BIOCHIMICA ET BIOPHYSICA ACTA 435 BBA 55269 FATTY ACID BIOSYNTHESIS II. FATE OF FATTY ACIDS SYNTHESISED BY SUBCELLULAR FRACTIONS OF LACTATING-RABBIT MAMMARY GLAND* STUART SMITH** AND R. DILS Department of Medical Biochemistry and Pharmacelogy. University of Birmingham, Birmingham (Great Britain) (Received June 15th, 1966) SUMMARY I. Subcellular fractions of lactating-rabbit mammary gland have been incu- bated under optimum conditions for fatty acid synthesis and the nature and subcel- lular site of the products examined. 2. When “microsomal + particle-free supernatant” fractions were incubated with labelled acetate, a large proportion of the radioactivity was associated with newly synthesised glycerides in the microsomal fraction. Radioactive unesterified fatty acids were found to be located in the particle-free supernatant. 3. Particle-free supernatant fractions alone synthesised exclusively unesterified fatty acids. 4. Though both the mitochondrial and microsomal fractions bind synthesised fatty acids, the microsomal fraction stimulated both fatty acid and glyceride syn- thesis to a greater extent than did the mitochondrial fraction. 5. It has been shown that whereas certain free fatty acids inhibited fatty acid synthesis, the corresponding glycerides had little inhibitive effect. 6. The results support the view that the microsomal stimulation of fatty acid synthesis is due, at least in part, to a relief of the feedback inhibition excercised by the products of fatty acid synthesis. INTRODUCTION The microsomal stimulation of fatty acid synthesis by soluble extracts of a number of tissues has been well documented - 1 lo. That the microsomal fraction might convert inhibitory products of fatty acid synthesis to complex lipids has proved a popular theory of microsomal action. HOWARD AND LOWENSTEIN~~ found that 3-gly- cerophosphate stimulated fatty acid synthesis in extracts of rat liver only in the * No. I in this series was ref. IO. ** Present address, Department of Biochemistry, Hadassah Medical School, P.O. Box I 172, The Hebrew University, Jerusalem, Israel. Biochim. Biophys. Acta, 125 (x966) 435-444
Transcript
BIOCHIMICA ET BIOPHYSICA ACTA 435
BBA 55269
FATTY ACID BIOSYNTHESIS
II. FATE OF FATTY ACIDS SYNTHESISED BY SUBCELLULAR
FRACTIONS OF LACTATING-RABBIT MAMMARY GLAND*
STUART SMITH** AND R. DILS
Department of Medical Biochemistry and Pharmacelogy. University of Birmingham, Birmingham (Great Britain)
(Received June 15th, 1966)
SUMMARY
I. Subcellular fractions of lactating-rabbit mammary gland have been incu- bated under optimum conditions for fatty acid synthesis and the nature and subcel- lular site of the products examined.
2. When “microsomal + particle-free supernatant” fractions were incubated with labelled acetate, a large proportion of the radioactivity was associated with newly synthesised glycerides in the microsomal fraction. Radioactive unesterified fatty acids were found to be located in the particle-free supernatant.
4. Though both the mitochondrial and microsomal fractions bind synthesised fatty acids, the microsomal fraction stimulated both fatty acid and glyceride syn- thesis to a greater extent than did the mitochondrial fraction.
5. It has been shown that whereas certain free fatty acids inhibited fatty acid synthesis, the corresponding glycerides had little inhibitive effect.
6. The results support the view that the microsomal stimulation of fatty acid synthesis is due, at least in part, to a relief of the feedback inhibition excercised by the products of fatty acid synthesis.
INTRODUCTION
The microsomal stimulation of fatty acid synthesis by soluble extracts of a number of tissues has been well documented - 1 lo. That the microsomal fraction might convert inhibitory products of fatty acid synthesis to complex lipids has proved a popular theory of microsomal action. HOWARD AND LOWENSTEIN~~ found that 3-gly- cerophosphate stimulated fatty acid synthesis in extracts of rat liver only in the
* No. I in this series was ref. IO. ** Present address, Department of Biochemistry, Hadassah Medical School, P.O. Box I 172, The Hebrew University, Jerusalem, Israel.
