Levels Of Oleate Research Articles

Analysis of the microbial community structure and characteristic flavor of traditional fermented bean paste.

Bean paste is a traditional Chinese fermented condiment, which is loved by consumers for its pleasant flavor and nutritional properties. However, the microbial communities and related flavor compounds in bean paste remain unclear. We studied the correlation between the core functional microorganisms and flavor compounds in bean paste samples at different fermentation stages. Bacterial and fungal communities changed significantly during fermentation. The results indicated that the dominant bacterial genera were Staphylococcus, Lactococcus, and Bacillus. The dominant fungi were Aspergillus and Lichtheimia. During the early and late stages of bean paste fermentation, alcohols, hydrocarbons, esters, and phenols were the main flavor compounds detected. The types and proportions of alcohols and esters in the bean pastes fermented for different durations were similar, whereas the relative contents of phenols and hydrocarbons were higher in the later stages of fermentation. Correlation analysis revealed that core functional microorganisms played a key role in the fermentation process, and the potential correlation between bacterial genera and flavors was stronger than that of fungal genera. Staphylococcus abundance was significantly correlated with the flavor levels of ethyl oleate, ç-pinene, diallyl disulfide, á-myrcene, D-limonene, and tetradecanoic acid, ethyl ester. The abundance of Aspergillus and Lichtheimia was significantly correlated with the flavor levels of á-myrcene, ç-pinene, and DL-3-phenyllactic acid. This study explored the relationship between microorganisms and flavor compounds in bean paste to deepen our understanding of the mechanism of flavor formation for bean paste.

New insights into changing honey bee (Apis mellifera) immunity molecules pattern and fatty acid esters, in responses to Ascosphaera apis infection

Monitoring of metabolite changes could provide valuable insights into disturbances caused by an infection and furthermore, could be used to define the status of an organism as healthy or diseased and define what could be defensive elements against the infection. The present investigation conducted a gas chromatography–mass spectrometry (GC/MS) for haemolymph of larval honey bees (Apis mellifera L.) infected with the fungal pathogen Ascosphaera apis in comparison with control haemolymph non-infected insects. Results revealed that the pathogen caused a general disturbance of metabolites detected in the haemolymph of the honey bee. The majority of metabolites identified before and after infection were fatty acid esters. The disease caused an elevation in levels of methyl oleate, methyl palmitate, and methyl stearate, respectively. Further, the disease drove to the disappearance of methyl palmitoleate, and methyl laurate. Conversely, methyl linolelaidate, and ethyl oleate were identified only in infected larvae. A high reduction in diisooctyl phthalate was recorded after the infection. Interestingly, antimicrobial activities were confirmed for haemolymph of infected honey bee larvae. In spite of the presence of some previously known bioactive compounds in healthy larvae there were no antimicrobial activities.

Breeding of high seed oil oleate levels into Upland cotton from wild Gossypium barbadense L. germplasm

AbstractA high seed oil oleate trait identified in wild Gossypium barbadense L. cotton accession GB713 (PI 608139) was bred into Upland cotton (Gossypium hirsutum L.) cultivar Sure‐Grow 747 (SG747, PVP 9800118). Starting from the F2 generation of an initial cross between GB713 and SG747, plants were selected by measuring the seed oil fatty acid distributions by gas chromatography. Selected F3 plants were crossed to M Rk‐Rn 6, a line previously created from GB713 and SG747 for improved nematode resistance. High‐oleate plants were selected in the F2 population. Selected plants were then crossed to HOa1 (a ∼35% oleate line derived from M Rk‐Rn 1, a sibling line to M Rk‐Rn 6), and high‐oleate plants were again selected in the F2 population. F3 plants were re‐selected for the high‐oleate trait, and four selected F4 lines were evaluated in 2022 field plots. The four lines had oleate levels between 52% and 57%, approximately triple that found in normal cottonseed oil (15%–19%) and higher than in either the high‐oleate wild GB713 parent (∼40%) or in the previously released HOa cotton plant lines (33%–35%). The increased oleate levels occurred with an approximately equal reduction in linoleic acid. Other seed and fiber properties were largely like those of SG747, although some residual GB713 character existed within the lines. The resulting oil composition should be ideal for use in deep fat frying, with the high level of oleic acid improving the oil's oxidative stability but with sufficient linoleic acid to maintain frying flavor.

