BCAA-nitrogen flux in brown fat controls metabolic health independent of thermogenesis.

Brown adipose tissue (BAT) is best known for thermogenesis. Rodent studies demonstrated that enhanced BAT thermogenesis is tightly associated with increased energy expenditure, reduced body weight, and improved glucose homeostasis. However, human BAT is protective against type 2 diabetes, independent of body weight. The mechanism underlying this dissociation remains unclear. Here, we report that impaired mitochondrial catabolism of branched-chain amino acids (BCAAs) in BAT, by deleting mitochondrial BCAA carriers (MBCs), caused systemic insulin resistance without affecting energy expenditure and body weight. Brown adipocytes catabolized BCAA in the mitochondria as nitrogen donors for the biosynthesis of non-essential amino acids and glutathione. Impaired mitochondrial BCAA-nitrogen flux in BAT resulted in increased oxidative stress, decreased hepatic insulin signaling, and decreased circulating BCAA-derived metabolites. A high-fat diet attenuated BCAA-nitrogen flux and metabolite synthesis in BAT, whereas cold-activated BAT enhanced the synthesis. This work uncovers a metabolite-mediated pathway through which BAT controls metabolic health beyond thermogenesis.

Ketone flux through BDH1 supports metabolic remodeling of skeletal and cardiac muscles in response to intermittent time-restricted feeding.

Time-restricted feeding (TRF) has gained attention as a dietary regimen that promotes metabolic health. This study questioned if the health benefits of an intermittent TRF (iTRF) schedule require ketone flux specifically in skeletal and cardiac muscles. Notably, we found that the ketolytic enzyme beta-hydroxybutyrate dehydrogenase 1 (BDH1) is uniquely enriched in isolated mitochondria derived from heart and red/oxidative skeletal muscles, which also have high capacity for fatty acid oxidation (FAO). Using mice with BDH1 deficiency in striated muscles, we discover that this enzyme optimizes FAO efficiency and exercise tolerance during acute fasting. Additionally, iTRF leads to robust molecular remodeling of muscle tissues, and muscle BDH1 flux does indeed play an essential role in conferring the full adaptive benefits of this regimen, including increased lean mass, mitochondrial hormesis, and metabolic rerouting of pyruvate. In sum, ketone flux enhances mitochondrial bioenergetics and supports iTRF-induced remodeling of skeletal muscle and heart.

Isolation of endophytic fungi from Cotoneaster multiflorus and screening of drought-tolerant fungi and evaluation of their growth-promoting effects.

In the context of climate change and human factors, the drought problem is a particularly serious one, and environmental pollution caused by the abuse of chemical fertilizers and pesticides is increasingly serious. Endophytic fungi can be used as a protection option, which is ecologically friendly, to alleviate abiotic stresses on plants, promote plant growth, and promote the sustainable development of agriculture and forestry. Therefore, it is of great significance to screen and isolate endophytic fungi that are beneficial to crops from plants in special habitats. In this study, endophytic fungi were isolated from Cotoneaster multiflorus, and drought-tolerant endophytic fungi were screened by simulating drought stress with different concentrations of PEG-6000, and the growth-promoting effects of these drought-tolerant strains were evaluated. A total of 113 strains of endophytic fungi were isolated and purified from different tissues of C. multiflorus. After simulated drought stress, 25 endophytic fungi showed strong drought tolerance. After ITS sequence identification, they belonged to 7 genera and 12 species, including Aspergillus, Fusarium, Colletotrichum, Penicillium, Diaporthe, Geotrichum, and Metarhizium. According to the identification and drought stress results, 12 strains of endophytic fungi with better drought tolerance were selected to study their abilities of dissolving inorganic phosphorus and potassium feldspar powder and producing indole-3-acetic acid (IAA). It was found that the amount of dissolved phosphorus in 7 strains of endophytic fungi was significantly higher than that of CK, and the content of soluble phosphorus was 101.98-414.51 µg. ml-1; 6 endophytic fungi had significantly higher potassium solubilization than CK, and the content of water-soluble potassium ranged from 19.17 to 30.94 mg·l-1; 6 strains have the ability to produce IAA, and the yield of IAA ranged between 0.04 and 0.42 mg. ml-1. This study for the first time identified the existence of endophytic fungi with drought tolerance and growth-promoting function in C. multiflorus, which could provide new direction for plant drought tolerance and growth promotion fungi strain resources. It also provides a theoretical basis for the subsequent application of endophytic fungi of C. multiflorus in agricultural and forestry production to improve plant tolerance.

