Large enrichments in fatty acid 2H/1H ratios distinguish respiration from aerobic fermentation in yeast Saccharomyces cerevisiae.

Shifts in the hydrogen stable isotopic composition (2H/1H ratio) of lipids relative to water (lipid/water 2H-fractionation) at natural abundances reflect different sources of the central cellular reductant, NADPH, in bacteria. Here, we demonstrate that lipid/water 2H-fractionation (2εfattyacid/water) can also constrain the relative importance of key NADPH pathways in eukaryotes. We used the metabolically flexible yeast Saccharomyces cerevisiae, a microbial model for respiratory and fermentative metabolism in industry and medicine, to investigate 2εfattyacid/water. In chemostats, fatty acids from glycerol-respiring cells were >550‰ 2H-enriched compared to those from cells aerobically fermenting sugars via overflow metabolism, a hallmark feature in cancer. Faster growth decreased 2H/1H ratios, particularly in glycerol-respiring cells by 200‰. Variations in the activities and kinetic isotope effects among NADP+-reducing enzymes indicate cytosolic NADPH supply as the primary control on 2εfattyacid/water. Contributions of cytosolic isocitrate dehydrogenase (cIDH) to NAPDH production drive large 2H-enrichments with substrate metabolism (cIDH is absent during fermentation but contributes up to 20 percent NAPDH during respiration) and slower growth on glycerol (11 percent more NADPH from cIDH). Shifts in NADPH demand associated with cellular lipid abundance explain smaller 2εfattyacid/water variations (<30‰) with growth rate during fermentation. Consistent with these results, tests of murine liver cells had 2H-enriched lipids from slower-growing, healthy respiring cells relative to fast-growing, fermenting hepatocellular carcinoma. Our findings point to the broad potential of lipid 2H/1H ratios as a passive natural tracker of eukaryotic metabolism with applications to distinguish health and disease, complementing studies that rely on complex isotope-tracer addition methods.

Evaluation of enzyme-constrained genome-scale model through metabolic engineering of anaerobic co-production of 2,3-butanediol and glycerol by Saccharomyces cerevisiae.

Enzyme-constrained genome-scale models (ecGEMs) have potential to predict phenotypes in a variety of conditions, such as growth rates or carbon sources. This study investigated if ecGEMs can guide metabolic engineering efforts to swap anaerobic redox-neutral ATP-providing pathways in yeast from alcoholic fermentation to equimolar co-production of 2,3-butanediol and glycerol. With proven pathways and low product toxicity, the ecGEM solution space aligned well with observed phenotypes. Since this catabolic pathway provides only one-third of the ATP of alcoholic fermentation (2/3 versus 2 ATP per glucose), the ecGEM predicted a growth decrease from 0.36 h-1 in the reference to 0.175 h-1 in the engineered strain. However, this <3-fold decrease would require the specific glucose consumption rate to increase. Surprisingly, after the pathway swap the engineered strain immediately grew at 0.15 h-1 with a glucose consumption rate of 29 mmol (g CDW)-1 h-1, which was indeed higher than reference (23 mmol (g CDW)-1 h-1) and one of the highest reported for S. cerevisiae. The accompanying 2,3-butanediol- (15.8 mmol (g CDW)-1 h-1) and glycerol (19.6 mmol (g CDW)-1 h-1) production rates were close to predicted values. Proteomics confirmed that this increased consumption rate was facilitated by enzyme reallocation from especially ribosomes (from 25.5 to 18.5 %) towards glycolysis (from 28.7 to 43.5 %). Subsequently, 200 generations of sequential transfer did not improve growth of the engineered strain, showing the use of ecGEMs in predicting opportunity space for laboratory evolution. The observations in this study illustrate both the current potential, as well as future improvements, of ecGEMs as a tool for both metabolic engineering and laboratory evolution.

The bloodstream form of Trypanosoma brucei displays non-canonical gluconeogenesis.

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.

Regulation of metabolism and proton motive force generation during mixed carbon fermentation by an Escherichia coli strain lacking the FOF1-ATPase.

