[18F](2S,4R)-4-Fluoroglutamine as a New Positron Emission Tomography Tracer in Myeloma

The high glycolytic activity of multiple myeloma (MM) cells is the rationale for use of Positron Emission Tomography (PET) with 18F-fluorodeoxyglucose ([18F]FDG) to detect both bone marrow (BM) and extramedullary disease. However, new tracers are actively searched because [18F]FDG-PET has some limitations and there is a portion of MM patients who are negative. Glutamine (Gln) addiction has been recently described as a typical metabolic feature of MM cells. Yet, the possible exploitation of Gln as a PET tracer in MM has never been assessed so far and is investigated in this study in preclinical models. Firstly, we have synthesized enantiopure (2S,4R)-4-fluoroglutamine (4-FGln) and validated it as a Gln transport analogue in human MM cell lines, comparing its uptake with that of 3H-labelled Gln. We then radiosynthesized [18F]4-FGln, tested its uptake in two different in vivo murine MM models, and checked the effect of Bortezomib, a proteasome inhibitor currently used in the treatment of MM. Both [18F]4-FGln and [18F]FDG clearly identified the spleen as site of MM cell colonization in C57BL/6 mice, challenged with syngeneic Vk12598 cells and assessed by PET. NOD.SCID mice, subcutaneously injected with human MM JJN3 cells, showed high values of both [18F]4-FGln and [18F]FDG uptake. Bortezomib significantly reduced the uptake of both radiopharmaceuticals in comparison with vehicle at post treatment PET. However, a reduction of glutaminolytic, but not of glycolytic, tumor volume was evident in mice showing the highest response to Bortezomib. Our data indicate that [18F](2S,4R)-4-FGln is a new PET tracer in preclinical MM models, yielding a rationale to design studies in MM patients.

Glycolytic Reprogramming in Silica-Induced Lung Macrophages and Silicosis Reversed by Ac-SDKP Treatment

Glycolytic reprogramming is an important metabolic feature in the development of pulmonary fibrosis. However, the specific mechanism of glycolysis in silicosis is still not clear. In this study, silicotic models and silica-induced macrophage were used to elucidate the mechanism of glycolysis induced by silica. Expression levels of the key enzymes in glycolysis and macrophage activation indicators were analyzed by Western blot, qRT-PCR, IHC, and IF analyses, and by using a lactate assay kit. We found that silica promotes the expression of the key glycolysis enzymes HK2, PKM2, LDHA, and macrophage activation factors iNOS, TNF-α, Arg-1, IL-10, and MCP1 in silicotic rats and silica-induced NR8383 macrophages. The enhancement of glycolysis and macrophage activation induced by silica was reduced by Ac-SDKP or siRNA-Ldha treatment. This study suggests that Ac-SDKP treatment can inhibit glycolytic reprogramming in silica-induced lung macrophages and silicosis.

A non-canonical convergence of carbohydrate and glutamine metabolism is required after metabolic rewiring in 3D environment

The fate of pyruvate, which is modulated mitochondrial pyruvate carrier (MPC) activity, is a defining metabolic feature in many cancers. Diffuse large B-cell lymphomas (DLBCLs) are a genetically and metabolically heterogenous cancer. Although MPC expression and activity differed between DLBCL subgroups, mitochondrial pyruvate oxidation was uniformly minimal. Mitochondrial pyruvate was instead robustly consumed by glutamate pyruvate transaminase 2 to support α-ketoglutarate production as part of glutamine catabolism. This led us to discover that glutamine exceeds pyruvate as a carbon source for the TCA cycle, but, MPC function is required to enable GPT2-mediated glutamine catabolism. Furthermore, we found that MPC inhibition only decreased DLBCL proliferation in a solid culture environment, but not in a suspension environment. Thus, the non-canonical connection between the consumption and assimilation of carbohydrates and glutamine in DLBCLs enables their proliferation in a solid 3D environment.

Formate-Dependent Acetogenic Utilization of Glucose by the Fecal Acetogen Clostridium bovifaecis

