Differences In Metabolic Flux Research Articles

Abstract 13646: APOL1 Nephropathy Risk Variant Alters Macrophage Metabolism

The common G1 and G2 variants in the APOL1 gene, specific to patients with African ancestry, increase the risk of developing chronic kidney disease and stroke. These variants also confer a survival benefit against African trypanosomiasis, but the effects of G1 and G2 variants on macrophage function remain unclear despite their role in innate immunity. Crucial to both innate immunity and tissue homeostasis, macrophages can promote cardiometabolic disease if dysregulated. Others have shown that metabolic changes can alter macrophage phenotype and function, and the G1 and G2 variants have been linked to mitochondrial dysfunction in epithelial cells. We therefore hypothesize that APOL1 risk variants promote cardiometabolic disease traits by altering macrophage metabolism and, thus, function. To test our hypothesis, we established CRISPR-Cas9 genome-edited induced pluripotent stem cell (iPSC) lines with the G0/G0 (major allele) genotype and the G1/G1 genotype on isogenic backgrounds. We then differentiated these iPSC lines into macrophage-like cells (iPSDMs) and evaluated their phenotype and function at baseline and under stimulation with 5 - 50 ug/mL interferon-γ, a potent inducer of APOL1 expression. We measured inflammatory markers via QPCR and ELISA, mitochondrial oxygen flux with the Agilent Seahorse XF Analyzer, and intracellular hydrophilic metabolites via LCMS. We found that G1/G1 iPSDMs express higher levels of pro-inflammatory markers and exhibited lower basal respiration as well as lower ATP production compared to G0/G0 controls. Because APOL1 risk variants have been linked to defective autophagy and ER stress, we treated the G1/G1 iPSDMs with rapamycin (autophagy inducer) or TUDCA (ER stress inhibitor) but found that they did not abrogate these genotype-specific differences in metabolic flux. Upon examination of metabolomics profiles of G1/G1 vs G0/G0 iPSDMs, differentially abundant metabolites were enriched for SMPDB pathways including spermidine biosynthesis (needed for anti-inflammatory reprogramming), gluconeogenesis, Warburg effect, homocysteine degradation, and fatty acid oxidation. Taken together, G1/G1 iPSDMs exhibit metabolic differences that may contribute to their proinflammatory phenotype.

ΔFBA-Predicting metabolic flux alterations using genome-scale metabolic models and differential transcriptomic data.

Genome-scale metabolic models (GEMs) provide a powerful framework for simulating the entire set of biochemical reactions in a cell using a constraint-based modeling strategy called flux balance analysis (FBA). FBA relies on an assumed metabolic objective for generating metabolic fluxes using GEMs. But, the most appropriate metabolic objective is not always obvious for a given condition and is likely context-specific, which often complicate the estimation of metabolic flux alterations between conditions. Here, we propose a new method, called ΔFBA (deltaFBA), that integrates differential gene expression data to evaluate directly metabolic flux differences between two conditions. Notably, ΔFBA does not require specifying the cellular objective. Rather, ΔFBA seeks to maximize the consistency and minimize inconsistency between the predicted flux differences and differential gene expression. We showcased the performance of ΔFBA through several case studies involving the prediction of metabolic alterations caused by genetic and environmental perturbations in Escherichia coli and caused by Type-2 diabetes in human muscle. Importantly, in comparison to existing methods, ΔFBA gives a more accurate prediction of flux differences.

A biophotonic approach to measure pH in small volumes in vitro: Quantifiable differences in metabolic flux around the cumulus-oocyte-complex (COC).

Unfertilised eggs (oocytes) release chemical biomarkers into the medium surrounding them. This provides an opportunity to monitor cell health and development during assisted reproductive processes if detected in a non-invasive manner. Here we report the measurement of pH using an optical fibre probe, OFP1, in 5 μL drops of culture medium containing single mouse cumulus oocyte complexes (COCs). This allowed for the detection of statistically significant differences in pH between COCs in culture medium with no additives and those incubated with either a chemical (cobalt chloride) or hormonal treatment (follicle stimulating hormone); both of which serve to induce the release of lactic acid into the medium immediately surrounding the COC. Importantly, OFP1 was shown to be cell-safe with no inherent cell toxicity or light-induced phototoxicity indicated by negative DNA damage staining. Pre-measurement photobleaching of the probe reduced fluorescence signal variability, providing improved measurement precision (0.01-0.05 pH units) compared to previous studies. This optical technology presents a promising platform for the measurement of pH and the detection of other extracellular biomarkers to assess cell health during assisted reproduction.

