Background/Objective: In a subset of participants from the CALERIE Phase 2 study we evaluated the effects of 2y of ~25% Calorie Restriction (CR) diet on IgG N-glycosylation (GlycAge), plasma and complement C3 N-glycome as markers of aging and inflammaging. Methods: Plasma samples from 26 participants in the CR group who completed the CALERIE2 trial and were deemed adherent to the intervention (~>10 % CR at 12 mo) were obtained from the NIA AgingResearchBiobank. Glycomic investigations using UPLC or LC-MS analyses were conducted on samples from baseline (BL), mid-intervention (12 mo) and post-intervention (24 mo), and changes resulting from the 2y CR intervention were examined. In addition, anthropometric, clinical, metabolic, DNA methylation (epigenetic) and skeletal muscle transcriptomic data were analyzed to identify aging-related changes that occurred in tandem with the N-glycome changes. Results: Following the 2y CR intervention, IgG galactosylation was higher at 24mo compared to BL (p = 0.051), digalactosylation and GlycAge (the IgG-based surrogate for biological age) were not different between BL and 12mo or BL and 24mo, but increased between 12mo and 24mo (p = 0.016, 0.027 respectively). GlycAge was also positively associated with TNF-alpha; and ICAM-1 (p=0.030, p=0.017 respectively). Plasma highly branched glycans were decreased by the 2y intervention (BL vs 24 mo: p=0.013), but both plasma and IgG bisecting GlcNAcs were increased (BL vs 24mo: p<0.001, p = 0.01 respectively). Furthermore, total complement C3 protein concentrations were reduced (BL vs 24mo: p <0.001), as were Man9 glycoforms (BL vs 24mo: p<0.001), and Man10 (which is glucosylated) C3 glycoforms (BL vs 24mo: p = 0.046). Conclusions: 24-mos of CR was associated with several favorable, anti-aging, anti-inflammatory changes in the glycome: increased galactosylation, reduced branching glycans, and reduced GlycAge. These promising CR effects were accompanied by an increase in bisecting GlcNAc, a known pro-inflammatory biomarker. These intriguing findings linking CR, clinical, and glycomic changes may be anti-aging and inflammatory, and merit additional investigation.

TP, HD, JS, TS, AFH and GL are working for or have stakes in Genos Ltd. D.W.B. is listed as an inventor on a Duke University and University of Otago invention, DunedinPACE, that was licensed to a commercial entity.

NCT00427193

Funding disclosures: This research was supported by US National Institute on Aging grants U01AG020487, R33AG070455, and R01AG061378. SK was supported from training grant NHLBIK12HL141956 from the National Institutes of Health. NEL was supported by NIGMH (R35GM119850) and the Novo Nordisk Foundation (NNF20SA0066621), and VBK was funded by R01AG054840 and by P30 AG028716.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The IRB at Pennington Biomedical Research Center (PBRC), Baton Rouge, LA; Tufts University (Tufts), Boston, MA; and Washington University School of Medicine, St. Louis, MO gave ethical approval for work done in original study and was coordinated by the IRB at Duke Clinical Research Institute, Durham, NC. The current analysis was conducted after obtaining an exemption from the University of Arizona IRB committee, due to lack of direct involvement of human participants.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

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I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

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Data and select biological samples are available from the NIA AgingResearchBiobank upon request (https://agingresearchbiobank.nia.nih.gov/studies/calerie/)