Biochim. Biophys. Acta, 125 (x966) 435-444
pence of the microsomal fraction. ALw wing rat-liver preparations, LOMX. ~RARAY AND CHAIKQW* found that addition of the microsomal fr&ion increased the proportion of both glyceride and phmphohpid synthesised from acetate.
‘She work described here was urdetiaken to test this theory in the case of lxtating-rabbit mammary gland and in particular to determine whether those fatty &Is show3 to inhibit fatty a,cid aynth&s were remwed by the micresomal f&x&on.
The sources of tnabxials have been &scribed lA. exaspt for the silicic acid (roa mesh A.R.. Ratch s&+7) which was obtained from Mallinckxodt Chemical Works, U.S.A. and alumiaium oxide (“Carnag” M.F.C., 100-200 mesh) which was manufac- turexl by CAMAG Chemie-Erzeuguisse wnd Absorptioustec&uik of Mettens. SW&W- land, for HopI& and %%&xrns, Er~glartil.
Preparation of lactating-rabbit mamlmw-gland homogrxates and sub-cellulsr fracizions has been described*~~~. Ia the text. Mit = mitochondria3 fraction, Mic = r&osomal fraction and Sup = par&lo-free. stqmnatant.
Incubations contained (final volume r,o mIf:r~o m.M potassium phasphtlte {pH &6), IO m&I sediunx [r-=C@cetate (2.22 - IO* disint.~mb.& 0.15 mM CoA, 25 mH ISHCUr 6.6 mgii sodium citrate, 3.3 mkf MuC19, 0.2 m&I NABP* aud x.5 m&f glucos$ &phosPbate. Incubations with particle-free superxatant fractious contained 5.0 mM ATP, thase with particulate fractions ccmtied 15-o mM ATP. A&?F incubating for 1 b at 3~“~ incubation systems were quickly cooled to oQ and &.luted to a volumr+ of 5.0 ml with 0.3 M s~crese,
Each diluted incubation system was centrifuged to give a 5.0 - ro* x Q * miar pediment {T), a 5.2 - xd x g - min c+et&mnt (IX) and a 6-z - rod x g * min supematant
(III). These valu@ are the same as those used for the initial preparation of the Nit, Mic and Sup fractions. As no attempt wus made to charact&e the post-itrcuba- tionaf fractions, they will be refermd to by their sedimentation characteristics and not as mitochondrial, microsomal and supernatant fractions.
Total lipi& were extra&xl by a; method based on that dexr~bed by l?i?~X, LEES AND %4?AXf-%%NLEY “. 5 vd. chloraform-methanol (2 : X, V/v) were added tcJ each sample and tra&erred to a. separat.ia$ funnel. Each fla& ww washed out with ~5 vol. chiorofo~~~e~au~~water fa : I : ad, Y Iv) and the wa.&ings transferred to the separating funnels. 0.2 vol. “upper phase”* were then add& and the funnels shaken far z min, The contents were centrifuged and the lower layer plus interfacial flti dried over anhydrous MgSO,. After removal of the MgSO, by centrifugstion,
FATE OF SYNTHESISED FATTY ACIDS 437
the extracts were taken to dryness under vacuum at 60” and redissolved in 40-60” light petroleum.
Alumina chromatogra$hy Total lipid extracts from incubation systems were fractionated on aluminum
oxide by a technique based on the method of TRAPPE’~-‘~, as described by CLARK
AND H~~BSCHER~'.
A slurry of 13.4 g aluminium oxide (100-200 mesh} in 40-60” light petroleum was poured into a glass column (1.x cm diameter) and allowed to settle. Lipid ex- tracts were applied to the columns in 6 ml of 40-60” light petroleum and washed down with 5-10 ml 40-60” light petroleum. Hydrocarbons were eluted with 50 ml carbon tetrachloride*, tri-, di-, and monoglyceride plus cholesterol with 60 ml chloro- form, and choline-containing phospholipids with 60 ml chloroform-methanol (I : I,
v/v). The material remaining on the column after elution with these solvents was assumed to contain free fatty acids. In some cases, portions of the eluates were re- tained for gas-liquid radiochromatography l8 and silicic acid chromatography.