FlySilico: Flux balance modeling of Drosophila larval growth and resource allocation

Organisms depend on a highly connected and regulated network of biochemical reactions fueling life sustaining and growth promoting functions. While details of this metabolic network are well established, knowledge of the superordinate regulatory design principles is limited. Here, we investigated by iterative wet lab and modeling experiments the resource allocation process during the larval development of Drosophila melanogaster. We chose this system, as survival of the animals depends on the successful allocation of their available resources to the conflicting processes of growth and storage metabolite deposition. First, we generated “FlySilico”, a curated metabolic network of Drosophila, and performed time-resolved growth and metabolite measurements with larvae raised on a holidic diet. Subsequently, we performed flux balance analysis simulations and tested the predictive power of our model by simulating the impact of diet alterations on growth and metabolism. Our predictions correctly identified the essential amino acids as growth limiting factor, and metabolic flux differences in agreement with our experimental data. Thus, we present a framework to study important questions of resource allocation in a multicellular organism including process priorization and optimality principles.

Distinctive Patterns of Flavonoid Biosynthesis in Roots and Nodules of Datisca glomerata and Medicago spp. Revealed by Metabolomic and Gene Expression Profiles.

Plants within the Nitrogen-fixing Clade (NFC) of Angiosperms form root nodule symbioses with nitrogen-fixing bacteria. Actinorhizal plants (in Cucurbitales, Fagales, Rosales) form symbioses with the actinobacteria Frankia while legumes (Fabales) form symbioses with proteobacterial rhizobia. Flavonoids, secondary metabolites of the phenylpropanoid pathway, have been shown to play major roles in legume root nodule symbioses: as signal molecules that in turn trigger rhizobial nodulation initiation signals and acting as polar auxin transport inhibitors, enabling a key step in nodule organogenesis. To explore a potentially broader role for flavonoids in root nodule symbioses across the NFC, we combined metabolomic and transcriptomic analyses of roots and nodules of the actinorhizal host Datisca glomerata and legumes of the genus Medicago. Patterns of biosynthetic pathways were inferred from flavonoid metabolite profiles and phenylpropanoid gene expression patterns in the two hosts to identify similarities and differences. Similar classes of flavonoids were represented in both hosts, and an increase in flavonoids generally in the nodules was observed, with differences in flavonoids prominent in each host. While both hosts produced derivatives of naringenin, the metabolite profile in D. glomerata indicated an emphasis on the pinocembrin biosynthetic pathway, and an abundance of flavonols with potential roles in symbiosis. Additionally, the gene expression profile indicated a decrease in expression in the lignin/monolignol pathway. In Medicago sativa, by contrast, isoflavonoids were highly abundant featuring more diverse and derived isoflavonoids than D. glomerata. Gene expression patterns supported these differences in metabolic pathways, especially evident in a difference in expression of cinnamic acid 4-hydroxylase (C4H), which was expressed at substantially lower levels in D. glomerata than in a Medicago truncatula transcriptome where it was highly expressed. C4H is a major rate-limiting step in phenylpropanoid biosynthesis that separates the pinocembrin pathway from the lignin/monolignol and naringenin-based flavonoid branches. Shikimate O-hydroxycinnamoyltransferase, the link between flavonoid biosynthesis and the lignin/monolignol pathway, was also expressed at much lower levels in D. glomerata than in M. truncatula. Our results indicate (a) a likely major role for flavonoids in actinorhizal nodules, and (b) differences in metabolic flux in flavonoid and phenylpropanoid biosynthesis between the different hosts in symbiosis.

Transcriptomic buffering of cryptic genetic variation contributes to meningococcal virulence

BackgroundCommensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence.ResultsDespite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region.ConclusionsOur data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis.

Genetic Variation in Choline-Metabolizing Enzymes Alters Choline Metabolism in Young Women Consuming Choline Intakes Meeting Current Recommendations.

