H OH metabolites OH Article Azoxymethane Alters the Plasma Metabolome to a Greater Extent in Mice Fed a High-Fat Diet Compared to an AIN-93 Diet Huawei Zeng 1,* , Shahid Umar 2, Zhenhua Liu 3 and Michael R. Bukowski 1 1 Grand Forks Human Nutrition Research Center, Agricultural Research Service, United States Department of Agriculture, Grand Forks, ND 58203, USA; [email protected] 2 Department of Surgery and University of Kansas Cancer Center, Kansas City, KS 66160, USA; [email protected] 3 School of Public Health and Health Sciences, University of Massachusetts, Amherst, MA 01003, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-701-795-8465 Abstract: Consumption of a high-fat diet (HFD) links obesity to colon cancer in humans. Our data show that a HFD (45% energy fat versus 16% energy fat in an AIN-93 diet (AIN)) promotes azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation in a mouse cancer model. However, the underlying metabolic basis remains to be determined. In the present study, we hy- pothesize that AOM treatment results in different plasma metabolomic responses in diet-induced obese mice. An untargeted metabolomic analysis was performed on the plasma samples by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). We found that 53 of 144 identified metabolites were different between the 4 groups of mice (AIN, AIN + AOM, HFD, HFD + AOM), and sparse partial least-squares discriminant analysis showed a separation between the HFD and HFD + AOM groups but not the AIN and AIN + AOM groups. Moreover, the concentrations of dihy- drocholesterol and cholesterol were inversely associated with AOM-induced colonic ACF formation. Citation: Zeng, H.; Umar, S.; Liu, Z.; Bukowski, M.R. Azoxymethane Functional pathway analyses indicated that diets and AOM-induced colonic ACF modulated five Alters the Plasma Metabolome to a metabolic pathways. Collectively, in addition to differential plasma metabolomic responses, AOM Greater Extent in Mice Fed a High-Fat treatment decreases dihydrocholesterol and cholesterol levels and alters the composition of plasma Diet Compared to an AIN-93 Diet. metabolome to a greater extent in mice fed a HFD compared to the AIN. Metabolites 2021, 11, 448. https:// doi.org/10.3390/metabo11070448 Keywords: aberrant crypt foci; azoxymethane; colon cancer; high-fat diet; inflammation; metabolome; obesity Academic Editor: Victor Gault Received: 7 May 2021 Accepted: 2 July 2021 1. Introduction Published: 9 July 2021 Consumption of a high-fat diet (HFD) links obesity to colon cancer in humans [1,2]. The global obesity epidemic has, in part, been attributed to the adoption of Western lifestyle Publisher’s Note: MDPI stays neutral practices, including increased consumption of high-energy diets [1–3]. Diet-induced obesity with regard to jurisdictional claims in published maps and institutional affil- is now established as a risk factor for cancer, and overweight and obesity affect two-thirds iations. of Americans and an estimated 2.3 billion people worldwide [4]. In mice, consumption of a HFD can lead to the accumulation of excess body fat that is associated with adipose tissue dysfunction and a chronic state of low-grade inflammation known to promote tumor development [5,6]. However, there are few mechanistic studies on early colonic tumorigenesis concerning the underlying metabolic regulation in the context of HFD- Copyright: © 2021 by the authors. induced obesity. Licensee MDPI, Basel, Switzerland. We recently reported that in a mouse model, a HFD promotes colonic aberrant crypt This article is an open access article distributed under the terms and foci (ACF, putative preneoplastic lesions) formation accompanied by increased systemic conditions of the Creative Commons levels of proinflammatory cytokines [7]. We therefore posit that determining plasma Attribution (CC BY) license (https:// metabolomic responses may lead to a greater understanding of the metabolic