Ceramide-mediated gut dysbiosis enhances cholesterol esterification and promotes colorectal tumorigenesis in mice.

Colorectal cancer (CRC) severely threatens human health and life span. An effective therapeutic strategy has not been established because we do not clearly know its pathogenesis. Here, we report that ceramide and sterol O-acyltransferase 1 (SOAT1) have roles in both spontaneous and chemical-induced intestinal cancers. We first found that miRNA-148a deficiency dramatically increased mouse gut dysbiosis through upregulating ceramide synthase 5 (Cers5) expression, which promoted ceramide synthesis afterward. The newly generated ceramide further promoted both azoxymethane/dextran sodium sulfate-induced (AOM/DSS-induced) and ApcMin/+ spontaneous intestinal tumorigenesis via increasing mouse gut dysbiosis. Meanwhile, increased level of ceramide correlated with the significant enhancements of both ß-catenin activity and colorectal tumorigenesis in a TLR4-dependent fashion. Next, we found a direct binding of ß-catenin to SOAT1 promoter to activate transcriptional expression of SOAT1, which further induced cholesterol esterification and colorectal tumorigenesis. In human patients with CRC, the same CERS5/TLR4/ß-catenin/SOAT1 axis was also found to be dysregulated. Finally, the SOAT1 inhibitor (avasimibe) showed significant levels of therapeutic effects on both AOM/DSS-induced and ApcMin/+ spontaneous intestinal cancer. Our study clarified that ceramide promoted CRC development through increasing gut dysbiosis, further resulting in the increase of cholesterol esterification in a SOAT1-dependent way. Treatment with avasimibe to specifically decrease cholesterol esterification could be considered as a clinical strategy for effective CRC therapy in a future study.

Performing under pressure: esterification activity of dry fermented solids in subcritical and supercritical CO2.

OBJECTIVE: Lipases are often used in immobilized form, but commercial immobilized lipases are costly. An alternative is to produce lipases in solid-state fermentation, dry the solids and then use the "dry fermented solids" (DFS) directly. We produced DFS by growing Burkholderia contaminans on a mixture of sugarcane bagasse and sunflower seed meal and used the DFS to esterify oleic acid with ethanol in subcritical and supercritical CO2 at 40 °C. RESULTS: Compared to a control without CO2 at atmospheric pressure, subcritical CO2 at 30 bar improved esterification activity 1.2-fold. Higher pressures, including supercritical pressures up to 150 bar, reduced activity to less than 80% of the control. At 30 bar, the esterification activity was improved a further 1.8-fold with the addition of 9% water (i.e. 9 g water per 100 g oleic acid) to the reaction medium. CONCLUSION: A subcritical CO2 atmosphere, with the addition of a small amount of water, improved the esterification activity of DFS containing lipases of Burkholderia contaminans.

Cell wall remodeling and vesicle trafficking mediate the root clock in Arabidopsis.

In Arabidopsis thaliana, lateral roots initiate in a process preceded by periodic gene expression known as the root clock. We identified the vesicle-trafficking regulator GNOM and its suppressor, ADENOSINE PHOSPHATE RIBOSYLATION FACTOR GTPase ACTIVATION PROTEIN DOMAIN3, as root clock regulators. GNOM is required for the proper distribution of pectin, a mediator of intercellular adhesion, whereas the pectin esterification state is essential for a functional root clock. In sites of lateral root primordia emergence, both esterified and de-esterified pectin variants are differentially distributed. Using a reverse-genetics approach, we show that genes controlling pectin esterification regulate the root clock and lateral root initiation. These results indicate that the balance between esterified and de-esterified pectin states is essential for proper root clock function and the subsequent initiation of lateral root primordia.

Esterification of side-chain oxysterols by lysosomal phospholipase A2.

