Synthetic Vesicles for Sustainable Energy Recycling and Delivery of Building Blocks for Lipid Biosynthesis†.

ATP is a universal energy currency that is essential for life. l-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow l-arginine/l-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use l-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder-utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.

Expression and characterisation of human glycerol kinase: the role of solubilising agents and molecular chaperones.

BACKGROUND: Glycerol kinase (GK; EC 2.7.1.30) facilitates the entry of glycerol into pathways of glucose and triglyceride metabolism and may play a potential role in Type 2 diabetes mellitus (T2DM). However, the detailed regulatory mechanisms and structure of the human GK are unknown. METHODS: The human GK gene was cloned into the pET-24a(+) vector and over-expressed in Escherichia coli BL21 (DE3). Since the protein was expressed as inclusion bodies (IBs), various culture parameters and solubilising agents were used but they did not produce bioactive His-GK; however, co-expression of His-GK with molecular chaperones, specifically pKJE7, achieved expression of bioactive His-GK. The overexpressed bioactive His-GK was purified using coloumn chromatography and characterised using enzyme kinetics. RESULTS: The overexpressed bioactive His-GK was purified apparently to homogeneity (∼295-fold) and characterised. The native His-GK was a dimer with a monomeric molecular weight of ∼55 kDa. Optimal enzyme activity was observed in TEA buffer (50 mM) at 7.5 pH. K+ (40 mM) and Mg2+ (2.0 mM) emerged as prefered metal ions for His-GK activity with specific activity 0.780 U/mg protein. The purified His-GK obeyed standard Michaelis-Menten kinetics with Km value of 5.022 µM (R2=0.927) for its substrate glycerol; whereas, that for ATP and PEP was 0.767 mM (R2=0.928) and 0.223 mM (R2=0.967), respectively. Other optimal parameters for the substrate and co-factors were also determined. CONCLUSION: The present study demonstrates that co-expression of molecular chaperones assists with the expression of bioactive human GK for its characterisation.

Evolution and function analysis of glycerol kinase GlpK in Pseudomonasaeruginosa.

Pseudomonas aeruginosa is a Gram-negative bacterium capable of widespread niches, which is also one of the main bacteria that cause patient infection. The metabolic diversity of Pseudomonas aeruginosa is an essential factor in adapting to a variety of environments. Based on the previous studies, adaptive genetic variation in the glycerol kinase GlpK, the glycerol 3-phosphotransferase, contributes to the fitness of bacteria in human bodies, such as Mycobacterium tuberculosis and Escherichia coli. Thus, this study aimed to explore the molecular evolution and function of glpK in P. aeruginosa. Using extensive population genomic data, we have identified the prevalence of two glpK copies in P. aeruginosa that clustered into distinct branches, which were later known as Clade 1 and 2. The evolution analysis revealed that glpK in Clade 1 derived from an ancestral P. aeruginosa species and the other from an ancient horizontal gene transfer event. In addition, we confirmed that the GlpK in Clade 2 still retained glycerol kinase activity but was much weaker than that of GlpK in Clade 1. We demonstrated the importance of the critical amino acid Q70 in GlpK glycerol kinase activity by point mutation. Furthermore, Co-expression network analysis implied that the two glpK copies of P. aeruginosa regulate separate networks and may be a strategy to improve fitness in P. aeruginosa.

An obesogenic diet impairs uncoupled substrate oxidation and promotes whitening of the brown adipose tissue in rats.

