Comp. Biochem. Physiol. Vol. 103B,No. 1, pp. 93-100, 1992 Printed in Great Britain
0305-0491/92$5.00+ 0.00 © 1992PergamonPress Ltd
COMPARISON OF GLYCEROLIPID BIOSYNTHESIS IN HOMOGENATES FROM HUMAN, OVINE, BOVINE AND RAT ADIPOSE TISSUE I N VITRO D. C. RULE
Department of Animal Science, University of Wyoming, Laramie, WY 82071, U.S.A. Tel.: (307) 766-3404; Fax: (307) 766-5098 (Received I0 February 1992; accepted 11 March 1992) Abstract--1. Assay conditions were compared for glycerolipid biosynthesis in homogenates prepared from human abdominal, ovine and bovine subcutaneous, and rat epididymal adipose tissues. 2. In contrast to other species, longer incubation time and greater homogenate concentration resulted in decreased glycerolipid biosynthesis with rat adipose tissue homogenates. 3. Species differences were observed in concentration-dependency for ATP and fatty acids (palmitate, oleate and palmitoleate). 4. Results indicated that glycerolipid biosynthesis transpired at different rates in the four species, and that ovine and human adipose tissue homogenates had similar properties.
(G3P) in a reaction catalyzed by glycerophosphate acyltransferase (GPAT; EC 2.3.1.15). A second esterification occurs, producing phosphatidate (a phospholipid, PL), the phosphate of which is subsequently hydrolyzed in a reaction catalyzed by phosphatidate phosphohydrolase (PPH, EC 3.1.3.4) producing diacylglycerol (DG). A third esterification occurs in a reaction catalyzed by diacylglycerol acyltransferase (DGAT) (EC 2.3.1.20) to produce TG. Although the pathway by which fatty acids are esterified to G3P appears to be the same in adipose tissue of most mammals, most research on regulation of this pathway has been conducted using rat adipose tissue. Similarities in catecholamine regulation of GLBS between rat and swine adipose tissue have been reported (Rule et al., 1987). Because several enzymes are involved in this pathway, and because the enzymes are membrane-bound and the substrates are large, relatively hydrophobic molecules, the particular assay condition employed can markedly change the rate and products of GLBS assays. In previous studies detailed observations regarding assay conditions for GLBS in swine adipose tissue homogenates documented such changes (Rule et aL, 1988a,b). The purpose of the present study was to understand the potential of adipose tissue GLBS as a biomedical model system by comparing the effects of varied assay conditions in homogenates prepared from human (Homo sapiens), ovine (Ovis aries), bovine (Bos taurus) and rat (Rattus rattus) adipose tissue.
INTRODUCTION The use of various species for biomedical research should require similarities between the model and the particular species of interest. For example, swine have been used as models for dietary protein and fat studies because of the similarity in human and swine digestive tracts (Diersen-Schade et al., 1985; Diersen-Schade et al., 1986). Also, swine, as well as rabbits and some non-human primates, are well suited as models for atherosderosis research (Armstrong and Heistad, 1990). For the interaction of dietary lipids and subsequent blood lipid effects the male Golden Syrian hamster has been used because of its similarity to humans regarding diet-induced LDL-cholesterol changes (Dietschy, 1985; Turley et aL, 1987). Cellular metabolism studies conducted with animal models for biomedical application should also consider similarities or differences between species. For example, de novo fatty acid biosynthesis occurs primarily in the liver in humans (Shrago et al., 1971) and chickens (O'Hea and Leveille, 1969a), but it occurs in adipose tissue of cattle (Hood et al., 1972), sheep (Ingle et al., 1972), swine (O'Hea and Leveille, 1969b; Mersmann et al., 1973), dogs (Baldner et al., 1985) and cats (Richard et al., 1989). In rats and mice, this process occurs in both liver and adipose tissue (Leveille, 1966). Although site differences exist for fatty acid biosynthesis in a number of species, glycerolipid biosynthesis (GLBS) commonly occurs in adipose tissue (Bell and Coleman, 1980; Bell and Coleman, 1983), liver (Nimmo, 1980), and gut (Brindley, 1985) of mammals. In adipose tissue GLBS is primarily involved in triacylglycerol (TG) production using the following metabolic pathway (Nimmo, 1980): activation of fatty acid by thioesterification to CoA, which is catalyzed by acyl--CoA synthetase (EC 6.2.1.3) and requires A T P - M g for energy, is followed by esterification of acyl--CoA to glycerol-3-phosphate
METHODS
Reagents Coenzyme A (CoA, sodium salt, C-3144), ATP (grade II disodium salt, A-3377), ethylenediaminetetraacetic acid (EDTA, EDS), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, H-3375), dithiothreitol (DTT, D0632), bovine serum albumin (BSA, fraction V, fatty acid 93
94
D.C. RULE
poor, A-6003), palmitic acid (P-5917), oleic acid (0-3879), palmitoleic acid (P-0875) and glycerol-3-phosphate (G3P, disodium salt, G-2138) were purchased from Sigma Chemical Co. (St Louis, MO). 14C-G3P(L-[14C(U)]-glycerol-3-phos phate, disodium salt, NEC-608) was purchased from New England Nuclear, (Boston, MA). All other reagents were reagent grade quality.
