Glycerol Uptake Research Articles

Glycerol metabolism and transport in yeast and fungi: established knowledge and ambiguities.

There is huge variability among yeasts with regard to their efficiency in utilizing glycerol as the sole source of carbon and energy. Certain species show growth rates with glycerol comparable to those reached with glucose as carbon source; others are virtually unable to utilize glycerol, especially in synthetic medium. Most of our current knowledge regarding glycerol uptake and catabolic pathways has been gained from studying laboratory strains of the model yeast Saccharomyces cerevisiae. The growth of these strains on glycerol is dependent on the presence of medium supplements such as amino acids and nucleobases. In contrast, there is only fragmentary knowledge about S. cerevisiae isolates able to grow in synthetic glycerol medium without such supplements as well as about growth of non-Saccharomyces yeast species on glycerol. Thus, more research is required to understand why certain strains and species show superior growth performance on glycerol compared with common S.cerevisiae laboratory strains. This mini-review summarizes what is known so far about the gene products and pathways involved in glycerol metabolism and transport in yeast and fungi as well as the regulation of these processes.

Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production

Ustilago trichophora RK089 has been found recently as a good natural malic acid producer from glycerol. This strain has previously undergone adaptive laboratory evolution for enhanced substrate uptake rate resulting in the strain U. trichophora TZ1. Medium optimization and investigation of process parameters enabled titers and rates that are able to compete with those of organisms overexpressing major parts of the underlying metabolic pathways. Metabolic engineering can likely further increase the efficiency of malate production by this organism, provided that basic genetic tools and methods can be established for this rarely used and relatively obscure species.Here we investigate and adapt existing molecular tools from U. maydis for use in U. trichophora. Selection markers from U. maydis that confer carboxin, hygromycin, nourseothricin, and phleomycin resistance are applicable in U. trichophora. A plasmid was constructed containing the ip-locus of U. trichophora RK089, resulting in site-specific integration into the genome. Using this plasmid, overexpression of pyruvate carboxylase, two malate dehydrogenases (mdh1, mdh2), and two malate transporters (ssu1, ssu2) was possible in U. trichophora TZ1 under control of the strong Petef promoter. Overexpression of mdh1, mdh2, ssu1, and ssu2 increased the product (malate) to substrate (glycerol) yield by up to 54% in shake flasks reaching a titer of up to 120gL−1. In bioreactor cultivations of U. trichophora TZ1 Petefssu2 and U. trichophora TZ1 Petefmdh2 a drastically lowered biomass formation and glycerol uptake rate resulted in 29% (Ssu1) and 38% (Mdh2) higher specific production rates and 38% (Ssu1) and 46% (Mdh2) increased yields compared to the reference strain U. trichophora TZ1. Investigation of the product spectrum resulted in an 87% closed carbon balance with 134gL−1 malate and biomass (73gL−1), succinate (20gL−1), CO2 (17gL−1), and α-ketoglutarate (8gL−1) as main by-products.These results open up a wide range of possibilities for further optimization, especially combinatorial metabolic engineering to increase the flux from pyruvate to malic acid and to reduce by-product formation.

Adiponectin regulates aquaglyceroporin expression in hepatic stellate cells altering their functional state.

Obesity is a major risk factor for liver fibrosis and tightly associated with low levels of adiponectin. Adiponectin has antifibrogenic activity protecting from liver fibrosis, which is mainly driven by activated hepatic stellate cells (HSC). Aquaporins are transmembrane proteins that allow the movement of water and, in case of aquaglyceroporins (AQPs), of glycerol that is needed in quiescent HSC for lipogenesis. Expression of various AQPs in liver is altered by obesity; however, the mechanisms through which obesity influences HSCs activation and AQPs expression remain unclear. This study aimed to identify obesity-associated factors that are related to HSC AQPs expression activation and lipid storage. Correlations between serum adipokine levels and hepatic AQPs gene expression were analyzed from a cohort of obese patients. AQP and fibrotic gene expression was determined in a HSC line (LX2) and in a hepatocyte cell line (HepG2) after stimulation with adiponectin using quantitative real-time polymerase chain reaction. We found that serum adiponectin significantly correlated with liver AQP3, AQP7, AQP9 gene expressions. In vitro, adiponectin induced upregulation of AQP3 gene and AQP3 protein expression in human HSCs, but not in hepatocytes, while AQP7, AQP9 remained undetectable. Accordingly, HSC stimulated with adiponectin increased glycerol uptake, lipogenic gene expression, and lipid storage while downregulating activation/fibrosis markers. These findings demonstrate that adiponectin is a potent inhibitor of HSC activation and induces AQPs expression. Thus, low serum levels of adiponectin could be a mechanism how obesity affects the functional state of HSC, thereby contributing to obesity-associated liver fibrosis.

