bims-mepmim 2022-10-09 papers

bims-mepmim

Biomed News

on Metabolites in pathological microenvironments and immunometabolism

Issue of 2022–10–09
twenty papers selected by
Erika Mariana Palmieri, NIH/NCI Laboratory of Cancer ImmunoMetabolism

Differential glutamine metabolism in the tumor microenvironment - studies in diversity and heterogeneity: A mini-review.
Lipid metabolic features of T cells in the Tumor Microenvironment.
Metabolic flux analysis: a comprehensive review on sample preparation, analytical techniques, data analysis, computational modelling, and main application areas.
Enforcing GLUT3 expression in CD8+ T cells improves fitness and tumor control by promoting glucose uptake and energy storage.
GAB functions as a bioenergetic and signalling gatekeeper to control T cell inflammation.
Metabolic alterations of short-chain fatty acids and TCA cycle intermediates in human plasma from patients with gastric cancer.
Mitochondrial fatty acid β-oxidation is important for normal osteoclast formation in growing female mice.
Coordinated metabolic transitions and gene expression by NAD+ during adipogenesis.
ACSL3 regulates lipid droplet biogenesis and ferroptosis sensitivity in clear cell renal cell carcinoma.
Hepatic glycerol shunt and glycerol-3-phosphate phosphatase control liver metabolism and glucodetoxification under hyperglycemia.
Two independent respiratory chains adapt OXPHOS performance to glycolytic switch.
Inter-organellar and systemic responses to impaired mitochondrial matrix protein import in skeletal muscle.
The structure of succinyl-CoA synthetase bound to the succinyl-phosphate intermediate clarifies the catalytic mechanism of ATP-citrate lyase.
CD36-mediated metabolic crosstalk between tumor cells and macrophages affects liver metastasis.
Adipocyte coculture induces a pro-inflammatory, fibrotic, angiogenic, and proliferative microenvironment in uterine leiomyoma cells.
Aurora A-mediated pyruvate kinase M2 phosphorylation promotes biosynthesis with glycolytic metabolites and tumor cell cycle progression.
Succinyl-CoA-based energy metabolism dysfunction in chronic heart failure.
Preparation of mitochondrial damage-associated molecular patterns from mouse liver tissue.
Nuclear TCA cycle reactions.
Cholesterol-induced robust Ca oscillation in astrocytes required for survival and lipid droplet formation in high-cholesterol condition.

Front Oncol. 2022 ;12 1011191

Differential glutamine metabolism in the tumor microenvironment - studies in diversity and heterogeneity: A mini-review.

Michael D Claiborne, Robert Leone.

  Increased glutamine metabolism is a hallmark of many cancer types. In recent years, our understanding of the distinct and diverse metabolic pathways through which glutamine can be utilized has grown more refined. Additionally, the different metabolic requirements of the diverse array of cell types within the tumor microenvironment complicate the strategy of targeting any particular glutamine pathway as cancer therapy. In this Mini-Review, we discuss recent advances in further clarifying the cellular fate of glutamine through different metabolic pathways. We further discuss potential promising strategies which exploit the different requirements of cells in the tumor microenvironment as it pertains to glutamine metabolism in an attempt to suppress cancer growth and enhance anti-tumor immune responses.

Keywords:  cancer; glutamine; immunooncology; metabolism; tumor microenvironment

DOI:  https://doi.org/10.3389/fonc.2022.1011191
Lipids Health Dis. 2022 Oct 06. 21(1): 94

Lipid metabolic features of T cells in the Tumor Microenvironment.

Wanshuang Lou, Chaoju Gong, Zhuoni Ye, Ynayan Hu, Minjing Zhu, Zejun Fang, Huihui Xu.

  The tumor microenvironment (TME) is characterized by discrete changes in metabolic features of cancer and immune cells, with various implications. Cancer cells take up most of the available glucose to support their growth, thereby leaving immune cells with insufficient nutrients to expand. In the relative absence of glucose, T cells switch the metabolic program to lipid-based sources, which is pivotal to T-cell differentiation and activation in nutrient-stressed TME. Although consumption of lipids should provide an alternative energy source to starving T cells, a literature survey has revealed that it may not necessarily lead to antitumor responses. Different subtypes of T cells behave differently in various lipid overload states, which widely depends upon the kind of free fatty acids (FFA) engulfed. Key lipid metabolic genes provide cytotoxic T cells with necessary nutrients for proliferation in the absence of glucose, thereby favoring antitumor immunity, but the same genes cause immune evasion in Tmem and Treg. This review aims to detail the complexity of differential lipid metabolism in distinct subtypes of T cells that drive the antitumor or pro-tumor immunity in specific TME states. We have identified key drug targets related to lipid metabolic rewiring in TME.