First Report of Leaf Spot Caused by Botrytis cinerea on Strawberry in China.

In the winter of 2022, circular or irregular leaf spots were observed on strawberry (Fragaria × ananassa) planted in commercial fields (cultivar 'xuetu', 'mengzhifu') in Yinzhou, Ningbo, Zhejiang, China (N29°48'48″, E121°39'47″), with disease incidence ranging from 10 to 15% in a field approximately 0.67 ha in size. The estimated crop loss associated with this disease was ~10%. Symptoms included circular or irregular lesions with brown halos and wheel marks, which eventually developed into leaf blight and petiole decay, but spore masses were seldom found on the leaf surface. In severe cases, leaves withered and abscissed. To isolate the causal agent, ten diseased leaves from ten different plants were collected, surface-sterilized with 75% ethanol for 50 s, rinsed twice with sterile distilled water, cut into small pieces (0.5 cm × 0.5 cm), and plated on potato dextrose agar (PDA), then incubated at 25°C in darkness for 5 days. Isolates , which displayed one kind of colony morphology were consistently obtained from each of the ten samples, and 58 single-conidium isolates with the same colony morphology were obtained. The isolation frequency was 58 of 60 samples. The colonies that grew on PDA produced white mycelia, which sporulated after 1 week, producing typical Botrytis-like gray spores. Three isolates (NBCM-1, NBCM-2, NBCM-3) were selected for identification and pathogenicity assays. Conidia were round to ellipsoid, 9.2 to 14.3 µm long (n=50), and 6.4 to 9.2 µm wide (n=50). Sclerotia were not observed on PDA. Based on these characteristics, the pathogen was tentatively identified as Botrytis cinerea (Zhang 2001). PCR was conducted for each of the three isolates to amplify the G3PDH, HSP60, RPB2, NEP1, and NEP2 genes, which are typically used for molecular identification of Botrytis species (Staats et al. 2005; Liu et al. 2016). The resulting amplicons were sequenced, and the sequences were processed using BLAST in the National Center for Biotechnology Information. Sequences of the three isolates were deposited in GenBank (accession nos. OR052082 to OR052086, OR493405 to OR493414). BLASTn analyses showed that isolates were 99 to 100% identical to B.cinerea reported causing leaf spot on strawberry in California; accession numbers MK919496 (G3PDH, 883/883 bp), MK919494 (HSP60, 992/992 bp), and MK919495 (RPB2, 1081/1081 bp). The resulting concatenated data set of G3PDH-HSP60-RPB2-NEP1-NEP2 was used to conduct a multilocus phylogenetic analysis (MLSA) using the maximum likelihood method. The MLSA tree indicated that the three isolates belonged to Botrytis cinerea. To test for pathogenicity, three 1-month-old strawberry (cultivar 'xuetu') plants were inoculated with each isolate (NBCM-1, NBCM-2, NBCM-3). A noninoculated control (sterile water only) was also included. The strawberry plants were inoculated by spraying with conidia suspension (1.0 × 105/ml) until run-off. Inoculations with sterile water served as controls. All plants were kept at 28/25°C (day/night), under a 12:12-h light/dark photoperiod. All plants were covered with transparent plastic bags to maintain humidity for the first 48 h, after which the bags were removed. After 4 to 7 days, leaf spot symptoms similar to those observed in the field were observed in all inoculated plants, while the controls remained healthy. The experiment was repeated three times. The pathogen was reisolated from the inoculated leaves and again identified as B. cinerea, with the same methodology used for the initial identification. Leaf spot caused by B. cinerea on strawberry was recently reported in California (Mansouripour and Holmes 2020) and Florida (Marin and Peres 2022). To our knowledge, this is the first report of B. cinerea causing leaf spot on strawberry in China. The pathogen is also the causal agent of Botrytis fruit rot on strawberry. Given the high variability of this pathogen (Marin and Peres 2022), further studies on its occurrence, spread, management, and control are required. The identification of this pathogen provides a basis for further research on its management and control.

Prevalence of propionic acidemia in China.

