Effect of AMO 1618 on cholesterol and fatty acid metabolism in chickens and rats

Effect of AMO 1618 on cholesterol and fatty acid metabolism in chickens and rats

Atherosclerosis, 46 (1983) 203-216 Elsevier Scientific Publishers Ireland, 203 Ltd. Effect of AM0 16 18 on Cholesterol and Fatty Acid Metabolism in ...

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Atherosclerosis, 46 (1983) 203-216 Elsevier Scientific Publishers Ireland,

203 Ltd.

Effect of AM0 16 18 on Cholesterol and Fatty Acid Metabolism in Chickens and Rats * Asaf A. Qureshi I, Naji Abuirmeileh **, Warren C. Burger ‘, Zafeer Z. Din ’ and Charles E. Elson ’ I USDA-ARS, Barley and Malt Laboratory, 501 N. Walnut St., Madison, WI 53705 and ’Department of Agronomy, and ’ Department of Nutritional Sciences, University of Wisconsin, Madison, WI 53706 (U.S.A.) (Received 16 July, 1982) (Revised, received 4 October, 1982) (Accepted 4 October, 1982)


AM0 16 18 (2-isopropyl-4-dimethylamino-5-methylphenyl1-piperidine carboxylate methyl chloride) was added to corn-soy based diets and fed to 9-week-old female chickens for 3 weeks to measure the inhibition of hepatic /?-hydroxy-P-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol 7o-hydroxylase. Dose-related decreases in the activities of these two enzymes were obtained (2.5- 15 ppm) of AM0 1618. Decreases in plasma total cholesterol, chol-HDL, and chol-LDL levels were observed, but the decreases in chol-LDL were substantially larger than those of chol-HDL in both chicken and rat. Assays of livers from rats fed 20 ppm AM0 1618 for 3 days had 24% less HMG-CoA reductase activity and 67% less cholesterol 7o-hydroxylase activity than the controls. Plasma cholesterol in these animals was reduced 26%; the ratio of total cholesterol : chol-HDL was reduced from 3.27 to 2.67 and the chol-LDL : chol-HDL ratio was reduced from 1.96 to 1.14 as a result of the relatively brief treatment. Fatty acid synthetase (FAS) and other key lipogenic enzymes increased 1.5-4-fold in both the chicken and rat. The inhibition of HMG-CoA reductase and the induction of FAS by AM0 1618 were tested in vitro, using lo-100 pg (28-280 pmoles) for 15 min with isolated hepatocytes from chicken and rat. Linear responses

This investigation was supported in part by Hatch-funds No. 1718 of the Research Division, College of Agricultural and Life Sciences, University of Wisconsin-Madison. * Cooperation investigation between the Science and Education Administration, U.S. Department of Agriculture, and College of Agriculture and Life Sciences, University of Wisconsin, Madison. ** Present address: Director, Department of Biological Sciences, Yarkmuk University, Irbid, Jordan. ~~21-91~~/83/~-~$03.0

0 1983 Elsevier Scientific





in activity were dose-dependent 85 pmoles AM0 1618, 5-120 pound acts at the cellular level which recommend it for testing Key words:

and increased with duration of incubation (30 pg or min) in both species. The results suggest the comand AM0 1618 appears to possess several properties as a cholesterol-lowering agent in humans.

AM0 1618 - Chicken - Cholesterol 7a-hydroxylase thetase - HMG-CoA reductase - Lipid metabolism growth retardant - Rat

- Fatty acid syn- Liver - Plant


A number of plant products exert a marked influence on metabolic systems of higher animals. These include plant growth retardants (abscisic acid, limonene, carvacrol), coumarin, caffeine, digitalis and, of course, the majority of the vitamins. Sterol biosynthesis in plants and plant growth and development are regulated by natural growth promoters and retardants, some of which are biologically active in animal cell systems [l-3]. These agents have stirred little interst insofar as their eliciting hypocholesterolemic responses in animals. Besides these plant regulators, plants also contain agents which are antagonists of gibberellic acid [4,5]. Water-extracts of plant tissues contain conjugated forms of these growth regulators (e.g. auxins, cytokinins); biologically active free forms are soluble in water, and gibberellic acid is soluble in less polar solvents. Many of the natural retardants are made more potent by the addition of a quaternary ammonium side chain (Fig. 1). AM0 1618 (2-isopropyl-4-dimethyl-


+JI:_I_N~Me:;~o-_N~ Me








I dH*










Fig. 1. Structural formulae of representative






growth retardants from each of the major groups.


