Dibutyryl Cyclic Adenosine 3',5-Monophosphate and Brain Lipid Metabolism BENJAMIN WEISS and R ICHARD L. STI LLER, Division of Neuroscience, New York State Psychiatric Institute and Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032

ABSTRACT

It was f o u n d tha t d ibutyry l cyclic adenos ine 3 ' , 5 ' - m o n o p h o s p h a t e exe r t ed its greatest suppressive e f fec t upon tota l l ipid synthes is in 7-10 day old rat bran s l i c e s i n c u b a t e d in the presence of 2 -1 4 C - a c e t a t e , U-14C-D-glucose, and 3-14C-DL-serine. F rac t iona t ion of the neutra l and polar lipids disclosed that the fo rma t ion of each of their respect ive com- ponen t s was r educed to varying ex ten t s , t hus suggesting tha t cyclic adenosine 3 ' , 5 ' -m onophospha t e , arising f rom the

TABLE I

Effect of Age upon Incorporation of 2 -14C-Acetate into Total Lipids of Brain Slices in the Presence of Dibutyryl Cyclic Adenosine 3',5'monophosphate a

Age Control

Total lipid

Experimental R e d u c t i o n

Days 103 cpm 103 cpm Percent 1 b 25 23 8 3 50 44 12 5 148 118 20 6 230 120 48 7 229 108 53 8 205 91 56 9 240 97 60

10 210 93 56 14 170 103 40 15 180 120 34 21 68 63 7 25 63 63 0

Adult b 23 20 13

aln each experiment, slices were prepared from a single rat brain after separation into hemispheres. Slices (3-4) each weighing ca. 100 mg were obtained from each hemisphere which served as a control and experimental. The slices were distributed among 3-4 serum bottles (15 ml) equipped with rubber septums and containing 2 ml Krebs-Ringer bicarbonate solu- tion; each bottle received ca. 100 mg tissue. Each group of bottles contained 1.25 ~tCi of 2-14C-sodium acetate. Both dbcAMP and theophylline were present at final concentrations of 1 mM in each experimental bottle. The bottles were flushed for several min with 95% 02-5% CO 2 incubated with gyrotory shaking at 100 rpm in a water bath at 37 C for 2 hr. The reaction was terminated by addition of l0 volumes of metha- nol after which the total lipids were removed. (See text for details.)

bone hemisphere from each of 2 brains and 1 O0 mg from each hemisphere of a single brain were used from 1 day old and adult rats, respectively.

hydrolys is o f d ibutyry l cyclic adenosine t ! 3 , 5 - m o n o p h o s p h a t e , may con t ro l the

synthesis of brain lipids by regulating the ut i l izat ion of ace ty l -coenzyme A.

I NTRODUCTI ON t t

C y c l i c a d e n o s i n e 3 , 5 - m o n o p h o s p h a t e (cAMP) in brain has been the subject of consid- erable s tudy. The func t ion of biogenic amines, depolarizing agents, and o the r factors , such as his tamine, norep inephr ine , and theophy l l ine on the cont ro l of cAMP levels in incuba ted brain slices has been ex ami n ed extensively (1-5). The phosphory la t ion of brain microsomal or r ibo- somal prote ins has been shown to be s t imula ted by a cAMP-media ted pro te in kinase in rat brain microsomes (6-8). A l though the role of cAMP on the regulat ion of l ipolyt ic activity in various tissues has been well d o c u m e n t e d (9,10), l i t t le is k n o w n regarding its e f fec t u p o n brain lipid metabol ism. The s tudy herein r epo r t ed was unde r t aken to examine the act ion of exogenous c A M P , N , 6 0 2 ' - d i b u t y r y l cyclic adenosine 3 ' , 5 " m o n o p h o s p h a t e (dbcAMP), and agents k n o w n to affect the levels o f cAMP in tissues on the synthesis o f lipids in rat brain slices.