Proton FOF1-ATPase is the key enzyme in E. coli under fermentative conditions. In this study the role of E. coli proton ATPase in the µ and formation of metabolic pathways during the fermentation of mixture of glucose, glycerol and formate using the DK8 (lacking FOF1) mutant strain was investigated. It was shown that the contribution of FOF1-ATPase in the specific growth rate was ∼45 %. Formate was not taken up in the DK8 strain during the initial hours of the growth. The utilization rates of glucose and glycerol were unchanged in DK8, however, the production of succinate, lactate and ethanol was decreased causing a reduction of the redox state up to -450 mV. Moreover, the contribution of FOF1-ATPase in the interplay between H+ and H2 cycles was described depending on the bacterial growth phase and main utilizing substrate. Besides, the H2 production rate in the DK8 strain was decreased by ∼60 % at 20 h and was absent at 72 h. Δp was decreased from -157 ± 4.8 mV to -140 ± 4.2 mV at 20 h and from -195 ± 5.9 mV to -148 ± 4.4 mV at 72 h, compared to WT. Taken together it can be concluded that during fermentation of mixed carbon sources metabolic cross talk between FOF1-ATPase-TrkA-Hyd-Fdh-H is taking place for maintaining the cell energy balance via regulation proton motive force.

Impact of cell wall polysaccharide modifications on the performance of Pichia pastoris: novel mutants with enhanced fitness and functionality for bioproduction applications.

BACKGROUND: Pichia pastoris is a widely utilized host for heterologous protein expression and biotransformation. Despite the numerous strategies developed to optimize the chassis host GS115, the potential impact of changes in cell wall polysaccharides on the fitness and performance of P. pastoris remains largely unexplored. This study aims to investigate how alterations in cell wall polysaccharides affect the fitness and function of P. pastoris, contributing to a better understanding of its overall capabilities. RESULTS: Two novel mutants of GS115 chassis, H001 and H002, were established by inactivating the PAS_chr1-3_0225 and PAS_chr1-3_0661 genes involved in ß-glucan biosynthesis. In comparison to GS115, both modified hosts exhibited a looser cell surface and larger cell size, accompanied by faster growth rates and higher carbon-to-biomass conversion ratios. When utilizing glucose, glycerol, and methanol as exclusive carbon sources, the carbon-to-biomass conversion rates of H001 surpassed GS115 by 10.00%, 9.23%, and 33.33%, respectively. Similarly, H002 exhibited even higher increases of 32.50%, 12.31%, and 53.33% in carbon-to-biomass conversion compared to GS115 under the same carbon sources. Both chassis displayed elevated expression levels of green fluorescent protein (GFP) and human epidermal growth factor (hegf). Compared to GS115/pGAPZ A-gfp, H002/pGAPZ A-gfp showed a 57.64% higher GFP expression, while H002/pPICZα A-hegf produced 66.76% more hegf. Additionally, both mutant hosts exhibited enhanced biosynthesis efficiencies of S-adenosyl-L-methionine and ergothioneine. H001/pGAPZ A-sam2 synthesized 21.28% more SAM at 1.14 g/L compared to GS115/pGAPZ A-sam2, and H001/pGAPZ A-egt1E obtained 45.41% more ERG at 75.85 mg/L. The improved performance of H001 and H002 was likely attributed to increased supplies of NADPH and ATP. Specifically, H001 and H002 exhibited 5.00-fold and 1.55-fold higher ATP levels under glycerol, and 6.64- and 1.47-times higher ATP levels under methanol, respectively, compared to GS115. Comparative lipidomic analysis also indicated that the mutations generated richer unsaturated lipids on cell wall, leading to resilience to oxidative damage. CONCLUSIONS: Two novel P. pastoris chassis hosts with impaired ß-1,3-D-glucan biosynthesis were developed, showcasing enhanced performances in terms of growth rate, protein expression, and catalytic capabilities. These hosts exhibit the potential to serve as attractive alternatives to P. pastoris GS115 for various bioproduction applications.

Enhanced interleukin-6 in human adipose tissue vein after sprint exercise: Results from a pilot study.

