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D . degree- in Dairy Science g/V/fim Major professor . “INK OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to Book drop to remove this checkout from your record. REC M @0193 THE REGULATION OF FATTY ACID OXIDATION IN BOVINE MAMMARY TISSUE Gary Patrick Dimenna A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Science and Institute of Nutrition 1978 ABSTRACT THE REGULATION OF FATTY ACID OXIDATION IN BOVINE MAMMARY TISSUE By Gary Patrick Dimenna Oxidation of fatty acid was studied in bovine mammary tissue slices in order to evaluate their potential contribu- tion to energy metabolism. Mammary tissue slices were incu— bated in bicarbonate buffer with palmitateCl-luc], and l“€02 was counted as an index of oxidation. Lipids were extracted from the tissue and esterified lipids determined by thin-layer chromatography. Rates of palmitate oxidation increased with time of incubation in mammary tissue. This phenomenon is not an artifact of the incubation system or due to substrate solubility, as rates of palmitate oxidation were constant in rat kidney cortex slices. Preincubating mammary tissue with or without unlabelled palmitate revealed that increasing rates of palmitate oxidation is not due to utilization of eruiogenous fatty acids. Fatty acid oxidation in mammary tissue is probably not COrltrolled by tissue fatty acid concentration. Palmitate at 0.26 mM, equivalent to arterial fatty acid concentration arm: less than tissue fatty acid concentration, gave maximal Gary Patrick Dimenna rates of oxidation. Half-maximal rates of oxidation were obtained at 0.1 mM palmitate. Rates of palmitate oxidation increased with time at all concentrations tested. Rates of fatty acid oxidation decreased with increasing chain length: acetate > octanoate > palmitate or oleate. Rates of oxidation of only long—chain fatty acids increased over time, which could not be explained by carnitine palmitoyltransferase (CPT) activity. The B-oxidation enzymes may restrict fatty acid oxidation as oxidation of palmitate [l-luC] > palmitateEU-luC]. Acetate inhibited palmitate oxidation (75%) but not esterification, suggesting that acetate inhibits palmitate oxidation by substrate competition at the mitochondrial level or via malonyl-CoA inhibition of OPT. Rates of palmitate oxidation increased with time in the presence of acetate. Glucose inhibited palmitate oxidation (67%) and stimula- ted palmitate esterification. Low palmitoyl-CoA levels would favor glyceride synthesis over oxidation, since the apparent Km for palmitoyl—CoA of the glycerol 3-phosphate (G3P) acyltransferases is lower than that of OPT. Thus, glucose presumably diverts palmitate from oxidation to glycero—lipids. In some experiments, rates of palmitate oxidation increased over time in the presence of glucose, which could not be explained by glucose depletion from the media. Decreasing rates of glycero-lipid formation over time could quantitatively account for increasing rates of palmitate oxidation. Gary Patrick Dimenna Clofenapate, a glyceride synthesis inhibitor, decreased triacylglycerol formation, increased intracellular fatty acid accumulation, and marginally increased palmitate oxida- tion. However, rates of palmitate oxidation increased 180% in the presence of carnitine and Clofenapate, suggesting the existence of multiple controls of palmitate oxidation. Glucose did not affect octanoate oxidation, but stimula- ted octanoate esterification, possibly resulting from separate microsomal and mitochondrial pools of octanoate. Octanoate activated in the mitochondria is not accessible to acylation of G3P. Octanoate activated at the microsomes is accessible to acylation of G3P. Fasting appeared to decrease the absolute rates of palmitate oxidation. However, fatty acid oxidation as a proportion of the total oxidative metabolism was not deter- mined and may have increased. Therefore, fatty acid esterification and oxidation seem to compete for available acyl—CoA with esterification being favored. Also, acetate inhibits palmitate oxidation presum- ably by substrate competition at the mitochondrial level or Via malonyl—CoA inhibition of OPT. It can be estimated that long—chain fatty acids can potentially account for 6-lO% of the oxidative metabolism of mammary tissue. DEDICATION To Niki and Debi ii ACKNOWLEDGMENTS I thank my advisor, Dr. Roy Emery, for his encouragement and many helpful suggestions during my stay at Michigan State, and members of my guidance committee, Drs. Thomas, Romsos, Bieber, and Mellenberger, for their appraisal of this disser— tation. Also, I express my gratitude to Jim Liesman for his assistance in the lab. And last but not least, a sincere debt of gratitude is owed to my wife, Debi, for her constant support and encourage- ment during the last four years, and to our daughter, Niki, who made it all worthwhile. TABLE OF CONTENTS Page ILIST OF TABLES ........................................ vi ILEST OF FIGURES ....................................... viii INTRODUCTION .......................................... 1 ILITERATURE REVIEW ..................................... 2 LIPID ABSORPTION AND DIGESTION ................... 2 Ruminal Biohydrogenation of Unsaturated Fatty Acids ................................... 3 Fatty Acid Absorption ......................... 3 Removal and Transport of Absorbed Lipids ...... A THE PATHWAYS AND CONTROL OF FATTY ACID UTILIZATION ...................................... 5 CELLULAR UPTAKE OF BLOOD FATTY ACIDS ............. 5 Fatty Acid Activation ......................... 8 Glyceride Synthesis... ........................ ll Carnitine Acyltransferase ..................... 16 The B—oxidation Pathway ....................... 23 Oxidation of Unsaturated and Odd-Chain Fatty Acids ................................... 28 w-oxidation of Fatty Acids ....... . ............. 29 Peroxisomal Fatty Acid Oxidation .............. 30 Relationship Between B-oxidation and the Citric Acid Cycle ............................. 32 Partitioning of Fatty Acids Between Oxidation and Esterification ............................ 35 iv TABLE OF CONTENTS (cont'd.) POSSIBLE CONTROL POINTS OF LONG-CHAIN FATTY, Page ACID OXIDATION IN RUMINANT MAMMARY TISSUE ........ 39 CONCLUDING REMARKS ............................... A2 MATERIALS AND METHODS ................... _ .............. AA PROCEDURE FOR MEASURING FATTY ACID OXIDATION AND ESTERIFICATION IN BOVINE MAMMARY TISSUE SLICES... AA Preparation of Buffer and Radioactive Substrates for Incubation ..................... AA Tissue Collection and Preparation for Incubation .............. p ...................... A5 Determination of CO2 .......................... A6 Source of Animals ............................. A7 Lipid Extraction of the Tissue Slices ......... A8 Fatty Acid Esterification ..................... A9 CARNITINE PALMITOYLTRANSFERASE ACTIVITY (CPT).... 50 Sources of Reagents ........................... SO Isolation of Mitochondria ..................... 51 CPT Assay ..................................... 52 STATISTICAL ANALYSIS ............................. 53 RESULTS AND DISCUSSION ................................ 5A CONCLUSIONS ........................................... 92 APPENDIX TABLES ....................................... 96 BIBLIOGRAPHY .......................................... 107 Table ll. 12. 13. 1A. 15. LIST OF TABLES Mammary Metabolism of Glucose, Acetate, and Fatty Acids in Fed and Fasting Lactating Goats.... Palmitate Oxidation Versus Time of Incubation in Bovine Mammary Tissue Slices ...................... Palmitate Oxidation Versus Time of Incubation in Rat Kidney Cortex Slices .......................... Palmitate Oxidation Versus Palmitate Concentration ..................................... Fatty Acid Oxidation Versus Chain Length .......... Effect of Preincubating Mammary Tissue Slices in Buffer With No Substrate on Palmitate Oxidation... Effect of Preincubating Mammary Tissue Slices With Unlabelled Palmitate on Palmitate Oxidation.. Oxidation of PalmitateEl—luC] Versus PalmitateEU-l c] .................................. Effect of Glucose on Palmitate Oxidation .......... Effect of Glucose Concentration on Palmitate Oxidation ......................................... Effect of Acetate on Palmitate Oxidation .......... Effect of Acetate and TOFA on Palmitate Oxidation. Effect of Glucose and Acetate on Palmitate Oxidation and Esterification ...................... Effect of Glucose on Octanoate Oxidation and Esterification .................................... Effect of Incubation Time and Glucose on Carnitine Palmitoyltransferase Activity in Mammary Tissue Slices ............................................ vi Page A1 55 56 58 59 61 62 65 67 68 69 71 73 8O 82 IQIST OF TABLES (cont'd.) Thable 1.6} 137. 3.8. 2L9. 2C). 2].. AJ_. A12. !\3. ALI. A55. A65. A7. Page Effect of Clofenapate and Glucose on Palmitate Oxidation and Esterification ....................... 8A Effect of Carnitine, Glucose, and Clofenapate on Palmitate Oxidation ............................. 86 Effect of B-hydroxybutyrate on Palmitate Oxidation .......................................... 87 Effect of Lactate and Glucose on Palmitate Oxidation and Esterification ....................... 88 Effect of Fasting on Palmitate Oxidation in Mammary Tissue Slices .............................. 90 Effect of Fasting on Milk and Milk Fat Production.. 91 Composition of Incubation Media .................... 96 The Effect of Ethanol on Palmitate Oxidation ....... 97 The Effect of Collecting and Rinsing Tissue in Warm Versus Cold Buffer on Palmitate Oxidation ..... 98 Palmitate Oxidation With Source of Cows ............ 99 Effect of Glucose and Acetate on Palmitate Oxidation and Esterification ....................... 100 Effect of Glucose on Octanoate Oxidation and Esterification ..................................... 103 Effect of Lactate and Glucose on Palmitate Oxidation and Esterification ....................... 105 vii LIST OF FIGURES Figure Page 1. Pathway for the biosynthesis of triacylcerol... 12 2. Mechanism for the transport of fatty acyl-CoA esters across the mitochondrial inner-membrane Via the enzyme palmitoyl-COA: L—carnitine 0-pa1mitoyltransferase ......................... l8 3. The B—oxidation pathway ........................ 2A A. w—oxidation pathway of fatty acids ............. 31 5. Double-receprocal plots of substrate concentration versus carnitine palmitoyltransferase activity in mammary mitochondria ................................... 76 6. Conversion of palmitate to C02 and glycero-lipids versus time ..................... 78 viii INTRODUCTION Milk synthesis is a very energy—demanding process. High- producing dairy cows during peak of lactation are often in a state of negative energy balance. The concentration of fat in milk is nearly constant throughout lactation, and milk fat represents a loss of energy to the cow. Therefore, by in— creasing the oxidative metabolism of fatty acids in the mammary gland more energy would be available for the produc- tion of more milk and milk protein. This would be especially useful to high-producing cows at peak of lactation, since production may be limited by the extent of energy deprivation. However, Annison et a1. (1967) could not detect the occurr- ence of fatty acid oxidation in the mammary gland of fed goats. Thus, in order to assess the feasability of stimulating fkitty acid oxidation in the mammary gland, two questions were Iwiised at the onset. To what extent does mammary tissue from feci lactating cows oxidize fatty acid, and what factor(s) r’egmlates fatty acid oxidation in bovine mammary tissue? LITERATURE REVIEW This review will discuss the regulation of fatty acid oxidation in mammalian tissues. A brief introduction on the modification of dietary lipids in the gastrointestinal tract of ruminants is in order, since these modified lipids will eventually serve as an energy source for some tissues. Characterization of the pathways involved in the metabolism of absorbed fatty acids and their regulation will follow. This review will be centered on the regulation of fatty acid oxidation in non—ruminant organs, as little is known of fatty acid oxidation in the ruminant mammary gland. In conclusion, long-chain fatty acids as a possible source of energy for the mammary gland will be discussed, integrating the previous aspects of this review. ILIPID ABSORPTION AND DIGESTION Practical dairy rations normally contain less than A% faiz. Most of the fatty acids in the diet are in glyceride CCHnbination and are unsaturated (Garton, 1960). Ruminal Imicroorganisms extensively modify the dietary lipids, by hydlnolysis of the glycerides, fermentation of the liberated glyxzerol, and hydrogenation of the unsaturated fatty acids (Gadnton, 1960). As a result, ruminant tissue lipids contain a liarge proportion of saturated fatty acids even though the 3 fat1357 acids found in pasture grasses are mainly polyunsatur- ateci.. Ruminal Biohydrogenation of Unsaturated Fatty Acids Attention was first directed towards the influence of the rumen on dietary lipids by Reiser (1951), who showed that when linseed oil was incubated with sheep rumen contents, the lino lenic acid content was reduced from approximately 30% to 5%. This was attributed to hydrogenation of linolenate by rumen bacteria. Mills et a1. (1970) studied the in vitro IUNirNogenation of linolenic, linoleic and oleic acids by a Chfiini—negative rumen micrococcus. Positional and geometrical iscyniers of the fatty acids were formed as intermediates. Theesse isomers are detectable in ruminant depot fat and milk (GELI‘ton, 1960). Polan et al. (196A) found a synergism be- tWEeern