Hao1 Is Not a Pathogenic Factor for Ectopic Ossifications but Functions to Regulate the TCA Cycle In Vivo

Ossification of the posterior longitudinal ligament (OPLL), a disease characterized by the ectopic ossification of a spinal ligament, promotes neurological disorders associated with spinal canal stenosis. While blocking ectopic ossification is mandatory to prevent OPLL development and progression, the mechanisms underlying the condition remain unknown. Here we show that expression of hydroxyacid oxidase 1 (Hao1), a gene identified in a previous genome-wide association study (GWAS) as an OPLL-associated candidate gene, specifically and significantly decreased in fibroblasts during osteoblast differentiation. We then newly established Hao1-deficient mice by generating Hao1-flox mice and crossing them with CAG-Cre mice to yield global Hao1-knockout (CAG-Cre/Hao1flox/flox; Hao1 KO) animals. Hao1 KO mice were born normally and exhibited no obvious phenotypes, including growth retardation. Moreover, Hao1 KO mice did not exhibit ectopic ossification or calcification. However, urinary levels of some metabolites of the tricarboxylic acid (TCA) cycle were significantly lower in Hao1 KO compared to control mice based on comprehensive metabolomic analysis. Our data indicate that Hao1 loss does not promote ectopic ossification, but rather that Hao1 functions to regulate the TCA cycle in vivo.

Enhanced 3-Hydroxypropionic Acid Production From Acetate via the Malonyl-CoA Pathway in Corynebacterium glutamicum

Acetate is an economical and environmental-friendly alternative carbon source. Herein, the potential of harnessing Corynebacterium glutamicum as a host to produce 3-hydroxypropionic acid (3-HP) from acetate was explored. First, the expression level of malonyl-CoA reductase from Chloroflexus aurantiacus was optimized through several strategies, strain Cgz2/sod-N-C* showed an MCR enzyme activity of 63 nmol/mg/min and a 3-HP titer of 0.66 g/L in flasks. Next, the expression of citrate synthase in Cgz2/sod-N-C* was weakened to reduce the acetyl-CoA consumption in the TCA cycle, and the resulting strain Cgz12/sod-N-C* produced 2.39 g/L 3-HP from 9.32 g/L acetate. However, the subsequent deregulation of the expression of acetyl-CoA carboxylase genes in Cgz12/sod-N-C* resulted in an increased accumulation of intracellular fatty acids, instead of 3-HP. Accordingly, cerulenin was used to inhibit fatty acid synthesis in Cgz14/sod-N-C*, and its 3-HP titer was further increased to 4.26 g/L, with a yield of 0.50 g 3-HP/g-acetate. Finally, the engineered strain accumulated 17.1 g/L 3-HP in a bioreactor without cerulenin addition, representing the highest titer achieved using acetate as substrate. The results demonstrated that Corynebacterium glutamicum is a promising host for 3-HP production from acetate.

Metabolic and Physiological Regulation of Aspartic Acid-Mediated Enhancement of Heat Stress Tolerance in Perennial Ryegrass

Aspartate is the most critical amino acid in the aspartate metabolic pathway, which is associated with multiple metabolic pathways, such as protein synthesis, nucleotide metabolism, TCA cycle, glycolysis, and hormone biosynthesis. Aspartate also plays an important role in plant resistance to abiotic stress, such as cold stress, drought stress, salt stress or heavy metal stress. This study found that the chlorophyll content and antioxidant active enzyme content (SOD, CAT, POD and APX) of perennial ryegrass treated with 2 mM aspartate were significantly higher than those treated with water under heat stress. The electrolyte leakage rate, MDA content and peroxide levels (O2− and H2O2) of perennial ryegrass treated with aspartate were significantly lower than those of perennial ryegrass treated with water, indicating that exogenous aspartate increases the content of chlorophyll, maintain the integrity of cell membrane system, and enhances SOD-CAT antioxidant pathway to eliminate the oxidative damage caused by ROS in perennial ryegrass under heat stress. Furthermore, exogenous aspartate could enhance the TCA cycle, the metabolism of the amino acids related to the TCA cycle, and pyrimidine metabolism to enhance the heat tolerance of perennial ryegrass.