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140 E 120
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Tissues
Adipose tissues were obtained from single sites for each species. Abdominal adipose tissue was obtained from two human subjects who underwent routine, abdominal surgery. Patient consent forms were prepared according to guidelines set forth by both the University of Wyoming and the Ivinson Memorial Hospital Institutional Review Boards. Subjects were otherwise healthy, middle-aged females. Ovine subcutaneous adipose tissue was obtained from the tail head area immediately after sacrifice of three Suffolk castrated lambs that weighed 66.5 + 4.2 (SEM) kg. Bovine subcutaneous adipose tissue also was obtained immediately after sacrifice from the tail head area of three Hereford x Angus steers that weighed 486.9 __+11.9 kg. Sheep and cattle were sacrificed by stunning followed by exsanguination in the Animal Science Department abbatoir under supervision of a Wyoming Department of Agriculture inspector. Rat epididymal adipose tissue was dissected from 300 to 500 g, male Sprague-Dawley rats after euthanization with chloroform. Adipose tissue from five rats was pooled to obtain enough homogenate for experiments. Additional rats were sacrificed to obtain enough adipose tissue for experimental replication in the later experiments. Tissues from each species were placed in phosphate-buffered saline at 4°C for transport to the laboratory. Homogenates of adipose tissue were prepared essentially as described by Rule et al. (1988a); 1.0g of adipose tissue was homogenized in 1.5 ml of homogenization buffer (0.15 M KCI, 10 mM HEPES, 1 mM EDTA and 1 mM DTT, pH 7.4, 4°C). The supernatant fat-cake was removed after centrifugation at 700g, and 5-ml aliquots of each aqueous infranatant fraction were snap-frozen in liquid nitrogen and stored at -70°C for up to 2 months. For each preparation 5- and 60-min GLBS assays were conducted to assess activity in fresh homogenates. This method of storage preserved enzyme activity, as it did for porcine homogenates (Rule et al., 1988b). Glycerolipid biosynthesis assay
Glycerolipid biosynthesis assays for each species initially were based on homogenate, substrate, cofactor and buffer concentrations reported previously for swine subcutaneous adipose tissue homogenates (Rule et al., 1988a). In 0.4 ml of total assay volume there were: 0.3 ml of 700g supernatant fraction (homogenate), 17.5mM G3P, 0.3/~Ci ~4C-G3P, 0.3 mM potassium palmitate, 1.0 mM DTT, 0.16 mM CoA, 12.0 mM ATP, 0.5 mg/ml BSA, 12.0 mM Mg C12, 50.0 mM HEPES, and 20.0 mM K2HPO4. All buffer stock solutions were pH 7.4 and all substrate and cofactor stock solutions were pH 7.0. A substrate and cofactor premix was prepared so that 0.1 ml was combined with 0.3 ml of homogenate in a 16 × 125 mm test tube at 37°C in duplicate to start the reaction. Reactions were terminated by adding 3.0 ml of 1 : 2 chloroform: methanol (v/v) and 5 sec of vortex-mixing. Details of glycerolipid extraction and separation of glycerolipids by thin-layer chromatography have been described previously (Rule et al., 1988a). Quantitation of 14C was accomplished by liquid scintillation spectrometry to calculate G3P incorporation into glycerolipids. Data were expressed on a mg of protein basis. Total homogenate protein was determined on TCA-precipitated material by using the biuret procedure (Gomall et al., 1949). For all assays, observations were obtained from preparations from two humans, three ovines, three bovines, and three rats or a pool of tissue from three to five rats.
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Fig. 1. Effect of incubation time on glycerolipid biosynthesis. Values are means of triplicate observations plus or minus the standard error of the mean and expressed per mg of homogenate protein. Assays contained 17.5 mM G3P, 50mM HEPES, 0.98mM DTT, 0.16mM CoA, 12mM ATP, 0.5mg/ml BSA, 20mM K2HPO4, 12mM MgCI2, 0.3 mM palmitate and assays for human, ovine, bovine and rat preparations contained 1.2 + 0.33 (SE), 1.87 + 0.08, 1.09 4- 0.17 and 0.84 4- 0.05 mg of protein, respectively.