Single nucleotide polymorphisms (SNPs) in choline metabolizing genes are associated with disease risk and greater susceptibility to organ dysfunction under conditions of dietary choline restriction. However, the underlying metabolic signatures of these variants are not well characterized and it is unknown whether genotypic differences persist at recommended choline intakes. Thus, we sought to determine if common genetic risk factors alter choline dynamics in pregnant, lactating, and non-pregnant women consuming choline intakes meeting and exceeding current recommendations. Women (n = 75) consumed 480 or 930 mg choline/day (22% as a metabolic tracer, choline-d9) for 10–12 weeks in a controlled feeding study. Genotyping was performed for eight variant SNPs and genetic differences in metabolic flux and partitioning of plasma choline metabolites were evaluated using stable isotope methodology. CHKA rs10791957, CHDH rs9001, CHDH rs12676, PEMT rs4646343, PEMT rs7946, FMO3 rs2266782, SLC44A1 rs7873937, and SLC44A1 rs3199966 altered the use of choline as a methyl donor; CHDH rs9001 and BHMT rs3733890 altered the partitioning of dietary choline between betaine and phosphatidylcholine synthesis via the cytidine diphosphate (CDP)-choline pathway; and CHKA rs10791957, CHDH rs12676, PEMT rs4646343, PEMT rs7946 and SLC44A1 rs7873937 altered the distribution of dietary choline between the CDP-choline and phosphatidylethanolamine N-methyltransferase (PEMT) denovo pathway. Such metabolic differences may contribute to disease pathogenesis and prognosis over the long-term.

Comparison of Protein Expression Profiles of Novel <i>Halomonas smyrnensis</i> AAD6<sup>T</sup> and <i>Halomonas salina</i> DSMZ 5928<sup>T</sup>

In this work, the protein pattern of novel Halomonas smyrnensis AAD6T was compared to that of Halomonas salina DSMZ5928T, which is the closest species on the basis of 16S rRNA sequence, to understand how AAD6T differs from type strains. Using high resolution NEPHEGE technique, the whole cell protein composition patterns of both Halomonas salina DSMZ5928T and H. smyrnensis AAD6T were mapped. The expressed proteins of the two microorganisms were mostly located at the acidic side of the gels, at molecular weight values of 60 to 17 kDa, and at isoelectric points 3.8 to 6.0, where they share a significant number of common protein spots. Identification and characterization of protein spots via whole genome sequencing data indicated that these two microorganisms used similar pathways, especially TCA cycle, for their survival; in other words, for their energy requirements. On the other hand, the protein expression differences in AAD6T and H. salina DSMZ 5928T showed that they prefer different metabolic pathways for lipid biosynthesis and in adaptation to extreme environments. Thus, we suggested that phylogenetic dissimilarities between these microorganisms could be related to the protein expression differences; in other words, metabolic flux differences in AAD6T and H. salina DSMZ 5928T. This is the first study to explain the dissimilarities of phenotypic characters and DNA-DNA hybridization between type strain and novel strain AAD6T by using protein expression differences.

In vivo metabolic flux profiling with stable isotopes discriminates sites and quantifies effects of mitochondrial dysfunction in C. elegans