Physiological Biochemistry-Combined Transcriptomic Analysis Reveals Mechanism of Bacillus cereus G2 Improved Salt-Stress Tolerance of Glycyrrhiza uralensis Fisch. Seedlings by Balancing Carbohydrate Metabolism

Salt stress severely threatens the growth and productivity of Glycyrrhiza uralensis. Previous results found that Bacillus cereus G2 enhanced several carbohydrate contents in G. uralensis under salt stress. Here, we analyzed the changes in parameters related to growth, photosynthesis, carbohydrate transformation, and the glycolysis Embden-Meyerhof-Parnas (EMP) pathway-tricarboxylic acid (TCA) cycle by G2 in G. uralensis under salt stress. Results showed that G2 helped G. uralensis-accumulating photosynthetic pigments during photosynthesis, which could further increase starch, sucrose, and fructose contents during carbohydrate transformation. Specifically, increased soluble starch synthase (SSS) activity caused to higher starch content, which could induce α-amylase (AM) and β-amylase (BM) activities; increased sucrose content due to the increase of sucrose synthase (SS) activity through upregulating the gene-encoding SS, which decreased cell osmotic potential, and consequently, induced invertase and gene-encoding α-glucosidase that decomposed sucrose to fructose, ultimately avoided further water loss; increased fructose content-required highly hexokinase (HK) activity to phosphorylate in G. uralensis, thereby providing sufficient substrate for EMP. However, G2 decreased phosphofructokinase (PFK) and pyruvate kinase (PK) activities during EMP. For inducing the TCA cycle to produce more energy, G2 increased PDH activity that enhanced CA content, which further increased isocitrate dehydrogenase (ICDH) activity and provided intermediate products for the G. uralensis TCA cycle under salt stress. In sum, G2 could improve photosynthetic efficiency and carbohydrate transformation to enhance carbohydrate products, thereby releasing more chemical energy stored in carbohydrates through the EMP pathway-TCA cycle, finally maintain normal life activities, and promote the growth of G. uralensis under salt stress.

Glucose feeds the TCA cycle via environmental ethanol in fermenting yeast

Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is catabolized by glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate fuel. Individual cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Here we show that similar uncoupling occurs in the yeast Saccharomyces cerevisiae. Even in fermenting yeast that are net releasing ethanol, media 13C-ethanol rapid enters and is oxidized to acetaldehyde and acetyl-CoA. This is evident in exogenous ethanol being a major source of both cytosolic and mitochondrial acetyl units. 2H-tracing reveals that ethanol is also a major source of both NADH and NADPH, and this role is augmented under oxidative stress conditions. Thus, uncoupling of glycolysis from the oxidation of glucose-derived carbon via rapid reversible reactions is an ancient and conserved feature of eukaryotic metabolism.

My biochemical journey from a Cambridge undergraduate to the discovery of phosphotyrosine

The most notable moment in my career as a biochemist was the discovery of phosphotyrosine, a somewhat serendipitous finding that turned out to have some very important consequences, notably, in human cancer. My career as a biochemist which has spanned nearly 60 years, began when I was 16. At the time, I was in the sixth form at Felsted School, a boarding school in Essex England, and my biology master, David Sturdy, elected to teach me some extracurricular biochemistry, giving me one-on-one tutorials on glycolysis and the TCA cycle. These early biochemistry lessons turned out to be invaluable because I was able to regurgitate them to answer a question in the University of Cambridge scholarship exam in the autumn of 1960. As a result, I was lucky enough to be awarded an Exhibition at Gonville and Caius College, the college where my father had studied for a medical degree during World War II. When I arrived in Cambridge in October 1962 to read natural sciences (see Figure 1), it was a natural choice to take biochemistry as one of my three required first-year courses. The Part I biochemistry course was taught by a series of excellent lecturers, including Philip Randle (a prominent diabetes researcher who described the Randle Cycle), Brian Chappell (who discovered mitochondrial transporters) and Asher Korner (a pioneer of cell free systems to study protein synthesis). It quickly became clear that biochemistry was an exciting subject, and Brian Chappell, my biochemistry supervisor at Caius, made supervisions a lot of fun. I also took Part I courses in invertebrate zoology and, importantly, organic chemistry, which gave me insights into how the metabolites we were learning about in biochemistry worked as chemicals.

Disruption of the TCA cycle reveals an ATF4-dependent integration of redox and amino acid metabolism

The Tricarboxylic Acid Cycle (TCA) cycle is arguably the most critical metabolic cycle in physiology and exists as an essential interface coordinating cellular metabolism, bioenergetics, and redox homeostasis. Despite decades of research, a comprehensive investigation into the consequences of TCA cycle dysfunction remains elusive. Here, we targeted two TCA cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), and combined metabolomics, transcriptomics, and proteomics analyses to fully appraise the consequences of TCA cycle inhibition (TCAi) in murine kidney epithelial cells. Our comparative approach shows that TCAi elicits a convergent rewiring of redox and amino acid metabolism dependent on the activation of ATF4 and the integrated stress response (ISR). Furthermore, we also uncover a divergent metabolic response, whereby acute FHi, but not SDHi, can maintain asparagine levels via reductive carboxylation and maintenance of cytosolic aspartate synthesis. Our work highlights an important interplay between the TCA cycle, redox biology and amino acid homeostasis.