Nutritional characterization and effect of cooking on phenolic compounds, antioxidant capacity and sensory acceptability of commercial wild rice (Zizania aquatica L.)

Wild rice shows a relevance in the gastronomic area due to its nutritional value, nutty flavor, long grain appearance, dark color, and texture described by the firm outer layer and soft inside of the grain. Wild rice is rich in proteins and phytochemicals associated with health-promoting effects and can be used as cooked rice or ingredient in food products. Rice preparation involves hydrothernic treatment that affects its composition as observed in the traditional rice (Oryza sativa). However, the effect of cooking on phytochemicals and antioxidants properties has not yet been explored in wild rice. The present work reports a nutritional characterization of two commercial brands of wild rice grains (Zizania aquatica L.) from three different harvesting years, and the effect of cooking process on phenolic compounds, antioxidant properties and sensory acceptability. Raw wild rice showed contents of proteins from 12.4 to 13.3 g/100g and lipids from 0.79 to 1.10 g/100g, values which were at least 30% higher and 60% lower, respectively, than those reported to common white rice. The levels of oleate, linoleate and linoleate were 0.66–0.81%, contributing with at least 70% of unsaturated fatty acids. The contents of phenolics (flavonoids, proanthocyanins and anthocyanins) and antioxidant capacity were higher than those reported in traditional rice (Oryza sativa), and their levels remained about 74 and 58%, respectively, even after cooking. Sensory evaluation of raw and cooked wild rice point out that color and taste were the attributes best evaluated, showing a 87% of acceptability.

Evaluation of ethyl oleate and potassium carbonate in drying on vine methodology of raisin making under tropical conditions of India

Different levels of ethyl oleate and potassium carbonate were tried to produce raisins by following DOV method. The application of ethyl oleate and potassium carbonate is found suitable for faster drying of grapes (Vitis virifera L.) in DOV method and able to produce quality raisins. However, application of solution (15 ml ethyl oleate and 25g potassium carbonate in 1L water) on first day and on 4th day (11 ml ethyl oleate and 18 g potassium carbonate in 1L water) was found most effective than other treatments. Further, studies on canopy management, load management and refinement of other agro-techniques are needed to obtain quality raisins by adopting DOV under tropical conditions of India

Oleic Acid Glycerides Content in the Oils of Maize Endospermic Mutants and Its Dependence on Temperature During Ripening

Abstract Identification of sources of increased content of oleic acid glycerides (oleates) and determination of the nature of its variability are prerequisites for improving oil quality. The purpose of this study was to establish the variability of the content of oleic acid glycerides (oleates) in maize oil depending on the genotype and ripening temperature. The experiments were carried out on a series of common maize inbreds and inbred-carriers of the su1, ae, and su2 mutations, which were grown for three years. The fatty acid composition of the oil was analysed by the Peisker gas chromatographic method. Genotype–environment interactions were assessed using the Eberhard– Russell algorithm. It was found that the carriers of the su1 and su2 mutations had the highest oleate content. The oleate content of maize oil generally increased with increasing ripening temperature. Unrelated inbreds based on one mutation significantly differed in the genetically determined level of oleate and showed non-identical responses to the ripening temperature. In inbreds of the first type, the content of oleates changed significantly with temperature fluctuation, while inbreds of the second type provided a fairly stable level of the trait under these conditions.

Using HMGB1 Protein as a Surrogate Marker for Lung Cancer in High‐Risk Populations

Lung cancer is the leading cause of cancer‐related death, independent of gender or ethnicity, with a 5‐year overall survival of 17.1%. Although screening protocols for early detection exist, lung cancer is often diagnosed at an advanced stage. This is due, in part, to many patients experiencing barriers to healthcare access which contribute to delays in diagnosis. Supra‐normal biosynthesis of saturated (SFA) and unsaturated fatty (UFA) acids is a hallmark of cancer. In fact, several lines of evidence demonstrate that increased lipid desaturation by stearoyl‐CoA desaturase (SCD1) promotes lung cancer growth. We hypothesize that increased intracellular UFA, such as oleate (OA), promotes cancer cell growth by reducing cancer associated inflammation. To test this hypothesis we evaluated the effect of OA and inhibition of OA synthesis on proinflammatory High Mobility Group Box Protein 1 (HMGB1). Decreasing OA synthesis reduced intracellular HMGB1 expression, but increased its release from cells. Given our data, alterations in OA levels appear to have a substantial effect on localization of HMGB1. Further work is necessary to define the connections between SCD1, OA and HMGB1 release in lung cancer cells. This knowledge will provide insight into how fat regulates cancer development.Support or Funding InformationThe University of Minnesota School of Medicine New Faculty Start up and College of Saint Scholastica McNair Scholars Program.

