Comp. Biochem. Physiol. Vol. 103B,No. 3, pp. 511-516, 1992
0305-0491/92 $5.00+ 0.00 Pergamon Press Ltd
Printed in Great Britain
TRIACYLGLYCEROL BIOSYNTHESIS IN BOVINE LIVER A N D SUBCUTANEOUS ADIPOSE TISSUE J. J. WILSON,C. R. YOUNO and S. B. SmTH Texas Agricultural Experiment Station, Texas A&M University, College Station, TX 77843-2471, U.S.A. (Tel. 409-845-3939; Fax 409-845-9454) (Received 1 May 1992; accepted 5 June 1992)
Abstract--1. Adipose tissue from Angus and Brahman steers incubated with [l-~4C]palmitate in the absence and presence of glucose exhibited a greater rate of lipid production than liver (P < 0.05). 2. Homogenates of adipose tissue used in the glycerol-3-phosphate acyltransferase assay exhibited a greater glycerolipid specific activity (nmol lipid/mg protein/30 rain) when compared to liver (P < 0.05). 3. The inverse was true for liver homogenates when calculated for tissue activity (nmol lipid/g tissue/30 rain). 4. Lysophosphatidate was produced in greater (P < 0.05) amounts than all other glycerolipids in the glycerol-3-phosphate acyltransferase assay. 5. The activity of phosphatidate phosphohydrolase in liver homogenates displayed greater rates than their respective adipose tissue homogenates. 6. Diaeylglycerol aeyltransferase activity was greater in adipose tissue homogenates compared to liver homogenates.
INTRODUCTION
MATERIALSAND METHODS
Adipose tissue is the primary site of fatty acid biosynthesis in ruminants. Studies /n vivo of Ingle et aL (1972) suggest that in the non-lactating sheep, adipose tissue is responsible for more than 90% of fatty acid biosynthesis and that the primary metabolic function of ruminant liver is gluconeogenesis. In vitro studies support this conclusion in cattle, sheep and goats, as rates of fatty acid synthesis in adipose tissue slices are more than 20-fold greater than rates in liver slices, with the final product being tdaeyiglycerols (Hood et al., 1972; Ingle et al., 1972; Palmquist, 1975; Christie et al., 1976; Deeth and Christie, 1979). The microsomal phosphatidic phosphohydrolase system is generally assumed to be the rate-limiting step in the pathway leading to triacylglycerol formation (Vavrecka et al., 1969). Others have shown that glycerol-3-phosphate acyltransferase is ratelimiting when animals are subjected to starvation and refeeding (Jamdar and osborn, 1982). Also, the activities of the esterification enzymes depend heavily upon cell size (Etherton and Allen, 1980a; Jamdar and Osborne, 1981), tissue depot and precursors used (Declercq et al., 1982), the concentrations of cofactors like ATP, coenzyme A (CoA) and MgCI2 (Raju and Six, 1975) and the amount of acyl-CoA (Lederer and Hers, 1985). Because of the paucity of information concerning triacylglycerol biosynthesis in cattle, the objective of this study was to compare the activities of enzymes responsible for fatty acid esterification. The activities were measured in the presence and absence of acylglycerol precursors, in both liver and adipose tissues and correlated to triacylglycerol biosynthesis in subcellular fractions. 511
Materials
Biochemicals were purchased from Sigma Chemical Co. (St Louis, MO). Radioisotopes were purchased from Amersham Corp. (Arlington Heights, IL). All other chemicals were purchased from Fisher Scientific (Houston, TX) and were reagent grade or greater. Animals and procedures Purebred Angus (Bos taurus) (N = 2) and Brahman (Bos indicus) (N = 4) steers (18 months of age) were fed a
high-energy ration consisting of 10% cottonseed hulls, 11% cottonseed meal and 72% ground milo, plus a vitamin and mineral supplement for 100 days at the Texas A&M University, McGregor Research Facility. The cattle were transported to and slaughtered at the Rosenthal Meat Science and Technology Center, Texas A&M University, College Station. Liver and subcutaneous adipose tissue samples were obtained within 60 min after the animal was exsanguinated. The animals suffered no unusual stress. Samples were placed in a solution of warm, oxygenated Krebs-Henseleit Ca2+free bicarbonate buffer (KRB) with 5mM glucose and transferred to the laboratory for processing. All samples were removed from the animal and in the laboratory for processing within 1 hr from the time the animal was stunned. Preparation of tissue incubations
Liver and adipose tissue slices (100-150mg) were incubated in vitro as described by Etherton and Allen (1980a), with modifications as needed. Half of the liver and adipose tissue slices were incubated in 20-ml scintillation vials, in triplicate, in 3 ml of KRB buffer (pH 7.4) that contained 120mM NaC1, 4.8raM KCI, 1.2mM MgC12, 1.2mM KH2PO4, 25 mM NaHCO3, 2 #Ci [1-14C]palmitic acid, 1 mU/ml insulin, 5 mM glucose, 10 mM Hepes (N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid), 0.75 mM palmitate (potassium salt) and BSA (30 mg/ml). The second half of the tissue sample was placed in a similar 3-ml mixture without glucose. All tissue samples were incubated for
