Specific effects of 9- and 13- hydroxyoctadecadienoic acids (9- and 13- hodes) on human monocyte activation and macrophage differentiation

In diabetes, activation of monocytes, macrophage differentiation in arterial walls, and foam cell formation are increased contributing to macrovascular complications. Mechanisms regulating these processes are incompletely understood. HODEs are oxidised linoleic acid (LA) derivatives produced non-enzymatically from LDL (oxLDL) or by enzyme 15- lipoxygenase. 9-HODE, (n-6) is pro-inflammatory and may act through the GPR132 receptor, while 13-HODE, (n-7), may have protective effects similar to palmitoleate (also n-7). We studied effects of long chain fatty acids (LCFA) HODEs, LA and α-linolenic acid (C18, n-3, ALA) on differentiation and gene expression in THP-1 cells. Monocytes were fully differentiated into macrophages with 100nM phorbol myristate acetate (PMA), while 1 nM PMA synergized with 30 µM LCFAs increased expression of activation marker CD11b, macrophage morphology and lipid accumulation (oil red-O). Monocyte cell number was decreased by HODEs (p < 0.001) but not LA or ALA. Decreased cell viability was confirmed, and shown to be due to apoptosis (caspase 3/7 activation) rather than cytotoxicity. Monocyte activation (PMA 100 nM or PMA 1nM + 30µM HODEs) markedly increased expression of lipogenic genes FABP4 and PPARγ. Genes involved in reverse cholesterol transport (ABCA1 and SCRB) were activated by HODEs. These effects were not seen to be induced by ALA and LA. Stearoyl CoA desaturase, increased in insulin resistance, was decreased in monocytes by 13-HODE, but increased during activation and in macrophages by 9-HODE. 9-HODE specifically increased foam cells (lipid droplets) in differentiated macrophage cultures. Possible mediators of fatty acid effects include long-chain fatty acid receptors (GPR120 and GPR132) and PPARγ. GPR120 was predominantly expressed in monocytes and GPR132 in macrophages (PCR and immuno-histochemistry) with 9-HODE increasing GPR132 expression. A decrease in expression of GPR120 and an increase in GPR132 was observed when treated with HODEs synergized with 1nM PMA. 9- and 13-HODE have specific effects on monocyte activation, macrophage differentiation, lipid transport and signaling genes compared to LA and ALA. Ongoing work with receptor activators/inhibitors and gene silencing will clarify which signaling pathways are involved in the actions of long-chain fatty acid mediators