Butyrivibrio fibrisolvens, Peptostreptococcus elsdenii 8Lm3. a.Selenomonas species for the biohydrogenation of liI1c>lenic acid to stearic acid. Rumen protozoa are also able tO liydrogenate linoleic acid to stearic acid (Chalupa and Kutxzhes, 1967). Thus, rumen microorganisms reduce dietary unserturated fatty acids to their saturated isomers which everitually pass to the intestines for absorption. E2E£gy_Acid Absorption In the ruminant nonesterified fatty acids are the major Clafss of lipids appearing in the small intestine. The fatty aciCls found in the intestinal lumen of the ruminant are assOciated with solid surfaces. The transfer of fatty acids :fincnn solid surfaces into micellar solution is accomplished tny' biliary and pancreatic secretions (Harrison and Leat, 119'72). Ruminant bile is composed of cholesterol, fatty zacxids and phospholipids. The predominant phospholipid is r>knosphatidyl choline (Leat, 1965). Lysophosphatidyl choline ar1d.fatty acids, arising from the action of pancreatic ratiospholipases on phosphatidyl choline, act in concert with ‘trie bile salts to solubilize the fatty acids into a micellar 1>Piase (Harrison and Leat, 1975). The major site of fatty éiczid absorption is the ileum (Leat, 1965). After the fatty acids of the micellar dispersion are aat>sorbed by the intestinal mucosal cell, triglycerides are £33rnthesized predominantly by the g1ycerol-3—phosphate path- VVE13'(Cunningham and Leat, 1969). The monoglyceride pathway 1:3 present in adult sheep (Cunningham and Leat, 1969), but 15C would be of little importance quantitatively since vir— t1ially no monoglyceride is absorbed from the intestines of I’Lllninants (Harrison and Leat, 1975). Therefore, fatty acid absorption by the intestinal In'Llczosal cells in ruminants involves fatty acid solubiliza- t3i.on by biliary and pancreatic secretions, uptake by the mLleosal cells, and esterification to g1ycerol-3-phosphate iri the mucosal cell. ‘Bfimoval and Transport of Absorbed Lipids The long-chain fatty acids enter the circulatory system Tin the form of chylomicrons or very low density lipoproteins (VLDL), density less than 1.006, from the digestive tract 5 \riga the thoracic duct. The VLDL is absorbed by.the intestinal lqyrnph, and passes into the thoracic duct via the intestinal diicrt (Felinski et al., 196A; Palmquist, 1976). In ruminants, isrfiiglyceride accounts for approximately 70% of lymph lipids aijci phospholipids as 20% (Palmquist, 1976). In sheep the iflxow of bile phosphatidyl choline is theoretically sufficient th account for most of the lymph phospholipids (Harrison and Inezat, 1975). The results of Palmquist and Mattos (1978) Sligggests that 76% of absorbed lipid is taken up directly by tlurs mammary gland of the lactating cow. In the liver the :fELtty acids derived from blood are reesterified to glycerol. T1213 majority of the triglycerides then re—enter the plasma as ‘VIQIH.(d.< 1.006) and low density lipoprotein (1.006 < d < l -<3A0)(Palmquist, 1976). ITIIIE PATHWAYS AND CONTROL OF FATTY ACID UTILIZATION nggiiuiar Uptake of Blood Fatty Acids Blood fatty acids, which are of quantitative importance fCDJ? cellular energy production in some tissues, exist in two COInpartments: nonesterified fatty acid bound to albumin and tr’iglyceride fatty acids in a lipid—protein complex (VLDL and OISIUer lipoproteins). In mammalian species the majority of tllfsse fatty acids are of long-chain length (Spector, 1971). Approximately 80% of blood triglycerides are completely 1'lIVdrolyzed during uptake by the mammary gland (Emery, 1973). Inhis hydrolysis is catalyzed by the enzyme lipoprotein lipase CLPL)(EC 3.1.1.3). As a result, fatty acid concentration in ceagoillary fluid is increased. The process is partially rwexfersible giving a degree of equilibration between plasma 'tznjmglycerides and fatty acids (Emery, 1973). The activity (Df‘ lipoprotein lipase is modified by apoprotein fractions of lgiraoprotein C. Apo C—II and maybe C—I activate LPL and apo (Z-JIII inhibits activity (Jensen and Pitas, 1976). Super et 231.- (1976) noted that activator concentration is related to tiles concentration of plasma total lipids in cows. Bovine Huarnmary LPL activity is also responsive to diet and hormones. Nkarnmary lipoprotein lipase activity is decreased in cows fed a. rnilk fat depressing ration (Emery, 1973). Prolactin and Ip1?c>lactin plus insulin enhances LPL activity in bovine Insinnmary tissue explants (Emery, 1973). Fasting, diabetes, eiceercise and epinephrine treatment increase LPL activity in IPEiI: heart (Neely and Morgan, 197A). In summation, the lipo- pr?<>tein lipase enzyme is of quantitative importance for tissue uptake of fatty acid from blood triglyceride, especially irl the mammary gland, and is highly regulable. Circulating nonesterified fatty acid is transported as a tigghtly bound, non—covalent complex with albumin (Spector, 159'71). The binding is reversible and some fatty acid exists fr‘eee in solution in equilibrium with albumin—bound fatty acid. Irj~sssue uptake of nonesterified fatty acid is not dependent ‘IDCDn metabolic energy or mediated enzymatically. In order to I39 absorbed the circulating fatty acid must dissociate from tflle carrier protein. Cells rapidly take up fatty acid and this alters the steady state distribution between bound and UDDCDLlnd fatty acid, which causes additional amounts of fattspr acid to dissociate from albumin (Spector, 1971). The rate of fatty acid uptake is a function of plasma conxzeantration in intact animals (Neely and Morgan, 197A) and in :iri vitro systems (Eaton and Steinburg, 1961). However, in ziri vitro systems uptake can be affected by factors other thari simply concentration. Ockner et al. (1972) isolated a cyt<>g>lasmic low molecular weight protein termed fatty acid binc1j;ng protein (FABP) or Z protein, which binds fatty acids. FABI? has a high affinity for fatty acids, and the relative bindijxng increases with increasing chain length. FABP is presseent in the cytoplasm of rat liver, myocardium, skeletal mus<3]_e, intestinal mucosa, adipose tissue and kidney (Mishkin et aLl., 1972). Flavispidic acid competes with fatty acid for the ITABP and decreases oleate uptake in isolated rat hepa- tocyizes (Wu-Rideout et a1., 1976). Uptake of fatty acid in rat enorta pieces is dependent on fatty acid chain length, decrwaasing as chain length increases (Hashimoto and Dayton, 1971) . Glucose enhances palmitate uptake in the perfused rat theart (Shipp, 196A). However, the rate of uptake was unaff‘ected by fasting in a perfused rat liver system (McGarry and Fkoster, 1971). Kidneys possess a unique pathway for fatty Uptakfe, which is dependent on potassium. Indeed, palmitate uptakfé is enhanced in rat renal cortex slices by addition of pOtaSESium (Wagner and Heinemann, 1977). Even though tissue uptake of fatty acid bound to albumin is not dependent on metabOlic energy or mediated enzymatically, nevertheless some studies suggest that nonesterified fatty acid uptake is regul ated. Fat 1: y Acid Activation The following reaction accounts for activation of fatty acids in mammalian tissues: ATP + fatty acid + CoASHé—a acyl-CoA + AMP + PPi The reaction is catalyzed by acyl-CoA synthetases (acid:CoA ligases, (AMP forming) EC 6.2.1.1-3). It requires Mg2+ and is readily reversible (Groot et al., 1976). The reaction involves formation of an enzyme-bound acyl adenylate inter- mediate. Pyrophosphate hydrolysis, catalyzed by inorganic pyrophosphatase (EC 3.6.1.1), shifts the equilibrium in favor of acyl-CoA formation. In addition to the ATP-dependent fatty acid activation, a mammalian acyl-CoA synthetase SDGCific for GTP catalyzes the reaction: GTP + fatty acid + CoASHH acyl-CoA + GDP + Pi The enzyme is also MgZI-dependent (Groot et al., 1976). HoweVer, this enzyme is of little physiological significance (Neely and Morgan, 197A). Thus, fatty acid activation is an enzymatic process dependent on metabolic energy. Enzymes that activate short—, medium-, and long-chain fatty acids have been described (Groot et al., 1976). Acetyl- COA Synthetase is present in all tissues and it also activates pI“Opionate. Quraishi and Cook (1972) found that about two- thirds of the enzyme activity is localized in the cytoplasm and One—third in the mitochondria of bovine heart and mammary gland, in kidney equally divided, and in lung and liver predominantly in the mitochondria. However, in the liver the activity of acetyl-CoA hydrolase is greater than the synthe- tase. Groot (1976) isolated a prOpionyl-COA synthetase from guinea pig liver mitochondria. Acetate, propionate and butyrate are activated, but specificity for propionate is highest. Butyryl-CoA synthetase, an enzyme with the highest Vmax toward butyrate, has been purified from beef heart mitochondria (Webster et al., 1965). A medium-chain acyl—CoA synthetase has been purified from beef liver particles (Mahler et al., 1953). This enzyme has broad substrate specificities, but the Km for octanoate is the lowest (0.15 mM). Medium—chain fatty acids (MCFA) are activated exclusively in the mitochondrial matrix (Aas and Bremer, 1968). Long-chain fatty acids (LCFA) can be activated by this enzyme, which could explain the carnitine— independent LCFA fatty acid oxidation found by Van T01 and Halsmann (1970). In heart mitochondria LCFA oxidation is totally carnitine—dependent. A long-chain acyl—CoA synthetase is present in microsomes and the outer mitochondrial membrane of mammalian tissues. This enzyme has broad substrate specificities with a higher Specificity for palmitate (Groot et al., 1976). In rat liver microsomes the enzyme has Km values of 1-3 11M, 7.2-9.5 uM and 0-29-0.A mM for LCFA, CoASH and ATP (Groot et al., 1976). In isOlated rat liver mitochondria the Km values are 50 Ml and 7'0 uM for palmitate and ATP (Van T01 and HIulsmann, 1970). Thus, enzymes specific for activation of short-, medium-, and long—chain fatty acids exist and their intracellular lO l<>calization is of metabolic importance. Pande (1973) found that low concentrations of palmitoyl- Cc1A.inhibit the activity of long-chain acyl-CoA synthetase in a :reversible manner. The inhibition was competitive with reaspect to 00A. The reported apparent Ki for palmitoyl—CoA wars nearly equal to the apparent Km for CoA. This mechanism wriuld serve to control intracellular levels of long-chain axzyl-CoA esters within desirable limits. Adenosine and AMP are potent competitive inhibitors with rwespect to ATP (Van T01 and Hulsmann, 1970). Levels of AMP acne increased and LCFA oxidation is impaired in hypoxic tnearts, thus AMP may have an energy-sparing effect and may lgimit the undesirable accumulation of long-chain acyl¢CoA essters (Neely and Morgan, 197A). On the contrary, AMP has besen shown to accelerate activation and oxidation of short- aiid medium-chain fatty acids in a rat heart homogenate (Neely 811d Morgan, 197A). Chagoya de Sanchez et a1. (1977) admin— irstered high doses of adenosine, and found decreased rates Of‘ hepatic LCFA oxidation via an inhibition of the extra- mistochondrial acyl-CoA synthetase. Pande (1971) revealed that the ability of isolated miJSQChondria from several rat tissues to activate LCFA was mu£fln greater than that required to support maximum rates of fa.‘tty acid oxidation, and concluded that this enzyme does not lirnit fatty acid oxidation. Aas and Daae (1971) measured r'a-tes of LCFA activation in liver, heart and adipose tissue Of‘ rats fed a stock diet, fasted, fasted refed-fat, fasted ll rwefed-carbohydrate, and fat-fed. LCFA activation was sZLightly depressed only in the liver of the carbohydrate— rwafed rats. Thus, long-chain acyl-CoA synthetase is a cxonstitutive enzyme in mammals. (Elyceride Synthesis Activated fatty acids, acyl-CoA esters, are at a taranchpoint between acylation to sn-glycerol—3-phosphate (G3P) or carnitine. Patton (1975) found an intensely Ilabelled pool of phosphatidic acid in mammary tissue within 113 minutes after an IV injection of P—32. Askew et a1. (il97la) showed that monopalmitin was an ineffective acyl arzceptor in bovine mammary homogenates, while palmitate, st3earate, oleate and linoleate were esterified to G3P at rertes consistent with their concentration in milk (Askew et al.., 1971b). These results suggest that the G3P pathway is tkue major pathway for the formation of milk fat triglycerides. A1130, since triglycerides account for approximately 98% of mile fat, this section will be limited to discussing the corrtrols of triglyceride synthesis via the G3P pathway. Triglyceride synthesis by the G3P pathway is shown in Figtune 1. Glucose serves as the source of G3P in mammary tiSEMie, since glycerol kinase levels are low (Baldwin and Millxigan, 1966). Phosphatidic acid synthesis proceeds thrcnigh a sequential acylation of G3P mediated by two distinct acyltransferases (Monroy et al., 1973; Yamashita and Numa, 1972). The enzyme phosphatidate phosphohydrolase converts phOSFfliatic acid to diacylglycerol, which is subsequently l2 Twang—chain acyl-CoA sn-glycerol-3-phosphate Lysophosphatidic Acid (+CoA) Inong—chain acyl-CoA-—————>\L’ (2) Phosphatidic Acid (+CoA) (3) 1,2-Diacyl-sn—glycerol (+Pi) Lcnng—chain acyl-CoA——————€> (A) Triacylglycerol (+CoA) Fimgure l. Pathway for the biosynthesis of triacylcerol. (l) acyl-CoA:sn—glycerol-3-phosphate O-acyltransferase (EC) 2.3.1.15); (2) acyl-00A:sn—monoacylglycerol-3-phosphate O-euzyltransferase; (3) L—d—phosphatidate phosphohydrolase (EC 3.1.3.14); and (A) acyl—GOA:sn—diacylglycerol O‘aCyltransferase (EC 2.3.1.20). l3 acylated to triacylglycerol. The acyltransferases exist tboth in the microsomes and mitochondria of rat liver (Shephard and HUbscher, 1969; Zborowski