Effects of prostaglandin E2 on rat skin: inhibition of sterol ester biosynthesis and clearing

Severe scaly lesions in the skin, especially in the were accompanied by growth retardation, impaired feet and tail, of the rat were induced by feeding a diet deficient fertility, increased water consumption, and diminished in essential fatty acids (EFA). Analysis of the fatty acids in skin urine production^ B~~~ and B~~~ proposed that linoleic lipids of these EFA-deficient rats showed a marked increase of and possibly linolenic acids were essential fatty acids monoenoic acids (1 6 : 1 and 18 : 1) and eicosatrienoic acid (EFA). The typical skin lesions therefore Serve to reflect (20 : 3), with concomitant decreases of dienoic acid (1 8 : 2) a derangement of metabolism associated with EFA deand tetraenoic acid (20 : 4). Topical application of prostaglanficiency. Apart from the macroscopic symptoms chardin E2 (PGE2) to the scaly lesions resulted in clearance of the lesions, but did not significantly alter the composition of fatty acterized by Severe scaling in the skin, very little is known acids in the &in. Intraperitoneal injection of P G E ~ had no about the biochemical events occurring in this tissue in observable effect on the skin lesions. Furthermore, incubation of EFA deficiency. skin specimens from the EFA-deficient rats with 14C-labeled Although arachidonic acid is not a normal component glucose showed a 4-5-fold increase of incorporation of glucose in vegetable fats, it is considered to be the principal uncarbon into lipid fractions, particularly the sterol esters, and a saturated fatty acid required by the animal organism 3-4-fold increase in pentose cycle activity. Addition of PGEz (3, 4). This line of thought is strengthened by reports to the incubation mixture resulted in approximately 70% infrom several investigators (5-8) who have shown that hibition of sterol ester biosynthesis by skin of the EFA-deficient linoleic and y-linolenic acids undergo transformations rats. These results suggest that the effects of PGEz in clearing in the animal organism to give arachidonic acid. Widmer the scales may be associated with its inhibitory effect on aband Holman (9) reported increased levels of arachidonate normal sterol esterification in the skin of the EFA-deficient in the heart and liver of fat-deficient rats fed diets suprats. plemented with linoleic acid. I n control rats that reSupplementary key words safflower oil . coconut oil ceived stearate and oleate, no increase in pOlyUnSatU. polyunsaturated fatty acids . eicosatrienoic acid . neurated acids in these organs could be detected. Greentral lipids . polar lipids . sterol esters . keratinization berg e t al. (10) reported that methyl arachidonate POST H E NUmITIONAL REQUIREMENT of mammalian species for certain fatty acids was first recognized in young rats and described by Burr and Burr (l, 2)' Thest? Preliminary reports of this work were presented at the 162nd meeting of the American Chemical Society, held in Washington, D.C., September 1971, and at the Conference on Prostaglandins in Cellular Biology and the Inflammatory Process, held in Carmel, investigators demonstrated that rats maintained on a fat-free diet over a long period developed an abnormality characterized by Scaliness Of the dOrsa1 skin, the feet, and the tail, which later became necrotic. These signs 458 Journal of Lipid Research Volume 13, 1972 Calif., October 1971. Abbreviations: PGE2, prostaglandin El: (1 1,154ihydroxy-9oxoprosta-5,13-dienoic acid); PGE1, prostaglandin E1 (11,154ihydroxy-9-oxoprosta-13-enoiC acid); EFA, essential fatty acid (s) ; GLC, gas-liquid chromatography. by gest, on O cber 9, 2017 w w w .j.org D ow nladed fom sessed a biopotency three to five times that of linoleic acid in correcting EFA deficiency. Recent studies have demonstrated that some of the EFA can be transformed enzymatically into prostaglandins by homogenates of vesicular glands from sheep (1 1-1 3) and adipose tissue of rats (14). A study in our laboratory has also demonstrated that arachidonic acid is transformed into PGEz by homogenates of rat skin (1 5). It is therefore interesting to investigate if prostaglandins play any role in the syndrome of EFA deficiency. This communication describes our observations that PGEz applied topically can clear the cutaneous lesions induced by feeding the rat a diet deficient in EFA. The facts that arachidonic acid is a precursor for the biosynthesis of PGE, and that PGEz exerts a curative effect on skin lesions in EFA deficiency suggest that the biochemical action of PGE2 in the skin may in some