Regulation of stearoyl-coa desaturase by hydroxyoctadecadienoic acids

Introduction: Beneficial effects of polyunsaturated fatty acids (PUFAs) include improved lipid profile and insulin sensitivity, leading to decreased cardiovascular risk. PUFAs regulate cell growth, differentiation and alter gene expression including stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in formation of monounsaturated fatty acids (MUFA). Increased SCD levels have been correlated with decreased fatty acid oxidation, increased triglyceride synthesis and thus obesity and insulin resistance. The mechanisms by which PUFAs regulate SCD are not understood. The PUFAs 9 and 13 hydroxyoctadecadienoic acids (HODEs), components of oxLDL, are oxidized derivatives of linoleic acid. Oxidized lipids are known to modulate lipid metabolism in adipocytes and macrophages. Macrophages are involved in pathogenesis of atherosclerosis, obesity and type 2 diabetes. We investigated effects of HODEs, linoleic acid (LA, n-6) and linolenic acid (ALA, n-3) on monocytes and macrophages – in particular, their effect on modulation of SCD. \ud \ud Methods: THP-1cells, a human monocyte-macrophage cell line was used. Cells were cultured in RPMI medium with 10% FBS, L-glutamine and antibiotics. To obtain macrophages, cells were treated with phorbol myristate acetate (PMA) for 36 hrs and rested for 24hrs in low serum medium before PUFA treatment. For experiments 1 million cells were treated with 30uM of LA, ALA, 9- and13- HODEs. For differentiation experiments, THP-1 cells with or without 1nM PMA ± PUFA were incubated for 24 hrs. Cells were harvested after 24 - 48 hrs and RNA extracted. Expression of SCD was determined by real-time RT PCR. \ud \ud Results: In monocytes, expression of SCD was not altered significantly by ALA, LA, or 9-HODE but 13-HODE specifically decreased SCD to 40 – 50% of control values. By contrast, in macrophages 13-HODE was without effect (as were ALA and LA), but SCD expression was increased specifically by 9-HODE. The latter also specifically up-regulated expression of its putative receptor G protein coupled receptor 132 (GPR 132) in macrophages. Exposure of monocytes to PMA led to macrophage differentiation in a dose-dependent manner – confirmed by morphology, lipid accumulation and macrophage markers CD 11b and scavenger receptors as well as increased SCD (greater than 2-fold). Cells were fully differentiated with 100 nM PMA and this was not affected by any of the four PUFAs. With 1 nM PMA cells were partially differentiated and SCD expression was inhibited by ALA, LA or 13-HODE but not by 9-HODE. \ud \ud Significance: Increased SCD expression during differentiation of monocytes into macrophages may contribute to insulin resistance and increased cardiovascular risk. HODEs appear to modulate expression of this key enzyme. HODEs are produced by non-enzymatic oxidation of LA in the vessel wall, or in macrophages by the action of the enzyme 15-lipoxygenase. The structural differences between the two HODEs and with the parent LA are relatively subtle, but their effects distinct. Evidence is emerging that 13-HODE (an n-7 fatty acid) has protective and antiinflammatory actions while 9-HODE (an n-6 fatty acid) is pro-atherogenic and pro-inflammatory. In this study, 13-HODE decreased SCD in monocytes and during macrophage differentiation. This latter effect was not found with 9-HODE which increased macrophage expression of SCD and GPR 132. The mechanism of these distinctive effects of HODEs on mediators of insulin resistance and atherogenesis require further study

Specific effects of 9- and 13- hydroxyoctadecadienoic acids (9- and 13- hodes) on human monocyte activation and macrophage\ud differentiation

In diabetes, activation of monocytes, macrophage differentiation in arterial walls, and foam cell formation are increased contributing to macrovascular complications. Mechanisms regulating these processes are incompletely understood. HODEs are oxidised linoleic acid (LA) derivatives produced non-enzymatically from LDL (oxLDL) or by enzyme 15- lipoxygenase. 9-HODE, (n-6) is pro-inflammatory and may act through the GPR132 receptor, while 13-HODE, (n-7), may have protective effects similar to palmitoleate (also n-7). We studied effects of long chain fatty acids (LCFA) HODEs, LA and α-linolenic acid (C18, n-3, ALA) on differentiation and gene expression in THP-1 cells.\ud \ud Monocytes were fully differentiated into macrophages with 100nM phorbol myristate acetate (PMA), while 1 nM PMA synergized with 30 µM LCFAs increased expression of activation marker CD11b, macrophage morphology and lipid accumulation (oil red-O). Monocyte cell number was decreased by HODEs (p < 0.001) but not LA or ALA. Decreased cell viability was confirmed, and shown to be due to apoptosis (caspase 3/7 activation) rather than cytotoxicity. Monocyte activation (PMA 100 nM or PMA 1nM + 30µM HODEs) markedly increased expression of lipogenic genes FABP4 and PPARγ. Genes involved in reverse cholesterol transport (ABCA1 and SCRB) were activated by HODEs. These effects were not seen to be induced by ALA and LA. Stearoyl CoA desaturase, increased in insulin resistance, was decreased in monocytes by 13-HODE, but increased during activation and in macrophages by 9-HODE. 9-HODE specifically increased foam cells (lipid droplets) in differentiated macrophage cultures. Possible mediators of fatty acid effects include long-chain fatty acid receptors (GPR120 and GPR132) and PPARγ. GPR120 was predominantly expressed in monocytes and GPR132 in macrophages (PCR and immuno-histochemistry) with 9-HODE increasing GPR132 expression. A decrease in expression of GPR120 and an increase in GPR132 was observed when treated with HODEs synergized with 1nM PMA.\ud \ud 9- and 13-HODE have specific effects on monocyte activation, macrophage differentiation, lipid transport and signaling genes compared to LA and ALA. Ongoing work with receptor activators/inhibitors and gene silencing will clarify which signaling pathways are involved in the actions of long-chain fatty acid mediators