Registration of four upland cotton germplasm lines with elevated levels of seed oil oleic acid

AbstractFour upland cotton (Gossypium hirsutum L.) germplasm lines, HOa1 (Reg. no. GP‐1072, PI 691652), HOa2 (Reg. no. GP‐1073, PI 691653), HOa3 (Reg. no. GP‐1074, PI 691654), and HOa4 (Reg. no. GP‐1075, PI 691655), with elevated levels of seed oil oleic acid were developed and released by the USDA‐ARS. High seed oil levels of oleic acid have been reported for a small number of G. barbadense L. accessions. Included among these accessions is GB713, which is also known as a source of reniform nematode (Rotylenchulus reniformis Linford and Oliveira) resistance and has been used to breed this trait into an upland cotton background. Root‐knot nematode resistance was bred into the GB713‐based reniform‐resistant lines to yield germplasm with resistance to both nematodes. Testing the nematode‐resistant lines for seed oil fatty acid composition revealed elevated levels of oleic acid in some seeds. Selection and propagation of individual plants with the highest oleate levels indicated that the trait was stable for three successive generations with approximately double the level of oleic acid found in normal cottonseed oil. Oils with high levels of oleic acid are considered to be healthier and have improved oxidative stability at elevated temperature, making them preferred for some cooking and frying applications.

Hepatic Stearoyl-CoA desaturase-1 deficiency-mediated activation of mTORC1- PGC-1\u03b1 axis regulates ER stress during high-carbohydrate feeding

Stearoyl CoA desaturase 1 (SCD1) is a key enzyme in lipogenesis as it catalyzes the synthesis of monounsaturated fatty acids (MUFAs), mainly oleate (18:1n9) and palmitoleate (16:1n7) from saturated fatty acids (SFA), stearate (18:0) and palmitate (16:0), respectively. Studies on SCD1 deficiency in mouse models demonstrated beneficial metabolic phenotypes such as reduced adiposity and improved glucose tolerance. Even though, SCD1 represents a potential target to resolve obesity related metabolic diseases; SCD1 deficiency causes endoplasmic reticulum (ER) stress and activates unfolded protein response (UPR). The induction of ER stress in response to SCD1 deficiency is governed by the cofactor, PGC-1α. However, the mechanism by which SCD1 deficiency increases PGC-1α and subsequently induces ER stress still remains elusive. The present study demonstrates that despite reduced lipogenesis, liver specific SCD1 deficiency activates the mechanistic target of rapamycin complex 1 (mTORC1) along with induction of PGC-1α and ER stress. Further, mTORC1 inhibition attenuates SCD1 deficiency-mediated induction of both PGC-1α and ER stress. Similar observations were seen by restoring endogenously synthesized oleate, but not palmitoleate, suggesting a clear mTORC1-mediated regulation of ER stress during SCD1 deficiency. Overall, our results suggest a model whereby maintaining adequate levels of hepatic oleate is required to suppress mTORC1-mediated ER stress. In addition, the activation of mTORC1 by SCD1 deficiency reveals an important function of fatty acids in regulating different cellular processes through mTORC1 signaling.

