bims-mepmim Biomed News
on Metabolites in pathological microenvironments and immunometabolism
Issue of 2022‒08‒28
29 papers selected by
Erika Mariana Palmieri
NIH/NCI Laboratory of Cancer ImmunoMetabolism

  1. Cancer Gene Ther. 2022 Aug 23.
      Metabolic reprogramming is a hallmark of cancer development, progression, and metastasis. Several metabolic pathways such as glycolysis, tricarboxylic acid (TCA) cycle, lipid metabolism, and glutamine catabolism are frequently altered to support cancer growth. Importantly, the activity of the rate-limiting metabolic enzymes in these pathways are specifically modulated in cancer cells. This is achieved by transcriptional, translational, and post translational regulations that enhance the expression, activity, stability, and substrate sensitivity of the rate-limiting enzymes. These mechanisms allow the enzymes to retain increased activity supporting the metabolic needs of rapidly growing tumors, sustain their survival in the hostile tumor microenvironments and in the metastatic lesions. In this review, we primarily focused on the post translational modifications of the rate-limiting enzymes in the glucose and glutamine metabolism, TCA cycle, and fatty acid metabolism promoting tumor progression and metastasis.
  2. Mol Cancer Res. 2022 Aug 22. pii: MCR-21-1032. [Epub ahead of print]
      Acute myeloid leukemia (AML) is a hematological malignancy metabolically dependent on oxidative phosphorylation and mitochondrial electron transport chain (ETC) activity. AML cells are distinct from their normal hematopoietic counterparts by this metabolic reprogramming, which presents targets for new selective therapies. Here, metabolic changes in AML cells after ETC impairment are investigated. Genetic knockdown of the ETC complex II (CII) chaperone protein SDHAF1 (succinate dehydrogenase assembly factor 1) suppressed CII activity and delayed AML cell growth in vitro and in vivo. As a result, a novel small molecule that directly binds to the ubiquinone binding site of CII and inhibits its activity was identified. Pharmacological inhibition of CII induced selective cell death in AML cells while sparing normal hematopoietic progenitors. Through stable isotope tracing, results show that genetic or pharmacological inhibition of CII truncates the tricarboxylic acid cycle (TCA) and leads to anaplerotic glutamine metabolism to reestablish the truncated cycle. The inhibition of CII showed divergent fates of AML cells since they lacked the metabolic plasticity to adequately utilize glutamine metabolism, resulting in preferential depletion of key metabolites in the TCA cycle and death; normal cells were unaffected. These findings provide insight into the metabolic mechanisms that underlie AML's selective inhibition of CII. Implications: This work highlights the effects of direct CII inhibition in mediating selective AML cell death and provides insights into glutamine anaplerosis as a metabolic adaptation that can be therapeutically targeted.
  3. BMB Rep. 2022 Aug 23. pii: 5675. [Epub ahead of print]
      Ferroptosis is a type of programmed cell death distinct from apoptosis or necroptosis. Ferroptosis is well characterized by an iron-dependent accumulation of lipid peroxides and disruption of cellular membrane integrity. Many metabolic alterations can prevent or accelerate ferroptosis induction. Recent advances in analytical techniques of mass spectrometry have allowed high-throughput analysis of metabolites known to be critical for understanding ferroptosis regulatory metabolism. In this review, we introduce mass spectrometry-based analytical methods contributing to recent discovery of various metabolic pathways regulating ferroptosis, focusing on cysteine metabolism, antioxidant metabolism, and poly-unsaturated fatty acid metabolism.
  4. Proc Natl Acad Sci U S A. 2022 Aug 30. 119(35): e2211310119
      Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Astrocytes are the most abundant glial cells in the CNS, and their dysfunction contributes to the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). Recent advances highlight the pivotal role of cellular metabolism in programming immune responses. However, the underlying immunometabolic mechanisms that drive astrocyte pathogenicity remain elusive. Nicotinamide adenine dinucleotide (NAD+) is a vital coenzyme involved in cellular redox reactions and a substrate for NAD+-dependent enzymes. Cellular NAD+ levels are dynamically controlled by synthesis and degradation, and dysregulation of this balance has been associated with inflammation and disease. Here, we demonstrate that cell-autonomous generation of NAD+ via the salvage pathway regulates astrocyte immune function. Inhibition of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the salvage pathway, results in depletion of NAD+, inhibits oxidative phosphorylation, and limits astrocyte inflammatory potential. We identified CD38 as the main NADase up-regulated in reactive mouse and human astrocytes in models of neuroinflammation and MS. Genetic or pharmacological blockade of astrocyte CD38 activity augmented NAD+ levels, suppressed proinflammatory transcriptional reprogramming, impaired chemotactic potential to inflammatory monocytes, and ameliorated EAE. We found that CD38 activity is mediated via calcineurin/NFAT signaling in mouse and human reactive astrocytes. Thus, NAMPT-NAD+-CD38 circuitry in astrocytes controls their ability to meet their energy demands and drives the expression of proinflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, MS. Our results identify candidate therapeutic targets in MS.
