Acetyl-CoA carboxylase inhibition alters tubulin acetylation and aggregation in thrombin-stimulated platelets

Introduction Acetyl-CoA carboxylase (ACC), the first enzyme regulating lipid synthesis, promotes thrombus formation by increasing platelet phospholipid content. Inhibition of its activity decreases lipogenesis and increases the content in acetyl-CoA which can serve as a substrate for protein acetylation. This posttranslational modification plays a key role in the regulation of platelet aggregation, via tubulin acetylation. Purpose To demonstrate that ACC inhibition may affect platelet functions via an alteration of lipid content and/or tubulin acetylation. Methods Platelets were treated 2 hours with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. Platelet functions were assessed by aggregometry and flow cytometry. Lipogenesis was measured via 14C-acetate incorporation into lipids. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Protein phosphorylation and acetylation were evaluated by western blot. Results Treatment with CP640.186 drastically decreased platelet lipogenesis. However, the quantitative lipidomics analyses showed that preincubation with the compound did not affect global platelet lipid content. Interestingly, this short-term ACC inhibition was sufficient to increase tubulin acetylation level, at basal state and after thrombin stimulation. It was associated with an impaired platelet aggregation, in response to low thrombin concentration, while granules secretion was not affected. Mechanistically, we highlighted a decrease in Rac1 activity, associated with a reduced phosphorylation of its downstream effector PAK2. Surprisingly, actin cytoskeleton was not impacted but we evidenced a significant decrease in ROS production which could result from a decreased NOX2 activity. Conclusion Pharmacological ACC inhibition decreases platelet aggregation upon thrombin stimulation. The mechanism depends on increased tubulin acetylation, with subsequent alteration of the Rac1/PAK2/NOX2 signaling pathway FUNDunding Acknowledgement Type of funding sources: Other. Main funding source(s): Fonds pour la formation à la Recherche dans l'Industrie et l'Agriculture (FRIA)

Evidence for functional G-coupled protein receptors 43 and 120 in subcutaneous and intramuscular adipose tissue of Angus crossbred steers

We conducted three independent experiments to demonstrate functional G-coupled protein receptor 43 (GPR43) and GPR120 in bovine intramuscular (i.m.) and subcutaneous (s.c.) adipose tissues. We hypothesized that media volatile fatty acids and long-chain fatty acids would affect cAMP-activated protein kinase-alpha (AMPKα) protein expression and cAMP concentrations differently in i.m. and s.c. adipose tissue. Experiment 1: Oleic acid (18:1n-9) decreased phosphorylated AMPKα protein (p-AMPKα) and the p-AMPKα/AMPKα protein ratio in i.m. preadipocytes, increased the p-AMPKα/AMPKα protein ratio in bovine satellite cells, and had no effect in s.c. preadipocytes. Experment 2: Ex vivo explants from the 5 th-8 th longissimus thoracic rib muscle section of Angus crossbred steers were cultured 48 h in media containing 0.25 µM ciglitizone, 5 mM glucose, and 5 mM acetate, in the absence or presence of 100 µM oleic acid. Oleic acid increased acetate incorporation into fatty acids and GPR43 gene expression in i.m. adipose tissue (P < 0.05), but oleic acid had no effect on fatty acid synthesis or GPR43 expression in s.c. adipose tissue. Experiment 3: Fresh s.c. and i.m. adipose tissue from the 5 th-8 th longissimus thoracic rib muscle section of Angus crossbred steers was transferred immediately to 6-well culture plates containing 3 mL of KHB/Hepes/5 mM glucose. Samples were pre-incubated with 0.5 mM theophylline plus 10 μM forskolin for 30 min, after which increasing concentrations of acetate or propionate (0, 10 -3, 10 -2.3, and 10 -3 M) in the absence or presence of 100 μM oleic acid or 100 µM palmitic acid (16:0) were added to the incubation media. Acetate had no effect on forskolin-stimulated cAMP production in s.c. adipose tissue but decreased cAMP in i.m. adipose tissue (P < 0.05); this indicates a functional GPR43 receptor in i.m. adipose tissue. The combination of 10 -2 M acetate and oleic acid decrease cAMP production in s.c. adipose tissue, consistent with GPR120 receptor activity, but oleic acid and palmitic acid attenuated the depression of cAMP production caused by acetate in i.m. adipose tissue. Palmitic acid depressed cAMP production in s.c. adipose tissue, and increased cAMP production in i.m. adipose tissue (P < 0.05). Propionate had no effect on cAMP production in s.c. or i.m. adipose tissue. These results provide evidence for functional GPR43 receptors in i.m. adipose tissue and GPR120 receptors in s.c. adipose tissue, both of which would suppress lipolysis.