Hydroxyoctadecadienoic acids (HODEs) regulate fatty acid binding protein-4 (FABP4) secretion in human monocytes and macrophages

9-HODE is an oxidation product of linoleic acid (LA, C18:2), is pro-inflammatory, and is a ligand for GPR132 which is involved in atherogenesis. By contrast, 13-HODE is protective and acts through PPARgamma. Plasma FABP4 is increased in diabetes and coronary artery disease. 9-HODE increases FABP4 expression in macrophages. We investigated whether GPR132 is a monocyte activation marker in diabetes, and if it mediates the effect of 9-HODE on FABP4. Monocyte populations from 31 type 2 diabetic patients and controls were studied using FACS. Plasma cytokines were measured using bead arrays and ELISA. THP-1 cells were used to investigate regulation of FABP4 secretion. Diabetic subjects had increased circulating CD14+, CD14+CD36+, CD14+CD11b+, CD14+CD54+ cells (p<0.01), and also increased GPR132 mRNA expression in CD14+ monocytes (p < 0.01). Levels of GPR132 expression did not correlate with any of the above cell populations, or with increased plasma levels of FABP4, sTNF-R, osteoprotegerin, MCP-1, resistin or leptin. FABP4 mRNA expression was markedly increased in both THP-1 monocytes and macrophages (differentiated with 100nM PMA) by 9-HODE and 13-HODE (all p < 0.001). 9-HODE (p < 0.01) and 13-HODE (p < 0.05) also increased GPR132 expression. The stimulatory effect of HODEs was replicated by the PPARgamma agonist rosiglitazone (p<0.001). The PPARgamma antagonist T0070907 decreased the effect of all three ligands (p<0.001). Similar effects on FABP4 protein secretion were documented (ELISA). LA and alpha-linolenic acid (C18:3) were without effect on FABP4 mRNA or protein. GPR132 gene silencing using siRNA had no effect on increased FABP4 expression in response to 9-HODE, 13-HODE, or rosiglitazone. In conclusion, GPR132 is an independent activation marker for monocytes, but does not mediate the increase in FABP4 expression induced by 9-HODE. FABP4 secretion is regulated through PPARgamma. Study of the signaling functions of fatty acids may lead to new treatments for diabetes and atherosclerosis

Correction and response to: Grommets in HBOT patients: GA vs. LA, unanswered questions

[Extract] We thank Gibbs and Commons for their interest in our paper. There is a key difference between the datasets for Commons et al and our study. Our data set, has grouped five years of data according to the calendar year. This is different from Commons et al's study population recruited between 01 June 2009 and 31 May 2010. We feel this may explain the difference of one case between the two papers in 2010. Our data collection used the standard clinic and operating theatre databases, and we were advised that there was no searchable clinical code for grommet procedures undertaken with local anaesthetic (LA) in the outpatient clinic. The alternative, to review many hundreds of patients, was considered beyond the study's scope. Instead, the TTH Hyperbaric Medicine Unit (HMU) database was used to recruit cases and cross checked with operating theatre data. We have since re-investigated the operating theatre database to identify any additional bilateral grommet procedures during 2008 to 2012 and cross checked these with the HMU database. This has identified one to four additional patients per year in the general anaesthesia (GA) group and one additional LA patient that meet the criteria for recruitment into the study. There was one further unconfirmed patient from each of 2008 and 2010, whose charts were unavailable for this response, and have not been included in this amendment. The corrected Figure 1 reflects these changes. Despite the additional cases, the frequency spike during 2010 remains. A published audit of the number of middle ear barotrauma (MEBT) cases between 2007-2010 also reports an increased incidence of MEBT in 2009-2010 compared with previous years at our unit. Possible reasons for this may be the introduction of new technology at the unit, in the form of the digital Macro View™ otoscope during this period, leading to a possible change in clinical practice and an increased detection of MEBT, or a lower threshold for ENT referral for grommet placement

Hydroxyoctadecadienoic acids (HODEs) increase apoptosis in human THP1 monocytes and macrophages