    Keywords:  Nicotinamide adenine dinucleotide; astrocyte; multiple sclerosis; neuroinflammation; tryptophan catabolism
  5. Biochimie. 2022 Aug 20. pii: S0300-9084(22)00216-4. [Epub ahead of print]
      The central control of energy homeostasis is a regulatory axis that involves the sensing of nutrients, signaling molecules, adipokines, and neuropeptides by neurons in the metabolic centers of the hypothalamus. However, non-neuronal glial cells are also abundant in the hypothalamus and recent findings have underscored the importance of the metabolic crosstalk and horizontal lipid flux between glia and neurons to the downstream regulation of systemic metabolism. New transgenic models and high-resolution analyses of glial phenotype and function has revealed that glia sit at the nexus between lipid metabolism and neural function, and therefore may markedly impact the brain's response to dietary lipids or the supply of brain-derived lipids. Glia comprise the main cellular compartment involved in lipid synthesis, lipoprotein production, and lipid processing in the brain. In brief, tanycytes provide an interface between peripheral lipids and neurons, astrocytes produce lipoproteins that transport lipids to neurons and other glia, oligodendrocytes use brain-derived and dietary lipids to myelinate axons and influence neuronal function, while microglia can remove unwanted lipids in the brain and contribute to lipid re-utilization through cholesterol efflux. Here, we review recent findings regarding glial-lipid transport and highlight the specific molecular factors necessary for lipid processing in the brain and, and how dysregulation of glial-neuronal metabolic crosstalk contributes to imbalanced energy homeostasis. Furthering our understanding of glial lipid metabolism will guide the design of future studies that target horizontal lipid processing in the brain to ameliorate the risk of developing obesity and metabolic disease.
    Keywords:  Astrocytes; Fatty acids; Glia; Lipid; Metabolism; Microglia; Neurons; Tanycytes
  6. Nat Microbiol. 2022 Sep;7(9): 1348-1360
      Urinary tract infections are among the most common human bacterial infections and place a significant burden on healthcare systems due to associated morbidity, cost and antibiotic use. Despite being a facultative anaerobe, uropathogenic Escherichia coli, the primary cause of urinary tract infections, requires aerobic respiration to establish infection in the bladder. Here, by combining bacterial genetics with cell culture and murine models of infection, we demonstrate that the widely conserved respiratory quinol oxidase cytochrome bd is required for intracellular infection of urothelial cells. Through a series of genetic, biochemical and functional assays, we show that intracellular oxygen scavenging by cytochrome bd alters mitochondrial physiology by reducing the efficiency of mitochondrial respiration, stabilizing the hypoxia-inducible transcription factor HIF-1 and promoting a shift towards aerobic glycolysis. This bacterially induced rewiring of host metabolism antagonizes apoptosis, thereby protecting intracellular bacteria from urothelial cell exfoliation and preserving their replicative niche. These results reveal the metabolic basis for intracellular bacterial pathogenesis during urinary tract infection and identify subversion of mitochondrial metabolism as a bacterial strategy to facilitate persistence within the urinary tract.
  7. J Biol Chem. 2022 Aug 18. pii: S0021-9258(22)00844-4. [Epub ahead of print] 102401
      Hepatic steatosis associated with high fat diets (HFD), obesity and type 2 diabetes is thought to be the major driver of severe liver inflammation, fibrosis, and cirrhosis. Cytosolic acetyl-coenzyme A (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 is 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, while 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 (SREBP1c) 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 due to its increased conversion to Malonyl CoA (MalCoA) and palmitate. Together, these data indicate that in HFD-fed obese mice, hepatic DNL is not limited by its immediate substrates AcCoA or MalCoA, but rather by activities of DNL enzymes.
  8. Cell Signal. 2022 Aug 18. pii: S0898-6568(22)00205-4. [Epub ahead of print] 110443
      Recent studies have reported that Angiotensin II (Ang II) contributes to podocyte injury by interfering with metabolism. Glycolysis is essential for podocytes and glycolysis abnormality is associated with glomerular injury in chronic kidney disease (CKD). Glycerol-3-phosphate (G-3-P) biosynthesis is a shunt pathway of glycolysis, in which cytosolic glycerol-3-phosphate dehydrogenase 1 (GPD1) catalyzes dihydroxyacetone phosphate (DHAP) to generate G-3-P in the presence of the NADH. G-3-P is not only a substrate in glycerophospholipids and glyceride synthesis but also can be oxidated by mitochondrial glycerol-3-phosphate dehydrogenase (GPD2) to regenerate DHAP in mitochondria. Since G-3-P biosynthesis links to glycolysis, mitochondrial metabolism and lipid synthesis, we speculate G-3-P biosynthesis abnormality is probably involved in podocyte injury. In this study, we demonstrated that Ang II upregulated GPD1 expression and increased G-3-P and glycerophospholipid syntheses in podocytes. GPD1 knockdown protected podocytes from Ang II-induced lipid accumulation and mitochondrial dysfunction. GPD1 overexpression exacerbated Ang II-induced podocyte injury. In addition, we proved that lipid accumulation and mitochondrial dysfunction were correlated with G-3-P content in podocytes. These results suggest that Ang II upregulates GPD1 and promotes G-3-P biosynthesis in podocytes, which promote lipid accumulation and mitochondrial dysfunction in podocytes.
    Keywords:  Angiotensin II; GPD1; Glycerol-3-phosphate; Lipid accumulation; Mitochondrial dysfunction; Podocyte
  9. J Mass Spectrom. 2022 Aug;57(8): e4880
      Altered lipid metabolism is one of the hallmarks of cancer. Cellular proliferation and de novo synthesis of lipids are related to cancer progression. In this study, we evaluated the lipidomic profile of two-dimensional (2D) monolayer and multicellular tumor spheroids from the HCT 116 colon carcinoma cell line. We utilized serial trypsinization on the spheroid samples to generate three cellular populations representing the proliferative, quiescent, and necrotic regions of the spheroid. This analysis enabled a comprehensive identification and quantification of lipids produced in each of the spheroid layer and 2D cultures. We show that lipid subclasses associated with lipid droplets form in oxygen-restricted and acidic regions of spheroids and are produced at higher levels than in 2D cultures. Additionally, sphingolipid production, which is implicated in cell death and survival pathways, is higher in spheroids relative to 2D cells. Finally, we show that increased numbers of lipids composed of polyunsaturated fatty acids (PUFAs) are produced in the quiescent and necrotic regions of the spheroid. The lipidomic signature for each region and cell culture type highlights the importance of understanding the spatial aspects of cancer biology. These results provide additional lipid biomarkers in colon cancer cells that can be further studied to target pivotal lipid production pathways.
