Free Fatty Acid Pool Research Articles

Lipidomic analysis of human TANGO2-deficient cells suggests a lipid imbalance as a cause of TANGO2 deficiency disease

TANGO2 deficiency disease (TDD) is a multisystem disease caused by variants in the TANGO2 gene. Symptoms include neurodevelopmental delays, seizures and potentially lethal metabolic crises and cardiac arrhythmias. While the function of TANGO2 remains elusive, vitamin B5/pantothenic acid supplementation has been shown to alleviate symptoms in a fruit fly model and has also been used with success to treat individuals suffering from TDD. Since vitamin B5 is the precursor to the lipid activator coenzyme A (CoA), we hypothesized that TANGO2-deficient cells would display changes in the lipid profile compared to control and that these changes would be rescued by vitamin B5 supplementation. In addition, the specific changes seen might point to a pathway in which TANGO2 functions. Indeed, we found profound changes in the lipid profile of human TANGO2-deficient cells as well as an increased pool of free fatty acids in both human cells devoid of TANGO2 and Drosophila harboring a previously described TANGO2 loss of function allele. All these changes were reversed upon vitamin B5 supplementation. Pathway analysis showed significant increases in triglyceride as well as in lysophospholipid levels as the top enriched pathways in the absence of TANGO2. Consistent with a defect in triglyceride metabolism, we found changes in lipid droplet numbers and sizes in the absence of TANGO2 compared to control. Our data will allow for comparison between other model systems of TDD and the homing in on critical lipid imbalances that lead to the disease state.

Sterols, free fatty acids, and total fatty acid content in the massive Porites spp. corals cultured under different pCO2 and temperature treatments

Lipids may serve as energy reserves to support coral calcification, allow acclimation to higher temperatures, and are implicated in the control of CaCO3 precipitation. Here, we report the lipid composition of the soft tissues (including host and symbionts) of 7 massive Porites spp. coral colonies (4 × P. lutea and 3 × P. murrayensis), which were cultured under different pCO2 concentrations (180, 260, 400 and 750 µatm) and at two temperatures (25 ℃ and 28 ℃), below the thermal stress threshold. We report the fatty acid methyl esters (FAME), free fatty acid (FFA) to total fatty acid content, sterol and wax ester profiles, and identify two ketones (n-alkanone) and three long chain aldehyde (n-alkanal) derivatives. Increasing seawater temperature significantly increases the contributions of FFAs to the total lipids, of C18:2 and C20:0 to the total FFA pool, of C14:0 to total FAME, and of campesterol to total sterol. The temperature increase also reduces the contributions of unusual fatty acid derivatives to total lipids, of C14:0, C15:0, C16:0 and C17:0 saturated free fatty acids to total FFAs, and of C16:0 FA to total FAME. Fatty acids are implicated in the control of membrane structure fluidity and the observed changes may promote acclimation and thermostability as temperature varies. Seawater pCO2 has no significant effect on the composition of tissue lipids with the exception that the contribution of C14:0 FA to total lipid content is significantly lower at 180 µatm compared to 260 and 750 µatm. Decreased contribution of total sterols and unusual fatty acid derivatives and increased contribution of total FFAs to total lipids are observed in the fastest calcifying coral (a P. lutea specimen) compared to the other corals, under all pCO2 and temperature conditions. Although a rapid calcifier this genotype has been shown previously to exhibit pronounced abnormal changes in skeletal morphology in response to decreased seawater pCO2. Variations in tissue lipid composition between coral genotypes may influence their resilience to future climate change.

Different Types of Dietary Fat and Fructose Interactions Result in Distinct Metabolic Phenotypes in Male Mice

