Anaerobic Glucose Utilization Research Articles

Noninvasive assessment of metabolic turnover during inflammation by in vivo deuterium magnetic resonance spectroscopy.

Inflammation and metabolism exhibit a complex interplay, where inflammation influences metabolic pathways, and in turn, metabolism shapes the quality of immune responses. Here, glucose turnover is of special interest, as proinflammatory immune cells mainly utilize glycolysis to meet their energy needs. Noninvasive approaches to monitor both processes would help elucidate this interwoven relationship to identify new therapeutic targets and diagnostic opportunities. For induction of defined inflammatory hotspots, LPS-doped Matrigel plugs were implanted into the neck of C57BL/6J mice. Subsequently, 1H/19F magnetic resonance imaging (MRI) was used to track the recruitment of 19F-loaded immune cells to the inflammatory focus and deuterium (2H) magnetic resonance spectroscopy (MRS) was used to monitor the metabolic fate of [6,6-2H2]glucose within the affected tissue. Histology and flow cytometry were used to validate the in vivo data. After plug implantation and intravenous administration of the 19F-containing contrast agent, 1H/19F MRI confirmed the infiltration of 19F-labeled immune cells into LPS-doped plugs while no 19F signal was observed in PBS-containing control plugs. Identification of the inflammatory focus was followed by i.p. bolus injection of deuterated glucose and continuous 2H MRS. Inflammation-induced alterations in metabolic fluxes could be tracked with an excellent temporal resolution of 2 min up to approximately 60 min after injection and demonstrated a more anaerobic glucose utilization in the initial phase of immune cell recruitment. 1H/2H/19F MRI/MRS was successfully employed for noninvasive monitoring of metabolic alterations in an inflammatory environment, paving the way for simultaneous in vivo registration of immunometabolic data in basic research and patients.

Energy substrate metabolism, mitochondrial structure and oxidative stress after cardiac ischemia-reperfusion in mice lacking UCP3

Myocardial ischemia-reperfusion (IR) injury may result in cardiomyocyte dysfunction. Mitochondria play a critical role in cardiomyocyte recovery after IR injury. The mitochondrial uncoupling protein 3 (UCP3) has been proposed to reduce mitochondrial reactive oxygen species (ROS) production and to facilitate fatty acid oxidation. As both mechanisms might be protective following IR injury, we investigated functional, mitochondrial structural, and metabolic cardiac remodeling in wild-type mice and in mice lacking UCP3 (UCP3–KO) after IR. Results showed that infarct size in isolated perfused hearts subjected to IR ex vivo was larger in adult and old UCP3–KO mice than in equivalent wild-type mice, and was accompanied by higher levels of creatine kinase in the effluent and by more pronounced mitochondrial structural changes. The greater myocardial damage in UCP3–KO hearts was confirmed in vivo after coronary artery occlusion followed by reperfusion. S1QEL, a suppressor of superoxide generation from site IQ in complex I, limited infarct size in UCP3–KO hearts, pointing to exacerbated superoxide production as a possible cause of the damage. Metabolomics analysis of isolated perfused hearts confirmed the reported accumulation of succinate, xanthine and hypoxanthine during ischemia, and a shift to anaerobic glucose utilization, which all recovered upon reoxygenation. The metabolic response to ischemia and IR was similar in UCP3–KO and wild-type hearts, being lipid and energy metabolism the most affected pathways. Fatty acid oxidation and complex I (but not complex II) activity were equally impaired after IR. Overall, our results indicate that UCP3 deficiency promotes enhanced superoxide generation and mitochondrial structural changes that increase the vulnerability of the myocardium to IR injury.

Evaluation of Anaerobic Glucose Utilization by Escherichia coli Strains with Impaired Fermentation Ability during Respiration with External and Internal Electron Acceptors

Evaluation of Anaerobic Glucose Utilization by Escherichia coli Strains with Impaired Fermentation Ability during Respiration with External and Internal Electron Acceptors

Thermodynamic approach to estimating reactions and stoichiometric coefficients of anaerobic glucose and hydrogen utilization

