Animals

Young (4 mo) and old (18 mo) male and female CB6F1 mice were obtained from the NIA Aged Rodent Colony. All animals were housed at standard temperature (~22°C) and humidity-controlled conditions under a 14L:10D photoperiod and provided ad libitum access to water and a low-fat-purified diet upon arrival (10% calories from fat D12450H Research Diets Inc). In vivo L2-Cmu validation studies were performed in 4–5-mo-old C57BL/6J male mice (Jackson Labs, Bar Harbor, ME). All experiments were approved by the Institutional Animal Care and Use Committee at the Albert Einstein College of Medicine (Protocol #20140107).

L2-Cmu development and validation

To target IGF-1R action in mice, we utilized the mAb, L2-Cmu (Amgen Inc, Thousand Oaks, CA), which is a murinized IgG1 version of the fully human L2-C mAb previously reported by Calzone et al.25. Specifically, L2-Cmu was engineered as a human/murine chimera (i.e., reverse chimeric antibody), such that the complementary-determining regions (CDRs) were engineered into a new framework in which the entirety of the variable regions are human and the constant regions are mouse. Unlike the human IgG1 constant region of L2-C, which might induce effector functions, the murine IgG1 constant region would not be expected to elicit effector function activity. Validation of L2-Cmu was confirmed by Biacore analysis and in murine fibroblasts (NIH-3T3) in vitro. For NIH-3T3 experiments, cells were grown in DMEM plus 10% fetal bovine serum (Invitrogen). At t = −4 h, cells were serum starved and at t = −1 h, pre-treated with vehicle, control IgG1, or L2-Cmu (100 µg/mL). Vehicle IGF-1 (5 nM) or IGF-2 (20 nM) was then added to the media for 2 min and cells were then rapidly lysed in ice-cold buffer to assess receptor activation as described below.

For in vivo validation of L2-Cmu blockade, C57BL/6J mice were sedated with 2% isoflurane for surgical placement of an indwelling catheter into the right internal jugular vein45. Animals were then assigned to receive either an initial i.p. injection of L2-Cmu (n = 6) at a dose of 20 mg/kg or vehicle (n = 9) 5 days after surgery. On day 7, animals were fasted at 0900 h and then received a second i.p. injection of either L2-Cmu or vehicle. Approximately 4 h later, animals were assigned to receive either a peripheral saline (Control n = 4) or IGF-1 i.v. infusion (5 µg total dose) over 1 min at a rate of 100 µL/min, resulting in three experimental groups: Control (vehicle + saline; n = 4), IGF-1 (vehicle + IGF-1; n = 5), and IGF-1 plus L2-Cmu (n = 5 for lung and n = 6 for heart). Animals were then quickly sacrificed 1 min after completion of the infusion, and tissues rapidly excised, snap-frozen, and stored at −80 °C for receptor activation assays.

Experimental design and treatment

CB6F1 mice were assigned to receive either weekly i.p. injections of vehicle or L2-Cmu (20 mg/kg) once per week and were monitored for interim survival to 24 mo of age (n = 24–38 per group) and sacrificed for blood, tissue, and histopathologic analysis. For hormone and metabolite analyses, animals were fasted at 0800 h, and trunk blood was routinely collected 4 h later at 1200 h by decapitation without anesthesia, and plasma was separated from red cells by centrifugation (1500 × g × 4 °C × 15 min) and stored at −80 °C. For the survival study, animals were treated with i.p. vehicle or L2-Cmu until natural death (females only; n = 45 group). Animals deemed severely moribund and anticipated to not survive another 24 h were immediately euthanized and this was considered the time of death. In addition, histopathology was conducted at 24 mo of age or end of life, as described below.