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30 min at 37°C while shaking (120 strokes/min). The incubations were terminated after 30 min by the addition of 3 ml 0.31 M trichloroacetic acid (TCA), followed by removal of tissue from the incubation media. Samples were rinsed first with 9 ml KRB and then rinsed with 9 ml 0.15 M NaC1 and placed in screw cap tubes with 10 ml chloroform:methanol (CHCI3:CH3OH, 2:1, v/v) under nitrogen and allowed to stand overnight. All tissue samples were homogenized at rheostat setting No. 7 for 60 sec with a Kinematic homogenizer (model pt 10-35, Kinematic Inc., Rockville Center, NY). Free fatty acids were separated from the glycerolipids by the addition of 5 ml 0.38 M Na2CO3 and shaking of the tubes for 20 min at high speed, centrifuging for 20 min at 700g and removing the top layer (Na2CO 3 plus nonesterified fatty acids) by aspiration. The CHC13:CH3OH layer was extracted two more times with Na2CO 3 and filtered through a glass microfiber filter (Whatman GF/C, 2.4 cm, Whatman Ltd., Maidstone, England). When lipid products were determined, the CHC13:CH3OH phase was dried under nitrogen, and the volume adjusted to 2 ml with CHC13:CH3OH. Aliquots of 50#1 for adipose tissue and 100 #1 for liver were separated into glycerolipid classes by thin-layer chromatography (TLC) on 250-micron silica gel-G plates. Glycerolipids were visualized by exposure to iodine vapors after development with petroleum ether/diethyl ether/acetic acid (90/10/4, v/v/v) for neutral lipids and with dichloromethane/methanol/3 M NH4OH (65/35/9, v/v/v) for phospholipids. Glycerolipids classes were identified by comparison to standards run on the same TLC plates as the samples and the corresponding spots were scraped into scintillation vials with 5 ml scintillation fluid for liquid scintillation counting. The glycerolipids remaining after removal of samples for TLC were dried, resuspended in 10 ml scintillation fluid and counted to obtain total lipids produced. Preparation o f liver and adipose tissue homogenates and enzyme assays Liver and adipose tissue samples were obtained as stated for incubations. Tissue samples were scissor-sliced prior to homogenization. The liver and adipose tissue homogenates were prepared with 3 vol (w/v) 0.15 M KCI: I0 mM HEPES: 1 mM EDTA: 1 mM dithiothreitol (DTT) (pH 7.4) using a Kinematic homogenizer at rheostat setting No. 8 (3 x 20 see) on ice for adipose tissue and with a loose-fitting teflon pestle in a glass mortar at setting No. 7 (4 x 15 sec) on ice for liver tissue. All tissue samples were centrifuged at 700 g for 30 min to remove cellular debris. The remaining supernate was centrifuged at 11,000 g for 30 min to remove the mitochondrial pellet. Microsomal pellets were obtained by centrifuging the supernate twice at 104,000 g for 60 min. All fractions were kept on ice during processing and centrifuged at 5°C to inhibit the degradation of desired fractions and to preserve enzyme activity. All pellets were rinsed and resuspended in 5 mi of the homogenization buffer, kept on ice and used fresh. All subcellular fractions were analyzed for protein content by the procedure of Lowry et al. (1951). Homogenates used in the glycerol-3-phosphate acyltransferase (EC 2.3.1.15) assay were assayed as described by Lamb and Fallon (1974) with modifications, in a reaction mixture containing 150mM HEPES (pH 7.4), 10mM MgC12, 6 m M ATP, 8 0 # M CoA, I mM DTT, 0.6mM palmitate, 10mM glycerol-3-phosphate, 0.5#Ci L-[U~4C]glycerol-3-phosphate and 0.2 mg BSA. Reactions were started by either the addition of 0.3 ml of mitochondrial or microsomal enzyme fraction or 0.I ml of supernate to the appropriate assay tubes (Liver: mitochondria, 3.4-5.4 mg/ tube; mierosomes, 1.9-2.6 rag/tube; supernate, 1.3-1.9 mg/ tube. Adipose: mitochondria, 0.20-0.50mg/tube; microsomes, 0.12-0.58 mg/tube; supernate, 0.11-0.30 mg/tube). The final volume was adjusted to 1.5 ml and the samples were incubated for 30min at 37°C while shaking (120
strokes/min). Assays were terminated by the addition of 3 ml CHC13:CHaOH (2:1, v/v) with thorough mixing. After 10 min, 1 ml CHCI3, 0.5 ml 2 M KCI:0.3 N HC1 and 0.9 ml distilled~leionized H20 were added and the solution was mixed and then centrifuged at 700g for 10min at room temperature. The upper aqueous phase was aspirated and discarded and the lower phase was washed three times with 2 ml 0.2 N HCI:CH3OH (1:I) to remove labeled glycerol3-phosphate (G-3-P) that was not incorporated into glycerolipids during the incubation. A 100 #1 aliquot was separated into glycerolipid classes by TLC as described above. Glycerolipids were visualized by exposure to iodine vapors and the appropriate spots were scraped into scintillation vials for counting of radioactivity. Phosphatidate phosphohydrolase (EC 3.1.3.4) activity was assessed by the method of Lamb and Fallon (1974) with an aqueous dispersion of phosphatidate as substrate. The release of inorganic orthophosphate by the enzyme was measured as outlined by Sanui (1974). The procedure of Manley et al. (1974) with modifications was used for evaluating diacylglyceride acyltransferase (EC 2.3.1.20) activity in the conversion of diacylglycerol to triacylglycerol for both tissues. The reaction mixture contained 150mM HEPES (pH 7.4), 10mM MgCI 2, 6 m M ATP, 8 0 # M CoA, 1 mM DTI', 0.2mg BSA, I mM oleic acid, 0.5 #Ci [l-~4C]oleic acid and 3 mM 1,2-dioleyl-glycerol in Tween-20. Reactions were started by the addition of 0.3 or 0.1 ml of the appropriate fraction to a final volume of 1.6 ml. Assays were allowed to run for 30 min at 37°C while shaking (120 strokes/min). The reactions were terminated by the addition of 2 ml 0.31 M TCA and mixed for 30 seconds. Two ml CHC13:CH3OH (2:1, v/v) with 0.01#mol/ml triolein were added to the reaction mixture to trap the esterified lipids in the organic phase. The tubes were shaken to obtain a better mixture of the CHCI3:CH3OH. Subsequently, 2 ml 0.38 M Na2CO3 were added to extract the labeled