AbstractAnaerobic digestion plays an important role in the gastrointestinal tract and in organic waste treatment. Thermodynamic analysis based on the reaction Gibbs free energy can be used to predict the favorability of some reactions occurring during anaerobic digestion. In this study, we used a thermodynamic approach to evaluating reactions and stoichiometric coefficients of the anaerobic process of in vitro rumen microbiota. The favorability of glucose, butyrate, propionate, and hydrogen utilizations was analyzed by calculating the Gibbs free energy change of each reaction. A previously published Gibbs free energy dissipation method was also used to calculate stoichiometric coefficients of the total metabolism reaction of glucose and hydrogen utilization. For glucose utilization in which the metabolism follows several different pathways, the fraction of glucose following each pathway is estimated by considering the number of electron transfer attributed throughout the catabolism reaction. Glucose utilization always occurs in the system, and the syntrophic correlation among butyrate, propionate, and hydrogen utilizations run well with propionate utilization following the alternative pathway that yields lower hydrogen. The approach applied in this research significantly reduces the stoichiometric coefficients that must be predicted in kinetic modeling. To verify the calculation result, the yield coefficients obtained were then applied in the previous mechanistic model of in vitro rumen microbiota, and the results were compared to the experimental data from literature.

Study of the Potential of the Reversal of the Fatty-Acid Beta-Oxidation Pathway for Stereoselective Biosynthesis of (S)-1,3-Butanediol from Glucose by Recombinant Escherichia coli Strains

The possible contribution of collateral enzymes to the formation of the key precursor metabolite, 3-hydroxybutyryl-CoA, has been evaluated in a recombinant Escherichia coli strain engineered for 1,3-butanediol biosynthesis from glucose via the inverted fatty-acid beta-oxidation pathway. Inactivation of the 3-hydroxyadipyl-CoA dehydrogenase gene, paaH, did not prevent 1,3-butanol biosynthesis during anaerobic glucose utilization by a strain with an intact, essential gene, fabG. This gene encodes 3-ketoacyl-ACP reductase, which can catalyze the conversion of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. The subsequent inactivation in the strain of the fadB gene, which encodes (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase of the fatty-acid beta oxidation led to the cessation of 1,3-butanediol synthesis. The respective diol was also not found among the products secreted by the strain possessing the intact fabG and paaH genes upon the individual deletion of the fadB gene. It was established that the collateral enzymes did not participate in the formation of 3-hydroxybutyryl-CoA in the studied strains, and the respective CoA derivative was synthesized solely by the (S)-specific enzyme of the fatty-acid beta-oxidation pathway. The results indicate that reversal of the fatty-acid beta oxidation pathway can ensure the enantioselective biosynthesis of the (S)-stereoisomer of 1,3-butanediol in engineered E. coli strains.

Оценка анаэробной утилизации глюкозы штаммами Escherichia coli с нарушенной способностью к брожению при дыхании с внешним и внутренним акцептором электронов

The characteristics of anaerobic glucose utilization and metabolite production by recombinant Escherichia coli strains with impaired fermentation ability upon respiration with pyruvate as an internal and nitrate as an external electron acceptor have been studied. It was found that respiration processes utilizing pyruvic acid as an endogenous electron acceptor and leading to the lactate and alanine formation were capable of mutual interference. After elimination of ammonium ions from the medium, the native activity levels of respiratory lactate dehydrogenases Did and LldD in E. coli strains deficient in the mixed acid fermentation pathways can almost completely compensate for the loss of activity of respiratory alanine dehydrogenase DadA, but are insufficient to maintain the entire intracellular redox balance. The addition of nitrate ions in the medium abolished alanine production by the strains despite the availability of ammonium ions, while the functionality of respiratory reduction of endogenous pyruvate to lactate retained in the studied strains even in the presence of a strong exogenous oxidant. Respiration with external electron acceptor provoked the activation of the oxidative tricarboxylic acid cycle in the strains. Anaerobic glucose utilization by the strain with interrupted tricarboxylic acid cycle increased during nitrate respiration, but remained restricted by the excessive generation of reducing equivalents in the residual reactions of the cycle. Escherichia coli, glucose, fermentation, respiration, pyruvate, lactate, alanine, nitrate, ammonium. The work was supported by a grant from the Russian Foundation for Basic Research (project #18-04-01222).

