Elaidate, an 18-carbon trans-monoenoic fatty acid, but not physiological fatty acids increases intracellular Zn(2+) in human macrophages.

Artificial trans fatty acids promote atherosclerosis by blocking macrophage clearance of cell debris. Classical fatty-acid response mechanisms include TLR4-NF-κB activation, and Erk1/2 phosphorylation, but these may not indicate long-term mechanisms. Indeed, nuclear NF-κB was increased by 60 min treatment by 30 µM of the 18 carbon trans unsaturated fatty acid elaidic acid (elaidate), the physiological cis-unsaturated fatty acid oleic acid (oleate), and the 18 or 16 carbon saturated fatty acids stearic and palmitic acid (stearate or palmitate). However, except for stearate, effects on related pathways were minimal at 44 h. To determine longer term effects of trans fatty acids, we compared mRNA expression profiles of (trans) elaidate to (cis) oleate, 30 µM, at 44 h in human macrophages. We found that elaidate changed Zn(2+) -homeostasis gene mRNAs markedly. This might be important because Zn(2+) is a major regulator of macrophage activity. Messenger RNAs of seven Zn(2+) -binding metallothioneins decreased 2-4-fold; the zinc importer SLC39A10 increased twofold, in elaidate relative to oleate-treated cells. Results were followed by quantitative PCR comparing cis, trans, and saturated fatty acid effects on Zn(2+) -homeostasis gene mRNAs. This confirmed that elaidate uniquely decreased metallothionein expression and increased SLC39A10 at 44 h. Further, intracellular Zn(2+) was measured using N-(carboxymethyl)-N-[2-[2-[2(carboxymethyl) amino]-5-(2,7,-difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)-phenoxy]-ethoxy]-4-methoxyphenyl]glycine, acetoxymethyl ester (FluoZin-3-AM). This showed that, at 44 h, only cells treated with elaidate had increased Zn(2+) . The durable effect of elaidate on Zn(2+) activation is a novel and specific effect of trans fatty acids on peripheral macrophage metabolism.

Elaidate, an 18-carbon trans-monoenoic fatty acid, inhibits ß-oxidation in human peripheral blood macrophages.

Consumption of trans-unsaturated fatty acids promotes atherosclerosis, but whether degradation of fats in macrophages is altered by trans-unsaturated fatty acids is unknown. We compared the metabolism of oleate (C18:1Δ9-10 cis; (Z)-octadec-9-enoate), elaidate (C18:Δ9-10 trans; (E)-octadec-9-enoate), and stearate (C18:0, octadecanoate) in adherent peripheral human macrophages. Metabolism was followed by measurement of acylcarnitines in cell supernatants by MS/MS, determination of cellular fatty acid content by GC/MS, and assessment of ß-oxidation rates using radiolabeled fatty acids. Cells incubated for 44 h in 100 µM elaidate accumulated more unsaturated fatty acids, including both longer- and shorter-chain, and had reduced C18:0 relative to those incubated with oleate or stearate. Both C12:1 and C18:1 acylcarnitines accumulated in supernatants of macrophages exposed to trans fats. These results suggested ß-oxidation inhibition one reaction proximal to the trans bond. Comparison of [1-(14)C]oleate to [1-(14)C]elaidate catabolism showed that elaidate completed the first round of fatty acid ß-oxidation at rates comparable to oleate. Yet, in competitive ß-oxidation assays with [9,10-(3)H]oleate, tritium release rate decreased when unlabeled oleate was replaced by the same quantity of elaidate. These data show specific inhibition of monoenoic fat catabolism by elaidate that is not shared by other atherogenic fats.

Evidence for physical association of mitochondrial fatty acid oxidation and oxidative phosphorylation complexes.

Fatty acid ß-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are key pathways involved in cellular energetics. Reducing equivalents from FAO enter OXPHOS at the level of complexes I and III. Genetic disorders of FAO and OXPHOS are among the most frequent inborn errors of metabolism. Patients with deficiencies of either FAO or OXPHOS often show clinical and/or biochemical findings indicative of a disorder of the other pathway. In this study, the physical and functional interactions between these pathways were examined. Extracts of isolated rat liver mitochondria were subjected to blue native polyacrylamide gel electrophoresis (BNGE) to separate OXPHOS complexes and supercomplexes followed by Western blotting using antisera to various FAO enzymes. Extracts were also subjected to sucrose density centrifugation and fractions analyzed by BNGE or enzymatic assays. Several FAO enzymes co-migrated with OXPHOS supercomplexes in different patterns in the gels. When palmitoyl-CoA was added to the sucrose gradient fractions containing OXPHOS supercomplexes in the presence of potassium cyanide, cytochrome c was reduced. Cytochrome c reduction was completely blocked by myxothiazol (a complex III inhibitor) and 3-mercaptopropionate (an inhibitor of the first step of FAO), but was only partially inhibited by rotenone (a complex I inhibitor). Although palmitoyl-CoA and octanoyl-CoA provided reducing equivalents to OXPHOS-containing supercomplex fractions, no accumulation of their intermediates was detected. In contrast, short branched acyl-CoA substrates were not metabolized by OXPHOS-containing supercomplex fractions. These data provide evidence of a multifunctional FAO complex within mitochondria that is physically associated with OXPHOS supercomplexes and promotes metabolic channeling.