Brown adipose tissue (BAT) is rich in mitochondria containing uncoupling protein 1 (UCP1), and dissipates energy through thermogenesis. However, even though BAT mass and its UCP1 content increase in rodents chronically fed a high-fat sucrose-enriched (HFS) diet, marked expansion of adiposity still occurs in these animals, suggesting insufficient BAT-mediated HFS diet-induced thermogenesis. Thus, the objective of this study was to investigate the metabolic and molecular mechanisms that regulate BAT thermogenesis in HFS-induced obesity. To accomplish this, rats were fed either a standard chow or HFS diet for 8 weeks. Subsequently, glucose and fatty acid metabolism and the molecular mechanisms underlying these processes were assessed in freshly isolated primary BAT adipocytes. Despite increasing BAT mass and its UCP1 content, the HFS diet reduced uncoupled glucose and palmitate oxidation in BAT adipocytes. It also markedly diminished tyrosine hydroxylase content and lipolysis in these cells. Conversely, glucose uptake, lactate production, glycerol incorporation into lipids, palmitate incorporation into triacylglycerol (TAG), phosphoenolpyruvate carboxykinase and glycerol kinase levels, and lipoprotein lipase and cluster of differentiation 36 gene expression were increased. In summary, a HFS diet enhanced glyceroneogenesis and shifted BAT metabolism toward TAG synthesis by impairing UCP1-mediated substrate oxidation and by enhancing fatty acid esterification in intact brown adipocytes. These adaptive metabolic responses to chronic HFS feeding attenuated BAT thermogenic capacity and favoured the development of obesity. KEY POINTS: Despite increasing brown adipose tissue (BAT) mass and levels of thermogenic proteins such as peroxisome proliferator-activated receptor γ coactivator 1α, carnitine palmitoyltransferase 1B and uncoupling protein 1 (UCP1), an obesogenic high-fat sucrose-enriched (HFS) diet attenuated uncoupled glucose and fatty acid oxidation in brown adipocytes. Brown adipocytes diverted glycerol and fatty acids toward triacylglycerol (TAG) synthesis by elevating the cellular machinery that promotes fatty acid uptake along with phosphoenolpyruvate carboxykinase and glycerol kinase levels. The HFS diet increased glucose uptake that supported lactate production and provided substrate for glyceroneogenesis and TAG synthesis in brown adipocytes. Impaired UCP-1-mediated thermogenic capacity and enhanced TAG storage in BAT adipocytes were consistent with reduced adipose triglyceride lipase and tyrosine hydroxylase levels in HFS diet-fed animals.

Elevating Phospholipids Production Yarrowia lipolytica from Crude Glycerol.

Phospholipids (PLs) are a class of lipids with many proven biological functions. They are commonly used in lipid replacement therapy to enrich cell membranes damaged in chronic neurodegenerative diseases, cancer, or aging processes. Due to their amphipathic nature, PLs have been widely used in food, cosmetic, and pharmaceutical products as natural emulsifiers and components of liposomes. In Yarrowia lipolytica, PLs are synthesized through a similar pathway like in higher eukaryotes. However, PL biosynthesis in this yeast is still poorly understood. The key intermediate in this pathway is phosphatidic acid, which in Y. lipolytica is mostly directed to the production of triacylglycerols and, in a lower amount, to PL. This study aimed to deliver a strain with improved PL production, with a particular emphasis on increased biosynthesis of phosphatidylcholine (PC). Several genetic modifications were performed: overexpression of genes from PL biosynthesis pathways as well as the deletion of genes responsible for PL degradation. The best performing strain (overexpressing CDP-diacylglycerol synthase (CDS) and phospholipid methyltransferase (OPI3)) reached 360% of PL improvement compared to the wild-type strain in glucose-based medium. With the substitution of glucose by glycerol, a preferred carbon source by Y. lipolytica, an almost 280% improvement of PL was obtained by transformant overexpressing CDS, OPI3, diacylglycerol kinase (DGK1), and glycerol kinase (GUT1) in comparison to the wild-type strain. To further increase the amount of PL, the optimization of culture conditions, followed by the upscaling to a 2 L bioreactor, were performed. Crude glycerol, being a cheap and renewable substrate, was used to reduce the costs of PL production. In this process 653.7 mg/L of PL, including 352.6 mg/L of PC, was obtained. This study proved that Y. lipolytica is an excellent potential producer of phospholipids, especially from waste substrates.