free fatty acids. The mixture was shaken for 10min at high speed and centrifuged at 700g for 10min after which time the top layer was discarded and the remaining sample was extracted twice with Na2COj. The CHCI3:CH3OH layer was dried under nitrogen and then resuspended in 0.5 ml chlorofoi'm:methanol from which a 150#1 sample was taken for TLC. After development in petroleum ether/diethyl ether/acetic acid (70/30/1, v/v/v), the triacylglycerides were identified by exposure to iodine vapors, scraped and quantitated in the liquid scintillation counter as before. Statistical analyses and expression of rates Statistical analyses were made using GLM procedures of SAS (1986) for a 2 (tissue depot)x 2 (substrate)x 5 (glycerolipid) design for the tissue incubations. The enzyme analysis was for a 2 (tissue)x 5 (glycerolipid) design to determine major end product and for a 2 (tissue)x 3 (centrifugal fraction) design to determine major enzyme activity. Tissue depot, substrate, glycerolipid and centrifugal fraction were used as the main effects to analyze the data. When the main effects were significant, mean separation was accomplished using the least squares means test (Montgomery, 1984). All data are tested at P < 0.05. Rates of palmitate esterification/n vitro and enzyme activities are reported as nmol substrate incorporated/rag protein/30 rain (specific activity) or as nmol substrate incorporated/g tissue/30 min (tissue activity). For specific activities of subcellular fractions, rates are expressed per respective amounts of mitochondrial, microsomal or supernatant protein. RESULTS Tissue incubations Esterification o f palmitate to triacylglycerols indicated t h a t the tissue was metabolically active at
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Triacylglycerol biosynthesis Table 1. Liver and subcutaneous adipose tissue slices incubated with and without glucose Tissue
Incubations conditions
Subcutaneous adipose tissue + glucose glucose -
Activity basis mg protein" g tissueb 797 711
2889 2401
4 23 157
735 972 467
0.0002 0.8351 0.7298
0.0015 0.7953 0.4289
Liver + glucose - glucose SEc Significance Tissued Substrate Interaction
"Least squares means for fatty acyI-CoA esterification; values are expressed as nmol glycerol-3-phosphate incorporated/rag protein/30 rain. bLeast squares means for fatty acyI-CoA esterification; values are expressed as nmol glycerol-3-phosphate incorporated/g tissue/ 30 rain. cSE = Pooled standard error of the mean (N = 6). dTissue ffi Subcutaneous adipose tissue esterified glycerolipids at a greater rate than liver.
the time of the incubations. The tissue produced primarily diacylglycerol as the final glycerolipid product if the tissue sample was not incubated within I hr after death (data not shown). Tissues used for the study in this report were incubated before this apparent 60 min critical period. As reported previously for de novo fatty acid synthesis (Hanson and Ballard, 1967; Ingle et aL, 1972), adipose tissue was more active than liver (P < 0.05) for either specific activity (nmol palmitate incorporated/rag protein/30min) or tissue activity (nmol palmitate incorporated/g tissue/30 rain). The addition of glucose did not affect the esterification rates for palmitate incorporation in either tissue (Table 1). Glycerolipid fractions produced during the/n vitro incubations, as determined by TLC separation, were the only tested main effect that generated a significant test value (P < 0.05) (Table 2). Triacylglycerols were in greater proportion (P < 0.05) than any other glycerolipid produced. Regardless of tissue or incubation conditions, virtually no phosphatidate accumulated in tissue incubations, indicating that the phosphatidic phosphohydrolase enzyme was not limiting triacylglycerol synthesis (Table 2). Enzyme assays
When compared to Lamb and Fallen (1974) for rat, the glycerol-3-phosphate assay liver cell fractions Table 2. Glycerolipid fractions (% of total) of tissue incubations separated by thin-layer chromatography Glycerolipid Monoaeylglycerol Diacylglycerol Triacylglycerol Lysophosphatidate SEa Significance Glycerolipidb
% of Total 1.9a~ 9.0d 83.7c 5.3 ~ 2.5 O.0001
" S E f P o o l e d standard error of the mean (N = 6). Data are pooled across tissue. ~ M e a n s within a column lacking a common superscript letter differ (P < 0.05).
Table 3. Glycerol-3-phosphate esterification into glycerolipids by homogenates from bovine liver and adipose tissue Tissue
Lipid fractions
Subcutaneous adipose homogenates Monoacylglyceride Diacylglyceride Triacyiglyceride Lysophosphatidate Phosphatidate Liver homogenates Monoacylglyceride Diacylglyceride Triacylglyceride Lysophosphatidate Phosphatidate SEc Significance Tissued Glycerolipide Interaction
Activity basis mg protein* g tissueb 20 25 32 163 76
42 47 23 134 99
36 8 8 71 12 19
475 118 157 741 110 88
0.0037 0.0001 0.0518
0.0001 0.0006 0.0056
'Least squares means for fatty acyl-CoA esteriflcation; values are expressed as nmol glycerol-3-phosphate incorporated/mg protein/30 rain. bLeast squares me.arts for fatty acyI-CoA esterification; values are expressed as nmol glycerol-3-phosphate incorporated/g tissue/30 min. cSE ffi Pooled standard error of the mean (N ffi 6). dSignificant ( P < 0 . 0 5 ) differences exist for nmol glycerol-3phosphate incorporated/rag protein/30 min and nmoI glycerol-3phosphate incorporated/g tissue/30 min. ~Lysophosphatidate was found in greater (P < 0.05) amounts than all other glycerolipids.