    Keywords:  TME; acidosis; cancer; fatty acid/metabolism; hypoxia; lipid droplets; mass spectrometry; serial trypsinization; spheroids; triacylglycerol
  10. Int J Mol Sci. 2022 Aug 14. pii: 9114. [Epub ahead of print]23(16):
      Atherosclerosis is a cardiovascular disease caused mainly by dyslipidemia and is characterized by the formation of an atheroma plaque and chronic inflammation. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that induces the degradation of the LDL receptor (LDLR), which contributes to increased levels of LDL cholesterol and the progress of atherosclerosis. Given that macrophages are relevant components of the lipidic and inflammatory environment of atherosclerosis, we studied the effects of PCSK9 treatment on human macrophages. Our data show that human macrophages do not express PCSK9 but rapidly incorporate the circulating protein through the LDLR and also activate the pro-inflammatory TLR4 pathway. Both LDLR and TLR4 are internalized after incubation of macrophages with exogenous PCSK9. PCSK9 uptake increases the production of reactive oxygen species and reduces the expression of genes involved in lipid metabolism and cholesterol efflux, while enhancing the production of pro-inflammatory cytokines through a TLR4-dependent mechanism. Under these conditions, the viability of macrophages is compromised, leading to increased cell death. These results provide novel insights into the role of PCSK9 in the crosstalk of lipids and cholesterol metabolism through the LDLR and on the pro-inflammatory activation of macrophages through TLR4 signaling. These pathways are relevant in the outcome of atherosclerosis and highlight the relevance of PCSK9 as a therapeutic target for the treatment of cardiovascular diseases.
    Keywords:  LDL; PCSK9; ROS; TLR4; atherosclerosis; cholesterol; macrophage
  11. PNAS Nexus. 2022 Jul;1(3): pgac142
      Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by the deficiency of mitochondrial protein frataxin, which plays a crucial role in iron-sulphur cluster formation and ATP production. The cellular function of frataxin is not entirely known. Here, we demonstrate that frataxin controls ketone body metabolism through regulation of 3-Oxoacid CoA-Transferase 1 (OXCT1), a rate limiting enzyme catalyzing the conversion of ketone bodies to acetoacetyl-CoA that is then fed into the Krebs cycle. Biochemical studies show a physical interaction between frataxin and OXCT1 both in vivo and in vitro. Frataxin overexpression also increases OXCT1 protein levels in human skin fibroblasts while frataxin deficiency decreases OXCT1 in multiple cell types including cerebellum and skeletal muscle both acutely and chronically, suggesting that frataxin directly regulates OXCT1. This regulation is mediated by frataxin-dependent suppression of ubiquitin-proteasome system (UPS)-dependent OXCT1 degradation. Concomitantly, plasma ketone bodies are significantly elevated in frataxin deficient knock-in/knockout (KIKO) mice with no change in the levels of other enzymes involved in ketone body production. In addition, ketone bodies fail to be metabolized to acetyl-CoA accompanied by increased succinyl-CoA in vitro in frataxin deficient cells, suggesting that ketone body elevation is caused by frataxin-dependent reduction of OXCT1 leading to deficits in tissue utilization of ketone bodies. Considering the potential role of metabolic abnormalities and deficiency of ATP production in FRDA, our results suggest a new role for frataxin in ketone body metabolism and also suggest modulation of OXCT1 may be a potential therapeutic approach for FRDA.
    Keywords:  Friedreich's ataxia; OXCT1; frataxin; ketone body
  12. Metabolites. 2022 Aug 18. pii: 760. [Epub ahead of print]12(8):
      Glycogen is a readily deployed intracellular energy storage macromolecule composed of branched chains of glucose anchored to the protein glycogenin. Although glycogen primarily occurs in the liver and muscle, it is found in most tissues, and its metabolism has been shown to be important in cancers and immune cells. Robust analysis of glycogen turnover requires stable isotope tracing plus a reliable means of quantifying total and labeled glycogen derived from precursors such as 13C6-glucose. Current methods for analyzing glycogen are time- and sample-consuming, at best semi-quantitative, and unable to measure stable isotope enrichment. Here we describe a microscale method for quantifying both intact and acid-hydrolyzed glycogen by ultra-high-resolution Fourier transform mass spectrometric (UHR-FTMS) and/or NMR analysis in stable isotope resolved metabolomics (SIRM) studies. Polar metabolites, including intact glycogen and their 13C positional isotopomer distributions, are first measured in crude biological extracts by high resolution NMR, followed by rapid and efficient acid hydrolysis to glucose under N2 in a focused beam microwave reactor, with subsequent analysis by UHR-FTMS and/or NMR. We optimized the microwave digestion time, temperature, and oxygen purging in terms of recovery versus degradation and found 10 min at 110-115 °C to give >90% recovery. The method was applied to track the fate of 13C6-glucose in primary human lung BEAS-2B cells, human macrophages, murine liver and patient-derived tumor xenograft (PDTX) in vivo, and the fate of 2H7-glucose in ex vivo lung organotypic tissue cultures of a lung cancer patient. We measured the incorporation of 13C6-glucose into glycogen and its metabolic intermediates, UDP-Glucose and glucose-1-phosphate, to demonstrate the utility of the method in tracing glycogen turnover in cells and tissues. The method offers a quantitative, sensitive, and convenient means to analyze glycogen turnover in mg amounts of complex biological materials.
