Study Of Metabolic Regulation Research Articles

SCOUR: a stepwise machine learning framework for predicting metabolite-dependent regulatory interactions

BackgroundThe topology of metabolic networks is both well-studied and remarkably well-conserved across many species. The regulation of these networks, however, is much more poorly characterized, though it is known to be divergent across organisms—two characteristics that make it difficult to model metabolic networks accurately. While many computational methods have been built to unravel transcriptional regulation, there have been few approaches developed for systems-scale analysis and study of metabolic regulation. Here, we present a stepwise machine learning framework that applies established algorithms to identify regulatory interactions in metabolic systems based on metabolic data: stepwise classification of unknown regulation, or SCOUR.ResultsWe evaluated our framework on both noiseless and noisy data, using several models of varying sizes and topologies to show that our approach is generalizable. We found that, when testing on data under the most realistic conditions (low sampling frequency and high noise), SCOUR could identify reaction fluxes controlled only by the concentration of a single metabolite (its primary substrate) with high accuracy. The positive predictive value (PPV) for identifying reactions controlled by the concentration of two metabolites ranged from 32 to 88% for noiseless data, 9.2 to 49% for either low sampling frequency/low noise or high sampling frequency/high noise data, and 6.6–27% for low sampling frequency/high noise data, with results typically sufficiently high for lab validation to be a practical endeavor. While the PPVs for reactions controlled by three metabolites were lower, they were still in most cases significantly better than random classification.ConclusionsSCOUR uses a novel approach to synthetically generate the training data needed to identify regulators of reaction fluxes in a given metabolic system, enabling metabolomics and fluxomics data to be leveraged for regulatory structure inference. By identifying and triaging the most likely candidate regulatory interactions, SCOUR can drastically reduce the amount of time needed to identify and experimentally validate metabolic regulatory interactions. As high-throughput experimental methods for testing these interactions are further developed, SCOUR will provide critical impact in the development of predictive metabolic models in new organisms and pathways.

RNA-Seq and UHPLC-Q-TOF/MS Based Lipidomics Study in Lysiphlebia japonica

Lipids play an important role in energy storage, membrane structure stabilization and signaling. Parasitoids are excellent models to study lipidomics because a majority of them do not accumulate during their free-living life-stage. Studies on parasitoids have mostly focused on the changes in the lipids and gene transcripts in hosts and little attention has been devoted to lipidomics and transcriptomics changes in parasitoids. In this study, a relative quantitative analysis of lipids and their gene transcripts in 3-days-old Lysiphlebia japonica larva (3 days after spawning) and pupae were performed using liquid chromatography, mass spectrometry and RNA-seq. Thirty-three glycerolipids and 250 glycerophospholipids were identified in this study; all triglycerides and the vast majority of phospholipids accumulated in the pupal stage. This was accompanied by differentially regulated lipid uptake and remolding. Furthermore, our data showed that gene transcription was up-regulated in key nutrient metabolic pathways involved in lipid synthesis in 3-days-old larvae. Finally, our data suggests that larva and pupa of L. japonica may lack the ability for fatty acids synthesis. A comprehensive, quantitative, and expandable resource was provided for further studies of metabolic regulation and molecular mechanisms underlying parasitic response to hosts defense.

Methods for studying the metabolic basis of Drosophila development.

The field of metabolic research has experienced an unexpected renaissance. While this renewed interest in metabolism largely originated in response to the global increase in diabetes and obesity, studies of metabolic regulation now represent the frontier of many biomedical fields. This trend is especially apparent in developmental biology, where metabolism influences processes ranging from stem cell differentiation and tissue growth to sexual maturation and reproduction. In this regard, the fruit fly Drosophila melanogaster has emerged as a powerful tool for dissecting conserved mechanisms that underlie developmental metabolism, often with a level of detail that is simply not possible in other animals. Here we describe why the fly is an ideal system for exploring the relationship between metabolism and development, and outline a basic experimental strategy for conducting these studies. WIREs Dev Biol 2017, 6:e280. doi: 10.1002/wdev.280 For further resources related to this article, please visit the WIREs website.