Fat and fructose are the two major components over-represented in the Western diet. The aim of this study was to determine the combined effects of different types of dietary fat and fructose on the development of nonalcoholic fatty liver disease (NAFLD) in a murine model. Eight-week-old male C57BL/6J mice were fed with high-fat diet enriched with saturated fat (HSF), or omega-6 polyunsaturated fat (n6HUSF), or omega-3 polyunsaturated fat (n3HUSF) with 42% of calories derived from the fat. Fructose supplementation was given via 10% fructose (w/v) in the drinking water ad libitum for 20 weeks. While both HSF and n6HUSF fed mice developed obesity, HSF fed mice exhibited severe hepatic steatosis associated with hepatomegaly and liver injury. Fructose feeding promotes the development of liver fibrosis in HSF fed mice. n6HUSF fed mice were characterized with moderate hepatic steatosis, accompanied with hypertriglyceridemia and hyperlipidemia. Notably, fructose supplementation led to remarkable glucose intolerance in n6HUSF fed mice compared to controls. Hepatic lipidomic analysis revealed that the total saturated fatty acids and total monounsaturated fatty acids were significantly increased by fructose in the free fatty acid pool in HSF fed mice. Moreover, fructose supplementation increased hepatic and plasma cholesterol levels in the HSF fed mice. Our data suggest that excess energy from HSF intake results in fat storage in the liver, likely due to impaired triglyceride secretion; whereas excess energy from n6HUSF diet is stored in the periphery. Both effects are exacerbated by fructose supplementation. n3HUSF is beneficial, even consumed with fructose.

Steatohepatitises: etiological variants, principles of diagnosis and management

Steatohepatitises is an etiologically heterogeneous group of pathological changes in the liver, which are characterized by the inflammatory infiltration of the hepatic parenchyma with underlying fatty degeneration of hepatocytes. Whatever is the etiological cause, the clinical significance of steatohepatitis involves the formation of liver fibrosis and, as a result, an increased risk of developing liver cirrhosis and hepatocellular carcinoma, which are life-threatening conditions. It is common practice to identify the following etiological variants of steatohepatitis: metabolic (55–65% of cases), alcoholic (45–55% of cases) and drug-induced (approximately 5% of cases). The pathogenetic basis of metabolic steatohepatitis lies in the mechanisms of increased lipolysis, excess free fatty acid pool and reduced β-oxidation stemming from obesity and insulin resistance. Pathogenetic factors mediating the development of alcoholic steatohepatitis are the toxic activity of acetaldehyde and increased CYP2E1 activity. Intake of some hepatotoxic drugs increases lipogenesis in hepatocytes and disrupts the electron transport chain, which leads to the formation of liver steatosis followed by transformation into steatohepatitis. Whatever is the etiological varient, steatohepatitis is asymptomatic in the prevailing majority of cases. However, some patients may present complaints of weakness, discomfort, or indolent pain in the right hypochondrium. A detailed history taking is essential for the establishment of the etiological cause of liver damage. Laboratory tests allow to diagnose steatohepatitis in increased levels of hepatic transaminases, usually not exceeding 2–3 times the normal values. In addition to liver enzymes, increased levels of alkaline phosphatase and GGTP can also be observed in steatohepatitis. Ultrasound imaging is the most accessible instrumental tool in clinical practice to establish the primary diagnosis of hepatic steatosis. Indirect elastometry is an equally informative non-invasive method for diagnosing steatohepatitis, which allows to measure both the degree of steatosis (the function of determining the ultrasonic controlled attenuation parameter (CAP) and liver fibrosis.

Lipid Metabolism Interplay in CRC-An Update.

Colorectal cancer (CRC) to date still ranks as one of the deadliest cancer entities globally, and despite recent advances, the incidence in young adolescents is dramatically increasing. Lipid metabolism has recently received increased attention as a crucial element for multiple aspects of carcinogenesis and our knowledge of the underlying mechanisms is steadily growing. However, the mechanism how fatty acid metabolism contributes to CRC is still not understood in detail. In this review, we aim to summarize our vastly growing comprehension and the accompanied complexity of cellular fatty acid metabolism in CRC by describing inputs and outputs of intracellular free fatty acid pools and how these contribute to cancer initiation, disease progression and metastasis. We highlight how different lipid pathways can contribute to the aggressiveness of tumors and affect the prognosis of patients. Furthermore, we focus on the role of lipid metabolism in cell communication and interplay within the tumor microenvironment (TME) and beyond. Understanding these interactions in depth might lead to the discovery of novel markers and new therapeutic interventions for CRC. Finally, we discuss the crucial role of fatty acid metabolism as new targetable gatekeeper in colorectal cancer.