Propionic acidemia (PA) is a rare autosomal recessive congenital disease caused by mutations in the PCCA or PCCB genes. Elevated propionylcarnitine, 2-methylcitric acid (2MCA), propionylglycine, glycine and 3-hydroxypropionate can be used to diagnose PA. Early-onset PA can lead to acute deterioration, metabolic acidosis, and hyperammonemia shortly after birth, which can result in high mortality and disability. Late-onset cases of PA have a more heterogeneous clinical spectra, including growth retardation, intellectual disability, seizures, basal ganglia lesions, pancreatitis, cardiomyopathy, arrhythmias, adaptive immune defects, rhabdomyolysis, optic atrophy, hearing loss, premature ovarian failure, and chronic kidney disease. Timely and accurate diagnosis and appropriate treatment are crucial to saving patients' lives and improving their prognosis. Recently, the number of reported PA cases in China has increased due to advanced diagnostic techniques and increased research attention. However, an overview of PA prevalence in China is lacking. Therefore, this review provides an overview of recent advances in the pathogenesis, diagnostic strategies, and treatment of PA, including epidemiological data on PA in China. The most frequent variants among Chinese PA patients are c.2002G > A in PCCA and c.1301C > T in PCCB, which are often associated with severe clinical symptoms. At present, liver transplantation from a living (heterozygous parental) donor is a better option for treating PA in China, especially for those exhibiting a severe metabolic phenotype and/or end-organ dysfunction. However, a comprehensive risk-benefit analysis should be conducted as an integral part of the decision-making process. This review will provide valuable information for the medical care of Chinese patients with PA.

Pathophysiological mechanisms of complications associated with propionic acidemia.

Propionic acidemia (PA) is a genetic metabolic disorder caused by mutations in the mitochondrial enzyme, propionyl-CoA carboxylase (PCC), which is responsible for converting propionyl-CoA to methylmalonyl-CoA for further metabolism in the tricarboxylic acid cycle. When this process is disrupted, propionyl-CoA and its metabolites accumulate, leading to a variety of complications including life-threatening cardiac diseases and other metabolic strokes. While the clinical symptoms and diagnosis of PA are well established, the underlying pathophysiological mechanisms of PA-induced diseases are not fully understood. As a result, there are currently few effective therapies for PA beyond dietary restriction. This review focuses on the pathophysiological mechanisms of the various complications associated with PA, drawing on extensive research and clinical reports. Most research suggests that propionyl-CoA and its metabolites can impair mitochondrial energy metabolism and cause cellular damage by inducing oxidative stress. However, direct evidence from in vivo studies is still lacking. Additionally, elevated levels of ammonia can be toxic, although not all PA patients develop hyperammonemia. The discovery of pathophysiological mechanisms underlying various complications associated with PA can aid in the development of more effective therapeutic treatments. The consequences of elevated odd-chain fatty acids in lipid metabolism and potential gene expression changes mediated by histone propionylation also warrant further investigation.

Glutathione limits RUNX2 oxidation and degradation to regulate bone formation.

Reactive oxygen species (ROS) are natural products of mitochondrial oxidative metabolism and oxidative protein folding. ROS levels must be well controlled, since elevated ROS has been shown to have deleterious effects on osteoblasts. Moreover, excessive ROS is thought to underlie many of the skeletal phenotypes associated with aging and sex steroid deficiency in mice and humans. The mechanisms by which osteoblasts regulate ROS and how ROS inhibits osteoblasts are not well understood. Here, we demonstrate that de novo glutathione (GSH) biosynthesis is essential in neutralizing ROS and establish a proosteogenic reduction and oxidation reaction (REDOX) environment. Using a multifaceted approach, we demonstrate that reducing GSH biosynthesis led to acute degradation of RUNX2, impaired osteoblast differentiation, and reduced bone formation. Conversely, reducing ROS using catalase enhanced RUNX2 stability and promoted osteoblast differentiation and bone formation when GSH biosynthesis was limited. Highlighting the therapeutic implications of these findings, in utero antioxidant therapy stabilized RUNX2 and improved bone development in the Runx2+/- haplo-insufficient mouse model of human cleidocranial dysplasia. Thus, our data establish RUNX2 as a molecular sensor of the osteoblast REDOX environment and mechanistically clarify how ROS negatively impacts osteoblast differentiation and bone formation.