Continuous bioproduction of 1,3‐propanediol from biodiesel raw glycerol: Operation with free and immobilized cells of Clostridium butyricum DSM 10702

AbstractThis work investigates the bioproduction of 1,3‐propanediol (1,3‐PD) from biodiesel derived raw glycerol with free and immobilized cells of Clostridium butyricum DSMZ 10702. The cultures were carried out under continuous conditions in a stirred tank bioreactor with suspended cells and a fluidized‐bed bioreactor with immobilized cells. The microorganisms were immobilized by entrapment in calcium alginate gel beads. The effect of dilution rate and glycerol concentration in the feed on 1,3‐PD production was investigated for both bioreactor configurations. Results showed that 1,3‐PD concentration and glycerol uptake decreased when dilution rate increased. Maximum product concentration was similar in both bioreactors, 24.3 g/L and 20.5 g/L for suspended cells and Ca‐alginate immobilized cells, respectively, for a glycerol feed concentration of 50 g/L and a dilution rate of 0.05 h−1. The higher productivities were observed for a dilution rate of 0.15 h−1 for the suspended culture bioreactor and 0.2 h−1 for the immobilized cell bioreactor. Although maximum productivity was similar in both systems, 2.8 g 1,3‐PD/(L · h), cell immobilization was shown to be more resistant to washout at higher dilution rates.

Glycerol-fed microbial fuel cell with a co-culture of Shewanella oneidensis MR-1 and Klebsiella pneumonae J2B.

Glycerol is an attractive feedstock for bioenergy and bioconversion processes but its use in microbial fuel cells (MFCs) for electrical energy recovery has not been investigated extensively. This study compared the glycerol uptake and electricity generation of a co-culture of Shewanella oneidensis MR-1 and Klebsiella pneumonia J2B in a MFC with that of a single species inoculated counterpart. Glycerol was metabolized successfully in the co-culture MFC (MFC-J&M) with simultaneous electricity production but it was not utilized in the MR-1 only MFC (MFC-M). A current density of 10mA/m(2) was obtained while acidic byproducts (lactate and acetate) were consumed in the co-culture MFC, whereas they are accumulated in the J2B-only MFC (MFC-J). MR-1 was distributed mainly on the electrodein MFC-J&M, whereas most of the J2B was observed in the suspension in the MFC-J reactor, indicating that the co-culture of both strains provides an ecological driving force for glycerol utilization using the electrode as an electron acceptor. This suggests that a co-culture MFC can be applied to electrical energy recovery from glycerol, which was previously known as a refractory substrate in a bioelectrochemical system.

The expression of glycerol facilitators from various yeast species improves growth on glycerol of Saccharomyces cerevisiae

Glycerol is an abundant by-product during biodiesel production and additionally has several assets compared to sugars when used as a carbon source for growing microorganisms in the context of biotechnological applications. However, most strains of the platform production organism Saccharomyces cerevisiae grow poorly in synthetic glycerol medium. It has been hypothesized that the uptake of glycerol could be a major bottleneck for the utilization of glycerol in S. cerevisiae. This species exclusively relies on an active transport system for glycerol uptake. This work demonstrates that the expression of predicted glycerol facilitators (Fps1 homologues) from superior glycerol-utilizing yeast species such as Pachysolen tannophilus, Komagataella pastoris, Yarrowia lipolytica and Cyberlindnera jadinii significantly improves the growth performance on glycerol of the previously selected glycerol-consuming S. cerevisiae wild-type strain (CBS 6412-13A). The maximum specific growth rate increased from 0.13 up to 0.18h−1 and a biomass yield coefficient of 0.56gDW/gglycerol was observed. These results pave the way for exploiting the assets of glycerol in the production of fuels, chemicals and pharmaceuticals based on baker's yeast.