Identification of a mutant from Arachis veigae with enhanced seed oleic and very long-chain fatty acid content

High oleate is an important seed quality trait frequently incorporated in peanut varieties. Crop wild relatives (CWR) are potentially useful genetic resources for cultivar improvement through genetic introgression; but for wild peanut species, many chemical or nutritional traits are not well characterized. A mutant from Arachis veigae S. H. Santana & Valls (2n = 2x = 20), with increased oleic and very long chain (C ≥ 22) fatty acid content was identified from screening 209 accessions of 45 species using gas chromatography (GC). The A. veigae (formerly A. sylvestris) accession, VVeSv 8373 (PI 688970) contained 55.5% oleic acid in seeds, significantly higher than the average (18.3%) of other accessions within the same species and also significantly higher than the average (37.0%) of all wild peanut accessions evaluated. A C37T substitution was identified by sequencing the coding region of FAD2H, resulting in the nonsense mutation of Q13* (a premature stop codon). This functional mutation may significantly reduce the fatty acid desaturase (FAD) activity and result in the enhanced oleate level. Arachis veigae also contained a high percentage of very long-chain (C ≥ 22) fatty acids, and their variation identified in this study is also discussed and compared with other species. The mutant with such an altered fatty acid composition may be useful for potentially improving seed or food nutrition quality.

Expression of the Arabidopsis WRINKLED 1 transcription factor leads to higher accumulation of palmitate in soybean seed.

SummarySoybean (Glycine max [L.] Merr.) is a commodity crop highly valued for its protein and oil content. The high percentage of polyunsaturated fatty acids in soybean oil results in low oxidative stability, which is a key parameter for usage in baking, high temperature frying applications, and affects shelf life of packaged products containing soybean oil. Introduction of a seed‐specific expression cassette carrying the Arabidopsis transcription factor WRINKLED1 (AtWRI1) into soybean, led to seed oil with levels of palmitate up to approximately 20%. Stacking of the AtWRI1 transgenic allele with a transgenic locus harbouring the mangosteen steroyl‐ACP thioesterase (GmFatA) resulted in oil with total saturates up to 30%. The creation of a triple stack in soybean, wherein the AtWRI1 and GmFatA alleles were combined with a FAD2‐1 silencing allele led to the synthesis of an oil with 28% saturates and approximately 60% oleate. Constructs were then assembled that carry a dual FAD2‐1 silencing element/GmFatA expression cassette, alone or combined with an AtWRI1 cassette. These plasmids are designated pPTN1289 and pPTN1301, respectively. Transgenic events carrying the T‐DNA of pPTN1289 displayed an oil with stearate levels between 18% and 25%, and oleate in the upper 60%, with reduced palmitate (<5%). While soybean events harboring transgenic alleles of pPTN1301 had similar levels of stearic and oleate levels as that of the pPTRN1289 events, but with levels of palmitate closer to wild type. The modified fatty acid composition results in an oil with higher oxidative stability, and functionality attributes for end use in baking applications.

High-oleate yeast oil without polyunsaturated fatty acids

BackgroundOleate-enriched triacylglycerides are well-suited for lubricant applications that require high oxidative stability. Fatty acid carbon chain length and degree of desaturation are key determinants of triacylglyceride properties and the ability to manipulate fatty acid composition in living organisms is critical to developing a source of bio-based oil tailored to meet specific application requirements.ResultsWe sought to engineer the oleaginous yeast Yarrowia lipolytica for production of high-oleate triacylglyceride oil. We studied the effect of deletions and overexpressions in the fatty acid and triacylglyceride synthesis pathways to identify modifications that increase oleate levels. Oleic acid accumulation in triacylglycerides was promoted by exchanging the native ∆9 fatty acid desaturase and glycerol-3-phosphate acyltransferase with heterologous enzymes, as well as deletion of the Δ12 fatty acid desaturase and expression of a fatty acid elongase. By combining these engineering steps, we eliminated polyunsaturated fatty acids and created a Y. lipolytica strain that accumulates triglycerides with > 90% oleate content.ConclusionsHigh-oleate content and lack of polyunsaturates distinguish this triacylglyceride oil from plant and algal derived oils. Its composition renders the oil suitable for applications that require high oxidative stability and further demonstrates the potential of Y. lipolytica as a producer of tailored lipid profiles.

