Mitochondrial Β-oxidation Pathway Research Articles

Imatinib therapy of chronic myeloid leukemia significantly reduces carnitine cell intake, resulting in adverse events

ObjectiveA prominent, safe and efficient therapy for patients with chronic myeloid leukemia (CML) is inhibiting oncogenic protein BCR::ABL1 in a targeted manner with imatinib, a tyrosine kinase inhibitor. A substantial part of patients treated with imatinib report skeletomuscular adverse events affecting their quality of life. OCTN2 membrane transporter is involved in imatinib transportation into the cells. At the same time, the crucial physiological role of OCTN2 is cellular uptake of carnitine which is an essential co-factor for the mitochondrial β-oxidation pathway. This work investigates the impact of imatinib treatment on carnitine intake and energy metabolism of muscle cells. MethodsHTB-153 (human rhabdomyosarcoma) cell line and KCL-22 (CML cell line) were used to study the impact of imatinib treatment on intracellular levels of carnitine and vice versa. The energy metabolism changes in cells treated by imatinib were quantified and compared to changes in cells exposed to highly specific OCTN2 inhibitor vinorelbine. Mouse models were used to test whether in vitro observations are also achieved in vivo in thigh muscle tissue. The analytes of interest were quantified using a Prominence HPLC system coupled with a tandem mass spectrometer. ResultsThis work showed that through the carnitine-specific transporter OCTN2, imatinib and carnitine intake competed unequally and intracellular carnitine concentrations were significantly reduced. In contrast, carnitine preincubation did not influence imatinib cell intake or interfere with leukemia cell targeting. Blocking the intracellular supply of carnitine with imatinib significantly reduced the production of most Krebs cycle metabolites and ATP. However, subsequent carnitine supplementation rescued mitochondrial energy production. Due to specific inhibition of OCTN2 activity, the influx of carnitine was blocked and mitochondrial energy metabolism was impaired in muscle cells in vitro and in thigh muscle tissue in a mouse model. ConclusionsThis preclinical experimental study revealed detrimental effect of imatinib on carnitine-mediated energy metabolism of muscle cells providing a possible molecular background of the frequently occurred side effects during imatinib therapy such as fatigue, muscle pain and cramps.

Abstract 15278: Cardiac Assessments and Manifestations Among Individuals With Long-Chain Fatty Acid Oxidation Disorders: A Retrospective Claims Database Analysis

Introduction: Long-chain fatty acid oxidation disorders (LC-FAOD) are inherited autosomal recessive defects resulting in enzyme deficiencies in the carnitine cycle or the mitochondrial β-oxidation pathway leading to deficiencies in mitochondrial energy metabolism. Cardiac manifestations, specifically cardiomyopathy and arrhythmias, are common and are leading causes of mortality in individuals with LC-FAOD. Goal: The goal of this study was to better understand cardiac manifestations and long-term outcomes in this group of disorders. Methods: The analysis was done using IQVIA PharMetrics® claims data. Individuals with confirmed LC-FAOD were identified and a non-LC-FAOD comparators group was created using 1:20 exact matching on age, gender, payer type, and enrollment start year. Cardiac-related complications were examined using ICD-10 diagnosis codes at any billing position during the study period. Cardiac diagnoses and assessments were evaluated using Current Procedural Terminology (CPT®) codes in a non-inpatient setting. Results: A total of 337 pediatric (peds) and 86 adult individuals with LC-FAOD were included in the study. Cardiomyopathy (peds: 8.0%, adults: 10.5%) and arrhythmias (peds: 8.9%, adults: 14.0%) were common among individuals with LC-FAOD and were significantly higher than comparators. LC-FAOD adults had significantly more cardiac conditions compared with pediatric patients and comparators. The other most common cardiac conditions among adults with LC-FAOD were chronic ischemic heart disease (18.6%), non-rheumatic mitral or tricuspid valve disorders (11.6% and 12.8%, respectively), heart failure (10.5%), atrioventricular and left bundle-branch block (7.0%), and cardiomegaly (5.1%). Individuals with LC-FAOD had significantly more cardiac related procedures than comparators. EKGs (peds: 35.9%; adults: 77.9%) and echocardiograms (peds: 44.8%; adults: 53.5%) were common among individuals with LC-FAOD. Conclusions: This analysis shows the importance of assessing the cardiac status of individuals with LC-FAOD. More analyses and natural history studies are needed to inform healthcare providers of cardiac risks associated with LC-FAOD and reduce mortality in these individuals.

Intestinal metabolomics of juvenile lenok (Brachymystax lenok) in response to heat stress.

