Reactive nitrogen species inhibit branched chain alpha-ketoacid dehydrogenase complex and impact muscle cell metabolism.

Branched chain α-ketoacid dehydrogenase complex (BCKDC) is the rate-limiting enzyme in branched chain amino acid (BCAA) catabolism, a metabolic pathway with great importance for human health. BCKDC belongs to the mitochondrial α-ketoacid dehydrogenase complex family, which also includes pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex. Here, we revealed that BCKDC can be substantially inhibited by reactive nitrogen species (RNS) via a mechanism similar to what we recently discovered with pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex-RNS can cause inactivating covalent modifications of the lipoic arm on its E2 subunit. In addition, we showed that such reaction between RNS and the lipoic arm of the E2 subunit can further promote inhibition of the E3 subunits of α-ketoacid dehydrogenase complexes. We examined the impacts of this RNS-mediated BCKDC inhibition in muscle cells, an important site of BCAA metabolism, and demonstrated that the nitric oxide production induced by cytokine stimulation leads to a strong inhibition of BCKDC activity and BCAA oxidation in myotubes and myoblasts. More broadly, nitric oxide production reduced the level of functional lipoic arms across the multiple α-ketoacid dehydrogenases and led to intracellular accumulation of their substrates (α-ketoacids), decrease of their products (acyl-CoAs), and a lower cellular energy charge. In sum, this work revealed a new mechanism for BCKDC regulation, demonstrated that RNS can generally inhibit all α-ketoacid dehydrogenases, which has broad physiological implications across multiple cell types, and elucidated the mechanistic connection between RNS-driven inhibitory modifications on the E2 and E3 subunits of α-ketoacid dehydrogenases.

Reactive nitrogen species inhibit branched chain alpha-ketoacid dehydrogenase complex and impact muscle cell metabolism.

Branched chain α-ketoacid dehydrogenase complex (BCKDC) is the rate limiting enzyme in branched chain amino acid (BCAA) catabolism, a metabolic pathway with great importance for human health. BCKDC belongs to the mitochondrial α-ketoacid dehydrogenase complex family, which also includes pyruvate dehydrogenase complex (PDHC) and oxoglutarate dehydrogenase complex (OGDC). Here we revealed that BCKDC can be substantially inhibited by reactive nitrogen species (RNS) via a mechanism similar to what we recently discovered with PDHC and OGDC - modifying the lipoic arm on its E2 subunit. In addition, we showed that such reaction between RNS and the lipoic arm of the E2 subunit can further promote inhibition of the E3 subunits of α-ketoacid dehydrogenase complexes. We examined the impacts of this RNS-mediated BCKDC inhibition in muscle cells, an important site of BCAA metabolism, and demonstrated that the nitric oxide production induced by cytokine stimulation leads to a strong inhibition of BCKDC activity and BCAA oxidation in myotubes and myoblasts. More broadly, nitric oxide production reduced the level of functional lipoic arms across the multiple α-ketoacid dehydrogenases and led to intracellular accumulation of their substrates (α-ketoacids), reduction of their products (acyl-CoAs), and a lower cellular energy charge. This work revealed a new mechanism for BCKDC regulation, demonstrated its biological significance, and elucidated the mechanistic connection between RNS-driven inhibitory modifications on the E2 and E3 subunits of α-ketoacid dehydrogenases. Together with previous work, we revealed a general mechanism for RNS to inhibit all α-ketoacid dehydrogenases, which has numerous physiological implications across multiple cell types.

Asparagine starvation suppresses histone demethylation through iron depletion.

Intracellular α-ketoglutarate is an indispensable substrate for the Jumonji family of histone demethylases (JHDMs) mediating most of the histone demethylation reactions. Since α-ketoglutarate is an intermediate of the tricarboxylic acid cycle and a product of transamination, its availability is governed by the metabolism of several amino acids. Here, we show that asparagine starvation suppresses global histone demethylation. This process is neither due to the change of expression of histone-modifying enzymes nor due to the change of intracellular levels of α-ketoglutarate. Rather, asparagine starvation reduces the intracellular pool of labile iron, a key co-factor for the JHDMs to function. Mechanistically, asparagine starvation suppresses the expression of the transferrin receptor to limit iron uptake. Furthermore, iron supplementation to the culture medium restores histone demethylation and alters gene expression to accelerate cell death upon asparagine depletion. These results suggest that suppressing iron-dependent histone demethylation is part of the cellular adaptive response to asparagine starvation.

Nitric oxide-driven modifications of lipoic arm inhibit α-ketoacid dehydrogenases.