amino-5methylphenyl1-piperidine carboxylate methyl chloride), a quaternary ammonium derivative of a natural product, carvacrol, has been reported to be involved not only in the inhibition of sterol biosynthesis in plants, but it is also a very strong growth retardant [6]. It inhibits the cyclization of geranyl-geranyl pyrophosphate for the formation of copalyl pyrophosphate and Kaurene in the biosynthesis of the gibberellins [4]. Furthermore, AM0 1618 has been reported to inhibit HMG-CoA reductase and squalene-2,3-epoxide cyclase in tobacco seedlings [7] and also inhibits at least one step between acetate and mevalonate in cell-free preparations of rat liver [6]. There is no report on the effects of AM0 1618 in vivo or in vitro on subsequent steps in the regulation of lipid metabolism in animal systems, especially on the concentration of plasma cholesterol levels of high density lipoprotein (HDL) and low density lipoprotein (LDL). Plasma cholesterol levels are thought to be directly related to the risk factors for the incidence of atherosclerosis in humans, specifically the cholesterol associated with the low density lipoproteins (LDL). The present study describes in detail the effect of different concentrations of AM0 16 18 in corn-based diets fed to female chickens for 3 weeks on the activities of enzymes involved in cholesterol and fatty acid synthesis and on the levels of cholesterol in plasma, HDL, and LDL. The short-term effect on the above parameters and the key lipogenic enzymes of AM0 1618supplemented chow diet fed to rats is also described. Finally, the inhibitory effects of this compound on cholesterol biosynthesis will be demonstrated in isolated hepatocytes of rats and chickens. Materials and Methods * Experimental materials were purchased from the following sources: acetyl-CoA, malonyl-CoA, RS-mevalonic acid, glucose-6-phosphate, dithiothreitol, DL-isocitrate, NADP+, NADPH, NADH, ATP, glucose-6-phosphate dehydrogenase, cysteamine, Tween-80, triethanolamine hydrochloride, sodium malate, coenzyme A, malate dehydrogenase, nicotinamide, 6-phosphogluconate and DL-3-hydroxy-3-methylglutarylCoA, Sigma Chemical Co., St. Louis, MO; cholesterol, Aldrich Chemical Co., Milwaukee, WI, was recrystallized twice in glacial acetic acid; 7a-hydroxycholesterol (5-cholesten-3P,7a-diol) and 7a-ketocholesterol (5-cholesten-3&ol-7-one) Steraloids, Inc., Wilton, NH; EDTA, Fisher Scientific Co., Itasca, IL; bovine serum albumin, Nutritional Biochemicals Corp., Cleveland, OH; and DL-3-hydroxy-3-methyl-[3“C]glutaryl-CoA (specific activity, 26.3 mCi/mmole), [4-14C]cholesterol (specific activity 50-60 mCi/mmole) and Aquasol (scintillation solution), New England Nuclear, Boston, MA; and AM0 1618, Calbiochem-Behring Corp., La Jolla, CA. All other chemicals were of analytical grade.

* Mention of a trademark or proprietary produce does not constitute a garantee product by the U.S. Department of Agriculture and does not imply its approval other products that may also be suitable.

or warranty of the to the exclusion of


Experiment I: Effect of different concentrations of AM0 1618 on the hepatic enzyme activities of lipid metabolism in chickens Thirty-six 9-week-old White Leghorn crossbred female chickens weighing approximately 569-626 g, were purchased locally. Six chickens were fed corn-based diet as a control; the rest of the birds were divided into groups of 6 which were each fed different dosages of AM0 1618 (2.5, 5.0, 7.5, 10.0, and 15 mg/kg feed). The diets were fed for 21 days; the birds were housed individually, in confinement with a 24-h light period, and with diets and water provided ad libitum. After 16 days of feeding all the birds were fasted for 24 h and refed for 72 h. The birds were then killed by severing the carotid arteries and blood was collected. Each liver was removed, washed, held on ice, weighed and then prepared for the analysis as described below. The experiment was done 2 or 3 times by using the same procedure. Experiment 2: Effect of AM0 1618 on the hepatic enzyme activities of lipid metabolism in rats Twelve 8-week-old Sprague-Dawley male rats, weighing approximately 180-190 g, were divided into 2 groups of 6 per treatment. The first group was fed the control (Purina chow) diet; the second group was fed AM0 1618 (20 mg/kg feed) in the powdered Purina chow diet; the calculated intake of the drug was 0.3 mg/day. For three days the rats were exposed to a 10 h light period (6 am-4 pm) and a 14 h dark period with free access to water at all times. At the end of the feeding period the rats were killed by cervical dislocation and blood was collected; the liver was removed, washed, kept on ice, weighed and worked up as described below. Experiment 3: Preparation of isolated chicken and rat liver cells for in vitro assays The isolated liver cells were prepared from livers of chickens and rats which had been fasted for 40 h and refed corn-soy and Purina chow diets, respectively, for 60 h prior to killing and liver perfusion in order to test the effects of AM0 1618 on lipid metabolism. In the preparation of birds for liver perfusion, bile duct canulation was omitted, and perfusion of the liver was made through the portal vein. The recirculating perfusion system was similar to that described by Zahlten and Stratman (8) which gave good yields of viable cells (2-4 X 10’ cells per liver in the present experiment); the cell viability was determined by the dye exclusion method (0.004% erythrosin B) which showed 72-78% viable cells. Calcium-free perfusate buffer: Krebs-improved Ringer I (K-RI) buffer was prepared from the following solutions: 80 ml 0.154 M NaCl, 4 ml 0.154 M KCl, 3 ml H,O, 1 ml 0.154 M KH,PO,, 1 ml 0.154 M MgSO,. 7 H,O, 21 ml 1.3% NaHCO,, 4 ml 0.16 M Na-pyruvate, 7 ml 0.1 M Na-fumarate, 4 ml 0.16 M Na-L-glutamate, and 5 ml 0.3 M glucose. Calcium-free incubation buffer: Krebs-Heneleit (KH) buffer had the following composition: 100 ml 0.154 M NaCl, 4 ml 0.154 M KCl, 3 ml H,O, 1 ml 0.154 M KH,PO,, 1 ml 0.154 M MgS0,.7 H,O, 21 ml 1.3% NaHCO, and 1.5% gelatin.


