The activation of supraspinal GPR40/FFA1 receptor signalling regulates the descending pain control system.

BACKGROUND AND PURPOSE: The ω-3 polyunsaturated fatty acids exert antinociceptive effects in inflammatory and neuropathic pain; however, the underlying mechanisms remain unclear. Docosahexaenoic acid-induced antinociception may be mediated by the orphan GPR40, now identified as the free fatty acid receptor 1 (FFA1 receptor). Here, we examined the involvement of supraspinal FFA1 receptor signalling in the regulation of inhibitory pain control systems consisting of serotonergic and noradrenergic neurons. EXPERIMENTAL APPROACH: Formalin-induced pain behaviours were measured in mice. Antinociception induced by FFA1 receptor agonists was examined by intrathecal injections of a catecholaminergic toxin, 5-HT lowering drug or these antagonists. The expression of FFA1 receptor protein and c-Fos was estimated by immunohistochemistry, and the levels of noradrenaline and 5-HT in the spinal cord were measured by LC-MS/MS. KEY RESULTS: FFA1 receptors colocalized with NeuN (a neuron marker) in the medulla oblongata and with tryptophan hydroxylase (TPH; a serotonergic neuron marker) and dopamine ß-hydroxylase (DBH; a noradrenergic neuron marker). A single i.c.v. injection of GW9508, a FFA1 receptor agonist, increased the number of c-Fos-positive cells and the number of neurons double-labelled for c-Fos and TPH and/or DBH. It decreased formalin-induced pain behaviour. This effect was inhibited by pretreatment with 6-hydroxydopamine, DL-p-chlorophenylalanine, yohimbine or WAY100635. Furthermore, GW9508 facilitated the release of noradrenaline and 5-HT in the spinal cord. In addition, GW1100, a FFA1 receptor antagonist, significantly increased formalin-induced pain-related behaviour. CONCLUSION AND IMPLICATIONS: Activation of the FFA1 receptor signalling pathway may play an important role in the regulation of the descending pain control system.

The Incorporation and Accumulation of Supplemented Fatty Acids in Mucor hiemalis HA-30.

Accumulation of free unsaturated fatty acids, added individually to the medium, into cellular triacylglycerol (TG) were examined using a fungus, Mucor hiemalis HA-30, which could produce the eicosapentaenoic acid (20:5n-3)-enriched TG. Linoleic (18:2n-6), γ-linolenic (18:3n-6), arachidonic acids (20:4n-6), and 20:5n-3 were favored for accumulation in TG and each fatty acid accumulated in TG at about 80% purity. However, docosahexaenoic acid (22:6n-3) was not efficiently accumulated in TG and its content was about 60%. Incorporation and accumulation of various fatty acids added to the cultivation medium were investigated using a model fatty acids mixture. Generally, unsaturated fatty acids were efficiently incorporated into cells more than saturated fatty acids. Fatty acids with odd-numbered double bonds such as 18:3n-3, 18:3n-6, and 20:5n-3 showed the higher incorporation efficiency compared with those with even-numbered double bonds such as 18:2n-6, 20:4n-6, and 22:6n-3. On the other hand, saturated and monounsaturated fatty acids of 16:0, 18:0, and 18:1 were preferentially accumulated in microbial cells over other fatty acids.

Production of Eicosapentaenoic Acid-Enriched Triacylglycerol by Mucor hiemalis HA-30.

Microorganisms that accumulated the eicosapentaenoic acid (EPA)-enriched triacylglycerol (TG), were screened for using yeast-malt medium containing 1% free EPA. The best strain was identified as Mucor hiemalis HA-30. The optimum culture conditions for the accumulation of EPA-enriched TG were : 3% soluble starch, 0.5% polypeptone, 0.3% yeast extract, 0.5% free EPA, and pH 6.0 at 25°C. After the cultivation, 1.77 mg/ml of the TG with EPA purity of 79% was obtained. The EPA content in TG increased in conjunction with the EPA content of the supplemented free fatty acids or ethyl esters. Free EPA were more efficiently incorporated than the ethyl esters. Trieicosapentaenoyl glycerol (EPA, EPA, EPA) accounted for 73% of total TGs.

Hydrolysis and synthesis of wax esters by different systems of carp hepatopancreas preparation.

It was found that the supernatant of hepatopancreas homogenate of the carp, Cyprinus carpio, contained enzymes involved in the hydrolysis and esterification of wax esters. The enzymes were salted out by 30% saturation with ammonium sulfate. This fraction also contained the highest activities of lipase and esterase. The enzyme for the hydrolysis of wax esters, together with lipase and esterase, was eluted in tube numbers 20 to 22 (void volume) from a Sephadex G-200 column. On the other hand, the enzyme for the synthesis of wax esters was eluted in tube numbers 33 to 35. It thus appears that the two activities belong to different enzyme proteins. The synthetic enzyme was activated by the addition of ATP, CoA, and NADH.