"Metformin Impairs Intestinal Fructose Metabolism".

Objective: To investigate the effects of metformin on intestinal carbohydrate metabolism in vivo. Method: Male mice preconditioned with a high-fat, high-sucrose diet were treated orally with metformin or a control solution for two weeks. Fructose metabolism, glucose production from fructose, and production of other fructose-derived metabolites were assessed using stably labeled fructose as a tracer. Results: Metformin treatment decreased intestinal glucose levels and reduced incorporation of fructose-derived metabolites into glucose. This was associated with decreased intestinal fructose metabolism as indicated by decreased enterocyte F1P levels and diminished labeling of fructose-derived metabolites. Metformin also reduced fructose delivery to the liver. Proteomic analysis revealed that metformin coordinately down-regulated proteins involved carbohydrate metabolism including those involved in fructolysis and glucose production within intestinal tissue. Conclusion: Metformin reduces intestinal fructose metabolism, and this is associated with broad-based changes in intestinal enzyme and protein levels involved in sugar metabolism indicating that metformin's effects on sugar metabolism are pleiotropic.

A high-fat diet increases hepatic mitochondrial turnover through restricted acetylation in a NAFLD mouse model.

Lysine acetylation of proteins has emerged as a key posttranslational modification (PTM) that regulates mitochondrial metabolism. Acetylation may regulate energy metabolism by inhibiting and affecting the stability of metabolic enzymes and oxidative phosphorylation (OxPhos) subunits. Although protein turnover can be easily measured, due to the low abundance of modified proteins, it has been difficult to evaluate the effect of acetylation on the stability of proteins in vivo. We applied 2H2O-metabolic labeling coupled with immunoaffinity and high-resolution mass spectrometry method to measure the stability of acetylated proteins in mouse liver based on their turnover rates. As a proof-of-concept, we assessed the consequence of high-fat diet (HFD)-induced altered acetylation in protein turnover in LDL receptor-deficient (LDLR-/-) mice susceptible to diet-induced nonalcoholic fatty liver disease (NAFLD). HFD feeding for 12 wk led to steatosis, the early stage of NAFLD. A significant reduction in acetylation of hepatic proteins was observed in NAFLD mice, based on immunoblot analysis and label-free quantification with mass spectrometry. Compared with control mice on a normal diet, NAFLD mice had overall increased turnover rates of hepatic proteins, including mitochondrial metabolic enzymes (0.159 ± 0.079 vs. 0.132 ± 0.068 day-1), suggesting their reduced stability. Also, acetylated proteins had slower turnover rates (increased stability) than native proteins in both groups (0.096 ± 0.056 vs. 0.170 ± 0.059 day-1 in control, and 0.111 ± 0.050 vs. 0.208 ± 0.074 day-1 in NAFLD). Furthermore, association analysis revealed a relationship between the HFD-induced decrease in acetylation and increased turnover rates for hepatic proteins in NAFLD mice. These changes were associated with increased expressions of the hepatic mitochondrial transcriptional factor (TFAM) and complex II subunit without any changes to other OxPhos proteins, suggesting that enhanced mitochondrial biogenesis prevented restricted acetylation-mediated depletion of mitochondrial proteins. We conclude that decreased acetylation of mitochondrial proteins may contribute to adaptive improved hepatic mitochondrial function in the early stages of NAFLD.NEW & NOTEWORTHY This is the first method to quantify acetylome dynamics in vivo. This method revealed acetylation-mediated altered hepatic mitochondrial protein turnover in response to a high-fat diet in a mouse model of NAFLD.

Pyruvate-supported flux through medium-chain ketothiolase promotes mitochondrial lipid tolerance in cardiac and skeletal muscles.

Even-chain acylcarnitine (AC) metabolites, most of which are generated as byproducts of incomplete fatty acid oxidation (FAO), are viewed as biomarkers of mitochondrial lipid stress attributable to one or more metabolic bottlenecks in the ß-oxidation pathway. The origins and functional implications of FAO bottlenecks remain poorly understood. Here, we combined a sophisticated mitochondrial phenotyping platform with state-of-the-art molecular profiling tools and multiple two-state mouse models of respiratory function to uncover a mechanism that connects AC accumulation to lipid intolerance, metabolic inflexibility, and respiratory inefficiency in skeletal muscle mitochondria. These studies also identified a short-chain carbon circuit at the C4 node of FAO wherein reverse flux of glucose-derived acetyl CoA through medium-chain ketothiolase enhances lipid tolerance and redox stability in heart mitochondria by regenerating free CoA and NAD+. The findings help to explain why diminished FAO capacity, AC accumulation, and metabolic inflexibility are tightly linked to poor health outcomes.