Myoglobin, expressed in brown adipose tissue of mice, regulates the content and activity of mitochondria and lipid droplets

The identification of novel physiological regulators that stimulate energy expenditure through brown adipose tissue (BAT) activity in substrate catalysis is of utmost importance to understand and treat metabolic diseases. Myoglobin (MB), known to store or transport oxygen in heart and skeletal muscles, has recently been found to bind fatty acids with physiological constants in its oxygenated form (i.e., MBO2). Here, we investigated the in vivo effect of MB expression on BAT activity. In particular, we studied mitochondrial function and lipid metabolism as essential determinants of energy expenditure in this tissue. We show in a MB-null (MBko) mouse model that MB expression in BAT impacts on the activity of brown adipocytes in a twofold manner: i) by elevating mitochondrial density plus maximal respiration capacity, and through that, by stimulating BAT oxidative metabolism along with the organelles` uncoupled respiration; and ii) by influencing the free fatty acids pool towards a palmitate-enriched composition and shifting the lipid droplet (LD) equilibrium towards higher counts of smaller droplets. These metabolic changes were accompanied by the up-regulated expression of thermogenesis markers UCP1, CIDEA, CIDEC, PGC1-α and PPAR-α in the BAT of MB wildtype (MBwt) mice. Along with the emergence of the “browning” BAT morphology, MBwt mice exhibited a leaner phenotype when compared to MBko littermates at 20 weeks of age. Our data shed novel insights into MB's role in linking oxygen and lipid-based thermogenic metabolism. The findings suggest potential new strategies of targeting the MB pathway to treat metabolic disorders related to diminishing energy expenditure.

Fatty Acid Production and Direct Acyl Transfer through Polar Lipids Control TAG Biosynthesis during Nitrogen Deprivation in the Halotolerant Alga Dunaliella tertiolecta.

The aims of this work were to evaluate the contribution of the free fatty acid (FA) pool to triacylglyceride (TAG) biosynthesis and to try to characterize the mechanism by which FA are assimilated into TAG in the green alga Dunaliella tertiolecta. A time-resolved lipidomic analysis showed that nitrogen (N) deprivation induces a redistribution of total lipidome, particularly of free FA and major polar lipid (PL), in parallel to enhanced accumulation of polyunsaturated TAG. The steady-state concentration of the FA pool, measured by prolonged 14C-bicarbonate pre-labeling, showed that N deprivation induced a 50% decrease in total FA level within the first 24 h and up to 85% after 96 h. The abundance of oleic acid increased from 50 to 70% of total free FA while polyunsaturated FA (PUFA) disappeared under N deprivation. The FA flux, measured by the rate of incorporation of 14C-palmitic acid (PlA), suggests partial suppression of phosphatidylcholine (PC) acyl editing and an enhanced turnover of the FA pool and of total digalactosyl-diacylglycerol (DGDG) during N deprivation. Taken together, these results imply that FA biosynthesis is a major rate-controlling stage in TAG biosynthesis in D. tertiolecta and that acyl transfer through PL such as PC and DGDG is the major FA assimilation pathway into TAG in that alga and possibly in other green microalgae. Increasing the availability of FA could lead to enhanced TAG biosynthesis and to improved production of high-value products from microalgae.

1881-P: Separate Free Fatty Acid (FFA) Pools Are Involved in Muscle Lipid Utilization

Despite discrepant phenotypes, the insulin resistant (IR) and trained states have high levels of intramyocellular lipid. We hypothesize that higher muscle lipid turnover in the trained state may explain this discrepancy. Obese, IR subjects and trained subjects were fasted overnight (Table1). To characterize muscle lipid turnover, we conducted a pulse-chase experiment using sequential [U-13C]palmitate and [9-2H]palmitate infusions (6 h each) with a 1 hour overlap to label endogenous and exogenous FFA pools. Biopsy#1 (Bx#1 -s/p 14 hr fast) was performed at the time of the overlap (last hour of the 6 h [U-13C]palmitate infusion, after 1 h of starting the [9-2H]palmitate). Bx#2 was performed at the study conclusion (last hour of the [9-2H]palmitate infusion-s/p 20 hr fast). Plasma and muscle samples were analyzed for palmitate enrichment and concentration. Regardless of phenotype, muscle triglyceride (IMTG) synthesis at Bx#1 was not different as assessed by either [U-13C]palmitate or [9-2H]palmitate; IMTG synthesis assessed by [9-2H]palmitate was 9-fold higher than [U-13C]palmitate. At Bx#2, IMTG breakdown as assessed by [U-13C]palmitate was similar between groups, whereas [9-2H]palmitate showed IMTG synthesis in the obese, IR group and IMTG breakdown in the trained group. IMTG synthesis was higher with recent (1 hour) FFA exposure than more prolonged (6 hour) FFA exposure supporting separate FFA pools in lipid utilization. Disclosure L.S. Chow: None. D.G. Mashek: None. M.D. Jensen: None. Funding National Institutes of Health (R01DK098203, UL1TR000114)