BACKGROUND: Low-volume sprint exercise is likely to reduce body fat. Interleukin (IL-6) may mediate this by increasing adipose tissue (AT) lipolysis. Therefore, the exchange of AT IL-6 and glycerol, a marker of lipolysis, was examined in 10 healthy subjects performing three 30-s all-out sprints. METHODS: Blood samples were obtained from brachial artery (a) and a superficial subcutaneous vein (v) on the anterior abdominal wall up to 9 min after the last sprint and analysed for IL-6 and glycerol. RESULTS: Arterial IL-6 increased 2-fold from rest to last sprint. AT venous IL-6 increased 15-fold from 0.4 ± 0.4 at rest to 7.0 ± 4 pg × mL-1 (p < 0.0001) and AT v-a difference increased 45-fold from 0.12 ± 0.3 to 6.0 ± 5 pg x mL-1 (p < 0.0001) 9 min after last sprint. Arterial glycerol increased 2.5-fold from rest to 9 min postsprint 1 (p < 0.0001) and was maintained during the exercise period. AT venous and v-a difference of glycerol increased 2-fold from rest to 9 min postsprint 1 (p < 0.0001 and p = 0.01, respectively), decreased until 18 min postsprint 2 (p < 0.001 and p < 0.0001), and then increased again until 9 min after last sprint (both p < 0.01). CONCLUSIONS: The concurrent increase in venous IL-6 and glycerol in AT after last sprint is consistent with an IL-6 induced lipolysis in AT. Glycerol data also indicated an initial increase in lipolysis after sprint 1 that was unrelated to IL-6. Increased IL-6 in adipose tissue may, therefore, complement other sprint exercise-induced lipolytic agents.

Dynamic flux balance analysis of 1,3-propanediol production by clostridium butyricum fermentation.

To study the relationship between the yield of 1,3-propanediol (1,3-PDO) and the flux change of the Clostridium butyricum metabolic pathway, an optimized calculation method based on dynamic flux balance analysis was used by combining genome-scale flux balance analysis with a kinetic model. A more comprehensive and extensive metabolic pathway was obtained by optimization calculations. The primary extended branches include: the dihydroxyacetone node, which enters the pentose phosphate pathway; the α-oxoglutarate node, which has synthetic metabolic pathways for glutamic acid and amino acids; and the serine and homocysteine nodes, which produce cystathionine before homocysteine enters the methionine cycle pathway. According to the expanded metabolic network, the flux distribution of key nodes in the metabolic pathway and the relationship between the flux distribution ratio of nodes and the yield of 1,3-PDO were analyzed. At the dihydroxyacetone node, the flux of dihydroxyacetone converted to dihydroxyacetone phosphate was positively correlated with the yield of 1,3-PDO. As an important intermediate product, the flux change in the metabolic pathway of α-oxoglutarate reacting with amino acids to produce glutamic acid is positively correlated with the yield. When pyruvate was used as the central node to convert into lactic acid and α-oxoglutarate, the proportion of branch flux was negatively correlated with the yield of 1,3-PDO. These studies provide a theoretical basis for the optimization and further study of the metabolic pathway of C. butyricum.

Glycerol-3-Phosphate Acyltransferase GPAT9 Enhanced Seed Oil Accumulation and Eukaryotic Galactolipid Synthesis in Brassica napus.

Glycerol-3-phosphate acyltransferase GPAT9 catalyzes the first acylation of glycerol-3-phosphate (G3P), a committed step of glycerolipid synthesis in Arabidopsis. The role of GPAT9 in Brassica napus remains to be elucidated. Here, we identified four orthologs of GPAT9 and found that BnaGPAT9 encoded by BnaC01T0014600WE is a predominant isoform and promotes seed oil accumulation and eukaryotic galactolipid synthesis in Brassica napus. BnaGPAT9 is highly expressed in developing seeds and is localized in the endoplasmic reticulum (ER). Ectopic expression of BnaGPAT9 in E. coli and siliques of Brassica napus enhanced phosphatidic acid (PA) production. Overexpression of BnaGPAT9 enhanced seed oil accumulation resulting from increased 18:2-fatty acid. Lipid profiling in developing seeds showed that overexpression of BnaGPAT9 led to decreased phosphatidylcholine (PC) and a corresponding increase in phosphatidylethanolamine (PE), implying that BnaGPAT9 promotes PC flux to storage triacylglycerol (TAG). Furthermore, overexpression of BnaGPAT9 also enhanced eukaryotic galactolipids including monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), with increased 36:6-MGDG and 36:6-DGDG, and decreased 34:6-MGDG in developing seeds. Collectively, these results suggest that ER-localized BnaGPAT9 promotes PA production, thereby enhancing seed oil accumulation and eukaryotic galactolipid biosynthesis in Brassica napus.