Changes in the metabolic profile within the intestine of lenok (Brachymystax lenok) when challenged to acute and lethal heat stress (HS) are studied using no-target HPLC-MS/MS metabonomic analysis. A total of 51 differentially expressed metabolites (VIP > 1, P < 0.05) were identified in response to HS, and 34 occurred in the positive ion mode and 17 in negative ion mode, respectively. After heat stress, changes in metabolites related to glycolysis (i.e., alpha-D-glucose, stachyose, and L-lactate) were identified. The metabolites (acetyl carnitine, palmitoylcarnitine, carnitine, and erucic acid) related to fatty acid β-oxidation accumulated significantly, and many amino acids (L-tryptophan, D-proline, L-leucine, L-phenylalanine, L-aspartate, L-tyrosine, L-methionine, L-histidine, and L-glutamine) were significantly decreased in HS-treated lenok. The mitochondrial β-oxidation pathway might be inhibited, while severe heat stress might activate the anaerobic glycolysis and catabolism of amino acid for energy expenditure. Oxidative damage in HS-treated lenok was indicated by the decreased glycerophospholipid metabolites (i.e., glycerophosphocholine, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine) and the increased oxylipin production (12-HETE and 9R, 10S-EpOME). The minor oxidative pathways (omega-oxidation and peroxisomal beta-oxidation) were likely to be induced in HS-treated lenok.

Tissue-Specific and Differential Cold Responses in the Domesticated Cold Tolerant Fugu

Domestication can be defined as the artificial selection in animals to achieve morphological, physiological, and developmental conformity to human needs, with the aim of improving various limitations in species under a human feeding environment. The future sustainability of aquaculture may rely partly on the availability of numerous domesticated fish species. However, the underlying adaptive mechanisms that result in the domestication of fish are still unclear. Because they are poikilothermic, temperature is a key environmental element that affects the entire life of fish, so studying the association between physiological and behavioral changes in low-temperature domesticated fish can provide a model for understanding the response mechanisms of fish under cold stress. Through 5 generations and 10 years of artificial selection at low temperatures, we used cold-tolerant fugu as a biological model to compare transcriptome changes in brain and liver tissues to study the effects of cold stress on fish. It was found that the expression of genes such as apoptosis, p53, oxidative phosphorylation, and mitochondrial β-oxidation in the brain of cold-tolerant fugu was significantly lower than the wild type due to cold stress, while excessive energy metabolism would lead to the production of reactive oxygen species (ROS) and exacerbate the brain damage, thus causing rollover and coma. Meanwhile, under cold stress, the signaling pathways involved in glycogenolysis and lipid metabolism, such as insulin signaling, adipocytokines, and mTOR signaling pathways, were significantly up-regulated in the liver of cold-tolerant fugu. Although the mitochondrial β-oxidation pathway was increased in cold-tolerant fugu liver tissues, the transcriptome was not enriched in apoptotic. These phenomena predict that in response to low-temperature conditions, cold-tolerant fugu employs a dynamic inter-organ metabolic regulation strategy to cope with cold stress and reduce damage to brain tissues.

P–307 Fatty acid degradation during in vitro decidualization of human endometrial stromal cells

