Figure 1.
Serum accumulation of NEFA and acylcarnitine metabolites in CAF compared to SC-fed.
Aged-matched male rats were fed diets for 10 weeks and serum was isolated in 6 hour-fasted rats. (n = 8 SC, 9 CAF). Amino acids and acylcarnitine mitochondrial intermediates were measured by LC-MS/MS. Non-esterified fatty acids (NEFA) were measured enzymatically. Metabolites indicate fold change of metabolites from CAF-fed serum compared to mean of SC-fed serum (CAF/SC) for each metabolite, which indicate accumulation (red) of many NEFAs and acylcarnitine species with decreases (green) in amino acids, arachidonoyl carnitine (C20∶4), and short chain acylcarnitines in the serum from CAF-fed rats (FDR 3.88%). See Table S4 for full names of metabolites.
Figure 2.
Serum myristate correlates to Metabolic Syndrome measures: weight gain, HOMA-IR, and blood glucose.
Aged-matched male rats were fed diets for 10 weeks and serum was isolated in 6 hour-fasted rats and metabolites measured as in Figure 1 (n = 8 SC, 9 CAF). A) Serum concentrations of individual NEFAs indicate that of the eight fatty acids measured, seven were significantly elevated in CAF-fed rats versus SC controls. (*p = 0.04, #p = 0.01, ∧p<0.0001). B–D) Serum saturated fatty acid myristic acid (C14∶0) significantly correlated with weight gain (B), HOMA-IR (C) and blood glucose at sacrifice (D).
Figure 3.
Lipotoxicity and mitochondrial dysfunction induced by CAF diet in muscle.
Aged-matched male rats were fed diets for 15 weeks and tissue was isolated from animals sacrificed after a 6 hour fast. A) Muscle triglyceride levels were doubled in CAF-fed rats compared to SC-fed controls (*p<0.02, n = 4 SC, 5 CAF). B) Acylcarnitines accumulate in muscle of CAF-fed rats compared to SC controls (*p≤0.02). Metabolites measured and tissue isolation as in Figure 1. (n = 8 SC, 9 CAF for B). See Table S4 for full names of metabolites.
Figure 4.
Severe mitochondrial dysfunction in CAF-fed white adipose tissue.
A) Diet-induced mitochondrial dysfunction was evident in CAF-fed rat epididymal adipose, which was greater than dysfunction in HFD-fed animals. CAF-diet increased levels of multiple acylcarnitines when compared to HFD-, LFD-, and SC- fed animals demonstrating mitochondrial dysfunction (*p≤0.03 SC v. CAF; ∧p<0.02 LFD v HFD). B) Lauroyl carnitine (“LC", C12-AC) specifically elevated by CAF diet (A) is significantly correlated to crown like structure (CLS) accumulation, a hallmark of adipose inflammation. Aged-matched male rats were fed diets for 15 weeks and tissue was isolated from animals sacrificed after a 6 hour fast. (n = 4, SC, LFD and HFD, n = 5 CAF). See Table S4 for full names of metabolites.
Figure 5.
Lauroyl carnitine drives pro-inflammatory macrophage response.
A) Primary bone marrow derived macrophages (BMDM) were treated with media alone for unpolarized macrophages “M0", 5 ng/mL LPS plus 10 ng/mL interferon gamma (IFNγ) to drive the pro-inflammatory “M1" phenotype [40], or 20 and 200 µM doses of lauroyl L-carnitine (“LC") for 24 hours. Western immunoblot using antibodies against phosphorylated AMP-activated protein kinase (AMPK), total AMPK, or actin. B) Bands are quantified using Image J and shown as pAMPK/AMPK normalized to actin. C) BMDM were treated for 24 hours with M1-polarizing cytokines (LPS/ IFNγ) or 10–1000 µM LC. Secreted cytokines were profiled from BMDM using Proteome Profiler Array Mouse Cytokine array (*p<0.05 relative to M0).
Figure 6.
Cafeteria diet drives accumulation of oxidative intermediates and pro-inflammatory lipid mediators.
Rats were fed SC or CAF diets for 10 weeks and at sacrifice serum and was isolated for metabolomic analysis including total and individual non-esterified fatty acids (NEFA), acylcarnitine and amino acid metabolite profiling of serum and muscle. To compare the effects of traditional defined lard-based diets with a CAF model on adipose metabolism, rats were fed SC, low fat and high fat lard-based defined diets, or CAF diet for 15 weeks and metabolites were analyzed. A. Metabolomic profiling has revealed that CAF diet-induced obesity drives accumulation of non-esterified fatty acids (NEFA), triglycerides (TG), and fatty acid β-oxidation intermediates in serum, muscle and for the first time white adipose tissue. CAF-mediated effects in adipose are more exaggerated than lard-based HFD effects. B. Lauroyl carnitine, an adipose-derived biomarker identified through metabolomic profiling was demonstrated to drive polarization of primary bone marrow derived macrophages (BMDM) towards the pro-inflammatory “M1" phenotype. Taken together, CAF diet proved to be a rapid and dramatic inducer of insulin resistance, components of Metabolic Syndrome, and metabolic biomarkers. In CAF-diet-induced obese adipose, lauroyl carnitine was identified as a potential mediator between metabolism of saturated fatty acids and the pro-inflammatory response.