Ischemia promotes acyl-CoAs dephosphorylation and propionyl-CoA accumulation.

INTRODUCTION: Our untargeted metabolic data unveiled that Acyl-CoAs undergo dephosphorylation, however little is known about these novel metabolites and their physiology/pathology relevance. OBJECTIVES: To understand the relationship between acyl-CoAs dephosphorylation and energy status as implied in our previous work, we seek to investigate how ischemia (energy depletion) triggers metabolic changes, specifically acyl-CoAs dephosphorylation in this work. METHODS: Rat hearts were isolated and perfused in Langendorff mode for 15 min followed by 0, 5, 15, and 30 minutes of global ischemia. The heart tissues were harvested for metabolic analysis. RESULTS: As expected, ATP and phosphocreatine were significantly decreased during ischemia. Most short- and medium-chain acyl-CoAs progressively increased with ischemic time from 0 to 15 min, whereas a 30-minute ischemia did not lead to further change. Unlike other acyl-CoAs, propionyl-CoA accumulated progressively in the hearts that underwent ischemia from 0 to 30 min. Progressive dephosphorylation occurred to all assayed acyl-CoAs and free CoA regardless their level changes during the ischemia. CONCLUSION: The present work further confirms that dephosphorylation of acyl-CoAs is an energy-dependent process and how this dephosphorylation is mediated warrants further investigations. It is plausible that dephosphorylation of acyl-CoAs and limited anaplerosis are involved in ischemic injuries to heart. Further investigations are warranted to examine the mechanisms of acyl-CoA dephosphorylation and how the dephosphorylation is possibly involved in ischemic injuries.

First Report of Pantoea ananatis Causing Crown Necrobiosis on Strawberry in China.

Strawberry (Fragaria × ananassa) is an economically important crop in Zhejiang, China. In the autumn of 2021, crown necrobiosis and angular leaf spot was observed in commercial strawberry fields (cultivar 'fenyu') in Cixi, Ningbo, Zhejiang, China (N30°9'55″, E121°21'13″). The disease incidence ranged from 5 to 8 % in the field, but could reach 50 to 60 % in some heavily affected plastic tunnels. In the affected field, this disease could reduce strawberry production by 50%. Early symptoms were water-soaked lesions around the vein of the abaxial leaves; subsequently, reddish-brown irregular spots and coalesced lesions developed. In humid conditions, a sticky bacterial ooze exuding from lesions was observed. Finally, the crown of the diseased plant was necrotized, and several pockets were observed inside the crown after dissection. To isolate the causal agent, the infected leaves and crown tissues from six different plants were surface-sterilized with 75% ethanol for 1 min, rinsed twice with sterile distilled water, cut into small pieces, and soaked in 5 ml of sterile distilled water for 20 min. The supernatant from the cut-up pieces was serially diluted and spread on nutrient agar medium. After 2 to 3 days at 28℃, several yellow colonies were grown on the medium. The colonies from five infected plants were gram-negative, anaerobic rods, yellow, viscous, and gloss, which are typical characteristics of Erwinia anana (Wells et al. 1986). To confirm the identity of the causal bacteria, PCR was conducted for six randomly selected colonies to amplify 16S rRNA (Monciardini et al. 2002), fusA, and gyrB (Stice et al. 2002). The amplicons were sequenced and blasted, and the results showed that the six colonies were identical. The 16S rRNA, fusA, gyrB sequences of the isolate CM3 were deposited in GenBank with accession number ON754076.1, OP587277, and OP587278; BLAST search showed 99.93% (1445 bp out of 1446 bp), 100% (746 bp out of 746 bp), 99.64% (1371 bp out of 1376 bp) similarity with strains of Pantoea ananatis (KT741001.1, MH015093.1 and CP066803.1 accessions, respectively). The resulting concatenated data set of 16S rRNA-fusA-gyrB was used to build a multilocus phylogenetic analysis (MLSA) by maximum likelihood criteria. The MLSA tree indicated that the isolate CM3 belonged to Pantoea ananatis. The isolate's identity was further confirmed by P. ananatis-specific primers pagyrB-F/R (Xiao et al. 2022). Thus, this isolate was designated as P. ananatis CM3. To fulfill Koch's postulates, two old leaves were broken off each of the ten 2-month-old strawberry (cultivar 'fenyu') plants to create wounds, each plants was sprayed with a cell suspension of P. ananatis (107CFU/ml, 0.5 ml) on the stem base. Ten plants were sprayed with water to serve as a control. All plants were kept at 28/25°C (day/night) under a 12-h/12-h photoperiod. All plants were covered with transparent plastic bags to maintain humidity. After 48 h, the bags were removed. After 2 weeks, water-soaked lesions on some leaves were observed similar to those in the field . Three to five weeks after inoculation, the crown of the inoculated plants was necrotized, which was similar to the symptoms in the field. No symptoms were observed in the control plants. The experiment was repeated three times. The bacteria were successfully reisolated from the inoculated crown tissues and leaves and confirmed as CM3 according to the same methodologies used for the initial identification. Bacterial leaf blight in strawberry caused by Pantoea ananatis has been reported in Nova Scotia, Canada, and Egypt (Bajpai et al. 2019; Abdel-Gaied et al. 2022). To our knowledge, this is the first report of Pantoea ananatis causing crown necrobiosis on strawberry in China. This report provides a basis for further research on this disease and its management and control.

