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Transcriptome‐based drug screening is emerging as a powerful tool to identify geroprotective compounds to intervene in age‐related disease. We hypothesized that, by mimicking the transcriptional signature of the highly conserved longevity intervention of FOXO3 (daf‐16 in worms) overexpression, we could identify and repurpose compounds with similar downstream effects to increase longevity. Our in silico screen, utilizing the LINCS transcriptome database of genetic and compound interventions, identified several FDA‐approved compounds that activate FOXO downstream targets in mammalian cells. These included the neuromuscular blocker atracurium, which also robustly extends both lifespan and healthspan in Caenorhabditis elegans. This longevity is dependent on both daf‐16 signaling and inhibition of the neuromuscular acetylcholine receptor subunit unc‐38. We found unc‐38 RNAi to improve healthspan, lifespan, and stimulate DAF‐16 nuclear localization, similar to atracurium treatment. Finally, using RNA‐seq transcriptomics, we identify atracurium activation of DAF‐16 downstream effectors. Together, these data demonstrate the capacity to mimic genetic lifespan interventions with drugs, and in doing so, reveal that the neuromuscular acetylcholine receptor regulates the highly conserved FOXO/DAF‐16 longevity pathway. [ABSTRACT FROM AUTHOR]

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]

Cellular metabolism is important for determining cell function and shaping immune responses. Studies have shown a crucial role for stromal cells in steering proper immune responses in the lymph node microenvironment. These lymph node stromal cells (LNSCs) tightly regulate immune tolerance. We hypothesize that malfunctioning LNSCs create a microenvironment in which normal immune responses are not properly controlled, possibly leading to the development of autoimmune diseases such as rheumatoid arthritis (RA). Therefore, we set out to determine their metabolic profile during health and systemic autoimmunity. We included autoantibody positive individuals at risk of developing RA (RA-risk individuals), RA patients and healthy volunteers. All study subjects underwent lymph node biopsy sampling. Mitochondrial function in cultured LNSCs was assessed by quantitative PCR, flow cytometry, Seahorse and oleate oxidation assays. Overall, mitochondrial respiration was lower in RA(-risk) LNSCs compared with healthy LNSCs, while metabolic potential was only lower in RA LNSCs. To maintain basal mitochondrial respiration, all LNSCs were mostly dependent on fatty acid oxidation. However, RA(-risk) LNSCs were also dependent on glutamine oxidation. Finally, we showed that RA LNSCs have impaired metabolic flexibility. Our results show that the metabolic landscape of LNSCs is not only altered during established disease, but partly already in individuals at risk of developing RA. Future studies are needed to investigate the impact of restoring metabolic capacity in LNSC-mediated immunomodulation and disease progression. [ABSTRACT FROM AUTHOR]

Abstract: Peroxisomes play an important role in bile acid biosynthesis because the last steps of the synthesis pathway are performed by the β-oxidation system located inside peroxisomes. As a consequence, C27-bile acid intermediates accumulate in several peroxisomal disorders. It has been suggested that C27-bile acids are especially toxic and contribute to the liver disease associated with peroxisomal disorders. For this reason, we investigated the toxicity of C27-bile acids and the underlying mechanisms. We studied the effects of conjugated and unconjugated C27-bile acids on cell viability, mitochondrial respiratory chain function and production of oxygen radicals in the rat hepatoma cell line McA-RH7777. Cell viability decreased progressively after incubation with increasing concentrations of different bile acids with dihydroxycholestanoic acid (DHCA) being clearly the most cytotoxic bile acid. In addition, the different bile acids caused a dose-dependent decrease in ATP synthesis by isolated mitochondria oxidizing malate and glutamate. Finally, there was a dose-dependent stimulation of ROS generation in the presence of C27-bile acids. In conclusion, our studies showed that C27-bile acids are more cytotoxic than mature C24-bile acids. In addition, C27-bile acids are potent inhibitors of oxidative phosphorylation and enhance mitochondrial ROS production by inhibiting the respiratory chain. [Copyright &y& Elsevier]

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]

