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Nicotinoproteins are a group of NAD+-dependent dehydrogenases that bind NAD+ tightly and catalyze reactions without using free NAD+. In this study, we investigated the role of the unique insertion loop in nicotinoproteins. Carveol dehydrogenase (CADh), a short-chain dehydrogenase/reductase (SDR) nicotinoprotein, and β-hydroxybutyrate dehydrogenase from Alcaligenes faecalis (AfBHBDh), a non-nicotinoprotein counterpart, were used as model enzymes. An insertion loop-deleted mutant, CADh Δ39–49, was constructed. An insertion loop from Mycobacterium paratuberculosis CADh (MpCADh) was introduced into AfBHBDh to generate the two mutants. The results showed that CADh Δ39–49 lost NAD+ tight binding capacity and could not utilize free NAD+. In contrast, the AfBHBDh mutants showed no dye-mediated dehydrogenase activity. Moreover, the KM and KD values for NAD+ were higher than those of the wild-type enzyme. Docking simulations revealed a stronger binding affinity between NAD+ and the mutants than with the wild-type AfBHBDh. Taken together, these results suggest that the insertion loop interferes with NAD+ entry into the active site of the enzyme while creating a more energetically favorable binding environment. This loop is necessary but alone is insufficient to achieve NAD+ tight binding. This study deepens understanding of NAD+ binding in SDR nicotinoproteins and provides insights for SDR enzyme engineering.

Background 3-Hydroxy butyrate dehydrogenase 2 (BDH2) is a short-chain dehydrogenase/reductase family member that plays a key role in the development and pathogenesis of human cancers. However, the role of BDH2 in gastric cancer (GC) remains largely unclear. Our study aimed to ascertain the regulatory mechanisms of BDH2 in GC, which could be used to develop new therapeutic strategies. Methods Western blotting, immunohistochemistry, and RT-PCR were used to investigate the expression of BDH2 in GC specimens and cell lines. Its correlation with the clinicopathological characteristics and prognosis of GC patients was analysed. Functional assays, such as CCK-8 and TUNEL assays, transmission electron microscopy, and an in vivo tumour growth assay, were performed to examine the proliferation, apoptosis, and autophagy of GC cells. Related molecular mechanisms were clarified by luciferase reporter, coimmunoprecipitation, and ubiquitination assays. Results BDH2 was markedly downregulated in GC tissues and cells, and the low expression of BDH2 was associated with poor survival of GC patients. Functionally, BDH2 overexpression significantly induced apoptosis and autophagy in vitro and in vivo. Mechanistically, BDH2 promoted Keap1 interaction with Nrf2 to increase the ubiquitination level of Nrf2. Ubiquitination/degradation of Nrf2 inhibited the activity of ARE to increase accumulation of reactive oxygen species (ROS), thereby inhibiting the phosphorylation levels of Akt Ser473 and mTOR Ser2448 . Conclusions Our study indicates that BDH2 is an important tumour suppressor in GC. BDH2 regulates intracellular ROS levels to mediate the PI3K/Akt/mTOR pathway through Keap1/Nrf2/ARE signalling, thereby inhibiting the growth of GC.

As of April 18, 2020, over 2,000,000 patients had been diagnosed with coronavirus disease-2019 (COVID-19) globally, and more than 140,000 deaths had been reported. The clinical and epidemiological characteristics of adult patients have been documented recently. However, information on pediatric patients is limited. We describe the clinical and epidemiological characteristics of pediatric patients to provide valuable insight into the early diagnosis and assessment of COVID-19 in children. This retrospective, observational study involves a case series performed at 4 hospitals in West China. Thirty-four pediatric patients with COVID-19 were included from January 27 to February 23, 2020. The final follow-up visit was completed by March 16, 2020. Clinical and epidemiological characteristics were analyzed on the basis of demographic data, medical history, laboratory tests, radiological findings, and treatment information. Data analysis was performed for 34 pediatrics patients with COVID-19 aged from 1 to 144 months (median 33.00, interquartile range 10.00-94.25), among whom 14 males (41%) were included. All the patients in the current study presented mild (18%) or moderate (82%) forms of COVID-19. A total of 48% of patients were noted to be without a history of exposure to an identified source. Mixed infections of other respiratory pathogens were reported in 16 patients (47%). Comorbidities were reported in 6 patients (18%). The most common initial symptoms were fever (76%) and cough (62%). Expectoration (21%), vomiting (12%), and diarrhea (12%) were also reported in a considerable portion of cases. A substantial increase was detected in serum amyloid A for 17 patients (among 20 patients with available data; 85%) and in high-sensitivity C-reactive protein for 17 patients (among 29 patients with available data; 59%), whereas a decrease in prealbumin was noticed in 25 patients (among 32 patients with available data; 78%). In addition, significant increases in the levels of lactate dehydrogenase and α-hydroxybutyrate dehydrogenase were detected in 28 patients (among 34 patients with available data; 82%) and 25 patients (among 34 patients with available data; 74%), respectively. Patchy lesions in lobules were detected by chest computed tomographic scans in 28 patients (82%). Ground-glass opacities, which were a typical feature in adults, were rare in pediatric patients (3%). Rapid radiologic progression and a late-onset pattern of lesions in the lobules were also noticed. Lesions in lobules still existed in 24 (among 32 patients with lesions; 75%) patients that were discharged, although the main symptoms disappeared a few days after treatment. All patients were discharged, and the median duration of hospitalization was 10.00 (8.00-14.25) days. The current study was limited by the small sample size and a lack of dynamic detection of inflammatory markers. Our data systemically presented the clinical and epidemiological features, as well as the outcomes, of pediatric patients with COVID-19. Stratified analysis was performed between mild and moderate cases. The findings offer new insight into early identification and intervention in pediatric patients with COVID-19.