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In order to understand the interspecific and ecological relationships of Michelia odora (extremely small population) community and strengthen the protection of wild M. odora resources in Junzifeng Nature Reserve, we studied the niche characteristics and interspecific associations of dominant tree species. The results showed that M. odora, Machilus chekiangensis, Schima superba, and Alniphyllum fortunei had obvious niche breadth advantages, which were the constructive species of the community. Among the 190 groups of species pairs among the 20 dominant tree species, 50.5% of species pairs had niche overlap value greater than 0.5. The degree of ecological niche differentiation among species was general. M. odora had large niche overlap with other 19 species, indicating a competitive risk when resources were insufficient. The overall associations of dominant tree species were significantly positive, indicating the community was at the late stage of relatively stable succession. The results ofχ2 test, asso-ciation coefficient, and Pearson correlation coefficient showed that all the significance ratios of interspecific association were lower, and that the independence among species was relatively strong. There was a positive correlation between interspecific association and niche overlap. The M. odora community was relatively mature, with full utilization of resources and stable interspecific relationship. To promote the rejuvenation and create a good habitat of M. odora population, the population size with large overlap with M. odora niche and significant negative association could be appropriately limited, while that with positive interaction could be increased.

The intestine is a major contributor to circulating succinate in mice.

The tricarboxylic acid (TCA) cycle is the epicenter of cellular aerobic metabolism. TCA cycle intermediates facilitate energy production and provide anabolic precursors, but also function as intra- and extracellular metabolic signals regulating pleiotropic biological processes. Despite the importance of circulating TCA cycle metabolites as signaling molecules, the source of circulating TCA cycle intermediates remains uncertain. We observe that in mice, the concentration of TCA cycle intermediates in the portal blood exceeds that in tail blood indicating that the gut is a major contributor to circulating TCA cycle metabolites. With a focus on succinate as a representative of a TCA cycle intermediate with signaling activities and using a combination of gut microbiota depletion mouse models and isotopomer tracing, we demonstrate that intestinal microbiota is not a major contributor to circulating succinate. Moreover, we demonstrate that endogenous succinate production is markedly higher than intestinal succinate absorption in normal physiological conditions. Altogether, these results indicate that endogenous succinate production within the intestinal tissue is a major physiological source of circulating succinate. These results provide a foundation for an investigation into the role of the intestine in regulating circulating TCA cycle metabolites and their potential signaling effects on health and disease.

Paradoxical activation of transcription factor SREBP1c and de novo lipogenesis by hepatocyte-selective ATP-citrate lyase depletion in obese mice.

