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Peroxisomal acyl-CoA oxidases catalyze the first step of beta-oxidation of a variety of substrates broken down in the peroxisome. These include the CoA-esters of very long-chain fatty acids, branched-chain fatty acids and the C27-bile acid intermediates. In rat, three peroxisomal acyl-CoA oxidases with different substrate specificities are known, whereas in humans it is believed that only two peroxisomal acyl-CoA oxidases are expressed under normal circumstances. Only three patients with ACOX2 deficiency, including two siblings, have been identified so far, showing accumulation of the C27-bile acid intermediates. Here, we performed biochemical studies in material from a novel ACOX2-deficient patient with increased levels of C27-bile acids in plasma, a complete loss of ACOX2 protein expression on immunoblot, but normal pristanic acid oxidation activity in fibroblasts. Since pristanoyl-CoA is presumed to be handled by ACOX2 specifically, these findings prompted us to re-investigate the expression of the human peroxisomal acyl-CoA oxidases. We report for the first time expression of ACOX3 in normal human tissues at the mRNA and protein level. Substrate specificity studies were done for ACOX1, 2 and 3 which revealed that ACOX1 is responsible for the oxidation of straight-chain fatty acids with different chain lengths, ACOX2 is the only human acyl-CoA oxidase involved in bile acid biosynthesis, and both ACOX2 and ACOX3 are involved in the degradation of the branched-chain fatty acids. Our studies provide new insights both into ACOX2 deficiency and into the role of the different acyl-CoA oxidases in peroxisomal metabolism. [ABSTRACT FROM AUTHOR]

Objective Cardiac hexokinase II (HKII) can translocate between cytosol and mitochondria and change its cellular expression with pathologies such as ischemia–reperfusion, diabetes and heart failure. The cardiac metabolic consequences of these changes are unknown. Here we measured energy substrate utilization in cytosol and mitochondria using stabile isotopes and oxygen consumption of the intact perfused heart for 1) an acute decrease in mitochondrial HKII (mtHKII), and 2) a chronic decrease in total cellular HKII. Methods/results We first examined effects of 200 nM TAT (Trans-Activator of Transcription)-HKII peptide treatment, which was previously shown to acutely decrease mtHKII by ~ 30%. In Langendorff-perfused hearts TAT-HKII resulted in a modest, but significant, increased oxygen consumption, while cardiac performance was unchanged. At the metabolic level, there was a nonsignificant (p = 0.076) ~ 40% decrease in glucose contribution to pyruvate and lactate formation through glycolysis and to mitochondrial citrate synthase flux (6.6 ± 1.1 vs. 11.2 ± 2.2%), and an 35% increase in tissue pyruvate (27 ± 2 vs. 20 ± 2 pmol/mg; p = 0.033). Secondly, we compared WT and HKII +/− hearts (50% chronic decrease in total HKII). RNA sequencing revealed no differential gene expression between WT and HKII +/− hearts indicating an absence of metabolic reprogramming at the transcriptional level. Langendorff-perfused hearts showed no significant differences in glycolysis (0.34 ± 0.03 μ mol/min), glucose contribution to citrate synthase flux (35 ± 2.3%), palmitate contribution to citrate synthase flux (20 ± 1.1%), oxygen consumption or mechanical performance between WT and HKII +/− hearts. Conclusions These results indicate that acute albeit not chronic changes in mitochondrial HKII modestly affect cardiac oxygen consumption and energy substrate metabolism. [ABSTRACT FROM AUTHOR]