Sensory motor neuropathy is associated with various inherited disorders including Charcot-Marie-Tooth disease, X-linked adrenoleukodystrophy/adrenomyeloneuropathy and Refsum disease. In the latter two, the neuropathy is thought to result from the accumulation of specific fatty acids. We describe here three patients with elevated plasma concentrations of pristanic acid (a branched-chain fatty acid) and C27-bile-acid intermediates. Two of the patients suffered from adult-onset sensory motor neuropathy. One patient also had pigmentary retinopathy, suggesting Refsum disease, whereas the other patient had upper motor neuron signs in the legs, suggesting adrenomyeloneuropathy. The third patient was a child without neuropathy. In all three patients we discovered a deficiency of α-methylacyl-CoA racemase (AMACR). This enzyme is responsible for the conversion of pristanoyl-CoA and C27-bile acyl-CoAs to their (S)-stereoisomers, which are the only stereoisomers that can be degraded via peroxisomal β-oxidation. Sequence analysis of AMACR cDNA from the patients identified two different mutations that are likely to cause disease, based on analysis in Escherichia coli. Our findings have implications for the diagnosis of adult-onset neuropathies of unknown aetiology. [ABSTRACT FROM AUTHOR]

Barth syndrome (BTHS) is a rare disorder caused by mutations in the TAFAZZIN gene. Previous studies from both patients and model systems have established metabolic dysregulation as a core component of BTHS pathology. In particular, features such as lactic acidosis, pyruvate dehydrogenase (PDH) deficiency, and aberrant fatty acid and glucose oxidation have been identified. However, the lack of a mechanistic understanding of what causes these conditions in the context of BTHS remains a significant knowledge gap, and this has hindered the development of effective therapeutic strategies for treating the associated metabolic problems. In the current study, we utilized tafazzin-knockout C2C12 mouse myoblasts (TAZ-KO) and cardiac and skeletal muscle tissue from tafazzin-knockout mice to identify an upstream mechanism underlying impaired PDH activity in BTHS. This mechanism centers around robust upregulation of pyruvate dehydrogenase kinase 4 (PDK4), resulting from hyperactivation of AMP-activated protein kinase (AMPK) and subsequent transcriptional upregulation by forkhead box protein O1 (FOXO1). Upregulation of PDK4 in tafazzin-deficient cells causes direct phospho-inhibition of PDH activity accompanied by increased glucose uptake and elevated intracellular glucose concentration. Collectively, our findings provide a novel mechanistic framework whereby impaired tafazzin function ultimately results in robust PDK4 upregulation, leading to impaired PDH activity and likely linked to dysregulated metabolic substrate utilization. This mechanism may underlie previously reported findings of BTHS-associated metabolic dysregulation. [ABSTRACT FROM AUTHOR]

Carnitine derivatives of disease‐specific acyl‐CoAs are the diagnostic hallmark for long‐chain fatty acid β‐oxidation disorders (lcFAOD), including carnitine shuttle deficiencies, very‐long‐chain acyl‐CoA dehydrogenase deficiency (VLCADD), long‐chain 3‐hydroxyacyl‐CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MPTD). The exact consequence of accumulating lcFAO‐intermediates and their influence on cellular lipid homeostasis is, however, still unknown. To investigate the fate and cellular effects of the accumulating lcFAO‐intermediates and to explore the presence of disease‐specific markers, we used tracer‐based lipidomics with deuterium‐labeled oleic acid (D9‐C18:1) in lcFAOD patient‐derived fibroblasts. In line with previous studies, we observed a trend towards neutral lipid accumulation in lcFAOD. In addition, we detected a direct connection between the chain length and patterns of (un)saturation of accumulating acylcarnitines and the various enzyme deficiencies. Our results also identified two disease‐specific candidate biomarkers. Lysophosphatidylcholine(14:1) (LPC(14:1)) was specifically increased in severe VLCADD compared to mild VLCADD and control samples. This was confirmed in plasma samples showing an inverse correlation with enzyme activity, which was better than the classic diagnostic marker C14:1‐carnitine. The second candidate biomarker was an unknown lipid class, which we identified as S‐(3‐hydroxyacyl)cysteamines. We hypothesized that these were degradation products of the CoA moiety of accumulating 3‐hydroxyacyl‐CoAs. S‐(3‐hydroxyacyl)cysteamines were significantly increased in LCHADD compared to controls and other lcFAOD, including MTPD. Our findings suggest extensive alternative lipid metabolism in lcFAOD and confirm that lcFAOD accumulate neutral lipid species. In addition, we present two disease‐specific candidate biomarkers for VLCADD and LCHADD, that may have significant relevance for disease diagnosis, prognosis, and monitoring. [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]

