... of the secretory. I The abbreviations used are: VLDL, very-low-density lipoproteins; ... sured enzymatically (30), after removal of phospholipid and saponi- fication (28). ... triglycerides and phospholipid was determined by thin-layer chro- matography of the chloroform extract in hexane:diethyl ether:acetic ... Student's t test.
THE JOURNAL OF BIOLOGICAL CHEMISTRY ic: 1985 by The American Society of Biological Chemists, Inc.
Contributions of Fatty Acid and SterolSynthesis to Triglyceride and Cholesterol Secretion by - the Perfused Rat Liver inGenetic Hyperlipemia andObesity* (Received for publication, June 29, 1984)
Michael J. Azain, Nobuhiro FukudaS, Fei-Fei Chao, Munehiko Yamamoto@, and Joseph A. Ontkoll From the Cardiovascular Research Program, Oklahoma Medical Research Foundation and the Departmentof Biochemistry and Molecular Biology, College of Medicine, University of Oklahoma, Oklahoma City, Oklahoma 73104
The relative importance of fatty acid synthesis in One of the most prominent characteristics of the genetically triglyceridesecretion by perfused livers from lean obese Zucker rat is the markedly elevated level of plasma (normal control) and obese Zucker rats was investi- triglyceride (1-4). It is established that the hepaticsecretion gated. Livers from fed animals were perfused in a of triglyceride-rich lipoproteins is elevated in the obese rat recirculating system with tritiated water and a con- (5-7). The lipoproteinlipase-catalyzed removal of triglyceride stant infusion of oleic acid. Triglyceride secretion was from the circulation appears to be normally operative in these 5 times greater and cholesterol secretionwas 35% animals @-lo), with hypertriglycer~demia resuiting from satgreater in the obese rat livers. The very-low-density uration of this removal process (11). lipoproteinhypersecreted by perfusedliversfrom The free fatty acids which are esterified in the liver to form obese rats contained more apolipoprotein B and exhibited an increased B-48/B-11)0 ratio. Apo-B was also triglyceridesmay be subsequently secreted in VLDL’ (12). elevated in the hypertriglyceridemic plasma of obese These fatty acid substrates are derivedfroma number of rats in both fed and fasting states. Thevery-low-den- sources, both endogenous and exogenous to the liver (13, 14). sity lipoprotein isolated therefrom was likewise char- The exogenous fatty acids are those supplied in the diet, and acterized by an increased B-48/B-100 ratio. Ketogen- those mobilized from adipose tissues. The endogenous origiesis was depressed 40% in the obese rat livers and nate from de nouo synthesis from amino acid and carbohyof triglyceride inthe liver increased hepatic malonyl-CoA was implicated in this drate precursors (15,16). The stores alteration.The de novo synthesisandsecretion of are another potential endogenous source of VLDL triglycernewly synthesized cholesterol was moderately in- ide. However, since thesetriglycerides are in continuous turncreased in the perfused livers fromobese rats. Tritium over and must be replaced (17, 18),they do not represent a incorporation into fatty acidswas 15 times greater in primary source of fatty acids. An understanding of the relative the obese genotype. Most of the synthesized fatty acids contrib~tionof these various sources of fatty acids to the remained in the liver and were recovered after perfu- formation of secretory triglyceride by the obese rat liver is sion in trigIyceride and phospholipids. Newly synthe- important for an overall understanding of the mechanismsof sized fatty acids accounted for only 3 and 15%of the triglyceride secreted by the lean and obese rat livers, VLDL hypersecretion in thisgenetically obese syndrome. In the obese rat liver, both the endogenous and exogenous respectively. A large portion of the secreted triglycacid eride fatty acids was derived from endogenous liver sources of fatty acids areelevated. The plasma free fatty lipids. When the turnover of newly synthesized fatty level is 2 times greater (3, 19, 20), while the liver triglyceride obese rat (7, 21). acids in these pools was considered, the contributionof concentration is 10 times greater in the Furthermore, the rate of de nouo synthesis of fatty acids in de novo fatty acid synthesis to triglyceride secretion was estimated to be 9% in the lean and 44% in the the obese rat liver is substantially higher than in the liver of obese rat livers. Therefore, the altered partition of free the lean rat, whether measured in uiuo (22,23) or in vitro (24fatty acids (Fukuda, N., Azain, M. J., and Ontko, J. A. 27). Thus, there is an ample supply of VLDL precursors (1982) J. B i d . Chern. 