Emergence of omega-3 fatty acids in biomedical research.

Shortly after the discovery that linoleic acid was an essential fatty acid in 1930, α-linolenic acid also was reported to prevent the fatty acid deficiency syndrome in animals. However, several prominent laboratories could not confirm the findings with α-linolenic acid, and as a result there was a loss of interest in omega-3 fatty acids in lipid research. Even the findings that a prostaglandin can be synthesized from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is necessary for optimum retinal function generated only limited interest in omega-3 fatty acids. The breakthrough came in the 1970s when Dyerberg and Bang reported that the low incidence of atherosclerotic coronary disease in Greenland Eskimos was due to the high marine lipid content of their diet. They subsequently found that EPA, which was increased in Eskimo plasma, inhibited platelet aggregation, and they concluded that the low incidence of coronary artery disease was due to the anti-thrombotic effect of EPA. This stimulated widespread interest and research in EPA and DHA, leading to the present view that, like their omega-6 counterparts, omega-3 fatty acids have important physiological functions and are essential fatty acids.

Proteasomal modulation of cellular SNAT2 (SLC38A2) abundance and function by unsaturated fatty acid availability.

Expression and activity of the System A/SNAT2 (SLC38A2) amino acid transporter is up-regulated by amino acid starvation and hypertonicity by a mechanism dependent on both ATF4-mediated transcription of the SLC38A2 gene and enhanced stabilization of SNAT2 itself, which forms part of an integrated cellular stress response to nutrient deprivation and osmotic stress. Here we demonstrate that this adaptive increase in System A function is restrained in cells subjected to prior incubation with linoleic acid (LOA, an unsaturated C18:2 fatty acid) for 24 h. While fatty acid treatment had no detectable effect upon stress-induced SNAT2 or ATF4 gene transcription, the associated increase in SNAT2 protein/membrane transport activity were strongly suppressed in L6 myotubes or HeLa cells preincubated with LOA. Cellular ubiquitination of many proteins was increased by LOA and although the fatty acid-induced loss of SNAT2 could be attenuated by proteasomal inhibition, the functional increase in System A transport activity associated with amino acid starvation/hypertonicity that depends upon processing/maturation and delivery of SNAT2 to the cell surface could not be rescued. LOA up-regulated cellular expression of Nedd4.2, an E3-ligase implicated in SNAT2 ubiquitination, but shRNA-directed Nedd4.2 gene silencing could not curb fatty acid-induced loss of SNAT2 adaptation. However, expression of SNAT2 in which seven putative lysyl-ubiquitination sites in the cytoplasmic N-terminal domain were mutated to alanine protected SNAT2 against LOA-induced proteasomal degradation. Collectively, our findings indicate that increased availability of unsaturated fatty acids can compromise the stress-induced induction/adaptation in SNAT2 expression and function by promoting its degradation via the ubiquitin-proteasome system.

Key roles for GRB2-associated-binding protein 1, phosphatidylinositol-3-kinase, cyclooxygenase 2, prostaglandin E2 and transforming growth factor alpha in linoleic acid-induced upregulation of lung and breast cancer cell growth.

The distribution of omega-6 and omega-3 polyunsaturated fatty acid (PUFA) intake in Western diets is disproportionate, containing an overabundance of the omega-6 PUFA, linoleic acid (LA; C18:2). Increased enrichment with LA has been shown to contribute to the enhancement of tumorigenesis in several cancer models. Previous work has indicated that phosphatidylinositol 3-kinase (PI3K) may play a key role in LA-induced tumorigenesis. However, the modes by which LA affects carcinogenesis have not been fully elucidated. In this study, a mechanism for LA-induced upregulation of cancer cell growth is defined. LA treatment enhanced cellular proliferation in BT-474 human breast ductal carcinoma and A549 human lung adenocarcinoma cell lines. Enrichment of LA increased cyclooxygenase (COX) activity and led to increases in prostaglandin E2 (PGE2), followed by increases in matrix metalloproteinase (MMP) and transforming growth factor alpha (TGF-α) levels, which are all key elements involved in the enhancement of cancer cell growth. Further investigation revealed that LA supplementation in both BT-474 breast and A549 lung cancer cell lines greatly increased the association between the scaffolding protein GRB2-associated-binding protein 1 (Gab1) and epidermal growth factor receptor (EGFR), although Gab1 protein levels were significantly decreased. These LA-induced changes were associated with increases in activated Akt (pAkt), a downstream signaling component in the PI3K pathway. Treatment with inhibitors of EGFR, PI3K and Gab1-specific siRNAs reversed the upregulation of pAkt, as well as the observed increases in cell proliferation by LA in both cell lines. A549 xenograft assessment in athymic nude mice fed high levels of LA exhibited similar increases in EGFR-Gab1 association and increased levels of pAkt, while mice fed with high levels of the omega-3 PUFA, docosahexaenoic acid (DHA; C22:6), demonstrated an opposite response. The involvement of Gab1 in LA-induced tumorigenesis was further defined utilizing murine cell lines that express high levels of Gab1. Significant increases in cell proliferation were observed with the addition of increasing concentrations of LA. However, no changes in cell proliferation were detected in the murine paired cell lines expressing little or no Gab1 protein, establishing Gab1 as major target in LA-induced enhancement of tumorigenesis.

