Glycerol Synthesis Research Articles

De novo synthesis of milk triglycerides in humans

Mammary gland (MG) de novo lipogenesis contributes significantly to milk fat in animals but little is known in humans. To test the hypothesis that the incorporation of (13)C carbons from [U-(13)C]glucose into fatty acids (FA) and glycerol in triglycerides (TG) will be greater: 1) in milk than plasma TG, 2) during a high-carbohydrate (H-CHO) diet than high-fat (H-FAT) diet, and 3) during feeding than fasting. Seven healthy, lactating women were studied on two isocaloric, isonitrogenous diets. On one occasion, subjects received diets containing H-FAT or H-CHO diet for 1 wk. Incorporation of (13)C from infused [U-(13)C]glucose into FA and glycerol was measured using GC-MS and gene expression in RNA isolated from milk fat globule using microarrays. Incorporation of (13)C2 into milk FA increased with increased FA chain length from C2:0 to C12:0 but progressively declined in C14:0 and C16:0 and was not detected in FA>C16. During feeding, regardless of diets, enrichment of (13)C2 in milk FA and (13)C3 in milk glycerol were ∼ 3- and ∼ 7-fold higher compared with plasma FA and glycerol, respectively. Following an overnight fast during H-CHO and H-FAT diets, 25 and 6%, respectively, of medium-chain FA (MCFA, C6-C12) in milk were derived from glucose but increased to 75 and 25% with feeding. Expression of genes involved in FA or glycerol synthesis was unchanged regardless of diet or fast/fed conditions. The human MG is capable of de novo lipogenesis of primarily MCFA and glycerol, which is influenced by the macronutrient composition of the maternal diet.

Parameter Estimation by Using an Improved Bee Memory Differential Evolution Algorithm (IBMDE) to Simulate Biochemical Pathways

Assessing and estimating essential parameters for a metabolic pathway by using a mathematical model is a significant step in Systems Biology. However, estimating process often faces numerous obstacles, for example when the number of unknown parameters escalates or data has noise, gets trapped in local minima and or having repeated exploration of poor solution during search process. Thus, this study proposes an improved Bee Memory Differential Evolution algorithm (IBMDE), which is a combination of the Differential Evolution algorithm (DE), the Kalman Filter (KF), the Artificial Bee Colony algorithm (ABC), and a memory feature to solve the aforementioned problems. The implemented metabolic pathways for this improved estimation algorithm were glycerol and pyruvate synthesis pathways. IBMDE was successful in generating the estimated optimal kinetic parameter values with noticeable reduction in errors (81.36% and 99.46% respectively) and faster convergence times (6.19% and 15.72% respectively) compared to DE, the Genetic Algorithm (GA), the Nelder Mead (NM), and the Simulated Annealing (SA). The results indicated that, most importantly, the kinetic parameters produced by IBMDE had enhanced the production of desired metabolites than the other estimation algorithms. Besides that, the results also demonstrated the reliability of IBMDE as an estimation algorithm in terms of lower error.

Activation of salt shock response leads to solubilisation of mutant huntingtin in Saccharomyces cerevisiae.

Formation of cytoplasmic and nuclear aggregates is a hallmark of Huntington's disease (HD). Inhibition of aggregation of mutant huntingtin has been suggested to be a feasible approach to slow down the progress of this neurodegenerative disorder. Exposure to environmental stimuli leads to the activation of the stress response machinery of the cell. In this work, we have investigated the effect of salt shock on the aggregation of mutant huntingtin (103Q-htt) in a yeast model of HD. We found that at an optimum concentration of NaCl, the protein no longer formed aggregates and existed in the soluble form. This led to lower oxidative stress in the cell. Salt shock resulted in the synthesis of the osmolyte glycerol, which was partially responsible for the beneficial effect of stress. Surprisingly, we also found increase in the synthesis of another osmolyte, trehalose. Using deletion strains, we were able to show that the effect on solubilisation of mutant huntingtin is due to the synthesis of optimum amounts of both osmolytes. Stress-induced effect was monitored on gene expression. Genes related to proteins of the osmosensory pathway were upregulated on exposure to salt while those coding for stress response proteins were downregulated when solubilisation of mutant huntingtin occurred. Our study shows that activation of stress response elements can have beneficial effect in the solubilisation of huntingtin in a yeast model of HD.

