Untargeted Metabolomic Profiling of Early Diet Energy Intake in C57BL/6 N Mice Reveals Differences Associated with Diet and Aberrant Crypt Foci

Abstract

Abstract Objectives A high-fat (H) diet leads to obesity, a known risk factor for colorectal cancer (CRC). In contrast, calorie restriction (E) is associated with reduced CRC risk. However, the metabolome associated with H vs. E-associated CRC risk has never been directly compared. The different influences of these diets on the proximal (PC), medial (MC), and distal (DC) colon metabolome has also not been studied. Thus, the objective is to elucidate metabolites associated with abberant crypt foci (ACF) number, a marker of CRC risk, in each colon region after consumption of H, E, or a normocaloric control diet (C). Methods 3-week-old C57BL/6 N mice were fed a C, E, or H initiation diet for 13 weeks. In weeks 16–21, animals were injected with azoxymethane to initiate ACF formation, and switched to a C, E, or H progression diet (for a total of 9 diet groups: CC, CH, CE, HH, HC, HE, EE, EC, EH). Polar extracts of the colon regions (i.e., PC, MC, and DC) were analyzed using ultra-high performance liquid chromatography-high resolution mass spectrometry method (HRMS) and 1H NMR metabolomics methods. Linear models assessed the main effects of ACF, initiation diet, progression diet, as well as the diet * ACF interaction, on relative metabolite concentration in each colon region. Results Following HILIC-HRMS analysis of extracts in positive and negative ionization mode, 492 and 415 metabolites were detected, respectively. Linear models revealed 21 metabolites were significantly associated with initiation E diet * ACF (8 unique to MC, 13 unique to PC), 14 with initiation H diet * ACF (only in DC), 27 with progression H diet * ACF (14 unique to DC, 2 to MC, 11 to PC) and 20 with progression E diet * ACF (17 unique to DC, 1 to PC, and 1 common to both). Pathway integration and authentication of tentative metabolite identities with chemical standards is underway. Conclusions Diet * ACF interaction significantly influences multiple metabolite concentrations. Little to no overlap is observed between metabolites associated with ACF in a given colon region and the other regions tested, revealing that the diet * ACF interaction is region-specific. Future studies in humans will determine if these metabolites may serve as early biomarkers for CRC diagnosis. Funding Sources Sample analyses were supported by NIH Award Number Grant P30 CA016058, OSU, and OSUCCC.