Energy use and management of energy reserves in hatchling turtles (Chrysemys picta) exposed to variable winter conditions

The painted turtle (Chrysemys picta) is an especially useful organism in the study of metabolic regulation during dormancy because it is sustained by finite energy reserves from hatching until emerging from its nest, about nine months later. In this study we subjected overwintering C. picta hatchlings to 4, 10, or 15°C, temperatures simulating cold, mild, and warm winters, respectively, to investigate how various energy reserves are impacted by differential metabolic demands. An energy budget based on seasonal changes in caloric content showed that these turtles consumed an average of 0.39, 0.75, or 1.21kJg−1, respectively, during the 6-month period of simulated hibernation. These estimates of energy use agreed reasonably well with estimates based solely on respirometric data. Unexpectedly, turtles in autumn contained little residual yolk, none of which was consumed by turtles in the cold- and mild-winter groups, this finding contradicting the widely held belief that residual yolk plays an important, direct role in the survival of turtles that overwinter inside their natal nest. By contrast, a marked reduction in dry mass of both liver and carcass attested to their importance in fueling metabolism and, indeed, catabolism of substrates from these components accounted for 31–52 and 35–63%, respectively, of the energetic cost of overwintering. The greater dependence on carcass reserves and relatively poor physiological condition of turtles in the mild- and warm-winter groups implies that metabolic demands imposed by high environmental temperatures would likely constrain post-emergence fitness.

Metabolic control analysis of developing oilseed rape (Brassica napus cv Westar) embryos shows that lipid assembly exerts significant control over oil accumulation

Metabolic control analysis allows the study of metabolic regulation. We applied both single- and double-manipulation top-down control analysis to examine the control of lipid accumulation in developing oilseed rape (Brassica napus) embryos. The biosynthetic pathway was conceptually divided into two blocks of reactions (fatty acid biosynthesis (Block A), lipid assembly (Block B)) connected by a single system intermediate, the acyl-coenzyme A (acyl-CoA) pool. Single manipulation used exogenous oleate. Triclosan was used to inhibit specifically Block A, whereas diazepam selectively manipulated flux through Block B. Exogenous oleate inhibited the radiolabelling of fatty acids from [1-(14)C]acetate, but stimulated that from [U-14C]glycerol into acyl lipids. The calculation of group flux control coefficients showed that c. 70% of the metabolic control was in the lipid assembly block of reactions. Monte Carlo simulations gave an estimation of the error of the resulting group flux control coefficients as 0.27±0.06 for Block A and 0.73±0.06 for Block B. The two methods of control analysis gave very similar results and showed that Block B reactions were more important under our conditions. This contrasts notably with data from oil palm or olive fruit cultures and is important for efforts to increase oilseed rape lipid yields.

The Use of Natural Genetic Diversity in the Understanding of Metabolic Organization and Regulation

The study of metabolic regulation has traditionally focused on analysis of specific enzymes, emphasizing kinetic properties, and the influence of protein interactions and post-translational modifications. More recently, reverse genetic approaches permit researchers to directly determine the effects of a deficiency or a surplus of a given enzyme on the biochemistry and physiology of a plant. Furthermore, in many model species, gene expression atlases that give important spatial information concerning the quantitative expression level of metabolism-associated genes are being produced. In parallel, “top-down” approaches to understand metabolic regulation have recently been instigated whereby broad genetic diversity is screened for metabolic traits and the genetic basis of this diversity is defined thereafter. In this article we will review recent examples of this latter approach both in the model species Arabidopsis thaliana and the crop species tomato (Solanum lycopersicum). In addition to highlighting examples in which this genetic diversity approach has proven promising, we will discuss the challenges associated with this approach and provide a perspective for its future utility.

Novel root culture system using a recessive mutant with a rooty phenotype

Hairy roots induced by Agrobacterium rhizogenes-mediated transformation are widely used for the study of metabolic regulation and large-scale metabolite production. We developed an alternative root culture using an allelic mutant of Arabidopsis superroot 1 (sur1). When the mutant was cultured in liquid modified Heller's medium developed for hairy root cultures, it grew rapidly into a globe-shaped rooty phenotype and was easily subcultured in the medium. This mutant is advantageous as the desired characteristics can likely be maintained via seeds because the rooty phenotype is recessive. To verify this postulate, heterozygous sur1 plants were crossed with fatty acid desaturase ( fad) mutants. The sur1 homozygous mutants under fad2 and fad3 background accumulated high amounts of oleic acid and linolenic acid, respectively, and the mutant crossed with a FAD3 overexpression transformant accumulated high amounts of a-linolenic acid. Thus, the sur1 root culture system is an alternative tool for studying metabolic regulation and the production of useful compounds in roots.

