scholarly journals Roles of key active-site residues in flavocytochrome P450 BM3

1999 ◽  
Vol 339 (2) ◽  
pp. 371-379 ◽  
Author(s):  
Michael A. NOBLE ◽  
Caroline S. MILES ◽  
Stephen K. CHAPMAN ◽  
Dominikus A. LYSEK ◽  
Angela C. MACKAY ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Active Site ◽  
Side Chain ◽  
Acid Oxidation ◽  
Dodecanoic Acid ◽  
Wild Type ◽  
Active Site Residues ◽  
P450 Bm3

The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 µM, kcat 3960 min-1; Y51F mutant, Km 432 µM, kcat 6140 min-1; wild-type, Km 288 µM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (ΔG‡) resulting from a smaller ΔG of substrate binding. The side chain of Phe-42 acts as a phenyl ‘cap ’ over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2.08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 µM, kcat 14620 min-1; compared with values of 4.7 µM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a ‘fast ’ to a ‘slow ’ rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat ‘fast ’, 760 (1620) min-1; kcat ‘slow ’, 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole > 10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid > 30-fold more tightly than wild-type.

scholarly journals Peroxisome Proliferator-Activated Receptor α Mediates the Effects of High-Fat Diet on Hepatic Gene Expression

Endocrinology ◽  
2006 ◽  
Vol 147 (3) ◽  
pp. 1508-1516 ◽  
Author(s):  
David Patsouris ◽  
Janardan K. Reddy ◽  
Michael Müller ◽  
Sander Kersten
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Microarray Analysis ◽  
High Fat Diet ◽  
Acid Oxidation ◽  
Peroxisome Proliferator ◽  
Dependent Manner ◽  
Wild Type ◽  
High Fat ◽  
Fat Feeding

Peroxisome proliferator-activated receptors (PPARs) are transcription factors involved in the regulation of numerous metabolic processes. The PPARα isotype is abundant in liver and activated by fasting. However, it is not very clear what other nutritional conditions activate PPARα. To examine whether PPARα mediates the effects of chronic high-fat feeding, wild-type and PPARα null mice were fed a low-fat diet (LFD) or high-fat diet (HFD) for 26 wk. HFD and PPARα deletion independently increased liver triglycerides. Furthermore, in wild-type mice HFD was associated with a significant increase in hepatic PPARα mRNA and plasma free fatty acids, leading to a PPARα-dependent increase in expression of PPARα marker genes CYP4A10 and CYP4A14. Microarray analysis revealed that HFD increased hepatic expression of characteristic PPARα target genes involved in fatty acid oxidation in a PPARα-dependent manner, although to a lesser extent than fasting or Wy14643. Microarray analysis also indicated functional compensation for PPARα in PPARα null mice. Remarkably, in PPARα null mice on HFD, PPARγ mRNA was 20-fold elevated compared with wild-type mice fed a LFD, reaching expression levels of PPARα in normal mice. Adenoviral overexpression of PPARγ in liver indicated that PPARγ can up-regulate genes involved in lipo/adipogenesis but also characteristic PPARα targets involved in fatty acid oxidation. It is concluded that 1) PPARα and PPARα-signaling are activated in liver by chronic high-fat feeding; and 2) PPARγ may compensate for PPARα in PPARα null mice on HFD.

scholarly journals Kinetic characteristics of propofol-induced inhibition of electron-transfer chain and fatty acid oxidation in human and rodent skeletal and cardiac muscles

2019 ◽  
Author(s):  
Tomáš Urban ◽  
Petr Waldauf ◽  
Adéla Krajčová ◽  
Kateřina Jiroutková ◽  
Milada Halačová ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Mitochondrial Membrane ◽  
Acid Oxidation ◽  
Kinetic Characteristics ◽  
Inner Mitochondrial Membrane ◽  
Electron Transfer Chain ◽  
Respiratory Complexes ◽  
Transfer Chain

