Effect of (-)-hydroxycitrate on fatty acid synthesis by rat liver in vivo.

Abstract Incorporation of 3H from 3H2O was used to measure the rate of fatty acid synthesis in rat liver. (—)-Hydroxycitrate strongly inhibits fatty acid synthesis in vivo.

* This work was supported by a grant from the National Science Foundation.
It is Publication 751 from the Graduate Department of Biochemistry, Brandeis University. The technical assistance of E. Gorin in part of this work is acknowledged with thanks. 1 (-)-Hydroxycitrate is n,n,-hydroxycitrate, or its equivalent erythro-n,-hydroxycitrate.
isotonic NaCl. Most of these injections were given intraperitoneally, but some administrations were made into the tail vein for comparison. After 45 min each rat was given 0.2 ml of a solution containing about 1 mCi of 3HsO by injection into a tail vein, The solution had a pH of about 7.4, and was made approximately isotonic by addition of sodium chloride. The rats were killed by decapitation 45 or 60 min after administering 8H~0, blood was collected, and the liver was excised quickly and weighed. Immediately thereafter the liver was subjected to one of two treatments. It was either dropped into 19 ml per g of liver of chloroform-methanol (3: 1, by volume) and homogenized with a Sorvall Omni-Mixer homogenizer (an overhead type of blender), or it was wrapped into aluminum foil and pressed quickly between two blocks of Dry Ice. In the latter case the liver was stored at -20" and homogenized in chloroform-methanol at a later date. Duplicates of either 10 or 20 ml of the clear chloroform-methanol extract were evaporated to dryness and saponified with 2 ml of 5 N NaOH at 90" for 2 hours.
Acidification and extraction were carried out as described previously (3). The extracted fatty acids were counted in 0.4% 2,5-diphenyloxazole and 0.01% 1,4-bis[2-(5-phenyloxazolyl)]benzene in toluene. The specific radioactivity of the body water of each animal was determined by counting a suitably diluted aliquot of its plasma. Aqueous samples were counted in the scintillator described by Bray (4) ; the results so obtained were converted to the all toluene scintillator mentioned above. In several experiments the radioactivity of the nonsaponifiable fraction was examined. It contained less than 5% of the total radioactivity incorporated into fatty acids.
The aH~O method for studying fatty acid synthesis described above is based on work of Fain and Scow (5). According to Jungas (6), the SHsO method yields an average of 0.87 atom of *H incorporated per carbon atom incorporated into palmitate. The isotope effects, exchange reactions, and the metabolic pathways which lead to this particular fraction have been discussed in detail by Jungas (6), and need not be repeated here. Suffice it to say that the isotope effects and exchange reactions which occur are approximately constant and are not of practical consequence to the present paper. In what follows results are expressed as micromoles of 8H~0 incorporated into long chain fatty acid per g of fresh weight of liver per hour. This can be converted to micromoles of acetyl group incorporated by dividing by 0.87 (or multiplying by 1.15).
Estimates of rates of fatty acid synthesis in v&o have been obtained in a number of ways. Most commonly, the rate of incorporation of a labeled precursor such as "C-acetate or 14C-  I  I  I  I  I  I  I   II  I  I  I  I  I  IO  20  30  40  50  60  70  00  90   TInI<  after  Inlectlng  shown are from a single experiment. Each rat received 0.2 ml of 3Hz0 in isotonic NaCl solution into a tail vein 3.5 to 5 hours after starting the last feeding schedule. This approach is open to serious criticism because it provides only a measure of the rate at which a particular radioactive precursor is incorporated into fatty acids. This is a fraction of the total rate, a fraction which may vary depending on the pool size of the precursor in question and on the relative rates of removal of the precursor via other metabolic pathways. By far the best estimate of the total rate of fatty acid synthesis is obtained by the use of tritium-labeled water as radioactive precursor (48), since the incorporation of this precursor is largely independent of the source of the acetyl groups which are incorporated into fatty acids.
Materials-I am greatly indebted to Mr. Y. S. Lewis for a generous gift of (-)-hydroxycitric lactone. This material was prepared as described previously (9). The lactone was hydrolyzed to hydroxycitrate by heating with 3 eq of NaOH at 80" for 30 min.