Abstract Study question Is the activity of the β-oxidation pathway, involved in the degradation of fatty acids, modified during in vitro decidualization of human endometrial stromal cells (HESC)? Summary answer The level of expression of fatty acid´s transporters suggests that the activity of the mitochondrial β-oxidation pathway is increased during in vitro decidualization of HESC. What is known already The differentiation of endometrial stromal cells (ESC), named decidualization, is essential for the proper formation of the materno-fetal interphase. One important feature of decidualization is the increased glucose consumption. In the endometrium, glucose is incorporated into ESC by glucose-transporters (GLUT). Fatty acids are another important energy source in living cells. Fatty acids are transported into mitochondria by the carnitine-palmitoyl-transferases 1 and 2 (CPT1 and 2) and are degraded there through the β-oxidation pathway. It has been described that the inhibition of CPT1 affects ESC decidualization. However, it is unknown whether the turn-over of fatty acids degradation is modified during decidualization. Study design, size, duration This study was performed using primary HESC. Endometrial biopsies (mid-late proliferative-phase) were obtained from healthy-regularly-cycling women (33.6±2.2 years-old) after written informed consent was obtained (protocol approved by Ethics committee no. S–239/2005). HESC were decidualized (D) in vitro with a decidualization-cocktail (containing: medroxyprogesterone acetate, estradiol and 8-Bromo-cyclic adenosine monophosphate) for 6 days. Non-decidualized (ND) controls were treated with vehicle solutions. Cell-culture supernatant and cell extracts were collected for the evaluation of protein/gene expression and metabolite content. Participants/materials, setting, methods Decidualization was evaluated by measuring prolactin (PRL) protein levels in cell-culture supernatant (mU/l). Changes in mRNA expression levels of GLUT1, CPT1A and CPT2 were evaluated by real-time polymerase chain reaction (RT-PCR). Analysis was performed by the ΔΔCt method (internal control: RPLP0) (fold change -FC- in D compared to ND cells). Contents of acylcarnitines were evaluated by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) (nmol/mg of total protein). N = 5, mean±SEM. Paired Student’s t-test was used for statistical analysis. Main results and the role of chance PRL protein levels in cell-culture supernatant were significative increased in HESC treated with the decidualization-cocktail compared to ND cells (ND 16.80±0.73 mU/l; D 684.20±219.80 mU/l, *p&amp;lt;0.05). This result confirmed the decidualized state of HESC upon in vitro treatment with the decidualization-cocktail. Additionally, the mRNA expression level of GLUT1 was highly upregulated in D compared to ND cells (FC 10.02±2.90, ***p&amp;lt;0.001), consistent with the increase in glucose consumption characteristic of decidualization. Once confirmed the decidualized state of HESC, the mRNA expression levels of CPTA1 and CPT2 were evaluated. The mRNA expression levels of both fatty acid´s transporters were upregulated in D compared to ND cells (CPTA1: FC 1.84±0.44, **p&amp;lt;0.01; CPT2: FC 2.04±0.49, **p&amp;lt;0.01). Finally, the content levels of different acylcarnitines, intermediate metabolites of the β-oxidation degradation of fatty acids, were evaluated. The concentrations of acetyl- (C2) and butyryl- (C4) acylcarnites were decreased in D compared to ND cells [(C2: ND 1.37±0.10 nmol/mg of total protein; D 1.06±0.20 nmol/mg of total protein, *p&amp;lt;0.05), (C4: ND 0.03±0.01 nmol/mg of total protein; D 0.01±0.00 nmol/mg of total protein, *p&amp;lt;0.05)]. The content levels of other intermediate acylcarnitines measured from cell extracts had no differences between D and ND cells (p &amp;gt; 0,05). Limitations, reasons for caution This study was performed in vitro using primary HESC treated with a decidualization-cocktail. The interconnection of different metabolic pathways within a living cell is very complex. Further studies are necessary to define whether the different intermediate metabolites of the mitochondrial β-oxidation pathway are being used by related-metabolic pathways during decidualization. Wider implications of the findings: The regulation of the energy metabolism and its interconnection with other important intra-cellular metabolic pathways is of great importance for cellular function. Our results contribute to highlight the importance of the regulation of fatty acids degradation during decidualization. Further insights into HESC metabolism could facilitate the improvement of womeńs health. Trial registration number Not applicable

P-307 Fatty acid degradation during in vitro decidualization of human endometrial stromal cells

Abstract Study question Is the activity of the β-oxidation pathway, involved in the degradation of fatty acids, modified during in vitro decidualization of human endometrial stromal cells (HESC)? Summary answer The level of expression of fatty acid´s transporters suggests that the activity of the mitochondrial β-oxidation pathway is increased during in vitro decidualization of HESC. What is known already The differentiation of endometrial stromal cells (ESC), named decidualization, is essential for the proper formation of the materno-fetal interphase. One important feature of decidualization is the increased glucose consumption. In the endometrium, glucose is incorporated into ESC by glucose-transporters (GLUT). Fatty acids are another important energy source in living cells. Fatty acids are transported into mitochondria by the carnitine-palmitoyl-transferases 1 and 2 (CPT1 and 2) and are degraded there through the β-oxidation pathway. It has been described that the inhibition of CPT1 affects ESC decidualization. However, it is unknown whether the turn-over of fatty acids degradation is modified during decidualization. Study design, size, duration This study was performed using primary HESC. Endometrial biopsies (mid-late proliferative-phase) were obtained from healthy-regularly-cycling women (33.6±2.2 years-old) after written informed consent was obtained (protocol approved by Ethics committee no. S-239/2005). HESC were decidualized (D) in vitro with a decidualization-cocktail (containing: medroxyprogesterone acetate, estradiol and 8-Bromo-cyclic adenosine monophosphate) for 6 days. Non-decidualized (ND) controls were treated with vehicle solutions. Cell-culture supernatant and cell extracts were collected for the evaluation of protein/gene expression and metabolite content. Participants/materials, setting, methods Decidualization was evaluated by measuring prolactin (PRL) protein levels in cell-culture supernatant (mU/l). Changes in mRNA expression levels of GLUT1, CPT1A and CPT2 were evaluated by real-time polymerase chain reaction (RT-PCR). Analysis was performed by the ΔΔCt method (internal control: RPLP0) (fold change -FC- in D compared to ND cells). Contents of acylcarnitines were evaluated by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) (nmol/mg of total protein). N = 5, mean±SEM. Paired Student’s t-test was used for statistical analysis. Main results and the role of chance PRL protein levels in cell-culture supernatant were significative increased in HESC treated with the decidualization-cocktail compared to ND cells (ND 16.80±0.73 mU/l; D 684.20±219.80 mU/l, *p&amp;lt;0.05). This result confirmed the decidualized state of HESC upon in vitro treatment with the decidualization-cocktail. Additionally, the mRNA expression level of GLUT1 was highly upregulated in D compared to ND cells (FC 10.02±2.90, ***p&amp;lt;0.001), consistent with the increase in glucose consumption characteristic of decidualization. Once confirmed the decidualized state of HESC, the mRNA expression levels of CPTA1 and CPT2 were evaluated. The mRNA expression levels of both fatty acid´s transporters were upregulated in D compared to ND cells (CPTA1: FC 1.84±0.44, **p&amp;lt;0.01; CPT2: FC 2.04±0.49, **p&amp;lt;0.01). Finally, the content levels of different acylcarnitines, intermediate metabolites of the β-oxidation degradation of fatty acids, were evaluated. The concentrations of acetyl- (C2) and butyryl- (C4) acylcarnites were decreased in D compared to ND cells [(C2: ND 1.37±0.10 nmol/mg of total protein; D 1.06±0.20 nmol/mg of total protein, *p&amp;lt;0.05), (C4: ND 0.03±0.01 nmol/mg of total protein; D 0.01±0.00 nmol/mg of total protein, *p&amp;lt;0.05)]. The content levels of other intermediate acylcarnitines measured from cell extracts had no differences between D and ND cells (p &amp;gt; 0,05). Limitations, reasons for caution This study was performed in vitro using primary HESC treated with a decidualization-cocktail. The interconnection of different metabolic pathways within a living cell is very complex. Further studies are necessary to define whether the different intermediate metabolites of the mitochondrial β-oxidation pathway are being used by related-metabolic pathways during decidualization. Wider implications of the findings The regulation of the energy metabolism and its interconnection with other important intra-cellular metabolic pathways is of great importance for cellular function. Our results contribute to highlight the importance of the regulation of fatty acids degradation during decidualization. Further insights into HESC metabolism could facilitate the improvement of womeńs health. Trial registration number not applicable

