Lf90ii

Prior to euthanasia, body composition was measured by time domain nuclear magnetic resonance (TD-NMR) spectroscopy (LF90II, Bruker). Whole-body fat and lean body mass were expressed as a percentage of body weight. Fasting NEFA levels were measured using a colorimetric assay (ab65341, Abcam) according to the manufacturer’s instructions. Samples were run in triplicate and absorbance read on a SpectraMax ID3 microplate reader.

At week 8, fat and lean body masses were measured by ¹H minispec system (LF90II, Bruker Optik, Germany) in mice. In addition, using PhenoMaster/LabMaster System (TSE Systems GmbH, Bad Homburg, Germany), four mice randomly chosen from each group were individually placed in metabolic cages to measure O₂ consumption and the respiratory exchange rate. Mice were monitored for 96 hr, and data were collected at intervals of 27 min after a 2‐days adaptation period. Parameters were compared between HFD and HFD‐S or HFD‐ML without considering body weight differences.

Whole body fat and lean body mass were determined by TD-NMR (LF90II, Bruker). Cell size distribution in hematoxylin-eosin (H&E)-stained sections of iSAT captured on a Thorlabs Tide Whole slide-scanning microscope was quantified using Adiposoft Software (Image J). After a 6-h fast, insulin sensitivity was assessed by injecting 0.5 IU/kg insulin (IP) and sampling blood glucose from the tail at baseline and at 15, 30, 45, 60, and 90-min postinjection. At euthanasia, trunk blood was collected immediately upon decapitation with a heparinized syringe and plasma frozen for later analyses. Colorimetric or fluorescent assays were used to assess plasma concentrations of triglycerides (ALPCO), nonesterified free fatty acids (ALPCO), total cholesterol (ALPCO), and Resistin (Abcam).