Higher Long-chain Fatty Acids Research Articles

P612: CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)-DERIVED T-CELLS HAVE DISTURBED FATTY ACID METABOLISM, POSSIBLY CONTRIBUTING TO T-CELL DYSFUNCTION

Background: T-cells from CLL patients are dysfunctional and display reduced activation, proliferation and cytotoxicity upon in-vitro activation. Whilst the role of glucose metabolism in activated T cells is extensively studied, the contribution of other energy sources is less clear. Fatty acid (FA) and lipid uptake in T cells involves the scavenger membrane transporter CD36. CD36-mediated lipid transport has been identified to (metabolically) modulate intratumoral T cells (both Treg and cytotoxic CD8+ T cells). However, lipid metabolism and a potential role for CD36 has not been studied in the context of T cell dysfunction in CLL Aims: To examine the role of FA metabolism and the fatty acid transporter CD36 in acquired T-cell dysfunction in CLL. Methods: Untreated CLL patients with a white blood cell count (WBC) greater than 20×109 cells/L and, as control, age-matched healthy donors (HD) were studied. T cells were analyzed by flow cytometry, confocal microscopy or extracellular flux analysis (Seahorse), either directly after thawing or after a 2 day culture with or without αCD3/αCD28 antibodies. Results: Analysis of CD36+ vs CD36- CD4 and CD8 T-cells from HD revealed that CD36+ cells had higher long-chain fatty acid (LCFA) and glucose uptake, and higher expression of the glucose transporter GLUT-1 and the rate-limiting mitochondrial FA transporter CPT1α (Fig. 1A). The cells expressing CD36 were also more likely to increase their CD25 expression and mitochondrial mass upon stimulation, indicating a metabolic and functional advantage potentially conferred by the surface expression of CD36. The absolute amount and percentage of T cells (CD4 and CD8) expressing FA transporter CD36 was lower in CLL T-cells as compared to HD (Fig. 1B). The levels of LCFA uptake and expression of CPT1α were comparable, indicating that the lower amount of CD36 might successfully cater to the FA demands of resting CLL T cells(Fig. 1C). However, while stimulation of HD T-cells markedly upregulated CD36 abundance, LCFA uptake and CPT1α, concomitantly with the activation marker CD25, CLL-derived T-cells failed to mount a similar response (Fig. 1D). This lack of induction of CD36 expression was also observed in CLL-derived T cells that increased CD25, which implies that this response is independent of T-cell activation. The transcription factors peroxisome proliferator-activated receptors (PPAR) α and γ are important regulators of FA metabolism. We observed reduced expression of these transcription factors in resting CLL T cells. However, distinct from CD36, CLL T cells were indistinguishable from HD T-cells in their ability to increase levels of both PPARα and PPARγ upon stimulation, indicating that alternative mechanisms might be implicated in the regulation of CD36 and FA metabolism in CLL T cells. Image:Summary/Conclusion: Collectively, our results show that disturbances in FA metabolism are present on multiple levels in CLL T cells and that lack of LCFA uptake into CLL T-cells could represent a limiting step in FA metabolism. We also highlight CD36+ cells as a metabolically distinct T-cell subtype that is largely absent in CLL T-cells, independently of their activation status. We are currently investigating strategies to increase CD36 expression and FA metabolism in general in CLL T cells, which could ameliorate CLL T cell dysfunction and provide targets to improve autologous T cell therapies.

Risk of Disease Recurrence and Mortality Varies by Type of Fat Consumed before Cancer Treatment in a Longitudinal Cohort of Head and Neck Squamous Cell Carcinoma Patients