Keywords:  CD36; Lipid; PD-L1/2; T-cells; Tumor microenvironment (TME)

DOI:  https://doi.org/10.1186/s12944-022-01705-y
RSC Adv. 2022 Sep 05. 12(39): 25528-25548

Metabolic flux analysis: a comprehensive review on sample preparation, analytical techniques, data analysis, computational modelling, and main application areas.

Bruna de Falco, Francesco Giannino, Fabrizio Carteni, Stefano Mazzoleni, Dong-Hyun Kim.

Metabolic flux analysis (MFA) quantitatively describes cellular fluxes to understand metabolic phenotypes and functional behaviour after environmental and/or genetic perturbations. In the last decade, the application of stable isotopes became extremely important to determine and integrate in vivo measurements of metabolic reactions in systems biology. 13C-MFA is one of the most informative methods used to study central metabolism of biological systems. This review aims to outline the current experimental procedure adopted in 13C-MFA, starting from the preparation of cell cultures and labelled tracers to the quenching and extraction of metabolites and their subsequent analysis performed with very powerful software. Here, the limitations and advantages of nuclear magnetic resonance spectroscopy and mass spectrometry techniques used in carbon labelled experiments are elucidated by reviewing the most recent published papers. Furthermore, we summarise the most successful approaches used for computational modelling in flux analysis and the main application areas with a particular focus in metabolic engineering.

DOI: https://doi.org/10.1039/d2ra03326g
Front Immunol. 2022 ;13 976628

Enforcing GLUT3 expression in CD8+ T cells improves fitness and tumor control by promoting glucose uptake and energy storage.

Elisabetta Cribioli, Greta Maria Paola Giordano Attianese, Pierpaolo Ginefra, Amandine Signorino-Gelo, Romain Vuillefroy de Silly, Nicola Vannini, Christoph Hess, Melita Irving, George Coukos.

  Despite the tremendous success of adoptive T-cell therapies (ACT) in fighting certain hematologic malignancies, not all patients respond, a proportion experience relapse, and effective ACT of most solid tumors remains elusive. In order to improve responses to ACT suppressive barriers in the solid tumor microenvironment (TME) including insufficient nutrient availability must be overcome. Here we explored how enforced expression of the high-affinity glucose transporter GLUT3 impacted tumor-directed T cells. Overexpression of GLUT3 in primary murine CD8+ T cells enhanced glucose uptake and increased glycogen and fatty acid storage, and was associated with increased mitochondrial fitness, reduced ROS levels, higher abundance of the anti-apoptotic protein Mcl-1, and better resistance to stress. Importantly, GLUT3-OT1 T cells conferred superior control of B16-OVA melanoma tumors and, in this same model, significantly improved survival. Moreover, a proportion of treated mice were cured and protected from re-challenge, indicative of long-term T cell persistence and memory formation. Enforcing expression of GLUT3 is thus a promising strategy to improve metabolic fitness and sustaining CD8+ T cell effector function in the context of ACT.

Keywords:  T cell engineering; adoptive cell therapy; glucose; immunotherapy; metabolism; tumor

DOI:  https://doi.org/10.3389/fimmu.2022.976628
Nat Metab. 2022 Oct 03.

GAB functions as a bioenergetic and signalling gatekeeper to control T cell inflammation.

Siwen Kang, Lingling Liu, Tingting Wang, Matthew Cannon, Penghui Lin, Teresa W-M Fan, David A Scott, Hsin-Jung Joyce Wu, Andrew N Lane, Ruoning Wang.

γ-Aminobutyrate (GAB), the biochemical form of (GABA) γ-aminobutyric acid, participates in shaping physiological processes, including the immune response. How GAB metabolism is controlled to mediate such functions remains elusive. Here we show that GAB is one of the most abundant metabolites in CD4+ T helper 17 (TH17) and induced T regulatory (iTreg) cells. GAB functions as a bioenergetic and signalling gatekeeper by reciprocally controlling pro-inflammatory TH17 cell and anti-inflammatory iTreg cell differentiation through distinct mechanisms. 4-Aminobutyrate aminotransferase (ABAT) funnels GAB into the tricarboxylic acid (TCA) cycle to maximize carbon allocation in promoting TH17 cell differentiation. By contrast, the absence of ABAT activity in iTreg cells enables GAB to be exported to the extracellular environment where it acts as an autocrine signalling metabolite that promotes iTreg cell differentiation. Accordingly, ablation of ABAT activity in T cells protects against experimental autoimmune encephalomyelitis (EAE) progression. Conversely, ablation of GABAA receptor in T cells worsens EAE. Our results suggest that the cell-autonomous control of GAB on CD4+ T cells is bimodal and consists of the sequential action of two processes, ABAT-dependent mitochondrial anaplerosis and the receptor-dependent signalling response, both of which are required for T cell-mediated inflammation.

DOI: https://doi.org/10.1038/s42255-022-00638-1
Life Sci. 2022 Sep 28. pii: S0024-3205(22)00710-X. [Epub ahead of print]309 121010

Metabolic alterations of short-chain fatty acids and TCA cycle intermediates in human plasma from patients with gastric cancer.

