The kinetics of rat liver and heart mitochondrial beta-hydroxybutyrate dehydrogenase.
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ABSTRACT:
SUBMITTER: Tucker GA
PROVIDER: S-EPMC1186666 | biostudies-other | 1979 Jun
REPOSITORIES: biostudies-other
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Project description:Extrahepatic tissues which oxidise ketone bodies also have the capacity to accumulate them under particular conditions. We hypothesised that acetyl-coenzyme A (acetyl-CoA) accumulation and altered redox status during low-flow ischaemia would support ketone body production in the heart. Combining a Langendorff heart model of low-flow ischaemia/reperfusion with liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), we show that β-hydroxybutyrate (β-OHB) accumulated in the ischaemic heart to 23.9 nmol/gww and was secreted into the coronary effluent. Sodium oxamate, a lactate dehydrogenase (LDH) inhibitor, increased ischaemic β-OHB levels 5.3-fold and slowed contractile recovery. Inhibition of β-hydroxy-β-methylglutaryl (HMG)-CoA synthase (HMGCS2) with hymeglusin lowered ischaemic β-OHB accumulation by 40%, despite increased flux through succinyl-CoA-3-oxaloacid CoA transferase (SCOT), resulting in greater contractile recovery. Hymeglusin also protected cardiac mitochondrial respiratory capacity during ischaemia/reperfusion. In conclusion, net ketone generation occurs in the heart under conditions of low-flow ischaemia. The process is driven by flux through both HMGCS2 and SCOT, and impacts on cardiac functional recovery from ischaemia/reperfusion.
| S-EPMC8423437 | biostudies-literature
Project description:BACKGROUND: The interconversion of two important energy metabolites, 3-hydroxybutyrate and acetoacetate (the major ketone bodies), is catalyzed by D-3-hydroxybutyrate dehydrogenase (BDH1: EC 1.1.1.30), a NAD+-dependent enzyme. The eukaryotic enzyme is bound to the mitochondrial inner membrane and harbors a unique lecithin-dependent activity. Here, we report an advanced purification method of the mammalian BDH applied to the liver enzyme from jerboa (Jaculus orientalis), a hibernating rodent adapted to extreme diet and environmental conditions. RESULTS: Purifying BDH from jerboa liver overcomes its low specific activity in mitochondria for further biochemical characterization of the enzyme. This new procedure is based on the use of polyclonal antibodies raised against BDH from bacterial Pseudomonas aeruginosa. This study improves the procedure for purification of both soluble microbial and mammalian membrane-bound BDH. Even though the Jaculus orientalis genome has not yet been sequenced, for the first time a D-3-hydroxybutyrate dehydrogenase cDNA from jerboa was cloned and sequenced. CONCLUSION: This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain. In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal. Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.
| S-EPMC2572057 | biostudies-literature
Project description:Disordered hepatic glucagon response contributes to hyperglycemia in diabetes. The regulators involved in glucagon response are less understood. This work aims to investigate the roles of mitochondrial β-oxidation enzyme HADHA and its downstream ketone bodies in hepatic glucagon response. Here we show that glucagon challenge impairs expression of HADHA. Liver-specific HADHA overexpression reversed hepatic gluconeogenesis in mice, while HADHA knockdown augmented glucagon response. Stable isotope tracing shows that HADHA promotes ketone body production via β-oxidation. The ketone body β-hydroxybutyrate (BHB) but not acetoacetate suppresses gluconeogenesis by selectively inhibiting HDAC7 activity via interaction with Glu543 site to facilitate FOXO1 nuclear exclusion. In HFD-fed mice, HADHA overexpression improved metabolic disorders, and these effects are abrogated by knockdown of BHB-producing enzyme. In conclusion, BHB is responsible for the inhibitory effect of HADHA on hepatic glucagon response, suggesting that HADHA activation or BHB elevation by pharmacological intervention hold promise in treating diabetes.
