regeneration by endoneurial injection of [14C]acetate and by in vitro incubation of rat sciatic endoneurium with. [14C]acetate, [3H]galactose or [3H]glucose.
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Metabolism of Peripheral Nerve Monogalactosylceramides 1 Jeffrey K. Yao* Department of Veterans AffairsMedical Center, Highland Drive, Pittsburgh,Pennsylvania 15206, and Western Psychiatric Instituteand Clinic, Universityof Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
The metabolism of hydroxy galactocerebrosides {GalCeOH) and nonhydroxy galactocerebrosides {GalCe) was investigated during nerve development, degeneration and regeneration by endoneurial injection of [14C]acetate and by in vitro incubation of rat sciatic endoneurium with [14C]acetate, [3H]galactose or [3H]glucose. After endoneurial microinjection, [14C]acetate was found to be incorporated first into GalCe-OH and later, and to a much lesser degree, into GalCe. The ratio of 14C-labeled GalCeOH to GalCe decreased with time and remained fairly constant after 4 hr. On the other hand, in vitro incorporation of [14C]acetate resulted in higher 14C-labeling in GalCe and monogalactosyl diacylglycerol {MGDG} and lower 14C-labeling in GalCe-OH, diminishing with incubation time. After 24 hr, only GalCe and MGDG were labeled. When [3H]galactose or [3H]glucose, instead of [14C]acetate, were used as precursor in vitro, a similar preference for labeling of GalCe-OH was demonstrated in regenerating nerve. These data suggest that hydroxy fatty acids and hydroxy ceramides are the preferred substrates in peripheral nervous system for the sphingosine acyltransferase and the UDP~ galactosyltransferase reactions, respectively. The ahydroxylation system did not appear to be fully functional under in vitro conditions. The biosynthesis of GalCe-OH was greatly enhanced during nerve fiber regeneration and decreased rapidly with increasing age. This suggests that a close interrelation exists between a-hydroxylation and peripheral nerve myelination. Lipids 24, 837-841 {1989).
galactocerebrosides coincides with axonal degeneration and demyelination {3}.These results suggest that the biosynthesis of endoneurial galactolipids take place preferentially at the time when the peripheral nerve is undergoing active myelination. The main biosynthetic pathway of peripheral nerve galactocerebrosides appears to involve the transfer of galactose to ceramide by the UDPgalactose:ceramide galactosyltransferase reaction {4}. On the other hand, glucocerebrosides, which are the major monohexosylceramides of extraneural tissues, are not considered constituents of the myelin membrane. Recently, we have shown {5) that the normally inactive pathway for the biosynthesis of glucocerebroside and oligohexosylceramides is activated in adult rat sciatic endoneurium following permanent nerve transection. Furthermore, only glucocerebroside homologues, not galactocerebroside, are synthesized in purified Schwann cell culture {Yao and Yoshino, unpublished data}. Thus, it is likely that the cells responsible for glucocerebroside biosynthesis are Schwann cells as these comprise 90% of the total endoneurial cell area in the distal nerve segment at 35 days after transection {6}. However, this does not exclude the possibility that other cell types, such as macrophages, may contribute to glucocerebroside biosynthesis during nerve fiber degeneration. To further evaluate the metabolic role of galactosylceramides in peripheral nerve myelination, the metabolism of galactolipids was investigated by in vivo and in vitro incorporation of [14C]acetate, [3H]galactose or [3H]glucose into rat sciatic endoneurium during nerve development, degeneration and regeneration.