L2-Cmu pharmacokinetic and chronic exposure assessments

For PK studies, CB6F1 mice (n = 4–5 per sex, per timepoint) were injected i.p. with L2-Cmu (20 mg/kg) and sacrificed at either 6 h, 24 h, 3 days or 7 days later, and plasma was isolated from whole blood. A baseline group was included, which was injected with vehicle 6 h prior to sacrifice. For determining chronic exposure levels, plasma was obtained 48 h following dose 24 of vehicle or L2-Cmu by i.p. injection in male Con (n = 9) and L2-Cmu (n = 34) and female Con (n = 9) and L2-Cmu (n = 30) mice. L2-Cmu plasma concentrations were measured using an ELISA method with an LLOQ of 0.1 µg/mL and assay range of 0.1–20 µg/mL. Briefly, the ELISA plate (Corning 3690) was coated with murine anti-AMG 479, clone 3 at 2 µg/mL in 1× PBS and incubated overnight at 4 °C. Standards and quality controls were prepared by spiking L2-Cmu into mouse plasma. The ELISA plate was washed and blocked with I-BlockTM (Applied Biosystems) buffer (1× PBS + 0.2% I-Block + 0.05% Tween20) for 1 h at room temperature. Mouse plasma was used as a diluent for samples when necessary. The standards and samples were then diluted 1:4 in assay buffer (I-Block buffer + 5% BSA). The diluted standards and samples were loaded into the ELISA plate and allowed to incubate for 1.5 h. After a wash step, a horseradish peroxidase (HRP) conjugated murine anti-AMG 479, clone B35 at 400 ng/mL was added to the plate and incubated for 1.5 h. After a final wash step, a tetramethylbenzidine (TMB) peroxidase substrate solution (KPL Inc.) was added to each well and absorbance of the developed color was measured by a colorimetric plate reader (Molecular Device).

Metabolic phenotyping

Body weight was monitored on a weekly basis and body composition was assessed at 3 mo intervals by qMR (ECHO MRS; Echo Medical Systems). Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed (n = 12 per group, per sex) to assess glucose metabolism and insulin sensitivity 46. For GTTs, animals were fasted for 4 h and a baseline blood glucose measurement was made prior to administering a 2 mg/kg i.p. glucose injection. Blood glucose was subsequently monitored at 15, 30, 60, 90, and 120 min post injection with a glucose meter (Bayer Contour). ITTs were performed in random-fed mice, early in their light cycle (~0700h–0800h)46. Following a baseline glucose measurement, mice were injected. i.p. with 0.75 U/kg insulin and blood glucose was measured at 15, 30, 45, and 60 min later.

Energy expenditure, substrate utilization, food intake, and spontaneous activity were determined45,47, based upon O 2 consumption and CO 2 production, using a Mouse CLAMS System (Columbus Instruments, Columbus, OH). In brief, animals (n = 8 per group, per sex) were placed into individual cages at their standard temperature and photoperiod and allowed to acclimate for at least 72 h prior to the experiment, and data were collected over a 24 h period.

Functional healthspan assessments

At 23–24 mo of age, motor coordination, strength, and endurance were evaluated in mice using a battery of healthspan assessments. Neuromuscular function was determined via balance beam in females [Young (n = 8), Old Con (n = 12), and Old mAb (n = 15)] and males [Young (n = 8), Old Con (n = 9), and Old mAb (n = 9)]. In brief, animals were first familiarized with walking across a 4 ft plank prior to testing three round beams of increasing difficulty (1″ easy; 0.75″ medium, 0.5″ difficult), with light and food cues as motivation to cross, and the number of slips were counted while traversing the beam48. Forelimb grip strength was determined by allowing animals to clasp a suspended wire and the time to release was recorded in females [Young (n = 8), Old Con (n = 12), and Old mAb (n = 15)] and males [Young (n = 8), Old Con (n = 10), and Old mAb (n = 9)]. Exercise capacity was determined by a single maximal exercise test to voluntary fatigue on a motorized treadmill (Exer 3/6, Columbus Instruments) in females [Young (n = 10), Old Con (n = 17), and Old mAb (n = 22)] and males [Young (n = 10), Old Con (n = 9), and Old mAb (n = 9)]. Mice were first familiarized to the treadmill for three non-consecutive days for 5 min at a walking speed (8 m/min). Animals were then challenged with a graduated fatigue test, beginning at 10 m/min and 4% grade for 3 min, and increasing in speed by 2 m/min every 2 min to a max speed of 16 m/min until exhaustion.

Cardiovascular phenotyping

Systolic and diastolic function was evaluated following 5–6 mo of treatment19,42. In brief, mouse electrocardiography was measured in females [Young (n = 6), Old Con (n = 8), and Old mAb (n = 7)] and males [Young (n = 5), Old Con (n = 6), and Old mAb (n = 5)], using a Visual Sonic Vevo2100 imaging system (FUJIFILM VisualSonics Inc, Toronto, ON). Cardiac left ventricular dimensions were obtained under M-mode, and left ventricle ejection fraction (EF) and fractional shortening (FS) were calculated accordingly. Left ventricular diastolic function, presented as the E/A ratio, was generated based on transmitral blood flow measured under Color Doppler mode. At sacrifice, heart tissue was immediately harvested, and the heart was perfused and fixed with 10% neutral-buffered formalin (NBF) for 24 h. Following fixation, hearts were embedded in paraffin and 5 μm sections were mounted onto treated slides, and stained with hematoxylin and Eosin (H&E) and co-stained with Masson’s trichrome. Tissue fibrosis was quantified in females [Young (n = 4), Old Con (n = 5), and Old mAb (n = 5)] and males [Young (n = 8), Old Con (n = 8), and Old mAb (n = 8)], by counting blue stained interstitial collagen within three random fields using Image J and values were averaged.