Calcium and energy transfer

A recent article in The Journal of Physiology by Gueguen et al. (2005) shows that an efficient metabolic coupling between mitochondrial creatine kinase (mtCK) or adenylate kinase (AK2), and ADP rephosphorylation via mitochondrial respiration is tissue specific: it is functioning in skinned muscle of oxidative type I and type IIa but not in IIx type muscles, and this coupling can be modified by calcium. We would like to add some critical considerations which may contribute to solving some of discrepancies in this paper, and point out some methodological difficulties in studies of the functional coupling phenomenon. Calcium, ADP, AMP and creatine are known to act as important regulators of energy flux in muscle cells. High energy phosphates may be channelled out of mitochondria via direct ATP flux or by mtCK or AK2 via a phosphoryl-creatine (PCr) shuttle (Wallimann et al. 1992; Dzeja & Terzic, 2003; Saks et al. 2004). While all types of isolated mitochondria studied before show apparent Km (ADP) of 5–17 μm, this parameter is tissue specific in permeabilized cells in situ: Gueguen et al. (2005) showed that it is about the same in fast twitch muscle fibres of IIx type, but very high (212 μm) in slow twitch oxidative fibres of the type I and intermediate in the mixed types of fibres IIax and IIb where two apparent Km are observed. These results are in good agreement with earlier data (Veksler et al. 1995; Kuznetsov et al. 1996; Ventura-Clapier et al. 1998; Saks et al. 2004). Since the fibre diameter is usually bigger in fast glycolytic muscles, these data show very clearly the differences in the intracellular organization between different muscle types, and the very high apparent Km values are caused by specific local restrictions of the diffusion of ADP inside the type I oxidative fibres (Abraham et al. 2002; Vendelin et al. 2004). In their studies Gueguen et al. (2005) have made another remarkable observation: when the calcium concentration in the medium was elevated from 0.1 to 0.4 μm, the affinity of respiration for exogenous ADP was increased due to activation of actomyosin MgATPase. The increased affinity for exogenous ADP increased activation of respiration at submaximal ADP concentrations to the extent usually seen in the presence of creatine. Furthermore, calcium-induced activation of actomyosin ATPase also prevented the stimulation of respiration by creatine and AMP. The authors concluded from these results that mtCK and AK2 are no longer functionally coupled with mitochondrial respiration. This is a very surprising conclusion since under high work load, that is under elevated energy demand, the flux through the mtCK reaction was shown in vivo to increase significantly, e.g. during sea urchin sperm activation (Dorsten et al. 1997) and in muscle during increase of contraction frequency when directly assessed by the in vivo18O labelling technique (Pucar et al. 2001; Janssen et al. 2003), or alternatively, to decrease when demand was lowered (Joubert et al. 2002). In addition, calcium has been shown to strengthen the interaction between mtCK and voltage-dependent anion channels (VDAC) (Schlattner et al. 2001), which also would favour the formation of a VDAC–mtCK–adenine nucleotide translocase (ANT) multienzyme channelling complex, and thus facilitate the export of PCr from mitochondria. The experimental conditions described in the paper by Gueguen et al. (2005) are far from physiological conditions in the sense that the activating steady state level of calcium was continuously maintained at a high level and thus no relaxation was possible. Under these conditions, production of ATP by the activated mitochondrial respiration evidently results in hypercontraction of fibres, complete disturbance of myofibrillar architecture. This will also change the diffusion restrictions for ADP and results in a strong decrease of apparent Km for exogenous ADP (Andrienko et al. 2003). The rather artifactual system may explain the observations made under calcium addition. The fact that in the presence of vanadate, where the fibres are prevented from hypercontraction, calcium no longer ablates creatine-stimulated respiration (see Fig. 7) is direct proof that the observed phenomenon is related to supercontraction of the fibres, much more than to calcium itself. Under these conditions, exogenous ADP added already maximally activates respiration, and there will be no way for creatine to increase respiration further even if functional coupling is intact. For exactly the same reason, the very high apparent affinity of type II fibres for ADP makes it impossible to see the functional coupling between mtCK or AK2 and respiration from measurements at the fixed and relatively high ADP concentration of 0.1 mm (Fig. 5). To study functional coupling under these conditions, more sophisticated approach should be used (Moreadith & Jacobus, 1982). Clearly, metabolic microcompartments depend on the structural organization and integrity of the cells, and these considerations can no longer be ignored in studies of metabolic regulation of the muscle cell respiration (Schlattner & Wallimann, 2004).