Glucocorticoids decrease thermogenic capacity and increase triacylglycerol synthesis by glycerokinase activation in the brown adipose tissue of rats.

Although it is well established that glucocorticoids inactivate thermogenesis and promote lipid accumulation in interscapular brown adipose tissue (IBAT), the underlying mechanisms remain unknown. We found that dexamethasone treatment (1 mg/kg) for 7 days in rats decreased the IBAT thermogenic activity, evidenced by its lower responsiveness to noradrenaline injection associated with reduced content of mitochondrial proteins, respiratory chain protein complexes, noradrenaline, and the ß3 -adrenergic receptor. In parallel, to understand better how dexamethasone increases IBAT lipid content, we also investigated the activity of the ATP citrate lyase (ACL), a key enzyme of de novo fatty acid synthesis, glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway, and the three glycerol-3-P generating pathways: (1) glycolysis, estimated by 2-deoxyglucose uptake, (2) glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase activity and pyruvate incorporation into triacylglycerol-glycerol, and (3) direct phosphorylation of glycerol, investigated by the content and activity of glycerokinase. Dexamethasone increased the mass and the lipid content of IBAT as well as plasma levels of glucose, insulin, non-esterified fatty acid, and glycerol. Furthermore, dexamethasone increased ACL and G6PD activities (79% and 48%, respectively). Despite promoting a decrease in the incorporation of U-[14 C]-glycerol into triacylglycerol (~54%), dexamethasone increased the content (~55%) and activity (~41%) of glycerokinase without affecting glucose uptake or glyceroneogenesis. Our data suggest that glucocorticoid administration reduces IBAT thermogenesis through sympathetic inactivation and stimulates glycerokinase activity and content, contributing to increased generation of glycerol-3-P, which is mostly used to esterify fatty acid and increase triacylglycerol content promoting IBAT whitening.

Improvement of the catalytic performance of glycerol kinase from Bacillus subtilis by chromosomal site-directed mutagenesis.

Glycerol kinase is the key enzyme in glycerol metabolism, and its catalytic efficiency has an important effect on glycerol utilization. Based on an analysis of the glycerol utilization pathway and regulation mechanism in B. subtilis, we conducted site-directed mutagenesis of the key glycerol kinase gene (glpK) on the chromosome to improve the glycerol utilization efficiency of Bacillus subtilis. Recombinant wild-type Bacillus subtilis glycerol kinase (BsuGlpKWT) and two mutants (BsuGlpKM270I and BsuGlpKS71V) were successfully overexpressed in Escherichia coli BL21(DE3) and purified by Ni-IDA metal chelate chromatography. The specific activity of the BsuGlpKM270I mutant (62.6 U/mg) was significantly higher (296.2%) than that of wild-type BsuGlpKWT (15.8 U/mg). By contrast, the mutant BsuGlpKS71V (4.89 U/mg) exhibited lower (69.1%) activity than BsuGlpKWT, which suggested that variant S71V exhibited reduced catalytic efficiency for the substrate. Furthermore, the mutant strain B. subtilis M270I was constructed using a markerless delivery system, and exhibited a higher specific growth rate (improved by 11.3%, from 0.453 ± 0.012 to 0.511 ± 0.017 h-1) and higher maximal biomass (cell dry weight increased by 16%, from 0.577 ± 0.033 to 0.721 ± 0.015 g/L) than the parental strain with a shortened lag phase (2 ~ 4 h shorter) in M9 minimal medium with glycerol. These results indicate that the mutated glpK resulted in improved glycerol utilization, which has broad application prospects.

The reliance of glycerol utilization by Cupriavidus necator on CO2 fixation and improved glycerol catabolism.