You Lee Kim, Wonwoong Lee, So Hyeon Chung, Byeong Min Yu, Yong Chan Lee, Jongki Hong.

   AIMS: Short-chain fatty acids (SCFAs) are produced by gut microbiota from dietary fiber. Since absorbed SCFAs could be introduced into the tricarboxylic acid (TCA) cycle in host cells, the relationships between SCFAs and TCA cycle intermediates might influence to energy metabolism in the human body. For this reason, information on profile changes between SCFAs and TCA cycle intermediates could help unveil pathological mechanisms of gastric cancer.
MAIN METHODS: A gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed to simultaneously determine SCFAs and TCA cycle intermediates in human plasma from patients with chronic superficial gastritis (CSG), intestinal metaplasia (IM), and gastric cancer. We applied a tetra-alkyl ammonium pairing method to prevent loss of volatile SCFAs and base decarboxylation of TCA cycle intermediates during sample preparation. To assess gastric diseases, metabolic alterations of SCFAs and TCA cycle intermediates in human plasma with gastric disorders were analyzed by their plasma levels.
KEY FINDINGS: Significantly different metabolic alterations based on the plasma levels of SCFAs and TCA cycle intermediates were investigated in cancer metabolic pathways. Not only propionate and butyrate, mainly produced by gut microbiota, were significantly decreased, but also cis-aconitate, α-ketoglutarate, and fumarate were significantly increased in plasma with IM or gastric cancer, compared to CSG. Further, based on ratios of product to precursor, three metabolic pathways (succinate/propionate, succinate/α-ketoglutarate, and cis-aconitate/citrate) were supposed to be distorted between gastric diseases.
SIGNIFICANCE: In conclusion, propionate, cis-aconitate, α-ketoglutarate, and fumarate could be used to assess the progression of gastric cancer.

Keywords:  Gas chromatography; Gastric cancer; Human plasma; Short-chain fatty acids; TCA cycle intermediates; Tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.lfs.2022.121010
Front Physiol. 2022 ;13 997358

Mitochondrial fatty acid β-oxidation is important for normal osteoclast formation in growing female mice.

Priyanka Kushwaha, Nathalie S Alekos, Soohyun P Kim, Zhu Li, Michael J Wolfgang, Ryan C Riddle.

  Skeletal remodeling is an energy demanding process that is linked to nutrient availability and the levels of metabolic hormones. While recent studies have examined the metabolic requirements of bone formation by osteoblasts, much less is known about the energetic requirements of bone resorption by osteoclasts. The abundance of mitochondria in mature osteoclasts suggests that the production of an acidified micro-environment conducive to the ionization of hydroxyapatite, secretion of matrix-degrading enzymes, and motility during resorption requires significant energetic capacity. To investigate the contribution of mitochondrial long chain fatty acid β-oxidation to osteoclast development, we disrupted the expression of carnitine palmitoyltransferase-2 (Cpt2) in myeloid-lineage cells. Fatty acid oxidation increases dramatically in bone marrow cultures stimulated with RANKL and M-CSF and microCT analysis revealed that the genetic inhibition of long chain fatty acid oxidation in osteoclasts significantly increases trabecular bone volume in female mice secondary to reduced osteoclast numbers. In line with these data, osteoclast precursors isolated from Cpt2 mutants exhibit reduced capacity to form large-multinucleated osteoclasts, which was not rescued by exogenous glucose or pyruvate, and signs of an energetic stress response. Together, our data demonstrate that mitochondrial long chain fatty acid oxidation by the osteoclast is required for normal bone resorption as its inhibition produces an intrinsic defect in osteoclast formation.

Keywords:  CPT2; bone; glucose; lipid metabolism; osteoclast

DOI:  https://doi.org/10.3389/fphys.2022.997358
J Cell Biol. 2022 12 05. pii: e202111137. [Epub ahead of print]221(12):

Coordinated metabolic transitions and gene expression by NAD+ during adipogenesis.

Edgar Sánchez-Ramírez, Thi Phuong Lien Ung, Alejandro Alarcón Del Carmen, Ximena del Toro-Ríos, Guadalupe R Fajardo-Orduña, Lilia G Noriega, Victor A Cortés-Morales, Armando R Tovar, Juan José Montesinos, Ricardo Orozco-Solís, Chiara Stringari, Lorena Aguilar-Arnal.