Shift‐workers show an increased incidence of type 2 diabetes mellitus (T2DM). A possible mechanism is the disruption of the circadian timing of glucose homeostasis. Skeletal muscle mitochondrial function is modulated by the molecular clock. We used time‐restricted feeding (TRF) during the inactive phase to investigate how mistimed feeding affects muscle mitochondrial metabolism. Rats on an ad libitum (AL) diet were compared to those that could eat only during the light (inactive) or dark (active) phase. Mitochondrial respiration, metabolic gene expressions, and metabolite concentrations were determined in the soleus muscle. Rats on AL feeding or dark‐fed TRF showed a clear daily rhythm in muscle mitochondrial respiration. This rhythm in mitochondrial oxidative phosphorylation capacity was abolished in light‐fed TRF animals and overall 24h respiration was lower. The expression of several genes involved in mitochondrial biogenesis and the fission/fusion machinery was altered in light‐fed animals. Metabolomics analysis indicated that light‐fed animals had lost rhythmic levels of α‐ketoglutarate and citric acid. Contrastingly, lipidomics showed that light‐fed animals abundantly gained rhythmicity in levels of triglycerides. Furthermore, while the RER shifted entirely with the food intake in the light‐fed animals, many measured metabolic parameters (e.g., activity and mitochondrial respiration) did not strictly align with the shifted timing of food intake, resulting in a mismatch between expected metabolic supply/demand (as dictated by the circadian timing system and light/dark‐cycle) and the actual metabolic supply/demand (as dictated by the timing of food intake). These data suggest that shift‐work impairs mitochondrial metabolism and causes metabolic inflexibility, which can predispose to T2DM. [ABSTRACT FROM AUTHOR]

Background: Hyperlysinemia is an autosomal recessive inborn error of L-lysine degradation. To date only one causal mutation in the gene encoding a-aminoadipic semialdehyde synthase has been reported. We aimed to AASS better define the genetic basis of hyperlysinemia. Methods: We collected the clinical, biochemical and molecular data in a cohort of 8 hyperlysinemia patients with distinct neurological features. Results: We found novel causal mutations in in all affected individuals, including 4 missense mutations, AASS 2 deletions and 1 duplication. In two patients originating from one family, the hyperlysinemia was caused by a contiguous gene deletion syndrome affecting and AASS and PTPRZ1 Conclusions: Hyperlysinemia is caused by mutations in AASS and PTPRZ1 hyperlysinemia is generally considered a benign AASS metabolic variant, the more severe neurological disease course in two patients with a contiguous deletion syndrome may be explained by the additional loss of PIPRZ1. Our findings illustrate the importance of detailed PTPRZ1 biochemical and genetic studies in any hyperlysinemia patient. [ABSTRACT FROM AUTHOR]

Abstract: Isoprenoids constitute an important class of biomolecules that participate in many different cellular processes. Most available detection methods allow the identification of only one or two specific nonsterol isoprenoid intermediates following radioactive or fluorescent labeling. We here report a rapid, nonradioactive, and sensitive procedure for the simultaneous detection and quantification of the eight main nonsterol intermediates of the isoprenoid biosynthesis pathway by means of tandem mass spectrometry. Intermediates were analyzed by HPLC–MS/MS in the multiple reaction monitoring mode using a silica-based C18 HPLC column. For quantification, their stable isotope-labeled analogs were used as internal standards. HepG2 cells were used to validate the method. Mevalonate, phosphomevalonate, and the six subsequent isoprenoid pyrophosphates were readily determined with detection limits ranging from 0.03 to 1.0μmol/L. The intra- and interassay variations for HepG2 cell homogenates supplemented with isoprenoid intermediates were 3.6–10.9 and 4.4–11.9%, respectively. Under normal culturing conditions, isoprenoid intermediates in HepG2 cells were below detection limits. However, incubation of the cells with pamidronate, an inhibitor of farnesyl pyrophosphate synthase, resulted in increased levels of mevalonate, isopentenyl pyrophosphate/dimethylallyl pyrophosphate, and geranyl pyrophosphate. This method will be suitable for measuring profiles of isoprenoid intermediates in cells with compromised isoprenoid biosynthesis and for determining the specificity of potential inhibitors of the pathway. [Copyright &y& Elsevier]

Abstract: X-linked adrenoleukodystrophy (X-ALD) is a progressive neurodegenerative disorder characterized by the accumulation of saturated and mono-unsaturated very long-chain fatty acids (VLCFA) and reduced peroxisomal VLCFA β-oxidation activity. In this study, we investigated the role of VLCFA biosynthesis in X-ALD fibroblasts. Our data demonstrate that elongation of both saturated and mono-unsaturated VLCFAs is enhanced in fibroblasts from patients with peroxisomal β-oxidation defects including X-ALD, and peroxisome biogenesis disorders. These data indicate that enhanced VLCFA elongation is a general phenomenon associated with an impairment in peroxisomal β-oxidation, and not specific for X-ALD alone. Analysis of plasma samples from patients with X-ALD and different peroxisomal β-oxidation deficiencies revealed increased concentrations of VLCFAs up to 32 carbons. We infer that enhanced elongation does not result from impaired peroxisomal β-oxidation alone, but is due to the additional effect of unchecked chain elongation. We demonstrate that elongated VLCFAs are incorporated into complex lipids. The role of chain elongation was also studied retrospectively in samples from patients with X-ALD previously treated with “Lorenzo’s oil.” We found that the decrease in plasma C26:0 previously found is offset by the increase of mono-unsaturated VLCFAs, not measured previously during the trial. We conclude that evaluation of treatment protocols for disorders of peroxisomal β-oxidation making use of plasma samples should include the measurement of saturated and unsaturated VLCFAs of chain lengths above 26 carbon atoms. We also conclude that chain elongation offers an interesting target to be studied as a possible mode of treatment for X-ALD and other peroxisomal β-oxidation disorders. [Copyright &y& Elsevier]