Post-translational modification by acetylation regulates the mitochondrial carnitine/acylcarnitine transport protein.

The carnitine/acylcarnitine transporter (CACT; SLC25A20) mediates an antiport reaction allowing entry of acyl moieties in the form of acylcarnitines into the mitochondrial matrix and exit of free carnitine. The transport function of CACT is crucial for the β-oxidation pathway. In this work, it has been found that CACT is partially acetylated in rat liver mitochondria as demonstrated by anti-acetyl-lys antibody immunostaining. Acetylation was reversed by the deacetylase Sirtuin 3 in the presence of NAD+. After treatment of the mitochondrial extract with the deacetylase, the CACT activity, assayed in proteoliposomes, increased. The half-saturation constant of the CACT was not influenced, while the V max was increased by deacetylation. Sirtuin 3 was not able to deacetylate the CACT when incubation was performed in intact mitoplasts, indicating that the acetylation sites are located in the mitochondrial matrix. Prediction on the localization of acetylated residues by bioinformatics correlates well with the experimental data. Recombinant CACT treated with acetyl-CoA was partially acetylated by non-enzymatic mechanism with a corresponding decrease of transport activity. The experimental data indicate that acetylation of CACT inhibits its transport activity, and thus may contribute to the regulation of the mitochondrial β-oxidation pathway.

Abstract 1014: The anti-Warburg agent BPM 31510 arbitrates fatty acid metabolism in eliciting an anticancer response

Abstract De-regulated lipid homeostasis is a key feature of the altered metabolic phenotype observed in cancer, particularly in the adipose-rich microenvironment of breast tumors. Mitochondria serve as a central hub for lipid metabolism through mitochondrial β-oxidation pathways and biosynthesis through production of citric acid cycle-derived citrate and the activity of ATP citrate lyase, the rate-limiting enzyme in lipid biosynthesis. We have previously demonstrated BPM 31510 ability to effectuate Warburg switch from glycolysis to mitochondrial oxidative phosphorylation resulting in activation of apoptosis in multiple cancers including triple negative breast cancer. The present investigated the role of BPM 31510 in influencing lipid metabolism in cancer cells to arbitrate its anti-cancer effect. Cell viability was assessed in MDA-MB231 and SkBr-3 breast cancer cells exposed to BPM 31510 alone or in combination with fatty metabolism inhibitors including C75, etomoxir, and trimetazidine, inhibitors of fatty acid synthase (FASN), carnitine palmitoyltransferase 1 (CPT-1), and β-oxidation, respectively, as well as a more pleiotropic fatty acid metabolism modulator, metformin. Breast cancer cells were more sensitive to BPM 31510 when treated in combination with C75, etomoxir, and trimetazidine as indicated by a left-shift in the BPM 31510 dose-response curve in both MDA-MB231 and SkBr-3 cells. In contrast, combined treatment with metformin did not alter cytotoxic responses to BPM 31510, indicating specificity for responses in fatty acid metabolism pathway modulation. Interestingly, BPM31510 treatment was associated with a dose- and time-dependent increase in mRNA expression of fatty acid metabolism gene products (FASN, CPT1, ACSL1) and accumulation of the triglyceride backbone, glycerol, in MDA-MB-231 cells. Structural lipidomic analysis used to assess the metabolic fate of BPM 31510 liposomal formulation components demonstrated that these were readily incorporated into prevalent diacyl-glyerol (DAG) and triacyl-glycerol (TAG) species along with de novo fatty acid species such as palmitate. Together, these results demonstrate that BPM 31510 alters endogenous and exogenous lipid homeostasis in breast cancer cells and rationale for potential combination with fatty acid metabolism inhibitors for anti-cancer therapy. The results expands on BPM 31510 anti-cancer mechanism, suggesting a central role in arbitrating the convergence of glycolysis, glucose and fatty acid oxidation pathways within the cancer cell metabolism network. Citation Format: Bianca Jambhekar, Tulin Dadali, Anne R. Diers, Fei Gao, Hannah Rockwell, Emily Chen, Stephane Gesta, Vivek K. Vishnudas, Michael A. Kiebish, Niven R. Narain, Rangaprasad Sarangarajan. The anti-Warburg agent BPM 31510 arbitrates fatty acid metabolism in eliciting an anticancer response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1014.