generated slightly higher rates of glycerolipid production for bovine subcellular fractions. Subcutaneous adipose tissue homogenates produced glycerolipids at a greater rate (P < 0.05) than liver tissue homogenates (diacylglycerol, triacylglycerol, lysophosphatidate and phosphatidate) when the rates were calculated as specific activity (nmol G-3-P incorporated/rag protein/30 min) (Table 3). There tended to be a tissue x glycerolipid interaction for data expressed as specific activity. Lysophosphatidate was the major glycerolipid produced in adipose tissue homogenates, whereas both lysophosphatidic acid and monoacylglycerol were abundant products in liver homogenates. Both tissue homogenates produced minor amounts of diacylglycerol and triacylglycerol. When the rates were calculated as tissue activities (nmol G-3-P incorporated/g tissue/30min), liver homogenates generated a greater (P < 0.05) rate of esterification of fatty acids than subcutaneous adipose tissue for all acylglycerol fractions. The tissue x glycerolipid interaction was significant; lysophosphatidate was the major glycerolipid produced in adipose tissue homogenates, whereas both lysophosphatidic acid and monoacylglycerol were abundant products in liver homogenates. The conversion of lysophosphatidate to phosphatidate by the acylation of a second fatty acyl-CoA was limiting in both tissues as indicated by the accumulation of lysophosphatidate (Table 3). In addition to glycerolipid fractions, subcellular distribution of enzyme activities was also determined (Table 4). Tissue and subcellular fraction main effects were significant (P < 0.05) for data expressed as specific activity. Adipose tissue exhibited greater specific activity than did liver. In both tissues, the
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Table 4. Glycerol-3-phosphate esterification into glycerolipids by enzyme fractions from liver and adipose tissue homogenates Activity basis Tissue Enzyme fraction mg proteina g tissue b Subcutaneous adipose tissue Mitochondria 92 93 Microsomes 172 83 Supernate 43 170 Liver Mitochondria 55 934 Microsomes 72 493 Supernate 43 170 SEe 26 133 Significance Tissue d 0.0090 0.0008 Fractionse 0.0028 0.0513 Interaction 0.4102 0.0165 aLeast squares means for fatty acyl-CoA esterification; values are expressed as nmol glycerol-3-phosphate incorporated/mg protein/30 min. bLeast squares means for fatty acyl-CoA esterification; values are expressed as nmol glycerol-3-phosphate incorporated/g tissue/30 min. 'SE = Pooled standard error of the mean (N = 6). aSignificant (P < 0.05) differences exist for nmol glycerol-3-phosphate incorporated/rag protein/30 rain and nmol glycerol-3phosphate incorporated/g tissue/30 min. CEnzyme fractions are significant (P < 0.05) for nmol glycerol-3phosphate incorporated/rag protein/30 min only, specific activity o f t h e m i c r o s o m e s exceeded t h a t o f the m i t o c h o n d r i a l a n d s u p e r n a t a n t fractions. Liver tissue activity exceeded a d i p o s e tissue activity for m i t o c h o n d r i a a n d m i c r o s o m e s . T h e tissue x f r a c t i o n i n t e r a c t i o n w a s significant ( P < 0.05) for t h e tissue activities ( T a b l e 4). I n a d i p o s e tissue, t h e greatest activity was o b s e r v e d in t h e s u p e r n a t a n t fraction, w h e r e a s in liver, t h e g r e a t e s t activity w a s o b s e r v e d in mitochondria. P h o s p h a t i d a t e p h o s p h o h y d r o l a s e activity was a n a lyzed as n m o l i n o r g a n i c o r t h o p h o s p h a t e (Pi) cleaved f r o m p h o s p h a t i d a t e acid d u r i n g a 30 m i n assay. Liver tissue h o m o g e n a t e s e x h i b i t e d g r e a t e r ( P < 0 . 0 5 ) tissue activity o f p h o s p h a t i d a t e p h o s p h o h y d r o l a s e t h a n d i d a d i p o s e tissue h o m o g e n a t e s , b u t specific Table 5. Phosphatidate phosphohydrolase activity of subcutaneous adipose and liver tissue homogenates Activity basis Tissue Enzyme fraction mg proteina g tissue b Subcutaneous adipose tissue Mitochondria 206 140 Microsomes 149 90 Supernate 23 138 Liver Mitrochondria 357 3017 Microsomes 146 596 Supernate 20 1101 SEc 73 482 Significance Tissue a 0.4225 0.0009 Fractionse 0.0045 0.0392 Interaction 0.4852 0.0467 aLeast squares means for inorganic orthophosphate (Pi) released; values are expressed as nmol Pi released/mg protein/30 min. bLeast squares means for Pi released; values are expressed as nmol P~ released/g tissue/30 min. cSE = Pooled standard error of the mean (N = 6). dSignificant (P < 0.05) tissue differences exist for nmol Pi released/g tissue/30 min. eEnzyme fraction differences are significant (P < 0.05) for nmol Pi released/rag protein/30 rain and nmol Pi released/g tissue/30 min.
Table 6. Oleic acid incorporation into glycerolipids by enzyme fractions from liver and adipose tissue homogenates Activity basis Tissue Enzyme fractions mg protein~ g tissue b Subcutaneous adipose tissue Microsomes 19 76 Supernate 5 28 Microsomes + Supernate 7 30 Liver Microsomes 2 38 Supernate 1 50 Microsomes + Supernate 1 32 SEc 1 8 Significance Tissue d 0.0011 0.5060 Fractions¢ 0.0189 0.0375 Interaction 0.0267 0.0217 aLeast squares means for nmol oleic acid incorporated/mg protein/30 min. bLeast squares means for nmol oleic acid incorporated/g tissue/30 min. cSE = Pooled standard error of the mean (N = 2). dSignificant (P < 0.05) differences exist for nmol oleic acid incorporated/mg tissue/30 min. CEnzyme fraction differences are significant (P < 0.05) for nmol oleic acid incorporated/mg protein/30min and nmol oleic acid incorporated/g tissue/30 min.