Genetic construction of recombinant Pseudomonas chlororaphis for improved glycerol utilization

Abstract This study is to use genetic engineering to improve the glycerol metabolic capability of Pseudomonas chlororaphis which is capable of producing commercially valuable biodegradable poly(hydroxyalkanoate) (PHA) biopolymers and biosurfactant rhamnolipids (RLs). In the study, the glycerol uptake facilitator or aquaglyceroporin gene ( glpF ) and the glycerol kinase ( glpK ) gene were PCR-cloned from E. coli , inserted into a shuttle vector pBS29P2- gfp , and expressed in P. chlororaphis by a Pseudomonas promoter P2. The P. chlororaphis recombinants were then tested for cell growth and glycerol metabolism in chemically defined medium containing 0.5% and 1.0% (v/v) glycerol. The simultaneous expression of glpF and glpK resulted in a shorter lag time for cell growth and a more immediate glycerol consumption by P. chlororaphis . In conclusion, the recombinant P. chlororaphis that grows more efficiently in glycerol is expected to improve the technoeconomics of PHA and RL production using the surplus bioglycerol byproduct stream from biodiesel production.

Elucidating the Effect of Glycerol Concentration and C/N Ratio on Lipid Production Using Yarrowia lipolytica SKY7

The high demand for renewable energy and increased biodiesel production lead to the surplus availability of crude glycerol. Due to the above reason, the bio-based value addition of crude glycerol into various bioproducts is investigated; among them, microbial lipids are attractive. The present study was dedicated to find the optimal glycerol concentration and carbon/nitrogen (C/N) ratio to produce maximum lipid using Yarrowia lipolytica SKY7. The glycerol concentration (34.4 to168.2g/L) and C/N ratio (25 to 150) were selected to investigate to maximize the lipid production. Initial glycerol concentration 112.5g/L, C/N molar ratio of 100, and with 5%v/v inoculum supplementation were found to be optimum for biomass and lipid production. Based on the above optimal parameters, lipid concentration of 43.8%w/w with a biomass concentration of 14.8g/L was achieved. In the case of glycerol concentration, the maximum Yp/s (0.192g/g); Yx/s (0.43g/g) was noted when the initial glycerol concentration was 112.5g/L with C/N molar ratio 100 and inoculum volume 5%v/v. The glycerol uptake was also noted to increase with the increase in glycerol concentration. At low C/N ratio, the glycerol consumption was found to be high (79.43g/L on C/N 25) whereas the glycerol consumption was observed to decrease when the C/N ratio was raised to 150 (40.8g/L).

Glucose-mediated regulation of glycerol uptake in Rhodosporidium toruloides: Insights through transcriptomic analysis on dual substrate fermentation.

Rhodosporidium toruloides is a robust oleaginous yeast that can accumulate lipids to more than 70% of its dry cell mass. Even though it is extensively studied for its fermentation of substrates like glucose and glycerol, limited information is available about its metabolism of mixture of glucose and glycerol. During growth on mixture of glucose and glycerol a typical diauxic growth and higher lipid yields were observed. To understand this phenomenon, RNA-seq analysis was implemented to study the gene expression profiles during growth on mixtures mainly to elucidate regulation of glycerol metabolism. Insights into lipid biosynthesis on mixed substrates are provided at a systems level. Among others, transcriptional profiles showed that glycerol might be produced intracellularly and glycerol kinase (GUT1) and glycerol 3-phosphate dehydrogenase (GUT2) enzymes were not downregulated in the presence of glucose. Transcriptional analysis also showed that the regulation of glycerol uptake in the presence of glucose at transcriptional level is different from that observed in Saccharomyces cerevisiae.

MAP Kinase Hog1 Regulates Metabolic Changes Induced by Hyperosmotic Stress.