Galactocerebrosides and galactosulfatides are the major glycolipids of peripheral nerve myelin {1), which is an ex- MATERIALS AND METHODS tension of the plasma membrane of Schwann cells {2}. Previous studies on peripheral nerve glycolipid composi- Materials. [:4C]Acetate, [3H]galactose and [3H]glucose tion {3}have shown that hydroxycerebrosides and hydrox- were purchased from Amersham {Arlington Heights, IL). ysulfatides are the main constituents of glycolipids of im- Unlabeled galactose was obtained from Sigma Chemical mature rat sciatic endoneurium. The ratio of hydroxy to Co. {St. Louis, MO). High-performance thin-layer chromanonhydroxy cerebrosides decreases rapidly as myelina- tography {HPTLC} plates {LHP-K with preadsorbent tion proceeds, and then remains fairly constant through- area} were from Whatman Chemical Separation {Clifton, out adulthood. More than 50% of the adult content of N J}. Sep-Pak silica gel cartridges and cartridge rack were endoneurial galactolipids is reached before 21 days of age. from Waters Associates {Milford, MA}. EN3HANCE In the absence of myelin assembly, e.g., in Wallerian was from New England Nuclear {Boston, MA}. Lipid standegeneration, the maximum decrease of endoneurial dards were from Supelco {Bellefonte, PA}. All solvents ~HPLC grade} were from Burdick and Jackson Laboratories {Muskegon, MD. Endoneurial injection of [14C]acetate. Using a nanoliter :A preliminaryreport of this work was presented at the 19th Annual Meeting of the American Societyfor Neurochemistry, New pump {model 1400 EC, W-P Instruments, Inc., New Orleans, LA, March 7, 1988. Part of this work was done while the Haven, CT}, the procedure used for endoneurial microinauthor was associated with the Mayo Clinic, Rochester, MN. jection was essentially the same as that described pre*Address correspondenceto: Dr. Jeffrey K. Yao, PsychiatryService viously {4,7}. [1-14C]Acetate {54-60 mCi/mmol} was 116A6, DVA MedicalCenter, HighlandDr., Pittsburgh, PA 15206. diluted in Evans blue solution {0.5 mg/ml} to yield a conAbbreviations: HPTLC, high-performance thin-layer chromatog- centration of 1.1 • 107 dpm/~l. Under the dissecting raphy; GalCe, galactocerebroside; GalCe-OH, hydroxy galac- microscope, the micropipette tip was placed inside the tocerebroside; MGDG,monogalactosyldiacylglycerol;Su, sulfatide; sciatic endoneurium and 0.4 ~l of [14C]acetate solution Su-OH, hydroxy sulfatide;C, cholesterol;NL, non-polarlipids; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, was injected at a rate of 0.1 ~l/min. Unless otherwise inphosphatidylserine;PC, phosphatidylcholine;Sp, sphingomyelin;TG, dicated, one-month-old male Sprague-Dawley rats were triacylglycerol. used throughout this study. LIPIDS, Vol. 24, No. 10 (1989)
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J.K. YAO Tissue preparation. Approximately 1 cm of sciatic RESULTS nerve, including the injection site, was removed at various times after injection. Each group consisted of four or more Endoneurial microinjection and in vitro incubation of nerve samples. Immediately after removal, the sciatic [14C]acetate. When [14C]acetate was injected into the ennerve was desheathed on a cold plate under a dissecting doneurial portion of one-month-old rat sciatic nerve, apmicroscope. The desheathed portion was blotted dry with proximately 9% of labeled lipids were found to be glycofilter paper before the wet weight was taken. The en- lipids (4). Following separation of 14C-labeled glycolipids doneurium was then lyophilized overnight to obtain the from nonpolar lipids and phospholipids on HPTLC and dry weight. as revealed by fluorography (Fig. 1), [14C]acetate was Surgicalprocedure. Wallerian degeneration and regen- found to be incorporated first into GalCe-OH and later, eration were produced in rat sciatic nerve by either crush although to a much lesser degree, into GalCe. Six hours injury or transection according to the method described after injection, only minute amounts of 14C-labeled previously (8). Reattachment of transected nerve was ac- sulfatides were detected on the fluorogram when the cording to the procedure of nerve xenograft described by HPTLC plate was exposed to Kodak X-Omat S film for Dyck et al. (9). 