Linoleic acid: between doubts and certainties.

Linoleic acid is the most abundant polyunsaturated fatty acid in human nutrition and represents about 14 g per day in the US diet. Following the discovery of its essential functions in animals and humans in the early 1920's, studies are currently questioning the real requirement of linoleic acid. It seems now overestimated and creates controversy: how much linoleic acid should be consumed in a healthy diet? Beyond the necessity to redefine the dietary requirement of linoleic acid, many questions concerning the consequences of its excessive consumption on human health arise. Linoleic acid is a direct precursor of the bioactive oxidized linoleic acid metabolites. It is also a precursor of arachidonic acid, which produces pro-inflammatory eicosanoids and endocannabinoids. A majority of the studies on linoleic acid and its derivatives show a direct/indirect link with inflammation and metabolic diseases. Many authors claim that a high linoleic acid intake may promote inflammation in humans. This review tries to (i) highlight the importance of reconsidering the actual requirement of linoleic acid (ii) point out the lack of knowledge between dietary levels of linoleic acid and the molecular mechanisms explaining its physiological roles (iii) demonstrate the relevance of carrying out further human studies on the single variable linoleic acid.

Wing (Ib) cells in frog taste discs detect dietary unsaturated fatty acids.

The effects of unsaturated fatty acids on membrane properties were studied using conventional whole-cell patch-clamp recording of isolated wing (Ib) cells in bullfrog (Lithobates catesbeianus) taste discs. Applying arachidonic acid to the bath induced monophasic inward currents in 60% of wing cells and biphasic inward and outward currents in the other cells. The intracellular dialysis of arachidonic acid did not induce an inward current; however, it enhanced a slowly developing Ba(2+)-sensitive outward current. The effects of various unsaturated fatty acids were explored under the condition of Cs(+) internal solution. Linoleic and α-linolenic acids induced large inward currents. Oleic, eicosapentaenoic and docosahexaenoic acids elicited the same inward currents as those of arachidonic acid. Wing cells, under the basal condition with Cs(+) internal solution, displayed a small inward current of -1.1±0.1pA/pF at -50mV (n=40), in which the peak existed at a membrane potential of -49mV. Removing external Ca(2+) further increased the inward current by -2.9±0.3pA/pF at -50mV (n=4) from the basal current and the peak was located at -55mV. External linoleic acid (50µM) also induced a similar inward current of -5.6±0.6pA/pF at -50mV (n=19) from the basal current and the peak was located at -61mV. External Ca(2+)-free saline and linoleic acid induced similar current/voltage (I/V) relationships elicited by a ramp voltage as well as voltage steps. Linoleic acid-induced currents were not influenced by replacing internal EGTA with BAPTA, whereas inward currents disappeared under the elimination of external Na(+) and addition of flufenamic acid. These results suggest that dietary unsaturated fatty acids may depolarize wing (Ib) cells, which affects the excitability of these cells.

Role of arachidonic acid metabolism in Stat5 activation induced by oleic acid in MDA-MB-231 breast cancer cells.