Upon hyperosmotic stress, the High Osmolarity Glycerol (HOG) pathway is activated, resulting in phosphorylation of the stress-activated protein kinasei Hog1 (O'Rourke et al., 2002; Saito and Posas, 2012; Brewster and Gustin, 2014). The Hog1/mitogen-activated protein kinase-mediated signaling cascades are essential for sensing hyperosmotic stress and for transmitting these signals to the nucleus to modulate gene expression (O'Rourke et al., 2002; Saito and Posas, 2012; Brewster and Gustin, 2014). MAPK-mediated cascade pathways are composed of three serine/threonine protein kinases (MAPK kinase kinase, MAPK kinase, and MAPK). This signal transduction pathway is well-conserved in eukaryotes (O'Rourke et al., 2002; Saito and Posas, 2012; Brewster and Gustin, 2014). Phosphorylated Hog1 stimulates expression of genes encoding enzymes involved in glycerol production and uptake (O'Rourke et al., 2002; Saito and Posas, 2012; Gomar-Alba et al., 2013; Lee et al., 2013; Babazadeh et al., 2014; Brewster and Gustin, 2014). The glycerol production starts with the reduction of the glycolytic intermediate di-hydroxyl-acetone phosphate (DHAP) to glycerol-3-phosphate (G3P) catalyzed by the NAD+-dependent glycerol-3-phospate dehydrogenase (GPD; Ansell et al., 1997; Pahlman et al., 2001; Valadi et al., 2004). Increased expression of GPD1 enhances glycerol production under hyperosmotic stress (Albertyn et al., 1994; Rep et al., 1999). In addition, Hog1 appears to stimulate the 6-phosphofructo-2-kinase Pfk26, which produces fructose-2,6-diphosphate (F26DP), an allosteric activator of the glycolytic enzyme phosphofructokinase (Pfk1; Dihazi et al., 2004). Hog1 is rapidly phosphorylated upon curcumin treatment, an active polyphenol derived from the spice turmeric (Azad et al., 2014). Trichosporonoides oedocephalis is known to produce large amounts of polyols (erythritol and glycerol). Tohog1 null mutation increased erythritol production and decreased glycerol production, respectively (Li et al., 2016). Therefore, Hog1 is considered to have an important role in the metabolic control, however little is known regarding the changes of metabolites induced by Hog1 from a metabolic perspective. Nuclear magnetic resonance (NMR) spectroscopy is a rapid method that requires minimal sample preparation (Beckonert et al., 2007; Kim et al., 2010). In addition, NMR is a useful technique for structure elucidation due to its various two-dimensional measurements, which makes NMR an ideal tool for the identification and quantification of metabolites (Beckonert et al., 2007; Kim et al., 2010).

Surfactant mediated enhanced glycerol uptake and hydrogen production from biodiesel waste using co-culture of Enterobacter aerogenes and Clostridium butyricum

In the present study, Tween 80, a non-ionic surfactant, has been used for enhanced hydrogen production by crude glycerol bioconversion using co-culture of Enterobacter aerogenes and Clostridium butyricum. The purpose of introducing the surfactant was to decrease the crude glycerol viscosity, so that apparent solubility and bioavailability of glycerol could be improved at the expenses of pretreatment steps. Experiments were planned using central composite design (CCD); crude glycerol and Tween 80 concentrations were optimized whereas, hydrogen production, glycerol utilization and viscosity of the media were considered as responses. The response surface for quadratic model showed, Tween 80 concentration had significant effect (p < 0.05) on all the three responses. Using the optimized conditions at 17.5 g/L crude glycerol and 15 mg/L Tween 80, hydrogen production reached a maximum of 32.1 ± 0.03 mmol/L of medium. The increase in hydrogen production was around 1.25-fold in presence of Tween 80 in comparison to its absence with 25.56 ± 0.91 mmol/L production. Selected optimum conditions were also validated against absence of crude glycerol (4.69 ± 0.76), with pretreated crude glycerol (20.06 ± 0.51) and across mono-culture system (15.43 ± 0.79 to 22.14 ± 0.94). Introduction of Tween 80 to the fermentation medium improved the glycerol utilization rate, resulting in increased hydrogen production and eliminated pretreatment steps.

Phylogenetic diversity and ecophysiology of Candidate phylum Saccharibacteria in activated sludge.