Impact of Extracellular Fatty Acids and Oxygen Tension on Lipid Synthesis and Assembly in Pancreatic Cancer Cells.

Lipid oxidation and biosynthesis are crucial for cell survival, especially for rapidly proliferating cancer cells in a heterogeneous metabolic environment. The storage of high-energy lipid reservoirs competitively advantages the cancer cell over non-neoplastic tissue. Disrupting lipid biosynthetic processes, through modulation of fatty acid (FA) esterification or de novo lipogenesis (DNL), is of interest in drug discovery. Mimicking the in vivo environment in vitro is also vital for testing the efficacy of potential drug compounds. We present here a stable isotope tracer-based approach for examining the impact of exogenous FA and oxygen tension on the pathways that affect lipid biosynthesis, including the rates of metabolic flux. By applying tandem mass spectrometry (MS/MS) analyses to studies using parallel tracers, we characterized the impact of FA bioavailability on the positional enrichment within specific lipids. Our observations suggest that adding bioavailable FA as a carbon source preferentially biases the cellular metabolism away from DNL and toward esterification of free fatty acid pools. Additionally, we have found that this FA addition, under hypoxic conditions, led to a biased increase in the total triglyceride pool (nearly 5-fold, as compared to phospholipids), regardless of the isotope tracer utilized. We discuss the implications of this metabolic flexibility on studies that aim to characterize apparent drug efficacy.

Hyperalphalipoproteinemic scavenger receptor BI knockout mice exhibit a disrupted epidermal lipid barrier

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters (CE) from high-density lipoproteins (HDL). An impaired SR-BI function leads to hyperalphalipoproteinemia with elevated levels of cholesterol transported in the HDL fraction. Accumulation of cholesterol in apolipoprotein B (apoB)-containing lipoproteins has been shown to alter skin lipid composition and barrier function in mice. To investigate whether these hypercholesterolemic effects on the skin also occur in hyperalphalipoproteinemia, we compared skins of wild-type and SR-BI knockout (SR-BI−/−) mice. SR-BI deficiency did not affect the epidermal cholesterol content and induced only minor changes in the ceramide subclasses. The epidermal free fatty acid (FFA) pool was, however, enriched in short and unsaturated chains. Plasma CE levels strongly correlated with epidermal FFA C18:1 content. The increase in epidermal FFA coincided with downregulation of cholesterol and FFA synthesis genes, suggesting a compensatory response to increased flux of plasma cholesterol and FFAs into the skin. Importantly, the SR-BI−/− epidermal lipid barrier showed increased permeability to ethyl-paraminobenzoic acid, indicating an impairment of the barrier function. In conclusion, increased HDL-cholesterol levels in SR-BI−/− mice can alter the epidermal lipid composition and lipid barrier function similarly as observed in hypercholesterolemia due to elevated levels of apoB-containing lipoproteins.