    Keywords:  13C6-glucose; glycogen turnover; microwave-assisted hydrolysis; stable isotope resolved metabolomics (SIRM)
  13. Antioxidants (Basel). 2022 Jul 29. pii: 1487. [Epub ahead of print]11(8):
      α-ketoglutarate dehydrogenase complex (KGDHc), or 2-oxoglutarate dehydrogenase complex (OGDHc) is a rate-limiting enzyme in the tricarboxylic acid cycle, that has been identified in neurodegenerative diseases such as in Alzheimer's disease. The aim of the present study was to establish the role of the KGDHc and its subunits in the bioenergetics and reactive oxygen species (ROS) homeostasis of brain mitochondria. To study the bioenergetic profile of KGDHc, genetically modified mouse strains were used having a heterozygous knock out (KO) either in the dihydrolipoyl succinyltransferase (DLST+/-) or in the dihydrolipoyl dehydrogenase (DLD+/-) subunit. Mitochondrial oxygen consumption, hydrogen peroxide (H2O2) production, and expression of antioxidant enzymes were measured in isolated mouse brain mitochondria. Here, we demonstrate that the ADP-stimulated respiration of mitochondria was partially arrested in the transgenic animals when utilizing α-ketoglutarate (α-KG or 2-OG) as a fuel substrate. Succinate and α-glycerophosphate (α-GP), however, did not show this effect. The H2O2 production in mitochondria energized with α-KG was decreased after inhibiting the adenine nucleotide translocase and Complex I (CI) in the transgenic strains compared to the controls. Similarly, the reverse electron transfer (RET)-evoked H2O2 formation supported by succinate or α-GP were inhibited in mitochondria isolated from the transgenic animals. The decrease of RET-evoked ROS production by DLST+/- or DLD+/- KO-s puts the emphasis of the KGDHc in the pathomechanism of ischemia-reperfusion evoked oxidative stress. Supporting this notion, expression of the antioxidant enzyme glutathione peroxidase was also decreased in the KGDHc transgenic animals suggesting the attenuation of ROS-producing characteristics of KGDHc. These findings confirm the contribution of the KGDHc to the mitochondrial ROS production and in the pathomechanism of ischemia-reperfusion injury.
    Keywords:  DLD; DLST; KGDHc; OGDHc; RET; ROS; antioxidant systems; cellular respiration; ischemia-reperfusion; mitochondria; oxoglutarate dehydrogenase complex; reactive oxygen species; reverse electron transfer; succinate; transgenic animal; α-glycerophosphate; α-ketoglutarate dehydrogenase complex
  14. Front Bioeng Biotechnol. 2022 ;10 943906
      Cancer cells reprogram their metabolism to meet their growing demand for bioenergy and biosynthesis. The metabolic profile of cancer cells usually includes dysregulation of main nutritional metabolic pathways and the production of metabolites, which leads to a tumor microenvironment (TME) having the characteristics of acidity, hypoxic, and/or nutrient depletion. Therapies targeting metabolism have become an active and revolutionary research topic for anti-cancer drug development. The differential metabolic vulnerabilities between tumor cells and other cells within TME provide nanotechnology a therapeutic window of anti-cancer. In this review, we present the metabolic characteristics of intrinsic cancer cells and TME and summarize representative strategies of nanoparticles in metabolism-regulating anti-cancer therapy. Then, we put forward the challenges and opportunities of using nanoparticles in this emerging field.
    Keywords:  cancer treatment; metabolic reprograming; metabolism; nanomedicine; tumor microenvironment
  15. NMR Biomed. 2022 Aug 23. e4817
      Advanced imaging technologies, large-scale metabolomics and the measurement of gene transcripts or enzyme expression all enable investigations of intermediary metabolism in human patients. Complementary information about fluxes in individual metabolic pathways may be obtained by ex vivo 13 C NMR of blood or tissue biopsies. Simple molecules such as 13 C-labeled glucose are readily administered to patients prior to surgical biopsies, and 13 C-labeled glycerol is easily administered orally to outpatients. Here we review recent progress in practical applications of 13 C NMR to study cancer biology, the response to oxidative stress, gluconeogenesis, triglyceride synthesis in patients, as well as new insights into compartmentation of metabolism in the cytosol. The technical aspects of obtaining the sample, preparing material for analysis, and acquiring the spectra are relatively simple. This approach enables convenient, valuable and quantitative insights into intermediary metabolism in patients.
    Keywords:  13C; NMR; cancer; glucose; glycerol; metabolic syndrome; stable isotope
  16. Acta Biochim Pol. 2022 Aug 26.
      BACKGROUND: Colorectal cancer is the most-incidence associated extremely high mortality rate worldwide. The overexpression of estrogen-related receptor α (ERRα) is contributing to a poor prognosis. Obtaining a better understanding of the mechanisms of ERRα in colorectal cancer is important for developing cancer therapies.METHODS: Western blotting and qRT-PCR were used to determine the protein and mRNA levels of ERRα, OUTB1, and solute carrier family 7 member 11 (SLC7A11) in HCT-116 cells. Short hairpin RNA (shRNA) was used to knockdown ERRα in HCT-116 cells. The level of reactive oxygen species (ROS), the nicotinamide adenine dinucleotide phosphate NADP+/NADPH, and the oxidized glutathione (GSSG)/reduced glutathione (GSH) ratio were measured by HPLC-MS to determine the redox state in HCT-116 cells. Lastly, tumor xenograft experiments were carried out to determine the effect of glucose transporter (GLUT) inhibitor.