MATERIALS AND METHODS

All chemicals were reagent grade; solvents were distilled before use. Sprague-Dawley rats o f e i ther sex were purchased f rom Marland Farms, Hewit t , N.J. cAMP and dbcAMP were ob ta ined f rom Sigma Chemical Co., 2 - t4C-So - dium acetate , 24.4/aCi/mg, U-14C-D-glucose, 26.5/aCi/mg and 3 -14C-DL-serine, 119/~Ci/mg were purchased f rom New England Nuclear, Boston, Mass. Lipid s tandards were f rom Ap- plied Science Laborator ies , State College, Pa., and Supelco, Bel lefonte , Pa.

Silicic acid (Mall inckrodt , St. Louis, Mo.), washed several t imes wi th ch lo ro fo rm: methano l (C:M) (2 :1) by suspens ion and centr igugat ion was dried overnight at 110 C; 6.0 g por t ions in ch lo ro fo rm were packed in to co lumns , 1.5 x 50 cm. Acid-, alkali-, and water -washed diethyl- aminoethyl-cel lu lose (DEAE) , 10 g por t ions , was packed in to co lumns af ter convers ion to the aceta te by t r ea tmen t wi th glacial acetic acid overnight . The co lumns were washed succes-

514

BRAIN LIPID

sively with methanol until free from acid and 10 column volumes (CV) of chloroform (11).

After incubation of the brain slices (12), the reaction was terminated by addition of 10 vol- umes of methanol. The brain slices from the same hemisphere in aqueous methanol were combined and homogenized in a teflon glass homogenizer. After centrifugation of the ho- mogenate, the precipitate was rehomogenized 3 times in the same manner with 20 ml portions of C:M (2:1). The combined aqueous methanol and C:M supernates were adjusted to a final ratio of C:M:water of 8:4:3 to form a partition (13). After removal of the upper layer, the lower phase was repartitioned against an equal volume of fresh upper layer. The residue ob- tained after concentration of the lower phase under reduced pressure was dried over phospho- rus pentoxide, weighed, and dissolved in chloro- form. An aliquot, dried under a stream of nitro- gen, was dissolved in 10 ml Omnifluor cotaining 0.1 ml solubilizer (Nuclear Chicago, Chicago, Ill.) and counted in a Packard Tri-Carb liquid scintillation spectrometer. The remainder of the chloroform solution containing ca. 60 mg lipid, derived from 4-5 pooled hemispheres, was ap- plied to a silicic acid column. The neutral and polar lipids were eluted from the column with 10 CV of chloroform and methanol, respec- tively. Upon removal of solvent, the residues were dried, weighed, and stored in chloroform at -25 C until ready for use after assay of an aliquot for radioactivity.

Neutral lipids, 5-10 rag, were applied to four 0.25 mm Silica Gel G coated thin layer plates and developed with petroleum ether:diethyl ether:glacial acetic acid (90:10:1). After drying the plate in a hood at room temperature for ca. 30 min until no odor of acetic acid was appar- ent, chromatography was repeated in the same d i m e n s i o n (14,15) with petroleum ether: diethyl ether:glacial acetic acid (70:20:4). Cho- lesterol, mono-, and dimyristin, and tripalmitin were used as neutral lipid standards. Bands were detected by spraying either with water or 0.01% Rhodamine 6G in acetone and viewed under UV light; they were removed by succes- sive treatments with hot methanol and twice with hot C:M (2:1) with centrifugation. After concentration of the combined supernates, the residues were counted. Ca. 50 mg polar lipids were applied from C:M (9:1) to a DEAE-cellu- lose column. After successive passage of 20 CV, each of C:M (9:1) and C:M (7:3), the combined eluates were concentrated, and the dried res- idues were weighed and dissolved in C:M (2:1). An aliquot was assayed for radioactivity, and another was analyzed by thin layer chromatog- raphy (TLC) in the same manner as used for the

METABOLISM 515

neutral lipids, except that the solvent system was c h l o r o f o r m : m e t h a n o l : c o n e . NH4OH (65:25:4). The polar lipid standards were cho- line lysophosphatide, sphingomyelin, sefine-, choline-, and ethanolamine phosphatides and cerebroside. Bands, detected by spraying with water, were isolated and counted in the same manner as described for the neutral lipids.