This technique, based on the method of BARRON AND HANAHAN~*, was used
to separate 14C-labelled mono-, di- and triglycerides synthesised by subcellular frac-
tions. 30 g silicic acid was washed according to the method prescribed by BARRON
AND HANAHAN~~, slurried in n-hexane (redistilled) and transferred to a glass column (2.5 cm diameter). Solvents were applied to the column under a positive nitrogen pressure, Neutral lipids were fractionated according to the following scheme: Sterol esters were eluted with 150 ml benezene in hexane, 15 T$ (V/V) ; triglyceride #ZGS un- esterified fatty acid with 150 ml diethyl ether in hexane, 5% (v/v); free sterol with 150 ml diethyl ether in hexane, 15% (v/v); diglyceride with I50 ml diethyl ether in
hexane, 35 y. (v/v) and monoglyceride with 150 ml diethyl ether.
SCHEME OF POST-INCUBATIONAL PROCEDURE
Procedure Fractions used Fractious obtained
Subcell&r fractionation Post-incubational systems
(
5.0 . 10~ x g . min sediment (duplicates)
(I) 6.2 . 10~ x g + min sediment (11) 6.2 . IO6 X g I min supernatant (111)
No subcellular fractionation Post-incubational systems Intact fractions o (duplicates)
Chloroform-methanol o, I, II and III extraction
Total lipids
Alumina chromatography Total lipids f
Phosphatidyl choline (glycerides and cholesterol)
Saponification, acidification, o, I, II and III extraction, radiochromato-
Individual radioactive fatty acids
graphy and liquid scintilla- tion
* Stored over aluminium oxide to remove sulphur compounds.
Biaclaim. Biophys. Acta, 125 (1966) 435-444
438 S. SMITH, R. DILS
RESULTS
Incubations were carried out in quadruplicate and analysed according to the scheme shown above. After incubating at 370 two incubations were subjected to the “subcellular fractionation” procedure to give Fractions I, II and III. The other two incubations were not subjected to subcellular fractionation (intact Fraction 0). Chlo- roform-methanol was used to extract total lipid from portions of Fractions o, I, II and III. Alumina chromatography of these total lipid extracts separated phos- phatidylcholine from (glycerides+cholesterol), leaving free fatty acids on the column. The remaining portions of Fractions o, I, II and III were saponified and acidified and the total radioactivity of the fatty acids determined by liquid scintillation. The radioactivity of the individual fatty acids was determined by gas-liquid radio- chromatography.
Additional analysis of the glyceride fraction by silicic acid chromatography was carried out only on the glyceride fraction isolated from the Mic Sup system which had not been subjected to the post-incubational subcellular fractionation.
Zero-time incubation blanks were carried through the entire scheme (except gas-liquid radiochromatography) and thus the values for unreacted [rJ4C]acetate were subtracted from the appropriate determinations. All results are given as average of the two duplicates f the deviation from the mean, except for gas-liquid radio- chromatography data which are the results of single determinations.
Table I shows the recovery of radioactive fatty acids synthesised by different combinations of the cell fractions. The most active fatty acid-synthesising system was that containing the microsomal and supernatant fractions. The Mit plus Sup system incorporated about the same amount of acetate as did the Sup fraction alone. The Mit plus Mic plus Sup system was slightly less active than the Mic plus Sup
system. Subcellular fractionation of the incubation systems revealed that, in those
which included a microsomal fraction, a large proportion of the 14C-labelled fatty acids was recovered in the 6.2 - xo6 x g * min sediment (Fractions II).
It appeared as though breakdown of the Mit fraction occurred during the in- cubation of the Mit plus Sup system as shown by the ‘Glabelled fatty acid sedi-
mented at 6.2 * 10~ x g. min (Fraction II). Alternatively, this radioactivity may have been associated with microsomal contamination of the original mitochondrial fraction.
As has been shown beforesylO, the Mic Plus Sup sytem synthesised a larger proporation of long-chain fatty acids than did either the Sup fraction alone or the Mit plus Sup system.