Genome-Wide Analysis of Glycerol-3-Phosphate Acyltransferase (GPAT) Family in Perilla frutescens and Functional Characterization of PfGPAT9 Crucial for Biosynthesis of Storage Oils Rich in High-Value Lipids.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step in triacylglycerol (TAG) biosynthesis. However, GPAT members and their functions remain poorly understood in Perilla frutescens, a special edible-medicinal plant with its seed oil rich in polyunsaturated fatty acids (mostly α-linolenic acid, ALA). Here, 14 PfGPATs were identified from the P. frutescens genome and classified into three distinct groups according to their phylogenetic relationships. These 14 PfGPAT genes were distributed unevenly across 11 chromosomes. PfGPAT members within the same subfamily had highly conserved gene structures and four signature functional domains, despite considerable variations detected in these conserved motifs between groups. RNA-seq and RT-qPCR combined with dynamic analysis of oil and FA profiles during seed development indicated that PfGPAT9 may play a crucial role in the biosynthesis and accumulation of seed oil and PUFAs. Ex vivo enzymatic assay using the yeast expression system evidenced that PfGPAT9 had a strong GPAT enzyme activity crucial for TAG assembly and also a high substrate preference for oleic acid (OA, C18:1) and ALA (C18:3). Heterogeneous expression of PfGPAT9 significantly increased total oil and UFA (mostly C18:1 and C18:3) levels in both the seeds and leaves of the transgenic tobacco plants. Moreover, these transgenic tobacco lines exhibited no significant negative effect on other agronomic traits, including plant growth and seed germination rate, as well as other morphological and developmental properties. Collectively, our findings provide important insights into understanding PfGPAT functions, demonstrating that PfGPAT9 is the desirable target in metabolic engineering for increasing storage oil enriched with valuable FA profiles in oilseed crops.

Promoting FADH2 Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli.

Hydroxytyrosol is a natural polyphenolic compound widely used in the food and drug industries. The current commercial production of hydroxytyrosol relies mainly on plant extracts, which involve long extraction cycles and various raw materials. Microbial fermentation has potential value as an environmentally friendly and low-cost method. Here, a de novo biosynthetic pathway of hydroxytyrosol has been designed and constructed in an Escherichia coli strain with released tyrosine feedback inhibition. By introduction of hpaBC from E. coli and ARO10 and ADH6 from Saccharomyces cerevisiae, the de novo biosynthesis of hydroxytyrosol was achieved. An important finding in cofactor engineering is that the introduction of L-amino acid deaminase (LAAD) promotes not only cofactor regeneration but also metabolic flow redistribution. To further enhance the hydroxylation process, different 4-hydroxyphenylacetate 3-monooxygenase (hpaB) mutants and HpaBC proteins from different sources were screened. Finally, after optimization of the carbon source, pH, and seed medium, the optimum engineered strain produced 9.87 g/L hydroxytyrosol in a 5 L bioreactor. This represents the highest titer reported to date for de novo biosynthesis of hydroxytyrosol in microorganisms.

New Cytotoxic Monoalkyl Glycerol Ether from the Red Sea Soft Coral Nephthea mollis.

A new monoalkyl glycerol ether, 3-(n-henicosyloxy)propane-1,2-diol (1), was isolated from the CH2 Cl2 /MeOH crude extract of the Red Sea soft coral Nephthea mollis. Additionally, three known related analogs were identified: chimyl alcohol (2), batyl alcohol (3), and 3-(icosyloxy)propane-1,2-diol (4). The chemical structure of 3-(n-henicosyloxy)propane-1,2-diol was determined using advanced spectroscopic analyses, including 1D, 2D Nuclear Magnetic Resonance (NMR), Electron Ionization mass spectra (EI-MS), and High-Resolution Electron Spray Ionization mass spectra (HR-ESI-MS) analyses. Furthermore, the identification of chimyl alcohol, batyl alcohol and 3-(icosyloxy)propane-1,2-diol was achieved by studying their EI mass fragmentation analyses and comparing their mass data with those previously reported in the literature. The cytotoxic activity of the Nephthea mollis crude extract and 3-(n-henicosyloxy)propane-1,2-diol was evaluated against five human cancer cell lines: HepG2 (hepatocellular carcinoma), MCF-7 (breast carcinoma), NCI-1299 (lung carcinoma), HeLa (cervical cancer cell), and HT-29 (colon adenocarcinoma). Moreover, 3-(n-henicosyloxy)propane-1,2-diol revealed moderate cytotoxicity against the HeLa cell lines with an IC50 value of 24.1 µM, while showing inactivity against the remaining cell lines (IC50 >100 µM).