A Fox2-dependent fatty acid ß-oxidation pathway coexists both in peroxisomes and mitochondria of the ascomycete yeast Candida lusitaniae.

It is generally admitted that the ascomycete yeasts of the subphylum Saccharomycotina possess a single fatty acid ß-oxidation pathway located exclusively in peroxisomes, and that they lost mitochondrial ß-oxidation early during evolution. In this work, we showed that mutants of the opportunistic pathogenic yeast Candida lusitaniae which lack the multifunctional enzyme Fox2p, a key enzyme of the ß-oxidation pathway, were still able to grow on fatty acids as the sole carbon source, suggesting that C. lusitaniae harbored an alternative pathway for fatty acid catabolism. By assaying 14Cα-palmitoyl-CoA consumption, we demonstrated that fatty acid catabolism takes place in both peroxisomal and mitochondrial subcellular fractions. We then observed that a fox2Δ null mutant was unable to catabolize fatty acids in the mitochondrial fraction, thus indicating that the mitochondrial pathway was Fox2p-dependent. This finding was confirmed by the immunodetection of Fox2p in protein extracts obtained from purified peroxisomal and mitochondrial fractions. Finally, immunoelectron microscopy provided evidence that Fox2p was localized in both peroxisomes and mitochondria. This work constitutes the first demonstration of the existence of a Fox2p-dependent mitochondrial β-oxidation pathway in an ascomycetous yeast, C. lusitaniae. It also points to the existence of an alternative fatty acid catabolism pathway, probably located in peroxisomes, and functioning in a Fox2p-independent manner.

Insights into mitochondrial fatty acid synthesis from the structure of heterotetrameric 3-ketoacyl-ACP reductase/3R-hydroxyacyl-CoA dehydrogenase.

Mitochondrial fatty acid synthesis (mtFAS) is essential for respiratory growth in yeast and mammalian embryonic survival. The human 3-ketoacyl-acyl carrier protein (ACP) reductase (KAR) of mtFAS is a heterotetrameric α2β2-assembly composed of 17β-hydroxysteroid dehydrogenase type-8 (HSD17B8, α-subunit) and carbonyl reductase type-4 (CBR4, β-subunit). Here we provide a structural explanation for the stability of the heterotetramer from the crystal structure with NAD(+) and NADP(+) bound to the HSD17B8 and CBR4 subunits, respectively, and show that the catalytic activity of the NADPH- and ACP-dependent CBR4 subunit is crucial for a functional HsKAR. Therefore, mtFAS is NADPH- and ACP dependent, employing the 3R-hydroxyacyl-ACP intermediate. HSD17B8 assists in the formation of the competent HsKAR assembly. The intrinsic NAD(+)- and CoA-dependent activity of the HSD17B8 subunit on the 3R-hydroxyacyl-CoA intermediates may indicate a role for this subunit in routing 3R-hydroxyacyl-CoA esters, potentially arising from the metabolism of unsaturated fatty acids, into the mitochondrial β-oxidation pathway.