Experimental procedure The animals were anesthesized with 50 mg/kg sodium pentobarbital by intraperitoneal injection in rat and intravenously in the wing vein in chicken and placed on its back on a support rack and secured in place with tape across each limb, with the head slanted down. The abdominal skin was cut through by lengthwise incision using a scalpel or scissors and the skin was peeled away from the muscle to each side. A midline incision was then made through the musclar layer up to the point where the diaphragm begins. The exposed muscles and organs were swabed with saline solution. The intestine was displaced to the right. During the rest of the procedure, the liver was bathed with 37°C (rat) or 42’C (chicken) perfusion buffer. The animal was then heparinized to prevent blood clotting. A loose tie was placed around the inferior vena cava above the place where the artery branches off to the kidney, The splenic vein was tightened with a knot, the thoracic cavity was opened to expose the superior vena cava and a loose tie was placed around it. The buffer pump was started so that calcium free K-RI buffer which was equilibrated to 37°C in the rat (42°C in chicken) and gassed constantly by 95% 0, : 5% CO, just slightly dripping out of the syringe. The inferior vena cava was cut well below the 1st loose tie to allow blood to escape, a hole was made in the ventricle of the heart and a needle inserted through the heart into the superior vena cava and the 2nd loose tie was tightened. The 1st loose tie was then tightened so that a closed circuit was made. This perfusion was conducted for 10 min without added collagenase; then 40 mg collagenase (the best results were obtained by using hte preparation made by Worthington; dissolved in 10 ml 0.154 M NaCl) was added and perfusion was continued for 7 min. After digestion, the liver was removed and transferred into a plastic beaker containing 50 ml perfusion buffer at room temperature. The liver was minced with a scissor and the crude suspension gassed for 2 min with 95% 0, : 5% CO, and filtered through one layer of cheesecloth into a second plastic beaker. The crude cell suspension containing hepatocytes and nonparenchymal cells was transferred to a centrifuge. The cells were counted in a Neuhauer hemocytometer (total number of viable cells 37-40 X 106) and protein was estimated by the Biuret method (total protein 200-225 mg). The total volume was adjusted according to the number of viable cells (5-6 X 106) or protein concentration (30 mg/0.9 ml) used per incubation. These cells were incubated with AM0 1618 (dissolved in saline solution) in a final volume of 1 ml at 37’C and 42°C for 15 min of rats and chickens, respectively. After incubation, the assay mixture was centrifuged at 5000 X g for 2 min at 4°C and supernatant discarded. Homogenizing buffer (0.4 ml) was added, the cells were homogenized and processed to obtain the cytosolic fraction. Preparation of tissues for analyses Homogenates of the liver and the sedimented hepatocytes from in vitro assays were prepared in 0.1 M potassium phosphate buffer, pH 7.4 containing 4 mM MgCl,, 1 mM EDTA and 2 mM dithiothreitol. Livers were chopped and suspended in the buffer (1 : 2, w/v). Homogenization was done with a Polytron homogenizer.