way mediate the normal process of keratinization. In further experiments we found, among other abnormalities, that the skin of the EFA-deficient rats synthesizes greater amounts of lipids from glucose than normal, especially the sterol esters. We also found that PGE2 markedly inhibits the biosynthesis of sterol esters by skin of the EFA-deficient rats. I t is tempting to associate the curative effects of PGEz with its inhibitory effect on the synthesis of sterol esters in the skin. To our knowledge, this is the first observation of a biochemical event relating PGE2 to EFA deficiency. MATERIALS AND METHODS Constituents of the basal diet were vitamin-test casein, salt mix (Wesson modification), and sucrose, purchased from General Biochemicals, Chagrin Falls, Ohio, and vitamin diet fortification mixture and cellulose, purchased from Nutritional Biochemicals, Cleveland, Ohio. Hydrogenated coconut oil and safflower oil were obtained from General Biochemicals and Nutritional Biochemicals, respectively. [U-14C]Glucose (specific activity, 14.7 mCi/mmole), [1-14C]glucose (specific activity, 5.6 mCi/mmole), [6-14C]glucose (specific activity, 4.85 mCi/mmole), and 14C-labeled lipids, [1-14C]stearic acid, [1J4C]palmitic a id, [4-14C]cholesterol, and [1-14C]cholesteryl palmitate, were from New England Nuclear Corp., Boston, Mass. Methyl esters of fatty acids (purity 95-99y0) were obtained from Lipid Organic Research Elysian, Minn. Sephadex (G-25 coarse) was purchased from Pharmacia Fine Chemicals, Inc., Piscataway, N.J.; Unisil, from Clarkson Chemical Co., Williamsport, Pa. ; and Florisil, from Floridin Co., Pittsburgh, Pa. Squalene, tripalmitin, dipalmitin, diolein, monopalmitin, and monoolein were from Pierce Chemical Co., Rockford, Ill. Hyamine hydroxide was from Packard Instrument Co., Downers Grove, Ill. Gentamicin sulfate was purchased from Schering Corp., Bloomfield, N.J. ; and PGEz was a gift from Dr. John Pike of the Upjohn Co., Kalamazoo, Mich. Reagents were of analytical grade and solvents were redistilled before use. Induction of EFA deficiency in rats Male weanling Sprague-Dawley rats 15-20 days old and weighing 28-36 g were used in these experiments. Induction of EFA deficiency was according to the procedure described by Aaes-Jflrgensen and Holman (16). The experimental group of animals was fed, ad lib., the basal diet supplemented with hydrogenated coconut oil; the diet of the control animals was supplemented with safflower oil. The animals were kept in individual cages in a room with controlled humidity and temperature (17). They were weighed and inspected for signs of deficiency at weekly intervals. The tail, forelegs, and hind legs were scored separately according to the dermal score described by Norby (1 8). Treatment of normal and EFA-deficient rats with PGEI For systemic administration PGEz was dissolved in sterile 0.9% NaC1, and for each kilogram of body weight 0.1 ml of the solution containing 1 mg of PGEz was injected intraperitoneally to the rat each day. For topical treatment, PGE2 was dissolved in a mixture of propylene glycol-ethanol 3 :7 (v/v), and 0.1 ml of the solution containing 25, 50, or 100 pg of PGE2 was applied daily with a small brush to the dorsal or plantar surface of either a forelimb or hind limb. Care was taken that the solution was spread evenly without running. The ethanol was allowed to evaporate so that a thin film of the residual solution remained on the skin. The therapeutic effectiveness of the PGE2 was evaluated at weekly intervals according to the dermal score. Analysis of fatty acids in skin of control and EFA-deficient rats Skin specimens were removed from the shaved area of the posterior dorsum from normal and EFA-deficient rats as reported previously (19). The skin specimens were minced and homogenized in a mixture of chloroform-methanol 2 : 1 (20) with a Polytron (model PT 10) homogenizer. Tissue debris was removed by filtration on sintered glass funnels. The filtrate was evaporated in a rotary evaporator. The total lipids were transesterified by refluxing under nitrogen with 30 vol of a 5% solution of HC1 in methanol. The methyl esters were analyzed by GLC, using an F & M model 402 with a hydrogen flame ionization detector. Glass columns, 182 cm X 3.5 mm, were packed with 3% diethylene glycol succinate polyester (DEGS) or 3y0 OV-101 Ziboh and Hsia Effects of Prostaglandin E, on Rat Skin 459 by gest, on O cber 9, 2017 w w w .j.org D ow nladed fom silicone copolymer coated on Supelcoport, 80-100 mesh (Supelco, Inc., Bellefonte, Pa.). When the DEGS column was used, the temperatures a t the injection