While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO2) fixation and refixation. As mixotrophic growth occurs in the wildtype due to low consumption rates of glycerol, CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle is essential. The deletion of both cbbX copies encoding putative RuBisCO-activases (AAA + ATPase) resulted in a sharp slowdown of growth and glycerol consumption. Activase activity is necessary for functioning carboxylation by RuBisCO. Each of the two copies compensates for the loss of the other, as suggested by observed expression levels. The strong tendency towards autotrophy supports previous investigations of glycerol growth and emphasizes the versatility of the metabolism of C. necator H16. Mixotrophy with glycerol-utilization and CO2 fixation with a high dependence on the CBB is automatically occurring unless transportation and degradation of glycerol are optimized. Parallel engineering of CO2 fixation and glycerol degradation is suggested towards application for value-added production from crude glycerol. KEY POINTS: • Growth on glycerol is highly dependent on efficient carbon fixation via CBB cycle. • CbbX is essential for the efficiency of RuBisCO in C. necator H16. • Expression of glycerol degradation pathway enzymes accelerates glycerol utilization.

Enhancing protein perdeuteration by experimental evolution of Escherichia coli K-12 for rapid growth in deuterium-based media.

Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affects growth of cells. Here, we have studied growth of Escherichia coli MG1655, a wild-type laboratory strain of E. coli K-12, in deuterated glycerol minimal medium. The growth rate and final biomass in deuterated medium is substantially reduced compared to cells grown in ordinary medium. By using a multi-generation adaptive laboratory evolution-based approach, we have isolated strains that show increased fitness in deuterium-based growth media. Whole-genome sequencing identified the genomic changes in the obtained strains and show that there are multiple routes to genetic adaptation to growth in deuterium-based media. By screening a collection of single-gene knockouts of nonessential genes, no specific gene was found to be essential for growth in deuterated minimal medium. Deuteration of proteins is of importance for NMR spectroscopy, neutron protein crystallography, neutron reflectometry, and small angle neutron scattering. The laboratory evolved strains, with substantially improved growth rate, were adapted for recombinant protein production by T7 RNA polymerase overexpression systems and shown to be suitable for efficient production of perdeuterated soluble and membrane proteins for structural biology applications.

Deletion of pleiotrophin impairs glucose tolerance and liver metabolism in pregnant mice: Moonlighting role of glycerol kinase.

Pleiotrophin is a pleiotropic cytokine that has been demonstrated to have a critical role in regulating energy metabolism, lipid turnover and plasticity of adipose tissue. Here, we hypothesize that this cytokine can be involved in regulatory processes of glucose and lipid homeostasis in the liver during pregnancy. Using 18-days pregnant Ptn-deficient mice, we evaluated the biochemical profile (circulating variables), tissue mRNA expression (qPCR) and protein levels of key enzymes and transcription factors involved in main metabolic pathways. Ptn deletion was associated with a reduction in body weight gain, hyperglycemia and glucose intolerance. Moreover, we observed an impairment in glucose synthesis and degradation during late pregnancy in Ptn-/- mice. Hepatic lipid content was significantly lower (73.6%) in Ptn-/- mice and was associated with a clear reduction in fatty acid, triacylglycerides and cholesterol synthesis. Ptn deletion was accompanying with a diabetogenic state in the mother and a decreased expression of key proteins involved in glucose and lipid uptake and metabolism. Moreover, Ptn-/- pregnant mice have a decreased expression of transcription factors, such as PPAR-α, regulating lipid uptake and glucose and lipid utilization. Furthermore, the augmented expression and nuclear translocation of glycerol kinase, and the decrease in NUR77 protein levels in the knock-out animals can further explain the alterations observed in hepatic glucose metabolism. Our results point out for the first time that pleiotrophin is an important player in maintaining hepatic metabolic homeostasis during late gestation, and further highlighted the moonlighting role of glycerol kinase in the regulation of maternal glucose homeostasis during pregnancy.

Glycerol suppresses glucose consumption in trypanosomes through metabolic contest.

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as "catabolite repression," allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named "metabolic contest" for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This "metabolic contest" depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met.

Application of a New Engineered Strain of Yarrowia lipolytica for Effective Production of Calcium Ketoglutarate Dietary Supplements.