and Wojtczak, 1969) sand mammary gland (Pynadath and Kumar, 196A). Phosphatidate gahosphohydrolase is distributed between the soluble and Inicrosomal fractions (Sturton et al., 1978). Phosphatidic eacid may be deacylated by the microsomal enzyme phospholipase A (Sturton et al., 1978). The first two acyl transferases nuiy not be evenly distributed between the mitochondrial and mixzrosomal fractions, as lysophosphatidic acid is the prtinciple product formed from the acylation of G3P by paLhnitoyl-carnitine in rat liver mitochondria while phos- phartidic acid is principally formed in microsomes (Daae, 19772). Thus, triglyceride synthesis by the G3P pathway in miczrosomes and mitochondria occurs by sequential acylation of C33P to form phosphatidic acid, hydrolysis of phosphatidate folLlowed by acylation of the resultant diglyceride to form triglyceride . The enzyme of triacylglycerol synthesis are modified by a nunflaer of effectors. Magnesium ions have been shown to inhiiiit the first acylation step in rat liver microsomes (Fallxan and Lamb, 1968) and the second acylation reaction in bovinee mammary gland (Kinsella, 1976) and to activate phos- phOhthrolase in rat adipocytes (Moller et al., 1977) guinea pig Hmunmary gland (Kuhn, 1967) and bovine mammary gland (MarShaJd.and Knudsen, 1977). However, partially purified Slycerophosphate acyltransferase from rat liver mitochondria lA 2+, which can be substituted by Mg2+, Mn2+, and requires Ca (202* (Yamashita and Numa, 1972). Phosphatidic acid inhibits trie first acylation reaction, thus regulating its own fkormation (Monroy et al., 1973; Shapiro and Tzur, 1968). TTie inhibitory effects of palmitoyl—CoA on acylation are realieved by albumin (Gross and Kinsella, 197A). Daae (1972) srnowed that the mitochondrial acylation of G3P was more serisitive to inhibition by acyl-carnitines than the microsomal sysrtem. The regulation of glyceride synthesis by G3P will be ctisczussed in a later section. Synthesis of fatty acids occurs on a soluble acyl carurier protein (ACP) and the products of this process are aCDfil thioesters of ACP. ACP has been shown to transfer its acyCL group to G3P in E. coli and clostriduim butyricum (GOIxfliine and Ailhaud, 1971). Palmitoyl-CoA competes eff'ecztively with palmitoyl—ACP for the sn-2 position of the Elycleeride molecule. The significance of this system in mammflEilian tissues is unknown. Glyceride synthesis in particulate systems is stimulated IU’ axidition of a particle-free supernatant (PFS). Vavrecka et £11. (1969) attributed this stimulation to the presence of phc”Sphatidate phosphohydrolase in the soluble fraction. Serfiim proteins can bind the products of synthesis, thereby rerrloving product inhibition (Shapiro and Tzur, 1968). Brindley et a1. (1967) found that unsaturated LCFA stimulate glywaeride synthesis, and attributed the stimulatory action of FEE; to the presence of unsaturated fatty acids, which would 15 allow synthesis of a more balanced product. Thus, the stimulatory effect of PFS on glyceride synthesis may be a result of enzyme (phosphatidate phosphohydrolase) addition, unsaturated LCFA, or simply removal of end-product. Fatty acids tend to be utilized differently depending upon fatty acid chain length and degree of unsaturation. . Slakey and Lands (1968) discovered a non—random distribution of fatty acids between the 1— and 3-positions on triacyl- glycerol in rat liver. The esterification of fatty acids at each position proceeds with a specificity that is not correlated with the composition of the other positions of the molecule. The fatty acid specificity for the formation Of milk triacylglycerols is particularly intriguing. Palmitoyl-COA is the preferred substrate for the initial acYlation of G3P, and the rate of palmitoyl-CoA acylation is 8 to 10 times greater that with myristoyl-, stearoy1-, OP Oleoyl-CoA. However, with l-palmitoyl sn—glycero-3—P as an acyl acceptor all acyl-CoA esters were easily esterified (Kinsella and Gross, 1973). Short-chain fatty acids, Synthesized in the mammary epithelial cell, are preferentially esterified to position sn—3 of milk fat triglycerides (Breckenridge and Kuksis, 1969). Oleic acid produced from desaturation of stearic acid in situ is also preferentially estSrified to the third position, and desaturase activity is 001"related with the rate of triglyceride synthesis (Kinsella, 1972). Results of Askew et al. (1971a) suggested that ability to acylate the sn-3 position in a bovine mammary l6 homogenate is difficult to achieve. However, Marshall and Knudsen (1977) found that microsomal 1,2-diacylglycerol acyltransferase from bovine mammary gland incorporated equal molar amounts of diglyceride, short-chain acyl-CoA or palmitoyl—COA. Thus, glyceride synthesis may be controlled in part by substrate specificity. The addition of sugars to the diet increases the formation of liver triglycerides (Lamb and Fallon, 197A). This increase is correlated temporally with an increase in microsomal and soluble phosphatidate phosphohydrolase. Under similiar dietary conditions, the ratio of phosphohydrolase: deacylase activity is enhanced (Sturton et al., 1978). These results suggest that the rate at which phosphatidate is converted to diacylglycerol or recycled back to G3P partly controls hepatic triacylglycerol synthesis. Mtine Acyltransferase The stimulatory action of carnitine on fatty acid oxida- tion is well documented. Carnitine is an obligatory require— merit for oxidation of long-chain fatty acids in isolated mitochondria of heart and skeletal muscle (Bode and Klingen- bul‘g, 196A), heart homogenate (Passeron et al., 1968), and heart slices (Fritz, 196A). Liver and kidney mitochondria can oxidize LCFA without carnitine, however, the oxidation Of their corresponding acyl—carnitine esters is much greater (Bode and Klingenburg, 196A). Since fatty acyl-00A is impermeable to the mitochondrial inner—membrane, acyl-00A esters are compartmentalized in cells between the l7 eextramitochondrial compartment, site of fatty acid activation, sand the intramitochondrial compartment, site of fatty acid coxidation (Beattie, 1968). In order to traverse this imper- Ineable barrier acyl-carnitine derivatives, which may be permeable to this barrier, are formed from acyl-CoA and carnitine. This transfer is catalyzed by carnitine acyl- transferase (also called carnitine palmitoyltransferase, CPT) as presented in Figure 2. Two pools of carnitine palmitoyltransferase (CPT-I and CPT-II) activities exist in mitochondria. CPT-I is associated with the outer membrane or the outer aspect of the inner membrane, and CPT—II is tightly bound to the inner membrane and available only to CoA in the mitochondrial matrix. CPT- II shows greater chain—length substrate specificity for the transfer of long-chain acyl groups from acyl-carnitine derivatives to CoA, while CPT-I shows broad chain-length substrate specificity (Kopec and Fritz, 1973; Yates and Garland, 1970; West et al., 1971; Brosnan and Fritz, 1971). Some now believe that the mitochondrial inner—membrane is as impermeable to carnitine and acyl-carnitine esters as to CoA and its esters. A process of exchange diffusion between carnitine and acyl-carnitine across the mitochondrial inner-membrane, analogous to that causing ATP-ADP exchange, has been proposed for the transfer of acyl units (Ramsay and Tubbs, 197A; Pande, 1975). This transport is facilitated by the presence of a translocase system in mitochondria. Using isopycnic sucrose density gradient centrifugation methods, CPT is exclusively mitochondrial (Markwell et al., 18 I ; LCFA Glyceride ERA Activating Synthesizing 1\ System System \IAcyl-CoA/i;~aGP ATP + CoA + Mg2+ (pool 1) /I Carnitine /I Carnitine Acyltransferase -———€>Acy1-CoA II (pool 2) Figure 2. Mechanism for the transport of fatty acyl—Coa esters across the mitochondrial inner—membrane via the enzyme palmitoyl-CoA: L-carnitine O-palmitoyltransferase (trivial name carnitine palmitoyltransferase (CPT) EC 2.3. 1.21)(Fritz and Yue, 1963). 19 l 9 7 3). A carnitine acetyltransferase was extracted from pig heart and partially purified (Fritz et al., 1963). The substrates, acetyl—CoA, propionyl—CoA, and butyryl-CoA react at approximately equal rates, while activity with palmitoyl—COA is nil. This enzyme was postulated to facilitate acetyl-00A movement across mitochondrial membranes (Fritz and Yue, 196A). Snoswell and Henderson (1970) post— ulated that the presence of carnitine acetyltransferase in large amounts in sheep liver allows the "acetyl pressure" in the starved condition to be shifted from the vital CoA system to the carnitine system. Acetyl-carnitine would then serve as a storage form of acetyl—CoA, thus making more CoA available for gluconeogenesis from propionate. Carnitine ace't 3'1-transferase is located in mitochondrial and microsomal fractions (Markwell et al., 1973; Markwell and Bieber, 1976). Microsomal carnitine acetyltransferase could be involved in providing a source of acetyl at sites of acetylation reactions. A partially purified preparation of carnitine palmitoyl- transferase revealed the existence of an additional protein fPa¢tion, which displayed a high substrate specificity tOWavI’Cls the transfer of medium-chain acyl—carnitine deriva— tives (Kopec and Fritz, 1971). This fraction was tentatively designated carnitine octanoyltransferase. Clofibrate, a hypolipidemic drug, causes variable increases in carnitine ac_3’3—’ISI~ansferase activities in liver dependent on the chain length of the substrates. Factors for increase in specific 20 activity are 5.3, 2.8 and 1.7 for the transfer of short- Chain, medium—chain and long-chain acyl-carnitine derivatives (solberg et al., 1972). Therefore, three separate carnitine acyltransferases exist in mitochondria. When attaching biological significance to the short— and medium—chain acyltransferases, one must keep in mind that short—chain and medium-chain fatty acids are activated in the matrix of the mitochondria. Thus, the carnitine acyltransferase step would be bypassed. The fact that the effect of carnitine is more pronounced in the oxidation of LCFA lends credence to this point (Bremer, 1962). Shepherd et a1. (1966) discovered that isolated rat liver mitochondria respire faster in the presence of palmitoyl- carnitine than with palmitoyl-CoA plus carnitine as substrates. This led to the tentative hypothesis that the CPT enzyme is rate—limiting for fatty acid oxidation. 0n the contrary, by using much lower substrate concentrations equal rates of respiration are obtained with palmitoyl-carnitine versus palmitoyl—CoA plus carnitine (Pande, 1971). In addition, several investigators have shown that CPT activity is several— fOld greater than the maximum ability for B-oxidation of Palmitoyl-carnitine in isolated mitochondrial preparations (Bremer. and Norum, 1967b; Pande, 1971; Cederbaum et al., 1975; Van '1‘01 and Hulsmann, 1969). In vitro studies showed that palmitoyl—carnitine at a concentration of 2 11M, well below tisslle concentrations of acid-insoluble carnitine (255—A26 Mu mOleS/g of 1iver)(Pearson and Tubbs, 1967), resulted in maximum oxygen uptake rates (Bremer and Norum, 1967a), and 21 high concentrations of carnitine inhibit palmitoyle-carnitine transferase at low palmitoyl-CoA levels (Bremer and Norum, 19 67a). With this in mind it is being accepted that CPT is not rate—limiting for the oxidation of fatty acids. In support of this latter concept, the CPT reaction is freely reversible in vitro (Norum, 196A), and if there is sufficient CPT in the cell, the ratio of long-chain acyl-CoA/ CoA will vary with the ratio of long—chain acyl-carnitine/ carnitine (Bremer, 1967). Indeed, long—chain acyl—CoA and acyl—carnitine tissue levels are increased during fasting, fasted refed-fat and diabetes (Tubbs and Garland, 196A; Bohmer, 1967 5 Greenbaum et al., 1971). Therefore, the ratios of long— chain acyl-CoA/CoA and long-chain acyl—carnitine/carnitine are in equilibrium in vivo. Long—chain acyl-CoA increased from 53 to 110 (TM in livers of rats after fasting (Tubbs and Garland, 196A), and long—chain acyl-carnitine increased from 70 to 200 uM in hearts of rats after fasting (Marquis and Fritz, 196A). These results suggest that the capacity of the CPT enzyme does not limit fatty acid oxidation. The B- OXidation process itself may be limiting presumably Via the availability of free CoA for the formation of intramitochon- drial long—chain acyl-CoA, since long—chain acyl—carnitine accumulates (Bremer, 1967). The Km for palmitoyl-CoA for the CPT enzyme is 31 11M (Norum, 196A), and a concentration of 53 uM for long—chain a0yl~CoA in liver has been reported (Tubbs and Garland, 1964). Thus, the concentration of long—chain acyl-CoA may play a 22 regulatory role in the formation of palmitoyl—carnitine. Bremer and Norum (1967b) found that palmitoyl—CoA is a competitive inhibitor of carnitine for CPT in vitro, but inhibition did not occur with protein-bound palmitoyl-CoA as one would expect to find in the cell. do A number of studies ]_end support for the original hypothesis that CPT activity ma3r ‘be rate-limiting in vivo. The results of Norum (1965) suggggests that CPT activity is enhanced by fasting, fat- feeding, and diabetes. The increase in activity is not due to de vovo enzyme synthesis, but activation of preformed protein. Rates of ketogenesis in perfused livers from fed rats diminished with oleic acid and octanoylcarnitine as substrates in comparison to fasting, whereas octanoic acid supported high rates of ketogenesis irrespective of the nutritional state of the donor animal (McGarry and Foster, 197A). This suggests that CPT-I is not coupled to CPT-II, and the ability of both LCFA and