With an expanding aging population burdened with comorbidities, there is considerable interest in treatments that optimize health in later life. Acarbose (ACA), a drug used clinically to treat Type 2 diabetes (T2DM) can extend mouse lifespan, with greater effect in males than in females. Utilizing a genetically heterogeneous mouse model, we tested the ability of ACA to ameliorate functional, pathological and biochemical changes that occur during aging, and determined which of the effects of age and drug were sex-dependent. In both sexes, ACA prevented age-dependent loss of body mass, in addition to improving grip strength, balance/coordination on an accelerating rotarod, and rotarod endurance. Age-related cardiac hypertrophy was seen only in male mice, and this male-specific aging effect was attenuated by ACA. ACA-sensitive cardiac changes were associated with reduced activation of cardiac growth promoting pathways and decreased abundance of peroxisomal proteins involved in lipid metabolism. ACA further ameliorated age-associated changes in cardiac lipid species, particularly lysophospholipids – changes which have previously been associated with aging, cardiac dysfunction and cardiovascular disease in humans. In the liver, ACA had pronounced effects on lipid handling in both sexes, reducing hepatic lipidosis during aging and shifting the liver lipidome in adulthood, particularly favoring reduced triglyceride accumulation. Our results demonstrate that ACA, already in clinical use for T2DM, has broad-ranging anti-aging effects in multiple tissues, and may have the potential to increase physical function and alter lipid biology to preserve or improve health at older ages.

Lysis/Digestion - Mouse heart tissue previously probe sonicated in 90 ul of 0.1% RapiGest in 100 mM Tris with phosphatase inhibitors was resuspended in 30 ul of 3X lysis buffer for a final concentration of 5% SDS, 50mM Triethylammonium bicarbonate (TEAB), 2mM MgCl2, 1X HALT phosphatase and protease inhibitors and vortexed. Protein concentration was measured with a BCA assay. Homogenate of 50 ug was added to a process control of 800 ng of yeast enolase protein (Sigma) which was then reduced with 20 mM DTT and alklyated with 40 mM IAA. Lysates were then prepared for S-trap column (Protifi) binding by the addition of 1.2% phosphoric acid and 350 ul of binding buffer (90% Methanol, 100 mM TEAB). The acidified lysate was bound to column incrementally, followed by 3 wash steps with binding buffer to remove SDS and 3 wash steps with 50:50 methanol:chloroform to remove lipids and a final wash step with binding buffer. Trypsin (1:10) in 50mM TEAB was then added to the S-trap column for digestion at 47°C for one hour. Hydrophilic peptides were then eluted with 50 mM TEAB and hydrophobic peptides were eluted with a solution of 50% acetonitrile in 0.2% formic acid. Elutions were pooled, speed vacuumed and resuspended in 0.1% formic acid. A heavy labeled Peptide Retention Time Calibrant (PRTC) mixture (Pierce) was added to each sample. Liquid Chromatography and Mass Spectrometry - One ug of each sample with 150 femtomole of PRTC was loaded onto a 30 cm fused silica picofrit (New Objective) 75 µm column and 3.5 cm 150 µm fused silica Kasil1 (PQ Corporation) frit trap loaded with 3 µm Reprosil-Pur C18 (Dr. Maisch) reverse-phase resin analyzed with a Thermo Easy nano-LC 1200. The PRTC mixture is used to assess quality of the column before and during analysis. Four of these quality control runs are analyzed prior to any sample analysis and then after every six sample runs another quality control run is analyzed. Buffer A was 0.1% formic acid in water and buffer B was 0.1% formic acid in 80% acetonitrile. The 40-minute QC gradient consists of a 0 to 16% B in 5 minutes, 16 to 35% B in 20 minutes, 35 to 75% B in 1 minute, 75 to 100% B in 5 minutes, followed by a wash of 9 minutes and a 30 minute column equilibration. The 110-minute sample LC gradient consists of a 2 to 7% for 1 minutes, 7 to 14% B in 35 minutes, 14 to 40% B in 55 minutes, 40 to 60% B in 5 minutes, 60 to 98% B in 5 minutes, followed by a 9 minute wash and a 30 minute column equilibration. Peptides were eluted from the column with a 50°C heated source (CorSolutions) and electrosprayed into a Thermo Orbitrap Fusion Lumos Mass Spectrometer with the application of a distal 3 kV spray voltage. For the quality control analysis, a cycle of one 120,000 resolution full-scan mass spectrum (350-2000 m/z) followed by a data-independent MS/MS spectra on the loop count of 76 data-independent MS/MS spectra using an inclusion list at 15,000 resolution, AGC target of 4e5, 20 sec maximum injection time, 33% normalized collision energy with a 8 m/z isolation window. For the sample digest, first a chromatogram library of 6 independent injections is analyzed from a pool of all samples within a batch. For each injection a cycle of one 120,000 resolution full-scan mass spectrum with a mass range of 100 m/z (400-500 m/z, 500-600 m/z…900-1000 m/z) followed by a data-independent MS/MS spectra on the loop count of 26 at 30,000 resolution, AGC target of 4e5, 60 sec maximum injection time, 33% normalized collision energy with a 4 m/z overlapping isolation window. The chromatogram library data is used to quantify proteins from individual sample runs. These individual runs consist of a cycle of one 120,000 resolution full-scan mass spectrum with a mass range of 350-2000 m/z, AGC target of 4e5, 100 ms maximum injection time followed by a data-independent MS/MS spectra on the loop count of 76 at 15,000 resolution, AGC target of 4e5, 20 sec maximum injection time, 33% normalized collision energy with an overlapping 8 m/z isolation window. Application of the mass spectrometer and LC solvent gradients are controlled by the ThermoFisher XCalibur data system. Data Analysis - Thermo RAW files were converted to mzML format using Proteowizard (version 3.0.19045) using vendor peak picking and demultiplexing. Chromatogram spectral libraries were created using default settings (10 ppm tolerances, trypsin digestion, HCD b- and y-ions) of Walnut in EncyclopeDIA (version 0.9.0) using the Uniprot mouse canonical FASTA. Quantitative spectral libraries were created by mapping spectral to the chromatogram spectral library using EncyclopeDIA requiring a minimum of 3 quantitative ions and filtering peptides at a 1% FDR using Percolator 3.01. The quantitative spectral library is imported into Skyline with the mouse uniprot FASTA as the background proteome to map peptides to proteins. The mzML data is imported and all data is TIC normalized and calibrated to the batch reference.

Each sample batch was 24 individual mouse heart tissue samples, representing 4 randomly selected mice from 6 groups, and 2 pooled references for a total of 26 samples per batch. The first reference was a pool composed of 32 samples, 4 randomly selected mice from 8 groups (2 of the 8 groups not represented in this study). The second reference was a pool composed of 24 samples, 4 randomly selected mice from 6 groups (none of the 6 groups represented in this study). Both references were homogenized, aliquoted, frozen, and used to compare between batches and as a “universal reference” for comparison between similar proteomic studies. Six groups are: 1) Male-Sham-Young-Control, 2) Male-Sham-Old-Control, 3) Male-Sham-Old-Aca, 4) Female-Sham-Young-Control, 5) Female-Sham-Old-Control, 6) Female-Sham-Old-Aca. Reference 1 - Sham and Reference 2 - Cast