S54 Preservation of mitochondrial oxidative capacity in critically ill patients balances reduction in mitochondrial biogenesis

Background and Aims In severe sepsis, early mitochondrial dysfunction in skeletal muscle is associated with decreased biogenesis and adverse patient outcome. We hypothesised that reduction in mitochondrial content during critical illness would be balanced by enhanced capacity to produce ATP achieved by switching from glucose to fatty acid oxidation as the preferred metabolic substrate. Methods 30 critically ill patients (70% male, age 56.4 ± 19.7 years, APACHE II score 22.4 ± 6.6) were prospectively recruited Results There were reduction in both skeletal muscle mtDNA (p = 0.04) and PGC1-α (p = 0.02) from day 1 to day 7, with fold change in PGC1α correlated with the fold change in mtDNA (r 2 = 0.65, p 0.1). Protein levels of mitochondrial respiratory complexes I-V did not change significantly from day 1 to day 7 and mtDNA fold change did not correlate with fold change in complex I-V (p > 0.1). There was a weak positive correlation between feeding (protein delivered) and fold change in complex I (r 2 = 0.20, p = 0.014) and complex III protein level (r 2 = 0.20, p = 0.016). Overall levels of mitochondrial β-oxidation pathway proteins (CPT1, MCAD, ETF, DecR1) did not change whilst peroxisomal MEFII increased significantly (p Conclusion These data suggest decreased mitochondrial biogenesis over the first week of critical illness. The increase in β-oxidation proteins combined with the lack of change in respiratory chain complex concentrations during the first week suggests that the oxidative capacity of the remaining mitochondria was enhanced with increased capacity to metabolise fatty acids. These data support our hypothesis that the decreased cellular energy utilisation accompanying decreased mitochondrial biogenesis is offset by increased capacity to produce ATP through the β-oxidation of fats.

Selective estrogen receptor modulation attenuates proteinuria-induced renal tubular damage by modulating mitochondrial oxidative status

Proteinuria is an independent risk factor for progressive renal diseases because it initiates or aggravates tubulointerstitial injury. Clinically, females are less susceptible to progression of chronic kidney disease; however, the mechanisms underlying the renoprotective effect of estrogen receptor stimulation have yet to be clarified. Recently, inflammasome-dependent inflammatory responses were shown to be triggered by free fatty acids, and mitochondria-derived reactive oxygen species were shown to be required for this response. Albumin-bound free fatty acids trigger inflammasome activation through mitochondrial reactive oxygen species production in human proximal tubule epithelial cells in vitro, an effect inhibited by raloxifene. Female ICR-derived glomerulonephritic mice (mice with hereditary nephritic syndrome) were ovariectomized and treated with raloxifene, a selective estrogen receptor modulator. Ovariectomized mice showed activation of tubular inflammasomes and elevated levels of inflammasome-dependent cytokines. Raloxifene attenuated these changes ameliorating tubulointerstitial damage, reduced production of reactive oxygen species, averted morphological changes, and improved respiratory function in mitochondria. The expression of genes that encode rate-limiting enzymes in the mitochondrial β-oxidation pathway was reduced by ovariectomy but enhanced by raloxifene. Thus, inflammasomes may be a novel and promising therapeutic target for proteinuria-induced renal injury.

Exploiting endobiotic metabolic pathways to target xenobiotic antioxidants to mitochondria

Oxidative stress plays a role in a range of human disease entities. Hence, strategies to target antioxidants to mitochondria are an active area of investigation. Triphenylphosphonium cation-based antioxidants and SS-peptides have been described and show significant uptake by mitochondria and effectiveness in animal models of conditions linked to oxidative stress. We tested the hypothesis that the mitochondrial β-oxidation pathway could be exploited to activate the antioxidant phenolic and methimazole prodrugs. Most compounds studied underwent mitochondrial biotransformation to release their antioxidant moieties, and some were cytoprotective in a hypoxia–reoxygenation model in rat cardiomyocytes. These results demonstrate the feasibility of exploiting mitochondrial bioactivation reactions for targeted drug delivery.

Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans

An understanding of the connections between metabolism and elaboration of virulence factors during host colonization by the human-pathogenic fungus Cryptococcus neoformans is important for developing antifungal therapies. Lipids are abundant in host tissues, and fungal pathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. In addition, lipids are important signaling molecules in both fungi and mammals. In this report, we demonstrate that defects in the peroxisomal and mitochondrial β-oxidation pathways influence the growth of C. neoformans on fatty acids as well as the virulence of the fungus in a mouse inhalation model of cryptococcosis. Disease attenuation may be due to the cumulative influence of altered carbon source acquisition or processing, interference with secretion, changes in cell wall integrity, and an observed defect in capsule production for the peroxisomal mutant. Altered capsule elaboration in the context of a β-oxidation defect was unexpected but is particularly important because this trait is a major virulence factor for C. neoformans. Additionally, analysis of mutants in the peroxisomal pathway revealed a growth-promoting activity for C. neoformans, and subsequent work identified oleic acid and biotin as candidates for such factors. Overall, this study reveals that β-oxidation influences virulence in C. neoformans by multiple mechanisms that likely include contributions to carbon source acquisition and virulence factor elaboration.