BackgroundThe associations between specific types of fat and head and neck squamous cell carcinoma (HNSCC) recurrence and mortality rates have not yet been examined. ObjectivesThe purpose of this study was to determine how intakes of various fat subtypes before cancer treatment are associated with recurrence and mortality in adults diagnosed with HNSCC. MethodsThis was a secondary analysis longitudinal cohort study of data collected from 476 newly diagnosed patients with HNSCC. Patients completed baseline FFQs and epidemiologic health surveys. Recurrence and mortality events were collected annually. Fat intakes examined included long-chain fatty acids (LCFAs), unsaturated fatty acids (FAs), PUFAs, ω-3 (n–3) PUFAs, ω-6 (n–6) PUFAs, MUFAs, animal fats, vegetable fats, saturated FAs, and trans fats. Associations between fat intake (categorized into tertiles) and time to event were tested using multivariable Cox proportional hazards models, adjusting for age, sex, smoking status, human papillomavirus status, tumor site, cancer stage, and total caloric intake. Intake of fats was compared with the lowest tertile. ResultsDuring the study period, there were 115 recurrent and 211 death events. High LCFA intake was associated with a reduced all-cause mortality risk (HR: 0.55; 95% CI: 0.34, 0.91; P-trend = 0.02). High unsaturated FA intake was associated with a reduced all-cause mortality risk (HR: 0.62; 95% CI: 0.40, 0.97; P-trend = 0.04) and HNSCC-specific mortality risk (HR: 0.51; 95% CI: 0.29, 0.90; P-trend = 0.02). High intakes of ω-3 PUFAs (HR: 0.56; 95% CI: 0.35, 0.91; P-trend = 0.02) and ω-6 PUFAs (HR: 0.57; 95% CI: 0.34, 0.94; P-trend = 0.02) were significantly associated with a reduced all-cause mortality risk. There were no significant associations between other fat types and recurrence or mortality risk. ConclusionsIn this prospective survival cohort of 476 newly diagnosed patients with HNSCC, our data suggest that HNSCC prognosis may vary depending on the fat types consumed before cancer treatment. Clinical intervention trials should test these associations.

Effect of Sub-Stoichiometric Fe(III) Amounts on LCFA Degradation by Methanogenic Communities.

Long-chain fatty acids (LCFA) are common contaminants in municipal and industrial wastewater that can be converted anaerobically to methane. A low hydrogen partial pressure is required for LCFA degradation by anaerobic bacteria, requiring the establishment of syntrophic relationships with hydrogenotrophic methanogens. However, high LCFA loads can inhibit methanogens, hindering biodegradation. Because it has been suggested that anaerobic degradation of these compounds may be enhanced by the presence of alternative electron acceptors, such as iron, we investigated the effect of sub-stoichiometric amounts of Fe(III) on oleate (C18:1 LCFA) degradation by suspended and granular methanogenic sludge. Fe(III) accelerated oleate biodegradation and hydrogenotrophic methanogenesis in the assays with suspended sludge, with H2-consuming methanogens coexisting with iron-reducing bacteria. On the other hand, acetoclastic methanogenesis was delayed by Fe(III). These effects were less evident with granular sludge, possibly due to its higher initial methanogenic activity relative to suspended sludge. Enrichments with close-to-stoichiometric amounts of Fe(III) resulted in a microbial community mainly composed of Geobacter, Syntrophomonas, and Methanobacterium genera, with relative abundances of 83–89%, 3–6%, and 0.2–10%, respectively. In these enrichments, oleate was biodegraded to acetate and coupled to iron-reduction and methane production, revealing novel microbial interactions between syntrophic LCFA-degrading bacteria, iron-reducing bacteria, and methanogens.

Enhanced Methanization of Long-Chain Fatty Acid Wastewater at 20°C in the Novel Dynamic Sludge Chamber–Fixed Film Bioreactor

Lipid-containing wastewaters, such as those arising from dairy processing, are frequently discharged at temperatures ≤20°C. Their valorization at low ambient temperatures offers opportunities to expand the application of high-rate anaerobic wastewater treatment towards achieving energy neutrality by minimizing the energy demand for heating. Lipid hydrolysis generates long chain fatty acids (LCFA), which incur operational challenges and hinder stable bioreactor operation by inducing sludge flotation and washout, coupled with the added challenge of treatment at lower temperature (20°C). These challenges are tackled together uniquely during the treatment of LCFA-rich synthetic dairy wastewater (SDW) (33% COD-LCFA) through de-novo formed microbial granular sludge within the dynamic sludge chamber fixed film (DSC-FF) reactor. The novel reactor design facilitated sludge retention for the entire operational period of 150 days by containing settled, floating and LCFA-encapsulated granular sludge and biofilm within a single module. High COD removal efficiencies (87-98%) were achieved in the three replicated DSC-FF reactors, along with complete LCFA removal at 18-72 h HRT (LCFA loading rate of 220-890 mgCOD-LCFA/L·d) and partial LCFA removal at 12 h HRT (LCFA loading rate of 1333 mgCOD-LCFA/L·d). The high removal efficiencies of unsaturated and saturated LCFAs achieved are reported for the first time during continuous anaerobic wastewater treatment at low temperatures (20°C). Moreover, de novo granulation was achieved within 8 d from a combination of inoculum mixtures at a high LCFA concentration (33% COD-LCFA) in SDW. The results demonstrate the feasibility of the DSC-FF reactor for treating LCFA-rich wastewaters at discharge temperatures and offer potential for expanded and more energetically productive anaerobic valorization of lipid-rich wastewater.