Candidate phylum Saccharibacteria (former TM7) are abundant and widespread in nature, but little is known about their ecophysiology and detailed phylogeny. In this study phylogeny, morphology and ecophysiology of Saccharibacteria were investigated in activated sludge from nine wastewater treatment plants (WWTPs) from Japan and Denmark using the full-cycle 16S rRNA approach in combination with microautoradiography (MAR) and fluorescence in situ hybridization (FISH). Phylogenetic analysis showed that Saccharibacteria from all WWTPs were evenly distributed within subdivision 1 and 3 and in a distinct phylogenetic clade. Three probes were designed for the distinct saccharibacterial groups, and revealed morphotypes representing thin filaments, thick filaments and rods/cocci. MAR-FISH results showed that most probe-defined Saccharibacteria utilized glucose under aerobic-, nitrate reducing- and anaerobic conditions. Some Saccharibacteria also utilized N-acetylglucosamine, oleic acid, amino acids and butyrate, which are not predicted from available genomes so far. In addition, some filamentous Saccharibacteria exhibited β-galactosidase and lipase activities determined using a combination of enzyme-labeled fluorescence and FISH (ELF-FISH). No uptake of acetate, propionate, pyruvate, glycerol and ethanol was observed. These results indicate that Saccharibacteria is a phylogenetically diverse group and play a role in the degradation of various organic compounds as well as sugar compounds under aerobic-, nitrate reducing- and anaerobic conditions.

A Systematic Characterization of Aquaporin-9 Expression in Human Normal and Pathological Tissues.

AQP9 is known to facilitate hepatocyte glycerol uptake. Murine AQP9 protein expression has been verified in liver, skin, epididymis, epidermis and neuronal cells using knockout mice. Further expression sites have been reported in humans. We aimed to verify AQP9 expression in a large set of human normal organs, different cancer types, rheumatoid arthritis synovial biopsies as well as in cell lines and primary cells. Combining standardized immunohistochemistry with high-throughput mRNA sequencing, we found that AQP9 expression in normal tissues was limited, with high membranous expression only in hepatocytes. In cancer tissues, AQP9 expression was mainly found in hepatocellular carcinomas, suggesting no general contribution of AQP9 to carcinogenesis. AQP9 expression in a subset of rheumatoid arthritis synovial tissue samples was affected by Humira, thereby supporting a suggested role of TNFα in AQP9 regulation in this disease. Among cell lines and primary cells, LP-1 myeloma cells expressed high levels of AQP9, whereas low expression was observed in a few other lymphoid cell lines. AQP9 mRNA and protein expression was absent in HepG2 hepatocellular carcinoma cells. Overall, AQP9 expression in human tissues appears to be more selective than in mice.

The effect of age and unilateral leg immobilization for 2 weeks on substrate utilization during moderate-intensity exercise in human skeletal muscle.

This study aimed to provide molecular insight into the differential effects of age and physical inactivity on the regulation of substrate metabolism during moderate-intensity exercise. Using the arteriovenous balance technique, we studied the effect of immobilization of one leg for 2weeks on leg substrate utilization in young and older men during two-legged dynamic knee-extensor moderate-intensity exercise, as well as changes in key proteins in muscle metabolism before and after exercise. Age and immobilization did not affect relative carbohydrate and fat utilization during exercise, but the older men had higher uptake of exogenous fatty acids, whereas the young men relied more on endogenous fatty acids during exercise. Using a combined whole-leg and molecular approach, we provide evidence that both age and physical inactivity result in intramuscular lipid accumulation, but this occurs only in part through the same mechanisms. Age and inactivity have been associated with intramuscular triglyceride (IMTG) accumulation. Here, we attempt to disentangle these factors by studying the effect of 2weeks of unilateral leg immobilization on substrate utilization across the legs during moderate-intensity exercise in young (n=17; 23±1years old) and older men (n=15; 68±1years old), while the contralateral leg served as the control. After immobilization, the participants performed two-legged isolated knee-extensor exercise at 20±1W (∼50% maximal work capacity) for 45min with catheters inserted in the brachial artery and both femoral veins. Biopsy samples obtained from vastus lateralis muscles of both legs before and after exercise were used for analysis of substrates, protein content and enzyme activities. During exercise, leg substrate utilization (respiratory quotient) did not differ between groups or legs. Leg fatty acid uptake was greater in older than in young men, and although young men demonstrated net leg glycerol release during exercise, older men showed net glycerol uptake. At baseline, IMTG, muscle pyruvate dehydrogenase complex activity and the protein content of adipose triglyceride lipase, acetyl-CoA carboxylase 2 and AMP-activated protein kinase (AMPK)γ3 were higher in young than in older men. Furthermore, adipose triglyceride lipase, plasma membrane-associated fatty acid binding protein and AMPKγ3 subunit protein contents were lower and IMTG was higher in the immobilized than the contralateral leg in young and older men. Thus, immobilization and age did not affect substrate choice (respiratory quotient) during moderate exercise, but the whole-leg and molecular differences in fatty acid mobilization could explain the age- and immobilization-induced IMTG accumulation.