The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol-oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO3, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 µg/g of kynurenic acid, which increases the health-promoting value of the product.

GlcA-mediated glycerol-3-phosphate synthesis contributes to the oxidation resistance of Aspergillus fumigatus via decreasing the cellular ROS.

Fungi activate corresponding metabolic pathways in response to different carbon sources to adapt to different environments. Previous studies have shown that the glycerol kinase GlcA that phosphorylates glycerol to the intermediate glycerol-3-phosphate (G3P) is required for the growth of Aspergillus fumigatus when glycerol is used as the sole carbon source. The present study identified there were two putative glycerol kinases, GlcA and GlcB, in A. fumigatus but glycerol activated only glcA promoter but not glcB promoter, although both glcA and glcB could encode glycerol kinase. Under normal culture conditions, the absence of glcA caused no detectable colony phenotypes on glucose and other tested carbon sources except glycerol, indicating dissimilation of glucose and these tested carbon sources bypassed requirement of glcA. Notably, the oxidative stress agent H2O2 on the background of glucose medium clearly induced GlcA expression and promoted G3P synthesis. Deletion and overexpression of glcA elicited sensitivity and resistance to oxidative stress agent H2O2, respectively, accompanied by decrease and increase of G3P production. In addition, the sensitivity to oxidative stress in the glcA mutant was probably associated with dysfunction of mitochondria with a decreased mitochondrial membrane potential and an abnormal accumulation of the cellular reactive oxygen species (ROS). Furthermore, overexpressing the glycerol-3-phosphate dehydrogenase GfdA thatcatalyzes the reduction of dihydroxyacetone phosphate (DHAP) to G3P rescued phenotypes of the glcA null mutant to H2O2. Therefore, the present study suggests that GlcA-involved G3P synthesis participates in oxidative stress tolerance of A. fumigatus via regulating the cellular ROS level.

Structural Characterization of Glycerol Kinase from the Thermophilic Fungus Chaetomium thermophilum.

Glycerol is an organic compound that can be utilized as an alternative source of carbon by various organisms. One of the ways to assimilate glycerol by the cell is the phosphorylative catabolic pathway in which its activation is catalyzed by glycerol kinase (GK) and glycerol-3-phosphate (G3P) is formed. To date, several GK crystal structures from bacteria, archaea, and unicellular eukaryotic parasites have been solved. Herein, we present a series of crystal structures of GK from Chaetomium thermophilum (CtGK) in apo and glycerol-bound forms. In addition, we show the feasibility of an ADP-dependent glucokinase (ADPGK)-coupled enzymatic assay to measure the CtGK activity. New structures described in our work provide structural insights into the GK catalyzed reaction in the filamentous fungus and set the foundation for understanding the glycerol metabolism in eukaryotes.

Designing next generation recombinant protein expression platforms by modulating the cellular stress response in Escherichia coli.

BACKGROUND: A cellular stress response (CSR) is triggered upon recombinant protein synthesis which acts as a global feedback regulator of protein expression. To remove this key regulatory bottleneck, we had previously proposed that genes that are up-regulated post induction could be part of the signaling pathways which activate the CSR. Knocking out some of these genes which were non-essential and belonged to the bottom of the E. coli regulatory network had provided higher expression of GFP and L-asparaginase. RESULTS: We chose the best performing double knockout E. coli BW25113ΔelaAΔcysW and demonstrated its ability to enhance the expression of the toxic Rubella E1 glycoprotein by 2.5-fold by tagging it with sfGFP at the C-terminal end to better quantify expression levels. Transcriptomic analysis of this hyper-expressing mutant showed that a significantly lower proportion of genes got down-regulated post induction, which included genes for transcription, translation, protein folding and sorting, ribosome biogenesis, carbon metabolism, amino acid and ATP synthesis. This down-regulation which is a typical feature of the CSR was clearly blocked in the double knockout strain leading to its enhanced expression capability. Finally, we supplemented the expression of substrate uptake genes glpK and glpD whose down-regulation was not prevented in the double knockout, thus ameliorating almost all the negative effects of the CSR and obtained a further doubling in recombinant protein yields. CONCLUSION: The study validated the hypothesis that these up-regulated genes act as signaling messengers which activate the CSR and thus, despite having no casual connection with recombinant protein synthesis, can improve cellular health and protein expression capabilities. Combining gene knockouts with supplementing the expression of key down-regulated genes can counter the harmful effects of CSR and help in the design of a truly superior host platform for recombinant protein expression.