RESULTS

Optimum age, concentration of components, and choice of buffers were determined prior to investigating the pattern of lipid metabolism. The greatest difference in incorporation of 14C_acetat e into the total lipids of brain slices incubated in the presence and absence of both dbcAMP and theophyltine occurred in rats 7-10 days old (Table I). The effective concentration of dbcAMP was determined by varying its levels in the incubation medium through a range of .001 mM-10 mM. Although 0.1 mM was ade- quate in many cases, for purposes of certainty, the working concentration was maintained at 1 mM. The results obtained with cAMP were not consistent and reproducible. In similar manner, the opt imum concentration of theophylline was found to be 1 raM. In a comparison of buffers in the absence of dbcAMP and theophylline, the u t i l i z a t i o n of isotope with 0.25 M Na2HPO4, pH 7.4, was as effective as Krebs- Ringer bicarbonate, whereas only 30% of the activity was obtained with 0.154 M sodium cit- rate, pH 7.4. Variation of the pH by ca. 1 unit below or above pH 7.4, eg. 0.154 M hepes, pH 6.4, 0.154 M glycylglycine, pH 6.6 or 0.154 M Tris, pH 8.4 sharply reduced the utilization to 10-15%.

The effects of various ions and compounds upon the incorporation of 2-14C-acetate into the total lipids of brain slices also were studied. The experimental conditions were the same as those described in the legend of Table I, except for the presence of dbcAMP and theophylline in both control and experimental flasks and for the addition or omission of the compounds under examination. CsC1, LiC1, RbC1, and NaF, each at 100 mM concentration, reduced from 23 to 68% the incorporation of isotope into the total lipids. In a typical experiment, in the pres- ence of Li +, the lipids obtained from control and experimental determinations contained 91 x 103 cpm and 30 x 103 cpm, respectively, which represented a reduction in isotope utili- zation of 67%. The effect shown by Li + was ca. 3 ,times greater than that of Cs + and Rb + which gave reductions of 23 and 24%, respectively. Fluoride (68% reduction) was comparable to Li +, whereas CN- at 10 mM concentration

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BRAIN LIPID METABOLISM 517

TABLE III

Fractionation of 14C-Labeled Neutral Lipids from Rat Brain Slices a

Neutral Lipids Mono- and applied recovered diglycerides Cholesterol Trig|ycerides

cpm Substrate 10 mg

Percent b cpm Percent c cpm Percent c cpm Percent c

2-14.Acetat e Control 192,600 83 60,750 38 70,340 44 28,770 18 Experimental 181,000 87 88,180 56 55,115 35 14,1"/0 9

U-14C-D-Glucose Control 62,100 95 11,200 19 27,700 47 20,050 34 Experimental 43,500 88 15,310 40 15,690 41 7,2'70 19

14C.DL_Serine Control 15,400 90 5,260 38 6,090 44 2,490 18 Experimental 10,900 90 5,980 61 2,840 29 980 10

aThe neutral lipids obtained by silicic acid chromatography of the total lipids were fractionated into their components by thin layer chromatography. (See text for details.)

bRepresents the sum of the radioactivities of all fractions divided by the total radioactivity applied to the thin layer plate.

eRepresents the percent of total radioactivities of all components.

closed down the cell a lmost comple te ly (97% reduct ion) . The inhibi t ion of 14 C uptake into the total lipids by Cs +, Li +, and Rb + was slight- ly tess when dbcAMP and theophyl l ine were omi t t ed f rom b o t h cot ro l and exper imenta l flasks. Little or no effect was observed with these ions at 10 mM concentra t ion. The aggre- gate omission of K +, Ca +2, and Mg +2 from the Krebs-Ringer bicarbonate solut ion reduced the isotope uptake by 31%, whereas their individual effects were variable; a response of 70% was given by e thy lenediamine te t raacet ic acid (EDTA). DL-Epinephrine (0.1 raM) iodoace ta te (5 mM) and puromycin (2.5 mM) inhibi ted the isotope ut i l izat ion by 50, 45 and 30%, respec- tively.