Comparative Toxicities of Salts on Microbial Processes in Soil

ABSTRACTSoil salinization is a growing threat to global agriculture and carbon sequestration, but to date it remains unclear how microbial processes will respond. We studied the acute response to salt exposure of a range of anabolic and catabolic microbial processes, including bacterial (leucine incorporation) and fungal (acetate incorporation into ergosterol) growth rates, respiration, and gross N mineralization and nitrification rates. To distinguish effects of specific ions from those of overall ionic strength, we compared the addition of four salts frequently associated with soil salinization (NaCl, KCl, Na2SO4, and K2SO4) to a nonsaline soil. To compare the tolerance of different microbial processes to salt and to interrelate the toxicity of different salts, concentration-response relationships were established. Growth-based measurements revealed that fungi were more resistant to salt exposure than bacteria. Effects by salt on C and N mineralization were indistinguishable, and in contrast to previous studies, nitrification was not found to be more sensitive to salt exposure than other microbial processes. The ion-specific toxicity of certain salts could be observed only for respiration, which was less inhibited by salts containing SO42−than Cl−salts, in contrast to the microbial growth assessments. This suggested that the inhibition of microbial growth was explained solely by total ionic strength, while ion-specific toxicity also should be considered for effects on microbial decomposition. This difference resulted in an apparent reduction of microbial growth efficiency in response to exposure to SO42−salts but not to Cl−salts; no evidence was found to distinguish K+and Na+salts.

Impact of anthracene on the arbuscular mycorrhizal fungus lipid metabolism

Anthracene, a low-molecular-weight polycyclic aromatic hydrocarbon (PAH) originating mainly from anthropogenic activities, represents one of the major persistent organic pollutants frequently detected in polluted soils. A few studies have reported the negative effect of PAH on the main steps of the arbuscular mycorrhizal fungi (AMF) life cycle resulting from lipid peroxidation; however, little is known regarding the impact of anthracene on extraradical AMF lipid metabolism. Radiolabelling experiments showed significant decreases of [1-14C]acetate incorporation into the sterol precursors (4,4-dimethylsterols and 4α-methylsterols) and in the total phospholipids (PL) of Rhizophagus irregularis (Blaszk., Wubet, Renker & Buscot) extraradical mycelium when grown in the presence of anthracene. These findings suggested a slowing down of the sterol and total PL biosynthesis pathways in AMF treated with anthracene. The negative impact of the organic pollutant on AMF membrane lipid biosynthesis may explain the growth inhibition of the fungus after PAH exposure. This study increases the understanding of the biochemical mechanisms involved in PAH ecotoxicity on AMF.

Active Hydrocarbon Biosynthesis and Accumulation in a Green Alga, Botryococcus braunii (Race A)

ABSTRACT Among oleaginous microalgae, the colonial green alga Botryococcus braunii accumulates especially large quantities of hydrocarbons. This accumulation may be achieved more by storage of lipids in the extracellular space rather than in the cytoplasm, as is the case for all other examined oleaginous microalgae. The stage of hydrocarbon synthesis during the cell cycle was determined by autoradiography. The cell cycle of B. braunii race A was synchronized by aminouracil treatment, and cells were taken at various stages in the cell cycle and cultured in a medium containing [ 14 C]acetate. Incorporation of 14 C into hydrocarbons was detected. The highest labeling occurred just after septum formation, when it was about 2.6 times the rate during interphase. Fluorescent and electron microscopy revealed that new lipid accumulation on the cell surface occurred during at least two different growth stages and sites of cells. Lipid bodies in the cytoplasm were not prominent in interphase cells. These lipid bodies then increased in number, size, and inclusions, reaching maximum values just before the first lipid accumulation on the cell surface at the cell apex. Most of them disappeared from the cytoplasm concomitant with the second new accumulation at the basolateral region, where extracellular lipids continuously accumulated. The rough endoplasmic reticulum near the plasma membrane is prominent in B. braunii , and the endoplasmic reticulum was often in contact with both a chloroplast and lipid bodies in cells with increasing numbers of lipid bodies. We discuss the transport pathway of precursors of extracellular hydrocarbons in race A.