Epidemiological studies and animal models suggest an association between high levels of dietary fat intake and an increased risk of breast cancer. In breast cancer cells, the free fatty acid oleic acid (OLA) induces proliferation, migration, invasion and an increase of MMP-9 secretion. However, the role of OLA on Stat5 activation and the participation of COX-2 and LOXs activity in Stat5 activation induced by OLA remain to be investigated. We demonstrate here that stimulation of MDA-MB-231 breast cancer cells with 100 µM OLA induces Stat5 phosphorylation at Tyr-694 and an increase of Stat5-DNA complex formation. The Stat5 DNA-binding activity requires COX-2, LOXs, metalloproteinases and Src activities. In addition, OLA induces cell migration through a Stat5-dependent pathway. In summary, our findings establish that OLA induces cell migration through a Stat5-dependent pathway and that Stat5 activation requires AA metabolites in MDA-MB-231 breast cancer cells.

A critical tyrosine residue determines the uncoupling protein-like activity of the yeast mitochondrial oxaloacetate carrier.

The mitochondrial Oac (oxaloacetate carrier) found in some fungi and plants catalyses the uptake of oxaloacetate, malonate and sulfate. Despite their sequence similarity, transport specificity varies considerably between Oacs. Indeed, whereas ScOac (Saccharomyces cerevisiae Oac) is a specific anion-proton symporter, the YlOac (Yarrowia lipolytica Oac) has the added ability to transport protons, behaving as a UCP (uncoupling protein). Significantly, we identified two amino acid changes at the matrix gate of YlOac and ScOac, tyrosine to phenylalanine and methionine to leucine. We studied the role of these amino acids by expressing both wild-type and specifically mutated Oacs in an Oac-null S. cerevisiae strain. No phenotype could be associated with the methionine to leucine substitution, whereas UCP-like activity was dependent on the presence of the tyrosine residue normally expressed in the YlOac, i.e. Tyr-ScOac mediated proton transport, whereas Phe-YlOac lost its protonophoric activity. These findings indicate that the UCP-like activity of YlOac is determined by the tyrosine residue at position 146.

TRPM5 is critical for linoleic acid-induced CCK secretion from the enteroendocrine cell line, STC-1.

Fatty acid-induced stimulation of enteroendocrine cells leads to release of the hormones such as cholecystokinin (CCK) that contribute to satiety. Recently, the fatty acid activated G protein-coupled receptor GPR120 has been shown to mediate long-chain unsaturated free fatty acid-induced CCK release from the enteroendocrine cell line, STC-1, yet the downstream signaling pathway remains unclear. Here we show that linoleic acid (LA) elicits membrane depolarization and an intracellular calcium rise in STC-1 cells and that these responses are significantly reduced when activity of G proteins or phospholipase C is blocked. LA leads to activation of monovalent cation-specific transient receptor potential channel type M5 (TRPM5) in STC-1 cells. LA-induced TRPM5 currents are significantly reduced when expression of TRPM5 or GPR120 is reduced using RNA interference. Furthermore, the LA-induced rise in intracellular calcium and CCK secretion is greatly diminished when expression of TRPM5 channels is reduced using RNA interference, consistent with a role of TRPM5 in LA-induced CCK secretion in STC-1 cells.

Acyl-CoA synthetase 1 is required for oleate and linoleate mediated inhibition of cholesterol efflux through ATP-binding cassette transporter A1 in macrophages.

Diabetes and insulin resistance increase the risk of cardiovascular disease caused by atherosclerosis through mechanisms that are poorly understood. Lipid-loaded macrophages are key contributors to all stages of atherosclerosis. We have recently shown that diabetes associated with increased plasma lipids reduces cholesterol efflux and levels of the reverse cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) in mouse macrophages, which likely contributes to macrophage lipid accumulation in diabetes. Furthermore, we and others have shown that unsaturated fatty acids reduce ABCA1-mediated cholesterol efflux, and that this effect is mediated by the acyl-CoA derivatives of the fatty acids. We therefore investigated whether acyl-CoA synthetase 1 (ACSL1), a key enzyme mediating acyl-CoA synthesis in macrophages, could directly influence ABCA1 levels and cholesterol efflux in these cells. Mouse macrophages deficient in ACSL1 exhibited reduced sensitivity to oleate- and linoleate-mediated ABCA1 degradation, which resulted in increased ABCA1 levels and increased apolipoprotein A-I-dependent cholesterol efflux in the presence of these fatty acids, as compared with wildtype mouse macrophages. Conversely, overexpression of ACSL1 resulted in reduced ABCA1 levels and reduced cholesterol efflux in the presence of unsaturated fatty acids. Thus, the reduced ABCA1 and cholesterol efflux in macrophages subjected to conditions of diabetes and elevated fatty load may, at least in part, be mediated by ACSL1. These observations raise the possibility that ABCA1 levels could be increased by inhibition of acyl-CoA synthetase activity in vivo. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Healing fats of the skin: the structural and immunologic roles of the omega-6 and omega-3 fatty acids.

