Animals

All procedures were approved by the Institutional Animal Care and Use Committee at the University of Colorado Boulder (Protocol No. 2539) and adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

A total of 50 C57Bl/6N male mice were purchased for this study from Charles River at 8 wk of age. Mice were housed in a conventional facility on a 12-h light/dark cycle. Mice were given ad libitum access to drinking water and fed either standard rodent chow diet (SC; n = 26; Envigo, Cat. No. 7917: https://insights.envigo.com/hubfs/resources/data-sheets/7017-datasheet-0915.pdf; Envigo, Madison, WI) or a customized WD (n = 24; Envigo Teklad TD.96132: https://insights.envigo.com/hubfs/resources/data-sheets/96132.pdf; Envigo). WD chow was designed to approximately match the 75th percentiles of US diets for total fat and sugar intake based on NHANES data (43, 44). Macronutrient content of the two diets is provided in Table 1. Per the manufacturers, content of TMAO precursors choline and l-carnitine were comparable between the two diets, although the manner in which these were provided did differ (see Table 1 caption) and choline from phosphatidylcholine in the ingredients added to the WD to increase fat content were not accounted for. Mice were multihoused until the beginning of the intervention (∼5 mo of age), at which point they were single-housed (at least 1 wk before baseline testing) to avoid mouse-to-mouse effects on the gut microbiome (mice are coprophagic) and to ensure accurate quantification of water and food intake.

Macro- and select micronutrient content of diets

To achieve higher fat content of the WD, hydrogenated vegetable shortening and beef tallow (both 100 g/kg) were added. The following were added to the WD to increase sugar content and made up all kcal from carbohydrates: sucrose (183 g/kg), cornstarch (160 g/kg), and maltodextrin (120 g/kg). The majority of fiber in both diets was insoluble fiber. Approximately 772 mg/kg of choline in the SC was provided as supplemental choline chloride; the remainder was in other ingredients. Choline content of the WD was provided in a vitamin mix (choline from phosphatidylcholine in added fat ingredients not accounted for). Both diets contained very low amounts of l-carnitine from fishmeal (SC) or casein (WD). Both diets were irradiated. SC, standard chow; WD, Western diet.