Metabolomic analysis

We used Biocrates AbsoluteIDQ p180 kit to analyze cardiac metabolites from female mice [Young (n = 7), Old Con (n = 8), and Old mAb (n = 7)] with UPLC-MS/MS Xevo TQ, Waters, Pittsburgh, PA, USA) in the Einstein Stable Isotope and Metabolomics Core, according to the manufacturer’s instructions (BIOCRATES Life Sciences AG, Innsbruck, Austria). Heart tissue samples were weighed, homogenized with 8 times of 2.5 mM ammonium acetate in methanol, and 20 µL of the extraction from each sample was used for the assay. A pooled quality control (QC) sample was added to the sample list. This QC sample was plated at different positions on the 96-well plate and injected six times to calculate the coefficient of variation (CV) for data quality control, and undetectable metabolites were excluded from the analysis. The dataset was then imported into R software [R version 3.4.2] and normalized using log transformation for multivariate analysis, unsupervised principle component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA).

Histopathology

Complete histopathology was performed in 24-mo-old female [Old Con (n = 16), and Old mAb (n = 16)] and male mice [Old Con (n = 15), and Old mAb (n = 17)] following 6 mo of mAb treatment, as well as in female mice at death from the longevity study [Old Con (n = 30), and Old mAb (n = 20)]. In brief, a gross evaluation was conducted when possible and then a complete necropsy was performed. Tissues were infiltrated with paraffin and H&E sections were obtained. Slides were shipped to the University of Texas at San Antonio Pathology Core and evaluated by two pathologists who were blinded to the experimental groups (Y.I. and G.B.H.). Diagnosis of each histopathological change was made using histological classifications for aging mice33,49,50. In brief, a list of lesions was compiled for each mouse that included both neoplastic and non-neoplastic diseases. Based on these histopathological data, tumor burden, disease burden, and severity of lesions in each mouse were assessed.

Tumor burden was calculated as the sum of the different types of tumors in each mouse. The disease burden was similarly calculated as the sum of the histopathological changes in a mouse and severity of neoplastic and renal lesions was assessed using an established grading system. The probable cause of death was determined independently by both pathologists based on the severity of the pathology found at necropsy. In cases with neoplastic lesions, mice with Grade 3 or 4 lesions were categorized as death by neoplastic disease. In more than 90% of cases, there was agreement by the two pathologists. In cases where the two pathologists did not agree or where disease did not appear severe enough, the cause of death was categorized as unknown.

Blood measures

Clinical blood chemistries and related measures were determined in whole blood and serum of female [Old Con (n = 7) and Old mAb (n = 8)] and male mice [Old Con (n = 7) and Old mAb (n = 8)] by Antech Diagnostics (New Hyde Park, NY). Basal insulin was measured in plasma from female [Young (n = 16), Old Con (n = 26), and Old mAb (n = 25)] and male mice [Young (n = 17), Old Con (n = 32), and Old mAb (n = 28)] using a bead-based assay for mouse insulin (EMD Millipore, Inc) and detection was performed on a Bio-Plex MAGPIX Multiplex Reader (Biorad Inc., Hercules, CA). Plasma IGF-1 levels were measured using the Mouse/Rat IGF-1 Quantikine ELISA Kit (MG100; R&D Systems) in plasma from female [Young (n = 8), Old Con (n = 16), and Old mAb (n = 15)] and male mice [Young (n = 8), Old Con (n = 15), and Old mAb (n = 16)]. In addition, a MAGPIX Multiplex Reader was used to measure 25 inflammatory mediators simultaneously in plasma from female [Young (n = 8), Old Con (n = 15), and Old mAb (n = 16)] and male mice [Young (n = 8), Old Con (n = 14), and Old mAb (n = 16)], including: G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, CXCL-10, CXCL-1, MCP-1, MIP-1α, MIP-1β, MIP-2, RANTES, and TNF-α (MCYTOMAG-70K-PMX; EMD Millipore, Billerica, MA).