Linoleic acid (18:2omega6) and alpha-linolenic acid (18:3omega3) represent the parent fats of the two main classes of polyunsaturated fatty acids: the omega-6 (n-6) and the omega-3 (n-3) fatty acids, respectively. Linoleic acid and alpha-linolenic acid both give rise to other long-chain fatty acid derivatives, including gamma-linolenic acid and arachidonic acid (omega-6 fatty acids) and docosahexaenoic acid and eicosapentaenoic acid (omega-3 fatty acids). These fatty acids are showing promise as safe adjunctive treatments for many skin disorders, including atopic dermatitis, psoriasis, acne vulgaris, systemic lupus erythematosus, nonmelanoma skin cancer, and melanoma. Their roles are diverse and include maintenance of the stratum corneum permeability barrier, maturation and differentiation of the stratum corneum, formation and secretion of lamellar bodies, inhibition of proinflammatory eicosanoids, elevation of the sunburn threshold, inhibition of proinflammatory cytokines (tumor necrosis factor-alpha, interferon-gamma, and interleukin-12), inhibition of lipoxygenase, promotion of wound healing, and promotion of apoptosis in malignant cells, including melanoma. They fulfill these functions independently and through the modulation of peroxisome proliferator-activated receptors and Toll-like receptors.

Age dependence of plasma phospholipid fatty acid levels: potential role of linoleic acid in the age-associated increase in docosahexaenoic acid and eicosapentaenoic acid concentrations.

Limited information is available with respect to the association between age and the plasma phospholipid fatty acid profile. Therefore we investigated the association between plasma phospholipid fatty acid status and age after correction for sex, smoking, alcohol use, BMI and fish intake. Plasma phospholipid fatty acid composition was measured and information on fish intake and other potential covariates was collected in 234 participants of the Maastricht Aging Study. The participants were healthy individuals of both sexes with an age range between 36 and 88 years. Hierarchical linear regression analyses were applied to study the relationship between age and fatty acid concentrations. After correction for fish consumption and other relevant covariates, a significant positive relationship was observed between age of the subjects and their plasma phospholipid concentrations of DHA (22 : 6n-3, P = 0.006) and EPA (20 : 5n-3; P = 0.001). Age contributed 2.3 and 3.9 % to the amount of explained variance, respectively. The higher n-3 long-chain PUFA status at advanced age was confirmed by lower concentrations of their putative 'shortage marker' Osbond acid (ObA, 22 : 5n-6; P = 0.022 for the relationship with age after correction for covariates and fish intake, R2 0.022). Concentrations of linoleic acid (LA; 18 : 2n-6) were negatively associated with age (P < 0.001; R2 0.061). In conclusion, DHA and EPA concentrations appeared to be higher in older age groups, partly because of a higher fish intake and partly because of another age-associated mechanism, possibly involving the well-known competition with LA.

Melatonin, sleep disturbance and cancer risk.

The pineal hormone melatonin is involved in the circadian regulation and facilitation of sleep, the inhibition of cancer development and growth, and the enhancement of immune function. Individuals, such as night shift workers, who are exposed to light at night on a regular basis experience biological rhythm (i.e., circadian) disruption including circadian phase shifts, nocturnal melatonin suppression, and sleep disturbances. Additionally, these individuals are not only immune suppressed, but they are also at an increased risk of developing a number of different types of cancer. There is a reciprocal interaction and regulation between sleep and the immune system quite independent of melatonin. Sleep disturbances can lead to immune suppression and a shift to the predominance in cancer-stimulatory cytokines. Some studies suggest that a shortened duration of nocturnal sleep is associated with a higher risk of breast cancer development. The relative individual contributions of sleep disturbance, circadian disruption due to light at night exposure, and related impairments of melatonin production and immune function to the initiation and promotion of cancer in high-risk individuals such as night shift workers are unknown. The mutual reinforcement of interacting circadian rhythms of melatonin production, the sleep/wake cycle and immune function may indicate a new role for undisturbed, high quality sleep, and perhaps even more importantly, uninterrupted darkness, as a previously unappreciated endogenous mechanism of cancer prevention.