High-level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient wax ester synthesis enzymes

BackgroundBiotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived wax esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of wax esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a wax synthase (WS) in seeds are sufficient.ResultsFor optimization of the enzymatic activity and subcellular localization of wax ester synthesis enzymes, two fusion proteins were created, which showed wax ester-forming activities in Saccharomyces cerevisiae. To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi (AbWSD1) and Marinobacter aquaeolei (MaWS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for wax ester production. The tested seven enzyme combinations resulted in different yields and compositions of wax esters. Expression of a FAR of Marinobacter aquaeolei (MaFAR) with AbWSD1 or MaWS2 led to a high incorporation of C18 substrates in wax esters. The MaFAR/TMMmAWAT2-AbWSD1 combination resulted in the incorporation of more C18:1 alcohol and C18:0 acyl moieties into wax esters compared with MaFAR/AbWSD1. The fusion protein of a WS from Simmondsia chinensis (ScWS) with MaFAR exhibited higher specificity toward C20:1 substrates in preference to C18:1 substrates. Expression of MaFAR/AbWSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total wax esters in seed oil, which was much higher than the 15 mol% reached by MaFAR/AbWSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina, lines expressing MaFAR/ScWS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed−1 of wax esters, containing 27–34 mol% oleyl oleate.ConclusionsThe overall yields and the compositions of wax esters can be strongly affected by the availability of acyl-CoA substrates and to a lesser extent, by the characteristics of wax ester synthesis enzymes. For synthesis of oleyl oleate in plant seed oil, appropriate wax ester synthesis enzymes with high catalytic efficiency and desired substrate specificity should be expressed in plant cells; meanwhile, high levels of oleic acid-derived substrates need to be supplied to these enzymes by modifying the fatty acid profile of developing seeds.

Oil Content and Fatty Acids Composition during Seeds Storage of Two Ecotypes of &lt;i&gt;Jatropha curcas &lt;/i&gt;L&lt;i&gt;.&lt;/i&gt; from Mali

To study the oil contents and fatty acid composition among the samples of Jatropha curcas L. from Mali, the seeds were collected from Katibougou (ecotype I) and Teriyabougou (ecotype II) center. Soxhlet extraction methods were used to determine the oil content of J. curcas seeds. Two different periods were used for various analyses and each analysis was duplicated. The oil concentration ranged in the first and second time of extraction from 62.95±0.24 % to 61.11±.18 % (ecotype I) and 65.9±0.76 % to 65.06±0.36% (ecotype II) respectively. The fatty acid profiles were developed by gas chromatography-mass spectrometry (GC-MS). Sixteen fatty acids were identified in quantifiable amount in the seed oils. Among these, saturated and unsaturated fatty acids were obtained with a predominance of unsaturated fatty acids. The oleic acid and linoleic acid were the dominant fatty acids present in the J. curcas seed oil of Mali. Their ratio (Oleic/Linoleic) varies from 0.87±0.02% to 1.22±0.02% (ecotype I) and 0.82±0.06% to 1.13±0.11% (ecotype II). This ratio possesses a high level of methyl oleate (monounsaturated fatty acid) which might have an excellent effect on biodiesel properties.

Elongation of Long‐Chain Fatty Acid Family Member 6 (Elovl6)‐Driven Fatty Acid Metabolism Regulates Vascular Smooth Muscle Cell Phenotype Through AMP‐Activated Protein Kinase/Krüppel‐Like Factor 4 (AMPK/KLF4) Signaling

BackgroundFatty acids constitute the critical components of cell structure and function, and dysregulation of fatty acid composition may exert diverging vascular effects including proliferation, migration, and differentiation of vascular smooth muscle cells (VSMCs). However, direct evidence for this hypothesis has been lacking. We investigated the role of elongation of long‐chain fatty acid member 6 (Elovl6), a rate‐limiting enzyme catalyzing the elongation of saturated and monounsaturated long‐chain fatty acid, in the regulation of phenotypic switching of VSMC.Methods and ResultsNeointima formation following wire injury was markedly inhibited in Elovl6‐null (Elovl6−/−) mice, and cultured VSMCs with siRNA‐mediated knockdown of Elovl6 was barely responsive to PDGF‐BB. Elovl6 inhibition induced cell cycle suppressors p53 and p21 and reduced the mammalian targets of rapamycin (mTOR) phosphorylation and VSMC marker expression. These changes are ascribed to increased palmitate levels and reduced oleate levels, changes that lead to reactive oxygen species (ROS) production and resulting AMP‐activated protein kinase (AMPK) activation. Notably, Elovl6 inhibition robustly induced the pluripotency gene Krüppel‐like factor 4 (KLF4) expression in VSMC, and KLF4 knockdown significantly attenuated AMPK‐induced phenotypic switching of VSMC, indicating that KLF4 is a bona fide target of AMPK.ConclusionsWe demonstrate for the first time that dysregulation of Elovl6‐driven long‐chain fatty acid metabolism induces phenotypic switching of VSMC via ROS production and AMPK/KLF4 signaling that leads to growth arrest and downregulation of VSMC marker expression. The modulation of Elovl6‐mediated cellular processes may provide an intriguing approach for tackling atherosclerosis and postangioplasty restenosis.

