Supplementary data: Age, Diet, and Cyp2b status (Cyp2b-null) contribute to changes in lipidomic profiles
The effects of age (9 months old), high-fat diet (4.5 months old), and the loss of Cyp2b9, Cyp2b10, and Cyp2b13 (Cyp2b-null mice) on the male murine hepatic lipidome was compared. Hierarchical clustering and principal component analysis show that age perturbs phospholipid profiles and serum lipid markers the most compared to healthy, young mice; followed by a high-fat diet and then loss of Cyp2b. WT and Cyp2b-null male mice were divided into groups and fed either commercially available diets; normal chow diet (ND; 2018S-Envigo Teklad Diet) or a high-fat diet (HFD; Envigo TD.06414) for 10 weeks. Mice were 4.5 months old at the end of the HFD study and referred to as ND-fed young or HFD-fed young; WT or Cyp2b-null mice. An additional experimental group of WT and Cyp2b-null male mice were fed a ND until they reached 9 months (termed old). Serum biomarkers and liver triglycerides (TAG) were measured. Liver and inguinal white adipose tissue (WAT) were excised, weighed, and divided by total body weight to determine the hepatosomatic index (HSI) and white adipose somatic index (WSI). Mass spectrometry performed using a Sciex AC LC system and Sciex QTrap5500 mass spectrometer. Phospholipid species were identified and quantified from the livers of mice (n=3 for young (4.5 mo) ND and HFD-fed mice; n=5 for old from each experimental group by LC-MS/MS at the Emory Integrated Lipidomics Core (EILC) as described in their online protocols. Supplementary File 1. Lipidomic data from all treatment groups. Lipid species were identified from liver samples of male mice from arachidonic (ARA), linoleic (LA), α-linolenic (ALA) and docosahexaenoic acid (DHA). Treatment groups are ND-fed young (4.5 mo), ND-fed old (9 mo), and young mice fed a HFD for 10 weeks. All data is in relative percent. Supplementary File 2. Data used to make Figure 4 principal component analysis (PCA) biplot. Supplementary File 3. (A) Lipids that are statistically different by Student's t-tests between Cyp2b-null and WT mice in the different age and diet groups. (B) Important or significantly perturbed phospholipid species in Cyp2b-null mice compared to WT mice identified by both random forest analysis and Student’s t-tests in young (4.5 mo) ND-fed, young (4.5 mo) HFD-fed, and old (9 mo) mice. Supplementary File 4. Perturbed hepatic phospholipid species in Cyp2b-null mice within each group. (A) Venn diagram of important lipid species between Cyp2b-null and WT mice of each featured group based on random forest analysis (MDA > 0). (B) Venn diagram of significantly increased (red) or decreased (blue) lipid species in Cyp2b-null mice of each featured group as determined by Student’s t-tests (p < 0.05). Supplementary File 5. Data used to make principal component analysis (PCA) biplot for Figure 5b. (A-top) Data. (B-bottom) PCA contribution graph below containing the top 10 contributing variables of the PCA plot based on loading strength expressed in percent contribution.