In both the Mit @us Sup and the Mic plus Sup incubation systems, most of the synthesised CaEo was located in the 6.2 . 10~ x g . min supernatant (Fraction III), whereas most of the radioactive long-chain fatty acids C,a:o-C1s:o were recovered from the particulate fractions I and II. The variation in recoveries of total radio- activity after “post-incubational” subcellular fractionation may well be due to the difficulties in dealing with the very small amounts of material present (e.g. about I mg particulate protein).
Results of the alumina chromatography carried out on lipid extracts of the incubation systems are shown in Table II.
Biochk Biophys. Acta, 125 (1966) 435-444
FATE OF SYNTHESISED FATTY ACIDS 439
SUBG~LLULBIC DTSTEIBUTICIN OF l*C-~~~~~~~~ PATTY ACIDS SYXTHESISED FROM [I-K]ACETATE
The ratio of Mit : Mic : Sup protein in the nuclei-free homogenate was about x.5 : x.5 : 4.15. Fraction L = 5 ’ 10~ x g * II-&I sediment, II = 6.2 * fo6 x g * min sediment, III = 6.2 + IO& x g * min supernatant {see SCHEME OF E’QST-INCUBATION PROCEDURES).
Fr&ions Total cccetatc: Past-incubationsl -4cetate inco@ovaled per fatty acid (disint.jmin in.cubuted incorporated Jraction isdatd x IO-~)
t Tzris represents the total activity recovered from the unfractionated incub&ions (see SCHEME OF PSXT- INCW3ATfONAL PRQCEDURES).
Comparing subcellular systems not fractionated after incubation, 36% of the radioactivity from the Mic @us Sup system was located in the glyceride fraction*. In contrast, only so/a of the activity incorporated by the Sup fraction alone was recovered in the glyceride fraction, i.e. the Mic $JEZGS Sup system incorporated 50 times as much acetate into the glycerides fraction as did the Sup fraction alone. Although ~5 yO of the acetate incorporated by the Nit $tzts Sup system was located as glyceride, this represented only one quarter of the total acetate incorporated into glycerides by the Mic $ki Sup fraction.
Subcehular fractionation of incubation systems containing particulate fractions showed that most of the 14C-labelled glycerides sedimented at 5.2 * IO@ x g * min supematant (Fraction III). Incorporation of acetate into choline phospholipids ap- proximately paralleled that into glycerides, but at a much lower level.
Separation of the radioactive glycerides synthesised by the Mic p&s Sup system was carried out on silicic acid columns. These glycerides were obtained by alumina chromatography (marked** in Table II). Unfurtunately, a pour separation of sterof and diglyceride was obtained, but the ratio in this sample of triglyceride : (stem1 + diglyceride) : monoglyceride was 2.4: J : oay. When, after saponification, incubation
* Calculated as tatal glyceride radioactivity from unfractionated incubation system : total
acetate radioactivity incorporated = ??.? x xaaoj6. 8705
IG.M.&, $3c3rthns t3f a-se glJKer& and p3mqs-A~ !zhlaa (GWed by ah- mina chromat~phy of the WC $ks Sup system) were saponj&d, the fatty a&% extracted and examtied by gas-liquid radiochromatography, Wkr-e;as only about 27 and 55% respectively of the C,:, and C a: O recovered from Fraction II was esterified, all the C,:,C,,:,t was recovered in the glyctida #MS choline phospholipid fractions (Table III). However, most of the radioactivity recovered from Raction III was associated with unesteS& fafty acid.