Caveolin-1 regulates hormone resistance through lipid synthesis, creating novel therapeutic opportunities for castration-resistant prostate cancer.

Caveolin-1 (Cav-1) is overexpressed in aggressive and metastatic prostate cancer (PCa) and induces PCa cell proliferation. Androgens mediate lipid synthesis through acetyl-CoA carboxylase-1 (ACC1) and fatty acid synthase (FASN). We investigated the Cav-1-mediated lipid synthesis in the development of castration resistance, and identified novel therapeutic opportunities. Using the PBCre+;Ptenloxp/loxp;PBCav-1+ mouse model we found that Cav-1 induction increased cancer incidence and growth, and ACC1-FASN expression in intact and castrated mice. We demonstrated that Cav-1 regulated ACC1 and FASN expression in an AR-independent way and increased palmitate synthesis using western blot analysis, qRT-PCR and mass spectrometry in vitro. By using FASN siRNA and C-75, we found that FASN inhibition was more effective in Cav-1-overexpressing cells. This inhibition was abrogated by ACC1si RNA, revealing the role of malonyl-CoA, an ACC1 product, as a mediator of cytotoxicity. Cav-1 was associated with ACC1 in human tumors and ACC1, FASN, and Cav-1 expression were increased in metastatic PCa compared to primary tumors and normal prostate epithelium. Palmitoleate and oleate levels were higher in BMA from patients with metastatic PCa who responded poorly to abiraterone acetate. Our findings suggest that Cav-1 promotes hormone resistance through the upregulation of ACC1-FASN and lipid synthesis under androgen deprivation, suggesting that FASN inhibition could be used to treat PCa that demonstrates Cav-1 overexpression.

ANT1-mediated fatty acid-induced uncoupling as a target for improving myocellular insulin sensitivity.

Aims/hypothesisDissipating energy via mitochondrial uncoupling has been suggested to contribute to enhanced insulin sensitivity. We hypothesised that skeletal muscle mitochondria of endurance-trained athletes have increased sensitivity for fatty acid (FA)-induced uncoupling, which is driven by the mitochondrial protein adenine nucleotide translocase 1 (ANT1).MethodsCapacity for FA-induced uncoupling was measured in endurance-trained male athletes (T) and sedentary young men (UT) in an observational study and also in isolated skeletal muscle mitochondria from Zucker diabetic fatty (ZDF) rats and C2C12 myotubes following small interfering RNA (siRNA)-mediated gene silencing of ANT1. Thus, fuelled by glutamate/succinate (fibres) or pyruvate (mitochondria and myotubes) and in the presence of oligomycin to block ATP synthesis, increasing levels of oleate (fibres) or palmitate (mitochondria and myotubes) were automatically titrated while respiration was monitored. Insulin sensitivity was measured by hyperinsulinaemic–euglycaemic clamp in humans and via insulin-stimulated glucose uptake in myotubes.ResultsSkeletal muscle from the T group displayed increased sensitivity to FA-induced uncoupling (p = 0.011) compared with muscle from the UT group, and this was associated with elevated insulin sensitivity (p = 0.034). ANT1 expression was increased in T (p = 0.013). Mitochondria from ZDF rats displayed decreased sensitivity for FA-induced uncoupling (p = 0.008). This difference disappeared in the presence of the adenine nucleotide translocator inhibitor carboxyatractyloside. Partial knockdown of ANT1 in C2C12 myotubes decreased sensitivity to the FA-induced uncoupling (p = 0.008) and insulin-stimulated glucose uptake (p = 0.025) compared with controls.Conclusions/interpretationIncreased sensitivity to FA-induced uncoupling is associated with enhanced insulin sensitivity and is affected by ANT1 activity in skeletal muscle. FA-induced mitochondrial uncoupling may help to preserve insulin sensitivity in the face of a high supply of FAs.Trial registrationwww.trialregister.nl NTR2002