Side-chain oxysterols produced from cholesterol either enzymatically or non-enzymatically show various bioactivities. Lecithin-cholesterol acyltransferase (LCAT) esterifies the C3-hydroxyl group of these sterols as well as cholesterol. Lysosomal phospholipase A2 (LPLA2) is related to LCAT but does not catalyze esterification of cholesterol. First, esterification of side-chain oxysterols by LPLA2 was investigated using recombinant mouse LPLA2 and dioleoyl-PC/sulfatide/oxysterol liposomes under acidic conditions. TLC and LC-MS/MS showed that the C3 and C27-hydroxyl groups of 27-hydroxycholesterol could be individually esterified by LPLA2 to form a monoester with the C27-hydroxyl preference. Cholesterol did not inhibit this reaction. Also, LPLA2 esterified other side-chain oxysterols. Their esterifications by mouse serum containing LCAT supported the idea that their esterifications by LPLA2 occur at the C3-hydroxyl group. N-acetylsphingosine (NAS) acting as an acyl acceptor in LPLA2 transacylation inhibited the side-chain oxysterol esterification by LPLA2. This suggests a competition between hydroxycholesterol and NAS on the acyl-LPLA2 intermediate formed during the reaction. Raising cationic amphiphilic drug concentration or ionic strength in the reaction mixture evoked a reduction of the side-chain oxysterol esterification by LPLA2. This indicates that the esterification could progress via an interfacial interaction of LPLA2 with the lipid membrane surface through an electrostatic interaction. The docking model of acyl-LPLA2 intermediate and side-chain oxysterol provided new insight to elucidate the transacylation mechanism of sterols by LPLA2. Finally, exogenous 25-hydroxycholesterol esterification within alveolar macrophages prepared from wild-type mice was significantly higher than that from LPLA2 deficient mice. This suggests that there is an esterification pathway of side-chain oxysterols via LPLA2.

A Thermostable Monoacylglycerol Lipase from Marine Geobacillus sp. 12AMOR1: Biochemical Characterization and Mutagenesis Study.

Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine Geobacillus sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in Escherichia coli as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth. The recombinant GMGL shows the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C. The GMGL is active on monoacylglycerol (MAG) substrate but not diacylglycerol (DAG) or triacylglycerol (TAG), and produces MAG as the single product in the esterification reaction. Modeling structure analysis showed that the catalytic triad is formed by Ser97, Asp196 and His226, and the flexible cap region is constituted by residues from Ala120 to Thr160. A mutagenesis study on Leu142, Ile145 and Ile170 located in the substrate binding tunnel revealed that these residues were related with its substrate specificity. The kcat/Km value toward the pNP-C6 substrate in mutants Leu142Ala, Ile145Ala and Ile170Phe increased to 2.3-, 1.4- and 2.2-fold as compared to that of the wild type, respectively.

Cholesterol Esterification Inhibition Suppresses Prostate Cancer Metastasis by Impairing the Wnt/ß-catenin Pathway.

Dysregulation of cholesterol is a common characteristic of human cancers including prostate cancer. This study observed an aberrant accumulation of cholesteryl ester in metastatic lesions using Raman spectroscopic analysis of lipid droplets in human prostate cancer patient tissues. Inhibition of cholesterol esterification in prostate cancer cells significantly suppresses the development and growth of metastatic cancer lesions in both orthotopic and intracardiac injection mouse models. Gene expression profiling reveals that cholesteryl ester depletion suppresses the metastatic potential through upregulation of multiple regulators that negatively impact metastasis. In addition, Wnt/ß-catenin, a vital pathway for metastasis, is downregulated upon cholesteryl ester depletion. Mechanistically, inhibition of cholesterol esterification significantly blocks secretion of Wnt3a through reduction of monounsaturated fatty acid levels, which limits Wnt3a acylation. These results collectively validate cholesterol esterification as a novel metabolic target for treating metastatic prostate cancer. Mol Cancer Res; 16(6); 974-85. ©2018 AACR.

Embryonic cholesterol esterification is regulated by a cyclic AMP-dependent pathway in yolk sac membrane-derived endodermal epithelial cells.

During avian embryonic development, endodermal epithelial cells (EECs) absorb yolk through the yolk sac membrane. Sterol O-acyltransferase (SOAT) is important for esterification and yolk lipid utilization during development. Because the major enzyme for yolk sac membrane cholesteryl ester synthesis is SOAT1, we cloned the avian SOAT1 promoter and elucidated the cellular functions of SOAT1. Treatments with either glucagon, isobutylmethylxanthine (IBMX), an adenylate cyclase activator (forskolin), a cAMP analog (dibutyryl-cAMP), or a low glucose concentration all increased SOAT1 mRNA accumulation in EECs from Japanese quail, suggesting that SOAT1 is regulated by nutrients and hormones through a cAMP-dependent pathway. Activity of protein kinase A (PKA) was increased by IBMX, whereas co-treatment with the PKA inhibitor, H89 negated the increase in PKA activity. Cyclic AMP-induced EECs had greater cholesterol esterification than untreated EECs. By promoter deletion and point-mutation, the cAMP-response element (-349 to -341 bp) was identified as critical in mediating transcription of SOAT1. In conclusion, expression of SOAT1 was regulated by a cAMP-dependent pathway and factors that increase PKA will increase SOAT1 to improve the utilization of lipids in the EECs and potentially modify embryonic growth.