the medium-chain carnitine ester to gain entry into the mitochondria is restricted in the fed state. An inhibitor Of Carnitine acyltranserase, (+)—decanoyl—carnitine, decreases ketOgenesiS in vivo (McGarry and Foster, 1973) and changes the pattern of oleate metabolism in livers from that of fasted rats to that demonstrated by livers from normal animals (oleate is virtually completely esterified)(McGarry et al., 1973). The authors concluded that no fundamental defect exists in the triglyceride—synthesizing capability of the liVer‘ in the fasted state, and that fatty acid oxidation is under strict dietary and hormonal control exerted primarily 23 by :rnegulation of an early step in the oxidation sequence, prc>tiably the carnitine acyltransferase. The relationship betzvmeen fatty acid oxidation and esterification will be disscrussed later in more detail. New and exciting evidence suggests that malonyl-CoA, an fightermediate in fatty acid synthesis, at physiological coriczentrations inhibits ketogenesis from oleate and the sitzee of inhibition is carnitine acyltransferase (McGarry et £11., 1977). Subsequently, malonyl-CoA was found to be a cornlaetitive inhibitor of ketogenesis in isolated hepatocytes wiisli a Ki of 2 nmol/g wet weight of cells (malonyl-CoA coriizent in hepatocytes from meal-fed rats was 1A.8 nmols/g we1: 'weight of cells)(Cook et al., 1978). This suggests that fatstzy acid synthesis and oxidation are incompatible. Thee B-oxidation Pathway B-oxidation is the main catabolic pathway for fatty acids (Spector, 1971; Wakil, 1970; Green and Allman, 1968). This P813frway, as shown in Figure 3, derives its name from the fact thert the oxidative attack occurs at carbon atom 3 of the fatrtfig acid, the 8 carbon atom. An acyl-CoA ester is degraded by- Sliccessive cleavage of 2-carbon atom fragments from the Cart>omwl.end of the fatty acid chain. The enzymes that catéllyze these reactions are located in the mitochondrial matI‘ixin close association with the inner-membrane (Beattie, 1968), The acyl-CoA dehydrogenase catalyzes the oxidation of Saturated acyl-CoA derivatives to the corresponding trans-d,8- 2A H H I | ,,0 R _— T — C - C ~ S - CoA H H H H __ I .0 Ikczyl CoA FAD ,>-R _ C = C - C ~ S - 00A (1) trans—a,B-unsaturated acyl—00A (2) j, H20 H H l | ,,0 R - C - f - C ~ S - CoA OH H /,0 Rtein, the electron transferring flavoprotein, which is dilr°ectly linked to cytochrome b of the mitochondrial electron trwaaasport system (Wakil, 1970). The trans a,B-unsaturated aOEIZL—COA ester is subsequently stereospecifically hydrated bY’ eenoyl-CoA hydrase (also referred to as crotonase) to the L(-+-)—B-hydroxyacyl—COA derivative. Rate of reaction decreases mal?1cedly with increasing chain length. The Vmax with CIVDt30nyl-COA (the CA derivative) is approximately l50-fold gr%351ter than with the 016 derivative as substrate (Waterson and Hill, 1972) . The L(+)-B—hydroxyacyl—CoA derivative is oxidized by NAD 3J1 tzhe presence of the L-B-hydroxyacyl—CoA dehydrogenase to the? corresponding B-ketoacyl-CoA derivative. The enzyme is Spe‘Cific for the L-form, and is active on the various chain- 1erugth fatty acyl derivatives (Wakil, 1970). The last step in the thiolytic cleavage of the B-ketoacyl- CoA to acetyl-00A and a saturated acyl-CoA(n_2). The thiolase 26 enzyme has broad substrate specificity, and reacts equally with B—ketoacyl-CoAs of various chain lengths. Although the thiolase reaction is reversible, the equilibrium (Keq = 6 x 10“) greatly favors acetyl—CoA formation (Wakil, 1970). When acyl—CoA derivatives enter the B-oxidation pathway, their oxidation was once thought to commence without regula- tion. However, studies now indirectly suggest the contrary. Intact liver mitochondria oxidizing labelled palmitate (Lopes-Cardozo et al., 1978) or labelled palmitoyl-carnitine (Stanley and Tubbs, 197A) accumulate substantial amounts of saturated acyl-CoA intermediates. The amount of these intermediates decreases with increasing chain length of the substrate (Stanley and Tubbs, 1975). The acyl-CoA inter- mediates accumulate slowly, while acetyl-CoA is produced linearly with time. These separate pools of "free acyl—00A" intermediates arise via "leakage" from the true intermediates Of B—oxidation, which are restricted in amount and have priveleged access to the enzymes of B-oxidation (Stanley and Tubbs, 1975). The quantity of intermediates formed depended on tOtal flux of acyl units through B-oxidation. These results indicate that acyl-00A dehydrogenase is rate limiting W, or it may reflect some degree of organization of the enZZVI'nes of B—oxidation. Korsrud et a1. (1977) found that the Vmax of the acyl-00A dehydrogenase decreases with in— creaLSing chain length, which indicates a possible control point for the oxidation of long—chain fatty acids. In accordance, intact liver mitochondria oxidizing octanoate did 27 not accumulate intermediates of the B—oxidation pathway (Stewart et al., 1973). Bremer and Wojtczak (1972) revealed that a high NADH/NAD ratio blocked B-oxidation in liver mitochondria with the SLibsequent accumulation of B-hydroxypalmitoyl—carnitine. It was concluded that the NAD-linked oxidation of the B-hydroxy- palmitoyl-carnitine is more easily suppressed than the flavoprotein-linked oxidation of palmitoyl-CoA. Acetoacetyl-CoA strongly inhibits the hydroxyacyl-CoA dehydrogenase (Schifferdecker and Schulz, 197A) and enoyl—CoA hydrase enzymes (Waterson and Hill, 1972). Inhibition is non— competitive with respect to dehydrogenation but competitive with respect to hydration. The inhibition of enoyl—CoA hydrase is particularly interesting. Due to a combination of (its cascading substrate specificity and its marked susceptibility to acetoacetyl-CoA, enoyl-CoA hydrase could be rate limiting for the oxidation of long—chain substrates, but not medium— and short-chain acyl-CoA substrates (Waterson and Hill, 1972). Pent-Aeenoic acid is a hypoglycemic compound structurally r’ela.ted to the active metabolite of hypoglycin from the fruit Of lChe ackee. Pent-A—enoic acid is a potent inhibitor of fatty acid oxidation in intact mitochochondria by its SpeCific inhibition of the thiolase reaction (Holland et al., 1973) . 28 ‘ggxidation of Unsaturated and Odd—Chain Fatty Acids The oxidation of unsaturated fatty acids by the B——oxidation pathway involves two additional enzymic reactions, :issomerization and epimerization (Wakil, 1970). The oxidation c>f‘ linoleic acid, for example, involves successive cleavage caf‘ acetyl units via B-oxidation to the formation of cis-A3a6- (3].2—CoA. This compound is then isomerized to the trans—A2- erioyl derivative by AB-cis-AB—trans-enoyl-COA, as shown in tile following reaction: 0 II M/__ :— C-SCOA I W COS-CoA Trice product enters the B-oxidation scheme at the enoyl-CoA hyxirese step and undergoes two cycles of B-oxidation, forming cisr—A2-octenoyl-COA. This is then hydrated by enoyl-CoA hyttrase to the D-B-hydroxyacyl-CoA epimerase to the L(+) antiqoode, as shown in the following reaction: OH 0 o H H W C-SCoA ’ We (1) “"/\ L<+) D(-) SCoA The Iw—B—hydroxyacyl—COA is the substrate for L—B-hydroxyacyl- CoA ciehydrogenase shown in Figure 3. The isomerase and epiflmirase enzymes most likely do not regulate oxidation to any eXterrt, since palmitate, stearate, oleate, linoleate, and lir1ncentrations. They are oxidized by the B—oxidation pathway ‘tco acetyl—00A and one equivalent of propionyl—CoA. Propionyl— (3CDA is then converted to succinyl-CoA (Wakil, 1970). co-—Oxidation of Fatty_Acids Fatty acid oxidation via the w-oxidation pathway has icesceived a renewed interest. Bjorkhem (1976) demonstrated triat w—oxidation of stearic acid in liver is enhanced by :Eaisting, and dicarboxylic acid administration to starved cor’ diabetic rats lowers the concentration of blood ketones (ldéida and Usami, 1977; Wada et al., 1971). Hemmelgarn et a1. (1977) estimated that A0% of the irii;tial oxidation of fatty acids in the nonketotic diabetic Trad: is via w-oxidation and the remainder is B-oxidation, whuile Wada and Usami (1977) estimate that about 15% of the fairty acids are subjected to m-oxidation and then B-oxidation in.;fasted or diabetic rats. Thus, a discussion of the meckianism of w-oxidation and its implication is warranted. The w—oxidation pathway is shown in Figure A. The omega methsfl group of fatty acid is directly hydroxylated in the liver? microsomal fraction. The reaction requires NADP and 02 811d is catalyzed by omega fatty acid hydroxylase (Robbins, 1968:). The omega hydroxy fatty acid is oxidized to the omega ketC> fatty acid by a liver—soluble supernatant fraction requiring NAD (Robbins, 1968). Bjorkhem (1973) attributes I3“? stimulatory action of the supernatant fraction to the presence of alcohol dehydrogenase, which could protect the omeEa-hyroxylase from product inhibition by oxidation of 3O ‘bkie omega-hydroxy fatty acid. Finally, the omega keto fatty £1C3id is oxidized to the dicarboxylic acid by a liver-soluble slipernatant fraction requiring NAD (Robbins, 1968). The lieexadecanedioic acid (dicarboxylic acid) can be activated by ]?Elt liver mitochondria in presence of 00A, ATP, and Mg2+. .A. carnitine ester is formed by the action of hexadecanoyl-CoA: czarnitine O—hexadecanoyltransferase (EC 2.3.1.-). The rate cof‘ this reaction is an order of magnitude lower than the :reate of formation of palmitoyl-carnitine, and is increased toyr fasting, diabetes, and clofibrate feeding. However, 02 liprtake with hexadecandioyl-carnitine as the substrate is lxovv and transitory in liver and heart mitochondria (Pettersen, 1973% The four terminal carbon atoms of fatty acids become zaceeto acetyl-CoA during B-oxidation, while those of dicarboxy- lic: acids become succinyl-CoA. Wada et a1. (1971) speculated thert the significance of w—oxidation is to produce succinyl— CoA. from fatty acids in the mitochondria. Succinyl—CoA pPOChJction facilitates the citric acid cycle and diverts acetsrl-COA from ketogenesis to oxidation. A net synthesis of succcinyl-COA could also result in a net synthesis of glucose. Hemflualgarn et al,(l977) suggested the w-oxidation could DOSEribly function to detoxify long-chain fatty acids, since it 1&3 a microsomal process. E§££E£I§Omal Fatty Acid Oxidation A new development in fatty acid metabolism is the dis— Covery of fatty acid oxidation in peroxisomes. Rat liver peroxisomes oxidize palmitoyl-CoA to acetyl-00A reducing O2 to 31 CH3 NADPH2 ' CH20H CHO (CH2) 0 (CH2) NAD \ (CH ) 2 \ l“(cytoplasrfifl 2 1A COOH (microsomesT7 COOH COOH Palmitate CHO NAD COOH -oxidation I 2 1A ( t l )7. I 2 1A COOH cy op asm COOH _—————-€>Succinyl-CCA Hexadecanedioic Acid Figure A. w-oxidation pathway of fatty acids. 32 H202 and three moles of NAD (Lazarow and De Duve, 1976; Lazarow, 1977). The activity of this system is increased approximately one order of magnitude by feeding hypolipidemic drugs (clofibrate, Wy-lA,6A3, and tibric acid). These drugs induce a marked proliferation of peroxisomes, while only stimulating catalase activity 50%. Catalase can constitute as much as 16% of the peroxisomal protein. Lazarow (1978) has recently discovered the presence of the B-oxidation enzymes in a purified peroxisomal preparation. The system has a high substrate specificity for long—chain acyl—CoA and is inactive towards butyryl-CoA. Palmitoyl—CCA is oxidized I to acetyl—00A. Thus, the oxidation of palmitoyl-CoA in peroxisomes involves the enzymes of B-oxidation and incomplete cleavage of palmitoyl-CoA to three acetyl—CoAs. .Relationship Between B-oxidation and the Citric Acid Cycle The complete oxidation of fatty acids to CO2 involves a Ccnicerted operation between the B-oxidation and citric acid Cyrzle pathways. Acetyl-CoA formed from B-oxidation of fatty atxids is at a branchpoint between ketogenesis and oxidation Viél the citric acid cycle (TCA). The rate of ketogenesis in liver is correlated with fatty acid concentration, while complete oxidation to 002 is moderately affected (Ontko and Jackson, 196A). This implies that the capacity of the TCA CYcle limits acetyl-CoA oxidation, and ketosis is a result of incPeased fatty acid flux to the liver. However, kidney COI‘tex slices oxidized palmitate completely to C02 at all concentrations tested (Lee et al., 1962). Thus, TCA cycle 33 capacity for oxidation of acetyl-00A appears to vary among organs. As rat liver oxidizes fatty acids the intramitochondrial NADH/NAD ratio rises. The substrate pairs malate/oxaloacetate and NAD/NADH are in equilibrium, and an increased NADH/NAD ratio would favor malate formation at the expense of oxaloa- cetate. Thus, a lack of oxaloacetate would divert acetyl-00A from citrate synthesis to acetoacetate synthesis (Lopes— Cardozo and Van Den Bergh, 197Ab; Wieland, 1968). Van Tol (1970) showed that carnitine addition to isolated liver mitochondria enhanced ketogenesis and inhibited CO2 production from palmitate, which resulted in an increased NADH/NAD ratio a decreased TCA cycle activity and lowered intramitochOndrial oxaloacetate levels. Williamson et a1. (1969) perfused livers ‘with oleate and found increased rates of ketogenesis, but :flux through the citrate synthase reaction was unaltered by Oxaloacetate a-ketoglutarate < citrate 6 _I Succinate and acyl-CoA compete at the flavoprotein level. Succinate can suppress B—oxidation independent of a high NADH/NAD ratio. Both the acyl-CoA and succinate dehydrogena- tions are followed by NAD—linked dehydrogenations, and accumulation of NADH prevents their oxidation. Malate dehydrogenation is more displaced to the hydroxy side than B-OH—acyl-CCA dehydrogenation and will be relatively more inhibited by NADH. Under conditions of relatively high ATP/ADP ratio in the 06211 the electron transport Chain will be rate limiting, and nurtual inhibitions of the two cycles will be observed. thixtable and Wakil (1971) found maximum palmitate oxidation ai: an energy charge of 0.65 in mitochondrial incubations. Since the B—oxidation cycle is less subject to suppression it will tend to dominate under conditions of increased acyl- carnitine availability, resulting in acetyl—00A accumulation With a.concomitant decreased rate of oxaloacetate formation. 