Mitochondrial respiratory chain (RC) disease diagnosis is complicated both by an absence of biomarkers that sufficiently divulge all cases and limited capacity to quantify adverse effects across intermediary metabolism. We applied high performance liquid chromatography (HPLC) and mass spectrometry (MS) studies of stable-isotope based precursor–product relationships in the nematode, C. elegans, to interrogate in vivo differences in metabolic flux among distinct genetic models of primary RC defects and closely related metabolic disorders. MethodsC. elegans strains studied harbor single nuclear gene defects in complex I, II, or III RC subunits (gas-1, mev-1, isp-1); enzymes involved in coenzyme Q biosynthesis (clk-1), the tricarboxylic acid cycle (TCA, idh-1), or pyruvate metabolism (pdha-1); and central nodes of the nutrient-sensing signaling network that involve insulin response (daf-2) or the sirtuin homologue (sir-2.1). Synchronous populations of 2000 early larval stage worms were fed standard Escherichia coli on nematode growth media plates containing 1,6-13C2-glucose throughout their developmental period, with samples extracted on the first day of adult life in 4% perchloric acid with an internal standard. Quantitation of whole animal free amino acid concentrations and isotopic incorporation into amino and organic acids throughout development was performed in all strains by HPLC and isotope ratio MS, respectively. GC/MS analysis was also performed to quantify absolute isotopic incorporation in all molecular species of key TCA cycle intermediates in gas-1 and N2 adult worms. ResultsGenetic mutations within different metabolic pathways displayed distinct metabolic profiles. RC complex I (gas-1) and III (isp-1) subunit mutants, together with the coenzyme Q biosynthetic mutant (clk-1), shared a similar amino acid profile of elevated alanine and decreased glutamate. The metabolic signature of the complex II mutant (mev-1) was distinct from that of the other RC mutants but resembled that of the TCA cycle mutant (idh-1) and both signaling mutants (daf-2 and sir-2.1). All branched chain amino acid levels were significantly increased in the complex I and III mutants but decreased in the PDH mutant (pdha-1). The RC complex I, coenzyme Q, TCA cycle, and PDH mutants shared significantly increased relative enrichment of lactate+1 and absolute concentration of alanine+1, while glutamate+1 enrichment was significantly decreased uniquely in the RC mutants. Relative intermediary flux analyses were suggestive of proximal TCA cycle disruption in idh-1, completely reduced TCA cycle flux in sir-2.1, and apparent distal TCA cycle alteration in daf-2. GC/MS analysis with universally-labeled 13C-glucose in adult worms further showed significantly increased isotopic enrichment in lactate, citrate, and malate species in the complex I (gas-1) mutant. ConclusionsStable isotopic/mass spectrometric analysis can sensitively discriminate primary RC dysfunction from genetic deficiencies affecting either the TCA cycle or pyruvate metabolism. These data are further suggestive that metabolic flux analysis using stable isotopes may offer a robust means to discriminate and quantify the secondary effects of primary RC dysfunction across intermediary metabolism.

Diverging pathways: Differential benzenoid and phenylpropanoid volatile production in Phlox subulata L. cultivars

Previous research into the genetic mechanisms of benzenoid and phenylpropanoid volatile biosynthesis has suggested the potential for metabolic flux, in which phenylalanine substrate dedicated to one pathway branch (i.e., benzenoid production) could alter volatile production in other pathways (i.e., phenylpropanoid production). However, little research has been conducted in planta to verify the validity of this hypothesis. We examined the emission rates of representative benzenoid and phenylpropanoid volatiles from seven cultivars of Phlox subulata L. to determine if cultivars had metabolic flux differences in terms of these two compound categories. Cultivars that produced large quantities of methyl benzoate and benzaldehyde were found to emit little or no phenylacetaldehyde and 2-phenylethanol, and vice versa. These results suggest that P. subulata cultivars experience phenylalanine substrate flux directed toward one pathway and away from the alternate branch. Such a pattern may be the result of differential selection pressures, in which gene expression has been altered to direct flux away from either benzenoid or phenylpropanoid production. Moreover, if these patterns hold true in wild populations, metabolic flux may lead to differential pollinator behavior and further phenotypic evolution. Future research using molecular tools could verify the role of metabolic flux in determining scent phenotypes and pinpoint the exact nature of the genetic mutations leading to phenotypic differences in odor. Simplified metabolic flux pattern found in Phlox subulata ; phenylalanine substrate can be directed toward phenylpropanoid (phenylacetaldehyde and 2-phenylethanol) or benzenoid (benzaldehyde and methyl benzoate) production. • Phlox subulata cultivars emit benzenoid and phenylopropanoid volatiles. • Plants with high benzenoid emission emit little/no phenylpropanoids and vice versa. • This pattern suggests differential phenylalanine flux through biochemical pathways. • Such a pattern may result from differential selection and gene expression.