Hepatic steatosis associated with high-fat diet, obesity, and type 2 diabetes is thought to be the major driver of severe liver inflammation, fibrosis, and cirrhosis. Cytosolic acetyl CoA (AcCoA), a central metabolite and substrate for de novo lipogenesis (DNL), is produced from citrate by ATP-citrate lyase (ACLY) and from acetate through AcCoA synthase short chain family member 2 (ACSS2). However, the relative contributions of these two enzymes to hepatic AcCoA pools and DNL rates in response to high-fat feeding are unknown. We report here that hepatocyte-selective depletion of either ACSS2 or ACLY caused similar 50% decreases in liver AcCoA levels in obese mice, showing that both pathways contribute to the generation of this DNL substrate. Unexpectedly however, the hepatocyte ACLY depletion in obese mice paradoxically increased total DNL flux measured by D2O incorporation into palmitate, whereas in contrast, ACSS2 depletion had no effect. The increase in liver DNL upon ACLY depletion was associated with increased expression of nuclear sterol regulatory element-binding protein 1c and of its target DNL enzymes. This upregulated DNL enzyme expression explains the increased rate of palmitate synthesis in ACLY-depleted livers. Furthermore, this increased flux through DNL may also contribute to the observed depletion of AcCoA levels because of its increased conversion to malonyl CoA and palmitate. Together, these data indicate that in fat diet-fed obese mice, hepatic DNL is not limited by its immediate substrates AcCoA or malonyl CoA but rather by activities of DNL enzymes.

Statin therapy inhibits fatty acid synthase via dynamic protein modifications.

Statins are a class of drug widely prescribed for the prevention of cardiovascular disease, with pleiotropic cellular effects. Statins inhibit HMG-CoA reductase (HMGCR), which converts the metabolite HMG-CoA into mevalonate. Recent discoveries have shown HMG-CoA is a reactive metabolite that can non-enzymatically modify proteins and impact their activity. Therefore, we predicted that inhibition of HMGCR by statins might increase HMG-CoA levels and protein modifications. Upon statin treatment, we observe a strong increase in HMG-CoA levels and modification of only a single protein. Mass spectrometry identifies this protein as fatty acid synthase (FAS), which is modified on active site residues and, importantly, on non-lysine side-chains. The dynamic modifications occur only on a sub-pool of FAS that is located near HMGCR and alters cellular signaling around the ER and Golgi. These results uncover communication between cholesterol and lipid biosynthesis by the substrate of one pathway inhibiting another in a rapid and reversible manner.

SLC38A2 provides proline to fulfill unique synthetic demands arising during osteoblast differentiation and bone formation.

Cellular differentiation is associated with the acquisition of a unique protein signature that is essential to attain the ultimate cellular function and activity of the differentiated cell. This is predicted to result in unique biosynthetic demands that arise during differentiation. Using a bioinformatic approach, we discovered that osteoblast differentiation is associated with increased demand for the amino acid proline. When compared to other differentiated cells, osteoblast-associated proteins, including RUNX2, OSX, OCN, and COL1A1, are significantly enriched in proline. Using a genetic and metabolomic approach, we demonstrate that the neutral amino acid transporter SLC38A2 acts cell-autonomously to provide proline to facilitate the efficient synthesis of proline-rich osteoblast proteins. Genetic ablation of SLC38A2 in osteoblasts limits both osteoblast differentiation and bone formation in mice. Mechanistically, proline is primarily incorporated into nascent protein with little metabolism observed. Collectively, these data highlight a requirement for proline in fulfilling the unique biosynthetic requirements that arise during osteoblast differentiation and bone formation.

Bones have diverse roles in the body, such as supporting weight, allowing movement and protecting internal organs. Regardless of their location, all bones in the body are formed and maintained by specialized cells called osteoblasts. To produce the different components of bone, osteoblasts need a constant supply of amino acids, the building blocks of proteins. If these nutrients are limited, this may lead to weak and fragile bones that can fracture more easily. Naïve cells in the bone, which are yet to have a defined role, also require large amounts of amino acids to develop into fully functioning osteoblasts. Previous studies have found that specific amino acids (like glutamine and asparagine) are particularly important for forming the proteins in bone. However, it was unclear which amino acids are critical for the development of osteoblasts. To investigate, Shen et al. studied naïve cells that had been extracted from the embryos of mice and developed into osteoblasts in the laboratory. They found that the developing osteoblasts produced proteins enriched in proline, and that naïve cells required large amounts of this amino acid as they turned into osteoblasts. Genetic analysis revealed that osteoblasts carry the gene for a protein called SLC38A2, which has been shown to transport proline into other types of cells. Shen et al. then used gene editing tools to delete this transporter from the osteoblasts of mice. The mutated mice could not efficiently produce proline-rich proteins during embryonic development and formed less bone. These findings highlight that proline is important for developing osteoblasts and synthesizing the products of bone. Further research is needed, but it is possible that dietary supplements of proline may be beneficial for maintaining or promoting bone formation in adulthood. This could help individuals that have more fragile bones, such as the elderly or patients with bone diseases, like osteoporosis.