RESULTS Incubation time
Effects of incubation time on G L B S in adipose tissue from the human, rat, ovine and bovine are illustrated in Fig. 1. Initial (5 min) values were lowest for the bovine, intermediate for the human and ovine, and highest for the rat adipose tissue homogenates. Distinct linear increases in G L B S were observed for bovine, ovine, human, and rat adipose tissue homogenates. Rates of bovine, ovine and human G L B S remained linear and distinctly different for 20 min. F o r the rat, rates were much greater than those of the other three species from 5 to 10min. At 120 and 60 min G L B S was maximal for human and ovine adipose tissue homogenates, respectively, whereas at 180 min G L B S in the bovine was still increasing, though at a reduced rate. In the rat maximal G L B S was observed at 30 min, which was followed by a 20% decrease at 60 min. The major similarity between species in this experiment was the initial rate of G L B S for the human and ovine homogenates. This similarity, however, was followed by a marked contrast in maximal G L B S between these two species with the human G L B S being nearly twice that of the ovine at 120 min. The contrasts between all species would not appear to have been caused by protein concentration in the homogenate because there were 2.1, 1.2, 1.02 and 1.04 mg per assay tube for the ovine, human, bovine and rat, respectively. Possibly, relative concentrations of G P A T per mg of protein were different, or G L B S inhibition or end-product degradation occurred. Although G3P incorporation decreased between 30 and 60 rain for rat adipose tissue homogenates, the proportion of T G produced increased from 38 to 54% and the proportion of PL decreased from 50 to 33% (Table 1). In the human, from 30 to 120 min the proportion of G3P incorporated into T G increased from 43 to 51% while PL decreased by an equal proportion from 48 to 40%; this occurred without
Glycerolipid biosynthesis in adipose tissue Table 1. Effect of incubation time on synthesis of TG, DG and PL in homogenates of human, ovine, bovine and rat adipose tissue* Species Glycerolipid Timer Human Ovine Bovine Rat (percent) TG 5 14.6 3.4 8.4 8.3 30 42.7 9.2 5.3 36.9 120 51.3 18.6 7.1 53.5 SE:~ 4.1 1.5 4.0 2.3 DG 5 8.0 14.7 6.8 8.9 30 9.2 16.6 6.8 13.0 120 8.5 15.7 6.6 13.3 SE 1.0 0.6 1.2 0.8 PL 5 77.1 79.7 70.0 82.5 30 48.0 73.5 83.0 49.8 120 39.9 65.4 85.0 33.0 SE 4.0 1.8 1.9 2.8 *Triacylglycerol = TG, diacylglycerol = DG (1,2- and 1,3isomers combined), and phospholipid = PL. For human, bovine, ovine, and rat assays, protein contents per assay were 1.20, 2.10, 1.02, and 1.04 mg, respectively. i'Values for time represent minutes of incubation. For rat homogenates, values corresponding to 120 min were for 60 min of incubation. :[:SE = pooled standard error for time within each species and glycerolipid. further increases in total GLBS. In the ovine, from 30 to 120 min of incubation, little additional change in total G L B S occurred; however, the proportion of T G increased from 9 to 19% and PL decreased from 74 to 65%. Finally, D G represented 16% of glycerolipids synthesized at 120 min in ovine adipose tissue homogenates, which was nearly twice that o f human homogenates at 120 min (9%) and similar to that of rat homogenates at 60 min (13%). This may indicate that D G A T was rate-limiting for T G synthesis and (or) T G degradation to D G exceeded the rate of further degradation to glycerol. With bovine adipose tissue homogenates, G L B S increased steadily through 180 min of incubation. In contrast to the other three species, with bovine adipose tissue homogenates the 160 -
(A)
Table 2. Effect of homogenate concentration on synthesis of TG, DG, and PL in human, ovine, bovine and rat adipose tissue* Species Glycerolipid Volume~"Human TG
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The effects on G L B S of the amount of homogenate in the assay are shown in Fig 2A and B. A t 10 min, incorporation of G3P did not change with increased bovine homogenate (Fig. 2A). The G L B S rates with 100 #1 of homogenate were 25 and 34% of the rates with 300/d of homogenate for human and ovine, respectively, for 10-min assays. At each volume of homogenate the ovine had greater 10-min G L B S activity than with the human or the bovine. If
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proportions of PL, T G and D G were 83, 5 and 7%, respectively, at 30 min and did not change during the remaining incubation time.