Lipid metabolism disorders contribute to hepatotoxicity of triclosan in mice

Previous in vivo exposure studies focused mainly on nuclear receptors involved in hepatotoxicity of triclosan (TCS). As liver plays a vital role in metabolic processes, dysregulations in lipid metabolism have been identified as potential drivers of pathogenesis. Investigation of changes in lipid metabolism might widen our understanding of toxicological effects as well as the underlying mechanism occurring in the liver. In this study, we comprehensively assessed the effect of TCS exposure on hepatic lipid metabolism in mice. Our results showed that TCS induced significant changes in hepatic free fatty acid pool by upregulation of fatty acid uptake and de novo fatty acid synthesis. Besides, hepatic levels of lipids, including acyl carnitine (AcCa), ceramide (Cer), triacylglycerols (TG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE) were also increased, together with upreguation of genes associated to TG synthesis, fatty acid oxidation and inflammation in TCS exposure group. These changes in lipid homeostasis could contribute to membrane instability, lipid accumulation, oxidative stress and inflammation. Our results suggested that TCS exposure could induce hepatic lipid metabolism disorders in mice, which would further contribute to the liver damage effects of TCS.

Drop-in biofuel production using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica

BackgroundOleaginous yeasts are potent hosts for the renewable production of lipids and harbor great potential for derived products, such as biofuels. Several promising processes have been described that produce hydrocarbon drop-in biofuels based on fatty acid decarboxylation and fatty aldehyde decarbonylation. Unfortunately, besides fatty aldehyde toxicity and high reactivity, the most investigated enzyme, aldehyde-deformylating oxygenase, shows unfavorable catalytic properties which hindered high yields in previous metabolic engineering approaches.ResultsTo demonstrate an alternative alkane production pathway for oleaginous yeasts, we describe the production of diesel-like, odd-chain alkanes and alkenes, by heterologously expressing a recently discovered light-driven oxidase from Chlorella variabilis (CvFAP) in Yarrowia lipolytica. Initial experiments showed that only strains engineered to have an increased pool of free fatty acids were susceptible to sufficient decarboxylation. Providing these strains with glucose and light in a synthetic medium resulted in titers of 10.9 mg/L of hydrocarbons. Using custom 3D printed labware for lighting bioreactors, and an automated pulsed glycerol fed-batch strategy, intracellular titers of 58.7 mg/L were achieved. The production of odd-numbered alkanes and alkenes with a length of 17 and 15 carbons shown in previous studies could be confirmed.ConclusionsOleaginous yeasts such as Yarrowia lipolytica can transform renewable resources such as glycerol into fatty acids and lipids. By heterologously expressing a fatty acid photodecarboxylase from the algae Chlorella variabilis hydrocarbons were produced in several scales from microwell plate to 400 mL bioreactors. The lighting turned out to be a crucial factor in terms of growth and hydrocarbon production, therefore, the evaluation of different conditions was an important step towards a tailor-made process. In general, the developed bioprocess shows a route to the renewable production of hydrocarbons for a variety of applications ranging from being substrates for further enzymatic or chemical modification or as a drop-in biofuel blend.

Placental lipoprotein lipase activity is positively associated with newborn adiposity

IntroductionRecent data suggest that in addition to glucose, fetal growth is related to maternal triglycerides (TG). To reach the fetus, TG must be hydrolyzed to free fatty acids (FFA) and transported across the placenta, but regulation is uncertain. Placental lipoprotein lipase (pLPL) hydrolyzes TG, both dietary chylomicron TG (CM-TG) and very-low density lipoprotein TG (VLDL-TG), to FFA. This may promote fetal fat accretion by increasing the available FFA pool for placental uptake. We tested the novel hypothesis that pLPL activity, but not maternal adipose tissue LPL activity, is associated with newborn adiposity and higher maternal TG. MethodsTwenty mothers (n = 13 normal-weight; n = 7 obese) were prospectively recruited. Maternal glucose, insulin, TG (total, CM-TG, VLDL-TG), and FFA were measured at 14–16, 26–28, and 36–37 weeks, and adipose tissue LPL was measured at 26–28 weeks. At term delivery, placental villous biopsies were immediately analyzed for pLPL enzymatic activity. Newborn percent body fat (newborn %fat) was assessed by skinfolds. ResultsPlacental LPL activity was positively correlated with birthweight (r = 0.48;P = 0.03) and newborn %fat (r = 0.59;P = 0.006), further strengthened by correcting for gestational age at delivery (r = 0.75;P = 0.0001), but adipose tissue LPL was not. Maternal TG and BMI were not correlated with pLPL activity. Additionally, pLPL gene expression, while modestly correlated with enzymatic activity (r = 0.53;P < 0.05), was not correlated with newborn adiposity. DiscussionThis is the first study to show a positive correlation between pLPL activity and newborn %fat. Placental lipase regulation and the role of pLPL in pregnancies characterized by nutrient excess and fetal overgrowth warrant further investigation.