257, 14066-14072) andinsynthesized in, and supplied to, the liver of the obese Zucker creased fatty acid synthesis are both major determi- rat, which may contribute to t.he enhanced rate of VLDL nants of the hypersecretion of triglyceride-rich lipo- secretion. proteins by the liver in the genetically obese Zucker We have characterized the secretion of exogenously suprat. plied free fatty acid in VLDL triglyceride from perfused livers of lean and obese rats (7). These studies demonstrated that while fatty acid uptake by perfused liversof the two genotypes was similar, the partitionof fatty acid substratebetween the intracellular pathways and sites of utilization was altered. *This work was supported by Grants HL 23181 and HL 32609 from the National Institutes of Health. The costs of publication of The obese rat livers secreted 3 times as much of the exogethis article were defrayed in part by the payment of page charges. nously supplied oleic acid substrate in VLDL triglyceride as This article must therefore be hereby marked“advertisement”in did the livers from lean animals. In the lean ratliver system, accordance with 18 U.S.C. Section 1734 solely to indicat,e this fact.. 29 of the 72 pmol (40%) of triglyceride fatty acid secreted $ Present address: Laboratory of Biochemistry and Nutrition, Dewere derived from the exogenously supplied fatty acid, while, partment of Agricultural Chemistry, Faculty of Agriculture, Univerin the obese, 83 of the 330 pmol(26%) of thesecretory sity of the Ryukyus, Okinawa, Japan. $ Present address: Department of Chemistry, Kurume IJniversity School of Medicine, Kurume, Japan. ll To whom correspondence should be addressed.
__I The abbreviations used are: VLDL, very-low-density lipoproteins; apo-B, apolipoprotein B.
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Hepatic Lipogenesis and VLDL Production by Zucker Rats triglyceride fatty acid originated from this source. Thus, although the total triglyceride secreted by the obese liverswas 4 times greater, only one-fourth of this circulating triglyceride could be attributed to theexogenousfatty acid supplied during the perfusion period. This implies that endogenous sources of fatty acids are quanti~tivelymore important in the production of t~glyceride-richlipoproteins in the genetically obese rat than in the lean controls. The purpose of the present study was to determine the relative roles of hepatic fatty acid synthesis in hepatic VLDL production in these animals. By studying de nouo fatty acid synthesis from tritiated water under the same experimental conditions employed in our previous study (7), we have assessed the relative importance of the various sources of fatty acids incorporated into VLDL triglyceride by the livers from both the normal and obese genotypes. EXPERIMENTAL PROCEDURES
Animals-Male lean and obese Zucker rats were obtained from Hoffmann-LaRoche Inc. and the Department of Nuclear Medicine, University of Massachusetts, Worcester, MA. Rats were housed as described (7) andgiven Purina Laboratory Chow and water ad libitum. On the days of perfusion, rats were given Nembutal (5 mg/100 g of body weight, intraperitoneally) between 9:00 and 9:30 a.m. for isolation of livers and perfusion. Brief ether anesthesia was employed when livers were freeze clamped in uiuo for malonyl-CoA assay. Liuer Perfusion-The perfusion equipment and procedures were the same as described previously (7, 28). Briefly, livers were perfused with Krebs-Henseleit buffer (pH 7.4), which contained 25 mM glucose, 1.5% fatty acid free bovine serum albumin (Fraction V, Miles Laboratories, Inc., Elkhart, IN) and 25% washed aged human red blood cells. Initially, 20 mCi of 'HZO was added to 200 ml of medium, 120 ml ofwhich was placed in the perfusion reservoir. The specific activity of the 3H20 was determined by analysis of the perfusate a t 45 min. Typically, the specific activity was 2.5 X 108 dpmlml. Five ml of 20 XIM oleate in 0.9% NaCl was added as a priming dose to thereservoir a t zero time. The same oleate solution was infused a t a rate of 90 pmol/h throughout the perfusion. At 45-min intervals, 18 ml of perfusate was removed for analysis and replaced with an equal volume of fresh medium. Bile was collected from a bile duct cannula. Liver function was assessed by several criteria: liver appearance, perfusate and bile flow rates, kinetics of triglyceride secretion and ketone body production, and the P-hydr0xybutyrate:acetoacetateratio. At the end of perfusion, livers were blotted dry, extraneous tissue was rapidly removed, and the livers were placed in a tared beaker containing ice-cold saline and weighed. The livers were then homogenized in 4 volumes of saline. The perfusates were centrifuged at 10,000 X g for 15 min to sediment blood cells. The resulting homogenates and cell-free