Adipocytes are the main cell type in adipose tissue, which is a critical regulator of metabolism, highly specialized in storing energy as fat. Adipocytes differentiate from multipotent mesenchymal stromal cells (hMSCs) through adipogenesis, a tightly controlled differentiation process involving close interplay between metabolic transitions and sequential programs of gene expression. However, the specific gears driving this interplay remain largely obscure. Additionally, the metabolite nicotinamide adenine dinucleotide (NAD+) is becoming increasingly recognized as a regulator of lipid metabolism, and a promising therapeutic target for dyslipidemia and obesity. Here, we explored how NAD+ bioavailability controls adipogenic differentiation from hMSC. We found a previously unappreciated repressive role for NAD+ on adipocyte commitment, while a functional NAD+-dependent deacetylase SIRT1 appeared crucial for terminal differentiation of pre-adipocytes. Repressing NAD+ biosynthesis during adipogenesis promoted the adipogenic transcriptional program, while two-photon microscopy and extracellular flux analyses suggest that SIRT1 activity mostly relies on the metabolic switch. Interestingly, SIRT1 controls subcellular compartmentalization of redox metabolism during adipogenesis.

DOI: https://doi.org/10.1083/jcb.202111137
Cancer Metab. 2022 Oct 03. 10(1): 14

ACSL3 regulates lipid droplet biogenesis and ferroptosis sensitivity in clear cell renal cell carcinoma.

Timothy D Klasson, Edward L LaGory, Hongjuan Zhao, Star K Huynh, Ioanna Papandreou, Eui Jung Moon, Amato J Giaccia.

   BACKGROUND: Clear cell renal cell carcinoma (ccRCC), the predominant subtype of kidney cancer, possesses characteristic alterations to multiple metabolic pathways, including the accumulation of cytosolic lipid droplets. However, the pathways that drive lipid droplet accumulation in ccRCC cells and their importance to cancer biology remain poorly understood.
METHODS: We sought to identify the carbon sources necessary for lipid droplet accumulation using Oil red O staining and isotope-tracing lipidomics. The role of the acyl-CoA synthetase (ACSL) family members, an important group of lipid metabolic enzymes, was investigated using siRNA and drug mediated inhibition. CTB and XTT assays were performed to determine the effect of ACSL3 knockdown and lipid starvation on ccRCC cell viability and shRNA was used to study the effect of ACSL3 in an orthotopic mouse model. The relationship between ferroptosis susceptibility of ccRCC and ACSL3 controlled lipid metabolism was examined using CTB and FACS-based assays. The importance of 5-LOX in ferroptosis susceptibility in ccRCC was shown with XTT survival assays, and the expression level and predictive value of 5-LOX in TCGA ccRCC data was assessed.
RESULTS: We found that ccRCC cells obtain the necessary substrates for lipid droplet accumulation by metabolizing exogenous serum derived lipids and not through de novo lipogenesis. We show that this metabolism of exogenous fatty acids into lipid droplets requires the enzyme acyl-CoA synthetase 3 (ACSL3) and not other ACSL family proteins. Importantly, genetic or pharmacologic suppression of ACSL3 is cytotoxic to ccRCC cells in vitro and causes a reduction of tumor weight in an orthotopic mouse model. Conversely, ACSL3 inhibition decreases the susceptibility of ccRCC cells to ferroptosis, a non-apoptotic form of cell death involving lipid peroxidation. The sensitivity of ccRCC to ferroptosis is also highly dependent on the composition of exogenous fatty acids and on 5-lipoxygenase (5-LOX), a leukotriene producing enzyme which produces lipid peroxides that have been implicated in other cancers but not in ccRCC.
CONCLUSIONS: ACSL3 regulates the accumulation of lipid droplets in ccRCC and is essential for tumor growth. In addition, ACSL3 also modulates ferroptosis sensitivity in a manner dependent on the composition of exogenous fatty acids. Both functions of ACSL3 could be exploited for ccRCC therapy.

Keywords:  5-lipoxygenase (5-LOX); Acyl-CoA synthetase 3 (ACSL3); Clear cell renal cell carcinoma (ccRCC); Ferroptosis; Lipid droplets; Lipid metabolism

DOI:  https://doi.org/10.1186/s40170-022-00290-z
Mol Metab. 2022 Oct 02. pii: S2212-8778(22)00178-8. [Epub ahead of print] 101609

Hepatic glycerol shunt and glycerol-3-phosphate phosphatase control liver metabolism and glucodetoxification under hyperglycemia.

Anfal Al-Mass, Pegah Poursharifi, Marie-Line Peyot, Roxane Lussier, Isabelle Chenier, Yat Hei Leung, Anindya Ghosh, Abel Oppong, Elite Possik, Yves Mugabo, Rasheed Ahmad, Robert Sladek, S R Murthy Madiraju, Fahd Al-Mulla, Marc Prentki.