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16.9 45.3 53.8 6.4 12.6 6.9 7.8 2.7 70.2 47.7 38.3 3.6
*Triacylglycerol = TG, diacylglycerol = DG (1,2- and 1,3isomers combined), and phospholipid = PL. For human, ovine, bovine, and rat assays, protein contents per I00 #1 of homogenate were 0.40, 0.93, 0.35, and 0.34mg, respectively. ?Volumes are #1. For rat homogenates, volumes were 200, 250, and 300/~1 instead of 100, 200, and 300/d, respectively. ~SE = pooled standard error for volume within each species and glycerolipid.
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Fig. 2. Effect of homogenate volume on glycerolipid biosynthesis. A. Assays were for 10 min for human, ovine and bovine, and 30 min for rat assays. B. Assays were for 2 hr. All data are means of triplicate assays plus or minus the standard error except for the rat in which tissue from several animals was pooled. All assays contained 17.5 mM G3P, 50mM HEPES, 0.98 mM DTT, 0.16 mM CoA, 12 mM ATP, 0.5 mg/ml BSA, 20 mM K2HPO4, 12 mM MgC12, 0.3 mM palmitate and protein concentrations per assay for human, ovine, bovine and rat preparations were 4, 9.3, 3.5 and 5.1/~g//d, respectively. CBPB 1 0 3 / 1 ~
D. C. RULE
96
expressed per mg of protein, similar values for human and ovine homogenates would have occurred because for the ovine with 100 pl of homogenate there were 0.93 mg of protein and for the human, 0.40 mg. There were 0.35 mg of protein for the bovine with 100 pl of homogenate. With rat adipose tissue homogenates, however, a markedly different response occurred. F r o m 100 to 150 #1 of homogenate G L B S increased by 1 7 ° , and then decreased linearly by 64% with 300/~1 of homogenate. The decreased incorporation represented an inhibition of G L B S and (or) extensive irreversible loss of glycerolipid. In Fig. 2B effects of homogenate volume after 120 min of incubation are presented. For the bovine homogenate, a linear increase in G L B S was observed. F o r both human and ovine homogenates, there was a rapid linear increase followed by a gradual reduction in rate to apparent asymptotes. The major contrast observed between ovine and human G L B S was in the proportions of TG, D G and PL produced (Table 2). With increasing homogenate volume the proportion of PL decreased and that of T G and D G increased in both species. However, with the ovine homogenate at 300ktl, T G and D G were 19 and 17%, respectively, whereas T G and D G were 54 and 8%, respectively, for human homogenates at 300 pl. The data suggest different rates of PPH in the two homogenates, with that in the human being greater than that in the ovine. Overall, the effects of time and homogenate volume on assay of G L B S indicated several marked contrasts between the four species under investigation. O f the four species, ovine and human adipose tissue responded to these two assay conditions most similarly; however, glycerolipid proportions were quite different. Homogenates prepared from rat adipose tissue gave negative results because of the inhibition and (or) degradation of end-product of G L B S with both incubation time and homogenate volume.
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COA (mU) Fig. 4. Effect of CoA on glycerolipid biosynthesis. Values are means of triplicate assays plus the standard error except for rat assays in which tissues from several animals were pooled. Data are expressed per mg of homogenate protein. Assays contained 17.5 mM G3P, 50 mM HEPES, 0.98 mM DTT, 12mM ATP, 0.5mg/ml BSA, 20mM K2HPO 4, 12 mM MgCI 2 and 0.3 mM palmitate. Protein contents for human, ovine, bovine and rat assays were 1.2 + 0.33 (SE), 1.87 + 0.08, 1.09 + 0.17 and 0.84 mg, respectively.
Assay component concentration Neither BSA at 0.25, 0.50 or 1.0 mg/ml nor H E P E S at 25, 50 or 1 0 0 m M changed the rate of G L B S in adipose tissue homogenates from any of the species studied. The C o A concentration (Fig. 3) did not affect G L B S in homogenates from human, ovine or bovine adipose tissue. For the rat homogenates, G L B S decreased by 17% when C o A was increased from 0.16 to 0.32 mM. Because rat adipose tissue had 140
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Fig. 3. Effect of ATP on glycerolipid biosynthesis. Values are means of triplicate incubations plus or minus the standard error and expressed per mg of homogenate protein. Assays contained 17.5 mM G3P, 50 mM HEPES, 0.98 mM DTT, 0.16mM CoA, 0.5 g/ml BSA, 20mM K2HPO 4 and 0.3 mM palmitate; MgCI 2 was varied equimolar with ATP concentration. Protein contents for human, ovine, bovine and rat assays were 1.20 _ 0.33 (SE), 1.60 _ 0.1, 1.06 _ 0.18 and 0.83 +__0.07 mg, respectively.