AbstractIntroductionPropofol causes a profound inhibition of fatty acid oxidation (FAO) and reduces spare electron transfer chain (ETC) capacity in a range of human and rodent cells and tissues – a feature that might be related to the pathogenesis of Propofol Infusion Syndrome. We aimed to explore the mechanism of propofol-induced alteration of bioenergetic pathways by describing its kinetic characteristics.MethodsWe obtained samples of skeletal and cardiac muscle from Wistar rat (n=3) and human subjects: vastus lateralis from hip surgery patients (n=11) and myocardium from brain-dead organ donors (n=10). We assessed mitochondrial functional indices using standard SUIT protocol and high resolution respirometry in fresh tissue homogenates with or without short-term exposure to a range of propofol concentration (2.5-100 μg/ml). After finding concentrations of propofol causing partial inhibition of a particular pathways, we used that concentration to construct kinetic curves by plotting oxygen flux against substrate concentration during its stepwise titration in the presence or absence of propofol. By spectrophotometry we also measured the influence of the same propofol concentrations on the activity of isolated respiratory complexes.ResultsWe found that human muscle and cardiac tissues are more sensitive to propofol-mediated inhibition of bioenergetic pathways than rats tissue. In human homogenates, palmitoyl carnitine-driven respiration was inhibited at much lower concentrations of propofol than that required for a reduction of ETC capacity, suggesting FAO inhibition mechanism different from downstream limitation or carnitine-palmitoyl transferase-1 inhibition. Inhibition of Complex I was characterised by more marked reduction of Vmax, in keeping with non-competitive nature of the inhibition and the pattern was similar to the inhibition of Complex II or ETC capacity. There was no inhibition of Complex IV nor increased leak through inner mitochondrial membrane with up to 100 μg/ml of propofol. If measured in isolation by spectrophotometry, propofol 10 μg/ml did not affect the activity of any respiratory complexes.ConclusionIn human skeletal and heart muscle homogenates, propofol in concentrations that are achieved in propofol-anaesthetized patients, causes a direct inhibition of fatty acid oxidation, in addition to inhibiting flux of electrons through inner mitochondrial membrane. The inhibition is more marked in human as compared to rodent tissues.

Exercise, FGF21, and PGC-1 : roles in hepatic metabolism

2014 ◽  
Author(s):  
◽  
Justin Andrew Fletcher
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Gene Transcription ◽  
Fibroblast Growth Factor 21 ◽  
Acid Oxidation ◽  
Wild Type ◽  
Hepatic Fatty Acid ◽  
Exercise Induced ◽  
Hepatic Fatty Acid Oxidation ◽  
Mitochondrial Adaptations

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The liver is instrumental in maintaining euglycemia during times of fasting and exercise, and in-turn exercise is a stimulus that challenges the liver and results in hepatic mitochondrial adaptations. Mechanisms responsible for these improvements in mitochondrial function are not currently known. Fibroblast growth factor 21 (FGF21), a powerful metabolic regulator, is one potential mechanism responsible for exercise- induced hepatic mitochondrial adaptations. Previous studies show that FGF21 modulates hepatic fatty acid oxidation (FAO), gluconeogenesis, ketogenesis, and TCA cycle flux, in addition to gene transcription of proteins important to these processes. The purpose of the first objective in the current study was to examine whether FGF21 is necessary for exercise to induce hepatic mitochondrial adaptations in mice. A second objective was to determine if PGC--1? is responsible for the upregulation of genes important to metabolic processes in response to FGF21 signaling. We mechanistically assessed the necessity of FGF21 for exercise-induced hepatic mitochondrial adaptations by providing wild-type and FGF21 knockout mice with running wheels for 8 weeks to promote physical activity. A major finding in the current study is that the FGF21KO mice experience a hepatic fatty acid oxidation deficit compared to the wild-type group and that 8 weeks of voluntary wheel running normalized FAO in the FGF21KO mice. The role of PGC-1[alpha] in FGF21 regulation of gene transcription was also assessed by continuously administering FGF21 (1 mg/kg), or saline into wild-type or liver specific PGC--1[alpha] heterozygous mice (LPGC--1[alpha]) for 4 weeks. It was found that female mice did not express a phenotype effect; however, in male mice hepatic FAO was significantly blunted in the LPGC-1[alpha] mice, yet FGF21 administration was able to elevate FAO regardless in both genotypes. Collectively, this data suggests that FGF21 is necessary for the expression and content of certain genes or proteins, but that VWR is able to circumvent the absence of FGF21 and normalize hepatic FAO. Furthermore, a reduction in hepatic PGC-1[alpha] does not appear to influence the ability of FGF21 to regulate hepatic FAO.