Electrogenic Staphylococcus epidermidis colonizes nasal cavities and alleviates IL-6 progression induced by the SARS2-CoV nucleocapsid protein.

AIM: Certain probiotic bacteria have been shown to possess an immunomodulatory effect and a protective effect on influenza infections. Using the Staphylococcus epidermidis K1 colonized mice model, we assessed the effect of nasal administration of glycerol or flavin mononucleotide (FMN) on the production of interleukin (IL)-6 mediated by the severe acute respiratory syndrome coronavirus 2 (SARS2-CoV) nucleocapsid protein (NPP). METHODS AND RESULTS: FMN, one of the key electron donors for the generation of electricity facilitated by S. epidermidis ATCC 12228, was detected in the glycerol fermentation medium. Compared to the S. epidermidis ATCC 12228, the S. epidermidis K1 isolate showed significant expression of the electron transfer genes, including pyruvate dehydrogenase (pdh), riboflavin kinase (rk), 1,4-dihydroxy-2-naphthoate octaprenyltransferase (menA), and type II NADH quinone oxidoreductase (ndh2). Institute of cancer research (ICR) mice were intranasally administered with S. epidermidis K1 with or without pretreatment with riboflavin kinase inhibitors, then nasally treated with glycerol or FMN before inoculating the NPP. Furthermore, J774A.1 macrophages were exposed to NPP serum and then treated with NPP of SARS2-CoV. The IL-6 levels in the bronchoalveolar lavage fluid (BALF) of mice and macrophages were quantified using a mouse IL-6 enzyme-linked immunosorbent assay kit. CONCLUSIONS: Here, we report that nasal administration of NPP strongly elevates IL-6 levels in both BALF and J774A.1 macrophages. It is worth noting that NPP-neutralizing antibodies can decrease IL-6 levels in macrophages. The nasal administration of glycerol or FMN to S. epidermidis K1-colonized mice results in a reduction of NPP-induced IL-6 production.

Sir2 and Glycerol Underlie the Pro-Longevity Effect of Quercetin during Yeast Chronological Aging.

Quercetin (QUER) is a natural polyphenolic compound endowed with beneficial properties for human health, with anti-aging effects. However, although this flavonoid is commercially available as a nutraceutical, target molecules/pathways underlying its pro-longevity potential have yet to be fully clarified. Here, we investigated QUER activity in yeast chronological aging, the established model for simulating the aging of postmitotic quiescent mammalian cells. We found that QUER supplementation at the onset of chronological aging, namely at the diauxic shift, significantly increases chronological lifespan (CLS). Consistent with the antioxidant properties of QUER, this extension takes place in concert with a decrease in oxidative stress. In addition, QUER triggers substantial changes in carbon metabolism. Specifically, it promotes an enhancement of a pro-longevity anabolic metabolism toward gluconeogenesis due to improved catabolism of C2 by-products of yeast fermentation and glycerol. The former is attributable to the Sir2-dependent activity of phosphoenolpyruvate carboxykinase and the latter to the L-glycerol 3-phosphate pathway. Such a combined increased supply of gluconeogenesis leads to an increase in the reserve carbohydrate trehalose, ensuring CLS extension. Moreover, QUER supplementation to chronologically aging cells in water alone amplifies their long-lived phenotype. This is associated with intracellular glycerol catabolism and trehalose increase, further indicating a QUER-specific influence on carbon metabolism that results in CLS extension.

Glycerol as a precursor for hepatic de novo glutathione synthesis in human liver.