Biochemical characterization of transducin, the G-protein of bovine retina

This article describes part of a graduate course in Biochemistry which involves the practical study of metabolic regulation in eukaryotic cells. This comes after three months of lectures on this topic and in the laboratory practical students partially purify and characterise transducin from bovine retina.

Locust flight metabolism studied in vivo by 31P NMR spectroscopy

Flight metabolism of locusts has been extensively studied, but biochemical and physiological methods have led to conflicting results. For this reason the non-invasive and non-destructive method of 31P NMR spectroscopy was used to study migratory locusts, Locusta migratoria, at rest and during flight. 1. In the flight muscle of resting locusts the ratio of phosphoarginine to ATP was the same whether determined by NMR (1.76) or biochemically, but the NMR-visible content of inorganic phosphate (Pi) was only 40% of ATP, i.e., much lower than total Pi as determined biochemically. This suggests that most of the Pi in flight muscle is not free, and hence not available as substrate or effector for cytosolic enzymes. Similarly, the free content of ADP and AMP in resting muscle was calculated to be much lower than the total content. 2. Flight brought about a marked increase in Pi and a decrease in phosphoarginine in flight muscle although there was no change in intracellular pH. 3. At the initiation of flight a new ‘steady state’ of ATP, Pi, and phosphoarginine was rapidly established and minimal changes occurred after the first 2 s of flight. 4. From the free contents of ATP and phosphoarginine in working flight muscle the flight-induced fractional increases in free ADP and free AMP were calculated to be 5.0-fold and 27.4-fold, respectively. As Pi, ADP, and AMP are substrates and potent effectors of enzymes, the flight-induced increase in their contents is likely to have marked effects on metabolic flux in working muscle. 5. After short-term flight as well as prolonged flight, phosphoarginine, ATP, and Pi returned rapidly to their preflight levels, indicating that metabolic recovery from flight is rapid. 6. The locust appears to be an appropriate model for the study of metabolic regulation in aerobic muscle during exercise.

Metabolic Studies in Human Subjects

Details of the major pathways of metabolism have been elucidated, in large measure through work on experimental animals. It is unlikely that they differ qualitatively between mammalian species. The more important challenge facing researchers today is that of metabolic regulation, and of the integrated control of metabolic pathways in the whole organism. These aspects may well differ quantitatively or even qualitatively between species. In this review, methods for studying metabolism and metabolic regulation in humans are described. There are both scientific and practical advantages to performing studies of metabolic regulation in humans. The scientific advantages are clear from some fundamental differences in metabolic regulation between rats and humans, such as the importance of de novo lipogenesis to the deposition of body fat, and the metabolism of atherogenic triacylglycerol-rich lipoproteins. The practical advantages result mainly from the size of a human compared with that of most laboratory animals, enabling large blood samples to be obtained, and several measurement techniques to be applied at one time. Reasons for the persistence of animal experimentation as the norm, rather than the exception, among life science researchers are discussed.

Hypoglycin, the famous toxin of the unripe Jamaican ackee fruit

Unripe fruit of the Jamaican ackee tree, Blighia sapida, contains hypoglycin, l-(methylenecyclopropyl)alanine), a hypoglycaemic toxin which has caused an estimated 5000 deaths. Methylenecyclopropylacetyl-CoA, a metabolite of hypoglycin, inactivates several, but not all, flavoprotein acyl-CoA dehydrogenases causing widespread disturbances of the oxidation of fatty acids and several amino acids leading to secondary inhibition of gluconeogenesis. Despite intensive study for three decades the mechanism of hypoglycin poisoning is still only partly understood. H. S. A. Sherratt reviews these complex effects which have implications for toxicology and the study of metabolic regulation and of many inborn errors of metabolism.

Sensitivity of pathway rate to activities of substrate-cycle enzymes: Application to gluconeogenesis and glycolysis