Abstract 17085: Metabolic Signature of Human Right Ventricular Heart Muscle in Pulmonary Arterial Hypertension and Comparison to Two Animal Models

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Giovanni E Davogustto ◽  
Megha Talati ◽  
Niki Penner ◽  
Kelsey Tomasek ◽  
Yan Ru Su ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Metabolic Changes ◽  
Acid Oxidation ◽  
Long Chain Fatty Acids ◽  
Wild Type ◽  
Human Right ◽  
Metabolic Signature ◽  
Pulmonary Arterial ◽  
Long Chain

Introduction: Molecular studies of the human right ventricle (RV) in pulmonary arterial hypertension (PAH) are lacking. Metabolic changes in the failing RV vary across different animal models and have not been directly compared with the human RV. We hypothesized that the BMPR2 murine model of PAH would closely recapitulate metabolic changes in the human PAH RV Methods: We performed metabolomic profiling of 596 compounds (Metabolon) on: RV specimens from patients with PAH and non-PAH controls (n=3 per group), BMPR2 mice (n=15), wild-type mice after pulmonary artery banding (PAB) (n=7), and wild-type control mice (WT) (n=7). Normalized metabolites per group were compared by Welch’s t-test between two groups, and two-way ANOVA for >2 groups, followed by adjustment for multiple comparison analysis Results: Principal component analysis (PCA) revealed markedly different metabolic profiles between PAH and controls ( Figure 1A ), with significant changes in 131 biochemicals in PAH. We observed an increase in glycolysis as evident by lactate accumulation and reduction in glycolytic intermediaries. We also observed a substantial reduction in fatty acid oxidation in the PAH RV, characterized by markedly reduced acylcarnitines and accumulation of long chain fatty acids, lysolipids, and glycerol compounds ( Figure 1B ). The BMPR2 model significantly reproduced the direction of difference in 43/131 metabolites and the PAB model 29/131. Both models were associated with an increase in glycolysis but only the BMPR2 model showed evidence of impaired fatty acid oxidation (accumulation of long-chain fatty acids, lysolipids, glycerol, and monoacylglycerols), as observed in the human PAH RV ( Figure 1C ) Conclusions: The failing RV of patients with PAH has a distinct metabolic signature characterized by increased glycolysis and impaired fatty acid oxidation. The BMPR2 model of PAH recapitulates more of the key metabolic changes observed in humans compared with a model of isolated pressure overload. The BMPR2 model may be preferable for metabolic studies of the failing RV

Control of cardiac pyruvate dehydrogenase activity in peroxisome proliferator-activated receptor-α transgenic mice

2003 ◽  
Vol 285 (1) ◽  
pp. H270-H276 ◽  
Author(s):  
Teresa A. Hopkins ◽  
Mary C. Sugden ◽  
Mark J. Holness ◽  
Ray Kozak ◽  
Jason R. B. Dyck ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Pyruvate Dehydrogenase ◽  
Glucose Oxidation ◽  
Pyruvate Dehydrogenase Kinase ◽  
Acid Oxidation ◽  
Peroxisome Proliferator ◽  
Wild Type ◽  
Peroxisome Proliferator Activated Receptor ◽  
Oxidation Rates

The pyruvate dehydrogenase enzyme complex (PDC) is rate limiting for glucose oxidation in the heart. Inhibition of PDC by end-product feedback and phosphorylation by pyruvate dehydrogenase kinase (PDK) operate in concert to inhibit PDC activity. Because the transcriptional regulator peroxisome proliferator-activated receptor (PPAR)-α increases PDK expression in some tissues, we examined what role PPAR-α has in regulating glucose oxidation in hearts from mice overexpressing PPAR-α (MHC-PPAR-α mice). Glucose oxidation rates were decreased in isolated working hearts from MHC-PPAR-α mice compared with wild-type littermates (428 ± 113 vs. 771 ± 63 nmol · g dry weight-1 · min-1, respectively), which was accompanied by a parallel increase in fatty acid oxidation. However, there was no difference in PDC activity between MHC-PPAR-α and wild-type animals, even though the expression of the PDK isoform PDK1 was increased in MHC-PPAR-α mice. Glucose oxidation rates in both MHC-PPAR-α and wild-type mouse hearts were decreased after 48-h fasting (which increases PPAR-α expression) or by treatment of mice with the PPAR-α agonist WY-14,643 for 1 wk. Despite this, PDC activity in both animal groups was not altered. Taken together, these data suggest that glucose oxidation rates in the heart can be dramatically altered independent of PDK phosphorylation and inhibition of PDC by PDK. It also suggests that PPAR-α activation decreases glucose oxidation in hearts mainly by decreasing the flux of pyruvate through PDC due to negative feedback of PDC by fatty acid oxidation reaction products rather than by the phosphorylated state of the PDC complex.