Effect of reduced heifer nutrition during in utero and post-weaning development on glucose and acetate kinetics

Energetic efficiency was evaluated in composite bred heifers born from dams receiving 1·8 or 1·2 kg/d winter supplementation for approximately 80 d before parturition. Heifers were then developed post-weaning and randomly assigned to heifer development treatments of either control (100 %; ad libitum; n 8/year) or restricted (80 %; fed 80 % of supplementation fed to controls adjusted to a common body weight: n 8/year) in a 2-year study. A glucose tolerance test (GTT) and acetate irreversible loss test (AILT) were administered to heifers at the termination of a 140 d development period when the heifers were approximately 403 d of age and consumed a silage-based diet, and again at 940 d of age when pregnant with their second calf and grazing dormant forage. No differences were measured (P>0·08) for dam winter nutrition or heifer development treatment for baseline serum metabolites or measures in either the GTT or the AILT. However, changes in baseline serum concentrations (P>0·05) were different between metabolic challenges, which occurred at different stages of development. No difference in acetate disappearance (P = 0·18) and half-life (P = 0·66) was measured between the two metabolic challenges. A trend for glucose half-life to be shorter in heifers born from dams receiving in utero winter treatments that supplied 1·2 kg/d of winter supplementation was observed (P = 0·083). Heifers developed with lower total DM intake during a 140 d development period had similar glucose and acetate incorporation rates as ad libitum-fed heifers when evaluated at two different production stages.

Contrasting Soil pH Effects on Fungal and Bacterial Growth Suggest Functional Redundancy in Carbon Mineralization

ABSTRACT The influence of pH on the relative importance of the two principal decomposer groups in soil, fungi and bacteria, was investigated along a continuous soil pH gradient at Hoosfield acid strip at Rothamsted Research in the United Kingdom. This experimental location provides a uniform pH gradient, ranging from pH 8.3 to 4.0, within 180 m in a silty loam soil on which barley has been continuously grown for more than 100 years. We estimated the importance of fungi and bacteria directly by measuring acetate incorporation into ergosterol to measure fungal growth and leucine and thymidine incorporation to measure bacterial growth. The growth-based measurements revealed a fivefold decrease in bacterial growth and a fivefold increase in fungal growth with lower pH. This resulted in an approximately 30-fold increase in fungal importance, as indicated by the fungal growth/bacterial growth ratio, from pH 8.3 to pH 4.5. In contrast, corresponding effects on biomass markers for fungi (ergosterol and phospholipid fatty acid [PLFA] 18:2ω6,9) and bacteria (bacterial PLFAs) showed only a two- to threefold difference in fungal importance in the same pH interval. The shift in fungal and bacterial importance along the pH gradient decreased the total carbon mineralization, measured as basal respiration, by only about one-third, possibly suggesting functional redundancy. Below pH 4.5 there was universal inhibition of all microbial variables, probably derived from increased inhibitory effects due to release of free aluminum or decreasing plant productivity. To investigate decomposer group importance, growth measurements provided significantly increased sensitivity compared with biomass-based measurements.

The effect of cadmium on lipid and fatty acid biosynthesis in tomato leaves

This research aims to examine the effect of cadmium uptake on lipid composition and fatty acid biosynthesis, in young leaves of tomato treated seedlings (Lycopersicon esculentum cv. Ibiza F1). Results in membrane lipids investigations revealed that high cadmium concentrations affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the unsaturated fatty acid content, resulting in a lower degree of fatty acid unsaturation. The level of lipid peroxides was significantly enhanced at high Cd concentrations. Studies of the lipid metabolism using radioactive labelling with [1-14C]acetate as a major precursor of lipid biosynthesis, showed that levels of radioactivity incorporation in total lipids as well as in all lipid classes were lowered by Cd doses. In total lipid fatty acids, [1-14C]acetate incorporation was reduced in tri-unsaturated fatty acids (C16:3 and C18:3); While it was enhanced in the palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0) and linoleic (C18:2) acids. [1-14C]acetate incorporation into C16:3 and C18:3 of galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] and some phospholipids [phosphatidylcholine (PC) and phosphatidylglycerol (PG)] was inhibited by Cd stress. Our results showed that in tomato plants, cadmium stress provoked an inhibition of polar lipid biosynthesis and reduced fatty acid desaturation process.