Insight into the Role of Facultative Bacteria Stimulated by Microaeration in Continuous Bioreactors Converting LCFA to Methane.

Conversion of unsaturated long chain fatty acids (LCFA) to methane in continuous bioreactors is not fully understood. Palmitate (C16:0) often accumulates during oleate (C18:1) biodegradation in methanogenic bioreactors, and the reason why this happens and which microorganisms catalyze this reaction remains unknown. Facultative anaerobic bacteria are frequently found in continuous reactors operated at high LCFA loads, but their function is unclear. To get more insight on the role of these bacteria, LCFA conversion was studied under microaerophilic conditions. For that, we compared bioreactors treating oleate-based wastewater (organic loading rates of 1 and 3 kg COD m-3 d-1), operated under different redox conditions (strictly anaerobic-AnR, -350 mV; microaerophilic-MaR, -250 mV). At the higher load, palmitate accumulated 7 times more in the MaR, where facultative anaerobes were more abundant, and only the biomass from this reactor could recover the methanogenic activity after a transient inhibition. In a second experiment, the abundance of facultative anaerobic bacteria, particularly Pseudomonas spp. (from which two strains were isolated), was strongly correlated ( p < 0.05) with palmitate-to-total LCFA percentage in the biofilm formed in a continuous plug flow reactor fed with very high loads of oleate. This work strongly suggests that microaeration stimulates the development of facultative bacteria that are critical for achieving LCFA conversion to methane in continuous bioreactors. Microbial networks and interactions of facultative and strict anaerobes in microbial communities should be considered in future studies.

The inclusion of low quantities of lipids in the diet of ruminants fed low quality forages has little effect on rumen function

Biohydrogenation within rumen fluid (RF) proceeds to varying degrees depending on retention time (RT) and type of basal diet, especially the profile of fatty acid (FA) being hydrogenised. The objective of this study was to examine the FA profile and the RT of liquid in the rumen of steers fed a low crude protein (CP) tropical grass (Chloris gayana hay, 38g CP, 17g crude lipid and 752g neutral detergent fibre (aNDFom)/kg dry matter (DM)) supplemented with various lipids. Five rumen cannulated Bos indicus cross, five-year-old steers (799±15kg live weight (LW)) were allocated to a 5×5 Latin square design. The treatments were control, hay only, or the addition of 30g/kg hay DM of lipid sources: Coconut (high lauric acid), cottonseed and soybean (high linoleic acid) or fish oil (high long chain FA (LCFA)). Retention time decreased with addition of soybean oil (14h) but no differences between other treatments (mean 17h). Coconut oil increased lauric and myristic acids in RF. There were no changes in total saturated FA (TSFA) in RF, with exception of a lower concentration for fish oil treatment. Addition of fish oil also decreased the concentration in RF of stearic and linolenic acid, but no differences to coconut and cottonseed treatments for linolenic acid. Fish oil also resulted in higher LCFA, linoleic and total unsaturated FA (TUFA), but no differences to soybean oil for the latter two acids. The conjugated linoleic acid (CLA) was only different in RF between cottonseed and fish oil treatments. Differences in FA profile of oils were only partially translated into the FA profile in RF of steers fed a tropical hay, without great changes in the proportion of CLA isomers observed.