Bdoeklm. Siop&~s. A&, lra~ (~966) 435-444
FATE OF SYNTHESISED FATTY ACIDS 441
TABLE III
CHAIN-LENGTH OF FATTY ACIDS E~CORPORATED INTO GLYCERIDES AND PHOSPH~LIPIDS BY THE MICK~S~MAL @us SUPERNATANT FRACTIONS
Fractions used are marked in Table II by *. Figures in parentheses represent the percentage of the total activity. __---- Post- Lipid from Radioactivity reccvered in each fatty acid ~d~si~~.~rni~ x I@)
inc~~a~i~a~ a~~rn~na COhWM
$:o 6:o 8:0 IO..0 13:o I‘#..0 16:o Total fraction ___ 6.2 . 10~ x g. min glyceride 43 sediment i.e. (2.2)
Fraction II choline phospholipids
(:) Total radioactivity as esterified fatty acid 43
Total acetate incorporated 157 (see SCHEME)
6.2 . IO@ x g * min glyceride supematant i.e. (;
Fraction III choline phospholipids Total acetate incorporated (see SCHEME) 840
109 (5.5)
(0; 109
198
552
‘13 713 (5.7) (36)
(i?o) 122
117
(51:)
145 338
575 342 (29) (17)
2.59 544
‘49
90 2129
=?o 2610
(51.:)
208
trace
*o9 M37
To determine whether the fatty acids synthesised by the soluble supernatant fraction were present mainly as free fatty acids, acyl-CoA’s or acyl-protein, a separate experiment was devised. Supernatant protein was incubated with malonyl-CoA, NADPH and a [l*C]acetyl-CoA-generating system. The contents of each tube was separated into a free fatty acid fraction (petroleum-soluble), an acyl-CoA fraction (ammonia-solubles and a residial protein fraction as shown in Table IV.
In the absence of any added NADPH, there was very little radioactivity re- covered in any of the fractions. When NADPH was added, but malonyl-CoA omitted, there was a IO-fold increase in the radioactivity recovered in each fraction. When both NADPH and malonyl-CoA were included, there was a large increase in the radio- activity recovered in the free fatty acid fraction.
Although this experiment does not of course exclude the possibility that the fatty acids are synthesised initially as acyl-CoA derivatives or acyl-protein, it does establish the majority of the fatty acid accumulates in the unesterified form.
The final phase of this work was to determine whether fatty acid synthesis in this system was inhibited by unesterified fatty acid and if so, whether conversion of the product to the glyceride form was likely to relieve this inhibition.
LEVY~~ has shown that the C,,: ,,-C,,:, fatty acids are potent inhibitors of rat mammary-gland acetyl-CoA carboxylase (acetyl-CoA:CO, ligase (ADP), (EC 6.4. I.z)), whereas C,,:, and C,,:, had little effect.
Our experiment showed that (a) whereas a long-chain fatty acid inhibited fatty acid synthesis, the corresponding triglyceride has less inhibitory effect (Fig. I) and (b) that in the presence of the Mic fraction, these inhibitive fatty acids could be esterified.
A remarkable point about the results in Table III is the similarity in the dis-
Biochim. Biophys. Acta, 125 (1966) 435-444
TABLE IV
NATDRE OF THE PRODUCT ACClJMULATING DURING FATTY ACID SYNTHESLS IN THE PARTICLE-FREE
SUPERNATANT
Incubations contained (final volume 1.0 ml), IQO mM potassium phosphate (pH 6.6). z mM GSH, I mM NADPH, 0.75 mM malonyl-GoA, 0.17 mM sodium [I-Wjacetate (5 J&Z), IO mM ATP, 0.x5 miM CoASH, 3.5 mM MnC.1, and 0.5 mg particle-free supernatant protein (Sup>. After incubating for 15 min at 3T0, I ml 8% perchloric acid was added and the tubes left at ow for 15 min. After removing free fatty acids by extracting four times with 4 ml 4o-60~ light petroleum, residual acetate was removed from the precipitate by washing thrice with 3 ml 5% perchloric acid. Radio- activity of the washings was monitored by liquid scintillation using Triton X-roo-xylene phosphor, (I:z, v/v). Acyl-CoA’s were then extracted from the precipitate by the method described by LYNEN, HOPPER-KESSIEL AND EGGER~R”“. Blank values obtained from incubatians without pro- tein were subtracted in all cases. Values are mean & S.D., No. of determinations in parentheses. Figures in brackets are per cent radioactivity recovered. - &?&eons ~u~~o~c~~~~~~ i&.&L iw&\
Fig. I. Effect of unesterified and esterified fatty acids on fatty acid synthesis. Incubations con- tained 3.16 mg (Mic+Sup) protein and [~J*C]acetate (4.0 PC). The Mic:Sup protein ratio in the tissue homogenate was z : 2.2, Incubation systems were saponified, acidified, the fatty acids ex- tracted and radioactivity determined. o-0, Tributyrin; A-A, sodium butyrate; O-O, trimy- &tin; x - x , sodium myristate.
tributian of radioactivity in the fatty acids of glycerides and choline phospholipids. As the distribution of radioactivity in the glyceride fatty acids is different from that of the total fatty acids synthesised, there appears to be a degree of specificity for chain-length exhibited by the esterifying system.
