Heterologous Expression of Two Malate Transporters From an Oleaginous Fungus Mucor circinelloides Improved the Lipid Accumulation in Mucor lusitanicus

The fungus, Mucor lusitanicus, is of great interest for microbial lipids, because of its ability to accumulate intracellular lipid using various carbon sources. The biosynthesis of fatty acid requires the reducing power NADPH, and acetyl-CoA, which is produced by the cleavage of citrate in cytosol. In this study, we employed different strategies to increase lipid accumulation in the low lipid-producing fungi via metabolic engineering technology. Hence, we constructed the engineered strain of M. lusitanicus CBS 277.49 by using malate transporter (mt) and 2-oxoglutarate: malate antiporter (sodit) from M. circinelloides WJ11. In comparison with the control strain, the lipid content of the overexpressed strains of mt and sodit genes were increased by 24.6 and 33.8%, respectively. These results showed that mt and sodit can affect the distribution of malate in mitochondria and cytosol, provide the substrates for the synthesis of citrate in the mitochondria, and accelerate the transfer of citrate from mitochondria to cytosol, which could play a significant regulatory role in fatty acid synthesis leading to lipids over accumulation.

Characterization of cellular development and fatty acid accumulation in thraustochytrid Aurantiochytrium strains of Taiwan

Long-chain saturated and polyunsaturated fatty acids of two new thraustochytrid isolates cultured from Taiwan mangroves, Aurantiochytrium sp. IMB169 and Aurantiochytrium sp. IMB171, were characterized through their cell growth and development in relation to their intracellular lipid accumulation using transmission electron microscopy. Flow cytometry in combination with the lipophilic fluorescent dye BODIPY 505/515 was used to stain and characterize intracellular lipid bodies in the two isolates. The transmission electron microscopy and flow cytometry analyses revealed a progressive accumulation of lipid products in IMB169 and IMB171. Further, selective BODIPY stained cells were successfully separated and enriched using flow cytometry at single cell level. Among the two isolates, IMB169 was found to produce a high level of docosahexaenoic acid. The qualitative and analytical results obtained using electron microscopy and flow cytometry studies were validated by gas chromatography (GC). In addition, a quantitative baseline was established using cell growth, flow cytometry and GC analyses for developing an efficient bioprocessing methodology to selectively enrich thraustochytrids phenotypes with desirable characteristics.

Overexpression of lipA or glpD_RuBisCO in the Synechocystis sp. PCC 6803 Mutant Lacking the Aas Gene Enhances Free Fatty-Acid Secretion and Intracellular Lipid Accumulation

Although engineered cyanobacteria for the production of lipids and fatty acids (FAs) are intelligently used as sustainable biofuel resources, intracellularly overproduced FAs disturb cellular homeostasis and eventually generate lethal toxicity. In order to improve their production by enhancing FFAs secretion into a medium, we constructed three engineered Synechocystis 6803 strains including KA (a mutant lacking the aas gene), KAOL (KA overexpressing lipA, encoding lipase A in membrane lipid hydrolysis), and KAOGR (KA overexpressing quadruple glpD/rbcLXS, related to the CBB cycle). Certain contents of intracellular lipids and secreted FFAs of all engineered strains were higher than those of the wild type. Remarkably, the KAOL strain attained the highest level of secreted FFAs by about 21.9%w/DCW at day 5 of normal BG11 cultivation, with a higher growth rate and shorter doubling time. TEM images provided crucial evidence on the morphological changes of the KAOL strain, which accumulated abundant droplets on regions of thylakoid membranes throughout the cell when compared with wild type. On the other hand, BG11-N condition significantly induced contents of both intracellular lipids and secreted FFAs of the KAOL strain up to 37.2 and 24.5%w/DCW, respectively, within 5 days. Then, for the first time, we shone a spotlight onto the overexpression of lipA in the aas mutant of Synechocystis as another potential strategy to achieve higher FFAs secretion with sustainable growth.

Persistent Effect of Advanced Glycated Albumin Driving Inflammation and Disturbances in Cholesterol Efflux in Macrophages

Advanced glycated albumin (AGE-albumin) impairs cholesterol efflux and contributes to inflammation in macrophages. The current study evaluated: (1) the persistence of the deleterious effect of AGE-albumin in cholesterol efflux and in inflammation, and (2) how metabolic control in diabetes mellitus (DM) contributes to attenuate the deleterious role of AGE-albumin in macrophage cholesterol homeostasis. Methods: AGE-albumin was produced in vitro or isolated from uncontrolled DM subjects’ serum before (bGC) and after improved glycemic control (aGC). Albumin samples were incubated with bone marrow-derived macrophages and 14C-cholesterol efflux or LPS- induced cytokine secretion were determined immediately, or after cell resting in culture media alone. The ABCA-1 degradation rate was determined after cell incubation with cycloheximide, and ABCA1 protein level by immunoblot. Oil Red O staining was used to assess intracellular lipid accumulation. Results: A persistent effect of AGE-albumin was observed in macrophages in terms of the secretion of inflammatory cytokines and reduced cholesterol efflux. HDL-mediated 14C-cholesterol efflux was at least two times higher in macrophages treated with aCG-albumin as compared to bGC-albumin, and intracellular lipid content was significantly reduced in aGC-albumin-treated cells. As compared to bGC-albumin, the ABCA-1 protein content in whole cell bulk was 94% higher in aCG-albumin. A 20% increased ABCA-1 decay rate was observed in macrophages treated with albumin from poorly controlled DM. AGE-albumin has a persistent deleterious effect on macrophage lipid homeostasis and inflammation. The reduction of AGEs in albumin ameliorates cholesterol efflux.