Background The 3-hydroxybutyrate dehydrogenase 1 ( BDH1 ) mainly participates in the regulation of milk fat synthesis and ketone body synthesis in mammary epithelial cells. In our previous study, BDH1 was identified as a key candidate gene regulating lipid metabolism in mammary glands of dairy goats by RNA-seq. This study aimed to investigate the effect of BDH1 on lipid metabolism in mammary epithelial cells of dairy goats (GMECs). Results The results suggest that BDH1 plays a significant role in reducing triacylglycerol content and lipid droplet accumulation in GMECs ( p  < 0.05). Overexpression of BDH1 significantly decreased the expression of lipid metabolism-related genes ( SREBF1 and GPAM ) and reduced the levels of C14:0 and C17:1, while increasing FABP3 expression and C10:0 concentration ( p  < 0.05). Interference with BDH1 significantly increased the expression of SREBF1 and GPAM and the concentration of C14:0, C15:1, and C20:1, but significantly decreased FABP3 and C18:0 ( p  < 0.05). Treatment of GMECs with β-hydroxybutyric acid (R-BHBA) significantly decreased the expression of FASN , ACACA , LPL , SREBF1 , FABP3 , ACSL1 , GPAM , DGAT1 , and triacylglycerol content, while significantly increasing the expression of BDH1 ( p  < 0.05). Interference with BDH1 rescued the reduction of cellular TAG content and the expression of FASN , LPL , SREBF1 , ACSL1 , and GPAM in BHBA-treated GMECs. Conclusion In conclusion, BDH1 negatively regulates lipid metabolism in mammary glands of dairy goats. Furthermore, it may mitigate the inhibitory effect of R-BHBA on lipid metabolism in GMECs. Graphical Abstract BDH1 serves as a negative regulator of milk lipid synthesis in GMECs, and BDH1 counteracts the inhibitory effect of R-BHBA on lipid synthesis in mammary epithelial cells of dairy goats. Highlights • BDH1 has a negative regulatory effect on lipid metabolism in GMECs. • R-BHBA negatively regulated milk fat metabolism and TAG synthesis in GMECs. • R-BHBA and BDH1 positively regulated the milk lipid metabolism of dairy goats.

Background Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells. Methods To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo , a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model. Results The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo , the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival. Conclusion In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro , suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways.

T follicular helper (Tfh) cells are a subset of CD4+ T cells that are essential in the pathogenesis of systemic lupus erythematosus (SLE). Notably, iron is required for activated CD4+ T lymphocytes to sustain high proliferation and metabolism. In this issue of the JCI, Gao et al. showed that CD4+ T cells from patients with SLE accumulated iron, augmenting their differentiation into Tfh cells and correlating with disease activity. Using human cells and murine models, the authors demonstrated that miR-21 was overexpressed in lupus T cells and inhibited 3-hydroxybutyrate dehydrogenase-2 (BDH2). The subsequent loss of BDH2 drove labile iron to accumulate in the cytoplasm and promoted TET enzyme activity, BCL6 gene demethylation, and Tfh cell differentiation. This work identifies a role for iron in CD4+ T cell biology and the development of pathogenic effectors in SLE. We await future investigations that could determine whether modulating iron levels could regulate Tfh cells in human health and disease.

Background 3-Hydroxybutyrate dehydrogenase type 2 (BDH2) is known to catalyse a rate-limiting step in the biogenesis of the mammalian siderophore and regulate intracellular iron metabolism. Here we aim to explore the expression and possible function of BDH2 in nasopharyngeal carcinoma (NPC). Methods The transcription and protein expression of BDH2 in NPC were determined by both real-time RT-PCR and immunohistochemistry staining assays. Cell proliferation, migration and invasion were evaluated by MTT assay, wound-healing assay and Transwell assay, respectively. The profile of genes regulated by restoring BDH2 expression in NPC cells was analysed by cDNA microarray. The level of iron in NPC cells was detected by iron colorimetric assay. Results The expression of BDH2 was significantly downregulated in NPC. Ectopic expression of BDH2 inhibited NPC cell proliferation and colony formation. Meanwhile, BDH2 suppressed the migration and invasion of NPC cells by reversing the epithelial–mesenchymal transition (EMT). In addition, a higher level of BDH2 decreased the growth and metastasis of NPC cells via reducing intracellular iron level. Conclusions Our findings suggest that BDH2 may be a candidate tumour-suppressor gene in NPC. Decreasing intracellular iron could be an effective therapeutic approach for NPC.