Pyruvate dehydrogenase complex (PDHC) and oxoglutarate dehydrogenase complex (OGDC), which belong to the mitochondrial α-ketoacid dehydrogenase family, play crucial roles in cellular metabolism. These multi-subunit enzyme complexes use lipoic arms covalently attached to their E2 subunits to transfer an acyl group to coenzyme A (CoA). Here, we report a novel mechanism capable of substantially inhibiting PDHC and OGDC: reactive nitrogen species (RNS) can covalently modify the thiols on their lipoic arms, generating a series of adducts that block catalytic activity. S-Nitroso-CoA, a product between RNS and the E2 subunit's natural substrate, CoA, can efficiently deliver these modifications onto the lipoic arm. We found RNS-mediated inhibition of PDHC and OGDC occurs during classical macrophage activation, driving significant rewiring of cellular metabolism over time. This work provides a new mechanistic link between RNS and mitochondrial metabolism with potential relevance for numerous physiological and pathological conditions in which RNS accumulate.

Macrophages undergo functionally significant reprograming of nucleotide metabolism upon classical activation.

During an immune response, macrophages systematically rewire their metabolism in specific ways to support their diversve functions. However, current knowledge of macrophage metabolism is largely concentrated on central carbon metabolism. Using multi-omics analysis, we identified nucleotide metabolism as one of the most significantly rewired pathways upon classical activation. Further isotopic tracing studies revealed several major changes underlying the substantial metabolomic alterations: 1) de novo synthesis of both purines and pyrimidines is shut down at several specific steps; 2) nucleotide degradation activity to nitrogenous bases is increased but complete oxidation of bases is reduced, causing a great accumulation of nucleosides and bases; and 3) cells gradually switch to primarily relying on salvaging the nucleosides and bases for maintaining most nucleotide pools. Mechanistically, the inhibition of purine nucleotide de novo synthesis is mainly caused by nitric oxide (NO)-driven inhibition of the IMP synthesis enzyme ATIC, with NO-independent transcriptional downregulation of purine synthesis genes augmenting the effect. The inhibition of pyrimidine nucleotide de novo synthesis is driven by NO-driven inhibition of CTP synthetase (CTPS) and transcriptional downregulation of thymidylate synthase (TYMS). For the rewiring of degradation, purine nucleoside phosphorylase (PNP) and uridine phosphorylase (UPP) are transcriptionally upregulated, increasing nucleoside degradation activity. However, complete degradation of purine bases by xanthine oxidoreductase (XOR) is inhibited by NO, diverting flux into nucleotide salvage. Inhibiting the activation-induced switch from nucleotide de novo synthesis to salvage by knocking out the purine salvage enzyme hypoxanthine-guanine phosporibosyl transferase (Hprt) significantly alters the expression of genes important for activated macrophage functions, suppresses macrophage migration, and increases pyroptosis. Furthermore, knocking out Hprt or Xor increases proliferation of the intracellular parasite Toxoplasma gondii in macrophages. Together, these studies comprehensively reveal the characteristics, the key regulatory mechanisms, and the functional importance of the dynamic rewiring of nucleotide metabolism in classically activated macrophages.

Asparagine bioavailability regulates the translation of MYC oncogene.

Amino acid restriction has recently emerged as a compelling strategy to inhibit tumor growth. Recent work suggests that amino acids can regulate cellular signaling in addition to their role as biosynthetic substrates. Using lymphoid cancer cells as a model, we found that asparagine depletion acutely reduces the expression of c-MYC protein without changing its mRNA expression. Furthermore, asparagine depletion inhibits the translation of MYC mRNA without altering the rate of MYC protein degradation. Of interest, the inhibitory effect on MYC mRNA translation during asparagine depletion is not due to the activation of the general controlled nonderepressible 2 (GCN2) pathway and is not a consequence of the inhibition of global protein synthesis. In addition, both the 5' and 3' untranslated regions (UTRs) of MYC mRNA are not required for this inhibitory effect. Finally, using a MYC-driven mouse B cell lymphoma model, we found that shRNA inhibition of asparagine synthetase (ASNS) or pharmacological inhibition of asparagine production can significantly reduce the MYC protein expression and tumor growth when environmental asparagine becomes limiting. Since MYC is a critical oncogene, our results uncover a molecular connection between MYC mRNA translation and asparagine bioavailability and shed light on a potential to target MYC oncogene post-transcriptionally through asparagine restriction.

A matter of time: temporal structure and functional relevance of macrophage metabolic rewiring.