Peroxisomal β-oxidation is an essential step in bile acid synthesis, since it is required for shortening of C27-bile acid intermediates to produce mature C24-bile acids. D-Bifunctional protein (DBP) is responsible for the second and third step of this β-oxidation process. However, both patients and mice with a DBP deficiency still produce C24-bile acids, although C27-intermediates accumulate. An alternative pathway for bile acid biosynthesis involving the peroxisomal L-bifunctional protein (LBP) has been proposed. We investigated the role of LBP and DBP in bile acid synthesis by analyzing bile acids in bile, liver, and plasma from LBP, DBP, and LBP:DBP double knock-out mice. Bile acid biosynthesis, estimated by the ratio of C27/C24-bile acids, was more severely affected in double knock-out mice as compared with DBP-/- mice but was normal in LBP-/- mice. Unexpectedly, trihydroxycholestanoyl-CoA oxidase was inactive in double knock-out mice due to a peroxisomal import defect, preventing us from drawing any firm conclusion about the potential role of LBP in an alternative bile acid biosynthesis pathway. Interestingly, the immature C27-bile acids in DBP and double knock-out mice remained unconjugated in juvenile mice, whereas they occurred as taurine conjugates after weaning, probably contributing to the minimal weight gain of the mice during the lactation period. This correlated with a marked induction of bile acyl-CoA:amino acid N-acyltransferase expression and enzyme activity between postnatal days 10 and 21, whereas the bile acyl-CoA synthetases increased gradually with age. The nuclear receptors hepatocyte nuclear factor-4α farnesoid X receptor, and peroxisome proliferator receptor a did not appear to be involved in the up-regulation of the transferase. [ABSTRACT FROM AUTHOR]

Summary Metabolic reprogramming has emerged as a crucial regulator of immune cell activation, but how systemic metabolism influences immune cell metabolism and function remains to be investigated. To investigate the effect of dyslipidemia on immune cell metabolism, we performed in-depth transcriptional, metabolic, and functional characterization of macrophages isolated from hypercholesterolemic mice. Systemic metabolic changes in such mice alter cellular macrophage metabolism and attenuate inflammatory macrophage responses. In addition to diminished maximal mitochondrial respiration, hypercholesterolemia reduces the LPS-mediated induction of the pentose phosphate pathway (PPP) and the Nrf2-mediated oxidative stress response. Our observation that suppression of the PPP diminishes LPS-induced cytokine secretion supports the notion that this pathway contributes to inflammatory macrophage responses. Overall, this study reveals that systemic and cellular metabolism are strongly interconnected, together dictating macrophage phenotype and function. Graphical Abstract Highlights • Systemic metabolism affects immune cell metabolism • Hypercholesterolemia suppresses the PPP and Nrf2 pathway in macrophages • PPP inhibition and hypercholesterolemia deactivate inflammatory macrophage responses • The Nrf2 pathway regulates the PPP in an LXR-independent manner The link between systemic and cellular metabolism is a neglected aspect in immunometabolism. Baardman et al. show that hypercholesterolemia alters macrophage metabolism and phenotype. The suppressed pentose phosphate pathway (PPP) in those "foam cell" macrophages attenuates inflammatory responses, signifying that systemic and cellular metabolism together regulate macrophage function. [ABSTRACT FROM AUTHOR]

The structural disruption of the mitochondrial inner membrane in hepatocytes lacking functional peroxisomes along with selective impairment of respiratory complexes and depletion of mitochondrial DNA was previously reported. In search for the molecular origin of these mitochondrial alterations, we here show that these are tissue selective as they do neither occur in peroxisome deficient brain nor in peroxisome deficient striated muscle. Given the hepatocyte selectivity, we investigated the potential involvement of metabolites that are primarily handled by hepatic peroxisomes. Levels of these metabolites were manipulated in L-Pex5 knockout mice and/or compared with levels in different mouse models with a peroxisomal β-oxidation deficiency. We show that neither the deficiency of docosahexaenoic acid nor the accumulation of branched chain fatty acids, dicarboxylic acids or C27 bile acid intermediates are solely responsible for the mitochondrial anomalies. In conclusion, we demonstrate that peroxisomal inactivity differentially impacts mitochondria depending on the cell type but the cause of the mitochondrial destruction needs to be further explored. [ABSTRACT FROM AUTHOR]