Abstract Barth syndrome (BTHS) is a rare X-linked disorder that is characterized by cardiac and skeletal myopathy, neutropenia and growth abnormalities. The disease is caused by mutations in the tafazzin (TAZ) gene encoding an enzyme involved in the acyl chain remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, this leads to decreased levels of mature CL and accumulation of the intermediate monolysocardiolipin (MLCL). At a cellular level, this causes mitochondrial fragmentation and reduced stability of the respiratory chain supercomplexes. However, the exact mechanism through which tafazzin deficiency leads to disease development remains unclear. We therefore aimed to elucidate the pathways affected in BTHS cells by employing proteomic and metabolic profiling assays. Complexome profiling of patient skin fibroblasts revealed significant effects for about 200 different mitochondrial proteins. Prominently, we found a specific destabilization of higher order oxidative phosphorylation (OXPHOS) supercomplexes, as well as changes in complexes involved in cristae organization and CL trafficking. Moreover, the key metabolic complexes 2-oxoglutarate dehydrogenase (OGDH) and branched-chain ketoacid dehydrogenase (BCKD) were profoundly destabilized in BTHS patient samples. Surprisingly, metabolic flux distribution assays using stable isotope tracer-based metabolomics did not show reduced flux through the TCA cycle. Overall, insights from analyzing the impact of TAZ mutations on the mitochondrial complexome provided a better understanding of the resulting functional and structural consequences and thus the pathological mechanisms leading to Barth syndrome. Highlights • Barth syndrome patient cells were analyzed by proteomic and metabolic profiling. • Complexome profiling revealed effects on 200 different mitochondrial proteins. • Cristae organization and cardiolipin trafficking complexes were prominently changed. • 2-Oxoglutarate and branched-chain ketoacid dehydrogenase were destabilized. • Metabolic flux distribution was not reduced. [ABSTRACT FROM AUTHOR]

Aims Mitochondrial fatty acid oxidation (FAO) is an important energy provider for cardiac work and changes in cardiac substrate preference are associated with different heart diseases. Carnitine palmitoyltransferase 1B (CPT1B) is thought to perform the rate limiting enzyme step in FAO and is inhibited by malonyl-CoA. The role of CPT1B in cardiac metabolism has been addressed by inhibiting or decreasing CPT1B protein or after modulation of tissue malonyl-CoA metabolism. We assessed the role of CPT1B malonyl-CoA sensitivity in cardiac metabolism. Methods and results We generated and characterized a knock in mouse model expressing the CPT1BE3A mutant enzyme, which has reduced sensitivity to malonyl-CoA. In isolated perfused hearts, FAO was 1.9-fold higher in Cpt1b E3A/E3A hearts compared with Cpt1b WT/WT hearts. Metabolomic, proteomic and transcriptomic analysis showed increased levels of malonylcarnitine, decreased concentration of CPT1B protein and a small but coordinated downregulation of the mRNA expression of genes involved in FAO in Cpt1b E3A/E3A hearts, all of which aim to limit FAO. In vivo assessment of cardiac function revealed only minor changes, cardiac hypertrophy was absent and histological analysis did not reveal fibrosis. Conclusions Malonyl-CoA-dependent inhibition of CPT1B plays a crucial role in regulating FAO rate in the heart. Chronic elevation of FAO has a relatively subtle impact on cardiac function at least under baseline conditions. [ABSTRACT FROM AUTHOR]