The response of macrophages to stimulation is a dynamic process which coordinates the orderly adoption and resolution of various immune functions. Accumulating work over the past decade has demonstrated that during the immune response macrophage metabolism is substantially rewired to support important cellular processes, including the production of bioactive molecules, intercellular communication, and the regulation of intracellular signaling and transcriptional programming. In particular, we discuss an important concept emerging from recent studies - metabolic rewiring during the immune response is temporally structured. We review the regulatory mechanisms that drive the dynamic remodeling of metabolism, and examine the functional implications of these metabolic dynamics.

Switching to the cyclic pentose phosphate pathway powers the oxidative burst in activated neutrophils.

Neutrophils are cells at the frontline of innate immunity that can quickly activate effector functions to eliminate pathogens upon stimulation. However, little is known about the metabolic adaptations that power these functions. Here we show rapid metabolic alterations in neutrophils upon activation, particularly drastic reconfiguration around the pentose phosphate pathway, which is specifically and quantitatively coupled to an oxidative burst. During this oxidative burst, neutrophils switch from glycolysis-dominant metabolism to a unique metabolic mode termed 'pentose cycle', where all glucose-6-phosphate is diverted into oxidative pentose phosphate pathway and net flux through upper glycolysis is reversed to allow substantial recycling of pentose phosphates. This reconfiguration maximizes NADPH yield to fuel superoxide production via NADPH oxidase. Disruptions of pentose cycle greatly suppress oxidative burst, the release of neutrophil extracellular traps and pathogen killing by neutrophils. Together, these results demonstrate the remarkable metabolic flexibility of neutrophils, which is essential for their functions as the first responders in innate immunity.

Discovering How Heme Controls Genome Function Through Heme-omics.

Protein ensembles control genome function by establishing, maintaining, and deconstructing cell-type-specific chromosomal landscapes. A plethora of small molecules orchestrate cellular functions and therefore may link physiological processes with genome biology. The metabolic enzyme and hemoglobin cofactor heme induces proteolysis of a transcriptional repressor, Bach1, and regulates gene expression post-transcriptionally. However, whether heme controls genome function broadly or through prescriptive actions is unclear. Using assay for transposase-accessible chromatin sequencing (ATAC-seq), we establish a heme-dependent chromatin atlas in wild-type and mutant erythroblasts lacking enhancers that confer normal heme synthesis. Amalgamating chromatin landscapes and transcriptomes in cells with sub-physiological heme and post-heme rescue reveals parallel Bach1-dependent and Bach1-independent mechanisms that target heme-sensing chromosomal hotspots. The hotspots harbor a DNA motif demarcating heme-regulated chromatin and genes encoding proteins not known to be heme regulated, including metabolic enzymes. The heme-omics analysis establishes how an essential biochemical cofactor controls genome function and cellular physiology.

Metabolomic and Lipidomic Analysis of Bone Marrow Derived Macrophages.

Macrophages are highly plastic immune cells that are capable of adopting a wide array of functional phenotypes in response to environmental stimuli. The changes in macrophage function are often supported and regulated by changes in cellular metabolism. Capturing a comprehensive picture of metabolism is vital for understanding the role of metabolic rewiring in the immune response. Here we present a method for systematically quantifying the abundance of metabolites and lipids in primary murine bone marrow derived macrophages (BMDMs). This method simultaneously extracts polar metabolites and lipids from BMDMs using a rapid two-phase extraction procedure. The polar metabolite fraction and lipid fraction are subsequently analyzed by separate liquid chromatography-mass spectrometry (LC-MS) methods for optimized coverage and quantification. This allows for a comprehensive characterization of cellular metabolism that can be used to understand the impact of a variety of environmental stimuli on macrophage metabolism and function.

Promoter demethylation of the asparagine synthetase gene is required for ATF4-dependent adaptation to asparagine depletion.

Tumor cells adapt to nutrient-limited environments by inducing gene expression that ensures adequate nutrients to sustain metabolic demands. For example, during amino acid limitations, ATF4 in the amino acid response induces expression of asparagine synthetase (ASNS), which provides for asparagine biosynthesis. Acute lymphoblastic leukemia (ALL) cells are sensitive to asparagine depletion, and administration of the asparagine depletion enzyme l-asparaginase is an important therapy option. ASNS expression can counterbalance l-asparaginase treatment by mitigating nutrient stress. Therefore, understanding the mechanisms regulating ASNS expression is important to define the adaptive processes underlying tumor progression and treatment. Here we show that DNA hypermethylation at the ASNS promoter prevents its transcriptional expression following asparagine depletion. Insufficient expression of ASNS leads to asparagine deficiency, which facilitates ATF4-independent induction of CCAAT-enhancer-binding protein homologous protein (CHOP), which triggers apoptosis. We conclude that chromatin accessibility is critical for ATF4 activity at the ASNS promoter, which can switch ALL cells from an ATF4-dependent adaptive response to ATF4-independent apoptosis during asparagine depletion. This work may also help explain why ALL cells are most sensitive to l-asparaginase treatment compared with other cancers.