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0305-0491/92$5.00+ 0.00 © 1992PergamonPress Ltd
COMPARISON OF GLYCEROLIPID BIOSYNTHESIS IN HOMOGENATES FROM HUMAN, OVINE, BOVINE AND RAT ADIPOSE TISSUE I N VITRO D. C. RULE
Department of Animal Science, University of Wyoming, Laramie, WY 82071, U.S.A. Tel.: (307) 766-3404; Fax: (307) 766-5098 (Received I0 February 1992; accepted 11 March 1992) Abstract--1. Assay conditions were compared for glycerolipid biosynthesis in homogenates prepared from human abdominal, ovine and bovine subcutaneous, and rat epididymal adipose tissues. 2. In contrast to other species, longer incubation time and greater homogenate concentration resulted in decreased glycerolipid biosynthesis with rat adipose tissue homogenates. 3. Species differences were observed in concentration-dependency for ATP and fatty acids (palmitate, oleate and palmitoleate). 4. Results indicated that glycerolipid biosynthesis transpired at different rates in the four species, and that ovine and human adipose tissue homogenates had similar properties.
(G3P) in a reaction catalyzed by glycerophosphate acyltransferase (GPAT; EC 2.3.1.15). A second esterification occurs, producing phosphatidate (a phospholipid, PL), the phosphate of which is subsequently hydrolyzed in a reaction catalyzed by phosphatidate phosphohydrolase (PPH, EC 3.1.3.4) producing diacylglycerol (DG). A third esterification occurs in a reaction catalyzed by diacylglycerol acyltransferase (DGAT) (EC 2.3.1.20) to produce TG. Although the pathway by which fatty acids are esterified to G3P appears to be the same in adipose tissue of most mammals, most research on regulation of this pathway has been conducted using rat adipose tissue. Similarities in catecholamine regulation of GLBS between rat and swine adipose tissue have been reported (Rule et al., 1987). Because several enzymes are involved in this pathway, and because the enzymes are membrane-bound and the substrates are large, relatively hydrophobic molecules, the particular assay condition employed can markedly change the rate and products of GLBS assays. In previous studies detailed observations regarding assay conditions for GLBS in swine adipose tissue homogenates documented such changes (Rule et aL, 1988a,b). The purpose of the present study was to understand the potential of adipose tissue GLBS as a biomedical model system by comparing the effects of varied assay conditions in homogenates prepared from human (Homo sapiens), ovine (Ovis aries), bovine (Bos taurus) and rat (Rattus rattus) adipose tissue.
INTRODUCTION The use of various species for biomedical research should require similarities between the model and the particular species of interest. For example, swine have been used as models for dietary protein and fat studies because of the similarity in human and swine digestive tracts (Diersen-Schade et al., 1985; Diersen-Schade et al., 1986). Also, swine, as well as rabbits and some non-human primates, are well suited as models for atherosderosis research (Armstrong and Heistad, 1990). For the interaction of dietary lipids and subsequent blood lipid effects the male Golden Syrian hamster has been used because of its similarity to humans regarding diet-induced LDL-cholesterol changes (Dietschy, 1985; Turley et aL, 1987). Cellular metabolism studies conducted with animal models for biomedical application should also consider similarities or differences between species. For example, de novo fatty acid biosynthesis occurs primarily in the liver in humans (Shrago et al., 1971) and chickens (O'Hea and Leveille, 1969a), but it occurs in adipose tissue of cattle (Hood et al., 1972), sheep (Ingle et al., 1972), swine (O'Hea and Leveille, 1969b; Mersmann et al., 1973), dogs (Baldner et al., 1985) and cats (Richard et al., 1989). In rats and mice, this process occurs in both liver and adipose tissue (Leveille, 1966). Although site differences exist for fatty acid biosynthesis in a number of species, glycerolipid biosynthesis (GLBS) commonly occurs in adipose tissue (Bell and Coleman, 1980; Bell and Coleman, 1983), liver (Nimmo, 1980), and gut (Brindley, 1985) of mammals. In adipose tissue GLBS is primarily involved in triacylglycerol (TG) production using the following metabolic pathway (Nimmo, 1980): activation of fatty acid by thioesterification to CoA, which is catalyzed by acyl--CoA synthetase (EC 6.2.1.3) and requires A T P - M g for energy, is followed by esterification of acyl--CoA to glycerol-3-phosphate
METHODS
Reagents Coenzyme A (CoA, sodium salt, C-3144), ATP (grade II disodium salt, A-3377), ethylenediaminetetraacetic acid (EDTA, EDS), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, H-3375), dithiothreitol (DTT, D0632), bovine serum albumin (BSA, fraction V, fatty acid 93
94
D.C. RULE
poor, A-6003), palmitic acid (P-5917), oleic acid (0-3879), palmitoleic acid (P-0875) and glycerol-3-phosphate (G3P, disodium salt, G-2138) were purchased from Sigma Chemical Co. (St Louis, MO). 14C-G3P(L-[14C(U)]-glycerol-3-phos phate, disodium salt, NEC-608) was purchased from New England Nuclear, (Boston, MA). All other reagents were reagent grade quality.
