Metabolomic profiling reveals mitochondrial-derived lipid biomarkers that drive obesity-associated inflammation

Abstract

Obesity has reached epidemic proportions worldwide. Several animal models of obesity exist, but studies are lacking that compare traditional lard-based high fat diets (HFD) to "Cafeteria diets" (CAF) consisting of nutrient poor human junk food. Our previous work demonstrated the rapid and severe obesogenic and inflammatory consequences of CAF compared to HFD including rapid weight gain, markers of Metabolic Syndrome, multi-tissue lipid accumulation, and dramatic inflammation. To identify potential mediators of CAF-induced obesity and Metabolic Syndrome, we used metabolomic analysis to profile serum, muscle, and white adipose from rats fed CAF, HFD, or standard control diets. Principle component analysis identified elevations in clusters of fatty acids and acylcarnitines. These increases in metabolites were associated with systemic mitochondrial dysfunction that paralleled weight gain, physiologic measures of Metabolic Syndrome, and tissue inflammation in CAF-fed rats. Spearman pairwise correlations between metabolites, physiologic, and histologic findings revealed strong correlations between elevated markers of inflammation in CAF-fed animals, measured as crown like structures in adipose, and specifically the pro-inflammatory saturated fatty acids and oxidation intermediates laurate and lauroyl carnitine. Treatment of bone marrow-derived macrophages with lauroyl carnitine polarized macrophages towards the M1 pro-inflammatory phenotype through downregulation of AMPK and secretion of pro-inflammatory cytokines. Results presented herein demonstrate that compared to a traditional HFD model, the CAF diet provides a robust model for diet-induced human obesity, which models Metabolic Syndrome-related mitochondrial dysfunction in serum, muscle, and adipose, along with pro-inflammatory metabolite alterations. These data also suggest that modifying the availability or metabolism of saturated fatty acids may limit the inflammation associated with obesity leading to Metabolic Syndrome.

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 , 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.