Certain fatty acids function as signaling molecules. HODEs are stable oxidation products of linoleic acid (LA; C18:2), are abundant in atherosclerotic plaque, and known to signal through GPR132 (9-HODE only) or PPARgamma (9-HODE and 13-HODE). Macrophage apoptosis is an important process, contributing to atherosclerosis progression. Both GPR132 and PPARgamma were expressed in THP1 (RT-PCR and immunohistochemistry) with expression of both increased when cells were differentiated into macrophages (PMA). In 24-hour cultures, 9-HODE but not 13-HODE or LA decreased cell number (68%, p<0.001). We aimed to determine whether this was due to apoptosis and how it was mediated. Using a caspase 3/7 assay, 9-HODE and 13-HODE (30-100mM) but not LA increased caspase activity in monocytes and macrophages, with 9-HODE being more potent (p<0.001). This was accompanied by decreased cell viability (ATP generation assay, both p<0.001). FACS was used to quantify cells that were either viable or apoptotic (7AAD and annexin V positive). There was a time-dependent (over 24 hours) increase in apoptotic cells with 9-HODE and 13-HODE (both p<0.001), with 9-HODE being more potent (p<0.001). The effect of HODEs was replicated with campothecin (10mM) but not with the PPARgamma agonist rosiglitazone (1mM). The pro-apoptotic effects of HODEs were abolished by addition of the caspase inhibitor DEVA-CHO but not affected by the PPARgamma antagonist T0070907. In a gel-based assay, DNA fragmentation was apparent with campothecin and 9-HODE but not with LA or 13-HODE. GPR132 expression was silenced using siRNA oligonucleotides. There was no evidence of decreased effect of either 9-HODE or 13-HODE with GPR132 silencing. In conclusion, HODEs, and particularly 9-HODE, are potent regulators of macrophage apoptosis. They do not appear to be signaling through GPR132 or PPARgamma, both of which have regulatory roles in atherosclerosis. Further study of their mode of action may lead to identification of novel therapeutic targets for atherosclerosis

Nature’s Selection of Geranyl Group as a tRNA Modification: The Effects of Chain Length on Base-Pairing Specificity

The recently discovered geranyl modification on the 2-thio position of wobble U34 residues in tRNA^Glu, tRNA^Lys, and tRNA^Gln in several bacteria has been found to enhance the U:G pairing specificity and reduce the frameshifting error during translation. It is a fundamentally interesting question why nature chose a C10 terpene group in tRNA systems. In this study, we explore the significance of the terpene length on base-paring stability and specificity using a series of 2-thiouridine analogues containing different lengths of carbon chains, namely, methyl- (C1), dimethylallyl- (C5), and farnesyl-modified (C15) 2-thiothymidines in a DNA duplex. Our thermal denaturation studies indicate that the relatively long chain length of ≥ C10 is required to maintain the base-pairing discrimination of thymidine between G and A. The results from our molecular dynamics simulations show that in the T:G-pair-containing duplex, the geranyl and farnesyl groups fit into the minor groove and stabilize the overall duplex stability. This effect cannot be achieved by the shorter carbon chains such as methyl and dimethylallyl groups. For a duplex containing a T:A pair, the terpene groups disrupt both hydrogen bonding and stacking interactions by pushing the opposite A out of the helical structure. Overall, as the terpene chain length increases, the xT:G pair stabilizes the duplex, whereas the xT:A pair causes destabilization, indicating the evolutionary significance of the long terpene group on base-pairing specificity and codon recognition

The vitamin K-dependent Gla proteins and risk of type 2 diabetes

[Extract] Recently Haase et al [1] reported that maternal low-protein diet in rats was associated with decreased beta cell mass and decreased expression of growth arrest specific protein 6 (GAS6) in islets of Langerhans, and that incubation of neonatal islets with GAS6 enhanced beta cell proliferation. Low beta cell mass early in life is associated with increased future risk of type 2 diabetes. Consistent with a protective effect of GAS6, the c.834+7G>A polymorphism is associated with type 2 diabetes risk in humans [2]. The AA phenotype is associated with higher levels of GAS6, lower glucose levels, and decreased diabetes risk. Currently used drugs affecting the incretin axis have stimulated interest in therapeutic approaches that either preserve or enhance beta cell mass or function, and GAS6 (secreted by alpha cells) is, therefore, an attractive therapeutic target