RESULTS
In the experiment shown in Fig. 1 the rate of fatty acid synt,hesis in rat liver was measured 3.5 to 5 hours after starting the last feeding schedule.
It is seen that the rate is proportional to time for at least 90 min after injecting zHzO. The rate reaches a maximum within 2 hours after the start of the a-hour feeding schedule.
It remains at or near maximum for the following 3 hours and then slowly decreases (Fig. 2). The points show the mean, the standard error, and t.he nnmbcr of animals per point.. The solid point denotes control animals which received isotonic N&l in place of (-)-hydroxycitrate. The hollow pink show animals which received hydrosyc*itrate.
In the case of animals which received inhibitor and 3H20 at zero time, the animals were first given (-)-hytiroxycitrate intraperitoneally and t.hen SH,O intravenously within 30 to 60 sec.
An intraperitonesl dose of as little as 0.1 mrnole per kg of body weight inhibits fatty acid synthesis by 25 to 300/& This is equivalent to about 2.9 mg of hydr0xycitrat.e per 150-g rat.
Fifty per cent inhibition is obtained with 0.28 mmole per kg of body weight (Fig. 3). A possible esplanation for that results shown in Fig. 3 is that hydroxycitrate causes the removal of rlewly synt.hesized fatty acid from the liver without, inhibiting fatty acid synthesis.
To test this possibility the carcass less the liver was analyzed for 3H20 incorporation into fatty acids in the presence and absence of (-)-hydroxycitrate. The results showed that fatty acid synthesis is inhibited to about the same extent in the carcass as in the liver.
The inhibiting effect of hydroxycitrate comes into play quite quickly, being at or near maximum within a matter of minutes aft.er (-)-hydroxycitrate is given intraperitoneally (Fig. 4). In the experiment shown, fatty acid synthesis was measured over a period of 45 min.
This suggest.s that immediately after giving hydroxycitrate t.he rate of fatty acid synthesis may have been normal, but that it must then have decreased rapidly as the inhibitor was taken up by the liver cells. The longest interval between administering hydroxycitrate and starting the mensure-me& of fatty acid synthesis shown in Fig. 4 was 50 min. Here too, the measurement of the rate of fatty acid synthesis lasted for 45 min.
This means that the inhibition of fatty acid synthesis remained strong between 50 and 95 min after administering hydroxycitrate.
Four additional experiments also showed that the inhibition of fatty acid synthesis remains strong 2 to 3 hours after giving hydroxycitrate. DISCUSSIOS (-)-Hydroxycitrate is a very strong inhibitor of citrat.e cleavage enzyme (I) and of fatty acid synthesis in vitro (10). The presrnt paper shows that (-)-hydroxycitrate is also an excelknt inhibitor of fatty acid synthesis in z&o.
The Ki of (-)-hydroxycitrate in the reaction catalyzed by citrate cleavage enzyme is between 0.2 and 0.6 C(M depending on conditions (1). The amount of (-)-hydroxycitrate required for 50% inhibition of fatty acid synthesis in uioo, namely 0.28 mmole per kg of body weight, is roughly 700 times larger than the K; for citrate-cleavage enzyme.
It seems likely that one of the factors limiting the effectiveness of (-)-hydroxycitrate in ctio is its passage across the cell membrane.
A possibility which was discussed previously (lo), and which should not be overlooked here, is that the inhibition of citrate cleavage enzyme by (-)-hgdroxycitrate in &o switches on a pathway for the generation of extramitochondrial acetyl-(loA which does not involve citrate. Such a pathway would circumvent the inhibited citrate cleavage reaction and this would manifest itself as an apparently lower rffectivenrss of (-)-hydroxycitrate than might otherwise be expected. There is at present no evidence favoring such an alternate pathway in nonruminant mammals. Another possibility which should be considered is that (-)-hydroxycitrate inhibits the transfer of citrate from tllc mitochondrial mat.rix into the cytoplasm.
Experiments in which the excretion of citrate formed in liver mitochondria was measured in the presence and absence of (-)-hydroxycitrste suggest that this type of inhibition does not occur, or if it occurs that it cannot bc large (10).
Measurements by l)rs. G. It. W'illiams and IS. II. Robinson show that (-)-hydroxycitrate does not, exchange with intramitochondrial citrate, and that. it does not inhibit the egress of citrate from mitochondria previously loaded with labeled citrate. 2 When the dose of (-)-hydroxycitrate was 1 mrnole per kg of body weight the inhibition of fatty acid synthesis was about 75% (Fig. 3). The control rate of fatty acid synthesis was about 60 pmoles of 3H20 incorporated into fatty acids per g of liver per hour. This is equivalent to 69 prnoles of acetyl group incorporated per g of liver per hour, or 8 pmoles per liver per min (assuming t.he liver to weigh 7 g). An inhibition of 757, of this rate corresponds t.o 6 pmoles of acetyl group per liver per min being prevented from going into fatty acids. In other words, this amount of carbon must be channeled into alternative products.
The nature of these products is presently under investigation.