The 100,000 X g supernates (cytosol fraction) and the microsomal fractions were stored at -20°C until they were assayed for enzymatic activities [9,10]. Protein concentrations were estimated by a modification of the Biuret method using the bovine serum albumin as a standard [ 111.

cholesterol biosynthesis, oxidation, and concentration: (A) Assays for /3-hydroxy#Lmethylglutaryl coenzyme A reductase and cholesterol 7a-hydroxylase Assays for HMG-CoA reductase and cholesterol 7a-hydroxylase were carried out The final essentially as described previously [ 10,12,13] with some modifications. volumes of incubation were 150 ~1 and 1.0 ml using 300 pg and 50 pg of microsomal protein, incubated at 42°C (chicken) and 37°C (rat) for 30 min and 15 min, respectively. Furthermore, in a number of HMG-CoA reductase assays, after the sedimentation of the denatured protein, the supernatant was saturated with anhydrous Na,SO,. The tubes were washed 3 times with 1 ml toluene (containing 0.8% 2.5Diphenyloxazole = PPO), and the wash solutions transferred to vials. Three ml of toluene (containing 0.8% PPO) were then added directly to the vials (total volume = 6 ml) and radioactivity was measured. Enzyme activity was expressed as nmoles mevalonic acid synthesized/min/mg microsomal protein. The results obtained using this method are comparable with those of reported method [ 121. Estimation of cholesterol and triglycerides in plasma and liver Plasma and liver cholesterol and triglyceride concentrations were estimated using Worthington ‘Cholesterol Reagent’ and ‘Triglycerides Reagent’ sets obtained from Worthington Diagnostics Division of Millipore Corporation, Freehold, NJ. Low density and very low density lipoproteins (VLDL) were isolated from the serum (100 ~1) by precipitation with a mixture of phosphotungstic acid 9.7 mM (10 ~1) + MgCl, 0.4 M (10 ~1). After standing for 5 min at room temperature, the mixtures were centrifuged at 2000 X g for 10 min, the supernatant was removed and was used to determine the level of cholesterol in HDL. The precipitate was dissolved in 0.1 M sodium citrate buffer (100 ~1) and the level of cholesterol (LDL + VLDL) was calculated by using the above method. The value of chol-LDL for most of this work was calculated from total-chol - chol-HDL - triglycerides/5 according to the reported method [ 14,151. (B) Spectrophotometric assays for lipogenic enzymes The activities of fatty acid synthetase [lo], glucose-6-phosphate dehydrogenase [ 161, 6-phosphogluconate dehydrogenase [ 161, malic enzyme [ 171 and citrate-cleavage enzyme in the 100000 X g (cytosolic protein) supernatant fraction were assayed spectrophotometrically at 25°C. These assays were monitored at 340 nm with the recorder set for 0.1 A full scale. The reactions in 0.5 ml were initiated by the addition of 25-250 pg cytosolic protein to the reaction mixture and carried out at room temperature. The absorbance of 1 mM NADPH or NADH is 6.22. Enzyme activities reflect nmoles NADPH or NADH (or NADP+ ) oxidized (or reduced)/min/mg cytosolic fraction.


Results and Discussion Chickens were fed a normal corn-soy diet consisting of corn (56.5%) and soybean (35.0%) as the major source of protein (Table 1). This diet was supplemented with AM0 1618 concentrations of 2.5, 5.0, 7.5, 10.0, and 15.0 mg/kg feed, Weight gain, feed consumption, and feed conversion (after feeding for 3 weeks) are shown in Table 2. Weight gain was suppressed with increasing concentrations of AM0 1618. The birds which received the higher doses of the compound were found more active, but none of them died prior to killing and no visible evidence of change was noted in any organs upon killing. Feed consumption was maximally suppressed (4%) in the group that received AM0 16 18 at 15 mg/kg of feed and suppression of weight gain was also maximal (19%) in this group, when compared to the control birds. A dose-related decrease in activity was observed with the rate-limiting enzymes for the synthesis (HMG-CoA reductase) and for the degradation (cholesterol 7a-hydroxylase) of cholesterol over the range of AM0 16 18 concentrations used (Table 3). to -6O%, respectively, Values ranged from - 14% to -45% and from -21% compared to the control group. The latter results may reflect only an in vivo response to the lowering of the substrate pool in liver effected by the inhibition of the biosynthetic activities. These effects were accompaned by significant decreases in plasma cholesterol levels (- 8% to - 22%), compared to the control (Table 3). Of more importance is the strong suppressive effect AM0 16 18 feeding had on plasma chol-LDL levels. Whereas chol-HDL levels were decreased by 4-2 1% over the range of AM0 1618 concentrations employed, chol-LDL levels were reduced by 19-54%. These values are reflected in the ratios of total cholesterol to chol-HDL, which were essentially unaffected, and the chol-LDL to chol-HDL ratios, which dropped from







Diet a

Corn (9.2 %) Soybean meal (44%) Meat scrap Alfalfa meal ( 17%) Dicalcium phosphate Calcium carbonate Mineral mixture b Vitamin mixture ’

56.5 35.0 5.0 1.0 1.0 0.5 0.5 0.5





a The above diet was supplemented with AM0 1618 at 3.5, 5.0, 7.5, 10, and 15 mg/kg feed for dietary groups 2-6, respectively. Five percent granite grit was also incorporated at the expense of each diet. b Contains/kg sodium chloride (NaCl) 2.0 mg, zinc sulfate (ZnSO,) 50.0 mg, and manganese dioxide (MnO,) 50.0 mg. ’ Contains/kg vitamin A 2000 IU, vitamin D, 200 ICU, vitamin E 10 IU, vitamin K 5.0 mg, choline 1.3 g, thiamin 1.8 mg, niacin 27 mg, riboflavin 3.6 mg, pyridoxine 3.0 mg, calcium pantothenate 10.0 mg, vitamin B,, 10.0 mg, lysine-HCl (hydrochloride) 1.0 g, methionine 0.72 g.