Intracerebroventricular ghrelin treatment affects lipid metabolism in liver of rainbow trout (Oncorhynchus mykiss)

We aimed to elucidate in rainbow trout (Oncorhynchus mykiss) the effects of central ghrelin (GHRL) treatment on the regulation of liver lipid metabolism, and the possible modulatory effect of central GHRL treatment on the simultaneous effects of raised levels of oleate. Thus, we injected intracerebroventricularly (ICV) rainbow trout GHRL in the presence or absence of oleate and evaluated in liver variables related to lipid metabolism. Oleate treatment elicited in liver of rainbow trout decreased lipogenesis and increased oxidative capacity in agreement with previous studies. Moreover, as demonstrated for the first time in fish in the present study, GHRL also acts centrally modulating lipid metabolism in liver, resulting in increased potential for lipogenesis and decreased potential for fatty acid oxidation, i.e. the converse effects to those elicited by central oleate treatment. The simultaneous treatment of GHRL and oleate confirmed these counteractive effects. Thus, the nutrient sensing mechanisms present in hypothalamus, particularly those involved in sensing of fatty acid, are involved in the control of liver energy metabolism in fish, and this control is modulated by the central action of GHRL. These results give support to the notion of hypothalamus as an integrative place for the regulation of peripheral energy metabolism in fish.

Quantitative assay of capreomycin oleate levels in a drug formulation for inhalation with a fully validated HPLC method

Capreomycin sulfate (CS), a mixture of 4 closely related compounds (powder mainly comprised of 2 forms), commonly injected intramuscularly is intended to be administer by inhalation for the treatment of pulmonary tuberculosis.In order to increase the drug residence time in the lung, capreomycin hydrophobicity was enhanced by substituting sulfate with oleate, thus obtaining capreomycin oleate (CO). The generation of a more hydrophobic ion-pair allows the reduction of the drug solubilisation in the bronchoalveolar fluids as well as its systemic absorption.The aim of the present study was to quantify CO in an in-house prepared drug formulation for inhalation. In this regard, a Hydrophilic Liquid Interaction Chromatography (HILIC) method was optimized with acetonitrile (ACN)/water containing eluents and a diol-type stationary phase. The optimal eluent composition [ACN/water-80/20 (v/v), 20mM ammonium formate, 3.0 wspH] produced a good separation (α equal to 1.15) between the two main peaks. The developed HILIC method succeeded in the quantitative assay of CO in the drug formulation and was fully validated. Very good precision and accuracy in the short- and long-period along with appreciably low LOD and LOQ values (respectively 1.75 and 5.25μg/mL) turned out.

Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells

Lysosomes are lined with a glycocalyx that protects the limiting membrane from the action of degradative enzymes. We tested the hypothesis that Niemann-Pick type C 1 (NPC1) protein aids the transfer of low density lipoprotein-derived cholesterol across this glycocalyx. A prediction of this model is that cells will be less dependent upon NPC1 if their glycocalyx is decreased in density. Lysosome cholesterol content was significantly lower after treatment of NPC1-deficient human fibroblasts with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside, an inhibitor of O-linked glycosylation. Direct biochemical measurement of cholesterol showed that lysosomes purified from NPC1-deficient fibroblasts contained at least 30% less cholesterol when O-linked glycosylation was blocked. As an independent means to modify protein glycosylation, we used Chinese hamster ovary ldl-D cells defective in UDP-Gal/UDP-GalNAc 4-epimerase in which N- and O-linked glycosylation can be controlled. CRISPR generated, NPC1-deficient ldl-D cells supplemented with galactose accumulated more cholesterol than those in which sugar addition was blocked. In the absence of galactose supplementation, NPC1-deficient ldl-D cells also transported more cholesterol from lysosomes to the endoplasmic reticulum, as monitored by an increase in cholesteryl [(14)C]-oleate levels. These experiments support a model in which NPC1 protein functions to transfer cholesterol past a lysosomal glycocalyx.