   OBJECTIVE: Glycerol-3-phosphate (Gro3P) phosphatase (G3PP) hydrolyzes Gro3P to glycerol that exits the cell, thereby operating a "glycerol shunt", a metabolic pathway that we identified recently in mammalian cells. We have investigated the role of G3PP and the glycerol shunt in the regulation of glucose metabolism and lipogenesis in mouse liver.
METHODS: We generated hepatocyte-specific G3PP-KO mice (LKO), by injecting AAV8-TBG-iCre to male G3PPfl/fl mice. Controls received AAV8-TBG-eGFP. Both groups were fed chow diet for 10 weeks. Hyperglycemia (16-20 mM) was induced by glucose infusion for 55 h. Hepatocytes were isolated from normoglycemic mice for ex vivo studies and targeted metabolomics were measured in mice liver after glucose infusion.
RESULTS: LKO mice showed no change in body weight, food intake, fed and fasted glycemia but had increased fed plasma triglycerides. Hepatic glucose production from glycerol was increased in fasted LKO mice. LKO mouse hepatocytes displayed reduced glycerol production, elevated triglyceride and lactate production at high glucose concentration. Hyperglycemia in LKO mice led to increased liver weight and accumulation of triglycerides, glycogen and cholesterol together with elevated levels of Gro3P, dihydroxyacetone phosphate, acetyl-CoA and some Krebs cycle intermediates in liver. Hyperglycemic LKO mouse liver showed elevated expression of proinflammatory cytokines and M1-macrophage markers accompanied by increased plasma triglycerides, LDL/VLDL, urea and uric acid and myocardial triglycerides.
CONCLUSIONS: The glycerol shunt orchestrated by G3PP acts as a glucose excess detoxification pathway in hepatocytes by preventing metabolic disturbances that contribute to enhanced liver fat, glycogen storage, inflammation and lipid build-up in the heart. We propose G3PP as a novel therapeutic target for hepatic disorders linked to nutrient excess.

Keywords:  Glucodetoxification; Glycerol shunt; Glycerol-3-phosphate phosphatase; Hepatocytes; Liver; NAFLD; cholesterol; glycogen; inflammation; lipogenesis; triglycerides

DOI:  https://doi.org/10.1016/j.molmet.2022.101609
Cell Metab. 2022 Sep 28. pii: S1550-4131(22)00395-3. [Epub ahead of print]

Two independent respiratory chains adapt OXPHOS performance to glycolytic switch.

Erika Fernández-Vizarra, Sandra López-Calcerrada, Ana Sierra-Magro, Rafael Pérez-Pérez, Luke E Formosa, Daniella H Hock, María Illescas, Ana Peñas, Michele Brischigliaro, Shujing Ding, Ian M Fearnley, Charalampos Tzoulis, Robert D S Pitceathly, Joaquín Arenas, Miguel A Martín, David A Stroud, Massimo Zeviani, Michael T Ryan, Cristina Ugalde.

  The structural and functional organization of the mitochondrial respiratory chain (MRC) remains intensely debated. Here, we show the co-existence of two separate MRC organizations in human cells and postmitotic tissues, C-MRC and S-MRC, defined by the preferential expression of three COX7A subunit isoforms, COX7A1/2 and SCAFI (COX7A2L). COX7A isoforms promote the functional reorganization of distinct co-existing MRC structures to prevent metabolic exhaustion and MRC deficiency. Notably, prevalence of each MRC organization is reversibly regulated by the activation state of the pyruvate dehydrogenase complex (PDC). Under oxidative conditions, the C-MRC is bioenergetically more efficient, whereas the S-MRC preferentially maintains oxidative phosphorylation (OXPHOS) upon metabolic rewiring toward glycolysis. We show a link between the metabolic signatures converging at the PDC and the structural and functional organization of the MRC, challenging the widespread notion of the MRC as a single functional unit and concluding that its structural heterogeneity warrants optimal adaptation to metabolic function.

Keywords:  COX7A1–2; SCAFI/COX7RP/COX7A2L; bioenergetics; glycolysis; metabolic switch; mitochondria; oxidative metabolism; pyruvate dehydrogenase; respiratory chain organizations; respiratory supercomplexes

DOI:  https://doi.org/10.1016/j.cmet.2022.09.005
Commun Biol. 2022 Oct 05. 5(1): 1060

Inter-organellar and systemic responses to impaired mitochondrial matrix protein import in skeletal muscle.

Nirajan Neupane, Jayasimman Rajendran, Jouni Kvist, Sandra Harjuhaahto, Bowen Hu, Veijo Kinnunen, Yang Yang, Anni I Nieminen, Henna Tyynismaa.

Effective protein import from cytosol is critical for mitochondrial functions and metabolic regulation. We describe here the mammalian muscle-specific and systemic consequences to disrupted mitochondrial matrix protein import by targeted deletion of the mitochondrial HSP70 co-chaperone GRPEL1. Muscle-specific loss of GRPEL1 caused rapid muscle atrophy, accompanied by shut down of oxidative phosphorylation and mitochondrial fatty acid oxidation, and excessive triggering of proteotoxic stress responses. Transcriptome analysis identified new responders to mitochondrial protein import toxicity, such as the neurological disease-linked intermembrane space protein CHCHD10. Besides communication with ER and nucleus, we identified crosstalk of distressed mitochondria with peroxisomes, in particular the induction of peroxisomal Acyl-CoA oxidase 2 (ACOX2), which we propose as an ATF4-regulated peroxisomal marker of integrated stress response. Metabolic profiling indicated fatty acid enrichment in muscle, a shift in TCA cycle intermediates in serum and muscle, and dysregulated bile acids. Our results demonstrate the fundamental importance of GRPEL1 and provide a robust model for detecting mammalian inter-organellar and systemic responses to impaired mitochondrial matrix protein import and folding.