.I ;

: -I :

G tl :

3 +I t-4

? 1


1.12 in the controls to 0.65 in the birds fed 10 and 15 mg/kg of the test compound (Table 3). The levels of liver cholesterol (Table 3) also showed a dose-related decrease with higher concentrations of AM0 1618 compared to control. The decreased cholesterol biosynthesis by AM0 1618 was accompanied by induction of fatty acid synthetase (FAS; 2-3-fold) and the other key lipogenic enzymes: glucose-6-phosphate dehydrogenase (G6PDH), 2-4-fold, 6-phosphogluconate dehydrogenase (6PGDH), 1.2- 1.8fold, malic enzyme (ME), 1.1 -2.2-fold, and citrate-cleavage enzyme (CCE), 1.2- 1.6fold compared to the control (Table 3). The maximum induction was found in glucose-6-phosphate dehydrogenase, the major NADPH generating system in the cytoplasm. The dose-related increase in the activities of all these enzymes with increasing concentrations of AM0 1618 indicate that all these enzymes respond in a similar manner to this compound. Addition of AM0 1618 (20 mg/kg feed) to the diets of g-week-old rats fed only for 3 days cause significant decreases in the activities of HMG-CoA reductase ( - 24%) and cholesterol 7a-hydroxylase ( - 67%) as shown in Table 4. The decrease in the latter activity probably reflects the decrease in the cholesterol pools of plasma and liver. As was observed in the chicken, there was a slight decrease in the level of chol-HDL ( - lo%), but the total plasma cholesterol ( - 26%) and chol-LDL ( - 47%) were significantly decreased (Table 4). In the plasma of the treated rats, cholLDL/chol-HDL ratio was 42% lower while the total cholesterol/chol-HDL decreased only 18%. These changes were accompanied by a 20% decrease in liver cholesterol concentration. The marked decrease in cholesterol biosynthesis in rats fed AM0 1618 accompanied a 4-fold induction in the activities of fatty acid synthetase, 6-fold in G6PDH, 2-fold in 6PGDH, Q-fold in ME, and 3-fold in CCE as shown in Table 4. These 2-6-fold inductions of hepatic lipogenic enzymes in rat compared to chicken by the treatment of AM0 1618 indicate that the liver is the major active site for the fatty acid synthesis. There exists one report that AM0 1618 has no effect in vivo and in vitro on cholesterol biosynthesis by Sabine et al. [20]. They injected the drug, 140 pg/kg body weight intraperitoneally (i.p.) into fed, male rats during the low phase of the diurnal rhythm of cholesterol synthesis. Their in vivo estimations were based on the i.p. injection of [l- 14C]acetate either 2 or 12 h following AM0 1618 administration. Then, after 2 or 12 h, the rats were killed and radiolabel in the digitonin precipitate of the liver extract was measured. Their in vitro estimates were based on the incubation of liver slices, taken 2 or 12 h after AM0 1618 administration, with [ 1-‘4C]acetate for 30 mm or 2 h. These investigators expected to find an increase in radiolabel incorporation, a finding consistent with the response effected by another plant growth retardant, phosfon. Finding no increase in radiolabel incorporation (data not shown) they reported that AM0 1618 has no effect on cholesterol biosynthesis. In view of the significant effects obtained when AM0 1618 was fed to rats or chickens, we tried intraperitoneal injections of AM0 1618 (110 pg/kg body weight in 1 ml saline) in 180 g rats which had received the chow diet for 1 week,






0 (corn-soybean)

2.5 mg

1 2 3 4 5 6 7 8 9 10 11 12 13

430 0.85 192 80 90 2.40 1.12 283 45.3 9.2 15.6 206.8 73.2

370 *24* (86)’ 0.67* 0.01 * (79)

HMG-CoA reductase ’ Cholesterol 7a-hydroxylase d Plasma cholesterol e Plasma cholesterol-HDL ’ Plasma cholesterol-LDL e Total Chol/Chol-HDL Chol-LDL/chol-HDL Liver cholesterol f Fatty acid synthetases Glucose-6-phosphate DH h 6-Phosphogluconate DH h Malic enzyme h Citrate-cleavage enzyme ’

530 (100)’ + 0.03 (100) * 2 (100) + 3 (100) & 4(100) (100) (100) + 4 (100) f 4.0 (100) + 1.0 (100) * 1.0 (100) ?c 7.0 (100) rt 3.8 (100)