A biorefinery approach to microbial oil production from glycerol by Rhodotorula glutinis

The use of biodiesel-derived glycerol as a carbon source for microbial oil production is a biorefinery engineering strategy that aims to reduce the glycerol surplus and make the microbial oil process more cost-effective. In this work, glycerol was used as the sole carbon source for the cultivation of the oleaginous yeast Rhodotorula glutinis along with only yeast extract as a nutrient supply and without pH control. Shake-flask cultivations showed that the specific growth rate and glycerol consumption of Rhodotorula glutinis were higher at lower glycerol concentrations (≤40 g L−1), while higher C/N atom ratios enhanced oil content. The present study extends the knowledge on the influence of the aeration rate and oxygen supply in cellular growth rate and microbial oil production, providing a wiser use of glycerol as an attempt to further reduce process costs. Cultivations at different air flow rates were performed in a 2 L bioreactor and showed that a low aeration rate of 0.5 L min−1 gave the best glycerol and nitrogen uptake rates, resulting in the highest growth (5.3 g L−1) and oil mass fraction (33% of the dry cell weight). A further 68% increase in cellular growth (16.8 g L−1) and a 34% oil mass fraction of the dry cell weight was achieved after applying a feeding strategy targeting combined growth and oil production.

The proliferation impairment induced by AQP3 deficiency is the result of glycerol uptake and metabolism inhibition in gastric cancer cells.

Gastric cancer is a big threat to human health. Effective therapeutic cancer target remains to be discovered. Aquaporin 3 (AQP3) belongs to a family of transmembrane channels that are important in transporting water, glycerol, and other small molecules across the cell membrane. Glycerol that is transported by AQP3 is necessary for cell energy generation and lipid synthesis which fulfill the cell biological processes. Previous studies have shown that AQP3 is implicated in disease progression in several cancer types. However, whether AQP3-regulated glycerol uptake and metabolism were involved in cancer progression remains to be further studied. Our study demonstrated that the expression of AQP3 was positively correlated with glycerol level in human gastric cancer tissues. AQP3 inhibition induced proliferation impairment in gastric cancer cells both in vitro and in vivo. AQP3 inhibition that induced glycerol uptake reduction and glycerol administration would rehabilitate the cell proliferation. The energy and lipid production decreased when AQP3 was knocked down since the cellular glycerol level and several lipogenesis enzymes were downregulated. PI3K/Akt signaling pathway, which was involved in the impaired lipid and ATP production, was also inhibited after AQP3 knockdown. Our study indicated that the energy and lipid production inhibition, which were responsible for gastric cancer cell proliferation impairment, were induced by glycerol uptake reduction after AQP3 knockdown.

Physiological and genetic differences amongst Rhodococcus species for using glycerol as a source for growth and triacylglycerol production.

We analysed the ability of five different rhodococcal species to grow and produce triacylglycerols (TAGs) from glycerol, the main byproduct of biodiesel production. Rhodococcus fascians and Rhodococcus erythropolis grew fast on glycerol, whereas Rhodococcus opacus and Rhodococcus jostii exhibited a prolonged lag phase of several days before growing. Rhodococcus equi only exhibited poor growth on glycerol. R. erythropolis DSMZ 43060 and R. fascians F7 produced 3.9-4.3 g cell biomass l(-1) and 28.4-44.6% cellular dry weight (CDW) of TAGs after 6 days of incubation; whereas R. opacus PD630 and R. jostii RHA1 produced 2.5-3.8 g cell biomass l(-1) and 28.3-38.4% CDW of TAGs after 17 days of growth on glycerol. Genomic analyses revealed two different sets of genes for glycerol uptake and degradation (here named clusters 1 and 2) amongst rhodococci. Those species that possessed cluster 1 (glpFK1D1) (R. fascians and R. erythropolis) exhibited fast growth and lipid accumulation, whereas those that possessed cluster 2 (glpK2D2) (R. opacus, R. jostii and R. equi) exhibited delayed growth and lipid accumulation during cultivation on glycerol. Three glycerol-negative strains were complemented for their ability to grow and produce TAGs by heterologous expression of glpK2 from R. opacus PD630. In addition, we significantly reduced the extension of the lag phase and improved glycerol assimilation and oil production of R. opacus PD630 when expressing glpK1D1 from R. fascians. The results demonstrated that rhodococci are a flexible and amenable biological system for further biotechnological applications based on the reutilization of glycerol.