Re-charging your fats: Charmm36 parameters for neutral lipids triacylglycerol and diacylglycerol

Neutral lipids (NLs) are an abundant class of cellular lipids. They are characterized by the total lack of charged chemical groups in their structure, and, as a consequence, they play a major role in intracellular lipid storage. NLs that carry a glycerol backbone, such as triacylglycerols (TGs) and diacylglycerols (DGs), are also involved in the biosynthetic pathway of cellular phospholipids, and they have recently been the subject of numerous structural investigations by means of atomistic molecular dynamics (MD) simulations. However, conflicting results on the physicochemical behavior of NLs were observed depending on the nature of the atomistic force field used. Here, we show that current phospholipid-derived CHARMM36 parameters for DGs and TGs cannot reproduce adequately interfacial properties of these NLs, due to excessive hydrophilicity at the glycerol-ester region. By following a CHARMM36-consistent parameterization strategy, we develop new parameters for both TGs and DGs that are compatible with both cutoff- based and Particle Mesh Ewald (PME) schemes for the treatment of Lennard Jones interactions. We show that our new parameters can reproduce interfacial properties of NLs and their behavior in more complex lipid assemblies. We discuss the implications of our findings in the context of intracellular lipid storage and NLs cellular activity.

Posidonia oceanica (L.) Delile Extract Reduces Lipid Accumulation through Autophagy Activation in HepG2 Cells

Posidonia oceanica (L.) Delile is a marine plant traditionally used as an herbal medicine for various health disorders. P. oceanica leaf extract (POE) has been shown to be a phytocomplex with cell-safe bioactivities, including the ability to trigger autophagy. Autophagy is a key pathway to counteract non-alcoholic fatty liver disease (NAFLD) by controlling the breakdown of lipid droplets in the liver. The aim of this study was to explore the ability of POE to trigger autophagy and reduce lipid accumulation in human hepatoma (HepG2) cells and then verify the possible link between the effect of POE on lipid reduction and autophagy activation. Expression levels of autophagy markers were monitored by the Western blot technique in POE-treated HepG2 cells, whereas the extent of lipid accumulation in HepG2 cells was assessed by Oil red O staining. Chloroquine (CQ), an autophagy inhibitor, was used to study the relationship between POE-induced autophagy and intracellular lipid accumulation. POE was found to stimulate an autophagy flux over time in HepG2 cells by lowering the phosphorylation state of ribosomal protein S6, increasing Beclin-1 and LC3-II levels, and decreasing p62 levels. By blocking autophagy with CQ, the effect of POE on intracellular lipid accumulation was clearly reversed, suggesting that the POE phytocomplex may reduce lipid accumulation in HepG2 cells by activating the autophagic process. This work indicates that P. oceanica may be considered as a promising molecule supplier to discover new natural approaches for the management of NAFLD.

Adam21 is dispensable for reproductive processes in mice

Background As a group of membrane-anchored proteins, the proteins containing a disintegrin and metalloprotease domain (ADAMs) control many biological processes, especially for male fertility. Mouse Adam21 was previously found to be specifically expressed in the somatic cells and germ cells of testes, but its functional role during spermatogenesis and male reproductive processes is still unknown. Methods Adam21-null mice were created using the CRISPR/Cas9 system. Quantitative real-time PCR was used for analyzing of gene expression. Histological, cytological and immunofluorescence staining were performed to analyze the phenotypes of mouse testis and epididymis. Intracellular lipid droplets (LDs) were detected by Oil red O (ORO) staining and BODIPY staining. Fertility and sperm characteristics were also detected. Results Here, we successfully generated an Adam21 conventional knockout mouse model via CRISPR/Cas9 technology so that we can explore its potential role in male reproduction. We found that male mice lacking Adam21 have normal fertility without any detectable defects in spermatogenesis or sperm motility. Histological analysis of the seminiferous epithelium showed no obvious spermatogenesis difference between Adam21-null and wild-type mice. Cytological analysis revealed no detectable defects in meiotic progression, neither Sertoli cells nor Leydig cells displayed any defect compared with that of the control mice. All these results suggest that Adam21 might not be essential for male fertility in mice, and its potential function still needs further investigation.

Renal Lipid Metabolism Abnormalities in Obesity and Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma is the most common and deadly type of cancer affecting the kidney, and is characterized histologically by large intracellular lipid deposits. These deposits are thought to result from lipid metabolic reprogramming occurring in tumor cells, but the exact mechanisms and implications of these metabolic alterations are incompletely understood. Obesity is an independent risk factor for clear cell renal cell carcinoma, and is also associated with lipid accumulation in noncancerous epithelial cells of the proximal tubule, where clear cell renal cell carcinoma originates. This article explores the potential link between obesity-associated renal lipid metabolic disturbances and lipid metabolic reprogramming in clear cell renal cell carcinoma, and discusses potential implications for future research.