35 Partitioning of Fatty Acids Between Oxidation and Esterifica- tion. It is well documented that the concentration of fatty acids in the milieu is a major determinant of the rate of fatty acid utilization. However, enhanced uptake of fatty acid by the liver is not sufficient in itself to initiate maximum ketogenesis. Fasting increases fatty acid oxidation and decreases fatty acid incorporation into glycerides in liver slices (Rubenstein and Rubenstein, 1966), in hemidia- phragm pieces (Fritz and Kaplan, 1961), liver cells (Ontko, 1972), and in perfused livers (McGarry and Foster, 1971), but in perfused hearts from rats previously starved fatty acid incorporation into glycerides is elevated (Neely and Morgan, 197A). Fasting markedly enhances fatty acid oxidation in the goat mammary gland, but marginally decreases milk fat ,production (Annison et al., 1968). It appears likely that a Inajor determinant of the rate of oxidation is competition for tile fatty acid substrates between the B-oxidation and glycer- ixie synthesis pathways. McGarry and Foster (1971) perfused livers from fasted enid nonfasted rats with labelled oleate and octanoate. Fasting markedly enhanced ketogenesis and depressed ester- ification from oleate, while octanoate metabolism was minimally affected. Octanoate is not directly used for glyceride synthesis in the rat liver. However, de novo Synthesized fatty acids (02-016) are used for glyceride SYchesis in ruminant mammary tissue (Breckenridge and Kuksis, 1969). Antiketogenic compounds (fructose, glycerol, 36 lactate and ethanol) in the perfusate reversed oleate metabolism from that observed in the fasting state. The major effect of these compounds was to promote esterification which was quantitatively sufficient to account for the diminished production of labelled ketones. These antiket- ogenic agents did increase the concentration of glycerol—3- phosphate (G3P) in livers from fasted rats. However, the concentration of G3P was not altered by fasting alone when compared to controls, even though the rate of esterification increased five-fold in the fed state. It is of interest to note that glucose in the perfusate had no effect on any parameter measured. Glucose plus insulin does enhance palmitate esterification in the diaphragm muscle (Bodel et al., 1962), but not in kidney cortex slices (Lee et al., 1962) and heart slices (Fritz, 196A). The glycerol effect was confirmed by Ontko (1972). Prager and Chitko (1976) have since shown that fructose inhibits fatty axzid oxidation by competitive substrate inhibition. Sorbitol hams antiketogenic effects in liver, presumably via formation of‘ G3P (Loten et al., 1966; Exton and Edson, 196A). Glucagon increased palmitate oxidation and decreased palmitate incorporation into triglyceride in liver from rats pPeViously fasted—refed carbohydrate. Glucagon enlarges the content of long—chain acyl—CoA which may directly inhibit the glyceride synthesizing enzymes (Christiansen, 1977). Glucagon may exert its effect through CAMP (Klausner et al., 1978). Fatty acid flux through the carnitine palmitoyltransferase 37 reaction is enhanced by glucagon, which is partly mediated by elevated tissue carnitine concentrations with glucagon treatment (McGarry et al., 1975). However, an increased cellular concentration of carnitine could not account for the altered fatty acid metabolism in liver cells from fasted rats (Christiansen et al., 1976). The distribution of fatty acid between oxidation and esterification is likely to be influenced by the relative activities of the carnitine palmitoyltransferase and glycerophosphate acyltransferases enzymes. Starvation or fat-feeding decreased glycerophosphate acyltransferase activity in rat liver only and increased carnitine palmitoyl- transferase activity in liver and heart. Carbohydrate re- feeding reversed these enzyme changes (Aas and Daae, 1971). Thus, these enzymes may establish the metabolic fate of fatty acids in the liver. No corresponding enzyme changes occurred ir1 adipose tissue indicating that the regulation of fatty ax:id metabolism in adipose tissue and heart is mainly mediated tlrrough the availability of fatty acids and competing sub— stxrates (Aas and Daae, 1971). At low acyl-CoA concentrations tflie acylation of glycerophosphate is the preferred reaction, probably because this reaction has the lower Km for palmitoyl- CoA (Borrebaek et al., 1976). In agreement, dietary conditions that enhance glyceride synthesis also reduce tissue levels of acyl—GOA esters. Thus, the partitioning of fatty acids between oxidation and esterification may be affected by addition of precursors for G3P and the relative activities of the carnitine and G3? 38 acyltransferases. The apparent Km's for G3P for glycero- phosphate acylation in liver mitochondria and microsomes are 0. 5 and 1.7 mM (Van T01, 1974), while the concentration of G3}? in liver is about 85 11M (Greenbaum et al., 1971). So increases in G3P concentration should augment glyceride synthesis. Also, the relative activities of the carnitine and glycerophosphate acyltransferases are important in this partitioning mechanism, especially in organs which are both highly lipogenic and oxidative, such as the liver. Organs such as heart and kidney have very low rates of glycerophos— phate acylation in comparison to carnitine acylation, while adipose is the reverse (Borrebaek et al., 1976). Again, in these organs fatty acid concentration in the milieu is the major determinant of utilization in the cell. Both enzymes demonstrate high activity in the liver. Flavaspidic acid, an inhibitor of fatty acid for the fatty acid binding protein, depresses oleate esterification bUt increases oxidation in rat hepatocytes (Wu—Hideout et al., 1976). Octanoate utilization is not altered. This suggests that as fatty acids are taken-up by the cell, their primary fate is esterification. However, this may simply be an 1mphysiological effect of binding by protein, since albumin addition lowers long-chain fatty acid oxidation (Lopes- Cardozo and Van Den Bergh, 1974a). 39 POSSIBLE CONTROL POINTS OF LONG-CHAIN FATTY ACID OXIDATION IN RUMINANT MAMMARY TISSUE Annison and coworkers (l96u, 1967, and 1968) provided quantitative data in an elaborate series of experiments on the metabolism of substrates by the mammary gland of lactating ruminants. These data were obtained by using isotope dilution coupled with measurements of milk secretion rate, mammary blood flow, and arteriovenous (AV) differences of substrates across the mammary gland. These data are summarized in Table l. Acetate and glucose are of primary importance for oxidative metabolism in the whole animal and the mammary gland. Fatty acids are important oxidative substrates in the fasted animal. Glucose uptake by the udder was 60-85% of the total glucose entering the circulation, and lactose accounted for the greatest proportion of the glucose uptake. Acetate, besides being a substrate for oxidative metabolism, and B- hYdroxybutyrate are the precursors for the Cu to C16 fatty acids synthesized de novo in the mammary gland. It is inter- eSting to note that glucose, acetate and plasma fatty acids account for less than 50% of the total CO2 output by the Udder of the fed goat. Acetate and glucose alone accounted for 30-75% of the total CO2 from the udders of fed cows (Bickerstaffe et al., 1971;). These data imply that acetate is the preferred oxidative substrate in the mammary gland, and that fasting releases inhibition of long-chain fatty acid oxidation. However, indirect evidence suggests that long—chain fatty acids may U0 be important oxidative substrates in the mammary gland of the fed ruminant. The total long-chain acyl-carnitine level (Ml uM) in mammary tissue of lactating goats is higher than in skeletal muscle, heart, kidney cortex and liver (Snoswell and Linzell, 1975). Also, carnitine palmitoyltransferase activity is significant in mammary tissue from lactating ewes (Snoswell and Linzell, 1975). Thus, the level of long—chain acyl—carnitine in mammary tissue is higher than the level in tissues in which fatty acids are quantitatively important oxidative substrates. The data in Table 1 suggest several possible points of control of long-chain fatty acid oxidation in mammary tissue. Acetate, the preferred oxidative substrate, could inhibit long-chain fatty acid oxidation via substrate competition, either by entry into the citric acid cycle or competition for available CoA. Acetate inhibits palmitate oxidation in rat skeletal muscle, presumably via competition between the two for available CoA (Karlsson et al., 1977). Another possible control would simply be an increased substrate availability to the udder, since starvation markedly enhances plasma fatty acid uptake. This would seem unlikely, Siruze the mammary gland also actively extracts fatty acids fPCHn blood triglycerides. The rate of plasma triglyceride Uptéike by the udder of fed cows is about 200 mg/min (Bicker- staffife et al., 1974), and mammary AV differences of plasma trigfilycerides in the fasted goat are 30% of those of fed goats; (Annison et al., 1968). Askew et al. (1971a) found .Amomav .Hm um COchc< Eopm mpmom .Amomav .Hm um QOchc< Eopm mama: .Azomav Haomcfiq new somwcc< Eogm apmom .oSmmfiu wxlcfls\ws mm commopoxo oxmpo: powwow .pcmeB hoop waCHE\mE mm ommmmpoxm bump appcmH Ml w o m m.: m.m onopmmmv I Hv Hv H.ov m.o :Apomv opmoao o z o e.m m.o mmempmwmv I Hv Hv H.ov m.o :Aemmv mpmcmmpm mm m w . o.m w.m mfiempmmmc I av HV. H.ov 2.0 :Acomo mpmoflsamm mm Ha OH m.H m.H mxempmmmc a: . am am m.mm e.m mxemmc mpmpmoa N a a :.ma m.H mflempmmmc em mm OH m.mm m.: mfiemmv mmoozfio amen: amen: Hmefica macs: mmxmpap mpmm mpwspmnsm an emNHeon Aavaoo 0» mumppmnzm page: poem mason: co a no soapspfinpcoo .mpmoc wcfipmpomq wcfipmmm com com CH mofio< eppmm pom .mpmpoo< .omoosflo mo EmHHoompmz hpmesmzll.a canoe M2 fatty acid concentrations of A mM in bovine mammary tissue. So it appears unlikely that fatty acid is in limited supply for fatty acid oxidation. Plasma fatty acids are quantitatively transferred to milk triglyceride fatty acids, and blood glucose is the precursor of the glycerol in milk triglycerides. Thus, long— chain fatty acid oxidation in mammary tissue may be limited by the competing glyceride synthesis pathway. However, long- chain fatty acid oxidation in mammary tissue of the goat is stimulated by fasting, but milk fat secretion is 81% of that in the fed animal (Annison et al., 1968). Thus, during fasting when fatty acid oxidation is stimulated, fatty acid esterification to G3P remains relatively constant. Assuming that the carnitine palmitoyltransferase (CPT) enzyme in rat liver mitochondria is similar to that of mammary gland, the possibility exists that carnitine may be limiting for fatty acid oxidation in the mammary gland. The apparent Km for carnitine is 0.25 mM for the liver mitochon— drial CPT enzyme (Bremer and Norum, 1967a). Free carnitine concentrations of 0.1 mM in cow's milk and 0.25 mM in goat mammary tissue have been reported (Snoswell and Linzell, 1975). Thus, mammary tissue levels of free carnitine may not be optimal for carnitine palmitoyltransferase activity. CONCLUDING REMARKS The control of fatty acid oxidation is multi—faceted. A‘Vailability of fatty acid is a major determinant on the rate Of‘ fatty acid oxidation in some tissues, such as kidney. In 43 organs such as liver with appreciable rates of glycerophos— phate and carnitine acylation, competition between oxidation and esterification determines the intracellular fate of fatty acids. The possibility of the carnitine transport mechanism as a rate-limiting step is still controversial. In ketogenic organs fatty acid oxidation is coupled to the citric acid cycle, and the capacity of the citric acid cycle can determine the ultimate fate of the end-product of B—oxidation. Just where the control(s) of fatty acid oxidation in the bovine mammary gland lies is presently unknown. MATERIALS AND METHODS PROCEDURE FOR MEASURING FATTY ACID OXIDATION AND ESTERIFICATION IN BOVINE MAMMARY TISSUE SLICES Preparation of Buffer and Radioactive Substrates for Incubation Tissue slices were incubated in Krebs-Ringer bicarbonate (KRB) buffer (Umbreit et al., 1972). The buffers were made the night before use, and pH was adjusted to 7.4 prior to collection of tissue. The composition of KRB buffer used for incubation is shown in Appendix Table l. The CaCl2 concentra- tion was halved relative to that recommended by Umbreit et al._ (1972), due to its insolubility at recommended concentrations. The incubation buffer contained bovine serum albumin (BSA Fraction V, fatty acid-poor, Sigma Chemical 00.). It was determined that palmitate oxidation was not affected by vary- ing the BSA concentration in the media. In consideration of the four strong fatty acid binding sites on BSA and that in ‘the majority of published studies on fatty acid oxidation the rmolar ratio of fatty acidzBSA ranges from 2.0 to 7.0, it was (fiecided to keep the fatty acidzBSA molar ratio constant at “.0. Stock solutions of palmitateEl-luc], palmitateEU-luC], anri oleateEl-luC](free acids, Amersham Corp.) were stored in berizene at A0, octanoateEl-luC](sodium salt, Amersham