regulation creativecommons.org/licenses/by/ concerning a HFD and colonic ACF formation. As small-molecule metabolites exhibit 4.0/). an organisms’ physiological condition at a given moment, these metabolites represent Metabolites 2021, 11, 448. https://doi.org/10.3390/metabo11070448 https://www.mdpi.com/journal/metabolites Metabolites 2021, 11, 448 2 of 12 a dynamic situation in response to biochemical and pathological changes [8,9]. Thus, untargeted metabolite profiling (e.g., metabolomics) is an effective approach that aids in determining these underlying biochemical changes [10]. It is known that a HFD promotes AOM-induced ACF formation in a mouse model, and differential metabolites were iden- tified in the serum of colon cancer patients compared to the healthy controls [7,11,12]. Therefore, we hypothesize that AOM treatment may cause different plasma metabolomic responses in AOM mouse colon cancer models fed with the HFD compared to the AIN. 2. Results 2.1. Identified Metabolites and Their Group Separation The plasma samples were collected at the end of the study (Week 14) [7]. We identified 144 metabolites (Table S1) from 555 discrete signals detected in the plasma by using GC- TOFMS. There were 53 of 144 metabolites significantly different between the 4 groups of mice (AIN, AIN + AOM; HFD, HFD + AOM), and the relative values for these 53 metabolites compared to the AIN group are shown in Table1. Table 1. The effect of HFD feeding and AOM treatment on the abundance of plasma metabolites. Metabolites AIN AIN + AOM HFD HFD + AOM Palmitoleic acid 1.00 ± 0.40 a 1.11 ± 0.27 a 0.19 ± 0.05 b 0.30 ± 0.11 b Myristic acid 1.00 ± 0.18 a 1.02 ± 0.10 a 0.57 ± 0.06 c 0.74 ± 0.12 b Oleic acid 1.00 ± 1.46 bc 0.73 ± 1.32 c 1.99 ± 0.42 ab 2.71 ± 0.95 a Malic acid 1.00 ± 0.26 a 1.23 ± 0.42 a 0.63 ± 0.08 b 0.60 ± 0.12 b Beta-sitosterol 1.00 ± 0.35 b 0.46 ± 0.11 c 2.22 ± 0.65 a 0.64 ± 0.10 bc Oxoproline 1.00 ± 0.14 b 1.08 ± 0.15 b 1.13 ± 0.17 b 1.42 ± 0.21 a Alpha-tocopherol 1.00 ± 0.23 b 0.67 ± 0.10 c 1.94 ± 0.46 a 0.84 ± 0.17 bc Alpha-ketoglutarate 1.00 ± 0.22 a 1.15 ± 0.34 a 0.72 ± 0.09 b 0.63 ± 0.13 b Fumaric acid 1.00 ± 0.14 a 1.17 ± 0.32 a 0.76 ± 0.05 b 0.75 ± 0.11 b Citric acid 1.00 ± 0.24 ab 1.08 ± 0.14 a 0.68 ± 0.07 c 0.88 ± 0.10 b 3-hydroxybutyric acid 1.00 ± 0.64 c 1.32 ± 0.62 bc 2.08 ± 0.50 a 1.80 ± 0.57 ab Saccharic acid 1.00 ± 0.18 c 1.14 ± 0.21 bc 1.49 ± 0.36 a 1.33 ± 0.21 ab Citrulline 1.00 ± 0.21 a 0.87 ± 0.17 ab 0.77 ± 0.13 bc 0.64 ± 0.12 c 2-hydroxyglutaric acid 1.00 ± 0.13 ab 1.12 ± 0.17 a 0.86 ± 0.11 b 0.87 ± 0.14 b 2-aminobutyric acid 1.00 ± 0.37 b 0.87 ± 0.36 b 0.88 ± 0.24 b 1.58 ± 0.54 a Palmitic acid 1.00 ± 0.15 ab 1.09 ± 0.14 a 0.86 ± 0.08 b 0.88 ± 0.13 b 3-ureidopropionate 1.00 ± 0.29 b 1.35 ± 0.49 b 1.19 ± 0.47 b 2.02 ± 0.55 a Myo-inositol 1.00 ± 0.24 b 0.97 ± 0.13 b 1.37 ± 0.32 a 1.09 ± 0.27 b Dihydrocholesterol 1.00 ± 0.26 b 0.48 ± 0.12 c 1.89 ± 0.55 a 0.48 ± 0.12 c Glucuronic acid 1.00 ± 0.33 c 1.98 ± 0.51 ab 1.32 ± 0.37 bc 2.48 ± 0.96 a Lysine 1.00 ± 0.37 a 0.77 ± 0.22 ab 0.57 ± 0.25 b 0.58 ± 0.15 b Cholesterol 1.00 ± 0.14 b 0.79 ± 0.14 c 1.32 ± 0.22 a 0.81 ± 0.12 c Isothreonic acid 1.00 ± 0.09 b 1.07 ± 0.08 b 1.22 ± 0.20 a 1.03 ± 0.06 b Uracil 1.00 ± 0.33 b 1.04 ± 0.15 b 0.95 ± 0.17 b 1.29 ± 0.23 a Ornithine 1.00 ± 0.27 b 1.39 ± 0.62 b 1.05 ± 0.46 b 2.04 ± 0.33 a Succinic acid 1.00 ± 0.21 ab 1.27 ± 0.47 a 0.87 ± 0.11 b 0.87 ± 0.24 b N-acetyl-d-tryptophan 1.00 ± 0.22 a 1.00 ± 0.26 a 0.68 ± 0.22 b 0.87 ± 0.18 ab Pyruvic acid 1.00 ± 0.45 ab 1.15 ± 0.65 a 0.58 ± 0.18 b 0.67 ± 0.34 b 2,3-dihydroxybutanoic acid 1.00 ± 0.19 b 1.11 ± 0.12 ab 1.00 ± 0.12 b 1.29 ± 0.24 a Linoleic acid 1.00 ± 0.26 b 1.10 ± 0.26 ab 0.99 ± 0.20 b 1.41 ± 0.50 a Beta-alanine 1.00 ± 0.32 a 0.97 ± 0.33 a 0.60 ± 0.21 b 0.86 ± 0.23 ab Maleimide 1.00 ± 0.13 b 0.88 ± 0.14 b 1.26 ± 0.14 a 0.85 ± 0.14 b Isocitric acid 1.00 ± 0.17 a 1.15 ± 0.23 a 0.77 ± 0.11 b 1.08 ± 0.14 a Metabolites 2021, 11, 448 3 of 12 Table 1.