Protein isolation and western blotting

For standard western blotting, tissues were lysed in RIPA buffer and extracted protein content was determined using the BCA protein assay (Sigma, St. Louis, Mo) with BSA as a standard 45,51. In brief, protein was separated on Bis Tris Stain-Free gels (4–20%) and electrophoresed at 120 V constant for 90 min (n = 8 per group, per sex)45,51. Prior to transfer, stain-free gels were imaged on a Biorad Chemidoc MP Imaging System (Biorad, Hercules, CA) to confirm equal protein load, and were then wet transferred onto PVDF membranes at 100 V constant for 1 h and equal transfer was routinely confirmed by Ponceau S stain. Following block in 5% milk or BSA, membranes were incubated with an appropriate primary antibody from Cell Signaling (Danvers, MA) against pAktSer473 (1:1000; #4060), total Akt (1:1000; #4691), p-p44/42MAPKThr202/Tyr204 (1:1000; #9101) total p44/42 MAPK (1:1000; #4695), pS6 (1:1000; #5364), Total S6 (1:1000; #2217), total IGF-1R (1:1000; #9750), and InsRβ (1:1000; #3025) overnight at 4 °C. Following a 1 h incubation with the appropriate secondary antibody, Clarity Western ECL Substrate was applied to the membrane and bands were visualized using a Biorad Chemidoc MP to first pixel saturation and densitometry performed using Image Lab (Biorad, Hercules, CA). Complete, uncropped western blot images from figures are provided in Supplementary Fig. 7.

Immunoprecipitation

For immunoprecipitation assays, NIH-3T3 cell or tissue protein, respectively, was extracted with a non-denaturing cell or tissue extraction buffer (Invitrogen/ThermoFisher, Carlsbad, CA). Immunoprecipitation was then performed using the Catch and Release Immunoprecipitation Kit (EMD Millipore), according to the manufacturer’s instructions with 250 µg of total protein from cells or 400 µg from tissues, and 1 µg of an anti-IGF-1R antibody (#9750, Cell Signaling). Following electrophoresis and transfer, membranes were blotted with either a pTyr antibody (1:1000; #8954, Cell Signaling) for IGF-1R activation, or an anti-IGF-1R antibody (#9750, Cell Signaling) for total levels. For IGF-1R/InsR HybridR activation, IGF-1R immunoprecipitates were probed with an antibody against the InsR specific pTyr1334 residue (#44809G, Invitrogen/ThermoFisher)52, and total HybridR determined by immunoblotting with an Anti-InsRβ antibody (#3025).

Statistics

All values are presented as means ± SE. Longitudinal data were assessed using a linear mixed effects model, with group as a categorical variable and time as a continuous variable, and cross sectional data were assessed either by independent samples t-test or one-way ANOVA. When a significant effect in the model was observed, planned two-group contrasts (Tukey Honest Significant Difference [HSD] method) were applied. Normality assumption was examined prior to analysis and data were log transformed when appropriate to ensure normality of distribution. When the normality assumption was uncertain, as was determined for plasma cytokines, non-parametric Kruskal–Wallis test and Mann–Whitney U test were used. The total frequency and grade of pathologic lesions were compared between genotypes using a chi-square test. When the expected frequencies were too small in any group (n < 5), the Fisher’s exact test was used instead. Survival analysis was performed by the UAB Comparative Data Analytics Core. As animals were obtained in six separate batches due to operational limitations and grouped in cages, the survival curves were plotted using the Kaplan–Meier method. The treatment effect was then evaluated using Cox proportional hazard regression, with cage assignment included as a random effect and batch as a covariate, using statistical software R (version 3.4.1) with package “coxme”. For testing the difference in mean and median lifespan between groups, we used linear mixed model with linear quantile mixed-effects model, which included cage assignment as a random effect and batch as a covariate, respectively, using package “nlme” and “lgmm” in statistical software R. Effects on maximum lifespan were determined by setting the threshold for lifespan to the 90th percentile for both groups combined53. All other statistical analyses were performed using either SPSS (SPSS Inc, Chicago, IL) or JMP software version 9 (SAS Institute Inc., Cary, NC). For metabolites, a nominal P ≤ 0.05 and FDR ≤ 0.05 was considered significant, and a P ≤ 0.05 and FDR ≤ 0.15 considered marginally significant. For all other analyses, P ≤ 0.05 was considered statistically significant.

Data availability

All data supporting the findings of this study are included in this published article and its Supplementary Information files, and are available from the corresponding author upon request. Datasets for survival, metabolomics, and cytokines are available here: [https://doi.org/10.17605/OSF.IO/8QGX9]

Code availability

The R code for survival and metabolomic analyses are available here: [https://doi.org/10.17605/OSF.IO/8QGX9]