BACKGROUND: Glycerol is a substrate for gluconeogenesis and fatty acid esterification in the liver, processes which are upregulated in obesity and may contribute to excess fat accumulation. Glycine and glutamate, in addition to cysteine, are components of glutathione, the major antioxidant in the liver. In principle, glycerol could be incorporated into glutathione via the TCA cycle or 3-phosphoglycerate, but it is unknown whether glycerol contributes to hepatic de novo glutathione biosynthesis. METHODS: Glycerol metabolism to hepatic metabolic products including glutathione was examined in the liver from adolescents undergoing bariatric surgery. Participants received oral [U-13C3]glycerol (50 mg/kg) prior to surgery and liver tissue (0.2-0.7g) was obtained during surgery. Glutathione, amino acids, and other water-soluble metabolites were extracted from the liver tissue and isotopomers were quantified with nuclear magnetic resonance spectroscopy. RESULTS: Data were collected from 8 participants (2 male, 6 female; age 17.1 years [range 14-19]; BMI 47.4 kg/m2 [range 41.3-63.3]). The concentrations of free glutamate, cysteine, and glycine were similar among participants, and so were the fractions of 13C-labeled glutamate and glycine derived from [U-13C3]glycerol. The signals from all component amino acids of glutathione - glutamate, cysteine and glycine - were strong and analyzed to obtain the relative concentrations of the antioxidant in the liver. The signals from glutathione containing [13C2]glycine or [13C2]glutamate derived from the [U-13C3]glycerol drink were readily detected, and 13C-labelling patterns in the moieties were consistent with the patterns in corresponding free amino acids from the de novo glutathione synthesis pathway. The newly synthesized glutathione with [U-13C3]glycerol trended to be lower in obese adolescents with liver pathology. CONCLUSIONS: This is the first report of glycerol incorporation into glutathione through glycine or glutamate metabolism in human liver. This could represent a compensatory mechanism to increase glutathione in the setting of excess glycerol delivery to the liver.

Breast cancer cell-derived exosome-delivered microRNA-155 targets UBQLN1 in adipocytes and facilitates cancer cachexia-related fat loss.

Cachexia occurrence and development are associated with loss of white adipose tissues, which may be involved with cancer-derived exosomes. This study attempted to characterize the functional mechanisms of breast cancer (BC) cell-derived exosome-loaded microRNA (miR)-155 in cancer cachexia-related fat loss. Exosomes were incubated with preadipocytes and cellular lipid droplet accumulation was observed using Oil Red O staining. Western blotting evaluated the cellular levels of lipogenesis marker peroxisome proliferator activated receptor gamma (PPARγ) and adiponectin, C1Q and collagen domain containing (AdipoQ). Differentiated adipocytes were incubated with exosomes, and phosphate hormone sensitive lipase (P-HSL), adipose triglyceride lipase (ATGL) and glycerol were detected in adipocytes, in addition to uncoupling protein 1 (UCP1) and leptin levels. A mouse model of cancer cachexia was established where cancer exosomes were injected intravenously. The changes in body weight and tumor-free body weights were recorded and serum glycerol levels and lipid accumulation in adipose tissues were determined. Also, the relationship between miR-155 and UBQLN1 was predicted and verified. BC exosome treatment reduced PPARγ and AdipoQ protein levels, promoted the levels of P-HSL and ATGL proteins, facilitated glycerol release, increased UCP1 expression and lowered leptin expression in adipocytes. Exosomal miR-155 inhibited lipogenesis in preadipocytes and boosted the browning of white adipose tissues. miR-155 downregulation alleviated cancer exosome-induced browning of white adipose tissues and fat loss. Mechanistically, miR-155 targeted UBQLN1, and UBQLN1 upregulation reversed the impacts of cancer exosomes. miR-155 loaded by BC cell-derived exosomes significantly affects white adipose browning and inhibition of cancer-derived exosomes.

Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis.

The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the cytosolic reaction is ∼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.

Mycobacterial phosphodiesterase Rv0805 is a virulence determinant and its cyclic nucleotide hydrolytic activity is required for propionate detoxification.

Cyclic AMP (cAMP) signaling is essential to Mycobacterium tuberculosis (Mtb) pathogenesis. However, the roles of phosphodiesterases (PDEs) Rv0805, and the recently identified Rv1339, in cAMP homeostasis and Mtb biology are unclear. We found that Rv0805 modulates Mtb growth within mice, macrophages and on host-associated carbon sources. Mycobacterium bovis BCG grown on a combination of propionate and glycerol as carbon sources showed high levels of cAMP and had a strict requirement for Rv0805 cNMP hydrolytic activity. Supplementation with vitamin B12 or spontaneous genetic mutations in the pta-ackA operon restored the growth of BCGΔRv0805 and eliminated propionate-associated cAMP increases. Surprisingly, reduction of total cAMP levels by ectopic expression of Rv1339 restored only 20% of growth, while Rv0805 complementation fully restored growth despite a smaller effect on total cAMP levels. Deletion of an Rv0805 localization domain also reduced BCG growth in the presence of propionate and glycerol. We propose that localized Rv0805 cAMP hydrolysis modulates activity of a specialized pathway associated with propionate metabolism, while Rv1339 has a broader role in cAMP homeostasis. Future studies will address the biological roles of Rv0805 and Rv1339, including their impacts on metabolism, cAMP signaling and Mtb pathogenesis.