Obesity is accompanied by the presence of chronic low-grade inflammation manifested by infiltration of macrophages into adipose tissue. Mannose-binding lectin (MBL), a soluble mediator of innate immunity, promotes phagocytosis and alters macrophage function. To assess the function of MBL in the development of obesity, we studied wild-type and MBL-/- mice rendered obese using a high-fat diet (HFD). Whereas no gross morphological differences were observed in liver, an HFD provoked distinct changes in the adipose tissue morphology of MBL-/- mice. In parallel with increased adipocyte size, MBL-/- mice displayed an increased influx of macrophages into adipose tissue. Macrophages were polarized toward an alternatively activated phenotype known to modulate apoptotic cell clearance. MBL deficiency also significantly increased the number of apoptotic cells in adipose tissue. Consistent with these observations, recombinant MBL enhanced phagocytic capacity of the stromal vascular fraction isolated from adipose tissue and modulated uptake of apoptotic adipocytes by macrophages. Despite changes in macrophage abundance and polarity, the absence of MBL did not affect systemic insulin resistance. Finally, in humans, lower levels of circulating MBL were accompanied by enhanced macrophage influx in subcutaneous adipose tissue. We propose a novel role for MBL in the recognition and clearance of apoptotic adipocytes during obesity. [ABSTRACT FROM AUTHOR]

Background: Hyperlysinemia is an autosomal recessive inborn error of L-lysine degradation. To date only one causal mutation in the AASS gene encoding α-aminoadipic semialdehyde synthase has been reported. We aimed to better define the genetic basis of hyperlysinemia.Methods: We collected the clinical, biochemical and molecular data in a cohort of 8 hyperlysinemia patients with distinct neurological features.Results: We found novel causal mutations in AASS in all affected individuals, including 4 missense mutations, 2 deletions and 1 duplication. In two patients originating from one family, the hyperlysinemia was caused by a contiguous gene deletion syndrome affecting AASS and PTPRZ1.Conclusions: Hyperlysinemia is caused by mutations in AASS. As hyperlysinemia is generally considered a benign metabolic variant, the more severe neurological disease course in two patients with a contiguous deletion syndrome may be explained by the additional loss of PTPRZ1. Our findings illustrate the importance of detailed biochemical and genetic studies in any hyperlysinemia patient. [ABSTRACT FROM AUTHOR]

Peroxisome biogenesis disorders (PBDs) and D-bifunctional protein (D-BP) deficiency are two types of inherited peroxisomal disorders. Patients with a PBD lack functional peroxisomes and patients with D-BP deficiency lack the enzyme, which is responsible for the second and third step of the peroxisomal β-oxidation. The clinical presentation of these peroxisomal disorders is severe and includes several neurological abnormalities. The pathological mechanisms underlying these disorders are not understood and no therapies are available. Because peroxisomes have been associated with oxidative stress, as oxygen radicals are both produced and scavenged in peroxisomes, we have investigated whether oxidative stress is involved in the pathogenesis of PBDs and D-BP deficiency. We found in D-BP-deficient patients increased levels of thiobarbituric acid-reactive substances (TBARS) and 8-hydroxydeoxyguanosine (8-OHdG), which are markers for lipid peroxidation and oxidative DNA damage, respectively, whereas the levels of the lipophilic antioxidants α-tocopherol and coenzyme Q10 were decreased. In addition, we found in skin fibroblasts from D-BP-deficient patients an imbalance between the activities of the peroxisomal H2O2-generating straight-chain acyl-CoA oxidase (SCOX) and the peroxisomal H2O2-degrading enzyme catalase. In conclusion, we have found clear evidence for the presence of increased oxidative stress in patients with D-BP deficiency, but not in patients with a PBD. [Copyright &y& Elsevier]

Nicotinamide adenine dinucleotide (NAD+) homeostasis is constantly compromised due to degradation by NAD+-dependent enzymes. NAD+ replenishment by supplementation with the NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can alleviate this imbalance. However, NMN and NR are limited by their mild effect on the cellular NAD+ pool and the need of high doses. Here, we report a synthesis method of a reduced form of NMN (NMNH), and identify this molecule as a new NAD+ precursor for the first time. We show that NMNH increases NAD+ levels to a much higher extent and faster than NMN or NR, and that it is metabolized through a different, NRK and NAMPT-independent, pathway. We also demonstrate that NMNH reduces damage and accelerates repair in renal tubular epithelial cells upon hypoxia/reoxygenation injury. Finally, we find that NMNH administration in mice causes a rapid and sustained NAD+ surge in whole blood, which is accompanied by increased NAD+ levels in liver, kidney, muscle, brain, brown adipose tissue, and heart, but not in white adipose tissue. Together, our data highlight NMNH as a new NAD+ precursor with therapeutic potential for acute kidney injury, confirm the existence of a novel pathway for the recycling of reduced NAD+ precursors and establish NMNH as a member of the new family of reduced NAD+ precursors. [ABSTRACT FROM AUTHOR]