Fatty acid elongase 6 plays a role in the synthesis of long-chain fatty acids in goat mammary epithelial cells.

In nonruminants, it is well established that elongation of very long-chain fatty acid-like fatty acid elongase 6 (ELOVL6) catalyzes the synthesis of C18:0 from C16:0 in lipogenic tissues like adipose and liver. However, the role of ELOVL6 in regulating lipid metabolism in ruminant mammary gland remains unknown. In the present study, ELOVL6 was overexpressed or knocked down via adenoviral transfection to assess its role in goat mammary epithelial cells. Results revealed that ELOVL6 overexpression had a weak effect on the expression of genes related to triacylglycerol (TAG) synthesis and desaturation. Overexpression of ELOVL6 increased the content of C18:0 at the expense of C16:0, and increased the elongation index of C16:0. Overexpression of ELOVL6 had no significant effect on the elongation index of C16:1n-7 and the desaturation indices of C16:0 and C18:0. Knockdown of ELOVL6 had a negative effect on mRNA expression of the esterification genes GPAM and diacylglycerolacyltransferase 2 (DGAT2) and TAG concentration; however, it increased the concentration of C16:0 and decreased C18:1n-7 and C18:1n-9 in goat mammary epithelial cells. Accordingly, downregulation of ELOVL6 significantly decreased the elongation indices of C16:0 and C16:1n-7. The lack of change in the desaturation indices of C16:0 and C18:0 upon knockdown of ELOVL6 was consistent with the minor change in SCD1 expression. In conclusion, these are the first results highlighting an important role of ELOVL6 in long-chain fatty elongation and TAG synthesis in ruminant mammary cells.

Knockdown of triglyceride synthesis does not enhance palmitate lipotoxicity or prevent oleate-mediated rescue in rat hepatocytes.

Experiments in a variety of cell types, including hepatocytes, consistently demonstrate the acutely lipotoxic effects of saturated fatty acids, such as palmitate (PA), but not unsaturated fatty acids, such as oleate (OA). PA+OA co-treatment fully prevents PA lipotoxicity through mechanisms that are not well defined but which have been previously attributed to more efficient esterification and sequestration of PA into triglycerides (TGs) when OA is abundant. However, this hypothesis has never been directly tested by experimentally modulating the relative partitioning of PA/OA between TGs and other lipid fates in hepatocytes. In this study, we found that addition of OA to PA-treated hepatocytes enhanced TG synthesis, reduced total PA uptake and PA lipid incorporation, decreased phospholipid saturation and rescued PA-induced ER stress and lipoapoptosis. Knockdown of diacylglycerol acyltransferase (DGAT), the rate-limiting step in TG synthesis, significantly reduced TG accumulation without impairing OA-mediated rescue of PA lipotoxicity. In both wild-type and DGAT-knockdown hepatocytes, OA co-treatment significantly reduced PA lipid incorporation and overall phospholipid saturation compared to PA-treated hepatocytes. These data indicate that OA's protective effects do not require increased conversion of PA into inert TGs, but instead may be due to OA's ability to compete against PA for cellular uptake and/or esterification and, thereby, normalize the composition of cellular lipids in the presence of a toxic PA load.

Elucidation of a key position for acyltransfer activity in Candida parapsilosis lipase/acyltransferase (CpLIP2) and in Pseudozyma antarctica lipase A (CAL-A) by rational design.