perfusates were used for lipid radioactivity, triglyceride, cholesterol, and ketone body determinations. Lipid Analysis-Lipids from liver homogenates and perfusates were extracted and washed according to Folch et al. (29). The extracts were evaporated to dryness and redissolved in a known volume of chloroform which contained 1%ethanol. An aliquot of this chloroform extract was used for cholesterol analysis (7). Triglyceride was measured enzymatically (30), after removal of phospholipid and saponification (28). For isolation of fatty acids and digitonin-precipitable sterols, another aliquot of the chloroform extract was evaporated and saponified in 2.0 ml of ethanol and 0.4 ml of 4 N KOH for 30 min a t 60-70 "C. The nonsaponifiable material was removed bythree hexane washes. The combined washes were evaporated under nitrogen and resuspended in ethano1:acetone (kl, v/v) after the addition of one drop of20% acetic acid in water. One mg of cold cholesterol was added t.o each sample, followed by 1 ml of 0.5% digitonin in ethano1:water ( k l , v/v). The digitonin precipitate was washed in acetone:~ethyl ether(1:2, v/v) and again in diethyl ether. The digitonide was then dissolved in methanol and transferred to a counting vial. Fatty acids were extracted from the acidified saponification solution with three hexane washes. The pooled hexane extracts were hackwashed with water, transferred to counting vials, and evaporated before counting. The incorporation of de nouo synthesized fatty acids into perfusate triglycerides and phospholipid was determined by thin-layer chromatography of the chloroform extract in hexane:diethyl ether:acetic
175
acid (80201) on Silica Gel 60 G plates containing Ultraphor (31). The triglyceride and phospholipid bands were detected by ultraviolet light and scraped into ch1oroform:methanol (2:l) and chloroform:methanol:acetic acid:water (50391:10), respectively. Water (one-third volume) was added to these extracts and thelipids in the chloroform layer were dried, saponified and processed as described above for the de~rmination of tritium inco~oration into fatty acids. All radioactive samples were counted in Insta-Gel (PackardInstrument Co., Inc., Downer's Grove, IL) in a PackardLiquid Scintillation Counter with automatic external standa~ization.Gpmwere converted to dpm based on the counting efficiencies of a series of quenched tritium standards. Cakulations-Dpm of tritium were converted to pmol of 3H20 incorporated from the specific radioactivities of the perfusates determined a t 45 min. Denouo synthesis of C , units was calculated according to Brunengraber et al. (32) for fatty acids (pmol of 3H20 X 1.15) and cholesterol (pmol of 3H20 X 1.31).Triglyceride synthesis from C2 units was estimated by dividing pmol of Cz units by 8 to obtain fatty acid and by 3 for triglyceride. Cholesterol synthesis was calculated by dividing pmol of C2 units by 13.5. Other Amlyses-Acetoacetate and B-hydroxybutyrate were measured enzymatically in perchloric acid extracts as described previously (7). Hepatic malonyl-CoA wasassayed by the radioisotopic procedure of McGarry et al. (33). Apolipoprotein B (apo-B) was measured by electroimmunoassay (34). The two major species of apo-B, termed herein B-48 and B-100 (35), were separated (31). The relative amounts of these proteins were estimated by densitometry. Statistics-The significance of genotypic differences was determined by the Student's t test with a two-tailed measurement of p values. The significance of differences in percentage values was determined using the arcsin conversion of proportions (36) and the Student's t test. RESULTS
The lean and obese Zucker rats and theperfused liversare describedin Table I. Bodyweight and liverweightwere si~ificantlyincreased in the obese group. Bile production and perfusate flow rates were similar in lean and obese rat livers. Liver lipids were determined after 225 min of perfusion. Liver triglycerideswere markedly elevatedin the obese group. The cholesterol concentration was unaltered. Total cholesterol per liver was increased in the obese rat liver, but, owing tothe large variation, the difference was not significant, although it was in the previous study (7). The rates of ketone body production by the perfused livers are shown in Fig. 1. Ketogenesis was significantly greater in TABLEI Characteristics of the animals and perfused livers Lean (4)"
Body weight (g) 367.3 f 18.3* Liver weight (g)' 13.2 f 0.9 Relative liver size (g/lOO g)d 3.6 f 0.3 3.9 Bile production (pl/liver)f 1241 f 96 1452 Perfusate flow (ml/minP 19.0 t 0.3 Liver triglyceride" pmol/g 12.7 39.0 f 0.7 18.4 pmol/lwer 168.0 f785.6 Liver choIesteroP rmol/g 7.3 k 0.2 7.3 pmol/lwer 97.5 t 9.0
Obese (4)
p
520.5 k 24.5
