Late-life targeting of the IGF-1 receptor improves healthspan and lifespan in female mice

Abstract

Diminished growth factor signaling improves longevity in laboratory models, while a reduction in the somatotropic axis is favorably linked to human aging and longevity. Given the conserved role of this pathway on lifespan, therapeutic strategies, such as insulin-like growth factor-1 receptor (IGF-1R) monoclonal antibodies (mAb), represent a promising translational tool to target human aging. To this end, we performed a preclinical study in 18-mo-old male and female mice treated with vehicle or an IGF-1R mAb (L2-Cmu, Amgen Inc), and determined effects on aging outcomes. Here we show that L2-Cmu preferentially improves female healthspan and increases median lifespan by 9% (P = 0.03) in females, along with a reduction in neoplasms and inflammation (P ≤ 0.05). Thus, consistent with other models, targeting IGF-1R signaling appears to be most beneficial to females. Importantly, these effects could be achieved at advanced ages, suggesting that IGF-1R mAbs could represent a promising therapeutic candidate to delay aging.

Fig. 1

L2-Cmu is an antagonist to the murine IGF-1R and hybrids. a NIH-3T3 cells were pre-treated with 100 μg/mL Control IgG1 or L2-Cmu for 1 h prior to addition of vehicle, 5 nM IGF-1 or 20 nM IGF-2 for 2 min. Cell lysates were then immunoprecipitated for IGF-1Rβ and blots were probed for pTyr and total IGF-1Rβ levels. b, c Dose response competition assays with murine IGF-1R(ECD)-mFc. The ability of L2-Cmu to block human Ru-labeled IGF-1 or IGF-2 to the murine IGF-1R extracellular domain was evaluated in the IGEN format. d NIH-3T3 cells were pre-treated with vehicle or L2-Cmu (100 µg/mL) 1 h prior to addition of media (Con), or IGF-1 (5 nM) for 2 min to assess activation of IGF-1R and IGF-1R/InsR Hybrids. Data show that L2-Cmu reduced activation of IGF-1Rs, HybridRs by ~65% as compared to IGF-1 alone (n = 4 per condition). e, f For in vivo validation, mice were pre-treated with vehicle or L2-Cmu (20 mg/kg) i.p. and then challenged with an i.v. bolus of saline or 5 µg IGF-1 to assess the ability of L2-Cmu to modulate IGF-1R activation in vivo for a total of 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). L2-Cmu significantly inhibited IGF-1R activation in lung and heart. Bars represent mean ± s.e.m. Dot plots overlaid on bar graphs represent individual data points. Different letters denote a significant difference between treatments by Tukey HSD, P ≤ 0.05

Fig. 2

L2-Cmu does not perturb glucose homeostasis in aged mice. a–d Glucose and insulin tolerance in 24-mo-old female and male mice is not adversely effected by 6 mo mAb treatment (n = 12 per group, per sex). e–h Chronic L2-Cmu treatment led to a non-significant (main effect, P = 0.097), numerical increase in plasma IGF-1 level in old females [Young (n = 8), Old Con (n = 16), and Old mAb (n = 15)], while no significant difference was observed for IGF-1 levels in male mice [Young (n = 8), Old Con (n = 15), and Old mAb (n = 16)]. Likewise, insulin levels were not significantly different in female [Young (n = 16), Old Con (n = 26), and Old mAb (n = 25)], or male mice [Young (n = 17), Old Con (n = 32), and Old mAb (n = 28)]. i, j L2-Cmu treatment in males and females prevented the age-related increase in hypothalamic IGF-1Rs and reduced cortical levels in males only (P ≤ 0.05), but had no effect on IGF-1Rs in lung or pancreas of either sex. k, l Further, L2-Cmu treatment had no effect on InsR levels in examined tissues of either sex (n = 8 per group, per sex). Bars represent mean ± s.e.m. Dot plots overlaid on bar graphs represent individual data points. NS not significant. Different letters denote a significant difference between groups by Tukey HSD, P ≤ 0.05