Multiple routes of chemosensitivity to free fatty acids in humans.

Selected free fatty acids (FFAs) are documented effective somatosensory and olfactory stimuli whereas gustatory effects are less well established. This study examined orthonasal olfactory, retronasal olfactory, nasal irritancy, oral irritancy, gustatory, and multimodal threshold sensitivity to linoleic, oleic, and stearic acids. Sensitivity to oxidized linoleic acid was also determined. Detection thresholds were obtained using a three-alternative, forced-choice, ascending concentration presentation procedure. Participants included 22 healthy, physically fit adults sensitive to 6-n-propylthiouracil. Measurable thresholds were obtained for all FFAs tested and in 96% of the trials. Ceiling effects were observed in the remaining trials. Greater sensitivity was observed for multimodal stimulation and lower sensitivity for retronasal stimulation. There were no statistically significant correlations for linoleic acid thresholds between different modalities, suggesting that each route of stimulation contributes independently to fat perception. In summary, 18-carbon FFAs of varying saturation are detected by multiple sensory systems in humans.

Response to conjugated linoleic acid in dairy cows differing in energy and protein status.

The trans-10, cis-12 conjugated linoleic acid (CLA) isomer inhibits milk fat synthesis, whereas milk yield and synthesis of other milk components generally remain unchanged in established lactation. However, in some CLA studies increases in milk yield, milk protein yield, or both have been observed in cows limited in energy, either in early lactation or when grazing pasture. Our objective was to evaluate the performance and monitor peripheral tissue responses to homeostatic signals regulating lipolysis and glucose uptake with CLA supplementation when cows were limited in metabolizable energy in combination with moderate or excess metabolizable protein supply. Holstein cows (n = 48; 112 +/- 5 d in milk; mean +/- SE) were provided ad libitum access to a diet that met energy and protein requirements for a 16-d standardization interval. Based on performance during this interval, the Cornell Net Carbohydrate and Protein System was used to design energy-limiting rations that provided 80% of metabolizable energy requirements, and these were fed throughout the treatment periods. Cows were randomly allocated to 4 treatments, in a 2-period crossover design. Treatments were 1) moderate metabolizable protein (MP) supply, 2) moderate MP supply + CLA, 3) excess MP supply, and 4) excess MP supply + CLA. Moderate and excess MP supply were at 88 and 117%, respectively, of the MP requirement established during the standardization period, as estimated by the Cornell Net Carbohydrate and Protein System. Each experimental period comprised 16 d, with crossover of CLA within each protein level. The lipid-encapsulated CLA supplement provided 12 g/d of trans-10, cis-12 CLA. Conjugated linoleic acid treatment reduced milk fat yield by 21% but increased milk yield and milk protein yield by 2.6 and 2.8%, respectively. Milk yield and content and yield of both milk protein and fat were unaltered by either protein treatment alone or in combination with CLA. Basal concentrations of glucose, insulin, and nonesterified fatty acids were unaffected by CLA supplementation. The fractional rate of glucose clearance in response to an insulin challenge and the nonesterified fatty acid response to an epinephrine challenge were also not altered by either CLA treatment or MP supply. Overall, the results demonstrate that CLA supplementation when cows are energy-limited allows for repartitioning of nutrients, resulting in increased yields of milk and milk protein, and this can occur without changes in whole-body glucose homeostasis and adipose tissue response to lipolytic stimuli.