| S-EPMC8770464 | biostudies-literature
Project description:Mitochondrial dysfunction is considered to be an important component of many metabolic diseases yet there is no reliable imaging biomarker for monitoring mitochondrial damage in vivo. A large prior literature on inter-conversion of β-hydroxybutyrate and acetoacetate indicates that the process is mitochondrial and that the ratio reflects a specifically mitochondrial redox state. Therefore, the conversion of [1,3-13 C]acetoacetate to [1,3-13 C]β-hydroxybutyrate is expected to be sensitive to the abnormal redox state present in dysfunctional mitochondria. In this study, we examined the conversion of hyperpolarized (HP) 13 C-acetoacetate (AcAc) to 13 C-β-hydroxybutyrate (β-HB) as a potential imaging biomarker for mitochondrial redox and dysfunction in perfused rat hearts. Conversion of HP-AcAc to β-HB was investigated using 13 C magnetic resonance spectroscopy in Langendorff-perfused rat hearts in four groups: control, global ischemic reperfusion, low-flow ischemic, and rotenone (mitochondrial complex-I inhibitor)-treated hearts. We observed that more β-HB was produced from AcAc in ischemic hearts and the hearts exposed to complex I inhibitor rotenone compared with controls, consistent with the accumulation of excess mitochondrial NADH. The increase in β-HB, as detected by 13 C MRS, was validated by a direct measure of tissue β-HB by 1 H nuclear magnetic resonance in tissue extracts. The redox ratio, NAD+ /NADH, measured by enzyme assays of homogenized tissue, also paralleled production of β-HB from AcAc. Transmission electron microscopy of tissues provided direct evidence for abnormal mitochondrial structure in each ischemic tissue model. The results suggest that conversion of HP-AcAc to HP-β-HB detected by 13 C-MRS may serve as a useful diagnostic marker of mitochondrial redox and dysfunction in heart tissue in vivo.
| S-EPMC6525062 | biostudies-literature
Project description:Aldehyde dehydrogenase activities in liver mitochondria isolated from rats given ethanol at hourly intervals by gastric intubation showed a brief lag period followed by a rapid increase in specific activities until a maximum was attained at about 3h.
| S-EPMC1163729 | biostudies-other
Project description:(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a phosphatidylcholine-requiring tetrameric enzyme with two thiol groups (SH-1 and SH-2) per protomer. By first protecting the more rapidly reacting thiol group (SH-1) with diamide [1,1'-azobis-(NN'-dimethylformamide), DM] to form DM(SH-1)BDH, SH-2 can be selectively derivatized by reaction with maleimide reagents such as 4-maleimido-2,2,6,6-tetramethyl-piperidine-N-oxyl (MSL), which gives DM(SH-1)MSL(SH-2)BDH. Reduction with dithiothreitol (DTT) regenerates SH-1, yielding MAL(SH-2)BDH (where MAL is the diamagnetic reduction product of MSL-BDH and DTT). The enzymic activity of DM(SH-1)BDH is decreased to approx. 4% relative to the native purified enzyme, and the apparent Km for substrate, KmBOH, is increased approx. 100-fold. Reduction of DM(SH-1)BDH with DTT regenerates SH-1 and restores normal enzymic function. Modification of SH-2 with piperidinylmaleimide [MAL(SH-2)BDH] diminishes enzymic activity to approx. 35% of its original value, but has no significant effect on apparent KmBOH. The doubly derivatized enzyme, DM(SH-1)MSL(SH-2)BDH, has lower enzymic activity [about half that for DM(SH-2)BDH] and a yet higher apparent KmBOH than DM(SH-1)BDH. Derivatization of SH-2 with different maleimide reagents results in diminished activity approximately proportional to the size of the maleimide substituent, suggesting that this inhibition is steric. Whereas modification of SH-1 results in marked changes in kinetic parameters (increased apparent Km and reduced apparent Vmax), derivatization of SH-2 has a lesser effect on enzymic function. Thus SH-1 is postulated to be closer to the active centre than is SH-2, although neither is involved in catalysis, since: (1) the activity of the derivatized enzyme is not abolished; and (2) activity can be enhanced by increasing substrate (and cofactor) concentrations.