Corp.) 1J1 absolute ethanol at NO, and acetate[l-luC](sodium salt, .Mmersham Corp.) in distilled water at —150. AA U5 Palmitate and oleate were prepared for incubation as follows: 1) 1.0 ml of labelled fatty acid in benzene (about 0.01 mCi) was dried under N 2) unlabelled fatty acid (free 2, acid, Sigma Chem. Co.) was added to desired concentration; and 3) the mixture was dissolved in 1.0 ml of absolute ethanol and stored at 40 prior to use. A 0.01 ml aliquot of the fatty acid in ethanol solution was added to 3 ml of KRB buffer in 25 ml flasks in a shaking water bath (37°, 60 cycles/min, Dubnoff Metabolic Shaker). The flasks were then gassed for 10—15 sec with 02:002 (95:5) and capped with rubber stoppers fitted with plastic center wells (Kontes of Vineland, New Jersey) containing a fluted filter paper 2 mm2. Gassing the flasks with 02:002 prior to incubation stimulated palmitate oxidation (about 25%) at 180 and 240 min of incubation. The flasks were then left shaking for approximately one hour to allow dissolution of fatty acid. Originally, palmitate was added to the incubation as an ammonium salt according to Ontko and Jackson (1964). However, using ethanol as a carrier for fatty acid was easier, and rates of palmitate oxidation were equivalent (Appendix Table 2). Also, palmitate oxidation was not affected by increasing volumes of ethanol. Eissue Collection and Preparation for Incubation Mammary tissue pieces, weighing 30 to 50 g, were excised from udders of lactating Holstein cows killed by a bolt gun. The tissue pieces were immediately placed in an insulated flask containing warm (37o) KRB buffer (2 units oxytocin/lOO nfl-, Sigma Chem. Co.). Tissue was then rinsed in warm KRB 46 buffer (no oxytocin), cubed, trimed to minimize non-parenchymal tissue, and sliced using a Stadie-Riggs microtome. The Stadie- Riggs was grooved to give slices of approximately 0.5 mm in thickness. The slices were placed in a common flask containing warm buffer, then rinsed several times with warm buffer. The data in Appendix Table 3 show that collecting and rinsing tissue in warm buffer as opposed to cold buffer enhanced palmitate oxidation. Slices were gently blotted to remove excess fluid, trimed to minimize non-parenchymal tissue, weighed (usually 60 to 80 mg) on a Mettler balance, then added to incubation flasks to commence incubation. Rates of palmitate oxidation replicated well with tissue slices ranging from A0 to 100 mg. Treatments within experiments were run in quadruplicate flasks, and values were averaged. Determination of C02 Production of CO2 was corrected against blanks (flasks incubated for 0 min with tissue). Incubation was terminated after various times by injecting 0.3 m1 of 5 N H2S0u into the media, followed by injection of 0.3 ml of methyl benzathonium hydroxide (tradename Hyamine Hydroxide, Sigma Chem. Co.) into the center well to trap C02. The flasks were shaken for an additional hour, after which the center well and its contents were transferred to scintillation vials. Samples were counted in a Nuclear-Chicago model 720 liquid scintillation counter for two-ten minute counts in 10 ml of Aqueous Counting 47 Scintillant (ACS, Amersham Corp.). Counting efficiency was 87.5%, using [inc] benzoic acid (New England Nuclear) as an internal standard, and 82.5% in the presence of 0.3 ml of Hyamine Hydroxide. The rate of palmitate [l-luC] conversion to 1 CO2 was calculated as follows: l4C02 _ (dpm in sample) - (dpm in blank) (pmoles/mg-min) (Specific activity added)(mg tissue)(min) Radioactivity in the blanks was 10-50% of that obtained in samples incubated for the shorter incubation times, and approximately 10% of that obtained for the longer incubation times. To determine concentration of glucose remaining in incu- bation media after C02 collection, 1.0 ml of media was neutralized and analyzed for glucose by using a coupled enzymatic assay (kit no. ll5—A, Sigma Chem. Co.). Source of Animals All of the animals used in this study were lactating Holstein cows either from the university herd or a local abattoir. The cows from the university herd could be divided into two groups. One group consisted of cows killed in early lactation (10 to 16 days postpartum) from two separate physiology experiments. The other group of cows from the university herd were cull cows. These cows were in middle to late lactation and culled for various reasons, mostly infer— tility and mastitis. Briefly, the physiology experiments involved: 1) prepar- tum injections of an ergot derivative or an ergot derivative 48 plus prolactin and 2) serum hormone patterns in cows nursing calves. Rates of palmitate oxidation for all groups of cows are summarized in Appendix Table A. Palmitate oxidation was not affected by any of the treatments in the physiology experiments and these cows are grouped as early lactation. Lipid Extraction of the Tissue Slices After the center wells were transferred to scintillations vials, the tissue slices from quadruplicate flasks were pooled and rinsed four times in distilled water to remove "loosely attached" fatty acids. The tissue slices were then extracted for lipids by the method of Folch et al. (1957). Tissue slices were homogenized in chloroformzmethanol (2:1, 2 ml per tissue slice) with a Polytron homogenizer (Brinkman Instruments). The homogenate was filtered, and the homogenizer and filter paper washed twice with 5 m1 of chloroformzmethanol (2:1). The volume of the total extract was recorded and a 5.0 ml aliquot was counted and counting efficiency was 75%, and the efficiency of recovery of palmitate [l-luC] by the Folch method was 95%. The rate of uptake was calculated as follows: dpm in total dpm in totaj + extract of - extract of rate of Uptake = sample _ lank CO2 (pmoles/mg-min) (specific activity added)(mg)(min)production I7Otal recovery of dpm as CO2 plus tissue plus media was 93- 987. of that added. To the lipid extract 0.2 volumes of 0.05 M KCl were added, vortexed three times, and centrifuged (IEC model K A9 centrifuge, Damon/IEC Division). The upper phase (water phase) was drawn off with a disposable Pasteur pipet, and the chloroform phase (lower phase) was washed again. The two upper phases were combined, volume recorded, and a 0.5 ml aliquot counted in 10 ml of ACS. Counting efficiency of 0.5 m1 of the upper phase was 79.3%. Accumulation of water- soluble intermediates was less than 1% of the total activity in tissue, and therefore, is not presented. Excess anhydrous Na2804 was added to the chloroform phase to remove "trapped" water. The chloroform solution was centrifuged, the super- natant drawn off into scintillation vials, dried under N2, and resuspended in 0.3 to 0.5 ml of chloroformzmethanol (2:1) for thin-layer chromatography. Fatty Acid Esterification Esterified fatty acid fractions were determined by thin- layer chromatography (TLC). A 30—50 pl aliquot of the lipid extract in chloroformzmethanol was spotted on silica gel-60 TLC plates (0.25 mm gel thickness, precoated on glass, E.M. Merck, Co.). The TLC plates were develOped 50 to 60 min in a glass tank with hexanezdiethyl ether:g1acial acetic acid 1 (70 30:2). A neutral lipid standard mix (Sigma Chem. Co.) Containing monoglyceride, 1,2— and 1,3-dig1ycerides, fatty ‘&Cid, and triglyceride was co—chromatographed. The plates Wfire then dried and sprayed with 0.2% dichloroflourescein in etfluanol for visualization. The spots were scraped-off and COunted in 10 ml of A08 to determine percentage distribution. Silice size of the spots varied, quenching and efficiency of eJJJtion of radioactivity from the gel was determined. Spots 50 of varying but known size and radioactivity were dried, sprayed, scraped—off, and counted. Quenching was nil and recovery of radioactivity was essentially 100%. Rates of accumulation for each lipid fraction were calculated as follows: - Rate (dpm in total extract)(% of radioactivity (pmols/mg-min) _ on TLC plate) (Specific activity added)(mg tissue)(min) The rates for fatty acid were corrected against blanks, since fatty acid was the only labelled lipid in the blanks. Incubation media, from an experiment with tissue slices incubated in the presence of glucose and palmitateEl-luc] for 60 and 180 min, was extracted for lipids and the extract was chromatographed. Fatty acid was the only lipid fraction detected, thus data reported in the text apply to tissue slices only. CARNITINE PALMITOYLTRANSFERASE ACTIVITY (CPT) CPT activity was assayed spectrophotometrically in mammary mitochondria according to Bieber et al. (1972). This assay is based on the principle that DTNB (5,5'—dithiobis- (2—nitrobenzoic acid)) traps coenzyme A liberated from palmi- 't0yl—CoA by the action of CPT. SOxurces of Reagent s Tris (Tris hydroxymethyl aminomethane), EDTA (ethylened- iantine tetraacetic acid), D—mannitol, HEPES (N-2-hydroxyethy- lpiperazine-N'-2-ethanesu1fonic acid) buffer, Triton X—100, DTBFB, and S-palmitoyl—CoA (free acid) were supplied by Sigma CheEmical 00., sucrose by Mallinckrodt, NaEDTA by Fisher 51 Scientific Co., and sodium bicarbonate by J.T. Baker Chemical Co. L(-)—carnitine was a generous gift of Dr. L. L. Bieber of the Biochemistry Department of Michigan State University. Isolation of Mitochondria Mammary mitochondria from three cows were assayed for CPT with varying concentrations of carnitine and palmitoyl- CoA, in order to determine appropriate substrate concentra- tions. Mammary tissue was excised from lactating cows at a local abattoir and placed in cold (40) buffer (0.25 M sucrose, 0.2 mM EDTA, pH 7.5 with 150 mM Tris buffer). Tissue was finely minced with scissors and homogenized in 10 volumes (wt/v) of buffer in a Ten-Broeck ground, glass homogenizer. Homogenates were centrifuged at U0 for 12 min at 750 x g. The supernatant was centrifuged at 40 for 12 min at 6700 x g. The pellet was resuspended and the 750 x g and 6700 x g centrifugations repeated twice. The mitochondrial pellet was suspended in 5 ml of buffer (70 mM sucrose, 220 mM mannitol, 2 mM HEPES buffer, pH 7-4, 1 mM EDTA) and stored frozen. For isolation of mitochondria from tissue slices the above procedure was modified somewhat. Since mitochondrial Yield was low from slices, the 750 x g and 6700 x g centrifu- égations were not repeated. The mitochondrial pellet, however, “Has washed 3 times with cold homogenization buffer, resuspended irl 0.3 ml of suspension buffer, and stored frozen. 52 CPT Assay Stock solutions of reagents (1% Triton X—100, 100 mM 1(—)-carnitine, 0.7 mM palmitoyl—CoA, and 2.5 mM DTNB in 0.01 M NaHCO3 pH 7.0 were prepared and stored frozen. A stock solution of buffer (2.3 M Tris, 0.022 M NaEDTA, pH 8.0) was prepared the day of assay. Reaction was started by adding 100 pl of a reagent and buffer mix solution, water, and mitochondrial suspension (kept on ice). Final reaction volume was 200 pl and the reaction was monitored at #12 nm (Gilford Spectrophotometer model 2A00-S) at room temperature. For the CPT assay in tissue slice mitochondria 60 ul of water and 40 ul of mitochondria were added to the cuvette. The final concentrations of reagents during the assay were: 116 mM Tris-HCl 0.1 mM NaEDTA, 0.1% Triton X—100, 35 uM palmitoyl-CoA, 0.25 mM DTNB, and 1.25 mM l(-)-carnitine. The change in optical density was determined for the first minute of the reaction, because the assay is linear for only 2-5 min at 250. To determine palmitoyl-CoA hydrolase activity, duplicate samples were assayed in the absence of carnitine. CPT activity was obtained by subtracting the initial rate of formation of CoA in the absence of carnitine from the rate in the presence of carnitine, an extinction coefficient of '13,600 for DTNB was used to determine enzyme rates. Enzyme activity was linear with protein concentrations tCD 0.24 mg/ml. Protein was determined by the method of LJO‘wrey et al. (1951). 53 STATISTICAL ANALYSIS Experiments were blocked according to treatment and time. Statistical evaluations were by analysis of variance, orthogonal contrasts and t-tests. For some sets of data there was mild heterogeneity of variance even after log transformation. However, standard statistical tests are known to be robust against departures from the assumption of equal variance. RESULTS AND DISCUSSION When investigating controls in vitro on a particular metabolic pathway, it is imperative to establish maximum reaction conditions for the incubation system to be used. As previously stated, palmitate oxidation per mg tissue was not affected by amount of tissue, BSA, and ethanol in the incubation media within limits used, and only slightly en— hanced by gassing the media prior to incubation. However, the rates of palmitate oxidation in mammary tissue slices were found to increase in a curvilinear fashion over time, shown in Table 2. The values shown are rates of palmitate oxidation obtained at the particular incubation time. Thus, mammary tissue can oxidize fatty acid, but rates of oxidation increase over time of incubation in vitro. Using typical rates of palmitate oxidation at 180 min for mammary tissue slices obtained from cows in which total udder weight was determined, it was estimated that palmitate oxida- tion could potentially account for 15 ml of 02 consumption by the whole udder per min using an R0 of 0.70 for the oxidation of lipids. This rate of 0 consumption is 6.2% of the rate 2 <10) <12) (6) *Rates at 180>30 (P<0.10), 240>30 (P<0.01), 180>60 (P<0.05), and 240>60 (P<0.01). lPalmitate at 0.26 mM. 2Rates expressed as pmoles/mg-min. 3Standard deviation. “Number of replicates. As mentioned, rates of palmitate oxidation increase with time of incubation, and further investigation of this time effect may provide insight concerning regulation of fatty acid Oxidation in the mammary gland of the cow. However, certain questions must first be adequately recognized and answered. Is the effect of incubation time on palmitate oxidation a Inatter of substrate solubility or an artifact of the incubation System? Are endogenous pools of fatty acid utilized in pre- ference to the exogenous pools? In order to determine if this increasing oxidation rate With time was unique to mammary tissue or an artifact of the 56 incubation system, the rate of palmitate oxidation was deter- mined in rat kidney cortex slices in the same manner as that determined for mammary tissue slices. The data in Table 3 show that rates of palmitate oxidation in rat kidney cortex slices were constant over time. The maximum rate of palmitate oxidation (2.9 pmoles/mg—min) observed in mammary tissue slices (Table 2) is comparable to rates found in rat kidney cortex slices (3.5-4.2 pmoles/mg—min). Barac-Nieto (1976) found that rat kidney cortex slices oxidize 0.98 mM palmitate at a constant rate of 8.3 pmoles/mg—min, which is comparable to the rates shown in Table 3 with 0.26 mM palmitate. Thus, increasing rates of palmitate oxidation with increasing time of incubation is not an artifact of the incubation conditions, and mammary tissue slices oxidize palmitate at rates similar to those of rat kidney cortex slices. Table 3.