E
140 E 120
60
_Ra
Tissues
Adipose tissues were obtained from single sites for each species. Abdominal adipose tissue was obtained from two human subjects who underwent routine, abdominal surgery. Patient consent forms were prepared according to guidelines set forth by both the University of Wyoming and the Ivinson Memorial Hospital Institutional Review Boards. Subjects were otherwise healthy, middle-aged females. Ovine subcutaneous adipose tissue was obtained from the tail head area immediately after sacrifice of three Suffolk castrated lambs that weighed 66.5 + 4.2 (SEM) kg. Bovine subcutaneous adipose tissue also was obtained immediately after sacrifice from the tail head area of three Hereford x Angus steers that weighed 486.9 __+11.9 kg. Sheep and cattle were sacrificed by stunning followed by exsanguination in the Animal Science Department abbatoir under supervision of a Wyoming Department of Agriculture inspector. Rat epididymal adipose tissue was dissected from 300 to 500 g, male Sprague-Dawley rats after euthanization with chloroform. Adipose tissue from five rats was pooled to obtain enough homogenate for experiments. Additional rats were sacrificed to obtain enough adipose tissue for experimental replication in the later experiments. Tissues from each species were placed in phosphate-buffered saline at 4°C for transport to the laboratory. Homogenates of adipose tissue were prepared essentially as described by Rule et al. (1988a); 1.0g of adipose tissue was homogenized in 1.5 ml of homogenization buffer (0.15 M KCI, 10 mM HEPES, 1 mM EDTA and 1 mM DTT, pH 7.4, 4°C). The supernatant fat-cake was removed after centrifugation at 700g, and 5-ml aliquots of each aqueous infranatant fraction were snap-frozen in liquid nitrogen and stored at -70°C for up to 2 months. For each preparation 5- and 60-min GLBS assays were conducted to assess activity in fresh homogenates. This method of storage preserved enzyme activity, as it did for porcine homogenates (Rule et al., 1988b). Glycerolipid biosynthesis assay
Glycerolipid biosynthesis assays for each species initially were based on homogenate, substrate, cofactor and buffer concentrations reported previously for swine subcutaneous adipose tissue homogenates (Rule et al., 1988a). In 0.4 ml of total assay volume there were: 0.3 ml of 700g supernatant fraction (homogenate), 17.5mM G3P, 0.3/~Ci ~4C-G3P, 0.3 mM potassium palmitate, 1.0 mM DTT, 0.16 mM CoA, 12.0 mM ATP, 0.5 mg/ml BSA, 12.0 mM Mg C12, 50.0 mM HEPES, and 20.0 mM K2HPO4. All buffer stock solutions were pH 7.4 and all substrate and cofactor stock solutions were pH 7.0. A substrate and cofactor premix was prepared so that 0.1 ml was combined with 0.3 ml of homogenate in a 16 × 125 mm test tube at 37°C in duplicate to start the reaction. Reactions were terminated by adding 3.0 ml of 1 : 2 chloroform: methanol (v/v) and 5 sec of vortex-mixing. Details of glycerolipid extraction and separation of glycerolipids by thin-layer chromatography have been described previously (Rule et al., 1988a). Quantitation of 14C was accomplished by liquid scintillation spectrometry to calculate G3P incorporation into glycerolipids. Data were expressed on a mg of protein basis. Total homogenate protein was determined on TCA-precipitated material by using the biuret procedure (Gomall et al., 1949). For all assays, observations were obtained from preparations from two humans, three ovines, three bovines, and three rats or a pool of tissue from three to five rats.
Human
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Fig. 1. Effect of incubation time on glycerolipid biosynthesis. Values are means of triplicate observations plus or minus the standard error of the mean and expressed per mg of homogenate protein. Assays contained 17.5 mM G3P, 50mM HEPES, 0.98mM DTT, 0.16mM CoA, 12mM ATP, 0.5mg/ml BSA, 20mM K2HPO4, 12mM MgCI2, 0.3 mM palmitate and assays for human, ovine, bovine and rat preparations contained 1.2 + 0.33 (SE), 1.87 + 0.08, 1.09 4- 0.17 and 0.84 4- 0.05 mg of protein, respectively.