Ligand- and species-dependent activation of PPARalpha.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is mainly expressed in liver and involved in lipid metabolism. Oxidation of certain fatty acids in peroxisomes is under PPARalpha control. A wide variety of lipid molecules activate PPARalpha as well as the fibric acid derivative clofibrate. In the present study, we evaluated the differential activation of PPARalpha with several agonist ligands through its expression and DNA binding in both rat (McA-RH7777) and human (HepG2) hepatoma cell lines. In McA-RH7777 cells, clofibrate alone mediated a higher induction of PPARalpha expression than linoleic acid. In contrast, linoleic acid was the most effective ligand in HepG2 cells and treatment with clofibrate plus linoleic acid did not further increase PPARalpha expression. PPRE-binding activity of PPARalpha in ligand-treated cells was also increased in a parallel manner. We suggest that ligand-induced PPARalpha activation might give rise to differential species-dependent responses.

The role of omega-3 fatty acids in the secondary prevention of cardiovascular disease.

It has long been recognized from epidemiological studies that Greenland Eskimos have substantially reduced rates of acute myocardial infarction (MI) compared with Western controls. From these epidemiological observations, the benefits of fatty fish consumption have been explored in cell culture and animal studies, as well as randomized controlled trials investigating the cardioprotective effects of omega-3 fatty acids. Dietary omega-3 fatty acids seem to stabilize the myocardium electrically, resulting in reduced susceptibility to ventricular arrhythmias, thereby reducing the risk of sudden death. These fatty acids also have potent anti-inflammatory effects, and may also be antithrombotic and anti-atherogenic. Furthermore, the recent GISSI-Prevention study of 11 324 patients showed a marked decrease in risk of sudden cardiac death as well as a reduction in all-cause mortality in the group taking a highly purified form of omega-3 fatty acids, despite the use of other secondary prevention drugs, including beta-blockers and lipid-lowering therapy. The use of omega-3 fatty acids should be considered as part of a comprehensive secondary prevention strategy post-myocardial infarction.

Association between E-cadherin expression by human colon, bladder and breast cancer cells and the 13-HODE:15-HETE ratio. A possible role of their metastatic potential.

The relationship between 15(S)-HETE and 13(S)-HODE from different human tumor cells exposed to n-6 and n-3 essential fatty acids (EFAs) and E-cadherin expression was studied. Colon cancer cells (HRT-18) exposed to gamma linoleic acid (18:3n-6, GLA) and eicosapentaenoic (20:5n-3, EPA) (50microM) showed an increased expression of E-cadherin. Breast cancer (MCF-7) exposed to EPA showed an increment whereas GLA had no effect on E-cadherin expression. No expression of E-cadherin was observed for urothelial cancer (T-24) after GLA or EPA treatment. Significant levels of 15(S)-HETE and 13(S)-HODE were detected after GLA or EPA treatment for all tumor lines. E-cadherin expression was inversely proportional to the 13(S)-HODE:15(S)-HETE ratio when cells were pretreated with GLA or EPA. Nevertheless, the liberation of these metabolites seems to be independent of the E-cadherin expression. The increase in the13(S)-HODE:15(S)-HETE correlates to a decrease in the expression of E-cadherin. Both factors may play a role in metastasis development.

Colonic anti-inflammatory mechanisms of conjugated linoleic acid.

Conjugated linoleic acid (CLA) is a mixture of positional (e.g. 7,9; 9,11; 10,12; 11,13) and geometric (cis or trans) isomers of octadecadienoic acid. This compound was first shown to prevent mammary carcinogenesis in murine models. Later investigations uncovered a number of additional health benefits, including decreasing atherosclerosis and inflammation while enhancing immune function. The mechanisms of action underlying these biological properties are not clearly understood. The aim of this review is to highlight recent advances in CLA research related to experimental inflammatory bowel disease. In addition, two possible mechanisms of action (i.e. endoplasmic and nuclear) were discussed in detail in the context of enteric inflammatory disorders. Conjugated linoleic acid was first implicated in down-regulating the generation of inducible eicosanoids (i.e. PGE(2) and LTB(4)) involved in early micro-inflammatory events (endoplasmic). More recently, CLA has been shown to modulate the expression of genes regulated by peroxisome proliferator-activated receptors (PPARs; nuclear). In pigs, prolonged dietary CLA treatment stimulated the expression of PPAR-gamma in the muscle. Thus, evidence supporting both mechanistic theories of CLA acting through eicosanoid synthesis and PPAR activity is available. The further understanding of the anti-inflammatory mechanisms of action of CLA may yield novel nutritional therapies for enteric inflammation.