A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction

ObjectiveRegulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo actions of ACSL4 are unknown. The purpose of our studies was to determine the in vivo role of adipocyte ACSL4 in regulating obesity-associated adipocyte dysfunction. MethodsWe developed a novel mouse model with adipocyte-specific ablation of ACSL4 (Ad-KO) using loxP Cre recombinase technology. Metabolic phenotyping of Ad-KO mice relative to their floxed littermates (ACSL4floxed) was performed, including body weight and body composition over time; insulin and glucose tolerance tests; and energy expenditure, activity, and food intake in metabolic cages. Adipocytes were isolated for ex vivo adipocyte oxygen consumption by Clark electrode and lipidomics analysis. In vitro adipocyte analysis including oxygen consumption by Seahorse and real-time PCR analysis were performed to confirm our in vivo findings. ResultsAd-KO mice were protected against DIO, adipocyte death, and metabolic dysfunction. Adipocytes from Ad-KO mice fed high-fat diet (HFD) had reduced incorporation of AA into phospholipids (PL), free AA, and levels of the AA lipid peroxidation product 4-hydroxynonenal (4-HNE). Additionally, adipocytes from Ad-KO mice fed HFD had reduced p53 activation and increased adipocyte oxygen consumption (OCR), which we demonstrated are direct effects of 4-HNE on adipocytes in vitro. ConclusionThese studies are the first to elucidate ACSL4's in vivo actions to regulate the incorporation of AA into PL and downstream effects on DIO-associated adipocyte dysfunction. By reducing the incorporation of AA into PL and free fatty acid pools in adipocytes, Ad-KO mice were significantly protected against HFD-induced increases in adipose and liver fat accumulation, adipocyte death, gonadal white adipose tissue (gWAT) inflammation, and insulin resistance (IR). Additionally, deficiency of adipocyte ACSL4 expression in mice fed a HFD resulted in increased gWAT adipocyte OCR and whole body energy expenditure (EE).

Insulin induces a shift in lipid and primary carbon metabolites in a model of fasting-induced insulin resistance.

Prolonged fasting in northern elephant seals (NES) is characterized by a reliance on lipid metabolism, conservation of protein, and reduced plasma insulin. During early fasting, glucose infusion previously reduced plasma free fatty acids (FFA); however, during late-fasting, it induced an atypical elevation in FFA despite comparable increases in insulin during both periods suggestive of a dynamic shift in tissue responsiveness to glucose-stimulated insulin secretion. To better assess the contribution of insulin to this fasting-associated shift in substrate metabolism. We compared the responses of plasma metabolites (amino acids (AA), FFA, endocannabinoids (EC), and primary carbon metabolites (PCM)) to an insulin infusion (65 mU/kg) in early- and late-fasted NES pups (n = 5/group). Plasma samples were collected prior to infusion (T0) and at 10, 30, 60, and 120 min post-infusion, and underwent untargeted and targeted metabolomics analyses utilizing a variety of GC-MS and LC-MS technologies. In early fasting, the majority (72%) of metabolite trajectories return to baseline levels within 2 h, but not in late fasting indicative of an increase in tissue sensitivity to insulin. In late-fasting, increases in FFA and ketone pools, coupled with decreases in AA and PCM, indicate a shift toward lipolysis, beta-oxidation, ketone metabolism, and decreased protein catabolism. Conversely, insulin increased PCM AUC in late fasting suggesting that gluconeogenic pathways are activated. Insulin also decreased FFA AUC between early and late fasting suggesting that insulin suppresses triglyceride hydrolysis. Naturally adapted tolerance to prolonged fasting in these mammals is likely accomplished by suppressing insulin levels and activity, providing novel insight on the evolution of insulin during a condition of temporary, reversible insulin resistance.

Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae

BackgroundIn vivo production of fatty acid-derived chemicals in Saccharomyces cerevisiae requires strategies to increase the intracellular supply of either acyl-CoA or free fatty acids (FFAs), since their cytosolic concentrations are quite low in a natural state for this organism. Deletion of the fatty acyl-CoA synthetase genes FAA1 and FAA4 is an effective and straightforward way to disable re-activation of fatty acids and drastically increase FFA levels. However, this strategy causes FFA over-accumulation and consequential release to the extracellular medium, which results in a significant loss of precursors that compromises the process yield. In the present study, we aimed for dynamic expression of the fatty acyl-CoA synthetase gene FAA1 to regulate FFA and acyl-CoA pools in order to improve fatty alcohol production yields.ResultsWe analyzed the metabolite dynamics of a faa1Δ faa4Δ strain constitutively expressing a carboxylic acid reductase from Mycobacterium marinum (MmCAR) and an endogenous alcohol dehydrogenase (Adh5) for in vivo production of fatty alcohols from FFAs. We observed production of fatty acids and fatty alcohols with different rates leading to high levels of FFAs not being converted to the final product. To address the issue, we expressed the MmCAR + Adh5 pathway together with a fatty acyl-CoA reductase from Marinobacter aquaeolei to enable fatty alcohol production simultaneously from FFA and acyl-CoA, respectively. Then, we expressed FAA1 under the control of different promoters in order to balance FFA and acyl-CoA interconversion rates and to achieve optimal levels for conversion to fatty alcohols. Expressing FAA1 under control of the HXT1 promoter led to an increased accumulation of fatty alcohols per OD600 up to 41% while FFA levels were decreased by 63% compared with the control strain.ConclusionsFine-tuning and dynamic regulation of key metabolic steps can be used to improve cell factories when the rates of downstream reactions are limiting. This avoids loss of precursors to the extracellular medium or to competing reactions, hereby potentially improving the process yield. The study also provides knowledge of a key point of fatty acid regulation and homeostasis, which can be used for future design of cells factories for fatty acid-derived chemicals.

Effects of prolonged darkness and temperature on the lipid metabolism in the benthic diatom Navicula perminuta from the Arctic Adventfjorden, Svalbard

The Arctic represents an extreme habitat for phototrophic algae due to long periods of darkness caused by the polar night (~4 months darkness). Benthic diatoms, which dominate microphytobenthic communities in shallow water regions, can survive this dark period, but the underlying physiological and biochemical mechanisms are not well understood. One of the potential mechanisms for long-term dark survival is the utilisation of stored energy products in combination with a reduced basic metabolism. In recent years, water temperatures in the Arctic increased due to an ongoing global warming. Higher temperatures could enhance the cellular energy requirements for the maintenance metabolism during darkness and, therefore, accelerate the consumption of lipid reserves. In this study, we investigated the macromolecular ratios and the lipid content and composition of Navicula cf. perminuta Grunow, an Arctic benthic diatom isolated from the microphytobenthos of Adventfjorden (Svalbard, Norway), over a dark period of 8 weeks at two different temperatures (0 and 7 °C). The results demonstrate that N. perminuta uses the stored lipid compound triacylglycerol (TAG) during prolonged dark periods, but also the pool of free fatty acids (FFA). Under the enhanced temperature of 7 °C, the lipid resources were used significantly faster than at 0 °C, which could consequently lead to a depletion of this energy reserves before the end of the polar night. On the other hand, the membrane building phospho- and glycolipids remained unchanged during the 8 weeks darkness, indicating still intact thylakoid membranes. These results explain the shorter survival times of polar diatoms with increasing water temperatures during prolonged dark periods.

CHANGES IN BLOOD FREE FATTY ACID CONTENT AMONG CHILDREN WITH LATENT FORM OF CHRONIC RENAL FAILURE

Aim. To assess the status of blood free fatty acid balance in children with megaureter diagnosed latent form of chronic renal failure. Materials and methods. The study design: prospective, controlled, nonrandomized, one-centered. The study included 40 children, divided into 2 groups. The main group ( n = 22) - patients after surgical intervention for megaureter with developing renal dysfunction. The comparison group - 18 relatively healthy children with a mean age of 6,1 ± 1,3 years. Results. It was revealed by the authors that in patients of the main group statistically significantly raised the content of a number of fatty acids. When conducting correlation analysis between the levels of blood total protein, erythrocytes and leukocytes with free fatty acid pool, it was detected that there are reliable, differently directed strong correlations between some of them. Conclusions. Children, who underwent surgical intervention for megaureter followed by development of latent chronic renal failure, differ from healthy children by both total protein content and qualitative composition of blood free fatty acids.