Pervasive inflammatory activation in patients with deficiency in very-long-chain acyl-coA dehydrogenase (VLCADD).

OBJECTIVES: Very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is a disorder of fatty acid oxidation. Symptoms are managed by dietary supplementation with medium-chain fatty acids that bypass the metabolic block. However, patients remain vulnerable to hospitalisations because of rhabdomyolysis, suggesting pathologic processes other than energy deficit. Since rhabdomyolysis is a self-destructive process that can signal inflammatory/immune cascades, we tested the hypothesis that inflammation is a physiologic dimension of VLCADD. METHODS: All subjects (n = 18) underwent informed consent/assent. Plasma cytokine and cytometry analyses were performed. A prospective case analysis was carried out on a patient with recurrent hospitalisation. Health data were extracted from patient medical records. RESULTS: Patients showed systemic upregulation of nine inflammatory mediators during symptomatic and asymptomatic periods. There was also overall abundance of immune cells with high intracellular expression of IFNγ, IL-6, MIP-1ß (CCL4) and TNFα, and the transcription factors p65-NFκB and STAT1 linked to inflammatory pathways. A case analysis of a patient exhibited already elevated plasma cytokine levels during diagnosis in early infancy, evolving into sustained high systemic levels during recurrent rhabdomyolysis-related hospitalisations. There were corresponding activated leukocytes, with higher intracellular stores of inflammatory molecules in monocytes compared to T cells. Exposure of monocytes to long-chain free fatty acids recapitulated the cytokine signature of patients. CONCLUSION: Pervasive plasma cytokine upregulation and pre-activated immune cells indicate chronic inflammatory state in VLCADD. Thus, there is rationale for practical implementation of clinical assessment of inflammation and/or translational testing, or adoption, of anti-inflammatory intervention(s) for personalised disease management.

Evaluation of mitochondrial bioenergetics, dynamics, endoplasmic reticulum-mitochondria crosstalk, and reactive oxygen species in fibroblasts from patients with complex I deficiency.

Mitochondrial complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) disorders in humans. In order to benchmark the effects of CI deficiency on mitochondrial bioenergetics and dynamics, respiratory chain (RC) and endoplasmic reticulum (ER)-mitochondria communication, and superoxide production, fibroblasts from patients with mutations in the ND6, NDUFV1 or ACAD9 genes were analyzed. Fatty acid metabolism, basal and maximal respiration, mitochondrial membrane potential, and ATP levels were decreased. Changes in proteins involved in mitochondrial dynamics were detected in various combinations in each cell line, while variable changes in RC components were observed. ACAD9 deficient cells exhibited an increase in RC complex subunits and DDIT3, an ER stress marker. The level of proteins involved in ER-mitochondria communication was decreased in ND6 and ACAD9 deficient cells. |ΔΨ| and cell viability were further decreased in all cell lines. These findings suggest that disruption of mitochondrial bioenergetics and dynamics, ER-mitochondria crosstalk, and increased superoxide contribute to the pathophysiology in patients with ACAD9 deficiency. Furthermore, treatment of ACAD9 deficient cells with JP4-039, a novel mitochondria-targeted reactive oxygen species, electron and radical scavenger, decreased superoxide level and increased basal and maximal respiratory rate, identifying a potential therapeutic intervention opportunity in CI deficiency.

Metabolomic profiling of fatty acid and amino acid metabolism in youth with obesity and type 2 diabetes: evidence for enhanced mitochondrial oxidation.

OBJECTIVE: We compared acylcarnitine (AcylCN) species, common amino acid and fat oxidation (FOX) byproducts, and plasma amino acids in normal weight (NW; n = 39), obese (OB; n = 64), and type 2 diabetic (n = 17) adolescents. RESEARCH DESIGN AND METHODS: Fasting plasma was analyzed by tandem mass spectrometry, body composition by dual energy X-ray absorptiometry and computed tomography, and total-body lipolysis and substrate oxidation by [(2)H(5)]glycerol and indirect calorimetry, respectively. In vivo insulin sensitivity (IS) was assessed with a 3-h hyperinsulinemic-euglycemic clamp. RESULTS: Long-chain AcylCNs (C18:2-CN to C14:0-CN) were similar among the three groups. Medium- to short-chain AcylCNs (except C8 and C10) were significantly lower in type 2 diabetes compared with NW, and when compared with OB, C2-, C6-, and C10-CN were lower. Amino acid concentrations were lower in type 2 diabetes compared with NW. Fasting lipolysis and FOX were higher in OB and type 2 diabetes compared with NW, and the negative association of FOX to C10:1 disappeared after controlling for adiposity, Tanner stage, and sex. IS was lower in OB and type 2 diabetes with positive associations between IS and arginine, histidine, and serine after adjusting for adiposity, Tanner stage, and sex. CONCLUSIONS: These metabolomics results, together with the increased rates of in vivo FOX, are not supportive of defective fatty acid or amino acid metabolism in obesity and type 2 diabetes in youth. Such observations are consistent with early adaptive metabolic plasticity in youth, which over time-with continued obesity and aging-may become dysfunctional, as observed in adults.