RESULTS Incubation time
Effects of incubation time on G L B S in adipose tissue from the human, rat, ovine and bovine are illustrated in Fig. 1. Initial (5 min) values were lowest for the bovine, intermediate for the human and ovine, and highest for the rat adipose tissue homogenates. Distinct linear increases in G L B S were observed for bovine, ovine, human, and rat adipose tissue homogenates. Rates of bovine, ovine and human G L B S remained linear and distinctly different for 20 min. F o r the rat, rates were much greater than those of the other three species from 5 to 10min. At 120 and 60 min G L B S was maximal for human and ovine adipose tissue homogenates, respectively, whereas at 180 min G L B S in the bovine was still increasing, though at a reduced rate. In the rat maximal G L B S was observed at 30 min, which was followed by a 20% decrease at 60 min. The major similarity between species in this experiment was the initial rate of G L B S for the human and ovine homogenates. This similarity, however, was followed by a marked contrast in maximal G L B S between these two species with the human G L B S being nearly twice that of the ovine at 120 min. The contrasts between all species would not appear to have been caused by protein concentration in the homogenate because there were 2.1, 1.2, 1.02 and 1.04 mg per assay tube for the ovine, human, bovine and rat, respectively. Possibly, relative concentrations of G P A T per mg of protein were different, or G L B S inhibition or end-product degradation occurred. Although G3P incorporation decreased between 30 and 60 rain for rat adipose tissue homogenates, the proportion of T G produced increased from 38 to 54% and the proportion of PL decreased from 50 to 33% (Table 1). In the human, from 30 to 120 min the proportion of G3P incorporated into T G increased from 43 to 51% while PL decreased by an equal proportion from 48 to 40%; this occurred without
Glycerolipid biosynthesis in adipose tissue Table 1. Effect of incubation time on synthesis of TG, DG and PL in homogenates of human, ovine, bovine and rat adipose tissue* Species Glycerolipid Timer Human Ovine Bovine Rat (percent) TG 5 14.6 3.4 8.4 8.3 30 42.7 9.2 5.3 36.9 120 51.3 18.6 7.1 53.5 SE:~ 4.1 1.5 4.0 2.3 DG 5 8.0 14.7 6.8 8.9 30 9.2 16.6 6.8 13.0 120 8.5 15.7 6.6 13.3 SE 1.0 0.6 1.2 0.8 PL 5 77.1 79.7 70.0 82.5 30 48.0 73.5 83.0 49.8 120 39.9 65.4 85.0 33.0 SE 4.0 1.8 1.9 2.8 *Triacylglycerol = TG, diacylglycerol = DG (1,2- and 1,3isomers combined), and phospholipid = PL. For human, bovine, ovine, and rat assays, protein contents per assay were 1.20, 2.10, 1.02, and 1.04 mg, respectively. i'Values for time represent minutes of incubation. For rat homogenates, values corresponding to 120 min were for 60 min of incubation. :[:SE = pooled standard error for time within each species and glycerolipid. further increases in total GLBS. In the ovine, from 30 to 120 min of incubation, little additional change in total G L B S occurred; however, the proportion of T G increased from 9 to 19% and PL decreased from 74 to 65%. Finally, D G represented 16% of glycerolipids synthesized at 120 min in ovine adipose tissue homogenates, which was nearly twice that o f human homogenates at 120 min (9%) and similar to that of rat homogenates at 60 min (13%). This may indicate that D G A T was rate-limiting for T G synthesis and (or) T G degradation to D G exceeded the rate of further degradation to glycerol. With bovine adipose tissue homogenates, G L B S increased steadily through 180 min of incubation. In contrast to the other three species, with bovine adipose tissue homogenates the 160 -
(A)
Table 2. Effect of homogenate concentration on synthesis of TG, DG, and PL in human, ovine, bovine and rat adipose tissue* Species Glycerolipid Volume~"Human TG
100 200 300 SE~/ 100 200 300 SE 100 200 300 SE
DG
PL
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6.0 8.3 7.8 1.9 4.9 5.4 6.4 0.4 83.9 85.4 84.7 2.2
The effects on G L B S of the amount of homogenate in the assay are shown in Fig 2A and B. A t 10 min, incorporation of G3P did not change with increased bovine homogenate (Fig. 2A). The G L B S rates with 100 #1 of homogenate were 25 and 34% of the rates with 300/d of homogenate for human and ovine, respectively, for 10-min assays. At each volume of homogenate the ovine had greater 10-min G L B S activity than with the human or the bovine. If
Od
~
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Assay homogenate concentration
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proportions of PL, T G and D G were 83, 5 and 7%, respectively, at 30 min and did not change during the remaining incubation time.