14 days. On the other hand, in vitro incorporation of In vitro radiolabeled precursor incorporation. The [14C]acetate (Fig. 2) resulted in higher 14C-labeling in method used for the in vitro incubation of sciatic en- GalCe and MGDG, and lower 14C-labeling in GalCe-OH, doneurium with [1-14C]acetate, [3H]galactose or [3H]- diminishing with incubation time. After 24 hr, only GalCe glucose was essentially the same as described by Yao and and MGDG were found to be labeled from [~4C]acetate. Poduslo (5). Following dissection and weighing, the No radioactivity was found in the region of glucocerebrodesheathed nerve was immediately placed in Lab-Tek side following borate-impregnated HPTLC. tissue culture chamber/slides (Miles Scientific, Naperville, IL) which contained 0.5 ml of modified Krebs glucose solution (10). Incubation medium was mixed with 2.5 gCi of the sodium salt of [1-14C]acetic acid (~60 mCi/mmol), h .15 .5 1 2 4 6 62/~Ci of [1-3H]glucose (19.4 mCi/mg) or 58 /~Ci of NL ~ ~ ~ ~ ~ t._ [3H]galactose (58 mCi/mg) plus 71 ~g of unlabeled galac--._. tose just before addition of nerve sample. Each nerve was G a l C e - "'--'" ~ incubated in a metabolic incubator at 37 ~ under a mix- GalCe-OH ture of 95% 02 and 5% CO2. After various incubation PE ~ ~ ~ , ~ times, the nerve segments were removed and washed five P I-"-----~ -.~ ~ ~ PS times with ice-cold unlabeled medium to ensure complete IR removal of free labeled precursor. The labeled nerve segspPC ~ ~ ~ ~ ment was then lyophilized to obtain the dry weight. Lipid extraction and separation. The lipids were extracted from lyophilized tissues according to the procedure described previously (8,11}. An aliquot was counted with a Beckman liquid scintillation counting FIG. 1. Fluorograms of HPTLC of total 14C-labeled lipids followsystem (model LS 5801) to obtain the total incorporation ing endoneurial microinjection of [14C]acetate into rat sciatic nerve of [~4C]acetate, [3H]galactose or [3H]glucose into en- after various periods of time (hr). Each sample represents total 14Cdoneurial lipids. Radiolabeled lipid profiles of endo- labeled lipids extracted from 0.05 mg dry wt of endoneurium, except 0.15 hr (sample was extracted from 0.15 mg). After TLC developneurium were analyzed by HPTLC using the procedure ment, the plate was exposed to Kodak X-Omat S film at --70~ for described by Yao and Rastetter (12). Separation of G alCe four days. O = origin. and glucocerebroside was achieved on borate-impregnated plates (13) using chloroform/methanol/water (60:17:2, v/v/v) as development solvent (14}. Fluorography was used to identify the radiolabeled lipids following HPTLC h 2 6 24 (4), thus ensuring the accuracy and resolution of radioactivity distribution. Quantification of fluorograms was done using a LKB Ultrascan Laser Densitometer and O O Q Apple IIe computer. Isolation of neutral glycosphingolipids from total lipid extracts was achieved by column separation on a Sep-Pak silica cartridge (12). Chemical reactions~ To remove any phospholipids or glycolipids containing ester-linked acyl moiety, the isolated glycolipids were subjected to mild alkaline r o d e methanolysis according to the procedure described by Vance and Sweeley (15). To determine the radioactivity distribution among different moieties, the labeled glycosphingolipids were reacted with 0.5 N methanolic HC1 at 100~ for 3 hr (16). The procedure used to extract FIG. 2. Effect of incubation time on peripheral nerve galactolipid biosynthesis. Following in vitro incorporation of [z4C]acetate into fatty acid methyl esters, methyl glycosides, and sphingo- rat sciatic endoenurium after various time periods, ca. 10,000 sine bases was essentially the same as described by cpm/sample were applied to a thin-layer plate which was exposed Desnick et al. (17}. to Kodak X4)mat S f i l l at --70~ for five days. h = incubation time. LIPIDS, Vol. 24, No. 10 (1989)
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GALACTOLIPIDS METABOLISM IN PERIPHERAL NERVE
In vitro incubation of [3H]galactose. The incorporation of [3H]galactose into endoneurial lipids was substratedependent with an optimal concentration of 0.2 ~mol/mg dry weight of endoneurium. There was a linear relationship between incorporation and incubation time up to 24 hr. [3H]Galactose was rapidly incorporated into endoneurial GalCe-OH, and later into GalCe and MGDG {Fig. 3). Approx. 80% of radioactivity incorporated into endoneurial glycolipids were found in the carbohydrate moiety. In addition, 3H-labeled nonpolar lipids and phospholipids were also identified by fluorography {Fig. 3). The ratio of 8H-labeled GalCe-OH to GalCe decreased with incubation time and remained fairly constant after 6 hr (Fig. 4). A similar change in the ratio of ~4C-labeled GalCe-OH to GalCe with time was also demonstrated after endoneurial microinjection of [14C]acetate into rat sciatic nerve {Fig. 4), except that GalCe-OH was predominantly labeled early in the experiment. Effect of age on galactolipid biosynthesis. In vitro incorporation of [3H]galactose into endoneurial lipids of rat sciatic nerve decreased rapidly with increasing age (Fig. 5). Approximately 70% of the labeled lipids were found to be galactolipids in rats four days of age as compared with