--Pa1mitate Oxidation Versus Time of Incubation in Rat Kidney Cortex Slices. Time 30 60 120 180 240 Palmitatel to 0022 3.5 3.5 5.0 4.2 3.9 SD3 0.5 0.3 1.5 0.2 - NLl (2) <2) <2) <2) <1) 1 Palmitate at 0.26 mM Rates expressed as pmoles/mg-min. 3Standard deviation. “Number of replicates. 2 57 The data in Tables 2 and 3 were taken from eXperimentS' in which palmitate was added to start the incubation. Pal— mitate is insoluble in aqueous media, and dissolution of fatty acid by binding to protein is not an immediate process. Hence, one can envision that as palmitate becomes available with time in a physically suitable form to the tissue, its rate of oxidation will increase. The results shown in Table 3 and data presented in the remainder of the text from experiments in which approximately one hour was allowed for binding of palmitate to protein in the media prior to start of incubation by adding tissue show that palmitate oxidation is enhanced by incubation time. Thus, the effect of incubation time can not be explained by substrate solubility. It should be noted that data in Tables 2 and 3 were from tissue slices that were weighed at the end of the incubation. The data presented in the remainder of the text are from experiments in which the tissue slices were weighed prior to start of incubation. Thus, these values may not be directly comparable to those obtained in the following experiments. The effect of substrate concentration on palmitate oxidation in mammary tissue slices is shown in Table A. A palrutate concentration of 0.26 mM gave maximum rates of oxidation at both times. This level of palmitate would correspond to arterial plasma fatty acid concentrations in the fed cow (Bickerstaffe et al., 1974). Also, rate of palmitate oxidation doubled between 60 and 180 min at all Concentrations tested. Half-maximal velocity was reached 58 at 0.1 mM palmitate for both times. Therefore, maximal rates of palmitate oxidation are obtained with physiological fatty acid concentrations (0.26 mM) at both times, and the effect of incubation time is independent of this substrate concentration which_normally would be expected in mammary tissue. Thus, it seems that in mammary tissue fatty acid oxidation is not regulated by fatty acid concentration. Table A.——Pa1mitate Oxidation Versus Palmitate Concentration. Time 1 Concentration 60 180 '—-pmo1es/mg—min-— 0.065 mM 0.31 0.6a 0.13 0.5X 1.2bY 0.26 . 0.8X 1.8Cy 0.33 0.8X 1.8Cy abcMeans in columns with different superscripts are different P<0.10. XyMeans in rows with different superscripts are different P<0.02. 1Standard error of difference between means is 0.26, and number of replicates is A. A comparison was made of the effect of fatty acid chain length on fatty acid oxidation as revealed in Table 5. Palrfitate, oleate and octanoate oxidations were determined ESimultaneously in four experiments, while the rate reported fYIr acetate oxidation was from three independent experiments. que rates reported here are not on an equivalent carbon—atom baisis. The data clearly show that oxidation decreases, as 59 chain length increases up to C16. Acetate is oxidized at substantially greater rates than is palmitate, oleate or octanoate, which agrees with Bickerstaffe et a1 (1974) who found that acetate is the preferred oxidative substrate in the bovine mammary gland. Swenson and Dimick (1974) found substantial rates of medium-chain fatty acid oxidation in the goat mammary gland. Annison et a1. (1967) could not detect oxidation of long—chain fatty acids in the mammary gland of the fed goat. Palmitate and oleate are oxidized at equal rates irrespective of chain length and unsaturation. The oxidation of octanoate and acetate marginally changes with time, while oxidation of palmitate and oleate is doubled by incubation time. Table 5.--Fatty Acid Oxidation Versus Chain Length. Time 1 Fatty Acid 60* 180 —-pmoles/mg—min--- Palmitate 0.72a 1.3a Oleate 0.7a 1.3a Octanoate 9.1b 11.3b Acetate 49.73 57.73 abMeans in columns with different superscripts are different P<0.01. :lPalmitate, oleate, and octanoate at 0.26 mM, acetate at 0.6 mM. 2Standard error of difference between means is 0.46, and number 0 7 (P<0.01). **At 180 min 1.9>1.4 (P<0.05). abMeans in rows with different superscripts are different P<0.01. 1Standard error of difference between means is 0.21, and number of replicates is 3. It should be noted here that determination of C02 specific activity over time would yield more definitive answers to the I above hypothesis. However, attempts to determine the C02 specific activity in earlier unrelated experiments using NaOH afS a C02 trap were unsuccessful. To test the validity of the previous hypothesis in another mailner, mammary tissue slices were again preincubated for 62 various times, but unlabelled palmitate was present during preincubation after which palmitate [l—luC] was added. The presence of palmitate during preincubation should block palmitate oxidation by replenishing endogenous pools of fatty acid. The results are shown in Table 7. Rate of palmitate oxidation tripled between 60 and 180 min with 0 min preincu- bation. However, preincubation of the tissue slices for 60 min with unlabelled substrate maximally stimulated palmitate oxidation and abolished the effect of incubation time. Thus, increasing rates of palmitate oxidation are independent of the presence of substrate prior to incubation. This would negate the hypothesis that utilization of endogenous fatty 14 acids limits C-palmitate oxidation in vitro. Table 7.-—Effect of Preincubating Mammary Tissue Slices With Unlabelled Palmitate on Palmitate Oxidation. - “ Time Preincubation 60“ 180 ----- pmoles/mg-min--- 0 min 0.6la 1.8b 30 1.4 2.0 60 2.1 2.0 90 2.2 2.0 ¥I At 60 min 2.1>O.6 (P<0.05). abMeans in rows with different superscripts are different P<0.05. 1Standard error of difference between means is 0.39, and number of replicates is 2. 63 In summation, the observed effect of incubation time on fatty acid oxidation can not be explained by substrate solubility, concentration, or presence of endogenous fatty acids, and is unique to bovine mammary tissue when compared to the rat kidney. Experiments were designed to indirectly test whether the entire palmitate molecule is oxidized to 002 in mammary tissue slices. This hypothesis was evaluated by comparing the oxidation of palmitateEl-luC] to palmitateEU-luC]. If fatty acid oxidation is limited by the B-oxidation enzymes in the mitochondria and/or peroxisomes, then greater rates of oxidation with palmitateEl—luc] in comparison to palmitate [U—luC] would be expected. If flux through the citric acid cycle limits palmitate oxidation to C02, then a greater rate of accumulation of non—acid labile, water-soluble intermediates (mostly ketone bodies and citric acid cycle intermediates) U-luC] versus would result from the oxidation of palmitate[ palmitateEl-luC]. The data in Table 8 indeed reveal that oxidation of palmitateEl—luC] is greater than palmitateEU-luc]. Also, time of incubation appears to have a more pronounced effect on palmitateEU-luC] oxidation, nearly a three—fold increase. Rates of accumulation of non—acid labile, water- soluble intermediates are low with both palmitate isotopes. Differences between the two isotopes in rates of uptake are Statistically insignificant. Thus, it appears that the enzymes of 8-oxidation may limit I palmitate oxidation. In agreement, Stanley and Tubbs (1975) 64 found substantial amounts of saturated acyl-CoA intermediates in liver mitochondria oxidizing palmitoyl—carnitine, and con— cluded that acyl-CoA dehydrogenase may be rate—limiting for fatty acid oxidation. Harper and Saggerson (1976) showed that in isolated rat adipocytes three palmitate isotopes were oxidized at different rates and in the following order: palmitateEl-luC] > palmitateEU-luC] > palmitateEl6—luC]. Waterson and Hill (1972) showed that enoyl-CoA hydrase demon- strates much greater substrate specificity for short- and medium—chain substrates than for long—chain substrates. This coupled with the data in Table 5, which shows greatly enhanced rates of octanoate oxidation as opposed to palmitate and oleate, suggests that the B-oxidation enzymes are rate-limit- ing. Long—chain fatty acyl-CoA is at a metabolic branchpoint between the pathways of esterification and oxidation. The effect of metabolites, which serve as precursors to g1ycerol- 3-phosphate (G3P),on fatty acid oxidation is well documented in non-ruminant tissues. In general, when one pathway is favored it is at the expense of the other pathway. In the bovine blood glucose is the precursor for G3P in the mammary gland. Therefore, stimulation of fatty acid esterification should influence fatty acid oxidation. The influence of glucose on fatty acid oxidation was investigated, and the re— sults are shown in Table 9. Glucose at a physiological concentration (2.8 mM = 50 mg/100 ml) markedly inhibits palmitate oxidation, and is especially effective when palmi- tate oxidation is maximally stimulated by doubling its 65 .25 mm.o as masaasasa: .mmptanEpopcfi oHQSHOmImemz n m3m .COHposoopd moo mo mums + oommflp Ca mpfi>fiuom mo coapmH58500m mo mums n oxmuoom .: ma moprHHaop Mo Lopez: cow .ma.o com «as.m .HH.o mam m3 new .oxmpd: .moo hoe momma semapon mommLmMMHo mo whoppo osmosmpma .mo.ovm pcopmmeao 0mm mpoflpomLmQSm pcoanMHo Spas mzop CH mcwozmx .Ho.ovm pcohmmmfio mam mpdfipommeSm ucmhmemao Qua: mcssaoo CH mcmozom H.ov a.o m.m m.aa sem.o gem.o moaausaeeseHEHss H.ov H.o sa.m x:.ea ssm.H ass.o afloaanaiaaseaEHsg IIIIIIIIIIIIIIIIIIIIIIIIII CHEIwE\moHoanlillIIIIIIIIIIIIIIIIII owe om owe om ems om amoeonH m.am3 , m.mesaas awed .Aoealpuseseasasi nsnat> mosasaatrsaaeHsa co soaaseaxo-u.w wanes 66 concentration. In agreement, glucose causes a 50% reduction in palmitate oxidation in chick embryo heart cells (Rosenthal and Warshaw, 1973), and glucose plus insulin blocks palmitate oxidation in rat hemi-diaphragm pieces (Fritz and Kaplan, 1961). Glucose apparently blocks fatty acid oxidation by supplying more G3P, which in turn would deplete the fatty acyl-CoA pool available for oxidation. Reimer et a1. (1975) showed that increased G3P levels derived from an increased glucose turnover, due to insulin, stimulates fatty acid eSterification. Rates of palmitate oxidation increase over time, although not statistically significant, with glucose present. It is unlikely that this is due to a depletion of glucose from the media as incubation progresses, as 1.4 mM glucose remains in the media after 180 min. The results in Table 10 show that palmitate oxidation is maximally inhibited by glucose at 1.4 mM in the media. Thus, rates of palmitate oxidation tend to increase with time even in the presence of glucose, which can not be explained on the basis of glucose concentration. Accordingly, acetate may inhibit long—chain fatty acid oxidation in the mammary gland by competing with fatty acids for available coenzyme A in the mitochondrial- matrix space, entry into the citric acid cycle, or at the oxidative phosphorylation level. Henceforth, this will be termed substrate competition at the mitochondrial level. Acetate may also compete with fatty acids for available coenzyme A in the cytoplasm. Also, acetate may inhibit fatty acid oxidation via malonyl—CoA which inhibits carnitine palmitoyltransferase activity. ¢& Table 9.4—Effect of Glucose on Palmitate Oxidation. Time __ Treatments 60 ' ’ ' " 180* -------- pmoles/mg-min------- 0.13 mM Palmitate 0.6al 1.3b 0.13 mM P + Glucose2 0.2 0.5 0.26 mM Palmitate 0.9a 2.2b 0.26 mM P + Glucose 0.3 0.6 (2.2)3 (1.4)3 *At 180 min 1.3>‘O.5 (P 0.6 (P<0.01), and 2.2> 1.3 (PHooc Hcooo one .Aoom: Eopmzm CpHs oo mo 53m on mH oxmpao m .o ma noosoHHoca Co cones: one .mm.o one .ae.o .mo.o .mm.o .mm.o .oo.o .eo.o .mo.H .OH.o one \\\ 1 I ~r- 1 I -.04 0.04 0.08 0.12 0.16 1 (Palmitoyl -CoA), pm 008- (b) 0.08- ] 0 Rate 0.04.1 . Km=07mM Vmax: 33.3 nmoles CoAI mg- min (Carnitine), mM Figure 5.--Double—receprocal plots of substrate concentration versus carnitine palmitoyltransferase activity in mammary mitochondria. 