Analysis of Arginine Metabolism Using LC-MS and Isotopic Labeling.

Arginine metabolism is linked to several important metabolic processes, and reprogramming of arginine metabolism occurs in various physiological and pathological conditions. Here we describe a method, using a LC-MS-based metabolomics and 15N4-arginine tracing approach, to quantitatively analyze arginine metabolism. This method can reliably quantify the abundance of important intermediates and fluxes of major metabolic reactions in arginine metabolism in a variety of cultured mammalian cell models.

Two-stage metabolic remodelling in macrophages in response to lipopolysaccharide and interferon-γ stimulation.

In response to signals associated with infection or tissue damage, macrophages undergo a series of dynamic phenotypic changes. Here we show that during the response to LPS and interferon-γ stimulation, metabolic reprogramming in macrophages is also highly dynamic. Specifically, the TCA cycle undergoes a two-stage remodeling: the early stage is characterized by a transient accumulation of intermediates including succinate and itaconate, while the late stage is marked by the subsidence of these metabolites. The metabolic transition into the late stage is largely driven by the inhibition of pyruvate dehydrogenase complex (PDHC) and oxoglutarate dehydrogenase complex (OGDC), which is controlled by the dynamic changes in lipoylation state of both PDHC and OGDC E2 subunits and phosphorylation of PDHC E1 subunit. This dynamic metabolic reprogramming results in a transient metabolic state that strongly favors HIF-1α stabilization during the early stage, which subsides by the late stage; consistently, HIF-1α levels follow this trend. This study elucidates a dynamic and mechanistic picture of metabolic reprogramming in LPS and interferon-γ stimulated macrophages, and provides insights into how changing metabolism can regulate the functional transitions in macrophages over a course of immune response.

Translating formative research findings into a behaviour change strategy to promote antenatal calcium and iron and folic acid supplementation in western Kenya.

The World Health Organization now recommends integrating calcium supplements into antenatal micronutrient supplementation programmes to prevent pre-eclampsia, a leading cause of maternal mortality. As countries consider integrating calcium supplementation into antenatal care (ANC), it is important to identify context-specific barriers and facilitators to delivery and adherence. Such insights can be gained from women's and health workers' experiences with iron and folic acid (IFA) supplements. We conducted in-depth interviews with 22 pregnant and post-partum women and 20 community-based and facility-based health workers in Kenya to inform a calcium and IFA supplementation programme. Interviews assessed awareness of anaemia, pre-eclampsia and eclampsia; ANC attendance; and barriers and facilitators to IFA supplement delivery and adherence. We analyzed interviews inductively using the constant comparative method. Women and health workers identified poor diet quality in pregnancy as a major health concern. Neither women nor health workers identified pre-eclampsia, eclampsia, anaemia or related symptoms as serious health threats. Women and community-based health workers were unfamiliar with pre-eclampsia and eclampsia and considered anaemia symptoms normal. Most women had not received IFA supplements, and those who had received insufficient amounts and little information about supplement benefits. We then developed a multi-level (health facility, community, household and individual) behaviour change strategy to promote antenatal calcium and IFA supplementation. Formative research is an essential first step in guiding implementation of antenatal calcium supplementation programmes to reduce pre-eclampsia. Because evidence on how to implement successful calcium supplementation programmes is limited, experiences with antenatal IFA supplementation can be used to guide programme development.

A Novel in Vivo Model for Assessing the Impact of Geophagic Earth on Iron Status.

The causes and consequences of geophagy, the craving and consumption of earth, remain enigmatic, despite its recognition as a behavior with public health implications. Iron deficiency has been proposed as both a cause and consequence of geophagy, but methodological limitations have precluded a decisive investigation into this relationship. Here we present a novel in vivo model for assessing the impact of geophagic earth on iron status: Gallus gallus (broiler chicken). For four weeks, animals were gavaged daily with varying dosages of geophagic material or pure clay mineral. Differences in haemoglobin (Hb) across treatment groups were assessed weekly and differences in liver ferritin, liver iron, and gene expression of the iron transporters divalent metal transporter 1 (DMT1), duodenal cytochrome B (DcytB) and ferroportin were assessed at the end of the study. Minimal impact on iron status indicators was observed in all non-control groups, suggesting dosing of geophagic materials may need refining in future studies. However, this model shows clear advantages over prior methods used both in vitro and in humans, and represents an important step in explaining the public health impact of geophagy on iron status.