Performing transesterifications in aqueous media is becoming a priority challenge in lipid biotechnology in order to develop more eco-friendly and efficient biocatalytic processes in systems containing both polar and apolar substrates. In this context, our group has explored for several years the high potential of the lipase/acyltransferase CpLIP2 from Candida parapsilosis and of several of its homologs, that catalyze efficiently acyltransfer reactions in lipid/water media with high water activity (aw>0.9). The discovery of a new member of this group, CduLAc from Candida dubliniensis, with a higher acyltransferase activity than CpLIP2, has provided a new insight on structure-function relationships in this group. Indeed, the comparison of sequences and 3D models, especially of CpLIP2 and CduLAc, with those of the phylogenetically related lipase A from Pseudozyma antarctica (CAL-A), allowed elucidating a key structural determinant of the acyltransferase activity: serine S369 in CpLIP2 and its equivalents E370 in CAL-A and A366 in CduLAc. Mutants obtained by rational design at this key position showed significant changes in acyltransfer activity. Whereas mutation S369E resulted in an increase in the hydrolytic activity of CpLIP2, S369A increased alcoholysis. More strikingly, the single E370A mutation in CAL-A drastically increased the acyltransferase activity of this enzyme, giving it the character of a lipase/acyltransferase. Indeed, this single mutation lowered the methanol concentration for which the initial rates of alcoholysis and hydrolysis are equal from 2M in CAL-A down to 0.3M in its mutant, while the exceptional stability of the parental enzyme toward alcohol and temperature was conserved.

Application of a statistically enhanced, novel, organic solvent stable lipase from Bacillus safensis DVL-43.

This paper presents the molecular identification of a newly isolated bacterial strain producing a novel and organic solvent stable lipase, statistical optimization of fermentation medium, and its application in the synthesis of ethyl laurate. On the basis of nucleotide homology and phylogenetic analysis of 16S rDNA sequence, the strain was identified as Bacillus safensis DVL-43 (Gen-bank accession number KC156603). Optimization of fermentation medium using Plackett-Burman design and response surface methodology led to 11.4-fold increase in lipase production. The lipase from B. safensis DVL-43 exhibited excellent stability in various organic solvents. The enzyme retained 100% activity after 24h incubation in xylene, DMSO and toluene, each solvent being used at a concentration of 25% (v/v). The use of partially purified DVL-43 lipase as catalyst in the synthesis of ethyl laurate, an esterification product of lauric acid and ethanol, resulted in 80% esterification in 12h under optimized conditions. The formation of ethyl laurate was confirmed using TLC and (1)H NMR. Organic solvent stable lipases exhibiting potential application in enzymatic esterification are in great demand in flavor, fine chemicals and pharma industries. We could not find any report on lipase production from B. safensis strain and its application in esterification.

Methods of downstream processing for the production of biodiesel from microalgae.

Despite receiving increasing attention during the last few decades, the production of microalgal biofuels is not yet sufficiently cost-effective to compete with that of petroleum-based conventional fuels. Among the steps required for the production of microalgal biofuels, the harvest of the microalgal biomass and the extraction of lipids from microalgae are two of the most expensive. In this review article, we surveyed a substantial amount of previous work in microalgal harvesting and lipid extraction to highlight recent progress in these areas. We also discuss new developments in the biodiesel conversion technology due to the importance of the connectivity of this step with the lipid extraction process. Furthermore, we propose possible future directions for technological or process improvements that will directly affect the final production costs of microalgal biomass-based biofuels.

Effect of the amyloidogenic L75P apolipoprotein A-I variant on HDL subpopulations.

BACKGROUND: Hereditary amyloidosis due to mutations of apolipoprotein A-I (apoA-I) is a rare disease characterized by the deposition of amyloid fibrils constituted by the N-terminal fragment of apoA-I in several organs. L75P is a variant of apoA-I associated with systemic amyloidosis predominantly involving the liver, kidneys, and testis, identified in a large number of unrelated subjects. Objective of the present paper was to evaluate the impact of the L75P apoA-I variant on HDL subpopulations and cholesterol esterification in carriers. METHODS AND RESULTS: Plasma samples were collected from 30 carriers of the amyloidogenic L75P apoA-I (Carriers) and from 15 non affected relatives (Controls). Carriers displayed significantly reduced plasma levels of HDL-cholesterol, apoA-I, and apoA-II compared to Controls. Plasma levels of LpA-I, but not LpA-I:A-II, were significantly reduced in Carriers. HDL subclass distribution was not affected by the presence of the variant. The unesterified to total cholesterol ratio was higher, and cholesterol esterification rate and LCAT activity were lower in Carriers than in Controls. CONCLUSIONS: The L75P apoA-I variant is associated with hypoalphalipoproteinemia, a selective reduction of LpA-I particles, and a partial defect in cholesterol esterification.