77 The rates of g1ycero-lipid accumulation tend to decline over time. One can envision that as less palmitoyl-CoA is being esterified to G3P more palmitoyl-CoA would be available for oxidation, resulting in increasing rates of oxidation with time. The shaded areas in Figure 6 represent the devia- tion from linearity over time in the formation of glycero— lipid and 002. The decline in glycero-lipid formation could quantitatively account for the increasing rates of palmitate oxidation in the absence of glucose. By the same reasoning, rates of palmitate oxidation should increase even more with time in the presence of glucose, since rates of glycero-lipid formation decrease with time (P<0.05) in the presence of glucose more than in its absence. However, rates of C02 production were less than predicted by the presumed increase in palmitoyl—CoA availability. However, increasing rates of palmitate oxidation over time is consistent with the hypothetical limitation of oxidation of palmitate esterification. Medium~chain fatty acids can be activated in the intra— mitochondrial space, and the resultant CoA esters are not accessible to acylation of G3P. Since fatty acid esterification is an extra-mitochondrial process, glucose should not affect medium-chain fatty oxidation. Therefore, mammary tissue slices were incubated with octanoateEl-luC] with or without glucose and rates of octanoate oxidation and esterification were determined. The data are shown in Table 14. The rates of octanoate esterification shown in Table 14 are the means of '78 1800 - 1800 1400 1200 1000 pmoles/ mg mm mm «m mm l 60 120 180 lfime Figure 6.—-Conversion of palmitate to CO and glycero-lipids versus time. The shaded areaé represent the deviation from linearity over time in the forma- tion of glycero-lipid and 002. 79' rates obtained at 60 and 180 min, and the complete data is presented in Appendix Table 6. The results reveal that glu- cose does not influence octanoate oxidation, nor do rates of octanoate oxidation change with time. Contrarily, glucose markedly enhances octanoate esterification in all of the lipid fractions, and the rates of glycero-lipid accumulation with octanoate are similar to those found with palmitate (Table 13). Marshall and Knudsen (1977) found that microsomal bound 1,2-diacylg1ycerol acyltransferase from mammary tissue of lactating cows incorporates equal molar amounts of butyryl- CoA, hexanoyl—CoA, or palmitoyl-CoA to microsomal-bound diglyceride. In addition, McGarry and Foster (1971) demon— strated that octanoate oxidation and esterification in the isolated perfused rat liver is unaffected by antiketogenic compounds. Thus, the ability of the mammary gland to esterify large quantities of short- and medium—chain fatty acids is unique. The predominant lipid fraction synthesized is TG, which is expected since medium-chain fatty acids are preferen- tially acylated to the sn-3 position (Breckenridge and Kuksis, 1969) and is the final step in TG formation (Marshall and Knudsen, 1977). Therefore, glucose does not block octanoate oxidation but enhances octanoate esterification. These results suggest the existence of two large segregated pools of octanoyl—CoA at two sites of activation, microsomes and intra- mitochondria. Glucose, by virtue of its ability to supply G3P, presumably increases octanoate esterification at the microsomal site only. .EE w.m UCm om.o pm omooCHc oCm oCCOCCCQOm moo 1 sec HsHoHoa u see Hogans ACHEVAoCmme mEVApocom mpH>Huom OHCHoodmv Sop HmeHCH HmeHC CH ACHEIwE\noHoEov fiasco anae Eco Hcoooc I AcosHo use so as so Hosnnao ca soc Hsooov n ooom uoHom enema CmHCHHoomCuCH mo CoHCmHCECoom Co pump on opmHCono on com: mm: COHmecm wCHBOHHoe on om .ooNHHHpC was oCCOCmpoo cocoa mo pmozm .: mH moumoHHooC Co access can .sm.m coo .zm.H . m.H .Hm.H .mH.o .mo.o .mo.o .mo.H .me.H one «a one «H0 «H2 «we .00 no: "Hm .oxmpao a oo 90% mCmoE Coospoo ooCmComeHo Co mCOCCo oCmoCmpmH .mo.ovm pCoComeHo oCm mpCHComCooCm pCoCoCMHo Csz mCECHoo CH maoz 80 mo .Hoo.ovm pCoComCHo oCm mpaHComCoQCm uCoCoCeHo CpHs mCECHoo CH mCmosz m.ma om.MH oo.mH om.HH oz.H nm.o 25.0 o.sH m.OH o.m momooCHc + 0 :.ml Us.w o:.w om.s mm.o mH.o mm.o m.mH m.oH m.w oCmOCmpoo IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII CHEIwE\moHoEQIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII . o m a m Cos ooh m.H0.7 (P<0.05), 1 4>0 4 (P<0.05), 1.0>0.4 (P<0.10). abMeans in rows with different superscripts are different P<0.05. lL-carnitine at 2.0 mM, a generous gift of Dr. L. L. Bieber, Biochemistry Department, Michigan State University. 2Standard error of difference between means is 0.16, and number of replicates is 2. slices. This was approached in two ways. In one manner, the influence of B-hydroxybutyrate (BOHB) on palmitate oxidation was studied since the enzyme BOHB dehydrogenase is a mitochon— drial enzyme in bovine mammary tissue (Bauman et al., 1970), and the other manner was by addition of l—lactate since lactate dehydrogenase is a cytoplasmic enzyme. Rates of palmitate oxidation were unaffected by 0.2 mM and 0.4 mM BOHB, as shown in Table 18. Thus, it would appear that palmitate oxidation is insensitive to mitochondrial redox state. However, BOHB dehydrogenase activity and BOHB éflld acetoacetate levels were not determined. BOHB also serves as a primer for fatty acid synthesis in mammary tissue. Thus, 13MB extent to which BOHB entered the mitochondria and effect- ively altered the redox state was unknown. 87 Table l8.—-Effect of B-hydroxybutyrate on Palmitate Oxidation. Time' Treatments 60 180 ------ pmoles/mg-min---—- Palmitate 1.1al 2.0b P + 0.2 mM 80HB3 1.2a 1.8b P + 0.4 mM BOHB 1.4a 1.9b abMeans in rows with different superscripts are different P<0.02. 1Standard error of difference between means is 0.18, and number of replicates is 4. 3BOHB from Sigma Chem. Co. and palmitate at 0.26 mM. Lactate, however, markedly inhibits palmitate oxidation, shown in Table 19. The rates of palmitate esterification are averages of rates obtained at 60 and 180 min and the complete data is presented in Appendix Table 7. Since these experi— ments were replicated only twice, statistically significant differences between treatment means were difficult to achieve, but trends do exist for increasing esterification rates with lactate. Rate of palmitate oxidation tends to increase with time in presence of lactate, and rates of palmitate esterifi— cation to glycero—lipid sharpely decrease between 60 and 180 Inin (15.3 vs 11.5). Palmitate oxidation is typically enhanced 'by time in the absence of lactate and glucose. This is in spite of increasing rates of palmitate esterification (rates :for g1ycero-lipid at 60 and 180 min are 7.7 and 9.6). The :inclusion of glucose was for comparative purposes and the eeffects of glucose on oxidation and esterification of 88 .SE m.m pCm om.o pm omOOCHw UCm mumpHEHmo ..oo .EoCo meHm EOCC “SE o.m um mumpomHIH .ao .qz .09 .oo .o: .qa .oxcoop m .m mH mopmoHHooC co access can om.o com .es.m .mm.m .mo.m .oo.o .mo.o .mm.o .mm.m .mm.o one .am one . o 0 COM mCmoE Coospmn ooCoComeHo mo mCOCCo oCmpCmpm H .OH.ovm pCoCoemHo oCm muoHComCmCCm pCoCoCmHo Csz mCECHoo CH mCmozoo .mo.ovm pCoCommHo mam mpCHComCoCCm pCoCommHo CpHs msop CH mCmozzx .mo.ovm pCoComeHo oCm mpCHComCooCm pCoCoCCHo CpHs mCECHoo CH mCmoz om m.o| ow.mH om.mH m.OH oH.m m.o m.H m.mH 93.0 m.o omooCHo + m m.on tow.mH oom.HH m.m oom.m m.o >.H o.:H no.0 m.o mopmpomq + m m.o| om.w o:.s o.o oH.H m.o :.H H.oH mm:.H xo.o opmpHEHmm lllllllllllllllllllllllllllllllll CHEIwE\moHoEoIIIlulu:IIIIIIIIIIIIIIIIIIIII H octanoate > palmitate or oleate. Palmitate at 0.26 mM which is the arterial plasma fatty acid concentra— tion, gave maximum rates of oxidation, and half—maximal rates of oxidation were obtained at 0.1 mM palmitate. Rates of palmitate oxidation increased with time of incubation at all concentrations of palmitate tested.) Also, increasing rates of fatty acid oxidation with time are unique to long—chain fatty acids, which can not be ex— plained by activity of carnitine palmitoyltransferase, as enzyme activity was constant over time and maximal enzyme activity was greater than rates of palmitate oxidation. Rates Of palmitateEl—luC] oxidation were greater than rates of palmitate[U-lu0] oxidation, suggesting that the B-oxidation enzymes may limit fatty acid oxidation. 92 93 Rates of palmitate oxidation are greatly diminished by acetate and glucose at physiological concentrations. Acetate inhibits palmitate oxidation but not palmitate esterification. This suggests that acetate inhibits palmitate oxidation by substrate competition at the mitochondrial level or via malonyl-CoA inhibition of carnitine palmitoyltransferase, and not by competition with palmitate for available CoA in the cytoplasm. TOFA, an inhibitor of acetyl-CoA carboxylase, did not relieve acetate inhibition of palmitate oxidation, suggesting that acetate is not affecting palmitate oxidation via malonyl-CoA. However, the extent to which TOFA altered intracellular metabolism was not determined. In some experi- ments, rates of palmitate oxidation increased with time in the presence of acetate, which could not be explained by acetate depletion from the media. Glucose inhibits palmitate oxidation and markedly en- hances palmitate esterification to G3P. In some experiments, rates of palmitate oxidation tend to increase with time in the presence of glucose, which apparently is not due to glucose depletion from the media as the glucose concentration remaining in the media is sufficient to induce maximum depression of palmitate oxidation by glucose. At low palmitoyl-CoA levels acylation of G3P would be favored over that of carnitine, since the apparent Km of 21 uM palmitoyl-CoA for carnitine palmitoyltransferase is greater than the apparent Km for 4 uM palmitoyl-CoA for G3P acyltrans- ferase as found by Kinsella and Gross (1973). Thus, glucose 94 inhibits palmitate oxidation presumably by decreasing fatty acyl—CoA available for oxidation by stimulating palmitate‘ esterification of G3P. Glucose does not affect octanoate oxidation, even though octanoate esterification to G3P was stimulated. This could be a reSult of two relatively large pools of octanoate at the microsomes and mitochondria. Octanoate activated at the microsomes is accessible to acylation of G3P. Octanoate activated in the mitochondria is not accessible to acylation of G3P. Rates of g1ycero-lipid formation from palmitate tend to decrease with time of incubation. The decreasing rates of glycero—lipid formation could quantitatively account for increasing rates of palmitate oxidation with time. Clofenapate, an inhibitor of glyceride synthesis in rat liver, was used as a model for studying how modifying palmitate esterification would affect oxidation. Clofenapate decreased rates of glycero-lipid accumulation and marginally stimulated palmitate oxidation. Clofenapate was also partially effective in relieving glucose inhibition of palmitate oxidation. Clofenapate enhanced the accumulation of intracellular fatty acid, which was assumed to be composed of mostly acyl-CoA esters. Addition of carnitine stimulated palmitate oxidation in tissue slices incubated in the presence or absence of clofenapate, suggesting that carnitine levels are limiting to fatty acid oxidation. However, this would be contradictory to data showing rates of palmitate oxidation increasing with time. 95 B-hydroxybutyrate addition did not affect palmitate oxidation. Lactate decreased palmitate oxidation, but stimulated palmitate esterification. Thus, lactate, a potential G3P precursor, decreased palmitate oxidation presumably by stimulating esterification. The extent to which cellular redox state was influenced by B-hydroxybutyrate and lactate was not determined. Rates of palmitate oxidation in mammary tissue slices from cows fasted for 72 hr were lower than those obtained from fed cows. Milk fat production was not affected by fasting, suggesting that lack of stimulatory effect of fasting on palmitate oxidation is a result of constant glyceride synthesis. Fatty acid oxidation as a proportion of the total oxidative metabolism was not determined and may have increased. In summation, the pathways of fatty acid esterification and oxidation seem to compete for available acyl—CoA with esterification being favored. Increasing rates of palmitate oxidation with time could be explained by simultaneously decreasing rates of glycero—lipid formation. Also, acetate inhibits palmitate oxidation presumably by substrate compe— tition at the mitochondrial level or by malonyl-CoA inhibition of carnitine palmitoyltransferase. It can be estimated that long-chain fatty acids can supply as much as 6-10% of the oxidative metabolism of mammary tissue. APPENDICES 96 Table Al.--Composition of Incubation Media. Volume Solution 100.0 ml 0.154 M NaCl 4.0 0.154 M K01 1.5 0.11 M CaCl 1.0 0.154 M KH2PO4 1.0 0.154 M MgSOu7H20 21.0 0.154 M NaHC03 (gas with CO. for 1 hour) 1 BSA 50 mg Penicillin/liter2 2 50 mg Streptomycin/liter 3 1.0 ml Fungizone/liter lMolar ratio of FAzBSA kept constant at 4.0. 2Purchased from Sigma Chemical Co. 3Purchased from Grand Island Biological Co. 97 Table A2.-—The Effect of Ethanol on Palmitate Oxidation. Timel Treatments:2 '60' 120 180 -----pmoles/mg-min ----- Expt. I NHu-Palmitate 2.4 Palm. in 10 ul Ethanol 2.6 Expt. II Vol. Ethanol: 2 ul 0.4 0.8 6 ul 0.4 1.0 10 ul 0.4 1.1 Expt. III Vol. Ethanol: 10 ul 0.7 1.2 1.2 20 ul 0.7 1.1 1.3 30 ul 0.7 1.0 1.0 1 Times of incubation were 60, 120, and 180 min and the rates of palmitate oxidation shown are those obtained at the particular incubation time. 2Each experiment represents one tissue sample. 98 Table A3.——The Effect of Collecting and Rinsing Tissue in Warm Versus Cold Buffer on Palmitate Oxidation. 'Time~ Treatments 607 180 -—pmoles/mg—min—— Cold 0.21 0.3 Warm 0.3 0.7 1Average of two experiments. 99 Table A4.--Palmitate Oxidation With Source of Cows. 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