Long chain fatty acids (LCFA) evolution for inhibition forecasting during anaerobic treatment of lipid-rich wastes: Case of milk-fed veal slaughterhouse waste

A detailed study of a solid slaughterhouse waste (SHW) anaerobic treatment is presented. The waste used in this study is rich in lipids and proteins residue. Long chain fatty acids (LCFA), coming from the hydrolysis of lipids were inhibitory to anaerobic processes at different degrees. Acetogenesis and methanogenesis processes were mainly affected by inhibition whereas disintegration and hydrolysis processes did not seem to be affected by high LCFA concentrations. Nevertheless, because of the high energy content, this kind of waste is very suitable for anaerobic digestion but strict control of operating conditions is required to prevent inhibition. For that, two inhibition indicators were identified in this study. Those two indicators, LCFA dynamics and LCFA/VSbiomass ratio proved to be useful to predict and to estimate the process inhibition degree.

Anaerobic Codigestion of Sludge: Addition of Butcher's Fat Waste as a Cosubstrate for Increasing Biogas Production.

Fat waste discarded from butcheries was used as a cosubstrate in the anaerobic codigestion of sewage sludge (SS). The process was evaluated under mesophilic and thermophilic conditions. The codigestion was successfully attained despite some inhibitory stages initially present that had their origin in the accumulation of volatile fatty acids (VFA) and adsorption of long-chain fatty acids (LCFA). The addition of a fat waste improved digestion stability and increased biogas yields thanks to the higher organic loading rate (OLR) applied to the reactors. However, thermophilic digestion was characterized by an effluent of poor quality and high VFA content. Results from spectroscopic analysis suggested the adsorption of lipid components onto the anaerobic biomass, thus disturbing the complete degradation of substrate during the treatment. The formation of fatty aggregates in the thermophilic reactor prevented process failure by avoiding the exposure of biomass to the toxic effect of high LCFA concentrations.

Microbiome response to controlled shifts in ammonium and LCFA levels in co-digestion systems

Anaerobic co-digestion using protein-rich and lipid-rich co-substrates is limited by the accumulation of ammonia and long chain fatty acids (LCFAs), which are important inhibitors of the anaerobic microorganisms. This work aimed to study the microbial community dynamics during gradual and abrupt increase in ammonium and LCFAs concentrations by applying several molecular techniques, as well as during gradual decrease. For this purpose, two anaerobic reactors co-digesting three agro-industrial wastes underwent abrupt and gradual changes of ammonium and LCFAs concentrations. Both variations provoked volatile fatty acids (VFAs) accumulation, mainly acetic acid up to 4.5gL−1. High ammonium levels were correlated to an increase in Pseudomonadaceae, Carnobacteriaceae and Clostridiadaceae families and to a drop in Syntrophomonadaceae. However, high LCFA levels provoked an increase in the Anaerobaculaceae and Peptococcaceae families. Both perturbations resulted in greater variations in the archaeal domain, going from Methanosaeta dominance in steady state to hydrogenotrophic pathway during the disturbance periods. During the abrupt changes, Bacteria domain experienced a minimal change, which indicates the adaptation bacterial populations to high ammonium and LCFAs levels. Species belonging to Porphyromonadaceae and Tissierellaceae families linked to VFAs consumption rose their presence during the recovery period. This study identifies a subset of microbial communities linked to high ammonia and LCFA concentrations, useful for optimizing the high-rate co-digestion processes dealing with lipid and protein-rich co-substrates.

Mechanism, kinetics and microbiology of inhibition caused by long-chain fatty acids in anaerobic digestion of algal biomass.