Distinct Roles of Glutamine Metabolism in Benign and Malignant Cartilage Tumors With IDH Mutations.

Enchondromas and chondrosarcomas are common cartilage neoplasms that are either benign or malignant, respectively. The majority of these tumors harbor mutations in either IDH1 or IDH2. Glutamine metabolism has been implicated as a critical regulator of tumors with IDH mutations. Using genetic and pharmacological approaches, we demonstrated that glutaminase-mediated glutamine metabolism played distinct roles in enchondromas and chondrosarcomas with IDH1 or IDH2 mutations. Glutamine affected cell differentiation and viability in these tumors differently through different downstream metabolites. During murine enchondroma-like lesion development, glutamine-derived α-ketoglutarate promoted hypertrophic chondrocyte differentiation and regulated chondrocyte proliferation. Deletion of glutaminase in chondrocytes with Idh1 mutation increased the number and size of enchondroma-like lesions. In contrast, pharmacological inhibition of glutaminase in chondrosarcoma xenografts reduced overall tumor burden partially because glutamine-derived non-essential amino acids played an important role in preventing cell apoptosis. This study demonstrates that glutamine metabolism plays different roles in tumor initiation and cancer maintenance. Supplementation of α-ketoglutarate and inhibiting GLS may provide a therapeutic approach to suppress enchondroma and chondrosarcoma tumor growth, respectively. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Complete chloroplast genome sequence of Schima superba (Teaceae).

Schima superba is the dominant species of subtropical evergreen broad-leaved forest which has the characteristics of ecological fire prevention function. In this study, we report the complete chloroplast genome sequence of S. superba. The cp genome was 157,205 bp in length with a GC content of 37.40%, including a large single-copy (LSC 87,161 bp), a small single-copy (SSC 18,092 bp), and a pair of inverted repeats (IR 25,976 bp). The genome encoded 133 functional genes, including 88 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The phylogenetic analysis showed that S. superba was closely related to Schima sinensis, Schima multibracteata, Schima crenata, and Schima remotisertata.

Overcompensation of CoA Trapping by Di(2-ethylhexyl) Phthalate (DEHP) Metabolites in Livers of Wistar Rats.

Di(2-ethylhexyl) phthalate (DEHP) is commonly used as a plasticizer in various industrial and household plastic products, ensuring widespread human exposures. Its routine detection in human bio-fluids and the propensity of its monoester metabolite to activate peroxisome proliferator activated receptor-α (PPARα) and perturb lipid metabolism implicate it as a metabolic disrupter. In this study we evaluated the effects of DEHP exposure on hepatic levels of free CoA and various CoA esters, while also confirming the metabolic activation to CoA esters and partial ß-oxidation of a DEHP metabolite (2-ethyhexanol). Male Wistar rats were exposed via diet to 2% (w/w) DEHP for fourteen-days, following which hepatic levels of free CoA and various CoA esters were identified using liquid chromatography-mass spectrometry. DEHP exposed rats showed significantly elevated free CoA and increased levels of physiological, DEHP-derived and unidentified CoA esters. The physiological CoA ester of malonyl-CoA and DEHP-derived CoA ester of 3-keto-2-ethylhexanoyl-CoA were the most highly elevated, at eighteen- and ninety eight-times respectively. We also detected sixteen unidentified CoA esters which may be derivative of DEHP metabolism or induction of other intermediary metabolism metabolites. Our results demonstrate that DEHP is a metabolic disrupter which affects production and sequestration of CoA, an essential cofactor of oxidative and biosynthetic reactions.