    RESULTS: Knockdown of ERRα decreased the expression of OTUB1 and SLC7A11 in HCT-116 cells. SLC7A11 overexpression induced NADPH-dependent redox system collapse. Aberrant expression of ERRα significantly reduced NADPH level and resulted in collapse of the redox system under glucose deprivation. Furthermore, ERR overexpression of ERRα sensitized cancer cells to inhibition of GLUTs. Treatment with GLUT inhibitor significantly reduced tumor volume after 6 weeks of tumor xenograft experiment. Our study demonstrates that the over-expression of ERRα causes redox system collapses via regulating the expressions of OUTB1 and SLC7A11.
    CONCLUSION: Up-regulation of SLC7A11 mediates the disruption of cell metabolism and the balance of redox state in colorectal cancer. Additionally, the GLUT inhibitor significantly reduces colorectal tumor volume, suggesting that the GLUT inhibitor could serve as a potential therapy for colorectal treatment.
  17. Elife. 2022 Aug 23. pii: e79422. [Epub ahead of print]11
      Pyruvate kinase (PK) and the phosphoenolpyruvate (PEP) cycle play key roles in nutrient-stimulated KATP channel closure and insulin secretion. To identify the PK isoforms involved, we generated mice lacking β-cell PKm1, PKm2, and mitochondrial PEP carboxykinase (PCK2) that generates mitochondrial PEP. Glucose metabolism generates both glycolytic and mitochondrially-derived PEP, which triggers KATP closure through local PKm1 and PKm2 signaling at the plasma membrane. Amino acids, which generate mitochondrial PEP without producing glycolytic fructose 1,6-bisphosphate to allosterically activate PKm2, signal through PKm1 to raise ATP/ADP, close KATP channels, and stimulate insulin secretion. Raising cytosolic ATP/ADP with amino acids is insufficient to close KATP channels in the absence of PK activity or PCK2, indicating that KATP channels are primarily regulated by PEP that provides ATP via plasma membrane-associated PK, rather than mitochondrially-derived ATP. Following membrane depolarization, the PEP cycle is also involved in an 'off-switch' that facilitates KATP channel reopening and Ca2+ extrusion, as shown by PK activation experiments and β-cell PCK2 deletion, which prolongs Ca2+ oscillations and increases insulin secretion. In conclusion, the differential response of PKm1 and PKm2 to the glycolytic and mitochondrial sources of PEP influences the β-cell nutrient response, and controls the oscillatory cycle regulating insulin secretion.
    Keywords:  cell biology; mouse
  18. J Neurosci. 2022 Aug 19. pii: JN-RM-0193-22. [Epub ahead of print]
      Electrical activity in neurons is highly energy demanding and accompanied by rises in cytosolic Ca2+ Cytosolic Ca2+, in turn, secures energy supply by pushing mitochondrial metabolism either through augmented NADH transfer into mitochondria via the malate aspartate shuttle (MAS) or via direct activation of dehydrogenases of the TCA cycle after passing into the matrix through the mitochondrial Ca2+ uniporter (MCU). Another Ca2+-sensitive booster of mitochondrial ATP synthesis is the glycerol-3-phosphate shuttle (G3PS) whose role in neuronal energy supply has remained elusive. Essential components of G3PS are expressed in hippocampal neurons. Single neuron metabolic measurements in primary hippocampal cultures derived from rat pups of either sex reveal only moderate, if any, constitutive activity of G3PS. However, during electrical activity neurons fully rely on G3PS when MAS and MCU are unavailable. Under these conditions, G3PS is required for appropriate action potential firing. Accordingly, G3PS safeguards metabolic flexibility of neurons to cope with energy demands of electrical signaling.SIGNIFICANCE STATEMENT:Ca2+ ions are known to provide a link between the energy-demanding electrical activity and an adequate ATP supply in neurons. To do so, Ca2+ acts both, from outside and inside of the mitochondrial inner membrane. Neuronal function critically depend on this regulation and its defects are often found in various neurological disorders. Although interest in neuronal metabolism increases, many aspects thereof have remained unresolved. In particular, a Ca2+-sensitive NADH shuttling system, the glycerol-3-phosphate shuttle, has been largely ignored with respect to its function in neurons. Our results demonstrate that this shuttle is functional in hippocampal neurons and safeguards ATP supply and appropriate action potential firing when malate aspartate shuttle and mitochondrial Ca2+ uniporter are unavailable, thereby ensuring neuronal metabolic flexibility.
  19. Liver Int. 2022 Aug 25.
      Acyl-CoA thioesterase 9 (ACOT9) is a critical regulator of cellular utilization of fatty acids by catalyzing the hydrolysis of acyl-CoA thioesters to non-esterified fatty acid and coenzyme A (CoA). Recently, ACOT9 was reported to participate in the pathogenesis of non-alcoholic liver disease (NAFLD), which arises from aberrant lipid metabolism and serves as a risk factor for hepatocellular carcinoma (HCC). However, the functions of ACOT9 in carcinogenesis and aberrant lipid metabolism in HCC remain unexplored. Here, we found that ACOT9 expression is significantly elevated in HCC at least in partial due to the down-regulation of miR-449c-3p. Upregulation of ACOT9 is closely associated with poor prognosis for patients with HCC. Knockdown of ACOT9 expression in HCC cells significantly decreased cell proliferation, colony formation, migration and invasion, mainly through suppression of G1-to-S cell cycle transition and epithelial-to-mesenchymal transition (EMT). By contrast, forced ACOT9 expression promoted HCC growth and metastasis. In addition, we found that ACOT9 reprogrammed lipid metabolism in HCC cells by increasing de novo lipogenesis. Furthermore, we demonstrated that increased lipogenesis was involved in ACOT9-promoted HCC growth and metastasis. Altogether, we demonstrate that ACOT9 plays a critical oncogenic role in the promotion of tumor growth and metastasis by reprogramming lipid metabolism in HCC, indicating ACOT9 as a potential therapeutic target in treatment of HCC.