177 77 73 2.29 0.94 261 92.6 20.0 18.2 236.2 90.1

+4* (92) *3 (96) +4* (81) * (95) * (84) +3* (92) + 8.0 * (204) f 2.9 * (220) + 1.0 * (116) i 13 * (114) fl0 * (123)


5.0 mg b 343 *22* (80)’ 0.58* 0.07 * (68) 162 + 4* (84) 70 f2’ (88) 64 f2* (71) 2.31 * (96) 0.91 * (81) 258 &2* (91) 100.7 + 6.0 * (222) 27.9 + 2.0 * (303) 22.4 f 2.0 * (144) 306.9 f 9.0 * (148) 101.2 + 7.0 * (138)

a Feeding period was 3 weeks; Time of killing was 0800 h; Data expressed as means+ SD; n = 6 chickens/group. b AM0 1618 (2-Isopropyl-4-dimethylamino-5-methylphenyl-l-piperidine carboxylate methyl chloride) 2.5-15 mg of AM0 1618/kg feed. ’ p-Hydroxy-P-methylglutaryl-CoA reductase, pmoles of mevalonic acid synthesized/min/mg of microsomal fraction. of microsomal fraction. d p moles of [ I4Clcholesterol converted into [ I4C]7a-hydroxycholesterol/min/mg

fasted for 48 h and then refed chow diet for 36 h prior to injection. After 12 h the animals were killed at the high phase of the diurnal rhythm of cholesterol synthesis and the livers examined for HMG-CoA reductase and FAS as before. The results (data not shown) were similar to those summarized in Table 4 for rats that had been fed 20 ppm AM0 1618 for 3 days. Thus there appears to be no barrier to AM0 1618 or an active altered form of the compound reaching the liver via this route; however, this demonstration of the inhibition of cholesterol biosynthesis at cellular level is not necessarily related to the failure of Sabine et al. [20] to demonstrate a change in the retention of cholesterol in the tissue. The effects of AM0 1618 on lipid metabolism were tested in isolated hepatocytes of chickens and rats, which were incubated with lo-100 pg (28-280 pmoles) AM0 1618 for 15 min. A dose-related decrease in the activity of the HMG-CoA reductase and increase in FAS were obtained with increasing concentration of AM0 1618 (Table 5) or time of incubation (using 30 pg = 85 pmoles, 5-120 min, Table 6) in both chicken and rat hepatocytes. These data confirmed the earlier in vivo studies which indicate that AM0 1618 can also be used as a reference compound for checking the viability of isolated hepatocytes which are prepared from chickens and


1 L

3 4 5 6 7 8 9 10 11 12 13

7.5 mg b

10.0 mg b

15.0 mg

320 k23 * (74)’ 0.46 + 0.13 * (54)

262 +20 * (61)’ 0.45+ 0.08 * (53)

237 +17* (55)’ 0.27k 0.03 * (32)

158 68 56 2.32 0.82 250 121.8 30.8 23.4 310.3 105.0

156 66 44 2.36 0.66 240 132.7 36.6 23.8 374.0 106.5

150 63 41 2.38 0.65 230 149.5 37.8 27.7 445.0 117.4

*3* *3* *4* * * k 3 * f 4.0 * 3.0 + 2.0 k23.0 k 3.8

(82) (85) (62) (97) (73) (88) +(269) * (335) *(150) *(150) * (143)

_+2* *4* *3* * f 3 * + 2.0 k 2.0 + 1.0 k34.0 f 6.0

* * * * *

(81) (83) (49) (98) (59) (85) (293) (398) (153) (181) (146)

*3* *2* *2* * k f * + f *

3 * 4.0 * 3.0 * 1.5 * 9.5 * 5.4 *

(78) (79) (46) (99) (58) (81) (330) (411) (178) (215) (160)

e The concentrations of all parameters are expressed as mg/lOO ml of plasma. f The concentration of cholesterol is expressed as mg/lOO g of liver. s nmoles of NADPH oxidized/min/mg of cytosolic fraction. h nmoles of NADP+ reduced/min/mg of cytosolic fraction. I nmoles of product formed/min/mg of cytosolic fraction. ’ Percentages of respective control activity data are in parentheses. * Significantly different from control (P < 0.01).

rats in order to test the inhibition of cholesterol biosynthesis. These results indicate that the plant growth retardant AM0 16 18 elicits responses similar to those gained using unidentified substances from dormant plant bodies such as seeds, bulbs, and roots [2 l-231. This compound provides potentially a means for lowering chol-LDL levels by decreasing cholesterogenesis in humans. More significant is the selective effect that this compound has on chol-LDL levels in both the chicken and the rat when it is fed at very low levels. Concentrations of IO-15 ppm AM0 1618 in the diets reduced the chol-LDL : chol-HDL ratio from 1.12 to 0.65 in chickens and at 20 ppm from 1.96 to 1.14 in rats. The in vitro data suggest that AM0 1618 acts at the cellular level and, although the minimal in vivo response time remains to be determined, the response appears to be rapid since the present rat feeding trial was terminated after only 3 days (Table 5). Acknowledgements The authors wish to thank Dr. Burt Olson for the Isocap/300 Nuclear Liquid Scintillation counter. We also thank Faye Roed for her excellent editorial assistance.