BackgroundOleaginous microalgae contain a high level of lipids, which can be extracted and converted to biofuel. The lipid-extracted residue can then be further utilized through anaerobic digestion to produce biogas. However, long-chain fatty acids (LCFAs) have been identified as the main inhibitory factor on microbial activity of anaerobic consortium. In this study, the mechanism of LCFA inhibition on anaerobic digestion of whole and lipid-extracted algal biomass was investigated with a range of calcium concentrations against various inoculum to substrate ratios as a means to alleviate the LCFA inhibition.ResultsWhole algal biomass of Nannochloropsissalina represents high lipid content algal biomass while lipid-extracted residue represents its low lipid counterpart. The anaerobic digestion experiments were conducted in a series of serum bottles at 35 °C for 20 days. A kinetic model, considering LCFA inhibition on hydrolysis, acidogenesis as well as methanogenesis steps, was developed from the observed phenomenon of inhibition factors as a function of the LCFA concentration and specific biomass content or calcium concentration. The results showed that inoculum to substrate ratio had a stronger effect on biogas production than calcium, and calcium had no effect on biogas production when inoculum concentration was extremely low. The microbial community analysis by high-throughput Illumina Miseq sequencing indicated that diversity of both bacterial and methanogenic communities decreased with elevation of lipid concentration. Hydrolytic bacteria and aceticlastic methanogens dominated bacterial and archaea communities, respectively, in both high and low LCFA concentration digesters.ConclusionsThis study demonstrated that inoculum concentration has a more significant effect on alleviating LCFA inhibition than calcium concentration, while calcium only played a role when inoculum concentration met a threshold level. The model revealed that each functional microbial group was subject to different levels of LCFA inhibition. Although methanogens were the most susceptible microbes to LCFA inhibition, the inhibition factor for hydrolytic bacteria was more highly affected by inoculum concentration. The microbial community analysis indicated that the bacterial community was affected more than the methanogenic community by high LCFAs concentration. Syntrophic acetogens were sensitive to high LCFA concentrations and thus showed a decreased abundance in such an environment. Graphical abstractProposed mechanism of calcium mitigated LCFA inhibition

Long-chain acyl coenzyme A synthetase 1 overexpression in primary cultured Schwann cells prevents long chain fatty acid-induced oxidative stress and mitochondrial dysfunction.

High circulating long chain fatty acids (LCFAs) are implicated in diabetic neuropathy (DN) development. Expression of the long-chain acyl-CoA synthetase 1 (Acsl1) gene, a gene required for LCFA metabolic activation, is altered in human and mouse diabetic peripheral nerve. We assessed the significance of Acsl1 upregulation in primary cultured Schwann cells. Acsl1 overexpression prevented oxidative stress (nitrotyrosine; hydroxyoctadecadienoic acids [HODEs]) and attenuated cellular injury (TUNEL) in Schwann cells following 12 h exposure to LCFAs (palmitate, linoleate, and oleate, 100 μM). Acsl1 overexpression potentiated the observed increase in medium to long-chain acyl-carnitines following 12 h LCFA exposure. Data are consistent with increased mitochondrial LCFA uptake, largely directed to incomplete beta-oxidation. LCFAs uncoupled mitochondrial oxygen consumption from ATP production. Acsl1 overexpression corrected mitochondrial dysfunction, increasing coupling efficiency and decreasing proton leak. Schwann cell mitochondrial function is critical for peripheral nerve function, but research on Schwann cell mitochondrial dysfunction in response to hyperlipidemia is minimal. We demonstrate that high levels of a physiologically relevant mixture of LCFAs induce Schwann cell injury, but that improved mitochondrial uptake and metabolism attenuate this lipotoxicity. Acsl1 overexpression improves Schwann cell function and survival following high LCFA exposure in vitro; however, the observed endogenous Acsl1 upregulation in peripheral nerve in response to diabetes is not sufficient to prevent the development of DN in murine models of DN. Therefore, targeted improvement in Schwann cell metabolic disposal of LCFAs may improve DN phenotypes.

Endurance of methanogenic archaea in anaerobic bioreactors treating oleate-based wastewater

Methanogenic archaea are reported as very sensitive to lipids and long chain fatty acids (LCFA). Therefore, in conventional anaerobic processes, methane recovery during LCFA-rich wastewater treatment is usually low. By applying a start-up strategy, based on a sequence of step feeding and reaction cycles, an oleate-rich wastewater was efficiently treated at an organic loading rate of 21 kg COD m(-3) day(-1) (50 % as oleate), showing a methane recovery of 72 %. In the present work, the archaeal community developed in that reactor is investigated using a 16S rRNA gene approach. This is the first time that methanogens present in a bioreactor converting efficiently high loads of LCFA to methane are monitored. Denaturing gradient gel electrophoresis profiling showed that major changes on the archaeal community took place during the bioreactor start-up, where phases of continuous feeding were alternated with batch phases. After the start-up, a stable archaeal community (similarity higher than 84 %) was observed and maintained throughout the continuous operation. This community exhibited high LCFA tolerance and high acetoclastic and hydrogenotrophic activity. Cloning and sequencing results showed that Methanobacterium- and Methanosaeta-like microorganisms prevailed in the system and were able to tolerate and endure during prolonged exposure to high LCFA loads, despite the previously reported LCFA sensitivity of methanogens.