activities o f liver a n d a d i p o s e tissue were n o t different (P=0.4225) ( T a b l e 5). T h e r e were significant differences b e t w e e n s u b c e l l u l a r f r a c t i o n s for rates expressed e i t h e r as specific activity or tissue activity. The mitochondrial tissue f r a c t i o n hydrolyzed p h o s p h a t i d a t e at a faster rate t h a n t h e m i c r o s o m a l o r s u p e r n a t a n t f r a c t i o n s ( T a b l e 5), a n d the liver mitochondrial enzyme fraction displayed greater p h o s p h a t i d a t e p h o s p h o h y d r o l a s e specific activity ( P < 0.05) t h a n the o t h e r f r a c t i o n s ( T a b l e 5). T h e tissue x s u b c e l l u l a r f r a c t i o n i n t e r a c t i o n for tissue activity was significant, i n d i c a t i n g t h a t liver mitochondrial phosphatidate phosphohydrolase activity was g r e a t e r t h a n p h o s p h a t i d a t e p h o s p h o h y d r o l a s e activity in liver m i c r o s o m e s or s u p e r n a t e , w h e r e a s t h e r e was n o difference in p h o s p h a t i d a t e p h o s p h o h y d r o l a s e activity a m o n g a d i p o s e tissue f r a c t i o n s ( T a b l e 5). S u b c u t a n e o u s a d i p o s e tissue h o m o g e n a t e s h a d greater (P
0305-0491/92 $5.00+ 0.00 Pergamon Press Ltd
Printed in Great Britain
TRIACYLGLYCEROL BIOSYNTHESIS IN BOVINE LIVER A N D SUBCUTANEOUS ADIPOSE TISSUE J. J. WILSON,C. R. YOUNO and S. B. SmTH Texas Agricultural Experiment Station, Texas A&M University, College Station, TX 77843-2471, U.S.A. (Tel. 409-845-3939; Fax 409-845-9454) (Received 1 May 1992; accepted 5 June 1992)
Abstract--1. Adipose tissue from Angus and Brahman steers incubated with [l-~4C]palmitate in the absence and presence of glucose exhibited a greater rate of lipid production than liver (P < 0.05). 2. Homogenates of adipose tissue used in the glycerol-3-phosphate acyltransferase assay exhibited a greater glycerolipid specific activity (nmol lipid/mg protein/30 rain) when compared to liver (P < 0.05). 3. The inverse was true for liver homogenates when calculated for tissue activity (nmol lipid/g tissue/30 rain). 4. Lysophosphatidate was produced in greater (P < 0.05) amounts than all other glycerolipids in the glycerol-3-phosphate acyltransferase assay. 5. The activity of phosphatidate phosphohydrolase in liver homogenates displayed greater rates than their respective adipose tissue homogenates. 6. Diaeylglycerol aeyltransferase activity was greater in adipose tissue homogenates compared to liver homogenates.
INTRODUCTION
MATERIALSAND METHODS
Adipose tissue is the primary site of fatty acid biosynthesis in ruminants. Studies /n vivo of Ingle et aL (1972) suggest that in the non-lactating sheep, adipose tissue is responsible for more than 90% of fatty acid biosynthesis and that the primary metabolic function of ruminant liver is gluconeogenesis. In vitro studies support this conclusion in cattle, sheep and goats, as rates of fatty acid synthesis in adipose tissue slices are more than 20-fold greater than rates in liver slices, with the final product being tdaeyiglycerols (Hood et al., 1972; Ingle et al., 1972; Palmquist, 1975; Christie et al., 1976; Deeth and Christie, 1979). The microsomal phosphatidic phosphohydrolase system is generally assumed to be the rate-limiting step in the pathway leading to triacylglycerol formation (Vavrecka et al., 1969). Others have shown that glycerol-3-phosphate acyltransferase is ratelimiting when animals are subjected to starvation and refeeding (Jamdar and osborn, 1982). Also, the activities of the esterification enzymes depend heavily upon cell size (Etherton and Allen, 1980a; Jamdar and Osborne, 1981), tissue depot and precursors used (Declercq et al., 1982), the concentrations of cofactors like ATP, coenzyme A (CoA) and MgCI2 (Raju and Six, 1975) and the amount of acyl-CoA (Lederer and Hers, 1985). Because of the paucity of information concerning triacylglycerol biosynthesis in cattle, the objective of this study was to compare the activities of enzymes responsible for fatty acid esterification. The activities were measured in the presence and absence of acylglycerol precursors, in both liver and adipose tissues and correlated to triacylglycerol biosynthesis in subcellular fractions. 511
Materials
Biochemicals were purchased from Sigma Chemical Co. (St Louis, MO). Radioisotopes were purchased from Amersham Corp. (Arlington Heights, IL). All other chemicals were purchased from Fisher Scientific (Houston, TX) and were reagent grade or greater. Animals and procedures Purebred Angus (Bos taurus) (N = 2) and Brahman (Bos indicus) (N = 4) steers (18 months of age) were fed a
high-energy ration consisting of 10% cottonseed hulls, 11% cottonseed meal and 72% ground milo, plus a vitamin and mineral supplement for 100 days at the Texas A&M University, McGregor Research Facility. The cattle were transported to and slaughtered at the Rosenthal Meat Science and Technology Center, Texas A&M University, College Station. Liver and subcutaneous adipose tissue samples were obtained within 60 min after the animal was exsanguinated. The animals suffered no unusual stress. Samples were placed in a solution of warm, oxygenated Krebs-Henseleit Ca2+free bicarbonate buffer (KRB) with 5mM glucose and transferred to the laboratory for processing. All samples were removed from the animal and in the laboratory for processing within 1 hr from the time the animal was stunned. Preparation of tissue incubations
Liver and adipose tissue slices (100-150mg) were incubated in vitro as described by Etherton and Allen (1980a), with modifications as needed. Half of the liver and adipose tissue slices were incubated in 20-ml scintillation vials, in triplicate, in 3 ml of KRB buffer (pH 7.4) that contained 120mM NaC1, 4.8raM KCI, 1.2mM MgC12, 1.2mM KH2PO4, 25 mM NaHCO3, 2 #Ci [1-14C]palmitic acid, 1 mU/ml insulin, 5 mM glucose, 10 mM Hepes (N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid), 0.75 mM palmitate (potassium salt) and BSA (30 mg/ml). The second half of the tissue sample was placed in a similar 3-ml mixture without glucose. All tissue samples were incubated for
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30 min at 37°C while shaking (120 strokes/min). The incubations were terminated after 30 min by the addition of 3 ml 0.31 M trichloroacetic acid (TCA), followed by removal of tissue from the incubation media. Samples were rinsed first with 9 ml KRB and then rinsed with 9 ml 0.15 M NaC1 and placed in screw cap tubes with 10 ml chloroform:methanol (CHCI3:CH3OH, 2:1, v/v) under nitrogen and allowed to stand overnight. All tissue samples were homogenized at rheostat setting No. 7 for 60 sec with a Kinematic homogenizer (model pt 10-35, Kinematic Inc., Rockville Center, NY). Free fatty acids were separated from the glycerolipids by the addition of 5 ml 0.38 M Na2CO3 and shaking of the tubes for 20 min at high speed, centrifuging for 20 min at 700g and removing the top layer (Na2CO 3 plus nonesterified fatty acids) by aspiration. The CHC13:CH3OH layer was extracted two more times with Na2CO 3 and filtered through a glass microfiber filter (Whatman GF/C, 2.4 cm, Whatman Ltd., Maidstone, England). When lipid products were determined, the CHC13:CH3OH phase was dried under nitrogen, and the volume adjusted to 2 ml with CHC13:CH3OH. Aliquots