Functionalized Ionic Liquid‐Catalyzed 1‐Feruloyl‐sn‐glycerol Synthesis

AbstractFeruloyl Glycerol (FG) is a potential antioxidant and UV absorbing ingredient in food and cosmetic industries. Transesterifications of ethyl ferulate (EF) with glycerol to synthesize FG were performed using different functionalized ionic liquids (1‐butylsulfonic‐3‐methylimidazolium tosylate, [BSO3HMIM]TS; 1‐propylsulfonic‐3‐methylimidazolium tosylate, [PSO3HMIM]TS; 1‐butylsulfonic‐3‐methylimidazolium trifluoromethanesulfonate, [BSO3HMIM]OTF; 1‐butylsulfonic‐3‐methylimidazolium hydrogen sulfate, [BSO3HMIM]HSO4; N‐methylimidazolium hydrogen sulfate, [HMIM]HSO4; 1‐butyl‐3‐methylimidazolium hydroxide, [BMIM]OH; 1‐butyl‐3‐methylimidazo tetrachloride molysite, [BMIM]FeCl4; and 1‐hexyl‐3‐methylimidazo tetrachloride molysite, [BMIM]FeCl4) as catalysts, respectively. High EF conversion (98.0 ± 1.5 %), 1‐FG (1‐feruloyl‐sn‐glycerol) yield (88.7 ± 1.1 %) and reaction selectivity for 1‐FG (90.5 ± 2.1 %) were obtained using [BSO3HMIM]TS as catalyst. The activation energy (Ea), the Michaelis–Menten kinetic constant (Km), and the maximum initial reaction rate (vmax) of the transesterification are 65.9 ± 3.3 kJ/mol, 1.8 ± 0.1 mol/L, and (1.6 ± 0.4) × 10−2 mol/(L min), respectively. Effects of catalyst loading, reaction temperature, and the molar ratio of EF to glycerol on EF conversion and reaction selectivity for 1‐FG (1‐FG yield/EF conversion) were also investigated.

The Role of Pyruvate Dehydrogenase Kinase in Diabetes and Obesity

The pyruvate dehydrogenase complex (PDC) is an emerging target for the treatment of metabolic syndrome. To maintain a steady-state concentration of adenosine triphosphate during the feed-fast cycle, cells require efficient utilization of fatty acid and glucose, which is controlled by the PDC. The PDC converts pyruvate, coenzyme A (CoA), and oxidized nicotinamide adenine dinucleotide (NAD+) into acetyl-CoA, reduced form of nicotinamide adenine dinucleotide (NADH), and carbon dioxide. The activity of the PDC is up- and down-regulated by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase, respectively. In addition, pyruvate is a key intermediate of glucose oxidation and an important precursor for the synthesis of glucose, glycerol, fatty acids, and nonessential amino acids.

Efficient synthesis of glycerol carbonate/glycidol using 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) based ionic liquids as catalyst

Transesterification of dimethyl carbonate with glycerol to glycerol carbonate has been catalyzed efficiently using basic ionic liquids as catalysts. Activity of all the ILs tested is very high and the best result (96% conversion with 82% selectivity to glycerol carbonate and 18% selectivity to glycidol) was obtained using IL1 as catalyst. The effect of catalyst loading has significant influence on the selectivity pattern. The higher activity of the ionic liquid is explained with a plausible mechanism based on the co-operative effect of both cation and anion.

Os2 MAP kinase-mediated osmostress tolerance in Penicillium digitatum is associated with its positive regulation on glycerol synthesis and negative regulation on ergosterol synthesis

High osmolarity glycerol (HOG) pathway is ubiquitously distributed among eukaryotic organisms and plays an important role in adaptation to changes in the environment. In this study, the Hog1 ortholog in Penicillium digitatum, designated Pdos2, was identified and characterized using a gene knock-out strategy. The ΔPdos2 mutant showed a considerably increased sensitivity to salt stress and cell wall-disturbing agents and a slightly increased resistance to fungicides iprodione and fludioxonil, indicating that Pdos2 is involved in response to hyperosmotic stress, regulation of cell wall integrity and sensitivity to fungicides iprodione and fludioxonil. Surprisingly, the mutant was not affected in response to oxidative stress caused by H2O2. The average lesion size in citrus fruits caused by ΔPdos2 mutant was smaller (approximately 25.0% reduction) than that caused by the wild-type strain of P. digitatum at 4 days post inoculation, which suggests that Pdos2 is needed for full virulence of P. digitatum. Interestingly, in the presence of 0.7M NaCl, the glycerol content was remarkably increased and the ergosterol was decreased in mycelia of the wide-type P. digitatum, whereas the glycerol content was only slightly increased and the ergosterol content remained stable in the ΔPdos2 mutant, suggesting that Pdos2-mediated osmotic adaption is associated with its positive regulation on glycerol synthesis and negative regulation on ergosterol synthesis.