scholarly journals The Influence of Shc Proteins on the Whole Body Energetic Response to Calorie Restriction Initiated in 3-Month-Old Mice

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Jennifer H. Stern ◽  
Kyoungmi Kim ◽  
Jon J. Ramsey
Keyword(s):  
Body Weight ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Calorie Restriction ◽  
Metabolic Response ◽  
Whole Body ◽  
Acid Oxidation ◽  
Wild Type ◽  
The Impact ◽  
Old Mice

There is increasing evidence that Shc proteins play a role in energy metabolism, and we have previously reported that knockdown of Shc proteins influences the energetic response to acute (3 days) calorie restriction (CR) in 18-month-old mice. Whether Shc proteins play a role in the metabolic response to CR in younger mice has yet to be elucidated. Hence, we sought to determine the impact of 3 days and longer term (2 months) CR on energy expenditure (EE) and respiratory quotient (RQ) in 3 month-old Shc knockout (ShcKO) and wild-type (WT) mice. ShcKO mice decreased (P < 0.001) EE normalized for body weight (EEBW) by 3 days of CR, while no such change was observed in WT animals. However, both ShcKO and WT mice decreased (P < 0.001) EEBW at 2 months of CR and there were no differences in body weight between the ShcKO and WT mice at either 3 days or 2 months of CR. Consistent with increased fatty acid oxidation, only ShcKO mice maintained decreased (P < 0.001) 24 h RQ through 2 months of CR, suggesting that they were able to maintain increased fatty acid oxidation for a longer period of time than WT mice. These results indicate that Shc proteins may contribute to some of the acute energetic responses to CR.

scholarly journals Prevention of Adipose Tissue Depletion during Food Deprivation in Angiotensin Type 2 Receptor-Deficient Mice

Endocrinology ◽  
2006 ◽  
Vol 147 (11) ◽  
pp. 5078-5086 ◽  
Author(s):  
Laurent Yvan-Charvet ◽  
Patrick Even ◽  
Noël Lamandé ◽  
Pascal Ferré ◽  
Annie Quignard-Boulangé
Keyword(s):  
Adipose Tissue ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Physiological Role ◽  
Acid Oxidation ◽  
Ang Ii ◽  
Wild Type ◽  
Angiotensin Type 2 Receptor

Angiotensin (Ang) II is produced locally in various tissues, but its role in the regulation of tissue metabolism is still unclear. Recent studies have revealed the role of type 2 Ang II receptor (AT2R) in the control of energy homeostasis and lipid metabolism. The contribution of the AT2R to adaptation to starvation was tested using AT2R-deficient (AT2Ry/−) mice. Fasted AT2Ry/− mice exhibited a lower loss of adipose tissue weight associated to a decreased free fatty acid (FFA) release from stored lipids than the controls. In vitro studies show that Ang II causes an AT1R-mediated antilipolytic effect in isolated adipocytes. AT1R expression is up-regulated by fasting in both genotypes, but the increase is more pronounced in AT2Ry/− mice. In addition, the increased muscle β-oxidation displayed in AT2Ry/− mice on a fed state, persists after fasting compared with wild-type mice. In liver from fed mice, AT2R deficiency did not modify the expression of genes involved in fatty acid oxidation. However, in response to fasting, the large increase of the expression of this subset of genes exhibited by wild-type mice, was impaired in AT2Ry/− mice. Taken together, decreased lipolytic capacity and increased muscle fatty acid oxidation participate in the decreased plasma FFA observed in fasted AT2Ry/− mice and could account for the lower FFA metabolism in the liver. These data reveal an important physiological role of AT2R in metabolic adaptations to fasting.

scholarly journals Kinetic characteristics of propofol-induced inhibition of electron-transfer chain and fatty acid oxidation in human and rodent skeletal and cardiac muscles

PLoS ONE ◽  
2019 ◽  
Vol 14 (10) ◽  
pp. e0217254
Author(s):  
Tomáš Urban ◽  
Petr Waldauf ◽  
Adéla Krajčová ◽  
Kateřina Jiroutková ◽  
Milada Halačová ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Acid Oxidation ◽  
Kinetic Characteristics ◽  
Electron Transfer Chain ◽  
Cardiac Muscles ◽  
Transfer Chain
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