/3-Hydroxy-/3-methylglutaryl-CoA reductase Cholesterol ‘la-hydroxylase ’

370 * (100) 4.9* 0.5(100)

Plasma total cholesterol Plasma cholesterol-HDL Plasma cholesterol-LDL

d d d

Total Chol/Chol-HDL Chol-LDL/ChoI-HDL Plasma



Fatty acid synthetase ’ Glucose-6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase Malic enzymes Citrate cleavage enzyme h a b ’ d e

98 30 59

+ 3 (100) * 2 (100) * 3 (100)

3.21 1.96

(100) (100)


280 +I6 * 1.6& 0.3 * 72 27 31

f4’ k 2’ + 2*

2.67 * 1.14 *

*20 (100) + 6.0(100)

149 + 2.8(100) 251 k31 (100) 71.4+ 18 (100) 35.6+ 14 (100) 30.2 f 7.0( 100)



Purina Chow + AM0 1618 b (20 mg/kg feed)


504 118




3 (100)


Liver cholesterol e Liver triglycerides ’

Chow (control)



(76) (33)

(74) (90) (53) (82) (58)

4 *


405 186

*20 * f 7 *

(80) (158)

591 727 137 162 89

f73 +48 +28 * 12 f19

(397) (290) (192) (455) (294)

* * * * *

Time of killing was 1600 h; data expressed as means f SD; n = 6 rats/group. p moles of mevalonic acid synthesized/min/mg of microsomal fraction. pmoles of [ t4C]cholesterol converted into [ “C]7a-hydroxycholesterol/min/mg of microsomal The concentrations of all parameters were expressed as mg/lOO ml of plasma. The concentrations of cholesterol and triglycerides were expressed as mg/lOO g of liver. ’ qmoles of NADPH oxidized/min/mg of cytosolic fraction. s nmoles of NADP+ reduced/min/mg of cytosolic fraction. h nmoles of product formed/min/mg cytosolic fraction. I Percentage of respective control activity are in parentheses. * Significantly different from control (P < 0.01).




AM0 1618 concentration ng/mI




0 10 20 30 40 50 100

0 28 56 85 110 140 280

24 (100)” 20 (83) 18 (75) 16 (67) 15 (63) 15 (63) 14 (58)

43 36 35 34 30 31 28

(1OO)d (84) (81) (79) (70) (72) (65)


accid synthetase’



22.3 27.5 36.9 44.0 45.3 45.2 44.8

14.2 18.1 19.2 21.3 25.6 26.0 25.6

(100) d (123) (165) (197) (203) (203) (201)

(100) d (127) (135) (150) (180) (183) (180)

a Eight-week-old female chickens and 6-week old male Sprague-Dawley rats were fed standard corn-soy and Purina chow diets, respectively. They were fasted for 40 h and refed for 60 h prior to the preparation of liver perfusion. Incubation period was 15 min. Values represent means of 2 replicates within incubation set. b nmoles of mevalonic acid synthesized/min/mg of microsomal fraction. ’ nmoles of NADPH oxidized/min/mg of cytosolic fraction. d Percentages of respective control activity data are in parentheses.


0 5 10 20 40 60 120

&Hydroxy-p-methylglutaryl-CoA reductase b Chicken


33 30 27 23 21 18 18

28 26 24 21 20 18 17

(1OO)d (91) (82) (70) (64) (55) (55)

(100) d (93) (86) (75) (71) (64) (61)

Fatty acid synthetase’ Chicken


20.6 23.5 29.8 31.7 35.3 35.8 36.5

17.8 21.7 26.2 29.4 31.0 31.2 31.4

(100) d (114) (145) (154) (171) (174) (177)

( 100) d (122) (147) (165) (174) (175) (176)

a Eight-week-old female chicken and 6-week-old male Sprague-Dawley rats were fed standard corn-soy and Purina chow diets, respectively. They were fasted for 40 h and refed for 60 h prior to the preparation of liver perfusion. Each incubation contains 30 ng (85 pmoles) of AM0 1618. Values represent means of 2 replicates within incubation set. b pmoles of mevalonic acid synthesized/min/mg of microsomal fraction. ’ nmoles of NADPH oxidized/min/mg of cytosolic fraction. d Percentages of respective control activity data are in parentheses.