Fig. 3

L2-Cmu preferentially improves female healthspan. Following 6 mo of mAb treatment, several functional measures of healthspan were assessed, including exercise tolerance in female [Young (n = 8), Old Con (n = 16), and Old mAb (n = 15)] and male mice [Young (n = 10), Old Con (n = 9), and Old mAb (n = 9)], grip strength in female [Young (n = 8), Old Con (n = 12), and Old mAb (n = 9)] and male mice [Young (n = 10), Old Con (n = 10), and Old mAb (n = 9)], and balance beam in female [Young (n = 8), Old Con (n = 12), and Old mAb (n = 15)] and male mice [Young (n = 8), Old Con (n = 9), and Old mAb (n = 9)]. a–c In females, mAb led to significant improvements in exercise tolerance (~50% over Old Con), grip strength (two-fold over Old Con), and motor coordination. d–f In males, L2-Cmu improved exercise tolerance by ~28%, but did not lead to improvements in grip strength, while motor coordination was only modestly improved on a medium difficulty beam. Bars represent mean ± s.e.m. Dot plots overlaid on bar graphs represent individual data points. Different letters denote a significant difference between groups by Tukey HSD, P ≤ 0.05

Fig. 4

L2-Cmu prevents age-related diastolic dysfunction in females. a–c Cardiac aging in CB6F1 female mice is characterized by a decline in diastolic function (E/A ratio), increased LVPWd, and accumulating amounts of fibrosis in the myocardium. However, 6 mo L2-Cmu treatment, beginning at 78 wks of age, was able to preserve diastolic function, reduce LVPWd measures in females [Young (n = 6), Old Con (n = 8), and Old mAb (n = 7)], and largely prevented the accumulation of cardiac fibrosis [Young (n = 4), Old Con (n = 5), and Old mAb (n = 6)]. d Metabolomic analysis of heart tissue in female mice [Young (n = 7), Old Con (n = 8), and Old mAb (n = 7)] revealed that the young and aged metabolome are distinct (see also Supplementary Fig. 5), and predominantly characterized by differences in glycerophospholipids, and to a lesser extent ceramides, while amino acids, biogenic amines, and acylcarnitines were largely unaffected. However, mAb treatment tended to oppose the age-related alterations in several metabolites, including the rise of several glycerophospholipids, resulting in a more youthful metabolomic signature in heart. Further metabolomic data analyses can be found in in Supplementary Fig. 5 and the dataset here: [10.17605/OSF.IO/8QGX9]. e–g Remarkably, the favorable effects of L2-Cmu in males were absent for E/A ratio [Young (n = 5), Old Con (n = 6), and Old mAb (n = 5)] and fibrosis [Young (n = 8), Old Con (n = 8), and Old mAb (n = 8)], suggesting that the beneficial effects of modulating IGF-1 signaling in heart is specific to females. Bars represent mean ± s.e.m. Dot plots overlaid on bar graphs represent individual data points. Different letters denote a significant difference between groups by Tukey HSD, P ≤ 0.05

Fig. 5

Sex differences in inflammatory markers with IGF-1R mAb treatment in aged mice. a, b A 25-plex cytokine/chemokine panel was performed on plasma obtained 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)]. Data were treated as nonparametric values and analyzed by the Kruskal–Wallis procedure and the Mann–Whitney U-test when appropriate. Any value below the lower limit of detection of the assay was replaced by the minimal detectable concentration (MOD)/√2 for the specific analyte. Therefore, undetectable values were treated as a tie for purposes of statistically ranking data. For generation of the heatmaps, values were normalized against Young Controls and log transformed. Group mean ± s.e.m. for all analytes are provided in Supplementary Tables 6, 7 and raw data found here: [10.17605/OSF.IO/8QGX9]. In females, several inflammatory mediators, including IL-6, IL-12p-40, and MIP-1α, were significantly increased with aging, but were largely restored to more youthful levels with mAb treatment. Meanwhile, systemic inflammation in old male mice was markedly exacerbated by mAb treatment, as indicated by a marked increase in the majority of measured analytes over age-matched controls. *P < 0.05 for Old mAb versus Old Controls

Fig. 6

Late-life IGF-1R modulation improves female lifespan. a Chronic mAb treatment beginning at 78 wks of age (~18 mo) in CB6F1 female mice tended to cause a slight and persistent reduction in body weight (group effect, P = 0.056). b, c This reduction in body weight was attributed to a reduction in lean mass (P = 0.052), rather than adiposity, as assessed by qMR. d Importantly, late-life mAb treatment was able to significantly improve mean (P = 0.023) and median lifespan (P = 0.03), as well as survival after 78 weeks of age (HR = 0.622, P = 0.029; n = 45 per group), but did not improve maximum lifespan. Details regarding these analyses and related R code can be found in the Statistics section and here: [10.17605/OSF.IO/8QGX9. Bars are means ± s.e.m. Dot plots overlaid on bar graphs represent individual data points. NS not significant. *Significantly different from Controls by independent samples t-test, P = 0.052