Regulation of pyruvate metabolism in metabolic-related diseases

Pyruvate is an obligatory intermediate in the oxidative disposal of glucose and a major precursor for the synthesis of glucose, glycerol, fatty acids, and non-essential amino acids. Stringent control of the fate of pyruvate is critically important for cellular homeostasis. The regulatory mechanisms for its metabolism are therefore of great interest. Recent advances include the findings that (a) the mitochondrial pyruvate carrier is sensitive to inhibition by thiazolidinediones; (b) pyruvate dehydrogenase kinases induce the Warburg effect in many disease states; and (c) pyruvate carboxylase is an important determinate of the rates of gluconeogenesis in humans with type 2 diabetes. These enzymes are potential therapeutic targets for several diseases.

Glycerol and ethylene glycol co-mediated synthesis of uniform multiple crystalline silver nanowires

The large scale synthesis of multiple crystalline silver nanowires (NWs) with uniform diameter were carried out by using glycerol and ethylene glycol (EG) as co-mediated solvents in the presence of poly(vinyl pyrrolidine) (PVP). Experimental results and structural characterizations reveal that Ag NWs are evolved from the multiple crystalline seeds initially generated by the reduction of AgNO3 with EG and glycerol. Owing to the different reduction ability and viscosity of EG and glycerol, which play an important role for controlling the nucleation at the beginning of reaction, glycerol with high viscosity slows down the migration velocity of Ag0 in favor of forming the uniform Ag NWs with small diameter (40 nm) in the presence of PVP molecules selectively adsorbed on the surface of Ag seeds. The yield of the Ag NWs is dependent on the volume ratio of EG and glycerol. In the absence of EG, large amount of Ag nanoparticles (NPs) and few Ag NWs were created. In contrast, Ag nanorods and polyhedral particles are prepared in the case of no glycol added. This paper provides a new approach for the large scale synthesis of Ag NWs with uniform diameter by simply adjusting the solvent components. Furthermore, V-shaped Ag nanostructure was obtained and the possible growth mechanism was discussed.

Study of the potential of the air lift bioreactor for xylitol production in fed-batch cultures by Debaryomyces hansenii immobilized in alginate beads

Cell immobilization has shown to be especially adequate for xylitol production. This work studies the suitability of the air lift bioreactor for xylitol production by Debaryomyces hansenii immobilized in Ca-alginate operating in fed-batch cultures to avoid substrate inhibition. The results showed that the air lift bioreactor is an adequate system since the minimum air flow required for fluidization was even lower than that leading to the microaerobic conditions that trigger xylitol accumulation by this yeast, also maintaining the integrity of the alginate beads and the viability of the immobilized cells until 3 months of reuses. Maximum productivities and yields of 0.43 g/l/h and 0.71 g/g were achieved with a xylose concentration of 60 g/l after each feeding. The xylose feeding rate, the air flow, and the biomass concentration at the beginning of the fed-batch operation have shown to be critical parameters for achieving high productivities and yields. Although a maximum xylitol production of 139 g/l was obtained, product inhibition was evidenced in batch experiments, which allowed estimating at 200 and 275 g/l the IC50 for xylitol productivity and yield, respectively. The remarkable production of glycerol in the absence of glucose was noticeable, which could not only be attributed to the osmoregulatory function of this polyol in conditions of high osmotic pressure caused by high xylitol concentrations but also to the role of the glycerol synthesis pathway in the regeneration of NAD(+) in conditions of suboptimal microaeration caused by insufficient aeration or high oxygen demand when high biomass concentrations were achieved.

Direct Synthesis of Sorbitol and Glycerol from Cellulose over Ionic Ru/Magnetite Nanoparticles in the Absence of External Hydrogen

A sweet catalyst: A catalyst formed of Ru/functionalized silica-coated magnetite nanoparticles is highly efficient in the one-pot production of sorbitol and glycerol, starting from cellulose and in the absence of an external hydrogen source. The ease of recoverability of the catalyst from the solid residues, and its reuse without loss of activity or selectivity for several runs, is an important green element of the process.

Analysis of osmoadaptation system in budding yeast suggests that regulated degradation of glycerol synthesis enzyme is key to near-perfect adaptation.