High fructose diet suppresses exercise-induced increase in AQP7 expression in the in vivo rat heart.

Objective:Cardiac uptake of fructose is thought to be mediated by glucose transporter 5 (GLUT5), whereas the uptake of glycerol is facilitated by aquaporin 7 (AQP7). We aimed to investigate the effect of a high-fructose diet (HFD) on GLUT5 and AQP7 levels in the rat heart subjected to exercise.Methods:Male Sprague–Dawley rats were allocated to control (C; n=11), exercise (E; n=10), HFD (n=12), and HFD plus exercise (HFD-E; n=12) groups. HFD was started 28 days before euthanasia. From day 24 to 27, rats were subjected to moderate exercise, followed by vigorous exercise on day 28 (groups E and HFD-E). Cardiac GLUT5 and AQP7 mRNA levels were determined using RT-PCR. The protein contents of GLUT5 and AQP7 were immunohistochemically assessed. Paired-t, ANOVA with Bonferroni, Kruskal–Wallis, and Bonferroni-corrected Mann–Whitney U tests were used for statistical analysis.Results:GLUT5 mRNA expression and protein content did not differ between the groups. AQP7 mRNA levels significantly increased (4.8-fold) in group E compared with in group C (p<0.001). Compared with group C, no significant change was observed in AQP7 mRNA levels in groups HFD and HFD-E. The AQP7 staining score in group E was significantly higher than that in groups C (p<0.001), E (p<0.001), and HFD-E (p<0.001).Conclusion:Our study indicates that exercise enhances cardiac AQP7 mRNA expression and protein content. However, HFD prevents the exercise-induced increase in cardiac AQP7 expression. This inhibitory effect may be related to the competition between fructose and glycerol as energy substrates in the rat heart subjected to 5 days of physical exercise. (Anatol J Cardiol 2016; 16: 916-22)

Sexual Dimorphism of Adipose and Hepatic Aquaglyceroporins in Health and Metabolic Disorders.

Gender differences in the relative risk of developing metabolic complications, such as insulin resistance or non-alcoholic fatty liver disease (NAFLD), have been reported. The deregulation of glycerol metabolism partly contributes to the onset of these metabolic diseases, since glycerol constitutes a key substrate for the synthesis of triacylglycerols (TAGs) as well as for hepatic gluconeogenesis. The present mini-review covers the sex-­related differences in glycerol metabolism and aquaglyceroporins (AQPs) and its impact in the control of adipose and hepatic fat accumulation as well as in whole-body glucose homeostasis. Plasma glycerol concentrations are increased in women compared to men probably due to the higher lipolytic rate and larger AQP7 amounts in visceral fat as well as the well-known sexual dimorphism in fat mass with women showing higher adiposity. AQP9 represents the primary route for glycerol uptake in hepatocytes, where glycerol is converted by the glycerol-kinase enzyme into glycerol-3-phosphate, a key substrate for de novo synthesis of glucose and TAG. In spite of showing similar hepatic AQP9 protein, women exhibit lower hepatocyte glycerol permeability than men, which might contribute to their lower prevalence of insulin resistance and NAFLD.

Challenges and achievements in the therapeutic modulation of aquaporin functionality.

Aquaporin (AQP) water and solute channels have basic physiological functions throughout the human body. AQP-facilitated water permeability across cell membranes is required for rapid reabsorption of water from pre-urine in the kidneys and for sustained near isosmolar water fluxes e.g. in the brain, eyes, inner ear, and lungs. Cellular water permeability is further connected to cell motility. AQPs of the aquaglyceroporin subfamily are necessary for lipid degradation in adipocytes and glycerol uptake into the liver, as well as for skin moistening. Modulation of AQP function is desirable in several pathophysiological situations, such as nephrogenic diabetes insipidus, Sjögren's syndrome, Menière's disease, heart failure, or tumors to name a few. Attempts to design or to find effective small molecule AQP inhibitors have yielded only a few hits. Challenges reside in the high copy number of AQP proteins in the cell membranes, and spatial restrictions in the protein structure. This review gives an overview on selected physiological and pathophysiological conditions in which modulation of AQP functions appears beneficial and discusses first achievements in the search of drug-like AQP inhibitors.