Glycerol kinase stimulates uncoupling protein 1 expression by regulating fatty acid metabolism in beige adipocytes.

Browning of adipose tissue is induced by specific stimuli such as cold exposure and consists of up-regulation of thermogenesis in white adipose tissue. Recently, it has emerged as an attractive target for managing obesity in humans. Here, we performed a comprehensive analysis to identify genes associated with browning in murine adipose tissue. We focused on glycerol kinase (GYK) because its mRNA expression pattern is highly correlated with that of uncoupling protein 1 (UCP1), which regulates the thermogenic capacity of adipocytes. Cold exposure-induced Ucp1 up-regulation in inguinal white adipose tissue (iWAT) was partially abolished by Gyk knockdown (KD) in vivo Consistently, the Gyk KD inhibited Ucp1 expression induced by treatment with the ß-adrenergic receptors (ßAR) agonist isoproterenol (Iso) in vitro and resulted in impaired uncoupled respiration. Gyk KD also suppressed Iso- and adenylate cyclase activator-induced transcriptional activation and phosphorylation of the cAMP response element-binding protein (CREB). However, we did not observe these effects with a cAMP analog. Therefore Gyk KD related to Iso-induced cAMP products. In Iso-treated Gyk KD adipocytes, stearoyl-CoA desaturase 1 (SCD1) was up-regulated, and monounsaturated fatty acids such as palmitoleic acid (POA) accumulated. Moreover, a SCD1 inhibitor treatment recovered the Gyk KD-induced Ucp1 down-regulation and POA treatment down-regulated Iso-activated Ucp1 Our findings suggest that Gyk stimulates Ucp1 expression via a mechanism that partially depends on the ßAR-cAMP-CREB pathway and Gyk-mediated regulation of fatty acid metabolism.

Affinity shift of ATP upon glycerol binding to a glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1.

Glycerol kinase (GK) is a key enzyme of glycerol metabolism. It participates in glycolysis and lipid membrane biosynthesis. A hexamer of GK from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1(Tk-GK) was identified as a substrate-binding form of the enzyme. Here, the X-ray crystal structure analysis and the biochemical analysis was done and the relationships between its unique oligomer structure and substrate binding affinity were investigated. Wild type GK and mutant K271E GK, which disrupts the hexamer formation interface, were crystallized with and without their substrates and analyzed at 2.19-3.05 Å resolution. In the absence of glycerol, Tk-GK was a dimer in solution. In the presence of its glycerol substrate, however, it became a hexamer consisting of three symmetrical dimers about the threefold axis. Through glycerol binding, all Tk-GK molecules in the hexamer were in closed form as a result of domain-motion. The closed form of Tk-GK had tenfold higher ATP affinity than the open form of Tk-GK. The hexamer structure stabilized the closed conformation and enhanced ATP binding affinity when the GK was bound to glycerol. This molecular mechanism is quite simple activity regulation mechanism among known GKs.

Hepatic glycerol metabolism is early reprogrammed in rat liver cancer development.