DOI: https://doi.org/10.1038/s42003-022-04034-z
Acta Crystallogr F Struct Biol Commun. 2022 Oct 01. 78(Pt 10): 363-370

The structure of succinyl-CoA synthetase bound to the succinyl-phosphate intermediate clarifies the catalytic mechanism of ATP-citrate lyase.

Ji Huang, Marie E Fraser.

  Succinyl-CoA synthetase (SCS) catalyzes a three-step reaction in the citric acid cycle with succinyl-phosphate proposed as a catalytic intermediate. However, there are no structural data to show the binding of succinyl-phosphate to SCS. Recently, the catalytic mechanism underlying acetyl-CoA production by ATP-citrate lyase (ACLY) has been debated. The enzyme belongs to the family of acyl-CoA synthetases (nucleoside diphosphate-forming) for which SCS is the prototype. It was postulated that the amino-terminal portion catalyzes the full reaction and the carboxy-terminal portion plays only an allosteric role. This interpretation was based on the partial loss of the catalytic activity of ACLY when Glu599 was mutated to Gln or Ala, and on the interpretation that the phospho-citryl-CoA intermediate was trapped in the 2.85 Å resolution structure from cryogenic electron microscopy (cryo-EM). To better resolve the structure of the intermediate bound to the E599Q mutant, the equivalent mutation, E105αQ, was made in human GTP-specific SCS. The structure of the E105αQ mutant shows succinyl-phosphate bound to the enzyme at 1.58 Å resolution when the mutant, after phosphorylation in solution by Mg2+-ATP, was crystallized in the presence of magnesium ions, succinate and desulfo-CoA. The E105αQ mutant is still active but has a specific activity that is 120-fold less than that of the wild-type enzyme, with apparent Michaelis constants for succinate and CoA that are 50-fold and 11-fold higher, respectively. Based on this high-resolution structure, the cryo-EM maps of the E599Q ACLY complex reported previously should have revealed the binding of citryl-phosphate and CoA and not phospho-citryl-CoA.

Keywords:  ATP-citrate lyase; catalytic intermediates; cryogenic electron microscopy; succinyl-CoA synthetase; succinyl-phosphate

DOI:  https://doi.org/10.1107/S2053230X22008810
Nat Commun. 2022 Oct 02. 13(1): 5782

CD36-mediated metabolic crosstalk between tumor cells and macrophages affects liver metastasis.

Ping Yang, Hong Qin, Yiyu Li, Anhua Xiao, Enze Zheng, Han Zeng, Chunxiao Su, Xiaoqing Luo, Qiannan Lu, Meng Liao, Lei Zhao, Li Wei, Zac Varghese, John F Moorhead, Yaxi Chen, Xiong Z Ruan.

Liver metastasis is highly aggressive and treatment-refractory, partly due to macrophage-mediated immune suppression. Understanding the mechanisms leading to functional reprogramming of macrophages in the tumor microenvironment (TME) will benefit cancer immunotherapy. Herein, we find that the scavenger receptor CD36 is upregulated in metastasis-associated macrophages (MAMs) and deletion of CD36 in MAMs attenuates liver metastasis in mice. MAMs contain more lipid droplets and have the unique capability in engulfing tumor cell-derived long-chain fatty acids, which are carried by extracellular vesicles. The lipid-enriched vesicles are preferentially partitioned into macrophages via CD36, that fuel macrophages and trigger their tumor-promoting activities. In patients with liver metastases, high expression of CD36 correlates with protumoral M2-type MAMs infiltration, creating a highly immunosuppressive TME. Collectively, our findings uncover a mechanism by which tumor cells metabolically interact with macrophages in TME, and suggest a therapeutic potential of targeting CD36 as immunotherapy for liver metastasis.

DOI: https://doi.org/10.1038/s41467-022-33349-y
Biochim Biophys Acta Mol Basis Dis. 2022 Sep 28. pii: S0925-4439(22)00235-6. [Epub ahead of print] 166564

Adipocyte coculture induces a pro-inflammatory, fibrotic, angiogenic, and proliferative microenvironment in uterine leiomyoma cells.

Sadia Afrin, Malak El Sabah, Ahmed Manzoor, Mariko Miyashita-Ishiwata, Lauren Reschke, Mostafa A Borahay.

