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.

Glutamine is required for M1-like polarization in response to Mycobacterium tuberculosis infection

In response to Mycobacterium tuberculosis infection, macrophages mount early proinflammatory and antimicrobial responses similar to those observed in M1 macrophages classically activated by LPS and IFN-γ. A metabolic reprogramming to HIF-1-mediated uptake of glucose and its metabolism by glycolysis is required for M1-like polarization, but little is known about other metabolic programs driving M1-like polarization during M. tuberculosis infection. Identification and quantification of labeling patterns of U 13 C glutamine and U 13 C glucose-derived metabolites demonstrated that glutamine, rather than glucose, is catabolized in both the oxidative and reductive TCA cycle of M1-like macrophages, thereby generating signaling molecules that include succinate, biosynthetic precursors such as aspartate, and the antimicrobial metabolite itaconate. This conclusion is corroborated by diminished M1 polarization via chemical inhibition of glutaminase (GLS), the key enzyme of the glutaminolysis pathway, and by genetic deletion of GLS in infected macrophages. Furthermore, characterization of the labeling distribution pattern of U 15 N glutamine in M1-like macrophages indicates that glutamine serves as a nitrogen source for the synthesis of intermediates of purine and pyrimidine metabolism plus amino acids including aspartate. Thus, the catabolism of glutamine, as an integral component of metabolic reprogramming in activating macrophages, fulfills the cellular demand for bioenergetic and biosynthetic precursors of M1-like macrophages. Knowledge of these new immunometabolic features of M1-like macrophages is expected to advance the development of host-directed therapies that will enhance bacterial clearance and prevent immunopathology during tuberculosis.

Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho-signaling where GSK3 inhibition inactivates energy-sensing by AMP-kinase through dephosphorylation of the AMP-kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti-Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

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.

Metabolic Adaptions/Reprogramming in Islet Beta-Cells in Response to Physiological Stimulators—What Are the Consequences

Irreversible pancreatic β-cell damage may be a result of chronic exposure to supraphysiological glucose or lipid concentrations or chronic exposure to therapeutic anti-diabetic drugs. The β-cells are able to respond to blood glucose in a narrow concentration range and release insulin in response, following activation of metabolic pathways such as glycolysis and the TCA cycle. The β-cell cannot protect itself from glucose toxicity by blocking glucose uptake, but indeed relies on alternative metabolic protection mechanisms to avoid dysfunction and death. Alteration of normal metabolic pathway function occurs as a counter regulatory response to high nutrient, inflammatory factor, hormone or therapeutic drug concentrations. Metabolic reprogramming is a term widely used to describe a change in regulation of various metabolic enzymes and transporters, usually associated with cell growth and proliferation and may involve reshaping epigenetic responses, in particular the acetylation and methylation of histone proteins and DNA. Other metabolic modifications such as Malonylation, Succinylation, Hydroxybutyrylation, ADP-ribosylation, and Lactylation, may impact regulatory processes, many of which need to be investigated in detail to contribute to current advances in metabolism. By describing multiple mechanisms of metabolic adaption that are available to the β-cell across its lifespan, we hope to identify sites for metabolic reprogramming mechanisms, most of which are incompletely described or understood. Many of these mechanisms are related to prominent antioxidant responses. Here, we have attempted to describe the key β-cell metabolic adaptions and changes which are required for survival and function in various physiological, pathological and pharmacological conditions.

San-Huang-Yi-Shen Capsule Ameliorates Diabetic Nephropathy in Rats Through Modulating the Gut Microbiota and Overall Metabolism