In a study of metabolic regulation, it is frequently useful to consider the degree to which an enzyme can influence the rate of its pathway. The most productive expression of rate-controlling influence is the fractional change in pathway rate per fractional change in enzyme activity (called control strength or sensitivity coefficient). We have developed a system for considering how a substrate-cycle enzyme's control strength depends on its flux and reaction order and on related features of other enzymes of its pathway. We have applied this system to the gluconeogenic pathway of rat liver and the glycolytic pathway of bovine sperm, where enough fluxes and reaction orders have been published to allow valid estimates of several control strengths. In normal fed animals where gluconeogenesis is slow and unidirectional substrate-to-product and product-to-substrate fluxes are comparable, all substrate-cycle limbs have very high and similar control strengths regardless of their flux rates and positions in the pathway. The activity of a step affects all substrate-cycle control strengths similarly as it affects unidirectional end-to-end fluxes relative to net rate. Control strengths of non-substrate-cycle enzymes are negligible compared to those of substrate cycles. In fasting animals, on the other hand, where unidirectional Pyr----Glc flux is much greater than Glc----Pyr flux, upstream enzymes (near Pyr) have a regulatory advantage over downstream enzymes (near Glc). In this circumstance, control strength of each substrate-cycle enzyme is inversely related to rate limitingness between its substrate and the pathway substrate. Because the Pyr/PEP cycle is significantly rate limiting, the control strength of the Pyr----PEP limb is much greater than that of pyruvate kinase and all downstream enzymes. In the glycolytic pathway of bovine sperm, strong product inhibition of hexokinase detracts greatly from its rate limitingness and control strength, which are very small despite its position at the beginning of the pathway and its large free energy. Because the glucose-transport-hexokinase segment is not rate limiting, phosphofructo 1-kinase has almost as much control strength as it would have as the first enzyme of the pathway, and because the F6P/FDP cycle is only moderately rate limiting, Fru-1,6-P2ase and enzymes further downstream have substantial control strengths. When glycolysis is accelerated by stimulation of phosphofructo 1-kinase, control strength shifts from phosphofructo-1-kinase and all downstream enzymes to the transporthesokinase segment.(ABSTRACT TRUNCATED AT 400 WORDS)

Failure of somatostatin to modify effect of glucagon on carbohydrate metabolism in the dog.

Somatostatin is widely used to inhibit insulin and glucagon release by the pancreas in studies of metabolic regulation in vivo. To determine whether the peptide can directly modify the metabolic effects of an increment in glucagon in overnight-fasted conscious dogs, glucagon was increased in the presence (+S) or absence (-S) of somatostatin. Either somatostatin (+S; 0.8 microgram . kg-1 . min-1) or a two-stage pancreatectomy (-S) was used to inhibit the endocrine pancreas, and at the same time replacement infusions of insulin (285-300 microU . kg-1 .min-1) and glucagon (0.65 ng . kg-1 . min-1) were given. After a 40-min control period the plasma glucagon level was raised fourfold in the presence of fixed basal insulin. Plasma insulin in both groups were similar [11 +/- 2 (+S) and 9 +/- 1 (-S) microU/ml]. Glucagon rose from 64 +/- 11 to 225 +/- 19 and 92 +/- 11 to 219 +/- 20 pg/ml in the +S and -S groups, respectively. Tracer-determined ([3-3H]glucose) glucose production rose by 5.28 +/- 1.02 (+S) and 4.25 +/- 1.12 (-S) mg . kg-1 . min-1 at 15 min and fell similarly over 3 h in both groups. Plasma glucose rose similarly in both groups peaking at 195 +/- 15 (+S) and 174 +/- 8 (-S) mg/dl. Plasma alanine fell similarly over 3 h [133 +/- 35 (+S) and 138 +/- 42 (-S) mumol/liter]. Conversion of [14C]alanine and [14C]-lactate to [14C]glucose rose progressively over 3 h in both groups, eventually being elevated by 210 +/- 58 (+S) and 148 +/- 48% (-S). We conclude that in the dog somatostatin at the dose used does not alter the effect of an increment in glucagon on carbohydrate metabolism.

Intracellular Regulatory Mechanisms

The study of metabolic regulation in microorganisms has revealed several simple but efficient regulatory circuits. In one, the operation of an entire sequence of enzymes is controlled by the activity of the initial enzyme which contains a specific inhibitor site. When this site is combined with the endproduct of the sequence, the catalytic site is rendered inactive. In another, the formation of an entire sequence of enzymes is controlled by means of a cytoplasmic mediator which blocks the transcription of the genetic message (repression) when activated by the endproduct, or which allows the transcription (induction) when activated by the substrate of the first enzyme in the sequence. Additional circuits have been proposed for the regulation of RNA and DNA synthesis. The same regulatory devices could account, in part, for intracellular metabolic control in more complex animal and plant forms. However, superimposed upon these simple control circuits will be found others which take advantage of the greater degree of organization in these cells and of the possibilities for regulating gene function that are provided by the chromosomes. The pattern of proteins with special control sites, such as have evolved in the relatively simple controls found in bacteria, may also be found essential for intercellular controls involving nervous and humoral mechanisms.