   OBJECTIVE: Obesity and its consequences are among the biggest challenges facing the healthcare system. Uterine leiomyomas are the most common gynecologic tumors. The risk of leiomyoma increases with obesity, but the underlying mechanisms of this association remain unclear. The aim of the present study to determine the cellular and molecular mechanisms by which adipocyte contributes to both leiomyoma tumor initiation and promotion.
METHODS: Primary myometrium and leiomyoma cells were isolated from patients who underwent a hysterectomy or myomectomy. Pro-inflammatory, fibrotic, and angiogenic factors were measured using a multiplex cytokine array in human primary and immortalized myometrial and leiomyoma cells cocultured with human adipocyte (SW872) cells, or in animal ELT3 leiomyoma cells cocultured with 3 T3-L1 adipocytes. The free fatty acids (FFAs) and fatty acid-binding protein 4 (FABP4) levels were measured using immunofluorescence assays. Other protein abundances were determined using western blots. The expression levels of TNF-α, MCP-1, phospho-NF-κB, TGFβ3 and VEGF-A in lean and obese in different leiomyoma patients were determined by immunofluorescence staining.
RESULTS: Adipocytes promote inflammation, fibrosis, and angiogenesis in uterine leiomyoma cells by upregulating associated factors, such as IL-1β, TNF-α, MCP-1, GM-CSF, TGF-βs, PLGF, VEGF, HB-EGF, G-CSF and FGF2. Coculture led to the transfer of FFAs and FABP4 from adipocytes to leiomyoma cells, suggesting that adipocytes may modulate metabolic activity in these tumor cells. Increased levels of FFA and FABP4 expressions were detected in obese leiomyoma tissue compared to lean. The adipocyte-leiomyoma cell interaction increased the phospho-NF-κB level, which plays a key role in inflammation, restructuring metabolic pathways, and angiogenesis. Obese leiomyoma patients expressed a higher amount of TNF-α, MCP-1, phospho-NF-κB, TGFβ3 and VEGF-A than lean leiomyoma patients, consistent with in vitro findings. Furthermore, we found that adipocyte secretory factors enhance leiomyoma cell proliferation by increasing PCNA abundance. Finally, the inhibition of the inflammatory factors TNF-α, MCP-1, and NF-κB abrogated the adipocyte coculture-induced proliferation of leiomyoma cells.
CONCLUSIONS: Adipocytes release inflammatory, fibrotic, and angiogenic factors, along with FFAs, which contribute to a tumor-friendly microenvironment that may promote leiomyoma growth and can represent a new target for leiomyoma prevention and treatment.

Keywords:  Adipocyte; Angiogenesis; Fibrosis; Inflammation; Proliferation; Uterine leiomyoma

DOI:  https://doi.org/10.1016/j.bbadis.2022.166564
J Biol Chem. 2022 Oct 02. pii: S0021-9258(22)01005-5. [Epub ahead of print] 102561

Aurora A-mediated pyruvate kinase M2 phosphorylation promotes biosynthesis with glycolytic metabolites and tumor cell cycle progression.

Ya Jiang, Ting Wang, Dandan Sheng, Chaoqiang Han, Tian Xu, Peng Zhang, Weiyi You, Weiwei Fan, Zhiyong Zhang, Tengchuan Jin, Xiaotao Duan, Xiao Yuan, Xing Liu, Kaiguang Zhang, Ke Ruan, Jue Shi, Jing Guo, Aoxing Cheng, Zhenye Yang.

  Cancer cells have distinctive demands for intermediates from glucose metabolism for biosynthesis and energy in different cell cycle phases. However, how cell cycle regulators and glycolytic enzymes coordinate to orchestrate the essential metabolic processes are still poorly characterized. Here, we report a novel interaction between the mitotic kinase, Aurora A, and the glycolytic enzyme, pyruvate kinase M2 (PKM2), in the interphase of the cell cycle. We found Aurora A mediated phosphorylation of PKM2 at threonine 45. This phosphorylation significantly attenuated PKM2 enzymatic activity by reducing its tetramerization, and also promoted glycolytic flux and the branching anabolic pathways. Replacing the endogenous PKM2 with a non-phosphorylated PKM2 T45A mutant inhibited glycolysis, glycolytic branching pathways, and tumor growth in both in vitro and in vivo models. Together, our study revealed a new pro-tumor function of Aurora A through modulating a rate-limiting glycolytic enzyme, PKM2, mainly during the S phase of the cell cycle. Our findings also showed that although both Aurora A and Aurora B kinase phosphorylate PKM2 at the same residue, the spatial and temporal regulations of the specific kinase and PKM2 interaction are context-dependent, indicating intricate interconnectivity between cell cycle and glycolytic regulators.

Keywords:  Aurora A; PKM2; biosynthesis; cell cycle progression; phosphorylation

DOI:  https://doi.org/10.1016/j.jbc.2022.102561
Proc Natl Acad Sci U S A. 2022 Oct 11. 119(41): e2203628119

Succinyl-CoA-based energy metabolism dysfunction in chronic heart failure.