Astrocyte arachidonate and palmitate uptake and metabolism is differentially modulated by dibutyryl-cAMP treatment

Astrocytes play a vital role in brain lipid metabolism; however the impact of the phenotypic shift in astrocytes to a reactive state on arachidonic acid metabolism is unknown. Therefore, we determined the impact of dibutyryl-cAMP (dBcAMP) treatment on radiolabeled arachidonic acid ([1-14C]20:4n-6) and palmitic acid ([1-14C]16:0) uptake and metabolism in primary cultured murine cortical astrocytes. In dBcAMP treated astrocytes, total [1-14C]20:4n-6 uptake was increased 1.9-fold compared to control, while total [1-14C]16:0 uptake was unaffected. Gene expression of long-chain acyl-CoA synthetases (Acsl), acyl-CoA hydrolase (Acot7), fatty acid binding protein(s) (Fabp) and alpha-synuclein (Snca) were determined using qRT-PCR. dBcAMP treatment increased expression of Acsl3 (4.8-fold) and Acsl4 (1.3-fold), which preferentially use [1-14C]20:4n-6 and are highly expressed in astrocytes, consistent with the increase in [1-14C]20:4n-6 uptake. However, expression of Fabp5 and Fabp7 were significantly reduced by 25% and 45%, respectively. Acot7 (20%) was also reduced, suggesting dBcAMP treatment favors acyl-CoA formation. dBcAMP treatment enhanced [1-14C]20:4n-6 (2.2-fold) and [1-14C]16:0 (1.6-fold) esterification into total phospholipids, but the greater esterification of [1-14C]20:4n-6 is consistent with the observed uptake through increased Acsl, but not Fabp expression. Although total [1-14C]16:0 uptake was not affected, there was a dramatic decrease in [1-14C]16:0 in the free fatty acid pool as esterification into the phospholipid pool was increased, which is consistent with the increase in Acsl3 and Acsl4 expression. In summary, our data demonstrates that dBcAMP treatment increases [1-14C]20:4n-6 uptake in astrocytes and this increase appears to be due to increased expression of Acsl3 and Acsl4 coupled with a reduction in Acot7 expression.

Baker's Yeast Deficient in Storage Lipid Synthesis Uses cis-Vaccenic Acid to Reduce Unsaturated Fatty Acid Toxicity.

The role of cis-vaccenic acid (18:1n-7) in the reduction of unsaturated fatty acids toxicity was investigated in baker's yeast Saccharomyces cerevisiae. The quadruple mutant (QM, dga1Δ lro1Δ are1Δ are2Δ) deficient in enzymes responsible for triacylglycerol and steryl ester synthesis has been previously shown to be highly sensitive to exogenous unsaturated fatty acids. We have found that cis-vaccenic acid accumulated during cultivation in the QM cells but not in the corresponding wild type strain. This accumulation was accompanied by a reduction in palmitoleic acid (16:1n-7) content in the QM cells that is consistent with the proposed formation of cis-vaccenic acid by elongation of palmitoleic acid. Fatty acid analysis of individual lipid classes from the QM strain revealed that cis-vaccenic acid was highly enriched in the free fatty acid pool. Furthermore, production of cis-vaccenic acid was arrested if the mechanism of fatty acids release to the medium was activated. We also showed that exogenous cis-vaccenic acid did not affect viability of the QM strain at concentrations toxic for palmitoleic or oleic acids. Moreover, addition of cis-vaccenic acid to the growth medium provided partial protection against the lipotoxic effects of exogenous oleic acid. Transformation of palmitoleic acid to cis-vaccenic acid is thus a rescue mechanism enabling S. cerevisiae cells to survive in the absence of triacylglycerol synthesis as the major mechanism for unsaturated fatty acid detoxification.