9 10 II 12


14 15 16

17 18 19 20 21 22 23

Khan. A.A., The Physiology and Biochemistry of Seed Dormancy and Germination, North-Holland Publ. Co., New York, NY, 1977. Bewley, J.D. and Black, M., The Physiology and Biochemistry of Seeds in Relation to Germination, Springer-Verlag, New York, NY, 1978. Moore, J.C., Biochemistry and Physiology of Plant Hormones, Springer-Verlag, New York, NY, 1979. Reeve. D.R. and Crozier, A., The analysis of gibberellins by high performance liquid chromatography. Cambridge University Press, London, In: J.R. Holden (Ed.), Isolation of Plant Growth Substances, 1978, pp. 41-77. Saunders, P.F.. The identification and quantitative analysis of abscisic acid in plant extracts. In: J.R. Holden (Ed.), Isolation of Plant Growth Substances, Cambridge University Press. London, 1978, pp. 115-134. Paleg, L.G., Site of action of plant growth retardants on cholesterol biosynthesis by cell-free rat liver preparations, Aust. J. Biol. Sci., 23 (1970) I1 15. Douglas, T.J. and Paleg, L.G., Inhibition of sterol biosynthesis by 2-isopropyl-4-dimethyl-amino-5methylphenyl-1-piperidine carboxylate methyl chloride in tobacco and rat liver preparations, Plant Physiol., 49 (1972) 417. Zahlten. R.N. and Stratman, F.W., The solution of hormone-sensitive rat hepatocytes by a modified enzymatic technique, Arch. Biochem. Biophys., 163 (1974) 600. Qureshi, A.A.. Burger, W.C., Prentice, N., Bird, H.R. and Sunde, M.L., Regulation of lipid metabolism in chicken liver by dietary cereals, J. Nutr.. 110 (1980) 388. Qureshi, A.A., Burger, W.C., Elson, C.E. and Benevenga, N.J., Effects of cereals and culture filtrate of Trichoderma uiride on lipid metabolism of swine, Lipid, 17 (1982) 924. Gornall. A.G.. Bardawill. C.J. and David, M.M., Determination of serum proteins by means of the biuret reaction, J. Biol. Chem., 177 (1949) 751. Shapiro, D.J., Norstrom, J.L., Mitchelen, J.J., Rodwell, V.W. and Schimke, R.T., Microassay for P-hydroxy-P-methylglutaryl-CoA reductase in rat liver and in L-cell fibroblasts, Biochim. Biophys. Acta, 370 (1974) 369. Carlson, S.E., Mitchell, A.D. and Goldfarb, S., Sex-related differences in diurnal activities and development of hepatic microsomal /I-hydroxy-/3-methylglutaryl-CoA reductase and cholesterol ‘la-hydroxylase, Biochim. Biophys. Acta, 531 (1978) 115. Kostner, G.M., Enzymatic determination of cholesterol in high-density lipoprotein fractions prepared by polyanion precipitation, Clin. Chem., 22 (1976) 695. Lobos-Virella, M.F., Stone, P., Ellis, S. and Colwell, J.A., Cholesterol determination in high density lipoproteins separated by three different methods, Clin. Chem., 23 (1977) 882. Bottomley, R.H., Pitot, H.C., Potter, V.R. and Morris, H.P., Metabolic adaptations in rat hepatomas, Part 5 (Reciprocal relationship between threonine dehydratase and glucose-6-phosphate dehydrogenase), Cancer Res., 23 (1963) 400. Hsu, R.H. and Lardy, H.A., Pigeon liver malic enzyme isolation crystallization and some properties, J. Biol. Chem., 245 (1963) 520. Innoue, H., Suzuke. F.. Fukunishi, K., Adachi, K. and Takeda, Y., Studies on ATP-citrate lyase of rat liver - Purification and some properties, J. Biochem., 60 (1966) 543. Snedecor, G.W. and Cochran, W.G., Statistical Methods, 6th edition, The Iowa State University Press. Ames, IA, 1971, pp. 258-298. Sabine, J.R., Paleg, L.G. and Douglas, T.J., The effects of a plant-growth retardant, phosfon, on mammalian lipid metabolism in vivo, Aust. J. Biol. Sci., 26 (1973) 113. Qureshi, A.A., Burger, W.C., Din, Z.Z. and Elson, C.E., Suppression of cholesterol biosynthesis by dietary high-protein barley flour, Amer. Sot. Clin. Nutr., May (1982) (Abstract 47). Din, Z.Z., Ahmad, Y., Elson, C.E. and Qureshi, A.A., Inhibition of lipid metabolism by garlic and its fractions in chicken liver, FASEB Proceedings, 41 (1982) 544 (Abstract 1599). Qureshi, A.A., Abuirmeileh. N., Din, Z.Z., Elson, C.E., Burger, W.C. and Ahmad, Y., Suppression of cholesterogenesis and lipoproteins by dietary ginseng and its fractions in chicken liver, Atherosclerosis. Submitted.