L-FABP T94A decreased fatty acid uptake and altered hepatic triglyceride and cholesterol accumulation in Chang liver cells stably transfected with L-FABP.

Liver fatty acid-binding protein (L-FABP, FABP1) is a highly conserved key factor in lipid metabolism. This study was undertaken to verify whether the T94A mutation in the L-FABP gene affects fatty acid uptake and intracellular esterification into specific lipid pools. Candidate SNPs were recreated using site-directed mutagenesis and tested for physical function in stably transfected Chang liver cell lines. We found that the T94A mutant of L-FABP lowered FFA uptake but had no effect on FFA efflux. L-FABP T94A-expressing cells showed decreased triglyceride content and increased cholesterol accumulation compared to the wild-type control for cells incubated with an FFA mixture (oleate: palmitate, 2:1 ratio). In conclusion, our study provided additional indications of the functional relevance of the L-FABP T94A SNP in hepatic fatty acid and lipid metabolism in humans.

Hepatic long-chain acyl-CoA synthetase 5 mediates fatty acid channeling between anabolic and catabolic pathways.

Long-chain acyl-CoA synthetases (ACSLs) and fatty acid transport proteins (FATPs) activate fatty acids (FAs) to acyl-CoAs prior to their downstream metabolism. Of numerous ACSL and FATP isoforms, ACSL5 is expressed predominantly in tissues with high rates of triacylglycerol (TAG) synthesis, suggesting it may have an anabolic role in lipid metabolism. To characterize the role of ACSL5 in hepatic energy metabolism, we used small interference RNA (siRNA) to knock down ACSL5 in rat primary hepatocytes. Compared with cells transfected with control siRNA, suppression of ACSL5 expression significantly decreased FA-induced lipid droplet formation. These findings were further extended with metabolic labeling studies showing that ACSL5 knockdown resulted in decreased [1-(14)C]oleic acid or acetic acid incorporation into intracellular TAG, phospholipids, and cholesterol esters without altering FA uptake or lipogenic gene expression. ACSL5 knockdown also decreased hepatic TAG secretion proportionate to the observed decrease in neutral lipid synthesis. ACSL5 knockdown did not alter lipid turnover or mediate the effects of insulin on lipid metabolism. Hepatocytes treated with ACSL5 siRNA had increased rates of FA oxidation without changing PPAR-α activity and target gene expression. These results suggest that ACSL5 activates and channels FAs toward anabolic pathways and, therefore, is an important branch point in hepatic FA metabolism.

Saccades in adult Niemann-Pick disease type C reflect frontal, brainstem, and biochemical deficits.

BACKGROUND: The autosomal recessive disorder Niemannn-Pick type C (NPC) presents in adulthood with psychosis or cognitive deficits associated with supranuclear gaze palsies. While saccadic innervation to the extraocular muscles is generated in the brainstem, the frontal lobes play an integral role in the initiation of volitional saccades and the suppression of unwanted reflexive saccades. No study has examined the frontally driven volitional control of saccadic eye movements in NPC. OBJECTIVE: To examine self-paced and antisaccades as well as reflexive saccades in adult patients with NPC, a disorder known to affect brainstem and frontal cortical function. METHODS: Three biochemically confirmed adult patients with NPC were compared with 10 matched controls on horizontal saccadic and antisaccadic measures using an infrared limbus eye tracker. Patients' cholesterol esterification and filipin staining, Mini-Mental State performance, and NPC symptom level were rated. RESULTS: Reflexive saccade latency ranged from shorter to longer than normal, reflexive saccade gain was reduced, asymptotic peak velocity was reduced, fewer self-paced saccades were generated, and increased errors on antisaccades were made by patients compared to controls. Patients with more severe biochemical, cognitive, and symptom deficits performed most poorly on brainstem and frontal ocular motor measures. Paradoxically, less severe illness was associated with an abnormally reduced saccadic latency. CONCLUSIONS: Ocular motor measures provide an index of disease severity in Niemannn-Pick type C (NPC) and may be a useful adjunct for monitoring the illness progress and medication response. Reduced saccadic latency may result from inadequate fixation input from abnormally functioning frontal eye fields in NPC.