Evidence shows that oral glycerol supplementation during the early stages of rat liver cancer reduces the growth of preneoplastic lesions. Besides, human hepatocellular carcinoma (HCC) cells display decreased expression of glycerol channel aquaporin 9 (AQP9) and also diminished glycerol-3-phosphate (G3P) content. According to this, we analyzed glycerol metabolism during the initial stages of rat liver carcinogenesis. Wistar rats were subjected to a 2-phase model of hepatocarcinogenesis (initiated-promoted, IP group) or left untreated (control, C group). Different features of glycerol metabolism were compared between both groups. IP animals showed increased plasma free glycerol levels and liver AQP9 protein expression. Also, IP rats showed increased glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH) hepatic activities. Gluconeogenesis from glycerol both in vivo and in isolated perfused liver was higher in rats having liver preneoplasia. Nevertheless, preneoplastic foci notably reduced AQP9 and GK protein expressions, displaying a reduced ability to import glycerol and to convert it into G3P, as a way to preserve preneoplastic hepatocytes from the deleterious effect of G3P. In conclusion, the metabolic shift that takes place in the initial stages of liver cancer development comprises an increased hepatic utilization of glycerol for gluconeogenesis. Enhanced glucose production from glycerol is mostly carried out by the surrounding non-preneoplastic tissue and can be used as an energy source for the early transformed liver cells.

Glycerol kinase enhances hepatic lipid metabolism by repressing nuclear receptor subfamily 4 group A1 in the nucleus.

Glycerol kinase (GYK) plays a critical role in hepatic metabolism by converting glycerol to glycerol 3-phosphate in an ATP-dependent reaction. GYK isoform b is the only glycerol kinase present in whole cells, and has a non-enzymatic moonlighting function in the nucleus. GYK isoform b acts as a co-regulator of nuclear receptor subfamily 4 group A1 (NR4A1) and participates in the regulation of hepatic glucose metabolism by protein-protein interaction with NR4A1. Herein, GYK expression was found to upregulate the expression of NR4A1-mediated lipid metabolism-related genes (SREBP1C, FASN, ACACA, and GPAM) in HEK293T and L02 cells, and in mouse in vivo studies. GYK expression increased blood levels of cholesterol, triglyceride, and high-density lipoprotein cholesterol, but not low-density lipoprotein cholesterol levels. It enhanced the transcriptional activity of Nr4a1 target genes by negatively cooperating with NR4A1 and its enzymatic activity or by other undefined moonlighting functions. This enhancement was observed in both normal and diabetic mice. We also found a feed-forward regulation loop between GYK and NR4A1, serving as part of a GYK-NR4A1 regulatory mechanism in hepatic metabolism. Thus, GYK regulates the effect of NR4A1 on hepatic lipid metabolism in normal and diabetic mice, partially through the cooperation of GYK and NR4A1.

Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.

African trypanosomiasis, sleeping sickness in humans or nagana in animals, is a potentially fatal neglected tropical disease and a threat to 65 million human lives and 100 million small and large livestock animals in sub-Saharan Africa. Available treatments for this devastating disease are few and have limited efficacy, prompting the search for new drug candidates. Simultaneous inhibition of the trypanosomal glycerol kinase (TGK) and trypanosomal alternative oxidase (TAO) is considered a validated strategy toward the development of new drugs. Our goal is to develop a TGK-specific inhibitor for coadministration with ascofuranone (AF), the most potent TAO inhibitor. Here, we report on the identification of novel compounds with inhibitory potency against TGK. Importantly, one of these compounds (compound 17) and its derivatives (17a and 17b) killed trypanosomes even in the absence of AF. Inhibition kinetics revealed that derivative 17b is a mixed-type and competitive inhibitor for TGK and TAO, respectively. Structural data revealed the molecular basis of this dual inhibitory action, which, in our opinion, will aid in the successful development of a promising drug to treat trypanosomiasis. Although the EC50 of compound 17b against trypanosome cells was 1.77 µM, it had no effect on cultured human cells, even at 50 µM.-Balogun, E. O., Inaoka, D. K., Shiba, T., Tsuge, C., May, B., Sato, T., Kido, Y., Nara, T., Aoki, T., Honma, T., Tanaka, A., Inoue, M., Matsuoka, S., Michels, P. A. M., Watanabe, Y.-I., Moore, A. L., Harada, S., Kita, K. Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.