Metabolomic profiling of amino acids and ß-cell function relative to insulin sensitivity in youth.

CONTEXT: In longitudinal studies of adults, elevated amino acid (AA) concentrations predicted future type 2 diabetes mellitus (T2DM). OBJECTIVE: The aim of the present investigation was to examine whether increased plasma AA concentrations are associated with impaired ß-cell function relative to insulin sensitivity [i.e. disposition index (DI)], a predictor of T2DM development. DESIGN, SETTING, AND PARTICIPANTS: Metabolomic analysis for fasting plasma AAs was performed by tandem mass spectrometry in 139 normal-weight and obese adolescents with and without dysglycemia. First-phase insulin secretion was evaluated by a hyperglycemic (∼225 mg/dl) clamp and insulin sensitivity by a hyperinsulinemic-euglycemic clamp. DI was calculated as the product of first-phase insulin and insulin sensitivity. RESULTS: DI was positively associated with branched-chain AAs (leucine/isoleucine and valine; r = 0.27 and 0.29, P = 0.001), neutrally transported AAs (phenylalanine and methionine; r = 0.30 and 0.35, P < 0.001), basic AAs (histidine and arginine; r = 0.28 and 0.23, P ≤ 0.007), serine (r = 0.35, P < 0.001), glycine (r = 0.26, P = 0.002), and branched-chain AAs-derived intermediates C3, C4, and C5 acylcarnitine (range r = 0.18-0.19, P ≤ 0.04). CONCLUSION: In youth, increased plasma AA concentrations are not associated with a heightened metabolic risk profile for T2DM; rather, they are positively associated with ß-cell function relative to insulin sensitivity. These contrasting observations between adults and youth may be a reflection of developmental differences along the lifespan dependent on the combined impact of the aging process together with the impact of progressive obesity.

Increased levels of plasma acylcarnitines in obesity and type 2 diabetes and identification of a marker of glucolipotoxicity.

Dysregulation of fatty acid oxidation (FAO) is recognized as important in the pathophysiology of obesity and insulin resistance (IR). However, demonstrating FAO defects in vivo in humans has entailed complex and invasive methodologies. Recently, the identification of genetic blocks in FAO has been vastly simplified by using tandem mass spectrometry (MS/MS) of dried bloodspots to specify acylcarnitine (AcylCN) alterations characteristic for each disorder. This technology has recently been applied to examine FAO alterations in human and animal models of obesity and type 2 diabetes mellitus (T2DM). This study focused on characterizing AcylCN profiles in human plasma from individuals with obesity and T2DM during fasting and insulin-stimulated conditions. Following an overnight fast, plasma was obtained from lean (n = 12), obese nondiabetic (n = 14), and T2DM (n = 10) participants and analyzed for AcylCN using MS/MS. Plasma samples were also obtained at the end of a 4-h insulin-stimulated euglycemic clamp. In obesity and T2DM, long-chain AcylCNs were similarly significantly increased in the fasted state; free-CN levels were also elevated. Additionally, T2DM subjects of comparable BMI had increased short- and medium-chain AcylCNs, both saturated and hydroxy, as well as increased C(4)-dicarboxylcarnitine (C(4)DC-CN) that correlated with an index of poor glycemic control (HbA(1c); r = 0.74; P < 0.0001). Insulin infusion reduced all species of plasma AcylCN but this reduction was blunted in T2DM. Plasma long-chain AcylCN species are increased in obesity and T2DM, suggesting that more fatty acids can enter mitochondria. In T2DM, many shorter species accumulate, suggesting that they have a generalized complex oxidation defect.

Participation of two members of the very long-chain acyl-CoA synthetase family in bile acid synthesis and recycling.

Bile acids are synthesized de novo in the liver from cholesterol and conjugated to glycine or taurine via a complex series of reactions involving multiple organelles. Bile acids secreted into the small intestine are efficiently reabsorbed and reutilized. Activation by thioesterification to CoA is required at two points in bile acid metabolism. First, 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid, the 27-carbon precursor of cholic acid, must be activated to its CoA derivative before side chain cleavage via peroxisomal beta-oxidation. Second, reutilization of cholate and other C24 bile acids requires reactivation prior to re-conjugation. We reported previously that homolog 2 of very long-chain acyl-CoA synthetase (VLCS) can activate cholate (Steinberg, S. J., Mihalik, S. J., Kim, D. G., Cuebas, D. A., and Watkins, P. A. (2000) J. Biol. Chem. 275, 15605-15608). We now show that this enzyme also activates chenodeoxycholate, the secondary bile acids deoxycholate and lithocholate, and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid. In contrast, VLCS activated 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoate, but did not utilize any of the C24 bile acids as substrates. We hypothesize that the primary function of homolog 2 is in the reactivation and recycling of C24 bile acids, whereas VLCS participates in the de novo synthesis pathway. Results of in situ hybridization, topographic orientation, and inhibition studies are consistent with the proposed roles of these enzymes in bile acid metabolism.