Biochim. Biophys. Rcta, 1%~ (x966) 435-444
FATE OF SYNTHESISED FATTY ACIDS 443
DISCUSSION
BORTZ AND LYNENZZ, using a purified preparation of rat-liver acetyl-CoA car- boxylase, found that only acyl-CoA derivatives inhibited the system ; neither albumin- bound fatty acids nor free fatty acids inhibited. We have shown that whereas a long- chain free fatty acid (C,,,,) inhibited fatty acid synthesis from acetate, the corres- ponding triglyceride (trimyristin) had a much smaller inhibitive effect. Our experi- ment (Fig. I) wan performed in the presence of ATP, CoA, Mns+ ions and the micra- somal fraction, so there is the possibility that the added free fatty acid was converted to the acyl-CoA derivative and this was responsible for the inhibition. Although the microsomal fraction can convert myristic acid to glyceride, the amount added fo.z5- 5.0 ,umoles) probably exceeded the esterifying capacity of the microsomal fraction so that the residual myristic acid (or myristyl-CoA) was available to exert an inhihi- tive effect.
ABRAHAM, MATTHEs AND CHAIKOFF~, using rat-liver preparations have shown that although microsomes will bind synthesised fatty acids, the stimulation of acetate incorporat~un by the addition of microsomes to the particle-free supernatant did not depend solely on binding capacity; mitochondria, which did not stimulate fatty acid synthesis, also bound fatty acids to a similar extent. In agreement with these findings, the results presented here show that both mitochondrial and microsomal fractions bound synthesised fatty acids. However, the microsomal fractions stimulated both fatty acid and glyceride synthesis to a greater extent than did the mitochondrial fraction.
If then, the binding of fatty acids by protein does not in itself promote further fatty acid synthesis, what is the mechanism of the albumin stimulation of fatty acid synthesis 7 BORTZ AND LYNEN 22 and recently HIBBITT~~ have shown that acetyl-CoA carboxyIase from rat liver and bovine mammary gland respectively are not inhibited by albumin-bound fatty acids. On the other hand, KORCHAK AND MASORO~~, using
rat-liver preparations have reported that albumin increased rather than decreased the
inhibition {by fatty acids) of lipogenesis from acetyl-CoA. Perhaps the type of Site
on the albumin molecule involved in the binding of the fatty acid is an important
factor here: albumin is known to possess several types of binding site with different afhnities for fatty acids of different chain-length25.
Although we have shown that the presence of the micrasomal fraction caused an increase in the level of glyceride biosynthesis, we have not actually shown that this was accompanied by a decrease in the level of free fatty acid. Therefore it might be argued that the increased level of fatty acid synthesis in the presence of the mi- crosomal fraction was the primary effect and that this consequently resulted in a raised level of glyceride synthesis. Alternatively of course, the increased glyceride synthesis might be the primary effect, relieving the feedback inhibition and increasing the levef of fatty acid s_ynthesis as a secondary effect.
In pursuit of this point we have investigated other possible primary courses of the microsomal stimulation of fatty acid synthesis and in particular have studied the subcellular distribution of the enzymes involved in fatty acid synthesis in the lacta- ting-rabbit mammary gland. The results are reported on in the following paper.
444 S. SMITH, R. DILS
ACKNOWLEDGEMENTS
We thank Professor A. C. FRAZER for his support and encouragement, Mr. R. DAINTY and Miss D. RICHARDS for their excellent assistence with the chromatography, and the Medical Research Council of Great Britain for a grant to purchase the Radio- panchromatograph and for financial support for one of us (S.S.).
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