The wt recovered in the neutral and polar lipids was ca. 100 • 3% of the total lipids, whereas the to ta l radioact ivi ty recovered in these fractions was ca. 100 • 10% (Table II). The wt of the to ta l and neutral lipids in the controls f rom all three 14C-precursors were slightly greater than those in the exper imenta l determinat ions , whereas the corresponding wt differences in the polar lipids were reversed but to a smaller ex ten t . In all instances, the specific activities of the controls were greater than their corresponding experimentals . The percent de- crease in incorpora t ion of i sotope be tween control and exper imenta l determinat ions wi th all of the substrates was greater than the corresponding changes in lipid wt, except for the neutral lipids f rom ~4C-acetate. The per- centage of neutra l and polar lipid wt f rom 14C-acetate and f rom 14C-DL-serine were dis- p ropor t iona te with the radioactivit ies in these

fract ions. The percentage of radioactivi t ies in the neutral and polar lipids of b o t h controls and exper imenta ls f rom U -14C-D-glucose corre- sponded wi th their respective wt percentages.

The radioact ivi ty recovered in the TLC sepa- rations of the neutral l ipid componen t s was 83-95% of that applied to the plates (Table III). The radioactivit ies of the choles terol and tri- glyceride controls derived f rom all 3 precursors were 20-65 percent greater than their corre- sponding exper imentals . The mon0- and diglyc- erides, however , showed a reversal wi th the ex- perimentals showing 10-30% greater act ivi ty than the controls. Generally, the largest and smallest f ract ions were choles terol and triglyc- erides, respectively.

The radioact ivi ty recovered in the TLC sepa- rations of the polar l ipid componen t s was in the same range, 80-89%, as that obta ined for the neutral lipids (Table IV). The radioactivit ies of the controls of all l ipid componen t s ob ta ined f rom ei ther of the 3 14 C-precursors was 20-60% greater than their corresponding exper imenta l s ; the except ion was the cerebroside derived f rom 14C_DL_serine" The largest componen t was cho- line phosphat ide; f rom 65-86% of the to ta l ra- dioactivi ty recovered was accounted for by bo th choline- and e thano lamine phosphatides.

DI SCUSSI ON

The increase or decrease in the rate of cellu- lar metabol i sm elici ted by many hormones is media ted by cAMP. The gray mat te r of the cen- tral nervous system f r o m mammal ian tissues has one of the highest levels of adenyl cyclase and

LIPIDS, VOL. 9, NO. 8

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phosphod ie s t e r a se (16) . In ra t b ra in ce rebe l lum slices, the adeny l cyclase r ecep to r s are no t de- t ec tab le for at least the first 3 days p o s t p a r t u m (17) and are local ized pr imar i ly in the mi ta- chondr ia l and mic rosoma l f rac t ions w i th the highest specif ic ac t iv i ty in t hose s ub f r ac t i ons con ta in ing nerve endings (18) . The grea tes t e f fec t shown by dbcAMP on the suppress ion of l ipid synthes is in the p resen t e x p e r i m e n t s (Ta- ble I) did no t coincide w i t h the peak per iod , 14-21 days, of mye l ina t i on . In the absence of d b c A M P (con t ro l s ) , t he bes t p r epa ra t i ons s h o w e d on ly 9.0% iso tope u t i l i za t ion . Since cAMP is re la t ively i m p e r m e a b l e to the cell, as are m a n y p l io sphory la t ed c o m p o u n d s , and sub- jec t to rapid hydro lys i s by phosphod ie s t e r a se , a t t e m p t s to o b t a i n slices or h o m o g e n a t e s act ive in the presence of cAMP were unsuccessfu l .