/"
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16.9 45.3 53.8 6.4 12.6 6.9 7.8 2.7 70.2 47.7 38.3 3.6
*Triacylglycerol = TG, diacylglycerol = DG (1,2- and 1,3isomers combined), and phospholipid = PL. For human, ovine, bovine, and rat assays, protein contents per I00 #1 of homogenate were 0.40, 0.93, 0.35, and 0.34mg, respectively. ?Volumes are #1. For rat homogenates, volumes were 200, 250, and 300/~1 instead of 100, 200, and 300/d, respectively. ~SE = pooled standard error for volume within each species and glycerolipid.
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Fig. 2. Effect of homogenate volume on glycerolipid biosynthesis. A. Assays were for 10 min for human, ovine and bovine, and 30 min for rat assays. B. Assays were for 2 hr. All data are means of triplicate assays plus or minus the standard error except for the rat in which tissue from several animals was pooled. All assays contained 17.5 mM G3P, 50mM HEPES, 0.98 mM DTT, 0.16 mM CoA, 12 mM ATP, 0.5 mg/ml BSA, 20 mM K2HPO4, 12 mM MgC12, 0.3 mM palmitate and protein concentrations per assay for human, ovine, bovine and rat preparations were 4, 9.3, 3.5 and 5.1/~g//d, respectively. CBPB 1 0 3 / 1 ~
D. C. RULE
96
expressed per mg of protein, similar values for human and ovine homogenates would have occurred because for the ovine with 100 pl of homogenate there were 0.93 mg of protein and for the human, 0.40 mg. There were 0.35 mg of protein for the bovine with 100 pl of homogenate. With rat adipose tissue homogenates, however, a markedly different response occurred. F r o m 100 to 150 #1 of homogenate G L B S increased by 1 7 ° , and then decreased linearly by 64% with 300/~1 of homogenate. The decreased incorporation represented an inhibition of G L B S and (or) extensive irreversible loss of glycerolipid. In Fig. 2B effects of homogenate volume after 120 min of incubation are presented. For the bovine homogenate, a linear increase in G L B S was observed. F o r both human and ovine homogenates, there was a rapid linear increase followed by a gradual reduction in rate to apparent asymptotes. The major contrast observed between ovine and human G L B S was in the proportions of TG, D G and PL produced (Table 2). With increasing homogenate volume the proportion of PL decreased and that of T G and D G increased in both species. However, with the ovine homogenate at 300ktl, T G and D G were 19 and 17%, respectively, whereas T G and D G were 54 and 8%, respectively, for human homogenates at 300 pl. The data suggest different rates of PPH in the two homogenates, with that in the human being greater than that in the ovine. Overall, the effects of time and homogenate volume on assay of G L B S indicated several marked contrasts between the four species under investigation. O f the four species, ovine and human adipose tissue responded to these two assay conditions most similarly; however, glycerolipid proportions were quite different. Homogenates prepared from rat adipose tissue gave negative results because of the inhibition and (or) degradation of end-product of G L B S with both incubation time and homogenate volume.
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COA (mU) Fig. 4. Effect of CoA on glycerolipid biosynthesis. Values are means of triplicate assays plus the standard error except for rat assays in which tissues from several animals were pooled. Data are expressed per mg of homogenate protein. Assays contained 17.5 mM G3P, 50 mM HEPES, 0.98 mM DTT, 12mM ATP, 0.5mg/ml BSA, 20mM K2HPO 4, 12 mM MgCI 2 and 0.3 mM palmitate. Protein contents for human, ovine, bovine and rat assays were 1.2 + 0.33 (SE), 1.87 + 0.08, 1.09 + 0.17 and 0.84 mg, respectively.
Assay component concentration Neither BSA at 0.25, 0.50 or 1.0 mg/ml nor H E P E S at 25, 50 or 1 0 0 m M changed the rate of G L B S in adipose tissue homogenates from any of the species studied. The C o A concentration (Fig. 3) did not affect G L B S in homogenates from human, ovine or bovine adipose tissue. For the rat homogenates, G L B S decreased by 17% when C o A was increased from 0.16 to 0.32 mM. Because rat adipose tissue had 140
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Fig. 3. Effect of ATP on glycerolipid biosynthesis. Values are means of triplicate incubations plus or minus the standard error and expressed per mg of homogenate protein. Assays contained 17.5 mM G3P, 50 mM HEPES, 0.98 mM DTT, 0.16mM CoA, 0.5 g/ml BSA, 20mM K2HPO 4 and 0.3 mM palmitate; MgCI 2 was varied equimolar with ATP concentration. Protein contents for human, ovine, bovine and rat assays were 1.20 _ 0.33 (SE), 1.60 _ 0.1, 1.06 _ 0.18 and 0.83 +__0.07 mg, respectively.
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