Defects in Mitochondrial and Peroxisomal β-Oxidation Influence Virulence in the Maize Pathogen Ustilago maydis

An understanding of metabolic adaptation during the colonization of plants by phytopathogenic fungi is critical for developing strategies to protect crops. Lipids are abundant in plant tissues, and fungal phytopathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. Previously, we demonstrated a role for the peroxisomal β-oxidation enzyme Mfe2 in the filamentous growth, virulence, and sporulation of the maize pathogen Ustilago maydis. However, mfe2 mutants still caused disease symptoms, thus prompting a more detailed investigation of β-oxidation. We now demonstrate that a defect in the had1 gene encoding hydroxyacyl coenzyme A dehydrogenase for mitochondrial β-oxidation also influences virulence, although its paralog, had2, makes only a minor contribution. Additionally, we identified a gene encoding a polypeptide with similarity to the C terminus of Mfe2 and designated it Mfe2b; this gene makes a contribution to virulence only in the background of an mfe2Δ mutant. We also show that short-chain fatty acids induce cell death in U. maydis and that a block in β-oxidation leads to toxicity, likely because of the accumulation of toxic intermediates. Overall, this study reveals that β-oxidation has a complex influence on the formation of disease symptoms by U. maydis that includes potential metabolic contributions to proliferation in planta and an effect on virulence-related morphogenesis.

Very Long Chain Fatty Acid β-Oxidation in Astrocytes: Contribution of the ABCD1-Dependent and -Independent Pathways

Very long chain fatty acid (VLCFA) metabolism in astrocytes is important for the maintenance of myelin structure in central nervous system. To analyze the contribution of the ABCD1-dependent and -independent pathways to VLCFA metabolism in astrocytes, we prepared human glioblastoma U87 cells with a silencing of ABCD1 and primary astrocytes from abcd1-deficient mice, and measured fatty acid β-oxidation in the presence or absence of a potent inhibitor of carnitine palmitoyltransferase I, 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA). In U87 cells, C24:0 β-oxidation was decreased to ca. 70% of the control in the presence of POCA, and the activity was further decreased to ca. 20% by the silencing of ABCD1. In mouse primary astrocytes, C24:0 β-oxidation was also decreased to ca. 70% of the control in the presence of POCA. The C24:0 β-oxidation in Abcd1-deficient primary astrocytes was ca. 60% of the wild-type cells and the activity was further decreased to ca. 25% in the presence of POCA. Compared to human skin fibroblasts, in which VLCFA β-oxidation is not significantly inhibited by POCA, approximately one-third of the overall VLCFA β-oxidation was inhibited in both types of astrocytic cells. These results suggest that VLCFA is indeed β-oxidized in ABCD1-dependent pathway, but the ABCD1-independent peroxisomal and mitochondrial β-oxidation pathways significantly contribute to VLCFA β-oxidation in astrocytic cells.

Putting bioactivation reactions to work: Targeting antioxidants to mitochondria

The goal of this research was to test the hypothesis that bioactivation reactions could be exploited to deliver and activate mitochondria-targeted antioxidant prodrugs. The concept that bioactivation reactions could be used for prodrug delivery and activation has received little attention. Most bioactivation reactions result in the conversion of the parent drug to a reactive electrophilic metabolite, but bioactivating enzymes that catalyze elimination or hydrolytic reactions may offer potential for targeted drug delivery. Because mitochondria are the major cellular source of reactive oxygen species, there is much interest in targeting antioxidants to mitochondria. Previous studies showed that the mitochondrial fatty acid β-oxidation pathway biotransforms a range of xenobiotic alkanoates, including ω-(phenyl)alkanoates and ω-(phenoxy)alkanoates. 5,6-Dichloro-4-thia-5-hexenoate, the desamino analog of S-(1,2-dichlorovinyl)- l-cysteine, is biotransformed by the fatty acid β-oxidation pathway. Hence, the prodrugs ω-(phenoxy)alkanoates, 3-(phenoxy)acrylates, and ω-(1-methyl-1 H-imidazol-2-ylthio)alkanoates were expected to undergo biotransformation by the mitochondrial β-oxidation pathway to release phenolic antioxidants and the antioxidant methimazole (Roser et al., Bioorg. Med. Chem. 18 (2010) 1441–1448). The rates of biotransformation of ω-(phenoxy)alkanoates varied with the structure, and bulky substituents on the phenoxy moiety reduced rates of biotransformation; this was attributed to substrate limitations imposed by the medium-chain acyl-CoA dehydrogenase. Hence, 3-(2,6-dimethylphenoxy)acrylate was prepared; it was expected that, after conversion to its CoA thioester, 3-(2,6-dimethylphenoxy)acryloyl-CoA would be a substrate for enoyl-CoA hydratase. This expectation was correct: 3-(2,6-dimethylphenoxy)acrylate was an excellent substrate. ω-(1-Methyl-1 H-imidazol-2-ylthio)alkanoates were also good substrates for the β-oxidation pathway. Significantly, 3-(2,6-dimethylphenoxy)propanoate, 3-(2,6-dimethylphenoxy)acrylate, and 3-(1-methyl-1 H-imidazol-2-ylthio)propanoate were cytoprotective in a hypoxia-reoxygenation model in rat cardiomyocytes. These results demonstrate the feasibility of exploiting bioactivation reactions for targeted drug delivery.