San-Huang-Yi-Shen capsule (SHYS) has been used in the treatment of diabetic nephropathy (DN) in clinic. However, the mechanisms of SHYS on DN remain unknown. In this study, we used a high-fat diet (HFD) combined with streptozotocin (STZ) injection to establish a DN rat model. Next, we used 16S rRNA sequencing and untargeted metabolomics to study the potential mechanisms of SHYS on DN. Our results showed that SHYS treatment alleviated the body weight loss, hyperglycemia, proteinuria, pathological changes in kidney in DN rats. SHYS could also inhibite the oxidative stress and inflammatory response in kidney. 16S rRNA sequencing analysis showed that SHYS affected the beta diversity of gut microbiota community in DN model rats. SHYX could also decrease the Firmicutes to Bacteroidetes (F to B) ratio in phylum level. In genus level, SHYX treatment affected the relative abundances of Lactobacillus, Ruminococcaceae UCG-005, Allobaculum, Anaerovibrio, Bacteroides and Candidatus_Saccharimonas. Untargeted metabolomics analysis showed that SHYX treatment altered the serum metabolic profile in DN model rats through affecting the levels of guanidineacetic acid, L-kynurenine, prostaglandin F1α, threonine, creatine, acetylcholine and other 21 kind of metabolites. These metabolites are mainly involved in glycerophospholipid metabolism, tryptophan metabolism, alanine, aspartate and glutamate metabolism, arginine biosynthesis, tricarboxylic acid (TCA) cycle, tyrosine metabolism, arginine and proline metabolism, arginine and proline metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, and D-glutamine and D-glutamate metabolism pathways. Spearman correlation analysis showed that Lactobacillus, Candidatus_Saccharimonas, Ruminococcaceae UCG-005, Anaerovibrio, Bacteroides, and Christensenellaceae_R-7_group were closely correlated with most of physiological data and the differential metabolites following SHYS treatment. In conclusion, our study revealed multiple ameliorative effects of SHYS on DN including the alleviation of hyperglycemia and the improvement of renal function, pathological changes in kidney, oxidative stress, and the inflammatory response. The mechanism of SHYS on DN may be related to the improvement of gut microbiota which regulates arginine biosynthesis, TCA cycle, tyrosine metabolism, and arginine and proline metabolism.

Metabolomic Signatures for the Effects of Weight Loss Interventions on Severe Obesity in Children and Adolescents

Childhood obesity has increased worldwide, and many clinical and public interventions have attempted to reduce morbidity. We aimed to determine the metabolomic signatures associated with weight control interventions in children with obesity. Forty children from the “Intervention for Children and Adolescent Obesity via Activity and Nutrition (ICAAN)” cohort were selected according to intervention responses. Based on changes in body mass index z-scores, 20 were responders and the remaining non-responders. Their serum metabolites were quantitatively analyzed using capillary electrophoresis time-of-flight mass spectrometry at baseline and after 6 and 18 months of intervention. After 18 months of intervention, the metabolite cluster changes in the responders and non-responders showed a difference on the heatmap, but significant metabolites were not clear. However, regardless of the responses, 13 and 49 metabolites were significant in the group of children with obesity intervention at 6 months and 18 months post-intervention compared to baseline. In addition, the top five metabolic pathways (D-glutamine and D-glutamate metabolism; arginine biosynthesis; alanine, aspartate, and glutamate metabolism; TCA cycle; valine, leucine, and isoleucine biosynthesis) including several amino acids in the metabolites of obese children after 18 months were significantly changed. Our study showed significantly different metabolomic profiles based on time post obesity-related intervention. Through this study, we can better understand and predict childhood obesity through metabolite analysis and monitoring.

New Discoveries and Ambiguities of Nrf2 and ATF3 Signaling in Environmental Arsenic-Induced Carcinogenesis

Environment exposure to arsenic had been linked to increased incidents of human cancers. In cellular and animal experimental systems, arsenic has been shown to be highly capable of activating several signaling pathways that play critical roles in cell growth regulation, malignant transformation and the stemness of cancer stem-like cells. Emerging evidence indicates certain oncogenic properties of the Nrf2 transcription factor that can be activated by arsenic and many other environmental hazards. In human bronchial epithelial cells, our most recent data suggested that arsenic-activated Nrf2 signaling fosters metabolic reprogramming of the cells through shifting mitochondrial TCA cycle to cytosolic glycolysis, and some of the metabolites in glycolysis shunt the hexosamine biosynthesis and serine-glycine pathways important for the energy metabolism of the cancer cells. In the current report, we further demonstrated direct regulation of oncogenic signals by arsenic-activated Nrf2 and connection of Nrf2 with ATF3 stress transcription factor. Meanwhile, we also highlighted some unanswered questions on the molecular characteristics of the Nrf2 protein, which warrants further collaborative efforts among scientists for understanding the important role of Nrf2 in human cancers either associated or not to environmental arsenic exposure.