Evolutionary adaptations of gene structure and expression in natural populations in relation to a changing environment: A multidisciplinary approach to address the million-year saga of a small fish

We have used an experimentally based strategy to address molecular mechanisms underlying adaptation in Fundulus heteroclitus. In an attempt to falsify the hypothesis that selection is a major driving force in the maintenance of genetic diversity, we employed a multidisciplinary approach including allelic isozyme and mtDNA phylogeography, kinetic analyses of allelic isozymes, analysis of variation in coding and regulatory DNA sequences, metabolic biochemistry, organismal physiology, and selection experiments. Observed differences in gene structure and expression led us to make testable predictions about differences in metabolic flux, whole organism performance, and differential survival between allotypes. We have shown that variation in the lactate dehydrogenase-B (Ldh-B) protein results in differences in physiological function and is correlated with differences in survival at high temperatures. Recent work has investigated the role of variation in Ldh-B expression. There are differences in the levels of Ldh-B protein, mRNA, and transcription rate. We have addressed the mechanisms responsible for differences in transcription rate by a combination of sequence comparison, DNase I footprinting, and functional analyses both in vitro and in vivo. We have shown that variation in the regulatory sequence of Ldh-B is responsible for the differences in transcription rate between populations and that the patterns of variation are inconsistent with a neutral model of molecular evolution. This functional differentiation, coupled with departures from neutral expectations, suggests that natural selection has acted on the regulation of Ldh-B. This article illustrates the value of a multidisciplinary approach in addressing problems in gene structure, expression, and evolutionary adaptation.

Evolutionary adaptations of gene structure and expression in natural populations in relation to a changing environment: A multidisciplinary approach to address the million‐year saga of a small fish

We have used an experimentally based strategy to address molecular mechanisms underlying adaptation in Fundulus heteroclitus. In an attempt to falsify the hypothesis that selection is a major driving force in the maintenance of genetic diversity, we employed a multidisciplinary approach including allelic isozyme and mtDNA phylogeography, kinetic analyses of allelic isozymes, analysis of variation in coding and regulatory DNA sequences, metabolic biochemistry, organismal physiology, and selection experiments. Observed differences in gene structure and expression led us to make testable predictions about differences in metabolic flux, whole organism performance, and differential survival between allotypes. We have shown that variation in the lactate dehydrogenase-B (Ldh-B) protein results in differences in physiological function and is correlated with differences in survival at high temperatures. Recent work has investigated the role of variation in Ldh-B expression. There are differences in the levels of Ldh-B protein, mRNA, and transcription rate. We have addressed the mechanisms responsible for differences in transcription rate by a combination of sequence comparison, Dnase I footprinting, and functional analyses both in vitro and in vivo. We have shown that variation in the regulatory sequence of Ldh-B is responsible for the differences in transcription rate between populations and that the patterns of variation are inconsistent with a neutral model of molecular evolution. This functional differentiation, coupled with departures from neutral expectations, suggests that natural selection has acted on the regulation of Ldh-B. This article illustrates the value of a multidisciplinary approach in addressing problems in gene structure, expression, and evolutionary adaptation. J. Exp. Zool. 282:71–94, 1998. © 1998 Wiley-Liss, Inc.

Drosophila alcohol dehydrogenase polymorphism and carbon-13 fluxes: opportunities for epistasis and natural selection.

The influence of genetic variations in Drosophila alcohol dehydrogenase (ADH) on steady-state metabolic fluxes was studied by means of 13C NMR spectroscopy. Four pathways were found to be operative during 8 hr of ethanol degradation in third instar larvae of Drosophila. Seven strains differed by 18-25% in the ratio between two major pathway fluxes, i.e., into glutamate-glutamine-proline vs. lactate-alanine-trehalose. In general, Adh genotypes with higher ADH activity exhibit a twofold difference in relative carbon flux from malate into lactate and alanine vs. alpha,alpha-trehalose compared to low ADH activity genotypes. Trehalose was degraded by the pentose-phosphate shunt. The pentose-phosphate shunt and malic enzyme could supply NADPH necessary for lipid synthesis from ethanol. Lactate and/or proline synthesis may maintain the NADH/NAD+ balance during ethanol degradation. After 24 hr the flux into trehalose is increased, while the flux into lipids declines in AdhF larvae. In AdhS larvae the flux into lipids remains high. This co-ordinated nature of metabolism and the genotype-dependent differences in metabolic flux may form the basis for various epistatic interactions and ultimately for variations in organismal fitness.