    Keywords:  ACOT9; HCC; growth; lipid metabolism; metastasis
  20. Biology (Basel). 2022 Jul 28. pii: 1132. [Epub ahead of print]11(8):
      Several intermediate metabolites harbour cell-signalling properties, thus, it is likely that specific metabolites enable the communication between neighbouring cells, as well as between host cells with the microbiota, pathogens, and tumour cells. Mitochondria, a source of intermediate metabolites, participate in a wide array of biological processes beyond that of ATP production, such as intracellular calcium homeostasis, cell signalling, apoptosis, regulation of immune responses, and host cell-microbiota crosstalk. In this regard, mitochondria's plasticity allows them to adapt their bioenergetics status to intra- and extra-cellular cues, and the mechanisms driving such plasticity are currently a matter of intensive research. Here, we addressed whether mitochondrial ultrastructure and activity are differentially shaped when human monocytes are exposed to an exogenous source of lactate (derived from glycolysis), succinate, and fumarate (Krebs cycle metabolic intermediates), or butyrate and acetate (short-chain fatty acids produced by intestinal microbiota). It has previously been shown that fumarate induces mitochondrial fusion, increases the mitochondrial membrane potential (Δψm), and reshapes the mitochondrial cristae ultrastructure. Here, we provide evidence that, in contrast to fumarate, lactate, succinate, and butyrate induce mitochondrial fission, while acetate induces mitochondrial swelling. These traits, along with mitochondrial calcium influx kinetics and glycolytic vs. mitochondrial ATP-production rates, suggest that these metabolites differentially shape mitochondrial function, paving the way for the understanding of metabolite-induced metabolic reprogramming of monocytes and its possible use for immune-response intervention.
    Keywords:  Krebs cycle; glycolysis; innate immunity; mitochondria; mitochondrial reprogramming; short-chain fatty acids
  21. Neurochem Res. 2022 Aug 23.
      Ketogenic diets and medium-chain triglycerides are gaining attention as treatment of neurological disorders. Their major metabolites, β-hydroxybutyrate (βHB) and the medium-chain fatty acids (MCFAs) octanoic acid (C8) and decanoic acid (C10), are auxiliary brain fuels. To which extent these fuels compete for metabolism in different brain cell types is unknown. Here, we used acutely isolated mouse cerebral cortical slices to (1) compare metabolism of 200 µM [U-13C]C8, [U-13C]C10 and [U-13C]βHB and (2) assess potential competition between metabolism of βHB and MCFAs by quantifying metabolite 13C enrichment using gas chromatography-mass spectrometry (GC-MS) analysis. The 13C enrichment in most metabolites was similar with [U-13C]C8 and [U-13C]C10 as substrates, but several fold lower with [U-13C]βHB. The 13C enrichment in glutamate was in a similar range for all three substrates, whereas the 13C enrichments in citrate and glutamine were markedly higher with both [U-13C]C8 and [U-13C]C10 compared with [U-13C]βHB. As citrate and glutamine are indicators of astrocytic metabolism, the results indicate active MCFA metabolism in astrocytes, while βHB is metabolized in a different cellular compartment. In competition experiments, 12C-βHB altered 13C incorporation from [U-13C]C8 and [U-13C]C10 in only a few instances, while 12C-C8 and 12C-C10 only further decreased the low [U-13C]βHB-derived 13C incorporation into citrate and glutamine, signifying little competition for oxidative metabolism between βHB and the MCFAs. Overall, the data demonstrate that βHB and MCFAs are supplementary fuels in different cellular compartments in the brain without notable competition. Thus, the use of medium-chain triglycerides in ketogenic diets is likely to be beneficial in conditions with carbon and energy shortages in both astrocytes and neurons, such as GLUT1 deficiency.
    Keywords:  Astrocytes; Decanoic acid; Epilepsy; Ketone bodies; MCFA; Octanoic acid
  22. JCI Insight. 2022 Aug 23. pii: e161783. [Epub ahead of print]
      Energy metabolism failure in proximal tubule cells (PTC) is a hallmark of chronic kidney injury. We combined transcriptomic, metabolomic and lipidomic approaches in experimental models and patient cohorts to investigate the molecular bases of the progression to chronic kidney allograft injury initiated by ischemia-reperfusion injury (IRI). The urinary metabolome of kidney transplant recipients with chronic allograft injury and who experienced severe IRI was significantly enriched with long chain fatty acids (FA). We identified a renal FA-related gene signature with low levels of Cpt2 and Acsm5 and high levels of Acsl4 and Acsm5 associated with IRI, transition to chronic injury, and established CKD in mouse models and kidney transplant recipients. The findings were consistent with the presence of Cpt2-, Acsl4+, Acsl5+, Acsm5- PTC failing to recover from IRI as identified by snRNAseq. In vitro experiments indicated that endoplasmic reticulum (ER) stress contributes to CPT2 repression, which, in turn, promotes lipids accumulation, drives profibrogenic epithelial phenotypic changes, and activates the unfolded protein response. ER stress through CPT2 inhibition and lipid accumulation, engages an auto-amplification loop leading to lipotoxicity and self-sustained cellular stress. Thus, IRI imprints a persistent FA metabolism disturbance in the proximal tubule sustaining the progression to chronic kidney allograft injury.