Regulation of fatty acid transport: from transcriptional to posttranscriptional effects

Long-chain fatty acids (LCFA) are essential for many cellular functions. In metabolically active tissues such as heart and skeletal muscle their principal role is to supply oxidative substrates. LCFA serve also as structural components of various lipids and proteins, and function in intracellular signaling. Although LCFA can enter into the cells via simple diffusion (Hamilton and Kamp 1999; Hamilton et al. 2002), LCFA utilization is not simply a function of blood LCFA levels, as plasmalemmal LCFA transport has been shown to be regulated at the tissue level (Turcotte et al. 1994; Coburn et al. 2000; Hajri et al. 2001). In particular, the membrane proteins such as FAT/CD36, FABPpm and FATP’s were shown to facilitate LCFA movement across the plasma membranes in skeletal muscle as well as in heart (Bonen et al. 2002; Schaffer 2002). Fatty-acid transporters are regulated proteins whose expression is modulated through transcriptional and post-transcriptional mechanisms. Recently, it was shown that LCFA protein-mediated transport is modulated in tissues with high LCFA utilization rates: a) by transcription factors such as peroxisome proliferator-activated receptors, b) by the activation of different intracellular signaling pathways, such as of AMP–activated protein kinase, mitogen activated protein kinase or phosphoinositide 3-kinase pathways (acute and chronic activation), and c) finally by conditions that alter lipid metabolism, namely diabetes mellitus or high-fat diet (for review: Koonen et al. 2005; Bonen et al. 2002, 2004a). In addition, it appears that posttranscriptional mechanisms may be involved in the regulation of fatty-acid transport proteins, without concomitant changes in mRNA or total protein expression.

Biosorption of long-chain fatty acids in UASB treatment process

Biosorption of long-chain fatty acids (LCFA) in the upflow anaerobic sludge blanket (UASB) treatment process was investigated using batch tests and continuous reactor runs. Batch experiments were conducted, for characterization of the biosorption, with two active and one inactivated (autoclaved) sludge granules as sorbents and with a single (oleic acid) or a mixture of LCFA (LCFA m; 50% oleic, 35% palmitic and 15% stearic acid) as sorbates. The LCFA m showed a faster adsorption onto the granules than oleic acid. With the active sludge granules, adsorption was followed by desorption. Methane production increased significantly, either simultaneously (at lower LCFA concentrations) or succeedingly (at higher concentrations) with desorption. The desorption was mediated by biological activity, since it did not prevail with inactivated granules or with active granules inhibited at higher LCFA m concentrations. The inactivated granules had a slightly higher initial biosorption capacity. Increased LCFA concentrations resulted in more LCFA adsorbed and greater inhibition of their biodegradation. A hypothesis is proposed to explicate the relationship between biosorption, desorption and biodegradation of LCFA by sludge granules: adsorption is a prerequisite for biodegradation while desorption is a consequence of biodegradation. Isothermal studies with oleate showed that the apparent biosorption could be described by the physical multilayer adsorption theory and the sorption isotherm derived was consistent with the Freundlich model. The quantitative relation between LCFA biosorption and granular sludge flotation was investigated in a UASB reactor fed with LCFA m. Sludge flotation depended on the LCFA m loading rate rather than on their concentration. The higher the loading implemented, the more flotation occurred and the shorter time required for complete flotation of the sludge bed. Flotation started when the LCFA m loading rate exceeded 0.09 g COD/g VSS·d, while complete flotation occurred at the loading rates exceeding 0.2 g COD/g VSS·d. These results suggest that sludge bed wash-out is likely to be encountered before inhibition of methanogenesis during the treatment of LCFA-containing wastewaters by the UASB process.