ObjectiveHypoxia plays a critical role in the pathophysiology of cardiomyopathy, myocardial infarction, and heart failure. Promoting ketone metabolism has been shown to be beneficial for myocardial cells under hypoxic conditions. However, the expression and regulatory mechanisms of key enzymes in the ketone pathway under hypoxic conditions are still unclear. This study aimed to investigate the effects of hypoxia on the expression of key enzymes in the ketone metabolic pathway and the underlying regulatory mechanisms involved.MethodsH9C2 myocardial cells were cultured for 6 h in an oxygen-glucose-deprived state, and the expression of various genes was detected by quantitative real-time PCR. ELISA and lactate dehydrogenase (LDH) cytotoxicity assay were used to measure CoAs, itaconic acid, and LDH levels, respectively, and the dependence of gene expression on hypoxia-inducible factor-1 alpha (HIF-1α) was evaluated using the inhibitor LW6.ResultsH9C2 cardiomyocytes exhibited increased ketone body metabolism in response to hypoxia. Hypoxia induced the expression of the ketone body enzymes succinyl-CoA:3-oxoacid CoA transferase (SCOT/OXCT1), 3-hydroxybutyrate dehydrogenase 2 (BDH2), and acyl-CoA: cholesterol acyltransferase 1 (ACAT1) in cardiomyocytes, with a concomitant increase in the level of acyl-CoA and a decrease in the level of succinyl-CoA. The HIF-1α inhibitor LW6 could partially reverse the expression of BDH2 and ACAT1, as well as the levels of succinyl-CoA. Interestingly, however, hypoxia-induced SCOT/OXCT1 expression was not regulated by the HIF-1α inhibitor. In addition, hypoxia promoted the expression of inflammatory factors.ConclusionThese data confirm the critical role of ketone metabolism in myocardial hypoxia and help to elucidate the pathophysiology of cardiomyopathy, myocardial infarction and heart failure.

Background: Type 2 diabetes mellitus (T2DM) is typically characterized by the dysregulation of metabolic remodeling. As a systemic metabolic disease, T2DM can affect the mass and function of skeletal muscle by inducing impaired energy metabolism, mitochondrial dysfunction, and chronic low-grade inflammation. β-Hydroxybutyrate dehydrogenase 1 (BDH1) is a rate-limiting enzyme involved in ketone body metabolism, and its activity is down-regulated in various models of diabetic complications. Aerobic exercise (AE) is recognized as an effective intervention to promote energy homeostasis and alleviate metabolic stress. Whether its protective effect on skeletal muscle in T2DM involves the regulatory control of BDH1 expression remains unclear. Methods: Wild-type (WT) and systemic BDH1 knockout (BDH1−/−) male C57BL/6J mice were used to establish the sedentary control (SED) and AE models of T2DM by providing a high-fat diet combined with streptozotocin injection. The indicators related to metabolic remodeling were detected by hematoxylin and eosin staining, immunofluorescence staining, quantitative real-time PCR, and Western blot assays. Results: After 8 weeks of AE, we found that AE improved glycolipid metabolic disorders and mitochondrial quality control in the gastrocnemius muscle of T2DM mice by up-regulating BDH1, thereby alleviating oxidative stress, inflammation, and fibrosis. Compared with the WT mice, the BDH1−/− T2DM mice in the SED group exhibited more severe phenotypic impairment. The metabolic improvement effect of AE was attenuated in the BDH1−/− mice. Conclusions: BDH1 is a key effector enzyme that may mediate the AE-induced improvement in metabolic remodeling in the gastrocnemius muscle of mice with T2DM.

Mitochondria are the site of iron utilization, wherein imported iron is incorporated into heme or iron–sulfur clusters. Previously, we showed that a cytosolic siderophore, which resembles a bacterial siderophore, facilitates mitochondrial iron import in eukaryotes, including zebrafish. An evolutionarily conserved 3-hydroxy butyrate dehydrogenase, 3-hydroxy butyrate dehydrogenase 2 (Bdh2), catalyzes a rate-limiting step in the biogenesis of the eukaryotic siderophore. We found that inactivation of bdh2 in developing zebrafish embryo results in heme deficiency and delays erythroid maturation. The basis for this erythroid maturation defect is not known. Here we show that bdh2 inactivation results in mitochondrial dysfunction and triggers their degradation by mitophagy. Thus, mitochondria are prematurely lost in bdh2-inactivated erythrocytes. Interestingly, bdh2-inactivated erythroid cells also exhibit genomic alterations as indicated by transcriptome analysis. Reestablishment of bdh2 restores mitochondrial function, prevents premature mitochondrial degradation, promotes erythroid development, and reverses altered gene expression. Thus, mitochondrial communication with the nucleus is critical for erythroid development.