Activation of Alternative Respiration with Internal Electron Acceptor during Anaerobic Glucose Utilization in Escherichia coli Strains with Impaired Fermentation Ability

The activation of alternative respiration with an internal electron acceptor during anaerobic glucose utilization in E. coli strains with impaired fermentation ability has been studied. It was found out that respiration processes utilizing pyruvic acid as an endogenous electron acceptor can markedly contribute to the maintenance of the anaerobic redox balance in E. coli strains deficient in mixed acid fermentation pathways. The sequential inactivation of the pathways of anaerobic dissimilation of pyruvate and impairment of the functionality of the reductive branch of the tricarboxylic acid cycle led to an increase in the contribution (from 11 to 54%) of the respiratory formation of lactic acid and alanine to the biosynthesis of the reduced products of anaerobic glucose utilization by the strains. Analysis of the enantiomeric composition of the lactic acid and alanine secreted by the strains demonstrated that D-lactate dehydrogenase (Dld), L-lactate dehydrogenase (LctD), and D-alanine dehydrogenase (DadA) participated in the biosynthesis of the respective compounds.

Активация альтернативного дыхания с внутренним акцептором электронов при анаэробной утилизации глюкозы у штаммов Escherichia coli с нарушенной способностью к брожению

The activation of alternative respiration with an internal electron acceptor upon anaerobic utilization of glucose in Escherichia coli strains with the impaired fermentation ability has been studied. It was found out that the respiration processes utilizing pyruvic acid as an endogenous electron acceptor can markedly contribute to the maintenance of the anaerobic redox balance in E. coli strains deficient in mixed acid fermentation pathways. The sequential inactivation of the pathways of anaerobic dissimilation of pyruvate and impairment of the functionality of the reductive branch of the tricarboxylic acid cycle led to the increase in the contribution of the respiratory formation of lactic acid and alanine to the biosynthesis of the reduced products of anaerobic glucose utilization by the strains from 11 to 54%. The analysis of the enantiomeric composition of the lactic acid and alanine secreted by the strains demonstrated that D-lactate dehydrogenase (Dld), L-lactate dehydrogenase (LctD), and D-alanine dehydrogenase (DadA) participated in the biosynthesis of the respective compounds. Escherichia coli, glucose, fermentation, respiration, metabolic engineering, pyruvate, lactate, alanine. The work was supported by a grant from the Russian Foundation for Basic Research (Project no. 18-04-01222)

Исследование потенциала обращения пути бета-окисления жирных кислот для стереоселективного биосинтеза (S)-1,3-бутандиола из глюкозы рекомбинантными штаммами Escherichia coli

A possible contribution of collateral enzymes to the formation of key precursor metabolite, 3-hydroxybutyryl-CoA, in a recombinant Escherichia coli strain engineered for 1,3-butanediol biosynthesis from glucose through the inverted fatty acid beta-oxidation pathway has been evaluated. The inactivation of the 3-hydroxyadipyl-CoA dehydrogenase gene, paaH, did not prevent the 1,3-butanol biosynthesis during anaerobic glucose utilization by the strain with the intact essential gene fabG coding for 3-ketoacyl-ACP reductase, which can catalyze the conversion of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. The subsequent inactivation in the strain of fadB gene coding for (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase of the fatty acid beta-oxidation led to the abolishment of the 1,3-butanediol synthesis. The respective diol was also not found among the products secreted by the strain possessing the intact fabG and paaH genes upon an individual deletion of fadB gene. It was established that the collateral enzymes did not participate in the formation of 3-hydroxybutyryl-CoA in the studied strains and the respective CoA-derivative was synthesized solely by the (S)-specific enzyme of the fatty acid beta-oxidation pathway. The obtained results indicate that the reversal of the fatty acid beta-oxidation pathway can ensure the enantioselective biosynthesis of the (S)-stereoisomer of 1,3-butanediol in engineered E. coli strains. 1,3-butanediol, fatty acid beta-oxidation, Escherichia coli, glucose, metabolic engineering, stereoisomer. The work was carried out with financial support Russian Foundation for Fundamental Research (No. 18-29-08059).

Inactivation of Malic Enzymes Improves the Anaerobic Production of Four-Carbon Dicarboxylic Acids by Recombinant Escherichia coli Strains Expressing Pyruvate Carboxylase

The genes maeA and maeB, encoding NADH- and NADPH-dependent malic enzymes, have been deleted in a recombinant Escherichia coli strain with inactivated mixed-acid fermentation pathways and a modified system of glucose transport and phosphorylation upon the heterological expression of the pyruvate carboxylase gene. During anaerobic glucose utilization, the parental strain synthesized malic, fumaric, and succinic acids as the main fermentation end products, while pyruvic acid was accumulated as the main by-product resulting from the functioning of the pyruvate–oxaloacetate–malate–pyruvate futile cycle. Upon individual deletions of the maeA and maeB genes, the mutant strains converted glucose into four-carbon dicarboxylic acids with increased efficiency still secreting notable amounts of pyruvic acid. The combined inactivation of both malic enzymes in the constructed strain significantly elevated the portion of malic, fumaric, and succinic acids among the fermentation end products with a concomitant decrease in the secretion of pyruvic acid and other by-products due to the abolishment of the action of the futile cycle competing with the target biosynthetic processes.