| S-EPMC1137735 | biostudies-other
Project description:β-hydroxybutyrate is the main ketone body generated by the liver under starvation. Under these conditions, it can sustain ATP levels by its oxidation in mitochondria. As mitochondria can modify its shape and function under different nutritional challenges, we study the chronic effects of β-hydroxybutyrate supplementation on mitochondrial morphology and function, and its relation to exercise capacity. Male C57BL/6 mice were supplemented with β-hydroxybutyrate mineral salt (3.2%) or control (CT, NaCl/KCl) for six weeks and submitted to a weekly exercise performance test. We found an increase in distance, maximal speed, and time to exhaustion at two weeks of supplementation. Fatty acid metabolism and OXPHOS subunit proteins declined at two weeks in soleus but not in tibialis anterior muscles. Oxygen consumption rate on permeabilized fibers indicated a decrease in the presence of pyruvate in the short-term treatment. Both the tibialis anterior and soleus showed decreased levels of Mitofusin 2, while electron microscopy assessment revealed a significant reduction in mitochondrial cristae shape in the tibialis anterior, while a reduction in the mitochondrial number was observed only in soleus. These results suggest that short, but not long-term, β‑hydroxybutyrate supplementation increases exercise capacity, associated with modifications in mitochondrial morphology and function in mouse skeletal muscle.
| S-EPMC7400376 | biostudies-literature
Project description:Mitochondrial dynamics and metabolites reciprocally influence each other. Mitochondrial-derived vesicles (MDVs) transport damaged mitochondrial components to lysosomes or the extracellular space, and they are an emerging mechanism that regulates mitochondrial quality. While many metabolites are known to modulate mitochondrial dynamics, it is largely unclear whether they are involved in MDVs generation. Here, we discovered that the major component of ketone body, β-hydroxybutyrate (BHB), improved mitochondrial quality control by facilitating the biogenesis of MDVs. Mechanistically, BHB supplementation drove lysine β-hydroxybutyrylation (Kbhb) of SNX9, a key regulator of MDV biogenesis, at three specific lysine residues. Kbhb increased SNX9 interaction with inner mitochondrial membrane (IMM)/matrix proteins and promoted the formation of IMM/matrix MDVs. Lastly, we found that SNX9 Kbhb not only played a critical role in maintaining mitochondrial homeostasis in cells, but also protected mice from alcohol-induced liver injury. Altogether, our research uncovers that metabolites influence the formation of MDVs by directly engaging in post-translational modifications of key protein machineries and establishes a new framework for understanding how metabolites regulate mitochondrial quality
2023-09-21 | PXD045541 |
Project description:ObjectiveThroughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. Therefore, we sought to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB).MethodsUsing targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, and complementary physiological approaches in novel cell type-specific knockout mice, we quantified the roles of hepatocyte D-β-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD+/NADH-dependent oxidation/reduction of ketone bodies.ResultsExogenously administered AcAc is reduced to D-βOHB, which increases hepatic NAD+/NADH ratio and reflects hepatic BDH1 activity. Livers of hepatocyte-specific BDH1-deficient mice did not produce D-βOHB, but owing to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-βOHB interconversion. Compared to littermate controls, hepatocyte-specific BDH1 deficient mice exhibited diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis, but normal hepatic energy charge overall. Glycemic recovery after acute insulin challenge was impaired in knockout mice, but they were not more susceptible to starvation-induced hypoglycemia.ConclusionsKetone bodies influence liver homeostasis. While liver BDH1 is not required for whole body equilibration of AcAc and D-βOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production. Therefore, through oxidation/reduction of ketone bodies, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis.
| S-EPMC8259407 | biostudies-literature
Project description:Colorectal cancer (CRC) is among the most frequent forms of cancer, and new strategies for its prevention and therapy are urgently needed1. Here we identify a metabolite signalling pathway that provides actionable insights towards this goal. We perform a dietary screen in autochthonous animal models of CRC and find that ketogenic diets exhibit a strong tumour-inhibitory effect. These properties of ketogenic diets are recapitulated by the ketone body β-hydroxybutyrate (BHB), which reduces the proliferation of colonic crypt cells and potently suppresses intestinal tumour growth. We find that BHB acts through the surface receptor Hcar2 and induces the transcriptional regulator Hopx, thereby altering gene expression and inhibiting cell proliferation. Cancer organoid assays and single-cell RNA sequencing of biopsies from patients with CRC provide evidence that elevated BHB levels and active HOPX are associated with reduced intestinal epithelial proliferation in humans. This study thus identifies a BHB-triggered pathway regulating intestinal tumorigenesis and indicates that oral or systemic interventions with a single metabolite may complement current prevention and treatment strategies for CRC.
| S-EPMC9448510 | biostudies-literature