A l t h o u g h the ion ic e n v i r o n m e n t in f luences the f o r m a t i o n and ac t ion o f cAMP, as well as a f fec t ing r eac t ions n o t m e d i a t e d by cAMP, gen- era l iza t ions as to the ef fec ts of ions on c o m p l e x sys tems, such as b ra in slices, are diff icul t . I t appears t h a t Cs § Li +, a n d Rb + exer t t he i r ef- fects u p o n l ip id m e t a b o l i s m i n d e p e n d e n t of the presence of exogenous dbcAMP, a l though , pos- sibly, these ions may act via t he cell 's endoge- nous cAMP. The ef fec t of F-, wh ich s t imula tes adenyl cyclase, as well as t h a t of CN- and iodo- ace ta te , mos t l ikely resul ts f r o m the i r roles as e n z y m e inh ib i t io rs . E p i n e p h r i n e and p u r o m y - cin, b o t h of w h i c h increase cAMP, e l ic i ted re- sponses cons i s ten t w i t h the i r k n o w n behav io r in l ipolysis (10) . O the r agents e x a m i n e d for the i r e f fec ts on 2-14C-aceta te u t i l i za t ion were cho- late, de soxycho la t e , glucagon, and insul in , all of which y ie lded var iable results . T heophy l l i ne alone showed an e f fec t b u t m u c h less t h a n in the presence of dbcAMP.

In spite of the e x p e c t e d d i f fe ren t ia l loss of l ipid due to leakage dur ing p r epa r a t i on of the slices, the recoveries of t o t a l l ip id (Table II) in the con t ro l s were cons i s t en t ly greater t h a n the expe r imen ta l s by 4-9%. The r e d u c t i o n in iso- tope u p t a k e p r o d u c e d by dbcAMP occur red t h r o u g h o u t all c o m p o n e n t s , excep t the m o n o - and diglycerides, of the neu t r a l and po la r frac- t ions , a l t h o u g h to d i f fe ren t e x t e n t s (Tables III , IV), thus , suggest ing t ha t cAMP may be invo lved in the synthes i s of l ip id by regula t ing the u t i - l iza t ion of ace ty l - coenzyme A (CoA) . Tha t the decrease in i so tope c o n t e n t and l ipid wt af- f ec ted by dbcAMP is n o t due to inc reased util i- za t ion b u t r a t h e r to decreased i n c o r p o r a t i o n was shown by the u n c h a n g e d CO2 p r o d u c t i o n dur ing i n c o r p o r a t i o n of 2-14C-aceta te i n to cho-

METABOLISM 519

les terol and f a t t y acids by ra t l iver slices (9) . Since n o discrete b a n d for ser ine p h o s p h a t i d e was obse rved in the TLC, if p resen t , some m a y have been processed a long w i t h the sph ingo- mye l in f rac t ion . The h igher values f o u n d for the m o n o - and diglyceride f r ac t ions in the ex- pe r imen ta l s der ived f r o m all 14C-precursors (Table I I I ) are at var iance w i th the s u r r o u n d i n g data, and any e x p l a n a t i o n would be speculat ive . A l t h o u g h the ef fec ts exe r t ed by cAMP may vary wi th in the b ra in ' s h e t e r o g e n e o u s cell popu- la t ion , i t appears t h a t i t may med ia t e a ma jo r role in cell m e m b r a n e synthes is , as well as regu- late the ra te and i n t e n s i t y of n e u r o n a l act iv i ty .

ACKNOWLEDGMENT

This study was supported in part by Public Health Research Grant NB06300-07 from the National Insti- tute of Neurological Diseases and Stroke.

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[ Received December 28, 1973]

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