Metabolic engineering of Escherichia coli for the production of an antifouling agent zosteric acid.

Zosteric acid (ZA) is a Zostera species-derived, sulfated phenolic acid compound with antifouling activity and has gained much attention due to its nontoxic and biodegradable characteristics. However, the yield of Zostera species available for ZA extraction is limited by natural factors, such as season, latitude, light, and temperature. Here we report the development of metabolically engineered Escherichia coli strains capable of producing ZA from glucose and glycerol. First, intracellular availability of the sulfur donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) was enhanced by knocking out the cysH gene responsible for PAPS consumption and overexpressing the genes required for PAPS biosynthesis. Co-overexpression of the genes encoding tyrosine ammonia-lyase, sulfotransferase 1A1, ATP sulfurylase, and adenosine 5'-phosphosulfate kinase constructed ZA producing strain with enhanced PAPS supply. Second, the feedback-resistant forms of aroG and tyrA genes (encoding 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase and chorismate mutase, respectively) were overexpressed to relieve the feedback regulation of L-tyrosine biosynthesis. Third, the pykA gene involved in phosphoenolpyruvate-consuming reaction, the regulator gene tyrR, the competing pathway gene pheA, and the ptsHIcrr genes essential for the PEP:carbohydrate phosphotransferase system were deleted. Moreover, all genes involved in the shikimate pathway and the talA, tktA, and tktB genes in the pentose phosphate pathway were examined for ZA production. The PTS-independent glucose uptake system, the expression vector system, and the carbon source were also optimized. As a result, the best-performing strain successfully produced 1.52 g L-1 ZA and 1.30 g L-1p-hydroxycinnamic acid from glucose and glycerol in a 700 mL fed-batch bioreactor.

Increased Aquaporin-7 Expression Is Associated with Changes in Rat Brown Adipose Tissue Whitening in Obesity: Impact of Cold Exposure and Bariatric Surgery.

Glycerol is a key metabolite for lipid accumulation in insulin-sensitive tissues. We examined the role of aquaporin-7 (AQP7), the main glycerol channel in adipocytes, in the improvement of brown adipose tissue (BAT) whitening, a process whereby brown adipocytes differentiate into white-like unilocular cells, after cold exposure or bariatric surgery in male Wistar rats with diet-induced obesity (DIO) (n = 229). DIO promoted BAT whitening, evidenced by increased BAT hypertrophy, steatosis and upregulation of the lipogenic factors Pparg2, Mogat2 and Dgat1. AQP7 was detected in BAT capillary endothelial cells and brown adipocytes, and its expression was upregulated by DIO. Interestingly, AQP7 gene and protein expressions were downregulated after cold exposure (4 °C) for 1 week or one month after sleeve gastrectomy in parallel to the improvement of BAT whitening. Moreover, Aqp7 mRNA expression was positively associated with transcripts of the lipogenic factors Pparg2, Mogat2 and Dgat1 and regulated by lipogenic (ghrelin) and lipolytic (isoproterenol and leptin) signals. Together, the upregulation of AQP7 in DIO might contribute to glycerol influx used for triacylglycerol synthesis in brown adipocytes, and hence, BAT whitening. This process is reversible by cold exposure and bariatric surgery, thereby suggesting the potential of targeting BAT AQP7 as an anti-obesity therapy.

Non-Transport Functions of Aquaporins.

Although it has been more than 20 years since the first aquaporin was discovered, the specific functions of many aquaporins are still under investigation, because various mice lacking aquaporins have no significant phenotypes. And in many studies, the function of aquaporin is not directly related to its transport function. Therefore, this chapter will focus on some unexpected functions of aquaporins, such the decreased tumor angiogenesis in AQP1 knockout mice, and AQP1 promotes cell migration, possibly by accelerating the water transport in lamellipodia of migrating cells. AQP transports glycerol, and water regulates glycerol content in epidermis and fat, thereby regulating skin hydration/biosynthesis and fat metabolism. AQPs may also be involved in neural signal transduction, cell volume regulation, and organelle physiology. AQP1, AQP3, and AQP5 are also involved in cell proliferation. In addition, AQPs have also been reported to play roles in inflammation in various tissues and organs. The functions of these AQPs may not depend on the permeability of small molecules such as water and glycerol, suggesting AQPs may play more roles in different biological processes in the body.