Effect of Anaplerotic Pathways Activation on CO2-dependent Anaerobic Glucose Utilization by Escherichia coli Strains Deficient in the Main Pathways of Mixed Acid Fermentation

The effect of anaplerotic pathways activation on CO2-dependent anaerobic glucose utilization by Escherichia coli strains deficient in the main fermentation pathways and possessing a modified system of glucose transport and phosphorylation was studied. Intracellular CO2 generation in the strains was ensured resulting from oxidative decarboxylation of pyruvic acid by pyruvate dehydrogenase. Sodium bicarbonate dissolved in the medium was used as an external source of CO2. The genes of heterologous pyruvate carboxylase and native NADH-dependent malic enzyme were overexpressed in the strains to allow anaplerotic carboxylation of pyruvic acid to oxaloacetic or malic acid. The ability of the strains to reoxidize NADH utilizing carboxylation products was additionally increased due to enhanced expression of malate dehydrogenase gene. In the case of endogenous CO2 formation, the activation of anaplerotic pathways did not cause a notable increase in the anaerobic glucose consumption by the constructed strains. At the same time, the expression of pyruvate carboxylase led to a pronounced decrease in the secretion of pyruvic acid with the concomitant increase in the yield of four-carbon metabolites. Further enhancement of NADH-dependent malic enzyme expression provoked activation of a pyruvate–oxaloacetate–malate–pyruvate futile cycle in the strains. The availability in the medium of the external CO2 source sharply increased the anaerobic utilization of glucose by strains expressing pyruvate carboxylase. The activity of the futile cycle has raised with the increased malic enzyme expression and dropped upon enhancement of malate dehydrogenase expression. As a result, the efficiency of CO2-dependent anaerobic glucose utilization coupled to the formation of four-carbon carboxylation products increased in the studied strains resulting from the primary anaplerotic conversion of pyruvic acid into oxaloacetic acid followed by the involvement of the precursor formed in NADH-consuming biosynthetic reactions dominating over the reactions of the revealed futile cycle.

Effect of extra- and intracellular sources of CO2 on anaerobic utilization of glucose by Escherichia coli strains deficient in carboxylation-independent fermentation pathways

The effect of extra- and intracellular CO2 sources on anaerobic glucose utilization by Escherichia coli strains deficient in the main pathways of mixed acid fermentation and possessing a modified system of glucose transport and phosphorylation was studied. Intracellular CO2 generation in the strains was ensured resulting from the oxidative decarboxylation of pyruvic acid by pyruvate dehydrogenase. Endogenous CO2 formation by pyruvate dehydrogenase stimulated anaerobic glucose consumption by the strains due to the involvement in the fermentation process of condensation reactions between oxaloacetic acid and acetyl-CoA. The availability of an external CO2 source (dissolved in medium sodium bicarbonate) promoted utilization of carbohydrate substrate by favoring the predominant participation in the fermentation of reactions directly dependent on phosphoenolpyruvate carboxylation. The positive effect of the availability of exogenous СО2 was sharply decreased in recombinant strains with the impaired functionality of the reductive branch of the tricarboxylic acid cycle. As a result, intracellular СО2 generation coupled to acetyl-CoA formation promoted anaerobic glucose utilization by cells of the corresponding mutants more markedly than the presence in the medium of dissolved sodium bicarbonate.