Shingo Takada, Satoshi Maekawa, Takaaki Furihata, Naoya Kakutani, Daiki Setoyama, Koji Ueda, Hideo Nambu, Hikaru Hagiwara, Haruka Handa, Yoshizuki Fumoto, Soichiro Hata, Tomoka Masunaga, Arata Fukushima, Takashi Yokota, Dongchon Kang, Shintaro Kinugawa, Hisataka Sabe.

  Heart failure (HF) is a leading cause of death and repeated hospitalizations and often involves cardiac mitochondrial dysfunction. However, the underlying mechanisms largely remain elusive. Here, using a mouse model in which myocardial infarction (MI) was induced by coronary artery ligation, we show the metabolic basis of mitochondrial dysfunction in chronic HF. Four weeks after ligation, MI mice showed a significant decrease in myocardial succinyl-CoA levels, and this decrease impaired the mitochondrial oxidative phosphorylation (OXPHOS) capacity. Heme synthesis and ketolysis, and protein levels of several enzymes consuming succinyl-CoA in these events, were increased in MI mice, while enzymes synthesizing succinyl-CoA from α-ketoglutarate and glutamate were also increased. Furthermore, the ADP-specific subunit of succinyl-CoA synthase was reduced, while its GDP-specific subunit was almost unchanged. Administration of 5-aminolevulinic acid, an intermediate in the pathway from succinyl-CoA to heme synthesis, appreciably restored succinyl-CoA levels and OXPHOS capacity and prevented HF progression in MI mice. Previous reports also suggested the presence of succinyl-CoA metabolism abnormalities in cardiac muscles of HF patients. Our results identified that changes in succinyl-CoA usage in different metabolisms of the mitochondrial energy production system is characteristic to chronic HF, and although similar alterations are known to occur in healthy conditions, such as during strenuous exercise, they may often occur irreversibly in chronic HF leading to a decrease in succinyl-CoA. Consequently, nutritional interventions compensating the succinyl-CoA consumption are expected to be promising strategies to treat HF.

Keywords:  5-aminolevulinic acid; heart failure; mitochondria; oxidative phosphorylation; succinyl-CoA

DOI:  https://doi.org/10.1073/pnas.2203628119
STAR Protoc. 2022 Sep 30. pii: S2666-1667(22)00618-9. [Epub ahead of print]3(4): 101738

Preparation of mitochondrial damage-associated molecular patterns from mouse liver tissue.

Lauren P Westhaver, Sarah Nersesian, Adam Nelson, Leah K MacLean, Emily B Carter, Jeanette E Boudreau.

  Mitochondrial damage-associated molecular patterns (mitoDAMPs) are released from cells dying uncontrolled, non-apoptotic deaths, usually secondary to disease or trauma. Here, we describe preparation of mitoDAMPs from mouse liver, but this protocol can be adapted for preparation of mitoDAMPs from other species and tissues. Tissues are dissociated and then processed to isolate mitochondria. Mitochondria are then sonicated and mitoDAMPs are collected by ultracentrifugation. This procedure produces μg quantities of mitoDAMPs and facilitates research to understand their impacts in health and disease. For complete details on the use and execution of this protocol, please refer to Westhaver et al. (2022).

Keywords:  Cell Biology; Cell isolation; Cell separation/fractionation; Cell-based Assays; Immunology; Metabolomics

DOI:  https://doi.org/10.1016/j.xpro.2022.101738
Nat Cell Biol. 2022 Oct 06.

Nuclear TCA cycle reactions.

Melina Casadio.

DOI: https://doi.org/10.1038/s41556-022-01006-7
iScience. 2022 Oct 21. 25(10): 105138

Cholesterol-induced robust Ca oscillation in astrocytes required for survival and lipid droplet formation in high-cholesterol condition.

Chihiro Adachi, Shio Otsuka, Takafumi Inoue.

  Cholesterol, one of the major cell membrane components, stabilizes membrane fluidity and regulates signal transduction. Beside its canonical roles, cholesterol has been reported to directly activate signaling pathways such as hedgehog (Hh). We recently found that astrocytes, one of the glial cells, respond to Hh pathway stimulation by Ca signaling. These notions led us to test if extracellularly applied cholesterol triggers Ca signaling in astrocytes. Here, we found that cholesterol application induces robust Ca oscillation only in astrocytes with different properties from the Hh-induced Ca response. The Ca oscillation has a long delay which corresponds to the onset of cholesterol accumulation in the plasma membrane. Blockade of the Ca oscillation resulted in enhancement of astrocytic cell death and disturbance of lipid droplet formation, implying a possibility that the cholesterol-induced Ca oscillation plays important roles in astrocytic survival and cholesterol handling under pathological conditions of cholesterol load such as demyelination.

Keywords:  Biological sciences; Molecular neuroscience; Neuroscience

DOI:  https://doi.org/10.1016/j.isci.2022.105138