji/ImageJ.
For brain-section staining26,33, mice received daily intraperitoneal injections of BrdU at 50 mg per kg body weight for 3 days and were euthanized on day 4. Mice were perfused with 10 ml of PBS containing 10 U ml−1 heparin, followed by 40 ml of PBS containing 4% paraformaldehyde. Brains were dissected and post-fixed overnight at 4 °C in PBS containing 4% paraformaldehyde. They were then incubated overnight at 4 °C in PBS containing 15% sucrose and subsequently overnight in PBS containing 30% sucrose. Brains were sectioned coronally at 40 μm using a cryomicrotome (Leica) and stored in cryoprotective medium.
For immunostaining, brain sections were treated with 2 N HCl at 37 °C for 30 min to detect BrdU. Sections were blocked for 2 h at room temperature and incubated overnight at 4 °C with anti-BrdU (Abcam, ab6326, 1:500), anti-DCX (Abcam, ab18723, 1:750), anti-HSP60 (CST, 12165, 1:500), anti-MCM2 (BD Biosciences, 610700, 1:250) and anti-SIRT7 (21st Century Biochemical, custom antibody, 1:500). The next day, sections were incubated with the corresponding secondary antibodies (Thermo Fisher Scientific, A11006, A32731, A32733 and A-11029; 1:1,000).
Immunopositive cells in the granule cell and subgranular cell layers of the dentate gyrus were counted in every sixth coronal hemibrain section. The mean number of positive cells per section was calculated, multiplied by the total number of sections and then multiplied by 2 to estimate total cell numbers across both dentate gyri. Double-positive cells were identified by co-localization of HSP60 or SIRT7 and MCM2 signals surrounding the same DAPI-stained nucleus. The proportion of activated neural stem cells expressing HSP60 or SIRT7 was calculated as the ratio of HSP60+MCM2+ or SIRT7+MCM2+ double-positive cells to the total number of MCM2+ cells.
Senescence-Associated β-Galactosidase Staining
Senescence-associated β-galactosidase (SA-β-gal) staining was performed using the Senescence beta-Galactosidase Staining Kit (CST, 9860). Briefly, cryosections were fixed with the supplied fixative for 15 min at room temperature. Following fixation and washing, sections were incubated overnight at 37 °C in freshly prepared β-galactosidase staining solution. Images were acquired with a Zeiss Axio Imager M2 microscope. Five randomly selected fields per mouse were analysed using Fiji/ImageJ.
Colony-Forming Unit Assay
A total of 2 × 104 bone marrow cells were resuspended in MethoCult GF M3434 medium (StemCell Technologies) and cultured in a cell incubator. Colony-forming units (CFUs) were counted on day 12. Bright-field images were captured using a Zeiss Axio Imager M2 microscope, and colony areas were quantified with Fiji/ImageJ.
Flow Cytometry and Hematopoietic Stem Cell Sorting
For hematopoietic stem cell (HSC) analysis, bone marrow cells were obtained by crushing long bones in staining medium consisting of PBS with 2% FBS. Cells were stained for 20 min at 4 °C with APC/Cy7-conjugated lineage antibodies, KIT-APC, SCA1-PB, CD48-FITC and CD150-PE antibodies (BioLegend, 101226, 108424, 116223, 100222, 100414, 100714, 103224, 105812, 108120, 103404, 115904; 1:100). Lineage markers included MAC1 (CD11b), GR1 (Ly-6G/C), Ter119 (Ly-76), CD3, CD4, CD8a (Ly-2) and B220 (CD45R) (BioLegend).
For the analysis of lineage-biased HSCs, bone marrow cells were stained for 20 min at 4 °C with PerCP/Cy5.5-conjugated lineage antibodies, KIT-APC/Cy7, SCA1-PB, CD150-PE, CD135-APC and CD34-FITC antibodies (BioLegend, 101228, 108428, 116228, 100218, 100434, 100734, 103236, 105826, 108120, 115904, 135310; eBioscience, 11-0341-81; 1:100).