of 50#1 for adipose tissue and 100 #1 for liver were separated into glycerolipid classes by thin-layer chromatography (TLC) on 250-micron silica gel-G plates. Glycerolipids were visualized by exposure to iodine vapors after development with petroleum ether/diethyl ether/acetic acid (90/10/4, v/v/v) for neutral lipids and with dichloromethane/methanol/3 M NH4OH (65/35/9, v/v/v) for phospholipids. Glycerolipids classes were identified by comparison to standards run on the same TLC plates as the samples and the corresponding spots were scraped into scintillation vials with 5 ml scintillation fluid for liquid scintillation counting. The glycerolipids remaining after removal of samples for TLC were dried, resuspended in 10 ml scintillation fluid and counted to obtain total lipids produced. Preparation o f liver and adipose tissue homogenates and enzyme assays Liver and adipose tissue samples were obtained as stated for incubations. Tissue samples were scissor-sliced prior to homogenization. The liver and adipose tissue homogenates were prepared with 3 vol (w/v) 0.15 M KCI: I0 mM HEPES: 1 mM EDTA: 1 mM dithiothreitol (DTT) (pH 7.4) using a Kinematic homogenizer at rheostat setting No. 8 (3 x 20 see) on ice for adipose tissue and with a loose-fitting teflon pestle in a glass mortar at setting No. 7 (4 x 15 sec) on ice for liver tissue. All tissue samples were centrifuged at 700 g for 30 min to remove cellular debris. The remaining supernate was centrifuged at 11,000 g for 30 min to remove the mitochondrial pellet. Microsomal pellets were obtained by centrifuging the supernate twice at 104,000 g for 60 min. All fractions were kept on ice during processing and centrifuged at 5°C to inhibit the degradation of desired fractions and to preserve enzyme activity. All pellets were rinsed and resuspended in 5 mi of the homogenization buffer, kept on ice and used fresh. All subcellular fractions were analyzed for protein content by the procedure of Lowry et al. (1951). Homogenates used in the glycerol-3-phosphate acyltransferase (EC 2.3.1.15) assay were assayed as described by Lamb and Fallon (1974) with modifications, in a reaction mixture containing 150mM HEPES (pH 7.4), 10mM MgC12, 6 m M ATP, 8 0 # M CoA, I mM DTT, 0.6mM palmitate, 10mM glycerol-3-phosphate, 0.5#Ci L-[U~4C]glycerol-3-phosphate and 0.2 mg BSA. Reactions were started by either the addition of 0.3 ml of mitochondrial or microsomal enzyme fraction or 0.I ml of supernate to the appropriate assay tubes (Liver: mitochondria, 3.4-5.4 mg/ tube; mierosomes, 1.9-2.6 rag/tube; supernate, 1.3-1.9 mg/ tube. Adipose: mitochondria, 0.20-0.50mg/tube; microsomes, 0.12-0.58 mg/tube; supernate, 0.11-0.30 mg/tube). The final volume was adjusted to 1.5 ml and the samples were incubated for 30min at 37°C while shaking (120
strokes/min). Assays were terminated by the addition of 3 ml CHC13:CHaOH (2:1, v/v) with thorough mixing. After 10 min, 1 ml CHCI3, 0.5 ml 2 M KCI:0.3 N HC1 and 0.9 ml distilled~leionized H20 were added and the solution was mixed and then centrifuged at 700g for 10min at room temperature. The upper aqueous phase was aspirated and discarded and the lower phase was washed three times with 2 ml 0.2 N HCI:CH3OH (1:I) to remove labeled glycerol3-phosphate (G-3-P) that was not incorporated into glycerolipids during the incubation. A 100 #1 aliquot was separated into glycerolipid classes by TLC as described above. Glycerolipids were visualized by exposure to iodine vapors and the appropriate spots were scraped into scintillation vials for counting of radioactivity. Phosphatidate phosphohydrolase (EC 3.1.3.4) activity was assessed by the method of Lamb and Fallon (1974) with an aqueous dispersion of phosphatidate as substrate. The release of inorganic orthophosphate by the enzyme was measured as outlined by Sanui (1974). The procedure of Manley et al. (1974) with modifications was used for evaluating diacylglyceride acyltransferase (EC 2.3.1.20) activity in the conversion of diacylglycerol to triacylglycerol for both tissues. The reaction mixture contained 150mM HEPES (pH 7.4), 10mM MgCI 2, 6 m M ATP, 8 0 # M CoA, 1 mM DTI', 0.2mg BSA, I mM oleic acid, 0.5 #Ci [l-~4C]oleic acid and 3 mM 1,2-dioleyl-glycerol in Tween-20. Reactions were started by the addition of 0.3 or 0.1 ml of the appropriate fraction to a final volume of 1.6 ml. Assays were allowed to run for 30 min at 37°C while shaking (120 strokes/min). The reactions were terminated by the addition of 2 ml 0.31 M TCA and mixed for 30 seconds. Two ml CHC13:CH3OH (2:1, v/v) with 0.01#mol/ml triolein were added to the reaction mixture to trap the esterified lipids in the organic phase. The tubes were shaken to obtain a better mixture of the CHCI3:CH3OH. Subsequently, 2 ml 0.38 M Na2CO3 were added to extract the labeled free fatty acids. The mixture was shaken for 10min at high speed and centrifuged at 700g for 10min after which time the top layer was discarded and the remaining sample was extracted twice with Na2COj. The CHCI3:CH3OH layer was dried under nitrogen and then resuspended in 0.5 ml chlorofoi'm:methanol from which a 150#1 sample was taken for TLC. After development in petroleum ether/diethyl ether/acetic acid (70/30/1, v/v/v), the triacylglycerides were identified by exposure to iodine vapors, scraped and quantitated in the liquid scintillation counter as before. Statistical analyses and expression of rates Statistical analyses were made using GLM procedures of SAS (1986) for a 2 (tissue depot)x 2 (substrate)x 5 (glycerolipid) design for the tissue incubations. The enzyme analysis was for a 2 (tissue)x 5 (glycerolipid) design to determine major end product and for a 2 (tissue)x 3 (centrifugal fraction) design to determine major enzyme activity. Tissue depot, substrate, glycerolipid and centrifugal fraction were used as the main effects to analyze the data. When the main effects were significant, mean separation was accomplished using the least squares means test (Montgomery, 1984). All data are tested at P < 0.05. Rates of palmitate esterification/n vitro and enzyme activities are reported as nmol substrate incorporated/rag protein/30 rain (specific activity) or as nmol substrate incorporated/g tissue/30 min (tissue activity). For specific activities of subcellular fractions, rates are expressed per respective amounts of mitochondrial, microsomal or supernatant protein. RESULTS Tissue incubations Esterification o f palmitate to triacylglycerols indicated t h a t the tissue was metabolically active at
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Triacylglycerol biosynthesis Table 1. Liver and subcutaneous adipose tissue slices incubated with and without glucose Tissue
Incubations conditions
Subcutaneous adipose tissue + glucose glucose -
Activity basis mg protein" g tissueb 797 711
2889 2401
4 23 157
735 972 467
0.0002 0.8351 0.7298
0.0015 0.7953 0.4289
Liver + glucose - glucose SEc Significance Tissued Substrate Interaction
"Least squares means for fatty acyI-CoA esterification; values are expressed as nmol glycerol-3-phosphate incorporated/rag protein/30 rain. bLeast squares means for fatty acyI-CoA esterification; values are expressed as nmol glycerol-3-phosphate incorporated/g tissue/ 30 rain. cSE = Pooled standard error of the mean (N = 6). dTissue ffi Subcutaneous adipose tissue esterified glycerolipids at a greater rate than liver.