In order to maintain its turgor pressure at a desired homeostatic level, budding yeast, Saccharomyces cerevisiae responds to the external variation of the osmotic pressure by varying its internal osmotic pressure through regulation of synthesis and transport of the intracellular glycerol. Hog1PP (dually phosphorylated Hog1), a final effector in the signalling pathway of the hyper osmotic stress, regulates the glycerol synthesis both at transcriptional and non-transcriptional stages. It is known that for a step-change in salt concentration leading to moderate osmotic shock, Hog1PP activity shows a transient response before it returns to the vicinity of pre-stimulus level. It is believed that an integrating process in a negative feedback loop can be a design strategy to yield such an adaptive response. Several negative feedback loops have been identified in the osmoadaptation system in yeast. However, the precise location of the integrating process in the osmoadaptation system which includes signalling, gene regulation, metabolism and biophysical modules is unclear. To address this issue, we developed a reduced model which captures various experimental observations of the osmoadaptation behaviour of wild type and mutant strains. Dynamic simulations and steady state analysis suggested that known information about the osmoadaptation system of budding yeast does not necessarily give a perfect integrating process through the known feedback loops of Hog1PP. On the other hand, regulation of glycerol synthesising enzyme degradation can result in a near integrating process leading to a near-perfect adaptation.

Glycerol synthesis in freeze-resistant rainbow smelt: towards the characterization of a key enzyme glycerol-3-phosphatase

Rainbow smelt (Osmerus mordax) synthesize high amounts of glycerol in winter as a cryoprotectant through the direct dephosphorylation of glycerol-3-phosphate by a phosphatase, glycerol-3-phosphatase (G3Pase). Such a protein is well described in a few species including fungi, bacteria and plants but never studied beyond tissue homogenates in any animal species. Purification, identification and characterization of this enzyme is thus crucial for a better comprehension of the biochemical adaptation in rainbow smelt in response to low temperature and more generally of the biochemical mechanisms involved in glycerol synthesis in animals. This work presents the first attempt to purify G3Pase from smelt liver, the main site of glycerol synthesis for the whole animal. A partial purification was performed, and some characteristics of the protein determined, including optimal pH, K(m) and cation requirements. Smelt G3Pase is most likely a low molecular weight, Mg⁺-dependent and cytosolic phosphatase.

Increasing ethanol titer and yield in a gpd1Δ gpd2Δ strain by simultaneous overexpression of GLT1 and STL1 in Saccharomyces cerevisiae

We have investigated whether simultaneous modification of cofactor metabolism and glycerol in a strain of Saccharomyces cerevisiae can eliminate glycerol synthesis during ethanol production. Two strains, S812 (gpd1Δ gpd2Δ PGK1p-GLT1) and LE17 (gpd1Δ gpd2Δ PGK1p-GLT1 PGKp-STL1) were generated that showed a 8 and 8.2 % increase in the ethanol yield, respectively, compared to the wild type KAM-2 strain. The ethanol titer was improved from 90.4 g/l for KAM-2 to 97.6 g/l for S812 and 97.8 g/l for LE17, respectively. These results provide a new insight into rationalization of metabolic engineering strategies for improvement of ethanol yield through elimination of glycerol production.

Genome-wide transcriptional profiling and enrichment mapping reveal divergent and conserved roles of Sko1 in the Candida albicans osmotic stress response

Candida albicans maintains both commensal and pathogenic states in humans. Here, we have defined the genomic response to osmotic stress mediated by transcription factor Sko1. We performed microarray analysis of a sko1Δ/Δ mutant strain subjected to osmotic stress, and we utilized gene sequence enrichment analysis and enrichment mapping to identify Sko1-dependent osmotic stress-response genes. We found that Sko1 regulates distinct gene classes with functions in ribosomal synthesis, mitochondrial function, and vacuolar transport. Our in silico analysis suggests that Sko1 may recognize two unique DNA binding motifs. Our C. albicans genomic analyses and complementation studies in Saccharomyces cerevisiae showed that Sko1 is conserved as a regulator of carbohydrate metabolism, redox metabolism, and glycerol synthesis. Further, our real time-qPCR results showed that osmotic stress-response genes that are dependent on the kinase Hog1 also require Sko1 for full expression. Our findings reveal divergent and conserved aspects of Sko1-dependent osmotic stress signaling.