Reduced Fatty Acid Use from CD36 Deficiency Deteriorates Streptozotocin-Induced Diabetic Cardiomyopathy in Mice

Cardiac dysfunction is induced by multifactorial mechanisms in diabetes. Deranged fatty acid (FA) utilization, known as lipotoxicity, has long been postulated as one of the upstream events in the development of diabetic cardiomyopathy. CD36, a transmembrane glycoprotein, plays a major role in FA uptake in the heart. CD36 knockout (CD36KO) hearts exhibit reduced rates of FA transport with marked enhancement of glucose use. In this study, we explore whether reduced FA use by CD36 ablation suppresses the development of streptozotocin (STZ)-induced diabetic cardiomyopathy. We found that cardiac contractile dysfunction had deteriorated 16 weeks after STZ treatment in CD36KO mice. Although accelerated glucose uptake was not reduced in CD36KO-STZ hearts, the total energy supply, estimated by the pool size in the TCA cycle, was significantly reduced. The isotopomer analysis with 13C6-glucose revealed that accelerated glycolysis, estimated by enrichment of 13C2-citrate and 13C2-malate, was markedly suppressed in CD36KO-STZ hearts. Levels of ceramides, which are cardiotoxic lipids, were not elevated in CD36KO-STZ hearts compared to wild-type-STZ ones. Furthermore, increased energy demand by transverse aortic constriction resulted in synergistic exacerbation of contractile dysfunction in CD36KO-STZ mice. These findings suggest that CD36KO-STZ hearts are energetically compromised by reduced FA use and suppressed glycolysis; therefore, the limitation of FA utilization is detrimental to cardiac energetics in this model of diabetic cardiomyopathy.

Activation of the TCA Cycle to Provide Immune Protection in Zebrafish Immunized by High Magnesium-Prepared Vibrio alginolyticus Vaccine

Vaccines are safe and efficient in controlling bacterial diseases in the aquaculture industry and are in line with green farming. The present study develops a previously unreported approach to prepare a live-attenuated V. alginolyticus vaccine by culturing bacteria in a high concentration of magnesium to attenuate bacterial virulence. Furthermore, metabolomes of zebrafish immunized with the live-attenuated vaccines were compared with those of survival and dying zebrafish infected by V. alginolyticus. The enhanced TCA cycle and increased fumarate were identified as the most key metabolic pathways and the crucial biomarker of vaccine-mediated and survival fish, respectively. Exogenous fumarate promoted expression of il1β, il8, il21, nf-κb, and lysozyme in a dose-dependent manner. Among the five innate immune genes, the elevated il1β, il8, and lysozyme are overlapped in the vaccine-immunized zebrafish and the survival from the infection. These findings highlight a way in development of vaccines and exploration of the underlying mechanisms.

Succinyl-CoA-based energy metabolism dysfunctions in chronic heart failure

Heart failure (HF) is a leading cause of death and repeated hospitalizations1. HF progression generally involves mitochondrial dysfunction2-4. However, how mitochondria react to chronic HF remains unclear. Here, we show the molecular basis of mitochondrial dysfunction in chronic HF, which is characterized by altered succinyl-CoA metabolism. In myocardial mitochondria of coronary ligated mice, heme synthesis and ketolysis, and enzymes using succinyl-CoA in these events were upregulated, and enzymes synthesizing succinyl-CoA at the tricarboxylic acid (TCA) cycle were also increased. Intriguingly, the ADP-specific, but not the GDP-specific, subunit of succinyl-CoA synthetase, which uses succinyl-CoA in the TCA cycle, was decreased. Myocardial succinyl-CoA levels were significantly reduced in chronic HF, impairing mitochondrial oxidative phosphorylation (OXPHOS). Consequently, the administration of 5-aminolevulinic acid (ALA)5, an intermediate in the pathway from succinyl-CoA to heme synthesis, prevented HF progression in mice. Previous reports also support the presence of succinyl-CoA metabolism abnormalities in HF patients6,7. Our results indicated that changes in succinyl-CoA usage in various energy production systems in myocardial mitochondria is characteristic to chronic HF, and that although similar alterations occur in healthy conditions, such as during strenuous exercise, they may often occur irreversibly in HF. Moreover, nutritional interventions compensating the metabolic changes are likely to provide effective methods to treat HF.