the time of the incubations. The tissue produced primarily diacylglycerol as the final glycerolipid product if the tissue sample was not incubated within I hr after death (data not shown). Tissues used for the study in this report were incubated before this apparent 60 min critical period. As reported previously for de novo fatty acid synthesis (Hanson and Ballard, 1967; Ingle et aL, 1972), adipose tissue was more active than liver (P < 0.05) for either specific activity (nmol palmitate incorporated/rag protein/30min) or tissue activity (nmol palmitate incorporated/g tissue/30 rain). The addition of glucose did not affect the esterification rates for palmitate incorporation in either tissue (Table 1). Glycerolipid fractions produced during the/n vitro incubations, as determined by TLC separation, were the only tested main effect that generated a significant test value (P < 0.05) (Table 2). Triacylglycerols were in greater proportion (P < 0.05) than any other glycerolipid produced. Regardless of tissue or incubation conditions, virtually no phosphatidate accumulated in tissue incubations, indicating that the phosphatidic phosphohydrolase enzyme was not limiting triacylglycerol synthesis (Table 2). Enzyme assays
When compared to Lamb and Fallen (1974) for rat, the glycerol-3-phosphate assay liver cell fractions Table 2. Glycerolipid fractions (% of total) of tissue incubations separated by thin-layer chromatography Glycerolipid Monoaeylglycerol Diacylglycerol Triacylglycerol Lysophosphatidate SEa Significance Glycerolipidb
% of Total 1.9a~ 9.0d 83.7c 5.3 ~ 2.5 O.0001
" S E f P o o l e d standard error of the mean (N = 6). Data are pooled across tissue. ~ M e a n s within a column lacking a common superscript letter differ (P < 0.05).
Table 3. Glycerol-3-phosphate esterification into glycerolipids by homogenates from bovine liver and adipose tissue Tissue
Lipid fractions
Subcutaneous adipose homogenates Monoacylglyceride Diacylglyceride Triacyiglyceride Lysophosphatidate Phosphatidate Liver homogenates Monoacylglyceride Diacylglyceride Triacylglyceride Lysophosphatidate Phosphatidate SEc Significance Tissued Glycerolipide Interaction
Activity basis mg protein* g tissueb 20 25 32 163 76
42 47 23 134 99
36 8 8 71 12 19
475 118 157 741 110 88
0.0037 0.0001 0.0518
0.0001 0.0006 0.0056
'Least squares means for fatty acyl-CoA esteriflcation; values are expressed as nmol glycerol-3-phosphate incorporated/mg protein/30 rain. bLeast squares me.arts for fatty acyI-CoA esterification; values are expressed as nmol glycerol-3-phosphate incorporated/g tissue/30 min. cSE ffi Pooled standard error of the mean (N ffi 6). dSignificant ( P < 0 . 0 5 ) differences exist for nmol glycerol-3phosphate incorporated/rag protein/30 min and nmoI glycerol-3phosphate incorporated/g tissue/30 min. ~Lysophosphatidate was found in greater (P < 0.05) amounts than all other glycerolipids.