Metabolic engineering of Escherichia coli for 1,3-butanediol biosynthesis through the inverted fatty acid β-oxidation cycle

The feasibility of 1,3-butanediol biosynthesis through the inverted cycle of fatty acid β-oxidation in Escherichia coli cells was investigated by the rational metabolic engineering approach. CoA-dependent aldehyde dehydrogenase MhpF and alcohol dehydrogenases FucO and YqhD were used as terminal enzymes catalyzing conversion of 3-hydroxybutyryl-CoA to 1,3-butanediol. Constitutive expression of the corresponding genes in E. coli strains, which are deficient in mixed acid fermentation pathways and expressing fad regulon genes under control of P trc-ideal-4 promoter, did not lead to the synthesis of 1,3-butanediol during anaerobic glucose utilization. Additional inactivation of fadE and ydiO genes, encoding acyl-CoA dehydrogenases, also did not cause synthesis of the target product. Constitutive expression of aceEF-lpdA operon genes encoding enzymes of pyruvate dehydrogenase complex led to an increase in anaerobic synthesis of ethanol. Synthesis of 1,3-butanediol was observed with the overexpression of acetyl-CoA C-acetyltransferase AtoB. Constitutive expression of atoB gene in a strain with a basal expression of alcohol/aldehyde dehydrogenase leads to synthesis of 0.3 mM of 1,3-butanediol.

A new F-18 labeled PET tracer for fatty acid imaging

In the presence of acute or chronic myocardial ischemia, myocardial energy metabolism is shifted from the use of aerobic fatty acids (FA) substrate to anaerobic glucose utilization resulting in decreased FA uptake in ischemic regions. This phenomenon may persist as long as 24-30 hours following an acute ischemic event, despite restoration of blood flow and resolution of symptoms and is referred as “ischemic memory”.1 Ischemic memory imaging can be particularly useful in various clinical settings2 as in the case of patients presenting to the emergency department with acute chest pain syndrome.3 We present the first human images of CardioPET (trans-9-F-18-fluoro-3,4-methyleneheptadecanoic acid, FCPHA)4 a F-18 labeled modified FA currently being evaluated in a phase II multicentre clinical trial in patients with a positive SPECT stress test for ischemia. A 69-year-old man with typical angina was evaluated using bicycle exercise stress-rest Tc-99m-Sestamibi SPECT (Figure 1). As part of a research protocol, prior to coronary angiography, the patient underwent repeat bicycle stress CardioPET imaging at a similar heart rate. CardioPET was injected 8 minutes after peak exercise stress test. CardioPET images (Figure 2) revealed alterations of fatty acid uptake in regions without abnormalities on Tc-99m-Sestamibi-SPECT (Figure 3). It better identified the extent of significant coronary stenosis than did Tc-99m-Sestamibi SPECT as demonstrated by coronary angiography (Figure 4). In addition, regional time-activity curves obtained from the CardioPET data suggested dynamic time-dependent changes in fatty acid retention in ischemic regions different from the non-ischemic regions that need to be further investigated (Figure 5). These preliminary findings suggest a promising role of this new F-18 labeled tracer of FA uptake for the identification of functionally significant coronary artery disease. FA uptake and retention in various pathophysiological conditions may be better understood using the quantitative capabilities of PET technology. Figure 1 Attenuation corrected stress-rest Tc-99m-Sestamibi perfusion SPECT images. Sestamibi was injected at the peak of the stress test (160 watts, 131 bpm, 86% of maximal predicted heart rate and blood pressure of 210/100 mm Hg). ... Figure 2 CardioPET images. CardioPET enters the myocardium by the same mechanism as naturally occurring free FAs and then undergoes partial metabolism before being trapped in the myocytes. The tracer (262 MBq) was injected 8 minutes after peak ... Figure 3 Slice by slice comparison of the stress perfusion SPECT, CardioPET, and rest perfusion SPECT images allowing to appreciate the more extensive defects on the cardioPET scan in relation to the Tc-99m-Sestamibi stress perfusion SPECT scan Figure 4 Coronary angiography. Coronary angiography revealed multi-vessel disease with a severe (76%-95%) ostial lesion of the circumflex artery (yellow arrow), 2 successive severe (76%-95%) medial lesions of the left anterior descending artery, a severe (76%-95%) ... Figure 5 CardioPET time-activities curves. The graph shows the evolution of the activity in the different regions of the left ventricle (lateral, posterior, inferior, septal, anteroseptal, and anterior) as a function of the time after tracer injection. Counts ...