port, column, and detector were 190, 140, and 190"C, respectively; for the OV-101 column the temperatures were 210, 180, and 23OoC, respectively. The nitrogen flow was 35 ml/min. The retention time was standardized by reference mixtures A, B, C, and D as specified by NIH. The methyl esters were identified by internal standards of reference methyl esters of fatty acids (purity 95-99y0) obtained from the Hormel Institute. Quantification was by triangulation, and the fatty acid composition was expressed as percentages of the total fatty acids. Preparation and incubation of skin specimens with differentially labeled glucose Skin specimens from normal and EFA-deficient rats were prepared as described in a previous publication (1 9). Paired skin specimens (approximately 60-80 mg) were incubated in 2.0 ml of Krebs-Ringer bicarbonate solution (pH 7.4) gassed with 95% 0 2 and 5% C02 and containing gentamicin sulfate (200 pg) and [U-14C]glucose (5.0 pCi), [1J4C]glucose (2.5 pCi), or [6-14C]glucose (2.5 pCi). PGE2 (1 pg) was added to one of each pair of incubation flasks. For collection of 14C02, the method of Snyder and Godfrey (21) was modified: a polyethylene center well (Kontes Glass Co., Vineland, N.J.) was attached to a rubber cap (serum stopper) which was fitted to the top of a 10-ml Erlenmeyer flask containing the incubation mixture. After shaking at 37°C for 120 min, 0.3 ml of 1 M Hyamine hydroxide was introduced by injection through the rubber cap into the center well for trapping the COZ, and 0.4 ml of 6 N HzS04 was added to the incubation mixture to stop the reaction and to liberate 14C02. The vessel was left at room temperature for 4 hr, after which the center well was carefully removed and placed directly into a counting vial for radioassay. Assay of 1% in lipid fractions For the determination of 14C incorporated into lipids, after the incubation with 14C-labeled glucose, the skin specimen was rinsed gently with 0.9% saline and homogenized in a mixture of chloroform-methanol 2 : 1 with the Polytron homogenizer. Tissue debris was removed by filtration on a sintered glass funnel and was washed with the solvent mixture until no radioactivity could be detected in the wash. The filtrate was taken to dryness in a rotary evaporator. Nonlipid radioactive contaminant in the extract was removed by gel filtration on a column of Sephadex G-25 (coarse) as described by Siakotos and Rouser (22). The 14C in the column effluent was assayed by liquid scintillation counting. After evaporation of the solvents, the recovered lipid mixture was dissolved in chloroform and percolated onto a column containing 5 g of Unisil (100-200 mesh) suspended in chloroform, according to the procedure described by Borgstrom (23). Neutral lipids were eluted with chloroform (200 ml) and polar lipids with methanol (200 ml). Aliquots from each fraction were assayed for radioactivity. The neutral lipids were dissolved in a small amount of hexane and were chromatographed further on 5 g of Florisil hydrated with 7% water and suspended in hexane, according to the procedure described by Carroll (24). Lipids were successively eluted from the column by the following solvents : (A) 15 ml of hexane ; (B) 50 ml of 5% ether in hexane; (C) 75 ml of 15% ether in hexane; (D) 60 ml of 250/, ether in hexane; (E) 40 ml of 50% ether in hexane; (F) 40 ml of 2% methanol in ether; and (G) 50 ml of 4Oy0 acetic acid in ether. The effluents were collected in 5-ml fractions, and aliquots were assayed for radioactivity. When reference compounds were chromatographed, squalene was eluted by (A), sterol and wax esters by (B), tripalmitin by (C), cholesterol by (D), dipalmitin and diolein by (E), monopalmitin and monoolein by (F), and palmitic acid and stearic acid by (G). Thin-layer chromatography For further resolution of fraction B, which usually contained both sterol esters and wax esters, thin-layer chromatography on silica gel G was performed according to the method of Schmid and Mangold (25), using the solvent system of hexane-diethyl ether 95 : 5 (v/v), with cholesteryl palmitate and stearyl palmitate as references. The 14C-labeled sterol esters were then eluted from the plates and saponified under nitrogen in 20% methanolic KOH at 80°C overnight in a closed vessel. The mixture was acidified with HCl and extracted with dichloromethane. The 14C was quantitatively recovered and the residue was subjected to thin-layer chromatography in the solvent system of isooctane-chloroformacetic acid 20:4:1 (v/v/v) as described by Pocock, Marsden, and Hamilton (26), with cholesterol and palmitic acid as references.