    Keywords:  Bioenergetics; Nephrology; Transplantation
  23. J Biochem. 2022 Aug 22. pii: mvac063. [Epub ahead of print]
      Lipid burden in macrophages driven by oxidized LDL (oxLDL) accelerates the foam cell formation and the activation of sterile inflammatory responses aggravating the atherosclerosis. However, there is limited information on the mediators and the pathways involved in the possible survival responses, especially at the initial phase, by lipid-burden in macrophage cells on encountering oxLDL. The present study was designed to assess the expression status of major mediators involved in the NLRP3 inflammasome pathway of sterile inflammation and the cellular responses in oxLDL-challenged cultured RAW 264.7 macrophage cells. Ox-LDL-treated RAW 264.7 macrophage cells displayed a decreased expression of the key sterile inflammatory mediators, TLR4, TLR2, ASC, NLRP3 and IL-18 at protein and transcript levels; however, displayed increased level of IL-1β, RAGE and TREM1 at protein level. Biological responses including lipid uptake, lipid peroxidation, cellular hypertrophy, mitochondrial density, and mitochondrial membrane potential were significantly increased in oxLDL-treated macrophages. Moreover, superoxide production was significantly decreased in the oxLDL-treated macrophages compared to the control. Overall, the findings revealed the expression status of key sterile mediators and the macrophage response during the initial phase of oxLDL exposure tend toward the prevention of inflammation. Further understanding would open novel translational opportunities in the management of atherosclerosis.
    Keywords:  Atherosclerosis; Lipid-burden; Macrophages; Oxidized-LDL; Sterile inflammation
  24. Curr Opin Chem Biol. 2022 Aug 23. pii: S1367-5931(22)00084-9. [Epub ahead of print]70 102199
      Human physiological activities and pathological changes arise from the coordinated interactions of multiple molecules. Mass spectrometry (MS)-based multi-omics and MS imaging (MSI)-based spatial omics are powerful methods used to investigate molecular information related to the phenotype of interest from homogenated or sliced samples, including the qualitative, relative quantitative and spatial distributions. Molecular network strategy provides efficient methods to help us understand and mine the biological patterns behind the phenotypic data. It illustrates and combines various relationships between molecules, and further performs the molecule identification and biological interpretation. Here, we describe the recent advances of network-based analysis and its applications for different biological processes, such as, obesity, central nervous system diseases, and environmental toxicology.
    Keywords:  Mass spectrometry; Mass spectrometry imaging; Metabolomics; Molecular network strategy; Spatial omics
  25. Cell Stem Cell. 2022 Aug 19. pii: S1934-5909(22)00304-6. [Epub ahead of print]
      Skeletal muscle regeneration depends on the correct expansion of resident quiescent stem cells (satellite cells), a process that becomes less efficient with aging. Here, we show that mitochondrial dynamics are essential for the successful regenerative capacity of satellite cells. The loss of mitochondrial fission in satellite cells-due to aging or genetic impairment-deregulates the mitochondrial electron transport chain (ETC), leading to inefficient oxidative phosphorylation (OXPHOS) metabolism and mitophagy and increased oxidative stress. This state results in muscle regenerative failure, which is caused by the reduced proliferation and functional loss of satellite cells. Regenerative functions can be restored in fission-impaired or aged satellite cells by the re-establishment of mitochondrial dynamics (by activating fission or preventing fusion), OXPHOS, or mitophagy. Thus, mitochondrial shape and physical networking controls stem cell regenerative functions by regulating metabolism and proteostasis. As mitochondrial fission occurs less frequently in the satellite cells in older humans, our findings have implications for regeneration therapies in sarcopenia.
    Keywords:  Drp1; OXPHOS; aging; metabolism; mitochondria; mitochondrial dynamics; mitophagy; muscle regeneration; muscle stem cells; satellite cells
  26. Mol Metab. 2022 Aug 19. pii: S2212-8778(22)00146-6. [Epub ahead of print] 101577
      BACKGROUND: Peroxisomes are single membrane-bound organelles named for their role in hydrogen peroxide production and catabolism. However, their cellular functions extend well beyond reactive oxygen species (ROS) metabolism and include fatty acid oxidation of unique substrates that cannot be catabolized in mitochondria, and synthesis of ether lipids and bile acids. Metabolic functions of peroxisomes involve crosstalk with other organelles, including mitochondria, endoplasmic reticulum, lipid droplets and lysosomes. Emerging studies suggest that peroxisomes are important regulators of energy homeostasis and that disruption of peroxisomal functions influences the risk for obesity and the associated metabolic disorders, including type 2 diabetes and hepatic steatosis.SCOPE OF REVIEW: Here, we focus on the role of peroxisomes in ether lipid synthesis, β-oxidation and ROS metabolism, given that these functions have been most widely studied and have physiologically relevant implications in systemic metabolism and obesity. Efforts are made to mechanistically link these cellular and systemic processes.