Adapting the Trials of Improved Practices (TIPs) approach to explore the acceptability and feasibility of nutrition and parenting recommendations: what works for low-income families?

Interventions to prevent childhood obesity must consider not only how child feeding behaviours are related to child weight status but also which behaviours parents are willing and able to change. This study adapted Trials of Improved Practices (TIPs) to assess acceptability and feasibility of nutrition and parenting recommendations, using in-depth interviews and household trials to explore families' experiences over time. A diverse sample of 23 low-income parents of 3-11-year-olds was recruited following participation in nutrition and parenting education. Parents chose nutrition and parenting practices to try at home and were interviewed 2 weeks and 4-6 months later about behaviour change efforts. Qualitative analysis identified emergent themes, and acceptability and feasibility were rated based on parents' willingness and ability to try new practices. The nutrition goal parents chose most frequently was increasing children's vegetable intake, followed by replacing sweetened beverages with water or milk, and limiting energy-dense foods. Parents were less inclined to reduce serving sizes. The parenting practices most often selected as applicable to nutrition goals were role-modelling; shaping home environments, often with other adults; involving children in decisions; and providing positive feedback. Most recommendations were viewed as acceptable by meaningful numbers of parents, many of whom tried and sustained new behaviours. Food preferences, habits and time were common barriers; family resistance or food costs also constrained some parents. Despite challenges, TIPs was successfully adapted to evaluate complex nutrition and parenting practices. Information on parents' willingness and ability to try practices provides valuable guidance for childhood obesity prevention programmes.

Bioavailability of iron in geophagic earths and clay minerals, and their effect on dietary iron absorption using an in vitro digestion/Caco-2 cell model.

Geophagy, the deliberate consumption of earth, is strongly associated with iron (Fe) deficiency. It has been proposed that geophagy may be practiced as a means to improve Fe status by increasing Fe intakes and, conversely, that geophagy may cause Fe deficiency by inhibiting Fe absorption. We tested these hypotheses by measuring Fe concentration and relative bioavailable Fe content of 12 samples of geophagic earth and 4 samples of pure clay minerals. Further, we assessed the impact of these samples on the bioavailability of Fe from an Fe-rich test meal (cooked white beans, WB). Fe concentrations were measured with inductively coupled plasma atomic emission spectroscopy. Fe bioavailability was determined using an in vitro digestion/Caco-2 cell model in which ferritin formation was used as an index of Fe bioavailability. Geophagic earth and clay mineral samples were evaluated with this model, both alone and in combination with WB (1 : 16 ratio, sample : WB). Median Fe concentration of the geophagic earth was 3485 (IQR 2462, 14 ,571) µg g⁻¹ and mean Fe concentration in the clay minerals was 2791 (±1782) µg g⁻¹. All specimens had Fe concentrations significantly higher (p ≤ 0.005) than the Fe concentration of WB (77 µg g⁻¹). Ferritin formation (i.e. Fe uptake) in cells exposed to geophagic earths and clay minerals was significantly lower than in cells exposed to WB (p ≤ 0.05) and Fe uptake responses of 11 of the 16 samples were not significantly different from the blank, indicating no bioavailable Fe. When samples were combined with WB, 5 of 16 had mean ferritin levels that were significantly lower (p ≤ 0.05, one tail) than the WB alone, indicating that the samples inhibited Fe uptake from the WB. None of the ferritin responses of cells exposed to both WB and earth/clay were significantly higher than WB alone. Thus, although geophagic earths and mineral clays are high in total Fe, very little of this Fe is bioavailable. Further, some geophagic earth and clay mineral samples inhibit Fe absorption from foods. In vivo research is warranted to confirm these observations and to determine if geophagic earth samples can be a source of Fe and/or inhibit Fe absorption.

Soil consumed by chacma baboons is low in bioavailable iron and high in clay.

Despite widespread consumption of soil among animals, the role of geophagy in health maintenance remains an enigma. It has been hypothesized that animals consume soil for supplementation of minerals and protection against toxins. Most studies determine only the total elemental composition of soil, which may not reflect the amount of minerals available to the consumer. Our aim was to test these hypotheses by evaluating the bioavailability of iron in soil consumed by chacma baboons, using a technique that simulates digestion and adsorption. Our results indicate that, despite variation in absolute iron concentration of soil samples, actual iron bioavailability was low while clay content was quite high. This suggests that iron supplementation is unlikely to be the primary motivation for geophagy in this population, and that detoxification is a plausible explanation. This study demonstrates that more research on bioavailability and clay composition is needed to determine the role geophagy plays in health maintenance.