For MitoSOX analysis, cells were incubated with 5 µM MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific) for 30 min at 37 °C in the dark after HSC staining with the indicated antibodies (BioLegend, 101226, 108424, 116223, 100222, 100414, 100714, 103224, 105812, 108120, 115916; 1:100).
For peripheral-blood lineage analysis, blood was collected from the submandibular vein into EDTA-treated tubes (BD). Samples were lysed with 500 µl of 1× BD FACS lysing solution for 5 min at room temperature. Lysis was stopped by adding 3 ml of PBS. Cells were then stained with MAC1-PE, GR1-FITC, B220-APC and CD3-PB antibodies (BioLegend, 101208, 108406, 103212, 100214; 1:100) for 20 min at 4 °C. Data were acquired using an LSRFortessa flow cytometer (BD) and analysed with FlowJo (v.10.9.0).
For HSC sorting, bone marrow cells were lysed with ACK buffer and enriched for KIT+ cells using KIT microbeads (Miltenyi Biotec). KIT-enriched cells were stained for 20 min at 4 °C with APC-Cy7-conjugated lineage antibodies, KIT-APC, SCA1-PB, CD48-FITC and CD150-PE antibodies (BioLegend), followed by staining with propidium iodide at 0.5 µg ml−1. Live HSCs were sorted using a FACSAria cell sorter (BD).
Statistical Analysis
Sample sizes were selected according to the principle of using the minimum number of mice required to achieve sufficient statistical power and were comparable to those reported in the literature for the same assays. Mice were randomized into treatment groups. Investigators were blinded to treatment during data collection and analysis of mice and tissue samples, except for calorie-restricted mice, whose feeding regimens were distinguishable. Measurements were obtained from distinct samples.
Experiments were repeated twice, except for Fig. 3. Findings in Fig. 3 were supported by experiments assessing ageing hallmarks, genetic regulators of ageing and nutrient sensors in Figs. 2 and 4 and Extended Data Figs. 3–6. Statistical analyses were performed using GraphPad Prism 10, Excel (v.16.84) and jamovi (v.2.7.26).
Data normality was assessed using the Shapiro–Wilk test. For normally distributed data, homoscedasticity was evaluated using an F-test or Brown–Forsythe test. Non-normally distributed continuous data involving two groups were analysed using the Mann–Whitney U-test. Student’s t-test was used for data with equal variances, whereas Welch’s t-test was used for data with unequal variances.
For continuous data involving three groups, non-parametric data were analysed using the Kruskal–Wallis test followed by Dunn’s multiple-comparison test. Normally distributed data with equal variance were analysed using one-way ANOVA followed by Tukey’s multiple-comparison test. Normally distributed data with unequal variances were analysed using Welch’s ANOVA followed by Games–Howell’s multiple-comparison test. Covariate-adjusted analyses were performed using ANCOVA.
Count data were analysed using generalized linear models with Poisson or negative binomial distributions when overdispersion was present. Percentage data were analysed using beta regression. Glucose-tolerance test data were analysed using two-way repeated-measures ANOVA followed by Sidak-adjusted pairwise comparisons between groups at each timepoint. Lifespan data were analysed using the log-rank test. Categorical distribution data were analysed using the Fisher–Freeman–Halton exact test.
Weekly body-weight data were analysed using linear mixed-effects models followed by Holm-adjusted pairwise comparisons between groups at each timepoint. Other longitudinal continuous data were analysed using linear mixed-effects models to assess ageing trajectories. Group-specific slopes were calculated and compared pairwise using Tukey adjustment. Longitudinal percentage data were analysed using β mixed-effects models to assess ageing trajectories, with pairwise slope comparisons performed using Tukey adjustment. Data are presented as means, and error bars represent the s.e.m.
Research Reporting Summary
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