    MAJOR CONCLUSIONS: Circulating plasmalogens, a form of ether lipids, have been identified as inversely correlated biomarkers of obesity. Ether lipids influence metabolic homeostasis through multiple mechanisms, including regulation of mitochondrial morphology and respiration affecting brown fat-mediated thermogenesis, and through regulation of adipose tissue development. Peroxisomal β-oxidation also affects metabolic homeostasis through generation of signaling molecules, such as acetyl-CoA and ROS that inhibit hydrolysis of stored lipids, contributing to development of hepatic steatosis. Oxidative stress resulting from increased peroxisomal β-oxidation-generated ROS in the context of obesity mediates β-cell lipotoxicity. A better understanding of the roles peroxisomes play in regulating and responding to obesity and its complications will provide new opportunities for their treatment.
    Keywords:  Diabetes; Fatty liver; Lipid metabolism; Obesity; Peroxisomes; Plasmalogen
  27. Mol Cell Biochem. 2022 Aug 27.
      Atherosclerotic morbidity is significantly higher in the diabetic population. Hyperglycemia, a typical feature of diabetes, has been proven to accelerate foam cell formation. However, the molecular mechanisms behind this process remain unclear. In this study, LPS and IFN-γ were used to convert THP-1-derived macrophages into M1 macrophages, which were then activated with ox-LDL in either high glucose or normal condition. We identified lipids within macrophages by Oil red O staining and total cholesterol detection. The genes involved in lipid absorption, efflux, inflammation, and metabolism were analyzed using qRT-PCR. The mechanisms of high glucose-induced foam cell formation were further investigated through metabolomics and transcriptomics analysis. We discovered that high glucose speed up lipid accumulation in macrophages (both lipid droplets and total cholesterol increased), diminished lipid efflux (ABCG1 down-regulation), and aggravated inflammation (IL1B and TNF up-regulation). Following multi-omics analysis, it was determined that glucose altered the metabolic and transcriptional profiles of macrophages, identifying 392 differently expressed metabolites and 293 differentially expressed genes, respectively. Joint pathway analysis suggested that glucose predominantly disrupted the glycerolipid, glycerophospholipid, and arachidonic acid metabolic pathways in macrophages. High glucose in the glyceride metabolic pathway, for instance, suppressed the transcription of triglyceride hydrolase (LIPG and LPL), causing cells to deposit excess triglycerides into lipid droplets and encouraging foam cell formation. More importantly, high glucose triggered the accumulation of pro-atherosclerotic lipids (7-ketocholesterol, lysophosphatidylcholine, and glycerophosphatidylcholine). In conclusion, this work elucidated mechanisms of glucose-induced foam cell formation via a multi-omics approach.
    Keywords:  Foam cell formation; High glucose; Lipid metabolism disorders; Sequence analysis
  28. EMBO J. 2022 Aug 23. e111528
      The regulation of cellular energy metabolism is central to most physiological and pathophysiological processes. However, most current methods have limited ability to functionally probe metabolic pathways in individual cells. Here, we describe SPICE-Met (Single-cell Profiling and Imaging of Cell Energy Metabolism), a method for profiling energy metabolism in single cells using flow cytometry or imaging. We generated a transgenic mouse expressing PercevalHR, a fluorescent reporter for cellular ATP:ADP ratio. Modulation of PercevalHR fluorescence with metabolic inhibitors was used to infer the dependence of energy metabolism on oxidative phosphorylation and glycolysis in defined cell populations identified by flow cytometry. We applied SPICE-Met to analyze T-cell memory development during vaccination. Finally, we used SPICE-Met in combination with real-time imaging to dissect the heterogeneity and plasticity of energy metabolism in single macrophages ex vivo and identify three distinct metabolic patterns. Functional probing of energy metabolism with single-cell resolution should greatly facilitate the study of immunometabolism at a steady state, during disease pathogenesis or in response to therapy.
    Keywords:  OXPHOS; energy; glycolysis; imaging; immunometabolism
  29. Oxid Med Cell Longev. 2022 ;2022 1198607
      Endothelial cell senescence is the main risk factor contributing to vascular dysfunction and the progression of aging-related cardiovascular diseases. However, the relationship between endothelial cell metabolism and endothelial senescence remains unclear. The present study provides novel insight into fatty acid metabolism in the regulation of endothelial senescence. In the replicative senescence model and H2O2-induced premature senescence model of primary cultured human umbilical vein endothelial cells (HUVECs), fatty acid oxidation (FAO) was suppressed and fatty acid profile was disturbed, accompanied by downregulation of proteins associated with fatty acid uptake and mitochondrial entry, in particular the FAO rate-limiting enzyme carnitine palmitoyl transferase 1A (CPT1A). Impairment of fatty acid metabolism by silencing CPT1A or CPT1A inhibitor etomoxir facilitated the development of endothelial senescence, as implied by the increase of p53, p21, and senescence-associated β-galactosidase, as well as the decrease of EdU-positive proliferating cells. In the contrary, rescue of FAO by overexpression of CPT1A or supplement of short chain fatty acids (SCFAs) acetate and propionate ameliorated endothelial senescence. In vivo, treatment of acetate for 4 weeks lowered the blood pressure and alleviated the senescence-related phenotypes in aortas of Ang II-infused mice. Mechanistically, fatty acid metabolism regulates endothelial senescence via acetyl-coenzyme A (acetyl-CoA), as implied by the observations that suppression of acetyl-CoA production using the inhibitor of ATP citrate lyase NDI-091143 accelerated senescence of HUVECs and that supplementation of acetyl-CoA prevented H2O2-induced endothelial senescence. Deficiency of acetyl-CoA resulted in alteration of acetylated protein profiles which are associated with cell metabolism and cell cycle. These findings thus suggest that improvement of fatty acid metabolism might ameliorate endothelial senescence-associated cardiovascular diseases.