Differential response of Dunaliella salina and Dunaliella tertiolecta isolated from brines of Sambhar Salt Lake of Rajasthan (India) to salinities: A study on growth, pigment and glycerol synthesis

Differential response of Dunaliella salina and Dunaliella tertiolecta isolated from brines of Sambhar Salt Lake of Rajasthan (India) to salinities: A study on growth, pigment and glycerol synthesis

Targeting Pyruvate Carboxylase Reduces Gluconeogenesis and Adiposity and Improves Insulin Resistance

We measured the mRNA and protein expression of the key gluconeogenic enzymes in human liver biopsy specimens and found that only hepatic pyruvate carboxylase protein levels related strongly with glycemia. We assessed the role of pyruvate carboxylase in regulating glucose and lipid metabolism in rats through a loss-of-function approach using a specific antisense oligonucleotide (ASO) to decrease expression predominantly in liver and adipose tissue. Pyruvate carboxylase ASO reduced plasma glucose concentrations and the rate of endogenous glucose production in vivo. Interestingly, pyruvate carboxylase ASO also reduced adiposity, plasma lipid concentrations, and hepatic steatosis in high fat–fed rats and improved hepatic insulin sensitivity. Pyruvate carboxylase ASO had similar effects in Zucker Diabetic Fatty rats. Pyruvate carboxylase ASO did not alter de novo fatty acid synthesis, lipolysis, or hepatocyte fatty acid oxidation. In contrast, the lipid phenotype was attributed to a decrease in hepatic and adipose glycerol synthesis, which is important for fatty acid esterification when dietary fat is in excess. Tissue-specific inhibition of pyruvate carboxylase is a potential therapeutic approach for nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes.

Synthesis and characterisation of galactosyl glycerol by β-galactosidase catalysed reverse hydrolysis of galactose and glycerol

A study of an enzymatic method for the production of galactosylglycerol is described. The effects of enzyme sources, enzyme amount, reaction temperature, reaction time, substrate ratio of glycerol to galactose, buffer content and pH on galactosylglycerol yield (mg/ml) were investigated. Under the optimum reaction conditions of β-galactosidases from Kluyveromyces lactis 240 U/ml, temperature 40°C, time 24h, buffer amount 60% (percent volume of buffer to that of substrates, pH 6.5), and the substrate molar ratio of 10 (glycerol16mmol:galactose 1.6mmol), the yield of galactosylglycerol was up to 116.47mg/ml (galactose conversion 55.88%). The product was purified by activated charcoal and Sephadex G-15 column chromatography, up to 96%. The purified galactosylglycerol was fully characterised by MS and NMR, and identified as a mixture of (2R)- and (2S)- 3-O-β-D-galactopyranosyl-glycerol.

Molecular Clone and Expression of a NAD+-Dependent Glycerol-3-Phosphate Dehydrogenase Isozyme Gene from the Halotolerant alga Dunaliella salina

Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD+-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.

Ultrasonic pretreatment for lipase-catalyzed synthesis of 4-methoxy cinnamoyl glycerol

• Ultrasonic pretreatment lipase-catalyzed synthesis of 4-methoxy cinnamoyl glycerol (4MCG) was studied. • The enzymatic method using ultrasonic pretreatment significantly improved overall esterification reaction rate and monoester (4MCG) yield. • The ultrasonic pretreatment process did not damage the lipase activity. 4-Methoxy cinnamoyl glycerol (4MCG) is a very promising UV filters material in personal care products. In order to effectively improve the yield of 4MCG, a systematic study on ultrasonic pretreatment enzymatic esterification for 4MCG products was carried out. An ultrasonic frequency of 35 kHz, ultrasonic power of 150 W and ultrasound irradiation time of 1.5 h was determined to guarantee satisfactory degree of esterification and lipase activity. The optimum production was achieved in organic solvent system at 65 °C with 4MCA to glycerol molar ratio of 1:5, enzyme amount of 15 mg/mL, resulting in a monoester yield of above 66% and 55% after 48 h and 24 h of reaction under ultrasonic pretreatment, respectively. The experimental kinetic data were studied. The reactions were modeled by a system of sequential first-order rate expressions, kinetic parameters were estimated from experimental data fit to the model equations. Results show that the monoester yield in the ultrasonic pretreatment process (24 h) were above 1.5-fold as that in mechanical stirring process without essential damaging to lipase activity. The enzymatic method using ultrasonic pretreatment was obviously superior to the mechanical stirring for enzymatic method and chemical method in terms of conversion rate and the monoester yield. These results are of great significance for applying ultrasonic pretreatment method to prepare 4MCG.