generated slightly higher rates of glycerolipid production for bovine subcellular fractions. Subcutaneous adipose tissue homogenates produced glycerolipids at a greater rate (P < 0.05) than liver tissue homogenates (diacylglycerol, triacylglycerol, lysophosphatidate and phosphatidate) when the rates were calculated as specific activity (nmol G-3-P incorporated/rag protein/30 min) (Table 3). There tended to be a tissue x glycerolipid interaction for data expressed as specific activity. Lysophosphatidate was the major glycerolipid produced in adipose tissue homogenates, whereas both lysophosphatidic acid and monoacylglycerol were abundant products in liver homogenates. Both tissue homogenates produced minor amounts of diacylglycerol and triacylglycerol. When the rates were calculated as tissue activities (nmol G-3-P incorporated/g tissue/30min), liver homogenates generated a greater (P < 0.05) rate of esterification of fatty acids than subcutaneous adipose tissue for all acylglycerol fractions. The tissue x glycerolipid interaction was significant; lysophosphatidate was the major glycerolipid produced in adipose tissue homogenates, whereas both lysophosphatidic acid and monoacylglycerol were abundant products in liver homogenates. The conversion of lysophosphatidate to phosphatidate by the acylation of a second fatty acyl-CoA was limiting in both tissues as indicated by the accumulation of lysophosphatidate (Table 3). In addition to glycerolipid fractions, subcellular distribution of enzyme activities was also determined (Table 4). Tissue and subcellular fraction main effects were significant (P < 0.05) for data expressed as specific activity. Adipose tissue exhibited greater specific activity than did liver. In both tissues, the
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Table 4. Glycerol-3-phosphate esterification into glycerolipids by enzyme fractions from liver and adipose tissue homogenates Activity basis Tissue Enzyme fraction mg proteina g tissue b Subcutaneous adipose tissue Mitochondria 92 93 Microsomes 172 83 Supernate 43 170 Liver Mitochondria 55 934 Microsomes 72 493 Supernate 43 170 SEe 26 133 Significance Tissue d 0.0090 0.0008 Fractionse 0.0028 0.0513 Interaction 0.4102 0.0165 aLeast squares means for fatty acyl-CoA esterification; values are expressed as nmol glycerol-3-phosphate incorporated/mg protein/30 min. bLeast squares means for fatty acyl-CoA esterification; values are expressed as nmol glycerol-3-phosphate incorporated/g tissue/30 min. 'SE = Pooled standard error of the mean (N = 6). aSignificant (P < 0.05) differences exist for nmol glycerol-3-phosphate incorporated/rag protein/30 rain and nmol glycerol-3phosphate incorporated/g tissue/30 min. CEnzyme fractions are significant (P < 0.05) for nmol glycerol-3phosphate incorporated/rag protein/30 min only, specific activity o f t h e m i c r o s o m e s exceeded t h a t o f the m i t o c h o n d r i a l a n d s u p e r n a t a n t fractions. Liver tissue activity exceeded a d i p o s e tissue activity for m i t o c h o n d r i a a n d m i c r o s o m e s . T h e tissue x f r a c t i o n i n t e r a c t i o n w a s significant ( P < 0.05) for t h e tissue activities ( T a b l e 4). I n a d i p o s e tissue, t h e greatest activity was o b s e r v e d in t h e s u p e r n a t a n t fraction, w h e r e a s in liver, t h e g r e a t e s t activity w a s o b s e r v e d in mitochondria. P h o s p h a t i d a t e p h o s p h o h y d r o l a s e activity was a n a lyzed as n m o l i n o r g a n i c o r t h o p h o s p h a t e (Pi) cleaved f r o m p h o s p h a t i d a t e acid d u r i n g a 30 m i n assay. Liver tissue h o m o g e n a t e s e x h i b i t e d g r e a t e r ( P < 0 . 0 5 ) tissue activity o f p h o s p h a t i d a t e p h o s p h o h y d r o l a s e t h a n d i d a d i p o s e tissue h o m o g e n a t e s , b u t specific Table 5. Phosphatidate phosphohydrolase activity of subcutaneous adipose and liver tissue homogenates Activity basis Tissue Enzyme fraction mg proteina g tissue b Subcutaneous adipose tissue Mitochondria 206 140 Microsomes 149 90 Supernate 23 138 Liver Mitrochondria 357 3017 Microsomes 146 596 Supernate 20 1101 SEc 73 482 Significance Tissue a 0.4225 0.0009 Fractionse 0.0045 0.0392 Interaction 0.4852 0.0467 aLeast squares means for inorganic orthophosphate (Pi) released; values are expressed as nmol Pi released/mg protein/30 min. bLeast squares means for Pi released; values are expressed as nmol P~ released/g tissue/30 min. cSE = Pooled standard error of the mean (N = 6). dSignificant (P < 0.05) tissue differences exist for nmol Pi released/g tissue/30 min. eEnzyme fraction differences are significant (P < 0.05) for nmol Pi released/rag protein/30 rain and nmol Pi released/g tissue/30 min.
Table 6. Oleic acid incorporation into glycerolipids by enzyme fractions from liver and adipose tissue homogenates Activity basis Tissue Enzyme fractions mg protein~ g tissue b Subcutaneous adipose tissue Microsomes 19 76 Supernate 5 28 Microsomes + Supernate 7 30 Liver Microsomes 2 38 Supernate 1 50 Microsomes + Supernate 1 32 SEc 1 8 Significance Tissue d 0.0011 0.5060 Fractions¢ 0.0189 0.0375 Interaction 0.0267 0.0217 aLeast squares means for nmol oleic acid incorporated/mg protein/30 min. bLeast squares means for nmol oleic acid incorporated/g tissue/30 min. cSE = Pooled standard error of the mean (N = 2). dSignificant (P < 0.05) differences exist for nmol oleic acid incorporated/mg tissue/30 min. CEnzyme fraction differences are significant (P < 0.05) for nmol oleic acid incorporated/mg protein/30min and nmol oleic acid incorporated/g tissue/30 min.
activities o f liver a n d a d i p o s e tissue were n o t different (P=0.4225) ( T a b l e 5). T h e r e were significant differences b e t w e e n s u b c e l l u l a r f r a c t i o n s for rates expressed e i t h e r as specific activity or tissue activity. The mitochondrial tissue f r a c t i o n hydrolyzed p h o s p h a t i d a t e at a faster rate t h a n t h e m i c r o s o m a l o r s u p e r n a t a n t f r a c t i o n s ( T a b l e 5), a n d the liver mitochondrial enzyme fraction displayed greater p h o s p h a t i d a t e p h o s p h o h y d r o l a s e specific activity ( P < 0.05) t h a n the o t h e r f r a c t i o n s ( T a b l e 5). T h e tissue x s u b c e l l u l a r f r a c t i o n i n t e r a c t i o n for tissue activity was significant, i n d i c a t i n g t h a t liver mitochondrial phosphatidate phosphohydrolase activity was g r e a t e r t h a n p h o s p h a t i d a t e p h o s p h o h y d r o l a s e activity in liver m i c r o s o m e s or s u p e r n a t e , w h e r e a s t h e r e was n o difference in p h o s p h a t i d a t e p h o s p h o h y d r o l a s e activity a m o n g a d i p o s e tissue f r a c t i o n s ( T a b l e 5). S u b c u t a n e o u s a d i p o s e tissue h o m o g e n a t e s h a d greater (P