ABSTRACT Acetogenic bacteria are a diverse group of anaerobes that use the reductive acetyl coenzyme A (acetyl-CoA) (Wood-Ljungdahl) pathway for CO2 fixation and energy conservation. The conversion of 2 mol CO2 into acetyl-CoA by using the Wood-Ljungdahl pathway as the terminal electron accepting process is the most prominent metabolic feature for these microorganisms. However, here, we describe that the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. Genome analysis of Clostridium bovifaecis revealed that it contains almost the complete genes of the Wood-Ljungdahl pathway but lacks the gene encoding formate dehydrogenase, which catalyzes the reduction of CO2 to formate as the first step of the methyl branch of the Wood-Ljungdahl pathway. The lack of a gene encoding formate dehydrogenase was verified by PCR, reverse transcription-PCR analysis, enzyme activity assay, and its formate-dependent acetogenic utilization of glucose on DNA, RNA, protein, and phenotype level, respectively. The lack of a formate dehydrogenase gene may be associated with the adaption to a formate-rich intestinal environment, considering the isolating source of strain BXX. The formate-dependent acetogenic growth of Clostridium bovifaecis provides insight into a unique metabolic feature of fecal acetogens. IMPORTANCE The acetyl-CoA pathway is an ancient pathway of CO2 fixation, which converts 2 mol of CO2 into acetyl-CoA. Autotrophic growth with H2 and CO2 via the acetyl-CoA pathway as the terminal electron accepting process is the most unique feature of acetogenic bacteria. However, the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. The formate-dependent acetogenic growth of Clostridium bovifaecis was associated with its lack of a gene encoding formate dehydrogenase, which may result from adaption to a formate-rich intestinal environment. This study gave insight into a unique metabolic feature of fecal acetogens. Because of the requirement of formate for the acetogenic growth of certain acetogens, the ecological impact of acetogens could be more complex and important in the formate-rich environment due to their trophic interactions with other microbes.

Loss of MiR-192 Initiates a Hyperglycolysis and Stemness Positive Feedback in Hepatocellular Carcinoma

Background: Emerging studies revealed that cancer stem cells (CSCs) possessed peculiar metabolic properties, which however remained largely unknown in hepatocellular carcinoma (HCC). Genetic silencing of liver-abundant miR-192 was a key feature for multiple groups of CSC-positive primary HCCs. We thus aimed to investigate essential metabolic features of hepatic CSCs via using HCCs with miR-192 silencing as a model. Methods: Data integration analyses of miR-192 with metabolome and mRNA transcriptome in HCC cohort 1 were performed to investigate miR-192 related metabolic features. Cellular and molecular assays were performed to examine whether and how miR-192 regulated the identified metabolic feature. Co-culture systems consisting of HCC and non-HCC cells were established to explore effects of the metabolomic property on stemness features in HCC cells via interacting with non-HCC cells. Results: High expression of glycolysis-related metabolites and genes presented in HCCs with low miR-192 and CSC-positive HCCs in two independent HCC cohorts. miR-192 knock-out cells displayed CSC features and miR-192 loss led to an enhanced glycolytic phenotype via targeting three glycolysis regulators, i.e., Glut1, Pfkfb3, and c-Myc. Meanwhile, c-Myc suppressed miR-192 transcription, ensuring a low-miR-192/high-c-Myc loop to maintain hyperglycolysis. Moreover, over-produced lactic acid from hyperglycolytic HCC cells stimulated the Erk phosphorylation of co-cultured non-HCC cells partially via NDRG3 and MCT1, which in turn promoted cell malignancy and stemness of HCC cells. Conclusions: In CSC-positive HCCs, miR-192 loss enhanced glycolysis via a c-Myc/miR-192/glycolysis regulators signal loop, allowing HCC cells to actively coordinate with their environment non-HCC cells for further increased stemness and malignancy.

Comparative transcriptomic and metabolomic analysis reveals pectoralis highland adaptation across altitudinal songbirds

Background Pectoralis phenotypic variation plays a fundamental role in locomotion and thermogenesis in highland birds. However, its regulatory and metabolic mechanisms remain enigmatic to date. Here, we integrated phenomic, transcriptomic and metabolomic approaches to determine muscle variation and its underpinning mechanisms across altitudinal songbirds. Results Phenomics revealed that all highland birds had considerable increases in muscle oxidative capacity, capillarity, and mitochondrial abundance. Correspondingly, transcriptomic analyses found that differentially expressed genes in modules associated with phenotypes enriched in blood vessel, muscle structure development, and mitochondrial organization. Despite similar traits and functional enrichments across highland birds, different mechanisms drove their occurrence in part for their own various evolutionary histories. Importantly, a metabolic feature shared by highland birds is the improvement in insulin sensitivity and glucose utilization through activating insulin signaling pathway, which is vital to increase muscle oxidative capacity and maintain metabolic homeostasis. Nevertheless, fatty acid biosynthesis and oxidation are enhanced in species with a long evolutionary history, also differing from ketone body metabolism in recently introduced colonizer. Conclusions Our study represents a vital contribution to reveal the regulatory and metabolic basis of pectoralis variation across altitudinal songbirds.