Mitochondrial biotransformation of ω-(phenoxy)alkanoic acids, 3-(phenoxy)acrylic acids, and ω-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids: A prodrug strategy for targeting cytoprotective antioxidants to mitochondria

Mitochondrial reactive oxygen species (ROS) generation and the attendant mitochondrial dysfunction are implicated in a range of disease states. The objective of the present studies was to test the hypothesis that the mitochondrial β-oxidation pathway could be exploited to deliver and biotransform the prodrugs ω-(phenoxy)alkanoic acids, 3-(phenoxy)acrylic acids, and ω-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids to the corresponding phenolic antioxidants or methimazole. 3- and 5-(Phenoxy)alkanoic acids and methyl-substituted analogs were biotransformed to phenols; rates of biotransformation decreased markedly with methyl-group substitution on the phenoxy moiety. 2,6-Dimethylphenol formation from the analogs 3-([2,6-dimethylphenoxy]methylthio)propanoic acid and 3-(2,6-dimethylphenoxy)acrylic acid was greater than that observed with ω-(2,6-dimethylphenoxy)alkanoic acids. 3- and 5-(1-Methyl-1H-imidazol-2-ylthio)alkanoic acids were rapidly biotransformed to the antioxidant methimazole and conferred significant cytoprotection against hypoxia-reoxygenation injury in isolated cardiomyocytes. Both 3-(2,6-dimethylphenoxy)propanoic acid and 3-(2,6-dimethylphenoxy)acrylic acid also afforded cytoprotection against hypoxia-reoxygenation injury in isolated cardiomyocytes. These results demonstrate that mitochondrial β-oxidation is a potentially useful delivery system for targeting antioxidants to mitochondria.

PPAR.BETA./.DELTA. regulates the human SIRT1 gene transcription via Sp1

NAD-dependent deacetylase SIRT1 is known to be activated by caloric restriction and is related to longevity. A natural polyphenolic compound resveratrol is also shown to increases SIRT1 activity and extends lifespan. However, the transcriptional regulation of SIRT1 gene has not completely examined in the context of metabolism. Thus, in this study, we characterized the 5' -flanking region of human SIRT1 gene. We first found that representative metabolic hormones and related factors (glucocorticoid, glucagon/cAMP, and insulin) did not show significant effect on SIRT1 gene transcription. PPARalpha and PPARgamma1 without/with their specific ligands did not have significant effect as well. In contrast, expression of PPARbeta/delta (PPARdelta markedly increased the 5' -promoter activity of SIRT1 gene, which was further amplified by the addition of GW501516, a selective PPARdelta agonist. Deletion/mutation mapping analyses failed to identify PPAR binding element but revealed the presence of canonical Sp1 binding site, which was conserved among species. The Sp1 site is functional, because Sp1 overexpresson significantly enhanced SIRT1 promoter activity, and the binding of Sp1 to the element was confirmed by EMSA and ChIP assays. Interestingly, specific Sp1 antagonist mithramycin completely abolished the PPARdelta-mediated induction of SIRT1 gene transcription. Altogether, our data suggest the predominant role of PPARdelta in the transcriptional regulation of SIRT1 gene. Furthermore, the effects of PPARdelta seem to be mediated by Sp1. We assume that, in vivo, starvation increases lipolysis-derived free fatty acid and activates PPARdelta and the resultant increase in SIRT1 expression, in addition to the activation by NAD and AMPK, facilitates the deacetylation of a variety of proteins involved in mitochondrial beta-oxidation pathway and cell survival.

A single acyl-CoA dehydrogenase is required for catabolism of isoleucine, valine and short-chain fatty acids in Aspergillus nidulans

An acyl-CoA dehydrogenase has been identified as part of the mitochondrial β-oxidation pathway in the ascomycete fungus Aspergillus nidulans. Disruption of the scdA gene prevented use of butyric acid (C 4) and hexanoic acid (C 6) as carbon sources and reduced cellular butyryl-CoA dehydrogenase activity by 7.5-fold. While the mutant strain exhibited wild-type levels of growth on erucic acid (C 22:1) and oleic acid (C 18:1), some reduction in growth was observed with myristic acid (C 14). The Δ scdA mutation was found to be epistatic to a mutation downstream in the β-oxidation pathway (disruption of enoyl-CoA hydratase). The Δ scdA mutant was also unable to use isoleucine or valine as a carbon source. Transcription of scdA was observed in the presence of either fatty acids or amino acids. When the mutant was grown in medium containing either isoleucine or valine, organic acid analysis of culture supernatants showed accumulation of 2-oxo acid intermediates of branched chain amino acid catabolism, suggesting feedback inhibition of the upstream branched-chain α-keto acid dehydrogenase.