Increased anaerobic metabolism is a distinctive signature in a colorectal cancer cellular model of resistance to antiepidermal growth factor receptor antibody

Cetuximab is a chimeric antibody approved for the treatment of metastatic colorectal cancer that selectively targets epidermal growth factor receptor (EGFR) signaling. Treatment efficacy with this drug is often impaired by acquired resistance and poor information has been accumulated on the mechanisms underlying such a phenomenon. By taking advantage of a syngenic cellular system of sensitivity and acquired resistance to anti-EGFR therapy in the colorectal carcinoma GEO cell line, we profiled protein expression differences between Cetuximab-sensitive and -resistant cells. Combined 2D DIGE and MS analyses revealed a main proteomic signature resulting from selective deregulation of various metabolic enzymes, including glucose-6-phosphate dehydrogenase, transketolase, lactate dehydrogenase B, and pyruvate dehydrogenase E1, which was also confirmed by Western blotting experiments. Lactate dehydrogenase B downregulation has been already related to an increased anaerobic utilization of glucose by tumor cells; accordingly, we verified that Cetuximab-resistant cells have a significantly higher production of lactate. Resistant cells also showed decreased nicotinamide adenine dinucleotide phosphate (NADPH) levels. Observed protein deregulations were not related to functional alterations of the hypoxia-inducible factor 1-associated pathways. Our data demonstrate that increased anaerobic metabolism is a prominent feature observed in the GEO syngenic model of acquired resistance to anti-EGFR therapy in colorectal cancer.

Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization

Phosphoenolpyruvate (PEP) is an important precursor for anaerobic production of succinate and malate. Although inactivating PEP/carbohydrate phosphotransferase systems (PTS) could increase PEP supply, the resulting strain had a low glucose utilization rate. In order to improve anaerobic glucose utilization rate for efficient production of succinate and malate, combinatorial modulation of galactose permease (galP) and glucokinase (glk) gene expression was carried out in chromosome of an Escherichia coli strain with inactivated PTS. Libraries of artificial regulatory parts, including promoter and messenger RNA stabilizing region (mRS), were firstly constructed in front of β-galactosidase gene (lacZ) in E. coli chromosome through λ-Red recombination. Most regulatory parts selected from mRS library had constitutive strengths under different cultivation conditions. A convenient one-step recombination method was then used to modulate galP and glk gene expression with different regulatory parts. Glucose utilization rates of strains modulated with either galP or glk all increased, and the rates had a positive relation with expression strength of both genes. Combinatorial modulation had a synergistic effect on glucose utilization rate. The highest rate (1.64 g/L h) was tenfold higher than PTS(-) strain and 39% higher than the wild-type E. coli. These modulated strains could be used for efficient anaerobic production of succinate and malate.

Lipoic acid inhibits leptin secretion and Sp1 activity in adipocytes

Lipoic acid (LA) is an antioxidant with therapeutic potential on several diseases such as diabetes and obesity. Hyperleptinemia and oxidative stress play a major role in the development of obesity-linked diseases. The aim of this study was to examine in vivo and in vitro the effects of LA on leptin production, as well as to elucidate the mechanisms and signalling pathways involved in LA actions. Dietary supplementation with LA decreased both circulating leptin, and adipose tissue leptin mRNA in rats. Treatment of 3T3-L1 adipocytes with LA caused a concentration-dependent inhibition of leptin secretion and gene expression. Moreover, LA stimulated the anaerobic utilization of glucose to lactate, which negatively correlated with leptin secretion. Furthermore, LA enhanced phosphorylation of Sp1 and inhibited Sp1 transcriptional activity in 3T3-L1 adipocytes. Moreover, LA inhibited Akt phosphorylation, a downstream target of phosphatidylinositol 3-kinase (PI3K). Treatment with the PI3K inhibitor LY294002 mimicked LA actions, dramatically inhibiting both leptin secretion and gene expression and stimulating Sp1 phosphorylation. All of these data suggest that the phosphorylation of Sp1 and the accompanying reduced DNA-binding activity are likely to be involved in the inhibition of leptin induced by LA, which could be mediated in part by the abrogation of the PI3K/Akt pathway.

Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing Saccharomyces cerevisiae

BackgroundFermentation of xylose to ethanol has been achieved in S. cerevisiae by genetic engineering. Xylose utilization is however slow compared to glucose, and during anaerobic conditions addition of glucose has been necessary for cellular growth. In the current study, the xylose-utilizing strain TMB 3415 was employed to investigate differences between anaerobic utilization of glucose and xylose. This strain carried a xylose reductase (XYL1 K270R) engineered for increased NADH utilization and was capable of sustained anaerobic growth on xylose as sole carbon source. Metabolic and transcriptional characterization could thus for the first time be performed without addition of a co-substrate or oxygen.ResultsAnalysis of metabolic fluxes showed that although the specific ethanol productivity was an order of magnitude lower on xylose than on glucose, product yields were similar for the two substrates. In addition, transcription analysis identified clear regulatory differences between glucose and xylose. Respiro-fermentative metabolism on glucose during aerobic conditions caused repression of cellular respiration, while metabolism on xylose under the same conditions was fully respiratory. During anaerobic conditions, xylose repressed respiratory pathways, although notably more weakly than glucose. It was also observed that anaerobic xylose growth caused up-regulation of the oxidative pentose phosphate pathway and gluconeogenesis, which may be driven by an increased demand for NADPH during anaerobic xylose catabolism.ConclusionCo-factor imbalance in the initial twp steps of xylose utilization may reduce ethanol productivity by increasing the need for NADP+ reduction and consequently increase reverse flux in glycolysis.

Prenatally Induced Changes in Muscle Structure and Metabolic Function Facilitate Exercise-Induced Obesity Prevention

Effective regulation of energy metabolism is vital for the maintenance of optimal health, and an inability to make these dynamic adjustments is a recognized cause of obesity and metabolic disorders. Epidemiological and experimental studies have highlighted the role of prenatal factors in the disease process, and it is now generally accepted that maternal nutrition during pregnancy significantly influences intrauterine development, shaping postnatal health. Consequences of impaired nutrition during fetal development include intrauterine growth restriction (IUGR) and subsequent obesity development in adult life. We have previously shown that prenatal undernutrition has a lasting effect on behavior, with IUGR offspring expressing a higher preference for voluntary exercise, and moderate daily exercise preventing obesity development. The present study investigated skeletal muscle structure in IUGR offspring and how moderate daily exercise drives changes in metabolic pathways that promote obesity prevention. Pregnant Wistar rats were either fed chow ad libitum or undernourished, generating control or IUGR offspring respectively. Although red muscle structure indicated higher oxidative capacity in IUGR offspring, obesity prevention was not due to increased fatty acid oxidation, indicated by decreased peroxisomal proliferator-activated receptor-gamma coactivator 1 and carnitine-palmitoyltransferase 1 expression. In contrast, increased protein kinase Czeta expression and glycogen content in white muscle of exercised IUGR offspring suggests an enhanced capacity for anaerobic utilization of glucose. Furthermore, exercise-induced lactate accumulation was effectively prevented by stimulation of a lactate shuttle, driven by the increases in monocarboxylate transporters-4 and -1 in white muscle. This enhanced metabolic flexibility in IUGR offspring may facilitate muscle contractile performance and therefore support moderate daily exercise for effective obesity prevention.

Conjugated linoleic acid inhibits glucose metabolism, leptin and adiponectin secretion in primary cultured rat adipocytes

Conjugated linoleic acid (CLA) supplementation has been reported to induce insulin resistance in animals and humans, however, the underlying mechanisms remain unclear. The aim of this study was to examine the direct effects of CLA on leptin and adiponectin secretion, two hormones with actions known to influence insulin sensitivity. Isolated rat adipocytes were incubated with CLA (1–200 μM) in the absence and presence of insulin (1.6 nM). CLA inhibited both basal and insulin-stimulated leptin gene expression and secretion (−30 to −40%, P < 0.05–0.01). CLA also inhibited basal adiponectin production (−20 to −40%, P < 0.05–0.01), but not in the presence of insulin. CLA (50–200 μM) decreased basal glucose uptake ( P < 0.05–0.01) and significantly increased the proportion of glucose metabolized to lactate ( P < 0.01). Insulin treatment partially prevented the inhibitory effects of CLA on glucose uptake and induced a significant increase ( P < 0.05–0.01) in the percentage of glucose metabolized to lactate. A strong inverse relationship was observed between the increase in the anaerobic utilization of glucose and the decreases of both leptin and adiponectin secretion. In addition, lipolysis and the expression of the adipogenic transcription factor PPARγ were decreased by CLA. These results indicate that CLA inhibits leptin and adiponectin secretion and suggest that increased anaerobic metabolism of glucose may be involved in these effects. The inhibition of PPARγ could also mediate the inhibition of adiponectin induced by CLA. Furthermore, the inhibition of leptin and adiponectin production induced by CLA may contribute to insulin resistance observed in CLA-treated animals and humans.